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Problem: Congenital Trypanosoma cruzi (T. cruzi) infection has been associated with changes in the levels of TNF-α and IFN-γ during the pregnancy. Therefore, we propose to study the participation and dynamics of proinflammatory cytokines in the infection process of placental explants infected by T. cruzi in vitro., Method of Study: Chorionic villous explants (CVE) obtained of human term placentas (n = 8) from normal pregnancies were cultured with 10 5 trypomastigotes/mL of Tulahuen strain DTU VI for 0, 2, 4, 16, 24, 48 and 72 h. Explants were treated with sulfasalazine (SULF) (5 mM) and N-acetyl-cysteine (NAC) (15 mM), as inhibitors molecules of NF-κB pathway, or LPS (1 μg/mL) for 24 and 72 h p.i. Motile trypomastigotes were counted in culture supernatants. Immunohistochemistry and ELISA for TNF-α, IFN-γ, IL-1β, IL-4, and IL-10 were performed in CVE and culture supernatants respectively. The parasite load was measured by RT-qPCR., Results: T. cruzi invades the chorionic villi from 4 h p.i. increasing significantly its DNA at 48 and 72 h p.i. of culture (parasite multiplication phase). They were detected in stromal cells, which was related to elevation of TNF-α, IL-1β, IFN-γ, and IL-10. The inhibition of NF-κB activity in the explants decreased the production of the analyzed cytokines, showing elevated levels of T. cruzi DNA during the multiplication phase of the parasite., Conclusions: Placental tissue modifies the secretion of pro-inflammatory cytokines during the phase of parasite multiplication, but not during the invasion phase, which in turns modifies the level of infection via the signaling pathway NF-κB., (© 2023 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: Trypanosoma cruzi crosses the placental barrier and produces the congenital transmission of Chagas disease (CD). Structural alterations of the chorionic villi by this parasite have been described in vitro, but little is known about trophoblast turnover in placentas from women with CD., Objective: To analyze the proliferation and fusion processes in placentas from women with CD., Methods: Archived human term placenta paraffin-embedded blocks were used, from women with CD (CDP), and no pathology (NP). Immunohistochemistry tests were performed for Ki67 to calculate the proliferation index (PI) of cytotrophoblast (CTB) and Syncytin-1, a fusion marker of syncytiotrophoblast (STB). Hematoxylin/Eosin stained sections were employed to analyze STB percentages, STB detachment areas and syncytial knots quantity. Non parametric Student's t-tests were performed (p < 0.05)., Results: Syncytial knots and STB detachment significantly increased in placental villi from the CDP group. STB percentage was significantly lower in the CDP group as well as the PI and Syncytin-1 expression significantly decreased in these placentas, compared with control (NP)., Conclusion: Dynamic of trophoblast turnover is altered in placentas from women with CD. These changes may lead into a gap in the placental barrier possibly allowing the parasite entry into the chorionic villi.

Problem: The innate immune response of the placenta may participate in the congenital transmission of Chagas disease through releasing reactive oxygen and nitrogen intermediates., Method of Study: Placental explants were cultured with 1 × 10 6 and 1 × 10 5 trypomastigotes of Tulahuen and Lucky strains and controls without parasites, and with the addition of nitric oxide synthase inhibitor Nω-Nitro-l-arginine methyl ester (l-NAME) and N-acetyl cysteine (NAC) as the reactive oxygen species (ROS) scavenger. Detachment of the syncytiotrophoblast (STB) was examined by histological analysis, and the nitric oxide synthase, endothelial (eNOS), and nitrotyrosine expressions were analyzed by immunohistochemistry, as well as the human chorionic gonadotrophin (hCG) levels in the culture supernatant through ELISA assays. Parasite load with qPCR using Taqman primers was quantified., Results: The higher number of T. cruzi (10 6 ) increased placental infection, eNOS expression, nitrosative stress, and STB detachment, with the placental barrier being injured by oxidative stress., Conclusion: The higher number of parasites caused deleterious consequences to the placental barrier, and the inhibitors (l-NAME and NAC) prevented the damage caused by trypomastigotes in placental villi but not that of the infection. Moreover, trophoblast eNOS played a key role in placental infection with the highest inoculum of Lucky, demonstrating the importance of the enzyme and nitrosative-oxidative stress in Chagas congenital transmission., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

The antiparasitic activity of 3-hydroxykynurenine (3-HK), one of the major tryptophan catabolites of the kynurenine pathway, against both Trypanosoma cruzi evolutive forms that are important for human infection, trypomastigotes (Tps) and amastigotes (Am), possible targets in the parasite and the drug toxicity to mammalian cells have been investigated. 3-HK showed a potent activity against Am with IC 50 values in the micromolar concentration range, while the IC 50 values to cause Tps death was ∼6000-times higher, indicating that the replicative form present in the vertebrate hosts is much more susceptible to 3-HK than bloodstream Tps. In addition, 3-HK showed activity against Tps and Am, at concentrations that did not exhibit toxicity to mammalian cells. Ultrastructural analysis and flow cytometry studies indicated that Am and Tps mitochondrion and nuclei contain 3-HK targets. The potency and selectivity of 3-HK, which is generated during T. cruzi infection in human and mice, suggest that 3-HK may be a suitable candidate for drug research and development for Chagas disease.

American trypanosomiasis has long been a neglected disease endemic in LatinAmerica, but congenital transmission has now spread Chagas disease to cause a global health problem. As the early stages of the infection of placental tissue and the vertical transmission by Trypanosoma cruzi are still not well understood, it is important to investigate the relevance of the first structure of the placental barrier in chorionic villi infection by T. cruzi during the initial stage of the infection. Explants of human chorionic villi from healthy pregnant women at term were denuded of their syncytiotrophoblast and co-cultured for 3h, 24h and 96h with 800,000 trypomastigotes (simulating acute infection). T. cruzi infected cells were identified by immunohistochemistry for cytokeratin-7 (+cytotrophoblast) and CD68 (+macrophages), and the infection was quantified. In placental tissue, the parasite load was analyzed by qPCR and microscopy, and the motile trypomastigotes were quantified in culture supernatant. In denuded chorionic villous, the total area occupied by the parasite (451.23μm 2 , 1.33%) and parasite load (RQ: 87) was significantly higher (p<0.05) than in the entire villous (control) (5.98μm 2 , 0.016%) (RQ:1) and with smaller concentration of nitric oxide. Stromal non-macrophage cells were infected as well as cytotrophoblasts and some macrophages, but with significant differences being observed. The parasite quantity in the culture supernatant was significantly higher (p<0.05) in denuded culture explants from 96h of culture. Although the human complete chorionic villi limited the infection, the detachment of the first structure of the placenta barrier (syncytiotrophoblast) increased both the infection of the villous stroma and the living trypomastigotes in the culture supernatant. Therefore structural and functional alterations to chorionic villi placental barrier reduce placental defenses and may contribute to the vertical transmission of Chagas., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Trypanosoma cruzi, the etiologic Chagas' disease agent, induces changes in protein pattern of the human placenta syncytiotrophoblast. The glucose transporter protein-1 (GLUT1) is the primary isoform involved in transplacental glucose transport. We carried out in vitro assays to determine if T. cruzi infection would induce changes in placental GLUT1 protein expression under normal and high concentration of glucose. Using Western blot and immunohistological techniques, GLUT1 expression was determined in normal placental villi cultured under normal or high concentrations of glucose, with or without in vitro T. cruzi infection, for 24 and 48 hours. High glucose media or T. cruzi infection alone reduced GLUT1 expression. A yet more accentuated reduction was observed when infection and high glucose condition took place together. We inform, for the first time, that T. cruzi infection may induce reduction of GLUT1 expression under normal and high glucose concentrations, and this effect is synergic to high glucose concentrations.

Congenital Chagas disease, a neglected tropical disease, endemic in Latin America, is associated with premature labor and miscarriage. During vertical transmission the parasite Trypanosoma cruzi (T. cruzi) crosses the placental barrier. However, the exact mechanism of the placental infection remains unclear. We review the congenital transmission of T. cruzi, particularly the role of possible local placental factors that contribute to the vertical transmission of the parasite. Additionally, we analyze the different methods available for studying the congenital transmission of the parasite. In that context, the ex vivo infection with T. cruzi trypomastigotes of human placental chorionic villi constitutes an excellent tool for studying parasite infection strategies as well as possible local antiparasitic mechanisms.

Background: 3-Hydroxy Kynurenine (3-HK) administration during the acute phase of Trypanosoma. cruzi infection decreases the parasitemia of lethally infected mice and improves their survival. However, due to the fact that the treatment with 3-HK is unable to eradicate the parasite, together with the known proapoptotic and immunoregulatory properties of 3-HK and their downstream catabolites, it is possible that the 3-HK treatment is effective during the acute phase of the infection by controlling the parasite replication, but at the same time suppressed the protective T cell response before pathogen clearance worsening the chronic phase of the infection. Therefore, in the present study, we investigated the effect of 3-HK treatment on the development of chronic Chagas' disease., Principal Findings: In the present study, we treated mice infected with T. cruzi with 3-HK at day five post infection during 5 consecutive days and investigated the effect of this treatment on the development of chronic Chagas disease. Cardiac functional (electrocardiogram) and histopathological studies were done at 60 dpi. 3-HK treatment markedly reduced the incidence and the severity of the electrocardiogram alterations and the inflammatory infiltrates and fibrosis in heart and skeletal muscle. 3-HK treatment modulated the immune response at the acute phase of the infection impairing the Th1- and Th2-type specific response and inducing TGF-β-secreting cells promoting the emergence of regulatory T cells and long-term specific IFN-γ secreting cells. 3-HK in vitro induced regulatory phenotype in T cells from T. cruzi acutely infected mice., Conclusions: Our results show that the early 3-HK treatment was effective in reducing the cardiac lesions as well as altering the pattern of the immune response in experimental Chagas' disease. Thus, we propose 3-HK as a novel therapeutic treatment able to control both the parasite replication and the inflammatory response.

Indoleamine 2,3-dioxygenase (IDO) is an inflammatory cytokine-inducible rate-limiting enzyme of the tryptophan (Trp) catabolism, which is involved in the inhibition of intracellular pathogen replication as well as in immunomodulation. Here we demonstrated the effect of IDO-dependent Trp catabolism on Trypanosoma cruzi resistance to acute infection. Infection with T. cruzi resulted in the systemic activation of IDO. The blocking of IDO activity in vivo impaired resistance to the infection and exacerbated the parasite load and infection-associated pathology. In addition, IDO activity was critical to controlling the parasite's replication in macrophages (Mos), despite the high production of nitric oxide produced by IDO-blocked T. cruzi-infected Mos. Analysis of the mechanisms by which IDO controls the parasite replication revealed that T. cruzi amastigotes were sensitive to L-kynurenine downstream metabolites 3-hydroxykynurenine (3-HK) and 3-hydroxyanthranilic acid, while 3-HK also affected the trypomastigote stage. Finally, 3-HK treatment of mice acutely infected with T. cruzi was able to control the parasite and to improve the survival of lethally infected mice. During infection, IDO played a critical role in host defense against T. cruzi; therefore, the intervention of IDO pathway could be useful as a novel antitrypanosomatid therapeutic strategy.

Chagas disease is caused by Trypanosoma cruzi, which can be transmitted to the fetus via the transplacental route. Factors that may be involved in transplacental transmission include parasite strain and placental immunological competence. The aim of this work was to compare the biological differences between two subpopulations of T. cruzi with respect to their interaction with the human placenta in vitro. We found that the Tulahuen strain (sublineage TcIIe) and another strain isolated from a congenitally infected newborn child had similar rates of productive infection in human chorionic villi in vitro, with similar deleterious nitric oxide levels between the two strains. We also found that the congenital T. cruzi stock had a greater ability than the Tulahuen strain to survive in the placental deleterious media, with the difference acquiring more importance considering the low reproductive rate of both subpopulations of T. cruzi within placental cells. As the presence of T. cruzi is a necessary condition to produce congenital transmission, we propose that the different survival rates of strains of T. cruzi in an adverse placental environment offer an opportunity for the parasite to infect the placenta in order to produce a sustainable infection during pregnancy, with the subsequent possibility of infecting the fetus.

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

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

Unlabelled: We hypothesize that a sustained infection of Trypanosoma cruzi into placental tissue might be diminished. Human placental chorionic villi and VERO cells as controls were co-cultured with T. cruzi. Parasites occupied 0.0137% at 3h, 0.0224% (24h), and 0.0572% (72 h) of the total chorionic villi area analyzed and some few placental samples were negative to parasite DNA, whereas 52% of VERO cells were infected at 3h and parasites occupied 0.57%, at 24h the parasite area was of 2.78% and at 72 h was of 3.32%. There were no live parasites in placenta-T. cruzi culture media at 72 h of co-culture. There were significantly increased dead parasites when T. cruzi was treated with unheated culture media coming from placental explants and fewer dead parasites when pre-heated culture media were employed., Conclusion: Low productive infection by T. cruzi into placental tissue associated with no viable parasites in the culture media partially due to placental thermo labile substances.

The R13 peptide sequence (EEEDDDMGFGLFD) that corresponds to the C-terminal region of Trypanosoma cruzi ribosomal P1 and P2 proteins differs from the eukariotic P concensus sequence EESDDDMGFGLFD (H13) only in a nonconservative amino acid substitution. The immunization of BALB/c mice with R13 synthetic peptide coupled to a carrier protein (OVA) induces specific (anti-R13) and autoreactive (anti-H13 and anti-heart) antibodies as well as heart functional alterations. Since aged human and experimental animals are impaired in their responses to most foreign antigens but they produce greater amounts of autoantibodies, in this work we used aged mice as an experimental model able to exaggerate the autoimmune component of the R13-induced response in case it was present. We studied whether these antibodies generated in the absence of the parasite would induce pathological changes in heart tissues. The levels of antibodies against R13 (foreign antigen) and H13 (autoantigen) studied comparatively in 2- and 12-month-old mice 10 days after the third immunization with R13 coupled to OVA were, as we expected for a foreign antigen, higher in almost all sera from 2-month-old mice tested than in sera from 12-month-old mice. Besides, these specific and cross-reactive antibody response remain elevated as long as 150 days post third immunization. In addition, the isotype pattern that recognizes R13 and the self-sequence H13 showed no differences between sera from young and aged mice. Moreover, when ECG traces were obtained from immunized mice, the heart functional alterations observed at 10 days continued at 80 and 150 days after the third immunization, showing an association with the levels of antibodies. In addition, despite the fact that the heart tissue morphology showed no alterations 10 days post third immunization, several abnormalities in the tissue architecture were revealed at 80 and 150 days post third immunization. This report demonstrates the biological relevance of R13-induced cross-reactive antibodies in some of the electrophysiologic and histological changes found in T. cruzi-infected mammalians., (Copyright 2000 Academic Press.)

Five subfractions were collected from six term placentas by mincing and differential centrifugation: homogenate, nuclear, mitochondrial, lysosomal, and supernatant. The effect of each subfraction on Trypanosoma cruzi was assessed by trypan blue exclusion, relative infectivity of mice, and penetration of susceptible cultured VERO cells. Ultrastructural changes in trypomastigotes were identified after high cell mortality was shown by dye exclusion following treatment with lysosomal and supernatant fractions. Trypomastigotes treated with other subfractions or preheated subfractions, those recovered from infected VERO cells, and controls remained unaffected. This was confirmed by the ability of treated trypomastigotes to infect mice or to penetrate susceptible cultured VERO cells. There were a 48% decrease in parasitemia and fewer myocardial lesions in Balb/c mice following treatment with the lysosomal subfraction compared to homogenate and controls. VERO cells were invaded about half as often after lysosomal treatment compared to controls (P < 0. 05); an 11% decrease in cell invasion following homogenate treatment was not significant. Placental lysosomal enzyme activity was unaffected by trypomastigotes. Human placentas contain one or more heat-labile substances in lysosomal and supernatant subfractions which inhibit or injure trypomastigotes of T. cruzi in cell-free systems., (Copyright 2000 Academic Press.)

Adenosine, derived from hydrolysis of 5'-AMP by 5'-nucleotidase activity, may be involved in coupling coronary blood flow to cardiac function and metabolism; it has been postulated as a cardioprotective substance in ischemic myocardium. The stimulation of beta-adrenergic receptors produces an increase in adenosine by 5'-AMP hydrolysis. In addition, it has been demonstrated that in Chagas' disease there is decreased cardiac perfusion. We show in this paper by histochemical and densitometric procedures that ecto-5'-nucleotidase activity increases in ventricles of acutely Trypanosoma cruzi-infected mice and that the density of beta-adrenergic receptors is significantly diminished with affinity similar to controls, showing that a compensatory mechanism was absent. The increase of ecto-5'-nucleotidase in heart myocytes from infected mice may produce cardioprotective adenosine that may be independent of beta-adrenergic function, based on the hypoperfusion conditions of acute chagasic cardiomyopathy.

Ganglioside treatment of mice during their acute infection with Trypanosoma cruzi promoted long-term survival and clearance of parasites from the bloodstream and organs. Additionally, such treatment completely prevented the clinical manifestations of the infection, and progression into the chronic stages of the disease, for at least 18 months post-infection. Trypanosoma cruzi must invade nucleated cells to survive and reproduce within the mammalian host, and it has been suggested that ganglioside treatment inhibits the parasite's phospholipase A2 enzymes (PLA2), which are involved in membrane destabilization. However, since total brain gangliosides were not toxic to the parasite, either in xenic or axenic cultures, it seems unlikely that their action in vivo relates to their inhibition of PLA2. Other possible mechanisms of action are discussed.

The kinetic properties of plasma placental alkaline phosphatase patients with Chagas' disease were studied. When Cl2Mg was used as activator the same increase of activity (17-20%) was found in the chagasic and non chagasic groups. The enzyme was not inhibited by F-ion in any of the groups. No significant differences were detected between the two groups (chagasic and non chagasic) when the enzyme was treated with inhibitors such as EDTA and L-phenylalanine. However, when the CN-ion was used, the enzyme of the normal pregnant women followed a Michaelian curve, whereas in the chagasic group a sigmoideal plot was observed. Thus, the Hill coefficient was 1.1 for the normal group and over 1.5 for the chagasic.

A structural, cytochemical, stereological, and biochemical study of lysosomes and a lysosome marker, the enzyme acid phosphatase, was performed, both in placentas at term from chagasic pregnant women without fetal infection and in normal placentas at term cocultured in vitro with Trypanosoma cruzi. It was found that in placentas from chagasic women lysosomes were normally distributed in the trophoblast. Stereological analysis showed that lysosomes and cytochemical acid phosphatase (AcP) activity were increased in the trophoblast of chagasic placentas. AcP activity increased in subcellular fractions of the isolated trophoblast from chagasic placentas, and the lysosomal fraction of those placentas exhibited the highest value of enzymatic activity in comparison to controls (P < 0.05). No differences in AcP activity were observed between homogenates of normal placentas cocultured with T. cruzi and controls. These data suggest that the placental lysosome population might be involved in the process of placental infection by T. cruzi.

Various authors have demonstrated that coronary blood vessels could have some participation in the pathogenesis of the cardiac alterations of Chagas' disease. The purpose of this work was to detect structural and cytochemical modifications of blood vessels in human chagasic placentas at term with optical and electron microscopy due their possible participation in the pathogenesis of the congenital transmission of the disease. In two of the six chagasic placentas at term from pregnant women with positive serology, there was diminution and occlusion of the lumen of the chorionic villi blood vessels, with hialine aspect of their walls. An increase of acid phosphatase activity in the endothelium was also observed with electron microscopy. The diminished blood vessel lumen could be due to smooth muscle and endothelium participation.

To analyze the interaction between normal human placentas with Trypanosoma cruzi, optical and electron microscopy of chorionic villi stroma cocultured in vitro with 1.5 x 106 Tulahuen strain Trypomastigotes of the T. cruzi for 1 h, 3 hs and 12 hs in Eagle minimal essential medium were done. An agglutination of chorionic villi in experimental cultures (with T. cruzi) from 1 h cultures was observed that was not present in control ones. this phenomenon resisted soft mechanical agitation to separate the isolated villi. Microscopical observations of stromal villi showed edema, separated structures and increment of Hofbauer cells as found by qualitative analysis. The chorionic villi agglutination could be caused by glycoproteins' modifications of the trophoblast, which in turn could be caused by secreted products of T. cruzi, as other authors have postulated in various cells' types. The increment of Hofbauer cells could represent a regulator mechanism of the placenta to equilibrate the intracellular water of the villi stroma.

The purpose of the present work was to analyze the presence of lysosomes in the syncytiotrophoblast of seven cultured and uncultured normal human placentas at term. An ultrastructural and ultracytochemical study for acid phosphatase was made by means of two techniques that employ different substrates, as well as morphometric studies. Two of the placentas were maintained "in vitro" for 48 h previous to their analysis. Scarce lysosomal dense bodies were located in the supranuclear region in relation to endocytotic vacuoles, specially in thinned off areas of the syncytiotrophoblast. The lysosomal population represented a 2.8% to 4.0% of the syncytial area. According to these results, it is suggested that human term placental lysosomes would participate in materno--fetal exchange.

Human term placental villi cultured "in vitro" were maintained with bloodstream forms of Trypanosoma cruzi during various periods of time. Two different concentrations of the parasite were employed. Controls contained no T. cruzi. The alkaline phosphatase activity was determined in placental villi by electron microscopy and its specific activity in the culture medium by biochemical methods. Results showed that the hemoflagellate produces a significant decrease in enzyme activity as shown by both ultracytochemical and specific activity studies and this activity was lower in cultures with high doses of parasites. The above results indicate that the reduction in enzyme activity coincides with the time of penetration and proliferation of T. cruzi in mammalian cells. These changes may represent an interaction between human trophoblast and T. cruzi.