The role of cytokines in the control of tissue parasitism and pathogenesis of experimental Chagas' disease was investigated. Wild-type and different cytokine as well as inducible nitric oxide synthase (iNOS) knockout mice were infected with the Colombian strain of Trypanosoma cruzi, and the kinetics of tissue parasitism, inflammatory reaction, parasitemia, and mortality were determined. We demonstrate the pivotal role of the interleukin (IL)-12/interferon (IFN)-gamma/iNOS axis and the antagonistic effect of IL-4 in controlling heart tissue parasitism, inflammation, and host resistance to acute infection with T. cruzi. Further, the heart and central nervous system were shown the main sites of reactivation of T. cruzi infection in mice lacking functional genes for IFN-gamma and IL-12, respectively. Our results also show that in contrast to IFN-gamma knockout (KO) mice, splenocytes from IL-12 KO mice infected with T. cruzi produced low levels of IFN-gamma upon stimulation with antigen. Consistently, high levels of anti-T. cruzi IgG2a antibodies were detected in the sera from IL-12 KO, but not from IFN-gamma KO mice, infected with the Colombian strain of T. cruzi. Thus, our results suggest that the level of IFN-gamma deficiency is a major determinant of the site of reactivation of T. cruzi infection in immunocompromised host.
In a previous study we had shown that membrane cholesterol removal induced unregulated lysosomal exocytosis events leading to the depletion of lysosomes located at cell periphery. However, the mechanism by which cholesterol triggered these exocytic events had not been uncovered. In this study we investigated the importance of cholesterol in controlling mechanical properties of cells and its connection with lysosomal exocytosis. Tether extraction with optical tweezers and defocusing microscopy were used to assess cell dynamics in mouse fibroblasts. These assays showed that bending modulus and surface tension increased when cholesterol was extracted from fibroblasts plasma membrane upon incubation with MβCD, and that the membrane-cytoskeleton relaxation time increased at the beginning of MβCD treatment and decreased at the end. We also showed for the first time that the amplitude of membrane-cytoskeleton fluctuation decreased during cholesterol sequestration, showing that these cells become stiffer. These changes in membrane dynamics involved not only rearrangement of the actin cytoskeleton, but also de novo actin polymerization and stress fiber formation through Rho activation. We found that these mechanical changes observed after cholesterol sequestration were involved in triggering lysosomal exocytosis. Exocytosis occurred even in the absence of the lysosomal calcium sensor synaptotagmin VII, and was associated with actin polymerization induced by MβCD. Notably, exocytosis triggered by cholesterol removal led to the secretion of a unique population of lysosomes, different from the pool mobilized by actin depolymerizing drugs such as Latrunculin-A. These data support the existence of at least two different pools of lysosomes with different exocytosis dynamics, one of which is directly mobilized for plasma membrane fusion after cholesterol removal.
BackgroundmRNAs are highly versatile, non-toxic molecules that are easy to produce and store, which can allow transient protein expression in all cell types. The safety aspects of mRNA-based treatments in gene therapy make this molecule one of the most promising active components of therapeutic or prophylactic methods. The use of mRNA as strategy for the stimulation of the immune system has been used mainly in current strategies for the cancer treatment but until now no one tested this molecule as vaccine for infectious disease.ResultsWe produce messenger RNA of Hsp65 protein from Mycobacterium leprae and show that vaccination of mice with a single dose of 10 μg of naked mRNA-Hsp65 through intranasal route was able to induce protection against subsequent challenge with virulent strain of Mycobacterium tuberculosis. Moreover it was shown that this immunization was associated with specific production of IL-10 and TNF-alpha in spleen. In order to determine if antigen presenting cells (APCs) present in the lung are capable of capture the mRNA, labeled mRNA-Hsp65 was administered by intranasal route and lung APCs were analyzed by flow cytometry. These experiments showed that after 30 minutes until 8 hours the populations of CD11c+, CD11b+ and CD19+ cells were able to capture the mRNA. We also demonstrated in vitro that mRNA-Hsp65 leads nitric oxide (NO) production through Toll-like receptor 7 (TLR7).ConclusionsTaken together, our results showed a novel and efficient strategy to control experimental tuberculosis, besides opening novel perspectives for the use of mRNA in vaccines against infectious diseases and clarifying the mechanisms involved in the disease protection we noticed as well.
Iogurtes com adição de doce de araticum, buriti, cagaita, jatobá, mangaba e pequi foram processados, verificando-se sua vida-de-prateleira, preferência e aceitação. Três formulações de iogurtes foram elaboradas, contendo 15%, 20% e 25% dos doces para os iogurtes de pequi e jatobá e 20%, 25% e 30% dos doces para os demais iogurtes. O doce dos frutos foi adicionado ao iogurte natural na temperatura de ± 20°C e homogeneizado. Após a preparação, as amostras foram resfriadas a ± 4°C e submetidas à análise de preferência. A amostra preferida foi submetida à análise de aceitação com 100 julgadores não-treinados com idade entre 8 e 65 anos, mediante escala hedônica de nove pontos. Verificou-se a vida-de-prateleira dos iogurtes elaborados durante 7 dias por meio de análise sensorial com equipe composta por 7 julgadores treinados, análise físico-química para verificação do pH e acidez titulável e análise microbiológica para verificação de coliformes totais, coliformes a 45°C, contagem de estafilococos coagulase positiva e presença de Salmonella sp. em 25 g. Os resultados indicaram boa aceitação dos iogurtes, sendo que o de sabor araticum (com 25% do doce) apresentou maior média (7,4). Já o iogurte sabor jatobá com 20% de doce revelou a menor média (5,49). As análises físico-químicas e sensoriais mostraram que os produtos apresentaram-se estáveis sob refrigeração durante 5 a 6 dias, caracterizando-os como produto de consumo rápido. As análises microbiológicas mostraram que as boas práticas de higiene foram adequadas para garantir a qualidade microbiológica dos produtos.
PALAVRAS-CHAVE: IOGURTE; ANÁLISE SENSORIAL; VIDA-DE-PRATELEIRA; FRUTOS DO CERRADO.
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