Pro-inflammatory cytokines are modified during the multiplication of Trypanosoma cruzi within the placental chorionic villi and are associated with the level of infection via the signaling pathway NF-κB.

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 105 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-1ß, 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-1ß, 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.

3-Hydroxykynurenine, a Tryptophan Metabolite Generated during the Infection, Is Active Against Trypanosoma cruzi.

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 IC50 values in the micromolar concentration range, while the IC50 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.

Mechanism of Trypanosoma cruzi Placenta Invasion and Infection: The Use of Human Chorionic Villi Explants.

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.

In Vitro Infection of Trypanosoma cruzi Causes Decrease in Glucose Transporter Protein-1 (GLUT1) Expression in Explants of Human Placental Villi Cultured under Normal and High Glucose Concentrations.

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.

3-Hydroxy kynurenine treatment controls T. cruzi replication and the inflammatory pathology preventing the clinical symptoms of chronic Chagas disease.

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-ß-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-dioxigenase (IDO) is critical for host resistance against Trypanosoma cruzi.

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.

Placental infection by two subpopulations of Trypanosoma cruzi is conditioned by differential survival of the parasite in a deleterious placental medium and not by tissue reproduction.

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.

Treatment with benznidazole or thioridazine in the chronic phase of experimental Chagas disease improves cardiopathy.

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.

Alkaline phosphatase activity in plasma of pregnant chagasic patients

The Kinetic properties of plasma placental alkaline phosphatase patients with Chagas' disease were studied. When Cl2 Mg was used as activator the same increase of activity (17-20 per cent) 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-phenylamine. 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.

Placenta humana chagásica: alteración estructural y citoquímica de vasos sanguíneos / Human chagasic placenta: structural and cytochemical changes of blood vessels

Diversos autores han demostrado que los vasos sanguíneos coronarios pueden tener alguna participación en la patogenia de las alteraciones cardíacas en la enfermedad de Chagas. El objetivo del presente trabajo fue detectar alteraciones estructurales y citoquímicas en vasos sanguíneos de placentas humanas a término mediante microscopía óptica y electrónica, considerando la posibilidad de su participación en la patogenia del pasaje transplacentario del agente causal del Chagas. En dos de seis placentas chagásicas provenientes de embarazadas a término con serologías positivas para enfermedad de Chagas, se halló estrechamiento u oclusión de vasos fetales de las vellosidades coriales con aspecto hialinizado de sus paredes mediante microscopía óptica y actividad de fosfatasa ácida aumentada en el endotelio vascular mediante ultracitoquímica. El estrechamiento de la luz vascular mediante ultracitoquímica. El estrechamiento de la luz vascular podría deberse a la participación del músculo liso y del endotelio.

Aumento de celulas de Hofbauer en placentas humanas cocultivadas in vitro con Trypanosoma cruzi / Increment of Hofbauer cells in human placentas cocultured in vitro with Trypanosoma cruzi

Con el objetivo de analizar la interacción de placentas humanas normales con el Trypanosoma cruzi, se estudió mediante el microscopio óptico y electrónico el estroma de vellosidades coreales cocultivadas in vitro con 1,5 x 106 formas tripomastigotes de la cepa Tulahuen del Trypanosoma cruzi durantre 1h, 3hs y 12hs, en medio mínimo esencial de Eagle bajo condiciones adecuadas. Se observó en los cultivos experimentales (con T. cruzi) una aglutinación de las muestras placentarias que fue más evidente a partir de los 60 min y que resistió la separación mediante agitación suave. En el estroma de las vellosidades coriales se apreció separación de sus estructuras, edema vellositario y aumento de las células de Hofbauer de acuerdo a los análisis cuantitativos realizados. La aglutinación de las vellosidades puede deberse a modificaciones de los componentes glucoproteicos del trofoblasto por acción de productos secretados por el parásito, como ya ha sido señalado por otros autores en diversos tipos celulares. El aumento de las células de Hofbauer podría representar un mecanismo regulador de la placenta para equilibrar el nivel de agua del estroma vellositario

Quantitative ultrastructural and ultracytochemical analysis of lysosomes in the trophoblast of human placentas at term

El objetivo del presente trabajo fue analizar la presencia de lisosomas en el sinciciotrofoblasto de placentas humanas a término cultivadas y no cultivadas. Se realizó el estudio ultraestructural y ultracitoquímico para fosfatasa ácida mediante dos técnicas que emplean distintos sustratos y análisis morfométricos en 7 placentas humanas normales a término. Dos de ellas se mantuvieron previamente en cultivos de tejido durante 48 h. El estudio reveló la presencia de escasos cuerpos densos lisosomales con localizaciones supranucleares en relación con vacuolas de endocitosis y principalmente en regiones adelgazadas del sinciciotrofoblasto. La población lisosomal representó un 2,8% a 4,0% de la superficie sincicial. De acuerdo a los resultados, se sugiere que los lisosomas de placentas humanas e términos participarían en el intercambio materno fetal

Trypanosoma cruzi: modification of alkaline phosphatose activity induced by trypomastigotes in cultured human placental villi

Vilos placentários a termo foram mantidos "in vitro" em interaçäo com formas tripomastigotas sangüíneas de Trypanosoma cruzi, durante diversos períodos de tempo. Foram utilizadas concentraçöes diferentes de parasitas. Os controles näo continham T. cruzi. Determinou-se a atividade de fosfatase alcalina em vilos placentários mediante microscopia eletrônica e sua atividade específica no meio de cultura, mediante métodos bioquímicos. Os resultados mostraram que o hemoflagelado produz uma diminuiçäo significante da atividade enzimática tanto pelos estudos ultracitoquímicos como de atividade específica e esta atividade de fosfatase alcalina foi menor em culturas com altas doses de parasitas. Estes resultados säo indicadores de que a reduçäo de atividade enzimática coincide com o tempo de penetraçäo e proliferaçäo do T. cruzi nas células. Estas mudanças podem representar uma interaçäo entre o trofoblasto humano e o T. cruzi