April 2000

1788

IDENTIFICATION OF HEUCOBACTER MUSTELAE VIRULENCEFACTORS BY SCREENING OF A RANDOM INSERTIONAL MU·TANT LIBRARY.Tadhg 0 Croinin, Billy Bourke, Christina M. Vandenbrouke, BrendanDrumm, Johannes G. Kusters, The Childrens Research Ctr, The ConwayInstitute, U C D, Dublin, Ireland; The Conway Institute, Dept of Paediat­rics U C D, Dublin, Ireland; Dept of Med Microbiology, Vrije Univ,Amsterdam, Netherlands.

BACKGROUND Helicobacter mustelae infection of ferrets has been as­sociated with gastritis, duodenal ulcer disease and gastric cancer. Althoughexperimental Helicobacter pylori infections have been developed, naturalinfection has only been described in humans and non-human primates.Therefore infection of ferrets with H mustelae is recognised as an impor­tant natural animal model of H pylori infection in humans. The aim of thisstudy was to develop a method for generating insertion mutants in Hmustelae with a view to identifying genes important in a natural Helico­bacter infection. METHODS H mustelae chromosomal DNA was digestedwith Clal and self ligated to create circular DNA. This DNA was thenre-digested with Bgl II and recircularized by ligation with the apha3kanamycin resistance cassette. The resultant apha-3 containing circles werethen naturally transformed into H mustelae strain NCTC 12032. Success­ful introduction gives rise to homologous cross-over with correspondinggenes in the chromosome resulting in disruption of a gene and conferringkanamycin resistance. Mutants were selected on blood agar plates contain­ing kanamycin. RESULTS Natural transformation of H mustelae with theconstructs resulted in 500 kanamycin resistant transformants per p.,g DNA.This indicates that H mustelae is naturally competent for transformationwith DNA. A Southern blot with 12 randomly selected transformantsprobed with the kanamycin cassette showed that the constructs integratedinto different sites in the H. mustelae chromosome suggesting randomintegration. An initial mutant library of 500 mutants was created andscreened for the absence of virulence factors. Screening for urease activityusing Christensens broth has revealed two urease deficient mutants. CON­CLUSION In this study we describe a method for generating a mutantlibrary in H. mustelae. The ability to screen for H mustelae mutantsattenuated in virulence both in vitro and in vivo represents an importantopportunity to investigate the pathogenesis of gastric Helicobacter speciesin their natural hosts.

1789

LOCALIZATION OF ANTIGEN PRESENTING CELLS (APCS) INHEUCOBACTER PYLORI (HP) INFECTED GASTRIC MUCOSAWITH OR WITHOUT INTESTINAL METAPLASIA.Tatsuhiko Suzuki, Katsuaki Kato, Yuuji Kubota, Naohiro Dairaku, KenjiNoguchi, Yutaka Konno, Hitoshi Sekine, Shuichi Ohara, TakayoshiToyota, Tooru Shimosegawa, Tohoku Univ Sch of Medicine, Sendai,Japan.

Backgraunsd and aims : HP infection is known to induce specific immuneresponse in the gastric mucosa. The immune response is triggered bypresentation of antigen peptides on the MHC assembly of APCs with apromotive assistance of costimulatory factors such as B7-1 (CD80) and -2(CD86). The counter receptor of T cells for the activation is a cell surfacemolecule CD28. The present study was aimed to clarify the localization ofAPCs and their relation with T cells in the HP infected human gastricmucosa. Materials and Methods : Gastric mucosal specimens were ob­tained from 10 HP-negative individuals and 20 HP-positive patients withchronic gastritis including 10 cases undergone HP eradication therapy, andwere processed for the immunohistochemistry of MHC class II antigen(HLA-DR), CD80, CD86 and CD28. Results : The HP-infected gastricmucosa showed the enhanced expression of HLA-DR in the gastric epi­thelial cells, inflammatory cells, vascular endothelial cells and interstitialcells in the lamina propria, whereas the immunoreactivities of CD80 andCD86 were detected mainly in mononuclear cells which infiltratied into thelamina propria, but were not detected in other type cells. The concomitantexpression of HLA-DR and CD80 was confirmed in the mononuclear cellsby the conforcal laser scanning microscopy. Furthermore, double immu­nostaining of CD80 and CD28 demonstrated that the CD28-positive cellsintimate contact with the CD80-positive cells. The appearance of intestinalmetaplasia was related to the absence of HP organisms and decreasedactivity of inflammation. Tere was tendency that the expression ofHLA-DR was weak and the number of CD80-positive cells and those withCD28 was low in the area with intestinal metaplasia. AfterHP eradicationtherapy, the expressions of HLA-DR and CD80 were clearly decreased.The labeling index of CD80-positive cells was significantly higher in theHP-positive cases than the HP-negative cases and was decreased to thesame level of the HP-negative cases after bacterial eradication. Conclu­sions : Our findings suggested that the mononuclear cells in the laminapropria may mainly act as APes in HP-infected gastric mucosa, and theirtriggered immune response might be involved in the mucosal immuneresponse in the inflamed gastric mucosa to invasive antigens related to HPorganisms.

AGAA329

1790

DNA VACCINES ENCODING HEAT SHOCK PROTEIN A AND B OFH. PYWRI STRONGLY SUPPRESS GASTRIC MUCOSAL COLONI·ZATION OF H. PYWRI AND INFLAMMATION IN MICE.Isami Todoroki, Takashi Joh, Katsushi Watanabe, Kyoji Seno, MakotoSasaki, Hiromi Kataoka, Hideo Suzuki, Huminori Okumura, YasuhideTakezono, Tsutomu Mizoshita, Yoshihumi Yokoyama, Makoto Itch,Nagoya City Univ Med Sch, Nagoya, Japan.

Aim; In recent years, administration of plasmid DNA (DNA vaccine) wasdemonstrated to induce both humoral and cellular immunity, and becamea very useful approach against several pathogens. In this study, we inves­tigated the effect of DNA vaccines encoding H. pylori -heat shock proteins(HspA, HspB) on immune responses against Hipylori in mice. Methods;HspA and HspB from Hipylori Sydney strain (SSI) were cloned intoplasmid vector, pcDNA3.1 carrying CMV promoter (HspA-DNA andHspB-DNA). Female C57BU6 mice aged 5 weeks were immunized bysingle injection of 10 p.,g HspA-DNA, HspB-DNA or mixture of HspA­DNA and HspB-DNA (mixed Hsp-DNA) into subcutaneous tissue ofabdomen. Plasmid DNA lacking the inserted Hsp were injected as acontrol. Three months after immunization, blood was taken, and then micewere given challenge with one orogastric doses of live HipyloriSS1 (100million organisms/dose). After a further 6 months, the animals were killedand assessed for Hipylori infection. Bacteria in stomach were counted ascolony forming unit (c.f.u.) on Hipylori selective plates. To detect anti­Hipylori specific serum IgG antibody, an enzyme-linked immunosorbentassay (ELISA) using plates coated with solid whole cells of H.pylori wasperfomed. Each stomach section was examined histologically to evaluatethe intensity of the mucosal inflammation and to grade the number ofIl.pylori. Results; Three months after vaccination, specific antibody againstHpylori were detected in the serum of all groups (114 mice for HspA­DNA, 3/5 mice for HSPB-DNA and 5/5 mice for mixed Hsp-DNA). DNAvaccine dramatically suppressed the numbers of live bacteria in micestomach. Six months after inoculation, 127,790±42,21O/mm2 (100%) forcontrol mice, I9,060 ±6,480/mm2 (15.0±3.0%) for HspA-DNA,771O±1970/mm2 (6.0±1.6%) for HspB-DNA, and only205±35/mm2(0.16±0.03%) for mixed Hsp-DNA were detected. Histological analysis ofthe stomach demonstrated that only a small number of inflammatory cellswere observed in DNA vaccinated mice, while a lot of inflammatory cellsinfiltrated into mucosal and submucosal layer of the stomach in controlmice. Conclusion; These results demonstrated that DNA vaccine encodingH.pylori-HspA or HspB strongly suppress gastric mucosal colonization ofH. pylori and inflammation, indicating that DNA vaccine may represent apromising approach against H.pylori in humans

1791

GASTRIC MUCOSA MAYBE A PRIMING SITE AGAINST HEL­ICOBACTER PYWRI IN THE PEYER'S PATCH·DEFICIENTMICE.Suguru Uose, Kazuichi Okazaki, Toshiki Nishi, Andra's Debreceni, Ka­zushige Uchida, Hiroshi Nakase, Masaya Ohana, Yumi Matsushima, MakiInai, Tsutomu Chiba, Kyoto Univ, Kyoto, Japan.

Aim: Peyer's patch plays an important role in priming lymphocytes withlumenal antigens. Although normal gastric mucosa contains no lymphoidtissues, it is well-known that lymphfollicles are often observed in Helico­bacter pylori (Hp) positive gastritis. It is still unknown whether the gastricmucosa can be a priming site of antigens or not. To clarify it, we examinedimmune responses to Hp using Peyer's patch-deficient mice (PPD-mice),which were made by injection of anti-IL-? receptor antibody to theirmother during pregnancy. Materials and Methods: Anti-ll>? receptor an­tibody was injected to normal Balb/c mice on the 14th day of pregnancy.Deficiency of Peyer's patch of the neonates was confirmed by whole­mount immunohistochemical staining. Live Hp (1X 108

) or the comparabledose of sonicated Hp were orally administrated to the PPD- and normalBalb/c mice. We measured anti-Hp antibody by the ELISA method usingdiluted serum (I :40). Fresh frozen sections of the stomach of these micewere used for immunohistochemical staining of Ia antigen or co-stimula­tory molecules (CD80 and CD86). Results: The levels of serous anti-Hpantibody in the PPD-mice infected with live Hp (O.D.; m±SE: 0.?6±O.11)was significantly higher than that in the PPD-mice administered withsonicated Hp (O.26±O.05),but not significantly different from the controls;normal mice with sonicated Hp (O.72±0.16) or live Hp (O.60±0.10 ).Immunohistochemical staining showed that the expression of Ia antigen,CD80 and CD86 in the gastric mucosa are observed only in the miceinfected with live Hp. Conclusion: Our findings suggested that the majorpriming site against live Hp seemed to be gastric mucosa in PPD-mice. InHp infection, gastric mucosa may be a priming site as well as Peyer'spatch.