ARG cytosolic localization in <i>arg</i>⁻/+<i>arg</i>ΔSKL add-back opposed to ARG glycosomal compartmentalization in WT and <i>arg</i>⁻/+<i>ARG</i> add-back.

<p>Cytological preparations probed for ARG immunolocalization via electron microscopy of <i>L. amazonensis</i> wild type (WT), <i>ARG</i> knockout (<i>arg</i>⁻), and the add-backs <i>arg</i>⁻/+<i>ARG</i> and <i>arg</i>⁻/+<i>arg</i>ΔSKL promastigotes. Images are representative of at least 8 different sections. Black arrows indicate membrane-surrounded organelles that present the same microscopic pattern as glycosomes. Parasite nuclei (N). Scale bar: 250 nm.</p

ARG absence or improper sub-cellular location impairs <i>L. amazonensis in vitro</i> infectivity.

<p>Peritoneal macrophages infected with <i>L. amazonensis</i> wild type (WT, white bars), <i>ARG</i> knockout (<i>arg</i>⁻, black bars) and the add-backs <i>arg</i>⁻/+<i>ARG</i> (grey bars) and <i>arg</i>⁻/+<i>arg</i>ΔSKL (dashed bars) mutants for 48 and 72 hours. (A) Means (+/− SD) of the rate of macrophage infection from two experiments. (B) Means (+/− SD) of amastigotes per infected macrophage from two experiments. At least 150 macrophages were analyzed for each experiment. ^# indicates values with no significant difference (One-way ANOVA). * p<0.005 and ** p<0.05 (t test).</p

L-arginine cellular concentration is increased in <i>arg</i>⁻ and <i>arg</i>⁻/+<i>arg</i>ΔSKL compared with WT and <i>arg</i>⁻/+<i>ARG</i>.

<p>L-arginine cellular concentration in <i>L. amazonensis</i> wild type (WT), <i>ARG</i> knockout (<i>arg</i>⁻) and the add-backs <i>arg</i>⁻/+<i>ARG</i> and <i>arg</i>⁻/+<i>arg</i>ΔSKL, determined by HPLC analyses of cellular extracts from 10⁷ parasites. Values are the means (+/−SD) of a representative experiment performed in triplicate.</p>*<p>indicates values significantly different compared with WT (p<0.05, t test).</p>§ and #<p>indicates values with no significant difference compared with WT and <i>arg</i>⁻, respectively.</p

<i>L. amazonensis</i> ARG remains compartmentalized during macrophage <i>in vitro</i> infection.

<p>Cytological preparation probed for arginase immunolocalization via electron microscopy. (A) J774A 1 macrophages infected for 24 hours with <i>L. amazonensis</i> promastigotes in the stationary phase (4 different sections). (B) <i>L. amazonensis</i> promastigotes. Black arrows indicate compartmentalized ARG labeling. Parasite nuclei (N), macrophage nuclei (MN) and the kinetoplast (K) are indicated. Scale bar: 200 nm.</p

<i>L. amazonensis in vivo</i> infectivity is impaired by lack of ARG and incorrect location.

<p>BALB/c mice were infected in the posterior left footpad with 10⁶ stationary phase promastigotes cells of <i>L. amazonensis</i> wild type (WT), <i>ARG</i> knockout (<i>arg</i>⁻) and the add-backs <i>arg</i>⁻/+<i>ARG</i> and <i>arg</i>⁻/+<i>arg</i>ΔSKL parasites, and lesion sizes were monitored weekly. Data are presented as the means (+/− SD) of 5 infected mice and are representative of at least 2 different strains of null and add-backs mutants.* p<0.005 (Two-way ANOVA).</p

<i>L. amazonensis</i> ARG protein level and enzymatic activity are impaired by arginase mislocation.

<p>(A) <i>ARG</i> mRNA copies were determined by real time PCR and normalized by <i>GAPDH</i> mRNA copies for <i>L. amazonensis</i> wild type (WT), <i>ARG</i> knockout (<i>arg</i>⁻), and the add-backs <i>arg</i>⁻/+<i>ARG</i> and <i>arg</i>⁻/+<i>arg</i>ΔSKL strains. The obtained values are the means (+/− SD) of at least 2 independent experiments each performed in duplicate. The values for <i>arg</i>⁻/+<i>ARG</i> and <i>arg</i>⁻/+<i>arg</i>ΔSKL are significantly different from those for <i>arg</i>⁻ and WT (p<0.0015*, t test). (B) ARG enzymatic activity from protein extracts of the same strains was determined and is expressed as nmol/min/mg. The obtained values are the means (+/− SD) of 3 independent experiments each performed in triplicate. The values for <i>arg</i>⁻/+<i>ARG</i> are significantly different from those for <i>arg</i>⁻ and <i>arg</i>⁻/+<i>arg</i>ΔSKL (p<0.0001**, t test). (C) ARG and Orc1 (loading control) levels were determined by western blotting of total cell lysates from the same strains before and after treatment with MG-132 50 µM for 24 hours.</p

Effects of JES6-1 treatment on the early CD4⁺ T cell response to <i>P. chabaudi</i> malaria.

<p>(A) C57BL/6 mice were treated with JES6-1 mAb on days 0, 2 and 4 p.i. with 10⁶ PRBC. On day 7 p.i., splenic CD4⁺ T cells were analyzed for CD122, CD25, CD69 and CD44 expression and cell size (FSC). Data show gated CD4⁺ T cells expressing high levels of activation markers and large size (n = 3–4). (B) Non-stimulated (basal) and anti-CD3 mAb stimulated IFN-γ and IL-17 production was evaluated in CD4⁺ T cells from the same groups of mice. (C) Numbers of CD4⁺ T cells per spleen were determined in the same groups of mice. (D) On day 4 p.i., PRBC-stimulated CD4⁺ T cell proliferation was analyzed <i>in vitro</i> in the presence or absence of JES6-1 mAb. Histograms show CFSE fluorescence in gated CD4⁺ T cells. The means ± SD (n = 3–4) of the percentages of replicating (CFSE^LO) cells are shown. In A–D, significant differences compared experimental conditions *p<0.05 with cells from non-infected (NI) mice; **p<0.05 with cells from non-treated (NT) mice; and #p<0.05 with non-stimulated cells. Data are representative of three separate experiments.</p

Effects of JES6-1 treatment on the splenic CD4⁺CD25⁺FoxP3⁺ cell population during <i>P. chabaudi</i> malaria.

<p>(A) C57BL/6 mice were infected with 10⁶ PRBC. On day 7 p.i., CD4⁺ T cells were analyzed for CD25 and FoxP3 expression and cell size (FSC). Dot plots represent gated CD4⁺ T cells. Dot plots and histograms show a representative mouse from each group. Numbers inside dot plots refer to means ± SD (n = 4) of cell percentages in each gate. Histograms show gated CD4⁺CD25⁺FoxP3⁺ and CD4⁺CD25⁺FoxP3⁻ cells in relation to CD4⁺CD25⁻FoxP3⁻ cells. Numbers inside histograms refer to means ± SD (n = 4) of large cell percentages. (B) On days 0, 2 and 4 p.i., C57BL/6 mice were treated with JES6-1 mAb. Data represent the means ± SD (n = 4) of CD4⁺CD25⁺FoxP3⁺ cell percentages and numbers per spleen on days 7 and 20 of infection. In A–B, significant differences compared experimental conditions *p<0.05 with cells from non-infected (NI) mice; **p<0.05 with cells from non-treated (NT) mice; and #p<0.05 with CD25⁺FoxP3⁺ cells. Data are representative of two separate experiments.</p

Effects of JES6-1 treatment on splenic CD4⁺ T cell phenotype and proliferative response to PRBC during chronic <i>P. chabaudi</i> malaria.

<p>(A) C57BL/6 mice were treated with JES6-1 mAb on days 0, 2 and 4 p.i. with 10⁶ PRBC. On day 20 p.i., CD62L and CD45RB expression was evaluated in gated CD4⁺ T cells. Numbers inside dot plots refer to means ± SD (n = 3–4) of cell percentages in each gate. (B) CD69 expression was analyzed in gated CD4⁺ T cells from the same groups of mice. (C) <i>In vitro</i> proliferative response of PRBC-stimulated CD4⁺ cells cultured in the presence or absence of JES6-1 mAb. CD4⁺ T cells were obtained from 30-day infected mice treated <i>in vivo</i> or not with JES6-1 mAb on days 0, 2 and 4 of infection. Histograms show CFSE fluorescence in gated CD4⁺ T cells. The means ± SD (n = 3–4) of the percentages of replicating (CFSE^LO) cells are shown. In A–C, significant differences compared experimental conditions *p<0.05 with cells from non-infected (NI) mice; **p<0.05 with non-treated (NT) mice or cells; and # p<0.05 with non-treated (NT) cells from JES6-1-treated mice. Data are representative of two separate experiments.</p

Effects of JES6-1 treatment on the regulation of acquired immune responses to <i>P. chabaudi</i> malaria.

<p>(A) C57BL/6 mice were treated with JES6-1 mAb on days 0, 2 and 4 p.i. with 10⁶ PRBC. On day 30 p.i., TNF-α and IFN-γ production was evaluated in spleen cell supernatants (mean ± SD, n = 4). (B) Serum titers of parasite-specific IgG2a were determined in the same groups of mice. (C) Parasitemia curves were evaluated in C57BL/6 mice injected with spleen cells from JES6-1-treated mice and non-treated (NT) mice on day 30 p.i. and challenged with 10⁸ PRBC (mean ± SD, n = 4). In A–B, significant differences compared experimental conditions *p<0.05 with non-infected (NI) mice; and **p<0.05 with non-treated (NT) mice. In C, significant differences compared to experimental conditions *p<0.05 with mice transferred with cells from non-treated (NT) mice. Data are representative of two separate experiments.</p