RESUMEN
Placental barrier regulates maternal-fetal interchange protecting the baby from damage caused by substances found in the uterine environment or circulating in the vascular system. Organophosphate (OP) pesticides are a paramount group of environmental pollutants used in intensive agriculture for protection against diseases and pests. While many studies have reported an increased risk of pregnancy alterations in pregnant women exposed to OPs, few have analyzed the effects caused by these pesticides in the placenta. Herein, we evaluated the effects of chlorpyrifos (CPF), one of the most widely used OP insecticides, on human placenta using in vitro and ex vivo exposure models. Villous cytotrophoblast cells isolated from normal human term placentas maintained their cell viability, differentiated into syncytiotrophoblast-like structures, and increased the expression of ß-hCG, ABCG2, and P-gp in the presence of CPF at concentrations of 10 to 100µM. The same doses of CPF induced marked changes in chorionic villi samples. Indeed, CPF exposure increased stroma cell apoptosis, altered villi matrix composition, basement membrane thickness, and trophoblastic layer integrity. Histomorphological and ultrastructural alterations are compatible with those found in placentas where maternal-placenta injury is chronic and able to impair the placental barrier function and nutrient transport from mother to the fetus. Our study shows that placental ex vivo exposure to CPF produces tissue alterations and suggest that human placenta is a potential target of CPF toxicity. In addition, it highlights the importance of using different models to assess the effects of a toxic on human placenta.
Asunto(s)
Cloropirifos/toxicidad , Inhibidores de la Colinesterasa/toxicidad , Vellosidades Coriónicas/efectos de los fármacos , Insecticidas/toxicidad , Trofoblastos/efectos de los fármacos , Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/metabolismo , Apoptosis/efectos de los fármacos , Membrana Basal/efectos de los fármacos , Membrana Basal/ultraestructura , Bioensayo , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Gonadotropina Coriónica Humana de Subunidad beta/metabolismo , Vellosidades Coriónicas/metabolismo , Vellosidades Coriónicas/ultraestructura , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Proteínas de Neoplasias/metabolismo , Embarazo , Reproducibilidad de los Resultados , Medición de Riesgo , Células del Estroma/efectos de los fármacos , Células del Estroma/ultraestructura , Factores de Tiempo , Técnicas de Cultivo de Tejidos , Pruebas de Toxicidad/métodos , Trofoblastos/metabolismo , Trofoblastos/ultraestructuraRESUMEN
The transcription factor Krüppel-Like Factor 6 (KLF6) has important roles in cell differentiation, angiogenesis, apoptosis, and proliferation. Furthermore, there is evidence that KLF6 is required for proper placental development. While oxygen is a critical mediator of trophoblast differentiation and function, the involvement of oxygen in the regulation of KLF6 expression remains unexplored. In the present study we examined the expression of KLF6 in placental tissue from uncomplicated and preeclamptic pregnancies, the latter often characterized by an inadequately perfused placenta. We also determined the effect of hypoxia and the involvement of Hypoxia-Inducible Factor 1α (HIF-1α) on the expression of KLF6 in cultured trophoblast cells and placental tissues. Results revealed that villous, interstitial and endovascular extravillous cytotrophoblasts from placentas from normal and preeclamptic pregnancies express KLF6. In addition, KLF6 immunoreactivity was higher in the placental bed of preeclamptic pregnancies than in those of uncomplicated pregnancies. We demonstrated that hypoxia induced an early and transient increase in KLF6 protein levels in HTR8/SVneo extravillous cytotrophoblast cells and in placental explants. Reoxygenation returned KLF6 protein to basal levels. Moreover, hypoxia-induced up-regulation of KLF6 expression was dependent on HIF-1α as revealed by siRNA knockdown in HTR8/SVneo cells. These results indicate that KLF6 may mediate some of the effects of hypoxia in placental development. The regulation of KLF6 protein levels by oxygen has significant implications for understanding its putative role in diseases affected by tissue hypoxia.
Asunto(s)
Hipoxia/metabolismo , Factor 6 Similar a Kruppel/metabolismo , Placenta/metabolismo , Preeclampsia/metabolismo , Trofoblastos/metabolismo , Línea Celular , Femenino , Regulación de la Expresión Génica , Humanos , Placentación/fisiología , Embarazo , Regulación hacia ArribaRESUMEN
A survey of existing data suggests that trophoblast cells produce factors involved in extracellular matrix degradation. In this study, we correlated the expression of cathepsins D and B in the murine ectoplacental cone with the ultrastructural progress of decidual invasion by trophoblast cells. Both proteases were immunolocalized at implantation sites in lysosome-endosome-like compartments of trophoblast giant cells. Cathepsin D, but not cathepsin B, was also detected ultrastructurally in extracellular compartments surrounded by processes of the invading trophoblast containing extracellular matrix components and endometrial cell debris. The expression of cathepsins D and B by trophoblast cells was confirmed by RT-PCR in ectoplacental cones isolated from implantation chambers at gestation day 7.5. Our data addressed a positive relationship between the expression and presence of cathepsin D at the extracellular compartment of the maternal-fetal interface and the invasiveness of the trophoblast during the postimplantation period, suggesting a participation of invading trophoblast cells in the cathepsin D release. Such findings indicate that mouse trophoblast cells might exhibit a proteolytic ability to partake in the decidual invasion process at the maternal-fetal interface.
Asunto(s)
Catepsina B/metabolismo , Catepsina D/metabolismo , Movimiento Celular , Implantación del Embrión , Intercambio Materno-Fetal , Trofoblastos/citología , Trofoblastos/enzimología , Animales , Catepsina B/genética , Catepsina D/genética , Femenino , Inmunohistoquímica , Ratones , Embarazo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Trofoblastos/ultraestructuraRESUMEN
Hyperglycemia occurs in a variety of conditions such as overt diabetes, gestational diabetes and mild hyperglycemia, all of which are generally defined based on the oral glucose tolerance test and glucose profiles. Whereas diabetes has received considerable attention in recent decades, few studies have examined the mechanisms of mild hyperglycemia and its associated disturbances. Mild gestational hyperglycemia is associated with macrosomia and a high risk of perinatal mortality. Morphologically, the placenta of these women is characterized by an increase in the number of terminal villi and capillaries, presumably as part of a compensatory mechanism to maintain homeostasis at the maternal-fetal interface. In this study, we analised the expression of VEGF and its receptors VEGFR-1 (Flt-1) and VEGFR-2 (KDR) in placentas from mildly hyperglycemic women. This expression was compared with that of normoglycemic women and women with gestational and overt diabetes. Immunohistochemistry revealed strong staining for VEGF and VEGFR-2 in vascular and trophoblastic cells of mildly hyperglycemic women, whereas the staining for VEGFR-1 was discrete and limited to the trophoblast. The pattern of VEGF and VEGF-receptor reactivity in placentas from women with overt diabetes was similar to that of normoglycemic women. In women with gestational diabetes, strong staining for VEGFR-1 was observed in vascular and trophoblastic cells whereas VEGF and VEGFR-2 were detected only in the trophoblast. The expression of these proteins was confirmed by western blotting, which revealed the presence of an additional band of 75 kDa. In the decidual compartment, only extravillous trophoblast reacted with all antibodies. Morphological analysis revealed collagen deposition around large arteries in all groups with altered glycemia. These findings indicate a placental response to altered glycemia that could have important consequences for the fetus. The change in the placental VEGF/VEGFR expression ratio in mild hyperglycemia may favor angiogenesis in placental tissue and could explain the hypercapillarization of villi seen in this gestational disturbance.
Asunto(s)
Diabetes Gestacional/metabolismo , Hiperglucemia/metabolismo , Placenta/metabolismo , Factor A de Crecimiento Endotelial Vascular/biosíntesis , Receptor 1 de Factores de Crecimiento Endotelial Vascular/biosíntesis , Receptor 2 de Factores de Crecimiento Endotelial Vascular/biosíntesis , Adulto , Femenino , Humanos , Inmunohistoquímica , Placenta/patología , Embarazo , Embarazo en Diabéticas/metabolismo , Trofoblastos/metabolismoRESUMEN
Enzymes are crucial for the metabolism of macromolecular substrates. In the great majority of cells, most enzymes are constitutive. Nevertheless, inducible enzymes can predominate, determining specialized cell functions. Within this context, histochemistry/immunohistochemistry and biochemistry were used to investigate expression of peroxidase and reduced nicotinamide-adenine dinucleotide phosphate (NADPH)-oxidase, as well as the expression and activity of cathepsin D and acid phosphatase, in trophoblast cells within the endotheliochorial labyrinth and marginal hematoma of the term cat placenta. In the marginal hematoma, elevated Cathepsin D expression and activity was accompanied by erythrophagocytosis. In contrast, acid phosphatase activity was much more intense in the labyrinth, where metabolic exchanges occur. Peroxidase and NAD(P)H-oxidase were predominantly active in trophoblast cells within endosomal vesicles of different placental compartments, indicating that, although reactive oxygen species might participate in endosomal/lysosomal processes, they are not territorially specific or functional markers. These findings highlight differential characteristics of cathepsin D and acid phosphatase activity within each placental compartment, thereby contributing to the comprehension of the territorial role played by the placenta and facilitating future metabolic studies.
Asunto(s)
Fosfatasa Ácida/metabolismo , Catepsina D/metabolismo , Placenta/enzimología , Animales , Gatos , Cesárea/veterinaria , Femenino , Inmunohistoquímica , EmbarazoRESUMEN
The binucleate trophoblast giant cells (BNC) of the water buffalo, Bubalus bubalis, placenta were studied, with emphasis on the synthesis of BNC-specific proteins. Placentomal tissues of 27 water buffalos (2-10 months of pregnancy) were processed for light and electron microscopy. The frequency of BNCs was 20% of the trophoblastic cells in 2-3-month placentas and increased to 27% in the later stages. Ultrastructurally, binucleate cells displayed a prominent granular endoplasmic reticulum and Golgi apparatus, typical of cells involved with protein synthesis and exportation. The buffalo BNCs contained periodic acid-Schiff (PAS)-positive granules and reacted with antisera against bovine placental lactogen, prolactin-related protein-I, and pregnancy-associated glycoproteins. Lectin histochemistry with Dolichos biflorus agglutinin, Vicia villosa agglutinin, and Phaseolus vulgaris leucoagglutinin showed specific staining of BNCs. Different stages of BNC migration and fusion with uterine epithelial cells were observed. Trinucleate feto-maternal hybrid cells were the typical outcome of cell fusions. These cells underwent degeneration, with typical morphological features of apoptosis. The results revealed a strong homology between water buffalo and cattle BNCs concerning cell morphology, protein expression, glycosylation pattern, and characteristics of cell migration and fusion.
Asunto(s)
Búfalos/anatomía & histología , Células Gigantes/diagnóstico por imagen , Placenta/citología , Trofoblastos/ultraestructura , Animales , Bovinos , Fusión Celular , Movimiento Celular , Núcleo Celular/ultraestructura , Células Epiteliales/ultraestructura , Femenino , Células Gigantes/metabolismo , Glicoproteínas/metabolismo , Aparato de Golgi/metabolismo , Aparato de Golgi/ultraestructura , Inmunohistoquímica , Microscopía Electrónica de Transmisión , Placenta/metabolismo , Lactógeno Placentario/metabolismo , Embarazo , Proteínas Gestacionales/metabolismo , Trofoblastos/metabolismo , Trofoblastos/fisiología , UltrasonografíaRESUMEN
Use of serological tests in the diagnosis of infectious diseases in wild animals has several limitations, primarily the difficulty of obtaining species-specific reagents. Wild canids, such as maned wolves (Chrysocyon brachyurus), are highly predisposed to infection by Toxoplasma gondii and, to a lesser extent, to Neospora caninum. The aim of the present study was to evaluate homologous, heterologous, and affinity conjugates in enzyme-linked immunosorbent assays (ELISAs) and indirect fluorescent antibody tests (IFATs) for detecting immunoglobulin (Ig) G antibodies against T. gondii and N. caninum in maned wolves. Serum samples were obtained from 59 captive animals in Brazil and tested by ELISA for T. gondii serology and IFAT for N. caninum serology using 3 different enzymatic and fluorescent conjugates: homologous (guinea pig anti-maned wolf IgG-peroxidase and -fluorescein isothiocyanate [FITC]), heterologous (rabbit anti-dog IgG-peroxidase and -FITC), and affinity (protein A-peroxidase and -FITC). Seropositivity to T. gondii was comparable among the homologous (69.5%), heterologous (74.6%), and affinity (71.2%) enzymatic conjugates. A significant positive correlation was found between the antibody levels determined by the 3 enzymatic conjugates. The highest mean antibody levels (ELISA index = 4.5) were observed with the protein A-peroxidase conjugate. The same seropositivity to N. caninum (8.5%) was found with the homologous and heterologous fluorescent conjugates, but protein A-FITC was not able to detect or confirm any positive samples with homologous or heterologous conjugates. Our results demonstrate that homologous, heterologous, and affinity conjugates might be used in ELISA for serological assays of T. gondii in wild canids, whereas for N. caninum infection, only the homologous or heterologous fluorescent conjugates have been shown to be useful.
Asunto(s)
Anticuerpos Antiprotozoarios/sangre , Canidae/parasitología , Coccidiosis/veterinaria , Neospora/inmunología , Toxoplasma/inmunología , Toxoplasmosis Animal/diagnóstico , Marcadores de Afinidad/normas , Animales , Animales de Zoológico , Brasil/epidemiología , Coccidiosis/diagnóstico , Coccidiosis/epidemiología , Coccidiosis/inmunología , Ensayo de Inmunoadsorción Enzimática/veterinaria , Técnica del Anticuerpo Fluorescente Indirecta/veterinaria , Inmunoglobulina G/sangre , Toxoplasmosis Animal/epidemiología , Toxoplasmosis Animal/inmunologíaRESUMEN
The main purpose of the present study was to investigate the occurrence of antibodies against T. gondii and N. caninum in captive maned wolves from Brazil, considering that little information is available at the literature about infections by these parasites in this wild animal. Serum samples were obtained from 59 maned wolves originated from six zoos and from one ecological reserve of the southeastern and midwestern regions of Brazil. To detect IgG antibodies against T. gondii, an ELISA protocol was used and the results were expressed as ELISA reactivity indexes (EI). Serology for N. caninum was carried out by indirect fluorescent antibody test (IFAT) and cut-off titers were established at 1:25 dilution. From the total of the analyzed samples, 44 (74.6%) were seropositive for T. gondii and only 5 (8.5%) for N. caninum. Seropositivity for T. gondii ranged from 0 to 100% in the seven different origin locals, with rates over 50% among the six zoos, whereas no positivity was found in the samples from ecological reserve. For N. caninum, seroprevalence varied from 0 to 50% in the different locals, with the highest rates also detected in zoos. Seroprevalence for T. gondii was strongly related with age, with rates significantly higher among adult wolves (91.7%) when compared to newborn or young animals. Seropositive samples for N. caninum were found predominantly in adult wolves. For both parasites, seroprevalence did not show a significant distinction in relation to gender. Although seroprevalence for T. gondii was significantly higher when compared to N. caninum in the Brazilian captive maned wolves tested, these findings reflect the great exposure of this species to T. gondii and, in lower extension, to N. caninum. Also, the present study demonstrated for the first time the presence of antibodies to N. caninum in wild life from South America.
Asunto(s)
Anticuerpos Antiprotozoarios/sangre , Coccidiosis/veterinaria , Neospora/inmunología , Toxoplasma/inmunología , Toxoplasmosis Animal/epidemiología , Lobos/parasitología , Factores de Edad , Animales , Animales Salvajes/parasitología , Animales de Zoológico/parasitología , Brasil/epidemiología , Coccidiosis/epidemiología , Ensayo de Inmunoadsorción Enzimática/métodos , Ensayo de Inmunoadsorción Enzimática/veterinaria , Femenino , Técnica del Anticuerpo Fluorescente Indirecta/veterinaria , Masculino , Estudios SeroepidemiológicosRESUMEN
This study analyses the manner in which trophoblast cells adhere to uterine epithelium and the subsequent interactions that contribute to the establishment of epitheliochorial placentation in the alpaca Lama pacos. Specimens at the luteal and follicular phases and at 22, 26, 30 and 45 days of pregnancy (op) were processed for morphological studies. On day 15 op, the blastocysts are completely free within the uterine lumen, with implantation starting around day 20. On days 22 and 26 of gestation, the trophoblast is apposed to the epithelial surface of the uterus, with areas of contact and adhesion by means of complex interdigitation. Implantation sites occur prevalently in the left uterine horn, but an expanded trophoblast also occupies large extensions of the right horn, where the maternofetal interaction shows peculiar areas of apposition. As development continues, attachment areas become more extensive. On days 30 and 45, many secretory granules can be seen in the uterine epithelium, while giant multinucleate cells appear interposed between the remaining trophoblast cells, showing intense alkaline phosphatase activity, deposits containing iron and PAS-positive granules. Placental lactogen hormone is not present within the cytoplasm of the binucleate or multinucleate trophoblast cells. By day 30 of gestation, the trophoblast layer is lined by an extraembryonic connective tissue that by day 45 is well vascularized, thus indicating the starting point of placental formation. Fetal and maternal capillaries indent the epithelium and the trophoblast, narrowing the specialized areas of exchange, which occur along the entire maternofetal interface, characterizing the diffuse nature of this placenta.
Asunto(s)
Camélidos del Nuevo Mundo/anatomía & histología , Camélidos del Nuevo Mundo/embriología , Corion/embriología , Intercambio Materno-Fetal/fisiología , Preñez/fisiología , Trofoblastos/citología , Animales , Corion/citología , Corion/fisiología , Implantación del Embrión/fisiología , Endometrio/citología , Endometrio/fisiología , Femenino , Edad Gestacional , Técnicas para Inmunoenzimas , Lactógeno Placentario/metabolismo , Embarazo , Trofoblastos/fisiologíaRESUMEN
Cryopreservation with storage at very low temperatures is essential to make full use of this technology for both biological and commercial reasons. However, most mammalian cells will die if exposed to these temperatures unless they are exposed to cryoprotectant solutions and cooled and warmed at specific rates. Lowering temperature below 0 degree C introduces the risk of intracellular ice formation, which likely increases rapidly as the temperature falls. Evidence indicates that ice formation during cooling can cause significantly more damage than ice formation during warming. Comparisons of the toxicity of various cryoprotectants indicated that ethylene glycol (EG) is a nontoxic compound for murine and bovine embryos. The 3.6 M EG solution resulted in similar high survival rates when compared with nonfrozen embryos; deleterious effects of high concentrations of EG became apparent at 7.2 M. The use of EG provides a nontoxic method for the rapid and simplified controlled freezing of in vivo bovine compact morulae-early blastocyst, avoiding the risk of injury caused by high concentrations of cryoprotectants usually required for vitrification. However, in vivo embryos used for freezing and thawing require further studies to understand the ultrastructural changes during the freezing procedure with EG as the single cryoprotectant, especially between Holstein and Nelore cows. This paper describes the ultrastructure of bovine compact morulae-early blastocysts derived by in vivo methods from Holstein and Nelore cows to investigate the fresh morphology as well as that after exposure to cryoprotectant and cryopreservation by conventional slow freezing, quick freezing (nitrogen vapor), and vitrification.
Asunto(s)
Blastocisto/ultraestructura , Bovinos/embriología , Bovinos/fisiología , Criopreservación/veterinaria , Crioprotectores/toxicidad , Mórula/ultraestructura , Animales , Blastocisto/efectos de los fármacos , Bovinos/clasificación , Criopreservación/métodos , Embrión de Mamíferos/citología , Embrión de Mamíferos/efectos de los fármacos , Embrión de Mamíferos/ultraestructura , Femenino , Congelación , Masculino , Microscopía Electrónica/veterinaria , Mórula/efectos de los fármacos , Factores de TiempoRESUMEN
The pattern of expression of a variety of placental nitric oxide synthase isoforms has contributed to elucidating the regulatory mechanisms of nitric oxide (NO) synthesis during gestation. The maintenance of vascular tone, attenuation of vasoconstriction, prevention of platelet and leukocyte adhesion to the trophoblast surface, and possible participation in uterine blood flow seem to be the main functions of NO generated at the fetal-maternal interface in humans and mice. Extending this knowledge to other rodent species commonly used as laboratory animals, in this study we focus on NADPH-diaphorase activity and the distribution of nitric oxide synthase isoforms (NOS) in the trophoblast cells of Calomys callosus during different phases of pregnancy. NADPH-diaphorase activity was evaluated cytochemically and the presence of NOS isoforms detected by immunohistochemistry. These techniques were performed on pre- and postimplantation embryos in situ and in vitro, as well as in placentae on d 14 and 18 of pregnancy. Neither NADPH-diaphorase activity nor inducible or endothelial NOS isoforms were found in pre-implanting embryos except after culturing for at least 48 h, when some of the embryonic cells were positive for the diaphorase reaction. On d 6.5 of pregnancy, trophoblast cells showed intense diaphorase activity both in situ and under in vitro conditions. A positive reaction was also found in the different placental trophoblast cells on d 14 and 18 of pregnancy. The inducible NOS (iNOS) isoform, but not the endothelial isoform, was immunodetected in trophoblast cells from the placenta and from postimplantation embryos in situ and under in vitro conditions. These results strongly suggest the production of NO by the iNOS isoform in the trophoblast of Calomys callosus after embryo implantation. The data also emphasise a possible role for the trophoblast in producing and releasing cytotoxic molecules at the fetal-maternal interface.
Asunto(s)
Arvicolinae/metabolismo , Dihidrolipoamida Deshidrogenasa/metabolismo , Implantación del Embrión/fisiología , Isoenzimas/metabolismo , Óxido Nítrico Sintasa/metabolismo , Trofoblastos/enzimología , Animales , Células Cultivadas , Femenino , Edad Gestacional , Histocitoquímica , Inmunohistoquímica , Fagocitosis , Placenta/enzimología , Embarazo , Útero/enzimologíaRESUMEN
While considerable progress has been made in elucidating nitric oxide (NO) regulatory mechanisms in the later stages of gestation, much less is known about its synthesis and role during embryo implantation. Thus, to evaluate the participation of the trophoblast in the production of NO during this phase, this study focused on NADPH-diaphorase activity and the distribution of NO synthase isoforms (NOS) using immunohistochemistry in pre- and postimplantation mouse embryos in situ and in vitro, as well as on NO production itself, measured as total nitrite, in trophoblast culture supernatants (Griess reaction). No NADPH-diaphorase activity was found in preimplanting embryos except after culturing for at least 48 h, when a few trophoblastic giant cells were positive. Conversely, postimplantation trophoblast cells either lodged into the implantation chamber (in situ) or after culturing (in vitro) showed intense NADPH-diaphorase activity. Also in the postimplantation trophoblast, the endothelial and inducible NOS (eNOS and iNOS) isoforms were immunodetected, under both in situ and in vitro conditions, although in different patterns. Extracts of ectoplacental cone also revealed bands of 135 and 130 kDa on SDS-PAGE that reacted with anti-eNOS and anti-iNOS, respectively, on Western blot. Analysis of the culture supernatant demonstrated that the nitrite concentration was 1) proportional to the number of cultured trophoblast cells, 2) almost completely abolished in the presence of N(omega)-nitro-L-arginine methyl ester, and 3) increased 2-fold in cultures stimulated with gamma-interferon. These results strongly suggest the production of NO from constitutive and inducible isoforms of NOS by the implanting mouse trophoblast. They also emphasize the possibility of the participation of these cells in vasodilatation and angiogenesis, and in cytotoxic mechanisms involved in the intense phagocytosis of injured maternal cells, which occur during the implantation process.
Asunto(s)
Implantación del Embrión/fisiología , Óxido Nítrico/biosíntesis , Preñez/metabolismo , Trofoblastos/metabolismo , Animales , Blastocisto/enzimología , Western Blotting , Células Cultivadas , Electroforesis en Gel de Poliacrilamida , Femenino , Inmunohistoquímica , Macrófagos/enzimología , Ratones , NADPH Deshidrogenasa/análisis , NADPH Deshidrogenasa/metabolismo , Óxido Nítrico Sintasa/biosíntesis , Óxido Nítrico Sintasa de Tipo II , Óxido Nítrico Sintasa de Tipo III , Placenta/citología , Placenta/metabolismo , Embarazo , Trofoblastos/enzimologíaRESUMEN
The potential of the RH strain of Toxoplasma gondii to invade trophoblast cells of the cricetid rodent Calomys callosus in a congenital infection in the initial third of pregnancy was investigated in this study using morphological and immunocytochemical approaches. The animals were intraperitoneally inoculated on the 1st day of pregnancy and the infection was observed on day 7. Various numbers of parasites could be observed inside the parasitophorous vacuoles in trophoblastic cells under light and electron microscopy. The trophoblast cells showed characteristics of healthy cells, and no alteration other than parasite vacuoles in their cytoplasm could be detected. Polyclonal or monoclonal anti-T. gondii antibodies (respectively, anti-T. gondii components and the major surface parasite antigen p30) labeled both the parasite surface and parasitophorous vacuole membranes, regardless of the number of parasites inside the compartment. In addition, p30-containing trails were detected in the extracellular matrix surrounding trophoblastic cells similar to those found with other parasites during locomotion and the invasion process. Our results show the ability of T. gondii to infect trophoblast cells during the early blastocyst-endometrial relationship and open new possibilities for more accurate study of the invasion process of this parasite and the role of the trophoblast as an embryo defense barrier.
Asunto(s)
Preñez , Toxoplasmosis/parasitología , Trofoblastos/parasitología , Animales , Antígenos de Protozoos/análisis , Modelos Animales de Enfermedad , Femenino , Masculino , Ratones , Miometrio/citología , Miometrio/parasitología , Embarazo , Proteínas Protozoarias/análisis , Roedores/parasitología , Toxoplasma/inmunología , Toxoplasmosis/patologíaRESUMEN
PROBLEM: To determine whether any blood plasma factor may play a regulatory role in trophoblast phagocytosis in rodent early pregnancy. METHOD OF STUDY: The effects of alloplasma on the phagocytosis of cultured mouse trophoblast cells (TCs) were evaluated using erythrocytes as target cells, in the presence of 10% fresh, normal plasma; 10% heat-inactivated plasma; 10% component 3 (C3)-depleted plasma; or medium alone. The possible activation of C3 complement, the phagocytosis of zymosan bound or unbound to C3b, and immunoreactivity to C3b receptor were also estimated. Phagocytic activity was expressed as the percentage of phagocytic TCs, and as the number of phagosomes/TCs. RESULTS: The use of complement sufficient plasma significantly enhanced the phagocytosis of the TCs while the use of heat-inactivated plasma eliminated the erythrophagocytosis. Very low levels of phagocytic activity were seen when the plasma was C3-complement deficient. Phagocytosis of C3b-bound zymosan was remarkable in comparison to zymosan alone, and immunoreactivity to C3b-receptors was seen on the TCs. CONCLUSION: These results indicate the participation of thermosensitive molecules mediating the phagocytosis of TCs and suggest, as in macrophages, a role for C3-C3b in this process.
Asunto(s)
Activación de Complemento , Complemento C3/fisiología , Fagocitosis , Trofoblastos/inmunología , Animales , Femenino , Inmunohistoquímica , Masculino , Ratones , Ratones Endogámicos AKR , Embarazo , Zimosan/metabolismoRESUMEN
The massive infiltration by polymorphonuclear leukocytes (PMN) soon after skin infection with Leishmania major suggests that PMN could participate in reducing parasite load and controlling the spreading of leishmanial infection. Yet, direct evidence for the participation of PMN in host defense against L. major was lacking. We investigated L. major infection in susceptible and resistant mice treated with the monoclonal (mAb) antibody RB6-8C5 that depletes the population of mature neutrophils and eosinophils. Both BALB/c and C57BL/6 mice depleted of PMN show accelerated parasite spreading and more severe footpad swelling than similarly infected untreated mice. In addition, significant higher parasite numbers were found in the lesion draining lymph nodes from PMN-depleted C57BL/6 mice. Histopathological analysis of the paw confirmed neutrophils containing ingested parasites as the dominant cell type in the infiltrate of the first days after infection and the nearly absolute neutrophil depletion in mAb-treated mice. Our data show the importance of PMN in early control of parasite load and parasitism spreading in cutaneous leishmaniasis.
Asunto(s)
Leishmania major/inmunología , Leishmaniasis Cutánea/inmunología , Neutrófilos/inmunología , Animales , Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Monoclonales/inmunología , Pie/parasitología , Granulocitos/inmunología , Leishmania major/aislamiento & purificación , Leishmaniasis Cutánea/parasitología , Leishmaniasis Cutánea/patología , Recuento de Leucocitos , Ganglios Linfáticos/parasitología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Neutrófilos/parasitologíaRESUMEN
The influence of nitric oxide (NO) on eosinophil infiltration into the airways was investigated in rats actively sensitized with ovalbumin. The animals were treated chronically with the NO synthase inhibitor, N omega-Nitro-L-arginine methyl ester (L-NAME; 75 mumol rat-1 day-1), for 4 weeks. Bronchoalveolar lavage was performed at 6, 24, 48 and 72 h after intratracheal injection of ovalbumin. Intratracheal challenge of the sensitized rats with ovalbumin caused a significant increase in total leucocyte infiltration in bronchoalveolar lavage fluid both 24 and 48 h post-ovalbumin injection. Neutrophils and eosinophils peaked, respectively, at 24 h (29%) and 48 h (30%) in bronchoalveolar lavage fluid whereas the mononuclear cell did not differ significantly from the counts in non-sensitized rats at any time. At both 6 and 24 h post-ovalbumin injection, the chronic treatment of the animals with L-NAME affected neither the total nor the differential leucocyte content. However, at 48 h post-ovalbumin challenge, the total cell count was reduced by approximately 48% in the L-NAME-treated animals and this was associated with a marked inhibition (81%) of the eosinophil influx. Histological examination of the lungs from these animals (48 h post-ovalbumin challenge) also showed a prominent reduction (69.5%; P < 0.05) of the eosinophil infiltration in the respiratory segments. Our results demonstrate that NO plays a pivotal role in the eosinophil infiltration in airways of actively sensitized rats.
Asunto(s)
Asma/fisiopatología , Inhibidores Enzimáticos/farmacología , Eosinófilos/patología , Óxido Nítrico/fisiología , Ovalbúmina/farmacología , Animales , Asma/inducido químicamente , Asma/inmunología , Líquido del Lavado Bronquioalveolar/citología , Líquido del Lavado Bronquioalveolar/inmunología , Inhibidores Enzimáticos/inmunología , Eosinófilos/efectos de los fármacos , Eosinófilos/inmunología , Histocitoquímica , Pulmón/efectos de los fármacos , Pulmón/inmunología , Pulmón/patología , Masculino , NG-Nitroarginina Metil Éster/farmacología , Ovalbúmina/inmunología , Ratas , Ratas WistarRESUMEN
Para a realizaçäo deste estudo foram coletados os estômagos de 36 animais, 28 queixadas e 8 catetos. Através da porçäo torácica da aorta, a artéria celíaca recebeu injeçäo de neoprene-látex 650 corado com o objetivo de preencher ramificaçöes arteriais deste vaso que se dirigiam aos compartimentos do estômago. Em seguida, as peças foram fixadas em soluçäo aquosa a 10 por cento para serem cuidadosamente dissecadas e analisadas. Os resultados mostraram que este órgäo, em ambas as espécies, encontra-se suprido pela artéria celíaca em 100 por cento das observaçöes, sendo que nos queixadas, a trifurcaçäo deste vaso, originando as artérias esplênica, gástrica esquerda e hepática, ocorreu com maior freqüência (71,41 por cento ñ7,5), enquanto nos catetos o referido vaso originou principalmente (80 por cento ñ 14,14) o tronco gastroesplênico e a artéria hepática individual
Asunto(s)
Animales , Masculino , Femenino , Arterias , EstómagoRESUMEN
Phorbol myristate acetate (PMA) and all-trans-retinal (retinal) were evaluated as possible phagocytic stimulators of cultured, post implantation, trophoblast cells. Ectoplacental cones dissected from 7.5 day-old mouse embryos provided the source of trophoblastic cells. Co-cultures were performed using stimulated and non-stimulated trophoblast cells and erythrocytes under standard conditions. Phagocytic activity was expressed as the total number of phagocytic cells per ectoplacental cone, and as phagosomic vacuoles per trophoblast giant cell, either in the presence or absence of the stimulators. Both chemical agents had similar effects, less than 12 h after stimulation, statistically significant numbers of erythrophagosomes appear in the trophoblast giant cells (TGC). These findings demonstrate that TGC, like neutrophils and macrophages, can be activated to phagocytosis by exogenous factors. This enhanced activity may result from the generation and release of reactive oxygen species. In conclusion, our data suggest that, because stimulation was provided, the remarkable in vivo phagocytic activity of the trophoblast can be maintained under in vitro conditions, allowing study of the pathways and regulatory steps involved in this process.
Asunto(s)
Eritrocitos , Fagocitosis , Retinaldehído/farmacología , Acetato de Tetradecanoilforbol/farmacología , Trofoblastos/fisiología , Animales , Núcleo Celular/ultraestructura , Células Cultivadas , Retículo Endoplásmico/ultraestructura , Femenino , Uniones Intercelulares/ultraestructura , Ratones , Microscopía Electrónica , Mitocondrias/ultraestructura , Trofoblastos/efectos de los fármacos , Trofoblastos/ultraestructuraRESUMEN
Ectoplacental cones isolated from embryos on the seventh day of pregnancy were transplanted beneath the hepatic capsule of recipient adult animals to document the morphological patterns of vascular invasion by the trophoblast in the absence of the maternal environment and the influence of its peculiar vasculature. Females, and females and males of Calomys callosus, a cricetid rodent, were used, respectively, as embryo donors and recipient animals. Three to 5 days later, the grafted regions were excised and processed for light and electron microscopy. Invasion of the liver parenchyma by the trophoblast progressed along the vascular beds, associated with gradual phagocytosis of hepatic cells, greatly favoring the morphological characterization of invasive steps exhibited by the trophoblast to access the different kinds of vessels, to trespass the various vascular components and the different levels of the surrounding hepatic parenchyma. It is possible that either in utero during the establishment of embryomaternal circulation in early pregnancy or ex utero under experimental conditions, the trophoblast exhibits similar vascular invasion behavior. In view of this, our findings may contribute to a better understanding of trophoblast cell migration to the maternal blood supply as well as the role of the trophoblast in the establishment of the placental circulation during pregnancy.
Asunto(s)
Transferencia de Embrión , Circulación Hepática , Hígado/cirugía , Trofoblastos/fisiología , Animales , Vasos Sanguíneos/fisiología , Vasos Sanguíneos/ultraestructura , Femenino , Hígado/ultraestructura , Masculino , Roedores , Trofoblastos/ultraestructuraRESUMEN
The capability of the mouse embryo to generate reactive oxygen species (ROS) was examined. Post-implantation embryos were carefully harvested on Day 8 of pregnancy and the production of ROS was quantified using luminol-sensitized chemiluminescence. The embryos were stimulated with either phorbol myristate acetate (PMA) or all-trans-retinal (retinal) and the reaction kinetics were followed over 10 min. ROS secretion was directly proportional to the number of embryos and was suppressed 56% by superoxide dismutase (SOD), 25% by mannitol and as little as 16% by catalase. Embryos deprived of trophoblast showed no light emission suggesting that the source of ROS generation is the trophoblast. Dihydronicotinamide adenine dinucleotide (NADH)-dependent oxidase activity in the plasma membrane of the trophoblast surface was demonstrated by cytochemical methods. The release of ROS into the extracellular medium during the phagocytic process has been related to the cytolytic effect exhibited by these molecules and, perhaps by this means, the trophoblast can play an active role in the phagocytosis of maternal cells during the process of embryo implantation.