The effects of inflammation on connexin 43 in chronic Chagas disease cardiomyopathy.

Background: Cardiac arrhythmias are the main cause of sudden death due to Chronic Chagasic Cardiomyopathy (CCC). Here we investigated alterations in connexin 43 (Cx43) expression and phosphorylation in cardiomyocytes as well as associations with cardiac arrhythmias in CCC. Methods: C57Bl/6 mice infected with Trypanosoma cruzi underwent cardiac evaluations at 6 and 12 months after infection via treadmill testing and EKG. Histopathology, cytokine gene expression, and distribution of total Cx43 and its phosphorylated forms Cx43S368 and Cx43S325/328/330 were investigated. Human heart samples obtained from subjects with CCC were submitted to immunofluorescence analysis. In vitro simulation of a pro-inflammatory microenvironment (IL-1ß, TNF, and IFN-γ) was performed in H9c2 cells and iPSC-derived cardiomyocytes to evaluate Cx43 distribution, action potential duration, and Lucifer Yellow dye transfer. Results: Mice chronically infected with T. cruzi exhibited impaired cardiac function associated with increased inflammation, fibrosis and upregulated IL-1ß, TNF, and IFN-γ gene expression. Confocal microscopy revealed altered total Cx43, Cx43S368 and Cx43S325/328/330 localization and phosphorylation patterns in CCC, with dispersed staining outside the intercalated disc areas, i.e., in lateral membranes and the cytoplasm. Reduced co-localization of total Cx43 and N-cadherin was observed in the intercalated discs of CCC mouse hearts compared to controls. Similar results were obtained in human CCC heart samples, which showed Cx43 distribution outside the intercalated discs. Stimulation of human iPSC-derived cardiomyocytes or H9c2 cells with IL-1ß, TNF, and IFN-γ induced alterations in Cx43 localization, reduced action potential duration and dye transfer between adjacent cells. Conclusion: Heart inflammation in CCC affects the distribution and phosphorylation pattern of Cx43, which may contribute to the generation of conduction disturbances in Chagas disease.

Pirfenidone Prevents Heart Fibrosis during Chronic Chagas Disease Cardiomyopathy.

Cardiac fibrosis is a severe outcome of Chagas disease (CD), caused by the protozoan Trypanosoma cruzi. Clinical evidence revealed a correlation between fibrosis levels with impaired cardiac performance in CD patients. Therefore, we sought to analyze the effect of inhibitors of TGF-ß (pirfenidone), p38-MAPK (losmapimod) and c-Jun (SP600125) on the modulation of collagen deposition in cardiac fibroblasts (CF) and in vivo models of T. cruzi chronic infection. Sirius Red/Fast Green dye was used to quantify both collagen expression and total protein amount, assessing cytotoxicity. The compounds were also used to treat C57/Bl6 mice chronically infected with T. cruzi, Brazil strain. We identified an anti-fibrotic effect in vitro for pirfenidone (TGF-ß inhibitor, IC50 114.3 µM), losmapimod (p38 inhibitor, IC50 17.6 µM) and SP600125 (c-Jun inhibitor, IC50 3.9 µM). This effect was independent of CF proliferation since these compounds do not affect T. cruzi-induced host cell multiplication as measured by BrdU incorporation. Assays of chronic infection of mice with T. cruzi have shown a reduction in heart collagen by pirfenidone. These results propose a novel approach to fibrosis therapy in CD, with the prospect of repurposing pirfenidone to prevent the onset of ECM accumulation in the hearts of the patients.

Identification of host antioxidant effectors as thioridazine targets: Impact on cardiomyocytes infection and Trypanosoma cruzi-induced acute myocarditis.

Phenothiazines inhibit antioxidant enzymes in trypanosomatids. However, potential interferences with host cell antioxidant defenses are central concerns in using these drugs to treat Trypanosoma cruzi-induced infectious myocarditis. Thus, the interaction of thioridazine (TDZ) with T. cruzi and cardiomyocytes antioxidant enzymes, and its impact on cardiomyocytes and cardiac infection was investigated in vitro and in vivo. Cardiomyocytes and trypomastigotes in culture, and mice treated with TDZ and benznidazole (Bz, reference antiparasitic drug) were submitted to microstructural, biochemical and molecular analyses. TDZ was more cytotoxic and less selective against T. cruzi than Bz in vitro. TDZ-pretreated cardiomyocytes developed increased infection rate, reactive oxygen species (ROS) production, lipid and protein oxidation; similar catalase (CAT) and superoxide dismutase (SOD) activity, and reduced glutathione's (peroxidase - GPx, S-transferase - GST, and reductase - GR) activity than infected untreated cells. TDZ attenuated trypanothione reductase activity in T. cruzi, and protein antioxidant capacity in cardiomyocytes, making these cells more susceptible to H2O2-based oxidative challenge. In vivo, TDZ potentiated heart parasitism, total ROS production, myocarditis, lipid and protein oxidation; as well as reduced GPx, GR, and GST activities compared to untreated mice. Benznidazole decreased heart parasitism, total ROS production, heart inflammation, lipid and protein oxidation in T. cruzi-infected mice. Our findings indicate that TDZ simultaneously interact with enzymatic antioxidant targets in cardiomyocytes and T. cruzi, potentiating the infection by inducing antioxidant fragility and increasing cardiomyocytes and heart susceptibility to parasitism, inflammation and oxidative damage.

The mitochondrial uncoupler 2,4-dinitrophenol modulates inflammatory and oxidative responses in Trypanosoma cruzi-induced acute myocarditis in mice.

By uncoupling oxidative phosphorylation, 2,4-dinitrophenol (DNP) attenuates reactive oxygen species (ROS) biosynthesis, which are known to aggravate infectious myocarditis in Chagas disease. Thus, the impact of DNP-based chemotherapy on Trypanosoma cruzi-induced acute myocarditis was investigated. C56BL/6 mice uninfected and infected untreated and treated daily with 100 mg/kg benznidazole (Bz, reference drug), 5 and 10 mg/kg DNP by gavage for 11 days after confirmation of T. cruzi infection were investigated. Twenty-four hours ​after the last treatment, the animals were euthanized and the heart was collected for microstructural, immunological and biochemical analyses. T. cruzi inoculation induced systemic inflammation (e.g., cytokines and anti-T. cruzi IgG upregulation), cardiac infection (T. cruzi DNA), oxidative stress, inflammatory infiltrate and microstructural myocardial damage in untreated mice. DNP treatment aggravated heart infection and microstructural damage, which were markedly attenuated by Bz. DNP (10 mg/kg) was also effective in attenuating ROS (total ROS, H2O2, and O2-), nitric oxide (NO), lipid (malondialdehyde - MDA) and protein (protein carbonyl - PCn) oxidation, TNF, IFN-γ, IL-10, and MCP-1/CCL2, anti-T. cruzi IgG, cardiac troponin I levels, as well as inflammatory infiltrate and cardiac damage in T. cruzi-infected mice. Our findings indicate that DNP aggravated heart infection and microstructural cardiomyocytes damage in infected mice. These responses were related to the antioxidant and anti-inflammatory properties of DNP, which favors infection by weakening the pro-oxidant and pro-inflammatory protective mechanisms of the infected host. Conversely, Bz-induced cardioprotective effects combined effective anti-inflammatory and antiparasitic responses, which protect against heart infection, oxidative stress, and microstructural damage in Chagas disease.

Oxidative stress, cardiomyocytes premature senescence and contractile dysfunction in in vitro and in vivo experimental models of Chagas disease.

AIMS: The relationship between redox imbalance and cardiovascular senescence in infectious myocarditis is unknown. Thus, the aim of this study was to investigate whether cardiomyocytes parasitism, oxidative stress and contractile dysfunction can be correlated to senescence-associated ß-galactosidase (SA-ß-Gal) activity in Trypanosoma cruzi-infection in vitro and in vivo. METHODS: Uninfected, T. cruzi-infected untreated and benznidazole (BZN)-treated H9c2 cardiomyocytes and rats were investigated. Parasitological, prooxidant, antioxidant, microstructural, and senescence-associated markers were quantified in vitro and in vivo. RESULTS: T. cruzi infection triggered intense cardiomyocytes parasitism in vitro and in vivo, which was accompanied by reactive oxygen species (ROS) upregulation, lipids, proteins and DNA oxidation in cardiomyocytes and cardiac tissue. Oxidative stress was parallel to microstructural cell damage (e.g., increased cardiac toponin I levels) and contractile dysfunction in cardiomyocytes in vitro and in vivo, whose severity accompanied a premature cellular senescence-like phenotype revealed by increased senescence-associated ß-galactosidase (SA-ß-Gal) activity and DNA oxidation (8-OHdG). Cellular parasitism (e.g., infection rate and parasite load), myocarditis and T. cruzi-induced prooxidant responses were attenuated by early BZN administration to interrupt the progression of T. cruzi infection, protecting against SA-ß-gal-based premature cellular senescence, microstructural damage and contractile deterioration in cardiomyocytes from T. cruzi-infected animals. CONCLUSION: Our findings indicated that cell parasitism, redox imbalance and contractile dysfunction were correlated to SA-ß-Gal-based cardiomyocytes premature senescence in acute T. cruzi infection. Therefore, in addition to controlling parasitism, inflammation and oxidative stress; inhibiting cardiomyocytes premature senescence should be further investigated as an additional target of specific Chagas disease therapeutics.

Macrophages Mediate Healing Properties of Fenofibrate in Experimental Chagasic Cardiomyopathy.

Chronic cardiomyopathy is one of the most relevant outcomes of Chagas disease associated with parasite persistence and exacerbated inflammatory response. Fenofibrate, a third generation fibric acid derivative and peroxisome proliferator-activated receptor-α ligand, is involved in the regulation of inflammatory response. However, the participation of macrophages in this scenario has not been elucidated. Here we show, for the first time, that macrophages play a fundamental role in the fenofibrate-mediated modulation of heart pro-inflammatory response and fibrosis caused by the infection with Trypanosoma cruzi. Furthermore, macrophages are required for fenofibrate to improve the loss of ventricular function and this restoration correlates with an anti-inflammatory microenvironment. Understanding the contributions of macrophages to the healing properties of fenofibrate reinforces its potential use as a therapeutic drug, with the aim of helping to solve a public health problem, such as chronic Chagas disease.

Long-term cardiology outcomes in children after early treatment for Chagas disease, an observational study.

BACKGROUND: Parasite persistence after acute infection with Trypanosoma cruzi is an important factor in the development of Chagas disease (CD) cardiomyopathy. Few studies have investigated the clinical effectiveness of CD treatment through the evaluation of cardiological events by long term follow-up of treated children. Cardiological evaluation in children is challenging since features that would be diagnosed as abnormal in an adult's ECG may be normal, age-related findings in a pediatric ECG trace. The objective was to evaluate cardiac involvement in patients with Chagas disease with a minimum follow-up of 6 years post-treatment. METHODOLOGY: A descriptive study of a cohort of pediatric patients with CD treated with benznidazole (Bz) or nifurtimox (Nf) was conducted. Children (N = 234) with at least 6 years post CD treatment followed at the Parasitology and Chagas Service, Buenos Aires Children's Hospital (Argentina) were enrolled. By convenience sampling, children who attended a clinical visit between August 2015 and November 2019 were also invited to participate for additional cardiovascular studies like 24-hour Holter monitoring and speckle-tracking 2D echocardiogram (STE). Benznidazole was prescribed in 171 patients and nifurtimox in 63 patients. Baseline parasitemia data was available for 168/234 patients. During the follow-up period, alterations in routine ECG were observed in 11/234 (4.7%, 95% CI [2-7.4%]) patients. In only four patients, with complete right bundle branch block (cRBBB) and left anterior fascicular block (LAFB), ECG alterations were considered probably related to CD. During follow-up, 129/130 (99%) treated patients achieved persistent negative parasitemia by qPCR. Also decrease in T.cruzi antibodies titers was observed in all patients and negative seroconversion occurred in 123/234 (52%) patients. CONCLUSIONS: A low incidence of cardiological lesions related to CD was observed in patients treated early for pediatric CD. This suggests a protective effect of parasiticidal treatment on the development of cardiological lesions and highlights the importance of early treatment of infected children. TRIAL REGISTRATION: ClinicalTrials.gov NCT04090489.

Correlation of TcII discrete typing units with severe chronic Chagas cardiomyopathy in patients from various Brazilian geographic regions.

BACKGROUND: Chagas disease (ChD) is caused by Trypanosoma cruzi. The genetic structure of the species is divided into seven distinct genetic groups, TcI to TcVI, and Tcbat, which have shown differences in terms of geographic distribution, biological properties, and susceptibility to drugs. However, the association between genetic variability and clinical forms of ChD has not yet been fully elucidated. The predominance of TcII and TcVI discrete typing units (DTUs) (genetic groups) is known to occur in several Brazilian regions and is associated with both the domestic and the wild cycles of ChD. Thus, this study aimed to verify the genotypes of the parasites present in 330 patients with chronic Chagas cardiomyopathy (CCC) from different Brazilian states attended at the Clinical Hospital of the Ribeirão Preto Medical School and to assess the existence of a correlation between the clinical forms with the main cardiovascular risk factors and the genetics of the parasite. METHODOLOGY PRINCIPAL FINDINGS: All patients with CCC were clinically evaluated through anamnesis, physical examination, biochemical tests, 12-lead electrocardiogram, echocardiogram and chest X-ray. Peripheral blood (5 mL) was collected in guanidine/ethylenediaminetetraacetic acid from each patient for DNA extraction and real-time polymerase chain reaction (PCR) for Chagas disease and genotyping of the parasite in the 7 DTUs. Parasite genotyping was performed using conventional multilocus PCR. Samples of only 175 patients were positive after amplification of the specific genes contained in the T. cruzi genotyping criteria. TcII (64/175), TcVI (9/175), and TcI (3/175) DTUs were predominant, followed by TcII/TcV/TcVI (74/175), and TcII/TcVI (23/175). The TcIII and TcIV DTU´s was detected in only one sample of CCC patients. CONCLUSIONS/SIGNIFICANCE: Our data corroborate previous findings, indicating the predominance of the TcII genotype in patients with CCC of Brazilian origin. Moreover, this study pioneered disclosing a direct correlation between the TcII DTU and severe CCC.

Insights from the use of erythropoietin in experimental Chagas disease.

In addition to the long-established role in erythropoiesis, erythropoietin (Epo) has protective functions in a variety of tissues, including the heart. This is the most affected organ in chronic Chagas disease, caused by the protozoan Trypanosoma cruzi. Despite seven million people being infected with T. cruzi worldwide, there is no effective treatment preventing the disease progression to the chronic phase when the pathological involvement of the heart is often observed. Chronic chagasic cardiomyopathy has a wide variety of manifestations, like left ventricular systolic dysfunction, dilated cardiomyopathy, and heart failure. Since Epo may help maintain cardiac function by reducing myocardial necrosis, inflammation, and fibrosis, this study aimed to evaluate whether the Epo has positive effects on experimental Chagas disease. For that, we assessed the earlier (acute phase) and also the later (chronic phase) use of Epo in infected C57BL/6 mice. Blood cell count, biochemical parameters, parasitic load, and echocardiography data were evaluated. In addition, histopathological analysis was carried out. Our data showed that Epo had no trypanocide effect nor did it modify the production of anti-T. cruzi antibodies. Epo-treated groups exhibited parasitic burden much lower in the heart compared to blood. No pattern of hematological changes was observed combining infection with treatment with Epo. Chronic Epo administration reduced CK-MB serum activity from d0 to d180, irrespectively of T. cruzi infection. Likewise, echocardiography and histological results indicate that Epo treatment is more effective in the chronic phase of experimental Chagas disease. Since treatment is one of the greatest challenges of Chagas disease, alternative therapies should be investigated, including Epo combined with benznidazole.

Trypanosoma cruzi-specific CD8+ T cells and other immunological hallmarks in chronic Chagas cardiomyopathy: Two decades of research.

Trypanosoma cruzi, the causal agent of Chagas disease, has coexisted with humans for thousands of years. Therefore, the parasite has developed several mechanisms of antigenic variability that has allowed it to live inside the cells and evade the host immune response. Since T. cruzi displays an intracellular cycle-stage, our research team focused on providing insights into the CD8+ T cells immune response in chronic Chagas cardiomyopathy. We began our work in the 2000s studying parasite antigens that induce natural immune responses such as the KMP11 protein and TcTLE, its N-terminal derived peptide. Different approaches allowed us to reveal TcTLE peptide as a promiscuous CD8+ T cell epitope, able of inducing multifunctional cellular immune responses and eliciting a humoral response capable of decreasing parasite movement and infective capacity. Next, we demonstrated that as the disease progresses, total CD8+ T cells display a dysfunctional state characterized by a prolonged hyper-activation state along with an increase of inhibitory receptors (2B4, CD160, PD-1, TIM-3, CTLA-4) expression, an increase of specific terminal effector T cells (TTE), a decrease of proliferative capacity, a decrease of stem cell memory (TSCM) frequency, and a decrease of CD28 and CD3ζ expression. Thus, parasite-specific CD8+ T cells undergo clonal exhaustion, distinguished by an increase in late-differentiated cells, a mono-functional response, and enhanced expression of inhibitory receptors. Finally, it was found that anti-parasitic treatment induces an improved CD8+ T cell response in asymptomatic individuals, and a mouse animal model led us to establish a correlation between the quality of the CD8+ T cell responses and the outcome of chronic infection. In the future, using OMICs strategies, the identification of the specific cellular signals involved in disease progression will provide an invaluable resource for discovering new biomarkers of progression or new vaccine and immunotherapy strategies. Also, the inclusion of the TcTLE peptide in the rational design of epitope-based vaccines, the development of immunotherapy strategies using TSCM or the blocking of inhibitory receptors, and the use of the CD8+ T cell response quality to follow treatments, immunotherapies or vaccines, all are alternatives than could be explored in the fight against Chagas disease.

Distinct CD4-CD8- (Double-Negative) Memory T-Cell Subpopulations Are Associated With Indeterminate and Cardiac Clinical Forms of Chagas Disease.

CD4-CD8- (double-negative, DN) T cells are critical orchestrators of the cytokine network associated with the pathogenic inflammatory response in one of the deadliest cardiomyopathies known, Chagas heart disease, which is caused by Trypanosoma cruzi infection. Here, studying the distribution, activation status, and cytokine expression of memory DN T-cell subpopulations in Chagas disease patients without cardiac involvement (indeterminate form-IND) or with Chagas cardiomyopathy (CARD), we report that while IND patients displayed a higher frequency of central memory, CARD had a high frequency of effector memory DN T cells. In addition, central memory DN T cells from IND displayed a balanced cytokine profile, characterized by the concomitant expression of IFN-γ and IL-10, which was not observed in effector memory DN T cells from CARD. Supporting potential clinical relevance, we found that the frequency of central memory DN T cells was associated with indicators of better ventricular function, while the frequency of effector memory DN T cells was not. Importantly, decreasing CD1d-mediated activation of DN T cells led to an increase in IL-10 expression by effector memory DN T cells from CARD, restoring a balanced profile similar to that observed in the protective central memory DN T cells. Targeting the activation of effector memory DN T cells may emerge as a strategy to control inflammation in Chagas cardiomyopathy and potentially in other inflammatory diseases where these cells play a key role.

Aspirin-triggered resolvin D1 reduces parasitic cardiac load by decreasing inflammation in a murine model of early chronic Chagas disease.

BACKGROUND: Chagas disease, caused by the protozoan Trypanosoma cruzi, is endemic in Latin America and is widely distributed worldwide because of migration. In 30% of cases, after years of infection and in the absence of treatment, the disease progresses from an acute asymptomatic phase to a chronic inflammatory cardiomyopathy, leading to heart failure and death. An inadequate balance in the inflammatory response is involved in the progression of chronic Chagas cardiomyopathy. Current therapeutic strategies cannot prevent or reverse the heart damage caused by the parasite. Aspirin-triggered resolvin D1 (AT-RvD1) is a pro-resolving mediator of inflammation that acts through N-formyl peptide receptor 2 (FPR2). AT-RvD1 participates in the modification of cytokine production, inhibition of leukocyte recruitment and efferocytosis, macrophage switching to a nonphlogistic phenotype, and the promotion of healing, thus restoring organ function. In the present study, AT-RvD1 is proposed as a potential therapeutic agent to regulate the pro-inflammatory state during the early chronic phase of Chagas disease. METHODOLOGY/PRINCIPAL FINDINGS: C57BL/6 wild-type and FPR2 knock-out mice chronically infected with T. cruzi were treated for 20 days with 5 µg/kg/day AT-RvD1, 30 mg/kg/day benznidazole, or the combination of 5 µg/kg/day AT-RvD1 and 5 mg/kg/day benznidazole. At the end of treatment, changes in immune response, cardiac tissue damage, and parasite load were evaluated. The administration of AT-RvD1 in the early chronic phase of T. cruzi infection regulated the inflammatory response both at the systemic level and in the cardiac tissue, and it reduced cellular infiltrates, cardiomyocyte hypertrophy, fibrosis, and the parasite load in the heart tissue. CONCLUSIONS/SIGNIFICANCE: AT-RvD1 was shown to be an attractive therapeutic due to its regulatory effect on the inflammatory response at the cardiac level and its ability to reduce the parasite load during early chronic T. cruzi infection, thereby preventing the chronic cardiac damage induced by the parasite.

Inhibition of calcium/calmodulin (Ca2+ /CaM)-Calcium/calmodulin-dependent protein kinase II (CaMKII) axis reduces in vitro and ex vivo arrhythmias in experimental Chagas disease.

Chagasic cardiomyopathy (CCC) is one of the main causes of heart failure and sudden death in Latin America. To date, there is no available medication to prevent or reverse the onset of cardiac symptoms. CCC occurs in a scenario of disrupted calcium dynamics and enhanced oxidative stress, which combined, may favor the hyper activation of calcium/calmodulin (Ca2+ /CaM)-calcium/calmodulin-dependent protein kinase II (CaMKII) (Ca2+ /CaM-CaMKII) pathway, which is fundamental for heart physiology and it is implicated in other cardiac diseases. Here, we evaluated the association between Ca2+ /CaM-CaMKII in the electro-mechanical (dys)function of the heart in the early stage of chronic experimental Trypanosoma cruzi infection. We observed that in vitro and ex vivo inhibition of Ca2+ /CaM-CaMKII reversed the arrhythmic profile of isolated hearts and isolated left-ventricles cardiomyocytes. The benefits of the limited Ca2+ /CaM-CaMKII activation to cardiomyocytes' electrical properties are partially related to the restoration of Ca2+ dynamics in a damaged cellular environment created after T. cruzi infection. Moreover, Ca2+ /CaM-CaMKII inhibition prevented the onset of arrhythmic contractions on isolated heart preparations of chagasic mice and restored the responsiveness to the increase in the left-ventricle pre-load. Taken together, our data provide the first experimental evidence for the potential of targeting Ca2+ /CaM-CaMKII pathway as a novel therapeutic target to treat CCC.

α-Gal immunization positively impacts Trypanosoma cruzi colonization of heart tissue in a mouse model.

Chagas disease, caused by the parasite Trypanosoma cruzi, is considered endemic in more than 20 countries but lacks both an approved vaccine and limited treatment for its chronic stage. Chronic infection is most harmful to human health because of long-term parasitic infection of the heart. Here we show that immunization with a virus-like particle vaccine displaying a high density of the immunogenic α-Gal trisaccharide (Qß-αGal) induced several beneficial effects concerning acute and chronic T. cruzi infection in α1,3-galactosyltransferase knockout mice. Approximately 60% of these animals were protected from initial infection with high parasite loads. Vaccinated animals also produced high anti-αGal IgG antibody titers, improved IFN-γ and IL-12 cytokine production, and controlled parasitemia in the acute phase at 8 days post-infection (dpi) for the Y strain and 22 dpi for the Colombian strain. In the chronic stage of infection (36 and 190 dpi, respectively), all of the vaccinated group survived, showing significantly decreased heart inflammation and clearance of amastigote nests from the heart tissue.

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.

Polymorphism in the catalytic subunit of the PI3Kγ gene is associated with Trypanosoma cruzi-induced chronic chagasic cardiomyopathy.

Chagas disease is caused by the protozoan parasite Trypanosoma cruzi. During the chronic phase of disease, while most infected people do not present symptoms, characterizing the asymptomatic form, some patients develop the cardiac form or chronic chagasic cardiomyopathy, which is considered the most severe manifestation of this disease. Considering that the activation of the PI3Kγ signaling pathway is essential for an efficient immune response against T. cruzi infection, we evaluated the PIK3CG C > T (rs1129293) polymorphism in exon 3 of this gene, which encodes the catalytic subunit of PI3Kγ. The PIK3CG CT and TT genotypes were found to be associated with an increased risk of developing the cardiac form of the disease rather than the asymptomatic or digestive forms. In conclusion, the presence of the T allele at single or double doses may differentiate the cardiac from other clinical manifestations of Chagas disease. This finding should help in further studies to evaluate the mechanisms underlying the differential association of PIK3CG in Chagas disease.

Dual chemotherapy with benznidazole at suboptimal dose plus curcumin nanoparticles mitigates Trypanosoma cruzi-elicited chronic cardiomyopathy.

Curcumin (Cur) is a natural polyphenolic flavonoid isolated from the rhizomes of Curcuma longa. Its anti-inflammatory and cardioprotective properties are increasingly considered to have beneficial effects on the progression of cardiomyopathy associated with Chagas disease, caused by Trypanosoma cruzi. However, the Cur therapeutic limitation is its bioavailability and new Cur nanomedicine formulations are developed to overcome this obstacle. In this research, we provide evidence showing that oral therapy with a suboptimal dose of the standard parasiticidal drug benznidazole (BZ) in combination with Cur-loaded nanoparticles is capable of reducing myocardial parasite load, cardiac hypertrophy, inflammation and fibrosis in mice with long-term infection by T. cruzi. Treatment with BZ plus Cur was highly effective in downregulating myocardial expression of proinflammatory cytokines/chemokines (IL-1ß, TNF-α, IL-6, CCL5), and the level/activity of matrix metalloproteinases (MMP-2, MMP-9) and inducible enzymes (cyclooxygenase, nitric oxide synthase) implicated in leukocyte recruitment and cardiac remodeling. Oral administration of a Cur-based nanoformulation displays potential as a complementary strategy to the conventional BZ chemotherapy in the treatment of chronic Chagas heart disease.

Exercise training improves microvascular function in patients with Chagas heart disease: Data from the PEACH study.

BACKGROUND: Chagas heart disease (CHD) impairs the systemic microvascular function. We investigated the effects of exercise training on cutaneous microvascular function among patients with CHD. METHODS: Patients from the PEACH study were randomly assigned to a supervised exercise training 3 times/week for 6 months (Trained; n = 10) or a control group (Untrained; n = 8). Both groups underwent evaluation of microvascular function before, and at 3- and 6-months of follow-up. Cutaneous vascular conductance (CVC) was assessed in the skin of the forearm using laser speckle contrast imaging coupled with iontophoresis of acetylcholine (ACh), sodium nitroprusside (SNP) and during post-occlusive reactive hyperemia (PORH). RESULTS: At 3-months of follow-up, no difference was detected between groups in CVC responses to ACh (p = 0.50), SNP (p = 0.26) and HRPO (p = 0.65). However, at 6-months of follow-up, trained vs. untrained patients improved CVC induced by SNP-iontophoresis (0.19 ± 0.10 vs. 0.14 ± 0.15 APU.mmHg-1; p = 0.05) and PORH (0.63 ± 0.15 vs. 0.48 ± 0.18 APU.mmHg-1; p = 0.05). CVC response to ACh-iontophoresis was similar between groups (0.19 ± 0.11 vs. 0.22 ± 0.17 APU.mmHg-1; p = 0.38). CONCLUSION: Exercise training performed during 6 months improved the cutaneous microvascular function of CHD patients. Further studies evaluating the mechanism involved in this response are warranted.

Case Report: COVID-19 and Chagas Disease in Two Coinfected Patients.

American trypanosomiasis, also named Chagas disease (CD), is an anthropozoonosis caused by the protozoan parasite Trypanosoma cruzi. The disease affects millions of people worldwide, leading yearly to approximately 50,000 deaths. COVID-19, generated by SARS-CoV-2, can lead to lymphopenia and death. We hereby describe the first report of two patients with CD and COVID-19 coinfection, from hospitalization until patients' death.

Circulating Trypanosoma cruzi load and major cardiovascular outcomes in patients with chronic Chagas cardiomyopathy: a prospective cohort study.

OBJECTIVES: To analyse the effect of parasite load assessed by quantitative reverse transcription PCR (RT-qPCR) in serum on the prognosis of patients with chronic Chagas cardiomyopathy (CCM) after a 2-year follow-up. METHODS: Prospective cohort study conducted between 2015 and 2017. One hundred patients with CCM were included. Basal parasitaemia levels of Trypanosoma cruzi (T. cruzi) were measured using a quantitative polymerase chain reaction (qPCR) test. The primary composite outcome (CO) was all-cause mortality, cardiac transplantation and implantation of a left ventricular assist device. Secondary outcomes were the baseline levels of serum biomarkers and echocardiographic variables. RESULTS: After a 2 years of follow-up, the primary CO rate was 16%. A positive qPCR was not associated with a higher risk of the CO. However, when parasitaemia was evaluated by comparing tertiles (tertile 1: undetectable parasitaemia, tertile 2: low parasitaemia and tertile 3: high parasitaemia), a higher risk of the CO (HR 3.66; 95% CI 1.11-12.21) was evidenced in tertile 2. Moreover, patients in tertile 2 had significantly higher levels of high-sensitivity troponin T and cystatin C and more frequently exhibited an ejection fraction <50%. CONCLUSION: Low parasitaemia was associated with severity markers of myocardial injury and a higher risk of the composite outcome when compared with undetectable parasitaemia. This finding could be hypothetically explained by a more vigorous immune response in patients with low parasitaemia that could decrease T. cruzi load more efficiently, but be associated with increased myocardial damage. Additional studies with a larger number of patients and cytokine measurement are required to support this hypothesis.

OBJECTIFS: Analyser l'effet de la charge parasitaire évaluée par PCR quantitative de transcription inverse (RT-qPCR) dans le sérum sur le pronostic des patients atteints de cardiomyopathie chronique de Chagas (CCM) après un suivi de deux ans. MÉTHODES: Etude de cohorte prospective menée entre 2015 et 2017. Une centaine de patients atteints de CCM ont été inclus. Les niveaux de parasitémie basale de Trypanosoma cruzi (T. cruzi) ont été mesurés en utilisant un test de réaction en chaîne de la polymérase quantitative (qPCR). Le principal résultat composite (RC) était la mortalité toutes causes, la transplantation cardiaque et l'implantation d'un dispositif d'assistance ventriculaire gauche. Les critères secondaires étaient les niveaux de base des biomarqueurs sériques et des variables échocardiographiques. RÉSULTATS: Après 2 ans de suivi, le taux de RC primaire était de 16%. Une qPCR positive n'était pas associée à un risque plus élevé de RC. Cependant, lorsque la parasitémie était évaluée en comparant les tertiles (tertile 1: parasitémie indétectable, tertile 2: parasitémie faible et tertile 3: parasitémie élevée), un risque plus élevé de RC (HR: 3,66; IC95%: 1,11-12,21) a été mis en évidence dans le tertile 2. De plus, les patients du tertile 2 avaient des niveaux significativement plus élevés de troponine T et de cystatine-C à haute sensibilité et présentaient plus fréquemment une fraction d'éjection <50%. CONCLUSION: Une faible parasitémie était associée à des marqueurs de sévérité des lésions myocardiques et à un risque plus élevé de résultat composite par rapport à une parasitémie indétectable. Cette découverte pourrait être hypothétiquement expliquée par une réponse immunitaire plus vigoureuse chez les patients présentant une faible parasitémie qui pourrait diminuer la charge de T. cruzi plus efficacement mais être associée à une augmentation des lésions myocardiques. Des études supplémentaires avec un plus grand nombre de patients et une mesure des cytokines sont nécessaires pour étayer cette hypothèse.