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Phlebotomine vectors, sand flies of the order Diptera, are known to transmit Leishmania parasites as well as RNA viruses (arboviruses) to humans. The arbovirus, Icoaraci Phlebovirus (BeAN 24262 - ICOV), used in this study was isolated from Nectomys rodents, a mammalian species that is the same natural sylvatic reservoir of Leishmania (Leishmania) amazonensis. This Leishmania species is distributed in primary and secondary forests in Brazil and other countries in America and causes localized and diffuse anergic skin lesions. In our recent studies, we observed an aggravation of the protozoan infection by ICOV through the modulation of cytokine expression, such as IL-10 and IFN-β, enhancing the parasite load and possibly the pathogenesis. Efficient viral production and quantitation had to be developed and standardized to ensure that immuno-molecular assays provide consistent and reproducible viral infection results. The standardization of these procedures becomes a particularly useful tool in research, with several applications in understanding the interaction between the host cell and Phlebovirus, as well as co-infections, allowing the study of intracellular signaling pathways. Here, we detail a protocol that allows the production and quantitation of the Icoaraci Phlebovirus using BHK-21 cells (baby hamster kidney cells) and subsequent infection of peritoneal macrophages from C57BL/6 mice.

In cutaneous leishmaniasis, Leishmania amazonensis activates macrophage double-stranded, RNA-activated protein kinase R (PKR) to promote parasite growth. In our study, Leishmania major grew normally in RAW cells, RAW-expressing dominant-negative PKR (PKR-DN) cells, and macrophages of PKR-knockout mice, revealing that PKR is dispensable for L. major growth in macrophages. PKR activation in infected macrophages with poly I:C resulted in parasite death. Fifty percent of L. major-knockout lines for the ecotin-like serine peptidase inhibitor (ISP2; Δisp2/isp3), an inhibitor of neutrophil elastase (NE), died in RAW cells or macrophages from 129Sv mice, as a result of PKR activation. Inhibition of PKR or NE or neutralization of Toll-like receptor 4 or 2(TLR4 or TLR2) prevented the death of Δisp2/isp3. Δisp2/isp3 grew normally in RAW-PKR-DN cells or macrophages from 129Sv pkr−/−, tlr2−/−, trif−/−, and myd88−/− mice, associating NE activity, PKR, and TLR responses with parasite death. Δisp2/isp3 increased the expression of mRNA for TNF-α by 2-fold and of interferon β (IFNβ) in a PKR-dependent manner. Antibodies to TNF-α reversed the 95% killing by Δisp2/isp3, whereas they grew normally in macrophages from IFN receptor–knockout mice. We propose that ISP2 prevents the activation of PKR via an NE-TLR4-TLR2 axis to control innate responses that contribute to the killing of L. major.—Faria, M. S., Calegari-Silva, T. C., de Carvalho Vivarini, A., Mottram, J. C., Lopes, U. G., Lima, A. P. C. A. Role of protein kinase R in the killing of Leishmania major by macrophages in response to neutrophil elastase and TLR4 via TNFα and IFNβ.

Submitted by Jonathan Barbosa (jon_crf@hotmail.com) on 2012-02-29T13:55:31Z No. of bitstreams: 1 Souza15_2008.pdf: 319631 bytes, checksum: 2a139348b18e3814e1c0b4bb40b792f6 (MD5) Approved for entry into archive by Catarina Soares(cfsoares@inmetro.gov.br) on 2012-03-15T13:16:25Z (GMT) No. of bitstreams: 1 Souza15_2008.pdf: 319631 bytes, checksum: 2a139348b18e3814e1c0b4bb40b792f6 (MD5) Made available in DSpace on 2012-03-15T13:16:25Z (GMT). No. of bitstreams: 1 Souza15_2008.pdf: 319631 bytes, checksum: 2a139348b18e3814e1c0b4bb40b792f6 (MD5) Previous issue date: 2008 The actin cytoskeleton controls pivotal cellular processes such as motility and cytokinesis, as well as cell-cell and cellsubstrate interactions. Assembly and spatial organization of actin filaments are dynamic events regulated by a large repertoire of actin-binding proteins. This report presents the first detailed characterization of the Trypanosoma cruzi actin (TcActin). Protein sequence analysis and homology modelling revealed that the overall structure of T. cruzi actin is conserved and that the majority of amino-acid changes are concentrated on the monomer surface. Immunofluorescence assays using specific polyclonal antibody against TcActin revealed numerous rounded and punctated structures spread all over the parasitic body. No pattern differences could be found between epimastigotes and trypomastigotes or amastigotes. Moreover, in detergent extracts, TcActin was localized only in the soluble fraction, indicating its presence in the G-actin form or in short filaments dissociated from the microtubule cytoskeleton. The trypanosomatid genome was prospected to identify actin-binding and actin-related conserved proteins. The main proteins responsible for actin nucleation and treadmilling in higher eukaryotes are conserved in T. cruzi. 11 p. : il.

5 p.-3 fig., We previously showed that intranasal (i.n.) vaccination with pCIneo plasmid encoding the leishmanial LACK gene (pCIneo-LACK) induces long-lasting protective immunity against cutaneous leishmaniasis in mice. In this work, we proposed to investigate whether the efficacy of i.n. pCIneo-LACK is extensive to visceral leishmaniasis. BALB/c mice received two i.n. doses of 30 μg pCIneo-LACK prior to intravenous (i.v.) infection with Leishmania chagasi. Vaccinated mice developed significantly lower parasite burden in the liver and spleen than control mice receiving empty pCIneo or saline. The spleen cells of vaccinated mice produced significantly increased IFN-γ and IL-4 concomitant with decreased IL-10 production during infection. Serum levels of specific IgG were elevated whereas TNF-α were decreased as compared with controls. These results show that the practical needle-free i.n. pCIneo-LACK vaccine displays potential broad-spectrum activity against leishmaniasis.

Submitted by Boris Flegr (boris@uerj.br) on 2021-01-07T15:13:48Z No. of bitstreams: 1 Luiz Gonzaga da Cunha Junior Tese completa.pdf: 970132 bytes, checksum: 955c51984deacbbba85ecb88e900b8f3 (MD5) Made available in DSpace on 2021-01-07T15:13:48Z (GMT). No. of bitstreams: 1 Luiz Gonzaga da Cunha Junior Tese completa.pdf: 970132 bytes, checksum: 955c51984deacbbba85ecb88e900b8f3 (MD5) Previous issue date: 2014-09-30 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior ExoU, a cytotoxin produced by the opportunistic pathogen Pseudomonas aeruginosa and translocated into the cytosol of host cells via a type III secretion system, is associated with severity of acute infections. In the present work, the effect of ExoU in the activation of oxidative stress and antioxidant response was evaluated in cultures of human respiratory epithelial cells infected with P. aeruginosa PA103 strain (producer of ExoU), or with its isogenic mutants, the ExoU-deficient PA103∆exoU or the exoU-depleted mutant complemented with an exoU gene with a site-specific mutation in the PLA2 catalytic site, PA103∆UT/S142A. Analysis of dosages of lipid hydroperoxides and isoprostanes, considered markers of oxidative stress, revealed that ExoU promoted an increase in their concentrations. It was also observed that ExoU stimulated the production of reactive oxygen species, nitric oxide and peroxynitrite in infected cells, as well as the expression of iNOS and eNOS, but not nNOS. Furthermore, ExoU was responsible for the increased activity of SOD1 and the reduced levels of GSH, but did not affect the activity of catalase or NQO1. In the in vivo model, the analysis of malondialdehyde, a byproduct of lipid peroxidation of membranes, showed increased production of this compound in the lungs of mice infected with the ExoU-producing strain compared to the lungs of mice infected with the mutant strain. Together, these results show that ExoU activates the production of reactive oxygen and nitrogen species, leading to lipid peroxidation and modulating the antioxidant defense system. ExoU, uma citotoxina produzida pelo patógeno oportunista Pseudomonas aeruginosa e translocada para o citosol de células hospedeiras via sistema de secreção do tipo III, é associada à gravidade de infecções agudas. No presente trabalho, o efeito de ExoU na ativação do estresse oxidativo e da resposta antioxidante foi avaliado em culturas de células epiteliais respiratórias humanas infectadas com a cepa PA103 de P. aeruginosa (produtora de ExoU), com a mutante deletada no gene exoU, PA103∆exoU, ou com a mutante complementada com exoU sem atividade tipo fosfolipase A2, PA103∆UT/S142A. Análises das dosagens de hidroperóxidos lipídicos e isoprostanos, considerados marcadores de estresse oxidativo, revelaram que ExoU promoveu um aumento em suas concentrações. Foi observado, também, que ExoU estimulou a produção de espécies reativas de oxigênio, óxido nítrico e peroxinitrito nas células infectadas, assim como a expressão de iNOS e eNOS, mas não de nNOS. Além disso, ExoU foi responsável pelo aumento da atividade de SOD1 e pela redução dos níveis de GSH, mas não afetou a atividade da catalase ou de NQO1. No modelo in vivo, a dosagem de malondialdeído, um subproduto da lipoperoxidação de membranas, evidenciou uma maior produção deste composto no pulmão de camundongos infectados pela cepa produtora de ExoU, em comparação ao pulmão de camundongos infectados pela cepa mutante. Em conjunto, estes resultados mostram que ExoU ativa a produção de espécies reativas de oxigênio e nitrogênio, levando à peroxidação lipídica e modulando o sistema de defesa antioxidante