New and improved anti-microbial therapies are needed due to increasing immunocompromise populations and increasing spread of drug-resistant strains of microorganisms. Phagocytes (PC), important in pathogen clearance, can be inhibited so effectively by pathogens that virulence increases as PCs become the infectious agent reservoir and/or as susceptibility to secondary infections rises. Phagocytosis and endocytosis are among the important cellular functions suppressed by many notorious human pathogens, including influenza virus (IV), M. leprae and M. tuberculosis. These intracellular vesicle-based transport processes are central to both normal PC function and to most therapies, including drug delivery, modulation of activation and antigen processing. Not enough is known about either the specific phagosomal-endosomal processes affected or the physiological and biochemical mechanisms pathogens utilize to rationally design interventions to restore PC function. The major goal of this proposal is to determine what physiological changes occur in which phagosome-endosome processes resulting in failure of these anti-microbial cellular activities during IV interaction with a well-characterized phagocytic cell the J774.1 murine macrophage. Information on macrophage- pathogen effects is lagging that for other PCs, but during immunosuppression they can become the predominate remaining PC. IV is an important disease and many of its effects on PCs are similar to those of bacteria, but it is simpler and therefore the prospect of identifying important PC inhibitor mechanism(s) and improving anti-viral therapies is increased.
The specific aims are: 1) Identify the extent and type of changes in macrophage intracellular trafficking pathways after exposure to IV. 2) Determine what changes occur in the intracellular physical-chemical environment of macrophages and intracellular compartments during early influenza virus exposure. 3) Investigate the effects of agents which either mimic or antagonize the IV-caused alterations in macrophage endosome and phagosome function. Quantitative digital video microscopy will be used to examine effects of IV on the J774 intracellular vesicle- based transport functions including the uptake and movement between compartments of molecular markers for specific endocytic and phagocytic functions. fluorescent probes will also be used to monitor the physical- chemical state of phagosomes and endosomes. It is expected that both the methodologies and cell biological findings will be applicable to understanding PC inhibition by other virus types, bacteria and parasites.
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