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Cerebral malaria (CM) is a severe neurological complication of malaria infection that results from interrelated pathologies. Despite extensive research efforts, the mechanism of the disease is not completely understood. Clinical studies, postmortem analysis, and animal models have been the main research arenas in CM. In this thesis, shotgun proteomics approach was used to further understand the pathology of human and experimental CM. The mechanism by which CM turns fatal is yet to be identified. A clinical proteomics study was conducted on pooled plasma samples from children with reversible or fatal CM from the Gambia. The results show that depletion of coagulation factors and increased levels of circulating proteasomes are associated with fatal pediatric CM. This data suggests that the ongoing coagulation during CM might be a disseminated intravascular coagulation state that eventually causes depletion of the coagulation factors leading to petechial hemorrhages. In addition, the mechanism(s) by which blood transfusion benefits CM in children was investigated. To that end, the concentration and multimerization pattern of von-willebrand factor, and the concentration of haptoglobin in the plasma of children with CM who received blood transfusions were measured. In addition to clinical studies, experimental cerebral malaria (ECM) in mice has been long used as a model for the disease. A shotgun proteomics workflow was optimized to identify the proteomic signature of the brain tissue of mice with ECM.Because of the utmost importance of membrane proteins in the pathology of the disease, sample fractionation and filter aided sample preparation were used to recover them. The proteomic signature of the brains of mice infected with P. berghei ANKA that developed neurological syndrome, mice infected with P. berghei NK56 that developed severe malaria but without neurological signs, and non-infected mice, were compared to identify CM specific proteins. Among the differentially expressed proteins in mice that developed neurological signs, coagulation and acute-phase proteins were enriched. The data are in accordance with data from the clinical study. Taken together, the results support the role of coagulation and platelets adhesion in the pathogenesis of the disease.

Seulement 1 à 3% des cas de paludisme dégénèrent en NP. Cependant, les séquelles à long termes touchent 3 à 10% des adultes et 25% des enfants et ces personnes présentent des déficits cognitifs notamment au niveau de l'apprentissage. Au niveau de l'armée française, ce sont 15 000 militaires qui sont exposés chaque année dans les zones impaludées, avec au moins 350 accès palustres mais surtout 1 à 2 morts chaque année.Dans le but de lutter contre P. falciparum mais aussi diminuer les séquelles dans le cas du NP deux molécules ont été étudiées. D'une part, l'atorvastatine (AVA) et d'autre part le bleu de méthylène (BM). Les données, précédemment publiées, ont montré l'efficacité de l'AVA non seulement comme antibactérien, antiviral ou antiparasitique mais aussi comme modulateur de l'immunité et adjuvant potentiel pour les antipaludiques actuels. Le BM est un antipaludique qui jusqu'ici présentait une voie de synthèse avec métaux lourds. C'est une nouvelle voie de synthèse sans métaux lourds et une efficacité démontrée qui nous ont incités à étudier cette molécule.Pour estimer l'efficacité de l'AVA et du BM en association avec les antipaludiques actuels nous avons successivement testé ces molécules dans un modèle in vitro (micro test isotopique simplifié) contre P. falciparum, puis dans un modèle murin de neuropaludisme en utilisant des souris C57BL/6N infectées avec Plasmodium berghei. Un gain d'efficacité a été observé en associant l'AVA ou le BM avec les antipaludiques contre P. falciparum mais aussi une protection par rapport au NP chez les souris traitées par les associations. Le BM protégeant aussi bien contre le paludisme simple que le NP. / Only 1 to 3% of malaria infections turn into CM. Meanwhile, long term neurological sequelae range from 3 to 10 % in adults and 25% of child survivors present long term cognitive impairments. In a military framework, there are 15 000 soldiers localized in endemic malaria areas, with at less 350 infection cases giving clinical malaria syndrome but mainly 2 deaths each year.In the aim to fight against P. falciparum but also to decrease sequelae related to CM, two molecules were studied. In one hand, atorvastatin (AVA) and in the other hand methylene blue (MB). AVA is a synthetic inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A (3HMG-CoA) reductase used in the treatment of hypercholesterolemia. Previous data, reported in numerous articles support the efficacy of AVA not only as antimicrobial, antiviral or antiparasitic agent but also as immune system modulator and potential adjuvant in vitro for common antimalarial drugs. BM is an antimalarial drug which until now had a synthesis pathway with heavy metals. It is a new synthesis pathway without heavy metals and an efficacy demonstrated which encouraged us to study this molecule.To evaluate AVA and BM efficacies in combination with common antimalarial drugs, we successively tested these molecules in an in vitro model (simplified isotopic microtest) against P. falciparum, then in experimental cerebral malaria using C57BL/6N mice infected with Plasmodium berghei. An increased efficacy was observed when AVA or MB is associated with common antimalarial drugs against P. falciparum but also a protection against CM in mice treated by drugs combinations. MB protects against malaria but also CM.

Malaria affects 200 million people annually, resulting in 584,000 - 1,238,000 deaths. The majority of these deaths occur in children, less than 5 years of age, in sub-Saharan Africa and are due to cerebral malaria (CM), a neuropathology induced primarily by the species Plasmodium (P.) falciparum. The pathogenesis of CM remains poorly understood and the mechanisms involved in acquired protection against the syndrome in malaria-endemic regions are undefined. Utilising the well characterised P. berghei ANKA experimental infection model of cerebral malaria (ECM), results presented in this thesis show that the development of ECM is associated with the accumulation and arrest of pathogenic CD8+ T cells within the perivascular spaces of the brain. Accumulation of activated CD8+ T cells, without arrest, was observed in the perivascular spaces of the brains of mice infected with the non-ECM causing P. berghei NK65 strain. These data show that the behaviour of intracerebral CD8+ T cells specifies their pathogenic function during malaria infection. The development of ECM was associated with extensive disruption to the BBB, which developed in the absence of extensive CD8+ T cell-dependent endothelial cell apoptosis. We modified the ECM model, establishing an infection-drug cure strategy, to investigate the immunological basis of parasite exposure-induced resistance to ECM development. Three rounds of infection-drug cure promoted resistance to ECM, which was associated with reduced intracerebral expression of genes involved in defence response, regulation of apoptosis, chemotaxis, CTL activity, antigen processing and presentation and cell adhesion, compared with ECM susceptible mice. Additionally, CD8+ T cell activation was suppressed in exposure-induced resistant mice and was associated with the antibody dependent expansion of a splenic plasmacytoid DC population, with a regulatory phenotype. The infection-induced protection against ECM was critically dependent upon secreted antibody production. A long standing problem in studying the immune response to malaria infection has been the inability to track parasite-specific CD4+ T cell responses. To address this, we generated and validated new transgenic P. berghei parasites expressing the model antigen, ovalbumin (OVA), either in the parasite cytoplasm or on the parasitophorous vacuole membrane (PVM). We found that cellular location and expression level of the antigen influence the induction and magnitude of parasite-specific T-cell responses. These parasites thus provide knowledge on the factors that influence the recognition of parasite antigens by the immune system and represent useful tools to study the development and function of antigen-specific T-cell responses during malaria infection. The results in this thesis improve our understanding of the events that lead to the development of CM, and the host immune responses that develop following parasite exposure to protect against it. The results should contribute towards the rational development of adjunctive therapies and effective vaccines for human CM.

Cerebral malaria (CM) is a severe complication of Plasmodium falciparum infection, predominantly experienced by children in sub-Saharan Africa. Patients with CM are comatose and often convulse, develop retinal haemorrhages and motor abnormalities. Recent histological studies on brain tissue obtained from patients who have died from CM have identified heterogeneity in brain pathology. As a result, CM is considered to be a complex disease that may be comprised of a number of syndromes. Patients admitted to hospital with CM are treated with anti-malaria drugs; however, even in the best equipped hospitals, a large number of CM patients die within the first 24-hours following hospital admission before the anti-malarial treatment can have an effect. For this reason, it is critical that the mechanisms leading to CM are elucidated in order to develop effective adjunct therapies. Experimental cerebral malaria (ECM) caused by P. berghei ANKA (PbA) infection of susceptible mice displays many features of human CM. A key feature of this model is the pivotal role of the host immune response in pathogenesis, particularly the involvement of T cells. Evidence, predominantly from ECM studies, suggests that tumour necrosis factor (TNF) superfamily (TNFSF) members play critical roles in the immunopathology associated with CM. The first hypothesis investigated in this thesis was that key immune response pathways contribute to the development of CM and, despite the heterogeneity observed between CM patients, common pathways exist that may be targeted to prevent CM. The second hypothesis tested was that members of the TNF superfamily modulate the immune response to infection and are involved in the development of pathology observed in severe malaria (SM). In order to investigate the above hypotheses, three projects were carried out. First, we examined the great heterogeneity in brain expression profiles between ECM-susceptible CBA/CaH (CBA) and C57BL/6 (B6) mice at the peak of disease, as well as the significant differences in circulating cytokine expression and expansion of microglia in brain tissue. We found that, despite these differences, common therapeutic and preventative strategies existed to disrupt the development of ECM in the two ECM-susceptible mouse strains. Second, studies in ECM mice have identified T cells and TNFSF members, TNF and lymphotoxin (LT)-a, as critical mediators of ECM pathology. We extend these studies to examine the role of the TNFSF member LIGHT in ECM. Specific blockade of LIGHT signalling through its receptor, LTβR, in PbA-infected B6 mice abrogated the hallmark features of ECM brain pathology and improved the control of parasite growth. Importantly, specific blockade of LIGHT-LTβR signalling caused the expansion of splenic monocytes and an overall enhanced capacity to remove and process antigen during infection. Together, this study discovered a novel pathogenic role for LIGHT and LTβR in ECM and identified this TNF family receptor-ligand interaction as a potential target for therapeutic intervention in SM. Finally, we investigated the role of LTa in human SM and, more specifically, CM. We tested whether the polymorphisms within the gene encoding LTa (LTA) were associated with susceptibility to SM in Papuan Highland children and adults who had migrated from an area without malaria pressure to a region where malaria is endemic. Despite a lack of association between single nucleotide polymorphisms (SNPs) in the LTA/TNF locus and susceptibility to SM in Papuan Highland children and adults, we found a significant association between a SNP in the LTa-related gene encoding galactin-2 (LGALS2) and susceptibility to CM in children, but not adults in this study population. Interestingly, no association was found between this SNP and susceptibility to CM in Tanzanian children originating from and living in a malaria endemic region. These results suggest that there may be differences in the mechanisms leading to CM in adults and children, as well as between individuals from malaria endemic and non-endemic areas. Together, the findings outlined in this thesis are important to both the understanding of the underlying mechanisms leading to CM and to the development of improved interventions and adjunct therapies.

Magister Public Health - MPH / Background: Cerebral malaria (CM) kills up to 25% of its patients and about one third of its survivors develop neurological impairments (NIs). With advancements in diagnostic and management techniques for CM, more children are likely to survive. The increase in the number of CM survivors may increase the prevalence of children with NIs. In Malawi, rehabilitation for children with NIs is mostly institution-based with erratic community-outreach services, resulting in poor long-term outcomes. To date, community-based rehabilitation (CBR), a comprehensive rehabilitation approach that also addresses socio-economic impact of NIs and may supplement institution-based rehabilitation services, has not been well explored and documented. Presented here are experiences and perceptions on CBR programmes for NIs following CM in Blantyre, Malawi.

A new era of malaria eradication programs relies on increased knowledge of the parasite through sequencing of the Plasmodium genome. Programs call for re-orientation at specific epidemiological markers as regions move from control towards pre- and total elimination. However, relatively little is known about the effects of intervention strategies on the parasite population or if the epidemiological cues correspond to effects on the parasite population. We hypothesized that genomic tools could be used to track population changes in Plasmodium falciparum to detect significant shifts as eradication programs apply interventions. Making use of new whole-genome sequencing data as well as GWAS and other studies, we used SNPs as biological markers for regions associated with drug resistance as well as a set of neutral SNPs to identify individual parasites. By utilizing tools developed as proxy for full genomic sequencing of the human pathogen Plasmodium falciparum, we characterized and tracked parasite populations to test for changes over time and between populations. When applied to markers under selection - those associated with reduced antimalarial drug sensitivity - we were able to track migration of resistance-associated mutations in the population and identify new mutations with potential implications for resistance. Using a population genetic analysis toolbox to study changes in neutral allele frequencies in samples from the field, we found significant population changes over time that included restricted effective population size, reduced complexity of infections, and evidence for both clonal and epidemic propagation of parasites.

Severe manifestations of malaria, including cerebral malaria (CM) and respiratory distress, result in approximately three million deaths annually worldwide. Currently, relatively little is known about severe disease pathogenesis. The development and outcome of severe malaria is determined by host-pathogen interactions, a complex interface of genetics and immune responses. Hypothetically, a spectrum of genetic susceptibility and resistance to severe disease exists within the host population, and malaria infection results in diverse host and parasite responses that impact disease outcome. The aim of this study was to identify differential host and parasite responses in a murine model of severe malaria, Plasmodium berghei ANKA (PbA), in CM-susceptible and CM-resistant mice; and to analyze host genetics in patients with severe disease due to Plasmodium falciparum. In vivo, expression microarray analysis showed that, in malaria target organs, differential responses were related to immune response – primarily interferon and complement pathways – and apoptosis. Histopathological examination of the brain confirmed an increased prevalence of apoptosis in CM-susceptible mice. Further examination of the role of complement in CM-susceptibility determined that early complement 5 (C5) activation conferred susceptibility to CM, and that C5 deficiency conferred resistance, which could be recapitulated by antibody blockade of activated C5 or its receptor in susceptible mice. Additionally, single nucleotide polymorphism (SNP) studies identified that complement receptor 1 SNPs were associated with disease severity in patients with P. falciparum malaria. PbA parasites displayed a unique transcriptional signature in each tissue examined (brain, liver, spleen and lung), showed differential gene expression between CM-resistant and susceptible hosts, and were most prominent in lung tissue. Closer examination of lung involvement in PbA infection revealed that PbA-infected C57BL/6 mice develop acute lung injury (ALI), defined by disruption of the alveolar-capillary membrane barrier. ALI susceptibility did not correlate with CM susceptibility, but was influenced by peripheral parasite burden and CD36-mediated parasite sequestration in the lung. PbA provides a clinically relevant experimental model for CM and ALI, through which important disease mechanisms can be identified and modulated. Ideally, the use of such models aids in the discovery of disease biomarkers and novel therapeutic strategies, which may be applied to human severe and cerebral malaria.

Severe manifestations of malaria, including cerebral malaria (CM) and respiratory distress, result in approximately three million deaths annually worldwide. Currently, relatively little is known about severe disease pathogenesis. The development and outcome of severe malaria is determined by host-pathogen interactions, a complex interface of genetics and immune responses. Hypothetically, a spectrum of genetic susceptibility and resistance to severe disease exists within the host population, and malaria infection results in diverse host and parasite responses that impact disease outcome. The aim of this study was to identify differential host and parasite responses in a murine model of severe malaria, Plasmodium berghei ANKA (PbA), in CM-susceptible and CM-resistant mice; and to analyze host genetics in patients with severe disease due to Plasmodium falciparum. In vivo, expression microarray analysis showed that, in malaria target organs, differential responses were related to immune response – primarily interferon and complement pathways – and apoptosis. Histopathological examination of the brain confirmed an increased prevalence of apoptosis in CM-susceptible mice. Further examination of the role of complement in CM-susceptibility determined that early complement 5 (C5) activation conferred susceptibility to CM, and that C5 deficiency conferred resistance, which could be recapitulated by antibody blockade of activated C5 or its receptor in susceptible mice. Additionally, single nucleotide polymorphism (SNP) studies identified that complement receptor 1 SNPs were associated with disease severity in patients with P. falciparum malaria. PbA parasites displayed a unique transcriptional signature in each tissue examined (brain, liver, spleen and lung), showed differential gene expression between CM-resistant and susceptible hosts, and were most prominent in lung tissue. Closer examination of lung involvement in PbA infection revealed that PbA-infected C57BL/6 mice develop acute lung injury (ALI), defined by disruption of the alveolar-capillary membrane barrier. ALI susceptibility did not correlate with CM susceptibility, but was influenced by peripheral parasite burden and CD36-mediated parasite sequestration in the lung. PbA provides a clinically relevant experimental model for CM and ALI, through which important disease mechanisms can be identified and modulated. Ideally, the use of such models aids in the discovery of disease biomarkers and novel therapeutic strategies, which may be applied to human severe and cerebral malaria.

Orientador: Fabio Trindade Maranhão Costa / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-10T03:13:18Z (GMT). No. of bitstreams: 1 Blanco_YaraCarollo_M.pdf: 1932159 bytes, checksum: 7fa3ee714af53f1bf4da1434c08a29f5 (MD5) Previous issue date: 2007 / Resumo: A malária é sem dúvida a doença parasitária mais importante do mundo, infectando 300-500 milhões de pessoas e levando a óbito cerca de 1-2 milhões de indivíduos anualmente. Malária cerebral (MC) é o mais importante tipo de complicação nas infecções maláricas. MC é uma síndrome multifatorial, entretanto sua patogênese ainda não foi totalmente elucidada. Diversos estudos mostram a adesão de eritrócitos infectados (EI) no endotélio microvascular do hospedeiro. No entanto, várias evidências indicam que um desbalanço da resposta imune e da homeostase, acarretando numa elevada expressão de citocinas pró-inflamatórias também desempenham um papel importante na patogênese da MC. O modelo utilizando camundongos C57BL/6 infectados com Plasmodium berghei ANKA (PbA) parece ser o que mais se assemelha a MC humana, já que em ambos casos ocorre um aumento significativo de citocinas próinflamatórias, da destruição neuronal, da hemorragia cerebral e da concentração de lactato. A terapia de oxigenação hiperbárica (HBO) tem sido amplamente utilizada como potencializador de cicatrização e contra microrganismos anaeróbicos. No entanto, nos últimos anos, diversos estudos têm mostrado que o oxigênio pressurizado é capaz de proteger contra a destruição da barreira hemato-encefálica (BHE), inibir a destruição neuronal, reduzir a produção de citocinas pró-inflamatórias e a expressão ICAM-1, receptor leucocitário, e de EI. Neste estudo, avaliamos os efeitos da HBO na prevenção dos sintomas da MC em camundongos infectados com PbA e nos eritrócitos expostos diretamente ao oxigênio hiperbárico. A utilização do oxigênio hiperbárico mostrou-se capaz de reduzir significativamente a taxa de mortalidade associada à malária cerebral experimental, assim como uma redução da queda da temperatura corporal e dos níveis de parasitemia. Além disso, nossos resultados demonstram que a terapia com oxigênio hiperbárico impede a destruição da BHE. Testes In vitro mostraram que a exposição direta de eritrócitos sadios e de eritrócitos infectados (EI), á HBO não é tóxica e nem capaz de alterar a viabilidade e infectividade de EI. Por último, mostramos que o tratamento com HBO realizado após o quarto dia de infecção (quando a parasitemia já é patente), retarda o aparecimento dos sintomas neurológicos da malária cerebral e morte associada / Abstract: Malaria is the world¿s most nefarious infectious disease, affecting 300-500 million people and leads to death of 1-2 million individuals per year. Cerebral malaria (CM) is the most serious complications that might occur during a malarial infection. CM is a multifactorial syndrome; however its pathogenesis is still a matter of debate. Several studies show that in adhesion of infected erythrocytes (IE) to the host microvascular endothelium is involved. Nevertheless, several bodies of evidence indicate that a unbalance in host immune response and homeostasis, leading to the expression at higher levels of proinflammatory cytokines. Infection of C57BL/6 mice with IE of Plasmodium berghei ANKA (PbA) is the largely used model for CM, as a significant augmentation in the expression levels of proinflammatory cytokines and lactate, neuronal damage and cerebral hemorrhage also occurs. Hyperbaric oxygen therapy (HBO) has been widely used against anaerobic microorganism and as an adjunct therapy in surgeries. However, in the last year several studies have shown that HBO is able to prevent brain-blood-barrier (BBB) breakdown, inhibit neural damage and the syntheses of proinflammatory cytokines and the expression of ICAM-1, a the host receptors involved in the adhesion of leukocytes and IE. Herein, we evaluated the HBO effects in preventing CM specific clinical signs in PbA-infected mice and directly against IE of PbA. The data provided by us demonstrated that HBO treatment reduces significantly CM associated mortality and clinical outcomes, such as hypothermia, BBB dysfunction and is able to inhibit parasite burden early on the infection. In vitro analysis showed that directly exposure to HBO, up to six hours, is not harmful to health erythrocytes neither inhibit parasite development and infectivity. Finally, HBO treatment was able to delay significantly the CM neurological signs and death, even thought administrated after parasite establishment / Mestrado / Mestre em Parasitologia