717 Expression of Homeodomain Protein Pdx-I in Mouse Stomach Sachiyo Nomura, James R. Goldenring BACKGROUND: Recent studies have implicated an antral phenotype in the fundic region with pathogenesis of gastric cancer. Pdx-1 is a homeodomain transcription factor developmentally expressed in the pancreas and the duodenum. Neonatal pdx-1-/- mice die due to an absence of the pancreas. In stomach, Pdx-1 was also reported antral endocrine cells and targeted deletion of the pdx-1 gene in mice resulted in animals with a decrease in gastrin cells. We have now reevaluated the cell lineages expressing Pdx-1 in the stomach using a combination of kacZ expression mapping and immunocytochemistry. METHODS: Pdx-1 expression in the stomachs of heterozygote knock-in mice expressing the lacZ gone under control of pdx- I promotor was analyzed following perfusion fixation and incubation of frozen sections with Xgal. Dual immunostaining was performed with antibodies agantst spasmolytic polypeptide (TFF2) and gastrin. RESULTS: The Pdx-1 was expressed in epithelial cells of pyloric glands. While gastrin cells did stain for LacZ (Pdx-1 expression), the vast majority of LacZ expressing cells in the deep antral glands were not stained with gastrin antibodies. Rather these deep antral cells expressing LacZ were stained with antibodies against TFF2. Surface antral cells were also stained with LacZ In addition, TFF2/LacZ dual stained glands were also observed in the distal fundic region as glands without gastrin cell staining, in the distal fundic glands, no LacZ staining of surface cells was observed. LacZ/TFF2 dual staining cells were also observed throughout the Brunner's glands. TFF2 staining mucous neck cells in the fundus did not express LacZ. LacZ expressing cells were observed in the most proximal 1-2 glands of the fundus just distal to the forestomach. We also observed scattered LacZ expressing cells in the deep regions of fundic glands. CONCLUSION: While gastrin cells do express Pdx-1, the maJOrity of the Pdx-1 expressing cells in the deep antral glands are TFF2- expressing mucous cells. These results suggest that Pdx-1 expressing cells are responsible for production of the entire repertoire of antral lineages. However, in distal fundic glands, Pdxl expression was only observed in deep cells and not in surface cells. Thus Pdx-1 co- expression with TFF2 marks the subset of transitional glands between the fundus and the antrum. In addition, Pdx-1 expression was also present in a limited number of glands in the proximal fundns and in scattered locations in the fundus. Pdx-1 may be an important marker of antral phenotype in the stomach. 718 The Zinc-Finger Transcription Factor KIf4 Is Required for Normal Gastric Epithelial Proliferation and Differentiation In Vivo Jonathan P. Katz, Nathalie Perreault, Bree G. Goldstein, Klans H Kaestner Background and aims: Klf4 (formerly GKLF) is a zinc-finger transcription factor expressed in the epithelia of the skin, lungs, gastrointestinal tract, and several other organs A growing body of evidence supports an important role for KIf4 in epithelial proliferation and differentia- tion. Mice homozygous for a null mutation in Klf4 die within 15 hours after birth and show selective perturbation of late-stage differentiation of the epidermis and alterations in the terminal differentiation of goblet cells in the colon. Due to the early lethality of the Klf4/ mice, the role of Klf4 in the gastric epithelium at later stages has not been explored. Methods: Using tissue-specific gene ablation, we have generated adult mice lacking KIf4 in the gastric epithelium (Foxa3-Cre/Klf4~'r176 mice). Foxa3-Cre/Klf4 I~ mice and controls were sacrificed on postnatal days 1, 60, 180, and 365. Stomachs were examined grossly for evidence of gastric tumors, and gastric tissue was harvested for histology, immnnohistochem- istry, RNA, and protein analyses. Results: Foxa3-Cre/Klf4 ~~ mice survive to at least one year of age and appear to grow normally. We show that Klf4 is successfully deleted in the gastric epithelium by immunohisto- chemical staining with anti-Klf4 antibodies and by RNase protection analysis (RPA) of whole gastric tissue. By RPA, Foxa3-Cre/Klf4 I~176 mice demonstrate a 75% decrease in expression of the Klf4 target gone p21w'~Fvc~v~ (p<0.05) and a 25% increase in expression of Klf5 (p<0.05). Histologically, Foxa3-Cre/Klf4 ~p mice have severe abnormalities of the gastric mucosa, with thickened epithelia, distorted glands, and the presence of large epithelial cysts. These findings are similar to those seen in Men~trier's disease, a premalignant disorder of the stomach which has been linked to enhanced epidermal growth factor receptor (EGFR) signaling. Conclusions: Adult mice lacking Klf4 in the gastric mucosa develop severe gastric epithelial abnormalities reminiscent of M~nEtrier's disease and have decreased expression of p2t w~t~ oP~ and K/fS. The similarities to M~n6trier's disease suggest that alterations in EGFR signaling may also play a role in the gastric epithelial abnormalities of these mice. Thus, using the Foxa3-Cre/Klf4 I~~ mice, we have gained insight into the role of Klf4 in the gastric epithelium in vivo and a greater understanding of the mechanisms underlying gastric epithelial prolifera- tion and carcinogenesis. 719 Aspirin, But Not NO-Releasing Aspirin (NCX-4016), Interacts with Seleetive COX-2 Inhibitors to Aggravate Gastric Damage and Inflammation John L. Wallace, Andrea Mencarelfi, Piero Del Soldato, Stelano Fiorucci We have recently demonstrated that aspirin administration to rats results causes a marked increase in production by the stomach of lipoxin A4 (or 'aspirin-triggered lipoxin; ATL). ATL, which has protective effects on the stomach, is formed via cyclooxygenase (COX)-2. Blockade of ATL formation with a selective COX-2 inhibitor results in a marked increase the severity of aspirin-induced gastric damage (Gastroenterology 2002; 123: t598). NCX- 4016 has been shown to block platelet aggregation as effectively as aspirin, without causing gastric injury. In the present study, we compared the effects of aspirin and NCX-4016, given alone or with celecoxib, on gastric injury and inflammation, and on ATL formation by the stomach. Methods: Rats were treated orally with aspirin (10-100 mg/kg) or eqnimolar doses of NCX-4016. At the same time, they received celecoxib (10 mg/kg) or vehicle i p Three hours later, gastric damage was blindly scored and ATL formation was measured To examine effects on gastric inflammation, ASA or NCX-4016 was given daily for 5 days, with celecoxib gaven on the final day. Results: Celecoxib significantly exacerbated aspirin-induced gastric damage at all doses tested, increasing the gastric damage scores by 2- to 3-fold. In contrast, NCX-4016 did not cause significant gastric damage, given alone or with celecoxib. Aspirin, but not NCX-4016, caused significant ATL formation by the stomach, which was blocked by celecoxib. After 5 days of administration of ASA, significant gastric inflammation was detected: double the myeloperoxidase activity (MPO) of controls. Celecoxib further increased neutrophil infiltration (2.5-fold). Daily administration of NCX-4016 did not cause significant increases in MPO activity, even when celecoxlb was co-administered. In contrast to the single administration studies, gastric ATL formation was sigmficantly increased by 5 day dosing of aspirin or NCX-4016, and in both cases it was suppressed by celecoxib. Conclusions: These results confirm that celecoxib inhibits the formation of a gastroprotective substance (ATL). While the combination of aspirin + celecoxib leads to substantially more gastric damage and gastritis than seen with either drug alone, the combination of NCX-4016 + celecoxib does not result in significant gastric injury or inflammation 720 pr-Nsaids Have Reduced Toxicity in a Rodent Model of Joint Inflammation Arda Yalvac, Lenard M. Lichtenberger Background: The phospholipid, phosphatidylcholine (PC) has been demonstrated by our and other laboratories to protect the gastroduodenal mucosa against the injurious actions of nonsteroidal anti-inflammatory drugs (NSA1Ds) in laboratory animals and hmnan subjects [Nature Medicine 1:154-8, 1995, Amer. J. Castro 94:1818-22,1999]. It has also been reported that peripheral inflammation in rats increases the sensitivity of the GI tract to the injurious actions of NSAIDs [ Dig. Dis. Sci. 46:1690-00,2001 ]. Aim: In the current study we decided to use the above animal model of joint inflammation to compare ulcerogenic potential of indomethacin (lndo) vs PC-lndo in sensitized Lewis (Lw) female rats, pre-injected with two preparations of Complete Freunds Adjuvant (CFA) from either a commercial (C) suppher or laboratory (k) prepared, possessing different levels of immunogemcity. Methods: CFAc or CFAL were injected into the hindpaw o[ Lw rats, and 14 days later the rats were intragastrically administered Indo, PC-Indo (20 mg of NSAID/kg) or vehicle and 4 hrs later the rats were euthanized and the ankle thickness and gastric lesion score (ram2) measured by caliper We also included groups of control (uninflamed) rats that were not injected with CFA and subsequently challenged with Indo or PC-indo. Results: Under the above conditions Indo failed to induced gastric ulceratmn in control Lw rats, whereas in rats pre-administered CFAL, lndo induced marked gastric ulceration (144 +/- 2.2) which was significantly greater than the gastric lesions of rats treated with PC-Indo (5.5 +/- 1.1) or vehicle (0.1 +/- 0.1). Interestingly, in rats who were pre-injected with CFAc, lndo induced a smaller degree of gastric injury (2.6 +/- 0.9), whereas no detectable lesions could be detected in rats challenged with PC-lndo The injurious potential of Indo appeared to be associated with the pbtency of the immunogen to induce ankle inflammation, with CFA~ inducing a 201%, and CFAc inducing a 35.2% increase in ankle thickness over control values. Conclusion: The ability of the NSAID, Indo to induce acute gastric injury in rats appears to be exacerbated by the presence of peripheral inflammation induced by CFA, and this ulcerogeinc effect can be significantly attenuated if rats are administered an equivalent dose of PC-lndo. These results, in this relevant animal model, support earlier evidence of the increased GI safety of PC- NSAIDs in the treatment of inflammatory disorders. 721 NSAID-Mediated Cyclooxygenase-2 Transcript Stabilization via Sustained p38 Activation Randy C Mifflin, Jamal 1. Saada, John F. Di Mari, Patrick A. Adegboyega, Don W. Powel[ Background: Intestinal myofibroblasts (IMFs) are important mucosal targets and sources of proinflammatory cytokines and lipid mediators. We have shown previously that IL-1 induces cyclooxygenase-2 (COX-2) expression in cultured human IMFs. Aspinn (ASA, 1.0 to 5.0 raM) and other nonsteroidal anti-inflammatory drugs (NSAIDs) also induce COX-2 expression and NSAID plus IL-1 results in synergistic induction of COX-2 expression after 24 hours Although our prior studies have identified NSA1D-mediated augmemation of p38 stress activated protein kinase activity, as an important component of this induction, the precise mechanism whereby ASA increases COX-2 expression has not been identified. Aim: The purpose of this study was to determine how NSAID-indnced p38 activation enhances COX- 2 expression in human intestinal myoflbroblasts. Methods: Nuclear mn off analysis was performed on cultured IMFs to determine the rate of transcription of the COX-2 gene following treatment with IL-1 or Ik-1 plus NSAID Pulse-chase studies using the transcrip- tional inhibitor 5,6-dichloro-l-B-D- ribofuranosyl- benzimidazole (DRB) in combination with Northern blotting were used to determine COX-2 mRNA decay rates following treatment with IL-1 or Ik-i plus NSAID. Microarray analysis of IMF RNA samples isolated 4, 8, or 24 hours following Ik-1 or lk-1 plus NSAID treatment was also performed to identify other genes likewise regulated by NSAIDs. Results: The COX-2 transcriptional rate, measured 4- 6 hours after treatment, was only 1.5 to 2 fold higher in cells treated with IL-1 (500 pg/ ml) plus ASA (5.0 raM) than in cells treated with Ik-1 alone. In contrast, ASA treatment resulted in a dramatic stabilization of the COX-2 message. The COX-2 mRNA half-fife in IL-1 treated cells was one hour. The COX-2 mRNA half-fife in Ik-I plus ASA treated cells was in excess of 5 hours. Microarray analysis revealed 12 other genes whose expression was enhanced 2.5 fold or greater by ASA. Most of these also encode mRNAs with relatively short half-lives whose stability is also subject to regulation. Examples include TNF-alpha, ICAM1, IL-8, and 1L-6. Conclusion: The synergistic induction of COX-2 expression seen following IL-1 and ASA treatment results from ASA-mediated maintenance of p38 activity resulting in prolonged stabilization of the COX-2 message. NSAIDs have the potential to induce a program of gene expression based upon stabilization of mRNAs encoding important intra- and extracellular signaling intermediates. Supported by the NIDDK and CCFA. AGA Abstracts A-92