Thyrotropin Binding Inhibitor Immunoglobulin Activities Research Articles

Changes in epitopes for thyroid-stimulating antibodies in Graves' disease sera during treatment of hyperthyroidism: therapeutic implications.

To determine whether there are changes in epitope recognition by stimulating TSH receptor antibodies (TSHRAbs) during treatment of hyperthyroidism and to evaluate the clinical relevance of such changes, we serially measured the activity of IgG preparations from 39 patients with Graves' disease over an 8-month period. To measure epitope changes of the stimulating TSHRAbs, we used Chinese hamster ovary (CHO) cells transfected with wild-type human TSHR (hTSHR) or TSHR chimeras with residues 90-165 (Mc2) substituted by equivalent residues of the rat LH/CG receptor. When initially examined, 37 of the 39 patients had significant stimulating TSHRAb activity measured with wild-type CHO-hTSHR cells. Serial measurements of stimulating TSHRAb activity in Mc2 chimera-transfected cells divided the 39 patients into three distinct groups. Thus, 10 patients (heterogeneous epitope group) exhibited low but significant activity in Mc2 chimera assays at the start of the study; 10 patients who were initially negative in Mc2 chimera assays remained negative (persistently homogeneous epitope group); and 19 patients who were initially negative in Mc2 chimera assays became transiently or persistently positive during treatment, despite a simultaneous decrease in TSHRAb activity measured with wild-type TSHR (changing epitope group). The functional stimulating TSHRAb epitope thus changed from residues 90-165 to residues outside this region in the last group, which comprises nearly two-thirds of the initially Mc2-negative patients (19 of 29) and one-half of all patients (19 of 39). Patients in the changing epitope group responded more quickly and to lower doses of methimazole than patients in the persistently homogeneous epitope group, behaving in this respect exactly as the patients in the heterogeneous epitope group. Additionally, although the decrease in stimulating TSHRAb activities during the 8-month treatment period was similar in the two groups, the thyrotropin binding inhibitor immunoglobulin (TBII) activities decreased more rapidly in patients in the persistently homogeneous epitope group than in patients in the changing epitope group (P < 0.05). There were no differences in initial stimulating TSHRAb or TBII activities, degree of hyperthyroidism, goiter size, or prior duration of symptoms between the persistently homogeneous epitope group and changing epitope group. In summation, we show that the epitopes of stimulating TSHRAbs in Graves' disease patients may change during their clinical course or treatment period, and that the change is from antibodies recognizing N-terminal TSHR residues 90-165 to antibodies recognizing other regions of the TSHR. We also show that the development of stimulating TSHRAbs with this heterogeneous epitope or their presence at the initial screening for disease activity seems to be associated with increased responsiveness to antithyroid drug therapy. We suggest, therefore, that Mc2 chimera assays may be useful to predict the response of patients to antithyroid drug therapy.

Possible binding site of thyrotropin binding inhibitor immunoglobulin (TBII) on the thyrotropin (TSH) receptor, which is different from TSH binding site

A synthetic decapeptide, P-194, which has the sequence No. 103 to 111 of hTSH receptor structure with an additional N-terminal tyrosine, did not bind TSH nor affected its receptor binding and thyroid stimulating activity. Preincubation of P-194 with sera from thyroid patients caused a significant decrease in TBII activity in almost all 12 TBII positive sera and an increase of thyroid stimulating activity in 3 of 7 Graves' IgG studied. In addition, [125I] P-194 bound to serum IgG fraction from thyroid patients with a positive correlation with TBII (N = 35, r = 0.509, p less than 0.01). The P-194 portion may be, at least a part of, TBII binding site distinct from the TSH binding site on the TSH receptor.

Changes in the Properties of the Thyrotropin Receptor Antibody in Patients with Graves` Disease after Radioiodine Treatment

We investigated the effect of a single dose of 131I upon thyrotropin receptor antibodies (TRAb) in 21 patients with Graves’ disease. The thyrotropin receptor antibodies were assessed by parallel measurements of thyrotropin binding inhibitor immunoglobulin (TBII), thyroid stimulating antibody (TSAb) and thyroid stimulation blocking antibody (TSBAb) in serum by radoreceptor assay, stimulation of adenlate cyclase and inhibition of TSH-stimulated adenylate cyclase activation in FRTL-5 cells, respectively. Prior to radioiodine treatment, TBII was detected in all 21 patients and TSAb in 19 patients. After radioiodine treatment, TBII activities did not change during the 12-months observation period, but in eight patients TSAb activities gradually decreased and were undetectable at the end of the 12-month observation period. Persistence of TSAb was not associated with the clinical outcome. Eight patients developed hypothyroidism within one year after radioiodine treatment. Three of the hypothyroid patients developed TSBAb, and the appearance of TSBAb coincided with the development of hypothyroidism. These results suggest that TSBAb might develop after radioiodine treatment in a minority of patients with Graves’ disease and that the appearance of TSBAb, in addition to radiation-induced thyroid destruction, might be involved in the development of hypothyroidism following radioiodine treatment.

Changes in Thyrotropin Receptor Antibody after Subtotal Thyroidectomy in Graves' Disease: Comparison with the Degree of Lymphocytic Infiltration in the Thyroid

To evaluate changes in TSH receptor antibody after surgery in Graves' disease and its relationship with the degree of lymphocytic infiltration, serial serum levels of TSH receptor antibody were measured before and after the subtotal thyroidectomy in 50 patients with Graves' disease. In 22 (44%) out of 50 patients, thyrotropin binding inhibitor immunoglobulin (TBII) levels gradually decreased and disappeared completely within 12 months after surgery (TBII disappearing group). Twenty-eight (56%) patients showed persistent TBII activity and their levels were not changed until 12 months after surgery (TBII persistent group). The changes of thyroid stimulating antibody (TSab) levels were very similar to those of TBII in both groups. The thyroidal lymphocytic infiltration was more prominent in the TBII disappearing group. The degree of the decrease of TBII levels after surgery correlated with the grade of thyroidal lymphocytic infiltration. There was no significant difference of TSH receptor antibody (both TBII and TSab) levels between the thyroid and peripheral venous blood. These data suggest that the persistence or disappearance of TSH receptor antibody after surgery may reflect the difference between patients in whom the thyroid is the major site of TSH receptor antibody and those in whom additional sites of TSH receptor antibody synthesis exist.

Clinical significance of anti-TSH antibody in sera from patients with Graves’ disease and other thyroid disorders

In a survey of patients having anti-TSH antibody (TSH Ab), data from 167 subjects were collected from 8 Japanese Institutions. They were divided into a high TSH Ab group consisting of 63 cases; since the means of assay was via a subnormal thyrotropin binding inhibitor immunoglobulin (TBII) assay, this group had TBII values less than -20%. An additional low TSH Ab group was made up of 104 cases. Out of a total of 11,211 patients, the incidence of TSH Ab for the high and low groups were 0.57% and 13.4%, respectively. More than 95% of these TSH Ab carriers had Graves' disease or some other autoimmune thyroid disorder, and anti-thyroglobulin and anti-thyroid microsomal antibodies were detected similarly in both groups. It was significant that TSH receptor antibodies could also be detected in both groups, namely, thyroid stimulating antibody and long acting thyroid stimulator (LATS) in 4 of 9 patients in the high TSH Ab group and TBII in 55 of 104 in the low TSH Ab group, respectively. The high TSH Ab levels tended to persist, but 26% of cases showed disappearance or appearance of the antibody during the observation period. In one Graves' patient, a moderate TBII activity (64.2%) was followed by markedly elevated TSH Ab (TBII: -83.4%) within 2 months. The TSH Ab in the low TSH Ab group disappeared in most cases. Also, fluctuations in TSH Ab did not always parallel those seen for TBII and reciprocal fluctuation pattern (transient or otherwise) were observed in 33%. In conclusion, anti-TSH antibody is produced frequently in patients with either Graves' disease or some other autoimmune thyroid disorder.(ABSTRACT TRUNCATED AT 250 WORDS)

Clinical studies on abnormal thyroid stimulators in patients with Graves' disease. I. A sensitive assay for thyroid-stimulating antibodies using cultured porcine thyroid cells and polyethylene glycol precipitation of serum

The activities of thyroid-stimulating antibody (TSAb) in serum from patients with Graves' disease were measured by a sensitive assay, using cultured porcine thyroid cells and the precipitation from serum with polyethylene glycol (PEG), and the activities were compared with those of thyrotropin binding inhibitor immunoglobulin (TBII), measured by the commercial assay kit. Porcine thyroid cells after digestion were cultured for 15-18 hours with TSH of 1-10,000 microU/ml or the precipitations of sera from normal subjects and patients with Graves' disease or Hashimoto's thyroiditis, and then the cAMP levels in the culture medium were determined by the commercial RIA assay kit (Yamasa). The precipitation was obtained by adding 0.5 ml of 30% PEG solution to 0.5 ml serum, and was resuspended with 0.6 ml of Hanks' medium without NaCl, containing 1.5% bovine serum albumin, 20mM Hepes and 0.5 mM 3-isobutyl-1-methylxanthine. The precipitation contained about 85% of immunoglobulin and 63% of albumin of the original amount of the serum, as well as substantial TSH, when the original serum contained TSH more than 40 microU/ml. When the PEG precipitations from 10 normal subjects were incubated with the thyroid cells of 4 X 10(5) cells, the cAMP releases into the medium ranged from 83 to 124%, when the mean value was calculated as 100%. Therefore, the cAMP release of more than 130% of the amount released into the culture medium incubated with normal IgG was judged as positive TSAb activity. The minimum detectable quantities were regarded as about 5 microU/ml TSH equivalent. TSAb and TBII activities were detected in 48 (92%) and 50 (96%) of 52 patients with untreated hyperthyroid Graves' disease, respectively, and either TSAb or TBII activities were detected in 16 (80%) of 20 patients with Graves' disease maintained in a clinically euthyroid state by treatment with antithyroid drugs. TBII was positive in 10(50%) of these patients. Some patients showed distinct discrepancies in these two activities, although there was a significant positive correlation between TSAb and TBII activities (r = 0.53, p less than 0.01) in patients with untreated Graves' disease. In these patients, TSAb activities showed a significant positive correlation with values for 99mTc thyroid uptake, determined 30 min after the injection. However, they did not show any significant correlation with serum T4 or T3 concentrations. Similarly, TBII showed significant correlations with goiter size and 99mTc thyroid uptake. To conclude, the present assay for TSAb is sensitive and reproducible.(ABSTRACT TRUNCATED AT 400 WORDS)

Clinical significance of elevated labeled TSH binding (LTB) activity in sera of patients with Graves' disease and other thyroid disorders.

Labeled TSH binding (LTB) of individual serum samples was monitored simultaneously using the thyrotropin binding inhibitor immunoglobulin (TBII) assay. In 643 TBII determinations, 86 sera (13.4%) showed elevated LTB. The incidence of elevated LTB in active Graves' patients (17.5%) was much higher than that of inactive Graves' patients (6.7%). After TBII activities were corrected by LTB, 79% of the active Graves' patients who had negative raw TBII were found to be positive. In patients with untreated active Graves' disease, the detectability of TBII increased from 85% to 91% after LTB correction, while those in inactive Graves' and other thyroid disorders did not increase so much (1.6 and 0%, respectively). Further, most of elevated LTB seen in other thyroid disorders were found to be different from those in Graves' disease by heat stability experiment. Serial observations of LTB and TBII in 24 Graves' patients showed 2 patterns. Parallel alterations were observed in 13 patients and reciprocal alterations in 11 patients. Patients showing parallel alteration had smaller goiter and were more sensitive to antithyroid drugs than those showing the latter pattern.

Pathophysiological role of thyroid blocking antibody in patients with primary hypothyroidism.

The pathophysiological role of thyroid blocking antibody (TBAb) in patients with adult primary hypothyroidism and the mechanism of TBAb action were studied. A sensitive bioassay for TBAb, which inhibits the TSH-induced cAMP accumulation, was established using normal human thyroid cells in culture. Thirty-four patients with primary hypothyroidism consisting of 17 goitrous and 17 non-goitrous patients were examined. Two out of 17 goitrous patients (11.8%) and three out of 17 non-goitrous patients (17.6%) were TBAb positive. There were no significant differences between TBAb positive and negative patients in terms of the severity of hypothyroidism or the titers of MCHA or TGHA. Four out of the five TBAb-positive IgGs had strongly positive thyrotropin binding inhibitor immunoglobulin activities. All five TBAb-positive IgGs inhibited the cAMP increase induced by Graves' IgG, but did not affect the action of either prostaglandin E1 or cholera toxin. However, three TBAb positive IgG also inhibited the cAMP increase induced by forskolin. These findings indicate: 1) TBAb is present in hypothyroid patients with autoimmune thyroiditis and TBAb may play a role in the pathophysiology of these patients. 2) TBAb may inhibit the action of TSH not only at the level of the TSH receptor, but also at a different site from the TSH receptor.

Detection of anti-thyroid plasma membrane antibody in patients with various thyroid diseases by solid phase enzyme-immunoassay

Antibodies against thyroid cell surface antigens have been demonstrated by indirect immunofluorescence on viable cells and by mixed hemadsorption using monolayer cell culture. Recently quantitative assays using thyroid plasma membrane or cultured thyroid cell were also reported. The present study reports a novel quantitative assay for the detection of anti-thyroid plasma membrane antibody (APA) using solubilized and immobilized thyroid plasma membrane. Thyroid plasma membrane purified by sucrose density gradient centrifugation was solubilized with Triton X-100 and coupled to CNBr activated Sepharose 4B. Sera from patients with various thyroid diseases were incubated with this solid phase. Five microliters of serum was sufficient for the assay. After extensive washing, immunoglobulin G (Ig G) or immunoglobulin M (Ig M) bound to thyroid plasma membrane was detected by horseradish-peroxidase labeled rabbit anti-human Ig G or Ig M antibody. Significantly elevated values of Ig G class APA (Ig G-APA) were detected in the sera from patients with Graves' or Hashimoto's disease. Some of the patients with thyroid adenoma also showed positive Ig G-APA, although all of the patients with thyroid cancer had negative Ig G-APA. A significant correlation between Ig G-APA and anti-thyroid microsomal antibody (AMA) among patients with autoimmune thyroid diseases was observed. No significant correlation, however, was found between anti-thyroglobulin antibody (ATA) and Ig G-APA. A large amount of thyroglobulin (Tg), which was reported to have the receptor in thyroid plasma membrane, inhibited the binding of Ig G-APA does dependently. Ten micrograms of ATA and one milligram of bovine serum albumin, however, had no influence on the binding. Ig G-APA also significantly correlated with thyrotropin binding inhibitor immunoglobulin (TBII). The binding, however, was not inhibited by one hundred milliunits of TSH. These findings suggested that most of Ig G-APA was bound to different sites from TSH receptor, although some part of Ig G-APA might have TBII activity. Ig M class APA (Ig M-APA) among various thyroid diseases was also detected. In Graves' disease, more than sixty percent of patients had positive Ig M-APA. There was no significant difference in the value of Ig M-APA between untreated and treated patients. Positive Ig M-APA was found even in some euthyroid patients. Ig M-APA in Graves' disease correlated with neither AMA nor TBII. Some of patients with Hashimoto's disease and subacute thyroiditis also showed positive Ig M-APA.(ABSTRACT TRUNCATED AT 400 WORDS)