Labeling of beta-endorphin and beta-lipotropin with iodine 125

5 micrograms of human beta-endorphin were labelled with 2 mCi 125I by the chloramine T technique. After two gel filtrations on Sephadex G-15 and on Sephadex G-50 in phosphate buffer with EDTA, Trasylol and mercapto-ethanol, a pure tracer was obtained with a specific activity about 150 microCi/ug. Kept at + 4 degrees C, the tracer remained utilizable for 30 days without loss of immunoreactivity. The labelling with lactoperoxydase and the use of another gel filtration method (filtration on Aca 202) gave a 125I beta-END tracer with the same immunoreactivity. The binding of this tracer to the antibody of an anti-beta-END antiserum diluted at 1/8000 was 32% with a non specific binding of 2%. 5 micrograms of human beta-lipotropin were labelled with 0.5 mCi 125I by the lactoperoxydase method. After two gel filtrations on Sephadex G-25 and on Sephadex G-75 in phosphate buffer with EDTA, Trasylol and mercapto-ethanol, a pure tracer with a specific activity of 140 microCi/micrograms was obtained. It remained utilizable for 30 days when kept at + 4 degrees C. Gel filtration on Aca 202 did not give good purification, while gel filtration on Aca 54 was good but slower than on Sephadex G-75. The binding to antibody in absence of unlabelled beta-LPH was 32% for an anti-beta-LPH antiserum diluted at 1/4000. The non specific binding was 2.5%

Radioimmunoassays of unextracted gonadotrophins in timed fractions of 24-hour urine: morning increase of gonadotrophin excretion, a circadian pattern in relation to puberty

Highly sensitive and specific radioimmunoassays to measure urinary FSH and LH were developed and applied to unextracted urine. The use of radioimmunoassays of gonadotrophins in 0.1 ml urine gave results which were significantly correlated (p < 0.001) with those obtained in acetone precipitates of urine. Gonadotrophin levels determined in unextracted urine were not influenced by variations in urine pH and by dilution. Urinary immunoreactive FSH appeared closely similar to pituitary standard, whereas LH was heterogeneous and partly different from the purified pituitary preparation. The urinary excretion of gonadotrophins was studied in 16 normal and 50 diabetic children and adolescents in relation to different time periods throughout day and night and to pubertal development. Diabetic subjects were shown to be similar to control subjects with respect to the daily excretion of gonadotrophins and creatinine. In both sexes, a sleep-wake pattern of FSH and LH excretion was observed, the lowest values being obtained during the night and the highest during the morning between 8 and 12 h. This circadian rhythm was found even in prepubertal children. Marked changes seem to occur throughout puberty: Gonadotrophin levels excreted in morning urine increase mostly until midpuberty (stage 3) whereby the circadian pattern is most apparent: Night excretion of gonadotrophins remains at a prepubertal level until midpuberty and slightly increases in late puberty (stage 4-5). Consequently, the differences between night and morning values of gonadotrophin excretion are less evident in late pubertal subjects (stage 5), especially in girls. © 1980 S. Karger AG, Basel

Production of interferon gamma by peripheral blood mononuclear cells from normal subjects and from patients with rheumatoid arthritis

A radioimmunoassay for human interferon gamma (IFN-gamma) has been carried out using a recombinant glycosylated interferon (Hu IFN-gamma) as tracer, the N.I.H. reference preparation (Gg 23-901-530) and a polyclonal rabbit antiserum. The assay is highly specific for IFN-gamma: there is no cross-reaction either with interferons alpha and beta, Interleukins 1 and 2, tumor necrosis factor alpha and beta or with various brain peptides. The sequential saturation procedure allowed a sensitivity of 0.4 U/ml with intra and between assay coefficients of variation less than 8 and 12%, respectively. The in-vitro production of IFN-gamma by peripheral blood mononuclear cells (P.B.M.C.) was also measured. In unstimulated cultures, IFN-gamma production remained undetectable, i.e. below the 0.4 U/ml sensitivity level. After stimulation of P.B.M.C. from normal subjects with increasing amounts of PHA, both the 3H-thymidine incorporation and IFN-gamma release followed bell-shaped curves. There was no significant difference of 3H-thymidine incorporation between PHA stimulated cultures (0.2 and 2.5 ug/ml) from normal subjects (36 cases) and those with active (16 cases) or non-active (14 cases) rheumatoid arthritis. At two PHA concentrations of 0.2 and 2.5 ug/ml, mononuclear cells from patients with active disease produced significantly less IFN-gamma than those from either controls or cases with non-active disease

Production of tumour necrosis factor-alpha, interferon-gamma and interleukin-2 by peripheral blood mononuclear cells of subjects suffering from rheumatoid arthritis

Using radio-immunoassay methods, the production of tumour necrosis factor-alpha (TNF-alpha), interleukin-2 (IL-2), and interferon-gamma (IFN-gamma) released by peripheral blood mononuclear cells (PBMC), maintained in culture and stimulated by phytohemagglutinin (PHA), was measured in normal subjects and patients with active or inactive rheumatoid arthritis (RA). Results indicated a dissociation between mitogenic response and secretion of mediators by PBMC under the influence of PHA in both normal controls and in patients with rheumatoid arthritis (RA). While [3H]thymidine incorporation was characterized by a rather bell-shaped curve with increasing concentrations of PHA, IL-2 and TNF-alpha displayed a linear dose-dependent increase. [3H]thymidine uptake by PBMC was in the same range in normal subjects as in patients with active and inactive RA, although cytokine secretion differed. The PBMC of patients with active RA produced less TNF-alpha, IL-2, and IFN-gamma than did those of the controls. In cases of inactive RA, the secretory response varied from subject to subject; mean values did not differ from those of normal subjects, except for those of IL-2 (p less than 0.01). The significance and the clinical relevance of these findings are discussed

In vitro effects of thymopentin on the gamma-interferon production by peripheral blood mononuclear cells from normal subjects and from patients with rheumatoid arthritis

The in vitro production of gamma-interferon (gamma-IFN) by peripheral blood mononuclear cells (MNC) was measured using a specific radioimmunoassay in 16 patients presenting with active rheumatoid arthritis (RA), in 14 patients with inactive disease, and in 36 control subjects (CS). Unstimulated cultures produced undetectable levels of gamma-IFN and did not incorporate tritiated thymidine. In response to phytohemagglutinin (PHA) 0.2 microgram/ml, MNC from active RA produced 9 times less, and under PHA 2.5 micrograms/ml, 4 times less gamma-IFN than did MNC from inactive RA or from CS. The uptake of tritiated thymidine was, however, similar in the 3 groups. In unstimulated cultures of the 3 groups, thymopentin (TP-5), at all concentrations tested, did not influence either the levels of gamma-IFN or the uptake of tritiated thymidine. In the presence of PHA 0.2 microgram/ml and TP-5, lambda-IFN levels were increased in CS, unchanged in inactive RA and reduced in active RA, whereas no changes were observed in the uptake of tritiated thymidine. Our results show that under our experimental conditions, TP-5 was able to increase the levels of gamma-IFN produced by normal MNC in vitro, but could not reverse the profound defect observed in active RA