Student Theses and Dissertations

Date of Award

1997

Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

RU Laboratory

Cohn-Steinman Laboratory

Abstract

This thesis investigates how human mononuclear phagocytes regulate the growth of mycobacteria. Previous studies carried out in our laboratory suggested that cell turnover [apoptosis and necrosis] within a granuloma may contribute to the control of mycobacterial infection. The experiments described here demonstrated that both apoptosis [Fas-mediated] and necrosis [perforin-induced] of infected human monocytes do not affect the survival of intracellular mycobacteria in an in vitro infection model. To determine the role of apoptosis and necrosis on mycobacterial infection in vivo, perforin gene-disrupted mice and Fas receptor-defective mice were experimentally infected with mycobacteria. The loss of either intact Fas pathway or perforin gene did not render mice more susceptible to mycobacterial infection, confirming the findings in vitro. Further studies in vitro of the killing of intracellular mycobacteria indicated an oxidative [H2O2-induced] killing mechanism. This may be physiologically significant since release of H2O2 has been documented following phagocytosis of mycobacteria by human monocytes. In vitro, there was an inverse relationship between intracellular growth rate and susceptibility to H2O2 To determine whether different species of mycobacteria vary in their susceptibility to oxidative killing, Mycobacterium tuberculosis [H37Ra] and a clinical isolate of M. avium-M.intracellulare [MAI] were grown in cultured human monocytes, and the intracellular growth rate, susceptibility to killing by H2O2, and cytokine induction were assessed. M. tuberculosis H37Ra replicated with a generation time of 48h while the isolate of MAl did not replicate in freshly explanted monocytes [generation time of 408h]. MAl was more sensitive to oxidative killing than H37Ra, although both mycobacterial species were equally sensitive to H2O2 treatment in cell-free culture media and in sonicated cell suspensions. Maturation of human monocytes in culture for 8 days prior to infection with H37Ra resulted in more restricted intracellular growth and greater susceptibility to H2O2-induced killing of the bacilli. The patterns of cytokine production in response to H37Ra infection were different in freshly explanted and 8d monocytes, suggesting that the cytokines produced by infected monocytes may contribute to the regulation of intracellular mycobacterial growth.

Comments

A thesis presented to the faculty of The Rockefeller University in partial fulfillment of the requirements for the degree of Doctor of Philosophy

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