FIG. 5.
Effect of hemozoin on NOS2 mRNA expression. NOS2 mRNA was quantified for three individuals by real-time RT-PCR. PBMCs (106 cells/ml) were obtained from healthy, malaria-naïve U.S. donors and cultured with medium alone, LPS (100 ng/ml) and IFN-γ (200 U/ml), or LPS and IFN-γ in the presence of hemozoin (10, 1.0, or 0.1 μg/ml). Cells were collected at 48 h for NOS2 mRNA determination by real-time RT-PCR. Data were compared using the ΔΔCT method, where the endogenous control gene (β-actin) cycle threshold (CT) value was subtracted from the experimental gene (NOS2) CT for each sample. The change in CT (ΔCT) for each experimental sample was then subtracted from the ΔCT of the unstimulated (medium alone) control sample. Change (n-fold) was expressed as 2−ΔΔCT relative to results from unstimulated conditions. Values are the means ± SEMs of results from three samples. Statistical significance was determined by the Mann-Whitney U test. *, P value of <0.05 compared to results for LPS+IFN-γ stimulation conditions.