Epidermolysis bullosa acquisita antigen is the globular carboxyl terminus of type VII procollagen.

Epidermolysis bullosa acquisita (EBA) is a severe, chronic blistering disease of the skin. EBA patients have circulating and tissue-bound autoantibodies to a large (Mr = 290,000) macromolecule that is localized within the basement membrane zone between the epidermis and dermis of skin, the site of blister formation. The "EBA antigen" is known to be distinct from laminin, heparan sulfate proteoglycan, fibronectin, the bullous pemphigoid antigen, elastin, and collagen types I, II, III, IV, and V. Sera from patients with EBA, two monoclonal antibodies to the EBA antigen, and a monoclonal antibody to the carboxyl terminus of type VII procollagen identically label human amnion and skin by immunofluorescent and immunoelectron microscopy. Western immunoblots of the EBA antigen extracted from skin and of type VII procollagen labeled with the above sera and antibodies are identical. None of the sera or antibodies labels Western blots of pepsinized type VII collagen which is missing the globular amino and carboxyl terminal domains. These data show that the EBA antigen is the carboxyl terminus of type VII procollagen.

Major proteins induced and down-regulated by interferons in human cultured cells: identification of a unique set of proteins induced by interferon-alpha in epithelial, fibroblast, and lymphoid cells.

In all, 40 major polypeptides ranging in molecular weights from 14.5 to 83 kDa were shown to be induced by IFNs alpha (also by IFN-alpha 2b and beta in a few cases) and gamma in human cultured cells of epithelial (transformed amnion cells (AMA)), fibroblast (proliferating and quiescent MRC-5 fibroblasts), and lymphoid origin (Molt-4). With the exception of a heat shock protein (IEF14 or hs x 70) and two tropomyosins (IEFs 52x and 55), none of these proteins corresponded to polypeptides (proliferation-sensitive or others) previously identified and catalogued by us. IFN-alpha induced the highest number of polypeptides in lymphoid cells, while the response to IFN-gamma was more pronounced in cultured epithelial and fibroblast cells. Several of the polypeptides induced by IFNs alpha and gamma were synthesized (albeit at different rates) by the control untreated cells, and in some cell types such as normal human peripheral blood mononuclear cells many were expressed at high levels. Only IFN-alpha-induced a unique set of proteins (alpha 1, 51 kDa; alpha 2, 15 kDa; alpha 19, 78 kDa; and gamma 10, 83 kDa) in all cultured cell types studied, implying that response to this IFN involves a shared biochemical pathway(s). Both IFN-alpha (also IFN-alpha 2b) and beta induced an identical group of proteins in AMA cells in agreement with the fact that type I IFNs share common receptors. IFNs alpha and gamma induced a few common polypeptides, but only gamma 10 (83 kDa) showed increased synthesis in all cell types exposed to either of these IFNs. A total of 28 major cellular polypeptides were down-regulated by IFNs in the various cell type studied. Different sets of proteins were affected, however, in each system, emphasizing the complexity of the mechanisms underlying the action of these factors. Treatment of synchronized G1 AMA cells with IFNs alpha, beta, or gamma (500 IU/ml, final concentration) did not inhibit their progression from G1 to S-phase as determined by indirect immunofluorescence using PCNA autoantibodies specific for cyclin. These observations were in line with the fact that IFNs did not affect dividin or cyclin(PCNA) synthesis (S-phase specific proteins) at least within the first 17 hr after their addition.

Identification of a 37 kilodalton protein at the epidermal basement membrane by an antiserum to human amnion.

A protein which is recognized by an antibody to human amniotic epithelial basement membrane was identified at the basal lamina of human epidermis by immunohistology. This protein was localized at the lamina lucida of human epidermal basement membrane by immunoelectron microscopy. Studies of normal human keratinocyte cultures and epidermal wound healing suggested that the protein was probably produced by keratinocytes. By immunoblotting, a basic apparent isoelectric pH (pHiapp = 7.3) protein band of 37 kD was seen. These data indicate that this 37 kD protein, clearly different from other known basement membrane components, is present in simple and stratified epithelia of ectodermal origin, and is associated with hemidesmosomes.

Specific cells of human amnion selectively localize prolactin.

The presence of PRL in high concentration in human amniotic fluid has been related to changes in water transport across amnion but not chorion leave. The cellular composition of human amniotic epithelium has been reported to include at least two structurally distinct cell types, known as light cells and dark cells, that differ in their ability to transport large molecules. In the present study, human amnion obtained from term cesarean section was evaluated through the use of autoradiography as to its ability to localize hormones of similar and variant molecular sizes. Amniotic epithelium exposed to [125I]human PRL, [125I]human Gh, [125I]human beta-endorphin, [125I]bovine FSH, and sodium 125I alone was found to display a selectivity in its ability to localize [125I]human PRL only. Furthermore, the selective localization of [125I]human PRL was found to be specific to the light cells, because dark cells failed to localize any of the other tracers used. These data provide additional evidence in support of the recently proposed concept that term human amniotic epithelium consists of at least two functionally distinct cell types. Furthermore, the high levels of PRL in amniotic fluid may have a specific physiologic role in the amniotic membrane during human gestation.

Amniotic membrane production of prostaglandin F2 alpha is reduced in dysfunctional human labor: results of in vivo and in vitro studies.

Mobilization of arachidonic acid from glycerophospholipids and prostaglandin (PG) release from fetal membranes were studied in women with dysfunctional labor in the absence of cephalopelvic disproportion or fetal malposition. Using superfusion of intact amnion and chorion, we found a slight decrease in PGE and a more significant decrease in PGF release by the amniotic side of the fetal membrane obtained from women with dysfunctional labor compared to that in women with normal labor (PGE: normal labor, 2992 pg/cm2.h; dysfunctional labor, 1846 pg/cm2.h; P less than 0.05; PGF: normal labor, 662 pg/cm2.h; dysfunctional labor, 204 pg/cm2.h; P less than 0.02). Release of both prostanoids was significantly greater from the amniotic side in tissues obtained after labor compared to that in prelabor tissue. Analysis of arachidonic acid (by gas liquid chromatography) and phospholipid content (by two-dimensional thin layer chromatography) confirmed metabolic disposal of arachidonic acid from the amnion after the onset of labor. However, no difference in either phospholipid or phospholipase A2-releasable arachidonic acid of individual phospholipid classes was found in amnion tissue from women with normal and dysfunctional labor, suggesting similar activities of phospholipase A2 in these two groups. The finding of decreased free and phospholipase A2-releasable arachidonic acid of the total lipid extract of the amnion of women with dysfunctional labor could suggest further metabolic exhaustion of the substrate or failure of liberation of this fatty acid from glycerophospholipids by enzymes other than phospholipase A2, such as phospholipase C or diacyl and monoacylglycerolipases.

Human relaxin in the amnion, chorion, decidua parietalis, basal plate, and placental trophoblast by immunocytochemistry and northern analysis.

Immunocytochemistry and Northern analysis were used to show that relaxin is a product of intrauterine tissues of pregnancy. In addition, tissues from a patient without ovaries had similar results on both immunocytochemistry and Northern analysis as tissues from intact patients. The parietal decidua was clearly the major source of relaxin within the uterus and the relaxin mRNA (1.2 kilobases) from this tissue was detected with a 48-mer oligonucleotide probe designed to hybridize with both H1 and H2 relaxin gene transcripts. The mRNA isolated from the placental trophoblast was slightly smaller (1.1 kilobases), and the placental basal plate which has both maternal and fetal cells contained relaxin mRNAs of both sizes. Two monoclonal antibodies (Mabs) raised to synthetic human relaxin (H2) gave different patterns of localization in the fetal membranes, decidua and placenta. One Mab (RLX8) stained the chorionic cytotrophoblast in the fetal membranes and all of the cells in the placental basal plate. The other Mab (RLX6) stained the chorionic cytotrophoblast in some instances and selectively stained the decidua-like cells of the placental syncytiotrophoblast, whereas Mab RLX8 failed to detect this relaxin. Tissues obtained after spontaneous labor and delivery contained significantly less relaxin mRNA than tissues obtained at elective cesarean section without labor, but their hormone contents, as judged by immunocytochemistry, were not different. We conclude that the relaxin gene (H2) is expressed in intrauterine tissues, but that expression and hormone synthesis are not ubiquitous. Whether the relaxin gene H1 is expressed has not been determined.

Uterine estrogen receptors are increased by RU486 in late pregnant rhesus macaques but not after spontaneous labor.

Progesterone withdrawal as a mechanism for parturition in primates is controversial. The progesterone antagonist RU486, given in late pregnancy to rhesus monkeys at a dose of 47 mmol/kg.day (20 mg/kg.day), causes an increase in uterine activity, but not the expected increase in amniotic fluid prostaglandins or cervical dilatation. We, therefore, studied the effect of RU486 on estrogen receptor (ER) localization and concentration in reproductive tract tissues in rhesus monkeys during late gestation and after spontaneous labor at term. Distribution of ER in pregnant uterine tissues was studied by immunocytochemical techniques and quantified by a biochemical assay, both of which employed a monoclonal antibody specific for ER. ER was not present in amnion and chorion by immunocytochemical investigation; however, a significant increase in receptor staining was seen in decidua and myometrium after RU486 treatment compared to that in both pregnant control tissues and parturient tissues. Sucrose gradient assay of nuclear (n) and cytosolic (c) ER revealed a low level of ER (expressed as fmol of estradiol bound/mg of DNA) in pregnant and parturient decidua (pregnant: nER = 7.3 +/- 2.4, cER = 17.1 +/- 6.4; parturient, nER = 7.7 +/- 3.1, cER = 16.4 +/- 8.8) and myometrium (pregnant: nER = 21.7 +/- 4.1, cER = 20.8 +/- 5.3; parturient: nER = 30.0 +/- 2.8, cER = 10.7 +/- 6.7). In contrast, tissues collected from RU486-treated animals contained high levels of ER in decidua (nER = 52.3 +/- 16.8, cER = 240.5 +/- 145.3) and myometrium (nER = 77.0 +/- 19.2; cER = 66.5 +/- 31.6). We conclude that 1) the increase in ER in decidua and myometrium after RU486 treatment is the result of a decrease in the inhibitory action of progesterone on ER and documents the progesterone receptor antagonism by RU486 during induced myometrial contractility in late pregnant rhesus monkeys; 2) the absence of ER from amnion and chorion indicates that the normally observed increase in prostaglandin production by rhesus fetal membranes during labor is not mediated by ER; and 3) the absence of a change in the concentration of ER in decidua and myometrium from pregnant control monkeys and those in spontaneous labor indicates that an increase in ER (and, by inference, a withdrawal of receptor-mediated progesterone inhibition) is not part of the normal events in preparation for parturition in primates.

Use of actinomycin D for the specific quenching of fluorescence of deoxyribonucleic acid in cells stained with acridine aminoderivatives.

Actinomycin D specifically quenches the fluorescence of acridine orange and quinacrine bound to deoxyribonucleic acid in cytologic preparations, but does not change the fluorescence of these fluorochromes bound to RNA. The following fluorescence-cytochemical applications of techniques based on these findings can be suggested: (a) distinction between deoxyribonucleic acid and ribonucleic acid; (b) detection of double-stranded virus ribonucleic acid; (c) approximate estimation of the lengths of A-T sequences in deoxyribonucleic acid molecules.

Factors affecting the extraneuronal inactivation of noradrenaline in cardiac and smooth muscle.

1. The relation between density of adrenergic innervation, noradrenaline (NA) accumulation (as seen with the fluorescence histochemical method) in tissues incubated in a high concentration of NA, and monoamine oxidase (MAO) and catechol-O-methyl transferase (COMT) activities, were examined in a wide range of tissues from different species.2. Evidence was obtained to support the proposal that accumulation of NA in the non-innervated smooth muscle of the human umbilical artery and chick amnion is associated with very low activities of COMT within muscle cells.3. The wide variation in tissue accumulation of NA in adrenergically innervated muscles was confirmed. For example, in the rabbit atrium and rat vas deferens, there was high NA accumulation in vascular smooth muscle but not in other muscle cells. In the mouse vas deferens there appeared to be preferential NA accumulation in the outer longitudinal muscle in comparison with the circular muscle. In the ventricle of the rat and mouse individual muscle cells showed different degrees of accumulation of NA. Many unidentified fluorescent cells were revealed in the submucosa of the guinea-pig ureter following loading with NA. The highest activities of COMT were found in the rat vas deferens and the lowest in the rabbit vascular tissues; the highest activity of MAO was found in the guinea-pig ileum, and the lowest in the rat aorta.4. No simple relation between tissue activities of MAO and COMT and degree of NA accumulation was found. Possible directions for further investigation of the problem are discussed.

Distribution of prostaglandins E2 and F2 alpha within the foetoplacental unit throughout human pregnancy.

The concentrations of prostaglandin E2 and F2 alpha have been measured by radioimmunoassay in portions of cord, placenta, amnion, chorion, decidua and myometrium. The samples were obtained at defined periods of pregnancy, and the results have been compared with those obtained from the analyses of endometrial and myometrial tissue removed from women during the secretory phase of a menstrual cycle. The results showed that during pregnancy the mean concentration of prostaglandin E2 was higher (27-518%) than the corresponding value for prostaglandin F2 alpha in all tissues. At term the concentration of prostaglandin E2 (ng/100 mg wet weight of tissue, mean +/- S.D.) was higher in the umbilical cord (5-54 +/- 0-88), decidua (4-02 +/- 1-78) and myometrium (4-19 +/- 1-06), than in the amnion (2-25 +/- 1-27), chorion (1-64 "/- 0-63) or placenta (1-04 +/- 0-25). During labour there was a significant rise (P less than 0.0005, Student's 't' test) in the concentration in decidua (10-76 +/- 4-45), and to a lesser extent (P less than 0-05) in the myometrium (5-84 +/- 2-65) and amnion (4-77 +/- 2-51). The overall concentration in decidua during the first trimester (3-09 +/- 1-02) was significantly lower (P less than 0-005) than in endometrial tissue(16-82 +/- 10-13). The concentration was lower in myometrial tissue from non-pregnant subjects (2-90 +/- 2-21), than in the corresponding tissue removed at term (4-19 +/- 1-06) or during labour 5-84 +/- 2-65). The results for prostaglandin F2 alpha showed a similar pattern, but the values were significantly lower in the umbilical cord, and the percentage changes in concentration in the decidua and myometrium were of a higher magnitude.

Immunohistochemical localization of relaxin, prolactin and prostaglandin synthase in human amnion, chorion and decidua.

Relaxin, prolactin and prostaglandin synthase were localized by the avidin-biotin immunoglucose oxidase method in human amnion, chorion and decidua. Specimens from ten normal spontaneous deliveries and four elective Caesarean section deliveries with no labour were compared. Relaxin was found more consistently in the cells of the chorionic cytotrophoblast than in the cells of the parietal decidua adherent to the fetal membranes. Only half the tissues after spontaneous delivery contained positive relaxin-stained cells, whereas all the tissues from elective Caesarean sections contained cells positively stained with antiserum to relaxin. In both series of tissues prolactin was localized predominantly in the parietal decidual cells and was very infrequently found in the chorionic cytotrophoblast. Polyclonal antiserum to prostaglandin synthase was used to identify those cells producing prostaglandin in amnion, chorion and decidua. The cells of the amnion and chorion showed positive immunolocalization with no differences between tissues collected before or after labour. Double immunostaining using avidin-biotin immunoperoxidase for prolactin, followed by avidin-biotin immunoglucose oxidase for prostaglandin synthase, produced identical results in the same series of tissues examined with the single-staining method.

Detection of oncomodulin, an oncodevelopmental protein in human placenta and choriocarcinoma cell lines.

Oncomodulin was detected by immunocytochemical means in human placenta and found to occur in the cytotrophoblastic shell during implantation, in Langhans type villar cytotrophoblastic cells, and in extravillar cytotrophoblast or intermediate trophoblast. The presence of oncomodulin during first and early second trimester was confirmed by immunoradiometric assay (IRMA). In term placenta oncomodulin appeared in intermediate trophoblast cells, and was largely absent from the villi which lack cytotrophoblast. Furthermore, oncomodulin was abundant in the choriocarcinoma cell line BeWo, and detectable in JAR but not JEG-3. The function of this oncodevelopmental calcium-binding protein in normal development or in neoplasia is not known.

Epidermal growth factor receptors in rat placenta, amnion and yolk sac: characteristics of specific binding are dependent on gestational age.

Studies of [125I]-EGF binding to the rat placenta, amnion and yolk sac were carried out on days 14, 17 and 20 of gestation. In the placenta EGF binding was detectable on all 3 days; in the amnion EGF binding was undetectably low on day 14 but was present on days 17 and 20, while in the yolk sac EGF binding was undetectably low on all 3 days. Although Scatchard analysis of EGF binding to placental tissue raised the possibility of high and low affinity receptors, a statistical analysis of the ligand binding data was consistent with the presence of only one type of EGF receptor. The overall affinity of the receptors did not change with stage of gestation. However, the concentration of EGF receptors was lower in placental tissue on day 17 than on days 14 or 20 of gestation; the receptor concentrations were similar on days 14 and 20. It is suggested that EGF binding to the placenta, amnion, and yolk sac may reflect the levels of cell proliferation in those tissues in the latter part of gestation.

Antenatal diagnosis of thalassemia major in Sardinia.

In this report, we summarized our experience, carried out in Sardinia, with antenatal diagnosis in one thousand pregnancies in which the fetus was at risk for homozygous beta-thalassemia. In the majority of these cases, the thalassemia lesion segregating in the family was the nonsense mutation at the codon corresponding to amino-acid 39. At the outset (976 cases) we used globin chain synthesis analysis by column chromatography on fetal blood obtained by placental aspiration, and recently (24 cases) we employed the synthetic oligonucleotide method on amniocyte DNA. Apart from 126 pregnancies still in progress, in all the other cases the diagnosis has been confirmed. In the majority of the cases (99%), we obtained sufficient fetal blood for the analysis. The fetal mortality associated with placental aspiration was 6.1%. The biochemical analysis gave reliable results. We had two misdiagnoses (0.2%): one due to a nonglobin protein comigrating with the beta chains and the other for a misclassification of the type of thalassemia segregating in the family. The oligonucleotide method gave clear-cut results in all the cases tested. The method was sensitive enough to detect the mutation directly in the DNA isolated from 20-25 ml of amniotic fluid in 75% of the pregnancies tested. In one case, we successfully employed this method for the analysis of the DNA isolated from chorionic villi. The oligonucleotide method seems to be the best procedure for monitoring the pregnancies at risk for beta-thalassemia in places where one or a few beta-thalassemia lesions are prevalent.

Collagen content of human amniotic membranes: effect of gestation length and premature rupture.

The collagen content of amniotic membranes was measured in samples obtained at delivery from patients with and without premature rupture of the membranes (PROM). In samples from patients with PROM the collagen content (343 microgram/mg) was significantly lower than in samples from patients without PROM (373 microgram/mg) (P less than .005). The collagen content decreased between 32 and 40 weeks' gestation from 446 to 362 microgram/mg (r = .588; P less than .001) in patients without PROM and from 393 to 332 microgram/mg (r = -.362; P less than .05) in patients with PROM. The latent period between membrane rupture and delivery was not associated with a decrease in collagen content. The changes in amnion collagen during gestation and the differences observed with PROM suggest that weakening of the amnion in preparation for rupture may be determined partly by factors controlling the synthesis and degradation of collagen.

Lecithin:sphingomyelin ratio in pregnancies complicated by insulin-dependent diabetes mellitus.

One hundred forty-one samples of amniotic fluid in 105 pregnancies from 95 insulin-dependent diabetics were analyzed for the lecithin:sphingomyelin (L:S) ratio. Fetal pulmonary maturity seemed to progress at a normal rate. The mean L:S ratio was not significantly different in diabetic and nondiabetic patients at various stages of gestation. Only 3 cases of respiratory distress syndrome (RDS) were observed in 77 infants with an L:S ratio of 2.0 or more delivered of diabetic mothers within 72 hours of amniocentesis. This 3.9% incidence of RDS in infants of diabetic mothers with L:S ratios of more than 2.0 was not significantly higher than the 1.5% incidence in the nondiabetic mothers. It is concluded that by using the method of Gluck, an L:S ratio of 2.0 or greater at 36 weeks' gestation or later is a reliable indicator of fetal lung maturity in insulin-dependent diabetic pregnancies, and abdominal delivery after 36 weeks' gestation does not increase the risk of RDS in a diabetic pregnancy with an L:S ratio at 2.0 or more.

Premature labor. I. Prostaglandin precursors in human placental membranes.

Amnion and chorion from premature and term placentas after both spontaneous labor and elective cesarean section were assayed for phospholipids and fatty acid composition by 2-dimensional thin-layer chromatography and gas-liquid chromatography. In preterm placental phospholipids, phosphorus concentrations were higher in amnion than in chorion, whereas at term the membranes were similar owing to an increase in phospholipid concentration in the chorion late in gestation. PHosphatidylcholine (PC) accounted for 47% of the total phospholipid phosphorus, followed by sphingomyelin at 20%, phosphatidylethanolamine (PE) at 15%, phosphatidylserine (PS) at 12%, and phosphatidylinositol (PI) at 5%. These percentages were similar for amnion and chorion and they did not change during gestation. The percentage of arachidonic acid (AA) was higher in PS (40 to 65%) than in PE (30 to 53%) and PC (10 to 13%). The percentage of AA was significantly higher in PC, PE, and PS from the amnion in premature pregnancies than in those of the premature chorion. At term, these amnionic and chorionic phospholipids had similar concentrations of AA owing to a significant increase in AA in the chorion late in gestation. Amnionic PE from term and preterm elective cesarean section had a significantly higher percentage of AA than that from preterm and term labor. These data suggest that AA is consumed during labor and that amnionic phospholipids, particularly PE, may be its principal source. The amnion seems to be more important for the storage of AA than the chorion, particularly in preterm pregnancies in which the concentrations of phospholipids and the percentages of AA in PC, PE, and PS were significantly higher than in the chorion.

A high performance liquid chromatography method for the analysis of 35S-cystine: application to the diagnosis of cystinosis.

An HPLC method for the measurement of radioactively labelled cystine is described. This method has been applied to studies of the uptake and retention of 35S-cystine by cultured cells. Radioactive cystine was measured, as a proportion of the non-protein labelled products in cultured cells incubated with medium containing 35S-cystine. Cells from healthy individuals contained less than 7% cystine whereas cells from cases of cystinosis contained at least 19% cystine. The method has been applied to the prenatal diagnosis of cystinosis. The use of flow radioactivity detection provides the advantages of rapid diagnosis and quantitation of metabolites.

The low hydroxyproline content of prematurely ruptured human fetal membranes.

Since collagen is one of the main factors responsible for the mechanical properties of soft tissue, we have determined the hydroxyproline content of the placental amnion, the free amnion and the chorion of 32 unruptured and 25 prematurely ruptured human fetal membranes. The hydroxyproline content of prematurely ruptured membranes was approximately 50% lower than in unruptured membranes. Hydroxyproline (microgram/mg lyophilized tissue), reported as mean +/- SD, was: 19.46 +/- 3.60 vs 37.53 +/- 8.93 for placental amnion; 16.97 +/- 3.93 vs 33.00 +/- 8.25 for free amnion, and 7.74 +/- 3.00 vs 13.23 +/- 3.95 for the chorion. This finding and the known decrease of hydroxyproline content of the amnion towards the end of gestation in normally evolving pregnancies suggest that an abnormally low collagen content may be the general cause of prematurely ruptured human fetal membranes.

[Characterization of smooth muscle organs by determination of cellular myosin content]. / Charakterisierung glattmuskulärer Organe mit Hilfe der Bestimmung des Myosingehaltes der Zellen.

The mean relative content of myosin of cells from smooth muscular organs was estimated using immunocytochemical methods after enzymatical tissue disintegration. The cells were classified according to myosin content. Aortas and urinary bladders of newborn rats and chicken amnions were used as models. Cells of a permanent cell strain, being free of smooth muscular myosin served as negative controls. It could be shown, that the content of myosin, estimated by immunocytochemical methods correlates with the state of development of smooth muscular organs.