RESUMEN
RTS,S is the first malaria vaccine recommended for implementation among young children at risk. However, vaccine efficacy is modest and short-lived. To mitigate the risk of cerebral malaria (CM) among children under the age of 5, it is imperative to develop new vaccines. EVs are potential vaccine candidates as they obtain the ability of brain-targeted delivery and transfer plasmodium antigens and immunomodulators during infections. This study extracted EVs from BALB/c mice infected with Plasmodium yoelii 17XNL (P.y17XNL). C57BL/6J mice were intravenously immunized with EVs (EV-I.V. + CM group) or subcutaneously vaccinated with the combination of EVs and CpG ODN-1826 (EV + CPG ODN-S.C. + CM group) on days 0 and 20, followed by infection with Plasmodium berghei ANKA (P.bANKA) on day 20 post-second immunization. We monitored Parasitemia and survival rate. The integrity of the Blood-brain barrier (BBB) was examined using Evans blue staining.The levels of cytokines and adhesion molecules were evaluated using Luminex, RT-qPCR, and WB. Brain pathology was evaluated by hematoxylin and eosin and immunohistochemical staining. The serum levels of IgG, IgG1, and IgG2a were analyzed by enzyme-linked immunosorbent assay. Compared with those in the P.bANKA-infected group, parasitemia increased slowly, death was delayed (day 10 post-infection), and the survival rate reached 75 %-83.3 % in the EV-I.V. + ECM and EV + CPG ODN-S.C. + ECM groups. Meanwhile, compared with the EV + CPG ODN-S.C. + ECM group, although parasitemia was almost the same, the survival rate increased in the EV-I.V. + ECM group.Additionally, EVs immunization markedly downregulated inflammatory responses in the spleen and brain and ameliorated brain pathological changes, including BBB disruption and infected red blood cell (iRBC) sequestration. Furthermore, the EVs immunization group exhibited enhanced antibody responses (upregulation of IgG1 and IgG2a production) compared to the normal control group. EV immunization exerted protective effects, improving the integrity of the BBB, downregulating inflammation response of brain tissue, result in reduces the incidence of CM. The protective effects were determined by immunological pathways and brain targets elicited by EVs. Intravenous immunization exhibited better performance than subcutaneous immunization, which perhaps correlated with EVs, which can naturally cross BBB to play a better role in brain protection.
Asunto(s)
Barrera Hematoencefálica , Eritrocitos , Vesículas Extracelulares , Malaria Cerebral , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Oligodesoxirribonucleótidos , Plasmodium berghei , Animales , Malaria Cerebral/inmunología , Malaria Cerebral/parasitología , Malaria Cerebral/prevención & control , Plasmodium berghei/inmunología , Vesículas Extracelulares/inmunología , Eritrocitos/parasitología , Eritrocitos/inmunología , Barrera Hematoencefálica/inmunología , Ratones , Oligodesoxirribonucleótidos/administración & dosificación , Vacunas contra la Malaria/inmunología , Vacunas contra la Malaria/administración & dosificación , Femenino , Encéfalo/parasitología , Encéfalo/inmunología , Encéfalo/patología , Citocinas/metabolismo , Citocinas/sangre , Plasmodium yoelii/inmunología , Anticuerpos Antiprotozoarios/sangre , Anticuerpos Antiprotozoarios/inmunología , Parasitemia/inmunología , Modelos Animales de Enfermedad , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunologíaRESUMEN
Malaria can have severe long-term effects. Even after treatment with antimalarial drugs eliminates the parasite, survivors of cerebral malaria may suffer from irreversible brain damage, leading to cognitive deficits. Angiotensin II, a natural human peptide hormone that regulates blood pressure, has been shown to be active against Plasmodium spp., the etiologic agent of malaria. Here, we tested two Ang II derivatives that do not elicit vasoconstriction in mice: VIPF, a linear tetrapeptide, which constitutes part of the hydrophobic portion of Ang II; and Ang II-SS, a disulfide-bridged derivative. The antiplasmodial potential of both peptides was evaluated with two mouse models: an experimental cerebral malaria model and a mouse model of non-cerebral malaria. The latter consisted of BALB/c mice infected with Plasmodium berghei ANKA. The peptides had no effect on mean blood pressure and significantly reduced parasitemia in both mouse models. Both peptides reduced the SHIRPA score, an assay used to assess murine health and behavior. However, only the constrained derivative (Ang II-SS), which was also resistant to proteolytic degradation, significantly increased mouse survival. Here, we show that synthetic peptides derived from Ang II are capable of conferring protection against severe manifestations of malaria in mouse models while overcoming the vasoconstrictive side effects of the parent peptide.
Asunto(s)
Antimaláricos , Malaria Cerebral , Animales , Ratones , Humanos , Malaria Cerebral/tratamiento farmacológico , Malaria Cerebral/prevención & control , Malaria Cerebral/parasitología , Angiotensina II/farmacología , Angiotensina II/uso terapéutico , Modelos Animales de Enfermedad , Antimaláricos/farmacología , Antimaláricos/uso terapéutico , Péptidos/farmacología , Péptidos/uso terapéutico , Plasmodium berghei/fisiología , Ratones Endogámicos C57BLRESUMEN
Cerebral malaria is the deadliest complication that can arise from Plasmodium infection. CD8 T-cell engagement of brain vasculature is a putative mechanism of neuropathology in cerebral malaria. To define contributions of brain endothelial cell major histocompatibility complex (MHC) class I antigen-presentation to CD8 T cells in establishing cerebral malaria pathology, we developed novel H-2Kb LoxP and H-2Db LoxP mice crossed with Cdh5-Cre mice to achieve targeted deletion of discrete class I molecules, specifically from brain endothelium. This strategy allowed us to avoid off-target effects on iron homeostasis and class I-like molecules, which are known to perturb Plasmodium infection. This is the first endothelial-specific ablation of individual class-I molecules enabling us to interrogate these molecular interactions. In these studies, we interrogated human and mouse transcriptomics data to compare antigen presentation capacity during cerebral malaria. Using the Plasmodium berghei ANKA model of experimental cerebral malaria (ECM), we observed that H-2Kb and H-2Db class I molecules regulate distinct patterns of disease onset, CD8 T-cell infiltration, targeted cell death and regional blood-brain barrier disruption. Strikingly, ablation of either molecule from brain endothelial cells resulted in reduced CD8 T-cell activation, attenuated T-cell interaction with brain vasculature, lessened targeted cell death, preserved blood-brain barrier integrity and prevention of ECM and the death of the animal. We were able to show that these events were brain-specific through the use of parabiosis and created the novel technique of dual small animal MRI to simultaneously scan conjoined parabionts during infection. These data demonstrate that interactions of CD8 T cells with discrete MHC class I molecules on brain endothelium differentially regulate development of ECM neuropathology. Therefore, targeting MHC class I interactions therapeutically may hold potential for treatment of cases of severe malaria.
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Malaria Cerebral , Ratones , Humanos , Animales , Malaria Cerebral/patología , Malaria Cerebral/prevención & control , Células Endoteliales/patología , Encéfalo/patología , Barrera Hematoencefálica/patología , Linfocitos T CD8-positivos , Endotelio/patología , Ratones Endogámicos C57BL , Modelos Animales de EnfermedadRESUMEN
Every year, thousands of children, particularly those under 5 years old, die because of cerebral malaria (CM). Following conventional treatment, approximately 25% of surviving individuals have lifelong severe neurocognitive sequelae. Therefore, improved conventional therapies or effective alternative therapies that prevent the severe infection are crucial. Omega-3 (Ω-3) polyunsaturated fatty acids (PUFAs) are known to have antioxidative and anti-inflammatory effects and protect against diverse neurological disorders, including Alzheimer's and Parkinson's diseases. However, little is known regarding the effects of Ω-3 PUFAs against parasitic infections. In this study, C57BL/6 mice received supplemental treatment of a fish oil rich in the Ω-3 PUFA, docosahexaenoic acid (DHA), which was started 15 days prior to infection with Plasmodium berghei ANKA and was maintained until the end of the study. Animals treated with the highest doses of DHA, 3.0 and 6.0 g/kg body weight, had 60 and 80% chance of survival, respectively, while all nontreated mice died by the 7th day postinfection due to CM. Furthermore, the parasite load during the critical period for CM development (5th to 11th day postinfection) was controlled in treated mice. However, after this period all animals developed high levels of parasitemia until the 20th day of infection. DHA treatment also effectively reduced blood-brain barrier (BBB) damage and brain edema and completely prevented brain hemorrhage and vascular occlusion. A strong anti-inflammatory profile was observed in the brains of DHA-treated mice, as well as, an increased number of neutrophil and reduced number of CD8+ T leukocytes in the spleen. Thus, this is the first study to demonstrate that the prophylactic use of DHA-rich fish oil exerts protective effects against experimental CM, reducing the mechanical and immunological events caused by the P. berghei ANKA infection.
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Ácidos Grasos Omega-3 , Malaria Cerebral , Niño , Humanos , Ratones , Animales , Preescolar , Aceites de Pescado/farmacología , Ácidos Docosahexaenoicos/farmacología , Ácidos Docosahexaenoicos/uso terapéutico , Malaria Cerebral/prevención & control , Malaria Cerebral/tratamiento farmacológico , Ratones Endogámicos C57BL , Ácidos Grasos Omega-3/uso terapéutico , Antiinflamatorios/farmacología , Antiinflamatorios/uso terapéuticoRESUMEN
Cerebral Malaria (CM) is associated with the complex neurological syndrome, whose pathology is mediated by severe inflammatory processes following infection with Plasmodium falciparum. Coenzyme-Q10 (Co-Q10) is a potent anti-inflammatory, anti-oxidant, and anti-apoptotic agent with numerous clinical applications. The aim of this study was to elucidate the role of oral administration of Co-Q10 on the initiation or regulation of inflammatory immune response during experimental cerebral malaria (ECM). For this purpose, the pre-clinical effect of Co-Q10 was evaluated in C57BL/6 J mice infected with Plasmodium berghei ANKA (PbA). Treatment with Co-Q10 resulted in the reduction of infiltrating parasite load, greatly improved the survival rate of PbA-infected mice that occurred independent of parasitaemia and prevented PbA-induced disruption of the blood-brain barrier (BBB) integrity. Exposure to Co-Q10 resulted in the reduction of infiltration of effector CD8 + T cells in the brain and secretion of cytolytic Granzyme B molecules. Notably, Co-Q10-treated mice had reduced levels of CD8 +T cell chemokines CXCR3, CCR2, and CCR5 in the brain following PbA-infection. Brain tissue analysis showed a reduction in the levels of inflammatory mediators TNF- α, CCL3, and RANTES in Co-Q10 administered mice. In addition, Co-Q10 modulated the differentiation and maturation of both splenic and brain dendritic cells and cross-presentation (CD8α+DCs) during ECM. Remarkably, Co-Q10 was very effective in decreasing levels of CD86, MHC-II, and CD40 in macrophages associated with ECM pathology. Exposure to Co-Q10 resulted in increased expression levels of Arginase-1 and Ym1/chitinase 3-like 3, which is linked to ECM protection. Furthermore, Co-Q10 supplementation prevented PbA-induced depletion of Arginase and CD206 mannose receptor levels. Co-Q10 abrogated PbA-driven elevation in pro-inflammatory cytokines IL-1ß, IL-18, and IL-6 levels. In conclusion, the oral supplementation with Co-Q10 decelerates the occurrence of ECM by preventing lethal inflammatory immune responses and dampening genes associated with inflammation and immune-pathology during ECM, and offers an inimitable opening for developing an anti-inflammatory agent against cerebral malaria.
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Malaria Cerebral , Ratones , Animales , Malaria Cerebral/tratamiento farmacológico , Malaria Cerebral/prevención & control , Arginasa , Modelos Animales de Enfermedad , Ratones Endogámicos C57BL , Antiinflamatorios/farmacología , Antiinflamatorios/uso terapéutico , Inmunidad , Plasmodium bergheiRESUMEN
Heme-biosynthetic pathway of malaria parasite is dispensable for asexual stages, but essential for mosquito and liver stages. Despite having backup mechanisms to acquire hemoglobin-heme, pathway intermediates and/or enzymes from the host, asexual parasites express heme pathway enzymes and synthesize heme. Here we show heme synthesized in asexual stages promotes cerebral pathogenesis by enhancing hemozoin formation. Hemozoin is a parasite molecule associated with inflammation, aberrant host-immune responses, disease severity and cerebral pathogenesis. The heme pathway knockout parasites synthesize less hemozoin, and mice infected with knockout parasites are protected from cerebral malaria and death due to anemia is delayed. Biosynthetic heme regulates food vacuole integrity and the food vacuoles from knockout parasites are compromised in pH, lipid unsaturation and proteins, essential for hemozoin formation. Targeting parasite heme synthesis by griseofulvin-a FDA-approved antifungal drug, prevents cerebral malaria in mice and provides an adjunct therapeutic option for cerebral and severe malaria.
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Malaria Cerebral , Parásitos , Animales , Griseofulvina/farmacología , Hemo/metabolismo , Hemoglobinas , Malaria Cerebral/tratamiento farmacológico , Malaria Cerebral/prevención & control , Ratones , Parásitos/metabolismoRESUMEN
1,8-Cineole is a naturally occurring compound found in essential oils of different plants and has well-known anti-inflammatory and antimicrobial activities. In the present work, we aimed to investigate its potential antimalarial effect, using the following experimental models: (1) the erythrocytic cycle of Plasmodium falciparum; (2) an adhesion assay using brain microvascular endothelial cells; and (3) an experimental cerebral malaria animal model induced by Plasmodium berghei ANKA infection in susceptible mice. Using the erythrocytic cycle of Plasmodium falciparum, we characterized the schizonticidal effect of 1,8-cineole. This compound decreased parasitemia in a dose-dependent manner with a half maximal inhibitory concentration of 1045.53 ± 63.30 µM. The inhibitory effect of 972 µM 1,8-cineole was irreversible and independent of parasitemia. Moreover, 1,8-cineole reduced the progression of intracellular development of the parasite over 2 cycles, inducing important morphological changes. Ultrastructure analysis revealed a massive loss of integrity of endomembranes and hemozoin crystals in infected erythrocytes treated with 1,8-cineole. The monoterpene reduced the adhesion index of infected erythrocytes to brain microvascular endothelial cells by 60%. Using the experimental cerebral malaria model, treatment of infected mice for 6 consecutive days with 100 mg/kg/day 1,8-cineole reduced cerebral edema with a 50% reduction in parasitemia. Our data suggest a potential antimalarial effect of 1,8-cineole with an impact on the parasite erythrocytic cycle and severe disease.
Asunto(s)
Antimaláricos , Edema Encefálico , Malaria Cerebral , Animales , Antimaláricos/química , Antimaláricos/farmacología , Antimaláricos/uso terapéutico , Modelos Animales de Enfermedad , Células Endoteliales , Eucaliptol/farmacología , Malaria Cerebral/tratamiento farmacológico , Malaria Cerebral/parasitología , Malaria Cerebral/prevención & control , Ratones , Ratones Endogámicos C57BL , Monoterpenos/farmacología , Monoterpenos/uso terapéutico , Parasitemia/tratamiento farmacológico , Parasitemia/parasitología , Plasmodium berghei , Plasmodium falciparumRESUMEN
BACKGROUND: Cerebral malaria is one of the most severe complications attributed to protozoal infection by Plasmodium falciparum, gaining prominence in children mortality rates in endemic areas. This condition has a complex pathogenesis associated with behavioral, cognitive and motor sequels in humans and current antimalarial therapies have shown little effect in those aspects. Natural products with antioxidant and anti-inflammatory properties have become a valuable alternative therapeutic option in the treatment of distinct conditions. In this context, this study investigated the neuroprotective effect of Euterpe oleracea (açai) enriched diet during the development of experimental cerebral malaria induced by the inoculation of Swiss albino mice with Plasmodium berghei ANKA strain. METHODS: After Plasmodium infection, animals were maintained on a feeding with Euterpe oleracea enriched ration and parameters such as survival curve, parasitemia and body weight were routinely monitored. The present study has also evaluated the effect of açai-enriched diet on the blood-brain barrier leakage, histological alterations and neurocognitive impairments in mice developing cerebral malaria. RESULTS: Our results demonstrate that between 7th-19th day post infection the survival rate of the group treated with açai enriched ration was higher when compared with Plasmodium-infected mice in which 100% of mice died until the 11th days post-infection, demonstrating that açai diet has a protective effect on the survival of infected treated animals. The same was observed in the brain vascular extravasation, where Evans blue dye assays showed significantly less dye extravasation in the brains of Plasmodium-infected mice treated with açai enriched ration, demonstrating more preserved blood-brain barrier integrity. Açai-enriched diet also attenuate the histopathological alterations elicited by Plasmodium berghei infection. We also showed a decrease of the neurological impairments arising from the exposure of cerebral parenchyma in the group treated with açai diet, ameliorating motor and neuropsychiatric changes, analyzed through the SHIRPA protocol. CONCLUSION: With these results, we conclude that the treatment with açai enriched ration decreased the mortality of infected animals, as well as protected the blood-brain barrier and the neurocognitive deficits in Plasmodium-infected animals.
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Euterpe , Malaria Cerebral/dietoterapia , Malaria Cerebral/prevención & control , Fármacos Neuroprotectores/uso terapéutico , Fitoterapia , Alimentación Animal , Animales , Síntomas Conductuales/etiología , Síntomas Conductuales/prevención & control , Barrera Hematoencefálica , Femenino , Frutas , Malaria Cerebral/fisiopatología , Masculino , Ratones , Plantas Medicinales , Plasmodium bergheiRESUMEN
The deadliest complication of infection by Plasmodium parasites, cerebral malaria, accounts for the majority of malarial fatalities. Although our understanding of the cellular and molecular mechanisms underlying the pathology remains incomplete, recent studies support the contribution of systemic and neuroinflammation as the cause of cerebral edema and blood-brain barrier (BBB) dysfunction. All Plasmodium species encode an orthologue of the innate cytokine, Macrophage Migration Inhibitory Factor (MIF), which functions in mammalian biology to regulate innate responses. Plasmodium MIF (PMIF) similarly signals through the host MIF receptor CD74, leading to an enhanced inflammatory response. We investigated the PMIF-CD74 interaction in the onset of experimental cerebral malaria (ECM) and liver stage Plasmodium development by using a combination of CD74 deficient (Cd74-/- ) hosts and PMIF deficient parasites. Cd74-/- mice were found to be protected from ECM and the protection was associated with the inability of brain microvessels to present parasite antigen to sequestered and pathogenic Plasmodium-specific CD8+ T cells. Infection of WT hosts with PMIF-deficient sporozoites or infection of Cd74-/- hosts with WT sporozoites impacted the survival of infected hepatocytes and subsequently reduced blood-stage associated inflammation, contributing to protection from ECM. We recapitulated these finding with a novel pharmacologic PMIF-selective antagonist that reduced PMIF/CD74 signaling and fully protected mice from ECM. These findings reveal a conserved mechanism for Plasmodium usurpation of host CD74 signaling and suggest a tractable approach for new pharmacologic intervention.
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Antígenos de Diferenciación de Linfocitos B/química , Linfocitos T CD8-positivos/inmunología , Antígenos de Histocompatibilidad Clase II/química , Inflamación/prevención & control , Hígado/patología , Factores Inhibidores de la Migración de Macrófagos/antagonistas & inhibidores , Malaria Cerebral/prevención & control , Plasmodium berghei/fisiología , Animales , Antígenos de Diferenciación de Linfocitos B/fisiología , Antígenos de Histocompatibilidad Clase II/fisiología , Inflamación/etiología , Inflamación/metabolismo , Inflamación/patología , Hígado/inmunología , Hígado/parasitología , Factores Inhibidores de la Migración de Macrófagos/metabolismo , Malaria Cerebral/etiología , Malaria Cerebral/metabolismo , Malaria Cerebral/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
Epidemiological studies provide compelling evidence that glucose-6-phosphate dehydrogenase (G6PD) deficiency individuals are relatively protected against Plasmodium parasite infection. However, the animal model studies on this subject are lacking. Plus, the underlying mechanism in vivo is poorly known. In this study, we used a G6pd-deficient mice infected with the rodent parasite Plasmodium berghei (P.berghei) to set up a malaria model in mice. We analyzed the pathological progression of experimental cerebral malaria (ECM) and acute liver injury in mice with different G6pd activity infected with P.berghei. We performed dual RNA-seq for host-parasite transcriptomics and validated the changes of proinflammatory response in the murine model. G6pd-deficient mice exhibited a survival advantage, less severe ECM and mild liver injury compared to the wild type mice. Analysis based on dual RNA-seq suggests that G6pd-deficient mice are protected from ECM and acute liver injury were related to proinflammatory responses. Th1 differentiation and dendritic cell maturation in the liver and spleen were inhibited in G6pd-deficient mice. The levels of proinflammatory cytokines were reduced, chemokines and vascular adhesion molecules in the brain were significantly down-regulated, these led to decreased cerebral microvascular obstruction in G6pd-deficient mice. We generated the result that G6pd-deficiency mediated protection against ECM and acute liver injury were driven by the regulatory proinflammatory responses. Furthermore, bioinformatics analyses showed that P.berghei might occur ribosome loss in G6pd-deficient mice. Our findings provide a novel perspective of the underlying mechanism of G6PD deficiency mediated protection against malaria in vivo.
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Deficiencia de Glucosafosfato Deshidrogenasa/complicaciones , Deficiencia de Glucosafosfato Deshidrogenasa/metabolismo , Glucosafosfato Deshidrogenasa/metabolismo , Parasitosis Hepáticas/complicaciones , Parasitosis Hepáticas/prevención & control , Malaria Cerebral/complicaciones , Malaria Cerebral/prevención & control , Animales , Biomarcadores , Biopsia , Barrera Hematoencefálica/metabolismo , Citocinas/metabolismo , Modelos Animales de Enfermedad , Susceptibilidad a Enfermedades , Activación Enzimática , Perfilación de la Expresión Génica , Deficiencia de Glucosafosfato Deshidrogenasa/etiología , Hemólisis , Mediadores de Inflamación/metabolismo , Parasitosis Hepáticas/metabolismo , Parasitosis Hepáticas/patología , Malaria Cerebral/metabolismo , Ratones , Plasmodium bergheiRESUMEN
Cerebral malaria (CM) is a neurological complication derived from the Plasmodium falciparum infection in humans. The mechanisms involved in the disease progression are still not fully understood, but both the sequestration of infected red blood cells (iRBC) and leukocytes and an exacerbated host inflammatory immune response are significant factors. In this study, we investigated the effect of Monocyte Locomotion Inhibitory Factor (MLIF), an anti-inflammatory peptide, in a well-characterized murine model of CM. Our data showed that the administration of MLIF increased the survival and avoided the neurological signs of CM in Plasmodium berghei ANKA (PbA) infected C57BL/6 mice. MLIF administration down-regulated systemic inflammatory mediators such as IFN-γ, TNF-α, IL-6, CXCL2, and CCL2, as well as the in situ expression of TNF-α in the brain. In the same way, MLIF reduced the expression of CD31, CD36, CD54, and CD106 in the cerebral endothelium of infected animals and prevented the sequestration of iRBC and leucocytes in the brain microvasculature. Furthermore, MLIF inhibited the activation of astrocytes and microglia and preserved the integrity of the blood-brain barrier (BBB). In conclusion, our results demonstrated that the administration of MLIF increased survival and conferred neuroprotection by decreasing neuroinflammation in murine CM.
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Antiinflamatorios/administración & dosificación , Malaria Cerebral/prevención & control , Fármacos Neuroprotectores/administración & dosificación , Oligopéptidos/administración & dosificación , Animales , Astrocitos/efectos de los fármacos , Astrocitos/inmunología , Encéfalo/efectos de los fármacos , Encéfalo/inmunología , Encéfalo/patología , Modelos Animales de Enfermedad , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/inmunología , Femenino , Humanos , Mediadores de Inflamación/antagonistas & inhibidores , Mediadores de Inflamación/metabolismo , Malaria Cerebral/inmunología , Malaria Cerebral/parasitología , Malaria Cerebral/patología , Ratones , Microglía/efectos de los fármacos , Microglía/inmunología , Plasmodium berghei/inmunologíaRESUMEN
CD47 is an antiphagocytic "don't eat me" signal that inhibits programmed cell removal of self. As red blood cells (RBCs) age they lose CD47 expression and become susceptible to programmed cell removal by macrophages. CD47-/- mice infected with Plasmodium yoelii, which exhibits an age-based preference for young RBCs, were previously demonstrated to be highly resistant to malaria infection. Our study sought to test the therapeutic benefit of CD47 blockade on ameliorating the clinical syndromes of experimental cerebral malaria (ECM), using the Plasmodium berghei ANKA (Pb-A) murine model. In vitro we tested the effect of anti-CD47 mAb on Plasmodium-infected RBC phagocytosis and found that anti-CD47 treatment significantly increased clearance of Plasmodium-infected RBCs. Infection of C57BL/6 mice with Pb-A is lethal and mice succumb to the clinical syndromes of CM between days 6 and 10 postinfection. Strikingly, treatment with anti-CD47 resulted in increased survival during the cerebral phase of Pb-A infection. Anti-CD47-treated mice had increased lymphocyte counts in the peripheral blood and increased circulating levels of IFN-γ, TNF-α, and IL-22. Despite increased circulating levels of inflammatory cytokines, anti-CD47-treated mice had reduced pathological features in the brain. Survival of ECM in anti-CD47-treated mice was correlated with reduced cellular accumulation in the cerebral vasculature, improved blood-brain barrier integrity, and reduced cytotoxic activity of infiltrating CD8+ T cells. These results demonstrate the therapeutic benefit of anti-CD47 to reduce morbidity in a lethal model of ECM, which may have implications for preventing mortality in young African children who are the highest casualties of CM.
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Antígeno CD47/antagonistas & inhibidores , Interacciones Huésped-Parásitos , Malaria Cerebral/patología , Animales , Anticuerpos Monoclonales/inmunología , Antígeno CD47/inmunología , Eritrocitos/parasitología , Humanos , Malaria Cerebral/prevención & control , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , FagocitosisRESUMEN
Cerebral malaria (CM) is a life-threatening diffuse encephalopathy caused by Plasmodium falciparum, in which the destruction of the blood-brain barrier (BBB) is the main cause of death. However, increasing evidence has shown that antimalarial drugs, the current treatment for CM, do little to protect against CM-induced BBB damage. Therefore, a means to alleviate BBB dysfunction would be a promising adjuvant therapy for CM. The adhesion molecule CD146 has been reported to be expressed in both endothelial cells and proinflammatory immune cells and mediates neuroinflammation. Here, we demonstrate that CD146 expressed on BBB endothelial cells but not immune cells is a novel therapeutic target in a mouse model of experimental cerebral malaria (eCM). Endothelial CD146 is upregulated during eCM development and facilitates the sequestration of infected red blood cells (RBCs) and/or proinflammatory lymphocytes in CNS blood vessels, thereby promoting the disruption of BBB integrity. Mechanistic studies showed that the interaction of CD146 and Galectin-9 contributes to the aggregation of infected RBCs and lymphocytes. Deletion of endothelial CD146 or treatment with the anti-CD146 antibody AA98 prevents severe signs of eCM, such as limb paralysis, brain vascular leakage, and death. In addition, AA98 combined with the antiparasitic drug artemether improved the cognition and memory of mice with eCM. Taken together, our findings suggest that endothelial CD146 is a novel and promising target in combination with antiparasitic drugs for future CM therapies.
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Barrera Hematoencefálica , Antígeno CD146 , Malaria Cerebral , Animales , Modelos Animales de Enfermedad , Células Endoteliales , Galectinas/uso terapéutico , Malaria Cerebral/tratamiento farmacológico , Malaria Cerebral/prevención & control , RatonesRESUMEN
BACKGROUND: Cerebral malaria (CM) is associated with morbidity and mortality despite the use of potent anti-malarial agents. Brain endothelial cell activation and dysfunction from oxidative and inflammatory host responses and products released by Plasmodium falciparum-infected erythrocytes (IE), are likely the major contributors to the encephalopathy, seizures, and brain swelling that are associated with CM. The development of adjunctive therapy to reduce the pathological consequences of host response pathways could improve outcomes. A potentially protective role of the nuclear factor E2-related factor 2 (NRF2) pathway, which serves as a therapeutic target in brain microvascular diseases and central nervous system (CNS) inflammatory diseases such as multiple sclerosis was tested to protect endothelial cells in an in vitro culture system subjected to tumour necrosis factor (TNF) or infected red blood cell exposure. NRF2 is a transcription factor that mediates anti-oxidant and anti-inflammatory responses. METHODS: To accurately reflect clinically relevant parasite biology a unique panel of parasite isolates derived from patients with stringently defined CM was developed. The effect of TNF and these parasite lines on primary human brain microvascular endothelial cell (HBMVEC) activation in an in vitro co-culture model was tested. HBMVEC activation was measured by cellular release of IL6 and nuclear translocation of NFκB. The transcriptional and functional effects of dimethyl fumarate (DMF), an FDA approved drug which induces the NRF2 pathway, on host and parasite induced HBMVEC activation was characterized. In addition, the effect of DMF on parasite binding to TNF stimulated HBMVEC in a semi-static binding assay was examined. RESULTS: Transcriptional profiling demonstrates that DMF upregulates the NRF2-Mediated Oxidative Stress Response, ErbB4 Signaling Pathway, Peroxisome Proliferator-activated Receptor (PPAR) Signaling and downregulates iNOS Signaling and the Neuroinflammation Signaling Pathway on TNF activated HBMVEC. The parasite lines derived from eight paediatric CM patients demonstrated increased binding to TNF activated HBMVEC and varied in their binding and activation of HBMVEC. Overall DMF reduced both TNF and CM derived parasite activation of HBMVEC. CONCLUSIONS: These findings provide evidence that targeting the NRF2 pathway in TNF and parasite activated HBMVEC mediates multiple protective pathways and may represent a novel adjunctive therapy to improve infection outcomes in CM.
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Antiinflamatorios/farmacología , Dimetilfumarato/farmacología , Células Endoteliales/parasitología , Malaria Cerebral/prevención & control , Malaria Falciparum/prevención & control , Plasmodium falciparum/efectos de los fármacos , Factor de Necrosis Tumoral alfa/metabolismo , Antioxidantes/metabolismo , Encéfalo/efectos de los fármacos , Encéfalo/parasitología , Niño , Preescolar , Células Endoteliales/efectos de los fármacos , Humanos , Lactante , Plasmodium falciparum/fisiologíaRESUMEN
The replication cycle and pathogenesis of the Plasmodium malarial parasite involves rapid expansion in red blood cells (RBCs), and variants of certain RBC-specific proteins protect against malaria in humans. In RBCs, bisphosphoglycerate mutase (BPGM) acts as a key allosteric regulator of hemoglobin/oxyhemoglobin. We demonstrate here that a loss-of-function mutation in the murine Bpgm (BpgmL166P) gene confers protection against both Plasmodium-induced cerebral malaria and blood-stage malaria. The malaria protection seen in BpgmL166P mutant mice is associated with reduced blood parasitemia levels, milder clinical symptoms, and increased survival. The protective effect of BpgmL166P involves a dual mechanism that enhances the host's stress erythroid response to Plasmodium-driven RBC loss and simultaneously alters the intracellular milieu of the RBCs, including increased oxyhemoglobin and reduced energy metabolism, reducing Plasmodium maturation, and replication. Overall, our study highlights the importance of BPGM as a regulator of hemoglobin/oxyhemoglobin in malaria pathogenesis and suggests a new potential malaria therapeutic target.
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Anemia/etiología , Anemia/prevención & control , Bisfosfoglicerato Mutasa/deficiencia , Malaria Cerebral/enzimología , Malaria Cerebral/prevención & control , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Secuencia de Bases , Bisfosfoglicerato Mutasa/química , Bisfosfoglicerato Mutasa/genética , Bisfosfoglicerato Mutasa/metabolismo , Estabilidad de Enzimas , Eritrocitos/enzimología , Eritrocitos/parasitología , Eritropoyesis , Matriz Extracelular/metabolismo , Femenino , Células HEK293 , Humanos , Malaria Cerebral/complicaciones , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Mutación/genética , Parásitos/crecimiento & desarrollo , Plasmodium/crecimiento & desarrollo , PolicitemiaRESUMEN
Due to the lack of efficiency to control malaria elicited by sub-unit vaccine preparations, vaccination with live-attenuated Plasmodium parasite as reported 70 years ago with irradiated sporozoites regained recently a significant interest. The complex life cycle of the parasite and the different stages of development between mammal host and anopheles do not help to propose an easy vaccine strategy. In order to achieve a complete long-lasting protection against Plasmodium infection and disease, we considered a genetically attenuated blood stage parasite in the hmgb2 gene coding for the high-mobility-group-box 2 (HMGB2). This Plasmodium protein belongs to the HMGB family and hold as the mammal proteins, a double life since it acts first as a nuclear factor involved in chromatin remodelling and transcription regulation and second, when secreted as an active pro-inflammatory alarmin protein. Even though the number of reports on whole living attenuated blood stage parasites is limited when compared to attenuated sporozoites, the results reported with Plasmodium KO parasites are very encouraging. In this report, we present a novel strategy based on pre-immunization with Δhmgb2PbNK65 parasitized red blood cells that confer long-lasting protection in a murine experimental cerebral malaria model against two highly pathogenic homologous and heterologous parasites.
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Proteína HMGB2/genética , Malaria Cerebral/prevención & control , Plasmodium berghei/genética , Animales , Anopheles/inmunología , Linfocitos T CD8-positivos/inmunología , Protección Cruzada/inmunología , Modelos Animales de Enfermedad , Eritrocitos/parasitología , Femenino , Proteína HMGB2/metabolismo , Inmunización/métodos , Vacunas contra la Malaria/inmunología , Malaria Cerebral/parasitología , Ratones , Ratones Endogámicos C57BL , Plasmodium berghei/patogenicidad , Esporozoítos/genética , Vacunación/métodos , Vacunas Atenuadas/genética , Vacunas Atenuadas/inmunologíaRESUMEN
Excessive inflammatory immune responses during infections with Plasmodium parasites are responsible for severe complications such as cerebral malaria (CM) that can be studied experimentally in mice. Dendritic cells (DCs) activate cytotoxic CD8+ T-cells and initiate immune responses against the parasites. Batf3-/- mice lack a DC subset, which efficiently induces strong CD8 T-cell responses by cross-presentation of exogenous antigens. Here we show that Batf3-/- mice infected with Plasmodium berghei ANKA (PbA) were protected from experimental CM (ECM), characterized by a stable blood-brain barrier (BBB) and significantly less infiltrated peripheral immune cells in the brain. Importantly, the absence of ECM in Batf3-/- mice correlated with attenuated responses of cytotoxic T-cells, as their parasite-specific lytic activity as well as the production of interferon gamma and granzyme B were significantly decreased. Remarkably, spleens of ECM-protected Batf3-/- mice had elevated levels of regulatory immune cells and interleukin 10. Thus, protection from ECM in PbA-infected Batf3-/- mice was associated with the absence of strong CD8+ T-cell activity and induction of immunoregulatory mediators and cells.
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Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/deficiencia , Encéfalo/inmunología , Células Dendríticas/inmunología , Malaria Cerebral/prevención & control , Plasmodium berghei/patogenicidad , Proteínas Represoras/deficiencia , Linfocitos T Citotóxicos/inmunología , Animales , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Barrera Hematoencefálica/inmunología , Barrera Hematoencefálica/parasitología , Encéfalo/metabolismo , Encéfalo/parasitología , Células Cultivadas , Células Dendríticas/metabolismo , Células Dendríticas/parasitología , Modelos Animales de Enfermedad , Femenino , Granzimas/inmunología , Granzimas/metabolismo , Interacciones Huésped-Parásitos , Interferón gamma/inmunología , Interferón gamma/metabolismo , Interleucina-10/inmunología , Interleucina-10/metabolismo , Malaria Cerebral/inmunología , Malaria Cerebral/metabolismo , Malaria Cerebral/parasitología , Ratones Endogámicos C57BL , Ratones Noqueados , Plasmodium berghei/inmunología , Proteínas Represoras/genética , Bazo/inmunología , Bazo/metabolismo , Bazo/parasitología , Linfocitos T Citotóxicos/metabolismo , Linfocitos T Citotóxicos/parasitologíaRESUMEN
Malaria, a mosquito-borne infectious disease caused by parasites of the genus Plasmodium continues to be a major health problem worldwide. The unicellular Plasmodium-parasites have the unique capacity to infect and replicate within host erythrocytes. By expressing variant surface antigens Plasmodium falciparum has evolved to avoid protective immune responses; as a result in endemic areas anti-malaria immunity develops gradually over many years of multiple and repeated infections. We are studying the role of Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) expressed by asexual stages of P. falciparum responsible for the pathogenicity of severe malaria. The immunopathology of falciparum malaria has been linked to cyto-adhesion of infected erythrocytes to specific host receptors. A greater appreciation of the PfEMP1 molecules important for the development of protective immunity and immunopathology is a prerequisite for the rational discovery and development of a safe and protective anti-disease malaria vaccine. Here we review the role of ICAM-1 and EPCR receptor adhering falciparum-parasites in the development of severe malaria; we discuss our current research to understand the factors involved in the pathogenesis of cerebral malaria and the feasibility of developing a vaccine targeted specifically to prevent this disease.
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Interacciones Huésped-Parásitos/inmunología , Malaria Cerebral/inmunología , Malaria Cerebral/parasitología , Malaria Falciparum/inmunología , Malaria Falciparum/parasitología , Plasmodium falciparum/inmunología , Variación Antigénica , Antígenos de Protozoos/química , Antígenos de Protozoos/inmunología , Humanos , Inmunidad , Vacunas contra la Malaria/inmunología , Malaria Cerebral/prevención & control , Malaria Falciparum/prevención & control , Proteínas Protozoarias/química , Proteínas Protozoarias/inmunología , Relación Estructura-ActividadRESUMEN
BACKGROUND: The development of Plasmodium resistance to the last effective anti-malarial drugs necessitates the urgent development of new anti-malarial therapeutic strategies. To this end, plants are an important source of new molecules. The objective of this study was to evaluate the anti-malarial effects of Terminalia albida, a plant used in Guinean traditional medicine, as well as its anti-inflammatory and antioxidant properties, which may be useful in treating cases of severe malaria. METHODS: In vitro antiplasmodial activity was evaluated on a chloroquine-resistant strain of Plasmodium falciparum (K-1). In vivo efficacy of the plant extract was measured in the experimental cerebral malaria model based on Plasmodium berghei (strain ANKA) infection. Mice brains were harvested on Day 7-8 post-infection, and T cells recruitment to the brain, expression levels of pro- and anti-inflammatory markers were measured by flow cytometry, RT-qPCR and ELISA. Non-malarial in vitro models of inflammation and oxidative response were used to confirm Terminalia albida effects. Constituents of Terminalia albida extract were characterized by ultra-high performance liquid chromatography coupled with high resolution mass spectrometry. Top ranked compounds were putatively identified using plant databases and in silico fragmentation patterns. RESULTS: In vitro antiplasmodial activity of Terminalia albida was confirmed with an IC50 of 1.5 µg/mL. In vivo, Terminalia albida treatment greatly increased survival rates in P. berghei-infected mice. Treated mice were all alive until Day 12, and the survival rate was 50% on Day 20. Terminalia albida treatment also significantly decreased parasitaemia by 100% on Day 4 and 89% on Day 7 post-infection. In vivo anti-malarial activity was related to anti-inflammatory properties, as Terminalia albida treatment decreased T lymphocyte recruitment and expression of pro-inflammatory markers in brains of treated mice. These properties were confirmed in vitro in the non-malarial model. In vitro, Terminalia albida also demonstrated a remarkable dose-dependent neutralization activity of reactive oxygen species. Twelve compounds were putatively identified in Terminalia albida stem bark. Among them, several molecules already identified may be responsible for the different biological activities observed, especially tannins and triterpenoids. CONCLUSION: The traditional use of Terminalia albida in the treatment of malaria was validated through the combination of in vitro and in vivo studies.
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Antiinflamatorios/farmacología , Antimaláricos/farmacología , Malaria Cerebral/prevención & control , Extractos Vegetales/farmacología , Especies Reactivas de Oxígeno/farmacología , Terminalia/química , Animales , Antimaláricos/química , Femenino , Ratones , Ratones Endogámicos C57BL , Extractos Vegetales/química , Plasmodium berghei/efectos de los fármacos , Plasmodium falciparum/efectos de los fármacosRESUMEN
Cerebral malaria is a lethal complication of malaria infection characterized by central nervous system dysfunction and is often not effectively treated by antimalarial combination therapies. It has been shown that the sequestration of the parasite-infected red blood cells that interact with cerebral vessel endothelial cells and the damage of the blood-brain barrier (BBB) play critical roles in the pathogenesis. In this study, we developed a ferritin nanozyme (Fenozyme) composed of recombinant human ferritin (HFn) protein shells that specifically target BBB endothelial cells (BBB ECs) and the inner Fe3O4 nanozyme core that exhibits reactive oxygen species-scavenging catalase-like activity. In the experimental cerebral malaria (ECM) mouse model, administration of the Fenozyme, but not HFn, markedly ameliorated the damage of BBB induced by the parasite and improved the survival rate of infected mice significantly. Further investigations found that Fenozyme, as well as HFn, was able to polarize the macrophages in the liver to the M1 phenotype and promote the elimination of malaria in the blood. Thus, the catalase-like activity of the Fenozyme is required for its therapeutic effect in the mouse model. Moreover, the Fenozyme significantly alleviated the brain inflammation and memory impairment in ECM mice that had been treated with artemether, indicating that combining Fenozyme with an antimalarial drug is a novel strategy for the treatment of cerebral malaria.