Do Brazilian scientific journals promote the adherence of Chagas disease researchers to international ethical principles?

The ethical aspects of the Brazilian publications about human Chagas disease (CD) developed between 1996 and 2010 and the policy adopted by Brazilian medical journals were analyzed. Articles were selected on the SciELO Brazil data basis, and the evaluation of ethical aspects was based on the normative contents about ethics in research involving human experimentation according to the Brazilian resolution of the National Health Council no. 196/1996. The editorial policies of the section “Instructions to authors” were analyzed. In the period of 1996-2012, 58.9% of articles involving human Chagas disease did not refer to the fulfillment of the ethical aspects concerning research with human beings. In 80% of the journals, the requirements and confirmation of the information about ethical aspects in the studies of human CD were not observed. Although a failure in this type of service is still observed, awareness has been raised in federal agencies, educational institutions/research and publishing groups to standardize the procedures and ethical requirements for the Brazilian journals, reinforcing the fulfillment of the ethical parameters, according to the resolution of NHC no. 196/1996

Cyclic AMP decreases the production of NO and CCL2 by macrophages stimulated with Trypanosoma cruzi GPI-mucins.

Glycosylphosphatidylinositol-anchored mucinlike glycoproteins (tGPI-mucin) present on the surface of the cellular membrane of Trypanosoma cruzi forms activate toll-like receptors 2 (TLR2) on the surface of immune cells and induce the release of several mediators of inflammation which may be relevant in the context of Chagas disease. Here, we evaluated the ability of tGPI-mucins to activate murine peritoneal macrophages to induce nitric oxide (NO) and monocyte chemoattractant protein-1 (MCP-1/CCL2). We also investigated the ability of compounds which increase or mimic cyclic adenosine monophosphate (AMP) to modulate the production of NO and CCL2. Our data show that elevation of intracellular levels of cyclic AMP prevents the release of NO and CCL2 induced by tGPI-mucins in macrophages. Overall, the release of CCL2 was decreased to a greater extent and at lower concentrations of cyclic AMP-modifying agents than the production of NO. It is suggested that the elevation of cyclic AMP during T. cruzi infection may modify the release of proinflammatory mediators and alter significantly the course of T. cruzi infection

Enalapril prevents cardiac immune-mediated damage and exerts anti- Trypanosoma cruzi activity during acute phase of experimental Chagas disease.

Chagas heart disease (CHD), caused by Trypanosoma cruzi infection, is a significant cause of morbidity and mortality in South and Central America. Enalapril, an angiotensin converting enzyme (ACE) inhibitor, is an important drug used to ameliorate heart functional capacity and its remodelling in individuals presenting CHD. In this study, we evaluated the effects of enalapril on systemic and cardiac immune response during experimental acute CHD. C57BL ⁄ 6 mice infected with 50 trypomastigote forms of T. cruzi (Colombian strain) were treated daily with enalapril (25 mg ⁄ kg) and, after 30 days, a reduction in seric levels of IFNgamma, TNF-alpha, CCL5⁄RANTES and nitric oxide, but not in that of IL-10, was detected. This imbalance of cytokines reflects in a reduction of heart mononuclear infiltration and in an increasing of cardiac mast cells. Enalapril also presents a new and interesting in vitro and in vivo anti- T. cruzi activity probably acting on parasite oxidative pathway via cytochrome-P450. Our data show that enalapril exerts an important anti-T. cruzi and anti-inflammatory activity during acute CHD reducing inflammatory cells and, possibly, preventing fibrotic process in the chronic phase. Nevertheless, further studies are still necessary to clarify the mechanisms by which this drug is acting on the parasites and on the immune pathways

Cardiomyopathy prognosis after benznidazole treatment in chronic canine Chagas' disease.

Objectives: To evaluate the effects of benznidazole on Chagas’ disease cardiac prognosis using an experimental dog model of infection. Methods: A total of 28 dogs were divided into three groups: 10 were infected with Trypanosoma cruzi and treated benznidazole during the chronic phase, 10 were infected but untreated, and 8 were non-infected/ healthy. The trypanocidal efficacy was measured by parasite kDNA detection in blood and cardiac tissue samples. The effects of benznidazole in ameliorating the cardiac systolic function were evaluated by echodopplercardiogram. Results: The benznidazole initially induced a potent suppression of parasitaemia in treated animals. However, 12 months post-treatment, the parasite kDNA detections were similar between infected groups. In the baseline echocardiographic parameters there was no variation among all animals. Similarly, 1 month post-treatment there was no significant difference among healthy and infected animals with regard to systolic function. At 12 months post-treatment, an increase in cardiac chamber size related to cardiomegaly was detected among treated and untreated animals, but not in the healthy controls. Interestingly, in spite of both groups of infected animals developing a decrease in their systolic cardiac function, this decline was slightly less in the treated animals. We also evaluated levels of tumour necrosis factor-a and interleukin-10 in peripheral blood mononuclear cell culture supernatant. Cytokine profiles were similar between infected animal groups and correlated with alterations in cardiac function. Conclusions: The temporary suppression of the T. cruzi infection induced by benznidazole treatment was efficient in reducing systolic cardiac function alterations, but not in preventing the development of cardiomyopathy

Carvedilol : decomposition kinetics and compatibility with pharmaceutical excipients.

Carvedilol (CARVE) is an important cardiovascular drug with limited bioavailability. To improve its therapeutic performance, the investigation of new dosage forms is of great interest due its relevance in clinical applications. Therefore, the aim of this work was to evaluate the stability of CARVE and its drug–excipient compatibility to support its pharmaceutical development. Kinetic analysis under isothermal conditions using thermogravimetry was performed to determine the activation energy of CARVE through an Arrhenius plot. Differential scanning calorimetry, Fourier transform infrared spectroscopy, and optical microscopy were used to test binary mixtures of CARVE and selected excipients. The activation energy of CARVE was 81.2 kJ mol-1, and from the compatibility studies, all the excipients showed strong thermal interactions, presenting changes in the melting profile of the drug. In addition, analytical assays revealed no physical or chemical changes; because of this, all eight excipients studied are considered compatible and are recommended in formulations containing CARVE. All the evidence together attests to the low chemical reactivity of CARVE and provides useful information for the development of new pharmaceutical formulations containing CARVE

Benznidazole therapy during acute phase of chagas disease reduces parasite load but does not prevent chronic cardiac lesions.

The goals of this study were to evaluate the efficacy of benznidazole (Bz) treatment in decreasing of the parasitic load during the acute phase of experimental Chagas disease and to analyze its influence in the development of cardiac chronic alterations in mice inoculated with drug-resistant Trypanosoma cruzi strains. Our results showed that the early Bz treatment (started at 4th day of infection) was efficient in reducing the parasite load in animals from both acute and chronic phase of the infection. Moreover, this reduction in the parasite load could not be associated with the intensity of the cardiac chronic lesions. The histopathological evaluation of cardiac tissue of Bz-treated mice showed three different patterns of response: (1) presence of a small number of inflammatory cells and fibrotic area similar to noninfected mice; (2) similar intensity of inflammatory infiltrate and smaller fibrotic area in relation to nontreated animals; (3) similar intensity of inflammatory infiltrated and fibrosis area among the Bz-treated and nontreated animals. Each specific pattern was obtained with different T. cruzi strain, suggesting that the pattern of the heart lesions in chronic phase of Bz-treated animals was T. cruzi strain dependent but not related with drug resistance levels

Trypanosoma cruzi : genetic diversity influences the profile of immunoglobulins during experimental infection.

The clonal evolution model postulated for Trypanosoma cruzi predicts a correlation between the phylogenetic divergence of T. cruzi clonal genotypes and their biological properties. In the present study, the linkage between phylogenetic divergence of the parasite and IgG, IgG1, IgG2a and IgG2b response has been evaluated during the acute and chronic phases of the experimental infection. Eight laboratory-cloned stocks representative of this phylogenetic diversity and including the lineages T. cruzi I (genotypes 19 and 20), T. cruzi II (genotype 32) and T. cruzi (genotype 39) have been studied. The results showed that the pattern of humoral immune response was correlated with T. cruzi genotype, and that stocks included in genotype 20 were responsible for the high IgG response in the acute and chronic phases. Moreover, T. cruzi I lineage was more efficient in over-expressing all subclasses of specific anti-parasite IgG than either T. cruzi II or T. cruzi lineages. Curiously, the alteration in the pattern of antibodies induced by Benznidazole treatment was related to the phase of the infection but not to the genotype of the parasite. The data suggest that genotypes of T. cruzi are able to drive levels/subclasses of specific IgG, hence giving rise to further concerns about the sensitivity of serological assays in the diagnosis of human Chagas disease

Protein deficiency alters CX3CL1 and endothelin-1 in experimental Trypanosoma cruzi-induced cardiomyopathy.

objective Chagas heart disease is developed as a result of the infection with Trypanosoma cruzi. Protein malnutrition contributes to secondary immunodeficiency. The aim of this study was to investigate the role of a low protein diet on the production of endothelin-1 and CX3CL1 in blood and cardiac tissue samples in an experimental model with T. cruzi infection. methods Fisher rats were submitted to low protein (6%) and normal protein (15%) diets and then infected with the Y strain of T. cruzi. At days 15 and 120, parasites and immune cells were evaluated. results The low protein diet reduced body weight and circulating serum proteins, but promoted elevation of CX3CL1 and endothelin-1 levels in infected animals, which were unable to control blood parasitemia replication. In heart tissue, the low protein diet reduced cardiac CX3CL1, endothelin-1 and leucocyte infiltration in the acute phase, in particular CD68 and CD163 macrophage phenotypes. conclusion Together, these results highlight the participation of endothelin-1 and CX3CL1 in the inflammatory process of Chagas diesease, both being mediators partially controlled by the host nutritional status

Use of fluorescence in a modified disector method to estimate the number of myocytes in cardiac tissue.

Fundamento: Métodos convencionais de disector atualmente requerem consideráveis custos financeiros, técnicos e operacionais para estimar o número de células, incluindo cardiomiócitos, em uma área de 3D. Objetivo: Usar a microscopia de fluorescência em um método de disector modificado para determinar o número de miócitos no tecido cardíaco em condições normais e patológicas. Métodos: O estudo empregou ratos Wistar machos com quatro meses de idade e peso de 366,25 ± 88,21 g randomizados em grupos controles (GC, n = 8) e infectados (GI, n = 8). Os animais do GI foram inoculados com cepa Y de T. cruzi (300.000 tripomastigotas/50 g). Após oito semanas, os animais foram pesados e sacrificados. Os Ventrículos Esquerdos (VE) foram removidos para análise estereológica da densidade numérica de cardiomiócitos (Nv [c]) e o número total dessas células no VE (N [c]). Esses parâmetros foram estimados usando um disector fluorescente (DF) e comparados com os métodos convencionais de disector óptico (DO) e disector físico (DFi). Resultados: Em ambos os métodos de disector, os animais do GI apresentaram queda significativa de Nv[c] e N[c] em comparação com os animais do GC (p > 0,05). Uma correlação forte, igual ou superior a 96%, foi obtida entre DF, DO e DFi. Conclusão: O método DF parece ser igualmente confiável para determinar Nv[c] e N[c] em condições normais e patológicas, apresentando algumas vantagens em relação aos métodos convencionais de disector: redução de cortes histológicos e imagens na análise estereológica, redução do tempo de análise das imagens, a construção de DF em microscópios simples, utilizando o modo de epifluorescência, distinção de planos de disector em ampliações inferiores.Background: Conventional disector methods currently require considerable financial, technical and operational costs to estimate the number of cells, including cardyomyocytes, in a 3D area. Objective: To use fluorescence microscopy in a modified disector method to determine the number of myocytes in cardiac tissue in normal and pathological conditions. Methods: The study employed four-month-old male Wistar rats with weight of 366.25 ± 88.21g randomized in control (CG, n=8) and infected (IG, n=8) groups. IG animals were inoculated with T. cruzi Y strain (300,000 trypomastigotes/50g wt). After eight weeks, the animals were weighted and euthanized. The left ventricles (LV) were removed for stereological analysis of numerical density of cardiomyocytes (Nv[c]) and total number of these cells in the LV (N[c]). These parameters were estimated using a fluorescent disector (FD) and compared with the conventional optical (OD) and physical (PD) disector methods. Results: In both disector methods, IG animals presented significant decrease of Nv[c] and N[c] compared to CG animals (P< 0.05). There was no significant difference in these variables despite the disector method applied in CG and IG animals (P> 0.05). A strong correlation, equal or above 96%, was obtained between FD, OD and PD. Conclusion: The FD method seems to be equally reliable to determine Nv[c] and N[c] in normal and pathological conditions and presents some advantages compared to conventional disector methods: reduction of histological slices and images in the stereological analysis, reduction of time to analyze the images, construction of FD in simple microscopes using the epifluorescence mode, distinction of disector planes in lower magnifications

Trypanosoma cruzi antigens induce inflammatory angiogenesis in a mouse subcutaneous sponge model.

Acute inflammation and angiogenesis are persistent features of several pathological conditions induced by biological agents leading to the resolution of local and systemic events. Glycoproteins derived from the protozoan Trypanosoma cruzi are suggested to mediate angiogenesis induced by inflammatory cells with still undescribed mechanisms. In this study, we investigated the effects of total antigen from trypomastigote forms of T. cruzi (Y strain), inoculated in sponges 24 h after implantation inmice, on angiogenesis, inflammatory cell pattern and endogenous production of inflammatory and angiogenicmediators on days 1, 4, 7 and 14 post-implant. There was an increase in hemoglobin content and in the number of blood vessels associated with T. cruzi antigen stimuli on the 14th day, assessed by the hemoglobin of the implants and by morphometric analysis. However, these antigens were not able to increase type I collagen content on the 14th day. Parasite antigens also induced high production of vascular endothelial growth factor (VEGF) and inflammatory mediators TNF-alpha, CCL2 and CCL5 on the 7th day in sponges when compared to the unstimulated group. Neutrophils and macrophages were determined by measuring myeloperoxidase (MPO) and N-acetyl-β-D-glucosaminidase (NAG) enzyme activities, respectively. Only NAG was increased after stimulation with antigens, starting from day 4 and peaking at day 7. Together, these data showed that antigens fromthe Y strain of T. cruzi are able to promote inflammatory neovascularization probably induced by macrophage-induced angiogenic mediators in T. cruzi antigen-stimulated sponges in Swiss mice