AGA abstracts 612-S988

Association Between Prior Diagnosis of an ABA or NSAID-Requldng Condition and Esophageal Cancer Condition OR 95% C,L Acute Myocardial infarction 0.71 0.64-0.79 Ischemlc Heart Disease 0~72 0.66-0.79 Rheumatoid arthritis 0.77 0.63-0.92 Osteoarthdtis 0.88 0.63-0.94 Any ASA or NSAID-Requiring Condition 0,74 0.70-0.77 Any ABA-requiring Condition 0.75 0.72-0~79 Any NSAID-requidng Condition 0.86 0.82-031 OR=odds ratio C.l.=confldence Interval

Placebo Exisulind20Omg Exisulind400mg p-value Median Polyp Size (mrn=) n = 43 n = 38 n = 33 Baseline 16 20 20 Final 12 12 12 Median Change from Baseline' -4 -4 -10 0.03(P % Change from Baseline 25% 20% 50% _ T~ic Response¢ n (%) n(%) n (%) Responders 13 (30.2) 13 (34,2) 18 (54.6) 0.038b.d *Complete 8 (18.6) 4 (10,5) 9 (27.3) =Partial 5 (11.6) 9 (23.7) 9 (27,3) Stable Disease 18 (41.9) 17 (44.7) 12 (39.4) Progressive Disease 12 (27.9) 8 (21,1) 2 (6.1) 0.017 = Median of the differences between baseline and final polyp size for individual patients. b4O0mg v$ placebo - Wilcoxon Rank-BumTest. =CR: complete resolution PR: ;~50% reduction in polyp size SD: < 50% reduction and <25% increase in polyp size PD: ~_.25% increase in polyp size dComparisonbased on number of patients with PR or CR,

614 Neoplasia-Associated Expression of MUC5AC Mucin Is Reduced in Adenomas by Treatment with Calcium and Vitamin D Robert S. Bresalier, Peter R. Holt, Kai-Feng Liu, Detroit, MI; New York, NY

612

A large body of observational and laboratory data suggests an inverse relationship between calcium and vitamin D intake and carcinogenesis in the colon, and a recent prospective randomized trial demonstrated a reduction in adenoma recurrence in patients taking ~lcium supplementation vs placebo. The MUC5AC gene is normally expressed in human gastric epithelium, but not in the normal colon. We and others have demonstrated aberrant expression of MUC5AC mucin in colorectal adenomas and cancers. Aim: To compare the effects of oral calcium carbonate (1,500 mg TID to deliver 1,800 mg Ca2+ daily) in combination with vitamin D3 (400 IU QD) versus oral placebo administered over 6 months on polyp mucosal histopathology including expression of the MUC5AC gene product. Methods: Double blind, randomized, placebo-controlled trial with 6 month intervention in average-risk adenomabearing patients. Adenomas -< 9 mm in size within reach of the flexible sigmoidoscopy (60 cm) were biopsied to remove half of the polyp, and the remainder left in situ and marked by india ink tattoo. Residual polyps were removed at a 6 month exit exam. Biopsies of normal rectal mucosa were obtained at baseline and 6 months. MUC5AC expression was assessed immunohistochemically with MAb 45M1.20 patients were randomized and 19 received both exams (11 treatment, 8 control). Mean polyp size at baseline was 7.2+/-1.6 mm, with no difference between groups. Results: Normal rectal mucosa rarely expressed MUC5AC mucin (<1% of glands) in 5/19 (20%) patients at baseline, while there was strong expression in 17/19 (90%) adenomas (28-36% of glands) (p<0.O5). After treatment with calcium and vitamin D there was no change in normal mucosa, but 9/11 (82%) adenomas exhibited reduced expression of MUC5ACcompared to baselinewith total loss of expression in 5 cases. In contrast 2/8 (25%) exhibited reduced expression in the control group (2/3 had increased expression) (p<0.05 treatment vs control). The percent of MUC5ACpositive glands in adenomas was also significantly reduced in the treatment group (9.0+/-3.0 exit vs 21.8+/-9.4 baselinemean +/- SEM) but not in controls (36.5 +/-8.0 exit vs 28.5 +/-7 baseline) (p = O.O3). There was no change in another neoplasia-related protein (galectin-3). Conclusion: Calcium and vitamin D treatment in adenoma-bearing patients results in a phenotype more closely approximating normal with regard to expression of the MUC5AC mucin gene product.

Chemoprevention of Esophageal Squamous Cancer: Randomized, PlaceboControlled Trial in a High-Risk Population Paul Limburg, Wenqiang Wei, Dennis Ahnen, Youlin Oiao, Ernest Hawk, Guoqing Wang, Carol Giffen, Mark Roth, Edward Korn, Zhiwei Dong, Philip Taylor, Sanford Dawsey, Rochester, MN; Beijing, China; Denver, CO; Rockville, MD

Background: Esophageal squamous cancer (ESC) is a leading cause of malignant death in Linxian, China. Previous data from our group support the use of endoscopic therapy to prevent ESCamong patients with histologically severedysplasia. However,appropriate ESC prevention strategies for patients with moderate dysplasia (MD) and mild dysplasia (mD) remain undefined. Aim: To assess celecoxib and selenomethionine as potential ESC chemoprevention agents among Linxian residents with MD or roD. Methods: EGD screening of 2,213 asymptomatic adults, aged 34-68 years, was conducted in 4/99 (tO). Consenting subjects (n=360) were enrolled based on tO histology. Pre-trsatment EGDs were repeated in 10/99 (t6) to confirm eligibility and document baseline lesions. Intervention groups were then randomly assigned using a 2x2 factorial study design, stratified by gender: celecoxib 200 mg bid, selenomethionine 200 p,g qd, both agents, or placebo. After a lO-month intervention (12/9910/00), post-treatment EGDswere performed (t18). The primary endpoint was defined a priori as change in histologic severity of esophagealsquamous dysplasia at t18 versus t6. Results: Subject withdrawals included 11 (3%) with adverse events, 75 (21%) with MD at t6, and 36 (10%) with no EGD at t18. Within the final analytic cohort (n =238), celecoxib was not associated with statistically significant changes in dysplasia severity (see Table). Selenomethionine had no apparent effect among subjects with MD at t6, but a statistically significant (p=O.02) improvement was noted among subjects with mD at t6. Conclusions: In this lO-month, randomized, placebo-controlled trial in Linxian, china, selenomethionine 200 I~g qd was associated with a statistically significant improvement in esophagealsquamous dysptasia among subjects with mD at baseline. Celecoxib 200 mg bid did not appear to alter dysplasia severity. Basedon these data, further investigation of selenomethionineas a potential ESC chemoprevention agent appears warranted. Off-treatment follow-up of this cohort is ongoing.

615 Incidence of Necrotizing Enterocolitis in Preterm Infants Is Feeding Volume Dependent while Maturation of Small Intestinal Motor Patterns Is Not Carol L. Berseth, Houston, TX

Change In Severity of Esophageal Dysplasla, by InterventionAgent Celecoxlb Sdenornsthlonine Regreasion:Stable:Progression* Regraselon :Stsble:Progression* Dyspleaia Grade at t6 Active Placebo P Value** Active Placebo P Value" AgSubjects(n=238) 43:59:19 47:50:20 0.78 53:53:17 37:56:22 0.08 MD only (n=123) 28:32:4 27:28:4 1.00 30:28:6 25:32:2 1.00 mD only (n=t15) 15:27:15 20:22:16 0.71 23:25:11 12:24:20 0.02 *based on each subject's worst histologlc diagnosis at t18 vs. t6 EGD; **two-sided, two sample permutationt.test

Background. When compared to preterm infants given no feedings, infants given small enteral feedings have more mature small intestinal motor patterns and reach full enteral feedings sooner. Previous studies have shown that preterm infants given larger enteral feedings reach full enteral feedings soonerthan infants given smaller feedings; however, larger enteralfeedings may precipitate necrotizing enterocolitis (NEC). The purpose of this study was to compare the maturation of small intestinal motor patterns, feeding outcomes, and the incidence of NEC in preterm infants fed small (Minimal) volumes or larger (Advancing) volumes. Methods. 141 preterm infants less than 32 wk gestation were fed the first 10 feeding days using Minimal or Advancing volumes. Advancing-fed infants were fed 20 ml/kg/d the first feeding day; volumes were increased by an additional 20 ml/kg/d until 160 ml/kg/d (i.e. full feeding volume) was reached and maintained. Minimal-fed infants were fed 20 ml/kg/d for 10 d. All were fed using 4 hr cyles consisting of 2 hr of feeding infusion followed by 2 hr of no feeding. On the 11th feeding day, blood was drawn after an over-night fast for determination of plasma concentrations of gastrin and motilin, and small intestinal manometry was performed. Feeding volumes for Minimal-fed infants were then increased daily by increments of 20 ml/kg/d until 160 ml/kg/d was reachedand maintained. Feedingcharacteristics were tracked prospectively. Results. Although plasma gastrin and motilin concentrations were higher in Advancing-fed infants than Minimal-fed infants, characteristics of motor patterns were similar in the two groups, as shown in the table below (mean_+SD). Advancing-fed infants reached full enteral feedings sooner, but the incidence of NEC was significantly higher in these infants compared to Minimal-fed infants. The time required to establish oral feedings and to be discharged home was similar in the two groups, as was the incidence of late sepsis and Cholestatic jaundice. Conclusion. Since the incidence of NEC is feeding volume dependent and maturation of motor patterns is not, it is not necessary to increase feeding volumes during the first 10 feeding days.

613 Aspirin and NSAID-Requiring Conditions and the Risk of Esophageal Carcinoma David Kearney, Casey Crump, Charles Maynard, Seattle, WA

BACKGROUND: COX-2 expression is increased in Barrett's esophagus and esophageal CA. Prior studies suggest that aspirin (ASA)and NSAID use decreases the risk of esophageal CA. AIM: To determine if conditions commonly prescribed ASA or NSAIDs are associated with a reduced risk of esophagealCA. METHODS:A case-control study. Incident cases of esophageal CA (years 1995-1999, ICD9 codes 150.x, 151.0, N=10,821) were identified using VHA administrative databasesthat record inpatient and outpatient diagnosis codes. Controls were persons without a diagnosis of esophageal CA who were frequency matched for age (10 controls/case, N = 97,474). The occurrence of conditions that commonly require ASA or NSAID use were recorded from 1986 onward and were compared for cases and controls. ASArequiring conditions were defined as: coronary artery disease (ICD9 codes 410, 411,413, 414), peripheral vascular disease (ICD9 code 443), arterial embolism and thrombosis (ICD9 code 444). NSAID-requiring conditions were defined as: Rheumatoid disease (ICD9 code 714), osteoarthritis (ICD9 code 715), arfhropathies (ICD9 code 716), ankylosing spondylitis (ICD9 code 720), spondytosis (ICD9 code 721), disorders of the back (ICD9 code 724). Logistic regression (with adjustment for age, sex, race & duration of care in the VHA) was performed to assess the association between ASA or NSAID-requiring conditions and esophageal CA. RESULTS: 98% of subjects were male. Casesand controls had similar demographic features. A diagnosis of any ASA or NSAID-requiring condition was associated with a significant reduction in the risk of esophageal cancer (OR 0.74, 95% C.I. 0.70-0.77), with a similar reduction in risk when limited to diagnoses of adenocarcinoma (OR 0.73 95% C.I. 0.64-0.84). CONCLUSION:Clinical conditions that are commonly prescribed ASA or NSAIDs are associated with a reduced risk of esophageal cancer. These findings support a role of ASA or NSAIDs as chemopreventive agents for esophageal cancer.

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Outcome N Wk gestationalage Gastdn, pg/ml Motilin, pglml MMC'e(%) Maturefed pattem (%) NED(%) Day full enteralfeeds Day full oral feeds

Minimal 71 28--2 1354-66 244--177 58 77 1.4 39_+18 72+31

Advancing 70 28+2 2264-136 358±170 49 66 10 25_+13 62_+29

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P Value

Improved Outcome of Multivisceral Transplantation in Children - Single Center experience of 26 cases Naveen Mittal, Timothy Kato, John Thompson, Barbara Miller, Phillip Ruiz, Patricia Cantwell, Seigo Nishida, Andreas Tzakis, Miami, FL

NS <_001 -<.01 NS NS .027 -<,001 NS

Aim: To analyzeoutcome of multivisceral transplant (MVTx)with stomach, pancreas, liver and small intestine in children at our center. Methods: Retrospective analysis of pediatric MVTx cases was performed at the University of Miami between April 1996 and Nov 2001. The results were compared in three separate periods: Period I ( 04/96-12/97, N =8), Period II ( 1/98-12/80, N=5)and Period III ( 1/01-11/01, N=13). Results: A total of 60 MVlx were performed at the University of Miami of which 26 were in children ( Mean Age-4 years; Median Age-1 year).2 cases had MVTx without liver and another 6 cases of MVTx had kidney(s) included. Causes of intestinal failure included megacystis microcolon syndrome/inteatinal pseudo-obstruction(N=10), necrotizing enterocolitis(N=4), gastroschisis(N=4), intestinal atresia(N= 3), others (N = 5).lmmunosuppression was based on tacrolimus with addition of Daclizumab in Period II and II1.3 patients in Period Itl had induction with Campath. Graft biopsy was performed in Period I on clinical suspicion but survillience graff biopsy were performed in Period II and III. Zoom endoscopy was available in Period II and III.Acturial patient survival at 6months/1-year/3-year are 62%/62%/37%, 80%/60%/40%, 92%/NNNA in Period I, II and III respectively. Currently 5 patients have survived more than 3 years. All survivors for 6 months are off TPN. They tolerate regular diet with normal gastric emptying and have no clinical signs of pancreatic exocrine or endocrine insufficiency. Including autopsy findings, no patient exhibited graft rejection limited to the stomach or pancreas. Biliary tract complication has not occured. 2 patients developed stricture at esophago-gastrotomy and needed dilatation. Conclusion: MVTx offers excellant functional outcome with reasonable chance of survival in children with liver and intestine failure. Additional morbidity due to inclusion of pancreas and stomach has not been observed in our series. Early post-operative survival has improved in our recent cases.

616 Efficacy of Montelukast in the Treatment of Children with Eosinophilic GaslroJnlestioal Disease Jon A. Vanderhoof, Rosemary J. Young, Omaha, NE Purpose: Eosinophi~ic inflammation is being seen with increasing frequency in children with a variety of gastrointestinal symptoms. Esophageal infiltrates commonly cause dysphagia, small bowel infiltrates result in diarrhea and abdominal pain, and distal colonic infiltrates are occasionally seen with constipation. The cause is often thought to be allergic, but dietary restriction is often difficult or only partially helpful. Medical therapy with cromolyn is often disappointing, and steroids, while effective, frequently result in unacceptableuntoward effects. Because of the demonstrated efficacy of Montelukast in allergic pulmonary diseases with known eosinophilic inflammation, we retrospectively assessed our experience with this agent in our pediatric patients with eosinophilic inflammatory disorders. Methods: We retrospectively evaluated the records of 8 children (age range 2-17 years) with documented eosinophilic inflammatory lesions in their gastrointestinal tracts. All had failed dietary restrictions or oral cromolyn therapy. Some had benefited from corticosteriods but were concerned about persistent adverseeffects. Four patients had primary esophagealdiseaseand presentedwith dysphagia, two had small bowel disease presenting with diarrhea, and 2 had colonic involvement and presented primarily with constipation. Children under age 5 received montelukast, 5 mg/ day, once daily in the morning, with children over age 5 receiving 10 rag/day. Results: All patients demonstrated marked improvement in symptoms within 2-3 weeks of initiation of therapy. All have remainedasymptomatic (mean follow-up of 1 year) on continued montelukast therapy. No attempts to wean patients from treatment have been made. Conclusion: Montelukast may be useful in the treatment of certain children with eosinophilic inflammatory lesions in the gastrointestinal tract. It appears to be effective in disorders effecting different levels of the gastrointestinal tract. Further double blind, placebo controlled studies are needed to assess the role of this agent in eosinophilic gastrointestinal disorders.

619 The Sucrose Breath Test (SBT): A Novel Test for Assessment of Small Bowel Mucosal Function (SBMF) and Damage Ross But~er, Nicole Pelton, Betty Zacharakis, Cuong D. Tran, David Tivey, Geoffrey Davidson, Adelaide, South Australia, Australia BACKGROUND:Measurementof small intestinal mucosal damageas a diagnostic or prognostic marker often requires an invasive procedure. A non-invasive test which could determine the severity of gut damage would be useful in gut conditions e.g. inflammatory bowel disease, coeliac disease, gastroenteritis and in chemotherapy-induced mucositis. Sucrase is a brush border enzyme that maintains similar activity once the small intestinal (SI) mucosa has matured. This study evaluated the potential for naturally enriched sucrose as a probe to determine SBMF. METHODS: Studies were carried out in n= 10 young adult subjects. A close response study with the sucrase-isomaltase inhibitor acarbose was performed. Acarbose was administered at the same time as the sucrose probe 13C02 was measured at 15 rain intervals for 240 rains using an isotope ratio mass spectrometer. Preliminary studies determined that a 20g dose of sucrose was sufficient to provide an adequate signal sufficient for assessment of sucrase activity. Two patients with known sucrase-isomaltase deficiency (SIM) were also tested. Traditional assessment of sucrose malabsorption using breath H2 was performed simultaneously. RESULTS: 1. With increasing acarbose administration (0,25, 50 100 and 200rag) the peak height of breath 13C02 dropped significantly (p<0.01) in a dose response fashion to 70%, 50%, 25% and 10% respectively compared to control subjects with no acarbose. 2. For the two patients with SIM no signal was seen in the first 90 rains. Thereafter both H2 and 13C02 were detected showing malabsorption and colonic metabolism of the sucrose. CONCLUSION:The SBT is an accurate non-invasive marker of changes to the sucrase activity in the small intestine of healthy humans. It also provides an easy to assess integrated measure of the total activity of this brush border enzyme. In the future this test may also provide a means to assess the extent of SIM directly.

617 Pro-Inflammatory Cytokine Production in the Duodenal and Colonic Mucosa of Children with Autistic Spectrum Disorder (ASD) and a Novel Entere-Colitis Paul Ashwood, John Walker-Smith, Simon Murch, Andrew Wakefield, London, UK Background: Previously we, and others, have reported a novel lymphocytic enterocolitis in children with ASD. Systemic immune responses consistent with a dysregulated innate immune response, with raised PBMC-TNFo(production (J. Neuroimmunol. 2001;120:170-9), and TH2 skewing (J. Neuroimmunol. 1998;85:106-9) have been reported in similarly affected children. Aim: To characterize the pro-inflammatory intracellular cytokine production in the duodenal and colonic mucosa of children with (ASD) and entero-colitis. Methods: The study compared colonic biopsies from ASD children (n=9) with histologically and developmentally normal controls (n = 7) and histologically inflamed controls (n = 10) comprising 5 Crohns disease, 1 ulcerative colitis and 4 with indeterminate colitis. In addition, duodenal biopsies from ASD children (n=9) and histologically normal controls (n=8) were compared. Single cell suspensions isolated from mucosal biopsies were prepared for flow cytometry. The epithelial compartment was isolated following washes in EDTA and the lamina propria, by incubation in collagenase (2mg/ml). Detection of CD3+ cells, spontaneously producing intracellular TNFo(, IL-2, IL-4, IFN-y, was performed by multi-colour flow cytometry using flurochromeconjugated monoclonal antibodies. Controls included isotype-matched antibodies, and stimulated and unstimulated PBMCs for defining gated regions. A minimum of 10,000 events was measured per sample. Conclusion: This study demonstrates a significant elevation of proinflammatory cytokine production (TNFc(and INF,y) in the small and large intestinal mucosa of children with ASD and entero-colitis.

620 Dose Response of PEG 3350 for the Treatment of Childhood Fecal Impaction Nader N. Youssef, John M, Peters, Wendy Henderson, Sandra Shultz-Peters, Oanielle K. Lockhart, Carlo Di Lorenzo, Pittsburgh, PA BACKGROUND:Guidelinesfor the treatment of functional constipation in children recommend removal of fecal impaction before starting maintenance treatment (J Pediatr Gastroenterol Nutr 1999; 29:612-26). Our aims were 1) to investigate the efficacy and safety and 2) to determine the optima[ dose of polyathy~eneglycol (PEG) 3350 in the treatment of fecal impaction in children. METHODS: New patients referred for evaluation of constipation and who had evidence of fecal impaction were enrolled in a prospective, double blind, parallel, randomized study of four different doses of PEG 3350 (0.25 g/kg/d, 0.5 g/kg/d, 1.0 g/kg/d, and 1.5 g/kg/d) for treatment of fecal impaction. All children had history of constipation for > 3 months with passage of stool <3/week. Thirty-five of forty patients had also had daily soiling. The median duration of symptoms at time of presentation was 39.6 months (range 6-120 mo). PEG 3350 was given for three consecutive days and patients were examined 5 days after initiation of treatment to assess clearance of impaction. The primary outcome measure was clearance of fecal impaction. Presence or absence of fecal impaction was assessed by abdominal and rectal examination. Laboratory evaluation of electrolytes and serum osmolarity was done before and after completion of treatment. RESULTS:Forty patients completed the study (27 male, median age 7.5, range 3.3 to 13.1 yr). Fecal disimpaction was achieved in 75% of children. There was a significant difference between higher doses (1.0 and 1.5 g/kg/d) and lower doses (0.25 and 0.5 g/kg/d) in the rate of disimpaction (95% versus 55%, p
Results: Mean percentageof CD3~ lymphocytesproducingcytoklnes COLON TNFo IL.2 IL-4 IFNy DUOD TNF_.o IL-2 IL-4 IFNy Laminapropda CD3 cells (%) ASD 31"" 4* 5* 14" 27" 2" 3* 8* NIC 1 0 1 1 3 0 1 1 IC 10 2 2 13 ND ND ND ND Epithelial CD3 cells (%) ASD 33* ** 4 2 19 30* 4" 9 10" NIC 3 1 1 1 2 1 1 1 IC 4 2 2 10 ND ND ND ND NlCfnon-inflammatory conb'ol; IC = inflammatory control; ND not done; *p<0.05vs NIC; ~p<0.O5 vs IC

AGA A b s t r a c t s

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loss, small intestinal villus atrophy, a decrease in brush-border enzymes, reduced enterocyte proliferation, and an increased number of goblet cells; Changes in the colon was mild. Interestingly, IL-4 receptor ~ was continuously expressed in the proliferating zone of the small intestinal crypts. Treatment of TCR~ recipients of IFN-yz-RBN~T cells with anti-lL-4 mAb abrogated both the wasting disease and villus atrophy. Our studies have thus demonstrated a novel function of RBH~Th2 cells, i.e., their involvement in small intestinal epithelial transformation and wasting disease.We employed an inflammation model in this present study, however, our result showed the possibility that Th2-type CD4÷ T cells and their derived cytokines also play critical roles in turnover and differentiation of enterocytes for maintaining the normal architecture of the small and large intestines.

(13%). Diarrhea and bloating were more prevalent (p<0.02) in the higher dose (1.0 and 1.5 g/kg/d) then in the lower doses (0.25 and 0.5 g/kg/d) groups. There were no significant changes in laboratory tests after treatment. All children were compliant with the treatment and said they would repeat the same regimen if they developed a subsequent fecal impnction. CONCLUSIONS: PEG 3350 is safe and effective in the treatment of childhood fecal impaction. Doses of 1.0 to 1.5 g/kg/day are more effective than lower doses but can be associated with diarrhea and bloating. Administration of PEG 3350 is well accepted in children. 621 Expression of a Novel Chemokine Ligand-Receptor Pair in Intestinal Epithelial Cells (IEC): CXCL16 Induced Signals Regulate Epithelial Restitution Stephan A. Brand, Takanori Sakaguchi, Xiubin Gu, Hans-Christian Reinecker, Boston, MA

624 Contribution of the Galanin-1 Receptor (GIR) to the Excess Colonic Fluid Secretion in Marine DSS-Induced Colitis Kristina A. Matkowskyj, Richard V. Benya, Chicago, IL

Background: CXCL16 is a transmembrane protein with a chemokine domain at the end of a mucin-like stalk. CXCL16 has been identified as a specific ligand for Bonzo (CXCR6), a HIV co-receptor. CXCL16 can mediate chemoattraction and induce cellular responses via cell-cell interactions. In this study we determined the signal transduction of CXCL16 and its receptor Bonzo in the regulation of lEG restitution. Methods: CXCL16 and Bonzo mRNA expression in human and murine intestine was determined by Northern Blotting and RT-PCR. Bonzo protein expression in human colonic mucosa was determined by immunohistochemistry. Regulation of CXCL16and Bonzo expression by TNF-~, IL-113, IFN-~,,and LPS was assessed in intestinal model epithelia. CXCL16 induced signal transduction was analyzed by electromobility assays and by immunoblotting using phospho-specific antibodies for MAP-kinases and Akt. Cell proliferation and intestinal epithelial cell restitution assays were performed with lEG-6 cells. Results: CXCL16 and Bonzo were constitutively and segment specifically expressed in the mouse intestine. CXCL16 mRNA expression was highest in the duodenum and jejunum and decreasedtowards the distal colon. In contrast; Bonzo mRNA expression increased from the proximal towards the distal intestinal tract, with highest expression in the distal colon. Bonzo was expressed apically and laterally by primary human colonic epithelial cells. Bonzo and CXCL16 mRNA were constitutively expressed in IEC lines (T-84, HT29, Caco-2, SW480, IEC6, CMT93) and, in contrast to IL-8, only modestly regulated by pro-inflammatory stimuli. CXCL16 binding to Bonzo on lEG activated ERK-1/2-, p38- and SAPK/JNK-MAP kinases and Akt, but failed to induce NF-KB activation. The activation of ERK-1/2 was MEK-1 dependent but PI3-kinase independent, while the activation of Akt was PI3-kinase and partially MEK-1 dependent. The CXCL16 mediated activation of these pathways resulted in a 55% increase in cell migration in restitution assays (p<0.002). This effect was not mediated by cell proliferation or induction of TGF-13expression. Conclusions: CXCL16 binding to Bonzo activates distinct signaling pathways and promotes intestinal epithelial cell migration. CXCL16 may have an important role in intestinal homeostasis by mediating the re-establishment of the intestinal barrier under conditions of enhanced epithelial cell loss.

Background & Aim: Galanin is present in enteric nerve terminals lining the GI tract that in the colon acts by binding to the G1R subtype. We have shown that the G1R is not normally expressed by epithelial cells lining the colon, but is up-regulated by enteric pathogens and causes the excess colonic fluid secretion observed in infectious diarrhea (Nature Med 2000; 6:1048). Since this up-regulation is mediated bythe transcription factor NF-KB, also important in the pathophysiology of UC and Crohns disease, we investigated the role of the G1R in mediating IBD-associated diarrhea. Methods: Colitis in wild type (WT) and G1R knockout mice (G1R-~-)was induced by spiking their drinking water with 2% DSS. Quantitative immunohistochemistry (O-IHC) was performed as previously described (J Histochem Cytochem 2000; 48: 303) using an antibody specific for the G1R. Colonic fluid secretion was determined by closed loop assay. Results: G1R is not detectable in colonic epithelial cells not exposed to DSS (Table). In contrast, constant exposure to DSS resulted in a progressive increase in G1R expression in only wr mice, with maximal levels observed 6 days after DSS initiation. The overall increase in colonic fluid secretion in WI mice paralleledthe increase in G1R expression, and was potentiated by exogenous 1 I~M galanin. In contrast, G1R-~-mice showed no increase in colonic fluid secretion despite their developing colitis indistinguishable from that of WT mice. Conclusion: G1R up-regulation represents a novel mechanism accounting for the increased colonic fluid secretion, or diarrhea, observed in IBD. G1R Expression and Colonic Fluid Secretion in DSS Treated Mice on Day0 and 6 of Treatment Colonic Fluid Colonic Fluid G1R Expression Saline Control 1 pM Gal DO 1.8+/-0.4 29+/-4 28+/-2 WT D6 262.7+/-11.6 68+/-7 156+/-12 G1R"~DO 1.6+/-0.3 28+/-3 29+/-3 G1W- D6 1.6+/-0.5 30+/-4 31+/-3 G1R expressed as eulpix, while colonic fluid is expressed as mg/cm

622 Lipoxin A4 Analogs Attenuate Induction of Intestinal Epithelial Pro-Inflammatory Gene Expression and Reduce the Severity of DSS-Induced Colitis Andrew T. Gewirtz, Lauren Collier, Andrew N. Young, Torsten Kucharzik, Ifor R. Williams, Andrew S. Neish, James L. Madara, Atlanta, GA

925 Neutralization of IL-18 Exacerbates Ulceration and Acute Inflammation in a Murine Model of Epithelial Injury and Repair Basak Coruh, Christopher A. Moskaluk, Kevin M. Overman, Brian D. Weber, Giorgio Sendali, Raffaella Faggioni, Theresa T. Pizarro, Charlottesville, VA; Thousand Oaks, CA

Background: The anti-inflammatory eicosanoid lipoxin A4 (LXA4), and its stable analogs, down-regulate secretion of interleukin-8 and subsequently recruitment of neutrophits by intestinal epithelia. Aim: Examine the overall effect of an LXA4 analog on epithelial gene expression, explore the potential mechanisms underlying effects on gene expression, and determine whether such effects result in attenuation of an in vivo model of colitis. Methods: Polarizedmodel intestinal epithelia were prepared by culturing T84 cells on permeablesupports. Expression of 7000 genes was quantitated via microarray mRNA analysis. NF-KB activity was measured via a reporter gene assay. Effects of LXA4 analog on colitis were examined using the well characterized DSS colitis model. Mice were given 4% DSS in the!r drinking water, which also contained vehicle (0.05% ethanol) or LXA4 analog (10 mg/ml of 15-epi-16parafluorophenoxy-LXA4). Weight and bleeding (gross and occult) were assessed daily. Results: LXA4 analog, alone, did not significantly affect expression of any of the >7000 genes analyzed. However, LXA4 analog pre-treatment attenuated induction of approximately 50 percent of the 125 genes upregulated in response to the gastroenteritis-causing pathogen S. typhimurium. A major subset of genes whose induction was reduced by LXA4 analog pretreatment are regulated by NF-KB suggesting LXA4 analog was influencing the activity of this transcription factor. Nanomolar concentrations of LXA4 analog reduced NF-KB-mediated transcriptional activation and inhibited induced degradation of IKB~x. LXA4 analog did not affect earlier stimulus-induced signaling events that lead to IKB~ degradation such as S. typhimurium-induced epithelial Ca+ + mobilization or TNF~-induced phosphorytation of IKBc~. In mice, oral administration of LXA4 analog (15-epi-16-para-fluoro-phenoxy-LXA4, 10 mg/day) significantly reduced the weight loss, hematochezia,and mortality that characterize DSS colitis. Conclusion: LXA4 analog-mediated down-regulstion of pro-inflammatory gene expression via inhibition of the NF-KB pathway can be therapeutic for diseases characterized by mucosal inflammation.

Introduction: Our group has previously shown that IL-18, a potent Th-1 pnlarizing cytokine, exhibits protective effects in the acute phase of gut inflammation, in contrast to its recognized pathogenic role in more chronic phases of disease. In fact, the intestinal epithelium has the ability to produce large amounts of IL-18 in response to acute activation or injury, while in more chronic states, IL-18 expression is predominantly observed within lamina propria macrophages and dendritic cells. IL-18 binding protein (IL-18BP) is a naturally occurring protein that effectively inhibits the bioactivity of IL-18 by preventing the interaction of the IL18 ligand with its endogenous receptor. Aim: The aim o;f the present study was to determine the cellular source of IL-18BP within the gut mucosa and to evaluate the effects of its administration on mediating mucosal immune responses in an acute model of DSS colitis. Methods: 5% DSS was administered to IL-18 KO and WT mice for 5d followed by a 7d recovery period. Mice were treated with recombinant IL-1BBP-Fc or Fc control (5 mg/kg, i.p.) twice during the experimental protocol. Water intake, hemoccult status and weight were monitored daily, and colonic tissues were collected for histologic and cytokine analyses. RTPCR was performed on isolated intestinal epithelial cells (IEC)and lamina propria mononuclear cells (LPMC) to detect the expression of IL-18BP mRNA. Results: Expression of IL-18BP mRNA transcripts was increased in LPMC compared to IEC within the gut mucosa. Histologic assessment demonstrated a significant increase in colonic ulceration of WT mice treated with IL-18BP-Fc compared to control (4.50 _+ 1.50 vs. 0.75 _+ 0.48, p<0.03) and was similar to that observed in IL-18 KO mice (4.14 _+ 0.94) Although chronic inflammation did not appear to be affected, acute colonic inflammation was significantly increased in WT mice compared to control following IL-18BP treatment (5.50 +_ 0.50 vs. 3.00 _+ 0.41, p<0.03), and again was comparable to IL-18 KO mice (5.29 _+ 0.84). Conclusions: Our results demonstrate that IL-18BP is predominantly expressed by LPMC within the intestinal mucosa, and that administration of this natural inhibitor of IL-18 results in increased surface ulceration and exacerbation of inflammation. These data strongly support the protective role of IL-18 in the acute phase of mucosal immune responses to gut epithelial injury.

623 Small Intestinal Transformation and Wasting Disease Induced in T Cell Deficient Mice by Adoptive Transfer of CD45RB "~ Th2 Cells Taeko Dohi, Kohtaro Fujihashi, Yuko Shirai, Yuki I. Kawamura, Rie Kato, Jerry R. McGhee, Tokyo, Japan; Birmingham, AL It is established that the transfer of CD4+CD45+RBHi T cells (RBH~T cells) to either SOlD or RAG-~- mice results in a severe colitis mediated by T helper-type 1 (Thl) cells. We have modified this approach to address the role of RBH~T cells genetically predisposed to Th2 (interferon-',/defective: IFN-yz) responses when transferred to T cell receptor (TCR) 13and chain-defective (TCR-~-)mice. Wild type (WT) T cells induced wasting diseasewith hypertrophic duodenitis in addition to colitis. In contrast, IFN-~,-~- RBH~T cells induced severe weight

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some of which resolve after acid suppression. Larger studies are neededto validate these findings.

Azulfidine and Olsalzine Inhibit T Cell Induced NFKappaB Activation in Intestinal Epithelial Cells and Prevent iNOS-Mediated Crypt Cell Apoptosis. Ramanarao V. Dirisina, Navdha Mittal, Ziad Alnadjim, Terrence A. Barrett, Chicago, IL

Endoscopic Criteria Pin-point vessels above SCJ (n(%)) Branching vessels below SCJ (n(%)) Serrated SCJ (n(%)) Triangular indentation into SCJ (n(%)) Obscured palisade vessels above SCJ (n(%)) Villiform mucosa below SCJ (n(%))

Inflammatory bowel disease is associated with increased expression of inducible nitric oxide synthase (iNOS) and apoptosis of the crypt epithelial cells in small and large bowel. Recent data indicate that azulfidine (SSA) as well as olsalzine (DLZ) decreases activation of NFkB, a major molecular regulator of pro-inflammatory cytokine/chemokine sythesis in epithelial cells. In the current study we utilize a model for examining the effects of T cell activation on the intestinal epithelial barrier. Treatment of mice with anti-CD3 mAb activates T cells in the intestine leading to rapid cytokine (TNFaipha, IFN-gamma) synthesis and epithelial NFkB activation (lhr), TNF-mediated diarrhea (3-9hr), crypt iNOS expression (3-6 hr) and crypt cell apoptosis (24 hr) mediated by Fas and TNF receptors (Alnadjim et al, DDW, 2001). To address the role of NFkB in crypt cell apoptosis, mice were treated with SSA for 4 days prior to T cell activation. Data indicate that SSA abrogates epithelial NFkB activation (EMSA) and iNOS induction (Western) induced by T cell activation without affecting TNF-mediated diarrhea (Wt./ length measurements). SSA decreasedcrypt cell apoptosis by 55% in the terminal ileum and large bowel (p
ENRD (n=6) Controls Pre-esom- Pest-eaom(n=10) eprazole eprazole 1(10) 3(50) 1(17) 2(20) 1(17) 2(33) 0(0) 1(17) 1(17) 5(50) 5(83) 5(83)

6(46) 12(92) ENRD (n=lt)

3(30) 4(67) 3(50) 8(80) 6(100) 6(100) Controls Pre-esom. Post-eaom' (n=9) eprazole eprazole

Histological Criteria Basal cell thickness (% of whole epithelial thickness, median(IQR)) 10(5.5-15) 6(5-10) 15(10-20) Length of papillae (%of whole epithelial thickness, median(IQR)) 50(36.7-60) 50(35.5-60) 60(50-60) Dilatation of intercellular spaces (Grade*, median(IQR)) 2(1 3) 2(2-2) 3(3-3) • Grade 0= none; Grade lfslight focal; Grade 2=focal; Grade 3=marked focal

3(2-5) 40(30-40) 1(1-2!

629 Prevalence of Extra-Esophageal Manifestations in Gastroesophageal Reflux Disease (GERD) - An Analysis Based on the ProGERD Study Daniel Jaspersen, Michael Kulig, Joachim Labenz, Andreas Leodolter, Tore Lind, Wolfgang Meyer-Sabellek, Michael Vieth, Stefan Willich, Dirk Lindner, Manfred Stolte, Peter Malfertheiner, Fulda, Germany; Berlin, Germany; Sieged, Germany; Magdeburg, Germany; MGIndal, Sweden; Wedel, Germany; Bayreuth, Germany

Introduction: Gastroesophagealreflux disease (GERD) is a common condition, with heartburn as a predominant symptom affecting a large proportion of the general population (17-40%). Up to 50% of patients with GERD suffer from symptoms other than heartburn which are regarded as extra-esophageal.Aim: To estimate the prevalenceof extra-esophagealdisorders (EEDs) in a population with either erosive reflux disease (ERD) or non-erosive reflux disease (NERD). Subjects and Methods: ProGERD is a prospective, multicentre, open cohort study following patients for 5 years after initial successful treatment. 6215 patients (3303 male, 2912 female; mean age 54 years) presenting with heartburn have been included (Table 1). All patients underwent upper endoscopy and completed a questionnaire. The association of EED with several potential factors was described by p-values and odds ratios (with 95% confidence limits) from univariate (x2-test) and multivariate (stepwise logistic regression) analyses. Results: EEDswere detected in 39.1% of patients with ERD (n = 3245) and in 35.4% of patients with NERD. As judged from multivariate analysis, age (OR = 1.30; 95%CI 1211.41 for steps by 20 years), ERD with LA grade C/D (0R=1.39; 95% CI 1.17-1.65) and duration of GERDdisease>1 year (DR = 1.20; 95% CI 1.07-1.34) were significantly associated with EEDs, whereas gender was not. Patients with ERD of LA grade A or B did not have a significantly higher risk than patients with NERD. Conclusion: The development of EEDs in patients with GERD may be associated with higher age, severity of the disease (erosive reflux disease of LA grade C/D) and disease duration (>1 year). Sponsored by a grant from AstraZeneca

627 Clinical Outcome of Conventional and Laparoscopic Nissen Fundoplication at Two Years: Results of a Randomised Clinical Trial Janiek Bais, Hein G. Gooszen, Utrecht, Netherlands

The results of conventional (CN) and laparoscopic Nissen fundoplication (LN) were compared in a randomised clinical trial (RCT). A total of 103 patients, most of them with refractory gastro-oesophageal reflux disease (CORD) were included: 56 CN and 47 LN: 33 women and 70 men, mean age 41 yrs (range 18 - 72). After inclusion and operation of 103 patients with a follow-up of at least three months, an interim analysis was conducted and further inclusion was stopped, because in the LN group significantly more patients had reached a study end point (dysphagia, recurrent CORD and wrap herniation, p < 0.001). Seven patients needed reoperation or dilatation for severe dysphagia all after LN, two had recurrent CORD after LN and one after CN and in two other patients the wrap had herniated into the thorax early after LN. All patients were followed up by a mailed questionnaire, focusing on general health, reflux symptoms and quality of life assessed with the SF 20 questionnaire and VAS-score, at two years after operation. The effect on the different parameters was included in this report. The response rate was 91%. Twelve patients (3 CN, 9 LN) were reoperated within the first year after surgery. In the second year another 5 patients (4 CN, 1 LN) were reoperated.(indications: dysphagia (7), re-GDRD (7), epigastric pain (3) and cicatricial hernia (1). Dysphagia was more present in the LN group. Reoperation had a good effect on reflux symptoms and general health in 8 of the 12 patients (66% success). After two years 93% of the CN patients had complete or significant symptom relief and 96% in the LN group. Success, improvement in both general health and reflux symptoms, was obtained in 38/41 (93%) patients after CN and in 46/53 (87%) patients after LN. The effect of operation on general health, CN > LN at 3 months (p=O.O03) and at two years (p=O.04). For the VAS score and the effect of surgery on symptoms : LN = CN for the groups as a whole. For the different items of the SF 20 : LN = CN. In this study, LN was not superior to CN. Considering that the reoperation rate is about three times higher in LN and that success of reoperation is only 66%, it must be indirectly concluded that laparoscopic antireflux surgery is expert surgery. In low-volume antireflux centres conventional Nissen may lead to better results.

Table 1: Baseline demographic and clinical charsctedstlcs of the study population NERD ERD All patients Ho. o1 subjects N 2970 3245 6215 Gender: male 13371450% 1966f60.6% 3303/53.1% female 16331550% 1279/39.4% 2912146.9% Age(years) mean+SD 53.0+14.3 54.5±13.7 53.8±14.0 Body mass index (BMI) (hg/r¢~) mean ± SD 26.6± 4.2 27.3 ± 4.0 27.0 ~ 4.1 Duration of GERD disease (years) mean± SD 5.1 ± 7.1 6.0 ± 7.8 5.6 + 7.5

63O Gastric Pacing in Morbid Obese Patients Improves Lower Esophageal Sphincter Function and Abnormal Esophageal pH Pattern Claudia Knippig, Stefanie Wolf, Jochen weigt, Andreas Leodolter, Hans Lippert, Peter Malfertheiner, Magdeburg, Germany

628 Endoscopy-Negative Reflux Disease (ENRD): High-Resolution Endoscopic and Histological Signs William Tam, Adders Edebo, Marco Bruno, Michael Vieth, Annmarie Van Berkel, Lars Lundell, John Dent, Guido Tytgat, Manfred Stolte, Mark Schoeman, Adelaide, Australia; Goteborg, Sweden; Amsterdam, Netherlands; Bayreuth, Germany

Introduction: Gastric pacing is a new experimental operative option for weight reduction in obese patients. GERD is an associated disease in morbid obesity. The aim of this study is to examine the effect of gastric pacing on weight reduction and GERD. Methods: 30 patients with a BMI>40 fulfilling WIlD criteria fur operativetherapy for weight reduction are randomized for a) gastric pacemaker (Transcend" ,JGS), switched on after 4 weeks, b) gastric pacemaker switched off (sham operated) or c) lap band procedure (standard procedure). Patients are evaluated at baseline and after 6 months for weight, gastric emptying time by 13C-octaonicbreath test, upper GI-endoscopy, 24-hour-pH-Metry and esophageal manometry. Diagnosis of endoscopy negative gastroesophageal reflux disease (Engerd) was based on symptoms and abnormal pH-metry according to DeMeester score in absence of esophageal lesions. Results 16 patients (BMI 46+3, 3 male, 13 female, 34+6 years) have been operated: 5 patients received a lap band, 11 a gastric pacemaker (7 paced patients, 4 sham operated). Mean weight loss of paced patients was 7,3+8 kg vs. 1,2+0,5 kg in the sham operated group. Five paced patients had Engerd at baseline. After six months pacing an increase of the lower esophagealsphincter pressure (17 + 6 vs 29 + 15mmHg, p<0,05) and normalisation of DeMeester score from 40,1 +64 to 8,2+3,5 (n.s.), leading to normal DeMeester Score was seen in all paced patients independently of weight toss. Gastric emptying was delayed at baseline in all patients and did not improve under electrical stimulation (t50:174 + 25mid vs. 178+39mid, n.s.). Conclusion: Gastric pacing has a positive effect on weight reduction

We have previously used high-resolution magnification endoscopy (Fujinon EG485Zt-t, 850kpixel) to develop proposed novel endoscopic criteria for non-erosive esophageal injury from reflux (Gastrointest. Endosc. 2001;53(5):ABl19). Aim: To evaluate differences in endoscopic and histological signs betweenENRDand controls. Methods: 13 patients with heartburn and pathological 24h esophagealacid exposure, but with no erosions on standard endoscopy, and 10 asymptomatic controls with normal acid exposure, underwent high-resolution magnification chromoendoscopy with Lugol's iodine. Endoscopic images were randomly selected for blinded assessment. Biopsies taken at the squamo-columnar junction (SCJ) were examined for changes in squamous mucosa. 6 patients underwent repeat endoscopy and biopsy after esomeprazole 40rag daily for 4 wks. Results: (Table, n(%), median(IOR)) Pin-point vessels and triangular indentation of the SCJ upwards into squamous mucosa were seen more commonly in ENRD compared to controls. A serrated SCJ was uncommon in ENRD,and was not seen in controls. Villiform mucosa below the SCJ was seen equally frequently in the 2 groups. Histology showed a trend to increased basal cell thickness in ENRD compared to controls. After acid suppression, some endoscopic and histological signs resolved. Conclusions: Subtle endoscopic and histological differences can be found betweenENRDand controls,

AGA A b s t r a c t s

ENRD (n=13) 5(46) 4(31) 2(15) 11(85)

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in morbid obese patients. The positive effect on Engerd may be explained by electrical entrainment of the lower esophageal sphincter but requires further examination.

633 Differential Productionof IL-12 and IL-10 by Colonic Dendritic Cells (DC) in Response to Bacterial Stimuli Rachael J. Rigby, Ailsa L. Hart, Michael A. Kamm, Stella C. Knight, Andrew J. Stagg, London, UK Intestinal dendritic cells (DC) sample luminal contents and play a central role in the regulated responseto the commensal gut flora. This is mediated in part by cytokine production following exposure to bacterial products, and results in a balance between pro- and anti-inflammatory responses. Cytokine production by murine colonic DC was assessed after stimulation by a cell wall component of either Gram-negative bacteria or a probiotic bacterium. Methods: DC were identified as CD11c+ MHC class II + cells in mononuclear cell preparations obtained by collagenase digestion of colonic tissue. Production of IL-12, IL-IO and IL-4 in response to LPS (1 p.g/ml), cell walls from Bifidobacteria Iongum(B. Iongum) (at the equivalent of 107 per ml) or medium alone was determined by intracellular staining. DC were also isolated by immunomagnetic separation on the basis of CD11c expression. Results: Approximately 3% of colonic cells were DC. They were CD40+ CD80+ CD86+ and stimulated a primary mixed leucocyte reaction following overnight culture. A small proportion (<5%) of unstimulated DC produced IL-12. LPS, hut not B. Iongum, increased the proportion of IL-12 producing DC by 4-16 fold. In contrast, IL-IO was not detected in unstimulated cuJturesor in response to LPS, but was induced in over 50% of DC in the presenceof B. Iongum. Addition of LPS to B. Iongum stimulated cultures, downregulated production of IL-lO by approximately 50% indicating crossregulation of the responses induced by these two stimuli. IL-4 production by DC was not detected under any conditions. Conclusions: Colonic dendritic cells, which are early and central regulators of mucosal immunity, respond to bacterial stimulation with the production of both pro-and anti-inflammatory cytokines. However, different bacteria or bacterial components stimulate opposing responses, and therefore have the potential to determine the subsequent immune response. This provides further supportive evidence for the use of probiotic bacteria in altering gut immune regulation.

631 The SymptomAssociation Probability (SAP) is Superior to the Symptom Index (SI) for Attributing Symptomsto Gastroesophageal Reflux: Validation Using Outcome from Laparoscopic Antireflux Surgery (LARS) Sergio Diaz, Ruben Aymerich, Ray E. Clouse, Kenneth E. Freedland, Chandra Prakash, Nathaniel J. Soper, St. Louis, MO ..... BACKGROUND: Predictors of outcome from LARS are important for best patient selection. Calculating the statistical probability of reflux-related symptoms (the SAP) during ambulatory pH monitoring has appeal as a predictor but is less commonly used than the SI and has not been validated. METHODS:Esophagealphysiological data, clinical information, and symptom scores were collected prospectively from 79 pts (37M/42F; age 47 +- 12 yr) with abnormal acid exposure times undergoing JARS. SAP and SI were calculated pre-op for the principal symptom at the time of pH monitoring (Weusten et aL, 1994). Symptom levels were recorded on and off medical therapy pre-op and at short-term (35 +_5 d) and distant (252 _+20 d) post-op follow-up. Outcome was measured as change in principal or global esophageal symptoms; stepwise regression models were used to determine outcome contributors. RESULTS: Comparing the 2 tests, the SAP significantly predicted principal symptom improvement at short-term follow-up (p=O.02) and showed a trend toward improvement in global esophageal symptoms at distant follow-up (p = 0,08). The SI had no predictive value. Effects of the SAP were strong in males, predicting improvement in the principal symptom at distant follow-up (p95% or p
634 Dissecting the Host Signaling Pathways Induced by EPEC and EHEC:EPECRequires N-WASP,but not WIP, for Nck Recruitment and Actin Pedestal Formation. Fuminao Takeshima, Tomoyuki Shibata, Ines Anton, Ching-Hui Liu, Rail Geha, Scott B. Snapper, Boston, MA Background and Aims: EPECand EHECactivate intracellular signaling pathways by introducing virulent proteins into the host cells employing a type III secretion system. During infection, the bacterial membrane protein intimin mediates attachment tO host cells by binding to the type III secreted protein Tir, which functions as the receptor for intimin. The interaction between intimin and Tir stimulates host cell signaling events that lead to actin pedestal formation beneath the adherent bacteria. Recent studies have demonstrated that Nck and NWASP are required for EPEC-inducedpedestal formation downstream of Tir. Tit shares a high degree of homology with the vaccinia protein A36R which is essential for actin-based motility. Recent studies have also implicated Nck, WlP and N-WASP in vaccinia motility. We therefore sought to determine the role of Nck, N-WASP, and WlP in intracellular signaling cascades that lead to pedestal formation of EPEC and EHEC. Methods: N-WASP-deficient (KO) WIP KO, and wild type (WT) control fibroblasts were generated by standard methods in our laboratories. A dominant negative (DN) Nck plasmid (containing three mutated SH3 domains) was provided by Dr. B. Mayer. In all infection assays, cells were infected for 6 hours. Pedestals and bacteria were visualized by microscopy using rhodamine-phalloidin and DAPI staining, respectively. Protein localization was demonstrated by immunostaining with an anti-Nck antibody or an anti N-WASP antibody, or via transfection with a WIP-GFP expression vector. Results: In WT cells, N-WASP and WlP are recruited to the pedestaltip of both EPEC and EHEC infected cells. However, Nck is recruited to the tip of pedestals induced only by EPEC and not by EHEC. EPEC and EHEC induce pedestal formation equally on both WT (EPEC: 99.6% +/-0.57%; EHEC: 90.3% +/- 2.51%) and WlP KO (99.7+/-0.58% or 90.1% +/4.7%) cells. DN Nck expression in WT cells prevents pedestal formation induced by EPEC but not by EHEC. EPECand EHECfail to recruit Nck and WlP in N-WASP KO cells. However, in WlP KO cells, EPEC recruits both N-WASP and Nck, whereas EHEConly recruits N-WASP. Finally, in cells expressing DN Nck, N-WASP and WIP are both recruited to EHEC but not EPEC. Conclusions: 1. Actin pedestal formation of EPEC and EHEC is independent of WlP. 2. EPECand EHECutilize different signaling cascades downstream of Tir to activate N-WASP and Induce pedestal formation. 3. EPECmay utilize a complex of Nck and N-WASP to induce pedestal formation.

632 Continued Use of Acid SuppressantMedicatiun Following Antireflux Surgery Christopher A. DiRe, Kevin G. Billingsley, Jeffrey A. Todd-Stenberg, Jason A. Dominitz, Seattle, WA Objective: Antireflux surgery has beengaining popularity since the introduction of laparoscopic technique. However, there are few large-scale multicenter studies assessing the durability. We evaluatedthe continued use of antireflux medication following fundoplication surgery in the VA population. Methods: Procedures and diagnoses were determined using VA administrative databases (3/1991-2/2001). The inclusion criteria were: 1) antireflux surgery from 3/1/19912/28/2000, 2) minimum of one outpatient visit at least six months after surgery, 3) at least one outpatient clinic during 10/1/1998-2/28/2001 (when pharmacy records are available).The exclusion criteria were: 1) those with a diagnosis of achalasiaat the time of the fundoplication (n = 88), and 2) those with a diagnosis of esophagealcancer within one year of the fundoplication (n = 62). We measured use of more than 2 prescriptions for acid suppressant or promotility medications more than 6 months after fundoplication. Results: Antireflux surgery was performed in 3405 veterans during the study period. 12.7% died at some point during followup. Study inclusion criteria were met in 2383 patients. The mean (sd) age at operation was 53.3 yrs (13) with a range of 22-86 yrs. 94% were male, 88% were white, 5% were black, and 4% were Hispanic. The median follow-up was 4.9 years. 406 patients had surgery during the time that complete pharmacy records were available, with a minimum of one year of follow-up (recent cohort), allowing for complete analysis of drug use in the perioperative period. Table 1 shows medication use after surgery. Overall, 49.2% had received at least 3 prescriptions for either an H2RA, prnmotility agent, or PPI, including at least one prescription more than 6 months after surgery. Analysis of the recent cohort reveals that GI prescriptions are very common in the 3 months following surgery, then decline in the 3-6 month window, only to increase after 6 months (e.g. PPI use fails from 39.7% to 7.9%, then rises to 14.8%). Conclusion: Antireflux surgery in the VA population is initially associated with a decline in acid suppression or promotility medication usage. This effect does not appear to be durable. Approximately 50% of surgical patients receive multiple prescriptions for reflux medications at a median of five year of follow-up. This study was funded by a grant from the American College of Gastroenterology.

635 The Probiotic E. coil Nissle 1917 (Mutaflor®) Induces Defensins in Intestinal Epithelial Cells: A Novel Mechanism Of Action Jan Wehkamp, Juergen Harder, Birte Wehkamp- von meissner, Lars Schwichtenberg, Klaus Fellermann, Klaus R. Herrlinger, Jens M. Schroeder, Eduard F. Stange, Stuttgart, Germany; Kiel, Germany Introduction: Recently, the results of several confirmatory clinical trials demonstrated that treatment with the non-pathogenic E.coli Nissle 1917 (Mutaflor~) is equivalent to mesalazine in maintaining remmission in ulcerative colitis. However, little is known about the mechanism of action of probiotics. The aim of our study was to compare the probiotic E.coli Nissle 1917 with other E.cufi strains with respect to innate defense mechanisms in the mucosal ceil Methods: E.coli strain Nissle 1917 as well as E.coli K12, E.coli EPEC and four other wildtype E. coli strains isolated from human feces were grown in Trypticase soy broth medium and incubated with the intestinal epithelial cell lines Caco-2 as well as HT29. Thereafter, we isolated mRNA and performed RT-realtime PCR using a lightcycler with specific primer pairs for the antimicrobial peptides human beta defensin 1 (HBD-1), human beta defensin 2 (HBD-2) and human beta defensin 3 (HBD-3). Resutts: Our results demonstrated that HBD-1 was not regulated by any of the tested £colistrains. HBD-2 could he induced up to 70 fold exclusively by the non-pathogenic £coli strain Nissle 1917 and not by any of the other strains. The effect

Table 1: Proportionof patientsusingmedicationsmorethan 6 monthspost-surgery (n=2383) Antacid Promotility H2RA PPI 5.6% 9,2% 22.7% 3h.0%

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was time (optimum after 6h of stimulation) and dose (minimal dose a~prox.:lOpotT) dependent in both, Caco-2 and HT29 cells, respectively. The observed transcriptional induction of the defensin HBD-3, as well, could be induced in HT 29 cells only by E.coliNissle 1917. Conclusion: The probiotic E. coil strain Nissle 1917 is able to induce the production of the antimicrobial peptides HBD-2 and HBD-3 in intestinal epithelial cell lines in contrast to other E. coil strains.Investigations with more strains and other bacterial species are currently under way. This represents a novel hypothesis of its mode of action. It is likely that this effect contributes to an enhancement of the mucosal barrier towards the load of luminal bacteria, leading to an effective maintenance of remission in inflammatory bowel disease.

shown to have a major role in RV diarrhea. Expression of receptors for the neuropeptide galanin (gal) was increased in colonic epithelial cells exposedto bacterial pathogens,a process initiated by NF-kB activation resulting in chloride and fluid secretion. As rotavirus activates NF-kB in vitro, we hypothesized that RV infection may increase diarrhea in a murine model by increasing the epithelial response to gal. Results: Murine pups age 10 days were orally infected with simian RV, (RRV, 5x107 infectious virions). At 24 hrs p.i., small intestine was removed, and NF-kB activation was confirmed by immunohistochemistry in epithelial cells. Gal-1 receptor expression was also increased at villous tips. To determine if gal-1 receptor upregulation contributes to RV diarrhea, gal-induced fluid secretion in closed smalFintestinal loops from similarly infected C576/8J mice was compared after 1 hr to non-infected controls. Results are expressedas mg/cm of intestine, normalized for pup weight. Infected mice yielded a ratio 2.82_+0.09 vs 2.24_+0.09 for uninfected controls (n=4, p_<.05), supporting the hypothesis that gal-1 receptor regulation has a role in RV diarrhea. To confirm this finding, we infected as above mice lacking the gal-1 receptor gene. Liquid stools were identified in 35/35 C576/6J controls, but in only 3/22 knockout mice. Conclusion: These findings confirm that the galanin system is an important regulator of fluid secretion during RV infection. The mechanism by which the virus induces this important host response may prove central to efforts at improved control of viral diarrhea.

636 Atypical PKC~ Regulates EPEC-Induced Inflammation through Interaction with IKB Kinase Suzana Savkovic, Athanasia Koutsouris, Gall Hecht, Chicago, IL Enteropathogenic E.coli (EPEC) infection of intestinal epithelial cells (IEC) activates transcription factor NF-KB thus inducing inflammation. Phosphorylation and proteasomal degradation of IKB~ frees NF-KB allowing it to translocate to the nucleus. While extracellular-regulated signal kinases (ERK) participate in EPEC-inducedNF-KB activation, other signaling molecules, such as atypical PKC;, are also involved. The aim of this study was to define the mechanisms by which PKC~ regulates EPEC-induced inflammation in IEC. Cultured human IEC, T84 and Caco, and EPEC strain E2348/69 were used for these studies. To determine whether EPEC activates PKCZ~in IEC, cellular fractionation, immunostaining and kinase activity assays were performed. In uninfected cells, the ratio of the cytosolic to membrane-associated PKCI; was 9:1. Following EPEC infection, PKCI; translocation from the cytosol to the membrane progressively increased to a ratio of 6:4 at 60 min. Immunostaining confirmed the cytosolic location of PKCt; in uninfected tEC and redistribution to the peripheral membrane after 30 min of infection. Kinase activity assays using immunoprecipitated PKCI; and myelin basic protein as a suhstrate showed that PKC; activity increased 1.4+/-0.2 fold by 15 min postinfection, reached a plateau of 2.9+/-1.2 fold after 30 min, and remained activated for up to 60 rain (n-4). Rottlerin (IO0~M), which blocks atypical PKC isoforms, prevented PKGZ, translocation as well as IKBc~ phosphorylation suggesting a connection between these two molecules. Pretreatment of cells with the myristoylated PKCI; pseudosubstrate (201~M, Bigsource, int.), that inhibits activity by binding to the active center of PKC; molecule, significantly attenuated (60+/-20%; n =4) EPEC-induced IKB~ phosphorylation. PKC~ can interact with and activate I~B kinase (tKK), the complex responsible for phosphorylation of IKB. Coimmunoprecipitation studies revealed a 1.4-fold increase in PKCI; and IKK association at 30 rain and a 3-fold increaseat 60 rain post-infection. Kinaseactivity assays using immunoprecipitatecl PKC~IKK complexes from IEC infected for 30 min and recombinant IKBe as a substrate, yielded a 2.5 fold increase in phosphorylation of this specific substrate. Based on these findings, we conclude that EPEC activates PKCZ~in host IEC and employs this molecule as part of the pro-inflammatory signaling cascade. The mechanism by which PKC~ stimulates this response appears to be through its interaction with and activation of IKK.

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Activation of Mutant Ki-ras in Murine Pancreatic Ductal Cells Induces Cell Cycle Regulators Franz S. Schreiher, Hideki Harada, Michael I. Boretti, Keith J. Gooch, Anil K. Rustgi, Philadelphia 19104, PA; Philadelphia, PA Introduction: The presumed precursor lesions of pancreatic ductal adenocarcinoma are classified as pancreatic intraepithelial neoplasia (PantN 1-3) and correlate with specific genetic alterations. Activating point mutation in cndon 12 of the Ki-ras oncogene occurs early and is the most common known genetic alteration. Using K19 Ki-ras transgenic mice as a platform, we have developed techniques to isolate and characterize pancreatic ductal cells from these mice and age-matched wild-type littermates. Methods: Pancreatic interlobular duct fragments were isolated and used as a source to expand ductal cells. These were characterized using light and electron microscopy, immunocytochemistry (Keratin 19) and Western Blotting (Carbonic Anhydrase II). To further characterizeboth cell types population doubling time, flow cytometry and immunobt~tting were obtained, the latter for evidence of activated Ki-ras, ERK 1/2, cyclin D1, Akt, p16, p19Arf and p27Kip1 and p53. Results: Cells derived from Ki-ras and wild-type mice showed morphological and biochemical features typical for ductal cells. Population doubling time and cell cycle analysis revealed no differences. Expression of activated Ki-ras as well as cell cycle regulators p16/p19Arf and p27Kip1 were increased in pancreatic ductal cells derived from Ki-ras transgenic mice in comparison to ductal cells obtained from wildtype mice. There was no differential expression of the other proteins tested. Conclusion: Our innovative derivation and characterization of mouse pancreatic ductal cell lines indicate that effects of oncogenic Ki-ras are counterbalanced by upregulation of the cell cycle regulators p16/p19Arf and p27Kip1. This may explain why Ki-ras activation in primary or normal ceils induces senescenceand at the same time, is necessary but not sufficient to cause pancreatic ductal adenocarcinoma in vivo.

637 Clostridium difficile Toxin B Is an Enterotoxinfor Human intestine In Vivo Tor Savidge, Weihua Pan, Paul Newman, Michael O'Brien, Charalabos Pothoulakis, Charlestown, MA; Boston, MA

640 Mitogenic and Anti-ApoptoticRole of Constitutive NF-kB/Rel Activity in Pancreatic Cancer Susanne Liptay, Leopold Ludwig, Christoph K. Weber, Martin Wagner, Guido Adler, Roland M Schmid, 89075 UIm, Germany; 89081 Ulm, Germany

Background and Objectives: Clostridium difficile represents the major cause of antibioticassociatedcolitis and diarrhea in health care facilities in the US and abroad. Intestinal pathology in response to this microbe is mediated by the release of two specific exotoxins, toxin A and toxin B. Aifhough both toxins can induce cytotoxic changes (cell rounding) in cultured cells and human colonic mucosa, toxin A, but not toxin B, is an enterotoxin in animal intestine. Our aim in this study was to determine if C. difficile toxin B is an enterotoxin in human intestine in vivo using a murine chimeric model for human intestine. Methods: Subcutaneous human fetal intestinal xenografts were generated in SCID mice. in situ hybridization using species-specific human and mouse DNA probes (performed at light and ultrastructural levels), demonstrated that the epithelium in these grafts was well differentiated and exclusively of human origin. After 10 weeks~the xenografts were injected with buffer or buffer containing 10 mg of purified toxin A or toxin B (n = 6/group). After 6 hrs, mucosal permeability to FITClabeled sulfonic acid, histologic changes and mucosal levels of the human cytokine IL-8 were measured. Results: Toxin A and toxin B induced a 20.6-fold and 4.3-fotd increase, respectively, in mucosal permeability. Toxin A and B-injected grafts also exhibited acute mucosal inflammation with epithelial cell damage, congestion and edema of the lamina propria; and a 3.4-fold and 5.2-fold potymorphonucleocyte (PMN) infiltration of the intestinal mucosa. A likely cause for this PMN influx was a 6.2-fold and 4.8-fold increase (for toxin A and toxin B, respectively) in tissue IL-8 levels measured by ELISA. Conclusions: Our results demonstrate that C. difficile toxin B, like toxin A, now needs to be regarded as a human-specific enterotoxin. The failure of anima~ intestine to respond to toxin B probably reflects the absence of toxin B receptors on intestinal epithelial cells of animal origin. Future therapeutic or vaccine strategies aimed at C. difficile-induced disease therefore also need to target both toxins. Supported by the Crohns and Colitis Foundation, and the National Institutes of Health (DK33506; DK40561)

Background & Aims: Nuclear factor kappa B (NF-kB) is a central regulator of the immune response and apoptosis. NF-kB/Relhas been implicated in the pathogenesis of certain tumors and found to be constitutively activated in human pancreatic cancer. Methods: MiaPaCa2, Pancl, and AsPc3 cells were used for in vitro studies. NF-kB/Rel activation was detected using EMSAs and Ik8-kinase assays. Subunit composition was identified using supershiff assays. Transcriptional activity was quantitated with luciferase assays.A cell permeablepeptide or a super-lkBa was used to block NF-kB activation. Apoptosis was assayed with annexin V binding. Cells stably expressing super-lkBa were generatedand assayedfor anchorage dependent and independent growth. Results: RelA is present in the nucleus in primary human pancreatic cancer samples as well as in pancreatic cancer cell lines. NF-kB/Relbinding activity consists of NF-kBI(p50) and RelA(p65). Constitutive NF-kB/Rel activity correlates with tkBkinase (IKK) activity and can be blocked by dominant negative mutants of IKKb and to a lesser extend by IKKa. Constitutive NF-kB/Rel activity and the transactivation potential of RelA(p65) can be inhibited by dominant negative mutant Ras, the PI3-kinase inhibitor LY294002 or dominant negative mutant Akt-kinase. Inhibition of constitutive IKK or NF-kB/ Rel activity increasedthe number of apoptotic cells. Stably expressing a nondegradableform of IkBa inhibited anchorage-dependentand -independent proliferation in MiaPaCa2and Pancl ceils. Conclusion: Our data demonstrate that a Ras / PI3-kinase/ Akt / IKK dependent pathway contributes to constitutive NF-kB/Rel activity in pancreatic cancer. Inhibition of NF-kB/Rel activity reveals a mitogenic and anti-apoptotic role for NF-kB/Rel in pancreatic cancer.

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Galanin-1 Receptor Expression Contributesto Rotavirus Diarrhea Robert D. Shaw, Scott J. Hernpson, Kristina A. Matkowskyj, Richard V. 8enya, Northport, NY; Chicago, IL

Analysis of the Molecules Smad4 - Independently-Regulated by TGF-Beta in Smad4Null Human Pancreatic Cancer Cells Hideaki Ijichi, Motoyuki Otsuka, Tsuneo Ikenoue, Keisuke Tateishi, Takayuki Kawakami, Goichi Togo, Fumihiko Kanai, Naohiko Seki, Yasushi Shiratori, Masao Omata, Tokyo, Japan; Chiba, Japan

Background: Rotavirus (RV) is the most important worldwide cause of severe viral diarrhea, especially in children. Infection of villus epithelial cells results in diarrhea in 12-24 hrs, although epithelial damage does not appear to be severe. Mechanisms to explain the pathogenesis of diarrhea include chloride secretion induced by a non-structural viral protein that regulates a calcium dependent pathway, altered epithelial barrier function, loss of mucosal surface and/ or reduced epithelial differentiation, among others. Recently, the enteric nervous system was

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Background & Aim: Transforming growth factor-beta (TGF-beta)-Smadsignaling pathway is known to have a growth inhibitory effect on the human epithelial cells, and is considered to function as a tumor suppressor, it is reported that Smad4 gene is mutated or deleted in 50% of pancreatic cancer patients, and we previously reported that many pancreatic cancer cell

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lines had impaired TGF-beta-Smad pathway due to functionally inactivated Smad4. In this study we investigated the target molecules Smad4-independentlyinduced by TGF-beta,which might be involved in Smad-independentpathway induced by TGF-beta.Methods: We performed cDNA microarray analysis using in-house cDNA chip containing 2280 named genes for screening the target molecules regulated by TGF-beta. A Smad4-null pancreatic cancer cell line BxPC-3was transfected with Smad4 expression vector or an empty vector, then incubated with or without 10 ng/ml of TGF-betafor 2 houre. 2 ~g of mRNA of each sample was extracted and used for hybridization to the chip. Semi-quantative RT-PCR and Western blotting was performed to confirm the microarray results. Transcriptional inducibility by TGF-betawas also examined by luciferase assay. Results: Among the 2280 genes, 2 clones were upregulated more than two times by TGF-beta, not only in Smad4-transfected cells but also in Smad4null ones. The RT-PCR and Western blotting showed that only one clone, p21/WAF1, was upregulated, p21/WAF1 promoter lesion (2.3kb) was not obviously activated by TGF-beta in the luciferase assay in Smad4-nufl cells. Conclusions: G1-S arrest induced by TGF-beta is mediated by cyclin-dependent kinase (CDK) inhibitors including p21/WAF1, which has been considered as one of the major target of TGF-beta-Smad pathway. In this study, however, p21/WAF1 could be upregulated by TGF-betain Smad4-null cells. The upregulation might not be due to transcriptional inducibility, suggesting that stability of mRNA might be involved. The clones considered as Smad4-dependentlyupregulated ones were 3 among 2280 according to the same microarray analysis. Thus, it is suggested that Smad-independent signaling pathway induced by TGF-beta might exist as widely as Smad-dependent pathway.

In vivoresults: TU-Volume (cmm) Met-Score(pts.) Ascites (n I n) Survival(n I n) WeigM (gram) VEGFP (pglml) MVD (/0.74 qmm) *=p
control 1550 ÷/- 131 17.8 ÷/- 1.i 4t8 1/ 8 27.3 +/- 0.6 59.5 +/- 5.8 70.9 +/- 5.7

IM862 1113 ÷/- 208 7.7 +t- 2,0* 215 6 / 8* 29.4 +/- 0.7* 30.5 +/- 1.6" 27.5 +/- 2.6*

644 Eradication of ChemoresistontAneuploid Pancreatic AdenosquamousCa Characterised by Overexpression of K-Ras and Hypermethylationof CpG Islands of p16(INK4a) after Chemogene Tx with DooetaxeI,Vinorelbine and Recombinant Adenoviral Type 5 Transfection of p16 cDNA Termed as seviNA-22/3 John N. Giannios, Athens, Greece Adenosquamous Ca is an aggressive and highly metastatic variant of adenocarcinoma with both glandular and squamous differentiation.Usually,it occurs in chemoradiated patients.Tumour cells were obtained from a resected pancreatic adenosquamous Ca which already had metastasizedto regional lymph nodes.Methylation-specific PCR (MSP) detected methylated DNA template of pt6.The methylated CpG islands in a promoter of p16 inhibited transcription by preventing RNA polymerase and the RNA transcription machinery from producing messenger RNA leading to gene inactivation.SSCPanalysis has detected mutated K-Ras.We constructed an adenovirus p16 expression vector and we inserted p16cDNA into a cassette cosmid containing an adenovirus type 5 genome.Subsequently,we produced a recombinant adenovirus termed as seviNA-22/3 by cotransfection of expression cosmid and adenovirus DNA-terminal protein complex into cells by calcium phosphate precipitation.After one hour treatment with seviNA-22/3,pancreatic Ca cells expressed high levels of p16 gene mRNA according to Northern blot hybridization analysis.Theadenoviral mediated genetransfer of wt p16-1NK4A formed a heterodimer with cyclin-dept kinase 4 and 6 preventing their interaction with cyclin D. PCR analysis has showed downregulation of cyclin D1 and K-Ras. Subsequent treatment with docetaxel and vinoreibine has exerted a synergistic antimitotic effect exhibited by BrdU and Ki-67.Flow-cytometry has reported diploid DNA. A biochemical assay showed activation of caspase-3/CPP32pathway which led to electron cytological signs of D2 apoptotic stage forming apoptosomes which were phagocytosed by adjacent tumour cells leading to a bystander killing effect.Concluding,by restoring wild-type p16 protein in chemoresistant adenosquamous carcinoma cells, we have achieved to block cyclin Df which led to inactivation of K-Ras allowing induction of apoptosis after the synergistic antimltotic action of docetaxel and vinorelbine.

642 Reactive OxygenSpecies (ROS) as Survival Factor in Human Pancreatic Cancer Cells Eva Vaquero, Kyung Joon Nam, Stephen J. Pandol, Anna S. Gukovskaya, Los Angeles, CA Background & Aims: Prooxidant state is a common feature in cancer cells. However, the functional role of ROS in cell survival is not well established. ROS can induce apoptosis but also stimulate anti-apoptotic signaling pathways. Growth factors and extracetlular matrix (ECM) are known to promote cell survival. Our aim was to determine the role of ROS in the effect of growth factors and ECM on apoptosis in human pancreatic cancer cells. Methods: MIA PaCa-2 cells were cultured for 72 h on polyHEMA (a nonadherent substrate) in serum-free medium; on potyHEMA in medium with 15% fetal bovine serum (FBS) or with insulin growth factor I (IGF-I; 100 ng/ml); and on laminin or fibronectin in serum-free medium. Apoptosis was analyzedby measuring phosphatidylserineexternalizationwith FACSand oligonucleosomal DNA fragmentation with ELISA; intracellular ROS were measured with FACS by using the redox-sensitive probe DCFH-DA;cytochrome c and Smac/DIABLO releasein cytosolic fractions and heat shock proteins (HSPs) in total cell lysates were detected by immunoblotting. Results: Laminin, fibronectin, FBS, and IGF-I dramaticafly stimulated intracellular ROS production compared to cells deprived of ECM and growth factors. The superoxide scavenger tiron (10 mM) and the NADPH oxidase inhibitor diphenylene iodonium (DPI; 15 p,M), but neither the xanthine oxidase (XO) inhibitor allopurinol (lmM) nor the nitric oxide synthase (NOS) inhibitor L-NAME (1 raM), blocked endogenous ROS generation. This indicates NADPH oxidase, but neither XO nor NOS, as an intracellular ROS source. Tiron and DPI, but not allopurinol and L-NAME, markedly induced apoptosis in cells cultured either on laminin or with serum and to a lesser extent in ECM and serum deprived cells. For example, tiron increased apoptosis by 3.3 _+ 0.04 fold in laminin-attached cells and by 1.6 _+ 0.1 fold in cells cultured without ECM and serum. These results suggest anti-apoptotic role for ROS in MIA PaCa-2cells. Tiron and DPI increased mltochondrial cytochrome c and Smac/DIABLO release suggesting that apoptosis induced by ROS depletion is mediated by mitochondrial dysfunction. MIA PaCa-2 cells displayed substantial levels of HSPs 27 and 70, which were reduced by antioxidants, suggesting HSPs as an anti-apoptotic mediator of ROS in MIA PaCa-2cells. Conclusion: ECM proteins and growth factors induce intracellular ROS generation in pancreatic cancer cells promoting their survival by inhibiting apoptosis. The results suggest antioxidants as potential therapy for pancreatic cancer.

645 The Long-Term Benefit of Therapeutic Drug Monitoring in IBD Patients Receiving AZA/6-MP Chinyu Su, Sunny Wang, Julius Deren, Andrew Weinberg, Gary R. Lichtenstein, Philadelphia, PA Background: Measuring 6-thioguanine (6-TG) levels has been suggested to be an important tool in optimizing therapy with azathioprine(AZA) or 6-mercaptopurine (6-MP) in inflammatory bowel disease (IBD) patients. No study has examined if long-term benefit is achieved by using this test Aim: To determine the impact Of measuring 6-TG levels on long-term outcomes in patients receiving AZA/6-MP. Methods: Patients with IBD on AZN6-MP had RBC 6-TG levels measured by Prometheus laboratories (San Diego,CA).Diseaseactivity was classified as active or remission. Outcomes during the follow-up period were assessed using a scoring system, assigning 1 point to each of the followings: surgery, hospitalization, dose increase, addition of IBD medications, discontinuation of AZN6-MP, and death, and deducting 1 point for discontinuation of other IBD medications upon achieving remission. Outcomes betweengroups were compared using the two-sample t-test. Results: 53 patients had 79 metabolite studies (mean 1.5 tests per patient, range 1-4). 18 patients (34%) had 6-TG levels > 230* on their initial studies, and 35 patients (66%) had levels < 230. None had leukopenia (WBC < 3.0 X 10 3/UL). Six patients had 6-MMP levels > 5700 (3 with 6-TG > 230). The mean followup duration was 2.8 years (range 0.1-4). There was no significant difference in the outcome scores when patients were stratified based upon 1) the subsequent dose adjustment (changed vs. unchanged) and 2) the 6-TG level (< or > / = 230). There is a trend toward a worse outcome in patients with active disease at the time of 6-TG studies compared to those in remission (mean outcome scores 1.38 vs. 0.69, p = 0.07). Importantly, cOmparing patients whose AZN6-MP dose was adjusted following the 6-TG test with patients whose dose was unchanged,there was no difference in their outcomes within each of the following 3 subgroups of patients: 1) in remission and 6TG < 230, 2) with active diseaseand 6-TG < 230, and 3) with active diseaseand 6TG > 230. Conclusions:Contrary to conventional wisdom, measurementof 6-TG levels and subsequent dose adjustment do not appear to influence patients' outcomes in our study. Patients in remission may have improved long-term outcomes than those with clinically active disease regardless of their 6-TG levels and subsequent dose change. Further studies are necessary to develop specific guidelines regarding the use of therapeutic drug monitoring, particularly focusing on its long-term clinical benefit. *6-TG and 6-MMP levels are expressed in units of pmole/8XlO 8 RBC

643 The Angiogenesis Inhibitor IM862 Increases Survival in Pancreatic Cancer Despite Delayed Onset of Therapy Hubert G. Hotz, Parkash S. Gill, Rizwan Masood, Henriette Graeubig, Birgit Hotz, Heinz J. Buhr, D-12200 Berlin, Germany; LOS Angeles, CA Background: IM862 is a dipeptide of L-glutamyI-L-tryptophan with antiangiogenic properties and potential antitumor activity. The aim of the present study was to evaluate the effect of IM862 on proliferation of human pancreatic cancer (PaCa) cells in vitro and to assess the efficiency of IM862 in an orthotopic nude mouse model of human pancreatic cancer after a delayed onset of therapy. Methods: In vitro: Three human PaCa cell lines (MIAPaCa-2, undifferentiated; AsPC-1, poorly differentiated; HPAF-2, moderately differentiated) were exposed to increasing concentrations (1 p.g/ml- 1000 t~g/ml) of IM862. Cell proliferation was assessed after 3 days by cell count and MTl-assay. In vivo: 1 cubic mm fragments of sc. AsPC-1 donor tumors were implanted into the pancreas of nude mice which received either IM862 (100 mg/kg) or vehicle (control) by daily intraperltoneal injection. Treatment started 6 weeks after tumor induction and continued for a maximum period of 8 weeks. Volume of primary tumor (TU-Votume), metastatic spread (Met-Score), development of ascltes, and animal weight were determined at autopsy. Plasma levels of vascular endothelial growth factor (VEGFP) were measured by ELISA. Microvessel density (MVD) was analyzed in CD31-stained tumor sections. Results: In vitro: Proliferation of all PaOacells was significantly inhibited only at high concentrations of IM862 (> 100 ~.g/ml). In vivo: table. Conclusions: IM662 increases survival in a clinically relevant model of human pancreatic cancer even after delayed onset of therapy, mainly by reducing metastatic spread. In vitro data, decreased VEGFPlevels, and reduced MVD indicate that IM862 inhibits tumor angiogenesis rather than proliferation of PaCa cells. Therapy with IM862 was not associated with systemic side effects such as weight loss.

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646

with the treatment of IBD with steroids or azathioprine are evident at different times and are matched by a corresponding fall in the faecal calprotectin, a surrogate marker of intestinal inflammation. Azathioprine, hut not steroids, is often associated with normalisation of faecal calprotectins, which may explain their efficacy in prevention of disease relapse

Measurement ol 6-TG Levels in Patients with IBD: Are All Groups Identical? Chinyu Su, Sunny Wang, Julius Deren, Andrew Weinberg, Gary R, Lichtenstein, Philadelphia, PA Background: Previous studies have suggested that a 6-thioguanine (6-TG) level > 230* correlates with clinical remission in patients with Crohn's disease(CD). No study has compared the utility of metabolite studies between patients with luminal, fistulizing CD and ulcerative colitis (UC), particularly in clinical practice where the decision to measure 6-TG level is often dependent on patients' clinical status. Aim: To determine the ability of 6-TG levels to predict disease activity in patients with luminal CD, fistulizing CD, and UC. Methods: 6-TG levels in IBD patients receiving AZA/6-MP were measured by Prometheus laboratories (San Diego, CA). Disease activity was classified as mild, moderate, severe activity or remission (ACG Criteria). The association between 6-TG level and diseaseactivity was analyzedby the Fisher's exact test. Results: 79 metabolite studies were analyzed:27 in patients with luminal CD, 18 with fistulizing CO, and 34 with UC. The mean doses of AZA/6-MP were 1.7/1.1, 1.3/1.0, and 1.3/1.1 mg/kg/d in patients with luminal CD, fistulizing CD, and UC, respectively. In patients with luminal CD in remission (n=7), mild (n=lO), moderate (n=5), and severe activity (n=5), the proportions of studies with 6-TG level <230 were 14%, 70%, 80%, and 100%, respectively. In contrast, these numbers were 67%, 57%, 86%, and 0%, in patients with fistulizing CD in remission (n=3), mildly (n=7), moderately (n=7), and severely active (n=l), and 60%, 86%, 56%, and 0% in UC patients in remission (n=lO), mildly (n=14), moderately (n = 9), and severelyactive (n = 1), respectively.There was a significant association between 6-TG level and disease activity for the luminal CD patients (p=O.01), but not for the fistulizing CD (p=0.55) or the UC patients (p=0.14). The sensitivity, specificity, and positive predictive value of using a 6-TG level above >230 to predict clinical remission are 86%, 80%, 60% for luminal CD, 33%, 66%, 17% for fistulizing CD, and 40%, 71%, 36% for UC. Conclusions: Although the 6-TG level > 230 is associated with remission in patients with luminal CD, their association is suboptimal in patients with fistulizing CD or UC. In clinical practice, up to 67% and 60% of patients in clinical remission with fistulizing CD and UC, respectively, will have "subtherapeutic" 6-TG levels. The clinical utility of obtaining metabolite studies in these patients in clinical remission requires further examination. *6-TG levels are expressed in units of pmole/8X10 8 RBC

649 Inappropriately High 1,25-Dihydroxyvitamin D Expression:A Marker for Crohn's Disease and Low Bone Mineral Density Maria T. Abreu, Vitaly Kantorovich, Robin Matuk, Eric Vasiliauskas, Sake Chen, Ying-Chao Lin, Ugis Gruntmanis, Daniel Zehnder, Martin Hewison, Huiying Yang, John Adams, Los Angeles, CA; Birmingham, AL Background: A high percentage of patients with Crohns disease (CD) have low bone mineral density (BMD) and this low BMD is not solely attributable to corticosteroid use. We hypothesized that patients with CD have elevated levels of 1,25-dihydroxyvitamin D (1,25-D) resulting in increased urinary calcium loss and low BMD. We further hypothesized that the increase in serum 1,25-D was secondary to increased 1-hydroxylation of 25-hydroxyvitamin D (25-D) by inflammatory cells in the intestine. Aim: To examine 1,25-D levels in patients with CD compared to patients with ulcerative colitis (UC) and the relationship between 1,25-D levels and BMD in patients with CD. Methods: An IRB-approved, retrospective review of medical records from patients with CD (n=98) or U C (n=21). Measurements of vit D, BMD and urinary calcium excretion (corrected for urinary creatinine) were noted. Charts were reviewed for corticosteroid use which was categorized as none, low (<6 months of exposure) or high (>6 months of exposure). Imm u nohistochemistry for the 25-D-1alpha-hydroxylase was performed on colonic biopsies from patients with CD and normal colons. Resutts: Inappropriately high levels of serum 1,25-D (> 60 pg/ml) were expressed in 49.0% (48/98) of patients with CD compared to only 9.5% (2/21) in UC. Mean serum 1,25-D levels were significantly higher in CD (60.04) versus UC (43.57) (p = 0.0004). In patients with CD, we found a significant negative correlation in 1,25-D levels and lumbar BMD (r= -0.416, p ~ 0.0012). There were no significant differences in 1,25-D levels between patients with low eorticosteroid use (mean 61.35) and high corticosteroid use (61.33). In patients with high corticosteruid use, 1,25-D levels were significant ly negatively correlated with lumbar 8MD (r = -0.49326, p = 0.0019) but not in patients with low corticosteroid use. These data suggest that high 1,25-D levels are independentlyassociated with low BMD. Immunohistochemistry revealedintestinal macrophagesexpressing 25-O-1alpha-hydroxylasein patients witti active colonic CO but not controls. Conclusions: These data suggest that, in addition to corticosteroid use, inappropriately high levels of 1,25-D may be another risk factor for the development of osteoporosis in patients with CD. The reason for el evated 1,25-D may be extrarenal production of the active vitamin O metabolite in the diseased intestine of patients with CO. High 1, 25-D may serve as an additional marker of CD. (Supported by AI40403 and GCRC NIH grant RRO0043))

647 Gene Profiling Reveals UnknownEnhancing and Suppressive Actions of Glucocorticoids on Immune Cells Denis P. Franchimont, Jerome Galen, Naoki Hiroi, Gregory Frey, Jacques Devi~re, John J. O'Shea, Georges Chrousos, Stefan Bornstein, Brussels, Belgium; Paris, France; Bethesda, MD Introduction. Glucocorticoids continue to be the major immunomodulatory agents used in clinical medicine today. However, their actions as antiinflammatory and immunosuppressive drugs are both beneficial and deleterious. A comprehensive analysis of gene regulation by GC has not yet been performed. Materials and Methods. We analyzed the effect of glucocorticolds on the gene expression profile of peripheral blood mononuclear cells from healthy donors, treated with dexamethasone.To accomplish this, we employed the novel technique of DNA microarray analysis, combined with quantitative TaqMan PCR and flow cytometry for confirmation. Expression of glucocorticoid dependent genes (n=25) was then examined in rested and TCR activated PBMCs using quantitative TaqMan PCR. Results. Glucocorticoids induced the expression of chemokine, cytokine and complement family members, as well as of newly discovered innate immune related genes, including scavenger and Toll like receptors. In contrast, glucocortieoids repressed the expression of adaptive immune related genes. Simultaneous inhibitory and stimulatory effects of glucocorticoids were found on inflammatory, T helper subsets and apoptosis related gone clusters. Intriguingly, in cells activated by T cell receptor cross-linking, glucocorticoids downregulated the expression of specific genes that were previously upregulated in resting cells, suggesting a potential new mechanism by which they exert positive and negative effects, depending on the stage of disease. Conclusion. Considering the broad and continuously renewed interest in glucocorficoid therapy, the gone expression profiles that we describe here will be useful in designing more specific and efficient treatment strategies.

650 Modilied Pouchilis Disease Activity Index (mPDAI): A New Approach to the Diagnosis of Pouchitis Be Shen, Bret Lashner, Jean-Paul Achkar, Jason T. Conner, Adrian H. Ormsby, Feza H. Remzi, Aaron Brzezinski, Charles L. Bevins, Victor Fazio, Cleveland, OH Pouchitis is the most common complication of ileal pouch-anal anastomosis for ulcerative colitis (UC). Our previous study suggested that symptom alone is not reliable for the diagnosis of pouchitis (Gastroenterology 2001 ;121:261-7). The most commonly used diagnostic instrument is the 1B-point PeAl consisting of 3 principal component scores: symptom, endoscopy, and histology. Our cost analysis showed that pouchoscopy without biopsy had a comparable cost per diagnosis of pouchitis when compared with a diagnostic therapeutic trial of antibiotics. However, it is not known whether pouchoscopy without biopsy can reliably diagnose pouchitis in symptomatic patients. Aim: We analyzed PDAI component scores in symptomatic patients with or without pouchitis to determine whether omitting histologic evaluation significantly affects the sensitivity and specificity of the diagnostic criteria. Methods: UC patients with symptoms (diarrhea, abdominal pain, rectal urgency, bleeding, or fever) were evaluated. Patients with PDAI > 7 were diagnosed as having pouchitis. Different diagnostic strategies were compared based on the PDAI scores. Receiver operating characteristic (ROC) curves were usedto determine the best cut-off points where sensitivity and specificity were maximized. Area under curve (AUC) measured how much these diagnostic strategies differed from each other. Results: 58 consecutive symptomatic patients were enrolled; 32 patients (55%) were diagnosed with pouchitis. The mean total PDAI, symptom, endoscopy, and histology scores in patients with or without pouchitis were 9.7 + 2.0 vs 4.5 + 1.2, 3.2 +_ 1.0 vs 2.1 __+1.2, 3.6 + 1.5 vs 0.2 _+ 0.4, and 2.9 + 0.9 vs 2.7 + 0.6, res-peetively.Using only symptom and ~doscopy scores (mPDAI) with~ cut-point o f ~ 5 led to an AUC of 0.99 with a sensitivity of 94% and specificity of 100%.

648 Faecal Calprotectin Falls in Responseto Treatment of Inflammatory Bowel Disease with Azathioprine or Steroids Simon Smale, Russell Foster, Ingvar Bjarnason, London, UK Background: Faecal calprotectin is a stable non-degraded protein ~argelyconfined to neutrophils. Levels of faecal calprotectin relate quantitatively to the degree of inflammation within the gastro-intestinal tract, correlate significantly with clinical disease activity scores in patients with inflammatory bowel disease (IBD) and provide predictive information of relapse in IBD. Corticosteroids and azathioprine differ in their ability to induce and maintain remission. We contrasted the effect of steroids or azathioprine in reducing both symptoms and levels of intestinal inflammation as measured by faecal calprotectin. Methods: 17 patients with relapse of IBD, as defined by a Crohn's disease activity Index (CDAI) of greater than 250 for those with Crohn's disease, increasein PowelI-Tuck index in those with ulcerative colitis, or chronic active disease necessitating administration of oral steroids (40rag initial dose) or azathoprine (2mg/kg). Patients provided samples for faecal calprotectin estimation at the time of relapse, after one week, one month and at three months. Results: The normal median for faecal calproteetin is 4 mg/L (range 0.5-11 rag/L). Of 11 patients treated with steroids mean calprotectin fell from 133 to 100 mg/L at one week (n = 8), 42 mg/L at one month of treatment (n =8), and 66 mg/L at three months (n =5). This was matched with a corresponding fall in the clinical disease activity scores. By comparison those treated with azathioprine had mean calprotectin values falling later and to a greater extent; from 77 mg/L at baseline to 66mg/I at one week, 56mg/I at one month and 13mg/I at three months. These patients also had a corresponding fall in disease activity scores. Conclusion: Clinical improvement associated

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Components

AUC

Sx + Endosc + Histol (PDAI) Sx + Endosc (mPDAI) Sx only (mPDAI)

1.00 0.99 0.76

ScaIe range 2-18 0-12 0-6

Pouchitis Cut-point > 7 > 5 > 3

Sensit.

Specif.

1.00 0.94 0.75

1.00 1.00 0.62

Conclusions:The mPDAI, consisting of symptom and endoscopy scores without histology, offers similar sensitivity and specificity when compared to the standard PDAI. In addition, the mPDAI offers better sensitivity and specificity when compared to symptom assessment alone. Omission of endoscopic biopsy and histology from PDAI would simplify pouchitis diagnostic criteria, reduce cost of diagnosis, and avoid delay in determining histology score, while providing equivalent sensitivity and specificity.

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654

(p = 0.001). 2 pts with hepatic metastasesdied due to the gastrinoma and none of the patients without hepatic metastases (p=O.03 4). CONCLUSIONS: These results demonstrate that hepatic metastasesoccur more commonly than previously reported in patients with duodenal gastrinomas. In contrast to pancreatic gastrinomas there is no correlation between the size of the duodenal gastrinoma and the occurrence of hepatic metastases. Hepatic metastases develop more frequently in patients without a prior duodenal gastrinoma resection: These results support the conclusion that all patients with ZES with or without MEN1 who might have a duodenal gastrinoma should undergo surgical exploration to prevent the development of hepatic metastases,which have beenshown to be the main determinant of long-term survival.

Expansion of Nestin-Positive Precursor Cells During Acinar-to-Ductal Metaplasia in Mouse Pancreas Yoshiharu Miyamoto, Ingrid M. Meszoely, Anna L Means, Bidyut Ghosh, Steven D. Leach, Baltimore, MD; Nashville, TN Acinar-to-ductal metaplasia may represent an initiating event during pancreatic cancer formation in both mice and humans. Based on the recent demonstration that multipotent precursor cells in rodent pancreas are marked by expression of the intermediate filament, nestin, we sought to determine the role of nestin-expressing intermediates in an in-vitro model of acinarto-ductal metaplasia. Methods: Acinar-to-ductal metaplasia was induced in primary explant cultures of mouse pancreas treated with recombinant human TGFalpha.Successful induction of acinar-to-ductal metaplasia was confirmed by immunoblot and immunofluorescent (IF) labeling for amylase and cytokeratin 20 (CK20). At daily intervals, nestin expression was determined by quantitative RT-PCRas well as IF labeling, with E14.5 mouse brain as a positive control. Both actin and GAPDH were used to confirm equal loading of RT-PCRconditions, and PCR reactions were sampled at after 20, 25, and 30 cycles to insure log-range detection of PCR product. All experiments were performed in triplicate. Results: TGFalpha-induced acinar-to-ductal metaplasia developed progressively over a five-day time course and was confirmed by phase contrast microscopy as well as recipiocal changes in the expression of amylase and CK20. Freshly isolated mouse pancreas demonstrated an abundance of acinar cells, a paucity of ductal epithelium, and little-to-no detectable nestin expression by either RT-PCR or IF. Three days later, there was strong nestin expression documented by both RTPCR and IF. At this time point, a small population of CK20-positive epithelium was present. By day five, nestin expression was again down-regulated, and the majority of the epithelium was now comprised of large CK20-positiveductal structures. Nestin expression was quantified by calculating the ratio of nestin:actio RT-PCR product (* indicates p<.05 vs. Day 0 by ANOVA; values represent mean+/-SEM of three independent experiments): Day O, 0.35--/0.5; Day 1, 0.67+/-.08; Day 2, 1.1 +/-0.2"; Day 3, 1.2+/02"; Day 4, 1.3+/-0.5"; Day 5, 0.8+/-0.1" Conclusions: The results demonstrate that TGFalpha-induced acinar-to-ductal metaplasia arises through a nestin-positive intermediate cell type. Given the known ability of nestin-positive precursor cells to generate both endocrine and exocrine cell types, these data suggest that early events in pancreatic cancer may involve activation of epithelial stem cells.

660 Recombinant Activated Factor VII (rFVIla) in the Treatment of Four Cases of Severe Hemorrhage from Portal Hypertension(PHi Rafael Romero, Francisco Pellicer, Manuel Jimenez, Manuel Gomez, Angel Sendon, Maria Dolores Guerrero, Juan Manuel Herrerias, Seville, Spain Objetives:Toevaluatethe efficacy of rFVila in achieving hemostasis in severe upper gastrointestinal bleeding (UGIB) resulting from PH. Methods:We studied the hemostatic effect of a single intravenous dose of 4.8 mg of rFVIla (Novoseven~; Novo Nordisk NS, Bagsvaerd, Denmark) given as a bolus over 3 minutes. All patients were experiencing severebleedingfrom esophageal or gastric varices and treated with somatostatin. The drug was administered through a compassionate use protocol, after written consent. Hemostasis was determined by endoscopy, hemodynamic, hematologic parameters and transfusional requirements, Results:Case I:A 50yr male with Child's B cirrhosis with bleeding from a deep ulcer due to recently variceal sclerosis. Hemodynamic instability persisted in spite of transfusion of 7 packed red blood cell (RBC) and Sengstaken.Affer administration of 90 i~g/bw of rFVIla 5 days later, hemostasis was obtained. Prothrombin time (PT) fell from 18.3" to 15.1" (normal 11-13"). No further transfusions were needed. He is now doing well after liver transplantation. Case 2:A 51-yr male with Child's A cirrhosis who rebind from esophageal varices following withdrawal of a Sengstaken. Hemoetasis was achieved within a few minutes after infusion of 56 i~g/bw of rFVIla and a firm clot was formed. Sclerosis was carried out later and he was discharged 9 days later. Case 3:A 67-yr male bled from gastric varices which were sclerosed and 8 packed RBC were transfused. He rebled 3 days later. Following infusion of 70p,g/bw of rFVIla bleeding stopped, which revealedan ulcer on the gastric varix. Two days later he rebind and aplenectomy for splenic vein trhombosis was carried out. Then, he was discharged doing well now. Case 4:A 55-yr male with Child's C cirrhosis bled from esophageal varices treated with sclerosis. A few days later rebled, but it was impossible to perform any procedure but Sengstaken placement due to the huge bleeding. In spite of balloon, bleeding persisted and 8 packed RBC were transfused in 14 hours. The balloon was removed but nothing was seen again. However, after 50 i~g/bw of rFVIla, bleeding stopped and we could see the esophagealvarices and perform sclerosis. PT passed from 44" to 24.6". The patient died from multisystemic failure. Conclusions: This is, to our knowledge, the first time where rR/lla has been used as a hemostatic agent in the treatment of acute GI bleeding.The results warrant further studies to confirm the efficacy, safety and cost-effectiveness in severe UGIB from PH.

655 Pancreatic Intraductal Papillary Mucinous Neoplasm (IPMN) with Cancer: LongTerm Survival and Prognostic Factors Dhiraj Yadav, Thomas C. Smyrk, Jonathan E. Clain, Laurence J. Miller, Randall K. Pearson, Suresh T. Chari, Rochester, MN Aims: To determine a) long-term survival in IPMN with in situ and invasive cancer and b) prognostic factors in IPMN with invasive cancer. Methods: In an ongoing effort, IPMN cases are being identified prospectively and retrospectively from PancreasClinic and Surgical Pathology databases at Mayo Clinic. The slides of 48 resected IPMN with cancer were reviewed by a single pathologist (TCP) to confirm diagnosis, classity cancer as in situ (n =10) or invasive (n =38), invasion as tubular (n=26) or muco-nodular (n=12), margin as negative (n=33) or showing dysplasia/cancer (n=5) and nodes as negative (n=26) or positive (n=12). Clinical (age, gender, extent of surgical resection) and follow-up data were obtained from patient records and/or by contacting patient. Recurrence was diagnosed by CT scan and histologically confirmed in majority of patients. We used Kaplan-Meier method for survival analysis and Cox-proportional hazards model to determine prognostic factors. Results: In IPMN with in situ cancer, 0/10 have developed recurrence or died of disease (mean followup 65 mo). In IPMN with invasive cancer the median survival was 41 mo and 5-year survival was 34%. On univariate analysis, positive margins, positive nodes and recurrence were predictors of death while type of invasion and extent of resection (limited vs total pancreatectomy) were not. On multivariate analysis, recurrencewas the only predictor of death ( p - 0.002). Median time to recurrence was 18 mo (range: 1-63) and median time to death after recurrence was 12 mo (range: 0-142). While 20/24 (80%) patients with recurrence have died of disease (median follow-up: 33 mo), only 3/14 (21%) patients without a recurrence have died, all of unrelated causes (median follow-up: 32 mo, p
661 The Cost Effectiveness o! Hepatic Venous Pressure Gradient (HVPG) Monitoring in Prevention of RecurrentVariceal Bleeding (RVB) Lewis Targownik, Brennan M. R. Spiegel, Hetal A. Karsan, lan M. Grainek, Los Angeles, CA Background: The risk of RVB is high following an initial variceal bleed in cirrhotic patients. Strategies to prevent RVB include endoscopic band ligation (EBL), and most recently the use of combination beta-blockers and nitrates (BBN). A significant decrease in HVPG (decline to <12mmHg or <80% of initial value) has been associated with a decreased rate of RVB in patients on BBN: HVPGmonitoring may be a useful adjunct to BBN therapy to identity treatment failures requiring alternative management. The study objective was to determine the cost effectiveness (C/E)of HVPG monitoring in prevention of RVB when BBN is used. Methods: Decision analysis with Markov modeling was usedto calculatethe C/E ratios of three competing strategies for the prevention of RVB in a patient with Child's ClassNB cirrhosis after successful hemostasis of an initial variceal bleed. The strategies were: 1) EBL, 2) BBN with HVPG monitoring to determine efficacy of therapy (BBN/HVPG+), and 3) BBN without HVPGmonitoring (BBN/HVPG-). Patients without a significant decrease in HVPG were offered EBL. Cost estimates were from a 3'd party payer perspective and time horizon was 2 years. Probability estimates were derived from a systematic review of the literature. Monthly rates of RVB were: EBL = 1.8%, BBN with significant decline in HVPG= 0.7%, BBN without significant decline in HVPG= 4.2%. 60% of patients on BBN were assumed to have a significant HVPG response. Mortality rate was 1A%/mo for all strategies. Compliance rates were: EBL- 80%, BBN = 70%, HVPG monitoring=65%. Primary outcomes were prevention of RVB and cost per RVB prevented. Results: Under base-case conditions, the BBN/HVPG+ strategy was the most effective for prevention of RVB (33% with bleed at 2 years vs. 38% EBL and 41% BBN/ HVPG-. BBN/HVPG- cost the least per RVB pre~'entedat $7215, compared tO $11,310 for BBN/HVPG+ and $13,614 for EBL. There was an incremental cost of $42,633 to prevent one additional RVB with the use of HVPG as an adjunct to BBN monitoring compared to BBN alone. In a compliant patient population (compliance with alltherapies > 90%), the incremental cost for this strategy was reducedto <$30,000 per RVB prevented. Conclusions: BBN therapy with HVPG monitoring appeared cost effective in preventing RVB. ICER was further improved in a highly compliant population, making the use of HVPG appealing as an adjunct to therapy in highly ~ommitted patients, e.g., patients awaiting OLT. Further prospective studies are warranted to confirm these findings.

656 Duodenal Gastrinomas Are More Aggressive Than Generally Thought: Results of a Prospective Study Fathia Gibril, Laurence Entsuah, Robert T. Jensen, Bethesda, MD BGD: The most frequent location of gastrinomas is the duodenum. Duodenal tumors are thought to be less aggressive than pancreatic gastrinomas based on limited data. Because patients with metastatic disease usually do not undergo surgical exploration and because duodenal tumors are commonly missed, their natural history is largely unknown. AIM: To evaluate the natural history of patients with duodenal gastrinomas. METHODS: 68 pts with duodenal gastrinomas were prospectively studied for a median of 10 yrs [range, 0.7-21.2]. Pts were evaluated every 3-6 mos with and every 12-18 mos without hepatic metastases using biochemical and imaging studies (MRI, US, CT, angiography and octreoscan since 1994). RESULTS: The mean age was 57.6 +/- 1.2 yrs and14% had MEN1.62 pts had a surgical resection and in 6 pts the tumor was diagnosed by biopsy (n = 4) or autopsy (n = 2). The mean tumor diameter was 1.2 +/- 0.1 cm with 75% equal or less than 1.3 cm and 9% > 2 cm. Thirteen (19%) pts had hepatic metastases with 6 (9%) at presentation and in 7 (10%) pts they developed during the study period. Tumor size did not correlate with the presence of liver metastases. Hepatic metastases developed in 3/56 (5%) pts post resection and in 4/6 (67%) pts without a resection (p = 0.0009). Patients with hepatic metastaseswere younger at onset of the disease (p=0.0087) and the mean fasting gastrin level was higher

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AGA A b s t r a c t s

healthcare utilization patterns. Conclusion: Significant unsuspected disparities exist in the diagnosis of HCV in Rispanics and may also be present in the treatment of HCV in blacks. Factors influencing these disparities likely include language and/or cultural differences in healthcare utilization. Identifying the barriers underlying these disparities is the first step toward improving both diagnostic and therapeutic efficacy and ultimately decreasing the disease burden imposed by HCV.

662 In Mouse Thoracic Aorta, Rite Acid interaction with Endothelial M3 Muscarinic Receptors Causes Nitric Cxide-Mediated Vasodilation Sandeep Khurana, Richard H. Kennedy, Masahisa Yamada, Jorgen Wess, Guofeng Xie, Jean-Pierre Raufman, Little Rock, AR; Bethesda, MD In rat thoracic aorta some bile acids, like deoxycholyltaurine (DCT), cause endothelium-dependent smooth muscle relaxation (Gastroenterology 120:A376, 2001). This effect is NO-mediated and inhibited by the acetylcholine:lithocholic acid hybrid, lithocholylcholine, an M3 muscarinic receptor antagonist (Gastroenterology 118:A82, 2000). Objective: To confirm the interaction of bile acids with muscarinic receptors, we evaluatedthe effects of DCT on thoracic aorta from wild type (WT) and M3 receptor knock out (M3KO) mice. Methods: Aortic segments from male mice (25 --33 gm) were mounted on strain gauges in baths containing Krebs buffer. DCT (0.1 p,M --1 mM) was added to baths containing murine aortic segments with endothelium that was intact or denuded by gentle rubbing. Segments were pretreated with 10 ~M phenylephrine (phe). Experiments were also conducted in the absence or presence of L-nitro arginine methyl ester (L-NAME),an inhibitor of NO synthese.The effects of acetylcholine (ACh), 0.01 p,M --30 p,M, were also examined on these segments. Results: In WT mice ACh caused dose dependent aortic smooth muscle relaxation in segments with intact, but not denuded, endothelium (p=O.O04, 2-way rpt ANOVA). Increasing doses of ACh had no effect on segments from M3KO mice with intact endothelium (p = 0.005). Increasing concentrations of DCT caused progressive relaxation of phe--constricted aortic segments. In WT mice there was a greater decrease in tension in segments with intact compared to those with denuded endothelium (p = 0.004). With DCTconcentrations of 0.1,1.0 and 10 p,M, the tension decreased more in segments with intact endothelium from w r mice (77 _+6%, 58 _+4% and 64 _+5%; mean _+ SD) compared to those from M3KO mice (99 _+5%, 91 _+4% and 85 _+4%) (p
Table 1 Ethnicity Healthcarewstem Patients diagnosed with HCV Patientstreatedfor HCV

White 25.2% 49.8% 61%

Black 14.1% 14.8% 5%

Other 8.3% 3.7% 7%

665 Keratin Mutations Predispose to Cryptogenicand NoncryptogenicLiver Disease Nam-On Ku, Jama Darling, Teresa L. Wright, Carlos O. Esquivel, Emmet B. Keiffe, Sheri Krams, M Omary, PaiD Alto, CA; San Francisco, CA Background: We previously described keratin 8 and 18 (K8/18) mutations in 6 of 55 patients with cryptogenic liver disease (two K8 Tyr53His, three K8 Gly61Cysand one K18 His127Leu), which were absent in 98 patients with noncryptogenic liver disease and in 86 randomly selected patients (N Eng J Med 344:1586-1587, 2001). The two K8 mutations interfered with the ability of keratin filaments to reorganizeproperly in response to oxidative and nonoxidative stresses. The identification of keratin mutations in patients supports the extensive body of transgenic animal data showing that keratins play an essential role in protecting hepatocytes from mechanical and nonmechanical stresses. Aim: Assess the role of keratin mutations in noncryptogenic liver disease and identity the frequency of keratin mutations in the general population. Methods: We screened for K8 and K18 mutations in genomic DNA isolated from 323 liver explants of patients with a wide range of noncryptogenic liver diseases, and from 319 normal blood-bank volunteers. Results: Seven unique K8 or 18 amino acid mutations were found in 11 independent liver explants of patients with biliary atresia, hepatitis C or B, alcohol, primary biliary cirrhosis, and acute fulminant hepatitis. Seven of these 11 patients had the previously described mutations in patients with cryptogenic liver disease: three K8 Tyr53®His, three K8 Gly61Cys and one K18 His127Leu. The 4 other mutations from the 11 patients are located in K8 head and K18 head or rod domains: K8 G52V; K18 TIO2A, R260Q, and G339R. Of the 319 normal blood samples, only one contained the K8 G61C mutation, and none contained the remaining unique mutations. Eleven individuals from the normal control group also harbored mutations at two additional sites (seven K8 162Vand four K18 $229T) that likely represent polymorphisms. These polymorphisms were also represented in three patients from the liver disease group (two with K18 $229T and one with K8 162V). Three silent mutations are also present in K8 head and 1(8/18 rod domains. Conclusions: Combining the data herein with our previous data shows that unique and likely functionaltering keratin mutations are present in 17 of 467 patients (3.6%) with various forms of liver disease, while only one of 319 normal volunteers (0.3%) harbored such mutations (p
663 Small and Multiple Gallstones Are Major Risk Factors for Acute Biliary Pancreatitis. Niels G. Venneman, Marc Besselink, Peter Go, Koop 8osscha, Hein G. Gooszen, Gerard P. vanBerge-Henegouwen,Karel J. van Erpecum, Utrecht, Netherlands; Nieuwegein, Netherlands Although acute gallstone pancreatitis is associated with considerable morbitlty and mortality, potential risk factors have not yet been clearly defined. Methods: We compared sizes and numbers (by ultrasound) of gallbladder and --if present-- bile duct stones (by ERCP) in 98 pts with acute pancreatitis, 57 pts with obstructive jaundice but without pancreatltis, 77 pts with acute cholecystitis, and 142 pts with uncomplicated symptomatic gallbladder stones. Gallstone sizes by ultrasound were highly correlated to sizes at subsequent surgery (n = 16: r= 0.92). Results: Pts with complicated gallstone disease were older (58 vs 47 yrs, P< 0.001) and more often males (44% vs 22%, P< 0.001) than pts without complications. Both pts with acute pancreatltis and pts with obstructive jaundice had more and smaller stones or sludge in their gallbladders than pts with acute cholecystitis or uncomplicated disease (smallest stone diameters: 3_+1, 3_+1, 8_+1, 9±1 mm resp., P< O.O01).ln contrast, bile duct stones (by ERCP)were smaller (smallest stone diameters: 3_+ 1 vs 9_+ 1 ram, P< 0.001) and sludge occurred more frequently (33% vs 17%, P= 0.03) in case of pancreatltis than obstructive jaundice. Multivariate analysis identified stone size and age as independent risk factors for pancreatitis. Conclusions: Small and multiple gallbladder stones are major risk factors for acute pancreatltis or obstructive jaundice. After migration to the bile ducts, small stones and sludge are more likely to induce pancreatitis, whereas larger stones may induce obstructive jaundice. Threshold for cholecystectomy should be low, particularly in case of small gaLIhledderstones.

666 Ornidazol for Prophylaxis of Postoperative Crohn's Disease: Final Results of a Double Blind Placebo Controlled Trial Paul Rutgeerts, Gert Van Assche, Geert D'Haens, Filip Baert, Maja Noman, Isolde Aerden, Karel Geboes,Andre D'Hoore, Freddy Penninckx, 3000 Leuven, Belgium Background and Aim: Crohn's disease almost inevitably recurs after surgery and efficacious prophylactic therapy is still lacking. We investigated the usefulness of Ornidazol prophylaxis in a placebo controlled double blind trial Methods: Eighty patients were randomized to Omidazol (Tiberal®, Roche, Switzerland) 1g/day or placebo for one year started within one week of curative ileal or ileocolonic resection with ileocolonic anastomosis. No other antiinflammatory drugs were permitted. Anti-diarrheals could be used by the patients. Ileocolonoscopy was performed at 3 and 12 months and clinical activity of the disease was assessed at 3,12,24 and 36 months after resection. Primary endpoints were endoscopic recurrence and symptomatic recurrence based on Il-r analysis at 12 months. Clinical relapse was defined as the appearanceof symptoms judged by the treating physician to represent a flare of active disease. At that time the study drug was stopped and other therapies started. The patients were further followed upto three years. Results: Two patients were excluded since they were not dosed because of withdrawal of informed consent. Ornidazol prophylaxis significantly reduced (table 1) the endoscopic recurrence rate both at 3 and 12 months and also the symptomatic recurrence rate was significantly decreasedat 12 months with Ornidazoltherapy. Smoking, duration of disease, extent of disease and intestinal resection, age and gender did not significantly influence outcome. There was a close relationship between development of severe Crohn's lesions in the neoterminal ileum as visualized at endoscopy and clinical relapse. After discontinuation of therapy the prophylactic effect disappearedat 24 and 36 months after surgery. Significantly more patients dropped out from the study because of side effects in the Ornidazol group. Conclusion: Prophylaxis with the Nitro-lmidazol antibiotic Ornidazol is efficacious in postoperative Crohn's disease and we advocate this therapy although tolerance may be a problem.

664 Ethnic Disparity in the Diagnosis and Treatment of Hepatitis C Virus (HCV) in a Large Urban Health System John F. Riopelle, Anisa K. Moore, Sharon Bahrych, Peter McNally, Vaman Jakribettuu, William Brown, Katherine Doll, Nell Toribara, Denver, CO Background: HCV disproportionately affects minority populations, with estimated seroprevalences of 2.1% in Hispanics, 3.2% in blacks, and 1.5% in whites. The patient population of our institution is largely minorities. Therefore, when analysis of the first 155 consecutive patients treated for HCV revealed a striking predominance of whites (Table 1), we examined the available demographic data to determine the cause for this marked disparity. Methods: Our database consisted of 666 patients who were diagnosed with HCV (ICD-9 codes 070.51 or 070.54) during the calendar year 2000. Age, sex, ethnicity, primary language, employment status, and household income were obtained from a self-reported cover sheet via retrospective electronic chart review. Results: See Table 1. These three groups were similar in average age and unemployment percentage.English was the primary languagefor 98% of black and white patients. However, 92.4% of Hispanic patients identified English as their primary language while only 7.6% listed Spanish, a surprising difference given that 31.3% of all Hispanics seen in our system speak Spanish only. Discussion: Analysis of our demographic patterns (Table 1) showed: (a) Hispanics are much less likely to be diagnosed with HCV, but once diagnosed they are appropriately referred for treatment, (b) whites are much more likely to be diagnosed and even more likely to be treated, and (c) black patients are diagnosed in proportion to their overall demographic representation but are less likely to receive treatment. Multiple factors probably contribute to these disparities. For example, the primary language data strongly suggests that HCV is underdiagnosedin Hispanics who speak primarily or exclusively Spanish. Also, blacks diagnosed with HCV may not seek treatment doe to cultural differences and

AGA Abstracts

Total Hispanic 149,765 52.4% 666 31.7% 155 27%

Results Omidazol 1g/day Placebo p value

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EndoscopicRecurrence 3 months 12 months 34% 54% 59% 79% 0.046 0.036

ClinicalRecurrence 12 months 24 months 36 months 8% 27% 35% 37% 45% 48% 0.002 0.1 0.27

667

unchanged/worse compared with index colonoscopy. Results: There were no differences between groups in any clinical, demographic, laboratory parameters, prior treatments, or duration of remission. After 1 year, 30 patients on AZA and 25 on BUD were in remission; 19 and 17 patients in the AZA and BUD groups, respectively, had ileocolitis. Healing of colonic lesions in AZA and BUD groups respectively was graded as: complete in 22/30 (73%) and 6/25 (24%); almost complete in 3/30 (10%) and 5/25 (24%); partial in 4/30 (13%) and 5/ 25 (20%); and unchanged/worsein 1/30 (4%) and 9/25 (36%) (2 = 15.83, p - 0.002). Healing of ileal lesions in AZA and BUD groups was graded respectively as: complete in 11/19(58%) and 2/17 (12%); almost complete in 4/19 (21%) and 3/17 (18%); partial in 3/19 (16%) and 4/17 (24%); and, unchanged/worse in 1/19 (5%) and 6/17 (35%) (x2 12.01, p=O.O17). The terminal ileum was not entered in 2 BUD patients. Areas of inflammatory ileal stenosis became patent in 14/19 AZA patients and 4/17 BUD patients (p = 0.02). Ileocecal valve lesions were seen in 12/19 patients on AZA and 15/17 patients on BUD; these were very mild in AZA group. Endoscopic complete/almost complete healing was associated with chronic inactive inflammation in AZA group; however, this was associated with underlying acute on chronic inflammation in the BUD group. Conclusion: After one-year maintenance therapy in clinically quiescent Crohn's disease, AZA was superior to BUD in improving endoscopic heating and histologic remission.

Once Daily High Dose Probiotic Therapy Maintains Remission and Improved Quality of Life in Patients with Recurrent or Refractory Pouchitis: A Randomised, PlaceboControlled, Double-Blind Trial Toshiki Mimura, Fernando Rizzello, Stephan Schreiber, lan C. Talbot, R J. Nicholls, Paolo Gionchetti, Massimo Campieri, Michael A. Kamm, Tokyo, Japan; Bologna, Italy; Kiel, Germany; London, UK Backgrounds and Aim: Most patients with acute pouchitis respond to antibiotics, but 10-15% experience recurrent or refractory pouchitis. This study aimed to evaluate the effectiveness of a probiotic therapy in maintaining the remission and quafity of life (QOL) in these patients. Methods: Patientswith active refractory or recurrent pouchitis, who were successfully brought into remission by a 4 week course of metronidazole and ciprofloxacin, were randomised into 2 groups: once daily VSL#3 6g or Placebo for one year or until relapse. VSL#3 is a probiotic preparation, containing 300 billion bacterial cells per gram of 4 strains of Lactobacilli, 3 strains of Bifidobacteria and one strain of Streptococcus. Symptomatic, endoscopic and histological evaluations were made before and 2 and 12 months after the randomisation or at the time of relapse, using the Pouchitis DiseaseActivity Index (POAI). Active refractory or recurrent pouchitis was defined as both PDAI score -> 7 ,range 0 (perfect) to 18 (worst),, and either a history of pouchitis at least twice in the previous one year or persistent pouchitis requiring continual intake of antibiotics. Remission was defined as clinical PDAI score _< 2 and endoscopic PDAI score _< 1. Relapsewas defined as the increase of clinical PDAI score -> 2 and the increase of endoscopic PDAI score >- 3 compared to the baseline score at entry. QOL was assessed with the Inflammatory Bowel Disease Questionnaire (IBDQ).RESULTS: Thirty-six patients (18 Male; median 36 years old) were randomised: 20 on VSL#3 and 16 on Placebo. 2 patients (10%) on VSL#3 relapsed, while 15 (94%) on placebo relapsed during the one-year study (p
670 Tacrolimus (FK506) for the Treatment of Perianal and EnterocutaneousFistulas in Patients with Crohn's Disease: A Randomized, Double-Blind, Placebo-Controlled Trial William J. Sandborn, Daniel H. Present~Kim L. Isaacs, Douglas C. Wolf, Eugene Greenberg, Stephen B. Hanauer, BrianG. Feagan,Therese Johnson, Galenko Joseph, Christopher F. Martin, Robert S. Sandier, Rochester, MN; New York, NY; Chapel Hill; NC; Atlanta, GA; Urbana, IL; Chicago, IL; London, ON, Canada

668 Randomized Double Blind Comparison of 4 mg/kg versus 2 mg/kg IV Cyclosporine in Severe Ulcerative Colitis Gert Van Assche, Geert D'Haens, Maja Noman, Martin Hiele, Katrien Asnong, Isolde Aerden, Caroline Swijsen, Joris Arts, Paul Rutgeerts, Leuven, 3000, Belgium Background and aim: High dose IV cyclosporine (cy-A) is efficacious in severe attacks of ulcerative colitis and most patients (pts) avoid colectomy. Cy-A carries a substantial burden of toxicity, mainly at high doses. The aim of the present study was to investigate a dose effect and therefore we compared 4 mg/kg with 2 mg/kg IV cy-A in pts admitted for severe UC flares. The hypothesis was that 4 mg/kg cy-A has a superior clinical efficacy and earlier response. Methods: 70 pts with severe UC requiring hospital admission were included and randomized to receive 2 or 4 mg/kg initial dose of cy-A ina continuous infusion over 24 hrs. An 8 day course of IV cy-A was given and plasma levels were maintained at 150-250 ng/ml (2 mg/kg group) and 250-350 (4 mg/kg) by an independent physician. Only pts with a Lichtiger CAI of >-10 were eligible and clinical response was defined as a drop below a score of 10 with a decrease of >-3 points. Sigmoidoscopy with colonic biopsies was performed at day 0 and 8. At day 8 pts were started on Neural® aiming at trough levels of 150-250 ng/ml. Results: 35 pts were included in each group. One patient (4 mg/kg) was withdrawn after the start of the first infusion due to an anaphylactic reaction, but all other pts completed the 8 (:lay IV cy-A course. 16/35 (4 mg) and 21/35 (2 mg) pts received concomitant steroid treatment. 10/ 35 (4 mg) and 9/35 (2 mg) of pts were on stable doses of azathioprine, all others were started at inclusion accept for known azathioprine intolerance. CAI response rates were 28/ 34 (4 mg/kg) and 29/35 (2 mg/kg). One patient had a colectomy in the first week, and 8 during 3 months follow up 5 (4 rag) and 3 (2 rag) . The median drop in CAI at day 8 was 7 in both groups. The median time to response was not different between the two groups 4 mg/kg: 4 days (2-8); 2 mg/kg 4 days (2-7) . 83% of pts with concomitant steroids responded versus 77% in non-steroid users. The mean daily dose of IV cy-A received over one week was: 2.65+_0.47 (4 mg/kg) and 1.82-+0.32 (2 mg/kg) mg/kg (P
Background: We evaluated the safety and efficacy Of tacrolimus for the treatment of perianal and enterocutanoeus fistulas in patients with CD. Methods: 48 patients with CD complicated by actively draining perianal or enterocutaneous fistulas were enrolled in a lO-week doubleblind trial and randomizedto oral tacrolimus 0.2 mg/kg/day or placebo.The doses of aminosalicylates, antibiotics, prednisone, and purine anti-metabolites were held constant. Patients treated with infllximab within 4 weeks were excluded. The primary outcome measure was improvement of open draining perianal or enterocutaneousfistulas (defined as > 50% reduction from baseline in the number of open draining fistulas for at least 4 weeks). A secondary outcome measure was closure of all fistulas for at least 4 weeks. Nephrotoxicity was defined as a rise in serum creatinine to a value > 1.5 mg/dL Results: The characteristics of the two groups were similar. 29 patients had previously received inffiximab. 2 patients (1 in each group, did not have a baseline assessment of fistula activity). Of the remaining 46 patients, 9/21 (43%) of tacrolimus-treated patients had improvement as compared with 2/25 (8%) of placebo-treated patients (p=O.O04). 2/21 (10%) of tacrolimus-treated patients had closure of all fistulas as compared with 2/25 (8%) of placebo-treated patients (p=0.86). Prior treatment with infliximab or failure to respond to infliximab did not influence improvement with tacrofimus. The frequency of selected adverse events in the tacrolimus and placebo groups were: any adverse event 95% vs 76% (p = 0.05); paresthesias57% vs 4% (p25% and >-70 points), and

669 Maintenance Therapy with Azathioprine Is Superior to Budesonide in Healing Endoscopic Lesions and Improving Histology in Clinically Quiescent Crohn's Disease Gerassimos J. Mantzaris, Kalliopi Petraki, Helen Chadio-lordanides, Angeliki Christidou, Alexander Karagiannidis, John Glarakis, Emmanuel Archavlis, George Triantafyllou, Athens, Greece Aim: To compare azathioprine (AZA) and hudesonide (BUD) in the endoscopic and histologic remission of Crohn's disease (CD). Methods: Sixty seven patients with inflammatory steroid dependent Crohn's ileocolitis or colitis in clinical remission (CDAI<150) for at least one month were randomized to receive AZA (2.2 mg/kg, n =34) or BUD (6-9 mg/day, n =33) for one year. Remission had been achieved on prednisolone before switching to AZA or BUD. Clinical examination, laboratory tests and CDAI measurementwere performed at baseline and at 2 month intervals for 1 year. Ileocolonoscopy with biopsies was performed at baseline and at 1 year. The one-year outcome of baseline lesions was graded as complete (no ulcers), almost complete (occasional ulcers/erosions), or partial healing (remaining big ulcers), and

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inflammatory bowel disease questionnaire (IBDG) scores. Conclusion: The ACCENT II trial is the largest trial in fistulizing CD and confirms that 3 doses of infliximab are effective in the short-term. Long-term response to maintenance therapy relative to maintenance placebo therapy will be reported.

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The aim of this study was to investigate the effects of high-frequency gastric electrical stimulation (GES) on symptoms, gastric slow waves and gastric emptying (GE) in diabetic patients with gastroparesis. Methods: 20 diabetic patients (6M, 14F, mean age: 37 years) with severe gastroparesis refractory to standard medical therapy were included. During the abdominal surgery one pair of electrodes was placed into the muscularis propria of the stomach along the greater curvature at about 10 cm proximal to the pylorus for electrical stimulation using a commercial pulse generator (Medtronic, Minneapolis, MN) permanently implanted in the abdominal wall. The study consisted of 1) assessment of severity of nausea and vomiting graded as 0 (none) to 4 (extremely severe) and nutritional status; 2) evaluation of 4-hour GEtest of a solid meal and surface electrogastrogram (EGG) recordings at baseline (before surgery) and after 6 and 12 months of GES (pulse width: 330 p.s, amplitude: 5 mA and frequency: 12 cpm) initiated within one week after surgery. Results: In comparison to baseline, severity of nausea and vomiting were significantly reduced after 6 and 12 months of GES (nausea: 3.5_+0.7 vs. 2.1 _+1.5 vs. 1.6_+1.4; vomiting: 3.0 + 1.3 vs. 1.6 + 1.5 vs. 1.5-+1.1; P10% at 4 hours). The mean percent of gastric retention at 4 hours was modestly reduced from 45-+ 30% at baselineto 29 -+26% at 6 months and to 37 _+36% after 12 months. The postprandial EGG power (amplitude) after 6 and 12 months of GES was substantially increased compared to baseline (0.4_+4.7 dB vs. 2.7_+4.9 dB vs. 1.5_+2.3 dB). On average, patients had gained 4 kg after 6 months of GES (P
Effects of High-Frequency Electrical Stimulation of the Stomach on Symptoms, Gastric Slow Waves and Gastric Emptying in Diabetic Gastroparesis Richard McCallum, Zhiyue Lin, Irene Sarosiek, Susan Shea, Jameson Forster, Suzanne Denton, Sara Durham, Kansas City, KS

Results of Long-Term Gastric Electrical Stimulation (GES) for Treatment of Gastroparesis Refractory to Standard Medical Therapy Richard McCallum, Jrene Sarosiek, Zhiyue Lin, Jameson Forster, Thomas Abell, Lesa Gann, Warren Starkebaum, Kansas City, KS; Jackson, MS; Little Rock, AR; Minneapolis, MN Purpose: The objective of this study was to evaluatethe long-term efficacy of high frequency gastric electrical stimulation for gastroparesis (GP) in an open label Compassionate Use Electrical Stimulation Study. Preliminary results at 6 and 12 months are reported here. Methods: 49 patients with chronic GP were enrolled from 2 centers [22 diabetic (8M, 14F), 19 idiopathic (2M, 17F), and 8 post surgical (3 fundoplication, 3 vagotomy, 2 partial gastrectomy) (1M, 7F)]; mean overall age was 42.6 years. Entry criteria were > 7 episodes/week of vomiting or nausea, delayed gastric emptying (GET) >10% at 4 hours determined by standardizedscintigraphic techniques, and refractoriness to prokinetic and antiemetic therapy. Chronic high frequency GES therapy was administered for 12 months via electrodes implanted by laparotomy (45 patients) or laparoscopy (4 patients) in the muscle wall of the antrum 10 cm from the pylorus and connected to an implanted stimulator placed in the abdominal wall (frequency = 0.2 Hz, intensity = 5 mA, pulse width = 330 msec). Total symptom score (TSS) (sum of vomiting, nausea, early satiety, bloating, fullness, and epigastric pain), quality of life scores (physical composite score, PCS; mental composite score, MCS), GET, and HbAlc in diabetics, were evaluated at baseline, 6 and 12 months. Results: Results are shown in table below. TSS, PCS, and MCS were significantly improved at 6 and 12 months compared to baseline scores. HbAlc was decreasedfor the diabetic group at 1 year. Gastric retention was not reduced. Key therapy-related complications were infections (5 events) at the implant site resulting in removal of the device in two patients. One patient died at 5 days from pulmonary embolism and one at 14 months due to suicide. Conclusions: Chronic high frequency GES therapy significantly improved TSS and quality of life in this heterogeneous GP patient population. HbAlc was also decreased in the diabetic subgroup. These data together with an earlier report (GE 120(5):A98, 2001), indicate that GES represents a major advancein Iongterm managementof GP patients refractory standard medical approaches.,Supportedin part by Medtronic,,. TSS (mean±SE) PCS (mesn±SE) MCS(mean+SE) HbAlc (mean+_SE) GET (%) 2 hr (median) GET (%) 4 hr (median) * p <.05 vs baseline

Baseline (49) 14.2~.9 23.5-+1,0 36.6--1.6 9.8+_0.5(19) 63.8 38,1

6 Months (n) 8.3"_+1.1(26) 31,4"_+2.6(22) 46,7*_+3.2(22) 8.9!-_0.7(10) 58.0 (23) 27.0 (23)

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12 Months (n) 7.2"-+1.7(19) 32.0*_+4.8(16) 45.2"+3.1 (16) 7.5"±0.6 (7) 74.0 (13) 40.0 (t 3)

Entrainment, but not Dominant Power of Gastric Electrical Activity, Depends on Variables of Pacing Stimulus Jinhong Xing, Edy Softer, Cleveland, OH Background: Gastric electrical stimulation (GES), using low frequency, long duration pulses (pacing stimuli) can entrain gastric slow waves, normalize gastric dysrhythmia and improve symptoms of gastroparesis. Optimal stimulus variables required for entrainment and their effect on gastric electrical activity (GEA) are not fully known. Aim: To study how variations of pacing stimulus affect entrainment and GEA in a canine model. Methods: 7 mongrel dogs were fitted each with four pairs of electrodes, implanted along the greater curvature of the stomach. Pairs were spaced 4cm apart, with the most distal one 2cm above the pylorus. GES was performed in fasted conscious animals by activating the most proximal pair of electrodes and GEA recorded by the other 3 pairs. After baseline recording, a series of pacing stimuli of 6 cpm (slightly above the normal frequency in dogs) and 12 cpm, with an amplitude of lmA, 2mA, 4mA and 6mA were applied in random. Pulse width (PW) was increased gradually until entrainment was achieved and recording continued for 10 more minutes. GES was discontinued for 3 minutes between sessions. PW required for entrainment, percent entrainment (PE, %) and the dominant power (DP) of the GEA were analyzed.Values of DP analyzed by ANOVA, p
673 Effect of Two-channel Gastric Electrical Stimulation on Vasopressin-induced Symptoms and Gastric Dysrhythmia in Dogs Jinsong Liu, Xian Qiao, Lijie Wang, Jiande Chert, Galveston, TX The aim of this study was to investigate two-channel GES on vasopressin-induced symptoms and gastric dysrhythmia. Method: Sevenhealthy female hound dogs (20-30kg) were implanted with 3 pairs of cardiac pacing wires along the great curvature of the stomach at an interval of 5cm with the distal pair 2cm above pylorus. The study was performed in 2 sessions on different days, each composed of 80-min in the fasting state. In the control session, vasopressin (O.5u/kg, in 20ml saline) was infused during the 2nd 20-min period. The protocol of the study session was the same as the control session except that two-channel GES was performed during the first three 20-min periods. The two-channel GES was performed via the two proximal pairs of electrodes. Long pulse stimulation was applied to channel 1 (pulse width: 500 ms, amplitude: 4mA, frequency: 5.5 cpm) and short pulse stimulation was applied to channel 2 (pulse width: 300 p,s, amplitude: 2 mA, frequency: 20 cpm). Motion sickness-like symptoms were scored during the study by their severity and/or frequency, including lick tongue, close eyes, yawn, belch, rapid breath, dry vomit, liquid vomit and defecate. Result 1) Vasopressin induced gastric dysrhythmia and motion sickness-like symptoms. The percentage of regular slow waves (4-6 cpm) decreased from 97%-+4% at baseline to 41%-+20% during the 20min infusion of vasopressin (p
Pulse width required for entrainment, percent entrainment and DP 6 cpm 12 cpm PW (ms) PE (%) DP (dB) PW (ms) PE (%)__ DP (dB) 1 rr~ 157.5±7.5 100.0±0.0 4.7±1,1"s 2mA 130.0_+11.3 100.0_+0.0 6.9+1.3~ 209.3+56.7 50.0-+0.0 6.2+2.7's 4rnA 94.8±10.8 100.0-+0.0 5.6-+1.7r~ 137.7±43.1 45.3_+4.7 5.7-+3.7"2 6 mA 76.8+60 100.0-+0.0 6.5+1.7r~ 100.0+_390 50.0~0.0 9.8±4.1"~ Compared to baseline (DP=6,3+1,6): ns, no significance. Data presented as mean-+SE,n=5 for 6 cpm, n--4 for 12 cpm.

676 Comparison of Hospitalizations, Medications, and Quality of Life in Patients Receiving Gastric Eleclrial Stimulation (GES) Therapy for Severe Gastroparesis Chris McEIhinney, Irene Sarosiek, Zhiyue Lin, Richard McCallum, Kansas City, KS Objective: GES therapy is availablefor patients diagnosed with severe gastroparesis, a difficult therapeutic challenge. In this study, we compare total number of hospitalization days, number of GI related medications and quality of life (QOL) in patients who have the GES (Medtronic, Inc.) placementfor gastroparesis (GP)for at least one year. Methods: This study included KUMC patients who were at least one year post-gastric stimulator implant. Days of hospitalization in the year prior to GES implant as well as all hospitalizations for one year after surgery were documented and compared. Daily prokinetics, antiemetics, and narcotics were also quantitated at the time of surgery and compared to data at 1 year. All patients were contacted by telephone

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and asked questions about their GI quality of life. Patients rated their well being on a 0-100 scale before their GP diagnosis (based on recall before onset of GP symptoms), at time of GES placement, and one year after placement. In addition, standard SF-36 QOL assessments were obtained before surgery and one year post-implant. Results: A total of 55 patients have been implanted with GES at KUMC, 25 of them have reached at least one-year of follow-up. Five of 25 patients were excluded from this interview: 3 patients died of unrelated causes and 2 patients had devices removed due to pocket infections. 19 out of 20 patients were interviewed for this study. The total number of days of hospitalization for the 19 patients one year prior to implant was 1146, compared to 330 (60 -+ 64 vs. 17_+22, P = .002) for the year after placement. 8 patients had no hospitalizations first year after implant. 16 were on at least 1 prokinetic (4/16 on 2) at baseline compared to 11 at 1 year (1.1 _+.6 vs..6_+ .5, P= .003). Antiemetics, and narcotics were used by 21 and 13 at time of surgery respectively and decreased to 16 and 7 at one year, respectively. Average QOL score was rated at 86.3% before GP diagnosis, decreased to 32.9% prior to placement and improved to 60.1% at 1 year after implant (P<.05 for all values). Also, SF-36 scores were significantly improved Conclusion:After 1 year of GEStherapy, 1) total number of hospitalization days was significantly reduced by 816 days. Assuming $1,500/ day hospitalization costs, this is a savings of $64,200/ patient/year. 2) Use of prokinetics and QOL significantly improved. 3) GES is a dramatic advance in therapy for 6P.

679 Pituitary Adenylate Cylclase-Activating Polypeptide Regulates the Histidine Decarboxylase PromoterVia Dual Signaling Mechanisms John McLaughlin, Natalie Sinclair, Rocchina Colucci, Rocchina Raychowdery, Timothy Wang, Theodore Koh, Salford, UK; Worcester, MA; Boston, MA Background: Recently,it has been Shownthat the enterochromaffin-like (EOL)cellof the gastric mucosa expressesthe receptor for pituitary adenylatecyclase-activating polypeptide (PACAP), and that PACAP can regulate ECL cell proliferation. In this study, we investigate whether PACAP can also regulate the expression of the major functional peptide of the ECL cell, histidine decarboxylase (HDC). Methods: Transient transfection studies were performed in PC12 cells stably transfected with the gastrin/CCK-B receptor. Full length 1.8 kb human HDC promoter-luciferase construct, or with serial deletion and mutant HI)C promoter-luciferase constructs were used. Cells were stimulated with either PACAP, gastrin, or both peptides. EMSA assays were also performed pn PACAP stimulated cells. Results: Promoter activation by PACAP was found to be temporally biphasic, with an early peak at 4 hours mediated by both the PKA and PKC signaling pathways, and a late peak at 24 hours that is PEA-dependent. While PACAPstimulation of the HDC promoter was synergistic with gastrin, deletional analysis, block mutational analysis and electrophoretic mobility shift assays demonstrated an independent pituitary adenylate cyclase-activating polypeptide response element (PRE) at 177 to 170 upstream from the transcriptional start site. Conclusions: PACAP mediates neural stimulation of HDC promoter activity in ECL cells through activation of both PEA and PKC signaling pathways. PACAP'seffect on HDCexpression are both complementaryto and distinct from gastrin.

677 Efficacy of Electrical Stimulation Therapy on Functional Constipation Hye-Sook Chang, Seung-Jae Myung, Suk-Kyun Yang, In Ja Yoon, Oh Ryoun Kwon, SukKyun Yang, Hwoon-Yong Jung, Weon-Seon Hung, Jin-Ho Kim, Young II Min, Seoul, Korea Background: Biofeedback therapy (BFT) is widely used for the management of constipation associated with pelvic floor dyssynergia. However, some patients cannot benefit by BFT alone. Of these patients, there is subgroup of patients who complain of absenceof desire to defecate, suggesting that the main pathophysiology of constipation may be impaired rectal sensation or increased rectal compliance. The aim of this study was to evaluatethe efficacy of electrical stimulation therapy (EST), that had been recognized as an effective treatment of urinary or fecal incontinence, for this subgroup of constipated patients with impaired rectal sensation. Methods: Of the 130 patients of functional constipation as defined by Rome II criteria, 12 patients were selected, who had impaired rectal sensation (rectal desire threshold > or = 100 mmHg) on anorectal function test. Seven patients (M:F=4:3, ages 23-71 years) were treated with EST (10-12 sessions, pulse generator; HMT, Seoul, Korea) and 5 patients (M:F= 3:2, ages 28-65 years) with BFT (10-14 sessions) according to the randomized order. Subjective symptoms using an organized questionnaire and the threshold for rectal sensation using balloon distension were evaluated before and after each therapy. Results: Symptoms of patients significantly improved from pre- to post therapy in both groups (data were not shown) (p
680 Lymphotnxin ~ Signaling Down-Regulates Cyclooxygenase-2 Expression in Mice and the Intestinal Cell Line HT-29 Teresa G. Tessner, Terrence E. Riehl, William F. Stenson, St. Louis, MO Background: The intracellular signaling events regulating the transient expression of cyclooxygenase-2 (Cox-2) by cytokines and growth factors have been extensively characterized. However, intestinal epithelial cells can also "constitutively" express Cox-2 in the case of colon cancer and inflammatory bowel disease.The mechanisms which allow the chronic expression of Cox-2 are unknown and are the subject of intense investigation. We have now identified both in vivo and in vitro systems in which chronic Cox-2 expression is regulated by the binding of lymphotoxin o~2 (LT~) to the LTI3receptor. Methods: Cox-2 immunohistochemistry was performed on sections of the small intestine of wild-type mice and mice in which production of the LT{x chain or the LTI3 receptor was genetically disrupted, and on wild-type mice treated with the LTI3 receptor-lgG fusion protein which prevents the binding of LTI3 to the LTI3 receptor. The level of Cox-2 protein and activated (phosphorylated) Akt in HT-29 cells treated with LTI3 was determined by Western blotting. PGE2 levels were assayed by enzyme immunoassay. Results: In normal mouse intestine there is no epithelial Cox-2 expression. However, mice in which the LTI3-LT~ receptor system is disrupted demonstrate constitutive expression of Cox-2 by villus epithelial cells. Concomitant with the enhanced expression of Cox-2 in LT~ -/- mice, there was a six-fold increase in PGE2 synthesis. The intestinal epithelial cell line HT-29 constitutively expresses Cox-2 and also expresses the LT~ receptor. Treatment of HT-29 ceils with LTI3 resulted in a time- and dose-dependent decrease in Cox2 expression and PGE~synthesis. Recently, phosphatidylinositol-3' kinase, an activator of Akt, was shown to be involved in the down-regulation of TNFc~-induced expression of Cox-2 in HT-29 cells (Weaver et al. 2001 Gastroenterology 120: 1117-1127). Therefore, we assessed the effect of LTt5 on Akt phosphorylation. LTI3 treatment of HT-29 cells resulted in a timedependent increase in Akt phosphorylation which coincided with the attenuation of Cox-2 expression. Conclusions: Our data demonstrate that signaling through the LT~ receptor ptays a crucial role in preventing the expression of Cox-2 in mouse intestine villus epithelial cells under basal conditions. This can be recapitulated in an intestinal cell line which exhibits high baseline expression of Cox-2. Activation of Akt appears to be a down-stream effect of LTI3 treatment of HT-29 cells and may be involved in turning off Cox-2 expression.

Mean rectal sensorythresholds(pro-therapyI post-therapy) ml 1st sensation Desire Urge Maximum EST 13_+7.6/ 1~0 133-+28/ 73+38"1" 189--68/ 140-+54" 214+53/ 189-+66 BFT 12_+4.5/10-+0 112+1t / 108_+27 170-+24/ 160_+39 224+39/ 200_+83 * p
681 An Upstream CRE-EBOXElement Is Essential for Gastrin-Dependent Activation of the Cox-2 Gene in Human Colon Cancer Cells Frank Schmitz, Stefan Juettner, Nikolaus Ansorge, Georgia Schaefer, Holger Knorth, Kirsten Mros, Rainer Lebert, Bertram Wiedenmann, Wolfgang E. Schmidt, Michael Hoecker, Bochum, Germany; Berlin, Germany Background. Cyclooxygenase-2 (COX-2) is the inducible key enzyme of arachidonic acid metabolism and enhanced expression of the COX-2 gone has been reported in colorectal cancerl Gastrin has been shown to exhibit mitogenic effects on the colonic epithelium in vivo and in vitro but the molecular basis of the growth factor action of gastrin is poorly understood. Aim. We sought to determine whether gastrin acts as a putative hormonal regulator of COX2 expression in human colorectaf cancer cells. Methods. The human colon cancer cell line Colo-320-B was stably transfected with the wild type human CCK-B/gastrin receptor. COX-2 mRNA expressionwas quantitated by RT-PCRin gastrin-stimulated and untreated cells. Cellular prostaglandin E2(PGE2)production was measured with an ELISA. Following transfection with COX-2-1uciferasereporter gene constructs, Colo-320-B cells were stimulated with gastrin and analyzedfor reporter gene activity. The gastrin responsive region of the COX-2 promotor was identified using a combination of 5' deletion analysis, element transfer experiments and sitedirected mutagenesis. Nuclearproteins binding to the proximal COX-2 promotor were identified employing standard electrophoretic mobility shift assays (EMSA). Results. Gastrin stimulated PGE2 secretion (2-3-fold) as well as COX-2 mRNA expression (2-fold) in Colo-320-B cells. Gastrin also potently transactivated the -963 bp COX-2 promotor/luciferase reporter gene in a time- and concentration-dependent manner. A maximal gastrin response was observed upon stimulation with 10 nM gastrin after 7 h of incubation, remained at the plateau-phase for further 3 h and declined back to baseline levels within 24 h of treatment. 5' deletion analysis

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revealed that a proximal promotor sequence spanning -68 to -50 bp is essential for gastrininduced COX-2transcription. Site-directed mutagenesisof this region revealedthat an overlapping CRE-Eboxelement at -57 to -48 bp mediates the effects of gastrin on the COX-2 gene. Transcription factors USF-1 and -2 were identified to bind to this promotor element by EMSA and immunosupershifting. Conclusions. Our study demonstrates for the first time that gastrin transactivates the COX-2 gene and stimulates PGE2secretion in human colon cancer cells. The effect of the peptide hormone is mediated via activation of a proximal CRE-Eboxelement of the COX-2 promotor. This observation provides a molecular model how gastrin can influence the pathogenesis of colorectal cancer.

684 Electrically Induced Calcium-Transients in Immunollistochemically Identified Myenteric Neurons Raf Bisschops, Pieter Vanden Berghe, Jozef Janssens, Jan Tack, Leuven, Belgium The myenteric plexus plays a key role in the control of gastrointestinal motility. Current models of peristalsis, derived from immunohistochemical (IH) and electrophysiological findings, presuppose a polarized spread of neuronal activity (ascending excitation and descending inhibition). Previously we showed that conf0cal imaging of intracellular calcium (Ca 2.)~can be used to monitor electrically induced neuronal activity in the myenteric plexus in situ. We observed a substantial amount of convergence and summation of responses to oral or aboral electrical stimulation (ETS) (Bisschops 2001), suggesting a complex input of ascending and descending pathways to individual neurons. The AIM of the present study was to investigate these responses in different classes of IH identified neurons. METHODS:Longitudinal musclemyenteric plexus preparations from guinea pig jejunum were incubated with Fluo-3-AM (30 p,M) in the presence of 10.6 M nifedipine. ETS was applied via two 50 i~m Oplatinum electrodes, placed on oral and aboral interganglionic fiber tracts of the same ganglion. (mean distance 139 _+ 7 p.m). Images were recorded on a Noran Oz confocal microscope. (Ca 2+)~ was measured using specifically developed software to correct for minimal residual tissue movement. Tissues were stained for caibindin (calb+) and calretinin (calr+). Data were analyzed using a Fisher's exact test. RESULTS : A high K+ (75mM) depolarization identified 65 neurons in 4 ganglia. In total 55% could be classified as calr + (n - 20) or calb + (n = 16) by correlating IH to confocal images of the ganglion. Orally or aborally applied ETS (30 V, 30 Hz, 3s) elicited a (Ca 6+)~transientin 58% of these neurons. The number of responding neurons was similar for calr+ (12/20) and calb+ (9/16) neurons (95% CI 0.61 1.87;p = 1.00). Convergence was present in 52% of the responding neurons: 6 calr + and 5 calb + neurons were activated by both oral and aborai ETS. Spatial summation recruited additional calr + and calb + neurons (5.5%). In non-identified neurons, aboral response rate tended to be lower (37.9%) and spatial summation tended to be more frequent (17.2%) than in calr+ or calb+ neurons. (95% CI 0.89-2.64 p=0.14; 0.07-1.54 p=0.23 resp). CONCLUSION: Using confocal (Ca 2+)~imaging we could not find a different response pattern to ETS in catr +, calb + or non-identified neurons. Thesefindings suggest a complex interaction of oral and aboral neural input to ascending excitatory interneurons, excitatory longitudinal motor neurons and sensory neurons.

682 Plasminogen Activator Inhibitor-2 (PAl-2) Is a Novel Target of Gastrin in Hypergastrinemia Graham J. Dockray, Elaine Hemers, Debbie Archer, Adelina Pagliocca, Chris Haigh, Rod Dimaline, Suhail Ahmed, Andrea Varro, Liverpool L69 3BX, UK Background & Aim. Gastrin regulates acid secretion, histamine secretion, proliferation and the organisation of the gastric epithelium. We have used a gene array to identify major, previously unidentified, gastrin-regulated genes that might be implicated in these functions. Methods. An array of 1176 genes associated with cancer was probed with samples from AGS-cells expressing the gastrin/CCK-B receptor and stimulated with gastrin. Western blot and ELISA of tissue and blood from hypergastrinemic patients was used to verify regulation by gastrin of candidate genes, and mechanisms of gene expression were studied using promoter-luciferase reporter constructs. Results. PAl-2 exhibited the largest change in expression on the gene array, and is a previously unknown, gastrin-regulated gene. There was significantly (p<0.05, t test) elevatedPAl-2 in plasma of hypergastrinemic patients (pernicious anaemia (PA): plasma gastrin, 1.20 _+ 0.16 riM, plasma PAl-2, 1.4_+0.3ng/ml, n = 9; gastrinoma in multiple endocrine neoplasia type 1 (MEN-l), gastrin, 0.53 +- 0.09 nM, PAl-2, 2.0_+0.2 ng/ml, n=4; controls: gastrin, 0.03_+0.01nM; PAl-2, 0.6 +- 02ng/ml, n=9). PAl-2 was also detected as a strong band in Western blots of gastric biopsies from 8 of 9 PA patients, 5 of 7 MEN-1 patients, but was at or below the limit of detection in 9 of 10 controls. In an invasion assay using Matrigel coated transwells, gastrin (lnM, 24hr) stimulated AGS cell invasion and this was potentiated by neutralising antibody to PAl-2 (control: <1cells/ field; gastrin, 205_+29; gastrin and PAl-2 Ab, 324+_33; p<0.05). In AGS cells transiently transfected with 2.34kb of the PAl-2 promoter in a luciferase reporter vector, gastrin (lnM) increased expression 14.0+_1.3 fold over control (p
685 involvement of an increase in [Ca2+]i and Chloride Channels in Generation of Regenerative Potentials in Guinea Pig Gastric Antrum George Hirst, Narelle Bramich, Frank Edwards, Parkville, Australia BACKGROUNDSlow waves recorded from guinea pig gastric antrum consist of two components, an initial component generated by myenteric ICC and a secondary component initiated by intramuscular ICC lying in the circular muscle layer. In isolated bundles of circular muscle, membrane depolarization initiates regenerative responses with the same properties as the secondary component of slow waves.AIMS To determine if an increasein internal concentration of calcium ions, [Ca~+]i, was associated with each regenerative response and the nature of the conductance change underlying them. METHODSGuinea-pigs (200 to 300g) were killed in accordance with practices laid down by the Animal Ethics Committee of the University of Melbourne. The antrum was removed, bathed in physiological saline and short single bundles of circular smooth muscle were isolated. Preparations were impaled with two independent electrodes, one was used to pass depolarizing current, the other to record evoked membrane potential changes. RESULTSIn preparations loaded with Fura-PE3,each regenerativepotential was found to be associated with a long lasting increase, duration 3 to 5 sec., in [Ca2+]i which started abruptly 1 second after the onset of the applied depolarizing step. Increases in [Ca2+]i but not regenerative potentials, were reduced in amplitude by nifedipine (lmicroM). Both regenerativepotentials and associated increases in [Ca2+]iwere abolished by buffering [Ca2+]i to low levels using MAPTA-AM (10 microM). When the membrane potential of the segments was changed, so allowing regenerative potentials to be initiated over a range of potentials, the relationship between their peak amplitudes and membrane potential was linear with an extrapolated reversal potential of -20 mV. Regenerative potentials were abolished by the chloride channel blockers 4,4'-diisothiocyano-2,2'-stilbene disulfonic acid, DIDS, (100 microM) or anthracene-9-carboxylicacid, 9-AC, (1 raM). CONCLUSIONSRegenerativepotentials, generated by intramuscular ICC, are associated with an increase in [Ca2+]i which appears to trigger an increase in chloride conductance.

683 The Human Gastrin-Releasing Peptide Receptor (hGRP-R) Gene Is Regulated by Cyclic AMP Dependent Signals Xiangping Ou, Dongmei Xiao, Horst C. Weber, Boston, MA The gastrin-releasing peptide (GRP), a bombesin-like regulatory peptide, plays an important rote as growth factor in human cancer by binding to its specific, high-affinity cell surface G6 protein-coupled receptor (GRP-R). This receptor is found aberrantly expressed in human cancer cells derived from the lung, colon, stomach and prostate. Previously, we charactedzed the human GRP-R gene structure and showed that its basal transcriptional activity in HuTu 80 cells requires both a TATA motif, and a cyclic AMP response element (CRE) located 112 bp upstream of the RNA start site (-112 bp). Hence, in this study we examined the cAMP dependent hGRP-R gene activation in duodenal cancer cells (HuTu 80) which natively express functional hGRP-R.Northern blots showed that agents increasing intracellular cAMP (forskolin [FSK], cAMP, IBMX) increased hGRP-R steady state mRNA expression in a time-dependent manner. To determine whether downstream targets of the cAMP dependent signaling pathway affected hGRP-R transcription, various hGRP-R promoter-luciferase reporter plasmids were tested for their responsiveness to FSK (25 ILM) stimulation (16 hrs prior to cell harvest) in transient transfections experiments. All promoter-reporter chimeras contained at least the minimal hGRP-R promoter sequence and resulted in a 2.5- fold (SEM: +/- 0.5) increased hGRP-R promoter activity compared to untreated controls in HuTu80 cells. Similarly, cotransfection with a protein kinase A (PKA) expression plasmid increased hGRP-R promoter activity. With electromobility shift assays (EMSA) we showed that the CRE site at -112 bp in the minimal hGRP-R promoter acts as a DNA-protein binding site. Competition experiments with CRE consensus and hGRP-R gene-specific CRE primers abolished this protein-DNA interaction dose-dependently, whereas SP-1, AP-2, and TFIID specific primers did not alter this DNA-protein interaction. Similarly, neither mutation nor deletion of the four core CRE nucleotides in the hGRP-R specific primers resulted in competition of the DNA-protein interaction. Incubation of nuclear extracts with an anti-phospho-CREBantibody prior to EMSAyielded a shift of this protein-DNA complex. Collectively, our results suggest that a protein of the CRE-binding transcription factor(s) (CREB)/ATF family interacts with the CRE site in the minimal hGRP-R promoter in duodenal cancer cells. Thus, the cAMP signaling pathway may play an important role in the aberrant expression of hGRP-R in human cancers.

AGA Abstracts

686

Chloride Channels in Interstitial Cells of Cajal Play a Major Role in Intestinal Pacemaker Activity Catherine Golden, Yaohui Zhu, John Malysz, Jing Ye, ,Jan D. Huizinga, Hamilton, ON, Canada BACKGROUND:The electrophysiological basis of gut pacemakeractivity is determined by the ion channels present in interstitial cells of Cajal (ICC). The objective of the present study was to elucidate a possible role for CI- channels. METHODS:The perforated patch technique was used to record whole cell currents in ICC, isolated from murine small intestine. Single channel activity was recorded using the inside out configuration. Intracellular electrodes were used to record slow wave activity. RESULTS: Isolated leg were obtained after 3 to 7 days in culture and recognized by morphology and ckit immunoreactivity. A ramp protocol over -100mV to +50mV, revealed both constitutively active and voltage activated currents. Cs+ was added to the pipette solution to eliminate K+ currents. Substituting extracellular chloride with methanesulfonic acid shifted the reversal potential from -20mV to + 20mV (n = 5). The CI- component of the intrinsically active current constituted 22.7-+ 2.8 % (n = 5) of the total current at positive holding potentials, with an average slope conductance of 53.04_+12.1 pS (n=5). The remaining portion of the total current consisted primarily of a non-selective cation current. At the single channel level, a CI- channel with a single channel conductance of 62 _+ 5 pS (n = 15) was active during rhythmic depolarizations of the ICC. At the tissue level, addition

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of the CI channel blockers, niflumic acid and DIDS, resulted in hyperpolarizations of 9.8 +_ 2.1 mV (n =7, P
for ERG protein. E4031 and cisapride were used to evaluate the effects of ERG channel blockade on electrical activity, with intracellular recording from guinea-pig GBSM, and on guinea-pig GBSM strip tension, RESULTS: Expression of ERG channel mRNA was found in both human and guinea-pig GBSM, and ERG immunoreactivity was observed in spindleshaped cells in GBSM bundles. Addition of lOOnM-lmicroM E4031 or cisapride caused a concentration-dependent prolongation of the plateau phase of spontaneous action potentials, usually resulting in multiple spikes before repolarization.After action potentials were abolished with 20microM diltiazem, 100-500 nM E4031 caused a membrane depolarization of 4-7mV. In muscle strip studies; E4031 slightly potentiated contractions elicited by bethanecol or electrical field stimulation. E4031 also induced phasic contractions in tissues that initially lacked them, and significantly increasedthe amplitude of phasic contractions in spontaneously active tissues (P = 0.001). CONCLUSIONS:ERG channels are expressed in the GBSM where they play an important role in regulating its activity, probably by contributing to repolarization of the plateau phase of the action potential.

687 OUlonium Bromide Inhibits Calcium Entry through Human Small Intestinal Smooth Muscle L-Type Calcium Channels Peter R. Strege, Stefano Evangelista, Michael G. Sarr, Gianrico Farrugia, Rochester, MN; Eirenze, Italy

690 Transfer of Highly Interleukin-7 Receptor Expressing Mucosal T Cells into Immunodeficient Mice Induced Chronic Severe Colitis Motomi Yamazaki, Eriko Okada, Tomoko Matsumoto, Ryuichi Okamoto, Tetsuya Nakamura, Takanori Kanai, Masanobu Tanabe, Tsutomu Takeuchi, Mamnru Watanabe, Tokyo, Japan

Introduction: Otilonium bromide (OB) is a quaternary ammonium salt used for treating irritable bowel syndrome as an antispasmodic. Pharmacokinetic studies show that OB is not systemically absorbed and its intestinal tissue levels are on the order of 1-10 FM. The cellular mechanism of action of OB is not well understood but it is known that Ca2+ entry into intestinal smooth muscle cells is required to trigger contractile activity with the main pathway for Ca2÷ entry being L-type Ca2+ channels. Aim-- To determine the effects of OB on L-type channels in human jejunal circular smooth muscle cells. Methods: Human jejunal smooth muscle cells were dissociated from circular muscle Strips obtained from normal human jejuna. Whole Cell currents were recorded using standard patch clamp techniques. An internal cellular solution containing 145 mM cesium was used to block outward potassium currents. External cellu ar solution was normal Ringer's solution with O, 0.09, 0.9, or 9 I~M OB. Cells were held at 100 mV between pulse protocols. Results: At concentrations at or below 0.09 I~M, OB had no effect on maximal peak inward Ca2÷ current. At 0.9 ~M OB inhibited mean (_+SEM) maximal inward Ca2+ current by 50% (-68_+11 to -36_+11 pA, n=6) and at 9 I~M by 90% (-77--10 to -8-+3 pA, n=7, p0.05). Conclusions: Otilonium bromide significantly inhibited Ca2÷ entry in human jejunal circular smooth muscle ceils at similar concentrations to therapeutic intestinal tissue levels. This effect may underlie the muscle relaxant effects of the drug. (Supported by NIH grant OK 52766, DK57061 and Menarini Ricemhe SPA).

BACKGROUND:We have demonstrated that intestinal epithelial cells produce interleukin-7 (IL7) and IL-7 servesas a regulatory factor for proliferation of mucosal lymphocytes expressing IL7 receptor (!I_-7R). Recent studies demonstrated that IL-7/IL-7R signal plays a crucial role in regulating the normal immune response inthe intestinal mucosa. Here, we demonstrated the pivotal role of mucosal IL-7R dependent signals in the development of chronic colitis. METHODS: CD4+ T cells were isolate d from coin nic LPLs of TCR~x-j- mice and IL-7Tg mice by magnetic cell sorting and IL-7R÷ T cells were then sorted using FACS. The purity of IL7R÷ T cells was confirmed by flow cytometry and was more than 97%. The purified 5 X 105 IL-7R + T cells were intraperitoneally transferred into RAG-2~- mice. Mice were killed 4-6 weeks later for analysis of colitic lesions. RESULTS: In TCRcc~- mice and IL-7Tg mice with the development of colitis, the lamina propria lymphocytes of colonic mucosa expressed ]L7R with high intensity. All recipient mice transferred with highly IL-TR expressing LPLs developed severe colitis in 4-6 weeks. Colonic inflammation was occurred more quickly and to severer extent in the recipient mice, as compared with in original mice. Histopathological examination of the colonic tissues revealed that inflammatory cell infiltration and goblet cell depletion was most prominent throughout the colon. Crypt abscesses, Paneth cell metaplasia and infiltration of eosinophils was also observed in the lesions. In contrast, transfer of CD4÷IL7R+ LPLs from wild type mice into RAG-2-~-mice never developed colitis during observation periods. CD4÷IL-7R LPLs of colitic mice also did not induce colitis. In chronic colitis lesion of RAG-2-!- mice transferred with CD4÷ IL-7R"i%PLs, IL-7R÷ T cells were remarkab I y infiltrated in the lamina propria. CONCLUSION:Highly IL-TR expressing T cells in the lamina propria of colonic mucosa mediated the development of chronic intestinal inflammation. Therefore, therapeutic approaches that target IL-7R-mediated immune responses are thought to be feasible.

6SS Calcium-Activated Chloride Current in Cultured Myenteric Neurons from Mouse Colon Sokhan Kang, Pieter Vanden Berghe, Terence K. Smith, Reno, NV Calcium activated chloride(ICl(Ca)) currents are activated by the r sen ntrace lu ar Ca2 close to the cell membrane. The physiological role of these channels in myenteric neurons remains to be determined. Owen et al (J. Neurophysiol. 55:115; 1986) suggested that Ca2+ activated chloride conductances may regulate the resting potential of some neurons. In addition, they maybe responsible for the afferdepolarization follow ng action potential firing in myenteric, tonic S-neurons (Smith et al., J. Physiol. 517: 817, 1999). However, no actual recording of Ic~(ca)has been reported in myenteric neurons. Aims: To investigate whether calcium-activated chloride current occurs in cultured mouse myenteric neurons. Methods: The colon was removed from adult mice, digested with collagenase and protease, and myenteric neurons cultured in 35mm dish. Neurons from proximal and distal colon were cultured separately. Ionic currents of myenteric neurons were recorded with 135mM CsCI- containing pipette. Results: Depolarizing voltage steps elicited a calcium current and a time dependant outward current, which was followed by a long-lasting inward tail (1=~)current following the repolarizing voltage step. I~,was reversed at a potential close to calculated ECI (-O.54mV), and abolished in external calcium free solution. Decreasingthe CI- concentration of the pipette solution with CsOH (ECI; -37.8mV) shifted the reversal potential of It,~. These results indicate that these currents are calcium-activatedchloride currents. Incidences of ICI-(Ca)were higher in proximal colon (45.8%, 11 of 24patches) than in distal colon (24.8%, 3 of 11 patches). Niflumic acid (lOmM), known ICl(Ca) blocker, reduced the amplitude of ItaJl from 1.36±0.63nA to 0.45±0.19nA. Nicardipine (1raM), an L type calcium channel blocker, had no effects on ICI(Ca), whereas, ~o-conotoxinGIVA(IOOnM), an N-type calcium channel blocker, reduced It,r~ 2.3±0.5nA to 0.31 ±0.21nA. FCCP, an inhibitor of mitochondrial Ca2+ uptake, prolonged the exponential decay time constant of I~from 405±25msec to 65-+15msec (p-
691 Differential Localization of Colitogenic CD4 T Cell Subsets Monospecific to a Micro Flora-Associated Antigen in a SCID-Transfer Model Yoshio Wakatsuki, Masaru Yoshida, Yasuhiko Shirai, Tomohiro Watanabe, Masashi Yamori, Toru Kita, Tsutomu Chiba, Kyoto, Japan Backgroundand Aims: Clonal expansion of T cells associateswith inflammatory bowel diseases in both human and experimental animals, implying antigenic activation of those T cells. We explored whether introduction of CO4 T cells specific to a microflnra is enough to initiate colitis and assessed the cytokine requirement for colitogenic CD4 T cells. Methods: SClD mice were reconstituted with CD4 T cells, either deficient in IL-4/IFN-gamma production or differentiated in vitro to Thl/Th2, all bearing a transgenic TcR specific to OVA and inoculated with an E.coli producing ovalbumin (ECOVA)via stomach. Clinical and histological manifestations of colitis were assessed. Results: Mice with ECOVA colonization and OVA-specific CD4 T cells developed colitis with histological features of focal infiltration by mononuclear cells, destruction of crypts and loss of goblet cells. The infiltration initiated in preexisting lymph follicles. Thl and IL-4 deficient T cells diffusely localized in lamina propria and submucosa whereas Th 2 and IFN-gamma deficient T cells preferentially localized to lymph follicles. Conclusions: A microbe-associated antigen, non-cross reactive to colonic tissue, can drive antigen specific CD4 T cells to cause colitis in SCID mice. Although IFN-gamma and IL-4 in the effecter CD4 T cells were not absolute requirements for the development of colitis, they appeared to regulate colitis in part by modulating migration of the effecter T cells in the colonic tissue. 692

689

Bacterial Antigen Specific T-Cell Activation Precedes Intestinal Inflammation in Enterococcus faecalis Monoassociated IL-IO Deficient Mice Sandra C. Kim, Susan L. Tonkonogy, Edward Balish, Ryan B. Sartor, Chapel Hill, NC; Raleigh, NC; Charleston, SC

Role of ERG K+ Channels in Electrical and Contractile Activity of Gallbladder Smooth Muscle (GBSM) Edward Parr, Maria Pozo, Burton Horowitz, Mark T. Nelson, Gary Mawe, Burlington, VT; Caceres, Spain; Reno, NV

Background: We previously demonstrated progressive colitis in gnotobiotic IL-IO deficient (-/-) mice monoassociated with non-pathogenic Enterococcus faecalis (El). Initial disease onset was noted 10-12 weeks after colonization. Aims: Determine whether immune activation precedes clinical and histologic inflammation by studying antigen-specific immune responses at different time points representing both pre-clinical/early (5-12 weeks) and advanced (30 + weeks) stages of disease. Methods: Germfree inbred 129 SvEv IL-10-/- and wild-type (WT) mice monoassociated at 12 weeks of age with a strain of E. faecalis were euthanized 5-40 weeks later.. Inflammation was determined in all sections of colon by blinded histologic scores (0 to 4). Spontaneous IL-12 secretion was quantified by ELISA from colonic culture

The ether a-go-go related gene (ERG) encodes a K+ channel with a recognized role in repolarization of the cardiac action potential, but more recent studies have suggested that it also has a role in regulating activity of various gastrointestinal organs. Our aim was to determine whether ERG is expressed in GBSM and what role it might play in the spontaneous electrical and contractile activities in GBSM. METHODS: RNA from dissected GBSM of human and guinea-pig was purified and reverse transcribed for RT-PCR analysis of ERG expression, and whole mount preparationsof guinea pig gallbladder were usedto evaluateimmunoreactivity

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AGA Abstracts

supernatants. CD4+ T-cells prepared from mesenteric lymph nodes (MLN) of moooassociated mice were stimulated with T-cell depleted antigen-presenting cells (APC) isolated from WT specific pathogen free (SPF) splenocytes and pulsed overnight with different bacterial lysates (E. faecalis, B. vulgatus,and E. co//). IFN-~Iwas measuredin these CD4* T-celI-APC cocuitures. Results: Colonic IL-12 was increased significantly at 5 weeks in IL-IO-/- Ef mice over WT Ef controls (p
IL-12(pglO,OSg) 1683±124" 24054-160" 1074-29

IFN-y (pg/ml) 1174_+199" 23173±422Y 130±76

death after engagement of their antigen receptor. Since integdn-mediated adhesion to the ECM in classical adherent non-immune cells modulates cell cycle progression and survival, we hypothesize that LPT ~ave adapted their behavior to this microenvironment, leading to their distinct phenotype. In contrast, we propose that if PBT are activated during their initial exposure to ECM, they will readily progress through the cell cycle. Methods: LPT and PBT, purified by negative selection from normal intestinal mucosa or blood, were stimulated with anti-CD3-coatedpolystyrene beads in the presenceor absenceof plate-immobilized fibronectin, collagen type I, or native ECM derived from human intestinal fibroblasts. Cells were stained with cyclin B1 antibody and propidium iodide (PI), and analyzed by flow cytometry. Results: Collagen type I and native ECM, but not fibronectin, co-stimulation with anti-CD3 induces a 4-5 fold increase in the proliferation, entry and progression of PBT through the cell cycle (32% of PBT in S/G2/M phase), over that observed with anti-CD3 alone (8% in S/G2/M). These effects on PBT were abrogated by neutralizing 131-integdnantibodies. None of the ECM proteins examined stimulated cell cycle entry or progression of LPT. In contrast, anti-CD3 activation of LPT in the presence of ECM, predominataly fibronectin, reduced activationinduced cell death by 40%, but did not change the rate of apoptosis in PBT. Conclusions: When PBT are activated through their antigen receptor, selected constituents of the ECM provide critical costimutatlon for entry and progression through the cell cycle. In contrast, LPT, which have already acclimated to interstitial tissue, rely on other components of the ECM for survival. These results demonstrate that distinct T cell populations respond differently to specific proteins of the ECM, and suggest that the maintenance of LPT longevity in the presence of continual activation by luminal antigens may be mediated by interactions with the ECM.

DC score 1,0±0,3 3.0±0.4" 0.674-0.1

693 Intestinal Intraepithelial Lymphocytes (IEL) Expressing CD8~z Homodimore with an ~[~ TCR are Preferentially Expanded as a Consequence of Deficiency of the CCR6 Chemokine Receptor Andreas Luegering, Torsten Kucharzik, James T. Hudson Ill, Ifor R. Williams, Atlanta, GA

696 Eradication of H. pylori to Prevent Recurrent Ulcer Complications Associated with Low-Doss Aspirin: A Long-Term Cohort Study. Francis K. L. Chan, Justin C. Y. Wu, Bing-Yee Suen, Wai Leung, Ka-Fai To, Lawrence C. T. Hung, Aric Hui, Yuk-Tong Lee, Henry L. - Y. Chan, Enders K. W. Ng, Sydney Chung, Joseph J. Y. Sung, Hong Kong, Hong Kong

Purpose: Absence of CCR6 in mice results in perturbations of the mucoeal immune system including an increase in intestinal IEL Using a new CCR6 mutant mouse that includes a knock-in of EGFP, we studied the pattern of CCR6 expression in T cells and dendritic cells (DC) and determined which IEL subsets are affected by lack of CCR6.Methods: Flow cytometry was used to characterize EGFP expression by cells from heterozygous CCR6/EGFPknock-in mice. IEL subsets in the small intestine were identified by staining with antibodies to CD4, CD8c~, CD813,c~13TCR and ~,~ TCR. Cytokine mRNA levels in small intestine were measured by ribonuclease protection assay (RPA). Electron miomgraphs of Peyer's patches (PP) were prepared to study M cell morphology. Results: In heterozygous knock-in mice, CDllb+ CD1lc + myeloid DC were almost all positive for EGFPin spleen and PP, indicating expression of CCR6. In contrast, CD8~+ lymphoid DC lacked CCR6 expression. DC in PP that do not express CD11b or CO8~ were also predominantly CCR6 positive. PP from CCR6 null mice contained significantly fewer B cell follicles (3.5 +_ 0.9) than heterozygous mice (5.3 _+ 1.9; p < 0.01). However, CCR6 deficient mice had a normal number of PP and cells with the characteristic features of M cells were present in the follicle-associated epithelium. The total number of IEL in the small intestine of CCR6 null mice was increased by 2.5 fold on average, with most of the increase in the ~ TCR+ fraction. Of the ~ TCR+ cells, CDSe~+ cells were expanded to the greatest extent with 3 to 5-fold increases in CD4- CD8e~+ cells and 4 to lO-fold increases in CD4+ C D 8 ~ + cells. C D 8 ~ + IEL from hetarozygous CCR6/ EGFPknock-in mice lacked EGFPexpression, indicating that these IEL subsets do not normally express CCR6. The levels of SCF, IL-7, and IL-15 mRNA were similar in the small intestine of CCR6 null and control mice by RPA, indicating that IEL expansion was not driven by increased local expression of growth factors known to be important for IEL development. Conclusions: Our results support the hypothesis that altered DC and B cell trafficking in CCR6 deficient mice indirectly leadsto altered developmentof intestinal IEL and preferentialexpansion of the ~13 TCR+ C D 8 ~ + subset of IEL

Background We previously reported that among patients with H. pylori (HP) infection and a history of ulcer bleeding who are taking low-dose aspirin, HP eradication is comparable to omeprazole in preventing recurrent ulcer bleeding in 6 months. It is uncertain whether eradication of HP alone will confer long-term protection against recurrent ulcer complications in these high-risk aspirin users. Aim To compare the long-term risk of ulcer complications in high-risk aspirin users after HP eradication with that of average-risk aspirin users. Methods We prospectively followed up two cohorts of aspirin users. The first cohort consisted of HPpositive patients who had ulcer bleeding documented by endoscopy. They received aspirin 80 mg once daily after HP eradication had been confirmed (High-dsk cohort). The second cohort consisted of patients who had no previous ulcer bleeding and were given aspirin 80 mg once daily (Average-risk cohort). Patients who used concomitant anti-ulcer agents, steroid, anticoagulant, other anti-platelet agents or non-aspirin NSAIDs were excluded. The endpoint was ulcer complications (bleeding or perforation) associated with low-dose aspirin. Results Between 1996 and 2001, 240 patients were enrolled in the high-risk cohort and 252 patients in the average-risk cohort (Table). 51% of patients in the average-risk cohort were infected with HP. Five patients in the high-dsk cohort and 5 in the average-risk cohort developed ulcer complications within 24 months. Conclusion Among patients with HP infection and a history of ulcer bleeding who continue to use low-dose aspirin, the long-term risk of ulcer complications after HP eradication is comparableto that of aspirin users without previous ulcer bleeding. High-risk Cohort Average-risk Cohort P Men % 70 43 0.001 Mean age (SO) 68 (10) 67 (9) 0.15 Smoking/Drinking % 14/2 9/2 0.14/0.75 Coronary head diseeae/CVA % 55/45 63/37 0.10 Median follow-up (range) months 17 (1 - 24) 18 (4 - 24) Probability of ulcer complications at 24 months (95% GI) 2.8 (0.2, 5.4) 2.6 (0.15, 5.0) 0.91# "Hazard ratio (95% el): unadjusted 1.07 (0.31, 3.70), adjusted for sex 1.16 (0.32,4.18)

694 A Critical Role for Leptin in Different Models of Experimental Colitis Britta Siegmund, Hans A. Lehr, Giamila Fantuzzi, Denver, CO; Mainz, Germany Leptin, the product of the oh gene, regulates the balance of Thl/Th2 cytokines and modifies T cell immunity. Leptin-deficient oh~ohas well as leptin receptor (Ob-Rb)-deficient db/db mice are resistant against acute and chronic OSS-ioduced colitis, as evaluated by body weight, diarrhea, bleeding, histology, induction of prointlammatory cytokines, STAT-3 activation in the colon and rate of epoptosis in lamina propria lymphocytes (LPL). Similar resistance could be observed in the model of TNBS-induced colitis, in which reduced cytokine production and T cell activation associated with increased apoptosis in LPL was observed in ob/ob mice. in vitro studies revealedthat leptin can directly induce STAT-3 activation in LPL and intraepithelial lymphocytss. To evaluate the role of T cells, we compared the effect of transfering CD4 CD45Rb"~" cells from WT and db/db mice into scid recipients, loifiai experiments indicate that colitis induction by CD4 CD45Rb"~h cells from db/db mice is significantly reduced when compared to the transfer of WT cells, suggesting that leptin receptor expression on T ceils is critical for development of intestinal inflammation. In conclusion, these results demonstrate that leptin represents a link between the endocrine and the immune system, which requires further investigations in experimental models and human IBD. Supported by a grant from the E. and E. L. Broad Foundation and the Deutsche Forschungsgemeinschaff DFG SI 749/2-1.

697 Long Term COX-2 Absence or Inhibition Is Associated with Small Bowel Inflammation Matthew Walley, Christof Hotz-Behofsits, Gudmundur Sigthorsson, Andrew Anthony, Robert Simpson, Ingvar Bjamason, London, UK, UK; London, UK BACKGROUND: COX-2 selective agents have a significantly better gastric tolerability profile than conventional NSAIDs. COX-2 selective agents also cause less small bowel damagewhen taken short term. However the long term consequences of COX-2 absence and inhibition are unknown. AIM: To assess and characterise small bowel integrity and inflammation in COX2 deficient (COX-2 KO) mice and the consequences of long term COX-2 inhibition in wild type animals. METHODS:Intestinal integrity was assessedby a 51CrEDTAintestinal permeability test, inflammation by measure of a faecal granulocyte marker protein (GMP), intestinal prostaglandin E2 (PGE2) levels by ELISA and the whole of the intestine was processed for microscopy in wild-type and COX-2 KO mice. Wild-type animals were fed celecoxib (> 100 mg/kg/day) for 3 months followed by histopathological examination of the intestine. RESULTS: Untreated COX-2 KO animals did not differ significantly from wild-type animals in respect of PGE2 levels (96 +/- 23% of control levels; n = 6) or intestinal permeability (24 hour urinary excretion of 51CrEDTA = 8.7 +/-1.1% versus 5.8 +/- 0.8% (n=12); p = 0.086), although 6 (50%) of the animals were above the normal range. The faecal GMP levels in COX-2 KO (15 +F 4 mg/L; n = 28) were significantly (p < 0.05) higher than that from the wild-type animals (6 +/- 1 mg/L; n=17). 11 (40%) had values above the normal range; 2 of 3 with GMP values above 50 mg/L had small bowel ulcers. 15% of the untreated COX-2 KO animals died suddenly due to small bowel perforation with areas of chronic multifocal mucosal necrosis and ulceration of the ileum (stomach was normal). 25% of wild-type animals fed celecoxib over 3 months developed intestinal symptoms and were found to have similar lesions.

695 Dual Function of the F.xtracnllular Matrix: Stimulatory for Cell Cycle Progression of Peripheral T Cells and Anti-Apoptotic for Mocosal T Ceils Kimberly Krivacic, Andrees Sturm, Claodio Fiocchi, Alan D. Levine, Cleveland, OH Entry of peripheral blood T cells (PBT) into tissue initiates a ~l-integfin-depeodent interaction with a network of glycoproteins and proteoglycans forming the interstitial extracellular matrix (ECM). Lamina propria T cells (LPT), intimately in contact with various components of the rnucosal ECM, exhibit specific phenotypic and functional characteristics, which are distinct from those of PBT, including low proliferative response and increased activation-induced cell

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CONCLUSIONS: Long term COX-2 deficiency and inhibition is associated with small bowel damage, COX-2 appears to have an important housekeeping function in the small intestine,

cancer risk. Physiological studies of COX-2 inhibitors on the human HP infected stomach have not been performed. AiM: To study the impact of a COX-2 specific inhibitor on gastric mucosal PG levels, HP gastritis, and proliferation, METHODS:20 HP infected (M:F, 8:12; age 38_+1.8) and 6 HP negative(4:2; age 36_+3.5) healthy human volunteers were treated with rofecoxib 25rag daily for 14d. Endoscopic mucosal biopsies at days 0 and t4 were evaluated for PGE2content by RIA, gastritis severity (Sydney classification), and proliferation with MIB1 staining. RESULTS: (mean +_ S.E.): Comparedto HP-negativesubjects, HP-infected subjects had significantly (p
698 Risk of Diverticular Bleeding with NSAIUs and Low-Dose Aspirin Byron Cryer, Anh D. Cung, Kevin C. Kelly, Rick A. Weideman, Dallas, TX BACKGROUND:The risk of upper gastrointestinal (GI) bleeding with aspirin (ASA) and other non-specific NSAIDs is well established. However, the risk of lower GI bleeding, specifically diverticular bleeding, with ASA and other NSAIDs is not well characterized. AIM: To assess the risks of diverticular bleeding in patients with verified colonic diverticula who take low dose ASA or NSNDs. METHODS: Identification of Cohort: We reviewed our computerized endoscopic GI data base to identify asymptomatic patients with colonic diverticula found during flexible sigmoidoscopy or colonoscopy as part of routine colon cancer screening at the Dallas VAMC from Jan 1996 to Dec 1998. Follow-Up of Cohort: 503 such patients with endoscopically-identified, asymptomatic diverticula were assessedfrom their ~ate of diverticula identification to June 2001. Patients were grouped by ASA or non-specific NSAID exposure during the follow-up interval: 1) No exposure, 2) non-specific NSND, 3) ASA 325 mg/day or less, or 4) concurrent NSAIDs and low-dose ASA. Cases of diverticular bleeding, defined as episodes of major hematocheziain which colonoscopic evaluation identified no other potential cause for lower GI bleeding other than diverticula, were identified by an observer blinded to NSAID exposure. Diverticular bleeding was expressed as incidence percent (events/pt-yrs of exposure) during the follow-up interval. RESULTS (mean -+ s.d.): Our cohort of 503 pts, mean age of 66.7-+9.0 (range 34-90) yrs, had a mean follow-up of 3.5_+2.0 yrs. Overall, 14 patients (2.8%) had a diverticular bleed. Rates of diverticular bleeding by NSAID exposure are shown in the Table. CONCLUSIONS:In those not taking NSAIDs, the risk for diverticular bleeding is low (0.4%). Non-ASA NSAID exposure alone (all forms of non-specific NSAIDs combined) does not significantly increase risk of diverticular bleeding while low-dose ASA more than triples bleeding risk, a statistically significant increase. The combination of lowdose ASA plus NSAID yields a 9-fold greater risk of diverticular bleeding compared to no exposure, Since the likelihood of diverticular bleeding is more strongly related to ASA than to NSAIDs, the mechanism for ASNNSAID-associated diverticular bleeding is likely due to an anti-platelet effect rather than to an NSAID-ulcerogenic effect in the colon. Diverticular Bleeding Rates by NSAID Exposure No Exposure NSAIOe No. of Events 4 1 Pt-Yrs 1048,9 130.0 Incidence % 0.38% 0.77% *=p
Low.Dose ASA 6 471.7 1,27%*

701 Prevalence of Gastric and Duodenal Ulcers during Treatment with 'Low-Dose' Aspirin Neville Yeomans, Christopher Hawkey, Angel Lanas, J6rgen Naesdal, Nicholas Talley, Alan Thomson, Melbourne, Australia; Nottingham, UK; Zaragoza, Spain; M61ndal,Sweden; Sydney, Australia; Edmonton, Canada Background and Aims: Case-control studies have shown an increased risk of ulcer hemorrhage with relative risks in the range 2-4 in patients taking vascular protective doses of aspirin. Since the underlying prevalence of endoscopic ulcers in such patients is unknown, we aimed to estimate this in 5 centers on three continents. Methods: Subjects on stable dosage with aspirin 75-325 mg/day (at least 5 days per week forat least the preceding 28 days) were invited to have an endoscopy to measure point prevalence of gastric and duodenal ulcers. Exclusion criteria included recent myocardial infarction, stroke or transient cerebral ischemic attacks (for reasonsof safety), consumption of other NSAIDsor corticosteroids, or concomitant treatment with gastroprotective drugs such as acid suppressants or prostaglandins. Ulcers were defined as mucosal breaks ->3mm diameter with distinct depth. H. pylori (Hp) status was assessed by rapid urease test and symptoms in the week prior to the baseline study were assessed by validated questionnaires. Subject recruitment was stratified into higher (age ->60 or prior peptic ulcer) and lower risk groups with each center to recruit not less than 30% in one group. Results: Of 189 subjects completing gastroscopy, 11% had one or more gastric ulcer (GU) or duodenal ulcer (DU): 11 GU, 8 DU, and 2 with both GU and OU. Median ulcer diameter was 5 ram; two patients had multiple ulcers. Gastric and duodenal erosions were common. Potential predictive factors for ulcers are listed in the Table. Ulcer patients were more likely to be infected with H. pylori (55% of GU, 88% of DU and 100% of GU/DU cf. 31% of controls), and were significantly older. Only a fifth of ulcer patients reported any epigastric symptoms (pain, discomfort or burning) in the week prior to gastroscopy, numerically (but not significantly) less than in patients without ulcer. There were significant differences in ulcer prevalence(p
ASA + NSAI0_D3 87.0 3,45%**

699 NSAIDs Inhibit Hypoxia-lnduced PI 3-Kinase Activation in Rat Gastric Endothelial Ceils: A Novel Target for NSAIDs inhibition of Hypoxia-lnduced Angiogenesis? Michael K. Jones, Kouji Tsugawa, Imre L. Szabo, Rama Pal, Andrzej S. Tarnawski, Irvine, CA BACKGROUND/AIMS: NSAIDs impair gastric erosion and ulcer healing, in part, through inhibition of angiogenesis but the molecular mechanism(s) of this action remain unknown, Hypoxia is a common cause of tissue injury including gastric ulcers and is also a strong inducer of angiogenesisvia activation of the gone encoding VEGF,the most potent angiogenic factor. NSAIDs may inhibit hypoxia-induced angiogenesis in gastric microvascular endothelial cells by blocking hypoxia-induced accumulation of HIF-I~, the transcription factor responsible for hypoxia-induced VEGF gene activation (Gastroenterology 2001;118".,4240). Since the PI 3-kinase/Akt pathway is involved in the hypoxia-induced expression of HIF-le (J. Cell. PhysioL 2001;188:223-35), we studied whether NSAIDs inhibit hypoxia-induced expression of HIF-lc~ in gastric microvascular endothelial cells through inhibition of hypoxia-induced PI 3-kinase/ Akt activation. METHODS: Rat gastric microvascular endothelial (RGMEC)cells were cultured under hypoxia or nnrmoxia (control) in the presence of vehicle (control), indomethacin (a non-selective NSAID), NS-398 (a selective COX-2 inhibitor) or wortmannin (a specific PI 3kinase inhibitor). STUDIES: 1) PI 3-kinase activity, 2) phosphorylation of Akt, 3) in vitro angiogenesis, 4) expression of HIF-lc~. RESULTS: Hypoxia resulted in a 70% (P
Group

Sex Ulcer Symptoms* Aspirin Hp +ve (female) history dose** Ulcer 24% 10% 19% 100 mg 87%~ No ulcer 38% 6% 31% 100 mg 31% * Epigastdc pain, burning or discomfort (any severity) in past week; **median; ***mean_+SE; lp<0,005; 2p<0.05

Age*** 67+_22 60+_1

702 Identification of an Apical Chloride/Bicarbonate (CI/HC03-) Exchanger in the Duodenum. Zhaohui Wang, Snezana Petrovic, Elizabeth Mann, Manoocher Soleimani, Cincinnati, OH HC03- secretion is the most important defensemechanism against acid injury in the duodenum. However, the identity of the transporter(s) mediating apical bicarbonate (HC03-) secretion in the duodenum remains unknown. A family of anion exchangerswhich include Down-Regulated in Adenoma (DRA or SLC26A3), Pendrin (PUS or SLC26A4), and the Putative Anion Transporter (PAT1 or SLC26A6) has recently been identified. DRA, Pendrin and PAT1 are expressed on the apical membrane domain of the epithelial cells of colon, kidney intercalated cells, and pancreatic duct, respectively. DRA and pendrin mediate CI-/OH-/HC03- exchange, however, the functional identity and distribution of PAT1 is not known. In these studies, we investigated the functional identity, tissue distribution, and subcellular localization of PAT1. Human and mouse full length PAT1 cDNAswere cloned from cultured pancreatic duct cells and duodenum, respectively, using primers designed based on published sequence (Genebank accession numbers AF279265 and AY032863). Functional expression studies in Xenopus oocytes injected with the human or mouse PAT1 cRNA demonstrated that in the presence of bicarbonate intracellular pH increased upon switching to a CFfree solution and returned to baseline after switching back to the CI-containing solution. In the absence of bicarbonate the rate of pHi alkalinization in response to CI- removal was minimal. These results are consistent with PAT1 functioning as a CI-/HC03- exchanger. The tissue distribution studies in mouse GI tract indicated that PAT1 is highly expressed in small intestine (duodenum, jejunum and ileum)

700 Impact of COX-2 Specific Inhibition on Human Hnlicobacter pylori (HP) Gastritis; Implications for UIcerogenesis and Carcinogenesis James Scheiman, Joel Greenson, Byron L. Cryer, Ann Arbor, MI; Dallas, TX Cyclooxygenase(COX)-2 is present at very low levels in normal gastric mucosa. HP infection increases COX-2 expression and gastric prostaglandin(PG) production. Non-selective COX inhibitors reduce PG's in HP infected mucosa, and although epidemiological studies support a potential role in cancer chemoprevention, ulceration risk precludestheir use. COX-2inhibitors cause minimal ulcer risk, even for patients with HP infection, and may have a role in reducing

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AGA Abstracts

(< 1 min) stimulated association of the Src family kinase, p6Os~c,with the EGFr in T84cells. This effect of VIP was mimicked by the cell-permeable analog of cAMP, Bt2cAMP/AM (3 ~M). The Src-tamily kinase inhibitor, PP2 (20 p,M) almost abolished EGFr phosphorylation in response to both VIP (n =3; p
but is vanishingly low in the colon, a pattern opposite that of DRA. PAT1 was also abundantly detected in stomach. Immunoblot analysis studies identified PAT1 as a ~90 kD protein in luminal membrane vesicles from the duodenum. Immunohistochemical studies localized PAT1 to the brush border membranes of the villus cells of the duodenum. We conclude that PAT1 is an apical Cl-/HC03- exchanger in the small intestine.

703 Functional Role of Specific Amino Acids in SLC19A2, a Human Thiamine Transporter: A Mutational Analysis Balamurugan Krishnaswamy, Harold M. Said, Long Beach, CA Background: Humans and other mammals cannot synthesize the water-soluble vitamin thiamine, and thus, must obtain the compound from exogenous sources via intestinal absorption. Recent studies from our laboratory have shown that the membrane thiamine transporter, SLC19A2, is expressed in different regions of the human GI-tract, thus suggesting a possible role for this transporter in normal intestinal thiamine uptake. To date, however, little is known about the structure/function relationship of this important membrane transporter. In this study, we examined the effect of mutating the only conserved transmembrane anionic residue (E138) and that of the putative glycosylation sites (N63 and N314) of the SLC19A2 protein on its ability to transport the cationic thiamine. We also examined the effect of introducing mutations in SLC19A2 (D93H, $143F, G172D) that are identical to those found in patients with thiamine-responsive megaloblastic anemia syndrome (TRMA) on its function. Methods: Site-directed mutagenesis; Vaccinia virus expression system; Northern, Western and 3Hthiamine uptake assays; and HeLa cells were used. Results: Cells transfected with all mutants and wild-type SLC19A2 expressed SLC19A2 mRNA. Mutating the conserved anionic residue (E138A) ted to a significant (p < 0.01) inhibition in thiamine (15 nM) uptake. Similarly, clinically relevant mutations in SLC19A2 led to a significant (p < 0.01) inhibition of thiamine uptake. In contrast, simultaneous mutation of the two potential N-linked glycosylation sites (N63Q, N314Q) of SLC19A2 did not alter its ability to transport thiamine; however, it led to a reduction in its apparent M.Wt. Western blot analysis using histidine-tagged mutants and wild-type SLC19A2 showed that all proteins were expressed in the membrane, not the cytoplasmic, fraction of HeLacells. Conclusions: 1) The conserved anionic residue in the predicted 4th transmembrane domain of the SLC19A2 is critical for its function, 2) Clinically relevant mutations in SLC19A2found in TRMA leads to mal-functioning of the transporter per se with no defect in translation/trafficking to cell membrane, and 3) Native SLC19A2is N-glycosylated, but this glycosylation is not important for its function. [Supported by grants from DVA and NIH (DK56061 and DK58057)].

706 Activation of NHE3 by Glucocorticoids Requires SGK1 C Chris Yun, Yueping Chert, Liudmita Cebotaru, Florian Lang, Atlanta, CA; Baltimore, MD; Tubingen, Germany The stimulative effect of glucocorticoids on intestinal salt and water absorption has been known for more than two decades. However, the molecular mechanisms underlying this activation remain elusive. Previous studies showed that methylprednisolone specifically increased NHE3rnRNA in ileum and kidney without affecting NHE1 mRNA levels. These results suggest that glucocorticoids activate NHE3 activity by the induction of NHE3 transcripts. We recently found that chronic incubation with dexamethasone activated NHE3 transport independent of gene induction, indicating that the transcriptional activation may not be the only determining factor in the NHE3 activation. OK cells are a well-defined cell model system for physiological characterization of NHE3. OK cells express NHERF1 but not NHERF2. Dexamethasone did not stimulate NHE3 activity in OK cells. However, we were able to reconstitute the dexamethasone-stimulationof NHE3 in OK ceils by expressing NHERF2. By a comparative search, we identified serum and glucocorticoid-regulated kinase I, SGK1, as a protein interacting with NHERF2. SGK1 is a serine/threonine kinase with 54% homology in its catalytic domain to the well-defined antiapoptotic kinase AKT/PKB. Recently SGK1 is shown to activate the epithelial Na channel (ENaC) in response to aldosterone, although SGK1 does not directly phosphorylate ENaC. Consistent with the physiological effect, SGK1 specifically interacted with NHERF2 but not with NHERF1.This interaction was mediated by the carboxyl terminal 4 aa of SGK1 interacting with the PDZ domains of NHERF2. Constitutive expression of SGK1 acutely activated NHE3 in PS120 fibroblasts. More importantly, expression of the kinase-dead SGK1 blocked stimulation of NHE3 by dexamethasone in OK cells. We also found that SGK1 directly phosphorylates NHE3 in vitro. These data demonstrated that glucocorticoids activation of NHE3 requires the activation of SGK1 and the presence of NHERF2acting as a scaffold protein.

704 Localization and Trafficking of Intestinal Adenosine 2b Receptor Shanthi V. Sitaraman, Didier Merlin, Lixin Wang, Torsten Kucharzik, James Madara, Atlanta, GA Background and Aims: We have previously shown that adenosine is formed in the intestinal lumen during active inflammation from neutrophil-derived 5'AMP. Acting through the adenosine A2b receptor (A2bR), the luminally-derived adenosine induces vectorial chloride secretion and a polarized secretion of interleukin-6 to the intestinal lumen. We and others have also shown that the A2bR is the only functional adenosine receptor present on colonic epithelial cells and intact human colonic mucosa. However, the localization and trafficking of the A2bR in epithelial cells is not known. Methods: The model intestinal epithelial cell line, T84 and Caco2-BBE cells stably transfected with GFP-A2b receptor were used to localize and study trafficking of the intestinal A2bR. Results: Using Western blot, cell surface biotinylation and confocal microscopy, we show that the A2bR is present in both the apical and basolateral membranes of colonic epithelia. The receptor density appears to be at least 10-fold greater on the basolateral membrane compared to the apical surface. Using cell surface biotinylation, we show that the apical or basolateral stimulation of the A2bR induces a time-dependent recruitment of the receptor to the plasma membrane and caveolar fractions. Further, the recruitment of the A2bR is polarized to the apical membrane whether the cells are stimulated with apical or basolateral adenosine. We next explored the possibility of interaction of A2bR with PDZ domain proteins that are emerging as important membrane-anchoringand organizing centers for regulatory complexes. Predictive computer modeling of the A2b receptor suggested that the A2bR may interact with PDZ domain containing protein, E3KARPvia a PDZ consensus sequence in the third intraeellular loop of the A2bR. Consistent with the modeling data, sitedirected mutagenesis of the putative PDZ-interacting amino acids in the third intracellular loop abolished cAMP response induced by adenosine. We also show that the A2bR can be co-immunoprecipitated with E3KARP upon agonist stimulation. Conclusions: These data suggest that the A2bR is recruited to the apical plasma membrane upon apical or basolateral agonist stimulation and may be anchored to the plasma membrane via its interaction with the PDZ domain protein, E3KARP. E3KARP is known to be associated with Ezrin and PKA in intestinal epithelial cells. Thus, A2bR upon agonist stimulation, may form a signaling complex for chloride secretion in the apical plasma membrane with E3KARP, Ezrin and PKA.

707 Serina/Threonine Protein Kinase Pathways Regulate the Induction of Guanylin and Urogoanylin during Osmotic Stress Kris A. Steinbrecher, Mitchell B. Cohen, Cincinnati, OH Guanylin and uroguanylin (Gn/Ugn) are guanylate cyclase C (GC-C) activating peptides that are secreted from the epithelial cells of the intestine, kidney, pancreas and salivary gland. These peptides elicit CI- and HC03- secretion via the CFTR; as such they may directly affect celt volume and intracellular inorganic ion concentration. We have previously shown that Gn/ Ugn are increased in a mouse model of osmotic diarrhea. Therefore, we hypothesized that Gn/Ugn might participate in the adaptive response to osmotic stress. To investigate the effect of cellular dehydration on Gn/Ugn levels, we identified an intestinal epithelial cell line that expresses both Gn/Ugn RNA. These HT29-18-N2 cells were exposed to hypertonic media and Gn/Ugn RNA levels were determined by Northern analysis. Gn/Ugn RNA levels were increased substantially by hypertonicity but only with solutes that were relatively impermeable to the cell membrane.The compatible osmolyte, betaine,blockedthis effect. Studies with actinomycin D and cycloheximide showed that this hypertonicity-mediated increase was transcriptional and did not require protein synthesis. Herbimycin A and the MAPK inhibitors SB-203580 and PD-98059 had no effect on basal or induced levels of Gn/Ugn. However, staurosporine, a serine/threonine kinase inhibitor, and prolonged exposure to phorbol ester, which results in depletion of protein kinase C (PKC) activity, reduced basal levels and completely blocked hypertonicity-related increases in Gn/Ugn RNA. A short exposure to phorbol ester, which activates PKC, resulted in large increases in Gn/Ugn RNA. Initial studies using bisindolylmaleimide, a highly specific PKC inhibitor, suggest that PKC is a major regulator of both basal and induced levels of Gn/Ugn. Collectively, these data support the conclusion that serine/ threonine protein kinase pathways regulate Gn/Ugn levels. This represents a novel association between this intracellular signaling network and the RNA abundance of these genes. Furthermore, PKC is the most likely candidate for the regulation of both basal and hypertonicitystimulated Gn/Ugn RNA levels in HT29-18-N2 cells. This connection fits well with the known effects of PKC isoforms on the osmotic stress response. Since PKC isoforms are also known to positively influence GC-C expression and activity, we speculate that this serine/threonine protein kinase family is central to the control of the physiological effects mediated by the GCC-Gn/Ugn signaling system.

705 Src Family Kinases Mediate cAMP-DependentCI Secretion in intestinal Epithelial Cells Lone S. Bertelsen, Kim E. Barrett, Stephen J. Keely, San Diego, CA Background: We have shown that activation of the epidermal growth factor receptor (EGFr; ErbB1) is required for the full expression of CI- secretory responses to cAMP-dependent agonists in intestinal epithelial cells. In the present study we set out to determine possible mechanisms underlying cAMP-dependent EGFr transactivation. Methods: CI- secretion was measured as changes in short circuit current (Is) across monolayers of T~ cells mounted in Ussing chambers. Immunoprecipitation (IP)/Western blot analysis was usedto determine EGFr phosphorylation and protein/protein interactions. Results: IP/Western blot studies revealedthat the cAMP-dependent secretagogue, vasoactive intestinal polypeptide (VIP; 100 nM), rapidly

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7O8

The Fur regulatory protein apparently is involved in regulation of at least two major ammoniaproducing enzymes of H. pylori, and amidase and urease activity are inversely linked. This suggests that Fur may be the master regulator of ammonia production by H. pylori, and indirectly modulates nitrogen metabolism and acid resistance of this bacterium. This novel type of amidase- and urease-regulation may be an adaptation to the variable and harsh conditions existing in the gastric mucosa.

Helicobacter pylori CagA Activation of MAPK Stimulates IL-8 and the Cell Cycle Songlin Zhang, Maura E. Munoz, Juanita L. Merchant, Ann Arbor, MI Background: HelicobacterpyloriCagA protein has been shown to be injected into human cell lines (AGS) through the TypelV secretory system and become tyrosine phosphorylated. Activation of host signal transduction pathways has been studied using wild type H. pylori and isogenic mutants of CagA. However, the effect of recombinant CagA alone on host cell signaling pathways has not been evaluated. Methods: Full length cagA was amplified from the H. pyloriSS1 strain and subcloned into both pCDNA3.1 and the Adeno-X Tet-Off expression vector. Fugene6was used to transiently transfect AGS cells. For adenoviral vectors, AGS cells were infected for 4 h and maintained in the gene-on or gene-off position using tetracycline for 48 h. Western blots were used to assay the levels of MAPK proteins, CagA, and cyclin DI. AGS cells were pulsed with BrdU 30 min before harvesting and analyzing the cell cycle by flow cytometry. Results: We showed that CagAexpression stimulates IL-8 promoter activity in a dose dependent manner using either transient transfection assays or inducible adenoviral expression vectors. Mutations of the IL-8 promoter showed that CagA activated IL-8 through the AP-1 and NFkB sites. Dominant negative Erk (DNErk), kinase deficient Raf (K375M Raf), ATP binding deficient Raf (KA Raf), and dominant negative HRas (15A Ras) blocked CagA activation. Furthermore, the Mekl/2 kinase inhibitor PD98059, but not the p38 inhibitor SB 203580, blocked the activation by CagA. Using western blots, we demonstrated that CagA increased phospho-Erk, c-los, and cyctin D1 levels and decreased p53 levels in AGS cells. Gel shift assays showed an increase in AP-1 transcription factor binding after inducible adenoviral CagA expression in AGS cells. Flow cytometry revealed that CagA expression significantly increased the number of cells in S phase. Conclusion: Recombinant CagA expressed in AGS cells stimulates cell proliferation and IL-8 expression via the MAPK pathway and represents the first documentation that recombinant CagA protein alone is sufficient to modulate the cell cycle.

711

A Novel Mechanism of Urease Activity Regulation by Helicobacterpylori in Acidic Media David R. Scott, Elizabeth A. Marcus, David L. Weeks, George Sachs, Los Angeles, CA Background & Aims: Helicobacter pylori, a neutralophile, uses acid neutralization by urease generated ammonia to combat gastric acidity, allowing gastric colonization. Both acute and chronic acid resistance mechanisms are present. Acute mechanisms of acid resistance could be due to surface urease and/or increased inner-membrane urea permeability via Urel. Slower mechanisms that have been suggested involve increased Ni2. insertion into apoenzyme (van Vflet et al; 2001, Infect. Immun. 69:4891-7) or increased enzyme synthesis by posttranscriptional regulation (Akada et al, 2000, Mol: Microbiol. 36:1071-1084). The aim of this study was to further define regulation of urease under acidic conditions. Methods: Surface bound urease was analyzed by measurement of free and bound urease after centrifugation through a ficoll step-gradient to prevent contamination by lysed bacteria and by quantitative urease immuno-staining of intact as compared to alcohol-fixed bacteria. Changes in urease synthesis or assembly were determined by incubation of the organisms at pH 5.5 or 7.0 for up to 3 hours in the absence and presence of urea without or with chloramphenicol, a protein synthesis inhibitor, or without or with dimethylglyoxime, a Ni2÷ chelator, and in urel positive and negative organisms. Results: The amount of surface urease was below detection limits using either centrifugation-washing or immuno-staining. Total bacterial urease activity was increased three to five-fold by incubation at pH 5.5 in the presence of chloramphenicol but not in Ni2+-freemedium or in urel knock-out organisms. There was also a three-fold increase in survival to acid shock in acid-adapted organisms. Conclusions: The data suggests that there is no surface bound ureaseand therefore no surface associated ureaseactivity. Extracellular ureasetherefore cannot be involved in the gastric habitation of H. pylori. A novel mechanism of regulation of intra-bacterial urease activity appears to be activation of intra-bacterial apoenzyme in acidic media, which is independent of protein synthesis but is dependent on the presence of NF+ and expression of Urel. This would allow habitation of the more acidic regions of the stomach. The intra-bacterial urease of H. pylori is therefore regulated in at least three ways, a rapid response via Urel activation and enhanced entry of urea, a more chronic response based on urease activation and presumably an even slower response based on alteration in mRNA levels and hence increase of protein synthesis.

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Helicobacterpylori CagA Protein Activates Serum ResponseElement via MAPK Signaling Pathway without Requiring Tyrosine Phosphorylation Yoshihiro Hirata, Shin Maeda, Ayako Yanai, Yuzo Mitsuno, Masao Akanuma, Haruhiko Yoshida, Yasushi Shiratori, Masao Omata, Tokyo, Japan Background: Type I strains of H. pylori activate various intracellular signaling pathways, including those leading to NF-K8 and SRE activation (Maeda et al. Gastroenterology 2000; 119:97, Mitsuno et al. Gut 2001; 49:18-22). These strains have cag PAl genes and transport CagA protein inside the host cells, where CagA can be tyrosine phosphorylated. However, little is known about the function of translocated CagA. Methods: CagA protein was expressed in HeLa cells by transfection and localization was visualized immunohistochemioally. Effects of CagA transfection on SRE- and NF-KB-dependenttranscription were analyzed by reporter assay using pSRE-Luc and pNFKB-Luc, respectively. Constructs with deletion or site-di[ected mutagenesis at tyrosine residues 899, 918, and 972 were also used. Dominant negative Ras and MEK1 inhibitor were used to assessthe involvement of MAPK signaling pathway. Results: Intracellular CagA expression activated SRE-dependenttranscription (40-fold of control) but had no effect on NF-KB-dependentone. SRE activation was abolished by N-terminal fragment(I-891) transfection, but not by C-terminal fragment(872-1186), although all CagAfragments were localized in cytosol. Site-directed mutagenesis at tyrosine residues diminished tyrosine phosphorylation but did not affect SRE activation. Immunoblot and EMSArevealedthat CagA transfection enhanced Elk1 phosphorylation and Elkl-DNA binding affinity, suggesting involvement of MAPK signaling pathway. Dominant negativeRasand MEK 1 inhibitor decreased CagA-mediated SRE activation by 50%. Conclusion: Transfected CagA enhanced Elk1 phosphorylation and activated SRE-dependenttranscription via MAPKsignaling pathway. C-terminal region of CagAwas responsible for the activation, however tyrosine phosphorylation was not required. The results suggest that transported CagAmay strongly affect SRE-dependentcellular responses, such as cell proliferation.

712 Urease Released from H. pylori Induces iNOS-Derived NO Production by Macrophages Alain P. Gobert, Benjamin D. Mersey, Yulan Cheng, Jamie C. Newton, Keith T. Wilson, Baltimore, MD BACKGROUND:Nitric oxide (NO) and its metabolites derived from the activation of inducible NO synthase (iNOS) are implicated in the pathogenesis of H. pylori (Hp) gastritis. We have reported that: 1) iNOS expression is upregulated in lamina propria mononuclear cells of Hp gastritis tissues, and 2) Hp stimulates iNOS expression and NO production in RAW 264.7 macrophages by an LPS-independentpathway involving soluble components. AIM: To identify the Hp-releasedfactor(s) that activates iNOS and NO production in macrophages. METHODS: RAW 264. 7 macrophages were co-cultured with Hp 3401 wild-type (WT), or the following isogonic mutants: vacA-, cagA-, picB-, and ureA-, in DMEM-IO% FBS. To separate bacteria from macrophages, Transwell filter supports (0.4 ~m pore size) were used. iNOS mRNA levels were assessed by Northern blotting and RT-PCR. iNOS protein was detected by Western blot. Concentrations of the NO metabolite, NO2,were measured in the co-culture supernatants by the Griess reaction. Water extracts of WT or mutant Hp strains, and recombinant urease were also used to stimulate macrophages. RESULTS: When macrophages were in contact with intact Hp WT or the 4 mutant strains, similar marked upregulation of iNOS mRNA was observed. However,when filters were used, the level of iNOS mRNA in macrophagesstimulated by Hp ureA- was decreased by 86% compared to ceils stimulated with Hp WT, while vacA, cagA, or picB deletion did not diminish the iNOS mRNA levels. The loss of iNOS induction with the ureA- mutant was confirmed by Western blotting. Hp in contact with RAW 264.7 cells induced 7- to 9-fold increases in NO~- release with either WT or mutant Hp strains; However, when Hp stra(ns were p(aced above the filters, NO2 production was significantly decreased by 58% with the ureA- strain compared to WT (p < 0.01), while there was no loss of NO2- production with the other mutant strains. Macropbage NO~ production was increased by 4-fold with 5 l~g/ml protein of Hp water extracts of WT, vacA-, cagA-, or picBstrains, but ureA- water extract failed to induce any NO2 production. Recominant urease (5 ~g/ml) stimulated significant increases in macrophage iNOS expression and NO2 production. CONCLUSIONS: When there is direct contact between Hp and macrophages, iNOS appears to be induced by non-specific pathways, but urease released by Hp is a very effective inducer of iNOS expression and NO production by macrophages.This result provides a new paradigm for Hp urease, implicating it in NO-dependent mucosal damage and carcinogenesis.

710 Expression of Amidase- and Urease-Mediated Ammonia Production in Helicobacter

pylori is Modulated by Fur

Arnoud H. M. Van Viler, Jeroen Stoof, Sophie W. Poppelaars, Ernst J. Kuipers, Johannes G. Kusters, Rotterdam, Netherlands Objective: Ammonia production is of vital importance to Helicobacter pylori, since ammonia is involved in acid resistance, nitrogen metabolism, chemotactic motility and tissue damage. In H. pylori, ammonia is enzymaticafly produced by hydrolysis of urea or amides through urease and amidase, respectively. Previous studies have indicated that activity of amidase is increased in a H. pylori urease mutant, and conversely urease activity is increased in an amidase mutant. However, key regulatory components in this balance of ammonia production have not yet been identified. We have previously shown that urease expression in H. pylori is modulated by Fur, the main iron-regulatory protein of H. pylori. In this study We have characterizedthe role of H. pyloriFur and iron in regulation of amidase and ureaseexpression. Methods: H. pylori 26695 and its isogenic fur mutant were grown in iron-restricted and ironreplete conditions, and amidase and urease expression and activity were monitored by enzyme assays, two-dimensional protein gel electrophoresis and Northern hybridization.Results: The AmiE amidase (HP0294) was identified on two-dimensional protein gels by MALDI-TOFmass spectrometry. AmiE protein was only detected in iron-restricted conditions, but was absent in iron-supplemented media. Amidase enzyme activity and levels of amiEmRNA were regulated likewise. Regulation was absent in an isogenic H. pylori fur mutant, demonstrating that Fur mediates AmiE regulation. Regulation of amidase is inversely correlated with urease activity; high amidase activity is associated with decreased urease activity, while low amidase activity was linked to increased urease activity. Conversely, nickel-supplementation of growth media, known to induce urease activity, resulted in decreased amidase activity. Conclusion: The regulation of amidase by iron and Fur in H. pylori contrasts with findings in other amidasepositive bacteria, where amidase expression is regulated in response to substrate availability.

713 Importance of Helicobacterpylori Outer Membrane Protein, oipA Yoshio Yamaoka, Shogo Kikuchi, Oscar Gutierrez, Michael S. Osato, David Y. Graham, Houston, TX; Aichi, Japan; Bogota, Colombia Background: There is continuing interest in identifying H. pylorivirulence factors that increase the riskfor clinical outcomes. Candidates proposed as disease-associatedvirulence factors include the cag PN, vacA, babA, iceA and oipA. However, as these are not independent of

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NF-Y represses expression of GC-C through interaction with the proximal promoter at a ciselement. Modulation of transcription by NF-Y may contribute to the tissue-specific expression of GC-C.

one another, it is not clear which factor(s) are the most important predictors of clinical outcome or severity of gastric inflammation. Methods: We examined 172 H. pylori; 86 from Colombia and 86 from the U.S. (43 with gastritis and 43 with DU for each), cag PAl, vacA, babA2and iceA status was evaluated by PCR and the oipA switch status ("on" or "off") was determined by PCR-based sequencing. A backward stepwise multiple logistic regression analysis was performed to determine which putative virulence factor(s) were the most discriminating for DU from gastritis. We used cag PAl, vacA (s and m), iceA, babA2 and oipA as explanatory variables. A similar regression analysis was also performed to determine which factors were related to severity of histology [H. pyloridensity, neutrophil infiltration, intestinal metaplasia (IM) and gastric atrophy] and mucosal interleukin (IL)-8 production using the putative virulence factors as well as country and clinical outcome as explanatory variables. Results: Only oipA "on" status was an independent determinant predictor of duodenal ulcer vs. gastritis [the U.S.: odds ratio (OR) = 6.64; 95% C.I. = 1.73-25.5, Colombia: OR = 4.0; 95% C.I. = 1.30-12.5]. oipA "on" status was significantly [partial regression coefficient (PRC) = 1.14 in the antrum and 0.97 in the corpus] (p
716 Cell-Specific Spatial Restriction Of Transcription Along the Gut Anterior-Posterior Axis Directed by the Lactase Promoter Zhi Wang, So Young Lee, Lynne C. Olds, Allen D. Cooper, Eric Sibley, Palo Alto, CA Background: Lactase gene expression is spatially restricted to the distal duodenum and jejunum of the small intestine during mammalian gut development. We aimed to characterize regions of the lactase promoter capable of regulating enterocyte-specific gene transcription along the antedor-postedor (A-P) gut axis. Methods: Two independenttransgenic mouse lines were generated harboring 1.5- and 2.0-kb of 5' flanking sequence of the rat lactase promoter cloned upstream of a firefly luciferase reporter gene. Transgene expression was quantified by luciferase assay of tissue tysates and by RT-PCR analysis of total RNA isolated from distinct small intestinal segments along the A-P axis. Cell-specific expression was localized by in situ hybridization and immuno-histochemical staining of intestinal sections from transgenic mice and wild-type controls. Results: Both the 1.5- and 2.0-kb lactase promoter-reporter constructs drive spatially restricted luciferase expression along the gut A-P axis, as detected by luciferase enzyme activity and mRNA abundance. The pattern of spatial restriction mimics that of the endogenous lactase gene with maximal levels of transgene expression (>1,000fold over background) in the distal duodenum and jejunum. Similar to that described for perinatal lactase expression, the zone of luciferasa reporter expression extends throughout the length of the gut in newborn transgenic mice and then becomes restricted to the middle of the small intestine by adulthood. Reporter gene expression is localized appropriately to epithelial cells lining the villi. Conclusions: Transgene expression recapitulates spatial restriction of lactase expression in two independent mouse lines and contrasts with the expression pattern previously described for a related construct. The results support the conclusion that lactase promoter sequences located 1.5-kb upstream of the transcription start site regulate spatial restriction of gene transcription in intestinal epithelial cells along the gut A-P axis during postnatal development.

714 A GC-Rich Region within the Human Intestinal Alkaline PhosphotaseGene Promoter Contains Multiple Cis-Elements Important for Transcriptional Activity Brian F. Hinnebusch, Madhu S. Malo, Joseph W. Henderson IV, Richard A. Hodin, Boston, MA Intestinal alkaline phosphatase (lAP) is an enterocyte differentitation marker. Previously, we have shown using DNAse I Footprinting that there are five discrete protein binding sites within the proximal lAP promoter region (named IF--I - - IF--V). In the present work, we have sought to determine the functional significance of this region of tandemly aligned cis-elements, as well as to characterize the nature of their cognate binding proteins. Transient transfections were performed in HT--29, Caco--2, HepG2,and COS--7 cells using Luclterase (LUC) reporter plasmids containing a 2.5 kb segment as well as multiple 5' deletions, of the human lAP 5' flanking region. A reporter construct containing an internal deletion of the segment containing IF--II, IF--Ill and IF--IV was also tested. Cells were co--transfected with the transcription factors gut--enriched Kruppel--like factor (KLF~) and the thyroid hormone receptor (TR). In some experiments, cells were differentiated by sodium tiutyrate treatment. Electromobility shift assays (EMSA) were performed with nuclear extract from HT--29 cells and radinlabeled oligomers corresponding to each DNA cis--element, lAP transactivation by either KLF--4 or butyrate required the GC--rich region containing IF--II, IF--Ill and IF--IV, since LUC activity in responseto both was decreasedby ~ 4 fold (p IF--II > IF--I > IF--IV with IF--V having negligible affinity for these proteins. We conclude that multiple, tandem cis--elements with differing affinities for transacting proteins exist within the proximal rAP promoter. These elements play a crucial role in lAP gene transcription in response to KLF--4 and in the context of differentiation, but thyroid hormone activates lAP independent of this GC--rich region.

717 Dual Mechanisms for the GATA-4 Activation of the Human Lactase-Phlorizin Hydrolase Promoter Herbert M. Van Wering, Evelien De Jong, Richard Grand, Francois Boudreau, Edmond H. H. M, Rings, Gary P. Swain, Stephen D. Krasinski, Boston, MA; Philadelphia, PA GATA4/5/6 transcription factor subfamily members are all expressed in the intestine and have been implicated as activators of intestinal gene promoters. We have previously shown that GATA-5 physically associates with HNF-I~ and cooperatively activates the lactase-phlorizin hydrolase (LPH) promoter (Gastroenterology120:A659, 2001). However, in the absence of HNF-lm GATA-5alone does not appreciablyactivate the human LPH promoter (Am. J. PhysioL 281:G69-G84, 2001). Based on the similar structural organization among the GATA4/5/6 subfamily, the hypothesis to be tested is that GATA-4 interacts with HNF-I~ by a mechanism similar to that of GATA-5. Thus, the function of critical domains in GATA-4 was mapped by first introducing mutations that delete or disrupt specific structures, and then characterizing specific functions such as physical association with HNF-I~ (GST pull-down assays), binding to DNA (EMSAs),and transcriptional activation in HeLacells (transient co-transfection assays). HeLa cells were used because GATA-specific and GATNHNF-I~ cooperative activation can be distinguished in cells that do not synthesize endogenous HNF-I~. Our data reveal that in contrast to GATA-5, GATA-4 activates the human LPH promoter independently of HNF-I~x by a mechanism that requires the activation domains and domains responsible for DNA binding. Similar to GATA-5, GATA-4 physically associates with HNF-I~ and cooperatively activates the human LPH promoter by a mechanismthat does not require the GATA-4activation domains or binding to DNA. Cooperative activation does require physical association between GATA4 and HNF-I~ as well as an intact HNF-1 binding site on the promoter. Physical interaction involves the C-terminal zinc finger of GATA-4 and homeodomain of HNF-lc~. Immunohistochemistry of GATA-4 and HNF-I~ in newborn mice revealsthat these factors are co-expressed in villus epithelial cells where lactase mRNA is highly expressed. CONCLUSIONS: GATA-4 demonstrates dual mechanisms of activation of the human LPH promoter that are characterized by: (1) HNF-l~-independent activation (in contrast to GATA-5), in which the DNA binding and activation domains are required, and (2) HNF-ls-dependent cooperative activation (similar to GATA-B), in which physical association is required but not the GATA-4 activation domains or GATA-4/DNAbinding. This study is the first to demonstrate distinct regulatory mechanisms for activation of the human LPH promoter by two conserved GATA factors.

715 Nuclear Factor Y Modulates Transcription of Guanylyl Cyclase C in Human Colon Carcinoma Cell Lines Matthew D. Di Guglielmo, Stephanie Schulz, Scott A. Waldman, Philadelphia, PA Background: Guanylyl cyclase C ((3C-C) is the receptor for the E. coil heat-stable enterntoxin, (STa), one of the leading causes of secretory diarrhea. GC-C is exclusively expressed by intestinal epithelial cells and primary and metastatic colorectal cancer cells. Aim: To examine the molecular mechanisms regulating intestine-specific expression of GC-C. Materials and Methods: Regulation of the GC-C proximal promoter was investigated employing DNase footprint analysis, electromobillty shift analysis (EMSA), GC-C promoter-luelterase reporter constructs, and co-transfection of reporter constructs with in vitro-expressed transcription factors. Where indicated, data were analyzed employing Student's t-Test. Results: DNase digestion of the proximal promoter of GC-C revealed four regions that were protected by intestinal nuclear proteins. Upon EMSA, these proteins formed complexes with a radiolabeled probe containing the consensus binding site for nuclear factor Y (NF-Y). A probe with a single base pair mutation in the NF-Y consensus site eliminated specific complex formation. The mobility of the specific complexes formed by wild-type probe and intestinal nuclear proteins was decreased (supershiffed) by specific antibodies to each of the three NF-Y subunlts. Transient transfection of T84 and Caco-2 (colorectal carcinoma cell lines) cells with luciferase reporter constructs controlled by the GC-C promoter revealedthat a single base pair mutation in the NF-Yconsensus binding site increasedluciferase expression -40% in Caco-2 (p
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718 The Lac Enhancer: An Intestinal Specific Enhancer from the Lactase Promoter Jesper T. Troelsen, Jorgen Olsen, Copenhagen, Denmark Lactasephlorizin hydrolase is expressedexclusively in the enterocytesand is in most mammals downregulated after weaning. Previous studies in transgenic mice have shown that gene regulatory elements neccesary for this expression pattern in pigs are located in 1 kb region upstream the transcriptional initiation site. The homeodomain transcription factors Cdx-2 and HNF-I's have been shown to interact and bind proximal promoter elements just upstream a TATA box. GATA-factors are also binding to an element in the proximal promoter and are probably also nessecary for the promoter activity. However the proximal promoter is not sufficient to drive a high expression of a reporter gene in differentiated intestinal cells. An upstream regulatory region, which we have named 'the lac enhancer', is located at position 894 to -798. This region is necessary to get high differentiation-dependent expression in intestinal cells. The lac enhancer was studied by mutation analysis, transfection experiments and electrophoretical mobility shift assays. The results from these experiments show that the lac enhancer is not a classical enhancer in the sense that it is not orientation-independent

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and it cannot be located 3' of a reporter gene, but it is able to activate transcription from a distant 5' position. Furthermore the ]ac enhancer is not promoter promiscuous as it is only able to enhance expression when it is located in front of an intestinal specific promoter such as the lactase or the sucrase-isomaltase promoter. In front of a SV40-derived promoter the lac enhancer has no stimilatory effect. The lac enhancer binds at least three transcription factors. HNFlalfa binds to the region -894 to -880 and an unknown factor binds to the region 833 to -814. A region -880 to -875 binds another unidentified factor. This element shows similarity to a cis-element located in the intestinal fatty acid binding protein promoter, which has been shown to be involved in repression of transcription in the intestinal crypt region. This is to our knowledge the first identification and characterization of an enhancer region that is necessary for intestinal differentiation-dependent transcription.

725 The Occurrence of Complications of Constipation and Bowel Surgery in Relation to Irritable Bowel Syndrome J Alexander Cole, Suzanne F. Cook, David P. Miller, Bruce Sands, Anuli N. Ajene, MeiSheng Duh, RhondaL. Bohn, Alexander M. Walker, Newton, MA; Research Triangle Park, NC; Boston, MA BACKGROUND:Bowel surgery and hospitalization for complications of constipation have been described in alosetron users for treatment of diarrhea-predominant irritable bowel syndrome (IBS). There are no epidemiologic data on the occurrence of these events among IBS patients. We estimated the incidence rates (IR) of these outcomes among patients with and without IBS before the introduction of alosetron. METHODS:We identified 87,449 people with an IBS diagnosis in a large national managed care.plan between January 1995 and December 1999. We classified person-time according to duration following an IBS diagnosis in the claims data. We estimated risk in the general population without an IBS diagnosis using a 1% random sample of the plan membership. We used the claims data to define complications of constipation and bowel surgery cases. We validated the definition using abstracted medical records for a sample of cases. We calculated incidence rates by age, gender, calendar year, and proximity to an IBS diagnosis and modeled their joint effects using Poisson regression. The three weeks following an IBS diagnosis were treated separately becausean IBS diagnosis was sometimes assigned at the same time as the outcomes. RESULTS: We identified 2,297 cases of complications of constipation and 15,209 cases of bowel surgery. Six months following an IBS diagnosis, the IR per 1,000 person-years (PY) was 1.39 for complications of constipation and 9.75 for bowel surgery. One year following an IBS diagnosis, the IR per 1,000 PY was 0.88 and 4.86 for complications of constipation and bowel surgery, respectively. Among people without an IBS diagnosis, the IR per 1,000 PY was 0.23 for complications of constipation and 1.35 for bowel surgery. People were 4.4 (95% CI 3.2 - - 6.0) times the risk of having complications of constipation and 4.9 (95% Cl 4:3 - - 5.5) times the risk of having bowel surgery six months following an 18S diagnosis. One year following an IBS diagnosis, people were 2.5 (95% CI 1.9 - - 3.3) and 2.2 (95% CI 2.0 - - 2.5) times the risk of developing complications of constipation and bowel surgery, respectively. There were 1.9% of complications of constipation and 0.7% of bowel surgery cases that occurred within three weeks of an IBS diagnosis. CONCLUSIONS: Risk in IBS patients persisted as the length of time after a diagnosis increased. These data indicate that IBS is associated with significant morbidity; IBS may not the benign disease as generally proposed.

719 Cux/CDP Represses the Sucrase-lsomaltase Gene by Interacting with the Novel Colonic Repressor Element CRESIP Francois Boudreau, Edmond H. H. M. Rings, Gary P. Swain, Angus M. Sinclair, EunRan Suh, Richard H. Scheuermann, Peter G. Traber, Philadelphia, PA; Dallas, TX BACKGROUND: Sucrase-lsomaltase (SI) is an enterocyte-specific gone that is expressed in a complex temporal pattern along the vertical and horizontal axis of the intestine. We have previously characterized a novel colonic element of the SI promoter (CRESIP) that conferred repression of SI during intestinal development. This site predicts a consensus site for a transcriptional repressor Cux/CDP. AIM: To investigate whether Cux/CDP can interact with the SI gone promoter and to assess mechanisms of SI gene repression in intestinal epithelial cells and Cux/CDP mutant mice. METHODSAND RESULTS: The four distinct DNA binding domains in Cux/CDP were studied for binding affinity to the CRESIP by EMSA. Only the Cut Repeat 3 (CR3) and homeodomain (HD) of Cux/CDP specifically interacted with CRESIP in vitro. Removal of the c-terminal region that comprises the repression domain and the HD of Cux/CDP did not abolish the interaction with CRESIP. Co-transfection of Cux/CDP and Cdx2 expression vectors reduced the Cdx2-dependentactivation of the SI promoter in preconfluent Caco2 cells. This effect was abolished when a mutated Cux/CDP protein or a SI promoter that contained mutations in the CRESIPwere used in the luciferase assay. Immunohistochemistry showed that Cux/CDP is localized in enterocytes of the crypt with a decreasing gradient of expression toward the villi of the jejunum. In contrast, Cux/CDPwas expressed predominantly in surface colonocytes with lower expression in the bottom of the colonic crypt. Western blot analysis showed an increasing gradient of Cux/CDP expression along the horizontal axis of the intestine. RNaseprotection assay was then used to monitor the expression of the SI gene in the intestine of CDPAHD mutant mice. SI mRNA level was induced early during postmatal intestinal development of Cux/CDPAHD mutant mice as compared to control heterozygoos mice. CONCLUSION:Cux/CDPinteracts with CRESIPto repress the SI gene promoter in vitro. The alteration of Cux/CDPprotein expression in mice confirms that it acts as a developmental repressor of SI in the intestine. Furthermore, these results suggest a role for Cux/CDP in the maintenance of the intestinal epithelium.

726 The Risk of Colonic Ischemia among Patients with Irritable Bowel Syndrome J Alexander Cole, Suzanne F. Cook, David P.. Miller, Bruce Sands, Anuli N. Ajene, MeiSheng Dub, Rhonda L. Bohn, Alexander M. Walker, Newton, MA; Research Triangle Park, NC; Boston, MA BACKGROUND: Colonic ischemia (also known as ischemic colitis) has been identified in patients who used alosetron fo[ treatment of diarrhea-predominant irritable bowel syndrome (IBS). The incidence of colonic Ischemia has not been described in the literature in relation to IBS, so the causal role of alosetron is a matter of speculation, We sought to estimate the incidence rate (IR) of colonic ischemia in patients With and without IBS before the introduction of alosetron. METHODS: We identified 87,449 persons with a diagnosis of IBS in a large national managed care plan between January 1995 and December 1999. We categorized each individual's person-time according to the length of time following an IBS diagnosis in the claims data.To estimate the risk among patients without IBS, we used a 1oYorandom sample of the plan membership. We used a colonic ischemia case definition using diagnoses, procedures, and drugs in the claims data. We validated and refined the definition by comparing claims data to abstracted medical records. We calculated incidence rates by age, gender, calendar year, and proximity to an IBS diagnosis. The joint effect of these predictors was then modeled using Poisson regression. The three weeks following an IBS diagnosis were treated separately because IBS and colonic ischemia diagnoses were sometimes assigned at the same time. RESULTS: We identified 714 cases of colonic ischemia, Most were women aged 30 to 69. Approximately 10% had a preceding IBS diagnosis. During the six months following an IBS diagnosis, the IR per 1,000 person-years (PY) was 0.49 for women and 0.47 for men. One year following an IBS diagnosis, the IR per 1,000 P¥ was 0.46 and 0.30 for women and men, respectively. By comparison, the IR per 1,000 PY among people without an IBS diagnosis was 0.09 for women and 0.05 for men. Adjusting for age, sex, and calendar year, people were 4,4 fold (95% CI 2.6 - - 7.4) increased risk of developing colonic ischemia within the six months following an IBS diagnosis. One year following an IBS diagnosis, people were 3.1 (95% CI 2.1 --4.6) times increased risk of developing colonic ischemia. 2.8% of colonic ischemia cases occurred within three weeks of an IBS diagnosis, and the great majority of these were within a few days. CONCLUSIONS:IBS is associated with a substantial elevation in risk for subsequently developing colonic ischemia. These data also suggest that some patients with colonic ischemia are misdiagnosed as having IBS. These relations warrant further investigation.

720 The Impact of Functional Gastrointestinal Disorders on Health-Related Quality o f Life: A Population-Based Case-Control Study Smita L. S. Halder, G Locke III, Nicholas J. Talley, Amy L. Weaver, Sara L. Felt, Alan R. Zinsmeister, Salford, UK; Rochester, MN; Penrdh, Australia Background: Health-Related Quality of Life (HRQoL) is clearly diminished in people with Functional Gastrointestinal Disorders (FGID) seen in referral centers. Whether people in the community with FGIO have diminished HRQoL is unclear. Aim: To determine if HRQoLdiffers among persons with and without FGID selected at random from the community. Methods: 904 Olmsted County, MN residents aged between 20 to 50 years were randomly selected to receive by mail the bowel disease questionnaire, a valid self report GI symptom measure. Those people reporting symptoms of either Non Ulcer Dyspepsia (NUD) or Irritable Bowel Syndrome (IBS) were considered as possible cases. Those reporting no abdominal pain and less than two other gastrointestinal symptoms were selected as possible controls. All eligible subjects were asked to be interviewed and complete a second symptom inventory, a battery of psychologic measures, and a HRQoL measure (SF-36). Those refusing to be interviewed were asked to complete the surveys by mail. The association between FGID and HRQoL was assessed by logistic regression adjusting for age, gender and psychological parameters including SCL-90, social support, and life experiences survey scores and history of abuse, Results: A total of 222 subjects participated (152 in person,70 by mail).: Mean age was 36 yrs; 58% female. The physical (PCS) and mental (MCS) composite scores from the SF-36 were both lower in the GI symptom groups than controls (Table). In the unadjusted regression model IBS was associated with the PCS (p
731 Identification of Enteric Primary Afferent Neurons in the Guinea-Pig Small Intestine Marcello Costa, Phil Davies, Kate Brody, Simon Brookes, Joel Bornstein, Adelaide, Australia; Melbourne, Australia

Qualityof Life: Age and GenderAdjustedPhysicaland MentalCompositescores GI group PhysicalCompositeScore MentalCompositeScore N Mean Std Deviation Median Mean Std Deviation Median HeelthyControls 110 52.7 7.3 54.8 54.6 5.8 55.7 ~ Any FGID 112 50.5 8.1 51.6 49.1 10.1 51.5 NUD 30 51.4 7.3 52.2 47.9 11.7 52.4 IBS 39 49.7 9.5 51.7 49.9 10.3 51.9 Both NUDIIBS 32 50.2 7,4 51,0 48,2 8.9 50.9 NeitherNUD/IBS 11 51.9 7.3 50.7 518 7.9 53.7

Electrophysiologicaland morphological evidencesuggest that enteric primary afferent neurons (EPANs) have Dogiel type II morphology and electrophysiological properties of AH neurons (1). The actual proportion of these neurons is not known, as only some of them contain calbindin (2). We have investigated whether antibodies to NeuN(3) identify enteric primary afferent neurons in the guinea-pig small intestine. We have used tissue from guinea-pigs of either sex (170-400g) killed humanely, and used multiple labelling immunohistochemistry, with antibodies to calbindin, calretinin, NOS, ChAT and NPY to mark the major classes of enteric neurons in combination with antibodies to NouN. NeuN neurons represented 38.0+/2.4 (SD) % of all myenteric neurons. Of these 62.8+/-2.9 oYoalso contained calbindin. All

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contained CHAT, hut none of the other neuronal markers. In order to establish if all the NeuN-immunoreactive neurons had the Dogiel type II shape and electrophysiological AH characteristics of EPANS, we performed random intracellular recording and biocytin filling of myenteric neurons subsequently visualised with straptavidin-AlexaFluor 488. Filled neurons were characterized electrophysiologically as S or AH, their shape was classified as Dogiel type I or type II and then immunoreectivity to Calbindin and NeuN was established. Blind analysis of 29 biocytin filled neurons, showed that all 15 AH neurons recorded had Dogiel type II shapes. 14/15 of these were NouN immunoreactive, whereas none of the 14 filled Sneurons, a~l of which had Dogiel type I shape, contained NeuN immunoreactivity. 8 of the NouN positive Dogiel type II/AH neurons contained calbindin and 6 did not. These results indicate that the enteric primary afferent neurons in the guinea-pig small intestine are identified by the marker NeuN.They represent a larger proportion of myenteric neurons than previously suspected. The nature and function of NeuN in these neurons remains to be established but it can be used to investigate afferent neural circuits in the enteric nervous system. 1. Furness, J.B.,et al (1998). Prog Neurobiol, 54, 1-18.2. Costa, M., et al (1996). Neuroscience,75, 949967. 3. Mullen, R.J., et al (1992). Development, 116, 201-211.

GABA acting via GABAAandGABAB,but not GABAc, Receptors is Important For Mucosal Reflexes to LM and CM of Guinea-Pig Distal Colon Heiton J. Reis, Marco A. Romano-Silva, Terence K. Smith, Reno, NV; 8elo Horizonte, Brazil ~/-aminobutyric acid (GABA) has been shown to be contained in a subset of myenteric neurons (Williamson et al. Cell Tiss Res: 284, 29 1996). Aims: To investigate the importance of GABA receptor subtypes in regulating both the initiation and output of enteric reflex pathways to both the longitudinal (LM) and circular muscle (CM) layers in distal colon. Methods: Guineapigs were killed by isofluorane inhalation followed by exsanguination. Segments of distal colon (7-8cm long) were removed and mounted in a partitioned chamber (Oh) which divided the stimulation site (STIM) from the recording site (REC) (Smith & McCarron, J Physiol. 512: 898, 1998). The oral or anal end of the preparation was opened and pinned with the mucosa uppermost. Oral or anal reflex responses,to mucosal stimulation, were measuredwith tension transducers attached to the LM and CM. To record pure LM responses the CM was dissected away to prevent mechanical interactions betweenthe two muscles (Smith & McCari'on, 1998). The organ bath contained oxygenated Krebs solution (37°0). Results: Oral and anal mucosal stimulation ~3 strokes) produced an anal relaxation and an oral contraction respectively. Phaclofen (lO~M), a GABAereceptor antagonist, added to the STIM Ch decreased the oral contraction (LM 38.7_+0.2% and CM 38.95_+0.5% of control) and anal relaxation (LM 40.36_+ 1.7% and CM 46.17_+0.7%) in both muscles by similar amounts. However, phaclofen had no effect on these responseswhen added to the recording chamber. In contrast, bicuculline 0OF,M), a GABAAantagonist, reduced both the reflex oral contraction and anal relaxation of the LM and CM when added to both the STIM Ch (oral contr: LM 21.0_+0.5, CM 32.5 _ 0.9% of control; anal relax: LM 22.1 _+2.1 and CM 41.9_+3.3%) and the REC Ch (oral contract: LM 9.4_ 2.6% and CM 29.9_+3.1; anal relax: LM 22.1 _+0.4% and CM 41.6_+3.0% of control (p
732 Multiple Sensory Mechanisms Activate Long Descending Enteric Vasodilator Reflexes in Guinea Pig Ileum David Reed, Stephen Vanner, Kingston, ON, Canada; Kingston, Canada Submucosal cholinergic vasodilator neurons innervating sabmucosal arterioles play a critical role in regulating mucosal blood flow but the enteric reflexes involved are unclear. This study examines sensory mechanisms which activate these reflexes and the pathways involved. An in vitro guinea pig ileal model was developed which enabled videomicroscopy recordings of submucosal arterioles in the mucosal strippad aborad segment of the preparation. Stimulation of the grad segment was conducted within the lumen of intact intestine or a flat sheet of the intestine (mucosa up). Sensory pathways were activated by balloon distention (in lumen of intact intestine or beneath the flat sheet) or brushing the mucosa (fiat sheet) with a fine brush (width=lcm). Nifedipine (II~M) was added to limit muscle contraction. Balloon distention in the lumen evoked dilator responses up to 2 cm from mid-balloon (n = 21). Tetmdotoxin blocked all responses (n= 10) and the muscarinic antagonist 4-DAMP blocked responses, consistent with the involvement of cholinergic submucosal vasodilator neurons. Selective surgical tesioning of the myentaric plexus between recording and stimulating sites abolished responses but submucosal lesioning had no effect. To explore the origin of sensory traosduction, preparations with or without mucosa were studied. Distention-evoked dilator responses were recorded in 100% of mucosa-on preparations and 54% of mucosa-off preparations (p=O.O02; n=31). The mean dilation of mucosa-on preparations was 38 _+ 3.3% (% of maximum) compared to 12 _+ 4.2% in mucosa off preparations (p
735 Neurochemical and Confocal Microscopic Analysis Reveals That Acid-Activated NHrergic Neurons of the Rat Gastric Myenteric Plexus Are Inhibitory Motor Neurons Rudolf Schicho, Marion Danzer, Maria A. Pabst, Peter Holzer, Irmgard Lippe, Graz, Austria Background: Challengeof the rat gastric mucosa with excess HCI (0.5 M) activates nitrergic, but not chollnergic, neurons of the myentedc plexus as visualized by expression of c-Fos. However, the functional implication of these neurons remained unknown. We therefore set out to analyze the neurochemical code of the activated neurons by immunocytochemical colocalization of c-Fos with nitric oxide synthase (NOS), neurupeptide Y (NPY), vasoactive intestinal polypeptide (VIP), enkephalin (ENK), gastdn-releasing peptide (GRP) and calbindin D-28k (CALB) and to trace the projection of their fibers by laser scanning confocal microscopy (LSCM). Methods: The stomachs were examined two hours after intragastric administration of HCI (0.5 M, 1 ml/lO0 g) to rats. Whole-mount preparations of the myenteric plexus and transverse sections of the gastric corpus wall were processedfo[ immunofluorescence multiple labeling with antibodies to c-Fos, NOS, NPY, VIP, ENK, GRP and CALB and examined by conventional fluorescence microscopy and LSCM. Results: Exposure of the gastric mucosa to HCI caused about 55 % of the nitrergic neurons in the myenteric plexus to express c-Fos. All neurons positive for c-Fos were immunoreective for NOS, NPY and VIP, but did not stain for GRP, ENK or CALB. The neurochemical code NOS/NPYNIP was also found in nerve fibers supplying the longitudinal muscle, the circular muscle and the muscularis mucosaeas revealed by LSCM. Conclusions: These data provide evidence that gastric mucosal acid challenge activates a distinct population of myenteric neurons characterized by the neurochemical code NOS/NPYNIP. The projection of nervefibers with this chemical code to the muscularis propda and muscularis mucosae suggests that the acid-activated neurons of the myenteric plexus are inhibitory motor neurons. Supported by FWF grants 14295 and 15452.

733 Chemical Coding of Putative Enteric Primary Afferent Neurons in the Mouse Myenteric Plexus Kate Brody, Marcello Costa, Simon Brookes, Adelaide, Australia In the mouse small intestine enteric primary afferent neurons have not yet been identified. NeuN has been recently established as a marker for enteric primary afferent neurons in the guinea-pig small intestine(I). In this study we have investigated whether antibodies to NeuN label mouse myenteric neurons that could represent primary afferent neurons. Adult BalbC mice were killed humanely, segments of small intestine were opened flat and fixed in Zamboni solution. A population of large myenteric neurons was immunoreective for NeuN. Immunoraactivity was faint in the cytoplasm but intense in the nuclei. NeuN immunoreacUve neurons represented9.6 +/- 3% of all myentedc neurons. None of the NeuN-immunoreective neurons contained NOS but all were faintly immunoreactive for CHAT. In the guinea-pig small intestine the enteric primary afferent neurons are large, contain ChAT and a large proportion contain calbindin (2). We have therefore performed triple labeling with NeuN, calbindin and calretinin. There were numerous calbindin immunoreactive myentaric neurons with both Dogiel type I and type It shape. There were also numerous calretinin neurons mostly Dogiel type I. Few of the neurons contained both calbindin and calratinin. The vast maiodty of NeuN neurons (78.4 +/- 8.8%) contained calbindin, calretinin or both and these all had a Dogiel type II shape. Conversely all Dogiel type II neurons with calcium binding proteins had NeuN. Almost half of NeuN neurons contained calbindin (44.9 + h 6.4%), about one third (30.2 + h 3.9%) contained both calbindin and calretinin and the remaining 3.3% +/- 2.3% contained only cairetinin, in conclusion, if NeuN labels enteric primary afferent neurons in the mouse, then these form a smaller proportion of myentefic neurones than in guinea-pig. In addition, on the basis of the two calcium binding proteins, there may he several suhpopulations of enteric primary afferent neurons. 1. M.Costa, P.Davies, K.M Brody, S.J.H.Brookes and J. Bornstein. Identification of enteric primary afferent neurons in the guinea-pig small intestine. AGA 2002. 2.Costa, M., S.J.H. Brookes, P.A. Steele,I. Gibbins, E. Burchar,and CJ. Kandiah.Neurochemical classification of myenteric neurons in the guinea-pig ileum. Neuroscience75: 949-967, 1996.

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736 An Arg-122-Cys Mutation in the Cationic Trypsinogen (PRSS1) Gene Causes Hereditary Pancreatitis and Reduces Trypsin Autoaativation and Antodegradation Peter Simon, Frank Ulrich Weiss, Miklos Sahin-Toth, JOrgen Schnekenhurger, Julia Mayerle, Wolfram Domschke, Markus M. Lerch, Muenster, Germany; Los Angeles, CA Hereditary pancreetitis has been found to be associated with various germline mutations in the cationic trypsinogen (PRSS1) gene. What remains unclear is the biological mechanism through which trypsinogen mutations cause pancreatitis. We have recomhinantly expressed cationic trypsinogen with a recently identified PRSSl mutation (R122C, Gastroenterology 2001 ;120:170) and have studied its enzyme kinetics in vitro in comparison to wild type trypsin. Methods: Recombinant human cationic trypsinogen cDNA was ligated into the modified trypsinogen expression vector pTrap-T7 under the control of the T7 promoter. The Cys-122 mutation was introduced by polymerase chain reaction mutagenesis. Wild type and Cys-122 cationic trypsinogen were expressed in E, coil BL21(DE3), inclusion bodies were isolated, and trypsinogens were re-folded and purified via ecotin affinity columns. Results: Recombinantly expressedR122C-mutanthuman trypsinogen was found to undergo greatly reducedautoactivation, enterokinase-inducedactivation and cathepsin B-induced activation. The Km of R122Ctrypsin was found to be unchanged but its kcat was reduced to 37% of the wild type. After correction for enterokinase activatable activity, and specifically in the absence of calcium, the R122C mutant was more resistant to autolysis than the wild type. Three dimensional modeling of R122C trypsin predicted an unimpaired active site, a destabilization of the calcium binding loop and the formation of disulfide bonds between unimpaired cysteine residues. Interestingly, in a family with hereditary pancreatitis which carries the R122C mutation the diagnosis cannot

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be made using the widely used Afl III restriction enzyme digest and we therefore propose a new assay based on restriction enzyme digest with BstU I as a a new screening method which detects three different mutations in the same codon. Conclusions: The R122Ctrypsinogen mutation has recently been identified in three unrelatedkindreds with hereditary pancreatitis and thus represent the forth most common PRSS1 mutation associated with the disease phenotype. The in vitro biochemistry of Cys-122 trypsin raises the question whether a gain or a loss of trypsin function initiates the onset of pancreatitis.

synthesis with stimulation at low and inhibition at high concentrations. Inhibition of protein synthesis can be due to an increase in phosphorylation of the eucaryotic initiation factor 2c~ subunit (elF2c~)and the inhibition of the activity of its associated guanine nucleotide exchange factor, elF2B, which is required for translation initiation. We therefore evaluated the effects of caerulein (CAE) induced AP on pancreatic protein synthesis in vivo, elE2B activity and the phosphorylation of elF2~. Methods: C5781/6J mice received IP caerulein (50 ~g/kg/h) to induce AP, confirmed by increased intracellular trypsin activity and serum amylase and by cytoskeletal disruption. Pancreatic protein synthesis was determined by the flooding dose technique using 3H-phenylalanine.Free phenylalanineconcentrations in plasma and pancreas were determined by HPLC. Changes in phosphorylation of translational effectnr proteins were measured by gel electrophoresis and Western blotting. Activity of elF2B was analyzed by measuring the rate of release of 3H-GDPfrom purified elF2. Results: Markers of AP were clearly observed after a single dose of caerulein which also inhibited pancreatic protein synthesis to 53.3% +/- 6.6 of control at 10 min, 39.7% +/- 2.9 at 30 min and 49.8% +F 7.2 at 60 rain. Protein synthesis was further reduced to 17.9% +/- 1.4 and 19.4% +/- 0.9 of control, after 3 and 5 hourly caerulein doses, respectively, e!F2B activity was reduced to 46.4% +/- 10.6 at 1 h and to 41.7% +/- 6.8 at 3 h, which correlated with an increase in the phosphorylation of its inhibitor eIF2~ that reached maximum levels (2.4 +/- 0.1 fold increase of control) at 1 h. By contrast, the relation of Other translational effectors including phosphorylation of the ribosomal protein $6, the eucaryotic initiation factor 4E (elF4E) and its binding protein (4E-BP) to this inhibition was not clear. Conclusions: Caerulein-induced AP was rapidly accompanied by inhibition of pancreatic protein synthesis. This inhibition most likely results from the inhibition of translation initiation triggered by increased eIF2c~ phosphorylation and reduction of elF2B activity

737 Model of Chronic Alcoholic Pancreatitis Ilya Gukovsky, Aurelia Lugea, Jason Cheng, Barbara A. French, Nora E. Riley, Samuel French, HidekazuTsukamoto, Stephen J. Pandol, Los Angeles, CA; Torrance, CA Background & Aims: Alcohol abuse is the major cause of chronic pancreatitis. A critical obstacle to our understanding of this disease is lack of animal models. Our objective was to develop such model. Methods & Results: Rats were pair-fed for 8 weeks Lieber-DeCarli (ethanol) or control diet. For the last 2 weeks, they received i.p. injections of cyclosporin A (CsA; 20 mg/kg once daily) or vehicle. 7 days betore the end of feeding, acute pancreatJtis was induced by 4 hourly Lp. injections of 20 ~g/kg cerulein; control rats received saline. 1 week after cerulein treatment the animals were killed and pancreas analyzed histologically and biochemically. None of the individual treatments produced pancreatic injury. In particular, with the control diet pancreas recovered almost completely 1 week after cerulein pancreatitis. However, the ethanol diet together with cerulein and CsA treatments resulted in severe pancreatic injury characterized by loss of parenchyma, widespread fibrosis, and massive inflammatory infiltration. Some damagewas observed with combinations of cerulein pancreatitis with either ethanol or CsA. Acinar cell loss was more pronounced in rats fed ethanol than the control diet. The combined treatment induced proliferation and activation of pancreatic stellate cells as shown by staining for desmin and smooth muscle actin, and by electron microscopy. Immunostaining showed that the inflammatory cells were macrophages and T lymphocytes. We observed necrosis and apoptosis of acinar cells, indicating the involvement of both pathways in parenchymal cell death. There was also an increase in PCNA staining of parenchymalcells suggesting the induction of regenerativeprocesses. Expressionof cytokines and extracellular matrix proteins was measured by RT-PCRand Western blotting, and activation of transcription factors NF-KB and AP-1, by gel-shift assay. Overall, the biochemical markers correlated with histological changes, in addition, some of them demonstrated differential contributions from the individual treatments. For example, cerulein by itself stimulated collagen I mRNA expression whereas CsA up-regulated TGFI31. Conclusion: We have developed a model of alcohol-mediated post-acute pancreatitis induced by synergistic effect of acute injury and ethanol diet (alone or in combination with CsA). It displays all 3 responses characteristic of the human disease: loss of parenchymal cells, fibrosis, and sustained inflammation. The model will allow investigations into the mechanisms by which alcohol sensitizes pancreas to chronic injury.

740 Acute PancreatiUs Induced by Retrograde Infusion of Radiologieal Contrast in Rats Is Attenuated by the Addition of an NK1 Receptor Antagonist to the Infusate: Implications for the Pathogenesisand Prevention of ERCP-InducedPancreatitis. Zhijun He, Tony E. Yusuf, John Winston, Pankaj J. Pasricha, Galveston, TX Background and Aim: Acute pancreatitis is a relatively uncommon but potentially serious complication of ERCP and related interventions. However, its pathogenesis remains poorly understood and attempts at prevention have met with partial success at best. Neurogenic inflammation mediated by substance P (SP)-NK1 receptor interaction has been implicated in the pathogenesis of acute secretagogue-mediatedpancreatitis. We hypothesizedthat a similar mechanism may play a role in ERCP-induced pancreatitis. Methods: A rat model simulating ERCP was set up by retrograde injection of the radiological contrast agent, meglumine, into the pancreatic duct under constant pressure monitoring. In another group of animals, the NKI-R antagonist CP-96345 (1 ~,M) was mixed in the contrast solution prior to ductal injection. Rats were sacrificed 24 hours later and serum amylase, pancreatic water content, and pancreatic myeloperoxidase (MPO) were measured. Results: The NK1-R antagonist CP96345 attenuatedthe severity of acute pancreatitis as measured by water content (%) (control: 73.69 _+ 1.72; contrast only:79.18 +_ 0.828*; contrast + 0P-96345:73.90 _+ 1.27**)and MPO(fold-increase) (control: 1.00; contrast only:12.51 _+ 3.50*;contrast + CP-96345 2.22 +_ 0.13"*) (ANOVA: * P < 0.05, compared with control group;** p < 0.05, compared with contrast group). Serum amylasewas elevatedto a similar extent in both groups with retrograde injection. Conclusion: Antagonism of the NK1 receptor ameliorates pancreatitis in this model without affecting the initial injury to parenchymal cells as reflected by the serum amylase level. Thesefindings suggest a role for a neurogenic contribution to ERCPinduced pancreatitis. Further, the simple addition of a specific NK1 receptor antagonist to the contrast agent may be a potentially useful way to prevent ERCP induced pancreatitis.

738 Disturbance of Intracellular Vesicle Transport during Acute Pancreatitis Is Associated with Negative Regulation of the Motorprotein Dynein J0rgen Schnekenburger, Ina-AlexandraWeber, Igor Buchwalow, Markus M. Lerch, 48149 Muenster, Germany The initial phaseof acute experimental pancreatitis is characterizedby a blockage0f exocytosis, an accumulation of zymogen granules and a disturbance of intracellular transport in pancreatic acinar cells. The cellular mechanisms responsible for the disturbed intra-cellular transport are not known but a disassembly of microtubules was suggested to be involved in this process. In an experimental model of pancreatitis we have studied the role of the tubulinassociated motorproteins dynein and kinesin, which stabilize the trans-Golgi-netwerk, can bind vesicular cargo, and can move secretory vesicles along micrntubules in an ATP-dependent manner. Methods: Pancreatitis was induced in male Wistar rats by infusion of 10 mg/kg/h cerulein for up to 48 hours. Rats were then sacrificed and the pancreas was removed and fixed in liquid nitrogen or formaldehyde. The ultrastructural localization of microtubules and motor proteins was determined by immunofluorescence microscopy, immunohistochemistry and EM. Their expression and association was studied by Western blot, tubulin polymerisation and immunoprecipitation with mono-specific antibodies. Results: Oynein and kinesin were strictly colocalized in cells of the exocrine pancreas and associated with the cytoskeleton protein tubulin. This association was maintained during acute pancreatitis and neither dynein, kinesin nor tubulin were degraded in the initial phase of an experimental pancreatitis. Under all conditions dynein and kinesin were exclusively found in the acinar cell trans-Golgi-network and not at the apical pole of pancreatic acinar cells. In the initial period of supramaximal cerulein stimulation the motorprotein dynein was found to be tyrosine-phosphorylated. Tyrosine phosphorylation had no effect on the dynein association with the cargo-receptor protein dynactin. The breakdown of the cytoskeletal structures during acute pancreatitis is not the result of the degradation of tubulin or its motorproteins. The rapid tyrosine-phosphorylation of dynein indicates a regulated inactivation of intracellular transport mechanisms. Oynein phosphorylation was shown in vitro to either inhibit its binding to dynactin or to inactivate its motor activity. Our results indicate a downregulation of force generating activity and provide insight into a new mechanism of vesicle transport regulation in an in vivo system.

741 Generation of Trypsinogee Activation Peptide in the Exocrine Pancreas by Different Mechanisms Walter Halangk, Thomas Reinheckel, Rainer Matthias, Thilo K~hne, Hans Lippert, Markus M. Lerch, Magdeburg, Germany; Freiburg, Germany; Muenster, Germany The intrapancreatic activation of trypsinogen is thought to he an initiating factor for the onset of pancreatitis and can be quantitated by measuring the generation of trypsinogen activation peptide (TAP). The extent of TAP generation has been found to reflect the clinical disease severity of acute pancreatitis. Here we show that the processing of trypsinogen and the generation of TAP can occur independently and in the complete absence of trypsin activity or trypsinogen activation. Methods: The processing of trypsinogen by purified cathepsins B (CTSB) and cathepsin L (CTSL)was characterizedby Western-blotting, N-terminal sequencing and mass spectrometry. Trypsin activity was quantitated using the fluorogenic substrate BOCGIn-Ala-Arg-AMCand TAP by ELISA (Biotrin). Intracellular trypsinogen activation was induced by supramaximal cerulein stimulation in CTSB-and CTSL-deficientmice in rive and in isolated pancreatic Iobules in vitro. Results: CTSB processed trypsinogen at the activation peptide cleavagesite and this resulted in the generation of trypsin activity as well as TAP (APFODDDK). In contrast, CTSL cleaved trypsinogen in position G26-G27 which produced a truncated and inactive trypsin as well as a non-immunoreactive, elongated TAP (APFDDDDK-IVG).Exposure of the elongated synthetic TAP-IVG to CTSB resulted in the additional, rapid and massive generation of immunoreactive TAP (APFDDDDK). In whole animal or lobule experiments, which were performed to test whether this mechanism is biologically relevant, we found ~hat supramaximal cerulein stimulation of the CTSL-deficient pancreas resulted in a higher trypsin activity but a lower TAP generation than in controls whereas CTS8 deletion reduced both events. Conclusion: CTSL must therefore be regarded as a trypsinogen-degrading enzyme that eliminates trypsinogen as substrate for activating enzymes such as CTSB. An initial cleavage by CTSL permits a subsequent exopeptidase cleavage by CTSB - which intum generates massive amounts of immunoreactive TAP in the absence of trypsin activity. This is the first biological condition in which the abundant generation of TAP neither reflects nor parallels trypsinogen activation, trypsin activity, or disease severity.

739 Caerulein-lnduced Acute Pancreatitis Inhibits Protein Synthesis in Mouse Pancreas by Inhibiting eIF2B Activity. Maria Dolors Sans, Matthew J. Oimagno, Louis G. D'Alecy, John A. Williams, Ann Arbor, MI Background & Aim: Experimental acute pancreatitis (AP) inhibits secretion but the effects on synthesis of digestive enzymes are not clear. Previous work from our laboratory has shown that cholecystokinin (CCK) in vitro exerts a biphasic effect on pancreatic acinar protein

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layers of the gastrointestinal tract, lmmunoreactivity for the neuronal marker, PGP-9.5 and histochemical staining for nitric oxide synthase revealedno disruption of the myenteric plexus in the small intestine. In studies on 11 cells (membrane potential -61.5 _+ 1.6 mV, mean _+ SEMi from 3, six-week old F/F mice, the electrical slow wave recorded in the circular muscle of the small intestine appeared to be normal (frequency 0.7 _+ 0.02 Hz, amplitude 23 _+ 1.7 mV). These results are consistent with an ICC network in mutant F/F mice that is intact and that functions normally. Electrical field stimulation under non-adrenergic, non-cholinergic conditions showed normal inhibitory junction potentials (amplitude = 8.9 -+ 1.0 mV) therefore neurotransmission seemedunimpaired in F/F mice. Similar numbers of ICC survived in primary culture when dissociated from jejunal muscle of either Y/Y or F/F, 9-11 day old mice (n = 3). Conclusion: We conclude that c-kit-induced PI3'K signaling is not required for normal differentiation and survival of ICC in F/F mice. Since PI3'K is not directly inhibited by th{s mutation, activation of PI3'K by other pathways may still be necessaryfor normal ICCfunction. Supported by Mayo Foundation and NIH grants DK52766, DK57061 and DK17238.

742 Localization and Trafficking of Receptor Activity Modifying Protein 1 (RAMP1) and Calcitonin Receptor like Receptor (CRLR) Dirk Roosterman, Helen Wong, Claire Bihoreau, Nigel W. Bunnett, Eileen Grady, San Francisco, CA; Los Angeles, CA; Paris, CA RAMP1 chaperones the CRLR from the endoplasmic reticulum to the plasma membrane, where RAMP1 and CRLR form a receptor for calcitonin gene related peptide (CGRP). Nothing is known about the cellular localization of RAMP1 in the gastrointestinal tract. We investigated the association of RAMP1 and CRLR in cell lines and examined RAMP distribution in the gut. We expressed in KNRK cells CRLR and RAMP1 tagged with GFP Or myc epitopes. A RAMP1 antibody was generated by immunizing rabbits with the C-terminal fragment Z-QSKRTEGIV. CRLRwas localized using GEP or by incubating cells with fluorescent Alexa-CGRP.Expression of RAMP1 was assessed by immunofluorescence and Western blotting. In ceils expressing CRLR alone or RAMP1 alone, CRLR and RAMP1 were confined to intracellular locations, and there was no detectable binding of Alexa-CGRP.When co-expressed, both CRLR and RAMP1 were colocalized at the cell surface. Alexa-CGRPbound to plasma membrane and induced internalization of CRLR and RAMP1 into the same endosomes, confirming the expression of a functional receptor. The RAMP1 antibody stained transfected cells and detected a protein of 22 kD by Western blotting. Signals were not observed in non-transfected cells and were abolished by pre-incubation with the peptide used for immunization, confirming selectivity. We localized RAMP1 in the rat by immunofluorescence. Immunoreactive RAMP1 was detected in longitudinal and circular muscle layers of the small and large intestines, in enteric neurons throughout the intestine, and in enterocytes. RAMP1 was also detected in cells at the base of fundic glands. Endothelial cells of arterioles and vcnules were also stained, as was arteriolar smooth muscle. RAMP1 was prominently localized to the plasma membrane of most cells, where it may interact with receptors. Some cells expressing RAMP1 were also in close proximity to nerve fibers containing CGRP,suggesting RAMP1 may be colocalizedwith CRLR. However,the widespread distribution of RAMP1 in multiple cell types suggests it also interacts with other receptors. Thus, RAMP1 may serve as a chaperonefor G-protein coupled receptors, thereby ensuring appropriate localization of functional receptors at the plasma membrane of many cell types. Supported by DK39957, DK52388.

745 Control of Oxidative Stress-Mediated Protein Kinase D Activation and Nuclear Translocatioe by Activation Loop Phosphor~letion Richard T. Waldron, Osvaldo Rey, Enrique Rozengurt, Los Angeles, CA Background: Protein kinase D (PKD), a widely expressed protein serine kinase, undergoes phosphorylation in the activation loop Ser744 and Ser748 during PKD activation that occurs in intact cells via PKC in response to GI peptides including bombesin. PKC-mediated PKD activation also controls the transient localization of PKD to the plasma membrane and subsequently, PKD nuclear accumulation in response to bombesin GPCR activation. Recently, oxidative stress was shown to activate PKD via a mechanism requiring PKC and involving tyrosine phosphorylation, but neither activation loop phosphorylation nor the subsequent PKD dynamic behavior associated with oxidative stress-mediated PKD activation, have yet been examined. Results: oxidative stress initiated by addition of H202 (0.15-1 mM) to Swiss 3T3 fibroblasts that stably overexpress both PKD and green fluorescent protein (PKD.GFP cells) induces PKD activation loop Ser744 and Ser748 phosphorylation dose- and time- dependently, as measured by Western blot analysis with phospho-specific antibodies that recognize phosphorylated Ser744 and 5er748 in PKD. Cell treatments with the specific src inhibitor PP-2, or with the specific PKC inhibitor, GEl, prevented phosphorylation of these residues during oxidative stress, demonstrating the involvement of tyrosine kinaseactivity of src-family tyrosine kinases and the requirement for PKC in this process. H202-induced oxidative stress also induced transient PKD translocation to the plasma membrane and subsequent regulated nuclear import, as shown either by monitoring movements of a PKD-GFP fusion protein by real-time fluorescence imaging or by studies of PKD immunocytochemical localization using an anti-PKD antibody. Conclusions: These results suggest the possibility that altered cell regulation observed in response to oxidative stress in the GI tract may proceed by a mechanism involving PKC-mediated PKD activation and dispatch of PKD to critical cellular locations including the nucleus.

745 Somatotropin Inhibits Intestinal Chloride Secretion: Mechanisms and Role in Crohn's Disease Therapy Jimmy Y. C. Chow, Kim E. Barrett, San Diego, CA Background: Somatotropin (growth hormone) stimulates intestinal growth, differentiation and calcium absorption. Recently, it has been shown to alleviate symptoms, including diarrhea, in patients with Crohn's disease. Therefore, the aim of the present study was to examine whether somatotropin inhibits chloride secretion induced by carbachol (a calcium-dependent secretagogue), and if so, the downstream effectors responsible. Methods: Studies were performed using T~ cells grown on permeable supports, and pretreated with somatotropin at various concentrations followed by carbachol (100 I~M). Chloride secretion was assessed as changes in short circuit current (Alsc) in Ussing chambers. AG1478 (an epidermal growth factor receptor (EGFr) inhibitor) was used to define a possible involvement of EGEr. In addition, SB203580 (a p38 inhibitor) and PD98059 (a MEK1 inhibitor) were usedto examineinvolvement of either p38 or ERK MAP kinases. Results: Somatotropin caused rapid tyrosine phosphorylation of a 180 kD protein, subsequently identified by immunoprecipitation and Western blotting as EGFr. Somatotropin (10 nM) inhibited carbachol-induced chloride secretion (L~lsc 46.7 _+ 10.0 vs. 21.1 _+ 1.5 p.A/cm2, means _+ SEM, n = 3, p < 0.05). However,higher concentrations of somatotropin were less effective. AG1478 pretreatment reversed the inhibitory effect of somatotropin. Alsc induced by carbachol plus somatotropin was 12.4 _+ 4.7 and 36.9 _+ 9.3 p.Ncm 2, n = 5, p < 0.05 in the absence and presence of AG1478, respectively. Somatotropin also induced activation of both p38 and ERK1/2 MAP kinases as assessed by Western blotting. However, AG1478 only reversed somatotropin-induced tyrosine phosphorylation of ERK1/2, without a significant effect on p38. Furthermore, PD98059, but not $8203580, reversedthe inhibitory effect of somatotropin on chloride secretion. Conclusion: Somatotropin pretreatment results in the inhibition of carbachol-induced chloride secretion in Ts, cells, which is accounted for by transactivation of EGEr and consequent recruitment of ERK1/2 kinases. Although somatotropin also activates p38, this kinase does not contribute to the inhibitory effect of somatotropin on secretion. Overall, these data shed light on mechanisms that may underlie the efficacy of somatotropin for symptomatic relief in Crohn's disease.

746 Participation of Amino Acids in the 2ndIntracellular Loop of the Gastrin-Releasing PepUde Receptor (GRP-R) in ReceptorActivation and Internalization Michael Schumann, Samuel A. Mantey, Kenji Tokita, Richard V. Benya, Robert T. Jensen, Bethesda, MD BACKGROUND:The GRP-R, similar to many G protein-coupled receptors, with agonist activation, undergoes rapid internalization, in general little is known about the receptor structural determinants of activation or internalization of the GRP-R, especially in receptor regions proximal to the third intracellular loop (IC3). Studies in a few other G protein-coupled receptors provide evidence that amino acids in the central portion of the 2"d intracellular loop can be particularly important in both activation and internalization.AIM: To determine whether residues in the central portion of the 2"~ intracellular loop (IC2) of the GRP-R are involved in receptor activation or internalization. METHODS: Using site-directed mutagenesis 6 point mutations (Ala substituted for amino acids (n=6), Set for Ala (n=l)) and one double mutation were made in the mGRP-R IC2. Stable transfectants in BALD cells were made as well as transient transfectants for some confirmatory studies in the same cells. Receptor density and affinity were assessed using ~251-Tyr'bombesin(Bn), activation by measuring increases in 3H-inositol phosphate (IP), and internalization using acid stripping. RESULTS: Wild type GRP-R had an affinity for Bn of 1.0+/-O.2nM and none of the mutants demonstrated a change in affinity. Three mutants (R145A, P146A, D148A) showed no change in ability of Bn to stimulate maximal increases in 3H-IP. However, with 4 mutants (A142S, 1143A,V144A, M147A) and the double mutant, VM144,147AA, maximal 3H-IP stimulation was reduced 67 +/-7, 63 +/-1 O, 83 +/-4, 85+/-3 and 85+/-1%, respectively. The ECsofor 3H-IP by Bn was unchanged except in 1143A in which it was shifted 3 times to the right. With internalization all mutants but two showed no change. R145A showed a 31% increase in maximal internalization and M147A showed a 20% slower rate of internalization, but normal maximal. Transient transfection studies showed similar results. CONCLUSIONS:Four hydrophobic residues (A143, 1143,V144, M147) in the central portion of the 2"~intracellular loop of the GRP-Rare essential for receptor activation. The presence of the positively charged Arg in position 145 had an inhibitory effect and a methionine at position 147 a stimulatory effect on internatization.The fact that 4 mutants had a marked decrease in agonist activation of PLC without an effect on internalization shows full activation of PLC is not essential for maximal internalization.

744 A Targeted Point Mutation that Inhibits Signaling between c-Kit and 1Phosphatidylinositol 3'-Kinase Does Not Affect Interstitial Cell of Cajal Survival or Function in Mice Simon J. Gibbons, Adam Rich, Marne A. Distad, Lei Sha, Steven M. Miller, John Malysz, Joseph H. Szurszewski, Peter 81ume-Jensen,Gianrico Farrugia, Rochester, MN; Randolph, MA Background: Normal function of the receptor tyrosine kinase c-kit is necessaryfor the development of interstitial cells of Cajal (ICC) in mice. Mutations in the genes encoding both c-kit and its ligand, stem cell factor, result in loss of ICC from the mouse gut and impaired motility. Activation of c-kit stimulates several downstream signaling molecules, including the 1-phosphatidylinositol 3'-Idnase (PI3'K) pathway. The signals required for normal ICC development are not well understood. We studied a gene-targeted mouse with tyrosine 719 mutated to phenylalanine in c-kit (Y719F). This disrupts binding of PI3'K to activated c-kit. Aims: To determine the effect of the Y719F mutation on ICC development, we have examined the functional properties and morphology of ICC in mice homozygous for the mutation (F/F mice). Also we studied, in primary culture the survival of ICC from both wild-type (Y/Y) and mutant mice. Results: The distribution and number of ICC in stomach, small intestine and colon was normal in 6-week old F/F mice when assessed by labeling with an anti-c-kit antibody (ACK2, n = 3 mice), c-kit-like immunoreactivity was observed in both plexuses and in the muscular

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proliferation or cell death rates by BrdU labeling and TUNELassays respectively. Conclusions: KIf4-/- mice show perturbation in goblet cell differentiation in the colon. While rare goblet cells are seen in KIf4-/- mice, numerous epithelial cells which express goblet cell markers but not normal goblet cell morphology are found, suggesting that KIf4 plays a critical role in maintaining or establishing the differentiated goblet cell phenotype in the colon.

Rab5a and Rablla Mediate Membrane Trafficking and Resensitization of ProteaseActivated Receptor 2 (PAR2) Dirk Roosterman, Fabien Schmidlin, Eileen Grady, Njgel W. Bunnett, San Francisco, CA The molecular mechanisms of desensitization and endocytosis of G-protein coupled receptors have been intensively investigated. Little is known about mechanisms of receptor trafficking from intracellular stores to the plasma membrane, which is required for resensitization. We investigated recycling and resensitizationof PAR2, a receptor for trypsin and mast cell tryptase. PAR2 was expressed in KNRKcells with an extracellular Flagepitope, which would be removed by proteolysis and detect only intact receptors, and an intracellular HAll epitope, which would detect intact and cleaved receptors. The role of rab5a and rablla was examined by expressing wild-type and dominant negative mutants (rab5aS34N, rabllaS25N). PAR2 and rabs were localized by confocal microscopy. Flow cytometry with Flag antibody was used to quantify intact PAR2 at the cell surface. Resensitization of protease signaling was determined by measuring intracellular Ca. Trypsin (10 nM) removed intact PAR2 (detected with Flag at 10 rain) from the cell surface, and stimulated endocytosis of cleaved PAR2 (detected with HAll at 10-60 rain) into vesicles that contained rab5a, and are thus early endosomes. At later times, cleaved PAR2 was detected in lysosomes. Trypsin induced mobilization of intact PAR2 from Golgi stores into vesicles containing rablla, which probably traffic from the Golgi to the plasma membrane. Rab5aS34N but not rabllaS25N caused retention of cleaved PAR2 at the plasma membrane, suggesting a role of rab5a but not rablla in endocytosis. Rab5aS34N and rabllaS25N caused retention of intact PAR2 in vesicles, and inhibited recovery of intact PAR2 at the cell surface. Dominant negative beta-arrestin319-418, which inhibits PAR2 endocytosis, also impaired trafficking of intact PAR2 to the cell surface. Flow cytometry indicated that rab5aS34N and rabllaS25N inhibited recovery of intact receptors at the cell surface by 17% and 35%, respectively. Similarly, Rab5aS34N and rabllaS25N inhibited resensitization of Ca signaling by 73% and 36%, respectively: Thus, beta-arrestin and rab5a are required for endocytosis of PAR2. This endocytosis, by an unknown mechanism, stimulates mobilization of PAR2 from Golgi stores to the cell surface. Rablla mediates translocation of PAR2 from the Golgi apparatus to the plasma membrane. Mobilization of PAR2 is required for resensitizationof responsesto proteasesand may be necessaryfor the sustained proinflammatory and hyperalgesic signaling by PAR2. Supported by DK43207, DK39957.

750 npo Encodes a Novel RNA-BindingProtein Essential for Cytodifferentiation of Digestive Organs Nan N. Mayer, Mark C. Fishman, Charlestown, MA Understanding the molecular control of organogenesis is a prerequisite for devising novel stem cell-based treatments. To identify genes essential for this process we performed a genetic screen in the zebrafish. We isolated a mutation, called nil per us (npo), that arrests organogenesis of the intestine, liver and pancreas at a discrete step: after anlage formation, but prior to organ cytodifferentiation. By positional cloning we identif!ed the npo gone as encoding a novel RNA binding protein that is dynamically expressed in the embryonic digestive organs. Overexpressionof Npo leads to hyperproliferation of the intestine, liver and pancreatic epithelium, in adult intestine npo is expressedat the base of the villus. These findings reveal a developmental checkpoint prior to cytodifferentiation that cannot be traversed without npo activity. The effects of npo overexpression and the location of npu expression in adult gut together suggest a role for npo as a positive regulator of organ precursor cell populations within both embryonic and developmental contexts.

751 Apobec-1 Regulates Intestinal Epithelial Stem Cell Survival after Radiation Injury in Association with Posttranscriptional Effects on COX-2 mRNA Stability Shrikant Anant, Dcbnath Mukbopadhyay, Courtney Houchen, William Stenson, Nicholas O. Davidson, St. Louis, MO Apobec-1 is an AU-rich RNA binding protein previously characterized in relation to its role as the catalytic deaminase within the apolipoproteinB RNA editosome. The primary target of apobec-1 is apoB RNA, whose transcript encodes an intestinal protein, apnB48, absolutely required for lipid transport and secretion. We have identified a new function for this protein. Multipotent stem cells, located near the base of intestinal crypts, undergo dose-dependent radiation injury. Lipopolysaccharide (LPS) protects cells from radiation injury, by increasing production of prostaglandin E2 (PGE2) through induction of cyclooxygenase-2 (COX-2). However, the mechanism of COX-2 induction by LPS is currently unknown. One mechanism Of COX-2 regulation is through posttranscriptional mRNA stability. We recently demonstrated that apobec-1, expressedexclusively in the human intestine, binds to AU-rich elements (AREs) in the 3-prime UTR and stabilizes c-myc. Since COX-2 3-prime UTR contains numerous AREs and a canonical apobec-1 binding site, we examined whether apobec-1 affects mRNA stability. We generated apobec-1-/- mice, backcrossed >10 generations into C57BL/6. Histological analysis of the intestine of apobec-1-/- mice demonstrated increased apoptotic crypts (3-fold) Comparedto wild type littermates, which increasedto >5-fold upon 12By F-irradiation. Crypt survival assessed 3.5 d after 12 Gy r-irradiation demonstrated a 50% reduction in surviving crypts in apnbec-1-/- mice. Administration of LPS 14 b prior to radiation injury did not protect crypt cells in apobec-1-/- mice, while complete protection was observed in wild type littermates. Both PGE2 and COX-2 levels in wild type mice increased upon LPS administration but no changes occurred in apobec-1-/- mice, suggesting that COX-2 is perturbed in apobec-1-/mice. To determine whether this is due to changes in COX-2 mRNA stability, we conducted mRNA turnover experiments using actinomycin D in transfected epithelial cells. The half-life of COX-2 mRNA changed from 60 rain to 150 rain in apobec-1 expressing versus vectortransfected cells. Further studies directly confirmed that recombinant apobec-1 binds COX2 3-prime UTR. These data suggest that apobec-1 plays a role in the protection of intestinal stem cells from radiation injury, by binding to COX-2 mRNA and altering its stability and translation, with downstream effects on PGE2 expression

748 Genetic Evidence for the Essential Role of a Novel j~-Spectrin ELF in the TGF-I] Signal Transduction Pathway and Gut Epithelial Cell Polarity Yi Tang, Varalakshmi Katuri, Allan Dillner, Bibhuti Mishra, Chu-Xia Deng, Lopa Mishra, Washington, DC; Bethesda, MD; Philadelphia, PA Abnormalities in the TGF-I~signal transduction are implicated in many pathological processes from autoimmunity to cancer predisposition, in particular gastrointestinal tumors. The pathway is also involved in key aspects of development, epithelial cell formation and differentiation. A crucial step upon TGF-131receptor activation is the recruitment of SMAD proteins to the receptor at the cell membrane. What then follows is a series of downstream biochemical events that eventually culminates in the activation of gone expression. Spectrins are key proteins involved in the support of general membrane integrity, stabilization of cell- cell interactions, the generation of functionally distinct membrane protein domains, and in Golgi transport. We present genetic and biochemical evidence that ELF, novel 13-Spectrinisoform, has a critical role in TGF-13signaling. To define the specific role of ELF in this pathway, the ELF gene was knocked out by homologous recombination in embryonic stem cells. We show that a mouse that carries a disruption of a novel 13-SpectrinELF has a phenotype very similar to that of mice carrying disruptions of Smad2/3 genes, with prominent gut and hepatic defects. Specifically, aberrant gut epithelial cell expression of Na,K ATPase, Microtubule Associated Protein-2 (MAP-2), SMAD3 and SMAD4 occurs, indicative of loss of cell polarization. Moreover, ELF and SMAD3 show similar tissue distribution and co-purify with each other. At a cellular level, ELF deficiency results in a defective response to TGF-I~. The absent p3TP-Lux reporter activation by TGF-[5 in the ELF mutant cells can be rescued by reconstitution of SMAD3 expression. The molecular basis for this defect may thus reside in the inability of SMAD3 protein to be targeted to the activated TGF-13receptor I cytoplasmic domain. These findings reveal a novel function of a spectrin molecule in the targeting of a key signaling protein to an activated TFG-13 I receptor. Our observations also demonstrate a pivotal role for ELF in gut epithelial cell polarization and differentiation.

752 Bone Marrow Derivation of Intestinal Subepithelial Myofibroblasts in the Mouse and Human Small Intestine and Colon Mairi Brittan, Toby Hunt, Rosemary Jeffery, Richard Poulsoml Stuart Forbes, Kairhaan Hodivala-Dilke, Malcolm Alison, Nicholas A. Wright, London, UK

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Introduction The intestinal crypts and gastric glands are enclosedwithin a protective fenestrated sheath of intestinal sub-epithelial myofibroblasts (ISEMFs). ISEMFs exist as a syncytium within the lamina propria, tightly apposed to the intestinal epithelial cells and are believed to play a vital role in epithelial:mesenchymal interactions. It has been suggested that ISEMFs originate from the neural crest, or locally from mesenchymal stem cells. Here we show that they can be derived from bone marrow stem cells. Methods We studied colons and small intestines of: (i) female mice that had received whole body irradiation followed by a bone marrow transplant from male donors, and (ii) gastrointestinal biopsies from female patients that had d ev eloped graft-versus-host disease following male bone marrow transplants. In situ hybridisation for the Y-chromosome combined with immunohistochemistry for various antigens were used to identify the presence of Y-chromosome positive cells of donor origin and to determine the phenotype of these cells. Results We found Y-chromosome positive cells in the intestines of female mice and humans, predominantly in the pericryptal region. The ISEMF phenotypewas characterised in the mouse by positive immunoreactivity for alphasmooth muscle actin (SMA). These cells are also negativefor desmin and have lost the mouse macrophage marker F4/80 antigen and the haematopoietic progenitor marker CD34. A few bone marrow-derived ISEMFswere present in the mouse intestine 7 days after bone marrow transplant. These were far more numerous at t4 days, and by 6 weeks, whole columns of pericryptal myofibroblasts were seen surrounding intestinal crypts in the colon and small intestine. Human female patients who had received a bone marrow transplant from male donors showed variation in the number of Y chromosome-positive myofibroblasts identified

The Zinc-Finger Transcription Factor KIf4 is required for Proper Goblet Cell Differentiation in the Colon Jonathan P. Katz, Nathalie Perreault, Bree G. Goldstein, Catherine S. Lee, Vincent W. Yang, Klaus H. Kaestner, Philadelphia, PA; Atlanta, GA Background and Aims: KIf4 is a zinc-finger transcription factor expressed in the epithelia of the skin, lungs, gastrointestinal tract, and several other organs. In the colon KIf4 RNA localizes to the upper region of the crypt epithelium. KIf4 has been shown in vitro to have a role in cell proliferation and/or differentiation. Mice homozygous for a null muetation in KIf4 die within 15 hours after birth and show selective perturbation of late-stage differentiation structures in the epidermis. We hypothesizedthat KIf4 might be critical in colonic epithelial cell proliferation and/or differentiation. Methods: KIf4-/- mice were generated by homologous recombination in mouse embryonic stem cells. KIf4-/- and wild-type controls were sacrificed shortly after birth and the colons removed for histology or isolation of RNA. In situ hybridization, RT PCR, immunohistochemistry, and electron microscopy were performed. Goblet cells and total epithelial cells were counted in a blinded fashion. Results: While all epithelial cell types are seen in the colon of KIf4-/- mice, KIf4-/- mice display a 90% decrease in the number of goblet cells, Levels of Muc2 and DRA mRNA are not significantly altered by RT PCR, but we demonstrate that Muc2 is not specific to goblet cells in KIf4-/- mice. Numerous epithelial cells are seen in KIf4-/- mice which express Muc2 and may be Alcian blue positive but lack the normal goblet cell morphology by electron microscopy. There are no changes in cell

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by SMA, and there was no obvious correlation betweenthe degreeof bone marrow engraftment and the time elapsed between transplant and intestinal biopsy. Discussion After bone marrow transplantation ISEMF regeneration has a major contribution from bone marrow cells, which thereby contribute to the turnover of the pericryptal myofibroblast sheath. We believe that this role of bone marrow in the axis of gut regeneration is biologically important and has therapeutic potential for gene delivery to the gut.

Does Nitric Oxide Inhibit Angiogenesis in Rat Gastric Microvascular Endothelial Cells? PKC and ERK2 as Targets Michael K. Jones, Kouji Tsugawa, I James Sarfeh, Andrzej S. Tamawski, Irvine, CA BACKGROUND/AIMS: Excessive production of nitric oxide (NO) by the endothelia of gastric mucosa{ and submucosal vessets is implicated as a causal factor in portal hypertensive (PHT) gastropathy and the increased susceptibility to injury. PHT gastric mucosa also displays an 8-fold reduction in angiogenic response to injury resulting in impaired injury healing (Gastroenterology 1994; 106:702). The role of NO in the regulation of angiogenesis is controversial since NO has been shown to have both pro- (J. Clin. Invest. 1998; 101.2567) and anti- (Br. J. PharmacoL 1994; 111:894-902)angiogenic effects. NO also inhibits PKC -induced VEGF gene expression by interfering with the AP-1 (c-Jun/c-Fos heterodimer) transcription activator (Nature Medicine 1997;3:879). The aims of the present study were to determine whether NO inhibits gastric angiogenesis and whether this is mediated through inhibition of PKC and/or ERK activation. METHODS: Rat gastric microvascular endothelial (RGMEC) ceils were cultured in the presence of vehicle (control) or the nitric oxide donor, S-nitro-N-acetylpenicillamine (SNAP). STUDIES: 1) in vitro angiogenesis- formation of capillary-like structures on matrigel - assessedby videoimageanalysis, 2) expressionof PKCand ERK2,and phosphorylation of ERK2 and c-jun, 3) PKC activity. RESULTS: SNAP, in the concentration range of O.5-4mM, dose-dedendentlyinhibited in vitroangiogenesis by up to 80% compared to controls (P
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FGFR-3 Regulates Epithelial Stem Ceil Numbers and Crypt Formation during Murine intectinal Development. Jenny M. Buzan, Alda Vidrich, Kirstin Skaar, Leigh Bradley, Chibuzo lie, Steven Cohn, Charlottesville, VA The rates of epithelial stem cell proliferation and differentiation of stem cell descendents are thought to regulate the number of crypts formed during intestinal development and the size of the replicating transit cell population within each crypt. We recently found that FGFR3 is expressed at high levels by replicating epithelial cells located in the lower intestinal crypts. The goal of this study was to determine the role of FGFR3-mediatedsignaling in regulating the dynamics of epithelial stem cell proliferation or differentiation during intestinal development. METHODS: The effect of FGFR3on crypt number was determined by histochemistry in mice that were homozygous for a disrupted FGFR3gene (FGFR3-/-)and wt/wt littermates. Epithelial cell proliferation was determined by BrdUrd-labeling. The microcolony assay was used to assess the number of clonogenic stem cells in FGFR3-/- and wt/wt mice. RESULTS: The growth of FGFR3-/- mice was reduced compared with wt/wt beginning at day 7. By day 14 the weight of FGFR3-/- mice was only about 53% of wlJwt (5.85-+0.25 g vs 11.0_+0.4 g). All differentiated cell types were representedin suckling FGFR3-/-mice. The number of BrdUrdlabeled cells per crypt was significantly reduced by day 21 in FGFR3-/- mice (4.8_+0.9 vs 10.6 _+1.7, p
756 NF-Ke-lnducing Kinase (NIK) Activates NF-KB Transcriptional Activity and Proinflammatory Gene Expression through an I KB Kinaso (IKK)-Independent Mechanism. Maria Russo, Brigitte Allard, Christian Jobin, Chapel Hill, NC Introduction: IL-ll~ and TNF activates the NF-KB pathway by inducing the IKB kinase (IKK) complex which is composed of the regulatory IKK~ and the catalytic IKKoEIKKI~ subunits. The IKK complex is activated by the upstream NIK kinase. Activated IKK phosphorylates IKB~ and triggers its proteolytic degradation, release of NF-KB which translocates to the nucleus and induces gene transcription. Aims: To characterizethe role of NIK in NF-KB activation and gene expression in intestinal epithelial cells (IEC). Methods: HT-29 and mouse embryonic fibroblast (MEF) isolated from wild type (wt) and IKKg gene deficient animals were used to assess the contribution of NIK in NF-KB activation. Adenovirat vectors (Ad5) encoding wt and dominant negative (dn) NIK (Ad5wtNIK and Ad5dnNIK), Ad5 myristylate Akt (Ad5myrAkt) and Ad5gB-Luciferase (Ad5KBLUC) were used to infect HT-29 cells and MEF. The Ad5GFP encoding for the green fluorescence protein was used as a control. IKB,x steady-state levels and its phosphorylated form were analyzed by Western blot. NF-KB transcriptional activity was determined by a luficerase assay. TNF gene expression was analyzed by RT-PCR using specific primers. NF-KB DNA binding activity was evaluated by electrophoretic mobility shift assay. IL-8 secretion was quantified from cell supernatant using an ELISA technique. Results: Ad5wtNIK strongly induced IKB
754 Restoration of Cytokine- or Bacterial-Induced Defects in Tight Junction Permeability by a Membrane Permeant Oligopeptide Jerrold R. Turner, Yevgeny Zolotarevsky, Gall Hecht, Athanasia Koutsouris, Randall J. Mrsny, Chicago, IL; Detroit, MI; South San Francisco, CA Epithelial tight junction (TJ) permeability is disrupted in several intestinal diseases. Models of this dysregulation include exposure of epithelial monolayers to pro-inflammatory Thl cytokines or infection by enteropathogenic E. coil (EPEC). The effects of EPEC infection on transepithelial electrical resistance (TER) require myosin II regulatory light chain (MLG) phosphorylation and contraction of perijunctional actomyosin, but the mechanisms by which cytokines alter TER are unknown. We have described a membrane Permeant Inhibitor of MLC Kinase (PIK), derived from the autoinhibitory region of MLC kinase, that specifically inhibits intestinal epithelial MLC kinase. PIK has sequence homology to the protein transduction domain of HIV-1 TAT, which crosses cell membranes freely. PIK crosses cell membranes, colocalizes with MLC kinase and ZO-1, and increases TER in epithelial monolayers. Thus, these studies were performed to determine if PIK could rectify TER decreases after EPEC infection or cytokine exposure. METHODS: Polarized monolayers of Caco-2 or T84 cells on Transwell supports were used. Intracellular MLC phosphorylation was determined in lysates of 32P-loadedmonolayers. PIK was added 1 hour after apical EPEC infection and TER was measured 3 hours later. TNF~ and IFN,ywere added to the basal chamber for 72 hours prior to apical PIK addition and TER was measured 1 hour later. RESULTS: Consistent with an ability to both cross cell membranes and inhibit MLC kinase, apical addition of PIK caused a 22%-+ 2% decrease in intracellular M LC phosphorylation in Caco-2 monolayers (p
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757 A Novel Nab-like GTPase Regulates Late EndosomeDevelopment. Xiubin Gu, Heidi M. Golden, Takanori Sakaguchi, Jim E. Casanova,Yen Feng, HansChristian Reinecker, Boston, MA; Charlottesville, VA Background: Degradativeand recycling endosomal processing is critical for the regulation of cytokine receptor signaling and antigen presentation. We have cloned and functionally expresseda novel small Rab-like GTPase(RIg) from human intestinal epithelial cells. Sequence homology of RIg with Rab proteins is limited to the GTPasedomain and a CAAX box. RIg belongs to a novel family of GTPases,which has not been functionally characterized. Methods: Tissue and cell line specific expression of RIg was determined by Northern and Western blotting. Flag-, myc-, and GFP-tagged dominant-negative and constitutive active GTPase mutants were generated by site directed mutagenesis. GTP binding to RIg was assessed with overlay assays. RIg induced vesicles were analyzed with lysotraker red and for the presence of lysosome associated membrane protein (Lamp) -1, Lamp-2, insulin-like growth factor II/ mannose 6-phosphate receptor (IGF-II/M6P) and for the late endosomal and lysosomal GTPase Rab-7 by immunostaining and confocal microscopy. GFP tagged wildtype, Rab-7, Rab7T22N, and Rab7067L were used in co-localization studies. Results: RIg was highly expressed in

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hematopoetic, neuronal and gastrointestinal organs with the exception of liver and kidney. Expression of Rig induced the formation of large intracellular vesicles in Cos-7, Hela, and Caco-2 cells. The formation of these vesicles was dependent on the GTPaseactivity of Rig, since constitutive active mutants induced larger, rapidly fusing structures, whereas dominantnegative mutants induced separatedand smaller vesicles. Rig induced vesicles were characterized by a low pH and the expression of Lamp-l, Lamp-2, and Rab7, but did not contain IGFII/M6P. GDP bound Rab7T22Nwas recruited from the cytosol into Rig positive late endosomes. In contrast, lysosnmes induced by wildtype Rab7 and constuitive active mutant Rab7Q76L, only partially overlapped with Rig induced vesicles. Conclusions: Rig is a novel small 6TPase in intestinal epithelial cells, which may regulate late endosomes to lysosomes transition by mechanisms, which partially overlap with those of Rab7 but diverge during the maturation of lysosomes. Through the regulation of the endocytic degradative compartment Rig may be able to regulate the processing of receptors and foreign materials or autophagocytosed intracellular eftector molecules.

760 The Tumor SuppressorSmad4 Induces Differentiation of Human Colon Cancer Cells through Regulation of Cadherin/Catenin Dependent Cell-Cell-Adhesion Anke Reinacher-Schick, Nicole Mueller, Anke Baar, Stephan Baldus, lrmgard SchwarteWaldhoff, Wolff Schmiegel, Bochum, Germany; Cologne, Germany Functional inactivation of the tumor suppressor Smad4 occurs in 1/3 of metastatic colnrectal cancers. The mechanisms underlying its tumor suppressor activity are not yet understood. Smad4 is a key transmitter of TGF-betasignals but there is evidencefor TGF-hetaindependent functions of Smad4. We have reported that cadherins may be a target of Smad4 in colon cancer cells in vitro. AIM: To investigate mechanisms of Smad4 tumor suppressor function in colon cancer cells in vitro and in vivo. METHODS:Stable reexpression of Smad4 in SW480 human colon cancer cells. Study of phenotyp by light and confocal microscopy after staining for cell adhesion molecules (cadherins, catenins) and differentiation markers (ZO-1, villin). Analysis of gene expression by Northern and Western blotting. Functional analysis of adhesion molecules in aggregation assays. Biochemicalassay for activity of the brush border enzyme Alkaline Phosphatase(ALP). Immunohistochemical examination of subcutaneous nude mouse tumors. RESULTS: Stable reconstitution of Smad4 in SW480 ceils caused a robust change in cell morphology to an epitheloid phenotypein vitro. Smad4 positive cells showed transcriptional upregulation of functionally active cadherins and formed tight cell-cell contacts. Beta- and alpba-catenin were recruited to the lateral plasma membrane in Smad4 positive clones as shown by immunofluorescence and biochemical fractionation further indicating functional activity of induced cadherins. Smad4 also caused recruitment of ZO-1 to the apicolateral membrane, upregulated villin and increased the activity of ALP all indicating an enterocytelike differentiation by Smad4. Importantly, Smad4 induced differentiation was strongly retained in vivo in nude mice. Smad4 negative SW480 cells formed undifferentiated solid tumors while Smad4 positive tumors were transient, displayed a growth pattern reminiscent of moderately differentiated adenocarcinomas with crypt-like structures strongly staining for E-cadherin. CONCLUSION: Here we present a novel mechanismof Smad4 tumor suppression in human colon carcinoma cells which involves induction of differentiation in vitro and in vivo. Cellular differentiation may be caused by transcriptional upregulation of cadherin adherens junction molecules associated with the establishment of mature cell-cell adhesion.

758 Regulated Expression of Noxl (NADPH Oxidasel) in Models of Altered Intestinal Cell Growth: Keratinocyte Growth Factor Increases Nox 1 Messenger RNA and Superoxide Production in Human Intestinal Epithelial Cells. L H Gu, Kathy K. Griendling, Sergey I. Dikalov, Bernard P. Lassegue, Dean P. Jones, J D. Lambeth, Thomas R. Zingier, Atlanta, GA Background: Reactiveoxygen species are important regulators of cell proliferation and redoxsensitive growth factor signaling. Noxl (NADPH oxidasel) is a recently cloned non-phagocyte NADPH oxidase that generates intracellular superoxide and H202 in vitro. Although noxl is highly expressed in colon, regulation of noxl expression and function in intestinal cells are unknown. Our aims were 1) to study gut noxl mRNA expression in models of altered intestinal cell growth; and 2) to test whether the gut-trophic peptide keratinocyte growth factor (KGF) regulates noxl mRNA and supernxide production in human gut epithelial cells. Methods: Rat cDNA-specific primers were used to detect a 703 bp noxl mRNA in rat tissue. Adult rats underwent 80% small bowel resection (SBR) or transection (control). Crypt depth and noxl mRNA expression (by RT-PCR) was determined in residual ileum after 7 days. Caco-2 cells were grown in MEM and noxl mRNA expression (2.4 kb) was determined at days 4, 7, 10 and 13 after plating by Northern blotting using a human noxl cDNA. In additional studies, Caco-2 cells were treated with KGF (10 ng/ml) for 24 h. The cysteine/cystine ratio in culture medium was varied to change extraceliular redox conditions from reducing to oxidizing during control and KGF treatments. Caco-2 cells cultured at physiologic redox were also treated with KGF (10 ng/ml) for 30 min or 24 h and cellular superoxide radical formation measured by electron spin resonance spectroscopy. Results: ileal noxl mRNA increased 2- to 3-fold in SBR versus transected control rats, in association with a 30% increase in crypt depth. In Cacn-2 cells, noxl mRNA levels markedly decreased between the proliferative phase (day 4) and the differentiated phase (day 13). KGF increased Caco-2 ceil noxl mRNA expression 4to 5-fold under oxidizing, physiologic and reducing extracellular redox conditions. KGE also increased superoxide formation in Caco-2 cells by 2.5-fold at both 30 rain and 24 h after treatment. Altered extracellular redox state alone did not regulate Caco-2 noxl mRNA expression. Conclusions: Oownregulationof noxl mRNA in contact-inhibited Caco-2 ceils and upregulation during adaptive ileal growth in vivo suggests a potential role for noxl in intestinal growth responses. Stimulation of noxl expression and superoxide formation by KGF may represent mechanisms of action for this growth factor in intestinal epithelial cells.

761 The HomeoboxTranscription Factor COX2 Interacts with the E3-Ubiquitin-Ligase HectH7 Jan Zinke, Sebastian Thaler, Joachim Mbssner, Eric Fearon, Karel Caca, Leipzig, Germany; Ann Arbor, MI CDX2 is a transcription factor, deriving its name from the related Drosophila homeotic gene caudal (Cad). CDX2 has a critical role in mammalian embryogenesis. In adult tissue, CDX2 expression is restricted to intestinal epithelial cells, where it is involved in enterocyte proliferation and differention, as well as regulating expression of intestinal-specific genes, including sucrase-isomaltase. Germline inactivation of one CDX2 ariel strongly predisposes mice to the development of hamartomatous polyps in the small intestine and colon. To address the role of CDX2 in intestinal polyp and potentially tumor formation by identifying novel COX2-binding proteins. In a yeast-two-hybrid screen with the CDX2 protein, among other interactions, we found 4 WW domains of a Hect domain protein binding to CDX2. Cloning of the open reading frame of this protein by RACEtechnique identified it as HectH7. HectH7 is a member of the Hect family of E3 Ubiquitin ligases involved in the proteasome mediated protein degradation pathway. However, no target proteins of HectH7 have been described so far. The genomic structure of HectH7 (24 exons) and chromosomal localization (8q21.3) were identified by screening a PAC library, PAC sequencing and FISH analysis. CDX2 binding to HeetH7 was confirmed through additional in vitro and in vivo studies~including combined immunoprecipitation and Western blot studies of colorectal cancer cell lines. Using GST pull-down assays with different deletion constructs HectH7 WW domain 1 and 2 could be identified as the preferential binding sites of CDX2. The PY motif of the CDX2 protein was identified as interacting region with HectH7. Northern blot analysis showed HectH7 expression in most tissues except lung and kidney, and in 11 of 12 colorectal cancer cell lines examined. In conclusion, our results indicate that CDX2 complexes with HectH7 in cells, and CDX2 may be the target for HectH7 mediated proteasomal degradation. Further studies are needed to address the role of HectH7 in CDX2 mediated proliferation.

759 Expression of Kinase-lnactive c-SRC Delays the Disruption and Accelerates the Assembly of Tight Junctions in Caco-2 Cell Monolayer R K Ran, R M. Ray, S Basuroy, Memphis, TN Src kinases are localized at the tight junctions (TJ) and adherensjunctions (AJ). We previously demonstrated that oxidative stress-induced increase in permeability is a tyrosine kinasedependent process. We also showed that oxidative stress activates c-Src and FAK. In the present study we determined the role of c-Src in disruption and assembly of TJ. Methods: Caco-2 cells were transfected with wild type c-Src (Src-wt) or kinase-inactive c-Src mutant (Sre-ki) or the vector. Clones were characterizedfor disruption of TJ by oxidative stress, and assembly of TJ in Ca~+-switch.Cellswere grown as monolayers on Transwell inserts. Oxidative stress was induced by administration of xanthine oxidase and xanthine (XO+X), which generated up to 17 ~M H202.In Ca2+-switch TJ was disrupted by EGTA, and assembly was induced by replacement of Ca~+. Paracellular permeability was analyzed by measuring TER, dilution potential and unidirectional flux of FITC-inulin. Disruption of TJ was examined by redistribution of occludin and 70-1 by confocal microscopy. Activation Ofc-Src was determined by immunecomplex Src-kinase assay and immunoblot analysis of Y418 phosphorylated cSrc using a specific antibody. Results. Treatment with XO+X induced a decrease in TER in vector-transfected cell monolayers. The rate of decrease in TER was significantly greater in Src-wt-transfected cells, while it was delayed in Src-ki-transfected cells. At 3 h of XO+X treatment, the TER values were reduced by 61 +/-4%, 85+/-5% and 13+/-2% in cells transfected with vector, Src-wt and Src-ki, respectively: XO+ X-induced decrease in dilution potential and increase in inulin flux were greater in Src-wt cells and lesser in Src-ki cells. XO + X induced a redistribution of occludin and ZO-1 from the intercellular junctions in vector and Src-wt cells, while distribution of these proteins in Src-ki cells was not altered. XO+X treatment resulted in activation of c-Src in vector and Src-wt cells, while Src activation was minimal in Src-ki cells. On the other hand,the rate of recovery of TER in Ca2+-switch experiment was significantly greater in Src-ki cells compared to vector and Src-wt cells. Relocation of occludin and ZO-1 at the intercellular junction was also much faster in Src-ki cells. Conclusion: These results indicate that expression of kinase-inactive c-Src delays oxidative stress-induced disruption of TJ, while it accelerates the assembly of TJ after Ca2+-switch. Supported by DK55532 and AA12307.

762 Over-Expression of Gut-Enriched KroppeI-Like Factor (GKLF or KLF4) in the Human Colon CancerCell Line RKO Leads to Reduced Tumorigenicity Duyen T. Dang, Xinming Chen, Long H. Dang, Vincent W. Yang, Baltimore, MD; Atlanta, GA BACKGROUND:KLF4 is a zinc finger-containing transcription factor, the expression of which is enriched in the post-mitotic ceils of the gut epithelium. Forced expression of KLF4 inhibits cell proliferation by blocking G1/S progression of the cell cycle. Recent studies indicate that expression of KLF4 is decreased in tumors of the intestinal tract, suggesting that KLF4 may have a tumor suppressor activity. AIM: To examine whether over-expression of KLF4 in RKO colon cancer cells, which do not express KLF4 (Oncogene20: 4884, 2001), will alter their in vitro and in vivo tumorigenicity. METHODS: We examined a recently established inducible system in RKO cells, in which expression of KLF4 is controlled by an inducible promoter that responds to the insect hormone ecdysone (JBC 276: 30423, 2001). RKO cells transfected with an empty vector were used as control. Four independent assays were used to assess the effect of KLF4 induct!on on tumor cells: cell proliferation, apoptosis, cell migration and in vivo tumorigenicity in nude mice. RESULTS: MTS assays were used to evaluate cell proliferation and revealed that induction of KLF4 prevented proliferation of RKO cells when compared to control. There was no evidence of significant fragmentation in either induced or control cells as determined by DNA ladder assays. Cell migration assays revealed only 3 +/- 1% of induced cells have migrated across a Transwell filter, to the bottom chamber after 6 hours when compared with 27 +/- 4 % of control cells (p < 0.01). In vivo tumorigenicity

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BAY 11-7082 and BAY 11-7085 (abbr.-7082, 7085), were tested on DLD1, HCT116,and HT29 CCa cell lines. Apoptosis was measured using flow cytometry to determine the percentage of annexin V-positive/propidium iodide-negative cells. NF-KBactivation levels were determined by electrophoretic mobility shift assays (EMSA). Tumorigenicity was measured by soft agar colony formation. Tumor seeding was simulated in athymic female mice by injecting 1 million CCa cells intraperitoneally (IP) 24 hours after pretreatment with 7085 (5mg/kg) or DMSO IP, and followed by 7085 or DMSO BIW for 21 days. RESULTS: 7082 and 7085 inhibited soft agar colony formation of DLD1 and HT29, but not HCT-116 cells. When adherent DLD, HCT116, and HT29 cells were treated with lO-20pM 7082 or 7085 <2% apoptosis occurred, but >78% apoptosis of DLD1 and HT29, but not HCT116 celts, occurred when they were transiently suspended (<15 minutes). All three cell lines had low constitutive levels of NFKB activation when adherent. However, readhesion of transiently suspended OLD1, HCT116, and HT29 cells caused a large activation of NF-KB for up to 6 hours, which was almost completely inhibited by 10-20~M 7082 or 7685. FLIP protein was expressed by DLD1 and HT29, but not HCT116cells. FLIP, TRAF1 and TRAF2, but not clAP1, protein levels decreased after 201~M7085 treatment of HT29 cells for 8 hrs. Based on this data, we hypothesizedthat NE-KB inhibitors could prevent colon cancer seeding in vivo. Of 6 athymic mice injected with HT29 cells IP and treated with 7685,0/6 and 2/6 mice had liver and peritoneal implants, respectively, whereas, 6/6 and 6/6 mice treated with DMSO formed liver and pedtoneal implants, respectively.Mice injected with HCT116cells IP formed similar numbers of peritoneal and liver implants regardless of treatment. A mouse pathologist blinded to treatment found no evidence of liver, lung, or kidney toxicity from 7085 after 32 days. CONCLUSIONS:CCa cell survival during readhesion following transient suspension is dependent on NF-KB and may require the presence of FLIP. This observation was extended in vivo by showing that a soluble NF-KB inhibitor prevented peritoneal implantation of CCa cells. NF-KB inhibitors may represent the first drugs to inhibit cancer cell adhesion and could thus prevent metastasis.

assays in nude mice revealed mean tumor volumes derived from implanted cells were 4 +F 1% of those found in control cells after 1 week of induction (p < 0.01) and 14 +/- 5 % of the mean volumes of control cells after 2 weeks of induction (p < 0.05). CONCLUSIONS: Over-expression of KLF4 in RKO colon cancer cells leads to a reduction of proliferation, migration and in vivo tumorigenicity of the tumor cells. Also, consistent with previous results, the mechanism of action of KLF4 does not involve apoptosis. These findings suggest that KLF4 is a cell cycle checkpoint protein that can reduce tumorigenicity.

763 Expression and Regulation of the Cyclooxyguuase2 (COX-2) Gone in Gastroenteropancreatic Neuroendocrine Tumor (GEP NET) Cells Stefan Juettner, Feng Gao, Thomas F. Meyer, Michael Naumann, Bertram Wiedenmann, Michael Hoecker, Berlin, Germany Background/Aims: COX-2 is the inducible key enzyme of arachidonic acid metabolism. Enhanced expression of COX-2appears to be crucially involved in the pathogenesis of inflammatory and neoplastic lesions. However,the pathophysiological role of COX-2 in gastroenteropancreatic NETs has not been determined yet. Therefore, we investigated COX-2 expression as well as the promotor elements and transcription factors regulating the COX-2 gene in neuroendocdne tumor cells. Methods: COX-2 expression was analyzed in 49 GEP NETs immunohistochemically as well as in QGP-1, SON and LCC-18 NET cell lines by western blot and RT-PCRassays. The promoter region responsible for COX-2 expression was characterized by 5' deletion analysis, element transfer experiments using a hctemlogous promoter system and site-directed mutagenesis employing COX-2-1uciferasereporter constructs. Nuclear proteins regulating the COX-2 gene in NET cell lines were identified employing standard EMSA ("Electrophoretic-Mobility-Shift-Assays") techniques. The effect of COX-2 inhibitors on NET cells was assessed in proliferation assays. Results: 43 of 49 GEP NETs (87,8 %) stained positive for COX-2. Of the three NET cell lines examined only OGP-1 and LCC-18 but not BON cells constitutively expressed the COX-2 gene. The COX-2-specific inhibitor NS-398 as well as Aspirin potently inhibited proliferation of QGP-1 cells dose-dependently. 5'-deletion analysis of COX-2 5'flanking DNA revealed that a proximal CRE-Eboxelement at -68 to -51 bp is essential for basal transcription of the COX-2 gene in neuroendocrine tumor celts. A single copy of the -68 to -38 bp elementconferred transcriptional upregulation to a heterologous promoter system. Finally, EMSA supershift analysis identified USF-1 and -2 as crucial nuclear proteins controlling COX-2 expression in NET cells. Conclusions: Our study for the first time demonstrates that GEP NETs overexpress the COX-2 gene, and indicates that COX-2 related prostanoids are involved in NET pathobiology. Moreover, we identified binding of USF-1/-2 transcription factors to the upstream CRE-Eboxat -57 bp to -48 as central mechanism for COX-2 gene expression in GEP NET cells. Further analysis of the pathogenic role of COX-2 in neuroendocrine tumors can offer novel therapeutic and/or diagnostic approaches.

766 Interergan Cress-Talk in GastroprotecUouInduced by Ischemic Preconditioning Against Ischemia/Repedusiou Injury. Role of Prostaglandins, Adenosine and Sensory Nerves Thomas M. Brzozowski, Peter C. Konturek, Zbigniew Sliwowski, Robert Pajdo, Stanislaw J. Konturek, Agata Ptak, Michal Pawlik, Eckhardt Hahn, Cracow, Poland; Erlangen, Germany Short repeatedgastric ischemic episodes protect gastric mucosa against the damage caused by subsequent prolonged ischemia/reperfusion (I/R) and this is called gastric ischemic preconditioning (IP). We studied whether brief ischemia of extragastrointestinal organs such as heart, liver or kidney could protect the gastric mucosa against damage induced by the prolonged I/R. The IP was induced in rats with or without sensory denervation by capsaicin (125 mg/kg s.c.), by short episodes of gastric ischemia (occlusion of celiac artery 1-5 times, 5 min each) applied 30 rain before prolonged (30 min) ischemia followed by 3h of reperfusion and compared with that achieved with short ischemia induced by clamping of left coronary artery, hepatic artery and left kidney artery followed 30 rain later by 3h of gastric I/R. The area of gastric lesions was determined by planimetry, gastric blood flow (GBF) measured by H2-gas clearance method and mucosal expression of COX-1 and COX-2 mRNA was analyzed by RT-PCR. I/R produced numerous gastric lesions and significant fall in the GBF and PGE2 generation. Short ischemic episodes (2x5 rain) of the heart and liver by themselves failed to cause gastric lesions but significantly attenuated those produced by gastric I/R similarly to that induced by gastric IP, whereas the kidney IP was not effective. These protective effects of cardiac, hepatic and gastric IP against gastric I/R were accompanied by an increase in the GBF, PGE2 generation and in the mucosal content of CGRP and abolished by capsaicin denervation but restored by administration of exogenous CGRP (10 ~.g/kg s.c.) to sensory denervated animals. Inhibitors of COX-1 and COX-2 such as indomethacin and Vioxx and 8phenylteophylline (8-SPT), an antagonist of adenosine A2 receptors, reversed the protective and hyperemic effects of cardiac, hepatic and gastric IP, and this was fully restored by addition of 16,16 dm PGE~and adenosine to COX-inhibitors and 8-SPT. Expression of COX-1 but not COX-2 mRNA was detected in intact mucosa and in that exposed to I/R with or without IP, whereas COX-2 was overexpressed only in the mucosa subjected to IP. We conclude that: 1) brief ischemia in remote organs such as heart and liver but not in the kidney is capable of protecting the stomach from injury as effectively as gastric IP and 2) PG derived from COX1 and COX-2, adenosine and sensory nerves appear to play a key role in the gastroprotective mechanism triggered by IP of remote organs.

764 intestinal Trefoil Factor (ITF) Expression by Rat Colon Cancer Cells Correlates with Aggressive Phenotype Xianyang Yio, Mark Babyatsky, Michael Linden, Jie-Yu Zhang, Jing Lin, Anli (;hen, Oiuxiang Fan, Natalie Dutheil, Katsunod Shinozake, Lawrence Werther, Steven Itzkowitz, New York, NY Background: We previously reported that ITF expression was associated with poor prognosis in gastric cancer patients. ITF is expressed by most colorectal carcinoma (CRC) tissues and metastases, but its role in CRC biology has not been established. Two subclones of a rat CRC cell line LMCR544 were selected previously for mucin associated STn antigen expression. The STn-negativesubclone (LMCR-) demonstrates a very aggressive clinical course in immunocompetent host BDIX rats, whereas the STn+ subclone (LMCR+) has very low tumor forming capability and manifests a much more indolent course. Aim: To compare ITF expression status in LMCR+ and LMCR- cells and correlate this with in vivo behavior of tumor cells. Methods: RT-PCRwas used to study native rat ITF expression. A construct consisting of 930 bp upstream from the transcriptional start site of the human ITF gene was used to direct expression of either a luciferase reporter or a TKGFP (fusion protein of HSV-thymidine kinase and green fluorescence protein) reporter. These were introduced into the cell lines to examine their transcriptional machinery for ITF.A recombinant adeno-associatedviral vector containing GFP(rAAV-GFP) was utilized to study the transducibility of these lines as models for gene therapy experiments. Results: Aggressive LMCR- cells expressed endogenous ITF and supported the transcription of hlTF in luciferace and GFP assays (Table). In contrast, indolent LMCR(+) cells did not express ITF or support IIF transcription. Both cell lines were transducible by rAAV-GFP. Conclusion: ITF expression correlates with aggressive behavior of rat colon cancer cells. These subclones are transducible with rAAM making them a useful model for studying tissue-specific gene therapy. (Supported by NIR RO1-CAB1363).

STn ag expression Mediansurvival(days) Native ITF expression hlTF930-iociferase hlTF93O-TKGFP rAAV-GFP

LMCR544 -,-inot tested .,../not tesled .qnot tested

LMCR+ +++ 42

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767 Caoo-2 Intestinal Epithelial Cell Motility May Be Regulated by an IntegrinDependent Signal Cascade Involving FAK, Src, PI 3-kinase, and ERK Matthew A. Sanders, Marc D. Basson, Detroit, MI Cell-matrix interactions initiate FAK and ERK signals that regulate gastrointestinal epithelial cell lamellipodial extension and motility, but the manner by which this occurs is unclear. We previously observed that collagen IV adhesion activates FAK, Src, ERK and PI 3-kinase. We now sought to order the signal cascade by which these events occur and elucidate their relationship to lamellipodial extension. All studies were performed at least three times with similar results. Both ~1(~1 and ~2l~1 integdns mediate Caco-2 adhesion to collagen IV. Treatment with a functional c~1 integdn antibody after adhesion partially blocked Caco-2 spreading across collagen IV. Blockade with an ~2 functional antibody alone had minimal effect, but combined ~1 and ~2 blockade essentiatty abtated Caco-2 cell spreading. Maximal antibody blockade of either ~1131 or ~21~1 integdn prior to adhesion only partially inhibited FAK and ERK activation, suggesting that collagen IV interactions can activate FAK and ERK via either integrin. No signal was seen with combined blockade since adhesion was completely inhibited. We further hypothesized that Caco-2 ERK activation requires a signal cascade which includes FAK, Src and PI 3-kinase. Src inhibition by PP2 did not affect FAK 397 autophosphorylation. However,transfection with mutant FAK lacking the 925 Src phosphorylation site attenuated ERK activation, suggesting that FAK-Src interaction is critical for complete ERK activation. Dominant negative Src transtection strongly inhibited cottagen IV-dependent Akt phosphorylation, a marker of PI 3-kinase activity, as well as ERK phosphorylation, which correlates with ERK activation. Dominant negative PI 3-kinase transfection also strongly

LMCR21 + + + -H-

765 Colon Cancer Cell Adhesion Stimulates NF-KB Mediated Survival. Scott K. Kuwada, Brad Trowbridge, Ernst J. Eichwald, Jinqiu Kuang, Phillip Gray, Raymond Daynes, Salt Lake City, UT The pteiomorphic transcription factor NF-KB is constitutively activated in several types of cancer cells and mediates the expression of several survival genes. HYPOTHESIS: NF-KB is a rational therapeutic target in colon cancer (CCa). METHODS:Two soluble NF-KB inhibitors,

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inhibited collagen IV dependent Akt phosphorylation and attenuated ERK phcsphorylation. These data suggest that cell-matrix interactions may regulate intestinal epithelial motility across a basementmembrane rich in collagen IV by a signal cascaderequiring el 131or cx2131 integrin heterodimeroccupancy,integrin-dependentFAKactivation, and subsequentactivation of ERK via Src and PI 3-kinase.

768 Altered Expression of Serum Response Factor during Gastric Ulcer Healing: Implications for Re-Epithelialization and Angiogenesis Jianyuan Chai, Dolgor Baatar, Woo S. Moon, Andrzej Tarnawski, Irvine, CA Gastric ulcer healing requires restorationof the muscularis mucosaeand the microvasculature (through angiogenesis),and filling of mucosal defect with the epithelial and connectivetissue cells. All these processesare tightly regulated by growth factors, e.g. EGF and VEGF.Serum responsefactor (SRF) is a transcription factor that controls immediateearly genesand muscle genes by binding to serum response element in the promoter regions of these genes. It is unknown whether SRF is expressedin normal or ulceratedgastric mucosa and/or participates in gastric ulcer healing. Methods: Gastric ulcer were produced in rats by focal serosal application of 100% acetic acid. Gastric tissue samples were collected 1, 3, 7 and 14 days after ulcer induction. Normal gastric tissue served as controls. Studies: 1) Ulcer size, 2) quantitative histology, 3) immunostaining for SRF, EGF and VEGFand signal quantification with videoimage,4) SRF protein levelsby Western blotting, 5) SRF mRNAby RT/PCR.Results: In normal gastric tissue, SRF was strongly expressed in muscularis mucosae, muscularis propria, supportive cells of submucosal vessels and microvessels,and to a lesser degree in neck and parietalcells. Western blot demonstratedstrong expressionof ~ 67kDa SRF protein. At 3 days, gastric ulcers were fully developed.Their size was reduced in a time dependent manner reflecting spontaneous healing. Gastric ulceration significantly reduced (by >50% p<0.001) SRF expressionat 3 days.At 7 - 14 days, SRF expressionhas significantly increased during ulcer healingand was abundantin myofibroblasts in granulationtissue (380% increase; p 10 fold increase; p
is uncertain. This study was aimed to determinethe expressionand localizationof EGF,EGFR, Cox2, bFGF, angiopoietinsl and 2 (ANG1, ANG2) and their receptor Tie2 mRNAs and proteins in normal and ulceratedhuman gastric mucosa. In addition,we studiedthe expression of serum response factor (SRF), a transcription regulator of numerous genes. Methods: Human biopsy specimensfrom normal gastric mucosa and from ulcer margins were obtained during endoscopy in 22 patients. Forty seven surgical specimens of gastric ulcers were retrievedfrom surgical pathologyarchives. Studies: 1) Expressionof EGF,TGFe, EGF-R,Cox2, as well as bFGF,ANG1, ANG2, and Tie2 mRNAs by RT/PCRand/or in-situ hybrid!zation, 2) expression of respective proteins and SRF by immunohistochemistry and Western blotting, 3) quantification of signal intensity with videoimage system. Results: Epithelium of the ulcer margin and regeneratingglands demonstrated overexpressionof EGF-R mRNA and protein (210-320% increase; p 410% (p300% (p
771 Delta (8) Isoform of PKC (PKC-8) Could Explain How Oxidants Disrupt the Microtubule (MT) Cytoskeleton and Injure Monolayers of Intestinal Epithelia Ali Banan, Jeremy Fields, Ashkan Farhadi, Lei Zhang, All Keshavarzian,Chicago, IL Gut barrier disruption and oxidant injury are key factors in the pathogenesisof inflammatory GI illnessessuch as inflammatory bowel disease(IBD). Using monolayers of intestinal (Caco2) cells, we showed that oxidants disassembleand injure the MT cytoskeleton and disrupt barrier integrity (BI). Since classical PKC isoform-S is translocated to particulatefractions by oxidant in our wild type (W7) cells, we sought to determinewhether PKC-5 is key in oxidantinduced injury to the cytoskeletonand monolayerBl. Methods: WTmonolayerswere incubated with oxidant (H202;HOCI) -+ PKC modulators. Other cells were transfected with varying levels of an inducible plasmid to stably over- or under-expressPKC-8 isoform. These clones were then exposedto oxidants _+ PKC modulators.Effects on monolayerBI (clearanceby fluorometry); MT cytoskeletal integrity ,laser confocal microscopy<,; PKC-5 subcellular distribution
769 Apical Acidification Decreases Gastric Mucusal Paracellular Permeability via Src Tyrosine Kinase Monica C. Chen, Enrique Rozengurt,Andrew H, Soil, Los Angeles, CA Background: Previous studies with canine gastric epithelial cells indicated that the apical surface constitutes an important barrier to luminal acid. An increasein transepithelialelectrical resistance (TER) occurs as the apical pH is decreasedfrom 6.5 to 2.0. This increasedTER is accompaniedby a decrease in mannitol flux, indicating that apical acidification decreases paracellular permeability. However, the mechanisms of regulation of TER by acid remain uncharacterized. Previous studies also indicated that Src-family tyrosine kinase plays an important role in secretin-mediatedregulation of paracellularpermeability.Aim and Methods: We examined the role of Src and the Src substrate Pyk2 in the TER response to an apical pH of 4.0 in caninegastric mucosal cells plated in Transwell inserts. We comparedthe change in TER to tyrosine phosphorylationof Src Tyr416 and Pyk2 in the presenceof PP2, a selective inhibitor of Src tyrosine kinase. Results: PP2 pretreatment reduced the apical acid-induced increase in TER by 37.5 + 13.2 % (n =4, p < 0,05). In addition, Western blots using a sitespecific phosphotyrosine antibody revealed that apical acidification increased Src Tyr416 autophosphorylation.Changeswere found in two Src isoforms at 62 kDa and 58 kDa. Maximal stimulation of phosphorylation was reached at ?_.5to 5 minutes and was sustained for 30 rain. Phosphorylationof Tyr416 was partially attenuated by pretreatment with PP2 at a dose highly specific for Src-family kinase (2 ;~M). Apical acidification also dramatically stimulated site-specific tyrosine phosphorylation of Pyk2. Phosphorylationof Tyr402 of Pyk2 was time dependent, reached maximal activation at 5 rain and was attenuated by PP2 pretreatment. PP3, the inactiveisoform of PP2,and AG1478, a highly specific EGERtyrosine kinaseinhibitor, did not block acid-induced increases in TER and tyrosine phosphorylation of Src or Pyk2. Conclusions:We found that apicalacidificationof caninegastric epithelialmonolayersproduced parallel increasesin TER and tyrosine phosphorylationof Src and Pyk2. Basedon the observation that PP2 inhibited both acid-induced increases in TER and in phesphorylationof Src and Pyk2, we speculatethat Src kinasefamily partially mediatesdynamictyrosine phosphorylation events requiredfor the decreasein paracellularpermeabilityin responseto apical acidification. Our findings suggest a novel role of Src-family tyrosine kinase in regulating apical resistance to acid.

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Infliximab in Steroid-resistant Ulcerative Colitis: A Randomised Controlled Trial Christopher S. J. Pi'obert, Stephen D. Hearing, Stephan Schreiber, Tanya Kuhhacher, Subrata Ghosh, Alastair Forbes, Bristol,, UK; Bristol, UK; Kiel, Germany; Edinburgh, UK; London, UK Introduction¶ Serum TNFcxis raised in ulcerative colitis (UC) (Murch 1991) and antibodies to TNFo~may ameliorate a rodent model of colitis (Ward 1993). The benefits of one such antibody, Infliximab, in Crohns diseaseare established.l] Methodsl] We conducted a RCT of Infliximab in the treatment of steroid-refractory UC. Patients were randomised to receive a blinded infusion of Infliximab 5mg/kg or placebo at week 0 and again at week 2. Disease activity (UCSS & Baron score), IBDQ, EuroQol and adverse events were recorded during 8 weeks follow-up. Remissionwas defined as UCSS -<2 and/or Baron score = O. Patients not in remission at week 6 were offered open,label Infliximab lOmg/kg and reviewed 2 weeks later.¶ Results¶ Completedata are currently availablefor 42 patients. After 2 weeks, Baron score of 0 was equally likely in the Infliximab and the placebogroups (3/22, 1/20 respectively, ns). After 6 weeks, remission (UCSS-<2) was no more likely in the Infliximab than placebo group (8/22 vs 6/20, ns respectively). The median improvement in UCSS was 3 for the Infliximab group and 2 for placebo group ins). A Baron score of 0 was no more likely in either group (5/22 vs 6/20, ns). The median improvement in the Baron score was 1 in both groups (12 patients in the Infliximab group improved, 3 deteriorated;11 in the placebogroup improved, 1 underwent co!ectomy). The median change in IBDG, from weeks 0 to 6, was similar in both groups (difference in medians9.5, 95% CI minus 18 to 39). The improvement in EuroGol was also similar (difference in medians 3, 95% CI minus 6 to 14). One placebo patient suffered septic complicationsduring this period. An open labelledinfusion of Infliximab was given to 20 patients with continued active disease. Remission was achieved in 3/10 patients initially treated with Infliximab and 1/9 patients tieated with placet~o(one patient not evaluated).llConclusion'llThesedata do not support the use of Infliximab in the management of steroid resistant ulcerative colitis.

770 Healing of Human Gastric Ulcers Triggers Activation of Genes Encoding EGF, Its Receptor, SRF, COX2, bFGF, Angiopoietins and Tie2 Andrzej Tarnawski, Jerald L. Jansen, Jay Kidao, Woo S. Moon, Jianyuan Chai, trvine, CA Growth factors induce cell proliferation, re-epithelializationand angiogenesis,all of which are essential for tissue regeneration. In animal models of gastric ulcer, epidermal growth factor (EGF) and its receptor (EGF-R) are upregulated (Gastroenterology 1992; J Physiol 2000), promoting re-epithelialization,while activation of pro-angiogenic growth factors stimulates neovascularization.The clinical relevanceof these findings for human gastric ulcer healing

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39 patients received i.v. hydrocortisone and 41 patients received placebo. The number of patients in both groups receiving concurrent immunosuppressants was similar at baseline, 17/39 (44%) u 17/41 (42%) p=0.9, and at follow-up. All patients were HACA-negativeat baseline and had similar HACA levels, 0.69_+0.13 v 0.61 _+0.05:p=0.5. At 8 weeks postinfusion, the hydrocortisone group had fewer HACA-positive patients, 8/34(24%) v 12/ 34(35%): p =0.07 and the HACA level was significantly lower compared to the placebo group, 2.0_+0.4 v 1t.6_+3.2: p=O.01. At 16 weeks, the hydrocortisone group had fewer HACApositive patients, 10/32 (31%) ~ 16/32 (50%): p = 0.03 and HACAlevels remainedsignificantly lower compared to the placebo group 1.6_+0.3 ~ 5.7_+2.0: p = 0.07. Analysis of the placebo group showed that concurrent immunosuppressants reduced HACAformation at 8 weeks (4/ 20(20%) u 8/14(57%): p=O.03) and at 16 weeks 3/14 (21%) ~ 13/20 (65%): p=O.01). CONCLUSION: Premedication with intravenous hydrocortisone significantly reduces the number of patients who become HACA-positive at 16 weeks.

773 Post Infusion Infliximab Levels Determine Duration of Response in Crohn's Disease and Are Directly Related to Infusion Reactions Maja Noman, Filip Baert, Severine Vermeire, Gert Van Assche, Geert D'Haens, An Carbonez, Paul Rutgeerts, Leuven, Belgium

Background and aims: The duration of response to Remicade® (Centocor) varies greatly and seems to decrease over time with repeated infusions when administered upon relapse of disease activity. Developmentof antibodies to infliximab (ATI) also called HACA (Human AntiChimeric Antibodies) is a risk factor for infusion reactions.There are no data on the relationship between ATI and post infusion infliximab levels and duration of response to the drug. We studied these parameters in a cohort of patients treated in an on demand schedule. Materials and Methods: 125 consecutive patients with refractory or fistulizing Crohn's disease treated with serial inffiximab infusions in an on demand schedule were systematically evaluated at regular intervals. Evaluations consisted of ATI and infliximab level determinations as well as recordings of clinical data, side effects (including infusion reactions) immediately before and 4 weeks after each infusion. Patients who had presented with an infusion reaction (IR) at a previous infusion were systematically pretreatedwith steroids and antihistamines (prophylaxis) prior to each subsequent infusion. Results: For the whole group higher infliximab levels 4 weeks after infusion predicted a significantly longer duration of response. There was a significant correlation between inflixirnab level at 4 weeks after infusion and the ATI level before this infusion (r2 = 0.35) if the ATI levels could be determined. No concomitant immune modulating therapy was the single important risk factor for higher ATI levels (RR is 2.6 (Cl 1.6-4.08) for ATI > 10) or for lower infliximab levels (RR 1.9 (CI 1.4-2.6) for infliximab < 12). IR dramatically decreasedthe median infliximab level and the time to relapse defined as to time to next infusion.Conclusions: Infliximab levels 4 weeks after infusion are positively related and ATI levels (if measurable)prior to an infusion are inversely related with the duration of clinical response. Infliximab is almost undetectableat 4 weeks after an IR. Once a patient has developed an IR the duration of response to infliximab will be short.

776 Endoscopic Healing after Infliximab Treatment for Crohn's Disease Provides a Longer Time to Relapse Geert R. D'Haens, Maja Noman, Filip Baert, Martin Hiele, Gert Van Assche, Marco Daperno, Paul Rutgeerts, Leuven, Belgium

Background: Biological therapy with antibodies to TNF (Infliximab, Centocor, Malvem, PA) induces significant healing of inflamed ileocolonic tissue in patients (pts) with refractory Crohn's disease (CD)(D'Haens, Castro 1999). It remains unclear, however, whether mucosal healing provides a better quality or duration of remission and if it should become a primary treatment objective in CO. Methods: Nineteen consecutive pts with CD who completed the Accent-I study, in which they received systematic q8w reinfusion of infliximab 5 or 10 rag/ kg or 'on demand' treatment with 5, 10 or 15 mg/kg for 40 weeks, were studied. At the end of the study (week 54) they underwent an ileocolonoscopy with recording of the endoscopic severity score COEIS and the clinical activity score CDAI. Patients were then followed with monthly CDAI measurements until clinical relapse. We determined the relationship between the endoscopic findings and the further clinical course of the patients (Spearman Rank correlations). Results: The mean CDEIS of the 19 pts (11 female/8 male, age 32 +/- 2 yrs, location: 2 ileal CD, 6 ileocolonic CD and 11 colonic CD) at the end of ACCENT-1 was 2.68 (range 0-21.7). Following this colonoscopy, the median time to relapse was 12 weeks (range 1- > 78 weeks), with a significant correlation between time to relapse and CDEIS (r= -0.45, p = 0.05). Nine pts who had complete disappearanceof ulcers remained without relapse for a median of 20 weeks (range 14- >78 weeks) following their last infliximab infusion; 6 pts had significant but incomplete endoscopic healing and had no relapse for a median of 19 weeks (17->50 weeks); 4 pts had no healing at all and developed clinical relapse after a median of only 4 weeks (0-8 weeks). Interestingly, the clinical score CDAI at the end of Accent 1 did neither correlate with the endoscopic appearance (r=0.16, p=O.5), nor with time to relapse (r-0.36, p=O.1), although pts with lower CDAI's tended to have a longer time to relapse. Concomitant immunomodulation did not affect endoscopic changes nor time to relapse. Conclusion: If significant mucosal healing can be achieved with infliximab therapy for CD, the time to relapse is significantly prolonged. The endoscopic appearanceis a better predictor of the further clinical course than COAl. Endoscopic healing should probably become a standard treatment goal in CD.

ATI levelweek 0 Infliximabweek 4 Timeto nextinfusion* never IR 3.1 +/- 9.03 14.09 +/- 7.32 68 days +/- 34 first IR 20.16 +/- 10.02 1.21 +/- 1.7 40 days +/- 13 no new IR with proph. 14.27+/-1191 12.89+/-8.81 36 days +/- 25 new IR with proph 16.21 +/- 6.04 1.0 -1- 0.63 30 days -,-/-28 Values are expressedus +/- SD. *Treatmentswith an inte~al 140 days betweentwo infusionswere omittedfromthe analysis.

774 High Incidence of Anergy Limits the Usefulness of PPD Screening for Tuberculosis (TB) Prior to Remicade® in InflammatoryBowel Disease (IBD) William S. Mow, Maria T. Abreu, Konstantinos A. Papadakis, Stephan R. Targan, Eric A. Vasfliauskas, Los Angeles, CA Backgroundand Aims: Reports of TB occurring in patients receiving Remicade® (infliximab) prompted recommendations that all patients be evaluatedfor the risk of latent TB. Tuberculin PPO skin testing has been proposed as an adequatescreening measure. The aim of this study was to evaluate the utility of PPD skin testing as a satisfactory screen for TB exposure in IBD patients. Methods: Sixty-one consecutive IBD patients (CD: 53, UC: 1, IC: 7; Mean age: 34 years [range 7 - 67 years]) at the Cedars-Sinai Medicat Center IBD Center being treated with or considered for Remicade® therapy underwent standard intradermal tuberculin PPD skin testing prior to or in between infusions of Remicade®. One or more control antigens (Candida, tetanus and/or mumps) were concurrently placed on fifty of these patients. Skin tests were read at 48-72 hours after placement and results recorded. Results: One of the 61 patients had a positive PPD. This patient had a prior history of a positive PPD and positive chest x-ray. Overall, 76% (37/50) of patients who had controls placed were anergic, failing to react to any of the control antigens. Twelve of 50 (24%) had positive reaction to Candid~, 1 of 10 (10%) reacted to mumps, and 1 of 2 (50%) to tetanus antigen. Conclusion: The high incidence of anergy observed in this study suggests that in IBD patients a negative PPD is an unreliable indicator for TB exposure. In areas in which TB is endemic, evaluation should include not just a PPD with control, but also a detailed history of travel and TB exposures, and of symptoms such as persistent cough or unexplained weight loss. A chest radiograph should be performed in all patients with suspicious histories or symptoms.

777 Symptomatic Luminal Stricture Underlies Infliximab Non-Response in Crohn's Disease (CO) Devang N. Prajapati, Kia Saeian, Joseph P. Kim, Subra Kugathasan, Josh F. Knox, Jeanne Emmons, David G. Binion, Milwaukee, Wl

Background and Aims: Infliximab is highly efficacious in the treatment of CO pts with refractory disease. However, a significant minority do not respond to infliximab, and this population is frequently referred to tertiary centers. There are no specific recommendations regarding the use of infliximab in CD pts with known stricture or obstructive symptoms. Clinical outcome in infliximab non-responders was evaluated at a referral center over a 3y period to determine contributing factors. Investigation was focused on the relationship between infliximab nonresponse and stenotic lesions in the GI tract. Methods: Infliximab treated CD pts evaluated at a tertiary center were analyzed. Infliximab outcome was classified as either response (i.e. corticosteroid cessation, decreased disease activity index, no surgery, over the subsequent 6 months) or non-response (inability to discontinue corticosteroid, no disease activity index improvement, and/or requirement for surgery within 6 months). Clinical outcome was assessed 6 months following initial infusion. Infliximab non-responders were further classified as: 1) stricture/obstruction requiring surgery; 2) loss of efficacy; 3) no efficacy and other. Patient demographics, duration and extent of disease as well as prior history of drug exposure were recorded. Results: Out of 235 adult CD pts evaluated, 98 (42%) received infflximab. 53/98 (54%) were long-term responders, while 45/98 pts (46%) failed to demonstrate sustained improvement following infliximab treatment. 27/45 non-responders (60%) were specifically referred for evaluation because of infliximab failure. 30/45 infliximab non-responders had underlying stricture and obstruction. 6/45 required surgery for refractory fistulizing disease, and 6 pts failed to respond to drug. 37/45 infliximab non-responders ultimately required surgery (76% luminal stricture, 14% refractory fistula, 10% fulminant disease). 28 strictures were located in small bowel or at enterocolonic anastomosis and 2 remaining strictures were found in the colon. A minority of infliximab non-responders either failed or lost response to drug. Conclusions: Infliximab non-response in CD pts is frequently associated with intestinal strictures and clinical obstruction requiring surgical intervention within 6 months of infusion. Our experiencesuggests that CD pts with symptomatic primary or anastomotic small intestinal strictures should undergo surgical evaluation prior to the initiation of infliximab therapy.

775 A Randomized, Double-Blind, Placebo-ControlledTrial of Intravenous HydrocorUsone in Reducing Human Anti-ChimericAntibody Following Infliximab Therapy Mazen Alsahli, Yoon-Tae jeen Jeen, Mark A. Peppercorn, Pierre Michetti, Richard J. Farrell, Boston, MA

BACKGROUND:Infliximab, a chimeric anti-TNF antibody is effective therapy for moderate-tosevere and fistulizing Crohn disease. However, in clinical trials, a significant proportion of patients develop human anti-chimeric antibodies (HACA)which may be associatedwith reduced efficacy and infusion reactions. AIMS: To determine whether premeditation with intravenous hydrecortisone can reduce HACA formation following infliximab therapy. METHODS: 80 patients with active Crohn disease referred for infliximab were randomized to receive either 200rag i.v. hydrocortisone or a placebo injection immediately prior to receiving their first 5mg/kg infusion as well as any subsequent infusions. Ouantitative HACAand Infliximab ELISA immunoassays (Prometheus Laboratories, Inc., San Diego, CA.) were performed blindly in duplicate on sera drawn prior to the first infusion, and at 8 and 16 weeks post-infusion. Becausethe presenceof infliximab interferes with the HACAassay, HACAresults were reported as follows: positive if the mean HACA result was >1.69 mg/ml and inftiximab level was < 1.4 mg/ml; negative if the mean HACA level was <1.69 mg/ml and infliximab level was <1.4 mg/ml and indeterminate if infliximab levels were >1.4 mg/ml. Patients were not routinely premedicated with Benadryl or Tylenol. HACA levels are expressed as Mean_+S.E. RESULTS:

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778 Helicobacter pylori (HP) Eradication Does Not Cause EsuphagiUs in Healthy Blood Donors: 8 Years Follow-Up Dino Vaira, Massimo Rugge, Chiara Ricci, Valentina Russo, [.uigi Gatta, Gioacchino Leandro, Marcello Menegatti, Maria Miglioli, Nimish Vakil, Bologna, Italy; Padova, Italy; Castellana Grotte (8A), Italy; Milwaukee, Wl

Background: there is controversy about whether HP eradication causes erosive esophagitis but most studies have been limited by short follow-up and pre-existing ulcer disease. Aim: to determine the incidence of endoscopic esophagitis in asymptomatic blood donors who underwent eradication therapy compared to those that did not. Methods: 147 asymptomatJc blood donors infected with HP underwent endoscopy in 1990-1 with histology, rapid urease test and culture. 73 subjects underwent successful eradication therapy immediately after the first endoscopy. In 2000, after a mean follow-up of 8,5 yrs; (range 6.25-9.33 yrs), the subjects were restudied with endoscopy and biopsy. Severity of esophagitis was determined by the Los Angeles (LA) classification. Results: At initial endoscopy, 0/147 HP positive BD had esophagitis. In 2000, 35 subjects had endoscopic esophagitis (24%). 14/74 (19%) were HP positive donors (LA grade A=13, LA C=1) and 21/73 (29%) in HP negative donors. (LA grade A n = 20, LAB n = 1). There was no significant difference in the prevalenceof moderate to severe corpus gastritis in subjects who did and did not develop esophagitis (p = 0,3998). Conclusions: 1. Eradicating HP does not lead to an increased incidence of esophagitis in healthy blood donors (OR 0.58, [C] 0.27-1.24]. 2. Corpus gastritis of moderate severity is significantly more common in subjects with esophagitis who are HP positive than those who are H pylori negative. 3. Corpus gastritis of moderate severity is more frequent in patients with HP infection without esophagitis. Corpus gastritis none/mild cases number (%) moderate/severe cases number (%) p~),004 Corpus gastritis none/mild cases number (% moderate/severe cases number (%) p<0,001

HP÷ve esophagitis * n= 14 6 (40) 9 (60)

HP-ve esophagi6s ÷ n=21 20 (95) 1 (5)

HP'~ve esophagitis33 (55) 27 (45)

HP-ve esophagitis • 52 (100) 0

(N = 940) duodenal ulcer disease and without baseline erosive esophagitis who were enrolled in 8 trials of esomeprazole or omeprazole dual and triple HP therapies had end-of-study endoscopies 4-30 weeks after completion of all active therapy. 533 patients had heartburn and regurgitation scores (4-point scale) assessedat baselineand 4 weeks after end of therapy. The 533 patients were divided into 2 groups: 1) No GERDsymptoms (heartburn, regurgitation) at baselineor before study (N = 127); 2) GERDsymptoms at baselineor before study (N = 406). HP status was assessed by endoscopic biopsy tests (rapid urease test, histology, culture) at baseline and at least 4 weeks after therapy. RESULTS(SEETABLE): Homogeneity was present across the 8 treatment studies (Breslow-Day test p=0.68) and between the studies with longer and shorter term follow-up (p =0.54), indicating validity of combining results of the individual studies. In the study with longest follow-up (28-30 weeks), erosive esophagitis developed in 2 (7%) of 28 patients with HP cure vs. 5 (7%) of 78 with persistent HP (OR = 1.09(95% CI 0.20 - 5.98)). CONCLUSIONS:H pylori eradication in patients with duodenal ulcer disease does not lead to the development of erosive esophagitis or the development of new symptomatic GERD. H. pylofi eradication may improve symptoms in patients with preexisting GERD. Development of New Erosive Esophagitis, New SymptomaticGERD, or Worsened _SymptomaticGERD with HP Eradicationvs, Persistent Infection HP Cure HP Persists OR (95% CI) New Erosive Esophagitis 24/621 (4%) 14/544(3%) 1,52 (0.78-2.97) New GERD 13/92 (14%) 7/35 (20%) 0.56 (0.24-1.82) Worsened GERD 20/269 (7%) 20/137 (15%) 0.47 (0,24-091)* * p = 0.02 for worsened GERD, HP curs vs. HP persists

781 Eradication of H. pylori Unleashes Post PPI Acid Hypersecretion Derek Gillen, Angela Wirz, Kenneth E. L. McCall, Glasgow, UK

779 Effect of H. pylori Eradication on Maintenance Treatment of Gastroesophageal Reflux Disease: A Double-Blind Placebo-Controlled Randomized Trial Justin C. Y. Wu, Jessica Y. L. Ching, Wai Leung, Yui Hui, Francis K. L. Chan, Carrian M. Y. Cheung, Joseph J. Y. Sung, Shatin, Hang Kong BACKGROUND: The role of H. pyiod (Hp) eradication in management of gastroesophageal reflux disease (GERD) is unclear. AIM: To study the effect of Hp eradication on GERD patients receiving maintenance medical therapy. METHODS: Consecutive patients with weekly reflux symptoms were prospectively recruited for endoscopy and symptom evaluation. Hp status was determined by biopsy urease test and histology. Grading of gastritis at antrum and body were assessed by updated Sydney classification. Exclusion criteria included gastric outlet obstruction, peptic ulcer, gastric surgery and previous use of PPI. Hp infected patients were assigned, in a double blind and randomized manner, to either HpE (1-week omeprazole (OM)based triple therapy) or Hp+ group O-week OM 20mg bid plus two placebos). Both groups then received OM 20rag daily for 8 weeks. Patients with complete healing of esophagitis and symptom relief were given OM 1grog daily as maintenance. Primary study endpoint was treatment failure, defined as either recurrence of esophagitis or symptom relapse requiring full-dose PPI. A logistic regression model was used to determine predictors of treatment failure (including assigned treatment, sex, age, intensity of gastritis, symptom severity, esophagitis and hiatus hernia). RESULTS: 93 patients (Mean age: 55.0+_13.8; M:F, 44:49)were randomizedto HpE (n = 45) and Hp + (n = 48). 30 (32%) had erosive esophagitis.The treatment failure rates of HpE vs Hp + were 41.0% vs 18.2% (log rank test: p = 0.018; odds ratio: 2.63, 95% C.I.: 1.11-6.20) at 26 weeks and 49.4% vs 23.7% (log rank test: p=O.O3;.odds ratio: 2.47, 95% C.I.: 1.05-5.83) at 52 weeks, respectively. Using multivariate analysis, Hp eradication was the only independent predictor for treatment failure (p=O.02; odds ratio: 3.17, 95% C.L: 1.14-8.81). CONCLUSION: Hp eradication leads to more difficult control of GERD in

maintenance therapy.

Propor~ of rei~e t~te at 1 yt; lOgrank=0.03;Upper]h~ FIp+~tow~lir~, H4~E

78O Effect of H. Pylori Eradication on Development of Erosive Esophagitis and GERD Symptoms in Double-Blind Prospective Studies Loren Laine, Jennifer Sugg, Los Angeles, CA; Wayne, PA

Whether H. pylori (HP) eradication increasesthe chance of developing GERDor endoscopicaliy documented erosive esophagitis is controversial. We determined the rate of new erosive esophagitis, new symptomatic GERD,and worsened symptomatic GERDin a post hoc analysis of double-blind trials of HP therapy. METHODS:1165 patients with past (N =225) or present

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Introduction: There is marked rebound acid hyperseoretion after omeprazole in H.pylori-ve but not +ve subjects. Oxyntic gastritis probably prevents it in the latter. Aim: To determine the effect of H.pylorieradication on rebound acid hypersecretion after omeprazole. Methods:17 healthy H.pylori +ve subjects had acid secretion studies prior to commencing omeprazole 40rag/day for 8 weeks. During the last week of omeprazole, they were randomised to a 1 week course of amoxycillin/cladthromycin or placebo. Further acid secretion studies were performed at 1,2,4,6&8 weeks after treatment. A further breath test was performed at 8 weeks to determine H.pylori status after treatment. Results: Marked rebound hypersecretion to physiological levels of gastrin was observed in the subjects who had their infection eradicated but not in those with persisting infection (Table). A similar trend was seen with iespect to supraphysiological gastrin stimulation. Summary: Eradication of H.pylori infection at time of stopping PPI therapy unleashes marked rebound acid hypersecretion which persists for at least 6 weeks. Rebound acid hypersecretion following PPI is not seen in those remaining H.pyloripositive. Conclusion: This acid hypersecretion induced by H.pylori eradication in patients previously receiving PPI therapy may explain the relation between H.pylorieradication and development of GERD. Submaximal acid output (mmol/h) Days Post-Orneprazole Day 7 Da1 4 y ~ Day 28 Day 42 Day 56 H. pylori 248* 28.7* 22.4 23.9* 25.8 eradicated (20.4-28.9) (22.8-30.0) (17.5-32.9) (20.8-29.1) (174-29.9) H.pylodnot 18.8(12.2-23.6) 13.1 16.0 17.2 18.1 15.0 'eradicated (9.6-20.8) (6.4-235) (12.7-23.1) (9.3-21.5) (10.3-22.5) Values are medians (quartiles); *significant versus pre at p
782 Paradoxical Dual Effect of Helicobacter pylori Eradication Therapy on Heartburn and Gastro-Esophageal Reflux: The Bristol Helicobacter Project Richard Harvey, J Lane, Jenny Donovan, Liam Murray, lan Harvey, Satheesh Nair, Matthias Egger, Bristol, UK

Helicobacterpylod eradication therapy has been reported in some studies to result in increased heartburn and acid reflux, but others have found either no effect or even a beneficial effect. We have investigated these contradictory findings, as part of the community-based Bristol Helicobacter Project. Methods 10,537 people aged 20-59 years, living in North East Bristol (UK), are participating in a community-based prospective double-blind randomised controlled trial of the effects of H. pylori eradication. 1634 of these had H. pylori infection on ~3C-urea breath testing, and 1558 were treated with either H. pylori eradication therapy (ranitidine bismuth citrate 400mg and clarithromycin 5OOmg twice daily for two weeks) or matching placebo. The prevalence,frequency and severity of heartburn and acid reflux were measured at baseline and two years after randomisation. Results Two years later there was 3.1% less heartburn and 2.5% less reflux (p = 0.04) after active treatment than after placebo. However, this very small net benefit concealed complex differential effects. Active treatment showed the most marked benefit in participants with mild or no GERDsymptoms at baseline, resulting in 6.8% less heartburn (p=O.02) and 4.3% less reflux (p=O.01) at 2 years. Of those with moderate initial symptoms, more improved after active treatment than after placebo, but also, paradoxically, more got worse, with little net benefit. Those with the most marked GERD symptoms at entry were more likely to be worse after active therapy. Risk factors for severe heartburn two years after treatment included GERD symptoms at entry (p=O.O01), obesity (p=O.O09), treatment with active H. pylori eradication therapy (p=0.022), and male sex (p = 0.046). Conclusions(1) H. pylori eradication therapy reduces GERDsymptoms. However, the net benefit is small because some subjects get worse, especially if they are obese, male or have troublesome GERD symptoms initially. (2) The contradictory results of previous studies may reflect differences in the selection or number of patients and in the definition of endpoints.

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Respondents with dyspeptic symptoms reported significantly higher rates of problems with mobility (39% vs. 61%, p-value 0.0013), self care (30% vs. 70%, p-value 0.0008), usual activities (39% vs. 61%, p-value 0.0001), pain/discomfort (32% vs. 68%, p-value 0.0001) and anxiety/depression (29% vs. 71%, p-value 0.0001) in comparison to those without dyspeptic symptoms. Patients with upper abdominal pain and heartburn more often reported problems with health status dimensions in comparison to those without upper abdominal pain or heartburn. Conclusions. Dyspeptic symptoms strongly impaired the health status of patients with cardiovascular disease.High degreesof problems with all health state dimensions were reported in patients with dyspeptic symptoms and cardiovascular disease.

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H. pylori Infection Is Not a Protective Factor for the Development of Barreft's Esophagus in Patients with Symptoms of Gastroesophageal Reflux Disease Andreas Leodolter, Michael Vieth, Dirk Lindner, Michael Kulig, Daniel Jaspersen, Joachim Labenz, Tore Lind, Wolfgang Meyer-Sabellek, Stefan Willich, Manfred Stolte, Peter Malfertheiner, Magdeburg, Germany; Berlin, Germany; Fulda, Germany; Siegen, Germany; MOndal, Sweden; Wedel, Germany; Bayreuth, Germany Background and Objective: The role of H. pylori infection in gastroesophageal reflux disease (GERD) and its complications is uncertain. A lower prevalence of gastric H. pylori infection in patients with Barrett's esophagus has been reported. The aim of our study was to identify predictive factors for 8arrett's esophagus in patients with GEROand in particular to test the hypothesis of whether H. pylori is an independent protective factor for Barrett's esophagus. Methods: A total of 6215 patients with symptoms of GERD were included in a prospective cohort study (ProGERD). All patients underwent endoscopy at baseline to identify stage of disease, non-erosive or erosive and if so, the grade of esophagitis. Biopsies were taken 2cm above the Z-line, from columnar metaplasia in the esophagus if present and from the stomach to assess H. pyloriin all patients. Factorsassociated with Barrett's esophagus were determined by stepwise multiple logistic regression analysis. Results: Barreft's esophagus was diagnosed in 506 patients (8.1%) based on the histological assessment of specialized columnar epithelium. In further 219 (3.5%) patients, columnar metaplasia was observed during endoscopy, but no specializedcolumnar epithelium was detected in the histology. Older age (age difference 20y, 0D=1.81, 95% CI 1.52-2.16), mare gender (OD = 1.24, 95% Cl 1.02-1.51), duration of GERD longer than one year (0D=1.59, 95% CI 1.29-1.97), smoking (1.28, 95% CI 1.051.54) and erosive GERD(OD = 3.85, 95% CI 3.07-4.83) were found to be independent predictive factors for 8arreft's esophagus. In the univariate analysis, H. pylori was significantly less prevalent in Barrett's patients older than 60 years (11.3% vs. 15.5%, p=O.01). In the multivariate analysis, H. pyloriwas not an independent protective factor (p =0.80) for Barrett's esophagus. Conclusion: Based on the multivariate analysis in this large cohort of patients, H. pylori is not a protective factor for the development of Barreft's esophagus in patients with symptoms of GERD.This finding contrasts with several study results based on univadate analysis only. Sponsored by a grant from AstraZeneca

786 Prevalence and Symptomatic Impact of Non-Erosive Reflux Disease in Functional Dyspepsia Jan Tack, Kwang Lee, Daniel Sifrim, Jozef Janssens, Leuven, Belgium As subset of functional dyspepsia (FO) patients respond to acid suppressive therapy (Talley et al., 1999). It has been suggested that these patients are suffering from non-erosive reflux disease, with atypical symptom manifestation, but the prevalenceof non-erosive reflux disease in typical FD and its relevance to symptoms have never been established. The aim of the present study was to study 24 hour pH monitoring in consecutive FD patients. Methods: 203 patients with epigastdc symptoms (138 women, age 45 _+1 years), with a negative upper g.i. endoscopy and without dominant symptoms of heartburn or pyrosis participated in the study. In all patients the severity (0-3, 0 = absent, 3 = severe) of 8 symptoms (pain, fullness, bloating, early satiety, nausea, vomiting, belching, epigastric burning) was scored and a 24 hour esophageal pH monitoring study was performed. All patients underwent a gastric emptying breath test and in 86 a gastric barostat study was performed, Results: Abnormal pH monitoring (acid exposure >5% of time) was found in 34 patients (16.7%). Demographic characteristics did not differ between both groups. The prevalenceof delayedgastric emptying, of hypersensitivity to gastric distention and of impaired accommodation to a meal did not differ between both groups. Patients with pathological acid exposure had a significantly higher prevalence of symptoms of postprandial pain (63 vs. 88%, p
784 The Effect of Reduced Quality of Life on the Subsequent Development of Dyspepsia and Irritable Bowel Syndrome: A Prospective Cohort Study Paul Moayyedi, Sara Duffett, Su Mason, Julia Brown, David Furman, Anthony Axon, Birmingham, UK; Leeds, UK

787 Dyspeptic Patients Exhibit Abnormal Duodenal Mucosal Defensive Responses to Topical Application of HCI as Measured by Endoscopic Reflectance Spectrophotometry (ERS). A Prospective, Randomized, Controlled Study Francisco C. Ramirez, James F. Holland, Felix W. Leung, Phoenix, AZ; Los Angeles, CA

Introduction: Dyspepsiaand irritable bowel syndrome (IBS) are associated with reduced quality of life (OoL). The temporal relationship between these events is unclear. We evaluated this in a cohort study. Methods: This cohort study was nested in a randomised controlled trial that evaluated the clinical benefit of H pylori screening and treatment in the community. Subjects betweenthe ages of 40-49 years were randomly selectedto attend their local general practice. H pylori status was assessed by 13C-urea breath test and infected individuals were randomised to eradication therapy or placeboand followed up for two years. QoL was assessed by the Psychological General Well Being Index (PGWBI), dyspepsia by the Leeds Dyspepsia Questionnaire and IBS by the presence of 3 or more Mannings criteria. Assessments were made at baseline and at two years. Reduced QoL was defined as a PGWBI of < 106 (the mean score at baseline). Results: 32,929 subjects were invited, 8,407 attended and were eligible, 1,769 were H pylori positive and had complete follow-up. Subjects that had dyspepsia or IBS at baseline were excluded. 71/576 (12%) of subjects with a PGWBI -> 106 that did not have dyspepsia at baseline had dyspepsia at two years compared with 76/388 (20%) of subjects with PGWBI < 106 (relative risk (RR) = 0.62; 95% confidence interval (CI) = 0.47 to 0.85; p=O.O03). 30/772 (4%) of subjects with a PGWBI > 106 that did not have IBS at baseline had IBS at two years compared with 67/622 (11%) of subjects with PGWBI < 106 (RR = 0.36; 95% CI = 0.24 to 0.55; p
BACKGROUND:The role of gastric acid (HCI) in dyspepsia is supported by clinical evidence of symptomatic improvement to anti-secretory therapy. RS measures indexes of hemoglobin oxygen saturation (IS02) and concentration (IHB). In vivo data showed that IS02 is linearly correlated with mucosal blood flow (blood flow in ml/min/lO0 gm = 4.6 + 1.8 X IS02). In vitro data showed that decrease in IHB is linearly correlated with mucus thickness (mucus thicknes in p,m = 21 + 2.3 decrease in IHB). HYPOTHESIS:Altered HCI-activatedmucosal defense mechanisms (mucosal blood flow and mucus production) are of pathogenic significance in dyspepsia. AIM: To determine the effect of I-ICI on duodenal IS02 and IHB by ERS. METHODS: Patients undergoing routine EGO had ERS measurements at baseline, 1 and 3 minutes after the instillation of 10 ml of a coded test solution: normal saline (NS) or 0.1 N HCI. Measurementswere performed via video endoscopy and using a Tissue Spectrum Analyzer TS-20O (Sumitnmo, Japan). After uncoding the treatment, patients were grouped according to the presence or absence of dyspepsia as the main indication for EGD. Data (as mean _+ SEM and % change from baseline) were analyzed using ANOVA with contrasts. RESULTS: Comparedwith non dyspeptic patients, dyspepsia did not alter baseline IS02, calcuratedblood flow or IHB. The effect of HCl is shown in the Table. Compared with NS, HCt produced a significant increase in duodenal IS02 and calculated blood flow, and a significant decrease in IHB (increase in mucus thickness), in non dyspeptic patients. In dyspeptic patients the favorable HCl-induced mucosal defensive responses were absent. CONCLUSIONS:The data indicate that dyspeptic patients have altered duodenal mucosal defensive responses to HCI. Endoscopic reflectance spectrophotometry may provide a useful tool to further study the pathogenic role of these defensive mechanisms in dyspepsia. Effect of HCl on Duodenal IS02, Calculated Blood Flow, and IHB Non Dyspeptic Non Dyspeptic Dyspeptic NS (n = 53) HCI (n = 55) NS (n = 7) % Change IS02 (1 mini 2.7_+0.7 5.0_+0.7* 1.8+1.5 % Change BF (1 mk~) 2.6+07 4.7+0.6* ~.7+ 1.4 % Change IHB (tmin) 6.2+1.6 -37+ 1.9" 2.4+4.9 % Change IHB (3 rain) -3.2-+ 1.6 - g,4-+2.4" -4.0+2,9 • vs NS, p <0.05, ANOVA with contrasts.

785 Quality of Life Implications in Patients with Cardiovascular Disease and Dyspeptic Symptoms. Robert Laheij, Leo Van Rossum, Jan Jansen, Freek Verheugt, Nijmegen, Netherlands Background. Medication use to treat and prevent ischemic heart disease may provokes gastrointestinal discomfort and complications. Objectives. To measure the health status of patients with cardiovascular disease and to evaluate the influence of dyspeptic symptoms by using the EuroOol-5D questionnaire. Methods. A total of 1000 consecutive patients who had been admitted between January and September 2001 at the Coronary-Care unit of the Heartcenter of the University Hospital Nijmegen, the Netherlands were sent a questionnaire on gastrointestinal symptoms (upper abdominal pain, heartburn, regurgitation, bloating, and nausea) and quality of life (EuroQol) questionnaire 2 weeks after discharge. Results. From 632 respondents 336 (53.2%) patients reported gastrointestinal symptoms: 77 with upper abdominal pain, 127 with heartburn, 130 with regurgitation, 191 with bloating and 138 patients with nausea. The mean self-rated quality of life status on visual analogue scale (0-100) of respondents without and with gastrointestinal symptoms was 70.0 (95%Confidenca Interval: 68-72) and 61.1 (95%Confidence Interval: 59-63), respectively (p-value 0.0001). The mean self rated health status decreased as the severity of the dyspeptic symptoms increased.

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Dyspeptic HCI (n = 5) 5.0_+2.0 4.7+1.8 -2.8+ 5.7 -5_+7.7

788 Comparison of Duodenal Acid Exposure in Functional Dyspepsia Patients and Healthy Controls Using 24-Hour Ambulatory Duodenal pH Monitoring Kwang-Jae Lee, 8runello Demarchi, Rita Vos, Ingrid Demedts, Jozef Janssens, Jan Tack, Leuven, Belgium Background: A recent study has reported that intraduodenal acid infusion induces nausea and that duodenal clearance of exogenous acid is decreased in functional dyspepsia (FD) patients (Samsom 1999). It is unknown whether spontaneous duodenal acid exposure is altered in FD. The aim of our study was 1)to compare duodenal acid exposure between FD and healthy controls and 2) to investigate duodenal clearance of exogenous acid and its

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influence by 5-HT3 receptor antagonism. Methods: Seven FD patients with prominent nausea (3 males: age 39_+9.6 yrs) and six controls (3 males: mean age, 34_+13.7 yrs) participated in the study. All patients had normal gastric emptying and a normal barostat study. An antimony pH sensor was endoscopically affixed to a mucosal fold in the first duodenal segment using a hemostatic clip. After endoscopy and a recovery period, duodenal pH was continuously recorded using an ambulatory data-logger and a standardized meal was administered at noon. The next morning, duodenal infusions of 5 mL of 0.1 N HCI or saline at a rate of 5 mL/min were given before and after i.v. administration of ondansetron 8 mg or placebo in a randomized double-blind design. Results: 24-hour, supine, fasting and postprandial 2-hour duodenal acid exposure was similar in FD and in controls. However, FD had a significantly higher acid exposure 2-4 hours postprandially, compared to controls. After duodenal acid infusion during fasting, acid clearance was similar in FD and controls (% time pH<4:19.5_+16.4 vs. 12.9_+14.1, NS; mean pH: 5.3_+0.9 vs. 5.8___1.3, NS). Ondansetron had no significant influence on clearance of exogenous acid (% time pH<4:13.1 _+13.9 vs. 24.7_+20.8, NS; mean pH: 5.8_+1.2 vs. 5.3_+1.5, NS). Conclusions: Compared to controls, FD patients with prominent nauseahave higher duodenal acid exposure 2 to 4 hours postprandially. FD patients have a normal clearance of exogenous acid. Administration of a 5-HT3 antagonist does not influence duodenal clearance of exogenous acid.

FD patients Period %timepH4 mean pH 24-boor 47.6±11.3 4.2+0.6 Supine 42.1±20.8 4.3±1.1 Fasting 16.3±5.2 5.9±0.7 PP* 0-2 hour 48.2±24.0 4.3±0.9 PP' 2-4 hour 70.2±6.6 3.2-1-0.3 *, Post.Prandial

Healthycontrols %timepH4 meanpH 47.6±10.9 4.3+08 37.8_+28.2 4.8+1.6 14.0±8.7 6.0--0.4 39.6+-13.2 4.3+0.5 54.5±5.5 4,1--0,2

p value NS NS NS NS <0.001

789 Effect of an Anticholinergic Agent on Gastric Sensory Thresholds in Functional Dyspepsia Mickael Bouin, France Lupien, Michel Boivin, Pierre Poitras, Montreal, QC, Canada Background: Visceral hypersensitivity and impaired gastric accommodation were both proposed as etiological mechanisms for functional dyspepsia (FD); both conditions could probably benefit from parietal relaxation.Aim: Evaluatethe effect of hyoscine (Buscopan~),an anticholinergic agent, on gastric tolerance to balloon distension in FD patients. Methods: We investigated 21 FD patients diagnosed according to Rome II criteria; 12 healthy subjects were used as controls. A latex balloon was positioned in the proximal stomach. Gastric distensions consisted of stepwise successive increments of 50 ml. In response to distension, patients reported first sensation, discomfort, and the maximum tolerated threshold (MTr). A) Dyspeptic patients were compared to normal controls. B) All dyspeptic patients underwent 2 series of distensions separated by 20 min. Before the second set of distensions, each patient received a placebo or hyoscine injection (20 mg, 1 ml). C) In hypersensitive patients (MTT < 600 ml), the response to distension was compared in absence or presence of hyoscine. Results are expressed as mean _+ SEM. Results: A) In dyspeptic patients, the 3 tested sensations were felt at lower volumes than in normal subjects. MTT below normal was seen in 15/21 patients. B) The placebo (n=11) and hyoscine (n=lO) groups were not different in terms of age, gender, weight, and height. First set of distensions: the 3 thresholds and gastric compliance were similar in both groups. Second set of distensions (after injection of saline or hyoscine): the sensory thresholds were not modified in the placebo group whereas they increased significantly in the hyoscine group. Gastric compliance increased with hyoscine. The results are shown in the table below. C) In 10 hypersensitivepatients, hyoscine administration restored MTr to normal values in 5 subjects, but had no effect in 3 others. Conclusions: A) Tolerance to gastric distension was impaired in FD patients. B) Antieholinergic agent hyoscine significantly improved gastric sensory thresholds in FD patients. C) Complianceof the gastric wall increased with hyoscine. D) The benefical effect of the anticholinergic seemed restricted to a subgroup of patients that remains to be characterized.

Normalsubjects 1~ perception (ml) : 392 _+37 Discomfort(ml) : 887 ± 82 MTr (ml) : 996264 Compliance (mllmmHg) : 38.8 ± 1

Placebo 225 ± 15 360 ± 20 540_+35 41.5 ± 1

Dyspepticpatients Hyoecine 260 ± 20 440 ± 30 725_+40 53.6 +- 1

p NS < 0.05 <0.05 < 0.05

790 Prokaryotic Activation of Beta-Catenin Signaling Jun Sun, Andrew S. Neish, Andrew T. Gewirtz, James L. Madara, Atlanta, GA Beta-catenin is a mutilfunctional protein involved in a variety of cellular processes including intercellular signaling and control of cell proliferation and is involved in colonic oncogenesis. We have shown that non-pathogenic salmonella, S. typhimurium PhoPc block the NF-kappa B pathway by inhibition of ubiquitination (Science.289:1560-3, 2000). Beta-cateninis regulated by ubiquitination and the ubiquitin ligase which ubiquitinates NF-kappaalso selectively ubiquitinates beta-catenin. Thus we hypothesized that such bacterial epithelial interactions would also influence beta-catenin signaling of epithelia. We utilized human epithelial Hela cells and T84 cells to analyzewhether this bacterium might activate the beta-catenin pathway. Treatment of cells with the proteasome inhibitor MG262 resulted in the appearance and accumulation of multi-ubiquitinated beta-catenin over 3-6 hours. Precolonization of ceils with PhoPc eliminated the appearance of the ubiquitinated forms, while colonization with wild type did not. Western blot analysis indicates that expression of beta-catenin protein decreases in cells colonized with pathogenic Salmonella typhimurium in a time-dependent fashion. In contrast, beta-catenin decreased over 6 hours, and then increased in cells colonized over 18 hours with non-pathogens PhoPc. Beta-catenintranslocation to the nucleus was detected by immunofluorescence staining in cells colonized with PhoPc for 18 hours. A nuclear complex of betacatenin acting as a coactivtor with lymphoid enhancerfactor/T cell factor (Lef/Tcf) transcription

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factors stimulates transcription of a variety of target genes. Transcription activation mediated by this complex was assayed by beta-catenin/Tcf response gone in PhoPc colonized cells, but not in wild type. Western blot analysis indicated that C-myc, one of the specific targets of beta-catenin/Tcf regulated transcription, increased after 18 hours of pre-colonization with non-virulent Salmonella. These suggested that bacterial epithelial interactions influence betacatenin signaling and may be involved in control of colonic cell growth and perhaps oncogenesis

791 The Role of Rho GTP-binding Proteins in Invasion of Biliary Epithelia by Cryptosporidium Parvum Xian-Ming Chen, Sing Q. Huang, Jason M. Vollenweider, Patrick L. Splinter, Mark A. McNiven, Nicholas F. Larrusso, Rochester, MN Cryptosporidium parvurn (CP) opportunistically infects intestinal and biliary epithelia causing considerable worldwide morbidity, especially in patients with AIDS. We previously demonstrated using an in vitro model of CP infection of cultured human biliary epithelial cells that CP activates C-src, a membrane-associatedtyrosine-kinase, and induces actin rearrangement at the epithelial cell-parasite interface. Since recent studies in other systems havedemonstrated that Rho GTP-binding proteins (e.g., RhoA, cdc42 and Rac) are involved in actin reorganization, we tested the HYPOTHESISthat C. parvum can activate Rho GTP-binding proteins via membrane-associated kinases to induce actin rearrangement and thus facilitate the organism's invasion of biliary epithelia. METHODS: Bile duct epithelial cells (i.e., cholangiocytes) derived from normal human liver and immortalized by SV40 transformation were exposed to freshly excysted CP sporozoites, and activation of Rho GTP-binding proteins and associated tyrosine kinases assessed by molecular and morphological approaches. RESULTS: By immunofluorescent microscopy, an increase of phosphorylated proteins was found in cholangiocytes directly adjacent to the invading CP organism. More specifically, RhoA and cdc42, but not Rac, accumulated in cholangiocytes at the parasite-host cell interface as assessed by immunofluorescent staining. By immunoelectron microscopy, RhoA and cdc42 associated gold particles decorated the parasitophorous vacuole and the dense-band,two structures consistently seen in chnlangiocytes at attachment sites of the organism. Transfeetion of cholangiocytes with a kinase inactive dominant negative mutant of C-src had no effect on CP-induced RhoA and cdc42 accumulation. In contrast, wortmannin and LY294002, two specific inhibitors of phosphatidylinositol 3-kinase (PI-3), a different membrane-associatedkinase, completely blocked cdc42, but not RhoA, accumulation at the parasite-host cell interface. Inhibition of PI-3 by wortmannin or LY294002 also decreased actin accumulation at the attachment site and decreased CP invasion of cholangiocytes by up to 60%. CONCLUSIONS: Our current and previous data suggest that CP attachment to biliary epithelia activates at least two host cell membrane associated kinases, C-src and PI-3, both of which are required for CP-induced actin reorganization and CP invasion.

792 Regulation of a Novel Colon-Specific Gene by Commensal Bacteria and Ligands for the PPAR? Nuclear Hormone Receptor Mei-Lun Wang, Weimian He, Claire M. Steppan, Elizabeth J. Brown, Han-Qing Jiang, John Cebra, Mitchell A. Lazar, Gary D. Wu, Philadelphia, PA It has long been hypothesizedthat colon-specific epithelial gene expression may be influenced by exogenousfactors such as normal enteric bacteria. We have recently identified an intestinal gone, RELM-13(also known as FIZZ2), that is related to an adipocyte-specific secreted protein called resistin. RELM-[~ is restricted primarily to the colon where it is expressed by the undifferentiated proliferating colonic epithelium. Immunohistochemistry shows that this protein is expressed in goblet cells located primarily in the distal half of the colon with trace levels detectable in the cecum and proximal colon. Remarkably, whereas resistin is secreted into the circulation, RELM-~ is secreted into the intestinal lumen. High levels of RELM-~ can be detected in the stool of both mice and humans where it exists as a homodimer under nonreducing conditions. Interestingly, the expression of RELM-{3 is dramatically reduced in germ-free mice where its expression is absent in the distal half of the colon. Endogenous RELM-I3 expression can be enhanced by TZD ligands for the nuclear receptor PPAR-'y in the LS174T colon cancer cell line, in vitro, and in the cecum and proximal colon of germ-free mice, in vivo. In addition, colonization of germ-free animals with a defined microflora induces RELM-13expression specifically in the cecum. Transfection studies using intestinal cell lines were used to identity transcriptional elements important in the regulation of this gone: RELMI~ was expressed specifically in the LS174T celt line whereas no expression was observed in multiple other cell lines. Primer extension analysis identified a single start of transcription in both normal mouse colon as well as in LS174T cells. Deletional analysis of the RELM-13 promoter using a luciferase reporter gone showed that a region located between -870 and 418 bp from the transcriptional start site is required for high level activation in this cell line. These studies provide the first direct evidence that gene expression by the colonic epithelium can be regulated by colonization with normal enteric bacteria as well as ligands for PPAR-%

793 Role of EHEC G157:H7 Virulence Factors and Epithelial Cell Signaling PathwaYsin the Upregulation of the Chemokine IL-8 M C. Berin, Arlette Darfeuille-Michaud, Laurence Egan, Martin F. Kagnoff, La Julia, CA EnterohemorrhagicEscherichia coil (EHEC)0157:H7 is a non-invasive, but epithelial adherent, enteropathogenthat causes diarrhea, hemorrhagic colitis, and can lead to the hemolytic uremic syndrome. The aim of this study was to define the role of EHECvirulence factors and signaling pathways in the upregulated expression by intestinal epithelial cells (lEG) of the neutrophil chemoattractant IL-8. Methods: Caco-2 or HCA-7 human IEC were infected with (a) wild-type (WT) EHECstrain 88-24 that expresses shiga-like toxin (Stx)-2; (b) an isogonic eae mutant deficient in intimin, which is required for intimate bacterial attachment and formation of attaching and effacing lesions (eae-); or (c) an isogenic mutant lacking Stx (stx-). IL-8 mRNA levels and protein secretion were determined by real-time PCR and ELISA. NF-KB activation

AGA Abstracts

was assessed by EMSA. IKB degradation and phosphorylation of p38 and ERK MAP kinases were determined by immunoblotting. Results: Upregulated expression of IL-8 mRNA and protein was similar in magnitude in IEC regardless of whether infection was with W-I EHEC or the stx- or eae- isogenic mutants. Moreover, WT EHEC and the eae- and stx- mutants each induced phosphorylation of the MAP kinases p38 and ERK and increased NF-KB binding activity. Inhibition of signaling through either p38 or ERK, or inhibition of NF-KB activation, abrogated EHEC-induced IL-8 secretion. However, inhibition of p38 or ERK did not inhibit EHEO-induced NF-KB activation. WT and stx- EHECreleased a soluble factor that resulted in MAP kinase phosphorylation, NF-KB activation, and IL-8 secretion from IEC, and this factor was demonstrated to be H7 flagellin. Moreover, addition of either H7 flagellin or EHECto the apical side of polarized IEC stimulated basolateral IL-8 release. Conclusion: EHEG infection of human IEC activates MAP kinase and NF-KB signaling pathways that culiminate in IL-8 secretion. Further, activation of these signaling pathways does not require the EHECvirulence factors Stx or intimin, whereas released H7 flagetlin plays a key role in the upregulation of IL-8 by lEG.

794 Retrograde Transport of Cholera Toxin into the ER of Host Epithelial Cells Yukako Fujinaga, Chiara Rodighiero, Anne A. Wolf, Larry Allen, Randall Holmes, Wayne 1. Lencer, Boston, MA; Denver, CO Cholera toxin (CT) exploits fundamental mechanisms of membrane dynamics for entry into intestinal epithelial cells. To induce disease, the ABs-subunit CT binds ganglioside GM1 on the apical membrane, enters the cell by endocytosis, and then moves retrograde through the Golgi into the ER where the enzymatic A-subunit unfolds and dislocates to the cytosol to induce toxicity. We propose that sorting into this pathway depends on the lipid-based membrane receptor GM1 and association with lipid rafts, a function of the CT B-subunit. To test this idea, we prepared a CT variant (CT-GS) with C-terminal extension of the B-subunit containing consensus N-glycosylation and tyrosine sulfation sites that report toxin entry into Golgi and ER. At 37°C, CT-GS was sulfated and N-glycosylated by intestinal T84 cells in a time course that corresponded closely to that of toxin action, assessed as cAMP-dependent CI- secretion. Both CT function and glycosylation of the B-subunit were completely inhibited at 4°C, by treatment with brefeldin A, and partially inhibited by depletion of T84 cells of cholesterol. Endo-H resistance was observed after 3 h providing evidence that the CT Bsubunif recycles slowly to medial Golgi after entry into the ER. The A-subunit was isolated in double-pull down experiments using concanavalin A-beads to precipitate glycosylated proteins, and then GMl-beads to pull down the CT B-subunit. Thus, CT can move retrograde into the ER as a fully folded protein. Mutation of the ER targeting motif KDEL on the CT Asubunit had little effect on toxin entry into ER as assessed in this assay. CT-B alone was sufficient for transport from the cell surface to the ER. To demonstrate specificity for GM1 in toxin trafficking, we utilized the closely related E. colitype II toxin LTIIb. LTIIb binds GDla and not GM1, does not associate with lipid rafts, and does not induce toxicity in T84 cells. LTIIb containing an identical C-terminal extension of the B-subunit entered T84 cells by endocytosis but did not traffic into Golgi or ER as assessed by the lack of sulfation or glycosylation, in contrast, both CT-GS and LTIIb-GS entered the ER of Veto cells. Both toxins fractionated w~th lipid rafts in this cell type and induced toxicity. Thus, binding of the CT Bsubunit to the membrane lipid GM1 provides a sorting motif that is specific and fully sufficient for retrograde trafficking into the biosynthetic pathway. Such sorting by GM1 likely depends on association with lipid rafts.

795 Clostridium difficile Toxin A Binds to a Receptor in Membrane Lipid Rafts and Induces ERK 1/2 Phosphorylation Ming Chert, Charalabos Pothoulakis, J Lamont, Boston, MA

Background: C. difficile toxin A is a major virulence factor for antibiotic-associated diarrhea and colitis. Sequenceanalysis reveals tl~at toxin A belongs to the clostridial and streptococcal family of ligand-binding toxins characterized by targe numbers of otigopeptide repeats in the C-terminus. The N-terminal portion of toxin A contains glucosyltransferase activity with target specificity toward the Rho family of GTPases while the C-terminus consists of 30 repetitive subunits that binds to the carbohydrate groups on the receptor(s). Cholera toxin binds to its receptor (GM1) in the detergent-insoluble membrane microdomains and enters cells via a lipid raft-dependent endocytosis. In this study, we examined whether toxin A enters host cells also via a lipid raft-dependent mechanism in a model colonic epithelial cell line (T84 cells). Methods: C-terminal peptides of toxin A containing either all 30 oligopeptide repeats (Cl: aa 1822-2710) or the first 10 repeats (CIl: aa 1822-2192) were cloned and expressed as GSTfusion proteins. T84 cells were exposed to either biotinylated toxin A, CII, or cholera toxin B subunit at 4°C for 30 rain. Cells were collected in buffer containing 1% Triton X-IO0 and subjected to sucrose equilibrium density centrifugation. Fractions containing equal amount of protein were analyzed by Western blot and probed with streptavidin conjugated with horseradish peroxidase.Erk 1/2 phosphorylation was detectedwith antibody against phosphorylated Erk 1/2 (Thr202/Tyr204). Results: Biotinylated toxin A, CII and cholera toxin B subunit were found in the Triton X lO0-insoluble lipid rafts localized at the 5% and 30% sucrose interface. Pretreating cells with filipin (a cholesterol binding reagent) or incubating TB4 cells at 37oCfor 1 hr abolished toxin A binding to lipid rafts. Filipin also blocked phosphorylation of Erk 1/2 in T84 cells exposed to either toxin A, GI or CII. Conclusions: (1) C. difficiletoxin A binds to receptor(s) located in lipid rafts leading to Erk 1/2 activation. (2) The first 10 oligopeptide repeats of toxin A C-terminus is sufficient to confer binding of holotoxin to its receptor. (3) Erk 1/2 activation by toxin A is independentof Rho glucosylation and is triggered by binding of toxin to its receptor. (Supported by the National Institute of Health grants DK34583)

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796 Artificial Neural Networks Distinguish Inflammatory Bowel Disease-Derived from Sporadic Colorectal Neoplastic Lesions Florin M. Selaru, Thomas C. Liu, Yan Xu, Jing Yin, Yuriko Mori, Fumiaki Sato, Kellie Perry, Andreea Olaru, Suna Wang, Tong-Tong Zou, Martha C. Kimos, Kena K. Oesai, Anatoly Leytin, David Shibata, John Abraham, Noam Harpaz, Stephen J. Meltzer, Baltimore, MD; New York, NY Study's purpose. Using current diagnostic methods, it is difficult to distinguish between neoplastic colorectal lesions arising in inflammatory bowel disease (IBDNs) and adenomas or cancers occurring in the sporadic setting (SAGs). The current study applied artificial neural networks (ANNs) to distinguish SACs from IBDNs based on global gene expression profiles. Methods. We hybridized cDNA microarrays, each containing 8,064 cDNA clones, to RNAs derived from 39 colorectal neoplastic specimens. Hierarchical clustering was performed, and an ANN was constructed, trained, and tested. The software program GeneFinderwas used to identity genes most relevantto the distinction betweenSACsand IBONs. Results. Hierarchical clustering and ANNs were used to analyze data from all 8,064 clones on the microarray. Clustering failed to correctly categorize the SACs and IBDNs. However, after being trained on a set of 5 IBDNs and 22 SACs, the ANN correctly diagnosed 12 of 12 blinded samples in a test set (3 IBDNs and 9 SACs). Next, using an iterative process based on GeneFinder,Cluster and MATLAB, the number of clones neededfor diagnosis was reduced from 8064 to 97. With this focused clone set, the ANN retained its capacity for correct diagnosis. Moreover, cluster analysis performed with these 97 clones now distinguished between the two types of lesion. Conclusions. Our results suggest that ANNs have the power to discriminate among subtly different clinical entities, such as IBDNs and SACs. Moreover, when combined with gene discovery software, ANNs help to identity genes important in making these diagnostic distinctions.

797 Significance of the Wnt-Palhway for Cancer Risk in Inflammatory Bowel Disease: Association of a Genetic Polymorphismin Human disheveled I with Dysplasia in Ulcerative Colitis Sonja M. S. Uthoff, Deidre Hilliard, Maurice Eichenberger, Hans-Peter Bruch, Susan Galandiuk, 23538 Luebeck, Germany; Louisville, KY OBJECTIVE: We wished to identity candidate genes in Crohn's disease and ulcerative colitis (UC) that could predict cancer risk in inflammatory bowel disease (IBD). We hypothesize that a selective polymorphism of the human disheveled 1 gene (OVL1), which is crucial in the wingless-type/beta-catenin signaling cascade, is associated with dyspiasia in UC. MATERIAL AND METHODS: Genomic DNA was obtained from peripheral blood leukocytes of patients with Crohn's disease, patients with either dysplastic or non-dysplastic UC and patients with sporadic colon cancer. Intron 1 of OVL1 was amplified and targeted for single nucleotide polymorphism (SNP) analysis. Temperature-modulatedheteroduplexchromatography (TMHC) was performed using partiafly denaturing high performance liquid chromatography (dHPLC) with resolution of the corresponding homoduplexes by ion-pair reversed phase fiquid chromatography, followed by dye-terminator sequencing. RESULTS: A heterozygous polymorphism (mutantJwild-type alleles *M/*W) within intron 1 of OVL1 was significantly over-represented among UC patients (66/101,65.3%), when compared with Crohn's disease (62/125, 49.6%), or population control subjects (36/53, 40.4%). Stratification by the presence or absence of dysplasia showed a stronger association of the genotype OVLI*M/*W with dysplastic UC (70.6%) than non-dysplastic UC (54.4%). The frequency of DVLI*M/*W in patients with sporadic colorectal cancer was lower (18/53, 34.0%) and concurred with that of the population controls. CONCLUSION: Our findings suggest that polymorphic genotypes within intronic regions of the OVL1 gene impact on individual susceptibility to colorectal cancer among IBD patients. A OVL1*M mutator allele in particular appears to differentially identify the dysplastic UC phenotype as opposed to non-dysplastic UC and Crohn's disease.

798 Unifocal Fiat Low-Grade Dysplasia (LGD) Carries Substantial Risk of Progressionto Advanced Neopiasia in Patients with Ulcerative Colitis (UC) Thomas Ullman, Victoria Croog, Noam Harpaz, Steven Itzkowitz, New York, NY Introduction: Flat LGD in UC often progressesto more advancedneoplasia, including colorectal cancer (CRC). Factors that predict progression are poorly defined. Multifocal LGD (mLGD) is commonly believedto confer high risk, whereas unifcoal LGD (uLGD) is thought by some to be a less ominous finding, allowing for continued cotonoscopic surveiflance instead of colectomy. However, this has never been formally tested. Aim: to determine whether patients with uLGD would progress to advanced neoplasia at a rate slower than those with mLGD. Methods: All patients from Jan 1994 to Oct 2001 with the coexistence of flat LGD and UC were identified by querying the Department of Pathology database. Patients with raised or polypoid dysplasia were excluded. The medical, colonoscopy, surgical, and pathology reports of all identified patients were reviewed, mLGD was defined as any case in which there were 2 or more loci of flat LGD. Progression was defined as any subsequent finding of high-grade dysplasia (HGD) or CRCat colonoscopy or colectomy. Patientswere censoredfor colectomy or last co~onoscopy without progression. Groups were analyzed by life-table methods. Results: We identified 53 patients with flat LGD. Average age was 54 years, and average duration of disease was 22 years. 36 had pan-colitis (extending proximal to the splenic flexure); the remaining had limited diseaseor unspecified extent. The median follow-up was 20 months. 14 patients demonstrated progression, with 9 proceeding to HGO and 5 going on to CRC; the mean time to advance was 28 months. Five-year rates of progression are described in the table. There was no difference in rate of progression for uLGD as compared to mLGD (p = 0.44 by log-rank test). With adjustment for potential confounders (disease duration, extent of colitis, and presence of PSC) in a multivariable model, there was still no difference (p=0.43). Conclusions: 1. Focality of dysplasia did not predict neoplastic progression in flat LGD. 2. Strong consideration should be given to colectomy for all patients with LGD, even those with uLGD, to reduce the risk of subsequent GRC.

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Five Year Progressionto Advanced Neoplasia 5-year rate Total 46% uLGD 51% mLGD 33%

+/- 95% CI +/- 24% ÷/- 28% +/- 41%

799 Microclonal Evolution and Chromosomal Instability within Colonic Epithelium David Crispin, Shawna Dziadon, Mary Bronner, Mary Emond, Teresa Brentnall, Peter Rabinovitch, Seattle, WA We have previously reported that Ulcerative Colitis (UC) patients with dysplasia or cancer have widespread chromosomal instability (GIN) that occurs even in the histologically normal appearing epithelium of their colons. Using fluorescence in situ hybridization (FISH)we sought to determine if this instability represented the outgrowth of clonal populations, and whether abnormalities at one locus made it more likely that an abnormality would be detected at another locus. To do this we examined the spatial relationship and range of abnormalities between individual cells within a crypt. METHODS:5 single crypts from histologically negative (2 crypts) and indefinite (3 crypts) sites in 2 patients were isolated by microdissection from biopsies obtained at colectomy. Individual crypts were fixed on glass slides then probed by FISH with probes at 17p13.1 (p53), 17 centromere, 11q13, and 11 centromere. For determination of spatial clustering, up to 600 cells were scored with two probes per crypt. Overall, more than 3000 nuclei were evaluated. RESULTS: Within single crypts, at least 10 categories of FISH abnormality (combinations of arm and centromere gains and losses) can be seen in cells, ranging in frequency from 1-10%. Furthermore, cells with particular FISH abnormalities were widely dispersed throughout the crypt with only small clusters of 2-4 cells with the same FISH abnormality. Cells with an abnormality on one chromosome (e.g. 17) were three times more likely to be abnormal at the other chromosome (11) (p=.OO01). DISCUSSION: If the chromosomal abnormalities seen in UC originate in stem cells, there would have to be a large number of such stem cells (50-100) in each crypt to account for the variability. Alternatively, chromosomal abnormalities may he acquired in daughter cells as they migrate up through the proliferative zone. In either case, CIN in UC can be present well before genetically abnormal cells are clonally expanded. 8OO Rate of Advanced Neoplasia at Colectomy for Flat Low-Grade Dysplasia (LGD) in Ulcerative Colitis (UC) Victoria Croog, Steven Itzkowitz, Noam Harpaz, Thomas UIIman, New York, NY Background: Little is known about the rate of synchronous colorectal cancer (CRC) in patients with UC found to have flat LGD at surveillance colonoscopy. Aim: to determine the rate of CRC at colectomy in patients who underwent surgery for the indication of LGD. Methods: All patients with UC and an endoscopic diagnosis of LGO from Jan 1994 and Oct 2001 were identified by review of Department of Pathology records. Those patients who subsequently went to surgery because of a diagnosis of flat LGD were selected for further analysis. The assignment of flat LGD was determined based on the reported findings of both the endoscopist and the pathologist. Patients with LGD who developed high-grade dysplasia (HGD) or CRC prior to surgery were excluded, as were patients with raised LGD. Findings at surgery were categorized based on worst grade of pathology, CRC > HGD > LGD > no dysplasia. Advanced neoplasia was defined as HGD or CRC. Surgery within 4 months of the initial finding of LGO was called "early," and any cancers found in this group were considered synchronous; surgery after 4 months was called "delayed." Results: 22 patients with UC who went to surgery for flat LGD were identified. Duration of colitis at the time of diagnosis of flat LGD ranged from 5 to 33 years. Time from initial diagnosis of flat LGD to surgery ranged from less than one month to 75 months with a median of 4 months. Pathology review of colectomy specimens identified 8 advanced neoplasms (2 CRC, 6 HGD). Of the remaining 14 patients, LGD was found in 13 (59%), and no dysplasia in one (4%). 11 patients underwent early colectomy, at which time 2 (18%) advanced neoplasms were detected (both HGD, no CRC). Of the 11 patients who underwent delayed colectomy, 6 (55%) had advanced pathology (2 CRC, 4 HGD). The table summarizes these findings. Conclusion: Advanced neoplasia in patients with UC who go to early colectomy for flat LGD is common. Furthermore, when surgery is delayed by four months or more, there is a greater risk of finding an advanced lesion at colectomy even in the absence of colonoscopic progression. This supports the practice of early intervention when flat LGO is found in the setting of UC. Pathologyat Colectomy Time to Surgery Early (n=t 1) Delayed(n=11)

Advanced CRC HGD O(0%} 2(18%) 2 (18%) 4 (36%)

No Advance LGD No D 8 (73%) 1 (9%) 5 (45%) 0 (0%)

801 Increased Risk of Malignancies in Patients with Ulcerative Colitis and Primary Sclerosing Cholangitis. A CameControl Study Alicia Sambuelli, Ruben Terg, Ema Coronel, Negreira M. Silvia, Anibal Gil, Silvina Blanco, Laura Moreno, Sergio Huernos, Zulema Kogan, Ana Cabanne, Ivan Doldan, Julio C. Bai, Buenos Aires, Argentina

were prospectively and systematically studied for presence of PSC according to conventional criteria. During this period, 32 patients fulfilled the criteria fur this association. The control population (n =64) was randomly selected from classical UC patients and consisted in two controls for each patient matched for sex, age, age at onset of UC and extension of the colonic compromise. Patients and controls underwent annual survey colonoscopies with multiple biopsies. The primary end point was the diagnosis of dysplasia or cancer. Secondat7 end points were the severity of thee clinical course (requirement o1surgery, use of intravenous steroids therapies, etc.) and diagnosis of other malignancies. RESULTS: During the followup, nine patients with UC and PSC (28%) and only one control (1.5%) developed malignancies (odds ratio: 24.6; 95% CI 2.9 to 205.4; p
803 RACK1, a Novel Src Substrate and Inhibitor of Src Tyrosine Kinase Activity and Cell Growth Betty Y. Chang, Rachel A. Harte, Christine A. Cartwright, Stanford, CA The specific activity of the Src tyrosine kinase is elevated in human colon carcinoma cells, and decreases as intestinal crypt cells differentiate. Thus, downregulation of Src activity appears to be important for differentiation~ and upregulation for growth and transformation of intestinal cells. To isolate proteins that interact with Src and potentially regulate its activity in intestinal cells, we used a yeast two-hybrid assay and identified RACK1 (a known PKCinteracting protein and a homolog of the [beta] subunit of G proteins) as a novel Src-SH2 binding protein. We found that RACK1 is an inhibitor of Src activity and cell growth. We showed that PKC activation induces the intracellular movement and co-localization of RACK1 and Src, and the tyrosine phoshorylation of RACK1. To determine whether RACK1 is a Src suhstrate, we recently assessed phosphorylation of RACK1 by various tyrosine kinases in vitro, and by kinase-active and inactive mutants of Src in vivo. We found that RACK1 is an in vitro substrate for Src and not for other tyrosine kinasestested. RACK1is also an endogenous substrate for Src. Moreover, Src activity is necessary for both the tyrosine phosphorylation of RACK1 and the binding of RACK1 to the SH2 domain of Src that occur following PKC activation. To identify the tyrosine(s) on RACK1 that is phosphorylated by Src, we generated and tested a series of RACK1 mutants. We found that Src phosphorylates RACK1 on Tyr 228 and/or Tyr 246, highly-conserved tyrosines located in the sixth WD repeat that are known to interact with the SH2 domain of Src. We think that RACK1 is an important Src substrate that signals downstream of growth factor receptor tyrosine kinases and is involved in the regulation of Src activity and intestinal cell growth. Understanding how inhibitors of mitogenic signals work to regulate intestinal cell growth and how loss of that inhibition results in uncontrolled cell growth and malignant transformation, could lead to development of novel strategies for colon cancer therapy.

BACKGROUND/AIMS: Primary sclerosing cholangitis (PSC) has been reported in some but not all studies to be a risk factor for the development of colorectal cancer in patients with ulcerative colitis (UC). It seems very possible that conflicting results might rise from potential bias in design of studies, if this increased risk is confirmed, implications in terms of screening of patients would be concrete. The aim of this study was to determine prospectively the natural history of a series consecutive of patients with the association of UC and PSC. MATERIAL & METHODS:From 1990 to 2600, 1044 consecutive patients diagnosed with UC

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804 Gene Hypermethylation Is a Major Determinant ot PhenotypicExpression in Colorectal Cancer Mark Norrie, Kay Cheong, Su Ku, Elisa Mokany, Alison Todd, Meagher Alan, Terrence O'Connor, Nicholas Hawkins, Robyn Ward, Sydney, Australia Introduction: There is increasing evidence that DNA CpG island hypermethylation is common in colorectal cancer and associated with tumor pathogenesis and phenotypic expression. Hypermethylation occurs not only at the promoter regions of known genes in association with transcriptional silencing, but also at intronic loci methylated only in tumors (MINT sites). There is evidence that hypermethylation of multiple genes is associated with a phenotypically distinct subgroup of tumors (CIMP phenotype). Thereforethe aim of this thesis was to examine the significance of gene methylation in colorectal cancer in relation to tumor pathogenesisand outcome. Therefore a series of 428 tumors were analyzed for hypermethylation of the genes hMLH1, p161NK4a, CDH1 and known MINT sites 1,2,12, 25 and 31. These results were correlated with other genetic markers, histological parameters, clinical data and gene expression. Method: Mutations in codon 12 of the K-ras gene were determined using REMSPCR. Microsatelllte status was delermined using the five NCI markers. All DNA samples for methylation analysis underwent prior bisulphite modification. The methylation status of the promoter regions of the hMLH1, p161NK4aand CDH1 genes was analysed using Methylation Specific PCR. Methylation of MINT sites was analysed by Combined Bisulphite Restriction Analysis. The expression of the hMLH1, p16, and p53 proteins was determined by immunohistochemistry on tissue paraffin sections. Results: Loss of gene expression correlated with promoter methylation for both hMLH1 and p161NK4a. Cell line and tumor data revealed a striking phenotypic pattern in association with gene methyiation. With the exception of MINT 25 and CDH1,methylation was strongly associatedwith rnicrosatellite instability (MSI), methylation at other loci, older age, females, right-sided tumors, wild-type p53 expression and poorly differentiated, mucinous tumors with intraepithelial lymphocytes (p< 0.05). MINT 25 was associated with microsatelllte stable tumors (p
8O5 The E3-Uhiquitin-Ligase HectH7 Activates CDX2 Transcriptional Activity Martina Heckel, Sylvia Heink, Sebastian Thaler, Martin Scheffner, Joachim MOssfler, Karel Caca, 04103 Leipzig, Germany; Leipzig, Germany; KGIn, Germany CDX2, a caudal-related homeobox gene is involved in enterocyte proliferation and differentiation. However, little is known about the intracellular signaling pathways that regulate CDX2 transcriptional activity. HectH7, a member of the Hect family of E3 Ubiquitin ligases involved in the proteasome mediated protein degradation pathway, was formerly identified as a CDX2 interacting protein in a Yeast-2-Hybrid-Screen. To further delineate the functional consequences of the CDX2-HectH7 interaction we investigated HectH7 ubiquitin-ligase activity, cellular localization and influence on CDX2 transcriptional activity. HectH7 ubiqultin-ligase activity was investigated by using thioester complex forming and ubiquitin binding assays, utilizing different E2's. A polyclonal rabbit HectH7-antibody was raised and immuno-purified by use of HectH7-GSTand HectH7-Hisfusion proteins. The cellular localization of HectH7 and co-localization with CDX2was examined by immunocytochemistry and confocal microscopy of overexpressed HectH7 and Cdx2 in the colorectal cancer cell lines Caco-2 and HCT116. The functional effects ol HectH7 on CDX2 transcriptional activity were tested using luciferase reporter assays with a CDX2 responsive SIF1-Reporter construct in Hela and DLD1 cancer cell lines. Thioester complex forming assays proved the E3-1igaseactivity of HectH7 with preferential utilization of UbcH7 as E2. Using in vitro translated HectH7 monoubiqultinated 35S-CDX2 adducts could be detected. In Caco-2 and HCT116, HectH7 showed a preferential nuclear localization and colocalization with CDX2. HectH7 increased CDX2 transcriptional activity on a SIF1 luciferase reporter construct in a dose-dependent manner. A site-directed mutation of the HectH7 ubiquitin-binding site abrogatedthe effect of HectH7on COX2transcriptional activity. Our results prove the ubiquitin ligase activity of HectH7 and support the interaction of HectH7-CDX2 in the nucleus. However, HectH7-CDX2 interaction may not preferentially lead to CDX2 degradation, but instead to an increase of the transcriptional activity of CDX2. This effect may be due to functional activation of CDX2 by ubiquitination or polyubiqultination and degradation of a CDX2 repressor.

807 Telomerase Induces Immortalization of Normal Human Esophageal Cells without Inactivation of the p16 Tumor SuppressorGene Hideki Harada, Hiroshi Nakagawa,Takaaki Mizushima, Kenji Oyama, Claudia Andl, Oliver Opitz, Anil Rustgi, Philadelphia, PA introduction: Normal human somatic cells have a finite life span and undergo replicative senescence after a limited number of cell divisions due to telomere shortening with each division. Senescenceis believedto be a mechanism to act as a checkpoint against tumorigenesis. Maintenanceof the telomere length is therefore required for immortalization and transformation of human cells. It has been reported that more than 80% of esophageal squamous cell carcinoma (ESCC) express telomerase. Expression and activation of telomerase might play a critical role in the development of ESCC. To clarify the contribution of telomerase in ESCC, we have transduced normal human esophageal keratinocytes (EPC2) with telomerase (hTERT). Methods: EPC2 cells are grown in Keratinocyte-SFM(GIBCO/BRL)until undergoing senescence at 42-44 population doublings (PDs), as confirmed by senescence associated beta-galactosidasestaining. PresenescentEPC2cells at 42 PDs were retrovirally infected with either pBABE vector expressing hygromycin resistance gene accompanied by hTERT (EPC2hTERT) or the vector alone (EPC2-hyg). Telomerase activity in EPC2-hTERTwas measured by the TRAP assay and the telomere length elongation was assayed by Southern blotting. Results: Whereas EPC2-hyg cells underwent senescence at 44 PDs, similar to the parental EPC2 cells, EPC2-hTERT overcame senescence with continued growth beyond 130 PUs, consistent with immortalization. It has been reported that p16 inactivation is required to immortalize human skin keratinocytes. Our western blotting showed, however, that the expression of p16 gradually increased in EPC2 and EPC-hyg and remained constant even in late PDs of EPC2-hTERT. DNA sequencing showed that the p53 tumor suppressor gene was not mutated in EPC2, EPC2-hyg and late PDs EPC2-hTERT. Moreover, late PDs of EPC2-hTERT had a normal karyotype with chromosome 20 trisomy in a small fraction of cells, whereas early PDs EPC2-hTERT had a normal karyotype. Interestingly, EPC2-hTERT showed rapid growth immediately after transduction with telomerase as supported by morphologic changes Conclusion: Taken together, telomerase can immortalize human esophageal keratinocytes and may be involved in cell growth, however, other genetic alterations are required for transformation. 808

TYPE 1 Anti-Neuronal Nuclear Antibodies (ANNA-l) Evoke Neuronal Apoptosisvia APAF-1 and Caspase-3 Activation in SH-SY5Y Cells. Roberto De Giorgio, Monica Bovara, Marco Canossa, Fabrizio De Ponti, Giovanni Barbara, Vincenzo Stanghellini, Marcello Tonini, Cesare Cremon, SiMa Allaria, Eduardo NobileOrazio, Rosanna Cogliandro, Roberto Corinaldesi, Bologna, Italy; Pavia, Italy; Milan, italy ANNA-1 are identified in pts with gut dysmotility in the context of a paraneoplasticsyndrome. Preliminary evidence suggests that ANNA-1 may impair enteric neuronal function, which provides the basis for occurrence of gastrointestinal motility disorders. To generate new insights into the mechanisms through which paraneoplastic anti-neuronal autoantibodies evoke neuronal damage, we evaluatedthe effects exerted by ANNA-1 in a neuroblastoma cell line. Our aims were to: 1) verify the cellular site of ANNA-1 binding; 2) perform a quantitative analysis of ANNA-l-induced neuronal apoptosis; 3) test whether ANNA-1 activate Apaf-1 and Caspase-3as mltochondria-dependentapoptotic pathway. METHODS:ANNA-1 were charaCterized by immunofluorescence and western blot from sera of four pts (2F, 2M; age range: 5564 yrs) with small cell lung carcinoma and a related paraneoplastic syndrome characterized by central nervous system disorders and severe gut dysmotility. Sera from blood donors served as controls. The neuroblastoma cell line SH-SY5Y was cultured in Dulhecco-modified Eagle medium containing 15% fetal calf serum (DMEM). Neurublasts exposed to DMEM containing 15% ANNA-1 or control sera for 12 and 48 hours were processedfor: a) immunofluorescence with anti-human fluorescein-conjugated IgG; b) TUNEL technique; c) Apaf-1 and Caspase-3immunocytochemistry using specific polyclonal antibodies. Ouantitativeassessment was performed by counting the number of TUNEL+ SH-SY5Y cell nuclei expressed as a percentage of positive cells/high magnification field compared to either control or dopamine (0.2 raM, used as a proapoptotic agent). RESULTS: After incubation of SH-SY5Y cells with ANNA-1 for 12 and 48 hrs, anti-human IgG showed intense immunolabeling around nuclei and, to a lower extent, in the cytoplasm of neuroblasts. A significantly higher percentage of SH-SY5Y cells exposed to ANNA-1 for 48 hrs showed TUNEL+ nuclei (34+/-5%)when compared to dopamine (20 +/-4%, p
806 Distinct Cliuicopathological Characteristics of Gastric Cancers of the Microsatellite Mutator Pheootype Kentaro Yamashita, Hiroyuki Yamamoto, Manuel Perucho, La Jotla, CA; Sapporo, Japan Background: Numerous studies have focused on colon cancer in regards to the properties of tumors displaying the microsatellite mutator phenotype (MMP), also known as microsatelllte instability (MSI). These studies have confirmed that colon cancer of the MMP shows distinct clinicopathological and genetic characteristics compared with tumors without the mutator phenotype. In contrast, MMP in gastric cancer has been less elaborated and recent results are still conflicting with previous reports that analyzed relatively small number of cases. Methods: MMP status was basically determined by two mononucleotide (BAT-26, APA3) markers. Frameshift mutations were analyzed in nine target genes (TGFI~RII, BAX, IGFIIR, hMSH3, hMSHG, caspase-5, MBD4, RAD50, and TCF4). The methylation status of mismatch repair gene hMLH1 and p16 suppressor gene was also examined in a subset of tumors. These genetic and epigenetic alterations were contrasted with clinicopathological parameters of these tumors. Results: A total of 446 unselected gastric cancer cases was classified as follows; 59 (13.3%) MMP+ (or MSI-H), 34 (7.8%) MMP+/- (or MSI-L), and 348 (78.9%) MMP- (or MSS). Compared with the MMP- group, MMP+ gastric cancers were associated with older patients (67.8 yrs vs. 62.1, P=O.O01), distal location (P=O.02), histological

AGA Abstracts

differentiation (P = 0.02), earlier TNM stage (P
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809 Colonic Manipulation Induces Remote Inflammatory DysmotiUtyof the Small Intestine via Gut Derived Endotoxin Andreas TOner, Christoph Schnurr, Sandra TOgel, BeverleyA. Moore, Anthony J. Bauer, Pittsburgh, PA Background: Surgical manipulation of the colon initiates inflammatory events within the colonic muscularis that participate in local postoperative dysfunction. The selective colonic manipulation induced inflammation is also associated with a significant delay in intestinal transit. Aims: To determine whether the observed delay in intestinal transit is caused by the downstream barrier of the manipulated colon or by inflammatory events within the small intestinal muscularis itself. And additionally, to investigate the potential role of gut derived endotoxin in the development of postoperative small intestinal dysfunction. Methods: SDRats (180-220g) underwent colonic manipulation. To investigate small intestinal transit independently from the motor function of the manipulated colon, a distal ileal loop ileostomy was performed before manipulation. The gut was decontaminated prior to manipulation using polymyxin B and neomycin. Mediator mRNA expression was determined by real-time RTPCR. Leukocyte extravasation was investigated in muscutaris whole-mounts. In vivo transit of FITC-dextranwas measured using geometric center analysis (GC). In vitro circular muscle contractility was assessed in a standard organ bath. Statistical analysis: unpaired Student t test, p
the contractions to ES but not those to CCh indicating an inhibitory action of these purines _on cholinergic nerve activity (ES 1Hz: inhibited from 51 -+ 5% to 13_+3% by 30 I~M adenosine, n = 8 and from 50 _+4% to 17 -+ 2% by 30 pM ATP, n = 7). The stable ATP-analoguee~methylene-ATP had no effect on the contractions to ES. The inhibitory action of adenosine and ATP on ES-induced contractions was mimicked by the stable adenosine-analoguemethyladennsine (3-30 p,M) and by the A1 receptor agonist CHA (10-100 nM) while it was prevented by the A~ receptor antagonist 8PT (10 pM) but not by the A~ receptor antagonist OMPX (1Hz: from 40-+3% to 38-+2% by 30 pM adenosine plus 10 I~M 8PT, n=6). In chronically inflamed ileum adenosine, methytadenosine, ATP (all 3-30 pM) and CHA (10-100 nM) all failed to inhibit the cholinergic nerve-mediated contractions to ES (for ES 1Hz: from 51-+5% to 48-+4%, n=6 by 30 p.M adenosine and from 52_+6 to 49-+7% by 30 I~M ATP, n =6). The e2 receptor agonist UK14304 (10 nM) inhibited the contractions to ES to a similar extent in control and inflamed ileum. Our results indicate that the A~ receptor-mediated inhibition of cholinergic transmission is disturbed in chronically inflamed ileum while the c~2 receptormediated inhibition is intact. This suggests a disturbed purinergic control of acetylcholine release during granulomatous intestinal inflammation.

812 He]icobacter pylori Infection Is Associated with Reduced nNos Gene Expressionand Defective Gastric Relaxation: Mediation by Interferon and Somatostatin. Ling Wang, II Song, G Reider, Matthew Dimagno; Juanita Merchant, Chung Owyang, Ann Arbor, MI Functional dyspepsia is common among patients with H. pylori (HP) infection. Many show defective gastric accommodation as well, which may play a role in functional dyspepsia. We hypothesizethat chronic infection with HP down regulates nNOS gene expression resulting in reduced gastric relaxation. This is mediated by interferon (IFN~,)which stimulates somatostatin release. Method: We employed C57BL/6 mice (8 wks old) infected with HP (SS1 strain). Gastric muscle strips were studied two months after infection. After precontraction with carbachol (10.6 m) in the presence of guanethidine, muscle responses to electrical field stimulation (EFS) were measured. In control, EFS (1-20 Hz, 80V 2 ms) produced frequency dependent relaxation consisting of a fast phasic relaxation followed by a sustained relaxation. The fast relaxation was abolished by the NOS inhibitor, L-NAME (100 p,M). Relaxation in response to EFS (1-20 Hz) was impaired in gastric muscle obtained from HP infected mice. At 5 Hz we observed a 33.9-+5.4% relaxation vs 73.8-+6.2% in controls, and a reduction of NOS activity (3H-citrullineassay). 3H-citrallineformation in responseto 5Hz was 31.9_+5.7% over basal compared to 71.5_+8.3% in controls. RT-PCR demonstrated that the expression of nNOS, eNOS and iNOS was suppressed by 76%, 64% and 71% with a similar degree of reduction of protein expression of the three NOS isoforms. To identity the isoforms of NOS mediating gastric relaxation, studies were done in nNOS(-), eNOS(-) and iNOS(-) mice. In nNOS(-), muscle strips from control and HP infected mice failed to relax in response to EFS and resembled the responses of wild type treated with L-NAME. No difference was observed between control and HP infected eNOS(-) or iNOS(-) mice. Gastric inflammation due to HP results in an increase of IFN~ expressing T lymphocytes and IFN~t stimulates snmatostatin (5OM) release from isolated D cells. Gastric strips obtained from C57BL/6 mice treated with IFN~, (250/kg delivered via micro-osmotic pump over 2 days) showed a 50% reduction in EFS induced relaxation and a 40% decrease in nNOS gene expression. Pretreatment with the somatostatin antagonist (20p,g/kg) significantly reversed the inhibiting effect of INF,,. Conclusion: HP infection markedly reduced gastric relaxation due to suppressed nNOSexpression and catalytic activity. This is likely to be mediated by enhanced INF.,/activity which stimulates the re!ease of SOM. SOM in turn inhibits the expression Of NOS.

810 Neuro-lmmune Interaction in Manipulated Intestine Triggers an Adrenergic Inhibitory Pathway Maintaining Prolonged Postoperative Ileus Wouter J. De Jonge, Renee M. Van Den Wijngaard, Roel J. Bennink, Sander Van Deventer, Guy E. Boeckxstaens,Amsterdam, Netherlands Background. Post-operative ileus directly following abdominal surgery results from activation of inhibitory neural pathways due to intestinal handling. The prolonged period of hypomntility however, may be mediated by a manipulation-induced inflammatory response in the intestine. We hypothesizedthat these intestinal inflammatory cells are able to activate neural pathways, thus inhibiting motility of the entire Gi-tract. Methods. The effect of laparotomy (L), or L combined with manipulation of the small intestine (L+ IM), on gastric emptying was determined in a murine model of post-operative ileus. After 6 and 24 h, gastric emptying was determined by scintigraphic imaging after oral gavage of a 99Tc labeled semi-liquid meal (1.5 % methylcellulose). After the gastric emptying studies, gastric and ileal tissue was isolated and assayed myeloperoxidase (MPO) activity. In addition, the neuromuscular function of gastric muscle strips was studied in organ baths. Results: L+IM, but not L alone, elicited an increase in MPO activity from 5.3+/-0.9 (6h after surgery), to 13.1 +/-2.9 U/g (24 h after surgery). No increased MPO activity was found in the stomach. The intestinal inflammation was paralleled by a delay in gastric emptying; gastric retention, measured 64 min after garage of the meal (Ret64) was significantly increased (p
813 Interleukin-9 Enhances Intestinal Muscle Contractility during Nematode infection Waliul I. Khan, Hirotada Akiho, Patricia A. Blennerhasset, Hiroshi Kaflbayashi, Melisande Richard, Jacques Van-Snick, Stephen Mi Co!lins, Hamilton, ON, Canada; Brussels, Belgium BACKGROUNDAND AIMS: In previous work, we have shown that infection of mice with the nematode Trichinella spiralis is accompanied by intestinal muscle hypercontractility which contributes to worm expulsion from the gut. We have also shown that the T helper 2 (Th2) cytokines IL-4 and IL-13 are present in the muscularis externa during infection and contribute to these changes via signal transducer and activator of transcription factor 6 (Stat6) dependent process. In contrast, another Th2 cytokine IL-5 contributes little. There is growing interest in another Th2 cytokine, IL-9, which acts on various cell types in a Stat6 independent way. However, its role in muscle function during nematode infection is unknown. In this study we investigated the role of IL-9 on intestinal muscle contractility and worm expulsion during T. spiralis infection. METHODS: C57BL/6 mice were infected with T. spiralis and treated with recombinant IL-9 (100 rig/day; i.p) everyday until sacrificed. Control mice received PBS. Mice were sacrificed at various time points to investigate worm recovery, intestinal muscle contractility, myeloperoxidase (MPO) activity, mouse mast cell protease-1 (MMCP-1), cytokine response and presence of IL-9 receptor in intestinal muscle by immunohistochemistry. RESULTS: IL-9 treatment acceleratedworm expulsion by 46% by day 14 post-infection (p.i). Infection induced intestinal muscle hypercontractility was further enhanced by 140% in IL-9 treated mice. (Carbachol induced muscle contraction was 3527 -+700 rag/ram2 in IL-9 treated mice compared to 1461 _+ 397mg/mm 2 in control on day 14 p.i., p
811 Granulomatous Intestinal Inflammation Disturbs Purinergic Control of Cholinergic Transmission Joris G. De Man, Tom G. Seerden, BenedicteY. De Winter; Arnold G. Herman, Erik A. Van Marck, Paul A. Pelckmans, Antwerp, Belgium Purines are neuroimmune modulators witt~ important physiological and pathological functions. It is not known whether the physiological role of purines in the enteric nervous system is affected by pathological conditions. We therefore studied the effect of chronic intestinal inflammation on the purinergic control of enteric cholinergic transmission. Swiss mice were infected with the parasite Schistosoma mansonL 16 weeks later, the ileum was removed and longitudinal muscle strips of age-matched control and infected mice were prepared and mounted in organ baths. Chronically inflamed ileum showed granulomas, blunted villi, increased wall thickness and increased MPO activity. Electrical stimulation (ES, 0.25-8Hz) and carbachol (CCh, 10-300nM) induced atropine-sensitive cholinergic contractions in control and inflamed ileum. The amplitude of the contractions to ES and CCh was higher in inflamed ileum. However, when the nerve-mediated contractions to ES were expressed as % of the direct smooth muscle contraction to CCh,the amplitude of the contractions to ES was similar in control and inflamed ileum. In control ileum, adenosine and ATP (both 3-30 p.M) inhibited

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HT29-F12, a subclone of HT29 colon cancer cell line, was also performed. (Results) Immunohistochemical analysis revealed that increased expression of ubiquitinylated protein was observed in epithelial cells of active UCpatients compared to normal control subjects. Ubiquitin mRNA and mono-ubiquitin protein expression by RT-PCR and Western blot analysis did not significantly differ from normal control. However increased amount of ubiquitin-protein conjugates (polyubiquitinyIated proteins) was observed in active UC mucosa. Total tkB~ protein expression was decreased,while ubiquitinylated IkB~ protein was increased. Additionally 26S proteasome and phospholylated IkBcL protein expression was also increased in active UC mueosa by Western blot analysis. Furthermore, similar findings were also demonstrated in the study using TNF-~x-stimulatedcolonic epithelial cell line. (Conclusion) These results suggest that ubiquitination and degeneration of IkBa could lead to NF-KB activation in the epithelial cells of UC and that the activation of ubiquitin-proteasome system through the luminal antigen and/or proinflammatory cytokine such as TNF-~ are responsiblefor regulating the inflammatory process in ulcerative colitis.

$814 CD2 Directed Immunotherapyof Transfer Colitis Nina N. Pawiowski, Hacer Kakirman, Anja A. KOhl, Sven Henschke, Sabine Ring, Martin Zeitz, JSrg C. Hoffmann, Berlin, Germany; Homburg, Germany Introduction: T cells play a central role in the immunopathogeness of inflammatory bowel disease. Previous studies have shown that anti CD2 mAb can induce T cell apoptosis, inhibit T cell proliferationl and can induce secretion of p~otectivecytokines, e.g. TGF-I3.We therefore asked whether anti-CO2 mAb have a positive effect on transfer colitis in mice. Methods: Transfer colitis was induced by transfer of purified ConA-CO4+ T cell blast into syngenic rag-1 deficient mice. The anti-CD2 monoclonal antibody (12-15) or control antibody (rat IgG) was given initially (400 I~g) and then weekly (200 t~g). Mice were sacrified at a weight loss >20% and/or obvious signs of pain and/or lethargy. At the end of the experiment, the colitis severity was jugded macroscopically and microscopically in a blinded fashion~Lamina propria lymphocytes (LPLs), mesenterial lymphnodes (mLN), Peyer Patches (PPs), and splenocytes were isolated, stimulated via CD3/CD28 and the cytokine profile was determined by ELISA. In addition, LPL proliferation was measured by 3H-thymidin incorporation. Results: All control mice (n = 13) died within 35 days, whereas 69% of anti-CD2 mAb treated animals (n = 16) survived beyond 35 days (p < 0.01). 50% of the 12-15 treated mice showed no clinical signs of colitis at the end of the experiment and survived Iongterm. The macroscopical and microscopical colitis scores of anti-CD2 mAb treated mice were lower than in the control group. Production of interferon-~, and IL-2 in CD3/CD28 stimulated LPLs was markedly reduced among the clinically normal, anti-CD2 mAb treated mice compared to the control group. Conclusion: The anti-CD2 mAb 12-15 prevents transfer colitis in a large propotion of treated animals. Some of these have no signs of inflammation, do not produce Thl cytokines, and show Iongterm survival. Future studies must be performed in order to explain the heterogenity among the anti-CD2 mAb treated colitis mice and to evaluate a CD2 directed immunotherapy in established colitis.

$817 Phospholipase A2-Aclivating Pratein--An Important Molecule Regulating COX-2 and TNFo~ Production during Inflammation Ashok K. Chopra, Johnny W. Peterson, Galveston, TX Background: Chronic inflammatory diseases,e.g., I BD appearto involve a multitude of signaling pathways, resulting in activation of various cytokines, complement cascades, apoptotic proteins, and eieosanoids. Most eicosanoids are formed from arachidonic acid (AA) generated from phospholipids by phospholipases (e.g., PLA2)through COX-1 and COX-2enzymes,which along with PLA2 are central targets for current IBD therapies. We have shown a role for phospholipaseA2-activatingprotein (PLAA) in regulating AA metabolism in stimulated eukaryotic ceils using antisense plaa cONA oligonucleotide. Pt.AA levels are also increased in IBD tissues and in DDS mouse model of colitis. We have cloned a cDNA encoding PLAA from a human monocytes. PLAA is a G-like protein that modulates AA metabolism, and therefore could be a target molecule for IBD therapy. We have shown that LPS and pro-inflammatory cytokines increase PLAA levels in stimulated macrophages with a concomitant increase in PGE2 and PLA2 activity. PI_AA (738 aa residues) shares homology with melittin (within aa residues 503-538), a 26 aa peptide, which others and we haveshown to regulate phospholipase D activity. Methods: Biopsy specimens from patients with various levels of inflammation were analyzed by Western blot analysis for levels of PLA2, COX-2 and PLA2 activity to establish the role of these mediators in clinical forms of inflammation. PI_AA peptides with homology to melittin were examined for their ability to modulate COX-2 and TNFc~production in macrophages. Results: Synthetic PI.AA peptide, like melittin, increased the expression of genes encoding both TNF~ and COX-2 in macrophages. More detailed analysis of PLAA peptide revealed that C-terminal region of this peptide (aa 515-538) was predominantly involved in TNF(x and COX-2 production as determined by Northern blot analysis and ELISA. The Nterminal region of PLAA peptide (aa 503-526) caused only a modest increase in the expression of genes encoding TNF~ and COX-2. Biopsy specimens from patients with inflammation showed elevated levels of PLAA, COX-2 and phospholipase activity as compared to noninflamed patients. Conclusions: We have identified an important region within PLAA that plays an important role in modulating pro-inflammatory cytokine and eicesanoid production. We believe that PI_AA could be potentially new target for the development of new therapeutic approaches to regulate phospholipases and subsequent PGE2production.

$815 Activated Platelets in the Circulation of Inflammatory Bowel Disease (IBD) Patients Express CD40 Ligand and Induce an Inflammatory Responsein the Intestinal Microvasculature Silvio Danese,Jeffrey Katz, Andreas Sturm, Gall West, Antonio Gasharrini, Claudio Fiocchi, Cleveland, OH; Rome, Italy BACKGROUND AND AIMS Platelets (PLT) dysfunction is a prominent feature of IBD as demonstrated by thrombocytosis, common thromboembolic events, mucossl microinfarctions, and the potential therapeutic value of heparin. Moreover, PLT circulate in an activate d state in IBD as shown by increased aggregation and enhanced P-selectin expression. Beside their pro-coagulant function, PLT also display potent pro-inflammatory activities mediated by a variety of molecules such as CD40 ligand (L). This allows PLT to a c tivate CD40-positive cells including human intestinal microvascular endothelial cells (HIMEC).Therefore, we investigated the expression of CO40Las a new marker of PLT activation in IBD and control subjects, and whether PLT can induce chemokine product ion and cell adhesion molecule (CAM) expression by HIMEC. METHODS 7 clinically active IBD and 6 healthy control subjects were studied. Flow cytometry measured CD40L and P-selectin expression by PLT at baseline and after thromhin stimulation. PLT were co-cu itured with INFg-treated CD40-positive HIMEC to induce IL-8 production which was measured in the supernatants by ELISA, and ICAM-1 and VCAM-1 expression which was assessed by flow cytometry. RESULTS PLT freshly isolated from IBD patients showed a sig nifi cantly higher CD40L and P-selectin expression than that of controls (both at *p
$818 Increased Expression of the Transcription Factor C/EBPp in the Inflamed Colon of IL-10 Deficient Mice Thomas Caspritz, Elisabeth Liebter-Tenorio, Michael Maehler, Maike Veen, Michael P. Manns, Michael Goeke, Hannover, Germany C/EBPI3 is a transcription factor known to be an important regulator of proinflammatory cytokines and acute phase proteins. We previously demonstrated regulation of C/EBP~ mRNA expression in intestinal epithelial cells by inflammatory mediators. Little published data is available about the expression of C/EBPI3in the intestine of patients with inflammatory bowel disease (IBD) or animal models of IBD. We therefore analyzed C/EBPI3expressionin the inflamed colon of IL-IO deficient mice. Methods: As animal model of IBD, II-l~'~cg"-mice on the C3H/HeJBir background were used at the age of 1, 3 and 6 months. C3H/HeJBir wild type mice served as controls. Total RNAwas extracted from the colon. C/EBI3mRNA expression was assessed by Northern blotting, signal intensity was normalized to the housekeepinggene 13-actin. C/EBPI3 protein expression was evaluated in paraplast sections of the whole colon by immunohistochemistry using the avidin-biotin-complex technique. Cell proliferation was evaluated by nuclear Ki-67 staining. Results: C/EBP~ mRNA expression was elevated in the inflamed colon of IL-IO deficient mice compared to wild type controls. Immunohistochemistry revealed increased nuclear C/EBPI3 protein staining in epithelial cells, especially in the lower half of crypts of 3-month old IL-IO deficient mice. In addition, pronounced C/EBPI3 staining was observed in inflammatory infiltrates in the lamina propria and submucosa of IL-IO knockout mice. Furthermore, C/EBPI3 expression was enhanced in areas of neoplastic proliferation of the epithelium in 6-month old IL-IO deficient mice as assessedby Ki-67 staining. Conclusions: Increased C/EBPI3 gene and protein expression is found in the inflamed colon of I1-1tm~cg"mice. increased C/EBP~ protein expression is observed mainly in the epithelium of the lower half of crypts, in inflammatory infiltrates, and in neoplastic areas of the epithelium. These results indicate that C/EBPI3 may play a role in the pathophysiology of mucosal inflammation in this IBD animal model

P-selectin and CD40L expression in resting and activated PLT of controland IBD subjects P.selectin CD40L Resting Activated Resting Activated Control (n=6) 13_+3 92_+5 3_+1 35+8 IBD (n=7) 35+8* 90_+8 12~3" 46_+18

$816 The Role of Epithelial Cells in Inflammatory Process of Ulcerative Colitis: UbiquiUnProteasome System and IkB Degeneration Haruhiko Ogata, Yusuke Kishi, Mamoru Watanabe, Takanori Kanai, Hiromasa Ishii, Yasushi 1wag, Nagamu Inoue, Osamu Hitotsumatsu, Toshifumi Hibi, Tokyo, Japan (Background and Aim) The transcriptional factor NF-~
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$819 Mesenchymal Cells Play a Modulatory Role in Lamina Propria Mononuclear Cell Induced Intestinal Barrier Disruption. Linette Willemsen, Katinka Schreurs, Hilde Kroes, Ernst Jan Spillenaar Bilgen, Sander Van Deventer, Ric Van Tol, Wageningen, Netherlands; Arnhem, Netherlands; Amsterdam, Netherlands Background. Activation of intestinal lamina propria mononuclear ceils (LPMC) may have detrimental effects on mucosal barrier integrity. Hence this mechanism contributes to the persistence of chronic inflammation in the context of increased exposure to microbial and/ or dietary stimuli. Here we evaluatedthe role of differentially stimulated LPMC in an innovative co-culture system of mucosal barrier formation. Methods.Wedevelopedan intestinal epithelial/ mesenchymal co-culture system of T-84 and CCD-18Co cells mimicking the in situ spatial and functional relation of these cell types. In mono and co-culture systems LPMC, either unstimulated or stimulated with PMA or ~CD2/~CD28, were introduced. Barrier integrity was determined by resistance (R; E~.cm2) and functional permeability (HRP flux; pmol.cm-2.h-1) at different time points. IFN-% TNF-~ and IL-IO production was also determined. Results. LPMC caused decreased barrier function with a more pronounced loss of barrier integrity in both co- and monocultures with PMA or ~CD2/c~CD28stimulated LPMC. In the co-cultures, where mesenchymal cells support the epithelium, the effect was found to be smaller and delayed. Incubation with PMA or ~CD2/~CD28 activated LPMC revealed a differential effect on loss of barrier function depending on the route of stimulation. Control incubations with PMA or c~CD2/~CD28alone did not change barrier integrity. LPMC derived cytokine levels were found to be similar for the same stimuli in different culture systems and viability of cultures was not affected. Additional analyses suggested the involvement of mesenchymal cell derived growth factors in barrier integrity. Conclusion. We found differences in loss of barrier function caused by LPMC to depend upon the presence of mesenchymal cells and the route of stimulation.

$820 Signal Transducer and Aclivator of Transcription 6 (STATB)-IndependentChanges in Muscarinic Receptor Properties in the Inflamed Murine Intestine Hirotada Akiho, Atheer AI-Kaabi, Patricia Blennerhassett, Stephen M. Collins, Hamilton, ON, Canada •

Backgrounc~ Inflammation of the proximal gastrointestinal tract during nematode infection is accompanied by smooth muscle hypercontractility to carbachol stimulation. We have also shown that this phenomenon is largely STAT6 mediated in muscle strips and isolated cells. In the present study, we have examined whether these changes are reflected in the ligandrecognition properties of the muscarinic receptor on routine smooth muscle. We therefore evaluated muscarinic receptor affinity on intestinal muscle cells isolated from STAT6+/+ or STAT6-/- mice in the presence or absence of Trichinella spiralis infection. Methods: Cells were isolated by collagenase digestion from longitudinal muscle myenteric plexus (LMMP). Muscarinic agonist receptor characteristics were examined by agonist displacement of Nmethyt-3H-scopolamine(NMS) binding. Results: Specific binding of 3H-NMSto isolated smooth muscle cells was concentration dependent over a range of O.l-lOnM. Scatchard analysis revealed dissociation constant (KD)and Bm~values for 3H-NMS of 2.6rim and 2.4 x 104 sites/ cell, respectively in control cells. No significant differences in Ko and Bm~for 3H-NMS were seen between STAT6+/+ and STAT6-/- mice. T spiralis infection was accompanied by a reduction in Ko to O.7nM (p
GFAP expressions after stimulation with IL-~. Conclusion: The ENS contains also a distinct GFAP-negative subpopulation of gila like it is known in the CNS. With a part of 38% of the whole enteric gila the GFAP-negativesubpopulatinn is a rather big group. Interestingly, this glia seems to be possible to express GFAP as a reaction to inflammatory cytokines. This shows, that inflammation, one of the important injuries in the gut induce GFAP upregulation similar to astrogliosis after injury in the CNS.As it has been shown that GFAP-positiveenteric gila cells are essential for the homoeostasis of the gut, characterization of factors that control the GFAP-positiveenteric gila may help in developing treatments for inflammation in the gut

$822 Expression and Localization of Intestinal Kallikrein, KallistaUn, and Kinin Receptors in Inflammatory Bowel Disease Antoni A. Stadnicki, Urszula M Mazurek, Danuta J. Plewka, Elzbieta U. Pastucha, Andrzej K. Plewka, Tadeusz M. Wilczok, Sosnowiec, Poland; Katowice, Poland Background/Objective. We have shown that intestinal tissue kallikrein (ITK), kinin forming enzyme is released during enterocolitis induced by peptidoglycan in Lewis rats. We also demonstrated that plasma level of kallistatin, a major tissue kallikrein inhibitor is reduced in patient with inflammatory bowel disease (IBD). Now we investigatethe expression and localization od ITK, kallistatain as well as B1 and B2 kinin receptors (B1R, B2R)in intestinal tissue of patients with IBO.Methods. ITK, kallistatn, B1R and B2R were visualized by immunohistochemical staining in tissue samples from patients with ulceraive colitis (UC), Crohn's disease, and normal controls.In the second series speciments of colonic biopsy during colonoscopy were obtained from active UC patients (n = 17), inactive UCpatients (n = 11), and noninflammatory controls(n=13). The expression of genes encoding ITK, kallistatn, B1R and BR2 was estimated by QRT-PCR TaqMan analysis. Results. TK was localized by immunostaining in goblet cells, whereas the kallistatin in epithelial cells of human intestine. Both proteins were also visualized in macrophagesinside granulomas in Crohn's disease.The B1R was immunolocalized in the basal part of epithelial cells,and in addition in fibroblast and inside granulnma in Crohn's disease. The B2R was demonstrated in the apices part of epithelial cells and:in endothelial cells of blood vessel. Mucosal biopsy speciments from UC active patients had significantly lower (p < 0.01) ITK mRNA levels (462_+ 324 mRNA copies/l~g RNA) as compared with controls (6704 _+ 3416), but not to UC inactive patients (5229_+ 4591). Tissue kallistatn mRNA level significantly decreased (p< 0.05) in UC active patients (3495_+ 937)as compared with controls(7812 + 2710), but not to UC inactive patients (6671 -+ 1936). In UC active patients the expression of genes encoding B1R was significantly higher (p < 0.002) as compared with expresson of B2R (ratio of B1R/B2R = 4.01), however in UC inactive patients B1R and B2R expression level did not differ significantly (ratio of BIR/B2R = 1.7). Conclusions. Alterations in the tissue kallikrein - kallistatn expression profile may contribute to intestinal inflammation. Expression of B1R in active UC intestine provides a structural basis for the kinins to mediate inflammation.

$823 Oxidant-induced injury to Monolayers of Human Intestinal Epithelia: Role of cGMP Pathway in Injury to Myosin Type II Cytoskeleton Robert Moghimi, All Banan, Jeremy Fields, Lei Zhang, All Keshavarzian,Chicago, IL Oxidative stress and mucosal barrier disruption are associated with the inflamed mucosa of inflammatory bowel disease. Nitric oxide (NO) is known to cause monolayer disruption, and activate soluble guanylyl cyclase (GC) resulting in cGMP production and activation of protein kinase G (PKG). Aims: We investigated the role of cGMP and PKG pathways in the disruption of Myosin Type II cytoskeleton under conditions of oxidant challenge in intestinal epithelial cell monolayers. Methods: Human colonic (Caco-2) monolayers were incubated (30 rain) with a range of concentrations of oxidant (H202;0.025 - 5.0 mM) _+ preincubation (15 min) with agents that mimic/activate or inhibit the GC-cGMP-PKG pathways. These included agents known to "~cGMP levels: dibutryl-cGMP (a membrane permeable cGMP analog, 5 -- 1000 I~M). Agents known to 3 cGMP levels: ODQ (a selective GC inhibitor, 1H:(1,2,4)-oxadiazolo(4,3,-a)-quinoxalin-l-one, 1-10 t~M) & KT5823 (a selective PKG inhibitor, 1 -50 I~M). We then performed detailed analysis of the myosin type II cytoskeleton (high-resolution laser scanning confocal microscopy) and monolayer barrier integrity (clearance of fluorescein sulfonic acid / FSA by fluorometry), N =6 per group. These outcomes were also correlated with measurements of intracellutar cGMP levels (ELISA). Results:
$821 Interleukin 113 Upregulates GFAP in Enteric Gila Georg B. T. Von Boyen, Guido Adler, Martin Steinkamp, Max Reinshagen, Karl-Herbert Schaefer, Joachim Kirsch, 89081 ulm, Germany; ulm, Germany; Mannheim, Germany Introduction: Loss of enteric glial fibrillary acidic protein (GFAP)-positive gila leads to a hemorrhagic jejuno-ileitis in a gila-knock-out mouse model. These findings demonstrate that the enteric gila plays an essential role in maintaining the integrity of the bowel and suggests that loss of gila-function may contribute to the cellular mechanisms of inflammatory bowel disease. The astrozytic gila in the CNS resembles the enteric glia. Two different classes of CNS astrocytes can be distinguished based on GFAP expression. In almost all types of injury of the CNS including inflammation with diffuse neuronal death, the amount of GFAP, as well as the number of GFAP-positive cells is consistently upregulated. In the present study we identified a GFAP-negativegila subtype in the enteric nervous system. We also investigated whether inflammation is capable upregulating GFAPin enteric gila. Methods: Myenteric plexus of newborn rats was dissociated and cultured as described previously. To study the effects of Interleukin-ll~ (IL-13) on GFAP in enteric gila the myenteric plexus cell cultures were incubated with rrlL-113and anti rrlL-13 in different concentration. Double immunofluorescence staining was done by combining rabbit (S-100) and mouse (GFAP)antibodies. The immunolabeled cells were examined and quantified by the scanning confocal microscopy. The amount of GFAPwith and without stimulation was determined by using western blot analysis. Results: Double immunofluorescence showed a subpopulation of enteric gila, which Was negative for GFAP(62% GFAP-positive,38% GFAP-negativeenteric gila). After stimulation with IL-13(100ng/ ml) the GFAP-positive enteric gila raised to 88% of all enteric gila. The upregulation was dose-dependentand could be antagonizedby anti IL-13.Westernblot analysis showed increased

$824 intestinal Epithelial and Myofibroblastic Cells Are Equally Susceptible to TNFo~and INF-,/for Laminin ProducUonand Apoptosis Caroline Francoeur, Elisabeth Herring, Pierre Vachon, Jean-Francois Beaulieu, Sherbrooke, Canada Altered cytokine and growth factor production as well as mucosal remodeling are welldocumented events occurring in chronic inflammatory bowel diseases. Our laboratory has recently shown that in Crohn's disease,the changes in the crypt-villus architecture that occur in the inflamed mucosa of the ileum are associated with a major redistribution of the laminins

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in the epithelial basement membrane (BM), particularly in the crypt region. In order to elucidate the mechanisms underlying this phenomenon, we have studied in vitro the effect of various pro-inflammatory cytokines on the expression of laminins in normal human intestinal crypt epithelial (HIEC) and myofibroblastic (HIM) cells, used as experimental models for the two cell types that contribute to epithelial BM laminin deposition in situ. When treated with a single cytokine, HIEC secrete small amounts of laminins. In the absence of serum, only TNFc~ (10 ng/ml) induces a slight increase in the secretion of laminin 5 and 10 (p
$825 Effects of Two Distinct Nuclear Factor-Kappa B luhibitors on Fas Ligand Plus Cytokine-lnduced Apoptosis in a Colonic Epithelial Cell Line Leo R. Fitzpatrick, Jian Wang, Truc Le, Rockville, MD Gliotoxin (CLIO) and Caffeic Acid Phenethyt Ester (CAPE) inhibit nuclear factor-kappaB (NFKB). These compounds were also effective, to varying degrees, in a rat model of inflammatory bowel disease (Fitzpatrick et al., 2000 and 2001 DDW meetings). However, the specific mechanism(s) involved are not well understood. Aims: Therefore, the aims of our study were: 1) To determine the efficacy of these compounds for inhibiting the relevant proteasome complex, which is involved in the NF-KB signaling pathway and 2) To determine if these compounds affected CH-11 Fas Ligand (F), plus Interferon-~/(I), plus TNF-~(T) (designated FIT) induced apoptosis of colonic epithelial cells. Methods: Proteasome inhibition was measured in a cell free system, with purified 20S proteasnme and an appropriate fluorogenic peptide substrate. Vehicle (0.2% DMSO), CLIO (1 p.g/ml), or CAPE (30 p.g/ml) were added to a 96well plate, before initiating the assay reaction (n = 4-8 per treatment group). The proteolytic activity of the 20S proteasome was measued by fluorescent spectroscopy. This activity is directly proportional to the amount of aminomethylcoumarin produced over a 30 minute incubation period. FIT-induced apoptosis (i.e., DNA fragmentation) was evaluated in a SW620 colonic epithelial cell line, by the cell death detection ELISA method. In these studies, vehicle, CLIO and CAPE (n = 8 per group) were used at the concentrations described above. SW620 .cell apoptosis was assessed after 24 hours. Results: CAPE effectively inhibted proteolytic activity of the 20S proteasome. Specifically, it reduced such activity by 72_+5% (p < 0.01 vs. vehicle treatment). In contrast, CLIO was a marginally effective proteasome inhibitor (20_+3% inhibition). FIT treatment resulted in a significant increase (p < 0.05 vs. vehicle) in the DNA fragmentation of SW620 cells. Both inhibitors attenuated this apoptosis. Interestingly, CLIO protected epithelial cells from FIT-induced apoptosis more effectively than did CAPE. Specifically, relative DNA fragmentation values for all treatments in SW620 cells were 1.0 -+0.1 (vehicle), 4.8-+0.4 (FIT), 1.6 -+0.2 (FIT+ CLIO) and 3.2_+0.3 (FIT+CAPE). Conclusion: NF-KB inhibitors showed different efficacies for inhibiting the 20S proteasome, as well as for protecting against FiT-induced apoptosis of colonic epithelial cells. Such differences may have relevance to the degree of efficacy demonstrated previously by these inhibitors, for the treatment of colonic inflammation.

$826 Abnormal Barrier Function in the Non-inflamed Ileum after Resection for Crohn's Disease: Primary or Secondary Defect? Johan D. Soderholm, Ping-Chang Yang, Cathy Streutker, Craig Paterson, Robert Riddell, Ken Croitoru, Derek M McKay, Philip M. Sherman, Mary H. Perdue, Linkoping, Sweden; Hamilton, ON, Canada;Toronto, ON, Canada Background: In a previous study we showed increased permeability to ovalbumin in the noninflamed ileum of patients with Crohn's disease (CD), possibly due to enhancedtranscytosis of proteins. Aim: Here, we examined if enhanced uptake of antigen into enterocyte endosomes was a primary defect or occurred only secondary to mild inflammation. Methods: Macroscopically non-inflamed mucosa from surgical specimens of distal ileum of patients with CD (n = 11) was studied, with ileal specimens trom colon cancer patients (n=9) as controls. Tissues were mounted in Ussing chambers. Endocytotic uptake into enterocytes was assessed after 5 rain of luminal exposure to horseradish peroxidase (HRP), by measuring the area of HRPcontaining endosomes in electron photomicrographs. Short-circuit current (Isc) was studied at baseline, in response to transmural nerve stimulation (TS) and glucose. Macroscopicatly normal tissues were grouped as non-inflamed or slightly inflamed (increased number of neutrophiis and/or mononuclear cells in the lamina propria or the epithelial layer) by light microscopy. Results are expressedas mean_+SEM. Results: HRP endosome area was significantly greater in non-inflamed ileum of CD (2.8 + 0.7 p,m2/3001~m2 apical cell area) than in control ileum (0.6 + 0.06 p.m2/3OOp,m2, p
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$827 Activation of Eotaxin-3/CCL26 Gene in Human Enterecytes Is Mediated by STAT6 Carine Blanchard, St~phane Durual, Emilie Boncoeur, Monique Estienne, Jean-Claude Cuber, Lyon, France Several inflammatory processes of the gut are characterizedby an accumulation of eosinophils and lymphocytes at sites of inflammation. Eotaxin-3/CCL26 is a member of the fatuity of CCchemokineswhich are known to be potent chemoattractants for eosinophils and lymphocytes. This chemokine was shown to be up-regulated by IL-4 and IL-13 in endothelial cells and dermal fibroblasts. The present study investigates the possibility that eotaxin-3 transcription could he stimulated in epithelial cells of the gut. IL-4 and IL-13 receptor subunits were identified by flow cytometry using specific antisera and by RT-PCR.The expression of eotaxin3 was evaluated by Northern-blot and semi-quantitative/quantitative RT-PCR. IL-4 and IL-13 up-regulated in a time- and dose-dependentfashion the expression of eotaxin-3 in the human intestinal cells HT29-CL19Aand T84 which both harbor an enterocyte phenotype, tn contrast, TNF~ could not act as an inducer on its own nor did it synergize with IL-4. Additionally, eotaxin-1 and -2 were weakly up-regulated in these cells upon IL-4 treatment. The activities of eotaxin-3 promoter luciferase constructs were dramatically increased by IL-4/-13 in these cetl lines. This effect was mediated by a binding site of transcription factor STAT6 located at -93 pb. The second STAT6 binding site located at -498 pb did not appear to enhance the activity of the proximal STAT6 binding site. Mobility shift and supershifts assays revealed that STAT6 binds to the consensus binding site of STAT6 Located in eotaxin-3 promoter. Mutation of the proximal STAT6 binding site abrogated up-regulation of eotaxin-3 promoter activity. The wild-type luciferase construct, but not the STAT6mutated construct was inducible by IL-4. Interestingly, a conditionally active form of STAT6 sufficed to activate the eotaxin-3 promoter, in IL-4 exposed epithelial ceils, the wild type luciferase construct was strongly repressed upon cotransfection of an expression vector containing a dominant negative form of STATB. These results indicate that, in epithelial cells of the gut, IL-4 and IL-13 activate eotaxin-3 gone expression in a STAT6 dependent fashion, thus suggesting that eotaxin-3 may exert a pivotal role in inflammatory processes of the gut involving type 2 cytokine

$828 Adhesion Molecule and Cytokine Expression in Cultured Human Intestinal Microvascular Endothelial Cells (HIMECs) from Patients with Inflammatory Bowel Disease (160) Stuart McDonald, lan Charles, Mike Lockyer, Stuart L. Bloom, London, UK Introduction.Endothelial cells secrete cytokines and express adhesion molecules to facilitate lymphocyte activation and extravasation into the lamina propria. Here we profile the expression of a variety of endothelial cytokines and adhesion molecules in control and {BD subjects. Methods.HIMECswere extracted from colon taken from the resection margin of patients with colonic carcinomas (n = 8) and inflamed resections from patients with ulcerative colitis (UC, n = 5) and Crohns disease (CD, n = 6). Reversetranscription-polymerase chain reaction (RTPCR) was performed on mRNA extracted from lx105 HIMECs from each group stimulated with either TNFa or IL-lb for 0,2,4,16 or 24 hrs. Gene specific primers for IL-1b,-2,-3,-4,5,-6,-7,-8,-10,-12 p40,-12p35 and -15, TNFa, TGFb, IFNg, VCAM-1, ICAM-1 and MAdCAM-1 were employed. IL-8 and MAdCAM-1 protein expression were quantified by ELISA and western blotting respectively. Results.HIMECsfrom control patients constitutively expressed IL-3,-8,15 and TGFb and after stimulation with TNFa or IL-1 increasing amounts of IL-lb, TNFa, IL6,-8, VCAM-1 and ICAM-I. Interestingly MAdCAM-1 mRNA and protein were not expressed in any conditions, although whole tissue mRNA and tissue sections clearly show it being expressed on endothelial cells in vivo. HIMECs from patients with UC and CD showed a similar pattern of cytokine and adhesion molecule expression in unstimulated conditions. TNFa-stimutated IBD HIMECs produced significantly more IL-6 mRNA and more IL-8 mRNA and protein than those from controls. The expression of VCAM-lmRNA in HIMECs from patients with either UC or CD was higher than controls but only after stimulation with TNFa for 24hrs. Conclusions. These data suggest that HIMECs are able to respond immunological mediators such as IL-1 and TNFa. TNFa-stimulated HIMECsfrom IBD patients produce more IL-8 and IL-6 than those from controls and this suggests that endothelial cells residing in the inflamed gut are chronically more sensitive to TNFwith regardsto their IL-6, IL-8 and VCAM1 production which may be genetic or conditioned. The result of this increasedexpression could lead to increased numbers of neutrophils and lymphocytes within the mucosa, which then contribute to the ongoing inflammation. The absenceof MAdCAM-1 in HIMECcultures suggests that IL-lb and T~IFaalone, or in combination with each other, are not sufficient to stimulate its expression.

$829 Differences in Expression and PhosphorylationKinetics of FAK and ERK between Control and Crohn's Disease Myofibroblasts during Migration Sandra N. Loeb, Katja Felgenhauer,Michael Cooke, Werner Falk, Juergen Schoelmerich, Cornelia M. Gelbmann, Gerhard Rogler, 93042 Regensburg, Germany; 30625 Hannover, Germany Background: Regulation of the migration of colonic myofibroblasts (CMF) is an important mechanism during wound healing or intestinal fibrosis. Focal adhesion kinase (FAK) and mitogen activated kinases (such as ERK1 and ERK2) play a central role during the induction of migration. Methods: Primary cultures of CMF were isolated from endoscopic biopsies of patients with Crohn's disease (CD:CMF), ulcerative colitis (UC-CMF) and control persons (control-CMF). Migration assays of CMF were performed in the modified 48-well Boyden chamber. The migrated cells were fixed, stained and counted in four high power fields (hpf) at a 400 fold magnification. FAK and ERK1 expression or phosphorylation in migrating CMF was determined by Western blotting. Results: CMF produce factors that stimulate their own migration, therefore medium conditioned of CMF for 48h was used to stimulate migration of CMF. Migration of CD- and UC-CMF was significantly reduced when compared to controlCMF (42 +/- 9 and 35 +/- 3 vs. 116 +/- 14 cells/hpf). Similarly, incubation of controlCMF with TNF (20ng/ml) for 3 days reduced their migratory response to 42,5% +/- 14,3%

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of normal level (p95% of cells were resting in the GO/G1 phase and <5% cells were dividing. This quiescent cell cycle profile changed in parallel with a progressive differentiation to a fibroblastlike morphology, and at 5 days the number of cells in the GO/G1phase decreased to 82% and those dividing increased to 18%. The number of c~-actin-pnsitivocells increased to 10, 50 and 100% at 4, 7, and 14 days, respectively. By immunoblotting collagen type I was absent at 4 days, appearing at 7-10 days, with a further increase when cells were exposed to TGF-13. CONCLUSIONS We describe the first, simple and reliable method for isolation of human intestinal steitate cells in a quiescent and undifferentiated state. This method allows to study the activation and differentiation from stellate cells to collagen-producing myofibroblasts. This mimics the in vivo events that lead to the development of intestinal fibrosis in response to intestinal inflammation. $831 Tropomyosin Expression in the Ileal Pouch: Relation with the Development of Pouchitis in UJceratJve CoJitJs. Livia Biancone, Giampiero Palmieri, Antonella Lombardi, Alfredo Colantoni, Elena Bonanno, Mario Erboso, Giovanna Del Vecchio Blanco, Piero Vavassori, Francesco Tonelli, Kiron Das, Francesco Pallone, Rome, Italy; Florence, Italy; New Brunswick, NJ Background. Bacterial-host interactions and apoptosis may induce the expression of cryptic cytoskeletal proteins on the cells surface. TMs are cytoskeletal proteins present in all eukariotic cells. TM isoform 5 (TM5) is expressed in normal colonic but not ileal epithelial cells. Serum and mucosal IgG to TMs are detected in 2/3 of Ulcerative Colitis (UC) patients, suggesting an autoantigenic role of TMs in UC. Whether changes of cytoskeletal proteins expression in the ileum are involved in pouchitis is unknown. Aims. To assess, in a prospective longitudinal study, the expression of TM on ileal epithelial cells at surgery and subsequentlyon development of pouchitis in UC. Methods. Population. In a prospective longitudinal study, 29 patients undergoing ileal pouch for UC were tested: 9 of them already completed the study follow up. Biopsies were taken from the ileum and rectum at surgery and at 6 months from the ileal pouch. Immunohistochemistry. All biopsies were stained by both haematoxylin and eosin for routine histology and by immunoperoxidase using an anti-TM5 IgM monoclonal antibody. The histological degree of pouchitis was assessed by the Pouchitis Disease Activity Index (PDAI). TM5 expression was scored as follows: O=no staining; 1 = focal staining in the goblet cells; 2=diffuse staining in the goblet ceils; 3=staining in the epithelial cells lining the lumen and the crypts. All sections were scored by 2 histopathologists. Results. At surgery. In all rectal samples, TM5 was expressed in the epithelial cells lining the lumen (score 3), while in all ileal samples TM5 was either not expressed or focally expressed only in the goblet ceils, but never at luminal surface (scores 0-2). 6 months. At 6 months, the ileum underwent morphological changes, becoming similar to the colon, showing different degree of shortening or reduced number of villi. These changes were associated with a rearrangement of TM5 expression in the ileum. TM5 expression indeed showed a diffuse staining in the goblet cells and also appeared in the non-goblet epithelial cells lining the crypts and the lumen (score 3). The TM5 score was related to the PDAI score at 6 months (r=0.84; p=O.OO8), but not at surgery (r=0.1O; p=0.79). Conclusions. TM5 expression in the ileal mucosa from UC shows a rearrangement and an increased expression in the ileal pouch after surgery. This event appears associated with morphological changes that may be related to "colonization" of the ileum and to the development of inflammatory changes.

S832 Alterations in Cytosolic or Soluble Phospoiipase A2 Modulates the Severity of Intestinal Inflammation in a Murine Model of Colitis Paul Beck, Josee Wong, Daniel Podolsky, J Bonventre, Calgary, AB, Canada; Boston, MA There are two main forms of phospholipase A2 (cytosolic (cPLA2) and soluble (sPLA2)) that regulate arachidonic acid production. The roles of prostaglandins and leukotrienes, produced via arachidonic acid, have been studied in inflammatory bowel disease (IBD) but are far from fully understood since inhibitors of cyclo-oxygenase(COX) such as NSAIDscan exacerbateIBD yet corticosteroids the main treatment of IBD inhibit pathways upstream of COX, furthermore lipoxygenase inhibitors have not been found to of benefit in IBD. The specific aims of this study were to address the role of cPLA2 and sPLA2 in regulating intestinal inflammation. Methods; Colitis was induced in adding 2.5% dextran sodium sulfate (DSS) to drinking water. All mice were of C57/BL6 or BALB-c background. To assess the role of cPLA2; mice with a targeted deletion of cPLA2 were generated on a sPLA2 deficient background (C57/B6) or sPLA2 replete background (BALB-c). To assess the role of COX, indomethacin was used and to assess the role of leukotrienes a specific lipoxygenase inhibitor MK0591 was used. Results; LTB4 and PGE were increased in OSS-induced colitis. Alterations in sPLA2 isoforms and cPLA2 were also noted. No significant differences in basal colonic expression of PGE and LTB4 were noted in the cPLA2-/- sPLA2 -/- mice or cPLA2 -/- sPLA2 + / + mice versus wild type controls. Inhibition of COX but not lipoxygenasemarked exacerbatedDSS-inducedcolitis. Mice deficient in sPLA2 or cPLA2 had more severe colitis than wild type controls and loss of both sPLA2 and cPLA2 dramatically increased the severity of colitis. Conclusion; Loss of sPLA2 or cPLA2 increases the severity of DSS-induced colitis. $833 EGF Protects Intestinal Barrier Integrity (BI) by Stabilizing F-Actin Dynamics: Role ot PKC-pl Isoform Signal Activation and Ca +2 Homeostasis All Banan, Ashkan Farhadi; Jeremy Z. Fields, Let Zhang, All Keshavarzian,Chicago, IL Using mnnolayers of intestinal (Caco-2) cells, we showed thai the stability of F-actin cytoskeleton is key to EGF protection against oxidant-induced barrier disruption & that EGF normalizes Ca--2, but the intracellula~"mechanism is elusive. PKC is key to protection and the I~1 isoform of PKC is abundant in our wild type (14q) ceils. HYPOTHESIS PKC-131 is essential to EGF protection of F-actin cytoskeleton & normalization of Ca+2 homeostasis. METHODS WTmonolayers were exposed to oxidant (H202) +- EGF or PKC or Ca+2 modulators. Other cells were transfected to stably over- or under-express PKC-131, & then pretreated with low or high doses of EGF or a PKC activator (OAG) before H~O~.Effects on actin integrity (laser confocal); BI (fluorometry); PKC-I~I subcellular distribution & activity (immunoblotting, ~71vitro assay); Ca÷2 efflux & influx; and F-actin & G-actin pools (western) were determined, N=6/grp. RESULTS ,A~, In WTmonolayers exposedto oxidant, pretreatment with EGFor PKC activators dose dependently 1) I"WTPKC-131 activity; 2) PCa÷2eftlux; 3) while 3 intracellular ,Ca+2,,~to control levels;4) 3 monomeric G-actin & Tstable F-actin; & 5) protected both actin cytoskeleton & BI. PKC inhibitors (e.g., GF 109203X) preventedthese effects. Incubation in Ca÷~ionophore A23187, similar to oxidants, caused the loss of actin stability & prevented protection by EGF or OAG. ,B,, Transfected monolayers stably over-expressing PKC-~I (+ 3.1 fold T), in contrast to WTones, were protected from 1-1202injury by low doses of EGFor OAG. In these transfected cells, pretreatment with even a low dose of EGF or OAG resulted in PCa ÷2 efflux; :1 rise in ,Ca÷2,,~; 3 G-actin; I F-actin; 1"integrity of actin & of BI to control levels. EGF or OAG also rapidly distributed the over-expressed PKC-I~I into.the membrane~+ cytoskeletal fractions, indicating I~1 activation. To further confirm role of PKC-131in EGF's effects on Ca÷2 & actin, ,C,, Stable inhibition of WT PKC-~I (91% 3) by anti-sense (AS) prevented all measures of EGF protection. Finally, similar to blunting effects of AS to [51, membrane Ca+LATPase inhibitors prevented protection by PKC-I~I over-expression (& EGF or PKC activator), and abolished its beneficial effects on Ca÷2fluxes. We show for the first time that 1) EGF protects the dynamic assembly of the F-actin cytoskeleton in intestinal monolayersthrough the activation of ~C-I~1 and normalization of ,Ca+2%2) PKC-I~I appears to be a key endogenous stabilizer of actin and Ca+2 homeostasis in cells. $834 Prostaglandin EP1 but Not IP Receptors Involved in Mucosal Blood Flow and Ulcerogenic Responses in the Stomach after Mild Injury. Yusaku Komoike, Masanori Takeeda,Shinichi Kato, Koji Takeuchi, Kyoto, Japan We previously found that endogenous prostaglandins (PC) produced by COX-1 play a role in maintaining the mucosal integrity of the stomach after exposure to 20 mM taurocholate (TC), by increasing the mucosal blood flow (GMBF). This response has been shown to be mediated by PG through EP1 receptors, yet the role for IP receptors remains unknown, in the present study, we investigated the role for IP receptors in gastric mucosal protection as well as functional responses induced by TC as a mild irritant using EP1- and IP-receptor knockout (-/-) mice. Methods: Male C57/BL6 mice fasted for 18 h were Used under urethane anesthetized conditions. The stomach was mounted on an ex-vivo chamber, perfused with 20 mM HCI, and the transmucosal PD, luminal acid loss, and gastric blood flow (GMBF) were simultaneously measured before and after exposure of the stomach to 20 mM TC for 20 rain. Mucosal PGE2 and PGI2 contents were measured by EIA. Results: Mucosal exposure to TC in wild-type mice caused a marked decrease in PD, followed by an increase in H+ loss and GMBF. The decreased PD was gradually normalized after removal of TC from the chamber, with minimal damage in the mucosa 1 h later. This hyperemic response was inhibited by indomethacin with no change in PD reduction or H+ loss, resulting in severe lesions in the mucosa. None of these responses induced by TC were altered in IP (-/-) mice. However, in mice lacking EP1 receptors, TC did not increase GMBF, despite causing PD reduction and acid loss, and resulted in severe damage in the mucosa, the responses being much the same as those observed in the animals pretreated with ONO-AE-829,the EP1 receptor antagonist (10 mg/kg, SC). Mucosal PGE2 was significantly increased after TC, similarly, in all groups of mice, while that of PGI2 showed slight but not significant increase in any groups. On the other hand, capsaicin applied to the chamber increased GMBF in wild-type mice, in an

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AGA Abstracts

with BCECF-AM,which was confined to the cytoplasm of the epithelial cells (see figure). On a conventional microscope, image analysis was used to measure pHi, which rapidly fell in response to luminal perfusion with a pH 2.2 solution, and recovered to baseline after removal of the acid stress. Conclusions: We have developed a system wherein pHi can be measured in mouse duodenal epithelium. We plan to use this system for measurements of pH~ and HCO3-measurements in genetically modified mice.

indomethacin-sensitive way, hut this response totally disappearedin IP (-/-) mice. Conclusion: These results suggest that 1) endogenous PGs may contribute to maintaining the mucosal integrity, mainly through activation of EP1 receptors, 2) IP receptors are not actively involved in adaptive protection and functional responses induced by a mild irritant in mouse stomachs, and 3) the presence of IP receptors is essential for gastric hyperemic action of capsaicin. $835 Cyclooxygenase Isozymes Involved in Adaptive Functional Responses in Rat Stomachs Following Mild Injury. Masanori Takeeda, Masahiro Matsumoto, Yusaku Komoike, Shinichi Kato, Koji Takeuchi, Kyoto, Japan Application of mild irritants to the stomach causes an increase of gastric mucosal blood flow (GMBF) and a decrease of acid secretion. They damage the surface epithelium, resulting in acid back-diffusion, yet they rarely cause gross damageand rather protect the stomach against noxious agents. It is known that these responses are mediated by endogenous PGs, yet the relative contribution of the COX-1 and COX-2 isozymes in these functional responses is not entirely clear, in the present study, we examined the effect of selective COX-1 and COX-2 inhibitors on various functional changes of the rat stomach following exposure to taurocholate (TC) as a mild irritant, in comparison with that of indomethacin, the nonselectiveCOX inhibitor. Methods: Under urethane anesthesia, a rat stomach was mounted in an ex-vivo chamber, perfused with saline or acid (50 mM HCI), and transmucosal PD, luminal pH, mucosal blood flow (GMBF), and acid secretion were measured before and after the application of 20mM TC for 30 min. Indomethacin (5 mg/kg), SC-560 (COX-1 inhibitor: 5 mg/kg) or rofecoxib (COX-2 inhibitor; 5 mg/kg) was given intraduodenally 1 h before TC treatment. Results: Mucosal application of TC caused a marked PD reduction, followed by decrease of acid secretion (increase of luminal PHi and increase of GMBF. Prior administration of indomethacin did not affect the PD reduction but significantly mitigated both acid and GMBF responses induced by TC. Although the PD response was similarly observed in the group tested with either SC-560 or rofecoxib, both acid and GMBF responses were significantly attenuated by SC-560 but not rofecoxib. Perfusion of the stomach with 50 mM HCI before and after TC caused minimal damage in the mucosa 1 h later, yet this treatment produced gross gastric lesions in the presenceof indomethacin or SC-560. Rofecoxib had no effect on the ulcerogenic response to TC plus 50 mM HCI. The mucosal PGE2 content was increased after exposure to TC, hut this PGE2 biosynthetic response was inhibited by both indomethacin and SC-560 but not rofecoxib. Conclusion: These results confirmed a mediator role for PGs in both acid and GMBF responses of the stomach following.barrier disruption, and suggested that COX1 but not COX-2 is a key enzyme for regulating the functional alteration of the stomach and for maintaining the mucosal integrity under adverse conditions.

$838 Colonic Mucosa Has More Sensitive I.lyperemic Response to Luminal pH Than Duodenal Mucosa through Vanilloid Receptor. Yasutada Akiha, Masahiko Nakamura, Hiromasa Ishii, Tokyo, Japan Hyperemic response is one of mucosal defense mechanisms in the gastrointestinal tract, especially in stomach and duodenum in response to luminal acid. We have reported that capsaicin pathway including vanilloid receptor (VR) on capsaicin-sensitiveafferents, calcitonin gone-related peptide (CGRP) and nitric oxide, is an acid sensing pathway to induce the hyperemia and mucus secretion in response to lumina~ acid in rat duodenum. We hypothesize that this acid sensing pathway is also present in the lower intestine, in which luminal pR range is mild acidic (pH 6.8-7.9 in ileum and pH 5.2-7.4 in colon). We examinedthe hyperemic responseto luminal acid in colon compared with duodenum in rats. Under urethaneanesthesia, proximal duodenal or proximal colonic mucosa was exposed and topically superfused with Krebs buffer through the chamber. Mucosal blood flow in duodenum or colon was measured with laser Doppler flowmetry. Various pH buffers were applied to the duodenum (pH 7.0, 4.5, 3.5 and 2.2) and the colon (pH 7.4, 7.0, 6.0 and 5.5) with or without capsazepine (CPZ), a selective VR antagonist. Immunofluorescence and western blot for VR-1 were performed to confirm VR-1 expression in duodenum and colon. In duodenum, the hyperemic response was observed with pH 3.5 and 2.2 superfusion, but not pH 4.5. CPZ (0.5 mM) abolished the hyperemia induced by pH 2,2. In contrast, the colonic hyperemia was observed with pH 6.0 and 5.5, which was inhibited with CPZ (0.01 raM). VR-l-positive nerves localized in the mucosa and myenteric plexus in both duodenum and colon. Western blot analysis showed the VR-1 expression in colon greater than in duodenum, Acid sensing pathway via VR is present in colon as well as duodenum with the different luminal pH range. Colonic mucosa is more sensitive to luminal acid than duodenal mucosa, probably due to the differences in the tight junction and mucus composition, suggesting the physiological role in the absorption of short fatty chain acids in the colon.

$836 Gastric Vasodilatory Action of Lafutidine, a Novel H2 Receptor Antagonist, Is Mediated by Capsaicin Sensitive Afferent Neurons and Gastric Mast Cells in Urothane Anesthetized Rats Hitoshi Aomi, Yoji Matsumoto, Keishi Kawakubo, Takayuki Matsumoto, Mitsuo lida, Fukuoka, Japan; Yamada, Japan Background: Lafutidine is a novel histamine H2-receptor antagonist with gastroprotective activity depending upon the activation of capsaicin sensitive afferent neurons (CSAN). Lafutidine given intragastrically caused a sustained dose-related increase in gastric mucosal blood flow that is no longer observed in deafferentated rats. 8i-directional communication between the nervous and immune systems has been established.Aims: To investigate in rats 1) whether lafutidine dilates the gastric mucosal arterioles and venules, and if so, 2) whether these effects are mediated by CSAN and mast ceils. Methods: Under urethane-anesthesia(125 g/kg), the stomach of male Wistar (250-300 g) rats was exposed and cut along the greater curvature. After resecting the anterior wall, the posterior wall was fixed in a plastic chamber with the serosal side up and superfused with a warm modified Krebs buffer solution. The basal part of the mucosal microcirculation was observed through a window in grandular stomach made by partial removal of the serosa,the smooth muscle and the suhmucosa. Changesin diameters of gastric mucosal microvessels induced by superfused lafutidine solution (1,10,100 and 1000 ~g/L) were measured for 15 rain. The influence of either capsaicin desentization (125 mg/kg, sc, -2 week) or ketotifen (a well-established mast cell stabilizer, 4 mg/kg/h, iv) was investigated. Values are expressed as means _+ SEM. Significance of differences was determined with ANOVAfollowed by Fishers PLSD. Results: Superfused Jafutidineinduced a doserelated dilation of gastric mucosal arterioles (1 i~g/L: 4 _+ 2 % (p>O.05), 10 p.g/L: 6 _+ 3 % (p>0.05), 100 p.g/L: 16 _+ 5 % (p
Acid-Inducedhyperemiain rat duodenumand colon duodenum colon superfusate blood flow (% basal) superfusate bloodflow (% basalt_ pH 7.0 97.6 ÷/- 3.6 pH 7.4 99.0 +/- 2.1 pH 4.5 97.3 ÷/- 1.3 pH 7.0 112.1 ÷/- 8.3 pH 3.5 110.4 +/- 3.7a pH 6.0 129.5 ÷/- P pH 2.2 141.9 +/- 4.2~ pH 5.5 137.3 +/- 9.P pH 2.2+CPZ 99.6 +/- 7.P pH 5.5+CPZ 109.1 +/- 7.P _._ 0p<0.O5vs. pH 7.0 in duodenumor pH 7.4 in colon,bp<0.05vs. pH 2.2 in duodenumor pH 5.5 in colon. $839 Human Esophageal Epithelium In Vitro:. A Novel Technique Using Endoscopic Biopsies David J. Pompa, Zachary Sellers, Petra Paulus, Jon I. Isenberg, Ravinder Mittal, Vijaya S. K. Pratha, San Diego, CA; La Jolla,, CA Background:Epithelialfactors involved in the pathogenesisof gastroesophagealreflux disease (GERD) remain poorly defined. While animal models of reflux diseaseexist, species differences limit the applicability of these studies to humans. Short circuit current (Isc),potential difference (PD), and tissue resistance (R) are important determinants of epithelial barrier function and are altered with m ucosal acidification of rabbit esophagus in vitro (Am J Castro 2001; 96:3062). Aims: 1) To validate a unique in vitro model of human esophageal epithelial function; and, 2) to examinethe effects of luminal acid exposure on esophagealepithelial function. Methods: Human subjects (n =6, 36-70 yr, 4 female) without heartburn and normal esophagealmucosa had 4-8 esophageal biopsies taken at endoscopy (distal 2 cm). Tissues were mounted in parallel in modified mini-Ussing chambers (short-circuit conditions, 2 ml, 0.031 cm2,37°C, modified Ringer's; mucosal bath HCO3-free)using validated methods (J Lab Clin Med 1998; 132:512). After a 30 min equilibration, tissues were exposed to acid (50 mM HCI, mucosal bath) or mucosal bath (control) for 15 rain, washed (10 ml, 2 min, HCO3--free),and observed an additional 30 min. PD and I,c were recorded q5 min and R calculated using Ohm's Law. Tissue viability was determined with ouabain addition (10 mM, serosal bath). Results: Basal

$837 Non-lnvasive Measurement of Duodenal Epithelial pH in Mice Masahiko Hirokawa, Osamu Furukawa, Shaoyou Chu, Marshall H. Montrose Jr, Jonathan D. Kaunitz, Los Angeles, CA; Indianapolis, IN Background: We have previously measured duodenal epithelial cell intracellular pH (pHi) in rats in our studies of mucosal defense mechanisms. We now report a technique in which the same measurementscan be made in mice. Methods: Under urethane(1.25 g/kg) anesthesia, the duodenum of C57BL mice was exteriorized and opened. The mucosa was placed on a perfusion chamber on the stage of a Zeiss Axiovert 200 or LSIV1-510confocal fluorescence microscope. The mucosa was loaded with the trapped, fluorescent, pH sensitive dye BCECFAM. pH~was measured by fluorescence ratio calculation. Results: Epithelial cells readily loaded

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PD, Isc, and R stabilized in 30 min (Table I) and did not change significantly over time (up to 120 min) in controls. Mucosal acidification resulted in an immediate and sustained drop in pH from 6.84_+0.06 to 1.93_+0.08. In contrast to controls, mucosal acidification resulted in a significant decline from basal levels in PD, 14, and R (table I). Following wash, prompt recovery of all parameters to near basal levels was seen (table I). Conclusions: These results suggest that: 1) human esophageal biopsies exhibit stable electrical parameters in vitro and remain viable > 75 min; and, 2) mucosal acid exposure, similar to rabbit mucosa, results in a significant, reversible decrease in R and other electrical parameters. This novel technique with human esophagealmucosa should enablestudy of esophagealepithelial function in GERD. Electrical parameters of esophagealepitheliumin vitro Basal % decline % decline (with (control) acid) PD (mV) 26+0.7 24.5±15.3 41.5+7 7* Isc (pEq/cm2-h) 08+0.2 22.~13.5 30.8+16.3" R (Q-cmz) 147.5_+31.3 6.8-+5.0 36.0_+6.0* *=p
$842 The Role of Gastric Potassium Channels KCNQ1 in Meal-Stimulated Gastric Acid Secretion and the Pathophysiologyof Gastroesophageal Reflux Disease Jerry O. Gardner, Sheila Rodriguez-Stanley, Malcolm Robinson, Philip B. Miner Jr, Chatham, NJ; Oklahoma City, OK Introduction. Cisapride is a prokinetic agent that reduces esophagealacid exposure in gastroesophageal reflux disease (GERD), an action that has always been attributed to effects of cisapride on gastroesophagealmotility. Recently, human gastric parietal cells have been found to possess KCNQ1 potassium channels that are involved in gastric acid secretion in animals. Cisapride can inhibit these channels in cardiac as well as other tissues. Aim. To assess the potential importance of gastric KCNQ1 potassium channels in the pathophysiology of GERD, we examined the effect of cisapride on meal-stimulated gastric acid secretion, gastric and esophagealacidity, and gastric emptying of solids in subjects with GERD. Methods. Subjects were 3 males and 8 females, ages 28-63, with symptomatic GERD. A low dose of cisapride (lOmg) or placebo was administered 30 minutes before a standard meal. Meal-stimulated gastric acid secretion was measured by in vivo gastric autotitration: a recently developed technique that measuresgastric secretion under physiological conditions. Gastric and esophageal pH were measured continuously from 1 hour before until 6 hours after the meal. Gastric emptying of solids was measured using a [130]octanoic acid breath test. Results. Cisapride decreased meal-stimulated gastric acid secretion from 47 (38-113) mmol/hr (median; interquartile range) to 38 (29-38) [P=0.0186]; decreased integrated gastric acidity from 385 to 193 mmol.hr/L [P = 0.0039]; and decreased integrated esophageal acidity from 3.27 to 1.08 mmol.hr/L [P=O.O010]. Cisapride did not significantly alter number of esophageal reflux episodes~ proportion of subjects who experienced esophageal reflux, or time esophageal pR<4. This low dose of cisapride also failed to alter the T1/2 or the lag phase of gastric emptying of solids. Conclusion. The present results indicate that gastric KCNQ1 potassium channels mediate meal-stimulated gastric acid secretion. Furthermore, the beneficial effects of cisapride in GERD are produced not by its effect on gastrointestinal motility, but from inhibition of meal-stimulated gastric acid secretion with a resulting decrease in postprandial gastric acidity and esophageal acid reflux.

% recovery post acid 80.3+10.0 91.5±7.1 82.9+6.1

$840 Reciprocal Paracrine Pathways Link Atrial Natriuretic PepUde (ANP) and Somatostatin Secretion in the Antrum of the Stomach William R. Gower Jr, Robert W. McCuen, John R. Dietz, Carol S. Landon, Mitchell L Schubert, Tampa, FL; Richmond, VA Rationale. Using molecular biological and immunohistochemica[ techniques, we have localized ANP, a 28-amino acid polypeptidefirst identified in cardiac atrial myocytes,to enterochromaffin (EC) cells in gastric antral mucosa and have demonstrated the presence of its receptor, NPRA, in antral mucosa. The physiologic role of ANP in this region of the stomach, however, is not known. Aim. The aim of the present study was to determine whether ANP regulates somatostatin secretion and whether somatostatin, in turn, can influence the secretion of ANP. Methods. Antral mucosal segments from rat antrum were superfused with Krebs buffer and the superfusate collected at 5-minute intervals for measurement of ANP and somatostatin by RIA. Results. Addition of ANP (0.1 pM to 0.1 p,M) caused a concentration-dependent increase in somatostatin secretion (ECso,0.3 nM; maximal increase, 60_+5% above basal level at 0.01 p,M); P
$843 Interactive Role of Prostaglandin, Nitric Oxide and Sensory Neurons in Duodenal I'1C03 Response Induced by Mucosal Acidification in Rats. Koji Takeuchi, Shigeru Kagawa, Hiroshi Mimaki, Masako Aoi, Kyoto, Japan Duodenal HC03-secretion increases in response tO mucosai acidification by luminal acid. This process is known to be mediated by prostaglandins (PC) and nitric oxide (NO) as well as capsaicin-sensitive afferent neurons, yet their interactive role in the HC03 response has not been much studied. In the present study, we examined the effects of indomethacin, NG-nitroL-arginine methyl ester (L-NAME) and sensory deafferentationon acid-induced HC03 secretion in the rat duodenum, together with the effect of acidification on mucosal PG generation as well as luminal release of NO. Methods: A proximal duodenal loop was perfused with saline, and the HC03-secretion was measured at pH 7.0 using a pH-stat method and by adding 10 mM HCI. Mucosal acidification was performed by exposing the loop to 10 mM HCI for 10 rain. Indomethacin was given SC 30 min before exposure to 10 mM HCl; while L-NAME was given IV 3 h before the acidification. Sensory deafferentation was performed by SC injection with a large dose of capsaicin. In addition, the HC03 stimulatory effect of NOR3 (a NO donor) or capsaicin was also examined. Results: Mucosal acidification increased the HC03-secretion with concomitant increase of mucosal PGE2content and luminal releaseof NO. Indomethacin significantly inhibited the acid-induced HC03-secretion as well as the PGE2 content, without influence on NO release. L-NAME as well as sensory deafferentation attenuated both the increase of PGE2 content and luminal NO following the acidification, resulting in a marked inhibition of the acid-induced HC03 response, and the effects of L-NAME were all antagonized by co-administration of L-arginine. On the other hand, the HC03 secretion was dose,dependently increased by mucosal exposure to NOR3 or capsaicin, with concomitant increase of PGE2 generation; these effects in both cases were mitigated in the presence of indomethacin, and the capsaicin effect was also inhibited by L-NAME as well as ruthenium red, the vanilloid receptor antagonist. Conclusion: These results suggest that 1) both PGE2, NO and sensory neurons are involved in the mechanism for the acid-induced duodenal HC03-secretion, 2) NO released locally in response to acid increases the HC03 secretion, partly by stimulating PG generation, and 3) the HCO3-stimulatory action of capsaicin is mediated by NO released through stimulation of sensory neurons.

$841 Cytoplasmic Clearance of Ca2+ from Intracellular Compartments in Gastric Parietal Cells: Dependence on Mitochondrial Function. Stephanie A. Chamberlain, Aldebaran M. Hofer, David I. Soybel, Boston, MA introduction: Optimal rates of H + secretion by the gastric parietal cell (PC) requires that Ca2+ be made availablefrom intracellutar compartments. Also required are efficient mechanisms for clearing GaS+from the cyoplasm. The Aim of this study was to evaluate the role of the endoplasmic reticulum (ER)and mitochondrial (MT) respiration in clearing Ca2÷ released from intracellular stores in the gastric PC. Methods: Rabbit gastric glands were isolated by collagenase digestion and loaded with the ratiometric Gas÷- sensing fluorescent dye, FURA2. Cytoplasmic Ca2÷ levels were monitored using digital image processing of fluorescence signals in individual cells (n=8 to 9 cells per gland). Glands were perfused for 5 min in 0 Ca2+ media and then exposed to no inhibitor or: 1) thapsigargin (]HPS,lp.M), an inhibitor of uptake mechanisms in the ER; 2} FCCP(IO~M), a protonophore that inhibits MT production of ATP by collapsing the mitochondrial proton gradient; or 3) the combination of 2pM Antimycin A and 5p.g/L oligomycin (AA/OL), agents which inhibit MT respiration and ATP production directly. Glands were then exposed to 5~M ionomycin, which releasesCa2+ from intracellular stores. Results: Under control conditions, ionomycin elicited increases in ceil Ca2÷ levels at 1 rain to levels that were 80 to lOOnM above baseline; these levels returned substantially toward baseline by 4 min, indicating effective cytoplasmic clearance. In response to THPS, a similar peak and fall of Ca2+~were also observed, indicating that inhibition of ER GaS+pumpsdoes not substantially alter cytoplasmic clearance. During exposure to FCCP,the initial ionomycin-induced increase in cell Gas* levets was not impaired by FCCP and was markedly exaggeratedby AA/OL. Strikingly, with MT inhibitors cell Gas÷ levels did not return to baseline; in response to AA/OL these levels continued to climb dramatically to levels lOfold higher than baseline. Conclusions: These findings indicate that inhibition of MT function does not acutely reduce cell Ca2÷andbut does inhibit its clearance from the cytoplasm. The exaggeratedresponse to ANOL suggests that PCs have additional reserves of cell Gas*, that may be released by ionomycin when MT respiration and ATP production are inhibited profoundly.

$844 K÷ and Cl Channels Mediate Shrinkage during Stimulation of Acid Secretion in Cultured Parietal Cells Oliver Bachmann, Alexander Heinzmann, Andreas Mack, Michael Gregor, Ursula Seidler, T(]bingen, Germany Background&Aims: We have recently shown that in isolated and cultured parietal cells, a rapid initial cell shrinkage occurs upon stimulation of acid secretion, followed by a regulatory volume increase (RVI), which is mainly mediated by Na+/H÷ and Cl/HCO3-exchange. The factors leading to the initial cell shrinkage are unknown. We hypothesizedthat either activation of basolateral K+ channels and/or of the apical K* and Cl conductances involved in acid secretion cause this cell shrinkage via KCl and volume loss. Methods: Volume changes in isolated and cultured rabbit parietal cells were monitored by confocal measurement of the changes in calcein concentration in the cytoplasm. Acid secretion was quantified using the ~4Caminopyrine uptake method. Results: Inhibition of basolateral K+ channels with barium led to a rapid cell swelling, indicating that Ba2+ sensitive K÷ channels are also open in resting parietal cells. The chromanol 293b, which inhibits the presumably apically located K+ channel KvLQT1, did by itself not induce cell swelling, but reduced the forskolin-induced cell shrinkage to a higher extent (69%) than the carbachol-inducedshrinkage (44%). Charybdotoxine(ChTX), which has been shown to inhibit Ca2÷ sensitive K+ channels, equally did not induce cell swelling, blocked the carbachol-induced cell volume decrease by 77%, and had no effect on

Control THPS FCCP ANOLIG Pre-lono 50+3 90-+10~ 57+4 67_+8 Iono I rain 120~7i 178_+1711[ 139-+12T 406_+4811[ Iono4 min 74+5t 132~2111] 133--161 807+73111 Resultsin nM, 1"p<0.O01comparedto pre-lono;l p<0.05comparedto controltissues by ANOVA.

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AGA A b s t r a c t s

forskolin-Jnduced shrinkage. The CI- channel blocker NPPBalso had no effect on cell volume in resting cells, but almost completelyinhibitedstimulation-associatedcell shrinkage. Inhibition of acid secretion by 293b, ChTX and NPPB followed the same pattern as inhibition of the initial cell volume loss after stimulation. Conclusion: Cell shrinkage associated with acid secretion is dependenton the activationol K* and C1-channelsby the respectivesecretagogues. Recently published data suggest that these channels may be at least in part located apically. K÷, CI- and water secretion into the secretory canaliculi are thus one likely mechanisms of stimulation-associated cell shrinkage in cultured parietal cells.

$848

$845

Transforming Growth Factor ~ (TGF~) Signaling and Transactivation after Complementation with a Single Copy Of TGF~ Receptor II (TGFL~RII)in DNA Mismatch-Restored Colon Cancer Cells Antonio Fiorino, N Munoz, Betty Cabrera, William M. Grady, John M. Carethers, San Diego, CA; Nashville, TN

Inhibition of Calcineurin (Protein Phosphatase-2B)Suppresses Ca2+-Mediated Acid Secretion in Rats Michiro Otaka, Satoshi Ito, Masaru Odashima, Mario Jin, Noriaki Konishi, Koga Komatsu, Sayuri Kato, Toshihiro Sato, Chieko Nakamura, Tamotsu Matsuhashi, Sumio Watanabe, Akita, Japan [Background] It has been suggestedthat calcineurin (CN, protein phosphatase-2B)regulates signal transduction, particularly in various secretory cells. In this study, we examinedwhether CN playsa role in stimulus-secretion coupling of gastric parietalcells. [Materials and Methods] Localization of CN in gastric epithelial cells was examined immunohistochemically.The role of CN in the acid secretion pathway of gastric parietal cells was assessed by evaluatingthe effect of FK5O6, a specific inhibitor of CN, on gastric acid secretion in pylorus-ligated rats. In addition, the effect of FK5O6 on secretagogue (carbachol, tetragastdn and histamine)stimulated acid secretionwas investigatedin lumen-perfusedrats. [Results] CNwas specifically expressedin gastric parietalceils and chief cells of the gastric mucosalepithelium immunohistochemically. FK506 dose-dependentlyinhibited gastric acid secretion in pylorus-ligated rats. In lumen-perfused rats, FK506 completely inhibited acid secretion prestimulated by carbachol and tetragastrin, agonists known to increasecytosolic Ca2+, but did not affect acid secretion prestimulated by histamine. [Conclusions] Our findings demonstratethat FK506 has a potent antisecretory effect in parietal cells through inhibition of only Ca2+-mediated acid secretion pathways. As EK506 is known to specifically inhibit CN, which plays an important role in signal transduction in various secretory cells, protein dephosphorylationsignaling might also be crucial for gastrin and M3 muscarine receptor-mediatedstimulation of proton pump. $846 Gene Expression by the Rat Parietal Cell Liana Asatryan, Mark Kidd, Yi Wen, Christian Prinz, David Scott, GeorgeSachs, Los Angeles, CA; New Haven, CT; Munich, Germany Background:The parietal cell (PC) plays a pivotal role in gastric acid secretion. While a few genes associated with PC function are known, most other genes have not been identified. We generateda naive PC gonedatabasefrom the rat using oligonucleetide-basedmicroarrays to define novel genes involved in PC function. Methods: Enriched PC populations (>80% pure) were obtainedfrom rat stomachsby Nycodenzgradient centrifugation following pronase digestion and elutriation. RNA was isolated, cRNA targets generated and hybridized to the Affymetrix RU34 GeneChip sets. The overall intensity of each gene was scaled using the GENECHIPsoftware and an Aftymetdx analysis was performed to identity expressedgenes. The data were normalizedto a panel of ribosomal housekeepinggenes. Results: 2778 gene transcripts were identified as expressed but 48% were EST sequences. As expected, genes encodingthe H÷/K--ATPaseheterodimerwere highly expressed(8-12 fold) as were mitochondrial genes (-12 fold), followed by genes encoding the proteasomalcomplex (-1.5 fold) and heat shock proteins. Other markers for PC included CCK2, muscarinic M3 and somatostatin receptors. Vesicular trafficking genes included SNAP 23, prenylated rab, rab proteins 5, 8, 13, 14 and 28, SNARE and clathrin-associatedproteins with SC2 having the highest level (-1.5 fold). Channelswere representedby potassium(KCNQ3,KvLQT1),chloride (RCL1, PKCregulated) channels. CCK1 receptors were absent. The database contained a n~mber of growth factor messages. Pepsinogenand mucin-5 RNA were also present but indicated low contamination. Neuroendocrinecell markers were absent (chromogranin, amine transporter). Although 78% of the genes overlapped with those in mouse (Mills JC et al, PNAS 2001), some gastrointestinal genes were present only in the rat database. Conclusion: Molecular profiles of rat gene databasereflected several known PC functions such as acid pumping and oxidative metabolism. Selectedtrafficking proteins were also found and a potential growthregulating phenotypeof PC was confirmed. Oxidativestress associatedgeneswere also highly expressed. Novel genes that are PC specific will help identity new molecular mechanisms within PC related to function, differentiation or growth.

Background&Aims:TGFI3signaling is a major growth suppressionpathwayfor colonocytesand loss of TGFI3responsivenessis involved in their neoplastictransformation. In the microsatellite unstable (MSI) and hMLHl-mutated cell line HCT116, both alleles of TGFI3RII, an essential component of the receptorfor TGF~, are inactivateddue to mutation of a polyAtract, resulting in a loss of TGFI3signaling. Transfer of chromosome 3 to HCT116 (HCT116+ ch3) corrects MSI becausehMLH1 is located on 3p21. Previouslywe demonstratedthat the wild-type allele of TGFI3RII (located on 3p22) was also present in HCT116+ch3, but cells did not show TGFI3 mediated growth inhibition. Our aim was to determine the mechanism through which HCT116+ ch3 had become resistant to TGFI3's effects on growth, despite having a wild-type TGFI3RIIallele. Methods:We assessedTGFI3RII/RIcomplexformation by immunoprecipltating the TGFI3RIreceptor.We assessedTGF-SMADsignaling by examiningSMAD2 phosphorylation after TGFI3stimulation. Nuclearaccumulation of SMADs in response to TGFI3was analyzed by Western blots of SMADs 2 and 3. TGFl~-inducedtranscriptional activation was assayed by transfecting cells with the luciferasereporter p3TP-luxcontainingTGFI3-induciblepromoter elements of plasminogen activator inhibitor 1. Growth inhibition was studied by clonagenic assays with and without TGFI3 stimulation. Results: TGFI3RII, which is absent in parental HCT116cells, was detectedin HCT116+ ch3 complexedwith TGFI~RIafter ligand stimulation. After TGF~ treatment, HCT116+ch3 cells (but not HCT116) phosphorylated SMAD2 and substantially increased the nuclear levels of SMADs 2 and 3. Transfection with p3TP-lux demonstrated a 6-fold increase in TGFI3 transactivation in HCT116+ch3 cells. Clonagenic assays,however,continuedto show no growth suppressionwith TGFI3treatment. Conclusions: Complementation with a single copy of TGFI3RII in DNA mismatch repair-corrected cells restores TGFI3frGFI3RII/RI complex formation, and restores signaling and nuclear trafficking of SMADs 2 and 3. TGF~ mediated transcription is also restored. Despite this, growth suppressionafter TGFI3treatment is absent.Thesefindings suggestthat there may be modulation of TGFI3signaling by other signaling pathwaysor interactionwith transcriptional cofactors in the nucleus that negatively modulate the growth suppressive effect of the TGF!3-SMAD pathway in colorectal cancer. $849 Phenotypic Alterations of Immortalized Pancreatic Epithelial Cells by Chronic TGF-I~ Treatment Are Associated with Upregulation of Cyclin D1 and Phosphon/lationof MAPK Daisuke Ito, Koji Fujimoto, Ryuichiro Doi, Masayuki Koizumi, Syoichiro Tsuji, Sanae Nakajima, Michiya Kawaguchi, Toshihiko Masui, Eiji Toyoda, Sidhartha Tulachan, Kazuhiro Kami, Tomohiko Mod, Masayuki Imamura, Kyoto, Japan

$847 Defects in TGF-I~ Signaling by SMAD4 and ELF-13Spectrins Are Associated with Gastric Carcinogenesis Varalakshmi Katuri, Yi Tang, Charles J. Fox, Allan Dillner, Pankaj Agarwal, Stuart Danovitch, Thomas Fleury, Bibhuti Mishra, Chu-Xia Deng, Anton N. Sidawy, Lopa Mishra, Philadelphia, PA; NW Washington, DC; Washington, DC; Bethesda,MD Strong evidence implicates the TGF-13signal transduction pathway in the suppression of gastric carcinogenesis.Mice that carry a heterozygousdisruption of the SMAD4 gene develop gastric cancer, thus implicating this downstream molecule, and central mediator in TGF-13 signaling, in this process. We attempt to address the potential mechanism by which this pathway participates in tumor suppression utilizing both biochemical and genetic methods. Human gastric biopsy specimenswere analyzedfor the expression of SMAD4 as well as ELF a novel I~-Spectrin implicated in TGF-13signaling. Both proteins were expressedin the same cells in normal tissue samples, and strikingly advancedgastric carcinoma tissues showed a reduced expression of both proteins. In normal cells, the two proteins appear to co-purity and colocalize upon stimulation with TGF-I3.This appearedto be abrogated in gastric cancer cells. These studies suggest that reduced expression of these two proteins may result in defective TGF-13signaling thereby fostering tumorigenesis. Further evidence is provided by

AGA Abstracts

intercrosses betweenSmad4+/- and elf +/- mice, that revealan exacerbatedgastric phenotype at an earlier age. In order to see how loss of this signal transduction pathway may lead to cancer, we analyzedthe activation of the SAPK kinase cascades in gastric cancer tissues derived from the Smad4+/- deficient mice. Remarkably, constitutive activation of the p38 MAP Kinase pathway was present only in these cells and not in controls. These results underscorethe critical role of TGF-~ signaling in tumor suppressionand suggestthat constitutive activation of p38 MAP Kinase may be a consequenceof the loss of this pathway.

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Introduction: The precise role of TGF-13in pancreaticcarcinogenesisremains unclear,although recent studies have demonstratedthat TGF-13expression is markedly enhanced in the ductal epithelial and stromal cells of pancreaticcarcinomasas well as chronic pancreatitis. Recently, there is increasing evidencethat loss of growth-inhibitory effects of TGF-~ appearsto be an important event that attends malignant transformation of epithelial cells. We hypothesized that chronic TGF-13treatment might alter the growth-inhibitory responsiveness of TGF-13 and induce malignant transformation in pancreatic epithelial cells. In this study, the novel immortalized pancreatic epithelial (IMPE) cells establishedfrom temperature-sensitiveSV40 Large-Tantigentransgenic mice was used. Methods:The IMPEcells proliferatecontinuously at the permissivetemperature (33°C), but proliferation ceasesat the non-permissivetemperature (39°C). (1) Selectionof 'TGF-~-resistant' IMPE cells (IMPE-Tr): IMPE-Tr cells wre generated by continuous exposure of 1 ng/ml TGF-13to the IMPE cells for >50 days. (2) Cell growth assay: IMPE or IMPE-Tr cells were incubatedwith medium containing various concentrations of TGF-I3at 33°C or 39°C for the indicatedtime. Cell number was counted by CoulterCounter. (3) Immunoblot analysis: Expressionsof TI311R,smad4 and cyclin D1 were examined.Further, MAPK activity was analyzed using an anti-active MAPK antibody. (4) In vitro tumorigenicity assay: The IMPE and IMPE-Tr cells were suspended in soft agar and colony formation was observed on day 14. Results: (1) In IMPE cells, TGF-13treatment at the concentration of 1 ng/ml inhibited the proliferation by 50% on day 4 and 75% on day 8 at 33°C, whereas its growth-inhibitory effects significantly decreasedin IMPE-Tr cells. (2) Although there was no difference in the expression of T~tlR and smad4, cyclin D1 expression showed about 2-fold increase in IMPE-Tr cells comparedto that of IMPE cells. Further, phosphorylationof MAPK was upregulatedtime-dependently by TGF-13treatment. (3) In soft agar, no colonies were detected in IMPE cells, whereas many small colonies were observedin IMPE-Tr cells. Conclusions: In this study, we found that continuous exposure of pancreatic epithelial cells to TGF13resulted in loss of its growth-inhibitory responseand acquisition of a tumorigenic phenotype that were associatedwith upreguiation of cyclin D1 and phosphorylation of MAPK.

corresponding controls. In addition, we have observed that serum or EGF/TGF-a induced activation of EGFR in HCT-116 cells was markedly inhibited by either the conditioned media containing ERRP or affinity purified ERRP. Conclusions: We conclude that ERRP is negative regulator of EGFRthat exerts its inhibitory effect by attenuating EGFRactivation and propose that ERRP is an effective inhibitor of epithelial cancer cell growth.

$850 Role of Membrane-Binding Metalloprotease (ADAM) in Transactivation of EGF Receptor (EGFR) Induced by IL-8 Which Stimulates Growth of Human Colon Carcinoma Ceils Yusuke Itoh, Takashi Joh, Katsushi Watanabe, Satoshi Tanida, Tadayuki Oshima, Kyoji Send, Yoshifumi Yokoyama, Shigeki Higashiyama, Makoto Itoh, Nagoya, Japan; Suita, Japan

$853

AIM: Interleukin-8 (IL-8) has been reported to promote tumor cell growth including colon cancer cell by binding its receptors (CXCR1 and CXCR2), which are members of the seven transmembrane G-protein coupled receptor (GPCR) family. Recently, stimulation of GPCR can induce shedding of Epidermal Growth Factor (EGF) ligands via activation of membranebinding metalloprotease, ADAM (a disintegrin and metalloprotease), and can transactivate EGF receptor (Nature 402; 884,1999). In this study, we investigated the mechanism of transactivation EGF receptor (EGFR)by IL-8 in human colon carcinoma cell in vitro. METHOD: The well-characterized human colon carcinoma cell line, Coco-2 was used in this study. Activated EGFRin Caco-2 was determined by immunoprecipitation and Western blot analysis using the antibodies against EGERand phosphorylated tyrosine. The data were quantified by NIH image. DNA synthesis was evaluated by Tritium-thymidine incorporation. Enzymeactivity of ADAM was blocked by specific inhibitor, KB-R7785 (JCB 151: 209, 2000). Phosphorylation of EGFRand Mitogen-activated Protein Kinase (MAPK) were inhibited by AG1478 and PD98059, respectivity. Action of heparin-binding EGF like growth factor (HB-EGF) was inhibited by neutralizing antibody. RESULT: IL-8 (2-2OOng/ml) increased DNA synthesis of Caco2 in a dose dependent manner. IL-8 (2Ong/ml) significantly induced EGFR phospholyration (2 to 6fold) at 5-60 rain after stimulation. Both actions of IL-8 were completely inhibited by ADAM inhibitor, 7785. DNA synthesis induced by IL-8 was also inhibited by EGFRinhibitor, AG1478 and MAPK inhibitor, PD98059. Neutralizing antibody against HB-EGF blocked transactivation of EGFR and DNA synthesis induced by IL-8. CONCLUSION: Our data indicate that IL-8 transactivates EGFR via activation of ADAM in Coco2 cells. HB-EGF plays an important role as a ligand for EGFR in this mechanism. This ADAM mediated transactivation pathway may be essential for IL-8 induced growth of colon cancer.

Nerve Growth Factor Influences Proliferation, Invasion, and Tumorigenicity of Pancreatic Cancer Cells Helmut Friess, Zhaowen Zhu, Jorg Kleeff, Murray Korc, Markus Buechler, Heidelberg, Germany; Irvine, CA Background & Aim: Nerve growth factor (NGF) exerts both stimulatory and inhibitory effects on neuronal and certain non-neuronal tumors depending on the underlying tumor type. Inasmuch as pancreatic nerves are enlarged in pancreatic cancer and almost all tumors show characteristically invasion of nerves by tumor cells, in the present study, the role of NGF in pancreatic cancer cell growth was further characterized. Methods: PANG-1 pancreatic cancer ceils were stably transfected with a full-length human beta-NGF expression vector. The in vitro and in vivo growth characteristics were analyzed by proliferation assays, invasion assays (Boyden chamber assays), and in a nude mouse tumor model Results: Parental and mock transfected PANG-1 cells exhibited exponential doubling times of approximately 48 h. In contrast, beta-NGFtransfected cells displayed significantly shorter doubling times of 24.2 h to 30.3 h. Parental and mock transfected PANG-1 cells exhibited colony-forming efficiencies of approximately 30%. In contrast, in transfected cells the colony-forming efficiencies were approximately 65%. In addition, the invasive ability was significantly higher in transfected cells compared with parental or mock transfected cells (pO.O5) and between beta-NGF transfected cells (p>O.05). Compared with parental and mock transfected PANC-1 cells, the potential of tumor cell growth was markedly enhanced in transfected PANC-1 cells, which had a higher tumor incidence (50% vs. 30%), shorter time to tumor appearance (average: 18 days vs. 26 days), and faster tumor growth. Conclusion: These results suggest that NGF might act in a paracrine and/or autocrine manner to enhance tumor growth and invasion in pancreatic cancers in vivo.

$851 EGFR Overexpression by Retroviral Transduction in Normal Human Esophageal Epithelial Cells Induces Increased Migration and Adhesion Takaaki Mizushima, Hiroshi Nakagawa,Claudia D. Andl, Kenji Oyama, Hideki Harada, Anil K. Rustgi, Philadelphia, PA

$854 Heregulin Activates Ras Down-Stream Signaling and Enhances Apoptotic Resistance in Caco-2 Cells: Roles ot Cox-2 and Akl Sharad Khare, Sonia Cerda, Reba Mustafi, Marc Bissonnette, Chicago, tL

Introduction: Epidermalgrowth factor receptor (EGFR)overexpression,through geneamplification, is associated with esophageal carcinogenesis, and other cancers as well. In particular, EGFR overexpression is an early event in the development and progression of squamous dysplasia. Yet, the biological consequences of EGFR overexpression in normal esophageal cells are unknown. Methods: Primary human esophageal cells (EPC) were established and characterized. Retroviral vectors harboring EGFR or GFP cDNAs were transduced into EPC cells to yield EPC-GFPand EPC-EGFR.Biochemical analysis of EGFRand downstream targets (Erk 1/2, Akt) was performed by Western blotting using phosphotyrosine antibodies and Erk 1/2 and Akt specific antibodies. Finally, cell migration on type 1 collagen using Boyden chamber methods and cell adhesion to extra cellular matrix coated plates were assayed. Results: The retrovirally mediated transduction of EPC cells with EGFR led to increased expression and tyrosine phosphorylation of EGFRIThis was accompanied by enhanced phosphorylation of Akt and Erk 1/2. Cell migration assays demonstrated that migrated cell counts of EPC-EGFR cells increased almost double compared with controls, and that LY29004 (phosphatidylinositol 3-OH kinase inhibitor) and G56976 (PKC inhibitor) but not U0126 (MEK1/ 2 inhibitor) reduced by 50% the migrated cell counts in a dose dependent fashion. Integrin neutralizing antibodies also reduced the migrated cell counts by 50-80% while high titer of the antibodies was required to inhibit cells overexpressing EGFR. Adhesion assays revealed that adhesion of EPC-EGFRcells increased30-50% compared with control cells. Conclusions: EGFRoverexpression leads to increased cell migration and adhesion in our novel cell culture model. The observed effects may be mediatedthrough both activation of the phosphatidylinositol 3-OH kinase and PKC pathway as well as integdn function. These novel mechanistic insights provide clues as to how EGFR may contribute to early events in esophageal carcinogenesis.

BACKGROUND:The azoxymethane (AOM) model of rat colon cancer has been widely used to study colonic carcinogenesis. In addition to mutations activating Ras, we have recently shown wild type Ras could be activated by growth factor signaling in AOM tumors. We have extended our studies to characterize in vitro the down-stream signaling of growth factoractivated Ras in Caco-2 cells, derived from a human colon cancer. These cells express wild type Ras and ErbB3 and ErbB2 receptors. METHODS:Proliferating Caco-2 cells were treated with 100 ng/ml heregulin and cell lysates prepared at the indicated times. The activities of ERK1, ERK2,p38 and Akt were assessedby Western blotting using phospho (active) antibodies. Cox-2, C/EBPI3, phosphorylated IKB~xand cyclin D1 proteins were measured by quantitative Western blotting. Ras activation (Ras-GTP) was assessed by a Ras pull-down assay using Sepharose beads conjugated to the Ras binding domain of c-Raf: For apoptosis studies, cells were plated overnight and then treated with heregulin, followed by butyrate 30 min later in sere free conditions. Cells were fixed 24 hrs later and apoptotic nuclei identified by TUNEL. RESULTS: Heregulin, an ErbB3 receptor ligand, activated Ras in a time-dependent manner with maximum activation by 10 min. Heregulin activated ERK1, ERK2, p38 and Akt, but not JNK. By 24 hrs heregulin increased Coco-2 proliferation and induced Cox-2, but not cyclin DI. Cox-2 induction was accompanied by increasedC/EBPI3and activation of NFKB (assessed by phosphorylated IKBc~), MAPK-regulated transcription factors implicated in Cox-2 gene expression. In addition, heregulin increased apoptotic resistance of Caco-2 cells to butyrate. CONCLUSIONS: Heregulin stimulates proliferation and activates Ras and several Ras downstream effectors, including ERKs, Akt, p38 and Cox-2. Several MAPK-regulatedtranscription factors implicated in Cox-2 gene expression are induced and/or activated by this growth factor. Heregulin signalingl moreover, increases apoptotic resistance to butyrate, which may involve Akt activation and Cox-2 induction.

$852 Inhibition of EGFR Actvation and Cell Proliferation by ERRP: A Novel Negative Regulator of EGFR Lathika Moragoda, Yingjie Yu, Arun K. Rishi, Zhi-Oiang Xiao, Adhip P. N. Majumdar, Detroit, MI

$855 Keratin 8 Associates with and Sequesters Raf Kinase during Interphaue in a Phosphorylation-DependentFashion Nam-On Ku, Haian Fu, M Omary, Paid Alto, CA; Atlanta, GA

Background: Receptortyrosine kinasefamily members are frequently implicated in experimental models of epithelial cell neoplasia as well as in human cancers. One of the best studied receptor signaling systems from this family is that controlled by the EGF-receptor (EGFR), whose expression and enzyme activity have been linked to a number of malignancies, including gastric and colon cancers. However, the intracellular events that regulate EGERhave not been fully elucidated. Recently, we described the isolation and characterization of a 1.95 kb cDNA done, referred to as ERRP (EGER Related Protein), from a rat gastric mucosal library that shows 85% nucleotide homology to the extra cellular domain of the rat EGFR (AJP:Cell Physiol. 280:C1083,2001). We have reported that transfection of ERRP cDNA into colon or prostate cancer cell lines not only inhibits EGFRactivation but also decreases proliferation in matrix-dependent and independentassays. Methods: To further study the functional properties of ERRP, we generated 55 kDa ERRP fusion protein in Drosophila expression system using pMTN5-HisA vector. Upon induction with CuS04, ERRP was found to be secreted in the medium. The media containing ERRP was used directly in the following experiments. ERRP was also affinity purified from CuS04 induced cell extracts using either a Ni-chelated or a His-antibody column. Results: Exposure of colon (HCT-116) or prostate (PC-3) cancer cells to either the conditioned media containing ERRP or affinity purified ERRP caused a marked inhibition of proliferation and stimulation of apoptosis, when compared with the

Background: Keratins 8 and 18 (K8/18) are cytoplasmic intermediate filament proteins of simple-type epithelia as found in the intestine, liver and pancreas.We previously showed that K18 phosphorylation regulates its association with the 14-3-3 protein family during mitosis in cultured enterocytes (EMBO J 17:1892-1906, 1998). The potential significance of keratin/ 14-3-3 binding is related to the phosphorylation-dependent association of 14-3-3 proteins with a wide range of signaling molecules including Raf-1 kinase, protein kinase C, and cdc25 phosphatase among others. Aims: (i) Identity the 14-3-3 domain that is important for keratin binding, and (ii) test the hypothesis that keratin binding to 14-3-3 proteins serves either as an adapter or anchor to bring together different 14-3-3 binding proteins, or serves to displace 14-3-3 binding proteins to allow their generalized cell availability. Methods: We used already characterized 14-3-3 mutants, which prevent Raf/14-3-3 binding, to test their binding to K8/ 18. We also used in vitro reconstitution, kinase assays, phosphopeptide mapping, and cell transfection with various Raf-1 and keratins constructs to address the above aims. Results: Immunoprecipitation of K8/18 from cultured human colonocytes or mouse liver, using a panel of anti-K8/18 antibodies, showed that Raf-1 kinase binds to K8/18 at a high stoichiometry during interphase. K8/18-Raf binding does not occur during mitosis when 14-3-3 binds to

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K8/t8. Raf-keratin binding occurs directly with K8 but not K18 or K19, is inhibited by keratin phosphorylation, maintain Raf Idnase activity, and is independent of the known K8 binding with heat shock protein 70. The same 14-3-3 charged residues (K49, R56, and R60) that regulate Raf-1 kinase binding to 14-3-3 proteins also regulate 14-3-3 binding to keratins. Conclusion: K8/18 associate with Raf-1 kinase in vivo during interphase then release the kinase during mitosis or mitogenic stimulation. This provides a unique way for the cytoskeleton to regulate mitogenic stimuation events and may provide a molecular explanation for the observed colonic hyperplasia that is noted in KS-null mice.

DCA, and CDCA stimulated HB-EGFexpression strongly. HB-EGF expression induced by LCA increased in a dose- and time-dependent manner. MIB-1 index also increased in a dosedependent manner up to 200r~M in TEl and up to lOOI~M in TE7. Conclusion: Bile acids may concern the growth of esophageal cancer because bile acids induced the expression of HBEGF and LCA promoted the proliferation of TEl and TE7.

$859 Active PI-3 Kinase/Akt2 Signaling Pathway Is Found in Lipid Rafts of the Caco-2 Colon Cancer Cell Line but Not Normal Intestinal Epithelial Cells: Possible Implications for Tumorigenesis Xuhang Li, Sharon Leu, Morris J. Birnbaum, Chris Shih, Mark Donowitz, Baltimore, MD; Philadelphia, PA Lipid rafts, also known as detergent resistant membranes (DRM), are involved in signal transduction and membrane protein trafficking. Although phosphotidyl-inositol 3-kinase (PI3K) has been shown to be associated with DRM, the significance of such an interaction in PI3Kmediated signal transduction remains unexplored. Since the PI3K/Akt signaling pathway is an important normal anti-apoptosis mechanism, we tested the hypothesis that DRM/lipid rafts may play an important role in the PI3K/Akt signaling pathways in both colon cancer and normal intestinal epithelial cells. Both Aktl and Akt2 were detected in the colon cancer cell line Caco-2 cells, whereas only Akt2 was present in ileal villus cells. Density gradient fractionation experiments showed that both PI3K and Akt2 were expressed mainly at the apical surface of normal rabbit ileal epithelial cells and Caco-2 cells. PI3K was present predominantly in the Triton X-lOO-resistant membranes of both cancer and normal epithelial cells. Akt2 was also mostly in the DRM of ileal villus cells but was equally distributed in the DRM and Triton XlO0-soluble (DS) pools in Caco-2 cells. EGF treatment stimulated Akt2 activity in the ileal brush border membranes (BBM). However, basal and EGF-stimulatedAkt2 activity could only be detected in the DS but not in the DRM of ileal BBM. In contrast, Akt2 from the DS and DRM pools isolated from total membranes of Caco-2 cells was equally active. Ftoatation experiments using Opti-Prep gradients showed that PI3K and Akt2 were associated with lipid rafts of the same density. However, only lipid raft-associated Akt2 from Caco-2, but not that from intestinal cells was active. To explain these differences in Akt2 activity, we studied PTEN, a phosphatase that counteracts PI3K function by dephosphorylating the 3'-phosphate of PI(3,4,5)P3 and thereby inhibits Akt activity. PTENwas also present in the lipid rafts of normal intestinal apical membranes, in Caco-2 cells, however, PTEN was excluded from lipid rafts. These results 1) show that a specific pool of PI3K and Akt2 is associated with lipid rafts in intestinal epithelial cells, 2) suggest that the co-localization of PI3K and PTEN in lipid rafts of ileum may explain why raft-associated ileal Akt2 is inactive, and 3) indicate that an active raft-associated pool of Akt2 in Caco-2 cells might be due to the lack of PTEN in lipid rafts. These results may explain the rarity of small intestinal cancer

$856 Difterentiation-inducing Factor-1 (DIF-1) SuppressesCell Growth by Inhibiting STAT3 Activity Via MEK-ERKDependent Pathway in Gastric Cancer Cells Naoki Kanda, Yositaka Konda, Masashi Kanai, Yoshio Izumi, Toshio Nakajima, Apichart Nanakin, Tsutomu Chiba, Kyoto, Japan Background: Differentiation-inducing factor-1 (DIF-1) is a putative morphogen isolated from Dictyostelium. DIF-1 has been reported to exhibit anti-tumor activity in several kinds of mammalian tumor ceils including human leukemia cells, however, the underlying mechanisms remain unknown. On the other hand, recent studies have indicated that constitutively activated STAT3 plays as an oncogene in breast cancer cells. Aim: To study whether DIF-1 possesses anti-tumor activity against gastric cancer, and the mechanisms of anti-tumor activity of DIF1 involve inhibition of STAT3 in these cultured cells. Method: To evaluate the effect of DIF1 stimulation on cell cycle, flow cytometric analysis was performed using AGS and MKN28 cells, and growth of these cells was assayed using WST-8 cell counting kit. To observe the phosphorylation status of STAT3,we performed Western blot analysis using specific antibodies against phosphorylated Ser-727 and Tyr-705 of human STAT3. To show the changes of the DNA binding activity of STAT3 induced by DIF-1 stimulation, electrophoretic mobility shift assay (EMSA) was done. Result: Addition of DIF-1 into culture medium increasedthe number of both AGS and MKN28 cells in GO/G1 and G2/M phases. Moreover, blockade of STAT3 by dominant-negative STAT3inhibited growth of AGS and MKN28 cells. OIF-1 selectively increased phosphorytation of Ser-727, but decreased Tyr-705 phosphorylation of STAT3 by MEK-ERK dependent manner. In addition, DIF-1 inhibited DNA binding activity of STAT3in gastric cancer cells. Conclusion: These results suggest that DIF-1 inhibited gastric cancer cell growth by inhibiting STAT3 activity. This is the first report of a possible anti-tumor agent that targets STAT3 activity.

$B57 interaction of Bile Acids with M3 Muscarinic Receptors on H508 Human Colon Cancer Cells Activates the MAP Kinase Pathway Kunrong Cheng, Piotr Zimniak, Jean-Pierre Raufman, Little Rock, AR

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Previously, we showed that conjugates of lithocholic acid stimulate H508 colon cancer cell proliferation by muscarinic mechanisms (Gastroenterology 118; A558, 2000). Muscarinic receptors are coupled to mitogen-activated protein kinase (MAPK) (Biochem. J. 348: 381, 2000). MAPKcan be phesphorylated by MEK and then activatetranscription by phosphorylation of kinases like ribosomal $6 kinase (p9ORSK).Objective:To determine the events downstream of the receptor that mediate bile acid-induced colon cancer cell proliferation. Methods: Colon cancer cell lines (H508 cells that express M3 muscarinic receptors and SNU-C4 that do not) were used to examine bile acid-induced changes in (a) cell proliferation as determined by the sulforhodamine B colorimetric assay and (b) downstream muscarinic signaling events as determined by phosphorylation of MAPK and p9ORSK. Potential cell toxicity was determined using trypan blue exclusion. Results: Lithocholyltaurine (LCT,300 ~M) and deoxycholylglycine (DCG, 300 p.M) increased H508 cell proliferation and p44/42 MAPK and pgORSKphosphorylation. These actions were inhibited by the muscarinic receptor inverse agonist atropine (1 ~M), the calcium chelator BAPTA {5 p.M) and a MEK inhibitor, PD98059 (10 t~M). LCT and DCG did not alter trypan blue exclusion. Similar concentrations of LCT and DCG did not alter proliferation of colon cancer cells (SNU-C4) that do not express M3 receptors. Acetylcholine (300 rtM) had the same actions on H508 cells as the bile acids. In contrast, like the bile acids, acetylcholine did not stimulate SNU-C4 celt proliferation. Conclusions: Interaction of bile acids, specifically lithocholyltaurine and deoxycholylglycine, with M3 muscarinic receptors stimulates colon cancer celt proliferation by inducing pgORSKphosphorylation via a calcium-, MEK- and MAPK-dependent pathway. Elucidation of this signaling pathway may suggest therapeutic targets to prevent colon cancer cell proliferation.

Genetic Control of Functional Activation of the Human IL4-Stat6 Signaling Pathway Wen Jie Zhang, Anna F. Tilberg, Walter A. Koltun, Hershey, PA Background: Signal transducer and activator of transcription 6 (Stat6) is involved in a signaling pathway that is activated by IL4. The IL4-Stat6 pathway is composed of at least 6 genes encoding IL4, IL4R,x, .~ chain, Jakl, Jak3 and Stat6. It plays an important role in biology by regulating gene activation in responsive cells to modify cell growth, differentiation and apoptosis. Mice lacking Stat6 exhibit defective Th2 cell development but enhancedtumor immunity. The pathway is active in a variety of cancer cells including colon cancer. Stat6 activation is undoubtedly determined by the genes involved but practical means to study it have been difficult. Methods: EBV-8 cell lines were stimulated with hlL4 and extracted nuclear proteins (2p.g/sample) were examined using EMSA in 5% gel with a ~P-labeled oligo (SBE1) and an antibody specific to Stat6. For consistency, all cell lines were referencedto a standard control. Optical densities (O.D.) were obtained for Stat6 bands and the results were expressed as a T/C 0.O. ratio (Test band O.D. vs Control band O.D.). Results: This semi-quantitative assay recognized 3 major Stat6 activation phenotypes assigned as Stat6"~" (full activation, T/C ratio >0.5), Stat6~°* (moderate activation, T/C ratio 0.1-0.5) and Stat6~ud~(defective activation, T/C ratio <0.1), respectively. Pedigree analyses in 5 informative families revealed Mendelian inheritance for these phenotypes and 2 STAT6A(Stat6 activation) genotypes were deduced as a and A (a determines an inactive Stat6 signaling while A determines an active Stat8 signaling). Homozygous STAT6A*aaexpresses a defective Stat6~" and homozygous STAT6A*AA gives rise to a fully activated Stat6"~0".Thus, heterozygous STAT6A*Aaencodes a moderate Stat6h°*.The deduced STAT6Agenotypes segregated and transmitted within family members in a Mendelian fashion, e.g., aa (Stat6~u~)x AA (Stat6"~") = Aa (Stat8~°*) and aa (Stat6"u~) x aa (Stat6n"") = aa (Stat6"u~). Conclusions: Using a genetic strategy to study functional activation, this is the first demonstration that the IL4-Stat6 signaling is genetically controlled in a Mendelianfashion. We propose that STAT6Agenotype is a collective determinant involving more than one locus. A major locus determinesthe majority of phenotypic expression and one or more minor loci contribute to minor phenotypic differences. This approach may lead to a new way for signal transduction research and the Stat6"°" cell lines may provide a natural human model for study in the same way as a Stat6+ mouse model.

$858 Bile Acids Induced the Expression of Heparin Binding Epidermal Growth Factor Like Growth Factor (HB-EGF) in Esophageal Cancer Cell Lines. Yukihiro Satoh, Hiroyuki Mutoh, Sayaka Honda, Yutaka Sekine, Akira Ohashi, Kiichi Tamada, Kentaro Sugano, Tochigi, Japan Background: Bile acids have been considered tumor promoters in the colon. However, the relationship between esophagealcancer and bile acids has not been clarified. Recent studies have demonstrated that heparin binding epidermal growth factor like growth factor (HB-EGF) plays a role in carcinogenesis and carcinoma progression in colon, breast, pancreatic cancer and hepatocellular carcinoma. The aim of this study is to investigate whether bile acids induce the expression of HB-EGF and stimulate the proliferation in human esophageal cancer cell lines. Methods: Human esophagealsquamous carcinoma cell ~ine(TEl) and human esophageal adenocarcinoma cell line (TE7) were stimulated 6 hours with 2001~M of each bile acid, chenodeoxycholic acid (CDCA), lithocholic acid (LCA), deoxycholic acid (DCA), cholic acid and ursodeoxycholic acid. Following stimulation, cells were harvested and the expression of HB-EGF mRNA was assessed by Northern blot. As for LCA stimulation, the relationships between expression of HB-EGF mRNA and time or dose were assessed. In order to examine the effect of LCA on the proliferation, cells were stimulated 6 hours by LCA (20, 100, 2001~M), and the proliferation was evaluated by immunohistochemistry using MIB-1 antibody. Result: All of bile acids examined induced the expression of HB-EGFin TEl and TE7. Especially, LCA,

AGA Abstracts

$861 HIF-IndepeudeutInduction of VEGF by Hypoxia in Colon Cancer Cells Xiaobo Zhang, Yusuke Mizukami, Daniel C. Chung, Boston, MA BACKGROUND:Vascular endothelial growth factor (VEGF)plays a pivotal role in the regulation of tumor angiogenesis. One of the most potent stimulators of VEGF expression is hypoxia, and this induction is typically mediated through hypoxia-inducible factor (HIF). The mechanisms that regulate VEGF expression in colon cancer are not well defined, and we sought to determine whether HIF also regulatesthe hypoxic induction of VEGFin colon cancer. METHODS: Hypoxic conditions were achieved by culturing cell lines in a sealed hypoxia chamber. Northern blotting was performed with a 400 bp human VEGFcDNA. VEGFprotein levels were determined

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by ELISA. Human VEGF gene promoter fragments were subcloned into the pGL2basic vector and transiently transfected utilizing cationic lipids. Reporter activity was normalized to the activity of a co-transfected pRL-CMV vector. Site directed mutagenesis was performed to selectively alter the HIF binding site in the VEGF promoter. RESULTS: Caco2 and Lovn ceils cultured in 0 % oxygen demonstrated a twofold upregulation of endogenous VEGF mRNA, and VEGF protein levels were upregulated 50%. A luciferase reporter construct containing 2.8 kb of the human VEGF gene promoter was similarly upregulated by hypoxia in Caco2 ceils. Both a 5'-deletion construct that eliminated the HIF binding site at -975 bp and a full-length VEGF promoter construct with a selectively mutated HIF site retained eumplete responsiveness to hypoxia in Caco2 cells. Control studies in Hela cells demonstrated no hypoxic induction of VEGF when this HIF site was mutated. The role of the K-ras signaling pathway in the regulation of VEGF by hypoxia was then determined. Expression of mutant Krasvat~2enhancedthe hypoxic induction of VEGFin an HIF-independentmanner in colon cancer cells. Neither an AP1 nor Ets binding element mediated this response. Inhibition of PI3K signaling with the dominant negative SRAp85 vector blocked this hypoxic induction by 40%, but no such inhibition was observed with a dominant negative Akt vector (AktK179A). CONCLUSIONS:The VEGF gene is upregulated by hypoxia in colon cancer ceils. In contrast to most other cell types, hypoxia-inducible factor (HIF) is not required for this upregulation. Signaling through K-ras enhances the HIF-independent induction of VEGF by hypoxia, and this effect appears to be mediated through PI3K but not Akt. These findings suggest a novel role for K-ras in the hypoxic induction of VEGF in colon cancer.

$864 Progastrin and Cyclooxygenase-2in Colorectal Cancer Peter C. Konturek, Wladyslaw 8ielanski, Stanislaw J. Konturek, Piotr Pierzchalski, Elzbieta Karczewska,Krzysztof Marlicz, Teresa Starzynska, Jens Rehfeld, Eckhardt Hahn, Erlangen, Germany; Cracow, Poland; Szczecin, Poland; Copenhagen, Denmark Colorectal cancers (CRC) are frequent all over the world and concern mostly the older population. The tumors have been attributed to genetic, dietary and other environmental factors, but recently also growth factors like gastrin, havebeen implicated in the carcinogenesis. Since Helicobacterpylori (Hp) infection is usually accompanied by increasedplasma concentrations of progastrin and amidated bioactive gastrin, that are also potent mitogens for gastric and colonic mucosa and inducers of cyclooxygenase-2 (COX-2), we decided 1) to compare plasma concentrations of progastrin and amidated gastrin in 50 CC patients before and 3-6 months after removal of the tumor, 2) to determine the tumor concentrations of gastrin peptides and the level of expression for gastrin mRNA and gastrin/CCKB receptor mRNA, 3) to examine the expression of cyclooxygenase COX-1 and COX-2 mRNA in CC tissue and 4) to compare the prevalence of Hp and its cytotoxic protein, CagA, add cytokines (TNFa, ILlb and IL-8) in CRCs, before and after removal of tumor. It was found that CRC, its resection margin and plasma contained severalfold higher levels of progastrin than of amidated gastrins and that the removal of the CRC tumor resulted in a marked reduction in plasma progastrin level without significantly affecting the ratio for progastrin versus amidated gastrins. The seroprevalenceof Hp, especially that expressing CagA, but not cytokines (except IL-II3) were significantly higher in CRC patients (84%) than in age- gender- and profession-matched controls (62%) and did not significantly change about 3-6 months after tumor resection. The gastrin and CCKB-R mRNA were detected in the cancer tissue and resection margin by RTPeR and, similarly, COX-2 mRNA was expressed in these tissues of most of CRCs. We conclude that; 1) CRC and its margin contain large amounts of progastrin and show gene expression of gastrin, CCKB-R and COX-2; 2) Removal of CRC markedly reduced the plasma concentrations of progastrin; 3) Hp infection is enhanced in CRC and this may contribute to colorectal cancerogenesisvia overproduction in cancer cells of progastrin and gastrin that in turn might upregulate COX-2 expression and subsequent stimulation of the tumor growth, angiogenesis and vascular invasion and 4) assessment of plasma progastrin concentrations might serve as CRC marker.

$862 Effects of Cimetidine and Ranitidine on the Growth of Colon Cancer in Wild and Histamine H2 Receptors KnockoutMice ; Role of IL-12 Kazuyoshi Tomita, Kuniharu Matsuno, Atsushi Yoshida, Takehiro ]watsubo, Kikuko Amagase, Susumu Okabe, Takeshi Watanabe, Kyoto, Japan; Fukuoka, Japan Background & Aims: Histamine H2,receptor antagonist cimetidine exerts anti-tumor effect in man and animals, probably by enhancing the host-immune response against tumor cells or by blocking the cell growth-promoting activity on histamine in cancer cells. We thus examined the anti-tumor effects of cimetidine and ranitidine in wild and histamine H2-receptor KO (H2RKO) mice and the underlying mechanism. Methods: Drugs were given orally twice daily to wild-type mice (C578L/6) and H2R-KO mice bearing colon cancer (colon 38) for 26 days. Control mice received vehicle alone. The volume (mms) of cancer was determined every 2 days throughout the experiments, and the expression of IL-12 mRNA and VEGF protein (RTPeR, western blot) was determined after the treatment. Cell growth of colon 38was assessed in in vitro study, by determing the incorporation of 3H-thymidine into DNA. Results: Both cimetidine (45, 90 and 180 mg/kg/day) and ranitidine (7.5, 15, 30 mg/kg/day) suppressed the growth of colon cancer dose-dependently in wild-type mice, beginning from 14 days after treatment until the end of study. The growth suppression was significant at 180 mg/kg/day for cimetidine and at 15, 30 mg/kg/day for ranitidine. In such wild-type mice, there was a tendency of elevation of IL-12 mRNA expression and suppression of VEGF protein expression in cancer tissues. In contrast, both eimetidine and ranitidine had no effect on cancer growth, IL-12 mRNA expression and VEGF protein expression in H2R-KO mice. Neither cimetidine nor ranitidine could exert any suppressive effect on the growth of colon cancer cells in in vitro study. Conclusions: The anti-colon cancer effects of cimetidine and randidine appear to be due to the increased tL-12 production and suppressed VEGFin cancer tissues, which may strengthen host immune responses and suppress angiogenesis in tumor.

$865 In Vitro Evidence of the Role of COX-2 in Attenuating Gastric Inflammation and

Promoting Gastric Carcinogenesis Ki-Baik Hahm, Sung-Won Cho, Jin-Hong Kim, Hee-JaeJog, Seong-Hyang Sohn, Hyuck-Jae Kwon, Young-Bae Kim, Byoung-Ok Ahn, Tae-Young Oh, Jeong-Sang Lee, Suwon, Kyunggi, South Korea; Yongin, Kyunggi, South Korea; Seoul, South Korea Although gastric adenocarcinoma is one of the most common malignancies in the world, little is known about its exact molecular processes of development and progression. Recent studies suggest that COX-2 is important in carcinogenesis of gastrointestinal cancers~especially involved in carcinogenesis in a mouse model of familial adennmatosis polyposis. To understand the role of COX-2 in gastric carcinogenesis and Helicobacter pylori-associated gastritis, we measuredCOX-2expression in 170 human gastric carcinoma tissues by immunnhistochemical analysis and compared the expression of COX-2 in paired tissues obtained from normal looking mucosa and cancer bearing mucosa. To know the further evidence of the involvement of COX-2 in gastritis and gastric carcinogenesis, we stablished stable cell lines overexpressing COX-2. After subcloning of COX-2 into pCB7 mammalian expression vector, two stable cell lines named MKN-28-COX-2 and MKN-45-COX-2 were generated by transfection of COX-2 cDNA. For knowing the effect of COX-2 on gastritis, we performed electrophoretic mobility shift assay of NF-KB, inflammation-associated transcription factor, and measured malondialdehyde levels and chemiluminescence activities in both mock-transfected MKN and MKN-COX-2cells after stimulation of H. pylori(1 X IO%FU/ml) and neutrophils (1 X 102 cells/ml). Marked attenuation of NF-KB bindings and generation of free radicals were observed in COX-2 overexpressed cells. Another sets of experiment including the growth inhibition by TGF-13treatment, Matrigel invasion assay, and apoptosis assay were done. COX2 showed advantagesof the escape from the growth inhibition by TGF-13through decreasing TGF-13RII expressions and increasedcell invasiveness.Conclusively, COX-2expression seems to be }nducedto attenuatethe degree of atrophic gastritis, initial eventsto gastric carcinogenesis and promoted gastric carcinogenesis.

$863 Mechanisms of Bombesin-lnduced Opposing Effects on the Growth of RIE/GRPR and MC-26 Cells Ji-Zhong Cheng, Yan-Shi Guo, Mark R. Hellmich, Courtney M. Townsend, Galveston, TX PURPOSE.Gastrin-releasingpeptide receptor (GRPR) distributes throughout the gastrointestinal (GI) system and overexpresses in colorectal carcinomas. Activation of GRPR by GRPor its homologue, bombesin (BBS), elicits a wide range of biological actions. However, the role of these peptides in growth regulation and/or differentiation of GI cells remains undefined. The objectives of this study were: 1) to determine whether BBS affects growth of a nontransformed rat intestinal epithelial cell line stably transfected with GRPR (RIE/GRPR) and a mouse colon carcinoma cell line which possesses native GRPR (MC-26); 2) to determine mechanisms involved. METHODS.Subconfluent monolayer RIE/GRPR and MC-26 cells were treated with BBS (100 nM) in serum-free conditions. Cell number in control and treated groups was counted using Coulter counter. Cell differentiation was assessed by intestinal alkaline phosphatase (lAP) activity assay. Expression of cell cycle inhibitors, WAF-1 and GADD45 (growth arrest and DNA damage-inducible gene), were determined by Northern and Western blots. The protein levels and promoter activity of Cyclin D~, a regulator of the cell cycle GcS phase checkpoint, were examined by Western blotting and luciferase assay, respectively. RESULTS.After 48 h treatment, BBS caused a depression in cell number of RIE/ GRPR cells by 42% and an increase of the MC-26 cell growth by 35%. BBS also stimulated the lAP activity in RIE/GRPRcells in a time-dependent fashion. Furthermore, BBS significantly increased the levels of WAF-1 mRNA and protein in RIE/GRPRcells, but decreasedthe levels of GADD45 mRNA and protein in MC-26 cells. In addition, Cyclin 0~ protein levels and its promoter activities by BBS administration were inhibited and stimulated in RIE/GRPR and M0-26 cells, respectively. CONCLUSIONS.BBS induces a growth inhibition and differentiation in non-transformed RIE/GRPRcells, which may be due to the increase of WAF-1 and inhibition of cyclin D~ expression in the cells. On the other hand, 8BS stimulates the growth of MC-26 colon cancer cells, which may result from the decrease of GADD45 level and elevation of cyclin D~ expression. The BBS-elicited opposing actions on the growth of different cell types reflect the existence of multiple coupling pathways of GRPR to intraeellular targets in intestinal cells.

$866 NSAIDS Inhibit the Proto-OncogeneAKT: Potential Mechanism for Modulation of Apoptosis and Proliferation Hemant K. Roy, William J. Karolski Jr, James Gulizia, Anne Ratashak, Dahn Clemens, Omaha, NE We have recently reported that AKT proto-oncogene (protein kinase B) is overexpressedearly in colon carcinogenesis (Roy et al., Carcinogenesis 2602) and is detectable in hepatomas. The anti-apoptotic effects of AKT are partly due to inactivation of glycogen synthase kinase (GSK)-313.AKT also enhancesproliferation through inhibition of p21 cip/waf nuclear translocarich, thus fostering G1-S phase progression. Since chemoprevention of GI malignancies by NSAIDS involves alterations in apoptosis a nd proliferation, we hypothesized that NSAIDS may inhibit AKr signaling. METHODS: Human colon cancer (HT-29) and hepatoma (HepG2) cell lines were treated with NSAIDS (either 150~M nabumetone or 800~M sulindac, respectively) for 5 days. FAGSanalysis was performed for cell cycle distribution and apoptosis (by subdiploid fraction and M30 labeling). Immunoblot and immunocytochemistry was conducted with standard techniques. AKT and GSK-3~ activity was determined by an immunoprecipitationkinase assay using enzyme-specific substrates. All data is expressed as % of vehicle control. RESULTS: NSAID treatment induced apoptosis in 38_+4% of HT-29 cells but <2% of HepG2 cells. Sulindac markedly decreasedHepG2proliferation (to 73.3 _+12.6%, p
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n d 75.9± 12 (P< 0.001) respectively, with a parallel decline in the activated enzyme (serine473 AKT) to 71_+14% and 79_+10%, respectively, P
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Mechanisms of Dendritic Cell Vaccine Failures in Colon Cancer-Bearing Mice John Y. - C. Kao, Chuan-Min Chen, Yusong Gong, Qiong-Duan Zheng, Jian-Jun Chen, Ann Arbor, MI

Signaling through RACK1, a CommonAdaptorfor the Src and PKC Protein Kinases Laura D. Clements, Kelly C. Lee, BeTtyY. Chang, Christine A. Cartwright, Stanford, CA The Src family of intracellular tyrosine kinases participates in diverse signaling pathways that regulate cell growth, differentiation, adhesion and architecture. We and others showed that downregulation of Src activity is important for differentiation and upregulation for growth and transformation of intestinal cells. Thus, it is important to search for mechanisms that regulate Src activity; in doing so, we will learn more about mechanisms that regulate intestinal cell growth. To isolate proteins that interact with Src and potentially regulate its activity in intestinal cells, we used a yeast two-hybrid assay and identified RACK1 as a novel Src substrate and binding partner, and an inhibitor of Src kinase activity and cell growth. RACK1 is one of a group of PKC-interacting proteins collectively called RACKs(_R.eceptorsfor Activated C Kinase). RACK1appearsto be required for the translocation of cytosolic 1311PKCto theplasma r~e-mbrane where isozyme-specific substrates are located. Previously, we found that PKC activation induces the intracellular movement and co-localization of RACK1 and Src at the plasma membrane and the tyrusine phosphorylation of RACK1. We have further characterized the mechanism by which RACK1 functions in Src and PKC signaling pathways. We found that RACK1, Src and 1311PKCare present in a trimolecular complex. We used specific peptides that inhibit or promote interactions between 1311PKC,RACK1 and/or Src (and thereby selectively modulate their function) to define the role of individual components of these signaling pathways. Our findings reveal an interesting intersection between Src and PKC signaling pathways that involve a common adaptor protein, RACK1. Because binding of RACK1 to Src inhibits Src activity, and Src activity appearsto regulate intestinal cell growth, modulating the interaction of Src with RACK1 may elucidate signaling pathways leading to colon carcinogenesis and could result in the identification of novel therapeutic targets.

$868 Endostatin is Expressed by Esophageal, Gastric and Pancreatic Cancers and is Regulated by Tumor Necrosis Factor Alpha Anant Desai, Roger Brammer, Simon Bramhall, Margaret Eggo, Birmingham, UK Background: Endostatin, a 22kDa carboxy terminal fragment of collagen XVlII is an extremely potent inhibitor of angiogenesis. Microvessel density is relatedto tumor invasivenessin gastric malignancies.In angiogenesis and apoptosis, tumor necrosis factor alpha (TNF alpha) is implicated as a local factor. Objectives:To investigate expression and regulation of endostatin in normal and neoplastic upper gastrointestinal tissues and the role of TNF alpha. Methods: Homogenatesof esophageal,gastric and pancreatictissues were screenedby Western blotting. Protein and mRNA expression in cancer cell lines were investigated for collagen XVIII and endostatin by Western blotting and RTPCR respectively. Cell lines were exposed to TNF alpha for 24 hours and apoptosis detected by DNA laddering. Regulation of endostatin processing was investigated by Western blotting. Results: Mature endostatin is expressed in esophageal, gastric and pancreatic cancers, but not in matched normal tissue. The 0E33 esophageal and SUIT2 pancreatic cell lines express collagen XVlll and endostatin. TNF alpha causes apoptosis in 0E33 cells and a dose dependent release of endostatin into the culture medium is seen. Conclusions: Human upper gastrointestinal tumors express endostatin where normal tissues do not. In the context of malignancy, the balance of pro- and antiangiogenic factors is in favor of the former. TNF alpha expressed locally will not only induce apoptosis but is able to partially redress this balance and prevent tumor-driven angiogenesis.

$869 Resveratrol Reverses Helicobacterpylori Fatty Acid Induced Stimulation of Cellular Proliferation: OpposingEffects of PKC and PKA Signaling Pathways Daniel Errampalli, Mary Jo Atten, Bashar M. Attar, Oksana Holian, Chicago, IL Cellular fatty acid composition aids in bacterial identification and often contributes to its virulence. Helicobacterpylori(Hp) produces a unique fatty acid, cis-9,10-methyteneoctadecanoic acid (MOA). Cis-unsaturated f a t acids are known tumor promoters, however, MOAs role in gastric carcinogenesis has yet to be elucidated. We have shown that resveratrol, a naturally occurring chemopreventive candidate, blocks cell cycle progression and inhibits proliferation of gastric adenocarcinoma cells by interfering with PKC mediated cellular signal transduction (8iochem Pharm 62:1423-32,2001). The present study addressesthe chemopreventive potential of resveratrol against a component of Hp and describes the action of MOA on DNA synthesis and on its engagement of PKC and PKA signaling cascades. The aim of this study is to dissect MOA elicited signal transduction events and describe their role in MOA-controlled proliferation of gastric cells, focusing on the interaction of cAMP and tumor promoter regulated cellular events. METHODS: Gastric adenocarcinoma SNU-1 cells were cultured in supplemented RPMI media. Cellular proliferation was assessed by 3H-thymidine incorporation into cellular DNA. Cytotoxicity of MOA was determined by release of LDH. Radioisotope transfer from 3,-32p-ATPto enzyme-preferredsubstrates was used to determine PKC and PKA phosphotransferaseactivities. RESULTS:MOA elicited a concentration dependent

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stimulation of DNA synthesis in quiescent SNU-1 cells, while exhibiting no cytotoxic action on these ceils. Increased DNA synthesis was closely associated to activation of PKA and translocation/activation of PKC from cell cytosol to plasma membranes. Resveratrol inhibited MOA-stimulated proliferation and directly inhibited plasma membrane translocated PKC activity, but did not suppress MOA-stimulated PKA activity. DISCUSSION: Hp-produced MOA may contribute to the carcinogenic potential of this bacterium. MOA stimulates cellular proliferation by unidirectional activation of ;ell signaling cascades that respond to growth factors and tumor promoters, whereas resveratrol suppresses only the tumor promotional signaling pathway (PKC), suggesting a dissociation of these two pathways in growth regulation of gastric cancer cells. Our findings provide further evidence to the chemopreventive potential of resveratrol in gastric carcinogenesis by demonstrating its action toward an integral component of Hp.

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Background: Colon cancer is the second leading cause of cancer-related mortality in the United States. Most patients with colon cancer present with advanced disease for which no effective therapy is available. Dendritic cell (OC) vaccine is a promising antitumor immunotherapy that has the potential to eradicate metastatic cancer. Although the majority of naive mice immunized with DC vaccine can reject lethal tumor challenges, the vaccine is much less effective in tumor-bearing mice, a situation that resembles patients with cancer. Therefore, it is critical to understand why DC vaccine fails to suppress tumor growth in tumor-bearing mice. Aims: We will address the following questions. 1) Can OC vaccine induce tumor-specific CTL activity in tumor-bearing mice? 2) Is a loss of tumor antigens responsible for failures to reject tumors? And 3) if CTL activity is found in tumor-bearing mice, is it able to prevent growth of metastatic lesions? Methods: Mouse colon adenocarcinoma cell line, CT26, grown in BALG/c, served as a mouse model of colon cancer. DC vaccines were generated by pulsing DCs with tumor lysate. Mice (3 groups) were inoculated s.c. with 0T26 on day O. CT26 lysatepulsed DCs (5x105 cells) were injected i.p. on day 5 and 10. In the first group, CTLs were isolated from spleen on day 21 and killing activity against CT26 was assessed. In the second group, tumors were harvested on day 21 and used as target cells for CT26-specific CTL killing assay to assess tumor antigen loss. Inthe third group, mice were challenged with tumor cells at a second site on Day 13. This group was designed to investigate whether DC vaccine can prevent distant metastasis. Results: 1) DO vaccine was able to induce tumor-specific CTL activity in animals with pro-established tumors, but failed to eradicate pre-established colon cancer. 2) Persistent tumors after DCvaccination were susceptible to CT26-specific CTL killing in vitro, suggesting no loss of tumor-specific antigens. 3) OC induced CTL activity in tumorbearing mice did not protect animals against tumor challenge at a second site. Conclusions: OC vaccine is able to induce tumor-specific CTL activity in tumor-bearing mice. These CTLs were not abte to inhibit primary tumors in vivo but has strong killing activity in vitro. Tumor growth despite existing tumor-specific CTLs suggests the presence of inhibitory factors suppressing in vivo CTL activity. Neutralization of immunosuppressive factors such as TGF13may lead to the development of a highly effective DC vaccine for the treatment of advanced colon cancer.

$871 EGFR Overexpression Induces Hyperproliferation of Primary Human Esophageal Cells in OrganotypicCulture Kenji Oyama, t-liroshi Nakagawa,Claudia O. Andl, Takaaki Mizushima, Hideki Harada, Anil K. Rustgi, Philadelphia, PA Introduction: Epidermal growth factor receptor (EGFR) is highly expressed in esophageal cancer, among other cancers. The morphological and biological effects induced by EGFR in squamous epithelial cells are not well understood, and impaired by use of cancer cells. Thus, we have pursued the mechanistic basis for EGFR'seffects in novel primary human esophageal cells in the setting of organotypic or three-dimensional culture (reconstruct) system. Methods: We transduced the EGFRand GFP genes with retroviral vectors in primary human esophageal epithelial cells. EGFRexpression and activation in the cells were assessedby western blotting. These cells were grown on a collagen gel containing human skin fibrohlasts being submerged in the medium for four days followed by cultivation at the air-liquid interface for seven days to induce terminal differentiation. We used AG1478, an EGFRspecific tyrosine kinase inhibitor. The reconstructs were evaluated by histotogy, immunuhistochemistry for EGFR and PCNA, and TUNELmethod for apoptosis. Results: Comparedwith normal and GFPtransduced primary human esophageal epithelial cells, EGFR overexpression resulted in a thicker epithelium consistent with hyperplasia. By adding AG1478 to media, the hyperplasia induced by EGFR was dramatically diminished such that the epithelium was thinner than that of normal or GFP cells. OverexpressedEGFR localized to the basal and suprahasal layers of the reconstituted epithelium with an increase in the number of PCNA-positivecells, but reduced by the addition of AG1476 in a dose-dependent fashion. The PCNA positive cells were confined in the basal layer only in the control reconstructs. No obvious difference was recognized in the numbers of apoptotic cells. Conclusion: EGFRoverexpression in primary human esophagealepithelial cells, achievedby retroviraltransduction, results in hyperplasiathat is mediated by a hyperproliferative response but not through induction of apoptosis. These results suggest an important role for EGFRin the basal cell compartment for maintaining a homeostasis betweenproliferation and differentiation.

$872 Suppression of Mouse Colon Tumor Progression and Augiogenesis by the INK4a/ ARF Locus Charlotte Y. Dai, Steven L. Gibson, Michael S. Gee, William M. Lee, Colleen Brensinoer, Emma E. Furth, Han-Woong Lee, Ror~aldA. Depinho, Greg H. Enders, Philadelphia, PA; Seoul, Korea; Boston, MA Background: Colorectal(colon) carcinnmais the second most common fatal human malignancy and has served as a prototype for studies of neoplastic progression. About half of colon tumors demonstrate methylation of the INK4a/ARFtumorsuppressor locus, which encodes two overlapping but structurally and functionally independent tumor suppressors, p161NK4a (p16) and p19ARF (ARF). Whether the locus suppresses colon carcinoma, however, has remained in doubt, and the specific steps of neoplastic progression countermanded by the locus are unknown for this or any tumor. We investigated the influence of the INK4a/AFIF locus on colon tumor progression in a mouse model of intestinal neoplasia. Methods: We employed mice of the C57BI/6 genetic background with Multiple intestinal Neoplasia (MIN), caused by a mutation of the adenomatouspolyposis colt (APC) gene. We generated MIN mice homozygous either for a wild type INK4a/ARFIocus(n =25) or a deletion of the INK4a/ ARF locus that abrogates both p16 and ARF function (n=29). An examiner blinded to the genotypes scored the size and coloration of colon tumors in three-month old mice. A pathologist analyzed 6 colon tumors of each genotype for histologic features of tumor progression. A total of 10 mice were injected intravenously with a fluorescein isothiocyanate-tomato lectin conjugate, to label vascular endothelial cells and allow colon tumor blood vessels to be visualized by confocal microscopy. Results: Colon tumors in INK4a/ARF-/-mice were moderately larger (p
$873 Microsatellite Instability (MSI-H) in Hyperplastic Polyposis (HPP) Associated Colorectal Cancer (CRC) Provides Evidence for the Serrated CRC Pathway Melanie Lockett, Wendy Smale, Josephine Lloyd, Sinead Burke, Kevin Pack, Wendy Atkin, lan Tomlinson, lan Talbot, Harrow, Middlesex, UK; London, UK BACKGROUND:MSI-H occurs in 10-15% sporadic CRC. The precursor is unknown but may be the hyperplastic polyp (HP) (serrated CRC pathway). In HPP there are multiple, large HPs and an increased CRC risk. If CRC in HPP showed MSI and the MSI-H phenotype (proximal, muttiple, CRCsin females with mucinous, undifferentiated histology and a prominent lymphocyte infiltrate), this would support the serrated CRC pathway. AIM: To describe the phenotype and microsatellite (MS) status of CRC in HPP to provide evidence for the serrated CRC pathway. METHODS: HPP patients were identified after a national call for cases and using the UK flexible sigmoidoscopy screening trial database.Clinical and histological features were described. Paraffin-embeddedtissue samples were microdissected and DNA was extracted. MSI analysis was performed by PCR at Bat25, Bat 26, MycL1, D2S123, APC, D15S221 and D17S250 and compared to genomic or normal tissue ONA. MS status was defined as follows: MSI-H = 3 unstable (= 1 mononucleotide marker); MSI-Low (MSt-L) 1-2 unstable; MS stable (MSS) O unstable. Tissue samples were analysed by immunohistochemistry for mismatch repair proteins hMLH1, hMSH2 and hMSH6. RESULTS: 42 HPP patients were identified, the largest series to date. 32 CRCs occurred in 18 patients (multiple CRCs in 6). 72% of the 18 CRC patients were male (median age 63 years male, 52 years female). None had a family history of CRC. 69% of CRCs were proximal to the splenic flexure, differentiation was poor in 31%, moderate in 47% and well in 9%. 22% showed mucinous or signet ring cell histology. 19% had a Crohns-like or conspicuous lymphocyte infiltrate. 6/22 (27%) CRCswere MSI-H and were proximal and showed hMLH1 loss. 5 (23%) were MSI-L and 50% MSS. CONCLUSIONS: Both MSI and chromosomal instability pathways play a role in HPP carcinogenesis. There is an increased prevalence of MSI CRC in HPP. This suggests that the HP is the precursor of MSI CRC in HPP. This supports the theory that the HP may also be the precursor of sporadic MSI-H CRC.

one in exon 8 of the gene, using radiolabeled primers. PCR products were resolved on denaturing gels, dried, and analyzed for mutant bands. Results: We analyzed 212 sporadic colorectal cancers, of which 47 were determined to be MSI-High. We identified 16 mutations in PTEN from MSI colorectal cancers (34%). The majority of mutations were found in exon 8 of PTEN (13/16, 81%). Overall, twelve (75%) of the mutations appearedto be heterozygous (indicating one wild-type and one mutated allele present), and four appeared homozygous (i.e. no wild-type allele present). All homozygous mutations were in the exon 8 polyadenine tract of PTEN. Conclusions: The tumor suppressor gene PTEN is a targeted gene for mutation in a third of microsate!lite unstable cancers. Most mutations occur in exon 8 of PTEN for unclear reasons since both coding polyadenine tracts are identical in length. The majority of the mutations identified appear to be heterozygous, and thus protein expression or PTEN function may haveto be assessedin conjunction with mutation assaysto determine inactivation of this protein in colorectal cancers and its potential involvement in colorectal tumorigenesis. Inactivation of PTEN may play a formative role in only a minority of colorectal cancers.

$875 Intrinsic Microsatellite DNA Replication Fidelity by Componentsof the Human DNA Mismatch Repair (MMR) System E J. Smith, Akihiro Tajima, Ryan T. Doctolero, Betty L. Cabrera, John M. Carethers, San Diego, CA Background&Aims: DNA MMR is an evolutionarily conserved postreplicative repair system that can correct base mispairs and insertion-deletion (I/D) loops of nucleotides at microsatellite sequences. Recognition fidelity in eukaryotes is determined by MMR heterodimers of hMSH2/ hMSH6, which may target base-basemispairs and single nucleotide I/D loops for repair, and hMSH2/hMSH3, which may target I/D loops >2 nucleotides. DNA loops form at microsatellite sequences, with the size of the I/D loop often related to the length of the microsatellite repeat. Our aim was to assess the recognition and repair of varied length-repeat microsatellites using human cell lines that are deficient in a component of MMR to determine the intrinsic microsatetlite repair fidelity by the human MMR system. Methods: We subcloned cells by flow cytometric sorting, expanded the clonal population in culture, extracted genomic DNA, and performed PCR at intrinsic mono (BAT25 and 26), di (D5S346, D17S250), tri (RB, REN), and tetranuclentide (HPRTII) microsatellites. Radiolabeled PCR products were then resolved on denaturing gels to assess alleles from each clone. A pooled cell population or the two predominant alleles among clones were taken as the normal alleles. PCR products were also sequenced to verify different alleles from the clones. Results: MMR-proficient SW480 clones did not have mutations at any microsatellite, hMLHl-defective cell clones HOT116and hMSH2defective LoVo clones demonstrated multiple mutations at every microsatellite, hMSH3-defective HCT116+ ch3 clones had no mutations among >100 clones at mononucleotide repeats, but demonstrated mutations at di (D5S346 2.9% of clones, D17S250 43%), tri (REN 25%) and tetranucleotide (HPRTII: 5%) repeats, hMSH6-defective DLD1 clones demonstrated mutations in mono (8AT25 41%, BAT26 26%) and dinucleotide (D17S250 28%) markers, but not at the tri and tetranucleotide repeats. Conclusions: Cells deficient in hMSH3 efficiently repair single nucleotide I/D loops, but not I/D loops >1 bp. Cells deficient in hMSH6 efficiently repair I/D loops >2 bp, but not I/D loops at mono or dinucleotide microsatellites. Efficient repair of I/D loops generated by intrinsic dinucleotide microsatellites may require the presence of both hMSH3 and hMSH6 proteins. The repair profile of hMSH2/hMSH3 and hMSH2,' hMSH6 seems to overlap at I/D loops >1 bp, and may explain the occasiona! occurrence of microsatellite instability in tumors of patients with hMSH6 germline mutations.

$876 hMLH-1 Protein Expression Is Reduced in Large Hyperplastic Polyps of the Colorectum Naomi Maeda, Yasushi Sano, Hiroshi Nagata, Koug-I Fu, Takayuki Yoshino, Tatsuya Okuno, Shigeharu Kato, Shigeaki Yoshida, Atsushi Ochiai, Takahiro FujiL Kashiwa Chiba, Japan; Tokyo, Japan Background and Aim: Patients meeting recommend criteria of hyperplastic polyposis have developed colorectal cancer and patients with large hyperplastic polyp (HP) were associated with synchronous colorectal cancers (PROs), but these incidences were low. These reports suggested that subsets of HPs might have malignant potential and risk factors for neoplastic progression include multiple, large, and proximally located HPs. The aim of this study was to clarify a role of hMLH1 and hMSH2 protein in HPs. Materials and Methods: A total of 51 HPs in 51 patients removed by polypectomy or endoscopic mucosal resection technique from January 1998 to July 2001, were assessed in this study. All HPs were classified into two groups; large HPs (>10 mm) and small HPs (<10 ram). Detection of hMLH1 and hMSH2 was performed on paraffin-embedded tissues with the use of an anti-hMLH1(G168-15, PharMingen) and anti- hMSH2 (NA27, Oncogene) monoclnnal antibody. Staining was considered assessablewhen nuclear staining was seen in either stromal or germinal follicle lymphocytes or in normal epithelial cells in the base of crypts. The absence of staining of all cells in one or more crypts was considered indicative hMLH1 or hMSH2 loss only when the basal cells of the crypts were assessable (as evidenced by adjacent muscularis mucosae) and when positive staining for hMLH1 and hMSH2 was present in adjacent stromal cells or in crypt epithelium. Result: A total of 11 HPs were in female and 40 were in male. A total of 15 HPs (right-sided colon: 13, left-sided colon and rectum: 2) were large HPs (mean size: 12.4 mm, mean age: 65 y.o.) and 36 HPs (right-sided: 14, left-sided and rectum: 22) were small HPs.(mean size 6.0 mm, mean age: 61 y.o.) Loss of hMLH1 protein expression were observed in a total of 12 large HPs (80%, p
$874 Mutations of the Tumor-SupressorGene PTEN in Microsatellite Unstable Colorectal Cancer Michelle Baun, Ryan T. Doctolero, Sherry C. Huang, Ajay Goei, Betty L. Cabrera, E J. Smith, Antonio Fiorino, John M. Carethers, San Diego, CA Background&Aims: PTEN is a cytoplasmic lipid phosphatase that antagonizes Akt/protein kinase B activity and therefore promotes programmed cell death and inhibits cellular migration and invasiveness. PTEN may also promote APC-mediated degradation of ~-catenin that is often interrupted in colorectal tumors. The coding region of PTEN has two potyA sequences that have been observed mutated in endometrial tumors with microsatellite instability (MSI). Our aim was to determine the frequency of PTEN mutations among MSI colorectal cancers as a potential mode for interrupting the APC/6-catenin gatekeeper pathway. Methods: We microdissected tumor and non-tumor DNA from formalin-fixed, paraffin-embedded colorectal cancers, and extracted the DNA. We used the NCl-recommended panel of 5 microsatellite markers, of which two markers must show novel alletes to classify the tumor as MSI-High and which is associated with inactivation of DNA mismatch repair function. We then amplified the DNA surrounding two hexapolyadeninetracts within PTEN, one located in exon 7 and

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$877 The Association of Hypermethylation with Colorectal Cancers Demonstrating Micro Satellite Instability (MSI) Barry M. Berger, Z J. Han, Pamela Shaw, Anthony P. Shuber, Maynard, MA BACKGROUND:MSI is a feature of up to 15% of sporadic colorectal carcinomas (CRCs) and is the hallmark of CRCsthat arise in patients with germline mutations responsiblefor hereditary non-polyposis colorectal cancer syndrome (HNPCC).Recentwork shows a significantly greater hypermethylation of MLH1 gene in sporadic CRCsthan in HNPCCassociated CRCs. A subset of these apparently sporadic MSI+ CRCs may represent previously undetected cases of HHpcc. Recent reports suggest screening tumor tissue from incident cases of CRC for MSI for evolving prognostic and therapeutic reasons. This will also raise the issue of determining which patients may have an HNPCC gerrnline mutation. MS[+ CRC patients then will likely need formal genetic counseling and further germline mutation testing, where appropriate. A test that could further define the risk that an MSI + CRC was HNPCCrelated could help make counseling more effective and downstream diagnostic workups more efficient. The ability of a hypermethylation assay to segregatesporadic MSI + CRCsinto subgroups was investigated. METHOD: DNA was isolated from micro-dissected paraffin embedded tissue sections (212) and archived frozen tumor tissue (100). Sixty-one MSI + CRCs were identified using a BAT26 mutation assay. CRC location was 87% proximal (53/61), 7 % distal (4/61) and 7% unknown (4/61). Hypermethylation was also assessedon normal peripheral blood lymphocyte DNA from a control group of 10 healthy subjects. The methylation status of the DNA from these cases and controls in the CpG islands of hMLH1 was determined by chemical treatment with sodium bisulfite and subsequent methylation-specific PCR (Herman JG et al, PNAS 1966 93:9821-26). RESULTS: No control specimen demonstrated hypermethylation (0/10). Hypermethylation was demonstrated in 77% (47/61) of MSI+ CRCs and was absent in 23%(14/61). Of the few left-sided MSI + CRCs, 3/4 demonstrated hypermethylation (75%). The presence of hypermethylation in the MSI+ CRCs was significantly different from the control specimens (p<.001). CONCLUSIONS:Hypermethylafionanalysis of a group of predominantly right-sided sporadic MSt + CRCsidentified a subset of patients without the hypermethylafion characteristic of sporadic CRCs.These may represent patients at higher risk for underlying HNPCC.If this proves out, this assay performed on MSI + CRCsmay allow for more specific patient triage for genetic counseling and further gene testing to identify new HNPCC kindreds.

$878 Steady State Changes of DNA Mismatch Repair Proteins in Gastric Cancer with Stable, Low or High Levels of Microsatellite Instability Antonia R. Sepulveda, Yuan Yao, Jae J. Kim, Pittsburgh, PA; Seoul, Korea Background: Defects in DNA mismatch repair (MMR) result in the development of a genetically unstable phenotype and render cells more susceptible to neoplastic transformation. About a third of gastric cancers display high-level microsatellite instability (MSI-H). MSI-H tumors show MSI in at least 30% of microsatellite loci when at least 5 defined markers are used. The remaining tumors show low-level instability (MSI-L) when less than 30% of markers show instability and another subset will show stable genotypes (MSS). MSI-H in sporadic gastric carcinomas is associated with loss of expression of hMLlll and rarely loss of hMSH2. The mechanisms underlying MSI-L are not known. Aims: To determine whether variations in the levels of mismatch repair proteins are associated with low and high level of microsatellite instability. Methods: The MSI status (MSI-High, MSI-Low or MSI-Stable) of sixteen different gastric cancer lines was determined using multiple clone analysis with a panel of five NClrecommended microsatellite markers. The steady state levels of MMR proteins (hMLH1, hMSH2, hMSH6, hPMS2 and hPMS1) were determined by western blot analysis. Results: The gastric cancer cell lines SNU-1 and SNU-638 showed MSI-H status, had decreased to essentially absent hMLHland hPMS2 and reduced hPMS1 levels.The MKN28, MKN87, KATOIII and SNU601 cell lines showed MSI-L status. The MMR protein levels of cells with MSI-L status were similar to the levels detected in cells with a stable phenotype. The BAT26 marker recognizedthe two lines with MSI-H but also the MSI-L fines MKN28 and SNU601. Conclusions: A marked decrease in the expression levels of MutL MMR proteins (hMLH1, hPMS2 and hPMS1) is associated with high levels of MSI in gastric cancer cells. The MSI-L status is not caused by significant changes in the levels of the main DNA mismatch repair proteins but might result from deficient MMR protein variants or as yet uncharacterizedfactors regulating human DNA mismatch repair.

$880 The Role of Rearrangements in the JC Virus (JCV) Promoter in Regulating Gene Expression Amiga Etbehti-Green, Loigi Ricciardiello, C Richard Boland, La Jolla, CA Introduction: JCV is a small DNA potyoma virus present in the GI tracts of most healthy people. JCV encodes a T antigen, which has led to the proposition that it may participate in transformation by inducing chromosomal instability (CIN). Expression of T-antigen in JCV is controlled by the transcription control region (TCR). The JCV strains we have found in the human gut can be categorized into two groups Mad-1 or rearranged based on their TCR sequences. The TCR of Mad-1 contains two copies of a 98 bp tandem repeat, while the rearranged Mad-1 strains contain deletions or duplications of the 98 bp tandem repeat. Mad1 TCR sequenceswere cloned from JCV obtained from the normal colon and colon cancers; deletions or additions of a 98 bp sequence in the Mad-1 promoter were selectively seen in colon cancers.Aims: To determine whether any TCR configuration selectivelysupports luciferase gene expression in colon cancer cells. Methods: Three different TCRs isolated from JCV found in normal colon and colon cancers were cloned into a pGL2 plasmid in front of a luciferase reporter gene and transfected into one cell fine with CIN (SW480) and one without CIN (HCT116). The three promoters are Pmad (Mad-l), PmadA (Mad-1 with an additional 98bp sequence)and PmadD (Mad1 with a deletion of one 98bp sequence). Results: Luciferase was expressed at different levels in colon cancer cells depending upon the cell line used and the TCR cloned into pGL2 plasmid, and are reported as the relative % activity of firefly luciferase/Renilla luciferase (the internal control)(see table). Conclusion: The PmadA TCR, which is rearranged with an extra 98 bp sequence, supports expression of the luciferase reporter gene 2 and 1.5 fold over that seen with PmadD, and 9 and 5 times that obtained with Pmad, in SW480 and HCT116 respectively. The 98 bp sequence contains consensus sequences that bind the transcription factors PurAIpha, YB1 and NF-1, and the rearranged promoters (PmadA and PmadD) were found in cancers but not in normal colon tissues: This suggests that the TCR rearrangementsfound in colon cancers may be of functional significance in carcinogenesis, and provide a possible explanation for a change in T antigen expression in neoplastic colons.

Promoter PmadA(Mad-1+98bp) PmadD(Mad-l-98bp) pGI.2 (vector, no TCR) Pmad(Mad-f) None(no transfection)

SW480 730 420 5 80 9

HCT116 520 350 1.6 100 4

$881 Genetic Abnormalities in an Aoinar Cell Tumar of the Pancreas. The Ro~e ol EUS Guided FNA Cytology and Microdissection Based Mutational Genotyping, Asif Khalid, Anthony J. Demetris, David C. Whitcomb, Eizaburo Sasatomi, Patricia Swalsky, Sydney Finkelstein, Pittsburgh, PA BACKGROUND: EUS guided FNA has high diagnostic accuracy for pancreatic neoplasm. However this accuracy varies depending on the type of pancreatic neoplasm encountered. Functional acinar cell tumors are rare, the role of EUS guided FNA and the accuracy of cell derived molecular diagnostics is unknown. AIMS: To determine the allelic profile of an acinar cell tumor. To compare microdissection based mutational genotyping in EUS-FNA derived samples and gross specimen. METHODS:A patient with a functional acinar cell tumor was identified. EUS guided FNA of the pancreatic tumor was performed. Upon death cancer and non-cancer tissue was obtained at rapid autopsy. Non-neoplastic (control) and cancer was microdissacted from unstained tissue sections. Aggregates of 100-200 representative cells were microdissected from each EUS-FNA cytology sample after removing the cover slip. Genomewide amplification was performed on the microdissected aggregateof cells to prepare sufficient DNA for broad panel microdissection based mutational analysis. The post genome wide amplification reaction was divided into aliquots for a panel of 15 separate allele loss markers targeting lp34, 5q21 at the APC locus, 9p21 at the p16 locus, 10q23 in proximity to PTEN, 17p13 at the p53 locus. LOll analysis utilized fluorescent capillary electrophoresis for quantitative determination. K-ras mutational analysis was also performed. RESULTS: FNA cytology suggested an acinar cell tumor. No LOH was seen in the control non-neoplastic samples. There was no K-ras point mutation in either of the samples. Extensive mutational change was found affecting lp34, 5q21, 9p21 and 17p13. This pattern of LOH was identical in the cytological material and surgical material confirming the accuracy and utility of microdissection based broad panel mutational analysis on cytological material. CONCLUSIONS:We for the first time describe the genetic abnormalities in an acinar cell carcinoma affecting lp34, APC, p16 and p53 loci, similar to ductal adenocarcinoma. K-ras mutations typical of a pancreatic ductal adenocarcinomawere absent. Our results also suggest a role for EUS guided FNA cytology material genotyping in atypical or inconclusive cases.

$879 The Frequency of Genetic Aberrations in Colorectal Carcinoma Approaches100% If Multiple Biopsies Are Analyzed Hans Olivecrona, Ulrik Lindforss, Henrik Zetterquist, Nikos Papadogiannakis,Huddinge, Sweden In the search of genetic markers of clinical relevancefor predicting the outcome after surgery for colorecta[ cancer (CRC), conflicting results are often obtained. For instance, differential conclusions of the clinical significance of loss of heterozygocity (LOH) at chromosome 18q have been presented, and this issue is still debated. We previously reported that the presence or not of LOll 18q and 17p in different invasive areas of the same colorectal tumors varies in a random fashion. Thus, single biopsies, which are almost exclusively used in this type of studies, do not reflect the true mutational status of a given tumor even if the specimen is microdissected. In order to estimate the true frequency of LOH at 5q, 17p, 18q and mutations in K-ras, ten colorectal tumors (stage I1-111)were completely divided into 5x5x5 mm cubes. From each cube DNA was extracted and analyzed consecutively until mutation or LOH was detected. Normal mucosa at least 10 cm from the tumors was used as control. DNA was PCR-amplified and analyzed by automated fragment analysis for loss of heterozygocity (LOH) at chromosome 5q14--22, 17p12--13, and 18q12--22. Point mutations of the K-ras gene were studied by temperature gradient gel electrophoresis. LOH at 18q were present in all ten tumors. LOH 17p were homozygous for our primers in three patients, and positive in the seven remaining tumors. LOH at 5q was present in 8/10 tumors, homozygous in one patient, and without loss in one. K-ras-mutations were detected in all of the tumors. LOH at 18q,

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17p, 5q and mutations in the K-ras gene reportedly occurs in about 35-70 % of the tumors in sporadic CRC.Thesefigures in general are obtained using single or afew biopsies. However, determination of the true frequencies of mutational activity in CRCare of significant importance if DNA analysis is to be considered in clinical practice, and especially if DNA analyzes are proposed as guidance for determination of adjuvant treatment modalities. Our results, based on DNA analysis of the complete tumors, suggests that the DNA aberrations studied are present in virtually all clinically significant CRC tumors or at least in a much higher proportion than generally accepted. Furthermore, the results demonstrates that conclusions from DNA analysis derived from single tumor biopsies are of limited value.

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$882 Chromosomal Aberrations Detected by ComparativeGenomic Hybridization (CGH) Are Frequent in Pancreatic Carcinoma but not in Chronic Pancreatitis Christina Schleicher, Christopher Poremba, Heiner H. Wolters, Thomas Mundel, Warner Boecker, Norbert Senninger, Mario Colombo-Benkmann, Munster, Germany Chronic pancreatitis is regarded as a potential preneoplasia for pancreatic cancer. However to date no molecular biological data are available to support this hypothesis. The objective of the present study was to investigate pancreatic adenocarcinomas of early and advanced stages and tissue from chronically inflamed pancreasfor analogous genomic alterations. DNA was isolated from deparaffinizedtissue of ductal (n =20) and mucinous (n = 7) adenocarcinomas as well as from chronically inflamed pancreas (n=7). Only microdissected samples which consisted of at least 70 % malignant cells (pancreatic cancer) or ductal epithelia (chronic pancreatitis) were selected for DNA isolation. DNA from pancreatic carcinoma or chronic pancreatitis were hybridized with normal reference DNA on unaltered metaphase chromosomes. Subsequentdiscrepancies betweensample and reference DNA were classified as gains or losses respectively. Tumors were classified according to the UlCC guidelines from 1997. Genomic imbalances were detected in 23 (85%) carcinomas of the pancreas in 2 or more chromosomal regions, no aberrations were detected in any sample from patients with chronic pancreatitis. In pancreatic cancer, losses of genetic material were observed more frequently (n = 69) than gains (n = 43). Deletions were found most frequently in 1p (61%), chromosome 22 (57%) and 19 (44%), 17p (30%) and 8p (17%), while gains involved 13q (44%), 8q (35%), 3q (17%) and 18p (17%). Loss of 8p only occurred in carcinomas of advancedstages (UlCC Ill/IV). However no additional correlation between type or number of detected genomic alterations with histological subtype (ductal vs. mucinous), tumour stages (UlCC I/ll vs. Ill/ IV) or pN-category (pNOvs. pN1) was identified. The identified chromosomal regions showing genetic alterations represent potential loci for new target genes, which seem to be of biological significance in the initiation and progression of adenocarcinomasof the pancreas.Since most of the relevant alterations could be detected in early as well as advanced tumour stages, genomic gains and losses can be regardedas early events in the tumorigenesis and progression of adenocarcinoma of the pancreas. In contrast such imbalances do not seem to play a role in chronic inflammatory alterations of ductal epithelia. Therefore our findings do not support the hypothesis that chronic pancreatitis is a precursor lesion for pancreatic cancer.

$883 Aberrant DNA Methylation Occurs early during the Adenoma-CarcinomaSequence. Mahan Ghiassi, Walter Smalley, Stephen J. Schultenover, Anthony Shuber; Sanford D. Markowitz, William M. Grady, Nashville, TN; Maynardl MA; Cleveland, OH Background: Geneticand epigeneticalterations occur during the adenoma-carcinomasequence during colon cancer formation. Specific genetic alterations, such as APC mutations, have been found to occur during the initation phase of this process. In fact, many of the genetic events appear to mediate specific progression events in colon cancer formation. Aberrant DNA methylation, a type of epigenetic alteration that causes the transcriptional repression of tumor suppressor genes, also occurs in colon cancer and has been shown to affect some genes; such as CDKN2Aand MGMT, in the pro-malignant phase of colon cancer development. In order to better understand the role of aberrant DNA methylation in colon cancer formation, we have determined the timing and frequency of DNA methylation of specific genes in the adenoma-carcinoma sequence in sporadic colon cancer formation. Aim: To determine the frequency of cancer-specific aberrant DNA methylation of CDKN2A, MGMT, and MLH1 in colonic adenomas. Methods:DNA was extracted from paraffin-embedded, formalin-fixed tissues obtained from individuals undergoing clinically indicated colonoscopy, The DNA was subjected to treatment using sodium bisulfite and then methylation-specific PCR using primers that assess the methylation status of CDKN2A, MGMT, and MLHI. The PCR products were visualized using ethidium bromide after agarose gel electrophoresis. Results: Aberrant DNA methylation of MGMT and CDKN2Awas found in tubular and tubulovillous adenomas. Fortytwo percent of adenomas showed methylation of CDKN2A(N = 10/24) and 29% of adenomas showed methyaition of MGMT (N = 6/21). No methylation of MLH1 was found in adenomas (N = 0/6). The frequency of methytation of CDKN2A was greater in tubulovillous adenomas than in tubular adenomas. The frequency of methylation of MGMT did not increase in more advanced adenomas. No methylation was detected in normal colon mucosa samples using these MSP assays (N = 20)consistent with the assays detecting tumor-specific methylation. Of interest, methylation of MGMT and CDKN2Awas also detected in some hyperplastic polyps in this study. Conclusions: Aberrant DNA methylation occurs frequently during the formation of colon adenomas. Approximately 42% of adenomas show methylation of either CKN2A, MGMT, or MLHI.

$884 The Role of hMLHi Promoter Hypermethylation in Drug Resistance to 5-Fluorouracil (5-FU) in Colorectal Cancer Cell Lines Christian N. Arnold, Ajay Goal, C Richard Boland, La Jolla, CA Background: Loss of ONA mismatch repair (MMR) due to hypermethylation of the hMLH1 gene promoter occurs in about 12% of sporadic colon cancer. Loss of MMR function results in drug resistance to a variety of clinically important chemotherapeutic drugs in in-vitro experimental models. It has been shown previously that drug resistance can be reverted for a number of anticancer drugs by the introduction of a wild type copy of the hMLH1 gone into the MMR deficient cell line HCT116. Aim: To test the hypothesis that demethylation of the hMLH1 promoter in MMR deficient colon cancer cells with hMLH1 promoter hypermethylation can restore MMR proficiency and restore drug sensitivity. Methods: Cell lines used were: SW48 (MMR deficient due to hMLH1 promoter hypermethylation), HCT116 (MMR deficient due to biallelic mutations in hMLH1), HCT116+chr3 (MMR proficient) and HCT116+chr2 (MMR deficient). After treatment with the demethylating agent 2'-deoxy-5-azacytidine (5DAC), mRNA expression was determined by RT-PCRfor hMLHI. Expression of MLH1 protein was determined by western blot. The hMLH1 promoter methylation status was investigated by methylation specific PCR.Cells were subsequentlytreated with 5-FU. Growth characteristics

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were ascertained by clonogenic assays. Results: After treatment with 5-DAC for 24h, hMLH1 promoter hypermethylation was successfully reverted for up to 15 days in SW48 cells, hMLH1 mRNA and protein expression were detected as early as 2 days after 5-DAC treatment and lasted for 15 days. Treatment with 5-DAC did not affect the methylation status of the hMLH1 promoter, mRNA and protein expression in the control cell lines. 5-DAC treatment alone did not markedly affect the growth of SW48 cells. However HCT116, HCT116+chr2, and HCT116+ chr3 cells showed a marked inhibition in growth rates after 5-DAC and combined 5-FU and 5-DAC treatment. 5-FU treatment alone strongly inhibited the colony forming ability of HCT116+ chr3 cells, while these effects were significantly less in all other cells. Combined 5-FU and 5-DAC treatment of SW48 resulted in a similar growth pattern to that seen in 5FU treated HCT116+chr3 cells. Conclusions: This is the first evidence that in vitro drug resistance to 5-FU due to hMLH1 promoter hypermethylation can be reversed by restoring MMR function through 5-DAC induced demethylation. Induction of expression in aberrantly methylated tumor suppressor or DNA repair genes could have an impact on the development of future chemotherapy regimen.

$885 Methylation Interferes with the Binding between CCAATBox of APC Promoter and the Transcription Factor CBF Resulting in Gene Silencing in Colorecta! Cancer Guoren Dang, Erik Porto, Geun-Am Song, Young S Kim, San Francisco, CA Background: The adenomatous polyposis coil (APC) gone is involved in apoptosis and cell cycle arrest. The loss of APC function is observed in most familial adenomatous polyposis associated cancer and sporadic colorectal cancer. The inactivation of the APC gone is caused predominantly by DNA mutations without affecting the mRNA levels. However, loss of APC mRNA expression concomittant with methylation of CpG sites in the promoter region has been recently reported in many tumors including colorectal cancer. In this study, we sought to investigate the molecular mechanisms involved in the inactivation of the APC gone by methylation of the promoter in colorectal cancer cells, Methods: Colorectal cancer cell lines were transfected with ptasmid constructs carrying the luciferase gone and different lengths of the APC promoter. The transfection assays were also performed after different regions of the APC promoter were in vitro methylated. Electrophoretic mobility shift assays (EMSA) and supershift assays were carried out to examine the binding of transcription factors to the promoter. Results: Transfection assays showed that cells transfected with the construct carrying a CCAATbox (located at -25 to -21, based on the transcription start site) expressed the highest luciferase activity. The luciferase activity fell significantly in the transfectants with a mutant CCAAT box. In the cells co-transfected with CCAAT box-containing construct and a plasmid expressing dominant negative CBF, the luciferase activity also decreased to 1/3. Cells transfected with plasmids carrying the APC promoter with CpG sites methylated at variant positions also showed a decrease in luciferase activities: The extent of the decrease was dependent upon the number and position of the methylated CpG sites. Methylation at CpG sites flanking the CCAATbox caused a significant decrease in luciferase activities, while methylation upstream of this region failed to reduce the luciferase activities, in EMSA and supershift assays, CBF bound to the CCAAT box in APC promoter. The binding was reduced to 1/2 to 1/3 after the CpG sites flanking CCAAT box were methylated. Conclusion: The regulation of APC expression is mediated by the binding of transcription factor CBF to the CCAAT box upstream of the transcription start site. Methylation of CpG sites flanking the CCAAT box interferes with CBF binding, resulting in the down-regulation of APC expression.

$886 APC Promoter Hypermethylation Contributes Strongly to Loss of APC Expression in Colorectal Cancers with Allelic Loss at 5q: A Study of CALGB9865 Christian N. Arnold, Ajay Goal, Carolyn Compton, Donna Niedzwiecki, Linda Wasserman, Robert J. Mayer, Monica M. Bertagnolli, C Richard Bnland, La Jolla, CA; Montreal, Canada; Durham, NC; Boston, MA Background: Germline mutations in the tumor suppressor gone APC are associated with FAP, and somatic mutations occur frequently in sporadic colon cancers. However, to abrogate APC function, the second allele must also be inactivated. This commonly occurs by a second mutation or by loss of heterozygosity (LOH). Recently it has been demonstrated that Apc promoter hypermethylation might play a role in silencing of the APC gone. Aims: Our aim was to investigate whether in tumors with allelic loss at the APC locus promoter hypermethylation influences APC expression. Methods: We performed our study on 138 sporadic colorectal cancers and matched normal tissue. LOH of the 5q locus was studied using the microsatellite marker D5S345. The APC promoter methylatinn pattern was determined by MSP following bisulfite modification of the DNA in 104 tumors and APC expression assayed by immunohistochemistry. Results: In the 138 tumors, 9.4% demonstrated LOH at 5q. APC expression was normal in 19.6% and reduced or absent in 80.4%. Of the neoplasms with 5q LOH, 23% showed normal staining, while 77% had reduced or absent APC expression. Of the cancers without 5q LOH, 84.2% had decreased or absent APC staining. Promoter hypermethylation was detected in 27% of all tumors. Aberrant APC methylation could be seen in 31% of the cancers with simultaneous 5q LOH. None of the tumors with normal APC staining and 5q LOH was methylated. However,40% of the cancers with LOH at 5q and reduced or absent APC expression were hypermethylated. 29% of the tumors without LOH at 5q and normal APC expression, and 25% of the tumors without 5q LOH and reduced or missing APC expression, were hypermethylatedrespectively.Of all tumors, 33% had normal APC expression and were negativefor 5q LOH and hypermethylation, respectively.Conclusions: The association between APC promoter methylation status, LOH on 5q, and reduced or lost APC expression suggests that hypermethylation of the APC promoter plays an important role as an additional inactivating event in silencing APC expression in colorectal neopiasia. This is the first report demonstrating that tumors with 5q LOH and reduced levels of APC expression have a much higher percentage of APC promoter hypermethylation compared to tumors with 5q LOH and normal expression. Furthermore our study indicates that the epigenetic modification of the APC promoter is not necessarily a biallelic event, which supports its role as a "second hit" in gone silencing.

AGA Abstracts

$887 NEG INDEF LGD HGD CA p14 PM 141219(6%) 2/16 (13%) 7/19 (37%) 4/11 (36%) 2/8 (25%) p16 PM 34/212 (16%) 8/16 (50%) 12/19 (63%) 4/10 (40%) 5/8 (63%} NEG: no dyeplssia (dyspl.); INDEF: indefinite for dyepl.; LGD: low grade, HGD: high grade dyspl.; CA: carcinoma p14 PM,xz NEG vs. others: p 2 NEG vs. others: p < 0.05

Promoter Hypermethylation of Cyclin D2 Gene in Gastric Cancer Jun Yu, Wai Leung, Matthias P. A. Ebert, Enders K. W. NO, Minnie Y. Y. Go, Sydney Chung, Peter Malfertheiner, Joseph J. Y. Sung, Hong Kong, Hong Kong; Magdeburg, Germany

Background: Overexpressionof cyclin D2 has been shown to play an important role in gastric cancer development. However, absence of cyclin D2 expression has been found in about 40% of gastric cancers. Our previous study showed that aberrant CpG island methylation, which results in transcriptional silencing of gene, was commonly found in gastric cancer. The methylation status of the cyclin D2 has not been clearly determined and there is no information on its role in gastric carcinogenesis. Methods: We examined the methylation status of the cyclin D2 in 5 gastric cancer cell lines (KATO III, AGS, NCI-N87, MKN45, MKN28) and 47 gastric cancer tissues. CpG island methylation status of the cyclin D2 gene was studied by methylation-specific polymerase chain reaction and mRNA expression was analyzed by RTPCR. Results: Twenty-three (23/47, 48.9%) gastric cancers and 4 (KATOlII, AGS, NCI-N87, MKN45) gastric cancer cell lines exhibited promoter hypermethylation in cyclin D2. In contrast, none of the 5 normal control gastric samples showed methylated DNA sequences. Loss of cyclin D2 mRNA expression was seen in 15 (15/23, 65.2%) cases with hypermethylated sequences and in 3 (3/24, 12.5%) unmethylated cases (p = 0.012). Three cell lines (KATOIII, AGS, NCI-N87) with hypermethylated cyclin D2 also lacked mRNA expression. There was no significant correlation between the cyclin D2 methylation status and clinicopathological parameters inlcuding age, tumor classification, lymph node metastasis and pathological staging. Conclusion: Promoter hypermethylation of the cyclin D2 is associated with loss of cyclin D2 expression in a subset of gastric cancer and may be an alternative pathway of tumorigenesis in the absence of cyclin D2 expression.

$890 Aberrant Methylation of ppENKin Mucinous Cystic Neoplasms of the Pancreas Sanjay Jagannath, Hideaki Niiyama, Matthew Yeatman, Noriyoshi Fukushima, Christophe Rosty, Marcia Irene Canto, Michael Goggins, Baltimore, MD

Introduction: Aberrant DNA methylation of CpG islands is a recognized epigenetic mechanism of inactivating tumor suppressor genes during tumorigenesis. We have previously shown that ppENKisthe most common aberrantly methylated gene in pancreatic adenocarcinoma,ppENK (met-enkephalin) suppresses the growth of pancreatic and other cancers and its aberrant methylation in pancreatic cancer results in transcriptional silencing. Pancreatic mucinous cystic neoplasms (MCNs) are uncommon pancreatic lesions that may progress to malignancy. Since aberrant methylation of ppENKis common in pancreatic adenocarcinoma but not found in histologically normal pancreas, we investigated the prevalence of aberrant ppENK methylation in pancreatic MCNs. Methods: We identified 26 patients with surgically resected pancreatic MCNs at The Johns Hopkins Hospital. The histology of each MCN was reviewed, archival specimens were obtained, and neoplastic epitheliawere microdissected from unstained paraffin sections. DNA was extracted from microdissected cells and subjected to bisulfite modification. Methylation-specific PCR was used to detect aberrant DNA methylation of the ppENKgene. For comparison, 15 pancreatic adenocarcinomas were assayed for aberrant methylation of ppENKina similar fashion. Results: Eleven(50%) of the MCN samples contained histologic evidenceof carcinoma and 5 (23%) contained histologic evidence of marked atypia. Amplifiable DNA was obtained from 22/26 cases, ppENKmethylation was present in 4/11 (36%) MCNs with carcinoma, and 3/5 (60%) MCNs with atypia. Methylation was detected in 1/6 (17%) MCNs without atypia, tn contrast, (14/15) 93% of pancreatic adenocarcinomas harbored methyLation of ppENK(34% vs. 93%, chi-square p < 0.0002). The sensitivity and specificity for detecting aberrant methylation in MCNs with carcinoma or marked atypia are 47% (7/15) and 83% (5/6), respectively. The positive and negative predictive values are 88% (7/8) and 39% (5/13), respectively. Conclusion: Some MCNs undergo aberrant methylation during neoplastic development. However, aberrant methylation of ppENKis less common in MCNs of the pancreasthan in 'usual' pancreatic adenocarcinomasuggesting that mechanisms other than aberrant methylation are important for neoplastic progression in MCNs. The identification of aberrant methylation of ppENKinfine needle aspirates of cystic lesions may be useful to predict the presence of neoplasia.

$888 Promoter Hypermethylatiou Associaled with SOCS-1Down Regulation in Gastric Cancer Ka-Fai To, Michael W. Y. Chan, Wai Leung, Enders K. W. Ng, Jun Yu, Francis K. L. Chart, Joseph J. ¥. Sung, NT, Hong Kong

Activation of JAK/STAT pathwaythrough interleukin-6 stimulation has been previously demonstrated. One of the downstream negative regulator of this pathway, SOCS-1 (suppressor of cytokine signalling), inhibits the phosphorylation of STAT3 protein. Recent findings demonstrated that hypermethylation of SOCS-1is associated with down regulation of SOCS-1 and subsequently activation of JAK/STAT pathway in hepatocellular carcinoma. We aimed to study the expression level and the methylation status of SOCS-1in gastric cancer cell lines and primary tumor tissues. Three gastric cancer cell lines (KATOIII, MKN28, AGS) and 14 gastric cancer samples (including 7 intestinal type, 4 diffuse type and 3 mixed type) were studied. Methylation status of SOCS-1was determined by methylation specific PCR (MSP). Complete methylation of SOCS-1was found in AGS and no methylation copies were detected in KATOIII or MKN28 cells while SOCS-1mRNA can be detected in KATO III and MKN28 but not in AGS cells. The phosphorylation status of STAT3 was also examined by western blotting. All three gastric cancer cell lines expressed STAT3 protein but phosphorylation of STAT3 can only be detected in AGS cells. In primary tumor tissues, hypermethylation of SOCS-Iwas detected in 3 out of 14 cases (21.4%). By comparing with paired normal gastric tissue, down regulation of SOCS-1mRNA was found in 2 out of 3 cases with hypermethylation of SOCS-1but not in all other samples. In conclusion, promoter hypermethylation is associated with transcription silencing of SOCS-1in gastric cancer. This may lead to disinhibition of JAK/STAT signalling pathway and being involved in gastric carcinogensis.

$891 identification of Genes with Significantly Differential Expression Levels in Adeeomalous vs. Normal and Cancerous Colonic Tissues. Thomas C. Liu, Florin M. Selaru, Tong-Tong Zou, Jing Ying, Yan Xu, Yuriko Mort, Fumiako Sato, Suna Wang, Andreea Olaru, Martha Kimos, Kellie Perry, David Shibata, John M. Abraham, Bruce D. Greenwald, Stephen J. Meltzer, Baltimore, MD

Background: The ability to genetically classify colonic adenomas as being similar to either normal colon or carcinoma could provide important prognostic information regarding their degree of malignant potential. Novel genomic techniques, such as cDNA microarrays coupled with bioinformatics analysis, may have the power to improve disease classification. Methods: cDNA microarrays containing 8,064 clones were probed with RNA from 9 adenomas (A), 9 normal colon samples (N), and 9 colon cancers (CC). Gene filtering techniques were used to identify genes whose expression levels were significantly increased or decreased in both As and CCs compared to Ns. A second set of genes was identified which were increased in CCs but unchanged in Ns and As. Data was analyzed with Genepix scanner and software and log transformed and mean centered with Cluster software. Significantly different genes were selected using the software program Significance Analysis of Microarrays (SAM). Results: 346 genes were significantly different in As and CCs compared to Ns, with a false discovery rate (FDR) of 2. 166 genes (FDR=2) were not different in As compared to Ns but were significantly different between As and CCs. Some of the more interesting genes are displayed in Table 1. Conclusions: cDNA microarray data, when analyzed by SAM, identified numerous genes with expression levels altered at different points in the colonic carcinogenetic cascade. Thus, this strategy may be useful in identifying potential molecular biomarkers of colonic neoplastic progression.

$889 Significance of Aberrant MethylaUon of Tumor Suppressor Genes (TSG) p161NK4a and p14ARF in Carcinogenesis of Ulcerative Colitis (UC) Chih-Jen Hsieh, E Jehle, V Gaco, T Okech, R Porschen, M Sarbia, A Donner, Michael Gregor, Bodo Klump, Tuebingen, Germany; Bremen, Germany; Duesseldorf, Germany

Background: UC predisposes for colorectal cancer (CRC). Colonoscopy and the detection of dysplastic lesions form the hallmarks of surveillance. The efficacy of surveillance is compromised by drawbacks of "dysplasia": (a) (multi-)focal distribution within the colon, (b) substantial intra-/inter-observer variability and (c) uncertainty regarding the prognostic significance especially of LGD. Hence, there is an interest in better markers of neoplastic transformation. Functional abrogation of the p53/MDM2- and/or the Rb/p16 tumor suppressive pathway is found in most human cancers. TSGs p14ARF and p161NK4aare major players within the respective pathways. We and others have shown that promoter methylation (PM) is a major mechanism of p16 inactivation - frequently and early detectable during carcinogenesis. Aim: To further characterize the role of p14/ARF and/or p161NK4aTSGs in carcinogenesis in UC. Methods: p14ARF and p161NK4aPM was analyzed in 273 tissue specimens of pats. with UC and compared with the histopathological diagnosis. A methylation-specific PCR protocol was applied. Thus, the prevalence and time of occurrence during neoplastic transformation was determined. A mapping of colectomy specimens in regard to the presence of dysplasia was performed in order to detect field effects and the distribution of lesions within the colon. Results: PM of both p14ARF & p161NK4awas significantly correlated with the presence of dysplasia. A substantial proportion already of LGD showed PM. p161NK4aPM was detected more frequently. Conclusion: This study demonstrates (1} the frequent PM of p14ARF and p16tNK4a, respectively in dysplastic lesions in UC, (2) the early occurrence of PM during neoplastic transformation and (3) the detection of INK4aJARFmethylation in non-dysplastic mucosal specimens from those pats. with neoplasia nearby. Herein, p16 PM seems to be of major significance. Thus, two promising molecular marker lesions have been characterized. Supported by a grant of the DeutscheKrebshilfe (70-2601). B.K. supported by the Interdisciplinary Center for Clinical Research of the University Tuebingen (IIIB2) and the IBD Competence Network Germany.

ALGA A b s t r a c t s

Table 1. Abbreviated List of Differentially Expressed Genes In Adenomat~us, Normal and Cancerous Colonic Tissues 46 Clones overexpressed in Cancer vs. 41 Clones Overexprsssed in Adenomas Adenomas and Normal Colon and Cancers re,Normal Colon 1. heat shock 27kD protein 2 1. GR01 oncogene 2. lysyl oxidase 2. B-factor, properdin 3. myosin IXB 3. gastrin-releasingpeptide 4. retinoic acid receptor, beta 4. CDC28protein kinase 2 305 Clones Underexpressed in Adenomas 120 Clones Underexprvssed in Cancer vs. Adenomas and Normal Colon and Cancers vs, Normal Colon 1. zinc finger protein202 1. adenylatecyclase 9 2. Rho GEF 5 2. histonedeacatylase3 3. iung cancer candidate - FUS1 3. sedne proteinaseinhibitor 4. endathefin convertingenzyme 1 4. metallothioneint H

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$892 N

Genomic Responseto Activation of PPAR~ in Human Coloractal Cancer Cells Rajnish A. Gupta, Raymond N. Dubois, Nashville, TN

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Peroxisome proliferator-activated receptor ~ (PPARB) is a ligand-activatedtranscription factor and a member of the nuclear hormone receptor superfamily. We and others have previously shown that PPARB is activated by the cyclooxygenase-2 (COX-2) derived eicosanoid prostacyclin and is over-expressedin human colorectal cancer. Moreover, the gene has been shown to be a target of the 13-cateninoncogene and genetic disruption of the receptor inhibits the tumorigenicity of a human colorectal cancer cell line. Despite these results implicating a prooncogenic role for PPARB in the development of colorectal cancer, little is known about the molecular pathways modulated by the receptor in colorectal cancer cells. Methods: Northern and western blot analysis was used to measure PPARB mRNA and protein levels in a panel of human colorectal cancer cell lines. Endogenous PPAR6 activity in these cell lines was measured using a luciferase-based PPAR reporter gene and the high affinity PPARBselective ligand GW1514. PPARB target genes were identified using spotted cDNA microarrays to compare the gene expression pattern of colorectal cancer cells treated with vehicle or GW1514. The induction or repression of putative gone candidates was confirmed using northern and western blot analysis. Co-transfection of a dominant-negative PPAR& was used to confirm the specificity of each induction/repression. Results: Genes specifically regulated by PPARB fell into several broad functional categories, including genes involved in fatty acid catabolism, peroxisome biogenesis, cell adhesion and cell migration, Of interest, PPARB also induced significant elevationsin several members of the forkhead protein family of transcription factors. Conclusions: A subset of PPARBregulated genes are involved in both adhesion and migration pathways. These changes may, in part, explain the ability of the receptor to promote the tumorigenicity of colon epithelial cells. For example, several forkhead proteins induced by PPARB are important components of sonic hedgehog signaling, a pathway that has been implicated in the promotion of epithelial to mesenchymal transition. A current focus is to determine the functional relevance of the target genes identified in this study to the biology of PPAR6 in colorectal cancer.

$893 Differential Gene Expression Analysis in a DPC4/Smad4 Model System Manfred Souquet, Irmgard Schwarte-Waldhoff, Wolff Schmiegel, Stephan A. Hahn, Bochum, Germany Introduction: Inactivation o1 the recently identified tumor suppressor gene DPC4/Smad4 was frequently identified in pancreatic tumors. DPC4 seems to play the role of a master switch in the TGF-beta signaling pathway. For detailed studies of the DPC4 function the pancreatic tumor cell line HS766T was reconstituted with DPC4. Near "physiological" expression levels were achieved and an in vivo tumor suppressive phenotype of this cell line could be demonstrated. Methods: Using Serial Analysis of Gone Expression (SAGE), expression profiles were generated for HS766T OPC4 plus and minus cells. We collected 105652 tag sequences representing 10118 individual nucleotide sequences (only tag sequences identified at least twice were counted as reliable), whereby each sequence is assumed to represent one gone. Results: By comparing the frequency of tag occurrence we found 1508 genes to be upregulated, 1482 genes down-regulated and 7128 genes not to be regulated at significat levels (<2). The majority of tags were identified only once in each library and are according to Monte Carlo simulations derived from low abundant genes (one tag/lO0.O00 tags collected, or <10-20 gene copies per cell). Genes up-regulated in DPC4-positive HS766 cells were among others TIMP-3 and lamin NC, whereas cathepsin H and CDC20were down regulated in HS766 ceils. Conclusion: Taken together, our detailed expression analysis in a DPC4 model system may help to improve our understanding of DPC4 mediated tumor suppression:

$894 Gene Expression Profiling of Aberrant Crypts in Colon Carcinogenesis Sandra Lechner, UIf MOIler-Ladner,8irgit Renke, Josef ROschoff, JOrgen Sch~lmerich~ Frank Kul[mann, Regensburg, Germany; Kassel, Germany Introduction: Aberrant crypts (ACF)are histologically well characterizedputative preneoplastic lesions in the colon. In contrast, the knowledge regarding the molecular events in ACFs is poor. It is especially not known, whether ACF are similar to adenoma at the molecular level. Methods: Frozensections (4-6 ~m) of three colonic specimen (in each case normal mucosa, aberrant crypts and adenoma) were investigated. Laser capture microdissection was used to isolate normal, aberrant and adenomatous crypts (with low-grade dysplasia) respectively. Nested RNA arbitrarily primed PCR (RAP-PCR) for differential display was used to screen mRNA-populations and to generate hybridization probes for cDNA expression arrays (Atlas Cancer cDNA Array, Clontech, Heidelberg). Evaluation of cDNA arrays was performed using the Atlaslmage2.O Software (Clontech). The intensities of gene expression signals of normal mucosa and adenomas were normalized to O and 100, respectively. The intensities of the gene expression signals of ACF in relation to normal mucosa (N) or adenoma were calculated as follows: (ACF-N)/((A-N)/IO0) Results: The analysis of the gene expression profiles of normal, aberrant and low-grade adenomatouscolonic crypts showed that ACF are more similar to adenomatous crypts than to normal crypts (Figure). Conclusions: Our findings show for the first time that the gene expression profile of ACF is more similar to adenoma than to normal mucosa. Therefore, ACFs can be classified as real preneoplastic lesions.

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$895 Alterations in Whole Coding Region (Exons 2-11) of the p53 Gene in Gastric Intestinal Metaplasia Detected by GeneChip p53 Assay Akashi Eda, Hiroyuki Mann, Shuichi Ueno, Hiroyuki Osawa, tchiro Yanaka, Kentaro Sugano, Tochigi, Japan Background-Gastricintestinal metaplasia is associated closely with the development of gastric cancer. To date, there have been no report about alteration in p53 whole exons (exons 2-11) on gastric intestinal metaplasia. Rapid mutation analysis of the p53 gene sequence (GeneChip p53 assay) has recently beendeveloppedutilizing an Oligonucleotideprobe array.This technique makes it possible to sequence p53 all coding region and a rapid and reasonably accurate approach for detecting p53 mutation. Aims-To ascertain whether p53 alterations except for the conserved regions (exons 5-9) occur during the early stages of gastric carcinogenesis. Methods-The p53 all coding lesion was sequenced in 48 endoscopic biopsy specimens with gastric intestinal metaplasia as documented by hematoxylin and eosin stain (43 patients with H. pylori-positive gastritis and five patients with H. pylori-negative gastritis) by using the p53 GeneChip assay. P53 mutations were also confirmed by the dyedeoxy terminator method. Results-There was no mutation in gastric mucosa not infected by Helicobacter pylori. The GeneChipassay detected 19 missensemutations, and 18 frameshift mutations in the specimens with gastric intestinal metaplasia. It is noteworthy that the mutations in the coding region except for conserved region (exon 5-8) were detected. Conclusion-In this study, we show that exons excluding exon 5-8 are also mutated in intestinal metaplasia, albeit in a small percentageof cases. Further studies are required to determine whether these mutations affect patient predisposition to gastric cancer.

$896 Gene Expression Pattern of Laser-Microdissected Epithelial Cells of Low Grade Colonic Adenomas Sandra Lechner, Ulf MDIler-Ladner, Birgit Renke, Josef Riischoff, J~rgen SchOImerich, Frank Kullmann, D-93042 Regensburg, Germany; D-34125 Kassel, Germany Introduction: Colorectal epithelial cells are prone to malignant transformation. Therefore, the :identification of gene expression differences in the process from normal colonic crypts to adenomas with low grade dysplasia is essential for further insights into tumorigenesis. Using a novel gene expression analysis strategy, a screening of expressed transcripts in small, histologically exactly defined tissue samples was performed. Methods: Frozen sections (4-6 p,m) of colonic tissue were fixed with ethanol/aceticacid (20/1) and laser capture microdissection was used to isolate normal and adenomatous crypts, respectively. Nested RNA arbitrarily primed PCR (RAP-PCR) for differential display was used to screen mRNA-populations and to generate hybridization probes for cDNA expression arrays (Atlas Cancer cDNA Array, Clontech, Heidelberg). Evaluation of cDNA arrays was performed using the Atlaslmage2.0 Software (Clontech). Differential expressionwas confirmed by immunohistochemistry. Results: The evaluation of gene expression profiles of normal versus adenomatous colonic crypts of six patients revealed at least in three patients a dysregulation of 1% of all analyzed genes (n = 588): P21-rac1 (4 of 6 upregulated), MAP-kinase p38c~ (3 of 6 upregulated), interferon gamma receptor (3 of 6 upregulated) FAST-kinase (3 of 6 downregulated), p53 (3 of 6 downregulated) and thrombospondin 2 (3 of 6 downregulated) could be identified to be dysregulated. Conclusions: For the first time, we present gene expression profiles of the dysplastic area of colonic adenomas using laser capture microdissection in combination with RAP-PCR and cDNA-array. An upregulation of proliferation associated genes (ras-oncogene .related p21-rac1 and p38c(), as well as the downregulation of apoptosis related genes (FASTkinase and p53) could be shown in adenomas with low-grade dysplasia. Based on the upregulation of p21-racl and p38(x, the activation of the MAP-kinase pathway appears to be an early event in colonic carcinogenesis.

$897 Gene Expression Profiles of Colorectal Cancers Resistant to Chemotherapy :Isabella T. Tai, Thuy T. Pham, Lan Bo Chen, Boston, MA BACKGROUND: Colorectal cancer (CRC) is the 3'~ most common cause of cancer-related death in the USA, with an overall 5-yr survival of only 61%.Chemotherapy is one of the treatment modalities used, which only results in a 30% reduction in mortality. Although many factors have been implicated in chemotherapy resistance,such as upregulation of MDR genes, p21 or mutations in K-ras, the general framework for the molecular basis of chemotherapy resistanceis still unclear.AIM: To obtain a genome-wide view of the changes in geneexpression in CRC resistant to chemotherapy. METHODS: Cells resistant to 5-FU, irinotecan (CPT-11), etoposide (ETO) and cisplatin (CIS) were developed using a poorly differentiated CRC cell line, MIPI01, following long-term exposure to each agent. The resistant cell lines achieved an 8-10 fold increasein IC50compared to the parental cells. Total mRNA isolated from resistant clones was compared to those isolated from the parental cells following hybridization to

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and is more important than either allelic losses or inactivating mutations. The significant correlation of PTENhypermethylation with MSI-H tumors strongly suggests that PTENis an important 'target', along with the hMLH1gone, in the evolution of MSI-H colorectal cancers and may confer the 'second hit' in a biallelic inactivation mechanism.

Affymetrix oligonucleotide Hg-U95Av2 microarrays (~12K genes). Data was analyzed using a model-based expression algorithm (dCHIP) and filtered (>2-fold change in gene expression; statistical significance, p
$900 TP53and P33~"glAre Co-Determinants of Prognosis in Colorectal Cancer. Ihab A. - R. M. Ahmed, Ghassan S. Nouman, Seamus B. Kelly, Nigel Bailey, John J. Anderson, Brian Angus, John Lunec, Newcastle -Upon-Tyne, UK; Newcastle upon Tyne, UK

ING1is a tumor suppressor gene mapping to chromosome 13q34. Over-expression of ING1 increases p53 dependant activation of p21Waf1.Thus, ING1expression is associated with inhibition of cell growth, while its down regulation is associated with increased cell growth and tumor progression. The aim of this study was to examine the relationship between p53, IN61 protein expression and clinical outcome in Dukes'C colorectal cancer in patients undergoing surgical resection of the primary tumor followed by 5-FU based adjuvant chemotherapy. Methods: Formalin-fixed paraffin-embedded tumour samples of 38 patients with Dukes'C colorectal cancer who underwent surgical resection of the primary tumour followed by 5-FU based adjuvant chemotherapy were analysed. The p331NG1nuclear expression was determined by immunohistochemistry using a newly developed monoclonal antibody (GN1). TP53gene mutations were determined using PCR-based direct DNA sequencing. Results: Low expression of p331NG1 was detected in 26.3% of samples. Mutant p53 was detected in 39.5% of samples. 22.2% of the patients with wild-type p53andnormal p33nuclear expression developed metastasis while 60% of the patients with either mutant p53 or low p33 nuclear expression or both developed metastasis (p = 0.02, Fisher's exact test). Kaplan-Meiersurvival analysis was carried considering time to metastasis and death. In either case, patients with mutant p53 and/or low p331NG1nuclear expression had an adverse survival outcome in respect to both metastasis and death (p=O.O07 and 0.019 respectively, log rank test). Conclusion: The results show that low p331NG1nuclear expression and/or mutant p53 is strongly associated with metastasis and adverse survival for patients with Dukes'C colorectal cancer undergoing surgical resection followed by 5-FU based adjuvant chemotherapy.

ClS 6.9 3.4 41.4 3.4

$898 Transcription Factor ZBP-89 Stabilizes Wild Type p53 and a Constitutively Active p53 Mutant Morihiro Okada, Juanita L. Merchant, Ann Arbor, MI BACKGROUND: ZBP-89 is a widely expressed Kr(Jppel-typezinc finger transcription factor that binds to GC-rich elements and represses or activates known target genes. Recently, we reported that ZBP-89 stabilizes wild-type p53, but not the transcriptionally inactive R273H p53 mutation (Mol.Cell.Siol.;21:p4670,2001). Therefore, we examinedwhether increasedZBP89 expression in human colon cancers correlates with p53 status. METHODS:Sections from 18 colon cancers were stained with ZBP-89 and p53(FL393) antibodies. The presence of p53 mutations in these sporadic cancers was assessed by both SSCP and direct sequencing of PCR amplimers. PCR on ONA extracted from paraffin sections was performed using primer sets that amplified exons 5-6 within the DNA binding region. Moreover, these exerts contain the greatest number of point mutations. Three of the p53 mutations detected in the sporadic cancers were subcloned into expression vectors. These 3 mutations plus 4 additional p53 "hot spots" were cotransfected with ZBP*89 into HCT116 p53 null cells to assess the effect of ZBP-89 on p53 mutant stability and regulation of a p53-dependent p21~' reporter. Levels of ZBP-89 and p53 were assessed by Western blot. RESULTS: In immunohistochemical analysis, ZBP-89 expression was elevated in 12/18(67%) samples. Of the 12 ZBP-89 positive colon cancers, 9/12(75%) also had elevated p53 expression, p53 was directly sequenced in 4 of the colon cancers staining for both ZBP-89 and p53. Three of the four cancers had point mutations of p53(A161T, Y236C and G266R) within these 4 exons and a fourth cancer expressed WT p53. When these 3 as well as 4 other mutation "hot spots"(V143A, R175H, R249S, R273H) were tested in cotransfection assays, we found that ZBP-89 was able to stabilize only wrp53 and the A161T mutation by increasing their protein levels 5-8 fold. However, ZBP-89 stabilization of the A161T mutation did not translate into a significant increase in p53 transcriptional activation. This appearedto be due to the fact that the A161T mutation was constitutively active. CONCLUSION:ZBP-89 appears to be capable of stabilizing only the forms of p53 that are transcriptionally active. ZBP-89 and p53 proteins are elevated in sporadic cancers regardless of whether p53 is mutated. These results indicate that there are mechanisms independent of those regulating ZBP-89 that modulate the levels of inactive mutant p53.

$901 Full-Length APC Expression is More Common in Proximal Colorectal Cancers (CRCs) but Nuclear Beta-Catenin is Universal in Cancers from all Colonic Sites. Michiko Iwamoto, Theodore R. Levin, Wei Zhao, Laurel Habel, Judy Morse, Steven Laken, Huang Xinen, Dennis Ahnen, Bethesda, MD; Walnut Creek, CA; Oakland, CA; Maynard, MA; Denver, CO Background: Bi-alletic inactivation of the APC gone or beta-catenin mutations are thought to be responsible for the nuclear accumulation of beta-catenin in CRCs. Almost all of the APC gene alterations described in CRC (truncating mutations, LOH, hypermethylation) would be expected to lead to the loss of full-length APC protein. We previously reported that 100% of a series of 83 colorectal cancers had nuclear beta-oatenin and 83% of them lacked fulllength APC protein by immunohistochemistry (IHC). There is considerable evidence that the mutational profile of colorectal cancers differs between the proximal and distal colon. Aim: To determine if full length APC protein is expressed differentially in a series of predominantly proximal cancersthat had beenmissed by FS. Methods: The Kaiser Permanente(KP) Colorectal Cancer Prevention (CoCaP) Program database was used to identify subjects with a negative screening FS in 1994-95 (without CRCor an adenoma). Recordswere linked to the KP Northern California Regional Cancer Registry, identifying cases with subsequent CRC diagnosed 6 months to 5.5 years after FS (mean=2.7 years). Tissue slides were re-reviewed; paraffin embedded blocks were retrieved, sectioned and beta-catenin and full-length APC and protein expression was determined by IHC. Results: Of 64,920 subjects, 161 were identified with a new diagnosis of CRC. A random sample of 125 cases was identified; histology was confirmed and sufficient tissue was available for IHC in 106; 84 were in the proximal colon. All 106 cancers had nuclear accumulation of beta-catenin but only 63% of the samples had loss of APC immunoreactivity, a rate that was significantly (p=O.O04) lower than the 83% in our previous series. The difference between the two studies is explained by the differences in the location of the cancers. In both series combined, APC immunoreactivity was significantly more likely to be present in proximal than in distal CRCs(42% vs 16%; p
$899 Frequent Inactivation of PTENby Promoter Hypermethylation and Its Association with Microsatellite Instability-High (MSI-H) in Sporadic Colorectal Cancers Ajay Goel, Christian N. Arnold, Donna Niedzwiecki, John M. Carethers, Linda Wasserman, Carolyn Compton, Robert J. Mayer, Monica M. Bertagnolli, C Richard Boland, La Jolla, CA; Durham, NC; Montreal, Canada; Boston, MA Introduction: Loss of PTENtumorsuppressor function has been implicated in the progression of several cancers. Allelic losses in proximity of the PTENlocus (10q23) occur in sporadic colon cancers, but bi-allelic inactivation has not beenfrequently demonstrated. Although PTEN would seem to be a candidate gene for colorectal carcinogenesis, previous studies have not revealed high frequency of somatic mutations in this gone. We hypothesize that alternative mechanisms of aflelic inactivation might be operative in the colon, which remain to be explored. Aim: To investigate whether promoter hypermethylation of PTENmight play a role in colon cancer, and if PTENinactivation might be related to recognized forms of genomio instability. Methods: We studied 146 sporadic colorectal tumors obtained from the CALGB-protoco19865 and UCSD Cancer Center, previously evaluated for their MSI status using five recommended markers. Tumors with instability in at least two markers were classified as MSI-H, whereas others were classified as MSI-L (unstable at one locus) or MSS (no instability). Methylation of PTENwas studied by bisulfite modification of genomic DNA followed by amplification of the promoter region in a methylation specific PCR assay. Immunnstaining of PTEN protein was also performed and results were correlated with the promoter methylation status. Results: Nineteenof 134 informative tumors (142%) showed hypermethylation of the PTENpromoter. Interestingly, PTENpromoter hypermethylation was significantly associated with the MSI-H tumors (34.1% of MSI-H versus 5.4% of MSI-IJMSS tumors; p
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$902 RACK1 Inhibits the Anchorage- and Serum-Independent Growth of v-Src Transformed Cells Betty Y. Chang, Salvador G. Gallardo, Christine A. Cartwright, Stanford, CA The specific activity of the Src tyrosine kinase is elevatedin most malignant and premalignant lesions of the colon, and decreasesas intestinal crypt cells differentiate. Thus, downregulation of Src activity appears to be important for differentiation, and upregulation for growth and transformation of intestinal ceils. Recently, we identified RACK1 (a known PKC-interacting protein and a homolog of the [beta] subunit of G proteins) as a novel Src substrate and binding partner, and an inhibitor of Src kinase activity and cell growth. To assessthe influence of RACK1 on cell transformation by v-Src, we stably expressed HA-RACK1 or vector alone in v-Src-transformed cells and analyzed the cells for anchorage-independentgrowth in soft agar, growth in low serum, focus formation on a monolayer of normal cells, and tumor formation in nude mice; all capabilities that fully transformed v-Src cells have and normal cells lack. We observed that HA-RACK1 expression in v-Src cells reduced the anchorageindependent growth of cells in soft agar (TABLE). Expression of HA-RACK1 in v-Src cells also reduced the ability of the cells to grow in low serum (0.2%), but had no effect on their ability to form foci on a monolayer of normal cells. Thus, HA-RACK1 expression may partially

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reverse the transformed phenotype of v-Src cells. We think that RACK1 is an important inhibitor of Src kinases, Src transformation and intestinal cell growth. Endogenous inhibitors of oncogenic kinases are potentially tumor suppressors; they represent exciting new targets for therapeutic intervention in colon cancer.

$906 Analysis of [~-Catenin Mutation in Serrated Adenomas of the Colorectum Taikan Yamamoto, Kazuo Konishi, Reiko Makino, Kazuhiro Kaneko, Toshinori Kurahashi, Hisako Nozawa, Atsushi Katagiri, Hiroaki Ito, Takashige Tomita, Toshiko Yamochi, Keiji Mitamura, Tokyo, Japan

Effect of HA-RACKoveruxprouslonon anchorage-independentgrowth of v-Src transformed cells no. cells seeded/plate no. colonies/plate vector.transfected HA.RACKl.transfscted 50 1 +#1 0 500 36+/-6 7+/-2 t0OO 74 +/- 6 14+#4 2000 175 +/. 10 32 +/- 4 5000 514 ~-/-51 117+/-14 2 ml of DMEM containing 0.5% agar and 10% FBS were added to 35 mm plates to form an agar base. Cells were seeded in 1 ml of DMEM containing 0.33% ager and 10% FBS onto the base, Colonies were counted 14 days later. Mean ~1. SEM for triplicate plates at each cell dilution,

Background and aims: Inactivation of the adenomatous polyposis coil (APC) suppressor gene is the first event in the carcinogenesis of the majority of colorectal tumors. APC mutations are associated with an accumulation of intracellular-catenin protein, which leads to loss of control of normal 13-cateninsignaling. Recently, it has been reported that mutation of serine/ threonine phosphorylation sites in exon 3 of [8-catenin has been identified in approximately half of colorectal tumors that lack APC mutations. Serrated adenoma has been recognizedto be a new subtype of colorectal adenomas. APC mutation is uncommon in the serrated adenomas. However, the contribution of 13-cateninmutation in these adenomas is unclear. The aim of this study was to clarify the contribution of lS-catenin mutation in development of serrated adenomas. Material and Methods: We analyzed 39 serrated adenomas, comparing with 63 traditional adennmas and 92 invasive carcinomas. These materials were resected endescopically or surgically. Informed consent was obtained from all patients. All serrated adenomas were histollogically diagnosed according to the criteria of Longacrn and FenoglioPreiser. Mutation of exon 3 of ~-catenin was examined using polymerase chain reactionsingle-strand conformation poiymnrphism (PCR-SSCP) analysis. Expression and localization of ~-catenin in cells were analyzed by immunohistochemical staining. Results: By PCR-SSCP analysis, no ~-catenin mutation was found in serrated adenomas, whereas 3 (5%) of 63 and 3 (4%) of 84 invasive carcinomas had I?,-cateninmutations. In serrated adenomas, 13-catenin was weakly positive in cytoplasm and no nuclear staining was observed. Cytoplasm of cells in the traditional adenomaswas strongly positive for 13-catenin,although nuclear was negative. Three (8%) of 39 serrated adenomas contained carcinoma, indicating their malignant potential and the existence of serrated adenoma-carcinomasequence, but neither mutation nor nuclear over-expression of beta-catenin was found in the carcinomatous lesions. However, nuclear over-expression of 13-cateninwas observed in 50% of invasive carcinomas. Conclusions: 13catenin mutation is unlikely to contribute to the serrated adenomas-carcinoma sequence.

$903 Genetic and Molecular Analysis of Chromosomal 22q Involved in Colorectal Cancer: Evaluation of Arhgap8 as a Candidate Gene Sergi Castellvi-Bel, Xavier Bessa, I-liroshi Nakagawa, Hideki Harada, Virginia Pinol, Maria Pellise, Josep I. Elizalde, Josep M Pique, Antoni Castells, Anil K. Rustgi, Barcelona, Spain; Philadelphia, PA

The development and progression of colorectal cancer (CRC) requires activation of oncogenes and inactivation of several tumor-suppressor genes. Recently, we have identified a minimal region of deletion on chromosome 22q in patients with CRC and breast cancer, suggesting the existence of one or more tumor-suppressor genes on such location. Our objective is to identify systematically tumor-suppressor gene(s) in this region. Methods: Using in silico cloning (GeneScan),we identified the ARHGAP8 gene on chromosomal region 22q13, which was verified by means of colon-specific cDNA library analysis. Positive clones were sequenced and aligned in order to obtain the ARHGAP8 full sequence. Since used libraries were not representativeenough, its full sequencewas established using 5'RACEtechniques from nontumor colon RNA. Results: It was determined that ARHGAP8 spans 160 kh on genomic DNA, and it is transcribed into a 1713 bp mRNA distributed in 15 exons. The encoded protein has 469 amino acids and it contains two remarkablefunctional domains, one of them corresponding to a RhoGAP domain which is similar to that of other proteins encoded by known tumorsuppressor genes (i.e. NF1 and TSC2). Mutational analysis was performed in 91 normaltumor paired samples from CRC and breast cancer patients, using the SSCP technique and followed by direct sequencing of all observed variants. This analysis showed several changes in the gene sequence,all of them corresponding to polymorphisms or rare variants with unlike pathogenic implication. Conclusions. Both the ARHGAP8gone sequenceand its localization on a chromosomal region frequently deleted in different neoplasms suggest it could be a novel tumor-suppressor gene, although sequence analysis of exons from tumors do not reveal mutations. It is possible that other mechanisms of inactivation are present and that the encoded protein product plays important roles in epithelial cell biology

$907 PTEN mRNA and Protein Expression in Colon Cancer Cell Lines is Regulated by the Hypermethylution of Precoding Region of the Gene Fumika Orii, Mitunori Honda, Kaori Fujiya, Toshifumi Ashida, Yasuko Miyoshi, Jun Sakamoto, Takahiro Ito, Kohtaroh Okamoto, Atsuo Maemoto, Takanori Fujiki, Mikihiro Fujiya, Yusuke Saitoh, Tokiyoshi Ayahs, Yutaka Kohgo, Asahikawa, Japan

Background/Aim: PTEN/MMAC1 (PTEN) gene product is an important phosphatase in cellular signaling pathways. We have previously reported that CpG island of the PTEN gone precoding region is methylated at various proportions in some colon cancer cell lines (Honda, et al, AGA 2000). However,expressions of PTENmRNA and proteins were not significantly correlated to the degree of CpG methylation. In this study, we have quantitatively analyzed PTEN mRNA and protein expressions by capillary PCR and flow cytometry, and got the evidence that PTEN gene expression is regulated by DNA hypermethyiation in colon cancer cell lines. Materials/ Methods: We have analyzed 5 colon cancer cell lines, T-84, HT-29, SW486, and CaC02 provided from ATCC. Expression of PTEN mRNA was confirmed by PCR-Southern blotting, and quantified by quantitative RT-PCR by LightCycler apparatus. PTEN protein expression was also confirmed by Western blot and immunostaining using ant-PTENmonoclonal antibody. Flow cytometrical quantification of PTEN protein expression was also performed with freshly permeabilized live cells. Analysis of DNA hypermethylation was analyzed by bisulfite gemonic sequence method, with direct-sequencing technique. Results. PTEN mRNA and protein were expressed,in all colon cancer cell line tested. Using bisulfite-converting reagents, results of genomic sequence showed that over 90% of CpG sequence within 800 bps PTEN gene in T84 cells were methylated. In contrast, CpGs in this region of GAG02cells was not methylated, and those of SW480 and HT-29 cells were partially (82.8%, 69%) methylated. Quantitative RT-PCR showed that demethylating agent 5-Azadeoxycitidine (5-AZA), added to the culture medium increased the PTEN mRNA expression in these cells. Flow cytometrical analysis also revealed the augmentation of PTEN protein expression in T-84, SW480 and HT-29 cells but not in GAG02 by 5-AZA treatment. Conclusions. These results indicate that expression of PTENgene is affected by hypermethytation of precoding region in some colon cancer cell lines.

$904 Causal Relationship between the Loss of runx3 Expression and Gastric Cancer Chouhei Sakakura, Katsumi Shimomura, Akeo Hagiwara, HisakazuYamagishi, ¥oshiaki lto, Kyoto, Japan

The human runt-related gene RUNX3/PEBP2aC, located On chromosome lp36, is a major mediator of signals elicited by members of the transforming growth factor-13(TGF-13)superfamity. Here we show that 45-60% of gastric cancer cell lines and surgically resected specimens do not significantly express RUNX3 due to a combination of hemizygous deletion and hypermethylation of the RUNX3 promoter region. Tumorigenicity of gastric cancer cell lines in nude mice was inversely relatedto their level of RUNX3 expression,and one gastric tumor associated mutation (R122C), occurring within the conserved Runt domain completely abolished the tumor suppressive effect of RUNX3. The results suggest that a tack of RUNX3 function is causally related to the genesis and progression of human gastric cancer.

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p53 Mutation Rate in Esophageal Carcinoma is Higher than Generally Accepted Stuart D. Oglesby, Emma Warbrick, David Johnston, John Dillon, Alastair Munro, Theodore Hupp, Alastair Thompson, Dundee, UK

Deleted in Osteosarcoma (DOS): A Novel Tumor Suppressor Gene Vijay Yajnik, Daniel Haber, Charlestown, MA

Introduction. p53 is a key regulator of cellular response to radiation and cytotoxic drugs. The currently accepted p53 mutation rate in esophagealcarcinoma is in the region of 50% (Beroud et al, Nuc Acid Res 1998). Until recently, sequencing has beenthe gold standard for identifying inactivating mutations. However,sequencingcan perform poorly when usedto analysematerial with a mixture of cell types such as tumour biopsies. The functional yeast assay (FYA) is a relatively new method of evaluating p53 mutations and outperforms sequencing when used on biopsy material. Duddy et al (J Mol Diagn 2000) found that sequencing p53 exons 5-8 identified only 54% of mutations detected by FYA in breast cancer. Robert et al (Carcionogenesis 2000) analysed 50 squamous and 6 adenocarcinomas of the esophagous using FYA and report a mutation frequency of 84%. However, in most western societies, the frequency of adenocarcinomanow exceedssquamous. We sought to identify the frequency of p53 mutations in carcinoma of the esophagus in a typical western population. Methods. Turnout biopsies were obtained from 20 consenting patients with esophageal carcinoma. The biopsies were assessed for p53 mutations using the FYA. Confirmation of p53 mutation was sought by sequencing plasmid recovered from single yeast colonies. The histological tumour type was recorded. Results. 14 of 20 turnouts were adenocarcinomas,all were identified as containing mutant p53 by FYA. The remaining 6 tumours were squamous and 4 of these (66%) were mutant. The overall p53 mutation rate was 90%, all mutations were confirmed by plasmid

Cancer progression is caused by accumulation of multiple mutations that provide selective advantage during cancer growth, invasion and metastasis. While gain of function mutations occur in oncogenes, many of the genetic events that underlie cancer appear to be inactivating, or loss of function mutations affecting tumor suppressor genes. To date, genes involved in advanced stages of cancer progression such as invasion, angiogenesis and metastasis have not been identified. They are likely to become evident over time with large-scale genome wide analysis. Using RepresentationalDifference Analysis (RDA) on mouse tumor model we found a region of homozygous deletion in a mouse osteosarcoma cell line. This region of deletion is homologous to human chromosome 7q31. We have cloned the gene residing in the deleted segment (DOS for deleted in osteosarcoma) from both mouse and humans. Human and mouse DOS protein sequence is 97% identical The protein has homology (approximately 30% identity) with three known gone in the database, namely, DOCK180, myoblast city and Cud 5. These three proteins are evolutionary conserved in both sequence and function and regulate actin cystokeletion during cell migrartion. Our data suggests that DOS may be involved in regutaing actin cytoskeleton in cell growth and cancer via small GTPase(s). We have found missense mutations in several human cancer cell lines and will discuss the functional significance of the mutations.

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sequencing. Discussion. Our study confirms a high p53 mutation frequency in esophageal carcinoma and also shows that this applies to adenocarcinomas. Previous studies attempting to correlate p53 status with treatment responsiveness have been inconclusive, this may in part be due to underreporting of p53 mutations.

(p
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Loss of Chromosome3p Heterozygosityin Human Gastric MALT Lymphoma Allan Weston, Snigdha Banerjee, Ram Sharma, Trang N. Tran, Rachel Cherian, Sushanta K. Banerjee, Kansas Ciy, MO

Lack of Axinl Mutations in Adenocarcinomas of the Gastro-EsophagealJunction Linetta B. Koppert, Bas P. L. Wijnhoven, Alhertina W. Van der Velden, Mustaffa Abbou, Hugo W. Tilanue, Winand N. M. Dinjene, Rotterdam, Netherlands

Genetic aberrations including chromosomal and microsatellite instability are associated with the development of various cancers including high-grade gastric MALT lymphoma. Previously we and other laboratories have documented that the aneuploidy of chromosome 3 was associated with the transformation of H. pylod-associated gastric MALT into low-grade Bcell gastric MALT lymphoma. To further explore the role of chromosome 3 and its associated specific abnormalities in gastric MALT lymphoma, we collected gastric MALT lymphoid tissues and associated adjacent non-lymphoid tissues and white blood cells from the 41 patients with gastric lymphoma and analyzedthe loss of heterozygosity (LOH). ONAfrom these tissues was isolated using DNA isolation kit and PCR was amplified using chromosome 3p-specific primers. Amplified DNA was digested using restriction enzyme Msp-1, and products were electrophoresed on a 3% agrose gel. Our results show that in 31% of patients heteruzygosity was lost due to loss or specific mutation in the chromosome 3p22-25. The LOR correlated strongly with high-grade gastric MALT lymphoma. We conclude that sub-microscopic instability of chromosome 3 may play a critical role in the development of gastric high-grade large B-cell lymphoma and that these genetic aberrations are responsible for lymphomagenesis in gastric cancer.

Despite the rising incidence of adenocarcinomas of the gastro-esophageal junction (GEJ), little is known about the molecular mechanisms underlying the origin of these tumors. We and others have reported nuclear 13-catenin expression in a high percentage of GEJ adenocarcinomas. This indicates activated Writ signaling in these tumors. Gene mutations resulting in nuclear !3-catenin expression have been described for APC, 13-catenin,Axinl and Axin2. Previously we performed mutation analyses in a large series of GEJ adenocarcinomas for exon 3 of the 13-cateningene and for the mutation cluster region of the APC gene. However, no mutations in these genes were found. These results prompted us to search for Axinl mutations in these tumors. We selected a series of 15 GEJ adenocarcinomas and 4 GEJ carcinoma cell lines with strong nuclear expression of I~,-catenin.DNA was isolated and PCRSSCP was performed for 23 fragments comprising all 10 Axinl exons. No tumor specific aberrations were found. We conclude that Axinl mutations do not contribute to the Wnt activation in GEJ adenocarcinomas.The mechanism of Writ activation in GEJ adenocarcinomas remains obscure.

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Reduction of the Mitochondrial Membrane Potential by a Novel ProapoptoticFactor 53BP2 Shinichi Kajino, Shinya Kohayashi, Teranishi Futoshi, Naoko Takahashi, Hirotaka Ohara, Shigeomi Shimizu, Makoto Itch, Takashi Okamoto, Nagoya, Japan; Osaka, Japan

Contiguous Hyperplastic Polyp (HP), Serrated Adenoma (SA) and Colorectal Cancer (CRC): Pathways and Timing Melanie Lockett, Victoria Johnson, Wendy Smale, Josephine Lloyd, Sinead Burke, lan Talbot, Emma Jaeger, lan Tomlinson, Wendy Atkin, Harrow, Middlesex, UK; London, UK BACKGROUND:HPs may progress to CRC via the SA (serrated CRC pathway). In hyperplastic polyposis (HPP) this occurs by microsatellite instability (MSl) and chromosomal instability (C[). In sporadic CRC, the serrated CP,C pathway has been suggested to lead to proximal CRCwith high levels of microsatellife instability (MSI-H). AIMS: To investigatethe heterogeneity of MSI and K-ras and p53 mutations in contiguous SNCRC and contiguous HP/SA to help understand the timings and genetic mechanisms driving the serrated pathway. METHODS: Paraffin-embeddedtissue blocks with contiguous SNCRC or contiguous HP/SA were selected. Using a haematoxylin-eosin stained section as a reference,tissue components were identified, micro-dissected and DNA was extracted. MSI analysis was done by PCR at Bat 25 and Bat 26 and by immunohistochemistry for the mismatch repair proteins hMLH1 and hMSH2. DNA samples were analysed for point mutations in co(ions 12 and 13 of the K-ras gene using minisequencing. Abnormal p53 accumulation was identified using immunohistochemistry. RESULTS: 11 contiguous SNCRC and 2 contiguous HP/SA were analysed. 10/11 SA/CRC samples were MS stable. MSI was seen in both SA & CRC components in 1/11. The CRC but not the SA showed loss of hMLHI. K-ras mutations were detected in 4/11 CRCs. 3/4 contiguous SAs had the same K-ras mutation (1/4 PCR failure). The remaining 7 SNCRCs were wild type. P53 results were available in 10. All 10 CRCs and 5/10 contiguous SAs showed accumulation of mutant p53. Neither of the two HP/SAs showed MSI or K-ras mutations. CONCLUSIONS:All contiguous SNCRC or HP/SA were concordant in MS and Kras status. This supports the serrated CRC pathway. K-ras mutation appears to be an early event whereas p53 over-expression (and presumably mutation) a late event. MSI was rare in this predominantly distal tumour series.

$911 Perinuclear Expression of CASK Is Associated with Reduced Syndecan-1 Expression and Tumor Invasion in Coloractal Cancers Mikihiro Fujiya, Toshifumi Ashida, Jiro Watari, Yasuko Miyoshi, Kaod Fujiya, Kohtaroh Okamoto, Atsuo Maemoto, Fomika Orii, Tokiyoshi Ayabe, Yusuke Saifoh, Yutaka Kohgo, Asahikawa, Japan

$914 Angiogenic Potential of HUVEC Cells Is Increased by Amidated and GlycineExtended Gastrin-17 Philip Clarke, Seen Evans, Daniel McWilliams, Susan Watson, Nottingham, UK

Background/aim: Calcium/calmudulin-dependent serine protein kinase (CASK), which is a member of the membrane-associatedguanylate kinase family, binds to heparansulfate proteoglycan syndecans and play an important role in cell adhesion. Recently, it was suggested that CASK migrate to the nuclei and binds to a specific DNA sequence (T-element) in a complex with a transcription factor Tbr-1 (Hsueh Y, et el. Nature 404, 2000). However, it is still unclear how CASKplays a role in malignant cells. We have reported that reducedexpression of syndeean-1was closely associated with the malignant behavior in cancer cells (Matsumoto A, et al. Int J Cancer, 1997) (Fujiya M, et al. Jpn J Cancer Research 92, 2001). The aim of this study was to elucidate the roles of CASK protein in colorectal cancer cells. Methods: Immunocytochemical staining was performed in colon cancer cell lines (HT29, T84, SW480, COLO320DM) using anti-CASKmonoclunal antibody (C63120). Sixty specimens resectedfrom CRC patients were examined by immunohistochmical staining using anti-CASK monoclonal antibody and anti-eyndecan-1 monoclonal antibody (B-B4). Clinicupathological findings were evaluatedaccording to TNM classification. Results: In normal colonic mucosa, CASK localized along the basal membrane. In contrast, CASK localized in cytoplasm or nuclei in all 4 cell fines. Out of 60 resected specimens, CASK expressions localized at basal side were observed in 18 cases (more than 75% of all cells were stained along the basal membrane), CASK expressions in cytoplasm or nuclei were observed in 36 cases (25-75%), only few membrane expressions of CASKwere detected in 6 cases (less than 25%). The specimens with membrane expressions of CASK showed significantly lower degree of T factor than those without the membrane expression (p
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Background and Aims: 53BP2 has been identified by its ability to bind the core domain of p53. The binding of 53BP2 to wild type p53 but not some mutant forms of p53 suggested that 53BP2 might he involved in biological actions of p53. Interestingly, 53BP2 interacts with apoptotic inhibitors Bcl-2 and p65 subunit of NF-KB. We previously reported that 53BP2 induced apoptosis even in a human pancreatic cancer cell line in which p53 gene is defective. Anti-apoptotic actions of Bcl-2 could be ascribed to its ability to interrupt the action of 538P2 in mitochondria. Therefore, we examined the action of 53BP2 on mitochondria and its membrane potentials. Methods: Effects of transient expression of 53BP2 and Bct-2 family proteins were examined in MIA PaCe-2, a pancreatic cancer cell line, and 293 renal cells. 53BP2-inducible 293 cells (293/53BP2), in which 53BP2 expression is induced by ponasterone A, was also used. To identify endogenous expression of 53BP2, cells were stained with the specific anti-53BP2 antibody. To identify the transfected cells and determine the intra-ceflular localization of 53BP2, we used the plasmid expressing EGFP fused with 53BP2 (pEGFP53BP2). In order to visualize mitochondria, we transfected a mitochondrial targeting plasmid, pDsRed2-Mito. We also stained the functional mitochondria by CMX-ROS or rhudamine 123 to detect the mitochondrial membrane potential. Confocalas well as fluorescence microscopes were used for these assays. Results: 1) Fluorescence microscopy showed that 53BP2 is localized in the cytoplaem of MIA PaCa-2 and 293 cells, not in the nucleus. 2) in some but not all cells, 53BP2 predominantly co-localized with mitochondria. 3) FIowcytometric analysis revealedobvious reduction of the mitochondriatransmembrane potential in the cellsexpressing 53BP2. 4) More importantly, this reduction of mitochondria transmembrane potential induced by 53BP2 was restored by co-expression with Bcl-2 and Bcl-xL and the apoptosis was efficiently blocked. Conclusions: 53BP2 appears to induce apoptosis through localizing to the mitochondria by reduction of its membrane potential. This effect was strongly inhibited by co-expression of anti-apoptotic Bcl-2 family members. Thus, anti-apoptotic actions of Bcl-2 could be ascribed, at least in a part, to its ability to interrupt the 53BP2 action in the mifochondria. More inquiry into the mechanism of 53BP2-induced apoptosis should further elucidate the mechanism of apoptotic control and promote a novel anti-cancer therapeutic strategy.

Introduction: Gastrin peptides directly and indirectly promote the growth of malignant cells. Gastrin modulates expression of heparin binding EGF (HB-EGF), which may play a role in angiogenesis.The aim of these studies was to determine whether gastrin modifies endothelial vessel formation of human umbilical vein endothelial (HUVEC)cells in in vitro culture. Methods: HUVEC cells were grown in a mixed culture system with irradiated feeder cells in 24-well plates. Amidated human gastrin-17 (G17) and glycine extendedG17 were added to the cultures at concentrations of 1OHM.Vascular endothelial growth factor (VEGF, 2ng/ml) and suramin (20mM) were used as positive and negative controls (n=2 wells were set up for each condition), respectively. Media supplementation took place on days 4 and 7. At day 8 the cultures were fixed with ethanol and stained using a CD31 monoclonal antibody. Image analysis was used to quantify tubule node formation. Gastrin receptor (CCK-2R) status of HUVEC cells from single culture was assessed by Real time PCR. Results: The mean nodes assessed for 2 separate experiments are shown below. There was found to be modest expression of CCK-2R with retained intron 4. Conclusion: G17 and GlyG17 are able to induce an angiogenic response in HUVEC cells which is equal in magnitude to that induced with VEGF, indicating the possible involvement of CCK-2R species.

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Condition Meannodes (SEM) Significancefrommediumcontrel(pvalue,MannWhitney) Assay I Assay2 Medium 15.6(1.2) 20.5(1.3) Suramin 5.9(0.5) 6.2(0.4) p=O00005, p=O0005 VEGF 29.5 (1.8) 33.5(1.7) p=O.ogo05,p=O.O05 (311 30.0 (2.1) 27.0(1.6) p=O.O0005,p~0.0062 GlyG17 31.0 (1.7) 35.0(1.7) p=O.O0005,p=O.O0005

Ca2+ and Protein Kinase C (PKC) Mediate Deoxycholic Acid (DCA)-Stimulated Prostaglandin E2 (PGE2) Production in Human Colonic Fibroblasts Eric J. Waffner, Yingting Zhu, Peter Lance, Michael E. Duffey, Buffalo, NY; Tucson, AZ We demonstrated previously dramatic stimulation of PGE2 synthesis by a secondary (DCA) but not a primary (choUc acid, CA) bile acid in fibroblasts from normal colon, and results were accentuated!n carcinoma-associatedfibroblasts. We have now investigated the signaling pathways for bile acid effects in CCD-18Cofibroblasts and primary fibroblast strains initiated from normal and adenocarcinomatous colon. PGE2 production was measured by radioimmunoassay. Changes in [Ca2 + ]i were estimated from alterations of F340/F380 ratio in individual fibroblasts loaded with Fura-2AM. Stimulation of PGE2synthesis following PKC activation was demonstrated using 12-O-tetradecanoylphorbol-13-acetate(TPA). Evidence of PKC-mediated transduction of DCA effects was obtained using non-specific protein kinase (staurosporine) and specific PKC (bisindoylmalemide, BIM) inhibitors. Stimulation of cAMP was measured by enzyme immunoassay using forskolin as a positive control. DCA (300 ?M) transiently increased fibroblast [Ca2+]i from 62 to 200 nM within 1 rain of exposure but CA had no such effect. Chelation of intracellular Ca2+ blocked DCA-stimulated PGE2synthesis. A Ca2 + ionophore, ionomycin (5 ?M), induced PGE2 levels 36-fold by 0.5 h following exposure. TPA (20 ng/ml) increased PGE2 levels 26-fold by 12 h. DCA-stimulated PGE2 synthesis was fully blocked by staurosporine or BIM. Neither DCA nor CA effected fibroblast cAMP production. In conclusion, increased [Ca2+ ]i was required for mediation of DCA-stimulatedPGE2synthesis and PKC was a further essential component of this signaling pathway. Colonic fibroblasts may be a major target for prostanoid biogenesis induced by fecal bile acids and, potentially, other noxious actions of these agents.

$915 Expansion of the Endothelial Surface by an Increase of Vessel Diameter during Tumor Angiogenesis in Experimental Pancreatic and Hepatocellular Cancer Eduard Ryschich, Jan Schmidt, Eduard Schmidt, Sasa-Marcel Maksan, Martha M Gebhard, Ernst Klar, 69120 Heidelberg, Germany; Mannheim, Germany Introduction: The vascular hypodensity as common feature of malignant tumors is well known. We suggested that the expansion of vessel diameter may reconstitute the oxygen and nutritient supply of the tumor. The aim of the present study was to compare the number and diameter of blood vessels in pancreatic and liver carcinoma with normal tissue. Methods: The tumor induction of pancreatic (DSLeA) or liver (MH 3924) carcinoma was performed in 12 male Lewis (pancreatic cancer) and 6 ACI (hepatoma) rats by an orthotopic innoculation of tumor cells (hepatoma) or solid tumor fragments (pancreatic cancer). 6 weeks (pancreatic cancer) or 12 days (hepatoma) after tumor implantation, the tumor microvasculature as well as normal pancreatic or liver blood vessels were investigated by intravital microscopy. The number of perfused blood vessels in tumor and healthy tissue were assessed by computer-assisted image analysis. Results: The vessel density in healthy pancreas (565(89)n/mm2) was significantly higher cornparedto pancreaticcancer (116(36)n/mm2) (p <0.001 ). Healthyliver showed also a significantly higher vessel density (689(36)n/mm2) compared to liver carcinoma (286(32)n/mm2) (p
normal pancreas 98 2 less than 1

pancreatic carcinoma 41 45 t3

normal liver 8 81 11

$918 ATRo and DNA-PK° Dependent Phosphorylationof Histone H2AX by ReplicationMediated DNA Double-Strand Break Induced by Camptothecin Takahisa Furuta, Christophe Redon, Duane Pilch, Olga Sedelnikova, Glenda Kohlhagen, Cordula U. Kirchgessner, Scott Kaufmann, William A. Cliby, William M. Bonner, Yves Pommier, Bethesda, MD; Stanford, CA; Rochester, MN Camptotecin analog (OPT) has been widely used for gastrointestinal cancer diseases. To elucidate the molecular mechanisms of cellular response to CPT is useful for improvement of cancer chemotherapy. CPT is a specific topoisomerase I (top1) inhibitor, which generates replication-mediated DSBs when DNA polymerase collides with CPT-trapped top1 cleavage complexes. When double-strand breaks (DSBs) are introduced into cellular DNA, histone H2AX is phosphorylated on a serine tour residues from C-terminus (.y-H2AX). We first examined whether CPT induces .y-H2AX in mammalian cells including the colon cancer cell line (HCT116). When HCT116 cells were treated with OPT, ~,-H2AX foci were formed in a dose- and time-dependent manner. These foci failed to form when cells were pretreated with the DNA replication inhibitor, aphidicolin, indicating that .~-H2AX formation resulted from CPT-induced replication-mediated DSBs. Consistently, ~-H2AX foci formation was enhanced by pretreatment with the S phase checkpoint abrogator, UCN-01. Next, we examined which kinases were involved in ~,-H2AX formation. Three kinases, ATR (ataxia teleangiectasia- and Rad3-related protein kinase), ATM (ataxia teleangiectasia mutated protein kinase) and DNAPK (DNA-dependent protein kinase) have bee n shown to be activated by DSBs. ~,-H2AXfoci were not observed in the ATR-dominant negativeGM847/ATRkdcells exposedto CPT,whereas -y-H2AX foci were observed in the parental GM847 cells. ,y-H2AX foci were generated in ataxia teleangiectasia(AT) cells (G M5849) by CPT treatment, although at slightly lower levels than in ATM-proficient GM637 cells. ~,-H2AXfoci were reduced in DNA-PKcs-deficient MO59J cells. The low intensity of ~/-H2AX foci in MO59J cells was restored by complementation with DNA-PK (MO59J/Fus1 cells: complemented with DNA-PK gone). These results indicate that, following CPT treatment, ATR is the most essential kinase for ~,-H2AX formation while DNA-PK and ATM only show supportive activity.

liver carcinoma 7 41 52

$916 Muc-1 Is a Novel Ligand for Galectin-3 and Is Up-Regulated in Invasive HT-29 Colorectal Cancer Cells. Nigel A. Andrews, Lu-Gang Yu, Oleg Gerasimenko, Jonathan Rhodes, Liverpool, UK Introduction:Galectins are a family (12 to date) of 13-galactoside-bindinglectins. It is unclear what are their functionally important natural ligands but Galeetin-3 expression is implicated in tumour invasion and metastasis.We haveinvestigatedthe ligand(s) for recombinant galectin3 in HT-29 colon cancer cells. Methods and Results: HT-29 cells were grown to 70% confluence, harvested, and a cytoplasmic extract prepared. The extract was then separated by SDS-4% polyacrylamide gel electrophoresis (PAGE), electroblotted onto a nitrocellulose membrane and probed using recombinant human galectin-3 protein followed by anti-galectim3 antibody overlay. A major ligand was then identified with a molecular weight of 420kDa. Since this corresponds to the mucin MUC-1, which is known to express the TF antigen (galactosel3 1,3 N-acetylgalactosamine(x), a known ligand for galectin-3, MUC-1 immunoprecipitate was prepared and probed as before. This confirmed binding of MUG-1 by recombinant galectin-3 protein (Fig 1). Lane a shows three proteins identified by galeetin-3 protein in HT-29 cytosolic extract, lane h shows two proteins identified by galectin-3 protein from MUC-1 immunoprecipitate.

$919 The Proto-OncogeneC-Mos Is Involved in Enterochromaffin-Like(ECL) Cell Transformation Toshinori Hinoue, Mark Kidd, Kevin Lye, Irvin Modlin, New Haven, CT

The two protein bands observed for MUC-1 are due to the two different alleles of MUC-I. HT-29 cells were separated into invasive and non-invasive cell types by their ability to migrate through a O.5mm Matrigel. Immuno-confocal microscopy of invasive HT-29 cells showed coIocalisation of MUG-1 and galectin-3 whereas MUC-1 expression was weak in the noninvasive cells. Conclusinns:The transmembrane TF-expressingmucin MUG-1 is a natural ligand for galectin3. Its increasedexpression is correlated with a more invasive phenotype in colon cancer cells.

Background: The proto-oncogene, c-mos, encodes a 39-kDa protein with serine/threonine kinase activity that is a key component of the MAP-kinase signaling pathway. It plays a vital role in oocyte maturation and, when expressed ectopically, can efficiently induce oncogenic transformation of somatic cells. In a transgenic mouse study, animals that over-expressed c-mos developed C-cell thyroid hyperplasia and neoplasia as well as adrenal tumors. Gastric carcinoid tumors originate from gastric neuroendocrine ECL cells, and in the rodent model (Mastomys), ECL cell neoplasia can be generatedby endogenous hypergastrinemia using the H2 receptor antagonist Ioxtidine for >16 weeks.The genetic lesion predisposing these animals to rapid carcinoid formation is undefined. Using gene-chip analysis we identified that c-mos was transcriptionally activated in the tumor ECL ce~ls.This study explores the possibility that alterations in this gene may play a role in neuroendocrine cell transformation. Methods: Preparations of naive (n = 4) and tumor (n = 5) ECL cells were obtained in an enriched form by a combination of pronase digestion, elutriation and Nycodenzgradient centrifugation, and RNA and protein were isolated. RT-PCR was used to evaluate alterations in c-mos gene expression between the naive and transformed state, and western blots were performed to examine protein expression. Immunohistochemistry was then undertaken to examine the distribution of c-Mos in oxntic mucosa at different stages of ECL cell proliferation. Finally, necroscopies (n = 17) were performed and the thyroids and adrenals examined for cell alterations. Results: 1) c-mos was identified by PCR in all tumor ECL cell preparations but was absent in naive ECL cells. 2) In agreement with the RT-PCR analysis, c-mos protein was detectable in transformed ECL cells. 3) The, c-mos immunoreactivity was rarely present in normal gastric mucosa but significantly increased during the development of neoplasia, and was specifically identified only in tumor ECL cells. 4) Eight (53%) of Mastomys evaluated

MUC-1

(Figure l) a

b

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had C-cell hyperplasiawhile 12% exhibited adrenal neuroblastomas. Conclusion: These observations suggest that c-mos may provide the underlying genetic mechanism for gastrin-driven ECL cell transformation.

$920 An N-Terminally Truncated Cytoplasmic Form of Oxygen-RegulatedProtein 150 (Orp150) Is Essential for Nuclear Locaiisation Sequence (NLS)-Dependent Nuclear Protein Import in Human intestinal Cancer Cells Lu-Gang Yu, Jonathan M. Rhodes, Liverpool, UK The classical NLS-dependent nuclear import system, which mediates import of large nuclear proteins, is fundamentally important for maintaining nuclear function. Our previous studies have shown that the inhibition of cell proliferation of mushroom Agaricus bisporus lectin (ABL) (Cancer Res. 1993,53:4627) is linked to its internalisation and selective blockade of NLS-dependent nuclear protein import (J.BioI.Chem.1999, 274:4890). One of the major intracellular ABL-binding ligands is an N-terminally truncated cytoplasmic form of Orp160 [Gastro.2001,120(supp11): 3579]. Orpl50 is a stress-related protein and is up regulated in tumours and highly expressed in cancer cell lines. In this study we investigated the role of Orpl50 in nuclear protein import. Nuclear protein import was studied in digitonin semipermeabilizedhuman cotorectal cancer HT29 and gastric cancer AGS cells using a fluoresceinconjugated synthetic NLS peptidefoovine albumin complex (NLS-BSA-FITC) as a transport marker. It was found that introduction of an anti-Orp160 antibody, but not other irrelevant antibodies, into the transport system resulted in 57% and 46% reduction of nuclear accumulation of NLS in HT29 and AGS cells respectively. Removal of cytosolic Orpl50 from the transport system caused over 40% reduction of NLS nuclear accumulation. The ras-related nuclear transport factor Ran was identified in the Orpl50 immunoprecipitate. Orp150 was also identified by immunoblotting in the immunoprecipitates of Ran but not in the immunoprecipitates of other Ran-associatedproteins (Ran-BP1, RCC1, Ran-GAP1 and NTF2). This suggests that the truncated cytoplasmic form of Orpl50 has a crucial role in NLS-dependent nuclear protein import, probably by interaction with Ran

$921 Differentiation-Dependent Expression of EGFR Related Protein (ERRP) in Caco-2 Cells and Colonic and Pancreatic Cancers. Maged K. Rizk, Raphaela Finkenauer, Kiran K. Negothu, Marc D. Basson, Adhip P. N. Maiumdar, Detroit, MI Members of the receptor tyrosine kinase family are frequently implicated in experimental models of epithelial cell neoplasia as well as in human cancers. One of the best studied receptor signaling systems from this family is that controlled by the EGF-receptor (EGFR), whose expression and enzymeactivity have been linked to a number of malignancies, including colorectal cancer. However, the intracellular events that regulate EGFR have not been fully elucidated. Recently, we isolated a negative regulator of EGFR, referred to as ERRP (AJP 280:C1083,2001). Overexpression of ERRP in colon cancer cells not only inhibits EGFR activation but also decreases proliferation in matrix-dependent and independent assays. To determine whether ERRP may play a role in intestinal epithelial differentiation, we examined its levels in Caco-2 cells, which are known to lose proliferative capacity and undergo differentiation on reaching confluence. ERRP and b-actin (internal control) were analyzed by Westernblot in pre-confluent, confluence and at 3-, 6- and 9-d post-confluent Caco-2 cells. In addition, the effect of sodium butyrate, which induces differentiation, on Caco-2 ERRP expression was studied. Although ERRP is expressed in Caco-2 cells during the proliferative phase, ERRP levels increased substantially and time-dependently (50-150%) during differentiation (i.e. at confluence and 3- and 6-d post-confluence), compared with pre-contluent levels. Exposure of Caco-2 cells to 10 mM butyrate for 24 h caused a 3-4-fold increase in ERRPlevels. In parallel immunohistochemical studies, ERRPwas expressed strongly in benign colonic epithelium but weakly in invasive human adenocarcinomas.Similarly, ERRPexpression correlated significantly (p
effects of pressure in ceils cultured on collagen I and IV, laminin or fibronectin. Thus, pressureinduced ERKactivation in vitro causestime-dependent, adhesion-requiring, matrix-independent proliferation of SW620 colon cancer cells. The function of the pressure-induced p38 signal awaits further study. These data suggest that physiologic and pathophysiologic changes in pressure may be sufficient to activate a specific MAPK signal stimulating neoplastic colonocyte proliferation.

$923 Novel Changes in APC Subcellular Distribution Underlie Hyperproliferation in Native Colonic Mucosa Shahid Umar, Famourou Kourouma, Andrew P. Morris, Joseph H. Sellin, Houston, TX Background: The adenomatous polyposis coil (APC) protein is implicated in the majority of hereditary and sporadic colon cancers. In a mouse model of colonic epithelial hyperproliferation/hyperplasia (TMCH; Castro A-297,1537, 2001), we showed increased expression of full length APC (p312)peaking at day 6 TMCH that returned to baseline by day 12. A novel 110 kDa N-terminal fragment (p110) first noticed at day 9, increased 5-fold by day 12, exhibited peptide map similarity with p312 and accumulated in the nucleus bound to 13-catenin.Aim: To determine whether alterations in APC expression are paralleled by changes in APC subcellular distribution during TMCH. Methods: Distal colonic crypts isolated from normal, day 6 and day 12 TMCHmice were immunofluorescently labeled by both N- and C-terminal APC antibodies (ofAPC,and oAPCc, respectively) and analysed by confocal microscopy. Results: In normal colonic crypts, cells within the base exhibited weak punctate staining with ~APCN. At day 6, a gradient of increasing crypt base:surfaceAPC immunoreactivity was seen along the subcellular apical membrane. The epithelial cells of the crypt also exhibited weak apical-lateral staining towards the surface, but goblet cells were devoid of APC signal. These results correlated with 4-fold increase in p312 protein abundance at day 6. At day 12, intense immunoreactivity extending throughout the longitudinal cellular axis of the crypt was observed. Nuclear staining was noted occasionally in basal crypt regions while goblet cells were negativefor APC. Staining with o~,PCcin normal crypt exhibited weak APC immunoreactivity. At day 6 TMCH, a gradient of staining similar to day 6 staining with ~APCN,was observed while day 12 crypt failed to show detectable immunoreactivity consistent with the decreasedp312 noted by Western blots. The dramatic increase in staining at day 12 with ~APCNthus represents p110 rather than p312.Conclusions: (i) Increasesin full length APC expression at day 6 correlate with increased staining intensities observed with both N- and C-terminal antibodies, (ii) Lack of APC immunoreactivity with ~APCcat day 12 correlates with its inability to detect p110 by Western blotting, (iii) p110 has a different distribution pattern both a~ongthe crypt axis and intracellularly compared to p312. Significance: The abundance of both wild type and low molecular forms of APC are subject to cell signaling and physiologic cues in native epithelia and may underlie functional changes.

$924 Paclitaxel and 5-Ruorouracil Enhance the Cell-cidal Effect of PhotodynamicTherapy in Cancer Cells In Vitro Seung Woo Park, Tae Yun Oh, Sun A Kim, Se Jun Lee, Jun Pyo Chung, Jae Book Chung, Jin Kyung Kang, Si Young Song, Seoul, South Korea Backrounds and Aims: Photodynamic therapy (PDT) is used for various cancers. The effect of PDT is based on the reactive oxygen species generated by the activated photosensitizer after light irradiation. However the maximal depth of tumor destruction is less than 15 mm, which can be a cause of recurrence. Combination of other drugs to potentiate the PDT effect can be a measure to enhance the effect. Therefore, to investigate whether combination of anticancer drug causing G1-S (5-FU) or G2-M (paclitaxel, Pct) arrest enhances the effect of PDT, we carried out in vitro study in cancer cell lines. Material and Methods: In vitro PDT was done using two cell lines (N-87~stric, YGIC-6B;bile duct cancer). Photofrin (Pf) and Verteporfin (Vf) were used as photosensitizers and PTH light source (1,000 W, Oriel Co.) with 630 nm narrow band pass filter was used. To estimate the enhancement by drug, varying light doses were combined with IC25 of each drug. Cell survival was analyzed by MTT assay and flow cytometry. Results: Both Pf and Vf revealed diffuse cytoplasmic and mitochondrial uptake, which was confirmed by confocal image after double staining with Rhodamine 123 and photosensitizers. PDTtreatment caused light dose dependentcell death,and flow cytometry revealedthat PDT caused prominent G2-M phase arrest. When we combined fixed drug dose (IC25) with PDT (IC25 and IC50), both Pct and 5-FU showed more than additive effect of cell death. To estimate PDT enhancement effect, we combined fixed drug doses (IC25) with increasing light doses of PDT in eachcell line. The survival data were normalizedto compensate the drug effect. PDT enhancement ratio (ER) was obtained by dividing light dose of IC25 and 50 in PDT alone by those of combined treatment. When the ER was more than 1.0, we could say the drug enhances the PDT effect. When Pct was combined to PDT, the ER was 126 (Vf) and 1.47 (Pf) in N87 cells, and was 124 (Vf) and 1.22 (Pf) in YGIC-6B cells. When 5FU was combined to PDT the ER was 1.35 (Vf) and 1.04 (Pf) in N87 cells, and was 1.15 (Vf) and 0.77 (Pf) in YGIC-6B cells. Therefore, while Pct markedly enhanced the PDT effect of both Vf and Pf, 5-FU enhanced the PDT effect only for Vf and the enhancement was modest. Flow cytometry also revealedmarked increment of subdiploid fraction when Pct was combined to PDT. Conclusion: Combination of Pct with PDT revealedstrong enhancementof PDT effect, which was modest when PDT was combined with 5-FU, and further study to elucidate the mechanism of such synergistic effect will be needed.

$922 Pressure Stimulates Proliferation in Human SW620 Colon Cancer Cells via ERK Activation. Mary F. Walsh, Russel K. - Y. Woo, Rainer Grotelneschen, Marc D. Basson, Detroit, MI Physical forces such as strain or pressure appear mitogenic in some cell types in vitro and pressure is mitogenic in rat bile duct epithelium in vivo. High fat-low fiber constipating diets may increase risk for human colon cancer but the mechanisms involved are unclear. We hypothesized that a moderate increase in pressure (15 mmHg over ambient) would promote colon cancer cell proliferation and speculated that MAPK activation might mediate this effect. Sub-confluent (50-60%) SW620 cells were exposed to increased pressure in a prewarmed pressurized box as previously described. Proliferation was assessed by cell counts 24 hours after application of 0-24 hours of 15 mmHg increased pressure, and MAPK by Western blotting for activated ERK 1,2, and p38. Increasing the duration of exposure (0, 3, 4.5, 6, 12, 24 hours) to pressure time-dependently stimulated proliferation. This reached statistical significance after 6 hours and yielded a 42 _+11% increase in cell number at 24 hours (n = 414, p
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caspase-3and p21waf/cip-1 documented a dramatic transcriptional downregulation of p21waf/ cip-1 exclusively in apoptosis susceptible HepG2cells. Reconstitution of p21waf/cip-1 rescued HepG2cells from IFl~-~/inducedapoptosis, indicating that p21waf/cip-1 reduction was required for apoptosis execution. CONCLUSION:Downregulation ot p21waf/cip-1 in HCCcells functions as a novel, critical determinant of alternative growth inhibitory pathways in response to IFN-,y

Immunohistochemical Expression of Infegrin ~5, ~6, ~xV, I~1, l~3 AND 134 Subunits in Gastric Cancer Chang Woo Chain, Seung Woo Park, Yule Shin, Kyung Hwa Park, Jae Bock Chung, Jin Kyung Kang, Si Young Song, Seoul, South Korea BACKGROUND: The integrins are a large family of cell adhesion receptors mediating cellmatrix adhesion. They seem to play a central role in cell survival and apoptosis, proliferation, differentiation, and migration in a way of cell to matrix or matrix to cell signaling. But the roles of integrin in carcinogenesis and tumor angiogenesis are largely unknown. So, we investigatedthe relationships betweenthe expressions of various integrin subunits and clinicopathologic factors and angiogenesis in gastric cancer. METHODS:Expression of integrin c~5, ~6, ~V, 131, 133, and 134 was analyzed by immunohistochemical staining in formalin-fixed paraffin embedded tissues obtained from 102 surgically resected gastric cancers. We also performed factor 8 immunostaining to evaluatetumor angiogenesis.Pathologicallythe tumors were categorized into differentiated (23) or undifferentiated (79) type. Pathologic T-stages were T1/T2 in 41 cases and T3/T4 in 61 cases and regional lymph nodes were positive in 55 cases. The stain was regarded as positive if more than 10% of the tumor cells were distinctly stained at the cell membrane or cytoplasm and positive cases were further divided into 1 + or 2+(if more than 50% of cells are positive). The tumor vessels were counted on a X200 microscopic field. RESULTS:Each integdn subunits were expressed at membrane or cytoplasm of the tumor cells. The positive rates of each subunits were as follows: c~5 24%; 131 8%; c~6 16%; 134 23%; c~V 29%, 133 34%. Compared to normal gastric mucosa, the expression of c~6 and 134 were frequently decreased, 62.7% for c~6 and 41.2% for 134, whereas other integrins showed no distinct difference between tumor and normal tissues. The expression pattern of each subunit was correlated well with each other according to the known pairs of integrin subunits, i.e. ~5131, ~6134, and cxV133(p
Background: Hepatitis C virus (HCV) infection may have a pathogenetic role in both mixed cryoglobulinaemia (MC)and development of B-cell non-Hodgkin lymphoma (NHL). Aims: Prevalence of MC was determined in patients with chronic hepatitis C and immunogtobulin heavy chain (IgH) rearrangement was studied to verify the clonality of lymphoproliferation in HCV infected patients. In addition, activation of nuclear factor kappa B (NFKB) was investigated in peripheral blood lymphocytes and liver biopsies. Patients and methods: A total of 90 patients with chronic HCV infection have been studied for MC and IgH rearrangement. Ten patients with chronic hepatitis C, 13 patients with B-cell NHL (among them 4 HCV positive) and 10 healthy subjects were examined for the activity of NFKB transcription factor using electrophoretic mobility shift assay (EMSA) and immunohistochemistry was performed in 8 liver biopsies from HCV positive patients. Results: MC was detected in 22/90 (24.4 %) patients, IgH rearrangement occured in 14/90 patients (15.5 %), 7 of them suffering from MC. NFKB specific oligonucleotide-protein complexes have been found in the lymphocyte extracts from 8/10 patients with chronic hepatitis C and in 10/13 patients with B-cell NHL. NFKB was detectable in 7/8 cases in liver biopsies by immunohistechemistry. Conclusions: IgH gene rearrangementis sensitive method for detecting occurrence of early stage of B-cell lymphoproliferative disease in HCV infection. Cryoglobulinaemia can be an intermediate step in HCVrelated B-cell proliferation and NHL. NFKB activation was shown in chronic hepatitis C similarly as that in B-cell NHL. Our findings support the hypothesis, that HCV plays an etiological role in the lymphoprolJferation leading to B-cell NHL. This study was supported by the Hungarian Ministry of Health

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Identification of Frameshift Intermediate Mutants in Human Colorectal Cancer Cells Chfistoph Gasche, Loki Natarajan, Ajay Goel, Jennifer Rhees, Dennis Young, Christian Arnold, Christina L. Chang, C Richard Boland, Vienna, Austria; La Jolla, CA

Expression of a Novel Human Tropomyosin Isoform, TC22, in Gastric Intestinal Metaplasia Associated with Gastric Carcinoma Sangeeta Das-Bhattacharya,Kelly Walton, Jiro Watari, Peter S. Amenta, Jim J. - C. Lin, Kiron M. Das, Newark, DE; New Brunswick, NJ; Iowa City, IA

Frameshift mutations at microsatellites are a time dependent function of poiymerase errors followed by failure of the mismatch repair (MMR) system. A cell culture system has been developed that allows the identification of both intermediate (i.e. post-replicational mutant cells before MMR) and definitive mutant cells. A plasmid was constructed that contained 13 repeats of a poly(dC-dA)-poly(dG-dT) immediately after the start codon of the EGFP gene, thereby shifting the EGFP gene out of its reading frame. The plasmid was introduced into human MMR deficient (HCT116; hMLH1 mutated) and MMR proficient (HCT116+ chr3 cells; hMLH1 wildtype) colorectal cancer cells. After stable transfection and single cell cloning, frameshift mutations that restored the EGFP reading frame were detected by FACS. Two distinct populations, M1 (dim) and M2 (bright), were identified (Fig). M2 cells accumulated in culture over time, but M1 cells did not. Single M1 and M2 cells were sorted by FACS, small colonies were grown, and DNA sequence analysis revealed that M2 cells displayed a 2bp deletion at the (CA)13 microsateflite. More interesting, 28% of M~ cells carried (CA)13(GT)12 heteroduplexesof the plasmid indicating that such cells are intermediate mutants that appear transiently after the polymerase error. The mutation rate in HCT116 cells (6.17"10-4) was 30 times higher than in HCT116+ chr3 (1.92"10-5; p
$927 p21waf/cip-1 Determines Cell Fate of Human Hepatocellular Carcinoma Cells in Response to Interferon-,,/ Katharina Detjen, Derek Murphy, Martina Welzel, Bertram Wiedenmann, Stefan Rosewicz, Berlin, Germany

$928 Immunoglobelin Heew Chain (IgH) Rearrangement, Activation of Nuclear Factor Kappa B (HFKB) and Mixed Cryoglobulinaemia in Chronic Hepatitis C Virus (HCV) Infection Beata Gasztonyi, Alajos Par, Katalin Kiss, Laszlo Kereskai, Jozsef Szeberenyi, Laszlo Pajor, Geza Hegedus, Gyula Mozsik, PGcs, Hungary

Objectives and Methods: Using a cDNA ~.ZAP library prepared from poly(A)+ RNAs of a human colon cancer cell fine T84, we have recently identified and cloned a novel human tropomyosin (hTM) isoform, termed TC22. The amino acid sequenceanalysis of TC22 demonstrated that it is identical to hTM isoform 5 except for the C terminal domain aa222-247 coding the exon 9. Using this C terminal peptide, we developed a monoclonal antibody, mAb, (TC22-4) which is specific to TC22 and does not react with any other hTM isoforms, including hTM5. Northern blot analysis with TC22 specific probes revealedthat normal epithelial tissue from colon and stomach does not express TC22, whereas transformed counterpart and tumor tissue expressed TC22. The TC22-4 mAb reacts with colon cancer (96%) and with benign adenomatous colonic polyp (35%), but not with normal colonic epithelium. To investigate if TC22 is expressedin gastric carcinoma and its pre-cancerousstate, gastric intestinal metaplasia (GIM), we have examined surgical specimens from 22 patients with gastric carcinoma. Paired samples of both cancer segment and gastric mucosa showing GIM away from the cancer regions were examined by a sensitive immunoperoxidase assay using TC22-4 mAb. Normal colon mucosa and a colon cancer were used as negative and positive controls respectively. Results: Nineteen of 22 gastric cancer tissue (86%) reacted with TC22-4 and 3 .did not. GIM areas away from the cancer segment of the same 19 patients also reacted with TC22-4 mAb. It is intriguing that the 3 gastric cancers which did not react with TC22-4, GIM areas away from cancer segment in these 3 patients, also did not react. The immunoreactivity was restricted to the cancer cells and in the metaplastic cells of GIM, including both goblet and non-goblet epithelial cells. Adjacent normal gastric mucosa and submucosal or muscle tissue of the stomach did not react with TC22-4. The reactivity within the epithelial cells was diffuse cytoplasmic, with dense strong globular dot-like,staining at supra-nuclear areas. The staining in the cancer area, in general,was more intensethan the GIM areas. Conclusion: The expression of a novel hTM isoform, TC22, is strongly associated with gastric cancer and pre-cancerous state of gastric carcinoma, GIM. The presence of TC22 may provide an important biomarker in the identification of high risk patients with GIM.

$930

BACKGROUNDand AIM: Currently, there is no effective treatment for advanced hepatocellular carcinoma (HCC). We therefore explored the molecular mechanisms of Interferon-~,(IFN-~,) mediated growth regulation in human HCC cell lines. METHODS:IFN-.y receptor expression, signal transduction and regulation of effectors were examined by RT-RCR, immunoprecipitation, immunoblotting and reportergene assays. Growth and apoptosis were determined based on cell numbers, cell cycle analyses, kinase assays, DNA fragmentation and PARP cleavage. RESULTS: HCCcell lines express functionally intact IFN-~,,receptors and downstream effectors. IFN-~, profoundly inhibited growth of HCC cells via two different mechanisms: inhibition of G1 cell cycle progression and induction of apoptosis. Analyses of SK-Hep-1 cells revealed a deficient cyclin D induction in IFN-,y treated cells, resulting in reduced activity of CDK4 and CDK2 kinases and pRB hypophosphorylation. In contrast, apoptosis prevailed in IFN-'ytreated HepG2 cultures. A survey of apoptosis relevant IFN-~-effectors including IRF-1, caspase-1,

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The P38 Kinase Inhibitor with SB203580 Inhibits Cell Proliferation and Induces GO/ G1 Arrest by Inhibiting Cyclin D1 Expressionin the Colonic Cancer Cell Line, HT29. Xian-Zhong Ding, Thomas E. Adrian, Chicago, Ih Three human MAP kinases have been identified; P42/44 MAP kinase, P38 MAP kinase and JNK/SAPK. Proliferation of colonic cancer cells is regulated by P42/44 MAP kinase. Growth factors, such as epidermal growth factor (EGF), stimulate ceil proliferation via activation of this signaling molecule while inhibition of P42/44 MAP kinase blocks colonic cancer cell growth. However,the role of P38 MAP kinase in colonic cancer cell growth is not established. Using the specific P38 kinase inhibitor, SB203580, we investigated the effects of P38 kinase on proliferation, cell cycle and cell cycle-regulating proteins in the colonic cancer cell I~ne, HT29. Our results demonstrate that SB203580 (1) concentration-dependently inhibits HT29 cell proliferation; (2) dramatically arrests HT29 cells to GO/G1phase; (3) significantly decreases cyclin D1 protein expression (4) has no effect on cyclin E or cyclin B; (5) has no effect on P21WAF or P27KIP; (6) has no effect on cyclin-dependent kinase 2 (CDK2), CDK4 and CDK6

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expression; (7) has no effect on phosphorylation of P42/44 MAP kinase. Based on these results, we speculate that SB203580 inhibits HT29 cell proliferation and GO/G1 arrest through decreasing cyclin-dependent kinase activity, due to inhibition of cyclin D1 expression. These findings point to P38 MAP kinase as an important growth regulatory pathway in colon cancer.

Caco-2 cell induced endothelial cell proliferation. This occurs in part by downregulation of VEGF, a key mediator of angiogenesis. Butyrate also may induce anti-angiogenic cytokine IL18, contributing to the decrease in endothelial cell proliferation.

$934 $931 Aberrant Methylation of the CDX1 CpG Island in Gastric Cancer Cell Lines Akashi Eda, Hiroyuki Osawa, Ichiro Yanaka, Kentaro Sugano, Tochigi, AE, Japan

Thioredoxin Reductase Expression in Colon Cancer: Discrepancy between In Vitro and In Vivo Findings Sandra Lechner, Uit M(iller-Ladner, Tanja SpOttl, Klaus Schlottmann, Josef Ritschoff, JOrgen SchOImerich, Frank Kullmann, D-93042 Regensburg, Germany; D-34125 Kassel, Germany

Background-TheCDXl and CDX2genes are intestinal transcription factors that may be involved in regulation of proliferation and differentiation of intestinal epithelial cells. We have reported that the expression of CDX2 was emerged at the stage of gastritis and preceded those of CDX1 during the progression of gastric intestinal metaplasia (J. Gastroenterol 2002; Vol. 37 No. 2 (in press), Gastroenterology 2001;120(Suppl 1):A-299). Furthermore, we generated a transgenic mouse in which intestinal metaplasia was induced by expressing CDX2 in the stomach(Gastroenterology 2001;120(Suppl 1):A+240). Therefore, we consider CDX2 expression may play a critical role in the development of intestinal metaplasia.A previous investigation showed that CDXl was expressed in the intestinal metaplasia, adenoma and carcinoma of stomach. Recently, however, lack of expression of CDX1 has been reported in a subset of gastric carcinoma celt lines whereas CDX2 was expressed in most gastric cancer cell lines. The precise molecular mechanisms by which COX1 expression is suppressed has not been elucidated yet. Aberrant CpG island methylation has been implicated in the transcriptional silencing of various genes in cancer. Nevertheless,there have been no reports about methylation status of CDX1 promoter region in gastric carcinoma cell lines. Aims-To examine the potential role of promoter methylation in the transcriptional silencing of CDX1 in gastric carcinoma cell lines. Methods-We examined the methylation status of the CDX1 5' CpG island in a series of tumor cell lines by combined bisulfite restriction analysis (COBRA) and bisulfite sequencing. Results-Detailed methylation mapping using bisulfite genomic sequencing revealed that hypermethytation of a region upstream of the CDX1 gene transcriptional start site was detected in five cell lines (MKN 28, MKN 45, MKN 7, MKN 74, AGS). In contrast, sparse methylation (26%) was detected in KATO II1. In KATO III cell, this methylation was predominantly in the 3' region of the island closest to the transcription start site. Expression could be restored by demethylation using the DNA methyltransferase inhibitor 5-aza-2'deoxycytidine (5-aza-CdR)in these cell lines. Conclusions-Thesefindings indicatethat methylation of promoter region is one possible mechanism by which CDX1 is silenced in gastric carcinoma cell lines.

Introduction: Thioredoxin and thioredoxin reductase (TR) are redox proteins that have been implicated in cellular events such as proliferation, transformation or apoptosis. Thus, they could play an important role in regulating cell growth and death. Analysis of the expression and localization of TR in different normal and cancer cell lines, and colon tissues (normal, neoplastic or inflamed) was performed using RT-PCR and in situ hybridization. Methods: Semiquantitative RT-PCRwas performed using genespecific primers for TR in order to quantity the amount of TR mRNA. Quantification of the PCR product was done by phosphorimaging. TR antisense and sense RNA probes (600 bp) were transcribed from human cONA and labelled with digoxigenin-uridine triphosphate. Hybridization was performed overnight at 50°C. To detect TR mRNA, an anti-digoxigenin antibody was applied and visualized with NBT/BCIP. In situ hybridization experiments with a TR antisense probe were performed using matched pairs of normal and colon cancer tissue, colon adenoma tissue and colon tissue from a patient with Crohns disease. Results: TR mRNA was expressed in all analyzedtissues with TR mRNA positive cells restricted to the stroma of colon crypts, being partly positive for CD3 or CD56 (double staining for TR and CO3/CD56). In neoplastic areas of colonic cancer tissue a loss of TR was obvious. None of the epithelial cells in colonic mucosa expressed TR mRNA, whereas more than 70% of HT-29 cells grown in monolayer were positive for TR. In contrast, HT-29 cells, grown as spheroids or as tumors in SCID mice, were negativefor TR. Conclusions: In contrast to the presented in vitro findings and previous studies, there is no evidence that TR plays a significant role in vivo in normal cell growth in colonic epithelial cells. In addition, the lack of TR in neoplastic areas of colon cancer tissue in comparison to non-neoplastic areas implicates that loss of TR might contribute to tumor promotion by reduceddefensemechanisms.

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$935

Role of Thymidine Phosphorylasein Fas-lndueed Apoptosis Shinichiro Mori, Shonsbin Takao, Yuko Mataki, Hiroyuki Shinchi, Takashi Aikou, Kagoshima, Japan

Thyroxine Modulates Ontogenetic Development of Intestinal Bile Acid Transport in Rats Iona M. Monteiro, Elmer S. David, Ying Jie L. Wu, Ronaldo P. Ferraris, Newark, NJ

PURPOSE.Thymidine Phosphorylase (TP) has angiogenic activity and promotes tumor growth in vivo. TP expression is associated with increase of angiogenesis and decreaseof apoptosis in pancreas carcinomas. TP expression is also associated with a poor prognosis in patients with ductal adenocarcinoma of pancreas. It suggests that TP has an important role in tumor growth and cell death. To clarity whether or not TP prevents apoptosis, we analyzedthe effect of TP on the cell death during Fas-induced apoptosis. MATERIALSAND METHODS.We used KBTP cells transfected with TP cONA and KBCV cells (a mock transfectant). Apoptosis was determined by staining Propidium Iodide, and mitochondrial membrane potential was analyzed by using Rhodamine123with FACSoan.Caspase-3,cytochrome c and caspase-8 protein levels in the cytosol were determined by western blot analysis. RESULT.OverexpressingTP decreased apoptosis, toss of mitochondrial membrane potential, and interfered with caspase-3 cleavage, cytochrome c releaseand caspase-8 cleavageduring Fas-inducedapoptosis. CONCLUSIONS. We demonstrate that overexpressing TP interferes caspase-8 cleavage in Fas-induced apoptosis. These findings indicate that TP has an important role in apoptotic cell death. It suggests that the inhibition of TP suppresses the progression of tumor by inducing apoptosis in TPoverexpressing solid tumors.

Plasma thyroxine concentrations rise sharply in rat pups during transition from suckling to weaning (~ 14 days of age) and precede the abrupt appearance of ileal sodium-dependent bile acid uptake at 17 days of age. Thyroxine regulates the appearanceof bile acid transport, but there has been no study on the effect of thyroxine removal prior to its naturally occurring surge. We therefore made pups hypothyroid by giving the dams 0.01% propylthiouracil as drinking water starting at 18-19 days of gestation until the end of the experiment. A second group of dams with euthyroid pups served as controls. One half of pups from each group received daily injections of thyroxine starting on day 6 of life and the other half were injected with vehicle. Hence there were four groups: hypothyroid injected with vehicle (HT+V), hypothyroid injected with thyroxine (HT+T), euthyroid injected with vehicle (ET+V) and euthyroid injected with thyroxine (ET+ T). Validation studies showed that in vitro taurocholate uptake in the neonatal ileum was carrier-mediated and had a Km of 0.52 mM, Jmax of 0.34 pmol/mg min and a Kd of 0.029 pmol/mg min raM. Taurocholate uptake was then measured at postnatal ages 12, 20 and 26 days in the jejunum and ileum of all treatment groups. The remainder of the ileum was frozen for Northern blot analysis for the sodium-dependent bile acid transporter (ASBT). Blood for thyroxine assay was collected at days 6, 12,20 and 26. Serum thyroxine levels were low in all groups at 12 days of life and continued to be low in HT+V pups at 20 and 26 days. Serum thyroxine levels increased in HT+T, ET+V, and ET+T at 20 and 26 days. Taurocholate uptake in the jejunum was low in all age and treatment groups. At 12 days of age, ileal ASBT mRNA abundance and taurocholate uptake were low in all groups. At 20 and 26 days, ileal ASBT mRNA abundance and taurocholate uptake remained low in the HT+V pups but increased (4-6X and 2-3X, respectively) in the HT+T, ET+T and ET+V pups. Glucose uptake did not vary among the four treatment groups indicating that thyroxine effects on taurocholate uptake were specific, Thus, the appearance of taurocholate transport in the ileum is inhibited in hypothyroid rat pups, suggesting that thyroxine plays a crucial role in the ontogeny of rat ileal bile acid transport. Becausechanges in ASBT mRNA abundance and activity are tightly correlated, thyroxine may regulate ASBT expression at the transcriptional level. (Supported by funds from the Foundation of UMDNJ and the NSF)

$933 Inhibition of Intestinal Tumor Cell Induced Proliferation of Endothelial Ceils by Botyrate Dimitrios Zgouras, Astrid W~.cbtersh~user,J•rgen Stein, FranMurt am Main, Germany Angiogenesis is of fundamental importance in severalphysiological and pathological processes, including tumor growth and metastasis. Proliferation of endothelial cells is an extremely important occurrence in angiogenesis. The aim of our study was to investigate the effects of butyrate on intestinal tumor cell (Caco-2)-induced proliferation of endothelial cells. Methods: Condiotioned media were obtained by Caco-2 cells, incubated with serum free medium containing either the solvent (PBS) or butyrate (2 or 4 mM). Proliferation of HUVEC cells was assesed by a BrdU incorporation assay. HUVEC cells were cultured in standard media until 90-100% confluent, and then subjected to serum-free media conditioned by Caco-2 cells. VEGFproduction in the supernatantof Caco-2 cells was quantified by use of a colorimetric ELISA. Furthermore IL-18 was detected by Western Blot analysis. Results: Conditioned medium collected from Caco-2 cells increased HUVECcell proliferation. VEGFsecretion raised simultaneously in Caco-2 ceils, starting from 153 +/- 39 pg after 12 h up to 556 +/- 114 pg after 24 h and 1.739 +/- 262 pg after 48 h. However HUVEC cell proliferation was decreased when celts were treated with conditioned media obtained from butyrate treated Caco-2 cells up to 46% of controls. In addition we observed that VEGF production was significantly diminished vs. controls (434 +/- 170 pg after 24 h vs. 556 +/- 114 in controls, p
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$936 Development of the Fetal Intestine in Glucocorticoid Receptor (GR) Deficient Mice Hans Gartner, Thomas J. Oesterreicher, Colleen Graul, Milton J. Finegold, Susan J. Henning, Houston, IX Although glucocorticoids are recognizedas playing an important role in postnatal development of the rodent intestine, to date there is conflicting evidence regarding the involvement of these hormones in prenatal intestinal development. This reflects the inherent difficulty of studies requiring endocrine manipulation in fetal animals. As an alternate approach we have used mice in which the gene for the glucocorticoid receptor (GR) is disrupted. Our specific hypothesis was that the fetal phase of intestinal development is glucocorticoid-independent, and thus would be unaffected by elimination of the GR. To investigate this hypothesis we first verified that the GR is normally expressed in the prenatal mouse intestine. To this end, fetal intestines were collected from C57BI/6 mice at E15.5, E17.5 and E18.5. RNA was extracted

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and GR mRNA was analyzed by both RT-PCR and Northern blotting. The results showed strong expression of GR mRNA at all ages. Our next step was to procure GR +/- mice from Dr. Genther SchOtz (Cole, et al. Genes Dev. 9:1608-1621, 1995) and to breed these onto a C57BI/6 background. Fetusesof heterozygote matings were then collected and genotyped by PCR. Lack of expression of native GR mRNA in -/- intestines was confirmed by RT-PCR. Morphologic maturation of the intestine was assessed by performing histology of -/- and +/ + liftermates during the late fetal period. Tissue collected at E13.5, E15.5, and E18.5 showed that small intestines from both wild type and GR-nul] animals underwent normal villus morphogenesis during this period. Eurthermore, staining for lactase activity by X-gal histochemistry at E18.5 showed an equally strong reaction in GR -/- intestine as in wild type intestine. Analyses of other aspects of intestinal maturation are in progress. Nevertheless, from the current data we conclude that the fetal phase of rodent intestine is indeed glucocorticold-independent. This work was supported by NIH grant number HD-14094.

$937 Musashi-1 and HES-1 Expression in Rodent Small Intestine. Takahisa Kayahara, Mitsutaka Sawada, Shigeo Takaishi, Hiroshi Seno, Hiroaki Fukuzawa, Katsumasa Suzuki, Mayumi Kawada, Hirokazu Fukui, Tsutomu Chiba, Kyoto-city, Japan Recently, there is increasing interest in intestinal epithelial stem cells. However, specific intestinal stem cell markers have not been identified. Musashi-l(Msi-1), the neural RNA-binding protein essential for neural cell development and required for asymmetric cell divisions in the Drosophila adult sensory organ development, is expressed predominantly proliferating multipotent neural precursor cells in rodents. Hes-1 is one of the basic helixloop-helix transcription factors that regulate mammalian CNS development, and its loss-andgain function phenotypes indicate that it negatively regulates neuronal differentiation. Both Msi-1 and HES-1 are known to he expressed in small intestine. In this study, we characterized Msi-1 or HES-1 expressing cells in the small intestine. Postnatal BALB/C mice and Wistar rats were used in this experiment. The day of birth was defined as postnatal day (P) O. The expression of Msi-1 and HES-1 in small intestine was confirmed by RT-PCR, and immunohistochemical staining was performed by using anti-MsJ-1 antibody. RT-PCR showed both Msi-1 and HES-1 were expressed in the small intestine throughout postnatal days. Immunohistochemical staining also showed Msi-1 was localized in only a few epithelial cells at basal part of crypt except Paneth cells. In contrast, HES-1 was localized in almost all crypt cells except Paneth cells. From our result, Msi-1 might be a more specific marker for immature cells in small intestine than HES-I. Moreover, HES-1 might be involved in upward differentiation of intestinal cells.

$938 Postnatal Changes in the Activation of NF-KB and Expression of NOS-2 and H0-1 in Rat Intestine Induced by LPS Kwo-Yih Yeh, Mary Yeh, D Nell Granger, Jonathan Glass, Shreveport, LA Background: Epithelial cell innate immunity plays a crucial role in mucosal host defense against invading pathogenic microbes. In mammals, the neonatal intestine may haveimmature epithelial innate immunity, as a variety of host defense components in maternal milk provide protection. Functional innate immunity is capable of sensing pathogenic bacteria and their products and activating defensive genesthrough various signaling pathways. Aims: To investigate postnatal development of the NF-KB pathway leading to inducible nitric oxide synthase (NOS-2) expression induced by LPS and to determine whether the inducible heine oxygenase (HO-1) is also upregulated for cell protection against increased nitric oxide (NO) production by NOS-2 and/or other reactive oxygen metabolites generated after LPS. Methods: Day 6,15, 24 or 60 rats were administered LPS (2 i~g/g), killed at defined times, and the intestinal mucosa was collected for determinations of NF-KB DNA binding activity by EMSA, NOS-2 and HO-1 mRNA and protein levels by Northern and Western analyses. The role of NF-KB in NOS-2 and HO-1 expression was analyzed in rats administered pyrrolidine dithiocarbamate (PDTC, iplO0 Fg/g) lh before LPS. Results: NF-KB DNA binding activity was low at 0-30 rain and increased 3-6 fold at 60 min after LPS in all ages of rats. At 120 rain, NF-KB activity increased further in day 6-15 rats, but decreased in day 24-60 rats. By super-shift assays, activated NF-KB-DNA complexes contained p65 NF-KB subunit only in day 24-60 rats. in all age groups of rats after a lag in which neither NOS-2 mRNA nor protein was detectable at 0 and 60 min, both appeared at 120 min and peaked at 240-360 min after LPS. Mucosal NOS-2 mRNA and protein levels were significantly lower in day 6-15 than day 24-60 rats. The induction of NOS-2 gene expression by LPS is at least in part mediated by NF-KB, as PDTC abolished NF-KB activation and reduced NOS-2 expression. Similar to NOS-2, HO-1 was upregulated by LPS in all ages of rats with significantly lower levels in day 6 than day 60 rats. In contrast to NOS-2, HO-1 expression was upregulated by PDTCalone and increased further by PDTCand LPS together. Conclusions: The NF-KB pathway that leadsto LPS induced expression of NOS-2 is incomplete in early postnatal rats and might compromise epithelial innate immunity. Up-regulation of HO-1 by LPS is independent of NF-KB and NO produced by increased NOS-2. The low responsiveness of HO-1 to LPS in early postnatal rats might limit the capacity of cell protection against oxidative stress.

$939 Mechanisms Underlying Thyroid Hormone Regulation of the Intestinal Alkaline Phosphatase Gene Madhu S. Malo, Wenying Zhang, Mario A. Abedrapo, Joseph W. Henderson IV, Richard A. Hodin, Boston, MA Thyroid hormone (T3) plays a pivotal role in mammalian gut development, most notably at the time of weaning when there are numerous changes,including an induction of the enterocyte differentiation marker intestinal alkaline phosphatase (lAP) gene. T3 generally mediates its cellular effects by binding to nuclear thyroid hormone receptors (TR) which interact with

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specific DNA cis-elements (TRE) located within the regulatory regions of target genes. In the present work, we investigated the molecular mechanisms involved in the T3 regulation of the lAP gone. METHODS:A 2.5 kb 5' flanking region of the human lAP gene (-2574 to -49 in relation to the ATG start codon) was subcloned into the pGL3-Basic Firefly luciferase reporter plasmid. Subsequently, a variety of plasmids (plAPs) were constructed carrying 5', 3', and internal deletions of the lAP regulatory region. The human colon carcinoma cell line Caco2 was transiently transfected with the plAP plasmids along with the Renilla luciferase plasmid, which was used as a control for calculating transfection efficiency. Cotransfections were carried out using plasmids expressing each of the three bona fide thyroid hormone receptors (TRc~I, TRI31 and TRI32), and the cells were then treated -/+ T3 (100 nM). RESULTS: In the presence of T3, the full-length plasmid (-2574/-49) showed 50, 35, and 25-fold activation mediated by TRI31, TRI32, and TRy1, respectively. We then used the 3' deletion constructs to determine the minimal proximal promoter elements required for lAP transcription. Examination of the various 5' deletion constructs led to the localization of a putative TRE in the 135 bp region between -750 and -616 (90 % decrease in luciferase activity), internal deletion of this 135 bp segment resulted in the loss of T3-induced and TR-mediated lAP activation, confirming that the TRE resided in this region. We then replaced this 135 bp segment with each of its three component segments. Only the segment between -660 and -616 was able to restore T3-induced and TR-mediated activity to the lAP plasmid, whereas the other two regions had no effect. CONCLUSION:The TRE within the human tAP gene has been localized to the region between -660 and -610, and sequenceanalysis of this region shows the presence of a 19 bp sequencethat has approximately 80% homology with the consensus TRE sequence. interestingly, this sequence has only three nucleotides separating its half-sites, indicating that this may be a novel TRE.

$94g Ectopic Expression of Ileal Bile Acid Binding Proteins in Jejunal Enterocytes Steve M. Cline, Michael W. Crossman, Cincinnati, OH Intestinal bile acid absorption is a complex transport process that mechanistically varies along the duodenal-colonic axis. Bile acids are translocated across the brush border membrane, trafficked vectorially to the basolateral membrane and are exported into the portal circulation. Two components of the ileal transport system, a sodium-dependent, and electrogenic, brush border membrane transporter (ASBT) and a cytosolic protein, known as the ileal lipid binding protein (ILBP) have been well characterized.Congenital or acquired defects affecting the ileum may result in surgical resection, which will lead to bile acid malabsorption. The adaptive capacity of the remnant intestine will not support this physiological pathway primarily due to absence of ASBT and ILBP expression in the jejunum. While suppression of ASBT and ILBP gene expression in the jejunal enterocyte obviates sodium-dependent bile acid transport, it is unknown whether their ectopic expression would be appropriately audited within the enterocyte. We hypothesized that the ectopic expression of transgenes encoding ASBT and ILBP Would exhibit appropriate subcellular localization and function within the jejunal enterocyte. Using well-characterized jejunal-specific regulatory elements from the Intestinal Fatty Acid Binding Protein Gene (Fabpi), transgenic mouse pedigrees were generated expressing ASBT and ILBP, respectively. Nucleotides minus 277 to + 28 of the Fabpi promoter have previously been demonstrated to target reporters to the proximal two thirds of the small intestine (jejunum). Developmental RNA and protein studies, revealedASBT and ILBP reporter expression in the jejunum up to the fourth postnatal week by Northern, RTPCR and immunoblot analyses. Immunohistochemical surveys on jejunum and control ileum, from transgenic pedigrees, reveal appropriate expression of ASBT at the brush border membrane and ILBP within the cytoplasm. Brush border membrane vesicles (BBMV) were prepared from jejunal and ileal mucosal scrapings using the Mg precipitation method. BBMV prepared from jejunum of ASBT transgenic mice displayed sodium-dependent uptake of tritiated taurocholate equal to vesicles prepared from ileum. These results demonstrate that ectopic ASBT expressed in jejunal enterocytes exhibit functional sodium-dependenttaurocholate transport activity. In conclusion, these studies demonstrate the feasibility of targeting useful foreign gene products to alternate locations along the cephalocaudal axis of the gut.

$B4i Indian HedgehogRegulates Colonic Epithelial Morphogenesisin the Adult Gijs R. Van Den Brink, James C. H. Hardwick, Berber E. Schepman, Corinne Nielsen, Sander Van Deventer, Drucilla J. Roberts, Maikel P. Peppelenbosch,Amsterdam, Netherlands; Boston, MA After the establishment of the gastrointestinal (GI) tract along all its axes of development, continuous renewal of GI epithelial cells in the adult occurs along a single vertical(or radial) axis. In this ongoing patterning, epithelial-stromal interactions play a key role. During development this kind of interaction is regulated by morphogens. We have investigated the expression the hedgehog (Hh) family of morphogens in the adult and previously described a role for Sonic hedgehog in the adult stomach (Van den Brink et al. Gastroenterology 2001, t21:317-328). We have now found that Indian Hedgehog (Ihh) is expressed in the adult colon and here explore the role of Ihh in the adult colon. We used both in situ-hibridization and immunohistochemistry to localize Ihh expression in adult human and rat tissue and examined the HT-29 model of colonic epithelial differentiation for Ihh expression. We treated 7 rats with th e Hh signaling inhibitor Cyelopamine and examined the colon for proliferation (BrdU, PCNA), differentiation and the expression of several Hh related molecules compared to 7 controls, we found that the terminally differentiated absorptive enterocyte produces both Ihh mRNA and protein. HT-29 cells do not normally produce Ihh but Ihh production can be induced in the butyrate model of HT-29 cell differentiation and correlates well with the degree of differentiation. Treatment of rats with the Hh inhibitor cyclopamine affected the expression of several transcription factors and morphogens previously linked to Hh signaling in other systems, and increased the expression of PCNA, Cyclin D1 and precursor cell proliferation (BrdU incorporation). Protein expression of the enterocyte marker Villin was significantly diminished whereasthat of a goblet cell marker (Intestinal Trefoil Factor) was strongly induced. Treatment of HT-29 ce!ls with recombinant hedgehog protein induced villin with similar efficiency as 5 mM buytrate that is normally used in this model. Our data indicate that Hh signaling negatively regulates colonic epithelial precursor cell proliferation. Furthermore, Ihh

AGA Abstracts

is both necessary and sufficient for enterocyte differentiation, whereas it negatively regulates differentiation of the goblet cell lineage. Our data therefore show that Ihh provides lineage instructive information to adult colonic epithelial cells. $942 The Expression of the Homeodomain Transcription Factors Cdx'l or Cdx2 Induces a Cadherin-Mediated Cell-Cell Adhesion in Human Colon Cancer Cells Matthew S. Keller, I Katherine Omoregie, John P. Lynch, Philadelphia, PA INTRODUCTION: Differentiation of intestinal epithelial cells as they ascend from the crypt base involves both new gene expression and morphoJogicalmaturation. While the mechanisms driving new gene expression have been well studied, those promoting column arization, acquisition of cell:cell junctions, and microvilli organization have not. The Cdx homeodomain transcription factors regulate intestine-specific gene expression and intestinal epithelial differentiation. We now report Cdx expression induces a c a dherin-dependent cell-cell adhesion and morphologic maturation in human COlD205 cancer cells. METHODS:The retroviral plasmid MSCV MIGR1 vector was used to deliver Cdxl or Cdx2 expression. Cells were studied unselected or selectedfor a uniformly inf ected population by GFPflow cytometry or antibiotic resistance. RESULTS: Within several days of Cdxl or Cdx2 expression, an obvious new homotypic cell-cell adhesion phenotype was noted only in the Cdx expressing (GFP +) cells. This was a specific effect o f Cdx that likely required new Cdx gene expression, as a Cdxl N-terminal truncation construct lacking a transcription-activation domain failed to elicit the effect. However, a C-terminal Cdxl truncation construct did induce the effect. This adhesion p hen ot ype was Ca+ +-dependent, and could be blocked by an E-cadherinblocking antibody. EM micrographs demonstrate induction of adherens and desmosomal junctions, as well as a columnar shape and an organized microvilli. CONCLUSIONS:Cdxl or Cdx2 express ion al one is sufficient to induce a cadherin dependent adhesion of cold 205 cells. This adhesion was associated with morphologic maturation of these poorly differentiated colon cancer cells. As cadherin-mediated cell-adhesion is known to promote inte s tin al eplth nlial differentiation and limit proliferation, ascertaining the mechanism for this novel Cdx effect may yield insight into processes normally occurring in the intestinal crypt. $943 Inhibition of GSK-3~ Attenuates Sodium Butyrete-Mediated Differentiation and Apoptosis in the HT29 Colon Cancer Cells Oing-Ding Wang, Xiao-Fu Wang, Robert P. Thomas, Stacey L. Walker, B Mark Evers, Galveston, TX

of actin controls. These data demonstrate a polyamine-dependent effect on 4E-BP1 gene transcription. Western blot analysis confirmed similar polyamine-dependent changes in 4EBP1 protein levels following the same treatment. A change in the repressor 4E-UP1 predicts a corresponding change in free, therefore active, eukaryotic initiation factor 4E (elF-4E). To ensure there was a functional change in free 4E-BP1 and elF-4E, a 7-methyI-GTP (7mGTP) sepharose pull down experiment was performed on DFMO treated cells. 7mGTP recognizes the cap binding protein elF-4e. These data showed a significant decrease in the amount of the translation repressor 4E-BP1 bound to the cap binding protein eIF-4E. These data demonstrate that putrescine regulatestranslation of numerous cell cycle progression proteins, growth factors and oncogenes by modulating transcription of the gene encoding 4E-BPI. $945 Role of the Notch Signalling Pathway in Gut Homeostasis Guy Sander, Barry C. Powell, Adelaide, Australia

Background:Themolecular mechanisms involved in establishing and maintaining the regional cellular complexity along the crypt-villus and duodenal-colonic axes of the gut are largely unknown. To address this issue we investigated Notch receptor and ligand expression in the adult rat gastrointestinal tract and show that this signalling pathway, previously shown to control the fate of other cell types, may play a role in gut homeostasis. Methods.Weinvestigated the expression of the receptors, Notch1, Notch2 and Notch3 and the ligands, Jagged1,Jagged2 and Delta1 throughout adult rat gut. Results:Transcriptswere detected for each gene by Northern blot analyses in the oesophagus, stomach, duodenum, jejunum, ileum, caecum and colon. In situ hybridisation revealed that the Notch receptors and ligands exhibit a complex expression pattern throughout the gut and are variously expressed in epithelial, immune, neuronal and endothelial cells. In stratified oesophagealtissue and in the adjacent keratinising region of the rat stomach, Notch1, Notch2, Jagged1 and Jagged2 were principally expressed in the basal epithelial cells. In the small and large intestine, Notch1, Jagged1 and Jagged2 were expressed in the lower half of the crypts, overlapping the cell proliferation marker, Histone H3. Curiously, Notch2 expression was restricted to few crypt cells. Notch1, Notch2 and Jagged2 were also expressed in the lamina propria. Furthermore, Notch1 and Notch2 but not the ligands Jagged1 and Jagged2 were expressed in Peyer's patch lymphocytes. Notch1, Jagged1 and Jagged2 were coexpressed in neuronal ganglion cells of the myenteric and submucesal plexus throughout the gut. Finally, Notch1, Jagged1 and Jagged2 were expressed in the arterial endothelium but not in veins. Oiscussion~rhecomplex expression pattern of the Notch receptors and tigands in various proliferative and post-mitotic cell types in the gut implies that Notch signalling has a significant role in maintaining gut homeostasis. S~16

The cellular mechanisms regulating intestinal differentiation and eventual apoptosis are not known. Recently, we have shown that inhibition of the phosphatidylinositol 3-kinase (PI3kinase) pathway enhancessodium butyrate (NaBD-mediated enterocyte-lika differentiation of the human colon cancer cell line HT29 as noted by increased activity and expression of the brush border enzyme, intestinal alkaline phosphatase (lAP). The purpose of our present study was to determine the role of glycogen synthase kinase-313(GSK-313), a kinase negatively regulated by the PI3-kinase pathway, in NaBT-induced differentiation and apoptosis of HT29 cells. METHODS. HT29 cells were treated with NaBT (5 mM) in the absence or presence of lithium chloride (LiCI), a specific GSK-3 inhibitor. Cells were harvested at 24 h after NaBT treatment, stained with propidium iodide for cell cycle analysis; cell extracts were analyzed for lAP activity, in addition, RNA was extracted and ribonuclease (RNase) protection assays performed using a labeled multiprobe (hCC-2; Pharmigen) which assessesmultiple cell cyclerelated genes including the Cdk inhibitor p21*~'. DNAfragmentation was evaluatedby examination of cytoplasmic histone-associated DNA fragments using a Cell Death ELISA kit. RESULTS. NaBT treatment alone induced cell cycle arrest at the Go/G1 phase, increased lAP activity and increased p21~t~ gene expression; treatment with LiCI alone increasedthe percentage of cells in S phase with no induction of either lAP activity or p21'~1 expression. The addition of LiCI with NaBT inhibited NaBT-mediated lAP induction in a dose-dependent fashion, but had no effect on the induction of p21*~fLSurprisingly, treatment with a combination of NaBT and LiCI induced a marked G2Mphasearrest accompanied by a decreaseof DNAfragmentation. CONCLUSIONS. Our results suggest that the regulation of intestinal cell differentiation by PI3-kinase may be mediated through the inhibition of GSK-313. Therefore, these findings identify a downstream target of PI3-kinasewhich may contribute to intestinal cell differentiation.

Role of Suppressor of Cytokine Signaling-2 (SOCS-2) in Intestinal Growth and Differentiation Carmen C. Zuniga, Megan Miller, James G. Simmons, P. Kay Lund, Christopher Greenhalgh, Joan K. Heath, Chapel Hill, NC; Parkville, Victoria, Australia BACKGROUND:Suppressors of cytokine signaling (SOCS) proteins were discovered as negative regulators of cytokine signaling via JAK-STAT pathways in immune cells. Recent findings in SOCS-2 knock-out mice indicate a role for SOCS-2 in limiting body growth and growth of several organs, but intestine was not examined (Metcalf et al., 2000). We tested a hypothesis that SOCS-2 inhibits intestinal growth in vivo and inhibits proliferation of intestinal epithelial cells in vitro. METHODS: In vivo studies compared length and weight of small intestine in SOCS-2 null and wild type (WT) mice and compared expression of SOCS-2 mRNA in the small intestine of unweaned and weaned mice. In vitro studies assessed the abundance of SOCS-2 mRNA in serum-deprwed Caco-2 calls over the course of 2-15 days in culture. These cells characteristically show an initial high growth phase between day 1 and day 5 in culture followed by reduced proliferation and increased differentiation between day 10 and day 15. 3H-thymidine incorporation and sucrasa mRNA abundance was assessed in Caco-2 cells transiently transfacted with a SOCS-2expression construct or empty vector. RESULTS:SOCS2 null mice showed a 13% increase in length (p
$944 Putrescine Modulates Transcription of the Translational Repressor 4E Binding Protein 1 Joseph F. Christian, Edward R. Seidel, Greenville, NC Polyamines have multiple physiological effects related to growth, differentiation and cell cycle progression. They, along with the polyamine synthetic enzyme ornithine decarboxylase(ODC), have also been implicated in cell transformation and cancer progression. Polyamines regulate the translation of the ODC mRNA as well as various other growth related proteins and oncogenes. Previous data suggested the mechanism by which putrescine regulates translation is thru modulation of the translational repressor molecule elF-4E binding protein 1 (4E-BP1). Polyamines alter both the steady-state level of 4E-BP1 mRNA and protein. The mechanism by which polyamines alter 4E-BP1 mRNA availability could be by either 1)enhancing mRNA stabilization or 2) stimulation of de novo transcription of the gene coding for 4E-BP1 mRNA. To measuremessagehalf-life, lEG-6cells were polyamine depleted with difluoromethlyornithine (DFMO) or treated with putrescine and both groups treated with actinomycin D. In control experiments the 4E-BP1 tl/2 was 6.9 hrs. There was no significant change in 4E-BP1 message half-life after either DFMO, tl/2 6.0 hrs, or putrescine, tl/2 6.4 hrs, suggesting polyamines do not modulate stability of the 4E-BP1 mRNA transcript. To determine the effects of polyamines on the rate of 4E-BP1 gene transcription, nuclear run-off assays were performed on nuclei isolated from DFMO or exogenous putrescine treated !EC-6 cells. Putrescina increased 4E-BP1 transcription by almost nine-fold whereas DFMO-induced polyamine depletion produced a 25-fold fall in the rate of transcription. There was no significant change in transcription

AGA A b s t r a c t s

$947 NoWASP is Required for the Development of a Polarized Epithelium. Tomoyuki Shibata, Ching-Hui Liu, Takanori Sakaguchi, Hans-Christian Reinecker, Scott B. Snapper, Boston, MA Background & Aims: N-WASP is a ubiquitously expressed protein that regulates the actin cytoskalaton. N-WASP is required for such diverse properties as cell motility, vesicle transport, and the pathogenesis of a variety of intracellular microbes. Our laboratory has been interested in the role of N-WASP in the development and function of the mammalian intestine. Since N-WASP-deficiency in mice results in early embryonic lethality, we have employed an in vitro differentiation model to study the role of N-WASP in epithelial development. During differentiation, embryonic stem (ES) cell-derived embryoid bodies (EBs) develop into two epithelial layers, the outer endoderm and the internal ectoderm, analogous to the visceral endoderm and embryonic ectoderm in the developing mouse embryo. Methods: N-WASPdeficient (KO) ES cells were previously generated in our laboratory. Wild-type (WT) and NWASP KO ES cells were cultured on mouse embryonic fibroblasts in media supplemented

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with 15% FCS, and leukemia inhibitory factor (LIF). To generate EBs, 2x10~ ES cells were cultured on gelatin coated 10 cm tissue culture dishes for three days, then transferred to suspension culture in lO-cm bacterial petri dishes in ES cell media that did not contain leukemia inhibitory factor (LIF). EBs were photographed and collected at 2 day intervals following the formation of a spherical structure (i.e., from day 4 to day 16). The formation of inner and outer structures were determined by histological analyses, To determine the ability of EBs to adhere to a non-coated surface, the percentage of spontaneously attached EBs was calculated at day 10. Results: N-WASP KO and WT ES cells could develop into early EBs. However, by day 4, Reicherts membrane-like structures were recognized in WT EBs but not in N-WASP KO EBs by inverted microscopy. Furthermore, hematoxilin & eosin staining of day 6 EBs revealed normal differentiation of both ectoderm and endoderm layers in WT but not in KO EBs. By day 10, large cystic cavities were found in 77% nt VVI EBs; none were identified in N-WASP KO EBs. in the adhesion analysis at day 10, the spontaneously attached numbers of EBs on surface of petri dishes were significantly different (WT: 20 +/- 6.8% versus KO: 5 +/- 3.7%, p
Glucagon-Like Peptide-2 Increases Glucose Uptake by Increasing SGLT-1 and GLUT2 Abundancein TPN-Fed Neonatal Pigs Anne L. Bartholome, Barbara Stoll, Douglas G. Burrin, Kelly A. Tappenden, Urbana, IL; Houston, TX Children with intestinal dysfunction, often require total parenteral nutrition (TPN) to meet their nutritional needs. Although TPN provides the necessary nutrition, it may limit intestinal adaptation, growth, and restoration of normal function. The trophic gut peptide, glucagonlike peptide-2 (GLP-2), which has been shown to reduce TPN associated atrophy and increase glucose uptake, may also enhance intestinal function. Therefore, we examined the effects of GLP-2 on the protein abundance of the brush border sodium-dependent glucose transporter, SGLT-1, and the basolateral sodium-independent glucose transporter, GLUT2. Thirty-two neonatal piglets were randomized to one of five diets for seven days: 1) enterally administered sow milk replacer (EN; n=6); 2) TPN+saline (TPN; n=9); 3) TPN + GLP-2 (3.125 nmol/kg BW; Low; n=5); 4) TPN +GLP-2 (6.25 nmol/kg BW; Med; n=5); and 5) TPN+GLP-2 (12.5 nmol/kg BW; High; n=7). The protein abundance of the apical sodium-dependent glucose transporter (SGLT-1) and the basolateral glucose transporter (GLUT2) were measured by Western blotting in the proximal jejunum, distal jejunum, proximal ileum and distal ileum. The SGLT-1 protein abundance in the EN group (P
$948 Insulin-Like Growth Factor I (IGF-I) Governs Epithelial Cell Migration during Mucosal Maturation Andrew C. Herman, Jessica B. Paxton, Phillip V. Gordon, Charlottesville, VA BACKGROUND:IGF-I is an important trophic factor for gut growth and development. We have previously demonstrated that IGF-I is abundant in the mesenchyme of the postnatal mouse ileum and that its localization is shifted from the mesenchymeto the mucosa during dexamethasone-induced maturation. To determine if IGF-I mediates quantifiable aspects of mucosal maturation, we chose to investigatethe maturation-linked phenomenonof acceleratedepithelial cell migration. We utilized both in vitro and in vivo models of ileal epithelial cell migration to test the hypothesis that IGF-I directly modulates epithelial cell crawling speed. METHODS: Rat ileal epithelial cells (IEC-18) were cultured to confluence in serum free media and then treated for 72 hours either with dexamethasone, a synthetic agonist of IGF-I (R3-1GF-1) or nothing. The cultures were wounded in a linear fashion then serial photomicrographs of individual cells migrating into the wound were taken over a 36-hour period and used to calculate the migration speeds in each condition. Likewise, heterozygous mice transgenic for human IGF-I were bred to yield litters containing wild type, heterozygous, and homozygous progeny. These newborns received intraperitoneal injections of bromo-deoxyuridine (BrdU) at 24 hours of life, were euthanized 48 hours later and their ileums were harvested for histology. BrdU immunu-laheling of nuclei was used to assess goblet cell clearancefrom the proximal villus in each of the transgene conditions, thereby assessingthe relationship between endogenous IGF-I and migration speed. RESULTS: Our in vitro migration assays show that dexamethasoneslows IEC-18 migration in a concentration dependent fashion, while R3-1GF1 accelerates IEC-18 migration in a concentration dependent fashion. Our in vivn assays demonstrate increased clearance of BrdU-labelled goblet cells with increasing gene dosage of human tGF-I. CONCLUSION: IGF-I governs ileal epithelial migration speed during mucosal maturation.

$951 Developmental Changes in Plasma GLP-2 and Tissue GLP-2 Receptor Expression Coincide with Rapid Intestinal Maturation in Young Pigs Yvette M. Petersen, Bolette Hartmann, Isabelle Le Huerou-Luron, Jens J. Hoist, Per T. Sangild, Frederiksberg, Denmark; CopenhagenN, Denmark; Rennes, France The structure and function of the small intestine develops rapidly in association with the nutritional transitions at: birth and weaning. The intestinotrophic hormone, glucagon-like peptide 2 (GLP-2), is released from intestinal L-cells in response to enteral nutrient intake. We investigated whether the developmental changes in plasma GLP-2 level and tissue GLP2 receptor (GLP-2R) expression are consistent with the hypothesis that GLP-2 may play a role in postnatal intestinal maturation. Blood samples were collected from fetal pigs (92% gestation, n=17) and at various ages after birth and weaning (n=8-13). Intestinal tissue was collected from 4 pigs in each age group. Bioactive GLP-2 (GLP-2~.33)was measured in plasma by RIA and GLP-2R mRNA abundancedetermined by semi-quantitative RT-PCR.GLP2 concentrations increased before birth and reached a maximum in young suckling pigs (Graph, means_+SE, *P
$949 Brush-Border Localization of Sodium/Glucose Cotransporter Protein in Crypt and Villus Neonatal Porcine Intestinal Epithelial Cells David M. Albin, Kelly A. Tappenden, Douglas G. Burrin, Dale Lackeyram, Ming Z. Fan, Urbana, IL; Houston, TX; Guelph, ON, Canada The small intestinal mucosa consists of crypt and villus regions where enterocytes undergo proliferation and differentiation, respectively. In order to better provide nutrients during critical periods, it is essential to have a thorough understanding of the physiological processes of the epithelial cells. Although it is usually assumed that only epithelial cells in the upper third of the villus are fully differentiated and capable of nutrient transport, neonates have high nutrient and energy requirements for normal growth and development. Thus, it is hypothesized that epithelial cells along the entire crypt-villus axis may be capable of nutrient transport to meet neonatal nutrient and energy requirements. A modified distended intestinal sac method was used to sequentially isolate neonatal porcine epithelial cells along the crypt-villus axis. Six 14-d old neonatal piglets, reared on formula for the final 7 d, were used. Small intestinal epithelial cells were harvested and fractionated into upper villus cells (U), mid-villus cells (M), and lower-villus/crypt cells (C). Total cellular homogenate and brush-border membranes from each fraction, using Western blotting techniques, were used to determine the abundance of the sodium/glucose cotransporter (SGLT-1) protein. The isolated brush-border membranes contained more (P < 0.0001) SGLT-1 protein than the homogenized cell suspensions (3.30 _+ 0.07 vs. 1.00 _+ 0.21 densitometry units), thereby validating the membrane isolation and Western blotting procedures. SGLT-1 protein abundancewas evenly found (P = 0.64) along the crypt-villus axis (U = 1.19 _+ 0.12; M = 1.00 _+ 0.13; C = 1.06 _+ 0.12). Cellular localization of SGLT-1 protein abundancedid not differ (P = 0.96) among fractions, indicating that intracellular and/or brush-border pools of SGLT-1 were not regionally associated. These data indicate that SGLT-1 protein is distributed evenly along the crypt-villus axis in the formulafed,.neonatal piglet, and not restricted to the upper villus. Immunohistochemical and mRNA analyses for SGLT-1 are underway to further provide evidence for these findings.

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$952 The Human Intestinal Xenograft Model to Study Functional Development of Premature Infant Gut: Role of Glucocorticoids N Nanthakumar, W Allan Walker, Boston, MA NecrotizingEnterocolitis (NEC) is an intestinal inflammatory disease predominantly associated with prematurity and treatment with exogenous glucocorticoids (GC), either prenatally or postnatally, results in a significant reduction in incidence. Since NEC is typically associated with an underdevelopedgut, GC may act to acceleratethe maturation of the intestine. However, the use of steroids in a pre- and postnatal setting is variably Successful in preventing NEC. This discrepancy could be due to a time-dependent restricted tissue responsiveness to GC during development. Previous studies havedemonstrated an age-dependentchange in responsiveness of the developing rat intestine to GC. Thus, we have hypothesizedthat glucocorticoids could facilitate the maturation of the gut only when administered within the limited period of responsiveness. In this study, using the xenograft model, we could recapitulate human in utero development and measure the optimal period of glucocorticoid sensitivity, using disaccharidases as markers.:By nine weeks post-transplantation, intestinal xenografts are morphologically and functionally developed and monitored up to six months. In both tissues, sucrase activity remains unchanged,hut lactase activity increases in a manner similar to that described in in utero development. Cortisone acetate treatment at 20 weeks acceleratedthe ontogeny of lactase but didn't alter sucrase activity in both tissues. Glucocorticoid sensitivity

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the ~1 chain is present early during intestinal development and restricted to the base of the intervillus epithelium between t2 and 15 weeks of gestation. From 16 weeks until the adult stage, the c~1 chain is gradually replaced by the c~2 chain at the bottom of the crypts. The ~3 and a5 chains are also detected at early stages of development at the base of all epithelial cells but become gradually excluded from the crypt area between 16 and 20 weeks. In the adult, these two laminins are predominantly found on the villus. RT-PCR analyses revealed that during development, the highest levels of c~1 transcript are found in the immature fetal intestine (12-15 weeks) while ~2, which is low at this stage, gradually increases toward midgestation and adult stages. A significant increase of the a3 mRNA level was observed between fetal and adult stages while ~4 and a5 remained constant. Analysis of the transcript levels of mid-gestation intestinal epithelial and mesenchymal fractions isolated by Matrisperse indicates that c~1 and ,x5 are produced by both epithelium and mesenchyme in the intact intestine while ~2 and ~4 are of predominantly mesenchymal origin and e3 is mostly originating from the epithelium. However, significant amounts of both the c~1 and c~2 chains were found to be expressed in the normal human intestinal crypt cell line HIEC suggesting a significant contribution of crypt cells to the production and deposition of these laminins in vivo. Taken together, the data indicate that the human intestinal epithelial BM contains multiple laminin chains, which are subject to both spatially and temporally regulated patterns of expression and of distinct epithelial and mesenchymal origin (Supported by CIHR)

is lost by 30 weeks post transplantation. Using lactase and ileal fatty acid binding protein as markers, we havealso shown that these changes are manifested at the level of mRNA. Biopsies from 1-2 year old infant intestine showed that all activities, except trehalase in the proximal intestine, corresponded to xenngrafts at 24-28 week post transplantation. These studies suggest that 20 week old jejunum and ileum have the ontogenic potential to follow normal intra- and extrauterine ontogeny as a xenograft and can help to explain a possible lack of steroid responsiveness in postnatal versus prenatal treatment due to a lack of tissue responsiveness after a specific gestational stage. Therefore, the xnnograff model could be useful in de!ineating additional markers that clearly define the period of glucocorticoid responsiveness and in understanding the pathophysiology of NEC in the developing human intestine during the second and third trimester.

$953 Reestablishment of Cell-Cell Contacts by Forced E-Cadherin Expression Is Not Sufficient to Trigger Differentiation in Human Intestinal Crypt Cells Fabrice Escaffit, Nathalie Perreault, Dominique Jean, EUsahethHerring, Claudine Rancourt, Nathalie Rivard, Jean-Francois Beaulieu, Sherbrooka, OC, Canada Regulation of a number of key biological processes such as cell growth, differentiation and morphogenesis involve cell-cell interactions. In epithelia, E-cadhedn, mainly located in the adherens junctions, regulates cell adhesivenessthrough calcium-dependent associations and has important roles by acting, for example, as a growth suppressor molecule. Likewise, abnormal expression of E-cedherin has been related to pathologies that involve a loss of cell polarization and/or differentiation. Recent observations suggest that E-cadherin could also be lacking from some epithelial progenitor cell types. In our laboratory, we have generatednormal human intestinal epithelial cell (HIEC) lines that exhibit great similarities to the lower crypt cells of the intact small intestine. HIECcells proliferate hut lack all polarizationand differentiation features typical to intestinal epithelial cells; they also express most of the cell junction complex molecules except E-cadhedn. The expression of Snail and E12 is observed at the transcript level suggesting the involvement of these factors with the lack of expression of E-cadhedn which could be related to the poorly differentiated phenotype of HIEC cells. To investigate this hypothesis, HIEC cells were infected with adenoviral or retroviral vectors in order to force the expression of E-cadherin (HIEC/E-cad+ cells). The newly expressed protein was mainly localized at the plasma membrane in association with ~-catenin, according to a correct localization of functional E-cadherin. The major phenotypical changes induced by this Ecadherin expression were a significant increase of 13-cateninexpression and the appearance of junction-like structures at the cell-cell interface. A reduction of cell proliferation and an increase in p21 expression and pRb phosphorylation were also observed in long term cultures of HIEC/E-cad+ cells. The expression of other cell cycle molecules (p27, p67) as well as intestinal cell markers (aminopepdidaseN, dipeptidylpeptidase IV, sucrase-isomaltase,alkaline phosphatase) were comparable to controls cells. These results show that re-introduction of E-cadherin induces the initiation of the polarization process and decreases cell growth in human intestinal cells but is not sufficient to trigger terminal differentiation. This suggests that other key complementary factors are involved in the behavior of HIEC cells.

$956 Extracellular Matrix (ECM) Influences IL-113-Medialed Chemokine Induction in Developing Human Intestinal Epithelium N Nanthakumar, Kdstin M. Spellmayer, Nassar Moiduddin, W Allan Walker, Boston, MA During inflammation, neutrophil recruitment to the epithelium is stimulated by IL-8 gradient and by ENA-78, if sustained recruitment is needed. Recent studies have demonstrated that ECM plays an important role in mucosal recruitment of lymphocytes during inflammation. The interaction of the epithelium with the ECM has been demonstrated to be essential for cellular differentiation and intestinal development. However,the role of ECM in the inflammatory response of the gut is less well known. Therefore, our aim was to elucidate the role of ECM in enterocyte secretion of IL-8 and ENA-78 in response to a specific pro-inflammatory signals such as IL-1B. We tested intestinal immune function in various human small intestinal epithelial models grown on Collagen I, Collagen IV, Laminin, and Fihronectin in response to IL-1B as measured by its ability to secrete IL-8 and ENA-78. Caco-2 and T-84 cells were used as a model for mature enterocytes and H4 cells (a primary non-malignant small intestinal fetal cell line) were used as a model for immature enterocytes. Caco-2 and T-84 cells secreted substantially less IL-8 (10 fold) and ENA-78 (3-4 fold) than 1-14cells. IL-8 secretion reached its maximum by 8 hours. However, ENA-78 secretion was delayed until after B hours reaching a maximum by 24 hours. With H4 cells grown on laminin the response to IL-1B was reduced by 50% for both chemnkines. In contrast, H4 cells grown on fibronectin enhanced only ENA78 section but on collagen-1 both IL-8 and ENA-78 was enhanced. However, collagen-IV has no effect on H4 cells in responseto IL-1B. The effect on T84 and Caco-2 cells was comparable but the capacity of these cells to secrete these chemnkines was substantially lower. Thus in mature enterocyte model cells, the role of ECM in IL-1B-mediated secretion of chemokines are much less pronounced than in immature cells. Since these ECM components are deposited differentially along the crypt -villus axis in various regions of the developing human intestine the effect of these matrices in eliciting an inflammatuc/ chemokine response may vary. Therefore, we conclude that an age-dependent change in inflammatory responses are also dependent on the composition of various ECM components contiguous with the epithelial monolayer and may play an important role in pathophysiolocial responses during inflammation in infants compared to adults.

$954 Developmental Patterns of Mucin Gene Expression in Human Fetal Small Intestinal Xenografts Maintained in Severe-CombinedImmunodefisieot Mice Made-Pierre Buisine, Jean-Pierre Aubert, W Allan Walker, Tor Savidge, Lille, France; Charlestown, MA Background and Objectives.The lack of a suitable animal model that expresseshuman intestinal mucin genes limits the study of mucin function. The aim of this study was to examine whether human fetal intestinal xenografts, known to model host-restricted interactions with human specific pathogens, express mucin genes in an appropriate developmental pattern when transplanted into scid mice. Methods. Expression profiles for 8 mucin genes were examined in human fetal ileal xenografts transplanted ectopically into scid mice for 10 weeks. In situ hybridization was performed on fetal, xenograff and adult intestinal tissue sections with 35Slabeled oligonucleotides specific to human tandem repeat sequencesfor MUC1, MUG2, MUC3, MUG4, MUC5AC, MUC5B, MUC6 and MUC7. Results. Hybridization patterns observed with the MUG2, MUC3, MUG4 and MUC5AC probes demonstrated that mucin gene expression in xenografted fetal intestine was comparable to third trimester fetal and/or adult tissues. MUC2 and MUC5AC were expressed in a developmental-specitic fashion. MUC5AC, expressed in first and early second trimester fetal bowel, was never detected in intestinal xenografts. MUC2 expression displayed a late fetal and/or edult-type hybridization pattern. MUC3 and MUC4 were not developmentally expressed. Conclusion. Appropriate developmental regulation of known intestinal mucin genes were recorded in ectopically grafted human fetal intestinal xenografts. Adult-like patterns of mucin gene expression in this model system will permit future studies aimed at characterizingcis/trans-acting factors that regulate mucin gennexpression and function during development, disease,wound healing and also in mucirepathogen interactions during host defense. Supported by the National Institutes of Health (DK33506; DK40561)

$957 Autoimmunily against Human Trepemyosin Isotorms (hTMs) in Ulcerative Colitis (UC): Localization ot Specific hTMs in the Intestine and F.xlraintestinal Organs Bhagyalakshmi Sastri, Zafar K. Mirza, Jim J. - C. Lin, Kiron M. Das, Bronx, NY; New Brunswick, NJ; iowa City, IA Background: Tropomyosins (TMs) are microfilament proteins present in all eukaryotic cells with organ specific isoforms. In humans, 5 major TM isoforms are described (hTM1-5): hTMs, particularly isoform 5 (hTM5) is capable of inducing autoantibodies and T cell response in ulcerative colitis (Gastroenterology 1998,114:912; Clinical Immunology 2001,101:289). Autoantihody response to hTM has also been reported in primary sclerosing cholangitis (Gut 2000;47:236). However,the cellular localization of hTMs in the intestine and in extraintestinal organs commonly involved in inflammatory bowel disease (IBD) is unknown. Aim: To investigate the distribution of hTMs in colon, small intestine and extraintestinal tissue from normal subjects. Methods: Using isoform specific monoclonal antibodies (mAb) against hTMs, by a sensitive immunopemxidase assay we examined the staining pattern in the normal colon (n=12), small intestine (n=14), skin (n=15), eye (n=12), and gallbladder (n=16) (total specimens n=71). The isoform specific mAbs used were: CG1 (IgG1) against hTM1, CG3 (IgM) against hTM5, LC1 (IgG1) against hTM5 and LC24 (IgG1) against hTM4. Results: The epithelium and not the connective tissue, blood vessels and smooth muscle of the colon, gallbladder, and skin express hTM5 isoform and not the other isoforms. In the eye, hTM5 was expressed only in the non-pigmented ciliary epithelium. The immunoreactivity in the epithelial cells was diffuse cytoplasmic and more dense along the periphery of the cells. In the colon, there is intense expression along the luminal (apical) surface. The hTM5 expression in the small intestinal epithelium is distinctly different than colon. Its reactivity in small intestinal enterocytes is faint and localized in the basal area without any apical staining, hTM1 is seen predominantly in the muscle and blood vessel wall. hTM4 is localized in the endothelial cells and in the colonic epithelium at the basement membranearea. Conclusion: The epithelium in the colon and extraintestinal organs commonly involved in IBD, show predominant expression of hTM5 isoform at both cytoplasmic and epithelial surface. Such a localization of hTM5 may allow its interaction with effector immune cells and may have significance in the immunopathogenesis of UC and its extraintestinal manifestations.

$955 Expression, Distribution and Cellular Origin of Lamioin e=l, eL2, e=3, e=4 and 0=5 Chains in the Developing Human Intestine Inga C Teller, Joelle Auclair, Elisabeth Herring, Hehong Ni, Caroline Francoeur, Ismo Virtanen, Jean-Francois Beaulieu, Sherbrooke, Canada; Helsinki, Finland Laminins are a multigene family of extracellular matrix molecules. Quantitatively,they are one of the most abundant glycoproteins present in the basement membrane (BM). Functionally, they can modulate several key biological activities, including cell adhesion, gene expression and cell survival. At present, five a, three ~ and two -/chains have been identified, which can associate to form 11 distinct BM heterotdmeric ~1~' laminins. In this study, we have examined the expression of the ~1-~5 laminin chains in the developing and adult human intestine at both the protein and transcript levels. Indirect immunoftuorescence revealed that

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$958 The Roles of TGFI~ and COX-2 in the Maintenance of Muscle Hypercontractility in a Morine Model of Post-Infective Irritable Bowel Syndrome Hirotada Akiho, Yikang Deng, Patricia Blennerhassett, Hiroshi Kanbayashi, Giovanni Barbara, Robert Degiorgio, Stephen Collins, Hamilton, ON, Canada; Bologna, Italy

Background~Aims: Acute gastroenteritis is a strong risk factor for the development of Irritable Bowel Syndrome (IBS). We have developedan animal model in which transient acute infection leads to persistent intestinal muscle hypercontractility which can be reversed using COX inhibitors (Barbara et al Gastroenterology 1997, 2001). Putative mediators of this response include IL-4, IL-13 or TGFI31. Recently, we showed that this hypercontractility is evident in muscle cells isolated from the gut 35 days post-infection (Akiho et al DDW 2001). The aim of this study was investigate the relationship between the above described cytokines and COX-2. Methods: mRNA or protein expression of cytokines or inflammatory mediators were examined from jejunal longitudinal muscle myenteric plexus of NIH Swiss mice infected with or without Trichinella spiralis, or from primary cultured longitudinal muscle cells of mice incubated with or without cytokines. Results: During the acute phase of infection there was significantly increased expression of Th2 cytokines, IL-4 or IL-13, TGFI31 and COX-2 mRNA in the muscle. Post infection, expression of Th2 cytokines returned to normal, but TGFI31 and COX-2 mRNA remained elevated in the muscle (p
(16.5 ng/ml _+5.6) in placebo-treated mice 7 days post colitis, values for SP increased to 46.0 ng/ml (-+23.9) in E2 treated mice. Moreover, at day 14 post-colitis SP/G3PDH mRNA ratio was 2.5-fold higher in E2-treated mice than in placebo-treatedmice. Conclusions: These results indicate that estrogen treatment induces a significant and persistent increase in colonic SP evident at least 2 weeks post colitis. It is possible that the risk of IBS post IBD may be higher in patients receiving estrogen supplements. Supported by Canadian Institutes of Health Research. $961 Immunoregulation of Somatostatin, Gastrin and Substance P by IFN,y and IL-4 in the Mouse Gastric Mucosa. Yana Zavros, Joel V. Weinstock, Juanita L. Merchant, Ann Arbor, MI; Iowa City, IA Background: Patients infected with Helicobacterpylori(H. pylori)have increasedIFN.y-expressing T lymphocytes (Thl immune response). Gastritis causes reciprocal changes in the neuroendocrine cell populations, gastrin (G)- and somatostatin (SOM)-secreting D-cells. Therefore, first we queried whether the proinflammatory cytokine IFN~ alone was sufficient to cause the pertubations observed in the neuroendocrine cells independent of the bacteria. SOM and substance P (SP) are short po!ypeptides that can mediate the expression of the Thl immune response. While SP stimulates, SOM inhibits IFN~ expression. The Th2 specific cytokine, IL4, is believed to antagonize the Thl immune response in part by inhibiting the release of SP. The role of SOM as a mediator of IL-4 suppression on IFN~, is less clear. Therefore, we studied the regulation of SOM and SP by IFN.y and IL-4 in vivo. Methods: PBS, IFN~,or iL4 was infused into C57BL/6 mice for 7 days. Plasma gastrin concentrations and tissue SOM content were measured by radioimmunoassay. Tissue sectjons were fixed for histology and morphometric analysis and immunostained for SOM and gaStrin. SP peptide levels were quantified by western blot analysis. Sections were stained f or H&E and PAS/alcian blue and graded on the intensity of gastritis. Results: After IFN~,infusion, tissue SOM content decreased by 50%; whereas, the number of G-cells and plasma gastrin increased 3-fold. In addition, SP protein expression increased si gnificantly by 5-fold. Histologic evaluation of the gastric mucosa revealedthe presence of lymphocytes, leukocytes and mucous gland metaplasia after 7 days of IFN,y infusion. In contrast, there were no inflammatory or metaplastic changes in the IL-4 treated mice. In addition, IL-4 significantly decreased gastrin concentrations (90%) and SP protein expression (50%). This correlated with a 2-fold increase in SOM. Conclusions: It is likely t hat the release of IFN~, from Thl lymphocytes mediates the hypergastdnemia observed during H. pyloriinfection. The effect of IFN~/on the G cell may be indirect through the releaseof SP. In contrast, IL-4 inhibits gastrin releasevia stimulation of SOM and inhibition of SP. $962 Par-2-1nduced Inflammation of Mouse Colon Involves Afferent Neurons and Nitric Oxide but Does Not Result from Increased Paracellular Permeability Nicolas Cenac, Rafael Carcia-Villar, Jean Fioramonti, Lionel Budno, ToulOuse, France Activation of colonic PAR-2 receptors provoked inflammation and increased mucosal permeability in mice colon. The mechanism of this inflammation is under debate but evidence suggests that it could be neurogenic (NANC neurons) and/or result from the opening of colonic tight junctions (TJs) with subsequent passageof exogenous macromolecules into the lamina propria. We aimed to further characterize PAR-2-induced inflammatory effect by investigating the involvement of (i) nitric oxide, (ii) capsaicin-sensitive afferent neurons, and (iii) a possible cause and effect relationship between the enhancement of paracellular permeability and mucosal inflammation. Methods: Thirteen groups of 8 male Swiss mice (2631 g) were infused intracolonically (ic, 50 FI) with either the PAR-2 activating peptide SLIGRL (100 Fg/mouse) or saline (controls), followed from 2 to 4 h later by 2.106 cpm 5~Cr-EDTA (750 ~l/h). Prior to these infusions (i) six groups receivedeither the iNOS inhibitor aminoguanidine (l.5mg/mouse, IP), the NOS inhibitor L-NAME (0.6 rag/mouse, IP) or vehicle; (ii) four groups were treated with capsaicin (0.75 mg/mouse twice/day for 2 days) or vehicle; (iii) three groups received the TJ blocker TAP (2,4,6-triaminopyrimidine; 200 raM, ic) or vehicle. At 4 h blood was collected for radioactivity counting (cpm/ml) and colons for myeloperoxidase assay (MPO; units/g protein) and histologic examination (microscopic damage score, MDS). Values are the mean _+ SEM for each group. Results: In all treated groups, ic SLIGRL increased MPO, MDS and paracellular permeability values. (i) Both L-NAME and aminoguanidine prevented SLIGRL-induced increases in MPO (75:7 _+ 16.4 and 98.1 ,+ 31.0 vs. 390 + 109), MDS (1.3 ,+ 0.5 and 1.9 _+ 0.6 vs. 4.8 +_ 0.6)and permeability (556 _+ 108 and 783 .+ 95 vs. 1703 _+ 328). (ii) Similarly capscaicin prevented SLIGRL-induced increases in MPO (46.1 _+ 9.4 vs. 596 _+ 195), MDS (0.8 _+ 0.2 vs. 5.1 ,+ 0.4) and permeability (666 + 125 vs. 1942 ,+ 215). (iii) In contrast, although TAP preventedSLIGRL-inducedpermeability increase (119 _+ 56 vs. 676 ,+ 95), it did not prevent increases in MPO (982 _+ 279 vs. 950 _+ 144) and MDS (4.9 _+ 0.3 vs. 5 _+ 0.8). Conclusion: Our data show that both nitric oxide and capsaicin-sensitive afferent neurons are involved in PAR-2-mediated colonic inflammation and increased paracellular permeability. Neverthelessthe inflammation process does not appear to result from the observed increase of permeability. $963 Interaction of Glucagon-Like Peptide-2 and Common Therapeutics in a Murine Model of Ulcerative Colitis Marie-Claude L'Heureux, Wendy Banff, Patricia L. Brubaker, Toronto, ON, Canada BACKGROUND:5-aminosalicylates (ASA) and curticosteroids (CS) are effective agents for the relief of the symptoms associated with ulcerative colitis: In contrast, glucagon-like peptide2 (GLP-2), a potent growth factor, has promising therapeutic properties because of its ability to stimulate intestinal growth and function. We have previously shown that GLP-2 enhances the reparative response to dextran sulfate (DS)-induced colitis in mice. We hypothesizedthat the intestinotropic effects of GLP-2 are enhanced by therapeutic agents commonly used to treat colitis. METHODS: Each therapeutic agent was administered alone or concomitantly with

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results show that both G~q and Gc~13/Rhocontribute to PKD activation through PKC-dependent signaling pathway and indicate that PKD activation is mediated by both Gc~qand G~x13in response to acute bombesin receptor stimulation.

GLP-2 in normal and DS (3.5-4%)-induced colitis CD1, female, 6 week old mice for a period of 10 days. The following groups were tested: control, PBS+/-GLP-2 (human Gly2-GLP-2: O.04mg/kg, sc, q12h), ASA+/-GLP-2 (sulfasalazine: lOOmg/kg, pc, q24h), CS+/-GLP-2 (methylprednisolone sodium succinate: lmg/kg, ip, q24h) and combination therapy, CS+ASA+/-GLP-2 (n=4-8/group). A) Whole body observations were recorded on a daily basis. Upon sacrifice, 1 cm intestinal segments were collected for measurements of B) morphometry and C)inflammation. RESULTS: A) GLP-2 increased survival of the DS-animais (PBS vs GLP-2: 44% vs 71%). ASA also increased survival to 75% and further enhanced the effect of GLP-2 on survival (ASA+ GLP-2: 88%). B) GLP-2 markedly increasedsmall intestinal growth in DS-colitis (PBS vs GLP-2: 0.84+/-0.04g vs 1.14+/-0.08g; p
$966 Agonict-Dependent, PKA-Mediated Phosphorylationof GRK2 Augments Homologous Desensitization of VPAC2 Receptors in Smooth Muscle Huiping Zbou, John R. Grider, Karnam S. Murtby, Richmond, VA Background: Smooth muscle of the gut expresses mainly VPAC2 receptors coupled via Gs to stimulation of cAMP and activation of cAMP-dependent protein kinase (PKA). Homologous desensitization of VPAC2 receptors, like that of secretin receptors, is probably mediated by receptor pbospborylation via G protein-coupled receptor kinase 2 (GRK2) and PKA. Aim: The aim of this study was to examine whether PKA could induce desensitization also by enhancing GRK-mediated desensitization in gastric smooth muscle cells. Methods: Studies were done in cultured muscle cells expressing wild-type GRK2, kinase-inactive GRK2 (K220R), and PKA site-deficient GRK2 (S685A). Mutations were confirmed by sequencing. ~2sI-VIPbinding and adenylyl cyclase activity were measured before and after treatment with 1 I~M VIP for 30 min (VPAC2-desensitizedcells). Results: In cells expressing wild-type GRK2, VIP induced phosphorylation of GRK2 in a concentration-dependentfashion. Phosphorylation was blocked by the selective PKA inhibitor, myristoylated PKI. Phosphorylation was also observed in cells expressing kinase-inactive GRK2, but not in cells expressing PKA site-deficient GRK2. In cells expressing wild-type GRK2, pretreatment with VIP inhibited ~251-VlPbinding by 86_+5% and cAMP production by 81 _+4%. The inhibition was greatly diminished in cells expressing kinaseinactive GRK2 (13_+6% and 20_+5% for binding and cAMP production, respectively). The inhibition was only partially blocked in cells expressing PKA site-deficient GRK2 (42_+5% and 46_+4% for binding and cAMP ptoducfion, respectively). Neither receptor-independent activation of PKA with forskolin nor activation of PKG with SNP bad any effect on GRK2 phosphorylatiun. The results implied that GRK2-mediated homologous desensitization of VPAC2 receptors was enhanced by PKA-dependent phosphorylation of GRK2. In support of this notion, VIP-induced GRK2 binding to I~'y was partly inhibited by PKI in cells expressing wild-type GRK2, and was decreased in cells expressing PKA site-deficient GRK2. The results implied that PKA-dependentphosphorylation of GRK2 increasesits binding to 13'yand facilitates GRK2 recruitment to the receptor. Conclusion: A novel mechanism of homologous desensitization of VAPC2 receptors by PKA has been identified whereby PKA enhances desensitization of occupied receptors by phosphorylation of GRK2. The mechanism is distinct from PKAmediated desensitization by direct receptor phosphorylation.

S964 A Cocktail of Pro-inflammatory Cytokines Increases CCK Release from STC-1 Cells via an Effect on Intracellular Calcium Fiona Leslie, John McLaughiin, Shazia Kazmi, Andrea Varro, Geoff Warhurst, Graham Dockray, David Thompson, Manchester, UK; Liverpool, UK The mechanisms by which GI mucosal inflammation produces anorexia is poorly understood, but modulation of enteroendocrine cell function is a plausible mechanism. Cbolecystokinin (CCK) produces symptoms of nausea and satiety when infused parenterally and is involved in IL-1 induced anorexia, an effect attenuated by CCK-Aantagonists and vagotomy implicating peripheral CCK release. The aim was to study the effect of pro-inflammatory cytokines on CCK secretion. Methods: The CCK secreting cell line, STC-1, was incubated with the pro-inflammatory cytokines IFN.y, IL-11~and TNF-~ alone or in combination. CCK secretion was measured by radioimmunoassay. As CCK secretion is mediated by increases in intracellular calcium ([Ca ~+]~),changes in [Ca 2+]~in responseto 70 mM K+ (a receptor-independentstimulus of secretion) were assessed using a dual wavelength ratio imaging technique (Fura-2). To investigate the effects on gene expression we studied the effects of combined cytokines on activity of CCK promoter-reporter constructs using a luciferase assay system and luminometry. Results: Pre-incubation with the single cytokines IFN--y, IL-113and TNF-~ for 2 hours produced small increases in basal CCK secretion. IFN-.y (6.25U/ml) produced a 38.8_+4.6% increase in CCK secretion compared to control (p = 0.02), IL-113(O.125ng/ml) prod uced a 16.9 _+1.5% increase (p = 0.005) and TNF-~ (5ng/ml) produced a 13.7_+1.83% increase (p = ns). However when a cocktail of IFN-% IL-11~and TNF-~ was pre-incubated together this produced an increase of 109.4 _+24.0% over basal secretion after 2 hours incubation (p = 0.001). This increase was maintained after 4 hours with a 70.7_+24.7% increase (p=O.02) but diminished at 8 hours to a 43.6_+8.0% increase (p=0.055). The cytokine cocktail had no immediate effects on basal [Ca 2,], but pre-incubation for 2 hours produced a 23.9_+2.9% (p=O.03) increase in the [Ca ~+]~response to K÷ compared to control. However the cytokine cocktail did not stimulate CCK reporter-promoter activrty during the same period of pre-incubafion. Conclusions: Incubation with a cocktail of the pro-inflammatory cytokines IFN-% IL-113and TNF-~ has a synergistic effect increasing basal CCK secretion in the STC-1 cell line. This occurs via a mechanism involving changes in intracellular calcium. This may have implications for symptom genesis, particularly anorexia in proximal Gl inflammation.

$967 Neurotensin Induces Protein Kinase C-Dependent Protein Kinase D Activation and DNA Synthesis in Human Pancreatic Carcinoma Cell Line PANC-1 Susbovan Guha, Osvaldo Roy, Enrique Rozengurt, Los Angeles, CA Purpose: Pancreatic cancer is a devastating disease with dismal worldwide survival rates. Neuropeptides and G protein-coupled receptors (GPCRs)are increasingly implicated in autocrine/paracrine stimulation of growth of cancer cells. Protein kinase C (PKC) plays a central role in signaling cascadesfrom GPCRsmediating cellular growth. Of all the GPCRs,functional Neurotensin (NT) receptors are significantly upregulated in human pancreatic ductal cancer cells. Here, using PANG-1 (human ductal pancreatic adenocarcinoma cell line) as a model system, we examinedwhether, NT can promote activation of protein kinase O (PKD), a member of novel family of serine-tbreonine kinases, and induce DNA synthesis that are both PKOdependent. Methodsand Results:Western Blots (WBs) demonstrate that multiple PKC isoforms are expressed in PANG-1 along with PKO. NT promoted maximal PKD activation after 15 rain in immune complexes of PANG-1 cells as detected by the in vitro kinase (IVK) assay. Half-maximal stimulation of PKD was achieved at 25 nM of NT. WBs with phospho-serine 916 antibody, which specifically measures PKD activity in intact cells, further substantiated the IVK results. PKD activation was blocked by prior treatment with the selective PKC inhibitors, GF-109203X and Ro 31-8220, in a dose-dependent manner. WBs with specific phosphoserine 744/748 antibody, which measures activation loop phosphorylation and transactivation of PKD in vivo, further supported a model of PKC-PKD phosphorylation cascade in PANC-1 cells. Stimulation with NT for 5 min induced rapid translocation of PKD from the cytosol to the plasma membrane as determined by immunocytochemistry and confocal microscopy. Interestingly, the reverse translocation of PKD from the plasma membrane to the cytosol that was nearly complete within 15 rain of NT stimulation is dependent on PKC activity. Finally, the results show that NT, at concentrations that induced PKC-PKD activation, also induced DNA synthesis in PANC-1 cells. Using a selective PKC inhibitor, GF-109203X and GF-V, an inactive analog of GF-1, the results demonstrate that ~T receptor mediated activation of ONA synthesis is PKC-dependentin PANC-1 cells. Conclusion: The results indicate, for the first time, the existence of a functional PKC-PKO signaling pathway downstream of NT receptors in human ductal pancreatic carcinoma cells and suggest that PKC isoforms mediate the mitogenJc signaling process initiated by NT.

$965 Activation of Protein Kinase D (PKD) by Bombesin through the Alpha Subunits of the Heterotrimeric G Proteins Gq and G13 Jingzhen Yuan, Lee W. Slice, Enrique Rozengurt, Los Angeles, CA BACKGROUND:PKO/PKC~ is a serinetthreouine protein kinase with distinct structural, enzymological and regulatory properties. Recently, activation of a number of receptors that couple to heterotrimeric G proteins including those for hombesin, bradykinin, endothelin and vasopressin have been shown to stimulate PKD activation in a variety of cell types. In order to clarity the mechanism(s) by which G protein-coupled receptors lead to PKD activation, we examined whether either Gc~q- or Rho and Gc~13-mediatedsignaling can promote PKD activation in intact cells and whether endogenous G~q or Rho and G~13 could mediate PKD activation in response to bombesin receptor stimulation. RESULTS: PKD immunoprecipitated from COS7 cells transiently transfected with a constitutively active ~ subunit of Gq (G~qO209L) or a constitutively active mutant of Rbo (RhoQ63L) or pOnco-Lbc exhibited a marked increase in basal activity. Addition of aluminum fluoride to cells co-transfected with PKD and wild type G~q or Gc~13 induced a marked increase in PKD activity. Co-transfection of Clostridium butulinum C3 toxin specifically blocked activation of PKD by either RhoO63L, or by pOncoLbc, or by aluminum fluoride-stimulated G~13 but did not block activation of PKD by G~q. Treatment with the protein kinase C (PKC) inhibitors GFI or Ro31-8220 prevented the increase in PKD activity induced by aluminum fluoride-stimulated G,~q or G~13. PKO activation in response to either G~q or G~13 or bombesin was completely prevented by mutation of Ser744 and SorT48 to Ala in the kinase activation loop of PKD. Expressionof a COOH-terminal fragment of G~q or G,x13,that acts in a dominant-negativefashion, attenuated PKD activation in response to agonist stimulation of bombesin receptor. Co-expression of Clostridium botulinum 03 toxin and a COOH-terminalfragment of G~q further attenuated or almost abolished PKD activation in response to agonist stimulation of bombesin receptor. CONCLUSION:Our

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$968 The Histamine H~ Receptor Regulates MAPK and c-los Transcription Via Src and Raf Dependent Mechanisms. Lidong Wang, Kenneth Butler, Chady Haurani, John Del Valle, Ann Arbor, MI Background: Previously, we have demonstrated that activation of the human histamine H2 receptor stimulates cell proliferation through a PKC, MAPK and c-fos dependent mechanism. We havealso shown that histamine can activate Ras, which in turn is important for H2receptor mediated cell growth. Despite these observations, it is not known whether Src and c-Raf, important regulators of MAPK and cell growth, play a role in histamines action. Aim: Determine whether Src and c-Raf are involved in histamine H2 receptor mediated activation of MAPK and cell growth. Methods: The human histamine H2 receptor cDNA was cloned into a pc DNA 3.1 expression vector and stably transfected into HEK-293 cells (HEK-H2 cells). Transcription

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of c-fos was measured by transiently transfecting HEK-293 cells with a construct containing the serum response element (base -357 to -276, SRE-Luc) of the c-fos gene promoter and measuring ligand stimulated luciferase activity. MAPK and Src activity was measured by immunoblotting techniques using phospho-p42MAPK/p44MAPK or phospho-Src (Tyr 416) antibody as probes. We used the potent and selective inhibitors of c-Raf (ZM336372) and Src tyrosine kinase (PP2) to examine the functional role of these pathways in HEK cells. Results: Histamine (10-4M)stimulated MAPK activity (848.4_+145% control; mean_+SE, n = 4, p
Vasopressin Induces Early Signaling Pathways in Intestinal Epithelial Cells Terence T. Chiu, Steven S. Wu, Enrique Rozengurt, Los Angeles, CA Background/Aims: We have previously reported that the vasoactive peptide angiotensin II can induce rapid calcium mobilization and striking Protein Kinase D (PKD) activation through a Protein Kinase C (PKC) dependent pathway. Since the nonapeptide vasopressin (VP) has previously been identified in rat and human small intestinal crypts, we hypothesized that functional endogenous receptors for VP are present in nontransformed epithelial lEO-18 cells, originally derived from rat ileal crypts. Here, we examined whether VP can stimulate early signaling pathways, including Ca2÷mobilization,PKD activation, and tyrosine phosphorylation of Pyk2 and Src. Results: Addition of 50 nM VP induced a rapid and transient increase in intracellular Ca2+ that reached peak values of ~1000 nM (992 _+ 38; n=6 ) which was completely abolished after pretreatment with selective V1A receptor antagonist, 13-Mercapto; {~,l~-cyclopentamethyleneproprionyl~,O-Me-Tyr2,ArgB-Vasopressin (50 nM). Treatment of lEO18 cells with VP (lOgnM) led to a rapid and striking activation of PKD. A detectable effect was obtained within 30 s and reached a maximum (~10 fold) after 1 min of VP stimulation. Half-maximal and maximal PKD activation was achieved at I nM and 10 nM, respectively. PKD activation induced by VP was abrogated by treatment with the selective PKC inhibitors, GF-I (2.5~M)and Ro 31-8220 (2.51~M), also in a dose-dependent manner. To measure the effect of VP on Pyk2 tyrosine phosphorylation, cells were treated with 50 nM VP, [ysed, and immunoprecipitated with antiphosphotyrosine antibody, followed by Western blot analysis with anti-Pyk2 antibody. VP induced a striking increase in the tyrosine phosphorylation of Pyk2, reaching maximum within 1 rain. Half-maximal and maximal activation were achieved at 3 nM and 10 nM, respectively. Analysis with a phosphospecific antibody demonstrated phosphorylation at Tyr-402, the major autophosphorylation site of Pyk2, in response to VP stimulation. The phosphorylation of Pyk2 at Tyr-492 creates a potential high affinity binding site for the SH2 domain of Src leading to Src activation. VP induced Src phosphorylation at Tyr-418 rapidly, reaching maximum within 1 rain. Thus, the kinetics of Src activation parallels that of Pyk2 tyrosine phosphorylation. Conclusion: VP activates Gq-coupled VlA receptors in IEC-18ceils, leading to rapid Ca2+ mobilization and PKC-dependentPKD activation. Furthermore, VP stimulates the tyrosine phosphorylation of the non-receptor tyrosine kinase Pyk2 and promotes Src activation.

$969 Substance P-Stimulated Interleukin-8 Expression in Human Colonic Epithelial Cells Involves Activation of MAP Kinases and Rho GTPases Dezheng Zhao, Sabina Kuhnt-Moore, Huiyan Zeng, Amy Pan, Jack S. Wu, Mary P. Moyer, Charalabos Pothoulakis, Boston, MA Background & Objectives:We and others previously showedthat interaction of the neuropeptide substance P (SP) and its neurokinin-1 receptor (NK1R) plays an important role in the pathophysiology of intestinal inflammation. The proinflammatory mechanism of SP involves stimulation of cytokine release, including interleukin-8 (IL-8), activation of mitogen activated protein (MAP) kinase Erk and p38MAPK and nuclear translocation of the transcription factor NFkappaB.Howeverthe mechanisms by which SP-NK1Rinteraction induces NF-kappaBactivation and IL-8 expression are not clear. In this study we sought to examine whether SP stimulates IL-8 expression in human colonocytes and investigate the molecular mechanism of this response. Methods: NCM460 non-transformed human colonocytes were stably transfected with NK1R-expressing retroviruses. IL-8 was determined by ELISA and MAP kinase activation by western blot analysis. The promoter activity of NF-kappaB,serum response element, and IL-8 was determined using luciferase reporter assays. Results: SP stimulated IL-8 secretion in a time and dose- (10-6 - 10-9 M) dependent fashion with a maximal activation (39-fold increase) 4 hrs after SP (10-7 M) exposure. SP also activated tL-8 promoter activity. SP exposure for 2 min stimulated Erk and p38 phosphorylation and pretreatment with PD98058 (a specific MEK inhibitor) or SB203580 (a specific p38MAP kinase inhibitor) significantly reduced SP-induced IL-8 secretion. Importantly, inhibition of endogenous Rho family proteins (RhoA, Racl and Cdc42) by their respectivedominant negativemutants significantly decreased SP-induced IL-8 secretion and IL-8 promoter activity. As well, SP rapidly (1 min) activated RhoA, Racl and Cdc42 and overexpression of the dominant negative mutants of RhoA, Racl and Cdc42 in NK1R cDNA-transfected NCM460 cells significantly inhibited SP-induced NFkappaB activation and serum response element-dependentreporter gene expression. Conclusions: SP-NK1R interaction in human colonocytes stimulates IL-8 gene transcription by a mechanism that involves activation of MAP kinases. This is the first report to demonstrate that SP-NK1R interaction activatesthe Rho family of proteins and that this activation is required for SP-mediated IL-8 production and NF-kappaB-dependentgone expression. Supported by a grant from the National Institutes of Health (DK-47343).

$972 Src Tyrosine Kinases Are Involved in CCK Stimulation of Tyrosine PhosphorylaUon of the Adapter Protein Crk but Not for ERK1/2 or JNK Activation in Rat Pancreatic Acini Alberto G. Andreolotti, Maria J. Bragado, Jose A. Tapia, Robert T. Jensen, Luis J. GarciaMatin, 10071 CACERES,Spain; 2089 Bethesda, MD BACKGROUND:Recentstudies show that similar to growth factors and other G-protein coupled receptors, CCK stimulates numerous intraoellular cascades including activation of ERK1/2 and JNK and tyrosine phosphorylation of adapter proteins such as Crk and p130Cas. With other stimulants of these cascades,activation of Src tyrosine kinases is a key intermediate; however, its role in CCK's ability to activate these cascades is unclear. AIM: To investigate whether CCK-inducedtyrosine phosphorylation of Crk is mediated by the Src family of tyrosine kinases and whether this intraceilular pathway is mediating the CCK activation of the MAPK signaling cascades in rat pancreatic acini. METHODS:Dispersed pancreatic acini were used. Cell lysates were subjected to SDS-PAGEfollowed by immunoblotting with specific antibodies. RESULTS: In dispersed rat pancreatic acini, 10 nM CCK-8 caused a time-dependent increase in the phosphorylation level of the Crk protein, showed by an electrophoretic mobility shift to a slower band. Crk phosphory~atJonreached a maximum within 5 min (65% of total Crk was shifted to the upper band) and remained elevated 40 min after CCK-8 stimulation (50% of total Crk). Pretreatment of pancreatic acini for 1 h with 20 p.M PP2, a specific inhibitor of the Src family of tyrosine kinases, caused a significant reduction (p
$970 Histamine H2 Receptor Stimulates c-los Transcription in Isolated Canine Parietal Cells via a Tyrosine Kinase, PKC and p38 Dependent Mechanism Lidong Wang, Meizhi Wang, Annie Hunsche, John Del Valle, Ann Arbor, MI Background: We have previously demonstrated that the human histamine H2receptor activates c-fos transcription in transfected HEK-293 cells via PKC and MAPK dependent pathways. Despite this observation, the link between H2 receptor activation and c-fos regulation in a primary cell system such as the parietal cell is unknown. Aim: Examinewhether the histamine H2 receptor regulates c-fos transcription in canine parietal cells and explore the mechanism by which it does so. Methods: Canine parietal cells were isolated by collagenase digestion, enriched to >90% homogeneity by counter flow elutriation/percoll density centrifugation and cultured on matrigel coated plates. Cultured parietal cells were transiently transfected with a construct containing the luciferase reporter gene coupled to the serum response element (SRE) of the c-fos gene promoter (base-357 to-276, SRE-Luc). Ligand mediated regulation of SRE-luciferase activity and c-fos mRNA were used as measures of c-fos transcription. Results: Histamine (10-4M) led to a 3 fold increase in c-fos mRNA and stimulated SRE-Luc activity to 317.5 _+40% of control (mean+_SE, n = 4, p<0.001) in transfected parietal cells. We explored the mechanisms by which histamine stimulates SRE-Luc activity through the use of several inhibitors of cell signaling. The tyrosine kinase inhibitor genistein (10-5M) inhibited histamine stimulated SRE-Luc activity by 69.8 +_9.3% (mean _+SE, n = 5, p
$973 The Ca2+-Sensing Receptor (CaSR) Modulates EGF-InducedProliferation in Cultures ot Normal Human Gastric Mucous Epithelial Cells Micheal J. Rutten, Kathy D. Bacon, Katie L. Marlink, Karin D. Rodland, Brett C. Sheppard, Karen E: Deveney, Clifford W. Deveney, Portland, OR; Richland, WA Background: We have previously shown that extracellular Ca2+ (Ca2+)o activated a Ca2÷sensing receptor (CaSR) and increased normal human gastric mucous epithelial cell growth. Epidermal growth factor (EGF) can activate the MAP-kinase signaling pathway which is important in regulating gastric mucosal growth. However, it is not known whether there is cross-talk between the CaSR and the EGF-MAP-kinasesignaling pathways in gastric mucous cells. The aim of the present study was to examine if cross-talk exists between the CaSRand EGF-signaling pathways in normal human gastric mucous epithelial cells. Methods: Human gastric mucous epithelial cells were isolated and cultured using H. pylofi-free surgical tissues. After 4-days in culture, cells were transfected with either a dominant-negative CaSR (R796W) or a pcDNA3 vector alone (control cells) using the Ca2÷-phosphatetransfection. Proliferation

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of cells at 24 and 48 hrs was measured by the MTT-assay. CaSR protein and p44/p42 MAPkinase activity (at 15 mini were analyzedby Western-immunoblotting. Intracellular Ca2+ [Ca2*]i mobilization was measured by spectrofluorimetry of Fura-2 loaded gastric cells. Results: In control gastric cells when shifted from 0.025 to 2 mM (Ca2+)oresulted in increases in [Ca2÷]~ from 108 _+3nM to 281 _+4 nM, a 6.7-fold increase in MAP-kinase activity, and a 1.73-fold increase in cell proliferation at 24-hrs. CaSR-mutantgastric cells shifted from 0.025 to 2 mM (Ca2÷)o had no change in [Ca~+]i, MAP-kinase activity, or cell proliferation. When control gastric cells were placed in 0.125 mM low Ca2÷-media,then EGF or TGF,x (20 nM) added, there were increases in [Ca2÷]~from 105 -+2rim to 163 -+4 nM, a 4.1-fold increase in MAPkinase activity, and a 1.67-fold increase in cell proliferation at 24 hours. In CaSR-mutant gastric cells in low Ca2+-media,the addition of EGF or TGFc~(20 nM) increased [Ca2+]ifrom only 106 _+3nM to 123 _+4 nM, a 1.3-fold increase in MAP-kinase activity, and a 1.2-fold increase in cell proliferation at 24-hrs. Compared to untreated cells, control gastric cells pretreated for 10 rain with 5 I~M of AG1478 (EGF-receptortyrosine kinase inhibitor) then shifted from 0.025 mM to 2 mM (Ca2÷)o,had attenuated changes in [Ca2÷]~from 104 +5nM to 139 _+4 nM, a 3.2-fold increase in MAP-kinase activity, and a 1.45-fold increase in cell proliferation at 24 hours. Conclusion: The CaSRand EGF-signalingpathways can be independent but can also cross-talk with each other which may be important in the regulation of gastric mucous epithelial cell growth. $974 IGF-I Elicits Growth of Human Intestinal Smooth Muscle by Sequential Activation of PI 3-Kinase, PDK-1 and p7gS6 Kinase John F. Kuemmerle, Richmond, VA BACKGROUND:We have previously shown that endogenous Insulin-like growth factor (IGF)I stimulates proliferation of human intestinal muscle cells by activating jointly erkl/2 and PI 3-kinase. PI 3-kinase causes rapid (5 mini and sustained (30 mini activation of Akt and p70S6 kinase. PI 3-kinase dependentactivation of p70S6 and growth are sensitive to rapamycin. AIM: To determine the sequenceof signaling events initiated by IGF-I leading to p70S6 kinase phosphorylation and proliferation of human intestinal smooth muscle cells. METHODS:Muscle cells isolated from the circular layer of normal human intestine were cultured in DMEM + 10% FBS and used at confluence in first passage. Cells were made quiescent by 24h incubation in serum-free DMEM and incubated with IGF-I for various times. Proliferation was measured by 3H-thymidine incorporation. Activation of phosphoinositide-dependent protein kinase-1 (PDK-1), Akt, and p70S6 kinase was measured using phosphorylation-state specific antibodies by Western blot and quantitated with densitometry. Muscle cells were transiently transfected with a dominant negative Akt mutant, Akt(K179M), or empty vector. RESULTS: Incubation of muscle cells with 100 nM IGF-Ifor 30 rain elicited Ser241-phosphorylation of PDK-1 (65_+13% above basal) that was abolished by a PI 3-kinase inhibitor, LY 294002 (10 I~M) or by a PDK1 inhibitor, Nc~-tosyI-Phechloromethy[ ketone (50 p,M), but was not affected by rapamycin (10 nM) or expression of dominant negativeAkt(K179M). IGF-I-inducedSer473-phosphorylation of Akt (77_+4% above basal) was abolished by the PDK-1 inhibitor, by expression of Akt(K179M), but not affected by rapamycin. IGF-I elicited phosphorylation of pTOS6 kinase on Thr389 (128_+16% above basal) and on Thr421/Ser424 (170_+14% above basal). IGF-I-inducad phosphorylation of both sites was abolished by the PDK-1 inhibitor, whereas Thr389 but not Thr421/Ser424 phosphorylation was abolished by expression of Akt(K179M) implying the operation of distinct Akt-dependentand Akt-independent mechanisms regulating p70S6 kinase phosphorylation. PI 3-kinase-dependenttGF-I-induced3H-thymidineincorporation (192_+25% above basal) was abolished by the PDK-1 inhibitor, or rapamyin but not affected by expression of Akt(K179M). CONCLUSIONS:(1) IGF-I-induced POK-l-dependent activation of Akt is not required for muscle cell growth. (2) IGF-I-induces muscle cell growth by sequential activation of PI 3-kinase, PDK-1, and p70S6 kinase.

The Secretin Receptor Requires Distinct C-Terminal PhosphoryletionSites for Internalization. Michael A. Shetzline, Robert H. Oakley, Durham, NC INTRODUCTION:G protein-coupled receptor (GPCR) internalization has been shown to initiate intracellular signaling and is likely important for physiological homeostasis. We have previously demonstrated that the secretin receptor is avidly internalizedwith arrestin after agonist stimulation. Receptor internalization may stimulate intracellular signaling important for agonistmediated exocytosis during cellular secretion. Arrestin has been shown to interact with serine/ threonine clusters in the carboxy-terminal (C-terminal) tail of certain GPCRs. The secretin receptor contains these clusters and these sites may be necessaryfor subsequent intracellu~ar signaling. METHODS:To investigatethe role of sedne/threonine phosphorylation of the secretin receptor, we used site-directed mutagenesis to produce secretin receptor constructs devoid of the sedne/threonine phosphorylation sites (C-terminal truncated, KAST-KAAA, THST-AHAA, ANAAA). These constructs contain a FLAG epitope that allows for fluorescent labeling and immunoprecipitation. HEK 293 cells were transiently transfected with wild-type or mutant receptor. Receptorsignaling was quantitated by measurementof cAMP accumulation. Receptor internalization was assessed by fluorescent-activated cell sorting (FACS), receptor phosphorylation was determined by metabolic labeling with P32 and immunoprecipitation of the epitopetagged receptor and receptor internalizationwas visualized by confocal microscopy. RESULTS: Substitution of alanine for serine/threonine in the tail did not alter cAMP accumulation. However, these mutants demonstrated reduced receptor internalization when exposed to agonist for 30 minutes as assessed by FACS analysis and loss of cell surface receptors. Similarly, confocal microscopy revealed less receptor/arrestin vesicle formation. CONCLUSIONS: Removal of the serine/threonine phosphorylation sites in the secretin receptor does not significantly alter surface receptor signaling, but reduces receptor internalization. Arrestin likely mediates secretin receptor internalization through interaction with distinct serine/threonine clusters in the C-terminal domain and these sites may be important for subsequent intracellular signaling. $977 Analysis of Downstream Effects of IL-11-1nduced Signaling in Human Colonic Epithelial Ceils Stephan Kiessling, Martin Hausmann, Werner Falk, Juergen Schoelmerich, Johannes Grossmann, Gerhard Rogler, Regensburg, Germany Background: A potential protective or anti-inflammatory effect of interleukin-11 (IL-11) for the intestinal mucosa has been postulated from several animal models of inflammatory bowel disease (IBD) and mucosal damage. Therefore, IL-11 was considered to be a promising drug candidate for the treatment of IBD. We could recently demonstrate functional Interleukin-11 receptor alpha (IL-11Ralpha) expression being restricted to epithelial cells of the human colonic mucosa. Stimulation with its cognate ligand induced signaling through phosphorylation of the Jak-STAT pathway. In the present study we investigated downstream effects of IL-11 induced signaling in human colonic epithelial cells (HT-29 and primary human colonic epithelial cells, CEC). Methods: To assess IL-11-induced anti-inflammatory effects, IL-8 secretion in supematants was measured by ELISA and degradation of IKB~ protein was investigated by Western blot. Metabolic and proliferative effects of IL-11 in colonic epithelial cells were analyzed by MTS-test (metabolic activity) and cell-cycle analysis (FACS). To investigate the activation of Protein kinase B (PKB/Akt), cytosolic extracts from IL-11 treated cells were analyzed using antibodies for the detection of tyrosine-phosphorylated Akt. Results: IL-11 Ralpha activation did not reduce IKB~ degradation and IL-8 secretion of TNF treated HT-29, indicating no anti-inflammatory effects of IL-11 in these cells. IL-11 pre-stimulation over several time periods resulted in no detectable increase of proliferation in HT-29 cells, despite clear activation of intracellular signaling pathways. However, IL-11 stimulation resulted in a dose dependent tyrosine phosphorylation of Akt, a key player in the survival of many cell types, in both HT-29 and CEC.Conclusion: IL-11 induces signaling through triggering activation of the Jak-STATpathway, without inducing anti-inflammatory or proliferative effects in colonic epithelial cells. The known beneficial effects of IL-11 therapy are likely to be mediated by epithelial cells via activation of the Akt-survival pathway, mediating anti-apoptotic effects to ensure mucosal integrity.

$975 Protein Kinase n (PKD) is a Downstream Target of Protein Kinase CO Jingzhen Yuan, David Bae, Andre Nel, Doreen Cantrell, Enrique Rozengurt, Los Angeles, CA; London, AA, UK BACKGROUND:PKD/PKCp, is a serine/threonine protein kinase with distinct structural, enzymological and regulatory properties. PKCe is a novel isoform of PKCs selectively expressed in lymphocytes and skeletal muscle. PKCe plays a particularly important role in T cell and B cell activation and in the protection of T cell from apoptosis. PKCe-mediatedsignaling pathways are also implicated in many important biological and/or pathophysiological responses in cells. Despite its potential importance, the downstream targets of PKCe remain poorly understood. In the present study, we examined whether PKD is a downstream target of the PKCO and whether PKD is regulated by PKCe through phosphorylation in the kinase activation loop Ser744 and Ser-748. RESULTS: Protein kinase D (PKD/PKC~) immunoprecipitated from either COS-7 cells or Jurkat T lymphocytes transiently transfected with a constitutively active mutant of PKCeAE (PKCOAE)exhibited a marked increase in basal activity. In contrast, co-expression of constitutively active mutant of PKC~ does not induce PKD activation in both types of cells. PKCeAEdoes not induce kinase activity in immunocomplexes of PKD kinase deficient mutants PKDK618N or PKDD733A. PKD activation in response to PKCOAEsignaling was completely prevented by treatment with the protein kinase C (PKC) inhibitors, GF I or Ro 31-8220, or by mutation of Ser-744 and Ser-748 to Ala in the kinase activation loop of PKD. CONCLUSION: Our results show that PKD is a downstream target of the e isoform of PKC in both COS-7 cells and lymphocytes and indicate that activation of PKD by PKCeis through phosphorylation in the kinase activation loop Ser-744 and Ser-748. The regulation of PKD by PKCe reveals a new pathway in the signaling network existing between multiple members of the PKC family and PKD.

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S978 Dynamic Localization and Function of Vesicle-Associated Membrane Protein-2 in Gastric Parietal Cells Serhan Karvar, Xueblao Yao, Joseph Duman, Kevin Hybiske, Yuechueng Liu, John Forte, Berkeley, CA; Oklahoma City, OK Background & Aims: The H,K-ATPase is the major vesicular cargo in the parietal cell vesicle trafficking pathwayassociated with the stimulation of acid secretion. Soluble n-ethylmaleimidesensitive factor attachment protein receptors (SNAREs) proteins are believed to regulate tubulovesicle trafficking and fusion during the secretory cycle of the parietal cell. Here we studied the intracallular distribution and functional importance of one SNARE, vesicle-associated membrane protein-2 (VAMP-2), in gastric parietal cells. Methods: Using a recombinant adenoviral expression system encoding VAMP-2 fused to the green fluorescent protein (GFP), we expressed the construct in primary cultures of rabbit parietal cells. Cells in a variety of functional states were analyzed by fluorescence microscopy. Streptolysin-O (SLO) permeabilized gastric glands were treated with tetanus toxin and acid secretion was measured. Results: In resting parietal cells GFP was detected throughout the cytoplasm in a pattern of distribution that was very similar to immunostaining for H,K-ATPase.After stimulation, we observed that the green fluorescent protein translocated to the apical plasma membrane along with the H,KATPase. A relatively high degree of co-localization was detected between GFP-VAMP-2 and H,K-ATPese was detected by immunocytochemistry. Tetanus toxin, which cleaves VAMP-2, inhibited cAMP/ATP-stimulated acid secretion by about 45% in SLO-permeabilized gastric glands. Conclusions: GFP-tagged recombinant proteins, expressed through adenoviral infection, can be used to efficiently study the functional dynamics of SNAREs in parietal cells.

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VAMP-2 is associatedwith tubulovesicle membranes in the parietal cell and plays a role in stimulation-associated membrane recruitment and acid secretion.

$979 Vasopressin-Mediated Mitogenic Signaling in Intestinal Epithelial Cells Terence T. Chiu, Chintda Santiskulvong, Enrique Rozengurt, Los Angeles, CA Background/Aims: We have recently identified the presence of endogenous receptors for arginine vasopressin (AVP), specifically V1A subtype, in nontransformed epithelial IEC-18 cells, originally derived from rat ileal crypts. In abstract #101703, we demonstratedthat AVP induced rapid Ca2+ mobilization, protein kinase C (PKC)-dependentprotein kinase D (PKD) activation, and tyrosine phosphoryiation of Pyk2 and Src. However,the role of AVP in other signaling pathwaysin IEC-18 cells is unknown. Here,we examinedwhether AVP can stimulate extracellular-relatedkinase (ERK) activation, DNA synthesis, and cell proliferation in IE0-18 cells. Results: Treatmentof serum-deprived IEC-18 cells with AVP (10 nM) led to a rapid and striking activation of ERK, which can be inhibited by selective V1A receptor antagonist 13Mercapto-13J~-cyclopentamethyleneproprionyY,O-Me-Tyr2,ArgLVasopressin (50 nM). A detectable effect was obtained within 30 s and reached a maximum (-10 fold) after 1 min of AVP stimulation. Half-maximal and maximal ERK activation was achievedat 1 nM and 10 nM, respectively. ERK activation induced by AVP was completed blocked by treatment (lh) with the selective MEK inhibitors, PD98059 (IOI~M) and U0126 (2.51~M).To investigatethe upstream pathway(s) leading to MEK activation, we examinedthe Contribution of PKC, Src, and EGFRto AVP-induced ERK activation. Pretreatment with selective PKC inhibitors, GF-I (2.5~M) and Ro 31-8220(2.51~M), Src-kinase inhibitor PP-2 (lO~M), or EGFRtyrosine kinase inhibitors, tyrphostin AG1478 (250 nM) and compound 56 (500 nM), abolishedAVP-induced ERK activation. We also demonstratedthat AVP,via V1A receptor, induced a striking increase in DNA synthesis in IEC-18 cells as judged by measuring tritiated-thymidine incorporation. A detectableeffect was obtained with 1 nM AVP and maximal effect (2.5 fold) was achieved at 10 nM In contrast, EGF (5ng/ml) led to only 1.6 fold increase. Furthermore, there is nearly a doubling in the cell number of cultures treated with AVP for 24h and 48h comparedto the controls. Pretreatmentof cell cultures with the MEK inhibitors, PD98059 (IO~M) and U0126 (2.5~M), attenuated(50%) AVP-induced tritiated-thymidine incorporation. Inhibition of PKC (GF-1), Src (PP-2), or EGFRtyrosine kinase (AG1478) also abrogatedAVP-inducedtritiatedthymidine incorporation. Conclusion: AVP acts as a potent growth factor for IEC-18 cells inducing DNA synthesis and cell proliferation through ERK-, PKC, EGFRtyrosine kinase- and Src-dependent pathways.

$980 Angiotensin II- and LysophosphatidicAcid-Induced Pyk2 Tyrosine Phosphorylation in Intestinal Epithelial Cells is Mediated By Rho/ROK Steven S. Wu, Terence Chiu, Enrique Rozengurt, Los Angeles, CA Background/Aims:Intestinalepithelialcell function is regulatedby a variety of stimuli including growth factors, cell adhesion,and stress. The cytosolic proline-rich tyrosine kinase2 (~k2), which is activatedby tyrosine phosphorylation,associateswith cytoskeletalproteins and also acts upstream of MAP kinase cascades; it thus potentially plays a role in cell motility and proliferation. We have previously shown that in intestinal epithelialcells, the G protein-coupled receptor (GPCR) agonists angiotensin II (Ang ll) and lysophosphatidic acid (LPA) rapidly induce Pyk2tyrosine phosphorylationin a manner dependenton Ca2+, protein kinaseC (PKC), and the integrity of the actin cytoskeleton. Since the small GTPaseRho, and its effector Rho kinase (ROK), are important upstream regulators of actin stress fiber assembly, our aim was to characterize the role of this pathway in Pyk2 phosphorylation. Methods/Results: Pyk2 phosphorylation was measured by immunoprecipitation and Western blotting with total or phospho-specificPyk2antibodies.As with overalltyrosine phosphorylationof Pyk2, phosphorytation at tyrosine 402 and tyrosine 580 (sites of Pyk2 auto- and trans-phosphorylation, respectively)were rapidly enhancedin responseto Ang II or LPA.When cells were preincubated for 24 h with Clostridium botulinum C3 exoenzyme,which catalyzesAOP-ribosylationand thereby inactivation of Rho, Ang II- and LPA-stimulated Pyk2 tyrosine phosphorylationwere inhibited. To further investigatethe effect of the Rho pathway on Pyk2, we focused on ROK, a downstream target of Rho. Pretreatmentwith the structurally unrelated ROK inhibitors HA1077 and Y-27632 each reduced both Ang II- and LPA-stimulated Pyk2 phosphorylation by 50%, while simultaneous inhibition of ROK, PKC, and Ca2+ completely abolished the Pyk2 response. Neither HA-1077 nor Y-27632 affected the PKC signaling or Ca2+ releaseinduced by Ang II or LPA, indicating that ROK contributed to Pyk2 tyrosine phosphorylation independently from PKC and Ca2+. Conclusion: Our results show that in IEC-18 cells, Ang II- or LPA-induced Pyk2 phosphorylation occurs via distinct PKC-, Ca2*-; and Rho/ROK-mediated pathways.Thesefindings identify a novel role for Rho upstreamof Pyk2 signaling, and suggest that Pyk2 representsa point of integrationfor PKC-,Ca2+-, and cytoskeleton-dependentsignals in intestinal epithelial cells.

$981 Overexpression of FGF Type I Receptor Enhances Surface Retention of Glypican-1 and FGF-2 Dependent Signaling. Kei Matsuda, Martha Lopez, Kimi Fukahi, Arthur Lander, Murray Korc, Irvine, CA Fibroblast growth factors (FGFs) are involved in various biological processesand have implicated in many human cancers. FGFsbind to a family of high affinity receptors(FGFRs). This binding often requires the presence of low affinity heparan sulfate proteoglycans (HSPGs) that act to enhance ligand presentation to the FGFRs. HSPBs have been implicated in the modulation of cell adhesion, migration, proliferation, and differentiation. HSPGsare able to cross-link FGFs with FGFRsto form activated ternary complexes, and regulation of these complexes may serve as a major control mechanism for FGF-2-mediatedactivation of FGFRs. We previously reported that glypican-1 is overexpressedin pancreatic and breast cancers, and that the presenceof glypican-1 is essentialfor the mdogenic effects of multiple heparinbinding growth factors. In the present study we examined the expression of FGFRs and

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glypicans, a family of HSPGs, in SNU-387 human hepatoceliularcarcinoma (HCC) cells. As determined by Northern blotting, quantitative, real-time RT-PCRand RNase protection, this cell line predominantly expressedthe 2 Ig-like form of FGFR-1 and exhibited high levels of glypican-1 and glypican-6 mRNA moieties,but low levels of the other membersof the glypican family. When parental SNU-387 cells were transfected to overexpressthe 3-1g like form of FGFR-1, there was marked retention of glypican-1 on the cell membrane in association with enhanced mitogenic signaling in response to FGF-2. Thus, there was enhanced tyrosine phosphorylation of the adaptor protein FRS-2, and increasedand more sustained activation of MAP kinase, jun kinase and p38 MAP kinase. These signaling eventswere associatedwith enhanced anchorage-dependentand -independentcellular proliferation, and were attenuated by pre:incubating transfected cells With phosphoinositide-specificphospholipase-C(PI-PLC), an enzyme that remOvesglypican-1 from the cell surface. Thesefindings suggest that high levels of the 3-1g loop form of FGFR-1 can lead to the cell-surface retention of glypican-1, which together may serve to amplify mitogenic signaling and promote the growth of certain HCC cells.

$982 Mitogenic Effect of Bombesin in Prostate Cancer Cells through MAP Kinase Pathway via Transactivation of EGF Receptor Dongmei Xiao, Xiangping Gu, Horst C. Weber, Boston, MA The amphibiantetradecapeptidebombesin(Bn) and its mammalian homologue,the 27 aminoacid regulatory gastrin-releasing peptide (GRP), have been shown to play an important role in human cancer as autocrine or paracrinegrowth factors through activation of specific, high affinity G protein-coupledGRP receptors (GRP-R). In the prostate gland, Bn-like peptidesare expressed and secreted by neurOendocrinecells and relate to prostate carcinogenesis and progression of the diseasevia paracrineinteractionwith its aberrantlyexpressedhuman GRPR. in this study, we found Bn-induced cell proliferation in androgen-independentPC3 and DU145, but not in androgen-dependentLNCaPcells in a dose dependentmanner, which was completely abolished by its specific GRP-R antagonist ME. Further studies showed this mitogenic effect of Bn on PC3 and DU145 prostate cancer cells was mediatedby the activated MAP kinase pathway. Bn induced MAP kinase (also called ERK1/2) phosphorylation in both PC3 and DU145 cells but not in LNCaPcells, which was completelyabolishedby pretreatment with the specific GRP-R antagonists ME and BW2258U89 or MEK1/2 inhibitor PD98059. Moreover, Bn-induced ERK1/2 phosphorylationwas demonstratedto occur at subnanomolar concentrations (0.1 riM) in a time-dependentmanner 1 min after Bn stimulation, reaching a maximum response 5 ~ 10 minutes later and declining to baselineafter 1 hour. To identify which upstream signaling pathways are involved in Bn-induced phosphorylation of ERK1/2, we demonstrated epithelial growth factor receptor (EGFR) transactivation was required for Bn-induced ERK1/2 phosphorylationand cell proliferation. Furthermore, the p6OSrc inhibitor PP2 nearly abolished Bn-induced EGFRtransactivation and ERK1/2 phosphorylation but not EGF-stimulated-EGFRactivation and ERK1/2phosporylation.Similarly, using the Ca2+ chelator BAPTNAM and the inhibition of intracellular Ca2+ releasewith Thapsigarginresulted in abolishing only the Bn-induced but not EGF-stimulatedERK1/2 phosphorylation. In contrast, both the PKC inhibitor GF109203X and the PKA inhibitor H89 had no effect on Bn-stimulated ERK1/2phosphoryiation.Theseresults indicatestrongiy that ligand-specifichGRP-Ractivation mediatesmitogenic properties in human prostatecancer by activatingthe MAP kinasepathway through transactivation of EGF receptor and this requires intracellular Ca2+ release and p6Osrc activation.

$983 Gastrin induces MAPK Activation and Proliferation in Renal Epithelial Cells Michael Blaeker, Hanna Michaelis, Martina Schulz, PhilomenaArrenberg,Sylvia Burghardt, Tammo Von Schrenck, Heiner Greten, Andreas De Weerth, Hamburg, Germany Background: We have recently shown that the G-protein coupled cholecystokinin2-receptor (CCK2R) is expressed in multiple cell types of the kidney in various species, suggesting a function for the CCK2R ligands CCK and/or gastrin in renal physiology and pathophysiology. Given the well establishedeffects of gastrin on cell proliferation in the gastrointestinaltract, we sought to investigate CCK2R expression as well as gastrin induced cell proliferation in two renal epithelial cell lines, MDCK-C7 and MDCK-C11. Moreover, to further elucidatethe intracellular mechanisms following stimulation of the CCK2R in these cell lines we studied activation of mitogen-activatedprotein kinase pathways. Methods: Expressionof the CCK2R in C7 and Cll cells was analyzedby RT-PCRand immunocytochemistry. C7 cells and Cll cells were grown in MEM-EARLE medium for three days in the absence of presence of epidermal growth factor (EGF), gastrin, and the CCK2R-specificantagonist L-365,260. Cell proliferation was assessedby cell counting as well as by measurementof BrdU-incorporation. Phosphorylationof Raf, MEK,and MAPKwas investigatedby Western blotting using phosphospecific antibodies. Results: Expression of the CCK2R in MDCK cells is detectable by RTPCR as well as by immunocytochemistry. Gastrin dose-dependentlystimulates MDCK cell proliferation, with a maximum of a 1.3-fold and 1.5-fold increase in the number of C7 cells and Cll cells, respectively,at 10-7 M. In addition, gastrin as well as EGF lead to a 1.4-fold stimulation of BrdU-incorporation.Thesegastrin-mediatedeffects are inhibited by the CCK2Rantagonist L=365,260. Gastrin induces time-dependentphosphorylation of Raf, MEK, as well as MAPK in both cell lines. Conclusion: Gastrin acts as a growth factor in renal epithelial cells by stimulation of the CCK2Rand subsequentphosphorylationof MAPKsignaling cascades.The proliferative effects of gastrin on renal epithelial cells might contribute to physiologic growth as well as growth after malignant transformation, i.e., in renal cell carcinoma. Conditions of hypergastrinemia,e.g. treatment with proton pump inhibitors, might be detrimental in patients with malignant kidney disease.

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$984 The PACAPType I Receptor (PAC1) is Expressed in Human Colonic Tumors and Is Coupled to Signaling Cascades Patrizia Germano, Robert Fan, Sang Le, Dean Yamaguchi, Mark Wu, Ken Tachiki, Joseph Pisegna, Los Angeles, CA Background: The Pituitary Adenylate Cyclase Activating Peptide (PACAP) type 1 receptor (PAC1) is a hepta-helical, G protein coupled receptor, that has been shown to be expressed on lung and breast cancers, and to be coupled to the growth of these tumors. We have developed a fluorescent PACAPcompound and rabbit specific polyclonal antibodies directed against the COOHterminus of PAC1, and demonstrated the specific binding of our FluorPACAPand anti-PAC1 antibodiesto PACl-transfected NIH3T3 cells by immunohistochemistry and cytofluorimetric analysis. Aims: The purpose of this study is to evaluatethe expression and the role of PAC1 in human colonic tumors. Methods: HCTS, HCT116, and FET tumor human colonic cell lines were cultured and PAC1 mRNA expression was detected by RTPCR. PAC1protein membraneexpressionon these cell lines was assessedby immunofluorescence, using Fluorescence-activatedcell sorter (FAGS)analysis and Confocal Laser Scanning Microscopy, using our Fluor-PACAPand anti-PAC1 polyclonal antibodies. Measurement of adenylylcyctasedose responseswas performedusing the Dowex-Aluminamethod.The ability of PACAP-27to stimulate intracellular phosphorylation pathways was measured using an in situ phosphotyrosineELISAassays. Results: Our data indicatethat native PAC1 receptors are abundantlyexpressedat both a mRNAand protein level in these tumor cell lines. Immunofluorescencestudies indicatethat the PAC1receptor is expressedon the cell surface of the human colonic tumor cells and that the receptor is rapidly (i.e. <5 min.). PACAP-38elevatedcAMP levels in a dose-dependentmanner in HCT8, HCT116, and FET with a halt-maximal (EC5O) stimulation of approximately 3 nM, 1.5 nM, and 1 nM, respectively. Furthermore, in situ phosphotyrosine direct ELISA assay showed maximum levels of tyrosine phosphorylation in responseto PACAP-38at 9 minutes for all these cell lines. Conclusions: The data show that PAC1 is expressed in human colonic tumors and in HCT8, HCT116, and FET cell lines, in which exhibits signal transduction pathways coupled to adenylyl cyclase and tyrosine phosphorylation. The presenceof PACAPreceptors on a wide range of human colon tumors suggests that PACAPhormone may play an important role in the regulation of their growth. These results might have important implications on potential strategiesfor treatment directed at blocking these signaling pathways in human colonic tumors. $985 HyperosmoUcStress Stimulates Src-DepondentTyrosine Phosphorylationof Focal Adhesion Kinase J Adrian Lunn, James Sinnett-Smith, Enrique Rozengurt,Los Angeles, CA

$987 Histamine 3 Receptors Inhibit Vagal Afferent Mechanoreceptors in the Ferret Proximal Stomach. Scott D. Staid, L Ashley Blackshaw, Adelaide,Australia Background:Histamine3 (H3)receptorshavebeenshownto possessantinociceptiveproperties and to inhibit gastric acid secretion in vivo. In the present study we aimed to determine whether H3 receptors could peripherally modulate vagal mechanosensitiveafferent nerves in an isolated gastric preparation from the ferret. Methods: The stomach and distal half of the esophagus from adult ferrets was removed and maintained in an organ bath perfused with Krebs solution and nifedipine (1 p~M)at 34°C. The vagus nerveswere preparedfor recordings of afferent nerve activity and action potentials recordedfollowing distensions of the proximal stomach with 2, 5 and 10 mls of air, before and after perfusion with the H3 receptorselectiveagoinst R-(~)-methyl-histamine (20-100 p,M). Results: Selectedvagal afferent fibers responded to distension (2 -10 mls air) of the stomach with a stimulus-dependent increase in afferentfiring frequency.Fiberscloseto the cardiaand lower esophagealsphincter (LES) also displayeduniform regular spontaneousdischarge.R-(a)-methylhistamine(IOOI~M)decreased basal activity of vagal afferent fibres in the cardia and LES, but did not modify the distensionevoked discharge of a separatepopulation of gastric mechanoreceptors(20 p,M). The effects of R-(~)-methylhistamine (IOOlLM) on spontaneousactivity were reversedfollowing treatment with the H3receptorantagonistclobenproprit (100 I~M). Conclusion:Theseresults demonstrate a direct inhibitory role for H3 receptors on a vagal afferent mechanoreceptorsin the ferret proximal stomach. H3receptor agonists may therefore havethe capacityto modulate sensory function in the gastric cardia. $988

Background and Significance Cells exposed to hyperosmotic stress (HS) undergo dramatic changes in celt shape and concomitant activation of p38 MAP kinase and stress-activated protein kinases(SAPK/JNKs)and ultimately undergoapoptosis. Cell signaling pathwaysmediating surviving HS have beenextensivelycharacterizedin yeast, however pathwaysmediating mammalian cell responseto HS remain poorly characterized.Previousstudies in mammalian cells have tended to focus on prolonged exposure to HS and on subsequentapoptosis. We investigatedeffects HS on focal adhesionkinase (FAK) suspectingthat such a focal adhesionlocalized molecule, able to regulate signals from integrin-extracellularmatrix (ECM) to actin cytoskeleton, might be a signal translator for HS. FAK is a non-receptortyrosine kinase first described as one of many proteins phosphorylatadon tyrosine in v-Src transformed chicken embryo fibroblasts. FAK localizesto focal adhesion plaques in v-Src transformed cells, and regulates cell movement and attachment. FAK has been shown to be a critical point of convergence in the action of integrins, oncogenic forms of Src, mitogenic neuropeptides, bioactive lipids, bacterial toxins, and growth factors. More recently, FAK activation has also beenshown to block p53 mediatedanoikis when epithelialcells are deprivedof their extracellular matrix attachments. Results-Using murine Swiss 3T3 fibroblasts and PanG1 (human pancreatic carcinoma) cells, we show that FAK is phosphorylatedon tyrosine in responseto hyperosmotic stress. The focal adhesion-essociatedmolecules, paxillin and p138 Crk-associated substrate (CAS), were also found to be phosphorylated in response to HS. Using the specific Src-family tyrosine kinaseinhibitor, PP2, and fibroblast cell lines deficient in multiple Src-family members,we show that this HS-inducedFAKactivation is pp60s~dependent.Using both actin-depolymerizingagents latrunculin A and cytochalasin D, we also show that intact actin cytoskeleton is necessaryfor FAK activation by HS. Finally, using structurally distinct, specific inhibitors of the Rho effector Rho-associatedKinase (ROK) we show that this HS activation of FAK is ROK independent.Conclusions- Hyperosmoticstress activates FAK via a novel, pp6OS"-dependentbut ROK-independentpathway. $986 Growth Promoting Effect of Muscarinic Acatylcholine Receptors in Colon Cancer Cells Yoshiaki Takeuchi, Satoshi Kusayanagi,Jun-lchi Ukegawa,Keiji Mltamura, Tokyo, Japan It has been reported that colon cancer cells express muscarinic acatylcholine receptors although their functional roles has been largely unknown. The aim of this study is to elucidate possible mechanisms responsible for growth promoting effect of muscarinic acetylcholine receptors in colon cancer cells. A colon cancer cell line, 184, derived from a human colon adenocarcinoma was used in our study. We first examined which subtypes of mussarinic acetylcholine receptors were expressed by RT-PCR, and found that only subtype 1 was expressed. The effect on cell proliferation was assessed by MTr assay, Carbacbol promoted dose dependently cell growth with maximal effect observed at a dose of O.lmM that was same potent as that of EGF. Pirenzepin, a highly specific antagonist to subtype 1, inhibited the stimulatory effect of carbachol,suggestingthat the growth promoting effect was mediated through receptors.We nexttested ERKsactivity using in vitro kinaseassayand immunoblotting of tyrosine phosphorytation.Carbacholstimulated ERKsactivity in a dose dependentmanner and this effect was also abolished in the presence of pirenzepin. Treatment of the T84 with PD-98059, a specific MEK inhibitor, blockedcarbacholactivation of ERKsand cell proliferation,

AGA Abstracts

indicatingthat ERKsare important mediators.EGFreceptorhas beenshownto betransactivat~l by G-protein-coupled receptors. Accordingly, we examinedtyrosine phosphorylation of EGF receptor by immunoblotting. Carbachol induced tyrosine phosphorylation of EGF receptor, which was functional in signal transduction since GRB2 coprecipitated EGF receptor and tyrosine phosphorylatedShc. When EGF receptor tyrosine kinase was blocked by treatment with either neutralizingantibody or AG 1478, carbacholstimulation of ERKsand cell proliferation were substantially inhibited. Taken together, our results indicate that growth promoting effect of muscarinic acetylcholinereceptor subtype 1 in colon cancer cells may be dependent on EGF receptor-ERKspathway and that EGF receptor not only receivesexternal stimuli but also relays intrinsic signal.

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Molecular Determinants of ArgLys ProhormoneProcessing David Oldfield, Masashi Matsushima, Chris J. Dickinson, Ann Arbor, MI; Kanagawa,Japan Background: Progastrin requires cleavage at a dibasic site (Lys74Lys75) by a prohormone convertase (PC) to convert gastrins of 34 residues (G34) to gastrins 17 amino acids (G17) in length. In a similar fashion, prosomatostatin (proSS) is cleaved at Arg~Lys78to produce somatostatin-14 ($14) from somatostatin-28 (S28)-see figure. Previously, we noted that ArgLys pairs were infrequent dibasic prohormone processing sites (<5% of total). Furthermore, mutation of preexisting dibasic sites to ArgLys often markedly impaired prohormone processing. Previously, we noted that mutation of the Lys74Lys75 site to Arg74Lys75 within progastrin markedly impaired progastrin processing and G17 production. Neverthelessthe Arg77Lys78site within proSSwas efficiently cleavedin the samecell lines. Thus, we hypothesized thatthe Pro and Arg residuesprecedingthe Arg~Lys7esite in proSSmay enhanceits processing. Thus, we expressed native and mutant progastrin cDNAs in an endocrine cell line (AtT-20) capableof recapitulatingprohormone processing reactions seen in vivo. We mutated residues within progastrin correspondingto the ProArgGlu residues precedingthe ArgnLys7scleavage site found in proSS and examined the effects on the production of G34 and G17. Peptide products in cell extracts were quantified using FPLCand radioimmunoassay.The ratio of G17 to G34 was taken as a measure of the efficiency of prohormone cleavage.Cleavageof native progastrin at the LysT"Lys7~site produced a G17:G34 ratio of 1:1. The ArgTZLys7s site within proSS was efficiently cleavedwith an S14:$28 ratio of 4:1. Gastrin Sequence G17:G34 AspProSerLysLys(wild) 1:1 AspProSerArgLys 1:3 ProProSerArgLys 2:1 AspArgSerArgLys 4:1 ProArgSerArgLys 20:1 Mutation of the nativeLysLyssite in progastrinto ArgLys markedlyimpaired gastrin processing (G17:634,1:1 to 1:3). Addition of upstream Pro or Arg residuesfound within proSS enhanced G17 production and Arg Lys processing (G17:G34, 2:1 and 4:1, respectively). Addition of both upstream Pro and Arg residues greatly enhanced processing (G17:G34, 20:1). This confirms that not only the nature of the dibasic pair found at a prohormone processing site hut also its surrounding amino acids effect the efficiency of cleavage.

(;17Y///A :~ii:

$989 A Reduced PseudopeptideBond in Xenin Prolongs Plasma Half-Life and Increases Potency In Vitro and In Vivo Gerhard E. Feurle, Marit Heger, Gerd Hamscher, Bonn, Germany Background:Xenin is a 25 aminoacid peptide produced by endocrine cells of the duodenal mucosa.Xenin acts as a prokinetic on interdigestive and postprandial jejunal motility and as a secretagogueon the exocrine pancreas.Asthe prokinetic property may he therapeutically useful, molecular modification to increasepotency are of interest.Methods:Thepentacosapeptide xenin and the C-terminal hexapeptidewithout and with a reduced pseudopeptidebond