Clinical applications of assays for thyrotropin-receptor antibodies in Graves' disease.

Graves'' disease is characterized by hyperthyroidism, diffuse goitre, infiltrative ophthalmopathy and, rarely, pretibial myxedema. In 1956 a substance capable of prolonged thyroid stimulation was discovered in the serum of some patients with Graves'' disease and termed long-acting thyroid stimulator (LATS). It was shown to be an antibody that could interact with the receptor for thyroid-stimulating hormone (TSH). The term LATS is usually reserved for the activity measured in a laborious in-vivo bioassay in mice. Today the activity of TSH-receptor antibodies (TSH-R Ab) can be measured by in-vitro bioassays or by radioreceptor assays. These assays are now becoming commercially available. TSH-R Ab assays may be useful in predicting the response to therapy for Graves'' disease, investigating euthyroid ophthalmopathy and predicting the likelihood of neonatal hyperthyroidism.     

Activities of citrate synthase, NAD+-linked and NADP+-linked isocitrate dehydrogenases, glutamate dehydrogenase, aspartate aminotransferase and alanine aminotransferase in nervous tissues from vertebrates and invertebrates.

Highly purified bovine TSH (thyroid-stimulating hormone) was labelled with 125I by using very low concentrations of chloramine-T. Human thyroid membranes prepared by discontinuous sucrose-density-gradient centrifugation were homogeneous on examination by electron microscopy. Incubation of radioiodinated TSH with the membranes showed that radioactivity could be bound to the membranes. Under the experimental conditions described here, binding was dependent on time and temperature and was a saturable phenomenon. Preincubation of the membranes with unlabelled hormone inhibited the subsequent binding of 125I-labelled TSH. Similarly, inhibition by the long-acting thyroid stimulator also showed a saturation behaviour. A rapid and sensitive method for the detection of the long-acting thyroid stimulator is described.     

Effects of Long-acting Thyroid Stimulator (LATS) and LATS Protector on Human Thyroid Adenyl Cyclase Activity

The long-acting thyroid stimulator (LATS) has been thought to be responsible for the hyperthyroidism of Graves''s disease. It is detected by its effect on the mouse thyroid gland but cannot be found in all patients with hyperthyroidism. In an attempt to clarify the problem of LATS-negative hyperthyroidism, serum was obtained from untreated patients and its effect in vitro on human thyroid tissue examined, using the activation of adenyl cyclase as a measure of stimulation. Human thyroid adenyl cyclase was activated by both thyroid-stimulating hormone (TSH) and LATS. Thyroid tissue obtained from patients with Graves''s disease was relatively less responsive to LATS than was non-toxic thyroid tissue. Of the 24 samples studied five contained LATS and all of these activated adenyl cyclase. The presence of LATS protector in LATS-negative hyperthyroid patients was confirmed but LATS-negative sera had no effect on human thyroid adenyl cyclase activity.     

Similar triiodothyronine releasing activity of thyroid stimulating antibodies in human and porcine thyroid slices

In an approach to addressing species specificity of thyroid stimulating antibodies (TSAb) stimulation of T3 release by Graves' sera was comparatively studied in human and porcine thyroid slices. A high sensitivity and specificity was found for the T3 bioassay independently on the use of human or porcine thyroid. Moreover, activity indices of the individual sera in both tissues were significantly correlated to each other and to circulating hormone levels in untreated disease. In conclusion, we suppose a lack of functionally relevant differences between target antigens, brought about probably by the TSH receptor itself and other membrane components, in human and porcine thyroid. Thus, for clinically applicable T3 releasing bioassay porcine thyroid may be alternatively used. In addition, this bioassay renders the advantage of reflecting the activity of disease.     

Thyroid follicular morphogenesis mechanism: organ culture of the fetal gland as an experimental approach

The morphological and physiological changes induced by organ culture and thyroid-stimulating hormone (TSH) stimulation in the rat fetal thyroid gland were studied. Organ culture increased Golgi activity which was further enhanced by TSH, subsequently facilitating the formation of intracellular lumina. TSH also raised the intracellular cAMP level. The intracellular lumina observed during follicular morphogenesis are structurally comparable to typical intracellular cavities formed in adult thyroid cells, which are considered as being the result of increased Golgi activity. The intracellular lumen, therefore, is probably not a physiologically significant step in thyroid morphogenesis.     

Thyrotropic activity of salmon pituitary glycoprotein hormones in the Hawaiian parrotfish thyroid in vitro

The thyrotropic activities of salmon pituitary extract, thyroid-stimulating hormone (TSH), gonadotropins (GTH), and glycoprotein fractions obtained during purification of salmon TSH and GTH were measured using the parrotfish thyroid culture system. Purified salmon TSH was approximately 1,000 times more potent than bovine TSH in stimulating thyroxine release into the culture medium. Most of the forms of salmon GTH had no thyrotropic activity. One of the forms of salmon GTH (GTH-F) and three chromatofocusing fractions (CF-B, -C, and -E) that were devoid of activity in the coho salmon in vivo had some thyrotropic activity in the parrotfish thyroid culture. Whether the activity of these fractions was due to contamination with TSH, less potent forms of TSH, or inherent thyrotropic activity of a form of GTH is discussed. These results indicate that the parrotfish thyroid culture system can be used to detect thyrotropic activity of fractions obtained during the purification of teleost TSH.     

Development of a human monoclonal antibody from a Graves' disease patient that identifies a novel thyroid membrane antigen

To investigate the interaction between antibodies and the thyroid gland in Graves' disease, PBL were harvested from seven Graves' disease patients and transformed into lymphoblasts by the addition of EBV in the presence of cyclosporine A. These lymphoblasts were cloned by limiting dilution and then assayed for binding activity to human thyroglobulin, thyroid-stimulating hormone, thyroid microsome, and thyroid as well as guinea pig fat cell membranes. Four patients' cells produced antibody that bound to at least one of the Ag; a single clone from one patient that bound equally well to both thyroid and guinea pig fat cell membranes (but not to other thyroid Ag) was selected for further evaluation. Fusion of these cells with SHM-D33 heteromyeloma cells yielded three cell lines that produced genetically identical mAb. Immunostaining of human thyrocytes with this mAb demonstrated an Ag present on both nuclear and cell membranes. This Ag was identified as an 18,000 m.w. protein band on Western blots of both human thyroid and guinea pig fat cell membranes. The mAb was also able to alter thyrocyte physiology as the short term incubation of this mAb with FRTL-5 cells in vitro inhibited thyroid-stimulating hormone-mediated production of cAMP. Thus, this mAb and the Ag it identifies may be relevant to Graves' disease.     

Molecular Mechanisms of the Relationship between Thyroid Dysfunctions and Diabetes Mellitus

Type 1 and type 2 diabetes mellitus (DM) are known to increase the incidence of thyroid gland (TG) dysfunctions. The review addresses the literature data and our experimental results on the molecular mechanisms that underlie thyroid disorders under DM. Most important of these mechanisms are the attenuation of thyrocyte adenylyl cyclase signaling system sensitivity to thyroid-stimulating hormone, the decrease in the number of thyroid hormone receptors in peripheral tissues, and the decline in activity as well as changes in the ratio of different deiodinase forms in these tissues. Decreased activity of D2 deiodinases, which convert thyroxine into the active form of triiodothyronine, is associated with the development of insulin resistance, while decreased activity of D3 deiodinases, which catalyze inactivation of triiodothyronine in pancreatic β cells, suppresses insulin secretion and leads to insulin deficiency. Thus, both the excess and the deficiency of thyroid hormones can entail diabetic pathology. Identification of thyroid disorders is of utmost importance for elaborating novel approaches to treat and prevent thyroid diseases associated with type 1 and type 2 DM.     

The Presence of Thyroid-Stimulation Blocking Antibody Prevents High Bone Turnover in Untreated Premenopausal Patients with Graves’ Disease

Osteoporosis-related fractures are one of the complications of Graves’ disease. This study hypothesized that the different actions of thyroid-stimulating hormone receptor (TSHR) antibodies, both stimulating and blocking activities in Graves’ disease patients might oppositely impact bone turnover. Newly diagnosed premenopausal Graves’ disease patients were enrolled (n = 93) and divided into two groups: patients with TSHR antibodies with thyroid-stimulating activity (stimulating activity group, n = 83) and patients with TSHR antibodies with thyroid-stimulating activity combined with blocking activity (blocking activity group, n = 10). From the stimulating activity group, patients who had matched values for free T4 and TSH binding inhibitor immunoglobulin (TBII) to the blocking activity group were further classified as stimulating activity-matched control (n = 11). Bone turnover markers BS-ALP, Osteocalcin, and C-telopeptide were significantly lower in the blocking activity group than in the stimulating activity or stimulating activity-matched control groups. The TBII level showed positive correlations with BS-ALP and osteocalcin levels in the stimulating activity group, while it had a negative correlation with the osteocalcin level in the blocking activity group. In conclusion, the activation of TSHR antibody-activated TSH signaling contributes to high bone turnover, independent of the actions of thyroid hormone, and thyroid-stimulation blocking antibody has protective effects against bone metabolism in Graves’ disease.     

Localization and role of leptin in the thyroid gland of the lizard Podarcis sicula (Reptilia, Lacertidae)

Leptin, the product of the ob gene, is a hormone secreted by adipocytes that regulates food intake and energy expenditure. The hypothalamus-pituitary-thyroid axis is markedly influenced by the metabolism status, being suppressed during food deprivation. The present study was designed to ascertain whether (1) lizard thyroid gland expresses the long form of leptin receptor (Ob-Rb) and (2) the leptin administration affects the thyroid gland activity in this species (and to verify whether leptin plays a similar role in reptiles as observed in the other vertebrates). The presence of leptin receptor in the thyroid gland of Podarcis sicula was demonstrated by immunohistochemical technique (avidin-biotin-peroxidase complex--ABC method). The role of leptin in the control of thyroid gland activity was studied in vivo using light microscopy (LM) technique coupled to a specific radioimmunoassay for thyroid-stimulating hormone (TSH) and thyroid hormones (T4 and T3). Leptin (0.1 mg/100 g body wt)/day increased T4 and T3 release for 3 days but decreased the plasma concentration of TSH; using LM clear signs of stimulation in the thyroid gland were observed. These findings suggest that systemic administration of leptin stimulates the morphophysiology of the thyroid gland in the lizard through a direct mechanism involving Ob-Rb.     

Concomitant association of thyroid sarcoidosis and Graves' disease

OBJECTIVE: Graves' disease (GD) with sarcoid involvement of the thyroid gland has rarely been reported. METHOD: We report a case of GD with thyroid sarcoidosis in a 28-year-old woman. Thyroid function was assessed by triiodothyronine (T(3)), thyroxine (T(4)), thyroid-stimulating hormone (TSH) and TSH receptor antibodies (TSH-R Ab). Thyroid scintigraphy, ultrasound and fine-needle aspiration biopsy were performed. The patient underwent surgery. RESULT: The patient had a nodular goiter. Serum T(3), T(4) and TSH-R Ab levels were elevated with suppressed TSH level. Scintigraphy showed diffuse activity as seen in GD, and ultrasound revealed that parenchyma was heterogenous. Sarcoidosis was discovered on routine chest X-ray. Although no sarcoid involvement was found on specimen, the thyroid gland showed non-caseating granulomas on histology. CONCLUSION: Since sarcoid involvement of the thyroid gland can cause hypofunction, we report the uncommon infiltration of sarcoidosis with hyperthyroidism.     

Inhibition of TSH binding to chicken thyroid by some cases of LATS (long acting thyroid stimulator)

TSH receptor antibody (TRAb) activity using chicken thyroid receptor (c-TRAb) and porcine thyroid receptor (p-TRAb) was determined by the incubation of 125I-bovine TSH with each receptor. Both c-TRAb and p-TRAb activity in LATS positive and negative Graves' sera were compared. 15 out of 39 LATS positive sera and 4 out of 46 LATS negative sera had positive c-TRAb activity. On the other hand, all LATS positive sera and 33 out of 46 LATS negative sera had positive p-TRAb activity. No relationship between c-TRAb and p-TRAb activity was observed, and there was also no correlation between c-TRAb and LATS activity. Changes in c-TRAb, p-TRAb and LATS activity in the clinical course of patients with Graves' disease were examined. These activities were parallel in some cases, but in others they were not. A weak c-TRAb activity was observed in 4 out of 29 Hashimoto's disease, but all cases with thyroid cancer and subacute thyroiditis showed no activity. Sera with positive c-TRAb activity did not stimulate chicken thyroid in chick bioassay. These results suggest that some cases of TRAb in Graves' disease (mainly LATS) inhibit TSH binding to chicken thyroid receptor (non-mammalian species) in the same way as mammalian thyroid, but may not have any stimulatory action on thyroid hormone synthesis. It is interesting to note that TRAb including LATS have the similar effect on TSH receptor even in nonmammalian species.     

Thyrotropin receptor non-mediated thyroid stimulating immunoglobulin in Graves' disease.

There exists a consensus that hyperthyroid Graves' disease is caused by thyrotropin receptor (TSH-R) autoantibodies. To test the possibility that the TSH-R is the sole antigen for thyroid stimulating antibodies (TSAb), we compared bioactivities of Graves' IgGs between non-thyroid mammalian cells transfected with human TSH-R cDNA and the reference thyroid bioassay. A Graves' IgG with TSH-binding inhibitor immunoglobulin (TBII) activity (89%) markedly stimulated cAMP formation in both CHO-K1 cells transfected with TSH-R cDNA (340 microU/ml of TSH equivalent) and rat thyroid cells, FRTL-5, (410 microU/ml of TSH equivalent). In contrast, a TBII negative (-1.5%) IgG from another patient with Graves' disease showed a strong thyroid stimulating activity (87 microU/ml of TSH equivalent) when FRTL-5 cells were used for the assay. But no stimulating activity was observed in this IgG when CHO-K1 cells transfected with TSH-R cDNA were used, suggesting a possible existence of TSH-R non-mediated thyroid stimulating immunoglobulin in some cases of Graves' disease.     

Primary cell culture from pig neonatal thyroid gland: Growth, folliculogenesis, and hormone activity

A comprehensive assay of proliferative and hormonal activity in primary cell cultures derived from neonatal pig thyroid was carried out for the first time. Morphology and basal and thyroid-stimulating hormone (TSH)-stimulated secretion of thyroxin were evaluated in cultures, depending on the initial material placed into the culture: single cells or follicular conglomerates. Spontaneous and under chronic TSH stimulation folliculogenesis and formation of dome structures were assessed in culture. It was demonstrated that the cells expressed β-III-tubulin during prolonged cultivation with nerve growth factor.     

Graves Ophthalmopathy After Radiation Treatment of Thyroid Cancer

ObjectiveTo describe a patient with metastatic thyroid cancer who developed Graves ophthalmopathy after treatment with radioiodine (I 131) and external beam radiation.MethodsWe present a case report that includes clinical, laboratory, and radiologic findings and a brief review of the literature.ResultsA 49-year-old woman who had had a total thyroidectomy and neck dissection followed by I 131 treatment 5 years earlier for papillary thyroid cancer presented for follow-up management after recent neck dissection for recurrent disease. Because she had thyroglobulin antibodies, she was again treated with I 131 after preparation with recombinant human thyroid-stimulating hormone. A post-treatment scan revealed uptake in the right iliac crest. A fludeoxyglucose F 18 positron emission tomography/computed tomography fusion scan revealed osseous metastases in the right pelvis, and external beam radiotherapy was delivered to this area. Approximately 5 months later, she developed periocular swelling and excessive tearing. Magnetic resonance imaging of the orbits revealed enlargement of the extraocular muscles. Serum thyroid-stimulating immunoglobulins were greatly elevated.ConclusionThis case corroborates an earlier report to suggest that radiation-associated thyroid injury in a patient with thyroid cancer may be followed by Graves ophthalmopathy and appearance of thyroid-stimulating immune-globulins in the serum. (Endocr Pract. 2008;14:419-421)     

Body dimensions and thyroid hormone levels in premenopausal and postmenopausal women from Austria

Correlations between thyroid hormone levels and body dimensions were investigated in a group of 124 premenopausal (ages 16–40 years) and 142 postmenopausal (ages 38–61 years) women from Vienna, Austria. Twenty-nine absolute body dimensions and thirteen anthropometric indices were correlated with the serum levels of thyroxine, triiodothyronine, thyroid-stimulating hormone, and thyroid-hormone-binding globulin as well as three hormone ratios (T3/T4, T4/TSH, T3/TSH). All hormones exhibited statistically significant correlations with 24 anthropometric variables and 11 indices. The correlations between thyroxine and triiodothyronine levels and the body measurements were predominantly positive in both proband groups. Higher thyroid hormone levels were associated positively with body dimensions, especially with the amount of subcutaneous fat tissue, in adult females independently of their menstrual status. The direction of the correlations between thyroid-stimulating hormone and body measures as well as anthropometric indices differed between the premenopausal and the postmenopausal women. While in premenopausal women mainly positive correlations between anthropometric characters and the level of thyroid stimulating hormone occur, in postmenopausal women most of these correlations are negative. This is probably due to the decrease of thyroid-stimulating hormone levels with increasing age, as well as with changes in body shape during the climacteric and after the menopause. © 1994 Wiley-Liss, Inc.     

Changes in tight junctions of thyroid epithelium with changes in thyroid activity

The morphology of the tight junction of rat thyroid epithelium was examined in freeze-fractured material fixed in glutaraldehyde and briefly glycerinated. In normal thyroids the overall appearance of this junctional specialization resembled that of other cell types in many respects. Short-term changes in thyroid activity and hypophysectomy for 3 wk did not obviously affect the appearance of tight junctions. Feeding of the goitrogen, thiouracil, which stimulates secretion of thyroid-stimulating hormone, resulted in the appearance of some very narrow and some very wide, tight junctions or sometimes junctions with both wide and narrow regions within the same cell.     

Cowden's Disease: Familial Goiter and Skin Hamartomas—A Report of Three Cases

Multiple hamartoma syndrome (Cowden''s disease) consists of characteristic skin lesions of the face, mucous membranes and distal extremities in association with a variety of benign and malignant internal tumors, especially of the thyroid and breast. We describe a family in which the father, daughter and son were found to have goiter associated with the skin lesions of Cowden''s disease. Review of the 40 reported cases of this syndrome indicates that thyroid disease occurs in two thirds of patients with Cowden''s disease and most often presents as goiter at an early age. Thyroid cancer has occurred in only three (7.5%) of the patients.Surgical removal of the large goiter of the son showed that it was composed of multiple encapsulated follicular adenomas and a few areas of lymphocytic thyroiditis. Studies of the thyroid tissue showed that peroxidase activity was decreased, the thyroglobulin had a reduced content of thyroxine and triiodothyronine (perhaps due to the therapeutic suppression of thyroid-stimulating hormone) and thyroxine 5''-monodeiodinase was greatly increased; increased outer ring monodeiodinase activity may be a characteristic of human follicular adenomas.     

Clinical usefulness of TSAb assay with high polyethylene glycol concentrations.

We previously demonstrated the stimulatory effect of polyethylene glycol (PEG) on thyroid-stimulating antibody (TSAb)-IgG-stimulated cAMP production (thyroid stimulating (TS) index) in porcine thyroid cell (PTC) assay. In the present study the clinical usefulness of the practical method using high PEG concentrations was examined. TS activity using PEG 22.5% precipitated fraction (PF) was significantly higher compared to standard TSAb activity using 12.5% PF from TSAb-positive serum, but the maximum TS activity was observed with PEG 12.5% PF + 4% PEG or PEG 22.5% PF + 2% PEG. In all cases of untreated Graves' patients, TSAb activity determined by PEG 22.5% PF was higher compared to standard TSAb activity using PEG 12. 5% PF from test serum, but the highest TSAb activity was observed by PEG 12.5% PF + 4% PEG without increased cAMP production to normal serum. TSAb was positive in 85% (40/47), 98% (46/47) and 100% (47/47) of untreated Graves' patients by the method of PEG 12.5% PF, PEG 22.5% PF and PEG 12.5% + 4% PEG, respectively. Increased TSAb activity by PEG 12.5% PF + 4% PEG method was also observed even if the standard TSAb activity using PEG 12.5% PF method was negative in the euthyroid states of Graves' patients during antithyroid drug therapy. The stimulatory effect of PEG on TS activity was not found in other thyroidal diseases [thyroiditis chronica (with high serum TSH), thyroid stimulation-blocking antibody (TSBAb)-positive sera (with low serum TSH), adenomatous goiter, subacute thyroiditis, and thyroid cancer]. The stimulatory effect of 5% PEG on TS activity produced directly by small amounts of Graves' serum (50 microl) was also found, although the sensitivity was lower than with PEG-precipitated IgG from 0.2 ml serum. The clinical usefulness of the sensitive TSAb assay using PEG-precipitated IgG or direct serum assay in the presence of high PEG concentrations was demonstrated.     

Atrial natriuretic peptide inhibits iodide uptake and thyroglobulin messenger ribonucleic acid expression in cultured bovine thyroid follicles

The involvement of atrial natriuretic peptide (ANP) in the regulation of thyroid gland is supported by the presence of high-affinity ANP receptors and the identification of the peptide in thyroid follicular cells. The aim of this work was to study the action of ANP on parameters of thyroid hormone biosynthesis and analyze the intracellular mechanism of the ANP action in cultured bovine thyroid follicles. The addition of ANP (0.1-10 nM) to the culture medium for 24 h inhibited the TSH (thyroid-stimulating hormone)-stimulated iodide uptake with a maximal inhibition at 1 nM ANP. When thyrocytes were incubated with 10 nM ANP the inhibitory effect slightly increased from 24 to 72 h. Thyroglobulin (Tg) mRNA expression was reduced by 1 and 10 nM ANP. After 24 h of treatment with the cGMP analogue, N(2),2'-O-dibutyrylguanosine 3':5'-cyclic monophosphate [(Bu)(2)cGMP] (0.1 and 1 mM), an inhibition of iodide uptake and Tg mRNA expression was obtained, evidencing a cGMP-mediated inhibitory signal in the thyroid cell. A reduction of the cAMP production was induced by incubation of thyroid follicles with 1 and 10 nM ANP for 24 h. Under a similar treatment the cGMP accumulation was increased only by 10 nM ANP. The inhibitory effect of ANP on Tg mRNA level was reverted in the presence of pertussis toxin, an inhibitor of the G(i)-protein-mediated reduction of the adenylate cyclase activity. These results indicate an inhibitory action of ANP on parameters of thyroid hormone biosynthesis. A G(i)-protein-mediated reduction of the cAMP production seems to be the main factor involved in the ANP action although a role of the cGMP pathway should not be discarded specially at high ANP levels.