gene per locus, 24 genes (57%) showed high-level expression (p<0.009, FDR<0.07). Nineof these (CPEB4, CCL2, ICAM3, RTL1, LTB, TNFSF8, CREM, LRRK2 and PTPN2) correspondto those previously reported as associated with CD in the meta-analysis, and 15 werenew additional candidate genes (LHX9, C11ORF10, PPP1R15A, SDF2L1, LIF, APOBEC3C,PHTF1, ARHGAP30, GMPPB, ANXA6, TRIB1, EGR2, CYLD, PTRF and C19ORF23). Of the18 genes (43%) showing lower-level expression (p<0.008, FDR<0.07), two are the same ofthe previously defined significant hits (SULT1A2 and SLC22A5), and 16 are new additionalgenes (PER3, ZBTB7B, PUS10, SLC9A2, TMEM171, CAST, SLC22A23, PLCB3, GPATCH1,CEBPA, SERBP1, AK3, GOT1, TSKU, THRA and PFKL). Conclusions: Sixty-seven deregulatedgenes in 71 loci were identified analyzing the gene expression in the colonic mucosa of CDpatients. Out of the 42 hit genes identified, 31 were new additional candidate deregulatedgenes in inflamed colonic mucosa and were reported as associated with active phase CD.Extensive resequencing, large-scale fine mapping and functional studies of these loci willbe required to elucidate the pathogenic mechanisms of CD.

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Autophagy Induces Immune Tolerance by Regulating Interactions BetweenDendritic Cells-Epithelial Cell in the GutCaterina Strisciuglio, Marjolijn Duijvestein, Anne Christine W. Vos, Auke Verhaar, ErasmoMiele, Riccardo Troncone, Gijs R. van den Brink, Daniel W. Hommes, Manon E.Wildenberg

Introduction Recently, various single nucleotide polymorphisms(SNPs)in the autophagyrelated genes ATG16L1 and IRGM have been associated with the development of Crohn'sdisease. These SNPs lead to decreased autophagic activity, suggesting a regulatory role forthis mechanism in immunity. In the intestine, DC are capable of sampling luminal antigenby protruding dendrites through the epithelial cell layer, while maintaining barrier integrity.It has been shown that the proper sampling of antigen, as well as the interactions betweenDC and the epithelium is crucial for maintenance of intestinal tolerance. We have previouslyshown that autophagy contributes to regulation of cell-cell interactions between dendriticcells (DC) and T cells and therefore, the aim of this study was to elucidate the role ofautophagy in the regulation of cell-cell interactions between DC and intestinal epithelium.Materials and methods An In Vitro model system for luminal sampling by DC was set up,in which an epithelial colon cancer cell line was cultured on one side of a transwell insert,and human dendritic cells (DC) on the other side of the insert, at the basolateral side ofthe epithelium. Modulation of autophagy was achieved using siRNA. In this assay, DCphenotype and luminal antigen sampling were measured by flow cytometry. The capacityof DC to form transepithelial protrusions was determined by confocal microscopy. ResultsIn the gut DC-epithelial interactions are characterized by the presence of tight junctionsand adhesion molecules. Interestingly, the adhesion molecule E-Cadherin partly localizedto autophagosomes and decreased autophagy resulted in increased levels of this protein,suggesting autophagy is involved in proper DC-epithelial interactions. Indeed, reducedautophagy in either DC, epithelial cells or both resulted in the decreased formation oftransepithelial protusions by DC as well as a reduction in antigen sampling. Moreover, whenautophagy was inhibited in either DC or epithelial cells in the co-culture model, DC expressedincreased levels of HLA-DR and costimulatory molecules. Furthermore, decreased levels ofautophagy resulted in an altered cytokine profile of DC and these cells induced significantlymore T-cell proliferation in an allogenic mixed lymphocyte reaction. This data suggest thatdecreased autophagy is also involved in increased maturation and increased immunoreactiv-ity. Conclusions Decreased autophagy is involved in the formation of proper interactionsbetween DC and intestinal epithelium. Autophagy deficiency leads to aberrant interactionsresulting in decreased antigen sampling, increased DCmaturation and a more pro-inflammat-ory type of DC. Therefore, autophagy-related SNPs may contribute to CD pathogenesisthrough dysregulation of the interactions between DC and epithelial cells resulting in a lackof immune tolerance.

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Human IL23R R381Q Genetic Variant Implicated in Crohn's DiseasePathogenesis is Hypomorphic Resulting in Reduced Function of the ReceptorHilary Clark, Svetlana Pidasheva, Sara Trifari, Jason Hackney, Yan Ma, Ashley Smith, SueSohn, Hergen Spits, Randall Little, Timothy Behrens, Lee Honigberg, Nico Ghilardi

The biggest challenge of the genome-wide association studies (GWAS) era is conductingfollow-up studies to identify and functionally analyze causal variants that are associated withcomplex diseases. GWAS in several populations have demonstrated significant associationsof the interleukin 23 receptor (IL23R) gene with Crohn's disease (CD) and psoriasis, sug-gesting that perturbation of the IL-23 signaling pathway is relevant to the pathophysiologyof these diseases. One particular variant, R381Q (rs11209026), confers strong protectionagainst development of CD, but the mechanism by which IL23R Q381 confers protectionis unresolved and remains a question of key importance. We investigated the effects of theR381Q IL23R variant in transformed cells and primary T cells from IL23R R381 and IL23RQ381 positive donors. We believe that the functional validation of GWAS findings is acritical step in the translation of genetics into novel therapeutics. Our study is the first toshow the alterations in function of IL23R Q381 allele, further strengthening the implicationfrom GWAS results. These data provide an explanation for the protective role of R381Q inCD and may lead to the development of personalized therapeutics for autoimmune disorderslike CD.

S-273 AGA Abstracts

Sa1289

Identification of Serum and Tissue Micro-RNA Expression Profiles in DifferentStages of the Inflammatory Bowel DiseaseMarisa Iborra, Francesca Bernuzzi, Belen Beltran, Antonino Spinelli, Pietro Invernizzi,Silvio Danese

Background: Inflammatory Bowel Disease (IBD), Crohn`s disease (CD) and ulcerative colitis(UC), is associated with the differential expression of genes involved in inflammation andimmune functions. MicroRNAs (miRNAs) are a class of small non-coding RNAs involvedin the control of gene expression at post-transcriptional level and are involved in theregulation of biological processes, as well as in the induction of chronic inflammatory diseasesand autoimmune diseases. Recently, the altered expression of miRNA has been associatedwith IBD. Aims: To establish specific miRNAs expression patterns in serum and mucosa ofCD and UC patients at different stages of the disease and to compare with healthy patients.Methods: Samples of serum and biopsies were obtained from 36 IBD patients (9 active CD(aCD), 9 inactive CD (iCD), 9 active UC (aUC) and 9 inactive UC (iUC)), and from 9healthy subjects (H). Blood samples were drawn at the time of the planed endoscopicprocedure. Three pools of 3 serum and biopsies samples were analysed for each group. TheRNA fraction highly enriched for small RNA species was isolated by the mirVanaTMPARISTMkit, according to the manufacturer's protocol. The small RNA fraction was reverse transcribedusing the miRNA Megaplex RT primers and TaqMan® microRNA Reverse Transcriptionkit. The cDNA obtained was amplified using to TaqMan® PreAmp Master Mix and MegaplexPreAmp Primers. Up to 700 miRNAs were evaluated by TaqManR Human miRNA Array.The values were calculated as DCt and U6 was the housekeeper gene. P<0.05 was consideredsignificant. Results: In aCD 21 serum miRNAs were exclusively regulated (H vs aCD). IniCD 85 serum miRNAs were exclusively regulated (H vs iCD). There were 37 serum miRNAscommonly expressed in the 3 groups (aCD-iCD-H). In the mucosa of aCD 8 miRNAs wereexclusively regulated (H vs aCD). In mucosa of iCD 11 miRNAs were exclusively regulated(H vs iCD). There were 6 tissue miRNAs commonly expressed in the 3 groups (aCD-iCD-H). In aUC 19 serum miRNAs were exclusively regulated (H vs aUC). In iUC 91 serummiRNAs were exclusively regulated (H vs iUC). There were 58 serum miRNAs commonlyexpressed in the 3 groups (aUC-iUC-H). In mucosa of aUC 38 miRNAs were exclusivelyregulated (H vs aUC). In mucosa of iUC 11 miRNAs were exclusively regulated (H vs iUC).There were 4 tissue miRNAs commonly expressed in the 3 groups (aUC-iUC-H). Conclusion:Our results suggest the existence of specific miRNA expression patterns associated to IBDand to their different stages. MiRNAs could be useful to distinguish IBD from healthycontrols. The different expression among CD, UC and H support the utility of miRNA aspossible biomarkers. It is necessary that our results are confirmed in larger samples andfurther studies based in the miRNA as therapeutic targets.

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Forward Genetic Analysis of Gut Homeostasis Using a Sensitized Screen inMiceKatharina Brandl, Wataru Tomisato, Bruce Beutler

Background: The epithelium of the gastrointestinal tract is occasionally subject to injury bytoxigenic enteric pathogens, and perhaps on occasion by microscopic particulates capableof wounding cells. However, homeostatic mechanisms normally restore the status quo ante,so that dysfunction is short-lived. These homeostatic mechanisms remain largely obscure.Their failure may lead to a chronic disease state, recognized clinically as inflammatory boweldisease (IBD). Methods: By exposing randomly mutagenized mice to brief treatments withdextran sodium sulfate (DSS) at a concentration that is harmless to wild type animals, wehave sought to identify genes with non-redundant function in homeostasis. A total of 5846mice have been screened by placing them on 1% DSS in drinking water for a period of 7days. During this time period all animals are weighed daily, and extreme outliers are retrievedfor further study. Results and Conclusion: A total of 17 transmissible mutations causinghypersensitivity to DSS have been detected. Of these, 6 have been identified, and fall into5 genes. Failure of epithelial integrity is sensed by Toll-like receptors (TLR) on epithelialcells, which signal to cause expression of epidermal growth factor ligands amphiregulin andepiregulin, promoting epithelial cell growth. Wound closure also requires water transportvia aquaporin 3, and an intact unfolded protein response, which depends on Mbtps1, alsoknown as the site-1 protease. Relatively common mutations affecting the structure of Mucin-2 enhance susceptibility to colitis, evidently by increasing ER stress. In addition, genes withunknown functions have been found to cause increased susceptibility to DSS colitis. Acoherent picture of the essential events required for homeostasis has begun to emerge.

Sa1291

Variants in the PTPN2 Gene Are Associated With Susceptibility to BothCrohn's Disease and Ulcerative Colitis in the German PopulationJohanna Wagner, Jürgen Glas, Julia Seiderer, Burkhard Göke, Thomas Ochsenkühn, JuliaDiegelmann, Stephan Brand

Introduction: Genome-wide association studies identified PTPN2 (protein tyrosine phosphat-ase, non-receptor type 2) as susceptibility gene for inflammatory bowel diseases (IBD).However, the exact role of PTPN2 in Crohn's disease (CD) and ulcerative colitis (UC) isstill controversial. Given different results of previous replication studies, we investigatedwhether genetic variants in this gene are associated with CD and UC in a large German IBDcohort. Methods: Genomic DNA from 2131 individuals of Caucasian origin including 906patients with CD, 318 patients with UC, and 907 healthy, unrelated controls was analyzedfor two SNPs in the PTPN2 region (rs2542151, rs7234029). From all study participants,blood samples were taken and genomic DNA was isolated from peripheral blood leukocytesusing the DNAbloodmini kit fromQiagen (Hilden, Germany) according to themanufacturer'sguidelines. Genotyping was performed by PCR and melting curve analysis using a pair offluorescence resonance energy transfer (FRET) probes in a LightCycler®480 Instrument(Roche Diagnostics, Mannheim, Germany). Results: Our analysis revealed a significant associ-ation of the PTPN2 SNP rs2542151 with susceptibility to both CD (p=1.95 x 10-5; OR

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s1.49 [1.34-1.79]) and UC (p=3.87 x 10-2, OR 1.31 [1.02-1.68]). Moreover, the PTPN2SNP rs7234029 demonstrated a significant association with susceptibility to CD (p=1.30 x10-3; OR 1.35 [1.13-1.62]) and a trend towards association with UC (p=7.53 x 10-2; OR1.26 [0.98-1.62]). Conclusion: Our data confirm the association of PTPN2 gene variantswith susceptibility to both CD and UC in the German population. Given the association ofPTPN2 variants with other autoimmune diseases such as type 1 diabetes and Graves‘ disease,PTPN2 gene variants may partially explain the increased susceptibility of IBD patients forother autoimmune diseases.

Sa1292

Response to Influenza Vaccine a/H1N1 2009 in Patients With InflammatoryBowel Disease on Anti TNF-Alpha TherapyGianluca Andrisani, Daniela Frasca, Alessandro Armuzzi, Alfredo Papa, Manuela Marzo,Carla Felice, Giammarco Mocci, Daniela Pugliese, Giovanna Vitale, Italo De Vitis, GianLudovico Rapaccini, Luisa Guidi

Aim: Patients with inflammatory bowel disease (IBD) are exposed to the same infectionsaffecting the community, in adjunct to the opportunistic infection related to the immunesuppression. Some of these infectious diseases may be prevented by the appropriate use ofa vaccination program. The immune response to vaccine in these patients is still unclear.Aim of the study was to assess the efficacy and safety of influenza A/H1N1 vaccine in patientswith IBD on anti TNF-alpha therapy. Materials and Methods: We enrolled 25 healthy controlsand 62 patients, 26 affected by Ulcerative Colitis (UC) and 36 by Crohn's disease (CD),all undergoing maintenance treatment with anti TNF-alpha. Patients were on anti TNFαmonotherapy (47) or on anti TNFα associated with immunosuppressor or corticosteroids(15). Mean age of patients was 45±16, of normal controls 38±14. All patients were vaccinatedwith the adjuvated vaccine A/ California/7/2009 (H1N1), Focetria®, Novartis. Sera wereobtained during the 2009/10 vaccination campaign before (T0) and 4-6 weeks after thevaccination (T1). Immune response to vaccination was measured by hemoagglutinationinhibition assay (HI). The results of HI are expressed as titers ≥1:40 at t1 (seroprotectionrate), as well as fold-increase after vaccination (titer at t1/titer at t0, seronversion rate),response rate (seroconversion or titer > 1:40 at T1 if lower at T0), geometric mean titer(GMT) and factor increase of GMT between T1 and T0. We recorded the activity indicesof disease before and after H1N1 vaccination. Results: IBD patients on anti TNF-alphaafter “pandemic” 2009 influenza vaccine obtained good HI seroprotection rates, while HIseroconversion rates and HI response rates were lower than HC (p<0.0001 by Fisher test).HI GMT and factor increase at T1 were significantly lower in IBD patients compared tohealthy controls (p<0.0001 and p=0.0026, respectively, by Mann Whitney test), and inpatients on combined therapy vs anti TNF-alpha monotherapy (p<0.022 and 0.034 respect-ively, by Mann Whitney test). Few patients in this study reported systemic adverse eventsafter the vaccination: 3% headache, 3% malaise and 2% shivering. None of them reporteda flare of the IBD. Activity indices showed no differences after vaccination. Conclusion: Inour patients the vaccine was safe and did not affect IBD activity. Moreover, patients withIBD undergoing anti TNF-alpha therapy showed a reduced response to the vaccine, ascompared to healthy controls, and among these patients those with anti TNF- α therapyand immunosuppressor were more impaired in the response to the vaccine but still able torespond. Vaccination is indicated to protect these patients. A double injection vaccinationschedule could be explored to improve the efficacy in the subgroup of patients on com-bined therapy.

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In Vivo and In Vitro Immune Response to Pandemic Influenza 2009 H1N1Vaccine in Inflammatory Bowel Disease Patients on Anti TNF-Alpha TreatmentGianluca Andrisani, Daniela Frasca, Alessandro Armuzzi, Alfredo Papa, Manuela Marzo,Carla Felice, Giammarco Mocci, Daniela Pugliese, Italo De Vitis, Gian LudovicoRapaccini, Luisa Guidi

Aim: Patients with inflammatory bowel disease (IBD) may be immunocompromised becauseof their treatment and for this reason, they might be considered as susceptible to and athigh- risk from complications of H1N1 virus infection. The immune response in thesepatients to vaccine is still unclear. Aim of this study was to assess the In Vivo and In Vitroresponse to the vaccine. Material and Methods: We enrolled 25 healthy controls and 16patients, 8 affected by Ulcerative Colitis (UC) and 8 by Crohn's disease (CD), all undergoingmaintenance treatment with anti TNF-alpha. Patients were on anti TNF-alpha monotherapy(n=10) or anti TNF-alpha associated with immunosuppressor or corticosteroids (combinedtherapy) (n=6). Mean age of patients was 46±15, of normal controls 38±14. All individualswere vaccinated with the monovalent vaccine A/ California/7/2009 (H1N1), Novartis. Bloodsamples were obtained during the 2009/10 vaccination campaign before (T0) and 4-6weeks (T1) after vaccination. Immune response to vaccination was measured In Vivo byhemoagglutination inhibition assay (HI) and ELISA. The results are expressed as fold-increaseafter vaccination (titer at T1/titer at T0). In Vitro, we evaluated the specific response of B cellsto the vaccine, as measured by AID (activation-induced cytidine deaminase) determination byqPCR. The results are expressed as fold-increase after vaccination. Results: Nine/10 (90%)of patients in monotherapy and 3/6 (50%) in combined therapy responded to vaccinationby HI, whereas 24/25 (96%) healthy controls responded (p=0.016 by Fisher test for combovs healthy controls). Six/10 (60%) patients in monotherapy also responded in H1N1-specificELISA for IgA and IgG, whereas 4/6 (66%) and 3/6 (50%) patients in combined therapyresponded in H1N1-specific ELISA for IgA or IgG, respectively. Six/10 (60%) patients inmonotherapy and 1/6 patients (17%) in combined therapy showed an In Vitro response asevaluated by AID by qPCR, as compared to 19/25 (76%) healthy controls ( p=0.013 byFisher test for combo vs healthy controls). The AID fold increase was significantly higherin the patients on monotherapy as compared with patients on combined treatment (p=0.0075 by Mann Whitney test). The fold increase AID and HI response were significantlycorrelated (p=0.001 by Spearman test, rho= 0.522). Conclusion: IBD patients showed reducedresponses to the vaccine both In Vivo and In Vitro as compared to healthy controls. AmongIBD patients, those in combined therapy showed the most significant reduction in the

S-274AGA Abstracts

response as compared to those undergoing monotherapy. We can propose the vaccinationto protect these immunocompromised patients.

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Infliximab Induces Regulatory Macrophages in Responders but Not in Non-RespondersAnne Christine W. Vos, Manon E. Wildenberg, Ingrid Arijs, Marjolijn Duijvestein, AukeVerhaar, Severine Vermeire, Gijs R. van den Brink, Paul J. Rutgeerts, Daniel W. Hommes

Introduction Regulatory macrophages play an important role in wound healing and guthomeostasis and have anti-inflammatory properties. We have previously shown that anti-TNF antibodies induce regulatory macrophages in an Fc region dependent manner In Vitro(Vos et al, Gastroenterology 2010). The aim was to examine the induction of regulatorymacrophages In Vivo, and to study a possible association between response and inductionof regulatory macrophages in patients treated with infliximab. Methods Colonic mucosalbiopsies were obtained during endoscopy from CD (n=6) and UC (n=4) patients before and4-6 weeks after first infliximab therapy. Response to infliximab was defined as endoscopicand histologic healing. Regulatory macrophages were defined as the proportion of CD206+(regulatory macrophagemarker) to CD68+macrophages (CD206+/CD68+) on immunohisto-chemistry. Results A significant (p = 0.0051) induction of regulatory macrophages wasobserved after treatment in responders to infliximab (n=5: 2 UC, 3 CD). Strikingly, thisinduction was absent in non-responders (n=5: 2UC, 3 CD). (Figure) The association betweenresponse and induction of regulatory macrophages was found in UC patients as well as inCD patients. Furthermore, non-responders had lower amounts of regulatory macrophagesat baseline (week 0), suggesting a defect in regulatory macrophage differentiation or recruit-ment in non-responders. Conclusion We show that infliximab induces regulatory macro-phages in responders but not in non-responders. This mechanism of action of infliximabmay play a role in mucosal healing in patients with IBD.

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Short and Long Term Outcome of Infliximab Scheduled Therapy for Acute,Severe Ulcerative Colitis. A Prospective, Open Label, Single-Centre, Two-YearStudyKonstantinos Papamichael, Emmanuel J. Archavlis, Alexandros Smyrnidis, George Agalos,Nikolaos Kyriakos, Panagiotis Konstantopoulos, Dimitrios M. Tzivras, Ioannis Drougas,Dimitrios Tsironikos, Gerassimos J. Mantzaris

Introduction: The role of infliximab (IFX) as rescue therapy for acute severe UC (AS-UC)is still under investigation. The aim of our study was to assess the short and long termoutcome of IFX rescue therapy in patients with AS-UC. Patients and method: Prospective,open label, single-centre, two-year study. Eligible were patients with AS-UC (as defined bythe Truelove & Witts criteria plus an endoscopic Mayo score 3), aged 18-70 years, whowere failing a 5-day intensive steroid regimen (Sweden index > 8 on day 3) and fulfilledthe safety requirements for anti-TNF therapy. Patients received a single dose of IFX (5 mg/kg i.v.). Azathioprine (AZA) was continued in prior AZA users. Patients who did not improveor deteriorated proceeded to colectomy. Responders received additional induction doses (5mg/kg at weeks 2 and 6) and then scheduled IFX maintenance therapy (5 mg/kg q8 weeks).Steroids were gradually tapered off. Patients were followed in the outpatient clinic withmonthly visits for two years or to treatment discontinuation. At each visit, clinical assessmentand laboratory tests were performed. At two years, colonoscopy was performed and endos-copic lesions were graded using the Mayo index. The primary end-points were steroid-freeremission (SFR) and complete mucosal healing (CMH) at 2 years after initiation of IFX.Results: Between January 2007 and September 2010, 37 patients [20 males, median age 33(range 18-65) years] who fulfilled the entry criteria received IFX rescue therapy. Twentytwo patients had extensive and 15 had left-sided UC of median duration 3.5 (range 0.5-16) years. Six patients were smokers; 8 had extra-intestinal manifestations. Twelve patients(32.5%) proceeded to colectomy: 5 (13.5%) did not respond to the first infusion, one (2.7%)developed severe allergic reaction during the second infusion and 6 (16.2%) relapsed beforethe fourth infusion after a partial response to IFX induction regimen. Twenty five patients(67.5%) patients achieved SFR at 6 months. In 12/25 (48%) patients who received combina-tion therapy AZA was discontinued after 6 months. During subsequent follow up 5 initiallyresponders were withdrawn for severe flare of UC (n=1) or adverse events to IFX (pneumonia,refractory psoriasiform rash, high blood pressure, and pericarditis, respectively). Five patientsneeded dose escalation of IFX (10mg/kg) and/or shortening of the time interval betweeninfusions to regain response. At the end of the two years follow up, 20 patents (54%) werein SFR. Colonoscopy revealed CMH (Mayo score 0) in 12 patients and near CMH (Mayo score1) in 8 patients. Conclusion: In this study which mirrors real life experience, approximately 2/3 of patients who received rescue IFX for AS-UC avoided emergency colectomy and over50% were maintained in long-term SFR and achieved CMH or near CMH.

Sa1296

Serum Proteome Profile of Crohn's Disease Patients Treated With Infliximab:A Pilot StudyMaria Gazouli, Gerassimos J. Mantzaris, Konstantinos Papamichael, ΑggelikiPapadopoulou, Konstantinos Vougas, Nicholas P. Anagnou, George T. Tsangaris

Background: Infliximab (IFX) is an effective treatment for Crohn's disease (CD) but consider-able proportion of patients may fail to respond primarily or lose response over time. Despiteintensive research robust factors to predict the response to IFX of patients with differentCD phenotypes have not yet been identified. Considering the cost and adverse events oftreatment this is an important unmet demand. Proteomics arose with new techniques todiscover protein biomarkers which may be useful for diagnosis. Method: In a pilot study,we analyzed the serum proteome profile of CD patients with various responses to IFXinduction therapy (5mg/kg iv at weeks 0, 2, 6). In a cohort of 60 CD patients we selected18 patients (6 females; 36.92±12.45ys) who were classified as complete, partial, or primary