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sthat NSAIDs cause lysosome malfunction) leads to autophagosome accumulation, cell death,and mucosal injury.

Mo2055

Anti-Inflammatory Effect of Black Raspberry Extract on Human IntestinalMicrovascular Endothelial Cells (HIMEC)Jamie Schmidt, Rituparna Medda,, Benjamin J. Link, Linghui Nie, Nebojsa Jovanovic,Orestis Lyros, Mary F. Otterson, Parvaneh Rafiee

Background and Aims: Gut microvascular endothelial cells play a critical role in intestinalphysiology and homeostasis, through their regulatory effect on vasorelaxation and tissueperfusion. Black raspberry is a dietary product and an effective chemopreventive agent inanimal models of malignancy, undergoing evaluation for the treatment of inflammatorybowel disease (IBD). However, the cellular and molecular mechanisms underlying the effectof BRP on HIMEC are not well defined. We examined the effect of black raspberry powder(BRP), on HIMEC expression of cellular adhesionmolecules (ICAM-1, VCAM and E-Selectin),NFκB and COX2 following TNF-α and LPS activation. Methods: All experiments wereapproved by the Institutional Review Board of the Medical College of Wisconsin. HIMECmonolayers passage 8-14 stimluated with TNF-α and LPS with or without BRP. COX2 geneand protein expression was determined byWestern blotting and Real-time PCR. Translocationof p65 subunit of NFκB to nucleus was examined by Fluoresence microscopy. Phosphoryl-ation of IκBα was detected by Western blotting. Expression of cellular adhesion molecules(CAMs) were determined using Real-time, Fluoresence microscopy and ELISA assay. Results:HIMEC at rest expressed low levels of COX2 gene and protein. Following TNF-α/LPSactivation of HIMEC both COX2 gene and protein were significantly increased. Immuno-staining revealed cytosolic and perinuclear expression of COX2 in activatedHIMEC. Unstimu-lated HIMEC expressed low level of ICAM-1, VCAM-1 and E-selectin which all were increasedfollowing TNF-α/LPS activation. CAMs expression was attenuated by BRP. Additionally inactivated HIMEC p65 subunit of NFκB was translocated to nucleus and the level of phospho-IκBα was increased in cytosol. BRP treatment of HIMEC dramatically suppressed levels ofCOX2 gene and protein compared to control HIMEC. NS398 specific inhibitor of COX2was used for comparison. TNF-α/LPS activation of HIMEC resulted in NFκB activation,nuclear localization of p65 subunit and increased IκBα phosphorulation compared to controlcells. BRP treatment of HIMEC effectively blocked NFκB activation in TNF-α/LPS activatedHIMEC. BRP alone had no effect on either COX2/CAMs expression or NFκB activation.Conclusions: Taken together, BRP possesses anti-inflammatory properties, which warrantsfurther investigation as adjuvant treatment of IBD.

Mo2056

Evaluation of Intestinal Epithelial and Stem Cell Injury and Protection UsingCrypt CultureXinwei Wang, Liang Wei, Brian Leibowitz, Wei Qiu, Lin Zhang, Jian Yu

Mouse models have been an important tool for our understanding of the pathways andagents that regulate intestinal injury. However the techniques required to specifically demon-strate the role of epithelial or stem cell components in injury remain laborious, and interpreta-tions are often complicated by the involvement of stromal and immune components. Recentadvancements prompted us to evaluate the feasibility of using crypt culture to study intestinalepithelial injury and protection. Crypt cultures were established according to a modifiedSato protocol and exposed to DNA damaging agents, small molecule inhibitors, or geneablation In Vitro. Cell death was evaluated by TUNEL and active caspase-3 staining usingparaffin-embedded sections. Cell proliferation was analyzed by BrdU and Ki-67 staining.DNA double-strand breaks and mitosis were analyzed by p-H2AX (Ser 139) and p-H3 (Ser10) staining, respectively. The growth and regeneration of crypts, or crypt budding, wasmonitored and photographed under an inverted microscope. Our data showed that knockoutof the Bcl-2 family member PUMA significantly blocked crypt apoptosis induced by radiationor Camptothecin and enhanced crypt budding, while having little or no effect on thespontaneous apoptosis or growth of untreated crypts. PUMA deficiency significantly protectedLgr5-expressing stem cells from DNA damage-induced apoptosis. Acute deletion of ADAR1,encoding a RNA editing enzyme, led to rapid crypt apoptosis within 24 hours of tamoxifenadministration. Irradiated crypt cultures exhibited delayed but enhanced budding in thepresence of GSK-3beta inhibitors, which is associated with minimal change in apoptosis butenhanced proliferation. Importantly, these observations are in agreement with data obtainedfrom whole animals. Using crypt culture, we established a specific role of PUMA and ADAR1in the survival of intestinal epithelial and stem cells, and the protective effects of GSK-3betainhibitors on irradiated crypts largely through an apoptosis-independent mechanism. Thesedata suggest crypt culture is a valid system for studying intestinal injury, protection andunderlying mechanisms, and complimentary to tissue or cell-type specific knockout mice.Future experiments will examine isolated single intestinal stem cell (ISC) cultures. Withfurther optimization in gene, siRNA or shRNA delivery and high throughput compoundscreening, this system will likely accelerate the discovery of novel pathways and smallmolecules for intestinal protection.

Mo2057

The Form of Dietary Protein is a Significant Factor in the Progress ofNecrotizing Enterocolitis and the Development of the Intestinal Epithelium inPremature RatsToshi Kinouchi, Chelsea Snarrenberg, Bohuslav Dvorak

Background: Protein hydrolysate formulas are sometimes used for preterm infants. Necrotiz-ing enterocolitis (NEC) is the most common intestinal disease in preterm infants, however,the effects of protein hydrolysate on the pathology of NEC are not clear. In addition, somereports suggested dietary protein may be involved in the developmental processes of theintestinal epithelium in infancy, but it remains uncertain if the form of dietary protein canaffect the processes. Objective: The aim of this study is to evaluate the effect of protein in

S-730AGA Abstracts

milk formula on NEC and the development of the intestinal epithelium using a rat NECmodel. Methods: An established rat model of NEC was used. Premature rats were hand-fedeither on formula with casein and whey protein (Prot) or on formula with whey proteinhydrolysate (Hydr), or dam-fed. All groups were exposed to asphyxia/cold stress to developNEC. The progress of NEC and the histology and functions of the intestinal epitheliumwere evaluated. Results: The pathology of NEC progressively developed and the incidenceof NEC increased to 62% after 96 h-feeding in the Prot group. On the other hand, in theHydr group, the incidence of NEC reached 100% in 24 h, and then the condition recovered.Luminal trypsin activity in the small intestine in the Hydr group was significantly low,except for the middle part of the small intestine, compared with the Prot group, suggestingluminal environment was physiologically different between the two groups. No significantdifferences were observed in bile acids concentrations in the ileum, pro-inflammatory mRNAlevels in the liver, and plasma concentrations of aminotransferases from the liver, whichcan affect the pathology of NEC, between the two groups. In the jejunum, after 96 h-feeding,villus length was short and cell size was large in the Hydr group, compared with the Protgroup. In the ileum, tissue weight to DNA and epithelial aminopeptidase activity weresignificantly high, and was epithelial lactase activity was significantly low in the Hydr group,compared with the Prot group. The jejunal concentration of cholecystokinin in the Hydrgroup was remarkably lower than that in the Prot group. All measurements regardinghistological and functional development of the intestinal epithelium in the Prot group wereequivalent to those in the dam-fed group. Conclusions: These results suggest the form ofdietary protein can affect the progress of NEC and the development of the intestinal epitheliumthrough the modification of luminal environment and cholecystokinin-signaling in pre-term infants.

Mo2058

Role of Cyclooxygenase Isoforms in Colonic Wall Fibrotic Remodeling in thePresence of Bowel InflammationRocchina Colucci, Chiara Ippolito, Cristina Segnani, Matteo Fornai, Luca Antonioli,Letizia Mattii, Amelio Dolfi, Corrado Blandizzi, Nunzia Bernardini

Background and aim. Fibrotic remodeling of gut wall can occur as a consequence of chronicinflammation, and can have serious clinical consequences. This process is characterizedby an excessive deposition of collagen and a rearrangement of other extracellular matrixcomponents. Cyclooxygenase isoforms (COX-1, COX-2) have been reported to be implicatedin fibrosis at extra-gastrointestinal districts, but data on their possible involvement in theremodeling of the inflamed bowel wall are lacking. The present study evaluates the effectsof cyclooxygenase inhibitors on the development of colonic fibrosis in a model of bowelinflammation. Methods. Colitis was induced in rats by intrarectal administration of 2,4-dinitrobenzenesulfonic acid (DNBS, 30 mg/rat in 0.25 ml ethanol 50%), and 6 days laterit was assessed by systemic [body and spleen weight] and tissue inflammatory parameters[macroscopic and microscopic damage]. In the last 3 days before colitis assessement, theanimals were treated daily with indomethacin (IND, non-selective COX-1/COX-2 inhibitor,2 mg/kg), SC-560 (SC, selective COX-1 inhibitor, 2.5 mg/kg), or celecoxib (CEL, selectiveCOX-2 inhibitor, 1 mg/kg), by intragastric gavage. At the time of sacrifice, COX-1, COX-2, collagen I and III, fibronectin, matrix metalloproteinase(MMP)-2 and MMP-9 proteinexpression were analyzed by western blot assays. COX-2 was also analyzed by immunohisto-chemistry. Collagen fibers (Van Gieson) and elastic fibers (orcein) were detected by histoch-emistry. Results. The induction of colitis by DNBS was demonstrated by body weightdecrease (-10±5 g), spleen weight increase (+22±3%), and macroscopic/microscopic scoring.Western blot analysis of inflamed colon showed an increased expression of COX-2 (9.5folds), collagen I (3.5 folds), collagen III (7.4 folds), fibronectin (10 folds), MMP-2 (2.1folds) and MMP-9 (2.8 folds); COX-1 was not affected. The enhanced expression of COX-2 was confirmed by immunohistochemistry. Histochemistry displayed an increased positivityfor collagen fibers (2 folds), in concomitance with a dramatic decrease in elastic fibers (-0.8 fold). Based on western blot, IND, SC and CEL counteracted the increased expressionof collagen III (from 7.4 to 3.9, 4.8, and 5.1 folds, respectively), fibronectin (from 10 to3.0, 3.8 and 3.6 folds, respectively) and, to a lesser extent, collagen I. Histochemistryconfirmed the inhibitory action of IND, SC and CEL on collagen fibers and showed theirreverting effect on the loss of elastic fibers. Conclusions. In the DNBS model of colitis,fibrotic bowel remodeling is characterized by an enhanced collagen/fibronectin depositionin parallel with an elastic fiber reduction. In this setting, both COX-1 and COX-2 appearto contribute to the fibrotic remodeling through an enhancement of collagen/fibronectinexpression and a decrease in elastic fiber amount.

Mo2059

Usefulness of Diamine Oxidase Activity for Evaluation of Intestinal MucosalDamage Due to Anticancer DrugsHiroshi Miyamoto, Soutaro Yamaguchi, Jinsei Miyosi, Yasuteru Fujino, HidetakaTakenaka, Hiromi Yano, Shinji Kitamura, Tetsuo Kimura, Koichi Okamoto, NaokiMuguruma, Toshiya Okahisa, Yutaka Nakaya, Tetsuji Takayama

[Aim] Intestinal mucosal damage due to anticancer drugs which induces malnutrition anddecrease of QOL is one of major problems during chemotherapy in patients withmalignancies.However, there is no method to evaluate objectively intestinal mucosal damage due toanticancer drugs, and therefore it is very difficult to estimate accurately it. Diamine oxidase(DAO) is an enzyme which exists specifically in villi tips of enterocytes of the small intestine.Since serum DAO activity decreases as intestinal mucosa is damaged, it is expected thatDAO activity is a good indicator which reflects sharply damage of small intestinal mucosa.The aim of this study is to investigate whether DAO activity is an indicator of gastrointestinaltoxicities in patients with chemotherapy. [Method] The subjects are 16 patients with unre-sectable advanced gastric cancer who received docetaxel, CDDP and S-1 (DCS) combinationchemotherapy (Takayama et al., Bri J Cancer, 2008) as a first line chemotherapy. DCStherapy is a modified regimen of docetaxel, CDDP and 5-FU combination chemotherapy,in which 5-FU was replaced by S-1. The serum DAO activity was measured according tothe methods of Takagi et al. (Clin Chim Acta, 1993) before chemotherapy, during chemother-apy and after drug holiday. Gastrointestinal toxicities were evaluated by NCI-CTC V3.0.