Phenotypic and genotypic characterization of two Toxoplasma gondii isolates in free-range chickens from Uberlândia, Brazil.

The aim of this study was to determine the seroprevalence of Toxoplasma gondii infection in free-range chickens from Uberlândia, Minas Gerais state, Brazil, and characterize the genotypic and phenotypic features of two isolates of this parasite, considering the importance of these hosts in the epidemiology of toxoplasmosis. Serum samples from 108 free-range chickens were obtained from ten different districts, and submitted to the modified agglutination test (MAT) for the presence of anti-T. gondii antibodies, and brain and heart tissue samples from infected chickens were processed for mouse bioassay. An overall seroprevalence of 71·3% was found and antibody titres ranged from 16 to 4096. After confirmation of seropositivity by mouse bioassay, the determination of the T. gondii genotypes of two isolates was performed by PCR-RFLP, using primers for the following markers: SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, new SAG2, Apico and CS3. These T. gondii isolates, designated TgChBrUD1and TgChBrUD2, were obtained from heart samples of free-range chickens. The TgChBrUD1 isolate belonged to ToxoDB PCR-RFLP genotype 11 and the TgChBrUD2 isolate belonged to ToxoDB PCR-RFLP genotype 6. Both isolates demonstrated high virulence in a rodent model, with the TgChBrUD1 isolate able to induce brain cysts, in accord with its pattern of multiplication rates in human fibroblast culture. Taken together, these results reveal high prevalence of T. gondii infection in free-range chickens throughout Uberlândia, indicating an important degree of oocyst environmental contamination and the existence of considerable risk for T. gondii transmission to humans by consumption of free-range chicken as a food source.

A quantitative PCR assay for antiviral activity screening of medicinal plants against Herpes simplex 1.

Herpes simplex virus one is one of the most prevalent pathogens worldwide. Strains resistant to current treatment have been reported, so it is necessary to search for new antiviral molecules. The most common method to quantify antiviral activity from natural products is the plaque reduction assay, a technically demanding method. In order to provide a simple alternative to this method, we have established a procedure for viral quantification by qPCR, and coupled with a cytotoxicity evaluation system using resazurin. In this way, it is possible to obtain both the estimation of cytotoxicity and the antiviral activity simultaneously, allowing rapid screening of plant extracts. Ten out of twenty-eight Paraguayan medicinal plant extracts evaluated using this method showed antiviral activity, and the EC50, CC50, and SI values were calculated for each extract. Our experience supports the employment of the described method for a rapid identification of plant extracts with antiviral activity.

BeWo trophoblast cell susceptibility to Toxoplasma gondii is increased by interferon-gamma, interleukin-10 and transforming growth factor-beta1.

The present study aimed to investigate BeWo trophoblast cell susceptibility to Toxoplasma gondii infection under stimulation with anti-inflammatory cytokines in comparison with HeLa cells. Both cell types were submitted to different treatments with recombinant cytokines [interleukin (IL)-10 and transforming growth factor (TGF)-beta1] or the respective antibodies (anti-IL-10 and anti-TGF-beta) before and after T. gondii infection. The effect of interferon (IFN)-gamma was also assessed alone or in combination with anti-inflammatory cytokines or the respective antibodies after the parasite infection. Cells were fixed, stained and parasites quantified under light microscopy to evaluate intracellular replication (mean number of parasites per cell in 100 infected cells) and infection index (percentage of infected cells per 100 examined cells). In contrast with HeLa cells, treatments with IL-10 or TGF-beta1 induced a considerable augmentation in both T. gondii intracellular replication and invasion into BeWo cells. In addition, treatment with IFN-gamma alone or associated with IL-10 or TGF-beta1 increased the same parameters in BeWo cells, whereas the opposite effect was observed in HeLa cells. When endogenous IL-10 or TGF-beta was blocked, both BeWo and HeLa cells were able to control the parasite infection only in the presence of IFN-gamma. Together, these results indicate that the higher susceptibility of BeWo cells to T. gondii may be due to immunomodulation mechanisms, suggesting that the role of trophoblast cells in maintaining a placental microenvironment favourable to pregnancy may facilitate the infection into the placental tissues.

Susceptibility to vertical transmission of Toxoplasma gondii is temporally dependent on the preconceptional infection in Calomys callosus.

Toxoplasma gondii is an obligate intracellular parasite that causes a variety of clinical syndromes, but the infection is more severe in immunocompromised individuals and in cases of congenital toxoplasmosis. This study aimed to verify if the susceptibility to vertical transmission of Toxoplasma gondii is temporally dependent on the preconceptional infection in Calomys callosus. Twelve C. callosus females were infected with 20 cysts of T. gondii ME49 strain and divided into three groups of four animals that were mated after approximately 10 days (group 1), 30 days (group 2), and 50 days (group 3) of infection. The animals were sacrificed from the 17th to 20th day of pregnancy, when placentas and embryos were collected for morphological and immunohistochemical studies, mouse bioassay for evaluating seroconversion and PCR for detecting parasite DNA. Serum samples from C. callosus females and mice used in bioassay were analysed for the detection of IgG antibodies to T. gondii by ELISA. Detection of T. gondii was observed by mouse bioassay and PCR in placentas and embryos from C. callosus females infected around 10 days pre-conception. However, only placentas, but not embryos, from females infected around 30 and 50 days pre-conception showed positivity for parasite DNA and seroconversion by mouse bioassay. In conclusion, this study model shows that vertical transmission of T. gondii may take place when maternal infection occurs within one month before conception, thus demonstrating the time of preconceptional seroconversion that rule out a risk of congenital toxoplasmosis.

BeWo trophoblasts are unable to control replication of Toxoplasma gondii, even in the presence of exogenous IFN-gamma.

The ability of RH strain of Toxoplasma gondii to invade and grow into BeWo cells was investigated in the present study using IFN-gamma, l-tryptophan, or alpha-methyl-tryptophan treatments. HeLa cells were used in the same conditions for comparison purposes. It was demonstrated that BeWo cells are more permissive to T. gondii infection, making them more susceptible to this pathogen when compared to HeLa cells. Infection rates of BeWo cells do not show any significant alteration in different protocols using IFN-gamma. In addition, BeWo treated with l-tryptophan was unable to significantly increase parasite growth. In contrast, HeLa cells treated with IFN-gamma or IFN-gamma plus l-tryptophan are able to impair or increase, respectively, parasite replication, providing evidence that this indoleamine-2,3-dioxygenase-dependent phenomenon is operant in these cells, whereas it is inactive in BeWo. Therefore, our data support the hypothesis that the immunological mechanisms controlling infection at the maternal-fetal interface are different from those occurring in the periphery. At the same time that operating regulatory mechanisms work inside and outside the cells located at that microenvironment to prevent maternal rejection of the concept, these events might facilitate the progression of infection caused by intracellular pathogens, as T. gondii.

A new steroidal saponin from Solanum sisymbriifolium roots.

Isonuatigenin-3-O-beta-solatriose (1) was isolated from the roots of Solanum sisymbriifolium. Its structure was determined by spectroscopic methods.

Trophoblast-macrophage crosstalk on human extravillous under Toxoplasma gondii infection.

INTRODUCTION: The interaction between human extravillous trophoblasts and macrophages has an important role in implantation and placentation. However, any dysfunction in this communication system is associated with pregnancy pitfalls, and a Toxoplasma gondii infection can be a potential problem in this crosstalk. Therefore, the aim of this study was to assess the influence of infected macrophages on cytokine production and the incidence of apoptosis in T. gondii-infected extravillous trophoblast cells. METHODS: HTR-8/SVneo cells were treated with supernatant from macrophages infected or not by T. gondii (conditioned medium) in order to analyze apoptosis and cytokine production in comparison to uninfected control conditions. RESULTS: The IL-6 secretion by HTR-8/SVneo cells increased synergistically by treatment with conditioned medium and T. gondii infection. The apoptosis index of HTR-8/SVneo cells was also upregulated by treatment with conditioned medium and infection. In addition, a low expression of Fas/CD95 and a high soluble FasL release were observed during infection, although no significant change was observed in the proliferation of T. gondii. DISCUSSION: The parasite modulates the high apoptosis index in HTR-8/SVneo cells in order to favor its establishment inside its host cells. On the other hand, the conditioned medium from uninfected macrophages restores the apoptosis rates, although the effect of the infection seems to be stronger. In conclusion, our results showed that T. gondii infection in human extravillous trophoblasts is able to modulate the trophoblast-macrophage crosstalk.

Trophoblastic invasion of the uterine epithelium in Calomys callosus (Rodentia, Cricetidae).

Implantation sites in the uterus of the cricetid rodent Calomys callosus were examined by light and electron microscopy at several points during day 4 of pregnancy to determine the changes occurring in the trophoblast and epithelial cells during the early blastocyst-endometrial relationship, particularly the epithelial removal phase. By morning, the blastocysts had already lodged in the antimesometrial region of the uterine crypts and the embryo-maternal interaction was limited to sparse contacts between the epithelial microvilli and the trophoblast surface. A few dense and altered epithelial cells were present. Three zones could be distinguished in the stromal region. In the subepithelial area, the stromal cells showed features similar to those found in other rodents during the early decidualization process. Effective epithelial invasion by the trophoblast occurred only in the evening, although at noon, apposition and attachment phases were identifiable. During invasion, the trophoblast processes were first seen insinuating between the epithelial cells, projecting towards the basal lamina, and finally replacing these cells. The epithelial cells in contact with the invading trophoblast exhibited severe disorganization similar to that found during the process of cell death by apoptosis. The removal of such cells occurred by trophoblast phagocytosis. Once in contact with the basal lamina, the trophoblast rapidly spread out both mesometrially and antimesometrially, phagocytosing the remaining epithelial cells. Trophoblast processes were also seen penetrating the basal lamina. Given that the epithelium breached and phagocytosed by the trophoblast habitually displays degenerative characteristics, we consider the mechanisms that might be involved in epithelial cell death and in the removal of the uterine epithelium, and the participation of the trophoblast in both these events.

Chemical composition of the essential oil from the leaves of Piper fulvescens, a plant traditionally used in Paraguay.

The essential oil from the leaves of the aromatic aniseed smelling plant Piper fulvescens, obtained by hydrodistillation, was investigated by GC and GC-MS, as well as by 13C-NMR after fractionation of the oil by column chromatography. A total of 37 constituents, representing 78.2% of the oil were identified. The main compounds were trans-anethole (26.4%) and ishwarane (12.1%), an unusual sesquiterpene hydrocarbon.

Hypotensive effect of crude root extract of Solanum sisymbriifolium (Solanaceae) in normo- and hypertensive rats.

The hypotensive effect of the crude hydroalcoholic extract from root of Solanum sisymbriifolium Lam. (Solanaceae) was investigated both in normotensive and hypertensive rats. The intravenous administration of the extract (50 and 100 mg/kg) produced a significant decrease in blood pressure in anaesthetized hypertensive (adrenal regeneration hypertension + deoxycorticosterone acetate (ARH + DOCA)) rats. Oral administration of the extract (10, 50, 100 and 250 mg/kg) also produced a dose-dependent hypotensive effect in conscious hypertensive animals. In anaesthetized normotensive rats, the extract (50 and 100 mg/kg, i.v.) also induced hypotension in a dose-dependent manner. Lastly, no significant effect on blood pressure was produced by the extract when administered orally (10, 50, 100, 250, 500 and 1000 mg/kg) to conscious normotensive rats.

Acute toxicity and general pharmacological effect on central nervous system of the crude rhizome extract of Kyllinga brevifolia Rottb.

Acute toxicity and general pharmacological activities of the crude hydro-alcoholic rhizome extract of Kyllinga brevifolia Rottb., a popular medicine used in Paraguay, were investigated on mice. The intraperitoneal LD50 was found to be 575 mg/kg. Oral administration of doses up to 3.0 g/kg did not provoke any toxic symptoms. Oral administration of 100 mg/kg of the extract induced a significant increase in gastrointestinal transit. In open field studies, a decrease of spontaneous locomotor activity, piloerection, passivity, palpebral ptosis, catatonia and a stereotyped behaviour was produced by the extract when administered orally (1, 10 and 100 mg/kg). A significant decrease in respiration rate was observed (1, 10 and 100 mg/kg, p.o.) using a continuous flow respiration system. Lastly, doses of 1, 10 and 100 mg/kg, p.o. of the extract produced a significant increase in the hypnotic effect induced by pentobarbital in a dose-dependent manner. The latest effects could probably explain its rational use in traditional medicine to alleviate stress or as a sedative agent.

Isolation of hypotensive compounds from Solanum sisymbriifolium Lam.

The crude hydroalcoholic root extract (CRE) of Solanum sisymbriifolium Lam. has formerly been shown to have hypotensive activity both in normo-and hypertensive rats. Hypotensive activity-guided fractionation of the CRE was performed in anaesthetized normotensive rats, which led to the isolation of the active principles. The intravenous (i.v.) and intraperitoneal (i.p.) values of the CRE in mice were found to be, respectively, 343 and 451 mg/kg, and no lethal effect was caused by doses up to 5.0 g/kg when administered by oral route. Depression of locomotion, increase of breathing rate and piloerection was observed in a general behavior test with doses up to 200 mg/kg i.p., and 1000 mg/kg p.o., respectively. Increase in the gastrointestinal transit was found using 0.1 g/kg, whereas at doses of 0.5 and 1 g/kg, no significant activity was observed in comparison with the control mice. Hexanic and butanolic fractions induced a remarkable hypotension in anaesthetized normotensive rats in doses of 1, 5, 7.5 and 10 mg/kg i. v. Two compounds isolated from the butanolic fraction induced a significant decrease of the blood pressure, HR, amplitude of the ECG and breathing rate when injected in a dose of 1 mg/kg i.v; and both systofic and diastolic, blood pressures were affected in a proportional mode. The hypotensive effect of the two compounds were not influenced by pretreatment with atropine and propranolol; and the pressor response to noradrenaline was not affected by any of them which suggests that neither a direct muscarinic activity, beta-adrenoceptor activation nor decrease of sympathetic vascular tone (sympatholitic activity) are probably involved in the mechanism of hypotension. The present study shows that the CRE of S. sisymbriifolium contains at least two hypotensive compounds whose characterization is under way.

A new lupane caffeoyl ester from Hippocratea volubilis.

The extract of the rootbark of Hippocratea volubilis afforded a new triterpene caffeoyl ester, lupeol caffeate (1). Its structure was determined by means of spectroscopic evidence.

Review: putative roles for the macrophage migratory inhibitory factor at the maternal fetal interface.

Complex and dynamic networks of molecules participate in the essential interactions between maternal organism, placenta and fetus in a healthy and successful pregnancy. Macrophage migratory inhibitory factor (MIF) is one of several molecules produced at implantation sites; MIF is mostly expressed by trophoblast cells. This has led to expectations of MIF's relevance as a partner in the maternal/fetal dialog. MIF is known by its biological interactions and functional roles as an activator of innate immunity, regulating subsequent adaptive responses, which include inhibition of migration of mononuclear cells in vitro, antagonism of glucocorticoids, and regulation of expression of Toll-like receptor 4. Beyond roles in the inflammatory response, MIF can interfere with proliferative activities in different cell types, as well as with cell death pathways. This intriguing factor found at the human, porcine, ovine, bovine and rodent maternal-fetal interfaces is present in a time- and spatially-dependent manner, indicating regulatory roles in the process of embryo implantation, placental development, maintenance of pregnancy and birth. Here, we will review MIF participation in placental physiology, including new evidence for a dialog with uterine cells, and a potential role in protection of uterine decidual cells.

Susceptibility to Toxoplasma gondii proliferation in BeWo human trophoblast cells is dose-dependent of macrophage migration inhibitory factor (MIF), via ERK1/2 phosphorylation and prostaglandin E2 production.

INTRODUCTION: Macrophage migration inhibitory factor (MIF) participates in the immune response to Toxoplasma gondii, triggers ERK1/2 and prostaglandin E2 (PGE2) activation, but there is limited information on these mechanisms in human trophoblast. The present study aimed to verify the role of MIF in the ERK1/2 phosphorylation and PGE2 production, as well as its effect on the susceptibility to T. gondii in BeWo cells. METHODS: BeWo cells were treated with increasing concentrations of recombinant MIF (rMIF) and/or T. gondii-soluble tachyzoite antigen (STAg) and analyzed for ERK1/2 phosphorylation and PGE2 production by Western blotting and ELISA, respectively. Cells were also treated with increasing concentrations of rMIF, rPGE2, or ERK1/2 inhibitor and tested for T. gondii proliferation. The supernatants of cells treated with rPGE2 were assayed for cytokine production by ELISA or CBA. RESULTS: ERK1/2 phosphorylation and PGE2 production increased when the cells were treated with low MIF concentrations while the parasitism control occurred only at high MIF concentrations. STAg was unable to change ERK1/2 phosphorylation or PGE2 release. BeWo cells demonstrated increased T. gondii proliferation and reduced production of pro-inflammatory cytokines when treated with PGE2, while PD98059 diminished the parasite proliferation. DISCUSSION: The intracellular mechanisms triggered by MIF are dose-dependent in BeWo cells, and PGE2 is an important factor for the persistence of T. gondii at the maternal fetal interface. CONCLUSION: MIF was unable to control T. gondii infection in BeWo cells at low concentrations since ERK1/2 and PGE2 expression were activated, demonstrating a critical effect of these mediators favoring parasite proliferation.

Toxicity assessment on trophoblast cells for some environment polluting chemicals and 17ß-estradiol.

The identification of reproductive toxicants is a major scientific challenge for human health. We investigated the effects of a selected group of environmental polluting chemicals mostly provided with estrogenic activity on the human trophoblast cell lines BeWo and HTR-8/SVneo. Cells were exposed for 24h to various concentrations (from 0.1 pM to 1 mM) of atrazine (ATR), diethylstilbestrol (DES), para-nonylphenol (p-NP), resveratrol (RES) and 17 ß-estradiol (E2) and assayed for cell viability and human beta-Chorionic Gonadotropin (ß-hCG) secretion. Decrease of cell viability as respect to control, vehicle-treated, cultures was obtained for all chemicals in the concentration range of 1 µM-1 mM in both cell types. A parallel decrease of ß-hCG secretion was observed in BeWo cells, at 1 µM-1 mM concentrations, with the only exception of ATR which caused an increase at concentrations up to 1mM. ß-hCG release was also unexpectedly inhibited by ATR, DES, p-NP and RES at non-toxic (pM-nM) concentrations. These findings raise concern about the negative, potential effects of various environmental polluting chemicals on pregnancy success and fetal health.

Trophoblast cells are able to regulate monocyte activity to control Toxoplasma gondii infection.

INTRODUCTION: Toxoplasma gondii is an intracellular parasite that causes severe disease when the infection occurs during pregnancy. Trophoblast cells constitute an important maternal-fetal barrier, with monocytes concentrating around them. Thus, interactions between trophoblasts and monocytes are important for maintaining a successful pregnancy, especially in cases of infection. This study aimed to evaluate the role of trophoblast cells (BeWo line) on monocyte (THP-1 line) activity in the presence or absence of T. gondii infection. METHODS: THP-1 cells were stimulated with supernatants of BeWo cells, previously infected or not with T. gondii, and then infected with parasites. The supernatant of both cells were collected and analyzed for cytokine production and T. gondii proliferation in THP-1 cells was determined. RESULTS: The results showed that after infection, the pattern of cytokines secreted by THP-1 and BeWo cells was characterized as a pro-inflammatory profile. Furthermore, supernatant of BeWo cells infected or not, was able to change the cytokine profile secreted by infected THP-1 cells, and this supernatant became THP-1 cells more able to control T. gondii proliferation than those that had not been stimulated. DISCUSSION: This effect was associated with secretion of interleukin (IL)-6 by the THP-1 cells and soluble factors secreted by BeWo cells, such as IL-6 and MIF. CONCLUSION: Together, these results suggest that trophoblast cells are able to modulate monocyte activity, resulting in the control of T. gondii infection and subsequent maintenance of pregnancy.

Differential apoptosis in BeWo cells after infection with highly (RH) or moderately (ME49) virulent strains of Toxoplasma gondii is related to the cytokine profile secreted, the death receptor Fas expression and phosphorylated ERK1/2 expression.

INTRODUCTION: Alterations of apoptosis are commonly associated with pregnancy complications and abortion. Modulation of apoptosis is a relevant feature of Toxoplasma gondii infection and it is related to parasite strain types. The aim of the present study was to evaluate the possible factors that are involved in the differential apoptosis of BeWo cells infected with distinct T. gondii strain types. METHODS: Human trophoblastic cells (BeWo cell line) were infected with RH or ME49 strains, the cytokine production was measured and the phosphorylation of anti-apoptotic ERK1/2 protein was analyzed. Also, cells were treated with different cytokines, infected with RH or ME49 strain, and analyzed for apoptosis index and Fas/CD95 death receptor expression. RESULTS: ME49-infected BeWo cells exhibited a predominantly pro-inflammatory cytokine profile, whereas cells infected with RH strain had a higher production of anti-inflammatory cytokines. Also, the incidence of apoptosis was higher in ME49-infected cells, which have been treated with pro-inflammatory cytokines compared to cells infected with RH and treated with anti-inflammatory cytokines. Moreover, Fas/CD95 expression was higher in cells infected with either ME49 or RH strain and treated with pro-inflammatory cytokines compared to anti-inflammatory cytokine treatment. The phosphorylation of ERK1/2 protein increased after 24 h of infection only with the RH strain. CONCLUSION: These results suggest that opposing mechanisms of interference in apoptosis of BeWo cells after infection with RH or ME49 strains of T. gondii can be associated with the differential cytokine profile secreted, the Fas/CD95 expression and the phosphorylated ERK1/2 expression.

Evaluation of vertical transmission of Toxoplasma gondii in Calomys callosus model after reinfection with heterologous and virulent strain.

Toxoplasma gondii is an obligate intracellular protozoan parasite that causes a variety of clinical syndromes, but the infection is severe in immunocompromised individuals and during pregnancy due to the possibility of transplacental transmission of the parasite causing congenital toxoplasmosis. Vertical transmission of the parasite usually occurs when females are primarily infected during pregnancy. Calomys callosus is resistant to T. gondii ME49 strain, which presents a moderate virulence and congenital disease occurs only during the acute phase of infection. The aim of this study was to determine whether vertical transmission occurs when females of C. callosus chronically infected with ME49 strain of T. gondii are reinfected with a highly virulent strain (RH, type I). Females were infected with cysts of the ME49 strain. On the 1st day of pregnancy, animals were reinfected with tachyzoites of the RH strain. In the 19th day of pregnancy, placentas and embryos were processed for morphological analysis, immunohistochemistry and for detection of the parasite by PCR and mouse bioassay. Morphological and immunohistochemical analyses revealed the presence of parasites only in placental tissues. Mouse bioassay results showed seroconversion only in mice that were inoculated with placental tissues. Also, T. gondii DNA was detected only in placental samples. Congenital toxoplasmosis does not occur in C. callosus females chronically infected with the moderately virulent ME49 strain of T. gondii and reinfected with the highly virulent RH strain, thus indicating that primary T. gondii infection before pregnancy leads to an effective long-term immunity preventing transplacental transmission to the fetus.

Azithromycin and spiramycin induce anti-inflammatory response in human trophoblastic (BeWo) cells infected by Toxoplasma gondii but are able to control infection.

Toxoplasma gondii is an important pathogen which may cause fetal infection if primary infection. Our previous studies have used human choriocarcinoma trophoblastic cells (BeWo cell line) as experimental model of T. gondii infection involving placental microenvironment. This study aimed to examine the effects of azithromycin and spiramycin against T. gondii infection in BeWo cells. Cells were treated with different concentrations of the macrolide antibiotics and analyzed first for cell viability using thiazolyl blue tetrazole (MTT) assay. As cell viability was significantly decreased with drug concentrations higher than 400 µg/mL, the concentration range used in further experiments was from 50 to 400 µg/mL. The number of infected cells and intracellular replication of T. gondii decreased after treatment with each drug. The infection induced up-regulation of the macrophage migration inhibitory factor (MIF), which was also enhanced in infected cells after treatment with azithromycin, but not with spiramycin. Analysis of the cytokine profile showed increase TNF-α, IL-10 and IL-4 production, but decreased IFN-γ levels, were detected in infected cells and treated with each drug. In conclusion, treatment of human trophoblastic BeWo cells with with azithromycin or spiramycin is able to control the infection and replication of T. gondii. In addition, treatment with these macrolides, especially with azityromycin induces an anti-inflammatory response and high MIF production, which can be important for the establishment and maintenance of a viable pregnancy during T. gondii infection.