Case Report: Burns Through Metal - Unique Pattern Of First Lightning Strike Injury In Israeli Army.

This case report presents a 19-year-old Israeli soldier who sustained injury as a result of a lightning strike during an outdoor military activity. The patient was found in a state of altered consciousness and respiratory distress. He suffered multiple second-degree burns to the neck, chest and abdomen area, corresponding to the locations of his metal identification tag, rifle and belt buckle. Lightning transmission through these metal objects caused considerable thermal burns at contact sites. The patient was treated conservatively until wound healing and stabilization of respiratory function. This is the first reported case of military personnel injured by lightning strike in Israel. Although rare in the Mediterranean region, safety guidelines and regulations should be implemented to avoid the associated serious and permanent injuries that may be caused by lightning strikes.

Nous présentons le premier cas décrit en Israël d'un soldat de 19 ans frappé par la foudre dans un exercice en extérieur. Il a été relevé avec des troubles de conscience et une détresse respiratoire. Il souffrait de brûlures du deuxième degré du cou, du thorax et de l'abdomen, au niveau de sa plaque d'identité, de la boucle de bretelle de son arme et de la boucle de ceinturon. Le passage de la foudre au niveau de ces éléments métalliques a dégagé une quantité considérable de chaleur. La prise en charge locale a été non invasive, concomitamment à la stabilisation de la fonction respiratoire. Bien que les orages soient rares au moyen- orient, des moyens de prévention primaire vis-à-vis du foudroiement doivent y être développés afin d'éviter les déficits permanents qu'ils sont susceptibles d'engendrer.

Preparation and culture of a serum-free human thyroid follicle system and its application for measuring thyroid hormone secretion, iodide uptake and organification, cyclic adenosine monophosphate formation, gene expression, and cell growth.

We describe a system of human thyroid follicles cultured in collagen suspended in serum-free medium. The method allows measurement of thyroid hormone secretion, iodide uptake and organification, cyclic adenosine monophosphate (cAMP) formation, gene expression, as well as cell growth. The system is superior to follicles freely suspended in cultured medium and also, as demonstrated by parallel experiments, to monolayer culture systems. A detailed description of the optimal conditions of the method is provided that, over a period of 8 years, has proven to be a powerful tool for measuring thyroid cell function, gene expression and cell proliferation.

Sulfate transport is not impaired in pendred syndrome thyrocytes.

Pendred syndrome is the most common form of syndromic deafness, characterized by dyshormonogenic goiter associated with sensory-neural deafness. The gene responsible for the disease (PDS) has been cloned, but its function is as yet unknown and the connection between thyroid goiter and sensory-neural deafness remains an enigma. PDS codes for a novel protein, pendrin, which is closely related to a number of sufate transporters. Mechanisms by which abnormal sulfate transport could deleteriously affect iodide organification have been proposed. We tested sulfate transport in thyrocytes obtained from Pendred syndrome patients and found that it was not defective. This suggests that pendrin in fact may not be a sulfate transporter, and emphasizes the importance of functional studies on this novel protein.

Regulation of HLA-DR and costimulatory B7 molecules in human thyroid carcinoma cells: differential binding of transcription factors to the HLA-DRalpha promoter.

The consequence of autoantigen presentation by thyroid cells is dependent on the magnitude of expression of both HLA class II antigens (mainly HLA-DR) and costimulatory molecules, such as B7 (CD80 and CD86). Autoimmune thyrocytes are induced to express HLA-DR by interferon-gamma (IFN-gamma). The costimulatory signal leading to autoantibody production or cytotoxic T-cell immune response could be provided by antigen presenting cells (APCs) attracted to the thyroid by the primary autoimmune stimulus. Malignant thyrocytes can express HLA-DR antigens either constitutively, as a result of a nonimmunologic stimulus, or on induction with IFN-gamma after triggering of an immune response. However, their ability to express B7 molecules, which may determine enhanced antitumoral immune response mainly in the absence of intrathyroidal macrophages, has not yet been studied. The regulation of HLA-DR gene expression in APCs, such as B cells, is mediated by a series of short DNA consensus sequences located in the promoter, termed the W, X, and Y boxes, which bind several known transcription factors. We have previously characterized the expression of HLA-DR in four human thyroid carcinoma cell lines and found differences between constitutive and high- or moderate-induced expression of the protein and mRNA. Evaluation of B7 expression on the surface of thyroid cancer cells and understanding the mechanisms of HLA-DR gene expression may help in designing efficient immune response to thyroid tumors. Using the electrophoretic mobility shift assay (EMSA), we have demonstrated differences between the four thyroid cell lines in the binding of transcription factors to each of the three boxes. The binding to the promoter in each of the cell lines resulted in a single band, probably representing a complex of proteins formed via protein-protein interactions. Using flow cytometry we have shown that the B7 molecule was absent in the four thyroid cell lines and could not be induced by IFN-gamma. The absence of surface B7 molecules from the malignant thyroid cells may lead to either suppression of antitumoral cytotoxic T cell response or demand the cooperation of infiltrating APCs to favor immune response. Differences previously found in HLA-DR expression in the four human malignant thyroid cell lines may be explained by the variation in the binding of transcription factors to the boxes in the HLA-DRalpha promoter. The binding patterns of nuclear proteins derived from the four thyroid cell lines or from the B lymphocyte cell line--Raji--to each of the boxes or to the whole promoter exhibit similarities, thus suggesting similar DNA-protein interactions.

Desialylated and deglycosylated human chorionic gonadotropin are superagonists of native human chorionic gonadotropin in human thyroid follicles.

Highly purified human chorionic gonadotropin (hCG) interacts with the thyrotropin (TSH) receptor and stimulates triiodothyronine (T3) secretion, iodide uptake and organification, and cyclic adenosine monophosphate (cAMP) formation in human thyroid follicles. Because of interest in the role of the carbohydrate component in the structure-function relationships of hCG we undertook to deplete hCG of its sialic acid or carbohydrate residues and assess the thyrotropic activity of the carbohydrate-modified forms. For this purpose, we used our assay system consisting of human thyroid follicles cultured and suspended in collagen gel in serum-free medium. Under these conditions, the cells are organized as follicular three-dimensional structures with normal polarity, enabling enhanced responsiveness to hormonal stimulation, and T3 secretion can be measured as a response parameter. Desialylated (ds)-hCG and deglycosylated (dg)-hCG dose-dependently stimulated T3 secretion, iodide uptake and organification, and in each case did so with about twice the intrinsic activity of native hCG. Indeed, removal of the sialic acid or carbohydrate residues from native hCG transformed it into a thyroid stimulator that elicited a maximal response in terms of iodide uptake, organification and T3 secretion by human thyroid follicles as high as TSH and almost twice as high as native hCG. Not only were ds-hCG and dg-hCG more intrinsically active than hCG, they were more than five times as potent. As with hCG, both ds-hCG and dg-hCG managed to elicit such responses in human thyrocytes while evoking minimal amounts of cAMP, illustrating the concept of cAMP superfluity and highlighting the potential pitfalls of using cAMP as a measure of hormonal bioactivity. hCG, and to a greater extent ds-hCG and dg-hCG, inhibited, as did TSH, gamma-interferon-induced human leukocyte antigen-DR (HLA-DR) expression in human thyrocytes, again reflecting the intrinsic thyrotropic activity of native hCG and its variants depleted of sialic acid or carbohydrate residues. In conclusion, this is the first report on the thyrotropic activity of ds-hCG and dg-hCG using the physiologically relevant hormonal end-point response, thyroid hormone secretion. The study was conducted in a serum-free culture system of human thyroid follicles and shows that removal of the sialic acid or carbohydrate residues from native hCG transform hCG variants into thyroid stimulating superagonists. The hCG variants inhibited, as did TSH, gamma-interferon-induced HLA-DR expression.

Pendred syndrome maps to chromosome 7q21-34 and is caused by an intrinsic defect in thyroid iodine organification.

Exactly 100 years ago, in 1896, Pendred first described the association of congenital deafness with thyroid goitre (MM#274600). The incidence of Pendred syndrome is estimated at 7.5-10/100,000, and may be responsible for as much as 10% of hereditary deafness. The cause of the congenital deafness in Pendred syndrome is obscure, although a Mondini type malformation of the cochlea exists in some patients. The reason for the association between the thyroid and cochlear defects is similarly obscure, leading some investigators to suggest that the two recessive defects may be occurring together by chance in highly consanguineous families. An in vivo defect in thyroid iodine organification in Pendred syndrome patients has been reported. However, the molecular basis of this defect is unknown and the presence of an intrinsic thyroidal defect has not been conclusively demonstrated. We have adopted a genetic linkage study as a first step towards identifying the gene. The availability of an inbred Pendred syndrome kindred allowed us to utilize an efficient DNA pooling strategy to perform a genome-wide linkage search for the disease locus. In this way, we have mapped the disease locus to an approximately 9-cM interval between GATA23F5 and D7S687 on chromosome 7. In addition, we demonstrate an intrinsic thyroid iodine organification defect in a patient's thyroid cells as the cause of the thyroid dysfunction.

Mutual antagonistic interactions between the thyrotropin (adenosine 3',5'-monophosphate) and protein kinase C/epidermal growth factor (tyrosine kinase) pathways in cell proliferation and differentiation of cultured human thyroid follicles.

Our aim has been to delineate the role of the major signal transduction pathways believed implicated in the control of thyroid function and growth: the cAMP-, epidermal growth factor (EGF) (tyrosine kinase)-, and protein kinase C (PKC)-mediated mechanisms. The experimental model used was our system of thyroid follicles of human origin cultured in suspension under serum-free conditions in which the follicular three-dimensional structure is retained. The phorbol ester 12-O tetradecanoylphorbol 13-acetate (TPA) time and dose dependently (10(-11)-10(-7) M) inhibited TSH-stimulated thyroid functions (cAMP formation, iodide uptake and organification, and T3 secretion). TPA also inhibited such forskolin- and 8-BrcAMP-stimulated effects, suggesting that the attenuation of the cAMP-dependent pathway occurs at steps both pre- and post-cAMP formation. The effects of TPA were mimicked by another PKC activator, phorbol 12,13-dibutyrate, but not by a phorbol ester that fails to activate PKC, 4 alpha-phorbol 12,13-didecanoate, and were reversed by staurosporine, a PKC inhibitor. The TPA actions seem, therefore, to be PKC-mediated. EGF exhibited a time- and dose-dependent (0.02-8 nM) restraining influence on the above TSH-stimulated differentiated functions, except for cAMP, which was enhanced. EGF also blunted such forskolin- and 8-BrcAMP-induced response parameters, suggesting inhibition at a post-cAMP locus. Regarding cell proliferation, during the initial stages of culture (day 2), TPA dose dependently (10(-11)-10(-7) M) attenuated cell proliferation, but subsequently (day 7 of culture) the same doses of TPA stimulated cell multiplication. The TPA-mitogenic and antimitogenic effects could not be mimicked by 4 alpha-phorbol-12,13-didecanoate and were reversed by staurosporine, thus indicating a PKC-mediated pathway for such TPA actions. EGF, on the other hand, only enhanced cell proliferation at a late stage (coincident with the TPA-mitogenic effect). TSH (0.5 U/liter) inhibited both the mitogenic and antimitogenic actions of TPA as well as the cell-proliferative influence of EGF. In conclusion, the data demonstrate mutual antagonistic interactions between the signal transduction pathways: the PKC and EGF (tyrosine kinase) pathways seem to inhibit TSH (cAMP)-mediated human thyroid cell differentiation, whereas TSH attenuates PKC-mediated thyroid cell mitogenesis and antimitogenesis as well as EGF-mediated cell proliferation.

Human chorionic gonadotropin stimulates thyroid hormone secretion, iodide uptake, organification, and adenosine 3',5'-monophosphate formation in cultured human thyrocytes.

Despite extensive studies, the issue of whether hCG possesses intrinsic thyrotropic activity remains unresolved. This is mainly because in the experimental systems used so far, the parameters measured did not include the thyroid-specific functions of iodine organification and the hormonal end-point response, T3 secretion, and cells of nonhuman origin were employed, constituting a major drawback in view of the wide variation in sensitivity of thyroid responsiveness to hCG in different species. We investigated the thyrotropic activity of hCG, using for this purpose a novel homologous assay system consisting of human thyroid follicles cultured suspended in collagen gel in serum-free medium. Under these conditions, the cells are organized as follicular three-dimensional structures with normal polarity, enabling enhanced responsiveness to hormonal stimulation. The parameters measured were the thyroid-specific functions of iodide uptake, organification, and T3 secretion, as well as formation of the second messenger, cAMP. Purified hCG (biological potency, 21,700 IU/mg; with no detectable TSH by immunoradiometric TSH assay) did indeed exhibit thyroid stimulatory activity. At doses ranging from 10-400 mg/L, hCG induced a dose-dependent increase in the parameters measured. The rise from basal to maximal levels achieved after hCG stimulation was 1.3 to 3.6 pmol/well for cAMP formation, 34 to 21,408 cpm/well for iodide uptake, 261 to 20,167 cpm/well for iodide organification, and 40 to 927 fmol/well for T3 secretion. Maximal levels elicited by hCG (200 mg/L) relative to maximal values achieved with bovine TSH were 49%, 56%, and 42% for iodide uptake, organification, and T3 secretion, respectively, and only 5% for cAMP. Iodide uptake proved to be the most sensitive indicator of the thyrotropic activity of hCG, with increases occurring at a concentration of 10 mg/L. Acting as a partial agonist, hCG was also capable of dose-dependently inhibiting TSH-stimulated cAMP formation. The free alpha- and beta- subunits of hCG, at doses as high as 200 mg/L, had no thyroid-stimulating effect. The present data thus clearly demonstrate that hCG is a human thyroid stimulator. Moreover, hCG managed to elicit substantial biological cell responses in human thyrocytes while evoking minimal amounts of cAMP, illustrating the concept of cAMP superfluity and highlighting the potential pitfalls of using cAMP as a reliable measure of hormonal bioactivity.

The IgG subclass distribution of TSH receptor blocking antibodies in primary hypothyroidism.

OBJECTIVE: TSH receptor stimulating antibodies are restricted to the IgG1 subclass suggesting an oligoclonal origin. We wished to determine whether thyroid stimulation blocking antibodies, which also bind to the TSH receptor but may cause hypothyroidism, are similarly IgG subclass restricted. DESIGN: Sera containing TSH receptor blocking antibody activity were separated into IgG subclasses by negative depletion of all other subclasses on affinity columns of subclass-specific monoclonal antibodies or protein A as appropriate. PATIENTS: Eleven patients from two centres were studied. All had autoimmune hypothyroidism and TSH receptor blocking antibodies but no thyroid stimulating antibodies. MEASUREMENTS: TSH receptor blocking antibody activity was measured by assessing inhibition of TSH-stimulated cAMP production by human thyroid cells (five Israeli samples) or by the FRTL-5 rat thyroid cell line (six Korean samples). RESULTS: IgG1 was the most important subclass containing TSH receptor blocking antibody activity but complete restriction to this subclass was never seen. Clearly detectable activity was found in the IgG2 subclass in eight patients, in the IgG3 subclass in three patients, and in the IgG4 subclass in six patients. The percentage recovery of activity in the sum of the separated fractions generally corresponded to the activity in whole immunoglobulin, being 117 +/- 66% in the nine patients in whom this could be assessed, although in one of these, the activities in the sum of the fractions was much higher (284%). CONCLUSIONS: Unlike thyroid stimulating antibodies, thyroid stimulation blocking antibodies are not subclass restricted and are therefore likely to have a polyclonal origin. The IgG subclass distribution of these blocking antibodies resembles that of thyroglobulin and thyroid peroxidase antibodies.

Changes in stimulating and blocking TSH receptor antibodies in a patient undergoing three cycles of transition from hypo to hyper-thyroidism and back to hypothyroidism.

We report a patient who underwent, over a mere 3-year period, three successive cycles of oscillation from hypo to hyper-thyroidism and back to hypothyroidism. This unusual sequence of events originated in a rare passage of primary hypothyroidism to hyper-thyroidism. The hyperthyroidism seemed typical of the autoimmune subgroup of toxic multinodular goitre. Stimulating and blocking TSH receptor antibody activities were measured (by cAMP functional bioassays using cultured human thyrocytes) during the course of the fluctuating phases of hypo and hyper-thyroidism. Measurement of such antibody activities revealed the coexistence of both stimulatory and blocking types of antibody in several serum samples from the patient. Throughout the whole course of alterations in thyroid function, thyroid stimulating antibodies were present. This was not the case with thyrotrophin receptor antibodies exhibiting TSH antagonist activity which seemed to appear and disappear. Monitoring such activity indicated that the emergence of blocking antibody seems to herald the onset of hypothyroidism.

Iodide uptake and organification, tri-iodothyronine secretion, cyclic AMP accumulation and cell proliferation in an optimized system of human thyroid follicles cultured in collagen gel suspended in serum-free medium.

An experimental system was established for measuring cell function and proliferation of human thyroid follicles cultured in collagen gel suspended in serum-free medium. Optimal culture conditions were defined and the system was characterized. The human thyrocytes were functional as indicated by their ability to respond to a TSH stimulus (as low as 1-10 microU/ml), in a time- and dose-dependent fashion, with at least a 15-fold increase in iodide uptake and organification, tri-iodothyronine (T3) secretion (demonstrated to derive from de-novo T3 biosynthesis) and cyclic AMP accumulation. Moreover, the same system allowed the measurement of cell proliferation (as indicated by thymidine incorporation and DNA content) following epidermal growth factor (EGF) and phorbol ester challenge under conditions of cell density and medium identical to those for the differentiated functions. The functional responses and cell proliferation were markedly higher compared with those of the same cells in the presence of serum or maintained in monolayer culture. Normal cell polarity, which critically determines functional capacity of thyroid follicles was maintained (as demonstrated by electron microscopy) by the use of collagen gel and serum-free medium. The use of thyroid cells of human origin assumes great importance in view of the wide species differences reported. Cryopreservation of cells rather than the necessity of using freshly derived cells confers greater convenience. The present model system provides a powerful tool for studying human thyroid physiology and pathophysiology.

Effects of gamma-interferon on DR antigen expression, growth, 3,5,3'-triiodothyronine secretion, iodide uptake, and cyclic adenosine 3',5'-monophosphate accumulation in cultured human thyroid cells.

We have examined, using the same system of human thyroid cells in culture, the effects of the cytokine human gamma-interferon (gamma IFN) on the expression of DR antigen, cell pro-liferation, cAMP accumulation, and the differentiated functions, iodide uptake and T3 secretion. gamma IFN elicited a dose- and time-dependent increase in DR expression, with a maximum effect on day 5 of culture. The cytokine, at the same concentrations and experimental conditions as those found to be effective in inducing DR antigen expression, caused on day 5 of culture a dose-dependent inhibition of [3H]thymidine incorporation, DNA content, cell count, as well as TSH-stimulated (but not basal) iodide uptake and T3 secretion. The gamma IFN suppressive influence on the differentiated functions was not merely due to a reduction in cell number, but was also apparent when results were expressed per micrograms DNA. Since the cytokine did not inhibit TSH- or forskolin-stimulated cAMP accumulation and showed a suppressive influence toward 8-bromo-cAMP- and forskolin-stimulated T3 secretion, its inhibitory effect seems to be exerted at a site located distal to cAMP formation. Although gamma IFN alone was devoid of any effect on cAMP accumulation, it enhanced forskolin-stimulated (as well as TSH-activated) cAMP in the presence of 3-isobutyl-1-methylxanthine, an inhibitor of cAMP degradation. Thus, it would seem that gamma IFN also exerts an influence on cAMP formation (rather than degradation) at a step subsequent to TSH binding to its receptor. The effects we observed seem specific to gamma IFN, since alpha IFN, although capable of inhibiting human thyrocyte multiplication, lacked any influence on DR antigen expression, cAMP accumulation, or T3 secretion by human thyroid cells. In what way, if any, is gamma IFN-induced DR antigen expression on human thyrocytes, an event believed to be critical in the pathophysiology of autoimmune thyroid disease, related to decreased thyroid function and growth is presently unknown.

A case of transient hypothyroidism: sequential serum measurements of autoantibodies inhibiting thyrotropin-stimulated thyroid cAMP production in a neonate.

Transient neonatal hypothyroidism has been observed in three successive offspring of a mother with autoimmune thyroiditis. Thyroxine replacement therapy was initiated in a 23-year-old woman with overt clinical and laboratory findings of non-goitrous primary hypothyroidism. While on such treatment, she gave birth to three infants manifesting hypothyroidism immediately after birth. The neonates were treated with thyroxine replacement therapy which was discontinued in the three siblings at ages 2 1/2 years, 3 1/2 years, and 13 months. Continuous observation following cessation of therapy revealed clinical and biochemical euthyroidism in the children. Thyroid scanning during the neonatal period in the first child failed to identify functional thyroid tissue, suggesting thyroid agenesis, whereas thyroid scan performed on subsequent follow-up revealed a normal gland. Sequential serum measurements of autoantibodies directed towards the thyrotropin receptor were made in the mother and third child by a cAMP bioassay. High titres (five-six fold above normal) of blocking antibodies (tested by measuring the inhibition of TSH-stimulated cAMP production of cultured human thyroid cells by serum immunoglobulin preparations) were present in the mother and newborn 10 days after birth. The levels remained persistently high in the mother, whereas they declined and were undetectable in the child at four months. Thyroid-stimulating immunoglobulin was absent in both mother and child. The data are compatible with transient neonatal hypothyroidism caused by transplacental transfer of antibodies which block thyroid response to TSH. The half-life of the maternally-derived blocking antibody in the infant was estimated as 1-2 months.(ABSTRACT TRUNCATED AT 250 WORDS)

Thyrotropin, acting at least partially via adenosine 3',5'-monophosphate, exerts both mitogenic and antimitogenic effects in cultured human thyroid cells.

The aim of this study was to examine the role of TSH and cAMP in human thyroid cell proliferation. For this purpose, human thyrocytes were cultured in the absence or presence of TSH, cAMP analogs, or forskolin, and the following parameters were measured during 7 consecutive days: [3H]thymidine incorporation into trichloroacetic acid-precipitable material, cell count (to assess cell proliferation), and cAMP accumulation. Cells proliferated during the first days in culture, reaching a maximum on day 5, followed by a decline in cell growth. TSH dose-dependently enhanced thyrocyte proliferation during the first few days of culture, but attenuated cell growth during the later stages of culture. The TSH mitogenic action was only apparent at a low cell density (10(4) cells/well in 96-well microtiter plates). TSH elicited a dose-dependent elevation of cAMP during all phases of cell culture. Furthermore, the cAMP analogs, 8-bromo-cAMP and dibutyryl cAMP, mimicked the thyrocyte proliferative and antiproliferative actions of TSH. Forskolin also mimicked the TSH mitogenic and antimitogenic effects while concomitantly elevating cAMP levels during both instances. The data, therefore, seem entirely consistent with the premise that the TSH stimulatory and inhibitory actions on human thyroid cell growth are mediated, at least partially, by cAMP. The dual actions of TSH as a mitogenic and antimitogenic factor may, thus, provide an experimental in vitro basis for the well established in vivo goitrogenic effect of TSH observed initially, followed by desensitization on prolonged exposure to the hormone.

Monoamine oxidase activity and triiodothyronine biosynthesis in human cultured thyroid cells.

1. The proposal that monoamine oxidase (MAO) is a source of peroxide in thyroid hormone biosynthesis has been examined by use of isolated cultured human thyroid cells which retain the ability to secrete triiodothyronine (T3) in response to thyroid stimulating hormone (TSH). 2. The results demonstrated the presence of MAO A and B in human thyroid cells which oxidized 5-hydroxytryptamine and 2-phenylethylamine, respectively, and were selectively inhibited by the MAO inhibitors clorgyline and (-)-deprenyl. 3. Addition of propylthiouracil to the culture system induced a 61% reduction in TSH-stimulated T3 secretion, indicating that the bulk of such secretion apparently derives from de novo iodothyronine synthesis. 4. The MAO A and B substrate, tyramine, was ineffective in stimulating T3 secretion. 5. The selective MAO inhibitors, clorgyline and (-)-deprenyl, alone and in combination, and in the presence and absence of tyramine, failed to inhibit basal as well as TSH-stimulated T3 secretion in cultured human thyrocytes. 6. It is therefore apparent that even though thyroid MAO A and B enzyme reactions result in the generation of H2O2, this H2O2 does not seem to play a significant role in T3 biosynthesis.

Triiodothyronine and 3',5'-cyclic AMP secretion by cultured human thyroid cells in response to thyrotropin and thyroid-stimulating immunoglobulin.

We have established a relatively simple and sensitive system for measuring T3 as well as cAMP secretion using cryopreserved human thyroid cells in culture. We defined optimal culture conditions and characterized the system. T3 secretion from human thyrocytes (only 1 x 10(5) cells/well) could be stimulated in a time- and dose-dependent fashion by both TSH (doses as low as 10 mU/l) and thyroid-stimulating immunoglobulin to levels 5- to 10-fold above baseline. The response to the thyroid stimulating agents was preserved for at least 3 weeks. Experiments with inhibitors of iodothyronine synthesis (propylthiouracil and methimazole) indicated that the bulk of the TSH-stimulated T3 secretion measured apparently derives from de novo iodothyronine biosynthesis rather than preformed T3. We utilized the system to investigate some aspects in the regulation of human thyrocyte T3 and cAMP secretion. Maximum stimulation of the thyroid hormone was achieved at TSH doses capable of evoking a further rise in levels of cAMP. A rise in cAMP accumulation was observed as early as 15 min following exposure to TSH, whereas it took 1-4 days to detect a significant increase in T3 secretion. Within 6 h of incubation, the bulk of TSH-stimulated intracellular cAMP was found released into the medium. 1-methyl-3-isobutylxanthine (MIX) caused a dose-related decrease (beyond 0.1 mmol/l MIX) in TSH-stimulated T3 secretion which contrasted with a concomitant expected increase in cAMP accumulation. Hence, as also observed in adrenal and testicular tissue, xanthines at high concentration seem to exhibit a dual action: potentiation of cAMP accumulation by inhibiting phosphodiesterase activity and a concomitant reduction of hormone formation.

Sensitization and desensitization of human thyroid cells in culture: effects of thyrotrophin and thyroid-stimulating immunoglobulin.

Using an in-vitro system of cultured human thyroid cells and cyclic AMP (cAMP) accumulation as an index of cell stimulation, we compared TSH and thyroid-stimulating immunoglobulin (TSI) with regard to thyrocyte sensitization and desensitization. The smallest dose of TSH (0.05 mU/ml) capable of stimulating thyroid cells was the same as the minimum dose required to induce desensitization upon subsequent rechallenge with the hormone. In contrast, about 30-fold higher doses of TSI were needed to cause cell refractoriness compared with doses capable of eliciting stimulation. Moreover, significant stimulation of the thyroid with TSI was apparent much later than with TSH. A longer time-lapse was also necessary for TSI to induce desensitization. Likewise, thyrocytes recovered more slowly from TSI compared with TSH desensitization. Although at high doses TSI induced homologous desensitization, at lower doses the antibody, unlike TSH, potentiated the cAMP response to subsequent exposure to the antibody. The stimulatory doses of TSI were in the range usually encountered in active Graves' disease, which may explain why prolonged TSI in vivo sustains a hyperthyroid condition. In addition, we found that under conditions in which TSH leads to desensitization of the cAMP response, the thyroid cells maintained their responsiveness in terms of triiodothyronine secretory activity. Pre-exposure of human thyrocytes to TSI induced heterologous desensitization towards the TSH-stimulated cAMP response. Moreover, addition of the antibody to maximally desensitizing doses of TSH decreased cell sensitivity to the hormone even further.(ABSTRACT TRUNCATED AT 250 WORDS)

Bioassay of thyroid-stimulating immunoglobulin in cryopreserved human thyroid cells: optimization and clinical evaluation.

We have explored the method of Rapoport et al. (J Clin Endocrinol Metab 1984;58:332-8) for the bioassay of thyroid-stimulating immunoglobulin (TSI) in cultured human thyroid cells, to optimize the assay and to evaluate its utility in clinical diagnosis and management of patients with autoimmune thyroid disease. Here we describe the procedure ultimately adopted, its major properties, and the results it has yielded in various clinical states. Clinical sensitivity of the assay was established by demonstrating TSI activity in all of 60 cases of active Graves' disease. We observed in these patients a nonlinear correlation between concentrations of TSI and of triiodothyronine, as well as between TSI concentration and the clinical severity of the thyrotoxicosis. Specificity of the assay was demonstrated by finding no TSI bioactivity in 13 patients with toxic adenoma, five with cold nodule, and 18 of 19 with nontoxic goiter. Remission of Graves' disease in 25 patients was invariably accompanied by undetectable concentrations of TSI; evidently this assay may be useful in identifying patients who are likely to go into remission. TSI activity was present in eight of 11 patients with euthyroid ophthalmopathy (unilateral and bilateral) associated with a normal response to the thyrotropin-releasing hormone test and absence of increased titers of antithyroid antibodies, suggesting that this assay may provide a powerful tool in the clinical diagnosis of this disorder.

Toxic multinodular goiter: a variant of autoimmune hyperthyroidism.

The aim of this study was to examine whether at least a subgroup of patients with toxic multinodular goiter may have autoimmune thyroid disease. Thyroid-stimulating immunoglobulin (TSI) activity, measured by a sensitive bioassay employing cultured human thyroid cells, was determined in patients with toxic multinodular goiter and other thyroid disorders. All patients with active Graves' disease (n = 47) had detectable serum TSI activity, whereas TSI was undetectable in patients with thyroid disease not believed to be of autoimmune origin: toxic adenoma (n = 13), cold nodule (n = 5), and nontoxic goiter (n = 19), with a single exception in the latter group. Toxic multinodular goiter (n = 26) was diagnosed based on clinical and laboratory evidence of hyperthyroidism associated with a multinodular goiter on palpation and scintiscan. The toxic multinodular goiter group was then subclassified according to scintiscan pattern (type A, diffuse but uneven distribution of technetium uptake; type B, multiple discrete nodules of varying size and function). All but 1 of the 11 TSI-positive toxic multinodular goiter patients had a type A scintiscan pattern. The patients with the type A scintiscan pattern were younger and more often had elevated antithyroid antibody titers, ophthalmopathy, and concurrent development of goiter and hyperthyroidism (rather than long-standing goiter preceding hyperthyroidism) compared to the type B patients. Thus, a subgroup of patients with clinically defined toxic multinodular goiter (type A) probably have autoimmune hyperthyroidism (a variant of Graves' disease), while in another subgroup (type B) hyperthyroidism is not related to an autoimmune etiology (a variant of toxic adenoma).