The ability of tumor necrosis factor (TNF) and lipopolysaccharide (LPS) to stimulate gene expression in bone marrow-derived macrophages (BMM) was first compared. It is demonstrated that they stimulated gene expression through distinct signal transduction pathways and that TNF stimulated gene expression through a protein kinase C (PKC)-dependent signal transduction pathway. The effect of the intracellular parasite of macrophages Leishmania donovani in BMM was then investigated. It is demonstrated that L. donovani impaired c-fos and TNF gene expression through two distinct mechanisms. The first one is indomethacin-reversible, and the second one involves the inhibition of diacylglycerol-induced PKC-dependent gene expression. A purified cell surface glycoconjugate of the parasite, termed lipophosphoglycan, selectively inhibited PKC-dependent gene expression in BMM. While the translocation of PKC from the cytosol to the membrane was normal, total cellular PKC enzyme activity was inhibited in the U937 human monocyte cell line pretreated with lipophosphoglycan.

The role of a parasite-produced or -induced soluble immunosuppressor in experimental kala azar was examined. It was found that in vivo infections with Leishmania donovani in the hamster (Mesocricetus auratus) produce a soluble immunosuppressor, which appears in the serum of the host and which reduces the proliferation of responding populations of murine splenocytes in a one-way mixed leukocyte reaction (MLR). The production in vitro infections of murine splenic macrophages from C57BL/6J ($Lsh sp{ rm s}$), C57L/J ($Lsh sp{ rm R}$) and BALB/c strains, the suppressive activity was not contained in either parasite-conditioned culture medium or in parasite extracts or from macrophages which have internalized killed parasites or inert particles and it is not blocked by the action of 2-mercaptoethanol or indomethacin in the culture medium. The suppressor was found to be able to selectively inhibit or reduce the proliferation of splenocytes of both the C57BL/6J and C57LN strains in a one way MLR, with the level of suppression being significantly greater upon splenocytes of the susceptible $Lsh sp{ rm s}$ strain. The suppression was dependent upon the genotype of the macrophages present in the responding population. The suppressor was also able to significantly inhibit the processing of human serum albumin by macrophages, to reduce the number of Ia ligands on the surfaces of macrophages and the production by these cells of IL-1 upon silica stimulation. / Significant reduction was also seen in the production of IL2 and in the expression of its receptor by PHA-stimulated T cells exposed to the suppressor. Partial purification and identification of the suppressor demonstrated that the suppressive activity was present in fractions between 30 and 50 kDa in size; the suppressor was also heat labile and freeze-thaw sensitive. The suppressive molecule(s) may therefore play a significant role in the establishment and pathology of L. donovani infections.

Prophylactic administration of the immunosuppressive drug cyclosporine A protects Balb/c mice from fatal Leishmania major infections. It is believed that distinct subpopulations of CD4+ T lymphocytes and their distinctive cytokines may determine susceptibility and resistance to leishmaniasis among inbred strains of mice. CsA may enhance disease resistance in Balb/c mice by modulating these T cell subsets and/or their cytokines. We have measured lymphoid cell production of IL-2, IL-4, and TNF-a in naturally resistant C57/Bl/6, CsA-treated Balb/c, and nontreated Balb/c mice during the course of L. major infection. CsA treatment inhibited IL-2 and IL-4 production for the first week of infection. Thereafter the cytokine production of all three groups of mice did not differ greatly except in week two when the treated mice produced significantly enhanced levels of IL-4. C57B1/6 mice did produce slightly more TNF-a than either group of Balb/c mice, but as the CsAprotected and diseased Balb/c mice produced similar amounts of this cytokine, the elevation in C57B1/6 animals probably reflects a strain-related difference rather than disease resistance. / Department of Biology

If used early in infection, prophylactic treatment with the immunomodulatory drug cyclosporin A of Leishmania ma'or infected Balb/c mice has been shown to enhance resistance of these mice to serious disease. It is thought that CsA treatment affects disease progression by altering the balance of specific T lymphocyte populations as well as the secretion of various cytokines. We have followed the levels of L3T4+ T cells, Ly-2+ T cells, and total T and B lymphocytes, as well as IL-4 in susceptible Balb/c mice, CsA-treated Balb/c mice, and naturally resistant C57B1/6 mice during the course of L. ma'or infection. The CsA-treated mice displayed a disease pattern similar to that of the C57B1/6 group throughout infection. Most importantly, CsA treatment appeared to inhibit IL-4 production early post infection in both spleen and lymph node, and also appeared to inhibit the dramatic early increase of L3T4+ (CD4+) T cells which is characteristic of the susceptible Balb/c mice. / Department of Biology

Leishmania protozoans are the etiologic agents of the disease leishmaniasis. The parasite exists in two morphological forms: promastigote and amastigote. Promastigotes are found in the gut of the sandfly vector while amastigotes reside inside the vertebrate macrophage. Leishmania, an obligate intracellular parasite, resists toxic reactive oxygen species (ROS) from both endogenous and exogenous sources. Like other protozoa, Leishmania lacks some of the antioxidant defence enzymes such as catalase and glutathione peroxidase (Gpx) that are usually found in aerobic cells. Instead they possess the antioxidant thiol compound trypanothione, in association with specific trypanothione linked antioxidant enzymes such as peroxidoxins. The transformation from promastigote to amastigote is a crucial step for parasite infection and survival. The molecular basis for this transformation is not clearly understood. Recently it was shown that the peroxidoxin gene is present in multiple copies in Leishmania. In the present study we examined the potential of antisense RNA and double stranded RNA (dsRNA) to perform functional knockout of the peroxidoxin gene. Towards that end antisense RNA and dsRNA expressing plasmids, targeting the peroxidoxin gene, were constructed. Leishmania promastigotes were subsequently transfected with these plasmids and the levels of peroxidoxin gene expression were studied. The results from this study suggest that there is no apparent reduction in either the levels of peroxidoxin mRNA or the protein in the transfected promastigotes as compared to the non-transfected cells.

Thesis (M.S.)--University of Texas at El Paso, 2008. / Title from title screen. Vita. CD-ROM. Includes bibliographical references. Also available online.

Proefschrift Universiteit van Amsterdam. / Met bibliogr., lit. opg. - Met samenvatting in het Nederlands.

Made available in DSpace on 2014-06-11T19:25:37Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-08-09Bitstream added on 2014-06-13T20:33:24Z : No. of bitstreams: 1 nardo_cdd_me_araca.pdf: 584996 bytes, checksum: d064cf941273079c61aca57c35d57979 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / A leishmaniose visceral é uma zoonose causada por um protozoário do gênero Leishmania, cujo agente etiológico no Brasil é a Leishmania chagasi. O primeiro caso canino autóctone no Estado de São Paulo, Brasil, foi identificado em 1998 no município de Araçatuba. Desde então a doença vem se espalhando por todo o Estado. O objetivo do presente estudo foi determinar a freqüência de ocorrência de anticorpos anti-Leishmania chagasi em amostras de soro de 584 cães de São José do Rio Preto, São Paulo, área não endêmica para a doença e que dista 160 Km de Araçatuba. Asoroprevalência foi de 0,86% por ELISA e 0,17% por imunocromatografia. Areação de imunofluorescência indireta foi utilizada em 138 amostras eidentificou 1,45% de soropositividade. O ELISA identificou cinco cães positivos,a RIFI dois e um cão foi identificado pela imunocromatografia. Um dos cãespositivo pela RIFI havia sido vacinado para leishmaniose visceral. Dois cãesque foram considerados positivos pelo ELISA indireto foram testados por PCRpara detecção da presença de Leishmania chagasi, mas os resultados foram negativos, não confirmando a doença. Somente um cão foi soropositivo em todos os métodos. Ele apresentava sinais clínicos inespecíficos e foi adquirido pelo proprietário em uma área endêmica para a doença. O diagnóstico não pôde ser confirmado por exame parasitológico direto ou PCR porque o cão morreu antes dos resultados dos exames sorológicos. Os resultados obtidos na população do presente estudo permitem concluir que São José do Rio Preto deve ser, ainda, área não endêmica para leishmaniose visceral canina. / Visceral leishmaniasis is a zoonosis caused by a protozoa of the genus Leishmania, whose etiological agent in Brazil is Leishmania chagasi. The first canine autochthonous case of the disease in the São Paulo State, Brazil, was identified in 1998 in Araçatuba. Since then, the disease has spread to great part of the State. The aim of the present study was to determine the frequency of occurrence of anti-Leishmania chagasi antibodies in serum samples of 584 dogs from São José do Rio Preto, São Paulo, a non endemic area for the disease, 160km far from Araçatuba. Seroprevalence was 0,86% by ELISA and 0,17% by imunochromatography. IFAT, used to test 138 samples, identified 1,45% of seropositivity. ELISA identified five positive dogs, IFAT two, and immunochromatography one. One of the dogs that were considered positive by IFAT has been vaccinated for visceral leishmaniasis. Two dogs that were considered positive by indirect ELISA were tested by PCR for the presence of leishmania chagasi, but the results were negative, not confirming the disease. Only one dog was seropositive by all methods. He presented inespecific clinical signs and was acquired by his owner in an endemic area for the disease. The diagnoses could not be confirmed by direct parasitological test or PCR, because the dog has died before serologic results. The population results achieved in this study allow us to conclude that São José do Rio Preto city still must be a canine visceral leishmaniasis non-endemic area.

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-β 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 β-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.

Made available in DSpace on 2014-06-11T19:27:18Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-08-13Bitstream added on 2014-06-13T19:35:13Z : No. of bitstreams: 1 souza_f_me_araca.pdf: 170217 bytes, checksum: 6f885ef38991b7f73f26fde178a12e06 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Os cães são o principal reservatório doméstico de Leishmania. (L.) chagasi. No hospedeiro vertebrado, incluindo o homem, o parasita pode causar leishmaniose visceral, transmitida aos humanos. As células T regulatórias (Treg) estão envolvidas na indução de supressão de mecanismos efetores relacionados à imunidade celular. Para investigar a possível implicação das células Treg durante a infecção por Leishmania spp., a presença de Treg no baço e no sangue periférico foi avaliada em cães naturalmente infectados e com sinais clínicos compatíveis com a doença. Quinze cães adultos provenientes do Centro de Controle de Zoonoses da Prefeitura Municipal de Araçatuba - SP, Brasil, com reação sorológica positiva para antígenos de Leishmania (L.) chagasi determinada por ELISA indireto e teste rápido rK39 (Kalazar Detect TM Rapid Test, InBios Inc-Seattle, WA-USA). Cinco cães saudáveis, provenientes de área não endêmica foram incluídos no estudo. As células T regulatórias foram quantificadas em amostras de baço e em células mononucleares do sangue periférico utilizando-se anticorpos monoclonais e citometria de fluxo. No baço, menor número de células Treg foi observado em cães infectados do que nos controles (p<0,05), enquanto que nenhuma diferença foi observada nas células mononucleares de sangue periférico. Esses resultados sugerem que as células Treg estão envolvidas na infecção por Leishmania spp. e que podem ter um papel na persistência do parasita e o estabelecimento da infecção crônica / Dogs are the main domestic reservoirs of L. (L.) chagasi. Once in the vertebrate host, the parasite may cause visceral leishmaniasis, which can also be transmitted to humans. Regulatory T cells (Treg) have been shown to be involved in the direct induction of immunosupression of effector cellular immune response. To investigate the possible involvement of T reg cells during Leishmania infection, the presence of Treg cells from the spleen and peripheral blood mononuclear cells (PBMC) of dogs naturally infected with L. (L.) chagasi, and showing clinical signs, was quantified. A total of 15 adult dogs from the Center for Zoonosis Control of Araçatuba - SP, Brazil, positive for Leishmania (L.) chagasi by indirect ELISA and TM rapid test rK39 (Kalazar Detect TM Rapid Test, InBios Inc-Seattleadult healthy dogs from non endemic area were included in the sspleen and PBMC were used for quantification of Treg by flow cytometmonoclonal antibodies. In spleen, Treg levels in infected dogs control group (p<0.05); in PBMC, no differences were observed betgroups. These results suggest that Treg population is involved in infection and may play a role in promoting parasite persistence achronic infection