0021-972X/78/4702-0361$02.00/0 Journal of Clinical Endocrinology and Metabolism Copyright © 1978 by The Endocrine Society
Vol. 47, No. 2 Printed in U.S.A.
Thyroid-Stimulating Antibodies in Patients with Autoimmune Disorders C. R. STRAKOSCH,* DIANNE JOYNER, AND J. R. WALL Department of Medicine, University of Queensland, Repatriation General Hospital, Greenslopes, Brisbane, Australia, 4120 ABSTRACT. A radioreceptor assay was used to measure thyroid-stimulating antibody (TSAb) in 1) patients with Graves' disease with untreated hyperthyroidism, selected for absence of clinically significant eye disease; 2) patients with Graves' ophthalmopathy, with and without previously treated hyperthyroidism; 3) patients with other thyroid disorders; 4) patients with other autoimmune disorders; and 5) normal subjects. TSAb was detected in 14 of 15 (93%) patients with Graves' hyperthyroidism and in 10 of 16 (63%) patients with Graves' ophthalmopathy. Of the patients with Graves' ophthalmopathy, TSAb was detected in 9 of 10 patients who had once been hyperthyroid and in only 1 of 6 patients who had never been hyperthyroid (euthyroid Graves' disease). TSAb was detected in 1 patient with
idiopathic Addison's disease (autoimmune adrenalitis) and in 1 patient with juvenile diabetes mellitus (both of whom were euthyroid), and borderline levels were found in 1 patient with Sjogren's syndrome and 1 patient with methyldopa-induced antired blood cell antibodies. TSAb was not detected in normal subjects or patients with other thyroid disorders. The conclusions are: 1) the test is very useful in the diagnosis of Graves' disease; 2) Graves' eye disease may be a frequently associated but separate disorder; and 3) because TSAb may be present in some euthyroid patients with other autoimmune disorders, TSAb production may occur primarily because of a disorder in the immune system. (J Clin Endocrinol :361, 1978)
I
T IS well established that Graves' disease is an autoimmune disorder in which thyroid-stimulating antibodies (TSAb) play an important role in the pathogenesis of the hyperthyroidism (1-3). These antibodies have the unique property of binding to, and stimulating, the TSH receptor on the plasma membrane component of the thyroid cell (1, 4). Competition between TSAb and TSH for these sites has led to the development of a radioreceptor assay for human TSAb (1, 4-6). In the original study of Smith and Hall, it was claimed that TSAb, as detected by this method, was present in 88% of patients with untreated Graves' hyperthyroidism (1). The prevalence was later increased to 100% (7). Subsequent studies by Schleusener and coworkers (8) and Bryson and coworkers (9) demonstrated TSAb in only 67% and 47% of cases, respectively. It is clearly important to determine the true prevalence of TSAb in Graves' hyperthyroidism because of the diagnostic potential of the test. A second important question to be answered is whether TSAb Received April 27, 1977. * To whom all correspondence and requests for reprints should be addressed.
can be present in patients who do not have Graves' hyperthyroidism, such as patients with other autoimmune disorders, in whom a variety of other antibodies are detected. LATS (measured by a bioassay) has been detected in an occasional patient with other autoimmune disease (10, 11), but this has not been confirmed using the more sensitive radioreceptor assay. This study was undertaken 1) to determine the prevalence of TSAb in patients with Graves' disease and 2) to investigate the occurrence of TSAb in euthyroid patients with other autoimmune disorders.
Clinical Subjects The study concerned: 1) 15 patients (13 females, 2 males), aged 21-69 yr (mean age, 44 yr), with untreated (hyperthyroid) Graves' disease, selected for absence of clinically significant ophthalmopathy [American Thyroid Association class 2 or less (12)]; 2) 16 patients (13 females, 3 males), aged 29-76 yr (mean age, 50 yr), with clinically significant Graves' ophthalmopathy (class 3 or more), 10 of whom had had hyperthyroidism (now treated) and 6 of whom had never been hyperthyroid (euthy-
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STRAKOSCH, JOYNER, AND WALL
roid Graves' disease); and 3) 17 patients (14 females, 3 males), aged 18-72 yr (mean age, 49 yr) with other thyroid disorders, namely, "cold" nodule (7 cases), Hashimoto's thyroiditis (4 cases), toxic nodule (3 cases), multinodular goiter (2 cases), and hydatidiform mole (1 case). The 3 patients with toxic nodule and the patient with hydatidiform mole were hyperthyroid at time of study. Twenty-seven patients (14 females, 13 males; aged 18-80 yr; mean age, 46 yr) with other autoimmune disorders were also studied: rheumatoid arthritis (10 cases), pernicious anemia (3 cases), Sjogren's syndrome (2 cases), juvenile diabetes mellitus (2 cases), idiopathic Addison's disease (2 cases), myasthenia gravis (2 cases), polymyositis (1 case), systemic lupus erythema tosus (1 case), Goodpasture's syndrome (1 case), idiopathic hypoparathyroidism (1 case), mucocutaneous candidiasis (1 case), and methyldopa-induced antired blood cell antibodies (1 case). Seven patients (2 females, 5 males; aged 53-79 yr; mean age 64 yr) with lepromatous leprosy were also studied. Twenty-one normal subjects (6 females, 15 males; aged 21-86 yr; mean age, 37 yr) were studied as controls.
Materials and Methods The method used for assay of TSAb was that of Smith and Hall (1), with minor modifications. Briefly, the immunoglobulin (Ig) fraction was isolated from serum by the addition of 3.75 M ammonium sulphate to a final concentration of 1.6 M. The precipitate was dissolved in distilled water and dialysed against 10 IDM Tris and 50 mM NaCl, pH 7.5, for 18 h. The protein concentration was measured by the optical density method and adjusted to 30 mg/ml. Human thyroid plasma membranes were obtained at operation from thyrotoxic thyroid tissue of patients with Graves' hyperthyroidism and stored at —20 C until use. Two grams of tissue was minced to a slurry with scissors, homogenised, passed through voile cloth, and further homogenised with 10 strokes of a glass-glass Dounce homogeniser. The homogenate was then centrifuged at 10,000 X g for 15 min and the pellet was suspended in buffer consisting of 10 mM Tris, 50 mM NaCl, and 1 mg/ml bovine serum albumin (BSA, pH 7.5), adjusted to a final concentration of 15 mg equivalents in 50 jul. Bovine TSH (a gift from Dr. Simon Manley) was iodinated using the lactoperoxidase method to a
JCE & M • 1978 Vol 47 • No 2
specific activity of about 80 /iCi/jug. The crude labeled TSH was then passed through a 50 x 1-cm Sephadex G-100 column and aliquots were purified by the membrane affinity method described by Smith and Hall (1). In the assay, 200 /xl test Ig preparation and 50 fxl thyroid plasma membrane suspension were added to plastic tubes and incubated in a water bath for 10 min at 37 C. Fifty microliters of [125I]TSH (approximately 10,000-20,000 counts/5 min) was then added and the tubes were incubated in the water bath for a further 90 min. Bound/free separation was obtained by passing a 200-/xl aliquot of the reaction mixture through SMI Sartorius cellulose nitrate filters which had been previously soaked in 5% BSA in 10 mM Tris, pH 7.5. The filters were counted in a y-counter for 5 min. The results were expressed as a TSAb index, namely: percentage [125I]TSH bound in the presence of test Ig — nonspecific binding percentage [I25I]TSH bound in the presence of pooled normal Ig - nonspecific binding The nonspecific binding was that binding which occurred in the presence of 1000 mlU TSH/ml and was from 10-12%. Binding of [125I]TSH in the presence of pooled normal Ig was from 35-40% which was 2-10% less than that which occurred in the presence of Tris-NaCl-BSA buffer alone. Tests were carried out in duplicate. Serum T4 levels were measured by the competitive protein-binding technique. Statistical analyses were carried out using the Mann-Whitney test.
Results The mean TSAb index in 21 normal subjects was 1.01 ± 0.08 SE (Fig. 1). The range was ...
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FIG. 1. Serum TSAb in 1) patients with Graves' hyperthyroidism selected for absence of eye disease; 2) patients with Graves' ophthalmopathy; 3) patients with other thyroid disease; 4) patients with Lepromatous leprosy, and 5) normal subjects. The results are expressed as TSAb indices. An index of 0.64 was considered positive. Horizontal bars represent mean values.
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TSAb IN AUTOIMMUNE DISORDERS 0.64-1.50; thus, for the purposes of this study, an index of less than 0.64 was considered positive for TSAb. The mean index of the 15 patients with hyperthyroid Graves' disease was 0.41 ± 0.05 SE. This is significantly less than the mean TSAb for normal subjects (Mann-Whitney, P < 0.001). Fourteen patients were regarded as TSAb-positive. The 16 patients with Graves' ophthalmopathy were found to have a mean TSAb of 0.55 ± 0.08 SE, which is significantly less than that of normals (P < 0.001). Of these patients, TSAb was found in 9 of 10 patients who had once had hyperthyroidism but were now euthyroid as a result of thyroidectomy or treatment with radioactive iodine, and in only 1 of 6 patients who had never been hyperthyroid (Fig. 2). The 27 patients with autoimmune disorders were found to have a mean TSAb index of 1.00 ± 0.07 SE, which is not significantly different from that of normals. Two patients, 1 with idiopathic Addison's disease and 1 with juvenile diabetes mellitus, were TSAb-positive, and 2 patients, 1 with Sjogren's disease and 1 with methyldopa-induced antired cell antibodies, had borderline levels
363
of TSAb. The mean TSAb index in the 17 patients with other thyroid disorders was 0.99 ± 0.05 SE, which is not significantly different from that of normals, and TSAb was not detected in any of these patients. The mean TSAb index in the 7 patients with leprosy was significantly higher (P < 0.01) than that of normals, at 1.13 ± 0.08 SE, and TSAb was not detected in any of these patients. All patients with other autoimmune disorders were clinically and biochemically euthyroid. Thyroid function tests were performed at the time of study in the patients with other autoimmune disorders in whom TSAb was detected. The results were: 1) patient with idiopathic Addison's disease who was taking oral contraceptives at the time—serum T4, 11.0 jtig/dl (normal range, 5-13 jug/dl); RT3U, 24% (normal range, 22-34%);! 2) patient with juvenile diabetes mellitus—serum T4, 7.2 /xg/ dl; RT3U, 28%; 3) patient with Sjogren's disease—serum T4, 6.1 jug/dl; RT3U, 29%; and 4) patient with antired cell antibodies—serum T4, 6.7 /xg/dl; RT3U, 28%. Discussion TSAb was demonstrated in 14 of 15 patients (93%) with untreated Graves' hyperthyroidism. This is similar to the findings of Mukhtar and his colleagues (7) who found TSAb in 100% of their patients. The smaller prevalences in other series (8, 9) could be due to technical factors or to different criteria for the diagnosis of Graves' disease. In our series, hyperthyroidism due to Graves' disease was initially diagnosed on clinical grounds and confirmed by demonstrating elevated serum thyroid hormone levels and a diffusely large thyroid on 131I scan, which was performed on all patients. The high prevalence of TSAb in our series confirms that the test may be very useful in the diagnosis of Graves' hyperthyroidism. Thus, absence of serum TSAb probably ex-
a) GRAVES'
b) OPHTHALMOPATHY
FIG. 2. Serum TSAb in patients with Graves' ophthalmopathy a) who had never been hyperthyroid (euthyroid Graves' disease) and b) who had once been hyperthyroid but were now euthyroid.
1 At follow-up 12 months after the study, the patient with Addison's disease, in whom high levels of TSAb were detected, complained of anxiety. Examination revealed a fine tremor and lig lag. Repeat thyroid function test results were: serum T4) 11.5 jug/dl; RT3U, 22%; and T3 RIA, 3.1 ng/dl (normal range, 1.0-2.0 ng/dl). TRH stimulation test confirmed hyperthyroidism.
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eludes the diagnosis. Furthermore, because disorders in whom TSAb was demonstrated ^ TSAb often disappears after treatment, espe- were clinically and biochemically euthyroid, cially after thyroidectomy (7), reappearance TSAb is presumably able to bind to the TSH A receptor (at least in vitro) but not cause stimmay indicate a relapse of the disease (1). TSAb was detected in 10 of 16 patients ulation of the thyroid gland. The development (63%) with Graves' ophthalmopathy. The of hyperthyroidism in these patients may deprevalence is similar to that obtained by Teng pend on additional factors. For example, it is and colleagues (13). TSAb was detected in 9 possible that excess TSAb may act as a of 10 of these patients who had once been "blocking" agent, or cell-mediated immune < hyperthyroid but were now euthyroid and in reactions against thyroid antigens may also be only 1 of 6 who had always been euthyroid. involved (3, 26). The finding of a group of euthyroid patients Acknowledgments with Graves' ophthalmopathy without TSAb lends support to the view that the eye disease We thank Dr. Simon Manley for his advice and for the may be a separate, but frequently associated, gift of TSH, Mr. Hambling for measurement of serum T4 V levels, and Dr. G. Cavaye for the statistical analyses. disorder. TSAb was also detected in one patient with References idiopathic Addison's disease and one patient B. R., AND R. HALL, Thyroid-stimulating immunowith juvenile diabetes mellitus. Although sub- 1. SMITH, globulins in Graves' disease, Lancet 2: 427, 1974. A clinical Graves' disease was not excluded in 2. KENDALL-TAYLOR, P., LATS and human-specific thyroid * stimulator; their relation to Graves' disease, J Clin Endocrithese patients, as serum T3RIA was not meanol Metab A: 319, 1975. sured and TRH tests were not performed, the 3. VOLPE, R., N. R. FARID, C. VON WESTARP, AND V. V. Row, The pathogenesis of Graves' disease and Hashimoto's thypatients were clinically euthyroid and TSAb roiditis, Clin Endocrinol 3: 239, 1974. was an incidental finding. It is well known that 4. MANLEY, S. W., J. R. BOURKE, AND R. W. HAWKER, The thyrotropin receptor in guinea-pig thyroid homogenate: interpatients with organ-specific autoimmune disaction with the long acting thyroid stimulator, J Endocrinol Y 61: 437, 1974. orders are likely to develop antibodies to other 5. MEHDI, S. Q., AND S. S. NUSSEY, A radio-ligand receptor organs. Graves' disease is associated with assay for the long acting thyroid stimulator, Biochem J 145: "f 105, 1975. other autoimmune diseases (10, 14-17) and 6. KOTULLA, P., F. ADLKOFER, E. AULBERT, H. MEINHOLD, AND LATS has been described in a few patients H. SCHLEUSENER, Radio ligand receptor assay for human thyroid stimulating immunoglobulins, Acta Endocrinol with other autoimmune disorders (10, 18). fSupplJ (Kbh) 82: 49, 1976. Demonstration of TSAb in patients with other 7. MUKHTAR, E. D., B. R. SMITH, G. A. PYLE, R. HALL, AND P. VICE, Relation of thyroid-stimulating immunoglobulins to autoimmune disorders suggests that TSAb, at thyroid function and effects of surgery, radioiodine and antileast in this situation, is not produced as a thyroid drugs, Lancet 1: 713, 1975. result of change in, or release of, thyroid an- 8. SCHLEUSENER, H., P. KOTULLA, H. SORGE, H. MEINHOLD, AND F. ADLKOFER, Relationship between thyroid status and -d tigen, but as a result of some abnormality of Graves' disease specific immunoglobulins, Acta Endocrinol [SupplJ (Kbh) 82: 25, 1976. the immune system, for example a deficiency 9. BRYSON, J. M., A. JOASOO, AND J. R. TURTLE, The thyrotroof suppressor T cell function (19, 20). pin receptor in human thyroid plasma membranes: effect of serum immunoglobulins, Acta Endocrinol (Kbh) 83: 528, Two other diseases in which increased prev1976. alences of autoantibodies are frequently 10. FREY, H. M. M., J. H. VOGT, AND J. NERUP, Familial poly' endocrinopathy, Acta Endocrinol (Kbh) 72: 401, 1973. found, although not primarily autoimmune, M., J. M. MCKENZIE, AND S.SALISBURY-MURPHY, ^, were also investigated. Firstly, a variety of 11. ZAKARIJA, LATS or thyroid-stimulating immunoglobulin as a prognostic index of relapse in hyperthyroidism of Graves' disease, Acta autoantibodies, particularly to adrenal and Endocrinol fSupplJ(Kbh) 82: 24, 1976. parathyroid gland are often detected in pa- 12. WERNER, S. C, Modification of the classification of the eye ( changes of Graves' disease: recommendations of the ad hoc tients with chronic mucocutaneous candidicommittee of the American Thyroid Association, J Clin Enasis (21). In the patient tested in this study, docrinol Metab 44: 203, 1977. TSAb was not detected. Secondly, leproma- 13. TENG, C. S., B. R. SMITH, B. CLAYTON, D. C. EVERED, F. CLARK, AND R. HALL, Thyroid stimulating antibodies (TSAb) ^ tous leprosy is also associated with a wide in ophthalmic Graves' disease. Correlation with other aspects of thyroid function, Acta Endocrinol [SupplJ (Kbh) 82: 23, variety of autoantibodies (22-25). Again, 1976. TSAb was not found in the seven patients 14. FURSZYFER, J., L. T. KURLAND, W. M. McCONAHEY, AND L. studied. Because patients with autoimmune R. ELVEBACK, Graves' disease in Olmsted Country, Minne-
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TSAb IN AUTOIMMUNE DISORDERS sota, 1935 through 1967, Mayo Clin Proc 45: 636, 1970. 15. ARDBMAN, S., I. CHANARIN, B. KRAFCHIK, AND W. SINGER,
Addisonian pernicious anaemia and intrinsic factor antibodies in thyroid disorders, Q J Med 35: 421, 1966. 16. MARSHALL, J. S., A. S. WEISBERGER, R. P. LEVY, AND R. T.
BRECKENRIDGE, Coexistent idiopathic thrombocytopenic purpura and hyperthyroidism, Ann Intern Med 67: 411,1967. 17. WALL, J. R., S. L. FANG, B. A. WALTERS, C. R. STRAKOSCH,
S. H. INGBAR, AND L. E. BRAVERMAN, Multi-system immunologically mediated disease, T lymphocyte deficiency and thyroid immunologic disease: a report of 4 cases, Aust NZ J Med, in press. 18. WALL, J. R., Immunological aspects of thyrotoxicosis, MD Thesis, Adelaide University, 1971. 19. TAYLOR, R. B., AND A. BASTEN, Suppressor cells in humoral immunity and tolerance, Br Med Bull 32: 153, 1976. 20. PENHALE, J. W., A. FARMER, R. P. MCKENNA, AND W.
J.
IRVINE, Spontaneous thyroiditis in thymectomised and irradiated Wistar rats, Clin Exp Immunol 15: 225, 1973.
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21. BLIZZARD, R. W., AND J. H. GIBBS, Candidiasis: studies pertaining to its association with endocrinopathies and pernicious anaemia, Pediatrics 42: 231, 1968. 22. BONOMO, L., AND F. DAMMACCO, Characterisation studies of thyroglobulin antibodies in leprosy. An immunological study of diethyl aminoethylcellulose chromatographic fractions, Immunology 13: 565, 1967. 23. MATHEWS, L. J., AND J. R. TRAUTMAN, Clinical and serological profiles in leprosy, Lancet 2: 915, 1965. 24. RUGE, H. G. S., G. FROMM, F. FUHNER, R. FARD, AND R. S.
B. GUITO, Serological findings in leprosy. An investigation into the specificity of various serological lists for syphilis, Bull WHO 23: 793, 1960. 25. WALL, J. R., AND D. J. M. WRIGHT, Testicular germinal cell antibodies in patients with lepromatous leprosy, Clin Exp Immunol 17: 51, 1974. 26. CALDER, E. A., AND W. J. IRVINE, Cell-mediated immunity and immune complexes in thyroid disease, J Clin Endocrinol Metab 4: 287, 1975.
Eighth International Thyroid Congress February 5-8, 1980, Sydney, Australia Initial announcements are at present being circulated. To receive the second announcement abstract and registration forms early in 1979, notify the Secretariat of your interest. This congress will be followed on the 10th of February by the Sixth International Congress of Endocrinology in Melbourne. Secretariat:
PO Box 783 Canberra City, A.C.T. 2601 Australia
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Vol. 47, No. 2 Printed in U.S.A.
Thyroid-Stimulating Antibodies in Patients with Autoimmune Disorders C. R. STRAKOSCH,* DIANNE JOYNER, AND J. R. WALL Department of Medicine, University of Queensland, Repatriation General Hospital, Greenslopes, Brisbane, Australia, 4120 ABSTRACT. A radioreceptor assay was used to measure thyroid-stimulating antibody (TSAb) in 1) patients with Graves' disease with untreated hyperthyroidism, selected for absence of clinically significant eye disease; 2) patients with Graves' ophthalmopathy, with and without previously treated hyperthyroidism; 3) patients with other thyroid disorders; 4) patients with other autoimmune disorders; and 5) normal subjects. TSAb was detected in 14 of 15 (93%) patients with Graves' hyperthyroidism and in 10 of 16 (63%) patients with Graves' ophthalmopathy. Of the patients with Graves' ophthalmopathy, TSAb was detected in 9 of 10 patients who had once been hyperthyroid and in only 1 of 6 patients who had never been hyperthyroid (euthyroid Graves' disease). TSAb was detected in 1 patient with
idiopathic Addison's disease (autoimmune adrenalitis) and in 1 patient with juvenile diabetes mellitus (both of whom were euthyroid), and borderline levels were found in 1 patient with Sjogren's syndrome and 1 patient with methyldopa-induced antired blood cell antibodies. TSAb was not detected in normal subjects or patients with other thyroid disorders. The conclusions are: 1) the test is very useful in the diagnosis of Graves' disease; 2) Graves' eye disease may be a frequently associated but separate disorder; and 3) because TSAb may be present in some euthyroid patients with other autoimmune disorders, TSAb production may occur primarily because of a disorder in the immune system. (J Clin Endocrinol :361, 1978)
I
T IS well established that Graves' disease is an autoimmune disorder in which thyroid-stimulating antibodies (TSAb) play an important role in the pathogenesis of the hyperthyroidism (1-3). These antibodies have the unique property of binding to, and stimulating, the TSH receptor on the plasma membrane component of the thyroid cell (1, 4). Competition between TSAb and TSH for these sites has led to the development of a radioreceptor assay for human TSAb (1, 4-6). In the original study of Smith and Hall, it was claimed that TSAb, as detected by this method, was present in 88% of patients with untreated Graves' hyperthyroidism (1). The prevalence was later increased to 100% (7). Subsequent studies by Schleusener and coworkers (8) and Bryson and coworkers (9) demonstrated TSAb in only 67% and 47% of cases, respectively. It is clearly important to determine the true prevalence of TSAb in Graves' hyperthyroidism because of the diagnostic potential of the test. A second important question to be answered is whether TSAb Received April 27, 1977. * To whom all correspondence and requests for reprints should be addressed.
can be present in patients who do not have Graves' hyperthyroidism, such as patients with other autoimmune disorders, in whom a variety of other antibodies are detected. LATS (measured by a bioassay) has been detected in an occasional patient with other autoimmune disease (10, 11), but this has not been confirmed using the more sensitive radioreceptor assay. This study was undertaken 1) to determine the prevalence of TSAb in patients with Graves' disease and 2) to investigate the occurrence of TSAb in euthyroid patients with other autoimmune disorders.
Clinical Subjects The study concerned: 1) 15 patients (13 females, 2 males), aged 21-69 yr (mean age, 44 yr), with untreated (hyperthyroid) Graves' disease, selected for absence of clinically significant ophthalmopathy [American Thyroid Association class 2 or less (12)]; 2) 16 patients (13 females, 3 males), aged 29-76 yr (mean age, 50 yr), with clinically significant Graves' ophthalmopathy (class 3 or more), 10 of whom had had hyperthyroidism (now treated) and 6 of whom had never been hyperthyroid (euthy-
361
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STRAKOSCH, JOYNER, AND WALL
roid Graves' disease); and 3) 17 patients (14 females, 3 males), aged 18-72 yr (mean age, 49 yr) with other thyroid disorders, namely, "cold" nodule (7 cases), Hashimoto's thyroiditis (4 cases), toxic nodule (3 cases), multinodular goiter (2 cases), and hydatidiform mole (1 case). The 3 patients with toxic nodule and the patient with hydatidiform mole were hyperthyroid at time of study. Twenty-seven patients (14 females, 13 males; aged 18-80 yr; mean age, 46 yr) with other autoimmune disorders were also studied: rheumatoid arthritis (10 cases), pernicious anemia (3 cases), Sjogren's syndrome (2 cases), juvenile diabetes mellitus (2 cases), idiopathic Addison's disease (2 cases), myasthenia gravis (2 cases), polymyositis (1 case), systemic lupus erythema tosus (1 case), Goodpasture's syndrome (1 case), idiopathic hypoparathyroidism (1 case), mucocutaneous candidiasis (1 case), and methyldopa-induced antired blood cell antibodies (1 case). Seven patients (2 females, 5 males; aged 53-79 yr; mean age 64 yr) with lepromatous leprosy were also studied. Twenty-one normal subjects (6 females, 15 males; aged 21-86 yr; mean age, 37 yr) were studied as controls.
Materials and Methods The method used for assay of TSAb was that of Smith and Hall (1), with minor modifications. Briefly, the immunoglobulin (Ig) fraction was isolated from serum by the addition of 3.75 M ammonium sulphate to a final concentration of 1.6 M. The precipitate was dissolved in distilled water and dialysed against 10 IDM Tris and 50 mM NaCl, pH 7.5, for 18 h. The protein concentration was measured by the optical density method and adjusted to 30 mg/ml. Human thyroid plasma membranes were obtained at operation from thyrotoxic thyroid tissue of patients with Graves' hyperthyroidism and stored at —20 C until use. Two grams of tissue was minced to a slurry with scissors, homogenised, passed through voile cloth, and further homogenised with 10 strokes of a glass-glass Dounce homogeniser. The homogenate was then centrifuged at 10,000 X g for 15 min and the pellet was suspended in buffer consisting of 10 mM Tris, 50 mM NaCl, and 1 mg/ml bovine serum albumin (BSA, pH 7.5), adjusted to a final concentration of 15 mg equivalents in 50 jul. Bovine TSH (a gift from Dr. Simon Manley) was iodinated using the lactoperoxidase method to a
JCE & M • 1978 Vol 47 • No 2
specific activity of about 80 /iCi/jug. The crude labeled TSH was then passed through a 50 x 1-cm Sephadex G-100 column and aliquots were purified by the membrane affinity method described by Smith and Hall (1). In the assay, 200 /xl test Ig preparation and 50 fxl thyroid plasma membrane suspension were added to plastic tubes and incubated in a water bath for 10 min at 37 C. Fifty microliters of [125I]TSH (approximately 10,000-20,000 counts/5 min) was then added and the tubes were incubated in the water bath for a further 90 min. Bound/free separation was obtained by passing a 200-/xl aliquot of the reaction mixture through SMI Sartorius cellulose nitrate filters which had been previously soaked in 5% BSA in 10 mM Tris, pH 7.5. The filters were counted in a y-counter for 5 min. The results were expressed as a TSAb index, namely: percentage [125I]TSH bound in the presence of test Ig — nonspecific binding percentage [I25I]TSH bound in the presence of pooled normal Ig - nonspecific binding The nonspecific binding was that binding which occurred in the presence of 1000 mlU TSH/ml and was from 10-12%. Binding of [125I]TSH in the presence of pooled normal Ig was from 35-40% which was 2-10% less than that which occurred in the presence of Tris-NaCl-BSA buffer alone. Tests were carried out in duplicate. Serum T4 levels were measured by the competitive protein-binding technique. Statistical analyses were carried out using the Mann-Whitney test.
Results The mean TSAb index in 21 normal subjects was 1.01 ± 0.08 SE (Fig. 1). The range was ...
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FIG. 1. Serum TSAb in 1) patients with Graves' hyperthyroidism selected for absence of eye disease; 2) patients with Graves' ophthalmopathy; 3) patients with other thyroid disease; 4) patients with Lepromatous leprosy, and 5) normal subjects. The results are expressed as TSAb indices. An index of 0.64 was considered positive. Horizontal bars represent mean values.
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TSAb IN AUTOIMMUNE DISORDERS 0.64-1.50; thus, for the purposes of this study, an index of less than 0.64 was considered positive for TSAb. The mean index of the 15 patients with hyperthyroid Graves' disease was 0.41 ± 0.05 SE. This is significantly less than the mean TSAb for normal subjects (Mann-Whitney, P < 0.001). Fourteen patients were regarded as TSAb-positive. The 16 patients with Graves' ophthalmopathy were found to have a mean TSAb of 0.55 ± 0.08 SE, which is significantly less than that of normals (P < 0.001). Of these patients, TSAb was found in 9 of 10 patients who had once had hyperthyroidism but were now euthyroid as a result of thyroidectomy or treatment with radioactive iodine, and in only 1 of 6 patients who had never been hyperthyroid (Fig. 2). The 27 patients with autoimmune disorders were found to have a mean TSAb index of 1.00 ± 0.07 SE, which is not significantly different from that of normals. Two patients, 1 with idiopathic Addison's disease and 1 with juvenile diabetes mellitus, were TSAb-positive, and 2 patients, 1 with Sjogren's disease and 1 with methyldopa-induced antired cell antibodies, had borderline levels
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of TSAb. The mean TSAb index in the 17 patients with other thyroid disorders was 0.99 ± 0.05 SE, which is not significantly different from that of normals, and TSAb was not detected in any of these patients. The mean TSAb index in the 7 patients with leprosy was significantly higher (P < 0.01) than that of normals, at 1.13 ± 0.08 SE, and TSAb was not detected in any of these patients. All patients with other autoimmune disorders were clinically and biochemically euthyroid. Thyroid function tests were performed at the time of study in the patients with other autoimmune disorders in whom TSAb was detected. The results were: 1) patient with idiopathic Addison's disease who was taking oral contraceptives at the time—serum T4, 11.0 jtig/dl (normal range, 5-13 jug/dl); RT3U, 24% (normal range, 22-34%);! 2) patient with juvenile diabetes mellitus—serum T4, 7.2 /xg/ dl; RT3U, 28%; 3) patient with Sjogren's disease—serum T4, 6.1 jug/dl; RT3U, 29%; and 4) patient with antired cell antibodies—serum T4, 6.7 /xg/dl; RT3U, 28%. Discussion TSAb was demonstrated in 14 of 15 patients (93%) with untreated Graves' hyperthyroidism. This is similar to the findings of Mukhtar and his colleagues (7) who found TSAb in 100% of their patients. The smaller prevalences in other series (8, 9) could be due to technical factors or to different criteria for the diagnosis of Graves' disease. In our series, hyperthyroidism due to Graves' disease was initially diagnosed on clinical grounds and confirmed by demonstrating elevated serum thyroid hormone levels and a diffusely large thyroid on 131I scan, which was performed on all patients. The high prevalence of TSAb in our series confirms that the test may be very useful in the diagnosis of Graves' hyperthyroidism. Thus, absence of serum TSAb probably ex-
a) GRAVES'
b) OPHTHALMOPATHY
FIG. 2. Serum TSAb in patients with Graves' ophthalmopathy a) who had never been hyperthyroid (euthyroid Graves' disease) and b) who had once been hyperthyroid but were now euthyroid.
1 At follow-up 12 months after the study, the patient with Addison's disease, in whom high levels of TSAb were detected, complained of anxiety. Examination revealed a fine tremor and lig lag. Repeat thyroid function test results were: serum T4) 11.5 jug/dl; RT3U, 22%; and T3 RIA, 3.1 ng/dl (normal range, 1.0-2.0 ng/dl). TRH stimulation test confirmed hyperthyroidism.
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STRAKOSCH, JOYNER, AND WALL
JCK & M Vol47
1978 No 2
eludes the diagnosis. Furthermore, because disorders in whom TSAb was demonstrated ^ TSAb often disappears after treatment, espe- were clinically and biochemically euthyroid, cially after thyroidectomy (7), reappearance TSAb is presumably able to bind to the TSH A receptor (at least in vitro) but not cause stimmay indicate a relapse of the disease (1). TSAb was detected in 10 of 16 patients ulation of the thyroid gland. The development (63%) with Graves' ophthalmopathy. The of hyperthyroidism in these patients may deprevalence is similar to that obtained by Teng pend on additional factors. For example, it is and colleagues (13). TSAb was detected in 9 possible that excess TSAb may act as a of 10 of these patients who had once been "blocking" agent, or cell-mediated immune < hyperthyroid but were now euthyroid and in reactions against thyroid antigens may also be only 1 of 6 who had always been euthyroid. involved (3, 26). The finding of a group of euthyroid patients Acknowledgments with Graves' ophthalmopathy without TSAb lends support to the view that the eye disease We thank Dr. Simon Manley for his advice and for the may be a separate, but frequently associated, gift of TSH, Mr. Hambling for measurement of serum T4 V levels, and Dr. G. Cavaye for the statistical analyses. disorder. TSAb was also detected in one patient with References idiopathic Addison's disease and one patient B. R., AND R. HALL, Thyroid-stimulating immunowith juvenile diabetes mellitus. Although sub- 1. SMITH, globulins in Graves' disease, Lancet 2: 427, 1974. A clinical Graves' disease was not excluded in 2. KENDALL-TAYLOR, P., LATS and human-specific thyroid * stimulator; their relation to Graves' disease, J Clin Endocrithese patients, as serum T3RIA was not meanol Metab A: 319, 1975. sured and TRH tests were not performed, the 3. VOLPE, R., N. R. FARID, C. VON WESTARP, AND V. V. Row, The pathogenesis of Graves' disease and Hashimoto's thypatients were clinically euthyroid and TSAb roiditis, Clin Endocrinol 3: 239, 1974. was an incidental finding. It is well known that 4. MANLEY, S. W., J. R. BOURKE, AND R. W. HAWKER, The thyrotropin receptor in guinea-pig thyroid homogenate: interpatients with organ-specific autoimmune disaction with the long acting thyroid stimulator, J Endocrinol Y 61: 437, 1974. orders are likely to develop antibodies to other 5. MEHDI, S. Q., AND S. S. NUSSEY, A radio-ligand receptor organs. Graves' disease is associated with assay for the long acting thyroid stimulator, Biochem J 145: "f 105, 1975. other autoimmune diseases (10, 14-17) and 6. KOTULLA, P., F. ADLKOFER, E. AULBERT, H. MEINHOLD, AND LATS has been described in a few patients H. SCHLEUSENER, Radio ligand receptor assay for human thyroid stimulating immunoglobulins, Acta Endocrinol with other autoimmune disorders (10, 18). fSupplJ (Kbh) 82: 49, 1976. Demonstration of TSAb in patients with other 7. MUKHTAR, E. D., B. R. SMITH, G. A. PYLE, R. HALL, AND P. VICE, Relation of thyroid-stimulating immunoglobulins to autoimmune disorders suggests that TSAb, at thyroid function and effects of surgery, radioiodine and antileast in this situation, is not produced as a thyroid drugs, Lancet 1: 713, 1975. result of change in, or release of, thyroid an- 8. SCHLEUSENER, H., P. KOTULLA, H. SORGE, H. MEINHOLD, AND F. ADLKOFER, Relationship between thyroid status and -d tigen, but as a result of some abnormality of Graves' disease specific immunoglobulins, Acta Endocrinol [SupplJ (Kbh) 82: 25, 1976. the immune system, for example a deficiency 9. BRYSON, J. M., A. JOASOO, AND J. R. TURTLE, The thyrotroof suppressor T cell function (19, 20). pin receptor in human thyroid plasma membranes: effect of serum immunoglobulins, Acta Endocrinol (Kbh) 83: 528, Two other diseases in which increased prev1976. alences of autoantibodies are frequently 10. FREY, H. M. M., J. H. VOGT, AND J. NERUP, Familial poly' endocrinopathy, Acta Endocrinol (Kbh) 72: 401, 1973. found, although not primarily autoimmune, M., J. M. MCKENZIE, AND S.SALISBURY-MURPHY, ^, were also investigated. Firstly, a variety of 11. ZAKARIJA, LATS or thyroid-stimulating immunoglobulin as a prognostic index of relapse in hyperthyroidism of Graves' disease, Acta autoantibodies, particularly to adrenal and Endocrinol fSupplJ(Kbh) 82: 24, 1976. parathyroid gland are often detected in pa- 12. WERNER, S. C, Modification of the classification of the eye ( changes of Graves' disease: recommendations of the ad hoc tients with chronic mucocutaneous candidicommittee of the American Thyroid Association, J Clin Enasis (21). In the patient tested in this study, docrinol Metab 44: 203, 1977. TSAb was not detected. Secondly, leproma- 13. TENG, C. S., B. R. SMITH, B. CLAYTON, D. C. EVERED, F. CLARK, AND R. HALL, Thyroid stimulating antibodies (TSAb) ^ tous leprosy is also associated with a wide in ophthalmic Graves' disease. Correlation with other aspects of thyroid function, Acta Endocrinol [SupplJ (Kbh) 82: 23, variety of autoantibodies (22-25). Again, 1976. TSAb was not found in the seven patients 14. FURSZYFER, J., L. T. KURLAND, W. M. McCONAHEY, AND L. studied. Because patients with autoimmune R. ELVEBACK, Graves' disease in Olmsted Country, Minne-
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TSAb IN AUTOIMMUNE DISORDERS sota, 1935 through 1967, Mayo Clin Proc 45: 636, 1970. 15. ARDBMAN, S., I. CHANARIN, B. KRAFCHIK, AND W. SINGER,
Addisonian pernicious anaemia and intrinsic factor antibodies in thyroid disorders, Q J Med 35: 421, 1966. 16. MARSHALL, J. S., A. S. WEISBERGER, R. P. LEVY, AND R. T.
BRECKENRIDGE, Coexistent idiopathic thrombocytopenic purpura and hyperthyroidism, Ann Intern Med 67: 411,1967. 17. WALL, J. R., S. L. FANG, B. A. WALTERS, C. R. STRAKOSCH,
S. H. INGBAR, AND L. E. BRAVERMAN, Multi-system immunologically mediated disease, T lymphocyte deficiency and thyroid immunologic disease: a report of 4 cases, Aust NZ J Med, in press. 18. WALL, J. R., Immunological aspects of thyrotoxicosis, MD Thesis, Adelaide University, 1971. 19. TAYLOR, R. B., AND A. BASTEN, Suppressor cells in humoral immunity and tolerance, Br Med Bull 32: 153, 1976. 20. PENHALE, J. W., A. FARMER, R. P. MCKENNA, AND W.
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IRVINE, Spontaneous thyroiditis in thymectomised and irradiated Wistar rats, Clin Exp Immunol 15: 225, 1973.
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21. BLIZZARD, R. W., AND J. H. GIBBS, Candidiasis: studies pertaining to its association with endocrinopathies and pernicious anaemia, Pediatrics 42: 231, 1968. 22. BONOMO, L., AND F. DAMMACCO, Characterisation studies of thyroglobulin antibodies in leprosy. An immunological study of diethyl aminoethylcellulose chromatographic fractions, Immunology 13: 565, 1967. 23. MATHEWS, L. J., AND J. R. TRAUTMAN, Clinical and serological profiles in leprosy, Lancet 2: 915, 1965. 24. RUGE, H. G. S., G. FROMM, F. FUHNER, R. FARD, AND R. S.
B. GUITO, Serological findings in leprosy. An investigation into the specificity of various serological lists for syphilis, Bull WHO 23: 793, 1960. 25. WALL, J. R., AND D. J. M. WRIGHT, Testicular germinal cell antibodies in patients with lepromatous leprosy, Clin Exp Immunol 17: 51, 1974. 26. CALDER, E. A., AND W. J. IRVINE, Cell-mediated immunity and immune complexes in thyroid disease, J Clin Endocrinol Metab 4: 287, 1975.
Eighth International Thyroid Congress February 5-8, 1980, Sydney, Australia Initial announcements are at present being circulated. To receive the second announcement abstract and registration forms early in 1979, notify the Secretariat of your interest. This congress will be followed on the 10th of February by the Sixth International Congress of Endocrinology in Melbourne. Secretariat:
PO Box 783 Canberra City, A.C.T. 2601 Australia
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