7 research outputs found

    Investigação sobre o papel dos antígenos parasitários na patogenia da miocardite crônica, em camundongos infectados pelo Trypanosoma Cruzi: a importância das células dendríticas intersticiais do miocárdio

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    Submitted by Repositório Arca ([email protected]) on 2019-08-06T11:25:52Z No. of bitstreams: 1 license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5)Approved for entry into archive by Ana Maria Fiscina Sampaio ([email protected]) on 2019-08-16T13:07:34Z (GMT) No. of bitstreams: 2 Renata Siqueira Portella Investigação...2006.pdf: 43905543 bytes, checksum: 63454418c6f4a53ce972927786a59f98 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5)Made available in DSpace on 2019-08-16T13:07:34Z (GMT). No. of bitstreams: 2 Renata Siqueira Portella Investigação...2006.pdf: 43905543 bytes, checksum: 63454418c6f4a53ce972927786a59f98 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2006Universidade Federal da Bahia. Faculdade de Medicina. Fundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, BA, Brasil.A doença de Chagas, evolui em diferentes fases: aguda, com sintomas gerais e comprometimento cardíaco devido ao parasitismo pelo T. cruzi, das miocélulas cardíacas e sua rotura o que determina intensa miocardite, pela presença dos parasitos e por mecanismos imunes. Após a fase aguda, os pacientes passam a uma forma indeterminada com escassos parasitos e lesões miocárdicas focais e discretas. Os pacientes na forma indeterminada, em 40 por cento dos casos, evoluem para uma forma crônica cardíaca. Nesta forma, os parasitos são raros nas miocélulas cardíacas, porém uma miocardite crônica difusa e fibrosante se instala, evoluindo para a insuficiência cardíaca e o óbito. A patogenia das lesões cardíacas é controvertida, sendo atribuída ao parasito o papel de estimular a resposta imune ou a um mecanismo de autoimunidade. No presente trabalho,utilizando-se o modelo do camundongo, procura-se investigar o papel dos antígenos parasitários na manutenção das lesões da fase crônica da infecção pelo T. cruzi, pela pesquisa de parasitos e de antígenos parasitários em focos inflamatórios ou pela demonstração de antígenos seqüestrados e expressos na membrana de células dendríticas intersticiais do miocárdio (CDls), pela marcação in situ, por métodos imunohistoquimicos, utilizando anticorpo anti-T.cruzi. Em camundongos cronicamente infectados, tratados com quimioterápico específico, as lesões histopatológicas regridem, permanecendo, entretanto um processo inflamatório residual. Foi desenvolvida uma investigação, com o objetivo de esclarecer, em camundongos cronicamente infectados, tratados e não tratados com o Benzonidàzol, a presença de parasitos ou de seus antígenos, em focos de destruição de miocélulas parasitadas ou capturadas por CDls, e a sua relação com a persistência de infiltrados inflamatórios residuais no coração. Foram utilizados camundongos Suíços infectados com a cepa Colombiana (resistentes ao Benzonidazol) ou com um clone da 21SF (susceptível) tratados na fase crônica a partir de 90 e 120 dias de infecção durante 90 dias. O inóculo para ambos os Grupos experimentais foi de 104 formas saguícolas do T. cruzi por via intraperitoneal. Os camundongos tratados foram sacrificados 150 ou 200 dias após o tratamento, juntamente com os controles não tratados. Foram realizados os testes de cura (parasitemia, subinoculação em camundongos recém-nascidos, hemocultura, sorologia). Secções de coração e de músculo esquelético foram fixadas e processadas para estudo histopatológico em secções coradas pela Hematoxilina e Eosina ou pelo método do Picro-Sirius para colágeno e para imunohistoquímica com anticorpos anti-T. cruzi e anti-MAC. Os resultados mostraram na infecção crônica pela cepa Colombiana lesões inflamatórias, moderadas a intensas, com necrose de miocélulas cardíacas e infiltrados mononucleares e fibrose intersticial difusa e focal. Nos tratados, houve regressão parcial das lesões permanecendo infiltrados inflamatórios residuais que variaram de discretos a moderadas e sinais de regressão da fibrose. Na infecção crônica pelo clone 21SF C3 havia miocardite crônica difusa discreta ou moderada na maioria dos casos. Nos tratados, houve decréscimo das lesões inflamatórias, com sinais de regressão da fibrose. Testes imunohistoquimicos com anticorpos anti- T.cruzi revelaram detritos parasitários positivos em focos inflamatórios. As CDls do miocárdio apresentaram em todos os casos, marcação positiva para antígeno do T. cruzi, comprovando o papel das células apresentadoras de antígenos na manutenção das lesões na fase crônica da infecção.Chagas’disease evolves through different phases; an acute phase, with systemic symptoms and cardiac involvement, due to Trypanosoma cruzi invasion of myocardiocytes and their rupture, which result in intense myocarditis, amplified by immune mechanisms. Following the acute phase, patients enter into an indeterminate form of the disease, when parasites are scarce and myocarditis becomes mild and focal; about 40% of patients progress to a diffuse chronic myocarditis, with fibrosis and few parasites, cardiac failure and death. The pathogenesis of cardiac lesions is disputed, especially regarding the role of the parasite and its antigens as opposed to auto-immunity. In the present study the importance of parasite antigens in maintaining the cardiac lesions during chronic infection with T. cruzi is investigated using the mice model. Search of parasites or parasite antigens within inflammatory foci, as well as the demonstration of antigens captured by interstitial dendritic cells of the heart (IDCs) and expressed at their membrane as revealed by in situ antigen immunolabeling were performed by using anti-7, cruzi antibodies. In chronically infected mice, treated with specific chemotherapy, a regression of the histopathological lesions was observed, although a residual inflammatory infiltrate remained. An investigation was made in chronically infected mice, either treated or not with Benznidazole, with the aim at clarifying whether the inflammatory infiltrates were related to the presence of parasites or to their antigens, either in the tissues or at the IDCs membrane. Chronically infected Swiss mice, either with the Colombian strain (resistant to Benznidazole) or with the clone 21 SF 03 (susceptible) were used. They were treated during the chronic phase (90 to 120 days of infection), for 90 days. Inocula for both groups were of 10“ blood forms of 7. cruzi, intraperitoneally injected. Treated mice were killed 150 or 200 days post-treatment, together with non-treated controls. Parasitemia, sub-inoculation into new-born mice, hemoculture, and serology were performed for cure tests. Fragments ofthe heart and skeletal muscles were fixed and processed for histopathological studies. The sections were stained with Hematoxilin and Eosin, Picro-Sirius-red method for collagen, and for immunohistochemistry with anti-7 cruzi and anti-MAC antibodies. Results revéaled that Colombian-stráin chronically infected mice presented inflammatory lesions varying from moderate to intense, with necrosis of cardiac myocells, mononuclear cells infiltration and diffuse and focal interstitial fibrosis. Treated mice showed partial regression of the inflammatory lesions, with the presence of focal or diffuse residual infiltrates, varying from mild to moderate, besides regression of fibrosis. Mild to moderate chronic diffuse myocarditis was present in most cases of the mice infected with the clone 21SF C3. Treated mice exhibited subsiding inflammatory lesions. Inflammatory lesions were residual and fibrosis showed signals of regression. Immunohistochemical tests with anti-7, cruzi antibodies revealed positively stained parasite debris inside the inflammatory foci. The IDCs of the heart presented in all cases positive immunolabeling for 7. cruzi antigens, confirming the participation of antigen presenting cells in the maintenance of chronic phase lesions of Chagas’ disease

    Presença de células dendríticas ativadas e das células T CD4+CD25hi na miocardite aguda e crônica da doença de chagas, em camundongos de linhagens resistente e susceptível

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    Submitted by Ana Maria Fiscina Sampaio ([email protected]) on 2014-02-07T18:23:30Z No. of bitstreams: 1 Renata Siqueira Portella.. Presença de células....pdf: 2025004 bytes, checksum: a85d3195f20f2a79bd6ff1b32049c260 (MD5)Made available in DSpace on 2014-02-07T18:23:30Z (GMT). No. of bitstreams: 1 Renata Siqueira Portella.. Presença de células....pdf: 2025004 bytes, checksum: a85d3195f20f2a79bd6ff1b32049c260 (MD5) Previous issue date: 2013Fundação Oswaldo Cruz. Centro de Pesquisa Gonçalo Moniz. Salvador, BA, Brasil / Universidade Federal da Bahia. Faculdade de Medicina. Salvador, BA, BrasilA doença de Chagas é caracterizada por apresentar duas fases com curso clínico bastante variável. Na fase aguda ocorre uma intensa miocardite, sendo o parasita facilmente detectado no sangue periférico e nos tecidos. A fase crônica cardíaca é caracterizada por uma cardiopatia, com intensa destruição das fibras cardíacas, presença de áreas de fibrose e escassos parasitas. Os mecanismos envolvidos na patogenia dessa miocardite ainda não são muito claros. Acredita-se que as células reguladoras e as células dendríticas estejam envolvidas nesse processo. Para compreender os mecanismos envolvidos, na resposta inflamatória à infecção pelo T. cruzi, resolvemos investigar a participação das células dendríticas, das células reguladoras e o perfil dos linfócitos T CD4+ e CD8+, utilizando duas linhagens de camundongos isogênicos, que apresentam diferentes graus de susceptibilidade a infecção. Em nossos resultados constatamos que os camundongos DBA/1 apresentaram maior sobrevida à fase aguda (90%), mesmo tratando os camundongos A (70%) com benzonidazol por três dias consecutivos, com o intuito de diminuir a carga parasitária prevenindo a alta mortalidade. A resistência dos DBA/1 e a susceptibilidade dos A, a infecção pelo T. cruzi, estaria relacionada ao perfil da resposta inflamatória e regulatória desenvolvida no decorrer da doença. Observamos que os camundongos DBA/1 possuem mais células dendríticas ativadas no baço e coração e mais células T CD4+CD25hi do que os camundongos da linhagem A. Essa diferença do perfil de resposta pode estar provocando uma maior expansão e diferenciação dos linfócitos T CD4+ pelas células dendríticas, levando ao controle da carga parasitaria.Chagas’diseasse, due to Trypanosoma cruzi infection is characterized by the development of two phases with a variable clinical course. During the acute phase there occurs a severe myocarditis with the parasite being easily detected in peripheral blood and tissues. The chronic cardiac phase is characterized as a chronic cardiopathy, when severe destruction of cardiac myocells and fibrosis are present and parasites are rare. The mechanisms involved in the pathogenesis of this myocarditis are somewhat obscure. It is believed that regulatory and dendritic cells play a role in this process. In an attempt to clarify the mechanisms involved in the inflammatory response to infection with T. cruzi we decided to investigate the participation of dendritic and regulatory cells and of TCD4 and TCD8 lymphocytes, using two strains of isogenic mice , which exhibit different degrees of susceptibility to infection. Our results have shown that the DBA/1 mice presented a higher survival (90%) in the acute phase than the A mice (70%), even when these were treated with Benznidazole for three consecutive days, with the objective of to reduce the parasitemia and the high mortality. Resistance of DBA/1 mice and susceptibility of A mice could be related to the evolution of the inflammatory and regulatory responses, during the infection. It was seen that DBA/1 mice disclosed a higher number of activated dendritic cells in the spleen and heart and a higher number of T CD4+CD25hi than the mice of A strain. This differences of the response could be influencing in the TCD4 differentiation and on the control of parasitic loa

    Effect of treatment with cyclophosphamide in low doses upon the onset of delayed type hypersensitivity in mice chronically infected with Trypanosoma cruzi: involvement of heart interstitial dendritic cells

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    Acute infection with Trypanosoma cruzi results in intense myocarditis, which progresses to a chronic, asymptomatic indeterminate form. The evolution toward this chronic cardiac form occurs in approximately 30% of all cases of T. cruzi infection. Suppression of delayed type hypersensitivity (DTH) has been proposed as a potential explanation of the indeterminate form. We investigated the effect of cyclophosphamide (CYCL) treatment on the regulatory mechanism of DTH and the participation of heart interstitial dendritic cells (IDCs) in this process using BALB/c mice chronically infected with T. cruzi. One group was treated with CYCL (20 mg/kg body weight) for one month. A DTH skin test was performed by intradermal injection of T. cruzi antigen (3 mg/mL) in the hind-footpad and measured the skin thickness after 24 h, 48 h and 72 h. The skin test revealed increased thickness in antigen-injected footpads, which was more evident in the mice treated with CYCL than in those mice that did not receive treatment. The thickened regions were characterised by perivascular infiltrates and areas of necrosis. Intense lesions of the myocardium were present in three/16 cases and included large areas of necrosis. Morphometric evaluation of lymphocytes showed a predominance of TCD8 cells. Heart IDCs were immunolabelled with specific antibodies (CD11b and CD11c) and T. cruzi antigens were detected using a specific anti-T. cruzi antibody. Identification of T. cruzi antigens, sequestered in these cells using specific anti-T. cruzi antibodies was done, showing a significant increase in the number of these cells in treated mice. These results indicate that IDCs participate in the regulatory mechanisms of DTH response to T. cruzi infection

    Treatment with benznidazole in association with immunosuppressive drugs in mice chronically infected with Trypanosoma cruzi: investigation into the possible development of neoplasias

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    Benznidazole is recommended in Brazil for the treatment of Trypanosoma cruzi infection in acute and early chronic phases of Chagas' disease. Observations by others have indicated a higher incidence of neoplasias in immunosuppressed patients, presenting Chagas' disease reactivation, submitted to treatment with benznidazole. In the present study, we investigated whether there is a potentiation in the generation of lymphomas in chronically infected mice, treated with immunosuppressive drugs and benznidazole. For this, 142 Swiss mice chronically infected with the 21 SF strain of T. cruzi and 72 normal Swiss mice were used. Both infected and normal mice were divided into experimental groups and submitted to one of the following treatment regimens: benznidazole alone; immunosuppressive drugs (azathioprine, betamethasone and cyclosporin); a combination of immunosuppressive drugs and benznidazole; and untreated controls. In the infected group treated with benznidazole, one mouse developed a non-Hodgkin's lymphoma. This finding has been interpreted as a spontaneous tumor of mice. The study of the chronically infected mice treated with the combination of immunosuppressive drugs and benznidazole demonstrated an absence of lymphomas or other neoplasias. These findings support the indication of benznidazole, as the drug of choice, for immunosuppressed patients that develop a reactivation of Chagas' disease

    Trypanosoma cruzi: parasite antigens sequestered in heart interstitial dendritic cells are related to persisting myocarditis in benznidazole-treated mice

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    We investigated whether sequestered Trypanosoma cruzi antigens found in heart interstitial dendritic cells (IDCs) contribute to the residual myocarditis found in mice following treatment with benznidazole, a specific chemotherapeutic drug. IDCs are antigen-presenting cells that are MHC-II-receptor dependent. Swiss mice were divided into two experimental groups: the 1st group was infected with the Colombian strain of T. cruzi, which is resistant to treatment with benznidazole, and the 2nd group was infected with clone 21SF-C 3, which has a medium susceptibility to the drug. Treatment of the Colombian strain group started on the 120th day post-infection and for the 21SF-C3 strain group treatment was started on the 90th day. In both groups, treatment lasted for 90 days. The animals were sacrificed either 150 or 200 days post-treatment. The myocardium was analysed by immunohistochemistry using anti-MAC3, 33D1, CD11b and CD11c monoclonal antibodies for IDCs or anti-T. cruzi purified antibodies. Parasite antigens were expressed on the IDC membranes in both treated and untreated mice. Myocarditis subsided following treatment, evidenced by both histological and morphometrical evaluation. A reduction in the number of IDCs carrying T. cruzi antigens in the treated group indicates that the elimination of parasites influences antigen presentation with concomitant decreases in inflammation. There is a correlation between the presence of T. cruzi antigens in these cells and the chronic focal, residual myocarditis seen in treated mice
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