Immunoglobulin G inhibitor of thyroid-stimulating antibody is a cause of delay in the onset of neonatal Graves' disease.

Studies were carried out with the serum IgG from a mother and her two children who developed neonatal Graves' disease several weeks after birth. The maternal IgG: (a) stimulated the human thyroid in vitro, but maximal stimulation was found only with dilution of the IgG; (b) was very potent in the long-acting thyroid stimulator (LATS)-protector assay, but only when an inhibitor of the system was diluted out; (c) inhibited a standard preparation of LATS in the mouse bioassay; (d) was biphasic in the thyrotropin-binding inhibition (TBI) assay, i.e., enhanced binding at low concentrations of IgG and inhibited binding at high levels. Enhancement in the TBI assay was found only with particulate preparations of human thyroid membranes as receptor and not when that material was solubilized, nor with guinea pig fat cell membranes as receptor. Serial blood samples from the second child were obtained at birth and until 3 mo of age. In the thyroid slice (cyclic AMP) assay system there was a negative dose-response relationship in testing IgG until age 45 d when it became positive, coinciding with the clinical recognition that hyperthyroidism had developed. The data are compatible with a concept that this mother's IgG contained thyroid-stimulating antibody (TSAb) and another moiety that inhibited TSAb through an action on the thyroid cell membrane, thus delaying the onset of hyperthyroidism in the neonate until the inhibiting IgG was metabolically cleared to an ineffective concentration.

Association of the long-acting thyroid stimulator protector with the immunoglobulin G fraction of serum from patients with thyrotoxicosis.

Both the long-acting thyroid stimulator and the long-acting thyroid stimulator protector are associated with the immunoglobulin G fraction of human serum proteins. Fractionation of globulins by (NH4)2SO4 precipitation and DEAE-cellulose chromatography showed that the protector activity was associated with subfractions of differing electrophoretic mobility. Attempts to concentrate the protector in immunochemically pure immunoglobulin G prepared in this way may fail because of loss of protector activity with other immunoglobulin G fractions. Gel filtration on Sephadex G-150 indicated that protector activity was associated with 7-S proteins.

Comparison of human and porcine thyroid membranes for radioreceptor assay of bovine thyrotrophin and thyrotrophin-binding inhibiting immunoglobulins.

A detailed comparison between the use of human and porcine thyroid membranes for the radioreceptor assay (RRA) of bovine TSH (bTSH) and thyrotrophin-binding inhibiting immunoglobulins (TBIIg) is reported. Bovine thyroid membranes were also investigated but were found to be far less satisfactory than either human or porcine thyroid membranes. The affinity constant (ka) of the interaction of bTSH with porcine thyroid membranes (Ka = 3.3 X 10(9) l/mol) measured b Scatchard analysis was higher than with human thyroid membranes (Ka = 2.1 X 10(8) l/mol). Porcine thyroid membranes were more sensitive for the assay of bTSH (detection limit 30 microunits, half-maximal inhibition 0.3 microunit) than human thyroid membranes (detection limit 200 microunits, half-maximal inhibition 7.4 mu.). Preincubation of membranes from either species with immunoglobulin rich in long-acting thyroid stimulator (LATS) inhibited the saturable binding of 125I-labelled TSH to a greater extent than did normal immunoglobulin. The binding of 125I-labelled TSH to porcine membranes was more sensitive to the inhibitory effect of LATS-immunoglobulin and was also less affected by normal immunoglobulin than was binding to human thyroid membranes. When assayed with each type of membrane preparation there was good correlation between the RRA of immunoglobulins prepared from patients with Grave's disease and from normal subjects (n = 18) (r = 0.85, P less than 0.001, n = 73). The incidence of positive TBIIg in untreated Grave's disease was greater for porcine than for human thyroid membranes.

Characteristics of thyrotrophin-stimulated cyclic AMP production in cultured human thyroid cells from thyrotoxic tissue and non-toxic goitres.

We have described a system for the maintenance in culture of isolated human thyroid cells from both thyrotoxic tissue and non-toxic goitres. The cells isolated from the two thyroid tissue types showed similar cyclic AMP response characteristics to TSH with large increases in intracellular and extracellular cyclic AMP after 20-min incubations. Maximal responses were obtained with 50 mu. TSH/ml and half-maximal responses at 1.0 mu. TSH/ml. With cell passage the cyclic AMP responses to TSH decreased in magnitude and sensitivity. As with other thyroid cultures, growth of the cells with TSH induced arrangement into follicular structures, whereas cells grown in the absence of TSH remained as a monolayer. Basal intracellular cyclic AMP levels were increased in a dose-related fashion in cells grown in the presence of graded concentrations of TSH and the maximal response to further additions of TSH was not greater than in control cultures.

A role for insulin-like growth factor-I in the regulation of human thyroid cell growth by thyrotrophin.

Human thyroid epithelial cells in monolayer culture were found to release radioimmunoassayable insulin-like growth factor-I (IGF-I) over a 48-h culture period in serum-free medium. In the presence of supraphysiological concentrations of TSH (1-100 mU/ml) known to be inhibitory to DNA synthesis by human thyroid cells, the release of IGF-I was found to be inhibited in six thyroid cultures studied. In only one out of the six was IGF-I release increased in the presence of physiological mitogenic concentrations of TSH (0.1-100 microU/ml). Human thyroid fibroblasts, established by long-term culture of thyroid epithelial cells under fibroblast-selective conditions, also secreted IGF-I which was unaffected by the presence of TSH at both low and high concentrations. Using a monoclonal antibody against human IGF-I, monolayer cultures of both human thyroid epithelial cells and human thyroid fibroblasts showed positive immunocytochemical staining for IGF peptide. However, fixed sections of intact thyroid tissue only showed positive staining for IGF peptide associated with the fibrous layers surrounding the thyroid follicle, with no staining of the follicular epithelial cells. The growth of human thyroid epithelial cells was also found to be increased by IGF-I (25-100 ng/ml) added in medium plus 1% fetal calf serum as assessed by the incorporation of [3H]thymidine into DNA. In the presence of a monoclonal antibody to IGF-I the increase in [3H]thymidine uptake in response to IGF-I was abolished as was that seen in response to TSH.(ABSTRACT TRUNCATED AT 250 WORDS)

Thyrotrophin and thyroid-stimulating immunoglobulins have similar characteristics in activating human thyroid membrane adenylate cyclase.

The activation of adenylate cyclase by TSH and thyroid-stimulating immunoglobulins (TSIg) was investigated in membranes prepared from non-toxic goitres. The assay conditions for maximal adenylate cyclase stimulation by TSH in the absence and presence of a cell cytosol preparation were determined. Cell cytosol had no detectable effect on the characteristics of activation by TSH although it increased the production of cyclic AMP in response to the hormone. In the presence of cell cytosol, membrane protein, pH and substrate concentration dependency of adenylate cyclase activation by both TSH and TSIg were similar. In a comparison of the time-courses of activation there was no apparent lag phase in the activation of adenylate cyclase by TSIg but the onset of activation was slower and linearity persisted longer than with TSH.

Relationship between growth and function of human thyroid cells in culture.

Subconfluent human thyroid cells in monolayer, isolated from thyrotoxic tissue or non-toxic goitres obtained at surgery, responded to the addition of epidermal growth factor (EGF) with an increase in cell growth as measured by increased incorporation of [3H]thymidine into trichloroacetic acid-precipitable material. The growth response to EGF was concentration-dependent and the characteristics of the responses were the same using EGF from murine or human sources. With concentrations which stimulated growth, EGF was found to inhibit human thyroid cell function as measured by the release of radioimmunoassayable tri-iodothyronine into the incubation medium. Thyrotrophin (TSH) was also found to stimulate human thyroid cell growth but at concentrations far lower than those used to stimulate thyroid cell function in this system. The effect of EGF on the differentiating action of TSH on human thyroid cells in culture was also investigated; the association of thyroid cells into two-dimensional follicular structures produced by the incubation of thyroid cells at a high cell density with TSH was found to be inhibited by the addition of EGF.

Interaction of epidermal growth factor with receptors on human and porcine thyroid membranes.

Recent evidence suggests that epidermal growth factor (EGF) may play an important role in the regulation of thyroid growth and function. We have examined the interaction of murine EGF (mEGF) with human and porcine thyroid membranes and compared this with the binding of bovine TSH (bTSH) using 125I-labelled hormones as tracers. The characteristics of the binding of mEGF were found to be similar for human and porcine thyroid membranes. Epidermal growth factor bound with high affinity (affinity constant = 1.4 X 10(9) l/mol); the density of binding sites was low compared with the TSH receptor. At 37 degrees C, the binding of 125I-labelled EGF was maximal at 1 h and was saturable in the presence of unlabelled EGF; half-maximal inhibition was at 1 ng EGF/tube (0.5 nmol/l) using 0.5 mg membrane protein/tube. Unlabelled bTSH had no effect on the binding of labelled EGF. Similarly, unlabelled EGF did not affect the binding of labelled TSH; hence it was concluded that mEGF and bTSH bound to independent sites. Epidermal growth factor had no effect on adenylate cyclase activity in membranes prepared from human non-toxic goitre; increasing concentrations of EGF did not affect basal, TSH-stimulated or fluoride-stimulated enzyme activity.

Evidence that both long-acting thyroid stimulator and long-acting thyroid stimulator-protector stimulate the human thyroid gland.

Thyroid-stimulating immunoglobulins were prepared from two potent sera, one contained long-acting thyroid stimulator (LATS) and the other contained both LATS and LATS-protector (LATS-P). The potencies of the immunoglobulin G (IgG) preparations were estimated in the McKenzie assay. The accumulation of cyclic AMP in mouse thyroid lobes was stimulated only by LATS--IgG; LATS-P--IgG was inactive. In contrast, both LATS-IgG and LATS-P--IgG were equally effective in slices of human thyroid.

Effects of human thyroid-stimulating hormone and immunoglobulins on adenylate cyclase activity and the accumulation of cyclic AMP in human thyroid membranes and slices.

The activation of adenylate cyclase and the accumulation of cyclic AMP resulting from the action of human thyroid-stimulating hormone (TSH), long-acting thyroid stimulator (LATS) or LATS-protector (LATS-P) have been investigated in preparations of human thyroid membranes and slices. Human TSH significantly increased adenylate cyclase activity in membranes from non-toxic goitres whereas LATS and LATS-P had no consistent effect. However, pre-incubation of goitrous membranes with LATS--immunoglobulin G inhibited the effect of TSH on adenylate cyclase. When thyroid membranes were prepared from the glands of patients with Graves's disease neither TSH nor thyroid-stimulating immunoglobulins (TSIg) stimulated adenylate cyclase significantly. Whether from non-toxic goitres or thyrotoxic tissue, the concentration of TSH needed to induce half of the maximum response was lower in thyroid slices than in membranes. Both LATS and LATS-P significantly stimulated the accumulation of cyclic AMP in slices of goitrous tissue but thyrotoxic tissue slices did not respond. In goitrous slices, submaximum concentrations of TSH and TSIg caused additive responses in the accumulation of cyclic AMP but TSIg did not increase the maximum response to TSH.

Human thyroid cells in monolayer retain the ability to secrete tri-iodothyronine in response to thyrotrophin.

Confluent monolayer cultures of human thyroid cells secreted low levels of immunoassayable tri-iodothyronine (T3) and this process could be stimulated by TSH in a concentration-dependent manner. The characteristics of the response to TSH were related to the age of the thyroid cell culture both in terms of the relative sensitivity to TSH and the quantity of T3 released. Cells which had been in culture for 2-3 days (primary cultures) secreted high levels of T3 under unstimulated and TSH-stimulated conditions with a median effective dose (ED50) for TSH of 0.030 mu. TSH/ml. However, cells which had been subcultured and consequently had been in culture for a longer period of 6-7 days secreted lower levels of T3 under basal and stimulated conditions. This was approximately 30% of that released from primary cultures with and ED50 for TSH of 0.1 mu. TSH/ml. Reorganization of human thyroid cells into follicular structures was seen during growth with TSH but these cultures showed little response to subsequent acute stimulation by TSH; the return of a diminished, less sensitive response to TSH was seen after a recovery period of 8 h. The time-course of T3 release was dependent on the TSH concentration with low TSH concentrations stimulating T3 secretion after increased incubation periods. Human thyroid cells had lost the ability to concentrate and organify free iodide after several days in culture but were still secreting T3. This indicates the presence of intracellular stores of T3 which are released on stimulation with TSH, rather than new synthesis of T3.

An investigation of the ability of TSH and Graves' immunoglobulin G to increase intracellular calcium in human thyroid cells, rat FRTL-5 thyroid cells and eukaryotic cells transfected with the human TSH receptor.

The purpose of this study was to determine if immunoglobulin G preparations (IgGs) from patients with Graves' disease can increase intracellular calcium in thyroid cells, as has been reported for TSH. Both TSH and Graves' IgGs (prepared by protein G affinity chromatography) increased calcium in a range of thyroid cells; however, the response seen, using Fura-2-loaded coverslips of cell monolayers, varied considerably. Chinese hamster ovary (CHO/JPO9) cells transfected with a high number of human TSH receptors showed the greatest response: TSH (10 mU/ml) increased calcium in 46% of experiments and 18 out of 25 (72%) Graves' IgGs increased calcium at 0.1 mg/ml (significantly greater, P < 0.001, than for control IgGs where cells responded to 2 out of 13 preparations). Rat FRTL-5 cells only responded to TSH in 22% of experiments and to 2 out of 8 (25%) of Graves' IgGs. Similarly, human thyroid cells responded to TSH in 22% of experiments and to 2 out of 9 (22%) of Graves' IgGs. (When studying cyclic AMP responses in JPO9 cells, much higher concentrations of Graves' IgGs were required (1-3 mg/ml). However, higher concentrations (0.3 mg/ml) of both Graves' IgGs, and to a lesser extent of control IgGs, were capable of increasing calcium in cells both with and without TSH receptors (control CHO cells and normal human dermal fibroblasts). We conclude that relatively low concentrations of patient IgGs can be distinguished from control IgGs in JPO9 cells on the basis of their ability to increase calcium, but that additionally all IgG preparations possibly contain another factor which can increase calcium in a range of cells independent of the presence of the TSH receptor.

Extracellular calmodulin and its association with epidermal growth factor in normal human body fluids.

In this study we describe the occurrence of a calmodulin-like protein in normal human biological fluids. Extraction of the calmodulin-like protein from breast milk, saliva, serum and urine provided an extract with enhanced calmodulin immunoreactivity which, in the case of milk and saliva, showed a protein band comigrating with authentic calmodulin (Mr 17,000) on sodium dodecylsulphate-polyacrylamide gel electrophoresis. However, in milk, saliva and serum a major protein band of Mr 14,000-15,000 was always observed, which we speculate may be related to calmodulin, possibly as a partially degraded form. Estimates of biologically active calmodulin in most normal extracellular fluids were of the order which we have found will stimulate cell division when added to the extracellular medium of cells in culture. Levels ranged from 0.03 nmol/l in urine to 18.6 nmol/l in breast milk, and exhibited a quantitative relationship (r = 0.79, P less than 0.01) to epidermal growth factor (EGF) levels in fluids. Where EGF concentrations varied from normal (increased in saliva 24 h after oral surgery and reduced in the urine of patients with renal failure) calmodulin concentrations were similarly affected. The presence of calmodulin in serum may in part be attributable to its release from platelets which are particularly rich in calmodulin. Release of calmodulin from the platelet was associated with that of EGF and other platelet products.

A possible role for calmodulin in human thyroid cell metabolism.

A protein which shared several characteristics with authentic calmodulin was extracted from human thyroid homogenates. The protein bound to fluphenazine--Sepharose and could be specifically eluted using EGTA. The eluted protein had a u.v. spectrum characteristic of calmodulin and migrated like authentic calmodulin with a calcium-dependent shift on sodium dodecyl sulphate polyacrylamide-gel electrophoresis. Calmodulin in thyroid cell extracts was shown to be biologically active, measured by its ability to activate a calmodulin-deficient cyclic GMP phosphodiesterase; this activation could be inhibited by trifluoperazine. A possible role for calmodulin in the action of TSH on the thyroid was demonstrated by studying the effects of phenothiazines and the naphthalene sulphonamide, W7, a more specific calmodulin inhibitor, on TSH-stimulated cyclic AMP levels in cultured thyroid cells. The phenothiazines and W7 were found to inhibit the accumulation of cyclic AMP in response to TSH in a concentration-dependent manner although low concentrations of W7 enhanced TSH-stimulated cyclic AMP accumulation.

Thyroid stimulating immunoglobulins, like thyrotropin activate both the cyclic AMP and the PIP2 cascades in CHO cells expressing the TSH receptor.

In human thyrocytes and in a permanent CHO cell line expressing the human thyroid stimulating hormone (TSH) receptor cDNA (JP09 cells), TSH activates both the cyclic AMP and the phosphatidylinositol 4,5-bisphosphate (PIP2) cascade, although the latter effect requires higher TSH concentrations. Thyroid stimulating autoantibodies (TSAb) activate also the human thyroid leading to the hyperthyroidism of Graves' disease. They bind to the TSH receptor and mimic the TSH stimulation of the gland by increasing intracellular cyclic AMP, but they do not enhance PIP2 hydrolysis in human thyroid slices. We show in this study that TSAb are able to activate the PIP2 cascade in JP09 cells, a cell line expressing high levels of TSH receptor. This suggests that the mechanism of action of TSAb on the TSH receptor is qualitatively similar to that of TSH.

Human infection in Tayside, Scotland due to Salmonella serotype Livingstone.

Livingstone was the third most common salmonella serotype isolated from cases of human salmonellosis in the Tayside region of Scotland in 1989-1991; latterly, it spread to Grampian region. The significant upsurge of Livingstone in these two Scottish regions was not matched by similar increases in its frequency of isolation from human cases of salmonellosis in other regions of Scotland or elsewhere in the UK. Although Salmonella Livingstone is usually associated in the UK with incidents of infection among poultry flocks, our detailed investigations found no clear evidence that poultry, eggs or poultry-related products were responsible for this outbreak. Most cases occurred in the summer months from July to September and many of the patients required hospital treatment. Other than one outbreak among geriatric patients in a long-stay hospital in north Tayside, most of the cases were sporadic. The extent of the outbreak, covering 3 years, was recognised mainly because Livingstone was previously an uncommon serotype in Tayside. There were few Livingstone isolations from non-human sources in Scotland in these same years. Possible sources of infection and predisposing factors among patients are discussed. Livingstone was not isolated in Scotland in 1992.

Monophasic group B Salmonella species infecting harbour porpoises (Phocoena phocoena) inhabiting Scottish coastal waters.

A monophasic strain of Salmonella group B having the antigenic structure 4, 12: a: - was isolated in culture from various tissues of 39 harbour porpoises (Phocoena phocoena). The tissue from which the organism was recovered most frequently was lung in 33 animals and intestine in 16 animals, but it was also isolated from heart valve, liver, kidney, spleen, mesenteric lymph node, pulmonary lymph node, hepatic lymph node, urethra, sheath and epididymis. As far as we are aware this is the first record of this strain from an animal source, raising the possibility that it may be host-adapted to harbour porpoises. The possible modes of transmission of monophasic group B Salmonella between porpoises are discussed.

A role for intracellular calcium and calmodulin in the release of triiodothyronine from human thyroid-cell monolayer cultures.

Human thyroid cells in monolayer responded to acute stimulation by TSH with an increase in the secretion of T3. This process appeared to be dependent on a rise in the cytosolic calcium concentration since the antagonist of intracellular calcium mobilization, TMB-8, was found to inhibit the release of T3 in response to TSH. The importance of intracellular calcium was further shown using the agent veratridine which increases the free calcium level within cells; veratridine potentiated the stimulation of T3 secretion by TSH and itself stimulated the release of T3 to a level higher than that seen in the presence of TSH alone. The calcium ionophore A23187 produced a biphasic effect on T3 secretion from human thyroid monolayers; at low concentrations, A23187 caused a decrease in both unstimulated and TSH-stimulated T3 secretion but above a concentration of 1 microM, T3 secretion was increased. The calmodulin antagonist W7 was found to inhibit T3 release in response to TSH, indicating a role for calmodulin in mediating the effects of intracellular calcium on T3 secretion.

Adrenal scintigraphy with 75Se selenonorcholestenol: a review.

We have reviewed 58 patients on whom adrenal scintigraphy has been performed using 75Se selenonorcholestenol. For 15 patients whose adrenal function was biochemically normal, the upper limit of normal of the 7 day adrenal uptake test was 0.45%, considerably higher than the generally accepted value of 0.3%. There is evidence from this group of patients that stress and obesity might account for uptakes in the range 0.3-0.45%. The sensitivity of the uptake test is poor, with 7 out of 23 patients with Cushing's syndrome having uptakes within the normal range. Scintigraphy of such patients may still be useful in differentiating between unilateral and bilateral adrenal involvement.