Suppression of the immune response by alpha-fetoprotein on the primary and secondary antibody response.

Mouse amniotic fluid was shown to contain a noncytotoxic inhibitor of primary gammaM and secondary gammaM, gammaG subclass splenic plaque forming cells in vitro to SRBC. The suppressive effect was not abolished by exhaustive dialysis or by absorption of mouse amniotic fluid (MAF) with SRBC. Polyacrylamide gel analysis showed that dialyzed MAF was composed of three major protein components, transferrin, albumin, and alpha-fetoprotein (AFP). The selective removal of each of these patients from MAF by affinity chromatography suggested that AFP was the immunosuppressive substance in MAF. This conclusion was verified by the demonstration that pure AFP suppressed in vitro antibody synthesis in microgram quantities whereas equivalent amounts of normal mouse serum, transferrin, or albumin did not. Dose-response studies showed that the effect of AFP in the isolated form was equivalent to the suppressive effect of comparable amounts of AFP in MAF. gammaA and gammaG plaque-forming cell (PFC) responses were suppressed by a significantly lower concentration of AFP than was the gammaM PFC response. The degree of suppression watration of AFP than was the gammaM PFC response. The degree of suppression was dependent on the time at which AFP was added to the cultures; MAF added to antigen-stimulated cultures up to 24 h after initiation of cultures was immunosuppressive whereas similar additions of MAF at 48 h after initiation or later did not suppress. The duration of exposure of spleen cells to MAF in cultures without antigen necessary to achieve suppression of a subsequent primary immune response was determine-d to be approximately 8 h. The results suggest that AFP may have an immunoregulatry function. This has potentially important implications in the maternal-fetal relationship, the immune capabilities of the fetus and newborn, and in certain malignant and nonmalignant diseases in which AFP is elevated.

Tay-Sachs disease: prenatal diagnosis.

Fifteen pregnant women with a 25 percent risk of delivering a child with Tay-Sachs disease were monitored by amniocentesis and hexosaminidase A assays of amniotic fluid, uncultured amniotic cells, and cultured amniotic cells. Tay-Sachs disease was diagnosed prenatally in six fetuses; the diagnosis was confirmed in one child after birth and in five fetuses after therapeutic abortion. Prenatal diagnosis indicated the absence of Tay-Sachs disease in nine other fetuses; this diagnosis was confirmed postnatally in six, three are still in utero.

Prenatal diagnosis of genetic disorders.

Sampling of amniotic fluid, visualization of the fetus, fetal blood sampling, and screening of maternal blood represent successive approaches to the diagnosis of specific genetic disorders in the second trimester of pregnancy. Clinical and ethical concerns about the appropriateness, safety, and efficacy of the techniques have led to multidisciplinary assessments at an early stage. A major growth in demand for medical and educational genetic services can be anticipated.

High fetal estrogen concentrations: correlation with increased adult sexual activity and decreased aggression in male mice.

In the house mouse (Mus musculus), fetuses may develop in utero next to siblings of the same or opposite sex. The amniotic fluid of the female fetuses contains higher concentrations of estradiol than that of male fetuses. Male fetuses that developed in utero between female fetuses had higher concentrations of estradiol in their amniotic fluid than males that were located between other male fetuses during intrauterine development. They were also more sexually active as adults, less aggressive, and had smaller seminal vesicles than males that had developed between other male fetuses in utero. These findings raise the possibility that during fetal life circulating estrogens may interact with circulating androgens both in regulating the development of sex differences between males and females and in producing variation in phenotype among males and among females.

Use of human surfactant low molecular weight apoproteins in the reconstitution of surfactant biologic activity.

Two low molecular weight (LMW) apoproteins were isolated from human pulmonary surfactant. SDS polyacrylamide gel analysis showed one protein (SP 18) to have an apparent molecular weight of 18,000 when unreduced and 9,000 D after reduction. The second protein (SP 9) migrated at approximately 9,000 D in the presence or absence of reducing agents. Both proteins contain a high number of hydrophobic amino acids. The NH2-terminal sequence of SP 18 was determined to be: NH2-phe-pro-ile-pro-leu-pro-tyr-. A cDNA clone isolated from a human adult lung cDNA library contained a long open reading frame encoding at an internal position the human SP 18 amino-terminal sequence. Mixtures of phospholipids (PL) and SP 9 and SP 18 were assessed for their capacity to reduce surface tensions on a pulsating bubble surfactometer. The addition of 1% apoprotein resulted in a reduction of surface tension after 15 s from 42.9 dyn/cm for PL alone to 16.7 and 6.3 dyn/cm for preparations containing SP 9 and SP 18, respectively. In vivo assessment of reconstituted surfactant activity was performed in fetal rabbits. Reconstituted surfactant consisting of PL + 0.5% SP 18 instilled intratracheally at delivery resulted in a marked increase in lung compliance, while the incorporation of 0.5% SP 9 yielded a moderate increase. These data show the ability to produce biologically active surfactant by the addition of isolated LMW apoproteins to defined PL.

Radioimmunoassay of growth hormone-dependent insulinlike growth factor binding protein in human plasma.

A specific radioimmunoassay has been established for a growth hormone-dependent insulinlike growth factor (IGF) binding protein (BP) from human plasma. Although the assay was directed against a 53-kD, acid-stable BP subunit, the main immunoreactive BP in the circulation had an apparent molecular mass of approximately 125 kD. Only higher primate species showed cross-reactivity, and IGF-I, IGF-II, and other peptides were without effect. Circulating BP levels in healthy subjects rose threefold from early childhood to puberty. In 65 adults aged 18 to 65, the mean level (+/- SD) was 6.12 +/- 1.43 micrograms/ml, and declined with age. Strong growth hormone-dependence of BP was also seen; there was a 2.2-fold increase in active acromegaly and a 50-80% reduction in growth hormone deficiency. Poorly controlled diabetic subjects had BP levels 40% below normal, whereas in renal failure and third-term pregnancy a mild elevation was seen. Measurement of BP may provide a useful adjunct to IGF assays in growth disorders.

Measurement, characterization, and source of somatostatin-like immunoreactivity in human amniotic fluid.

Somatostatin-like immunoreactivity (SLI) is widely distributed in tissues and biological fluids. To determine whether SLI is also present in amniotic fluid, samples obtained by amniocentesis from 30 normal and 27 abnormal pregnancies were studied by radioimmunoassay. Direct incubation of [(125)I-Tyr(1)]tetradecapeptide somatostatin (SRIF) with amniotic fluid resulted in 89% tracer degradation. Damage was reduced to <5% when samples were acidified and boiled before the assay. With this technique, SLI was detectable in all normal amniotic fluid samples; the mean level at 15-20 wk of gestation (320+/-55 pg/ml, n = 15) being 4.5 times higher than the mean at 32-43 wk (70+/-12 pg/ml, n = 15) (P < 0.001). In cases of preeclampsia (n = 6), gestational diabetes (n = 5), anencephaly (n = 1), and meningomyelocele (n = 1), SLI values were in the normal range, but in one juvenile diabetic and one patient with chronic renal failure, SLI was undetectable (<10 pg/ml). In a pair of monochorionic diamniotic twins, SLI levels were very different (33 and 197 pg/ml), which suggests that fetal factors are more important than materno-placental ones in determining amniotic fluid SLI. Serial dilutions of amniotic fluid showed parallelism with standard SRIF. When concentrates of pooled amniotic fluid were chromatographed on Sephadex G-25 columns, all SLI eluted in the void volume ahead of SRIF even after treatment with 8 M urea and dithiothreitol. This "big" SLI incubated in amniotic fluid showed 100% stability over 24 h at 37 degrees C, whereas SRIF was rapidly inactivated (t((1/2)) congruent with 7 min). Extracts of placenta and fetal membranes contained no SLI, but small amounts (6-20% of total amniotic fluid SLI) were found in cells from fresh fluid. Radioimmunoassay of SLI in extracts of seven paired cord arterial and venous plasma samples showed no arteriovenous gradient consistent with fetal origin of cord blood SLI. It is concluded that (a) amniotic fluid contains SLI which is of fetal origin and (b) normal levels vary with gestational age. The SLI has a higher molecular weight (>/=5,000) and is more stable in amniotic fluid than SRIF.

Exchange of carbon dioxide in the pregnant rhesus monkey: multicompartmental analysis of carbon dioxide kinetics.

The exchange of carbon dioxide in the pregnant rhesus monkey has been studied quantitatively using sodium bicarbonate-(14)C and applying the model of a system of seven compartments. The transfer rates among the various compartments, compartment sizes, and the rate of production of carbon dioxide by fetus and mother were determined with a computer programmed to fit the theoretical model to the data by adjusting the parameter values of the model until a "best fit" was obtained. It was confirmed that the exchange of carbon dioxide between fetal and maternal blood across the placenta is rapid, that between fetal blood and amniotic fluid is slow, and that there is no appreciable exchange between maternal blood and amniotic fluid. The mean net production of CO(2) by fetus was 0.476 +/-0.0402 mmoles/kg.min, and that by mother was 0.373 +/-0.0279 mmoles/kg.min.

Subhuman primate pregnancy complicated by streptozotocin-induced diabetes mellitus.

Polydipsia, polyuria, polyphagia, and glucosuria followed the administration of streptozotocin to 6 nonpregnant and 15 pregnant monkeys (Macaca mulatta) in the first trimester of pregnancy. The diabetogenic action of the drug was also reflected in an induced but variable deterioration in maternal intravenous glucose tolerance and a marked attenuation of maternal plasma insulin responsiveness to intravenous glycemic stimuli. The products of conception were examined in 29 pregnancies. The neonates and the placentas of the streptozotocin-treated pregnant animals were significantly heavier than average for the period of gestation, polyhydramnios was consistently present, and there was an increase in the incidence of third trimester stillbirths. The fetal and maternal plasma glucose, insulin, and growth hormone concentrations were examined after the intravascular administration of glucose or a solution of mixed amino acids to the fetus in the third trimester. The neonatal plasma responses to similar insulinogenic stimuli were also examined.Fetal and neonatal base line plasma insulin concentrations were significantly elevated compared to those of the controls. The administration of intravascular glucose to the fetus, mother, or neonate was associated with a prompt 2-to 5-fold increase in fetal or neonatal plasma insulin concentrations. These findings contrast to the unresponsiveness of the pancreatic islet tissue we reported in normal subhuman primate pregnancy. The intravascular infusion of a relatively low concentration of mixed amino acids (2 mg/min) to the conceptii from the streptozotocin-treated pregnancies was associated with an elevation in fetal and neonatal plasma insulin levels, whereas normal monkey fetuses and neonates required a 10-fold greater concentration of amino acids in the infusate for similar responses. The induced hyperaminoacidemia or hyperglycemia did not consistently alter plasma growth hormone concentrations in the conceptii from normal or streptozotocin-treated pregnancies. These data provide evidence that maternal glucose intolerance during pregnancy is associated with enhanced fetal and neonatal pancreatic islet cell responsiveness to glucose and mixed amino acids. Although the specific mechanism(s) that alters both the sensitivity and responsiveness of the normal pancreatic fetal islet to insulinogenic stimuli remains unclear, the data do indicate that insulin-dependent maternal hyperglycemia and hyperaminoacidemia, separately or in combination could contribute to the fetal hyperinsulinemia of pregnancies complicated by diabetes mellitus. Moreover, the overall experiences with these streptozotocin-treated animals suggest that a subhuman primate model may be available to examine directly the antenatal pathophysiology of abnormal carbohydrate metabolism.

Cachectin/tumor necrosis factor-alpha formation in human decidua. Potential role of cytokines in infection-induced preterm labor.

This study was conducted as part of an investigation to evaluate the hypothesis that bacterial toxins (LPS or lipoteichoic acid), acting on macrophage-like uterine decidua to cause increased formation of cytokines, may be involved in the pathogenesis of infection-associated preterm labor. We found that cachectin/tumor necrosis factor-alpha (TNF-alpha) was synthesized and secreted into the culture medium by human decidual cells and explants in response to treatment with LPS. LPS treatment also caused an increase in PGF2 alpha production by decidual cells and explants. In amnion cells in monolayer culture, TNF-alpha stimulated PGE2 formation, and TNF-alpha was cytostatic (inhibited [3H]thymidine incorporation into DNA) but not cytolytic in amnion cells. TNF-alpha was not detectable (less than 0.34 ng/ml) in the amniotic fluid of normal pregnancies at midtrimester or at term before or after the onset of labor (n = 44); but TNF-alpha was present at concentrations between 2.8 and 22.3 ng/ml in amniotic fluids of 4 of 20 pregnancies with intact membranes complicated by preterm labor (less than 34 wk gestational age). LPS was present in 10 of the 20 amniotic fluids of preterm labor pregnancies, including all four in which TNF-alpha was present. Bacteria were identified in only one of the four LPS-positive, TNF-alpha-positive fluids. Cytokine formation in macrophage-like decidua may serve a fundamental role in the pathogenesis of preterm labor, including increased prostaglandin formation and premature rupture of the membranes.

Arginine vasotocin in ovine fetal blood, urine, and amniotic fluid.

Arginine vasotocin ([8-arginine]-oxytocin) (AVT), the primary antidiuretic principle in submammalian vertebrates, has been reported to be present in mammalian pituitary and pineal glands. Although the most phyletically ubiquitous of the known neurohypophysial peptides, AVT is still not recognized as a mammalian hormone. We examined plasma, urine, and amniotic fluid from fetal lambs by radioimmunoassay (RIA) for evidence of AVT to assess the possibility of its being such a hormone. Measureable quantities of AVT-like immunoreactivity (irAVT) were observed in fetal plasma (2.4 +/- 0.2 pg/ml), urine (1.4 +/- 0.2 pg/ml), and amniotic fluid (1.9 +/- 0.2 pg/ml). Since the AVT antiserum shows minimal cross-reactivity with arginine vasopressin (AVP) and oxytocin (OT), measurements of AVP and OT concentrations in the same biological fluids also were conducted with specific antisera. The results suggest that the irAVT was not accountable on the basis of cross-reaction. To further verify the identity of the irAVT, a high pressure liquid chromatography system using RIA as a means of detection was developed. This system is sufficiently sensitive to allow the separation and quantitation of picogram quantities of the synthetic peptides AVT, AVP, and OT. In this system, the irAVT in fetal plasma, urine, and amniotic fluid appeared as a single peak coeluting with synthetic AVT. These results indicate that AVT is present in ovine fetal plasma and support the view that the fetus secretes this peptide. The physiological significance of circulating AVT remains to be defined.

Development of gamma G, gamma A, gamma M, beta IC-beta IA, C 1 esterase inhibitor, ceruloplasmin, transferrin, hemopexin, haptoglobin, fibrinogen, plasminogen, alpha 1-antitrypsin, orosomucoid, beta-lipoprotein, alpha 2-macroglobulin, and prealbumin in the human conceptus.

The synthesis of gammaG, gammaA, gammaM, beta(1C)/beta(1A), C'1 esterase inhibitor, ceruloplasmin, transferrin, hemopexin, haptoglobin, fibrinogen, alpha(1)-antitrypsin, orosomucoid, beta-lipoprotein, alpha(2)-macroglobulin, and prealbumin was studied in 15 normal human embryos and fetuses of 29 days to 18 wk gestation and in the yolk sacs of four embryos from 5.5 to 11.5 wk gestation using tissue culture in (14)C-labeled amino acids followed by radioimmunoelectrophoresis. The human embryo as early as 29 day gestation synthesized beta(1C)/beta(1A), C'1 esterase inhibitor, transferrin, hemopexin, alpha(1)-antitrypsin, beta-lipoprotein, alpha(2)-macroglobulin, and prealbumin in culture. At 32 days gestation ceruloplasmin and orosomucoid were also synthesized, but synthesis of fibrinogen was not observed before 5.5 wk. Synthesis of gammaM occurred as early as 10.5 wk gestation, and gammaG synthesis was found in cultures as early as 12 wk gestation; gammaA synthesis was not detected in any of the tissue cultures. With the exception of the gamma-globulins, each of the proteins studied was synthesized by the liver, but additional sites of synthesis for some of these proteins were also found. Synthesis of gammaG and gammaM occurred primarily in the spleen, but other sites of synthesis were noted as well. Changes in the concentrations of most of these proteins and plasminogen in embryonic and fetal serum from 5.5 to 41 wk gestation, in amniotic fluid from 6.5 to 38 wk gestation, and in the sera of neonates during the 1st 3 wk postpartum are described. Although gammaA, gammaM, ceruloplasmin, or haptoglobin were not detectable in some of the embryonic and fetal sera, gammaA and ceruloplasmin were both present as early as 6.5 wk gestation, haptoglobin by 9.5 wk gestation, and gammaM by 17 wk gestation. Each of the other proteins were present in all of the sera examined.

Absence of the cancer-associated factor with a molecular weight of 60,000 from the plasma of patients with a spectrum of nonneoplastic conditions.

We demonstrated previously (Cancer Res., 42: 4964-4969, 1982) that a tumor-associated factor was consistently present in the plasma of over 100 human cancer patients with tumors at 31 different sites. The plasma of healthy controls had very low activity in the biochemical assay. In the present study, we show by a combination of molecular sieving and assay of nuclear RNA transport that the tumor-associated factor, which has a molecular weight of 60,000, is undetectable in the plasma of healthy adults. The low activity reported earlier is due to three normal cell factors of markedly different molecular weight. Furthermore, the tumor factor is shown to be absent from the plasma of male and female patients hospitalized for a variety of nonmalignant surgical conditions. Only the plasma from patients who were pregnant, suffered from chronic renal failure, or had recent myocardial infarction gave false positives in the biochemical assay. However, in these cases, the activity was due to an increase in the normal tissue-associated factors and not to the appearance of the Mr 60,000 tumor-associated factor. The factor is present in amniotic fluid, confirming that it is a fetal factor which does not cross the placental barrier. Thus, it may be classified as an oncofetal factor. All four factors found in the plasma were identified in the cytosol from a human tumor. In summary, the tumor-associated factor appears to be tumor specific and can be unambiguously identified by bioassay of the plasma factors eluting from Sepharose CL-6 B columns in the Mr 60,000 region. It can also be identified by examination of sodium dodecyl sulfate:polyacrylamide gel electrophoretograms of the appropriate Sepharose CL-6 B fractions after removal of albumin.

Concanavalin A variants of the fetoacinar pancreatic protein in the developing human pancreas and other biological sources.

Two new sources for the fetoacinar pancreatic protein (FAP protein) are described in this study: amniotic fluids taken at 18 weeks gestation, and pancreatic juices from patients with pancreatic pathology. The FAP protein from different biological sources showed two kinds of molecular heterogeneity: (a) molecular weight, and (b) lectin-binding affinity. By Western blot the protein was shown to exist either as a doublet (the higher-Mr component at 110 kDa and the second in the range 80-100 kDa) or as a single band (110 kDa) depending on the source. By chromatography on Con A-Sepharose the protein could be separated into two variants, reactive and nonreactive. Most of the protein was present as the Con A-nonreactive variant. The Western-blot patterns of both variants in a given sample were identical. The FAP protein expression had an oncodevelopmental character; maximal concentration was seen in middle-gestation fetal pancreas extracts. Expression of the FAP protein Con A variants followed the same developmental pattern as that of total FAP protein, and their relative amounts remained almost constant during fetal growth. Evidence is given for the presence of lectin and molecular-weight heterogeneities of the protein as well as for the lack of a developmental pattern for the expression of these variants.

Isolation and characterization of a pulmonary glycoprotein from human amniotic fluid.

A glycoprotein of the molecular weight of 36 000 has been isolated from human amniotic fluid. The glycoprotein was found to contain sialic acid, galactose, mannose, fucose, glucosamine, hydroxyproline and relatively high amounts of glycine. End-group analyses resulted in a single NH2-terminal residue indicating that the glycoprotein was homogeneous. The data indicate that this unique collagen-like glycoprotein, which is immunologically identical to a major alveolar glycoprotein found in alveoli of patients with alveolar proteinosis, is also a major protein in the human amniotic fluid. The idea that the pulmonary constituents enter the amniotic fluid cavity during fetal lung development is also confirmed by this report.

Isolation of prolactin from human amniotic fluid.

Amniotic fluid, obtained from women at term, was centrifuged and concentrated by ultrafiltration. The concentrated amniotic fluid was purified by gel filtration on Sephadex G-100 and ion-exchanged chromatography on DEAE-cellulose. The fractions obtained during purification procedures were assayed for prolactin and somatotropin activities by respective radioimmunoassays. The prolactin-rich fraction obtained from ion-exchange chromatography was further purified by isoelectric focusing. A yield of 0.21 mg of highly pruified prolactin containing 40 units/mg was obtained from 1 liter of the amniotic fluid. The prolactin was free of somatotropin and human placental lactogen.

Binding of 17beta-estradiol by variants of alpha-fetoprotein in rat amniotic fluid.

Two variants of alpha-fetoprotein in rat amniotic fluid were separated by their different affinity for concanavalin A-Sepharose, which selectively binds alpha-D-manno-pyranosides and alpha-D-glucopyranosides. Both forms had the same mobility upon polyacrylamide gel electrophoresis. The binding of 17beta-estradiol per mg of alpha-fetoprotein, determined both immunologically and electrophoretically, was the same for both variants. These results indicate that a specific carbohydrate portion of the molecule is not necessary for steroid binding.

125I derivatives of prostaglandins. A novel approach in prostaglandin analysis by radioimmunoassay.

We report the production of radioactive iodinated (125 I) derivatives of prostaglandins E1, E2, F2alpha and their use in radioimmunological assays. Histamine or tyramine was coupled to the prostaglandins carboxyl group and the iodination was accomplished using the chloramine T method. The high specific radioactivity of these tracers and the resolution of the purification procedure allowed the detection of 0.5 pg of prostaglandins. A comparison with tritiated prostaglandin was made and showed a 10-fold gain in sensitivity. Furthermore in the case of the prostaglandin E1 system using 125I-labelled histamine or tyramine as tracer the cross reaction curves obtained were different from those obtained with [3H]prostaglandin E1; we suggest that the blocking of the carboxyl group alters the prostaglandin E1 structure, modifying its immunoreactivity.

Fatty acids bound to alpha-fetoprotein and albumin during rat development.

The time-course levels and composition of the fatty acids bound to rat alpha-fetoprotein (AFP) and albumin from several sources, were determined throughout development, and related to the intake of lipids from milk and the compositional changes in brain and liver fatty acids. The major fatty acids bound to AFP were acids bound to AFP were polyunsaturated and mainly docosahexaenoic acid (22:6(n-3], either from fetal serum (23.1%) or whole fetuses (21.6%), whereas palmitic (34.1%) and oleic (29.9%) acids were the main acids bound to albumin from the same sources. Amniotic fluid AFP contained less fatty acids (0.8 mol/mol protein) than that of fetal serum (1.4 mol/mol protein), and especially noticeable was a reduced amount of 22:6 (9.6%). Both AFP-concanavalin A microforms showed identical fatty acid composition. Levels of 22:6 bound to AFP decreased quickly after birth until a minimum at 8-10 days, increasing moderately thereafter. This minimum is coincident in time with a maximal accumulation of this fatty acid by brain and a loss of 22:6 by liver. Except for colostrum, levels of 22:6 in milk lipids were low and fairly constant, but always greater than those of its precursor, linolenic acid (18:3 (n-3]. These results support a specialized role of AFP in the plasma transport and tissue delivery of polyunsaturated fatty acids, and mainly docosahexaenoic acid.

The measurement of phosphatidate phosphohydrolase in human amniotic fluid.

Phosphatidate phosphohydrolase (EC 3.1.3.4) activity can be found in late gestational human amniotic fluid and is thought to originate in type II alveolar cells of the fetal lungs where it plays an important role in lung surfactant synthesis. In the present study, phosphatidate phosphohydrolase activity was detected and characterized in a 105 000 X g pellet of amniotic fluid using either [32P]phosphatidate or a water-soluble analog, 1-O-hexadecyl-rac-[2-(3)H]glycerol 3-phosphate as substrate. With either substrate, enzyme activity was optimal at pH 6.0. The soluble analog was hydrolyzed with a Km value of 163 micrometer and a V of 30 nmole/min per mg of protein, and offered several advantages over phosphatidate as a substrate for assaying phosphatidate phosphohydrolase in amniotic fluid. Using the synthetic analog, phosphatidate phosphohydrolase activity was measured in the 700 X g supernatant fraction of 30 human amniocentesis samples and compared with another index of fetal lung maturity, the phosphatidylcholine/sphingomyelin ratio. The results suggest that the new phosphohydrolase assay may be clinically useful in the assessment of fetal lung development.