Changes of TSH-Stimulation Blocking Antibody (TSBAb) and Thyroid Stimulating Antibody (TSAb) Over 10 Years in 34 TSBAb-Positive Patients with Hypothyroidism and in 98 TSAb-Positive Graves' Patients with Hyperthyroidism: Reevaluation of TSBAb and TSAb in TSH-Receptor-Antibody (TRAb)-Positive Patients

Two TRAbs: TSBAb and TSAb. TSBAb causes hypothyroidism. TSAb causes Graves' hyperthyroidism. TSBAb and TSAb block TSH-binding to cells as TRAb, measured as TSH-binding inhibitory immunoglobulin (TBII). We reevaluate TSBAb and TSAb. We studied TSBAb, TSAb, and TBII over 10 years in 34 TSBAb-positives with hypothyroidism and in 98 TSAb-positives with hyperthyroidism. Half of the 34 TSBAb-positives with hypothyroidism continued to have persistently positive TSBAb, continued to have hypothyroidism, and did not recover from hypothyroidism. Ten of the 98 TSAb-positives with hyperthyroidism continued to have positive TSAb and continued to have hyperthyroidism. TSBAb had disappeared in 15 of the 34 TSBAb-positives with hypothyroidism. With the disappearance of TSBAb, recovery from hypothyroidism was noted in 13 (87%) of the 15 patients. TSAb had disappeared in 73 of the 98 TSAb-positives with hyperthyroidism. With the disappearance of TSAb, remissions of hyperthyroidism were noted in 60 (82%) of the 73. Two of the 34 TSBAb-positives with hypothyroidism developed TSAb-positive Graves' hyperthyroidism. Two of the 98 TSAb-positive Graves' patients with hyperthyroidism developed TSBAb-positive hypothyroidism. TSBAb and TSAb are TRAbs. TSBAb-hypothyroidism and TSAb-hyperthyroidism may be two aspects of one disease (TRAb disease). Two forms of autoimmune thyroiditis: atrophic and goitrous. We followed 34 TSBAb-positive patients with hypothyroidism (24 atrophic and 10 goitrous) over 10 years. All of the 10 TSBAb-positive goitrous patients recovered from hypothyroidism and 19 (79%) of the 24 TSBAb-positive atrophic patients continued to have hypothyroidism.

It has been generally believed that Graves' patients have TSAb but do not have TSBAb, and that blocking antibody-(TSBAb-) positive patients with hypothyroidism have TSBAb but do not have TSAb. However, TSBAb-positive patients with hypothyroidism and TSAb-positive Graves' patients with hyperthyroidism could have both TSBAb and TSAb [13]. Some patients may have TSBAb and TSAb simultaneously or sequentially [13]. The balance of TSBAb and TSAb determines whether a patient has hypothyroidism or hyperthyroidism [13]. We have encountered TSBAb-positive patients with hypothyroidism, who developed TSAb-positive Graves' hyperthyroidism, and also TSAb-positive Graves' patients with hyperthyroidism, who developed TSBAbpositive hypothyroidism. Thyroid function can oscillate between hypothyroidism and hyperthyroidism as TSBAb or TSAb becomes dominant.
There are two forms of autoimmune thyroiditis: atrophic autoimmune thyroiditis and goitrous autoimmune thyroiditis [3]. It has become evident that hypothyroidism may occur as a result of the production of TSBAb. TSBAb has been said to cause hypothyroidism in the patients with atrophic autoimmune thyroiditis [3]. However, TSBAb has been found in patients with atrophic autoimmune thyroiditis, and also in patients with goitrous autoimmune thyroiditis [11]. TSBAb was detected in 25% of the patients with atrophic autoimmune thyroiditis and in 9% of those with goitrous autoimmune thyroiditis [3]. TSBAb causes hypothyroidism. With the disappearance of TSBAb, recovery from hypothyroidism has been reported [3]. Here, we followed 24 TSBAbpositive hypothyroid patients with atrophic autoimmune thyroiditis and 10 TSBAb-positive hypothyroid patients with goitrous autoimmune thyroiditis over 10 years. All of the 10 TSBAb-positive patients with goitrous autoimmune thyroiditis recovered from hypothyroidism and 19 (79%) of the 24 TSBAb-positive patients with atrophic autoimmune thyroiditis continued to have hypothyroidism.
We reevaluated TSBAb and TSAb in TRAb-positive patients. We studied serial changes of TSBAb and TSAb over 10 years in 34 TSBAb-positive patients with hypothyroidism and in 98 TSAb-positive Graves' patients with hyperthyroidism. With persistently positive TSBAb, recovery from hypothyroidism was not observed. With persistently positive TSAb, remissions of Graves' hyperthyroidism were not obtained. With the disappearance of TSBAb, recovery from hypothyroidism was seen. With the disappearance of TSAb, remissions of Graves' hyperthyroidism were also seen. Two of the 34 TSBAb-positive patients with hypothyroidism developed TSAb-positive Graves' hyperthyroidism. Two of the 98 TSAb-positive Graves' patients with hyperthyroidism developed TSBAb-positive hypothyroidism. TSBAb-positive hypothyroidism and TSAb-positive hyperthyroidism may be two aspects of one disease (TRAb disease).

Subjects and Method
2.1. Subjects. We studied 34 TSBAb-positive patients with hypothyroidism and 98 TSAb-positive Graves' patients with hyperthyroidism ( Table 1). The 34 TSBAb-positive patients with hypothyroidism were treated with thyroxine (T4) and the 98 TSAb-positive Graves' patients with hyperthyroidism were treated with antithyroid drugs (ATDs). Serial changes of TSBAb and TSAb over 10 years were studied in 34 TSBAbpositive patients with hypothyroidism (I) and in 98 TSAbpositive Graves' patients with hyperthyroidism (II). TSBAbpositive patients with hypothyroidism were diagnosed on the basis of the history, signs of hypothyroidism, and the laboratory findings, including positive TSBAb (>+40%) and decreased serum-free thyroxine (fT4) and free triiodothyronine (fT3) with high TSH [3,13]. The diagnosis of goitrous autoimmune thyroiditis was based on the finding of palpable goiter and that of atrophic autoimmune thyroiditis on the absence of goiter [3]. The 34 TSBAb-positive patients with hypothyroidism were treated with thyroxine (T4). Thyroxine was discontinued at 3 months after the disappearance of TSBAb. After the discontinuation of T4, the patients had been seen every 1-3 months. When the patients continued to be in euthyroid states and to have negative TSBAb and negative TBII for more than 1 year after the T4discontinuation, they were considered to have recovery from hypothyroidism; otherwise, they had recurrence [3]. When serum TSH became higher than 10 mIU/L, T4administration was restarted [3]. TSAb-positive Graves' patients with hyperthyroidism were diagnosed on the basis of the history, signs of hyperthyroidism with diffuse goiter, and the laboratory findings, including positive TRAb (TSAb and/or TBII) and elevated fT4 and fT3 with low TSH [1,2]. The 98 Graves' patients were treated with antithyroid drugs (ATDs). They had been treated with ATD over several years. ATD was discontinued at 6 months after the TSAb-disappearance. After the discontinuation of ATD, the patients had been seen every 1-3 months. When the patients continued to be in euthyroid states and to have negative TSAb and negative TBII for more than 1 year after the ATD-discontinuation, they were considered to be in remission; otherwise, they had recurrence [14]. When they had recurrence, ATD-treatment was restarted. We had followed these 34 TSBAb-positive patients with hypothyroidism and 98 TSAb-positive Graves' patients with hyperthyroidism over 10 years.
To measure TSBAb-activities, crude IgG was incubated with porcine thyroid cells in the presence of 25 μU bTSH (100 mU/L, final concentration), as before [3,[10][11][12][13]15]. Cyclic AMP production during 5 h incubation was measured. TSBAb-activity was expressed as percentage inhibition of bTSH-stimulated cAMP production by test IgG. TSBAbactivity was calculated as follows: [3,[10][11][12][13]15], where a: cAMP generated in the presence of normal IgG and bTSH, b: cAMP generated in the presence of normal IgG, and c: cAMP generated in the presence of test IgG and bTSH. Test IgG and normal IgG were the 12.5% PEG-precipitated fraction from test serum and normal human serum, respectively. TSBAb, described in this report, corresponds to TSBAb-A in the previous report [13]. TSBAb activities were studied in 95 normal subjects (normal values were less than +40%) [13]. TSBAb activities were more than +40% in all of the TSBAb-positive patients with hypothyroidism. TSAb activity was expressed as percentage cAMP production compared with the mean values for 125 normal subjects (normal values were less than 180%) [1,2,14]: where b: cAMP generated in the presence of normal IgG, and d: cAMP generated in the presence of test IgG.

Statistical Analysis and
Others. All samples were tested in duplicate or triplicate. Statistical analysis was performed using Student's t-test or χ 2 -test. P values less than 0.05 were considered to be statistically significant. Serum-free T3, -free T4, and TSH were determined by electrochemiluminescence immunoassays (ECLIAs) (Roche Diagnostics, Tokyo, Japan). Normal reference ranges are as follows: fT3 3.5-6.6 nmol/L, fT4 11.6-21.9 pmol/L, and TSH 0.4-4.20 mIU/L. The study plan was reviewed and approved by our institutional review committee. Written informed consent was obtained from the patient prior to publication of this paper.

98 TSAb-Positive Graves' Patients with Hyperthyroidism (II)
With the disappearance of TSAb, 60 (IIc1) (82%) of the 73 patients, in whom TSAb had disappeared (IIc), got remissions of Graves' hyperthyroidism. Figures 3d1 and  3d2 show the changes of TSAb and TSBAb, respectively, in the 2 patients with TSAb-positive Graves' hyperthyroidism, who developed TSBAb-positive hypothyroidism (IId) ( Table 1, IId). In these 2 patients, TSAb was dominant initially (Figure 3(d1)), and then TSBAb became dominant (Figure 3(d2)). The 2 patients had TSAb-positive Graves' hyperthyroidism and then developed TSBAbpositive hypothyroidism. They were treated with MMI, and then treated with T4. Figure 4 demonstrates the clinical course of one of these 2 patients with TSAb-positive Graves' hyperthyroidism, who developed TSBAb-positive hypothyroidism (Table 1 hypothyroidism. TSAb was dominant initially (Figure 4(a)), and then TSBAb became dominant (Figure 4(b)). She had TSAb-positive Graves' hyperthyroidism with undetectable serum TSH and then developed TSBAb-positive hypothyroidism with high TSH. She was treated with MMI and then treated with T4. She had a goiter over 10 years.

Discussion
We TSBAb blocks TSH-stimulation of the thyroid and causes hypothyroidism. TSAb stimulates the thyroid and causes Graves' hyperthyroidism. Both TSBAb and TSAb block TSHbinding to thyroid cells as TSH receptor antibodies (TRAbs), which have been measured as TSH-binding inhibitory immunoglobulin (TBII) [1][2][3]13]. TBII reflects TSBAb-and TSAb-activities. TBII measures the binding of antibody to TSH receptor by competition with radiolabeled TSH and does not distinguish between TSBAb and TSAb. TSBAb is measured as a TSH-stimulation blocking assay and TSAb as a stimulator assay. TSBAb is a blocking antibody [3,13] and TSAb is a stimulating antibody [1,2,13].
All of the 34 TSBAb-positive patients with hypothyroidism and all of the 98 TSAb-positive Graves' patients had positive TBII (TRAb). TSBAb and TSAb are TSH-receptor antibodies (TRAbs), which have been measured as TBII. TBII does not distinguish between TSBAb and TSAb. TBII reflects TSBAb-and TSAb-activities [1][2][3]13]. All of the 34 TSBAb-positive patients with hypothyroidism had strongly positive TSBAb. Some of them had positive TSAb [13]. All of the 98 Graves' patients had positive TSAb. Some of them had positive TSBAb [13]. TSBAb-positve patients with hypothyroidism had narrow distributions of TSBAb and TSAb, and Graves' patients with hyperthyroidism had wide distributions of TSBAb and TSAb [13]. TSBAb-positive patients with hypothyroidism have strongly positive TSBAb.
TBII reflects TSBAb-and TSAb-activities [1][2][3]13]. Some of the TBII-positive patients have hypothyroidism, and the other TBII-positive patients have hyperthyroidism. The former TBII is TSBAb, and the latter TBII is TSAb. The numbers of the former TSBAb-positive patients with hypothyroidism are less than those of the latter TSAbpositive Graves' patients with hyperthyroidism. All of the TSBAb-positive patients with hypothyroidism have high titers of TBII, which is TSBAb [3]. Almost all of the untreated Graves' patients with hyperthyroidism have TBII, which is TSAb [1,2]. TSBAb-(TRAb-) positive hypothyroidism and TSAb-(TRAb-) positive Graves' hyperthyroidism may be two aspects of one disease (TRAb disease).
Hypothyroidism may result from the production of TSBAb [3]. In 1992, we followed 21 TSBAb-positive patients with hypothyroidism over 10 years and found that with the disappearance of TSBAb, recovery from hypothyroidism was noted in 6 (40%) of the 15 TSBAb-positive patients [3]. Here, we followed 34 TSBAb-positive patients with hypothyroidism over 10 years and found that with the disappearance of TSBAb, recovery from hypothyroidism was noted in 13 (87%) of the 15 patients. The frequency of recovery from hypothyroidism with the disappearance of TSBAb in this paper is much higher than that in the previous one [3]. With the disappearance of TSBAb, recovery from hypothyroidism is observed. The production of TSBAb may subside, producing remissions of hypothyroidism.
It is important to know whether a patient with Graves' disease gets remission or not during ATD treatment. Disappearance of TSAb predicted the remissions of Graves' hyperthyroidism [14]. With the disappearance of TSAb, 36 (82%) of the 44 patients were reported to get remissions in the previous paper [14] and 60 (82%) of the 73 patients are reported to get remissions in this paper. Disappearance of TSAb predicts the remissions of Graves' hyperthyroidism.
In Japan, TRAb has been measured as TBII and TSAb [14]. TSAb is a bioassay, using porcine thyroid cells. We usually measure TSAb, using a commercially available kit [14]. In Japan, TSAb-assay kit is available, but TSBAbassay kit is not. When a patient has hypothyroidism with elevated TSH and positive TBII, this TBII is thought to be TSBAb. We usually do not measure TSBAb. Practically, when a patient with hypothyroidism has positive TBII, this TBII may be TSBAb. When a patient with hyperthyroidism has positive TBII, this TBII may be TSAb. TSAb and TSBAb can be used to document TRAb-function. TBII, measuring the antibody-binding to the receptor by competition with radiolabeled TSH, does not distinguish between TSAb and TSBAb. A positive TBII result in a patient with hypothyroidism is evidence for the presence of TSBAb. A positive TBII result in a patient with hyperthyroidism is evidence for the presence of TSAb. These bioassays (TSAb and TSBAb) are useful to detect transient neonatal hyperthyroidism and hypothyroidism [10] and are also important to confirm the causes of hyperthyroidism and hypothyroidism [13]. TBIIpositive patients may have TSBAb or TSAb. Thyroid function can oscillate between hypothyroidism and hyperthyroidism as TSBAb or TSAb becomes dominant. TSAb and TSBAb can be used to document TRAb-function [13].
There are two forms of autoimmune thyroiditis: atrophic autoimmune thyroiditis and goitrous autoimmune thyroiditis [3]. We followed 34 TSBAb-positive patients with hypothyroidism (24 patients with atrophic autoimmune thyroiditis and 10 with goitrous autoimmune thyroiditis) over 10 years. TSBAb has been found in patients with atrophic autoimmune thyroiditis, and also in patients with goitrous autoimmune thyroiditis [11]. All of the 10 TSBAbpositive patients with goitrous autoimmune thyroiditis recovered from hypothyroidism and 19 (79%) of the 24 with atrophic autoimmune thyroiditis continued to have hypothyroidism. With the disappearance of TSBAb, recovery from hypothyroidism has been seen. TSBAb-positive hypothyroid patients with goitrous autoimmune thyroiditis may recover from hypothyroidism, and those with atrophic autoimmune thyroiditis may continue to have hypothyroidism.