| Budget Amount *help |
¥2,700,000 (Direct Cost: ¥2,700,000)
Fiscal Year 1998: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 1997: ¥1,600,000 (Direct Cost: ¥1,600,000)
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| Research Abstract |
In our laboratory, Trypanosoma cruzi, the parasitic protozoan flagellate that causes Chagas' disease, and infected HeLa cells are utilized as a model system in which to investigate the cell biology and pathology of the host cells. We report here that T.cruzi infection inhibits Fas-mediated apoptosis in HeLa cells. Exponentially growing HeLa cells were inoculated into each well of a 24-well plate, and allowed to grow at 37ﾟC for 2 days. The cells were infected with T.cruzi trypomastigotes and further incubated for 4-5 days. Apoptosis was induced in control and infected HeLa cells, by the addition of anti-Fas monoclonal antibody. The cultures were further incubated, washed with PBS, and stained with Giemsa or Hoechst 33342 for observation under a microscope. To examine the translocation of phospholipids from the inside to the outside of the plasma membrane, Fas-induced HeLa cells were stained with a fluorescein-labeled probe specific for phosphatidylethanolamine and analyzed by flow cytometry. One day after the addition of anti-Fas antibody, most of the control HeLa cells appeared roundish and detached from the well. In contrast, some infected cells showed an apparently normal morphology, adhering to the well. Six hours after the induction of Fas-mediated apoptosis, nuclear staining with Hoechst 33342 showed that 59.5 % of control cells were undergoing apoptosis, whereas 30.0 % of the infected cells were apoptotic, indicating that T.cruzi infection markedly inhibits host-cell apoptosis. The control population of HeLa cells contained more fluorescein-positive cells than the infected cell population. This implies that the translocation of phosphatidylethanolamine from the inner side to the outer side of the plasma membrane takes place in an early stage of apoptosis. Inhibition of Fas-mediated apoptosis may constitute a novel immune escape mechanism for T.cruzi . Potentially critical molecules from parasite and host cells are now under investigation.
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