Screening Helicobacter pylori genes induced during infection of mouse stomachs Our efforts resulted in H. pylori specific antibiotic-based in vivo expression technology (IVET) systems that were used to identify in vivo induced (ivi) genes in mice and cultured macrophage. These IVET screenings identified 31 novel in vivo induced genes (ivi genes) which had not been previously reported. These novel ivi genes are possible additional virulence factors essential for colonization or pathogenesis of the stomach mucosa. They belong to several functional gene families, including several well-known virulence factors that are expressed by the bacterium in infected mouse stomachs. Virulence factors, vacA and cagA, were found in this screen and are known to play important roles in H. pylori infection, colonization and pathogenesis. Transcription profiles of all ivi genes were confirmed by real time PCR analysis of H. pylori RNA isolated from H. pylori infected RAW 264.7 macrophages. We compared the expression profile of H. pylori and RAW 264.7 coculture with that of H. pylori only. Some genes such as cag A, vac A, lpxC, murI, tlpC, trxB, sod B, tnpB, pgi , rbfA and infB showed a 2-20 fold upregulation. Statistically significant upregulation was obtained for all the above mentioned genes (P <0.05). tlp C, cag A, vac A, sod B, rbf A, inf B, tnpB, lpxC and murI were also significantly upregulated (P <0.01). Host mucosal and immune responses to Helicobacter pylori infection In collaboration with Dr. Griffin Rodgers research group, NHLBI, we investigated the role of olfactomedin 4 (OLFM4), an olfactomedin-related glycoprotein, in neutrophil bactericidal capability and host innate immunity. Neutrophils from OLFM4-/- mice have increased intracellular killing of Staphlocococus aureus and Escherichia coli in vitro. The OLFM4-/- mice also have enhanced in vivo bacteria clearance and are more resistant to sepsis when challenged with S. aureus or E. coli by intra-peritoneal injection. OLFM4 is found to interact with cathepsin C. We demonstrated that OLFM4 is a direct substrate of cathepsin C and inhibited cathepsin C activity in vitro and in vivo. The cathepsin C activity in neutrophils from OLFM4-/- mice is significantly higher than that in neutrophils from wild-type littermate mice. The activities of serine proteinases, neutrophil elastase, cathepsin G and proteinase 3, which require cathepsin C activity for processing and maturity, are also higher in OLFM4-/- neutrophils. These results indicate that OLFM4 is an important negative regulator of neutrophil bactericidal activity by restricting cathepsin C activity and its downstream granule-associated serine proteases. Ongoing research activities: OLFM4 is a novel anti-inflammatory mediator in H. pylori infection, which plays an important role in the regulation of host resistance and gastric inflammatory response to H. pylori infection. Our previous studies show that H. pylori colonization in the gastric mucosa of OLFM4-/- mice was significantly lower compared with wild-type littermates. Production and expression of proinflammatory cytokines/chemokines such as IL-1, IL-5, IL-12 p70, and MIP-1 were increased in OLFM4-/- mice compared with infected controls. However, the mechanism of its role in biological processes such as inflammation or other immune response is uncertain. In this study, we generated MyD88-/-/OLFM4-/- mice to investigate the potential role of MyD88 on the effect of OLFM4 in gastric mucosal responses to H. pylori infection. Our findings indicate that MyD88-/-/OLFM4-/- deficiency results in: 1) similar H. pylori colonization as wild-type littermates;2) less infiltration of inflammatory cells compared with OLFM4-/- gastric mucosa;and 3) enhanced immune and inflammatory responses. Expression of proinflammatory cytokines/chemokines such as IL-1, IL-5, IL-12 p70, and MIP-1 seen in OLFM4-/- mice was abrogated in MyD88-/-/OLFM4-/- mice. The loss of feedback inhibition of the NF-kappaB pathway and the resulting enhanced NF-kappaB activation in OLFM4-/- mice were reversed in MyD88-/-/OLFM4-/- mice, which may explain the observed decrease in inflammation and immune response and the subsequent more persistent colonization of H. pylori. Together these observations indicate that MyD88 is critical for the action of OLFM4 which exerts considerable negative influence on the host defenses against H. pylori infection mediated via NF-kappaB activation;reducing subsequent cytokine and chemokine production. These changes resulted in decreased OLFM4 inhibition of the host immune responses and enhanced of persistence of H. pylori colonization. Our recent preliminary studies in collaboration with Dr. Hubert C. Morses group indicated that IRF8 is upregulated in stomach tissues of H. pylori infected mice. Thus, examining IRF8 as a new regulatory factor in gastric epithelial cells will have major implications for understanding how pathogenic microorganisms affect IRF8 expression in mucosal cells and how IRF8 regulates the innate immune system. IRF8 is broadly expressed in hematopoietic lineage cells and plays an important role in innate and adaptive immunity. Recent findings suggest that IRF8 is also expressed in non-hematopoietic cells such as intestine, stomach, eye and brain. In this proposed study, we take advantage of our mouse models bearing genetically engineered IRF8 alleles to ask whether and how IRF8 is involved in gastric mucosal innate immunity against H. pylori infection. H. pylori infection damages the gastric epithelial barrier and triggers innate immune response. Adaptive immune responses are also elicited following infection, especially the induction of Th17 cells. In collaboration with Dr. Kamal Datta of Georgetown University Medical Center we assess the risk estimate of gastrointestinal cancer after combined radiation and Helicobacter pylori exposure. Exposure to low dose ionizing radiation from medical procedures and Helicobacter pylori infection has been strongly implicated in gastrointestinal (GI) cancer initiation and progression. A significant fraction of the population in the United States (33%) carries H. pylori infection and exposure to low dose ionizing radiation has increased due to surge in diagnostic and therapeutic procedures. Considering the high spontaneous incidence of GI cancer and higher probability of exposure to both agents, an even modest increase after combined radiation and H. pylori exposure could have a significant effect on health risk estimates for population in this country. However, any synergetic or additive effects of combined low dose radiation and H. pylori exposure on promoting GI tumorigenesis remains unexplored. Both radiation and H. pylori are Group I carcinogen according to the International Agency for Research on Cancer (IARC) and both the agents modulate immune response and induce persistent inflammation known for promoting cancer. The collaborative projects include: 1. To quantitatively determine H. pylori and radiation induced gastrointestinal tumor incidence and grade using the APCMin/+ tumor model. 2. To identify immune function and inflammatory pathway changes associated with exposure to combined H. pylori and radiation exposure and compared to those induced by H. pylori or radiation exposure alone. 3. To develop and compare toxicogenomic and toxicometabolomic signatures for combined H. pylori and low dose radiation exposure vs. H. pylori or radiation alone. Both H. pylori and radiation are known to induce cancer initiating and promoting events such as oxidative stress, inflammation, and genomic alterations. However, the precise mechanisms by which combined radiation and H. pylori exposure may cause stress and inflammation leading to carcinogenesis in GI tissues are still unresolved.

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