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Chagas disease is a major public health issue, affecting ∼10 million people worldwide. Transmitted by a protozoan named Trypanosoma cruzi , this infection triggers a chronic inflammatory process that can lead to cardiomyopathy (Chagas disease). Resolvin D1 (RvD1) is a novel proresolution lipid mediator whose effects on inflammatory diseases dampens pathological inflammatory responses and can restore tissue homeostasis. Current therapies are not effective in altering the outcome of T. cruzi infection, and as RvD1 has been evaluated as a therapeutic agent in various inflammatory diseases, we examined if exogenous RvD1 could modulate the pathogenesis of Chagas disease in a murine model. CD-1 mice infected with the T. cruzi Brazil strain were treated with RvD1. Mice were administered 3 μg/kg of body weight RvD1 intraperitoneally on days 5, 10, and 15 to examine the effect of RvD1 on acute disease or administered the same dose on days 60, 65, and 70 to examine its effects on chronic infection. RvD1 therapy increased the survival rate and controlled parasite replication in mice with acute infection and reduced the levels of interferon gamma and transforming growth factor β (TGF-β) in mice with chronic infection. In addition, there was an increase in interleukin-10 levels with RvD1 therapy in both mice with acute infection and mice with chronic infection and a decrease in TGF-β levels and collagen content in cardiac tissue. Together, these data indicate that RvD1 therapy can dampen the inflammatory response, promote the resolution of T. cruzi infection, and prevent cardiac fibrosis., (Copyright © 2020 American Society for Microbiology.)

Chagas disease affects millions of people, and it is a major cause of death in Latin America. Prevention and development of an effective treatment for this infection can be favored by a more thorough understanding of T. cruzi interaction with the microbiome of vectors and hosts. Next-generation sequencing technology vastly broadened the knowledge about intestinal bacteria composition, showing that microbiota within each host (triatomines and mammals) is composed by high diversity of species, although few dominant phyla. This fact may represent an ecological balance that was acquired during the evolutionary process of the microbiome-host complex, and that serves to perpetuate this system. In this context, commensal microbiota is also essential to protect hosts, conferring them resistance to pathogens colonization. However, in some situations, the microbiota is not able to prevent infection but only modulate it. Here we will review the role of the microbiota on the parasite-vector-host triad with a focus on the kinetoplastida of medical importance Trypanosoma cruzi. Novel strategies to control Chagas disease based on intestinal microbiome will also be discussed., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The M17 leucyl-aminopeptidase of Trypanosoma cruzi (LAPTc) is a novel drug target for Chagas disease. The objective of this work was to obtain recombinant LAPTc (rLAPTc) in Escherichia coli. A LAPTc gene was designed, optimized for its expression in E. coli, synthesized and cloned into the vector pET-19b. Production of rLAPTc in E. coli BL21(DE3)pLysS, induced for 20 h at 25 °C with 1 mM IPTG, yielded soluble rLAPTC that was catalytically active. The rLAPTc enzyme was purified in a single step by IMAC. The recombinant protein was obtained with a purity of 90% and a volumetric yield of 90 mg per liter of culture. The enzymatic activity has an optimal pH of 9.0, and preference for Leu-p-nitroanilide (appK M  = 74 µM, appk cat  = 4.4 s -1 ). The optimal temperature is 50 °C, and the cations Mg 2+ , Cd 2+ , Ba 2+ , Ca 2+ and Zn 2+ at 4 mM inhibited the activity by 60% or more, while Mn 2+ inhibited by only 15% and addition of Co 2+ activated by 40%. The recombinant enzyme is insensitive toward the protease inhibitors PMSF, TLCK, E-64 and pepstatin A, but is inhibited by EDTA and bestatin. Bestatin is a non-competitive inhibitor of the enzyme with a K i value of 881 nM. The enzyme is a good target for inhibitor identification.

Background: Chagas disease is still prevalent in rural areas of South America. In endemic areas of Bolivia, school children are screened for the program of Chagas disease eradication of the Ministry of Health, and positive children are treated. Here, we compared the fecal, oral and skin microbiomes of children with or without Chagas disease, and before and after benznidazol treatment of infected children., Methods: A total of 543 Bolivian children (5-14 years old) were tested for Chagas disease, and 20 positive children were treated with Benznidazole. Fecal samples and oral and skin swabs were obtained before and after treatment, together with samples from a group of 35 uninfected controls. The 16S rRNA genes were sequenced and analyzed using QIIME to determine Alpha diversity differences and community distances, and linear discriminant analyses to determine marker taxa by infection status or treatment., Results: Twenty out of 543 children screened were seropositive for Chagas disease (3.7%) and were included in the study, together with 35 control children that were seronegative for the disease. Fecal samples, oral and skin swabs were taken at the beginning of the study and after the anti-protozoa therapy with Benznidazole to the chagasic children. Infected children had higher fecal Firmicutes (Streptococcus, Roseburia, Butyrivibrio, and Blautia), and lower Bacteroides and also showed some skin -but not oral- microbiota differences. Treatment eliminated the fecal microbiota differences from control children, increasing Dialister (class Clostridia) and members of the Enterobacteriaceae, and decreasing Prevotella and Coprococcus, with minor effects on the oral and skin bacterial diversity., Conclusions: The results of this study show differences in the fecal microbiota associated with Chagas disease in children, and also evidence that treatment normalizes fecal microbiota (makes it more similar to that in controls), but is associated with oral and skin microbiota differences from control children. Since microbiota impacts in children, it is important to determine the effect of drugs on the children microbiota, since dysbiosis could lead to physiological effects which might be avoidable with microbiota restoration interventions., Competing Interests: The authors have declared that no competing interests exist.

The IL-17 family contributes to host defense against many intracellular pathogens by mechanisms that are not fully understood. CD8+ T lymphocytes are key elements against intracellular microbes, and their survival and ability to mount cytotoxic responses are orchestrated by several cytokines. Here, we demonstrated that IL-17RA-signaling cytokines sustain pathogen-specific CD8+ T cell immunity. The absence of IL-17RA and IL-17A/F during Trypanosoma cruzi infection resulted in increased tissue parasitism and reduced frequency of parasite-specific CD8+ T cells. Impaired IL-17RA-signaling in vivo increased apoptosis of parasite-specific CD8+ T cells, while in vitro recombinant IL-17 down-regulated the pro-apoptotic protein BAD and promoted the survival of activated CD8+ T cells. Phenotypic, functional, and transcriptomic profiling showed that T. cruzi -specific CD8+ T cells derived from IL-17RA-deficient mice presented features of cell dysfunction. PD-L1 blockade partially restored the magnitude of CD8+ T cell responses and parasite control in these mice. Adoptive transfer experiments established that IL-17RA-signaling is intrinsically required for the proper maintenance of functional effector CD8+ T cells. Altogether, our results identify IL-17RA and IL-17A as critical factors for sustaining CD8+ T cell immunity to T. cruzi .

The activation of macrophage respiratory burst in response to infection with Trypanosoma cruzi inflicts oxidative damage to the host's tissues. For decades, the role of reactive oxygen species (ROS) in the elimination of T. cruzi was taken for granted, but recent evidence suggests parasite growth is stimulated in oxidative environments. It is still a matter of debate whether indeed oxidative environments provide ideal conditions (e.g., iron availability in macrophages) for T. cruzi growth and whether indeed ROS signals directly to stimulate growth. Nitric oxide (NO) and ROS combine to form peroxynitrite, participating in the killing of phagocytosed parasites by activated macrophages. In response to infection, mitochondrial ROS are produced by cardiomyocytes. They contribute to oxidative damage that persists at the chronic stage of infection and is involved in functional impairment of the heart. In this review, we discuss how oxidative stress helps parasite growth during the acute stage and how it participates in the development of cardiomyopathy at the chronic stage.

Some 1,3-diarylureas and 1-((1,4-trans)-4-aryloxycyclohexyl)-3-arylureas (cHAUs) activate heme-regulated kinase causing protein synthesis inhibition via phosphorylation of the eukaryotic translation initiation factor 2 (eIF2) in mammalian cancer cells. To evaluate if these agents have potential to inhibit trypanosome multiplication by also affecting the phosphorylation of eIF2 alpha subunit (eIF2α), we tested 25 analogs of 1,3-diarylureas and cHAUs against Trypanosoma cruzi, the agent of Chagas disease. One of them (I-17) presented selectivity close to 10-fold against the insect replicative forms and also inhibited the multiplication of T. cruzi inside mammalian cells with an EC 50 of 1-3 µM and a selectivity of 17-fold. I-17 also prevented replication of African trypanosomes (Trypanosoma brucei bloodstream and procyclic forms) at similar doses. It caused changes in the T. cruzi morphology, arrested parasite cell cycle in G1 phase, and promoted phosphorylation of eIF2α with a robust decrease in ribosome association with mRNA. The activity against T. brucei also implicates eIF2α phosphorylation, as replacement of WT-eIF2α with a non-phosphorylatable eIF2α, or knocking down eIF2 protein kinase-3 by RNAi increased resistance to I-17. Therefore, we demonstrate that eIF2α phosphorylation can be engaged to develop trypanosome-static agents in general, and particularly by interfering with activity of eIF2 kinases.

Trypanosoma cruzi parasites are the causative agents of Chagas disease. These parasites infect cardiac and gastrointestinal tissues, leading to local inflammation and tissue damage. Digestive Chagas disease is associated with perturbations in food absorption, intestinal traffic and defecation. However, the impact of T. cruzi infection on the gut microbiota and metabolome have yet to be characterized. In this study, we applied mass spectrometry-based metabolomics and 16S rRNA sequencing to profile infection-associated alterations in fecal bacterial composition and fecal metabolome through the acute-stage and into the chronic stage of infection, in a murine model of Chagas disease. We observed joint microbial and chemical perturbations associated with T. cruzi infection. These included alterations in conjugated linoleic acid (CLA) derivatives and in specific members of families Ruminococcaceae and Lachnospiraceae, as well as alterations in secondary bile acids and members of order Clostridiales. These results highlight the importance of multi-'omics' and poly-microbial studies in understanding parasitic diseases in general, and Chagas disease in particular.

Chagas disease (CD) is a parasitic zoonosis endemic in most mainland countries of Central and South America affecting nearly 10 million people, with 100 million people at high risk of contracting the disease. Treatment is only effective if received at the early stages of the disease. Only two drugs (benznidazole and nifurtimox) have so far been marketed, and both share various limitations such as variable efficacy, many side effects, and long duration of treatment, thus reducing compliance. The in vitro and in vivo efficacy of poly-aggregated amphotericin B (AmB), encapsulated poly-aggregated AmB in albumin microspheres (AmB-AME), and dimeric AmB-sodium deoxycholate micelles (AmB-NaDC) was evaluated. Dimeric AmB-NaDC exhibited a promising selectivity index (SI = 3164) against amastigotes, which was much higher than those obtained for licensed drugs (benznidazole and nifurtimox). AmB-AME, but not AmB-NaDC, significantly reduced the parasitemia levels (3.6-fold) in comparison to the control group after parenteral administration at day 7 postinfection. However, the oral administration of AmB-NaDC (10-15 mg/kg/day for 10 days) resulted in a 75% reduction of parasitemia levels and prolonged the survival rate in 100% of the tested animals. Thus, the results presented here illustrate for the first time the oral efficacy of AmB in the treatment of trypanosomiasis. AmB-NaDC is an easily scalable, affordable formulation prepared from GRAS excipients, enabling treatment access worldwide, and therefore it can be regarded as a promising therapy for trypanosomiasis.

This work describes the antitrypanocidal activity of two hydroxamic acid derivatives containing o-ethoxy (HAD1) and p-ethoxy (HAD2) as substituent in the aromatic ring linked to the isoxazoline ring. HAD1 and HAD2 induced a significant reduction in the number of intracellular parasites and consequently showed activity on the multiplication of the parasite. Treatment of cardiomyocytes and macrophages with the compounds revealed no significant loss in cell viability. Ultrastructural alterations after treatment of cardiomyocytes or macrophages infected by Trypanosoma cruzi with the IC50 value of HAD1 revealed alterations to amastigotes, showing initial damage seen as swelling of the kinetoplast. This gave a good indication of the ability of the drug to permeate through the host cell membrane as well as its selectivity to the parasite target. Both compounds HAD1 and 2 were able to reduce the cysteine peptidases and decrease the activity of metallopeptidases.

Since the immune response mounted by the host to a particular microorganism might be influenced by the acquired immunological experience due to previous contact with other microorganisms, we performed a cross-sectional study to explore the pattern of Trypanosoma cruzi infection-related antibodies in T. cruzi-seropositive individuals presenting concomitant tuberculosis, or the antecedent of BCG vaccination. Sampled individuals were grouped as follows: patients with Chagas disease, not vaccinated with BCG, who further developed pulmonary tuberculosis; individuals with Chagas disease, BCG-vaccinated; and subjects with Chagas disease, presenting neither BCG scar nor tuberculosis disease. Non-vaccinated individuals or without tuberculosis, presented the highest values of anti-PH (P < 0.001), anti-FRA (P < 0.001), anti-p2β (P = 0.0023) and anti-B13 (P < 0.001) antibodies. The present findings constitute the first demonstration of the potential influence of concomitant tuberculosis on Chagas disease., (© FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

American trypanosomiasis, commonly known as Chagas disease, is a neglected tropical disease caused by the protozoan parasite Trypanosoma cruzi. The chronic form of the infection often causes debilitating morbidity and mortality. However, the current treatment for the disease is typically inadequate owing to drug toxicity and poor efficacy, necessitating a continual effort to discover and develop new antiparasitic therapeutic agents. The structure of T. cruzi histidyl-tRNA synthetase (HisRS), a validated drug target, has previously been reported. Based on this structure and those of human cytosolic HisRS, opportunities for the development of specific inhibitors were identified. Here, efforts are reported to identify small molecules that bind to T. cruzi HisRS through fragment-based crystallographic screening in order to arrive at chemical starting points for the development of specific inhibitors. T. cruzi HisRS was soaked into 68 different cocktails from the Medical Structural Genomics of Pathogenic Protozoa (MSGPP) fragment library and diffraction data were collected to identify bound fragments after soaking. A total of 15 fragments were identified, all bound to the same site on the protein, revealing a fragment-binding hotspot adjacent to the ATP-binding pocket. On the basis of the initial hits, the design of reactive fragments targeting the hotspot which would be simultaneously covalently linked to a cysteine residue present only in trypanosomatid HisRS was initiated. Inhibition of T. cruzi HisRS was observed with the resultant reactive fragments and the anticipated binding mode was confirmed crystallographically. These results form a platform for the development of future generations of selective inhibitors for trypanosomatid HisRS.

Unbiased phenotypic screens enable identification of small molecules that inhibit pathogen growth by unanticipated mechanisms. These small molecules can be used as starting points for drug discovery programs that target such mechanisms. A major challenge of the approach is the identification of the cellular targets. Here we report GNF7686, a small molecule inhibitor of Trypanosoma cruzi, the causative agent of Chagas disease, and identification of cytochrome b as its target. Following discovery of GNF7686 in a parasite growth inhibition high throughput screen, we were able to evolve a GNF7686-resistant culture of T. cruzi epimastigotes. Clones from this culture bore a mutation coding for a substitution of leucine by phenylalanine at amino acid position 197 in cytochrome b. Cytochrome b is a component of complex III (cytochrome bc1) in the mitochondrial electron transport chain and catalyzes the transfer of electrons from ubiquinol to cytochrome c by a mechanism that utilizes two distinct catalytic sites, QN and QP. The L197F mutation is located in the QN site and confers resistance to GNF7686 in both parasite cell growth and biochemical cytochrome b assays. Additionally, the mutant cytochrome b confers resistance to antimycin A, another QN site inhibitor, but not to strobilurin or myxothiazol, which target the QP site. GNF7686 represents a promising starting point for Chagas disease drug discovery as it potently inhibits growth of intracellular T. cruzi amastigotes with a half maximal effective concentration (EC50) of 0.15 µM, and is highly specific for T. cruzi cytochrome b. No effect on the mammalian respiratory chain or mammalian cell proliferation was observed with up to 25 µM of GNF7686. Our approach, which combines T. cruzi chemical genetics with biochemical target validation, can be broadly applied to the discovery of additional novel drug targets and drug leads for Chagas disease.

Cruzain is a primary cysteine protease expressed by the protozoan parasite Trypanosoma cruzi during Chagas disease infection, and thus, the development of inhibitors of this protein is a promising target for designing an effective therapy against the disease. In this paper, the mechanism of inhibition of cruzain by two different irreversible peptidyl halomethyl ketones (PHK) inhibitors has been studied by means of hybrid quantum mechanics/molecular mechanics-molecular dynamics (MD) simulations to obtain a complete representation of the possible free energy reaction paths. These have been traced on free energy surfaces in terms of the potential of mean force computed at AM1d/MM and DFT/MM levels of theory. An analysis of the possible reaction mechanisms of the inhibition process has been performed showing that the nucleophilic attack of an active site cysteine, Cys25, on a carbon atom of the inhibitor and the cleavage of the halogen-carbon bond take place in a single step. PClK appears to be much more favorable than PFK from a kinetic point of view. This result would be in agreement with experimental studies in other papain-like enzymes. A deeper analysis of the results suggests that the origin of the differences between PClK and PFK can be the different stabilizing interactions established between the inhibitors and the residues of the active site of the protein. Any attempt to explore the viability of the inhibition process through a stepwise mechanism involving the formation of a thiohemiketal intermediate and a three-membered sulfonium intermediate has been unsuccessful. Nevertheless, a mechanism through a protonated thiohemiketal, with participation of His159 as a proton donor, appears to be feasible despite showing higher free energy barriers. Our results suggest that PClK can be used as a starting point to develop a proper inhibitor of cruzain.

Many infectious diseases arise from co-infections or re-infections with more than one genotype of the same pathogen. These mixed infections could alter host fitness, the severity of symptoms, success in pathogen transmission and the epidemiology of the disease. Trypanosoma cruzi, the etiological agent of Chagas disease, exhibits a high biological variability often correlated with its genetic diversity. Here, we developed an experimental approach in order to evaluate biological interaction between three T. cruzi isolates belonging to different Discrete Typing Units (DTUs TcIII, TcV and TcVI). These isolates were obtained from a restricted geographical area in the Chaco Region. Different mixed infections involving combinations of two isolates (TcIII + TcV, TcIII + TcVI and TcV + TcVI) were studied in a mouse model. The parameters evaluated were number of parasites circulating in peripheral blood, histopathology and genetic characterization of each DTU in different tissues by DNA hybridization probes. We found a predominance of TcVI isolate in blood and tissues respect to TcIII and TcV; and a decrease of the inflammatory response in heart when the damage of mice infected with TcVI and TcIII + TcVI mixture were compared. In addition, simultaneous presence of two isolates in the same tissue was not detected. Our results show that biological interactions between isolates with different biological behaviors lead to changes in their biological properties. The occurrence of interactions among different genotypes of T. cruzi observed in our mouse model suggests that these phenomena could also occur in natural cycles in the Chaco Region.

Background: Clinical and experimental conflicting data have questioned the relationship between infectious agents, inflammation and dilated cardiomyopathy (DCM)., Objectives: The aim of this study was to determine the frequency of infectious agents and inflammation in endomyocardial biopsy (EMB) specimens from patients with idiopathic DCM, explanted hearts from different etiologies, including Chagas' disease, compared to donated hearts., Methods: From 2008 to 2011, myocardial samples from 29 heart donors and 55 patients with DCMs from different etiologies were studied (32 idiopathic, 9 chagasic, 6 ischemic and 8 other specific etiologies). Inflammation was investigated by immunohistochemistry and infectious agents by immunohistochemistry, molecular biology, in situ hybridization and electron microscopy., Results: There were no differences regarding the presence of macrophages, expression of HLA class II and ICAM-I in donors and DCM. Inflammation in Chagas' disease was predominant. By immunohistochemistry, in donors, there was a higher expression of antigens of enterovirus and Borrelia, hepatitis B and C in DCMs. By molecular biology, in all groups, the positivity was elevated to microorganisms, including co-infections, with a higher positivity to adenovirus and HHV6 in donors towards DCMs. This study was the first to demonstrate the presence of virus in the heart tissue of chagasic DCM., Conclusions: The presence of inflammation and infectious agents is frequent in donated hearts, in the myocardium of patients with idiopathic DCM, myocardial dysfunction related to cardiovascular diseases, and primary and secondary cardiomyopathies, including Chagas' disease. The role of co-infection in Chagas' heart disease physiopathology deserves to be investigated in future studies., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Introduction: Most studies that have evaluated the stomachs of patients with Chagas disease were performed before the discovery of Helicobacter pylori and used no control groups. This study compared the gastric features of chagasic and non-chagasic patients and assessed whether gastritis could be associated with Chagas disease., Methods: Gastric biopsy samples were taken from patients who underwent endoscopy for histological analysis according to the Updated Sydney System. H. pylori infection was assessed by histology, 16S ribosomal ribonucleic acid (rRNA) polymerase chain reaction (PCR), serology and the 13C-urea breath test. Patients were considered H. pylori-negative when all of these diagnostic tests were negative. Clinical and socio-demographic data were obtained by reviewing medical records and using a questionnaire., Results: The prevalence of H. pylori infection (70.3% versus 71.7%) and chronic gastritis (92.2% versus 85%) was similar in the chagasic and non-chagasic groups, respectively; such as peptic ulcer, atrophy and intestinal metaplasia. Gastritis was associated with H. pylori infection independent of Chagas disease in a log-binomial regression model. However, the chagasic H. pylori-negative patients showed a significantly higher grade of mononuclear (in the corpus) and polymorphonuclear (PMN) (in the antrum) cell infiltration. Additionally, the patients with the digestive form of Chagas disease showed a significantly lower prevalence of corpus atrophy than those with other clinical forms., Conclusions: The prevalence of H. pylori infection and of gastric histological and endoscopic features was similar among the chagasic and non-chagasic patients. Additionally, this is the first controlled study to demonstrate that H. pylori is the major cause of gastritis in patients with Chagas disease.

A synthetic glycoarray containing non-reducing α-galactopyranosyl moieties related to mucin O-glycans of the parasite Trypanosoma cruzi was evaluated by a chemiluminescent enzyme-linked immunosorbent assay with sera from patients with chronic Chagas disease. Our data revealed the disaccharide Galα(1,3)Galβ as the immunodominant glycotope, which may eventually be employed as a diagnostic antigen for Chagas disease.

Chagas disease (CD) is a chronic and endemic illness caused by the parasite Trypanosoma cruzi. Microvascular disturbances play an important role in the progress of the disease. The purinergic signaling system participates in regulatory functions, such as immunomodulation, neuroprotection, and thromboregulation. This study aimed to investigate the activities of purinergic system ecto-enzymes present on the platelet surface and the platelet aggregation profile from patients with indeterminate form of Chagas disease (IFCD). Thirty patients diagnosed with IFCD and 30 healthy subjects were selected. Ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase), ecto-nucleotide pyrophosphatase/phosphodiesterase (E-NPP), ecto-5'-nucleotidase (E-5'-NT) and ecto-adenosine deaminase (E-ADA) activities were measured in platelets isolated from these individuals as well as the platelet aggregation. Results demonstrated an increase of 21 % in the E-NPP activity and 30 % in the E-5'-NT activity in IFCD group (P < 0.05); however, a decrease of 34 % in the E-ADA activity was determined in the same group (P < 0.001). A significant decrease of 12.7 % and 12.8 % in the platelet aggregation of IFCD group in two different concentrations of ADP (5 and 10 μM) was observed, respectively (P < 0.05). Increased E-NPP and E-5-NT activities as well as decreased E-ADA activity in platelets of patients with IFCD contributed to decrease platelet aggregation, suggesting that the purinergic system is involved in the thromboregulation process in these patients, since adenosine (the final product of ATP hydrolysis) has cardioprotective and vasodilator effects that prevent the clinical progress of the disease.

Organ shortage is the first cause of death on liver transplant waiting lists. As a consequence, we recently decided to expand liver acceptance to those organs that could potentially transmit infectious diseases to their recipients. On January 2010, we initiated a prospective protocol using livers from Chagas-infected donors for transplanting uninfected recipients without using prophylactic therapy. During a 13-month period, 9 of 37 (24%) liver transplants were performed within this protocol. After transplant, each recipient was sequentially and strictly monitored for infection transmission using the Strout method and promptly treated with benznidazole if this occurs. During follow-up, two patients died without Chagas infection and only two (donor-derived T. cruzi transmission rate: 2/9; 22%) patients developed donor-derived Chagas transmission without clinical symptoms. The median follow-up time of the seven live patients was 15 months (range: 13-20). At present, all are symptoms-free with excellent allograft function and without evidence of Chagas disease. In conclusion, we consider that Chagas-infected donors are a promising source of liver grafts that could reduce the growing mortality on liver waiting lists in America. Relevant data from larger prospective studies are required to confirm these preliminary excellent results., (© Copyright 2012 The American Society of Transplantation and the American Society of Transplant Surgeons.)

Background: Chagas disease was originally reported in Panama in 1931. Currently, the best knowledge of this zoonosis is restricted to studies done in historically endemic regions. However, little is known about the distribution and epidemiology of Chagas disease in other rural areas of the country., Methods and Findings: A cross-sectional descriptive study was carried out between May 2005 - July 2008 in four rural communities of the Santa Fe District, Veraguas Province. The study included an entomologic search to collect triatomines, bloodmeal type identification and infection rate with trypanosomes in collected vectors using a dot- blot and PCR analysis, genotyping of circulating Trypanosoma cruzi (mini-exon gene PCR analysis) and the detection of chagasic antibodies among inhabitants. The vector Rhodnius pallescens was more frequently found in La Culaca and El Pantano communities (788 specimens), where it was a sporadic household visitor. These triatomines presented darker coloration and larger sizescompared with typical specimens collected in Central Panama. Triatoma dimidiata was more common in Sabaneta de El Macho (162 specimens). In one small sub-region (El Macho), 60% of the houses were colonized by this vector. Of the examined R. pallescens, 54.7.0% (88/161) had fed on Didelphis marsupialis, and 24.6% (34/138) of T. dimidiata specimens collected inside houses were positive for human blood. R. pallescens presented an infection index with T. cruzi of 17.7% (24/136), with T. rangeli of 12.5% (17/136) and 50.7% (69/136) were mixed infections. In 117 T. dimidiata domestic specimens the infection index with T. cruzi was 21.4%. Lineage I of T. cruzi was confirmed circulating in these vectors. A T. cruzi infection seroprevalence of 2.3% (24/1,056) was found in this population., Conclusions: This is the first report of Chagas disease endemicity in Santa Fe District, and it should be considered a neglected public health problem in this area of Panama.

Trypanosoma cruzi in order to complete its development in the digestive tract of Rhodnius prolixus needs to overcome the immune reactions and microbiota trypanolytic activity of the gut. We demonstrate that in R. prolixus following infection with epimastigotes of Trypanosoma cruzi clone Dm28c and, in comparison with uninfected control insects, the midgut contained (i) fewer bacteria, (ii) higher parasite numbers, and (iii) reduced nitrite and nitrate production and increased phenoloxidase and antibacterial activities. In addition, in insects pre-treated with antibiotic and then infected with Dm28c, there were also reduced bacteria numbers and a higher parasite load compared with insects solely infected with parasites. Furthermore, and in contrast to insects infected with Dm28c, infection with T. cruzi Y strain resulted in a slight decreased numbers of gut bacteria but not sufficient to mediate a successful parasite infection. We conclude that infection of R. prolixus with the T. cruzi Dm28c clone modifies the host gut immune responses to decrease the microbiota population and these changes are crucial for the parasite development in the insect gut.

Chagas' disease, caused by Trypanosoma cruzi, is considered a public health problem. Nowadays, chemotherapy is the only available treatment for this disease, and the drugs currently used, nifurtimox and benzonidazole, present high toxicity levels. Alternatives for replacing these drugs are natural extracts from Eugenia jambolana, a plant used in traditional medicine because of its antimicrobial and biological activities. An ethanol extract from E. jambolana was prepared. To research in vitro anti-epimastigote activity, T. cruzi CL-B5 clone was used. Epimastigotes were inoculated at a concentration of 1×10(5)/mL in 200 μL of tryptose-liver infusion. For the cytotoxicity assay J774 macrophages were used. To examine antifungal activity, Candida albicans, Candida tropicalis, and Candida krusei were used. This is the first record of trypanocide activity for E. jambolana. The effective concentration capable of killing 50% of the parasites was 56.42 μg/mL. The minimum inhibitory concentration was ≤1,024 μg/mL. Metronidazole showed a potentiation of its antifungal effect when combined with the ethanol extract of E. jambolana. Thus our results indicate that E. jambolana could be a source of plant-derived natural products with anti-epimastigote and antifungal modifying activity with moderate toxicity.

Trypanosoma cruzi phosphodiesterase (PDE) C (TcrPDEC), a novel and rather unusual PDE in which, unlike all other class I PDEs, the catalytic domain is localized in the middle of the polypeptide chain, is able to hydrolyze cyclic GMP (cGMP), although it prefers cyclic AMP (cAMP), and has a FYVE-type domain in its N-terminal region (S. Kunz et al., FEBS J. 272:6412-6422, 2005). TcrPDEC shows homology to the mammalian PDE4 family members. PDE4 inhibitors are currently under development for the treatment of inflammatory diseases, such as asthma, chronic pulmonary diseases, and psoriasis, and for treating depression and serving as cognitive enhancers. We therefore tested a number of compounds originally synthesized as potential PDE4 inhibitors on T. cruzi amastigote growth, and we obtained several useful hits. We then conducted homology modeling of T. cruzi PDEC and identified other compounds as potential inhibitors through virtual screening. Testing of these compounds against amastigote growth and recombinant TcrPDEC activity resulted in several potent inhibitors. The most-potent inhibitors were found to increase the cellular concentration of cAMP. Preincubation of cells in the presence of one of these compounds stimulated volume recovery after hyposmotic stress, in agreement with their TcrPDEC inhibitory activity in vitro, providing chemical validation of this target. The compounds found could be useful tools in the study of osmoregulation in T. cruzi. In addition, their further optimization could result in the development of new drugs against Chagas' disease and other trypanosomiases.

It is has been shown that the majority of T. cruzi strains isolated from Mexico belong to the T. cruzi I (TCI). The immune response produced in response to Mexican T. cruzi I strains has not been well characterized. In this study, two Mexican T. cruzi I strains were used to infect Balb/c mice. The Queretaro (TBAR/MX/0000/Queretaro)(Qro) strain resulted in 100% mortality. In contrast, no mortality was observed in mice infected with the Ninoa (MHOM/MX/1994/Ninoa) strain. Both strains produced extended lymphocyte infiltrates in cardiac tissue. Ninoa infection induced a diverse humoral response with a higher variety of immunoglobulin isotypes than were found in Qro-infected mice. Also, a stronger inflammatory TH1 response, represented by IL-12p40, IFNgamma, RANTES, MIG, MIP-1beta, and MCP-1 production was observed in Qro-infected mice when compared with Ninoa-infected mice. We propose that an exacerbated TH1 immune response is a likely cause of pathological damage observed in cardiac tissue and the primary cause of death in Qro-infected mice.

The cytokine-mediated stimulation of the hypothalamus-pituitary-adrenal (HPA) axis is relevant for immunoregulation and survival during bacterial endotoxemia and certain viral infections. However, only limited information is available regarding the effect of endogenous glucocorticoids on parasitic diseases. Here, we discuss evidence that the increased levels of corticosterone that occur following Trypanosoma cruzi infection in mice is an endocrine response that protects the host by impeding an excessive production of pro-inflammatory cytokines. Comparative studies between susceptible C57Bl/6J and resistant Balb/c mice indicate that the predisposition to the disease depends on the appropriate timing and magnitude of the activation of the HPA axis. However, this endocrine response also results in thymus atrophy and depletion of CD4(+)CD8(+) by apoptosis. On the other hand, using tumor necrosis factor (TNF)-receptor knockout mice, we found that TNF-alpha plays a complex role during this disease; it is involved in the mediation of cardiac tissue damage but it also contributes to prolonged survival. Taken together, this evidence indicates that a subtle balance between endocrine responses and cytokine production is necessary for an efficient defense against T. cruzi infection.

American Trypanosomiasis is caused by the hemoflagellate Trypanosoma cruzi (T. cruzi) and affects millions of persons causing variable degrees of digestive and heart disturbances. As far as we concerned, T. cruzi capacity to synthesize steroid hormones has not been investigated. Therefore, the aim of this work was to investigate the capacity of T. cruzi trypomastigotes to transform tritiated steroid precursors into androgens and estrogens. The T. cruzi Tulahuén strain was obtained from mice blood. The trypomastigotes were cultured for 6 and 24h in Dulbbeco's modified Eagle's medium plus FCS and antibiotics. Tritiated dehydroepiandrosterone or androstendione were added to the culture media and parasites were incubated for 6 or 24h. The cultures were centrifuged and ether extracted. The steroids were analyzed by thin layer chromatography (TLC) in two solvent systems. After incubation with 3H-androstenedione, T. cruzi trypomastigotes synthesized 3H-testosterone (T), 3H-17beta-estradiol (E2) and 3H-estrone (E1). Metabolism of 3H-DHEA by the parasites yielded 3H-androstendione and 3H-androstendiol at 6h of incubation. The recrystallization procedure further demonstrated the 3H-androstendiol and 3H-17beta-estradiol syntheses. Results indicate for the first time that T. cruzi trypomastigotes produce androgens and estrogens when incubated in the presence of steroid precursors and suggest the presence of active parasite steroidogenic enzymes.

Blood smear analysis is especially useful for diagnosing five infectious diseases: babesiosis, ehrlichiosis, relapsing fever due to Borrelia infection, malaria, and American trypanosomiasis (Chagas disease). It should be performed in patients with persistent or recurring fever or in those who have traveled to the developing world or who have a history of tick exposure, especially if accompanied by hemolytic anemia, thrombocytopenia, or hepatosplenomegaly.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) plays an important role in the life cycle of the Trypanosoma cruzi, and an immobilized enzyme reactor (IMER) has been developed for use in the on-line screening for GAPDH inhibitors. An IMER containing human GAPDH has been previously reported; however, these conditions produced a T. cruzi GAPDH-IMER with poor activity and stability. The factors affecting the stability of the human and T. cruzi GAPDHs in the immobilization process and the influence of pH and buffer type on the stability and activity of the IMERs have been investigated. The resulting T. cruzi GAPDH-IMER was coupled to an analytical octyl column, which was used to achieve chromatographic separation of NAD(+) from NADH. The production of NADH stimulated by d-glyceraldehyde-3-phosphate was used to investigate the activity and kinetic parameters of the immobilized T. cruzi GAPDH. The Michaelis-Menten constant (K(m)) values determined for d-glyceraldehyde-3-phosphate and NAD(+) were K(m) = 0.5 +/- 0.05 mM and 0.648 +/- 0.08 mM, respectively, which were consistent with the values obtained using the non-immobilized enzyme.

Both invasion of the host cell by T. cruzi and its establishment into the mammalian host are critical steps. In this review, the adhesion step and the intracellular survival in non-professional phagocytes are particularly focused on, with special emphasis on the role of Gp85/trans-sialidase (Gp85/TS) superfamily. Excellent reviews have been published lately, some covering other aspects of T. cruzi-host interaction and will be cited instead of the original articles due to limited number of listed references.

In the megaesophagus of Chagas' disease, chronic esophagitis is caused by stasis of swallowed food and saliva. In this environment, the overgrowth of aerobic and anaerobic bacteria, including nitrate-reducing bacteria, is observed. The reduction of nitrate into nitrite by the action of these bacteria has been associated with the formation of volatile nitrosamines in different situations of gastric bacterial overgrowth. We have hypothesized that this phenomenon could occur in the esophageal lumen of patients with megaesophagus. To evaluate the concentration of nitrite, the presence of volatile nitrosamines and the concentration of nitrate-reducing bacteria in the esophageal lumen of patients with non-advanced megaesophagus of Chagas' disease and in a group of patients without esophageal disease. Fifteen patients with non-advanced megaesophagus [megaesophagus group (MG)] and 15 patients without any esophageal disease [control group (CG)] were studied. Saliva samples were taken for nitrate and nitrite quantitative determination and esophageal stasis liquid samples were taken for nitrate and nitrite quantitative determination, volatile nitrosamines qualitative determination and reductive bacteria quantitative determination. MG and CG were equivalent in nitrate and nitrite saliva concentration and in nitrate esophageal concentration. Significant difference was found in nitrite (p = 0.003) and reductive bacteria concentration (p < 0.0001), both higher in MG. Volatile nitrosamines were identified in three MG patients and in none of the CG patients, but this was not significant (p = 0.113). There is a higher concentration of reductive bacteria in MG, responsible for the rise in nitrite concentration at the esophageal lumen and, eventually, for the formation of volatile nitrosamines.

The target cell F-actin disassembly, induced by a Ca2+-signaling Trypanosoma cruzi factor of unknown molecular identity, has been reported to promote parasite invasion. We investigated whether the metacyclic trypomastigote stage-specific surface molecule gp82, a Ca2+-signal-inducing molecule implicated in host cell invasion, displayed the ability to induce actin cytoskeleton disruption, using a recombinant protein (J18) containing the full-length gp82 sequence fused to GST. J18, but not GST, induced F-actin disassembly in HeLa cells, significantly reducing the number as well as the length of stress fibers. The number of cells with typical stress fibers scored approximately 70% in untreated and GST-treated cells, as opposed to approximately 30% in J18-treated samples, which also showed decreased F-actin content. J18, but not GST, inhibited approximately 6-fold the HeLa cell entry of enteroinvasive Escherichia coli (EIEC), which depends on actin cytoskeleton. Not only were fewer cells infected with bacteria in the presence of J18, there were also fewer bacteria per cell. The inhibitory activity of J18 was Ca2+ dependent. In co-infection experiments, preincubation of HeLa cells with EIEC drastically reduced gp82-dependent internalization of T. cruzi metacyclic forms. All these data, plus the finding that gp82-mediated penetration of metacyclic forms was associated with disrupted HeLa cell cytoskeletal architecture, indicate that gp82 promotes parasite invasion by disassembling the cortical actin cytoskeleton.

To study the influence of normal associated microbiota on systemic immunological responses during experimental Chagas' disease, germ-free and conventional NIH Swiss mice were infected with Y strain of Trypanosoma cruzi. Although no statistical differences in mortality and parasitaemia were found, conventional mice showed IFN-gamma, TNF-alpha and NO production (P < 0.05) by spleen cell cultures and higher blood levels of immunoglobulins of the IgG2a isotype (P < 0.05) when compared to their germ-free counterparts. Moreover, higher levels of IgG1 were also found in conventional animals. On the other hand, no differences in IL10 production were found between germ-free and conventional mice after infection (P < 0.05). Interestingly, spleen cell cultures from non-infected germ-free mice spontaneously produced higher levels of IL10 than cultures from conventional mice. Moreover, cultures from non-infected germ-free mice responded to T. cruzi antigens with IFN-gamma production, contrary to cultures from conventional animals. In conclusion, the presence of the normal microbiota skews the immune response towards production of inflammatory cytokines during experimental infection with T. cruzi in mice. However, the increase in production of cytokines that is linked to resistance to this parasite did not alter the outcome of infection significantly, probably due to high virulence of the Y strain., (Copyright 2004 SGM)

Objective: To study atheromas, Mycoplasma pneumoniae (M. pneumoniae), and Chlamydia pneumoniae (C. pneumoniae)., Methods: C. pneumoniae was studied with immunohistochemistry and M. pneumoniae with in situ hybridization (ISH), in segments of coronary arteries (SCA) as follows: group A - thrombosed ruptured plaques (TRP) of 23 patients who died due to acute myocardial infarction (AMI); group B - 23 nonruptured plaques (NRP) of group A patients; group C - NRP of 11 coronary patients who did not die due to AMI; and group D - 11 SCA from patients with dilated cardiomyopathy or Chagas' disease without atherosclerosis., Results: The mean number of C. pneumoniae+ cells/400x in groups A, B, C, and D was, respectively, 3.3 +/- 3.6; 1.0 +/- 1.3; 1.2 +/- 2.4; and 0.4 +/- 0.3; and the percentage of M. pneumoniae area was, respectively, 3.9 +/- 3.5; 1.5 +/- 1.6; 0.9 +/- 0.9; and 0.4 +/- 0.2. More M. pneumoniae and C. pneumoniae were found in of group A than in group B (P<0.01). Good correlation was seen between the area of the vessel and the M. pneumoniae area in the plaque (r = 0.46; P=0.001) and between C. pneumoniae+ cells and CD4+ T lymphocytes (r = 0.42; P<0.01). The number of C. pneumoniae+ cells correlated with CD20+ B cells (r=0.48; P<0.01)., Conclusion: M. pneumoniae and C. pneumoniae are more frequently found in TRP correlate with the intensity of the inflammation and diameter of the vessel (positive remodeling).

Elimination of vector populations through the use of insecticides is the principal means of controlling Chagas disease. Because of the limitations of insecticide use, we have been developing a new potential method of control, to be used in conjunction with insecticide programs, a method which utilizes genetically modified symbiotic bacteria. These transformed bacteria can express anti-parasitic agents in the gut of the bug where the trypanosomes also are found. Previous studies have shown that it is possible to transform Rhodococcus rhodnii with a shuttle plasmid that contains the gene for cecropin A, an insect anti-microbial peptide. The bacteria expressed this peptide and reduced or eliminated the number of trypanosomes in the bug Rhodnius prolixus [Proc. Natl. Acad. Sci. U.S.A. 94 (1997) 3274]. In an effort to improve efficacy and transformation stability, we have begun using plasmids that contain integrative elements from the L1 mycobacteriophage to insert DNA into the genome of the bacterium. The integrative plasmid pBP5 contains the attachment site (attP) and integrase gene (int) of the L1 mycobacteriophage, an antibiotic resistance gene and the lacZ gene. After transforming R. rhodnii with pBP5, nine positive clones were obtained and six different insertions sites were identified. In each clone, the integrative plasmid is inserted only once, the lacZ gene is expressed intensely and, all clones but one, remained stable for 100 generations of culture in the absence of antibiotic selection. In addition, the construct remains stable throughout the life cycle of the bug. These data demonstrate that L1 mycobacteriophage integrative plasmids are significantly more stable than episomally located plasmids used in previous studies and will be greatly beneficial for use in the transformation of symbiotic bacteria of Chagas disease vectors.

Background: The risk of development of spin cell carcinoma of the esophagus is 33 times higher in patients with chagasic achalasia. It is possible that the production of N-nitroso compounds in the esophageal lumen by of bacterial action in the stasis liquid that reduce nitrates from diet into nitrites may play a role in this process., Aim: To analyze qualitatively and quantitatively the microbiota in chagasic megaesophagus with special attention to bacteria capable of transforming nitto reduction., Patients: Fifteen patients (six men and nine women) were prospectively studied, with ages varying from 28 to 73 years. Patients were divided into three sub-groups according to Rezende et al. classification of esophageal dilation (grade I, grade II and grade III)., Method: The sample collection was performed using a method specially developed to avoid contamination with microorganisms of the oral cavity and oropharynx, using a Levine catheter n 14 and a 7,5 oro-traqueal tube., Results: Ninety three point three percent of the cultures were positive, with great bacterial variability and predominance of a variety of aerobic Gram-positive and anaerobic bacteria. The bacterial concentrations were generally more elevated in grade III in comparison to grade I and grade II. Among the microorganisms found, Staphylococcus sp, Corynebacterium sp, Peptostreptococcus sp e a Veillonella sp were those with the capability of nitrate reduction., Conclusion: It was concluded that patients with megaesophagus present some bacteria in the esophageal lumen that are able to reduce nitrates intro nitrites, an important step in the formation of N-nitroso compounds.

Our previous studies show that in mice immunized with the paraflagellar rod (PFR) proteins of Trypanosoma cruzi protective immunity against this protozoan parasite requires MHC class I-restricted T cell function. To determine whether PFR-specific CD8+ T cell subsets are generated during T. cruzi infection, potential CTL targets in the PFR proteins were identified by scanning the amino acid sequences of the four PFR proteins for regions of 8-10 amino acids that conform to predicted MHC class I H-2b binding motifs. A subset of the peptide sequences identified were synthesized and tested as target antigen in 51Cr-release assays with effector cells from chronically infected T. cruzi mice. Short-term cytotoxic T lymphocyte (CTL) lines specific for two of the peptides, PFR-1(164-171) and PFR-3(123-130), showed high levels of lytic activity against peptide-pulsed target cells, secreted interferon (IFN)-gamma in response to parasite-infected target cells, and were found to be CD8+, CD4-, CD3+, TCRalphabeta+ cells of the Tc1 subset. Challenge of PFR immunized CD8-/- and perforin-deficient (PKO) mice confirmed that while CD8+ cells are required for survival of T. cruzi challenge infection, perforin activity is not required. Furthermore, while lytic activity of PFR-specific CD8+ T cell lines derived from PKO mice was severely impaired, the IFN-gamma levels secreted by CTLs from PKO mice were equivalent to that of normal mice, suggesting that the critical role played by CD8+ T cells in immunity to the parasite may be secretion of type 1 cytokines rather than lysis of parasite infected host cells.

A systematic, house-based serological survey for Trypanosoma cruzi seroreactivity was conducted in three contiguous communities in Olopa municipality, Chiquimula Department, Guatemala. Blood samples from a total of 292 individuals in 63 households were examined by enzyme-linked immunosorbent assay. The seropositive rate ranged from 0% to 20.8% for the three communities, with a mean of 15.1%. Log-linear models showed that seroprevalence was significantly related to age (P < 0.005) but not to sex. However, when the age group with the lowest prevalence (1-9 years) was excluded from the analysis, age was not a significant factor (P = 0.55). Data from a stratified sample collected at the same time were combined with those of the systematic sample to analyze the relationship between seropositivity and possible explanatory variables. Log-linear models, based on 586 individuals in 129 households from the two surveys, revealed a significant positive association between seropositivity and thatched roofs (P = 0.01).

Data from the late eighties indicate that 1.6% of the Mexican population was infected with Chagas' disease and that transmission by way of blood transfusion was taking place in nearly every state, in areas of different sizes. The risk of transmission via that route has seldom been documented in Mexico, and for this reason a sentinel survey was conducted in 1994 in 18 blood banks belonging to the Ministry of Health and located in various states. The purpose of the study was to determine the risk of transmission via blood transfusion and to calculate the national prevalence of infection among potential donors, so as to have a set of general indicators of the prevailing disease burden and of the importance of this transmission route. Participants were selected on the basis of operating criteria: all government-run transfusion centers with the capacity to screen blood donors for at least one year and persons seeking to donate blood (n = 64,969) who satisfied the Official Mexican Standards (Norma Official Mexicana) for the therapeutic use of human blood and blood products. For the analysis of the results the centers were grouped according to migration flow in order to detect any possible influence the latter may have had on Chagas' disease transmission within the country. Screening was done with indirect hemagglutination using a reagent produced by the Instituto Nacional de Diagnóstico y Referencia Epidemiológicos and donated to the blood banks. Positive cases were confirmed by indirect immunofluorescence. Positive results were detected in 996 persons, for a prevalence of 1.5% (95% CI: 1.44 to 1.63). Concordance between the final results obtained by local labs and by the central lab was given by a kappa index of 0.87 (95% CI: 0.862 to 0.877). Cities having the highest emigration rates had three times the risk of transmission as compared to cities that drew immigrants (odds ratio = 2.82; 95% CI: 2.18 to 3.65). We recommend that mandatory serologic screening be enforced throughout Mexico, since migration makes it difficult to determine which areas are endemic.

We report two cases of Chagas' disease with megacolon who presented with chronic diarrhea. One of the patients also had evidence of malabsorption, such as steatorrhea and hypocalcemia. Barium meal follow-through showed remarkable dilation of the jejunum in both cases and, in one of them, an associated megaduodenum. Manometric studies of gastrointestinal motility showed abnormally slow propagation of the interdigestive migrating motor complex, which was also excessively prolonged. Cultivation of jejunal aspirates revealed strict anaerobic bacterial growth in both cases. Oral antibiotic therapy led to substantial improvement in symptoms. The two cases herein reported indicate that clinical manifestations of small bowel bacterial overgrowth, possibly caused by motor disturbances associated with megajejunum, may occasionally include the clinical picture of gastrointestinal involvement in Chagas' disease.

The author use the H2 breath test to study the small bowel microflora of chagasic patients with megaesophagus and/or megacolon. Compare this group with a control one. Find a significant increase (P < 0.05) in the small bowel flora of chagasic group. It is concluded that H2 breath is a simple and useful test to detect alteration in intestinal flora.