Helicobacter hepaticus -infected Rag 2 -/- mice emulate many aspects of human inflammatory bowel disease, including the development of colitis and colon cancer. To elucidate mechanisms of inflammation-induced carcinogenesis, we undertook a comprehensive analysis of histopathology, molecular damage, and gene expression changes during disease progression in these mice. Infected mice developed severe colitis and hepatitis by 10 wk post-infection, progressing into colon carcinoma by 20 wk post-infection, with pronounced pathology in the cecum and proximal colon marked by infiltration of neutrophils and macrophages. Transcriptional profiling revealed decreased expression of DNA repair and oxidative stress response genes in colon, but not in liver. Mass spectrometric analysis revealed higher levels of DNA and RNA damage products in liver compared to colon and infection-induced increases in 5-chlorocytosine in DNA and RNA and hypoxanthine in DNA. Paradoxically, infection was associated with decreased levels of DNA etheno adducts. Levels of nucleic acid damage from the same chemical class were strongly correlated in both liver and colon. The results support a model of inflammation-mediated carcinogenesis involving infiltration of phagocytes and generation of reactive species that cause local molecular damage leading to cell dysfunction, mutation, and cell death. There are strong correlations among histopathology, phagocyte infiltration, and damage chemistry that suggest a major role for neutrophils in inflammation-associated cancer progression. Further, paradoxical changes in nucleic acid damage were observed in tissue- and chemistry-specific patterns. The results also reveal features of cell stress response that point to microbial pathophysiology and mechanisms of cell senescence as important mechanistic links to cancer.

Background & Aims-Transgenic, insulin-gastrin (INS-GAS) mice have high circulating levels of gastrin. On a FVB/N background, these mice 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 non-helicobacter microbiota overgrowth. We determined if germ-free INS-GAS mice spontaneously develop GIN and if H. pylori accelerates GIN in gnotobiotic INS-GAS mice.

Helicobacter hepaticus causes chronic hepatitis and liver cancer in mice. It is the prototype enterohepatic Helicobacter species and a close relative of Helicobacter pylori, also a recognized carcinogen. Here we report the complete genome sequence of H. hepaticus ATCC51449. H. hepaticus has a circular chromosome of 1,799,146 base pairs, predicted to encode 1,875 proteins. A total of 938, 953, and 821 proteins have orthologs in H. pylori, Campylobacter jejuni, and both pathogens, respectively. H. hepaticus lacks orthologs of most known H. pylori virulence factors, including adhesins, the VacA cytotoxin, and almost all cag pathogenicity island proteins, but has orthologs of the C. jejuni adhesin PEB1 and the cytolethal distending toxin (CDT). The genome contains a 71-kb genomic island (HHGI1) and several genomic islets whose G؉C content differs from the rest of the genome. HHGI1 encodes three basic components of a type IV secretion system and other virulence protein homologs, suggesting a role of HHGI1 in pathogenicity. The genomic variability of H. hepaticus was assessed by comparing the genomes of 12 H. hepaticus strains with the sequenced genome by microarray hybridization. Although five strains, including all those known to have caused liver disease, were indistinguishable from ATCC51449, other strains lacked between 85 and 229 genes, including large parts of HHGI1, demonstrating extensive variation of genome content within the species.genomics ͉ pathogenicity island ͉ evolution

Inflammation associated with bacterial infections is a risk factor for cancers in humans, yet its role in breast cancer remains poorly understood. We have previously shown that innate immune inflammatory response against intestinal bacteria is sufficient to induce colon cancer. Here we report that infecting Rag2-deficient C57BL/6 Apc Min/+ mice with an intestinal bacterial pathogen, Helicobacter hepaticus, significantly promotes mammary carcinoma in females and enhances intestinal adenoma multiplicity by a tumor necrosis factor A (TNFA)-dependent mechanism. The mammary and intestinal tumor development as well as the increase in proinflammatory mediators is suppressed by adoptive transfer of interleukin 10-competent CD4 + CD45RB lo CD25 + regulatory (T R ) cells. Furthermore, prior exposure of donor mice to H. hepaticus significantly enhances antitumor potency of their T R cells. Interestingly, these microbially experienced T R cells suppress tumorigenesis more effectively in recipient mice irrespective of their tumor etiology. These data suggest that infections with enteric pathogens enhance T R -cell potency and protect against epithelial cancers later in life, potentially explaining paradoxical increases in cancer risk in developed countries having more stringent hygiene practices. The possibility that dysregulated gut microbial infections in humans may lead to cancer in anatomically distant organs, such as breast, highlights the need for novel immune-based strategies in cancer prevention and treatment. (Cancer Res 2006; 66(15): 7395-400)

Recombinase-activating gene-2-deficient (Rag2 ؊/؊ ) mice lacking functional lymphocytes provide a useful model of chronic inflammatory bowel disease-emulating events in human colon cancer. Infection of Rag2 ؊/؊ mice with Helicobacter hepaticus led to accumulation of macrophages and neutrophils in the colon, a process temporally related to up-regulation of tissue inducible nitric oxide synthase (iNOS) expression at the site of infection and increased nitric oxide (NO) production, as evidenced by urinary excretion of nitrate. Progressive development of increasingly severe inflammation, hyperplasia, dysplasia, and cancer accompanied these changes. Concurrent administration of an iNOS inhibitor prevented NO production and abrogated epithelial pathology and inhibited the onset of cancer. The presence of Gr-1 ؉ neutrophils and elevated tumor necrosis factor-␣ (TNF-␣) expression in colon were required for increased iNOS expression and cancer, whereas interleukin-10 (IL-10) down-regulated TNF-␣ and iNOS expression and suppressed cancer. Anti-inflammatory CD4 ؉ regulatory lymphocytes also down-regulated iNOS and reduced cancer formation. Collectively, these results confirm essential roles for inflammation, increased TNF-␣ expression, and elevated NO production in colon carcinogenesis.colorectal cancer ͉ IBD ͉ innate immunity C hronic Helicobacter pylori infection in humans leads to gastritis and has been established as a causative agent for human gastric cancer (1). Inflammatory bowel diseases (IBDs), such as Crohn's disease and ulcerative colitis, also increase risk for colon cancer (2, 3). Generation of nitric oxide (NO) by inducible NO synthase (iNOS) is a central feature of chronic inflammatory diseases in the gastrointestinal tract (4-9), but precise mechanistic roles for NO in colon cancer development remain undefined.Colon cancer patients exhibit evidence of nitrosative and oxidative stresses that increase cancer risk (10), resulting from mutagenic reactive oxygen and nitrogen species derived from NO generated by immune cells (6,(11)(12)(13)(14)(15). Roles for chronic bacterial infection in IBD and colon cancer have been identified recently in recombinase-activating gene-2-deficient mice (Rag2 Ϫ/Ϫ ), which completely lack functional lymphocytes (16-18). We have exploited this mouse model of chronic IBDassociated cancer for studies of the role of NO and its products because it emulates naturally occurring inflammatory events in humans (16,19,20).Rag2 Ϫ/Ϫ mice have been used to assess functions of lymphocytes by adoptive transfer. Populations of CD4 ϩ T cells with low or high expression of CD45RB (17,21, 22) or CD25 (16,18,23,24) prevent or accelerate colitis in these animals. In wild-type (wt) mice, protection against inflammatory pathology induced by bacterial infection has been attributed to interleukin-10 (IL-10) and IL-10-dependent functions of CD4 ϩ cells (18,20,25,26). Collectively, this evidence forms the rationale for the hypothesis that NO overproduction comprises a linkage between Helicobacter hepaticus-i...

Objectives Gastric colonisation with intestinal flora (IF) has been shown to promote Helicobacter pylori (Hp)-associated gastric cancer. However, it is unknown if the mechanism involves colonisation with specific or diverse microbiota secondary to gastric atrophy. Design Gastric colonisation with Altered Schaedler’s flora (ASF) and Hp were correlated with pathology, immune responses and mRNA expression for proinflammatory and cancer-related genes in germ-free (GF), Hp monoassociated (mHp), restricted ASF (rASF; 3 species), and specific pathogen-free (complex IF), hypergastrinemic INS-GAS mice 7 months postinfection. Results Male mice cocolonised with rASFHp or IFHp developed the most severe pathology. IFHp males had the highest inflammatory responses, and 40% developed invasive gastrointestinal intraepithelial neoplasia (GIN). Notably, rASFHp colonisation was highest in males and 23% developed invasive GIN with elevated expression of inflammatory biomarkers. Lesions were less severe in females and none developed GIN. Gastritis in male rASFHp mice was accompanied by decreased Clostridum species ASF356 and Bacteroides species ASF519 colonisation and an overgrowth of Lactobacillus murinus ASF361, supporting that inflammation-driven atrophy alters the gastric niche for GI commensals. Hp colonisation also elevated expression of IL-11 and cancer-related genes, Ptger4 and Tgf-β, further supporting that Hp infection accelerates gastric cancer development in INS-GAS mice. Conclusions rASFHp colonisation was sufficient for GIN development in males, and lower GIN incidence in females was associated with lower inflammatory responses and gastric commensal and Hp colonisation. Colonisation efficiency of commensals appears more important than microbial diversity and lessens the probability that specific gastrointestinal pathogens are contributing to cancer risk.

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