INCREASED EXPRESSION OF INTERLEUKIN-33 AND ITS RECEPTOR ST2 IN THE INTESTINAL MUCOSA AND SERUM OF IBD PATIENTS

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Abstracts / Digestive and Liver Disease 41S (2009), S1–S167

OC.12.4 ENTERIC GLIA STIMULATES INFLAMMATION-INDUCED RESPONSES IN HUMAN INTESTINE AND INTERACTS WITH IMMUNE CELLS VIA S100B PROTEIN C. Cirillo ∗ , G. Sarnelli, A. Mango, I. Esposito, M. Grosso, S. Masone, G. Aprea, R. Cuomo Università degli Studi di Napoli Federico II, Napoli Background and aim: Enteric glial cells (EGC) participate to gut homeostasis and intestinal inflammation. Human gut inflammation is characterized by EGC-derived S100B up-regulation. However, it is still unclear if EGC-response is directly involved in, or it represents an epiphenomenon of inflammation itself. To investigate: 1) the role of EGC-derived S100B protein in mediating inflammation and nitric oxide (NO) production; 2) EGC activation and responses to inflammatory stimuli in mucosa and in isolated myenteric ganglia and 3) EGC ability to stimulate immune cells proliferation. Material and methods: Rectal biopsies from 30 control subjects were stimulated with exogenous S100B to evaluate iNOS expression, NO production, lipid peroxidation and p38MAPkinase activation in the presence of anti-RAGE antibody, SB203580 (inhibitor of p38MAPkinase), TLCK (inhibitor of NF-κB transcription), respectively. Mucosal biopsies and isolated myenteric ganglia (obtained from the ileum of 10 surgical specimens) were stimulated with lipopolysaccharides (LPS) + interferon gamma (IFN-γ); a double labeling immunofluorescence using anti-S100B or anti-GFAP was performed to identify EGC expressing cfos in the nucleus, as a marker of cells activation. S100B mRNA, protein expression and secretion, iNOS expression and NO production were assessed either in the presence, or absence of anti-RAGE. Proliferation of human peripheral blood mononuclear cells (PBMC), isolated from healthy subjects, was checked by incubation with exogenous S100B. Results: In mucosal biopsies, exogenous S100B increased iNOS expression, NO production and lipid peroxidation through p38MAPkinase/ NF-κB activation and via RAGE. LPS + IFN-γ increased S100B mRNA, protein expression and secretion, together with enhanced iNOS expression and NO production. In isolated myenteric ganglia, LPS + IFN-γ incubation resulted in a consistent EGC’s activation (c-fos positive nuclei in S100B/GFAP positive cells), together with S100B up-regulation and increased NO production. Interestingly, exogenous S100B significantly increased PBMC proliferation and activation (increased NO and Tumor Necrosis Factor-alpha production) via RAGE. Conclusions: EGC are activated by inflammatory stimuli and participate to NO production through S100B up-regulation. Increased levels of EGC-derived S100B are able to stimulate immune cells in a RAGE-dependent pathway. # L. Inflammatory bowel diseases 1. Basic science

OC.12.5 RECEPTOR FOR ADVANCED GLYCATION END PRODUCTS (RAGE) EXPRESSION IN INFLAMMATORY BOWEL DISEASE (IBD) R. Ciccocioppo 1 , C. Falcone 2 , M.L. Russo 3 , C. Ubezio 1 , P. Buzzi 3 , V. Boccaccio 3 , G. Zanellati 3 , C. Alvisi 1 , O. Luinetti 1 , G.R. Corazza 1 1 Fondazione

IRCCS San Matteo, Pavia; 2 Istituto Casa di Cura Città di Pavia, Pavia; 3 Università degli Studi, Pavia Background and aim: RAGE belongs to the immunoglobulin superfamily of cell surface receptor and is expressed at low level in multiple tissues. When engaged by its ligands that accumulate during inflammation, RAGE-dependent transduction signals delay monocyte apoptosis and increase their differentiation into macrophages able to produce great amounts of pro-fibrotic and pro-inflammatory cytokines. These latter, in turn, activates the expression of RAGE resulting in a magnification of inflammatory process. We aimed, therefore, to study the expression

of RAGE and the cytokine pattern in mucosal mononuclear cells of IBD patients. Material and methods: Perendoscopic biopsies were obtained from 14 active Crohn’s disease patients (M/F ratio 7/7, mean age 38.7 years), 12 active ulcerative colitis patients (M/F ratio 8/4, mean age 40.8 years) and 10 sex/age matched healthy controls, and used to isolate lamina propria mononuclear cells. In IBD patients, mucosal samples were taken from both macroscopically diseased and healthy areas. mRNA transcript levels of the following molecules: RAGE, NF-κB, TGF-β, IL-1β, IL-6, TNF-α, INF-γ, and β-actin, were quantified by real-time RT-PCR (Light Cycler 2.0; Roche). Statistical analysis was performed by using Mann-Whitney U test and Spearman rank correlation test. Results: In mononuclear cells from diseased areas of IBD patients, a critical increase of mRNA transcript levels of RAGE (p<0.005), NF-κB (p<0.00001), IL-6 (p<0.001), TNF-α (p<0.001), and INF-γ (p<0.001) in comparison to controls was observed, with RAGE levels that strongly correlated with those of NF-κB and TNF-α (r = 0.71 and 0.64, respectively). By contrast, no modification of the expression of these molecules was observed in cells isolated from unaffected mucosa. Conclusions: Our results point on the presence of a peculiar inflammatory pattern in diseased mucosa of IBD patients in which the high levels of RAGE may be responsible for a continuous production of pro-inflammatory and pro-fibrotic cytokines, leading to perpetuation of tissue damage. # L. Inflammatory bowel diseases 1. Basic science

OC.12.6 INCREASED EXPRESSION OF INTERLEUKIN-33 AND ITS RECEPTOR ST2 IN THE INTESTINAL MUCOSA AND SERUM OF IBD PATIENTS L. Pastorelli ∗ ,1 , S.B. Hoang 1 , M. Vecchi 2 , T.T. Pizarro 1 1 University

of Virginia, Charlottesville, USA; 2 Università degli Studi di Milano, Milano Background and aim: Interleukin (IL)-33 is a novel member of the IL1 family of cytokines that induces Th2 cytokine production, epithelial hyperplasia and granulocyte infiltration. Evidence suggests that IL-33 and its receptor ST2 may play a critical role in several inflammatory and autoimmune disorders. To date, the role of IL-33/ST2 in the pathogenesis of IBD is unknown. The aim of the present study was to characterize the expression and localization of IL-33/ST2 in patients with IBD. Material and methods: Surgically resected, intestinal tissues were collected from both involved and non-involved areas of Crohn’s disease (CD) and ulcerative colitis (UC) patients, as well as non-inflamed controls (N=4/group), and immunostained for IL-33 and ST2. IL-33 and ST2 mRNA expression was assessed in gut mucosal biopsies (N=13/group) and isolated intestinal epithelial cells (IEC) (4-17/group) from the aforementioned groups. IL-33 serum levels were measured by ELISA in IBD patients and controls (N=23/group). Results: IHC analysis localized IL-33 to IEC and lamina propria (LP) infiltrates, with increased staining in UC compared to CD and control tissues, and in affected compared to non-affected areas. Staining for ST2 was localized to IEC, particularly in the apical portion of colonic crypts from non-inflamed controls, but was absent in IEC from involved areas of both UC and CD patients. LP leukocytes were markedly stained for ST2 in inflamed intestinal tissues, but not in controls, suggesting differential regulation of IL-33 and ST2 in different cell populations. IL-33 mRNA transcripts were significantly increased in biopsies from UC-involved (5.8-fold) compared to UC-non-involved (1.0-fold) and non-inflamed controls (set as 1.0); similar results were obtained for ST2 transcripts. IL-33 mRNA levels were 9.7-fold higher in IEC isolated from UC versus controls (p<0.05). Finally, IL-33 serum concentrations were elevated both in UC and CD patients (UC>CD) compared to controls (14.2 and 10.8 vs. 4.9 ng/ml, P<0.05). Conclusions: Our study shows that: 1) IL-33 is increased in the intesti-

Abstracts / Digestive and Liver Disease 41S (2009), S1–S167 nal mucosa and serum of UC patients; 2) IL-33 and ST2 are localized to both IEC and LP leukocytes; 3) Changes in the pattern of ST2 expression occur during IBD. Taken together, our study is the first to report that IEC are a primary source of IL-33 in the gut mucosa and that IL-33/ST2 may play a central role in the pathogenesis of IBD. # L. Inflammatory bowel diseases 1. Basic science

OC.13.1 PREVALENCE AND RISK FACTORS OF FUNCTIONAL DYSPEPSIA IN THE GENERAL POPULATION: LOIANOMONGHIDORO STUDY R.M. Zagari 1 , G. Law 2 , L. Fuccio 1 , L. Eusebi 1 , D. Forman 2 , F. Bazzoli 1 1 University

of Bologna, Bologna; 2 University of Leeds, Leeds, UK

Background and aim: Functional dyspepsia (FD) is the most common cause of dyspeptic symptoms, but its epidemiology in the community is still unclear. Our aim was to evaluate the prevalence and risk factors of FD in a representative sample of general population. Material and methods: The study population was sampled as part of the Multicentre Italian Study on Cholelithiasis. 1533 adults from two villages (total adult population, 6332) were approached to undergo symptoms assessment using a validated questionnaire, upper gastrointestinal endoscopy with gastric biopsy and 13C-urea breath test. Data were also collected on demographic and lifestyle characteristics and use of medication. Epigastric pain, post-prandial fullness and early satiety occurring for at least 12 weeks during the previous year without or with concomitant non prominent reflux symptoms (both mild and less frequent than twice a week) in absence of endoscopic organic lesions were defined as FD symptoms (Rome II criteria). A logistic regression analysis was used to estimate potential risk factors for FD using as controls asymptomatic subjects without organic lesions. Results: 1033 individuals were enrolled (67.4%). 244 (23.6%) subjects reported dyspeptic symptoms without (n.156) or with non prominent reflux symptoms (n.88). Of the 244 subjects, 62 (25.4%) had organic lesions including esophagitis, Barrett’s esophagus, peptic ulcer (PU), erosions and malignancy, while 182 (74.6%) had FD symptoms. Of the 182 subjects, 45 (24.7%) had epigastric pain, 45 (24.7%) epigastric pain and post-prandial fullness/early satiety and 92 (50.6%) post-prandial fullness/early satiety without epigastric pain. The prevalence of FD in the study population was 17.6% (182/1033) (95% CI, 15.1-20.4). Irritable bowel syndrome (OR 5.76; 95% CI,1.81-18.33), 1st degree family history (FH) for gastric cancer (OR 3.36; 95% CI,1.51-7.44) and two occupations, that of clerk (OR 4.78; 95% CI,1.33-17.10) and manual worker (OR 4.09; 95% CI,1.10-15.32), were associated with an increased risk of FD. A borderline association was found with the divorced status (OR 4.27; 95% CI,0.98-18.7). Age, sex, education level, BMI, smoking, alcohol, coffee, H. pylori infection, chronic gastritis and 1st FH for PU were not risk factors for FD. Conclusions: About 17% of adults in the community suffer from FD. Subjects with IBS, positive FH for gastric cancer, clerks and manual workers had an increased risk of FD. # M. Functional disorders 1. Dyspepsia

OC.13.2 BREATH METHANE EXCRETION AND SEVERITY OF SYMPTOMS IN FUNCTIONAL BOWEL DISORDERS M. Di Stefano ∗ , C. Mengoli, A. Zanaboni, P. Tana, F. Racca, S. Mazzocchi, G.R. Corazza 1st Department of Medicine, University of Pavia, Foundation IRCCS “S. Matteo” Hospital, Pavia Background and aim: Colonic methane (CH4) production is the main

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pathway of hydrogen (H2) consumption; on a stechiometric basis, it is able to reduce total gas volume. Breath CH4 excretion is detectable in 35-50% of subjects and little information is available on a putative role of CH4 in the physiopathology of functional symptoms. We have already shown that breath CH4 excretion does not correlate with either a specific clinical presentation or the severity of functional symptoms (Di Stefano et al, DDW 2008). The aim of this study was to assess whether colonic production of different breath CH4 excretion patterns correlate with the presence and severity of symptoms in patients with functional bowel disorders. Material and methods: 137 subjects, 10 healthy volunteers and 127 patients with functional bowel disorders (106 females, mean age 40±10), were enrolled. 10gr lactulose in 250ml water were given orally after fasting; breath samples were collected every 15 min for 7h for H2 and CH4 detection. H2 and CH4 production was evaluated by the area under the time-concentration curve calculation (AUC). A cumulative gas excretion area was calculated by the sum of H2 AUC and CH4 AUC. 30 patients showed both H2 and CH4 excretion with breath, 97 excreted only H2 (methane non-excretors). We subdivided the CH4 excretors into 3 patterns: subtype 1 = increase in H2 excretion and stable CH4 excretion (n=14); subtype 2 = reduction of CH4 excretion when H2 increases (n=10); subtype 3 = parallel increase of CH4 and H2 (n=6). The presence and severity of bloating, flatulence, abdominal pain and borborygmi were evaluated by a semiquantitative scale where 0 = no symptom, 3 = severe symptom. Results: Symptom severity did not differ among the three subtypes. Subtype 2 showed a cumulative AUC of H2 and CH4 significantly lower than both subtype 1 and subtype 3 (subtype 2: AUC = 16052±5818 ppmxmin; subtype 1: AUC = 27372±24389 ppmxmin; subtype 3: AUC = 27888±9358 ppmxmin; P<0.05 for all these comparisons) and higher than healthy volunteers (HV: AUC = 7425±4948 ppmxmin, P<0.05). A strong inverse correlation was found between the severity of each symptom and the cumulative excretion of gas: bloating R=0.79, P<0.005; abdominal pain R=-0.72, P<0.05; flatulence R=-0.75, P<0.05, barborygmi R=-0.45, P=0.05. Conclusions: In at least one subtype of methane-excretors the severity of symptoms is correlated with cumulative excretion of H2 and CH4. # M. Functional disorders 2. IBS

OC.13.3 RIMONABANT-INDUCED EARLY SATIETY IS ASSOCIATED WITH DECREASED GASTRIC COMPLIANCE AND INHIBITION OF GASTRIC ACCOMMODATION E. Scarpellini ∗ ,1 , K. Ameloot 2 , R. Vos 2 , P. Vandenberghe 2 , I. Depoortere 2 , J. Tack 2 1

Gemelli Hospital, Roma; 2 Leuven Hospital, Leuven, Belgium

Background and aim: Satiety is partly controlled by the gastric accommodation (GA) and by the activation of descending endogenous anti-nociceptive pathways, putatively through mediators like endocannabinoids. The endocannabinoid receptor antagonist rimonabant (RI) has been shown to decrease food intake, mainly due to an effect on the central nervous system. We want investigate how suppression of endocannabinoid signalling by RI would influence the gastric response to meal ingestion and the sensitivity to gastric distension in normal volunteers. Material and methods: 9 healthy subjects participated in a placebocontrolled, double blind, randomized, crossover gastric barostat study. After 3 days of pretreatment with RI 20 mg/day or placebo (PL), stepwise distensions were performed (2 mmHg steps at 2 minute intervals, until discomfort or pain). To quantify gastric sensitivity, upper abdominal sensation was scored on graded scales (0=no sensation, 6=maximal) at the end of every distension step. To quantify gastric accommodation (GA), the mean gastric volume over consecutive 5 min intervals was measured 30 min before and 60 min after a standardized