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.

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 ß-human chorionic gonadotropin (ß-hCG) and syncytin protein expression in HPCVE and BeWo cells using immunofluorescence and western blotting. Additionally, ß-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 ß-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.

Natural sesquiterpene lactones induce programmed cell death in Trypanosoma cruzi: a new therapeutic target?

BACKGROUND: Chagas disease or American Trypanosomiasis is caused by the flagellated protozoan parasite Trypanosoma cruzi (T. cruzi) and is recognized by the WHO as one of the world's 17 neglected tropical diseases. Only two drugs (Benznidazol, Bz and Nifurtimox, Nx) are currently accepted for treatment, however they cause severe adverse effects and their efficacy is still controversial. It is then important to explore for new drugs. PURPOSE: Programmed cell death (PCD) in parasites offers interesting new therapeutic targets. The aim of this work was to evaluate the induction of PCD in T. cruzi by two natural sesquiterpene lactones (STLs), dehydroleucodine (DhL) and helenalin (Hln) as compared with the two conventional drugs, Bz and Nx. MATERIAL AND METHODS: Hln and DhL were isolated from aerial parts of Gaillardia megapotamica and Artemisia douglassiana Besser, respectively. Purity of compounds (greater than 95%) was confirmed by (13)C-nuclear magnetic resonance, melting point analysis, and optical rotation. Induction of PCD in T. cruzi epimastigotes and trypomastigotes by DhL, Hln, Bz and Nx was assayed by phosphatidylserine exposure at the parasite surface and by detection of DNA fragmentation using the TUNEL assay. Trypanocidal activity of natural and synthetic compounds was assayed by measuring parasite viability using the MTT method. RESULTS: The two natural STLs, DhL and Hln, induce programmed cell death in both, the replicative epimastigote form and the infective trypomastigote form of T. cruzi. Interestingly, the two conventional antichagasic drugs (Bz and Nx) do not induce programmed cell death. A combination of DhL and either Bz or Nx showed an increased effect of natural compounds and synthetic drugs on the decrease of parasite viability. CONCLUSION: DhL and Hln induce programmed cell death in T. cruzi replicative epimastigote and infective trypomastigote forms, which is a different mechanism of action than the conventional drugs to kill the parasite. Therefore DhL and Hln may offer an interesting option for the treatment of Chagas disease, alone or in combination with conventional drugs.

Expression, functionality, and localization of apurinic/apyrimidinic endonucleases in replicative and non-replicative forms of Trypanosoma cruzi.

Trypanosoma cruzi is the etiological agent of Chagas disease. The parasite has to overcome oxidative damage by ROS/RNS all along its life cycle to survive and to establish a chronic infection. We propose that T. cruzi is able to survive, among other mechanisms of detoxification, by repair of its damaged DNA through activation of the DNA base excision repair (BER) pathway. BER is highly conserved in eukaryotes with apurinic/apirimidinic endonucleases (APEs) playing a fundamental role. Previous results showed that T. cruzi exposed to hydrogen peroxide and peroxinitrite significantly decreases its viability when co-incubated with methoxyamine, an AP endonuclease inhibitor. In this work the localization, expression and functionality of two T. cruzi APEs (TcAP1, Homo sapiens APE1 orthologous and TcAP2, orthologous to Homo sapiens APE2 and to Schizosaccaromyces pombe Apn2p) were determined. These enzymes are present and active in the two replicative parasite forms (epimastigotes and amastigotes) as well as in the non-replicative, infective trypomastigotes. TcAP1 and TcAP2 are located in the nucleus of epimastigotes and their expression is constitutive. Epimastigote AP endonucleases as well as recombinant TcAP1 and TcAP2 are inhibited by methoxyamine. Overexpression of TcAP1 increases epimastigotes viability when they are exposed to acute ROS/RNS attack. This protective effect is more evident when parasites are submitted to persistent ROS/RNS exposition, mimicking nature conditions. Our results confirm that the BER pathway is involved in T. cruzi resistance to DNA oxidative damage and points to the participation of DNA AP endonucleases in parasite survival.

Role of matrix metalloproteinases 2 and 9 in ex vivo Trypanosoma cruzi infection of human placental chorionic villi.

BACKGROUND: Chagas' disease is caused by the haemophlagelated protozoan Trypanosoma cruzi (T. cruzi). During congenital transmission the parasite breaks down the placental barrier. In the present study we analyzed the participation of matrix metalloproteases (MMPs) in the extracellular matrix (ECM) remodeling during T. cruzi ex vivo infection of human placental chorionic villi explants. METHODS: Chorionic villi from healthy woman placentas were incubated in the presence or absence of 105 or 106 T. cruzi trypomastigotes (Y strain) with or without the MMPs inhibitor doxycycline. Effective infection was tested measuring parasite DNA by real time PCR (qPCR). MMP-2 and MMP-9 expression were determined by western blotting and immunohistochemistry and their activities were measured by zymography. The effect of MMPs on ECM structure was analyzed histochemically. RESULTS: T. cruzi induces the expression and activity of MMP-2 and MMP-9 in chorionic villi. Inhibition of the MMPs prevents the tissue damage induced by T. cruzi and partially decreases the ex vivo infection of the chorionic villi. CONCLUSION: MMPs are partially responsible for the ECM changes observed in human chorionic villi during T. cruzi infection and participate in tissue invasion. On the other hand, MMPs may be part of a local placental antiparasitic mechanism.

DNA repair BER pathway inhibition increases cell death caused by oxidative DNA damage in Trypanosoma cruzi.

Trypanosoma cruzi, a parasitic protozoan, is the etiological agent of Chagas disease, an endemic and neglected pathology in Latin America. It presents a life cycle that involves a hematophagous insect and man as well as domestic and wild mammals. The parasitic infection is not eliminated by the immune system of mammals; thus, the vertebrate host serves as a parasite reservoir. Additionally, chronic processes leading to dysfunction of the cardiac and digestive systems are observed. To establish a chronic infection some parasites should resist the oxidative damage to its DNA exerted by oxygen and nitrogen free radicals (ROS/RNS) generated in host cells. Till date there are no reports directly showing oxidative DNA damage and repair in T. cruzi. We establish that ROS/RNS generate nuclear and kinetoplastid DNA damage in T. cruzi that may be partially repaired by the parasite. Furthermore, we determined that both oxidative agents diminish T. cruzi cell viability. This effect is significantly augmented in parasites subsequently incubated with methoxyamine, a DNA base excision repair (BER) pathway inhibitor, strongly suggesting that the maintenance of T. cruzi viability is a consequence of DNA repair mechanisms.

Chronic restraint stress in oral squamous cell carcinoma.

Pathogenic processes have been identified that could associate chronic stress and cancer, but these findings have not been observed in oral cancer. This study examined the role of chronic restraint stress on the incidence and severity of OSCC induced with 4-nitroquinoline-1-oxide (4-NQO) in the tongues of CF-1 mice. One hundred twenty CF-1 male mice were divided into 4 groups: (A) received two treatments - restraint stress and induction of chemical carcinogenesis (n = 50); (B) induction of chemical carcinogenesis, without restraint stress (n = 50); (C) restraint stress (n = 10); and (D) control (n = 10). After 30 weeks, tongues were dissected and analyzed by conventional histopathology. The severity of OSSC was analyzed according to the International Histological Classification of Tumors and Bryne's Multifactorial Grading System for the Invasive Tumor Front (ITF). Chronic stress induction was confirmed by plasma corticosterone levels. Results showed that chronic stress was induced with movement restriction (p ≤ 0.05, Mann-Whitney U-test). However, chronic stress did not increase the incidence (p > 0.05, Chi-square) or severity (p > 0.05, Mann-Whitney U-test) of the 4-NQO-induced OSSC in the tongues of CF-1 mice. These results suggest that there is no relationship between chronic stress (induced in mice by restraint) and the incidence and severity of OSSC.

Trypanosoma cruzi induces apoptosis in ex vivo infected human chorionic villi.

Chagas' disease, produced by the haemoflagellated protozoan Trypanosoma cruzi (T. cruzi), is one of the most frequent endemic diseases in Latin America. In spite that in the past few years T. cruzi congenital transmission has become of epidemiological importance, studies about this mechanism of infection are scarce. The placental tissue undergoes apoptosis throughout gestation, as part of its normal turnover. On the other hand, it is known that T. cruzi induces, delays or inhibits apoptosis in other mammalian tissues. In order to determine the effect of parasite invasion on normal apoptosis in the placenta, explants of human chorionic villi were incubated with 105 trypomastigotes for 24 h. Effective infection was tested by visualizing T. cruzi antigens in histological preparations and by PCR. Upon infection, apoptotic cell death was determined by light and transmission electron microscopy, TUNEL analysis, measurement of caspase-3 like activity and immunohistochemical detection of caspase 3 cleaved cytokeratin 18. Our results clearly show that T. cruzi induces apoptosis in the chorionic villi and suggest that this is one of mechanisms used by the parasite to insure infection and invasion of human placenta and fetus.

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.

Trypanosoma cruzi induces tissue disorganization and destruction of chorionic villi in an ex vivo infection model of human placenta.

Congenital Chagas' disease, endemic in Latin America and also present with lower frequency in other countries, is associated with premature labor, miscarriage, and placentitis. The mechanism of tissue invasion and infection of human placenta by the parasite Trypanosoma cruzi (T. cruzi) remains unclear. In order to explore some morphological aspects of this infection in the placenta, we incubated chorionic villous explants from normal human placentae ex vivo with the parasite and studied the resulting effects by immunohistochemical and histochemical methods. Infection of the chorionic villi with the parasite was confirmed by immunofluoresence and PCR. T. cruzi induces syncytiotrophoblast destruction and detachment, selective disorganization of basal lamina and disorganization of collagen I in the connective tissue of villous stroma. These effects are a function of the number of parasites used for the infection. Our results suggest a participation of the proteolytic activity of the parasite on the placental basal lamina and connective tissue in the mechanism of infection of the fetus by T. cruzi.

Mesocestoides corti: morphological features and glycogen mobilization during in vitro differentiation from larva to adult worm.

Mesocestodes corti has the capacity to develop from the tetrathyridium (larva) stage to adult worm in vitro by trypsin and serum stimulation. Consequently, it has been used as an experimental model system for studying cestode development, host-parasite relationships and anthelmintic drugs. We describe morphological features in 5 different developmental stages of M. corti obtained in vitro, including larvae from the peritoneal cavity of infected mice, trypsin- and serum-stimulated larvae, elongated parasites as well as segmented and mature worms. It is unambiguously confirmed that sexually mature worms are obtained as a result of this in vitro process of differentiation. Defined cellular regions are present in all stages of development studied, some of them surrounded by a basal lamina. Glycogen is present in the larvae obtained from the mouse peritoneal cavity and in parasites encapsulated in the mouse host liver. Glycogen distribution in the parasite changes on trypsin and serum stimulation to differentiate. We propose that changes in the distribution of neutral polysaccharides in the parenchyma of the parasite at different stages of development and degradation of polysaccharides in the transition from segmented to adult worm are related to energy needs necessary for the cellular processes leading to the mature specimen.

Localization, specific activity, and molecular forms of acetylcholinesterase in developmental stages of the cestode Mesocestoides corti.

The nervous system of flatworms is quite simple although there is increasing evidence indicating that it is chemically complex. Studies of the nervous system in these animals have only been performed in the larval stage or in the adult worms, which are easy to obtain in nature, while the description of the nervous system in developing stages of these organisms is missing. Mesocestoides corti is a parasitic platyhelminth whose larvae can be induced in vitro to develop to adult, sexually mature worms, opening the possibility of studying the nervous system of a flatworm in different stages of development. Here, we describe the presence, activity, location, and molecular forms of acetylcholinesterase (AChE) in different stages of development of M. corti, from the larvae to adult forms of this endoparasite, obtained in in vitro cultures after induction of the larval stage with trypsin. Our results point to AChE as a molecular marker of the nervous system in platyhelminthes. The change in molecular forms of this enzyme and the increase in its activity during development from larvae to adult worm may reflect the presence of a more complex nervous system, necessary to adjust and coordinate the movement of a much bigger structure. A relationship between the development of the reproductive apparatus in segmented and adult worms with a more complex nervous system in these stages is also apparent. Finally, our study opens the possibility of applying anti-AChE as more effective therapeutic strategies against cestode parasites.