In vivo and in vitro evaluation of the antimalarial activity of Caesalpinia pluviosa and its active fraction analysis

Orientador: Fabio Trindade Maranhão CostaDissertação (mestrado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: Para superar o problema do aumento de resistência às drogas, os medicamentos tradicionais são fontes importantes na investigação de potenciais novos antimaláricos. Caesalpinia pluviosa, mais conhecida como 'sibipiruna', é originária do Brasil e estudos mostraram que este gênero apresenta várias propriedades farmacológicas, incluindo a atividade antimalárica. O extrato bruto obtido da casca foi submetido ao fracionamento com diferentes solventes resultando em sete frações. Para avaliar a citotoxicidade do extrato e frações em células MCF-7 foi realizado o ensaio de MTT. Essas amostras foram testadas in vitro contra P. falciparum cloroquino sensível (3D7) e resistente (S20) e in vivo em camundongos infectados por P. chabaudi. A interação da fração etanólica 100% de C. pluviosa com o artesunato foi avaliado e análises de espectrometria de massas foram realizados. As frações etanólica 100% e metanólica 50% apresentaram atividade antimalárica significativa em concentrações não tóxicas, e o ensaio de interação medicamentosa do artesunato com a fração etanólica 100% foi sinérgico. Essa fração foi capaz de inibir significativamente a parasitemia dos animais de forma dose dependente após 4 dias de tratamento (0-3 dia pós infecção). Além disso, análise de espectrometria de massas revelou a presença do íon m/z 303.0450, sugerindo a presença de quercetina. No entanto, em uma segunda análise com o padrão de quercetina mostrou íons diferentes como m/z 137 e 153. Nossos resultados mostram que a fração etanólica 100% de C. pluviosa apresentou atividade antimalárica in vitro em concentrações não tóxicas e esse efeito foi potencializado com a presença de artesunato. Além disso, essa atividade antimalárica foi também sustentada após o tratamento in vivo de camundongos infectados. Finalmente, as análises de espectrometria de massas sugerem que um novo composto, provavelmente um isômero da quercetina, possa estar relacionado à atividade antimalárica da fração etanólica 100%Abstract: To overcome the problem of increasing drug resistance traditional medicines are an important source for investigation of potential new antimalarials. Caesalpinia pluviosa, commonly named 'sibipiruna', is originated from Brazil and studies showed that this genus present multiple therapeutic properties, including antimalarial activity. Crude extract obtained from stem bark was purified with different solvents, resulting in seven fractions. MTT assay was performed to evaluate cytotoxicity in MCF-7 cells. The crude extract and its fractions were tested in vitro against chloroquine-sensitive (3D7) and -resistant (S20) strains of Plasmodium falciparum and in vivo in P. chabaudi-infected mice. In vitro interaction with artesunate and C. pluviosa fraction was assessed and mass spectrometry analyses were conducted. At non-toxic concentrations the 100% ethanolic and 50% methanolic fractions presented significant antimalarial activity against both 3D7 and S20 strains, and drug interaction assays with artesunate showed a synergistic effect with 100% ethanolic fraction. This fraction was able to inhibit mice parasitemia significantly and in a dose dependent manner after 4 days treatment (0-3 post-infection). Moreover, mass spectrometry analyses revealed the presence of an ion corresponding to m/z 303.0450, suggesting the presence of quercetin. However, a second set of analyses, with the standard quercetin, showed distinct ions of m/z 137 and 153. Our findings show that the 100% ethanolic fraction of C. pluviosa exhibited antimalarial activity in vitro at non-toxic concentrations and this effect was potentiated with the presence of artesunate. Moreover, this antimalarial activity was also sustained in vivo after treatment of infected mice. Finally, mass spectrometry analyses suggest a new compound, most likely an isomer of quercetin, related with antimalarial activity of the 100% ethanolic fractionMestradoImunologiaMestre em Genética e Biologia Molecula

Violacein-Induced Chaperone System Collapse Underlies Multistage Antiplasmodial Activity

Antimalarial drugs with novel modes of action and wide therapeutic potential are needed to pave the way for malaria eradication. Violacein is a natural compound known for its biological activity against cancer cells and several pathogens, including the malaria parasite, Plasmodium falciparum (Pf). Herein, using chemical genomic profiling (CGP), we found that violacein affects protein homeostasis. Mechanistically, violacein binds Pf chaperones, PfHsp90 and PfHsp70-1, compromising the latter's ATPase and chaperone activities. Additionally, violacein-treated parasites exhibited increased protein unfolding and proteasomal degradation. The uncoupling of the parasite stress response reflects the multistage growth inhibitory effect promoted by violacein. Despite evidence of proteotoxic stress, violacein did not inhibit global protein synthesis via UPR activation - a process that is highly dependent on chaperones, in agreement with the notion of a violacein-induced proteostasis collapse. Our data highlight the importance of a functioning chaperone-proteasome system for parasite development and differentiation. Thus, a violacein-like small molecule might provide a good scaffold for development of a novel probe for examining the molecular chaperone network and/or antiplasmodial drug design.publishersversionpublishe

Coagulation disturbance in experimental cerebral malaria

Orientador: Fabio Trindade Maranhão CostaTese (doutorado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: A malária cerebral (MC) é a mais grave complicação resultante da infecção por Plasmodium falciparum, levando a óbito milhares de pessoas todos os anos. Apesar dos avanços científicos para desvendar os fatores responsáveis pela MC, sua patogênese não é bem compreendida. MC é uma síndrome multifatorial envolvendo o sequestro de parasitos na microvasculatura e o aumento da expressão de citocinas pró-inflamatórias. Estes fatores, apesar de não estar claro como estão orquestrados, acarretam em disfunção endotelial e coagulopatia, contribuindo de maneira significativa no comprometimento clínico. Sendo assim, o entendimento dos mecanismos relacionados à coagulopatia na malária cerebral abre novas perspectivas para o desenvolvimento de estratégias terapêuticas. Neste trabalho utilizamos o modelo de malária cerebral pela infecção de camundongos C57BL/6 por Plasmodium berghei ANKA (PbA). Com o intuito de assegurar se a malária cerebral experimental (MCE) estaria marcada por uma disfunção no processo de coagulação, similar às infecções em humanos, animais infectados com PbA foram submetidos à cirurgia para indução fotoquímica do trombo arterial e o tempo de formação do trombo foi determinado. Estes ensaios mostraram que na MCE existe um fenótipo pró-trombótico dependente de células imunocompetentes e cepa específica, uma vez que animais infectados por P. berghei NK65 (linhagem que não causa malária cerebral) não apresentaram disfunção equivalente. Ainda, a presença do parasito parece ser fundamental no processo de coagulopatia, assim como de linfócitos T CD8+. A disfunção da hemostasia também foi demonstrada no cérebro desses animais através do aumento da expressão de icam1, tf e epcr e do elevado tempo de retenção das plaquetas nos vasos cerebrais. Além disso, o aumento do complexo trombina-antitrombina e do tempo de lise da euglobulina, juntamente com os níveis elevados do inibidor do ativador do plasminogênio (PAI-1), indicam uma hipercoagulabilidade, com ativação da coagulação associada à diminuição da degradação de fibrina. Para determinar se este fenótipo estaria relacionado ao desenvolvimento da doença, os animais foram tratados com ácido tranexâmico (inibidor do sistema fibrinolítico) e posteriormente infectados por PbA. Os camundongos apresentaram uma morte prematura em relação aos não tratados, bem como um aumento da expressão de tnfa, ifng e prf1. Além disso, camundongos deficientes em PAI-1 (PAI-/-) infectados por PbA não sucumbiram a MCE e essa proteção foi associada com níveis baixos de moléculas inflamatórias e prevenção da ativação endotelial. Isso mostra que a MCE, assim como a disfunção endotelial, é dependente da presença de PAI-1. Coletivamente, estes achados abrem novas perspectivas para o entendimento fisiopatológico da malária cerebral e no desenho de novas abordagens terapêuticasAbstract: Cerebral malaria (CM) is the most severe complication resulting from P. falciparum infection which leads to death thousands of people every year. Despite scientific advances to unveil the factors responsible for CM, its pathogenesis is not well understood. CM is a multifactorial syndrome involving parasite sequestration in the microvasculature and increased expression of proinflammatory cytokines. Although it is not clear how these factors are orchestrated, they result in endothelial dysfunction and coagulopathy that contribute significantly in clinical aggravation. Thus, the understanding of coagulopathy mechanims related to cerebral malaria opens new perspectives for the development of therapeutic strategies. In this work we use the cerebral malaria model: C57BL/6 mice infected by Plasmodium berghei ANKA (PbA). To ensure that experimental cerebral malaria (ECM) was characterized by a dysfunction in the coagulation process, similar to human infections, PbA-infected mice were submitted to surgery for induction of photochemically arterial thrombus and time for thrombus formation was determined. These tests showed that the ECM has a prothrombotic phenotype dependent of immuno-competent cells and strain specific, since animals infected with P. berghei NK65 (non cerebral strain) showed no equivalent dysfunction. Furthermore, the presence of the parasite appears to be critical in the coagulopathy process as well as CD8+ T lymphocytes. The dysfunction of hemostasis was also demonstrated in the brain of these animals through increased expression of icam1, tf and epcr and elevated retention time of platelets in the brain vessels. Furthermore, the increase in thrombin-antithrombin and the euglobulin lysis time, together with high levels of inhibitor of plasminogen activator (PAI-1) indicate a hypercoagulability, with activation of coagulation associated with decreased fibrin degradation. To determine whether this phenotype could be related to the disease development, animals were treated with tranexamic acid (an inhibitor of the fibrinolytic system) and subsequently infected with PbA. The mice showed an early death compared to untreated group, as well as increased expression of tnfa, ifng and prf1. Furthermore, mice deficient in PAI-1 (PAI-1-/-) infected with PbA did not succumbed to ECM and protection was associated with low levels of inflammatory molecules and prevention of endothelial activation. These findings show that ECM, as well as endothelial dysfunction is dependent on the presence of PAI-1. Collectively, these findings open new perspectives for the understanding of the cerebral malaria pathophysiology and the design of new therapeutic approachesDoutoradoImunologiaDoutora em Genética e Biologia Molecular2010/18570-0FAPES

Platelet disturbances correlate with endothelial cell activation in uncomplicated Plasmodium vivax malaria.

Platelets drive endothelial cell activation in many diseases. However, if this occurs in Plasmodium vivax malaria is unclear. As platelets have been reported to be activated and to play a role in inflammatory response during malaria, we hypothesized that this would correlate with endothelial alterations during acute illness. We performed platelet flow cytometry of PAC-1 and P-selectin. We measured platelet markers (CXCL4, CD40L, P-selectin, Thrombopoietin, IL-11) and endothelial activation markers (ICAM-1, von Willebrand Factor and E-selectin) in plasma with a multiplex-based assay. The values of each mediator were used to generate heatmaps, K-means clustering and Principal Component analysis. In addition, we determined pair-wise Pearson's correlation coefficients to generate correlation networks. Platelet counts were reduced, and mean platelet volume increased in malaria patients. The activation of circulating platelets in flow cytometry did not differ between patients and controls. CD40L levels (Median [IQ]: 517 [406-651] vs. 1029 [732-1267] pg/mL, P = 0.0001) were significantly higher in patients, while P-selectin and CXCL4 showed a nonsignificant trend towards higher levels in patients. The network correlation approach demonstrated the correlation between markers of platelet and endothelial activation, and the heatmaps revealed a distinct pattern of activation in two subsets of P. vivax patients when compared to controls. Although absolute platelet activation was not strong in uncomplicated vivax malaria, markers of platelet activity and production were correlated with higher endothelial cell activation, especially in a specific subset of patients