Christian Castillo

The Interaction of Classical Complement Component C1 with Parasite and Host Calreticulin Mediates Trypanosoma cruzi Infection of Human Placenta

Background 9 million people are infected with Trypanosoma cruzi in Latin America, plus more than 300,000 in the United States, Canada, Europe, Australia, and Japan. Approximately 30% of infected individuals develop circulatory or digestive pathology. While in underdeveloped countries transmission is mainly through hematophagous arthropods, transplacental infection prevails in developed ones. Methodology/Principal Findings During infection, T. cruzi calreticulin (TcCRT) translocates from the endoplasmic reticulum to the area of flagellum emergence. There, TcCRT acts as virulence factor since it binds maternal classical complement component C1q that recognizes human calreticulin (HuCRT) in placenta, with increased parasite infectivity. As measured ex vivo by quantitative PCR in human placenta chorionic villi explants (HPCVE) (the closest available correlate of human congenital T. cruzi infection), C1q mediated up to a 3–5-fold increase in parasite load. Because anti-TcCRT and anti-HuCRT F(ab′)2 antibody fragments are devoid of their Fc-dependent capacity to recruit C1q, they reverted the C1q-mediated increase in parasite load by respectively preventing its interaction with cell-bound CRTs from both parasite and HPCVE origins. The use of competing fluid-phase recombinant HuCRT and F(ab′)2 antibody fragments anti-TcCRT corroborated this. These results are consistent with a high expression of fetal CRT on placental free chorionic villi. Increased C1q-mediated infection is paralleled by placental tissue damage, as evidenced by histopathology, a damage that is ameliorated by anti-TcCRT F(ab′)2 antibody fragments or fluid-phase HuCRT. Conclusions/Significance T. cruzi infection of HPCVE is importantly mediated by human and parasite CRTs and C1q. Most likely, C1q bridges CRT on the parasite surface with its receptor orthologue on human placental cells, thus facilitating the first encounter between the parasite and the fetal derived placental tissue. The results presented here have several potential translational medicine aspects, specifically related with the capacity of antibody fragments to inhibit the C1q/CRT interactions and thus T. cruzi infectivity.

Reorganization of Extracellular Matrix in Placentas from Women with Asymptomatic Chagas Disease: Mechanism of Parasite Invasion or Local Placental Defense?

Chagas disease, produced by the protozoan Trypanosoma cruzi (T. cruzi), is one of the most frequent endemic diseases in Latin America. In spite the fact that in the past few years T. cruzi congenital transmission has become of epidemiological importance, studies about this mechanism of infection are scarce. In order to explore some morphological aspects of this infection in the placenta, we analyzed placentas from T. cruzi-infected mothers by immunohistochemical and histochemical methods. Infection in mothers, newborns, and placentas was confirmed by PCR and by immunofluorescence in the placenta. T. cruzi-infected placentas present destruction of the syncytiotrophoblast and villous stroma, selective disorganization of the basal lamina, and disorganization of collagen I in villous stroma. Our results suggest that the parasite induces reorganization of this tissue component and in this way may regulate both inflammatory and immune responses in the host. Changes in the ECM of placental tissues, together with the immunological status of mother and fetus, and parasite load may determine the probability of congenital transmission of T. cruzi.

Trypanosoma cruzi induces trophoblast differentiation: A potential local antiparasitic mechanism of the human placenta?

INTRODUCTION The congenital transmission of Trypanosoma cruzi (T. cruzi) is responsible for one-third of new Chagas disease cases each year. During congenital transmission, the parasite breaks down the placental barrier formed by the trophoblast, basal laminae and villous stroma. The observation that only 5% of infected mothers transmit the parasite to the fetus implies that the placenta may impair parasite transmission. The trophoblast undergoes continuous epithelial turnover, which is considered part of innate immunity. Therefore, we propose that T. cruzi induces differentiation in the trophoblast as part of a local antiparasitic mechanism of the placenta. METHODS We analyzed β-human chorionic gonadotropin (β-hCG) and syncytin protein expression in HPCVE and BeWo cells using immunofluorescence and western blotting. Additionally, β-hCG secretion into the culture medium was measured by ELISA. We assessed the differentiation of trophoblastic cells in BeWo cells using the two-color fusion assay and by determining desmoplakin re-distribution. RESULTS T. cruzi trypomastigotes induce β-hCG secretion and protein expression as well as syncytin protein expression in HPCVE and BeWo cells. Additionally, the parasite induces the trophoblast fusion of BeWo cells. DISCUSSION T. cruzi induces differentiation of the trophoblast, which may contribute to increase the trophoblast turnover. The turnover could be a component of local antiparasitic mechanisms in the human placenta.

A local innate immune response against Trypanosoma cruzi in the human placenta: The epithelial turnover of the trophoblast

Congenital Chagas disease, caused by Trypanosoma cruzi, is partially responsible for the progressive globalization of Chagas disease despite of its low transmission rate. The probability of congenital transmission depends on complex interactions between the parasite, the maternal and fetus/newborn immune responses and placental factors, being the latter the least studied one. During transplacental transmission, the parasite must cross the placental barrier where the trophoblast, a continuous renewing epithelium, is the first tissue to have contact with the parasite. Importantly, the epithelial turnover is considered part of the innate immune system since pathogens, prior to cell invasion, must attach to the surface of cells. The trophoblast turnover involves cellular processes such as proliferation, differentiation and apoptotic cell death, all of them are induced by the parasite. In the present review, we analyze the current evidence about the trophoblast epithelial turnover as a local placental innate immune response.

Toxic and therapeutic effects of Nifurtimox and Benznidazol on Trypanosoma cruzi ex vivo infection of human placental chorionic villi explants

Nifurtimox (Nfx) and Benznidazole (Bnz) are the only available drugs in use for the treatment of Chagas disease. These drugs are recommended but not fully validated in evidence-based medicine and reports about the differential toxicity of both drugs are controversial. Here, we evaluated the toxic and therapeutic effects of Nfx and Bnz on human placental chorionic villi explants (HPCVE) during ex vivo infection of Trypanosoma cruzi, performing histopathological, histochemical, immunohistochemical as well as immunofluorescence analysis of the tissue. Additionally, we determined the effect of both drugs on parasite load by real time PCR. Bnz prevents the parasite induced tissue damage in ex vivo infected HPCVE compared to Nfx, which is toxic per se. The presence of T. cruzi antigens and DNA in infected explants suggests that these drugs do not impair parasite invasion into the HPCVE. Additionally, our results confirm reports suggesting that Bnz is less toxic than Nfx and support the need for the development of more effective and better-tolerated drugs.

Simvastatin Attenuates Endothelial Activation through 15-Epi-Lipoxin A4 Production in Murine Chronic Chagas Cardiomyopathy.

ABSTRACT Current treatments for chronic Chagas cardiomyopathy, a disease with high mortality rates and caused by the protozoan Trypanosoma cruzi, are unsatisfactory. Myocardial inflammation, including endothelial activation, is responsible for the structural and functional damage seen in the chronic phase. The clinical efficacy of benznidazole could be improved by decreasing chronic inflammation. Statins, which have anti-inflammatory properties, may improve the action of benznidazole. Here, the action of simvastatin in a murine model of chronic Chagas cardiomyopathy and the link with the production of the proresolving eicosanoid 15-epi-lipoxin A4, produced by 5-lipoxygenase, are evaluated. Simvastatin decreased the expression of the adhesion molecules E-selectin, intracellular adhesion molecule type 1 (ICAM-1), and vascular cell adhesion molecule type 1 (VCAM-1) in T. cruzi-infected mice. However, when this drug was administered to 5-lipoxygenase-deficient mice, the anti-inflammatory effect was not observed unless exogenous 15-epi-lipoxin A4 was administered. Thus, in chronic Chagas disease, 5-epi-lipoxin A4 induced by simvastatin treatment could improve the pathophysiological condition of patients by increasing the trypanocidal action of benznidazole.

Ex vivo infection of human placental chorionic villi explants with Trypanosoma cruzi and Toxoplasma gondii induces different Toll-like receptor expression and cytokine/chemokine profiles

Trypanosoma cruzi and Toxoplasma gondii present, respectively, low and high congenital transmission rates. The placenta as an immune regulatory organ expresses TLRs, leading to the secretion of cytokines. Both parasites are recognized by TLR‐2, TLR‐4, and TLR‐9. Here, we studied if the parasites induce differences in TLR protein expression, cytokine profiles, and whether receptor inhibition is related to parasite infection.

Trypanosoma cruzi infectivity assessment in "in vitro" culture systems by automated cell counting.

Chagas disease is an endemic, neglected tropical disease in Latin America that is caused by the protozoan parasite Trypanosoma cruzi. In vitro models constitute the first experimental approach to study the physiopathology of the disease and to assay potential new trypanocidal agents. Here, we report and describe clearly the use of commercial software (MATLAB(®)) to quantify T. cruzi amastigotes and infected mammalian cells (BeWo) and compared this analysis with the manual one. There was no statistically significant difference between the manual and the automatic quantification of the parasite; the two methods showed a correlation analysis r(2) value of 0.9159. The most significant advantage of the automatic quantification was the efficiency of the analysis. The drawback of this automated cell counting method was that some parasites were assigned to the wrong BeWo cell, however this data did not exceed 5% when adequate experimental conditions were chosen. We conclude that this quantification method constitutes an excellent tool for evaluating the parasite load in cells and therefore constitutes an easy and reliable ways to study parasite infectivity.

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.

IFPA Meeting 2013 Workshop Report III: maternal placental immunological interactions, novel determinants of trophoblast cell fate, dual ex vivo perfusion of the human placenta.

Workshops are an important part of the IFPA annual meeting as they allow for discussion of specialised topics. At IFPA meeting 2013 there were twelve themed workshops, three of which are summarized in this report. These workshops related to various aspects of placental biology but collectively covered areas of placental function, cell turnover and immunology: 1) immunology; 2) novel determinants of placental cell fate; 3) dual perfusion of human placental tissue.