Brief Report - Monoclonal Antibodies Illustrate the Difficulties in Measuring Blocking TSH Receptor Antibodies.

TSH receptor (TSHR) antibodies are the cause of Graves' disease and may also be found in patients with Hashimoto's thyroiditis. They come in at least three varieties: thyroid stimulating, thyroid blocking and neutral. The measurement of TSH receptor antibodies in Graves' disease and Hashimoto's thyroiditis is a common clinical activity and can be useful in diagnosis and prognosis. We show that it is not possible to detect the blocking variety of TSHR antibody in patients with Graves' disease because the stimulating antibody may overwhelm the measurement of blocking in the bioassays available for their measurement and may blind the valid interpretation of the results. To help explain this in more detail we show a series of studies with monoclonal TSHR antibodies which support this conclusion.

Screening of 23 candidate genes by next-generation sequencing of patients with permanent congenital hypothyroidism: novel variants in TG, TSHR, DUOX2, FOXE1, and SLC26A7.

PURPOSE: To date, many genes have been associated with congenital hypothyroidism (CH). Our aim was to identify the mutational spectrum of 23 causative genes in Turkish patients with permanent CH, including thyroid dysgenesis (TD) and dyshormonogenesis (TDH) cases. METHODS: A total of 134 patients with permanent CH (130 primary, 4 central) were included. To identify the genetic etiology, we screened 23 candidate genes associated with CH by next-generation sequencing. For confirmation and to detect the status of the specific familial variant in relatives, Sanger sequencing was also performed. RESULTS: Possible pathogenic variants were found in 5.2% of patients with TD and in 64.0% of the patients with normal-sized thyroid or goiter. In all patients, variants were most frequently found in TSHR, followed by TPO and TG. The same homozygous TSHB variant (c.162 + 5G > A) was identified in four patients with central CH. In addition, we detected novel variants in the TSHR, TG, SLC26A7, FOXE1, and DUOX2. CONCLUSION: Genetic causes were determined in the majority of CH patients with TDH, however, despite advances in genetics, we were unable to identify the genetic etiology of most CH patients with TD, suggesting the effect of unknown genes or environmental factors. The previous studies and our findings suggest that TSHR and TPO mutations is the main genetic defect of CH in the Turkish population.

Autoimmune gastritis.

Autoimmune gastritis (AIG) is an increasingly prevalent, organ-specific, immune-mediated disorder characterized by the destruction of gastric parietal cells, leading to the loss of intrinsic factor and reduced acid output. These alterations result in malabsorption of iron, vitamin B12 (pernicious anaemia) and potentially other micronutrients. For several years, most studies have focused on pernicious anaemia only, generating confusion between the two entities. In AIG, the gastric proton pump, H+/K+ ATPase, is the major autoantigen recognized by autoreactive T cells. The T cell-dependent activation of B cells stimulates the production of anti-parietal cell antibodies, the serological hallmark of AIG. The role of Helicobacter pylori infection in activating or favouring the autoimmune process is still uncertain. Early histopathological alterations allowing a more precise and prompt recognition have recently been described. AIG is burdened by a substantial diagnostic delay as it can present with varied clinical signs including, among others, gastrointestinal symptoms and neuropsychiatric manifestations. In advanced stages, AIG might progress to neuroendocrine tumours and gastric adenocarcinoma. Management includes early detection through a proactive case-finding strategy, micronutrient supplementation and endoscopic surveillance. This Primer comprehensively describes the most important insights regarding the epidemiology, pathophysiology, diagnosis and management of AIG, focusing on the most controversial, outstanding issues and future directions.

The success rate of radioactive iodine therapy for Graves' disease in iodine-replete area and affecting factors: a single-center study.

OBJECTIVE: Initial treatment options for Graves' disease include antithyroid drugs, radioactive iodine (RAI), and surgery. Because of similar effects of three modalities, initial treatment preferences differ by country to country. In Korea, iodine-replete area, RAI was used as a second-line option. For these reasons, the RAI success rate in Korea might differ from other countries. METHODS: A total of 247 patients were enrolled. The primary outcome was cumulative success rate in the first year, and the secondary outcome was factors that affected the success. Delayed response, which included patients who attained successful RAI during the follow-up, after the first year without further RAI, and factors that affected the delayed response were reviewed. RESULTS: The cumulative success rate in the first year was 62.8%. Higher RAI activity [odds ratio (OR) 2.56, P = 0.02], longer disease duration (OR 0.43, P = 0.01), larger goiter (OR 0.31, P = 0.01), and higher post-RAI fT4 (fT4 between upper normal limit (UNL) to 1.5 × UNL; OR 0.24; P < 0.01, 1.5 × UNL<; OR 0.08; P < 0.01) were associated with RAI success. Twenty-seven patients showed delayed response, and goiter size (P < 0.05), pre-RAI TSH receptor Ab (TRAb) level (P < 0.01) and post-RAI fT4 (P < 0.01) were associated. CONCLUSION: The success rate of the first RAI in Korea was lower than that in other countries. Delayed response was observed in patients with smaller goiter, low pre-RAI TRAb, and low post-RAI fT4. In these patients, clinical follow-up with monitoring could be an option, and decision of optimal timing of first RAI is crucial.

Expression of proteins related to thyroid hormone function in the uterus is down-regulated at the day of implantation in hypothyroid pregnant rats.

Hypothyroidism has been linked to infertility, but the mechanisms underlying infertility-related hypothyroidism have yet to be fully elucidated. Therefore, in this study, effects of hypothyroidism on expression of the proteins related to thyroid hormone function in the uterus, which were thought to play a role implantation, including thyroid hormone receptor (TR), thyroid stimulating hormone receptor (TSHR), retinoic acid receptor (RAR) and extracellular kinase (ERK) were identified. Pregnant female rats were rendered hypothyroid by giving methimazole (MMI), orally. Following hypothyroid induction, rats were grouped into control (non-treated) and received subcutaneous thyroxine at 20, 40, and 80 µg/kg/day for five consecutive days. At Day 6, which is the day of implantation (GD 6), rats were sacrificed and the number of embryo implantation site in the uterus was calculated. Then, uterine horns were harvested and expression of the above proteins and their mRNAs were identified by Western blotting and real-time PCR, respectively. In non-treated hypothyroid pregnant rats, the number of embryo implantation sites decreased as compared to euthyroid and hypothyroid rats receiving thyroxine treatment. Similarly, expression of TRα-1, TRß-1, TSHR, ERK1/2 and RAR proteins and mRNA in the uterus of non-treated hypothyroid rats also decreased (P < 0.05 when compared to euthyroid and thyroxine-treated hypothyroid rats). In conclusion, downregulated expression of the thyroid hormone related proteins in the uterus at the day of implantation might result in infertility as reported in hypothyroid condition.

Evidence of G-protein-coupled receptor and substrate transporter heteromerization at a single molecule level.

G-protein-coupled receptors (GPCRs) can constitute complexes with non-GPCR integral membrane proteins, while such interaction has not been demonstrated at a single molecule level so far. We here investigated the potential interaction between the thyrotropin receptor (TSHR) and the monocarboxylate transporter 8 (MCT8), a member of the major facilitator superfamily (MFS), using fluorescence cross-correlation spectroscopy (FCCS). Both the proteins are expressed endogenously on the basolateral plasma membrane of the thyrocytes and are involved in stimulation of thyroid hormone production and release. Indeed, we demonstrate strong interaction between both the proteins which causes a suppressed activation of Gq/11 by TSH-stimulated TSHR. Thus, we provide not only evidence for a novel interaction between the TSHR and MCT8, but could also prove this interaction on a single molecule level. Moreover, this interaction forces biased signaling at the TSHR. These results are of general interest for both the GPCR and the MFS research fields.

Mouse Thyroid Gland Changes in Aging: Implication of Galectin-3 and Sphingomyelinase.

Prevalence of thyroid dysfunction and its impact on cognition in older people has been demonstrated, but many points remain unclarified. In order to study the effect of aging on the thyroid gland, we compared the thyroid gland of very old mice with that of younger ones. We have first investigated the changes of thyroid microstructure and the possibility that molecules involved in thyroid function might be associated with structural changes. Results from this study indicate changes in the height of the thyrocytes and in the amplitude of interfollicular spaces, anomalous expression/localization of thyrotropin, thyrotropin receptor, and thyroglobulin aging. Thyrotropin and thyrotropin receptor are upregulated and are distributed inside the colloid while thyroglobulin fills the interfollicular spaces. In an approach aimed at defining the behavior of molecules that change in different physiopathological conditions of thyroid, such as galectin-3 and sphingomyelinase, we then wondered what was their behavior in the thyroid gland in aging. Importantly, in comparison with the thyroid of young animals, we have found a higher expression of galectin-3 and a delocalization of neutral sphingomyelinase in the thyroid of old animals. A possible relationship between galectin-3, neutral sphingomyelinase, and aging has been discussed.

Targeting thyroid diseases with TSH receptor analogs.

The thyroid-stimulating hormone (TSH) receptor (TSHR) is a major regulator of thyroid function and growth, and is the key antigen in several pathological conditions including hyperthyroidism, hypothyroidism, and thyroid tumors. Various effective treatment strategies are currently available for many of these clinical conditions such as antithyroid drugs or radioiodine therapy, but they are not devoid of side effects. In addition, treatment of complications of Graves' disease such as Graves' ophthalmopathy is often difficult and unsatisfactory using current methods. Recent advances in basic research on both in vitro and in vivo models have suggested that TSH analogs could be used for diagnosis and treatment of some of the thyroid diseases. The advent of high-throughput screening methods has resulted in a group of TSH analogs called small molecules, which have the potential to be developed as promising drugs. Small molecules are low molecular weight compounds with agonist, antagonist and, in some cases, inverse agonist activity on TSHR. This short review will focus on current advances in development of TSH analogs and their potential clinical applications. Rapid advances in this field may lead to the conduct of clinical trials of small molecules related to TSHR for the management of Graves' disease, thyroid cancer, and thyroid-related osteoporosis in the coming years.

Modern methods to investigate the oligomerization of glycoprotein hormone receptors (TSHR, LHR, FSHR).

As for other GPCRs, the oligomerization of glycoprotein hormone receptors (GPHRs) appears as critical event for receptor function. By means of modern techniques based on the BRET or FRET principle, GPHR oligomerization has been reported to explain several physiological and pathological conditions. In particular, the presence of oligomers was demonstrated not only in in vitro heterologous systems but also in in vivo tissues, and GPHR homodimerization appears associated with strong negative cooperativity, thus suggesting that one hormone molecule may be sufficient for receptor dimer stimulation. In addition, oligomerization has been reported to occur early during the posttranslational maturation process and to be involved in the dominant negative effect exerted by loss-of-function TSH receptor (TSHR) mutants, that are prevalently retained inside the cell, on the surface expression of wild-type receptors. This molecular mechanism thus explains the dominant inheritance of certain forms of TSH resistance. Here, we provide the description of the methods used in the original BRET, FRET, and HTRF-RET experiments.

Receptors for thyrotropin-releasing hormone, thyroid-stimulating hormone, and thyroid hormones in the macaque uterus: effects of long-term sex hormone treatment.

OBJECTIVE: Thyroid gland dysfunction is associated with menstrual cycle disturbances, infertility, and increased risk of miscarriage, but the mechanisms are poorly understood. However, little is known about the regulation of these receptors in the uterus. The aim of this study was to determine the effects of long-term treatment with steroid hormones on the expression, distribution, and regulation of the receptors for thyrotropin-releasing hormone (TRHR) and thyroid-stimulating hormone (TSHR), thyroid hormone receptor α1/α2 (THRα1/α2), and THRß1 in the uterus of surgically menopausal monkeys. METHODS: Eighty-eight cynomolgus macaques were ovariectomized and treated orally with conjugated equine estrogens (CEE; n = 20), a combination of CEE and medroxyprogesterone acetate (MPA; n = 20), or tibolone (n = 28) for 2 years. The control group (OvxC; n = 20) received no treatment. Immunohistochemistry was used to evaluate the protein expression and distribution of the receptors in luminal epithelium, glands, stroma, and myometrium of the uterus. RESULTS: Immunostaining of TRHR, TSHR, and THRs was detected in all uterine compartments. Epithelial immunostaining of TRHR was down-regulated in the CEE + MPA group, whereas in stroma, both TRHR and TSHR were increased by CEE + MPA treatment as compared with OvxC. TRHR immunoreactivity was up-regulated, but THRα and THRß were down-regulated, in the myometrium of the CEE and CEE + MPA groups. The thyroid-stimulating hormone level was higher in the CEE and tibolone groups as compared with OvxC, but the level of free thyroxin did not differ between groups. CONCLUSIONS: All receptors involved in thyroid hormone function are expressed in monkey uterus, and they are all regulated by long-term steroid hormone treatment. These findings suggest that there is a possibility of direct actions of thyroid hormones, thyroid-stimulating hormone and thyrotropin-releasing hormone on uterine function.

A TSHR-LH/CGR chimera that measures functional thyroid-stimulating autoantibodies (TSAb) can predict remission or recurrence in Graves' patients undergoing antithyroid drug (ATD) treatment.

CONTEXT: A functional thyroid-stimulating autoantibodies (TSAb) assay using a thyroid-stimulating hormone receptor chimera (Mc4) appears to be clinically more useful than the commonly used assay, a binding assay that measures all the antibodies binding to the thyroid-stimulating hormone receptor without functional discrimination, in diagnosing patient with Graves' disease (GD). OBJECTIVE: The objective of the study was to investigate whether an Mc4 assay can predict relapse/remission of hyperthyroidism after antithyroid drug (ATD) treatment in patients with GD. DESIGN: An Mc4 assay was used to prospectively track TSAb activity in GD patients treated with ATD over a 5-yr period. SETTING AND PATIENTS: GD patients from the Chieti University participated in this study. INTERVENTIONS: Interventions included the assessment of patients' sera using the Mc4 assay, the Mc4-derivative assay (Thyretain), and a human monoclonal thyroid-stimulating hormone receptor antibody, M22 assay. MAIN OUTCOME MEASURES: The Mc4 assay, a sensitive index of remission and recurrence, was used in this study. RESULTS: The TSAb levels significantly decreased only in the remitting group as evidenced by Mc4 assay values at the end of ATD (0.96 ± 1.47, 10.9 ± 26.6. and 24.7 ± 37.5 arbitrary units for the remitting, relapsing, and unsuspended therapy groups, respectively). Additional prognostic help was obtained by thyroid volume measurements at the end of treatment. Although not statistically significant, the Mc4 assay has a trend toward improved positive predictive value (95.4 vs. 84.2 or 87.5%), specificity (96.4 vs. 86.4 and 90.9%), and accuracy (87.3 vs. 83.3 and 80.9%) comparing the Mc4, Thyretain, and M22 assays, respectively. Thyretain has a trend toward improved negative predictive value (82.6 vs. 81.8 and 76.9%) and sensitivity (80 vs. 77.8 and 70%) comparing Thyretain, Mc4, and M22 assays, respectively. CONCLUSION: The Mc4 assay is a clinically useful index of remission and relapse in patients with GD. Larger studies are required to confirm these findings.

A TSHr-LH/CGr chimera that measures functional TSAb in Graves' disease.

CONTEXT: Stimulating thyrotropin receptor (TSHr) autoantibodies (TSAb) are the cause of hyperthyroidism in Graves' disease. In a patient's serum, TSAb can coexist with antagonist TSHr autoantibodies that block TSAb stimulatory activity (TSBAb); both can vary in amount and time. OBJECTIVE: The objective of the study was to create a functional assay that detects only TSAb, thus having an increased accuracy for diagnosing Graves' disease. DESIGN: A TSHr chimera (Mc4) that retains an agonist-sensitive TSAb epitope but replaces a TSBAb epitope was stably transfected in cells to establish the Mc4 assay. SETTING: The study was conducted at the Chieti University (Outpatient Endocrine Clinic) and the University of Pisa (the Department of Endocrinology). PATIENTS: The assay was validated using sera from 170 individuals with Graves' disease, Hashimoto's thyroiditis, and nonautoimmune hyperthyroidism and normal subjects from Chieti University. A second blinded study evaluated sera from 175 patients with autoimmune thyroid disease (mainly Graves' disease) from the University of Pisa. INTERVENTIONS: Interventions included the assessment of patients' sera using human wild-type TSHr (WT-TSHr), Mc4 chimera, and binding (TRAb) assays. MAIN OUTCOME MEASURES: The Mc4 assay has the best accuracy for diagnosing Graves' disease. RESULTS: The Mc4 assay has a better diagnostic accuracy than WT-TSHr and second-generation TRAb assays. Indeed, the sensitivity of the WT-TSHr, TRAb, and Mc4 assays was 97.3, 86.5, and 100%, respectively, whereas the specificity was 93.1, 97, and 98.5%, respectively. CONCLUSION: The Mc4 assay is a functional assay with improved sensitivity and specificity for the detection of TSAb and is clinically useful in diagnosing Graves' disease.

Pathogenesis of Graves' orbitopathy: a 2010 update.

The most important of the extra-thyroidal manifestations of Graves' disease, Graves' orbitopathy (GO), remains a vexing clinical problem. Treatment of severe active disease has been limited to steroids or radiotherapy. In the relatively rare case where vision is threatened, emergent decompression surgery can be performed. The proptosis, motility, or cosmetic concerns associated with stable GO are commonly remedied with surgical intervention. Substantial obstacles have prevented the development of specific medical therapies for GO, in large part resulting from poor understanding of disease pathogenesis and the absence of preclinical animal models. Fundamental aspects of GO's etiology have been uncovered from studies based in cell culture, extensive analysis of blood constituents, and detailed examination of orbital contents collected at the time of surgical intervention. Many of the published reports resulting from these studies are descriptive and all have failed to yield unifying concepts that integrate the anatomically divergent manifestations of Graves' disease. This brief review covers recent findings of several research groups. While major breakthroughs continue to occur in closely related autoimmune diseases, progress in identifying the pathogenic mechanisms relevant to GO has been limited. As emerging insights into human autoimmunity becomes applied to the study of Graves' disease, we anticipate that improved therapeutic strategies will find their way to our patients with GO.

[New assays of TSH-receptor antibodies: analytical and clinical performances in the diagnosis of Graves' disease]. / Nouveaux dosages des anticorps anti-récepteur de la TSH : performances analytiques et cliniques dans le diagnostic de la maladie de Basedow.

Graves' disease autoimmunity is attributable to the presence of serum antibodies (Ab) directed against the TSH receptor (TSHR) measured by a second generation (2G) assay using the human TRAK (hTRAK) with a high sensitivity in the diagnosis of Graves' disease. In this study, we have compared both analytical and clinical performances of hTRAK with those of five new methods using a porcine TSHR: two 2G methods and three assays using the monoclonal M22 directed against the TSHR pocket. We showed a bad reproducibility of these new methods with inter assay CVs higher than 10%. High clinical sensitivity and specificity that appeared similar to those of the hTRAK and next to 100% were observed except for a 2G method that failed to detect five Graves' patients. All these new methods should be avoided since they display a high variability despite their calibration against the same International Standard 90/672. The TRAKh using a human TSHR should be still used for a correct interpretation of results in the follow-up of Graves' disease.

TSH induces co-localization of TSH receptor and Na/K-ATPase in human erythrocytes.

Thyroid stimulating hormone (TSH) binds to a specific TSH receptor (TSHR) which activates adenylate cyclase and increases cAMP levels in thyroidal cells. Recent studies have reported the presence of TSH receptor in several extra-thyroidal cell types, including erythrocytes. We have previously suggested that TSH is able to influence the erythrocyte Na/K-ATPase ouabain binding properties through a receptor mediated mechanism. The direct interaction of TSH receptor with the Na/K-pump and a functional role of TSHR in erythrocytes was not demonstrated. The interaction of TSH receptor with Na/K-pump and a TSHR functional role are not yet demonstrated in erythrocytes. In this study, we examined the interaction between the two receptors after TSH treatment using immunofluorescence coupled to confocal microscopy and a co-immunoprecipitation technique. The cAMP dependent signalling after TSH treatment was measured to verify TSHR functionality. We found that TSH receptor and Na/K-ATPase are localized on the membranes of both erythrocytes and erythrocyte ghosts; TSH receptor responds to TSH treatment by increasing intracellular cAMP levels from two to tenfold. In ghost membranes TSH treatment enhances up to three fold co-localization of TSHR with Na/K-ATPase and co-immunoprecipitation confirms their direct physical interaction. In conclusion our results are compatible with the existence, in erythrocytes, of a functional TSHR that interacts with Na/K-ATPase after TSH treatment, thus suggesting a novel cell signalling pathway, potentially active in local circulatory control.

Clinical value of the first automated TSH receptor autoantibody assay for the diagnosis of Graves' disease (GD): an international multicentre trial.

BACKGROUND: Most recently, a new rapid and fully automated electrochemiluminescence immunoassay for the determination of TSH receptor autoantibodies (TRAb) based on the ability of TRAb to inhibit the binding of a human thyroid-stimulating monoclonal antibody (M22) has been established. OBJECTIVE: To evaluate this assay system in clinical routine based on an international multicentre trial and to compare the results with other established TRAb assays. PATIENTS AND MEASUREMENTS: Totally 508 Graves' disease (GD), 142 autoimmune thyroiditis, 107 subacute thyroiditis, 109 nonautoimmune nodular goitre, 23 thyroid cancer patients and 446 normal controls were retrospectively evaluated. RESULTS: ROC plot analysis revealed an area under curve of 0.99 (95% CI: 0.99-1.0) indicating a high assay sensitivity and specificity. The highest sensitivity (99%) and specificity (99%) was seen at a cut-off level of 1.75 IU/l. Here, the calculated positive predictive value was 95%, whereas the negative predictive value was 100%. Applying the ROC plot-derived cut-off of 1.75 IU/l we found a sensitivity for TRAb positivity within the group of newly diagnosed GD patients of 97% which is in accordance to the sum of different nonautomated porcine TSH receptor-based assays with a sensitivity of 94% indicating an excellent analytical performance of the new assay format. Detailed comparison of the automated and the sum of manual assays revealed a near identical specificity. CONCLUSION: Our results demonstrate that this new assay system has a high sensitivity for detecting GD and specificity for discriminating from other thyroid diseases. This assay may represent the future technology for rapid fully automated TRAb detection.

A fast method to detect cell surface expression of thyrotropin receptor (TSHr): the microchip flow cytometry analysis.

Loss-of-function mutations of the thyrotropin receptor (TSHr) may be responsible for congenital hypothyroidism or isolated hyperthyreotropinemia. To study cell surface expression of inactivating TSHr mutations detected in patients with isolated hyperthyreotropinemia (L252P, Q8fsX62, P27T, E34K, R46P, D403N, W488R, and M527T), we used the Agilent 2100 bioanalyzer to perform microchip flow cytometry analysis. The previously described TSHr inactivating mutation T477I was used as control. The level of receptor expression in COS-7 cells transfected with the T477I measured by binding assay was four times lower with respect to the wild-type TSHr. The very low expression of T477I was confirmed by fluorescence-activated cell sorting (FACS) analysis and by microchip flow cytometry analysis, suggesting that this method can be a reliable system to measure receptor cell surface expression. Other inactivating TSHr mutations were expressed in COS-7 cells for binding studies, FACS analysis, and microchip flow cytometry analysis. Binding studies showed that L252P, Q8fsX62, P27T, E34K, R46P, D403N, W488R, and M527T mutants had a low expression at the cell surface, as demonstrated by Bmax values. Data obtained by binding studies were in good agreement with data obtained by FACS analysis and microchip flow cytometry analysis. In conclusion, the low number of cells required for analysis and the ease of use make the microchip flow cytometry analysis a very reliable and favorable system to study cell surface expression of TSHr mutations.