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treatment with 150 μl of TRF significantly (p<0.05) reduced diarrhea, rectal bleeding andanimal weight loss, as well as colonic TNF-α and vimentin expression. Conclusions: TRFdid not prevent intestinal fibrosis in an optimized rat model. However, treatment with TRFhad anti-inflammatory effects, decreasing some of the clinical hallmarks of TNBS-inducedcolitis. We therefore can't rule out the possibility that treatment with TRF for longer periodsof time might improve intestinal fibrosis.

Su1953

ZP1848, a Novel GLP-2 Agonist, Provides a Wide Window of TherapeuticEfficacy in the Experimental Crohn's Disease ModelJolanta Skarbaliene, Yvette M. Petersen, Kennet N. Christjansen, Kirsten Ebbehoej, HenrikD. Pedersen, Christian Thorkildsen

INTRODUCTION: Glucagon-like peptide-2 (GLP-2) enhances intestinal repair and attenuatesinflammation in preclinical models of inflammatory bowel disease (IBD). This dual effectmakes GLP-2 agonists attractive therapeutic candidates in the treatment of IBD. ZP1848 isa novel, potent and biologically stable GLP-2 agonist that is currently in clinical developmentfor the treatment of Crohn's disease (CD). A therapeutic agent that could both effectivelyprevent inflammation onset and treat established inflammation would satisfy a significantunmet need in IBD therapy. Therefore, the investigation of the length of the potentialtherapeutic window of ZP1848 is of interest. AIMS & METHODS: The aim of this studywas to investigate the anti-inflammatory and intestinotrophic effects of ZP1848 in rats withindomethacin-induced small intestinal (SI) inflammation. ZP1848 treatment was initiatedat different stages of disease: Co-treatment (treatment initiated concomitantly with inductionof SI inflammation) or post-treatment (treatment initiated after established SI inflammation).SI concentrations of the inflammatory markers Alpha 1 acid glycoprotein (AGP) and Myelop-eroxidase (MPO) were used to assess the anti-inflammatory effect of ZP1848. SI mass andplasma citrulline concentrations were used to asses the intestinotrophic effect of ZP1848.SI inflammation was induced by indomethacin administration (7 mg/kg, s.c., 2 doses with24 hr interval). Rats were treated with ZP1848 (200 and 400 nmol/kg, s.c., b.i.d) for 14days. Groups of rats were sacrificed after 3, 6, and 14 days co-treatment with ZP1848, andafter 2, 5, and 14 days post-treatment with ZP1848. RESULTS: Indomethacin-induced SIinflammation was characterized by increased SI concentrations of AGP and MPO. Both co-and post-treatment with ZP1848 attenuated SI inflammation as evidenced by decreasedconcentrations of AGP and MPO. The magnitude of the effect of ZP1848 was both dose-dependent and site specific (jejunal vs. ileal). Moreover, both co- and post-treatment withZP1848 for 14 days significantly increased plasma citrulline concentrations (p<0.001; 200nmol/kg) in parallel with a significant increase in SI mass. CONCLUSION: Treatment withZP1848 initiated either together with induction of SI inflammation or after the establishmentof inflammation effectively attenuated inflammation in experimental model of CD indicatingthat ZP1848 may have wide window of therapeutic efficacy. The demonstration that ZP1848increased SI mass and plasma citrulline concentration indicates that, in addition to its anti-inflammatory properties ZP1848 restores SI functional mucosal mass and absorptive capacity.Our findings provide evidence that ZP1848 may have significant therapeutic benefits atdifferent stages of disease, which would be of significant value considering the unpredictablecourse of inflammation in CD.

Su1954

How Relevant is the Homing of Mesenchymal Stem Cells Into the InflamedGut to Their Therapeutic Efficacy in Experimental Colitis?Emanuela Sala, Massimo Locati, Achille Anselmo, Vincenzo Arena, Patrizia Naccarato,Alberto Malesci, Miquel Sans, Silvio Danese, Stefania Vetrano

Background and aim. Mesenchymal stem cells (MSCs) are multipotent cells present in bonemarrow and other adult tissues that have the capacity to differentiate into a variety of celltypes. In addition to multilineage differentiation capacity, MSCs express high migratorycapacity towards inflamed or remodelling tissues, and exert immunosuppressive and anti-inflammatory properties. The aim of this study was to investigate the therapeutic efficacyof MSCs in experimental colitis. Methods. Colitis was induced in C57B6 mice by 3% DSStreatment for 10 days. MSCs were isolated from bone marrow of WT and GFP transgenicC57B6 mice, cultured for 4 weeks and then sorted for Sca-1+, CD31-, cocktail lineage-surface markers. MSCs (3x106) were injected within the peritoneal cavity of mice at day 5of DSS treatment. Body weight and disease activity index (DAI) were evaluated daily andendoscopic and histological scores were calculated at day 10. To follow the migratory activityof the MSCs, GFP-MSCs were injected intraperitoneally in healthy or colitic mice at day 5and their presence was assessed by flow cytometry after 24 and 48 hours in the colon andmesenteric lymph nodes. Results. In control healthy animals GFP-MSCs did not egress fromthe peritoneal cavity. Conversely, in colitic mice a small fraction (< 1%) of GFP-MSCs wasdetected, with a peak value after 48 h of injection, selectively in inflamed tissues (colonand mesenteric lymph nodes), while no GFP-MSCs were found in non inflamed tissues.Treatment with MSCs significantly ameliorated acute DSS-induced colitis, in terms of weightloss, DAI, colon shortening, endoscopic and histological scores (p < 0.05 compared tountreated colitic mice for all parameters).On the day of sacrifice, colitic mice treated withMSCs demonstrated a large number of aggregated MSC cells along with macrophages andlymphocytes in the peritoneal cavity. Conclusions. Overall, our results confirm MSCs as atherapeutic tool for the treatment of inflammatory bowel diseases and show their selectivehoming in the inflamed colon and draining lymph nodes. Given the low homing frequencyof MSCs in the inflamed gut, it is possible to speculate that their efficacy could be independentfrom their homing properties, and could mainly involve other mechanisms, including thesecretion of soluble factors.

S-519 AGA Abstracts

Su1955

Dextran Sodium Sulfate Inhibition of Real-Time PCR Amplification andReversal With Poly-A Purification of DSS-Exposed mRNAThomas A. Kerr, Matthew A. Ciorba, Brian K. Dieckgraefe, Nicholas O. Davidson

INTRODUCTION: Dextran sodium sulfate (DSS) is used in experimental models of colonicinjury and neoplasia. Despite its widespread use, little has been published regarding thetechnical challenges associated with gene expression analysis in DSS-treated mice. We haveobserved impaired quantitative PCR (qPCR) amplification of cDNA derived from DSS-exposed tissue. This led us to hypothesize that DSS interferes with real-time qPCR geneexpression analysis. AIMS: To characterize the impact of DSS on qPCR gene expressionanalysis and develop a cost-effective solution to this technical challenge. METHODS: TotalRNA was extracted from control and 2.5% DSS-treated mice. cDNA was generated by reversetranscription and amplified by real-time qPCR. In-vitro DSS inhibition of qPCR amplificationwas measured by titrating DSS (from 0.2 nM to 20 um) into the qPCR reaction. Tissuedistribution of DSS was assessed by comparing differences in GAPDH qPCR crossing thresh-olds in various tissues procured from control and DSS-exposed mice. To separate DSS fromRNA and allow for gene expression analysis we used standard RNA wash protocols includingethanol precipitation, silica-column wash, and column and magnetic bead-based oligo-dTtechniques. RESULTS: Enteral DSS treatment led to delayed or absent qPCR amplificationof cDNA from multiple tissues. This was most pronounced in stomach where DSS exposurecompletely prevented qPCR amplification of tissue-derived GAPDH cDNA. GAPDH ampli-fication in spleen and colon was markedly delayed (>9 cycle increase in crossing threshold)compared to non-DSS exposed controls. qPCR amplification was delayed to a lesser extentin lung, kidney, small bowel and liver. DSS exposure did not impair qPCR amplificationfrom brain or heart. In-vitro titration of DSS into PCR amplification of control cDNA resultedin complete inhibition of amplification between 2 and 20 nM DSS. Ethanol precipitationand silica-based RNA wash techniques failed to reliably separate RNA from DSS. Columnand magnetic bead oligo-dT mRNA isolation permitted robust amplification of cDNA fromDSS-exposed tissue. Magnetic bead-based mRNA isolation was significantly less expensivethan column mRNA isolation (~$3 vs. $20/sample). CONCLUSIONS: DSS potently inhibitsqPCR amplification of cDNA derived from DSS-exposed tissue complicating gene expressionanalysis in DSS experimental models. RNA can be cost-effectively separated from DSS bymagnetic bead oligo-dT purification prior to cDNA synthesis. IMPLICATIONS: RNA expres-sion analysis from DSS-exposed tissue requires adequate RNA clean-up to provide optimaldata. Widespread tissue distribution of DSS, as evidenced by DSS interference with geneexpression analysis, suggests that extracolonic tissue injury may occur in DSS-treated mice.

Su1956

A Novel Toxoplasmosis Model for Gastrointestinal Complications inPregnancy and Drug EvaluationHelieh S. Oz, Thomas N. Tobin

Two billion people are globally infected with Toxoplasma, a common cause of foodborneand congenital illnesses, frequently with unknown health consequences. In the event ofstressors, dormant cysts are reactivated and trigger a life-threatening clinical disease. Atova-quone is a drug of choice; however, there is no safe and effective (FDA approved) therapyagainst congenital toxoplasmosis or to eliminate the persistent chronic infection. Thus,understanding the pathogenesis and identification of new therapeutic approaches are requiredto impact this disease. The objectives were to study pathogenesis of gastrointestinal complica-tions and pregnancy outcomes as consequences of toxoplasmosis. Methods: CD1 pro-grammed pregnant mice were infected with 50-3000 Tachyzoites from Type II strain (clonePTG). Dams were monitored for pain, distress and abortion. Samples were collected ongestation day 16. Results: Dams infected during the 2nd trimester demonstrated abdominalresponses (allodynia) to von Frey mechanical stimuli in a dose dependent manner. Infecteddams developed severe anemia (Controls 44.5+1.2 vs infected-dams 33+2 p<0.01), hydro-thorax, ascities, giant cell hepatitis (score 0-4:3.5+0.01) with influx of inflammatory andplasma cells, multinucleated dysplastic hepatocytes, necrosis and significant increases inliver enzymes. In addition, infected dams developed mild to severe pancreatitis. This wasconsistent with splenomegaly (X3 Fold), massive infiltration of epithelioid cells and loss ofgerminal structure in splenic tissues. Colonic length (10.4+0.2 vs 8.7+0.6cm) was significantlyshortened (p<0.01) in infected dams. Colonic pathology included shortening of cryptswith numerous microabscess formations, infiltration of lymphocytes, and macrophages.Atovaquone treatment partially but significantly protected the dams from some aspects ofthe disease. Conclusions: This pregnancy model can be useful in studying GI complicationsand drug discovery against toxoplasmosis. This study was supported by KY Science andTechnology 721-RFP-006(TT) and NIH-DE019177(HO)

Su1957

Dextran Sulphate Sodium-Induced Colitis in Interleukin-4 Deficient MiceChoi Jongkyoung, Seong-Joon Koh, Joo Sung Kim, Hyun Chae Jung

Background/Aims: The interleukin 4 (IL-4) plays a pivotal role in inflammatory Th 2 responseknown for controlling functions of B and T lymphocytes. In previous studies, IL-4 expressionwas enhanced in colonic tissue in patients with ulcerative colitis, not acute colitis. Therefore,we investigated a role of IL-4 in acute and chronic experimental colitis. Methods: Acutecolitis was induced in IL-4-/- and WT mice by administration 4% DSS in drinking waterfor 5 days. For chronic DSS colitis, mice were treated with 4 cycles of 2% DSS for 5 daysand 15 days of drinking water between each cycle. A clinical disease activity score includingbody weight, stool consistency and bloody diarrhea was evaluated daily. Colon length andhistological evaluation was performed to assess inflammation. The expression of cytokinesTNF-α and MIP-2 in colonic tissue was determined by real-time RT-PCR and colonicmyeloperoxidase (MPO) levels were assessed by ELISA. Results: In the acute colitis model,both groups developed diarrhea and rectal bleeding starting on day 4. During experiment,clinical disease activity showed no significant difference between two groups. At the endpoint, colon length and histopathology of colitis also had no difference between them. In

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