Clomipramine and benznidazole association for the treatment of acute experimental Trypanosoma cruzi infection

Alternative strategies are being designed to identify candidates among drugs already available on the market that could be used in combination to improve the efficacy of Chagas disease treatment. This work evaluates the effect of the association of clomipramine (CLO) with benznidazole (BZN) for the treatment of experimental Chagas disease in the acute stage, in Swiss albino mice infected with Trypanosoma cruzi Tulahuen strain. Infected mice were treated with CLO 5 mg/kg/day and BZN 50 and 100 mg/kg/day, each separately or together. Efficacy of the treatment was evaluated through parasitemia, survival, electrocardiography, histopathological studies, serological and PCR assays at 90 days post-infection (dpi). All treatments significantly (P < 0.05) reduced mortality and decreased parasitemia. Histopathological analysis of liver and kidneys of mice treated with CLO and the drug combination showed less injury than mice treated only with BZN. The lower dose of BZN (50 mg/kg/day) combined with CLO showed the same efficacy as the habitual dose of BZN (100 mg/kg/day) combined with CLO. The therapeutic results from the combination of BZN with CLO presented lesser side effects than the treatment with BZN.     

Protective Role of Acetylsalicylic Acid in Experimental Trypanosoma cruzi Infection: Evidence of a 15-epi-Lipoxin A4-Mediated Effect

Chagas'' disease, produced by Trypanosoma cruzi, affects more than 8 million people, producing approximately 10,000 deaths each year in Latin America. Migration of people from endemic regions to developed countries has expanded the risk of infection, transforming this disease into a globally emerging problem. PGE₂ and other eicosanoids contribute to cardiac functional deficits after infection with T. cruzi. Thus, the inhibition of host cyclooxygenase (COX) enzyme emerges as a potential therapeutic target. In vivo studies about the effect of acetylsalicylic acid (ASA) upon T. cruzi infection are controversial, and always report the effect of ASA at a single dose. Therefore, we aimed to analyze the effect of ASA at different doses in an in vivo model of infection and correlate it with the production of arachidonic acid metabolites. ASA decreased mortality, parasitemia, and heart damage in T. cruzi (Dm28c) infected mice, at the low doses of 25 and 50 mg/Kg. However, this effect disappeared when the high ASA doses of 75 and 100 mg/Kg were used. We explored whether this observation was related to the metabolic shift toward the production of 5-lipoxygenase derivatives, and although we did not observe an increase in LTB₄ production in infected RAW cells and mice infected, we did find an increase in 15-epi-LXA₄ (an ASA-triggered lipoxin). We also found high levels of 15-epi-LXA₄ in T. cruzi infected mice treated with the low doses of ASA, while the high ASA doses decreased 15-epi-LXA₄ levels. Importantly, 15-epi-LXA₄ prevented parasitemia, mortality, and cardiac changes in vivo and restored the protective role in the treatment with a high dose of ASA. This is the first report showing the production of ASA-triggered lipoxins in T. cruzi infected mice, which demonstrates the role of this lipid as an anti-inflammatory molecule in the acute phase of the disease.     

L-arginine supplementation increases cardiac collagenogenesis in mice chronically infected with Berenice-78 Trypanosoma cruzi strain

Chagas disease, caused by Trypanosoma cruzi, is a major neglected tropical disease that occurs mainly as chronic infection and systemic infection. Currently, there is no suitable and effective drug to treat this parasitic disease. Administration of nutrients with immunomodulatory properties, such as arginine and nitric oxide radicals, may be helpful as antiparasitic therapy. In this study, we evaluated the effects of arginine supplementation during the acute phase of infection under the development of chronic Chagas' heart disease in Swiss mice inoculated with the Berenice-78 strain of T. cruzi. The effectiveness of arginine was determined by daily detection of the parasite in the blood and long-term serum levels of nitric oxide and tumor necrosis factor-alpha, in addition to evaluation of heart tissue damage. Arginine could flatten parasitemia and prevent elevation of tumor necrosis factor-alpha in T. cruzi-infected mice. Regarding chronic inflammatory myocardial derangements, similar findings were verified among T. cruzi-infected groups. Arginine promoted collagenogenesis in the heart muscle tissue of T. cruzi-infected arginine-supplemented group. These data show the paradoxical benefits of arginine in improving the outcome of Chagas chronic cardiomyopathy.     

Oral Outbreak of Chagas Disease in Santa Catarina,Brazil: Experimental Evaluation of a Patient’s Strain

Chagas disease is a worldwide public health problem. Although the vectorial transmission of Chagas disease has been controlled in Brazil there are other ways of transmission, such as the ingestion of T. cruzi contaminated food, which ensures the continuation of this zoonosis. Here, we demonstrate the influence of the inoculation route on the establishment and development of the SC2005 T. cruzi strain infection in mice. Groups of Swiss mice were infected intragastrically (IG) or intraperitoneally (IP) with the T. cruzi SC2005 strain derived from an outbreak of oral Chagas disease. The results revealed that 100% of IP infected mice showed parasitemia, while just 36% of IG infected showed the presence of the parasite in blood. The parasitemia peaks were later and less intense in the IG infected mice. Mortality of the IP infected animals was more intense and earlier when compared to the IG infected mice. In the IP infected mice leucopenia occurred in the early infection followed by leucocytosis, correlating positively with the increase of the parasites. However, in the IG infected mice only an increase in monocytes was observed, which was positively correlated with the increase of the parasites. Histopathological analyses revealed a myotropic pattern of the SC2005 strain with the presence of inflammatory infiltrates and parasites in different organs of the animals infected by both routes as well as fibrosis foci and collagen redistribution. The flow cytometric analysis demonstrated a fluctuation of the T lymphocyte population in the blood, spleen and mesenteric lymph nodes of the infected animals. T. cruzi DNA associated with the presence of inflammatory infiltrates was detected by PCR in the esophagus, stomach and intestine of all infected mice. These findings are important for the understanding of the pathogenesis of T. cruzi infection by both inoculation routes.     

Efficacy of novel benznidazole solutions during the experimental infection with Trypanosoma cruzi

Chagas' disease is caused by the protozoan parasite Trypanosoma cruzi. About 8 million people throughout Latin America are infected causing approximately 10,000 deaths annually. Benznidazole, available as unique 100 mg tablets in many of the endemic countries, is currently the drug of choice for the specific treatment of this condition. Despite of the large number of pediatric patients infected, there are no commercial liquid dosage forms available to treat this trypanosomiasis. This work showed that novel benznidazole–water–polyethylene glycol 400 solutions are active against T. cruzi in a murine model of Chagas' disease. Present results constitute the first demonstration on the usefulness of benznidazole solutions in infected mice.     

The effect of BCG on the course of experimental Chagas' disease in mice

Experiments were done to determine the effect of BCG treatment on longevity, development of parasitemia, and in vivo distribution of ⁵¹Cr-labelled trypanosomes in C3H(He) female mice infected with a Brazil strain of Trypanosoma cruzi. BCG sensitization of mice was accomplished by a single IV injection of 3·0 mg (wet weight) of BCG. Twenty-one days after BCG injection mice were infected with 5 × 10⁴ blood-form trypomastigotes. Parasitemia determinations were made on alternate days during the experiment while in vivo distribution of exogenously supplied ⁵¹Cr-epimastigotes was made in groups of BCG or PBS stimulated mice on day 15 of the T. cruzi infection.It was found that BCG sensitization had no effect on longevity or parasitemia development in T. cruzi infected C3H(He) female mice. There were, however, some differences in the in vivo distribution of parasites between BCG treated and control mice. BCG stimulated mice accumulated greater numbers of radiolabelled trypanosomes in the kidneys and small intestines while PBS treated mice were found to have greater numbers of labelled parasites in the liver. Although no significant differences were observed in longevity of BCG or PBS treated mice, it was noted that BCG treated animals which were bled for parasitemia determinations lived significantly longer than those which were merely observed for longevity.     

Trypanosoma cruzi: effects of anti-thymocyte serum in mice and neonatal thymectomy in rats

CF₁ mice were given eight injections of normal rabbit serum (NRS), Hanks' balanced salt solution (HBSS), or rabbit anti-mouse thymocyte serum (ATS) beginning 3 days prior to and at 3-day intervals subsequent to intraperitoneal (ip) inoculation with 5 × 10⁴ trypomastigotes of a Brazil strain of Trypanosoma cruzi. Markedly enhanced parasitemia, increased numbers of tissue stages (amastigotes), and higher mortality occurred in ATS-treated mice as compared to NRS- or HBSS-treated controls. Administration of three injections of ATS at 3-day intervals during the latter stages of acute Chagas' disease, i.e., when numbers of parasites were declining, resulted in a transitory relapse (increase in numbers) of blood and tissue parasites. No relapse occurred in mice when ATS was administered at 3-day intervals over a period of 15 days during the subacute stage of the disease, i.e., after parasites had disappeared from the blood.Parasitemia and mortality were enhanced in neonatally thymectomized rats when compared to that observed in sham-operated and unoperated control rats following ip injection of 2 × 10⁵ trypomastigotes of T. cruzi. Serum obtained from thymectomized and control rats 5 weeks after inoculation with T. cruzi at a time when the blood of all animals had become microscopically negative for parasites were equally protective in passive transfer experiments, while serum from uninfected controls gave no protection.Gamma globulin levels significantly increased in thymectomized as well as intact rats by the third to fourth week of infection with T. cruzi, reached maximum concentrations in 5–6 wk, and remained elevated significantly at the twelfth week post infection as compared with uninfected controls. No significant changes occurred in total serum proteins or α and β fractions of any group, infected or uninfected.Total circulating leukocytes, especially lymphocytes, were diminished in mice and rats subjected to treatment with ATS or neonatal thymectomy.These data clearly indicate that neonatal thymectomy of rats and ATS treatment of mice suppress the acquired immune response to T. cruzi. Further, passive transfer experiments in rats confirm the protective role of circulating antibody in acquired immunity to Chagas' disease.     

Canova medication modifies parasitological parameters in mice infected with Trypanosoma cruzi

The goals of this study were to evaluate the effect of the Canova® medication, a homeopathic immune-system modulator, on the evolution of infection induced by the Trypanosoma cruzi Y strain in mice. The animals were divided into five groups: (i) untreated infected controls (I), (ii) infected animals treated with benznidazole (Bz), (iii) infected animals treated with the Canova medication (CM), (iv) infected animals treated with benznidazole and the Canova medication (Bz + CM), and (v) uninfected controls that received only the vehicle (grain alcohol) (C). The parameters evaluated were: parasitemia, mortality, control of cure, and tissue parasitism analysis. Our results showed that the evolution of the experimental infection was modified by treatment with CM, and that daily and consecutive doses were harmful to the animals, causing death in 100% of the infected animals in a brief period. The analysis of parasitism performed on the organs on the 12th day postinfection showed that in infected animals treated with CM, the number of amastigote/nests in the spleen was significantly reduced, while in cardiac tissue, intestine, and liver the number was significantly increased compared with infected control animals. These results indicate that CM has a negative influence on the host-parasite relationship, modifying the tropism of the parasite for tissues, and increasing the parasitemia peak in this experimental model.     

Involvement of ectonucleotidases and purinergic receptor expression during acute Chagas disease in the cortex of mice treated with resveratrol and benznidazole

Chagas disease (CD) is caused by the parasite Trypanosoma cruzi. CD affects people worldwide, primarily in tropical areas. The central nervous system (CNS) is an essential site for T. cruzi persistence during infection. The protozoan may pass through the blood–brain barrier and may cause motor and cognitive neuronal damage. Once in the CNS, T. cruzi triggers immune responses that the purinergic system can regulate. Treatment for CD is based on benznidazole (BNZ); however, this agent has negative side-effects and is toxic to the host. For this reason, we investigated whether resveratrol (RSV), a potent antioxidant and neuroprotective molecule, would modulate purinergic signaling and RSV alone or in combination with BNZ would prevent changes in purinergic signaling and oxidative damage caused by T. cruzi. We infected mice with T. cruzi and treated them with RSV or BNZ for 8 days. Increases in ATP and ADP hydrolysis by NTPDase in the total cortex of infected animals were observed. The treatment with RSV in infected group diminished ATP, ADP, and AMP hydrolysis compared to infected group. The combination of RSV + BNZ decreased AMP hydrolysis in infected animals compared to the INF group, exerting an anti-inflammatory effect. RSV acted as a neuroprotector, decreasing adenosine levels. Infected animals presented an increase of P2X₇ and A_2A density of purine receptors. RSV reduced P2X₇ and A_2A and increased A1 density receptors in infected animals. In addition, infected animals showed higher TBARS and reactive oxygen species (ROS) levels than control. RSV diminished ROS levels in infected mice, possibly due to antioxidant properties. In short, we conclude that resveratrol could act as a neuroprotective molecule, probably preventing inflammatory changes caused by infection by T. cruzi, even though the mice experienced high levels of parasitemia.     

Paradoxical effects of vitamin C in Chagas disease

Trypanosoma cruzi infection stimulates inflammatory mediators which cause oxidative stress, and the use of antioxidants can minimize the sequelae of Chagas disease. In order to evaluate the efficacy of vitamin C in minimizing oxidative damage in Chagas disease, we orally administered ascorbic acid to Swiss mice infected with 5.0?×?10⁴ trypomastigote forms of T. cruzi QM2 strain. These animals were treated for 60?days to investigate the acute phase and 180?days for the chronic phase. During the acute phase, the animals in the infected and treated groups demonstrated lower parasitemia and inflammatory processes were seen in more mice in these groups, probably due to the higher concentration of nitric oxide, which led to the formation of peroxynitrite. The decrease in reduced glutathione concentration in this group showed a circulating oxidant state, and this antioxidant was used to regenerate vitamin C. During the chronic phase, the animals in the infected and treated group showed a decrease in ferric reducing ability of plasma and uric acid concentrations as well as mobilization of bilirubin (which had higher plasma concentration), demonstrating cooperation between endogenous non-enzymatic antioxidants to combat increased oxidative stress. However, lower ferrous oxidation in xylenol orange concentrations was found in the infected and treated group, suggesting that vitamin C provided biological protection by clearing the peroxynitrite, attenuating the chronic inflammatory process in the tissues and favoring greater survival in these animals. Complex interactions were observed between the antioxidant systems of the host and parasite, with paradoxical actions of vitamin C.     

Histopathological and parasitological evidence of immunization of mice against challenge with 17 wild isolates of Trypanosoma cruzi

The possibility of preventing chronic infection by a battery of 17 wild isolates of Trypanosoma cruzi was studied in Swiss mice preimmunized with culture forms of an attenuated strain (TCC). Mice were challenged intradermally with low numbers of wild trypomastigotes obtained from naturally infected insect vectors captured within a 57,000 km² subtropical area in northern Argentina and which had not undergone any laboratory propagation. A significant degree of protection was observed in all cases, according to one or more parameters. Immunization reduced the level of parasitemia (P < 0.05) in infections caused by 4 out of 13 isolates, as evaluated by microscope counts performed on fresh blood mounts and in 10 out of 15 isolates as evaluated by xenodiagnosis. A lesser degree of histopathology (P < 0.05) was detected in the heart (7 out of 17 isolates), urinary bladder (10 out of 17 isolates) and skeletal muscle (10 out of 17 isolates). None of these parameters reflected infection or pathology in TCC-immunized, non-challenged mice. While antigenic variation frustrates vaccination against African trypanosomes, the effective protection shown here against 17 T. cruzi primary isolates indicates lack of antigenic variation and thus the possibility of effective vaccination in Chagas' disease.     

PPARγ ligand treatment inhibits cardiac inflammatory mediators induced by infection with different lethality strains of Trypanosoma cruzi

Trypanosoma cruzi (T. cruzi), the etiological agent of Chagas' disease, causes cardiac alterations in the host. Although the main clinical manifestations arise during the chronic stage, the mechanisms leading to heart damage develop early during infection. In fact, an intense inflammatory response is observed from acute stage of infection. Recently, peroxisome proliferator-activated receptors (PPARs) have attracted research interest due to their participation in the modulation of inflammation. In this work we addressed the role of 15-Deoxy-?^12,14 ProstaglandinJ2 (15dPGJ2), a PPARγ natural ligand in the regulation of inflammatory mediators, in acute and chronic experimental mouse models of Chagas' disease with the RA and K98 T. cruzi strains, respectively. This work demonstrates that 15dPGJ2 treatment inhibits the expression and activity of inducible nitric oxide synthase (NOS2) as well as TNF-α and IL-6 mRNA levels. Also, expression and activity of metalloproteinases 2 (MMP-2) and 9 (MMP9) were inhibited by 15dPGJ2. Moreover GW9662, a specific PPARγ antagonist, revealed the participation of other signaling pathways since, in GW9662 presence, 15dPJG2 had a partial effect on the inhibition of inflammatory parameters in the acute model of infection. Accordingly, NF-κB activation was demonstrated, assessing p65 nuclear translocation in the hearts of infected mice with both T. cruzi strains. Such effect was inhibited after 15dPGJ2 treatment. Our findings support the concept that in vivo PPARγ and NF-κB pathways are implicated in the inhibitory effects of 15dPGJ2 on inflammatory mediators at different times depending on whether the infection is caused by the lethal or non-lethal T. cruzi strain.     

Aptamer-Based Detection of Disease Biomarkers in Mouse Models for Chagas Drug Discovery

Drug discovery initiatives, aimed at Chagas treatment, have been hampered by the lack of standardized drug screening protocols and the absence of simple pre-clinical assays to evaluate treatment efficacy in animal models. In this study, we used a simple Enzyme Linked Aptamer (ELA) assay to detect T. cruzi biomarker in blood and validate murine drug discovery models of Chagas disease. In two mice models, Apt-29 ELA assay demonstrated that biomarker levels were significantly higher in the infected group compared to the control group, and upon Benznidazole treatment, their levels reduced. However, biomarker levels in the infected treated group did not reduce to those seen in the non-infected treated group, with 100% of the mice above the assay cutoff, suggesting that parasitemia was reduced but cure was not achieved. The ELA assay was capable of detecting circulating biomarkers in mice infected with various strains of T. cruzi parasites. Our results showed that the ELA assay could detect residual parasitemia in treated mice by providing an overall picture of the infection in the host. They suggest that the ELA assay can be used in drug discovery applications to assess treatment efficacy in-vivo.     

The Receptor Slamf1 on the Surface of Myeloid Lineage Cells Controls Susceptibility to Infection by Trypanosoma cruzi

Trypanosoma cruzi, the protozoan parasite responsible for Chagas'' disease, causes severe myocarditis often resulting in death. Here, we report that Slamf1−/− mice, which lack the hematopoietic cell surface receptor Slamf1, are completely protected from an acute lethal parasite challenge. Cardiac damage was reduced in Slamf1−/− mice compared to wild type mice, infected with the same doses of parasites, as a result of a decrease of the number of parasites in the heart even the parasitemia was only marginally less. Both in vivo and in vitro experiments reveal that Slamf1-defIcient myeloid cells are impaired in their ability to replicate the parasite and show altered production of cytokines. Importantly, IFN-γ production in the heart of Slamf1 deficient mice was much lower than in the heart of wt mice even though the number of infiltrating dendritic cells, macrophages, CD4 and CD8 T lymphocytes were comparable. Administration of an anti-Slamf1 monoclonal antibody also reduced the number of parasites and IFN-γ in the heart. These observations not only explain the reduced susceptibility to in vivo infection by the parasite, but they also suggest human Slamf1 as a potential target for therapeutic target against T. cruzi infection.     

Trypanosoma cruzi: allopurinol in the treatment of mice with experimental acute Chagas disease

The therapeutic effect of allopurinol was studied in an experimental Trypanosoma cruzi infection (Chagas disease) in outbred IVIC-NMRI and inbred C57B1/6J mice intraperitoneally inoculated with the parasites 2–6 days before drug treatment. Allopurinol protected against T. cruzi infection. This effect was evidenced by highly significant reductions in both parasitemias and mortality rates and increased survival time in allopurinol-treated animals compared with untreated infected mice. Allopurinol protected effectively when administered in 10 daily doses of 32–64 mg/kg body wt/day injected intraperitoneally. Using direct methods, parasitemia remained undetectable for at least 310 days. An indirect method, subinoculation to susceptible mice, showed a few circulating trypanosomes which decreased greatly in number after a second schedule of allopurinol treatment; finally no trypanosomes were detectable 275 days after treatment initiation. Allopurinol also induced a strong trypanostatic effect when tested in vitro on five different Trypanosoma cruzi strains (optimal inhibitory concentration: 3 μg/ml). These results suggest that allopurinol protects mice with acute Chagas infection by a direct trypanostatic effect. The low toxicity of this drug suggests its use in more chronic experimental Chagas infections.     

Synergy Testing of FDA-Approved Drugs Identifies Potent Drug Combinations against Trypanosoma cruzi

An estimated 8 million persons, mainly in Latin America, are infected with Trypanosoma cruzi, the etiologic agent of Chagas disease. Existing antiparasitic drugs for Chagas disease have significant toxicities and suboptimal effectiveness, hence new therapeutic strategies need to be devised to address this neglected tropical disease. Due to the high research and development costs of bringing new chemical entities to the clinic, we and others have investigated the strategy of repurposing existing drugs for Chagas disease. Screens of FDA-approved drugs (described in this paper) have revealed a variety of chemical classes that have growth inhibitory activity against mammalian stage Trypanosoma cruzi parasites. Aside from azole antifungal drugs that have low or sub-nanomolar activity, most of the active compounds revealed in these screens have effective concentrations causing 50% inhibition (EC₅₀''s) in the low micromolar or high nanomolar range. For example, we have identified an antihistamine (clemastine, EC₅₀ of 0.4 µM), a selective serotonin reuptake inhibitor (fluoxetine, EC₅₀ of 4.4 µM), and an antifolate drug (pyrimethamine, EC₅₀ of 3.8 µM) and others. When tested alone in the murine model of Trypanosoma cruzi infection, most compounds had insufficient efficacy to lower parasitemia thus we investigated using combinations of compounds for additive or synergistic activity. Twenty-four active compounds were screened in vitro in all possible combinations. Follow up isobologram studies showed at least 8 drug pairs to have synergistic activity on T. cruzi growth. The combination of the calcium channel blocker, amlodipine, plus the antifungal drug, posaconazole, was found to be more effective at lowering parasitemia in mice than either drug alone, as was the combination of clemastine and posaconazole. Using combinations of FDA-approved drugs is a promising strategy for developing new treatments for Chagas disease.     

The Effects of Bacterial Endotoxin on the Infection of Mice with Trypanosoma cruzi*

Mice (Rockland strain) infected with Trypanosoma cruzi strain Tulahuén were treated with Escherichia coli endotoxin before, simultaneously with, and after inoculation of the parasites. The peak parasitemias of endotoxin-treated mice were higher than those of nontreated infected animals, regardless of the time of endotoxin administration. Peak parasitemias occurred at the same time in infected nontreated mice as in animals given endotoxin before or simultaneously with the trypanosomes. If endotoxin was administered 24 hr after the infection, a delay in the peak parasitemia was noted. Changes in the survival time were not observed unless endotoxin was given 24 hr postinfection. Infected mice had an increasing susceptibility to the lethal effect of endotoxin. The LD₅₀ of endotoxin decreased from 675 μg for normal mice to 230, 92, and 18 μg for infected animals 1, 3, and 8 days after the infection, respectively. In the infected mice, the endotoxin-detoxifying ability of the spleen was found to be impaired.     

Inducible nitric oxide synthase in heart tissue and nitric oxide in serum of Trypanosoma cruzi-infected rhesus monkeys: association with heart injury

Background

The factors contributing to chronic Chagas'' heart disease remain unknown. High nitric oxide (NO) levels have been shown to be associated with cardiomyopathy severity in patients. Further, NO produced via inducible nitric oxide synthase (iNOS/NOS2) is proposed to play a role in Trypanosoma cruzi control. However, the participation of iNOS/NOS2 and NO in T. cruzi control and heart injury has been questioned. Here, using chronically infected rhesus monkeys and iNOS/NOS2-deficient (Nos2 ^−/−) mice we explored the participation of iNOS/NOS2-derived NO in heart injury in T. cruzi infection.

Methodology

Rhesus monkeys and C57BL/6 and Nos2 ^−/− mice were infected with the Colombian T. cruzi strain. Parasite DNA was detected by polymerase chain reaction, T. cruzi antigens and iNOS/NOS2⁺ cells were immunohistochemically detected in heart sections and NO levels in serum were determined by Griess reagent. Heart injury was assessed by electrocardiogram (ECG), echocardiogram (ECHO), creatine kinase heart isoenzyme (CK-MB) activity levels in serum and connexin 43 (Cx43) expression in the cardiac tissue.

Results

Chronically infected monkeys presented conduction abnormalities, cardiac inflammation and fibrosis, which resembled the spectrum of human chronic chagasic cardiomyopathy (CCC). Importantly, chronic myocarditis was associated with parasite persistence. Moreover, Cx43 loss and increased CK-MB activity levels were primarily correlated with iNOS/NOS2⁺ cells infiltrating the cardiac tissue and NO levels in serum. Studies in Nos2 ^−/− mice reinforced that the iNOS/NOS2-NO pathway plays a pivotal role in T. cruzi-elicited cardiomyocyte injury and in conduction abnormalities that were associated with Cx43 loss in the cardiac tissue.

Conclusion

T. cruzi-infected rhesus monkeys reproduce features of CCC. Moreover, our data support that in T. cruzi infection persistent parasite-triggered iNOS/NOS2 in the cardiac tissue and NO overproduction might contribute to CCC severity, mainly disturbing of the molecular pathway involved in electrical synchrony. These findings open a new avenue for therapeutic tools in Chagas'' heart disease.     

The onset of immune protection in acute experimental chagas' disease in C3H(HE) mice

The onset of protective immunity against Trypanosoma cruzi in mice was determined by adoptively immunizing newly infected recipients with spleen cells from normal or infected donor mice. It was found that spleen cells from animals with 3 day and 6 day infections did not provide protection but that spleen cells from infections of 9, 12, 15 and 18 days significantly increased longevity in infected recipient animals. The protective capacity per spleen cell was found to increase in proportion to the duration of infection of donor mice. It was further noted that immune protection, as reflected in increased longevity, did not result in decreased development of parasitemia. Immunized mice which demonstrated the greatest longevity developed parasitemias over twice that observed in contrrol groups.     

Coinfection with different Trypanosoma cruzi strains interferes with the host immune response to infection

A century after the discovery of Trypanosoma cruzi in a child living in Lassance, Minas Gerais, Brazil in 1909, many uncertainties remain with respect to factors determining the pathogenesis of Chagas disease (CD). Herein, we simultaneously investigate the contribution of both host and parasite factors during acute phase of infection in BALB/c mice infected with the JG and/or CL Brener T. cruzi strains. JG single infected mice presented reduced parasitemia and heart parasitism, no mortality, levels of pro-inflammatory mediators (TNF-α, CCL2, IL-6 and IFN-γ) similar to those found among naïve animals and no clinical manifestations of disease. On the other hand, CL Brener single infected mice presented higher parasitemia and heart parasitism, as well as an increased systemic release of pro-inflammatory mediators and higher mortality probably due to a toxic shock-like systemic inflammatory response. Interestingly, coinfection with JG and CL Brener strains resulted in intermediate parasitemia, heart parasitism and mortality. This was accompanied by an increase in the systemic release of IL-10 with a parallel increase in the number of MAC-3⁺ and CD4⁺ T spleen cells expressing IL-10. Therefore, the endogenous production of IL-10 elicited by coinfection seems to be crucial to counterregulate the potentially lethal effects triggered by systemic release of pro-inflammatory mediators induced by CL Brener single infection. In conclusion, our results suggest that the composition of the infecting parasite population plays a role in the host response to T. cruzi in determining the severity of the disease in experimentally infected BALB/c mice. The combination of JG and CL Brener was able to trigger both protective inflammatory immunity and regulatory immune mechanisms that attenuate damage caused by inflammation and disease severity in BALB/c mice.