On a particular method of isolation of the human placenta trophoblastic cells.

With the purpose of demonstrating the presence of different types of cells in the syncytial and cytotrophoblast of the human placenta, a new technique of cell isolation was performed by utilizing a light enzymatic digestion and a separation on density gradients. Normal human placentas of the first trimester of pregnancy have been studied. After an accurate and light washing in saline and anticoagulant substances, whole villi have been incubated in a trypsin solution for various periods of time at 40 degrees C in a thermostatic stirrer. Detached cells have been collected, rinsed and separated by means of different density gradients of Percoll (d = 1.038 and 1.080). Three cellular layers have been collected and processed for the studies at light and electron microscopy. The first layer was mostly composed by multinucleated elements with a morphological pattern closely related to the histological characteristics of the syncytiotrophoblast; the second fraction was composed by mononucleated elements with the structural findings of the Langhans' cells; the third layer was represented almost exclusively by blood cells. The obtained results demonstrated the high utility and accuracy of the suggested method of cell isolation.     

Immunobiology of the TAM receptors

Recent studies have revealed that the TAM receptor protein tyrosine kinases--TYRO3, AXL and MER--have pivotal roles in innate immunity. They inhibit inflammation in dendritic cells and macrophages, promote the phagocytosis of apoptotic cells and membranous organelles, and stimulate the maturation of natural killer cells. Each of these phenomena may depend on a cooperative interaction between TAM receptor and cytokine receptor signalling systems. Although its importance was previously unrecognized, TAM signalling promises to have an increasingly prominent role in studies of innate immune regulation.     

Characterization and differentiation of human first trimester placenta trophoblastic cells in culture.

A preparation of highly enriched isolated cytotrophoblasts was obtained from first trimester placenta using dispase incubation of villous tissue at 4 degrees C, followed by a spontaneous cell release at 37 degrees C. After 24 h of culture, 90-95% of the cells were immunostained by anticytokeratin antibody, showing their epithelial characteristic. After 48 h of culture, these cells differentiated into syncytiotrophoblast, as shown by optic and electron microscopic study. The secretion of hCG, and of its free alpha and beta subunits, and the secretion of hPL were studied as a function of cell culture time. While the level of secreted hCG and its free subunits was stable during 72 h of culture, the hPL level was undetectable during the first 48 h of culture, increasing continuously afterwards. Addition of dibutyryl cAMP from the start or after 96 h of cell culture induced an increase of hCG production and of its free subunits and also stimulated the secretion of hPL. This suggests that these cells maintained the capacity to respond to stimuli which increased intracellular cAMP level. Such a cell culture is of interest in further determining the mechanisms of early gestation involved in the differentiation and growth of placental cytotrophoblasts, and in the regulation of their endocrine functions.     

Some new findings about Hofbauer cells in the chorionic villi of the human placenta

The cytological structure of the Hofbauer cells was investigated in human placentas of the first and second trimesters of gestation. These cells are found in the stromal channel system of the chorionic villi core. Their walls, which are supported by collagen fiber bundles, are produced by reticulum cells and fibroblasts. The cytoplasmic processes of the Hofbauer cells are in contact with the walls of the channels without being associated with them by desmosomal complexes. Some of these cells have features in common with macrophages, such as cytoplasmic processes, larger vacuoles, many pinocytotic vesicles and intracytoplasmic granules. This system of vacuoles and vesicles enables micropinocytotic activity and phagocytosis. This type of Hofbauer cell resembles the typical macrophages. These cells may play a role in the regulation of stromal water content, transportation of ions and the flow of interstitial fluid. The most original finding of this study are long tubes observed in some Hofbauer cells and extending between the nucleus and the extracellular ground substance through the cytoplasm. One of these tubular formations resembles a cilium in structure with three limiting membranes and is filled with a slightly electron-dense substance. This type of Hofbauer cell may transport information between the nucleus and the extracellular ground substance by means of these tubular structures.     

Immunobiology of the human placenta: II. Localization of macrophages,in vivo bound IgG and C3

Human placentae ranging in gestational age from less than 4 weeks to full term were studied for the localization of IgGFc receptor bearing cells by the EA adsorption method. Most cells populating the area beneath the syncytiotrophoblast in trophoblastic villi, e.g., the stromal region, adsorbed EA. When cell suspensions of trophoblastic tissue were prepared and the individual cells were rosetted and characterized, most IgGFc receptor bearing cells were determined to be macrophages by morphology, phagocytosis and nonspecific esterase staining. A few of the syncytiotrophoblast cells also were IgGFc receptor positive. When esterase staining was applied to sections of trophoblastic tissue, the pattern of esterase positivity paralleled the pattern of EA adsorption in that most cells in trophoblastic villi were positive while decidual cells were negative. Furthermore, the pattern of IgG bound in vivo as determined by direct immunofluorescence paralleled the EA adsorption and esterase staining patterns. The bound IgG was removed by prolonged washing of the trophoblastic tissue at 37 °C which suggested that most IgG was bound to IgGFc receptors in trophoblastic villi. The results are discussed with respect to the possible functions of large numbers of macrophages located at the interphase between maternal and fetal circulations.     

Opioid receptors of human placental villi modulate acetylcholine release

The human placental villous tissue contains components of the cholinergic system and opioid receptors of the kappa type. In vitro stimulation of the villous tissue releases acetylcholine in organ baths. A selective kappa agonist, ethylketocyclazocine, inhibits the release of acetylcholine. This inhibition is reversed by the antagonist Mr 2266. The antagonist alone stimulates the release of acetylcholine 18-fold over control. These results demonstrate an interaction between the placental opioid receptors and the cholinergic system in a non-neural tissue. The modulation of acetylcholine release by endogenous opioid peptides could be one of the in vivo functions of placental opioid receptors.     

Quantification of tissue fibronectin from terminal villi of placenta.

Tissue fibronectin (TFn) was solubilized from the terminal villi of perfused human placentas by sequential chemical extractions and plasmin digestion. Alternatively, plasmin digestion of intact tissue solubilized all the TFn, which amounted to 1.8-2.9% of the dry weight of the villi. Concomitantly, 69% of the tissue was solubilized. The non-equilibrium competitive e.l.i.s.a. (enzyme-linked immunoabsorbent assay), in which the TFn was immunologically identical with plasma fibronectin (PFn), was used for the quantification of TFn. This study demonstrates that the bulk of TFn can be obtained in a form that can be quantified by e.l.i.s.a. and that TFn is immunologically identical with PFn. Thus the fibronectin molecule is not significantly altered as it is incorporated into the connective-tissue matrix and could exchange with PFn.     

Analysis of ultrashort feedback regulation in human placenta: synthesis and secretion of GnRH by human trophoblastic cells.

BACKGROUND: Gonadotropin-releasing hormone (GnRH) presumably controls placental growth and functions by autocrine/paracrine mechanisms, and is therefore an important part of the neuroendocrine network in human placenta. AIM: Our earlier work had indicated that GnRH was expressed in human placenta; in extension to these findings, we wanted to analyse synthesis and release of GnRH by trophoblastic cells. GnRH-associated peptide, co-linearly synthesised with GnRH, was used as indicator of actual peptide synthesis. METHOD: First, we immunised rabbits with lipopeptides containing partial sequences of GnRH-associated peptide (GAP) and developed antibodies for immunohistochemical staining. Second, we set up a competitive enzyme immunoassay to measure GnRH: Non-biotinylated GnRH, GnRH analogues or trophoblastic cell culture supernatants were used to inhibit binding of biotinylated des-pGlu1-GnRH to a monoclonal anti-GnRH antibody. RESULTS: a) Placental sections stained positive for GAP in the layers of trophoblastic cells. b) GnRH could be detect by a competitive EIA in supernatants of placental cultures in concentrations between 200 and 5 nM. CONCLUSIONS: GnRH is synthesised and released by trophoblastic cells.     

Identification of components of the extracellular matrix in trophoblastic cell columns of human placenta]

The distribution of five components of the extracellular matrix was studied in human placenta (9-12 and 39-40 weeks of gestation) by an indirect immunofluorescence method with polyclonal monospecific antibodies. In trophoblastic cell columns fibronectin, collagen types IV and V formed homogeneous deposits, whereas collagen types I and II comprised small conglomerates and scanty, discrete granules. The origin of these macromolecules was discussed.     

Kappa opioid receptors of human placental villi modulate acetylcholine release

Human placental villus tissue is non-innervated, yet it contains components of the opiate and cholinergic systems. We investigated whether opioids modulate a calcium dependent acetylcholine release from the villus tissue in a manner similar to that demonstrated by the parasympathetic nerve-smooth muscle junction. We reported that the kappa receptor agonist ethylketocyclazocine (EKC) inhibits acetylcholine release, and that the inhibition is reversed by the selective antagonist, Mr2266. Findings reported here substantiate the role of opioids as modulators of acetylcholine release from villus tissue. The nonselective agonist, morphine, also inhibits acetylcholine release. Inhibition caused by morphine is reversed by low concentrations of non-selective antagonists, naloxone and naltrexone. Naloxone at high concentrations potentiates the inhibition of acetylcholine release caused by morphine. In addition, the calcium channel blocker, diltiazem, was found to inhibit the release of acetylcholine. The combination of morphine and diltiazem resulted in a greater inhibition of acetylcholine release than by either alone. These results suggest that opiate cholinergic interactions occur in non-neural tissue with a mechanism similar to that known to occur at certain cholinergic synapses.     

Kinetic and immunologic evidence for the absence of glucose-6-phosphatase in early human chorionic villi and term placenta.

The existence of the enzyme glucose-6-phosphatase (G6Pase) in early and term human placenta was investigated by comparing the characteristics of placental microsomal glucose 6-phosphate (G6P) hydrolytic activity and liver G6Pase. Placental microsomes exhibited similar apparent Km values for G6P and beta-glycerophosphate in intact and deoxycholate-treated microsomes, heat stability at acidic pH, low latency of mannose 6-phosphate hydrolysis, very low activity of pyrophosphate: glucose phosphotransferase, and undetectable [U-14C]G6P transport into the placental microsomes, all of which indicated that specific G6Pase activity does not exist in placenta. Immunological evidence of the absence of both 36.5 kDa and T2 proteins, which represent the G6Pase catalytic protein and the phosphate/pyrophosphate transporter protein, respectively, confirmed that early and term human placenta are devoid of the multicomponent G6Pase enzyme.