in patients receiving azathioprine at the time of diagnosis of NH-LPD (in association in onecase with infliximab). Among these, 11 could be tested for EBV; seven of them were EBV-associated, including a fatal case of brain LPD, a fatal case of intestinal large B-cell lymphomaand two fatal cases of early post-mononucleosis LPD. Results will be updated in May 2008.Conclusion: Interim results of the CESAME cohort suggest an overall increased risk of LPDin IBD. The excess risk appears to be related to IT since ¾ of the incident cases of LPDoccurred in patients receiving azathioprine, with a fatal issue in almost half of the cases,and a frequent association with EBV infection. [1] Kandiel A et al., Gut 2005 ;54 :1121-5.

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Reconstitution of Physiological Immune Responses to Bacterial DNA DuringExperimental Colitis Can Be Achieved By Inhibition of Gsk3-βClaudia Hofmann, Nadja Dunger, Nicole Grunwald, Juergen Schoelmerich, Werner Falk,Florian Obermeier

Background: CpG motifs of bacterial DNA from the intestinal flora are protective againstexperimental colitis. In contrast bacterial DNA essentially contributes to the perpetuationof an already established chronic intestinal inflammation in a TLR9-dependent manner. Adisturbed regulation of TLR signal transduction resulting in the exclusive activation ofproinflammatory signaling pathways may therefore be critical for the perpetuation of chronicintestinal inflammation. Recently, glycogen synthase kinase 3-β (GSK3-β) was identified asan important regulator of TLR signaling mediating excessive inflammatory responses. Aimof this study was therefore to assess the role of GSK3-β in experimental colitis. Methods:For induction of chronic colitis, female Balb/c mice and TLR9-/- received 4 cycles of dextransodium sulphate (DSS) treatment. For the CD4+CD62L+ T-cell transfer model, splenicCD4+CD62L+ T-cells from Balb/c mice were isolated and injected i.p. in recipient C.B.-17SCID mice. After the onset of chronic colitis mice were treated for 5 days i.p. with PBS,CpG-ODN or GSK3-β-inhibitors (SB216763, LiCl) in both models. Evaluation of intestinalinflammation was performed by histologic analysis as well as by cytokine secretion ofmesenteral lymph node cells (MLN) after stimulation with anti-CD3 and IL-2 (determinedby Luminex). Results: GSK3-β inhibition in DSS-induced colitis resulted in a significantreduction of histologic scores compared to controls (SB216763: p=0.008 / LiCl: p=0.016).Treatment with CpG-ODN intensified DSS-induced inflammation. However, this effect wascompletely abolished by simultaneous administration of LiCl (p=0.006). Exclusive inhibitionof GSK3-β did not significantly influence production of proinflammatory cytokines, butresulted in significantly increased secretion of IL-10 (p=0.006). Strongly elevated productionof TNF and IL-6 after CpG-ODN treatment was reduced to control levels by blocking GSK3-β. In contrast, GSK3-β inhibition did not influence intestinal inflammation during DSS-induced colitis in TLR9-/- mice. Similarly, blockade of GSK3-β resulted in a significantdecrease of histologic scores in the T cell transfer model (p=0.01) with strongly elevatedlevels of IL-10 (p=0.007). Conclusion: Blockade of GSK3-β is capable of attenuating excessiveTLR9-mediated immune responses to bacterial DNA during chronic intestinal inflammationby reconstituting physiologic anti-inflammatory responses. GSK3-β inhibition therefore con-stitutes a promising therapeutic option for IBD.

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Erythrocytes-Mediated Delivery of Low Doses of Dexamethasone RevertSteroid-Dependency in Ulcerative Colitis. a Double-Blind, Sham-ControlledStudyFabrizio Bossa, Anna Latiano, Luigia Rossi, Mauro Magnani, Orazio Palmieri, BrunoDallapiccola, Sonja Serafini, Gianluca Damonte, Angelo Andriulli, Vito Annese

Introduction and Aim: Up to one fourth of patients with Ulcerative Colitis (UC) on steroidsbecame steroid-dependent within 1 year. Immunosuppressive drugs (ISD) are used as steroid-sparing agents, but they are ineffective or not tolerated in up to one third of cases. Weplanned a sham-controlled study to evaluate the systemic steroid sparing efficacy and safetyof erythrocytes-mediated delivery of low doses of dexamethasone (EDD) in steroid-dependentUC patients. Materials and Methods: Thirty patients in remission or mild clinical activity(14 males; mean age at diagnosis 35.5 +/- 13.4 yrs) steroid-dependent since 6-26 months,were randomly assigned to six monthly infusions of EDD (group A, n=15) or sham infusions(group B, n=15). Fifty millilitres of blood were drawn from all patients; dexamethasone 21-Phosphate (Dex 21-P) was encapsulated into autologous erythrocytes (group A) by meansof specific equipment, and re-infused into original donors. In group B (sham), erythrocyteswere re-infused without dexamethasone loading. Twenty-three patients (77%) were undera stable dose of ISD for at least 6 months, while the remaining 7 patients were intolerant.Results: At study inclusion, mean values of Powell-Tuck index (3.5 vs 4.1), Baron score(1.2 vs 1.3), ESR (25 vs 31 mmhr), CRP (1.1 vs 0.9 mg/dl), cortisol (13.5 vs 9.0 mg/dl),and prednisolone dose (21.2 +/- 8.9 vs 25.3 +/- 12.1 mg) were comparable in both groups.A mean dose of 11.5 +/-10 mg of Dex 21-P was loaded into the erythrocytes (group A) ateach treatment. Seventeen patients (13 in the group A, and 4 in the group B) completedthe six infusions of EDD or placebo (87% vs 26%, p<0.0001), while the others droppedout due to symptoms worsening. Oral steroids could be withdrawn in 11 patients (76%)of group A and 3 (20%) of group B (p<0.0001), within 2 and 3.5 months from the studyinclusion, respectively. At the end of the study (one month after the last infusion), the meanvalue of Powell-Tuck was 2 +/- 1.2 in group A and 6 +/- 3.9 in group B (p=0.001), whereasthe mean dose of steroids used was 4.2 +/- 9.3 mg vs 21.4 +/- 18.3 mg, in groups A andB, respectively (p=0.0049). At study inclusion, 25 patients (83%) had 1 or more steroid-related adverse events (SRAEs)(13 in the group A and 12 in the group B). During treatment,SRAEs disappeared in 8 (67%) and 2 (15%) patients of groups A and B, respectively(p<0.0001). Conclusions: The very low dose of EDD administered monthly was effectivein systemic steroids sparing, with disappearance of SRAEs in most cases. This treatmentmodality might represent a valuable option in steroid-dependent UC patients non-responderto ISD or as “bridge” therapy.

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Muc1 Limits H. pylori Infection By Acting As a Releasable DecoySara K. Linden, Kim M. Miles, Timothy H. Florin, Andre Dubois, Michael A. McGuckin

Background: The cell-surface mucin MUC1 is a dominant feature of the mucosal glycocalyxand extends further than most other cell-surface structures. We previously showed thatMUC1 limits the density of H. pylori infection in a murine infection model and that severegastritis develops in ∆MUC1 mice. Here, we determined changes in MUC1 expressionimmediately after infection in a primate model and explored the mechanism by which MUC1limits colonization. Materials and Methods: The MUC1 response to infection was assessedby immunohistochemistry in gastric biopsies from 11 rhesus monkeys before and 7 daysafter H. pylori inoculation. ELISA was used to assess binding of H. pylori to human MUC1.Viable microaerobic co-cultures of human MKN7 gastric carcinoma cells with H. pylori wereused to investigate the dynamic interactions between live bacteria and cellular MUC1 andto assess the influence of MUC1 on H. pylori induced apoptosis. Results: In rhesus monkeys,MUC1 was present on the apical surface of gastric epithelial cells and expression wassubstantially increased at the 7-day time point, particularly within the cytoplasm of surfaceepithelial cells (p >0.001). H. pylori bound MUC1 isolated from human gastric cells as wellas MUC1 shed into human gastric juice. H. pylori bound to the oligosaccharides Leb andsialyl-Lea on MUC1 via the blood-group binding adhesin (BabA) and sialic acid bindingadhesin (SabA), respectively. Both H. pylori carrying these adhesins, and 1 µm beads coatedwith MUC1 antibodies, caused a depletion of MUC1 from gastric epithelial cells, whichshed MUC1, demonstrating that MUC1 acts as a releasable decoy following engagement byparticulate ligands. In contrast, isogenic H. pylori without adhesins, or beads coated withisotype control antibodies, did not cause shedding of MUC1. Both H. pylori strain J99, whichcarries both the BabA and SabA adhesins, and the isogenic CagA deletion mutant bothcaused apoptosis in 30 vs 20% of MKN7 cells after 24h co-culture (p <0.05). In contrast,the isogenic BabA/SabA deletion mutant lacking the adhesins that interact with MUC1 didnot affect epithelial cell viability. MKN7 cells transfected with MUC1 siRNA had 80-90%less cell surface MUC1 and became apoptotic earlier after H. pylori infection than MKN7cells transfected with control siRNA. Conclusions: Taken together these data show thatMUC1 protects gastric cells from adhesin dependent H. pylori induced death. Gastric MUC1is upregulated in response to infection and protects the gastric mucosal epithelium from H.pylori-induced damage by acting as a releasable decoy.

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Mucosal Gene Signatures to Predict Response to Infliximab in Patients withInflammatory Bowel DiseaseIngrid Arijs, Leentje Van Lommel, Kristel Van Steen, Gert De Hertogh, Marc Ferrante,Marie Joossens, Karel Geboes, Gert A. Van Assche, Severine Vermeire, Frans C. Schuit,Paul J. Rutgeerts

Introduction and aim: Infliximab (IFX), the chimeric monoclonal antibody to TNFα, is aneffective therapy in inflammatory bowel disease (IBD). However, a subset of patients showsno effect to this agent and the molecular mechanism underlying its beneficial effects is stillunclear. The aim of this study is to identify gene signatures predictive of response to IFXand to identify new targets for treatment of IBD patients resistant to IFX therapy using high-density oligonucleotide arrays. Methods: Colonic mucosal biopsies were obtained from 41IBD (17 Crohns' disease (CD) and 24 ulcerative colitis (UC)) patients before and 4-6 weeksafter first IFX treatment. The response was assessed at 4-6 weeks after first IFX treatment,based on endoscopic and histologic findings. Endoscopic response was defined as completemucosal healing for CD and UC (Mayo endoscopic subscore of 0 or 1 (1)). Histologicresponse was defined as a decrease of at least 3 points for CD (2) and a decrease to grade0 or 1 for UC (3). Absence of response was defined as no mucosal healing for CD and UC(Mayo endoscopic subscore of 2 or 3) and no change or an increase in the histological score.Total RNA was isolated, labelled and hybridized to Affymetrix HGU133plus2.0 array. Datawas analyzed using Bioconductor software. Results: We identified 18 responders (10 CDand 8 UC), 15 non-responders (5 CD and 10 UC) and 8 partial (non-) responders (2 CDand 6 UC). For predicting response to IFX treatment, expression profiles before treatmentwere compared for responders and non-responders. Comparative analysis (false discoveryrate < 5% and > 2-fold change) identified a total of 1216 DE probe sets in CD, 8 DE probesets in UC and 833 DE probe sets in IBD. Class prediction analysis revealed that only 1probeset is necessary in CD to classify samples as responder or non-responder with anoverall misclassification error (ME) of 0 (0/15) and only 9 probe sets were necessary in UCwith ME of 0.11 (2/18). For IBD overall, only 1 probe set that represents a Th2 immunesystem pathway molecule allowed almost complete separation between responders and non-responders with a ME of 0.03 (1/33). Conclusion: We showed that transcriptional profilingof pre-treatment biopsies allows reliable prediction of response to IFX therapy. The techniqueallowed to identify a new target for treatment in IBD patients not responding to IFX therapy.Reference List: (1)Rutgeerts P, et al. N Engl J Med 2005;353:2462-2476. (2)D'Haens GR,et al. Gastroenterology 1998;114:262-267. (3)Geboes K, et al. Gut 2000;47:404-409.

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TNF-Induced Cell Shedding in the Small Intestine Causes NovelReorganization of Tight Junctional ProteinsAmanda M. Marchiando, Yanfang Guan, Aaron L. Hecht, Elizabeth M. Angus, MarshallH. Montrose, Jerrold R. Turner, Alastair J. Watson

Background: Small intestinal epithelial cells are continuously shed from the villus at steadystate. Villus epithelium is punctuated by gaps in the epithelial monolayer. These gaps aresealed by an unknown impermeable substance that maintains barrier function at these sites.Epithelial gaps are found in both mice and humans and can be distinguished from gobletcells. Phosphorylation of myosin light chains (a process previously associated with epithelialwound healing in the intestine) is found in 50% of cell shedding events suggesting that thegaps caused by shedding cells ultimately resolve by a wound healing mechanism. Aims:Our aim was to determine if tumor necrosis factor (TNF-α) increases the rate of cell shedding,

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