Impact of chlorpyrifos on human villous trophoblasts and chorionic villi.

Placental barrier regulates maternal-fetal interchange protecting the baby from damage caused by substances found in the uterine environment or circulating in the vascular system. Organophosphate (OP) pesticides are a paramount group of environmental pollutants used in intensive agriculture for protection against diseases and pests. While many studies have reported an increased risk of pregnancy alterations in pregnant women exposed to OPs, few have analyzed the effects caused by these pesticides in the placenta. Herein, we evaluated the effects of chlorpyrifos (CPF), one of the most widely used OP insecticides, on human placenta using in vitro and ex vivo exposure models. Villous cytotrophoblast cells isolated from normal human term placentas maintained their cell viability, differentiated into syncytiotrophoblast-like structures, and increased the expression of ß-hCG, ABCG2, and P-gp in the presence of CPF at concentrations of 10 to 100µM. The same doses of CPF induced marked changes in chorionic villi samples. Indeed, CPF exposure increased stroma cell apoptosis, altered villi matrix composition, basement membrane thickness, and trophoblastic layer integrity. Histomorphological and ultrastructural alterations are compatible with those found in placentas where maternal-placenta injury is chronic and able to impair the placental barrier function and nutrient transport from mother to the fetus. Our study shows that placental ex vivo exposure to CPF produces tissue alterations and suggest that human placenta is a potential target of CPF toxicity. In addition, it highlights the importance of using different models to assess the effects of a toxic on human placenta.

Involvement of the beta-adrenergic system in the cardiac chronic form of experimental Trypanosoma cruzi infection.

Changes in the cardiac beta-adrenergic system in early stages of Trypanosoma cruzi infection have been described. Here, we studied an early (135 days post-infection-p.i.) and a late stage (365 days p.i.) of the cardiac chronic form of the experimental infection (Tulahuen or SGO-Z12 strains), determining plasma epinephrine and norepinephrine levels, beta-receptor density, affinity and function, cardiac cAMP concentration and phosphodiesterase activity, cardiac contractility, and the presence of beta-receptor autoantibodies. Tulahuen-infected mice presented lower epinephrine and norepinephrine levels; lower beta-receptor affinity and density; a diminished norepinephrine response and higher cAMP levels in the early stage, and a basal contractility similar to non-infected controls in the early and augmented in the late stage. The Tulahuen strain induced autoantibodies with weak beta-receptor interaction. SGO-Z12-infected mice presented lower norepinephrine levels and epinephrine levels that diminished with the evolution of the infection; lower beta-receptor affinity and an increased density; unchanged epinephrine and norepinephrine response in the early and a diminished response in the late stage; higher cAMP levels and unchanged basal contractility. The SGO-Z12 isolate induced beta-receptor autoantibodies with strong interaction with the beta-receptors. None of the antibodies, however, acted a as beta-receptor agonist. The present results demonstrate that this system is seriously compromised in the cardiac chronic stage of T. cruzi infection.

Trypanosoma cruzi: altered parasites after in vitro treatment with gangliosides, a therapeutic agent in experimental Chagas' disease.

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.

Expression and localization of StarD7 in trophoblast cells.

The StAR-related lipid transfer (START) domain is defined as a motif of around 200 amino acids implicated in lipid/sterol binding. In a previous study, we identified the StarD7 transcript encoding one of the 15 family members with START domain present in the human genome. This transcript was found to be overexpressed in choriocarcinoma JEG-3 cells. In addition, we demonstrated that the recombinant StarD7 protein forms stable Gibbs and Langmuir monolayers at the air-buffer interface, showing marked surface activity and interaction with phospholipid monolayers, mainly with phosphatidylserine, cholesterol and phosphatidylglycerol. This study was undertaken to evaluate the expression and localization of StarD7 protein in trophoblastic samples. Here, we show for the first time the presence of StarD7 protein in human trophoblast cells. Western blot assays revealed a unique specific 34 kDa protein in JEG-3 cell line, choriocarcinoma tissue, complete hydatidiform mole, early and normal term placenta. Immunohistochemical data from early and normal term placentas and complete hydatidiform moles showed that this protein is abundant in the syncytiotrophoblasts, mainly at the apical side of the syncytium, with a weak and focal reaction in the cytotrophoblast cells. Furthermore, an increased StarD7 mRNA and protein expression, as well as a change in its sub-cellular localization was observed in in vitro differentiating cytotrophoblast isolated from normal term placenta. Taken together, these findings support and allow future studies to explore the possibility that StarD7 protein mediates transplacental lipid transport and/or is involved in syncytialization.

Trypanosoma cruzi: Cardiac mitochondrial alterations produced by different strains in the acute phase of the infection.

The parasite Trypanosoma cruzi is the causative agent of Chagas disease. T. cruzi invasion and replication in cardiomyocytes induce cellular injuries and cytotoxic reactions, with the production of inflammatory cytokines and nitric oxide, both source of reactive oxygen species. The myocyte response to oxidative stress involves the progression of cellular changes primarily targeting mitochondria. We studied the cardiac mitochondrial structure and the enzymatic activity of citrate synthase and respiratory chain CI-CIV complexes, in Albino Swiss mice infected with T. cruzi, Tulahuen strain and SGO Z12 isolate, in two periods of the acute infection. Changes in the mitochondrial structure were detected in both infected groups, reaching values of 71% for Tulahuen and 88% for SGO Z12 infected mice, 30 days post infection. The citrate synthase activity was different according to the evolution of the infection and the parasite strain, but the respiratory chain alterations were similar with either strain.

Role of placental barrier integrity in infection by Trypanosoma cruzi.

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 (451.23µm2, 1.33%) and parasite load (RQ: 87) was significantly higher (p<0.05) than in the entire villous (control) (5.98µm2, 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.

Thioridazine treatment prevents cardiopathy in Trypanosoma cruzi infected mice.

Trypanosoma cruzi trypanothione reductase is an enzyme that has been identified as a potential target for chemotherapy. Thioridazine inhibits it and prevented cardiopathy in mice infected with T. cruzi Tulahuen strain. As not all T. cruzi strains respond to treatment in the same way, an isolate from a chronic patient (SGO Z12) was used; parasitaemias were studied along with, survival, serology, electrocardiography, histology and cardiac beta receptor function. Parasitaemia in thioridazine (80 mg/(kg day) for 3 days) treated mice was less and lasted for a shorter period (P < 0.01), there were reduced electrocardiographic and histological alterations and significantly improved survival (80% of non-treated died). Treated mice had lower receptor affinity and higher density as a compensatory mechanism, modifying the course of T. cruzi infection (SGO Z12 isolate) and preventing the consequent cardiopathy.

Effects of clomipramine on Trypanosoma cruzi infection in mice.

Trypanosoma cruzi, widely distributed in Latin American countries, provokes Chagas disease, characterized by cardiomyopathy and mega-viscera. The drugs used currently for treatment of acute Chagas disease are highly toxic; the side-effects are undesirable and patients may abandon treatment. We have previously demonstrated that clomipramine (CLO) exerts trypanocidal effects upon epimastigotes and trypomastigotes in vitro with anticalmodulin activity. The present study analyses the effectiveness of CLO treatment in mice infected with a low number of T. cruzi, an animal model that reproduces acute, indeterminate and chronic phases of this trypanosomiasis. In this work, our results demonstrated that CLO 5 mg/kg daily for 30 days, or 2 doses of CLO 40 mg/kg given intraperitoneally at 1 h and 7 days after infection, was not toxic for the host, but was effective against the parasite in that parasitaemias became negative and only mild heart structural and electrocardiographic alterations were detected in the chronic phase in the group treated with CLO 5 mg/kg. In mice treated with CLO 40 mg/kg, none of these alterations was detected. Cardiac beta receptor density and affinity returned to normal in the chronic stage in both experimental groups. T. cruzi enzymes such as calmodulin and trypanothione reductase represent potential drug targets. It has been reported that both can be inhibited by CLO, a tricyclic drug used in clinical therapeutics. We have shown that CLO strongly decreased the mortality rate and electrocardiographic alterations; in addition cardiac beta receptor density and heart histology returned to, or close to, normality 135 days post infection. These results clearly demonstrated that CLO treatment modified significantly the natural evolution of T. cruzi infection.

Effect of gangliosides on Trypanosoma cruzi infection in mice.

Albino Swiss male mice were inoculated with Trypanosoma cruzi, Tulahuen strain trypomastigotes, and separated into three groups: control, without treatment; control, treated with Nifurtimox 25 mg/day; and experimental, treated with total brain gangliosides 1 mg/day, intramuscular. The treatment was started immediately after infection and maintained for 4 weeks. Parasitemia was determined twice a week and histopathological analyses of hearts were performed. The parasitemia was significantly lowered by the ganglioside treatment. All untreated mice died by day 14 post infection. Survival at day 30 was 96% for mice in the experimental group. Hearts from untreated animals showed acute chagasic myocarditis, while those from mice treated with gangliosides presented only minor mononuclear infiltration. The effect of gangliosides is probably due to interference of parasite penetration into the host cells.

Trypanosoma cruzi: increased 5'-nucleotidase activity associated with dysfunction of adrenergic receptors in acutely infected albino Swiss mice.

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.

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

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.

Trypanocidal effects of promethazine.

Promethazine is currently used for its antipsychotic and ansiolytic effects. It is a phenothiazine with anticalmodulin action, not toxic for human beings at therapeutic doses. The present results show that promethazine has trypanocidal effect on both epimastigote and trypomastigote stages of T. cruzi; two hundred microM inhibited epimastigote growth in culture medium and 2 microM immobilized and killed bloodstream trypomastigotes. When promethazine (55 mg/Kg/day) was used as treatment of T. cruzi infected mice, it proved effective in reducing parasitemia and it increased the survival of treated animals. Ultrastructural studies suggest that the lethal effect of this phenothiazine is related to a detergent effect that disrupts T. cruzi cell membrane.

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

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.

[Increase of Hofbauer cells in human placentas cocultured in vitro with Trypanosoma cruzi]. / Aumento de células de Hofbauer en placentas humanas cocultivadas in vitro con Trypanosoma cruzi.

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.

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

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.

Alkaline phosphatase activity in plasma of pregnant chagasic patients.

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.

Vaccination with Trypanosoma rangeli induces resistance of guinea pigs to virulent Trypanosoma cruzi.

Chagas' disease, endemic in Latin America, is spread in natural environments through animal reservoirs, including marsupials, mice and guinea pigs. Farms breeding guinea pigs for food are located in some Latin-American countries with consequent risk of digestive infection. The aim of this work was to study the effect of vaccination with Trypanosoma rangeli in guinea pigs challenged with Trypanosoma cruzi. Animals were vaccinated with fixated epimastigotes of T. rangeli, emulsified with saponin. Controls received only PBS. Before being challenged with T. cruzi, parasitemia, survival rates and histological studies were performed. The vaccinated guinea pigs revealed significantly lower parasitemia than controls (p<0.0001-0.01) and a discrete lymphomonocytic infiltrate in cardiac and skeletal muscles was present. In the chronic phase, the histological view was normal. In contrast, control group revealed amastigote nests and typical histopathological alterations compatible with chagasic myocarditis, endocarditis and pericarditis. These results, together with previous works in our laboratory, show that T. rangeli induces immunoprotection in three species of animals: mice, guinea pigs and dogs. The development of vaccines for use in animals, like domestic dogs and guinea pigs in captivity, opens up new opportunities for preventive tools, and could reduce the risk of infection with T. cruzi in the community.

Differential susceptibility of isolated human trophoblasts to infection by Trypanosoma cruzi.

The aim of the work was to analyze the susceptibility of the placental syncytiotrophoblast (STB) and cytotrophoblast (CTB) cells to infection by the causal agent of congenital Chagas' disease, Trypanosoma cruzi, and the possible parasite route for placental invasion. Monolayers of CTB and STB and VERO as control cells were used. The infection of STB was significantly lower that of the CTB and Vero cells (p < 0.05) which coincided with a significantly increased mortality of parasite cells in the culture medium and trypanocidal levels of nitric oxide. We conclude that the syncytiotrophoblast, the first placental barrier, is the main barrier of the chorionic villous that limits the infection by T. cruzi. This work opens the possibility of a new mechanism for placental infection when there are discontinuities in the first placental barrier.

IFPA Meeting 2010 Workshops Report II: Placental pathology; trophoblast invasion; fetal sex; parasites and the placenta; decidua and embryonic or fetal loss; trophoblast differentiation and syncytialisation.

Workshops are an important part of the IFPA annual meeting. At IFPA Meeting 2010 diverse topics were discussed in twelve themed workshops, six of which are summarized in this report. 1. The placental pathology workshop focused on clinical correlates of placenta accreta/percreta. 2. Mechanisms of regulation of trophoblast invasion and spiral artery remodeling were discussed in the trophoblast invasion workshop. 3. The fetal sex and intrauterine stress workshop explored recent work on placental sex differences and discussed them in the context of whether boys live dangerously in the womb.4. The workshop on parasites addressed inflammatory responses as a sign of interaction between placental tissue and parasites. 5. The decidua and embryonic/fetal loss workshop focused on key regulatory mediators in the decidua, embryo and fetus and how alterations in expression may contribute to different diseases and adverse conditions of pregnancy. 6. The trophoblast differentiation and syncytialisation workshop addressed the regulation of villous cytotrophoblast differentiation and how variations may lead to placental dysfunction and pregnancy complications.

PCR múltiple para la detección simultánea de los genes mecA y pvl en Staphylococcus spp / Multiplex PCR for simultaneous detection of mecA and pvl genes in Staphylococcus spp

Este estudio observacional descriptivo de corte transverso tuvo por objetivo estandarizar una PCR múltiple para la detección simultánea de los genes mecA y pvl en Staphylococcus spp. Se emplearon como cepas control: S. aureus ATCC 25923, S. aureus ATCC 43300 y un aislado de S. aureus portador de los genes mecA y pvl. La extracción de ADN se realizó por el método de ebullición. El límite de detección se estableció por medio de diluciones seriadas de ADN. Se determinó la aplicabilidad de la PCR múltiple testando 41 aislados de S. aureus y 51 Estafilococos coagulasa negativo (ECN) previamente caracterizados por métodos fenotípicos en noviembre del año 2009. Los productos de PCR fueron visualizados por electroforesis en gel de agarosa al 2% previa tinción con bromuro de etidio. Los productos de amplificación de la PCR múltiple presentaron tamaño esperado de 533pb y 433pb para los genes mecA y pvl respectivamente, con límites de detección de hasta 0,5 ng/µL. El gen mecA se detectó en 13 (31,7%) aislados de S. aureus y en 29 (56,7%) ECN. El gen pvl se detectó en 2 (4,9%) S. aureus y no fue detectado en ECN. La presencia del gen mecA tuvo 100% de concordancia con los métodos fenotípicos. Esta técnica es una herramienta útil en la confirmación de cepas de Estafilococos meticilino resistentes e identificación del gen pvl, además de ser relativamente sencilla con la ventaja de detectar ambos genes en una sola reacción