P191 SELECTIVE INDUCTION OF CROHN'S DISEASE (CD) AND ULCERATIVE COLITIS (UC) LAMINA PROPRIA MONONUCLEAR CELLS (LPMC) APOPTOSIS BY PROBIOTICS: INVOLVEMENT OF BACTERIAL SPHINGOMYELINASE (SMAse)

Abstracts of ECCO Congress, Innsbruck, Austria, 1—3 March 2007 P191 SELECTIVE INDUCTION OF CROHN' S DISEASE (CD) AND ULCERATIVE COLITIS (UC) LAMINA PROPRIA MONONUCLEAR CELLS (LPMC) APOPTOSIS BY PROBIOTICS: INVOLVEMENT OF BACTERIAL SPHINGOMYELINASE (SMAse) S. Angulo 1 , S. Danese 2 , N. Pultz 3 , M. Grazia Cifone 4 , A. Morales 5 , C. De Simone 4 , J.C. Fernandez-Checa 5, J. Panes 1 , C.J. Donskey 3 , C. Fiocchi 6 , M. Sans 1 . 1 Department of Gastroenterology, Hospital Clinic i Provincial/IDIBAPS, Barcelona, Spain; 2 Department of Gastroenterology, Istituto Clinico Humanitas, Milan, Italy; 3 Division of Infectious Diseases, Veterans Affairs Medical Center, Cleveland, USA; 4 Departamento de Medicina Experimental, Universita de l' Aquila, L' Aquila, Italy; 5 Consejo Superior de Investigaciones Cientificas, Barcelona, Spain; 6 Division Of Gastroenterology, Case Western Reserve University, Cleveland, USA Background: Probiotics appear to be beneficial in inflammatory bowel disease (IBD), but their mechanism of action is poorly understood. Lactobacillus brevis (LB) and Streptococcus thermophilus (ST), two of the probiotic bacteria included in VSL#3, have high levels of neutral SMAse, an enzyme leading to ceramide-mediated apoptosis. Aim: We investigated the ability of probiotics to induce apoptosis of immune cells as well as the influence of the degree of cell activation and the role of neutral SMAse on this form of apoptosis. Methods: LPMC were obtained from CD (n=10) and UC (n=7) patients, and normal colon (n=21), and peripheral blood mononuclear cells (PBMC) from healthy volunteers. Fresh LPMC and PBMC were used as resting or activated cells after stimulation with anti-CD3+CD28. Bacterial sonicates were obtained from LB, ST, and a non-pathogenic E. coli (EC) and S. faecalis (SF), as non-probiotic bacteria. LPMC and PBMC cells were exposed to bacterial sonicates (1 mg protein/mL), ceramide (30 g/mL), dihidroceramide (30 g/mL), or medium alone for 24 hours. In some experiments, sonicates were preincubated with GW4869, a specific inhibitor of neutral SMAse. Apoptosis was assessed by flow cytometry of propidium iodide-stained cells and expressed as percentage of apoptotic cells. SMAse activity was measured by TLC assay, using NBD-sphingomyelin as a substrate. Results: LB and ST sonicates induced significantly (p<0.01) more apoptosis in CD (55.1 4.7 and 32.7 3.3%) and UC (44.1 6.4 and 24.6 4.1) than in control LPMC (28.2 1.6 and 15.9 2.1%). EC and SF sonicates completely failed to induce apoptosis of all cell types. Stimulation with anti-CD3+CD28 induced a significant (p<0.01) and time-dependent increase in LB-induced apoptosis of both LPMC and PBMC. Ceramide induced significantly (p<0.05) more apoptosis in CD (30.8 3.1) and UC (24.1 3.1) than in control LPMC (16.3 1.1), whereas dihidroceramide had no effect. Inhibition of SMAse by GW4869 caused a dosedependent reduction of LB-induced apoptosis, which was significantly higher (p<0.005) in CD and UC than in control LPMC. SMAse activity of LB and ST was 10-fold that of EC and SF sonicates. SMAse activity of LB sonicate was completely abrogated by GW4869. Conclusions: Enhanced apoptosis of CD and UC LPMC appears to be a selective effect of the probiotics LB and ST, since other enteric bacteria like EC and SF fail to enhance apoptosis. Probiotic-induced apoptosis is modified by the degree of cell activation and bacterial SMAse plays a key role on this process. These results suggest that induction of immune cell apoptosis is one of the mechanisms mediating the therapeutic effects of probiotics in IBD.

P192 PRELIMINARY STUDY ON BACTERIAL TRANSLOCATION IN SERUM OF PATIENTS WITH INFLAMMATORY BOWEL DISEASE

53 ing analysis. Sequence alignments were carried out with NCBI database. Microbiological cultures were carried out among all the patients. Results: 3 out of 5 patients in Group I (60%), 3 out of 5 patients in Group II (60%), 6 out of 9 in Group III (66.6%) and none out of 4 in Group IV (0%) showed bactDNA in blood. Bacteria identifications included E. coli (4), Enterococcus (4), StaphylococcuS (3) and Streptococcus (1). Blood cultures were negative in all cases. Conclusion: Molecular detection tools in patients with IBD are able to identify a subgroup of patients with presence of circulating bacterial DNA that remains undetected by microbiological culture. Presence of bacterial translocation in patients with inactive CD, the surprising prevalence of bacterial DNA from Enterococcus in patients with active UC, and the likely existence of immune consequences associated to these facts require new investigations.

P193 PRO-INFLAMMATORY EFFECT OF HELICOBACTER PULLORUM ON HUMAN INTESTINAL CELL LINES D. Laharie 1 , C. Varon 2 , A. Duriez 2 , F. Zerbib 2 , A. Menard 2 , P. Lehours 2 , F. Megraud 2 . 1 Hopital Haut-Leveque; 2 Laboratoire de Bacteriologie, INSERM-ERI10, Universite Bordeaux 2, France Helicobacter pullorum (H. pullorum) is an entero-hepatic Helicobacter species from avian origin observed in patients with acute diarrhea and inflammatory bowel disease (IBD). Its direct effect on intestinal epithelial cells is still unknown. Aim: to determine whether H. pullorum exerts a direct effect on human epithelial intestinal cells and to characterize the bacterial tools and the signaling pathways involved. Methods: 1. Comparison on gastric epithelial cells (AGS) between H. pullorum and H. pylori (J99 and B38 strains) interleukin-8 (IL-8)-induced secretion assessed with ELISA. 2. Measurement on CaCo-2 and HT-29 intestinal cell lines of IL-8-induced production with avian and human H. pullorum strains. 3. Evaluation of H. pullorum-adherence on IL-8-induced secretion by intestinal epithelial cells using bacterial supernatants and Transwell inserts. 4. Determination of nuclear factor kappaB (NF-κB) role and activation with specific inhibitors (pharmacological - SN50 - and RNA-interference with siRNA) and immunofluorescence (IF). Results: 1. H. pullorum strains induce higher AGS-IL-8 levels than controls, intermediate between the potent inflammatory J99 and the mild inflammatory B38 H. pylori strains. 2. On HT-29 and CaCo-2 intestinal cells, H. pullorum-induced IL-8 secretion was increased with human or avian strains, higher than in controls. 3. Coculture with H. pullorum supernatants did not induced high IL-8 levels as compared to the whole bacteria. The same trend was observed with Transwell inserts. 4. NF-κB inhibition with SN50 and siRNA reduced H. pullorum-induced IL-8 production. H. pullorum induced a rapid nuclear translocation of NF-kB visualized by immunofluorescent staining. Conclusion: Human and avian H. pullorum strains induce IL-8 production on gastric and intestinal cell lines. This effect needs the bacterial adherence and implies NF-κB activation. These results confirm H. pullorum as a potent human pathogen which could be involved in acute and chronic digestive disease such as IBD.

P194 HPEPT1 SELECTIVELY TRANSPORTS MURAMYL DIPEPTIDE BUT NOT NOD1-ACTIVATING MURAMYL PEPTIDES

A. Gutiérrez, R. Francés, J. Such, M. Garmendia, M. Ndongo, R. Jover 1 , M. Pérez-Mateo. Servicio de Medicina Digestiva, Hospital General Universitario Alicante

S. Vavricka 1 , M. Ismair 1 , G. Kullak-Ublick 1 , M. Fried 1 , D. Mengin-Lecreulx 2 , S. Girardin 3 . 1 Division of Gastroenterology and Hepatology, University Hospital Zurich; 2 IBBMC, Université Paris-Sud; 3 Department of Laboratory Medicine and Pathobiology, University of Toronto

Background: The pathogenesis of inflammatory bowel disease (IBD) involves the interaction between genetic susceptibility, mucosal immunity and intestinal bacteria. Bacterial DNA (bactDNA) derived from luminal bacteria may contribute to the perpetuation of chronic intestinal inflammation. Blood microbiological cultures, though, are frequently negative in these patients and bacterial translocation episodes may be put out of sight. Aims: To evaluate the presence of bacterial translocation in patients with IBD by detection of bacterial DNA in blood. Patients and Methods: 23 patients with IBD not treated with antibiotics the month before inclusion were distributed into Group I: active Crohn' s disease (CD); Group II: inactive CD; Group III: active ulcerative colitis (UC); and Group IV: inactive UC. A blood sample was collected in endotoxin-free tubes and a broad-range PCR of a highly conserved region of the bacterial 16SrRNA gene was performed using the following primers: 5 -TTCCGGTTGATCCTG CCGGA-3 as forward, and 5 -GGTTACCTTGTTACGACTT-3 as reverse. Bacterial genomic fragments were purified and identified by nucleotide sequenc-

Muramyl peptides derived from bacterial peptidoglycan are detected intracellularly by Nod1 and Nod2, two members of the newly characterized Nodlike Receptor (NLR) family of pattern recognition molecules. In the absence of bacterial invasion into the host cytosolic compartment, it remains unclear if muramyl peptides can cross the plasma membrane and localize into the cytosol. We have recently demonstrated that the plasma membrane transporter, hPepT1, was able to translocate efficiently muramyl dipeptide (MDP), a specific Nod2-activating molecule, into host cells. We aimed to characterize the transport properties of hPepT1 towards a spectrum of muramyl peptides, including Nod1-activating molecules. To do so, we designed an original procedure based on the ectopic expression of hPepT1 in oocytes from Xenopus laevis. Our results demonstrated that hPepT1 transports MDP but no other Nod2activating molecule. Moreover, we observed that Nod1-stimulating muramyl peptides were not transported by hPepT1. Since hPepT1 expression is strongly associated with intestinal epithelial cells, where Nod1 and Nod2