Analise in silico e in vivo da diversidade nucleotidica em Coffea ssp.

Polimorfismos de modificações nucleotídicas (SNPs ? Single Nucleotide Polymorphisms, INDELs ? Insertion / Deletions) têm uma alta freqüência nos genomas da maioria dos organismos, incluindo plantas. Eles vêm se tornando a escolha principal de marcador para trabalhos de melhoramento, genotipagem e diagnóstico. A identificação destes polimorfismos irá fornecer marcadores que poderão ser utilizados para o mapeamento genético, estudos de genética de população e de associação. Portanto, os objetivos deste trabalho foram: 1) identificar in silico SNPs e INDELS existentes em seqüências de ESTs disponíveis; e 2) analisar a diversidade nucleotídica em Coffea spp. Um pipeline para identificação de SNPs e INDELs foi desenvolvido utilizando seqüências de ESTs disponíveis de Coffea spp. Foi utilizado uma estratégia para detecção de SNPs em dentro de 23.019 contigs. Um total 23.062 SNPs e 2.165 INDELS foram encontrados em 5184 contigs que continham pelo menos quatro ESTs. Analises in silico permitiram a identificação de diferentes alelos de C. canephora e C. eugenioides que estão presentes em C. arabica. A maioria dos ESTs de C. arabica vieram de apenas dois alelos, uma evidência molecular sobre a especiação de C. arabica. De acordo com essas análises cerca de 55% das seqüências de C. arabica são derivadas do genoma de C. eugenioides e 45% de C. canephora. Além disso, foi possível observar que o genoma de C. eugenioides contribui principalmente para genes relacionados a metabolismo basal, enquanto que os genes de C. canephora estão envolvidos com sinais de tradução e regulação da expressão gênica. Análises in vivo estão sendo realizadas através do sequenciamento de diversos genes em 24 genótipos de Coffea sendo 12 de C. arabica, 9 de C. canephora e três de outras espécies de Coffea, para uma analise maior da diversidade nucleotídica do gênero. Resultados referentes ao sequenciamento do gene de sacarose fosfato sintase (SPS) apresentaram 21 polimorfismos, sendo a maioria interespecíficos (C. arabica, C. canephora, C. eugenioides e C. racemosa). Para os genótipos de C. canephora foram observados nove polimorfismos intraespecíficos. Já os polimorfismos encontrados entre os genótipos de C. arabica forma os mesmos detectados entre C. canephora e C. eugenioides

Inducible Nitric Oxide Synthase in Heart Tissue and Nitric Oxide in Serum of Trypanosoma cruzi-Infected Rhesus Monkeys: Association with Heart Injury

Chagas disease, a neglected tropical disease caused by the protozoan Trypanosoma cruzi, afflicts from 8 to 15 million people in the Latin America. Chronic chagasic cardiomyopathy (CCC) is the most frequent manifestation of Chagas disease. Currently, patient management only mitigates CCC symptoms. The pathogenic factors leading to CCC remain unknown; therefore their comprehension may contribute to develop more efficient therapies. In patients, high nitric oxide (NO) levels have been associated with CCC severity. In T. cruzi-infected mice, NO, mainly produced via inducible nitric oxide synthase (iNOS/NOS2), is proposed to work in parasite control. However, the participation of iNOS/NOS2 and NO in T. cruzi control and heart injury has been questioned. Here, infected rhesus monkeys and iNOS/NOS2-deficient mice were used to explore the participation of iNOS/NOS2-derived NO in heart injury in T. cruzi infection. Chronically infected monkeys presented electrical abnormalities, myocarditis and fibrosis, resembling the spectrum of human CCC. Moreover, cardiomyocyte lesion correlated with iNOS/NOS2+ cells infiltrating the cardiac tissue. Our findings support that parasite-driven iNOS/NOS2+ cells accumulation in the cardiac tissue and NO overproduction contribute to cardiomyopathy severity, mainly disturbing the pathway involved in electrical synchrony in T. cruzi infection

CD8+ T-Cells Expressing Interferon Gamma or Perforin Play Antagonistic Roles in Heart Injury in Experimental Trypanosoma Cruzi-Elicited Cardiomyopathy

In Chagas disease, CD8+ T-cells are critical for the control of Trypanosoma cruzi during acute infection. Conversely, CD8+ T-cell accumulation in the myocardium during chronic infection may cause tissue injury leading to chronic chagasic cardiomyopathy (CCC). Here we explored the role of CD8+ T-cells in T. cruzi-elicited heart injury in C57BL/6 mice infected with the Colombian strain. Cardiomyocyte lesion evaluated by creatine kinase-MB isoenzyme activity levels in the serum and electrical abnormalities revealed by electrocardiogram were not associated with the intensity of heart parasitism and myocarditis in the chronic infection. Further, there was no association between heart injury and systemic anti-T. cruzi CD8+ T-cell capacity to produce interferon-gamma (IFNγ) and to perform specific cytotoxicity. Heart injury, however, paralleled accumulation of anti-T. cruzi cells in the cardiac tissue. In T. cruzi infection, most of the CD8+ T-cells segregated into IFNγ+ perforin (Pfn)neg or IFNγnegPfn+ cell populations. Colonization of the cardiac tissue by anti-T. cruzi CD8+Pfn+ cells paralleled the worsening of CCC. The adoptive cell transfer to T. cruzi-infected cd8−/− recipients showed that the CD8+ cells from infected ifnγ−/−pfn+/+ donors migrate towards the cardiac tissue to a greater extent and caused a more severe cardiomyocyte lesion than CD8+ cells from ifnγ+/+pfn−/− donors. Moreover, the reconstitution of naïve cd8−/− mice with CD8+ cells from naïve ifnγ+/+pfn−/− donors ameliorated T. cruzi-elicited heart injury paralleled IFNγ+ cells accumulation, whereas reconstitution with CD8+ cells from naïve ifnγ−/−pfn+/+ donors led to an aggravation of the cardiomyocyte lesion, which was associated with the accumulation of Pfn+ cells in the cardiac tissue. Our data support a possible antagonist effect of CD8+Pfn+ and CD8+IFNγ+ cells during CCC. CD8+IFNγ+ cells may exert a beneficial role, whereas CD8+Pfn+ may play a detrimental role in T. cruzi-elicited heart injury

A novel aspect of the structure of the avian thymic medulla.

We provide evidence for the compartmentalization of the avian thymic medulla and identify the avian thymic dendritic cell. The thymic anlage develops from an epithelial cord of the branchial endoderm. Branches of the cord are separated by primary septae of neural crest origin. The dilation of the primary septae produces the keratin-negative area (KNA) of the thymic medulla and fills the gaps of the keratin-positive network (KPN). Morphometric analysis indicates that the KNA takes up about half of the volume of the thymic medulla, which has reticular connective tissue, like peripheral lymphoid organs. The KNA receives blood vessels and in addition to pericytes, the myoid cells of striated muscle structure occupy this area. The myoid cells are of branchial arch or prechordal plate origin providing indirect evidence for the neural crest origin of the KNA. The marginal epithelial cells of the KPN co-express keratin and vimentin intermediate filaments, which indicate their functional peculiarity. The basal lamina of the primary septum is discontinuous on the surface of the KPN providing histological evidence for the loss of the blood-thymus barrier in the medulla. In the center of the KNA, the dendritic cells lie in close association with blood vessels, whereas the B-cells accumulate along the KPN. The organization of the KPN and KNA increases the "surface" of the so-called cortico-medullary border, thereby contributing to the efficacy of central tolerance

CC-chemokine receptors: a potential therapeutic target for Trypanosoma cruzi-elicited myocarditis

The comprehension of the pathogenesis of Trypanosoma cruzi-elicited myocarditis is crucial to delineate new therapeutic strategies aiming to ameliorate the inflammation that leads to heart dysfunction, without hampering parasite control. The augmented expression of CCL5/RANTES and CCL3/MIP-1alpha, and their receptor CCR5, in the heart of T. cruzi-infected mice suggests a role for CC-chemokines and their receptors in the pathogenesis of T. cruzi-elicited myocarditis. Herein, we discuss our recent results using a CC-chemokine receptor inhibitor (Met-RANTES), showing the participation of CC-chemokines in T. cruzi infection and unraveling CC-chemokine receptors as an attractive therapeutic target for further evaluation in Chagas disease

Subdominant/Cryptic CD8 T Cell Epitopes Contribute to Resistance against Experimental Infection with a Human Protozoan Parasite

During adaptive immune response, pathogen-specific CD8+ T cells recognize preferentially a small number of epitopes, a phenomenon known as immunodominance. Its biological implications during natural or vaccine-induced immune responses are still unclear. Earlier, we have shown that during experimental infection, the human intracellular pathogen Trypanosoma cruzi restricts the repertoire of CD8+ T cells generating strong immunodominance. We hypothesized that this phenomenon could be a mechanism used by the parasite to reduce the breath and magnitude of the immune response, favoring parasitism, and thus that artificially broadening the T cell repertoire could favor the host. Here, we confirmed our previous observation by showing that CD8+ T cells of H-2a infected mice recognized a single epitope of an immunodominant antigen of the trans-sialidase super-family. In sharp contrast, CD8+ T cells from mice immunized with recombinant genetic vaccines (plasmid DNA and adenovirus) expressing this same T. cruzi antigen recognized, in addition to the immunodominant epitope, two other subdominant epitopes. This unexpected observation allowed us to test the protective role of the immune response to subdominant epitopes. This was accomplished by genetic vaccination of mice with mutated genes that did not express a functional immunodominant epitope. We found that these mice developed immune responses directed solely to the subdominant/cryptic CD8 T cell epitopes and a significant degree of protective immunity against infection mediated by CD8+ T cells. We concluded that artificially broadening the T cell repertoire contributes to host resistance against infection, a finding that has implications for the host-parasite relationship and vaccine development