Leonardo J. M. Carvalho

Murine Cerebral Malaria Is Associated with a Vasospasm-Like Microcirculatory Dysfunction, and Survival upon Rescue Treatment Is Markedly Increased by Nimodipine

Brain hemodynamics in cerebral malaria (CM) is poorly understood, with apparently conflicting data showing microcirculatory hypoperfusion and normal or even increased blood flow in large arteries. Using intravital microscopy to assess the pial microvasculature through a closed cranial window in the murine model of CM by Plasmodium berghei ANKA, we show that murine CM is associated with marked decreases (mean: 60%) of pial arteriolar blood flow attributable to vasoconstriction and decreased blood velocity. Leukocyte sequestration further decreased perfusion by narrowing luminal diameters in the affected vessels and blocking capillaries. Remarkably, vascular collapse at various degrees was observed in 44% of mice with CM, which also presented more severe vasoconstriction. Coadministration of artemether and nimodipine, a calcium channel blocker used to treat postsubarachnoid hemorrhage vasospasm, to mice presenting CM markedly increased survival compared with artemether plus vehicle only. Administration of nimodipine induced vasodilation and increased pial blood flow. We conclude that vasoconstriction and vascular collapse play a role in murine CM pathogenesis and nimodipine holds potential as adjunctive therapy for CM.

Nitric Oxide Protection Against Murine Cerebral Malaria Is Associated With Improved Cerebral Microcirculatory Physiology

Cerebral malaria (CM) is a leading cause of death in Plasmodium falciparum infections. In the Plasmodium berghei ANKA (PbA) murine model, CM pathogenesis is associated with low nitric oxide (NO) bioavailability and brain microcirculatory complications, with a marked decrease in cerebral blood flow, vasoconstriction, vascular plugging by adherent cells, and hemorrhages. Using intravital microscopy through a closed cranial window, here we show that NO supplementation in the form of a NO donor (dipropylenetriamine NONOate [DPTA-NO]) prevented vasoconstriction and improved blood flow in pial vessels of PbA-infected mice. Arterioles and venules of smaller diameters (20-35.5 μm) showed better response to treatment than vessels of larger diameters (36-63 μm). Exogenous NO provided protection against brain hemorrhages (mean, 1.4 vs 24.5 hemorrhagic foci per section) and inflammation (mean, 2.5 vs 10.9 adherent leukocytes per 100 μm vessel length) compared with saline treatment. In conclusion, NO protection against CM is associated with improved brain microcirculatory hemodynamics and decreased vascular pathology.

Immunization of Saimiri sciureus Monkeys with Plasmodium falciparum Merozoite Surface Protein‐3 and Glutamate‐Rich Protein Suggests that Protection is Related to Antibody Levels

The immunogenicity and protective efficacy of various antigen‐adjuvant formulations derived either from the merozoite‐surface protein‐3 (MSP‐3) or the glutamate‐rich protein (GLURP) of Plasmodium falciparum were evaluated in Saimiri sciureus monkeys. These proteins were selected for immunogenicity studies based primarily on their capacity of inducing an antibody‐dependent cellular inhibition effect on parasite growth. Some of the S. sciureus monkeys immunized with MSP‐3212−380‐AS02 or GLURP27−500‐alum were able to fully or partially control parasitaemia upon an experimental P. falciparum [Falciparum Uganda Palo Alto (FUP‐SP) strain] blood‐stage infection, and this protection was related to the prechallenge antibody titres induced. The data are indicative that MSP‐3 and GLURP can induce protective immunity against an experimental P. falciparum infection using adjuvants that are acceptable for human use and this should trigger further studies with those new antigens.

Germinal center architecture disturbance during Plasmodium berghei ANKA infection in CBA mice

BackgroundImmune responses to malaria blood stage infection are in general defective, with the need for long-term exposure to the parasite to achieve immunity, and with the development of immunopathology states such as cerebral malaria in many cases. One of the potential reasons for the difficulty in developing protective immunity is the poor development of memory responses. In this paper, the potential association of cellular reactivity in lymphoid organs (spleen, lymph nodes and Peyers patches) with immunity and pathology was evaluated during Plasmodium berghei ANKA infection in CBA mice.MethodsCBA mice were infected with 1 × 106P. berghei ANKA-parasitized erythrocytes and killed on days 3, 6–8 and 10 of infection. The spleen, lymph nodes and Peyers patches were collected, fixed in Carsons formalin, cut in 5 μm sections, mounted in glass slides, stained with Lennerts Giemsa and haematoxylin-eosin and analysed with bright-field microscopy.ResultsEarly (day 3) strong activation of T cells in secondary lymphoid organs was observed and, on days 6–8 of infection, there was overwhelming activation of B cells, with loss of conventional germinal center architecture, intense centroblast activation, proliferation and apoptosis but little differentiation to centrocytes. In the spleen, the marginal zone disappeared and the limits between the disorganized germinal center and the red pulp were blurred. Intense plasmacytogenesis was observed in the T cell zone.ConclusionThe observed alterations, especially the germinal center architecture disturbance (GCAD) with poor centrocyte differentiation, suggest that B cell responses during P. berghei ANKA infection in mice are defective, with potential impact on B cell memory responses.

Immunization of Saimiri sciureus Monkeys with a Recombinant Hybrid Protein Derived from the Plasmodium falciparum Antigen Glutamate-Rich Protein and Merozoite Surface Protein 3 Can Induce Partial Protection with Freund and Montanide ISA720 Adjuvants

ABSTRACT The immunogenicity and efficacy of a hybrid recombinant protein derived from the N-terminal end of the glutamate-rich protein (GLURP) and the C-terminal portion of the merozoite surface protein 3 (MSP3) of Plasmodium falciparum was evaluated in Saimiri sciureus monkeys. The GLURP/MSP3 hybrid protein, expressed in Lactococcus lactis, was administered in association with alum, Montanide ISA720, or complete or incomplete Freund adjuvant (CFA/IFA) in groups of five animals each. The three formulations were shown to be immunogenic, but the one with alum was shown to be weak compared to the other two, particularly CFA/IFA, which provided very high antibody titers (enzyme-linked immunosorbent assay titers of >3,000,000 and immunofluorescence antibody test titers of 6,400). After a challenge infection with P. falciparum FUP strain, all five monkeys from the GLURP/MSP3-alum group showed a rapid increase in parasitemia, reaching 10% and were treated early. The two monkeys with the highest antibody titers in group GLURP/MSP3-Montanide ISA720 had a delay in the course of parasitemia and were treated late due to a low hematocrit. In the GLURP/MSP3-CFA/IFA group, parasitemia remained below this threshold in four of the five animals and, after it reached a peak, parasitemia started to decrease and monkeys were treated late. When all animals were grouped according to the outcome, a statistically significant association between high antibody titers and partial protection was observed. The challenge infection boosted the antibody titers, and the importance of this event for vaccine efficacy in areas where this parasite is endemic is discussed. In conclusion, these data suggest that GLURP and MSP3 can induce protection against malaria infection if antibodies are induced at properly high titers.

Exogenous nitric oxide decreases brain vascular inflammation, leakage and venular resistance during Plasmodium berghei ANKA infection in mice

BackgroundCerebral malaria (CM) is a lethal complication of Plasmodium falciparum infections. In the Plasmodium berghei ANKA (PbA) murine model, CM is associated with marked brain inflammation, increased expression of endothelial cell adhesion molecules and leukocyte and platelet accumulation in brain vessels, causing vascular occlusion and decreased blood flow, damaging the endothelium and leading to blood-brain barrier breakdown, leakage and hemorrhages. Exogenous nitric oxide (NO) administration largely prevents the syndrome. Here we evaluated whether the mechanism of action of NO in preventing murine CM is related to its anti-inflammatory properties and to protection of the endothelium.MethodsC57Bl/6 mice infected with PbA were treated twice a day with saline or dipropylenetriamineNONOate (DPTA-NO). Endothelial cell adhesion molecule (ICAM-1, VCAM, E- and P-selectin) expression in brain tissue on day 6 of infection was assessed in both groups by western blot. For intravital microscopy studies, DPTA-NO-treated and saline-treated mice with a previously implanted closed cranial window were injected with albumin-FITC, anti-CD45-TxR and anti-CD41-FITC antibodies on day 6 of infection for quantification of albumin leakage, leukocyte and platelet adherence in pial vessels.ResultsPbA-infected mice treated with the NO-donor DPTA-NO showed decreased expression of ICAM-1 and P-selectin, but not VCAM-1, in the brain, compared to saline-treated mice. DPTA-NO treatment also decreased the number of adherent leukocytes and platelets in pial vessels, particularly in venules 30-50 μm in diameter, decreased inflammatory vascular resistance and prevented the occurrence of arteriolar and venular albumin leakage observed in saline-treated PbA-infected mice, as assessed by intravital microscopy.ConclusionsThese results indicate that the protective effect of exogenous NO on murine CM is associated with decreased brain vascular expression of inflammatory markers resulting in attenuated endothelial junction damage and facilitating blood flow.

Artemether and artesunate show the highest efficacies in rescuing mice with late-stage cerebral malaria and rapidly decrease leukocyte accumulation in the brain.

ABSTRACT The murine model of cerebral malaria (ECM) caused by Plasmodium berghei ANKA (PbA) infection in susceptible mice has been extensively used for studies of pathogenesis and identification of potential targets for human CM therapeutics. However, the model has been seldom explored to evaluate adjunctive therapies for this malaria complication. A first step toward this goal is to define a treatment protocol with an effective antimalarial drug able to rescue mice presenting late-stage ECM. We evaluated the efficacy of artemisinin, artemether, artesunate, and quinine given intraperitoneally once a day, and combinations with mefloquine, in suppressing PbA infection in mice with moderate parasitemia. Artemether, artesunate, and quinine were then evaluated for efficacy in rescuing PbA-infected mice with ECM, strictly defined by using objective criteria based on the presentation of clinical signs of neurological involvement, degree of hypothermia, and performance in a set of six motor behavior tests. Artemether at 25 mg/kg presented the fastest parasite killing ability in 24 h and fully avoided recrudescence in a 5-day treatment protocol. Artemether and artesunate were equally effective in rescuing mice with late-stage ECM (46 and 43% survival, respectively), whereas quinine had a poor performance (12.5% survival). Artemether caused a marked decrease in brain leukocyte accumulation 24 h after the first dose. In conclusion, artemether and artesunate are effective in rescuing mice with late-stage ECM and decrease brain inflammation. In addition, the described protocols for more strict clinical evaluation and for rescue treatment provide a framework for studies of CM adjunctive therapies using this mouse model.

Nitric Oxide Synthase Dysfunction Contributes to Impaired Cerebroarteriolar Reactivity in Experimental Cerebral Malaria

Cerebrovascular dysfunction plays a key role in the pathogenesis of cerebral malaria. In experimental cerebral malaria (ECM) induced by Plasmodium berghei ANKA, cerebrovascular dysfunction characterized by vascular constriction, occlusion and damage results in impaired perfusion and reduced cerebral blood flow and oxygenation, and has been linked to low nitric oxide (NO) bioavailability. Here, we directly assessed cerebrovascular function in ECM using a novel cranial window method for intravital microscopy of the pial microcirculation and probed the role of NOS isoforms and phosphorylation patterns in the impaired vascular responses. We show that pial arteriolar responses to endothelial NOS (eNOS) and neuronal NOS (nNOS) agonists (Acetylcholine (ACh) and N-Methyl-D-Aspartate (NMDA)) were blunted in mice with ECM, and could be partially recovered by exogenous supplementation of tetrahydrobiopterin (BH4). Pial arterioles in non-ECM mice infected by Plasmodium berghei NK65 remained relatively responsive to the agonists and were not significantly affected by BH4 treatment. These findings, together with the observed blunting of NO production upon stimulation by the agonists, decrease in total NOS activity, augmentation of lipid peroxidation levels, upregulation of eNOS protein expression, and increase in eNOS and nNOS monomerization in the brain during ECM development strongly indicate a state of eNOS/nNOS uncoupling likely mediated by oxidative stress. Furthermore, the downregulation of Serine 1176 (S1176) phosphorylation of eNOS, which correlated with a decrease in cerebrovascular wall shear stress, implicates hemorheological disturbances in eNOS dysfunction in ECM. Finally, pial arterioles responded to superfusion with the NO donor, S-Nitroso-L-glutathione (GSNO), but with decreased intensity, indicating that not only NO production but also signaling is perturbed during ECM. Therefore, the pathological impairment of eNOS and nNOS functions contribute importantly to cerebrovascular dysfunction in ECM and the recovery of intrinsic functionality of NOS to increase NO bioavailability and restore vascular health represents a target for ECM treatment.

Efficacy of Different Nitric Oxide-Based Strategies in Preventing Experimental Cerebral Malaria by Plasmodium berghei ANKA

Background Low nitric oxide (NO) bioavailability plays a role in the pathogenesis of human as well as of experimental cerebral malaria (ECM) caused by Plasmodium berghei ANKA (PbA). ECM is partially prevented by administration of the NO-donor dipropylenetriamine NONOate (DPTA-NO) at high concentration (1 mg/mouse), which also induces major side effects such as a sharp drop in blood pressure. We asked whether alternative strategies to improve NO bioavailability with minor side effects would also be effective in preventing ECM. Methodology/Principal Findings Mice were infected with PbA and prophylactically treated twice a day with bolus injections of L-arginine, Nω-hydroxy-nor-Arginine (nor-NOHA), tetrahydrobiopterin (BH4), separately or combined, sodium nitrite, sildenafil or sildenafil plus DPTA-NO starting on day 0 of infection. L-arginine and BH4 supplementation, with or without arginase inhibition by nor-NOHA, increased plasma nitrite levels but failed to protect against ECM development. Accordingly, prophylactic treatment with continuous delivery of L-arginine using osmotic pumps also did not improve survival. Similar outcomes were observed with sodium nitrite sildenafil (aimed at inhibiting phosphodiesterase-5) or with DPTA-NO. However, sildenafil (0.1 mg/mouse) in combination with a lower dose (0.1 mg/mouse) of DPTA-NO decreased ECM incidence (82±7.4% mortality in the saline group and 38±10.6% in the treated group; p<0.05). The combined prophylactic therapy did not aggravate anemia, had delayed effects in systolic, diastolic and mean arterial blood pressure and induced lower effects in pulse pressure when compared to DPTA-NO 1 mg/mouse. Conclusions/Significance These data show that sildenafil lowers the amount of NO-donor needed to prevent ECM, resulting also in lesser side effects. Prophylactic L-arginine when given in bolus or continuous delivery and bolus BH4 supplementation, with or without arginase inhibition, were able to increase NO bioavailability in PbA-infected mice but failed to decrease ECM incidence in the doses and protocol used.

Aotus infulatus monkey is susceptible to Plasmodium falciparum infection and may constitute an alternative experimental model for malaria

Aotus is one of the WHO-recommended primate models for studies in malaria, and several species can be infected with Plasmodium falciparum or P. vivax. Here we describe the successful infection of the species A. infulatus from eastern Amazon with blood stages of P. falciparum. Both intact and splenectomized animals were susceptible to infection; the intact ones were able to keep parasitemias at lower levels for several days, but developed complications such as severe anemia; splenectomized monkeys developed higher parasitemias but no major complications. We conclude that A. infulatus is susceptible to P. falciparum infection and may represent an alternative model for studies in malaria.