293 Increased Intraepithelial Lymphocytes and Mast Cells with Depressed Serotonin Transporter in Duodenal Biopsies and Platelets of Patients with IBS and Diarrhoea (IBS-D): Correlation with Bowel Frequency Stephen J. Foley, Gulzar Singh, Laurie C. Lau, Andrew F. Walls, Geoffrey K. Holmes, Andrew Bennett, Charles Marsden, Robin C. Spiller INTRODUCTION: Several groups report increased mucosal inflammation and increased circulating cytokines, which both cell and animal studies show to depress SERT, in IBS. We therefore assessed mucosal and platelet SERT function together with 5HT & Mast Cell tryptase release (MCT) from duodenal biopsies which were also assessed for mast cells (MC), intra-epithelial lymphocytes (IEL) & 5HT-containing enterochromaffin cells (EC) compared to coeliac patients who are known to have mucosal inflammation with depressed SERT mRNA. AIMS & METHODS: 28 healthy volunteers, 20 patients with IBS-D & 20 with untreated coeliac disease (CD) completed bowel symptom questionnaires and underwent duodenal biopsy. 5HT & MCT release was assayed after 6 hour incubation. MC, IELs & ECs were identified by immuno-histochemistry. SERT and cytokeratin 20(CK20) mRNA were assessed by Taqman qRT-PCR. Platelet SERT function was assayed by H3-5HT uptake. Project supported by Novartis Pharma AG. RESULTS: IBS-D showed increase IELs, mast cell numbers and increased mast cell tryptase release (see Table). DIBS also showed depressed SERT/CK20(arbitrary units) at 0.19(0.1-0.43) versus 0.96(0.14-1.40) for HV, p<0.02. Platelet uptake of H3-5HT was also depressed at 12.8(8.3-23.2) versus 45.2(27.7-69.3)pmol/min/ mg protein for HV, p<0.01. CD had intermediate values, difference NS v HV. Mast cell numbers correlated (r=0.56, p=0.02) with stool frequency in IBS patients but not in coeliacs nor HV. IELs, in HV and IBS, correlated positively with mast cell numbers r=0.45 , p=0.001 and with EC count r=0.39, p=0.006 but negatively with SERT r=-0.37 ,p=0.01 and platelet 5HT uptake -0.53, p=0.001. CONCLUSIONS: IBS-D is characterised by increased duodenal IELs and mast cells associated with increased mast cell tryptase release. The depressed SERT in mucosa and platelets correlates with mucosal IELs and may be secondary to increased inflammatory mediators. Depressed SERT may contribute to increased 5HT availability and diarrhoeal symptoms. Mast cell numbers correlate with stool frequency suggesting mast cell products also play a part in symptoms. *p<0.03, **p<0.01 & ***p<0.001 all v HV a p<0.01 v IBS-D 294 Mast Cell Alterations in Post-Infectious Functional Gastrointestinal Disorders: Are They Related to the Disorder or to the Infection? Antonia Perello, Fermin Mearin, Josep Lloreta, Agustin Balboa, Monica Perona, Antonio Salas, Marc Perez-Oliveras, Jordi Coderch Functional gastrointestinal disorders (FGID) may appear after an acute infectious gastro- enteritis (AGE). The pathogenic mechanism is thought to be related to a persistent low grade inflammatory response in which mast cells participate. In a previous study, we showed that after a Salmonella infectious outbreak approximately 17% of patients developed post- infectious (PI)-FGID (Gastroenterology 2005;129:98-104). Aims: To investigate by electron microscopy mast cell presence, localization and activation in the cohort of patients with PI- FGID of our previous study. Methods: We included 16 patients with PI-FGID, 8 patients that had not developed FGID after the same infection (PI-controls) and 6 healthy-controls (without FGID or AGE). Gastric, right and left colon mucosal biopsies were obtained by endoscopy. Representative samples were analyzed by electron microscopy to obtain total mast cell counts, number of degranulating mast cells and number o mast cells located within 5μm of nerve fibres. Data were analyzed under blind conditions for the pathologist. Results: In the stomach both PI groups showed increased number of mast cells compared to healthy controls (p<0.05): total mast cells per field (PI-FGID 20.4±2.5 vs PI-controls 20.8±3.5 vs healthy-controls 8.5±2.9); degranulating mast cells per field (PI-FGID 17.2±2.2 vs PI-controls 19.5±3.4 vs healthy-controls 7.7±2.7); and number of mast cells within 5μm of nerve fibres (PI-FGID 5.6±1.2 vs PI-controls 6.6±1.5 vs healthy controls 2.5±1.1). Mast cell numbers in the right colon were also higher in both PI groups compared to the healthy group (p<0.05): number of total mast cells per field (PI-FGID 20.5±2.1 vs PI-controls 15.2±1.7 vs healthy-controls 9.9±2.9); degranulating mast cells per field (PI-FGID 16.3±1.8 vs PI- controls 11.5±1.7 vs healthy-controls 8.8±2.7); and number of mast cells within 5μm of nerve fibres (PI-FGID 9.7±1.3 vs PI-controls 8.0±1.3 vs healthy-controls 4.1±1.7). In the left colon the results were similar to the right colon. Conclusion: Three years after a Salmonella AGE, the observed increase in mast cell counts are not related to the presence of FGID but to the infection itself. A-41 AGA Abstracts 295 Dysfunctional T Cell Responses After Polyclonal Stimulation in Patients with Irritable Bowel Syndrome (IBS) Lena Ohman, Ann-Charlotte Lindmark, Stefan Isaksson, Iris Posserud, Hans Strid, Henrik Sjövall, Magnus Simren AIM: Several observations implicate that IBS may, in at least a subpopulation, be due to a low grade inflammation. We have therefore investigated phenotype of freshly isolated blood T cells and the function of these cells in response to polyclonal stimulation in IBS patients. METHOD: Blood samples were taken from 30 healthy subjects (mean age 39±10) and 74 IBS patients (mean age 34±16), defined by the Rome II criteria. Of the IBS patients 26 had predominantly diarrhoea (IBS-d), 11 predominantly constipation (IBS-c) and 37 alternating diarrhoea and constipation (IBS-a). Peripheral blood mononuclear cells were isolated and the cells were stimulated with CD3/CD28 antibodies. T cell phenotype was investigated by flow cytometry before and after 72 h polyclonal stimulation. T cell proliferation and cytokine secretion were analysed after 72 h culture. RESULTS: IBS patients had normal frequencies of blood T cells, whereas an increased frequency of the CD4 + T cells was expressing CD69 (11.9±15 vs 5.7±4.5, p=0.001), a marker for recent T cell activation, as compared to controls. Polyclonal stimulation of T cells demonstrated an abrogated down regulation of CD45RA on T cells in the IBS group (52.3±15.6 vs 33.0±18.7, p=0.001). Stimulated T cells from IBS patients proliferated less well as compared to controls (99822±41935 vs 137303±24263cpm, p=0.0001), and the decreased proliferation in IBS patients correlated to increased frequency of blood CD25 + regulatory T cells (R=0.373, P=0.02). Furthermore, the frequency of blood CD25 + regulatory T cells was negatively correlated to bowel habit satisfaction (R=0.296, P=0.02) and total IBS symptom severity(R=0.294, P=0.02). However, the frequencies of blood CD25 + regulatory T cells did not statistically differ between IBS and control subjects. The polyclonally stimulated T cell production of the cytokines IFN-γ and IL-10 did not differ between the study groups, although the IL-10 production varied substantially within the IBS group. High IL-10 producing IBS patients (n=25) were found to produce high amounts of IFN-γ (29372±17012 vs 20664±8256 pg/ml, p=0.03) and had a lower frequencies of blood CD25 + Treg cells (4.6±3.1 vs 10.9±14.4, p=0.02) as compared to IBS patients with a medium production of IL-10 (n=22). CONCLUSION: IBS patients have an increased basal immune activity of freshly isolated T cells, but an abrogated T cell response to polyclonal stimulation as compared to control subjects. The diminished T cell response was associated with an increased frequency of blood regulatory T cells which indicate that a subgroup of IBS patients may have an ongoing suppressed inflammatory response. 296 Expression Analysis of All Genes Implicated in Susceptibility to Crohn's Disease from Genome-Wide Association Studies Charles W. Lees, Colin L. Noble, Lauri Diehl, Jack Satsangi BACKGROUND. Genome-wide association studies in the UK, Belgium, Germany and N America in the past 12 months have identified several new genes directly implicated in the pathogenesis of Crohn's disease (CD). These include IL-23R, ATG16L1, IRGM, NKX2.3 and PTPN2. On-going meta-analysis of UK, Belgium and American datasets and subsequent replication studies promise to yield another several CD genes that did not meet genome- wide significance (p<10-7) on the individual scans. However, little is known of the direct functional relevance of many of these genes in CD and indeed UC. We therefore aimed to analyse the expression profiles of all these genes from our existing genome-wide microarray dataset. METHODS. Microarray expression data (Agilent platform) were available on colonos- copic biopsies from 53 CD and 67 UC patients and 33 healthy controls (HC). The major findings from this dataset have previously been presented (Noble et al, DDW 2007 & submitted). The Agilent probes for each gene were identified from existing databases. In addition to IL-23R, expression of IL-23A (p19), IL-23B / IL-12B (p40) and IL-12A (p35) were analysed. No microarray expression data were available for IRGM. For regions of extensive linkage disequilibrium, all genes in the area of association were analysed. The expression profiles for each gene were compared in CD vs. HC and UC vs. HC and then further analysed by the inflammatory status of the disease biopsies. RESULTS. The expression of the autophagy gene ATG16L1 was significantly lower in CD vs. HC biopsies regardless of inflammatory status (p=0.0017), but there was no difference between UC and HC. NKX2.3, a homeobox gene critical to normal intestinal development, was expressed at higher levels in inflamed and non-inflamed CD vs. HC (p<0.001) and inflamed UC vs. HC (p<0.0001), but not in non-inflamed UC biopsies. There was no difference in expression profiles of IL- 23R in inflamed or non-inflamed CD or UC samples and HC. However, IL-23A (p19) was increased in inflamed CD and UC compared with controls (p<0.0001). There were no significant differences in expression profiles of IL-12B (p40) or IL-12A (p35). PTPN2 did not show any consistent expression differences between CD, UC and HC. Data on all confirmed and replicated genes / regions from the on-going international meta-analysis will be presented. CONCLUSIONS. These expression data provide the first insights into the potential functional relevance of a series of exciting new discoveries in CD genetics based on genome-wide association scanning. Key expression differences are noted for ATG16L1 in CD, NKX2.3 in CD and UC and the IL-23 pathway in intestinal inflammation. 297 Replication of Signals from Recent Genome Wide Association Studies in Crohn Disease Identifies New Disease Genes for Ulcerative Colitis in a Large German Patient Panel Andre Franke, Tobias C. Balschun, Jürgen Hedderich, Tom Hemming Karlsen, Sandra May, Timothy T. Lu, Dörthe Schuldt, Philip C. Rosenstiel, Michael Krawczak, Stefan Schreiber Inflammatory bowel disease (IBD) comprises two related subphenotypes, namely Crohn disease (CD) and ulcerative colitis (UC). The molecular mechanisms of the pathogenesis of IBD are poorly understood. Starting from associations identified in several recent genome- wide association scans, we set out to replicate and extend these association signals in an AGA Abstracts