Functional human thyroid cells and their insulin-like growth factor-binding proteins: regulation by thyrotropin, cyclic 3',5' adenosine monophosphate, and growth factors.

The regulation of de novo synthesis of thyroid hormones in primary cultures of human thyroid cells has been examined and correlated with the regulation of the synthesis of the insulin-like growth factor-binding proteins (IGFBPs). In the serum-free culture medium, insulin and TSH (0.01-0.3U/L)were found to be obligatory additives for iodide uptake and organification. In the presence of TSH, cells reorganized into 3D follicles, which stored thyroglobulin. High concentrations of TSH ( > 1U/L), epidermal growth factor, protein kinase C activation with phorbol esters, and transforming growth factor beta 1 all were strongly inhibitory to iodide metabolism and thyroid hormone synthesis. Conditioned medium from the thyroid cell cultures contained at least 5 125I-IGF-labeled bands IGFBPs, including the two glycosylation variants of IGFBP-3. TSH, at concentrations optimal for iodide uptake, inhibited the secretion of all these binding proteins. These effects were mimicked by forskolin and the cell-permeable analog of cAMP, dibutyryl cAMP. The changes in IGFBP proteins were reflected by marked reductions in the steady-state levels of the messenger RNAs of IGFBP-3 and IGFBP-5. This reduction was less pronounced for IGFBP-4. In contrast, protein kinase C activation with phorbol esters and transforming growth factor beta, and high TSH concentrations enhanced IGFBP secretion. Steady-state levels of IGFBP-3 and IGFBP-5 messenger RNAs were elevated after treatment with transforming growth factor-beta and high TSH concentrations. This Study shows that enhanced production of IGFBPs is correlated with inhibition of thyroid function and that TSH, through cAMP, is one factor capable of inhibiting IGFBP production.

Relation between serum interleukin-6 and thyroid hormone concentrations in 270 hospital in-patients with non-thyroidal illness.

OBJECTIVES: Non-thyroidal illness (NTI) is frequently accompanied by alterations in circulating thyroid hormone concentrations, despite patients remaining clinically euthyroid. The mechanisms accounting for these changes in circulating thyroid hormone concentrations remain unknown. Much attention has focussed on the role of inflammatory cytokines which are known to be important mediators of disease. The aim of this study was to investigate the role of the cytokine interleukin-6 (IL-6) in alterations of thyroid hormone metabolism seen in NTI. DESIGN: Longitudinal study of hospital in-patients, correlating serum IL-6 concentrations with circulating thyroid hormone concentrations. PATIENTS: Two hundred and seventy in-patients recruited consecutively, excluding those with known or suspected thyroid disorder. The patients were divided into 5 subgroups reflecting the nature of their NTI and comprised 41 patients with liver disease, 99 with renal disease, 19 intensive care (ITU) patients, 22 with cardiac disease and 89 patients with general medical, or surgical conditions. MEASUREMENTS: Serum IL-6 concentrations were determined using a commercially obtained immunoassay (IL-6 Quantikine assay, R&D Systems, Abingdon, UK). Serum total T4 and total T3 were measured using chemiluminescent immunometric assays (Kodak Clinical Diagnostics Ltd, Amersham, UK) and serum TSH was measured using a third-generation chemiluminescent immunometric assay (Amerlite TSH 30, Kodak Clinical Diagnostics Ltd, Amersham, UK). RESULTS: Ninety-three patients studied (35%) had a serum T3 below the normal range (<1.0 nmol/l), 89 patients (33%) had a serum T4 below the normal range (<65 nmol/l) and in 58 patients (21%) both serum T3 and T4 were below the normal range. There was a significant negative correlation between serum total T3 and IL-6 (r = -0.219; P < 0.001) and total T4 and IL-6 (r = -0.32; P = 0.32), but not between TSH and IL-6 (r = -0.075; P = 0.22). The ITU patient subgroup had the highest IL-6 concentrations (229.3 +/- 48.1 ng/l, mean +/- standard error), whilst also having the lowest T3 (0.93 +/- 0.08 nmol/l), TSH (0.79 +/- 0.25 mU/l) and T4 concentrations (66.6 +/- 7.3 nmol/l). The subgroup of patients under general medical or surgical care had least disturbance of their T3 (low in 19%) and T4 (low in 8%) concentrations, whilst also having the lowest mean IL-6 concentration (39.0 +/- 5.3 ng/l). The renal patient subgroup, whilst including a high proportion of patients with low T3 (39%) and T4 (45%) concentration, demonstrated only modest elevation of IL-6 concentrations (mean 41.4 +/- 8.5 ng/l). CONCLUSIONS: Our data revealed a statistical relation between elevated serum IL-6 concentrations and alterations in circulating thyroid hormone concentrations seen in NTI; however, the findings in patients with renal disease suggest that circulating IL-6 is not the only factor responsible for alteration in thyroid hormone metabolism in NTI.

Serum thyroglobulin: an early indicator of autoimmune post-partum thyroiditis.

OBJECTIVE: The aim of this study was to assess whether autoimmune thyroid damage in post-partum thyroiditis was accompanied by a significant rise in the concentration of thyroglobulin in the serum and whether its measurement could be useful in the prediction of the risk and severity of an episode of post-partum thyroid dysfunction. PATIENTS: Fifty-one women, who had taken part in a larger survey of post-partum thyroiditis, were selected at random for this study. Fourteen women without elevated circulating thyroid autoantibodies and 21 with raised thyroid autoantibodies remained euthyroid throughout the post-partum year. A third group of 14 women had raised thyroid autoantibody levels and showed one or more episodes of thyroid dysfunction during the course of the first year post partum. MEASUREMENTS: Thyroid autoantibodies were measured by ELISA, free T3 and free T4 by the Amerlex M method and TSH by an immunoradiometric method. Serum thyroglobulin was measured by a method free from interference by circulating endogenous thyroglobulin autoantibodies. Thyroid ultrasonography was performed using a General Electric RT3600 scanner operating at 7.5 MHz. RESULTS: Fourteen control women had a mean serum thyroglobulin concentration of 3.3 micrograms/l (SD 4.4; range < 1-12 micrograms/l; 95% confidence interval up to 6.0 micrograms/l). Twenty-one thyroid autoantibody positive euthyroid women had a mean serum thyroglobulin level of 5.8 micrograms/l (SD 6.2; range < 1-36 micrograms/l) which was not significantly different from that seen in the control group. Sixteen thyroid autoantibody positive women who showed one or more episodes of thyroid dysfunction during the post-partum period had a mean serum thyroglobulin of 31 micrograms/l (SD 24.8; range up to 88 micrograms/l) and this was significantly elevated compared with both the control and antibody positive groups (P < 0.001). Serum thyroglobulin concentrations at 3 months post partum correlated with the degree of post-partum hypothyroidism (as indicated by the maximum TSH and the minimum free thyroxine concentrations post partum) and, in those cases where thyroid ultrasound examinations were performed, with the degree of lymphocytic infiltration of the thyroid gland. CONCLUSIONS: The data presented in this paper confirm the destructive nature of post-partum thyroiditis and indicate that the measurement of serum thyroglobulin concentration could assist in the identification of those women at risk of post-partum thyroiditis.

Comparison of second and third generation methods for measurement of serum thyrotropin in patients with overt hyperthyroidism, patients receiving thyroxine therapy, and those with nonthyroidal illness.

We compared serum TSH results determined in second and third generation assays in patients with thyroid disease and nonthyroidal illnesses (NTIs) to evaluate the usefulness of the more sensitive assay. We studied 19 subjects with untreated hyperthyroidism, 12 hyperthyroid subjects sampled at 4-week intervals after beginning carbimazole, 153 subjects receiving T4 replacement, and 300 hospital in-patients with a variety of NTIs. Serum TSH was measured, using a second generation immunometric method, together with free T4 and free T3. Samples with subnormal TSH (< 0.5 mU/L) were reassayed, using a more sensitive chemiluminescent immunometric method. Both assays revealed undetectable serum TSH levels in 18 of 19 overtly hyperthyroid patients. Undetectable TSH values (in both assays) were found in 30 of 33 patients with low serum TSH levels who were receiving treatment for hyperthyroidism, in association with normal thyroid hormone levels in 11. Undetectable TSH was evident in both patients receiving T4 and those with NTI, but use of the more sensitive assay led to a reduction in the number of subjects with undetectable TSH compared with the second generation results (T4-treated, 55 vs. 77 cases; NTI, 13 vs. 19 cases). There was a significant correlation between serum TSH and free T4 in the whole group on T4 (P < 0.001) and in those receiving T4 with low TSH (r = -0.33; P < 0.05); no significant correlation was evident in subjects with low serum TSH levels associated with NTI. An improvement in assay sensitivity led to a reduction in the number of patients being treated with T4 or with NTI in whom serum TSH was undetectable and, hence, an increase in those in whom overt hyperthyroidism could be excluded. Undetectable TSH results, even in a third generation assay, are not diagnostic of overt hyperthyroidism, but are also found in subjects with treated thyroid disease and NTI.

Basic fibroblast growth factor: an autocrine mitogen of rat thyroid follicular cells?

Basic fibroblast growth factor (FGF) is a mitogen for the rat thyroid cell line FRTL-5. A possible autocrine role for this growth factor has been investigated in rat thyroid follicular cells both in vitro and in vivo. We report here the synthesis and localisation of basic FGF and one of its high affinity receptors (flg) in FRTL-5 cells, shown by Northern hybridization analysis, Western blotting, and immunohistochemistry. Two major species of basic FGF mRNA of approximately 2.2 and 7.0 kilobases and one major species of flg mRNA of approximately 4.2 kilobases were identified in FRTL-5 cells. The basic FGF immunoreactivity observed histologically was attributed to a heparin-binding protein of approximately 20 kilodaltons mol wt. The physiological relevance of basic FGF to the thyroid is underlined by the demonstration of significant stores of immunoreactive protein, predominantly in the basement membrane of thyroid follicular cells, in paraffin sections of the normal rat thyroid, although basic FGF mRNA was not detected by in situ or Northern hybridization analysis. The mitogenic response of FRTL-5 cells to human recombinant basic FGF has been further characterized, and the factor shown to stimulate with an ED50 of 4 ng/ml. The mitogenic effects of exogenously supplied and endogenously produced basic FGF were shown to be potentiated by heparin. Examination of the mitogenic activity of both exogenous and endogenous basic FGF and its immunoneutralization in vitro suggests that locally produced basic FGF may be an important autocrine regulator of thyroid follicular cell growth.

Serum thyroglobulin measurements in thyroid cancer: evaluation of 'false' positive results.

OBJECTIVE: Serum thyroglobulin (Tg) should be undetectable in patients successfully treated for thyroid carcinoma. We have examined the course of disease in 19 patients with raised serum Tg (greater than 5 micrograms/l) on initial measurement but no other evidence of residual, recurrent or metastatic cancer. DESIGN: 416 patients from several centres were followed for periods between 1 and 9 years. Serum Tg was measured at 6-12-month intervals. PATIENTS: All had differentiated thyroid cancer, treated by partial or total thyroidectomy and/or 131I ablation, and were receiving suppressive thyroxine therapy. MEASUREMENT: Serum Tg was measured and clinical, X-ray and scan assessment made of presence or absence of residual, recurrent or metastatic cancer. RESULTS: Of 416 patients initially assessed, only 19 had Tg greater than 5 micrograms/l but no clinical or radiological evidence of disease. At follow-up, 11 patients had developed overt signs of malignancy; one had been treated with 131I with a subsequent fall in Tg; five had Tg between 5 and 20 micrograms/l with incompletely suppressed TSH levels; two subjects remained with slightly elevated Tg and undetectable TSH. CONCLUSION: Patients with elevated Tg require careful follow-up even in the apparent absence of disease. Moderate elevation of serum Tg may be due to inadequate thyroxine suppression therapy, assessed by detectable TSH values measured in a sensitive assay.

Basic fibroblast growth factor affects DNA synthesis and cell function and activates multiple signalling pathways in rat thyroid FRTL-5 and pituitary GH3 cells.

We have used a recombinant human basic fibroblast growth factor (basic FGF) to study its effects on cell proliferation, gene expression and accumulation of cyclic AMP (cAMP) and inositol phosphates in two well-characterized endocrine cell lines, FRTL-5 rat thyroid and GH3 rat pituitary cells. Basic FGF induced a dose-dependent increase in mitogenesis (assessed by measuring incorporation of [3H]thymidine) in FRTL-5 cells (40 ng basic FGF/ml increased mitogenesis above the control value by 2148 +/- 108% (mean +/- S.E.M.), but inhibited mitogenesis in GH3 cells at all doses (85 +/- 4% of control with 40 ng basic FGF/ml]. Thyroglobulin mRNA concentration was increased in FRTL-5 cells (126 +/- 6% of control with 40 ng basic FGF/ml) as was prolactin mRNA in GH3 cells (246 +/- 11% of control with 40 ng basic FGF/ml), but GH mRNA in GH3 cells was not significantly affected by any dose of basic FGF. Intracellular cAMP was reduced by basic FGF in both FRTL-5 and GH3 cells (40 ng bFGF/ml giving 80 +/- 5% of the control value in FRTL-5, and 67 +/- 15% of the control value in GH3 cells) despite increased levels when FRTL-5 cells were stimulated with 150 microU TSH/ml (5645 +/- 484% of control) or GH3 cells were stimulated by 10 mumol forskolin/l (3347 +/- 396% of control). In both FRTL-5 and GH3 cells, accumulation of [3H]inositol phosphates were increased by 40 ng basic FGF/ml (201 +/- 6 and 330 +/- 51% of control values respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

Thyrotrophin, forskolin and ionomycin increase cathepsin B mRNA concentrations in rat thyroid cells in culture.

In order to study the regulation of cathepsin B expression in the thyroid, cathepsin B mRNA concentrations were measured in rat thyroid cells (FRTL5) in culture. Northern blot analysis demonstrated that cathepsin B mRNA concentrations were increased in FRTL5 cells cultured for up to 6 days in TSH. The effect of TSH on cathepsin B mRNA concentrations was dose dependent over the range 25-150 mu units/ml. Cytoplasmic dot-blot analysis was used to characterize this effect further. The TSH-induced increase in cathepsin B mRNA concentrations (approximately fivefold over that in untreated cells) was partially mimicked by forskolin (approximately threefold) and ionomycin, while phorbol ester decreased cathepsin B mRNA concentrations. Similar changes were observed for thyroglobulin and actin mRNA concentrations. TSH had no effect on cathepsin B enzymatic activity or immunoreactive protein concentration. These results demonstrate (1) that cathepsin B expression in the thyroid is regulated in parallel with that of thyroglobulin and actin, and (2) that cyclic AMP- and Ca2+-dependent processes stimulate gene expression, while phorbol ester treatment inhibits gene expression in FRTL5 cells.

Evaluation of a sensitive chemiluminescent assay for TSH in the follow-up of treated thyrotoxicosis.

We have studied 21 patients treated for thyrotoxicosis to evaluate a highly sensitive chemiluminescent thyrotrophin (TSH) assay in the assessment of changing thyroid status. Serum TSH was generally suppressed with high serum thyroid hormone concentrations and invariably rose when free T4 and T3 fell substantially below the normal range. However TSH values in 14 of the 21 patients remained undetectable or subnormal for variable periods despite normal or even slightly subnormal free T4 and T3 values. The level of free T4 and T3 at which TSH concentrations rose was highly variable, suggesting differing degrees of hypothalamo-pituitary suppression. Sensitive TSH assays are likely to provide useful information regarding physiological regulation of TSH secretion in man, but our data indicate that in certain circumstances these assays used alone will be inadequate for biochemical assessment of thyroid status.

Serial serum thyroglobulin measurements in the management of differentiated thyroid carcinoma.

Serum thyroglobulin (Tg) was measured on repeated occasions in 416 patients with differentiated thyroid cancer for up to 7 years after initial therapy. All patients had thyroidectomy and/or ablative 131I therapy and all measurements were done while patients were receiving T4 replacement. Tg was measured using a double-antibody radioimmunoassay. Overall correlation between serum Tg concentration and presence or absence of cancer was 95.9%. At the time of initial measurement 295 patients had serum Tg less than 5 micrograms/l, and in latest analysis only 1.7% of these patients showed evidence of disease. Initially there were 19 patients of a total of 121 with serum Tg greater than 5 micrograms/l in whom no cancer was apparent. In eight of these 19 subjects recurrent or metastatic disease has been diagnosed up to 3.5 years after the first measurement indicating that in these cases serum Tg values were 'predictive'. Serum Tg appears to be a sensitive and specific means of detecting residual, recurrent or metastatic thyroid cancer and in most situations can replace routine, expensive and inconvenient radioactive thyroid scans; these should be performed when serum Tg values are elevated or when there is clinical evidence suggesting recurrence.

Clinical application of a sensitive non-isotopic immunometric assay of thyrotropin.

We have evaluated an immunometric assay of thyrotropin (TSH) based on enhanced chemiluminescence signal; its detection limit is 0.06 milli-int. unit/L. Values in 101 clinically euthyroid subjects with normal thyroid hormone concentrations ranged from 0.39 to 6.83 milli-int. units/L. TSH in 15 hypothyroid patients ranged from 10.3 to greater than 200 milli-int. units/L, whereas in 31 hyperthyroid subjects with increased concentrations of free thyroxin and free triiodothyronine, TSH was undetectable serum of all but one subject. Of 32 clinically and biochemically euthyroid patients with goiter, two had undetectable serum TSH and six had values below the normal range. In 19 clinically euthyroid patients from an intensive-care unit, TSH was undetectable in two and below the normal range in another two. This immunometric chemiluminescence assay distinguishes thyrotoxic from euthyroid subjects, but caution is required in interpreting TSH values alone in subjects with goiter or nonthyroidal illness.

Glycosylation of human thyroglobulin and characterization by lectin affinity electrophoresis.

Thyroglobulin, a 660 kDa glycoprotein, is the major product of protein synthesis in the thyroid gland. It has been suggested that modifications of thyroglobulin glycosylation occur in various thyroid disorders. In order to study possible changes in glycosylation of tissue thyroglobulin associated with thyroid disease, we have developed a lectin affinity electrophoresis system which allows characterization of small (less than 1 microgram) quantities of thyroglobulin. Human thyroglobulin was extracted and purified. Agarose gels were cast containing concanavalin A, Ricinus communis agglutinin, L-phytohaemagglutinin and pokeweed mitogen at various concentrations. Purified human thyroglobulin was serially diluted, loaded onto lectin gels and electrophoresed. Concanavalin A, R. communis agglutinin and phytohaemagglutinin all bound thyroglobulin in a concentration-dependent manner. Pokeweed mitogen did not bind thyroglobulin. Purified thyroglobulin was treated with neuraminidase and endoglycosidase H. Two-dimensional immunoelectrophoresis revealed the migration of thyroglobulin to be modified by neuraminidase but not by endoglycosidase H. Lectin affinity electrophoresis of purified human thyroglobulin with and without enzyme treatment indicated the presence of: oligomannose structures as shown by concanavalin A reactivity and modification by endoglycosidase H, and complex oligosaccharides as shown by affinity for R. communis agglutinin and modification by neuraminidase. These structures are in keeping with the proposed patterns of glycosylation of human thyroglobulin and indicate suitability of the method for characterizing the glycosylation of small quantities of thyroglobulin.

An enzyme-linked immunosorbent assay for the measurement of thyroglobulin in human serum using mouse monoclonal antibodies.

A robust, rapid, sensitive and specific enzyme-linked immunosorbent assay (ELISA), using an in-house immunoglobulin-A-sub-class mouse monoclonal human-thyroglobulin antibody (WNSM2), with a sensitivity of 1.51 pmol/l has been established for the measurement of thyroglobulin in serum. Standard curves in varying dilutions of human serum were similar to standard curves obtained in serum-free medium, thus demonstrating no significant cross-reactivity with any serum proteins other than thyroglobulin. Levels of serum thyroglobulin detected by ELISA correlated significantly (r = 0.93, P less than 0.001) with those from a standardized and well-characterized radioimmunoassay. The coefficients of variation within and between ELISA assays were 3.9 and 7.1% respectively. Thyroglobulin was detectable in 87% of 54 normal subjects who had no history of thyroid or autoimmune disease, the mean (+/- S.D.) for this group being 15 +/- 6.6 pmol/l with a range of 1.51-53 pmol/l. Using this assay, levels of thyroglobulin were shown to be significantly (P less than 0.002) increased in patients with untreated hyperthyroid Graves' disease compared with normal subjects.

The hypothalamic-pituitary-thyroid axis in type 1 diabetes: influence of diabetic metabolic control.

The influence of diabetic metabolic control on indices of thyroid function was studied in 9 euthyroid, insulin-dependent (Type 1) diabetics. During chronic poor metabolic control (mean fasting blood glucose 13 mmol/l and HbA1 concentrations 14.7%) serum T3 concentrations were low (P less than 0.01) while serum T4 and basal TSH concentrations were normal. After 6-8 weeks of improved metabolic control, mean HbA1 concentrations had fallen to 10.7% (P less than 0.01) and serum T3 concentrations had increased into the normal range. Serum T4 and basal TSH concentrations were unchanged. The serum TSH response to iv TRH remained normal throughout the study. In Type 1 diabetics, with chronic poor metabolic control, the serum T4 concentration and the TSH response to TRH are therefore appropriate indicators of thyroid function.

Control of thyroglobulin secretion from the rat thyroid gland.

Serum thyroglobulin (Tg), measured by radioimmunoassay, was high in 6-propylthiouracil (PTU)-treated rats but low in thyroxine (T4)-treated animals compared with euthyroid controls. Thyroid-stimulating hormone (TSH) stimulated Tg release in vitro from enzymatically dispersed normal rat thyroid cells in a dose-dependent manner. Thyroid cells prepared from T4-treated animals behaved similarly to cells from control rats, whereas in vitro basal release of Tg from thyroid cells prepared from PTU-treated animals was high and the response to TSH was lost. Our data confirm the TSH dependency of Tg release in vivo and in vitro and our system provides a means of studying the control of Tg secretion in vitro.

Should one measure serum thyroglobulin in the presence of anti-thyroglobulin antibodies?

Thyroglobulin auto-antibodies (anti-Tg) may affect measurement of serum thyroglobulin (Tg). We have assayed sera for Tg by RIA regardless of presence or absence of anti-Tg, and this study was designed to assess the influence of anti-Tg on the results. Binding of human anti-Tg by the second antibody in our RIA (sheep anti-rabbit immunoglobulin) is very low and sera containing anti-Tg of varying titres do not systematically affect assay of Tg regardless of concentration. In our follow-up study of patients with thyroid cancer, correlation between clinical state and Tg concentration is greater than 97% whether anti-Tg is present or absent. These results indicate that in our assay anti-Tg positive sera do not need to be excluded.