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TH1/TH17 Profiles in Crohn's Disease: A Cross Sectional Single Centre Studyin Postoperative Crohn's DiseaseAravinth U. Murugananthan, David Bernardo Ordiz, Phil Tozer, Cheng T. Tee, ElizabethR. Mann, Ailsa Hart, Naila Arebi, Stella C. Knight, Hafid O. Al-Hassi

Introduction: Th1 and Th17 pathways are implicated in Crohn's disease (CD). In operativeresection samples healthy ileum shows high TGFb levels in patients who develop recurrence,with TGFb being a known activator of the Th17 response. Other studies in CD show adominant Th1 cytokine profile, with high levels of IFNg, which reduce Th17 response andaugment Th1 response. The relationship of Th1/Th17 cytokine profiles in postoperative CDhas not been examined. We aimed to study tissue Th1/Th17 cytokine secretion after in-vitro biopsy culture in postoperative CD. Methods: Colonoscopy was undertaken in postoper-ative CD patients. Recurrence graded as no/minimal inflammation (Rutgeert Score [RS] ≤1)or progressive inflammation (RS ≥2). Ileal biopsies were cultured overnight and cell freesupernatants obtained. Supernatant cytokines (IL-2, IL-4, L10, IL17 TNF a, INFg and IL6)were assessed by flow cytometry using cytometric bead array TM(Becton Dickinson). Statist-ical analysis was via unpaired t-tests. Results: Consecutive patients attending endoscopy (n=24, 9m/15f) were identified. Mean age 45.0 yrs and time from I-C resection was 5.8 years;5 patients were smokers. Drugs were thiopurines 13, Infliximab 1 and nil 10. Endoscopicseverity was i0 n=5, i1 n=6, i2 n=5, i3 n=3, i4 n=5. Mean cytokine concentrations fromsupernatants are shown in the table. Comparison between RS ≤1 and ≥2 showed that pro-inflammatory cytokines IL17a (p<0.02) and IFNg (p<0.03) were significantly higher in RSi2-i4 neo terminal ileum as compared with those with RS i0-i1. The regulatory cytokine IL-10 was significantly higher in patients with RS≤1 (p<0.038). Conclusion: Cytokine profilesin those with RS >2, show higher levels of IL 17a and IFNg and reduced IL10 comparedto RS <1. This profile supports a Th17 and Th1 mediated response as one of the earlyinstigators of endoscopic progression in postoperative CD. Our observation is consistentwith recent findings of a T cell subset able to produce cytokines involved in both Th1 andTh17 responses. Previous therapies directed at Th1 pathway e.g. anti-IL-12p40 antibodyustekinumab and anti-IFNG Fontolizumab failed to show significant clinical benefit in CD.Given our findings targeting the Th17 response e.g. with anti-IL-23 antibodies and anti-IL17 may deliver improved therapeutic outcome.Cytokine concentrations per Rutgeert Score

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Protease-Activated Receptor 1, 2 but Not 4 Sensitizes Transient ReceptorPotential Vanilloid 4 in Human Intestinal Epithelial CellsEmilie D'Aldebert, Nicolas Cenac, Charlyne Casteras, Nathalie Vergnolle

In a previous study, our laboratory has demonstrated that Transient Receptor PotentialVanilloid 4 (TRPV4) was expressed and functional in human intestinal epithelial cells. TRPV4activation in those cells caused increases in intracellular calcium concentrations, chemokinerelease and its activation in mouse gastrointestinal tract caused colitis. TRPV4 appears tobe an important actor in intestinal inflammation. In an inflammatory context, many mediatorsare released, such as serine proteases, which will activate protease-activated receptors (PAR).We hypothesized that these proteases released during inflammation, through activation oftheir receptors the PARs, may sensitize TRPV4. Methods: TRPV4 response was evaluatedby measurement of calcium flow with the probe Fluo-8 in Normal derived Colon Mucosa(NCM460) cells. These cells were stimulated by PAR1-activating peptide (TFLLRN-NH2),PAR2-activating peptide (SLIGRL-NH2), PAR4-activating peptide (AYPGFK-NH2) or theirvehicle, 3 minutes before a second stimulation with the specific agonist of TRPV4, 4αPDD(4α-phorbol-12,13-didenoate) or its vehicle. Results: TRPV4 specific activation involved adose-dependent calcium flow in NCM460 cells. The response to 10 μM of 4αPDD wassignificantly increased when cells were pre-incubated with PAR1-activating peptide (100μM) or PAR2-activating peptide (10 μM). Intestinal epithelial cells did not respond to lowconcentration (1 μM) of 4αPDD. However, if cells were pre-treated with the PAR1 agonist(100 μM) or the PAR2 agonist (10 μM), they became responsive to low concentration (1μM) of the TRPV4 agonist 4αPDD. PAR4 agonist (200 μM) never modified TRPV4 response.Conclusions: Our study shows that activation of PAR1, PAR2 but not PAR4 potentiated thecellular response of TRPV4 but also sensitized the receptor. Overall, these results suggestthat TRPV4 signals and potentially its pro-inflammatory effects in intestinal epithelial cellscan be enhanced by proteases acting on PAR1 and PAR2.

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Icilin, an Agonist Cold Activated Transient Receptor Potential (TRP) ChannelsProtects Mice Against ColitisEric Hyun, Rithwik Ramachandran, Morley D. Hollenberg

Icilin is a known agonist for members of the transient receptor potential family of ionchannels (TRPA1 and TRPM8), which are reported to play a role in various physiologicalevents including temperature sensation. TRPA1 is activated by temperatures below 18°C,

S-840AGA Abstracts

whereas TRPM8 is reported to be activated at temperatures below 25°C. Since cold temper-ature is commonly used as local anti-inflammatory treatment, we hypothesized that icilinwould diminish colitis in mouse models of colonic inflammation. Colonic inflammation inC57BL6 mice was induced by treatment with either 2.5% dextran sodium sulphate (DSS)solution as drinking water or tri-nitro-benzene sulphonic acid (TNBS, 2mg in 100ul of 40%ethanol, intracolonically). Colitis was allowed to develop for 7 days and icilin (100μg in3% DMSO/saline, intraperitoneally) was administered daily. Seven days after induction ofcolitis, macroscopic damage score, bowel thickness and myeloperoxidase (MPO) activitywere measured. DSS or TNBS challenge induced potent colonic inflammation in mice leadingto significant increases in bowel thickness, macroscopic damage scores and MPO activity.However, DSS- or TNBS-induced increase in bowel thickness and macroscopic damagescores were significantly reduced in mice that received daily icilin treatment. Lastly, micechallenged with DSS showed significantly reduced MPO activity when treated with icilin ascompared to mice treated with vehicle. In conclusion, icilin confers protection against thedevelopment of colitis in mice. Thus, agents that can activate TRPA1 and/or TRPM8 mayrepresent novel therapeutics for inflammatory bowel disease in the future.

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Evaluation of p38 MAPK Pathway as a Molecular Signature in UlcerativeColitisXinmei Zhao, Fachao Zhi, Bin Kang, Bo Jiang, Siqi Liu, Youyong Lu

Early diagnosis and treatment of Ulcerative colitis (UC) is clinically challenging. In orderto overcome this problem, we explored the interrelated multiplex signaling pathway toidentify molecular signatures in UC by use of integrated strategy in proteomics. Twenty-four cases intestinal mucosa of UC and normal control underwent comparative proteomicanalysis. A total of 30 unique differential proteins were identified, including 16 up-regulatedand 14 down-regulated in UC group. A differential protein cluster, consisting of 11 proteinsinvolved in p38 mitogen-actived protein kinase (MAPK) pathway, was deduced and validatedby Western blot. Furthermore, the expression of three proteins elicited from the proteincluster, phosphorylated p38, MAWBP and galectin-3, as a molecular signature, were detectedby immunohistochemistry on 120 cases UC and normal samples. The molecular signaturewas significantly different between UC and normal samples (P<0.001), correlates with diseaseprogression of UC (P<0.01), and classified the UC risk with high sensitivity (95.00%±2.81%)and specificity (98.33%±1.65%). P38 MAPK pathway modulates the expression of the proteinclusters in macrophages RAW264.7 cells as evidenced by the alteration of the expressionpattern of the protein cluster with specific inhibitor SB203580. Therefore,molecular signatureof P38 MAPK pathway may serve as a promising biomarker in evaluating UC risk.

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Tryptophan Metabolome Signature Differences in Inflammatory BowelDiseasesYunki Yau, Sean S. Shin, Sonia Bustamante, Russell Pickford, Rupert W. Leong, Valerie C.Wasinger

BACKGROUND: Metabolism of the essential amino acid tryptophan via the kynureninepathway is associated with inflammatory neurotransmission and gut motility. Quinolinicacid (QA) and Picolinic acid (PA) are the terminal end products of this pathway. QA isknown to preferentially activate a glutamate receptor subtype, while PA induces macrophageinflammatory proteins preceding acute neutrophilic inflammation. Metabolomics is a noveltechnique in IBD research and we present here the first metabolome assay of human IBDplasma. METHODS: We quantified and compared QA and PA expression levels and per-formed a whole plasma metabolome assay between populations of blinded CD, UC, andhealthy controls using Gas Chromatography/Mass Spectrometry (GC/MS) and Liquid Chro-matography (LC)/MS, respectively. GC/MS was performed on a HP 6890 gas chromatographinterfaced to a mass selective detector, and a Ultra High Pressure Liquid Chromatograph(UHPLC) with C18 reverse-phase LC and Orbitrap MS instrument was utilized in scanningfor chemical identities between 50 - 1000 m/z for a whole metabolome assay. RESULTS:Nineteen UC, 16 CD and 9 control samples were analyzed. In CD, QA levels was significantlyincreased when compared to controls by a mean of 0.1348 pmol/ml (P=0.021), and by0.1401pmol/ml in active disease from non-active (p=0.027). Total kynurenine metabolitesin CD was also significantly increased against controls (mean increase of 0.1678 pmol/ml(P=0.015)), and once again in active disease when compared to non-active (0.1879 pmol/ml mean increase (p=0.009)). Kynurenine metabolites did not differ significantly betweenUC and controls, or with UC disease activity. SIEVE software quantitated, and Chemspidersearch engine identified a large number of differentially expressed metabolites from wholemetabolome assay. Of note, peroxisome dysfunction marker tetracosa hexaenoic acid (THA)was significantly more abundant in UC compared to CD (p<0.008). CONCLUSION: Signific-ant overexpression of QA in CD suggests an overactivation of receptors involved in the gut-brain axis and possible irregularities in interleukin 2 (IL-2) induced T cells. Furthermore,relative overabundance of THA in UC may have implications in disease-specific mucin deple-tion.

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Short and Long-Term Regulation of Intestinal Na+/H+ Exchange by TLR2José Miguel Cabral, Fernando Magro, Patricio Soares-da-Silva

Type 2 toll-like receptors (TLR2) are expressed in the cell membrane and recognize a widerange of pathogen-associated molecular patterns derived from bacteria as lipoteichoic acid(LTA), a Gram-positive bacteria component. Aims: To evaluate the effect of TLR2 activationby LTA on activity of type 1 and 2 Na+/H+ exchanger (NHE) in T84 intestinal epithelialcells. Matherial and methods: Intracellular pH levels and NHE activity were measured usingfluorimetric assays and protein expression was determined by western blot analysis. Allexperiments were performed in presence of S3226 (100 nM), a selective inhibitor of NHE3,to ensure an effective evaluation NHE1/NHE2 activity. Results: Short-term (0.5 h) TLR2