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from the ligature, resulting in a calibrated constriction of SMA. After the operation, the animals were fed normally and killed various days later up to 3 days. Various antibiotics, cyclooxygenase (COX) inhibitors and NO synthase (NOS) inhibitors were given PO or SC, for 2 days after the operation. PGE2 and AE1-329 (EP4 agonist) were given IV for 2 days after the operation. Mucosal PGE2 content was measured by EIA while NO content was measured by the Griess method. The expressions of COX-1/COX-2 and nNOS/ iNOS in the intestinal mucosa were examined by RT-PCR or Western Blotting. Results: The stenosis of SMA caused the mucosal ischemia and resulted in damage in the small intestine, especially the ileum, within 3 days. This model of enteritis was accompanied by enterobacterial invasion in the mucosa, the bacterial count being significantly increased 8 h after the operation. The development of these lesions was prevented by the repeated treatment with ampicillin (antibiotic), aztreonam (gram- negative bacterium specific antibiotic), L-NAME (nonselective NOS inhibitor) or aminoguanidine (selective iNOS inhibitor), while markedly aggravated by indomethacin (nonselective COX inhibitor) and rofecoxib (selective COX-2 inhibitor). In addition, the deleterious effect of indomethacin was abrogated by the co-administration of PGE2 or AE1-329 the EP4 agonist. The expressions of iNOS and COX-2 were both upregulated in the small intestine in a time-dependent manner following the onset of ischemia, together with the increase of mucosal NO and PGE2 contents. Conclusion: These results suggest that enterobacteria, especially gram-negative bacteria play a major pathogenic role in this model of ischemic enteritis, COX-2/PGE2 prevent the development of ischemic enteritis through EP4 receptors, and iNOS/NO is deleterious in the pathogenesis of these lesions.

AGA Abstracts

Bile Acids Decrease Mucin 2 and Trefoil Factor 3 in Neonatal Rat Ileal Explants Crystal Johnson, Mitchell A. Thomas, Bohuslav Dvorak, Melissa Halpern Background: We have recently shown that accumulation of ileal hydrophobic bile acids (BAs) contributes to ileal damage during the development of experimental necrotizing enterocolitis (NEC). Mucin 2 (Muc2), the predominant secretory mucin produced by intestinal goblet cells, has been shown to be altered by BAs, but results differ depending on the intestinal cell lines utilized and the pathological conditions studied. We have reported that Muc2 positive cells are significantly decreased in the ileum of neonatal rats with NEC and removal of ileal BAs from neonatal rats with NEC using cholestyramine normalizes these decreases. However, we do not know if BAs alone cause these changes, or if other goblet cell products beside Muc2 are similarly affected. To further examine how BAs affect goblet cells in the neonatal intestine, we utilized ileal explant cultures to perform ex vivo analyses using chenodeoxycholic acid (CDCA) and cholic acid (CA), BAs found in the ileum during the development of experimental NEC. Objective: To examine the effects of BAs on Muc2 and Trefoil Factor 3 (TFF3) in neonatal ileal explant cultures. Methods: Ileal tissue from 3-day old Sprague-Dawley rats was removed and cut into 0.5 cm sections. Each section was opened longitudinally on sterile Metricel Membrane Filters (Pall Corporation, Ann Arbor MI). The tissue was cultured in DMEM/F12 media alone or with 0.25 or 1.0 mM CDCA or CA. Tissue portions were then fixed in 70% ethanol, paraffin embedded and serial sectioned for immunohistologic evaluation of Muc2 and TFF3 positive cells using anti-Muc2 (Santa Cruz Biotechnology) and anti-TFF3 (provided by Dr. Daniel K Podolsky). Positive cells were counted from at least 10 villi and data are expressed as mean positive cells per 100 intestinal epithelial cells. Additional tissue portions from each animal were snap frozen and RNA was extracted. Real-Time PCR analyses were performed to determine expression of Muc 2. Results: Muc 2 and TFF3 positive cells were significantly decreased in neonatal ileal explants cultured with CDCA or CA for 2 hours in a dose-dependent manner. Decreased Muc2 and TFF3 positive cells were observed after as little as 15 minutes of exposure to BAs. Muc2 mRNA levels were not changed after ileal culture with BAs. Conclusions: BAs found in the ileum during the development of experimental NEC decrease goblet cell products Muc2 and TFF3 in neonatal ileal tissue. These decreases are likely a result of increased secretion rather than decreased production. Supported by the Arizona Biomedical Research Committee.

T1762 Diagnosis and Grading of Intestinal Acute Graft-Versus-Host Disease Following Allogeneic Stem Cell Transplantation By Colonoscopy Wolfgang Kreisel, Mirjam Dahlberg, Jan Harder, Annette Schmitt-Gräff, Juergen Finke Objective: About 40% of patients develop acute graft-versus-host disease (aGvHD) following allogeneic stem cell transplantation. It mainly involves skin, liver, and gut. Diagnosis and grading of intestinal aGvHD are based on clinical symptoms and are verified by histology. Since acute GvHD grade ≥2 requires intensive immunosuppression, a rapid diagnosis is crucial. At time of therapeutic decision making, however, histologic results often are pending. Previously we have described the macroscopical lesions of aGvHD of the lower gastrointestinal tract and a score from grades 1 to 4 (Kreisel W et al., Eur J Gastroenterol Hepatol 1994; 6: 723-729). Histological criteria for intestinal aGvHD have been described earlier (Sale GE et al. Am J Surg Pathol 1979; 3: 291-299. Epstein RJ et al. Gastroenterology 1980; 78: 764771. McDonald GB et al. Gastroenterology 1986; 90: 770-784). This study aimed to evaluate the macroscopical grading of aGvHD ≥2 in comparison with the histological grading ≥2, which is regarded as the gold standard for initiation of therapy. Patients and methods: We retrospectively evaluated 602 patients who underwent allogeneic stem cell transplantation at our haematological department from 08/2001 to 02/2008. In 153 patients a colonoscopy was performed due to suspected aGvHD. In 148 colonoscopies, macroscopical and histological evaluation could be evaluated. The median age of patients was 52.3 years (18.8-72.7) Main underlying diseases were: AML (n=63), ALL (n=13), MDS (n=21), NHL (n=19), MM (n=16). Fludarabine, BCNU, melphalane, and cyclophosphamide were used for conditioning. GvHD prophylaxis consisted of cyclosporine A, MMF, and MTX. In case of unrelated donor grafts anti-T-cell globulin was additionally administered Results: In no patient colonoscopy induced a complication such as major bleeding, perforation, or infections. The sensitivity of macroscopical diagnosis of aGvHD ≥2 in comparison with histology was 84.5% (49/58); the specificity was 82.2% (74/90). The positive predictive value was 75.4% (49/65); the negative predictive value was 89.2% (74/83). For diagnosis of aGvHD ≥3 the figures were: sensitivity 89.1% (41/46), specificity 84.3% (86/102), positive predictive value 71.9% (41/ 57), negative predictive value 94.5% (86/91). Conclusions: This by far largest study for evaluation of colonoscopy in acute graft-versus-host disease following allogeneic stem cell transplantation shows: (1) Colonoscopy can be safely performed even in patients with highgrade aGvHD. (2) Macroscopic grading, which can be obtained immediately, is a reliable method for objective and rapid diagnosis and grading of aGvHD ≥ grade 2.

T1760 Lower Electrical Potential Difference in the Rectum Is Easy and Good Measurement Marker to Assess Early Stage of Bacterial Translocation Nobuhiro Kurita, Mitsuo Shimada, Takashi Iwata, Masanori Nishioka, Kozo Yoshikawa, Motoya Chikakiyo Background: Early detection of bacterial translocation (BT) is important to prevent progress of severe infectious diseases. We reported preventive effects and mechanisms of Kampo medicine “Dai-kenchu-to” (DKT) for BT caused after 6 days fast (Yoshikawa K et al, Dig Did Sci 2008). The increased expressions of IFN-γ, TNF-α, IL-6 after fast were significantly decreased by DKT. The decreased numbers and heights of jejunal villi after fast were maintained by dose dependence of DKT. DKT also inhibited apotosis in the intestinal mucosa. A new clinically applicable monitoring system for BT is necessary. The aim of this study is to develop new methods to detect early stage of BT that used electrophysiological changes in intestinal mucosa. Methods: Wistar rats were divided three different groups: Control (n= 5), Fast (n=6) and Limited Diet (LD) groups (n=5). The Fast group received a dosage of 0.1ml/g with maintenance liquid containing 10% sugar under the skin for five days without oral ingestion. The LD group was provided with 0.04kcal/g powder meal and 0.1ml/g water orally for five days. The onset of BT was confirmed by being a 16S ribosomal DNA sequence of Bacteroides vulgates estimated by polymerase chain reaction (PCR) and bacterial culture in the mesenteric lymph nodes. Electrophysiological changes in the rectum were measured using a short circuit current amplifier (SCCA) and an Ussing Chamber. Epithelial sodium channels (ENaC) were quantified by real-time PCR. Results: Confirmation of BT: The 16S ribosomal DNA sequences of B. vulgatus appeared in the Fast group and no such sequences were found in the Control group. The incidence of positive culture in the mesenteric lymph nodes was 66% in the Fast group. Electrical changes measured using SCCA: The electrical potential difference (PDR) in the rectum of the Fast and LD group was significantly lower than that of the Control group. Electrical changes measured using Ussing chamber: The electrical resistance (TER) of the fast group was significantly lower than that of the Control group. After administration of amirolide, (an epithelial sodium channel blocker), the percentage of difference in electrical potential (ΔPDR) of the fast group was significantly higher than that of the control group. Epithelial sodium channels (ENaC): The α, β, and γ subunits of ENaCs of the Fast group significantly increased compared with those of the Control group. Conclusions: The electrical potential and resistance, and quantity of ENaC in the rectum reflected a failure of mucosal barrier function, which indicated a new method to assess bacterial translocation.

T1763 Targeted Deletion of Intestinal Epithelial Cxcr4 Modulates Epithelial Barrier Repair Through Altered ERK1/2 Signaling Noah P. Zimmerman, Rebecca A. Vongsa, Priscilla A. Johanesen, Michael K. Wendt, Nita H. Salzman, Michael B. Dwinell Background and Aims: The chemokine receptor CXCR4 and its cognate ligand CXCL12 are constitutively expressed by the gastrointestinal epithelium. In culture, the CXCR4-CXCL12 signaling axis regulates intestinal epithelial migration, barrier maturation and restitution, consistent with an In Vivo role for this axis in the maintenance of mucosal barrier integrity. To better understand the role of CXCR4 at the gastrointestinal epithelial surface, conditional knockout mice in which CXCR4 expression was ablated specifically in intestinal epithelial cells were established. Methods: Epithelial specific CXCR4 knockout mice were generated using the Cre/lox system. Animal weight, development and intestinal morphology were monitored using histological analysis, RT-PCR, immunostaining and immunoblot. Administration of dextran sodium sulfate (DSS) in drinking water was used to induce colitis. In Vitro analysis was conducted using lentiviral vectors to deplete IEC-6 cells of CXCR4. ERK1/ 2 activity was assessed using phosphospecific antibodies. Results: RT-PCR, immunoblot, and immunofluorescence microscopy confirmed that CXCR4 was specifically deleted in intestinal epithelial cells. Conditional CXCR4 knockout mice developed normally and were phenotypically and morphologically comparable to control animals. Chronic colitis induced by three bouts of 3% (w/v) DSS resulted in more consistent GALT formation and reepithelialization in knockout mice compared to controls. Heterozygous animals demonstrated greater healing of ulcers than control or homozygous CXCR4 deleted animals. Consistent with those data, knockdown of CXCR4 in cultured IEC-6 cells evoked more epithelial cell migration relative to controls. Notably, ERK1/2 phosphorylation was markedly decreased in CXCR4 depleted cells. Moreover, the site of ERK1/2 phosphorylation was reversed from surface colonocytes to crypt cells in CXCR4 deficient mice, suggesting an important role

T1761 Roles of Prostaglandins, Nitric Oxide and Enterobacteria in Pathogenesis of Ischemic Enteritis in Rats Yoshino Komatsu, Kikuko Amagase, Yuka Nakamori, Tohru Kotani, Koji Takeuchi Background/Aim: We recently established an animal model of ischemic enteritis that has a high clinical predictability, yet the pathogenic mechanism remains fully unexplored. In the present study, we investigated the roles of prostaglandins (PGs), nitric oxide (NO) and enterobacteria in the development of ischemic enteritis in rats. In addition, we also examined the mechanism by which PGs prevent the development of ischemic enteritis, including EP receptor subtypes. Methods: Male SD rats were used after 18 h fasting. Ischemic enteritis was induced by partial ligation of the superior mesenteric artery (SMA). Under ether anesthesia, SMA was isolated, and a stenosis was produced by placing a needle (23 guage) on the vessel and then ligature of both the vessel and needle, and then a needle was removed

AGA Abstracts

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for ERK signaling in barrier formation and ulcer healing. Conclusions: These data indicate that while exogenous CXCL12 is a potent motogen, sustained CXCR4 signaling is a key regulator of basal ERK1/2 activity. Conditional deletion of CXCR4 in epithelial cells leads to the marked spatial reversal in ERK1/2 activity within the crypt/surface axis and likely modification in barrier integrity through its impact on proliferation, migration and anoikis at sites of epithelial ulcers.

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BACKGROUND: Cystic fibrosis (CF) is caused by mutations in the CFTR gene that impair function of this cAMP-regulated Cl- channel. In the small intestine loss of CFTR function creates a dehydrated, acidic luminal environment which is believed to cause an accumulation of mucus, a phenotype characteristic of CF. We investigated whether lubiprostone, which activates the Clc2 Cl- channel, would improve the CF intestinal phenotype. METHODS: Cftrtm1UNC (CF) and wildtype (WT) littermate mice on the C57BL/6 background were used. Lubiprostone (10μg/kg-day) was administered by gavage for two weeks. Mucus accumulation was estimated from crypt lumen widths in Carnoy's-fixed, PAS/AB stained sections. Luminal bacterial load was measured by qPCR for the bacterial 16S gene. Gastric emptying and small intestinal transit were assessed by gavage of rhodamine-dextran. Analysis of intestinal transit was performed on the geometric center of the fluorescence (GCF)=Σ(fraction per segment x segment number). Gene expression was evaluated by Affymetrix Mouse430 2.0 microarray. RESULTS: Mucus accumulation in control CF mice, as measured by crypt lumen width, was 700% that of WT mice (P<0.001). Lubiprostone did not affect WT crypt width but, unexpectedly, increased CF crypt width 22% (P=0.001). Lubiprostone did not significantly affect bacterial load in WT mice, but decreased overgrowth in CF mice by 60% (P=0.005). Lubiprostone significantly increased gastric emptying in both WT (P<0.001; control=57±3%, treated=81±4%) and CF mice (P<0.001; control=48±4%, treated=75±5%). After lubiprostone small intestinal transit was significantly enhanced in WT mice (P=0.024; GCF in control= 4.18±0.22, treated=4.94±0.19) but the effect was not significant in CF mice (P=0.377; GCF in control=1.98±0.17, treated=2.28±0.21). Mast cell markers were elevated 30-40 fold in the control CF intestine and lubiprostone decreased their expression about 10-fold. CONCLUSIONS: These results indicate that lubiprostone has some benefits for the CF intestinal phenotype, especially on bacterial overgrowth and the innate immune response. The unexpected observation of increased mucus accumulation in the crypts of lubiprostone-treated CF mice suggests the possibility that lubiprostone increases mucus secretion. Increased mucus secretion caused by lubiprostone may be beneficial to other gastrointestinal conditions but could be counterproductive in CF where mucus is already excessive in the intestinal lumen. This study was supported by Takeda Pharmaceuticals and the Cystic Fibrosis Foundation.

T1764 A Protective Role of HSPS Against Various Gastrointestinal Diseases and Application of HSP-Inducing Drugs for These Disease Shintaro Suemasu, Tomoaki Ishihara, Teita Asano, Ken-ichiro Tanaka, Tohru Mizushima Background & Aims: It has been known for a long time that heat shock proteins (HSPs) are induced by various stressors in order to confer protection against such stressors. Furthermore, anti-inflammatory activity of HSPs, such as inhibitory effect of NF-kB was recently reported. A number of indirect lines of evidence suggested that HSPs protect gastric mucosa from irritant-induced lesions. For example, geranylgeranylacetone (GGA), a clinically used anti-ulcer drug was shown to have HSP-inducing activity. However, whether this activity of GGA is the main mechanism of its anti-ulcer activity has not been proved. Furthermore, the role of HSPs in other gastrointestinal diseases, such as small intestinal lesions and inflammatory bowel disease (IBD) was unclear. In this presentation, we overview our recent work on the role of HSPs in various gastrointestinal diseases by use of transgenic mice. Methods: Expression of proteins was monitored by real-time RT-PCR and immunohistochemical analyses. Results: Null mice lacking HSF1, a transcription factor for hsp genes or transgenic mice expressing HSP70 showed sensitive or resistant, respectively, phenotype to irritant-induced gastric and small intestinal lesions and mucosal apoptosis. HSF-1-null mice or transgenic mice expressing HSP70 also showed sensitive or resistant phenotype to DSSinduced colitis, an animal model of IBD. Increased expression of inflammatory cytokines and cell adhesion molecules associated with small intestinal lesions and colitis was stimulated or suppressed in HSF-1-null mice or transgenic mice expressing HSP70, respectively. In Vitro, expression of HSP70 in cultured cells caused resistant phenotype to irritants and suppression of expression of inflammatory cytokines and cell adhesion molecules. Orally administered GGA induced HSP70 at gastric, small intestinal and colonic mucosa in an HSF1-dependent manner. GGA suppressed gastric lesions in wild-type mice but not in HSF1-null mice. Furthermore, we found that GGA ameliorated the small intestinal lesions and colitis. Conclusions: Up-regulation of expression of HSP70 by genetic modification and GGA seems to prevent irritant-induced small intestinal lesions and DSS-induced colitis through both direct protection of intestinal mucosa and its anti-inflammatory activity. The HSP-inducing activity of GGA seems to mainly contribute to the drug's ameliorative effect against irritant-induced gastric lesions. Based on results in this study, we propose that nontoxic HSP70-inducers, such as GGA, would be therapeutically beneficial against irritantinduced small intestinal lesions and IBD.

T1767 Examination of the Depressed Gut Reveals Changes in Microbiota and Serotonin Levels Amber J. Park, Patricia Blennerhassett, Emmanuel Denou, Premysl Bercik, Stephen M. Collins Introduction: Infectious gastroenteritis is a recognized risk factor for subsequent development of Irritable Bowel Syndrome (post-infectious (PI)-IBS). Recent tragedies such as that which occurred in Walkerton, Ontario and others worldwide have highlighted some important risk factors including co-morbid anxiety and depression. However, the exact influence of pre-existing psychological perturbations on the basal state of the gastrointestinal tract is unclear. Further characterization of this relationship is needed to better understand the response of the depressed gut to infection, and the increased risk seen in these patients. Methods: Olfactory bulbectomy (OBx) is a well-characterized rodent model of co-morbid anxiety and depression. OBx was preformed on 8-10 week old male C57Bl/6 mice by making two burr holes in the skull overlying the olfactory bulbs. The bulbs are removed by suction, and the wound is treated with antibiotic and closed by suture. Sham animals were treated in the same manner with the bulbs left intact. At weeks 4 and 10 behavioral tests were completed including tail-suspension test (TST), step-down latency, and open-field activity. At week 10 the animals were anesthetized and euthanized by cervical dislocation. Brains were removed and OBx was confirmed using histological methods. Fecal samples were collected for DNA extraction and PCR-DGGE analysis of the microflora. Additionally, distal colon tissue was collected and levels of serotonin were measured by ELISA. Results: At week 4 OBx mice showed a significant increase in immobility duration as measured by TST. At weeks 4 and 10 OBx mice showed a significant decrease in latency to step-down. In the open-field apparatus, at weeks 4 and 10, OBx mice showed significant hyperactivity as measured by total distance traveled, as well as a decrease in center zone activity including rearing, distance traveled, and number of entries. Upon examination, the distal colon tissue of these mice showed a significant decrease in serotonin levels relative to sham operated controls. Additionally, PCR-DGGE analysis showed quantitative changes in the proportion of the phylum from the large bowel microbiota of OBx mice vs. Sham. Specifically, there was an increase of the Clostridia and Actinobacteria, and a decrease in Bacteroides. Summary: Pre-existing psychological perturbation appears to alter certain characteristic of the gut including serotonin contents and the proportion of specific phylum of bacteria. It remains to be determined whether or not these factors play a role in the response of the gastrointestinal tract to infectious bacteria, and the risk of subsequent development of PI-IBS.

T1765 Interluekin 11 Ameliorates Radiation Colitis By Enhanced Bone Marrow Contribution to Repair of Colonic Tissue in Rats Kazuko Shichijo, Makoto Ihara, Shiro Miura, Mutsumi Matsuu, Masahiro Nakashima, Toshiyuki Nakayama, Ichiro Sekine BACKGROUND: While radiation therapy is considered useful in the treatment of gynecological malignant tumors such as uterine cervical cancer, it may cause radiation enterocolitis which is characterized by prolonged clinical course and also by a latent period after irradiation. Interleukin-11 (IL-11) is a stromal cell-derived cytokine with several biological activities against hematopoietic cells. We have already shown that IL-11 suppresses radiation colitis in rats and bone marrow contributes seriously to regeneration of colonic epithelia after radiation damage. This study aimed to explore the ameliorating mechanism of IL-11 in radiation colitis on bone marrow contribution to repair of colonic tissue in rats. METHODS: 1) Female rats were transplanted with male Green Fluorescent Protein (GFP) transgenic bone marrow (BMT) after sublethal irradiation. 2) Rats received 200ug/kg/day IL-11 sc. or vehiclE. colitis was induced by a focal irradiation of 22.5 Gy X ray 5 weeks after the BMT. The ulceration ranged. Y-chromosome and 12-chrompsome signals in the colonic epithelium were detected by FISH (Y Cy3 / 12 FITC). FISH Index (signal number ratio of Y-chromosome and 12-chromosome in the 60x60um2 mucosa) was estimated 56 days after irradiation. 3) GFP protein expression in the colonic tissue was detected by western blotting methods. 4) Intestinal epithelial cell monolayer cultures (IEC18) treated with IL-11(100 ng/ml) were examined by a focal irradiation of 22.5 Gy X ray and co-cultured with GFP bone marrow cells. RESULTS: 1) IL-11 has a beneficial effect on mucosal parameters. The IL-11 reduced colonic ulcer index and accelerated regenerative surface epithelium expression 2) Transplanted female rats showed localization of Y-chromosome-containing cells and GFP positive cells in the lamina propria and the regenerative epithelium. FISH indices in regenerative epithelium from the transplanted female rats were 0.317+0.026 (59.9% vs. male) in the lower mucosa and 0.471+0.051(94.2% vs. male) in the upper mucosa. 3) By using western blotting method GFP expression in the damaged colon of IL-11 treated rats were higher than that of control. 4) In the IEC18 cells, IL-11 treatment enhanced transition of bone marrow-derived cells to colonic epithelia after radiation. Conclusion: Bone marrow-derived circulating stem cells of IL-11 treated rats much frequently engraft into the ulcerated area and differentiate into colonic epithelial cells. These data indicated that IL-11 enhanced bone marrow cells contribution to regeneration of colonic epithelia after radiation damage, and it is suggested that this could be exploited therapeutically

T1768 Generation of a Tightly Regulated Doxycycline-Inducible Model for Studying Mouse Intestinal Biology Sabrina Roth, Patrick Franken, Wendy van Veelen, Lau Blonden, Lalini Raghoebir, Ellen van Drunen, H. B. Beverloo, Ernst J. Kuipers, Robbert Rottier, Riccardo Fodde, Ron Smits Background: Mouse models are frequently being used to address basic research questions about intestinal development, tumor formation, and inflammatory bowel disease. Inducible transgenic mouse models represent unique experimental tools to study the expression of specific genes in a tightly regulated fashion. Ideally, such an inducible mouse model should drive homogeneous transgene expression in the tissue of interest exclusively upon induction and in a dosage-dependent manner. Furthermore, induction of gene expression should be reversible. Such a model has not been available for the intestinal tract. Therefore, we set out to generate a mouse model allowing robust induction of intestine-specific gene expression

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AGA Abstracts

AGA Abstracts

Effects of Lubiprostone On the Cystic Fibrosis Mouse Small Intestine Phenotype Robert C. De Lisle, Lauren Meldi, Eileen Roach