Experimental Chagas Research Articles

Trypanosoma cruzi: Altered parasites after in vitro treatment with gangliosides, a therapeutic agent in experimental Chagas’ disease

Biochemical and structural modifications were investigated in axenic cultured Trypanosoma cruzi after treatment with gangliosides. Fluorescence anisotropy showed dose dependent increments in parasite membranes of ganglioside treated epimastigotes. NADP-GDH activity increased in parasites treated at day 4 (13%), 7 (137.2%), and 14 (28.50%) while NAD-MDH but decreased from day 7 to 21 (−5.74%, −32.22%, −27.92%). Treated parasites presented electron-lucent vacuoles opposite to the cytostoma, multilamellar bodies and dilated mitochondrion cristae, disorganized kinetoplast and altered heterochromatin structure. Gangliosides inhibited fusogenic ability (80%) and PLA2 activity (>75%) from the parasite. The same occurred with anti-PLA2 antibodies. Trypomastigotes suffered loss of cytoplasmic material and organelles when GM1 was present in culture medium. We propose that exogenous gangliosides produced: altered lipid order, inhibited membrane enzymes, the parasite energy source shifted from glucose to amino acids, ending on a structural transformation which signals parasite cell death.

Trypanosoma cruzi: Dehydroepiandrosterone (DHEA) and immune response during the chronic phase of the experimental Chagas’ disease

Dehydroepiandrosterone (DHEA) has long been considered as a precursor for many steroid hormones. It also enhances the immune responses against a wide range of viral, bacterial, and parasitic pathogens. The aims of this work were to evaluate the influences of exogenous DHEA treatment on Wistar rats infected with the Y strain of Trypanosoma cruzi during the acute and its influence on the chronic phase of infection. Animals were subcutaneous treated with 40mg/kg body weight/day of DHEA. DHEA treatment promoted increased lymphoproliferative responses as well as enhanced concentrations of NO and IL-12. So, we point in the direction that our results validate the utility of the use of DHEA as an alternative therapy candidate against T. cruzi.

Why does GM1 induce a potent beneficial response to experimental Chagas disease?

Being one of the world's neglected diseases, Chagas has neither a vaccine nor a satisfactory therapy. Inoculation of murine models with the ganglioside GM1 has shown a strikingly nonlinear effect, leading to a strong decrease in parasite load at low doses but reverting to a load increase at high doses. Cardiocyte destruction concomitant with the disease is also significantly reduced by a moderate application of GM1. A mathematical model for the interaction between the parasite and the immune system is shown to explain these effects and is used to predict an optimal dosage that maximizes parasite removal with minimal cardiocyte destruction.

Changes in cellular contractility and cytokines profile during Trypanosoma cruzi infection in mice

Trypanosoma cruzi, an intracellular protozoan parasite infecting a wide variety of vertebrates, is the agent responsible for Chagas' disease. This pathology often results in severe inflammatory heart condition and it is one of the major causes of dilated cardiomyopathy leading to heart failure in Latin America. Nevertheless, little is known about the changes in isolate cardiac myocytes contractility during the development of this pathology. Here we report a relationship between cytokines profile of mice infected with T. cruzi and the modifications in the cellular contractility pattern. We found that cellular contractility, measured as fractional shortening, showed a complex behavior. The changes were evaluated during the acute phase (15, 30 and 45 dpi) and chronic phase (>90 dpi). The time to half contraction and relaxation were lengthier despite the number of days after infection or the heart region evaluated. The maximal contraction and relaxation velocities were significantly slower. The observed changes in cellular contractility were correlated with the presence of circulating IFN-gamma, TNF-alpha and MCP-1/CCL2 during the course of infection. Together, our data demonstrate that cellular contractility is altered in the three heart regions studied, and these alterations are observed at the very beginning of the parasitism and they remained until the chronic phase has been reached. Indeed, we propose a role for IFN-gamma, TNF-alpha and MCP-1/CCL2 in the mechanical heart remodeling during experimental Chagas' disease.

Trypanosoma cruzi and myoid cells from seminiferous tubules: interaction and relation with fibrous components of extracellular matrix in experimental Chagas' disease.

The main transmission route of Trypanosoma cruzi is by triatomine bugs. However, T. cruzi is also transmitted through blood transfusion, organ transplantation, ingestion of contaminated food or fluids, or is congenital. Sexual transmission, although suggested since the discovery of Chagas' disease, has remained unproven. Sexual transmission would require T. cruzi to be located at the testes and ovaries. Here we investigated whether T. cruzi is present in the gonads of mice infected with 10(4) T. cruzi trypomastigotes from the CL strain. Fourteen days after experimental infection, histopathological examination showed alterations in the extracellular matrix of the lamina propria of the seminiferous tubules. Furthermore, amastigotes were present in seminiferous tubules, within myoid cells, and in the adjacencies of the basal compartment. These results indicate that T. cruzi is able to reach seminiferous tubule lumen, thus suggesting that Chagas' disease could potentially be transmitted through sexual intercourse. Complementary studies are required to demonstrate that Chagas' disease can be transmitted by coitus.

Trypanosoma cruzi: The effects of zinc supplementation in the immune response during the course of experimental disease

Zinc is an essential nutritional component required for normal development and maintenance of immune functions. The possible effects of zinc in upregulating the host immune response during the acute and chronic phases of experimental Chagas’ disease were evaluated. In young, infected and Zn-supplemented animals, higher concentrations of IFN-γ and NO were observed. During the chronic phase, decreased concentrations of NO and IFN-γ were found for older infected animals that received Zn supplementation. For young animals, hearts from Zn-supplemented groups displayed reduced inflammatory infiltrate, heart weight and number of amastigote burdens. For older, infected and Zn-supplemented animals amastigote nests were absent with reduced inflammatory cell infiltrate. This study identifies a potentially novel therapeutic approach that could control the parasite load during acute phase of disease, consequently preventing the deleterious, parasite-elicited responses observed during chronic phase.

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: Effects of adrenalectomy during the acute phase of experimental infection

Glucocorticoid hormones have been implicated as an important modulator of Trypanosoma cruzi pathogenesis. Since adrenal steroid hormones play a fundamental role in modulating the immune response, we hypothesized that adrenalectomy affect the course of the experimental T. cruzi infection. This study was undertaken to determine the effects of adrenalectomy during the acute phase of T. cruzi infection. Blood and tissue parasitism, macrophages, nitric oxide (NO) production and IFN-γ were evaluated in male Wistar rats infected with the Y strain of T. cruzi. Our results show that adrenalectomized rats displayed increased number of blood and heart parasites accompanied by decreases in the total number of peritoneal macrophages and IFN-γ when compared to controls. Adrenalectomy also reduced the levels of NO released from peritoneal macrophages of infected animals. These results suggest that adrenal corticosteroid insufficiency due to adrenalectomy could be considered an important factor during development of acute phases of experimental Chagas’ disease, enhancing pathogenesis through disturbance of the host’s immune system.

The regulatory CD4+CD25+ T cells have a limited role on pathogenesis of infection with Trypanosoma cruzi

Recent reports have established an important role of CD4+CD25+ T cells in the immune regulation of infectious diseases, autoimmune disorders and cancer. In the present work, we investigated whether these cells had a regulatory role during Trypanosoma cruzi infection, using the Colombian strain. Inactivation of CD4+CD25+ cells in vivo conferred mice slightly more resistant to infection with the Colombian strain of T. cruzi, as evidenced by lower parasitemia and mortality rates. The augmented resistance to infection with Colombian strain did correlate with increased activation of effector CD4 cells. It was antibody-independent, since no difference in levels of IgM, IgG, IgG1 and IgG2a b recognizing T. cruzi antigens was observed throughout the infection of CD25-inactivated and control mice. Regarding pathogenesis, inflammatory infiltrate and frequency of CD4 and CD8 T cells or macrophages in the cardiac tissue was similar in both groups. Together, our data indicate that CD4+CD25+ cells have a limited role on host resistance during early T. cruzi infection. Despite exhaustive investigation, we did not observe any role for these regulatory cells in the pathogenesis of experimental chronic Chagas' disease.

Zinc supplementation increases resistance to experimental infection by Trypanosoma cruzi

It is well recognized that zinc is an essential trace element for all organisms, influencing growth and affecting the development and integrity of the immune system. It is also well known that the protective response against Trypanosoma cruzi depends on both innate and acquired immunity and for the control of the parasite load and host survival, the participation of special cells such natural killer (NK), T and B lymphocytes and macrophages are required. So the aims of this study were to evaluate the effects of zinc supplementation on the host's immune response infected with T. cruzi. Our data point in the direction that zinc supplementation triggered enhanced thymocyte and splenocyte proliferation as compared to unsupplied group of animals. It is also important to emphasize that interleukin-12 (IL-12) participates in the resistance to several intracellular pathogens including T. cruzi. Our findings demonstrate an enhanced production of IL-12 during the acute phase of infection in zinc-supplied groups. So we conclude that zinc supplementation leads to an effective host's immune response by up-modulating the host's immune response, thus contributing in the reduction of blood parasites and the harmful pathogenic effects of the experimental Chagas’ disease.

Trypanosoma cruzi: The role of PGE2 in immune response during the acute phase of experimental infection

Chagas disease is characterized by cardiac lesions and a high level of PGE2. Our objective was to investigate the role of PGE2 in cardiac lesions. BALB/c mice were infected with Trypanosoma cruzi (1 × 10 3 trypomastigote forms) and were treated daily with PBS, meloxicam (0.5 mg/kg) or etoricoxib (0.6 mg/kg). The animals were sacrificed on the 21st day of infection and we collected the cardiac tissue and spleen cells for tissue culture. We observed that treatment with COX-2 inhibitors was able to decrease synthesis of PGE2 by spleen cells. This reduction was accompanied by reduction of the inflammatory infiltrate, parasite nets, cardiac fibrosis and fewer COX-2 positive cells in cardiac tissue obtained from these animals. In conclusion, treatment with COX-2 inhibitors, and consequent inhibition of PGE2 synthesis, was able to reduce the cardiac damage observed during the acute phase of experimental Chagas disease, thus demonstrating the involvement of this mediator in the cardiac lesion.

Circulating antibodies against nicotinic acetylcholine receptors in chagasic patients.

Human and experimental Chagas' disease causes peripheral nervous system damage involving neuromuscular transmission alterations at the neuromuscular junction. Additionally, autoantibodies directed to peripheral nerves and sarcolemmal proteins of skeletal muscle have been described. In this work, we analyse the ability of serum immunoglobulin factors associated with human chagasic infection to bind the affinity-purified nicotinic acetylcholine receptor (nAChR) from electric organs of Discopyge tschudii and to identify the receptor subunits involved in the interaction. The frequency of serum anti-nAChR reactivity assayed by dot-blot was higher in seropositive chagasic patients than in uninfected subjects. Purified IgG obtained from chagasic patients immunoprecipitated a significantly higher fraction of the solubilized nAChR than normal IgG. Furthermore, immunoblotting assays indicated that alpha and beta are the main subunits involved in the interaction. Chagasic IgG was able to inhibit the binding of alpha-bungarotoxin to the receptor in a concentration-dependent manner, confirming the contribution of the alpha-subunit in the autoantibody-receptor interaction. The presence of anti-nAChR antibodies was detected in 73% of chagasic patients with impairment of neuromuscular transmission in conventional electromyographical studies, indicating a strong association between seropositive reactivity against nAChR and electromyographical abnormalities in chagasic patients. The chronic binding of these autoantibodies to the nAChR could induce a decrease in the population of functional nAChRs at the neuromuscular junction and consequently contribute to the electrophysiological neuromuscular alterations described in the course of chronic Chagas' disease.

Trifluralin Toxicity in a Chagas Disease Mouse Model

Even though trifluralin (alpha,alpha,alpha-2,6-dinitro-N-N-dipropyl-p-toluidine) is effective for the treatment of experimental Chagas disease, more preclinical toxicity studies need to be performed. Cell toxicity of trifluralin was studied in Hep-G2 and Vero C76 cells treated with 50 and 150 microM trifluralin. The results show that duplication time, amount of cellular protein and cell protein/DNA values were normal. Histological, haematological and chemical parameters were measured in CF1 mice after oral trifluralin administration. Acute toxic effects were assayed by administration of 50 or 200 mg/kg body weight daily for 30 days, and chronic effects by administration of 200 mg/kg body weight once a week for 90 days (n = 20). In the acute scheme treatment, hepatic (glutamic-pyruvic, glutamic-oxalacetic and alkaline phosphatase activities; proteins and albumin plasma concentrations) and pancreatic (amylase, glycaemia) functions were normal. Mean corpuscular volume, haemoglobin and haematocrit decreased. Creatine phosphokinase, lactate dehydrogenase and glutamic-oxalacetic activity increased, suggesting lesion in myocardial tissue. Histology was normal, excepting for the heart (mild myocarditis). Similar results were observed in acutely treated animals. There were no differences in body weight gain for treated mice compared to controls. In view of the published therapeutic effects of trifluralin on CF1 Chagas disease model and considering the present results, trifluralin seems to be a moderately toxic drug with a potential selective effect on the myocardium.

Treatment with benznidazole or thioridazine in the chronic phase of experimental Chagas disease improves cardiopathy

Mice infected with Trypanosoma cruzi Tulahuen strain or SGO-Z12 isolate were treated at 180 days post infection (p.i.) (i.e. chronic phase) with benznidazole (for 30 days) or thioridazine (for 12 days). Both drugs produced a decrease in electrocardiographic alterations, fewer modifications in the affinity and density of cardiac β-receptors, and few isolated areas of fibrosis in the heart, whereas untreated mice presented areas of necrosis and fibre fragmentation 350 days p.i. ( P < 0.01). Survival in treated mice was 100% for benznidazole and 88% for thioridazine, independent of the parasite strain; survival for untreated mice was 30% and 40% for Tulahuen strain and SGO-Z12 isolate, respectively ( P < 0.01). No cardiotoxic effects of thioridazine were detected at the dose and treatment schedule used. These results show the benefit of treatment in the chronic phase of Chagas disease and that thioridazine should be considered as a promising agent for the treatment of Chagas disease.

Caspase inhibition reduces lymphocyte apoptosis and improves host immune responses to Trypanosoma cruzi infection

In experimental Chagas' disease, lymphocytes from mice infected with Trypanosoma cruzi show increased apoptosis in vivo and in vitro. Treatment with a pan-caspase blocker peptide inhibited expression of the active form of effector caspase-3 in vitro and rescued both B and T cells from cell death. Injection of the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethyl ketone, but not a control peptide, reduced parasitemia and lymphocyte apoptosis in T. cruzi-infected mice. Moreover, treatment with caspase inhibitor throughout acute infection increased the absolute numbers of B and T cells in the spleen and lymph nodes, without affecting cell infiltrates in the heart. Following treatment, we found increased accumulation of memory/activated CD4 and CD8 T cells, and secretion of IFN-gamma by splenocytes stimulated with T. cruzi antigens. Caspase inhibition in the course of infection reduced the intracellular load of parasites in peritoneal macrophages, and increased the production of TNF-alpha and nitric oxide upon activation in vitro. Our results indicate that inhibition of caspases with a pan-caspase blocker peptide improves protective type-1 immune responses to T. cruzi infection. We suggest that mechanisms of apoptosis are potential therapeutic targets in Chagas' disease.

DNA damage and nitric oxide synthesis in experimentally infected Balb/c mice with Trypanosoma cruzi

This study aimed to evaluate whether experimental Chagas disease in acute phase under benznidazole therapy can cause DNA damage in peripheral blood, liver, heart, and spleen cells or induce nitric oxide synthesis in spleen cells. Twenty Balb/c mice were distributed into four groups: control (non-infected animals); Trypanosoma cruzi infected; T. cruzi infected and submitted to benznidazole therapy; and only treated with benznidazole. The results obtained with the single cell gel (comet) assay showed that T. cruzi was able induce DNA damage in heart cells of both benznidazole treated or untreated infected mice. Similarly, T. cruzi infected animals showed an increase of DNA lesions in spleen cells. Regarding nitric oxide synthesis, statistically significant differences ( p < 0.05) were observed in all experimental groups compared to negative control, the strongest effect observed in the T. cruzi infected group. Taken together, these results indicate that T. cruzi may increase the level of DNA damage in mice heart and spleen cells. Probably, nitric oxide plays an important role in DNA damaging whereas benznidazole was able to minimize induced T. cruzi genotoxic effects in spleen cells.

Immunoneuroendocrine Interactions in Chagas Disease

We investigated immunoneuroendocrine interactions in vivo and in vitro following infection by Trypanosoma cruzi, the causative agent of Chagas disease. In a first set of experiments, we studied the hypothalamus-pituitary-adrenal axis. Nests of parasites were seen in the adrenal gland, whereas T. cruzi-specific PCR gene amplification product was found in both the adrenal and pituitary glands of infected mice. These endocrine glands also revealed alterations including vascular stasis, increase in the deposition of extracellular matrix (ECM), as well as T cell and macrophage infiltration. Functionally, we found a decrease in corticotrophin-releasing hormone and an increase in corticosterone contents, in hypothalamus and serum, respectively, whereas no significant changes were seen in serum adrenocortricotropic hormone of infected animals. Nevertheless, the serum levels of interleukin-6 (known to directly stimulate glucocorticoid secretion) were increased, as compared to controls. Considering the presence of T cells within the nervous tissue of chagasic animals, we performed a number of in vitro experiments co-culturing spleen-derived T cells from control or infected mice, with neuronal cells (being or not being directly infected in vitro). In particular, we looked for ECM-mediated interactions, known to affect T cell migration. We found an increase in ECM deposition in infected cultures, as compared to controls. Moreover, adhesion of T cells was enhanced when neuronal cells were infected in vitro, or when T cells were derived from T. cruzi-infected mice, events that could be abrogated with anti-ECM antibodies. Together, the data summarized above clearly reveal that neuroendocrine axes are altered in experimental Chagas disease.

Blockade of endothelin ETA/ETB receptors favors a role for endothelin during acute Trypanosoma cruzi infection in rats

Endothelin has been implicated in the pathogenesis of experimental and human Chagas' disease (American trypanosomiasis). In the present study, we tested the effect of bosentan, an antagonist of both ETA and ETB endothelin receptors, on parasitemia, histopathology (heart and diaphragm), heart levels of tumor necrosis factor (TNF)-α, interleukin (IL)-10, interferon (IFN)-γ, CCL2, CCL3 and CCL5, and the serum levels of nitrate/nitrite (NOx). Bosentan treatment was accompanied by a significant increase in parasitemia and tissue parasitism or inflammation. In vehicle-treated rats, Trypanosoma cruzi infection increased the cardiac levels of TNF-α, IFN-γ and IL-10, at day 9 post inoculation, and the TNF-α remained elevated until day 13. The infection also caused a significant increase in the cardiac levels of the chemokines CCL2 (9, 13 and 18days) and CCL3 (13 and 18days). Bosentan-treatment had no significant effect on the infection-associated increase in IFN-γ and chemokine concentrations. There was a lower increase in IL-10 at day 9 and this was mirrored by a greater increase of TNF-α at day 13, in comparison with vehicle-treated rats. These latter findings correlated well with the enhanced inflammatory process in hearts of bosentan-treated infected rats. Bosentan treatment reduced the infection-associated increase in NOx serum concentration. Altogether, our data suggest that ET action on ETA and ETB receptors may play a role in the initial control of T. cruzi infection in rats probably by interfering in NO production.

Attenuation of Folic Acid-Induced Renal Inflammatory Injury in Platelet-Activating Factor Receptor-Deficient Mice

Platelet-activating factor (PAF), a potent lipid mediator with various biological activities, plays an important role in inflammation by recruiting leukocytes. In this study we used platelet-activating factor receptor (PAFR)-deficient mice to elucidate the role of PAF in inflammatory renal injury induced by folic acid administration. PAFR-deficient mice showed significant amelioration of renal dysfunction and pathological findings such as acute tubular damage with neutrophil infiltration, lipid peroxidation observed with antibody to 4-hydroxy-2-hexenal (day 2), and interstitial fibrosis with macrophage infiltration associated with expression of monocyte chemoattractant protein-1 and tumor necrosis factor-alpha in the kidney (day 14). Acute tubular damage was attenuated by neutrophil depletion using a monoclonal antibody (RB6-8C5), demonstrating the contribution of neutrophils to acute phase injury. Macrophage infiltration was also decreased when treatment with a PAF antagonist (WEB2086) was started after acute phase. In vitro chemotaxis assay using a Boyden chamber demonstrated that PAF exhibits a strong chemotactic activity for macrophages. These results indicate that PAF is involved in pathogenesis of folic acid-induced renal injury by activating neutrophils in acute phase and macrophages in chronic interstitial fibrosis. Inhibiting the PAF pathway might be therapeutic to kidney injury from inflammatory cells.

Treatment of Experimental Chronic Chagas Disease with Trifluralin

We tested trifluralin against Trypanosoma cruzi in a model of chronic Chagas disease in mice. CF1 mice (n=148) were intraperitoneally infected with 10(5) trypomastigotes of T. cruzi, H510C8C3 clone. One hundred mice were partially treated with benznidazole. Mortality was 100% at day 41 in the control group (n=48). At day 90 of the chronic disease (74% survival) mice were divided into three groups and treated orally with trifluralin (50 mg/kg/day, n=26), benznidazole (50 mg/kg/day, n=25) and vehicle (peanut oil; control group, n=23) for 60 days. Electrocardiography (under pentobarbital anaesthesia, 30 mg/kg/dose), serologic immunofluorescence and microstrout were performed at the beginning and at the end of the treatment. Mice were sacrificed at day 10 after treatment; cardiac tissue was studied histopathologically and polymerase chain reaction (PCR) was performed. Spontaneous mortality was 30.43%, 3.85% and 4% in the control, trifluralin and benznidazole groups, respectively (significant survival, P=0.03). Microstrouts were negative in all three groups. Negative immunofluorescence titers were 0%, 16% (P=0.05) and 29% (P<0.02) in the control, trifluralin and benznidazole groups, respectively. The prevailing electrocardiographic disorder was prolongation of the PR interval in the control group, which was not significantly altered in trifluralin- and benznidazole-treated mice, suggesting that trifluralin and benznidazole improve or even stop the damage caused by the disease on the conduction system. Trifluralin- and benznidazole-treated animals showed similar histologic patterns of myocarditis. PCR results were negative for benznidazole and trifluralin (100% and 70.8%, respectively). These results show the therapeutic potential of trifluralin in the treatment of chronic Chagas disease.