Search

Toxoplasmose congênita é uma doença de importância clínica que acomete tecidos fetais causando sérios problemas aos recém-nascidos. O conhecimento de gestantes sobre a forma de transmissão e consequências clínicas da doença pode diminuir casos da doença. O objetivo foi avaliar, por meio de um questionário, os conhecimentos e conduta de profissionais da saúde e gestantes atendidas nas Unidades de Saúde e nos Centros de Assistência Social de Ituiutaba em relação à toxoplasmose congênita e desenvolver ações educativas para orientação acerca das consequências para o recém-nascido. Participaram 149 gestantes e 12 profissionais da saúde, respondendo um questionário sobre formas de transmissão e profilaxia e assistindo uma palestra de orientação e esclarecimento. As gestantes participaram de oficinas para confecção de material informacional. A faixa etária média das gestantes foi 25 ±6,6 anos, sendo que 34,2% concluíram o ensino médio e 49,6% afirmaram ter ouvido falar da doença, sendo a principal forma de transmissão do parasito a vertical (37%) seguida do contato com as fezes contaminadas do gato (31,5%) e ingestão de carne crua ou mal cozida (29,5%). A maioria dos profissionais de saúde (66,6%) afirmou explicar a doença e formas de prevenção para as gestantes e todos as orientam sobre a soroconversão na gestação. Porém, algumas não são capazes de compreender a importância da doença, principalmente as consequências para o seu filho. Esse fato ficou perceptível durante as palestras e oficinas. Cabe aos profissionais de saúde aperfeiçoar o acompanhamento pré-natal das gestantes, alertando-as sobre os meios de transmissão e controle da toxoplasmose.

Aim The outbreak of the new coronavirus pandemic (SARS-CoV-2) was initiated in December 2019, and within a couple of months it became a global health emergency. Given the importance to assess the evolution and transmissibility of SARS-CoV-2 and to forecast the next scenario of the pandemic, mainly in countries with limited healthcare systems, we estimated the reproductive number (R0) of SARS-CoV-2 in Jammu and Kashmir (J&K), India, and a possible scenario for this pandemic in the region. Subject and methods We estimated the reproductive number (R0) of SARS-CoV-2 in its first outbreak stage in the northwestern region of Himalaya, India, and we also predicted new daily cases for the next 90 days using different R0, testing a plausible end of the SARS-CoV-2 outbreak. Results Our results showed a considerable increase in the number of cases, but with a tendency to asymptote. Anantnag, Bandipora, Baramulla, Shopian, and Srinagar districts showed more than 100 cases and Kulgam and Kathua districts showed strong growth of the number of cases from the beginning of May, without a tendency to normalization. The estimated R0 for the J&K region was 1.041; but by decreasing the RO by 10, 25, and 50%, we observed a great decrease in the daily number of new cases, especially by decreasing by 50%. Conclusion In this study, we indicate positive effects of the preventive measures, such as lockdown and social distancing, taken in the J&K region, showing a stabilization of the growth curves of new cases of SARS-CoV-2, which tends to a strong decrease over time as the R0 decreases.

Congenital toxoplasmosis is represented by the transplacental passage of Toxoplasma gondii from the mother to the fetus. Our studies demonstrated that T. gondii developed mechanisms to evade of the host immune response, such as cyclooxygenase (COX)-2 and prostaglandin E2 (PGE2) induction, and these mediators can be produced/stored in lipid droplets (LDs). The aim of this study was to evaluate the role of COX-2 and LDs during T. gondii infection in human trophoblast cells and villous explants. Our data demonstrated that COX-2 inhibitors decreased T. gondii replication in trophoblast cells and villous. In BeWo cells, the COX-2 inhibitors induced an increase of pro-inflammatory cytokines (IL-6 and MIF), and a decrease in anti-inflammatory cytokines (IL-4 and IL-10). In HTR-8/SVneo cells, the COX-2 inhibitors induced an increase of IL-6 and nitrite and decreased IL-4 and TGF-β1. In villous explants, the COX-2 inhibitors increased MIF and decreased TNF-α and IL-10. Furthermore, T. gondii induced an increase in LDs in BeWo and HTR-8/SVneo, but COX-2 inhibitors reduced LDs in both cells type. We highlighted that COX-2 is a key factor to T. gondii proliferation in human trophoblast cells, since its inhibition induced a pro-inflammatory response capable of controlling parasitism and leading to a decrease in the availability of LDs, which are essentials for parasite growth.

CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico FAPEMIG - Fundação de Amparo a Pesquisa do Estado de Minas Gerais Toxoplasma gondii é um protozoário parasito intracelular obrigatório capaz de infectar uma grande variedade de células. Este parasito apresenta uma grande habilidade de explorar diversas moléculas presentes na superfície das células e ultrapassar diferentes barreiras biológicas, e finalmente alcançar os tecidos considerados imunologicamente privilegiados, como a placenta. Embora esse órgão represente uma formidável barreira contra a infecção por T. gondii, a transmissão deste para o feto pode ocorrer em alguns casos. Entretanto, os mecanismos envolvidos na transmissão do parasito pela placenta ainda são mal compreendidos. Assim, o presente trabalho teve dois objetivos gerais: (i) avaliar o papel de ICAM-1 na passagem transplacentária de T. gondii em modelos animais murinos C57BL/6 WT e ICAM-1 knockouts (ICAM-1-/-); e (ii) caracterizar células trofoblásticas mesenquimais humanas (TSCT) para avaliar se elas recapitulam a sensibilidade de células trofoblásticas primárias humanas frente a infecção por T. gondii, bem como avaliar a expressão diferencial de genes entre células citotrofoblásticas (TSCYT) e sinciciotroblásticas (TSSYN) após a infecção. Para responder ao primeiro objetivo, fêmeas WT e ICAM-1-/- foram acasaladas e após a detecção do início da gestação, os animais foram infectados com 5 cistos da cepa ME-49 de T. gondii e divididos em diferentes grupos de acordo com o dia de infecção e eutanásia (8dg/8di, 14dg/7di, 19dg/12di e 19dg/8di). Fêmeas prenhes não infectadas foram usadas como controles (8dg/ni, 14dg/ni, 19dg/ni). Em todos os animais foram avaliados o número de sítios de implantação, e de reabsorção. Além do mais, foi coletado o soro para análise de citocinas por CBA; os órgãos maternos, incluindo cornos uterinos, e embriões/fetos e placentas foram coletados para avaliação do parasitismo por qPCR. Os resultados mostraram que fêmeas ICAM-1-/- infectadas no início da gestação (8dg/8di) e no final do terço médio de gestação (19dg/8di) tiveram maior comprometimento do sucesso gestacional se comparado às fêmeas WT. Em relação ao parasitismo, fêmeas ICAM-1-/- 8dg/8di e 14dg/7di apresentaram maior taxa de infecção por T. gondii no corno uterino gravídico e na placenta, respectivamente, ao comparar com fêmeas WT. Por outro lado, não foram verificadas diferenças significativas na carga parasitária no baço e fígado entre as fêmeas ICAM-1-/- e WT nos grupos 8dg/8di e entre os tecidos fetais dos animais ICAM-1-/- e WT nos grupos 14dg/7di. Adicionalmente, a dosagem de citocinas mostrou que IFN-γ, TNF- α, IL-6 e IL-10 foram moduladas pela infecção dependendo do tempo gestacional. Para a realização do segundo capítulo, células TSCYT foram cultivadas em cultura e diferenciadas em TSSYN e infectadas ou mock infectadas (apenas meio) com T. gondii. Em seguida, foi avaliada a porcentagem de diferenciação em células TSSYN, bem como a taxa de infecção entre as células TSCYT e TSSYN por meio ensaio de RT-qPCR. O sobrenadante foi coletado para dosagem da quimiocina CCL22. Em seguida, células infectadas e mock infectadas foram coletadas e submetidas à análise pelo sequenciamento de RNA. Os dados mostraram que TSSYN foram altamente refratárias à adesão e replicação de T. gondii quando comparado com as células TSCYT. Além do mais, os resultados mostraram que ambas as células não foram capazes de produzir CCL22 em reposta à infecção pelo parasito. Também, os dados de RNA-seq mostraram diferenças no transcriptoma entre TSSYN e TSCYT após a infecção com T. gondii, evidenciando que vias relacionadas ao metabolismo e ao ciclo celular foram reguladas negativamente em células TSSYN em comparação com TSCYT. Finalmente, o perfil transcricional de T. gondii identificou diferenças significativas na expressão de genes em parasitos que infectaram/ou estavam associados às células TSSYN em relação às células TSCYT, evidenciando alterações nos genes envolvidos na replicação do DNA e no metabolismo, principalmente nas vias relacionadas com a síntese de ácidos graxos. Em conjunto, nossos dados indicaram que células TSCT representam um promissor modelo no estudo das interações T. gondii/placenta, além disso, os resultados sugerem que a resistência das células TSSYN pode estar associada ao baixo metabolismo celular e baixa produção de nutrientes por tais células, conferindo um ambiente estressante para o desenvolvimento do parasito. Toxoplasma gondii is an intracellular protozoan parasite that is able to infect a wide variety of cells. This parasite has a great ability to explore various molecules present on the surface of cells and overcome different biological barriers, and finally reach the tissues considered immunologically privileged, such as placenta. Although this organ represents a formidable barrier against a T. gondii infection, its transmission to the fetus can occur. However, the mechanisms involved in the transmission of the parasite through the placenta are still poorly understood. Therefore, the present work had two major goals: (i) evaluate the role of ICAM-1 in the transplacental passage of T. gondii in murine animal models C57BL/6 WT and ICAM-1 knockouts (ICAM-1-/-); and (ii) characterize human mesenchymal trophoblastic cells (TSCT) to assess whether they recapitulate the sensitivity of primary human trophoblastic cells by T. gondii infection, as well as to evaluate the differential gene expression between cytotrophoblastic (TSCYT) and synciciotroblastic cells (TSSYN) after the infection. To answer the first objective, WT and ICAM-1-/- mice were mated and after detecting the first day of pregnancy, the animals were infected with 5 cysts of ME-49 strain of T. gondii and divided in different groups according to the day of infection and euthanasia (8dg / 8di, 14dg / 7di, 19dg / 12di and 19dg / 8di). Uninfected pregnant females were used as controls (8dg/ni, 14dg/ni, 19dg/ni). The number of implantation and resorption sites was evaluated. In addition, serum was collected for cytokine analysis; maternal organs, including uterine horns, and embryos/fetus and placentas were collected to assess the parasitism by qPCR. The results showed that ICAM-1-/- females infected at the beginning of pregnancy (8dg/8di) and at the end of the middle third of pregnancy (19dg/8di) had a greater impairment of gestational success compared to the WT females. Regarding to the parasitism, ICAM-1-/- 8dg/8di and 14dg/7di presented higher rates of infection in uterine horns and in the placenta, respectively, compared to WT. On the other hand, no significant differences were seen in the parasitism in the spleen and liver between ICAM-1-/- and WT in the 8dg/8di groups, and between fetal tissues of ICAM-1-/- and WT animals in the group 14dg/7di. Additionally, the cytokines IFN-γ, TNF-α, IL-6 and IL-10 were modulated by infection depending on the gestational time. For the second chapter, TSCYT cells were cultured and differentiated into TSSYN and infected or mock infected (only medium) with T. gondii. Then, the percentage of differentiation in TSSYN cells was evaluated, as well as the infection rate between TSCYT and TSSYN cells through the RT-qPCR assay. The supernatant from the different experimental conditions was collected to measure the chemokine CCL22. Then, infected and mock infected cells were collected and subjected to analysis by RNA sequencing. The data showed that TSSYN were highly refractory to the adhesion and replication of T. gondii when compared with TSCYT cells. Furthermore, the results suggested that both cells are not able to produce CCL22 in response to the parasite infection. Evaluating the RNA-seq data, we observe a huge difference in the transcriptome between TSSYN and TSCYT after the infection by T. gondii, showing that pathways related to metabolism and the cell cycle are negatively regulated in TSSYN cells compared to TSCYT. Finally, the transcriptional profile of T. gondii identified differences in gene expression in the parasites that infected/or were associated with TSSYN cells in relation to TSCYT cells, showing differences in genes involved in DNA replication and metabolism pathways, especially those related to the biosynthesis of fatty acids. Together, our data indicate that TSCT cells represent a promising model in the study of T. gondii/placenta interactions; moreover, we suggest that the resistance of TSSYN cells may be associated with low cell metabolism and low nutrient production by such cells, providing a stressful environment for the development of the parasite. Tese (Doutorado)

Toxoplasma gondii is a parasite able to infect various cell types, including trophoblast cells. Studies have demonstrated that interleukin (IL)-10, transforming growth factor (TGF)-β1 and interferon (IFN)-γ are involved in the susceptibility of BeWo trophoblast cells to T. gondii infection. Furthermore, T. gondii is able to adhere to the plasma membrane of host cells through intercellular adhesion molecule (ICAM)-1. Thus, the present study aimed to assess the role of IL-10, TGF-β1 and IFN-γ in the expression of ICAM-1 in BeWo and HeLa cells and to analyze the role of ICAM-1 in the adhesion and invasion of T. gondii to these cells under the influence of these cytokines. For this purpose, BeWo and HeLa cells were treated or not, before and after T. gondii infection, with rIL-10, rTGF-β1 or rIFN-γ. For the BeWo cells, rIL-10 and rTGF-β1 favored susceptibility to infection, but only rTGF-β1 and rIFN-γ increased ICAM-1 expression, and TNF-α release. On the other hand, rIFN-γ downregulated the expression of ICAM-1 triggered by T. gondii in HeLa cells, leading to control of the infection. Moreover, we observed that upregulation of ICAM-1, mediated by cytokine's stimulation, in BeWo and HeLa cells resulted in a high number rate of both parasite adhesion and invasion to these cells, which were strongly reduced after ICAM-1 neutralization. Likewise, the blockage of ICAM-1 molecule also impaired T. gondii infection in human villous explants. Taken together, these findings demonstrate that TGF-β1 and IFN-γ differentially regulate ICAM-1 expression, which may interfere in the adhesion/invasion of T. gondii to BeWo and HeLa cells for modulating susceptibility to infection.

Crosstalk between trophoblast and monocytes is essential for gestational success, and it can be compromised in congenital toxoplasmosis. Cell death is one of the mechanisms involved in the maintenance of pregnancy, and this study aimed to evaluate the role of trophoblast in the modulation of monocyte cell death in the presence or absence of Toxoplasma gondii infection. THP-1 cells were stimulated with supernatants of BeWo cells and then infected or not with T. gondii. The supernatants were collected and analyzed for the secretion of human Fas ligand, and cells were used to determine cell death and apoptosis, cell death receptor, and intracellular proteins expression. Cell death and apoptosis index were higher in uninfected THP-1 cells stimulated with supernatants of BeWo cells; however, apoptosis index was reduced by T. gondii infection. Macrophage migration inhibitory factor (MIF) and transforming growth factor (TGF)-β1, secreted by BeWo cells, altered the cell death and apoptosis rates in THP-1 cells. In infected THP-1 cells, the expression of Fas/CD95 and secretion of FasL was significantly higher; however, caspase 3 and phosphorylated extracellular-signal-regulated kinase (ERK1/2) were downregulated. Results suggest that soluble factors secreted by BeWo cells induce cell death and apoptosis in THP-1 cells, and Fas/CD95 can be involved in this process. On the other hand, T. gondii interferes in the mechanism of cell death and inhibits THP-1 cell apoptosis, which can be associated with active caspase 3 and phosphorylated ERK1/2. In conclusion, our results showed that human BeWo trophoblast cells and T. gondii infection modulate cell death in human THP-1 monocyte cells.

The combination of sulfadiazine and pyrimethamine plus folinic acid is the conventional treatment for congenital toxoplasmosis. However, this classical treatment presents teratogenic effects and bone marrow suppression. In this sense, new therapeutic strategies are necessary to reduce these effects and improve the control of infection. In this context, biogenic silver nanoparticles (AgNp-Bio) appear as a promising alternative since they have antimicrobial, antiviral, and antiparasitic activity. The purpose of this study to investigate the action of AgNp-Bio in BeWo cells, HTR-8/SVneo cells and villous explants and its effects against Toxoplasma gondii infection. Both cells and villous explants were treated with different concentrations of AgNp-Bio or combination of sulfadiazine + pyrimethamine (SDZ + PYZ) in order to verify the viability. After, cells and villi were infected and treated with AgNp-Bio or SDZ + PYZ in different concentrations to ascertain the parasite proliferation and cytokine production profile. AgNp-Bio treatment did not reduce the cell viability and villous explants. Significant reduction was observed in parasite replication in both cells and villous explants treated with silver nanoparticles and classical treatment. The AgNp-Bio treatment increased of IL-4 and IL-10 by BeWo cells, while HTR8/SVneo cells produced macrophage migration inhibitory factor (MIF) and IL-4. In the presence of T. gondii, the treatment induced high levels of MIF production by BeWo cells and IL-6 by HTR8SV/neo. In villous explants, the AgNp-Bio treatment downregulated production of IL-4, IL-6, and IL-8 after infection. In conclusion, AgNp-Bio can decrease T. gondii infection in trophoblast cells and villous explants. Therefore, this treatment demonstrated the ability to reduce the T. gondii proliferation with induction of inflammatory mediators in the cells and independent of mediators in chorionic villus which we consider the use of AgNp-Bio promising in the treatment of toxoplasmosis in BeWo and HTR8/SVneo cell models and in chorionic villi.

Aim: The outbreak of the new coronavirus pandemic (SARS-CoV-2) was initiated in December 2019, and within couple of months it has turned out to be a global health emergency. Given the importance to assess the evolution and transmissibility of SARS-CoV-2 and to forecast the next scenario of the pandemic in the future, mainly in countries with limited health care system, we estimated the reproductive number (R0) of SARS-CoV-2 in Jammu and Kashmir (J&K), India, and a possible scenario for this pandemic in the region.Subject and Methods: We estimated the reproductive number (R0) of SARS-CoV-2 in its first outbreak stage in the North western region of Himalaya, India, and we also predicted new daily cases for the next 90 days using different R0, testing a plausible end of SARS-CoV-2 outbreak;Results: Our results showed a considerable increase in the number of cases, but with a tendency to asymptote. Anantnag, Bandipora, Baramulla, Shopian, and Srinagar districts showed more than 100 cases and Kulgam and Kathua districts showed strong growth of the number of cases from the beginning of May, without a tendency to normalization. The estimated R0 for J&K region was 1.041; but by decreasing 10, 25 and 50% the R0, we observed a great decrease in the daily number of new cases, specially by decreasing 50%;Conclusion: Here, we indicate positive effects of the preventive measures, as lockdown and social distance, taken in J&K region, showing a stabilization of the growth curves of new cases of SARS-CoV-2, which tends to a strong decrease over time as the R0 decreases.

Activities of phospholipases (PLAs) have been linked to pathogenesis in various microorganisms, and implicated in cell invasion and so the interest in these enzymes as potential targets that could contribute to the control of parasite survival and proliferation. Chicken eggs immunized with BnSP-7, a Lys49 phospholipase A2 (PLA2) homologue from Bothrops pauloensis snake venom, represent an excellent source of polyclonal antibodies with potential inhibitory activity on parasite PLAs. Herein, we report the production, characterization and anti-parasitic effect of IgY antibodies from egg yolks of hens immunized with BnSP-7. Produced antibodies presented increasing avidity and affinity for antigenic toxin epitopes throughout immunization, attaining a plateau after 4weeks. Pooled egg yolks-purified anti-BnSP-7 IgY antibodies were able to specifically recognize different PLA2s from Bothrops pauloensis and Bothrops jararacussu venom. Antibodies also neutralized BnSP-7 cytotoxic activity in C2C12 cells. Also, the antibodies recognized targets in Leishmania (Leishmania) amazonensis and Toxoplasma gondii extracts by ELISA and immunofluorescence assays. Anti-BnSP-7 IgY antibodies were cytotoxic to T. gondii tachyzoite and L. (L.) amazonensis promastigotes, and were able to decrease proliferation of both parasites treated before infection. These data suggest that the anti-BnSP-7 IgY is an important tool for discovering new parasite targets and blocking parasitic effects.

During pregnancy, Toxoplasma gondii can triggers serious manifestations and potentially affect the fetal development. In this scenario, differences in susceptibility of trophoblast cells to T. gondii infection might be evaluated in order to establish new therapeutic approaches capable of interfering in the control of fetal infection by T. gondii. This study aimed to evaluate the susceptibility of cytotrophoblast, syncytiotrophoblast and extravillous trophoblast cells to T. gondii infection. Our data demonstrate that HTR-8/SVneo cells (extravillous trophoblast cells) present higher susceptibility to T. gondii infection when compared to syncytiotrophoblast and cytotrophoblast cells, whereas syncytiotrophoblast was the cell type more resistant to the parasite infection. Also, cytotrophoblast and syncytiotrophoblast cells produced significantly more IL-6 than HTR-8/SVneo cells. On the other hand, HTR-8/SVneo cells showed higher ERK1/2 phosphorylation than cytotrophoblast and syncytiotrophoblast cells. ERK1/2 inhibition reduced T. gondii infection and increased IL-6 production in HTR-8/SVneo cells. Thus, it is plausible to conclude that the greater susceptibility of HTR-8/SVneo cells to infection by T. gondii is related to a higher ERK1/2 phosphorylation and lower levels of IL-6 in these cells compared to other cells, suggesting that these mediators may be important to favor the parasite infection in this type of trophoblastic population.

During pregnancy, the placenta regulates the transfer of oxygen, nutrients, and residual products between the maternal and fetal bloodstreams and is a key determinant of fetal exposure to xenobiotics from the mother. To study the disposition of substances through the placenta, various experimental models are used, especially the perfused placenta, placental villi explants, and cell lineage models. In this context, nanotechnology, an area of study that is on the rise, enables the creation of particles on nanometric scales capable of releasing drugs aimed at specific tissues. An important reason for furthering the studies on transplacental transfer is to explore the potential of nanoparticles (NPs), in new delivery strategies for drugs that are specifically aimed at the mother, the placenta, or the fetus and that involve less toxicity. Due to the fact that the placental barrier is essential for the interaction between the maternal and fetal organisms as well as the possibility of NPs being used in the treatment of various pathologies, the aim of this review is to present the main experimental models used in studying the maternal-fetal interaction and the action of NPs in the placental environment.

Toxoplasma gondii is able to infect a wide range of vertebrates, including humans. Studies show that cyclooxygenase-2 (COX-2) is a modulator of immune response in multiple types of infection, such as Trypanosoma cruzi. However, the role of COX-2 during T. gondii infection is still unclear. The aim of this study was to investigate the role of COX-2 during infection by moderately or highly virulent strains of T. gondii in Calomys callosus rodents and human THP-1 cells. C. callosus were infected with 50 cysts of T. gondii (ME49), treated with COX-2 inhibitors (meloxicam or celecoxib) and evaluated to check body weight and morbidity. After 40 days, brain and serum were collected for detection of T. gondii by real-time PCR and immunohistochemistry or cytokines by CBA. Furthermore, peritoneal macrophages or THP-1 cells, infected with RH strain or uninfected, were treated with meloxicam or celecoxib to evaluate the parasite proliferation by colorimetric assay and cytokine production by ELISA. Finally, in order to verify the role of prostaglandin E2 in COX-2 mechanism, THP-1 cells were infected, treated with meloxicam or celecoxib plus PGE2, and analyzed to parasite proliferation and cytokine production. The data showed that body weight and morbidity of the animals changed after infection by T. gondii, under both treatments. Immunohistochemistry and real-time PCR showed a reduction of T. gondii in brains of animals treated with both COX-2 inhibitors. Additionally, it was observed that both COX-2 inhibitors controlled the T. gondii proliferation in peritoneal macrophages and THP-1 cells, and the treatment with PGE2 restored the parasite growth in THP-1 cells blocked to COX-2. In the serum of Calomys, upregulation of pro-inflammatory cytokines was detected, while the supernatants of peritoneal macrophages and THP-1 cells demonstrated significant production of TNF and nitrite, or TNF, nitrite and MIF, respectively, under both COX-2 inhibitors. Finally, PGE2 treatment in THP-1 cells triggered downmodulation of pro-inflammatory mediators and upregulation of IL-8 and IL-10. Thus, COX-2 is an immune mediator involved in the susceptibility to T. gondii regardless of strain or cell types, since inhibition of this enzyme induced control of infection by upregulating important pro-inflammatory mediators against Toxoplasma.

Macrophage migration inhibitory factor (MIF) is a potent pro-inflammatory cytokine, which mediates the regulation of diverse cellular functions. It is produced by extravillous trophoblastic cells and has been found to be involved in the pathogenesis of diseases caused by some protozoa, including Toxoplasma gondii. Previous studies demonstrated the ability of T. gondii to take advantage of MIF action in human trophoblast cells. However, MIF action in T. gondii-infected extravillous trophoblastic cells (HTR8/SVneo cell line) has not been fully investigated. The present study aimed to investigate the role of MIF in T. gondii-infected HTR8/SVneo cells and verify the intracellular signaling pathways triggered by this cytokine. We found that T. gondii increased MIF production by HTR8/SVneo cells, and by contrast, MIF inhibition, by ISO-1, led to a significant decrease in T. gondii proliferation and CD74 expression in HTR8/SVneo cells. Moreover, in infected HTR8/SVneo cells, the addition of recombinant MIF (rMIF) increased CD44 co-receptor expression, ERK1/2 phosphorylation, COX-2 expression, and IL-8 production, which favored T. gondii proliferation. Our findings indicate that T. gondii can use MIF to modulate important factors in HTR8/SVneo cells, being a possible explanation for the higher susceptibility of extravillous trophoblast cells than other trophoblast cell populations.

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.

Toxoplasma gondii (T. gondii) is an intracellular parasite responsible for causing toxoplasmosis. When infection occurs during pregnancy, it can produce severe congenital infection with ocular and neurologic damage to the infant. From the oral infection parasite reaches the intestine, causing inflammatory response, damage in tissue architecture and systemic dissemination. Macrophage migration inhibition factor (MIF) is a cytokine secreted from both immune and non-immune cells, including gut epithelial cells. MIF is described to promote inflammatory responses, to be associated in colitis pathogenesis and also to play role in maintaining the intestinal barrier. The aim of the present study was to evaluate the influence of the pregnancy and MIF deficiency on T. gondii infection in the intestinal microenvironment and to address how these factors can impact on the intestinal architecture and local cytokine profile. For this purpose, small intestine of pregnant and non-pregnant C57BL/6 MIF deficient mice (MIF-/-) and Wild-type (WT) orally infected with 5 cysts of ME-49 strain of T. gondii were collected on day 8th of infection. Intestines were processed for morphological and morphometric analyses, parasite quantification and for cytokines mensuration. Our results showed that the absence of MIF and pregnancy caused an increase in T. gondii infection index. T. gondii immunolocalization demonstrated that segments preferentially infected with T. gondii were duodenum and ileum. The infection caused a reduction in the size of the intestinal villi, whereas, infection associated with pregnancy caused an increase in villi size due to edema caused by the infection. Also, the goblet cell number was increased in the ileum of MIF-/- mice, when compared to the corresponding WT group. Analyses of cytokine production in the small intestine showed that MIF was up regulated in the gut of pregnant WT mice due to infection. Also, infection provoked an intense Th1 response that was more exacerbated in pregnant MIF-/- mice. We also detected that the Th2/Treg response was more pronounced in MIF-/- mice. Altogether, our results demonstrated that pregnancy and MIF deficiency interferes in the balance of the intestinal cytokines and favors a Th1-immflamatory profile, which in turn, impact in the development of pathology caused by T. gondii infection in the intestinal microenvironment.

Background Toxoplasma gondii is a protozoan parasite that causes congenital toxoplasmosis by transplacental transmission. Parasite strains are genetically diverse and disease severity is related to the genotype. In Uberlândia city, Brazil, two virulent strains were isolated: TgChBrUD1 and TgChBrUD2. Congenital toxoplasmosis is more prevalent in South America compared to Europe, and more often associated with severe symptoms, usually as a result of infection with atypical strains. Methods Considering that T. gondii has shown high genetic diversity in Brazil, the effectiveness of traditional treatment may not be the same, as more virulent strains of atypical genotypes may predominate. Thus, the aim of this study were to evaluate the Brazilian strain infection rate in human villous explants and the azithromycin efficacy with regard to the control of these strains compared to traditional therapy. Villi were infected with RH, ME49, TgChBrUD1 or TgChBrUD2 strains and treated with azithromycin, spiramycin or a combination of pyrimethamine plus sulfadiazine. The villous viability was analyzed by LDH assay and morphological analysis. Parasite proliferation, as well as production of cytokines was analyzed by qPCR and ELISA, respectively. Statistical analysis was performed using the GraphPad Prism 5.0. Results The treatments were not toxic and TgChBrUD1 infected villi showed a higher parasite burden compared with others strains. Treatments significantly reduced the intracellular proliferation of T. gondii, regardless of the strain. TgChBrUD1-infected villi produced a larger amount of MIF, IL-6 and TGF-β1 compared with other infected villi. Azithromycin treatment increased MIF production by RH- or TgChBrUD2-infected villi, but in ME49- or TgChBrUD1-infected villi, the MIF production was not altered by treatment. On the other hand, azithromycin treatment induced lower IL-6 production by ME49- or TgChBrUD1-infected villi. Conclusions Azithromycin treatment was effective against T. gondii Brazilian strains compared with conventional treatment. Also, the TgChBrUD1 strain replicated more in villi and modulated important cytokines involved in parasite control, showing that different strains use different strategies to evade the host immune response and ensure their survival.

Migration inhibitory factor (MIF) is a pro-inflammatory cytokine that plays important roles in physiology, pathology, immunology and parasitology, including the control of infection by protozoa parasites such as Toxoplasma gondii. As the MIF function in congenital toxoplasmosis is not fully elucidated yet, the present study brings new insights for T. gondii infection in the absence of MIF based on pregnant C57BL/6MIF-/- mouse models. Pregnant C57BL/6MIF-/- and C57BL/6WT mice were infected with 05 cysts of T. gondii (ME49 strain) on the first day of pregnancy (dop) and were euthanized at 8 dop. Non-pregnant and non-infected females were used as control. Our results demonstrated that MIF-/- mice have more accentuated change in body weight and succumbed to infection first than their WT counterparts. Otherwise, pregnancy outcome was less destructive in MIF-/- mice compared to WT ones, and the former had an increase in the mast cell recruitment and IDO expression and consequently presented less inflammatory cytokine production. Also, MIF receptor (CD74) was upregulated in MIF-/- mice, indicating that a compensatory mechanism may be required in this model of study. The global absence of MIF was associated with attenuation of pathology in congenital toxoplasmosis, but resulted in female death probably because of uncontrolled infection. Altogether, ours results demonstrated that part of the immune response that protects a pregnant female from T. gondii infection, favors fetal damage.

Activities of phospholipases (PLAs) have been linked to pathogenesis in various microorganisms, and implicated in cell invasion and so the interest in these enzymes as potential targets that could contribute to the control of parasite survival and proliferation. Chicken eggs immunized with BnSP-7, a Lys49 phospholipase A

Classical treatment for congenital toxoplasmosis is based on combination of sulfadiazine and pyrimethamine plus folinic acid. Due to teratogenic effects and bone marrow suppression caused by pyrimethamine, the establishment of new therapeutic strategies is indispensable to minimize the side effects and improve the control of infection. Previous studies demonstrated that enrofloxacin and toltrazuril reduced the incidence of Neospora caninum and Toxoplasma gondii infection. The aim of the present study was to evaluate the efficacy of enrofloxacin and toltrazuril in the control of T. gondii infection in human trophoblast cells (BeWo line) and in human villous explants from the third trimester. BeWo cells and villous were treated with several concentrations of enrofloxacin, toltrazuril, sulfadiazine, pyrimethamine, or combination of sulfadiazine+pyrimethamine, and the cellular or tissue viability was verified. Next, BeWo cells were infected by T. gondii (2F1 clone or the ME49 strain), whereas villous samples were only infected by the 2F1 clone. Then, infected cells and villous were treated with all antibiotics and the T. gondii intracellular proliferation as well as the cytokine production were analyzed. Finally, we evaluated the direct effect of enrofloxacin and toltrazuril in tachyzoites to verify possible changes in parasite structure. Enrofloxacin and toltrazuril did not decrease the viability of cells and villous in lower concentrations. Both drugs were able to significantly reduce the parasite intracellular proliferation in BeWo cells and villous explants when compared to untreated conditions. Regardless of the T. gondii strain, BeWo cells infected and treated with enrofloxacin or toltrazuril induced high levels of IL-6 and MIF. In villous explants, enrofloxacin induced high MIF production. Finally, the drugs increased the number of unviable parasites and triggered damage to tachyzoite structure. Taken together, it can be concluded that enrofloxacin and toltrazuril are able to control T. gondii infection in BeWo cells and villous explants, probably by a direct action on the host cells and parasites, which leads to modifications of cytokine release and tachyzoite structure.

Autophagy is a crucial and physiological process for cell survival from yeast to mammals, including protozoan parasites. Toxoplasma gondii, an intracellular parasite, typically exploits autophagic machinery of host cell; however host cell upregulates autophagy to combat the infection. Herein we tested the efficacy of Rottlerin, a natural polyphenol with autophagic promoting properties, against Toxoplasma infection on the chorioncarcinoma-derived cell line BeWo. We found that Rottlerin, at sub-toxic doses, induced morphological and biochemical alterations associated with autophagy and decreased Toxoplasma growth in infected cells. Although autophagy was synergically promoted by Toxoplasma infection in combination with Rottlerin treatment, the use of the autophagy inhibitor chloroquine revealed that Rottlerin anti-parasitic effect was largely autophagy-independent and likely mediated by the converging inhibitory effect of Rottlerin and Toxoplasma in host protein translation, mediated by mTOR inhibition and eIF2α phosphorylation. Both events, which on one hand could explain the additive effect on autophagy induction, on the other hand led to inhibition of protein synthesis, thereby depriving Toxoplasma of metabolically essential components for multiplication. We suggest that modulation of the competition between pathogen requirement and host cell defense might be an attractive, novel therapeutic approach against Toxoplasma infection and encourage the development of Rottlerin-based new therapeutic formulations.

The conventional treatment for toxoplasmosis with pyrimethamine and sulfadiazine shows toxic effects to the host, and it is therefore necessary to search for new drugs. Some studies suggest the use of statins, which inhibit cholesterol synthesis in humans and also the initial processes of isoprenoid biosynthesis in the parasite. Thus, the objective of this study was to evaluate the activity of the statins pravastatin and simvastatin in HeLa cells infected in vitro with the RH strain of T. gondii. HeLa cells (1×105) were infected with T. gondii tachyzoites (5×105) following two different treatment protocols. In the first protocol, T. gondii tachyzoites were pretreated with pravastatin (50 and 100μg/mL) and simvastatin (1.56 and 3.125μg/mL) for 30min prior to infection. In the second, HeLa cells were first infected (5×105) with tachyzoites and subsequently treated with pravastatin and simvastatin for 24h at the concentrations noted above. Initially, we evaluated the cytotoxicity of drugs by the MTT assay, number of tachyzoites adhered to cells, number of infected cells, and viability of tachyzoites by trypan blue exclusion. The supernatant of the cell cultures was collected post-treatment for determination of the pattern of Th1/Th2/Th17 cytokines by cytometric bead array. There was no cytotoxicity to HeLa cells with 50 and 100μg/mL pravastatin and 1.56 and 3.125μg/mL simvastatin. There was no change in the viability of tachyzoites that received pretreatment. Regarding the pre- and post-treatment of the cells with pravastatin and simvastatin alone, there was a reduction in adhesion, invasion and proliferation of cells to T. gondii. As for the production of cytokines, we found that IL-6 and IL-17 were significantly reduced in cells infected with T. gondii and treated with pravastatin and simvastatin, when compared to control. Based on these results, we can infer that pravastatin and simvastatin alone possess antiproliferative effects on tachyzoites forms of T. gondii, giving these drugs new therapeutic uses.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior O tratamento clássico para a toxoplasmose congênita é baseado na combinação de sulfadiazina e pirimetamina mais ácido folínico. Devido aos efeitos teratogênicos e a supressão da medula óssea causados pela pirimetamina, o estabelecimento de novos alvos terapêuticos é indispensável para minimizar os efeitos indesejáveis e melhorar o controle da infecção por T. gondii. Estudos prévios demonstraram que enrofloxacina e toltrazuril são capazes de controlar o parasitismo em infecções por Neospora caninum e Toxoplasma gondii. Dessa forma, o objetivo deste estudo foi avaliar a eficácia de enrofloxacina e toltrazuril no controle da proliferação de Toxoplasma gondii em células trofoblásticas humanas (linhagem BeWo) e em explantes de vilos placentários humanos de terceiro trimestre. Células BeWo e vilos foram tratados com diferentes concentrações de enrofloxacina, toltrazuril, sulfadiazina, pirimetamina ou associação (sulfadiazina + pirimetamina) com o objetivo de avaliar a viabilidade pelo ensaio de MTT ou LDH, respectivamente. Em seguida, células BeWo foram infectadas com as cepas RH (clone 2F1) ou ME-49 de T. gondii, enquanto que os vilos foram infectados somente com a cepa RH. Em seguida, células e vilos foram tratados ou não com os mesmos medicamentos e a proliferação intracelular do parasito foi analisada pelo ensaio da β-galactosidase (para a cepa RH) ou coloração com azul de toluidina (para a cepa ME-49) e o perfil de citocinas foi analisado. Enrofloxacina e toltrazuril não alteraram a viabilidade nas células BeWo e nos vilos quando administrados nas menores concentrações. Além do mais, as drogas diminuíram a proliferação intracelular de T. gondii independentemente da cepa utilizada em células BeWo e em explantes de vilos quando comparado as condições infectadas e não tratadas. Em células BeWo infectadas por RH, enrofloxacina induziu altos níveis de IL-6 e não modificou significativamente a produção de MIF, enquanto que ambas as citocinas IL-6 e MIF foram produzidas por células BeWo infectadas com a cepa ME-49 e tratadas com enrofloxacina e toltrazuril. Adicionalmente, em vilos placentários, enrofloxacina induziu alta produção de MIF. Assim, podemos concluir que enrofloxacina e toltrazuril foram capazes de controlar o parasitismo em células BeWo e em explantes de vilos placentários humanos. Provavelmente este controle do parasitismo mediado por estas drogas ocorra por modulação da resposta imune nas células BeWo e tecidos placentários, mas não exclui a possibilidade de ação direta no parasito, entretanto futuros experimentos devem ser conduzidos para verificar esta hipótese. The classical treatment for congenital toxoplasmosis is based on combination of sulfadiazine and pyrimethamine plus folinic acid. Due to teratogenic effects and bone marrow suppression caused by pyrimethamine, the establishment of new therapeutic targets is indispensable to minimize the undesirable effects and improve the control of the infection. Previous studies demonstrated that enrofloxacin and toltrazuril were able to control the infection triggered by Neospora caninum and Toxoplasma gondii. Therefore, the aim of this present study was evaluate the efficacy of enrofloxacin and toltrazuril in the control of T. gondii proliferation in human trophoblast cells (BeWo lineage) and in human villous explants from third trimester. BeWo cells and villous were treated with several concentrations of enrofloxacin, toltrazuril, sulfadiazine, pyrimethamine or association (sulfadiazine + pyrimethamine) in other to verify their viability by MTT or LDH assay, respectively. Next, BeWo cells were infected with T. gondii RH (2F1 clone) or ME49 strain, whereas villous were infected only with RH strain (2F1 clone), after, both cells and villous were treated or not with the same antibiotics and analyzed to T. gondii intracellular proliferation by beta-galactosidase assay (for RH strain) or blue toluidine staining (for ME49 strain). ELISA was performed in the supernatant to evaluate the cytokine profile. Enrofloxacin and toltrazuril did not change strongly the viability in cells and villous. Furthermore, the drugs decreased the parasite intracellular proliferation regardless T. gondii strain in BeWo cells and villous explants when compared to untreated and infected conditions. In BeWo cells infected by RH, enrofloxacin induced high levels of IL-6 low levels of MIF, while both cytokines were upregulated by enrofloxacin and toltrazuril in BeWo cells infected by ME49 strain. Additionally, in villous explantes, enrofloxacin induced high MIF production. Thus, enrofloxacin and toltrazuril were able to control the parasitism in BeWo cells and villous explants, and probably it occurs by modulation of immune response in these cells or tissues and direct action on parasite, but future experiments are necessary to verify this hypothesis. Dissertação (Mestrado)

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

Here we evaluate the effects of BpLec, a C-type lectin isolated from Bothrops pauloensis snake venom, on Toxoplasma gondii parasitism. BpLec (0.195-12.5 μg/mL) did not interfere with HeLa (host cell) viability by MTT assay, whereas higher doses decreased viability and changed HeLa morphology. In addition, the host cell treatment before infection did not influence adhesion and proliferation indexes. BpLec did not alter T. gondii tachyzoite viability, as carried out by trypan blue exclusion, but decreased both adhesion and parasite replication, when tachyzoites were treated before infection. Galactose (0.4 M) inhibited the BpLec effect on adhesion assays, suggesting that BpLec probably recognize some glycoconjugate from T. gondii membrane. Additionally, we performed cytokine measurements from supernatants collected from HeLa cells infected with T. gondii tachyzoites previously treated with RPMI or BpLec. MIF and IL-6 productions by HeLa cells were increased by BpLec treatment. Also, TGF-β1 secretion was diminished post-infection, although this effect was not dependent on BpLec treatment. Taken together, our results show that BpLec is capable of reducing T. gondii parasitism after tachyzoite treatment and may represent an interesting tool in the search for parasite antigens involved in these processes.

s / Placenta 36 (2015) 469e521 502 Methods: Histological and immunofluorescence analysis of Swiss Webster mouse placentas of 9 to 12 dpc, through serial sections and using different stains and antibodies to evaluate the presence, topography and origin of hematopoietic foci. Results: Only the labyrinth region seemed to have hematopoietic activity. Distinct erythroid cells were observed in fetal vessels consisting of larger acidophilic cells with more spherical shape than conventional erythrocytes probably seeded by yolk sac. These cells were often seen in cell division and attached to others and tomore immature cells in circulation. In some areas, they were surrounded by trophoblasts with no apparent vessels. Close to trophoblast giant cells (TGC), heterogeneous cellswere observed protruding toward fetal vessels, which were suggestive to be derived from trophoblast cells rather than fromtransendothelialmigration.Otherarrangements in the labyrinth showed erythroid cells with different levels of hemoglobinization forming agglomerates that resembled erythroid foci. Hematopoietic clusters similar to those of HSC formed by fetuses’ large vessels were also found near umbilical insertion. Immunostaining deepened the characterization of these arrangements, contributing to phenomena interpretation. Conclusions: The placenta is proposed to have hematopoietic potential producing blood cells by at least two different ways inside the labyrinth region. One might occurs near TGC and seems to be restricted to the erythroid lineage. Trophoblast cells are probably the precursors of these hematopoietic cells through migration into fetal circulation, where they proliferate and differentiate, and through their differentiation inside the trophoblast layer, forming erythropoietic foci. A possible second mechanism would occur near the umbilical cord and seems to be related to definitive hematopoiesis. Furthermore, it seems that the placenta not only generates hematopoietic cells but also promotes their expansion and/or differentiation before the fetal liver stage. PA.46. ENROFLOXACIN AND TOLTRAZURIL ARE ABLE TO CONTROL TOXOPLASMA GONDII INFECTION IN HUMAN TROPHOBLAST CELLS R.J. Silva , A.O. Gomes , J.R. Mineo , N.M. Silva , E.A.V. Ferro , B.F. Barbosa . 1 Laboratory of Reproduction Immunophysiology, Institute of Biomedical Science, Federal University of Uberlândia, Uberlândia, Brazil; 2 Laboratory of Immunoparasitology, Institute of Biomedical Science, Federal University of Uberlândia, Uberlândia, Brazil; 3 Laboratory of Immunopatology, Institute of Biomedical Science, Federal University of Uberlândia, Uberlândia