Ang IV has a greater effect than Ang II and Ang-(1–7).

<p>(A) Dose–response of the effect of Ang IV on the <i>P. falciparum</i> erythrocytic cycle. Parasite schizont forms were incubated with a fresh erythrocyte culture at 2% parasitemia in the absence or presence of increasing concentrations of Ang IV (10⁻¹²–10⁻⁶ M). Parasite invasion was determined as the number of intracellular rings after 24 h incubation as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0017174#s4" target="_blank">Materials and Methods</a> section (n = 5). (B) Parasite schizont forms were incubated with a fresh erythrocyte culture at 2% parasitemia in the absence or presence of 10⁻⁸ M Ang II, 10⁻⁸ M Ang-(1–7) or 10⁻⁸ M Ang IV. Parasite invasion was determined as the number of intracellular rings after 24 h incubation as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0017174#s4" target="_blank">Materials and Methods</a> section (n = 5). (C) The Ang IV-mediated effect was not prevented by AT₁, AT₂ or MAS receptor antagonists. The effect of Ang IV on parasite invasion was determined as described in (A). Where indicated, cultures were pre-treated with 10⁻⁶ M losartan, 10⁻⁷ M PD123319 or 10⁻⁷ M A779 before addition of Ang IV (10⁻⁸ M). Parasite invasion was determined as the number of intracellular rings after 24 h incubation in thick blood smears as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0017174#s4" target="_blank">Materials and Methods</a> section (n = 5). The results are expressed as means±SE. Statistically significant compared with *control value and #Ang II and Ang-(1–7) (<i>P</i><0.05).</p

Ang II inhibitory effect is maintained for 11 days.

<p>(A) Parasites were incubated with a fresh erythrocyte culture at 0.5% parasitemia in the absence (○) or presence (•) of 10⁻⁸ M Ang II, added daily for 4 consecutive days. Total parasitemia was determined on each day of the assay as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0017174#s4" target="_blank">Materials and Methods</a> section (n = 7). (B) The pre-incubation effect of Ang II was determined after treating the culture without (bar A) or with (bar B) 10⁻⁸ M Ang II for 4 days. The culture was then split and maintained for 11 days without Ang II (bar C). The total parasitemia was determined as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0017174#s4" target="_blank">Materials and Methods</a> section (n = 5). The results are expressed as means±SE. *Statistically significant compared with the control value (<i>P</i><0.05).</p

Ang-(1–7) has the same but non-additive effect as Ang II.

<p>(A) Dose-response of the effect of Ang-(1–7) on the <i>P. falciparum</i> erythrocytic cycle. Parasite schizont forms were incubated with a fresh erythrocyte culture at 2% parasitemia in the absence or presence of increasing concentrations of Ang-(1–7) (10⁻¹²–10⁻⁶ M). Parasite invasion was determined as the number of intracellular rings after 24 h incubation as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0017174#s4" target="_blank">Materials and Methods</a> section (n = 8). (B) Parasite schizont forms were incubated with a fresh erythrocyte culture at 2% parasitemia in the absence or presence of 10⁻⁸ M Ang II, 10⁻⁸ M Ang-(1–7) or both. Parasite invasion was determined as the number of intracellular rings after 24 h incubation as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0017174#s4" target="_blank">Materials and Methods</a> section (n = 8). (C) The Ang-(1–7)-mediated effect was avoided by the MAS receptor antagonist. The effect of Ang II in parasite invasion was determined as described in (B). Where indicated, cultures were pre-treated with 10⁻⁶ M losartan, 10⁻⁷ M PD123319 or 10⁻⁷ M A779 before addition of Ang-(1–7) (10⁻⁸ M). Parasite invasion was determined as the number of intracellular rings after 24 h incubation as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0017174#s4" target="_blank">Materials and Methods</a> section (n = 6). (D) The effect of Ang II and Ang-(1–7) on the erythrocytic cycle of malaria parasite obtained from thick blood smears for parasitemia determination by Diff-Quick staining (n = 8). The results are expressed as means±SE. Statistically significant compared with *control and #Ang-(1–7) values (<i>P</i><0.05). Magnification ×100.</p

Erythrocyte PKA is reduced by Ang II and Ang-(1–7) and is involved in infection.

<p>(A) The membrane fraction of erythrocytes was assayed for PKA activity as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0017174#s4" target="_blank">Materials and Methods</a> section, in the absence or presence of 10⁻⁸ M Ang II or 10⁻⁸ M Ang-(1–7). Where indicated, membranes were pre-treated with 10⁻⁶ M losartan, 10⁻⁷ M PD123319 or 10⁻⁷ M A779 before the addition of 10⁻⁸ M Ang II or 10⁻⁸ M Ang-(1–7) (n = 6). (B) Parasite schizont forms were incubated with a fresh erythrocyte culture at 2% parasitemia in the absence or presence of 10⁻⁷ M dAMPc, 10⁻⁷ M iPKA or 10⁻⁷ M PMA. Parasite invasion was determined as the number of intracellular rings after 24 h incubation as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0017174#s4" target="_blank">Materials and Methods</a> section (n = 7). The results are expressed as means±SE. Statistically significant compared with *control value, ^ψAng II, ^ΨAng-(1–7), ^#dAMPc (<i>P</i><0.05).</p

Ang II is promptly metabolized during the invasion assay.

<p>The time course of Ang II metabolism was monitored by mass spectroscopy (MALDI) in the supernatant of infected (•) and non-infected (○) erythrocytes incubated with 10⁻⁵ M Ang II. (A) Ang II, (B) Ang-(1–7) and (C) Ang IV levels. The peak areas for different angiotensin forms (Ang II, Ang-(1–7) and Ang IV) present in the same spectrum (masses 1046.19, 899.02 and 774.92 Da, respectively) were obtained and their sum was arbitrarily assigned as 100%. The results are expressed as means±SE. *Statistically significant compared with the control value (n = 4, <i>P</i><0.05).</p

Angiotensin receptors are present in erythrocyte membranes.

<p>(A) Membrane preparations of human erythrocytes (50, 150 or 200 µg total protein, lanes 1, 2 and 3, respectively) underwent immunoblotting for detection of AT₁, AT₂ and MAS receptors. The microsomal fraction of rat renal cortex was used as positive control (lane C) (n = 3). (B) The Ang II-mediated effect was partially abolished by AT₂ receptor antagonist. Where indicated, cultures were pre-treated with 10⁻⁶ M losartan, 10⁻⁷ M PD123319 or 10⁻⁷ M A779. Parasite invasion was determined as the number of intracellular rings after 24 h incubation as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0017174#s4" target="_blank">Materials and Methods</a> section (n = 6). The results are expressed as means±SE. Statistically significant compared with *control and #Ang II values (<i>P</i><0.05).</p

Losartan or captopril treatment inhibited <i>P.berghei</i> ANKA-induced splenic T cell activation.

<p>C57BL/6 mice were infected with <i>P. berghei</i> ANKA and treated with vehicle, losartan or captopril by gavage. T cells were isolated at day 6 post infection, stained with fluorescent antibodies, and analyzed by flow cytometry. (A) Representative dot plots of CD4⁺CD69⁺ and CD8⁺CD69⁺ T cells obtained from gated CD3⁺ cells. Percentage of CD4⁺CD69⁺ T cells (B) and CD8⁺CD69⁺ T cells (C). Parasitemia was determined by Giemsa-stained blood smears in vehicle- (solid line), losartan- (dotted line) or captopril- (dash line) treated mice (D). Mortality was checked daily in vehicle- (▪), losartan- (▴) or captopril- (▾) treated mice (E). Behavioral analysis was assessed as described in Materials and Methods (F). Evans blue dye extraction from the brain tissue to evaluate blood-brain barrier disruption (G). The results are expressed as means±SD. Statistically significant compared with values for *naive mice (<i>p</i><0.05) and #vehicle-treated mice infected with <i>P. berghei</i> ANKA (<i>p</i><0.05). Statistically significant compared with values for vehicle-treated infected mice at ^aday 5, ^bday 7 or day ^c10 post-infection.</p

Ang II influences the production of perforin by splenic CD8⁺ T cellsduring <i>P. berghei</i> ANKA infection.

<p>T cells from naive mice and mice infected with <i>P. berghei</i> ANKA treated with vehicle, losartan or captopril by gavage were isolated at day 6 post infection, stained with fluorescent antibodies and analyzed by flow cytometry. (A) Representative dot plots of perforin⁺CD8⁺ T cells obtained from gated CD3⁺ cells. The percentage (B) and absolute number (<b>C</b>) of perforin⁺CD8⁺ T cells were determined. The results are expressed as means±SD. Statistically significant compared with values for *naive mice (<i>p</i><0.05) and #vehicle-treated mice infected with <i>P. berghei</i> ANKA (<i>p</i><0.05).</p

Ang II induces the upregulation of CD11a expression in splenic T cells during <i>P.berghei</i> ANKA infection.

<p>C57BL/6 mice were infected with <i>P. berghei</i> ANKA and treated with vehicle, losartan or captopril by gavage. T cells were isolated at day 6 post infection, stained with fluorescent antibodies and analyzed by flow cytometry. (A) Representative dot plots of CD3⁺CD11a⁺ T cells. The percentage of CD11a⁺ T cells (B) and absolute number of CD11a⁺ T cells (C) were calculated. CD11a expression was analyzed by MIF on gated CD3⁺ T cells (D, E). The results are expressed as means±SD. Statistically significant compared with values for *naive mice (<i>p</i><0.05) and #vehicle-treated mice infected with <i>P. berghei</i> ANKA (<i>p</i><0.05).</p

Endogenous Ang II induces transmigration of T lymphocytes during <i>P.berghei</i> ANKA infection on contact with endothelial basal membrane proteins.

<p>5×10⁵ splenic T lymphocytes isolated from naive mice or mice infected with <i>P. berghei</i> ANKA treated with vehicle, losartan or captopril by gavage were added to the upper chamber of Transwell culture inserts (5.0 µm pore diameter) previously coated with fibronectin (10 µg/ml), laminin (10 µg/ml) or BSA (10 ug/ml) as negative control. The inserts were placed in individual wells of a 24-well cell culture plate containing 600 µl of assay medium in the absence or presence of 10⁻⁸ M Ang II as depicted in each panel and incubated for 3 h at 37°C in 5% CO₂. (A) Transmigrated cells through the fibronectin matrix (black bars) and through the laminin matrix (open bars), minus the number of cells that migrated from BSA×10⁴. The results are expressed as means±SE. Statistically significant compared with values for *naive mice (<i>p</i><0.05) and #vehicle-treated mice infected with <i>P. berghei</i> ANKA (<i>p</i><0.05). Transmigrated T lymphocytes from naive (black bars) or vehicle-treated mice infected with <i>P. berghei</i> ANKA (open bars) that received treatment in culture with 10^–6 M losartan or 10^–6 M captopril for 30 min before the transmigration assay, through the fibronectin matrix (B) and through the laminin matrix (C) minus the number of cells that migrated from BSA×10⁴. The results are expressed as means±SE. Statistically significant compared with values for *naive mice in the control condition (<i>p</i><0.05) and #vehicle-treated mice infected with <i>P. berghei</i> ANKA in the control condition (<i>p</i><0.05).</p