Is the human trophoblast in vitro an adaptive tissue for immunological influences?

The role of the extra-embryonic organs (e.g. placenta) in the feto-maternal immunological interactions is not clear. Trophoblast cells of human placenta were studied in vitro in relation to their adaptive capacities under the immunological influence of allogeneic leucocytes of pregnant women. It was shown that after an 18-h interaction some trophoblastic cells were still viable, they were rich in glycoproteins and RNA, and elicited a prominent activity of some enzymes. Mitosis was often seen. The authors suppose that the trophoblast is highly adaptable to some immunological influences in vitro.     

Effect of cyclic 3′:5′-AMP derivatives,prostaglandins and related agents on human chorionic gonadotropin secretion in human malignant trophoblast in culture

Summary The secretion of human chorionic gonadotropin (hCG) is stimulated by addition of N⁶, O^2′-dibutyryl cyclic 3′:5′-AMP (dbcAMP) or theophylline to normal term placenta and human malignant trophoblast cells in vitro. To understand better the specificity of this process. malignant trophoblast cultures were incubated with 3′:5′-cyclic AMP (cAMP) derivatives, prostaglandins and other agents for 1 to 3 days, and the secretion of radioimmuno-assayable hCG was measured. Whereas dbcAMP was the most potent agent in stimulating secretio of hCG, the N⁶- and O^2′-monobutyryl derivatives of cAMP and phosphodiesterase inhibitors (theophylline, papaverine, 3-isobutyl-1-methylxanthine) also increased the secretion of the hormone. A slight increase in hCG secretion was observed following addition of adenine. By contrast, butyrate, cAMP, cyclic 3′:5′-GMP (cGMP), dbcBMP, 5′-AMP, adenosine, L-epinephrine and prostaglandins E₁, E₂, F_1α and F_2α were ineffective. Particulate fractions from sonicates of malignant trophoblast cultures contained adenylate cyclase activity which was stimulated more than 10-fold by NaF, but not by either catecholamines or prostaglandins. The relatively specific stimulation of hCG secretion suggested that a regulatory process involving cAMP may have physiological significance in the trophoblast. This investigation was supported by Grant Nos. CA14232 and CA16539 awarded by the National Cancer Institute, DHEW.     

Caffeine reduces 11β-hydroxysteroid dehydrogenase type 2 expression in human trophoblast cells through the adenosine A(2B) receptor

Maternal caffeine consumption is associated with reduced fetal growth, but the underlying molecular mechanisms are unknown. Since there is evidence that decreased placental 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2) is linked to fetal growth restriction, we hypothesized that caffeine may inhibit fetal growth partly through down regulating placental 11β-HSD2. As a first step in examining this hypothesis, we studied the effects of caffeine on placental 11β-HSD2 activity and expression using our established primary human trophoblast cells as an in vitro model system. Given that maternal serum concentrations of paraxanthine (the primary metabolite of caffeine) were greater in women who gave birth to small-for-gestational age infants than to appropriately grown infants, we also studied the effects of paraxanthine. Our main findings were: (1) both caffeine and paraxanthine decreased placental 11β-HSD2 activity, protein and mRNA in a concentration-dependent manner; (2) this inhibitory effect was mediated by the adenosine A(2B) receptor, since siRNA-mediated knockdown of this receptor prevented caffeine- and paraxanthine-induced inhibition of placental 11β-HSD2; and (3) forskolin (an activator of adenyl cyclase and a known stimulator of 11β-HSD2) abrogated the inhibitory effects of both caffeine and paraxanthine, which provides evidence for a functional link between exposure to caffeine and paraxanthine, decreased intracellular levels of cAMP and reduced placental 11β-HSD2. Taken together, these findings reveal that placental 11β-HSD2 is a novel molecular target through which caffeine may adversely affect fetal growth. They also uncover a previously unappreciated role for the adenosine A(2B) receptor signaling in regulating placental 11β-HSD2, and consequently fetal development.     

Ultracytochemical localizations of adenylate cyclase,guanylate cyclase and cyclic 3′,5′-nucleotide phosphodiesterase activity on the trophoblast in the human placenta

Summary Ultracytochemical localizations of cyclic nucleotide-metabolizing enzymes, namely adenylate cyclase (AC), guanylate cyclase (GC) and cyclic 3,5-nucleotide phosphodiesterase (PDE), have been demonstrated in the human term placenta. AC activity was found positive on the basal plasma membrane of the syncytiotrophoblast and on the pinocytotic vesicle of the fetal capillary endothelial cell. GC activity was observed to be strong on the plasma membrane of the microvilli of the syncytiotrophoblast. The cAMP PDE activity was shown positive both on the basal plasma membrane and on the microvillous membrane, while cGMP PDE activity was exclusively confined to the microvilli of the syncytiotrophoblast. These observations suggest that the syncytiotrophoblast plays an important role in the cyclic nucleotide metabolism in the human term placenta and that there might be significant functional differences between its basal plasma membrane and its microvillous membrane.     

Expression of the Thomsen-Friedenreich antigen and of its putative carrier protein mucin 1 in the human placenta and in trophoblast cells in vitro

The Thomsen-Friedenreich (TF) antigen (or, more precisely, epitope Galbeta1-3GalNAcalpha-O-) has been known for a long time as a carcinoma-associated antigen. In normal tissues the occurrence of TF antigen is restricted to a few immunologically privileged areas. Here we report on the identification of the TF epitope and its putative carrier protein mucin 1 (MUC1) in human placental tissue, on isolated trophoblast cells in vitro and on trophoblast tumour cell lines BeWo and Jeg3. Cryosections of placental and decidual tissues of the first, second and third trimester were double stained with monoclonal antibodies directed against the TF epitope (IgM) and against MUC1 (IgG). In the first trimester of pregnancy we found strong expression of TF antigen and MUC1 at the apical side of the syncytiotrophoblast directed towards the maternal blood. This expression was consistent in the second trimester of pregnancy, and to a lesser degree in the third trimester. In addition, we found positive staining for TF antigen and MUC1 on extravillous trophoblast cells in the decidua during the first and second trimester of pregnancy. Trophoblast tumour cells of the cell line BeWo, which form a syncytium in vitro, were also positive for TF antigen and MUC1, whereas Jeg3 cells, which are unable to form a syncytium, expressed only MUC1. Freshly isolated trophoblast cells from first trimester placentas showed strong staining for MUC1; however, only a few of these cells (less than 1%) were positive for TF antigen, and might consist of digested fragments of the syncytium. In summary, TF antigen and MUC1 are expressed by the syncytiotrophoblast at the feto-maternal interface and by extravillous trophoblast cells invading the decidua, whereas villous cytotrophoblast cells in situ as well as freshly isolated trophoblast cells from first trimester placentas only express MUC1 but not TF antigen.     

Comparative study of placental protein 19, human chorionic gonadotrophin and pregnancy-specific β1-glycoprotein as immunohistochemical markers for extravillous trophoblast in pregnancy and trophoblastic disease

Summary PP19, a new placental tissue protein, has 1-1 electrophoretic mobility, a molecular weight of 36 500 and 3.9% carbohydrate. To study immunocytochemical PP19 localization in extravillous trophoblast, we obtained formalin-fixed specimens from extravillous tubal pregnancy at gestational weeks (GW) 7–9 (12 blocks); four early intrauterine pregnancies at GW 7–13 (12 blocks); four late pregnancies at GW 28–38 complicated with intramural uterine myoma, placenta increta and abruptio placenta (8 blocks); four invasive complete moles (9 blocks); and seven primary and metastatic gestational choriocarcinomas (12 blocks). Immunohistochemical staining was done for PP19, pregnancy-specific 1-glycoprotein (SP1) and human chorionic gonadotrophin (hCG) using the indirect-labeled antibody method [purified PP19 (Lot no. 225/242) and antibody against PP19 (Lot no. 632ZA) prepared by H. Bohn, antibodies against hCG (Behringwerke, Marburg, FRG) and SP1 (Dakopatts, Copenhagen, Denmark)]. In both early and late intrauterine pregnancies, the extravillous syncytiotrophoblastic cell (XST) showed positive staining for hCG and SP1 in the cytoplasm, as well as for PP19, which stained more intensively in the nucleus than in the cytoplasm. The three proteins were not seen in the evtravillous cytotrophoblastic cell (XCT) in the trophoblastic cell column and shell. The interstitial cytotrophoblast-like cell (ICT), which infiltrated into the decidua and myometrium, and their blood vessels, was immunoreactively positive for PP19 but negative for hCG and SP1 with the exception of SP1-positive ICT in the myometrium in late pregnancy. XST and ICT in the endosalpinx of tubal pregnancy stained for all three proteins. In invasive complete mole, XST stained for the three proteins, but ICT infiltrating into the decidua and myometrium stained more intensively for PP19 than for either hCG or SP1. XCT did not stain for the three proteins. Staining for the three proteins in gestational choriocarcinoma resembled that in invasive mole. By PP19 staining, XST and ICT infiltrating into surrounding tissue were clearly distinguishable from other cells of similar shape. PP19 staining thus can be a useful histochemical marker in assessing the cell viability of the trophoblastic tumor after chemotherapy.     

Decidual natural-killer-cell interaction with trophoblast: cytolysis or cytokine production?

At the implantation site, the uterine mucosa (decidua) is infiltrated by large numbers of natural killer (NK) cells. These NK cells are in close contact with the invading fetal trophoblast and we have proposed that they might be the effector cells that control the implantation of the allogeneic placenta. Recent characterization of NK cell receptors and their HLA class I ligands has suggested potential mechanisms by which NK cells might interact with trophoblast. However, what happens as a result of this interaction is not clear. The traditional method for investigating NK cell function in vitro is the protection from lysis of target cells by expression of HLA class I antigens. This might not be an accurate reflection of what happens in vivo. Another function of NK cells is the production of cytokines on contact with target cells. This could be an important outcome of the interaction between decidual NK cells and trophoblast. Decidual NK cells are known to produce a variety of cytokines; trophoblast cells express receptors for many of these cytokines, indicating that they can potentially respond. In this way, decidual NK cells have a significant influence on trophoblast behaviour during implantation.     

CUL1 promotes trophoblast cell invasion at the maternal–fetal interface

Human trophoblast progenitor cells differentiate via two distinct pathways, to become the highly invasive extravillous cytotrophoblast (CTB) cells (EVT) or fuse to form syncytiotrophoblast. Inadequate trophoblast differentiation results in poor placenta perfusion, or even complications such as pre-eclampsia (PE). Cullin1 (CUL1), a scaffold protein in cullin-based ubiquitin ligases, plays an important role in early embryonic development. However, the role of CUL1 in trophoblast differentiation during placenta development has not been examined. Here we show that CUL1 was expressed in CTB cells and EVT in the first trimester human placentas by immunohistochemistry. CUL1 siRNA significantly inhibited outgrowth of extravillous explants in vitro, as well as invasion and migration of HTR8/SVneo cells of EVT origin. This inhibition was accompanied by decreased gelatinolytic activities of matrix metalloproteinase (MMP)-9 and increased expression of tissue inhibitors of MMPs (TIMP-1 and -2). Consistently, exogenous CUL1 promoted invasion and migration of HTR8/SVneo cells. Notably, CUL1 was gradually decreased during trophoblast syncytialization and CUL1 siRNA significantly enhanced forskolin-induced fusion of choriocarcinoma BeWo cells. CUL1 protein levels in human pre-eclamptic placental villi were significantly lower as compared to their matched control placentas. Taken together, our results suggest that CUL1 promotes human trophoblast cell invasion and dysregulation of CUL1 expression may be associated with PE.     

Elevated concentration of TNF-α induces trophoblast differentiation in mouse blastocyst outgrowths

Elevated expression of tumour necrosis factor- (TNF-) is associated with adverse pregnancy outcome. This study has examined the expression of TNF- and its receptors (TNF-Rs) by mouse blastocysts and blastocyst outgrowths from day 4 to 9.5 of pregnancy and investigated the effects of elevated TNF- on the inner cell mass (ICM) and trophoblast cells of blastocyst outgrowths. RT-PCR demonstrated TNF- mRNA expression from day 7.5 to 9.5, TNF-R1 from day 6.5 to 9.5 and TNF-R2 from day 5.5 to 7.5 of pregnancy, and in situ hybridisation revealed the trophoblast giant cells (TGCs) of the early placenta as the site of TNF- expression. Day 4 blastocysts were cultured in a physiologically high concentration of TNF- (100 ng/ml) for 72 h to the outgrowth stage and then compared to blastocysts cultured in media alone. TNF--treated blastocyst outgrowths exhibited a significant reduction in ICM cells (mean ± SD 23.90±10.42 vs 9.37±7.45, t-test, P<0.0001) with no significant change in the numbers of trophoblast cells (19.97±8.14 vs 21.73±7.79, t-test, P=0.39). Within the trophoblast cell population, the TNF--treated outgrowths exhibited a significant increase in multinucleated cells (14.10±5.53 vs 6.37±5.80, t-test, P<0.0001) and a corresponding significant decrease in mononucleated cells (5.87±3.60 vs 15.37±5.87, t-test, P<0.0001). In summary, this study describes the expression of TNF- and its receptors during the peri-implantation period in the mouse. It also reports that elevated TNF- restricts ICM proliferation in the blastocyst and changes the ratio of mononucleated to multinucleated trophoblast cells. These findings suggest a mechanism by which increased expression of TNF- during trophoblast differentiation may be detrimental to pregnancy.This work was supported by the National Health and Medical Research Council of Australia     

Paleopediatrics: Or when did human infants really become human?

Modern human children take about twice as long as their closest biological relative, the chimpanzee, to mature. One standard explanation for the evolution of “delayed maturation” at an early stage of human evolution is that it provided the time necessary for immature individuals to learn complex skills, most notably those relating to tool-making abilities. However, after comparing dental maturational profiles of early hominids from South Africa (who apparently did make and use stone tools) (Susman [1994] Science 265:1570–1573) to those of extant humans and chimpanzees, we find no evidence to document an association between “delayed maturation” and tool-making abilities in the early stages of human evolution. This also suggests that the assumed association between prolonged childhood dependency and other behaviors often associated with the advent of tool-making such as cooperative hunting, food sharing, home bases, sexual division of labor, etc., is also suspect. Instead, we must look for other, or additional, selective pressures for the evolution of “delayed maturation,” which may postdate the australopithecine radiation. © 1995 Wiley-Liss, Inc.     

Binding of galectin-1 (gal-1) to the Thomsen–Friedenreich (TF) antigen on trophoblast cells and inhibition of proliferation of trophoblast tumor cells in vitro by gal-1 or an anti-TF antibody

Galectin-1 (gal-1), a member of the mammalian β-galactoside-binding proteins, recognizes preferentially Galβ1-4GlcNAc sequences of several cell surface oligosaccharides. We demonstrate histochemically that the lectin recognizes appropriate glycotopes on the syncytiotrophoblast and extravillous trophoblast layer from second trimester human placenta and on BeWo chorion carcinoma cells. Gal-1 binding to BeWo cells was diminished by the Thomsen–Friedreich (TF)-disaccharide (Galβ1-3GalNAc-) conjugated to polyacrylamide (TF–PAA). Gal-1 also inhibited BeWo cell proliferation in a concentration-dependent manner. Similar antiproliferative effects were also observed with an anti-TF monoclonal antibody (mAb, A78-G/A7). Therefore, we conclude that ligation of Galβ1-4GlcNAc and Galβ1-3GalNAc epitopes on BeWo cells may have regulatory effects on cell proliferation.     

What makes us human?

The sequence of chimpanzee chromosome 22 is starting to help us to define the set of genetic attributes that are unique to humans, but interpreting the biological consequences of these remains a major challenge.     

Meta-operation conflict resolution for human–human interaction in collaborative feature-based CAD systems

Conflicts resolution is one of the key issues in maintaining consistency and in supporting smooth human–human interaction for real-time collaborative systems. This paper presents a novel approach of meta-operation conflict resolution for feature-based collaborative CAD system. Although commutative replicated data type (CRDT) is an emerging technique for conflict resolution, it is not capable of resolving conflicts among meta operations for 3D CAD systems. By defining 3 types of meta operations, this work extends CRDT capability to meta operation conflict resolution from 1D to 3D applications. The paper defines the dependency, casuality, conflict and compatible relations specific for 3D collaborative CAD systems. The conflicts of feature-based operations are automatically detected by tracking topological entity changes with the assistance of a persistent data structure, topological entity structure tree (\(TES\_Tree\)). An efficient commutativity-based confliction combination method is proposed to preserve the design intention of each user in a transparent way and maintains the eventual consistent state of the system. The proposed methods are tested in a prototype system with case studies, time complexity analysis and correctness proof.     

Glycopeptides from human κ-chains

Glycopeptides have been isolated from tryptic digests of kappa-type light chains separated from human myeloma proteins obtained from the serum of two patients, Car and Rai. The glycopeptides are derived from the variable region of the chain in both cases, but from different sections. On the basis of homology it is deduced that glycopeptide from Car, kappaI type, is derived from position 25-31 whereas that from Rai, kappaII type, is from position 62-77, their sequences being respectively Ala-Ser-Gln-Asn-Ile-Ser and Phe-Ser-Gly-Ser-Gly-Ser-Gly(Thr,Asp)Phe-Thr-Leu-Asx-Ile-Ser-Arg. The significance of the results is discussed in connexion with the nature of the attachment site of carbohydrate to protein.     

Crystallization of human α-lactalbumin

Crystals of human milk α-lactalbumin have been grown from ammonium sulfate solutions in the presence of calcium ions at 35 °C. They belong to space group P2₁2₁2, with unit cell dimensions: $\text{a = 33.6}\text{A}\text{?}\text{, b = 69.9}\text{A}\text{?}\text{, c = 47.3}\text{A}\text{?}$ and have a single molecule in the asymmetric unit. The crystals diffract X-rays strongly to Bragg spacings of 2 Å and appear suitable for X-ray structural analysis.