Fig 7.

Primed phagocytes challenged with the pkh2-02Δ mutant produce increased levels of TNF-α and ROS relative to those challenged with the wild type. (A) The indicated strains were opsonized with MAb 18B7 for 1 h and cocultured with J774 cells that had been prestimulated with LPS and IFN-γ for 24 h. Supernatants were collected and analyzed for the presence of TNF-α as described in Materials and Methods. (B) Opsonized C. neoformans cells were cocultured with LPS- and IFN-γ-prestimulated J774 cells for 2 h. The nitrite concentration in the supernatants was determined using the Griess reagent kit. (C and D) The indicated strains were opsonized and cocultured with PMA-stimulated J774 cells. ROS were measured by luminal-enhanced chemiluminescence as described in Materials and Methods. For all panels, the solid bars indicate mean values and error bars indicate the standard deviation for at least two independent experiments performed in triplicate. Differences between strains were analyzed by Student's t test with statistical significance set at P < 0.05. NS, not significant.