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. 2011;6(11):e27714.
doi: 10.1371/journal.pone.0027714. Epub 2011 Nov 16.

Inhaled nitric oxide reduces endothelial activation and parasite accumulation in the brain, and enhances survival in experimental cerebral malaria

Lena Serghides  1 , Hani Kim, Ziyue Lu, Dylan C Kain, Chris Miller, Roland C Francis, W Conrad Liles, Warren M Zapol, Kevin C Kain
Affiliations

Inhaled nitric oxide reduces endothelial activation and parasite accumulation in the brain, and enhances survival in experimental cerebral malaria

Lena Serghides et al. PLoS One. 2011.

Abstract

The host immune response contributes to the onset and progression of severe malaria syndromes, such as cerebral malaria. Adjunctive immunomodulatory strategies for severe malaria may improve clinical outcome beyond that achievable with artemisinin-based therapy alone. Here, we report that prophylaxis with inhaled nitric oxide significantly reduced systemic inflammation (lower TNF, IFNγ and MCP-1 in peripheral blood) and endothelial activation (decreased sICAM-1 and vWF, and increased angiopoeitin-1 levels in peripheral blood) in an experimental cerebral malaria model. Mice that received inhaled nitric oxide starting prior to infection had reduced parasitized erythrocyte accumulation in the brain, decreased brain expression of ICAM-1, and preserved vascular integrity compared to control mice.Inhaled nitric oxide administered in combination with artesunate, starting as late as 5.5 days post-infection, improved survival over treatment with artesunate alone (70% survival in the artesunate only vs. 100% survival in the artesunate plus iNO group, p = 0.03). These data support the clinical investigation of inhaled nitric oxide as a novel adjunctive therapy in patients with severe malaria.

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Conflict of interest statement

Competing Interests: The authors have read the journal's policy and have the following conflicts: The Massachusetts General Hospital has licensed patents on inhaled NO to Linde Corp and Ikaria Corp. WMZ and his laboratory receive royalties. This does not alter the authors' adherence to all the PLoS ONE policies on sharing data and materials. All other authors have no commercial or other association that might pose a conflict of interest.

Figures

Figure 1
Figure 1. Prophylactic inhaled NO prolongs survival in mice infected with P. berghei ANKA.
Kaplan Myer survival curves are shown for C57BL/6 mice infected with 1x106 P. berghei 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).
Figure 2
Figure 2. Inhaled NO reduces systemic inflammation in mice infected with P. berghei ANKA.
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.
Figure 3
Figure 3. Inhaled NO modulates markers of endothelial activation in mice infected with P. berghei ANKA.
(A-C) Heparinized saphenous vein blood was collected on day 5 post-infection from infected mice prophylactically treated with either air or 80ppm NO. Plasma levels of sICAM-1 (A), vWF (B), and Ang-1 (C) were determined by ELISA. The line represents median levels of these factors measured in uninfected mice. (D) Quantitative real time PCR was performed on RNA isolated from brains collected from parasitemia-matched air or 80ppm NO treated mice on day 6 post-infection. The copy number for ang-1 and ang-2 were determined and the ratio between ang-2 and ang-1 is shown. The line represents the copy number ratio in uninfected mice. Statistical differences were assessed using Mann-Whitney. For (A) p = 0.0094, for (B) p = 0.0089, for (C) p = 0.039, for (D) p = 0.028.
Figure 4
Figure 4. Inhaled NO reduces PE accumulation and ICAM-1 expression in the brains of P. berghei ANKA infected mice.
(A) Luminometer imaging of brains collected on day 4 post-infection from mice infected with P. berghei 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 P. berghei 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.
Figure 5
Figure 5. Inhaled NO prevents loss of blood-brain barrier vascular integrity in mice infected with P. berghei ANKA.
(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.
Figure 6
Figure 6. Inhaled NO administered in combination with artesunate confers a survival advantage over artesunate treatment alone in experimental CM.
Mice were infected with P. berghei ANKA and were treated with either medical air (grey line), 10mg/kg artesunate (black line), or 10mg/kg artesunate plus 80 ppm NO (red line) starting on day 3 (A-C) or day 5.5 (D-F) post infection. Artesunate was given once daily by i.p. injection for 4 days starting on day 3, or once daily for one day starting on day 5.5. Air or 80 ppm NO was initiated on day 3 or day 5.5 and continued until the end of the experiment on day 15 post-infection. (A and D) Survival was monitored twice daily. Significant differences in survival were assessed by Log rank test. For Day 3: p = 0.049 for ART + air vs. ART + NO, n = 31 for ART + air, n = 30 for ART + NO, and n = 10 for air alone. For Day 5.5: p = 0.031 for ART + air vs. ART + NO, n = 10 for ART + air, n = 14 for ART + NO, and n = 8 for air alone. (B and E) Parasitemia did not differ significantly between the ART + air and the ART + NO groups. (C and F) Hematocrit was assessed on day 14 of infection in mice receiving ART + air or NO starting on day 3 (C) or 5.5 (F) post-infection. For Day 3: p<0.0001 for ART + air vs. ART + NO, n = 18 per group; for Day 5.5: p = 0.0002 for ART + air vs. ART + NO, n =  18 for ART + air, n = 23 for ART + NO; by Student's t-test. ART, artesunate, NO, nitric oxide.
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References

    1. Dorovini-Zis K, Schmidt K, Huynh H, Fu W, Whitten RO, et al. The neuropathology of fatal cerebral malaria in Malawian children. Am J Pathol. 2011;178:2146–2158. - PMC - PubMed
    1. John CC, Bangirana P, Byarugaba J, Opoka RO, Idro R, et al. Cerebral malaria in children is associated with long-term cognitive impairment. Pediatrics. 2008;122:e92–99. - PMC - PubMed
    1. Dondorp A, Nosten F, Stepniewska K, Day N White N; South East Asian Quinine Artesunate Malaria Trial (SEAQUAMAT) group. Artesunate versus quinine for treatment of severe falciparum malaria: a randomised trial. Lancet. 2005;366:717–725. - PubMed
    1. Dondorp AM, Fanello CI, Hendriksen IC, Gomes E, Seni A, et al. Artesunate versus quinine in the treatment of severe falciparum malaria in African children (AQUAMAT): an open-label, randomized trial. Lancet. 2010;376:1647–1657. - PMC - PubMed
    1. John CC, Kutamba E, Mugarura K, Opoka RO. Adjunctive therapy for cerebral malaria and other severe forms of Plasmodium falciparum malaria. Expert Rev Anti Infect Ther. 2010;8:997–1008. - PMC - PubMed

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