In Vivo Role of NO in Mucosal Inflammation and Cancer

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This Project will characterize the role of nitric oxide (NO) and NO-derived species in mouse models of inflammatory bowel disease (IBD) and cancer. These studies will use 129/SvEv Recombinase-activating gene 2 (Rag-2) knockout (KO) mice and C57BL/6 T cell receptor (TCR) alphabeta KO mice. Rag-2 KO mice on a 129/SvEv background develop severe IBD and cancer when infected with H. hepaticus. Histopathologic lesions and the pattern of macrophage and neutrophil infiltration will be correlated with biomarkers for nitration, oxidation, and halogenation of DNA and proteins in Rag-2 KO mice. These biomarkers will be further validated by comparing disease in naive and effector CD45RB high T cell-bearing Rag-2 KO mice, and by modulating NO production with N-methylarginine (NMA). TCR alphabeta KO mice develop severe IBD when infected with H. hepaticus. IBD in TCRbeta KO mice, like in Rag-2 KO mice, is a model for Crohn's disease (CD) and features activated macrophages and abundant interferon-gamma (IFN) and tumor necrosis factor-alpha (TNF). TCR alpha KO mice are a model for ulcerative colitis (UC), and are dependent on interleukin-4 (K,-4) but not IFN or TNF for the development of disease. Histopathologic lesions and cytokine levels will be correlated with biomarkers in TCR alpha and TCR beta KO mice. To further characterize the role of NO and NO-derived species in IBD, we will compare pharmacologic inhibition of inducible nitric oxide synthase (iNOS) with genetic inactivation of the enzyme by generating iNOS-deficient TCR alphabeta KO mice. In vitro analysis of somatic mutations arising in vivo in the presence and in the absence of NO and NO-derived species will also be performed. By using a novel IBD associated cancer model and models for UC and CD, we will be able to validate new biomarkers for nitration, oxidation, and halogenation of DNA and proteins. We will also gain a better understanding of the role of NO and NO-derived species across a broad range of carcinogenic events, including genotoxicity, cellular proliferation, cytotoxicity, and angiogenesis. The Specific Aims are: Specific Aim #1. Characterize the role of NO an oxidative stress in IBD and cancer in Rag-2 KO mice Specific Aim #2. Characterize the role of NO and oxidative stress in IBD in TCR alphabeta KO mice