Macrophage migration inhibitory factor (MIF) and pregnancy may impact the balance of intestinal cytokines and the development of intestinal pathology caused by Toxoplasma gondii infection.

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.

Experimental models of maternal-fetal interface and their potential use for nanotechnology applications.

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.

Brazilian strains of Toxoplasma gondii are controlled by azithromycin and modulate cytokine production in human placental explants.

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-ß1 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.

Azithromycin treatment is able to control the infection by two genotypes of Toxoplasma gondii in human trophoblast BeWo cells.

Trophoblast infection by Toxoplasma gondii plays a pivotal role in the vertical transmission of toxoplasmosis. Here, we investigate whether the antibiotic therapy with azithromycin, spiramycin and sulfadiazine/pyrimethamine are effective to control trophoblast infection by two Brazilian T. gondii genotypes, TgChBrUD1 or TgChBrUD2. Two antibiotic protocols were evaluated, as follow: i) pre-treatment of T. gondii-tachyzoites with selected antibiotics prior trophoblast infection and ii) post-treatment of infected trophoblasts. The infection index/replication and the impact of the antibiotic therapy on the cytokine milieu were characterized. It was observed that TgChBrUD2 infection induced lower infection index/replication as compared to TgChBrUD1. Regardless the therapeutic protocol, azithromycin was more effective to control the trophoblast infection with both genotypes when compared to conventional antibiotics. Azithromycin induced higher IL-12 production in TgChBrUD1-infected cells that may synergize the anti-parasitic effect. In contrast, the effectiveness of azithromycin to control the TgChBrUD2-infection was not associated with the IL-12 production. BeWo-trophoblasts display distinct susceptibility to T. gondii genotypes and the azithromycin treatment showed to be more effective than conventional antibiotics to control the T. gondii infection/replication regardless the parasite genotype.

IL10, TGF beta1, and IFN gamma modulate intracellular signaling pathways and cytokine production to control Toxoplasma gondii infection in BeWo trophoblast cells.

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.

Experimental infection of Calomys callosus with atypical strains of Toxoplasma gondii shows gender differences in severity of infection.

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.

Systematic Review and Meta-Analysis of Congenital Toxoplasmosis Diagnosis: Advances and Challenges.

Objective: To understand how congenital toxoplasmosis (CT) diagnosis has evolved over the years, we performed a systematic review and meta-analysis to summarize the kind of analysis that has been employed for CT diagnosis. Methods: PubMed and Lilacs databases were used in order to access the kind of analysis that has been employed for CT diagnosis in several samples. Our search combined the following combining terms: "congenital toxoplasmosis" or "gestational toxoplasmosis" and "diagnosis" and "blood," "serum," "amniotic fluid," "placenta," or "colostrum." We extracted data on true positive, true negative, false positive, and false negative to generate pooled sensitivity, specificity, and diagnostic odds ratio (DOR). Random-effects models using MetaDTA were used for analysis. Results: Sixty-five articles were included in the study aiming for comparisons (75.4%), diagnosis performance (52.3%), diagnosis improvement (32.3%), or to distinguish acute/chronic infection phases (36.9%). Amniotic fluid (AF) and placenta were used in 36.9% and 10.8% of articles, respectively, targeting parasites and/or T. gondii DNA. Blood was used in 86% of articles for enzymatic assays. Colostrum was used in one article to search for antibodies. In meta-analysis, PCR in AF showed the best performance for CT diagnosis based on the highest summary sensitivity (85.1%) and specificity (99.7%) added to lower magnitude heterogeneity. Conclusion: Most of the assays being researched to diagnose CT are basically the same traditional approaches available for clinical purposes. The range in diagnostic performance and the challenges imposed by CT diagnosis indicate the need to better explore pregnancy samples in search of new possibilities for diagnostic tools. Exploring immunological markers and using bioinformatics tools and T. gondii recombinant antigens should address the research needed for a new generation of diagnostic tools to face these challenges.

Effect of macrophage migration inhibitory factor (MIF) in human placental explants infected with Toxoplasma gondii depends on gestational age.

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-ß1, 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.

Polarisation of human macrophages towards an M1 subtype triggered by an atypical Brazilian strain of Toxoplasma gondii results in a reduction in parasite burden.

Toxoplasma gondii Nicolle et Manceaux, 1909, the etiologic agent of toxoplasmosis, was considered a clonal population with three distinct genetic lineages (I, II and III); however, sequence analysis of different strains has revealed distinct atypical genotypes. Macrophages are essential for immunity against toxoplasmosis and differential cell regulation may affect the course of the disease. In this context, our study aims to investigate the infection by TgChBrUD2, a highly virulent atypical Brazilian strain of T. gondii, on the activation and polarisation of human macrophages. Human macrophage-like cells obtained from THP-1 cells were infected with TgChBrUD2, RH or ME49 strains of T. gondii to evaluate the impact of parasite infection on macrophage polarisation. Our results indicate that the TgChBrUD2 and ME49 strains of T. gondii induced a classic activation of human macrophages, which was confirmed by the high rate of spindle-shaped macrophages, low amount of urea and increase in the levels of nitrite, as well as the down-regulation of M2-markers. In contrast, RH strain promoted an alternative activation of macrophages. The polarisation of human macrophages towards an M1 subtype mediated by TgChBrUD2 and ME49 strains resulted in a low parasite burden, with high levels of IL-6 and MIF. Finally, the M2 subtype triggered by the RH strain culminated in a lower intracellular proliferation index. We concluded that the atypical (TgChBrUD2) and clonal (ME49) strains are able to elicit an M1 subtype, which results in parasitism control, partially explained by the high levels of IL-6 and MIF produced during the infection by these genotypes. In contrast, the clonal (RH) strain promoted a macrophage polarisation towards an M2 subtype, marked by a high parasite burden, with a weak modulation of pro-inflammatory cytokines. Thus, atypical strains can present different mechanisms of pathogenicity and transmissibility compared to clonal strains, as well as they can use distinct strategies to evade the host's immune response and ensure their survival.

Cyclooxygenase (COX)-2 modulates Toxoplasma gondii infection, immune response and lipid droplets formation in human trophoblast cells and villous explants.

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-ß1. 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.

BEWO trophoblast cells and Toxoplasma gondii infection modulate cell death mechanisms in THP-1 monocyte cells by interference in the expression of death receptor and intracellular proteins.

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)-ß1, 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.

Erratum to "Macrophage migration inhibitory factor (MIF) and pregnancy may impact the balance of intestinal cytokines and the development of intestinal pathology caused by Toxoplasma gondii infection" [Cytokine: X 2 (2020) 100034].

[This corrects the article DOI: 10.1016/j.cytox.2020.100034.].

Cyclooxygenase (COX)-2 Inhibitors Reduce Toxoplasma gondii Infection and Upregulate the Pro-inflammatory Immune Response in Calomys callosus Rodents and Human Monocyte Cell Line.

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.

Clinical-epidemiologic aspects of ophidian accidents occurred in Triângulo Mineiro region, Minas Gerais State, Brazil: retrospective case series / Aspectos clínico-epidemiológicos dos acidentes ofídicos ocorridos na região do Triângulo Mineiro, Minas Gerais, Brasil: estudo retrospectivo

Ophidian accidents constitute a serious problem of public health in the tropical countries. In Central and South America, most of the accidents are caused by Bothrops (90.5%), followed by the Crotalus (7.7%), Lachesis (1.4%) and Micrurus (0.4%) genus. The aim of this work was to evaluate clinical-epidemiological aspects of ophidian accidents reported and treated at the Clinical Hospital at Federal University of Uberlândia, in the central region of Brazil. In this study, 641 medical records from January 1999 to December 2013 were analyzed. The results showed that the accidents were more common in the afternoon, from October to April. The major bite occurrence frequency was attributed to the Bothrops (54.76%), followed by Crotalus (30.58%) and Micrurus (1.40%) snakes. Most of the victims were males (80.34%). The main anatomical regions bitten were the lower and upper limbs, 65.67% and 30.58%, respectively. Approximately 80% of the victims were treated in the first 6 hours after the accident.

Os acidentes ofídicos constituem um sério problema de saúde pública em países tropicais. Nas Américas Central e do Sul, a maioria dos acidentes são causados pelo gênero Bothrops (90,5%), seguido por Crotalus (7,7%), Lachesis (1,4%) e Micrurus (0,4%). O objetivo deste trabalho foi avaliar os aspectos clínico-epidemiológicos dos acidentes ofídicos registrados e tratados no Hospital de Clínicas da Universidade Federal de Uberlândia, na região central do Brasil. Neste estudo, foram analisados 641 prontuários médicos de janeiro de 1999 a dezembro de 2013. Os resultados mostraram que os acidentes ofídicos foram mais comuns durante o período da tarde, de outubro a abril. A maior frequência de ocorrência das picadas foi atribuída às serpentes do gênero Bothrops (54,76%), seguido por Crotalus (30,58%) e Micrurus (1,40%). A maioria das vítimas foi do sexo masculino (80.34%). As principais regiões anatômicas acometidas foram os membros inferiores e superiores, 65,67% e 30,58%, respectivamente. Aproximadamente 80% das vítimas foram tratadas nas primeiras 6 horas após o acidente.