Efeito de diferentes formas de cultivo na ação do óxido nítrico na maturação e na integridade da membrana plasmática de complexos cumulus-oócito em bovinos

The aim of the present study was to evaluate the influence of different forms of in vitro culture on the nitric oxide action in maturation and membrane integrity on bovine cumulus-oocyte complex. No significant effect was observed between different forms of culture (mineral oil vs plate; P &gt; 0.05), as much for membrane integrity as for expansion of the CC. However, it was observed that oocytes of the groups control and 10-3 M of SNP, cultivated in plate, had presented greater percentage of cell with maintenance of membrane integrity than same treatments cultivated in drop. The addition of 10-3 M of SNP showed an inhibitory effect on the expansion and membrane integrity of CC and oocytes in both, culture in drops under oil and plate (P < 0.05). The culture form did not intervene with the extrusion of the first polar corpuscle and the addition of 10-3 M of SNP inhibited this extrusion in the both systems (P < 0.05). There was a dose-response effect on the concentration of NO in the maturation medium in both types of cultivation (P < 0,05), and this was higher in the culture medium under oil, except when added 10-3 M of SNP, treatment in which there was no difference in the types of cultivation employed. (PO objetivo do presente estudo foi avaliar se diferentes formas de cultivo interferem no efeito do óxido nítrico (NO) sobre a maturação e a integridade da membrana plasmática do complexo cumulus-oócito de bovinos. Para tanto, realizou-se cultivo em gotas sob óleo mineral ou em placas de quatro poços com a adição de diferentes concentrações de nitroprussiato de sódio (SNP, doador de óxido nítrico). Não foi observada diferença (P &gt; 0,05) entre as formas de cultivo quando se avaliou a integridade de membrana plasmática e a expansão das células do cumulus (CC). Contudo, os oócitos dos grupos controle e os cultivados na presença de 10-3 M de SNP, ambos cultivados em placa, apresentaram maior porcentagem de membrana íntegra do que os mesmos tratamentos cultivados em óleo mineral (P &lt; 0,05). Observou-se que a adição de 10-3 M de SNP diminuiu o grau de expansão das CC e de integridade da membrana plasmática dos oócitos, tanto no cultivo em gota sob óleo quanto em placa, diferindo dos outros grupos (P &lt; 0,05). Semelhante à expansão, a forma de cultivo não interferiu na extrusão do primeiro corpúsculo polar, sendo que a adição de 10-3 M de SNP inibiu a extrusão em ambos os sistemas (P &lt; 0,05). Houve um efeito dose-resposta na concentração de NO no meio de maturação em ambos os tipos de cultivo (P &lt; 0,05), sendo que esta foi maior no meio de cultivo sob óleo, exceção feita quando se adicionou 10-3 M de SNP, tratamento no qual não houve diferença nos tipos de cultivo empregados. Estes dados mostram que o sistema de cultivo não interferiu na ação do NO na maturação in vitro de COC bovinos, mas interfere na integridade da membrana plasmática do oócito

Pathogenic Mycobacterium bovis strains differ in their ability to modulate the proinflammatory activation phenotype of macrophages

Abstract Background Tuberculosis, caused by Mycobacterium tuberculosis or Mycobacterium bovis, remains one of the leading infectious diseases worldwide. The ability of mycobacteria to rapidly grow in host macrophages is a factor contributing to enhanced virulence of the bacteria and disease progression. Bactericidal functions of phagocytes are strictly dependent on activation status of these cells, regulated by the infecting agent and cytokines. Pathogenic mycobacteria can survive the hostile environment of the phagosome through interference with activation of bactericidal responses. To study the mechanisms employed by highly virulent mycobacteria to promote their intracellular survival, we investigated modulating effects of two pathogenic M. bovis isolates and a reference M. tuberculosis H37Rv strain, differing in their ability to multiply in macrophages, on activation phenotypes of the cells primed with major cytokines regulating proinflammatory macrophage activity. Results Bone marrow- derived macrophages obtained from C57BL/6 mice were infected by mycobacteria after a period of cell incubation with or without treatment with IFN-γ, inducing proinflammatory type-1 macrophages (M1), or IL-10, inducing anti-inflammatory type-2 cells (M2). Phenotypic profiling of M1 and M2 was then evaluated. The M. bovis strain MP287/03 was able to grow more efficiently in the untreated macrophages, compared with the strains B2 or H37Rv. This strain induced weaker secretion of proinflammatory cytokines, coinciding with higher expression of M2 cell markers, mannose receptor (MR) and arginase-1 (Arg-1). Treatment of macrophages with IFN-γ and infection by the strains B2 and H37Rv synergistically induced M1 polarization, leading to high levels of inducible nitric oxide synthase (iNOS) expression, and reduced expression of the Arg-1. In contrast, the cells infected with the strain MP287/03 expressed high levels of Arg-1 which competed with iNOS for the common substrate arginine, leading to lower levels of NO production. Conclusions The data obtained demonstrated that the strain, characterized by increased growth in macrophages, down- modulated classical macrophage activation, through induction of an atypical mixed M1/M2 phenotype

Pathogenic Mycobacterium bovis strains differ in their ability to modulate the proinflammatory activation phenotype of macrophages

Abstract\ud \ud Background\ud Tuberculosis, caused by Mycobacterium tuberculosis or Mycobacterium bovis, remains one of the leading infectious diseases worldwide. The ability of mycobacteria to rapidly grow in host macrophages is a factor contributing to enhanced virulence of the bacteria and disease progression. Bactericidal functions of phagocytes are strictly dependent on activation status of these cells, regulated by the infecting agent and cytokines. Pathogenic mycobacteria can survive the hostile environment of the phagosome through interference with activation of bactericidal responses. To study the mechanisms employed by highly virulent mycobacteria to promote their intracellular survival, we investigated modulating effects of two pathogenic M. bovis isolates and a reference M. tuberculosis H37Rv strain, differing in their ability to multiply in macrophages, on activation phenotypes of the cells primed with major cytokines regulating proinflammatory macrophage activity.\ud \ud \ud Results\ud Bone marrow- derived macrophages obtained from C57BL/6 mice were infected by mycobacteria after a period of cell incubation with or without treatment with IFN-γ, inducing proinflammatory type-1 macrophages (M1), or IL-10, inducing anti-inflammatory type-2 cells (M2). Phenotypic profiling of M1 and M2 was then evaluated. The M. bovis strain MP287/03 was able to grow more efficiently in the untreated macrophages, compared with the strains B2 or H37Rv. This strain induced weaker secretion of proinflammatory cytokines, coinciding with higher expression of M2 cell markers, mannose receptor (MR) and arginase-1 (Arg-1). Treatment of macrophages with IFN-γ and infection by the strains B2 and H37Rv synergistically induced M1 polarization, leading to high levels of inducible nitric oxide synthase (iNOS) expression, and reduced expression of the Arg-1. In contrast, the cells infected with the strain MP287/03 expressed high levels of Arg-1 which competed with iNOS for the common substrate arginine, leading to lower levels of NO production.\ud \ud \ud Conclusions\ud The data obtained demonstrated that the strain, characterized by increased growth in macrophages, down- modulated classical macrophage activation, through induction of an atypical mixed M1/M2 phenotype

Mycobacterium tuberculosis Strains of the Modern Sublineage of the Beijing Family Are More Likely To Display Increased Virulence than Strains of the Ancient Sublineage

Made available in DSpace on 2015-05-04T17:07:30Z (GMT). No. of bitstreams: 2 license.txt: 1914 bytes, checksum: 7d48279ffeed55da8dfe2f8e81f3b81f (MD5) lia_gomesetal_IOC_2014.pdf: 2345129 bytes, checksum: 1b32aaff28a0f9a34cdb2bdae8526c41 (MD5) Previous issue date: 2014Universidade Estadual do Norte Fluminense.Laboratório de Morfologia Animal e Patologia. Laboratório de Biologia do Reconhecimento. Campos, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Molecular Aplicada a Micobactérias. Rio de Janeiro, RJ, Brasil.Universidade de São Paulo. Instituto de Ciências Biomédicas. Departamento de Imunologia. São Paulo, SP, BrasilUniversidade Estadual do Norte Fluminense.Laboratório de Morfologia Animal e Patologia. Laboratório de Biologia do Reconhecimento. Campos, RJ, Brasil.Universidade Estadual do Norte Fluminense.Laboratório de Morfologia Animal e Patologia. Laboratório de Biologia do Reconhecimento. Campos, RJ, Brasil.Universidade Estadual do Norte Fluminense.Laboratório de Morfologia Animal e Patologia. Laboratório de Biologia do Reconhecimento. Campos, RJ, Brasil.Universidade Estadual do Norte Fluminense.Laboratório de Morfologia Animal e Patologia. Laboratório de Biologia do Reconhecimento. Campos, RJ, Brasil.Universidade Estadual do Norte Fluminense.Laboratório de Morfologia Animal e Patologia. Laboratório de Biologia do Reconhecimento. Campos, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Molecular Aplicada a Micobactérias. Rio de Janeiro, RJ, Brasil.St. Petersburg Pasteur Institute. Laboratory of Molecular Microbiology. St. Petersburg. Russian Federation.Universidade Estadual do Norte Fluminense.Laboratório de Morfologia Animal e Patologia. Laboratório de Biologia do Reconhecimento. Campos, RJ, Brasil.Strains of the Beijing genotype family of Mycobacterium tuberculosis are a cause of particular concern because of their increasing dissemination in the world and their association with drug resistance. Phylogenetically, this family includes distinct ancient and modern sublineages. The modern strains, contrary to the ancestral counterparts, demonstrated increasing prevalence in many world regions that suggest an enhanced bacterial pathogenicity. We therefore evaluated virulence of modern versus ancient Beijing strains with similar epidemiological and genotype characteristics. For this, we selected six strains that had very similar 24-locus mycobacterial interspersed repetitive-unit–variable-number tandem-repeat (MIRU-VNTR) typing profiles and belonged to the region of difference 181 (RD181) subgroup but differed using markers (mutT2 and mutT4 genes and NTF locus) that discriminate between modern and ancient Beijing sublineages. The strains were isolated from native patients in Brazil and Mozambique, countries with a low prevalence of Beijing strains. The virulence levels of these strains were determined in models of pulmonary infection in mice and in vitro macrophage infection and compared with that of a strain from Russia, part of the epidemic and hypervirulent Beijing clone B0/W148, and of the laboratory strain H37Rv. The results showed that two of the three modern Beijing strains were highly pathogenic, exhibiting levels of virulence comparable with that of the epidemic Russian strain. In contrast, all isolates of the ancient sublineage displayed intermediate or low virulence. The data obtained demonstrate that the strains of the modern Beijing sublineage are more likely to exhibit highly virulent phenotypes than ancient strains and suggest that genetic alterations characteristic of the modern Beijing sublineage favor selection of highly virulent bacteria

[pt] ESCRITA: CELEBRANDO UMA DÉCADA

The purinergic P2X7 receptor (P2X7R) is a sensor of extracellular ATP, a damage-associated molecule that is released from necrotic cells and that induces pro-inflammatory cytokine production and cell death. To investigate whether the innate immune response to damage signals could contribute to the development of pulmonary necrotic lesions in severe forms of tuberculosis, disease progression was examined in C57BL/6 and P2X7R-/- mice that were intratracheally infected with highly virulent mycobacterial strains (Mycobacterium tuberculosis strain 1471 of the Beijing genotype family and Mycobacterium bovis strain MP287/03). The low-dose infection of C57BL/6 mice with bacteria of these strains caused the rapid development of extensive granulomatous pneumonia with necrotic areas, intense bacillus dissemination and anticipated animal death. In contrast, in P2X7R-/- mice, the lung pathology presented with moderate infiltrates of mononuclear leukocytes without visible signs of necrosis; the disease attenuation was accompanied by a delay in mortality. In vitro, the hypervirulent mycobacteria grew rapidly inside macrophages and induced death by a P2X7R-dependent mechanism that facilitated the release of bacilli. Furthermore, these bacteria were resistant to the protective mechanisms elicited in macrophages following extracellular ATP stimulation. Based on this study, we propose that the rapid intracellular growth of hypervirulent mycobacteria results in massive macrophage damage. The ATP released by damaged cells engages P2X7R and accelerates the necrotic death of infected macrophages and the release of bacilli. This vicious cycle exacerbates pneumonia and lung necrosis by promoting widespread cell destruction and bacillus dissemination. These findings suggest the use of drugs that have been designed to inhibit the P2X7R as a new therapeutic approach to treat the aggressive forms of tuberculosis

Lung gross pathology in C57BL/6 and P2X7R^−/− mice on day 28 p.i. with hypervirulent mycobacteria.

<p>C57BL/6 and P2X7R^−/− mice were infected i.t. with approx. 100 bacilli of H37Rv <i>Mtb</i>, Beijing 1471 <i>Mtb</i> and MP287/03 <i>Mbv</i>. Non-infected mice were used as controls. (A) Representative images of the right lungs are shown. (B) Right lung weights and (C) relative masses (circles) were evaluated (<i>n</i> = 9). The mean values are represented by horizontal lines. The relative lung masses were calculated by the ratios of the mean values of the lung weights in the indicated groups and the control group. Significant differences were observed for the indicated groups (**<i>p</i><0.01 and ***<i>p</i><0.001). The data are representative of three separate experiments.</p

Lung infiltrating cells and cytokine production in C57BL/6 and P2X7R^−/− mice on day 28 p.i. with hypervirulent mycobacteria.

<p>C57BL/6 and P2X7R^−/− mice were infected i.t. with approx. 100 bacilli of H37Rv <i>Mtb</i>, Beijing 1471 <i>Mtb</i> and MP287/03 <i>Mbv</i>. Non-infected mice were used as controls. (A) The numbers of total, CD11b⁺, CD19⁺, CD4⁺ and CD8⁺ cells in the lungs are shown (means ± SD, <i>n</i> = 3). (B) IL-1β, IFNγ and IL-10 were quantified in the supernatants of lung cells after 48 h of culture (means ± SD, <i>n</i> = 3). Significant differences were observed for the indicated groups (*<i>p</i><0.05, **<i>p</i><0.01 and ***<i>p</i><0.001). The data are representative of three separate experiments.</p

Schematic illustration of a hypothetical model to explain the high and low involvement of the P2X7R during severe and mild TB.

<p>In severe TB, the rapid intracellular growth of hypervirulent mycobacteria results in massive macrophage damage. The eATP released by damaged cells may engage the P2X7R on their surfaces or on neighboring cells. The autocrine or paracrine P2X7R signaling cooperates with mycobacterial components exhibiting membrane-lysing activity and accelerates the necrotic death of infected macrophages and the spread of bacilli. The resistance of hypervirulent mycobacteria to the protective mechanisms elicited in macrophages by eATP contributes to disease dissemination. The release of large amounts of eATP triggers a vicious cycle that exacerbates the pulmonary recruitment of pathogen-permissive monocytes and macrophages and thereby leads to further intracellular bacillus growth. The suppressive environment that results from an excess of adenosine, a byproduct of eATP degradation, may also facilitate the survival of hypervirulent mycobacteria. According to this model, lung necrosis derives from programmed cell death that is triggered by P2X7R signaling. The modest levels of tissue damage induced by less virulent strains and the susceptibility of these mycobacteria to eATP-induced intracellular killing explain, respectively, the minor role and the protective effect of the P2X7R in the mild forms of TB.</p

Survival curves and bacillary burdens in the lungs, liver and spleen of C57BL/6 and P2X7R^−/− mice infected with hypervirulent mycobacteria.

<p>C57BL/6 and P2X7R^−/− mice were infected i.t. with approx. 100 bacilli of H37Rv <i>Mtb</i>, Beijing 1471 <i>Mtb</i> and MP287/03 <i>Mbv</i>. Non-infected mice were used as controls. (A) The infected mice were examined daily to determine the survival curves (<i>n</i> = 13–19). (B) The number of CFU/g of tissue was evaluated in the left lung, liver and spleen on day 28 p.i. (means ± SD, <i>n</i> = 9). Significant differences were observed for the indicated groups (*<i>p</i><0.05, **<i>p</i><0.01 and ***<i>p</i><0.001). The data are representative of three separate experiments.</p