Nitric oxide production and susceptibility to NO-mediated killing. A. Nitrite production in the presence of ALT expression. J774 cells were incubated with medium alone (-ve), or stimulated with IFN-γ (40 U/ml) and LPS (10 ng/ml) (+ve) with or without the arginine analogue N-monomethyl-D-arginine (D-NMMA, 1 mM), or pre-incubated with L. mexicana promastigotes for 4 h before stimulation. Nitrite accumulation in the medium over the subsequent 24 h or 48 h was used as an indicator of NO production and was assayed by the Griess reaction. Each bar represents mean ± standard deviation. B. Survival of transfected Leishmania in activated macrophages. Bone marrow-derived macrophages stimulated with 100 ng/ml LPS and the indicated concentrations of IFN-γ were infected with stationary phase promastigotes of wild-type L. mexicana or transfected alt-1 and alt-2 parasites at a ratio of 10 parasites per cell. After 72 h at 37°C the cells were stained with Giemsa. The percentage of macrophages infected with parasites was determined by counting 4 samples of 100 cells. Each bar represents mean ± standard error of the mean. C. Nitrite production in response to dose of exogenous IFN-γ. Supernatants from the experiment described above (Figure 3B) were recovered and assayed by the Griess reaction for accumulated nitrite. Mo denotes uninfected macrophages. Each bar represents the mean ± standard deviation.