Immunology of the thyrotropin receptor.

Antibodies to the thyrotropin receptor appear to be responsible for hyperthyroidism in Graves' disease. The antibodies, described as thyroid-stimulating antibodies (TSAb) mimic the effects of thyrotropin (TSH) by binding to the TSH receptor and activating adenylate cyclase. TSAb consist of an electrophoretically heterogeneous population of IgG and the thyroid-stimulating site is formed by combination of heavy and light chains in the Fab part of the molecule. Binding studies indicate that the TSAb molecule interacts monovalently with membrane bound TSH receptors and that TSAb consists of an antibody population which shows a restricted heterogeneity with regard to TSH receptor affinity. Studies in patients with Graves' disease and hyperthyroidism indicate that the levels of TSAb correlate well with thyroidal iodine uptake and the absence of pituitary control of thyroid function. However in some patients with ophthalmic Graves' disease or autoimmune thyroiditis there is evidence of serum antibodies which interact with the TSH receptor but are unable to stimulate thyroid function.     

Prognostic value of thyroid stimulating antibodies and TSH-binding inhibiting immunoglobulins in the follow-up of Graves' disease

Summary The prognostic value of the determinations of autoantibodies in Graves' disease is still questionable. So far, the role of different assay procedures used has not been intensively investigated. We simultaneously applied two different techniques, a radioreceptor assay and a T3 releasing in vitro assay, in the follow-up of patients with Graves' disease to directly compare the course of the antibody activities determined by these assays and to find out a prognostic significance of the composition of the antibody spectrum present. The initial activities of thyroid stimulating antibodies (TSAb) and TSH-binding inhibiting immunoglobulins (TBII) were not significantly correlated in patients before treatment. During a 12-month antithyroid medication antibody titres showed a concordant course in the majority of patients. In 6 of 25 patients, however, a discordant behaviour was clearly documented including dose-response curves. At the end of treatment, the patients could be divided into three groups: group I included 5 patients positive for both TSAb and TBII, group II 6 patients positive for TBII and negative for TSAb and group III 14 patients negative for both of them. During the following survey of 18 months all patients of group I, 2 patients of group II and 6 patients of group III experienced a relapse of hyperthyroidism. In conclusion, TSAb and TBII activities dissociate in some patients during antithyroid drug therapy. For the individual patient, the disappearance of both TSAb and TBII was no certain indicator for a longstanding remission of Graves' hyperthyroidism. The persistence of TSAb seems to be more reliably associated with persisting or rapidly relapsing disease than the persistence of TBII.Abbreviations cAMP Cyclic Adenosine Monophosphate - GD Graves' disease - T3 Triiodothyronine - T4 Tetraiodothyronine - TBII TSH-binding inhibiting immunoglobulins - TRH TSH releasing hormone - TSAb Thyroid stimulating antibodies - TSH Thyroid stimulating hormone     

Heterogeneity of thyrotropin binding inhibitor immunoglobulins in serum from untreated patients with hyperthyroid Graves' disease.

We have previously established an assay for the simultaneous assessment of thyrotropin (TSH) binding inhibitor immunoglobulin (TBII) and thyroid stimulating autoantibody activities in cultured rat thyroid cells (FRTL-5 cell), and found a discrepancy in some patients with untreated Graves' disease between the activities of TBII measured in FRTL-5 cells (TBII-rc) and in solubilized thyroid membranes (TBII-pm). In three selected patients with untreated Graves' disease, the different dose-response relationship between TBII-rc and TBII-pm clearly indicated the heterogeneous populations of TBII-pm in patients' sera, with different binding affinities for TSH receptor in intact cells.     

Immunological of the Thyrotropin Receptor

Antibodies to the thyrotropin receptor appear to he responsible for hyperthyroidism in Graves disease. The antibodies, described as thyroid-stimulating antibodies (TSAb) mimic the effects of thyrotropin (TSH) by binding to the TSH receptor and activating adenylate cyclase. TSAb consist of an electrophoretically heterogeneous population of IgG and the thyroid-stimulating site is formed by combination of heavy and light chains in the Fab part of the molecule. Binding studies indicate that the TSAb molecule interacts monovalently with membrane bound TSH receptors and that TSAb consists of an antibody population which shows a restricted heterogeneity with regard to TSH receptor affinity. Studies in patients with Graves disease and hyperthyroidism indicate that the levels of TSAb correlate well with thyroidal iodine uptake and the absence of pituitary control of thyroid function. However in some patients with ophthalmic Graves' disease or autoimmune thyroiditis there is evidence of serum antibodies which interact with the TSH receptor but are unable to stimulate thyroid function.     

Inverse correlation between activated T cells and TSH receptor antibodies in Graves' disease

TSH receptor antibodies and peripheral blood lymphocyte subsets have been measured in fourteen patients with untreated Graves' thyrotoxicosis. CD8 (suppressor) cells were reduced (p less than 0.01) and helper/suppressor cell ratio was increased in Graves' patients. Increased levels of 4F2 positive (activated) T cells were found in the patients compared to controls (p less than 0.001) and there was a negative correlation between 4F2 positive cells and TSH receptor antibodies (TBII). It may be possible, with multiple immunological markers, to identify different stages in the pathogenesis of autoimmune thyroid disease.     

Thyroid-stimulating antibodies in patients with long-term remission of Graves' hyperthyroidism

Summary The persistence of TSH receptor antibodies in Graves' disease despite the remission of hyperthyroidism has been described. Our study was designed to evaluate whether this extends to functionally active stimulators of the thyroid, since the occurrence of thyroid-stimulating antibodies (TSAb) in a euthyroid patient could well have important implications on our understanding of the pathogenetic role of such autoantibodies. Forty-four patients with a previous history of Graves' hyperthyroidism were reexamined after having been in long-lasting remission for 3 to 35 years (mean 8 years). Of the patients 16 had been treated by radioiodine, 17 by surgery, and 11 exclusively by antithyroid drugs. The determination of TSAb was based on T3 release from thyroid tissue in vitro to document the final response to these immunoglobulins. TSH-binding inhibiting immunoglobulins (TBII) were evaluated by a radioreceptor assay.TSAb were highly elevated in three of the 44 patients. These three patients showed a normal TSH response to i.v. TRH, suffered from endocrine ophthalmopathy, and had been treated by radioiodine for hyperthyroidism. TBII were found positive in seven patients including the three patients mentioned. The majority of patients positive for TSAb or TBII had been treated by radioiodine and none exclusively by antithyroid drugs.In conclusion, not only TBII but also T3 release-stimulating antibodies may occur in a minority of patients with long-term remission of Graves' hyperthyroidism. However, an absence of hyperthyroidism in these patients despite the presence of such thyroid stimulators seems to be only possible in association with a lack of functional responsiveness of the target organ due to previous administration of destructive therapies. Moreover, a major role of TBII in the absence of TSAb representing stimulatory inactive autoantibodies to the maintenance of remission was not apparent.Abbreviations cAMP cyclic adenosine monophosphate - T3 triiodothyronine - T4 tetraiodothyronine - TBII TSH-binding inhibiting immunoglobulins - TRH TSH-releasing hormone - TSAb thyroid-stimulating antibodies - TSH thyroidstimulating hormone     

Graves病患者治疗前、后血清甲状腺刺激抗体和TSH结合抑制免疫球蛋白活性的观察

本文研究了Ｇｒａｖｅｓ病（ＧＤ）患者在抗甲状腺药物（ＡＴＤ）治疗前、后血清甲状腺刺激抗体（ＴＳＡｂ）和ＴＳＨ结合抑制免疫球蛋白（ＴＢＩＩ）的变化，发现治疗前ＴＳＡｂ和ＴＢＩＩ的检出率分别为９１．７％和７９．２％，治疗后两抗体的活性及阳性率均显著下降，表明抗甲状腺药可改善ＧＤ患者的免疫异常。ＴＳＡｂ和ＴＢＩＩ活性不相关，提示ＴＳＨ受体抗体（ＴＲＡｂ）具有异质性。ＴＳＡｂ和ＴＢＩＩ活性与血清甲状腺激素水平无相关关系，说明体外测定的ＴＳＡｂ或／和ＴＢＩＩ活性并不能完全反映甲亢的严重程度。     

运用ROC曲线方法评价TRAb、TPO-Ab和TGA在Graves'病和HT鉴别诊断中的意义

目的:应用临床诊断性能(ROC)曲线方法评价TSH受体抗体(TRAb)、甲状腺过氧化物酶抗体(TPO-Ab)和甲状腺球蛋白抗体(TGA)在Graves'病(格雷夫斯病)和桥本甲状腺炎鉴别诊断中的意义.方法:以甲状腺细针穿刺细胞学检查结果作为诊断金标准,以采用化学发光法测定63例自身免疫性甲状腺病患者血清的TRAb、TP...     

Absence of gradient of thyrotropin receptor antibody and T cell subset distribution between thyroid and peripheral venous blood in patients with Graves'' disease prepared for surgery with carbimazole and potassium iodide.

The aim of the study was to examine tri-iodothyronine, thyroxine, thyroid antibody and thyrotropin receptor antibody levels in thyroid and peripheral venous blood at the time of surgery in patients with Graves' disease. T cell subset patterns in peripheral and thyroidal venous blood were compared to the distribution of T cell subset patterns within the thyroid gland itself. The results showed that at the time of surgery there were no significant differences in any of the parameters measured between thyroidal and peripheral venous samples. T cell subset patterns within the thyroid gland were subjectively similar to those in the venous samples.     

Graves病患者甲状腺和外周静脉血中甲状腺特异性抗体的对比研究

本文对比研究１１例Ｇｒａｖｅｓ病（ＧＤ）和９例非ＧＤ患者甲状腺静脉血（ＴＶＢ）和外周静脉血（ＰＶＢ）中甲状腺刺激抗体（ＴＳＡｂ）、甲状腺球蛋白抗体（ＴＧＡｂ）和甲状腺过氧化物酶抗体（ＴＰＯＡｂ）的活性和Ｔ３、Ｔ４浓度。结果显示：（１）抗体阳性的ＧＤ患者，其ＴＶＢ中ＴＳＡｂ、ＴＧＡｂ和ＴＰＯＡｂ水平均显著高于ＰＶＥ的，ＰＶＢ和ＴＶＢ的ＴＳＡｂ活性呈显著正相关，这提示甲状腺本身是甲状腺特异性抗体产生的主要部位；（２）ＧＤ组和非ＧＤ组ＴＶＢ和ＰＶＢ的血清Ｔ３、Ｔ４不形成浓度梯度；（３）ＴＳＡｂ、ＴＧＡｂ和ＴＰＯＡｂ活性及其在ＴＶＢ和ＰＶＢ之间的活性梯度，与ＴＶＢ和ＰＶＢ中Ｔ３、Ｔ４浓度均无相关关系。     

Glycosylated ectodomain of the human thyrotropin receptor induces antibodies capable of reacting with multiple blocking antibody epitopes

Recently, we showed that the glycosylated ectodomain of the human thyrotropin receptor (hET-gp) reacts with autoantibodies from autoimmune thyroid disease (AITD) patients' sera. To better understand the effects of glycosylation of thyrotropin receptor (TSHR) in antibody induction, we immunized rabbits with hET-gp protein. The rabbits developed relatively high titers of antibodies with highly potent TSH binding inhibitory immunoglobulin (TBII) and thyroid stimulatory blocking antibody (TSBAb) activities. Both the hET-gp and a nonglycosylated ectodomain of the human TSHR (hETSHR) protein significantly reversed the TBII as well as TSBAb activity. Based on the ability of synthetic peptides to significantly reverse the functional activity of these rabbit antisera, we identified three discrete regions of the TSH R, represented by amino acids 202-221, 292-311 and 367-386, as TBII epitopes and four regions represented by amino acids 352-371, 367-386, 382-401 and 392-415 as TSBAb epitopes. These data demonstrate that rabbit antibodies that bind to amino acids 367-386 mediate their TSBAb activity by inhibiting the binding of TSH to TSHR; whereas, antibodies to regions 352-415, excluding aa 367-386, exert their TSBAb activity by affecting a step subsequent to TSH binding. Coincident with the elevation of TBII and TSBAb activity, serum total T4 levels declined and thus suggested that the antibodies exerted functional effects on thyroid in vivo. Together, these data demonstrate that glycosylated hET-gp protein is a more potent immunogen and it can induce a broader antibody response directed against multiple TBII and TSBAb epitopes.     

Time-dependent effect of cyclosporin-A on the TSH-receptor antibody synthesis in patients with Graves' disease

Two patients with hyperthyroidism and Graves' ophthalmopathy were treated with cyclosporin A (CyA), in addition to methimazole, after failure of steroid therapy. Eye disease showed favorable responses and TSH receptor antibody concentration showed precipitous decline in concentrations compared to a gradual linear decline in antibody concentrations observed in 10 patients not treated with CyA. These results prompted us to investigate the in vitro influence of CyA on the synthesis of TSH receptor antibody by a patient's lymphocytes (with highest antibody concentration) in response to thyroid membrane antigen. CyA caused a dose-dependent reduction of TSH receptor antibody synthesis compared to control cultures. The effect of CyA was more marked when added to lymphocyte culture at the same time rather than 24 h after addition of antigen, consistent with CyA's interference of early T cell triggering by antigen. This study emphasizes the importance of helper T cells in synthesis of TSH-receptor antibody by cells and suggests that the drug may be therapeutically beneficial in severe Graves' ophthalmopathy and/or Graves' hyperthyroidism resistant to conventional treatment.     

Immunological of the Thyrotropin Receptor

Antibodies to the thyrotropin receptor appear to he responsible for hyperthyroidism in Graves disease. The antibodies, described as thyroid-stimulating antibodies (TSAb) mimic the effects of thyrotropin (TSH) by binding to the TSH receptor and activating adenylate cyclase. TSAb consist of an electrophoretically heterogeneous population of IgG and the thyroid-stimulating site is formed by combination of heavy and light chains in the Fab part of the molecule. Binding studies indicate that the TSAb molecule interacts monovalently with membrane bound TSH receptors and that TSAb consists of an antibody population which shows a restricted heterogeneity with regard to TSH receptor affinity. Studies in patients with Graves disease and hyperthyroidism indicate that the levels of TSAb correlate well with thyroidal iodine uptake and the absence of pituitary control of thyroid function. However in some patients with ophthalmic Graves' disease or autoimmune thyroiditis there is evidence of serum antibodies which interact with the TSH receptor but are unable to stimulate thyroid function.     

Immunoblotting for the detection of TSH receptor autoantibodies.

Immunoblotting was optimized to detect autoantibodies to TSH receptors from human and porcine thyroid tissue and to determine their epitope specificity. Autoantibodies to putative TSH receptor proteins in thyroid particulate membranes were detected in approximately 35% of sera from patients with Graves' disease. However, despite modifications to increase immunoblotting sensitivity and specificity, only a minority (less than 15%) of Graves' disease sera contained autoantibodies that identified epitopes within TSH affinity-purified human or porcine receptor proteins. In these sera there was no correlation between the TSH receptor antibody titre, determined by radioreceptor assay, and receptor epitope reactivity. The sensitivity of immunoblotting was limited by reduced transfer of purified receptor from the gel. However, in addition, the inability to immunoblot the purified receptor with a majority of Graves' sera, under conditions designed to enhance receptor renaturation, appears to reflect a strict conformational requirement for immunoreactivity. Immunoblotting of purified receptors therefore has a limited application in detecting, and defining the epitope reactivity of, TSH receptor autoantibodies.     

The pathogenetic connection between Graves' disease and chronic lymphocytic thyroiditis. (The role and incidence of thyroid stimulating antibodies)

Antibody-positivity to thyroid specific antigens (Htg, microsomal) and/or lymphocytic infiltration of the gland's parenchyma were observed in 207 (55%) of 377 patients with Graves's disease. Only in 48 (12.7%) of the cases were the findings in agreement with the criteria of chronic lymphocytic thyroiditis. Human thyroid stimulating antibody (HTSab) was detected in 135 (65%) of these 207 patients. In cases of Graves' disease associated with chronic lymphocytic thyroiditis, this proportion was found to be as high as 89.6% and attained even 100% in cases of Hashitoxicosis (39 patients). The presence of HTSab thus seems to form one of the features of patients with Hashitoxicosis. Infiltrative ophthalmopathy also showed a remarkably high incidence (59%) in this porcess. The typical prevalence of Graves' disease in females in the present material attained a 15:1 female-to-male ratio when the disease was associated with chronic lymphocytic thyroiditis. The results of the present study suggest that chronic lymphocytic thyroiditis associated with Graves' disease promotes the formation of thyroid stimulating antibodies.     

THE RELATION OF THYROID AUTO-IMMUNITY TO ROUND-CELLED INFILTRATION OF THE THYROID GLAND

The relationship between thyroid auto-immunity and the presence of lymphocytes in the thyroid gland has been analysed in patients with Graves' disease, toxic adenoma, and non-toxic nodular goitre. In all these conditions circulating antibody to thyroglobulin was significantly associated with lymphocytic infiltration. No similar correlation was established in the case of the complement-fixing antibody.     

Distribution of lymphocytic subpopulations infiltrated in thyroid glands of Graves' disease

We studied ninety cases of thyroid glands both histopathologically and by immunohistochemical methods in patients with Graves' disease using B and T cell markers to evaluate the role of lymphocytic subpopulation. Females were affected more frequently than males with a ratio of 6.5:1, and usually the females were younger than the males at the time of surgery. The heavier the lymphocytic infiltration, the higher was the percentage of germinal center formation or fibrosis. The degree of lymphocytic infiltration was also related to the titers of antithyroglobulin or antimicrosomal antibodies. T cells were mostly scattered individually or in small groups between the follicles; however, in the severely infiltrated group, the major pattern was in clusters. T8 positive cells were more abundant than T4 positive cells, and their distribution pattern was accordant with T11 positive cells. Immunoglobulin synthesizing B cells were positively stained in 47 of 94 cases tested and IgG was the most predominant. In the mild and moderate lymphocytic infiltration groups, IgM was mostly stained at the mantle zone or in the lymphoid cluster of the interfollicular stroma, whereas IgM positive cells were present exclusively in the germinal center of the severely infiltrated group. The results of our study indicate that the major lymphocyte subpopulation in Graves' disease is B lymphocytes, and the degree of T lymphocytic infiltration correlated better with titers of antimicrosomal antibody than antithyroglobulin.     

Current topics on immunological laboratory tests-thyroid diseases

Current topics in the field of thyroid disease are the development of the second generation assay for TSH receptor antibody (TRAb) using recombinant human TSH receptor and the appearance of antineutrophil cytoplasmic antibodies(ANCA) in Graves' disease patients treated with propylthiouracil(PTU). This new TRAb assay is very useful, since the sensitivity and the specificity were almost 100%, respectively, in the diagnosis of Graves' disease. Furthermore, a new coated tube assay for the detection of blocking TRAb has been developed by using TSH/LH receptor chimera. The prevalence of ANCA is high in Graves' disease patients treated with PTU, but the clinical significance of ANCA is under controversy, since only a part of them develop vasculitis, and recently it has been reported that ANCA is frequently positive in Graves' disease patients before the onset of methimazole treatment. The 7th version of guidelines for the diagnosis of thyroid disease have been prepared by the Japan Thyroid Association, and opens to public inspection. They show the importance of immunological laboratory tests in this field.     

Administration of thyroxine in treated Graves' disease. Effects on the level of antibodies to thyroid-stimulating hormone receptors and on the risk of recurrence of hyperthyroidism

BACKGROUND. Antibodies to thyroid-stimulating hormone (TSH) receptors that stimulate the thyroid gland cause hyperthyroidism in patients with Graves' disease, and their production during antithyroid drug treatment is an important determinant of the course of the disease. One factor that might contribute to the persistent production of antibodies to TSH receptors is stimulation of the release of thyroid antigens by TSH during antithyroid drug therapy. We therefore studied the effect of the suppression of TSH secretion by thyroxine on the levels of antibodies to TSH receptors after thyroid hormone secretion had been normalized by methimazole. METHODS AND RESULTS. The levels of antibodies to TSH receptors were measured during treatment with methimazole, either alone or in combination with thyroxine, in 109 patients with hyperthyroidism due to Graves' disease. The patients first received 30 mg of methimazole daily for six months. All were euthyroid after six months, and their mean (+/- SD) level of antibodies to TSH receptors decreased from 64 +/- 9 percent to 25 +/- 15 percent (P less than 0.01; normal, 2.9 +/- 1.4 percent). Sixty patients then received 100 micrograms of thyroxine and 10 mg of methimazole and 49 received placebo and 10 mg of methimazole daily for one year. In the thyroxine-treated group, the mean serum thyroxine concentration increased from 108 +/- 16 nmol per liter to 145 +/- 11 nmol per liter (P less than 0.01), and the level of antibodies to TSH receptors decreased from 28 +/- 10 percent to 10 +/- 3 percent after one month of combination therapy. In the patients who received placebo and methimazole, the mean serum thyroxine concentration decreased and the level of antibodies to TSH receptors did not change. Methimazole, but not thyroxine or placebo, was discontinued in each group 1 1/2 years after the beginning of treatment. The level of antibodies to TSH receptors further decreased (from 6.6 +/- 3.2 percent at the time methimazole was discontinued to 2.1 +/- 1.2 percent one year later) in the patients who continued to receive thyroxine, but it increased (from 9.1 +/- 4.8 percent to 17.3 +/- 5.8 percent during the same period) in the patients who received placebo. One patient in the thyroxine-treated group (1.7 percent) and 17 patients in the placebo group (34.7 percent) had recurrences of hyperthyroidism within three years after the discontinuation of methimazole. CONCLUSIONS. The administration of thyroxine during antithyroid drug treatment decreases both the production of antibodies to TSH receptors and the frequency of recurrence of hyperthyroidism.     

A non-isotopic screening test for antibodies to TSH receptor: dot immunobinding assay

A dot immunobinding assay (DIBA) for thyrotropin (TSH) receptor antibodies is described. The method depends on the detection of antibody binding to highly purified thyroid plasma membrane attached to nitrocellulose solid support by horse-radish peroxidase - conjugated anti-human IgG. The method can detect down to 0.75 mU LATS standard and 1/1000 dilution of Graves' serum or immunoglobulin fraction. The interaction is inhibited dose-dependently by bTSH but not by insulin or human chorionic gonadotropin. The DIBA results show close correlation to those of TRAK (TBII) but not to cyclic AMP generation assay. DIBA is reproducible when tested monthly for 4 months. Sera and immunoglobulins gave virtually the same results. The method has a sensitivity of 90%, validity of 90% and specificity of 80% for both. We have, thus developed a sensitive and reliable method for screening for TSH receptor antibodies which can be performed in routine clinical laboratories.