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

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

54 have been shown to play a key role, these observations suggest a distinct contribution of Nod1 and Nod2 in mucosal homeostasis following the cellular uptake of muramyl peptides by hPepT1.

P195 THE PRESENCE OF ESCHERICHIA COLI BACTERIOPHAGES IN HUMAN STOOL OF PATIENTS WITH INFLAMMATORY BOWEL DISEASE M. Lusiak-Szelachowska 1, B. Weber-Dabrowska 1 , A. Górski 1 , A. Annabhani 2, K. Blachut 2 , L. Paradowski 2 . 1 Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Center of Excellence, Wroclaw; 2 Department of Gastroenterology and Hepatology, Wroclaw Medical University Introduction: Bacteriophages are bacteria-specific viruses that attack bacteria and, after multiplication, lyse the host cell. Escherichia coli bacteriophages are widely distributed throughout the environment as well as in many human individuals and animals. Endogenous bacteriophages may play a valuable role in protecting against microbes. Aim: The aim of our study was to investigate the presence of coliphages in the human intestine and to compare their frequency in healthy individuals and patients with inflammatory bowel disease (IBD) (Crohn' s disease and ulcerative colitis) in a Polish population. Material and method: Stool samples were taken from 70 volunteers and 165 patients with IBD (48 with Crohn' s disease and 117 with ulcerative colitis). Coliphages were detected in human feces using the indicatory bacteria E. coli B and E. coli 1962 from the Institute' s collection. E. coli-specific phages were examined by the double-agar-layer method after 24 hours of incubation at 37°C of the stool sample with the bacterial host culture. The activity of phages was determined in immediate materials. Results: Coliphages for both E. coli B and E. coli 1962 strains were detected in: 30% of the stools of volunteers (mean concentration: 2.7×103 PFU/g in randomly chosen samples), 14.6% of samples from patients with Crohn' s disease (mean concentration: 2.9×105 PFU/g), 18.8% of samples from patients with ulcerative colitis (mean concentration: 3.6×103 PFU/g). Conclusion: Our results suggest that the frequency of samples positive for E. coli bacteriophages is higher in those from volunteers than in those from patients with IBD. The concentrations of coliphages in patients' feces were higher compared with healthy individuals. The results suggest that there may be associations between phage presence in the human gastrointestinal tract and its disorders.

P196 CYTOMEGALOVIRUS BLOCKS APOPTOSIS IN PATIENTS WITH ACTIVE ULCERATIVE COLITIS AND COLONIC VIRAL INFECTION R. Vega 1 , H. Keshaw 1 , E. Domenech 2 , I. Ojanguren 2 , M.A. Gassull 2 , A. Forbes 1 , R. Day 1 . 1 University College Hospital; 2 Hospital Universitari Germans Trias i Pujol Introduction: The immunological response of the colonic mucosa to cytomegalovirus (CMV) infection in patients with active ulcerative colitis (UC) remains unknown. TNF-related apoptosis-inducing ligand (TRAIL) mediated apoptosis is increased in patients with inflammatory bowel disease. Human CMV is known to block apoptosis in different types of cells despite an upregulation of TRAIL and TRAIL membrane receptors. Aim: To examine the apoptosis rates in active UC patients with and without CMVinfection; and investigate the expression of TRAIL and TRAIL R1 and R2 in the colonic tissue of these patients. Materials and Methods: Paraffin embedded colonic tissue specimens were obtained from 8 non-steroid resistant active UC patients, 8 steroid-resistant active UC, and 6 steroid-resistant active UC with colonic infection by CMV, which were included in a clinical prospective study to investigate the prevalence of CMV in UC. Apoptosis of epithelial and lamina propia cells was assessed by immunohistochemical staining for cleaved caspase-3. Immunohistochemical staining was also performed for TRAIL, TRAIL-R1 and R-2. Results: There was no significant difference in disease activity index, CRP and ESR among the patient groups when the biopsies were obtained. Histology assessment showed moderate to severe colonic inflammation. Colonic CMV infection in the samples was confirmed by immunohistochemical staining for CMV. TRAIL was strongly expressed in the epithelial cells in all samples, especially in the upper 1/3 area of the crypts. The expression of TRAILR2 in lamina propia was higher in infected patients compared to the patients without viral infection regardless to steroid response. There were no differences in TRAIL-R1 expression among the different groups. Apoptotic indices (proportion of caspase-3 positive cells from the total of cells counted) in intestinal epithelial cells were significantly lower (0.27±0.15) in patients with CMV infection compared with steroid-resistant patients (0.53±0.13) and nonsteroid-resistant patients (0.66±0.22) p<0.05. No significant differences in

Poster Presentations the apoptosis indices were seen in lamina propia cells among the different groups. Conclusions: Apoptosis in colonic epithelial cells decreases in CMV infected patients despite the presence of TRAIL and the expression of TRAIL-R2. This may be explained by the capacity of the human CMV to encode proteins, such as viral inhibitor of caspases-8-induced apoptosis (vICA) and viral mitochondrial inhibitor of apoptosis (vMIA), which directly interfere with the apoptotic signalling pathway in infected tissues. CMV may contribute to steroid resistance in UC infected patients.

P197 IS ESCHERICHIA COLI MORE THAN A COMMENSAL BACTERIUM IN THE INTESTINAL MUCOSA OF CROHN' S DISEASE PATIENTS? M. Martinez-Medina 1, X. Aldeguer 2 , F. Gonzalez-Huix 2 , D. Acero 2 , M. Lopez-Siles 1 , L.J. Garcia-Gil 1 . 1 University of Girona; 2 Hospital Josep Trueta The role of bacteria in Crohn' s disease (CD) has received special attention in the last few years. In particular, Escherichia coli (EC) has been suggested to play a role in the onset of the inflammatory response [1-4] and has recently been shown to be more prevalent in CD patients [5] In this work EC strains isolated from CD patients have been analyzed and compared with those from healthy controls in order to identify and characterize the pathogenic strains. A total of 1600 presumptive EC from fresh ileo-colonic biopsies corresponding to 8 CD patients, 10 healthy controls and 2 patients suffering from Ulcerative Colitis, were isolated. Transient and loosely attached bacteria were discarded by mild sonication. A mild osmotic shock was performed to release intracellular bacteria. EC was isolated on TBX agar medium and further confirmed by indole assay. The clonality of isolates was checked by PFGE using two enzymes (XbaI and SpeI). The presence of up to 18 virulence genes (cnf1, cnf2, hly, iuc, papG, sfa/foc, afa, cdtB, neuC, stx1, stx2, eae, bfp, ipaH, pCDV432, eltA and est) was tested by PCR. A total of 1600 presumptive EC from 8 CD patients, 10 healthy controls and 2 patients suffering from Ulcerative Colitis, were isolated. After analyzing the clonality of the isolates, a total of 31 different EC subtypes were obtained. On average, 1.93 ± 0.96 different strains per individual were found, with no significant differences between CD patients and controls. Except for 5 subtypes, which were found in two patients each, every EC clone were unique to each human carrier, which supports the idea that the host-microbe relationship in the gut ecosystem is controlled by complex factors. We have not found genetic relatedness among EC from CD patients in terms of presence of certain known virulence genes, neither by their PFGE profile. Our results so far indicate that there is not a common strain inhabiting the colonic mucosa of CD patients, as previously suggested. Nevertheless, the pathogenic features of the isolates are currently under study, since the possibility of certain common features of EC from CD patients in terms of pathogenicity cannot be ruled out. References: [1] Darfeuille-Michaud A et al, 2004. Gastroenterology; 127(2): 412-21. [2] Helen M. Martin et al, 2004. Gastroenterology;127(1): 80-93. [3] Masseret E et al, 2001. Gut; 48: 320-325. [4] Ryan M.D et al, 2004. Am J Gastroenterol; 99: 1539-1543. [5] Martinez-Medina et al. 2006. Inflammatory Bowel Diseases; 1136-1145

P198 GALECTIN-4 INDUCES T CELL APOPTOSIS AND AMELIORATES INTESTINAL INFLAMMATION IN AN EXPERIMENTAL MODEL OF MURINE COLITIS D. Paclik 1 , C. Guzy 1 , U. Berndt 1 , B. Wiedenmann 1, A. Dignass 2, H.-J. Gabius 3 , A. Sturm 1 . 1 Charite, Campus-Virchow Clinic; 2 Markus-Krankenhaus, Frankfurt; 3 Institut für Physiologische Chemie, Tierärztliche Fakultät, LMU, München Background: Galectins, a growing family of animal lectins, has recently attracted the interest of cell biologists and immunologists as master regulators of immune cell homeostasis. Within this family, Galectin-4 (Gal-4) is of particular interest with regard to the mucosal immune system since it is expressed in intestinal epithelial cells and the colonic mucosa. Thus, we aimed to explore the effect of Gal-4 in the human immune system and in an experimental model of colitis. Methods: PBT were stimulated with cross-linked anti-CD3/28 mAb and cultured in the presence or absence of Gal-4. Flow cytometry analysis was used to determine apoptosis (Annexin-V), necrosis (PI), cell activation (CD25), cell cycle progression (Cyclin A, B1) and expansion (CFDA). Cytokine secretion was determined by CBA analysis. Acute colitis was induced in BALB/c mice by adding 5% DSS to the drinking water. Mice were treated i.p. with either NaCl as control or Gal-4 (1 mg/kg BW) three times a day until they were sacrificed on day 8. The disease activity (DAI) and histological inflammation index were quantified by established scores. TUNEL assay and BrdU staining were performed to determine mucosal apoptosis and proliferation.