The phenotypic characteristics of polymorphonuclear neutrophils and their correlation with B cell and CD4+T cell subsets in thyroid-associated ophthalmopathy.

Introduction: Thyroid-associated ophthalmopathy (TAO) is considered to be an organ-specific autoimmune disease. Polymorphonuclear neutrophils (PMN) have been implicated in the pathogenesis of TAO. However, little is known about the role of PMN in the development of TAO, much less the relationship between PMN with B cells and CD4+T cells in TAO. Objective: This study aims to investigate the phenotypic characteristics of PMN and the relationship between PMN with CD4+T cell and B cell subsets in the pathogenesis of TAO. Methods: Blood routine information was collected from 135 TAO patients, 95 Grave's disease without TAO (GD) patients, and 116 normal controls (NC), while surface marker expression of PMN and the level of CD4+T cell and B cell subsets in peripheral blood from 40 TAO patients, 17 GD patients, and 45 NC was assessed by flow cytometry. Result: The level of PMN, CD62L+PMN, CD54+PMN, CD4+T cells, and Th17 cells displayed an increase in TAO patients than NC, while Treg cells were lower in the TAO group compared to NC. There was no statistical difference in Th1 and plasma cells among the groups. PMN were positively correlated with Th17 cells, but not the Th1, Treg, and plasma cells. Conclusion: In the present study, we found that the percentage of PMN and PMN subset cells was significantly higher in TAO than in NC, and PMN were positively correlated with Th17 cells. It suggests that PMN may be involved in the immunopathogenesis of TAO and modulate the Th17 cell response during this process.

Prognostic factors for thyroid-stimulating immunoglobulin normalization in moderate-to-severe Graves' orbitopathy: a 36-month longitudinal study.

PURPOSE: The primary objective of this study was to identify predictive factors linked to the normalization of thyroid-stimulating immunoglobulin (TSI) levels in patients diagnosed with active, moderate-to-severe Graves' orbitopathy (GO). The study also tracked the longitudinal changes in TSI levels over a 36-month period following treatment. METHODS: The study population consisted of individuals who were recently diagnosed with active, moderate-to-severe GO and received a 12-week course of intravenous methylprednisolone (IVMP) treatment. A subgroup of patients who did not respond to the initial treatment received an additional 20 Gy of radiation therapy (RTx). TSI levels were monitored at the time of diagnosis, after treatment, and subsequently every 6 months for 36 months. Normalization was defined as a TSI level below 140%. Patients were divdied into two groups with success and failure group depending on whether TSI became normal or not. RESULTS: Out of 83 patients, 36 (43.4%) achieved normalized TSI levels within two years post-IVMP treatment. Lower initial TSI levels (< 425%), absence of additional RTx, and early treatment initiation were associated with a higher likelihood of TSI normalization (P = 0.035, P = 0.028, P < 0.001, respectively). Notably, significant differences in TSI level reduction were observed from 18 months post-treatment between the two groups (P = 0.031). A TSI cutoff value of 413% was identified as predictive for normalization at 24 months (P = 0.002). CONCLUSION: This study is the first to identify key factors that influence normalization of TSI levels in moderate-to-severe Graves' Orbitopathy. It highlights the importance of early treatment decisions, particularly for patients with initial TSI levels above 425%. Despite the treatment, less than half of the patients achieved TSI normalization within 24 months, underscoring the need for additional research to explore the relationship between TSI levels and the clinical manifestations of chronic GO.

Clinical Significance of IgG4 Serum Concentration in Graves' Disease.

Elevated immunoglobulin G4 (IgG4) serum antibodies are an important feature of IgG4-related disease. However, IgG4 antibodies can play a role in autoimmune thyroid disorders. In this study, we aimed to evaluate the impact of serum IgG4 levels on clinical features of Graves' disease (GD). We recruited 60 patients with GD (48 patients without thyroid eye disease, 12 patients with moderate-to-severe Graves' orbitopathy [GO], and 25 healthy control subjects). The prevalence of high IgG4 serum concentration was 4.2% among GD patients without GO and 33.33% in patients with moderate-to-severe GO. The group with GO had significantly higher median IgG4 levels (87.9 mg/dL) than the control group (41.2 mg/dL, P = 0.034) and the GD without GO group (30.75 mg/dL, P < 0.001). Patients with thyroid nodules had lower IgG4 levels than patients without thyroid nodules, but the difference was not statistically significant (35.7 [24.8; 41.53] mg/dL vs. 43 [30.1; 92.7] mg/dL, P = 0.064). IgG4 as a diagnostic tool for moderate-to-severe GO had the following parameters: area under the curve (AUC): 0.851 (P < 0.001), at the cut-off value of 49 mg/dL, negative predictive value: 100%, positive predictive value: 48%, sensitivity: 100%, specificity: 73%. There were no significant differences between the high and normal IgG4 groups in thyroid hormones, antithyroid antibodies, and ultrasound features. Serum IgG4 levels are associated with some of the clinical features of GD and can help in the diagnostic process of the disease. More research is needed to better understand the pathophysiology of IgG4 involvement in GD.

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.

Epidemiology of thyroid-stimulating immunoglobulin in recent-onset symptomatic thyroid eye disease.

Purpose: This study aims to report correlations between thyroid-stimulating immunoglobulin (TSI) and both clinical and radiological parameters in recent-onset symptomatic thyroid eye disease (TED) patients. Methods: A prospective cohort study of TED patients managed at the Chinese University of Hong Kong from January 2014 to May 2022. Serum TSI levels were determined with the functional assay. Outcomes included the Clinical Activity Score (CAS), marginal reflex distance1 (MRD1), extraocular muscle motility restriction (EOMy), exophthalmos, and diplopia. The radiological assessment included cross-sectional areas and signal of extraocular muscles on STIR-sequence MRI. Results: A total of 255 (197 female) treatment-naive patients, with an average onset age of 50 ± 14 years (mean ± s.d.), were included. Elevated pre-treatment TSI level was observed in 223 (88%) patients. There was a weak positive correlation between TSI and CAS (r = 0.28, P = 0.000031), MRD1 (r = 0.17, P = 0.0080), and the size of the levator palpebrae superioris/superior rectus complex (r = 0.25, P = 0.018). No significant correlation existed between TSI and STIR signals. The AUC and optimal cut-off value for clinical active TED were 0.67 (95% CI: 0.60-0.75) and 284% (specificity: 50%, sensitivity: 85%). In total, 64 patients received intravenous methylprednisolone (IVMP) during the study interval, and they had a higher baseline TSI level than those who did not have IVMP (P = 0.000044). Serial post-IVMP TSI among the 62 patients showed a significant reduction compared to the baseline level (P < 0.001). Both the baseline and post-IVMP TSI levels, and percentages of TSI changes were comparable between patients who responded and did not respond to the first course of IVMP. Conclusion: TSI can be a serum biomarker for the diagnosis, prognosis, and treatment response of TED. Further validation should be warranted.

Plasma exosomes from patients with active thyroid-associated orbitopathy induce inflammation and fibrosis in orbital fibroblasts.

BACKGROUND: The pathogenesis of thyroid-associated orbitopathy (TAO) remains incompletely understand. The interaction between immunocytes and orbital fibroblasts (OFs) play a critical role in orbital inflammatory and fibrosis. Accumulating reports indicate that a significant portion of plasma exosomes (Pla-Exos) are derived from immune cells; however, their impact upon OFs function is unclear. METHODS: OFs were primary cultured from inactive TAO patients. Exosomes isolated from plasma samples of patients with active TAO and healthy controls (HCs) were utilized for functional and RNA cargo analysis. Functional analysis in thymocyte differentiation antigen-1+ (Thy-1+) OFs measured expression of inflammatory and fibrotic markers (mRNAs and proteins) and cell activity in response to Pla-Exos. RNA cargo analysis was performed by RNA sequencing and RT-qPCR. Thy-1+ OFs were transfected with miR-144-3p mimics/inhibitors to evaluate its regulation of inflammation, fibrosis, and proliferation. RESULTS: Pla-Exos derived from active TAO patients (Pla-ExosTAO-A) induced stronger production of inflammatory cytokines and hyaluronic acid (HA) in Thy-1+ OFs while inhibiting their proliferation. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis and single sample gene set enrichment analysis (ssGSEA) suggested that the difference in mRNA expression levels between Pla-ExosTAO-A and Pla-ExosHC was closely related to immune cells. Differential expression analysis revealed that 62 upregulated and 45 downregulated miRNAs in Pla-ExosTAO-A, with the elevation of miR-144-3p in both Pla-Exos and PBMCs in active TAO group. KEGG analysis revealed that the target genes of differentially expressed miRNA and miR-144-3p enriched in immune-related signaling pathways. Overexpression of the miR-144-3p mimic significantly upregulated the secretion of inflammatory cytokines and HA in Thy-1+ OFs while inhibiting their proliferation. CONCLUSION: Pla-Exos derived from patients with active TAO were immune-active, which may be a long-term stimulus casual for inflammatory and fibrotic progression of TAO. Our finding suggests that Pla-Exos could be used as biomarkers or treatment targets in TAO patients.

Single-cell BCR and transcriptome analysis reveals peripheral immune signatures in patients with thyroid-associated ophthalmopathy.

Thyroid-associated ophthalmopathy (TAO) is the most prevalent orbital disease in adults caused by an autoimmune disorder, which can lead to disfigurement and vision impairment. Developing effective treatments for this condition presents challenges due to our limited understanding of its underlying immune aberrations. In this study, we profiled the immune components in the peripheral blood of patients with TAO as well as healthy individuals, utilizing single-cell RNA sequencing and B-cell receptor repertoires (BCR) analysis. We observed a significant reduction in the proportions of regulatory B cells (Bregs) and type 2 conventional dendritic cells (DCs) in patients with TAO during the active phase. Conversely, there was a significant increase in the proportion of type 1 DCs. Further analysis of cell differentiation trajectory revealed potential impairment in the transition of B cells towards Breg phenotype during the active phase of TAO. Besides, the activation process of TAO appeared to involve inflammation and immune dysfunction, as indicated by the dynamic changes in the activities of key regulators. The abnormalities in the peripheral immune system, such as the reduced capacity of Bregs to suppress inflammation, were primarily driven by the enhanced interaction among Breg, DCs, and monocytes (i.e., CD22-PTPRC and BTLA-TNFRSF14). Collectively, our findings offer a comprehensive insight into the molecular regulation and cellular reconfiguration during the active phase of TAO at the single-cell level, in order to explore the pathogenesis of TAO and provide new ideas for the future treatment of TAO.

The circulating IL-35+ regulatory B cells are associated with thyroid associated opthalmopathy.

BACKGROUND: Thyroid-associated ophthalmopathy (TAO) is the most common orbital disease in adults, potentially leading to disfigurement and visual impairment. However, the causes of TAO are not fully understood. IL-35+B cells are a newly identified regulatory B cells (Bregs) in maintaining immune balance in various autoimmune diseases. Yet, the influence of IL-35+Bregs in TAO remains unexplored. METHODS: This study enrolled 36 healthy individuals and 14 TAO patients. We isolated peripheral blood mononuclear cells and stimulated them with IL-35 and CpG for 48 h. Flow cytometry was used to measure the percentages of IL-35+Bregs. RESULTS: The percentage of circulating IL-35+Bregs was higher in TAO patients, and this increase correlated positively with disease activity. IL-35 significantly increased the generation of IL-35+Bregs in healthy individuals. However, B cells from TAO patients exhibited potential impairment in transitioning into IL-35+Breg phenotype under IL-35 stimulation. CONCLUSIONS: Our results suggest a potential role of IL-35+Bregs in the development of TAO, opening new avenues for understanding disease mechanisms and developing therapeutic approaches.

Causality of blood metabolites and metabolic pathways on Graves' disease and Graves' ophthalmopathy: a two-sample Mendelian randomization study.

OBJECTIVES: There is ample that metabolic dysregulation is involved in Graves' disease (GD) and Graves' ophthalmopathy (GO). Recent studies have identified numerous metabolites associated with GD and GO. However, the causal impact of metabolites on GD and GO remains to be investigated. METHODS: This two-sample Mendelian randomization (MR) analysis investigated the causal relationships between 486 blood metabolites and GD and GO. Sensitivity analysis was also performed to examine heterogeneity and pleiotropy. RESULTS: MR analysis showed that 9 and 13 metabolites were associated with GD and GO, respectively, each meeting the nominal significance criteria (inverse variance weighted, p < 0.05). Additionally, four metabolic pathways were identified for each condition using network-based MetaboAnalyst 5.0. CONCLUSIONS: The metabolites and pathways discovered in this study could serve as circulating metabolic biomarkers for clinical screening and prevention of GD and GO. They can be also used for further studies on the mechanisms and drug targets in GD and GO.

Glucocorticoid-induced adrenal insufficiency after therapy with intravenous methylprednisolone in patients with moderate-to-severe and active Graves' orbitopathy: assessment with a low-dose corticotropin test.

PURPOSE: We aimed to assess adrenal function following treatment of moderate-to-severe and active Graves' orbitopathy (GO) with intravenous methylprednisolone (IVMP) in weekly pulses in a cumulative dose of 4.5 or 7.5 g. We evaluated the impact of IVMP pulses on adrenal reserve using a low-dose (1 µg) ACTH stimulation test (LDT) for the first time. METHODS: In this prospective study we evaluated adrenal function in 21 patients with moderate-to-severe and active GO treated with 12 weekly IVMP pulses according to the European Group on Graves' Orbitopathy (EUGOGO) recommendations. We assessed serum cortisol, plasma adrenocorticotropic hormone (ACTH), and dehydroepiandrosterone sulfate (DHEA-S) levels before the 1st and 12th IVMP pulse. We performed dynamic testing using LDT before the 12th IVMP pulse in all patients. In those who failed LDT, adrenal function was reassessed with LDT and the overnight metyrapone test after 4-7 weeks. RESULTS: Two patients failed to achieve serum cortisol levels ≥ 18.1 µg/dL at 30 and 60 min in LDT and were diagnosed with glucocorticoid-induced adrenal insufficiency (GC-induced AI). They were recommended to take hydrocortisone in situations of acute stress. Both patients were reassessed within 4-7 weeks after treatment cessation and showed an adequate response in LDT and overnight metyrapone test. We observed a statistically significant decrease in DHEA-S levels (p = 0.004) before the 12th IVMP pulse compared to baseline in all patients. CONCLUSION: For the first time, our research shows that administering IVMP in 12 weekly pulses can result in GC-induced AI. We suggest that patients should undergo careful evaluation for GC-induced AI, including LDT, after therapy with IVMP according to EUGOGO guidelines. Screening for altered adrenal reserve could prevent life-threatening complications, particularly during acute stress situations.

Glycemic Trends in Patients with Thyroid Eye Disease Treated with Teprotumumab in 3 Clinical Trials.

PURPOSE: Assess incidence, severity, and glucose excursion outcomes in thyroid eye disease (TED) patients receiving the insulin-like growth factor-1 receptor inhibitor teprotumumab from 3 clinical trials. DESIGN: Analysis of pooled glycemic data over time. PARTICIPANTS: Eighty-four teprotumumab- and 86 placebo-treated active TED patients from the phase 2 and phase 3 (OPTIC) controlled clinical trials and 51 teprotumumab-treated patients from the OPTIC extension (OPTIC-X) trial. METHODS: Eight intravenous infusions were given over 21 weeks. Phase 2 serum glucose was measured at weeks 1, 4, 15, and 21, with fasting measurements at weeks 1 and 4. Serum glucose was measured at each study visit in OPTIC and OPTIC-X, with fasting measurements at weeks 1 and 4 (in patients without diabetes) or all visits (in patients with diabetes). In all studies, hemoglobin A1c (HbA1c) was measured at baseline, 12, and 24 weeks plus weeks 36 and 48 in OPTIC-X. MAIN OUTCOME MEASURES: Serum glucose and HbA1c. RESULTS: In the phase 2 and 3 studies, 9 hyperglycemic episodes occurred in 8 teprotumumab patients; mean HbA1c level increased 0.22% from baseline to week 24 (to 5.8%; range, 5.0%-7.9%) versus 0.04% in patients receiving the placebo (to 5.6%; range, 4.6%-8.1%). At study end, 78% (59/76) of teprotumumab patients and 87% (67/77) of patients receiving placebo had normoglycemic findings. Normoglycemia was maintained in 84% (57/68) of patients receiving teprotumumab and 93% (64/69) of patients receiving placebo. Among baseline prediabetic patients, 43% (3/7) remained prediabetic in both groups, and 29% (2/7) of teprotumumab patients and 14% (1/7) of patients receiving placebo had diabetic findings at week 24. OPTIC-X patients trended toward increased fasting glucose and HbA1c whether initially treated or retreated with teprotumumab. Fasting glucose commonly rose after 2 or 3 infusions and stabilized thereafter. Most hyperglycemic incidents occurred in patients with baseline prediabetes/diabetes but were controlled with medication. No evidence was found for progression or increased incidence of hyperglycemia with subsequent doses. CONCLUSIONS: Serious glycemic excursions are uncommon in patients with normoglycemia before teprotumumab therapy. Patients with controlled diabetes or impaired glucose tolerance can be treated safely if baseline screening, regular monitoring of glycemic control, and timely treatment of hyperglycemia are practiced. FINANCIAL DISCLOSURE(S): Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.

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.

Integrated Proteomics and Metabolomics Analyses of Serum in Chinese Patients with Severe and Active Graves' Orbitopathy: A Cross-sectional Study.

OBJECTIVE: The present study aims to investigate the alterations of serum proteomic and metabolomic profiles in Chinese patients with severe and active Graves' Orbitopathy (GO). MATERIALS AND METHODS: Thirty patients with GO and 30 healthy volunteers were enrolled. The serum concentrations of FT3, FT4, T3, T4, and thyroid-stimulating hormone (TSH) were analyzed, after which TMT labeling-based proteomics and untargeted metabolomics were performed. Metabo- Analyst and Ingenuity Pathway Analysis (IPA) was used for integrated network analysis. A nomogram was established based on the model to explore the disease prediction ability of the identified feature metabolites. RESULTS: One hundred thirteen proteins (19 up-regulated and 94 down-regulated) and 75 metabolites (20 increased and 55 decreased) were significantly altered in GO compared to the control group. By combining the lasso regression, IPA network, and protein-metabolite-disease sub-networks, we extracted feature proteins (CPS1, GP1BA, and COL6A1) and feature metabolites (glycine, glycerol 3-phosphate, and estrone sulfate). The logistic regression analysis revealed that the full model with the prediction factors and three identified feature metabolites had better prediction performance for GO compared to the baseline model. The ROC curve also indicated better prediction performance (AUC = 0.933 vs. 0.789). CONCLUSION: A new biomarker cluster combined with three blood metabolites with high statistical power can be used to discriminate patients with GO. These findings provide further insights into the pathogenesis, diagnosis, and potential therapeutic targets for this disease.

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.

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.

Combination Model of Thyrotrophin Receptor Antibody and Volumetric Orbital Apex Crowding Index as an Indicator of Dysthyroid Optic Neuropathy.

BACKGROUND: Dysthyroid optic neuropathy (DON) is one of the most serious vision-threatening complications of thyroid eye disease (TED); however, accurate and established diagnostic tools for DON are yet lacking. The present study was aimed at identifying new diagnostic factors for the accurate diagnosis of DON. METHODS: This retrospective cross-sectional study included 25 TED patients (50 eyes) with enlarged extraocular muscles, no previous anti-inflammatory therapy, and the absence of other vision-affecting diseases between May 2017 and August 2019. Baseline data, such as gender, age, ophthalmological history, thyroid disease and management, TED history including clinical features, management, and long-term results, ophthalmological examinations, serology examinations, and single-photon emission computed tomography/computed tomography (SPECT/CT) results, were extracted. The diagnostic criteria were as follows: (1) best-corrected visual acuity (BCVA) loss coexisting with either of the following-increased latency or reduction of amplitude on visual evoked potential (VEP), impaired color vision, visual field defects, contrast sensitivity impairment, and optic disk swelling-and (2) Barrett's index ≥ 60% in CT. Univariate and multivariate logistic regression analyses assessed the differences in age, gender, eyes, medical history, clinical activity, thyroid hormone and antibodies, uptake ratio (UR) of extraocular muscles in SPECT/CT, and volumetric orbital apex crowding index (VACI) using the generalized estimation equation. Consequently, the receiver operating characteristic curve (ROC) of the significant factors was constructed. RESULTS: Univariate analysis revealed significant differences in the clinical activity, free triiodothyronine (FT3), free thyroxine (FT4), thyrotrophin receptor antibody (TRAb) levels, the UR of superior and medial rectus, and VACI between DON and TED (without DON) groups. Multivariate regression analysis revealed that TRAb and VACI were significantly different. ROC analysis showed that the univariate models of TRAb or VACI and the multivariate model were effective indicators of DON, while the multivariate model had the highest area under the ROC curve. CONCLUSION: A combination of TRAb and VACI is an effective indicator for DON.

Association between Clinical Activity Score and Serum Interleukin-6, Interleukin-8 and Interleukin-10 during Systemic Glucocorticoid Treatment for Active Moderate-to-Severe Graves' Orbitopathy.

Purpose: Some interleukins (ILs) play an important role in Graves' orbitopathy (GO) pathogenesis. We aimed to compare serum IL-6, IL-8 and IL-10 in GO patients, patients with Graves' disease (GD) without GO and healthy controls (HC); to follow IL changes during glucocorticoid (GC) treatment for GO; to examine associations between ILs and Clinical Activity Score (CAS).Materials and Methods: Thirty-one patients with active moderate-to-severe GO (GO(+) group), 30 patients with GD without GO (GO(-) group) and 30 HC were enrolled. At baseline, ILs were measured in all groups, CAS was evaluated in GO(+) patients, who were then treated with systemic GCs for 12 weeks. ILs and CAS were reassessed after the first week of treatment (W2) and at the end of the therapy (W12).Results: At baseline, IL-6 was significantly higher in GO(+) and GO(-) patients, IL-8 - higher in GO(-) patients and IL-10 - lower in GO(+) patients compared to HC. Baseline ILs did not correlate with CAS. At W2, all ILs and CAS decreased significantly. At W12, CAS decreased further, IL-6 remained low, IL-8 and IL-10 returned to baseline. CAS reduction correlated positively with IL-6 reduction at W12 (ρ = 0.38, p = .04).GO(+) patients with overall CAS reduction≥2 had higher baseline IL-6 (3.4 vs 2.6 pg/ml, p = .15), smaller IL-10 reduction at W2 (10.5 vs 18.2%, p = .09), lower IL-6 (1.4 vs 2.4 pg/ml, p < .01) and higher IL-6 reduction at W12 (48.6 vs 21.4%, p = .01) compared to patients with CAS reduction<2. Logistic regression analysis confirmed that overall CAS reduction≥2 was associated with higher baseline IL-6, lower IL-6 at W12 and smaller IL-10 reduction at W2 (R2 = 0.66).Conclusions: Higher baseline IL-6, lower IL-6 at W12 and smaller IL-10 reduction at W2 were associated with higher probability of significant overall CAS reduction. IL-6 might be a potential additional marker for assessing disease activity.

Effect of systemic steroid therapy in Graves' orbitopathy on regulatory T cells and Th17/Treg ratio.

PURPOSE: Glucocorticoids are a mainstay treatment for Graves' orbitopathy, yet their exact mechanisms of action remain unclear. We aimed to determine whether the therapeutic effects of systemic steroid therapy in Graves' orbitopathy are mediated by changes in regulatory T lymphocytes (Tregs) and T helper 17 lymphocytes (Th17). METHODS: We assessed Treg and Th17 levels in the peripheral blood of 32 patients with active, moderate-to-severe Graves' orbitopathy who received 12 weekly pulses of methylprednisolone, and determined their association with disease severity, disease activity, and treatment outcomes. The acute orbitopathy phase was confirmed based on clinical evaluation and magnetic resonance imaging, and assessed using the clinical activity score (CAS). The severity of the disease was classified according to ETA/EUGOGO guidelines, and quantified based on the total eye score. Treatment response was determined based on specific criteria (e.g., changes in CAS score, diplopia grade, visual acuity, etc.). Treg and Th17 cells were identified using flow cytometry. RESULTS: Methylprednisolone treatment improved the activity of the disease and altered the Th17/Treg balance (i.e., the percentage of Tregs decreased while the number of Th17 cells remained unchanged). There was no association between the Treg/Th17 ratio and the activity and severity of the disease or the treatment response. CONCLUSIONS: Therapeutic effects of steroid therapy in Graves' orbitopathy are not mediated by Treg and Th17 alterations in the peripheral blood. The decrease in peripheral Treg percentage is likely a consequence of the non-specific effects of steroids and does not impact clinical outcome.

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.

Beneficial effect of low-dose radioiodine ablation for Graves' orbitopathy: results of a retrospective study.

OBJECTIVE: Graves' orbitopathy (GO) reflects an autoimmune response against antigens expressed by the thyroid and orbital tissues. Elimination of thyroid antigens may be beneficial for GO. Total thyroid ablation (TTA) [thyroidectomy (Tx), followed by 30 mCi of radioiodine] was shown to exert a beneficial effect on GO following intravenous glucocorticoids (ivGC) compared with Tx alone. Here, we investigated retrospectively whether TTA performed with a 15 mCi of radioiodine still maintains advantages over Tx. METHODS: Thirty-two subjects, 13 treated with TTA (performed with 15 mCi of radioiodine) and 19 with Tx alone, all with moderately severe, active GO, treated with ivGC, were studied. The primary objective was the outcome of GO at 24 weeks based on a composite evaluation. RESULTS: The two groups did not differ at baseline in terms of sex, age, smoking habits, TSH, anti-TSH receptor autoantibodies, GO duration and eye features. The proportion of GO responders at 24 weeks was greater in the TTA (61.5%) than in the Tx group (26.3%, P = 0.046). In contrast, GO outcome at 48 weeks did not differ between the two groups (69.2% vs 52.6% of responder in TTA and Tx group, respectively). The outcome of the individual GO features did not differ between the two groups both a 24 and 48 months. CONCLUSIONS: The advantage of total thyroid ablation seems to be a more rapid response for GO to ivGC treatment. Prospective, randomized studies in a larger number of subjects are needed to confirm our findings.