Certain strains of mice, e.g. C57BL/6, are highly resistant to serious infections with the protozoan pathogen, Leishmania major, whereas other strains, e.g. BALB/c, are not. It has beenproposed that interferon gamma (IFN-y) is one of the most critical lymphokines produced in a protective response to these intracellular pathogens. IFN-y has been classified as a Thi lymphokine produced by the Thl subset of T lymphocytes which not only activates macrophages to kill the protozoa but also helps regulates the immune system overall to form a lasting immunity to the microorganism (4,19). Mice susceptible to L. major arethought to produce inadequate amounts of IFN-y and instead produce an excessive amount of a Th2 lymphokine, IL-4, produced by Th2 T cells. (6) We have previously found that prophylactic treatment with cyclosporine A (CsA), a T cell specific immunosuppressant, reduces the susceptibility of the BALB/c to L. major by either preventing disease entirely or delaying itsdevelopment significantly (19). In this murine model, it may be that CsA causes a switch from the production of the less protective Th2 lymphokines to the more protective Thl lymphokines. In order to test this hypothesis we examined the IFN-y produced by lymph node and spleen cells over time after infection in three groups of mice: C57BL/6, BALB/c and cyclosporine- protected BALB/c. Interestingly, cells taken from all three groups of mice were able to secrete IFN-y in vitro in response to co-culture with Leishmania, antigens. The pattern of secretion over time, however, varied and may indicate a significant difference in the animals' response to the pathogen. In addition to this work, we also examined the ability of another immunosuppressant, FK506, which is very similar in action to but much less toxic than CsA, to induce enhanced resistence to L. major. FK506 also appears to be effective in reversing the susceptibility of the BALB/c mice towards this pathogen with much less apparent toxicity. / Department of Biology

Article / Military personnel deployed to the Amazon Basin are at high risk for cutaneous leishmaniasis (CL). We responded to an outbreak among Peruvian Army personnel returning from short-term training in the Amazon, conducting active case detection, lesion sample collection, and risk factor assessment. The attack rate was 25% (76/303); the incubation period was 2–36 weeks (median = 8). Most cases had one lesion (66%), primarily ulcerative (49%), and in the legs (57%). Real-time polymerase chain reaction (PCR) identified Leishmania (Viannia) braziliensis (59/61 = 97%) and L. (V.) guyanensis (2/61 = 3%). Being male (risk ratio [RR] = 4.01; P = 0.034), not wearing long-sleeve clothes (RR = 1.71; P = 0.005), and sleeping in open rooms (RR = 1.80; P = 0.009) were associated with CL. Sodium stibogluconate therapy had a 41% cure rate, less than previously reported in Peru (70%; P < 0.001). After emphasizing pre-deployment education and other basic prevention measures, trainees in the following year had lower incidence (1/278 = 0.4%; P < 0.001). Basic prevention can reduce CL risk in deployed militaries.

Orientador: Katia Denise Saraiva Bresciani / Resumo: A Leishmaniose Visceral (LV) apresenta uma ampla distribuição geográfica em todos os continentes, representando um sério problema de Saúde Pública. Os flebotomíneos (Diptera, Psychodidae, Phlebotominae) são importantes insetos vetores de microrganismos patogênicos como Leishmania spp., Bartonella spp. e arbovírus (Vesiculovirus, Phlebovirus, Orbivirus), porém, estes insetos apresentam grande importância para a transmissão das leishmanioses em várias regiões do mundo, incluindo as Américas do Sul e Central. No Brasil, há duas espécies, até o momento, relacionadas com a transmissão da Leishmania infantum: a Lutzomyia longipalpis e a Lutzomyia cruzi. Infecções por Leishmania spp. são potencialmente zoonóticas e acometem homens e diversas espécies de animais silvestres e domésticos. Os cães são importantes hospedeiros, fontes de infecções e potenciais reservatórios, não só pelo estreito relacionamento ou convívio com os seres humanos, mas também por sua incapacidade imunológica em responder à doença com sucesso. O Ministério da Saúde no Brasil, preconiza para o diagnóstico da Leishmaniose Visceral Canina (LVC), o teste imunocromatográfico, como exame de triagem e o ensaio imunoenzimático, como confirmatório. Também recomenda o diagnóstico precoce e tratamento adequado dos casos humanos, o controle dos vetores, a eutanásia dos cães e atividades de educação em saúde nas áreas endêmicas para esta doença. Por tanto, foram instituídas ações de prevenção e controle da LV canina, na áre... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Visceral leishmaniasis (VL) has a wide geographical distribution on all continents, representing a serious Public Health problem. Sand flies (Diptera, Psychodidae, Phlebotominae) are important insect vectors of pathogenic microorganisms such as Leishmania spp., Bartonella spp. and arboviruses (Vesiculovirus, Phlebovirus, Orbivirus), however, these insects are of great importance for leishmaniasis transmission in various regions of the world, including South and Central America. In Brazil, there are two species related to the transmission of Leishmania infantum: Lutzomyia longipalpis and Lutzomyia cruzi. Leishmania spp. infections are potentially zoonotic and affect men and various species of wild and domestic animals. Dogs are important hosts, sources of infections and potential reservoirs, not only for their close relationship or contact with humans, but also for their immunological inability to successfully respond to the disease. The Health Ministry of Brazil recommends for the diagnosis of canine visceral leishmaniasis (CVL), the immunochromatographic test as a screening test and the enzyme immunoassay as confirmatory. It also recommends early diagnosis and appropriate treatment of human cases, vector control, dog euthanasia and health education activities in endemic areas for this disease. Therefore, prevention and control actions of canine VL were instituted in the urban area of municipality of Araçatuba, São Paulo, Brazil. / Doutor

A Leishmaniose é uma doença infecciosa causada por protozoários flagelados do gênero Leishmania. Os parasitas como a Leishmania braziliensis, sofrem várias mudanças morfológicas durante seu ciclo de vida, incluindo a troca de organismo hospedeiro. Durante essas mudanças, proteínas de choque térmico ou chaperones moleculares, como, por exemplo, a Hsp70, são expressas em grande quantidade. A função da Hsp70 é auxiliar no processo de enovelamento protéico, no transporte de proteínas entre as membranas e em muitas outras importantes funções celulares. A Hsp70 é auxiliada por várias proteínas denominadas como co-chaperone e a Hep1 (do inglês Hsp70-escort protein 1) é uma delas. Essa co-chaperone tem seu papel descrito principalmente em mitocôndrias como estabilizadoras da Hsp70 capazes de prevenir a sua agregação. O objetivo deste trabalho foi clonar, expressar, purificar e caracterizar as proteínas Hsp70 e Hep1 de L. braziliensis (LbHsp70 e LbHep1). Os ensaios preliminares mostraram que a LbHsp70 foi expressa de forma insolúvel, sendo necessário expressar a proteína em corpos de inclusão para tentativas de reenovelamento, afim de obter a mesma na fração solúvel. Apesar da LbHsp70 se apresentar na fração solúvel após o reenovelamento, a mesma foi purificada como agregado. Ainda na tentativa de obter a LbHsp70 na forma solúvel, a mesma foi co-expressa com a LbHep1 (expressa na forma solúvel), porém a LbHsp70 continuou na fração insolúvel do lisado bacteriano. Como a LbHep1 não apresentou a atividade esperada quando co-expressa com a LbHsp70 citoplasmática, foram feitos ensaios de co-expressão da LbHep1 com a Hsp70 mitocondrial humana, que é heterologamente expressa na forma de agregados, com o intuito de confirmar a atividade estabilizadora das Hep1 sobre as Hsp70 mitocondriais. Este experimento possibilitou a obtenção de ambas proteínas na fração solúvel, de acordo com dados apresentados na literatura para este sistema em outros organismos. Uma vez mostrada à funcionalidade da LbHep1, foi feita a caracterização desta proteína por métodos biofísicos como dicroísmo circular, espectrometria de fluorescência, cromatografia de exclusão molecular analítica e ultracentrifugação analítica. Os experimentos mostraram que a LbHep1 apresenta estrutura secundária composta principalmente de folhas-&beta; pregueadas e que o único triptofano está parcialmente exposto ao solvente. As análises hidrodinâmicas mostraram que a LbHep1 é assimétrica e em equilíbrio entre monômeros e dímeros. Por fim, dados de ultracentrifugação analítica indicam que a LbHep1 está em equilíbrio monômero-dímero. / Leishmaniasis is an infectious disease caused by flagellate protozoa of the genus Leishmania. The parasites such as Leishmania braziliensis undergo various morphological changes during its life cycle, including the exchange of the host organism. During these changes, heat shock proteins or molecular chaperones like Hsp70, for example, are expressed in large amounts. The function of Hsp70 is to assist in the process of protein folding, protein transport between the membranes and many other important cellular functions. The Hsp70 is assisted by several proteins called co-chaperones and the Hsp70-escort protein (Hep1) is one of them. This co-chaperone has been described based on its role as a stabilizer of mitochondrial Hsp70 preventing their aggregation. The objective of this study was to clone, express, purify and characterize the Hsp70 and Hep1 ortologues of Leishmania braziliensis (LbHsp70 and LbHep1). The preliminary tests showed that LbHsp70 was expressed in the insoluble form, being necessary to express the protein in inclusion bodies to attempt its refolding in order to get it in the soluble fraction. Despite LbHsp70 was obtained in the soluble fraction after refolding, it was purified as aggregates. Still trying to get the LbHsp70 in the soluble form, it was co-expressed with LbHep1 (always expressed in the soluble form), but LbHsp70 remained in the insoluble fraction of the bacterial lysate. As LbHep1 showed no expected activity when co-expressed with LbHsp70, which is citoplasmatic, we tested if LbHep1 was able to act on human mitochondrial Hsp70 which is expressed as aggregates in bacterial heterologous systems. Then, we co-expressed LbHep1 with human mitochondrial Hsp70 which allowed obtaining both proteins in the soluble fraction, in according to data presented in the literature. Once the functionality of LbHep1 was showed, we characterize this protein by biophysical methods such as circular dichroism, fluorescence spectrometry, molecular exclusion chromatography and analytical ultracentrifugation analysis. The experiments showed that the secondary structure features LbHep1 composed mainly of &beta;-sheets and that the only tryptophan is partially exposed to solvent. Hydrodynamic analysis showed that the protein is asymmetric and in equilibrium between monomers and dimers. Finally, analytical ultracentrifugation data indicate that LbHep1 is a system in equilibrium monomer-dimer.

Leishmanioses são um complexo de doenças infecciosas causadas por protozoários intramacrofágicos do gênero Leishmania, fatal se não tratadas adequadamente. Os antimoniais pentavalentes são os medicamentos de primeira escolha para o tratamento, apesar de sua toxicidade e seu mecanismo de ação pouco esclarecido. Uma terapia mais eficaz pode ser conseguida pelo direcionamento de fármacos antileishmania para os locais de infecção. Os lipossomas são vesículas lipídicas que promovem melhora na eficácia e na ação de fármacos na célula alvo. Os lipossomas são capturados preferencialmente pelas células do sistema mononuclear fagocitário (SMF). O objetivo deste estudo foi desenvolver uma formulação de antimoniato de meglumina lipossomal, constituído por fosfatidilserina e estudar sua farmacocinética em animais sadios para esclarecer seu metabolismo e distribuição. As análises quantitativas de antimônio em lipossomas demonstram que Análise por Ativação Neutrônica foi a técnica mais sensível com cerca de 100 % de precisão. Todas as formulações de lipossomas apresentaram um tamanho de diâmetro médio de 150 nm. A determinação da CE50 em macrófagos infectados mostrou que as formulações de antimoniato de meglumina encapsulado em lipossomas foram entre 10 - 63 vezes mais eficazes do que a fármaco livre, indicando maior índice de seletividade. Por microscopia de fluorescência, foi verificada uma maior internalização de lipossomas fluorescentes em macrófagos infectados durante um curto tempo de incubação em comparação com macrófagos não infectados. A biodistribuição do antimoniato de meglumina irradiado encapsulado em lipossomas contendo fosfatidilserina mostrou que a formulação lipossomal promoveu um direcionamento seletivo do antimônio para tecidos do SMF, além do que manteve as doses elevadas nos órgãos por um período prolongado. Em conclusão, estes dados sugerem que o antimoniato de meglumina encapsulado em lipossomas apresentou maior eficácia do que a fármaco não lipossomal contra a infecção por Leishmania. O desenvolvimento de formulações lipossomais pode ser uma nova alternativa para a quimioterapia de doenças infecciosas, especialmente Leishmanioses, já que são usados como sistemas carreadores para entrega sustentada e direcionada de fármacos ao local da infecção. / Leishmaniases are a complex of parasitic diseases caused by intramacrophage protozoa of the genus Leishmania, and is fatal if left untreated. Pentavalent antimonials, though toxic and their mechanism of action being unclear, remain the first-line drugs for treatment. Effective therapy could be achieved by delivering antileishmanial drugs to these sites of infection. Liposomes are phospholipid vesicles that promote improvement in the efficacy and action of drugs in target cell. Liposomes are taken up by the cells of mononuclear phagocytic system (MPS). The purpose of this study was to develop a preparation of meglumine antimoniate encapsulated in liposomes of phosphatidylserine and to study its pharmacokinetic in healthy mice to establish its metabolism and distribution. Quantitative analysis of antimony from liposomes demonstrated that Neutron Activation Analysis was the most sensitive technique with almost 100 % of accuracy. All liposome formulations presented a mean diameter size of 150 nm. The determination of IC50 in infected macrophage showed that liposome formulations were between 10 63 fold more effective than the free drug, indicating higher selectivity index. By fluorescence microscopy, an increased uptake of fluorescent-liposomes was seen in infected macrophages during short times of incubation compared with non-infected macrophages. Biodistribution studies showed that meglumine antimoniate irradiated encapsulated in liposomes of phosphatidylserine promoted a targeting of antimony for MPS tissues and maintained high doses in organs for a prolonged period. In conclusion, these data suggest that meglumine antimoniate encapsulated in liposomes showed higher effectiveness than the non-liposomal drug against Leishmania infection. The development of liposome formulations should be a new alternative for the chemotherapy of infection diseases, especially Leishmaniasis, as they are used to sustain and target pharmaceuticals to the local of infection.

A leishmaniose é uma doença tropical negligenciada que afeta milhares de pessoas podendo até levar a óbito em sua forma visceral. Durante o seu ciclo de vida, o parasita passa por diversas mudanças ambientais como mudança de temperatura e pH, principalmente quando da transfecção do inseto vetor para o hospedeiro mamífero. Tais mudanças geram estresse celular que pode levar proteínas ao enovelamento incorreto assim como a processos agregativos, sendo necessários sistemas de controle de qualidade proteico para manter a homeostase celular, do qual fazem parte as chaperonas moleculares. Chaperonas como a Hsp100, ajudam a manter a homeostase celular e a adaptação desempenhando um papel importante para protozoários como a Leishmania braziliensis, causador da leishmaniose. A Hsp100 tem papel desagregase, atuando com outras chaperonas moleculares para a extração de polipeptídios de agregados proteicos possibilitando seu desenovelamento e posterior reenovelamento, evitando seu efeito tóxico sobre a célula. A Hsp100 parece ser essencial para esses microrganismos, no entanto não há muito dados disponíveis para Hsp100 em Leishmania sp. e Plasmodium sp. Neste trabalho está descrito o protocolo para expressão e purificação da Hsp100 recombinante de L. braziliensis (rLbHsp100), assim como sua caracterização estrutural e funcional inicial in vitro. A proteína foi analisada por espectropolarimetria de dicroísmo circular, apresentando estrutura típica de proteínas ricas em hélices &alpha;, a fluorescência estática de triptofano demonstrou que a proteína possui estrutura terciária local com seus triptofanos parcialmente expostos ao solvente. Por cromatografia de exclusão molecular analítica, observou-se que a LbHsp100 se comporta como um oligômero cujo estado é influenciado tanto pela concentração proteica como pela presença de nucleotídeos adenosina. Análises por ultracentrifugação analítica evidenciaram que a rLbHsp100 em solução apresenta um equilíbrio de diversas espécies havendo deslocamento para um hexâmero de maneira concentração dependente. Análises de SAXS confirmaram a estrutura hexamérica e proporcionaram a obtenção de um modelo ab initio da proteína. Através de microscopia eletrônica de transmissão pode-se observar a forma toróide e a dispersividade do sistema. Por fim, atestou-se que a proteína foi obtida funcional com fraca atividade ATPásica, apresentando também interações com nucleotídeos adenosina (ATP e ADP) assim como com a suramina. / Leishmaniasis is a neglected tropical disease that affects thousands of people and may even lead to death in its visceral form. During its life cycle, the parasite undergoes several environmental changes such as temperature and pH changes, especially when transfecting from the vector insect into the mammalian host. Such changes generate a cellular stress that can lead to misfolding as well as to aggregative processes, therefore a protein quality control system is necessary to maintain cell homeostasis, which includes molecular chaperones. Chaperones such as Hsp100 can help maintain cellular homeostasis and adaptation playing an important role for protozoa such as Leishmania braziliensis, which causes leishmaniasis. The Hsp100 has a disaggregase action, acting with other proteins of the chaperone system to extract polypeptides from protein aggregates, allowing their unfolding and subsequent refolding, avoiding their toxic effect on the cell. Hsp100 appears to be essential for these microorganisms, however there is not much data available for Hsp100 in Leishmania sp. and Plasmodium sp. This work describes the protocol for expression and purification of the recombinant Hsp100 of Leishmania braziliensis (rLbHsp100), as well as its initial in vitro characterization. The protein was analyzed by circular dichroism spectropolarimetry, presenting a typical structure of ?-helix rich protein as well as a concentration-dependent structure gain, static fluorescence of tryptophan demonstrated that the protein has local tertiary structure with its tryptophans partially exposed to the solvent. Analytical size exclusion chromatography showed that LbHsp100 behaves as an oligomer whose state is influenced by both the concentration and the presence of adenosine nucleotides. Analysis by analytical ultracentrifugation has shown that the rLbHsp100 in solution exhibits an equilibrium of several species shifting towards a hexamer in a concentration dependent manner. SAXS analyzes confirm the hexameric structure and had provide an ab initio model for the protein. Transmission electron microscopy shows the toroidal form and dispersivity of the system. Finally, the obtained protein had showed catalytic function, and also interacted with adenosine nucleotides (ATP and ADP) as well as suramine.

Leishmanioses são um complexo de doenças infecciosas causadas por protozoários intramacrofágicos do gênero Leishmania, fatal se não tratadas adequadamente. Os antimoniais pentavalentes são os medicamentos de primeira escolha para o tratamento, apesar de sua toxicidade e seu mecanismo de ação pouco esclarecido. Uma terapia mais eficaz pode ser conseguida pelo direcionamento de fármacos antileishmania para os locais de infecção. Os lipossomas são vesículas lipídicas que promovem melhora na eficácia e na ação de fármacos na célula alvo. Os lipossomas são capturados preferencialmente pelas células do sistema mononuclear fagocitário (SMF). O objetivo deste estudo foi desenvolver uma formulação de antimoniato de meglumina lipossomal, constituído por fosfatidilserina e estudar sua farmacocinética em animais sadios para esclarecer seu metabolismo e distribuição. As análises quantitativas de antimônio em lipossomas demonstram que Análise por Ativação Neutrônica foi a técnica mais sensível com cerca de 100 % de precisão. Todas as formulações de lipossomas apresentaram um tamanho de diâmetro médio de 150 nm. A determinação da CE50 em macrófagos infectados mostrou que as formulações de antimoniato de meglumina encapsulado em lipossomas foram entre 10 - 63 vezes mais eficazes do que a fármaco livre, indicando maior índice de seletividade. Por microscopia de fluorescência, foi verificada uma maior internalização de lipossomas fluorescentes em macrófagos infectados durante um curto tempo de incubação em comparação com macrófagos não infectados. A biodistribuição do antimoniato de meglumina irradiado encapsulado em lipossomas contendo fosfatidilserina mostrou que a formulação lipossomal promoveu um direcionamento seletivo do antimônio para tecidos do SMF, além do que manteve as doses elevadas nos órgãos por um período prolongado. Em conclusão, estes dados sugerem que o antimoniato de meglumina encapsulado em lipossomas apresentou maior eficácia do que a fármaco não lipossomal contra a infecção por Leishmania. O desenvolvimento de formulações lipossomais pode ser uma nova alternativa para a quimioterapia de doenças infecciosas, especialmente Leishmanioses, já que são usados como sistemas carreadores para entrega sustentada e direcionada de fármacos ao local da infecção. / Leishmaniases are a complex of parasitic diseases caused by intramacrophage protozoa of the genus Leishmania, and is fatal if left untreated. Pentavalent antimonials, though toxic and their mechanism of action being unclear, remain the first-line drugs for treatment. Effective therapy could be achieved by delivering antileishmanial drugs to these sites of infection. Liposomes are phospholipid vesicles that promote improvement in the efficacy and action of drugs in target cell. Liposomes are taken up by the cells of mononuclear phagocytic system (MPS). The purpose of this study was to develop a preparation of meglumine antimoniate encapsulated in liposomes of phosphatidylserine and to study its pharmacokinetic in healthy mice to establish its metabolism and distribution. Quantitative analysis of antimony from liposomes demonstrated that Neutron Activation Analysis was the most sensitive technique with almost 100 % of accuracy. All liposome formulations presented a mean diameter size of 150 nm. The determination of IC50 in infected macrophage showed that liposome formulations were between 10 63 fold more effective than the free drug, indicating higher selectivity index. By fluorescence microscopy, an increased uptake of fluorescent-liposomes was seen in infected macrophages during short times of incubation compared with non-infected macrophages. Biodistribution studies showed that meglumine antimoniate irradiated encapsulated in liposomes of phosphatidylserine promoted a targeting of antimony for MPS tissues and maintained high doses in organs for a prolonged period. In conclusion, these data suggest that meglumine antimoniate encapsulated in liposomes showed higher effectiveness than the non-liposomal drug against Leishmania infection. The development of liposome formulations should be a new alternative for the chemotherapy of infection diseases, especially Leishmaniasis, as they are used to sustain and target pharmaceuticals to the local of infection.

Leishmania species are vector-borne protozoan parasites that cause a spectrum of human diseases, with an estimated 12 million people infected in 88 countries. Inflammation plays distinct roles in the different clinical syndromes. Visceral leishmaniasis, in which parasites migrate from the site of infection and proliferate in liver and spleen, is accompanied by systemic immune suppression. Cutaneous leishmaniasis, where parasites remain at the site of inoculation and create a long-term ulcer, is associated with vigorous systemic immunity to the parasite. The innate immune sensing pathways responding to Leishmania spp. parasites are not fully described. NLR proteins are a class of structurally related cytosolic proteins. The most well described NLRs form inflammasome complexes that generate strong inflammatory responses to “danger” signals. Other NLRs do not form inflammasomes and have anti-inflammatory functions. While NLR proteins are known to be important in the immune response to many pathogens, the roles NLR proteins in leishmaniasis have only begun to be investigated. We hypothesized that NLR proteins affect the pathogenesis of leishmaniasis through their ability to modulate inflammatory responses. We hypothesized that inflammasome activation in cutaneous leishmaniasis would be detrimental, leading to greater disease pathology, and that the potential anti-inflammatory functions of the non-inflammasome NLRs, NLRP6, NLRP10, and NLRP12, would be protective, reducing tissue damage. In contrast, we hypothesized that in visceral leishmaniasis greater inflammation due to activation of the inflammasome would be protective and control parasite replication, while the anti-inflammatory NLRs would be permissive to parasite replication in the liver and spleen by contributing to the immunosuppressive strategy of the parasite. We used knockout mouse strains lacking the inflammasome adaptor protein ASC, and several non-inflammasome forming NLRs, to investigate NLR proteins in murine models of visceral or cutaneous leishmaniasis. Our data showed that NLR proteins have important functions in visceral leishmaniasis, where they are essential for appropriate parasite homing and replication in the liver and spleen. In cutaneous leishmaniasis, we found that NLRP10 is essential for controlling inflammation in the skin, limiting lesion development and tissue damage at the site of infection. Taken together our findings show important functions for NLR proteins in leishmaniasis, influencing localized tissue specific inflammation, the adaptive immune responses, and clearance or long term residence of the parasite in the infected organs. This research underscores the importance of localized inflammation at the infection site to the pathogenesis and the course of leishmaniasis.

Trypanosomatids cause human diseases such as leishmaniasis and African trypanosomiasis. Trypanosomatids are protists from the order Trypanosomatida and include species of the genera Trypanosoma and Leishmania, which occupy a similar ecological niche. Both have digenic life-stages, alternating between an insect vector and a range of mammalian hosts. However, the strategies used to subvert the host immune system differ greatly as do the clinical outcome of infections between species. The genomes of both the host and the parasite instruct us about strategies the pathogens use to subvert the human immune system, and adaptations by the human host allowing us to better survive infections. We have applied unsupervised learning algorithms to aid visualization of amino acid sequence similarity and the potential for recombination events within Trypanosoma brucei's large repertoire of variant surface glycoproteins (VSGs). Methods developed here reveal five groups of VSGs within a single sequenced genome of T. brucei, indicating many likely recombination events occurring between VSGs of the same type, but not between those of different types. These tools and methods can be broadly applied to identify groups of non-coding regulatory sequences within other Trypanosomatid genomes. To aid in the detection, quantification, and species identification of leishmania DNA isolated from environmental or clinical specimens, we developed a set of quantitative-PCR primers and probes targeting a taxonomically and geographically broad spectrum of Leishmania species. This assay has been applied to DNA extracted from both human and canine hosts as well as the sand fly vector, demonstrating its flexibility and utility in a variety of research applications. Within the host genomes, fine mapping SNP analysis was performed to detect polymorphisms in a family study of subjects in a region of Northeast Brazil that is endemic for Leishmania infantum chagasi, the parasite causing visceral leishmaniasis. These studies identified associations between genetic loci and the development of visceral leishmaniasis, with a single polymorphism associated with an asymptomatic outcome after infection. The methods and results presented here have capitalized on the large amount of genomics data becoming available that will improve our understanding of both parasite and host genetics and their role in human disease.

Leishmaniasis is a spectrum of diseases caused by several species of protozoan parasites belonging to the genus, Leishmania. The disease, which is transmitted by Sandflies, ranges from self-healing cutaneous lesions to the life-threatening visceral leishmaniasis. Cutaneous leishmaniasis, caused by L. major, is the most common form of the disease. With no vaccine available for use in humans, cutaneous leishmaniasis remains a global public health problem. Since understanding the factors that regulate effective immunity to cutaneous leishmaniasis is critical for the development of an affective vaccine and treatment strategies, the overall aim of my thesis was to decipher the host and pathogen factors that regulate immunity in cutaneous leishmaniasis. Firstly, I show that parasite dose affects the expansion of different T cell subsets following L. major infection; with low dose infection inducing more CD8+ T cells while high dose infection induced more CD4+ T cells. However, although CD8+ T cells were important for optimal primary immunity following low dose infection, they where dispensable during secondary immunity. Secondly, I found that blockade of LIGHT, (lymphotoxin like, exhibits inducible expression and competes with HSV glycoprotein D for HVEM, a receptor expressed by T lymphocytes) significantly impaired DC maturation, expression of co-stimulatory molecules, and early cytokine production (IL-12 and IFN-γ) following L. major infection. Interestingly, LIGHT was completely dispensable during secondary immunity in wild type mice but was critical for effective secondary immunity in CD40 deficient mice. Thirdly,I compared disease progression and immune response in CD40 and CD40L deficient mice infected with L. major under identical experimental conditions. I found significant differences in disease progression and immune response between CD40KO and CD40L KO mice infected with virulent L. major and treated with recombinant IL-12. My data revealed a novel pathway (CD40L-Mac-1 interaction) for IL-12 production and resistance to Leishmania major. Collectively, this thesis provides novel insights into the mechanisms involved in the development and maintenance of protective immunity against cutaneous leishmaniasis, which could lead to the development of a more efficient and effective immunotherapeutic and/or vaccination strategies against the disease. / October 2015