Changes in gastric mucosal peroxisome proliferator-activated receptor γ (PPARγ) expression after Helicobacter pylori eradication

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luterleukin-6-1nduced Thrombopoiutin Mediates Inflammation-Associated Thrombocytosis Arthur Kaser, Gerald Brandacher,Wolfgang Steurer, Susanne Kaser, Felix A. Offner, Clemens Molnar, Univ Hosp Innsbruck, Innsbruck Austria; Michael B. Atkins, Beth Israel Deaconess Medical Ctr; James W. Mier, Harvard Medical Sch, Boston, MA; Herbert Tilg, Univ Hosp Innsbruck, Innsbruck Austria

Regulation of Somatostatin and Gautrin by luterferon~ in the Mouse Gastric Mucosa. Yana Zavros, Juanita L. Merchant, Univ of Michigan, Ann Arbor, MI Background: Patients infected with Helicobacter pylori show an increase in gastric mucosal lymphocytesand thus inflammation. Gastric inflammation, associatedwith bacterial infection, results in an increase of IFN~/-expressingT lymphocytes that is indicative of a cell-mediated (Thl) immune response. In addition, infection causes changes in neuroendocdnecell populations including gastrin (G)- and somatostatin(SOM)-secretingD-cells. Proinflammatorycytokines such as IFN'y and TNF~stimulate the release of gastrin and SOM from isolated G- and D-cells respectivelyin vitro. However, in vivo immunoregulation of SOM and gastrin has not been studied in the gastric mucosa. Methods: PBS or IFN~,were infused into C57BL/6 mice at a dose of 250 IU/kg (15 IU/mouse/day)for 2, 5 and 7 days. Plasmagastrin concentrations and tissue SUM content were measured by radioimmunoassay.Tissue sections that include both the fundic and antral regions were fixed for histology and morphometric analysis after immunostained for SOM and gastdn. Results: Within 2 days of infusion, tissue SOM content increasedabovethe PBScontrols in both the fundus (9-fold) and antrum (3-fold) and correlated with an increase in the number of D-cells. There was a reciprocal 2-fold decrease in plasma gastrin concentration with no change in G cells. After a 5 and 7 day infusion, tissue SOM content decreasedby 50% in both fundus and antrum; whereas the number ol G-cells and plasma gastdn concentration increased3-fold. Histologic evaluation revealedthe recruitment of lympbocytes and leukocytes in the gastric mucoea after 2, 5 and 7 days of infusion. Further, staining also revealedthe presenceof SOM-secratingmacrophagesat all time points. Conclusions: In the gastric mucoea, SOM and gastrin are regulated by IFN-y. It is likely that the release of IFN~/, consistent with a predominant Thl response contributes to the neuroendocrine changes observed during Helicobacter pylori infection.

Background.lnflammetion in generaland in particular in the gastrointestinaltract is commonly accompanied by thrombocytosis. Thrombocytosis of inflammation is thought to be related to increasedintedeukin-6 (IL-6) levels. Baselineplatelet production is dependenton thrombopoietin (TPO). Methods.Plasmalevels of TPO were determined by ELISA in samples from a human IL-6 clinical trial. TPO mRNA expression,TPO plasma levels, and platelet counts were determined in 057BL/1B mice receiving murine IL-6 intraperitoneally. Furthermore, C57BIJ 10 mice were additionally administered neutralizinganti-TPOantibody. Results.lL-6 treatment results in elevated TPO plasma levels in patients and C57BL/10 mice. IL-6 administration leads to upregulationof hepaticTPO mRNA expressionand elevatedplateletcounts in C57BL/ 10 mice. Neutralizationof TPO results in abrogation of the increase in platelet count in IL6-treated animals. Discussion.Ourdata indicate that thombocytosis in inflammation might be related to upregulation of TPO by the inflammatory mediator IL-6. We propose a novel regulatory pathwayfor megakaryopoiesisin inflammation, which differs from the situation in health, where plasma TPO levels are regulated mainly by platelets themselves.

3912 Recombinant Human Growth Hormone Downregulated mRNA Expression of Acute Phase Proteins Induced by IL-1,8 and IL-6 in Hep G2 Cell Line Xiaowu Wu, David N. Herndon, Steven E. Wolf, Shriners Hospitals for Children, Galveston, TX

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BACKGROUND:Previous in vivo studies showed that recombinant human growth hormone (rhGH) exerted inhibitory effects on gene expression of type I acute phase proteins lAPPs). In this study, we investigatedthe effects of rhGH on mRNA expression of APPs induced by recombinant human IL-1/~ and IL-6 (rhlL-1/~ and rhlL-6) in Hep G2 cells. METHODS:Hep G2 cells were grown to confluence in 6-well plates, and then treated with rhlL-1/~ (2ng/ml) and rhlL-6 (2Ong/ml) alone or combined with rhGH (2,~g/ml) for 0.25, 0.5,1, 2, 4,12 hours. Total RNA was extracted from the ceils after treatment, and levels of mRNA expression for ~-l-acid glycoprotein (Type I APP) and ~l-antitrypsin (Type II APP) were measured semiquantitatively by RT-PCR. The products of RT-PCRwere ran on the 1.5% agarose gel, and analyzed by Digital Imaging Analysis System. RESULTS: Expression of mRNA for ~-l-acid glycoprotein lAGP) was increasedand reachedpeak levelsat 1 hour after rhlL-1/~ stimulation, while mRNA expression for
Gastrin and Cyclooxygenaoe-2 (COX-2) Expression in Helicebacter pylori (Hp) Infected Gastric Ulceration Stanislaw J. Konturek, Dept of Physiology, Univ Sch of Medicine, Cracow Poland; Peter C. Konturek, Dept of Medicine, Univ of Erlangen-Nuramberg,Erlangen Germany; Wladyslaw Bielanski, Aiekcandra Duda, Monika Zuchowicz~Elzbieta Karczewska,Oept of Physiology, Univ Sch of Medicine, Cracow Poland; Eckhart G. Hahn, Dept of Medicine, Univ of Erlangen-Nuremberg, EdangenGermany Background: Gastric ulcerations (GU) have been linked to Hp infection that is accompanied by hypergastrinemiaand enhanced generation of prostaglandins (PG), both involved in the pathogenesisof peptic ulcer but no study was undertakento assessthe relationship between the Hp infection and coexpression of gastrin and COX-es, the rate limiting enzymes in the PG production. Aims: This study was designed 1. to determinethe gene expressionof gastrin and its receptors (CCKe-R) at the margin of GU and in gastric mucosa before and after successful Hp eradication; 2. to assess the plasma levels and gastric luminal contents of gastrin before and after Hp eradication and 3. to examinethe mRNA and protein expression of COX-1 and COX-2 as well as the PGE2generation in ulcer margin and gastric mucosa before and after the Hp eradication. Material and Methods: The trial material included 20 GU patient~ and 40 controls. Gene expressions of gaetdn, CCKB-R, COX-1 and CUX-2 were examined using RT-PCRwith/~actin as a reference and employing Western blot for COX-2 expression, while gastrin and PGE2were measured by RIA. All GUs were located within the antral mucosa. Results: The seroprevalence of Hp and CagA was significantly higher in GU(85%) than in controls (62.5%). Both gastdn and CCKe-RmRNA were detected by RTPCR in ulcer margin and gastrin mRNA was overexprassedin remaining antral mucosa, while CCKB-RmRNAwas overaxpressedin fundic mucosa. Similarly COX-2 mRNA and protein were found in margin of GU and in the Hp infected antral and fundic mucosa but not in the mucosa of Hp eradicated patients in whom ulcers completely healed and gastrin was detected only in antrum, CCI~-R only in corpus, while COX-1 - both in antrum and corpus. Hp positive GUs showed about 2-3 times higher levels of plasma immunoreactive gastrin and PGEzand about 50% higher luminal gastrin contents than the Hp negative controls and this increased plasma and luminal gastdn and tissue PGE2generation were normalized following the Hp eradication. A significant fall in gastrin and CCKB-RmRNA expressionwas noticed six weeks after Hp eradication in gastric antral and fundic mucosa,while COX-2 mRNA completely disappearedafter this treatment. Conclusions: 1) Hp infected GU margin coexpressesgastdn, its receptorsand CUX-2;2. Hp infection may contribute to GU via increased releaseof gastrin and PGE2,and 3. Gastrin produced in Hp infected antral mucosa seems to play a crucial role in the induction of COX-2 and PG generation at GU margin.

Fig. 2 GHand IL-lp Stimulation

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3913 Gastrin Induces IL-8 Expression in Gastric Epithelial Cells Through the Activation of NF-kappaB and AP-1 Yoshiji Miyazaki,Sbintaro Hiraoka, Shinji Kitamura, Miyuki Toyota, Tatsuya Kiyohara, Yutaka Nagasawa,Yasuhisa Shinomura, Yuji Matsuzawa,GraduateSch of Medicine, Osaka Univ, OsakaJapan Background and Aims: Hypergastrinemiais frequently observed in individuals with a chronic Helicobacterpylori infection. However,the pathophysiologicalsignificance of hypergastrinemia in gastric mucosal inflammation is unclear. The present study was designed to determine if gastrin induces the expression of IL-8 in human and rat gastric epithelial cells. Methods: Human and rat gastric epithelial cells, transfected with gastdn receptor, were stimulated with gastrin. The expression of IL-8 mRNA and release of its protein were then determined by Northern blot analysis and an enzyme-linked immunosorbent assay, respectively. Results: Gastrin induced the expressionof IL-8 transcripts and protein and this effect was synergistic with IL-1/~or TNF-e. A luciferase assay using IL-8 promoter genes showed that NF-KB is absolutely required and AP-1 is partly required for the maximum induction of IL-6 by gastrin. An electrophoraUcmobility shift assay revealedthat gastrin is capable of activating both NFKB and AP-1, and that the inhibition of NF-KB abrogated the gastdn induction of IL-B. The inhibition of either protein kinase C or mitogen-activated protein kinase kinase partially suppressed gastdn-induced IL-8 expression.Conclusions: These results suggest that gaetrin is capableof upregulatingI L-8 expressionin gastric epithelialcells and thereforemay contribute to the progression of the inflammatory process in the stomach.

3916 Changes in Gastric Mucosal Perexisome Praliferator-Activated Receptor ,y (PPAR~,) Expression After Helicobacler pylori Eradication Atsushi Nakajima, Emi Oeawa, Hisahiko Sekihara, Yokohama City Univ, YokohamaJapan; Sachiyo Nomura, Michio Kaminishi, Nobuyuki Metsuhashi, Univ of Tokyo, Tokyo Japan PURPOSE: Expression of peroxisome proliferator-activated receptor .y (PPAR-,/) has been demonstrated in some gastric cancer cells. Although PPAR~/ligandsare reportedto suppress cell proliferation in gastric cancer cell lines, the precise role of PPAR~,expressedin the gastric cancer cells is unknown. Since chronic atrophic gastritis, hyperplastic polyp, gastric adenoma and gastric cancer are all known to be associated with chronic Helicobacter pylori (Hp) infection, we investigatedgastric mucosalexpressionof PPAR~/beforeand after Hperedication to elucidatethe role of the receptorin those settings. METHODS:Hp eradicationwas performed in 9 patientswith chronic atrophic gastritis, 5 patients with gastric adenomas,and 10 patients with hyperplasUcpolyps. All patients were positive for Hp. Biopsy samples of the lesions obtained before and after the Hp eradication were subjected to immunohistochemistry for PPAR~t.All hyperplastic polyps regressedafter Hp eradication, while adenomasshowed little or no regression. RESULTS:Prior to Hp eradication,all samples of chronic atrophic gastritis and hyperplaeticpolyps were positive for PPAR-t, especially in infiltrating inflammatory cells.

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Positive staining was also observed in the nucleus and the cytoplasm of the epithelial cells. mainly in the basolateral aspect. Hp eradication in these patients abrogated the PPAR~t expression, Before Hp eradication, all samples of gastric adenomas showed strong staining for PPAR~,in the cytoplasm, especially in the apical aspect. Nuclear staining was observed in 4 out of 5 cases. Moderate staining was also observed in infiltrating inflammatory cells. After Hperadication, however, epithelial PPAR,~expressionwas not altered, although PPAR~, expression in inflifating inflammatory cells was decreased.CONCLUSION:Downregulationof PPAR~, after Hp eradication for chronic atrophic gastritis and hyperplastic polyp suggests that the PPAR~/expressedin these conditions plays a role in modifying inflammation. On the other hand, unchangedepithelial PPAR-/expressionafter Hperadicationfor gastric adenomas suggests a role of PPAR~,in tumorigenesis or cell differentiation. 3917 Different Effects of Chronic Helicabactor pylori Infection on Padofal and ECL Cells Between Wild Type and Gastrin-Doficiest Mice Duan Chen, Norwegian Univ of Science and Technology, Trondhaim Norway; Lennart FriisHansen, Univ of Copenhagen,CopenhagenDenmark; Xin Wang, Univ of Lund, Luod Sweden; Chun-Mei Zhao, Helge L. Waldum, Jostein Halgunset, NTNU,Trondheim Norway; Torkel Wadstrom, Univ of Lund, Lund Sweden BACKGROUND& AIM: Helicobactorpylori infection increasesgastric acid secretion in patients with duodenal ulcers but diminishes acid output in patients with gastric cancer and their relatives. Gastrin plays an important role in the regulation of function and differentiation/ growth of parietal and ECL cells. In the present study, we examined the effects of chronic H. pylori infection on the acid secretion and parietal and ECL cells in witd-tybe (WT) vs gastrin-deficient (knockout, KO) mice. METHODS:Both WT and KO mice were infected with H. pylori (CagApositive type I, 119/95) for 9 months. The infection was verified by immunoblot and culture. The acid output was measured in fasted mice by pyloric ligation model and expressed as nEq H÷. Parietal and ECL cells were examined by immunocytocbemistry (with antisera to cx-subunitof H+/K+-ATPaseor histamine and vesicle monoamine transporter-2) and quantitative electron microscopy. RESULTS:In WT mice, the acid output was impaired by H. pylori infection (14.6 _+ 2.2 vs 2.6 __. 0.6, p< 0.001), which was associated with a reduced proportion of secreting parietal cells (displaying secretory canaliculi). The parietal cell number was unaffected, but ECL cell number was increased 2-fold by the infection. Gastrin KO mice displayed ~ 30 % reduction in the number of parietal cells which was associated with a low acid secretion. However, the infection increasedthe acid output (1.0 _+ 0.2 vs 2.8 -+ 0.7, p
3919 Pharmacological Efficacy in Gastric Variceal Rebleeding and Survival: Including Multivariate Analysis Chun-Ying Wu, Hong-Zen Yeh, Gran-Hum Chen, Taichong Veterans Gen Hosp, Taichung Taiwan Roc Background: Therapy with/~-blocker and nitrate has been reported to improve survival of patients with bleeding esophagealvarices and to decrease esophagealrebleeding.However, there is little information availableconcerning the efficacy of these medicationson rebleeding risk and survival in gastric variceal bleeding after initial hemostasis. Methods: We conducted an open trial to realizethe roles of ~-blocker and nitrate in the long-term outcome of bleeding gastric varices.Eighty-threepatientswere included and evaluatedon the basis of age, gender, gastric variceal size, associated esophagealvariceal size, Child-Pugh classification, existence of hepatomaand podal vein thrombosis, ~blocker or nitrate therapy, and follow up histoacryl injection. Survival analysis and multivariate analysis with the Cox proportional hazardsmodel were performed to evaluatethe independentrisk factors. Results: Larger gastric variceal size has been shown to be the only risk factor for rebleeding (adjusted odds ratio, 4.50; 95% CI: 1.30-15.59). ~blocker or nitrate did not significantly reducethe incidence of rebleeding(adjusted odds ratio, 0.37; 95% CI: 0.08-1.66), but improved the overall survival after initial hamostasis by Kaplan-Meiermethod (PlO0/min), history of esophagealvarices, systolic BP 200mmHg) whilst taking negativechronotropes; intermediate risk: age >60 years, hemoglobin <11g/all, co-morbidity, melena, alcohol (excessive recent or chronic), NSAIDs, previous GI bleed or peptic ulceration, abnormal LFrs, postural hypotension (>lOmmHg), systolic BP >20mmHg below patient's normal; low risk: none of the aforementioned factors). RESULTS: 1349 episodes occurred during the study period (incidence of 100 per 100,000 per year). 2-week mortality was 84 (6.2%) overall with 66 (11.8%) of 561 high risk, 18 (2.5%) of 717 intermediaterisk and 0 (0%) of 71 low risk. This was significantly higher in the high nsk group than intermediate (p
3918 The Endoloopversus the Saeed Six Shooter RuhbarbandLigator in the Troofmest of Bleeding Esophageal Varices Maria Margaret B. Luspo-Ouiblat,Venancio I. Gloria, Virgilio P. Banez,MANILA DOCTORS Hosp, Manila Philippines The osefullnessof the endoloop in the control of esophagealvaricealbleed in cirrhotic patients still has to be evaluatedin well-controlled trials. Objectives:To compare the endoloop versus the saeed six-shooter rubber band ligator in terms of the control of active bleeding during the procedure,incidenceof rebleeding,duration of time before rebleeding,number of sessions before completeobliteration of varices,complications, number of hospitalizationdays, number of units of blood for transfusion, mortality secondaryto esophagealveitceal bleed,enitoscopists's easeof procedure,and cost. Methodology:This is a pioneeringprospectivecase-controlled trial in the Philippines. All consecutive, adult cirrhotic patients who qualified, with a history of variceal bleeding, admitted or seen on out-patient basis at 4 tertiary institutions were included. The diagnosisof cirrhosis was arrivedat through a combination of clinical, biochemical, and ultrasonographic findings. Out of the 37 patients, 7 were lost to follow-up. Of the remaining30 subjects,Group I (N = 13) underwentendoloopwhile Group II (N = 17) underwent rubber band ligation. Every 2 weeks thereafter, endoloop or RBL was performed till complete obliteration of varices. Group I and Group II were followed up prospectively for a mean duration of 215.35 _+ 154.39 days and 176.35 + 156.123 days, respectively.The end of the study was marked by rebleedingor mortality secondaryto variceal bleed as confirmed by a repeat endoscopy. Results: There was no statistically significant difference between Group I and Group II in terms of the control of active bleeding during the procedure, number of sessions for complete eradication, number of hospitalizationdays, number of units of blood for transfusion, complications, mortality secondaryto variceal bleed, and endoscopists ease of procedure. However,there was a statistically significant difference betweenthe two groups in terms of incidence of rebleeding (20% vs 40.9% respectively; p = 0.039); duration before rebleeding (39 _+ 39.5 days vs 12.9 _+ 30.2 days respectively;p = 0.014); and cost (P 3,318 _+ 603 vs P 11,000, respectively;p = 0.000). Conclusion: Patientstreated with the endoloop spend less, have less incidenceof rebleeding,with rebleedingoccuring after a longer duration of time comparedto the patients treated with the saeedsix shooter robber hand ligator. This is a preliminary report and the results involvinga largersamplesizeshall also later be presented.

3921 Is Iron Deficiency Investigated Appropriately? Alexandra Di Mambro, Trevor N. Brooklyn, Bristol Royal Infirmary, Bristol United Kingdom; Nell Haslam, Bury Gen Hosp, Bury United Kingdom BACKGROUND:Gastrointestinal disease often presents as iron deficiency anemia (IDA) and The British Societyof Gastroenterologists(BSG) has issuednationalguidelinesto aid investigation. We not infrequently encounter iron deficiency (ID) without anemia where there are no clear guidelines. We hypothesizedthat although the same pathologies are likely to underlie these patients, they were less likely to be investigated. AIM: To audit the investigation and underlying pathologies of ID and IDA DESIGN: Retrospectivecase-note audit of confirmed ID SUBJECTS: Patients 45 years and older, with ferritin levels below the normal range (2O/~/dl) METHOD:Caseswere identified from the hematologycomputer. Using a proforma, gastrointestinal investigations (upper and lower gastrointestinal endoscopy, barium studies, and serology for coeliacdisease),the specialtyof the investigatingphysician,and the diagnosis were recorded.The two groups were compared using odds ratios (OR) with 95% confidence intervals (CI). RESULTS:153 cases (102 female, 51 male) were identified, 69 with IDA and 84 with ID. The number of investigationsperformed, the number of patients not investigated, and the number in whom a diagnosis was established are tabulated below. (Table) ID is investigated less thoroughly than IDA, OR 2.07 (CI 1,08 to 3.97) and hence the odds of establishing a diagnosis differ significantly, OR 0.47 (CI 0.24 to 0.92). Of the 153 patients, 26 were underthe care of a gastroenterologyteam.24 (92%) of these patientswere investigated according to the guidelines for that of IDA. Of the remaining 127 patients only 64 (43%) were investigatedfor a cause. These differences are highly significant, OR 0.085 (CI 0.0019 to 0.37). CONCLUSION:IDis less thoroughly investigatedthan IDA, although we feel it should be investigated according to the BSG guidelines on IDA. The BSG guidelines do not appear to have disseminated beyond the gastroenterology community. As the majority of IDA is

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