- Email: [email protected]
P163 CRUCIAL ROLE OF THE PROTEIN C PATHWAY IN GOVERNING MICROVASCULAR INFLAMMATION IN INFLAMMATORY BOWEL DISEASE
Abstracts of ECCO Congress, Innsbruck, Austria, 1—3 March 2007
45
single subcutaneous injection of azoxymethane (AOM) at a dose of 10 mg/kg body weight. One week later the mice received 3% dextran sodium sulfate (DSS) in the drinking water continuously for up to 5 days. Specimens were taken from proximal and distal colon and rectum in weeks 6, 12 and 18 and macroscopic and microscopic changes of the colon mucosa were evaluated. In addition, immunohistochemical determination of beta-catenin was performed and the specimens were examined by scanning electron microscopy. Administration of DSS usually causes acute colitis in Balb/c mice but when combined with AOM the inflammation assumes a chronic form. Chronic colitis, low or high grade dysplasia or adenocarcinoma was visible in histological sections at week 6. At week 12 the incidence of macroscopically visible carcinomas reached nearly 100%. Strong nuclear and cytoplasmic expression of beta-catenin was observed in adenocarcinoma cells. Scanning electron microscopy confirmed changes in the position and structure of the crypts. These findings support the idea that chronic inflammation plays an important role in colon carcinogenesis. The experimental model used in our study may help in identification and modification of injurious substances.
lation in both CD patients and controls with a trend to lower expression in CD patients (P=0.079). Four hours after stimulation, expression returned to basal levels and a significant lower HNP1-3 expression was seen in CD patients (P=0.034). Furthermore, expression of MMP-8, -9, TIMP-1 and -2 mRNA in unstimulated neutrophils was similar in CD patients and controls. Only in the subgroup of patients carrying a CARD15 polymorphism, a significantly lower expression of TIMP-1 mRNA was observed (P=0.016). Conclusion: The reduced expression of HNP1-3 in CD patients can lead to impaired killing of phagocytized bacteria by neutrophils. Moreover, the decreased expression of TIMP-1 in neutrophils of CD patients with CARD15 polymorphisms might be implicated in a disturbed tissue remodeling effect
P160 MUCOSAL RELEASE OF PHAGOCYTE-SPECIFIC S100 PROTEINS IS STRONGLY ENHANCED DURING ACTIVE INFLAMMATORY BOWEL DISEASE
Open-labeled Phase I studies of visilizumab, a humanized non-FcR-binding anti-CD3 monoclonal antibody, in intravenous steroid-refractory ulcerative colitis and in inflammatory Crohn' s disease patients showed tolerability and clinical activity at doses 5-15 mg/kg administered on 2 consecutive days. Visilizumab caused rapid and reversible disappearance of peripheral blood T cells with rapid clinical improvement that in some patients persisted long after visilizumab was cleared from circulation. Preliminary data from two-third of dosed patient population suggest clinical response on day 30 may be related to peripheral blood CD8+ T cell count. We hypothesized that the clinical activity of visilizumab may be related to targeting several immune-mediated pathways important in IBD. Short-term exposure of resting PBMC to visilizumab in vitro induced mild T cell proliferation, cytokine and chemokine production and no measurable apoptosis. When added to coculture of PBMC and endothelial cells, visilizumab induced robust production of the chemokine IP-10 resulting in selective blockade of migration of CXCR3+ cells. Long-term exposure of resting PBMC to visilizumab led to preferential expansion of CD8+ T cells with regulatory function. Exposure of preactivated PBMC to visilizumab resulted in apoptosis of proliferating CXCR3+ T cells. Overall, our results suggest that visilizumab could act through multiple mechanisms — (1) elimination of proliferative activated CXCR3+ T cells through apoptosis, (2) blockade of migration of CXCR3+ T cells that may impair recruitment to the site of inflammation, and (3) the induction of CD8+ T cells with regulatory function. We propose that apoptosis of activated T cells may contribute to the rapid clinical response, while alterations in inflammatory T cell trafficking and induction of regulatory CD8+ cells may contribute to the long-term response. While these mechanisms remained to be confirmed in vivo, these data suggest that visilizumab could act by restoring the immune balance in inflamed mucosa through targeting several immunemediated pathways.
D. Foell 1 , Z. Ren 2 , H. Wittkowski 1 , J. Roth 1 , T. Borody 3 , R. Clancy 2 . 1 University of Muenster; 2 University of Newcastle; 3 Centre for Digestive Diseases, Five Stocks Objectives: Phagocyte-derived S100 proteins have pro-inflammatory properties and may serve as biomarkers for disease activity. S100A12 is a neutrophil activation marker, while S100A8/S100A9 (MRP8/14, calprotectin) is less specific and also expressed in monocytes and possibly epithelial cells. Our aim was to analyze the release of these molecules from inflamed tissue into the gut lumen. Methods: Bowel tissue from patients with Crohn' s disease (CD, n=29), ulcerative colitis (UC, n=30), irritable bowel syndrome (IBS, n=28), or without inflammation (n=30) was obtained during endoscopy and taken into culture. After 12h culture media were collected, centrifuged and stored at -80°C until required for immunoassays. Endoscopic, histological, and clinical disease activity measures were documented. Results: We found an increased spontaneous release of neutrophil activationmarker S100A12 from tissue in IBD compared to IBS and healthy controls (p<0.0001). The release of S100A12 into the supernatants was 30-fold enhanced in inflamed tissue when compared to non-inflamed tissue (mean 9.35 ng/ml vs. 0.3 ng/ml, p<0.0001). Although less difference was found for S100A8/S100A9, this protein complex was also found at higher concentrations in supernatants from inflamed tissue (8-fold, p<0.0001). The levels of these proteins were not influenced by the patients' mycobacterium avium paratuberculosis status. Conclusions: This is the first demonstration of direct release of phagocytederived S100 proteins from local tissues under inflammatory conditions, which may reflect secretion from infiltrating neutrophils (S100A12) and also monocytes or epithelial cells (S100A8/S100A9) during active disease. This release validates endoscopic assessment of activity and explains the correlation of fecal S100 levels with disease activity in IBD.
P161 DISTURBED EXPRESSION OF ALPHA-DEFENSINS AND TISSUE INHIBITOR OF MATRIX METALLOPROTEINASE-1 IN NEUTROPHILS OF CROHN' S DISEASE PATIENTS
P162 VISILIZUMAB TARGETS MULTIPLE IMMUNE-MEDIATED PATHWAYS V. Vexler, J. Sheridan, T. Tsao, J. Li, L. Zeng, Y. Mu, K. Lin, J. Shi, J. Woo. PDL BioPharma, Fremont, USA
P163 CRUCIAL ROLE OF THE PROTEIN C PATHWAY IN GOVERNING MICROVASCULAR INFLAMMATION IN INFLAMMATORY BOWEL DISEASE F. Scaldaferri 1, M. Sans 2 , C. Graziani 1, R. De Cristofaro 1, S. Vetrano 3 , B. Gerlitz 4 , A. Repici 3 , A. Gasbarrini 1, A. Malesci 3, J. Panes 2 , B.W. Grinnell 4 , S. Danese 3. 1 Intitute of Internal Medicine, Catholic University of Rome, Italy; 2 Division of Gastroenterology, Hospital Clinic, Barcelona, Spain; 3 Division of Gastroenterology, IRCCS Istituto Clinico Humanitas, Rozzano, Milan; 4 Biotechnology Discovery Research, Lilly Research Laboratories, Indianapolis USA
S. Bogaert, H. Peeters, D. Laukens, M. De Vos. University Hospital Ghent Background: Human neutrophil peptides (HNP1-3), which belong to the alpha-defensins, are major constituents of neutrophil granules and assist in killing phagocytized bacteria. Inappropriate infiltration and activation of neutrophils is involved in tissue damage and repair in Crohn' s disease (CD). Neutrophils express matrix metalloproteinases (MMP) -8, -9 and tissue inhibitors of MMP (TIMP) -1 and -2. The activity of MMPs is regulated by interaction with TIMPs. We wondered if neutrophils of CD patients show an altered expression of alpha-defensins, MMPs and TIMPs. Methods: Neutrophils of 10 healthy controls and 10 CD patients, of which 4 CD patients carrying one of the CD-associated CARD15 polymorphisms, were isolated with CD15 magnetic labelling. They were infected with a CDassociated adherent invasive Escherichia Coli (AIEC) strain LF82 for 1 or 4 hours. Quantitative real-time PCR was used to measure mRNA expression of HNP1-3, MMP-8, -9 and TIMP-1, -2. Gene expression was correlated with the CARD15 genotype. Results: Basal expression of HNP1-3 mRNA in unstimulated neutrophils was significantly lower in CD patients than in healthy controls irrespective of CARD15 status (P=0.034). Bacterial challenge with AIEC induced an upregu-
Endothelial protein C receptor (EPCR) and thrombomodulin (TM), expressed in the microvasculature, convert protein C (PC) to its activated form, which exerts potent anti-inflammatory activities. In several chronic inflammatory conditions, the PC pathway is down-regulated, leading to impaired generation of activated PC. Aim of this study was to explore the protein C pathway in inflammatory bowel disease (IBD). TM and EPCR expression levels were evaluated by immunohistochemistry in colonic sections from controls and IBD subjects, and by flow cytometry in human intestinal microvasculature endothelial cells (HIMEC), at baseline and after treatment with TNF-α, and IL-10. The capacity to generate activated PC by HIMEC was measured by colorimetric assay. The anti-inflammatory effects of recombinant activated PC was studied on TNF-α stimulated HIMEC, expression of VCAM-1 and ICAM-1 assessed by flow cytometry, chemokines measured by ELISA. Adhesion assays were used to evaluate the capacity of activated PC to inhibit T-cell adhesion to HIMEC. In vivo, the anti-inflammatory effect of activated PC was tested in the dextran sodium sulphate (DSS) model of colitis in mice and by intravital microscopy. IBD patients expressed lower levels of TM and EPCR in intestinal microvasculature than controls. In cell culture, inflammatory cytokines down regulated HIMEC expression of EPCR and TM, leading to a sig-
46
Poster Presentations
nificant (p<0.05) impaired capacity to activate PC. Recombinant activated PC had a potent anti-inflammatory effect, being able to down-regulate TNFα-dependent VCAM-1 and ICAM-1 expression (p<0.05), as well as chemokine production (p<0.05), and to inhibit T-cell adhesion to HIMEC (p<0.05). Finally, administration of activated PC was very effective in preventing experimental colitis and inhibited leukocyte adhesion to the inflamed intestinal vessels (p<0.05). The protein C pathway represents a new system involved in microvascular inflammation in the gut. Restoring the PC pathway may represent a new therapeutic approach to down play intestinal inflammation.
P164 INFLIXIMAB TREATMENT DECREASES SERUM OSTEOPROTEGERIN CONCENTRATION IN PATIENTS WITH CROHN' S DISEASE 1
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P. Miheller , G. Mûzes , K. Rácz , A. Blázovits , L. Herszényi , P. Lakatos , Z. Tulassay 1,3. 1 IInd Department of Medicine, Semmelweis University, Budapest; 2 Ist Department of Medicine, Semmelweis University, Budapest; 3 Hungarian Academy of Sciences, Gastroenterological and Endocrinological Research Group Background: Crohn' s disease (CD) is frequently complicated with low bone mineral density (BMD) and altered bone metabolism. Recent studies indicate increased serum osteoprotegerin (OPG) level in the affected patients. In vitro, OPG was secreted from the inflamed macrophages and dendrtitc cells. Infliximab (IFX) influences BMD favourably in CD but its effect has not yet been clarified. Aim: To assess the possible molecular pathways of IFX on bone metabolism in CD patients. Methods: Twenty nine patients with CD treated with 5 mg/kg IFX at baseline (day 1), week 2 (day 14) and week 6 (day 42) were included in the study. Serum samples were collected at the same time for measurements of biochemical markers of bone turnover (osteocalcin, OC, and β-crosslaps, bCL), and of their orchestrating cytokine elements osteoprotegerin (OPG) and soluble receptor activator of nuclear factor kappa-B ligand (sRANKL). OC, bCL, OPG and sRANKL were determined by enzyme-linked immunoassays. Results: OC concentration was 27.66±14.98 vs. 34.77±20.05 ng/ml (p<0.005) at baseline and day 42, respectively and sRANKL elevated from 0.0105±0.026 up to 3.63±1.49 ng/ml (NS) at the end of the study. bCL level was 0.633±0.563 vs. 0.539±0.24 ng/ml (NS) at baseline and day 42, respectively while OPG decreased from 3.633±1.499 to 3.342±1.61 ng/ml (p<0.05) on the same days. At the end of the study a positive correlation (0.572, p<0.05) was detected between bCL and OPG. Conclusions: In CD the elevated OPG could reflect a counterregulatory response to inflammatory cytokines. Furthermore, it may indicate T-cell activation. IFX therapy decreased both serum OPG and bCL concentrations of patiens, however, levels of OC and sRANKL were detected to be increased. Since IFX can inhibit the production of T-cell dependent cytokines, eg. IFNgamma, wich is a known potent inhibitor of osteoblast activity and osteoclastogenesis, our findings indicate its indirect effect on bone metabolism.
P165 INFLAMMATORY BOWEL DISEASE IN CGD REPRODUCES THE PATHOLOGICAL FEATURES OF CROHN' S DISEASE D.J.B. Marks 1 , K. Miyagi 1 , F.Z. Rahman 1 , N. Grosvenor 1 , S.L. Bloom 2 , A.W. Segal 1 . 1 University College London; 2 Department of Gastroenterology, University College Hospital Introduction: Patients with chronic granulomatous disease (CGD), a rare congenital disorder affecting 1/250,000 people and characterised by defective neutrophil function, frequently develop an inflammatory bowel disease similar to Crohn' s disease. Recently, Crohn' s patients have also been demonstrated to mount unusually weak innate immune responses, proposed to play a primary pathogenic role. Concordance between features of the bowel disease in these two conditions has never been formally evaluated. Methods: Retrospective case note analysis. Results: 25 adult patients with CGD were identified (11 X-linked, 12 autosomal recessive, 2 genotype unidentified). Mean age of diagnosis was 13.6 years (range: 0-41 years). 14 patients (56%) had experienced gastrointestinal symptoms in the preceding 3 years, most frequently diarrhoea (36%), abdominal pain (32%), constipation (28%), and bleeding per rectum (24%). 11 patients (44%) had documented gastrointestinal inflammation that was not secondary to infection. Mean age of onset of gastrointestinal disease was 19.2 years (range: 4-55 years). Disease manifested at all locations throughout the alimentary canal including the upper gastrointestinal tract (45%), small intestine (27%), colon (73%) and rectum (73%). All these patients had discontinuous inflammation and peri-anal involvement. Approximately half (45%) demonstrated epithelioid granulomata on histology. In addition, some patients developed intestinal fistulae (27%) or stenosis (9%). All 11 patients
(100%) fulfilled the Lennard-Jones criteria for the diagnosis of Crohn' s disease, and 3 patients were originally misdiagnosed as such prior to the recognition of the immunodeficiency phenotype. Partial therapeutic responses (by clinical and biochemical criteria) were observed with 5-aminosalicylates (5 patients), thalidomide (1 patient) and interferon-ã (1 patient), and complete remission achieved with azathioprine (1 patient), Infliximab (1 patient) and intestinal resection (1 patient). Conclusions: There are striking pathological similarities between the bowel diseases observed in CGD and Crohn' s disease. CGD patients appear particularly prone to developing peri-anal complications. †These authors contributed equally to this work
P166 LACTOBACILLUS PRECOLONIZATION OF SCID MOUSE MOTHERS PROTECT THEIR OFFSPRING AGAINST THE INFLAMMATION INDUCED BY DEXTRAN SULPHATE SODIUM TREATMENT H. Kozakova 1 , T. Hudcovic 1 , T. Hrncir 1 , R. Stepankova 1 , H. Tlaskalova-Hogenova 1 , B. Cukrowska 2 . 1 Institute of Microbiology AS CR; 2 Dept. Pathol., The Children' s Memorial Health Inst., Warsaw, Poland The aim of the study was to show whether primary colonization of SCID mice with Lactobacillus rhamnosus affects the development of the experimental ulcerative colitis. Immunodeficient SCID mice were born to ex-germ-free mothers monocolonized with L. rhamnosus. At the age of two months, monoassociated mice, as well as age-matched germ-free controls, were transferred in conventional conditions. One month later, mice received 3% DSS in drinking water for one week to develop enterocolitis. Clinical symptoms as bleeding, rectal prolapses, decreased length of colon and reduced body weight were found only in control mice. Impairment of the colon was evaluated histologically. The level of proinflammatory cytokine TNF-alpha was determined in cultivation medium of intestine pieces (jejunum, ileum, colon ascendens, colon descendes) and in supernatant of the cultivated spleen lymphocytes. L. rhamnosus-precolonized mice remained healthy without signs of the intestinal inflammation. The level of TNF-alpha produced by intestinal pieces of L. rhamnosus-precolonized mice was significantly reduced as compared with control mice. Cultivated spleen lymphocytes of L. rhamnosus-precolonized mice secreted decreased amounts of TNF-alpha as well. We concluded that primary colonization of mice with L. rhamnosus prevented development of DSS-induced inflammation in conventional SCID mice. Grant 303/05/2249 of the Czech Science Foundation.
P167 CHARACTERIZATION OF THE NEUROCHEMICAL AND NEUROMUSCULAR ADAPTIONS IN A RAT MODEL OF ULCERATIVE COLITIS M. Auli 1 , Y. Nasser 2, W. Ho 2 , C.M. Keenan 2 , J.F. Burgueño 1 , C. Romero 1 , K.A. Sharkey 2 , E. Fernandez 1. 1 Universitat Autònoma de Barcelona; 2 University of Calgary Intrarectal administration of Trichinella spiralis larvae in rats causes colonic inflammation with features similar to ulcerative colitis. Our aim was to characterize the functional and neurochemical changes occurring during T. spiralis-induced colitis and to study infiltrating cells within the myenteric plexus (MP). T. spiralis infected rats were studied 6 and 14 days postinfection (PI). Circular muscle strips were evaluated in contractility studies and whole-mount preparations of MP were used for immunohistochemistry. Myeloperoxidase (MPO) activity was assessed as an index of neutrophil infiltration and tissue macrophages were examined by immunohistochemisty. Rats had poorly formed stools and mucosal lesions after infection. This was associated with increased MPO activity and a dramatic increase in ED1immunoreactive macrophages in the MP of infected animals. Responses to acetylcholine and KCl were reduced in inflamed tissue, suggesting an impairment of contractility. In addition, there was markedly decreased spontaneous motor activity and a reduced sensitivity to the NOS inhibitor L-NOArg. Immunoreactivity for the neuronal marker PGP9.5 was reduced after infection (Control: 32.3±1.6, 6d PI: 24.1±5.2, 14d PI: 24.8±2.5 cells/ganglion; n=3/group). An increase in inducible nitric oxide synthase (NOS) immunoreactivity was observed in the mucosa and submucosa, together with a reduction in the number of neuronal NOS-immunoreactive cells in the MP (Control: 18.4±0.5, 6d PI: 8.7±0.3, 14d PI: 12.0±0.7 cells/ganglion, n=3/group; P<0.0001). In contrast, VIP, substance P and ChAT immunoreactivity appeared unchanged. Similarly, no differences in the number of calretinin or calbindin neurons were observed in the MP. Mucosal inflammation induced by T. spiralis in the colon causes profound neuromuscular adaptations. There is a neuronal loss in the MP and much of this is due to a reduction in NOS neurons. These changes appear to underlie alterations in gut motility. Macrophages are found extensively in the MP and might play a pivotal role in the neuromuscular alterations observed.
45
single subcutaneous injection of azoxymethane (AOM) at a dose of 10 mg/kg body weight. One week later the mice received 3% dextran sodium sulfate (DSS) in the drinking water continuously for up to 5 days. Specimens were taken from proximal and distal colon and rectum in weeks 6, 12 and 18 and macroscopic and microscopic changes of the colon mucosa were evaluated. In addition, immunohistochemical determination of beta-catenin was performed and the specimens were examined by scanning electron microscopy. Administration of DSS usually causes acute colitis in Balb/c mice but when combined with AOM the inflammation assumes a chronic form. Chronic colitis, low or high grade dysplasia or adenocarcinoma was visible in histological sections at week 6. At week 12 the incidence of macroscopically visible carcinomas reached nearly 100%. Strong nuclear and cytoplasmic expression of beta-catenin was observed in adenocarcinoma cells. Scanning electron microscopy confirmed changes in the position and structure of the crypts. These findings support the idea that chronic inflammation plays an important role in colon carcinogenesis. The experimental model used in our study may help in identification and modification of injurious substances.
lation in both CD patients and controls with a trend to lower expression in CD patients (P=0.079). Four hours after stimulation, expression returned to basal levels and a significant lower HNP1-3 expression was seen in CD patients (P=0.034). Furthermore, expression of MMP-8, -9, TIMP-1 and -2 mRNA in unstimulated neutrophils was similar in CD patients and controls. Only in the subgroup of patients carrying a CARD15 polymorphism, a significantly lower expression of TIMP-1 mRNA was observed (P=0.016). Conclusion: The reduced expression of HNP1-3 in CD patients can lead to impaired killing of phagocytized bacteria by neutrophils. Moreover, the decreased expression of TIMP-1 in neutrophils of CD patients with CARD15 polymorphisms might be implicated in a disturbed tissue remodeling effect
P160 MUCOSAL RELEASE OF PHAGOCYTE-SPECIFIC S100 PROTEINS IS STRONGLY ENHANCED DURING ACTIVE INFLAMMATORY BOWEL DISEASE
Open-labeled Phase I studies of visilizumab, a humanized non-FcR-binding anti-CD3 monoclonal antibody, in intravenous steroid-refractory ulcerative colitis and in inflammatory Crohn' s disease patients showed tolerability and clinical activity at doses 5-15 mg/kg administered on 2 consecutive days. Visilizumab caused rapid and reversible disappearance of peripheral blood T cells with rapid clinical improvement that in some patients persisted long after visilizumab was cleared from circulation. Preliminary data from two-third of dosed patient population suggest clinical response on day 30 may be related to peripheral blood CD8+ T cell count. We hypothesized that the clinical activity of visilizumab may be related to targeting several immune-mediated pathways important in IBD. Short-term exposure of resting PBMC to visilizumab in vitro induced mild T cell proliferation, cytokine and chemokine production and no measurable apoptosis. When added to coculture of PBMC and endothelial cells, visilizumab induced robust production of the chemokine IP-10 resulting in selective blockade of migration of CXCR3+ cells. Long-term exposure of resting PBMC to visilizumab led to preferential expansion of CD8+ T cells with regulatory function. Exposure of preactivated PBMC to visilizumab resulted in apoptosis of proliferating CXCR3+ T cells. Overall, our results suggest that visilizumab could act through multiple mechanisms — (1) elimination of proliferative activated CXCR3+ T cells through apoptosis, (2) blockade of migration of CXCR3+ T cells that may impair recruitment to the site of inflammation, and (3) the induction of CD8+ T cells with regulatory function. We propose that apoptosis of activated T cells may contribute to the rapid clinical response, while alterations in inflammatory T cell trafficking and induction of regulatory CD8+ cells may contribute to the long-term response. While these mechanisms remained to be confirmed in vivo, these data suggest that visilizumab could act by restoring the immune balance in inflamed mucosa through targeting several immunemediated pathways.
D. Foell 1 , Z. Ren 2 , H. Wittkowski 1 , J. Roth 1 , T. Borody 3 , R. Clancy 2 . 1 University of Muenster; 2 University of Newcastle; 3 Centre for Digestive Diseases, Five Stocks Objectives: Phagocyte-derived S100 proteins have pro-inflammatory properties and may serve as biomarkers for disease activity. S100A12 is a neutrophil activation marker, while S100A8/S100A9 (MRP8/14, calprotectin) is less specific and also expressed in monocytes and possibly epithelial cells. Our aim was to analyze the release of these molecules from inflamed tissue into the gut lumen. Methods: Bowel tissue from patients with Crohn' s disease (CD, n=29), ulcerative colitis (UC, n=30), irritable bowel syndrome (IBS, n=28), or without inflammation (n=30) was obtained during endoscopy and taken into culture. After 12h culture media were collected, centrifuged and stored at -80°C until required for immunoassays. Endoscopic, histological, and clinical disease activity measures were documented. Results: We found an increased spontaneous release of neutrophil activationmarker S100A12 from tissue in IBD compared to IBS and healthy controls (p<0.0001). The release of S100A12 into the supernatants was 30-fold enhanced in inflamed tissue when compared to non-inflamed tissue (mean 9.35 ng/ml vs. 0.3 ng/ml, p<0.0001). Although less difference was found for S100A8/S100A9, this protein complex was also found at higher concentrations in supernatants from inflamed tissue (8-fold, p<0.0001). The levels of these proteins were not influenced by the patients' mycobacterium avium paratuberculosis status. Conclusions: This is the first demonstration of direct release of phagocytederived S100 proteins from local tissues under inflammatory conditions, which may reflect secretion from infiltrating neutrophils (S100A12) and also monocytes or epithelial cells (S100A8/S100A9) during active disease. This release validates endoscopic assessment of activity and explains the correlation of fecal S100 levels with disease activity in IBD.
P161 DISTURBED EXPRESSION OF ALPHA-DEFENSINS AND TISSUE INHIBITOR OF MATRIX METALLOPROTEINASE-1 IN NEUTROPHILS OF CROHN' S DISEASE PATIENTS
P162 VISILIZUMAB TARGETS MULTIPLE IMMUNE-MEDIATED PATHWAYS V. Vexler, J. Sheridan, T. Tsao, J. Li, L. Zeng, Y. Mu, K. Lin, J. Shi, J. Woo. PDL BioPharma, Fremont, USA
P163 CRUCIAL ROLE OF THE PROTEIN C PATHWAY IN GOVERNING MICROVASCULAR INFLAMMATION IN INFLAMMATORY BOWEL DISEASE F. Scaldaferri 1, M. Sans 2 , C. Graziani 1, R. De Cristofaro 1, S. Vetrano 3 , B. Gerlitz 4 , A. Repici 3 , A. Gasbarrini 1, A. Malesci 3, J. Panes 2 , B.W. Grinnell 4 , S. Danese 3. 1 Intitute of Internal Medicine, Catholic University of Rome, Italy; 2 Division of Gastroenterology, Hospital Clinic, Barcelona, Spain; 3 Division of Gastroenterology, IRCCS Istituto Clinico Humanitas, Rozzano, Milan; 4 Biotechnology Discovery Research, Lilly Research Laboratories, Indianapolis USA
S. Bogaert, H. Peeters, D. Laukens, M. De Vos. University Hospital Ghent Background: Human neutrophil peptides (HNP1-3), which belong to the alpha-defensins, are major constituents of neutrophil granules and assist in killing phagocytized bacteria. Inappropriate infiltration and activation of neutrophils is involved in tissue damage and repair in Crohn' s disease (CD). Neutrophils express matrix metalloproteinases (MMP) -8, -9 and tissue inhibitors of MMP (TIMP) -1 and -2. The activity of MMPs is regulated by interaction with TIMPs. We wondered if neutrophils of CD patients show an altered expression of alpha-defensins, MMPs and TIMPs. Methods: Neutrophils of 10 healthy controls and 10 CD patients, of which 4 CD patients carrying one of the CD-associated CARD15 polymorphisms, were isolated with CD15 magnetic labelling. They were infected with a CDassociated adherent invasive Escherichia Coli (AIEC) strain LF82 for 1 or 4 hours. Quantitative real-time PCR was used to measure mRNA expression of HNP1-3, MMP-8, -9 and TIMP-1, -2. Gene expression was correlated with the CARD15 genotype. Results: Basal expression of HNP1-3 mRNA in unstimulated neutrophils was significantly lower in CD patients than in healthy controls irrespective of CARD15 status (P=0.034). Bacterial challenge with AIEC induced an upregu-
Endothelial protein C receptor (EPCR) and thrombomodulin (TM), expressed in the microvasculature, convert protein C (PC) to its activated form, which exerts potent anti-inflammatory activities. In several chronic inflammatory conditions, the PC pathway is down-regulated, leading to impaired generation of activated PC. Aim of this study was to explore the protein C pathway in inflammatory bowel disease (IBD). TM and EPCR expression levels were evaluated by immunohistochemistry in colonic sections from controls and IBD subjects, and by flow cytometry in human intestinal microvasculature endothelial cells (HIMEC), at baseline and after treatment with TNF-α, and IL-10. The capacity to generate activated PC by HIMEC was measured by colorimetric assay. The anti-inflammatory effects of recombinant activated PC was studied on TNF-α stimulated HIMEC, expression of VCAM-1 and ICAM-1 assessed by flow cytometry, chemokines measured by ELISA. Adhesion assays were used to evaluate the capacity of activated PC to inhibit T-cell adhesion to HIMEC. In vivo, the anti-inflammatory effect of activated PC was tested in the dextran sodium sulphate (DSS) model of colitis in mice and by intravital microscopy. IBD patients expressed lower levels of TM and EPCR in intestinal microvasculature than controls. In cell culture, inflammatory cytokines down regulated HIMEC expression of EPCR and TM, leading to a sig-
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Poster Presentations
nificant (p<0.05) impaired capacity to activate PC. Recombinant activated PC had a potent anti-inflammatory effect, being able to down-regulate TNFα-dependent VCAM-1 and ICAM-1 expression (p<0.05), as well as chemokine production (p<0.05), and to inhibit T-cell adhesion to HIMEC (p<0.05). Finally, administration of activated PC was very effective in preventing experimental colitis and inhibited leukocyte adhesion to the inflamed intestinal vessels (p<0.05). The protein C pathway represents a new system involved in microvascular inflammation in the gut. Restoring the PC pathway may represent a new therapeutic approach to down play intestinal inflammation.
P164 INFLIXIMAB TREATMENT DECREASES SERUM OSTEOPROTEGERIN CONCENTRATION IN PATIENTS WITH CROHN' S DISEASE 1
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P. Miheller , G. Mûzes , K. Rácz , A. Blázovits , L. Herszényi , P. Lakatos , Z. Tulassay 1,3. 1 IInd Department of Medicine, Semmelweis University, Budapest; 2 Ist Department of Medicine, Semmelweis University, Budapest; 3 Hungarian Academy of Sciences, Gastroenterological and Endocrinological Research Group Background: Crohn' s disease (CD) is frequently complicated with low bone mineral density (BMD) and altered bone metabolism. Recent studies indicate increased serum osteoprotegerin (OPG) level in the affected patients. In vitro, OPG was secreted from the inflamed macrophages and dendrtitc cells. Infliximab (IFX) influences BMD favourably in CD but its effect has not yet been clarified. Aim: To assess the possible molecular pathways of IFX on bone metabolism in CD patients. Methods: Twenty nine patients with CD treated with 5 mg/kg IFX at baseline (day 1), week 2 (day 14) and week 6 (day 42) were included in the study. Serum samples were collected at the same time for measurements of biochemical markers of bone turnover (osteocalcin, OC, and β-crosslaps, bCL), and of their orchestrating cytokine elements osteoprotegerin (OPG) and soluble receptor activator of nuclear factor kappa-B ligand (sRANKL). OC, bCL, OPG and sRANKL were determined by enzyme-linked immunoassays. Results: OC concentration was 27.66±14.98 vs. 34.77±20.05 ng/ml (p<0.005) at baseline and day 42, respectively and sRANKL elevated from 0.0105±0.026 up to 3.63±1.49 ng/ml (NS) at the end of the study. bCL level was 0.633±0.563 vs. 0.539±0.24 ng/ml (NS) at baseline and day 42, respectively while OPG decreased from 3.633±1.499 to 3.342±1.61 ng/ml (p<0.05) on the same days. At the end of the study a positive correlation (0.572, p<0.05) was detected between bCL and OPG. Conclusions: In CD the elevated OPG could reflect a counterregulatory response to inflammatory cytokines. Furthermore, it may indicate T-cell activation. IFX therapy decreased both serum OPG and bCL concentrations of patiens, however, levels of OC and sRANKL were detected to be increased. Since IFX can inhibit the production of T-cell dependent cytokines, eg. IFNgamma, wich is a known potent inhibitor of osteoblast activity and osteoclastogenesis, our findings indicate its indirect effect on bone metabolism.
P165 INFLAMMATORY BOWEL DISEASE IN CGD REPRODUCES THE PATHOLOGICAL FEATURES OF CROHN' S DISEASE D.J.B. Marks 1 , K. Miyagi 1 , F.Z. Rahman 1 , N. Grosvenor 1 , S.L. Bloom 2 , A.W. Segal 1 . 1 University College London; 2 Department of Gastroenterology, University College Hospital Introduction: Patients with chronic granulomatous disease (CGD), a rare congenital disorder affecting 1/250,000 people and characterised by defective neutrophil function, frequently develop an inflammatory bowel disease similar to Crohn' s disease. Recently, Crohn' s patients have also been demonstrated to mount unusually weak innate immune responses, proposed to play a primary pathogenic role. Concordance between features of the bowel disease in these two conditions has never been formally evaluated. Methods: Retrospective case note analysis. Results: 25 adult patients with CGD were identified (11 X-linked, 12 autosomal recessive, 2 genotype unidentified). Mean age of diagnosis was 13.6 years (range: 0-41 years). 14 patients (56%) had experienced gastrointestinal symptoms in the preceding 3 years, most frequently diarrhoea (36%), abdominal pain (32%), constipation (28%), and bleeding per rectum (24%). 11 patients (44%) had documented gastrointestinal inflammation that was not secondary to infection. Mean age of onset of gastrointestinal disease was 19.2 years (range: 4-55 years). Disease manifested at all locations throughout the alimentary canal including the upper gastrointestinal tract (45%), small intestine (27%), colon (73%) and rectum (73%). All these patients had discontinuous inflammation and peri-anal involvement. Approximately half (45%) demonstrated epithelioid granulomata on histology. In addition, some patients developed intestinal fistulae (27%) or stenosis (9%). All 11 patients
(100%) fulfilled the Lennard-Jones criteria for the diagnosis of Crohn' s disease, and 3 patients were originally misdiagnosed as such prior to the recognition of the immunodeficiency phenotype. Partial therapeutic responses (by clinical and biochemical criteria) were observed with 5-aminosalicylates (5 patients), thalidomide (1 patient) and interferon-ã (1 patient), and complete remission achieved with azathioprine (1 patient), Infliximab (1 patient) and intestinal resection (1 patient). Conclusions: There are striking pathological similarities between the bowel diseases observed in CGD and Crohn' s disease. CGD patients appear particularly prone to developing peri-anal complications. †These authors contributed equally to this work
P166 LACTOBACILLUS PRECOLONIZATION OF SCID MOUSE MOTHERS PROTECT THEIR OFFSPRING AGAINST THE INFLAMMATION INDUCED BY DEXTRAN SULPHATE SODIUM TREATMENT H. Kozakova 1 , T. Hudcovic 1 , T. Hrncir 1 , R. Stepankova 1 , H. Tlaskalova-Hogenova 1 , B. Cukrowska 2 . 1 Institute of Microbiology AS CR; 2 Dept. Pathol., The Children' s Memorial Health Inst., Warsaw, Poland The aim of the study was to show whether primary colonization of SCID mice with Lactobacillus rhamnosus affects the development of the experimental ulcerative colitis. Immunodeficient SCID mice were born to ex-germ-free mothers monocolonized with L. rhamnosus. At the age of two months, monoassociated mice, as well as age-matched germ-free controls, were transferred in conventional conditions. One month later, mice received 3% DSS in drinking water for one week to develop enterocolitis. Clinical symptoms as bleeding, rectal prolapses, decreased length of colon and reduced body weight were found only in control mice. Impairment of the colon was evaluated histologically. The level of proinflammatory cytokine TNF-alpha was determined in cultivation medium of intestine pieces (jejunum, ileum, colon ascendens, colon descendes) and in supernatant of the cultivated spleen lymphocytes. L. rhamnosus-precolonized mice remained healthy without signs of the intestinal inflammation. The level of TNF-alpha produced by intestinal pieces of L. rhamnosus-precolonized mice was significantly reduced as compared with control mice. Cultivated spleen lymphocytes of L. rhamnosus-precolonized mice secreted decreased amounts of TNF-alpha as well. We concluded that primary colonization of mice with L. rhamnosus prevented development of DSS-induced inflammation in conventional SCID mice. Grant 303/05/2249 of the Czech Science Foundation.
P167 CHARACTERIZATION OF THE NEUROCHEMICAL AND NEUROMUSCULAR ADAPTIONS IN A RAT MODEL OF ULCERATIVE COLITIS M. Auli 1 , Y. Nasser 2, W. Ho 2 , C.M. Keenan 2 , J.F. Burgueño 1 , C. Romero 1 , K.A. Sharkey 2 , E. Fernandez 1. 1 Universitat Autònoma de Barcelona; 2 University of Calgary Intrarectal administration of Trichinella spiralis larvae in rats causes colonic inflammation with features similar to ulcerative colitis. Our aim was to characterize the functional and neurochemical changes occurring during T. spiralis-induced colitis and to study infiltrating cells within the myenteric plexus (MP). T. spiralis infected rats were studied 6 and 14 days postinfection (PI). Circular muscle strips were evaluated in contractility studies and whole-mount preparations of MP were used for immunohistochemistry. Myeloperoxidase (MPO) activity was assessed as an index of neutrophil infiltration and tissue macrophages were examined by immunohistochemisty. Rats had poorly formed stools and mucosal lesions after infection. This was associated with increased MPO activity and a dramatic increase in ED1immunoreactive macrophages in the MP of infected animals. Responses to acetylcholine and KCl were reduced in inflamed tissue, suggesting an impairment of contractility. In addition, there was markedly decreased spontaneous motor activity and a reduced sensitivity to the NOS inhibitor L-NOArg. Immunoreactivity for the neuronal marker PGP9.5 was reduced after infection (Control: 32.3±1.6, 6d PI: 24.1±5.2, 14d PI: 24.8±2.5 cells/ganglion; n=3/group). An increase in inducible nitric oxide synthase (NOS) immunoreactivity was observed in the mucosa and submucosa, together with a reduction in the number of neuronal NOS-immunoreactive cells in the MP (Control: 18.4±0.5, 6d PI: 8.7±0.3, 14d PI: 12.0±0.7 cells/ganglion, n=3/group; P<0.0001). In contrast, VIP, substance P and ChAT immunoreactivity appeared unchanged. Similarly, no differences in the number of calretinin or calbindin neurons were observed in the MP. Mucosal inflammation induced by T. spiralis in the colon causes profound neuromuscular adaptations. There is a neuronal loss in the MP and much of this is due to a reduction in NOS neurons. These changes appear to underlie alterations in gut motility. Macrophages are found extensively in the MP and might play a pivotal role in the neuromuscular alterations observed.