Neutrophils (PMN) and macrophages provide host defense against infection by ingesting and killing microorganisms. These essential functions depend on the creation of a toxic intracellular environment created by the fusion of lysosomes with the organism containing phagosome. One of the goals of this project is to better define the events which regulate this fusion process. Changes in Ca++ concentration and pH have been intimately linked to PMN and macrophages activation and are a major focus of the proposed studies. We have identified a new Ca++ pumping enzyme in human PMN lysosomes which functions at physiologic Ca++, ATP, and H+ concentrations and have proposed that this Ca++ pump plays a major role in activating phagocytes. Using isotopic and fluorescent methods, we will characterize the regulation of calcium uptake by PMN lysosomes and the release of calcium from these organelles. These studies will provide information on normal control mechanisms for triggering phagocytes and will form the basis of a strategy for isolating this Ca++ -ATPase. A combination of affinity and standard chromatographic methods are proposed. Failure to create a phagolysosomal environment sufficiently toxic to kill pathogenic microorganisms results in morbid infectious diseases. Intrinsic cell defects are one class of this failure and the Chediak-Higashi syndrome is a prominent example. The hallmark of this human disorder is giant, abnormal lysosomes which fuse defectively with the phagosome. This results in recurrent, often lethal, infections. While the fundamental defect is unknown, we have recently described an abnormal Ca++ pump in CHS PMN lysosomes. Using human and beige mouse PMN and macrophages lysosomes, we will further examine the nature of this pump and determine whether this represents the first example of an abnormal ion translocating pump which is the proximate cause of human disease. Organisms, including Yersinia, Legionella, and Mycobacteria which survive and multiply within phagolysosomes, cause morbid infectious diseases. Using a model of Yersinia pestis organisms which have been genetically engineered to report the Ca++ and pH conditions in their environment, we have begun to define the ionic constitution of the phagolysosome. Our goal is to identify characteristics of the organism-containing phagolysosome which can be mainipulated as part of a new antimicrobial strategy. We will genetically engineer additional mutants of Y. pestis and use these probes to examine whether modifying the ionic environment alters the virulence of the pathogen.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI016732-11
Application #
3126800
Study Section
Bacteriology and Mycology Subcommittee 2 (BM)
Project Start
1981-01-01
Project End
1992-03-31
Budget Start
1991-04-15
Budget End
1992-03-31
Support Year
11
Fiscal Year
1991
Total Cost
Indirect Cost
Name
Tufts University
Department
Type
DUNS #
City
Boston
State
MA
Country
United States
Zip Code
02111
Katzen, N A; Segal, G M; Vannier, E et al. (1992) Comparison of granulocyte-macrophage colony stimulating factor and interleukin-1 production from human peripheral blood mononuclear cells as measured by specific radioimmunoassays. Eur Cytokine Netw 3:365-72
Georgilis, K; Noring, R; Steere, A C et al. (1991) Neutrophil chemotactic factors in synovial fluids of patients with Lyme disease. Arthritis Rheum 34:770-5
Georgilis, K; Steere, A C; Klempner, M S (1991) Infectivity of Borrelia burgdorferi correlates with resistance to elimination by phagocytic cells. J Infect Dis 163:150-5
Lindler, L E; Klempner, M S; Straley, S C (1990) Yersinia pestis pH 6 antigen: genetic, biochemical, and virulence characterization of a protein involved in the pathogenesis of bubonic plague. Infect Immun 58:2569-77
Klempner, M S; Noring, R; Mier, J W et al. (1990) An acquired chemotactic defect in neutrophils from patients receiving interleukin-2 immunotherapy. N Engl J Med 322:959-65
Styrt, B; Klempner, M S (1988) Lysosomotropic amines modulate neutrophil calcium homeostasis. J Cell Physiol 135:309-16
Styrt, B; Klempner, M S (1988) Effects of pH on killing of Staphylococcus aureus and Escherichia coli by constituents of the neutrophil phagolysosome. J Med Microbiol 25:101-7
Styrt, B; Pollack, C R; Klempner, M S (1988) An abnormal calcium uptake pump in Chediak-Higashi neutrophil lysosomes. J Leukoc Biol 44:130-5
Georgilis, K; Klempner, M S (1988) In vitro effects of omega-3 fatty acids on neutrophil intracellular calcium homeostasis and receptor expression for FMLP and LTB4. Inflammation 12:475-90
Styrt, B; Klempner, M S (1988) Lysosomotropic behavior of adrenergic antagonists in interactions with human neutrophils. Biochem Pharmacol 37:435-41

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