A vector for promoter trapping in Bacillus cereus

In view of the accumulating evidence for paracrine mechanisms regulating trophoblast function, we tested the hypothesis that placental macrophages affect trophoblast activity in a paracrine fashion. Trophoblast was isolated from 17 term placentas (-IP). One aliquot of cells was further immunopurified (+IP) using an HLA class I antibody. This increased the proportion of trophoblast (+IP >97%; -IP approximately 70%) as identified by rigorous immunocytochemistry. Most (approximately 70%) non-trophoblast cells in -IP were macrophages. The cells were cultured for 5 days with a daily medium change. In addition, +IP cells from seven placentas were cultured with lipopolysaccharide (LPS)-stimulated or -unstimulated macrophage-conditioned media. The concentrations of lactate, trophoblast-specific hormones, human chorionic gonadotropin-beta (hCG-beta) and human placental lactogen (hPL), of several prostanoids and of endothelin-1 and angiotensin II were determined in the culture media. The accumulated amounts of substances released into the culture media, corrected for the greater proportion of trophoblast in +IP cultures, were on average two- to threefold higher (hCG-beta: 18-fold) in +IP than in -IP, with the exception of endothelin-1,2 (no change), angiotensin II (-70%) and 6-keto-prostaglandin-F1alpha (-40%). [3H]leucine incorporation into the trichloroacetic acid (TCA)-precipitable pool measured on day 5 was twofold higher in +IP than in -IP. Addition of conditioned media reverted these changes. The data demonstrate that placental macrophages in culture affect trophoblast biosynthetic activity in a paracrine fashion. We conclude that macrophages are important regulators of trophoblast activity.     

Uptake and processing of 59Fe-labelled and 125I-labelled rat transferrin by early organogenesis rat conceptuses in vitro

The delivery of iron to the early organogenesis rat embryo has been studied, using 59Fe- and 125I-labelled rat transferrin. Rat conceptuses at 9.5 days postconception were cultured for 27 or 51 h in whole rat serum. Rat transferrin labelled with 59Fe was added for the final 0.1, 0.5, 6, 24 or 48 h of culture. Radioactivity accumulated progressively in both the embryo and the visceral yolk sac. Similar results were obtained when unconjugated 59Fe3+ was added to the rat serum used as culture medium. Both acid-soluble and acid-insoluble 59Fe were substantially present in the embryo and yolk sac after all exposure periods. When conceptuses were cultured in the presence of 125I-labelled rat transferrin, acid-soluble radioactivity was progressively released into the culture medium, but accumulation into the embryo and visceral yolk sac was slight and did not change with duration of exposure to the labelled protein. Similar findings were obtained using 125I-labelled bovine serum albumin. In these experiments, there was a close correspondence between the amount of iron accumulated by the embryo and visceral yolk sac in the final 24 h of a 51-h culture and the amount of transferrin converted into acid-soluble products in the same period. Visceral yolk sacs from 17.5-day pregnant rats were explanted and cultured in the presence of 59Fe-labelled rat transferrin, 125I-labelled rat transferrin or 125I-labelled bovine serum albumin, for periods up to 3 h. Again uptake of 59Fe increased with time of incubation, and the 125I-labelled proteins were digested to acid-soluble products which were released into the culture medium. The results indicate that transferrin delivers iron for incorporation into both the embryo and the visceral yolk sac, and are consistent with a mechanism involving receptor-mediated endocytosis of iron-laden transferrin by the cells of the visceral yolk sac. The transferrin itself appears to be quantitatively degraded, following delivery of iron to the yolk sac cells, a result that differs from findings in other cell types, in which the protein is not degraded but returns to the plasma membrane to participate in further cycles of iron acquisition and delivery.     

The intracellular degradation of 125I-EGF and the release of low-molecular degradation products under conditions of actin cytoskeleton disorganization

The influence of cytochalasin B (CB) on the epidermal growth factor (EGF) intracellular degradation and release of low-molecular products of degradation into the culture medium were investigated using two cell lines, A431 and 3T3. Investigated parameters were registered 3.0-4.5 h after the beginning of 125I-EGF endocytosis at 37 degrees C. With A431 cells it was shown that actin cytoskeleton disorganization significantly reduced the rate of 125I-EGF final degradation: a high amount of 125I-EGF, still retained cell-associated, and a reduced amount of low-molecular degradation products were released into the medium in experiments with CB addition. That is in agreement with our previous results (Barkan et al., 1988), revealing a long-term accumulation of EGF-rhodamine fluorescence in a juxtanuclear area of A431 cells after CB treatment. Nevertheless when similar experiments were performed on Swiss 3T3 cells, previously used as a model for demonstrating the inhibitory effect of CB on DNA synthesis stimulation by EGF (Barkan, Nikol'ski?, 1986), we could not find such a distinct influence of CB on the process of degradation of internalised 125I-EGF.     

Skin Langerhans cells failure to trap bacterial antigen in non-sensitized guinea-pig

We have studied by ultrastructural histo-autoradiography, in a primary immunological response, the behavior of three types of guinea-pig histiocytic cells exposed to 125I-flagellin, lymph node histiocytes, alveolar macrophages and skin Langerhans cells, making use of the experimental model of Nossal et al. (1964). Whereas latero cave lymph node histiocytes exposed to 125I-flagellin by in vivo injection of the labelled antigen into the hind foot of the guinea-pig trap the flagellin as do 20% of alveolar macrophages incubated in vitro in a culture medium containing 125I-flagellin, skin Langerhans cells exposed in vivo (intradermal and hypodermal injection of the antigen) and in vitro, as was done for alveolar macrophages, are never labelled. These results suggest that, despite the fact that it belongs to the mononuclear phagocytic system, the Langerhans cell is not a common essentially phagocytic macrophage but represents a cell lineage involved in more complex immune reactions requiring the cooperation of sensitized lymphocytes.     

Effect of various hormonal preparations on trophoblast growth in tissue culture

The application of different methods of tissue culture, in particular, investigations on trophoblast cells outside the mother's body, opens new possibilities for understanding pathogenesis of premature interruption of pregnancy caused by the hormonal and metabolic disorders. To study the effects of various hormones (Choriogonien, estrone and estradiol) on the growth and development of trophoblast cells the method of tissue culture was applied. 683 histological preparations, obtained after incubating trophoblast tissues with and without hormones were examined. It was established that choriogonine in doses of 5-100 IU/L stimulated growth in trophoblast cells but 200-400 IU/L of hormone did not increase this stimulating effect. Estrone and estradiol added to incubated tissues in doses of 60-120 IU/L brought about no changes or else had a weak effect on development of cultivated tissues. When choriogonine (or estrone) was added to the trophoblast tissues together with blood serum taken from women in danger of premature interruption of pregnancy the negative effect of the serum on the development of the trophoblast cells was prevented.     

Prevention of spinal anesthesia-induced hypotension in the elderly: comparison between preanesthetic administration of crystalloids, colloids, and no prehydration

The expression of tissue transglutaminase (tTG) was studied during the formation of the normal human placenta and in molar pregnancies and choriocarcinoma, in order to correlate its expression with the functional characteristics of the recognized trophoblast cell types. tTG expression was found to be developmentally regulated. Before 6-7 weeks' gestation, only the chorionic villous cytotrophoblast expresses tTG. Thereafter the overlying syncytiotrophoblast becomes positive. tTG expression is gradually downregulated in the intermediate trophoblast cells emerging from the tips of the chorionic villi invading the uterine tissue. In the decidual wall, the intermediate trophoblast does not express tTG, whereas scattered syncytial cells, the placental bed giant cells, express tTG. Villi from complete hydatidiform mole (CHM) show tTG expression in both the cyto- and the syncytiotrophoblast. The intermediate trophoblast cells from CHM show heterogeneous tTG expression, with a majority of negative cells, whereas extravillous syncytia always express tTG. In choriocarcinoma, the tumour cells show heterogeneous tTG expression, with a majority of positive cells. Analysis of tTG protein and mRNA in placental extracts by Western and Northern blotting did not provide evidence for expression of the truncated form of tTG found in some cell types. The regulated expression of tTG in the normal placenta suggests that the enzyme is involved in important trophoblastic functions and may participate in the control of invasion.     

Immunohistochemical identification of the receptor for urokinase plasminogen activator associated with fibrin deposition in normal and ectopic human placenta

The receptor for urokinase plasminogen activator (uPAR) is a key molecule in cell surface-directed plasminogen activation. uPAR binds urokinase plasminogen activator (uPA) and thereby focuses plasminogen activation on the cell surface. Plasmin dissolves fibrin deposits and facilitates cell migration during tissue repair processes by degrading the extracellular matrix. During human implantation and placental development, plasmin is considered important for both trophoblast migration/invasion and for fibrin surveillance. This study examined the expression of uPAR in normal and ectopic human placentae by immunohistochemistry. In first and third trimester normal placentae as well as in tubal ectopic placental tissues, a high uPAR expression was seen in the trophoblast associated with deposits of fibrin-type fibrinoid. Extravillous trophoblast of the basal plate, of the cell islands, and of the cell columns was also positive for uPAR in the first trimester whereas at term the expression of the protein was decreased. Moreover, uPAR immunostaining was observed in decidual cells throughout normal gestation and in endometrial tissues of patients with ectopic pregnancies. These findings suggest that uPAR participates in placental development and in trophoblast invasion particularly in the first trimester of pregnancy and that uPAR is involved in repair mechanisms of the trophoblast and fibrin surveillance.     

Culture of first-trimester and full-term human chorionic villus explants: role of human chorionic gonadotropin and human placental lactogen as a viability index

In long-term cultures of human chorionic villus explants, the viability of the tissue must be controlled to ensure the reliability of functional studies. Ionic levels (pH), gas concentrations (pO2, pCO2) and metabolic markers (glucose, lactate) in the culture medium are often utilized. Analyses of hormone, enzyme and protein levels are also frequently used to estimate viability. The purpose of this study was to evaluate whether in vitro release and immunoreactivity of human chorionic gonadotropin (hCG) and human placental lactogen (hPL) were correlated with the viability of first-trimester and full-term chorionic villus explants as determined by histopathology. Villus explants of first-trimester and full-term pregnancies were incubated in 6-well plates of RPMI medium which was supplemented with 10% fetal calf serum. Incubations were performed for 10 days, and the plates were kept at 37 degrees C under a water-saturated atmosphere containing 5% CO2 and 95% O2. The medium was replaced every day and samples of supernatant were frozen for later testing of hCG (first trimester) or hPL (full term), glucose consumption and lactate production. The tissue was also fixed and embedded for light-microscopic examination and immunocytochemistry. The hCG release remained stable during 6-7 days at a high level before decreasing, whereas hPL release decreased during the first 5-6 days then stabilized at a relatively low level. Only hCG kinetics were significantly different between tissue incubated with and without cycloheximide or iodoacetic acid. Both hCG and hPL immunoreactivity were not significantly different between tissue cultures with, and without, addition of cycloheximide or iodoacetic acid and even with morphological evidence of trophoblast and endothelial necrosis. The immunoreactivity for both hormones remains highly positive when the significant release has stopped, and does not reflect the tissue viability.     

Post-implantation mouse embryos have the capability to generate and release reactive oxygen species

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.     

The ultrastructure of intermediate type trophoblast cell

Transmission electron microscopy was used to clarify the detailed morphology of the "intermediate type" trophoblast cell in normal and tumor issue. 67 normal placental villi specimen, 10 placental bed specimens and 10 malignant mole, 10 hydatidiform mole, 5 choriocarcinoma specimen (the last three types taken before chemotherapy) were examined. Results showed that the transitional type trophoblasts of the placenta were developed from cytotrophoblasts through differentiation and fusion to syncytiotrophoblasts which showed features of maturation and aging, having features of cytotrophoblast nuclei and syncytiotrophoblast cytoplasm. The transitional trophoblast of placental bed showed similar morphology as that of transitional type cells of villi. The morphology of transitional type cells of villi. The morphology of transitional type cells of trophoblastic tumors had both normal morphology and cellular hyperplasia, atypia and features of tumor ultrastructure. The prominent feature was the high electron density of the granules and polymorphic cysts crowded in villi, demonstrating that the morphology of "intermediate type" trophoblasts in placental and tumor tissue are similar, whereas heterotype cellular morphology is present in varying degrees in tumor tissue.     

A role for scavenger receptors in phagocytosis of protein-coated particles in rainbow trout head kidney macrophages

In macrophages of higher vertebrates, Fc receptors and receptors for complement and other serum factors, are generally known to enhance the phagocytic process. In lower vertebrates like salmonid fishes, none of these or other phagocytic receptors have been thoroughly characterized. The purpose of this study was to elucidate to what extent these and other receptors are involved in the process of phagocytosis in rainbow trout (Oncorhynchus mykiss) head kidney macrophages. We used tosyl activated, paramagnetic dynabeads (2.8 microm in diameter), specifically coated with 125I labeled Atlantic salmon (Salmo salar) IgM or bovine serum albumin (BSA) as phagocytic probes. The effect of complement opsonization was also investigated by incubating the beads in serum. Our results indicate that neither the Fc- nor the complement-receptor(s) were important for phagocytosis of these beads. Our data support the idea that scavenger receptors are involved in phagocytosis in rainbow trout head kidney macrophages, as the use of a competitive scavenger receptor ligand extensively decreased degradation of the labeled protein coat on the beads.     

Distribution of type-I interferon-receptors in human first trimester and term placental tissues and on isolated trophoblast cells

PROBLEM: Type-I interferon (IFN) is the protein recognizing pregnancy in ruminants. Although IFN is secreted in early pregnancy, its role is not still clear in other species. Like other cytokines, IFN exerts its biological functions through specific membrane receptors. We have investigated the potential action of IFN in human pregnancy by studying the distribution of the receptors in the human placenta. METHOD: Reactivity to monoclonal antibodies (mAbs) to the type-I IFN-receptor (R) was analyzed by immunohistochemistry in human placental tissues and in cytospins of first trimester trophoblast cells. RESULTS: Type-I IFN-R immunoreactivity was observed mostly in first trimester villous cytotrophoblasts and in the cytotrophoblast cell columns. Trophoblast in the decidua, the epithelium of the uterine glands, and most of the isolated trophoblast cells were also immunoreactive. CONCLUSION: The expression of type-I IFN-R in the highly proliferating and migrating trophoblast suggests that this cytokine has a role in trophoblast growth and invasion.     

Metastasizing placental site trophoblastic tumor. An immunohistochemical and flow cytometric study of two cases

An immunohistochemical and flow cytometric DNA study of two cases of metastasizing placental site trophoblastic tumor are presented. One patient aged 29 died rapidly of widespread metastases despite hysterectomy and multiagent chemotherapy. The patient had a low level of serum HCG. The course was complicated by the presence of a nephrotic syndrome. The other patient aged 34 had a vaginal metastasis and high levels of serum HCG, and was alive without disease for 9 years after hysterectomy and chemotherapy. Histologically, these tumors were characterized by a monomorphic trophoblastic cell population, probably derived from intermediate trophoblast in the placental site. The mitotic rate in these tumors was 2-4/10 high-power fields. Immunohistochemically, many tumor cells contained human placental lactogen and placental alkaline phosphatase. Beta-unit chorionic gonadotropin was present in many cells of the second patient, and only focally in the first. All specimens including the curettaged and metastatic lesions revealed a diploid DNA content in both cases. Neither DNA ploidy nor S-phase fraction was associated with survival of patient. Since predicting the biologic behavior of placental site trophoblastic tumor is very difficult, making a correct diagnosis on endometrial curettings, hysterectomy, and monitoring serum HCG level is essential in patients with this tumor.     

Identification of ''tissue'' transglutaminase binding proteins in neural cells committed to apoptosis

Mice in which the gene that encodes the receptor (R) for leukemia inhibitory factor (LIF) has been deleted show abnormal growth and development of the placenta. This indicates that LIF plays an important role in placental development. The expression of LIF-R and LIF was examined in human trophoblast and decidua using in situ hybridization and immunocytochemistry. LIF-R mRNA and immunoreactivity was localized in villous and extravillous trophoblast throughout pregnancy, and in endothelial cells of the fetal villi. Strong expression of mRNA encoding LIF was detected in decidual leukocytes, which are abundant at the implantation site. Extravillous trophoblast, which invades the maternal decidua, therefore expresses LIF-R as it moves past decidual leukocytes, which express LIF mRNA. The effect of LIF on cultured human trophoblast was examined in vitro. Recombinant human LIF had no effect on [3H]thymidine incorporation by purified extravillous trophoblast, nor on expression of integrins alpha1, alpha5, or beta1 by isolated trophoblast. These results identify fetal endothelial cells and all cells of the trophoblast lineage as targets for the action of LIF in human placenta. Although its effects on trophoblast are not yet clear, LIF appears to mediate interactions between maternal decidual leukocytes and invading trophoblast. LIF may also play a critical role in controlling angiogenesis in the placental villi, since human fetal endothelial cells express LIF-R, and mice lacking a functional LIF receptor gene show altered vascular development in the placenta.     

The occurrence of protein kinase C theta and lambda isoforms in retina of different species

Salmonella species are intracellular facultative pathogens which survive within phagocytic cells such as macrophages and proliferate inside vacuoles of epithelial cells. Early reports suggested that the capacity for surviving within macrophages was due to the inhibitory effect on the phagosome-lysosome fusion event induced by intracellular Salmonella. However, recent cell biology data, obtained both with phagocytic and epithelial cells, have shown that Salmonella-containing phagosomes have large amounts of lysosomal membrane glycoproteins (lgp), major components of the lysosomal membrane. This apparent discrepancy has partly been clarified at least in epithelial cells: the Salmonella-containing phagosome fuses with lgp-rich compartment different from the classical mature lysosome, as they do not contain certain lysosomal enzymes and are not connected with the endocytic route. Therefore, Salmonella seems to use an alternative strategy not merely based on the inhibition of phagosome-lysosome fusion event. This strategy essentially involves acquisition of only certain lysosomal components to form a specialized phagosomal compartment in which to survive or proliferate intracellularly. These observations have also exemplified the potential use of intracellular bacterial pathogens as biological probes to understand normal biological aspects of the eukaryotic cell. The intracellular lifestyle of Salmonella will undoubtedly provide new insights into the process of lysosome biogenesis.     

Determination of 2-hydroxypropyl-gamma-cyclodextrin in plasma of cynomolgus monkeys after oral administration by gas chromatography-mass spectrometry

The presence of hCG beta-core fragment (beta-core) in the human placenta has been controversial. To clarify its presence in the villous tissue, first, we developed an enzyme immunoassay which is highly specific for beta-core. Then, we investigated the presence of beta-core immunoreactivity in the supernatants of placental organ culture and those of primary culture of trophoblasts as well as in the placental extracts at different stages of gestation. The immunoreactivity of beta-core was demonstrated in each sample, and the amount was at least 5% of intact hCG immunoreactivity. Immunohistochemical analysis of the placental tissue showed localization of beta-core immunoreactivity to the syncytiotrophoblast. Western blot analysis of the supernatants of organ culture as well as urine samples of pregnant women showed two major bands with molecular weight of approximately 15000. On the other hand, beta-core immunoreactivity in the pregnant sera ranged from 0.1 to 0.19% of intact hCG immunoreactivity. These results suggest the presence and possible secretion of beta-core fragment in the human placenta, and its immunoreactivity in the serum is likely to be reduced due to its short half life in the maternal blood.