Experimental Models of Mycobacterial Replication and Killing in Macrophages

<p>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 MAI did not replicate in freshly explanted monocytes [generation time of 408h]. MAI 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. </p>

Name thesis:degree_name

Doctor of Philosophy (PhD)

Level thesis:degree_level

Thesis

Year

1997

Author dc:creator

• Laochumroonvorapong, Pairote

Contributors dc:contributor

• Gilla Kaplan

• mycobacteria
• monocytes
• apoptosis
• oxidative killing
• hydrogen peroxide
• cytokines
• Life Sciences

Repository record dc:identifier

https://digitalcommons.rockefeller.edu/student_theses_and_dissertations/458

OAI identifier oai:identifier

oai:digitalcommons.rockefeller.edu:student_theses_and_dissertations-1463