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scolumns and EET, HETE and diHETE quantified by mass spectrometry after HPLC. In asecond set of experiments, by calcium flux quantification, agonist properties of thesesmetabolites and their commercially available standard (10μM) were studied on HEK-TRPV4transfected cells or on human epithelial cells line (Caco2 cells) treated or not by a silencerRNA against TRPV4. Results: 7 days treatment by DSS provoked an inflammatory reactioncharacterized by an increase in MPO activity, macro and microscopic damage score. TRPV4mRNA expression was increased in the colon of inflamed mice compared to control. TRPV4was localized predominantly on epithelial cells and was not observed on immune cells(CD45 positive cells). By LC-MS we observed a different profile of EET, HETE and diHETEexpression between control and inflamed colon characterized by an increase in EET concen-tration. Exposure of HEK-TRPV4 transfected cells to lipid extracts only from inflamed micecolon provoked an increase in calcium flux compared to none transfected cells. Moreover,in Caco-2 cells, 5, 6-EET induced calcium signal through TRPV4 activation. Conclusions:We have demonstrated in a mouse model of colitis that TRPV4 expression was increased.Moreover, we show an increase of TRPV4 agonist activity in lipid extracts from inflamedcolon. These results position this receptor channel as a new potential therapeutic target forcolonic inflammation.

S1738

Role of D2 Dopamine Receptors in the Pathogenesis of ExperimentalUlcerative Colitis: Implication of Colonic Vascular PermeabilityGanna Tolstanova, Xiaoming Deng, Klara Osapay, Tessa M. Kaplan, Brankica Paunovic, MMazen Jamal, Sandor Szabo, Zsuzsa Sandor

VEGF/VPF plays a role in the initiation and perpetuation of abnormal vascular response byenhancing colonic vascular permeability, leukocyte infiltration and angiogenesis in experi-mental ulcerative colitis (UC) (Tolstanova et al. J Pharmacol Exp Ther 2009;328:749-57).Dopamine through D2R inhibits VEGF/VPF-mediated microvascular permeability, prolifera-tion, migration of endothelial cells in tumor models. We hypothesized that activation ofD2R may play a role in UC pathogenesis. METHODS: A) Experimental UC was studied inIL-10 knockout (KO) mice (12 and 18 weeks old) and in rats 0.5, 2, 6 hr and 3 and 7days after 6% iodoacetamide (IA) enema. D2R agonists quinpirole and bromocriptine weregiven intragastrically for 5 days in rats and 13 days in IL-10 KO mice. Body weight anddiarrhea were recorded daily. Animals were euthanized on the 7th or 14th day. Colonicvascular and epithelial permeability was measured by Evans blue extravasation (mg/g wetcolon) and serum FITC-dextran concentration, respectively. Immunostaining with anti-factorVIII-related antigen was used for morphometric analysis of colonic angiogenesis. Expressionsof tyrosine hydroxylase (rate-limiting enzyme in dopamine synthesis) andD2Rweremeasuredby Western blot. B) Subsequently, a retrospective analysis was conducted in “the 2002National Inpatient Sample” database. RESULTS: A) Although colonic D2R protein expressionincreased, levels of tyrosine hydroxylase (rate-limiting enzyme in dopamine synthesis)decreased during IA-induced UC in rats and these proteins were almost undetectable in IL-10 KO vs. wild type mice. Treatment with quinpirole (1 mg/100g) or bromocriptine (1mg/100g, 5mg/100g) decreased the size of colonic lesions in rats with IA-induced UC from222.9±112.5 to 81.8±26.1 (p=0.002), 137.8±87.3 (p=0.08) and 102.5±53.1 mm2 (p=0.01),respectively, and significantly diminished blood vessel density. Quinpirole (1mg/100g)reduced colon wet weight in IL-10 KO mice from 0.27±0.03 to 0.22±0.03 g/10g (p=0.03). Quinpirole (1mg/100g) and bromocriptine (5mg/100g) also decreased colonic vascularpermeability from 18.6±4.3 to 12.3±1.0 and 13.7± 2.9 (p≤0.001), respectively, and didn'taffect colonic epithelial permeability. B) The patient data analysis demonstrated a diminishedrisk for UC (OR: 0.43; 95% CI 0.31-0.60) and regional enteritis (OR: 0.47; 95% CI 0.37-0.60) (p<0.001) in patients with schizophrenia (hyper-dopaminergic system). CONCLU-SIONS: D2R agonists decreased the severity of UC, in part, by attenuation of enhancedvascular permeability, prevention of excessive vascular leakage, and reduction of angiogenesiswith an apparent switch to normal angiogenesis.

S1739

Interleukin-23/TH17 Pathway Expression in Chronic Trichuris Muris Colitis:Lessons for Iatrogenic HelminthosisScott E. Levison, John McLaughlin, Leo Zeef, Joanne Pennnock

Background: Host response to Trichuris spp (whipworm) infection is variable, dictated byenvironment and host genetics. Following an acute infection, worms may be expelled(resistance) driven by a Th2 immune response. However, chronic infection or susceptibilitycan equally develop (Th1-driven) with resultant colonic inflammation. Trichuris muris(T.muris) provides the perfect platform to unravel colonic immuno-biology relevant tooutcome. Surprisingly, little is currently known of the IL-23/Th17 pathway in chronictrichuriasis susceptibility, a critical pathway in human intestinal inflammation. Aims: 1)Phenotype response to T.muris infection in resistant and susceptible mouse strains. 2)Evaluate colonic T-cell transcriptional activity and compare findings to human Crohn'sdisease (CD) data. Method: Susceptible (AKR) and resistant (BALB/c) mice were administered300 T.muris ova by oral gavage. Naïve, resistant and susceptible mice were compared atday 35 post-infection. Well-being, body weight and stools were assessed throughout. Wormcount, serum IgG, macroscopic and microscopic colonic changes were determined. Pooledcolonic mRNA (n=5) was applied to Affymetrix GeneChip Mouse Exon arrays, with 3technical replicates. Bio-informatic and ANOVA statistical analysis, qPCR replication, andin silico data mining was performed. Results: BALB/c exhibited ‘normal' macroscopic colonsand histology, and a strong Th2 immune response post-infection. Diarrhea, weight loss,and colonic shortening occurred in infected AKR, with moderate focal, transmural colonicinflammation and fat hypertrophy. Infected AKR demonstrated compositely upregulatedTh1 and Th17 transcript expression of cytokines (e.g. IFNγ, TNFα, IL-12, IL-17F, IL-6, IL-21) (>x1.2-fold upregulation c.f. naïve; p<0.05) and their receptors. Similarly upregulatedgene expression of ligand and downstream signalling molecules in the IL-23R/Th17 pathway(e.g. IL23α, IL-12β, IL-12Rβ1, TGFβ, TNFSF15, Stat3) demonstrated mechanistic geneexpression parallels with CD. Conclusion: Upregulated expression of Th17 and Th1 pathwaytranscripts were seen in chronic Trichuris colitis. The upregulation of Th17 differentiation,stabilization and amplification molecules shares mechanistic expression profiles of human

S-264AGA Abstracts

CD. With many biological commonalities to CD, T.muris offers a validated, immuno-competent and reproducible tool for colitis research, particularly in the testing and dissectionof the ‘Hygiene Hypothesis'. Furthermore, such immunological findings suggest iatrogenichelminthosis may potentially aggravate colonic inflammation if administered to an individualgenetically predisposed to Trichuris susceptibility.

S1740

Methyl Deficient Diet Influences Pro-Inflammatory Pathways and AggravatesExperimental Colitis in RatsMin Chen, Laurent Peyrin Biroulet, Amandine Georges, Florence Coste, Bing Xia,Namour Bernard, Jean-Louis Gueant

The pathomechanisms leading to Crohn disease result from complex interactions betweenenvironmental, pro-inflammatory, genetic and nutritional factors. Among nutritional andmetabolic factors, homocysteine and its nutritional (methyl donors) and genetic determinantshave received increasing attention over the past decade as potential contributors to thegreater risk of inflammatory bowel disease (IBD).We aimed to evaluate whether homocysteineand the deficiency in folate and vitamin B12 could be regarded as factors influencing oxidantstress and inflammatory pathways in the colon mucosa exposed to an environmental stress.We performed a study in rat pups from dame fed a methyl deficient diet (MDD, deprivedof folate, vitamin B12 and choline) from one month before gestation until pup's weaning.Pups were subjected to administration of Dextran sodium sulfate. The respective and com-bined effects of DSS and the MDD in the colon mucosa were evaluated on apoptosis, oxidantstress and inflammation pathways. Four groups were considered (n=12-16 per group): CDSS- (Control/DSS-), D DSS- (Deficient/DSS-), C DSS+ (Control/DSS+) and D DSS+ (Defi-cient/DSS+) MDD significantly decreased the plasma concentration of vitamin B12 (P<0.001)and folate (P<0.001) and increased the plasma concentration of homocysteine (7.8±0.9 vs22.6±1.2 , P<0.001). The severity of the colitis induced by DSS, reflected by disease activityindex (DAI), was dramatically higher in the group D DSS+ compared with each other group(P<0.001). The activities of SOD and GPX were unchanged in the deficient rats treated withDSS. Caspase3 and Bax were significantly decreased (P=0.012, and P=0.015, respectively)and the expression level of Bcl-2 was significantly increased (P=0.044) in D DSS+ comparedwith D DSS- rats; mRNA of TNF-alpha and immunoblots of p38, PLA2c and COX2 weresignificantly increased in the colon of rats from group D DSS+, compared with those of thegroup C DSS+ (P<0.001, n=15). In contrast, the expression of COX1 and LOX were notaffected. The expression of COX2 was confirmed in immunohistochemistry In conclusion,MDD is an aggravating condition for TNF-dependent hyper-expression of p38 and thesubsequent increased expression of PLA2c and COX2. In standard conditions, COX2 is notexpressed in the intestinal mucosa, while it is detected in colon mucosa of IBD patients.These results obtained in an animalmodel highlight a possible aggravating effect of hyperhom-ocysteinemia and folate and/or vitamin B12 deficiency in IBD, which is consistent with theassociation studies reported in patients. This suggests to pay attention and to correct theB12 and folate deficits in IBD.

S1741

Modulation of Local Urocortin 1 Expression Levels in a Rat Model of Crohn'sColitis: An Effect That Requires the Presence of CRF-R2 but Not CRF-R1Melanie Adams, Burcu Hasdemir, Min Liao, Aditi Bhargava

BACKGROUND. Inflammatory bowel disease (IBD) has a poorly defined, multi-factorialetiology. Urocortins (Ucns) and their receptors are emerging as potent immunoregulatorsin the gastrointestinal tract, where they can exert both pro- and anti-inflammatory effects.However, how Ucns and their receptors contribute to the genesis and progression of IBDis unknown. AIM. To examine the spatio-temporal expression of Ucn1 in an animal modelof Crohn's colitis and ascertain the receptor type that is involved in mediating the effectsof Ucns in the gut. METHODS. A rectal enema of TNBS was used to induce colitis in maleSprague-Dawley rats. Colons were harvested 1-15 days after a single bolus of TNBS, tissueswere evaluated byH&E staining, and changes in inflammationmeasured using amyeloperoxi-dase assay. RT-PCR and immunohistochemistry was used to determine the spatio-temporalexpression of Ucn1. Colonic expression of receptors was silenced using RNA interference(RNAi) by injecting siRNA in the colonic wall/lumen of anesthetized male rats (n=4-6/group),followed by a TNBS enema. Since activation of JNK and ERK is increased in gut tissue ofCrohn's patients, we examined activation of pERK/ERK2 signaling by western blot analysisin colon homogenates. RESULTS. Ucn1 transcripts and immunoreactivity were ubiquitousin epithelium, endothelium, smoothmuscle, immune cells andmyenteric neurons throughoutthe GI tract under basal conditions. During colitis, average Ucn1 mRNA expression increasedduring days 1-6, with significant increases on day 6. Thereafter, Ucn1 mRNA levels fellsharply below basal levels, and remained low until day 15. During colitis, all cell types thatexpressed Ucn-IR under basal conditions continued to express Ucn1-IR. Moreover, thenumbers of inflammatory cells increased dramatically in highly thickened submucosa andproliferating granulation tissue and mucosa, with Ucn1-IR being prominently expressed inthese de novo sites. To ascertain whether both receptors play a role in exerting effects ofUcn during colitis, we eliminated expression of Ucn receptors CRF-R1 and CRF-R2 by RNAi.Knockdown of CRF-R2 significantly reduced the spread of ulceration, and myeloperoxidaseactivity as compared to un-injected or dsRNA-injected controls or animals with CRF-R1knockdown. Average pERK expression, was bimodal: increasing after 1 day of TNBS enema,decreasing markedly at day 6, and then increasing until day 15. CONCLUSIONS. We showfor the first time that in TNBS-induced colitis in rats, average Ucn1 mRNA peaks at day 6,whereas pERK levels are at their nadir on day 6. Spread of ulceration is dependent on thepresence of CRF-R2 but not CRF-R1. Supported by NIH R01 DK080787 (AB).