Untreated Graves' Disease Patients Research Articles

The role of activated monocyte IFN/SIGLEC1 signalling in Graves' disease.

Graves' disease (GD) is characterized by dysregulation of the immune system with aberrant immune cell function. However, there have been few previous studies on the role of monocytes in the pathology of GD. The object of this study was to investigate whether and how monocytes participate in GD pathology. CD14+ monocytes were isolated from untreated initial GD patients and healthy controls. Then, RNA-seq was performed to investigate changes in global mRNA expression in monocytes and found that type I interferon (IFN) signalling was among the top upregulated signalling pathways in GD monocytes. Type I IFN-induced sialic acid-binding immunoglobulin-like lectin1 (SIGLEC1) expression was significantly upregulated in untreated GD patients and correlated with thyroid parameters. Patient serum SIGLEC1 concentrations were reduced after anti-thyroid drug treatment. Inhibiting SIGLEC1 expression could inhibit proinflammatory cytokine (IL-1β, IL-6, IL-8, IL-10 and M-CSF) expression in monocytes. In conclusion, our study suggested that type I IFN-mediated monocyte activation could have a deleterious effect on the pathogenesis of GD. These observations indicated that the inhibition of type I IFN-activated monocytes/macrophages could have a therapeutic effect on GD remission.

Clinical diagnostic performance of a fully automated TSI immunoassay vs. that of an automated anti‑TSHR immunoassay for Graves' disease: a Chinese multicenter study.

Both thyroid-stimulating immunoglobulins immunoassay (TSI IA) and thyrotrophin receptor antibody immunoassay (TRAb IA) are commonly used for the diagnosis of Graves' disease (GD). The aim of the present study was to compare the clinical diagnostic performance of these two methods. Sera were obtained from 1103 subjects presenting a variety of clinical conditions from three centers: 100 subjects with untreated GD, 200 with treated GD, 62 with autoimmune thyroid disease(AIT), 216 with other thyroid diseases (OTHER-T), 214 with non-thyroid autoimmune diseases (NTAD), 191 with other diseases (OD), and 120 healthy subjects (HS). Both TSI and TRAb IAs were performed for all 1013 serum samples. Bioassay was performed for 86 samples whose TSI results were inconsistent the TRAb assay results. Comparing untreated GD patients with the control groups (AIT, NTAD, OTHER-T, OD, and HS) resulted in an area under the curve (AUC) of 0.992 for the TSI IA and 0.989 for the TRAb IA with no statistically significant difference observed between these AUC values (P = 0.2733). The best TSI CDP (clinical decision point) value was 0.42 IU/L. The differences in sensitivity (100% vs. 95%, P = 0.7991) and specificity (97.1% vs. 97.6%, P = 0.9426) between the TSI and TRAb IA were not statistically significant. TSI IA had a higher agreement with the TSI bioassay than TRAb IA. The clinical diagnostic performance of the TSI IA for diagnosing Graves' disease was very similar to that of the TRAb IA. TSI IA can be used to diagnose GD in the Chinese.

SAT-422 Evaluation of the Siemens Thyroid Stimulating Immunoglobulin (TSI) Assay for Diagnosis and Prognosis of Graves’ Disease

Background: Hyperthyroidism due to Graves’ disease (GD) is an autoimmune condition caused by thyroid stimulating hormone receptor (TSHR) autoantibodies. Autoantibodies to the TSHR can stimulate or block thyroid hormone production, therefore testing specifically for stimulating antibodies would be beneficial for diagnosis of GD. Objectives: The primary objective of the first phase of this trial is to assess the diagnostic capability of the Siemens Thyroid Stimulating Immunoglobulin (TSI) immunoassay in diagnosing GD and to compare it with the Roche TSH Receptor Antibody (TRAb) assay. Design and Methods: Two hundred patients with suspected GD are being enrolled in this single-center multiphase prospective cohort study. Consenting patients undergo biochemical testing including thyroid stimulating hormone (TSH), free T3 (FT3) and T4 (FT4), TRAb and TSI measurements. GD diagnosis was confirmed by endocrinologists that were blinded to TSI results. Results: To date, 85 patients were included in the analysis, of which 66 were diagnosed with GD. For the primary analysis, all patients taking anti-thyroid drugs (ATD) at time of sample collection (n=14) were removed. The respective sensitivity, specificity, negative predictive value (NPV), and positive predictive value (PPV) for TSI was 98, 84, 94 and 94%, which were comparable to those generated by TRAb (98, 95, 95, and 98%). In patients with clinical findings of GD (ie. orbitopathy or goiter, n=33), both the TSI and TRAb assays had identical sensitivity and specificity at 96% and 80% respectively. In patients without orbitopathy or goiter (n=38), the TSI assay had perfect sensitivity and excellent specificity of 100% and 86% respectively (TRAb had 100% sensitivity and specificity). Sensitivity, specificity, NPV, and PPV were slightly lower for both TSI and TRAb in patients treated with ATDs compared to patients without treatment (TSI: 85, 84, 62, 95%; TRAb: 91, 95, 75, 98%). Of ten patients with GD and false negative TSI results, nine were on ATDs. Of this subset, four patients had discordant results between TSI (negative) and TRAb (positive). Notably, one of these patients had normalization of their FT3 and FT4 on the day of sample collection. Discussion and Conclusion: Based on our preliminary results, TSI is an excellent marker for diagnosing GD, particularly in untreated GD patients. The performance of the TSI assay has been comparable to the TRAb assay and correlates well with clinical findings. Discordant false negative results were only seen in patients on ATD. One potential explanation is that the TSI assay is detecting a decrease in stimulating autoantibodies when there is normalization of FT3 and FT4. Importantly, all discordant samples will be tested by a TSI bioassay to confirm diagnosis. Further patient enrollment is occurring, and prognostic assessment of these assays will soon be possible.

Prediction of response to medical therapy by serum soluble (pro)renin receptor levels in Graves' disease.

Antithyroid drugs are generally selected as the first-line treatment for Graves’ Disease (GD); however, the existence of patients showing resistance or severe side effects to these drugs is an important issue to be solved. The (pro)renin receptor [(P)RR] is a multi-functional protein that activates the tissue renin-angiotensin system and is an essential constituent of vacuolar H+-ATPase, necessary for the autophagy-lysosome pathway. (P)RR is cleaved to soluble (s)(P)RR, which reflects the status of (P)RR expression. In this retrospective study, we aimed to investigate whether serum s(P)RR concentration can be used as a biomarker to predict the outcome of antithyroid drug treatment in GD patients. Serum s(P)RR levels were measured in 54 untreated GD patients and 47 control participants. Effects of medical treatment with antithyroid drugs on these levels were investigated in GD patients. Serum s(P)RR levels were significantly higher in patients with Graves’ disease than in control subjects (P<0.005) and were significantly reduced after medical treatment for Graves’ disease. High serum s(P)RR levels were associated with resistance to antithyroid drug treatment, suggesting that serum s(P)RR concentration can be used as a useful biomarker to predict the outcome of antithyroid drug treatment in these patients. Patients with Graves’ disease with low body mass index showed higher levels of serum soluble (pro)renin receptor levels than those with high body mass index. In addition, in patients with Graves’ disease, serum triglyceride levels were negatively correlated with serum soluble (pro)renin receptor levels. All these data indicated an association between low nutrient condition due to hyperthyroidism and increased (pro)renin receptor expression in these patients, suggesting that (pro)renin receptor expression could be increased in the process of stimulating intracellular energy production via activating autophagy function to compensate energy loss.

MicroRNA-4443 Causes CD4+ T Cells Dysfunction by Targeting TNFR-Associated Factor 4 in Graves' Disease.

Aberrant CD4+ T cell function plays a critical role in the process of Graves' disease (GD). MicroRNAs (miRNAs) are important regulators of T cell activation, proliferation, and cytokine production. However, the contribution of miRNAs to CD4+ T cell dysfunction in GD remains unclear. To investigate how certain miRNA causes aberrant CD4+ T cell function in GD patients. We compared the expression pattern of miRNAs in CD4+ T cells from untreated GD (UGD) patients with those from healthy controls. The most significantly dysregulated miRNAs were selected and their correlations with clinical parameters were analyzed. The effect of miR-4443 on CD4+ T cells cytokines production and proliferation was assessed. The potential gene target was identified and validated. GD patients had unique pattern of miRNA expression profile in CD4+ T cells comparing to healthy subjects. miR-10a, miR-125b, and miR-4443 were the three most significantly dysregulated miRNAs. The elevated miR-4443 levels were strongly correlated with clinical parameters in an independent dataset of UGD patients (N = 40), while miR-4443 was normally expressed in GD patients with euthyroidism and negative TRAb level. We found that miR-4443 directly inhibited TNFR-associated factor (TRAF) 4 expression to increase CD4+ T cells cytokines secretion as well as proliferation through the NF-κB pathway. Furthermore, the TRAF4 levels in GD patients were inversely correlated with miR-4443, and knocking down TRAF4 had a similar effect with miR-4443 overexpression. The increased expression of miR-4443 induced CD4+ T cells dysfunction by targeting TRAF4, which may cause GD.

ITM2A Expands Evidence for Genetic and Environmental Interaction in Graves Disease Pathogenesis.

Graves disease (GD) is a common autoimmune disease triggered by genetic predisposition and environmental factors. However, the mechanisms of interaction between genetic and environmental factors contributing to the development of GD remain unknown. We aimed to identify GD susceptibility variants and genes on Xq21.1 locus and interpret the contribution of interaction between genetic predisposition on Xq21.1 and environmental factors to GD. We performed refining study on Xq21.1 in a 2-stage study and carried out expression quantitative trait locus analysis of the best association signal with GD. A total of 4316 GD patients and 4374 sex-matched controls were collected from the Chinese Han population by cooperation with multiple hospitals. We identified that rs3827440 or its linkage single nucleotide polymorphisms (SNPs) were probably the causal variant in the Xq21.1 locus, with the most substantial association with GD in our combined cohorts (P = 2.45 × 10-15). The genotypes of rs3827440 were correlated with the expression of ITM2A in monocytes and peripheral blood mononuclear cells (PBMCs) from healthy volunteers. Notably, the expression of ITM2A in monocytes after lipopolysaccharide (LPS) and interferon-γ (INF-γ) stimulation showed substantial difference among the volunteers that carried different genotypes of rs3827440 (P = 9.40 × 10-7 and P = 1.26 × 10-5 for 24 hours' LPS and INF-γ stimulation, respectively). Moreover, ITM2A expression was significantly decreased in PBMCs from untreated GD patients than that from controls. The results suggest that ITM2A might be a susceptibility gene for GD in the Xq21.1 locus, and environmental factors, such as viral and bacterial infections, probably contribute to GD pathogenesis by interacting with the risk SNP rs3827440 mediating the regulation of ITM2A expression.

Down-regulated resistin level in consequence of decreased neutrophil counts in untreated Grave's disease.

Resistin, belongs to cysteine-rich secretory protein, is mainly produced by circulating leukocytes, such as neutrophils monocytes and macrophages in humans. To date, few but controversial studies have reported about resistin concentrations in hyperthyroid patients, especially in Graves' disease (GD). We undertaked a controlled, prospective study to explore the serum resistin concentration in GD patients before and after -MMI treatment. In addition, we also investigated the main influencing factor on serum resistin level and discuessed the potential role of serum resistin plays in GD patients. 39 untreated GD (uGD) patients, including 8 males and 31 females, were enrolled in our investigation. All of these patients were prescribed with MMI treatment, in addition to 25 healthy controls. Anthropometric parameters and hormone assessment were measured. Enzyme-linked immunosorbent assay was used to detect serum resistin concentration in different stages of GD patients. Furthermore, neutrophil cell line NB4 with or without T3 treatment to detect the effect of thyroid hormones on resistin expression. The serum resistin level and neutrophil counts in untreated GD patients were significantly declined. And all of these parameters were recovered to normal after MMI treatment in ethyroid GD (eGD) and TRAb-negative conversion (nGD) patients. Resistin concentration exhibited a negative correlation with FT3 and FT4, but a positive correlation with absolute number of neutrophiles in uGD patients, whereas did not correlate with thyroid autoimmune antibodies and BMI. Neutrophile cell line, NB4, produced decreased expression of resistin when stimulated with T3. Our study showed a decrease of serum resistin level in GD patients and we suggested that the serum resistin might primarily secreted from circulating neutrophils and down-regulated by excessive thyroid hormones in GD patients.

Hyperthyroidism induced by Graves' disease reversibly affects skin microvascular reactivity.

The impact of hyperthyroidism induced by Graves' disease (GD) on skin microcirculation has not been established. We aimed to assess vascular reactivity in hyperthyroid GD patients before and during treatment. Laser Doppler flux (LDF) was measured in 31 newly diagnosed hyperthyroid GD patients with an increased TSH receptor stimulating antibody (TSAb) levels before the methimazole treatment; and again 5.8 ± 0.8 months later when euthyroidism had been established; and in 30 healthy age- and gender-matched controls. Postocclusive reactive hyperaemia (PRH) was assessed by a 3-min occlusion of the brachial artery. Baseline LDF on the finger pulp and on the volar forearm were significantly higher in untreated GD patients compared to treated GD patients and controls (p < 0.05 for both). On the finger pulp, the time to maximal LDF during PRH was significantly shorter in untreated GD patients compared to controls (p < 0.05). On the forearm, the duration of PRH was significantly longer in untreated GD patients compared to controls (p < 0.05). Positive correlations of triiodothyronine and TSAb with some indices of PRH were established in treated GD patients. Hyperthyroidism induced by GD reversibly affects skin microcirculation, presumably by increasing the vasodilator capacity. Potential involvement of TSAb might be implicated.

Increased IL-21/IL-21R expression and its proinflammatory effects in autoimmune thyroid disease

To determine the potential role of interleukin-21 (IL-21) / IL-21 receptor (IL-21R) in the pathogenesis of autoimmune thyroid disease (AITD) mainly known as Graves’ disease (GD) and Hashimoto’s thyroiditis (HT). IL-21 and IL-21R of peripheral blood samples and/or thyroid tissues from AITD patients and healthy controls were analyzed by ELISA, quantitative real-time polymerase chain reaction (qRT-PCR), flow cytometry and immunohistochemistry. In vitro, the mRNA and protein of inflammatory cytokines of cultured peripheral blood mononuclear cells (PBMCs) upon recombinant human IL-21 (rhIL-21) stimulation were detected. There was an increased serum concentration of IL-21 in untreated GD and HT patients, and IL-21+CD3+CD8−T cells were significantly increased in PBMCs of HT patients compared with healthy volunteers. The IL-21 mRNA expression in PBMCs increased dramatically in GD and HT patients, and marked augmentations of IL-21 and IL-21R mRNA in thyroid tissues of HT patients were observed. Immunohistochemical staining confirmed the expression of IL-21R protein in HT thyroid cells and lymphocytes. In vitro, PBMCs from GD cultured with rhIL-21 induced increased IL-17A but decreased IL-4 production, while from HT stimulated by rhIL-21 induced augmented production of IFN-γ. In conclusion, the expression of IL-21 and IL-21R were up-regulated in AITD and may be involved in the pathogenesis of the disease through augmenting aberrant immune cascade.

Increase of Interferon-γ Inducible CXCL9 and CXCL11 Serum Levels in Patients with Active Graves' Disease and Modulation by Methimazole Therapy

Chemokine (C-X-C motif) ligand (CXCL)9 and CXCL11 play an important role in the initial phases of autoimmune thyroiditis (AT); however, their serum levels in patients with Graves' disease (GD) have never been evaluated in relation to thyroid function and treatment. To evaluate CXCL9 and CXCL11 serum levels in GD and to relate these parameters to the clinical phenotype, we measured CXCL9 and CXCL11 serum levels in 91 GD patients; 91 AT, 34 nontoxic multinodular goiters (MNGs), 31 toxic nodular goiters (TNGs), respectively; and 91 healthy controls (age- and sex-matched). Mean CXCL9 and CXCL11 levels were higher in GD in comparison with controls, euthyroid AT, MNG, or TNG (p < 0.05, ANOVA; CXCL9: 274 ± 265, 76 ± 33, 132 ± 78, 87 ± 48, and 112 ± 56 pg/mL; CXCL11: 140 ± 92, 64 ± 20, 108 ± 48, 76 ± 33, 91 ± 41 pg/mL, respectively). Hyperthyroid GD patients had significantly higher CXCL9 or CXCL11 than euthyroid or hypothyroid GD patients. GD patients with untreated hyperthyroidism had higher CXCL9 or CXCL11 than hyperthyroid or euthyroid GD patients under methimazole (MMI) treatment. Comparable CXCL9 and CXCL11 levels were observed in newly diagnosed untreated hyperthyroid GD versus untreated patients with relapse of hyperthyroidism after a previous MMI course. Serum CXCL9 and CXCL11 levels are associated with the active phase of GD both in newly diagnosed and relapsing hyperthyroid patients. The reduction of serum CXCL9 and CXCL11 levels in treated patients with GD may be related to the immunomodulatory effects of MMI.

Chemokine (C-C Motif) Ligand 20, a Potential Biomarker for Graves' Disease, Is Regulated by Osteopontin

ContextGraves’ disease (GD) is a common autoimmune disease involving the thyroid gland. The altered balance of pro- and anti-inflammatory cytokines plays an important role in the pathogenesis of GD. Chemokine (C-C motif) ligand 20 (CCL20) is important for interleukin-17 (IL-17) signal activation and a potent chemoattractant for Th17 cells. Meanwhile, Osteopontin (OPN), a broadly expressed pleiotropic cytokine, has been implicated in GD through inducing Th1-involved response to enhance the production of proinflammatory cytokines and chemokines, but little is known about the role of OPN in regulating CCL20 and IL-17 signaling.ObjectiveThis study sought to explore the possibility of CCL20 level as a biomarker for GD, as well as investigate the role of OPN in regulating CCL20 production.MethodsFifty untreated GD patients, fifteen euthyroid GD patients, twelve TRAb-negative GD patients and thirty-five healthy control donors were recruited. OPN, CCL20 and other clinical GD diagnosis parameters were measured. CD4+T cells were isolated from peripheral blood mononuclear cells (PBMCs) using antibody-coated magnetic beads. Enzyme-linked immune-sorbent assay and quantitative polymerase chain reaction were used to determine CCL20 expression level.ResultsWe found that the plasma CCL20 level was enhanced in GD patients and decreased in euthyroid and TRAb-negative GD patients. In addition, CCL20 level correlated with GD clinical diagnostic parameters and plasma OPN level. Moreover, we demonstrated that recombinant OPN and plasma from untreated GD patients increased the expression of CCL20 in CD4+T cells, which could be blocked by OPN antibody. Furthermore, we found that the effect of OPN on CCL20 expression was mediated by β3 integrin receptor, IL-17, NF-κB and MAPK pathways.ConclusionsThese results demonstrated that CCL20 might serve as a biomarker for GD and suggested the possible role of OPN in induction of CCL20 expression.

Prevalence of echocardiographic criteria for the diagnosis of pulmonary hypertension in patients with Graves' disease: before and after antithyroid treatment.

Right-sided heart failure with clinical manifestation is only occasionally seen in patients with Graves' disease (GD). Recent studies revealed that pulmonary hypertension (PHT) detected by echocardiography was not rare in patients with GD. We performed this study to investigate the prevalence of PHT in patients with GD before and after antithyroid treatment, and to assess potential mechanisms from the relationship with clinical and echocardiographic features. Serial echocardiographic examinations were performed in 64 patients with newly diagnosed GD before and after antithyroid treatment to measure cardiac factors, such as pulmonary artery systolic pressure (PAPs), cardiac output, total vascular resistance, left ventricular filling pressure and right ventricular (RV) function. PHT was defined as PAPs of at least 35 mmHg. The prevalence of PHT in untreated GD patients was 44% (28 out of 64 patients). The presence of systemic hypertension was associated with PHT, especially with pulmonary venous hypertension. GD patients with PHT showed reduced RV function represented by higher RV myocardial performance index without difference of pulmonary vascular resistance, RV wall thickness and peak systolic velocity of free wall side of tricuspid annulus. Follow-up echocardiography was performed in 20 out of 28 GD patients with PHT, and PHT disappeared in all except one patient. PHT is a frequent and reversible complication in patients with GD. Our study suggests that PHT in GD may not be related to underlying autoimmune process and increased pulmonary blood flow from thyrotoxicosis might contributes to the pathogenesis of PHT related to GD.

The thyroid function of Graves' disease patients is aggravated by depressive personality during antithyroid drug treatment

BackgroundWe previously reported that depressive personality (the scores of hypochondriasis, depression and psychasthenia determined by the Minnesota Multiphasic Personality Inventory (MMPI)) and daily hassles of Graves' disease (GD) patients treated long trem with antithyroid drug (ATD) were significantly higher in a relapsed group than in a remitted group, even in the euthyroid state. The present study aims to examine the relationship among depressive personality, emotional stresses, thyroid function and the prognosis of hyperthyroidism in newly diagnosed GD patients.MethodsSixty-four untreated GD patients responded to the MMPI for personality traits, the Natsume's Stress Inventory for major life events, and the Hayashi's Daily Life Stress Inventory for daily life stresses before and during ATD treatment.ResultsIn the untreated thyrotoxic state, depressive personality (T-scores of hypochondriasis, depression or psychasthenia greater than 60 points in MMPI) were found for 44 patients (69%). For 15 (23%) of these patients, the scores decreased to the normal range after treatment. However, depressive personality persisted after treatment in the remaining 29 patients (46%). Normal scores before treatment were found for 20 patients (31%), and the scores were persistently normal for 15 patients (23%). The remaining 5 patients (8%) had higher depressive personality after treatment. Such depressive personality was not associated with the severity of hyperthyroidism. Serum TSH receptor antibody activity at three years after treatment was significantly (p = 0.0351) greater in the depression group than in the non- depression group. The remission rate at four years after treatment was significantly (p = 0.0305) lower in the depression group than in the non- depression group (22% vs 52%).ConclusionThe data indicate that in GD patients treated with ATD, depressive personality during treatment reflects the effect of emotional stress more than that of thyrotoxicosis and that it aggravates hyperthyroidism. Psychosomatic therapeutic approaches including antipsychiatric drugs and/or psychotherapy appears to be useful for improving the prognosis of hyperthyroidism.

A Novel Bioreporter Assay for Thyrotropin Receptor Antibodies Using a Chimeric Thyrotropin Receptor (Mc4) Is More Useful in Differentiation of Graves' Disease from Painless Thyroiditis Than Conventional Thyrotropin-Stimulating Antibody Assay Using Porcine Thyroid Cells

Graves' disease (GD) is caused by thyrotropin (TSH) receptor antibodies (TSHRAbs) that bind to TSHR and activate thyrocytes. The measurement of TSHRAbs therefore has been used to assist in the diagnosis and management of GD. In this study, we evaluated the clinical significance of a newly developed bioreporter assay for the detection of TSHRAbs (Thyretain). The Thyretain bioreporter assay utilizes a chimeric receptor (Mc4), in which residues 262-335 of TSHR are replaced with a rat lutropin-choriogonadtropin receptor segment. This bioreporter is designed to specifically detect stimulating TSHRAbs (Mc4-TSHRAbs). The Mc4-TSHRAb level of sera obtained from 110 normal healthy controls, 103, 99, and 50 patients with untreated GD, painless Hashimoto's thyroiditis (PT), and subacute thyroiditis (SAT) were 27.3% +/- 11.3%, 327.8% +/- 105.9%, 48.9% +/- 48.5%, and 24.9% +/- 13.4%, respectively. Compared with the Mc4-TSHRAb levels of patients with PT and SAT, and normal healthy controls, the Mc4-TSHRAb levels of untreated GD patients were significantly higher (p < 0.01). The sensitivity and specificity of the Thyretain bioreporter assay for GD and PT were 95.1% and 96.0%, respectively, at the optimal cut-off value of 128%. Measurement of TSHRAbs with a bioassay that uses porcine thyroid cells (TSH-stimulating antibody [TSAb]) showed a positive correlation (r = 0.472, p < 0.001) with the Thyretain assay for untreated GD, and strong positive correlation (r = 0.821, p < 0.001) for the entire untreated GD, PT, and SAT population. The positive rate of Mc4-TSHRAbs for GD was significantly higher than that of TSAb (95.1% vs. 89.3%, p < 0.05) and the negative rate of PT by Mc4-TSHRAbs was also significantly higher than that of TSAb (96.0% vs. 86.9%, p < 0.01). As a result, Mc4-TSHRAbs showed statistically better (p < 0.01) diagnostic accuracy in differentiating GD from PT than TSAb. These data suggest that the Thyretain bioreporter assay with a chimeric TSHR (Mc4) is more useful in the differential diagnosis of GD from PT than the bioassay with wild-type TSHR on porcine thyroid cells.

Changes of Serum Cytokines in Hyperthyroid Graves’ Disease Patients at Diagnosis and during Methimazole Treatment

Objective: Cytokines are involved in the pathogenesis of Graves’ disease (GD), but ambiguous serum cytokine results have been described. Methods: We studied the changes in serum interleukin (IL)-1β, soluble IL-2 receptor (sIL-2R), IL-5, IL-6 and tumor necrosis factor (TNF)-α concentrations in 29 untreated GD patients before and after restoration of euthyroidism with methimazole (MMI) treatment compared to 25 control subjects. Eleven out of 29 GD patients had active Graves’ ophthalmopathy (GO). Results: Compared to controls, untreated GD patients had significantly higher median levels of serum IL-1β (18.7 vs. 34.0 pg/ml), sIL-2R (292.5 vs. 1,585.0 pg/ml), IL-5 (1.0 vs. 9.0 pg/ml), IL-6 (3.0 vs. 5.0 pg/ml) and TNF-α (8.1 vs. 16.0 pg/ml). In euthyroidism following MMI treatment, concentrations of IL-1β (25.0 pg/ml), sIL-2R (362.0 pg/ml), IL-5 (3.0 pg/ml), IL-6 (3.0 pg/ml) and TNF-α (5.0 pg/ml) declined significantly and were similar to controls. The greatest reductions were noted in sIL-2R (76.9%), TNF-α (68.8%) and IL-5 (66.6%) levels. Serum sIL-2R, IL-5 and TNF-α levels in active GO patients were significantly elevated, but no significant differences were observed in GD patients without GO. Using a multiple linear regression analysis, serum IL-1β was significantly associated with free thyroxine, sIL-2R with triiodothyronine and serum thyrotropin receptor antibody (TRAb) and TNF-α with TRAb. Conclusion: These results support the notion that serum cytokines could be used as a marker of GD activity, and the decrease in cytokine levels might be related to the achievement of euthyroidism and the immunomodulatory effects of MMI treatment.

Serum Levels of Angiogenic Molecules in Autoimmune Thyroid Diseases and Their Correlation with Laboratory and Clinical Features

Because angiogenesis has a role in the pathogenesis of inflammatory conditions, we studied angiogenesis soluble markers in autoimmune thyroid diseases. The aim of the study was to measure concentrations of angiopoietins, Tie-2, and vascular endothelial growth factor in sera from autoimmune thyroid disease patients. Soluble Tie-2 (sTie-2), angiopoietin-1, angiopoietin-2, and vascular endothelial growth factor were quantified by ELISA in sera from 44 untreated Graves' disease (GD) patients, 25 untreated Hashimoto's thyroiditis (HT) patients, 13 non-GD hyperthyroid patients, and 22 age-matched controls. Subgroups of patients with active and non-active Graves' ophthalmopathy (GO) were analyzed. Correlations among these markers and clinical parameters were assessed by bivariate and multivariate analyses. STIE-2 levels were higher in GD or HT patients compared to controls (P < 0.01). In addition, serum Ang-2 concentrations were higher in untreated GD patients compared to controls, HT patients, or non-GD hyperthyroid patients (P < 0.01), and no difference was observed between HT patients and controls. Significant correlations were found between free T(4)/sTie-2 and free T(4)/Ang-2 levels (r = 0.464, P < 0.01; and r = 0.463, P < 0.01, respectively) as well as between sTie-2/anti-TSH receptor antibody (r = 0.527; P < 0.01) and sTie-2/Ang-2 (r = 0.563; P = 0.001). Furthermore, sTie-2 levels were significantly higher in patients with active GO when compared to those with inactive GO (P < 0.05). Interestingly, Ang-2 levels decreased significantly after treatment with antithyroid drugs (P < 0.01). Ang-2 and sTie-2 could participate in the pathogenesis of GD and potentially be used as markers of GO activity. Antithyroid drugs affect the angiogenic pattern in GD.

Intermethod Variability in TSH-Receptor Antibody Measurement: Implication for the Diagnosis of Graves Disease and for the Follow-Up of Graves Ophthalmopathy

We compared the analytical and clinical performance of 3 porcine thyroid receptor antibody (TRAb) methods (1 second- and 2 new third-generation systems) with the conventional TRAb assay based on the human recombinant TSH receptor (hTRAK). We obtained sera from 86 patients with untreated Graves disease (GD) and 71 healthy controls. We measured TRAb concentrations by radioreceptor assay using the hTRAK (Brahms) or the porcine TSH receptor (pRRA) from Beckman-Coulter, by electrochemiluminescence immunoassay (ECLIA) with the Elecsys/Cobas (Roche), and by ELISA using the Medizym TRAb clone (Medipan). Between-run assay imprecision was < or =10% and < or =7.6% for hTRAK and ECLIA, but reached 14% and 14.9% for ELISA and pRRA, respectively. Maximal specificity and sensitivity close to 100% were obtained for hTRAK, ECLIA, and ELISA. pRRA failed to detect positive TRAbs in 5 GD patients. Although calibrated against the same reference standard 90/672, the assays displayed a high intermethod variability. The results were significantly higher by ECLIA and lower by ELISA and pRRA compared with hTRAK. Patients with ophthalmopathy had higher TRAb results by ELISA and pRRA than those without eye disease. Second- and third-generation TRAb assays had similar diagnostic sensitivities in the diagnostic evaluation of GD. Despite the use of the same reference standard for calibration, high intermethod variability in TRAb assay results was seen in untreated GD patients. Assay harmonization is necessary for correct interpretation in the follow-up of Graves ophthalmopathy.

L‐selectin gene polymorphisms in Graves' disease

Graves' disease (GD) is an autoimmune disorder with genetic predisposition. There is strong evidence that members of the selectin family participate in the interaction between leucocytes and the endothelium, as well as in inflammatory cell recruitment. Moreover, a high serum level of a soluble form of L-selectin (SL-selectin) has been reported in untreated GD patients. However, the impact of L-selectin polymorphisms on GD has not yet been investigated. The aim of the present study was to elucidate whether L-selectin gene polymorphisms were associated with the development of GD. L-selectin gene polymorphisms were investigated in 230 Chinese GD patients and 208 healthy control subjects without antithyroid autoantibodies or a family history of autoimmune disorders. Two L-selectin SNPs were genotyped by the PCR-restriction fragment length polymorphism (PCR-RFLP) method. A C/T polymorphism at position -642 of the promoter region and a Pro213Ser (c.725 C-T) polymorphism in exon 6 were examined using PCR-RFLP. There was a significant increase in -642T allele frequency in GD patients compared with healthy controls (70 vs. 62%; P = 0.0126; P(c) = 0.0252). The frequency of the c.725C allele in exon 6 also appeared higher in GD patients than in controls. Haplotype analysis showed a significant decrease in the -642C/c.725T haplotype in GD patients (26 vs. 34%; P = 0.0095; Pc = 0.0190). However there was no association between polymorphisms and certain GD clinical phenotypes, including age of onset and ophthalmopathy. L-selectin gene polymorphisms are associated with GD susceptibility in Chinese patients.

Significance of thyroid blood flow as a predictor of methimazole sensitivity in untreated hyperthyroid patients with Graves' disease

Objective The peak systolic velocity (PSV) of the inferior thyroid artery (ITA) is increased in untreated hyperthyroid patients with Graves' disease (GD). We investigated the clinical significance of the ITA-PSV and its determinants in hyperthyroid GD patients. Patients and methods ITA-PSV, together with thyroid volume, was measured by ultrasonography in untreated hyperthyroid GD patients ( n = 49) and healthy subjects ( n = 22). Established markers of GD activity such as TSH receptor antibody (TRAb), thyroid stimulating antibody (TSAb), vascular endothelial growth factor (VEGF) and immunoglobulin E (IgE) were simultaneously determined. Results ITA-PSV, thyroid volume, VEGF and IgE were significantly higher in hyperthyroid GD patients than in normal subjects. ITA-PSV in hyperthyroid GD patients was correlated positively with serum levels of FT 3, FT 4 and IgE, smoking index and thyroid volume, and negatively with total, HDL- and LDL-cholesterols, but did not correlate significantly with age, triglyceride, TRAb, TSAb or VEGF. In stepwise regression analysis, ITA-PSV showed significant positive and negative associations with IgE and LDL-cholesterol, respectively, in hyperthyroid GD patients. In the pre-treatment hyperthyroid state, FT 4 and ITA-PSV, but not IgE, were found to be significantly and positively associated with the maintenance dose of methimazole (MMI) required to keep serum TSH within normal range for at least 12 months. Conclusion These results suggest that ITA-PSV in untreated hyperthyroid GD patients may reflect GD activity and thus MMI sensitivity.

A bioluminescence assay for thyrotropin receptor antibodies predicts serum thyroid hormone levels in patients with de novo Graves’ disease

TSH receptor antibodies (TBII) in Graves' disease (GD) may be TSH receptor stimulating (TSAb) and blocking (TBAb) antibodies. In commercially available assays however, only total TBII titres can be measured, without discriminating between TSAb and TBAb. To design a TBII bioassay to detect of TSAb and to correlate TSAb activity with severity of hyperthyroidism in de novo GD patients. Thirty-five patients with de novo GD and 27 controls. The JP-26-26 cell line, which constitutively expresses the TSH receptor (TSHR), was stably transfected with a cyclic adenosine monophosphate responsive element--luciferase construct. The clone B1 exhibited a near linear increase in luminescence from 0.2 mU/l to 50 mU/l bovine TSH and was used as a TBII bioassay. TBII, free T4 and TSH were measured in the sera of all patients and controls. In the sera of 35 GD patients, TBII titres did not correlate with serum free T4 concentrations. In contrast, a strong and highly significant correlation was found between TSHR stimulating activity (luminescence) as measured with the TBII bioassay and serum free T4 levels (R = 0.80, P < 0.001). Interestingly, the luminescence/TBII ratio had a wide range at low TBII titres, whereas high TBII titres were associated with a low degree of TSHR activation. The TBII bioassay also detected TBAb in GD patients who spontaneously developed hypothyroidism. The B1-TBII-bioassay as developed in our laboratory has a high sensitivity for the detection of TSAb in GD and predicts the severity of hyperthyroidism in untreated GD patients. In addition, we found that high TBII titres are associated with weak TSHR activation.