Search

In Brazil, where Chagas disease is endemic, the most frequent form of transmission of the parasite is the oral route, associated with greater severity and worse response to benznidazole (BZ), the drug used in its treatment. This study aimed to evaluate the impact of gastrointestinal infection (GI) and BZ treatment on the parasitological and histopathological parameters in mice inoculated with a strain of T. cruzi II. Swiss mice were inoculated by GI and intraperitoneal (IP) routes with 2x10 6 culture-derived metacyclic trypomastigotes of the Y strain (TcII) of T. cruzi and were treated with BZ in the acute phase of the infection. Fresh blood examination, qPCR, histopathological and biochemical evaluations (enzymatic dosages and oxidative stress-OS) were performed. BZ treatment of uninfected animals caused changes in the liver, increased the activity of aspartate aminotransferase and alanine aminotransferase enzymes and OS, showing that the drug alone affects this organ. Inflammation and necrosis in the cardiac tissue were less intense and deaths occurred later in animals inoculated via the GI route than the animals inoculated via the IP route. BZ reduced the intensity of tissue lesions and avoided lethality in animals inoculated via the GI route, and decreased parasitemia and OS in those inoculated via both routes. Although BZ alone caused liver damage, it was less intense than that caused by both routes of inoculation. Infection with the Y strain of T. cruzi II via the GI route proved to be less virulent and pathogenic and responded better to treatment than the infection acquired via the IP route., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Strains of Trypanosoma cruzi, etiological agent of Chagas disease, are classified into different discrete typing units that may present distinct dynamics of infection and susceptibility to benznidazole (BZ) treatment. Mice that were orally inoculated with T. cruzi IV strains exhibited a more intense course of infection compared with intraperitoneally inoculated mice, reflected by higher parasite loads. We evaluated the efficacy of BZ treatment in Swiss mice that were inoculated with T. cruzi IV strains from the Western Brazilian Amazon. The mice were orally (OR) or intraperitoneally (IP) inoculated with 2 × 10 6 culture-derived metacyclic trypomastigotes of the AM14, AM16, AM64, and AM69 strains of T. cruzi that were obtained from two outbreaks of orally acquired acute Chagas disease in the state of Amazonas, Brazil. The animals were treated with BZ (100 mg/kg/day for 20 days). Fresh blood examination, hemoculture, conventional and quantitative real-time polymerase chain reaction were performed to monitor the therapeutic effects of BZ. Significant reductions in five of 24 parameters of parasitemia and parasite load were found in different tissues in the OR group, indicating worse response to BZ treatment compared with the IP group, in which significant reductions in nine of those 24 parameters were observed. The cure rates in the OR groups ranged from 18.2% (1/11) to 75.0% (9/12) and in the IP groups from 58.3% (7/12) to 91.7% (11/12), for the AM14 and AM69 strains, respectively. These findings indicate that treatment with BZ had fewer beneficial effects with regard to reducing parasitemia and parasite load in different tissues of mice that were OR inoculated with four TcIV strains compared with IP inoculation. Therefore, the route of infection with T. cruzi should be considered when evaluating the therapeutic efficacy of BZ in patients with Chagas disease., (Copyright © 2021 Elsevier Inc. All rights reserved.)

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., (Copyright © 2021 Elsevier B.V. All rights reserved.)

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 (Qβ-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., Competing Interests: The authors have declared that no competing interests exist. Author Professor Egler Chiari was unable to confirm their authorship contributions. On their behalf, the corresponding author has reported their contributions to the best of their knowledge.

Chagas' disease and Lyme disease are two endemic, vector-borne zoonotic infectious diseases that impact multiple organ systems, including the heart. Chagas' cardiomyopathy is a progressive process that can evolve into a dilated cardiomyopathy and heart failure several decades after the acute infection; in contrast, although early-disseminated Lyme carditis has been relatively well characterized, the sequelae of Lyme disease on the heart are less well-defined. A century of research on Chagas' cardiomyopathy has generated compelling data for pathophysiological models, evaluated the efficacy of therapy in large randomized controlled trials, and explored the social determinants of health impacting preventative measures. Recognizing the commonalities between Chagas' disease and Lyme disease, we speculate on whether some of the lessons learned from Chagas' cardiomyopathy may be applicable to Lyme carditis., (Copyright © 2020. Published by Elsevier Inc.)

Chagas disease (CD) is endemic in Latin America. Drugs available for its treatment are benznidazole (BZ)/nifurtimox (NF), both with low efficacy in the late infection and responsible for several side effects. Studies of new drugs for CD among natural products, and using drug combinations with BZ/NF are recommended. Silibinin (SLB) is a natural compound that inhibits the efflux pump (Pgp) of drugs in host cell membranes, causes death of trypanosomatids, has anti-inflammatory activity, and was never assayed against T. cruzi. Here, in vitro and in vivo activities of SLB, SLB+BZ, and BZ against T. cruzi Y strain were evaluated. Cytotoxicity of SLB in VERO cells by the MTT method revealed IC50 of 250.22 μM. The trypanocidal activity evaluated by resazurin method in epimastigotes showed that SLB 25 μM inhibited parasite growth. SLB IC50 and selectivity index (SI) for amastigote were 79.81 μM and 3.13, respectively. SLB100+BZ10 showed higher parasite inhibition (91.44%) than SLB or BZ. Swiss mice infected with Y strain were treated with SLB, SLB+BZ, and BZ. Parasitemia was evaluated daily and 90, 180, and 240 days after treatment in surviving animals by hemoculture, blood qPCR, and after euthanasia, by qPCR in heart tissue. SLB monotherapy was not able to control the parasitemia/mortality of the animals. Parasitological negativation of 85.7-100% was observed in the experimental groups treated with SLB+BZ. Although SLB had shown activity against T. cruzi in vitro, it was not active in mice. Thus, the results of the therapeutic effect observed with SLB+BZ may be interpreted as a result from BZ action.

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., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Quantification of parasites in the context of Chagas disease is required to monitor the treatment with benznidazole, disease-associated cardiomyopathies and graft rejection after heart transplantation. As parasitological exams lack sensitivity, Real Time Polymerase Chain Reaction (rt-PCR) has emerged to evaluate the parasite load in blood samples and cardiac biopsies. However, despite its higher sensitivity, rt-PCR does not provide information on the location and distribution of amastigote nests within infected tissues, the characterization of inflammatory infiltrates or changes to tissue architecture. On the contrary, a sensitive immunohistochemistry technique (IHC) could fill these gaps. In the present study, a quantitative IHC exam was standardized and validated by testing adipose and cardiac tissues of experimentally infected mice containing variable parasite load levels of T. cruzi assessed by a sensitive Sybr Green rt-PCR with kDNA primers. Tissues were divided into four groups according to the parasite load: group A- 100 parasites/50 ng of DNA; group B -10 parasites; group C - around 1 parasite and group D - less than 1 parasite/50 ng/DNA. IHC was able to detect T. cruzi in the four groups, even in group D tissues containing fractions of a single parasite/50 ng of DNA sample according to rt-PCR. In conclusion, a highly sensitivity and reliable quantitative immunohistochemistry technique was developed and is proposed to estimate the percentage of T. cruzi-infected tissue area in chagasic patients presenting with cardiomyopathies, as a complementary test to rt-PCR., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Systematic microscopical observations on tissues from mice, inoculated with different Trypanosoma cruzi isolates, were carried out in order to assess whether the parasite expresses tissue-specific tropism, or if it can invade tissues pervasively within the mammal host. A total of ninety mice were included in the study. Sixty, subcutaneously-inoculated with 15 × 10 4 T. cruzi-blood trypomastigotes were dissected and examined daily for detecting and counting parasites during 12 days of acute infection. Additionally, two long-term experiments using mice inoculated with 5 × 10 3 metacyclic-forms were performed. A group of 10 mice inoculated intraperitoneally and another group of 20 mice inoculated intradermally. Results demonstrated that T. cruzi does not exhibit a tissue-specific tropism, revealing characteristics of a paninfective species able to invade tissues of ectodermal, mesodermal, and endodermal embryonic origin, irrespective of the parasite's lineage, infective form, route of entry, or size of the inoculum causing the host's infection. Details on T. cruzi-tissue invasion, tissue-parasite load during the course time, and the hypothetical potential pseudocyst/amastigote whole-body burden in the murine model is analyzed. The importance of the findings and its interpretation related to human Chagas disease and the tissue-parasite persistence is also discussed., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Trypanosoma cruzi is the causative agent of Chagas disease, which is endemic in Latin America and around the world through mother to child transmission. The heart is the organ most frequently affected in the chronic stage of the human infection and depends on mitochondria for the required energy for its activity. Cyclophilins are involved in protein folding and the mitochondrial isoform, Cyclophilin D (CyPD), has a crucial role in the opening of the mitochondrial permeability transition pore. In the present study, we infected CyPD deficient mice, with ablation of the Ppif gene, with T. cruzi parasites and the course of the infection was analyzed. Parasite load, quantified by PCR, was significantly lower in skeletal and cardiac tissues of Ppif - /- mice compared to wild type mice. In vitro cultured cardiomyocytes and macrophages from mice lacking CyPD exhibited lower percentage of infected cells and number of intracellular parasites than those observed for wild type mice. Although histopathological analysis of heart and mRNA of heart cytokines showed differences between T. cruzi-infected mice compared to the uninfected animals, no significant differences were found mice due to the ablation of the Ppif gene. Our results suggest that cells deficient for mitochondrial CyPD, inhibited for the mitochondrial membrane potential collapse, reduces the severity of parasite aggression and spread of cellular infection., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Trypanosoma cruzi is the etiologic agent of American trypanosomiasis and can infect humans and different species of domestic and wild animals. The marsupials are important wild reservoirs of T. cruzi, aiding in the maintenance of this agent in sylvatic and peri-domestic environments. The objective of this study was to report the parasitological and clinicopathological findings of a natural infection by T. cruzi in one specimen of Philander opossum that originated from the Brazilian Amazon. The animal was captured in a forest fragment near a rural community with reports of human Chagas disease. T. cruzi infection was diagnosed by blood smear examinations, blood culture, scent glands secretion culture, histopathological examination, and nested-PCR. Positive samples were subjected to PCR to characterize the discrete typing units (DTUs) of T. cruzi. Characteristic trypomastigotes of T. cruzi were observed in the blood smear, and spheromastigotes, epimastigotes, and trypomastigotes were visualized in the cultures. Non-suppurative myocarditis associated with amastigote clusters was the principal histopathological finding. DNA from T. cruzi was detected in samples of blood, blood cultures, scent glands secretion cultures, cardiac muscles, and the spleen. The TcI and the TcII/V/VI group DTUs were detected in blood culture and scent glands secretion cultures. Infection by T. cruzi can cause myocarditis in P. opossum and DTUs TcI and TcII/V/VI group mixed infection can be detected in the acute phase. P. opossum can be a source of infection for triatomine vectors and has the potential source for direct transmission of T. cruzi by secretions from the scent glands. These data are important to improve the understanding of the complex enzootic transmission cycle of T. cruzi in the Brazilian Amazon., (Copyright © 2020. Published by Elsevier B.V.)

Chagas disease (CD), caused by Trypanosoma cruzi , is a degenerative heart condition. In the present study, we investigated the role of poly [ADP-ribose] polymerase 1/activator protein 1 (PARP1/AP-1) in upregulation of profibrotic macrophages (Mϕ) and subsequent development of cardiac fibrosis in CD. We used in vitro and in vivo models of T. cruzi infection and chemical and genetic inhibition of Parp1 to examine the molecular mechanisms by which Mϕ might augment profibrotic events in CD. Cultured (RAW 264.7 and THP-1) Mϕ infected with T. cruzi and primary cardiac and splenic Mϕ of chronically infected mice exhibited a significant increase in the expression, activity, and release of metalloproteinases (MMP2, MMP9, and MMP12) and the cytokine transforming growth factor β (TGF-β). Mϕ release of MMPs and TGF-β signaled the cardiac fibroblast to myofibroblast differentiation, as evidenced by a shift from S100A4 to alpha smooth muscle actin (α-SMA) expression. Incubation of infected Mϕ with MMP2 and MMP9 inhibitors resulted in 60 to 74% decline in TGF-β release, and MMP9 and PARP1 inhibitors resulted in 57 to 70% decline in Mϕ TGF-β-driven cardiac fibroblast differentiation. Likewise, histological studies showed a 12- to 16-fold increase in myocardial expression of CD68 (Mϕ marker) and its colocalization with MMP9/TGF-β, galectin-3, and vimentin in wild-type mice with CD. In comparison, chronically infected Parp1 -/- mice exhibited a >50% decline in myocardial levels of Mϕ and associated fibrosis markers. Further study showed that PARP1 synergized with c-Fos and JunB AP-1 family members for transcriptional activation of profibrotic response after T. cruzi infection. We conclude that PARP1 inhibition offers a potential therapy for controlling the T. cruzi -driven fibroblast differentiation in CD through modulation of the Mϕ signaling of the AP-1-MMP9-TGF-β pathway. IMPORTANCE Cardiomyopathy is the most important clinical manifestation of T. cruzi -driven CD. Recent studies have suggested the detrimental role of the matrix metalloproteinases MMP2 and MMP9 in extracellular matrix (ECM) degradation during cardiac remodeling in T. cruzi infection. Peripheral TGF-β levels are increased in clinically symptomatic CD patients over those in clinically asymptomatic seropositive individuals. We provide the first evidence that during T. cruzi infection, Mϕ release of MMP2 and MMP9 plays an active role in activation of TGF-β signaling of ECM remodeling and cardiac fibroblast-to-myofibroblast differentiation. We also determined that PARP1 signals c-Fos- and JunB-mediated AP-1 transcriptional activation of profibrotic gene expression and demonstrated the significance of PARP1 inhibition in controlling chronic fibrosis in Chagas disease. Our study provides a promising therapeutic approach for controlling T. cruzi -driven fibroblast differentiation in CD by PARP1 inhibitors through modulation of the Mϕ signaling of the AP-1-MMP9-TGF-β pathway., (Copyright © 2020 Choudhuri and Garg.)

The receptor Signaling Lymphocyte-Activation Molecule Family 1 (SLAMF1) controls susceptibility to Infection by the lethal Trypanosoma cruzi Y strain. To elucidate whether genetic diversity of the parasite was related with disease susceptibility, we further analyzed the role of SLAMF1 using 6 different Trypanosoma cruzi strains including Y. The interaction of SLAMF1 receptor with T. cruzi was evidenced by fluorescence microscopy, flow cytometry and quantitative PCR. All the strains, except VFRA, showed a decrease in parasite load in infected macrophages in Slamf1-/- compared to BALB/c. In macrophages gene expression NADPH oxidase (NOX2), and reactive oxygen species (ROS) production increased in Slamf1-/- compared to BALB/c in 5 out of 6 strains. However, Slamf1-/-macrophages infected with VFRA strain exhibited a divergent behavior, with higher parasite load, lower NOX2 expression and ROS production compared to BALB/c. Parasitological and immunological studies in vivo with Y strain showed that in the absence of SLAMF1 the immune response protected mice from the otherwise lethal Y infection favoring a proinflammatory response likely involving CD4, CD8, dendritic cells and classically activated macrophages. In the case of VFRA, no major changes were observed in the absence of SLAMF1. Thus, the results suggest that the T. cruzi affects SLAMF1-dependent ROS production, controlling parasite replication in macrophages and affecting survival in mice in a strain-dependent manner. Further studies will focus in the identification of parasite molecules involved in SLAMF1 interaction to explain the immunopathogenesis of the disease., Competing Interests: The authors have declared that no competing interests exist.

The underlying pathogenic mechanisms of cardiomyopathy in Chagas disease are still unsolved. In order to better clarify the role of fat on the evolution of cardiomyopathy, the present study employed three murine models of chronic Trypanosoma cruzi infection: (1) aP2-RIDα/β transgenic mice (RID mice; an adipose tissue model which express a gain-of-function potent anti-inflammatory activity), (2) allograft inflammatory factor-1 knockout mice (Aif1 -/- ), and (3) a Swiss outbred mice. RID mice and non-transgenic mice (wild type, WT) were infected with blood trypomastigotes of Brazil strain. During the acute stage of infection, RID mice had lower parasitemia, lower heart inflammation, and a decrease in the relative distribution of parasite load from cardiac muscle tissue toward epididymal fat. Nevertheless, comparable profiles of myocardial inflammatory infiltrates and relative distribution of parasite load were observed among RID and WT at the chronic stage of infection. Aif1 -/- and Aif1 +/+ mice were infected with bloodstream trypomastigotes of Tulahuen strain and fed with high-fat diet (HFD) or regular diet (RD). Interestingly, Aif1 +/+ HFD infected mice showed the highest mortality. Swiss mice infected with blood trypomastigotes of Berenice-78 strain on a HFD had higher levels of TNFα and more inflammation in their heart tissue than infected mice fed a RD. These various murine models implicate adipocytes in the pathogenesis of chronic Chagas disease and suggest that HFD can lead to a significant increase in the severity of parasite-induced chronic cardiac damage. Furthermore, these data implicate adipocyte TLR4-, TNFα-, and IL-1β-mediated signaling in pro-inflammatory pathways and Aif-1 gene expression in the development of chronic Chagas disease.

Damaged cells release the pro-inflammatory signal ATP, which is degraded by the ectonucleotidases CD39 and CD73 to the anti-inflammatory mediator adenosine (ADO). The balance between ATP/ADO is known to determine the outcome of inflammation/infection. However, modulation of the local immune response in different tissues due to changes in the balance of purinergic metabolites has yet to be investigated. Here, we explored the contribution of CD73-derived ADO on the acute immune response against Trypanosoma cruzi parasite, which invades and proliferates within different target tissues. Deficiency of CD73 activity led to an enhanced cardiac microbicidal immune response with an augmented frequency of macrophages with inflammatory phenotype and increased CD8+ T cell effector functions. The increment of local inducible nitric oxide (NO) synthase (iNOS) + macrophages and the consequent rise of myocardial NO production in association with reduced ADO levels induced protection against T. cruzi infection as observed by the diminished cardiac parasite burden compared to their wild-type (WT) counterpart. Unexpectedly, parasitemia was substantially raised in CD73KO mice in comparison with WT mice, suggesting the existence of tissue reservoir/s outside myocardium. Indeed, CD73KO liver and visceral adipose tissue (VAT) showed increased parasite burden associated with a reduced ATP/ADO ratio and the lack of substantial microbicidal immune response. These data reveal that the purinergic system has a tissue-dependent impact on the host immune response against T. cruzi infection., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Chagas disease, caused by Trypanosoma cruzi, is a neglected tropical disease that affects 5-6 million people in endemic areas of the Americas. Presently, chemotherapy relies on two compounds that were proposed as trypanocidal drugs four decades ago: nifurtimox and benznidazole. Both drugs are able to eliminate parasitemia and to avoid seroconversion in infected people when used in the acute phase; however, their use in the chronic phase (the time when the majority of cases are diagnosed) is limited due to their serious side effects. Memantine is a glutamate receptor antagonist in the central nervous system of mammals that has been used for the treatment of Alzheimer's disease. Our group previously reported memantine as a trypanocidal drug that is able to induce apoptosis-like death in T. cruzi. In the present work, we further investigated the effects of memantine on the infection of RAW 264.7 macrophages and in vivo (in BALB/c mice). Here, we showed that memantine is able to diminish NO and Ca2+ entry in both LPS-activated and non-activated cells. These results, together with the fact that memantine was also able to reduce the infection of macrophages, led us to propose that this drug is able to activate a pro-oxidant non-NO-dependent cell defense mechanism. Finally, infected mice that were treated with memantine had diminished parasitemia, cardiac parasitic load, and inflammatory infiltrates. In addition, the treated mice had an increased survival rate. Taken together, these results indicate memantine to be a candidate drug for the treatment of Chagas disease., Competing Interests: The authors have declared that no competing interests exist.

Introduction: Trypanosoma cruzi is the causal agent of the American trypanosomiasis, an endemic disease in México. The commensal rodents Mus musculus and Rattus rattus are reservoirs of this parasite, which invades cardiac fibers and develops parasite nests causing various lesions. Histopathological studies in naturally infected rodents are scarce. Objective: To describe the types and frequencies of microscopic lesions in cardiac tissue of M. musculus and R. rattus infected with T. cruzi captured in Mérida, México. Materials and methods: The rodents were captured in suburban environments of Mérida. Cardiac tissue was extracted and processed by the paraffin inclusion technique and hematoxylin and eosin stained. The observation was made with a conventional microscope and all the lesions, as well as their degree, were identified. Results: Eight tissue samples of M. musculus and seven of R. rattus were studied. Parasite nests were found in 7/15, specifically 3/8 in M. musculus and 4/7 in R. rattus. The inflammatory infiltrate was the most frequent lesion. Other lesions were: Degeneration of cardiac fibers (8/15), congestion of blood vessels (6/15), and necrosis (5/15). Discussion: The lesions we observed have been described in experimental animal models and in humans with American trypanosomiasis. The inflammatory infiltrate has been identified as the most significant lesion in humans and reservoirs in the chronic stage of the disease. Conclusion: The lesions we described are associated with T. cruzi infection, which confirms that the rodents studied are reservoirs of this parasite.

CD8+ T lymphocytes play an important role in controlling infections by intracellular pathogens. Chemokines and their receptors are crucial for the migration of CD8+ T-lymphocytes, which are the main IFNγ producers and cytotoxic effectors cells. Although the participation of chemokine ligands and receptors has been largely explored in viral infection, much less is known in infection by Trypanosoma cruzi, the causative agent of Chagas disease. After T. cruzi infection, CXCR3 chemokine receptor is highly expressed on the surface of CD8+ T-lymphocytes. Here, we hypothesized that CXCR3 is a key molecule for migration of parasite-specific CD8+ T-cells towards infected tissues, where they may play their effector activities. Using a model of induction of resistance to highly susceptible A/Sn mice using an ASP2-carrying DNA/adenovirus prime-boost strategy, we showed that CXCR3 expression was upregulated on CD8+ T-cells, which selectively migrated towards its ligands CXCL9 and CXCL10. Anti-CXCR3 administration reversed the vaccine-induced resistance to T. cruzi infection in a way associated with hampered cytotoxic activity and increased proapoptotic markers on the H2KK-restricted TEWETGQI-specific CD8+ T-cells. Furthermore, CXCR3 receptor critically guided TEWETGQI-specific effector CD8+ T-cells to the infected heart tissue that express CXCL9 and CXCL10. Overall, our study pointed CXCR3 and its ligands as key molecules to drive T. cruzi-specific effector CD8+ T-cells into the infected heart tissue. The unveiling of the process driving cell migration and colonization of infected tissues by pathogen-specific effector T-cells is a crucial requirement to the development of vaccine strategies., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Trypanosoma cruzi, the causal agent of chronic Chagas cardiomyopathy, exhibits an important tropism for cardiac tissue. In consequence, T. cruzi experimental infection represents a unique model to study cardiac macrophage behavior and effector functions during either acute or chronic immune response. In this chapter we describe a protocol to isolate immune cells from T. cruzi-infected murine cardiac tissue and to determine the percentage, absolute number, phenotype, and functionality of monocytes and macrophages by using flow cytometry. Moreover, we describe the parameters to discriminate between resident and infiltrating mononuclear phagocytic cells within infected hearts. The investigations in this field will provide mechanistic insights about the roles of these innate immune cells in the context of a clinically relevant target tissue.

It is still unclear whether the progression of acute to chronic Chagas cardiomyopathy is predominantly associated with the limited efficacy of aetiological chemotherapy, or with the pharmacological resistance profiles and pathogenicity of specific Trypanosoma cruzi strains. Thus, we tested the hypothesis that parasitic load could be a limited target of aetiological chemotherapy to prevent chronic cardiomyopathy in dogs infected by different T. cruzi strains. Animals were infected with benznidazole-susceptible (Berenice-78) and -resistant (VL-10 and AAS) strains of T. cruzi. A quantitative real-time PCR strategy was developed to comparatively quantify the parasite load of the three different strains using a single standard curve. For dogs infected with the VL-10 strain, benznidazole treatment reduced cardiac parasitism during the acute phase of infection. However, similar parasite load and collagen deposition were detected in the myocardium of treated and untreated animals in the chronic phase of the infection. In animals infected with the AAS strain, benznidazole reduced parasite load, myocarditis and type III collagen deposition in the acute phase. However, increased type III collagen deposition was verified in the chronic phase. Dogs infected with the Berenice-78 strain showed a parasitological cure and no evidence of myocardial fibrosis. Parasitic load and cardiac fibrosis presented no correlation in acute or chronic phases of T. cruzi infection. Our findings in a canine model of Chagas disease suggest that parasite burden is a limited predictor for disease progression after treatment and show that benznidazole, although not inducing parasitological cure, is able to prevent total fibrosis in the early stages of infection, as well as complete prevention of cardiac damage when it eliminates parasites at the onset of infection., (Copyright © 2018 Elsevier B.V. All rights reserved.)

MicroRNAs (miRNAs) are a class of small noncoding RNAs (typically 19-23 nucleotides) which act by annealing to partially complementary binding sites present on the 3' untranslated regions (UTR) of messenger RNAs (mRNAs) leading to inhibition of protein translation or by inducing mRNA decay. Since their discovery, miRNAs have come to be recognized as master regulators of gene expression in plant and mammals, controlling tissue-specific protein expression. Up to one-third of mammalian mRNAs are susceptible to miRNA-mediated regulation. It has been shown that miRNAs are determinants of the physiology and pathophysiology of the cardiovascular system, and altered expression of muscle- and/or cardiac-specific miRNAs in myocardial tissue is involved in heart development and cardiovascular diseases, including myocardial hypertrophy, heart failure, and fibrosis. The analysis of miRNA expression pattern provides important information, as well as is a starting point to understand miRNA function in different tissues, during development, and in disease. Several techniques can be used for miRNA profiling analysis like high-throughput sequencing, microarrays, and real-time PCR using microfluidic low-density arrays. This chapter describes the complete methodology to perform miRNA profiling using the stem-loop reverse-transcription (RT)-based TaqMan ® MicroRNA low-density arrays (TLDA) method. This methodology was used to perform miRNA profiling in the heart of T. cruzi acutely infected mice.

Chagas disease is caused by infection with the protozoan Trypanosoma cruzi and affects over 8 million people worldwide. In spite of a powerful innate and adaptive immune response in acute infection, the parasite evades eradication, leading to a chronic persistent infection with low parasitism. Chronically infected subjects display differential patterns of disease progression. While 30% develop chronic Chagas disease cardiomyopathy (CCC)-a severe inflammatory dilated cardiomyopathy-decades after infection, 60% of the patients remain disease-free, in the asymptomatic/indeterminate (ASY) form, and 10% develop gastrointestinal disease. Infection of genetically deficient mice provided a map of genes relevant for resistance to T. cruzi infection, leading to the identification of multiple genes linked to survival to infection. These include pathogen resistance genes (PRG) needed for intracellular parasite destruction, and genes involved in disease tolerance (protection against tissue damage and acute phase death-DTG). All identified DTGs were found to directly or indirectly inhibit IFN-γ production or Th1 differentiation. We hypothesize that the absolute need for DTG to control potentially lethal IFN-γ PRG activity leads to T. cruzi persistence and establishment of chronic infection. IFN-γ production is higher in CCC than ASY patients, and is the most highly expressed cytokine in CCC hearts. Key DTGs that downmodulate IFN-γ, like IL-10, and Ebi3/IL27p28, are higher in ASY patients. Polymorphisms in PRG and DTG are associated with differential disease progression. We thus hypothesize that ASY patients are disease tolerant, while an imbalance of DTG and IFN-γ PRG activity leads to the inflammatory heart damage of CCC.

Elderly organisms are more susceptible to infectious diseases. However, the impact of aging on antiparasitic mechanisms, especially the nitric oxide pathway, is poorly understood. Using an integrated in vivo and in vitro model, we compared the severity of Trypanosoma cruzi infection in young and elderly (8 or 72 weeks old) mice. Forty C57BL/6 mice were randomized into four groups: Y-inf, young infected; Yn-inf, young uninfected; A-inf, aged infected; An-inf, aged uninfected. Parasitemia was measured daily, and animals were euthanized after 15 days of infection. Trypanosoma cruzi-induced inflammatory processes were analyzed in blood and heart samples, as well as in bone marrow-derived macrophages (BMDMs) co-cultured with splenocytes isolated from young or elderly mice. Our results indicated upregulated IgG2b and IL-17 production in elderly animals, which was not sufficient to reduce parasitemia, parasitic load and myocarditis to levels observed in young animals. The higher susceptibility of elderly mice to T. cruzi infection was accompanied by reduced cardiac inducible nitric oxide synthase (iNOS) gene expression, nitric oxide (NO) and IFN-γ levels, as well as an antagonistic upregulation of arginase-1 expression and arginase activity. The same responses were observed when BMDMs co-cultured with splenocytes from elderly mice were stimulated with T. cruzi antigens. Our findings indicate that elderly mice were more susceptible to T. cruzi infection, which was potentially related to an attenuated response to antigenic stimulation, inhibition of iNOS gene expression and NO production, and antagonistic upregulation of arginase gene expression and activity, which created favorable conditions for heart parasitism and myocarditis development., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Background: Heart transplantation has been shown to be a safe and effective intervention for progressive cardiomyopathy from chronic Chagas disease. However, in the presence of the immunosuppression required for heart transplantation, the likelihood of Chagas disease reactivation is significant. Reactivation may cause myocarditis resulting in allograft dysfunction and the rapid onset of congestive heart failure. Reactivation rates have been well documented in Latin America; however, there is a paucity of data regarding the risk in non-endemic countries., Methods: We present our experience with 31 patients with chronic Chagas disease who underwent orthotopic heart transplantation in the United States from 2012 to 2016. Patients were monitored following a standard schedule., Results: Of the 31 patients, 19 (61%) developed evidence of reactivation. Among the 19 patients, a majority (95%) were identified by laboratory monitoring using polymerase chain reaction testing. One patient was identified after the onset of clinical symptoms of reactivation. All subjects with evidence of reactivation were alive at follow-up (median: 60 weeks)., Conclusions: Transplant programs in the United States are encouraged to implement a monitoring program for heart transplant recipients with Chagas disease. Our experience using a preemptive approach of monitoring for Chagas disease reactivation was effective at identifying reactivation before symptoms developed., (Published 2018. This article is a U.S. Government work and is in the public domain in the USA.)

Chagasic heart disease develops in 30% of those infected with the protozoan parasite Trypanosoma cruzi , but can take decades to become symptomatic. Because of this, it has been difficult to assess the extent to which antiparasitic therapy can prevent the development of pathology. We sought to address this question using experimental murine models, exploiting highly sensitive bioluminescent imaging to monitor curative efficacy. Mice were inoculated with bioluminescent parasites and then cured in either the acute or chronic stage of infection with benznidazole. At the experimental endpoint (5 to 6 months postinfection), heart tissue was removed and assessed for inflammation and fibrosis, two widely used markers of cardiac pathology. Infection of BALB/c and C3H/HeN mice with distinct T. cruzi lineages resulted in greatly increased myocardial collagen content at a group level, indicative of fibrotic pathology. When mice were cured by benznidazole in the acute stage, the development of pathology was completely blocked. However, if treatment was delayed until the chronic stage, cardiac fibrosis was observed in the BALB/c model, although the protective effect was maintained in the case of C3H/HeN mice. These experiments therefore demonstrate that curative benznidazole treatment early in murine T. cruzi infections can prevent the development of cardiac fibrosis. They also show that treatment during the chronic stage can block pathology but the effectiveness varies between infection models. If these findings are extendable to humans, it implies that widespread chemotherapeutic intervention targeted at early-stage infections could play a crucial role in reducing Chagas disease morbidity at a population level., (Copyright © 2018 Francisco et al.)

Statins are inhibitors of cholesterol synthesis, but other biological properties, such as antimicrobial effects, have also been assigned to them, leading to their designation as pleiotropic agents. Our goal was to investigate the activity and selectivity of atorvastatin (AVA) against Trypanosoma cruzi by using in vitro models, aiming for more effective and safer therapeutic options through drug repurposing proposals for monotherapy and therapy in combination with benznidazole (BZ). Phenotypic screening was performed with different strains (Tulahuen [discrete typing unit {DTU} VI] and Y [DTU II]) and forms (intracellular forms, bloodstream trypomastigotes, and tissue-derived trypomastigotes) of the parasite. On assay of the Tulahuen strain, AVA was more active against intracellular amastigotes (selectivity index [SI] = 3). Also, against a parasite of another DTU (Y strain), this statin was more active (2.1-fold) and selective (2.4-fold) against bloodstream trypomastigotes (SI = 51) than against the intracellular forms (SI = 20). A cytomorphological approach using phalloidin-rhodamine permitted us to verify that AVA did not induced cell density reduction and that cardiac cells (CC) maintained their typical cytoarchitecture. Combinatory approaches using fixed-ratio methods showed that AVA and BZ gave synergistic interactions against both trypomastigotes and intracellular forms (mean sums of fractional inhibitory concentration indexes [∑FICIs] of 0.46 ± 0.12 and 0.48 ± 0.03, respectively). Thus, the repurposing strategy for AVA, especially in combination with BZ, which leads to a synergistic effect, is encouraging for future studies to identify novel therapeutic protocols for Chagas disease treatment., (Copyright © 2018 American Society for Microbiology.)

In the present work, we evaluated the effect of mixed Trypanosoma cruzi infections, studying the biological distribution of the different parasites in blood, heart and skeletal muscle during the acute phase. Albino Swiss mice were infected with different parasite strain/isolates or with a combination of them. The parasites in the different tissues were typified through specific PCR, population variability was analyzed through RFLP studies and parasitological and histopathological parameters were evaluated. We found a predominance of TcII and TcVI in all tissues samples respect to TcV and different parasite populations were found in circulation and in the tissues from the same host. These results verify the distribution of parasites in host tissues from early stages of infection and show biological interactions among different genotypes and populations of T. cruzi., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Chagas disease is caused by infection with the protozoan Trypanosoma cruzi (T. cruzi) and is an important cause of severe inflammatory heart disease. However, the mechanisms driving Chagas disease cardiomyopathy have not been completely elucidated. Here, we show that the canonical PI3Kγ pathway is upregulated in both human chagasic hearts and hearts of acutely infected mice. PI3Kγ-deficient mice and mutant mice carrying catalytically inactive PI3Kγ are more susceptible to T. cruzi infection. The canonical PI3Kγ signaling in myeloid cells is essential to restrict T. cruzi heart parasitism and ultimately to avoid myocarditis, heart damage, and death of mice. Furthermore, high PIK3CG expression correlates with low parasitism in human Chagas' hearts. In conclusion, these results indicate an essential role of the canonical PI3Kγ signaling pathway in the control of T. cruzi infection, providing further insight into the molecular mechanisms involved in the pathophysiology of chagasic heart disease.

Although many Trypanosoma cruzi (T. cruzi) strains isolated from a wide range of hosts have been characterized, there is a lack of information about biological features from vertically transmitted strains. We describe the molecular and biological characteristics of the T. cruzi VD strain isolated from a congenital Chagas disease patient. The VD strain was typified as DTU TcVI; in vitro sensitivity to nifurtimox (NFX) and beznidazole (BZ) were 2.88 μM and 6.19 μM respectively, while inhibitory concentrations for intracellular amastigotes were 0.24 μM for BZ, and 0.66 μM for NFX. Biological behavior of VD strain was studied in a mouse model of acute infection, resulting in high levels of parasitemia and mortality with a rapid clearence of bloodstream trypomastigotes when treated with BZ or NFX, preventing mortality and reducing parasitic load and intensity of inflammatory infiltrate in skeletal and cardiac muscle. Treatment-induced parasitological cure, evaluated after immunossupression were 41% and 35% for BZ and NFX treatment respectively, suggesting a partial response to these drugs in elimination of parasite burden. This exhaustive characterization of this T. cruzi strain provides the basis for inclusion of this strain in a panel of reference strains for drug screening and adds a new valuable tool for the study of experimental T. cruzi infection., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Background: Chagas disease is a neglected parasitic infection caused by the protozoan Trypanosoma cruzi (T. cruzi) that affects more than 6 million people, mainly in Latin America. Benznidazole is still the drug of choice in many countries to treat it in spite of its dosage regimen and adverse side effects such as such as allergic dermatitis, peripheral neuropathy and anorexia. Thus, novel, safer, and more efficacious treatments for such neglected infection are urgently required., Methodology: In this study, the efficacy of orally administered low doses of benznidazole (BNZ) nanoparticles was evaluated during the acute phase in mice infected with T. cruzi Nicaragua (TcN) that were immunosuppressed during the chronic stage of the disease. Moreover, the production of T. cruzi-specific antibodies, cardiac tissue inflammation and reactive oxygen species generation by Vero cells treated with both BNZ nanoparticles (BNZ-nps) and raw BNZ (R-BNZ) were also evaluated., Principal Findings: T. cruzi infected mice treated with 10, 25 or 50 mg/kg/day of BNZ-nps survived until euthanasia (92 days post infection (dpi)), while only 15% of infected untreated mice survived until the end of the experiment. PCR analysis of blood samples taken after induction of immunosuppression showed that a dosage of 25 mg/kg/day rendered 40% of the mice PCR-negative. The histological analysis of heart tissue showed a significant decrease in inflammation after treatments with 25 and 50 mg/kg/day, while a similar inflammatory damage was observed in both infected mice treated with R-BNZ (50 mg/kg/day) and untreated mice. In addition, only BNZ-nps treated mice led to lower levels of T. cruzi-specific antibodies to 50-100%. Finally, mammalian Vero cells treated with BNZ-nps or R-BNZ lead to a significant increase in ROS production., Conclusions: Based on these findings, this research highlights the in-vitro/in-vivo efficacy of nanoformulated BNZ against T. cruzi acute infections in immunosuppressed and non-immunosuppressed mice and provides further evidence for the optimization of dosage regimens to treat Chagas disease.

Background: Chagas' disease (CD) is caused by infection with the protozoan Trypanosoma cruzi. The disease can affect the heart and/or the gastrointestinal (GI) tract, but around 70% of infected individuals remain asymptomatic in the chronic form. Organ transplantation from T. cruzi-infected donors is often avoided because of the risk of disease transmission, previously reported after heart, kidney, or liver transplantation., Methods: We investigated by histology, immunohistochemistry, and polymerase chain reaction (PCR) the persistence of T. cruzi in samples of the heart, lung, liver, kidney, pancreas, adrenal gland, esophagus, and GI tract of 21 chronic chagasic patients., Results: Parasite persistence was detected in 12/21 (57.1%) heart samples, mainly by PCR-based assays. T. cruzi parasites were detected by histology and immunohistochemistry in smooth muscle cells of the central vein from 1/21 (4.8%) adrenal gland samples. No samples of the lung, liver, kidney, pancreas, esophagus, or GI tract were found to have parasites by histology, immunohistochemistry, or PCR., Conclusions: We concluded that, aside from the heart, the other solid organs of T. cruzi-infected donors can be used for transplantation with a lot of caution. Such organs are not safe in the view of previous reports of CD transmission, but seem to present a low T. cruzi load compared to the heart., (© 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: Previous studies showed that a naturally attenuated strain from Trypanosoma cruzi triggers an immune response mainly related to a Th2-type profile. Albeit this, a strong protection against virulent challenge was obtained after priming mice with this attenuated strain. However, this protection is not enough to completely clear parasites from the host. In T. cruzi infection, early Interferon-gamma (IFN-γ) is critical to lead type 1 responses able to control intracellular parasites. Therefore we evaluated whether the co-administration of a plasmid encoding murine IFN-γ could modify the immune response induced by infection with attenuated parasites and improve protection against further infections., Methods: C57BL/6J mice were infected intraperitoneally with three doses of live attenuated parasites in combination with plasmid pVXVR-mIFN-γ. Before each infection dose, sera samples were collected for parasite specific antibodies determination and cytokine quantification. To evaluate the recall response to T. cruzi, mice were challenged with virulent parasites 30 days after the last dose and parasite load in peripheral blood and heart was evaluated., Results: As determined by ELISA, significantly increase in T. cruzi specific antibodies response was detected in the group in which pVXVR-mIFN-γ was incorporated, with a higher predominance of IgG2a subtype in comparison to the group of mice only inoculated with attenuated parasites. At our limit of detection, serum levels of IFN-γ were not detected, however a slight decrease in IL-10 concentrations was observed in groups in which pVXVR-mIFN-γ was supplemented. To analyze if the administration of pVXVR-mIFN-γ has any beneficial effect in protection against subsequent infections, all experimental groups were submitted to a lethal challenge with virulent bloodstream trypomastigotes. Similar levels of challenge parasites were detected in peripheral blood and heart of mice primed with attenuated parasites alone or combined with plasmid DNA. Expansion of IgG antibodies was not significant in TCC+ pVXVR-mIFN-γ; however, the overall tendency to sustain a Th2 profile was maintained., Conclusions: Overall, these results suggest that administration of plasmid pVXVR-mIFN-γ could have beneficial effects on host specific antibody production in response to T. cruzi attenuated infection; however, this outcome is not reflected in an improved protection against further virulent infections.

Trypanosoma cruzi parasites are the causative agents of Chagas disease, a leading infectious form of heart failure whose pathogenesis is still not fully characterized. In this work, we applied untargeted liquid chromatography-tandem mass spectrometry to heart sections from T. cruzi-infected and uninfected mice. We combined molecular networking and three-dimensional modeling to generate chemical cartographical heart models. This approach revealed for the first time preferential parasite localization to the base of the heart and regiospecific distributions of nucleoside derivatives and eicosanoids, which we correlated to tissue-damaging immune responses. We further detected novel cardiac chemical signatures related to the severity and ultimate outcome of the infection. These signatures included differential representation of higher- vs lower-molecular-weight carnitine and phosphatidylcholine family members in specific cardiac regions of mice infected with lethal or nonlethal T. cruzi strains and doses. Overall, this work provides new insights into Chagas disease pathogenesis and presents an analytical chemistry approach that can be broadly applied to the study of host-microbe interactions.

A therapeutic vaccine for human Chagas disease is under development by the Sabin Vaccine Institute Product Development Partnership. The aim of the vaccine is to significantly reduce the parasite burden of Trypanosoma cruzi in humans, either as a standalone product or in combination with conventional chemotherapy. Vaccination of mice with Tc24 formulated with monophosphoryl-lipid A (MPLA) adjuvant results in a Th1 skewed immune response with elevated IgG2a and IFNγ levels and a statistically significant decrease in parasitemia following T. cruzi challenge. Tc24 was therefore selected for scale-up and further evaluation. During scale up and downstream process development, significant protein aggregation was observed due to intermolecular disulfide bond formation. To prevent protein aggregation, cysteine codons were replaced with serine codons which resulted in the production of a non-aggregated and soluble recombinant protein, Tc24-C4. No changes to the secondary structure of the modified molecule were detected by circular dichroism. Immunization of mice with wild-type Tc24 or Tc24-C4, formulated with E6020 (TLR4 agonist analog to MPLA) emulsified in a squalene-oil-in-water emulsion, resulted in IgG2a and antigen specific IFNγ production levels from splenocytes that were not significantly different, indicating that eliminating putative intermolecular disulfide bonds had no significant impact on the immunogenicity of the molecule. In addition, vaccination with either formulated wild type Tc24 or Tc24-C4 antigen also significantly increased survival and reduced cardiac parasite burden in mice. Investigations are now underway to examine the efficacy of Tc24-C4 formulated with other adjuvants to reduce parasite burden and increase survival in pre-clinical studies.

Chagas disease is a tropical parasitic disease caused by the protozoan Trypanosoma cruzi, which affects about ten million people in its endemic regions of Latin America. After the initial acute stage of infection, 60-80% of infected individuals remain asymptomatic for several years to a lifetime; however, the rest develop the debilitating symptomatic stage, which affects the nervous system, digestive system, and heart. The challenges of Chagas disease have become global due to immigration. Despite well-documented dietary changes accompanying immigration, as well as a transition to a western style diet in the Chagas endemic regions, the role of host metabolism in the pathogenesis of Chagas disease remains underexplored. We have previously used a mouse model to show that host diet is a key factor regulating cardiomyopathy in Chagas disease. In this study, we investigated the effect of a high-fat diet on liver morphology and physiology, lipid metabolism, immune signaling, energy homeostasis, and stress responses in the murine model of acute T. cruzi infection. Our results indicate that in T. cruzi-infected mice, diet differentially regulates several liver processes, including autophagy, a stress response mechanism, with corresponding implications for human Chagas disease patients., Competing Interests: STATEMENT None of the authors have conflict of interest.

During Trypanosoma cruzi infection, oxidative stress is considered a contributing factor for dilated cardiomyopathy development. In this study, the effects of astaxanthin (ASTX) were evaluated as an alternative drug treatment for Chagas disease in a mouse model during the acute infection phase, given its anti-inflammatory, immunomodulating, and anti-oxidative properties. ASTX was tested in vitro in parasites grown axenically and in co-culture with Vero cells. In vivo tests were performed in BALB/c mice (4-6 weeks old) infected with Trypanosoma cruzi and supplemented with ASTX (10 mg/kg/day) and/or nifurtimox (NFMX; 100 mg/kg/day). Results show that ASTX has some detrimental effects on axenically cultured parasites, but not when cultured with mammalian cell monolayers. In vivo, ASTX did not have any therapeutic value against acute Trypanosoma cruzi infection, used either alone or in combination with NFMX. Infected animals treated with NFMX or ASTX/NFMX survived the experimental period (60 days), while infected animals treated only with ASTX died before day 30 post-infection. ASTX did not show any effect on the control of parasitemia; however, it was associated with an increment in focal heart lymphoplasmacytic infiltration, a reduced number of amastigote nests in cardiac tissue, and less hyperplasic spleen follicles when compared to control groups. Unexpectedly, ASTX showed a negative effect in infected animals co-treated with NFMX. An increment in parasitemia duration was observed, possibly due to ASTX blocking of free radicals, an anti-parasitic mechanism of NFMX. In conclusion, astaxanthin is not recommended during the acute phase of Chagas disease, either alone or in combination with nifurtimox., (© J.M.E. Contreras-Ortiz et al., published by EDP Sciences, 2017.)

Trypanosoma cruzi causes a cardiac infection characterized by an inflammatory imbalance that could become the inciting factor of the illness. To this end, we evaluated the role of carvedilol, a beta-blocker with potential immunomodulatory properties, on the immune response in C57BL/6 mice infected with VL-10 strain of T. cruzi in the acute phase. Animals ( n = 40) were grouped: (i) not infected, (ii) infected, (iii) infected + carvedilol, and (iv) not infected + carvedilol. We analyzed parameters related to parasitemia, plasma levels of TNF, IL-10, and CCL2, and cardiac histopathology after the administration of carvedilol for 30 days. We did not observe differences in the maximum peaks of parasitemia in the day of their detection among the groups. The plasma TNF was elevated at 60 days of infection in mice treated or not with carvedilol. However, we observed a decreased CCL2 level and increased IL-10 levels in those infected animals treated with carvedilol, which impacted the reduction of the inflammatory infiltration in cardiac tissue. For this experimental model, carvedilol therapy was not able to alter the levels of circulating parasites but modulates the pattern of CCL2 and IL-10 mediators when the VL10 strain of T. cruzi was used in C57BL6 mice., Competing Interests: The authors declare that they do not have any conflict of interests regarding this publication.

A cynomolgus macaque received a heterotopic cardiac allograft as part of a transplant study, with monoclonal antibodies targeted to specific immune costimulation molecules (CD154, CD28) but no traditional immunosuppressive therapy after surgery. Clinical anemia was detected on postoperative day (POD) 35 and had worsened (Hgb, 2.3 g/dL; Hct = 7.3%) by POD 47, despite type-matched whole-blood transfusions. After a total of 4 blood transfusions, hematologic parameters were improved (Hgb, 5.9 g/dL; Hct, 18.7%). On POD 50, a peripheral blood smear revealed trypomastigotes, and qualitative RT-PCR of whole blood identified the organism as Trypanosoma cruzi. Although clinically stable initially, the macaque soon developed sufficient weight loss to necessitate euthanasia on POD 64. The final diagnosis was clinical anemia due to T. cruzi infection. This study represents the first reported case of Chagas disease after heart transplant in a NHP.

Background: Diverse cardiovascular signaling routes have been considered critical for Chagas cardiomyopathy caused by the protozoan parasite Trypanosoma cruzi. Along this line, T. cruzi infection and endothelin-1 (ET-1) have been shown to cooperatively activate the Ca 2+ /NFAT cascade in cardiomyocytes, leading to cyclooxygenase type 2 (COX-2) induction and increased release of prostanoids and prohypertrophic peptides., Purpose: To determine whether the well-known cardioprotective and anti-inflammatory effects of curcumin (Cur) could be helpful to interfere with this key machinery for pathogenesis of Chagas myocarditis., Study Design: Cur treatment was evaluated through in vivo studies using a murine model of acute T. cruzi infection and in vitro experiments using ET-1-stimulated and parasite-infected mouse cardiomyocytes., Methods: Cur-treated and untreated infected mice were followed-up to estimate survival postinfection and heart tissues from both groups were analyzed for inflammatory infiltration by histopathology, whereas parasite load, induction of arachidonic acid pathway and natriuretic peptide expression were determined by real-time PCR. Molecular analysis of Cur myocardial targets included intracellular calcium measurement, NFAT and COX-2 induction in transfected cells, and assessment of NFAT, COX-2 and microsomal prostaglandin E synthase-1 (mPGES-1) levels by immunoblotting, prostaglandin E 2 (PGE 2 ) by ELISA, b-type natriuretic peptide (BNP) by real-time PCR, and PGE 2 /EP4 receptor/BNP interaction by transwell experiments., Results: Cur treatment of acute Chagas mice enhanced survival and proved to hinder relevant inflammatory processes in the heart, including leukocyte recruitment, activation of the eicosanoid pathway and BNP overexpression, without modifying parasite burden in the organ. Cur was capable of blocking Ca 2+ -dependent NFATc1 transcriptional activity, COX-2 and mPGES-1 induction, and subsequent PGE 2 production in ET-1-stimulated and parasite-infected cardiomyocytes. Furthermore, the decline of cardiomyocyte-derived prostaglandin levels achieved upon Cur treatment impaired effective PGE 2 /EP4 receptor interaction, resulting in attenuated expression of BNP, a strong indicator of cardiac pathogenesis in Chagas disease, in both infected and uninfected cells., Conclusion: Our current study shows a putative mechanism of action of Cur involving inhibition of the Ca 2+ /NFAT-dependent, pathogenic COX-2/mPGES-1/PGE 2 pathway in T. cruzi-infected myocytes, underlying cardioprotection achieved in Cur-treated infected mice. With a view to the limited therapeutic possibilities available, Cur represents a promising approach for the treatment of Chagas heart disease., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

Trypanosoma cruzi has high biological and biochemical diversity and variable tissue tropism. Here we aimed to verify the kinetics of cytokine and chemokine in situ secretion in animals infected with two distinct T. cruzi strains after oral inoculation. Also, we investigated parasite migration, residence and pathological damage in stomach, heart and spleen. Our results showed that host immune response against T. cruzi infection is an intricate phenomenon that depends on the parasite strain, on the infected organ and on the time point of the infection. We believe that a wide comprehension of host immune response will potentially provide basis for the development of immunotherapeutic strategies in order to clear parasitism and minimize tissue injury. In this context, we find that KC poses as a possible tool to be used., (Copyright © 2016. Published by Elsevier B.V.)

Chagas disease, caused by Trypanosoma cruzi, results in an acute febrile illness that progresses to chronic chagasic cardiomyopathy in 30% of patients. Current treatments have significant side effects and poor efficacy during the chronic phase; therefore, there is an urgent need for new treatment modalities. A robust TH1-mediated immune response correlates with favorable clinical outcomes. A therapeutic vaccine administered to infected individuals could bolster the immune response, thereby slowing or stopping the progression of chagasic cardiomyopathy. Prior work in mice has identified an efficacious T. cruzi DNA vaccine encoding Tc24. To elicit a similar protective cell-mediated immune response to a Tc24 recombinant protein, we utilized a poly(lactic-co-glycolic acid) nanoparticle delivery system in conjunction with CpG motif-containing oligodeoxynucleotides as an immunomodulatory adjuvant. In a BALB/c mouse model, the vaccine produced a TH1-biased immune response, as demonstrated by a significant increase in antigen-specific IFNγ-producing splenocytes, IgG2a titers, and proliferative capacity of CD8(+) T cells. When tested for therapeutic efficacy, significantly reduced systemic parasitemia was seen during peak parasitemia. Additionally, there was a significant reduction in cardiac parasite burden and inflammatory cell infiltrate. This is the first study demonstrating immunogenicity and efficacy of a therapeutic Chagas vaccine using a nanoparticle delivery system.

Trypanosoma cruzi is mainly transmitted by blood-sucking triatomines, but other routes also have epidemiological importance, such as blood transfusion and congenital transmission. Although the possibility of sexual transmission of T. cruzi has been suggested since its discovery, few studies have been published on this subject. We investigated acquisition of T. cruzi by sexual intercourse in an experimental murine model. Male and female mice in the chronic phase of Chagas disease were mated with naive partners. Parasitological, serological and molecular tests demonstrated the parasites in tissues and blood of partners. These results confirm the sexual transmission of T. cruzi in mice., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The search for new compounds with trypanocidal activity is crucial for the treatment of Chagas' disease. Previous in vitro studies have shown that the diterpene 5-epi-icetexone (ICTX) is active against Trypanosoma cruzi. The aim of this work was to evaluate the effect of ICTX on the parasites in infected mice, in an experimental model that mimics the acute phase of the disease. Swiss albino mice were infected with T. cruzi and treated daily with 10mg/kg/day ICTX (i.p.). Infected mice and mice injected with either saline or the vehicle DMSO were used as controls. Animals' survival and parasitemia were monitored once a week and histological studies were made at necropsy by the 5th week after infection. It was observed that the administration of ICTX increased the survival of mice infected, and induced a significant decrease in the parasitemia, as compared to controls. A similar protective effect was observed when animals were treated orally with benznidazole (BZN, used as a control of antiparasitic effect). By the 5th week post-infection, the presence of amastigote nests was observed within the fibers of the cardiac and skeletal muscle in controls, but not in animals treated with either ICTX or BZN. In addition, inflammatory infiltrates were observed in the tissues of controls, but not in animals treated with the drugs. We conclude that ICTX has an antiparasitic effect against T. cruzi, thus constituting an interesting option for the treatment of Chagas' disease, alone or combined with other drugs., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Chagas disease, caused by Trypanosoma cruzi, is the main cause of dilated cardiomyopathy in the Americas. Antiparasitic treatment mostly relies on benznidazole (Bzl) due to Nifurtimox shortage or unavailability. Both induce adverse drug effects (ADE) of varied severity in many patients, leading to treatment discontinuation or abandonment. Since dosage may influence ADE, we aimed to assess Bzl efficacy in terms of parasiticidal and anti-inflammatory activity, using doses lower than those previously reported. BALB/c mice infected with the T. cruzi RA strain were treated with different doses of Bzl. Parasitaemia, mortality and weight change were assessed. Parasite load, tissue infiltrates and inflammatory mediators were studied in the heart. Serum creatine kinase (CK) activity was determined as a marker of heart damage. The infection-independent anti-inflammatory properties of Bzl were studied in an in vitro model of LPS-treated cardiomyocyte culture. Treatment with 25 mg/kg/day Bzl turned negative the parasitological parameters, induced a significant decrease in IL-1β, IL-6 and NOS2 in the heart and CK activity in serum, to normal levels. No mortality was observed in infected treated mice. Primary cultured cardiomyocytes treated with Bzl showed that inflammatory mediators were reduced via inhibition of the NF-κB pathway. A Bzl dose lower than that previously reported for treatment of experimental Chagas disease exerts adequate antiparasitic and anti-inflammatory effects leading to parasite clearance and tissue healing. This may be relevant to reassess the dose currently used for the treatment of human Chagas disease, aiming to minimize ADE.

Background: Chagas disease, caused by the protozoan Trypanosoma cruzi (T. cruzi), is a complex disease endemic in Central and South America. It has been gathering interest due to increases in non-vectorial forms of transmission, especially in developed countries. The objective of this work was to investigate if adipose tissue-derived mesenchymal stromal cells (ASC) can alter the course of the disease and attenuate pathology in a mouse model of chagasic cardiomyopathy., Methodology/principal Findings: ASC were injected intraperitoneally at 3 days post-infection (dpi). Tracking by bioluminescence showed that cells remained in the abdominal cavity for up to 9 days after injection and most of them migrated to the abdominal or subcutaneous fat, an early parasite reservoir. ASC injection resulted in a significant reduction in blood parasitemia, which was followed by a decrease in cardiac tissue inflammation, parasitism and fibrosis at 30 dpi. At the same time point, analyses of cytokine release in cells isolated from the heart and exposed to T. cruzi antigens indicated an anti-inflammatory response in ASC-treated animals. In parallel, splenocytes exposed to the same antigens produced a pro-inflammatory response, which is important for the control of parasite replication, in placebo and ASC-treated groups. However, splenocytes from the ASC group released higher levels of IL-10. At 60 dpi, magnetic resonance imaging revealed that right ventricular (RV) dilation was prevented in ASC-treated mice., Conclusions/significance: In conclusion, the injection of ASC early after T. cruzi infection prevents RV remodeling through the modulation of immune responses. Lymphoid organ response to the parasite promoted the control of parasite burden, while the heart, a target organ of Chagas disease, was protected from damage due to an improved control of inflammation in ASC-treated mice.

There are currently no biomarkers to assess which patients with chronic indeterminate Chagas disease will develop heart disease and which will spend their entire life in this state. We hypothetize that the parasite burden and Trypanosoma cruzi genotypes are related to the presence of heart disease in patients with Chagas disease. This study is aimed to investigate the parasite burden and T. cruzi genotypes in chagasic cardiopaths versus chagasic individuals without cardiac involvement according to the New York Heart Association. Patients with chronic Chagas disease, 50 with and 50 without cardiopathy (controls), groups A and B, respectively, were submitted to anamnesis, physical examination, and electrocardiogram. Echo-Doppler was performed for group A; all important known causes of cardiopathy were discarded. Xenodiagnosis, conventional PCR, and quantitative PCR were performed on patients of both groups. T. cruzi genotyping was done for 25 patients of group A and 20 of group B. The 50 cardiopaths had 80 electrocardiographic alterations, most of them in grade II of the New York Heart Association classification; 49 were classified in grade I by Echo-Doppler, and only one patient was in grade III. The difference in average parasitemia in patients of groups A and B was not significant. The most frequent T. cruzi DTU found was TcV. The parasite burden and genotype of the groups with and without cardiopathy were similar. Graphical abstract Imagen 1 Chronic chagas cardiopathy chest X-ray heart enlargement Figure 2 Chronic Chagas cardiopathy microaneurism of left ventricle. Cineangiography.

Introduction: Four species of triatomines have been reported in Nuevo León, northeast (NE) México, but Triatoma gerstaeckeri has only been recorded from a peridomestic dwelling., Objectives: To assess the natural infection index (NII) of Trypanosoma cruzi in triatomines and the infestation index (II) of T. gerstaeckeri collected in a suburban locality, and to collect histopathological data to understand tissue tropism of the regional T. cruzi strain (strain NE) obtained from the vectors collected after an experimental inoculation in Mus musculus ., Materials and Methods: Triatomines were collected from 85 houses and peridomiciles in Allende, Nuevo León. Stool samples were obtained to determine the T. cruzi NII and were used in an experimental mice infection., Results: A total of 118 T . gerstaeckeri were captured, and 46 (adults and nymphs) were collected inside the same house (II=1.17%). Thirty-seven reduvids were infected with T. cruzi (NII=31.3%). Tissue tropism of the T. cruzi NE strain was progressive in skeletal muscle, myocardial, and adipose tissues and was characterized by the presence of intracellular amastigotes and destruction of cardiac myocells., Conclusions: The presence of naturally infected domiciliary vectors is an important risk factor for public health in the region considering that these vectors are the principal transmission mechanism of the parasite. The T. cruzi NE strain has similar virulence to that of other Mexican and Texan strains and caused chagasic infections in 11 of 12 mice.

Purpose of Review: American trypanosomiasis, or Chagas disease, is a lifelong and persistent infection caused by the protozoan Trypanosoma cruzi and is the most significant cause of morbidity and mortality in South and Central America. Owing to immigration and additional risks from blood transfusion and organ transplantation, the number of reported cases of Chagas disease has increased recently in Europe and the USA. The disease is caused by a moderate to intense lasting inflammatory response that triggers local expression of inflammatory mediators and activates and recruits leukocytes to various tissues to eliminate the parasites., Recent Findings: This long-term inflammatory process triggers biochemical, physiological and morphological alterations and clinical changes in the digestive, nervous and cardiac (e.g. myocarditis, arrhythmias, congestive heart failure, autonomic dysfunctions and microcirculatory disturbances) systems. Indeed, the pathogenesis of Chagas disease is intricate and multifactorial, and the roles of the parasite and the immune response in initiating and maintaining the disease are still controversial., Summary: In this review, we discuss the current knowledge of 'strategies' employed by the parasite to persist in the host and host defence mechanisms against Trypanosoma cruzi infection, which can result in equilibrium (absence of the disease) or disease development, mainly in the cardiac systems.

Background: Chronic chagasic cardiomyopathy (CCC), the main clinical sign of Chagas disease, is associated with systemic CD8+ T-cell abnormalities and CD8-enriched myocarditis occurring in an inflammatory milieu. Pentoxifylline (PTX), a phosphodiesterase inhibitor, has immunoregulatory and cardioprotective properties. Here, we tested PTX effects on CD8+ T-cell abnormalities and cardiac alterations using a model of experimental Chagas' heart disease., Methodology/principal Findings: C57BL/6 mice chronically infected by the Colombian Trypanosoma cruzi strain and presenting signs of CCC were treated with PTX. The downmodulation of T-cell receptors on CD8+ cells induced by T. cruzi infection was rescued by PTX therapy. Also, PTX reduced the frequency of CD8+ T-cells expressing activation and migration markers in the spleen and the activation of blood vessel endothelial cells and the intensity of inflammation in the heart tissue. Although preserved interferon-gamma production systemically and in the cardiac tissue, PTX therapy reduced the number of perforin+ cells invading this tissue. PTX did not alter parasite load, but hampered the progression of heart injury, improving connexin 43 expression and decreasing fibronectin overdeposition. Further, PTX reversed electrical abnormalities as bradycardia and prolonged PR, QTc and QRS intervals in chronically infected mice. Moreover, PTX therapy improved heart remodeling since reduced left ventricular (LV) hypertrophy and restored the decreased LV ejection fraction., Conclusions/significance: PTX therapy ameliorates critical aspects of CCC and repositioned CD8+ T-cell response towards homeostasis, reinforcing that immunological abnormalities are crucially linked, as cause or effect, to CCC. Therefore, PTX emerges as a candidate to treat the non-beneficial immune deregulation associated with chronic Chagas' heart disease and to improve prognosis.

Infection with the protozoan parasite Trypanosoma cruzi may lead to a potentially fatal cardiomyopathy known as Chagas heart disease. This disease is characterized by infiltration of the myocardium by mononuclear cells, including CD4+ T cells, together with edema, myofibrillary destruction, and fibrosis. A multifaceted systemic immune response develops that ultimately keeps parasitemia and tissue parasitosis low. T helper 1 and other pro-inflammatory T cell responses are effective at keeping levels of T. cruzi low in tissues and blood, but they may also lead to tissue inflammation when present chronically. The mechanism by which the inflammatory response is regulated in T. cruzi-infected individuals is complex, and the specific roles that Th17 and T regulatory (Treg) cells may play in that regulation are beginning to be elucidated. In this study, we found that depletion of Treg cells in T. cruzi-infected mice leads to reduced cardiac parasitosis and inflammation, accompanied by an augmented Th1 response early in the course of infection. This is followed by a downregulation of the Th1 response and increased Th17 response late in infection. The effect of Treg cell depletion on the Th1 and Th17 cells is not observed in mice immunized with T. cruzi in adjuvant. This suggests that Treg cells specifically regulate Th1 and Th17 cell responses during T. cruzi infection and may also be important for modulating parasite clearance and inflammation in the myocardium of T. cruzi-infected individuals.