Autoimmune Thyroid Disease Patients Research Articles

A novel bioassay for thyroid-blocking immunoglobulins.

Thyroid-blocking immunoglobulins (TBI) are present in 10%-15% of patients with autoimmune thyroid disease (AITD). TBI affect thyroid function. The analytical performance of a novel TBI bioassay was evaluated. Sera from AITD patients were tested with a cell-based TBI reporter bioassay (Thyretain®) with the expression of a luciferase transgene as readout and a new "Turbo™" TBI bioassay with a readout based on a cyclic AMP-activated luciferase. All samples were also run on two TSH-R binding immunoassays. A Passing-Bablok regression, a Bland-Altman plot, and user/lot comparisons were performed. In addition, dose-response curves for Turbo and Thyretain were fitted using serial dilutions, and half-maximal and 80% inhibitory concentrations (IC50/IC80) were compared. Of 1,011 unselected AITD patients, 131 patients (212 samples) were TBI positive. Of the 212 samples, 149 (70.3%), 47 (22%), and 16 (7.5%) were hypothyroid, euthyroid, and hyperthyroid, respectively. The three thyrotropin receptor antibody (TSH-R-Ab) assays were negative in 90 controls devoid of autoimmune thyroid disorders. In contrast, the Turbo cyclic adenosine 3',5'-monophosphate (cAMP) TBI, Thyretain TBI, and the binding assays detected TBI in 212 (100%), 168 (79%), and 138/180 (65%) samples, respectively (p< 0.001). Turbo highly correlated with thyroid function (p< 0.001). The percentage inhibition in both Turbo and Thyretain correlated with TSH-R-Ab binding assay positivity (both p< 0.001). The two bioassays correlated (r = 0.8, p< 0.001), and the Bland-Altman plot displayed no significant bias (0.24). Values scatter with slight systemic deviation between TBI mean values of 10%-50% inhibition, with higher Turbo than Thyretain results. Intra-assay validation demonstrated adequate precision with a very low coefficient of variation (average CV 5.4%) and lower CV with samples with a high inhibitory effect (CVAverage= 1.7% for a sample with 95% inhibition Thyretain). CV did not differ between users (p = 0.35) and lots (p = 0.121). The IC50/IC80 values were 1.55 ng/mL/3.48 ng/mL for Turbo and 6.76 ng/mL/18.46 ng/mL for Thyretain, respectively, demonstrating the markedly higher sensitivity of Turbo. The novel, easy-to-perform, rapid, and reliable Turbo TSH-R blocking bioassay detected significantly more TBI than the established immunoassays, emphasizing its higher analytical performance and clinical utility in the management of patients with AITD.

The Absence of Thyroid-Stimulating Hormone Receptor Expression on Natural Killer T Cells: Implications for the Immune-Endocrine Interaction.

The expression of thyroid-stimulating hormone receptor (TSHR) has been documented on various immune cells, including B lymphocytes, T lymphocytes, Natural Killer (NK) cells, monocytes, and dendritic cells (DCs). Natural Killer T (NKT) cells serve as a crucial link between innate and adaptive immunity, playing significant roles in immunological interactions and autoimmune diseases. The aim of the present study was to evaluate the presence of TSHR on NKT cells. Our research involved patients with thyroid disease, as well as healthy controls. Peripheral blood mononuclear cells (PBMCs) and, thereafter, NKT cells were isolated from 86 patients with benign nodular thyroid disease with and without autoimmune thyroid disease (AITD) (28 and 56 cases, respectively), and TSHR expression was analyzed using fluorescence-activated cell sorting (FACS). In order to confirm the results, the reverse-transcription polymerase chain reaction (RT-PCR) method was used in cells obtained from healthy individuals. Our findings obtained with application of the FACS method revealed that TSHR is not expressed on NKT cells in either AITD or non-AITD patients, though TSHR was detected in the total PBMC population (TSHR+ cells 2.77%). The absence of TSHR on NKT cells was further confirmed with RT-PCR in healthy individuals (p < 0.0001). These results questioned the previously suggested direct influence of NKT cells on AITD development.

Unveiling the Role of Gut Microbiota and Metabolites in Autoimmune Thyroid Diseases: Emerging Perspectives.

Autoimmune thyroid diseases (AITDs) are among the most prevalent organ-specific autoimmune disorders, with thyroid hormones playing a pivotal role in the gastrointestinal system's structure and function. Emerging evidence suggests a link between AITDs and the gut microbiome, which is a diverse community of organisms that are essential for digestion, absorption, intestinal homeostasis, and immune defense. Recent studies using 16S rRNA and metagenomic sequencing of fecal samples from AITD patients have revealed a significant correlation between a gut microbiota imbalance and the severity of AITDs. Progress in animal models of autoimmune diseases has shown that intervention in the gut microbiota can significantly alter the disease severity. The gut microbiota influences T cell subgroup differentiation and modulates the pathological immune response to AITDs through mechanisms involving short-chain fatty acids (SCFAs), lipopolysaccharides (LPSs), and mucosal immunity. Conversely, thyroid hormones also influence gut function and microbiota composition. Thus, there is a bidirectional relationship between the thyroid and the gut ecosystem. This review explores the pathogenic mechanisms of the gut microbiota and its metabolites in AITDs, characterizes the gut microbiota in Graves' disease (GD) and Hashimoto's thyroiditis (HT), and examines the interactions between the gut microbiota, thyroid hormones, T cell differentiation, and trace elements. The review aims to enhance understanding of the gut microbiota-thyroid axis and proposes novel approaches to mitigate AITD severity through gut microbiota modulation.

7007 Analytical &amp; Clinical Performance of a Novel Sensitive Rapid Bioassay for Thyrotropin Receptor Antibodies

Abstract Disclosure: P.D. Olivo: Consulting Fee; Self; QuidelOrtho, CA USA. H. Kim: Employee; Self; QuidelOrtho. L. Miao: Employee; Self; QuidelOrtho. J. Houtz: Employee; Self; QuidelOrtho. M. Luffy: None. J. Wolf: None. G.J. Kahaly: Grant Recipient; Self; QuidelOrtho, USA. Background: The role of stimulatory anti-TSH-R-Ab in the pathogenesis of autoimmune hyperthyroidism is well established. A novel and rapid cell-based bioassay, referred to as Turbo TSITM, for measurement of thyroid-stimulating autoantibodies (TSAb) was recently developed. An assessment of the analytical &amp; clinical performance of this bioassay is described herein. Methods: Turbo TSI greatly reduces the complexity and time required to measure TSAb. It avoids the need for cell culture, has no wash or lysis steps, and has a total turn-around-time of less than 90 minutes. Thawed cells from Turbo TSI kits were treated with different concentrations of a World Health Organization (WHO) international standard (IS) TSI or TSAb-positive serum. TSAb was measured as a function of luciferase activity measured as relative light units (RLU) and converted into international units per liter (IU/L). Two users at two sites performed analytical performance studies on numerous samples, over multiple days. Results: The limit of blank, limit of detection and limit of quantitation were determined to be 0.007 IU/L, 0.014 IU/L, and 0.021 IU/L, respectively. Receiver operator characteristics (ROC) analysis determined the cut-off to be 0.0241 IU/L with an area under the curve of 0.984. The linear range was shown to be from 0.015 to 11.958 IU/L. The intra-laboratory precision was ≤15 percent CV. The overall reproducibility of the assay was ≤20 percent CV for five concentrations (0.06 to 5.16 IU/L). Interference and cross reactivity studies with a variety of substances showed that the assay was robust. Turbo TSI demonstrated 95.2% (95% CI 83.3-98.1) sensitivity and 94.8% (95% CI 90.9-97.1) specificity when compared with an FDA-cleared bioassay (Thyretain® TSI) using serum from 295 patients with autoimmune thyroid disease (AITD). When testing 172 unselected, consecutive TSAb+ AITD patients, a US based, prospective, multicenter, blinded study showed a 98% concordance between Turbo TSI and Thyretain® TSI. When comparing Turbo TSI, Thyretain TSI and a TSH-R-Ab binding immunoassay (Cobas) in 670 unselected, consecutive, treated and untreated AITD subjects, sensitivity was 83%, 79% and 68%, respectively (Turbo vs. Cobas, P&amp;lt;0.001). Furthermore, testing 84 patients with naïve, untreated Graves’ hyperthyroidism, detection of TSH-R-Ab was 100%, 98% and 92%, respectively (Turbo TSI vs binding assay, P&amp;lt;0.01). Finally, 95% vs. 73% of 332 patients with Graves’ orbitopathy were TSH-R-Ab+ with Turbo TSI vs. binding assay (P&amp;lt;0.001). Conclusions: The Turbo TSI bioassay exhibits excellent analytical &amp; clinical performance, a high level of reproducibility, and compares well with the FDA-cleared Thyretain® TSI. Presentation: 6/1/2024

Association of COVID-19 with thyroid dysfunction and autoimmune thyroid disease: A retrospective cohort study

BackgroundSince the end of the COVID-19 pandemic, the potential roles of thyroid-inflammatory derangements in driving or being associated with the prognosis of COVID-19 remain controversial. We aimed to clarify the association between COVID-19 infection and thyroid dysfunction, and highlight the impacts of subsequent autoimmune thyroid disease (AITD) on the prognosis of COVID-19. MethodsThe retrospective, multicenter, cohort study enrolled 2,339 participants with COVID-19 from three hospitals located in the north, middle, and south regions of Shaan Xi Province, China, between December 2022 and July 2023. 464 non-COVID-19 patients within the same period were supplemented, divided into groups with and without AITD. At hospital admission (baseline), 3- and 6-month follow-ups, we presented a dynamic description and correlation analysis of thyroid-inflammatory-autoimmune derangements in patients with AITD. ResultsA total of 2,082 COVID-19 patients diagnosed with AITD and 257 cases without AITD were included in the study, and 464 non-COVID-19 patients were supplemented, dividing into 14 AITD and 450 non-AITD cases. We found that COVID-19 infection was closely associated with thyroid dysfunction (χ2 = 1518.129, p = 0.000). AITD patients with COVID-19 showed a higher prevalence of symptoms and comorbidities and longer hospital stays at baseline than non-AITD patients with COVID-19 (p = 0.000, p = 0.000, and p = 0.000). The baseline free triiodothyronine (FT3), free thyroxine, and radioactive iodine uptake at 24 h in AITD cases significantly decreased (p = 0.000, p = 0.000, and p = 0.000), while thyroid stimulating hormone, thyroglobulin, reverse triiodothyronine (rT3), and thyroid antibodies varying elevated from the baseline to the follow-up (baseline: p = 0.000, p = 0.000, p = 0.000, p = 0.000, p = 0.000, and p = 0.000; 3-month follow-up: p = 0.000, p = 0.000, p = 0.000, p = 0.000, p = 0.030, and p = 0.000). C-reactive protein, calcitonin, interleukin-6, -8, -10, and tumor necrosis factor-α rose significantly at baseline (p = 0.000, p = 0.000, p = 0.000, p = 0.000, p = 0.000, and p = 0.000) in AITD. Interferon-α and interferon-γ at baseline showed a significant decrease (p = 0.000 and p = 0.000), and remained at low levels after 6 months (p = 0.000 and p = 0.000). FT3 and rT3 were positively and negatively correlated with hospitalization, respectively (r = -0.208 and 0.231; p = 0.000 and p = 0.000). ROC curves showed that FT3 and rT3 had better robustness in predicting severe COVID-19 prognosis (AUC = 0.801 and 0.705). Ordered logistic regression revealed that ORs were 0.370, 0.048, and 0.021 for AITD [(subacute thyroiditis, Grave's disease, and Hashimoto's thyroiditis compared to non-thyroidal illness syndrome (NTIS)] with COVID-19 risk, indicating that NTIS was the predominant risk factor for the severity of COVID-19. ConclusionsA robust association has been identified, wherein COVID-19 infection is closely associated with thyroid dysfunction, and the subsequent AITD may aggravate the poor prognosis of COVID-19.

US-based, Prospective, Blinded Study of Thyrotropin Receptor Antibody in Autoimmune Thyroid Disease.

Bioassays provide information on the functionality of thyrotropin receptor antibodies (TSH-R-Ab) and thus may offer more clinical utility than binding assays. In this prospective, blinded, US-based study, the clinical performance of several TSH-R-Ab assays was compared. US endocrinology clinic. One hundred sixty-two unselected, consecutive, well-documented patients with various thyroid diseases and healthy controls. Blinded TSH-R-Ab measurements. Sensitivity and specificity of 4 TSH-R-Ab assays. The 4 TSH-R-Ab assays were negative in all 42 patients without autoimmune thyroid disease (AITD). In 104 patients with Graves' disease (GD), irrespective of the disease duration, TSH-R-Ab positivity was present in 65 (63%), 67 (65%), and 87 (84%) for the Cobas and Immulite binding assays and stimulatory TSH-R-Ab [thyroid-stimulating immunoglobin (TSI)] bioassay, respectively (TSI vs Immulite P < .0025, TSI vs Cobas P < .0009). Fifteen newly diagnosed GD patients were all positive in the TSI bioassay, but only 11 (73%) were positive in the Cobas and Immulite binding assays. Nine GD patients with biochemical subclinical hyperthyroidism were TSI-positive but Immulite- and Cobas-negative. Two GD patients were blocking TSH-R-Ab [thyroid-blocking immunoglobin (TBI)]-positive and TSI-negative, and the Immulite and Cobas were positive in both. Additional serum samples from AITD patients that consisted of 30 TBI-positive and 10 TSI-positive samples were blindly tested in the binding assays. Only 6 of the 10 TSI-positive samples were positive in both binding assays, and 30 and 28 of the TBI-positive samples were positive in the Cobas and Immulite assays, respectively. Binding TSH-R-Ab assays are less sensitive than TSI bioassays and are not specific for stimulating antibodies. Measuring the function of TSH-R-Ab in a bioassay can provide useful information to clinicians.

Enzyme-linked immunosorbent assay of 3 Screen Islet Cell Autoantibody in patients with autoimmune thyroid disease.

In recent years, the emergence of multiplex technology that can simultaneously measure multiple anti-islet autoantibodies has become particularly valuable for the staging and early diagnosis of immune-mediated type 1 diabetes (T1D). While it has been established that 20%-30% of T1D patients suffer from autoimmune thyroid disease (AITD), there is limited available data regarding the presence of anti-islet autoantibodies in AITD patients. Among commercially available anti-islet autoantibodies, glutamic acid decarboxylase 65 autoantibodies (GADAs) are often the first marker measured in general clinical practice. To investigate the frequency of anti-islet autoantibodies in AITD patients. Our study involved four hundred ninety-five AITD patients, categorized into three distinct groups: AITD with T1D (n = 18), AITD with phenotypic type 2 diabetes (T2D) (n = 81), and AITD without diabetes (n = 396), and the enzyme-linked immunosorbent assay (ELISA) was employed to determine the frequencies of 3 Screen Islet Cell Autoantibody (3 Screen ICA), GADA, insulinoma-associated antigen-2 autoantibodies (IA-2As), and zinc transporter 8 autoantibodies (ZnT8As) within these groups. The frequency of 3 Screen ICA in AITD patients with T1D, T2D, and those without diabetes were 88.9%, 6.2%, and 5.1%, respectively, with no significant difference seen between the latter two groups. Notably, the frequency of 3 Screen ICA was 11.1% higher in AITD patients with T1D, 1.3% higher in AITD patients with T2D, and 1.1% higher in AITD patients without diabetes compared to GADA, respectively. Furthermore, 12.5%, 20.0%, and 20.0% of the 3 Screen ICA-positive patients were negative for GADA. Additionally, 1.3% of the AITD patients who tested negative for 3 Screen ICA in both the AITD with T2D and non-diabetic AITD groups were found to be positive for individual autoantibodies. Among the 3 Screen ICA-positive patients, there was a significantly higher proportion of individuals with multiple autoantibodies in AITD patients with T1D compared to those without diabetes (37.5% vs 5.0%, P < 0.05). However, this proportion was similar to that in AITD patients with T2D (20.0%). Nevertheless, there was no significant difference in 3 Screen ICA titers between AITD patients with T1D and those without diabetes (436.8 ± 66.4 vs 308.1 ± 66.4 index). Additionally, no significant difference in 3 Screen ICA titers was observed between Graves' disease and Hashimoto's thyroiditis in any of the groups. Our findings reveal that some AITD patients without diabetes exhibit 3 Screen ICA titers comparable to those in AITD patients with T1D. Thus, 3 Screen ICA outperforms GADA in identifying latent anti-islet autoantibody-positive individuals among AITD patients.

A Mendelian randomization study of the effect of selenium on autoimmune thyroid disease.

The impact of selenium on autoimmune thyroid disease (AITD) is a subject of ongoing debate. This study aimed to analyze the causal correlations of selenium with autoimmune thyroiditis (AIT), autoimmune hyperthyroidism (AIH), and Graves' disease (GD) by Mendelian randomization (MR). Single nucleotide polymorphisms related to selenium, AIT, AIH, and GD were sourced from the IEU Open GWAS project and FinnGen. Exposure-outcome causality was assessed using inverse variance weighted, MR-Egger, and weighted median. Horizontal pleiotropy was examined using the MR-Egger intercept, heterogeneity was evaluated with Cochran's Q test, and the robustness of the results was confirmed via leave-one-out sensitivity analysis. The MR analysis revealed that selenium did not exhibit a causal relationship with AIT (OR 0.993, 95% CI 0.786 to 1.108, p=0.432), AIH (OR 1.066, 95% CI 0.976 to 1.164, p=0.154), or GD (OR 1.052, 95% CI 0.984 to 1.126, p=0.138). Moreover, the MR-Egger intercept and Cochran's Q test demonstrated the absence of horizontal pleiotropy or heterogeneity in these results (p>0.05). Sensitivity analysis affirmed the robustness of these results. This MR analysis concluded that selenium was not linked to AIT, AIH, or GD risk. Therefore, indiscriminate selenium supplementation is not advisable for AITD patients without concurrent selenium deficiency.

Increased interleukin-9 and Th9 cells in patients with refractory Graves' disease and interleukin-9 polymorphisms are associated with autoimmune thyroid diseases.

Autoimmune thyroid diseases (AITDs) are prevalent disorders, primarily encompassing Graves' disease (GD) and Hashimoto's thyroiditis (HT). Despite their common occurrence, the etiology of AITDs remains elusive. Th9 cells, a new subset of CD4+T cells with immunomodulatory properties, have been linked to the development of various autoimmune diseases. However, research on the role of Th9 cells in AITDs is limited. We investigated the expression of Th9 cells,their functional cytokine IL-9, and transcription factor IRF4 in peripheral blood mononuclear cells (PBMCs) and plasma of AITD patients and healthy controls. Additionally, we explored the genetic association between four loci polymorphisms (rs31564, rs2069879, rs1859430, and rs2069868) of the IL-9 gene and AITDs. We reported, for the first time, that refractory GD patients exhibited elevated mRNA levels of IL-9 and IRF4 in PBMCs, increased IL-9 protein levels in plasma, and a higher proportion of Th9 cells in peripheral blood when compared to normal controls. Furthermore, human recombinant IL-9 protein was found to enhance IFN-g secretion in PBMCs from both GD patients and normal controls. At the genetic association level, after adjusting for age and sex, the rs2069879 polymorphism exhibited a significant association with AITDs under an additive model (P<0.001, OR= 0.05, 95% CI=0.03-0.08). Our results reveal that Th9 cells may exert a pivotal role in the pathogenesis and progression of refractory GD and HT, and IL-9 holds promise as a novel therapeutic target for the management of AITDs.

FRI537 Thyrotropin Receptor Blocking Antibodies - A Multicenter, 4-Assay Comparison

Abstract Disclosure: A. George: None. A.T. Bossowski: None. B. Hatun: None. J. König: None. M.A. Lupo: None. L. Frommer: None. J. Wolf: None. G.J. Kahaly: None. Background: Thyrotropin receptor (TSH-R) blocking autoantibodies (TBAb) are present in 10-15% of patients with autoimmune thyroid disease (AITD). TBAb are functional, impact thyroid function, and are clinically relevant. This multicenter study compares, in a large collective of AITD subjects, the sensitivity and specificity of four immunoassays for the measurement of TBAb and introduces a novel and ultra-rapid TBAb bioassay. Methods: Serum samples from AITD patients were tested with a TSH-R binding immunoassay (Cobas e411, Roche, Penzberg, Germany), an established cell-based TBAb reporter bioassay (Thyretain®, Quidel, San Diego, USA) with expression of a Luciferase transgene as readout and a new “TurboTM” TBAb bioassay (Quidel) with a readout that is based on a cyclic AMP-activated luciferase. A Passing-Bablok regression and intra- and inter-assay validations were performed. All samples were also tested for stimulating TSH-R-Ab (TSAb, Thyretain®, Quidel). Results: Of 1011, unselected, consecutive AITD patients (Graves’ disease and Hashimoto thyroiditis), 132 patients and 212 samples were TBAb positive. Median age was 32 years (25/75 percentile 13.25/46.5 years) and female: male ratio was 2.86:1. Of 212 samples, 149 (70.3%), 47 (23,1%) and 16 (7,5%) were hypothyroid, euthyroid and hyperthyroid, respectively. Thyroperoxidase and thyroglobulin antibodies were present in 136 (64.15%) and 70 (33.02%) samples, respectively. The four TSH-R-Ab assays were negative in 90 control subjects devoid of autoimmune thyroid and endocrine disorders. In contrast, the TurboTM cAMP TBAb, Luciferase TBAb and the Cobas binding assays detected TBAb in 212 (100%), 168 (79%) and 137/180 (76%) samples, respectively (Chi-square test p&amp;lt;0.001). The TurboTM TBAb bioassay highly correlated with thyroid function (p=0.007). Furthermore, the magnitude of percentage inhibition in both TurboTM and Luciferase TBAb bioassays correlated with TSH-R-Ab binding assay positivity (both p&amp;lt;0.001). Seventeen of 212 (8%) samples showed dual TSH-R-Ab positivity in the Turbo TBAb and TSAb bioassays, while 13 (6.1%) samples showed dual positivity for TSAb and TBAb in all three bioassays. The two TBAb bioassays correlated (Spearman’s r =0.59, p&amp;lt; 0.001). Single-user testing and intra-assay validation demonstrated adequate precision with very low coefficient of variation (CV 0.05) and low CV (0.14) at 92% and 44% inhibition (serum TBAb positivity), respectively. Conclusions: This is the largest reported collective of TBAb-positive samples in AITD patients, measured with four different immunoassays. TBAb markedly affects thyroid function. The novel, easy-to-perform, rapid and reliable TurboTM blocking bioassay detected significantly more TBAb than the established immunoassays demonstrating both its higher analytical performance and clinical utility in the management of AITD patients. Presentation: Friday, June 16, 2023

FRI540 A Novel, Rapid, Sensitive Bioassay For Thyrotropin Receptor Stimulatory Antibodies Improves Diagnosis &amp; Management Of Autoimmune Thyroid Disease

Abstract Disclosure: G.J. Kahaly: None. M.A. Lupo: None. A.T. Bossowski: None. L. Frommer: None. B. Hatun: None. A. George: None. J. Wolf: None. Background: Autoantibody mimicry of hormone action at the thyrotropin receptor (TSH-R) and aberrant signaling of TSH-R by autoantibodies (TSH-R-Ab) causes autoimmune thyroid disease (AITD), hyperthyroidism and hypothyroidism, both of which affect millions of patients worldwide. In this multicenter, single blind study, the specificity, sensitivity and performance of a novel bioassay for stimulatory TSH-R-Ab were tested. Methods: TSH-R-Ab were measured in a blinded manner with a TSH-R binding immunoassay (Cobas e411, Roche), and both stimulatory (TSAb) and blocking (TBAb) bioassays with luciferase release as readout (Thyretain®, Quidel) according to manufacturer’s instructions. TSH-R-Ab was also measured using the new TSAb bioassay (TurboTM). TurboTM utilizes a cAMP biosensor that is an engineered form of firefly luciferase in which the cAMP-binding domain of protein kinase A is fused between the N- and C-termini such that the luciferase is inactive until cAMP binds to the cAMP binding site. Using this biosensor, luciferase activity is proportional to intracellular cAMP levels. TurboTM detects TSAb in serum at room temperature, in a real-time homogeneous format, which delivers results in 60 minutes. It does not require cell culture, sample dilution, washing or cell lysis steps. Results: Eight-hundred forty-four samples of unselected, consecutive AITD patients and control subjects, devoid of autoimmune endocrine and thyroid disorders, were included. Mean age (SD) was 49 (14.7) years, range 1-89 years. Female: male sex ratio was 3.7:1. The four TSH-R-Ab assays were negative in all 174 controls. In contrast, the Turbo TSAb, Thyretain® TSAb and the binding assays detected TSAb in 557 of 670 (83%), 526/670 (78%) and 430/637 (67%) AITD samples, respectively (TurboTM bioassay versus binding immunoassay, chi-square test p&amp;lt;0.001). The results of the Turbo bioassay highly correlated with both thyroid function in Graves’ hyperthyroidism as well as with the clinical activity and severity of associated thyroid eye disease (p&amp;lt;0.001). Inter- (two users) and intra- (two lots) assay comparison for the Turbo bioassay showed coefficient of variations of only 4%, 3%, and 2%, respectively. The two TSAb bioassays, TurboTM and Thyretain®, correlated (Spearman’s r =0.7, p&amp;lt;0.001). Furthermore, the magnitude of serum levels in both TSAb bioassays correlated with TSH-R-Ab binding (both p&amp;lt;0.001). Finally, seventeen AITD samples showed dual TSH-R-Ab positivity in the TBAb and TSAb bioassays. Conclusions: The novel, rapid, easy-to-perform, sensitive and specific TurboTM bioassay performs better than the established TSH-R-Ab binding immunoassay and the Thyretain® bioassay. It provides clinically useful information for the accurate diagnosis and management of AITD patients. Presentation: Friday, June 16, 2023

Assessing the thyroid autoimmunity association with recurrent aphthous stomatitis

BackgroundRecent investigations have highlighted autoimmune origins and abnormal immune responses; particularly those related to T cell-mediated immunity and elevated T lymphocyte cells in the oral mucosa. Therefore, we investigated the relationship between recurrent aphthous stomatitis (RAS) and autoimmune thyroid diseases (ATDs) in an Iranian population.MethodsA cross-sectional study was performed on 102 patients diagnosed with ATD (cases) and 102 healthy patients (controls) who had been referred for the routine dental treatment. All participants were asked for the history of RAS and their age, gender, other systemic diseases, medications, and frequency of RAS in a year. Matching was performed based on the propensity scores for age and sex. In addition, the number of lesions in each recurrence in both groups was assessed and compared. The type of thyroid disease has been assessed for case participants and has been confirmed by the endocrinologist. The chi-square test, t-test, and Mann-Whitney U test were used to analyze the data using SPSS 18.ResultsPatients with ATD had higher RAS than healthy controls (P = 0.040). ATD patients had 1.93 times more risk for RAS, and the frequency of RAS in a year was 3.15times higher in these patients (P = 0.011). Moreover, the frequency of RAS was higher in patients with hypothyroidism than in those with hyperthyroidism. However, there were no significant differences in the size and the number of lesions between the groups.ConclusionThe risk and frequency of RAS were significantly higher in patients with ATDs. This would provide valuable insights into the underlying mechanisms and potential treatment strategies for both conditions.

Th1 or Th2 cytokines are correlated with Tregs and T cell subsets and pregnancy outcomes in patients with autoimmune thyroid disease during early, middle, late pregnancy, and postpartum period

Autoimmune thyroid disease (AITD) is a T lymphocytes-mediated autoimmune disorder affecting pregnant women. The current study sought to determine the correlations between T helper-1 (Th1)/T helper-2 (Th2) cytokines and regulatory T cells (Tregs) and T cell subsets and pregnancy outcomes in AITD patients during early pregnancy (T1), middle pregnancy (T2), late pregnancy (T3), and postpartum period (PP). A total of 60 patients with Graves’ disease, 60 patients with Hashimoto’s thyroiditis, and 30 healthy pregnant women were initially enrolled in the study. Thyroid hormones and antibodies, Th1 or Th2 cytokines, transforming growth factor-β, Tregs, CD4+ T helper cells (CD4+), CD8+ T helper cells (CD8+) levels were determined by means of Maglumi2000 automatic chemiluminescence instrument, enzyme-linked immunosorbent assay, and flow cytometry. Our findings demonstrated higher IFN-γ and IL-2 levels, along with lower IL-4, IL-10, TGF-β, Treg, and CD4+/CD8+ levels in AITD patients during T1, T2, T3, and PP. Furthermore, the TGF-β, Treg, and CD4+/CD8+ levels were lower in the IFN-γ/IL-2 high expression group but higher in the IL-4/IL-10 high expression group. The IFN-γ and IL-2 levels were higher, while IL-4 and IL-10 level were lower in AITD patients with adverse pregnancy outcomes. Lastly, Th1 cytokines were higher and Th2 cytokines were lower in AITD patients and elicited correlation with Tregs and CD4+/CD8+ levels. Collectively, our findings highlighted that up-regulation of Th1 cytokines may increase the percentage of adverse pregnancy outcomes in AITD patients.

Retrospective analysis of low-dose interleukin-2 therapy on chronic autoimmune thyroid disease with concurrent systemic lupus erythematosus

Introduction Low-dose interleukin-2 (IL-2) regulates the homeostasis of CD4+ T cells by modulating the proportions of effector and regulatory T cells, thus reducing disease activity in patients with systemic lupus erythematosus (SLE). However, to date, no research has been carried out on the efficacy of low-dose IL-2 for treating autoimmune thyroid disease (AITD). The aim of this study was to observe the effects of IL-2 on AITD patients with concurrent SLE, and explore potential mechanism of action. Methods A retrospective analysis was conducted on 29 SLE patients with concurrent AITD. Among them, 11 patients were in IL-2 therapy group and 18 patients without IL-2 treatment were considered as control group. Two groups had similar disease activities and were treated with comparable regular strategy. Free triiodothyronine (FT3), free thyroxine (FT4), thyroxine(T4), triiodothyronine(T3), thyroid stimulating hormone (TSH), thyroglobulin antibody (TG-Ab), thyroid peroxidase antibody (TPO-Ab) levels and immune cell subgroups were measured. Results After receiving low-dose IL-2 therapy, the TG-Ab and TPO-Ab levels decreased drastically (TG-Ab p = 0.008, TPO-Ab p = 0.007), and the majority of the AITD patients became seronegative, while there was no discernible change in control group. In IL-2 group, percentage of CD4+ T cells showed a significant increase after treatment (p = 0.029), with an upward trend in the ratio of regulatory T (Treg) cells to follicular helper T (Tfh) cells (Treg/Tfh). The percentage as well as absolute count of B cells demonstrated a decreasing trend. Conclusion Low-dose IL-2 may downregulate the levels of TG-Ab and TPO-Ab by modulating the immune balance of Treg/Tfh and B-cells, providing new avenue for clinical treatment of AITD.

Association between dental caries experience and salivary profile among autoimmune thyroid disease subjects - a cross-sectional comparative study.

Background: Autoimmune thyroid disease (AITD) is an inflammatory condition that primarily affects women between the ages of 30 and 50. It has been hypothesised that AITD causes salivary glands to produce less saliva due to its endocrine effects. Studies showing the effect of AITD on salivary glands are very scarce. The aim of this study was to compare AITD patients with controls who were of a similar age and gender in terms of salivary metrics and the prevalence of dental caries. Methods: 200 subjects in Bangalore city (100 AITD subjects and 100 healthy subjects as controls) participated in a cross-sectional study. Subjective oral dryness was assessed using Farsi's criteria. Salivary parameters and caries were assessed using GC Saliva-Check BUFFER kit and WHO caries criteria respectively. Descriptive and analytical statistics were done. Significant data was defined as a p value of <0.05. Results: When compared to controls, the AITD group had substantially more subjective mouth dryness and dental caries. Unstimulated (USFR) and stimulated salivary flow rate (SFR), pH and buffering capacity were reduced in AITD group whereas viscosity was high. There was a significant negative correlation between USFR, SFR, subjective oral dryness and dental caries. In a linear regression, there was no association between age, gender, socioeconomic status (SES), thyroid stimulating hormone (TSH), salivary viscosity, pH, buffering capacity and dental caries. Dental caries, USFR and SFR demonstrated a substantial relationship. Conclusions: The present research may indicate an underlying association between thyroid and salivary gland dysfunction resulting in oral dryness and high dental caries experience.

Autoimmune thyroid disease disrupts immune homeostasis in the endometrium of unexplained infertility women-a single-cell RNA transcriptome study during the implantation window.

Autoimmune thyroid disease (AITD) is known to be associated with unexplained infertility in women. Although the presence of antithyroid antibodies have been speculated to be a marker of an immune imbalance that might lead to implantation failure, its underlying mechanism influencing the endometrial receptivity remains to be elucidated. In this study, we used single-cell RNA sequencing (scRNA-seq) to dissect immune microenvironment in endometrium of AITD patients during window of implantation (WOI). We collected CD45+ immune cell populations of endometrium samples of unexplained infertile women with AITD (n=3), as well as samples of AITD- controls (n=3). The cells were then processed with 10X Genomics Chromium for further analysis. We characterized 28 distinct immune cell subtypes totally, and uncovered differences in the composition and gene expression patterns between AITD patients and controls. The proportions of T CD4+, cNK, ILC3, T CD8+ GZMK+, T CD8+ Cytotoxic and ILC3 CD3E - cells were increased, and CD366+ uNK1 was decreased in AITD+ patients. And the abnormal expression of GNLY and chemokines was observed in AITD patients. In addition, uNK and T CD8+ Cytotoxic cells showed lower cytotoxicity but activation of immune response. Genes enriched in cell adhesion of ILC3 and Tregs were downregulated, while the number of ILC3 and Tregs were increased. Immune imbalance exists in endometrium during WOI, which may impact embryo implantation.

Effective Inhibition of Thyroid Antigen Presentation Using Retro-Inverso Peptides in Experimental Autoimmune Thyroiditis: A Pathway Toward Immune Therapies of Thyroid Autoimmunity.

Background: Autoimmune thyroid diseases (AITD) represent the most common autoimmune diseases. However, current therapies focus on relieving the symptoms instead of curing AITD, and new therapies to reverse the autoimmune attack on the thyroid are needed. HLA-DRβ1-Arg74 is the key HLA class II allele that triggers AITD by presenting pathogenic thyroglobulin (Tg) peptides that activate thyroid self-reactive T cells. We hypothesized that blocking the presentation of Tg peptides to T cells within the HLA-DRβ1-Arg74 peptide binding cleft could reverse the autoimmune response to the thyroid in AITD. Methods: The approach we used to block Tg peptide presentation within HLA-DRβ1-Arg74 is to design retro-inverso D-amino acid (RID) peptides that have high affinity to the HLA-DRβ1-Arg74 peptide binding pocket. Results: By using computational approaches and molecular dynamics simulations, we designed two RID peptides, RT-15 and VT-15, that blocked peptide binding to recombinant HLA-DRβ1-Arg74 molecule, as well as T cell activation in vitro. Furthermore, RT-15 and VT-15 blocked in vivo T cell activation by thyroglobulin in humanized NOD-DR3 mice induced with experimental autoimmune thyroiditis. Conclusions: In summary, we discovered two RID peptides that block thyroglobulin peptide binding to HLA-DRβ1-Arg74 and their presentation to T cells in AITD. These findings set the stage for a personalized medicine therapeutic approach for AITD patients who carry the DRβ1-Arg74 allele. This antigen-specific therapeutic strategy can potentially be extended to other autoimmune diseases.

Medication Adherence in Children with Down Syndrome with Autoimmune Thyroid Disease During the COVID-19 Pandemic: Indonesian Study

Background: The coronavirus disease-19 (COVID-19) pandemic has significantly affected healthcare systems. Down syndrome (DS) is a chronic disease caused by trisomy of chromosome 21 which is associated with a variety of medical problems such as autoimmune thyroid disease (AITD) that necessitate comprehensive routine treatment. During the COVID-19 pandemic, there was an increasing an unavailability, which became an impediment to chronic disease patients’ drug consumption. Objectives: The purpose of this study was to examine the barriers to medication adherence faced by DS patients during the COVID-19 pandemic. Methods: An observational analytic study was conducted from January to July 2021 among parents of DS patients who registered in the pediatric endocrinology outpatient clinic of Dr. Soetomo General Hospital. Inclusion criteria include: The parents of DS with AITD patients aged 1 - 18 years who came to the pediatric endocrinology polyclinic, routinely took oral medication before March 2020, can fill out questionnaire forms independently, and signed the informed consent. Exclusion criteria were parents of DS patients who did not take regular medication, or started treatment after March 2020. Data were collected and analyzed using the Wilcoxon comparison test Results: There are 31 DS patients responded and completed the questionnaires. Adherence to hospital visits in DS with AITD patients before and during the COVID-19 pandemic showed significant differences (P = 0.001). The main barriers to follow-up visits during the pandemic were lockdown protocol which made travel difficult (28%). The compliance for taking medication was still high although 13 (41.9%) obtained the medicine without a prescription. Conclusions: Changes in terms of medication adherence during the pandemic have highlighted the importance of improving DS patient’s access to healthcare. Shifting medication counseling to the nearest primary health care provider with supervision from a tertiary referral specialist appears to be a reasonable and potentially cost-effective strategy in improving treatment adherence especially in a pandemic setting.

Association of ITM2A rs1751094 polymorphism on X chromosome in Korean pediatric patients with autoimmune thyroid disease.

Autoimmune thyroid disease (AITD) manifests with a female predominance, and much attention has been directed towards the integral membrane protein 2 A (ITM2A) gene located on the X chromosome. In a study of 166 pediatric patients with autoimmune thyroid disease (AITD), the ITM2A rs1751094 single-nucleotide polymorphism (SNP) was genotyped. The sample comprised 143 females and 23 males, with 67 patients diagnosed with Hashimoto chronic thyroiditis (HD) and 99 with Graves' disease (GD). In the 99 GD patients, 49 (49.5%) exhibited thyroid-associated ophthalmopathy (TAO). Among the 85 GD patients, 70.6% (60/85) were considered intractable GD. The results were compared to those from 198 healthy Korean individuals, including 97 females and 101 males. The frequency of the rs1751094 C allele and CC/AC genotype were higher in AITD, GD and HD patients compared to controls, while the frequency of the A allele and AA genotype were lower. The results were more pronounced in female AITD and GD patients compared to male patients. The association was also found in intractable GD and TAO patients. Target SNP fits Hardy-Weinberg equilibrium. These findings indicate that the ITM2A gene polymorphism on the X chromosome may contribute to the immunological basis of female-predominant AITD in Korean children.

Juvenile idiopathic arthritis patients with positive family history of autoimmune thyroid disease might benefit from serological screening: analysis of the international Pharmachild registry

BackgroundLittle is known about the association between juvenile idiopathic arthritis (JIA) and autoimmune thyroid disease (AITD) and therefore there are no indications for AITD screening in this population, which is possible using standard blood tests. The objective of this study is to determine the prevalence and predictors of symptomatic AITD in JIA patients from the international Pharmachild registry.MethodsOccurrence of AITD was determined from adverse event forms and comorbidity reports. Associated factors and independent predictors for AITD were determined using univariable and multivariable logistic regression analyses.ResultsThe prevalence of AITD after a median observation period of 5.5 years was 1.1% (96/8965 patients). Patients who developed AITD were more often female (83.3% vs. 68.0%), RF positive (10.0% vs. 4.3%) and ANA positive (55.7% vs. 41.5%) than patients who did not. AITD patients were furthermore older at JIA onset (median 7.8 years vs. 5.3 years) and had more often polyarthritis (40.6% vs. 30.4%) and a family history of AITD (27.5% vs. 4.8%) compared to non-AITD patients. A family history of AITD (OR = 6.8, 95% CI: 4.1 – 11.1), female sex (OR = 2.2, 95% CI: 1.3 – 4.3), ANA positivity (OR = 2.0, 95% CI: 1.3 – 3.2) and older age at JIA onset (OR = 1.1, 95% CI: 1.1 – 1.2) were independent predictors of AITD on multivariable analysis. Based on our data, 16 female ANA positive JIA patients with a family history of AITD would have to be screened during ±5.5 years using standard blood tests to detect one case of AITD.ConclusionsThis is the first study to report independent predictor variables for symptomatic AITD in JIA. Female ANA positive JIA patients with positive family history are at increased risk of developing AITD and thus might benefit from yearly serological screening.