Figure 5. Arg1 expression and polyamine content in L. donovani infected macrophages.

A) Arginase activity in macrophages isolated by adherence from single-cell suspensions from the spleens of control hamsters (0 days post-infection) and hamsters infected with L. donovani for 7, 14, 21, 42, and 56 days. The mean and SD (error bars) of the arginase activity in 100,000 cells, determined by assay of urea production, is shown from a single experiment that is representative of 2 independent experiments. B) Log₁₀ ratio of host arg1 to NOS2 mRNA in splenic macrophages of 4-week L. donovani infected mice and hamsters. The mRNA expression was determined by real time RT-PCR in groups of 5 animals and used to calculate the log ratios, shown as the mean and SD (error bars) from a single experiment that is representative of 2 independent experiments. The ratios were determined from the following raw data: Arg1 mRNA fold-increase with reference to the BHK cell calibrator (mean ± SD): uninfected mice, 5.7±5.9; uninfected hamster, 3,367±2,858, infected mice, 8.5±10; infected hamster, 131,984±56,407; iNOS mRNA fold-increase with reference to BHK cell calibrator (mean ± SD): uninfected mice, 133.34±182.4; uninfected hamster, 1.03±0.3, infected mice, 2,984±4,535; infected hamster, 19.56±10.87). C) Arginase activity in peritoneal macrophages isolated from mice and hamsters that were uninfected (open bars) or infected in vitro with L. donovani for 48 hrs (filled bars). The mean and SD (error bars) of the arginase activity, determined by assay of urea production, is shown from a single experiment that is representative of 2 independent experiments. D) Arginase activity in hamster peritoneal macrophages that were uninfected (Un) or infected in vitro with L. donovani (1∶5 macrophage:parasite ratio). Parasites were either unopsonized (None), opsonized with normal fresh complement-containing hamster serum (Comp), or opsonized with freeze-thawed hamster serum containing anti-Leishmania antibody (Ab). The mean and standard deviation (error bars) of the arginase activity in 200,000 cells of 6 different samples determined by assay of urea production, is shown from a single experiment that is representative of 2 experiments. Statistical comparisons are made to the control group. E) Expression of hamster arginase mRNA in hamster peritoneal macrophages that were uninfected (Un) or infected in vitro with L. donovani stationary-phase promastigotes for 24 hrs (Inf). The mean and SEM (error bars) of the fold-increase of arg1 mRNA relative to BHK cells, determined by real time RT-PCR in groups of 6 samples per experiment, is shown as data pooled from 4 independent experiments. F) The concentration of polyamines in uninfected (open bars) and 48-hour in vitro infected hamster peritoneal macrophages (filled bars) (n = 6 per group) is expressed as the mean and SD (error bars) of nmol polyamine per mg protein. The data shown are from a single experiment that is representative of 2 independent experiments. The statistical significance of differences in each of the panels is identified by asterisks (*, p<0.05; **, p<0.01; ***, p<0.001).