TSH-binding Inhibitory Immunoglobulin Research Articles

Graves' Hyperthyroidism Showing Transient Hypothyroidism during Interferon Therapy for Chronic Hepatitis Type C

We report a patient with Graves' disease in whom thyroid function was changed from initial hyperthyroidism to transient hypothyroidism and back to hyperthyroidism during interferon (IFN) therapy. A 43-year-old man was admitted to our hospital to receive IFN treatment for chronic active hepatitis (type C) in June 1998. His thyroid function was normal and testing for thyroid gland antibodies (TSH binding inhibitor immunoglobulins; TBII, anti-thyroglobulin antibodies; TgAb and anti-thyroid peroxidase antibodies; TPOAb) was negative before IFN therapy. The patient had neither history of thyroid disease nor any particular family history. He developed hyperthyroidism four months after its initiation of IFN therapy. When he was hyperthyroid, TBII, the activity of thyroid-stimulating antibodies (TSAb) and thyroid stimulation-blocking antibodies (TSBAb) were 40.2% (normal range, -15 approximately +15.0%), 1201% (normal range, <or=180%) and 52.0% (normal range, <or=45.6%), respectively. Thyroid 99mTc(technetium)-uptake ratio (Tc-UTR) was 1.07% (normal range, 0.5-3.0%). He transiently developed hypothyroidism in December 1998 and recurrent hyperthyroidism in February 1999. When he was hypothyroid, TBII, TSAb and TSBAb were 74.3%, 769% and 95.9%, respectively. To investigate the mechanism of his fluctuating thyroid status, we serially assessed the serum levels of cytokines (TNF-alpha, IFN-gamma, IL-1beta, IL-2, IL-4) and the soluble form of ICAM-1 (sICAM-1) as well as the activities of two types of TSH receptor antibodies (TRAb), TSAb and TSBAb, before and after IFN therapy. There were no characteristic changes of cytokines or sICAM-1 during the follow-up period. The transient hypothyroid state may be explained by two possible mechanisms: one may be due to the shift in the balance between the stimulating and blocking types of TRAb, and the other may be due to the complication of destructive thyroiditis that developed during IFN therapy.

Clinical results of anti‐inflammatory therapy in Graves’ ophthalmopathy and association with thyroidal autoantibodies*

Graves' ophthalmopathy (GO) is clinically associated with autoimmune thyroid disease, and autoantibodies to thyroidal antigens, especially to the TSH-receptor (TRAb), might be involved in the disease process. While there is mounting evidence that TRAb are associated with GO at the onset of the disease, so far no studies have looked at the association between thyroidal autoantibodies and the clinical outcome of GO therapy. The aim of this retrospective study was to evaluate whether TSH binding inhibitory immunoglobulins (TBII) and thyroid stimulating antibodies (TSAb) are still associated with the clinical activity and severity of GO after the completion of anti-inflammatory therapy. In addition, we wanted to elucidate whether thyroid peroxidase (TPO) or thyroglobulin (TG) autoantibodies (TPOAb and TGAb) are in any way related to GO. DESIGN PATIENTS AND MEASUREMENTS: Clinical activity score (CAS) and the severity of GO (modified NOSPECS score) were assessed in 108 patients with GO after steroid therapy and, if indicated, orbital irradiation. Patients were grouped according to their clinical presentation and autoantibody levels (TBII, TSAb, TPOAb and TGAb) were measured. After therapy for hyperthyroidism, all patients were clinically euthyroid but showed clear heterogeneity for GO 4-12 months after the end of anti-inflammatory therapy. Fifty-two patients had inactive GO, 41 had moderately active and 15 still had very active (non-responsive) GO. Concerning severity, 27 patients had mild GO, 64 moderately severe and 17 severe GO. TBII titres were still positive in 14 (93%) of 15 patients in the non-responsive group (CAS > 6) compared to 22 (42%) of 52 patients (P < 0.001) with post-therapeutic inactive GO (CAS </= 2). A similar result was seen between TBII levels and the NOSPECS score. The simultaneous presence of TBII and TSAb was associated with significantly higher activity and severity [odds ratios: 4.9 (activity); 9.0 (severity)] than the presence of TBII without measurable TSAb [odds ratios: 2.1 (activity), 2.0 (severity)] in comparison to absence of both antibodies. Only TBII and TSAb, but not TPOAb or TGAb medians, increased statistically significantly with CAS or NOSPECS scores. Both scores were positively associated with TBII (CAS: r = 0.31 P < 0.001; NOSPECS: r = 0.38, P < 0.0001) and, to a lesser degree, with TSAb (CAS: r = 0.27, P < 0.007, and NOSPECS: r = 0.29, P < 0.003). This association was independent of the treatment of hyperthyroidism, although highest levels of TBII were seen after radioiodine treatment. The NOSPECS score was negatively associated with TGAb (r =-0.27, P < 0.01) but not with TPOAb, while both showed no association with the CAS score. We conclude that the persistence of TBII and TSAb levels in patients with therapy-resistant disease in comparison to patients with inactive disease supports the role of TRAb in the pathogenesis of GO. Furthermore, the fact that, even after anti-inflammatory therapy, TBII and TSAb levels and prevalence still correlate with the severity and activity of GO suggests not only a trigger but also a possible role in the maintenance of the autoimmune process in the orbits.

Surgical treatment of Graves' disease in children.

Thyroidectomy may be indicated in children with Graves' disease who have adverse reactions to antithyroid drugs or who relapse after antithyroid drug therapy. We investigated the characteristics of childhood Graves' disease from the standpoint of surgical outcome. Between 1989 and 1998, 1897 patients with Graves' disease underwent thyroidectomy and their thyroid function could be evaluated 2 to 3 years after thyroidectomy. The patients were divided into three groups according to age at thyroidectomy: 74 patients were 15 years old or less (children), 345 patients 16 to 20 years of age (adolescents), and 1478 patients 21 years of age or more (adults). The children included higher proportions of patients who had a large goiter (> 100 g), high thyrotropin-binding inhibitory immunoglobulin (TBII) level (> 50%), and small remnant thyroid (< 4 g). At 2 to 3 years after thyroidectomy, the overt recurrence rate of the children, adolescents, and adults was 9.5%, 4.9%, and 5%, respectively. The cumulative recurrence-free rate of the children, adolescents, and adults at 5 years after thyroidectomy was 82%, 90%, and 92%, respectively. Surgical complications were more frequently observed in children. Considering the aggressiveness of childhood Graves' disease, subtotal thyroidectomy with thyroid remnant less than 3 g is the procedure of choice for preventing recurrent hyperthyroidism.

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.

バセドウ病と甲状腺癌合併症例の検討

We retrospectively reviewed our series of 390 consecutive patients operated on for Graves' disease from 1983 through 2002 at Tenri Hospital. Of the 390 patients, twelve (Group A) were found to have thyroid carcinoma, eleven papillary and one follicular, for an incidence of 3.1%. In 58% of these cases the tumors were less than 1cm in diameter and only one patient had recurrence in the cervical lymph nodes. All patients are alive without disease. There was no correlation between the TSH-binding inhibitory immunoglobulin titer and the progression of their carcinomas. Ninety-seven (Group B) patients who had been surgically treated for differentiated thyroid carcinoma from 1999 through 2002 were selected for comparison. The mean age at operation in Group A was 38.8 years, younger than that of Group B, 55.2 years. However, there was no difference in tumor size, and intrathyroidal, lymphatic and distant metastasis between the two groups. Our results suggest that Graves' disease is not associated with increased or rapid growth of concurrent thyroid carcinoma.

Graves' hyperthyroidism and thyroiditis in HLA-DRB1*0301 (DR3) transgenic mice after immunization with thyrotropin receptor DNA.

Familial and twin studies in Caucasians have established that the MHC class II allele HLA-DRB1*0301 (DR3) is a strong susceptibility gene in Graves' hyperthyroid disease (GD). To determine if a DR3 transgene could help establish an animal model for GD, we expressed DR3 molecules in class II-knockout NOD mice (H2Ag7-). DR3+g7- mice were given cardiotoxin prior to immunization on weeks 0, 3 and 6 with plasmid DNA encoding human thyrotropin receptor (TSHR). Two groups of mice were also coimmunized with plasmid DNA for IL-4 or GM-CSF. Serial bleeds on weeks 8, 11 and 14 showed that approximately 20% of mice produced thyroid-stimulating antibodies (Abs), and approximately 25% had elevated T4 levels. In particular, a subset displayed both signs of hyperthyroidism, resulting in approximately 30% with some aspect of GD syndrome. Additional mice had thyroid-stimulating blocking Abs and/or TSH-binding inhibitory immunoglobulins, while most mice showed strong labelling of TSHR+ cells by flow cytometry. Interestingly, lymphocytic infiltration with thyroid damage and Abs to mouse thyroglobulin were also noted. Vector controls were uniformly negative. Thus, DR3 transgenic mice can serve as a model for GD, similar to our earlier reports that this allele is permissive for the Hashimoto's thyroiditis model induced with human thyroglobulin.

Changes in thyroid function and immunological parameters long after subtotal thyroidectomy for Graves' disease.

To find out whether subtotal thyroidectomy results in long term stable functional and immunological remission in patients with Graves' disease. Retrospective study. Teaching hospital, Japan. 176 patients who underwent subtotal thyroidectomy for Graves' disease, 1970-79. Follow up surveys in 1984 and 1992. Changes in thyroid function, antibody titres, and lymphocyte subsets. 29/79 patients (39%) who were euthyroid in 1984 had evidence of thyroid dysfunction in 1992. Of the 8 patients with latent hypothyroidism in 1984, 3 (38%) had become euthyroid by 1992, and none required treatment. Of the 29 patients who were hypothyroid in 1984, 5 had latent hypothyroidism and 1 was euthyroid in 1992, and of the 18 patients with recurrent hyperthyroidism in 1984, 1 had become euthyroid by 1992. The number of positive titres to TSH-binding inhibitory immunoglobulin was significantly higher in the recurrence group (31/36, 86%) compared with the hypothyroid (7/26, 27%), latent hypothyroidism (8/37, 22%), and euthyroid (22/77, 29%) groups (p < 0.01). There were also significant differences in the mean (SD) number of Leu HLA DR subsets between the control (17 (3), n = 18) and recurrence (21 (6), n = 38), hypothyroid (22 (6), n = 35), latent hypothyroidism (22 (6), n = 22), and euthyroid (22 (9), n = 64) groups (p < 0.002). There were no differences in the number of T cell subsets among the groups. Treatment of Graves' disease by subtotal thyroidectomy does not necessarily result in stable late functional or immunological remission. Long term follow up of such patients may be necessary.

Seasonal Variation in Relapse Rate of Graves' Disease after Thionamide Drug Treatment

Controversy abounds on the issue of seasonal variation in new onset of Graves' disease, partly due to the difficulty of precisely dating the exact start of symptoms. To address the possible relationship between climatic changes and disease activity from a different perspective, we reviewed time of relapse during regular follow-up after successful drug treatment with thionamides. Retrospective analysis of a case series in a university clinic. We consecutively registered patients who experienced re-emergence of hyperthyroidism between 1992 and 2001 after successful antithyroid drug therapy. Excluded were subjects with superimposing painless thyroiditis, in postpartum, on immunomodulatory drugs, or off thionamides prematurely on their own volition. Fifty-two patients recurred 2 to 36 months after drug cessation. The frequency was higher in spring and summer (March to August) than in autumn and winter (September to February). With a new coated-tube radioreceptor assay, TSH binding inhibitor immunoglobulin activity was detected in sera from 87.5% of the reworsened patients. Graves' disease tends to relapse more frequently in spring and summer. Further clinical studies are warranted to clarify underlying mechanism (s) for this seasonal variation.

Un nouveau dosage des anticorps anti-récepteur de la TSH : le TRAK humain. Intérêt diagnostique et pronostique dans la maladie de Basedow

Hyperthyroidism in Gravesˈdisease is attributable to the presence of TSH-receptor antibodies : thyroid stimulating antibodies (TSAb) and TSH binding inhibiting immunoglobulins (TBII). A new assay for TBII using a recombinant human receptor is now commercially available (Dynotest TRAK human, Brahms). We have evaluated its analytical and clinical performances in the diagnostic and in the follow-up of Gravesˈdisease. We have also compared them to those obtained for porcine TRAK (TRAK assay) and for TSAb measured on cellular cultures. We studied here 154 patients with Gravesˈdisease before and after treatment with antithyroid drugs (ATD). The performances of TBII assay with human TRAK were higher than those obtained for porcine TRAK and sometimes for TSAb. Thus TBII shoud be now measured with this commercially kit. Moreover, this new assay can advantageously replace TSAb measurement in the diagnosis and in the follow-up of Gravesˈpatients treated with ATD.

Comparação entre diferentes métodos para avaliar a presença de auto-anticorpos dirigidos ao receptor de TSH em pacientes com moléstia de Graves-Basedow

Auto-anticorpos anti-receptores de TSH (TSHRAbs) foram avaliados por dois diferentes métodos [TRAb, como % de inibição de ligação do TSH por anticorpos séricos e produção de AMP cíclico em cultura de células CHO expressando o receptor de TSH humano recombinante (CHO-rhTSHR)] em 52 pacientes (36F/16M) com moléstia de Graves-Basedow (DGB), tanto antes do tratamento como aos 6 e 12 meses de terapia contínua com metimazol (40-60mg/dia) e L-tiroxina (100mig/dia); outros 20 pacientes tireotóxicos (12F/8M) foram tratados com doses individualizadas de radioiodo. Os TSHRAbs determinados pelo radioreceptorensaio foram positivos em 47/52 pacientes (90,4%) com valor médio±EPM de 56,7±3,9%, diminuindo significantemente aos 6 (40,5±3,2%) e 12 meses (43,5±4,7%) de terapêutica, bem como após radioiodo (30,7±4,5%). Os TSHRAbs foram discriminados pelo bioensaio em todos os 52 pacientes com DGB ativa (1122±409%). Após 6 e 12 meses de terapêutica houve decréscimo (não significante) dos valores iniciais. Nenhum indivíduo do grupo controle normal (n= 80) apresentou TSHRAbs detectado por qualquer dos métodos. Portanto, a sensibilidade da pesquisa dos anticorpos pelo bioensaio, nos 52 pacientes com DGB ativa, foi maior que nos mesmos indivíduos avaliados pelo radioreceptorensaio. Houve correlação positiva (r= 0,59; p&lt;0,001) entre TRAb e CHO-rhTSHR. Concluímos que a pesquisa dos TSHRAbs, realizada quer pelo radioreceptorensaio como pelo bioensaio, constitui recurso útil para se avaliar a atividade autoimune na DGB.

Clinical validity of a new commercial method for detection of TSH-receptor binding antibodies in sera from patients with Graves’ disease treated with antithyroid drugs

In this work, we compared the activities of TSH binding inhibitory immunoglobulin (TBII) results obtained with a new human TBII assay (h-TBII) using the human recombinant TSH receptor with thyroid stimulating antibodies (TSAbs). Sera were obtained from 90 patients with Graves’ disease before and after therapy with carbimazole (1-methyl-2-thio-3-carbethoxyimidazole). Before treatment, h-TBII were detected in 89/90 patients (98.9%) whereas TSAb activity was positive in 88/90 patients (97.7%). The two parameters fell during therapy. At the end of treatment, only h-TBII levels were significantly different between patients in remission and those in relapse ( Z=−2.212; P=0.0270). The relapse rate in the patients with positive antibodies at drug withdrawal was significantly increased ( χ 2=6.057; P=0.0139 for h-TBII and χ 2=8.988; P=0.0021 for TSAb). Most of patients (76%) relapsed during the 2 years following drug withdrawal. h-TBII or TSAb values were positive in 84.6% or in 80.8% of patients at the time of relapse. There was a significant correlation between the two antibody measurement methods before treatment, at drug withdrawal and at the time of relapse. These results indicate that the new TBII assay using human TSH receptor is as sensitive as the TSAb assay. Because of its much easier performance, it may advantageously replace TSAb measurement especially for the Graves’ disease diagnosis and in the prediction of short-term relapse at the end of treatment.

Detection of functionally different types of pathological autoantibodies against thyrotropin receptor in Graves' patients sera by luminescent immunoprecipitation analysis.

We describe a new method for the detection of different types of pathological autoantibodies against TSH receptor (TSHR) in Graves' patients sera by luminescent immunoprecipitation analysis. For this purpose three different chimeras composed of human TSHR and rat luteotropin/choriogonadotropin receptor (LH-CGR) were constructed, as was described previously (Tahara K, Ishikawa N, Yamamoto K, Hirai A, Ito K, Tamura Y, Yoshida S, Saito Y, Kohn LD. 1997 Thyroid 7:867-877). They were used in the immunoprecipitation reactions: (i) the wild type TSHR (for the detection of total TSHR autoantibodies), (ii) TSHR/LH-CGR chimera wherein TSHR amino acid residues 8-165 (epitopes for thyroid stimulating antibodies) are replaced by comparable LH-CGR residues, (iii) TSHR/LH-CGR chimera wherein TSHR amino acids 261-370 (epitopes for thyroid blocking antibodies) are replaced by comparable LH-CGR residues, and (iv) TSHR/LH-CGR chimera wherein TSHR amino acids 8-165 and 261-370 are replaced by comparable LH-CGR residues (for the detection of neutral TSHR autoantibodies). DNA encoding the N-terminal 725 (of 764) amino acids of wild type TSHR (or TSHR/LH-CGR chimera) was fused to the cDNA for the 550-amino acid firefly luciferase. The hybrid proteins were produced in HeLa cells using recombinant vaccinia viruses. All fusion proteins retained the enzymatic activity of firefly luciferase and TSHR-LUC interacted with TSH with the same affinity as wild type receptor. The luciferase tagged TSHR and TSHR/LH-CGR chimeras were used for the detection of different types of TSHR autoantibodies (i.e. total, neutral, thyroid stimulating and thyroid blocking) in 63 Graves' disease and 62 normal sera by immunoprecipitation analysis. The data demonstrated positive correlation between results of immunoprecipitation assay and results obtained using cAMP bioassay or assay for TSH binding inhibitory immunoglobulins in test sera.

Disseminated thyroid autonomy or Graves' disease: reevaluation by a second generation TSH receptor antibody assay.

The clinical diagnosis of disseminated autonomy (DISA) can only be established by exclusion of Graves' disease (GD). Both hyperthyroid conditions share the same scintigraphic appearance and can only be distinguished from each other clinically either by the presence or absence of endocrine ophthalmopathy (EO) or thyrotropin (TSH) binding inhibiting immunoglobulins (TBIIs). The purpose of this study was the reevaluation of thyroid autonomies originally classified as DISAs by a second-generation radioreceptor antibody assay (RAA) (DYNOtest TRAKhuman) (B.R.A.H.M.S. Diagnostika, Berlin, Germany). The analysis included 32 patients (female: n = 25, male: n = 7; mean age: 46 +/- 18 years) who were initially diagnosed with DISA. All patients were TSH receptor (TSHR) antibody (TRAb) negative by a conventional radioimmunoassay (RIA) (TSH-REZAK RIA) (Medipan Diagnostica, Selchow, Germany) during their first evaluation. The presence of EO was excluded by clinical signs in all patients. Surgery had been performed prior to our evaluation in 5 patients and after our survey in 1 patient. Four patients had been treated previously with 131I. Ten patients were treated with thionamides during our evaluation, and 13 had not been treated before. One hundred three patients who had either healthy thyroids, nontoxic goiters, or focal autonomies served as controls and were evaluated both by the TSH-REZAK assay and the DYNOtest TRAKhuman assay. Seven of thirty-two (22%) patients originally classified as DISA were TRAb positive in the second-generation assay. In this group, 5 of 7 patients had a total thyroid volume (TTV) <30 mL (positive predictive value [PPW] for TRAb positivity 71%), and 5 of 7 patients had a diffuse goiter (PPW for TRAb-positivity 71%). Six of seven patients were anti-thyroperoxidase (TPO) positive (PPW for TRAb positivity 85%). A hypoechoid pattern on ultrasound was present by visual analysis in 3 of 7 patients (PPW for TRAb positivity 43%). A 100% PPW for TRAb positivity could be obtained if a goiter <30 ml was combined with anti-TPO positivity, but this combination was present in only 4 of 7 (57%) patients. With the second-generation assay, one false positive test result was observed in the control group. Surgery was performed in 6 patients who were TRAb negative in both assays. In all these cases, the histologic findings were compatible with autonomous transformation of the thyroid. Our study demonstrates that a significant number (22%) of patients formerly classified as DISA may actually have GD. However, DISA still exists as a clinical entity, and its pathophysiological link to multifocal and unifocal autonomy should be further investigated.

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.

Differences between thyrotropin receptor antibody bioactivity and inhibition of 125I-bovine thyrotropin binding.

Thyrotropin (TSHR) receptor antibodies that bind to the TSHR without stimulating the TSHR have been identified with a direct binding assay. Moreover, TSHR antibodies that exhibit thyroid epithelial cell stimulation without inhibition of 125I-bovine thyrotropin (bTSH) binding and vice versa have been described. These data suggest that stimulation or blocking of the TSHR by stimulating (TSAB) or blocking (TSBAB) TSHR antibodies could be possible without detectable bTSH-displacement activity. However, to date, possible differences between TSAB or TSBAB activity and inhibition of 125I-bTSH binding have not been systematically investigated. Therefore we compared inhibition of 125I-bTSH binding and TSAB or TSBAB activity of sera from 113 patients with Graves' disease treated with antithyroid drugs. To exclude the different assay conditions of previous investigations as possible confounding factors, we determined TSAB or TSBAB and inhibition of 125I-bTSH binding (TBIIW) with the same Chinese hamster ovary (CHO) cells expressing the human TSHR. Furthermore inhibition of 125I-bTSH binding was also determined as thyrotropin-binding inhibitory immunoglobulin (TBII) with solubilized porcine thyroid membranes (TRAK, Brahms, Berlin Germany) and the highly sensitive recombinant human TSH receptor assay (hTRAK, Brahms, Berlin Germany). Only 78% (54/69) of TSAB-positive and 78% (21/27) of TSBAB-positive sera detected with JP26 cells exhibit inhibition of 125I-bTSH binding measured as TBII or TBIIW. Furthermore, 59% (10/17) of sera without TSAB and TSBAB activity revealed inhibition of 125I-bTSH binding measured as TBII or TBIIW. We found significant differences between TSHR bioactivities (TSAB or TSBAB) and inhibition of 125I-bTSH binding. Moreover, there was no agreement between the detectable TSHR bioactivities (TSAB or TSBAB) and their detectable inhibition of 125I-bTSH binding. Therefore, it is very likely that TSH displacement by TSHR antibodies and stimulation or blocking of the TSHR by TSHR antibodies are different functions that do not need to occur together.

Thyroid-stimulating antibody is related to Graves' ophthalmopathy, but thyrotropin-binding inhibitor immunoglobulin is related to hyperthyroidism in patients with Graves' disease.

We investigated the relationship between thyroid function or ophthalmopathy of Graves' disease and thyrotropin receptor antibodies (TRAb) in 155 untreated patients with Graves' hyperthyroidism. All patients were examined by ophthalmologists, and underwent computed tomography of the orbit and measurement of serum free triiodothyronine (FT3), free thyroxine (FT4), thyrotropin-binding inhibitor immunoglobulin (TBII), and thyroid stimulating antibodies (TSAb). Patients were divided into three groups according to the presence of orbital fat increase (OFI) and extraocular muscle enlargement (EME): 57 patients without OFI and EMO formed the no Graves' ophthalmopathy (NGO) group; 55 patients with OFI but without EMO formed the OF group; 43 patients with EME with or without OFI formed the EM group. The FT3, FT4, and thyroid weight increased in the order of the EME, NGO, and OFI groups. TSAb increased in the order of the NGO, OFI, and EME groups, and TSAb was significantly greater in the EME and OFI than in the NGO group. TBII was not significantly different among the three groups, but was lower in EME than NGO. There was a significant positive correlation between TBII and FT3 or FT4 in all patients combined as well as in all three groups, but correlation between TSAb and FT3 or FT4 was very weak in all groups, and that between TSAb and FT3 was not significant in the EM group In the relationship between ophthalmopathy and TRAb, the sum of the scores of eyelid swelling, proptosis, and extraocular muscle enlargement was taken as a measure of the overall severity of the Graves' ophthalmopathy (GO). TSAb was significantly correlated with the GO score, but there was no correlation between TBII and GO scores. In conclusion, TSAb was correlated with ophthalmopathy but TBII was related to hyperthyroidism.

The Hyperthyroid Fetus and Infant

After completing this article, readers should be able to: 1. Describe the etiology of transient and persistent neonatal hyperthyroidism. 2. Delineate the diagnostic tests for thyroid-stimulating hormone receptor antibodies. 3. Develop a treatment plan for fetal and neonatal hyperthyroidism and a management plan for subsequent pregnancies. 4. Provide guidelines for breastfeeding when the mother is receiving antithyroid medications. 5. Describe the long-term risks of neonatal hyperthyroidism. The first clinical description of neonatal hyperthyroidism was published in 1912, and the discovery of the transplacental passage of thyroid-stimulating immunoglobulins (TSI) provided an understanding for the etiology of this condition in 1964. There were, however, a few cases of persistent neonatal hyperthyroidism, initially described in 1976, that were inconsistent with the transient clinical course expected with a maternally acquired antibody. The etiology of persistent neonatal hyperthyroidism was clarified by the discovery of an activating germ-line mutation in the thyroid-stimulating hormone (TSH) receptor in 1995. When a mother has autoimmune thyroid disease, fetal and neonatal hyperthyroidism result from transplacental passage of TSI. The mother usually has a history of hyperthyroidism, but neonatal hyperthyroidism also has been reported in mothers who have hypothyroidism due to Hashimoto thyroiditis. The clinical status of the mother does not determine the infant’s risk for hyperthyroidism. The mother may have been treated with 131I or a subtotal thyroidectomy in the past and may be euthyroid or she may be receiving thyroid replacement therapy at the time of her pregnancy, but she still can be producing high titers of TSI. In a review of neonatal hyperthyroidism, mothers were clinically hyperthyroid during pregnancy in only 18 of 38 cases (47%). The prevalence of clinical hyperthyroidism in pregnancy has been reported to be 0.1% to 0.4% and is the most common endocrine disorder during pregnancy after diabetes. Clinical fetal and neonatal hyperthyroidism has been reported to occur …

Adjuvant Effects of Cholera Toxin b Subunit on Immune Response to Recombinant Thyrotropin Receptor in Mice

We had previously shown that BALB/c mice immunized with the extracellular domain of human thyrotropin receptor (ETSHR) developed moderate hyperthyroxinemia. The antibody responses in these mice were predominantly of the IgG1 subclass. Since cholera toxin B subunit (CT-B) has direct effects on the thyroid, and is known to activate B lymphocytes and cause enhanced IgG1 production, we tested the ability of CT-B to modulate the antibody response to ETSHR. CT-B is unique in that it not only elicits a strong immune response to itself, but more importantly, when given with other antigens acts as a potent adjuvant. In the present study, BALB/c mice given ETSHR with CFA or CT-B via ip route showed higher titers of antibodies to ETSHR when compared to mice similarly immunized with ETSHR alone, or with IFA. Antibodies in ETSHR+CT-B immunized mice were mostly of the IgG1 subclass and reacted predominantly with ETSHR peptides 1 (aa 22–41), 21 (aa 322–341), and 23 (352–371). In contrast, animals immunized with ETSHR+CFA showed IgG1, IgG2a and IgG2b responses and reacted with peptides 1 and 21. Furthermore, mice immunized with ETSHR along with CT-B showed significantly higher levels of thyrotropin (TSH) binding inhibitory immunoglobulins (TBII) compared to those that did not receive CT-B. None of the mice immunized with a control antigen showed antibody response to ETSHR. These results suggested that CT-B could enhance and modulate immune response to ETSHR.

The combination of absent thyroid peroxidase antibodies and high thyroid-stimulating immunoglobulin levels in Graves' disease identifies a group at markedly increased risk of ophthalmopathy.

Among Graves' Disease (GD) patients, we have observed an unexpectedly high prevalence of antithyroperoxidase antibody (TPOAb) and antithyroglobulin antibody (TgAb) negativity in those with severe ophthalmopathy. To study the possible role of thyroid autoantibodies in the pathogenesis of Graves' ophthalmopathy (GO), TPOAb, TgAb, thyroid-stimulating immunoglobulin (TSI), and thyrotropin-binding inhibitory immunoglobulin (TBII) levels were measured, and the presence or absence of GO was assessed by a single observer in 100 consecutive patients with newly diagnosed, untreated GD who were nonsmokers. Ophthalmopathy was present in 43 patients. TSI levels (p = 0.001), and the prevalence of TPOAb-negativity (p = 0.002) were significantly higher in patients with ophthalmopathy compared to those without. Logistic regression analysis showed that TSI levels (p = 0.005) and the absence of TPOAb (p = 0.0025) were independent predictors of GO. No correlation between TBII or TgAb and eye disease was found. The prevalence of GO increased with each quartile of TSI levels. The prevalence was 20%, 36%, 52%, and 64% in the first, second, third and fourth quartiles of TSI, respectively. The odds ratio of GO (with 95% confidence intervals) when TSI levels were above the median level (1640%) was 3.6 (1.5-8.0), when TPOAb was negative it was 5.0 (1.7-14.4), and with both risk factors it was 36.6 (4.3-313.5). The prevalence of ophthalmopathy in this last group was 92.9%. The combination of negative TPOAb and high TSI levels appears to be associated with a markedly increased risk of clinically evident ophthalmopathy.

Regulation of thyrotropin receptor protein expression in insect cells.

Expression of large quantities of conformationally intact thyrotropin receptor (TSHR) is essential to understand the structure-function relationship of the receptor. We expressed three different constructs of full-length human TSHR in insect cells: (a) a TSHR cDNA lacking signal sequence (TSHR-ns), (b) a TSHR cDNA containing human TSHR signal sequence (TSHR-hs) and (c) a TSHR cDNA with baculovirus envelope protein encoded signal sequence gp-67 (TSHR-gp). No unique protein band, corresponding to any of these recombinant proteins, was visible upon Coomassie Blue staining after SDS-PAGE. However, Western blot using TSHR specific monoclonal antibody showed unique bands around 80, 100 and 100 kDa in TSHR-ns, TSHR-hs and TSHR-gp virus infected insect cells respectively. All three full-length TSHR proteins could neutralize the TSH binding inhibitory immunoglobulin (TBII) activity from sera of experimental animals. However, only glycosylated proteins (TSHR-hs and TSHR-gp) neutralized the TBII activity of sera from autoimmune thyroid patients, confirming the importance of glycosylation for patient autoantibody reactivity. Expression levels of full-length TSHR proteins were much lower than the levels of similarly produced corresponding ectodomains of TSHR proteins. Southern blot and Northern blot analyses showed that DNA and RNA levels in full-length TSHR virus infected insect cells were comparable to the levels found in cells infected with viruses encoding only the ectodomain of TSHR. These data suggest that full-length TSHR expression is very low and is regulated at the translational level.