Dinâmica das integrações de minicírculos de kDNA de Trypanosoma Cruzi no genoma hospedeiro

Dissertação (mestrado)—Universidade de Brasília, Faculdade de Medicina, Programa de Pós-Graduação em Patologia Molecular, 2016.

SEROPREVALENCE OF T. cruzi INFECTION IN BLOOD DONORS AND CHAGAS CARDIOMYOPATHY IN PATIENTS FROM THE COAL MINING REGION OF COAHUILA, MEXICO

Context and Objective: Chagas disease is considered a worldwide emerging disease; it is endemic in Mexico and the state of Coahuila and is considered of little relevance. The objective of this study was to determine the seroprevalence of T. cruzi infection in blood donors and Chagas cardiomyopathy in patients from the coal mining region of Coahuila, Mexico.Design and Setting: Epidemiological, exploratory and prospective study in a general hospital during the period January to June 2011.Methods: We performed laboratory tests ELISA and indirect hemagglutination in three groups of individuals: 1) asymptomatic voluntary blood donors, 2) patients hospitalized in the cardiology department and 3) patients with dilated cardiomyopathy.Results: There were three levels of seroprevalence: 0.31% in asymptomatic individuals, 1.25% in cardiac patients and in patients with dilated cardiomyopathy in 21.14%.Conclusions: In spite of having detected autochthonous cases of Chagas disease, its importance to local public health remains to be established as well as the details of the dynamics of transmission so that the study is still in progress.

Selective Decrease of Components of the Creatine Kinase System and ATP Synthase Complex in Chronic Chagas Disease Cardiomyopathy

Background: Chronic Chagas disease cardiomyopathy (CCC) is an inflammatory dilated cardiomyopathy with a worse prognosis than other cardiomyopathies. CCC occurs in 30 % of individuals infected with Trypanosoma cruzi, endemic in Latin America. Heart failure is associated with impaired energy metabolism, which may be correlated to contractile dysfunction. We thus analyzed the myocardial gene and protein expression, as well as activity, of key mitochondrial enzymes related to ATP production, in myocardial samples of end-stage CCC, idiopathic dilated (IDC) and ischemic (IC) cardiomyopathies. Methodology/Principal Findings: Myocardium homogenates from CCC (N = 5), IC (N = 5) and IDC (N = 5) patients, as well as from heart donors (N = 5) were analyzed for protein and mRNA expression of mitochondrial creatine kinase (CKMit) and muscular creatine kinase (CKM) and ATP synthase subunits aplha and beta by immunoblotting and by real-time RT-PCR. Total myocardial CK activity was also assessed. Protein levels of CKM and CK activity were reduced in all three cardiomyopathy groups. However, total CK activity, as well as ATP synthase alpha chain protein levels, were significantly lower in CCC samples than IC and IDC samples. CCC myocardium displayed selective reduction of protein levels and activity of enzymes crucial for maintaining cytoplasmic ATP levels. Conclusions/Significance: The selective impairment of the CK system may be associated to the loss of inotropic reserve observed in CCC. Reduction of ATP synthase alpha levels is consistent with a decrease in myocardial ATP generation through oxidative phosphorylation. Together, these results suggest that the energetic deficit is more intense in the myocardium of CCC patients than in the other tested dilated cardiomyopathies.

Heterozygosity for the S180L Variant of MAL/TIRAP, a Gene Expressing an Adaptor Protein in the Toll-Like Receptor Pathway, Is Associated with Lower Risk of Developing Chronic Chagas Cardiomyopathy

Background. Chagas disease is caused by the protozoan parasite Trypanosoma cruzi. Among T. cruzi-infected individuals, only a subgroup develops severe chronic Chagas cardiomyopathy (CCC); the majority remain asymptomatic. T. cruzi displays numerous ligands for the Toll-like receptors (TLRs), which are an important component of innate immunity that lead to the transcription of proinflammatory cytokines by nuclear factor-kappa B. Because proinflammatory cytokines play an important role in CCC, we hypothesized that single-nucleotide polymorphisms (SNPs) in the genes that encode proteins in the TLR pathway could explain differential susceptibility to CCC among T. cruzi-infected individuals. Methods. For 169 patients with CCC and 76 T. cruzi-infected, asymptomatic individuals, we analyzed SNPs by use of polymerase chain reaction-restriction fragment length polymorphism analysis for the genes TLR1, TLR2, TLR4, TLR5, TLR9, and MAL/TIRAP, which encodes an adaptor protein. Results. Heterozygous carriers of the MAL/TIRAP variant S180L were more prevalent in the asymptomatic group (24 [32%] of 76 subjects) than in the CCC group (21 [12%] of 169) (chi(2) = 12.6; P = .0004 [adjusted P (P(c)) = .0084]; odds ratio [OR], 0.31 [95% confidence interval {CI}, 0.16-0.60]). Subgroup analysis showed a stronger association when asymptomatic patients were compared with patients who had severe CCC (i.e., patients with left-ventricular ejection fraction <= 40%) (chi(2) = 11.3; P = .0008 [P(c) = .017]; OR, 0.22 [95% CI, 0.09-0.56]) than when asymptomatic patients were compared with patients who had mild CCC (i.e., patients with left-ventricular ejection fraction >40%) (chi(2) = 7.7; P = .005 [P(c) = .11]; OR, 0.33 [95% CI, 0.15-0.73]). Conclusion. T. cruzi-infected individuals who are heterozygous for the MAL/TIRAP S180L variant that leads to a decrease in signal transduction upon ligation of TLR2 or TLR4 to their respective ligand may have a lower risk of developing CCC.

Development of chronic cardiomyopathy in canine Chagas disease correlates with high IFN-gamma, TNF-alpha, and low IL-10 production during the acute infection phase

When infected with Trypanosoma cruzi, Beagle dogs develop symptoms similar to those of Chagas disease in human beings, and could be an important experimental model for a better understanding of the immunopathogenic mechanisms involved in chronic chagasic infection. This study evaluates IL-10, IFN-gamma and TNF-alpha production in the sera, culture supernatant, heart and cervical lymph nodes and their correlation with cardiomegaly, cardiac inflammation and fibrosis in Beagle dogs infected with T. cruzi. Pathological analysis showed severe splenomegaly, lymphadenopathy and myocarditis in all infected dogs during the acute phase of the disease, with cardiomegaly, inflammation and fibrosis observed in 83% of the animals infected by T. cruzi during the chronic phase. The data indicate that infected animals producing IL-10 in the heart during the chronic phase and showing high IL-10 production in the culture supernatant and serum during the acute phase had lower cardiac alterations (myocarditis, fibrosis and cardiomegaly) than those with high IFN-gamma and TNF-alpha levels. These animals produced low IL-10 levels in the culture supernatant and serum during the acute phase and did not produce IL-10 in the heart during the chronic phase of the disease. Our findings showed that Beagle dogs are a good model for studying the immunopathogenic mechanism of Chagas disease, since they reproduce the clinical and immunological findings described in chagasic patients. The data suggest that the development of the chronic cardiac form of the disease is related to a strong Th1 response during the acute phase of the disease, while the development of the indeterminate form results from a blend of Th1 and Th2 responses soon after infection, suggesting that the acute phase immune response is important for the genesis of chronic cardiac lesions. Crown Copyright (C) 2009 Published by Elsevier B.V. All rights reserved.

Synanthropic rodent reservoirs of Trypanosoma (Schizotrypanum) cruzi in the valley of Caracas, Venezuela

Direct blood examination and xenodiagnosis of 47 synanthropic rodents (Rattus rattus, R. norvegicus, Mus musculus) captured in the valley of Caracas, Venezuela, revealed trypanosomal infections in 12 R. rattus, 10 with T. lewisi and 2 with T. cruzi. Of the latter the course of parasitemia, the pleomorphism of the bloodstream trypomastigotes, tissue tropism in naturally and experimentally infected rats and mice, host mortality, morphology of fecal parasites in Rhodnius prolixus used for xenodiagnosis, and infectivity of the bug feces for NMRI mice, were all characteristic of Trypanosoma (Schizotrypanum) cruzi. One rat, with a patent parasitemia, had numerous nests of amastigotes in cardiac muscle and moderate parasitism of the smooth muscle of the duodenum and of skeletal muscle. Mice inoculated with fecal flagellates from the bugs had moderate tissue tropism in the same organs and also in the colon and pancreas. The possible role of R. rattus in the establishment of foci of Chagas’ disease in Caracas is discussed

Quantitative analysis of cardiac lesions in chronic canine chagasic cardiomyopathy

Lesions observed in chronic chagasic cardiopathy frequently produce electrocardiographic alterations and affect cardiac function. Through a computerized morphometrical analysis we quantified the areas occupied by cardiac muscle, connective and adipose tissues in the right atrium of dogs experimentally infected with Trypanosoma cruzi. All of the infected dogs showed chronic myocarditis with variable reduction levels of cardiac muscle, fibrosis and adipose tissue replacement. In the atrial myocardium of dogs infected with Be78 and Be62 cardiac muscle represented 34 and 50%, fibrosis 28 and 32% and adipose tissue 38 and 18%, respectively. The fibrosis observed was both diffuse and focal and mostly intrafascicular, either partially or completely interrupting the path of muscle bundles. Such histological alterations probably contributed to the appearance of electrocardiographic disturbances verified in 10 out 11 dogs which are also common in human chronic chagasic cardiopathy. Fibrosis was the most important microscopic occurrence found since it produces rearrangements of collagen fibers in relation to myocardiocytes which causes changes in anatomical physiognomy and mechanical behavior of the myocardium. These abnormalities can contribute to the appearance of cardiac malfunction, arrythmias and congestive cardiac insufficiency as observed in two of the analyzed dogs. Strain Be78 caused destruction of atrial cardiac muscle higher than that induced by strain Be62.

Distribuição e infecção de triatomíneos por Trypanosoma do tipo cruzi na Ilha de São Luís-Maranhão

São apresentados dados referentes às espécies e infecção natural de triatomíneos na Ilha de São Luis-MA. Dos triatomíneos coletados foram encontradas as seguintes espécies: Rhodnius pictipes Rhodnius neglectus, Rhodnius nasutus, Triatoma rubrofasciata, Panstrongylus lignarus e Panstrogylus geniculatus. A presença de infecção natural por Trypanosoma do tipo cruzi foi detectada em 19,7% do total de triatomíneos sendo o R. pictipes a espécie mais homogênea em distribuição na Ilha e com índice de infecção natural de 38,8%.

Echocardiographic parameters associated with pulmonary congestion in Chagas cardiomyopathy

INTRODUCTION: Discrepancy between the intensity of pulmonary congestion and the grade of cardiomegaly seems to be a common finding of Chagas cardiomyopathy, in spite of significant systolic dysfunction of the left ventricle. Its mechanism has not been established. The aim of this study was to investigate pulmonary congestion and to analyze if it correlated with Doppler echocardiographic parameters in patients with Chagas dilated cardiomyopathy. METHODS: Fifty-five patients with positive serology tests for Trypanosoma cruzi and Chagas dilated cardiomyopathy were studied. Chest x-rays, Doppler echocardiogram and plasmatic brain natriuretic peptide levels were obtained in all patients. The degree of pulmonary venous vessels changes on chest x-ray was graded using a pulmonary congestion score, and then compared to Doppler echocardiographic parameters. RESULTS: Mean age was 48.5 ± 11.2 years and 29% were women. The majority (95%) of patients were in NYHA functional class I and II. Mild pulmonary congestion by chest x-ray was found in 80% of the patients. In a multivariate analysis, left ventricular ejection fraction, right ventricular TEI index and the color M-mode velocity correlated with the degree of pulmonary congestion. CONCLUSIONS: Pulmonary venous changes on chest x-rays are frequent, but usually mild in patients with Chagas dilated cardiomyopathy. The degree of pulmonary congestion correlates with Doppler echocardiographic left and right ventricular dysfunction and with color M-mode velocity.

Pathogenesis of chronic Chagas cardiomyopathy: the role of coronary microvascular derangements

There is ample experimental and clinical evidence of functional and structural microvascular abnormalities occurring in patients with Chagas cardiomyopathy, possibly due to the inflammatory process and/or autonomic disturbances caused by Trypanosoma cruzi infection. Those microvascular derangements are likely to constitute at least an ancillary factor that potentiates and amplifies the chronic inflammation in myocardial tissue. It is possible to devise appropriate therapeutic interventions aimed at reverting or slowing the progression of the microvascular abnormalities to positively affect the natural history of Chagas cardiomyopathy.

Defective production of interleukin 2 in patients with Chagas' disease: purified IL-2 augments in vitro response in patients with chagasic cardiomyopathy

The production of interleukin 2 (IL-2) by peripheral blood mononuclear cells, from patients with different clinical forms of Chagas disease and healthy controls, was evaluated after stimulation with Trypanosoma cruzi antigen, PPD and PHA. PHA induced higher production of IL-2 in infected patients than healthy controls. No diferences were found between infected groups. With PPD the trend was similar, the only difference was that asymptomatic infected patients (INF) showed higher levels of IL-2 production than patients with cardiomyopathy (CDM). With T. cruzi antigen, most patients showed little or no IL-2 production at 24 hr, a peak at 48 hr and an abrupt fall at 72 hr. A similar pattern of IL- 2 production was observed in INF and CDM. To evaluate the physiologic relevance of the deficit in IL-2 production, we studied the effect of non-mitogenic concentratios of IL-2 in the proliferative response to specific antigens. The addition of IL-2 only enhanced the proliferative response of CDM patients. These observations suggest that patients suffering Chagas' disease, particularly CDM, have a significant reduction in the capacity to produce IL-2. These findings could be of importance in the pathogenesis of Chagas' disease.

Immunopathology of cardiomyopathy in the experimental Chagas disease

The mechanisms by which Trypanosoma cruzi causes cardiomyopathy and induces neuronal destruction are discussed in this paper. The results suggest that autoimmunity in the chronic phase is the main cause of the progressive cardiac destruction, and that autoreactivity is restricted to the CD4+ T cell compartment. During the acute phase, the neuronal and cardiac fiber destruction occurs when ruptured parasite nests release T. cruzi antigens that bind to the cell surface in the vicinity which become targets for the cellular and humoral immune response against T. cruzi. The various factors involved in the genesis of autoimmunity in chronic T. cruzi infection include molecular mimicry, presentation of self-antigens and imbalance of immune regulation.

Increased plasma levels of tumor necrosis factor-alpha in asymptomatic/"indeterminate" and Chagas disease cardiomyopathy patients

We compared plasma tumor necrosis factor-alpha (TNF-alpha) levels among asymptomatic/"indeterminate" Chagas disease patients (ASY) and patients across the clinical spectrum of chronic Chagas disease cardiomyopathy (CCC). Idiopathic dilated cardiomyopathy (DCM) patients and normal controls (NC) were included as controls. ASY Chagas disease patients had significantly higher plasma TNF-alpha levels than NC. TNF-alpha levels among severe CCC patients with significant left ventricular (LV) dysfunction were similar to those of DCM patients, showing average 2-fold higher levels than CCC patients without LV dysfunction and ASY patients, and 8-fold higher levels than NC. In Chagas disease, chronic TNF-a production prior to heart failure may play a role in CCC progression.

Brain natriuretic peptide and left ventricular dysfunction in chagasic cardiomyopathy

Global left ventricular (LV) systolic dysfunction is the strongest predictor of morbidity and mortality in Chagas disease. Echocardiography is considered the gold standard for the detection of LV dysfunction, but not always available in endemic areas where chagasic cardiomyopathy is most common. Brain natriuretic peptide (BNP) is a neurohormone that has been recently described as a simple and inexpensive diagnostic and prognostic marker for patients with congestive heart failure. Chagasic patients (n = 63) and non-infected healthy individuals (n = 18) were recruited prospectively and underwent complete clinical examination, echocardiography and 24-h Holter monitoring. BNP was measured from thawed plasma samples using the Triage BNP test. We observed high levels of BNP in association with depression of LV ejection fraction, with increase of LV end-diastolic diameter and with LV premature complexes. An elevated concentration of BNP, defined as a concentration of 60 pg/ml or more, had a sensitivity of 91.7%, specificity of 82.8%, positive predictive value of 52.4%, and negative predictive value of 98% for detecting LV dysfunction (LV ejection fraction < 40%).BNP measurement using a simple, relatively inexpensive and rapid test has a promising role in identifying LV dysfunction associated with chagasic cardiomyopathy. Equally important, patients with Trypanosoma cruzi infection who have low levels of BNP level in plasma have a very low likelihood of severe cardiac involvement, and echocardiography is probably not necessary.

Clinical characterization of chronic chagasic cardiomyopathy in dogs

On the American continent, almost 15 million people are affected by Chagas disease, resulting in important economic and social damages. Dogs are considered to be an excellent experimental model to study Chagas' disease; as a result, in this research, the characterization of cardiovascular abnormalities was performed in dogs experimentally infected with Trypanosoma cruzi (the Colombian strain) that were at chronic stage. Thirteen adult female dogs were evaluated by electrocardiographic, echocardiographic, hematological and biochemical analyses in the chronic phase. For the electrocardiographic studies, respiratory sinus arrhythmia was the predominant rhythm during the entire research period (49.55% to 67%), with a low prevalence of right bundle branch block (0-13%) and first-degree atrioventricular block (0-14%). The spectral Doppler echocardio-graphy showed E and A mitral wave reversal (0.71±0.17), confirming the diastolic dysfunction present in all dogs. An increase in the enzymes activities was detected in the serum analysis, indicating myocardial injury by the infection. Six dogs died during the follow-up. In this way, the clinical characterization of experimentally infected dogs, as described here, increases the knowledge and allows for recognition of the behavioural modifications present in Chagas' disease in affected dogs.