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.

Transcriptome analysis of the effect of diosgenin on autoimmune thyroiditis in a rat model.

In a mouse model of Graves' disease (GD), diosgenin has been shown to have a therapeutic effect on GD by alleviating goitre. However, research on the effect of diosgenin on autoimmune thyroiditis (AIT) is lacking. In this study, transcriptomics was used to comprehensively analyse the protective effect of diosgenin against AIT in rats and the possible mechanism. The results showed that in the diosgenin-intervention group, compared to the model group, the expression of serum triiodothyronine, thyroxine, free triiodothyronine, and free thyroxine was decreased and that of thyroid-stimulating hormone was increased; these changes were accompanied by the downregulation of thyroglobulin, TSH receptor antibody and thyroid peroxidase expression in serum. Furthermore, transcriptome detection, RT-qPCR and immunohistochemistry verification revealed that in thyroid tissue, the relative mRNA and protein expression of cyclic adenosine 3',5'-monophosphate (cAMP), protein kinase A (PKA) and cAMP response element-binding protein (Creb) were increased and the mRNA expression of S100 calcium-binding protein A9 (S100A9) was decreased in the diosgenin groups. In summary, diosgenin alleviates the development of AIT, possibly via the activation of the cAMP/PKA/Creb pathway and downregulation of S100A9 gene expression.

Therapeutic plasma exchange for control of thyroid storm.

Therapeutic plasma exchange (TPE) for thyroid storm has recently been upgraded to a category II indication after decades though its recommendation level still remains at Grade 2C according to the American Society for Apheresis (ASFA). In the absence of prospective randomized controlled trials due to the rarity of thyroid storm, retrospective data from case series continue to elevate the clinical evidence supporting TPE as a life-saving modality for complicated thyroid storm patients. We report three cases of life-threatening thyroid storm from Graves' disease rescued by TPE via rapid reduction in circulating thyroid hormones. Each patient underwent TPE when it was judged that other thyroid storm treatment options were futile or unsafe. The first patient received 4 cycles of TPE while the second patient received 9 cycles of TPE, and the third patient received 2 cycles of TPE with satisfactory clinical improvement. Plasma FT4 and TSH receptor antibody levels of the first case declined by 41.3% and >50% respectively right after the first round of TPE; plasma FT4 of the second patient dropped by up to 31.6% during the course of TPE; plasma FT4 and TSH receptor antibody of the third patient declined by 66% and 56.2% respectively after the first cycle of TPE. This demonstrates the safety, efficacy, and feasibility of TPE in thyroid storm especially when other therapeutic interventions are contraindicated. TPE operates via the elimination of serum proteins-bound thyroid hormones, thyroid autoantibodies, cytokines, and catecholamines in addition to increasing unsaturated binding sites for thyroid hormones.

The association of TSH-receptor antibody with the clinical and laboratory parameters in patients with newly diagnosed Graves' hyperthyroidism: experience from a tertiary referral center including a large number of patients with TSH-receptor antibody-negative patients with Graves' hyperthyroidism.

INTRODUCTION: Although the TSH-receptor antibody (TRAb) plays a central role in the pathogenesis of Graves' disease (GD), the association between TRAb at first diagnosis and clinical and laboratory parameters is not well known. On the other hand, a minority of patients with GD may be TRAb negative, and there is a lack of adequate evidence to demonstrate the clinical and laboratory characteristics of these patients. Therefore, we aimed to investigate the association of TRAb at the initial diagnosis of GD with the clinical and laboratory parameters in a large number of patients with GD and to compare the clinical and laboratory parameters between patients with high TRAb levels and TRAb-negative patients. MATERIAL AND METHODS: This study included 440 patients [326 (74%) female, 114 (26%) male]. All patients were classified according to gender, age, smoking habit, and TRAb levels. RESULTS: TRAb levels were significantly higher in male compared to female patients and in smokers compared to non-smokers. Smoking male patients had the highest TRAb levels. In regression analysis, goiter size, male gender, cigarette smoking, Graves' orbitopathy, fT3, and anti-TPO antibody levels were independently associated with high TRAb levels, while age at diagnosis and fT4 levels were not independently associated with high TRAb levels. TRAb-negative GD was diagnosed in 80 (18%) patients. TRA-negative patients had markedly less severe clinical and laboratory hyperthyroidism compared to patients with high TRAb levels. Moreover, the smoking habit was significantly lower in patients with TRAb-negative GD. CONCLUSIONS: According to our study results, TRAb levels at the initial diagnosis of GD are differently associated with clinical and laboratory parameters. Male patients and smoking patients with GD tended to have markedly higher TRAb levels and more severe clinical hyperthyroidism. Therefore, besides other contributing factors, male gender and smoking may affect TRAb levels and consequently the severity of hyperthyroidism in patients with GD. Furthermore, male gender and smoking may have a synergistic effect on TRAb levels and consequently on the severity of hyperthyroidism in patients with GD.

Serum interferon levels associated with the disease activity in women with overt Graves' disease.

BACKGROUND: Inflammatory cytokines participate in immune reactions and the pathogenesis of autoimmunity. Herein, we quantified four groups of inflammatory cytokines, including interferons (IFNs), the tumor necrosis factor (TNF) superfamily (TNFSF), interleukin (IL)-related cytokines, and bone and extracellular matrix remodeling-related cytokines to determine their contributions in women with overt Graves' disease (GD). METHODS: Forty-three women with GD were enrolled in this cross-sectional study. Thirty-seven cytokines, thyroid-stimulating hormone (TSH), free thyroxine, and TSH receptor antibody (TSHRAb) were quantified. GD patients with a low TSH level at the time of sample collection were defined as having active GD. RESULTS: Patients with active GD had higher IFN-α2, IFN-γ, IFN-λ1, and IFN-λ2 levels than those with inactive GD. In addition, certain TNFSF cytokines, including soluble cluster of differentiation 30 (sCD30), TNFSF member 14 (TNFSF14), pentraxin (PTX)-3, soluble TNF receptor 2 (sTNF-R2), and thymic stromal lymphopoietin (TSLP) were higher in active GD than in inactive GD. Moreover, active GD patients had higher IL-2, IL-12(p40), osteocalcin (OCN), and matrix metalloproteinase (MMP)-3 than inactive GD patients. All IFNs except IFN-λ1 were correlated with TSHRAb titers. Moreover, TNFSF cytokines, consisting of B-cell-activating factor, sCD30, TNFSF14, PTX-3, sTNF-R2, and TSLP, were associated with TSHRAb levels. CONCLUSIONS: Serum IFNs could be the most remarkable cytokines in modulating the disease severity and TSHRAb titers in women with full-blown GD. Further molecular-based research to clarify the actual role of IFNs in the disease progression of GD is needed.

Reply to Drs. Kiaei and Molinaro Regarding the Publication "Comparison of a Bridge Immunoassay with Two Bioassays for Thyrotropin Receptor Antibody Detection and Differentiation".

Dear Editor,Drs. Kiaei and Molinaro 1 put forth two criticisms of the manuscript published by us 2. They state that the experimental design of this study is flawed and that the authors falsely claim that negative Thyretain™ TSI Reporter BioAssay results for two Graves' diseases patients undergoing drug treatments means the absence of stimulating antibodies. To substantiate this claim Drs. Kiaei and Molinaro point out that the manufacturer of the Thyretain TSI Reporter BioAssay clearly states in the package insert that "[t]he effects of various drug therapies on the performance of this Kit have not been established" 1. Second, the package insert explicitly states that "[a] negative result does not exclude the possibility of the presence of TSI" and results of the test should be interpreted in conjunction with information available from other clinical information, such as physical symptoms and thyroid hormone testing, as recommended by the American Thyroid Association (ATA)". Furthermore they state that the "authors of the manuscript did not consider the manufacturer's warning regarding the intended patient population and the ATA guidelines regarding the interpretation of the test results in conjunction with other clinical information. Instead, the authors based their conclusions on the negative Thyretain TSI Reporter BioAssay results and ignored the patients' clinical history of Graves' disease."

No detection of TSH or TSHR in oral lichen planus lesions in patients with or without hypothyroidism.

Objective: An association between hypothyroidism (HT) and oral lichen planus (OLP) has been reported. However, the mechanisms that could explain this association remain unresolved. This study aimed to evaluate the expression of thyroid-stimulating hormone (TSH) and thyroid-stimulating hormone receptor (TSHR) in healthy oral mucosa and in OLP lesions of individuals with and without HT.Material and methods: Immunohistochemical expression of TSH and TSHR was studied in oral mucosal biopsies obtained from 14 OLP patients with HT, 14 OLP patients without HT and 10 healthy controls without oral mucosal lesions. Gene expression of TSHR was investigated by using three different PCR techniques in oral mucosal samples from 7 OLP patients with HT, 3 OLP patients without HT, 9 healthy controls and in cultured human oral epithelial cells. Gene expression of TSH was examined by employing 2 PCR techniques in oral mucosal samples from 2 OLP patients with HT, 2 OLP patients without HT and 4 healthy controls.Results: TSH and TSHR stainings were negative in the studied oral mucosal specimens. Gene quantification assays demonstrated negative gene expression of TSH and TSHR in clinical and in vitro samples.Conclusions: These results suggest that TSH and TSHR may not be commonly involved in the pathogenetic mechanism that could explain the association between OLP and hypothyroidism.

Distinct pattern of peripheral lymphocyte subsets in Graves' disease with persistency of anti-TSHR autoantibodies.

Background: Graves' disease (GD) is characterized by the production of autoantibodies against the TSHR (TRAbs). With long-term treatment, serum concentrations of TRAbs decline but in some patients, despite being clinically stable, TRAbs persist for many years.Objective: To investigate whether GD patients with persistence of TRAbs constitute a subset of patients that could be identified by phenotypic analysis of circulating lymphocytes, suggesting disease heterogeneity.Materials and methods: Peripheral blood lymphocytes (including naïve, memory and effector T and B cells, Th17, regulatory T cells (Treg), recent thymic emigrants (RTEs) and double positive CD4+CD8+ (DP) cells) were analysed by flow cytometry in a cross-sectional study in 25 clinically stable GD patients, five patients at onset of GD disease and 40 healthy donors (HDs).Results: GD patients with persistence of TRAbs showed a lower percentage of Treg and lower absolute numbers of central and effector memory CD8+ T cells than HD. No differences in RTEs were found in peripheral blood from GD patients compared to HD. Stable GD patients had higher percentage of DP cells of effector phenotype than HD.Conclusions: Using extensive phenotypic analysis of lymphocyte subpopulations, it is possible to detect changes that help to identify patients with persistent TSHR antibodies and may contribute to understand why the autoimmune response is maintained.

Crystal structure of a ligand-free stable TSH receptor leucine-rich repeat domain.

The crystal structures of the thyroid-stimulating hormone receptor (TSHR) leucine-rich repeat domain (amino acids 22-260; TSHR260) in complex with a stimulating human monoclonal autoantibody (M22TM) and in complex with a blocking human autoantibody (K1-70™) have been solved. However, attempts to purify and crystallise free TSHR260, that is not bound to an autoantibody, have been unsuccessful due to the poor stability of free TSHR260. We now describe a TSHR260 mutant that has been stabilised by the introduction of six mutations (H63C, R112P, D143P, D151E, V169R and I253R) to form TSHR260-JMG55TM, which is approximately 900 times more thermostable than wild-type TSHR260. These six mutations did not affect the binding of human TSHR monoclonal autoantibodies or patient serum TSHR autoantibodies to the TSHR260. Furthermore, the response of full-length TSHR to stimulation by TSH or human TSHR monoclonal autoantibodies was not affected by the six mutations. Thermostable TSHR260-JMG55TM has been purified and crystallised without ligand and the structure solved at 2.83 Å resolution. This is the first reported structure of a glycoprotein hormone receptor crystallised without ligand. The unbound TSHR260-JMG55TM structure and the M22 and K1-70 bound TSHR260 structures are remarkably similar except for small changes in side chain conformations. This suggests that neither the mutations nor the binding of M22TM or K1-70TM change the rigid leucine-rich repeat domain structure of TSHR260. The solved TSHR260-JMG55TM structure provides a rationale as to why the six mutations have a thermostabilising effect and provides helpful guidelines for thermostabilisation strategies of other soluble protein domains.

Conversion to Graves disease from Hashimoto thyroiditis: a study of 24 patients

ABSTRACT Objective: The conversion of Hashimoto's thyroiditis (HT) to hyperthyroidism due to thyrotropin receptor antibodies is intriguing and considered rare. The contribution of TSH receptor blocking antibodies (TRAb), which may be stimulators (TSAb) or blockers (TBAb), is suspected. We describe clinical and biological variables in a series of patients switching from Hashimoto's thyroiditis to Grave's disease. Subjects and methods: Retrospective case study of 24 patients with Hashimoto's thyroiditis followed during 48 ± 36 months that developed later Graves' disease (GD). These variables were analysed in the hypo and hyperthyroid phase: age, sex, initial TSH, free triiodothyronine (fT3), free thyroxine (fT4), anti-TPO, TBII antibodies, parietal cell autoantibodies, time between hypo and hyperthyroidism, thyroid volume and levothyroxine doses (LT). Results: In HT, mean TSH was 9.4 ± 26.1 UI/L and levothyroxine treatment was 66.2 ± 30.8 µg/day. The switch to GD was observed 38 ± 45 months after HT diagnosis. As expected, we found significant differences on TSH, FT3, FT4 and TBAb levels. Three out of 14 patients had parietal cell autoantibodies. In two of these three cases there was an Helicobacter pylori infection. There were no significant differences between HT and GD groups with respect to thyroid volume. Conclusions: To our knowledge, large series documenting the conversion of HT to GD are scarce. Although rare, this phenomenon should not be misdiagnosed. Suspicion should be raised whenever thyroxine posology must be tapered down during the follow-up of HT patients. Further immunological and genetic studies are needed to explain this unusual autoimmune change.

Thyroid Stimulating Hormone Receptor Antibodies in Thyroid Eye Disease-Methodology and Clinical Applications.

BACKGROUND: Thyroid stimulating hormone receptor antibodies (TSHR-Ab) cause autoimmune hyperthyroidism and are prevalent in patients with related thyroid eye disease (TED). PURPOSE: To provide a historical perspective on TSHR-Ab and to present evidence-based recommendations for clinical contemporary use. METHODS: The authors review the recent literature pertaining to TSHR-Ab in patients with TED and describe the various immunoassays currently used for detecting TSHR-Ab and their clinical applications. RESULTS: We provide a historical summary and description of the various methods used to detect TSHR-Ab, foremost, the functional TSHR-Ab. Increasing experimental and clinical data demonstrate the clinical usefulness of cell-based bioassays for measurements of functional TSHR-Ab in the diagnosis and management of patients with autoimmune TED and in the characterization of patients with autoimmune-induced hyperthyroidism and hypothyroidism. Thyroid stimulating hormone receptor antibodies, especially the functional stimulating antibodies, are sensitive, specific, and reproducible biomarkers for patients with autoimmune TED and correlate well with clinical disease activity and clinical severity. Unlike competitive-binding assays, bioassays have the advantage of indicating not only the presence of antibodies but also their functional activity and potency. CONCLUSIONS: Measurement of TSHR-Ab (especially stimulating antibodies) is a clinically useful tool for the management of patients with TED.

Thyrotropin Receptor Antibodies-An Overview.

PURPOSE: Thyroid autoimmunity affects approximately 5% of the population, and its investigation relies heavily on the use of autoantibodies. Thyroid stimulating hormone receptor (TSHR) autoantibodies (TRAb) play a central role in the evaluation of Graves disease (GD), Graves ophthalmopathy (GO) and pretibial myxedema (PTM). However, there is still controversy regarding overall TRAb assay diagnostic accuracy and their prognostic utility. METHODS: We reviewed and analyzed the literature reporting TRAb assays and their clinical utility. RESULTS: Current assays measure the overall TRAb titer in a competitive manner (TSH binding inhibiting immunoglobulin assay) or biologic activity of the stimulating TSHR autoantibodies (thyroid stimulating immunoglobulin assay). Both types of assays have improved over time with advances in sensitivity and specificity. TRAb are particularly relevant in hyperthyroidism cases where use of iodinated contrast is not an option (e.g., pregnancy or recent use of iodinated contrast) or in cases of euthyroid eye disease, suspicious for GO. Third generation TRAb assays are useful for therapy selection in GD, prognostic predictions in GO and risk prediction for fetal and neonatal thyrotoxicosis. DISCUSSION: Given the pathogenic role of TRAb, we expect that the future will bring useful evidence regarding their predictive role with respect to efficacy of therapeutic modalities for GO and PTM. We also hope to better understand the role of blocking and neutral antibodies against TSHR, and harness that ability for modulation of thyroid function or therapy of differentiated thyroid carcinoma managed with TSH suppression. CONCLUSIONS: Thyroid autoimmune diseases have seen tremendous gains in understanding their pathophysiology, largely antibody mediated. Better TRAb testing is becoming a springboard for providing individualized patient care.

Serum Trace Elements Profile in Graves' Disease Patients with or without Orbitopathy in Northeast China.

OBJECTIVE: The purpose of the present study was to investigate serum trace elements in Graves' disease (GD) patients with or without orbitopathy in Northeast China. METHODS: Patients with newly diagnosed Graves' disease (HyGD) (n = 66), GD patients with euthyroid status or subclinical thyroidism after treatment (EUGD) (n = 55), GO patients with euthyroid status or subclinical thyroidism after treatment (GO) (n = 57), and normal controls (NC) (n = 66) were enrolled in this study. Serum trace elements were measured with ICP-MS. RESULTS: Serum selenium (Se) levels in EUGD group (median: 7.53 µg/dL), HyGD group (median: 6.76 µg/dL), and GO group (median: 7.40 µg/dL) were significantly lower than those in NC group (median: 9.20 µg/dL, all P < 0.01). Serum copper (Cu) levels in GO group (median: 95.93 µg/dL) were significantly lower than those in the NC group (median: 113.59 µg/dL, P = 0.015). After being adjusted for multivariables, thyroid-specific antibodies grade was associated with low Se levels. Hyperthyroidism and thyroid-specific antibodies grade were associated with high Cu levels. In addition, orbitopathy was associated with low Cu levels. CONCLUSIONS: Thyroid autoimmunity was associated with low Se levels. Hyperthyroidism and thyroid autoimmunity may be associated with relatively high serum Cu levels. Alternatively, ophthalmopathy may be related to low serum Cu levels.

Conversion to Graves disease from Hashimoto thyroiditis: a study of 24 patients.

OBJECTIVE: The conversion of Hashimoto's thyroiditis (HT) to hyperthyroidism due to thyrotropin receptor antibodies is intriguing and considered rare. The contribution of TSH receptor blocking antibodies (TRAb), which may be stimulators (TSAb) or blockers (TBAb), is suspected. We describe clinical and biological variables in a series of patients switching from Hashimoto's thyroiditis to Grave's disease. SUBJECTS AND METHODS: Retrospective case study of 24 patients with Hashimoto's thyroiditis followed during 48 ± 36 months that developed later Graves' disease (GD). These variables were analysed in the hypo and hyperthyroid phase: age, sex, initial TSH, free triiodothyronine (fT3), free thyroxine (fT4), anti-TPO, TBII antibodies, parietal cell autoantibodies, time between hypo and hyperthyroidism, thyroid volume and levothyroxine doses (LT). RESULTS: In HT, mean TSH was 9.4 ± 26.1 UI/L and levothyroxine treatment was 66.2 ± 30.8 µg/day. The switch to GD was observed 38 ± 45 months after HT diagnosis. As expected, we found significant differences on TSH, FT3, FT4 and TBAb levels. Three out of 14 patients had parietal cell autoantibodies. In two of these three cases there was an Helicobacter pylori infection. There were no significant differences between HT and GD groups with respect to thyroid volume. CONCLUSIONS: To our knowledge, large series documenting the conversion of HT to GD are scarce. Although rare, this phenomenon should not be misdiagnosed. Suspicion should be raised whenever thyroxine posology must be tapered down during the follow-up of HT patients. Further immunological and genetic studies are needed to explain this unusual autoimmune change.

Prevalence and clinical relevance of thyroid stimulating hormone receptor-blocking antibodies in autoimmune thyroid disease.

The prevalence and clinical relevance of thyroid stimulating hormone (TSH) receptor (TSHR) blocking antibodies (TBAb) in patients with autoimmune thyroid disease (AITD) was investigated. Serum TBAb were measured with a reporter gene bioassay using Chinese hamster ovary cells. Blocking activity was defined as percentage inhibition of luciferase expression relative to induction with bovine TSH alone (cut-off 40% inhibition). All samples were measured for TSHR stimulatory antibody (TSAb) and TSHR binding inhibiting immunoglobulins (TBII). A total of 1079 unselected, consecutive patients with AITD and 302 healthy controls were included. All unselected controls were negative for TBAb and TSAb. In contrast, the prevalence of TBAb-positive patients with Hashimoto's thyroiditis and Graves' disease was 67 of 722 (9·3%) and 15 of 357 (4·2%). Of the 82 TBAb-positive patients, thirty-nine (48%), 33 (40%) and 10 (12%) were hypothyroid, euthyroid and hyperthyroid, respectively. Ten patients were both TBAb- and TSAb-positive (four hypothyroid, two euthyroid and four hyperthyroid). Thyroid-associated orbitopathy was present in four of 82 (4·9%) TBAb-positive patients, with dual TSHR antibody positivity being observed in three. TBAb correlated positively with TBII (r = 0·67, P < 0·001) and negatively with TSAb (r = -0·86, P < 0·05). The percentage of TBII-positive patients was higher the higher the level of inhibition in the TBAb assay. Of the TBAb-positive samples with  > 70% inhibition, 87% were TBII-positive. Functional TSHR antibodies impact thyroid status. TBAb determination is helpful in the evaluation and management of patients with AITD. The TBAb assay is a relevant and important tool to identify potentially reversible hypothyroidism.

An improved method for the establishment of a model of Graves' disease in BALB/c mice.

The present study aimed to develop a stable Graves' disease (GD) model in BALB/c mice by immunization and electroporation (EP). A total of 90 mice were divided into experimental (n=50), control (n=20) and blank (n=20) groups. The recombinant plasmid pcDNA3.1/thyroid­stimulating hormone (TSH) receptor 268 was constructed and injected into the bilateral gastrocnemius of experimental group mice at weeks 1, 4, 7 and 10. Equal volumes of saline were injected into the control and blank groups at the same time. The experimental and control groups were subjected to EP at the same time and location to enhance immunization. The levels of total serum thyroxine (T4) and serum TSH were examined by radioimmunoassay and immunoradiometric assay, respectively. The levels of serum thyrotropin receptor N­terminal (TRAb N) and C­terminal (TRAb C) antibodies were assessed by ELISA. Whole body pertechnetate (99mTcO4­) imaging was performed. Mouse weight and thyroid morphology and pathology were analyzed. The GD BALB/c mouse model was successfully established, with a positive rate of 79.17% (38/48). T4 levels increased from baseline levels of 12.05±4.23 to 52.51±23.58 ng/ml by week 12 (P<0.0001). TSH levels decreased from baseline levels of 5.53±2.78 to 1.43±0.89 µIU/ml by week 12 (P<0.0001). TRAb N antibody levels increased from baseline levels of 0.006±0.002 to 0.278±0.106 mIU/ml by week 12 (P<0.0001). TRAb C antibody levels increased from baseline levels of 11.111±2.808 to 46.701±26.436 arbitrary units/ml by week 12 (P<0.0001). At week 21, TSH levels remained reduced compared with pre­immunization levels (P<0.0001). Although T4, and TRAb N and C levels decreased, they remained increased compared with preimmunization levels (P<0.0001, P<0.0001, P=0.001). There were no significant alterations in antibody levels between the control and blank groups. Following four immunizations, the uptake of 99mTcO4­ by the thyroid was significantly increased in the experimental group. The mean weight of the experimental mice was significantly reduced compared with the control and blank groups (all P<0.0001). Furthermore, the thyroid glands of the immunized mice were enlarged and exhibited lymphocyte infiltration, fewer colloid nodules and an increased height of epithelial cells. In conclusion, by injecting recombinant plasmid pcDNA3.1/TSHR268 and EP, a GD mouse model was successfully established.

Persistent Graves' hyperthyroidism despite rapid negative conversion of thyroid-stimulating hormone-binding inhibitory immunoglobulin assay results: a case report.

BACKGROUND: Graves' disease is an autoimmune thyroid disorder characterized by hyperthyroidism, and patients exhibit thyroid-stimulating hormone receptor antibody. The major methods of measuring circulating thyroid-stimulating hormone receptor antibody include the thyroid-stimulating hormone-binding inhibitory immunoglobulin assays. Although the diagnostic accuracy of these assays has been improved, a minority of patients with Graves' disease test negative even on second-generation and third-generation thyroid-stimulating hormone-binding inhibitory immunoglobulins. We report a rare case of a thyroid-stimulating hormone-binding inhibitory immunoglobulin-positive patient with Graves' disease who showed rapid lowering of thyroid-stimulating hormone-binding inhibitory immunoglobulin levels following administration of the anti-thyroid drug thiamazole, but still experienced Graves' hyperthyroidism. CASE PRESENTATION: A 45-year-old Japanese man presented with severe hyperthyroidism (serum free triiodothyronine >25.0 pg/mL; reference range 1.7 to 3.7 pg/mL) and tested weakly positive for thyroid-stimulating hormone-binding inhibitory immunoglobulins on second-generation tests (2.1 IU/L; reference range <1.0 IU/L). Within 9 months of treatment with oral thiamazole (30 mg/day), his thyroid-stimulating hormone-binding inhibitory immunoglobulin titers had normalized, but he experienced sustained hyperthyroidism for more than 8 years, requiring 15 mg/day of thiamazole to correct. During that period, he tested negative on all first-generation, second-generation, and third-generation thyroid-stimulating hormone-binding inhibitory immunoglobulin assays, but thyroid scintigraphy revealed diffuse and increased uptake, and thyroid ultrasound and color flow Doppler imaging showed typical findings of Graves' hyperthyroidism. CONCLUSIONS: The possible explanations for serial changes in the thyroid-stimulating hormone-binding inhibitory immunoglobulin results in our patient include the presence of thyroid-stimulating hormone receptor antibody, which is bioactive but less reactive on thyroid-stimulating hormone-binding inhibitory immunoglobulin assays, or the effect of reduced levels of circulating thyroid-stimulating hormone receptor antibody upon improvement of thyroid autoimmunity with thiamazole treatment. Physicians should keep in mind that patients with Graves' disease may show thyroid-stimulating hormone-binding inhibitory immunoglobulin assay results that do not reflect the severity of Graves' disease or indicate the outcome of the disease, and that active Graves' disease may persist even after negative results on thyroid-stimulating hormone-binding inhibitory immunoglobulin assays. Timely performance of thyroid function tests in combination with sensitive imaging tests, including thyroid ultrasound and scintigraphy, are necessary to evaluate the severity of Graves' disease and treatment efficacy.

Evaluation of the first fully automated immunoassay method for the measurement of stimulating TSH receptor autoantibodies in Graves' disease.

BACKGROUND: Thyroid stimulating hormone (TSH) receptor autoantibodies (TRAb) are pathogenetic and diagnostic hallmarks of Graves' disease (GD). Three varieties of TRAb have been described: stimulating (S-TRAb), blocking (B-TRAb), and apoptotic (A-TRAb) autoantibodies. Very recently, the first immunoassay method (Immulite TSI assay, Siemens Healthcare Diagnostics) declared to measure serum S-TRAb concentration, has been made available in an automated commercial platform. The aim of the study was to evaluate the ability of this new test to identify patients suffering from GD, in comparison with two current IMA methods for total TSH receptor autoantibodies (T-TRAb) measurement. METHODS: Sera of 383 subjects [72 patients with untreated GD, 55 patients with autoimmune thyroiditis (AIT), 36 patients with multinodular non-toxic goiter, 100 patients with other non-thyroid autoimmune diseases (NTAD) and 120 healthy subjects (HS)] were evaluated. RESULTS: The threshold obtained by receiver operating characteristic (ROC) analysis was 0.54 IU/L, very similar to that proposed by the manufacturer (0.55 IU/L). Thyroid-stimulating immunoglobulins (TSI) were positive in all GD patients and negative in all but three controls (clinical sensitivity and specificity of 100% and 98.7%, respectively). Passing and Bablok regression analysis and Bland-Altman plot showed an acceptable agreement between TSI Immulite assay and other two immunoassay methods (Cobas/Elecsys, Roche and TRAK RIA, BRAHMS Thermo Scientific). CONCLUSIONS: The diagnostic performance of fully automated Immulite TSI assay in GD patients is at least comparable to that of current TRAb immunoassays (IMAs) suggesting the possibility of including such assay in rapid and cost-saving diagnostic and monitoring algorithms. However, our results do not provide full evidence that this assay is specific for S-TRAb only, and future studies comparing Immulite TSI assay to stimulating activity are required.