Prophylactic inhaled NO prolongs survival in mice infected with <i>P. berghei</i> ANKA.

<p>Kaplan Myer survival curves are shown for C57BL/6 mice infected with 1x10⁶<i>P. berghei</i> ANKA PEs and treated in (A-C) with either 80 ppm NO (red) or air (black) starting one day prior to infection, or in (D-E) with either nitrite (administered in the drinking water at 500 mg/L of water, shown in red) or water (black). Survival (A and D) was assessed twice daily. Significant differences in survival were assessed by Log rank test. For (A) p = 0.0007, n = 18 in air and n = 20 in NO group, representative of 5 independent experiments. For (D) p = 0.0004, n = 10 per group, representative of 2 independent experiments. Parasitemia levels did not differ between the groups (B and E). Hematocrit did not differ between iNO- and air-treated groups (C).</p

PPARγ Agonists Improve Survival and Neurocognitive Outcomes in Experimental Cerebral Malaria and Induce Neuroprotective Pathways in Human Malaria

<div><p>Cerebral malaria (CM) is associated with a high mortality rate, and long-term neurocognitive impairment in approximately one third of survivors. Adjunctive therapies that modify the pathophysiological processes involved in CM may improve outcome over anti-malarial therapy alone. PPARγ agonists have been reported to have immunomodulatory effects in a variety of disease models. Here we report that adjunctive therapy with PPARγ agonists improved survival and long-term neurocognitive outcomes in the <i>Plasmodium berghei</i> ANKA experimental model of CM. Compared to anti-malarial therapy alone, PPARγ adjunctive therapy administered to mice at the onset of CM signs, was associated with reduced endothelial activation, and enhanced expression of the anti-oxidant enzymes SOD-1 and catalase and the neurotrophic factors brain derived neurotrophic factor (BDNF) and nerve growth factor (NGF) in the brains of infected mice. Two months following infection, mice that were treated with anti-malarials alone demonstrated cognitive dysfunction, while mice that received PPARγ adjunctive therapy were completely protected from neurocognitive impairment and from PbA-infection induced brain atrophy. In humans with <i>P. falciparum</i> malaria, PPARγ therapy was associated with reduced endothelial activation and with induction of neuroprotective pathways, such as BDNF. These findings provide insight into mechanisms conferring improved survival and preventing neurocognitive injury in CM, and support the evaluation of PPARγ agonists in human CM.</p></div

Inhaled NO prevents loss of blood-brain barrier vascular integrity in mice infected with <i>P. berghei</i> ANKA.

<p>(A) Photographs of brains from mice injected with Evans blue on day 6 post-infection. Brains from mice prophylactically treated with air (left panels) and with 80 ppm NO (right panels). The number on the lower left corner is the peripheral parasitemia for that mouse. (B) Quantification of total Evans blue extravasation in the brain as an indication of vascular leak. The line indicates median Evans blue values from uninfected mice. p<0.0001 by Mann-Whitney. n = 15 per group. (C) Number of hemorrhages quantified (blinded to the treatment group) from H&E stained sections of brains collected on day 6 post-infection from mice prophylactically treated with either air or 80 ppm NO. p = 0.0079 by Mann Whitney. n = 5 per group.</p

Inhaled NO reduces PE accumulation and ICAM-1 expression in the brains of <i>P. berghei</i> ANKA infected mice.

<p>(A) Luminometer imaging of brains collected on day 4 post-infection from mice infected with <i>P. berghei</i> ANKA–GFP–luciferase PEs and prophylactically treated with either air (left panels) or 80 ppm NO (right panels). The rainbow scale represents the relative level of luciferase activity (correlates with parasite load). The numbers on the lower left corner are the peripheral parasitemia levels for each mouse. The bottom panels are brains from a C57BL/6 mouse on the left and a BALB/c mouse on the right. (B) To quantify the brain bioluminescence, total flux (photons per second) was calculated for each brain. p = 0.023 by Mann-Whitney test, n = 17 per group. (C) Immunohistochemistry analysis of ICAM-1 on formalin fixed brain sections from parasitemia matched mice infected with <i>P. berghei</i> ANKA and prophylactically treated with either air (top panels) or 80 ppm NO (middle panels), or uninfected mice (bottom panels). (D) ICAM-1 mRNA expression displayed as copy number was quantified by quantitative real time PCR in the brains of parasitemia matched infected mice prophylactically treated with air or 80 ppm NO. The line represents the median copy number for uninfected mice. p =  0.028, by Mann-Whitney, n = 14 for air, n = 10 for NO.</p

Rosiglitazone adjunctive therapy improves survival in ECM.

<p>Survival curves (A and B) and parasitemia (C and D) for mice infected with PbA and receiving, no treatment (dashed line), 10 mg/kg artesunate plus saline (solid black line), or 10 mg/kg artesunate plus 2.5 mg/kg rosiglitazone (red line). The blue arrow indicates treatment initiation (day 3 post-infection for A and C, and onset of CM for B and D). In (A), p = 0.0042 for artesunate vs. artesunate + rosiglitazone; p<0.0001 for no treatment vs. artesunate + rosiglitazone, and for no treatment vs. artesunate, by Logrank test, N = 10/group. In (B), p = 0.0012 for artesunate vs. artesunate + rosiglitazone; p<0.0001 for no treatment vs. artesunate + rosiglitazone, and for no treatment vs. artesunate, by Logrank test, N = 40 for the artesunate and artesunate + rosiglitazone groups, N = 10 for the no treatment group. (C–D) Parasitemia curves did not differ significantly between artesunate and artesunate + rosiglitazone groups. Abbreviations: Art, artesunate; Rosi, rosiglitazone.</p

Inhaled NO reduces systemic inflammation in mice infected with <i>P. berghei</i> ANKA.

<p>Heparinized saphenous vein blood was collected on day 5 post-infection from mice that were prophylactically treated with either air or 80ppm NO. Plasma levels of (A) IFNγ, (B) TNF, and (C) MCP-1 were determined using the mouse inflammation cytometric bead array. Statistical differences were assessed by Mann-Whitney test. For (A) p = 0.0004, for (B) p = 0.0035, for (C) p = 0.018. Data are representative of 2 independent experiments. Plasma from uninfected mice was also tested as a control, and levels of all 3 cytokines were below the limit of detection.</p

Rosiglitazone adjunctive therapy increased BDNF and lowered the angiopoietin-2:angiopoietin-1 ratio in patient with falciparum malaria.

<p>BDNF, Ang-1, and Ang-2 levels were determined in plasma collected from patients with uncomplicated malaria randomised to atovaquone proguanil plus either rosiglitazone or placebo. (A) BDNF levels were normalized to levels measured prior to treatment initiation. Normalised BDNF levels for day 1-3 post-treatment initiation are shown. Comparisons between placebo vs. rosiglitazone by Kruskal-Wallis test with Dunn's post-test, N = 52/group. Comparison between baseline vs. treatment by Wilcoxon Signed Rank test: p<0.001, p = 0.0024, and p<0.0001 for placebo baseline vs. placebo day 1, day 2, and day 3 respectively; and p<0.0001, p = 0.84, and p = 0.94 for rosiglitazone baseline vs. day 1, day 2, and day 3 respectively. (B) The ratio of Ang-2 to Ang-1 in plasma assessed 3 days post-treatment initiation. Data shown are medians with range. All comparisons by Mann Whitney test. N = 67/group.</p

Rosiglitazone adjunctive therapy increases expression of neurotrophic factors in the brains of malaria-infected mice.

<p>(A–C) Mice infected with PbA were treated with artesunate plus saline (grey bars), or artesunate plus rosiglitazone (red bars) starting at the onset of CM signs. Expression of BDNF (A), NGF (B), and the BDNF receptor Trk-B (C) mRNA was assessed in brain homogenates collected from uninfected mice (day 0), infected mice prior to the initiation of therapy (day 5), and infected mice following treatment initiation (on day 7 and 9 post-infection). All comparisons by one-way ANOVA with Bonferroni's multiple comparison tests.</p

Rosiglitazone adjunctive therapy protects mice from malaria-induced brain atrophy.

<p>Following completion of all behavioural testing mouse brains were scanned using magnetic resonance imaging (MRI). Image registration and volumetric analysis of the brain volume of 62 distinct regions were performed. Linear regression analysis was used to identify areas that differed significantly between the mice treated with artesunate/mefloquine plus saline (grey bars), and the mice treated with artesunate/mefloquine plus rosiglitazone (red bars). (A) Identified brain regions are shown. Percentage change in brain structure volume is indicated by colour (see rainbow bar on the left). Significant differences were observed in (B) the corpus callosum, (C) the hippocampus, (D) the thalamus, (E) the midbrain, (F) the arbour vitae of the cerebellum, and (G) the globus pallidus of the basal ganglia. N = 13 for artesunate/mefloquine, N = 10 for artesunate/mefloquine + rosiglitazone. The median volume for uninfected mice is shown as a dashed line (N = 10 for uninfected control). Additional brain regions are shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003980#ppat.1003980.s008" target="_blank">Figure S8</a>.</p

Rosiglitazone adjunctive therapy increases brain expression of the anti-oxidant enzymes SOD-1 and catalase.

<p>Mice infected with PbA were treated with artesunate plus saline (grey bars), or artesunate plus rosiglitazone (red bars) starting at the onset of CM signs. Expression of SOD-1 (A) and catalase (B) mRNA was assessed in brain homogenates collected from uninfected mice (day 0), infected mice prior to the initiation of therapy (day 5), and infected mice following treatment initiation (on day 7 and 9 post-infection). Data were analysed by one-way ANOVA with Bonferroni's multiple comparison test, N = 6 per group.</p