Gastroesophageal reflux disease in monozygotic and dizygotic twins

246

Results: CD45RBh'0"CD4÷ cells from CD28-z- mice failed to induce colitis in SCID recipients. Of note, cotranster of CD25÷CD4÷ cells from CD28-z- mice effectively suppressed colitis induced by WT CD45RB~'~"cells in SCID recipients. Transmural inflammation and epithelial hyperplasiawere ellectively inhibited. Histological scores were significantly decreasedcompared with those in WT CD45RB~0"cell-reconstituted mice (p < 0.01). Lamina propria CD4+ T cells, when stimulated with Con A, also secreted lower levels of IL-2 and IFN-,ycompared to those from WT CD45RB"~"CD4÷ cell-reconstituted mice. In vivo treatment with anti-CTLA4 blocked the regulatory functions of CD25+CD4÷ cells from CD28 ~- mice. Conclusions: These data suggest that the CD28/CTLA-4-B7costimulatory pathway plays a different role in the induction of effector T cells responsible for intestinal mucosal inflammation and in the activation of regulatory T cells. B7-CD28 interaction plays a dominant role for pathogenic CD45RBh'ghcell activation, whereas B7-CTLA-4 costimulation is involved in CD25+CO4÷ cell activity. Thus, CTLA-4signaling plays a key rote in maintaining mucosallymphocytetolerance.

VLA-1 ImmunoneutralizaUon Protects Against Colon Damage in a Mouse Model of Colitis Stefano Fiorucci, Andrea Mencarelii, Silvia Baroni, Univ of Perugia, Perugia Italy; Antonin R. De Fougerolles,Victor E. Koteliansky,Biogen Inc, Cambridge,MA; Eleonora Distrutti, Antonio Morelli, Univ of Perugia, Perugia Italy; GiuseppeCirino, Uuiv of Naples, Naples Italy Background.Adhesiveinteractions play an important role in inflammatory bowel disease(IBD) by promoting leukocyteattachmentand extravasationfrom thevasculaturs into the peripheral tissues. However,the importanceof adhesion molecules within the extracellular matrix rich environment of peripheral tissues, in which cells must migrate and be activated, in this condition has not been well explored. VLA-1 is involved in targeting T lymphocytesat site of inflammation. Aim. To evaluatethe pathogeneticrole of VLA-1 and therapeutic effect of VLA1 immune-neutralizationon trinitrohenzene sulfonic acid(TNBS)-inducedcolitis in mice, a Thelper (Thl) cell-mediated model of chronic inflammation that shows a number of clinical, histological and immunological analogieswith Crohn s disease.Methods. Colitis was induced by injecting mice with 100/zl TNBS. Animals were treated by daily s.c. injection of 0.1, 0.3 or 1 mg/kg anti-VLA-1 monoclonal antibody (Biogen Inc., Cambridge, MA) or 1 mg/kg control mAb. Clinical (weight curve), histological (mucosal lymphocyte infiltration, colon wall thickness) and immunological studies (plasma and mecosal concentrationof TNFa, IFN,.tand IL-2) were evaluated 7 after TNBS administration. Production of Thl-like cytokines (IFN~/) from isolated lamina propria (LP} T cells was analyzed.Results.Administration of anti-VLA1 mAh to the TNBS-trsated mice at the concentration of 0.3 and 1 mg/kg led to a significant improvement in the clinical, histopathological and immunological aspects of the disease (P
244 Blocking ICOS in Combination with CD28 Impairs the Proper Activation of Aggressor Thl Cells but Is Not Necessary for Suppression by Regulatory T Cells. Ype P. De long, Beth Israel DeaconessMedical Ctr, Boston, MA; Anthony J. Coyle, Millenium Pharmaceuticals,Cambridge,MA; Svend T. Rietdijk, Beth Israel Deaconess Medical Ctr, Boston, MA; Emiko Mizoguchi, Atol K. Bhan, MA Gen Hosp, Boston, MA; Jose-Carlos Gutierrez-Ramos, Millenium Pharmaceuticals,Cambridge, MA; Cox Terhorst, Beth Israel DeaconessMedical Ctr, Boston, MA Background. Many autoimmune diseases, including inflammatory bowel disease, are likely due to an imbalancebetweenT helper 1 (Thl) and regulatory helper T (Treg) cells. All T cells are dependenton proper co-stimulstory signals apart from the main signal through the T cell antigen receptor. In these studies, the necessityfor two co-stimulatory signals, namely CD28 and Inducible Co-Stimulatory molecule (ICOS), on both Thl and Treg was investigated. Method~. The CD45Rhhi transfer model, which shares some features with Crohn's disease, was used. By taking cells from wild type or CD28 deficient mice, and by separating naive or Treg donor T cells, we assessedthe differential needs for CD28 co-stimulation on these cell types. In addition, blocking reagentsdirected against ICOSwere used. Diseasewas assessed by the percentweight loss, clinical diseaseactivity and pathological examinationof the colon. Resul~ Blocking ICOS on wild type Thl cells did not affect the outcome of disease, and CD28 deficient T cells induced only 33% less sever colitis, which did not reach statistical significance. However, blocking ICOSin the absence of CD28 resulted in a 61% decrease in colitis (p
247 Blocking IL-g/IL-6 Receptor Signaling Inhibits Leukocyte Recruitment and Promotes T Cell Apoptesis in Murine Colitis Hiroaki Ito, Mitsunari Yamamoto, Osaka Univ Graduate Sch of Medicine, Suita Japan; Tadamitsu Kishimoto, Osaka Univ, Sulfa Japan Background/Aims:Enhancedexpressionof adhesionmoleculesis important in the development of inflammation, and impaired T cell apoptosis may lead to its chronicity, both of which could be attributed to IL-6. Interestingly,IL-6/soluhle IL-6 receptor (slL-6R) complexescan stimulate even receptor-negativecells such as vascular endothelium. To test the therapeutic potential of IL-6 signaling blockade for Crohn's disease, we introduced anti-lL-6R mAb to a murine model of colitis. Methods: Colitis was induced in CB17-scid mice transferred CD45RB~ CO4* T cells from Balb/c mice. Anti-lL-6R mAb or control rat IgG was administered intraperitoneally after T cell transfer, followed by weekly injection. Body weight was monitored, colons were removed at 8 weeks, and colitis was assessedhistologically. Cytoklneexpressionwas determined by RT-PCR.Adhesion molecule expression was analyzed by immunohistochemistry, apoptotic cells were demonstrated by TUNEL method. Results: Mice treated with anti-lL-6R mAb showed normal growth while controls lost weight. Colitis was improved histologically. IFN--/, TNF-a, and IL-I~mRNAs were markedly reduced, but no difference was observed in the expression of IL-4, IL-IO, and TGF-/3. ICAM-1 and VCAM-1 expression in the colonic vascular endothelium was markedly suppressed by the treatment, whereas no significant difference was seen in MAdCAM-I. TUNEL+ apoptotic cells were very sparse despite massive infiltration of CD4÷ T cells in colitic mice, while increasedapoptosis was seen in mAb treated mice with apparently reduced number of CD4÷ T cells. Summary/Conclusions: Anti-lL-6 receptor mAb abrogated colitis in mice. It effectively blocked the expression of adhesion molecules thereby stopped leukocyte recruitment, and increasedapoptosis of T cells, which, otherwise would have promoted chronic inflammation. These results strongly suggest the therapeutic potential of anti-lL-6R mAb for human Crohn's disease.

245 Tronsgenic Mice Over-Expressing the B7 Related Protein-1 (B7RP-1) Develop an Intestinal Pathology Similar to Human Crohn's Disease A New Mouse Model of inflammatory Bowel Disease Fergus R. Byrne, Dept of Pathology, Aragon Inc, Thousand Oaks, CA; John S. Whodskey, Dept of Inflammation, Amgen Inc, Thousand Oaks, CA; Ulla Sarmiento, Giorgio Senaldi, Dept of Pathology,Amgen Inc, Thousand Oaks, CA; Ming Zhang, Kevin Gaida, Dept of Inflammation, Amgen Inc, Thousand Oaks, CA; Dimitry Danilenko, Dept of Pathology, Amgen Inc, Thousand Oaks, CA; Steven K. Yoshinaga, Dept of Inflammation, Amgen Inc, Thousand Oaks, CA ICOS(Inducible Co-Stimulator)and B7RP-1 (B7 RelatedProtein-I) representa newlydescdhed reccptor-ligand pair involved in co-stimulation of T cells by Antigen Presenting Cells (APCs). ICOS is expressedon activated and memory T cells and B7RP-1 is expressedon B cells and macrophages.Although structurally related,this pathway is independentof the interaction of CO28 with B7.1 and B7.2. In this study, transgenic mice over-expressing B7RP-1 fused to the Fc region of a human IgG1 were generated.An apolipoprotein E expression vector was used to direct over-expressionto the liver of the animals. Gross developmentof the transgenic mice was essentially normal but approximately33% of the transgenic mice were cachexicat the age of 5-6 months and most of these animals developedrectal prolapse by 10-12 months. Necropsy revealed proportionately larger spleens, small intestines and colons compared to non-transgenic littermate controls. Mucosal thickening was observed in a discontiguous manner along the length of the colon. Histologically, the most severe inflammatory changes were in the proximal and distal colon with milder changes in the small intestine. Colonictissue was markedlythickenedwith fissuring ulceration,transmural inflammation and hypertrophyof the colonic mucosa.The small intestinehad mild to markedmucosal hypertrophy.Hematologically, the sick transgenic mice exhibited increased levels of white blood cells, particularly neutrophils, and low levels of hemoglobin, Histologically, the penetrance of disease was approximately50% and this variability is likely relatedto differing levels of expression of the transgene as well as variations in the enteric microflora of the mice: Interestingly, penetrance appearedto be slightly higher in female animals over males. Extra-intestinalmanifestations of inflammatory processes such as reactive lymphoid hyperplasia in the spleen and lymph nodes was also observed in the affected animals. Severalof the transgenic animals exhibited hyperglobulinemia with increased levels of circulating IgE and this proportion appears to increase with age. These data suggest that the B7RP-1 transgenic mice exhibit a phenotype resembling human Crohn s Diseaseand that B7RP1 may play a role in the pathogenesisof IBD.

248

Gastroesophageal Reflux Disease in Monozygotic and OizygoUcTwins Alan J. Cameron, Mayo Clin, Rochester, MN; Jesper Lageroren, Christer Henriksson, Olof Nyrsn, Karolinska Inst, Stockholm Sweden; G Richard Locke lii, Daniel J. Schaid, Mayo Clin, Rochester, MN; Nancy L. Pedersen,Karolinska lust, Stockholm Sweden Background:A genetic component in the etiology of gastmesophagealreflux disease(GERD) has been proposed, but the results in two previous family studies were conflicting. Aim: To assess genetic factors by comparing the concordance for GERD in monozygotic (MZ: all genes shared) versus dizygotic (DZ: average50% of genes shared) twin pairs. Methods: Data were collected March 1998 - November 2000 as part of the Screening Acmes the Lifespan Twin Study.Twins age 55 and older in the population-basedSwedishTwin Registrywere invited to participate in a computer-assistedtelephone interview, conducted by trained professional interviewers. The reflux questions used (in Swedish) were based on previous work from our two institutions. GERDwas defined as the occurrence, at least weekly, of either a)heartburn, with antacid relief or radiation toward the neck; or b) acid regurgitation. Zygosity was determined by questions found 98% correct compared to analysis of 10 DNA polymorphisms. Results: 23,592 twins were interviewed, of whom 15.4% had GERD symptoms. GERDwas reported by 15.7% of twins age 55-64 and 15.1% age 65 and above, and by 14.6% of males and 15.9% of females. 15,784 twins were in informative pairs (Table). There were 2040 MZ pairs and 3029 same-sex DZ pairs. In both sexes, MZ pairs were concordant for GERD significantly more often than DZ pairs (Table). Conclusion: In the general population age 55

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and older, the influence of heredity in the etiology of GERD is about 30%. Supported by

251

grants from Astra Zeneca, NIH grant AG 08724 and the Swedish Council for the Planning and Coordinationof Research.

Male MZ Male DZ Female MZ Female DZ gnlike..an~DZ

Total pairs

Discordant pairs

860 1293 1180 1736 2823

172 317 264 427 730

Probandwh~e concordancer a t e 601232(26%) 78/395(20%) 120/384(31%) 112/539(21%) 160/890(18%)

Helicobacter pylori as a Protective Factor for Guctreesophageal Reflux and its Complications Hector Cardona, Oscar Gutierrez, William Otero, C Prieto, Univ Nacional de Colombia, Bogota Colombia

Tatrschodc correlation

Background: It remains unclear whether H.pylofi infection protects against gastroesophageal reflux disease or its complications such as Barrett s esophagus. Aim: To investigate the relationship betweengastric Helicohacterpylori infection and the presenceof gastroesophageal reflux (GER). Methods: During a 2 year period 1,400 consecutive outpatients referred for an upper gastrointestinal endoscopy were evaluated. Each patient completed a questionnaire related to age, gender and symptoms of GER including duration and frequency per week.12 biopsies were taken : 2 from the distal esophagus, 3 from the cardia, 3 from the body, 3 from the antrum for histological analysis and one for a rapid ureasetest. Caseswith histological absence of H. pylod (HpNo) were compared with infected controls (HpYes). Results: 348 patient were included in the study, mean age 52.4 + 17 y: 116 HpNO and 232 HpYes. There were no significant differences between the groups for age or sex. GER symptoms were more h'equent among HpNo (38.8%) compared to HpYes (19%) (p
0.30 (0.15-0.44) 0,11 (-0.03-0.25) 0.35(0.23-0.45) 0.13 (0.03-0.24) 0.05 (-0.02-0.14)

249 A Beneficial Effect of H. pylori Eradication in Reflux Patients in lhe Community. Robert C. Stuart, Carol F. Craig, Christine Morran, Glasgow Royal Infirmary, Glasgow United Kingdom; Harry Bums, Greater Glasgow Health Board, Glasgow United Kingdom; Ken Harden, Andrew Power, Glasgow Royal Infirmary, Glasgow United Kingdom; David Walsh, Greater Glasgow Health Board, Glasgow United Kingdom; A. John Morris, Glasgow Royal Infirmary, Glasgow United Kingdom Theoretical concerns have been raised about H pylorieradicationin patients with reflux disease. The effect of H py/or/eradication in patients in general practice with reflux is not known. These patients tend to present with heartburn as their main symptom or with oesophsgifis diagnosed on endoscopy. Aim: To assess an H pylori test and treat strategy in patients on maintenancetherapy with either uninvestigated heartburn or endoscopy positive oesophagifis. This was undertaken within the context of a community nurse led clinic during the ACID1 study. Methods: Two patient groups were studied: 1) Heartburn with no previous investigation (n = 204) & 2) Endoscopic oesuphagitis (n = 267). We defined maintenance therapy as ->3 prescriptions / year for PPI and/or H2RAtherapy. Patients in group 1 all reported heartburn as their main presenting symptom. H py/ori status was determined using ~3C urea breath testing. Symptoms were assessed before and 6 months after treatment, using the Glasgow Dyspepsia Severity Score (GDSS). A significant change in the GDSS was defined as a change of ->3 points. Results: Changes in GDSS and prescribing are shown in the table below. Conclusion: H pylori eradication in the community is of benefit in patients on therapy with uninvestigatedheartburn. In addition H pylori eradicationdoes not adversely affect patients with endoscopic oesophagitis and is beneficial in reducing prescribing costs.

252 Natural HIctofy and Acid Secretion of Reflux Esophagitis (RE) After H. pylori (HF) Eradication Tomoyuki Koike, Shuichi Ohara, Hitoshi Sekine, Katsunori lijima, Yasuhiko Abe, Masashi Kawamura, Yasushi Kitagewa, Katsuaki Kato, Tooru Shimosegewa, Tohoku Univ Graduate Sch of Medicine,SeodaiJapan Background and Aims: We havereported that prevalenceof HP infection and grade of atrophic gastritis were significantly lower, while acid secretion was significantly greater in the patients with RE than those without RE. (AJG 1999;94: 3468-72, Gastroentero11999;116: A217). We have also reported that increased gastric acid secretion after HP eradication may explain a reason for developing RE (Gastroenterol 1998;114: A183). Therefore, HP is considered to be a protective factor for the devel prospectively followed for more than 7 months after the clearance of HP. In addition, the natural history and acid secretion of these patients were compared with those of 105 patients with endoscopically diagnosed common RE and 105 control subjects who had no localized lesions in the upper gastrointestinal tract. Gastric acid secretion was assessed by endoscopic gestrin test (EGT) (AJG 1998;93: 2113-8). Results: RE developed in 11 patients (10.5 %) when examined at 7 months after the eradication therapy. Before HP eradication, there was no significant difference in the EGT value between the pa~ents who developedRE and the controls (2.27_+1.66 vs 1.86_+1.77 mEq/lOmin, P = 0.47 ). After HP eradication, the EGT value increased in the patients who developed RE (4.45_+1.87), and the value at 7 months was significantly higher than that of the controls (P < 0.0001). Although EGT value of the patients who developed RE after HP eradication was not significantly different from that of the endoscopically established common RE patients (3.65+2.17)(P = 0.24). the grade of RE in the former was judged to be relatively milder than that of the latter (A or B by the LA classification), and 66.7% of the former patients continued to have RE during the follow-up period of more than 7 months (13-49 months after eradication). Conclusion : The present data suggest that acid secretion of RE developed after HP eradication is almost the same level as that of common RE. Moreover, most RE which developed after HP eradication took a chronic course similar to that of common RE.

Effectsof Hperadicationon GDSSand drugtherapy. ..H.Ostatus Changein GDSS improved>3 deteriorated_>3 unchanged<3 Drug therapy t year post intervention % offtherapy % changedto PPI

heartburn eradicated negative

eredicted

ce~zOies negative

32.4% 8.8% 58.8%

21.7% 3.6% 74.4%

28.8% 4.5% 66.7%

31.1% 3.9% 65.0%

35.2%1" 4.6%

8.7%

14.6%

25.7%1" 4.1%

14.4% 4.1%

1"P<0.05,~z test.

250

H. pylori (Hp), Intragastric (IG) pH and Esophageal Acid Exposure (EAE) - The GERD Triangle. Virender K. Sharma, Univ of Arkansas for Medical Science, Little Rock, AR; Colin W. Howden, Northwestern Univ, Chicago, IL Background: Recent interest has focused on a possible preventive role of Hp in GERD. The inter-relation between Hp, IG pH and EAE is poorly understood. Aim: To assess the role of Hp on fG pH, EAE and response to antisecreturytherapy in patients with erosive esophagifis (EE). Methods: 41 EE patients (mean age = 46y; sd = 13; 54% men; 90% white) underwent dual probe pH testing. The distal probe was positioned in the gastric body / antrum and the proximal probe 5 cm above the GE junction. Biopsies for Hp were obtained from antrum and fundus. Repeat pH testing on lansoprazole 3grog qd was done at 1 (n=12) and 12 months (n = 9). Results: Gastric acid suppression with lansoprazolewas significantly superior in Hp+ than in Hp- subjects (mean IG pH = 5.8 and 3.7, P 4 for 85% and 43%, P 4 = 7.5 vs 6.9, respectively, P=O.85; # of GER episodes > 5 min = 2.3 vs 3.1, P=O.67). Change in EAE was no different between Hp+ and Hp subjects after 1 month of therapy (8 % time esophageal pH > 4 = 6.6 vs 4.0, respectively; P=0.58; 8 # of GER episodes > 5 min = 3.7 vs 2.3, P=0.54) or at 12 months post-therapy. Conclusions: Hp+ GERD patients are significantly older and have higher IG pH than those who are Hp-. However, they have a greater degree of EAE. This may be due to effects of age, Hp infection, or both on the lower esophageal sphincter. Acknowledgement: Data provided by TAP pharmaceutical.

Symptom Relief with Omeprezole in GERD is Not Impaired by H. pylori Eradication. Results of a Long-Term Prospective RandomizedTrial. Annemieke Cats, Netherlaeds Cancer Institute, Amsterdam Netherlands; Frits Hells, Isala Clinics, Zwolle Nethedands; Pleun Snel, Slotervaart Hosp, Amsterdam Netherlands; Elly C. Kliokenberg-Knol, Acad Hosp Vrije Univ, Amsterdam Netherlands; Jeroen J. Kolkman, Medicine Spectrum Twente, Enschede Netherlands; Henk Pm Festen, Bosch Medicentrum, Den Bosch Nethedands; Ernst J. Kuipers, Aced Hosp Rotterdam, Rotterdam Netherlands It has been reported that proton pump inhibitors have a stronger acid suppressive effect in H. py/ori(Hp) infected than uninfected subjects. This difference disappears after Hperadicetion. The aim of the present prospective open randomized study was therefore to evaluate whether Hp eradication increases reflux symptoms in GERD pts during omeprazole maintenance treatment (OMT). Hp-posltive reflux pts with reflux esophagitis who had been treated for >12 months with OMT were eligible for inclusion. They were randomized to Hp eradication with a 1-weak course of omeprazole, amoxicillin, and clarithromycin or omeprazole only. All pts continued OMT for 2 years. Pts were evaluated at baseline, and after 12 and 24 months using a validated reflux symptom questionnaire for complaints during the previous week on a scale. from 0-3 for each individual item. Patients were divided according to Hp status at 24 months in eradicated (E+) and non-eradicated (E-). Change from baseline in reflux score between groups was tested using the Wilcoxon rank sum test, In 84 patients (42 E +, 42 E-) symptom scores were obtained at baseline, and at 12 and/or 24 months. The mean-+SDage was 60_+12 yrs in E- and 63_+13 yrs in E+ patients. The 95% confidence intervals at baseline of the number of days of reflux symptoms (0, 1, 2-4, ~5 days), and the severity of pyrosis, regurgitation, retrosternal pain and dysphagia (none, mild, moderate, severe) during the previous week was not different between the groups, both at 12 and 24 months, nor was

Gastric pH and EAE in Hp+ and Hp- EE Patients Baseline (Mean) Age (year) % time esophageal pH<4.0 # GER episodes • 1 min # GER episodes • 5 min Mean IG pH % time IG pH >4.0

Hp+

Hp-

P

55.4 17.5 215 8.4 3.0 31.8

42.8 11.6 190 5.7 2.1 12.3

0.007 0.12 0.6 0.3 0.005 0.003

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