Immunological response to re-infections with clones of the Colombian strain of Trypanosoma cruzi with different degrees of virulence: influence on pathological features during chronic infection in mice

Re-infections with Trypanosoma cruzi are an aggravating factor for Chagas disease morbidity. The Colombian strain of T. cruzi represents multiclonal populations formed by clonally propagating organisms with different tropisms and degrees of virulence. In the present study, the influence of successive inoculations with clones of the Colombian strain, exhibiting different degrees of virulence, on chronic myocarditis and the humoral and cellular immune responses (Col-C1 high virulence, Col-C8 medium virulence and Col-C5 low virulence) were demonstrated. Mice from three groups with a single infection were evaluated during the acute (14th-30th day) and chronic phases for 175 days. An immunofluorescence assay, ELISA and delayed type hypersensitivity (DTH) cutaneous test were also performed. Mice with a triple infection were studied on the 115th-175th days following first inoculation. The levels of IgM and IgG2a were higher in the animals with a triple infection. DTH showed a higher intensity in the inflammatory infiltrate based on the morphometric analysis during a 48 h period of the triple infection and at 24 h with a single infection. The histopathology of the heart demonstrated significant exacerbation of cardiac inflammatory lesions confirmed by the morphometric test. The humoral responses indicate a reaction to the triple infection, even with clones of the same strain.

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

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.

In vitro and in vivo experimental models for drug screening and development for Chagas disease

Chagas disease, a neglected illness, affects nearly 12-14 million people in endemic areas of Latin America. Although the occurrence of acute cases sharply has declined due to Southern Cone Initiative efforts to control vector transmission, there still remain serious challenges, including the maintenance of sustainable public policies for Chagas disease control and the urgent need for better drugs to treat chagasic patients. Since the introduction of benznidazole and nifurtimox approximately 40 years ago, many natural and synthetic compounds have been assayed against Trypanosoma cruzi, yet only a few compounds have advanced to clinical trials. This reflects, at least in part, the lack of consensus regarding appropriate in vitro and in vivo screening protocols as well as the lack of biomarkers for treating parasitaemia. The development of more effective drugs requires (i) the identification and validation of parasite targets, (ii) compounds to be screened against the targets or the whole parasite and (iii) a panel of minimum standardised procedures to advance leading compounds to clinical trials. This third aim was the topic of the workshop entitled Experimental Models in Drug Screening and Development for Chagas Disease, held in Rio de Janeiro, Brazil, on the 25th and 26th of November 2008 by the Fiocruz Program for Research and Technological Development on Chagas Disease and Drugs for Neglected Diseases Initiative. During the meeting, the minimum steps, requirements and decision gates for the determination of the efficacy of novel drugs for T. cruzi control were evaluated by interdisciplinary experts and an in vitro and in vivo flowchart was designed to serve as a general and standardised protocol for screening potential drugs for the treatment of Chagas disease.

O modelo murino da miocardiopatia crônica chagásica / The murine of chagas chronic cardiomiopathy

Camundongos isogênicos das linhagens AKR e A/J e camundongos suiços näo isogênicos, cronicamente infectados com cepas do Trypanosoma cruzi de três diferentes Tipos, desenvolveram uma miocardite crônica difusa e focal com fibrose intersticial. Estas lesöes ocorreram em graus variáveis de caso para caso, representando o espéctro de lateraçöes observadas na doença de Chagas humana. Os camundongos cronicamente infectados apresentaram alta incidência de alteraçöes eletrocardiográficas predominando os distúrbios da conduçäo intraventricular, os bloqueios AV de 1§ e 2§ graus e distúrbios do rítmo cardíaco, semelhantes às observadas na cardiopatia chagásica humana. Ao lado disto, os animais apresentaram reaçöes sorológicas específicas consistentemente positivas, pelo teste de imunofluorescência indireta e de hemaglutinaçäo indireta. Estes achados indicam vários pontos de correlaçäo entre a doença de Chagas humana e a cardiopatia que se desenvolve no modelo murino da infecçäo crônica pelo Trypanosoma cruzi. Foi observada uma nítida influência da cepa do Trypanosoma cruzi na determinaçäo das lesöes da fase crônica tendo sido mais elevada a incidência de lesöes inflamatórias, ao mesmo tempo focais e difusas, em aurículas, nos camundongos isogênicos infectados com diversas cepas do Tipo III (Colombiana, PMN e as cepas de Montlvania). A influência da cepa na intensidade das lesöes inflamatórias e fibróticas do miocárdio foi comprovada pela análise estatística (teste do X²) que demonstrou lesöes significantemente mais intensas nos camundongos infectados com a cepa Colombiana. Por outro lado näo foi encontrada relaçäo significante entre a intensidade das lesöes e a duraçäo da infecçäo ou o nível dos inóculos através a análise estatística pelo coeficiente de Kruskal e goodman...