Both C57BL/KsJ (H2d haplotype) and CB10-H2 (H2b haplotype) mice are highly susceptible to congenital toxoplasmosis.

Congenital toxoplasmosis may cause abortion, neonatal death, or foetal abnormalities. Despite little information from human studies, a genetic influence over congenital disease was demonstrated and, host genome have been implicated to resistance/susceptibility to Toxoplasma gondii infection in both human and mice. It was previously shown that BALB/c mice (H2d) were more resistant to congenital toxoplasmosis than C57BL/6 mice (H2b). However, it is unclear whether these differences are attributable to the MHC haplotype or to other components of the mouse's genetic background. Therefore, in this work, we intend to address this question by investigating the pregnancy outcome in H2d -congenic C57BL/6 mice (C57BL/KsJ-H2d) and H2b-congenic BALB/c mice (CB10-H2-H2b). For this, animals were infected by intragastric route on the first day of pregnancy and examined on days 8 (8dP/8dI) or 18 (18dP/18dI) of gestation and infection. The pregnancy outcome, parasite burden, systemic cytokine profile and antibody response to infection were evaluated. Infected mice showed adverse pregnancy outcomes, in parallel low parasite detection in the uterus/placenta, being that the C57BL/KsJ showed the worst results in relation to CB10-H2 mice. Both mouse lineages showed an increase in IFN-γ and TNF levels systemically on 8dP/8dI and on 18dP/18dI, and C57BL/KsJ showed an increase in IL-6 levels in both gestation/infection periods. Additionally, C57BL/KsJ showed 7- and 7-fold increase in IL-6, 4- and 2.5-fold increase in IFN-γ and, 6- and 4-fold increase in TNF production on 8dP/8dI and 18dP/18dI, respectively in association with 1.5-fold decrease in TGF-ß levels on 8dP/8dI compared to CB10-H2 mice. In conclusion, the high IFN-γ and TNF serum levels observed in C57BL/KsJ (H2d) and CB10-H2 (H2b) mice were involved in the poor pregnancy outcomes in congenital toxoplasmosis. In addition, the higher IFN-γ, IL-6 and TNF levels detected in C57BL/KsJ in relation to CB10-H2 mice on 8dP/8dI seem to be related to the genetic background of C57BL/6J mice that may have contributed to the worse pregnancy outcome in this mouse lineage.

Rottlerin impairs early and late steps of Toxoplasma gondii infection in human trophoblast cells and villous explants.

Congenital toxoplasmosis, caused by the opportunistic protozoan parasite T. gondii, can cause stillbirths, miscarriages and fetal abnormalities, as well as encephalitis and chorioretinitis in newborns. Available treatment options rely on antiparasitic drugs that have been linked to serious side effects, high toxicity and the development of drug-resistant parasites. The search for alternative therapeutics to treat this disease without acute toxicity for the mother and child is essential for the advancement of current therapeutic procedures. The present study aimed to unravel the mode of the anti-T. gondii action of Rottlerin, a natural polyphenol with multiple pharmacological properties described. Herein, we further assessed the antiparasitic activity of Rottlerin against T. gondii infection on the human trophoblastic cells (BeWo cells) and, for the first time, on human villous explants. We found that non-cytotoxic doses of Rottlerin impaired early and late steps of parasite infection with an irreversible manner in BeWo cells. Rottlerin caused parasite cell cycle arrest in G1 phase and compromised the ability of tachyzoites to infect new cells, thus highlighting the possible direct action on parasites. An additional and non-exclusive mechanism of action of Rottlerin involves the modulation of host cell components, by affecting lipid droplet formation, mitochondrial function and upregulation of the IL-6 and MIF levels in BeWo cells. Supporting our findings, Rottlerin also controlled T. gondii proliferation in villous explants with low toxicity and reduced the IL-10 levels, a cytokine associated with parasite susceptibility. Collectively, our results highlighted the potential use of Rottlerin as a promising tool to prevent and/or treat congenital toxoplasmosis.

CCR5 contributes to adverse outcomes during malaria in pregnancy.

CCR5 is a chemokine receptor that mediates cell recruitment to sites of inflammation. It has been previously reported that the expression of CCR5 is increased in the placentas of women with malaria, a disease characterized by causing deliveries with low birth weight among other complications. CCR5 has been associated with pathology of protozoan infections during pregnancy but its role during malaria in pregnancy has not been elucidated. In the present work, we assessed the pregnancy outcome, placental structure, and levels of inflammatory markers of pregnant C57BL/6 and CCR5-/- mice infected or not with Plasmodium berghei NK65, with the purpose of determine the role of CCR5 in pregnancy associated malaria complications. We demonstrated that the expression of CCR5 mRNA increases in late pregnancy placentas of C57BL/6 when compared to uninfected controls. Infected pregnant C57BL/6 mice showed preterm birth, decreased fetal weight, placental inefficiency, and reduced placental vascular space. On the other hand, CCR5 deficiency led to increased levels of maternal parasitemia, reduced fetal weight and placental inefficiency compared to C57BL/6 mice. However, the infection did not cause additional changes in these parameters or in the incidence of preterm delivery in infected CCR5-/- mice in relation to C57BL/6 mice, showing that CCR5 may contribute to the adverse effects caused by infection during pregnancy. This improvement in pregnancy outcome, observed in infected CCR5-/- mice, was accompanied by lower placental levels of the inflammatory markers, such as TNF and NAG. Furthermore, it was observed that the placentas of CCR5-/- animals showed structural differences in relation to C57BL/6 mice, which could improve the efficiency of maternal-fetal exchanges, reflecting on fetal weight. Taken together, these results indicate that CCR5 expression contributes to the adverse outcomes caused by malaria in late pregnancy.

The ethanolic extract of the fungus Trichoderma stromaticum decreases the Toxoplasma gondii replication in vitro and ameliorates the experimental toxoplasmosis in vivo.

Trichoderma are fungi that are well-known to inhibit the growth of a variety of plant pathogens. Currently, there is an increasing search for new drugs to treat toxoplasmosis. The aims of this study were to investigate the effect of ExtTs in the control of Toxoplasma gondii proliferation in vitro and the course of toxoplasmosis in a mouse model. Firstly, the cytotoxicity of the ExtTs was evaluated by cultivating macrophages with different concentrations of the extract and cell viability was assessed by the MTT assay. Next, the infectivity of the T. gondii treated with extract was analyzed by infecting J774 macrophages. To evaluate the effect of the ExtTs in vivo, C57BL/6 mice were infected orally with T. gondii, ME-49, treated daily with ExtTs, and clinical, biochemical and histological changes were monitored. It was demonstrated that the extract did not affect the host cellular viability and, the treatment of parasites with ExtTs altered their morphology and decreased their ability to proliferate inside macrophages. Additionally, the treatment of mice with ExtTs decreased the parasitism and inflammation in the small intestine and liver of infected mice in parallel with increased IL-10/TNF ratio systemically and prevented alterations to serum VLDL and triglyceride levels. Thus, ExtTs could be considered an alternative/complementary therapy to control toxoplasmosis.

Effects of Notch signaling pathway inhibition by dibenzazepine in acute experimental toxoplasmosis.

Notch signaling pathway plays a crucial role in cellular fate across species, being important for the differentiation and development of several cell types. The aim of this study was to evaluate the effect of Notch inhibition pathway by dibenzazepine (DBZ) in histological and inflammatory alterations and, tissue parasitism in acute Toxoplasma gondii infection. For this, C57BL/6 mice were treated with DBZ before infection with T. gondii, and the small intestine, lungs and liver were analyzed. The genes related to Notch signaling pathway were assayed through qPCR in the organs, and cytokine measurement was performed in serum samples. In the small intestine, T. gondii infection impaired the Hes1 and Math1 mRNA expressions, increased the inflammation and decreased goblet and Paneth cell numbers. The DBZ-treatment was able to partially preserve these cells, however, the parasitism and inflammation were not altered. In parallel, the high IL-2, IL-6, TNF and, IFN-γ levels induced by infection were not changed with the DBZ treatment, with the IFN-γ levels even higher. In contrast, in the liver and lungs, the DBZ-treatment diminished parasitism and inflammation. Our results highlight that Notch pathway inhibition in T.gondii infection results in different parasitological and inflammatory outcomes depending on the organ analyzed.

Toxoplasma gondii Infection Decreases Intestinal 5-Lipoxygenase Expression, while Exogenous LTB4 Controls Parasite Growth.

5-Lipoxygenase (5-LO) is an enzyme required for the production of leukotrienes and lipoxins and interferes with parasitic infections. In vitro, Toxoplasma gondii inhibits leukotriene B4 (LTB4) production, and mice deficient in 5-LO are highly susceptible to infection. The aim of this study was to investigate the effects of the pharmacological inhibition of the 5-LO pathway and exogenous LTB4 supplementation during experimental toxoplasmosis. For this purpose, susceptible C57BL/6 mice were orally infected with T. gondii and treated with LTB4 or MK886 (a selective leukotriene inhibitor through inhibition of 5-LO-activating protein [FLAP]). The parasitism, histology, and immunological parameters were analyzed. The infection decreased 5-LO expression in the small intestine, and treatment with MK886 reinforced this reduction during infection; in addition, MK886-treated infected mice presented higher intestinal parasitism, which was associated with lower local interleukin-6 (IL-6), interferon gamma (IFN-γ), and tumor necrosis factor (TNF) production. In contrast, treatment with LTB4 controlled parasite replication in the small intestine, liver, and lung and decreased pulmonary pathology. Interestingly, treatment with LTB4 also preserved the number of Paneth cells and increased α-defensins expression and IgA levels in the small intestine of infected mice. Altogether, these data demonstrated that T. gondii infection is associated with a decrease in 5-LO expression, and on the other hand, treatment with the 5-LO pathway product LTB4 resulted in better control of parasite growth in the organs, adding to the knowledge about the pathogenesis of T. gondii infection.

Inhibition of Heme Oxygenase-1 by Zinc Protoporphyrin IX Improves Adverse Pregnancy Outcomes in Malaria During Early Gestation.

The enzyme heme oxygenase-1 (HO-1) has cytoprotective effects by catalyzing the degradation of heme to produce carbon monoxide, iron and biliverdin. Furthermore, HO-1 activity has been associated with successful pregnancy. On the other hand, in the context of certain inflammatory conditions, HO-1 can induce iron overload and cell death. To investigate the role of HO-1 in gestational malaria, pregnant BALB/c mice were infected with Plasmodium berghei ANKA in early, mid and late gestation. We found that malaria affected the pregnancy outcome in the three periods evaluated. However, only poor pregnancy outcomes in early pregnancy were related to HO-1 upregulation, iron overload, lipid peroxidation and necrosis of the decidua, which were prevented by HO-1 inhibition. In conclusion, HO-1 expression must be finely tuned in gestational malaria to avoid the deleterious effect of increased enzyme activity.

The imbalance of TNF and IL-6 levels and FOXP3 expression at the maternal-fetal interface is involved in adverse pregnancy outcomes in a susceptible murine model of congenital toxoplasmosis.

Vertical transmission of Toxoplasma gondii leads to adverse pregnancy outcomes depending on the time at which the infection occurs and the immunological state of the mother. C57BL/6 and BALB/c mice have been described as susceptible and resistant mouse lineages to congenital T. gondii infection, respectively. This study aimed to elucidate the systemic and local cytokine profile of pregnant mice infected with T. gondii and whether the expression of the transcription factor FOXP3, related to T regulatory cells, is associated with the resistance/susceptibility of these lineages of mice in the context of experimental congenital toxoplasmosis. For this purpose, C57BL/6 and BALB/c females were orally infected with the T. gondii ME-49 strain on the day of vaginal plug detection or day 14 of gestation, examined 7 or 5 days later, respectively, as models of early and late pregnancy. Cytokine levels were measured systemically and in the uterus/placenta. Additionally, the uterus/placenta were evaluated macroscopically for resorption rates and histologically for parasite and FOXP3 immunostaining. The FOXP3 protein expression was also evaluated by western blotting assay. It was found that, during early pregnancy, the infection leads to high IFN-γ, TNF and IL-6 levels systemically, with the TNF levels being higher in C57BL/6 mice. At the maternal-fetal interface, the infection induced high levels of IFN-γ in both mouse lineages; however, higher levels were observed in BALB/c, while high TNF and IL-6 levels were found in C57BL/6, but not in BALB/c mice. In contrast, in late gestation, T. gondii interfered less strongly with the cytokine profile. In early pregnancy, a reduction of FOXP3 expression at the maternal-fetal interface of infected mice was also observed, and the reduction was larger in C57BL/6 compared with BALB/c mice. Additionally, the parasite was seldom found in the uterus/placenta. Thus, the worse pregnancy outcomes observed in C57BL/6 mice were associated with higher TNF systemically, and TNF and IL-6 at the maternal-fetal interface, with lower FOXP3 expression.

Heme Oxygenase-1 Induction in Human BeWo Trophoblast Cells Decreases Toxoplasma gondii Proliferation in Association With the Upregulation of p38 MAPK Phosphorylation and IL-6 Production.

Heme oxygenase-1 (HO-1) enzyme exerts beneficial effects at the maternal-fetal interface, especially in trophoblasts, being involved in survival and maturation of these cell phenotypes. Trophoblast cells play essential roles throughout pregnancy, being the gateway for pathogens vertically transmitted, such as Toxoplasma gondii. It was previously shown that HO-1 activity was involved in the control of T. gondii infection in vivo; however, its contribution in trophoblast cells during T. gondii infection, remain undefined. Thus, this study aimed to investigate the influence of HO-1 in T. gondii-infected BeWo and HTR-8/SVneo human trophoblast cells. For this purpose, trophoblast cells were infected and the HO-1 expression was evaluated. T. gondii-infected BeWo cells were treated with hemin or CoPPIX, as inducers of HO-1, or with bilirubin, an end-product of HO-1, and the parasitism was quantified. The involvement of p38 MAPK, a regulator of HO-1, and the cytokine production, were also evaluated. It was found that T. gondii decreased the HO-1 expression in BeWo but not in HTR-8/SVneo cells. When treated with the HO-1 inducers or bilirubin, BeWo cells reduced the parasite proliferation. T. gondii also decreased the p38 MAPK phosphorylation in BeWo cells; on the other hand, HO-1 induction sustained its activation. Finally, the IL-6 production was upregulated by HO-1 induction in T. gondii-infected cells, which was associated with the control of infection.

IgE AND IgG antibody responses to Dermatophagoides pteronyssinus in dogs with demodicosis and atopic dermatitis / Respostas de anticorpos IgE E IgG específicos à Dermatophagoides pteronyssinus em soros de cães com demodicose e dermatite atópica

Canine demodicosis is a common inflammatory parasitic skin disease caused by Demodex mites. House dust mites, such as Dermatophagoides spp., play an important role in the pathogenesis of canine atopic dermatitis (AD). The goal of this experimental work was to investigate whether demodectic dogs could be previously exposed/sensitized to house dust mites' antigens. First the prevalence of demodicosis in a southeastern region of Brazil was investigated by analyzing clinical files of dogs that were admitted to a Veterinary Hospital. Subsequently, the IgG responses to Dermatophagoides pteronyssinus (Dp) and Dermatophagoides farinae (Df) and IgE to D.pteronyssinus (Dp) were evaluatedin two groups, AD or demodicosis dogs. Additionally, the major IgE-binding Dp proteins that are recognized by sera from dogs with demodicosis and AD were evaluated. A total of 2,599 clinical files were analyzed to identify the major parasitic skin diseases in dogs from this region, considering the age, sex and breed of the animals. The epidemiological study identified 111 animals with skin diseases; from these 20.7% presented demodicosis. Afterwards, serum samples were obtained from another groups of demodicosis, AD, and healthy dogs, and analyzed for Dp and Df-specific IgG, and IgE antibody levels, Dp IgG avidity by ELISA and IgE-binding Dp-specific proteins by immunoblot. IgG and IgE antibodies to Dp were detected in sera from additional groups of dogs with AD, demodicosis or healthy, with higher IgE levels to Dp in AD than demodectic or healthy dogs. IgG to Df was detected, despite with smaller levels compared to Dp in sera from demodectic dogs, and also in healthy dogs. Immunoblot showed IgE-binding to Dp proteins in sera of dogs with demodicosis and AD; with strong reactivity for the 72 and 116 kDa antigens detected by sera from demodicosis dogs. However, sera from healthy dogs >12 months old also presented reactivity to these bands. In conclusion, the detection of Dp-IgG and IgE antibodies in sera from demodectic dogs indicates previous exposure and sensitization to the house dust mite, respectively, more than cross-reactivity between demodex mites and Dp antigens detected by canine antibodies. Additionally, higher Dp-specific IgE levels were found in dogs with AD compared with those with demodicosis or healthy, suggesting that Dp-specific IgE could better discriminate dogs with AD from healthy ones or even those with demodicosis.

Demodicose canina é uma doença inflamatória comum da pele causada por ácaros do gênero Demodex. Ácaros da poeira doméstica como Dermatophagoides spp. desempenham papel importante na patogênese da dermatite atópica canina (DA). O objetivo desse trabalho experimental foi investigar se cães com demodicose poderiam ser previamente expostos/sensibilizados com antígenos de ácaros da poeira doméstica. A princípio, investigou-se a prevalência de demodicose em uma região sudeste do Brasil, analisando-se prontuários clínicos de cães admitidos em um Hospital Veterinário. Posteriormente, as respostas de IgG a Dermatophagoides pteronyssinus (Dp) e D. farinae (Df) e IgE a D. pteronyssinus (Dp) foram avaliadas em dois grupos, DA ou demodicose. Também foram avaliadas as principais proteínas Dp reconhecidas por anticorpo IgE presente em soros de cães com demodicose e DA. Um total de 2.599 prontuários clínicos foram analisados para identificar as principais doenças parasitárias da pele em cães dessa região, considerando a idade, sexo e raça dos animais. O estudo epidemiológico detectou 111 animais com doenças de pele e destes, 20,7% apresentavam demodicose. Posteriormente, amostras de soro foram obtidas de outros grupos de cães com demodicose, DA ou saudáveis, e analisadas quanto aos níveis de IgG e IgE específicos para Dp e Df, avidez de IgG a Dp por ELISA e proteínas específicas de Dp reconhecidas por IgE por immunoblot. Anticorpos IgG e IgE para Dp foram detectados em soros de grupos adicionais de cães com DA, demodicose ou saudáveis, com níveis mais altos de IgE para Dp na DA do que no soro de animais saudáveis. Níveis de IgG específicos para Df foram detectados, apesar serem menores em comparação com os detectados para Dp em soros de cães demodéticos, e também em cães saudáveis. A análise de immunoblot demonstrou detecção de IgE para proteinas de Dp em soros de cães com demodicose e DA; com forte reatividade para os antígenos de 72 e 116 kDa detectados por soros de cães com demodicose. No entanto, soros de cães saudáveis > 12 meses de idade também apresentaram reatividade a essas bandas. Em conclusão, a detecção de anticorpos Dp-IgG e IgE específicos em soros de cães demodéticos indica exposição prévia e sensibilização aos ácaros, respectivamente, mais do que reatividade cruzada entre ácaros Demodex e antígenos Dp detectados por anticorpos caninos. Além disso, níveis de Dp-IgE específicos mais elevados encontrados em cães com DA, sugerem que esses anticorpos poderiam discriminar melhor cães com DA daqueles saudáveis ou mesmo demodéticos.

The Availability of Iron Is Involved in the Murine Experimental Toxoplasma gondii Infection Outcome.

Iron is an important constituent of our environment, being necessary for both mammalian and pathogenic protozoa survival. Iron-containing proteins exert a wide range of biological processes such as biodegradation and biosynthesis, as well as immune function, fetal development, and physical and mental well-being. This work aimed to investigate the effect of iron deprivation in Toxoplasma gondii infection outcome. C57BL/6 mice were orally infected with T. gondii and treated with an iron chelator, deferoxamine, or supplemented with iron (ferrous sulfate), and the parasitism as well as immunological and histological parameters were analyzed. It was observed that the infection increased iron accumulation in the organs, as well as systemically, and deferoxamine treatment diminished the iron content in serum samples and intestine. The deferoxamine treatment decreased the parasitism and inflammatory alterations in the small intestine and lung. Additionally, they partially preserved the Paneth cells and decreased the intestinal dysbiosis. The ferrous sulfate supplementation, despite not significantly increasing the parasite load in the organs, increased the inflammatory alterations in the liver. Together, our results suggest that iron chelation, which is commonly used to treat iron overload, could be a promising medicine to control T. gondii proliferation, mainly in the small intestine, and consequently inflammation caused by infection.

Susceptibility of human villous (BeWo) and extravillous (HTR-8/SVneo) trophoblast cells to Toxoplasma gondii infection is modulated by intracellular iron availability.

Congenital toxoplasmosis is a serious health problem that can lead to miscarriage. HTR-8/SVneo is a first trimester extravillous trophoblast, while BeWo is a choriocarcinoma with properties of villous trophoblast cells. In the placenta, iron is taken up from Fe-transferrin through the transferrin receptor being the ion an important nutrient during pregnancy and also for Toxoplasma gondii proliferation. The aim of this study was to evaluate the role of iron in T. gondii proliferation in BeWo and HTR-8/SVneo cells and in human chorionic villous explants. The cells were infected with T. gondii, iron supplemented or deprived by holo-transferrin or deferoxamine, respectively, and parasite proliferation and genes related to iron balance were analyzed. It was verified that the addition of holo-transferrin increased, and DFO decreased the parasite multiplication in both trophoblastic cells, however, in a more expressive manner in HTR-8/SVneo, indicating that the parasite depends on iron storage in trophoblastic cells for its growth. Also, tachyzoites pretread with DFO proliferate normally in trophoblastic cells demonstrating that DFO itself does not interfere with parasite proliferation. Additionally, T. gondii infection induced enhancement in transferrin receptor mRNA expression levels in trophoblastic cells, and the expression was higher in HTR-8/SVneo compared with BeWo. Finally, DFO-treatment was able to reduce the parasite replication in villous explants. Thus, the iron supplementation can be a double-edged sword; in one hand, it could improve the supplement of an essential ion to embryo/fetus development, and on the other hand, could improve the parasite proliferation enhancing the risk of congenital infection.