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INFLUÊNCIA DE REINFECÇÕES SEQUENCIAIS COM CLONES DA CEPA COLOMBIANA DO TRYPANOSOMA CRUZI COM DIFERENTES GRAUS DE VIRULÊNCIA, SOBRE A MIOCARDITE CRÔNICA NO MODELO MURINO
Alternative title
Influence of sequential reinfections with diferent clones of the colombian strain of trypanosoma cruzi upon murine chronic myocarditisAuthor
Advisor
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Abstract in Portuguese
Reinfeções pelo Trypanosoma cruzi em pacientes residentes de areas endemicas tern sido mencionadas como fator agravante das manifesta96es cardiacas na doen9a de Chagas. Achados experimentais demonstram que animais quando submetidos a infec96es sucessivas apresentam rea96es inflamat6rias mais intensas quando comparados aqueles com infec9ao (mica. 0 estudo da triplice infec9ao com cepas de diferentes Biodemas demonstrou exacerba9ao das lesoes teciduais em musculo cardiaco e esqueletico provavelmente devido ao abrigo de diferentes popula96es (cepas). Neste trabalho, utilizando-se o modelo murino, procura-se investigar a influencia de infec96es sucessivas com clones de T. cruzi de um mesmo Biodema, com diferentes graus de virulencia, como fator agravante da miocardite cronica. Para o presente estudo foram utilizados camundongos sui9os infectados com os clones (Col-C1/Col-C5/Col-C8) sacrificados na fase aguda (14°; 20°; 25°; 30°) todos os grupos e na fase cronica da infec9ao (150 dias) para os grupos Col-C5 e Col-CB. O clone Col-C1 nao teve a fase cronica estudada devido a mortalidade total ao 30° dia de infec9ao. 0 grupo submetido a infec9ao triplice foi avaliado entre o 115° e o 130° dias da primeira infec9ao e aos 175 dias. 0 in6culo para todos os grupos experimentais (infec9ao unica e triplice infec9ao) foi de 5 x 104 formas tripomastigotas sanguicolas do T. cruzi por via intraperitoneal. Os camundongos sacrificados na fase aguda e na fase cronica em todos os grupos tiveram as sec96es de cora9ao e musculo esqueletico coletadas, fixadas e posteriormente processadas para o estudo histopatol6gico em sec9oes coradas pela Hematoxilina e Eosina ou pelo metodo do Picro-Sirius para investiga9ao da fibrose. lnvestigou-se tambem a resposta humeral pela sorologia (lmunofluorescencia indireta) e pela rea9ao de Elisa. Os camundongos sacrificados na fase aguda nos grupos experimentais submetidos a infec9ao unica demonstraram no geral lesoes inflamat6rias que variaram de discretas a moderadas, com necrose de miocelulas cardiacas e infiltrados inflamat6rios mononucleares e fibrose intersticial difusa e focal. Os camundongos com. triplice infec9ao demonstraram exacerba9ao das lesoes que variaram na maioria dos casos de moderadas a intensas no cora9ao e no musculo esqueletico, aos 25° e 30° dia ap6s a triplice infec9ao com rela9ao aos animais submetidos a infec9ao unica. Os camundongos com triplice infec9ao avaliados aos 175 dias havia miocardite cronica em todos os casos, com lesoes que variaram de discretas a moderadas. Os titulos sorol6gicos variaram de 1/10 a 1/640 nos grupos infec9ao unica e de 1/10 a 1/2560 nos animais submetidos a triplice infec9ao. A dosagem das imunoglobulinas lgM e lgG2a mostraram-se mais elevadas nos animais submetidos a triplice infec9ao e, a lgG1 abaixo dos niveis normais. A analise quantitativa dos infiltrados inflamat6rios (miocardio e do teste cutaneo de hipersensibilidade tardia) demonstrou diferen9a significativa nos animais submetidos a triplice infec9ao quando comparados com os animais submetidos a infec9ao unica. Os resultados confirmaram que os animais submetidos a multiplas infec96es apresentam exacerba9ao das lesoes inflamat6rias, maiores niveis de imunoglobulinas e de titulos sorol6gicos quando comparados aqueles com infec9ao unica. Concluimos que as reinfec9oes ao T. cruzi e um fator agravante para a evolu9ao da miocardiopatia chagasica no modelo murino da doen9a de Chagas.
Abstract
Reinfections with Trypanosoma cruzi in patients living in endemic areas have been mentioned as an aggravating factor of cardiac manifestations in Chagas disease. Experimental findings demonstrate that mice, when submitted to successive infections present more intense inflammatory reactions when compared to those with a single infection. The study of triple infection with strains from different Biodemas demonstrated exacerbation of tissue lesions in heart and skeletal muscle. In this work we seek to investigate the influence of successive infections with T. cruzi clones of the same Biodema, with different degrees of virulence using the murine model, as an aggravating factor of chronic myocarditis. For the present study, Swiss mice were infected with the clones (Col-C1(high virulence); Col-CS (medium virulence);Col-CB (low virulence). Infected mice of the three groups were sacrificed in the acute phase (14°; 20°; 25°; 30°) With the exception of mice infected with Col-C1, that did not survive until the chronic phase; those infected with Col-CS and Col-CB were sacrificed in the chronic hase of infection (150 days). The group submitted to triple infection was evaluated on the 11st , 130th and on the 175th day of the first infection. The inoculum for all experimental groups (single infection and triple infection) was of 5 x 104 tripomastigote blood forms of T. cruzi by intraperitonal route. In the acute and chronic phases sections of the heart and skeletal muscle were collected, fixed and then processed for the histopathological study in sections stained with Hematoxilin and Eosin or with the Picro-Sirius method, for fibrosis investigation. The humoral response was also investigated by serology (indirect immunofluorescence) and by Elisa reaction, and the delayed hypersensitivity by cutaneous test. The mice sacrificed at the acute phase, from the experimental groups submitted to single infection demonstrated, in general, inflammatory lesions that varied from mild to moderate, with necrosis of cardiac myocells and mononuclear inflammatory infiltrates, as well as diffuse and focal interstitial fibrosis. The mice with triple infection presented an exacerbation of lesions that varied, in the majority of the cases, from moderate to intense in the heart and skeletal muscle, on the 25th and 30th days after the triple infection, when compared with animals submitted to a single infection. Mice with triple infection evaluated on the 175th day had chronic myocarditis in all cases, with lesions that varied from mild to moderate. The serologic titles varied from 1/10 to 1/640 on the single infection groups and from 1/10 to 1/2560 on the animals submitted to triple infection. The dosage of immunoglobulins lgM and lgG2a were more elevated on animals submitted to triple infection. The evaluation of delayed type hypersensitivity was positive, with a higher intensity of the lesions in the period of 48 hours on the animals with triple infection and 24 hours on the single infection. Our results confirmed that the animals submitted to multiple infections presented exacerbation of inflammatory lesions, higher levels of immunoglobulin and serologic titles, when compared to those with single infection. We concluded that reinfections with T. cruzi are an aggravating factor for the evolution of Chagas myocardiopathy on the murine model of Chagas disease, even when reinfections were performed with clones of the same strain, with different degrees of virulence
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