Usefulness of the 2nd generation assay for anti-TSH receptor antibodies to differentiate relapse of Graves' thyrotoxicosis from development of painless thyroiditis after antithyroid drug treatment for Graves' disease

After antithyroid drug (ATD) treatment for Graves' disease, either a relapse of Graves' thyrotoxicosis or painless thyroiditis can develop. It is important to differentiate these two types of thyrotoxicosis because of the difference in required therapy. However, differentiation of thyrotoxicosis is usually difficult without radioactive iodine uptake (RAIU) which is not available in general practice. We investigated the clinical usefulness of the 2nd generation assay for anti-TSH receptor antibodies (TRAb) to differentiate these two types of thyrotoxicosis after ATD treatment for Graves' disease. We recruited 26 patients who developed thyrotoxicosis after ATD treatment for Graves' disease. These patients once became negative for TRAb and seemed to be in remission after ATD treatment. Upon development of thyrotoxicosis after ATD treatment, TSH, free T4, free T3 and TRAb were measured. TRAb were measured by the 2nd generation assay using recombinant human TSH receptors instead of porcine TSH receptors. Fourteen patients relapsed into Graves' thyrotoxicosis and 12 patients developed painless thyroiditis. Twelve (85.7%) of 14 patients with relapse of Graves' thyrotoxicosis were positive for TRAb. Eleven (91.7%) of 12 patients with development of painless thyroiditis after ATD treatment for Graves' disease were negative for TRAb. Levels of TRAb were significantly different between patients with relapse of Graves' thyrotoxicosis (4.86 +/- 6.45 IU/L) and those with painless thyroiditis (0.62 +/- 0.61 IU/L) (P<0.001). The 2nd generation assay for TRAb was useful to differentiate relapse of Graves' thyrotoxicosis from development of painless thyroiditis in patients who seemed to be in remission after ATD treatment for Graves' disease.     

Sensitive thyrotropin and thyrotropin-receptor antibody determinations one month after discontinuation of antithyroid drug treatment as predictors of relapse in Graves' disease.

OBJECTIVE: After primary successful antithyroid drug treatment (ATDT), Graves' disease has a relapse rate of 30% to 50%. Previous studies have evaluated age, gender, goiter volume, smoking habits, and the presence of thyrotropin-receptor antibodies (TRAb) as predictive markers to facilitate an individualized patient management. Despite higher sensitivity and specificity of the new second generation human TSH-receptor assay, the predictive value of TRAb for relapse of hyperthyroidism is still controversial. In a recent prospective multicenter study we have previously shown that suppressed or low TSH values predict both early (persistence) and late relapse of Graves' disease. We now present a more detailed analysis of the predictive value of TSH and TRAb for recurrent hyperthyroidism. METHODS: Four weeks after withdrawal of ATDT, 96 patients were available for thyroid function tests, including a sensitive third-generation TSH assay and a second-generation recombinant TSH receptor assay. Relapse of Graves' disease was evaluated for a total follow-up of 2 years. RESULTS: Within 2 years, 47 of 96 patients (49%) developed relapse of hyperthyroidism. Nine patients relapsed within the first 4 weeks after withdrawal of ATDT and were thus considered to have persistent Graves' disease. Ten of 15 other patients with TSH levels below 0.3 mU/L without overt hyperthyroidism relapsed within 2 years. Twenty-five of 65 patients with normal TSH (0.3-3.0 mU/L) and 3 of 4 patients with TSH values above 3 mU/L also had recurrent hyperthyroidism. After ATDT cessation, TSH had a positive predictive value of 70% and a negative predictive value of 62% (specificity 85%) for relapse of Graves 'disease. Mean TRAb levels in the group of patients with relapse were significantly higher (11.1 IU/L +/- 0.17) than TRAb values in the remission group (4.5 IU/L +/- 0.6), p < 0.001. Using a cutoff value of 1.5 IU/L, TRAb had low positive and negative predictive values of 49% and 54%, respectively (specificity, 14%), but with a cutoff level of 10 IU/L, predictive values improved to 83% and 62%, respectively (specificity, 92%). Combination of TSH and TRAb determinations did not further improve prediction of relapse. Other factors such as gender, age, goiter volume, smoking habits, presence of thyroid-associated ophthalmopathy, and urinary iodine excretion did not show a significant influence on relapse rate. CONCLUSION: Low TSH values 4 weeks after ATDT withdrawal predict relapse of Graves' disease, both early (persistence) and, to a lesser extend, within 2 years of follow-up. Also, TRAb above 10 IU/L found in a small subset of patients, correlated with a higher relapse rate.     

Clinical value of the first automated TSH receptor autoantibody assay for the diagnosis of Graves' disease (GD): an international multicentre trial

Background  Most recently, a new rapid and fully automated electrochemiluminescence immunoassay for the determination of TSH receptor autoantibodies (TRAb) based on the ability of TRAb to inhibit the binding of a human thyroid-stimulating monoclonal antibody (M22) has been established.
Objective  To evaluate this assay system in clinical routine based on an international multicentre trial and to compare the results with other established TRAb assays.
Patients and measurements  Totally 508 Graves' disease (GD), 142 autoimmune thyroiditis, 107 subacute thyroiditis, 109 nonautoimmune nodular goitre, 23 thyroid cancer patients and 446 normal controls were retrospectively evaluated.
Results  ROC plot analysis revealed an area under curve of 0·99 (95% CI: 0·99–1·0) indicating a high assay sensitivity and specificity. The highest sensitivity (99%) and specificity (99%) was seen at a cut-off level of 1·75 IU/l. Here, the calculated positive predictive value was 95%, whereas the negative predictive value was 100%. Applying the ROC plot-derived cut-off of 1·75 IU/l we found a sensitivity for TRAb positivity within the group of newly diagnosed GD patients of 97% which is in accordance to the sum of different nonautomated porcine TSH receptor-based assays with a sensitivity of 94% indicating an excellent analytical performance of the new assay format. Detailed comparison of the automated and the sum of manual assays revealed a near identical specificity.
Conclusion  Our results demonstrate that this new assay system has a high sensitivity for detecting GD and specificity for discriminating from other thyroid diseases. This assay may represent the future technology for rapid fully automated TRAb detection.     

Measuring TSH receptor antibody to influence treatment choices in Graves’ disease

TSH receptor antibody (TRAb) plays a key role in the pathogenesis of Graves’ disease (GD), and its levels correlate with the clinical course. The second‐ and third‐generation TRAb assays have >95% sensitivity and specificity for the diagnosis of GD and have improved the utility of TRAb to predict relapse. TRAb levels decline with antithyroid drug (ATD) therapy and after thyroidectomy. Its level increases for a year following radioactive iodine (RAI) therapy, with a gradual fall thereafter. TRAb level >12 IU/l at diagnosis of GD is associated with 60% risk of relapse at 2 years and 84% at 4 years. The prediction of risk of relapse improves further to >90% with TRAb >7·5 IU/l at 12 months or >3·85 IU/l at cessation of ATD therapy. TRAb tests are not expensive, and hence, TRAb measurements at presentation, after 12 months and/or 18 months (at cessation) of ATD therapy, could potentially guide treatment choices in GD. Elevated TRAb favours definitive treatment in the form of RAI or thyroidectomy, depending on the presence or absence of moderate‐to‐severe Graves’ ophthalmopathy (GO) and the ability to comply with radiation protection requirements. Use of ATDs in early pregnancy is associated with increased risk of congenital anomalies; early ablative treatment (RAI/surgery) should be considered in women of childbearing age at higher risk of relapse of GD. TRAb ≥5 IU/l in pregnant women with current or previously treated GD is associated with increased risk of foetal and neonatal thyrotoxicosis, and hence needs close monitoring. TRAb levels parallel the course of GO, and elevated TRAb is an indication for steroid prophylaxis to prevent progression of GO with RAI therapy.     

TSH-receptor antibody measurement for differentiation of hyperthyroidism into Graves' disease and multinodular toxic goitre: a comparison of two competitive binding assays

OBJECTIVES: Graves' disease is characterized by stimulating autoantibodies to the TSH-receptor (TRAb). The aim of this study was to compare the performance of a new TRAb assay based on competitive binding to recombinant human TSH-receptors (H-TRAb) with an assay employing purified porcine TSH-receptors (P-TRAb). Furthermore, to evaluate the applicability of the H-TRAb assay to discriminate between patients with hyperthyroidism due to Graves' disease (GD) and multinodular toxic goitre (MNTG). DESIGN AND MEASUREMENTS: H-TRAb and P-TRAb were measured in patients with newly diagnosed hyperthyroidism due to GD (n = 106) and MNTG (n = 94). For comparison, TRAb was measured in patients with primary autoimmune hypothyroidism, euthyroid subjects with an enlarged thyroid gland by ultrasound, and healthy controls (n = 100 for each group). Patients were consecutively included from a population survey. RESULTS: If the cut-off values recommended by the manufacturer for TSH-receptor antibody positivity were used for evaluation, the sensitivity of the H-TRAb assay vs. the P-TRAb assay in diagnosing GD was: 95.3/67.9% (P < 0.001). Specificity was (H/P-TRAb): 99/99%. The sensitivity of P-TRAb was increased if the upper 97.5% limit of measurements in controls was used as cut-off (H-TRAb vs. P-TRAb: 95.3/80.2%, P < 0.001). Specificity (H/P-TRAb): 98/98%. The difference between assay performance may partly be due to a better technical performance of the H-TRAb assay with more reliable results in the low range of measurements. However, even in GD patients with clearly measurable TRAb, 25% had a P-TRAb < 50% of the value expected from the H-TRAb measurement. This suggests that a subgroup of patients produce TRAb with a higher affinity for the human than the porcine TSH receptor. A relatively high proportion of patients with MNTG were TRAb positive (H-TRAb/P-TRAb: 17/9%). Characteristics of H-TRAb positive and negative MNTG patients were compared. There was no difference between size of thyroid gland and number of nodules by ultrasonography. H-TRAb positive patients had significantly higher serum T4 and T3 and a greater number were TPO-Ab positive. CONCLUSIONS: H-TRAb diagnosed Graves' disease with a high sensitivity and specificity than P-TRAb. The high occurrence of TRAb in multinodular toxic goitre might in part reflect an overlap between Graves' disease and multinodular toxic goitre in some patients.     

TSH-receptor antibodies determined by the first, second and third generation assays and thyroid-stimulating antibody in pregnant patients with Graves' disease

The measurement of TSH receptor antibody (TRAb) has been recommended to predict the risk of neonatal hyperthyroidism (NH) in pregnant women with Graves' disease (GD). For the first generation TRAb (TRAb1) assay with commercial kit (Brahms, Berlin, Germany; or Cosmic co., Tokyo, Japan) an arbitrary limit of 40 U/l or 50% was suggested to indicate risk when measured late in pregnancy. In order to substitute TRAb1 with the second generation TRAb using porcine TSH receptor (pTRAb2) and human recombinant TSH receptor (hTRAb2) and the third generation TRAb (TRAb3) assay for this purpose, we measured TRAb in these four methods late in pregnancy in a total of 62 pregnant women with Graves' disease. The data showed that no cases with TRAb1 >50% has been missed if the TRAb1 assay was replaced by the pTRAb2, hTRAb2 or TRAb3 assay using their equivalent cut-off value of 70%, 10 IU/l, and 75%, respectively, but that an additional group of women would have been included in the risk group, especially in the TRAb3 assay. Next, the effect of maternal TRAb on thyroid function of offspring was studied in the 47 pregnant women with GD (43 with TRAb1 <50% and 4 with TRAb1 >50% during late pregnancy). In 2 women who gave birth to hyperthyroid children at days 6 and 14 of life, the maternal sera had strongly positive levels of TRAb1 (73.5% and 84.1%), pTRAb2 (84.9% and 91.5%), hTRAb2 (40.68 IU/L and 89.70 IU/L) and TRAb3 (92.1% and 93.5%) late in pregnancy, with one case displaying high positive (1114.3%) thyroid stimulating antibody (TSAb) level and the other case had moderate positive (433%) TSAb level. Of the remaining 45 women, 43 had TRAb1 <50% and the other 2 had TRAb >50% including 1 with low TSAb positive and 1 with positive thyroid stimulating blocking antibody (TSBAb) and negative TSAb; all of them gave birth to euthyroid children. Finally, a serial study regarding TRAb in 23 women with Graves' disease during pregnancy showed that TRAb1, pTRAb2, hTRAb2, TRAb3 value and TSAb level decreased significantly as pregnancy progressed. In conclusion, the present study supported TRAb as a useful marker to predict the risk of NH.     

The significance of thyroid blood flow at the inferior thyroid artery as a predictor for early Graves' disease relapse

OBJECTIVE: We investigated the clinical usefulness of thyroid blood-flow measurement in predicting relapse of Graves' disease (GD) in comparison with known risk factors for GD relapse. MEASUREMENT: Thyroid blood flow was measured in pulsed Doppler mode at the inferior thyroid artery (ITA), and the peak systolic velocity (PSV) calculated. PATIENTS: ITA-PSV was measured in euthyroid GD patients (n = 79) immediately before withdrawal of anti-thyroid drug (ATD) and in healthy subjects (n = 17). RESULTS: In the 79 euthyroid GD patients, the values of free triiodothyronine (FT3), TSH receptor autoantibody (TRAb), ITA-PSV and thyroid volume were significantly higher in the relapse group (n = 40) than in the nonrelapse group (n = 39) and the Youden index of ITA-PSV was significantly higher than that of FT3, TSH, TRAb and vascular endothelial growth factor (VEGF). CONCLUSION: ITA-PSV may assist in the prediction of early GD relapse after ATD withdrawal.     

促甲状腺激素受体抗体放射受体分析法的建立及其临床应用

用0.5％ Triton X-100一次增溶猪甲状腺细胞膜,用固相Iodogen法标记bTSH再经受体亲合纯化获得了高比生物活性的可溶性TSH受体和~(125)I-bTSH,建立了TRAb放射受体分析法。制备的TSH受体和~(125)I-bTSH的结合可被非标记bTSH和Graves病患者血清中的TRAb呈剂量依赖性抑制。TRAb平均抑制率分别为15.4％和90.6％时,批间变异系数相应为12.6％和3.1％。试验和临床应用结果表明,本方法灵敏度高、特异性强、重复性好,且操作简便、快速,适于临床常规应用。     

Remission of Graves' hyperthyroidism and A/G polymorphism at position 49 in exon 1 of cytotoxic T lymphocyte-associated molecule-4 gene

We studied whether a patient with Graves' disease will go into remission during antithyroid drug (ATD) treatment. Remission of Graves' hyperthyroidism is predicted by a smooth decrease in TSH receptor antibody (TRAb) during ATD treatment. Cytotoxic T cell lymphocyte-associated molecule-4 (CTLA-4) may play an important role in the development of Graves' hyperthyroidism and in its remission. We studied A/G polymorphism at position 49 in exon 1 of the CTLA-4 gene in 144 Japanese Graves' patients. We intended to reveal the possible association of CTLA-4 gene polymorphism with the remission of Graves' hyperthyroidism. All patients with Graves' disease were treated with ATD. Thyroid-stimulating antibody and TSH binding inhibitory Ig were measured as TRAb. We analyzed CTLA-4 genotypes and alleles with PCR. We calculated the frequencies of CTLA-4 genotypes and alleles. A significant increase in the frequency of the G allele was seen in Graves' patients compared with controls (P = 0.0095). Graves' patients were divided into three groups (A, B, and C) according to time of TRAb disappearance after the start of ATD treatment. In group A patients TRAb had disappeared within 1 yr after the start of ATD treatment, in group B TRAb had disappeared between the beginning of the second year and the end of the fifth year of treatment, and in group C TRAb continued to be positive after 5 yr of ATD treatment. The frequencies of the GG genotype and the G allele were significantly higher in group C patients with persistently positive TRAb over 5 yr of ATD treatment than in the other groups (P < 0.0001). Group C patients did not have the AA genotype. The periods of time until remission were significantly shorter in the AA genotype. Graves' patients with the G allele need to continue ATD treatment for longer periods.     

Impact of smoking on the course of Graves' disease after withdrawal of antithyroid drugs.

Cigarette smoking has been reported to alter relapse rate in patients with Graves' disease (GD). However, the predictive effect of smoking in GD patients after withdrawal of antithyroid drug treatment (ATDT) is still controversial. A prospective multicenter trial has previously identified smoking as an independent risk factor for relapse. Based on this study, the present paper gives a more detailed analysis of the impact of smoking on the long-term course of GD after ATDT withdrawal. To this end, 86 smokers and 177 non-smokers were followed during two years after ATDT cessation. At the end of ATDT (visit 1) and four weeks later (visit 2) smokers had significant higher TSH receptor antibody (TRAb) levels than non-smokers (10.0 IU/L+/-1.6; mean+/-SEM vs. 6.4 IU/L+/-0.9; 11.0 IU/L+/-1.8 vs. 6.8 IU/L+/-0.8, p < 0.01, respectively). During follow-up, Kaplan Meier analysis showed a significantly higher relapse rate in smokers than non-smokers. A subset of GD patients with TRAb levels >10 IU/L had the highest risk to develop relapse during follow-up. Among them, smokers more often relapsed than non-smokers irrespective of TRAb levels, p < 0.01. Thus, in smokers with TRAb levels > or =10 IU/L the predictive values of a positive and negative test for relapse was 68% and 73%, respectively (specificity 95%). In conclusion, we identified two effects by which smoking alters the course of GD. First, smoking is implicated to elevate TRAb levels and therefore increase the risk for relapse during follow-up. Second, smoking is an independent risk factor to worsen the clinical course of both, GD patients with low and high immunological risk to experience relapse after a successful outcome of ATDT. Thus, our data suggest that smoking has modifying immunological consequences and an adverse impact on the course of GD after withdrawal of ATDT. Therefore, patients should be encouraged to stop smoking.     

TSH receptor antibody measurements and prediction of remission in Graves' disease patients treated with minimum maintenance doses of antithyroid drugs

Prediction of remission is one of the main problems of antithyroid drug (ATD) therapy for Graves' disease especially in patients who are treated with a minimum maintenance dose of ATD. We evaluated the ability of new sensitive TSH receptor antibody (TRAb) assays to predict remission in Graves' patients using two commercially available kits (TRAb-CT from Cosmic Corporation and TRAb-Dyno from Yamasa Corporation), compared to the original PEG assay. When a euthyroid state was achieved for more than 6 months with methimazole 5 mg/day or propylthiouracil 50 mg/day and thereafter for three months with 5 mg every other day or 50 mg every other day, respectively, we discontinued ATD medication. One year of observation after discontinuation of ATD was completed in 71 patients (60 females, median age 43 years, range 18-71), and TRAb values from these patients were analyzed in relation to prognosis. Twenty-six (37%) of the 71 patients had relapse of thyrotoxicosis and 45 remained euthyroid. The median TRAb levels in the relapse group were significantly higher than those in the remission group (P < 0.05). Relapse occurred in 15/51 patients negative by TRAb-CT, in 11/20 patients positive by TRAb-CT (chi2 = 4.1; P < 0.05), in 11/42 patients negative by TRAb-Dyno and in 15/29 patients positive by TRAb-Dyno (chi2 = 4.8; P < 0.05). By contrast, relapse occurred in 23/64 patients with negative TRAb by PEG assay and in 3/7 patients with PEG assay positive values (n.s.). All patients with TRAb-CT values of 30% inhibition or greater, or TRAb-Dyno values of 3.0 U/L or greater relapsed during the observation period. Thus, measurement of TRAb by the new sensitive assays is useful for prediction of remission in our patients.     

促甲状腺激素受体抗体检测的临床意义

50年前人们发现了促甲状腺激素受体抗体(TRAb),随着对促甲状腺激素受体(TSHR)结构和功能认识的不断更新,加之TSHR信号转导和与TRAb相互作用的逐步阐明,人们对TRAb及其临床应用的认识得到了进一步提高.TRAb的检测在Graves病(GD)及Graves眼病的诊断中有重要作用,并有效预测GD经抗甲状腺药物或放射性碘治疗后复发.其亦可应用于近期服碘的孕妇和乳母,因为甲状腺扫描对她们来说是禁忌的.另外,TRAb有助于胎儿、新生儿甲状腺功能亢进及其他类型的甲状腺毒症的诊断和鉴别诊断.目前,已有文献报道TRAb阳性的GD患者发生甲状腺肿瘤及不良预后之间的可能联系,但尚需更多的前瞻性研究来证实.

Abstract：

It has been 50 years since the discovery of thyrotropin receptor autoantibody (TRAb). Advances in the knowledge of thyrotropin receptor ( TSHR) structure and function, combined with the elucidation of TSHR signaling and TSHR-autoantibody interaction have greatly facilitated our understanding of TRAb and their clinical applications. Measurement of TRAb activity plays an important role in the diagnosis of Graves' disease ( GD) and Graves' opthalmopathy. It has also been well recognized that TRAb is an effective predictor of GD relapse or remission after antithyroid drug and radioactive iodine treatment. TRAb test is of particular help in pregnant women and lactating mothers with recent iodine load, where radioactive iodine or technetium tests are contraindicated. In addition, it is useful in the diagnosis and differential diagnosis of fetal and neonatal hyperthyroidism as well as some rare forms of thyrotoxicosis in clinical practice. Accumulating evidence also indicates the possible correlation between thyroid cancer occurring in GD patients with positive TRAb and adverse outcomes. However, further innovation and standardization of TRAb tests are required to help pave the way for clinical applications.     

促甲状腺激素受体抗体检测的临床意义

50年前人们发现了促甲状腺激素受体抗体(TRAb),随着对促甲状腺激素受体(TSHR)结构和功能认识的不断更新,加之TSHR信号转导和与TRAb相互作用的逐步阐明,人们对TRAb及其临床应用的认识得到了进一步提高.TRAb的检测在Graves病(GD)及Graves眼病的诊断中有重要作用,并有效预测GD经抗甲状腺药物或放射性碘治疗后复发.其亦可应用于近期服碘的孕妇和乳母,因为甲状腺扫描对她们来说是禁忌的.另外,TRAb有助于胎儿、新生儿甲状腺功能亢进及其他类型的甲状腺毒症的诊断和鉴别诊断.目前,已有文献报道TRAb阳性的GD患者发生甲状腺肿瘤及不良预后之间的可能联系,但尚需更多的前瞻性研究来证实.     

促甲状腺激素受体抗体检测的临床意义

50年前人们发现了促甲状腺激素受体抗体(TRAb),随着对促甲状腺激素受体(TSHR)结构和功能认识的不断更新,加之TSHR信号转导和与TRAb相互作用的逐步阐明,人们对TRAb及其临床应用的认识得到了进一步提高.TRAb的检测在Graves病(GD)及Graves眼病的诊断中有重要作用,并有效预测GD经抗甲状腺药物或放射性碘治疗后复发.其亦可应用于近期服碘的孕妇和乳母,因为甲状腺扫描对她们来说是禁忌的.另外,TRAb有助于胎儿、新生儿甲状腺功能亢进及其他类型的甲状腺毒症的诊断和鉴别诊断.目前,已有文献报道TRAb阳性的GD患者发生甲状腺肿瘤及不良预后之间的可能联系,但尚需更多的前瞻性研究来证实.     

Autoantibodies to the thyrotropin receptor

This review considers recent developments in our understanding of the properties of TRAb, particularly measurement of the antibodies and their sites of action and synthesis. Two new assay methods have allowed considerable improvements in the sensitivity, specificity, precision, and ease of measuring TRAb. In particular: 1) receptor assays based on inhibition of receptor-purified labeled TSH binding to detergent-solubilized TSH receptors and 2) bioassays based on stimulation of cAMP release from monolayer cultures of isolated thyroid cells. Detailed studies with the two assays indicate that TSH receptor antibodies nearly always act as TSH agonists in patients with a history of Graves' hyperthyroidism. Studies in areas of dietary iodine sufficiency suggest that measurement of the antibodies at various stages in the course of treating Graves' disease can be of value in predicting the outcome of therapy. However, in areas of iodine deficiency, difficulties in the ability of patients' thyroid tissue to recover from the effects of antithyroid drugs may prevent the receptor antibodies from causing a relapse of thyrotoxicosis. Consequently, the predictive value of receptor antibody measurements would be expected to be lower in these geographical areas. Although patients with a history of Graves' hyperthyroidism nearly always have TRAb which act as TSH agonists, about 20% of patients with frank hypothyroidism due to autoimmune destruction of the thyroid have TRAb which act as TSH antagonists (blocking antibodies). There is some evidence that these blocking antibodies can cause hypothyroidism particularly in the neonate. With regard to the site of synthesis of TRAb, there is now direct evidence that they are synthesized by thyroid lymphocytes, particularly the lymphocytes in close proximity to thyroid follicular cells. This is consistent with the well established effects of antithyroid treatment (drugs, radioiodine, or surgery) on TRAb levels in addition to their effects on thyroid hormone synthesis. Recent studies using affinity labeling with 125I-labeled TSH have enabled elucidation of the structure of the TSH receptor. TSH receptors in human, porcine, and guinea pig thyroid tissue have a two-chain structure in which the TSH binding site is formed on the outside surface of the cell membrane by a water-soluble A subunit (Mr approximately 50 K). The A subunit is linked by a disulfide bridge and weak noncovalent bonds to the amphiphilic B subunit (Mr approximately 30 K). This subunit, which penetrates the lipid bilayer, probably forms the site for interaction of the receptor with the regulatory subunits of adenylate cyclase.(ABSTRACT TRUNCATED AT 400 WORDS)     

Affinity purification and diagnostic use of TSH receptor autoantibodies from human serum

Purification of TSH receptor autoantibodies (TRAb) from the serum of patients with Graves' disease (GD) might help to elucidate the nature of these disease causing autoantibodies. We describe here for the first time the successful affinity purification of human TRAb.Affinity purification was performed in a four step procedure with human recombinant TSH receptor (TSH-R) expressed in K562 cells. Purification from six different serum pools from patients with GD and two individual sera (one with only thyroid stimulating antibodies (TSAb) one with only thyroid blocking antibodies (TBAb)) resulted in a purity of 39.2+/-3.8 IU/mg TRAb or 25.7+/-2.1 microg IgG/IU (about 3.5-13.7 microg TRAb/ml serum). The average enrichment based on the respective original serum was 3420-fold (range 1200-10,000). The kDa of the purified TRAb were in the range of 0.7-2.6 x 10(-10)M. All purified TRAb (except from the TBAb serum which showed blocking activity) showed a more than 1000-fold stronger stimulation in the TSAb bioassay based on the IgG content than the original serum, and similar stimulation based on international units (IU/l) TRAb. When labelled purified TRAb were used in a competitive assay as tracer instead of bovine TSH, their binding to the human recombinant TSH-R on tubes was displaced by 99 of 100 GD sera (selected for TBII activity). Correlation to the standard TSH tracer was r=0.92. Interestingly, the use of TRAb tracer derived from a patient with TSAb and a patient with TBAb gave virtually identical results (r=0.93) with these patients, suggesting similar if not identical binding sites for both TRAb subtypes.In conclusion, this is the first report on the purification of human TRAb from the serum of patients with GD. The purified TRAb are of low concentration with high affinity, strong TBII and TSAb activity. Further characterisation may allow new insights in TRAb epitope localisation, the pathology of GD and the differences between TSAb and TBAb. Also, their use as tracer in a competitive assay is the first report on a completely homogenous assay with high sensitivity for TSH-R autoantibodies.     

The incidence of gestational hyperthyroidism and postpartum thyroiditis in treated patients with Graves' disease.

Graves' disease (GD) is one of the most common thyroid diseases that cause hyperthyroidism. Gestational transient thyrotoxicosis (GTT) is nonautoimmune hyperthyroidism that occurs in women with a normal pregnancy. Postpartum transient thyroiditis (PTT) is a destructive thyroiditis induced by autoimmune mechanism in the postpartum period. Hyperthyroidism due to GD usually tends to improve during the course of gestation and exacerbate after delivery. When the patient with treated GD presents with thyrotoxicosis in the early pregnancy or in the postpartum period, differential diagnosis of exacerbation of GD with GTT or PTT is important because the latter disorders are fundamentally transient. To evaluate the incidence of GTT and PTT in a GD population, we investigated the thyroid functions, thyrotropin receptor antibodies (TRAb), and human chorionic gonadotropin (hCG) during pregnancy and for 1 year after delivery for 39 pregnancies in 34 women with GD. The incidence of GTT was 26% (10/39) of pregnancies. The peak value of hCG in the GTT group ([23.7 +/- 14.5] x 10(4) IU/mL, n = 9) was significantly higher than that in the non-GTT group ([13.3 +/- 4.7] x 10(4) IU/mL, n = 19). The incidence of PTT was 44% (17/39) of deliveries. The free triiodothyronine (FT(3))/free thyroxine (FT(4)) ratio of the exacerbation group of GD (3.1 +/- 1.0, n = 10) at the time of thyrotoxicosis after delivery was significantly higher than that of the PTT group (2.5 +/- 0.4, n = 16). The peak TRAb value of the exacerbation group of GD (72.5 +/- 121.7 IU/L, n = 10) at the time of thyrotoxicosis after delivery was also significantly higher than that of the PTT group (1.4 +/- 0.8 IU/L, n = 16). In conclusion, the high peak value of hCG is valuable for suspecting GTT, and the high FT(3)/FT(4) ratio is valuable for suspecting recurrence in the patients with GD. In both situations, changes of TRAb were also valuable in differentiating the recurrence of GD from GTT or PTT.     

Graves' ophthalmopathy in the absence of elevated free thyroxine and triiodothyronine levels: prevalence, natural history, and thyrotropin receptor antibody levels.

The aims of this study were to (a) determine the prevalence of patients without elevated thyroid hormone levels in Graves' ophthalmopathy (GO) using current generation free thyroid hormone assays, (b) measure the prevalence of thyrotropin receptor antibodies (TRAb) in these cases, and (c) identify possible predictors of hyperthyroidism. Over a 30-month period, 1020 cases of thyroid eye disease were evaluated, of which only 19 (1.9%) met the diagnostic criteria. Ten (1%) had subclinical thyrotoxicosis, 7 (0.7%) were euthyroid, and 2 (0.2%) were hypothyroid as determined by a third-generation thyrotropin (TSH) assay. TRAb levels were measured in 16 of these 19 patients. The prevalence of TRAb varied according to the assay used. Polyethylene glycol-extracted thyroid-stimulating immunoglobulin (PEG-TSI), unfractionated thyroid-stimulating immunoglobulin (uTSI), first-generation porcine TSH-binding inhibitory immunoglobulin (pTBII), and second-generation human TSH-binding inhibitory immunoglobulin (hTBII) assays were positive in 93.8%, 50%, 18.8%, and 81.3% of patients, respectively. TRAb was detected by at least one method in all patients. Patients were followed up for 15 to 45 months. Hyperthyroidism developed in 4 patients (25%). Suppressed TSH levels and elevated TBII were predictors of hyperthyroidism. When sensitive assays are used, the prevalence of GO patients without elevated thyroid hormone levels is extremely low. The sensitivities of assays for TRAb detection differ substantially in these cases. PEG extraction improves the detection rate of TSI (p = 0.02), and hTBII assays improve the detection of TBII in these patients (p = 0.002). The high prevalence of TRAb in such cases supports a role for these antibodies in the pathogenesis of thyroid-associated eye disease.     

Transient hyperthyroidism after withdrawal of antithyroid drugs in patients with Graves' disease

The development of silent thyroiditis in patients with a history of Graves' disease is common, especially in the postpartum period. We describe herein patients with Graves' disease who developed transient hyperthyroidism but not silent thyroiditis after withdrawal of antithyroid drug (ATD). If such patients are diagnosed as recurrence of Graves' disease, they may receive ATD or radioiodine therapy unnecessarily. We investigated the characteristics of these patients to prevent unnecessary therapy. We retrospectively studied 22 patients with Graves' disease who showed transient thyrotoxicosis after withdrawal of ATD. Two of 22 patients were male and the mean ages (+/- SD) were 33.7 +/- 12.6 yr. We observed these patients for 28.5 +/- 12.8 (mean +/- SD; range 12-53) months after transient thyrotoxicosis, and measured TSH, FT4, and TSH binding inhibitor immunoglobulin in sera. Radioiodine uptake was measured in 6 of them. The radioiodine uptake in the 4 patients was not suppressed (27.5%, 28.0%, 32.7%, 38.1%). These uptake levels indicate that their thyrotoxicosis was not caused by silent thyroiditis. Most of the 22 patients became euthyroid within 6 months. This study suggests a new therapeutic option as follows: in the case of young patients with mild thyrotoxicosis after withdrawal of ATD, physicians should follow them up for one month without medication unless they have unbearable symptoms or complications.     

Remission of Graves' hyperthyroidism predicted by smooth decreases of thyroid-stimulating antibody and thyrotropin-binding inhibitor immunoglobulin during antithyroid drug treatment.

It is important to know whether a patient with Graves' disease will get into remission or not during antithyroid drug (ATD) treatment. Thyrotropin (TSH) receptor antibodies (TRAb) cause Graves' disease. Thyroid-stimulating antibody (TSAb) and TSH-binding inhibitor immunoglobulin (TBII) have been measured as TRAb to diagnose Graves' disease and to follow Graves' patients. Smooth decreases of TSAb and TBII during ATD treatment predict the remission of Graves' hyperthyroidism. We followed serial changes of TSAb and TBII in 58 Graves' patients before, during, and after ATD treatment; TSAb was measured as a stimulator assay, using porcine thyroid cells, and TBII as a receptor assay. Patterns of TSAb and TBII changes during ATD treatment differ from one patient to another. TSAb and TBII activities decreased and disappeared in 52 (group A) but continued to be high in the other 6 (group B); 39 of the 52 group A patients achieved remission, but all of the 6 group B patients with persistently positive TSAb and TBII continued to have hyperthyroidism. No differences in the initial TSAb and TBII activities were noted between the 52 group A patients and the 6 group B patients. Of the 52 group A patients in whom TSAb and TBII had disappeared, 44 had smooth decreases of TSAb and TBII (group A1), and 8 had complex changes of TSAb and/or TBII (group A2); 36 of the 44 group A1 patients (82%) but only 3 of the 8 group A2 patients (37%) continued to be in remission more than 1 year after ATD discontinuation. The number of remission in group A1 was significantly larger than that in group A2. No differences in the initial TSAb and TBII activities were noted between group A1 and group A2. More than 80% of group A1 patients, who had smooth decreases of TSAb and TBII, continued to be in remission longer than 1 year. We demonstrated that smooth decreases of TSAb and TBII during ATD treatment predicted the remission of Graves' hyperthyroidism. The Graves' patients can be classified into A1, A2, and B groups according to the patterns of TSAb and TBII changes during ATD treatment. Group A1 patients, who had smooth decreases of TSAb and TBII, had higher rate of remission than the others. Smooth decreases of TSAb and TBII during the early phase of ATD treatment are a reliable predictor of the remission.