TSH Receptor Autoantibodies Research Articles

The TSH receptor antibody reactome contributes to retro-orbital inflammation

Abstract The Thyroid Eye Disease (TED) of Graves’ disease is associated with high titers of stimulating TSH receptor antibodies, retro-orbital inflammation, fibroblast release of cytokines and chemokines and adipogenesis which in turn leads to proptosis, muscle fibrosis and dysfunction. Part of this scenario is the induction of fibroblast proliferation and autophagy secondary to synergism between the TSH receptor (TSHR) and the IGF-1 receptor (IGF-1R). While TED is well associated with thyroid stimulating antibodies to the TSHR which is also well expressed on fibroblasts, in fact the TSHR reactome has a variety of TSHR antibodies with varying biological activity. Therefore, we have now evaluated the possible role of neutral TSHR antibodies (N-TSHR-mAb), directed at the hinge region of the TSHR, which do not induce cell proliferation but are known to have effects on multiple proteins in thyroid cells including stress related signaling molecules. We examined the consequences of an N-TSHR-mAb acting on TSHR expressing fibroblasts and found marked cell stress which initiated signaling pathways involving inflammosome activation. This response ended in widespread cell death by pyroptosis through activation of caspase 8 and gasdermin D. Hence, not only can stimulating TSHR autoantibodies influence TED inflammation but the N-TSHR antibodies, representing more of the reactome, may also exaggerate the retro inflammatory response seen in TED.

Circular RNA CircZNF644 Facilitates Circulating Follicular Helper T Cells Response in Patients with Graves' Disease.

Aberrant accumulation of circulating follicular helper T cells (cTfh) has been found in the peripheral blood mononuclear cells (PBMCs) of Graves' disease (GD) patients. However, the underlying mechanism that contributes to the imbalance of cTfh cells remains unknown. Previously, studies described a GD-related circular RNAs (circRNAs)-circZNF644 that might be associated with cTfh cells. This study aimed to investigate the role of circZNF644 on cTfh cells in GD patients. Here, we found that circZNF644 was highly stable expression in the PBMCs of GD patients, which was positively correlated with the serum levels of TSH receptor autoantibodies (TRAb). Knockdown of circZNF644 caused a reduction of the proportion of cTfh cells in vitro. Mechanistically, circZNF644 served as a ceRNA for miR-29a-3p to promote ICOS expression, resulting in increased cTfh cells. In the PBMCs of GD patients, circZNF644 expression was positively correlated with ICOS expression and the percentage of cTfh cells, but negatively related to miR-29a-3p expression. Additionally, a strong relationship between circZNF644 and IL-21 was revealed in GD patients, and silencing of circZNF644 inhibited IL-21 expression. Our study elucidated that elevated expression of circZNF644 is a key feature in the development of GD and may contribute to the pathogenic role of cTfh cells in GD.

Evaluation of macular choroidal and microvascular network changes by activity scores and serum antibodies in thyroid eye patients and healthy subjects.

To investigate the choroidal thickness and the microvascular network changes around the macula in thyroid eye disease (TED) patients at different stages and the relationship of those changes with risk factors, serum antibodies and the severity of TED. A total of 85 participants were enrolled. All participants underwent ophthalmology and endocrinology examinations. Subfoveal choroidal thickness (SFCT), superficial (s) and deep (d) foveal avascular zone (FAZ) area, mean (m) and central (c) superficial vascular density (SVD), deep vascular density (DVD) measurements of the enrolled cases were performed with Topcon swept source optical coherence tomography (OCT)/OCT angiography (OCTA) DRI OCT Triton. Multiple linear regression analysis was used to explore the associations between SFCT, FAZ area, SVD, DVD and the relevant factors of TED. Those with active TED patients had higher c-DVD and m-DVD levels (P<0.05), however there is no statistically significant difference in SFCT between active and stable TED patients. Among the serum antibodies, it was observed that s-FAZ and d-FAZ increased, c-SVD and m-SVD decreased in patients with high thyroid stimulating hormone-receptor autoantibodies (TRAB) level, whereas SFCT thickened in patients with high levels of both TRAB and human thyroglobulin (hTG). There was no significant difference in SFCT, FAZ, SVD and DVD measurement at gender, between hyperthyroid and euthyroid patients and among those with or without thyroid papillary carcinoma. The results show that both disease activation and serum antibodies differentially affect both superficial and deep retinal vascular density. It has also been shown that high serum antibody levels affect choroidal thickness independent of clinical activity.

Primary Thyroid Dysfunction Is Prevalent in Hidradenitis Suppurativa and Marked by a Signature of Hypothyroid Graves’ Disease: A Case–Control Study

Hidradenitis suppurativa (HS) is a chronic skin disease that can have an association with endocrine disorders. There is conflicting information in the literature regarding the role of the thyroid gland in HS. This study aimed to close this knowledge gap and investigate how thyroid disease is involved in patients with HS. We carried out a case–control study with a total of 160 patients, of whom 108 were patients with HS and 52 were controls matched for age and sex. Parametric and non-parametric methods were used to analyze the results. We calculated structural parameters of thyroid homeostasis to detect subclinical thyroid disease, non-thyroid disease syndrome and other forms. The severity of HS was not associated with thyroid hormone levels and antibodies (p &gt; 0.05). HS patients with or without hypothyroidism had decreased FT4 levels and a decreased thyroid secretory capacity (SPINA-GT). Titers of TSH receptor autoantibodies (TRAb) were significantly higher in smoking HS patients compared to non-smokers (median: 1.18 vs. 1.08; p = 0.042). The rate of subclinical hypothyroidism was significantly higher in HS patients; thus, subclinical hypothyroidism is an important comorbidity of HS (p &lt; 0.05). Further studies are needed to investigate whether the chronic inflammation of HS is a cause of increased rates of (subclinical) hypothyroidism.

OR03-06 Transmembrane Water Channels Within The Tsh Receptor

Abstract Disclosure: R. Latif: None. T.F. Davies: Advisory Board Member; Self; Board of Kronus Inc (Starr, ID, USA);. M. Mezei: None. Using our recently reported hydrated model of the TSH receptor transmembrane (TM) region (Endocrinology 2021,162:7;1-12), we have now identified a network of putative TM water channels within this structure. By analyzing the molecular simulation trajectory for water molecules crossing the plasma membrane embedded TSHR we found evidence of putative water channels suggestive of active transport. To ascertain the biological activity of such TSHR channels we examined the movement of water down osmotic gradients using calcein aceto-oxymethyl ester (Calcein-AM) fluorescence in rat thyrocyte cells (FRTL5). Calcein-AM is a membrane permeant fluorescent dye that is quenched in a concentration-dependent manner and is, therefore, an effective probe of cell volume changes that would result from water movement across the cell membrane. Since thyroid cells are known to contain aquaporin (AQP) channels, principally AQP4, we first blocked these channels (using 3μM of TGN020) prior to examining the effects of TSHR activation using TSH (10-103 µU/ml), stimulating TSHR monoclonal antibodies (mAbs MS1 and M22 at 10μg/ml and 1μg/ml respectively) and TSHR-specific small molecule allosteric agonists (MS437 and MS438 at 10µM) and measured the changes in fluorescence quenching. D-Mannitol at 400mM was used as the hypertonic medium to induce cell shrinkage measured by continuous fluorescence measurements with read intervals of 50msec. Analyzing the normalized fluorescence of cells treated with TSH in a dose-dependent manner we observed ∼25% quenching with 103 µU/ml of TSH. On blocking the binding of TSH using a TSHR blocking mAb K1-70 (1μg/ml), we found marked de-quenching. Furthermore, using equimolar concentrations of stimulating TSHR antibodies the quenching was even more significantly enhanced to 54-60%. These data suggested that ligands that activate the TSHR by binding to the TSHR ectodomain could influence the TM water transport. However, using the allosteric activators, which act at the TM, we also showed similar changes indicating this was not ectodomain dependent. These data confirm the presence of the modeled putative TSHR water channels indicating that TSH and TSHR autoantibodies have an important role in regulating TM water passage. Presentation: Thursday, June 15, 2023

Functional Water Channels Within the TSH Receptor - a new paradigm for TSH action with disease implications.

The TSH receptor (TSHR) transmembrane domain (TMD) lives in the plasma membrane consisting of lipids and water molecules. To understand the role of TSHR-associated water molecules we used molecular dynamic simulations of the TMD and identified a network of putative receptor associated transmembrane water channels. This result was confirmed with extended simulations of the full length TSHR with and without TSH ligand binding. While the transport time observed in the simulations via the TSHR protein was slower than via the lipid bilayer itself we found that significantly more waters traversed via the TSHR than via the lipid bilayer which more than doubled with the binding of TSH. Using rat thyroid cells (FRTL-5) and a calcein fluorescence technique we measured cell volumes after blockade of aquaporins 1 and 4, the major thyroid cell water transporters. TSH showed a dose-dependent ability to influence water transport and similar effects were observed with stimulating TSHR autoantibodies. Small molecule TSHR agonists, which are allosteric activators of the TMD, also enhanced water transport illustrating the role of the TMD in this phenomenon. Furthermore, the water channel pathway was also mapped across two activation motifs within the TSHR TMD suggesting how water movement may influence activation of the receptor. In pathophysiological conditions such as hypothyroidism and hyperthyroidism where TSH concentrations are highly variable this action of TSH may greatly influence water movement in thyroid cells and many other extrathyroidal sites where the TSHR is expressed thus affecting normal cellular function.

Expansion of the immature B lymphocyte compartment in Graves' disease.

The specific mechanisms driving autoimmunity in Graves' disease (GD) remain largely unknown. Kappa-deleting recombination excision circles (KRECs) are circular DNA molecules generated during B cell maturation in the bone marrow which provide a measure of B cell production and proliferation. We aimed to investigate the association between KRECs and B cell subpopulations, with thyroid status and clinical outcome in GD patients. Kappa-deleting recombination excision circles were measured by quantitative real-time PCR using a triple-insert plasmid control in 132 GD patients and 140 healthy controls. In addition, KRECs in GD patients on withdrawal of antithyroid drug (ATD) and 6-10 weeks later were analysed according to a clinical outcome at 1 year. Flow cytometry was performed on isolated CD19+ B cells to quantitate 7 B lymphocyte subpopulations in 65 GD patients. Circulating KRECs were higher in GD vs. controls (P = 1.5 × 10-9) and demonstrated a positive correlation to thyroid hormones and autoantibodies (free thyroxine: P = 2.14 × 10-5, rho = .30; free triiodothyronine: P = 1.99 × 10-7, rho = .37; thyroid stimulating hormone receptor autoantibodies: P = 1.36 × 10-5, rho = .23). Higher KRECs in GD patients 6-10 weeks after ATD withdrawal were associated with relapse of hyperthyroidism at 1 year (P = .04). The KRECs were positively correlated to the total CD19+ B cell count (P = 3.2 × 10-7). This study reports a robust association between KRECs and GD, highlighting the importance of B cells in the pathogenesis of GD and the influence of thyroid status on B cell activity. The findings indicate a potential role for KRECs as a marker of disease activity and outcome in GD.

Discovery of SYD5115, a novel orally active small molecule TSH-R antagonist

The thyrotropin receptor (TSH-R) regulates the thyroid gland and is normally activated by thyrotropin. In patients with Graves’ disease, TSH-R is also stimulated by stimulatory TSH-R autoantibodies leading to hyperthyroidism. In this paper, we describe the discovery of SYD5115 (67), a novel small molecule TSH-R antagonist with nanomolar potency. SYD5115 also blocks stimulating antibody induced synthesis of the thyroid hormone thyroxine (T4) in vivo, after a single oral dose. During optimization, several issues had to be addressed such as the low metabolic stability and the potential mutagenicity of our first series of compounds.

Utility of Antibodies in the Diagnoses of Thyroid Diseases: A Review Article.

Thyroid problems are among the most widespread endocrine illnesses, affectingindividuals in India andthe global population. A thyroid function test is used to diagnose, screen, and monitor patients. Hyperthyroidism is a clinical condition due to excessive circulation of thyroid hormone; in contrast, hypothyroidism is due to a deficiency of thyroid hormone. Graves' disease (GD) is a form of hyperthyroidism due to thyroid-stimulating hormonereceptor autoantibodies (TRAb), and anti-thyroid peroxidase antibodies (anti-TPO antibodies). The most common reason for hypothyroidism is Hashimoto's thyroiditis (HT), in which patients have thyroid receptor antibodies (TRAb), antibodies to thyroid peroxidase (TPO), and thyroglobulin antibodies.Many essential genes, including the thyroid-specific genes thyroglobulin (TSGT), TSH-receptor gene, human leukocyte antigen (HLA) genes, cytotoxic T lymphocyte-associated antigen (CTLA)genes, thyroglobulin gene, vitamin D receptor gene, and many immune-regulatory geneswere associated with autoimmune thyroid diseases' (AITDs') etiology. This review paper aims to determine if antibodies are beneficial in detecting autoimmune thyroid disease or not. We have also discussed the etiology of autoimmune thyroid illness, serum antibodies in autoimmune thyroid disease, pathophysiology, and TSH receptor features.

PSUN39 A rare presentation of autoimmune thyroid diseases and myasthenia gravis: A clinical case report

Abstract We report a case of a 37-years-old female that presented with a three-week history of diplopia, ptosis, muscle weakness, difficulty chewing, tachycardia, and fatigue; these clinical symptoms were concerning for acute myasthenia gravis crisis. The diagnosis of Myasthenia gravis was further confirmed by a positive immunology workup. Chest CT imaging during her assessment showed anterior mediastinal mass/enlarged thymus indicative of thymic hyperplasia. Medical therapy with intravenous immune globulin, pyridostigmine and further treatment with high-dose steroid. She had a positive response to treatment but still reported persistent symptoms of fatigue, tachycardia, and intermittent diplopia, raising suspicions for underlying Graves’ thyrotoxicosis. Thyroid function testing was performed which showed a very suppressed thyroid-stimulating hormone (TSH) and elevated Free t4 levels. Further workup for Graves thyroid autoimmune antibodies indicative of extremely elevated thyroid stimulating hormone receptor autoantibodies consistent with Graves’ disease. We initiated medical therapy for Graves’ disease with a beta-blocker and thioamide (methimazole). She endorsed progressive improvement in her symptoms following treatment for Grave's disease. She was discharged with high dose steroids, pyridostigmine, and appropriate therapy for Graves’ disease. She did well after her discharge from the hospital and six months afterwards she returned to the hospital for a robot-assisted right thymectomy. Final pathology report indicative of non-thymomatous myasthenia gravis. She continued to follow up with us in the endocrinology outpatient clinic. Her steroid therapy was tapered off and medical therapy for Graves’ disease was continued accordingly. She demonstrated a positive response to medical therapy with remarkable improvement in the remaining of her symptoms, especially her neurological and ocular symptoms after initiation of treatment for Graves’ disease. Conclusion we report two rare concomitant autoimmune diseases, myasthenia gravis, and Graves’ disease. Both conditions have similar overlapping clinical features making diagnosis and treatment challenging in clinical practice. It is well reported in clinical literature and studies that hyperthyroidism may worsen symptoms of MG and management of hyperthyroidism in the setting of MG may lead to worsening of myasthenia symptoms; hence, certain precautions should be taken to avoid worsening symptoms of myasthenia gravis. Therefore, we call for more case-control, case series and clinical trials to explore more clinical knowledge about the association between the two conditions and formulate better management strategies. Presentation: Sunday, June 12, 2022 12:30 p.m. - 2:30 p.m.

RF35 | PSAT269 Double-Blind, Randomized, Placebo-controlled, Multicenter, Phase 2b Study of Batoclimab in Active and Moderate-to-Severe Thyroid Eye Disease

Abstract Clinical manifestations of thyroid eye disease (TED) are mainly caused by the binding of immunoglobulin G (IgG) thyrotropin receptors (TSHR) autoantibodies (TSHR-Ab) on orbital fibroblasts. Batoclimab (IMVT-1401) is an anti-neonatal Fc receptor (FcRn) human monoclonal antibody that prevents normal IgG recycling, leading to reduced levels of circulating IgG. Thus, batoclimab may provide benefit for patients with TED by reducing pathogenic anti-TSHR-Ab. In this Phase 2b, double-blind, randomized study (NCT03938545), subjects with active and moderate-to-severe TED received weekly subcutaneous (SC) injections of batoclimab (680mg, 340mg, 255mg) or placebo for 12-weeksSixty-five subjects were randomized and received batoclimab (680mg [n=18], 340mg [n=19], 255mg [n=10]) or placebo (n=18). Recruitment and treatment were stopped because of an unanticipated increase in LDL cholesterol levels, not included in subject informed consent. Hence, forty-five of 77 planned subjects completed the 12-week treatment period. Serum IgG and anti-TSHR-Ab were reduced from baseline to Week-12 with batoclimab (680mg [-77.9%; -65.5%], 340mg [-65.8%; -70.1%], 255mg [-56.6%; -42.1%]) versus placebo (-4.67%; -3.23%), respectively. Substantial reductions were also observed at Week-12 in stimulatory anti-TSHR-Ab. Fifty-one of 65 subjects had proptosis ≥ upper level of normal at baseline. At Week-12 (primary outcome), greater proportions of 680mg (30%; p=0.2197) and 340mg (30.8%; p=0.1311) subjects were proptosis responders (≥2mm reduction) versus placebo (8.3%). Absolute risk reduction (ARR [95% CI]) versus placebo was 21.4 (-11.3, 54.1) for 680mg and 24.2 (-4.8, 53.2) for 340mg at Week 12. Earlier proptosis responses were statistically different from placebo for 680mg at Weeks-four (35.7%; ARR [95% CI] 36.0 [10.9, 61.1]), five (42.9%; 43.0 [17.1, 68.9]), and 11 (41.7%; 34.5 [3.4, 65.6]) and for 340mg at Weeks-five (29.4%; 29.6 [7.9, 51.3]), nine(35.7%; 30.6 [3.0, 58.2]), and 11 (46.2%; 40.2 [10.2, 70.1]) (all p&amp;lt;0.05). Changes in clinical activity at Week-12 were not significantly different from placebo.The proportion of subjects with an adverse event (AE) were similar across treatment groups; most were mild or moderate in severity. Two subjects reported serious AEs not related to treatment (optic neuropathy, autoimmune encephalitis [occurring after treatment period]). No subjects withdrew from the study due to an AE during the 12-week treatment period. There was a reversible dose-dependent reduction in serum albumin during the treatment period. Post-hoc lipid analysis demonstrated a reversible increase in mean serum LDL during treatment (680-mg: 58.7%; 340-mg: 30.8%; 255-mg: 19.0%) vs placebo (-3.9%),.In conclusion, batoclimab reduced circulating IgG and especially the pathogenic anti-TSHR-Ab. Although the proptosis response was not statistically significant at the primary endpoint, significance was seen at earlier time points. This is probably related to early study termination which reduced the numbers of patients available for efficacy assessment at the primary endpoint. Presentation: Saturday, June 11, 2022 1:00 p.m. - 3:00 p.m., Monday, June 13, 2022 12:54 p.m. - 12:59 p.m.

Changes in the T and B lymphocyte subset profiles upon treatment of patients with Graves’ disease with radioactive iodine

The aim of the present study was to evaluate the subpopulation profile of T and B lymphocytes, and their relationships during therapy of the patients with Graves’ disease (GD) treated by means of radioactive iodine. We have examined 36 women with verified diagnosis of GD. The contents of thyroid hormones were determined by immunoradiometric analysis. The levels of thyroid-stimulating hormone receptor autoantibodies (rTSH) were evaluated by enzyme-linked immunosorbent assay. On the basis of comprehensive pre-therapeutic examination, all patients were exposed to the fixed-activity therapy with radioactive iodine-131 at a dose of 400 to 700 MBq administered orally in isotonic aqueous solution of sodium iodide. 56 practically healthy women were examined as a control group. The phenotype of T and B cells in whole blood was studied by flow cytometry using direct immunofluorescence. It was shown that the patients, prior to treatment with radioactive iodine, had high levels of cellular functional activity, as determined by expression of CD25 antigen on T cells and CD23-antigen on B lymphocytes. Higher functional activity of the cells responsive for adaptive immunity in the patients with GD manifests in the presence of increased levels of autoantibodies to rTSH. By means of correlation analysis, we found that the patients with GD examined before the therapy had the thyroid status may determine the functional stimulation of T and B cells, thus increasing the levels of autoimmune processes. One month after radioiodine therapy (RIT), the GD patients, along with transient hyperthyroidism with increased concentration of autoantibodies to rTSH, showed a reduction of activated T lymphocyte contents (including T helpers and cytotoxic T cells) to control values. However, the level of cytotoxic T lymphocytes in the blood remained low, and the content of Treg cells was significantly increased in the patients. Decreased contents of B cells activated memory B cell to the control levels were found in patients with GD over 1 month after RIT when studying the phenotype of blood B lymphocytes. In this case, increased levels of naive B lymphocytes and B2 cells were detected, as well as decreased numbers of activated B1 lymphocytes. The observed changes in the subpopulation composition of T and B cells, and in their phenotype developed against the background of complete absence of relationships between the studied parameters, thus suggesting loss of thyroid control of immune processes and cooperative cell interaction during the development of the immune response. Generally, the phenotypic changes of T and B lymphocyte subsets in the blood of patients with GD through 1 month after treatment with radioactive iodine may reflect a trend for decreased functional activity of adaptive cellular immunity which may also account for inhibition of autoimmune processes.

Computational model of the full-length TSH receptor.

(GPCR)The receptor for TSH receptor (TSHR), a G protein coupled receptor (GPCR), is of particular interest as the primary antigen in autoimmune hyperthyroidism (Graves' disease) caused by stimulating TSHR antibodies. To date, only one domain of the extracellular region of the TSHR has been crystallized. We have run a 1000 ns molecular dynamic simulation on a model of the entire TSHR generated by merging the extracellular region of the receptor, obtained using artificial intelligence, with our recent homology model of the transmembrane domain, embedded it in a lipid membrane and solvated it with water and counterions. The simulations showed that the structure of the transmembrane and leucine-rich domains were remarkably constant while the linker region (LR), known more commonly as the 'hinge region,' showed significant flexibility, forming several transient secondary structural elements. Furthermore, the relative orientation of the leucine-rich domain with the rest of the receptor was also seen to be variable. These data suggest that this LR is an intrinsically disordered protein. Furthermore, preliminary data simulating the full TSHR model complexed with its ligand (TSH) showed that (a) there is a strong affinity between the LR and TSH ligand and (b) the association of the LR and the TSH ligand reduces the structural fluctuations in the LR. This full-length model illustrates the importance of the LR in responding to ligand binding and lays the foundation for studies of pathologic TSHR autoantibodies complexed with the TSHR to give further insight into their interaction with the flexible LR.

2021 Asia-Pacific Graves' Disease Consortium Survey of Clinical Practice Patterns in the Management of Graves' Disease.

Although Graves' disease (GD) is common in endocrine practices worldwide, global differences in diagnosis and management remain. We sought to assess the current practices for GD in countries across Asia and the Pacific (APAC), and to compare these with previously published surveys from North America and Europe. A web-based survey on GD management was conducted on practicing clinicians. Responses from 542 clinicians were received and subsequently analysed and compared to outcomes from similar surveys from other regions. A total of 542 respondents participated in the survey, 515 (95%) of whom completed all sections. Of these, 86% were medical specialists, 11% surgeons, and 3% nuclear medicine physicians. In addition to serum thyroid-stimulating hormone (TSH) and free thyroxine assays, most respondents would request TSH-receptor autoantibody (TRAb) measurement (68%) during initial work-up. Thyroid ultrasound is requested by about half of respondents (53%), while the use of nuclear medicine scans is limited. The preferred first-line treatment is anti-thyroid drug (ATD) therapy (79%) with methimazole (MMI) or carbimazole (CBZ), followed by radioiodine (RAI; 19%) and surgery (2%). In case of surgery, one-third of respondents would opt for a subtotal rather than a total thyroidectomy. In case of mild Graves orbitopathy (GO), ATDs (67%) remains the preferred treatment, but a larger proportion of clinicians prefer surgery (20%). For a patient with intention to conceive, the preferred treatment pattern remained unchanged, although propylthiouracil (PTU) became the preferred ATD-agent during the first trimester. In comparison to European and American practices, marked differences were noted in the relatively infrequent usage of nuclear medicine scans and the overall higher use of a ATDs and β-blockers and adjunctive ATD-treatment during RAI in the APAC-group. Although regional differences regarding the diagnosis and management of GD are apparent in this first pan-Asia-Pacific survey, this study reveals the overall approach to the management of this disease in Asia-Pacific generally tends to fall between the trends appreciated in the American and European cohorts.

Clinical evaluation of an automated TSI bridge immunoassay in the diagnosis of Graves’ disease and its relationship to the degree of hyperthyroidism

BackgroundThe 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.MethodsA 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.ResultsAmong 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 < 0.001) and FT3 levels (TSI: r = 0.288, TRAb: r = 0.360; all P < 0.001) in new-onset GD patients. The ROC analysis showed that the optimal TSI cut-off value was 0.577 IU/L for GD diagnosis in this Chinese population, with a sensitivity of 96.92% and a specificity of 97.25%, respectively. The optimal TRAb cut-off value of was 1.38 IU/L, with a sensitivity of 96.92% and a specificity of 99.08%. There were no significant differences between the cut-off values obtained through the ROC analysis and those provided by the manufacturer for both TSI and TRAb when calculating their sensitivity and specificity in diagnosing GD. Among the 8 newly diagnosed GD cases with discordant qualitative antibody results, TSI was more likely than TRAb to match the clinical diagnosis of GD (6 TSI-positive vs. 2 TRAb-positive patients).ConclusionThe 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.

Case series of Euthyroid Graves’ Ophthalmopathy in patients seronegative for TSH receptor autoantibody (TRAb) &amp; developed Hyperthyroidism in follow-up visits

We reported cases of Graves’ ophthalmopathy who presented solely with symptoms of the eyes and normal thyroid function tests, negative TSH receptor autoantibody (TRAb). These cases were referred to our hospital for unilateral or bilateral eye swelling with painful eye movements, with or without double vision, without any signs or symptoms of hyper- or hypothyroidism. Serum thyroid function tests and 99mTc uptake studies were within the normal range. Anti-thyroid autoantibodies (TRAb) were negative in all the cases. Since orbital CT scan and MRI gave typical results compatible with Graves’ ophthalmopathy and after exclusion of other possibilities, these patients were treated with corticosteroid pulse therapy and orbital radiation therapy, leading to a partial improvement of the symptoms. We followed up those cases with serial thyroid profile testing and TRAb estimation and they were subsequently developed clinical &amp; biochemical hyperthyroidism within the next 18 months follow-up. At that time, they also became TRAb positive. These cases give us insight into the potential pathophysiologic mechanism underlying Graves’ ophthalmopathy and cast light upon the difficulties of establishing the diagnosis in biochemically euthyroid &amp; thyroid autoantibody negative cases. Clinicians should be aware of the variable temporal relationship between the clinical expression of thyroid dysfunction and orbital disease in the natural course of Graves’ disease.

A case of graves' orbitopathy in a patient sero-negative for TSH receptor autoantibody

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

TSH-receptor autoantibodies in patients with chronic thyroiditis and hypothyroidism.

The reported prevalence of TSH-receptor (TSHR) autoantibodies (TRAb) in patients with chronic thyroiditis (CT) range from 0 to 48%. The objective was to study the prevalence of TRAb in patients with CT and hypothyroidism and to correlate it with gender, age, thyroid dimensions, TSH levels, and autoimmune diseases. The study comprised 245 patients with CT and hypothyroidism (median age 42 years, 193 females, 52 males) and 123 Italian healthy subjects matched for sex and age as controls. TRAb were tested with ELISA using a >2.5IU/L cut off for positivity. TSHR blocking (TBAb) and TSHR stimulating autoantibodies (TSAb) were measured in 12 TRAb-positive patients using bioassays with Chinese hamster ovary (CHO) cells expressing wild-type or R255D-mutated TSHR. TRAb positivity was found in 32/245 (13.1%) patients and significantly correlated (p<0.05) with TSH levels. TRAb positivity was significantly higher in males vs. females (p=0.034), in females 16-45 years of age vs. >45 years of age (p<0.05) and in patients with reduced vs. normal/increased thyroid dimensions (p<0.05). Linear regression analysis showed a correlation between TRAb concentrations with age (p<0.05) and TRAb concentrations with TSH (p<0.01). In bioassay with TSHR-R255D all 12 patients tested were TBAb-positive while 33% were also TSAb-positive suggesting the presence of a mixture of TRAbs with different biological activities in some patients. TRAb have been found in patients with CT and hypothyroidism. A mixture of TBAb and TSAb was found in some patients and this may contribute to the pathogenesis of thyroid dysfunction during the course of the disease.

Abstract #1153940: Specific Detection of TSAb and TBAb in Serum of Suspected AITD Patients using Two Rapid Homogenous Bioassays

Two rapid homogenous bioassays, named as Turbo TSI and Turbo TBI, have been developed to either quantitatively detect Thyroid Stimulating Antibodies (TSAb) or qualitatively detect Thyroid Blocking Antibody (TBAb) in Autoimmune Thyroid Disease (ATID) patient sera. The two bioassays do not require cell culture, sample dilution, washing or cell lysis steps, resulting in a dramatically reduced turnaround time to 60 minutes. This study was designed to demonstrate the high specificity of the two bioassays in comparison with the TSHR Autoantibody (TRAb) ELISA Kit (Kronus).

A Tear Metabolomic Profile Showing Increased Ornithine Decarboxylase Activity and Spermine Synthesis in Thyroid-Associated Orbitopathy.

About half of patients with Graves’ disease develop an orbitopathy related to an inflammatory expansion of the periorbital adipose tissue and muscles. We used a targeted metabolomic approach measuring 188 metabolites by mass spectrometry to compare the metabolic composition of tears in patients with active (n = 21) versus inactive (n = 24) thyroid-associated orbitopathy. Among the 44 metabolites accurately measured, 8 showed a significant alteration of their concentrations between the two groups. Two short-chain acylcarnitines, propionylcarnitine and butyrylcarnitine, and spermine showed increased concentrations in the tears of patients with active orbitopathy, whereas ornithine, glycine, serine, citrulline and histidine showed decreased concentrations in this group. In addition, the ratio putrescine/ornithine, representing the activity of ornithine decarboxylase, was significantly increased in patients with active compared to inactive orbitopathy (p = 0.0011, fold change 3.75). The specificity of this candidate biomarker was maintained when compared to a control group with unclassified dry eye disease. Our results suggest that the stimulation of ornithine decarboxylase by TSH receptor autoantibodies in orbital fibroblasts could lead to increased synthesis of spermine, through the increased activity of ornithine decarboxylase, that may contribute to periorbital expansion in Graves’ ophthalmopathy.