The association of IgG heavy-chain allotypes (Gm) with Graves' disease in Hungary

In an Eastern Hungarian population of patients, we sought to confirm an association with IgG heavy-chain allotypes (Gm) with Graves' disease. We found an increase in the phenotype fb (chi 2(1) = 4.7, p less than 0.05) among the patient group compared to controls. The influence of Ig-linked genes to Graves' disease susceptibility is, thus, dose-dependent. We found no interaction between fb homozygosity and HLA-DR3 positivity in Graves' disease susceptibility.     

Gm phenotypes in autoimmune thyroid disease

The Gm phenotype Gm f,b or Gm f,n,b was found in all forty patients with Graves' disease studied, contrasted with thirty-five out of forty controls and twenty out of thirty-one patients with thyroiditis. The difference between the two groups with autoimmune thyroid disease was significant. These results suggest that thyroid stimulating antibodies may be allotypically restricted.     

The putative anti-thyrotropin receptor antibodies of Graves' disease. I. Gm allotypes

The hyperthyroidism of Graves' disease is thought to be related to antithyrotropin receptor (TSH-R) antibodies. In order to study the degree of immunogenetic homogeneity of these antibodies, we carried out Gm typing of 'receptor-purified' IgG from patients with active Graves' disease and controls. The results were compared to those of serum, total IgG and IgG which failed to attach to TSH-R. We found that in five out of seven Gm heterozygote patients studied the receptor-purified antibodies were restricted to the products of one haplotype compared to three out of five similar controls. Such eluted antibodies were biologically active. Similar results in terms of immunogenetic restriction and activity were obtained when F(ab)2 preparations were used. An unexpected finding was that sera and IgG from normal persons attached to thyroid membranes and that the attachment occurred via F(ab)2. Normal whole serum and 'receptor-purified' IgG and F(ab)2 inhibited TSH binding in the receptor assay; however, this inhibition showed no specificity for TSH-R.     

Assays of TSH-receptor antibodies in 576 patients with various thyroid disorders: their incidence, significance and clinical usefulness

The incidence and the significance of TSH-receptor antibodies in Graves' disease and in various thyroid disorders have been evaluated. TSH-binding inhibiting antibodies (TBIAb) and thyroid stimulating antibodies (TSAb) were detected in a large proportion of Graves' disease patients (TBIAb in 68.8% and TSAb in 77.8%), in a small number of patients with idiopathic myxoedema or Hashimoto's thyroiditis, and were not detected in patients with endemic euthyroid goitre, differentiated thyroid carcinoma and toxic adenoma. Furthermore, TSH-receptor antibodies were present in some patients with toxic multinodular goitre (TBIAb in 12.7% and TSAb in 15.9%). When TSH-receptor and other thyroid autoantibodies were compared, it was found that 13 of the 15 Graves' patients with negative tests for thyroglobulin and thyroid microsomal antibodies were positive for TSH-receptor antibodies. On the other hand, 9 of the 11 patients with toxic multinodular goitre who had positive TSH-receptor antibody tests, also had serum thyroglobulin and/or thyroid microsomal antibodies. No significant differences in the prevalence of TSH-receptor antibodies were found in Graves' patients irrespective of the presence of ophthalmopathy or pretibial myxoedema. Elevated TBIAb activity at the end of anti-thyroid drug treatment was found in 52.9% of Graves' patients who subsequently relapsed, while in Graves' patients in remission TBIAb was always negative. TSH-receptor antibody results were not predictive of the outcome of radioiodine treatment in Graves' disease. Finally no correlation could be found between TBIAb and TSAb in Graves' disease and Hashimoto's thyroiditis. In conclusion: the high incidence of TSH-receptor antibodies in Graves' disease confirms their pathogenetic role in the development of hyperthyroidism; TSH-receptor antibodies in Graves' disease are not significantly associated with the presence of ophthalmopathy or pretibial myxoedema; TSH-receptor antibody assays may be useful for the diagnosis of Graves' disease in the absence of other signs of autoimmunity. TBIAb seems to be a good predictor of relapse in Graves' patients treated with anti-thyroid drugs; a fraction of toxic multinodular goitre could be a nodular variant of Graves' disease.     

Studies on thyroid cell surface antigens using cultured human thyroid cells.

Human thyroid cells in primary culture were used for studies of thyroid cell surface antibodies in patients with thyroid autoimmune disorders. Radioiodinated IgG preparations containing thyroid microsomal antibody (TMAb), thyroid stimulating antibody (TSAb) and/or thyroglobulin antibody (TgAb) were tested for binding to thyroid cells. Binding was observed with radioiodinated IgG from patients with Graves' disease, Hashimoto's thyroiditis and idiopathic myxoedema containing TMAb, irrespective of the presence of TSAb and TgAb, while negative results were obtained with normal IgG. A dose-dependent inhibition of binding to thyroid cells was produced by the addition of the corresponding unlabelled IgG preparations. Evidence for tissue specificity was provided by the absence of binding to human skin fibroblasts used as controls. Preabsorption with human thyroid microsomes completely abolished the binding to thyroid cells of a radioiodinated TMAb positive IgG preparation, while only incomplete removal of the reactivity to thyroid microsomes was produced by preabsorption with thyroid cells. These data suggest that some but not all microsomal antigenic determinants are expressed on the thyroid cell surface. Binding to thyroid cells was also observed with purified TgAb, indicating that thyroglobulin antigenic determinants are present on the surface of thyroid cells. No evidence of binding was obtained with a TSAb positive Graves' IgG preparation with undetectable TMAb and TgAb. Unlabelled IgG preparations containing TMAb from patients with either Hashimoto's thyroiditis or idiopathic myxoedema were shown to inhibit the binding to thyroid cells of radioiodinated TMAb positive Graves' IgG and vice versa. These data indicate that antibodies present in these thyroid autoimmune disorders share common thyroid cell surface antigens. However, the binding of radioiodinated IgG from a patient with idiopathic myxoedema was only partially inhibited by Graves' or Hashimoto's IgG, suggesting that some of the thyroid cell surface antibodies of idiopathic myxoedema may not be detectable in other thyroid autoimmune disorders.     

Comparative study on IgG and IgA antibodies against human thyroid and eye-muscle antigens in Graves' ophthalmopathy.

Circulating IgG and IgA anti-thyroid and anti-eye muscle antibodies were investigated in 87 patients with Graves' disease (60 cases with ophthalmopathy). The ELISA method was used. Both IgG and IgA antibodies were demonstrated against human thyroid and eye-muscle membrane or cytosol antigens. Anti-eye-muscle antibodies of the IgA type were observed more frequently than those of the IgG type (25 cases vs. 18 were demonstrated with membrane antigens and 37 cases vs. 23 with cytosol antigens). The respective distributions for thyroid antigens the cytosol fraction were 55 cases vs. 13 and 18 cases vs. 36. A significant difference was observed in the anti-thyroid IgG levels and the anti-eye-muscle membrane or cytosol levels between the patients with Graves' disease and those in control group (P less than 0.001). The difference in the IgA antibody to thyroid and eye-muscle antigens was significant between the patients with and without ophthalmopathy (P less than 0.002). The strong correlation between the levels of IgA antibodies to thyroid and those to the eye-muscle cytosol fractions might be connected with the theory of the common aetiology of the thyroid and eye diseases in Graves' ophthalmopathy (P less than 0.001). Circulating IgA anti-human thyroid and eye-muscle antibodies seemed to have a diagnostic relevance in the development of ophthalmopathy in Graves' ophthalmopathy.     

A study of human-human hybridomas from patients with autoimmune thyroid disease

Human-human B-cell hybridomas were established using peripheral blood lymphocytes from patients with autoimmune thyroiditis and Graves' disease. Peripheral mononuclear cells (PMC), with or without mitogen prestimulation, were fused with HGPRT-negative human myeloma cell lines (Gm4672 and Gm0462) using 44% polyethylene glycol. Developing hybridomas were screened by enzyme-linked immunosorbent assays (ELISAs) for human IgG and IgM and antibodies to human thyroglobulin (hTg) and microsomal antigen (M-Ag). A¹²⁵I-TSH binding inhibition assay was utilized for detecting antibodies to TSH receptor (TSH-R) protein. Hybridoma formation was observed only after prior mitogen stimulation of PMC. The amount of antibody secreted by the human-human hybridomas was highly variable (10 ng-100 µg/ml IgG/IgM). Nine and six-tenths percent of the hybrids secreted anti-hTg and 8.4% secreted anti-M-Ag. A 5% cloning efficiency was achieved, with detection of specific thyroid autoantibody secretion in one-third of the clones derived from positive hybridomas. Immunoglobulin secretion decreased with time and long-term stable clones were not achieved. Thyroid monoclonal autoantibodies to hTg, M-Ag, and TSH-R (IgG and IgM) detected during these studies were of a low affinity. In addition, antibodies were identified which exhibited marked specificity crossover between hTg, M-Ag, and nonthyroid antigens, suggesting the presence of recurrent epitopes. Such observations may help explain the multiplicity of thyroid autoantibodies in human thyroid disease and indicate a common defect in immunoregulation. We suggest that cross-reacting epitopes may be important in the derivation of thyroid-specific B-cell clones.     

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。     

Association of immunoglobulin Gm allotypes with antiglomerular basement membrane antibodies and their titer

We studied the immunoglobulin Gm allotypes in 41 patients with glomerular nephritis caused by autoantibodies to glomerular basement membrane (GBM). Gm phenotypes of all 41 patients were attributable to combinations of the 3 Gm haplotypes commonly found in Caucasoid populations; identified by the allotypes Gm 1,21 (ag), Gm 1,2,21 (axg), and Gm 3,5,11 (fb). The incidence of the putative haplotype Gm 1,2,21 (axg) was greatly increased in the patients being present in 22 of 41 (56%) of patients compared to 28 of 167 controls. (P_cor = 1.5 × 10⁻⁵). The increase in Gm 1,2,21 (axg) was attributable entirely to presumed heterozygotes with the phenotype Gm 1,2,21; 3,5,11 (axg; fb), with concomitant decreases in the frequencies of patients with the phenotypes Gm 1,21 (ax) and with Gm 3,5,11 (fb). Heterozygotes at Gm loci had higher titers of anti-GBM antibodies irrespective of the presence of Gm 1,2,21 (axg). Thus genes within or closely linked to the Gm complex in addition to HLA linked genes influence susceptibility to or clinical expression of anti-GBM disease.     

Carbimazole and the autoimmune response in Graves' disease

Microsomal antibodies and antibodies directed toward the receptor for thyroid-stimulating hormone (TSH) decreased in parallel while patients with Graves' disease were taking carbimazole, whereas no significant changes were observed during treatment with placebo or propranolol. The changes in autoantibody levels during carbimazole treatment were independent of changes in serum thyroxine and could have been due to a direct effect of the drug on autoantibody synthesis. Evidence for this suggestion was provided when low doses of methimazole (the active metabolite of carbimazole) were found to inhibit thyroid-autoantibody production in cultured lymphocytes. Since thyroid lymphocytes are probably a major site of thyroid-antibody synthesis in Graves' disease and methimazole is concentrated in the thyroid during treatment, a local action of the drug on antibody production seems likely. This possibility could be important in the use of carbimazole to control hyperthyroidism.     

Modulation of Fas-mediated apoptosis of human thyroid epithelial cells by IgG from patients with Graves' disease (GD) and idiopathic myxoedema

The expression of two autoimmune thyroid diseases, GD and idiopathic myxoedema, is associated with antibodies to the thyroid-stimulating hormone (TSH) receptor. Thyroid stimulating antibodies (TSAb) in GD are TSH agonists and cause hyperthyroidism as well as goitre, whereas thyroid stimulation blocking antibodies (TSBAb) in idiopathic myxoedema are TSH antagonists and cause hypothyroidism and thyroid atrophy. We investigated the effect of antibodies to TSH receptor on Fas-mediated apoptosis of thyroid epithelial cells (thyrocytes). Human IgG was isolated from healthy donors, patients with GD and idiopathic myxoedema. Human thyrocytes were obtained from surgical specimens. Thyrocytes were cultured in the presence or absence of human IgG with or without interferon-gamma (IFN-γ) or IL-1β for a specified time. After incubation, we examined the level of cAMP in cultured supernatants and both Fas and Bcl-2 expression on thyrocytes. In addition, we examined anti-Fas-mediated apoptosis of thyrocytes. Fas expression on thyrocytes was significantly down-regulated by Graves' IgG and TSH, although idiopathic myxoedema IgG did not affect Fas expression on thyrocytes. Idiopathic myxoedema IgG abrogated the effect of TSH on both cAMP production and inhibition of Fas expression on thyrocytes. Treatment of thyrocytes with IL-1β or IFN-γ caused a marked augmentation of Fas expression on thyrocytes. The increase of Fas expression of thyrocytes induced by IL-1β or IFN-γ was significantly suppressed in the presence of TSH or Graves' IgG. Anti-Fas-induced apoptosis of thyrocytes was observed in thyrocytes treated with IL-1β or IFN-γ, but was markedly inhibited in the presence of TSH or Graves' IgG. Furthermore, idiopathic myxoedema IgG abrogated most of the inhibitory effect of TSH on Fas-mediated apoptosis of thyrocytes treated with IL-1β or IFN-γ. Bcl-2 expression of thyrocytes did not change after stimulation with TSH, Graves' IgG, idiopathic myxoedema IgG, IL-1β or IFN-γ. These results suggest that TSAb found in Graves' patients may be potentially involved in the development of goitre by inhibition of Fas-mediated apoptosis of thyrocytes. In addition, TSBAb inhibit the action of TSH and increase the sensitivity toward Fas-mediated apoptosis of thyrocytes, inducing thyroid atrophy seen in patients with idiopathic myxoedema.     

An anti-idiotypic antibody against Graves' IgG

Rabbit antibodies were raised against Graves' IgG adsorbed onto a TSH-receptor affinity and eluted thereof by [3H]NaCl. The rabbit serum absorbed against normal human IgG (ARI) still bound to Graves', control and Hashimoto's IgG preparations but in the latter two, binding was inhibited by bTSH (10 mU/ml). In addition, ARI stimulated thyroid cell cyclic AMP accumulation in both human and rat thyroid cells. The ARI preparation may, therefore, contain an "internal image" anti-idiotype causing thyroid (Ab2) stimulation, a Graves' disease specific anti-idiotype whose binding with Ab2 inhibits its ability to bind TSH and anti-anti-idiotype (Ab3) to "internal image" Ab2. In further studies, Graves' specific cross-reactive idiotype was found in 10/11 IgGs from patients with active Graves' disease. This study emphasizes the workings of Jerne's immunologic network and the complexity of polyclonal "anti-idiotypic" antibodies.     

冷冻保存人甲状腺细胞的活力,功能和临床应用

本文研究冷冻保存不同时间甲状腺细胞的活力、功能及其在甲状腺刺激抗体（ＴＳＡｂ）检测中的应用。结果表明：冻存３、６和１２个月的正常或甲亢甲状腺细胞具有良好的活力，各冷冻时相的细胞对ＴＳＨ刺激生成ｃＡＭＰ的反应性无明显差异，且与未冷冻细胞的反应性相似。以冻存甲亢甲状腺细胞测定血清ＴＳＡｂ．在Ｇｒａｖｅｓ病（ＧＤ）未治组的检出率为０．９１３，而甲状腺腺瘤（甲瘤）组与对照组ＴＳＡｂ均为阴性。这提示冻存一年内的人甲状腺细胞仍保持较好的活性与功能，可以作为甲状腺功能和甲状腺疾病基础与临床研究的材料。     

Antibodies that promote thyroid growth. A distinct population of thyroid-stimulating autoantibodies

We used a strain of differentiated rat-thyroid cells in continuous culture (the FRTL-5 strain) to detect the presence of growth-promoting antibodies in serum samples from patients with autoimmune thyroid disease. We found that IgG preparations from 17 of 20 patients (85 per cent) with active Graves' disease and two of five patients (40 per cent) with Hashimoto's thyroiditis could augment thyroid-cell growth. In parallel with IgG-induced elevations in intracellular cyclic AMP levels in the same cell line, all 20 of the patients with active Graves' disease had thyroid-stimulatory antibodies. Patients' IgG preparations fell into three subclasses: those with both potent cyclic AMP stimulation and potent growth-promoting activity; those with potent cyclic AMP stimulation but low-level growth promotion; and those with potent growth promotion and low-level cyclic AMP action. Growth-promoting antibodies were not detected in patients with Graves' disease in remission (seven patients), nodular goiter (seven), subacute thyroiditis (five), or atrophic thyroiditis (one). Simultaneous assays of growth promotion and cyclic AMP stimulation may be useful in the care of patients with autoimmune thyroid disease.     

Thyroid antibodies are produced by thyroid-derived lymphocytes.

The significance of the characteristic lymphocytic infiltrate in the target organ in organ-specific autoimmune disease is unknown. We have demonstrated the production of thyroglobulin antibodies and immunoglobulins (IgG, IgM and IgA) by thyroid-derived lymphocytes in Graves' disease and Hashimoto's thyroiditis two plaque forming cell (PFC) assays. The thyroid appears to be an important site of thyroglobulin antibody production but the thyroid lymphocytes also contain many IgG PFCs of non-thyroglobulin specificity. Short-term culture and direct thyroglobulin antibody assay on micro-ELISA plates confirmed the results of the PFC assay. Therapies such as carbimazole may therefore be acting on a localized source of autoantibody production.     

Prediction of the course of Graves' disease after medical antithyroid treatment

The course of thyrotoxicosis in 33 patients with Graves' disease was evaluated clinically and biochemically (free thyroxine index, serum triiodothyronine, thyroid stimulating antibodies, (TSAb), thyroid stimulating hormone binding inhibiting immunoglobulins (TBII)). Relapse of the disease was found to be correlated to anamnestic information of thyrotoxicosis among first degree relatives (predictive value 90%) and to concomitantly raised levels of TSAb and TBII at the start of treatment (predictive value 71%). Mean duration of treatment of patients with long-lasting remission was 16.8 months. When comparing various information used to predict relapse of Graves' disease, anamnestic information of familial predisposition to thyrotoxicosis carries the highest predictive value.     

THE PUTATIVE ANTI-THYROTROPIN RECEPTOR ANTIBODIES OF GRAVES' DISEASE:I. Gm ALLOTYPES *

The hyperthyroidism of Graves' disease is thought to be related to antithyrotropin receptor (TSH-R) antibodies. In order to study the degree of immunogenetic homogeneity of these antibodies, we carried out Gm typing of ‘receptor-purified’ IgG from patients with active Graves' disease and controls. The results were compared to those of serum, total IgG and IgG which failed to attach to TSH-R. We found that in five out of seven Gm heterozygote patients studied the receptor-purified antibodies were restricted to the products of one haplotype compared to three out of five similar controls. Such eluted antibodies were biologically active. Similar results in terms of immunogenetic restriction and activity were obtained when F(ab)₂ preparations were used. An unexpected finding was that sera and IgG from normal persons attached to thyroid membranes and that the attachment occurred via F(ab)₂. Normal whole serum and ‘receptor-purified’ IgG and F(₂ inhibited TSH binding in the receptor assay; however, this inhibition showed no specificity for TSH-R.     

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.     

Adenovirus encoding the thyrotropin receptor A-subunit improves the efficacy of dendritic cell-induced Graves' hyperthyroidism in mice

Stimulating the immune system by in vivo expression of the thyrotropin receptor (TSHR) is an efficient means to induce Graves' disease experimentally. For example, BALB/c mice injected with dendritic cells (DCs) infected with adenovirus encoding the full-length TSHR (AdTSHR) develop hyperthyroidism, albeit at a low incidence (36%). Recent observations suggest that the shed TSHR A-subunit, rather than the full-length receptor, is the autoantigen responsible for initiating/enhancing immune responses leading to thyroid stimulating antibodies (TSAb) and hyperthyroidism. Therefore, we attempted to improve the efficacy of the DC-based approach for Graves' disease using adenovirus encoding the TSHR A-subunit (AdTSHR289). Three injections of DCs infected with AdTSHR289 induced hyperthyroidism in 70% of BALB/c mice, approximately twice the disease induction rate with AdTSHR. TSAb activity was detected in most hyperthyroid mice, whereas virtually all immunized mice developed antibodies that inhibit [125I]TSH binding to the TSHR or recognize linear or conformational epitopes on the TSHR. TSHR antibodies were of IgG1 and IgG2a, indicating mixed T-helper type 1 (Th1)/Th2 immune responses. In conclusion, immunization with DC infected with adenovirus expressing the TSHR A-subunit is a highly efficient protocol to induce Graves' hyperthyroidism in BALB/c mice. This improved model will permit studies of the pathogenic role and therapeutic potential of DCs in Graves' hyperthyroidism.