Cytotoxic and pathogenic properties of Klebsiella oxytoca isolated from laboratory animals.

Klebsiella oxytoca is an opportunistic pathogen implicated in various clinical diseases in animals and humans. Studies suggest that in humans K. oxytoca exerts its pathogenicity in part through a cytotoxin. However, cytotoxin production in animal isolates of K. oxytoca and its pathogenic properties have not been characterized. Furthermore, neither the identity of the toxin nor a complete repertoire of genes involved in K. oxytoca pathogenesis have been fully elucidated. Here, we showed that several animal isolates of K. oxytoca, including the clinical isolates, produced secreted products in bacterial culture supernatant that display cytotoxicity on HEp-2 and HeLa cells, indicating the ability to produce cytotoxin. Cytotoxin production appears to be regulated by the environment, and soy based product was found to have a strong toxin induction property. The toxin was identified, by liquid chromatography-mass spectrometry and NMR spectroscopy, as low molecular weight heat labile benzodiazepine, tilivalline, previously shown to cause cytotoxicity in several cell lines, including mouse L1210 leukemic cells. Genome sequencing and analyses of a cytotoxin positive K. oxytoca strain isolated from an abscess of a mouse, identified genes previously shown to promote pathogenesis in other enteric bacterial pathogens including ecotin, several genes encoding for type IV and type VI secretion systems, and proteins that show sequence similarity to known bacterial toxins including cholera toxin. To our knowledge, these results demonstrate for the first time, that animal isolates of K. oxytoca, produces a cytotoxin, and that cytotoxin production is under strict environmental regulation. We also confirmed tilivalline as the cytotoxin present in animal K. oxytoca strains. These findings, along with the discovery of a repertoire of genes with virulence potential, provide important insights into the pathogenesis of K. oxytoca. As a novel diagnostic tool, tilivalline may serve as a biomarker for K oxytoca-induced cytotoxicity in humans and animals through detection in various samples from food to diseased samples using LC-MS/MS. Induction of K. oxytoca cytotoxin by consumption of soy may be in part involved in the pathogenesis of gastrointestinal disease.

Helminth co-infection in Helicobacter pylori infected INS-GAS mice attenuates gastric premalignant lesions of epithelial dysplasia and glandular atrophy and preserves colonization resistance of the stomach to lower bowel microbiota.

Higher prevalence of helminth infections in Helicobacter pylori infected children was suggested to potentially lower the life-time risk for gastric adenocarcinoma. In rodent models, helminth co-infection does not reduce Helicobacter-induced inflammation but delays progression of pre-malignant gastric lesions. Because gastric cancer in INS-GAS mice is promoted by intestinal microflora, the impact of Heligmosomoides polygyrus co-infection on H. pylori-associated gastric lesions and microflora were evaluated. Male INS-GAS mice co-infected with H. pylori and H. polygyrus for 5 months were assessed for gastrointestinal lesions, inflammation-related mRNA expression, FoxP3(+) cells, epithelial proliferation, and gastric colonization with H. pylori and Altered Schaedler Flora. Despite similar gastric inflammation and high levels of proinflammatory mRNA, helminth co-infection increased FoxP3(+) cells in the corpus and reduced H. pylori-associated gastric atrophy (p < 0.04), dysplasia (p < 0.02) and prevented H. pylori-induced changes in the gastric flora (p < 0.05). This is the first evidence of helminth infection reducing H. pylori-induced gastric lesions while inhibiting changes in gastric flora, consistent with prior observations that gastric colonization with enteric microbiota accelerated gastric lesions in INS-GAS mice. Identifying how helminths reduce gastric premalignant lesions and impact bacterial colonization of the H. pylori infected stomach could lead to new treatment strategies to inhibit progression from chronic gastritis to cancer in humans.

An analysis of the role of the indigenous microbiota in cholesterol gallstone pathogenesis.

BACKGROUND AND AIMS: Cholesterol gallstone disease is a complex process involving both genetic and environmental variables. No information exists regarding what role if any the indigenous gastrointestinal microbiota may play in cholesterol gallstone pathogenesis and whether variations in the microbiota can alter cholesterol gallstone prevalence rates. METHODS: Genetically related substrains (BALB/cJ and BALB/cJBomTac) and (BALB/AnNTac and BALB/cByJ) of mice obtained from different vendors were compared for cholesterol gallstone prevalence after being fed a lithogenic diet for 8 weeks. The indigenous microbiome was altered in these substrains by oral gavage of fecal slurries as adults, by cross-fostering to mice with divergent flora at <1 day of age or by rederiving into a germ-free state. RESULTS: Alterations in the indigenous microbiome altered significantly the accumulation of mucin gel and normalized gallbladder weight but did not alter cholesterol gallstone susceptibility in conventionally housed SPF mice. Germ-free rederivation rendered mice more susceptible to cholesterol gallstone formation. This susceptibility appeared to be largely due to alterations in gallbladder size and gallbladder wall inflammation. Colonization of germ-free mice with members of altered Schaedler flora normalized the gallstone phenotype to a level similar to conventionally housed mice. CONCLUSIONS: These data demonstrate that alterations in the gastrointestinal microbiome may alter aspects of cholesterol gallstone pathogenesis and that in the appropriate circumstances these changes may impact cholesterol cholelithogenesis.

Colitis and colon cancer in WASP-deficient mice require helicobacter species.

BACKGROUND: Wiskott-Aldrich syndrome protein-deficient patients and mice are immunodeficient and can develop inflammatory bowel disease. The intestinal microbiome is critical to the development of colitis in most animal models, in which Helicobacter spp. have been implicated in disease pathogenesis. We sought to determine the role of Helicobacter spp. in colitis development in Wiskott-Aldrich syndrome protein-deficient (WKO) mice. METHODS: Feces from WKO mice raised under specific pathogen-free conditions were evaluated for the presence of Helicobacter spp., after which a subset of mice were rederived in Helicobacter spp.-free conditions. Helicobacter spp.-free WKO animals were subsequently infected with Helicobacter bilis. RESULTS: Helicobacter spp. were detected in feces from WKO mice. After rederivation in Helicobacter spp.-free conditions, WKO mice did not develop spontaneous colitis but were susceptible to radiation-induced colitis. Moreover, a T-cell transfer model of colitis dependent on Wiskott-Aldrich syndrome protein-deficient innate immune cells also required Helicobacter spp. colonization. Helicobacter bilis infection of rederived WKO mice led to typhlitis and colitis. Most notably, several H. bilis-infected animals developed dysplasia with 10% demonstrating colon carcinoma, which was not observed in uninfected controls. CONCLUSIONS: Spontaneous and T-cell transfer, but not radiation-induced, colitis in WKO mice is dependent on the presence of Helicobacter spp. Furthermore, H. bilis infection is sufficient to induce typhlocolitis and colon cancer in Helicobacter spp.-free WKO mice. This animal model of a human immunodeficiency with chronic colitis and increased risk of colon cancer parallels what is seen in human colitis and implicates specific microbial constituents in promoting immune dysregulation in the intestinal mucosa.

Prevalence of murine Helicobacter spp. Infection is reduced by restocking research colonies with Helicobacter-free mice.

Most academic research colonies of mice are endemically infected with enterohepatic Helicobacter spp. (EHS). We evaluated EHS prevalence in surveillance mice before and after a 10-y period of requiring that imported mice be free of EHS by embryo transfer rederivation or purchase from approved vendors. In 2009, composite fecal samples from CD1 surveillance mice representing colony health in 57 rooms located in 6 facilities were evaluated for EHS infection by using PCR assays. Fecal samples were screened with primers designed to detect all known EHS, and positive samples were further assayed by using primers specific for H. hepaticus, H. bilis, H. rodentium, and H. typhlonicus. Most EHS were detected in surveillance mice within the first month of dirty bedding exposure, with prevalence ranging from 0% to 64% as monoinfections or, more commonly, infections with multiple EHS. Compared with 1999 prevalence data, EHS remained endemic in colonies importing the lowest number of EHS-free mice. EHS were absent or the prevalence was greatly reduced in colonies receiving the highest percentage of EHS-free mice. This study demonstrates that the management decision to require exclusive importation of EHS-free mice reduced EHS prevalence on an institutional scale without intensive labor and expense associated with other techniques or interference with research objectives.

Animal models of Helicobacter-induced disease: methods to successfully infect the mouse.

Animal models of microbial diseases in humans are an essential component for determining fulfillment of Koch's postulates and determining how the organism causes disease, host response(s), disease prevention, and treatment. In the case of Helicobacter pylori, establishing an animal model to fulfill Koch's postulates initially proved so challenging that out of frustration a human volunteer undertook an experiment to become infected with H. pylori and to monitor disease progression in order to determine if it did cause gastritis. For the discovery of the organism and his fulfillment of Koch's postulates he and a colleague were awarded the Nobel Prize in Medicine. After H. pylori was established as a gastric pathogen, it took several years before a model was developed in mice, opening the study of the organism and its pathogenicity to the general scientific community. However, while the model is widely utilized, there are a number of difficulties that can arise and need to be overcome. The purpose of this chapter is to raise awareness regarding the problems, and to offer reliable protocols for successfully establishing the H. pylori mouse model.

Coinfection with Enterohepatic Helicobacter species can ameliorate or promote Helicobacter pylori-induced gastric pathology in C57BL/6 mice.

To investigate how different enterohepatic Helicobacter species (EHS) influence Helicobacter pylori gastric pathology, C57BL/6 mice were infected with Helicobacter hepaticus or Helicobacter muridarum, followed by H. pylori infection 2 weeks later. Compared to H. pylori-infected mice, mice infected with H. muridarum and H. pylori (HmHp mice) developed significantly lower histopathologic activity index (HAI) scores (P < 0.0001) at 6 and 11 months postinoculation (MPI). However, mice infected with H. hepaticus and H. pylori (HhHp mice) developed more severe gastric pathology at 6 MPI (P = 0.01), with a HAI at 11 MPI (P = 0.8) similar to that of H. pylori-infected mice. H. muridarum-mediated attenuation of gastritis in coinfected mice was associated with significant downregulation of proinflammatory Th1 (interlukin-1beta [Il-1ß], gamma interferon [Ifn-γ], and tumor necrosis factor-alpha [Tnf-α]) cytokines at both time points and Th17 (Il-17A) cytokine mRNA levels at 6 MPI in murine stomachs compared to those of H. pylori-infected mice (P < 0.01). Coinfection with H. hepaticus also suppressed H. pylori-induced elevation of gastric Th1 cytokines Ifn-γ and Tnf-α (P < 0.0001) but increased Th17 cytokine mRNA levels (P = 0.028) at 6 MPI. Furthermore, mRNA levels of Il-17A were positively correlated with the severity of helicobacter-induced gastric pathology (HhHp>H. pylori>HmHp) (at 6 MPI, r² = 0.92, P < 0.0001; at 11 MPI, r² = 0.82, P < 0.002). Despite disparate effects on gastritis, colonization levels of gastric H. pylori were increased in HhHp mice (at 6 MPI) and HmHp mice (at both time points) compared to those in mono-H. pylori-infected mice. These data suggest that despite consistent downregulation of Th1 responses, EHS coinfection either attenuated or promoted the severity of H. pylori-induced gastric pathology in C57BL/6 mice. This modulation was related to the variable effects of EHS on gastric interleukin 17 (IL-17) responses to H. pylori infection.

Helicobacter marmotae and novel Helicobacter and Campylobacter species isolated from the livers and intestines of prairie dogs.

Prairie dogs (Cynomys ludovicianus) are used to study the aetiology and prevention of gallstones because of the similarities of prairie dog and human bile gallstone composition. Epidemiological and experimental studies have suggested a connection between infection with Helicobacter species and cholesterol cholelithiasis, cholecystis and gallbladder cancer. Ten of the 34 prairie dogs in this study had positive Helicobacter species identified by PCR using Helicobacter genus-specific primers. Ten of 34 prairie dogs had positive Campylobacter species identified in the intestine by PCR with Campylobacter genus-specific primers. Six Helicobacter sp. isolates and three Campylobacter sp. isolates were identified taxonomically by 16S rRNA gene analysis. The prairie dog helicobacters fell into three clusters adjacent to Helicobacter marmotae. On the basis of 16S rRNA gene sequence analysis, three strains in two adjacent clusters were included in the species H. marmotae. Three strains were only 97.1 % similar to the sequence of H. marmotae and can be considered a novel species with the provisional designation Helicobacter sp. Prairie Dog 3. The prairie dog campylobacters formed a single novel cluster and represent a novel Campylobacter sp. with the provisional designation Campylobacter sp. Prairie Dog. They branched with Campylobacter cuniculorum at 96.3 % similarity and had the greatest sequence similarity to Campylobacter helveticus at 97.1 % similarity. Whether H. marmotae or the novel Helicobacter sp. and Campylobacter sp. identified in prairie dogs play a role in cholesterol gallstones or hepatobiliary disease requires further studies.

17 ß-estradiol suppresses Helicobacter pylori-induced gastric pathology in male hypergastrinemic INS-GAS mice.

Helicobacter pylori-associated gastric cancer is male predominant and animal studies suggest that sex hormones influence gastric carcinogenesis. We investigated the effects of 17ß-estradiol (E2) or castration on H.pylori-induced gastritis in male INS-GAS/FVB/N (Tg(Ins1-GAS)1Sbr) mice. Comparisons were made to previously evaluated sham (n = 8) and H.pylori-infected (n = 8), intact male INS-GAS mice which had developed severe corpus gastritis accompanied by atrophy, hyperplasia, intestinal metaplasia and dysplasia of the epithelium within 16 weeks postinfection (all P < 0.01). Castration at 8 weeks of age had no sparing effect on lesions in uninfected (n = 5) or H.pylori-infected mice (n = 7) but all lesion subfeatures were attenuated by E2 in H.pylori-infected mice (n = 7) (P < 0.001). Notably, inflammation was not reduced but glandular atrophy, hyperplasia, intestinal metaplasia and dysplasia were also less severe in uninfected, E2-treated mice (n = 7) (P < 0.01). Attenuation of gastric lesions by E2 was associated with lower messenger RNA (mRNA) expression of interferon (IFN)-γ (P < 0.05) and interleukin (IL)-1ß (P < 0.004), and higher IL-10 (P < 0.02) as well as decreased numbers of Foxp3(+) regulatory T cells when compared with infected intact males. Infected E2-treated mice also developed higher Th2-associated anti-H.pylori IgG1 responses (P < 0.05) and significantly lower Ki-67 indices of epithelial proliferation (P < 0.05). E2 elevated expression of mRNA for Foxp3 (P < 0.0001) and IL-10 (P < 0.01), and decreased IL-1ß (P < 0.01) in uninfected, intact male mice compared with controls. Therefore, estrogen supplementation, but not castration, attenuated gastric lesions in H.pylori-infected male INS-GAS mice and to a lesser extent in uninfected mice, potentially by enhancing IL-10 function, which in turn decreased IFN-γ and IL-1ß responses induced by H.pylori.

Lactobacillus reuteri promotes Helicobacter hepaticus-associated typhlocolitis in gnotobiotic B6.129P2-IL-10(tm1Cgn) (IL-10(-/-) ) mice.

To model inflammatory bowel disease, we assessed infection with Helicobacter hepaticus 3B1 (ATCC 51449) and a potential probiotic Lactobacillus reuteri (ATCC PTA-6475) in gnotobiotic B6.129P2-IL-10(tm1Cgn) (IL-10(-/-) ) mice. No typhlocolitis developed in germ-free controls (n=21) or in L. reuteri (n=8) or H. hepaticus (n=18) mono-associated mice for 20 weeks post-infection. As positive controls, three specific pathogen-free IL-10(-/-) mice dosed with H. hepaticus developed severe typhlocolitis within 11 weeks. Because L. reuteri PTA-6475 has anti-inflammatory properties in vitro, it was unexpected to observe significant typhlocolitis (P<0·0001) in mice that had been infected with L. reuteri followed in 1 week by H. hepaticus (n=16). The H. hepaticus colonization was not affected through 20 weeks post-infection but L. reuteri colonization was lower in co-infected compared with L. reuteri mono-associated mice at 8-11 weeks post-infection (P<0·05). Typhlocolitis was associated with an increased T helper type 1 serum IgG2c response to H. hepaticus in co-infected mice compared with H. hepaticus mono-associated mice (P<0·005) and similarly, mRNA expression in caecal-colonic tissue was elevated at least twofold for chemokine ligands and pro-inflammatory interleukin-1α (IL-1α), IL-1ß, IL-12 receptor, tumour necrosis factor-α and inducible nitric oxide synthase. Anti-inflammatory transforming growth factor-ß, lactotransferrin, peptidoglycan recognition proteins, Toll-like receptors 4, 6, 8 and particularly 9 gene expression, were also elevated only in co-infected mice (P<0·05). These data support that the development of typhlocolitis in H. hepaticus-infected IL-10(-/-) mice required co-colonization with other microbiota and in this study, required only L. reuteri. Although the effects other microbiota may have on H. hepaticus virulence properties remain speculative, further investigations using this gnotobiotic model are now possible.

Campylobacter troglodytis sp. nov., isolated from feces of human-habituated wild chimpanzees (Pan troglodytes schweinfurthii) in Tanzania.

The transmission of simian immunodeficiency and Ebola viruses to humans in recent years has heightened awareness of the public health significance of zoonotic diseases of primate origin, particularly from chimpanzees. In this study, we analyzed 71 fecal samples collected from 2 different wild chimpanzee (Pan troglodytes) populations with different histories in relation to their proximity to humans. Campylobacter spp. were detected by culture in 19/56 (34%) group 1 (human habituated for research and tourism purposes at Mahale Mountains National Park) and 0/15 (0%) group 2 (not human habituated but propagated from an introduced population released from captivity over 30 years ago at Rubondo Island National Park) chimpanzees, respectively. Using 16S rRNA gene sequencing, all isolates were virtually identical (at most a single base difference), and the chimpanzee isolates were most closely related to Campylobacter helveticus and Campylobacter upsaliensis (94.7% and 95.9% similarity, respectively). Whole-cell protein profiling, amplified fragment length polymorphism analysis of genomic DNA, hsp60 sequence analysis, and determination of the mol% G+C content revealed two subgroups among the chimpanzee isolates. DNA-DNA hybridization experiments confirmed that both subgroups represented distinct genomic species. In the absence of differential biochemical characteristics and morphology and identical 16S rRNA gene sequences, we propose to classify all isolates into a single novel nomenspecies, Campylobacter troglodytis, with strain MIT 05-9149 as the type strain; strain MIT 05-9157 is suggested as the reference strain for the second C. troglodytis genomovar. Further studies are required to determine whether the organism is pathogenic to chimpanzees and whether this novel Campylobacter colonizes humans and causes enteric disease.

Lack of commensal flora in Helicobacter pylori-infected INS-GAS mice reduces gastritis and delays intraepithelial neoplasia.

BACKGROUND & AIMS: Transgenic FVB/N insulin-gastrin (INS-GAS) mice have high circulating gastrin levels, and develop spontaneous atrophic gastritis and gastrointestinal intraepithelial neoplasia (GIN) with 80% prevalence 6 months after Helicobacter pylori infection. GIN is associated with gastric atrophy and achlorhydria, predisposing mice to nonhelicobacter microbiota overgrowth. We determined if germfree INS-GAS mice spontaneously develop GIN and if H pylori accelerates GIN in gnotobiotic INS-GAS mice. METHODS: We compared gastric lesions, levels of messenger RNA, serum inflammatory mediators, antibodies, and gastrin among germfree and H pylori-monoinfected INS-GAS mice. Microbiota composition of specific pathogen-free (SPF) INS-GAS mice was quantified by pyrosequencing. RESULTS: Germfree INS-GAS mice had mild hypergastrinemia but did not develop significant gastric lesions until 9 months old and did not develop GIN through 13 months. H pylori monoassociation caused progressive gastritis, epithelial defects, oxyntic atrophy, marked foveolar hyperplasia, dysplasia, and robust serum and tissue proinflammatory immune responses (particularly males) between 5 and 11 months postinfection (P<0.05, compared with germfree controls). Only 2 of 26 female, whereas 8 of 18 male, H pylori-infected INS-GAS mice developed low to high-grade GIN by 11 months postinfection. Stomachs of H pylori-infected SPF male mice had significant reductions in Bacteroidetes and significant increases in Firmicutes. CONCLUSIONS: Gastric lesions take 13 months longer to develop in germfree INS-GAS mice than male SPF INS-GAS mice. H pylori monoassociation accelerated gastritis and GIN but caused less severe gastric lesions and delayed onset of GIN compared with H pylori-infected INS-GAS mice with complex gastric microbiota. Changes in gastric microbiota composition might promote GIN in achlorhydric stomachs of SPF mice.

Persistent infection of rhesus monkeys with 'Helicobacter macacae' and its isolation from an animal with intestinal adenocarcinoma.

A novel helicobacter, 'Helicobacter macacae', was previously isolated from a colony of rhesus and cynomolgus monkeys in which diarrhoea from chronic idiopathic colitis was enzootic. A survey performed in a second colony of rhesus monkeys without a history of chronic diarrhoea determined that 57 % were faecal-culture positive for Helicobacter species. Ten years after the survey, one of the animals from which 'H. macacae' had been isolated, a 23-year-old, intact male rhesus monkey (Macaca mulatta), presented with partial inappetence and progressive weight loss. Subsequent evaluation of the monkey revealed anaemia, hypoproteinaemia, hypoalbuminaemia and a palpable abdominal mass. Contrast radiography suggested partial intestinal obstruction. The animal was euthanized and a diagnosis was made of intestinal adenocarcinoma of the ileocaecocolic junction with metastasis to regional lymph nodes and liver. Microaerobic culture of caecal tissue yielded a helicobacter organism identified as 'H. macacae' by 16S rRNA gene sequencing - the same species of bacteria isolated 10 years previously. The liver, small intestine and colon were also positive by PCR for Helicobacter species. Intestinal adenocarcinoma is the most common malignancy of aged macaques. Faeces or caecal tissue from five out of five monkeys that remained from the original cohort and that were colonized with 'H. macacae' in the initial survey were positive for the organism. The apparent persistence of 'H. macacae' in these animals, the isolation of the bacterium from animals with colitis and the recognition of the importance of inflammation in carcinogenesis raise the possibility of an aetiological role in the genesis of intestinal adenocarcinoma in aged rhesus monkeys.

Characterization of cytotoxic necrotizing factor 1-producing Escherichia coli strains from faeces of healthy macaques.

Twenty-five (27 %) of 92 clinically normal macaques were found to have beta-haemolytic Escherichia coli isolated from their faeces. Five of six isolates chosen for further characterization had multiple antibiotic resistance and were PCR-positive for cytotoxic necrotizing factor 1 (cnf1) with a demonstrated cytopathic effect in vitro. By repetitive element sequence-based PCR genotyping, genetic similarity was established for selected isolates. We believe this to be the first report of E. coli strains producing CNF1 in non-human primates.

Inflammation and foveolar hyperplasia are reduced by supplemental dietary glutamine during Helicobacter pylori infection in mice.

We recently showed that L-Gln protects cultured gastric cells from ammonia-induced cell death and predicted that Gln may also protect during Helicobacter pylori infection in vivo. Thus, the aim of this study was to test whether supplemental dietary Gln protects against H. pylori-associated pathology. For this, C57BL/6 mice were fed a purified diet consisting of 20.3% protein (1.9% Gln), 66% carbohydrate, and 5% fat or 25.3% protein (5% supplemental L-Gln; 6.9% total Gln), 61% carbohydrate, and 5% fat. After a 2-wk prefeeding period, mice were divided into sham-(uninfected) or H. pylori-infected groups. Body weight and food consumption were recorded weekly. Tissue histopathology, H. pylori colonization, serum IgG, and pro- and antiinflammatory cytokine mRNA expression were determined at 6, 12, and 20 wk postinfection (wkPI). Inflammation, antiinflammatory cytokine, and interleukin-1beta mRNA expression were significantly greater at 6 wkPI in H. pylori-infected mice fed supplemental Gln compared with those fed the control diet. At 20 wkPI, however, inflammation and foveolar hyperplasia were significantly lower in H. pylori-infected mice fed supplemental Gln compared with those fed the control diet. Body weight gain, food consumption, H. pylori colonization, and serum IgG did not differ in H. pylori-infected mice fed supplemental Gln compared with the control diet. Our data demonstrate that H. pylori-infected mice fed supplemental dietary Gln have reduced H. pylori-associated pathology in vivo that is accompanied by beneficial changes in the immune response to H. pylori early in infection. Thus, Gln supplementation may be an alternative therapy for reducing H. pylori-associated pathology.

Epithelial cell expression of BCL-2 family proteins predicts mechanisms that regulate Helicobacter pylori-induced pathology in the mouse stomach.

Corpus-predominant infection with Helicobacter pylori (HP) results in the activation of programmed cell death pathways in surface, parietal, and chief cells. At present, mechanisms that regulate these pathways to result in HP-associated pathology are not fully understood. Because it is not known which survival and death pathways are present in gastric epithelial cells, we used an antibody panel to evaluate the expression of BCL-2 family prosurvival proteins or multi-Bcl-2 homology (BH)-domains (group 1) or BH3-only (group-2) proapoptotic proteins in the stomachs of uninfected or HP-infected C57BL/6 mice. This strategy identified BCL-2, BAK, and BAD as the major prosurvival and proapoptotic proteins, in surface cells and BAD as the only BCL-2 family protein expressed in parietal cells. Chief cells express altogether different effectors, including BCL-X(L)/BCL-2, for survival but have no constitutively expressed proapoptotic proteins. In model chief cells, however, the group 1 proapoptotic protein BCL-X(S) was expressed after exposure to proinflammatory cytokines concomitant with reduced viability, demonstrating that chief cells can transcriptionally regulate the induction of proapoptotic proteins to execute apoptosis. During HP infection, no additional BCL-2 family proteins were expressed in epithelial cells, whereas those present either remained unchanged or were reduced as cell deletion occurred over time. Additional studies demonstrated that the posttranslational regulation of BAD in surface and parietal cells was negatively affected by HP infection, a result that may be directly related to an increase in apoptosis during infection. Thus, gastric epithelial cells express cell-specific prosurvival and proapoptotic pathways. From the results presented here, mechanisms that regulate HP-related changes in the survival and death profile of gastric epithelial cells can be predicted and then tested, with the ultimate goal of elucidating important therapeutic targets to inhibit the progression of HP-related pathology in the stomach.

Helicobacter hepaticus HHGI1 is a pathogenicity island associated with typhlocolitis in B6.129-IL10 tm1Cgn mice.

Helicobacter hepaticus strain 3B1 (H. hepaticus) contains a genomic island of approximately 71 kb, HHGI1, with some of the common features shared among known bacterial pathogenicity islands. In this study, we characterized the pathogenic potential of HHGI1 by infecting B6.129-IL10 tm1Cgn (IL10-/-) mice with an isogenic mutant (namely HhPAId1) lacking 19 predicted genes within HHGI1. In contrast to H. hepaticus (P<0.001), HhPAId1 did not cause typhlocolitis and hyperplasia in IL10-/- mice. Colonization levels of HhPAId1 were significantly higher in the cecum (P<0.007) and similar in the colon (P=0.27) when compared to H. hepaticus by 13 or 16 weeks post inoculation (WPI). The magnitude of the Th1-associated IgG2c response against HhPAId1 was less than that against H. hepaticus (P<0.004). There was no significant difference in Th2-associated IgG1 responses against these two strains. Cecal and colonic mRNA levels of proinflammatory cytokines IFN-gamma, TNF-alpha and IL-17a in the HhPAId1-infected mice were significantly lower than those in the H. hepaticus-infected mice (P<0.05) at 13 WPI. These results demonstrate that genes in the HHGI1 contribute to the pathogenicity of H. hepaticus, at least in part via up-regulation of proinflammatory mediators IFN-gamma, TNF-alpha and IL-17a.

Helicobacter hepaticus urease is not required for intestinal colonization but promotes hepatic inflammation in male A/JCr mice.

Urease activity contributes to bacterial survival in the acidic environment of the stomach and is essential for persistent infection by known gastric helicobacters such as the human pathogen Helicobacter pylori. Several enterohepatic Helicobacter species (EHS) that primarily infect the less acidic intestine also have very active urease enzymes. The importance of urease and its contribution to pathogenesis for these EHS are poorly understood. In this study, we generated a urease-deficient, isogenic mutant (HhureNT9) of Helicobacter hepaticus 3B1 (Hh 3B1), an EHS that possesses a urease gene cluster similar to that of H. pylori. Lack of urease activity did not affect the level of cecal colonization by HhureNT9 compared to Hh 3B1 in male A/JCr mice (P=0.48) at 4 months post-inoculation (MPI). In contrast, there was no HhureNT9 detected in the livers of any infected mice, whereas all livers from the Hh 3B1-infected mice were PCR-positive for Hh 3B1. The mice infected with HhureNT9 developed significantly less severe hepatitis (P=0.017) and also produced significantly lower hepatic mRNA levels of proinflammatory cytokines IFN-gamma (P=0.0007) and TNF-alpha (P<0.0001) compared to the Hh 3B1-infected mice. The Hh 3B1-infected mice developed significantly higher total IgG, Th1-associated IgG2a and Th2-associated IgG1 responses to infection. These results indicate that H. hepaticus urease activity plays a crucial role in hepatic disease but is not required for cecal colonization by H. hepaticus.

Isolation and characterization of a novel helicobacter species, "Helicobacter macacae," from rhesus monkeys with and without chronic idiopathic colitis.

Chronic idiopathic colitis is a common clinical entity in young captive rhesus monkeys. Eight isolates, cultured from five monkeys in colony 1 with endemic diarrhea and three from colony 2 without diarrhea, were grown under microaerobic conditions on selective agar and were classified by full 16S rRNA sequence, biochemical, and phenotypic analysis as a novel helicobacter, "Helicobacter macacae" (proposed name). All eight strains of H. macacae had 99.5% identical 16S rRNA sequences.

Protective role of 17 beta -estradiol against the development of Helicobacter pylori-induced gastric cancer in INS-GAS mice.

The incidence of gastric cancer is higher in men than women. Epidemiological studies suggest that female hormones reduce gastric cancer risk. We examined the effect of ovarian-dependent female hormones on Helicobacter pylori-induced gastric cancer in hypergastrinemic INS-GAS mice. Male and female sexually intact or ovariectomized (OVX) mice were inoculated with H.pylori SS1 or vehicle-only at 10 weeks of age, and tissues were evaluated at 16 or 28 weeks post-infection (WPI). A subset of OVX females were supplemented with 17beta-estradiol (E2), beginning at 16 WPI. Stomachs were evaluated by histopathology, Ki-67 proliferation index, H.pylori quantitative culture and quantitative polymerase chain reaction for messenger RNA expression of inducible nitric oxide synthase (iNOS) and inflammatory cytokines. Infected OVX females developed significantly more severe gastritis (P < 0.05) than infected intact females at both time points. E2 treatment in infected OVX females attenuated the severity of gastritis. Gastrointestinal intraepithelial neoplasia (GIN) developed in 42% of infected males and 10% of infected OVX females by 28 WPI, whereas infected intact females and E2-treated OVX females did not develop GIN. Infected OVX females showed significantly increased iNOS expression and epithelial cell proliferation when compared with intact, infected females. Likewise, interferon-gamma, tumor necrosis factor-alpha and interleukin-1beta (IL-1beta) expression in infected OVX females were significantly increased at 28 WPI when compared with intact counterparts. E2 treatment in infected OVX females significantly decreased IL-1beta expression, increased IL-10 expression and reduced epithelial cell proliferation. These results demonstrate a protective effect of E2 in H.pylori-induced gastric cancer in a mouse model.