Identification and functionalization of thyrotropin receptor antibodies with different antigenic epitopes.

One of the sensitive markers for autoimmune thyroid disease (AITD) clinical identification is thyroid-stimulating hormone receptor antibodies (TRAbs). To quickly distinguish TRAb with distinct antigenic epitopes, a straightforward and uncomplicated technique has not yet been created. The objective of this study is to search for molecular diagnostic targets for different types of AITD {Graves' disease (GD), Graves' orbitopathy (GO), GD with third-degree goiter [GD(3)], hypothyroidism combined with positive TRAb [HT(TRAb+)]} as molecular diagnostic targets. Following action on thyroid cells, differential genes (DEGs) generated by TRAb with distinct antigenic epitopes were detected and identified by RNA sequencing (RNA-Seq), bioinformatics analysis, and quantitative reverse transcription-polymerase chain reaction (RT-qPCR) in the serum of patients with AITD. Using the 5-ethynyl-2'-deoxyuridine (EdU) assay, the effect of coculturing thyroid cells with different antigenic TRAb epitopes on the cells' capacity to proliferate was investigated. Bioinformatics analysis and RT-qPCR validation identified one GD key gene alpha 2-HS glycoprotein (AHSG), two GO key genes [adrenoceptor alpha 1D (ADRA1D) and H2B clustered histone 18 (H2BC18)], two GD(3) key genes [suppressor of cytokine signaling 1 (SOCS1) and cytochrome b-245 beta (CYBB)], and one HT(TRAb+) key gene (MASP2). Correlation analysis and ROC curves showed that the abovementioned genes could be used as molecular diagnostic targets for different types of AITD. Finally, EdU results showed that TRAb inhibited thyroid cell proliferation in the HT(TRAb+) group compared with the normal control group, whereas the remaining three groups promoted thyroid cell proliferation, with a statistically significant difference (P < 0.05). We identified six key genes for different types of AITD, which have diagnostic value for different types of AITD. Meanwhile, we found that TRAbs with different antigenic epitopes in AITD have different biological functions.NEW & NOTEWORTHY We identified six molecular targets of different types of AITD [GD, GO, GD(3), and HT(TRAb+)], which have diagnostic value for different types of AITD. Meanwhile, we found that TRAb with different antigenic epitopes extracted from the sera of patients with AITD had different biological functions, which also provided a new idea for further research on the mechanism of action of TRAb with different antigenic epitopes in AITD.

Potential role of IGF-1R in the interaction between orbital fibroblasts and B lymphocytes: an implication for B lymphocyte depletion in the active inflammatory phase of thyroid-associated ophthalmopathy.

BACKGROUND: Thyroid eye disease (TED) is an inflammatory process involving lymphocyte-mediated immune response and orbital tissue damage. The anti-insulin-like growth factor-1 receptor (IGF-1R) antibodies produced by B lymphocytes are involved in the activation of orbital fibroblasts and the inflammatory process of orbital tissue damage in TED. The purpose of this study was to explore the role of IGF-1R in the mechanistic connection between orbital fibroblasts and B lymphocytes in TED. METHODS: Orbital fibroblasts sampled from orbital connective tissues and peripheral B lymphocytes isolated from peripheral blood, which were obtained from 15 patients with TED and 15 control patients, were co-cultured at a ratio of 1:20. The level of IGF-1R expression in orbital fibroblasts was evaluated by flow cytometry and confocal microscopy. Transient B lymphocyte depletion was induced with anti-CD20 monoclonal antibody rituximab, while the IGF-1R pathway was blocked by the IGF-1R binding protein. The expression levels of interleukin-6 (IL-6) and regulated upon activation, normal T cell expressed and secreted (RANTES) in the co-culture model were quantified via ELISA. RESULTS: IGF-1R expression was significantly elevated in TED orbital fibroblasts compared to that of controls. A 24-h co-culture of orbital fibroblasts with peripheral B lymphocytes induced elevated expression levels of IL-6 and RANTES in each group (TED patients and controls), with the highest levels occurring in TED patients (T + T group). Rituximab and IGF-1R binding protein significantly inhibited increased levels of IL-6 and RANTES in the co-culture model of TED patients. CONCLUSIONS: IGF-1R may mediate interaction between orbital fibroblasts and peripheral B lymphocytes; thus, blocking IGF-1R may reduce the local inflammatory response in TED. Rituximab-mediated B lymphocyte depletion played a role in inhibiting inflammatory responses in this in vitro co-culture model, providing a theoretical basis for the clinical application of anti-CD20 monoclonal antibodies in TED.

Immune-related visual dysfunction in thyroid eye disease: a combined orbital and brain neuroimaging study.

OBJECTIVES: To investigate the pathological interplay between immunity and the visual processing system (VPS) in thyroid eye disease (TED). METHODS: A total of 24 active patients (AP), 26 inactive patients (IP) of TED, and 27 healthy controls (HCs) were enrolled. Orbital magnetic resonance imaging (MRI) and resting-state functional MRI (rs-fMRI) were conducted for each participant. Multiple MRI parameters of the intraorbital optic nerve (ON) were assessed. The amplitude of low-frequency fluctuations (ALFF) and regional homogeneity (ReHo) were calculated. Correlation analyses were carried out on the above parameters and clinical characteristics. RESULTS: Visual functioning scores differentiated between the AP and IP groups. The ON subarachnoid space and ON sheath diameter were significantly higher in AP than in IP. Six vision-related brain regions were identified in TED patients compared with HCs, including right calcarine (CAL.R), right cuneus (CUN.R), left postcentral gyrus (PoCG.L), right middle temporal gyrus (MTG.R), left superior frontal gyrus (SFG.L), and left caudate (CAU.L). The brain activity of MTG.R, SFG.L, and CAU.L differentiated between the AP and IP groups. The correlation analysis revealed a close association among the vision-related brain regions, MRI parameters of ON, and clinical characteristics in AP and IP, respectively. CONCLUSIONS: Combined orbital and brain neuroimaging revealed abnormalities of the VPS in TED, which had a close correlation with immune statuses. Vision-related brain regions in TED might be possibly altered by peripheral immunity via a direct or indirect approach. CLINICAL RELEVANCE STATEMENT: The discovery of this study explained the disparity of visual dysfunction in TED patients with different immune statuses. With the uncovered neuroimaging markers, early detection and intervention of visual dysfunction could be achieved and potentially benefit TED patients. KEY POINTS: • Patients with different immune statuses of thyroid eye disease varied in the presentation of visual dysfunction. • The combined orbital and brain neuroimaging study identified six altered vision-related brain regions, which had a significant correlation with the MRI parameters of the intraorbital optic nerve and immunological characteristics. • Peripheral immunity might possibly give rise to alterations in the central nervous system part of the visual processing system via a direct or indirect approach.

IgG4 serum levels in Graves' orbitopathy.

OBJECTIVE: IgG4-related disease (IgG4-RD) can involve many organs, including thyroid and orbital tissues. A link between IgG4, Graves' disease (GD) and Graves' orbitopathy (GO) has been proposed, but results are conflicting. Here we investigated the possible association between IgG4 and GO. METHODS: Retrospective investigation in 297 patients with Graves' disease (GD), 152 with GO. PRIMARY OUTCOME: prevalence of IgG4 ≥ 135 mg/dL (cut-off for IgG4-RD). SECONDARY OBJECTIVES: (1) serum IgG4 concentrations; (2) IgG4/IgG ratio; (3) prevalence of IgG4/IgG ratio ≥ 8.0%; (4) relationship between IgG4 and eye features; (5) relationship between IgG4 and anti-TSH receptor antibodies (TRAbs). RESULTS: Because GO patients had lower FT3 concentrations, we evaluated the main objectives in the second and third FT3 quartiles subpopulation, in which there were no relevant differences between patients with (n = 81) or without GO (n = 67) for baseline parameters. Within this population, the prevalence of IgG4 levels ≥ 135 mg/dL did not differ between patients without and with GO (17.9% vs 17.3%). No difference was observed concerning IgG4 concentrations, prevalence of IgG4/IgG ≥ 8.0%, and IgG4/IgG ratio. There was no relationship between IgG4 and eye features and no correlation between IgG4 levels and TRAb was found. CONCLUSIONS: Our results suggest that, within GD, there is no relationship between serum IgG4 and GO.

Peripheral CD3+CD4+ T cells as indicators of disease activity in thyroid eye disease: age-dependent significance.

PURPOSE: To provide an in-depth analysis of the association of peripheral lymphocytes and the disease activity of thyroid eye disease (TED). METHODS: This retrospective study enrolled 65 active TED patients and 46 inactive TED patients. Comparative analyses of peripheral lymphocyte subsets were conducted between active and inactive patients. Subgroup analyses were performed based on sex, age, disease duration, and severity. Correlation analyses explored the associations between lymphocyte subsets and TED activity indicators. Prediction models for TED activity were established using objective indicators. RESULTS: Significantly elevated levels of CD3+CD4+ T cells were observed in active TED patients compared to inactive patients (P = 0.010). Subgroup analyses further revealed that this disparity was most prominent in females (P = 0.036), patients aged 50 years and younger (P = 0.003), those with long-term disease duration (P = 0.022), and individuals with moderate-to-severe disease (P = 0.021), with age exerting the most substantial impact. Subsequent correlation analysis confirmed the positive association between CD3+CD4+ T cells and the magnetic resonance imaging indicator of TED activity among patients aged 50 years and younger (P = 0.038). The combined prediction models for TED activity, established using objective indicators including CD3+CD4+ T cells, yielded areas under curve of 0.786 for all patients and 0.816 for patients aged 50 years and younger. CONCLUSIONS: Peripheral CD3+CD4+ T cells are associated with disease activity of TED, especially in patients aged 50 years and younger. Our study has deepened the understanding of the peripheral T cell profiles in TED patients.

2021 update on thyroid-associated ophthalmopathy.

PURPOSE: Our understanding of thyroid-associated ophthalmopathy (TAO, A.K.A Graves' orbitopathy, thyroid eye disease) has advanced substantially, since one of us (TJS) wrote the 2010 update on TAO, appearing in this journal. METHODS: PubMed was searched for relevant articles. RESULTS: Recent insights have resulted from important studies conducted by many different laboratory groups around the World. A clearer understanding of autoimmune diseases in general and TAO specifically emerged from the use of improved research methodologies. Several key concepts have matured over the past decade. Among them, those arising from the refinement of mouse models of TAO, early stage investigation into restoring immune tolerance in Graves' disease, and a hard-won acknowledgement that the insulin-like growth factor-I receptor (IGF-IR) might play a critical role in the development of TAO, stand out as important. The therapeutic inhibition of IGF-IR has blossomed into an effective and safe medical treatment. Teprotumumab, a ß-arrestin biased agonist monoclonal antibody inhibitor of IGF-IR has been studied in two multicenter, double-masked, placebo-controlled clinical trials demonstrated both effectiveness and a promising safety profile in moderate-to-severe, active TAO. Those studies led to the approval by the US FDA of teprotumumab, currently marketed as Tepezza for TAO. We have also learned far more about the putative role that CD34+ fibrocytes and their derivatives, CD34+ orbital fibroblasts, play in TAO. CONCLUSION: The past decade has been filled with substantial scientific advances that should provide the necessary springboard for continually accelerating discovery over the next 10 years and beyond.

Role of the mononuclear cell infiltrate in Graves' orbitopathy (GO): results of a large cohort study.

OBJECTIVE: The extent to which mononuclear cells and TSH-receptor autoantibodies (TRAb) contribute to Graves' orbitopathy (GO) is not completely defined. Here we investigated the relationship between the immunohistochemical phenotype of orbital infiltrating cells and GO features in a large number of patients. METHODS: We conducted an observational cohort study in 76 consecutive patients with GO (16 men and 60 women) who underwent orbital decompression over a period of 18 consecutive months. An ophthalmological evaluation was performed in all patients, as well as immunohistochemistry for CD3, CD4, CD8, CD56 (T-cell markers), CD25 (T and B-cell marker), CD20, CD19 (B-cell markers), and CD138 (plasmacell marker) in specimens collected at decompressive surgery. RESULTS: Having established cutoff values for each marker, cell infiltrates were found in 60 patients (78.9%; CD3: 39.4%, CD4 55.2%, CD8 50%, CD56: 0%, CD25: 28.9%, CD20: 51.3%, CD19: 25%, CD138: 26.3%). Eleven (14.4%) stained exclusively for CD138 (plasmacells). Patients with CD4-positive mononuclear cells had a significantly greater GO clinical activity score (CAS) (mean difference 1.07, 95% CI - 0.33 to - 1.82, P = 0.004 by univariate, P = 0.05 by multivariate analysis). CAS as well as the remaining GO features were not affected significantly by the mononuclear cell subpopulations in multivariate analyses. CONCLUSIONS: Mononuclear cell infiltrates are present in the majority of GO patients, with a small percentage represented exclusively by plasmacells. CD4 cells exert a major role on GO activity. These findings may represent a further advancement in the comprehension of GO pathogenesis.

Clinical value of functional thyrotropin receptor antibodies in Serbian patients with Graves' orbitopathy.

PURPOSE: Thyrotropin receptor autoantibodies (TSH-R-Ab) are heterogeneous in their biological function and play a significant role in the pathophysiology of both Graves' disease and Graves' orbitopathy (GO). The clinical significance and utility of determining functional TSH-R-Ab in a Serbian collective were evaluated. METHODS: 91 consecutive patients with GO were included in this study. Total TSH-R-Ab concentration, referred to as TSH-R binding inhibitory immunoglobulins (TBII) was detected using a competitive-binding immunoassay. Stimulating and blocking TSH-R-Ab (TSAb and TBAb) were measured with cell-based bioassays. RESULTS: Stimulating TSAb activity and TBII positivity were detected in 85 of 91 (93.4%) and 65 of 91 (71.4%) patients with GO (P < 0.001). Blocking TBAb activity was observed in only one patient who expressed dual stimulating and blocking TSH-R-Ab activity. The sensitivity rates for differentiating between clinically active versus inactive and mild versus moderate-to-severe GO were 100% and 100% for TSAb, respectively. In contrast, these were 82% and 87% only for TBII. Seven of eight (87.5%) and one of eight (12.5%) euthyroid patients with GO were TSAb and TBII positive, respectively (P < 0.031). TSAb serum levels significantly predicted GO activity compared to TBII (odds ratio, OR, 95%CI: 3.908, 95%CI 1.615-9.457, P = 0.003; versus 2.133, 0.904-5.032, P = 0.084, univariate analysis; and OR 4.341, 95%CI 1.609-11.707, P = 0.004; versus 2.337, 0.889-6.145, P = 0.085 multivariate analysis). CONCLUSION: Stimulating TSAb are highly prevalent in patients with GO and show superior clinical characteristics and predictive potential compared to the traditionally used TBII.

A cyclic peptide significantly improves thyroid function, thyrotropin-receptor antibodies and orbital mucine /collagen content in a long-term Graves' disease mouse model.

BACKGROUND: BALB/c mice which received long-term immunizations of adenovirus (Ad) expressing thyrotropin receptor A-subunits (TSHR) developed stable Graves' disease (GD). TSHR-derived cyclic peptide 19 (P19) was identified as effective therapy in this model. METHODS: In Ad-TSHR mice, we investigated shorter disease intervals up to 4 months for histological alterations of the orbits, fine tuning of anti-TSHR antibodies (Ab) and free thyroxine (fT4) hormone levels by using novel detection methods in an independent laboratory. Therapy (0.3 mg/kg P19 or vehicle) was given intravenously after the fourth Ad-TSHR immunization (week 11) and continued until week 19. RESULTS: Thyrotropin binding inhibitory immunoglobulins (TBII, bridge immunoassay), blocking (TBAb) and stimulating (TSAb) TSHR-Ab (both cell-based bioassays) and serum levels of fT4 were significantly elevated at week 11 in Ad-TSHR-immunized mice versus none in control mice. For the first time, TSAb, TBAb, and thyroperoxidase-Ab were detected in 17 of 19, 12/19 and 6/19 Ad-TSHR immunized mice, respectively at week 21. Also, for the first time, this study showed that P19 treatment markedly reduced serum TBII (p < 0.0001), serum fT4 (p = 0.02), and acidic mucins and collagen content in the orbital tissue of Ad-TSHR-immunized mice. CONCLUSION: P19 significantly improved thyroid function, confirming previous results in an independent second laboratory. A relevant shift of anti-TSHR antibody subpopulations in response to P19 therapy may help explain its immunological effects. Moreover, P19 exerted a beneficial effect on mucine and collagen content of orbital tissue. Hence, P19 offers a potential novel therapeutic approach for GD and associated orbitopathy.

Teprotumumab: The Dawn of Therapies in Moderate-to-Severe Thyroid-Associated Ophthalmopathy.

Thyroid-associated ophthalmopathy (TAO) is a potentially sight-threatening ocular disease. About 3-5% of patients with TAO have severe disease with intense pain, inflammation, and sight-threatening corneal ulceration or compressive optic neuropathy. The current treatments of TAO are often suboptimal, mainly because the existing therapies do not target the pathogenesis of the disease. TAO mechanism is unclear. Ocular fibrocytes express relatively high levels of the functional TSH receptor (TSHR), and many indirect evidences support its participation. Over expression of insulin-like growth factor-1 receptor (IGF-IR) in fibroblasts, leading to inappropriate expression of inflammatory factors, production of hyaluronic acid and cell activation in orbital fibroblasts are also possible mechanisms. IGF-1R and TSHR form a physical and functional signaling complex. Inhibition of IGF-IR activity leads to the attenuation of signaling initiated at either receptor. Teprotumumab (TMB) is a human immunoglobulin G1 monoclonal antibody, binding to IGF-IR. Recently two TMB clinical trials had been implemented in TAO patients, indicating dramatic reductions in disease activity and severity, which approved its use for the treatment of TAO in the US. This review summarizes the treatments of TAO, focusing on the pathogenesis of IGF-1R in TAO and its application prospects.

Predicting the Course of Graves' Orbitopathy Using Serially Measured TSH-Receptor Autoantibodies by Automated Binding Immunoassays and the Functional Bioassay.

The aim of the study was to investigate the use of serial measurements of TSH-receptor autoantibodies (TRAb) with the newest available assay technology to predict the course of Graves' Orbitopathy (GO) during the first 24 months from disease onset. Serial serum samples from patients with GO (103 mild/135 severe) were collected between 2007 and 2017 and retrospectively analyzed. The course of GO were classified into mild/severe 12 months after manifestation (severe: NOSPECS≥5; mild<5). TRAb were measured with automated binding immunoassays (IU/l): TRAb Elecsys (Cobas, Roche), TRAb bridge assay (IMMULITE, Siemens), and a cell-based bioassay (percent of specimen to reference ratio - SRR%) (Thyretain, Quidel). Variable cut off levels of measured TRAb were calculated at specificity of 90% from receiver operator curve (ROC) analysis for several timepoints during the course of GO. To select one: 5-8 months after first GO symptoms, which is the timepoint for usual referals for treatment mild course could be predicted at cut offs of 1.5 IU/l (Elecsys), 0.8 IU/l (Immulite) and 402% SRR (Thyretain) and the risc of severe course has to be anticipated if TRAb are above 11.6 IU/l (Elecsys), 6.5 IU/l (Thyretain), and 714% SRR (Thyretain). The Thyretain bioassay showed the highest diagnostic sensitivity (using the commercial cut off's) over the entire follow up period. TRAb measurements during the 24-month follow up of GO provide added value to the GO clinical activity and severity scores and should be used especially in the event of an unclear decision-taking situation with regard to therapy.

Predicting the Relapse of Hyperthyroidism in Treated Graves' Disease with Orbitopathy by Serial Measurements of TSH-Receptor Autoantibodies.

The aim of this study was to investigate the potential of the new TSH-receptor antibody (TRAb) assays to predict remission or relapse of hyperthyroidism in patients with Graves' disease (GD) and Graves' orbitopathy (GO). TRAbs were measured retrospectively in sera from a cohort of GD patients with GO (n=117; remission n=38 and relapse n=79-Essen GO biobank) with automated binding immunoassays: TRAb Elecsys (Cobas Roche) and TRAb bridge assay (IMMULITE, Siemens), and the TSAb (thyroid stimulating Ab) cell-based bioassay (Thyretain, Quidel Corp.). To identify relapse risk/remission of hyperthyroidism patients were followed up at least 10 months after the end of antithyroid drug therapy (ATD) therapy. ROC plot analysis was performed to calculate cut-off levels of TRAb and TSAb for prediction of relapse and remission of hyperthyroidism. Cut-off serum levels are provided for timepoints around 3, 6, 10, and 15 months after the beginning of ATD. Repeated measurements of TRAb increase the rate of relapses predictions to 60% (Elecsys), 70% (IMMULITE), and 55% (Thyretain). Patients with remission have consistently TRAb levels below the cut off for relapse in repeated measurements. The cell-based bioassay was the most sensitive - and continued to be positive during follow up [at 15 months: 90% vs. 70% (IMMULITE) and 65% (Elecsys)]. Identification of relapsing hyperthyroidism is possible with automated immunoassays and cell-based bioassay especially with serial TRAb measurements during the course of ATD therapy. Patient who need eye surgery may profit from an early decision towards definitive treatment.

Altered expression profile of BAFF receptors on peripheral blood B lymphocytes in Graves' disease.

BACKGROUND: B lymphocyte activating factor (BAFF) is a growth factor regulating B lymphocytes survival and maturation. Serum BAFF levels were elevated in patients affected with autoimmune thyroid diseases (AITD), including Graves' disease (GD) and Hashimoto's thyroiditis (HT). The aim of this study is to explore the association of expression levels of BAFF and its receptors with AITD. METHODS: Fifty-two GD patients, 39 Hashimoto's thyroiditis (HT) patients and 23 healthy controls (HC) were recruited in this study. Serum BAFF levels were measured by ELISA. Expression of BAFF receptors, including BAFF receptor 3 (BR3) and transmembrane activator and calcium-modulating and cyclophilin ligand interactor (TACI), on B lymphocytes were analyzed by flowcytometry. Effects of steroids on serum BAFF levels and expression of BR3 and TACI were also observed in 10 patients with Graves' orbitopathy (GO) receiving steroids therapy. RESULTS: Serum BAFF levels were significantly elevated from 0.93 ± 0.24 ng/ml in HC to 1.18 ± 0.33 ng/ml in GD (P = 0.0027) and 1.02 ± 0.24 ng/ml in HT (P = 0.0331). BR3 expression on peripheral B lymphocytes were elevated in GD (mean MFI: 4.52 ± 2.06 in GD vs. 3.00 ± 0.87 in HC, P = 0.0015), while TACI expression on peripheral B lymphocytes were decreased in GD without significance (mean MFI: 7.96 ± 4.06 in GD vs. 9.10 ± 3.37 in HC, P = 0.1285). Expression of BR3 and TACI was not changed significantly in HT patients. Steroids significantly suppressed serum BAFF concentrations (from 1.18 ± 0.27 ng/ml to 0.97 ± 0.10 ng/ml, P = 0.0364) and BR3 expression in GO patients (mean MFI from 6.26 ± 4.91 to 4.05 ± 1.58, P = 0.0083). CONCLUSIONS: Altered expression of BAFF and its receptor may mediate the autoimmunity in GD. Restoring the normal expression profile of receptors for BAFF could be a new strategy to treat GD.

Thyrotropin receptor antibodies and Graves' orbitopathy.

CONTEXT AND PURPOSE: The thyrotropin receptor (TSHR) is the key autoantigen in Graves' disease (GD) and associated orbitopathy (GO). Antibodies targeting the TSHR (TSHR-Ab) impact the pathogenesis and the course of GO. This review discusses the role and clinical relevance of TSHR-Ab in GO. METHODS: Review of the current and pertinent literature. RESULTS: GO is the most common extrathyroidal manifestation of GD and is caused by persistent, unregulated stimulation of TSHR-expressing orbital target cells (e.g. fibroblasts and pre-adipocytes). Serum TSHR-Ab and more specifically, the stimulatory Ab (TSAb) are observed in the vast majority of patients with GD and GO. TSHR-Ab are a sensitive serological parameter for the differential diagnosis of GO. TSHR-Ab can be detected either with conventional binding immunoassays that measure binding of Ab to the TSHR or with cell-based bioassays that provide information on their functional activity and potency. Knowledge of the biological activity and not simply the presence or absence of TSHR-Ab has relevant clinical implications e.g. predicting de-novo development or exacerbation of pre-existing GO. TSAb are specific biomarkers of GD/GO and responsible for many of its clinical manifestations. TSAb strongly correlate with the clinical activity and clinical severity of GO. Further, the magnitude of TSAb indicates the onset and acuity of sight-threatening GO (optic neuropathy). Baseline serum values of TSAb and especially dilution analysis of TSAb significantly differentiate between thyroidal GD only versus GD + GO. CONCLUSION: Measurement of functional TSHR-Ab, especially TSAb, is clinically relevant for the differential diagnosis and management of GO.

Correlation between serum anti-TSH receptor autoantibodies (TRAbs) and the clinical feature of Graves' orbitopathy.

BACKGROUND: Graves' orbitopathy (GO) is the most common extrathyroidal manifestation of Graves' disease (GD). Several studies support the involvement of TSH receptor autoantibodies (TRAbs) in the pathogenesis of GO, and a correlation between GO features and TRAbs has been reported, but not confirmed by all studies. Thus, we conducted a cross-sectional investigation to determine whether there is a correlation between TRAbs and the clinical features of GO in an initial phase of the eye disease. METHODS: Ninety consecutive patients with untreated GO (67 women and 23 men, age 48.9 ± 12.6 years) were included. Patients who had received treatments other than anti-thyroid drugs for hyperthyroidism or lubricants for GO were excluded. All patients underwent an endocrinological and ophthalmological evaluation, the latter including exophthalmometry, measurement of eyelid width, clinical activity score (CAS), visual acuity, assessment of diplopia, and NOSPECS score. TRAb levels were measured by a third-generation competitive immunoassay. RESULTS: There was a statistically significant, direct correlation between serum TRAb levels and CAS by linear regression analysis (R = 0.278, P = 0.007). The correlation was confirmed by a multiple regression analysis (R = 0.285; P = 0.006) including age and FT3 levels, which also correlated with CAS. There were no relationships between TRAbs and exophthalmometry, eyelid aperture, degree of diplopia, visual acuity, and NOSPECS score. CONCLUSIONS: The levels of TRAb in subjects with a recent-onset, untreated GO are directly correlated with the clinical activity of the disease, confirming a possible role of these antibodies in the pathogenesis of GO.

IgG4 as a Biomarker in Graves' Orbitopathy.

Immunoglobulin G4-related disease (IgG4-RD) is a chronic inflammatory disorder associated with fibrosis and abundant tissue lymphoplasmacytic infiltrations. It typically affects the pancreas, the salivary glands, and the retroperitoneal space. However, it might also involve multiple other organs, including the orbit and the thyroid. Recent studies have suggested that IgG4 plays a role in the pathophysiology of autoimmune thyroid diseases. This ultimately led to the establishment of new clinical entities called IgG4-related thyroid disease and thyroid disease with an elevation of IgG4. The aim of this paper is to describe the pathophysiological, histopathological, and clinical features of Graves' Disease (GD) and Graves' Orbitopathy (GO) with elevated IgG4 levels. Multiple studies have demonstrated higher IgG4 serum concentrations in GD patients than in healthy euthyroid controls. Depending on the studied population, elevated serum IgG4 levels occur in 6.4-23% (average: 10.3%) of all patients with GD, 8.3-37.5% (average: 17.6%) of patients with GO, and 0-9.8% (average: 5.4%) of patients with GD without GO, while GO patients comprise 37.5-100% (average: 65.8%) of all GD patients with elevated IgG4 levels. Characteristic features of GD with elevated IgG4 levels include lower echogenicity of the thyroid gland on ultrasound examination, peripheral blood eosinophilia, higher prevalence of orbitopathy, and better response to antithyroid drugs with a tendency to develop hypothyroidism when compared to patients with GD and normal levels of IgG4. Typical signs of GO accompanied by increased concentration of IgG4 include younger age at diagnosis, and more severe course of the disease with a higher Clinical Activity Score (CAS).. We strongly recommend considering the diagnosis of GO with elevated IgG4 in patients with an established diagnosis of GD, elevated serum IgG4 levels, and clinical features of ophthalmic disease overlapping with those of IgG4-related orbital disease.

Therapeutic Effect of α-MSH in Primary Cultured Orbital Fibroblasts Obtained from Patients with Thyroid Eye Disease.

Inflammation, hyaluronan production, and adipogenesis are the main pathological events leading to thyroid eye disease (TED). α-Melanocytemelanocyte-stimulating hormone (α-MSH) is a well-known tridecapeptidetreatment for several inflammatory disorders including sepsis syndrome, acute respiratory distress syndrome, rheumatoid arthritis, and encephalitis. Here, we investigated the effect of α-MSH treatment on TED. The 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and Lactate Dehydrogenase (LDH) assays were performed to analyze the effect of α-MSH on cell viability and it's toxicity. Using primary cultures of orbital fibroblasts from TED patients and non-TED as control, we examined the effects of α-MSH on proinflammatory cytokine production induced by interleukin (IL)-1ß, further analyzed by real-time reverse transcription-polymerase chain reaction (qPCR) and western blotting. Immunofluorescence staining assay and qPCR were performed to examine proopiomelanocortin (POMC) expression, the upstream neuropeptide of α-MSH in TED patients and non-TED control. Treatment with non-cytotoxic concentrations of α-MSH resulted in the dose-dependent inhibition of mRNA and protein levels (p < 0.05) for IL-1ß-induced inflammatory cytokines: IL-6, IL-8, MCP-1, ICAM-1, and COX-2. The expression of POMC mRNA and protein were significantly higher in TED patients compared to non-TED control (p < 0.05). Our data show significant inhibitory effects of α-MSH on inflammation, POMC production in orbital fibroblasts. At present, this is the first in vitro preclinical evidence of α-MSH therapeutic effect on TED. These findings indicate that POMC and α-MSH may play a role in the immune regulation of TED and can be a potential therapeutic target.

A case report of thyroid-associated Orbitopathy with elevated TPO antibodies.

BACKGROUND: Thyroid associated orbitopathy (TAO) is defined as an immune mediated inflammatory process affecting the extraocular muscles, connective and adipose tissue of uncertain etiopathogenesis. TAO are classically described in Grave's disease (GD) however it may occur in euthyroid and hypothyroid patients. Those patients usually test positive for Thyroid Stimulating Hormone receptor antibodies (TRAb). For instance, only few cases of severe Hashimoto's thyroiditis (HT) associated orbitopathy with negative TRAb are reported to date. CASE PRESENTATION: Herewith we report a rare case of a middle-aged female who presented with bilateral progressive upper and lower palpebral edema and a unilateral marked proptosis associated with asthenia, headache and decrease in visual acuity. Biological investigation was notable for high levels of anti-thyroid peroxidase antibodies (Anti-TPO) in an otherwise euthyroid patient with negative TRAb. Orbital Magnetic resonance imaging revealed edema of the extraocular muscles and inflammation of periorbital soft tissue. The patient received a treatment with intravenous methylprednisolone followed by oral treatment with prednisone. This regimen was both effective and safe with minimal metabolic side effects in our patient. CONCLUSION: Minor ocular manifestations of HT are common; however, severe sight threatening ophtalmopathy in the absence of TRAb is rare. Multiple differential diagnosis should be considered and investigated before diagnosing this rare entity. Management of similar cases is currently based on reports and no clear guidelines have been elaborated, corticosteroids is the mainstream of treatment with a potential benefit of selenium supplementation in mild to moderate cases.

Putative protective role of autoantibodies against the insulin-like growth factor-1 receptor in Graves' Disease: results of a pilot study.

BACKGROUND: The insulin-like growth factor-1 receptor (IGF-1R) is a key element in the pathogenesis of Graves' Orbitopathy (GO), but the role of IGF-1R autoantibodies (IGF-1RAbs) has not been established. METHODS: We designed a cross-sectional investigation to measure IGF-1RAbs in patients with Graves' disease (GD), with or without GO, who underwent radioiodine therapy followed by glucocorticoids (GC). Twenty-nine patients were included, 15 of which with GO. Patients were evaluated at baseline and three and 6 months after radioiodine. The primary objective was the prevalence of positive tests for IGF-1RAbs. The secondary objectives were: (1) IGF-1RAbs concentrations and their variations; (2) relationship between IGF-1RAbs and the features of GO; (3) relationship between IGF-1RAbs and anti-thyroid autoantibodies. RESULTS: IGF-1RAbs above the cut-off value were found only in one patient with GD without GO. IGF-1RAb levels were greater in patients with GD without GO, at baseline (P < 0.0001), and after three (P < 0.0001) and six (P = 0.0001) months. No correlations were observed between IGF-1RAbs and the features of GO, nor between IGF-1RAbs and anti-thyroglobulin or anti-thyroperoxidase autoantibodies. There was an inverse correlation between anti-TSH receptor autoantibodies (TRAbs) and IGF-1RAb levels in GD patients with GO at 6 months (P = 0.03). CONCLUSIONS: IGF-1RAbs appear to be greater in patients with GD without GO compared with those with GO, suggesting a putative protective role of IGF-1RAbs on the development of GO, in line with the beneficial effects of Teprotumumab on GO. The inverse correlation between IGF-1RAbs and TRAbs 6 months after radioiodine may reflect antigen spreading and/or GC treatment.

Role of a new bioassay for thyroid-stimulating antibodies (aequorin TSAb) in Graves' ophthalmopathy.

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