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Toxoplasma gondii is an obligate intracellular parasite that can cause variable clinical symptoms or can even be asymptomatic in immunocompetent individuals. More severe symptoms are observed in immunocompromised patients and congenital transmission of the parasite has been reported. The objective of this study was to evaluate the response of peripheral blood mononuclear cells (PBMC) in parturient and non-pregnant women exposed to live tachyzoites of T. gondii strain RH or ME49. PBMC were isolated from parturient and non-pregnant women with negative or positive serology for toxoplasmosis and cultured with live tachyzoites of the two T. gondii strains for 24 h. Next, the cell culture supernatants were collected and levels of CCL2, CCL5, IL-6, IL-10, IL-12, and TNF-α produced by PBMC after tachyzoite exposure were measured. Live tachyzoite forms of T. gondii significantly inhibited the synthesis of CCL2 in seropositive parturient women, whereas a stimulatory effect on CCL5 was observed in seronegative parturient women. Cells from T. gondii-seronegative non-pregnant women produced significantly higher levels of TNF-α and IL-12, demonstrating the proinflammatory profile induced by the presence of the parasite in culture. The results suggest that the immunomodulation seen during pregnancy contributes to the development of an environment that facilitates escape of the parasite from the immune response.

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.

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.

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

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