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A male junior high school student presented with failure to gain weight and acceleration of growth for 2 years. Free triiodothyronine and free thyroxine levels were elevated, and the thyroid-stimulating hormone (TSH) level was suppressed. TSH receptor antibody (TRAb) and thyroid-stimulating antibody were negative. On I-123 thyroid scintigraphy, iodine uptake was most pronounced in the upper pole of the right lobe. The patient was initially diagnosed with asymmetrical TRAb-negative Graves’ disease (GD). His thyroid hormone level normalised with potassium iodide (KI) alone, and he became TRAb-positive 4 months after the initiation of KI therapy. This case demonstrates a rare presentation of GD that was initially TRAb-negative, which had asymmetrical iodine uptake on a thyroid scan and was confirmed to be TRAb positivity during the follow-up. KI monotherapy could be one of the effective treatment options for GD that is initially TRAb-negative.

Graves' orbitopathy (GO), an extrathyroidal manifestation of Graves' disease, can seriously threaten a patient's quality of life. Given that immunosuppressive treatment during the early active phase of GO has been found to reduce both disease activity and severity, sensitive screening tests are needed.The present study included 86 patients with GO, in whom serum levels of thyroid-stimulating hormone (TSH), free triiodothyronine (T3), free thyroxine, thyroid-stimulating antibody, TSH receptor antibody, thyroid peroxidase antibody, thyroglobulin, and thyroglobulin antibody were measured within 2 months before magnetic resonance imaging (MRI) for orbit assessment.The thyroid-stimulating antibody/TSH receptor antibody ratio was able to distinguish MRI results with a correct classification rate of 81%. When focusing on patients without T3 predominant Graves' diseases, the ratio distinguished MRI results at a rate of 92%. Receiver operating characteristic curve analysis revealed a cutoff antibody ratio of 87, which yielded a sensitivity, specificity, positive likelihood ratio, and negative likelihood ratio of 91%, 95%, 18.2, and 0.0957, respectively, for distinguished MRI results.The thyroid-stimulating antibody/TSH receptor antibody ratio is a highly sensitive and specific indicator for active GO, especially in patients without T3 predominance, and serves as a good screening test for active GO in primary care settings.

Objective To investigate the consistency between thyrotropin receptor antibody (TRAb) and thyroid-stimulating antibody (TSAb) levels in patients with Graves disease (GD). Methods We performed a cross-sectional observational study to recruit eligible patients with GD who visited the outpatient endocrinology clinic for the purpose of evaluating the consistency between their TRAb and TSAb levels. Our cohort included 28 men and 99 women. Results The median levels of TRAb and TSAb were 5.65 IU/L and 3.76 IU/L, respectively, in the enrolled patients with GD. The levels of TRAb (5.03 vs 8.42 IU/L; P = .008) and TSAb (2.69 vs 5.37 IU/L; P = .008) in patients with adequate thyroid regulation were all lower than those in patients with inadequate thyroid regulation. Conclusions Although TRAb is closely related to TSAb, we observed high heterogeneity of TRAb due to relatively low consistency between the levels of the 2 antibodies.

Background The syndrome of thyrotoxicosis typically relies on radioactive iodine scans for establishing its etiology. Alternatively, the determination of TSH receptor antibodies (TRAb) helps to distinguish Graves' disease (GD) from thyrotoxic thyroiditis. Current assays are impacted by limitations in sensitivity and/or turnaround time. Therefore, we decided to test a new assay for the detection of TSH receptor stimulating antibodies (TSAb) and compare it with the clinically available assays. Methods We enrolled 110 individuals in 5 cohorts: patients with incident or recurrent Graves' disease (cohort 1); patients with thyroiditis, painless or subacute (cohort 2); patients with Graves' Orbitopathy/Thyroid Eye Disease (TED) in cohort 3; patients with Hashimoto's thyroiditis (cohort 4) and a control group of normal volunteers (cohort 5). The patients were tested with the two clinically available assays: Roche Elecsys anti-TSHR assay (ROC-TBII) from Roche Diagnostics and Thyretain™ TSI Reporter BioAssay Kit (QUI-TSI) from Quidel. In addition, the samples were tested with the aequorin TSAb assay (OTS-TSI) provided by Otsuka Pharmaceutical Co., Ltd. (Tokyo, Japan). The data was collected in a cross-sectional fashion before initiation of therapy. Results We had 36 cases of GD, 17 cases of thyroiditis, 27 cases of TED, 10 cases of Hashimoto's thyroiditis and 20 normal volunteers. OTS-TSI had 100% sensitivity and specificity in distinguishing GD from thyroiditis, identical with QUI-TSI but superior to ROC-TBII (sensitivity 86% and specificity 94.1%). OTS-TSI had 93% sensitivity and 100% specificity for the diagnosis of TED, compared with normal controls. QUI-TSI and ROC-TBII performed similarly in this analysis, demonstrating 82% sensitivity and 100% specificity. The range of detectable values for OTS-TSI was 20 - 29,000 mIU/L and the turnaround time was ≤ 6 hours, without the need for cell culture equipment. Conclusions. OTS-TSI performed excellently, though similarly to QUI-TSI, for the differential diagnosis of GD versus thyroiditis, while being superior in that respect to ROC-TBII. Furthermore, OTS-TSI has superior sensitivity to QUI-TSI and ROC-TBII for TED diagnosis, while retaining high specificity. It has a short turnaround time and avoids the need for cell culture and sterility. Larger studies in US populations are needed for its validation.

Objective Conventional diagnostic methods are limited in their ability to differentiate destructive thyroiditis from Graves’ disease. We hypothesised that serum diiodotyrosine (DIT) and monoiodotyrosine (MIT) levels could be biomarkers for differentiating destructive thyroiditis from Graves’ disease. Design Patients with destructive thyroiditis (n = 13) and Graves’ disease (n = 22) were enrolled in this cross-sectional study. Methods We assayed the serum DIT and MIT levels using liquid chromatography-tandem mass spectrometry. A receiver operating characteristic (ROC) curve analysis was used to determine the sensitivity and specificity of the serum DIT and MIT levels as biomarkers for differentiating destructive thyroiditis from Graves’ disease. Results The serum DIT and MIT levels were significantly higher in patients with destructive thyroiditis than in those with Graves’ disease. The ROC curve analysis showed that the serum DIT levels (≥359.9 pg/mL) differentiated destructive thyroiditis from Graves’ disease, significantly, with 100.0% sensitivity and 95.5% specificity (P < 0.001). The diagnostic accuracy of the serum MIT levels (≥119.4 pg/mL) was not as high as that of the serum DIT levels (sensitivity, 84.6%; specificity, 77.3%; P = 0.001). Conclusions The serum DIT levels may serve as a novel diagnostic biomarker for differentiating destructive thyroiditis from Graves’ disease.

Context Neuropsychiatric symptoms are common features of Graves disease (GD) in hyperthyroidism and after treatment. The mechanism behind these symptoms is unknown, but reduced hippocampal volumes have been observed in association with increased thyroid hormone levels. Objective This work aimed at investigating GD influence on regional medial temporal lobe (MTL) volumes. Methods Sixty-two women with newly diagnosed GD underwent assessment including magnetic resonance (MR) imaging in hyperthyroidism and 48 of them were followed up after a mean of 16.4 ± 4.2 SD months of treatment. Matched thyroid-healthy controls were also assessed twice at a 15-month interval. MR images were automatically segmented using multiatlas propagation with enhanced registration. Regional medial temporal lobe (MTL) volumes for amygdalae and hippocampi were compared with clinical data and data from symptom questionnaires and neuropsychological tests. Results Patients had smaller MTL regions than controls at inclusion. At follow-up, all 4 MTL regions had increased volumes and only the volume of the left amygdala remained reduced compared to controls. There were significant correlations between the level of thyrotropin receptor antibodies (TRAb) and MTL volumes at inclusion and also between the longitudinal difference in the levels of free 3,5,3′-triiodothyronine and TRAb and the difference in MTL volumes. There were no significant correlations between symptoms or test scores and any of the 4 MTL volumes. Conclusion Dynamic alterations in the amygdalae and hippocampi in GD reflect a previously unknown level of brain involvement both in the hyperthyroid state of the condition and after treatment. The clinical significance, as well as the mechanisms behind these novel findings, warrant further study of the neurological consequences of GD.

Background As thyroid-stimulating immunoglobulins (TSI) are a sign of Graves' disease (GD), measuring TSI titers is becoming increasingly important for GD diagnosis. This study evaluated the diagnostic accuracy of a new fully automated TSI immunoassay (Immulite™ TSI assay) in GD patients and compared it to the third generation thyroid-stimulating hormone receptor antibody (TRAb) electrochemiluminescence assay (Elecsys Anti-TSHR assay). Additionally, clinical characteristics associated with responsiveness to methimazole in patients with newly diagnosed GD were preliminarily explored. Methods This study involved 324 subjects, comprising patients with untreated GD (GD-UT), Graves’ ophthalmopathy (GO) patients, GD patients who had been treated for > 12 months (GD-T), autoimmune thyroiditis (AIT) patients, and healthy subjects (HS). The Immulite™ TSI and Elecsys Anti-TSHR assay were performed on all samples. According to their responsiveness to methimazole, the GD-UT patients were divided into rapid and slow responder groups, and their clinical characteristics were compared. Results A receiver operating characteristic (ROC) curve analysis of GD-UT patients showed that the optimal TSI cut-off value was 0.57 IU/L. Logistic regression revealed that age and initial FT4 and TSI levels in the middle-dose methimazole group were related to a rapid response, while the initial FT4 level, but not TSI, in the high-dose group was also associated with a rapid response. Conclusions The clinical diagnostic performance of the Immulite™ TSI assay for diagnosing GD was comparable to that of the Elecsys Anti-TSHR assay. The initial FT4 and TSI levels can be used as predictors of the responsiveness to methimazole in patients with newly diagnosed GD.

The thyrotropin receptor (TSHR) plays critical roles in thyroid growth and function and in the pathogenesis of several thyroid diseases including Graves' hyperthyroidism and ophthalmopathy, non-autoimmune hyperthyroidism and thyroid cancer. Several low-molecular weight compounds (LMWCs) and anti-TSHR monoclonal antibodies (mAbs) with receptor antagonistic and inverse agonistic activities have been reported. The former binds to the pocket formed by the receptor transmembrane bundle, and the latter to the extracellular TSH binding site. Both are effective inhibitors of TSH/thyroid stimulating antibody-stimulated cAMP and/or hyaluronic acid production in TSHR-expressing cells. Anti-insulin-like growth factor 1 inhibitors are also found to inhibit TSHR signaling. Each agent has advantages and disadvantages; for example, mAbs have a higher affinity and longer half-life but are more costly than LMWCs. At present, mAbs appear most promising, yet the development of more efficacious LMWCs is desirable. These agents are anticipated to be efficacious not only for the above-mentioned diseases but also for resistance to thyroid hormone and have utility for thyroid cancer radionuclide scintigraphy/therapy as a new theranostic.

Thyroid hormones are vital in metabolism, growth and development(1). Thyroid hormone synthesis is controlled by thyrotropin (TSH), which acts at the thyrotropin receptor (TSHR)(2). In patients with Graves’ disease, autoantibodies that activate the TSHR pathologically increase thyroid hormone activity(3). How autoantibodies mimic thyrotropin function remains unclear. Here we determined cryo-electron microscopy structures of active and inactive TSHR. In inactive TSHR, the extracellular domain lies close to the membrane bilayer. Thyrotropin selects an upright orientation of the extracellular domain owing to steric clashes between a conserved hormone glycan and the membrane bilayer. An activating autoantibody from a patient with Graves’ disease selects a similar upright orientation of the extracellular domain. Reorientation of the extracellular domain transduces a conformational change in the seven-transmembrane-segment domain via a conserved hinge domain, a tethered peptide agonist and a phospholipid that binds within the seven-transmembrane-segment domain. Rotation of the TSHR extracellular domain relative to the membrane bilayer is sufficient for receptor activation, revealing a shared mechanism for other glycoprotein hormone receptors that may also extend to other G-protein-coupled receptors with large extracellular domains.

Objective: Hysterosalpingography (HSG) performed with an iodine contrast media can cause thyroid dysfunction, including thyrotoxicosis and hypothyroidism. We investigated the association between the serum levels of thyroid-stimulating hormone receptor antibody (TRAb), an indicator of Graves’ disease, and abnormal thyroid function after performing HSG.Methods: The screening of TRAb was conducted in 362 patients who first visited the Tawara IVF Clinic between April and September 2018. The association between TRAb levels and the effects of HSG examinations on thyroid function were evaluated.Results: Of the 362 patients, 2 (0.55%) had high levels (>2.0 IU/L) of TRAb, whereas 18 (5.0%) had intermediate TRAb levels, ranging from 0.3 to 1.9 IU/L. Of the 98 women (including 7 of the 18 women with TRAb level 0.3–1.9 IU/L, and 91 of the 342 women with TRAb level

Background: The actions of thyrotropin-binding inhibitory immunoglobulins (TBIIs) against thyrotropin receptors in thyroid follicular cells have been studied as important etiological factors in Graves' disease (GD). The purpose of this study was to investigate changes in the TBII levels of patients undergoing total thyroidectomy (TTx) or radioactive iodine (RAI) therapy for GD refractory to antithyroid drugs (ATDs). Methods: We enrolled patients who underwent TTx or RAI for GD with previous ATD use between January 2011 and December 2017 at the Samsung Medical Center in Seoul, Korea. Thorough retrospective reviews of medical records were performed in 130 patients. Results: Patients with goiter, ophthalmopathy, high levels of TBIIs, and high doses of ATDs received TTx. Elderly patients with arrhythmia received RAI. We observed that TBII levels continued to decrease after TTx. On the contrary, TBIIs initially increased for 138 days (estimated median time) and then decreased slowly after RAI. A faster decline in TBII levels was observed in the TTx group than in the RAI group (p 4.5 IU/L) were present in 70 (82%) at 6 months, 57 (67%) at 1 year, and 3 (3%) at 2 years. In the TTx group, rapid decreases in TBII levels were observed in younger patients and those with lower baseline TBII levels. In the RAI group, smaller thyroid volume was correlated with more rapid decrease in TBII levels. Conclusions: The changes in TBII levels following TTx or RAI were different in patients with refractory GD. When deciding on TTx or RAI, this difference should be considered with patient age, severity of hyperthyroidism, goiter, ophthalmopathy, and future pregnancy plans (for young female patients).

To investigate the prognostic factors of extraocular muscle restriction in patients with thyroid eye disease (TED), 65 patients with TED and restrictive myopathy were evaluated. Demographics, clinical activity score (CAS), smoking status, thyroid disease status, thyroid hormone status, thyroid autoantibody status, orbital computed tomography (CT) scan at initial presentation, and treatment regimens were assessed. The movements of the most severely affected extraocular muscles were categorized into five grades. The patients were divided into the improved and the not-improved group based on the improvement in the limitation of the extraocular muscle excursion (LOM) throughout the follow-up, and the groups were compared using clinical factors. The mean LOM significantly improved from 2.3 ± 1.1 to 1.7 ± 1.2 after 1 year of follow-up. The excursion of the most restricted muscle improved in 32 patients but not in 33 patients during the follow-up. The initial concentration of the thyroid-stimulating antibody (TSAb) was significantly lower in the improved (229.3 ± 114.1) than in the not-improved group (345.0 ± 178.6) (P = 0.02) Age, sex, smoking status, CAS, thyroid status, and muscle thickness on the CT scan did not significantly differ in the groups. This study showed that the initial concentration of TSAb is a factor affecting the recovery of restrictive myopathy.

Background The rapid and accurate detection of thyroid-stimulating hormone (TSH) receptor antibodies has always been an urgent need for the clinical diagnosis and management of Graves’ disease (GD). We aimed to evaluate the use of an automated thyroid-stimulating immunoglobulin (TSI) bridge immunoassay in the diagnosis of GD and to analyze the relationship between TSI and the degree of hyperthyroidism. Methods A total of 227 new-onset GD patients, 29 Hashimoto thyroiditis, 43 non-autoimmune thyroid diseases and 37 euthyroid controls were consecutively recruited. All participants accepted the measurement of their serum thyroid function and thyroid-associated antibodies, including TSI being measured by an Immulite 2000 bridge immunoassay and TSH receptor autoantibodies (TRAb) being measured by a third-generation Roche electrochemiluminescence immunoassay. The quantitative consistency between the TSI and TRAb detection methods was analyzed by using Passing-Bablok regression and Bland–Altman plots. The diagnostic performance for GD was assessed by receiver operating characteristic (ROC) curve analysis. Results Among 227 GD patients (174 females and 53 males, with a mean age of 39 years), the quantitative TSI was positively correlated with TRAb (r = 0.8099). According to the cut-off values proposed by the manufacturers (TSI: 0.55 IU/L, TRAb: 1.75 IU/L), the positive rates of TSI and TRAb in new-onset GD patients were 96.92% and 95.15%, respectively. Both TSI and TRAb levels positively correlated with FT4 levels (TSI: r = 0.243, TRAb: r = 0.317; all P 3 levels (TSI: r = 0.288, TRAb: r = 0.360; all P Conclusion The automated TSI bridge immunoassay was positively correlated with thyroxine levels in new-onset GD patients and was more likely to be consistent with the clinical diagnosis of GD than with that of TRAb. The positive Immulite 2000 TSI cut-off value of 0.577 IU/L for GD diagnosis in the Chinese population were close to the value recommended by the manufacturer.

The thyroid-stimulating hormone receptor (TSHR) is a Class A G protein-coupled receptor (GPCR) that mediates signalling through the hypothalamic-pituitary-thyroid axis. Inappropriate activation of TSHR by autoantibodies or mutations, results in human disease such as Grave's disease and Hashimito's thyroiditis. Therefore, there is a need to develop novel therapeutics targeting the TSHR. Understanding the structure and mechanism of activation of this receptor would help elucidate the pathogenesis of disease and aid drug development. Here, we describe a method for the expression of the human TSHR in a mammalian cell line generated through a lentiviral expression system. The receptor is then purified by affinity chromatography in the ligand-free state and is suitable for structure determination by single-particle electron cryo-microscopy (cryo-EM).

ObjectivesThe clinical course of thyroid eye disease (TED) is heterogeneous and predicting patients who may develop the severe sequelae of the disease is difficult. In this study, we evaluated the longitudinal association between changes in serum thyroid-stimulating hormone (TSH) receptor antibody (TRAb) levels and course of disease activity and severity over time.DesignThis was a multicentre, prospective, observational study.SettingFifteen tertiary care oculoplastic service centres in Korea.ParticipantsSeventy-six patients with newly diagnosed TED were included and followed up for 12 months.MethodsWe evaluated clinical characteristics and serum TRAb levels at baseline, 6 and 12 months of TED diagnosis. Additionally, we analysed longitudinal associations between the serum TRAb levels and clinical activity score (CAS), no signs or symptoms, only signs, soft tissue involvement, proptosis, extraocular muscle involvement, corneal involvement, sight loss (NOSPECS) score and proptosis.ResultsThyroid-stimulating immunoglobulin (TSI) and TSH-binding inhibitory immunoglobulin (TBII) levels decreased during the 1-year follow-up, whereas disease activity measured using CAS decreased mainly in the first 6 months. Disease severity measured using NOSPECS score and proptosis remained unchanged. Moreover, inter-person differences in TBII levels were associated with CAS, NOSPECS score and proptosis over time, whereas inter-person differences in TSI levels were associated with NOSPECS score. Subgroup analysis of patients with a baseline CAS≥4 demonstrated that within-person changes in TSI levels affected the CAS and NOSPECS score.ConclusionsFollow-up measurement of serum TSI and TBII levels may help evaluate TED prognosis and enable accurate clinical decision-making.

Background Thyroid-stimulating hormone receptor (TSHR)-activating autoantibodies stimulate thyroid growth and hormone synthesis/secretion, causing hyperthyroidism of Graves’ disease (GD). TRAb measurement helps diagnose GD and is an important first test in evaluating hyperthyroidism according to the recent American Thyroid Association guidelines. We compared the performance of the BRAHMS TRAK Kryptor (Thermo Scientific) and Roche cobas TRAb immunoassays for use in GD. Method Method comparison (n = 40) and clinical agreement were assessed between the Kryptor, cobas e411, and cobas e601. The analytical performance of Kryptor and cobas e411 were assessed for within- and between-day imprecision across 20 days, linearity, functional assay sensitivity (FAS), dilution recovery, and cut-off verification. Results The Kryptor, e411, and e601 TRAb immunoassays correlated well (r > 0.95, overall percent agreement = 0.95, Cohen’s kappa = 0.90). With a total allowable error of 20%, percent bias was within 13%, which was minimally negative at 20 IU/L. The Kryptor, but not e411, was linear across the claimed analytical measuring range (AMR). The claimed functional assay sensitivity (FAS), which was close to the clinical GD cut-off 1.8 IU/L, was verified for Kryptor and e411. Conclusion Overall, our evaluation demonstrates acceptable comparability between TRAb immunoassays with in-house imprecision up to 13% and 10% on Kryptor and e411, respectively. While Roche has preferable calibration frequency and on-board reagent stability, both platforms demonstrate acceptable imprecision using patient samples at their claimed FAS, which is important for GD diagnosis. Diluted results (using a negative patient pool as diluent) exhibits proportional positive bias on the Kryptor relative to the Roche methods.

BackgroundThe ongoing coronavirus disease 2019 (COVID-19) pandemic has forced the development of vaccines. Reports have suggested that vaccines play a role in inducing autoimmune diseases (AIDs). Scattered cases have reported that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines may promote thyroid disease, including Graves’ disease (GD). However, the effect of inactivated SARS-CoV-2 vaccine on GD remains unclear. The aim of the present study was to investigate the response of thyrotropin receptor antibody (TRAB) to inactivated SARS-COV-2 vaccines.MethodsWe conducted a retrospective study to observe the differences in thyroid function and TRAB trends between pre-vaccination (n=412) and post-vaccination (n=231) groups at an interval of 2 months. We then retrospectively observed the differences in serum thyroid function and TRAB levels at 3 months before (n=280), 1 month before (n=294), 1 month after (n=306), and 3 months after (n=250) vaccination. Subsequently, 173 GD patients who were not vaccinated with inactivated SARS-COV-2 vaccines were selected for a prospective study. Thyroid function and TRAB assessment were performed before 3 and 1 months and 1 and 3 months after the first dose of vaccination and were then compared by repeated measures ANOVA to explore their dynamic changes.ResultsA retrospective study preliminarily observed that the trend of TRAB post-vaccination was opposite of that pre-vaccination (p=0.000), serum TRAB levels decreased before vaccination and increased after vaccination. In this prospective study, repeated measures ANOVA indicated significant differences in serum FT3 (p=0.000), FT4 (p=0.000), TSH (p=0.000), and TRAB (p=0.000) levels at different time points before and after vaccination. Serum TRAB levels showed dynamic changes that decreased significantly at 1 month before vaccination (p=0.000), no significant differences at 1 month after vaccination (p=0.583), and reflected an upward trend at 3 months after vaccination (p=0.034). Serum FT3 and FT4 levels showed similar trends to serum TRAB levels before and after vaccination. Instead, the serum TSH levels showed a continuous upward trend over time.ConclusionBased on the results obtained in both retrospective and prospective studies, we concluded that serum TRAB levels decreased less after inactivated SARS-CoV-2 vaccination and showed an upward trend, which may be related to humoral immunity induced by vaccination.

ObjectiveGraves’ disease (GD) is an organ-specific autoimmune disease characterized by the production of thyroid-stimulating antibodies (TSAb). The newly discovered CD4+ T helper cells, Th9 and Th17 lymphocytes, have been confirmed to be closely associated with a variety of immune diseases. However, relationships with the onset and development of GD remain unclear. The purpose of this study was to investigate the roles of Th9 and Th17 in the pathogenesis and prognosis of GD.PatientsWe recruited 26 patients with newly diagnosed GD, 45 patients with GD in remission, and 20 healthy individuals.MeasurementsThyroid function and autoantibodies were evaluated using chemiluminescence immunoassays. Th9 and Th17 cells were analyzed using flow cytometry. The expression of Foxo1, IRF-4, RORc, IL-9, and IL-17 mRNA was examined using real-time PCR, and IL-9 and IL-17 protein levels were measured using enzyme-linked immunosorbent assay.ResultsTh9, Th17, and characteristic cytokines IL-9 and IL-17 in the GD-untreated group were significantly higher than those in the control and remission groups. The above indexes significantly decreased in the remission group, with the levels in the TRAb− remission group being similar to those in the normal group, while in the TRAb+ remission group, levels were differentially increased. TRAb titer was positively correlated with the levels of Th9, Th17, and their functional cytokines.ConclusionsTh9 and Th17 cells may be involved in the pathogenesis and disease outcome of GD, which could provide a new direction for developing immunotherapy for patients with GD.

BACKGROUND: Graves' eye disease, also called Graves' orbitopathy (GO), is a potentially debilitating autoimmune disease associated with retro-orbital inflammation and tissue expansion, involving both fibroblasts and adipocytes, resulting in periorbital edema, worsening proptosis, and muscle dysfunction with diplopia and may ultimately threaten sight. Accumulating evidence has indicated that autoantibodies to the thyrotropin receptor (TSHR), which induce the hyperthyroidism of Graves' disease, also help mediate the pathogenesis of the eye disease in susceptible individuals through TSHR expression on retro-orbital cells. Since it has long been known that the effects of insulin-like growth factor 1 (IGF-1) and thyrotropin are additive, recent clinical trials with a human monoclonal IGF-1 receptor blocking antibody (teprotumumab; IGF-1R-B-monoclonal antibody [mAb]) have demonstrated its ability to induce significant reductions in proptosis, diplopia, and clinical activity scores in patients with GO. However, the molecular mechanisms by which such an antibody achieves this result is unclear. METHODS: We have used Li-Cor In-Cell Western, Western blot, and immunohistochemistry to define levels of different proteins in mouse and human fibroblast cells. Proteomic array was also used to define pathway signaling molecules. Using CCK-8 and BrdU cell proliferation ELISA, we have analyzed proliferative response of these cells to different antibodies. RESULTS: We now show that a stimulating TSHR antibody was able to induce phosphorylation of the IGF-1R and initiate both TSHR and IGF-1R signaling in mouse and human fibroblasts. IGF-1R-B-mAb (1H7) inhibited all major IGF-1R signaling cascades and also reduced TSHR signaling. This resulted in the antibody-induced suppression of autophagy as shown by inhibition of multiple autophagy-related proteins (Beclin1, LC3a, LC3b, p62, and ULK1) and the induction of cell death by apoptosis as evidenced by activation of cleaved caspase 3, FADD, and caspase 8. Furthermore, this IGF-1R-blocking mAb suppressed serum-induced perkin and pink mitophagic proteins. CONCLUSIONS: Our observations clearly indicated that stimulating TSHR antibodies were able to enhance IGF-1R activity and contribute to retro-orbital cellular proliferation and inflammation. In contrast, an IGF-1R-B-mAb was capable of suppressing IGF-1R signaling leading to retro-orbital fibroblast/adipocyte death through the cell-extrinsic pathway of apoptosis. This is likely the major mechanism involved in proptosis reduction in patients with Graves' eye disease treated by IGF-1R inhibition.

Antithyroid antibodies, which include thyroid-stimulating hormone receptor antibodies (TRAbs), thyroid peroxidase antibodies (TPOAbs), and thyroid globulin antibodies (TgAbs), are widely known for their tight association with thyroid autoimmune diseases. The variation in all three kinds of antibodies also showed different trends during and after pregnancy (Weetman, 2010). This article reviewed the the physiological changes, while focusing on the variation of thyroid antibodies concentration in women during and after pregnancy, and adverse consequences related to their elevation. Since abnormal elevations of these antithyroid antibodies may lead to adverse outcomes in both mothers and fetuses, special attention must be paid to the titer of the antibodies during pregnancy. The molecular mechanisms of the variations in those antibodies have yet to be explained. The frequency and timing of thyroid antibody measurement, as well as different reference levels, also remain to be elucidated.

Both thyroid-stimulating immunoglobulins immunoassay (TSI IA) and thyrotrophin receptor antibody immunoassay (TRAb IA) are commonly used for the diagnosis of Graves’ disease (GD). The aim of the present study was to compare the clinical diagnostic performance of these two methods. Sera were obtained from 1103 subjects presenting a variety of clinical conditions from three centers: 100 subjects with untreated GD, 200 with treated GD, 62 with autoimmune thyroid disease(AIT), 216 with other thyroid diseases (OTHER-T), 214 with non-thyroid autoimmune diseases (NTAD), 191 with other diseases (OD), and 120 healthy subjects (HS). Both TSI and TRAb IAs were performed for all 1013 serum samples. Bioassay was performed for 86 samples whose TSI results were inconsistent the TRAb assay results. Comparing untreated GD patients with the control groups (AIT, NTAD, OTHER-T, OD, and HS) resulted in an area under the curve (AUC) of 0.992 for the TSI IA and 0.989 for the TRAb IA with no statistically significant difference observed between these AUC values (P = 0.2733). The best TSI CDP (clinical decision point) value was 0.42 IU/L. The differences in sensitivity (100% vs. 95%, P = 0.7991) and specificity (97.1% vs. 97.6%, P = 0.9426) between the TSI and TRAb IA were not statistically significant. TSI IA had a higher agreement with the TSI bioassay than TRAb IA. The clinical diagnostic performance of the TSI IA for diagnosing Graves’ disease was very similar to that of the TRAb IA. TSI IA can be used to diagnose GD in the Chinese.

Objective Soluble CD40 ligand (sCD40L) is elevated in various autoimmune disorders, which may have diagnostic and therapeutic implications. The aims of the current study were to evaluate serum sCD40L concentrations in children with newly diagnosed Graves’ disease (GD) and to correlate its levels with patients’ clinical and laboratory parameters. Methods This study included 48 children with newly diagnosed GD and 48 healthy children. Serum thyroid-stimulating hormone (TSH) (TSH, fT4 and fT3), TSH receptor antibodies (TRAbs), high sensitivity C-reactive protein (hsCRP) and sCD40L levels and thyroid volume were measured. Results Compared to control subjects, children with GD had higher thyroid volume standard deviation scores (SDS) (p=0.001), and higher levels of hsCRP (p=0.001), TRAbs (p=0.001) and sCD40L (p=0.001). Significant correlations were found between sCD40L and age (p=0.01), thyroid volume SDS (p=0.001), hsCRP (p=0.01) and TRAbs (p=0.001). In multivariate analysis, sCD40L concentrations were correlated with TRAbs [odds ratio (OR)=3.1, 95% confidence intervals (CI): 2.2-2.7, p=0.001] and thyroid volume SDS (OR=2.1, 95% CI: 1.2-2.7, p=0.001). Conclusion This preliminary study has evidence of high concentrations of sCD40L in children with newly diagnosed GD and a correlation between sCD40L and both TRAbs and thyroid volume, which may indicate a biologically active role for sCD40L in the pathogenesis of GD.

BACKGROUND AND OBJECTIVE Thyrotoxicosis is associated with accelerated bone turnover and increases the risk of fractures and osteoporosis. Graves' disease is the most common cause of hyperthyroidism. However, studies that examined risk factors associated with fragility fractures only in patients with Graves' disease are limited. Here, we investigated whether the risk of vertebral fracture (VF) of postmenopausal Graves' disease patients is high and tried to identify the risk factors for VF in that population. DESIGN AND METHODS Forty-three postmenopausal women with Graves' disease were enrolled. Physical and biochemical indices, thyroid indices and bone mineral density (BMD) were measured, and lateral X-rays were obtained to evaluate VFs. Age- and sex-matched healthy individuals were enrolled as the control group (n = 86). RESULTS The prevalence of VFs (35% vs 17%, P

Studies on the TSH receptor (TSHR) have numerous practical applications in vitro and in vivo. For example human monoclonal autoantibodies (MAbs) to the TSHR are useful reagents for in vitro diagnostics. Measurement of TSHR autoantibodies (TRAbs) is helpful in diagnosis and management of autoimmune thyroid disease. Currently available highly sensitive and specific assays to measure TRAbs use the human TSHR MAb M22 instead of the TSH. Furthermore, preparations of the human TSHR MAb M22 are useful as the World Health Organisation International Standard for thyroid stimulating antibody and for calibration of the assays for measuring TRAbs. Preparations of thermostabilised TSHR extracellular domain have recently become available and this is likely to have an impact on improvements in specificity testing for TRAb assays. In addition the stable TSHR preparations have practical application for specific immunoadsorption of patient serum TRAbs. Human TSHR MAbs also have promising prospects as new therapeutics. Autoantibodies with TSHR antagonistic activities are “natural” inhibitors of TSHR stimulation and are expected to be helpful in controlling TSHR activity in patients with Graves’ disease, Graves’ ophthalmopathy and thyroid cancer.

Graves' disease is uncommon in children. The remission rate after antithyroid drugs (ATD) therapy is lower than in adults. We evaluated the clinical course of ATD therapy in children with Graves' disease in southern Taiwan to determine whether their biochemical markers could be used to predict remission in these patients. We retrospectively reviewed the clinical data of 53 children diagnosed with Graves' disease between 2009 and 2019. Clinical and biochemical parameters were analyzed for predictors of remission. About three‐fourths of the patients were female. Their median age at diagnosis was 13 years. No sex differences were found in most clinical characteristics. There was no correlation between thyroid‐stimulating hormone receptor antibody (TRAb) levels at diagnosis and thyroid function or adverse reactions to ATD. Relapse occurred in 62% of patients after discontinuation of first‐course ATD therapy. Three variables—good initial response to ATD, a decrease in TRAb levels during the first year after diagnosis, and a decrease in TRAb levels during the second year after diagnosis—were significant predictors of remission for more than 18 months. In conclusion, children with Graves' disease who had early ATD‐controlled Graves' disease and decreased TRAb levels during the first 2 years are likely to enter remission for more than 18 months.

Background: Thyrotropin (TSH) and thyroid-stimulating antibodies (TSAbs) activate TSH receptor (TSHR) signaling by binding to its extracellular domain. TSHR signaling has been studied extensively in animal thyrocytes and in engineered cell lines, and differences in signaling have been observed in different cell systems. We, therefore, decided to characterize and compare TSHR signaling mediated by TSH and monoclonal TSAbs in human thyrocytes in primary culture. Methods: We used quantitative reverse transcription-polymerase chain reaction to measure mRNA levels of thyroid-specific genes thyroglobulin (TG), thyroperoxidase (TPO), iodothyronine deiodinase type 2 (DIO2), sodium-iodide symporter (NIS), and TSHR after stimulation by TSH or two monoclonal TSAbs, KSAb1 and M22. We also compared secreted TG protein after TSHR activation by TSH and TSAbs using an enzyme-linked immunosorbent assay. TSHR cell surface expression was determined using fluorescence activated cell sorting (FACS). Results: We found that TSH at low doses increases and at high doses (>1 mU/mL) decreases levels of gene expression for TSHR, TG, TPO, NIS, and DIO2. The biphasic effect of TSH on signaling was not caused by downregulation of cell surface TSHRs. This bell-shaped biphasic dose–response curve has been termed an inverted U-shaped dose–response curve (IUDRC). An IUDRC was also found for TSH-induced regulation of TG secretion. In contrast, KSAb1- and M22-induced regulation of TSHR, TG, TPO, NIS, and DIO2 gene expression, and secreted TG followed a monotonic dose–response curve that plateaus at high doses of activating antibody. Conclusions: Our data demonstrate that the physiological activation of TSHRs by TSH in primary cultures of human thyrocytes is characterized by a regulatory mechanism that may inhibit thyrocyte overstimulation. In contrast, TSAbs do not exhibit biphasic regulation. Although KSAb1 and M22 may not be representative of all TSAbs found in patients with Graves' disease, we suggest that persistent robust stimulation of TSHRs by TSAbs, unrelieved by a decrease at high TSAb levels, fosters chronic stimulation of thyrocytes in Graves' hyperthyroidism.

Graves' ophthalmopathy (GO) is characterized by an autoimmune reaction against thyrotropin (TSH) receptors and is diagnosed by TSH receptor antibody (TRAb). A novel assay for thyroid-stimulating antibody (TSAb) was recently introduced using a frozen Chinese hamster ovary cell line expressing TSH receptors, cyclic adenosine monophosphate (cAMP)-gated calcium channel, and aequorin (aequorin TSAb). The aim of this study was to evaluate the role of aequorin TSAb in GO. We studied 136 Japanese patients with GO (22 euthyroid and 8 hypothyroid GO patients) at our hospital. TRAbs were estimated by first generation TRAb (TRAb 1st), second generation TRAb (hTRAb 2nd), conventional porcine TSAb, and the new aequorin TSAb assays. Aequorin TSAb, porcine TSAb, TRAb 1st, and hTRAb 2nd were positive in 125/136 (92%), 110/136 (81%), 81/130 (62%), and 93/114 (82%) patients, respectively. In patients with hyperthyroid GO, they were positive in 98/106 (98%), 96/106 (91%), 78/101 (77%), and 84/93 (90%) patients, respectively. In patients with euthyroid GO, they were positive in 19/22 (86%), 9/22 (41%), 1/21 (5%), and 6/17 (35%) patients, respectively. Aequorin TSAb levels were significantly related to TRAb 1st (r = 0.4172, p < 0.0001), hTRAb 2nd (r = 0.2592, p < 0.0001), and porcine TSAb (r = 0.4665, p < 0.0001). Clinical activity score (CAS) was significantly greater in patients with high titers of aequorin TSAb than in those with low titers. Aequorin TSAb levels were significantly related to the signal intensity ratio of the enlarged eye muscle and proptosis evaluated by MRI before steroid pulse therapy. Aequorin TSAb assay was more sensitive than the conventional assays, especially in euthyroid GO.

Objective: Antibodies (Abs) to the thyrotropin (TSH) receptor (TSH-R) play an important role in the pathogenesis of autoimmune thyroid disease (AITD). We define the complex terminology that has arisen to describe TSH-R-Abs, review the mechanisms of action of the various types of TSH-R-Abs, and discuss significant advances that have been made in the development of clinically useful TSH-RAb assays. Methods: Literature review and discussion. Results: TSH-R-Abs may mimic or block the action of TSH or be functionally neutral. Stimulating TSH-R-Abs are specific biomarkers for Graves disease (GD) and responsible for many of its clinical manifestations. TSH-R-Abs may also be found in patients with Hashimoto thyroiditis in whom they may contribute to the hypothyroidism of the disease. Measurement of TSH-R-Abs in general, and functional Abs in particular, is recommended for the rapid diagnosis of GD, differential diagnosis and management of patients with AITD, especially during pregnancy, and in AITD patients with extrathyroidal manifestations such as orbitopathy. Measurement of TSH-R-Abs can be done with either immunoassays that detect specific binding of Abs to the TSH-R or cell-based bioassays that also provide information on their functional activity and potency. Application of molecular cloning techniques has led to significant advances in methodology that have enabled the development of clinically useful bioassays. When ordering TSH-R-Ab, clinicians should be aware of the different tests available and how to interpret results based on which assay is performed. The availability of an international standard and continued improvement in bioassays will help promote their routine performance by clinical laboratories and provide the most clinically useful TSH-R-Ab results. Conclusion: Measurement of TSH-R-Abs in general, and functional (especially stimulating) Abs in particular, is recommended for the rapid diagnosis, differential diagnosis, and management of patients with Graves hyperthyroidism, related thyroid eye disease, during pregnancy, as well as in Hashimoto thyroiditis patients with extra-thyroidal manifestations and/or thyroid-binding inhibiting immunoglobulin positivity. Abbreviations: Ab = antibody; AITD = autoimmune thyroid disease; ATD = antithyroid drug; cAMP = cyclic adenosine 3′,5′-monophosphate; ELISA = enzyme-linked immunosorbent assay; GD = Graves disease; GO = Graves orbitopathy; HT = Hashimoto thyroiditis; MAb = monoclonal antibody; TBAb = thyrotropin receptor blocking antibody; TBII = thyroid-binding inhibiting immunoglobulin; TSAb = thyrotropin receptor–stimulating antibody; TSB-Ab or TRBAb = thyrotropin receptor–stimulating blocking antibody; TSH = thyrotropin; TSH-R = thyrotropin receptor

IntroductionNeonatal hyperthyroidism mainly occurring in the children born to mothers with Graves’ disease (GD). The influence of maternal GD on the newborn’s thyroid function includes not only hyperthyroidism, but also various forms of hypothyroidism. Maternally transferred thyrotropin receptor antibodies (TRAb), the antithyroid drug (ATD) administration during pregnancy and previous definitive treatment of GD (radioactive iodine therapy or thyroidectomy) in the mother impact the function of the fetal/neonatal thyroid. Some newborns born to mothers with GD may present central hypothyroidism (CeH) due to impaired regulation of the fetal hypothalamic-pituitary-thyroid axis. The aim of this study was to evaluate different types of thyroid dysfunction in babies with neonatal hyperthyroidism.Materials and MethodsMedical records of 14 infants with neonatal hyperthyroidism (13 born to mothers with GD, and one born to mother with Hashimoto thyroiditis) were analyzed.ResultsTransient hyperthyroidism was the main thyroid dysfunction in our study group. Overt hyperthyroidism with highly increased TRAb levels (mean 13.0 ± 7.0 IU/L) was diagnosed in 6 (43%) neonates. Another 6 (43%) babies presented hyperthyroidism with slightly increased fT4 and/or fT3 levels and TSH levels in the lower limit of the normal range coinciding with positive TRAb levels (mean 3.8 ± 1.6 IU/L). Normal thyroid hormone levels with TSH levels below the lower limit of the range were observed in 2 (14%) neonates. Four babies in the study group (28.5%) required further levothyroxine (L-T4) supplementation due to CeH or, in one case, due to primary hypothyroidism.ConclusionOur study highlights the need for prolonged monitoring of thyroid function in children born to mothers with GD. Diagnosis of CeH could be delayed due to its masking by transient hyperthyroidism. Prolonged thyroid-stimulating hormone suppression after TRAb elimination should be considered as a signal announcing CeH.

Graves’ ophthalmopathy (GO) is a complex inflammatory condition affecting the orbit and is often associated with Graves’ disease (GD). This study aims to determine the levels of thyroid-stimulating immunoglobulin (TSI) and thyroid-stimulating hormone receptor autoantibody (TSHR-ab) in the serum of patients with GO, compare it with the GD, and determine whether there is a correlation with the clinical course of GO. The cross-sectional study included 82 patients with GO, 81 patients with GD, and 75 healthy subjects. The ocular manifestations of GO were identified and evaluated by the clinical activity score (CAS) and severity of GO using the European Group of Graves’ Orbitopathy (EUGOGO). TSI and TSHR-ab levels in the serum of participants were determined with ELISA kits and correlated with clinical findings. A total of 238 participant’s data were analyzed. There were 14 patients (17%) with unilateral GO. The most common ocular signs were eyelid retraction 68 (82.3%) and proptosis 61 (74.4%). The mean CAS score was 2.65±1.64 in GO patients and was higher in men than women (P = 0.008). The mean of TSI was 37.95±35.41 in GO, 14.16±15.67 in GD, and 4.33±2.94 in healthy controls (PPP = 0.023). The area under the ROC curve (AUC) of TSI was 0.933 and selected 14.1 IU/ml was the optimal cutoff value (98.78% of sensitivity, 83.97% of specificity). Our study showed that TSI is significantly related to GO and the severity of GO. Therefore, TSI can be used as a predictor of severe GO to help in prognostication, follow-up and treatment planning.

Thyroid-stimulating hormone (TSH), through activation of its G-protein-coupled thyrotropin receptor (TSHR), controls the synthesis of thyroid hormone-an essential metabolic hormone

Graves’ Ophthalmopathy (GO) is an organ-specific autoimmune disease that is often characterized by infiltration of orbital tissues and is considered as the most common extra-thyroid manifestation of Graves’ disease (GD). Although genetic susceptibility has been found to be critical for the phenotype of GO, the associated risk alleles in a single gene are generally insufficient to cause the disease. Accruing evidence has shown that epigenetic disorders can act as the potentially missing link between genetic risk and clinically significant disease development. Abnormal epigenetic modifications can lead to pro-inflammatory cascades and activation of orbital fibroblasts (OFs) by promoting the various inflammatory response pathways and regulating the diverse signaling molecules that are involved in the fibrogenesis and adipogenesis, thereby leading to the significant expansion of orbital tissues, fibrosis and inflammation infiltration. Additionally, emerging evidence has shown that the gut microbiome can possibly drive the pathogenesis of GO by influencing the secretion of Thyrotropin receptor antibody (TRAb) and T-helper 17 (Th17)/regulatory T cells (Treg) imbalance. This paper describes the latest epigenetic research evidence and progress made in comprehending the mechanisms of GO development, such as DNA methylation, histone modification, non-coding RNAs, and the gut microbiome.

Objective To evaluate the analytical and clinical performance of two immunoassays for diagnosis of Graves’ disease (GD), the Immulite thyroid‐stimulating immunoglobulin (TSI), and Elecsys Anti‐TSH receptor (TSHR) assay. Methods Precision and analytical measurement range were assessed using pooled samples of patients. The comparison between the two methods was evaluated using 579 clinical samples, and receiver operating characteristic (ROC) curves were drawn using the final diagnosis as reference. Clinical sensitivity and specificity, accuracy, positive predictive value (PPV), and negative predictive value (NPV) were calculated for the two tests. Results The repeatability and intermediate imprecision coefficient of variation (CV%) of the TSI assay were 3.8% and 4.1% at 0.95 IU/L, and 3.5% and3.6% at 19.5 IU/L, respectively. The assays were linear over a range 0.27–38.5 IU/L. There was a high correlation between the quantitative results of the two methods (correlation coefficient r = 0.930). The cut‐off value obtained by ROC analysis for TSI assay was 0.7 IU/L with sensitivity of 93.7% and specificity of 85.1%. An overall qualitative agreement of 91.5% between two methods was observed. Among 44 patients with discordant qualitative results, the TSI assay provided more satisfactory results consistent with clinical diagnoses. Conclusion The TSI assay showed excellent analytical performance and provided a high PPV for GD., To evaluate the analytical performance and the diagnostic efficacy of the TSI assay, in comparison with that of Elecsys TSHR autoantibody (Anti‐TSHR) in a large cohort of serum samples obtained from Chinese Graves' disease (GD) patients. The TSI assay showed a relatively high positive predictive value (PPV) and negative predictive value (NPV) in identifying GD.