Microsporidia are obligate intracellular parasites that cause opportunistic and emerging infections in humans and animals. No universally effective drugs exist to treat microsporidiosis and very little is known about the immune response mechanisms employed by the host to control and kill microsporidia. Resistance to lethal disease depends upon CD8+ T cells and IFN-g since mice genetically deficient for either trait and persons with AIDS are susceptible to lethal disease. In addition, microsporidia often replicate in macrophages and disseminate to cause systemic infections via infected trafficking monocytes/macrophages, yet macrophages are the only cells presently known that can kill microsporidia. The hypothesis of this application is that CD8+ T cells and macrophages both function in resistance to microsporidiosis, and the specific aims of this proposal are: ? ? 1. Define the role of CD8+ T cells in controlling microsporidia infections. Sensitized antigen-specific murine CD8+ T cells will be assayed for their ability to kill extracellular microsporidia by direct contact or by release of cytolytic factors. Secondly, these CD8+ T cells will be assayed for their ability to kill microsporidia-infected host cells (macrophages or epithelial cells) as well as for their ability to kill the microsporidia within the infected target cells. MHC-restriction, antigen specificity, and the cytolytic mechanisms used to kill the target cell will be determined. Finally, the effector CD8+ T cells will be assayed for their ability to secrete signals (cytokines) to activate macrophage-mediated destruction of microsporidia. ? ? 2. Define the role of macrophages in controlling microsporidia. Macrophages will be activated with known mediators (IFN-g and LPS) or incubated with sensitized/effector CD8+ T cells (or their cytokines) to monitor microsporidia killing. Mechanisms by which the macrophages kill microsporidia will be determined, including the roles of reactive nitrogen intermediates (RNl's), reactive oxygen intermediates (ROl's), peroxynitrite, and iron starvation. ? ? 3. Define the interaction between CD8+ T cell and macrophage pathways. Based on the results of specific aims 1 and 2, adoptive transfer experiments will be performed to determine if the CD8+ T cell mechanisms and macrophage-mediated killing mechanisms of microsporidia function, a) through independent pathways (where each pathway is required), b) as backuphedundant mechanisms (where each pathway can compensate for the other if one pathway is lacking), and/or c) by directly interactive pathways such as the CD8+ T cells releasing signals for macrophages to kill microsporidia. ? ? These studies will employ three species of microsporidia which infect humans and that replicate in different sites within macrophage host cells. Encephalitozoon species replicate within host cell-derived parasitophorous vacuoles, Vitfaforma corneae replicates in direct contact with the host cell cytoplasm, and Trachipleistophora horninis replicates within parasite-derived sporophorous vesicles. In addition, mechanisms employed to kill both intracellular and extracellular microsporidia will be defined. This model will provide a better understanding of how the immune response functions to control microsporidia that replicate in different sites within a host cell.
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