Biomedical subjects

S R Crosby

Publications and source records attributed to S R Crosby.

27 records · Page 2Linked to original sources

PubMed1990

ACTH adrenal cell bioassay: improved sensitivity (12 ng/L) achieved by immunoextraction of ACTH from human plasma by a monoclonal antibody.

We have previously reported a bioassay for human plasma ACTH based upon trypsin dispersed guinea-pig adrenal cells which was sensitive to 100 ng/L ACTH in unextracted human plasma when measured against human pituitary ACTH (1-39) standard 74/555. We now present a bioassay of increased sensitivity (12 ng/L) which incorporates three major changes. The trypsin/trypsin inhibitor step in the cell dispersion protocol has been replaced with collagenase, donor calf serum (3%) has been incorporated into the standard curve and ACTH has been extracted from human plasma and dilutions of standard hormone by a sephacryl bound monoclonal antibody (2A3) directed towards the 25-39 sequence. The extracted standard curve has a detection limit of 6 ng/L and the cells can tolerate up to 50% plasma equivalent concentration. Thus, the improved assay has a detection limit of 12 ng/L ACTH in plasma. The assay can now measure bioactive plasma ACTH levels reliably in the normal range.

Adrenal Glands↗

PubMed1990

Defective glucocorticoid regulation of proopiomelanocortin gene expression and peptide secretion in a small cell lung cancer cell line.

A human small cell lung cancer cell line (COR L103) that actively expresses the proopiomelanocortin (POMC) gene has been used as a model of extrapituitary ACTH-secreting tumors to investigate the phenomenon of resistence of ACTH production to glucocorticoids. After both short term (24 h) and long term (10 days) exposure to hydrocortisone at concentrations of 500 and 1000 nM, the accumulation of intracellular POMC mRNA, ACTH, and ACTH precursor peptides in the culture medium was not suppressed. These finding contrast with those in the pituitary corticotroph cell line AtT20, in which POMC mRNA, ACTH, and ACTH precursors were suppressed under the same conditions. Two other genes that are regulated by glucocorticoids in other cell types, the tyrosine amino transferase gene and the glucocorticoid receptor gene, were expressed in COR L103 cells. However, neither gene appeared to be regulated by hydrocortisone in this small cell lung cancer cell line. Further studies demonstrated that glucocorticoid receptor binding could be detected in the nucleus and cytoplasm, with a Kd of 5 X 10(-9) M. It is concluded that nonsuppression of POMC by glucocorticoids is probably part of a more global defect of glucocorticoid signaling in these cells, but that this defect lies distal to steroid binding in the nucleus.

Adrenocorticotropic Hormone↗

PubMed1990

Comparison of ACTH and ACTH precursor peptides secreted by human pituitary and lung tumour cells in vitro.

The molecular forms of ACTH secreted by established human small cell lung cancer (SCLC) cells and primary cultures derived from a bronchial carcinoid tumour, a pituitary adenoma and hyperplastic pituitary tissue have been characterized by Sephadex G-75 chromatography and quantified with two novel immunoradiometric assays for ACTH and ACTH precursor peptides. Pro-opiomelanocortin (POMC; Mr 31,000) and pro-ACTH (Mr 22,000) were secreted by all cell types. No smaller peptides were identified in the culture media from SCLC and bronchial carcinoid cells, implying a deficiency in the enzymes and/or intracellular organelles required for extensive POMC processing. A more heterogeneous profile of ACTH-containing peptides was produced by cells of pituitary origin, indicating more extensive proteolytic processing of POMC. However, the major peptide secreted by cells from a large aggressive pituitary adenoma was unprocessed POMC (Mr 31,000). These results suggest that both lung and pituitary cells in vitro retain their in-vivo pattern of POMC processing and provide valuable models in which to study the regulation of ACTH synthesis and secretion.

Adenoma↗

PubMed1989

Small cell lung cancer cell lines secrete predominantly ACTH precursor peptides not ACTH.

A panel of 18 well characterised human small cell lung cancer (SCLC) cell lines was assessed for the production of adrenocorticotrophin (ACTH) and its precursor peptides, pro-opiomelanocortin (POMC) and pro-ACTH. These precursor peptides were measured directly using a novel two-site immunoradiometric assay (IRMA) based on monoclonal antibodies, in conjunction with a similar IRMA for ACTH 1-39. Significant concentrations of ACTH precursors were secreted by 10 of the 18 cell lines (56%). The low levels of ACTH immunoreactivity detected in seven cell lines could be accounted for by the known cross-reactivity of precursors in the ACTH IRMA. This suggests there is little, if any, processing of ACTH precursors to ACTH. Cell pellet extracts contained undetectable or low levels of ACTH precursors and ACTH, indicating that these peptides are not stored intracellularly. During the growth of the SCLC cells in vitro ACTH precursors accumulated progressively in the culture medium. Thus the combination of a direct assay for the ACTH precursors and the panel of SCLC cell lines provides a valuable in vitro model for the expression of POMC in human tumours.

Adrenocorticotropic Hormone↗

PubMed1989

Pro-opiomelanocortin gene expression and peptide secretion in human small-cell lung cancer cell lines.

Expression of the RNA coding for the ACTH-beta-lipotrophin precursor, pro-opiomelanocortin (POMC), has been demonstrated in five human small-cell lung cancer (SCLC) cell lines. Using Northern and slot-blot hybridization analysis of RNA and a bovine POMC cDNA as probe, the processed POMC RNA from SCLC cells was found to be approximately 1350 nucleotides in length, which is larger than that found in the normal human pituitary. Expression of the POMC gene was confirmed by measurement of ACTH precursors secreted by the cells, using a novel two-site immunoradiometric assay based on monoclonal antibodies, which directly quantifies both POMC and pro-ACTH but does not recognize ACTH. Levels of POMC in medium accumulated throughout the growth of the cells, in contrast to POMC RNA which showed a relatively constant level of expression. We conclude that human SCLC cell lines are valuable models for studying the aberrant expression and regulation of the human POMC gene.

Adrenocorticotropic Hormone↗

PubMed1988

Direct measurement of the precursors of adrenocorticotropin in human plasma by two-site immunoradiometric assay.

An immunoradiometric assay (IRMA) for the direct measurement of the precursors of ACTH in unextracted human plasma has been developed and evaluated clinically in normal subjects and patients with disorders of the hypothalamic-pituitary-adrenal axis. The IRMA is based on an iodinated monoclonal antibody to ACTH and a monoclonal antibody to gamma MSH coupled to Sephacryl S300. The assay detects only peptides containing both epitopes, i.e. POMC (31K) and pro-ACTH (22K). The reference standard was partially purified POMC from culture medium of human corticotroph adenoma cells. The detection limit (greater than +2.5SD of the 0 standard) was 2.0 pmol/L and the within-assay coefficient of variation was less than 10% between 29 and 2600 pmol/L. Plasma concentrations of ACTH precursor peptides in 11 normal subjects sampled at 0930 h ranged from 5-34 pmol/L. The concentrations in the patient groups studied were: 260-2300 pmol/L in 5 patients with the ectopic ACTH syndrome associated with small cell lung cancer, less than 2.0-104 pmol/L in 10 patients with pituitary-dependent Cushing's disease, 23 pmol/L in a patient with Nelson's syndrome, and 3.0-230 pmol/L in 5 patients with Addison's disease. We conclude that this IRMA offers a simple and reliable method for measuring ACTH precursors in unextracted plasma. The proportionately greater elevation of ACTH precursors compared to ACTH in patients with the ectopic ACTH syndrome associated with small cell lung cancer but not in pituitary-dependent Cushing's syndrome, suggests that this assay may be clinically useful.

ACTH Syndrome, Ectopic↗

PubMed1986

Characterisation of monoclonal and polyclonal antibodies to 19-linked conjugates of testosterone with corresponding radioligands.

Monoclonal antibodies to testosterone T were produced using testosterone 19-O-carboxymethyl ether (T19C) and testosterone 19-hemisuccinate (T19H) immunogens. All antibodies were characterised with iodinated derivatives of both T19C and T19H. Monoclonal antibodies derived from the T19C immunogen had similar titres and assay sensitivities with both T19-tracers. In contrast antibodies derived from the T19H immunogen bound the homologous but not the heterologous tracer. Individual antibodies showed a wide variation in cross-reactivity with 5 alpha-dihydrotestosterone, DHT (4.4-100%), androstenedione AN (0.5-100%) and progesterone, Po (0.08-5.4%). One antibody 3F11 derived from a T19C immunogen gave 50% displacement of tracer with 180 pgT/tube and low cross-reactivity of 12% with DHT, 3.0% with AN and 1.1% with Po. In general, assay sensitivity and antibody specificity were poorer with an [125I]-histamine conjugate of T-3-carboxymethyloxime than with T19 tracers. Radioimmunoassays for T in extracted human serum were developed with [125I]T19C as tracer and monoclonal antibody 3F11 (T19C immunogen) and rabbit antiserum T19H3R1 (T19H immunogen). Sensitivities of the extracted assays were 43 and 20 pg/tube respectively and results correlated well with those obtained after chromatographic separation of testosterone (r = 0.97 for both antibodies). We conclude that 19-linked derivatives of T are highly immunogenic for the production of specific testosterone antibodies. Selection of the appropriate iodinated tracer is essential to achieve optimal titre, assay sensitivity and specificity, since these characteristics vary widely with individual monoclonal antibodies, and classical bridge recognition is not observed.

Androstenedione↗

PubMed1985

A study of 19-O-carboxymethyl ether and 19-hemisuccinate derivatives of testosterone: their immunogenicity and use as iodinated radioligands for radioimmunoassay of testosterone.

Testosterone 19-O-carboxymethyl ether (T19C) and 19-hemisuccinate (T19H) derivatives were synthesised, and coupled to bovine serum albumin (BSA) or porcine thyroglobulin (PT) for immunogens or to iodohistamine for radioligands. The immunogenicity of these conjugates in mice was compared with those of testosterone 3-O-carboxymethyloxime and 15 beta-thioethyl conjugates. Of 10 immunogens studied, those linked to PT gave the highest antiserum titres and more sensitive standard curves. The site of conjugation (19 or 3 position) had little effect on immunogenicity. Cross-reactivity with 5 alpha-dihydrotestosterone (DHT) was in the range 22-100%, for the 19-linked immunogens. Antisera to T19C and T19H conjugated to PT were then raised in rabbits and characterised with 4 radioligands. Homologous assay systems in which the chemical bridge was identical in immunogen and 125I-radioligand gave the highest antiserum titres but the poorest assay sensitivity while those heterologous with respect to bridge or site gave the most sensitive standard curves. Rabbit antisera to both T19C and T19H immunogens showed good specificities with respect to DHT (range of cross reactions 0.78-21.1%), androstenedione, AN (range 0.45-2.3%) and progesterone, Po (range 0.05-1.4%) with all radioligands. The best assay system employed an antiserum to the T19H-PT immunogen with heterologous radioligand [125I]T19C. It had a detection limit of 15pg/tube and low cross-reactivity with DHT (0.78%), Po (0.3%) and AN (0.43%). We conclude that 19-linked derivatives of testosterone conjugated to PT are good immunogens and the antisera when combined with [125I]testosterone radioligands which are heterologous with respect to the chemical bridge, provide highly specific assays for testosterone with potential for clinical application.

Animals↗

PubMedDate not supplied

Cerebrospinal fluid levels of beta endorphin in painful and painless diabetic polyneuropathy.

beta endorphin (beta-EP) is an important modulator of central pain pathways. To examine whether changes in central production of beta-EP contribute to the pathogenesis of diabetic neuropathic pain, we compared the cerebrospinal fluid (CSF) levels of beta-EP and its precursor proopiomelanocortin (POMC) between 15 diabetic patients with chronic painful diabetic polyneuropathy, eight patients with severe painless diabetic neuropathy, and ten nondiabetic controls. Both peptides were measured by specific monoclonal antibody-based two-site immunoradiometric assays (IRMAs). In the diabetic patients with painful neuropathy, mean +/- SD CSF beta-EP concentrations (5.7 +/- 2.2 pmol/L) were comparable to those of the diabetic patients with painless neuropathy (6.0 +/- 2.3 pmol/L) and did not correlate with the severity of neuropathic pain. CSF beta-EP, but not POMC, concentrations were lower in the diabetic neuropathic patients overall (5.8 +/- 1.9 pmol/L) compared to the control subjects (7.6 +/- 2.2 pmol/L) (p < 0.05). CSF POMC showed no intergroup differences. However, POMC levels were 80-fold higher than those of beta-EP and should always be considered when interpreting immunoreactive beta-EP or other derivative peptide levels in CSF. We conclude that CSF beta-EP levels appear to be reduced in diabetic polyneuropathy but they do not relate to the presence of neuropathic pain. This might explain why opioid analgesics are of little, if any, help in alleviating diabetic neuropathic pain.

Cohort Studies↗