significantly lower in K-sup group than in the K-def group. Conclusion: Vitamin K has asuppressive effect on IL-6 production especially in B cells. Administration of vitamin K hasa protective effect against DSS colitis in association of down-regulation of IL-6 and vitaminK may have a potential for the treatment target of IBD.

Tu1670

WITHDRAWN

Tu1671

A Novel Biomarker for the Early Detection of Colorectal Polyps Prior toScreening EndoscopyAlexandra Wlodarski, Benno Weigmann, Raja Atreya, Stefan Tenzer, Hansjörg Schild,Markus F. Neurath

Introduction:Currently available biomarkers are aiming at the detection of advanced colo-rectal cancer (CRC) but are of limited value for the detection of premalignant lesions in thecolon prior to endoscopy. Here, we identified a precursor protein, denoted Daedalus, forcolorectal polyps by using mass spectrometry. Methods:Serum samples from patients wereobtained prior to screening colonoscopy for the detection of colorectal polyps and cancer.We purified low-abundance serum proteins from serum samples of healthy controls withoutpolyps, patients with colonic polyps and patients with colorectal cancer cancer by using anovel tandem immunoaffinity depletion system for enhanced detection of low abundanceproteins in humans. We then subjected proteins in the flow-through and eluted fractionsto tryptic digestion and subsequent analysis by label-free quantitative mass spectrometrywith the aim to identify proteins which were specific for the polyp and cancer samples andtherefore constitute potential biomarkers. After verification by Western blotting and ELISA,we used receiver operating characteristics (ROC) to calculate the diagnostic accuracy of theidentified proteins. Results:We identified several upregulated proteins in the fractionatedplasma samples by comparing cancer, polyp and control groups via quantitative massspectrometry. Western blot analysis with unfractionated plasma samples confirmed that theproteins Daedalus and CRP were significantly increased in the serum of cancer patients andpolyp patients compared to healthy controls. ELISA measurements of serum samples from57 patients yielded the same results. No differences between cancer and polyp patients werenoted. The area under a receiver operating characteristic (ROC) curve was 0.86 for Daedalusand 0.79 for CRP alone when we compared controls vs. polyps/ tumor group. For bothproteins in combination an AUC value of 0.93 could be achieved, which resulted in ahigh sensitivity of 92% for detection of colorectal polyps/tumors. Discussion:We identifiedbiomarkers which were able to distinguish control patients from either colorectal polyps orcolorectal cancer patients. To our knowledge, Daedalus is the first serum marker proteinwith high sensitivity and marked specificity for detection of colorectal polyps. Serum testingof Daedalus might allow the stratification of patients for surveillance colonoscopy.

Tu1672

IL-21 Production and Function in Peripheral Immune Cells From Crohn'sDisease PatientsJennifer H. Cox, Evan Thomas, Rebecca P. Wu, Kem Valliant-Saunders, Scott Hussell,Patricia Smith, Jason Stucky, Amanda Blasius, Kyle Serikawa, Hal Blumberg, Steve Levin,Dorthe Lundsgaard

Background: Interleukin-21 (IL-21) is a pleiotropic pro-inflammatory cytokine that is pro-posed to contribute to the pathogenesis of autoimmune inflammatory disorders includinginflammatory bowel diseases. In mice, IL-21 is described as a key regulator of T helper celldifferentiation and B cell maturation. However, there is limited published data evaluatingthe function of IL-21 in the human immune system when using patient material. In thepresent study, our aim was to investigate IL-21 and IL-21 receptor (IL-21R) expression andfunction in Crohn's disease (CD). Methods:We obtained peripheral blood cells from patientswith moderate to severe CD and age/gender-matched healthy controls. Patients had beentreated with less than 20 mg/day Prednisone or 6 mg/day Budesonide. Mucosal biopsieswere also collected from the inflamed colon and ileum of CD patients. Expression of IL-21and IL-21R was analysed by flow cytometry or qPCR. Results: In peripheral cells frompatients with CD and healthy controls, IL-21 is detectable by intracellular staining in CD4+and, to a lesser extent, CD8+ T cells. IL-21R is present on CD4+ T cells, CD8+ T cells, NKcells, B cells, and mature DCs. In vitro differentiation of CD4+ cells results in robustproduction of IL-21 by Th1 and Th17-polarized cells, but IL-21 is not produced by regulatoryT cells (Tregs). Levels of IL-21 correlate with both disease activity and production of IL-17A and IFN-γ. In contrast, there is an inverse relationship between IL-21 production andTreg frequency. Furthermore, IL-21 significantly reduces the frequency of induced Tregs incultures of Naïve CD4+ T cells from patients with CD and controls. Finally, an IL-21neutralizing antibody increases the frequency of Tregs under non-polarizing conditions.Mucosal biopsies from the inflamed colon and ileum of CD patients had detectable levelsof IL-21 and IL-21R mRNA, supporting a model in which IL-21 has activity in diseasedintestinal tissue. Conclusions: Together, these data demonstrate IL-21 production andresponsiveness in CD patients and provide evidence for an IL-21 mediated effect on Teffector/Treg balance. Therefore, targeting IL-21 activity with neutralizing antibodies has potentialas a novel therapeutic approach for the treatment of Crohn's disease.

S-819 AGA Abstracts

Tu1673

Corticotropin-Releasing Hormone Receptors Activate Inflammatory Pathwaysin Mesenteric Adipose TissueJill M. Hoffman, Christopher Fink, Aristea Sideri, Ivy Ka Man Law, IordanisKaragiannidis, Charalabos Pothoulakis

Background and Aims: The corticotropin-releasing hormone (CRH) peptide/receptor familymediates several gastrointestinal responses, including intestinal inflammation. In the intestine,CRH and 3 types of Urocortins (Ucn1-3) bind to two types of CRH receptors to exert theireffects; type 1 (CRHR1) and type 2 (CRHR2). Abdominal adipose tissue hyperplasia is ahallmark of Crohn's disease, and mounting evidence suggests that adipocyte-derived mole-cules are involved in intestinal inflammation. Human visceral and subcutaneous adipocytesexpress Ucn1 and CRH receptors, but expression in mesenteric fat, including mesentericadipocytes, has not been evaluated. Here we determined whether CRH peptides and receptorsare expressed in mesenteric adipose tissue and isolated preadipocytes and explored the novelhypothesis that adipose tissue CRH signaling is associated with the pathophysiology ofcolitis. Methods: Human preadipocytes were isolated from surgical specimens from patientsundergoing abdominal procedures and grown in culture. Isolated mesenteric preadipocyteswere stimulated (4 h) with CRH or the CRHR2-specific peptide Ucn2. RNA was thenextracted for qPCR and multiplex gene expression analysis, and cell lysates were processedfor phosphoprotein assays. qPCR was also performed on adipose tissue harvested from micefollowing acute (48 h) trinitrobenzene sulfonic acid (TNBS) colitis. Results: CRHR1, CRHR2and Ucn2 mRNA was detected in human mesenteric preadipocytes and mouse mesentericadipose tissue. Stimulation of human mesenteric preadipocytes with CRH increased mRNAlevels of the proinflammatory cytokines and chemokines TNF-α (2.2 fold), IL-8 (1.26 fold),CCL7 (1.94 fold) and CXCL1 (1.95 fold; p,0.05 for all). These effects were inhibited bypretreatment with the specific CRHR1 antagonist, antalarmin. Ucn2 stimulation of humanmesenteric preadipocytes decreased IL-8 (0.64 fold), CCL7 (0.37 fold) and CXCL1 (0.38fold; p,0.05 for all), increased the anti-inflammatory factor adiponectin (1.8 fold; p,0.05),and activated the serine/threonine-specific protein kinases Akt, p70S6K, c-Jun and GSK3B.CRHR2 mRNA levels were decreased in acute TNBS-colitis (0.46 fold; p,0.05). Conclusion:This is the first demonstration of CRH receptors and Ucn2 in human mesenteric preadipo-cytes, suggesting a local receptor action in adipose tissue function. Activation of CRHR1and 2 had opposing effects on cytokine expression in these cells. Decreased adipose tissueCRHR2 expression during experimental colitis supports a link between the inflamed intestineand the adjacent mesenteric fat. We suggest that CRH receptors in mesenteric adipose tissuemay participate in the pathophysiology of Inflammatory Bowel Disease. Supported by NIHPO-1 DK33506 (CP), P50 DK064539 (CP & IK), RC1 DK086150-01 (CP), The BroadMedical Foundation (IK), and the Blinder Research Foundation (JMH).

Tu1674

PAK1 Mediates NF-KB Signaling in Colitis and Colitis-Associated CancerKyle W. Dammann, Vineeta Khare, Michaela Lang, Nicolas Granofszky, Christoph Gasche

Background Inflammatory bowel disease such as ulcerative colitis (UC) is associated withan increased risk of developing colitis-associated cancer (CAC). We recently reported thatmesalamine, an anti-inflammatory drug used in the treatment of UC, acts via inhibition ofp-21 activated kinase-1 (PAK1) (Khare et al, Biochem Pharmacol, 2012). PAK1 is a serine/threonine kinase effector of the small Rho GTPases Rac1/Cdc42 and participates in tumor-promoting pathways including proliferation, invasion, and evasion of apoptosis; however,its role in intestinal inflammation is unknown. Here we investigated a potential role of PAK1on activation of NF-kB, p38 MAPK, and JNK. Methods PAK1 expression was analyzed usingIHC in UC and CAC samples and in the AOM/DSS mouse model. In vitro studies wereperformed in normal diploid human colon epithelial cells (HCEC-1CT) within an in vitroco-culture model of activated neutrophils and in the presence of inflammatory cytokines.Cells were treated with IFNγ (100ng/ml), IL-1β (10ng/ml), or TNFα (10ng/ml) for 5 minto 1 h. Whole cell lysates, cytoplasmic and nuclear fractions were analyzed by western blotfor total- and phospho-PAK1 (Thr423), and phosphorylation of downstream kinases suchas AKT1 (Ser473), JNK (Thr183/Tyr185), p38 (Tyr 182)MAPK as well as NF-kB translocation(nuclear p65). Overexpression of PAK1 wild type and kinase dead K299R mutant wasperformed for 72 h. PAK1 kinase activity was also inhibited using IPA-3. Results PAK1expression was up-regulated in inflamed mucosa and tumors both in human (UC and CAC)and mouse intestinal tissue. Total PAK1 expression increased in HCEC-1CT upon co-culturewith neutrophils from 60 min to 24 h. Co-culture also induced phosphorylation of p38and JNK within 60 min. Pro-inflammatory cytokines IFN γ, IL1β, and TNFα resulted inphosphorylation of PAK1 within 5 to 15 min. TNF α had the most profound effect startingat 5 min and peaking at 30 min. PAK1 activation was associated with phosphorylation ofJNK (15 min) and p38 (15, 30 min) and nuclear translocation of p65 (30 min). PAK1overexpression itself activated JNK andNF-kB in the absence of TNFα. Moreover, overexpres-sion of the kinase dead K299R mutant PAK1 or pre-treatment with the PAK1 kinase inhibitorIPA-3 abrogated the effect of TNFα on activation of JNK and NF-kB. Conclusions Our datademonstrate that PAK1 is overexpressed in human andmurine colitis and CAC. Both activatedneutrophils and inflammatory cytokines up-regulate PAK1 expression and activation. TNFαis a potent inducer of PAK1 activation. Most important, however, PAK1 mediates the effectsof TNFα on NF-kB signaling in colon epithelial cells and may be a potential target fortherapy that is already utilized by mesalamine.

Tu1675

Decreased Expression of miRNA-10a Predicts the Progress of Crohn's DiseaseZhanju Liu

AIMS: microRNA (miRNA)-10a has been reported to be decreased in inflamed mucosa ofcolitic mice and is involved in immunoregulation and intestinal mucosal homeostasis in thegut. In this study, expression of miRNA-10a was investigated in Crohn's disease (CD) toexplore the potential role in the pathogenesis of human CD. METHODS: Serum, peripheralblood mononuclear cells (PBMC) and intestinal mucosal biopsies were harvested from

AG

AA

bst

ract

s