Redox mechanisms in autoimmune thyroid eye disease.

Thyroid eye disease (TED) is an autoimmune condition affecting the orbit and the eye with its adnexa, often occurring as an extrathyroidal complication of Graves' disease (GD). Orbital inflammatory infiltration and the stimulation of orbital fibroblasts, triggering de novo adipogenesis, an overproduction of hyaluronan, myofibroblast differentiation, and eventual tissue fibrosis are hallmarks of the disease. Notably, several redox signaling pathways have been shown to intensify inflammation and to promote adipogenesis, myofibroblast differentiation, and fibrogenesis by upregulating potent cytokines, such as interleukin (IL)-1ß, IL-6, and transforming growth factor (TGF)-ß. While existing treatment options can manage symptoms and potentially halt disease progression, they come with drawbacks such as relapses, side effects, and chronic adverse effects on the optic nerve. Currently, several studies shed light on the pathogenetic contributions of emerging factors within immunological cascades and chronic oxidative stress. This review article provides an overview on the latest advancements in understanding the pathophysiology of TED, with a special focus of the interplay between oxidative stress, immunological mechanisms and environmental factors. Furthermore, cutting-edge therapeutic approaches targeting redox mechanisms will be presented and discussed.

Presentation of Graves' orbitopathy within European Group On Graves' Orbitopathy (EUGOGO) centres from 2012 to 2019 (PREGO III).

BACKGROUND: Graves' orbitopathy (GO) is subject to epidemiological and care-related changes. Aim of the survey was to identify trends in presentation of GO to the European Group On Graves' Orbitopathy (EUGOGO) tertiary referral centres and initial management over time. METHODS: Prospective observational multicentre study. All new referrals with diagnosis of GO within September-December 2019 were included. Clinical and demographic characteristics, referral timelines and initial therapeutic decisions were recorded. Data were compared with a similar EUGOGO survey performed in 2012. RESULTS: Besides age (mean age: 50.5±13 years vs 47.7±14 years; p 0.007), demographic characteristics of 432 patients studied in 2019 were similar to those in 2012. In 2019, there was a decrease of severe cases (9.8% vs 14.9; p<0.001), but no significant change in proportion of active cases (41.3% vs 36.6%; p 0.217). After first diagnosis of GO, median referral time to an EUGOGO tertiary centre was shorter (2 (0-350) vs 6 (0-552) months; p<0.001) in 2019. At the time of first visit, more patients were already on antithyroid medications (80.2% vs 45.0%; p<0.001) or selenium (22.3% vs 3.0%; p<0.001). In 2019, the initial management plans for GO were similar to 2012, except for lid surgery (2.4% vs 13.9%; p<0.001) and prescription of selenium (28.5% vs 21.0%; p 0.027). CONCLUSION: GO patients are referred to tertiary EUGOGO centres in a less severe stage of the disease than before. We speculate that this might be linked to a broader awareness of the disease and faster and adequate delivered treatment.

Optimizing levothyroxine treatment for subclinical hypothyroidism during pregnancy.

The onset of pregnancy places additional stress of the thyroid gland, which must produce additional thyroid hormones to support the developing foetus. Hypothyroidism, including subclinical hypothyroidism (SCH), may appear de novo at this time, or existing thyroid disease may become more severe. Accordingly, SCH is a relatively common complication of up to about 3% of pregnancies, with higher rates in some areas. There is strong evidence from systematic reviews and meta-analyses that uncontrolled SCH is associated with an increased risk of adverse pregnancy outcomes, including miscarriage, preeclampsia, and gestational diabetes. The evidence base also suggests that treatment with levothyroxine (LT4), optimized to control thyrotropin (TSH) to within its pregnancy-specific reference ranges reduces these risks. Current management guidelines provide a clear framework of intervention with LT4 in pregnant women with SCH, especially where TSH is high or where thyroperoxidase autoantibodies are present. Sub-optimal adherence to LT4 is common: it is important that patients take their LT4 correctly and that treating physicians and/or healthcare professionals manage these patients according to the latest management guidelines. The titration of LT4 is likely to occur within a range of LT4 daily doses between 25 µg and 75 µg for the majority of this population. LT4 is a narrow therapeutic index drug and small variations in dosage may produce a clinically significant change in thyroid status. Newer formulations of LT4, engineered to provide more precise and consistent dosing, and with a broad range of tablet strengths, may facilitate the precise titration of the LT4 dose for these patients.

Thyroid Inflammation and Immunity During the COVID-19 Pandemic: A Comprehensive Review and Case Study.

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to the development of various vaccines. Reports have emerged suggesting a possible association between SARS-CoV-2 vaccination and the onset of thyroid diseases. This review explores the clinical aspects of thyroid disorders following SARS-CoV-2 vaccination, including a case report of a patient with concomitant subacute thyroiditis (SAT) and Graves' disease (GD) with blocking thyrotropin receptor autoantibodies (TSH-R-Ab) following SARS-CoV-2 vaccination. SAT, characterized by transient inflammation of the thyroid gland, has been reported after SARS-CoV-2 vaccination. GD, an autoimmune hyperthyroidism, has also been observed post-vaccination, often with stimulating TSH-R-Ab. Graves' orbitopathy (GO) has been associated with SARS-CoV-2 vaccination in patients with a history of immune thyroid disease. The unique case underscores a very rare thyroid condition of functional hypothyroidism in possible relation to SARS-CoV-2 vaccination and the usefulness of functional analysis of TSH-R-Ab that can provide valuable insights into disease pathogenesis and help to guide treatment. This review highlights the need for continued monitoring and awareness of potential thyroid-related complications following SARS-CoV-2 vaccination.

Thyroid Eye Disease: Epidemiology, Natural History, and Risk Factors.

BACKGROUND: Thyroid eye disease (TED) is an autoimmune disorder of the orbit and the most frequent extrathyroidal manifestation of Graves' disease but it may rarely occur in euthyroid/hypothyroid patients with chronic autoimmune thyroiditis. EPIDEMIOLOGY: TED is a relatively infrequent disorder, particularly in its severe forms. Men tend to have more severe TED at an older age. The prevalence of TED is lower than in the past among patients with recent onset Graves' hyperthyroidism, and moderate-to-severe forms requiring aggressive treatments are no more than 5% to 6% of all cases. NATURAL HISTORY: After an initial inflammatory (active) phase and a plateau phase, TED stabilizes and eventually inactivates (inactive or burnt-out phase) after an estimated period of 18-24 months. Minimal-to-mild TED often remits spontaneously, but complete restitutio ad integrum almost never occurs when TED is more than mild. RISK FACTORS: Several risk factors contribute to its development on a yet undefined genetic background. Cigarette smoking is the most important of them, but thyroid dysfunction (both hyper- and hypothyroidism), radioactive iodine therapy (if not accompanied by low-dose steroid prophylaxis), elevated thyrotropin receptor antibodies, and, probably, hypercholesterolemia represent relevant modifiable risk factors. Early diagnosis, control and removal of modifiable risk factors, and early treatment of mild forms of GO (local treatment and selenium) may effectively limit the risk of progression to more severe forms.

Proof-of-concept and Randomized, Placebo-controlled Trials of an FcRn Inhibitor, Batoclimab, for Thyroid Eye Disease.

CONTEXT: Inhibition of the neonatal fragment crystallizable receptor (FcRn) reduces pathogenic thyrotropin receptor antibodies (TSH-R-Ab) that drive pathology in thyroid eye disease (TED). OBJECTIVE: We report the first clinical studies of an FcRn inhibitor, batoclimab, in TED. DESIGN: Proof-of-concept (POC) and randomized, double-blind placebo-controlled trials. SETTING: Multicenter. PARTICIPANTS: Patients with moderate-to-severe, active TED. INTERVENTION: In the POC trial, patients received weekly subcutaneous injections of batoclimab 680 mg for 2 weeks, followed by 340 mg for 4 weeks. In the double-blind trial, patients were randomized 2:2:1:2 to weekly batoclimab (680 mg, 340 mg, 255 mg) or placebo for 12 weeks. MAIN OUTCOME: Change from baseline in serum anti-TSH-R-Ab and total IgG (POC); 12-week proptosis response (randomized trial). RESULTS: The randomized trial was terminated because of an unanticipated increase in serum cholesterol; therefore, data from 65 of the planned 77 patients were analyzed. Both trials showed marked decreases in pathogenic anti-TSH-R-Ab and total IgG serum levels (P < .001) with batoclimab. In the randomized trial, there was no statistically significant difference with batoclimab vs placebo in proptosis response at 12 weeks, although significant differences were observed at several earlier timepoints. In addition, orbital muscle volume decreased (P < .03) at 12 weeks, whereas quality of life (appearance subscale) improved (P < .03) at 19 weeks in the 680-mg group. Batoclimab was generally well tolerated, with albumin reductions and increases in lipids that reversed upon discontinuation. CONCLUSIONS: These results provide insight into the efficacy and safety of batoclimab and support its further investigation as a potential therapy for TED.

Thyrotropin Receptor Antagonism by a Novel Small Molecule: Preclinical In Vitro Observations.

Background: Treatment of Graves' hyperthyroidism (GH) and Graves' orbitopathy (GO) is far from adequate, and hence, new substances that specifically target the autoantigens in GH/GO are warranted. This study determined the preclinical in vitro efficacy of SYD5115, a novel low-molecular-weight compound that inhibits the thyrotropin receptor (TSH-R). Methods: The TSH-R inhibiting capability of SYD5115 was tested through stimulation of wild-type and chimeric TSH-R expressed in Chinese hamster ovary (CHO) cells using two functional (stimulatory and blocking) cell-based TSH-R-Ab bioassays. TSH-R expressing human orbital fibroblasts, collected from GH+GO patients (GOF), were stimulated with the monoclonal antibody M22 or with stimulatory TSH-R-Ab (TSAb)-positive sera with cyclic adenosine monophosphate (cAMP) or hyaluronic acid (HA) release as readouts. The effect of SYD5115 on the viability of GOF was tested in 4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide and scratch cell growth assays. Results: SYD5115 significantly and dose dependently inhibited the TSH-R activation through M22 or TSAb-positive sera in all performed bioassays. Inhibition showed similar levels in the TSAb reporter bioassay and in the cAMP assay with GOF. The % inhibition and compound concentration showed a sigmoidal relationship, with all seven TSAb-positive sera markedly inhibited by SYD5115. An SYD5115 dose-dependent inhibition of M22 (10 ng/mL, 6 hours)-stimulated HA and/or cAMP-release from GOF was observed. Strong SYD5115-induced inhibitions of M22-stimulated cAMP production in GOF were registered with SYD5115 concentrations of 1 (p = 0.0029), 10 (p < 0.0001), 100 (p < 0.0001), 1,000 (p < 0.0001), and 10,000 (p < 0.0001) nM, respectively. SYD5115-induced inhibition of M22-stimulated HA production was noted with SYD5115 concentrations of 100 (p = 0.0392), 1000 (p = 0.0431), and 10,000 (p = 0.0245) nM, respectively. The inhibitory activity of SYD5115 was confirmed in a human osteosarcoma U2OS cell line stably expressing human TSH-R with cAMP as readout. SYD5115 induced 100% inhibition of the M22-induced cAMP levels with a potency of 193 nM. Compared with control, SYD5115 did neither impact the growth nor the migration of cultivated GOF. In addition, SYD5115 did not alter the viability of GOF. Conclusions: SYD5115 blocked M22- and TSAb-induced TSH-R activity with a nanomolar potency in TSH-R-overexpressed CHO cells as well as primary GOF, which demonstrates the ability of this small molecule to block TSH-R overactivity.

Rapamycin improves Graves' orbitopathy by suppressing CD4+ cytotoxic T lymphocytes.

CD4+ cytotoxic T lymphocytes (CTLs) were recently implicated in immune-mediated inflammation and fibrosis progression of Graves' orbitopathy (GO). However, little is known about therapeutic targeting of CD4+ CTLs. Herein, we studied the effect of rapamycin, an approved mTOR complex 1 (mTORC1) inhibitor, in a GO mouse model, in vitro, and in patients with refractory GO. In the adenovirus-induced model, rapamycin significantly decreased the incidence of GO. This was accompanied by the reduction of both CD4+ CTLs and the reduction of orbital inflammation, adipogenesis, and fibrosis. CD4+ CTLs from patients with active GO showed upregulation of the mTOR pathway, while rapamycin decreased their proportions and cytotoxic function. Low-dose rapamycin treatment substantially improved diplopia and the clinical activity score in steroid-refractory patients with GO. Single-cell RNA-Seq revealed that eye motility improvement was closely related to suppression of inflammation and chemotaxis in CD4+ CTLs. In conclusion, rapamycin is a promising treatment for CD4+ CTL-mediated inflammation and fibrosis in GO.

Pathophysiology of thyroid-associated orbitopathy.

Thyroid-associated orbitopathy, the most common extrathyroidal manifestation of Graves' disease, is characterized by orbital inflammatory infiltration and activation of orbital fibroblasts, which mediates de novo adipogenesis, excessive production of hyaluronan, myofibroblast differentiation and ultimately tissue fibrosis. Interactions among T cells, B cells, and orbital fibroblasts result in their activation and perpetuation of orbital inflammation as well as tissue remodelling. T helper 17 cells belong to a newly identified pathogenic CD4+ T cell subset which possesses prominent pro-inflammatory and profibrotic capabilities. Thyroid stimulating hormone receptor/insulin-like growth factor-1 receptor crosstalk and the downstream signalling pathways of both receptors represent the major mechanisms leading to activation of orbital fibroblasts. Thyroid stimulating hormone receptor autoantibody is the disease specific biomarker of great clinical relevance and utility. There is growing evidence that oxidative stress, gut microbiome and epigenetics also play a role in the pathogenesis and their manipulation may represent novel therapeutic strategies.

Recurrence risk of autoimmune thyroid and endocrine diseases.

BACKGROUND AND OBJECTIVE: The recurrence risk ratio (λ) expresses the risk ratio of index patients' first-degree relatives developing a disease as compared to the general population and is a quantitative measure of the genetic contribution to the disease. This paper offers the results of a specialized center as well as a review of the pertinent literature. METHODS: Data from 3315 consecutive subjects followed at an ORPHAN academic tertiary referral expert center for endocrine autoimmunity as well as 419 unrelated German families were collected. λ was assessed based on 806 well-documented subjects, 299 index patients with autoimmune glandular (AIGD) and non-endocrine diseases and 507 of their first-degree relatives (328 children, 179 siblings). RESULTS: As many as 36% of relatives of patients with autoimmune diseases (AID) were affected by various autoimmune conditions. Twenty-five percent and 23% of all relatives had an AIGD or an autoimmune thyroid disease (AITD), respectively. Furthermore, 29% and 25% of relatives of index cases with polyglandular (PGA) and monoglandular (MGA) autoimmunity were affected. The recurrence risk for AITD was increased 16-fold in both children and siblings compared to the general population (λ, 95% CI 16, 11-21 and 16, 12-19, respectively). Furthermore, λ for AITD/AIGD was 21.62 (95% CI 14.17-30.69)/17.57 (11.80-24.36) and 13.48 (8.42-20.52)/10.68 (6.76-16.02) for siblings of patients with PGA and MGA, respectively. Overall, a strong genetic component for AITD and AIGD with a significant genetic impact on the development of PGA was demonstrated. CONCLUSION: These novel results strongly recommend the screening for AITD and AIGD in children and siblings of index patients with AITD.

Use of levothyroxine in the management of hypothyroidism: A historical perspective.

The thyroid operates within a complex system of homeostatic regulation, where the level of thyrotropin (TSH) influences the rate of secretion of the principal thyroid hormones, thyroxine (T4) and triiodothyronine (T3). The devastating consequences of untreated thyroid dysfunction have been evident for centuries. Indeed, sources from antiquity described goitre and cretinism, two of the clinical sequelae of untreated overt thyroid disease. It was not until the first part of the 19th century that goitre and cretinism were first associated with iodine status; however, the endocrine function of the thyroid was not clearly identified until the early part of the 20th century. Three principal innovations in the 20th century supported the use of levothyroxine (LT4) replacement therapy for the management of hypothyroidism: a practical technique for the synthesis of LT4 suitable to support pharmaceutical use (late 1940s), the discovery that LT4 is converted to the active thyroid hormone, T3, in the peripheral tissues (1970), and the development of robust and sensitive assay methodology for measuring thyroid hormones in the blood (1960 onwards). Synthetic LT4, titrated to bring the level of TSH within a predefined "normal" reference range, is now established as the mainstay of treatment for hypothyroidism, and provides adequate restoration of thyroid hormone function for most people with this condition. Future research will explore further the nuances of the hypothalamic-pituitary-thyroid axis, and the place, if any, for T3 within the management of thyroid dysfunction.

A Multicenter, Single-Blind, Case-Control, Immunohistochemical Study of Orbital Tissue in Thyroid Eye Disease.

Background: Thyroid eye disease (TED) involves several pathogenic pathways and a battery of infiltrating mononuclear cells, cytokines, and chemokines in the orbit. Revealing the main molecules, which play a major role in the pathogenesis of TED, will help developing novel treatment strategies. Methods: In a multicenter, single-blind, case-control study, 60 tissue samples were collected during orbital decompression (44 TED patients) or non-TED related oculoplastic (16 controls) surgeries. Formalin-fixation and paraffin embedding preserved orbital tissue. Tissue sections were immunostained with 18 antibodies by the micro-polymer labeling technique. Immunostaining slides were scanned by Panoramic Desk and blindly evaluated by a user-independent viewer software. Results: Marked lymphocyte infiltration was observed in orbital tissue specimens of patients with clinically active TED (n = 22) and to a much lesser extent in inactive cases (n = 22), while it was absent in controls. Increased vascularity was noted in all samples, with orbital congestion in specimens of clinically active TED. Tissue fibrosis was present in TED samples but not in controls. Immunohistochemistry of orbital tissue clearly differentiated between TED and controls, as well as between active and inactive TED. In contrast to controls and with the exception of cluster of differentiation 20 (CD20), 17 out of 18 antibodies were highly expressed in orbital connective tissue of TED patients. Especially, thyrotropin receptor (TSH-R), insulin-like growth factor 1 receptor (IGF-1R), CD40, cluster of differentiation 40 ligand (CD40L), CD3, CD68, interleukin-17A (IL-17A), IL-23A, IL-1ß, IL-4, regulated on activation, normal T cell expressed and secreted (RANTES), macrophage chemoattractant protein 1 (MCP-1), IL-16, and B cell activating factor (BAFF) were overexpressed in clinically active TED (all p < 0.001). Also, the expression of CD40L, IL-17A, IL-23A, IL-6, IL-1ß, RANTES, and BAFF was very high (TED/control ratio >3), moderate (ratio >2), and low in active (p < 0.001), inactive TED and controls, respectively. The expression of TSH-R, IGF-1R, CD40, CD40L, CD3, CD68, CD20, IL-17A, IL-23A, RANTES, MCP-1, and BAFF positively and significantly correlated with both serum TSH-R stimulatory antibody concentrations and clinical activity scores while it negatively correlated with TED duration. Orbital irradiation decreased TSH-R (p < 0.001) and IGF-1R expression (p = 0.012); in contrast, neither smoking, age, nor gender did impact immunohistochemical staining. Conclusions: Adaptive and cell-mediated immunity, overexpression of TSH-R/IGF-1R and CD40/CD40L are the relevant pathomechanisms in TED. Targeting these key players in the active phase of the disease offers specific and novel treatment approaches.

A novel point-of-care device accurately measures thyrotropin in whole blood, capillary blood and serum.

OBJECTIVES: Point-of-care (POC) measurement of thyrotropin (TSH) may facilitate prompt diagnosis of thyroid dysfunction. We evaluated the analytical performance of a new POC TSH assay (Wondfo). METHODS: TSH measurements were made from 730 consecutive, unselected subjects in an outpatient setting, using Wondfo in whole blood, capillary blood and serum or automated reference equipment (serum only). RESULTS: TSH measurements were user-independent. Total intra-and inter-assay variation (CV%) was 12.1 and 16.2%, respectively. Total CV% was 10.6-22.6% and 14.5-21.6% in serum and whole blood, respectively. Linearity was very good. Recovery rate was 97-127%. Prolongation of incubation time increased TSH results of 12% (13%) and 33% (35%) after 2 and 5 additional minutes in serum (blood), respectively. When measured simultaneously in two Wondfo devices, the slope of the regression line was 1.03 (serum) and 1.02 (blood), with Spearman's correlation of 0.99 for both. TSH measurements between Wondfo and reference correlated strongly (r=0.93-0.96), though TSH measurements were lower with Wondfo (slopes of plots of measurements made using the two devices were 0.94 [serum vs. serum]; 0.83 [whole blood vs. serum] and 0.64 [capillary blood vs. serum]). Depending on sample material, TSH in capillary blood was lower vs. whole blood (slope: 0.82) and for whole blood vs. serum (Wondfo and reference method; slope: 0.69 and 0.83). Total haemolysis, but not elevated bilirubin or lipemia, disrupted TSH measurement. CONCLUSIONS: The Wondfo system was straightforward to use without need for specialist technicians and demonstrated analytic performance suitable for clinical use for the diagnosis of thyroid dysfunction.

Sensitivity of three thyrotropin receptor antibody assays in thyroid-associated orbitopathy.

Background: Thyrotropin receptor autoantibodies (TSH-RAb) are indispensable biomarkers in the laboratory assessment of thyroid-associated orbitopathy (TAO). Clinical sensitivity of three different assays for TSH-R-Ab determination was evaluated in patients with TAO. Methods: 87 consecutive TAO patients were enrolled and their serum samples analyzed in parallel with three assays. An ECLIA competitive binding and a chemiluminescent bridge immunoassay were used to measure total and binding TSH-R-Ab concentration, while their functional activity was determined using a stimulatory TSH-R-Ab (TSAb) cellbased bioassay. Results: Compared to the two binding assays (ECLIA p<0.001, bridge p=0.003), the TSAb bioassay was more sensitive pertaining to the positive detection of TSH-R-Ab in TAO patients. No difference (p=0.057) was noted between the ECLIA and bridge assays regarding sensitivity rate. All patients with active and/or moderate-to-severe TAO tested positive in the TSAb bioassay (100% and 100%, respectively), while the positivity rates for bridge and ECLIA binding assays were 89.7% and 82.1% for active TAO, and 90.2% and 86.3% for severe TAO, respectively. Negative predictive values of the bioassay, bridge, and ECLIA assays were 100%, 75%, and 71%, respectively for active TAO, and 100%, 86%, and 71%, respectively for moderate-to-severe TAO. The superiority of the bioassay was most prominent in euthyroid (ET) TAO. Positivity rates of the TSAb bioassay, bridge and ECLIA binding assays were 89.6%, 75%, and 64.6%, respectively for inactive TAO; 86.1%, 69.4%, and 52.8%, respectively for mild TAO; 87.5%, 62.5%, and 12.5%, respectively for euthyroid TAO. The bridge assay correlated better with the ECLIA binding assay (r=0.893, p<0.001), compared to the bioassay (r=0.669, p<0.001). Conclusions: In patients with TAO of various activity and severity, the TSAb bioassay demonstrates a superior clinical performance compared to both ECLIA and bridge binding assays.

Drug safety in thyroid eye disease - a systematic review.

INTRODUCTION: The autoimmune-induced thyroid eye disease (TED) is a frequent extrathyroidal manifestation of Graves' disease and less frequently of Hashimoto's thyroiditis. Pathognomonic clinical signs, i.e. exophthalmos, double vision, and inflammation of the orbital tissue cause physical, ophthalmic, and socio-psychological limitations. AREAS COVERED: PubMed and MeSH database were searched for specific guidelines, randomized controlled trials, prospective clinical studies, systematic reviews, and meta-analyses pertaining to the safety profile of currently administered immunosuppressive agents for the treatment of TED. Occurred adverse events (AE), severe AE (SAE), side effects (SE), and severe SE (SSE) were classified according to the standardized medical dictionary for regulatory activities (MedDRA). EXPERT OPINION: This novel systematic analysis offers an overview of potential AE, SAE, and SE for currently recommended immunosuppressive drugs for the treatment of TED. Nonspecific, anti-inflammatory drugs and more specific, targeted biologicals are treatment options for active and severe TED. Critical evaluation of the pertinent literature confirms an evidence-based, beneficial efficacy/risk ratio of the current first-line and second-line treatment recommendations endorsed by the European Society of Endocrinology. However, further large, well-conceived trials are mandatory to enhance our knowledge and experience with novel specific small molecules and/or monoclonal antibodies targeting the key autoantigens in TED.

Imaging of the medial rectus muscle predicts the development of optic neuropathy in thyroid eye disease.

Goal of the study was to evaluate bony orbit remodeling and extraocular muscle (EOM) volume in thyroid eye disease (TED) and their role as predicting factors for development of dysthyroid optic neuropathy (DON). Orbital computed tomography of 92 patients with TED with (76 orbits) or without DON (98 orbits) were retrospectively evaluated. Orbits (n = 40) of subjects without TED served as controls. Measurements of the bony orbit as well as EOM volume were incorporated into a generalized linear mixed model to predict DON. The angle of the medial orbital wall was significantly smaller (p < 0.001) in patients with TED (- 2.3 ± 3.6°) compared to patients with TED + DON (1.0 ± 4.1°). Both groups differed significantly from controls (- 4.2 ± 2.7°). Bowing of the medial orbital wall correlated positively with muscle volume (r = 0.564; p < 0.001). Total EOM volume was significantly larger in TED + DON (7.6 ± 2.5cm3) compared to TED only (5.6 ± 3.0cm3; p < 0.001) or controls (2.6 ± 0.5cm3). Multivariate analysis revealed the medial rectus muscle volume (TED: 1.06 ± 0.48cm3 vs. TED + DON: 2.16 ± 0.84cm3) as the strongest predictor, achieving a specifity of 86.7% and a sensitivity of 73.7% in diagnosing DON in univariate analysis. Though characterized by a wide range of variability, increased medial rectus muscle volume is the strongest predictor for DON in our patient cohort with TED when analyzing a single muscle.

Graves' Autoantibodies Exhibit Different Stimulating Activities in Cultures of Thyrocytes and Orbital Fibroblasts Not Reflected by Clinical Assays.

Background: The pathogenesis of Graves' hyperthyroidism (GH) and associated Graves' orbitopathy (GO) appears to involve stimulatory autoantibodies (thyrotropin receptor [TSHR]-stimulating antibodies [TSAbs]) that bind to and activate TSHRs on thyrocytes and orbital fibroblasts. In general, measurement of circulating TSHR antibodies by clinical assays correlates with the status of GH and GO. However, most clinical measurements of TSHR antibodies use competitive binding assays that do not distinguish between TSAbs and antibodies that bind to but do not activate TSHRs. Moreover, clinical assays for TSAbs measure stimulation of only one signaling pathway, the cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA) pathway, in engineered cells that are not thyrocytes or orbital fibroblasts. We determined whether measuring TSAbs by a cAMP-PKA readout in engineered cells accurately reveals the efficacies of stimulation by these antibodies on thyrocytes and orbital fibroblasts. Methods: We measured TSAb stimulation of normal human thyrocytes and orbital fibroblasts from patients with GO in primary cultures in vitro. In thyrocytes, we measured secretion of thyroglobulin (TG) and in orbital fibroblasts secretion of hyaluronan (hyaluronic acid [HA]). We also measured stimulation of cAMP production in engineered TSHR-expressing cells in an assay similar to clinical assays. Furthermore, we determined whether there were differences in stimulation of thyrocytes and orbital fibroblasts by TSAbs from patients with GH alone versus from patients with GO understanding that patients with GO have accompanying GH. Results: We found a positive correlation between TSAb stimulation of cAMP production in engineered cells and TG secretion by thyrocytes as well as HA secretion by orbital fibroblasts. However, TSAbs from GH patients stimulated thyrocytes more effectively than TSAbs from GO patients, whereas TSAbs from GO patients were more effective in activating orbital fibroblasts than TSAbs from GH patients. Conclusions: Clinical assays of stimulation by TSAbs measuring activation of the cAMP-PKA pathway do correlate with stimulation of thyrocytes and orbital fibroblasts; however, they do not distinguish between TSAbs from GH and GO patients. In vitro, TSAbs exhibit selectivity in activating TSHRs since TSAbs from GO patients were more effective in stimulating orbital fibroblasts and TSAbs from GH patients were more effective in stimulating thyrocytes.