Binding of human IgG and F(ab')2 and Fc fragments to cultured trophoblast cells from human term placenta.

Trophoblast cells, dispersed by trypsin digestion of human term placental villi and purified on Percoll gradient, were maintained in serum-containing medium as monolayer cultures up to 7 days. The initially mononucleated cells, probably cytotrophoblasts, differentiated in culture within 90 h to multinucleated syncytiotrophoblast-like cells. The enigmatic binding of human immunoglobulin G (hIgG) to these cells was studied and compared to the well-known binding of hIgG to cultured human monocytes. Binding of hIgG to cultured trophoblasts at 4 degreesC reached steady state by 0.5-1 h, increased about two- to threefold after 90 h in culture and was linear throughout all concentrations tested (0.00067-132 microM). Fc fragments and even F(ab')2 fragments were found to bind to a similar extent to trophoblasts as the complete hIgG molecules. In contrast, in experiments with cultured monocytes, saturation of hIgG binding could be demonstrated. The binding of complete hIgG molecules and of Fc fragments to monocytes was similar whereas binding of F(ab')2 fragments to monocytes was significantly lower. Our results suggest that, despite morphological and at least partially functional differentiation of trophoblast cells in primary culture, no measurable amounts of functional Fc receptor for monomeric hIgG was expressed.

Sterylsulfatase expression in normal and transformed human placental cells.

Isolated cytotrophoblast cells and choriocarcinoma cell lines are commonly applied in-vitro systems for the study of human placental endocrine function. We tested these normal and transformed placental cells for expression of the enzyme sterylsulfatase which is necessary for the production of free steroids from sulfoconjugated precursors in the placenta as well as in other human tissues, and compared the results with respective data obtained from term placental tissue. Specific sterylsulfatase activity was highest in placental homogenates but was lower by about a factor of 5 to 10 in homogenates of freshly isolated cytotrophoblast or JEG-3 cells and by about a factor of 100 in BeWo cell homogenates; the enzyme activity could not be detected in Jar cells. Sterylsulfatase mRNA levels as analyzed by Northern blotting roughly paralleled the levels of enzyme activity measured in cytotrophoblast, JEG-3, and BeWo cells; in Jar cells, RNA species complementary to the specific probe were clearly detectable but differed by size from the mRNA species found in the other cells. Our results indicate that sterylsulfatase activity is differently expressed in normal and transformed placental cells due to different rates or products of gene transcription in these cells.

Serum concentration and urinary excretion of "classical" estrogens, catecholestrogens and 2-methoxyestrogens in normal human pregnancy.

Catecholestrogens, 2-methoxyestrogens and "classical" estrogens (estrone, estradiol, estriol) were measured simultaneously in serum and urine samples of 220 pregnant women from the 8th week of pregnancy until to delivery. From these data we established the central 0.80 centile intervals as time specified reference intervals for each substance analyzed. Serum and urinary estradiol rise steadily during the progress of pregnancy, whereas estrone, catecholestrogens and 2-methoxyestrogens reach a plateau during the last trimester. These observations support the hypothesis, that the amount of the latter compounds may be regulated by separate mechanisms. The values of concentration and excretion of 2- and 4-substituted estrogens varied widely throughout pregnancy. Even very high or very low concentrations of these substances had no recognizable relation to the outcome of pregnancy. This supports the assumption that catecholestrogens and their methylethers are metabolites without any regulatory function in pregnancy.

[What benefit and harm can be expected from screening and routine examinations and from their omission?]. / Welcher Nutzen und welcher Schaden kann von Screening- und Routineuntersuchungen erwartet werden und von deren Unterlassung?

Benefit and harm of screening and routine tests or their omission are dealt with in four parts. In the first part methods are described to evaluate the diagnostic value of medical testing. The concepts of diagnostic sensitivity, diagnostic specificity, and pre- and posttest probability of a diagnosis are defined. It is then shown how these concepts intercorrelate and how their numerical values can be calculated ("Bayes" theorem"). In consideration of the above mentioned intercorrelations, the second and third parts deal with the diagnostic value of preoperative routine tests from an anaesthesiological viewpoint, and the diagnostic value of other screening and follow-up tests is discussed from a gynaecological point of view. Pre-operative laboratory tests are necessary, and necessary only then, if careful evaluation of patient history and physical examination reveal pathological findings or risk factors. The benefits from regular lab-screening tests and follow-up tests, as recommended to the gynaecologists, are low. This is due to the large share of "healthy" women among the gynaecological patients, as well as the fact that treatment of early detected recurrences shows no demonstrable advantage over treatment of later detected recurrences. In the fourth part, we show that no adverse forensic consequences are to be expected if diagnostic tests are omitted because of demonstrably low diagnostic value. In case of legal procedures against the physician, a medical expert will have to evaluate the diagnostic value of the omitted test objectively from an "ex-ante" point of view, using the methods defined in the first part.(ABSTRACT TRUNCATED AT 250 WORDS)

Human placental sterylsulfatase. Interaction of the isolated enzyme with substrates, products, transition-state analogues, amino-acid modifiers and anion transport inhibitors.

The enzymatic properties of a homogeneous sterylsulfatase preparation isolated from human term placenta were studied. The enzyme exhibited both arylsulfatase and sterylsulfatase activity: it catalysed the hydrolysis of sulfuric acid esters of (in the order of decreasing specific activity) non-steroidal phenols, of a phenolic steroid, and of neutral 3 beta-, 21- and (though at a very low rate) 17 beta-hydroxysteroids. However, among all the substrates tested only the 3-sulfates of phenolic and neutral steroids exhibited high affinity towards the sulfatase. Vitamin D3 sulfate was not hydrolysed by the sterylsulfatase but strongly inhibited its activity. The products of the catalytic reaction, free steroids or phenols as well as the sulfate anion or analogues thereof, likewise interfered with the enzyme's activity. Ki values of unconjugated steroids were ten- to hundredfold higher than Km values of the respective sulfoconjugates. Inorganic sulfate only slightly inhibited the sulfatase activity; its inhibitory potency, however, increased in a time-dependent manner when it was preincubated with the enzyme prior to assay. In contrast to sulfate, the hypothetical transition-state analogues sulfite and vanadate acted as strong inhibitors of the sulfatase activity. According to the results of an analysis of the effect of pH on sterylsulfatase kinetics, enzyme constituents with pK values of approximately 5.8 and 8.0 are involved in a general acid-base catalysed reaction. Treatment of the sulfatase with amino-acid side chain modifying reagents directed against arginine, cysteine, cystine, serine or tyrosine residues did not result in significant alteration of its activity. Diethyl-pyrocarbonate known to react primarily with histidyl groups, however, rapidly inactivated the enzyme; this inactivation reaction was markedly retarded in the presence of substrate. Histidine thus appears to be essential for the catalytic activity of the sulfatase. Taken together, the present results reveal a considerable similarity between the catalytic mechanism of human placental sterylsulfatase and the ones already proposed for the lysosomal arylsulfatases A and B. Taurocholate, salicylate, ouabain, and 4,4'-substituted stilbene-2,2'-disulfonates are well known inhibitors of carrier-mediated transport of anions across cellular membranes. With the exception of ouabain, these compounds likewise turned out to inhibit the enzymatic hydrolysis of steryl sulfates; the pattern of dose dependences of their interference with the sulfatase activity resembles the one reported for inhibition of anion transport. Since the sterylsulfatase in vivo strongly is associated with cellular membranes including the plasma membrane of the syncytiotrophoblast, this finding supports the speculation that similar molecular structures may be involved in both placental transport and hydrolysis of anionic steryl sulfates.

Urinary excretion of catecholestrogens, 2-methoxy-estrogens and "classical estrogens" throughout the normal menstrual cycle.

Catecholestrogens, 2-methoxyestrogens and "classical" estrogens (estrone, estradiol, estriol) were measured simultaneously in urine samples of healthy women during the menstrual cycle. All estrogen values reach a preovulatory maximum at the time of the LH peak and show a marked increase during the luteal phase as compared to the follicular phase. Catecholestrogens and estrone seem to behave similarly supporting the assumption that catecholestrogens are predominantly metabolites of estrone. Daily measurements of urinary estrogens show large interindividual variations of 2- and 4-hydroxyestrogens as well as very differing 2-hydroxy-/2-methoxyestrogen ratios. The results obtained support the assumption, that catecholestrogens and their methylethers are excretory products with no additional regulatory functions.

Human placental sterylsulfatase: immunocytochemical and biochemical localization.

Human placental sterylsulfatase was localised in situ by light and electron microscope immunocytochemical techniques as well as in homogenate and tissue extract fractions by enzyme assays. Light microscope observations on frozen sections of term and preterm placenta revealed sterylsulfatase immunoactivity primarily in the syncytiotrophoblast. Electron microscope observations confirmed the light microscope findings; in addition, they showed that the sulfatase is present in the endoplasmic reticulum of endothelial cells, too. In the syncytiotrophoblast, the enzyme was detectable in the cytoplasmic membrane of the nuclear evelope, in the membranes of the rough endoplasmic reticulum, in the plasma membrane with predominant localisation in coated pits, and in the membranes of endosomes and multivesicular bodies; little or no reactivity was detectable over the membranes of the Golgi complex and of lysosomes. Sterylsulfatase immunoactivity was absent in placentas with hereditary sterylsulfatase deficiency. The observations indicate that human placental sterylsulfatase is normally present in the membranes of compartments along the secretory pathway and the endocytic route of cells lining the fetal and maternal blood. Homogenates of normal term placenta as well as membrane vesicle preparations obtained by extraction of trophoblast tissue with isotonic saline were fractionated by differential centrifugation; the fractions were assayed for specific activities of sterylsulfatase and several marker enzymes of cellular topography. In agreement with our immunocytochemical findings, the results of these biochemical localisation experiments indicate the repeatedly described association of the placental sterylsulfatase with microsomal membranes but also point to the presence of the enzyme's activity in the microvillous plasma membrane of the syncytiotrophoblast. This localisation of sterylsulfatase may have functional implications in the placental uptake of circulating steroid sulfates.

2- and 4-iodinated estriol as indicator ligands for estriol radioimmunoassays with anti-estriol-C6 conjugate antiserum.

The suitability of 2- and 4-125I-estriol as indicator ligands for estriol determination in radioimmunoassays with anti-estriol-C6 conjugate antiserum was tested and compared to the one of 3H-estriol, estriol-6-(O-carboxymethyl)oxime-125I-histamine and a commercially available 125I-estriol derivative of unknown structure. An ideal radioiodinated tracer would react identically with the analyte and its tritiated analogue and the 4-monoiodo-estriol was found to fulfil these requirements as shown by the pattern of dilution and standard curves obtained with the various labeled ligands. This observation was corroborated by a comparison of the apparent estriol concentrations in human pregnancy sera determined with the different indicator ligands. The experimentally proven advantage of 4-monoiodo-estriol over other iodinated estriol derivatives verifies a hypothesis deduced previously from binding constants obtained with analogous estriol derivatives and the same antiserum.

The N-terminal amino-acid sequence of human placental sterylsulfatase.

The N-terminus of the recently isolated sterylsulfatase of human placental cellular membranes was sequenced. According to our results, the enzyme preparation proved to be homogeneous at least with respect to this part of the polypeptide chain; the n-terminal sequence of the sulfatase previously proposed by others, however, had to be revised partially.