Avaliação do tratamento com benznidazol sobre a infecção experimental de cães por cepas do Trypanosoma cruzi pertencentes a DTU I (Colombiana) ou DTU II (Y).

Programa de Pós-Graduação em Ciências Biológicas. Núcleo de Pesquisas em Ciências Biológicas, Pró-Reitoria de Pesquisa de Pós Graduação, Universidade Federal de Ouro Preto.Estima-se que 7 a 8 milhões de indivíduos estejam infectados pelo Trypanosoma cruzi, agente etiológico da doença de Chagas (DC) (W.H.O., 2014), em especial na América Latina, onde essa doença é endêmica (Schofield e Dias, 1999). Desde a descoberta da DC, em 1909 por Carlos Chagas, a heterogeneidade do parasito foi considerada um fator relacionado às diferentes formas clínicas da doença. A classificação dos subtipos do T. cruzi foi determinada conforme as características biológicas, parasitismo tecidual, morfologia, virulência e patogenicidade do parasito na infecção murina (Brener, 1973; Andrade, 1974). Sendo o T. cruzi foi agrupado em três fenótipos, denominados como Biodemas I, II e III (Andrade e Magalhães, 1997). De modo geral, o Biodema I, correspondente ao Zimodema Z2b, foi caracterizado por apresentar cepas predominantemente de forma fina e macrofagotrópica na fase inicial da infecção, com alta virulência com 100% de mortalidade em 12 dias, e pico de parasitemia e mortalidade entre o 7º e o 11º dia pós-infecção (d.p.i.). O Biodema II, correspondente ao Zimodema Z2, apresenta cepas de forma predominantemente larga, miotrópica, com tropismo pelo músculo cardíaco e taxa de multiplicação lenta, cujo pico de parasitemia ocorre de maneira irregular entre o 12º e o 20º d.p.i. Cepas pertencentes a este tipo de Biodema podem apresentar diferentes níveis de virulência. Já o Biodema III, correspondente ao Zimodema Z1, exibe cepas de forma predominantemente larga e miotrópica, com envolvimento de músculo esquelético, pico de parasitemia tardio entre o 25º e o 30º d.p.i. e baixa mortalidade aos 30 dias (Andrade e Magalhães, 1997). Posteriormente, o parasito foi agrupado em dois grupos principais denominados como T. cruzi I e T. cruzi II, que anteriormente se encaixavam na classificação como Biodema III (Zimodema Z1) e Biodema II (Zimodema Z2), respectivamente. As cepas híbridas e com caracterização incerta foram denominadas apenas como T. cruzi até a realização de novos estudos (Anonymous, 1999). Recentemente, as populações de T. cruzi foram reagrupadas, conforme a expressão fenotípica e as características moleculares, em unidade de tipagem (do inglês “Discrete Typing Units” - DTU), denominadas TcI, TcII, TcIII, TcIV, TcV e TcVI, as quais foram caracterizadas por marcadores genéticos (Zingales et al., 2009). Estudos geográficos e epidemiológicos apontam distribuições distintas dessas DTUs, sendo os TcI e TcII mais abrangentes, visto que o TcI é prevalente na América Central e na região norte do Brasil, e o TcII é encontrado predominantemente na América do Sul (Zingales et al., 2009). Essas distintas DTUs, aparentemente, apresentam diferentes padrões de tropismo e parasitismo tecidual (Vago et al., 2000). O padrão do parasitismo tecidual e o grau de resistência ao tratamento por sua vez tem relação com as propriedades biológicas do parasito, uma vez que de modo geral a infecção com o T. cruzi I e o T. cruzi II estão relacionadas com casos de cardiomiopatia chagásica crônica (CCC). Ainda, a DTU I foi considerada resistente frente ao único fármaco disponível comercialmente no Brasilpara o tratamento da DC, o Benznidazol (Bz). Por outro lado, o T. cruzi II foi considerado macrofagotrópico e parcialmente susceptível ao tratamento com Bz (Filardi e Brener, 1987; Toledo et al., 2003; Toledo et al., 2004). Em estudos clínicos randomizados, o tratamento com Bz na fase aguda favorece a redução da carga parasitária e sororeversão dos pacientes tratados (de Andrade et al., 1996; Coura et al., 1997; Sosa-Estani et al., 1998). Sugere-se que o tratamento etiológico com Bz deva ser iniciado nos estágios iniciais da DC, pois quando iniciado na fase aguda pode alcançar níveis de 70% de cura (Rodrigues Coura e de Castro, 2002). Outros trabalhos tem revelado que a intervenção com o tratamento de pacientes na forma indeterminada melhora o prognóstico da doença (Lana et al., 1992; Macchado-de-Assis et al., 2013). O Bz ativa a resposta imune inata com aumento dos níveis de monócitos, todavia com diminuição dos monócitos TNF-γ + e IL-12+ (Sathler-Avelar et al., 2009). Na resposta imune adaptativa, o tratamento com Bz favorece mecanismos imunomoduladores mediados por IL-10 produzido por linfócitos T CD4 e B, além de aumentar o número de células NK ativas no sangue periférico, que tem participação na produção e citocinas pró-inflamatórias e moduladoras (Sathler-Avelar et al., 2006). Isto demonstra a importância da inter-relação entre a resposta imune inata e adaptativa no controle da resposta imune exacerbada, que poderá acarretar na lesão tecidual (Sathler-Avelar et al., 2009). Por outro lado, os casos de falha terapêutica, aparentemente, dependem de mecanismos que interferem na expressão de genes relacionados à resistência de algumas cepas do parasito (Campos et al., 2014). Apesar de estar elucidado que o Bz alcança níveis de cura importantes nos pacientes tratados na fase aguda da DC, ainda se faz necessário compreender a participação das diferentes DTUs de T. cruzi na patogênese da doença, bem como elucidar as alterações imunológicas e histopatológicas cardíacas frente ao tratamento na infecção com cepas do T. cruzi que apresentam diferentes graus de susceptibilidade ao tratamento com Benznidazol. Portanto, neste trabalho avaliamos a infecção com duas cepas de DTUs distintas, a cepa Y, caracterizada como TcII e considerada parcialmente resistente ao tratamento com Bz no modelo murino e susceptível no modelo canino, e a cepa Colombiana, classificada como TcI e resistente à terapia com Bz nos modelos murino e canino (Filardi e Brener, 1987; Veloso et al., 2001; Guedes et al., 2002).Trypanosoma cruzi infection may be caused by different strains of distinct discrete typing units (DTUs), characterized by different biological and genetic patterns, which result in variable clinical forms of chronic Chagas disease. These biological differences of the parasite are apparently related to the distinct patterns of resistance to the Benznidazole treatment, the only drug available for Chagas disease treatment in Brazil. In randomized clinical trials, Benznidazole treatment presents a higher cure rate in the acute phase, with reduction of the parasite load and negative seroreversion of treated and cured patients, but there are still questions about the immunopathological changes resulting from this treatment. This study evaluates the immune response and cardiac lesions in dogs experimentally infected with different strains of distincts T. cruzi DTUs that showing different degrees of susceptibility to treatment with Benznidazole, namely Y and Colombian from TcI and TcII DTU, respectively. For this, mongrel dogs were experimentally infected with 2000 Y or Colombian T. cruzi trypomastigotes and undergoing Benznidazole treatment at oral dose of 7 mg/kg, divised in two by day, for 45 days. The infection with the Y strain led to increased levels of alanine aminotransferase and aspartate aminotransferase, leucopoenia, thrombopoenia, inversion of the ratio of CD4+/CD8+ T-lymphocytes and alterations in monocyte number. In addition, Y strain infection stimulated the production of IFN-γ by CD4+ and CD8+ T-lymphocytes and IL-4 by CD8+ T-cells. In the chronic phase, significant heart inflammation and fibrosis were observed. During infection with the Colombian strain, increased levels of alanine aminotransferase, erythrocytes, haematocrit and haemoglobin were observed. In addition, CD8+ T-lymphocytes isolated from the peripheral blood produced higher levels of IL-4. The latter results suggests that during the acute phase, infection with the Colombian strain may remain unnoticed by circulating mononuclear cells. In the chronic phase, a significant increase in the number of inflammatory cells was detected in the right atrium, demonstrating that strains of different DTUs interact differently with the host. When was evaluated the Benznidazole treatment of dogs infected with the Y strain it was observed increase levels of alanine aminotransferase, urea and hemoglobin, lymphopenia, neutropenia, monocytopenia and inversion of the ratio of CD4+/CD8+ T-lymphocytes. The infection with the Y strain and Benznidazole treatment stimulated the production of IFN-γ and IL-4 by CD4+ T-lymphocytes. In dogs infected with the Colombian strain and Benznidazole treatment, it was observed increase of alanine aminotransferase and hemoglobin, and decrease the monocyte number. So the treatment in Colombian infection controlled the increased amount of red blood cells. Moreover, in chronic phase, dogs with Y and with Colombian strains infection and Benznidazole treatment do not showed significant difference in the number of inflammatory cells and collagen area in the right atrium compared to the not infected and treated group. Therefore, the Benznidazole treatment despite having changed some cell populations in peripheral blood, had significant participation for decrease the IgG level and the inflammation and cardiac fibrosis. So, in this work, the Y strain was apparently first recognized by the immune system, while the Colombian strain escaped from the immune response in the acute phase of Chagas disease, which would impact the progression of cardiomyopathy in the chronic phase. Moreover, the Benznidazole treatment in acute experimental infection controlled the cardiac damage in chronic phase

Correction: Enteric Neuronal Damage, Intramuscular Denervation and Smooth Muscle Phenotype Changes as Mechanisms of Chagasic Megacolon: Evidence from a Long-Term Murine Model of Tripanosoma cruzi Infection.

[This corrects the article DOI: 10.1371/journal.pone.0153038.]

Enteric neuronal damage, intramuscular denervation and smooth muscle phenotype changes as mechanisms of chagasic megacolon : evidence from a long - term murine model of Tripanosoma cruzi infection.

We developed a novel murine model of long-term infection with Trypanosoma cruzi with the aim to elucidate the pathogenesis of megacolon and the associated adaptive and neuromuscular intestinal disorders. Our intent was to produce a chronic stage of the disease since the early treatment should avoid 100% mortality of untreated animals at acute phase. Treatment allowed animals to be kept infected and alive in order to develop the chronic phase of infection with low parasitism as in human disease. A group of Swiss mice was infected with the Y strain of T. cruzi. At the 11th day after infection, a sub-group was euthanized (acute-phase group) and another sub-group was treated with benznidazole and euthanized 15 months after infection (chronic-phase group). Whole colon samples were harvested and used for studying the histopathology of the intestinal smooth muscle and the plasticity of the enteric nerves. In the acute phase, all animals presented inflammatory lesions associated with intense and diffuse parasitism of the muscular and submucosa layers, which were enlarged when compared with the controls. The occurrence of intense degenerative inflammatory changes and increased reticular fibers suggests inflammatoryinduced necrosis of muscle cells. In the chronic phase, parasitism was insignificant; however, he architecture of Aüerbach plexuses was focally affected in the inflamed areas, and a significant decrease in the number of neurons and in the density of intramuscular nerve bundles was detected. Other changes observed included increased thickness of the colon wall, diffuse muscle cell hypertrophy, and increased collagen deposition, indicating early fibrosis in the damaged areas. Mast cell count significantly increased in the muscular layers. We propose a model for studying the long-term (15 months) pathogenesis of Chagasic megacolon in mice that mimics the human disease, which persists for several years and has not been fully elucidated. We hypothesize that the long-term inflammatory process mediates neuronal damage and intramuscular and intramural denervation, leading to phenotypic changes in smooth muscle cells associated with fibrosis. These long-term structural changes may represent the basic mechanism for the formation of the Chagasic megacolon

The TcI and TcII Trypanosoma cruzi experimental infections induce distinct immune responses and cardiac fibrosis in dogs

Trypanosoma cruzi infection may be caused by different strains with distinct discrete typing units (DTUs) that can result in variable clinical forms of chronic Chagas disease. The present study evaluates the immune response and cardiac lesions in dogs experimentally infected with different T. cruzi strains with distinct DTUs, namely, the Colombian (Col) and Y strains of TcI and TcII DTU, respectively. During infection with the Col strain, increased levels of alanine aminotransferase, erythrocytes, haematocrit and haemoglobin were observed. In addition, CD8+ T-lymphocytes isolated from the peripheral blood produced higher levels of interleukin (IL)-4. The latter suggests that during the acute phase, infection with the Col strain may remain unnoticed by circulating mononuclear cells. In the chronic phase, a significant increase in the number of inflammatory cells was detected in the right atrium. Conversely, infection with the Y strain led to leucopoenia, thrombopoenia, inversion of the ratio of CD4+/CD8+ T-lymphocytes and alterations in monocyte number. The Y strain stimulated the production of interferon-γ by CD4+ and CD8+ T-lymphocytes and IL-4 by CD8+ T-cells. In the chronic phase, significant heart inflammation and fibrosis were observed, demonstrating that strains of different DTUs interact differently with the host

Histopathological features and evidence of parasitism in the colons of Swiss mice infected with 50,000 trypomastigotes of the Y strain of <i>T</i>. <i>cruzi</i>.

<p>A, C, E, G, and H represent acute-phase aspects (11 d.a.i.). B, D, F, and I represent chronic-phase aspects (15 m.a.i.). (A) The colons of the control group of paired animals in the acute phase present normal cellularity and thickness. (B) In the chronic phase, no alterations were observed in the control group. (C) In acute-phase infected animals, a mononuclear inflammatory infiltrate was observed in the submucosal and muscular layers (arrow). In the myenteric plexus (arrowhead) and in the inner muscular layer, there is evidence of muscle fiber necrosis (thick arrow). This transmural pattern is merged with non-inflamed areas (not shown). (D) In contrast, in the chronic-phase infected group, mononuclear infiltrates are focal and more intense in the outer muscular layer in the periganglionar and perivascular areas (arrow). (E) Intense parasitism (inset,arrow) is associated with inflammatory infiltrates in the acute phase. (F) In the chronic phase, parasites are scarce and not associated with inflammatory foci (inset). (G) The acute-phase infected group showed strong iNOS positivity associated with inflammatory and degenerative changes in the myenteric plexus (arrowheads) compared (I) with the weak staining in inflammatory cells (arrow, and inset) in the chronic-phase infected group. A possible glial cell of ganglia (arrowhead) is also stained. (H) In addition, a greater amount of reticular fibers with thickened areas around the ganglia (arrow) were present in the acute phase. The results represent two independent experiments. A, B, C, D, I, H Magnification at 20x. Scale bar corresponds to 20 μm. E, F Magnification at 4x. Scale Bar corresponds to 100 μm. G Magnification at 40x. Scale bar corresponds to 10 μm. HE staining in A, B, C and D. Immunohistochemistry with anti-<i>T</i>. <i>cruzi</i> antibody in E, and F, and insets of E and F. Immunohistochemistry with anti-iNOS antibody in G and I. Silver Gomori staining in H.</p

Morphometric evaluation of colon wall thickness in Swiss mice in the acute and chronic phases of infection with 50,000 trypomastigotes of the Y strain of <i>T</i>. <i>cruzi</i>.

<p>(A and B) The chronic-phase infected group presented increased thickness of the total colon wall compared with the acute-phase infected group and with the control group. Values represent means ± SEM. *p = 0.001, **p = 0.026. N = 10 for each experimental and control group. The results represent two independent experiments. Black: infected groups; white: control groups.</p