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sbased on circulating IgG antibodies against food antigens was effective with respect to stoolfrequency, abdominal pain and general wellbeing. The mechanisms by which IgG antibodiesmight contribute to disease activity remain to be elucidated.

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Upregulation of E3 Ubiquitin Ligases Related to T Cell Anergy in CD4+ TCells Isolated from Patients with Ulcerative Colitis in RemissionSatoshi Egawa, Hideki Iijima, Shinichiro Shinzaki, Sachiko Nakajima, Jumpei Kondo,Shuji Ishii, Toshiyuki Yoshio, Tsutomu Nishida, Yoshimi Kakiuchi, Tatsuya Kanto,Masahiko Tsujii, Shingo Tsuji, Norio Hayashi

Background and Aims Insufficient tolerance against luminal antigens is suggested to causeinflammatory bowel diseases including ulcerative colitis (UC). T cell anergy is one of themechanisms regulating immune tolerance. Recently, some E3 ubiquitin ligases are reportedto downregulate downstream signals of T cell receptor and induce T cell anergy. The aimof this study was to investigate the state of T cell anergy in healthy subjects and UC patientsby quantitating the expression of E3 ubiquitin ligases, GRAIL, c-Cbl, Itch, all of which arereported to induce anergy on T cells. Patients and Methods Peripheral mononuclear cellswere obtained from 20 UC patients (active UC: 12, UC in remission: 8) and 10 healthyvolunteers (HV) by either centrifugal leukocytapheresis or sampling of peripheral blood.CD4+ T cells were isolated by magnetic cell sorter and expressions of GRAIL, c-Cbl, andItch mRNA in these cells were measured by real-time quantitative RT-PCR. The level ofGRAIL, c-Cbl, and Itch mRNA expression were compared between in active UC patients,UC patients in remission, and HV. The expression of these ubiquitin ligases were followedin 6 active UC patients who were treated by corticosteroid and centrifugal leukocytapheresis.Results Anergic CD4+ T cells obtained by ionomycin treatment In Vitro revealed higherGRAIL, c-Cbl, and Itch expression when compared with non-anergic T cells. GRAIL waspredominantly expressed on CD4+ T cells whereas c-Cbl and Itch were also expressed onnon-CD4+ T cell subsets. The GRAIL expression in CD4+ T cells of UC patients in remissionwas significantly higher than that of active UC patients or that of HV (p<0.01). The c-Cbland Itch expressions in CD4+ T cells of UC patients in remission were relatively higherthan that of active UC or that of HV, but there were no significant differences. Remarkablythe levels of GRAIL expression were significantly increased after the treatment in UC patientswhose clinical activities ewre improved by the treatment. However, the c-Cbl and Itchexpression did not increase after the treatment. Conclusion Among the 3 different E3ubiquitin ligases which induce T cell anergy, upregulation of GRAIL expression seems tobe associated with the induction of remission in UC patients. These results indicate thatGRAIL is a potential therapeutic target of inflammatory bowel disease.

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In Silico Analysis of T-Bet Binding Sites in Peripheral Blood MononuclearCells in Patients with Inflammatory Bowel Disease (IBD)Nielsen Q. Fernandez-Becker, Alan C. Moss

Background: T-bet (Tbx21) has recently been identified as a regulator of the mucosalimmune response to host bacteria. It has been suggested that relative T-bet deficiency inimmune cells may play a key role in the pathogenesis of IBD. Methods: NCBI microarraygene expression profiles (n=127) from peripheral blood mononuclear cells (PBMCs) ofpatients Crohn's disease (n=59), ulcerative colitis (n=26), and healthy individuals (n=42)were analyzed. Normalized expression values were compared using student's t-test. Geneswhose expression was altered in Crohn's PBMCs were examined for T-bet transcription factorbinding sites. Gene-gene interactions and biological processes were determined. Results: Theexpression of T-bet in PBMCs was significantly reduced in patients with Crohn's diseasecompared to controls (p<0.0001) and ulcerative colitis (p=0.005). There was no differencebetween T-bet expression in the PBMCs of controls and ulcerative colitis patients (p=0.3).In addition to T-bet, there were 52 other genes whose expression was significantly alteredonly in Crohn's disease PBMCs. Eight down-regulated genes contained transcription factorbinding sites (TFBSs) for T-bet; KLRF1, GZMK, S100A16, ABHD3, EZH1, NCAM1, RGL2,PILRB. Three of these (S100A16, ABHD3 and EZH1) contain a complex promoter modulethat consists of T-bet and Early Growth Response binding sites. Fifteen up-regulated genescontained T-bet binding sites; CLEC1B, TPM1, FHL2, EGR1, PTGS1, C21orf7, PPBP, PROS1GNG11, HIST1H2BN, GUCY1B3, ITGB5, EGR2, ITGB3, CA2. Ontological analysis revealedthat genes involved in innate immunity (8 genes, Z-score 4.11) and signal transduction (5genes, Z-score 2.65) are over-represented amongst those altered genes in PBMCs that containT-bet binding sites. Conclusion: Gene expression of T-bet is relatively reduced in the PBMCsof patients with Crohn's disease. A cohort of genes associated with innate immunity shareT-bet binding sites. This suggests a mechanism for T-bet's role in disease pathogenesis.

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Peripheral Blood from Crohn's Disease Patients Demonstrate Both IL-17Expressing CD4+ Lymphocytes and IL-17 and Interferon-Gamma DoubleExpressing CD4+ LymphocytesVinod K. Patel, Vieira L. Pedro, Gavin Screaton, Julian R. Walters, Subrata Ghosh

Introduction: IL-17 expressing T-lymphocytes (Th-17) have been recently identified asimportant effector cells in chronic inflammation. Development of IL-17 + T-lymphocytes isinfluenced by IL-23, TGF-β and IL-6. Crohn's disease is thought to be characterised by Th-1 lymphocytes with IFN-γ being the signature cytokine whereas ulcerative colitis is moreTh-2 like. These differences have been challenged and we aimed to explore whether Crohn'sdisease and ulcerative colitis differed in terms of peripheral blood population of Th-17lymphocytes. Methods: 9 patients with Crohn's disease and 8 patients with ulcerative colitiswere recruited and their clinical disease activity indices (CDAI, Mayo score) and C-reactiveprotein concentrations were recorded. Peripheral blood mononuclear cells were isolatedfrom freshly drawn blood. Th1, Th2 and Th17 cells were characterised by the evaluationof signature cytokines at the single cell level by FACS analysis after incubation with PMA/

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ionomycin and monensin. CD4+ and CD8+ subsets were separately analysed for Th1, Th2and Th17 lymphocyte subpopulations. Results: Using t-test analysis we found that IL-17+IFN γ- CD4+ T lymphocytes were a significantly higher subpopulation of lymphocytes inCrohn's disease compared with ulcerative colitis (2.01 ± 0.88% Vs 0.51 ± 0.43% p<0.001).. IL-17- IFNγ+ CD4+ T lymphocytes were a more numerous subpopulation and significantlyhigher in Crohn's disease than ulcerative colitis (14.17 ± 4.41% Vs 6.22 ± 2.44% p<0.0005).Double positive IL-17+ IFNγ+ T lymphocytes were fewer (1.57 ± 0.24% in Crohn's diseaseVs 0.07 ± 0.07% in ulcerative colitis p<0.01). IL-4+ CD4+ T lymphocyte subpopulationswere similar in Crohn's disease and ulcerative colitis (p=NS). IL-17+ CD8+ T lymphocyteswere not detected, but IL-17- IFNγ+ CD8+ lymphocytes were higher in Crohn's diseasethan ulcerative colitis (15.98 ± 6.88% Vs 5.26 ± 2.47% p <0.001). There was no correlationof IL-17+ T lymphocytes with clinical disease activity indices or with C-reactive proteinconcentrations. There were also no significant differences pre- and post- treatment. Conclu-sion: IL-17+ IFNγ- T lymphocytes can be detected in peripheral blood of human IBD inthe CD4+ lymphocyte compartment and are more numerous in Crohn's disease comparedwith ulcerative colitis. IL-17+ IFNγ+ T lymphocytes can also be detected in peripheral bloodof Crohn's disease patients in the CD4+ lymphocyte compartment, but not in ulcerative colitis.

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Adipokines and Immunity. On the Interplay Between Adipose Tissue and TLymphocytesArvind Batra, Besir Okur, Jakob Ihbe, Thorsten Stroh, Rainer Glauben, Inka Fedke,Martin Zeitz, Britta Siegmund

Background: In the recent years a close link between adipose tissue and immunity becameevident with adipose tissue beeing a potent producer of various immune regulatory mediatorsincluding leptin, TNFα and IL-6. Interestingly in Crohn's disease a hypertrophy of themesenteric fat with altered leptin mRNA expression is common. In the past it was describedthat the adipokine leptin is a strong promoter of Th1 cells. Here we assessed further rolesof leptin as well as of the adipokine adiponectin in T cell polarization with a focus on Th2T cells. To assess the role of leptin for Th2 cells in colonic inflammation In Vivo, the modelof oxazolone colitis in mice was employed. Methods: Naïve murine T cells were stimulatedin the presence or absence of the adipokines leptin or adiponectin at polarizing conditionsvia anti-CD3 anti-CD28 stimulation and proliferation and cytokine production were assessedby flow cytometry. To test the effect of leptin on Th2 cells In Vivo, the Th2-dependentcolitis model induced by rectal oxazolone application following immunization was employedin WT Bl6 mice and leptin-deficient ob/ob mice reconstituted or not with leptin. Results:Excess adiponectin did not affect proliferation or polarization if naïve T cells were stimulatedat polarizing conditions. However, high amounts of adiponectin increased the percentageof IFNγ producers at non-polarizing conditions. Leptin not only promoted induction ofIFNγ producing cells at Th1 polarizing conditions, but decreased the amount of IL-4 produ-cers under Th2 polarizing conditions. Additionally it favored proliferation of lymphocytesstimulated under non-polarizing or Th1 polarizing but negatively affected proliferation ofcells stimulated at Th2 polarizing conditions. Whereas in WT and leptin reconstituted ob/ob mice oxazolone treatment induced significant inflammation and shortening of the colon,no such effects were evident in ob/ob mice. While oxazolone treatment was paralleled byproduction of the Th2 cytokine IL-13 in MLN from WT and leptin reconstituted ob/ob mice,no increase in IL-13 production was present in leptin-deficient mice. Conclusion: Eventhough adiponectin is the most abundant adipokine in circulation, it's effects on immuneregulation considering T cell proliferation and polarization are solely marginal In Vitro.However, even though commonly described solely as a promoter of Th1 cell differentiation,leptin furthermore excerts regulatory effects on Th2 cells In Vitro and vivo. Hence, thedysregulation of leptin production present in the mesenteric fat of Crohn's disease patientsmight be of even more importance than initially expected.

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DNA Methylation Status of IFNG Correlates with pANCA Reactivity in UCPatientsRivkah Gonsky, Richard L. Deem, Carol J. Landers, Carrie A. Derkowski, Stephan R.Targan

BACKGROUND: pANCA reactivity is a well-studied serologic marker used to classify UC.Serum pANCA is generally indicative of aggressive disease and is associated with post-surgical development of pouchitis. Studies have suggested that ICAM-1 and HLA-DR genepolymorphisms are associated with pANCA positivity, although no functional differencescorrelated to pANCA status have been described. pANCA antibodies exhibit a subnuclearstaining pattern, which is sensitive to pretreatment with DNase I, suggesting that the triggeringantigen may be DNA-related. Indeed, histone H1 DNA binding protein has been proposedas a candidate antigen. Mucosal expression of pro-inflammatory cytokines plays a pivotalrole in IBD pathogenesis. Although enhanced Th1 cytokine expression, particularly IFN-γ,is commonly associated with CD, high levels of IFN-γ have been observed in the mucosafrom some UC patients.1 Epigenetic remodeling at the DNA level contributes to regulationof cytokine gene expression with a decrease in DNA methylation linked to enhanced cytokineproduction. AIM: To determine whether DNA methylation levels of IFNG are associatedwith specific pANCA reactivity profiles in IBD patients. METHODS: DNA from peripheralblood T cells of 60 IBD patients (30 with CD and 30 with UC) and 21 normal controls wasanalyzed using bisulfite pyrosequencing for eight DNA methylation sites within the IFNGlocus and pANCA reactivity was measured by ELISA. RESULTS: DNA from IBD patientsdisplayed significantly less methylation of the IFNG +122 bp site, directly adjacent to thefirst coding sequence, and the conserved -22 kb CNS promoter regulatory region, comparedto normal controls (p < 0.001). The pANCA reactivity profile was associated with the levelof DNA methylation in UC, but not in CD patients. Significantly less methylation over awide IFNG promoter region was detected in the pANCA- versus the pANCA+ UC populations(p < 0.002). In fact, the absolute level of pANCA reactivity directly correlated with theabsolute level of methylation of the IFNG promoter (p < 0.01 to p < 0.03). Additionally,UC patients displayed less methylation of the IFNG promoter compared to CD patients whenstratified based on pANCA reactivity (p < 0.02). CONCLUSION: The results demonstrate the