To further our understanding of host-pathogen interactions, we need to be able to dissect at the cellular and molecular levels the appropriate immune and neuronal elements involved in host protection. With the advent of flow cytometry, we can now evaluate the immune reactivity and function of specific lymphoid and leukocyte subpopulations. To be able to obtain sorted immune cell subsets will enhance our current NIH-funded programs by enabling us to analyze greater than 95% pure cell populations in cellular and molecular assays. To meet such need, we propose to purchase a FACSCalibur flow cytometry system whereby we will be able to obtain such sorted cell subsets. While characterizing distinct cell subsets from inductive and effector immune tissues can provide insights into the nature of the immune response, the focus of this grant application is to advance our capabilities to the next level of sophistication in this characterization. In this effort, the FACSCalibur will be able to obtain sorted cell populations for further analysis. For experimental vaccine efficacy studies to human pathogens, this system will enable us to obtain specific T cell subsets from peripheral and mucosal tissues, e.g., alpha/beta versus delta/gamma TCR, CD4+ versus CD8+ T cells, memory versus naive T cells plus those T cell subsets that express substance P receptors. Such capability will enable our studies to determine the T cell subsets that are specifically responding to vaccine antigen, and will enable us to follow the co-segregation of SP receptor-positive populations for cytokine determinations. We will also be able to follow Lymphoid cell trafficking as a consequence of infection or vaccine testing, and perform subsequent functional studies from these sorted populations. Further, we will be able to identify those host cells (marginal zone macrophages) which support adhesion of infectious agents, e.g., Candida, for subsequent elimination. We will be able to specifically sort those host cells infected with Candida, and identify both host and parasite ligand responsible for this adhesion event. Specific characterization of host defense function can be further developed through the analysis of NADPH oxidase-inhibitory peptides loaded into neutrophils to examine the role of NADPH-dependent oxidative killing. The FACSCalibur will allow us to track peptide-fluorochrome loading efficiency and quantitatively determine inhibitory dose-response relationships in individual leukocyte subpopulations. Thus, the FACSCalibur flow cytometry system will advance our institution's capabilities to analyze at the single cell level the function of immune lymphoid cells and leukocytes in various disease models.
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