Defense Date


Document Type


Degree Name

Master of Science


Microbiology & Immunology

First Advisor

Francine Marciano-Cabral


In the present study, we have examined the differences among Corynebacterium parvum-activated, thioglycollate-elicited, and resident macrophages from (C57BL/6 X C3H)F1 mice utilizing two microcytotoxicity assays. Macrophage and Naegleria fowleri were interchangeably employed as effector and target cells. To assess the ability of the macrophage to lyse N. fowleri, the amoebae were labeled with 5 μCi of 3H-uridine per 5 x 10^5 cells for 18 h prior to co-culturing with adherent macrophage monolayers. Although the macrophage-induced lysis was density and time dependent, C. parvum macrophages consistently demonstrated an increased cytotoxic response to N. fowleri after 24 h co-incubation at a 10:1 effector-to-target ratio. In contrast, thioglycollate and resident macrophages were unable to elicit a cytotoxic response to N. fowleri.

To ascertain the ability of N. fowleri to destroy the three macrophage populations, a 51cr post-labeling technique was employed. After 24 h of co-incubation, uptake of 51cr into remaining C. parvum-activated and thioglycollate-elicited macrophages exceeded that for resident macrophages. Parallel electron microscopic studies confirmed simultaneous destruction of both amoebae and macrophages. Concurrent studies utilizing cell-free lysates of N. fowleri displayed the same divergent spectrum of macrophage destruction for the different macrophage populations as was seen with N. fowleri trophozoites.

Catalase is an enzyme which breaks down hydrogen peroxide, a chemical released from activated macrophages, which could contribute to macrophage-induced cytotoxicity. Leupeptin is an inhibitor of some serine and cysteine proteases. Activated macrophages also secrete proteases which may contribute to macrophage-induced cytotoxicity. Catalase, leupeptin, or hydrogen peroxide were added to macrophage cultures or N. fowleri lysate prior to coincubation or during co-incubation to determine possible roles of hydrogen peroxide and proteases in macrophage susceptibility to N. fowleri lytic substances. Exogenously added hydrogen peroxide had no effect on resident macrophage survival. Catalase and leupeptin produced effects unique for the macrophage cell type, with survival closely correlating to the types and quantities of cytotoxic substances associated with the macrophage. Results suggest that macrophage susceptibility to N. fowleri lysate may be due to the absence or presence of two or more macrophage components.


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