M-CSF and GM-CSF regulation of STAT5 activation and DNA binding in myeloid cell differentiation is disrupted in nonobese diabetic mice.

Defects in macrophage colony-stimulating factor (M-CSF) signaling disrupt myeloid cell differentiation in nonobese diabetic (NOD) mice, blocking myeloid maturation into tolerogenic antigen-presenting cells (APCs). In the absence of M-CSF signaling, NOD myeloid cells have abnormally high granulocyte macrophage colony-stimulating factor (GM-CSF) expression, and as a result, persistent activation of signal transducer/activator of transcription 5 (STAT5). Persistent STAT5 phosphorylation found in NOD macrophages is not affected by inhibiting GM-CSF. However, STAT5 phosphorylation in NOD bone marrow cells is diminished if GM-CSF signaling is blocked. Moreover, if M-CSF signaling is inhibited, GM-CSF stimulation in vitro can promote STAT5 phosphorylation in nonautoimmune C57BL/6 mouse bone marrow cultures to levels seen in the NOD. These findings suggest that excessive GM-CSF production in the NOD bone marrow may interfere with the temporal sequence of GM-CSF and M-CSF signaling needed to mediate normal STAT5 function in myeloid cell differentiation gene regulation.

Second-trimester amniotic fluid or maternal serum interleukin-10 levels and small for gestational age neonates.

OBJECTIVE: To evaluate if interleukin-10 levels in either early second-trimester amniotic fluid (AF) or maternal serum can be utilized as a predictor of the subsequent occurrence of small for gestational age (SGA) infants after controlling for gestational age at delivery. METHODS: We identified patients who underwent genetic amniocentesis for standard genetic indications or maternal blood sampling for maternal serum alpha-fetoprotein (MSAFP)/triple screen between January 1992 and February 1995 with available follow-up delivery data. Small for gestational age was defined as birth weight less than the tenth percentile for gestational age. Control patients were matched for gestational age at delivery, maternal age, race, and parity with at least two controls for each study patient. We excluded patients with maternal immune disease, chronic hypertension, diabetes, asthma, congenital heart disease, multiple gestation, and fetuses with structural or chromosomal anomalies. Second-trimester AF and serum samples were assayed for interleukin-10. Potential confounding variables considered were MSAFP level, smoking history, pregnancy-induced hypertension, and neonatal gender. The interleukin-10 levels were normalized using natural log transformation for statistical analysis. Statistical analysis included chi 2, Fisher exact test, and analysis of variance, with P < .05 considered significant. RESULTS. From the AF data base, 18 patients (6%) delivered SGA neonates and were matched with 46 controls. From the maternal serum data base, 13 patients (7%) delivered SGA neonates and were matched with 45 controls. Neither AF nor maternal serum interleukin-10 levels were significantly different in patients subsequently delivering SGA neonates compared with controls (AF: median 21.0 pg/mL. [range 13.8-27.6] versus 17.5 pg/mL. [range 8.9-362.12], P = .18; serum: median 15.7 pg/mL [range 9.9-73.5] versus 18.7 pg/mL [range 9.7-71.7], P = .60, respectively). No significant differences were identified in gestational age at sampling, maternal smoking history, pregnancy-induced hypertension, or elevated MSAFP in patients delivering SGA neonates compared with controls (P > .05 for each). As expected, birth weight was significantly lower in patients delivering SGA neonates compared with controls (P < .001). CONCLUSION: Second-trimester AF or maternal serum interleukin-10 levels are not predictive of subsequent delivery of SGA infants.

Role of cytochrome P450 in the control of the production of erythropoietin.

Effects of agents affecting cytochrome P450 were studied on the production of erythropoietin (Epo) in cultures of the human hepatoma cell line HepG2. Epo was measured by radioimmunoassay of the culture media after 24 h of incubation. The addition of phenobarbital or 3-methylcholanthrene, which induce cytochrome P450, significantly enhanced the formation of Epo. Likewise, the thyroid hormones T3 and T4 stimulated the rate of the production of Epo. On the other hand, the formation of Epo was lowered following the addition of diethyldithiocarbamate or cysteamine chloride, which inhibit cytochrome P450. These findings support the idea that O2 sensitive hemoproteins of the microsomal mixed-functional oxidases play a role in the control of the synthesis of Epo.

GM-CSF induces STAT5 binding at epigenetic regulatory sites within the Csf2 promoter of non-obese diabetic (NOD) mouse myeloid cells.

Myeloid cells from non-obese diabetic (NOD) mouse and human type 1 diabetic (T1D) patients overexpress granulocyte-macrophage colony stimulation factor (GM-CSF). This overproduction prolongs the activation of signal transduction and activator of transcription 5 (STAT5) proteins, involved in GM-CSF-induced control of myeloid cell gene expression. We found that GM-CSF can regulate the binding of STAT5 on the promoter of its own gene, Csf2, within regions previously identified as sites of chromatin epigenetic modification important to the regulation of GM-CSF during myeloid differentiation and inflammation. We found multiple sequence polymorphisms within NOD mouse chromosome 11 Idd4.3 diabetes susceptibility region that alter STAT5 GAS binding sequences within the Csf2 promoter. STAT5 binding at these sites in vivo is increased significantly in GM-CSF-stimulated-bone marrow cells and in unactivated, high GM-CSF-producing macrophages from NOD mice as compared to non-autoimmune C57BL/6 mouse myeloid cells. Thus, GM-CSF overproduction by NOD myeloid cells may be perpetuating a positive epigenetic regulatory feedback on its own gene expression through its induction of STAT5 binding to its promoter. These findings suggest that aberrant STAT5 binding at epigenetic regulatory sites may contribute directly to immunopathology through cytokine-induced gene expression dysregulation that can derail myeloid differentiation and increase inflammatory responsiveness.

Isolation of a second rifamycin-binding from Escherichia coli by affinity chromatography.

When extracts of Escherichia coli are filtered through a Sepharose column containing covalently bound rifamycin a protein is bound which can be eluted either with a high concentration of urea or more specifically with low concentrations of rifamycins. Its Mr is 18,000 +/- 1,000 in the presence of dodecylsulfate, in its absence 36,000 +/- 3,000. The association constant of the protein for rifampicin is 2.4 +/- 0.5 x 10(-4) M with two binding sites per dimer as determined by equilibrium dialysis. Large amounts of this protein are released from the cells by an osmotic shock.

A new stable epithelial cell line (RK-L) from normal rat kidney.

A stable epithelial cell line has been established from the kidneys of a normal Sprague-Dawley rat. This line, termed RK-L, has a high proliferative capacity (minimal doubling time 12.3 h) and can be grown in medium containing 1% fetal bovine serum. Thus far, the line has been carried through more than 60 serial passages. The RK-L cells were found to display similarities with kidney tubule cells. Using light microscopy, confluent cultures were seen as pavement-like monolayers forming domes, which are thought to result from transepithelial fluid transport. Electron microscopy revealed polarized cells that had microvilli on the apical surface, junction complexes in the apical part of the lateral cell membrane, and a basal lamina-like layer. Pinocytotic activity was indicated by infoldings of the apical plasma membrane and the formation of vesicles. The RK-L line should prove useful for investigations of kidney tubule transport mechanisms.

Lecithin can be detected by volume-selected proton MR spectroscopy using a 1.5 T whole body scanner: a potentially non-invasive method for the prenatal assessment of fetal lung maturity.

The prenatal determination of fetal lung maturity is currently assessed by chemical analysis of surfactant associated lipids from aminotic fluid obtained by amniocentesis. This is an invasive procedure with rare but occasionally serious morbidity. Magnetic resonance spectroscopy (MRS) is a non-invasive method for the in vivo localization and identification of molecules with known resonance peaks at specified chemical shifts. In this report we examine the in vitro MRS of a lecithin/saline solution as well as term and preterm amniotic fluid samples with the use of a 1.5 T whole body scanner and a flexible surface coil. We found that amniotic fluid at term demonstrates a resonance peak at 3.2 ppm which was the same as the chemical shift of lecithin in saline. The lecithin peak is not observed in preterm amniotic fluid. This demonstrates the feasibility of using MRS with a whole body scanner to detect lecithin, one of the markers for fetal lung maturity.

Risk of abnormal triple screen for Down syndrome is significantly higher in patients with female fetuses.

Previous studies have shown that mid-trimester maternal serum alpha-fetoprotein (AFP) levels are significantly higher and human chorionic gonadotrophin (hCG) levels significantly lower in women with male compared with female fetuses. We have evaluated whether triple-screen criteria are more likely to identify women with female fetuses as at risk for Down syndrome. From the Georgetown University genetics database we obtained the absolute values and corresponding multiples of the median (MoM) for AFP, hCG and unconjugated oestriol (uE3) in singleton gestations for the period database November 1992 July 1996. A Down syndrome risk of 1/270 or greater at mid-trimester was considered as high risk. A total of 977 patients with triple screen and outcome information were identified, including 502 female and 475 male fetuses. Patients with female fetuses were significantly more likely to have lower serum AFP (p=0.003) and a positive triple screen for Down syndrome (72 (14 per cent) versus 45 (9 per cent), p<0.02) than those with male fetuses. The gestational age at triple screen, maternal serum hCG and uE3, race and diabetes were not significantly different between the two groups. Since Down syndrome is less common in female than male fetuses, and the rates of female and male Down syndrome fetuses detected by triple screen and subsequent amniocentesis are not significantly different, the excess of positive mid-trimester maternal serum triple screen in women with female fetuses is likely due to false-positive results.