Figure 10.

Effect of regulation of arginase-inducible nitric oxide synthase (iNOS) balance in Leishmania donovani survival inside macrophages. THP-1 monocyte-derived MΦs were either left untreated or preincubated with BEC (200 µM) or L-NIL (100 ng/ml) for 3 h followed by infection with L. donovani for 48 h (A,B,E,G,H). In another experiment, THP-1 MΦs were transfected with control siRNA, Arg-1, Arg-2, or iNOS siRNA followed by infection with L. donovani for 48 h (C,D,F,I,J). Intracellular parasites were visualized by staining the cells with Giemsa followed by optical microscopy at 100× oil immersion. The parasite load was measured by counting the number of intracellular amastigotes per 100 macrophages (A,C). The rate of infection was analyzed by counting the percent infected macrophages (B,D). Extracellular nitrite level was measured by Griess method using NaNO₂ as standard (E,F). The polyamines (putrescine and spermidine) were extracted by TCA precipitation followed by dansyl chloride derivatization, separation by reverse-phase high performance liquid chromatography as described in “Materials and Methods.” Dansylated putrescine (G,I) and dansylated spermidine (H,J) were quantified by fluorescence spectrometry. Each experiment was repeated three times. Each determination was made in triplicate and the values were expressed as mean ± SD for three independent experiments. Kruskal–Wallis with Dunn’s multiple comparison test was used to evaluate statistical significance for comparing three or more groups; *p < 0.05; **p < 0.005; ns, non-significant.