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sDNMT -1, -3A and -3B were un-methylated in CD affected, non affected and controls.DNMT3L was differentially methylated between DNA from matched affected and non-affected tissue in 17 confirmed CD cases and 11 control samples(CD Affected average=0.55CD non-affected average=0.33, p<0.0002. Control average=0.39, p<0.006, Student's t-test.Level of methylation of DNMT3L for each CpG site in CD affected and non-affected CDand controls revealed that DNMT3L is hypermethylated in affected CD (Fig 1). Relativegene expression of DNMT3L to GAPDH revealed that DNMT3L expression was less inCrohn's affected tissue samples compared with matched normal tissue controls. Conclusion:DNMT3L is important for normal gut function and inflammation in CD affects its gene expres-sion.

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Genes Regulated by Nkx2-3 in Sporadic and Inflammatory Bowel Disease-Associated Colorectal Cancer Cell LinesWei Yu, Zhenwu Lin, Danielle M. Pastor, John P. Hegarty, Xi Chen, Ashley A. Kelly,Yunhua Wang, Lisa S. Poritz, Walter Koltun

Nkx2-3 is a member of the Nkx family of homeodomain transcription factors. Recently, anassociation of Nkx2-3 to inflammatory bowel disease (IBD) has been identified by genome-wide association studies. Alterations in Nkx2-3 regulation have been described in B celllines and intestinal tissues from Crohn's disease (CD) patients and in colorectal cancer(CRC). However, related mechanisms are unknown. To better understand the function ofNkx2-3 in sporadic and inflammatory bowel disease (IBD)-associated CRC, we sought toidentify Nkx2-3-regulated genes in sporadic CRC (CRS61) and IBD-associated CRC (CRS4)cell lines by cDNA microarray. Cell lines were generated from tumor tissue from patientswith sporadic or IBD-associated colorectal cancer and confirmed to be epithelial and tumorig-enic. Small interfering RNA (siRNA)-mediated knockdown of Nkx2-3 in both cell lines wasachieved using the OligoEngine pSUPER RNAi System. High-density cDNA microarraysrepresenting over 25,000 genes with >48,000 probes were performed. Ingenuity PathwayAnalysis was used to identify gene networks according to biological functions and associatedpathways. Top 100 genes regulated by Nkx2-3 in each cell line were grouped primarilywithin the following 2 functional categories: (1) immune and inflammatory response; or (2)cell proliferation, growth and oncogenes. Thirty-nine genes were similarly regulated byNkx2-3 knockdown in both cell lines: 13 genes were down-regulated and 26 genes wereup-regulated in both lines. These genes included 19 inflammation-related and 9 proliferation-related genes. Among these genes, most affected pathways included antigen presentationand cell-cell signaling and interaction. Nineteen genes were differentially regulated by Nkx2-3 knockdown between the two lines. Eleven genes were down-regulated by Nkx2-3 knock-down in CRS4 cells, but up-regulated in CRS61 cells. In contrast, 8 genes were up-regulatedby Nkx2-3 knockdown in CRS4 cells, but down-regulated in CRS61 cells. Among thesegenes, 8 were inflammation-related and 9 were proliferation-related, with several involvedin WNT/β-catenin signaling. Our results indicate that Nkx2-3 may contribute to the patho-genesis of human CRC developed in the setting of IBD.

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Evidence for Stat4 as a Common Autoimmune Gene: Rs7574865 is AssociatedWith Colonic Crohn's Disease and Early Disease OnsetJulia Diegelmann, Jürgen Glas, Julia Seiderer, Melinda Nagy, Florian Beigel, SimonePfennig, Burkhard Göke, Thomas Ochsenkühn, Stephan Brand

Background & Aims: Recent studies demonstrated an association of STAT4 variants withsystemic lupus erythematosus (SLE) and rheumatoid arthritis (RA), indicating that multipleautoimmune diseases share common genetic and molecular pathways. We therefore investig-ated the influence of STAT4 variants on the susceptibility and phenotype of inflammatorybowel disease (IBD) in a large German patient and control cohort. Methods: Genomic DNAfrom 2704 individuals of Caucasian origin including 857 patients with Crohn's disease (CD),464 patients with ulcerative colitis (UC), and 1383 healthy, unrelated controls was analyzedfor seven SNPs in the STAT4 gene (rs11889341, rs7574865, rs7568275, rs8179673,rs10181656, rs7582694, rs10174238). In addition, a detailed genotype-phenotype analysiswas performed. Results: Our analysis revealed an association of the STAT4 SNP rs7574865with decreased susceptibility to CD (p=4.69 x 10-2, OR 0.86 [95%CI 0.74-0.99]). Comparedto carriers of the wild type genotype, the STAT4 SNP rs7574865 was significantly associatedwith early CD onset (p=0.021) and colonic CD (p=0.008; OR=4.60, 95% CI 1.63-12.96).For two other STAT4 variants, there was a trend towards protection against CD susceptibility(rs7568275, p=5.75 x 10-2, OR 0.86 [95 % CI 0.74-1.00]; rs10174238, p=5.67 x 10-2,OR 0.86 [95 % CI 0.75-1.00]). However, we did not observe any association with UCsusceptibility. Conclusion: Our results identified the STAT4 SNP rs7574865, which is alsoassociated with SLE and RA, as a disease-modifying gene variant in colonic CD. ThereforeSTAT4 may contribute to the increased susceptibility of autoimmune diseases in the IBDpopulation.

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The Three Common CARD15/NOD2 Polymorphisms Associated With Crohn'sDisease are Rare or Absent in a UK South Asian Crohn's Disease PopulationDavid G. Walker, Horace R. Williams, Panagiotis Pantelidis, Hiroe Sato, Kwok Lee,Andrew N. Milestone, John C. Chambers, Jaspal S. Kooner, Tim Orchard

INTRODUCTION: The CARD15/NOD2 gene on chromosome 16q (IBD1) was the firstsusceptibly gene identified for inflammatory bowel disease (IBD). Approximately 16-45%of Caucasian Crohn's disease (CD) patients are carriers of one of three major disease-associated variants (SNP8 [2104C/T], SNP12 [2722G/C] and SNP13 [3020Fs]), comparedto 7-19% of healthy Caucasian controls. Polymorphisms in this gene have been associatedwith disease phenotype and in particular ileal disease. We have recently shown phenotypicdifferences between Caucasian and South Asian patients with CD living in North WestLondon, UK (1). Colonic Crohn's disease is the most common phenotype in this population

S-684AGA Abstracts

and limited ileal disease is rare. Previous studies suggest that the frequency of CARD15/NOD2 polymorphisms is lower in East Asian populations (Japanese and Chinese). Thefrequency of these polymorphisms in the UK South Asian CD population has not beenpreviously studied. AIMS & METHODS: The study aim was to investigate the frequency ofthree CARD15/NOD2 polymorphisms associated with CD in a UK South Asian cohort (SNP8,SNP12, SNP13). South Asian CD patients were recruited from the IBD clinics of five hospitalsin North West London. Patients with all four grandparents originating from South Asia anda confirmed diagnosis of IBD were included in the study. Disease location (determined asmaximal extent of disease) and behaviour were defined in terms of the Montreal classification.The patients were genotyped using SSP-PCR. The results were compared with a cohort ofulcerative colitis (UC) patients and healthy South Asian controls using the Chi-square test.RESULTS: 230 South Asian IBD patients (67 CD: 163 UC) and 202 healthy South Asiancontrols were recruited. CARD15/NOD2 polymorphisms were present in only 2 CD patients(SNP12 in both patients); they both had stricturing ileocolonic disease. Only 1 UC patientwas positive for SNP12 and 1 for SNP13. CARD15/NOD2 polymorphisms were present in7 South Asian controls (6 SNP12 & 1 SNP8). There were no significant differences betweenthe CD and control cohort (P=0.85). CONCLUSION: The three NOD2 polymorphismsassociated with CD in the Caucasian population are rare or absent in the UK South AsianCD population. The lack of the three CARD15/NOD2 polymorphisms in this ethnic groupdemonstrates that this is not an important pathogenic mechanism in this population. Furtherstudies are required to determine whether further polymorphisms in this gene are importantin this population or whether other genes (ATG16L1 and IL23R) are important in determiningdiagnosis in South Asian patients. REFERENCES: 1. Walker DG et al. Gut 2009; 58, A60

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Is Celiac Disease Genetic Predisposition Related to Fructose or LactoseMalabsorption in Inflammatory Bowel Disease (IBD) Patients?Miguel Minguez, Ursula Estada, Marta M. Bosca, Joan Tosca, Isabel Pascual, PedroAlmela, Pilar Mas, Adolfo Benages

Hypothesis: Genetic predisposition to celiac disease (HLA DQ2 and DQ8) in IBD patientsdetermines a greater risk of lactose and fructose malabsorption. Objective: To determinethe haplotypes that show predisposition to celiac disease in IBD patients (Crohn's Disease(CD) and Ulcerative Colitis (UC)) and analyze if the haplotypes are associated to lactoseand/or fructose malabsorption. Methods: A prospective study was carried out including 144consecutive IBD patients (72 CD, 72 UC, 73 male and 71 female, mean age 44.5, range18-86). DQ2 and DQ8 haplotypes were determined. Lactose and fructose absorption breathtests were performed in inactive IBD phases, according to activity indexes (CDAI for CDpatients and Truelove-Witts for UC patients). The Chi-squared test was used for the statisticalanalysis, with p<0.05 as statistical significance. Results: 47 IBD patients (32.7%) had geneticpredisposition to celiac disease: 23 (16 %) DQA1*0501/DQB1*0201, 19 (13.2 %) DQA103/DQB1*0302 and 5 (3.5%) DQA1*0201/DQB1*0202 + DQA1*0505/DQB1*0301. No differ-ences were observed between CD and UC. 42 patients (29.2%) had lactose malabsorptionand 41 (28.5 %) had fructose malabsorption. 15 patients (12%) had malabsorption to bothsugars (fructose and lactose). Malabsorption is not related to having celiac disease-relatedHLA in IBD patients, which is observed when analyzing both globally and by subgroups(CD and UC) (Table). The higher prevalence of fructosa malabsorption in CD patients withrespect to UC patients (47,2% vs 17.1%; p< 0.001) is independent of the existence of HLApredisposing to celiac disease. Conclusion: The HLA associated to celiac disease in IBDpatients is not related to a higher prevalence of lactose or fructose malabsorption.

All results in %. P>0.05

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Measurement of Human Anti-Chimeric Antibodies (Haca) and InfliximabLevels in Patient Serum Using a Novel Homogeneous AssayShui-Long Wang, Linda Ohrmund, Sharat Singh

Background: Infliximab (IFX) is a chimeric monoclonal antibody against TNF-α that iseffective in treating autoimmune diseases such as rheumatoid arthritis (RA) and inflammatorybowel disease (IBD). Immunogenicity, which may reduce efficacy or induce adverse effects,was found in some patients treated with IFX through the detection of human anti-chimericantibodies (HACA). Monitoring of HACA development and IFX levels in individual patientsmay help optimize treatment and dosing of IFX. Current methods of detecting HACA arebased on a solid-phase assay and are limited by the fact that IFX in circulation may maskthe presence of HACA so that measurement can only be done after IFX has been cleared fromserum, typically 8 or more weeks following a dose of IFX. Detection is further confounded byrapid clearance of high molecular weight immune complexes. We evaluated a new methodto measure serum IFX and HACA levels in patients treated with IFX with high sensitivityand accuracy. Methods: A proprietary non-radiolabeled liquid-phase homogeneous assaywas developed to measure HACA and IFX levels in serum from patients treated with IFX.Large immune complexes (TNF/IFX or IFX/HACA), free TNF-α or IFX and the ratio ofbound/free could be resolved and calculated with high sensitivity. Serum concentrations ofIFX or HACA were calculated with the standard curve generated by incubating with differentconcentrations of IFX or anti-human IgG. Using this novel assay, we tested serum IFX andHACA levels from 20 patients with IBD who had relapsed from IFX treatment and comparedthe results with the traditional bridge ELISA assay. Results: Dose-response curves weregenerated from our novel assay with a low detection limit. Detection of HACA was demon-strated in the presence of an excess of IFX. Of the 20 serum samples from patients treatedwith IFX, 15 samples had the IFX levels above the detection limit. For HACA levels, 18

(90%) samples were positive while two were negative compared with 15 (75%) positiveand five negatives when the bridge ELISA assay was used. The three patients who werepositive for HACA in the homogenous assay also had low levels of IFX in their serum.Conclusions: A novel non-radiolabeled liquid-phase homogeneous assay with high sensitivity,accuracy and reproducibility has been developed to measure the IFX and HACA levels inserum from patients treated with IFX. This automated assay provides an important tool forclinicians to monitor and relate the clinical status of patients with their HACA and druglevels at any time during the course of treatment.

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Infliximab for Severe Ulcerative Colitis: Short-Term and One Year Outcome ofThree Dose Regimen. An Italian Multicentre Open-Label StudyRita Monterubbianesi, Marco Daperno, Alessandro Armuzzi, Livia Biancone, MariaCappello, Alessandro Lavagna, Vito Annese, Ambrogio Orlando, Angelo Viscido, GabrieleRiegler, Gianmichele Meucci, Claudio Papi, Raffaello Sostegni, Luisa Guidi, ManuelaMarzo, Carmelina Petruzziello, Sergio Peralta, Annalisa Aratari, Cosimo Prantera, AnnaKohn

Background and Aims:Infliximab(IFX) is used in moderate to severe Ulcerative Colitis (UC)but its role in severe acute attacks it's not still clear. A number of multicentre case seriesevaluated IFX infusions in hospitalized patients with steroid-refractory severe UC reportingconflicting results. In a previous study we confirmed the effectiveness of IFX with high rateof short-term success, suggesting that two or three infusions were more effective than asingle one to prevent early colectomy. Aim of the study was to evaluate the colectomy rateat two months and one year of a series of severe steroid refractory UC patients schedulatedfor a three-infusion regimen of IFX. Methods: From 2002 to 2008 132 patients (M/F 72/60, mean age 39[range14-76]) hospitalized in12 Italian gastroenterology referral centreswere started with a three-infusion regimen (0-2-6 weeks) of IFX 5 mg/kg for acute severeor moderately severe attack of UC. 69% of patients had extensive colitis, 47% had deepulcers at colonoscopy. All patients were candidates for colectomy because of resistance tointensive intravenous steroid treatment. Patients were evaluated for colectomy rate at 2months and at 1 year after the first IFX infusion. Results:Sixteen percent of patients (21/132) underwent colectomy in the first two months.77% of pts (101/132) completed theinduction regimen with 3 infusions of IFX. Ten patients didn't receive all the 3 scheduledinfusions:5 for serious adverse events,2 for hypersensibility reactions and 3 lost to followup.77% of patients (101/132) ended 1 year follow up.22% of patients (28/129) underwentcolectomy in the first year. Patients who survived colectomy received maintenance treatmentwith IFX (13%),Azathioprine (50%),IFX+ Azathioprine (32%).We observed serious sideeffects in 7.5% of patients,2 deaths included (1 for legionella pneumonia, 1 for suicide).Theother causes were:1 CMV infection,1 pneumocystis carinii pneumonia,1 TBC,1 pneumonia,1Herpes V infection, 1 sepsi (candida),1 CMV hepatitis with pneumonia,1 EBV hepatitis.Conclusions: This study confirms that IFX is an effective rescue therapy in patients withsevere UC unresponsive to i.v. steroid treatment. 78% of patients survive free from colectomyone year after the first IFX infusion under maintenance treatment with IFX alone, immunomo-dulators alone or both treatments associated. Safety issues has to be taken into account.

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Relationship Between Thiopurine Metabolite Levels and EndoscopicImprovement in Patients With Postoperative Moderate to Severe EndoscopicRecurrence of Crohn's DiseaseSieglinde Angelberger, Elke Schaeffeler, Alexander Teml, Wolfgang Petritsch, OlgaShonova, Milan Lukas, Simon Bar-Meir, Roland Greinwald, Ralph Mueller, Eduard Stange,Klaus R. Herrlinger, Matthias Schwab, Walter Reinisch

Introduction: Endoscopic recurrence of Crohn's disease (CD) after curative ileocecal resection,which is reported in up to 70% of patients within 1 year, precedes clinical relapse. Azathiop-rine (AZA) reduces the risk of both clinical and endoscopic recurrence. Aim: To evaluatethe association between thiopurine metabolite levels and endoscopic improvement in CDpatients with moderate to severe postoperative endoscopic recurrence. Methods: Patientstreated with AZA within the AZT-2 study, a prospective double-blinded trial comparing 2.0- 2.5mg/kg/d AZA versus 4.0 g/dmesalamine for prevention of clinical relapse in patients withpostoperative moderate to severe endoscopic recurrence, were included. Ileocolonoscopy wasperformed at baseline and at week 52 or at premature discontinuation (median 364 days,range: 176 - 381). Red blood cell concentrations (RBC) of 6-thioguanine nucleotides (6-TGN),6-methyl-mercaptopurine ribonucleotides (6-MMPR), and 6-methyl-thioguanine nucleotides(6-MTGN) were measured by high-performance liquid chromatography assay. A cut-offvalue of 6-TGN of ≥ 235 pmol/8x108 RBC was used as established in literature. 6-MMPRand 6-MTGN levels below and above the median were considered low and high, respectively.Endoscopic improvement was defined as a reduction of the endoscopic score according toRutgeerts by at least one point, and mucosal healing as endoscopic score of i0 (= no lesions).Results: Endoscopic data were available on 31 patients treated with azathioprine (f/m=13/18, median age: 32 years, median AZA dose: 2.24 mg/kg/d) for median 358 days. At lastobservation, the median levels of 6-TGN (n=26), 6-MMPR (n=27), and 6-MTGN (n=26)were 139.7, 1696.1 and 70.1 pmol/8x108 RBC, respectively. Overall, endoscopic scoreimproved from i3-i4 to i0-i2 (p=0.004). Mucosal healing was achieved in 6/31 (19.4%) ofpatients. High 6-TGN levels (n=8) were associated with a significantly higher drop in theRutgeerts score than low 6-TGN (n=18) levels (median drop of 3 vs 1, p=0.023). There wasno difference concerning mucosal healing (p=0.33). Furthermore, there was no correlationbetween 6-MMPR and 6-MTGN levels and endoscopic improvement. Conclusion: Our datasuggest that high 6-TGN levels may linked to endoscopic improvement in patients withpostoperative endoscopic recurrence of CD. Supported by Dr. Falk Pharma GmbH, Frei-burg, Germany.

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Adalimumab vs Infliximab Clinical Comparision: Longitudinal StudyFrancesca Zorzi, Sara Onali, Emma Calabrese, Carmelina Petruzziello, Elisabetta Lolli,Francesco Pallone, Livia Biancone

Background.The efficacy of Infliximab and Adalimumab in Crohn's Disease (CD), has beenKnown. No study compare two anti-TNFa treatments about disease activity and quality oflife.Aims. To compare clinical outcome and quality of life in CD pts treated with Infliximabor Adalimumab.Methods.In a longitudinal study 53 CD patients(pts.)were enrolled,24 pts(13M,median age 37 yrs,range(18-62)received Infliximab 5 mg/Kg e.v. at wks 0(T0),2(T2)and6(T3).29 pts(10 M, median age 39 yrs,range 23-59)received Adalimumab(160,80,40 mgs.c.)at wks 0(T0),2(T2)and4(T3).Indication to Infliximab included refractory disease(n=4),perianal disease(n=5)and severe CD(n=15);indication to Adalimumab included refractorydisease(n=8),perianal disease (n=7),severe CD (n=14). At each infusion all patients underwentclinical assessment(Crohn's Disease Activity Index:Remission CDAI<150;Response ΔCDAI>70 points),before and after treatments each pts had compile the inflammatory bowel diseasequestionary (IBDQ.Results.Treatment with Infliximab was completed in 16/24 (67%)pats,considered for the analysis. Reasons for drop out included infusion reaction (n=6)or needof surgery (n=2).Median value and range of CDAI, IBDQ at T0, T2, T3 in both groups oftreatments were reported in the table. The CDAI significantly reduced compare the T0 versus(vs) T2 and T3 in both groups (p<0.001). In infliximab group the percentage of clinicalimprovement,taken as percent of reduction of CDAI comparing T0 vs T2 and T3 arerespectively 27% and 24%, while the percentage of improvement in adalimumab groupcompare time 0 vs time 2 and time 3 are respectively 23% and 38%.The IBDQ valuesignificantly increase compare before and after treatments in both groups (p<0.001),thepercentage of improvement in infliximab group is 12% and in Adalimumab group is 27%.Inthe infliximab group,at wks 0, 9 pts had Joints complication that persist at 2wks in 6/9 ptsand in 8/9 pts at 6 wks. In the Adalimumab group, at wks 0 14 pts had Joints complicationthat that persist at 2wks in 11/14 pts and in 9/14 pts at 4 wks. No patients required majortreatment changes during the study period.Conclusions.In our CD pts, Infliximab wasassociated with a high frequency of adverse events;Infliximab and Adalimumab showed acomparable efficacy in term of CDAI and IBDQ improvements.

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Maintenance of Clinical Benefit in Crohn's Disease Patients AfterDiscontinuation of Infliximab: Long Term Follow-up of a Single Center CohortAlistair W. Waugh, Karen Wong, Richard N. Fedorak

Background: Tumor necrosis factor (TNF)-blockade with infliximab has offered significantadvances in the treatment of Crohn's disease (CD). While the therapeutic effect of infliximabis rapid and efficacious, there are long term issues of cost and safety. It remains unknownas to whether infliximab can be discontinued in some patients after induction of clinicalremission. Objective: The primary objective of this study was to assess, in CD patients whoobtained a stable infliximab-induced corticosteroid-free remission, the proportion of patientswho relapsed after infliximab discontinuation. The secondary objective was to examinefactors associated with this relapse. Methods: This is a longitudinal observational study ofa single IBD referral center cohort where a prospectively designed standardised follow-upschedule was used for assessment of all patients by experienced IBD gastroenterologists.Forty eight CD patients (M=22) in full clinical steroid-free remission who then discontinuedinfliximab were followed for up to 10 years (interquartile range 0.5 to 10.4 yrs). CD relapsewas defined as a physician or hospital visit with documented disease activity and therapeuticintervention with CD medications. Results: Mean age at CD diagnosis was 25±1.4 yrs. Firstinfliximab infusion occurred at age 37±1.8 yrs and infliximab discontinuance at 43±1.9 yrs.A 3 dose infliximab (5mg/kg) induction was followed by a median of 6 regular maintenanceinfliximab infusions before discontinuance. Reasons for discontinuance were: 51% patientpreference, 19% loss of insurance, 16% infusion reactions, and 14% pregnancy. At the timeof discontinuance 44% were receiving, and remained on, azathioprine, 19% methotrexateand 4% 6-mercaptopurine. Kaplan-Meier analysis of the proportion of patients with sustainedclinical benefit demonstrated that 50% of CD patients relapsed within 500 days after inflixi-mab discontinuance. Thirty five percent of patients remained well, and without relapse upto the end of the 10 year follow-up of the study, implying a subgroup of Crohn's patientsin whom infliximab induced a long lasting remission that was sustained after the infliximabtreatment was discontinued. In multivariate analysis; patient age, age at diagnosis, diseaselocation, number of infliximab doses, and concomitant medication did not affect the sustainedclinical benefit survival time after discontinuation. Conclusion: After a stable infliximab-induced corticosteroid remission one half of patients who discontinue infliximab will relapsewithin 500 days. Nevertheless, a small subgroup of patients (one third in this cohort)demonstrate the capacity for long term remission.

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Effect of Adalimumab Treatment on the Microbiota Recovery in the IntestinalMucosa of Crohn's Disease PatientsMaria Teresa Mas de Xaxars, L. Jesus Garcia-Gil, Margarita Martinez-Medina, MireiaLopez-Siles, David Busquets, Xavier Aldeguer Mante

Bacteria might play an important role in Crohn's Disease (CD). The density and diversityof the intestinal microbiota in CD patients differs from that in healthy subjects. Anti-TNFdrugs have been implemented in recent years as good alternative for CD treatment. The

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