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Effect of Macrophage Migration Inhibitory Factor (MIF) in Human Placental Explants Infected with Toxoplasma gondii Depends on Gestational Age
2011; Elsevier BV; Volume: 178; Issue: 6 Linguagem: Inglês
10.1016/j.ajpath.2011.02.005
ISSN1525-2191
AutoresAngélica de Oliveira Gomes, Deise Aparecida de Oliveira Silva, Neide Maria Silva, Bellisa de Freitas Barbosa, Priscila Silva Franco, Mariana Bodini Angeloni, Marise Lopes Fermino, Maria Cristina Roque‐Barreira, Nicoletta Bechi, Luana Paulesu, Maria Célia dos Santos, José Roberto Mineo, Eloísa Amália Vieira Ferro,
Tópico(s)Congenital Anomalies and Fetal Surgery
ResumoBecause macrophage migration inhibitory factor (MIF) is a key cytokine in pregnancy and has a role in inflammatory response and pathogen defense, the objective of the present study was to investigate the effects of MIF in first- and third-trimester human placental explants infected with Toxoplasma gondii. Explants were treated with recombinant MIF, IL-12, interferon-γ, transforming growth factor-β1, or IL-10, followed by infection with T. gondii RH strain tachyzoites. Supernatants of cultured explants were assessed for MIF production. Explants were processed for morphologic analysis, immunohistochemistry, and real-time PCR analysis. Comparison of infected and stimulated explants versus noninfected control explants demonstrated a significant increase in MIF release in first-trimester but not third-trimester explants. Tissue parasitism was higher in third- than in first-trimester explants. Moreover, T. gondii DNA content was lower in first-trimester explants treated with MIF compared with untreated explants. However, in third-trimester explants, MIF stimulus decreased T. gondii DNA content only at the highest concentration of the cytokine. In addition, high expression of MIF receptor was observed in first-trimester placental explants, whereas MIF receptor expression was low in third-trimester explants. In conclusion, MIF was up-regulated and demonstrated to be important for control of T. gondii infection in first-trimester explants, whereas lack of MIF up-regulation in third-trimester placentas may be involved in higher susceptibility to infection at this gestational age. Because macrophage migration inhibitory factor (MIF) is a key cytokine in pregnancy and has a role in inflammatory response and pathogen defense, the objective of the present study was to investigate the effects of MIF in first- and third-trimester human placental explants infected with Toxoplasma gondii. Explants were treated with recombinant MIF, IL-12, interferon-γ, transforming growth factor-β1, or IL-10, followed by infection with T. gondii RH strain tachyzoites. Supernatants of cultured explants were assessed for MIF production. Explants were processed for morphologic analysis, immunohistochemistry, and real-time PCR analysis. Comparison of infected and stimulated explants versus noninfected control explants demonstrated a significant increase in MIF release in first-trimester but not third-trimester explants. Tissue parasitism was higher in third- than in first-trimester explants. Moreover, T. gondii DNA content was lower in first-trimester explants treated with MIF compared with untreated explants. However, in third-trimester explants, MIF stimulus decreased T. gondii DNA content only at the highest concentration of the cytokine. In addition, high expression of MIF receptor was observed in first-trimester placental explants, whereas MIF receptor expression was low in third-trimester explants. In conclusion, MIF was up-regulated and demonstrated to be important for control of T. gondii infection in first-trimester explants, whereas lack of MIF up-regulation in third-trimester placentas may be involved in higher susceptibility to infection at this gestational age. Toxoplasma gondii is a protozoan parasite extremely adapted for infection in humans, which accounts for its ubiquitous distribution and high seroprevalence.1Tenter A.M. Heckeroth A.R. Weiss L.M. Toxoplasma gondii: from animals to humans.Int J Parasitol. 2000; 30: 1217-1258Crossref PubMed Scopus (2587) Google Scholar This parasite has developed mechanisms that make possible a long-lasting parasite-host interaction to ensure its survival without inducing life-threatening disease in the intermediate host.2Lang C. Gross U. Lüder C.G. Subversion of innate and adaptive immune responses by Toxoplasma gondii.Parasitol Res. 2007; 100: 191-203Crossref PubMed Scopus (105) Google Scholar In contrast, the immune system in immunocompetent hosts controls parasite replication through induction of antibody- and cell-mediated immune responses that preclude disease onset; however, it cannot abolish the infection.2Lang C. Gross U. Lüder C.G. 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Genotype of 86 Toxoplasma gondii isolates associated with human congenital toxoplasmosis, and correlation with clinical findings.J Infect Dis. 2002; 186: 684-689Crossref PubMed Scopus (381) Google Scholar The type II strain association with severe congenital toxoplasmosis depends on host-parasite interaction. Disease outcome depends not only on the intrinsic virulence of the infecting strain but also on the host immune response and specific susceptibility to infection.7Maubon D. Ajzenberg D. Brenier-Pinchart M.P. Dardé M.L. Pelloux H. What are the respective host and parasite contributions to toxoplasmosis.Trends Parasitol. 2008; 24: 299-303Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar Successful gestation is associated with nonrejection of paternal antigens by the mother, which is achieved through multiple immunologic mechanisms at the interface between the mother and the fetus. Typical examples include inactivation of natural killer cells through HLA-G expression,8Rouas-Freiss N. Gonçalves R.M. Menier C. Dausset J. Carosella E.D. Direct evidence to support the role of HLA-G in protecting the fetus from maternal uterine natural killer cytolysis.Proc Natl Acad Sci USA. 1997; 94: 11520-11525Crossref PubMed Scopus (616) Google Scholar tryptophan depletion by indoleamine 2,3-dioxygenase,9Munn D.H. Zhou M. Attwood J.T. Bondarev I. Conway S.J. Marshall B. Brown C. Mellor A.L. Prevention of allogeneic fetal rejection by tryptophan catabolism.Science. 1998; 281: 1191-1193Crossref PubMed Scopus (2210) Google Scholar expansion of the regulatoryT-cell subset during pregnancy,10Aluvihare V.R. Kallikourdis M. Betz A.G. Regulatory T cells mediate maternal tolerance to the fetus.Nat Immunol. 2004; 5: 266-271Crossref PubMed Scopus (1352) Google Scholar and maternal shift from a Th1 to a Th 2 immune response.11Krishnan L. Guilbert L.J. Russell A.S. Wegmann T.G. Mosmann T.R. Belosevic M. Pregnancy impairs resistance of C57BL/6 mice to Leishmania major infection and causes decreased antigen-specific IFN response and increased production of Th2 cytokines.J Immunol. 1996; 156: 644-652PubMed Google Scholar Hormones such as progesterone and glucocorticoids have an important role in Th2 immune responses.12Piccinni M.P. Giudizi M.G. Biagiotti R. Beloni L. Giannarini L. Sampognaro S. Parronchi P. Manetti R. Annunziato F. Livi C. Progesterone favors the development of human T helper cells producing Th2-type cytokines and promotes IL-4 production and membrane CD30 expression in established Th1 cell clones.J Immunol. 1995; 155: 128-133Crossref PubMed Google Scholar, 13Miyaura H. Iwata M. Direct and indirect inhibition of Th1 development by progesterone and glucocorticoids.J Immunol. 2002; 168: 1087-1094PubMed Google Scholar However, this type of immune response is not efficient against protozoan parasites, and the mother becomes more susceptible to pathogens such as T. gondii.14Luft B.J. Remington J.S. Effect of pregnancy on resistance to Listeria monocytogenes and Toxoplasma gondii infection in mice.Infect Immun. 1982; 38: 1164-1171PubMed Google Scholar Thus, when the parasitic infection occurs during pregnancy, the maternal body must control the infection and simultaneously create an environment of immune privilege for the fetus. To understand how the immune system can attempt to resolve this paradox, it was hypothesized that a proinflammatory cytokine that is normally expressed in pregnancy15Ietta F. Todros T. Ticconi C. Piccoli E. Zicari A. Piccione E. Paulesu L. Macrophage migration inhibitory factor in human pregnancy and labor.Am J Reprod Immunol. 2002; 48: 404-409Crossref PubMed Scopus (48) Google Scholar and can be induced rather than inhibited in the presence of glucocorticoid hormones16Calandra T. Bernhagen J. Metz C.N. Spiegel L.A. Bacher M. Donnelly T. Cerami A. Bucala R. MIF as a glucocorticoid-induced modulator of cytokine production.Nature. 1995; 377: 68-71Crossref PubMed Scopus (1063) Google Scholar might have an important role in this condition. A cytokine with these characteristics is macrophage migration inhibitory factor (MIF), first described as a factor produced by lymphocytes and associated with inhibition of random macrophage migration during delayed hypersensitivity responses.17David J. Delayed hypersensitivity in vitro: its mediation by cell-free substances formed by lymphoid cell-antigen interaction.Proc Natl Acad Sci USA. 1966; 56: 72-77Crossref PubMed Scopus (1108) Google Scholar, 18Bloom B.R. Bennett B. Mechanism of a reaction in vitro associated with delayed-type hypersensitivity.Science. 1966; 153: 80-82Crossref PubMed Scopus (1295) Google Scholar However, its real importance was neglected for a long time until the interest in MIF was intensified because of its wide range of biological functions. MIF has a multifaceted role in immune responses such as phagocytosis, inflammation, and inhibition of neutrophil apoptosis.19Amano T. Nishihira J. Miki I. Blockade of macrophage migration inhibitory factor (MIF) prevents the antigen-induced response in a murine model of allergic airway inflammation.Inflamm Res. 2007; 56: 24-31Crossref PubMed Scopus (32) Google Scholar, 20Baumann R. Casaulta C. Simon D. Conus S. Yousefi S. Simon H.U. Macrophage migration inhibitory factor delays apoptosis in neutrophils by inhibiting the mitochondria-dependent death pathway.FASEB J. 2003; 17: 2221-2230Crossref PubMed Scopus (111) Google Scholar In addition, it is an important factor in determining the outcome of infections caused by protozoan parasites such as Plasmodium chabaudi,21Martiney J.A. Sherry B. Metz C.N. Espinoza M. Ferrer A.S. Calandra T. Broxmeyer H.E. Bucala R. Macrophage migration inhibitory factor release by macrophages after ingestion of Plasmodium chabaudi-infected erythrocytes: possible role in the pathogenesis of malaria.Infect Immun. 2000; 68: 2259-2267Crossref PubMed Scopus (110) Google Scholar P. falciparum,22Chaisavaneeyakorn S. Lucchi N. Abramowsky C. Othoro C. Chaiyaroj S.C. Shi Y.P. Nahlen B.L. Peterson D.S. Moore J.M. Udhayakumar V. Immunohistological characterization of macrophage migration inhibitory factor expression in Plasmodium falciparum-infected placentas.Infect Immun. 2005; 73: 3287-3293Crossref PubMed Scopus (42) Google Scholar Leishmania major,23Jüttner S. Bernhagen J. Metz C.N. Röllinghoff M. Bucala R. Gessner A. Macrophage migration inhibitory factor induces killing of Leishmania major by macrophages: dependence on reactive nitrogen intermediates and endogenous TNF-alpha.J Immunol. 1998; 161: 2383-2390PubMed Google Scholar Trypanosoma cruzi,24Reyes J.L. Terrazas L.I. Espinoza B. Cruz-Robles D. Soto V. Rivera-Montoya I. Gómez-García L. Snider H. Satoskar A.R. Rodríguez-Sosa M. Macrophage migration inhibitory factor contributes to host defense against acute Trypanosoma cruzi infection.Infect Immun. 2006; 74: 3170-3179Crossref PubMed Scopus (69) Google Scholarand T. gondii,25Ferro E.A. Mineo J.R. Ietta F. Bechi N. Romagnoli R. Silva D.A. Sorda G. Bevilacqua E. Paulesu L.R. Macrophage migration inhibitory factor is up-regulated in human first trimester placenta stimulated by soluble antigen of Toxoplasma gondii, resulting in increased monocyte adhesion on villus explants.Am J Pathol. 2008; 172: 50-58Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 26Flores M. Saavedra R. Bautista R. Viedma R. Tenorio E.P. Leng L. Sánchez Y. Juárez I. Satoskar A.A. Shenoy A.S. Terrazas L.I. Bucala R. Barbi J. Satoskar A.R. Rodriguez-Sosa M. Macrophage migration inhibitory factor (MIF) is critical for the host resistance against Toxoplasma gondii.FASEB J. 2008; 22: 3661-3671Crossref PubMed Scopus (59) Google Scholar, 27Terrazas C.A. Juarez I. Terrazas L.I. Saavedra R. Calleja E.A. Rodriguez-Sosa M. Toxoplasma gondii: impaired maturation and pro-inflammatory response of dendritic cells in MIF-deficient mice favors susceptibility to infection.Exp Parasitol. 2010; 126: 348-358Crossref PubMed Scopus (29) Google Scholar and in helminth (Taenia crassiceps)28Rodríguez-Sosa M. Rosas L.E. David J.R. Bojalil R. Satoskar A.R. Terrazas L.I. Macrophage migration inhibitory factor plays a critical role in mediating protection against the helminth parasite Taenia crassiceps.Infect Immun. 2003; 71: 1247-1254Crossref PubMed Scopus (68) Google Scholar and bacterial (Salmonella typhimurium)29Koebernick H. Grode L. David J.R. Rohde W. Rolph M.S. Mittrücker H.W. Kaufmann S.H. Macrophage migration inhibitory factor (MIF) plays a pivotal role in immunity against Salmonella typhimurium.Proc Natl Acad Sci USA. 2002; 99: 13681-13686Crossref PubMed Scopus (106) Google Scholar infections. A previous study25Ferro E.A. Mineo J.R. Ietta F. Bechi N. Romagnoli R. Silva D.A. Sorda G. Bevilacqua E. Paulesu L.R. Macrophage migration inhibitory factor is up-regulated in human first trimester placenta stimulated by soluble antigen of Toxoplasma gondii, resulting in increased monocyte adhesion on villus explants.Am J Pathol. 2008; 172: 50-58Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar provided evidence that T. gondii–soluble antigen induces production and secretion of MIF by human first-trimester villus explants. The objective of the present study was to investigate the effect of MIF on susceptibility to T. gondii infection in first- and third-trimester human placental explants. Third-trimester placentas (36 to 40 weeks of gestation) were collected after elective cesarean section deliveries, and first-trimester placentas (9 to 12 weeks of gestation) were obtained after authorized termination of pregnancy in women seronegative for T. gondii or other infection. Placental tissues were washed in ice-cold sterile PBS (pH 7.2) and aseptically dissected using a microscope to remove endometrial tissue and fetal membranes up to 1 hour after collection. Terminal chorionic villus containing five to seven free tips per explant was collected as described previously.30Caniggia I. Taylor C.V. Ritchie J.W. Lye S.J. Letarte M. Endoglin regulates trophoblast differentiation along the invasive pathway in human placental villus explants.Endocrinol. 1997; 138: 4977-4988Crossref PubMed Scopus (141) Google Scholar The volume of villus explants was determined as previously described.24Reyes J.L. Terrazas L.I. Espinoza B. Cruz-Robles D. Soto V. Rivera-Montoya I. Gómez-García L. Snider H. Satoskar A.R. Rodríguez-Sosa M. Macrophage migration inhibitory factor contributes to host defense against acute Trypanosoma cruzi infection.Infect Immun. 2006; 74: 3170-3179Crossref PubMed Scopus (69) Google Scholar In brief, 800 μL medium was placed in a pipette and the villus explant was added, which became totally submerged in the medium. The amount of increased volume was assumed as the villus volume. Overall, the volume of villus explants was approximately 10 mm3. Explants were added to a 96-well plate (one per well) and cultured in 150 μL RPMI 1640 medium supplemented with 10% fetal calf serum and antibiotics (complete medium) for 24 hours at 37°C and 5% CO2. The study was approved by the institutional ethics committee. T. gondii RH strain tachyzoites were maintained in Swiss mice via intraperitoneal serial passage at 48-hour intervals.31Mineo J.R. Camargo M.E. Ferreira A.W. Enzyme-linked immunosorbent assay for antibodies to Toxoplasma gondii polysaccharides in human toxoplasmosis.Infect Immun. 1980; 27: 283-287PubMed Google Scholar Mouse peritoneal exudates were harvested in sterile RPMI 1640 medium and washed twice (720 × g for 10 minutes at 4°C) in medium. Tachyzoites were resuspended in medium, counted in a hemocytometric chamber, and used to infect a BeWo trophoblastic cell line (American Type Culture Collection, Manassas, VA). The parasites were maintained by passages in this cell line for posterior infection of placental explants. Villus explants were treated using various concentrations of recombinant MIF (5, 25, and 100 ng/mL), IL-12 (25 ng/mL), interferon-γ (IFN-γ; 25 ng/mL), transforming growth factor-β1 (TGF-β1; 1 and 10 ng/mL), or IL-10 (10 and 25 ng/mL). Alternatively, explants were treated with goat anti-human MIF antibody (10 μg/mL) or goat IgG (10 μg/mL) for 30 minutes to verify the effect of MIF blockage. Nontreated explants served as controls. Control and experimental conditions were conducted in parallel. After 24 hours of incubation with 5% CO2 at 37°C, explants were infected or not with T. gondii tachyzoites (1 × 106 parasites per well) and incubated for 24 hours. Villus explants were then washed with medium and again incubated in the presence or absence of the same stimulus for 24 hours as described. Villus explants were collected for morphologic analysis, immunohistochemistry (IHC) for MIF, MIF receptor (CD74), and T. gondii detection. Infected villus explants were processed using real-time PCR to determine parasite burden. Culture supernatants were collected and stored at −80°C for measurement of MIF and NO. MIF was measured in supernatants from villus explant cultures using a double-antibody sandwich enzyme-linked immunosorbent assay. In brief, plates were coated overnight with capture monoclonal antibody anti-human MIF (R&D Systems Europe Ltd., Abingdon, Oxfordshire, England), blocked, and incubated with samples in duplicate for 2 hours at room temperature. After washing, plates were incubated with biotinylated detection polyclonal antibody anti-human MIF (R&D Systems Europe Ltd.) for 2 hours at room temperature. The assay was developed using streptavidin–horseradish peroxidase (Zymed Laboratories, Inc., South San Francisco, CA), and revealed with 3,3′,5,5′-tetramethylbenzidine (Zymed Laboratories, Inc.). MIF concentration was determined via extrapolation from a standard curve obtained from known concentrations of rMIF cytokine standard (R&D Systems Europe Ltd.). Assay sensitivity was 18 pg/mL. Intra-assay and interassay coefficients of variation were 3.86% and 9.14%, respectively. NO release in supernatants from villus explant cultures was determined using the Griess method.32Green L.C. Wagner D.A. Glogowski J. Skipper P.L. Wishnok J.S. Tannenbaum S.R. Analysis of nitrate, nitrite, and [15N] nitrate in biological fluids.Anal Biochem. 1982; 126: 131-138Crossref PubMed Scopus (11142) Google Scholar In brief, samples were added in triplicate to 96-well plates and mixed 1:1 with 1% sulfanilamide dihydrochloride and 0.1% naphthylenediamide dihydrochloride in 2.5% H3PO4. Absorbance was read in a plate reader at 570 nm, and concentration was determined with reference to a standard curve of sodium nitrite with concentrations ranging from 5 to 200 μmol/L. Frozen villus explants were homogenized in radioimmunoprecipitation assay buffer [50 mmol/L Tris hydrochloride, 150 mmol/L NaCl, 1% (v/v) Triton X-100, 1% (w/v) sodium deoxycholate, and 0.1% (w/v) SDS; pH 7.5] plus protease inhibitor cocktail tablets (Roche Diagnostics GmbH, Mannheim, Germany). The homogenate was centrifuged at 15,000 × g for 15 minutes at 4°C. The supernatants were used for protein content measurement using the Bradford method.33Bradford M.M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.Anal Biochem. 1976; 72: 248-254Crossref PubMed Scopus (225018) Google Scholar Total protein concentration (mg/mL) was used to normalize data of MIF concentration (pg/mL) obtained at enzyme-linked immunosorbent assay, resulting in MIF concentration (pg/mL). To verify the integrity of villus explants, fragments of placental explants were fixed in 10% buffered formalin, dehydrated in increasing alcohol concentrations, and embedded in glycol methacrylate (Historesin; LKB Produkter AB, Stockholm, Sweden), and 2-μm sections were stained using 1% toluidine blue and analyzed at light microscopy. Alternatively, fixed placental explants were embedded in paraffin for IHC. For electron microscopy, explants were fixed in 2.5% glutaraldehyde plus 2% paraformaldehyde in 0.2 mmol/L PBS, dehydrated in acetone, and embedded in Epon 812 resin (Fluka Chemie GmbH, Buchs, Switzerland). Sections were stained with 2% uranile34Watson M.L. Staining of tissue sections for electron microscopy with heavy metals.J Biophys Biochem Cytol. 1958; 4: 475-478Crossref PubMed Scopus (2348) Google Scholar and 0.5% lead citrate35Reynolds E.S. The use of lead citrate at high pH as on electron opaque stain in electron microscopy.J Cell Biol. 1963; 17: 208-212Crossref PubMed Scopus (17969) Google Scholar contrast medium, and analyzed using an electron microscope (Zeiss EM 109; Carl Zeiss AG, Oberkochen, Germany). Paraffin-embedded explant tissues were cut into 4-μm sections. For antigen retrieval, sections were covered with trypsin solution (0.05% trypsin and 0.1% calcium chloride (Sigma-Aldrich Corp., St. Louis, MO) for 30 minutes at 37°C. Explant sections were incubated with 5% acetic acid at room temperature. For MIF detection, explants were treated with 2.5% normal rabbit serum in Tris-buffered saline solution to block nonspecific sites. Explants were incubated with goat polyclonal antibody anti-human MIF (R&D Systems Europe Ltd.). Negative controls were generated via replacement of the primary antibody with normal goat serum. Samples were washed in Tris-buffered saline solution and incubated with biotinylated rabbit anti-goat IgG (Jackson ImmunoResearch Europe, Ltd., Newmarket, Suffolk, England) for 1 hour at 37°C. Alternatively, for CD74 and T. gondii detection, explants were treated with 2.5% normal goat serum in Tris-buffered saline solution to block nonspecific sites. Explants were incubated with mouse anti-human CD74 (eBioscience, Inc., San Diego, CA), or mouse anti–T. gondii serum, respectively, overnight at 4°C. Negative controls were generated by replacement of the primary antibody with normal mouse serum. Samples were washed in Tris-buffered saline solution and incubated with biotinylated goat anti-mouse IgG (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) for 1 hour at 37°C. Amplifications were performed using streptavidin–biotinylated alkaline-phosphatase complex (ABC kit; Vector Laboratories, Inc., Burlington, CA), developed with Fast Red–naphtol (Sigma-Aldrich Corp.), and counterstained using Mayer's hematoxylin. Tissue parasitism was evaluated at IHC and real-time PCR. Infected and nontreated explants from first- and third-trimester gestations were evaluated at IHC. To score the parasite load in placental explants, quantification was performed in five noncontiguous sections 40 μm apart. The number of parasites in a whole villus explant was quantified. For each field, parasite immunolocalization was determined as follows: parasites attached to trophoblasts, parasites inside trophoblasts, and parasites inside the mesenchyme. The sum of parasites was considered the total parasite load per tissue. Because the villi were similar in size, it was possible to quantify approximately 10 microscopic fields per villus (original magnification, ×400). Variations in these values were adjusted to obtain 10 microscopic fields for each villus. All were composed of areas of connective tissue surrounded by trophoblasts. Placental explants previously stimulated and infected with T. gondii tachyzoites were collected for parasite quantification at real-time PCR using the 2−ΔΔCT method.36Bieche I. Parfait B. Le Doussal V. Olivi M. Rio M.C. Lidereau R. Vidaud M. Identification of CGA as a novel estrogen receptor–responsive gene in breast cancer: an outstanding candidate marker to predict the response to endocrine therapy.Cancer Res. 2001; 61: 1652-1658PubMed Google Scholar DNA extraction was performed using lysis buffer (10 mmol/L Tris hydrochloride 5 mmol/L EDTA, 0.2% SDS, and 200 mmol/L NaCl) plus proteinase K (20 mg/mL) and precipitation of DNA with isopropyl alcohol. PCR amplification and analysis were achieved using a sequence detector (ABI Prism 7500; Applied Biosystems, Inc., Foster City, CA). All reactions were performed using SYBR Green PCR Master Mix (Applied Biosystems, Inc.) with a 10-μL volume in each reaction, which contained 25 ng template cDNA, 2.5 pmol of each primer, and 5 μL SYBR Green. The cycles were processed according to the manufacturer′s instructions. Each sample was tested in duplicate, and all quantifications were normalized on the basis of human β-actin. Primers used for PCR amplification corresponded to the T. gondii B1 gene (amplicon 50 pb): forward, 5′-TTCAAGCAGCGTATTGTCGA-3′, and reverse, 5′-CATGAACGGATGCAGTTCCT-3′, and the β-actin human gene (amplicon 100 pb): forward, 5′-AAGGATTCCTATGTGGGCGA-3′, and reverse, 5′-TCCATGTCGTCCCAGTTGGT-3′. T. gondii RH strain tachyzoites (107 parasites) served as positive control of the reaction, and samples of noninfected placental explants as negative control. Data were analyzed using Data Analysis and Technical Graphics software (Origin version 6.0; Microcal Software, Inc., Northampton, MA). First- and third-trimester placental explants previously infected or not with T. gondii tachyzoites were collected for CD74 quantification at real-time PCR. Total RNA was isolated from the tissues using Trizol reagent (Life Technologies Corp., Carlsbad, CA) following the manufacturer's instructions. cDNA synthesis was performed in a final volume of 20 mL using ImProm-II Reverse Transcriptase (Promega Corp., Madison, WI). The reaction mixture contained 4 mg total RNA, 20 pmol oligo dT primer (Life Technologies Corp.), 40 U ribonuclease inhibitor (RNasin; Promega Corp.), 500 mmol/L dNTP mix, and 1 U reverse transcriptase in 1X reverse transcriptase buffer. cDNA was treated with 10 mg RNase (Gibco-BRL, Invitrogen Corp., Carlsbad, CA) and used immediately. PCR amplification and analysis were achieved using a sequence detector (ABI Prism 7500; Applied Biosystems, Inc.). All reactions were performed using SYBR Green PCR Master Mix (Applied Biosystems, Inc.), with a 25-mL volume in each reaction, which contained 2 mL template cDNA, 5 pmol of each primer, and 12.5 mL SYBR Green. Primers used for PCR amplification were β-actin (forward, 5′-AGCTGCGTTTTACACCCTTT-3′, and reverse, 5′-AAGCCATGCCAATGTTGTCT-3′) and CD74 (forward, 5′-CATGGATGACCAACGCGAC-3′, and reverse, 5′-TGTACAGAGCTCCACGGCTG-3′). The relative expression of each gene was obtained using the comparative CT method, and were normalized using β-actin as an endogenous control. For the infected samples, evaluation of 2−ΔΔCT indicates the fold change in gene expression relative to the uninfected control. Data are expressed as mean ± SEM of three independent experiments in triplicate. Differences between the means, from parametric data, were analyzed using one-way analysis of variance and the Bonferroni post hoc test. Alternatively, Student's t-test was used to compare the parasitism index between first- and third-trimester explants. All data were analyzed using commercially available software (PRISM version 4.0; GraphPad Software Inc., San Diego, CA). Differences were considered statistically significant at P < 0.05. To quantify and verify the expression and localization of MIF receptor in first- and third-trimester placental explants, CD74 was evaluated at real-time PCR and IHC. Compared with third-trimester explants, first-trimester placental explants expressed a larger amount of MIF receptor at PCR (Figure 1A). In addition, IHC revealed that the MIF receptor was localized primarily in the syncytiotrophoblast layer and mesenchymal cells such as Hofbauer cells in first-trimester explants (Figure 1B), whereas expression of MIF receptor was observed primarily in the syncytiotrophoblast layer in third-trimester explants (Figure 1C). To verify whether, compared with nontreated and noninfected tissues, placental explants infected with T. gondii tachyzoites or treated with several stimuli could increase MIF production, MIF levels were measured in supernatants from tissue cultures after 24 hours of treatment. Compared with controls, in first-trimester placental explants, MIF release was increased in the presence of T. gondii (P < 0.01) (Figure 2A). Stim
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