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Due to the lack of efficient antiparasitic therapy and vaccines, as well as emerging resistance strains, congenital toxoplasmosis is still a public health issue worldwide. The present study aimed to assess the effects of an oleoresin obtained from the species Copaifera trapezifolia Hayne (CTO), and an isolated molecule found in the CTO, ent-polyalthic acid (ent-15,16-epoxy-8(17),13(16),14-labdatrien-19-oic acid) (named as PA), against T. gondii infection. We used human villous explants as an experimental model of human maternal-fetal interface. Uninfected and infected villous explants were exposed to the treatments; the parasite intracellular proliferation and the cytokine levels were measured. Also, T. gondii tachyzoites were pre-treated and the parasite proliferation was determined. Our findings showed that CTO and PA reduced efficiently the parasite growth with an irreversible action, but without causing toxicity to the villi. Also, treatments reduced the levels of IL-6, IL-8, MIF and TNF by villi, what configures a valuable treatment option for the maintenance of a pregnancy in an infectious context. In addition to a possible direct effect on parasites, our data suggest an alternative mechanism by which CTO and PA alter the villous explants environment and then impair parasite growth, since the pre-treatment of villi resulted in lower parasitic infection. Here, we highlighted PA as an interesting tool for the design of new anti-T. gondii compounds., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

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 EC 50 , CC 50 , 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.

Introduction: Introduction: total antioxidant capacity (TAC) is considered as a reliable indicator of the antioxidant content of the diet and it is associated to a reduced risk of chronic disease. Objectives: to estimate the TAC related to the intake of fruits and vegetables in young adults from urban areas of Paraguay. Methods: a minimum of three 24-hour dietary recalls were recorded from to a random sample of 190 university students in Asunción-Paraguay, between 2012 and 2014, to determine the intake of fruits and vegetables, their TAC value and the contents of specific antioxidants, applying the United States Department of Agriculture (USDA) data bases. Results: the mean value of the total vegetable intake and its TAC value were 262 g/d and 3,093 oxygen radical absorbance capacity (ORAC), respectively, distributed as follows: 76.9 g/d from fruits (1,624 ORAC), 57.9 g/d from fruit juices (444 ORAC), 47.8 g/d from raw vegetables (314 ORAC), 75.1 g/d from cooked vegetables (604 ORAC) and 4.5 g/d from legumes (106 ORAC). The mean C-vitamin intake was 73.2 mg/d, and fl avonoids and carotenoids were present with 26.4 mg/d and 4,848 μg/d, respectively. Conclusions: the total fruit and vegetable intake, distributed by groups of foods, was low, considering the World Health Organization (WHO) fruit and vegetable recommendation. The TAC value related to vegetable intake was lower than those reported in other studies. The major contribution to both intake and antioxidants was provided by fruits, and the minor by legumes. Our results reveal the need to increase the vegetable intake above 400 g/d, including antioxidant-rich food sources.

This study was conducted to investigate annexin A1 (ANXA1) functions in human placental explants infected with Toxoplasma gondii (T. gondii). We examined the first and third trimester placental explants infected with T. gondii (n = 7 placentas/group) to identify the number and location of parasites, ANXA1 protein, potential involvement of formyl peptide receptors (FPR1 and FPR2), and COX-2 expressions by immunohistochemistry. Treatments with Ac 2-26 mimetic peptide of ANXA1 were performed to verify the parasitism rate (β-galactosidase assay), prostaglandin E 2 levels (ELISA assay), and ANXA1, FPR1 and COX-2 expression in third trimester placentas. Placental explants of third trimester expressed less ANXA1 and were more permissive to T. gondii infection than first trimester placentas that expressed more ANXA1. Ac 2-26 treatment increases endogenous ANXA1 and decreases parasitism rate, COX-2, and prostaglandin E 2 levels. Altogether, these data provide further insight into the anti-parasitic and anti-inflammatory effects of ANXA1 in placentas infected with T. gondii., (Copyright © 2018. Published by Elsevier Ltd.)

Toxoplasmosis affects a third of the global population and presents high incidence in tropical areas. Its great relevance in public health has led to a search for new therapeutic approaches. Herein, we report the antiparasitic effects of BnSP-7 toxin, a Lys49 phospholipase A2 (PLA2) homologue from Bothrops pauloensis snake venom, on Toxoplasma gondii. In an MTT assay, BnSP-7 presented significant cytotoxicity against host HeLa cells at higher doses (200 μg/mL to 50 μg/mL), whereas lower doses (25 μg/mL to 1.56 μg/mL) produced low cytotoxicity. Furthermore, the toxin showed no effect on T. gondii tachyzoite viability when evaluated by trypan blue exclusion, but decreased both adhesion and parasite proliferation when tachyzoites were treated before infection. We also measured cytokines in supernatants collected from HeLa cells infected with T. gondii tachyzoites previously treated with RPMI or BnSP-7, which revealed enhancement of only MIF and IL-6 cytokines levels in supernatants of HeLa cells after BnSP-7 treatment. Our results showed that the BnSP-7 PLA2 exerts an anti-Toxoplasma effect at a lower dose than that required to induce cytotoxicity in HeLa cells, and also modulates the immune response of host cells. In this sense, the anti-parasitic effect of BnSP-7 PLA2 demonstrated in the present study opens perspectives for use of this toxin as a tool for future studies on toxoplasmosis., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

After oral infection, Toxoplasma gondii invades intestinal cells, induces breakdown of intestinal physiology and barrier functions, and causes intestinal pathology in some animal species. Although parasites' invasion into host cells is a known phenomenon, the effects of T. gondii infection in the intestinal barrier are still not well established. To evaluate morphological and physiological modifications on the colorectal adenocarcinoma-derived Caco-2 cell line during T. gondii infection, microvilli, tight junction integrity, and transepithelial electrical resistance (TEER) were investigated under infection. It was observed that the dextran uptake (endocytosis) and distribution were smaller in infected than in noninfected Caco-2 cells. The infection leads to the partial loss of microvilli at the cell surface. Claudin-1, zonula occludens-1 (ZO-1), and occludin expressions were colocalized by immunofluorescence and presented discontinuous net patterns in infected cells. Immunoblotting analysis at 24 hr postinfection revealed decreasing expression of occludin and ZO-1 proteins, whereas claudin-1 presented similar expression level compared with noninfected cells. T. gondii decreased TEER in Caco-2 cells 24 hr after infection. Our results suggest that T. gondii infection may lead to the loss of integrity of intestinal mucosa, resulting in impaired barrier function., (© 2016 The Histochemical Society.)

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.

Background: Ocular toxoplasmosis is a prominent and severe condition of high incidence in Brazil. The current study provides new insights into the immunological events that can be associated with retinochoroiditis in the setting of congenital toxoplasmosis in human infants., Methods: Flow cytometry of intracytoplasmic cytokines in leukocyte subsets following in vitro short-term antigenic recall in infants with congenital T. gondii infection., Results: Our data demonstrates that whereas neutrophils and monocytes from T. gondii-infected infants display a combination of proinflammatory and regulatory cytokine profiles, natural killer cells showed a predominantly proinflammatory profile upon in vitro T. gondii stimulation. The proinflammatory response of CD4(+) and CD8(+) T cells, characterized by the production of interferon γ (IFN-γ) and interleukin 17 in patients with an active retinochoroidal lesion, revealed the presence of IFN-γ and tumor necrosis factor α during early and late immunological events. This specific proinflammatory pattern is associated with early events and active retinochoroidal lesion, whereas a robust monocyte-derived interleukin 10-mediated profile is observed in children with cicatricial ocular lesions., Conclusions: These findings support the existence of a progressive immunological environment concomitant with the initial, apical, and cicatricial phases in the process of retinochoroidal lesion formation in infants with congenital toxoplasmosis that may be relevant in the establishment of stage-specific clinical management., (© The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.)

This study intended to apply the flow cytometric analysis of IgA and IgG reactivity and intracytoplasmic cytokine analysis to understand and decode the clinical aspects of infants with ocular congenital toxoplasmosis. The Toxoplasma gondii-infected infants (TOXO) were subdivided according to their clinical aspects based on the absence (NRL), presence of active (ARL), active/cicatricial (ACRL) or cicatricial retinochoroidal lesions (CRL) and compared to non-infected controls (NI). The reactivity of anti-T. gondii IgG subclasses resembles the clinical aspects of ocular lesions. IgG and IgG1 discriminate infants with cicatricial lesions (ACRL and CRL) from both ARL and NLR. IgG2 and IgG3 are particularly higher in ACRL and CRL as compared to NLR. No differences were observed when IgG4 reactivity was evaluated. Thus, the results indicated that the reactivity patterns of IgA, IgG and IgG subclasses are able to discriminate ARL, ACRL and CRL from NLR or NI. IgA and IgG subclasses are relevant serological biomarkers with diagnostic and prognostic applicability, respectively. Moreover, IgA and IgG1 were closely related to cytokine production by innate/adaptive immunity cells. IgA reactivity was directly associated to TNF-α-derived from neutrophils, monocytes and CD8(+) T-cells, while IgG1 was inversely correlated with IFN-γ-producing CD4(+) and CD8(+) T-cells but positively correlated with IL-10(+) B-cells. These findings provide insights on the relationship between the cytokine production by innate/adaptive immunity and the antibody pattern of infants with ocular congenital toxoplasmosis. In addition, the present study supports the use of flow cytometric serology as a potential tool for the diagnosis and monitoring of ocular lesions in T. gondii-infected infants in the clinical setting., (Copyright © 2015 Elsevier B.V. All rights reserved.)

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., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Considering that Toxoplasma gondii has shown high genetic diversity in Brazil, the aim of this study was to determine whether Calomys callosus chronically infected by the ME-49 strain might be susceptible to reinfection by these Brazilian strains, including vertical transmission of the parasite. Survival curves were analyzed in non-pregnant females chronically infected with ME-49 and reinfected with the TgChBrUD1 or TgChBrUD2 strain, and vertical transmission was analyzed after reinfection of pregnant females with these same strains. On the 19th day of pregnancy (dop), placentas, uteri, fetuses, liver, spleen, and lung were processed for detection of the parasite. Blood samples were collected for humoral and cellular immune response analyses. All non-pregnant females survived after reinfection and no changes were observed in body weight and morbidity scores. In pregnant females, parasites were detected in the placentas of ME-49 chronically infected females and reinfected females, but were only detected in the fetuses of reinfected females. TgChBrUD2 reinfected females showed more impaired pregnancy outcomes, presenting higher numbers of animals with fetal loss and a higher resorption rate, in parallel with higher levels of pro-inflammatory cytokines and IgG2a subclass antibodies. Vertical transmission resulting from chronic infection of immunocompetent C. callosus is considered a rare event, being attributed instead to either reactivation or reinfection. That is, the pregnancy may be responsible for reactivation of the latent infection or the reinfection may promote T. gondii vertical transmission. Our results clearly demonstrate that, during pregnancy, protection against T. gondii can be breached after reinfection with parasites belonging to different genotypes, particularly when non-clonal strains are involved in this process and in this case the reinfection promoted vertical transmission of both type II and Brazilian T. gondii strains.

Considering that interleukin 10 (IL10), transforming growth factor beta1 (TGFB1), and interferon gamma (IFNG) are involved in the susceptibility of BeWo trophoblast cells to Toxoplasma gondii infection, the aim of the present study was to investigate the effector mechanisms triggered by these cytokines in the control of T. gondii in BeWo cells. For this purpose, infected/uninfected BeWo cells were treated with IL10, TGFB1 (50 ng/ml), and IFNG (20 or 100 ng/ml) in order to verify the phosphorylation of signal transducers and activators of transcription 1 (STAT1), STAT3, and Smad2, parasite intracellular proliferation, as well as the Th1/Th2/IL17A cytokine production. The treatment of BeWo cells with IL10 and TGFB1 favored T. gondii proliferation, and these findings were associated with STAT3 and Smad2 phosphorylation, respectively (P < 0.05). Also, these cytokine treatments were able to down-modulate TNF alpha (TNFA) and IL6 production (P < 0.05). Low concentration of IFNG was unable to control T. gondii infection but was able to trigger STAT1 phosphorylation and up-regulate IL6 and IL17A production; whereas a high concentration of IFNG was unable to activate STAT1 but down-modulated IL6 and TNFA and increased T. gondii proliferation (P < 0.05). IL10, TGFB1, and IFNG regulate a differential T. gondii proliferation in BeWo cells because they distinctly trigger intracellular signaling pathways and cytokine production, especially IL6 and TNFA. Our data open new windows to understand the mechanisms triggered by IL10, TGFB1, and IFNG at the maternal-fetal interface in the presence of T. gondii, contributing to recognizing the importance of these effector mechanisms involved in the vertical transmission of this parasite., (© 2015 by the Society for the Study of Reproduction, Inc.)

This study developed a remarkable methodological innovation (FC-ATE) which enables simultaneous detection of antibodies specific to the three evolutive forms of Trypanosoma cruzi: live amastigote (AMA), live trypomastigote (TRYPO), and fixed epimastigote (EPI) using a differential fluorescence staining as low (AMA), intermediate (TRYPO), and high (EPI). An outstanding performance (100%) was observed in the discrimination of the chagasic (CH) and non-chagasic (NCH) patients. In the applicability of FC-ATE in the diagnosis of Chagas disease, 100% of the CH samples presented positivity in the percentage of positive fluorescent parasites (PPFP) for all the three forms of T. cruzi. Moreover, 94% of the samples of NCH presented negative values of PPFP with AMA and TRYPO, and 88% with EPI. Samples from the NCH group with false-positive results were those belonging to the leishmaniasis patients. Considering the applicability of this technique in post-therapeutic monitoring of Chagas disease, 100% of non-treated (NT) and treated non-cured (TNC) samples were positive with the three T. cruzi evolutive forms, while a percentage of 100% from samples of the treated cured (TC) patients were negative with AMA, 93% with TRYPO and 96% with EPI. The comparison between FC-ATE and two other flow cytometric tests using the same samples of patients NT, TNC and TC showed that the three techniques presented different reactivities, although categorical correlation between the methodologies was observed. Taken together, the results obtained with the novel FC-ATE method have shown an outstanding performance in the diagnosis and post-therapeutic monitoring of Chagas disease., (Copyright © 2014 Elsevier B.V. All rights reserved.)

There is a significant genetic diversity of Toxoplasma gondii in Brazil. Two parasite isolates were recently obtained from chickens in Uberlândia, Minas Gerais state, Brazil, namely, TgChBrUD1 and TgChBrUD2. In this study, we investigated Calomys callosus susceptibility to these atypical T. gondii strains. Male and female animals were intraperitoneally infected with tachyzoites and monitored to evaluate body weight change, morbidity, and mortality. Immunohistochemical assay and qPCR were performed to determine the parasitism in liver, spleen, and brain. Our data showed that TgChBrUD2-infected males died earlier than TgChBrUD1-infected males and 100% of mortality was observed after 10 and 12 days of infection, respectively. Also, TgChBrUD1-infected females died earlier than TgChBrUD1-infected males and 100% of mortality was observed after 9 and 12 days of infection, respectively. Both strains were able to induce a decrease in body weight of males, but only the TgChBrUD1 strain induced an increase in body weight of females. TgChBrUD2-infected females had significantly higher parasite load in both liver and spleen in comparison to TgChBrUD1-infected females, but no significant difference was found between genders or strains when males were infected. There was higher parasitism in the liver than the brain from both males and females infected with either strain. In conclusion, C. callosus specimens are susceptible to both T. gondii atypical strains with differences between males and females in severity of infection. These findings open new prospects for understanding different aspects of T. gondii infection, including reinfection and vertical transmission with these atypical strains when utilizing this experimental model.

Background: Although Toxoplasma gondii infection is normally asymptomatic, severe cases of toxoplasmosis may occur in immunosuppressed patients or congenitally infected newborns. When a fetal infection is established, the recommended treatment is a combination of pyrimethamine, sulfadiazine and folinic acid (PSA). The aim of the present study was to evaluate the efficacy of azithromycin to control T. gondii infection in human villous explants., Methods: Cultures of third trimester human villous explants were infected with T. gondii and simultaneously treated with either PSA or azithromycin. Proliferation of T. gondii, as well as production of cytokines and hormones by chorionic villous explants, was analyzed., Results: Treatment with either azithromycin or PSA was able to control T. gondii infection in villous explants. After azithromycin or PSA treatment, TNF-α, IL-17A or TGF-β1 levels secreted by infected villous explants did not present significant differences. However, PSA-treated villous explants had decreased levels of IL-10 and increased IL-12 levels, while treatment with azithromycin increased production of IL-6. Additionally, T. gondii-infected villous explants increased secretion of estradiol, progesterone and HCG+β, while treatments with azithromycin or PSA reduced secretion of these hormones concurrently with decrease of parasite load., Conclusions: In conclusion, these results suggest that azithromycin may be defined as an effective alternative drug to control T. gondii infection at the fetal-maternal interface.

This work aimed to test the influence of heparin on the susceptibility of retinal cells to Toxoplasma gondii infection. Primary cultures of retinas from chick embryos of 8 (E8) or 11 (E11) days and fibroblasts (control) were used. To determine the influence of heparin in T. gondii infection, tachyzoites of the RH strain were treated with heparin before addition in the culture. A monoclonal anti-heparin antibody was used to analyze the heparin distribution on fibroblast and retinal cell surfaces. Our results showed that retinal cells (E8 and E11) had a higher infection rate than fibroblasts (91% and 24% versus 13%, respectively). Pre-treatment of T. gondii with heparin decreased infection of E8 retinal cells when compared with non-treated parasites (45% versus 91%, respectively), but not of E11 cells (35% versus 48%). In accordance, retinal cells presented an intense heparin staining by immunofluorescence assay. In conclusion, retinal cells from chick embryos were more susceptible to infection by T. gondii compared to fibroblasts and, pre-treatment of tachyzoites with heparin decreased the number of infected cells and parasite burden particularly for E8 retinal cells., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

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., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2013 IFPA and Elsevier Ltd. All rights reserved.)

Toxoplasma gondii is the main infectious cause of human posterior retinochoroiditis, the most frequent clinical manifestation of congenital toxoplasmosis. This investigation was performed after neonatal screening to identify biomarkers of immunity associated with immunopathological features of the disease by flow cytometry. The study included infected infants without NRL and with retinochoroidal lesions (ARL, ACRL, and CRL) as well as noninfected individuals (NI). Our data demonstrated that leukocytosis, with increased monocytes and lymphocytes, was a relevant hematological biomarker of ARL. Immunophenotypic analysis also revealed expansion of CD14(+)CD16(+)HLA-DR(high) monocytes and CD56(dim) cytotoxic NK-cells in ARL. Moreover, augmented TCRγ δ (+) and CD8(+) T-cell counts were apparently good biomarkers of morbidity. Biomarker network analysis revealed that complex and intricated networks underscored the negative correlation of monocytes with NK- and B-cells in NRL. The remarkable lack of connections involving B-cells and a relevant shift of NK-cell connections from B-cells toward T-cells observed in ARL were outstanding. A tightly connected biomarker network was observed in CRL, with relevant connections of NK- and CD8(+) T-cells with a broad range of cell subsets. Our findings add novel elements to the current knowledge on the innate and adaptive immune responses in congenital toxoplasmosis.

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., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Heme oxygenase-1 (HO-1) is an enzyme that catabolizes free heme, which induces an intense inflammatory response. The expression of HO-1 is induced by different stimuli, triggering an anti-inflammatory response during biological stress. It was previously verified that HO-1 is able to induce indoleamine 2,3-dioxygenase (IDO), an enzyme that is induced by IFN-γ in Toxoplasma gondii infection. To verify the role of HO-1 during in vivo T. gondii infection, BALB/c and C57BL/6 mice were infected with the ME49 strain and treated with zinc protoporphyrin IX (ZnPPIX) or hemin, which inhibit or induce HO-1 activity, respectively. The results show that T. gondii infection induced high levels of HO-1 expression in the lung of BALB/c and C57BL6 mice. The animals treated with ZnPPIX presented higher parasitism in the lungs of both lineages of mice, whereas hemin treatment decreased the parasite replication in this organ and in the small intestine of infected C57BL/6 mice. Furthermore, C57BL/6 mice infected with T. gondii and treated with hemin showed higher levels of IDO expression in the lungs and small intestine than uninfected mice. In conclusion, our data suggest that HO-1 activity is involved in the control of T. gondii in the lungs of both mouse lineages, whereas the hemin, a HO-1 inducer, seems to be involved in the control of parasitism in the small intestine of C57BL/6 mice.

This paper reports the effects of BnSP-7 toxin, a catalytically inactive phospholipase A2 from Bothrops pauloensis snake venom, on Leishmania (Leishmania) amazonensis. BnSP-7 presented activity against promastigote parasite forms both in the MTT assay, with IC50 of 58.7 μg mL(-1) of toxin, and a growth curve, inhibiting parasite proliferation 60-70% at concentrations of 50-200 μg mL(-1) of toxin 96 h after treatment. Also, the toxin presented effects on amastigotes, reducing parasite viability by 50% at 28.1 μg mL(-1) and delaying the amastigote-promastigote differentiation process. Ultrastructural studies showed that BnSP-7 caused severe morphological changes in promastigotes such as mitochondrial swelling, nuclear alteration, vacuolization, acidocalcisomes, multiflagellar aspects and a blebbing effect in the plasma membrane. Finally, BnSP-7 interfered with the infective capacity of promastigotes in murine peritoneal macrophages, causing statistically significant infectivity-index reductions (P < 0.05) of 20-35%. These data suggest that the BnSP-7 toxin is an important tool for the discovery of new parasite targets that can be exploited to develop new drugs for treating leishmaniasis.

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 β-estradiol (E2) and assayed for cell viability and human beta-Chorionic Gonadotropin (β-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 β-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. β-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., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Currently, toxoplasmosis is treated with sulfadiazine and pyrimethamine. However, this treatment presents several adverse side effects; thus, there is a critical need for the development and evaluation of new drugs, which do not present the same problems of the standard therapy. Enrofloxacin is a fluoroquinolone antibiotic known to control infection against several bacteria in veterinary medicine. Recently, this drug has demonstrated protective effects against protozoan parasites such as Neospora caninum. The present study aimed to determine the effect of enrofloxacin in the control of Toxoplasma gondii infection. For this purpose, human foreskin fibroblast (HFF) cells were infected with T. gondii RH strain and treated with sulfadiazine, penicillin/streptomycin, pyrimethamine, or enrofloxacin. Following treatment, we analyzed the infection index, parasite intracellular proliferation and the number of plaques. Additionally, tissue parasitism and histological changes were investigated in the brain of Calomys callosus that were infected with T. gondii (ME49 strain) and treated with either sulfadiazine or enrofloxacin. Enrofloxacin was able to reduce the infection index, intracellular proliferation and the number of plaques in HFF cells infected by T. gondii in comparison with untreated or penicillin/streptomycin-treated ones. Enrofloxacin was more protective against T. gondii in HFF infected cells than sulfadiazine treatment (P<0.001). In addition, pyrimethamine, enrofloxacin or the associations of sulfadiazine plus pyrimethamine, enrofloxacin plus sulfadiazine or enrofloxacin plus pyrimethamine-treatments were able to reduce the plaque numbers in HFF cells infected by T. gondii when compared to medium, penicillin/streptomycin or sulfadiazine alone. In vivo experiments demonstrated that enrofloxacin diminished significantly the tissue parasitism as well as the inflammatory alterations in the brain of C. callosus infected with T. gondii when compared with untreated animals. Based on our findings, it can be concluded that enrofloxacin is a potential alternative drug for the treatment of toxoplasmosis., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Congenital toxoplasmosis is associated with adverse pregnancy outcome. Despite the type 1 immune response, C57BL/6 mice are more susceptible than BALB/c mice to Toxoplasma gondii infection. Additionally, successful pregnancy appears to be correlated with type 2 T helper maternal immunity and regulatory T cells. In order to investigate the mechanisms of susceptibility/resistance to congenital toxoplasmosis in mice with different genetic backgrounds and the influence of inducible nitric oxide synthase in pregnancy outcome, groups of C57BL/6, BALB/c and C57BL/6 iNOS(-/-) females were orally infected with T. gondii ME-49 strain on day 1 of pregnancy and were sacrificed on day 8 p.i. and day 19 p.i. The uterus and placenta were evaluated for the foetal resorption rate, parasite load, immunological and histological changes. C57BL/6 mice presented inflammatory foci in the decidua (endometrium) of the uterus at a higher frequency than BALB/c mice on day 8 p.i., and a large number of pregnant C57BL/6 mice presented necrotic implantation sites. The parasite was seldom found in the uterus or placenta of either lineage of mice. Interestingly, there was no observed difference in inducible nitric oxide synthase expression in the uterus and placenta of infected mice. In addition, higher levels of TNF-α were detected in serum samples from C57BL/6 mice compared with BALB/c mice. Accordingly, C57BL/6 mice presented with levels of 90% abortion compared with 50% in BALB/c mice on day 19 p.i. C57BL/6 iNOS(-/-) mice showed low placental parasite counts and high absorption rates, similar to wild type mice. The data suggest that the impaired pregnancy outcome due to T. gondii infection in C57BL/6 mice could be associated with a higher inflammatory response leading to cell apoptosis and necrosis of implantation sites compared with BALB/c mice, and this phenomenon was not due to inducible nitric oxide synthase expression in the decidua., (Copyright © 2012 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.)

Background: T. cruzi strains have been divided into six discrete typing units (DTUs) according to their genetic background. These groups are designated T. cruzi I to VI. In this context, amastigotes from G strain (T. cruzi I) are highly infective in vitro and show no parasitemia in vivo. Here we aimed to understand why amastigotes from G strain are highly infective in vitro and do not contribute for a patent in vivo infection., Methodology/principal Findings: Our in vitro studies demonstrated the first evidence that IFN-γ would be associated to the low virulence of G strain in vivo. After intraperitoneal amastigotes inoculation in wild-type and knockout mice for TNF-α, Nod2, Myd88, iNOS, IL-12p40, IL-18, CD4, CD8 and IFN-γ we found that the latter is crucial for controlling infection by G strain amastigotes., Conclusions/significance: Our results showed that amastigotes from G strain are highly infective in vitro but did not contribute for a patent infection in vivo due to its susceptibility to IFN-γ production by host immune cells. These data are useful to understand the mechanisms underlying the contrasting behavior of different T. cruzi groups for in vitro and in vivo 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., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Autogenous bone tissue has regeneration potential; however, this capacity may not be sufficient in larger bone defects. The aim of this study is to histologically evaluate anorganic bovine bone grafts (GenOx Inorg) with or without platelet-rich plasma (PRP). Two bone lesions were created in calvaria of 12 rabbits. The 24 surgical lesions were separated into 3 groups: coagulous, anorganic, and anorganic with PRP. At the 4-week time point, the animals were euthanized and the grafted area removed, fixed in formalin 10% with phosphate buffered saline, 0.1 M, and embedded in paraffin. The histologic parameters analyzed were new bone filling the defect area, presence of giant cells and particles of the graft, and new bone formation associated with the particles. In the coagulous group, defects were filled with fibrous tissue that attached the periosteum and little bone neoformation in the periphery. In anorganic groups with or without PRP, little new bone formation in the periphery of the defect was observed; however, in the center of some defects there was new bone. Moderate presence of giant cells and little new bone formation was associated with the innumerous graft particles. Histologic results revealed no statistically significant differences among the defects new bone fill between the studied groups (P = .64). There was no significant difference in the number of giant cells (P = .60), graft particles (P = .46), and new bone formation around graft particles (P = .26), whether PRP was added or not. Anorganic bone, isolated or mixed with PRP, was biocompatible and osteoconductive, while maintaining bone volume.

The aim of the study is to assess the hypotensive properties of the hydro-ethanolic crude root extract (CRE), the n-butanol fraction (F(BtOH)) and nuatigenin-3-O-β-chacotriose, from Solanum sisymbriifolium Lam., in adrenal regeneration hypertension+deoxycorticosterone acetate (ARH+DOCA) rats, following a chronic administration. The roots of S. sisymbriifolium Lam. (Solanaceae) were extracted by reflux with ethanol-water 7:3 and the active extract was fractionated by bioassay-guided liquid-liquid separation. Nuatigenin-3-O-β-chacotriose (B(3-1)) was identified as the main hypotensive compound from the crude drug by spectroscopic methods. Immature Wistar rats of both sexes were submitted to both surgery and deoxycorticosterone acetate treatment to obtain adrenal regeneration hypertensive rats (ARH+DOCA). Different groups of experimentally induced hypertensive rats were randomly allotted and received during 16 weeks a daily oral administration of 1% saline solution (0.1 mL/100g body weigh), 100.0 mg/kg of CRE, 10.0, 30.0 and 50.0 mg/kg of F(BtOH), and 1.0 mg/kg of B(3-1), respectively. In addition, two groups of ARH+DOCA rats were randomly assigned to receive either B(3-1) (1.0 mg/kg/day) or 1% of saline solution (0.1 mL/100g body weight/day) for 7 weeks and then a cross over procedure was performed in order to complete the 16th-week treatment. After 16 weeks of oral administration of crude root extract (CRE), butanolic fraction (F(BtOH)) and nuatigenin-3-O-β-chacotriose (B(3-1)) a significant reduction of blood pressure value was induced in hypertensive animals (ARH+DOCA) in comparison to the control group receiving 1% saline solution, at the end of experiment. Administration of B(3-1) (1.0 mg/kg/day p.o.) to ARH+DOCA rats provoked a significant reduction of blood pressure, observed gradually from 5th week (p<0.05) to the end of the 16th week period of treatment (p<0.01). Moreover, in a cross over design it was observed that the reduction of blood pressure to normotensive condition is associated to B(3-1). The latest demonstrated that the blood pressure-lowering effect, in clearly hypertensive animals, is reversible and depend upon administration of nuatigenin-3-O-β-chacotriose (B(3-1)). Our results demonstrated that daily oral administration of CRE, F(BtOH) and nuatigenin-3-O-β-chacotriose from S. sisymbriifolium for a 16-week period exhibits an antihypertensive effect in experimentally hypertensive (ARH+DOCA) rats., (Copyright © 2011 Elsevier GmbH. All rights reserved.)

Because macrophage migration inhibitory factor (MIF) is a key cytokine in pregnancy and has a role in inflammatory response and pathogen defense, the objective of the present study was to investigate the effects of MIF in first- and third-trimester human placental explants infected with Toxoplasma gondii. Explants were treated with recombinant MIF, IL-12, interferon-γ, transforming growth factor-β1, or IL-10, followed by infection with T. gondii RH strain tachyzoites. Supernatants of cultured explants were assessed for MIF production. Explants were processed for morphologic analysis, immunohistochemistry, and real-time PCR analysis. Comparison of infected and stimulated explants versus noninfected control explants demonstrated a significant increase in MIF release in first-trimester but not third-trimester explants. Tissue parasitism was higher in third- than in first-trimester explants. Moreover, T. gondii DNA content was lower in first-trimester explants treated with MIF compared with untreated explants. However, in third-trimester explants, MIF stimulus decreased T. gondii DNA content only at the highest concentration of the cytokine. In addition, high expression of MIF receptor was observed in first-trimester placental explants, whereas MIF receptor expression was low in third-trimester explants. In conclusion, MIF was up-regulated and demonstrated to be important for control of T. gondii infection in first-trimester explants, whereas lack of MIF up-regulation in third-trimester placentas may be involved in higher susceptibility to infection at this gestational age., (Copyright © 2011 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

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., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Neospora caninum is an apicomplexan parasite, closely related to Toxoplasma gondii, and causes abortion and congenital neosporosis in cattle worldwide. Trophoblast cells act in mechanisms of innate immune defense at the fetal-maternal interface and no data are available about the interaction of Neospora with human trophoblasts. Thus, this study aimed to verify the susceptibility of human trophoblastic (BeWo) compared with uterine cervical (HeLa) cell lines to N. caninum. BeWo and HeLa cells were infected with different parasite:cell ratios of N. caninum tachyzoites and analyzed at different times after infection for cell viability using thiazolyl blue tetrazole and lactate dehydrogenase assays. Both cell lines were also evaluated for cytokine production and parasite infection/replication assays when pre-treated or not with Neospora lysate antigen (NLA) or human recombinant IFN-γ. Cell viability was increased up to 48 h of infection in both types of cells, suggesting that infection could inhibit early cell death and/or induce cell proliferation. Neospora infection induced up-regulation of the macrophage migration inhibitory factor (MIF), mainly in HeLa cells, which was enhanced by cell pre-treatment by NLA or IFN-γ. Conversely, parasite infection induced down-regulation of the transforming growth factor (TGF-β), mostly in BeWo cells, which was decreased with NLA or IFN-γ pre-treatment. HeLa cells were more susceptible to Neospora infection than BeWo cells and IFN-γ pre-treatment resulted in reduced infection indices in both cell lines. Control of parasite growth was mediated by IFN-γ through an indoleamine-2,3-dioxygenase-dependent mechanism in HeLa cells alone. Based on these results, we concluded that BeWo and HeLa cells are readily infected by N. caninum, although presenting differences in susceptibility to infection, cytokine production and cell viability. Thus, these host cells can be considered in comparative approaches to understand strategies used by N. caninum to survive at the maternal-fetal interface., (Copyright © 2010 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.)

In order to investigate the differential ALCAM, ICAM-1 and VCAM-1 adhesion molecules mRNA expression and the blood-brain barrier (BBB) permeability in C57BL/6 and BALB/c mice in Toxoplasma gondii infection, animals were infected with ME-49 strain. It was observed higher ALCAM on day 9 and VCAM-1 expression on days 9 and 14 of infection in the central nervous system (CNS) of C57BL/6 compared to BALB/c mice. The expression of ICAM-1 was high and similar in the CNS of both lineages of infected mice. In addition, C57BL/6 presented higher BBB permeability and higher IFN-gamma and iNOS expression in the CNS compared to BALB/c mice. The CNS of C57BL/6 mice presented elevated tissue pathology and parasitism. In conclusion, our data suggest that the higher adhesion molecules expression and higher BBB permeability contributed to the major inflammatory cell infiltration into the CNS of C57BL/6 mice that was not efficient to control the parasite., (Copyright 2010 Elsevier Inc. All rights reserved.)

Background: Macrophage migration inhibitory factor (MIF) has special pro-inflammatory roles, affecting the functions of macrophages and lymphocytes and counter-regulating the effects of glucocorticoids on the immune response. The conspicuous expression of MIF during human implantation and early embryonic development also suggests this factor acts in reproductive functions. The overall goal of this study was to evaluate Mif expression by trophoblast and embryo placental cells during mouse pregnancy., Methods: Mif was immunolocalized at implantation sites on gestation days (gd) 7.5, 10.5, 13.5 and 17.5. Ectoplacental cones and fetal placentas dissected from the maternal tissues were used for Western blotting and qRT-PCR assays on the same gestation days., Results: During the post-implantation period (gd7.5), trophoblast giant cells showed strong Mif reactivity. In later placentation phases (gds 10.5-17.5), Mif appeared to be concentrated in the junctional zone and trophoblast giant cells. Mif protein expression increased significantly from gd7.5 to 10.5 (p = 0.005) and from gd7.5 to 13.5 (p = 0.03), remaining at high concentration as gestation proceeded. Higher mRNA expression was found on gd10.5 and was significantly different from gd13.5 (p = 0.048) and 17.5 (p = 0.009)., Conclusions: The up-regulation of Mif on gd10.5 coincides with the stage in which the placenta assumes its three-layered organization (giant cells, spongiotrophoblast and labyrinth zones), fetal blood circulation begins and population of uNK cells reaches high proportions at the maternal counter part of the placenta, suggesting that Mif may play a role in either the placentation or in the adaptation of the differentiated placenta to the uterus or still in gestational immunomodulatory responses. Moreover, it reinforces the possibility of specific activities for Mif at the maternal fetal interface.

Toxoplasma gondii infection during pregnancy may cause severe consequences to the embryo. Current toxoplasmosis treatment for pregnant women is based on the administration of spiramycin or a drug combination as sulphadiazine-pyrimethamine-folinic acid (SPFA) in cases of confirmed fetal infection. However, these drugs are few tolerated and present many disadvantages due to their toxic effects to the host. The aim of this study was to evaluate the effectiveness of different treatments on the vertical transmission of T. gondii, including azithromycin, Artemisia annua infusion, spiramycin and SPFA in Calomys callosus as model of congenital toxoplasmosis. C. callosus females were perorally infected with 20 cysts of T. gondii ME49 strain at the day that a vaginal plug was observed (1st day of pregnancy - dop). Treatment with azithromycin, A. annua infusion, and spiramycin started at the 4th dop, while the treatment with SPFA started at the 14th dop. Placenta and embryonic tissues were collected for morphological and immunohistochemical analyses, mouse bioassay and PCR from the 15th to 20th dop. No morphological changes were seen in the placenta and embryonic tissues from females treated with azithromycin, spiramycin and SPFA, but embryonic atrophy was observed in animals treated with A. annua infusion. Parasites were found in the placenta and fetal (brain and liver) tissues of animals treated with SPFA, A. annua infusion and spiramycin, although the number of parasites was lower than in non-treated animals. Parasites were also observed in the placenta of animals treated with azithromycin, but not in their embryos. Bioassay and PCR results confirmed the immunohistochemical data. Also, bradyzoite immunostaining was observed only in placental and fetal tissues of animals treated with SPFA. In conclusion, the treatment with azithromycin showed to be more effective, since it was capable to inhibit the vertical transmission of T. gondii in this model of congenital toxoplasmosis.

Transplacental transmission of Toxoplasma gondii causes congenital toxoplasmosis, one of the most severe forms of infection. The ability of the parasite to survive intracellularly largely depends on the blocking of different proapoptotic signaling cascades of the host cells. During pregnancy, however, alterations in the incidence of apoptosis are associated with abnormal placental morphology and function. The aim of this study was to evaluate the incidence of apoptosis and cell proliferation in trophoblastic (BeWo cell line) and uterine cervical (HeLa cell line) cells infected with a highly virulent RH strain or a moderately virulent ME49 strain of T. gondii. BeWo and HeLa cells were infected with RH or ME49 tachyzoites (2:1 and 5:1; parasite:cell) or medium alone (control). After 2 h, 6 h and 12 h of incubation, cells were fixed in 10% formalin and analyzed by immunohistochemistry to determine the apoptosis (expression of cytokeratin 18 neo-epitope--clone M30) and cell in S phase (expression of proliferating cell nuclear antigen--PCNA) indices. RH strain-infected BeWo and HeLa cells showed a lower apoptosis index than non-infected controls, whereas a higher apoptosis index was found in ME49 strain-infected cells compared to controls. In addition, RH-infected cells displayed lower apoptosis index than ME49-infected cells, even though active caspase-3 was detected in both cell types infected with either RH or ME49 strains as well in non-infected cells in all analyzed times of infection. Also, the cell S phase indices were higher in ME49 strain-infected BeWo and HeLa cells as compared to non-infected controls and RH strain-infected cells. These results indicate that RH and ME49 strains of T. gondii possess opposing mechanism of interference in apoptosis and cell cycle S phase of both BeWo and HeLa cells and these differences can be associated to evasion strategies of the parasite to survive inside the host cells.

Toxoplasma gondii is a widely distributed obligatory intracellular parasite that causes severe disease to the fetus when transmitted during pregnancy. Drugs used to avoid congenital transmission have shown side effects, and their efficacy is controversial. The most widely used treatment for acute toxoplasmosis during pregnancy is pyrimethamine plus sulfadiazine, which has several side effects. In this work, we tested the efficacy of azithromycin in reducing congenital transmission of T. gondii in the large vesper mouse, Calomys callosus, a rodent. Females of C callosus were inoculated perorally with 20 cysts of ME49 strain of T. gondii on the day of fertilization, and fetuses were collected from the 15th to the 19th day of gestation. Azithromycin (300 mg/kg), in association with pyrimethamine (100 or 50 mg/kg) plus sulfadiazine (100 or 75 mg/kg) and folinic acid (15 mg/kg) (SPAf), or vehicle, were administered orally on different days after infection. Brain and ocular tissues were removed and processed for immunohistochemistry using a polyclonal antibody against T. gondii, or were processed for parasite DNA quantification. Toxoplasma gondii was detected in the brains of all females and in fetuses' eyes of C. callosus treated with SPAf. On the other hand, in females treated with azithromycin, there was a reduction of T. gondii in the brains of mothers, and no parasites were detected in eyes of fetuses, indicating that azithromycin may represent an alternative treatment for toxoplasmosis during pregnancy.

Considering that the treatment for toxoplasmosis is based on drugs that show limited efficacy due to their substantial side effects, the purpose of the present study was to evaluate the effects of Artemisia annua on in vitro and in vivo Toxoplasma gondii infection. A. annua infusion was prepared from dried herb and tested in human foreskin fibroblasts (HFF) or mice that were infected with the parasite and compared with sulfadiazine treatment. For in vitro experiments, treatment was done on parasite before HFF infection or on cells previously infected with T. gondii and the inhibitory concentration (IC(50)) values for each treatment condition were determined. Viability of HFF cells in the presence of different concentrations of A. annua infusion and sulfadiazine was above 72%, even when the highest concentrations from both treatments were tested. Also, the treatment of T. gondii tachyzoites with A. annua infusion before infection in HFF cells showed a dose-response inhibitory curve that reached up to 75% of inhibition, similarly to the results observed when parasites were treated with sulfadiazine. In vivo experiments with a cystogenic T. gondii strain demonstrated an effective control of infection using A. annua infusion. In conclusion, our results indicate that A. annua infusion is useful to control T. gondii infection, due to its low toxicity and its inhibitory action directly against the parasite, resulting in a well tolerated therapeutic tool.

The aim of the present work is to evaluate the putative antidepressant-like effects of a hydro-ethanolic extract (CEAp) and their fractions from the aerial parts of Aloysia polystachya (Griseb.) Moldenke (Verbenaceae) on the performance of male mice in the forced swimming test (FST). A single dose (100.0mg/kgp.o.) of CEAp, in male mice provoked a significant reduction of the immobility time (p<0.01). Such effect was also observed with short-term treatment (7 days) with single doses of 1.0 (p<0.01), 10.0 (p<0.05) and 100.0 (p<0.05)mg/kg/day of CEAp. Additionally, in a different set of experiments, repeated administration in a 24-h period (24, 18 and 1h before swimming test) with doses of 1.0 (p<0.05) and 10.0 (p<0.05)mg/kg p.o., of CEAp and 10.0mg/kgp.o., (p<0.05) of ethyl acetate fraction, provoked significant reduction of the immobility time of male mice in the FST. Moreover, it was noted important differences in the onset of the antidepressant-like effect in the FST, depending on the modality of treatment with CEAp (acute, short-term or repeated). Both, efficacy and potency were higher when repeated administration of CEAp was used, and surprisingly the dose of 10mg/kg (24, 18 and 1h before swimming test) was more effective than imipramine. In the same way, the short term administration (7 days) improved significantly efficacy and potency of the CEAp in comparison to a single dose treatment. The ethyl acetate fraction submitted to TLC demonstrated that main and minor components are phenolics and terpenes, respectively. In addition, this fraction gives a negative Shinoda's test for flavonoids. These results indicate an antidepressant-like profile of action for the hydro-ethanolic extract and the component(s) of the ethyl acetate fraction obtained from A. polystachya, which deserve further investigation.

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.

Considering the potential role of macrophage migration inhibitory factor (MIF) in the inflammation process in placenta when infected by pathogens, we investigated the production of this cytokine in chorionic villous explants obtained from human first-trimester placentas stimulated with soluble antigen from Toxoplasma gondii (STAg). Parallel cultures were performed with villous explants stimulated with STAg, interferon-gamma (IFN-gamma), or STAg plus IFN-gamma. To assess the role of placental MIF on monocyte adhesiveness to human trophoblast, explants were co-cultured with human myelomonocytic THP-1 cells in the presence or absence of supernatant from cultures treated with STAg (SPN), SPN plus anti-MIF antibodies, or recombinant MIF. A significantly higher concentration of MIF was produced and secreted by villous explants treated with STAg or STAg plus IFN-gamma after 24-hour culture. Addition of SPN or recombinant MIF was able to increase THP-1 adhesion, which was inhibited after treatment with anti-MIF antibodies. This phenomenon was associated with intercellular adhesion molecule expression by villous explants. Considering that the processes leading to vertical dissemination of T. gondii remain widely unknown, our results demonstrate that MIF production by human first-trimester placenta is up-regulated by parasite antigen and may play an essential role as an autocrine/paracrine mediator in placental infection by T. gondii.

Background: To study the possible role of aldosterone on thyroid hormone-induced myocardium hypertrophy, using spironolactone., Objective: To evaluate morphological changes in the myocardium induced by thyroid hormone and the possible effects of spironolactone use on these alterations., Methods: Forty Wistar rats were studied. The animals were allocated to four groups and were given: the vehicle used for dilution of the thyroid hormone (C); sodium levothyroxin at 50 microg/rat/day (H); spironolactone, 0.3 mg/kg/day (S); or thyroid hormone plus spironolactone (HS), at the same doses mentioned above, for 28 consecutive days. All the animals were weighed, had blood drawn for hormonal measurements and underwent ECG at the start and the end of the experiment. At the end of experiment all animals were euthanized, the weight of the left ventricle (LV) was determined and LV slices were obtained for morphological analysis., Results: There was an increase in T3 levels, decrease of body weight and higher heart rate in the animals from group H. The LV weight was significantly higher in the H e HS groups. The histometric analyses that measured the diameter of the myocytes showed higher values in group H and a progressive decrease in groups HS, S and C, with a significant difference among all the groups. The addition of spironolactone decreased the transversal myocyte hypertrophy by 14.6%., Conclusion: Rats treated with thyroid hormone present cardiac hypertrophy with increased LV weight and greater myocyte diameter. Spironolactone, when associated with thyroid hormone, can partially prevent this hypertrophy through mechanisms that are yet to be determined.

The immune response induced by Toxoplasma gondii is characterized by Th1 immune mechanisms. We previously demonstrated that C57BL/6 mice infested with Myocoptes musculinus and infected with T. gondii by intraperitoneal route undergo accelerated mortality according to Th2 immune mechanisms induced by the acarian. To evaluate whether infection with M. musculinus influences T. gondii-induced Th1 response in a resistant mouse lineage, BALB/c, which develops latent chronic toxoplasmosis in a way similar to that observed in immunocompetent humans, this study was done. The animals were infected with T. gondii ME-49 strain 1 month after M. musculinus infestation, being the survival and the immune response monitored. The double-infected displayed higher mortality rate if compared with the mono-infected mice. In addition, infection with M. musculinus changed the T. gondii-specific immune response, converting BALB/c host to a susceptible phenotype. Spleen cells had increased the levels of IL-4 in double-infected mice. This alteration was associated with severe pneumonia, encephalitis and wasting condition. In addition, a higher tissue parasitism was observed in double-infected animals. It can be concluded that infection with these two contrasting parasites, M. musculinus and T. gondii, may convert an immunocompetent host into a susceptible one, and such a host will develop severe toxoplasmosis.

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.

Infection with Toxoplasma gondii leads to a Th1 immune response. Alternatively, the acarian Myocoptes musculinus induces a disease in BALB/c mice that involves Th2 immune mechanisms. In this study, we investigated whether infestation by M. musculinus induces Th2 immune response in C57BL/6 mice and if this response influences the T. gondii-induced Th1 response when mice are inoculated by intraperitoneal or oral route. The animals were infected with M. musculinus and one month later with T. gondii ME-49 strain and the survival and immune response were monitored. The co-infected animals displayed higher mortality rate and the spleen cells showed a decreased IFN-gamma and elevated IL-4 and IL-5 production. These changes were associated with severe pneumonia and wasting condition. On the other hand, when mice were orally infected with 100 T. gondii cysts, co-infection prolonged the survival rates and ameliorated intestinal lesions in association with a significant drop in IFN-gamma levels in sera. These results indicate the interference of Th2 response induced by M. musculinus in a T. gondii-induced Th1 response. Altogether, these data demonstrate the profound interactions between the immune response induced against unrelated organisms T. gondii and M. musculinus, and suggest that this type of interactions may impact clinical disease.