Glomerular uptake of nucleosomes: evidence for receptor-mediated mesangial cell binding.

DNA-containing immune complexes (IC) are believed to have a central causal role in the glomerulonephritis of systemic lupus erythematosus. Extracellular DNA which provides the antigenic source for these ICs circulates as oligonucleosomes (ON). The in vivo glomerular uptake of radiolabeled ON in rats, as well as its binding by cultured rat mesangial cells, was examined. The data show that the binding of ON to kidney, and specifically glomeruli, was almost fourfold greater than that of purified DNA. Uptake appeared dose-dependent and saturable, while there were no differences in hepatic or splenic uptake. Most of the nucleosomal DNA recovered from glomeruli was TCA-precipitable, and on gel electrophoresis was about 100 to 300 bp, a size sufficient to allow formation of large ICs. In vitro studies demonstrated that ON are bound by cultured mesangial cells in a dose-dependent and saturable manner, with a dissociation constant of 1.25 x 10(-10) M/liter and 750 binding sites per cell. Autoradiography of cell cultures incubated with radiolabeled ON showed deposition along the plasma membrane which was inhibited by excess unlabeled ON. The data show that binding of ON to glomeruli exceeds that of purified DNA and may be mediated by histones. ON bind to mesangial cells in a receptor-mediated fashion. The data support the hypothesis of in situ formation of DNA-containing ICs and suggest a role for the mesangial cell in lupus glomerulonephritis.

The assessment of fetal pulmonary maturity by fluorescence polarization (FP-value) of pooled vaginal amniotic fluid associated with ruptured membranes.

This prospective study was undertaken to examine the reliability of fluorescence polarization (FP) readings on amniotic fluid collected from the posterior fornix of the vagina after membranes have ruptured. This method was chosen because it is as accurate as the L/S ratio, but it is simpler, faster and requires only 0.5 ml of sample. Forty-seven out of 55 patients were eligible for the study, for a success rate of 85%. Respiratory distress syndrome (RDS) occurred in one out of 29 neonates with FP in the mature or low risk group. In the high risk group for RDS, 7 out of 12 developed the syndrome. In 11 patients, FP-values obtained from transabdominal amniocentesis were not significantly different from those obtained from pooled vagina amniotic fluid once membranes were ruptured. Analysis of pooled vaginal amniotic fluid is simple, non-invasive and capable of being performed with a high rate of success. FP-values from properly collected pooled vaginal amniotic fluid can be used in the assessment of functional fetal lung maturity.

(3-sn-Phosphatidyl)cholines (lecithins) in amniotic fluid.

Many currently used thin-layer chromatographic methods for phospholipid assay rely on charring the developed plate in the presence of cupric acetate. Saturated acyl phospholipids do not react. We find that substitution of cupric sulfate results in detection of both saturated and unsaturated phospholipids. By exploiting the difference with the two reagents, one can separately estimate the amounts of saturated phospholipid. The method described here is reproducible, and we illustrate its use in determinations of (3-sn-phosphatidyl)cholines (lecithins) in amniotic fluid from problem pregnancies.

Identification of corticosteroid-binding globulin in human milk: measurement with a filter disk assay.

We have studied the concentration and properties of a protein which binds cortisol in human milk in samples obtained from women during the first 100 days after delivery. A filter disk assay was developed both for the measurement of plasma corticosteroid-binding globulin (CBG) and for the cortisol-binding protein in milk. The concentration of CBG in milk, expressed as its capacity to bind cortisol, is highest on the day of delivery, ca, 0.80 mug/dl, falls over the next 10 days to ca. 0.25 mug/dl, and remains at that level thereafter. If the concentration of CBG is expressed relative to the concentration of serum albumin in milk, it increases from day 1 to day 3 and then remains constant. A detailed comparison of CBG derived from milk and plasma showed that the two proteins co-migrated on Sephadex, sucrose gradient ultracentrifugation, and polyacrylamide gel electrophoresis. The two proteins had the same affinity for cotrisol, progesterone, 17-OH-progesterone, and dexamethasone. Furthermore, the binding activity of CBG in milk was neutralized with anti-CBG antibodies raised against CBG isolated from plasma. Unlike CBG, the concentration of cortisol in milk, 0.8-3.5 mug/dl, showed no systematic variation as a function of the postpartum day on which the sample was obtained.