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

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

Clinical significance of a new autoantibody against a human eye muscle soluble antigen, detected by immunofluorescence.

The clinical significance of a circulating autoantibody against a recently identified soluble human eye muscle-derived antigen was studied in patients with Graves' ophthalmopathy and autoimmune thyroid disorders. Tests were positive in 73% of patients with Graves' ophthalmopathy, including six of seven with no associated thyroid disease (euthyroid Graves' disease). Tests were also positive in 27% of patients with hyperthyroidism but no clinically apparent eye disease, in 13% of patients with Hashimoto's thyroiditis without eye disease, in two of 12 patients with subacute thyroiditis, in one of 20 patients with nonimmunological thyroid disorders but in none of 39 normal subjects. There were significant positive correlations between serum levels of the antibody (expressed as a titre) and the severity of the eye muscle component quantified as an index as well as the duration of the eye disease. Antibodies were detected in three of five patients with only lid lag and state who subsequently developed active ophthalmopathy, in six of nine patients who developed eye disease after treatment of their hyperthyroidism and in one of eight first degree relatives of patients with Graves' ophthalmopathy. In addition three of the 12 patients with autoimmune thyroid disease without apparent eye involvement, but positive antibody tests, have developed ophthalmopathy since the time of testing. These findings suggest that tests for antibodies against a soluble human eye muscle antigen may be useful clinically as a diagnostic test and to predict the onset of eye disease in predisposed patients and subjects.     

Antibodies in the serum of patients with autoimmune thyroid disorders react with a recombinant 98 amino acid fragment of a full length 64 kDa eye muscle membrane protein which is also expressed in the thyroid.

We have tested sera from patients with autoimmune thyroid disorders with or without ophthalmopathy for immunoreactivity, in a dot blot assay, against a recombinant 98 amino acid fragment of a cloned 64 kDa protein, D1, which is expressed in human eye muscle and thyroid, in the form of a Lac Z fusion protein. Tests were positive in 19 out of 40 patients with established thyroid-associated ophthalmopathy (TAO), in 12 out of 21 patients with Graves' hyperthyroidism (GH) without clinically evident ophthalmopathy, in 5 out of 10 patients with thyroid autoimmunity and lid retraction but no other signs of ophthalmopathy, in 4 out of 23 patients with Hashimoto's thyroiditis (HT) without evident ophthalmopathy and in 2 out of 18 patients with benign adenoma or multinodular goitre, but in only 2 out of 37 normal subjects tested. SDS-polyacrylamide gel electrophoresis and Western blotting for an antibody reactive with a 64 kDa antigen in pig eye muscle membranes was also carried out on sera from patients with TAO and GH. While immunoblotting for antibodies reactive with a 64 kDa protein was more often positive in patients with TAO, in whom 58% had serum antibodies which reacted with a 64 kDa protein, this was not the case in patients with GH without eye signs in whom the prevalence of positive immunoblot tests was 35%. Overall there was a fairly close correlation between the two tests although there were many exceptions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Extraocular muscle autoimmunity and orbital fat inflammation in thyroid-associated ophthalmopathy

The immunological basis for the ophthalmopathy associated with Graves’ hyperthyroidism is both poorly understood and controversial. The mechanism for its link with thyroid autoimmunity is unknown but likely to be due to autoimmunity against some thyroid and orbital tissue-shared antigen, such as the thyroid-stimulating hormone receptor, which is expressed on the orbital pre-adipocyte and extraocular muscle cell, or the putative ‘eye muscle cell membrane antigen’. Chronic upper-eyelid retraction, which sometimes occurs as a dominant feature of ophthalmopathy or as an isolated abnormality, is a common and related orbital disorder. Recent evidence that antibodies targeting the calcium-binding protein calsequestrin are specific and sensitive markers of eye muscle and upper-eyelid muscle damage has highlighted the need for diagnostic antibody tests in ophthalmopathy. In the context of this confusion, this review will address the nature of the autoimmune reactions in thyroid-associated ophthalmopathy, focusing on the eye muscle.     

A 63 kDa skeletal muscle protein associated with eye muscle inflammation in Graves' disease is identified as the calcium binding protein calsequestrin

It is generally accepted that thyroid-associated ophthalmopathy (TAO) is an autoimmune disease of the eye muscle (EM) and the surrounding orbital connective tissue in which circulating antibodies play an important role. Antibodies against EM membrane proteins of 63-67kDa mol. wt. seem to be the best markers of ophthalmopathy in patients with autoimmune thyroid disease. We purified a 63 kDa EM protein using SDS-polyacrylamide gel electrophoresis technology and TAO patients' sera as probes, digested the protein with cyanogen bromide and sequenced immunoreactive peptides. We also screened a human EM library with a rabbit antiserum against 63-65 kDa proteins and affinity purified antibodies from a TAO patient's serum that reacted with a 55 kDa EM membrane protein. From partial sequence information and from DNA sequencing of positive cDNA clones, the protein was identified as calsequestrin, a 63 kDa calcium binding protein localized in the sarcoplasmic reticulum of the muscle fiber. As determined by Northern blotting, calsequestrin was expressed in EM and other skeletal muscle but not thyroid or fibroblasts. Calsequestrin is different from the "64 kDa protein", which has been identified as succinate dehydrogenase flavoprotein subunit, which has a corrected mol. wt. of 67 kDa. Serum antibodies against calsequestrin were found in 40% of patients with clinically active TAO, but in only 4% of those with stable eye disease, and in 5% of normal subjects, by immunoblotting. Although it is possible that autoimmunity against calsequestrin plays a role in the progressive EM damage that characterizes ophthalmopathy it is more likely that the antibodies are secondary to a reaction against some other cell membrane protein, such as the novel thyroid and eye muscle shared protein G2s or the TSH receptor.     

Peripheral blood T lymphocyte sensitisation against calsequestrin and flavoprotein in patients with Graves' ophthalmopathy

Thyroid-associated ophthalmopathy (TAO) is an orbital autoimmune disorder of the extraocular and eyelid muscles and surrounding connective and adipose tissue. Although mononuclear cell infiltration of orbital tissue is a characteristic feature of TAO the likely role of T lymphocyte reactivity against eye muscle antigens in the initiation of eye muscle damage in TAO has not been explored in detail. Therefore, we tested for T lymphocyte sensitisation to three eye muscle antigens namely, calsequestrin, G2s and flavoprotein (Fp), in patients with Graves' ophthalmopathy (GO), Graves' hyperthyroidism (GH) without ophthalmopathy and age and sex matched normal subjects. T lymphocyte reactivity was determined in a proliferation assay, results being expressed as stimulation index (SI). Mean ( +/- SE) SI for patients with GO, but not GH without ophthalmopathy, were significantly greater than that for normal subjects for calsequestrin and Fp, but not G2s. Mean ( +/- SE) SI was also significantly increased in patients with active ophthalmopathy, but not chronic ophthalmopathy, compared to normal subjects, for calsequestrin and Fp, but not G2s. Overall, positive lymphocyte proliferation to calsequestrin was demonstrated in 59% of patients with GO and 33% of patients with GH, which was significantly greater than in normals for both groups. In conclusion, we have demonstrated significant T lymphocyte reactivity to calsequestrin and, to a lesser extent, Fp in patients with GO. Because calsequestrin is located in the cell membranes of the eye muscle cell during the myotubular stage of the cell cycle, its targeting might be the primary reaction which leads to extraocular muscle inflammation in patients with GH.     

Antibodies against 1D,a Recombinant 64-kDa Membrane Protein,Are Associated with Ophthalmopathy in Patients with Thyroid Autoimmunity

We have tested for serum antibodies reactive with ₁D, a recombinant 65-kDa human thyroid protein which is also expressed in eye muscle, in patients with thyroid autoimmunity and ophthalmopathy by immunofluorescence and SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. We also measured antibodies to a 64-kDa pig eye muscle membrane protein which is identified by SDS-PAGE and Western blotting, correlating the two reactivities. While antibodies to ₁D, expressed in Chinese hamster ovary (CHO) cell membrane, were detected in approximately 40% of patients with ophthalmopathy, in both tests the greatest prevalence, by immunofluorescence, 73%, was demonstrated in patients with Graves' hyperthyroidism without clinically evident eye disease, although only 50% of these patients were positive in immunoblotting. When the two tests for anti-₁D antibodies were compared, immunofluorescence appeared to be the more specific and immunoblotting appeared to be the more sensitive. The greatest prevalence of antibodies reactive with a 64-kDa pig eye muscle protein, 71%, was in patients with TAO of less than 1 year duration; tests were positive in 49% of patients with more chronic ophthalmopathy and in 50% of patients with Graves' hyperthyroidism without evident eye disease. Antibodies reactive with ₁ D were detected in 17% of normals by immunofluorescence and 24% by immunoblots, while antibodies reactive with the 64-kDa pig eye muscle protein were detected in only 10% of the normal subjects tested. Lesser prevalences of antibodies to the two 64-kDa proteins in patients with established eye disease suggest that such antibodies may be an early abnormality in patients with Graves' hyperthyroidism who are predisposed to develop ophthalmopathy. Although the association was not close, reactivity against ₁D by immunoblotting, but not immunofluorescence, was significantly correlated with reactivity to a 64-kDa eye muscle membrane protein by immunoblotting. On the other hand, when sera containing antibodies reactive with both ₁D and the 64-kDa eye muscle protein were incubated with CHO (₁D) cell membrane, reactivity against ₁D was absorbed while that against the eye muscle protein was not. The precise relationship between the two 64-kDa proteins can only be clarified by cloning the 64-kDa protein from an eye muscle expression library and comparing the sequences with those of ₁D.     

Peripheral blood lymphocyte transformation to G2s in patients with autoimmune thyroid disease with and without ophthalmopathy

Thyroid associated ophthalmopathy (TAO) is an autoimmune disease involving the extra ocular muscles and surrounding orbital connective and adipose tissues. The mechanism for the link between ophthalmopathy and thyroid autoimmunity is unknown but current evidence favors an immune reaction against a thyroid and orbital tissue shared antigen such as the novel protein G2s, which is highly expressed in both eye muscles and thyroid, or the TSH receptor (TSHR). Earlier, we showed that serum antibodies against G2s were closely linked to ophthalmopathy. Although lymphocytic infiltration of the eye muscles is a pathologic feature of TAO, it is unclear whether the reaction is in the eye muscle fiber or the surrounding connective tissue. We tested for peripheral blood mononuclear cell sensitization to G2s fusion protein in patients with TAO, Graves' hyperthyroidism or Hashimoto's thyroiditis without evident ophthalmopathy and normal subjects. Results were expressed as counts per min (cpm) and as stimulation indices (SI). Although proliferation tests were positive in 23% of patients with TAO, overall, there were no significant differences between the four groups. Tests were also positive in four out of seven patients with Hashimoto's thyroiditis, suggesting that immune reactivity against G2s could be a marker of this progressive thyroid disorder. There was no significant correlation between T cell reactivity to G2s and serum antibodies against the same protein, measured in enzyme linked immunosorbent assay. Failure to demonstrate significant T lymphocyte sensitization to G2s in the majority of patients with TAO may reflect the small number of sensitized T cells expected to be circulating in the peripheral blood which could be overcome by testing cloned orbital T cells, as available. Another possibility is that the T cell epitope(s) is not present on the 141 amino acid fragment of G2s that we have so far cloned. The finding of positive T cell tests in a small proportion of patients with ophthalmopathy suggests that future studies using cloned orbital T cells and full length G2s, or its dominant epitope, are indicated.     

Immunologically mediated cytotoxicity against human eye muscle and thyroid cells in euthyroid and thyrotoxic Graves' ophthalmopathy

We have studied the role of eye muscle and thyroid autoimmunity in patients with Graves' hyperthyroidism with or without ophthalmopathy in an area of relatively low iodine intake. Antibody dependent cell mediated cytotoxicity (ADCC) and complement mediated antibody dependent cytotoxicity (CMAC) against thyroid and eye muscle cells, and levels of antibodies against TSH receptor antigen and the thyroid microsomal antigen (thyroid peroxidase) were determined in three groups of patients: (1) thyrotoxic with exophthalmos (TX, n = 28), (2) thyrotoxic without ophthalmopathy (GR, n = 10), and (3) euthyroid ophthalmopathy (EU, n = 12). The thyroid glandular mass of the EU group was significantly less (P less than 0.01) compared with TX or GR. Mean (+/- SD) TSH receptor antibody (TRAb) level was 27 +/- 14% in EU which was significantly lower compared with TX (52.4 +/- 20%) and GR (59 +/- 18%). The prevalence of microsomal antibodies were similar and not significantly different in the three groups. On the other hand the prevalence of positive ADCC and CMAC tests was significantly greater, and at higher levels, in EU (ADCC THY CELLS 10.9 +/- 8.9% SL, ADCC Eye muscle = 25.9 +/- 20% SL, CMAC = 70.2 +/- 43% SL) and TX (ADCC THY CELLS = 9.3 +/- 9.2% SL, ADCC Eye muscle = 20.1 +/- 19% SL, CMAC = 62.4 +/- 30% SL) compared to GR (ADCC THY CELLS = 4.4 +/- 9.5% SL, ADCC Eye muscle = 7.7 +/- 6.7% SL, CMAC = 24.7 +/- 23% SL).(ABSTRACT TRUNCATED AT 250 WORDS)     

Graves眼病的治疗

Graves眼病又叫甲状腺相关性眼病 ,浸润性突眼 ,与甲状腺自身免疫有关 ,尤其是与 Graves甲亢有关 ,发病率占甲亢的 5 %～ 10 % ,男性多于女性。严重 Graves眼病的治疗是一个临床难题 ,因此人们不断研究新的治疗方法 ,企图攻克 Graves眼病这一课题。在此 ,将 Graves眼病的治疗作一简要的介绍 ,并展望 Graves眼病治疗的发展趋势。     

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

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

Antithyroglobulin monoclonal and autoantibodies cross-react with an orbital connective tissue membrane antigen: a possible mechanism for the association of ophthalmopathy with autoimmune thyroid disorders.

The possibility that Graves' ophthalmopathy and autoimmune thyroid disorders may be associated because of autoimmune reactions against antigens shared between human orbital and thyroid tissues was investigated using anti-thyroglobulin (Tg) monoclonal and autoantibodies. Eleven of 16 mouse monoclonal antibodies (MCAB) tested reacted, in an enzyme-linked immunosorbent assay (ELISA), with an antigen in human orbital connective tissue membranes (OCTmem), but not with the OCT soluble fraction, or with membrane or soluble fractions of human eye muscle, lacrimal gland or skin connective tissue. The anti-OCTmem activity was absorbed by OCTmem and Tg, but not by liver membranes or bovine serum albumin (BSA). In preliminary studies four out of 113 human MCAB against thyroid or orbital tissue antigens showed reactivity restricted to Tg and OCTmem. Sera from approximately 50% of patients with autoimmune thyroid disorders, with or without ophthalmopathy, also reacted with OCTmem. The autoantibody activity correlated closely with serum titres of antithyroglobulin but not with the presence, duration, or severity of the eye disease. The OCTmem reactivity was absorbed by Tg, thyroid membranes, and OCTmem but not liver membranes, membranes prepared from other orbital tissues, or BSA. The OCTmem-Tg shared antigen site appeared not to be native thyroglobulin since, (i) MCAB and serum autoantibodies did not react with the cytosol fraction of OCT, and (ii) because the membrane antigen was not solubilizable. Because not all patients with ophthalmopathy have detectable anti-Tg antibodies and, conversely, because not all patients with detectable anti-Tg antibodies develop ophthalmopathy it is unlikely that autoimmunity against a OCTmem-Tg shared antigen is the primary mechanism of Graves' ophthalmopathy, although this possibility has not been excluded. On the other hand the reaction of anti-Tg autoantibodies with OCT membranes may be a model for other autoimmune reactions against other thyroid-orbital tissue-shared antigens. While the pathogenesis of Graves' ophthalmopathy is likely to be multifactorial, humoral and cellular reactions against primary orbital antigens, thyroid-orbitol tissue shared antigens, or both, are likely to play important roles.     

Epitopes,immunoglobulin classes and immunoglobulin G subclasses of calsequestrin antibodies in patients with thyroid eye disease

A number of serum autoantibodies are associated with thyroid eye disease (TED), including those reactive against the calcium binding protein calsequestrin (CASQ). There are two isoforms of CASQ namely; CASQ1, found in skeletal, including extra ocular, muscle, and CASQ2, found in cardiac muscle. We determined (i) the reactivity profiles of CASQ1 and CASQ2 antibodies and (ii) the immunoglobulin (Ig) classes and IgG subclasses of CASQ1 antibodies, using enzyme-linked immunosorbent assay. Of the 20 patients with TED tested, 35% were positive for CASQ1 antibodies, 25% for CASQ2 antibodies and two patients (10%) were positive for both antibodies. Of the 12 patients with Hashimoto's thyroiditis and ophthalmopathy tested, 25% were positive for CASQ1 antibodies, 42% for CASQ2 antibodies and two patients (17%) were positive for both antibodies. CASQ1 antibodies were mainly of the IgG class and IgG1 and IgG3 subclasses. These results suggest that CASQ1 and CASQ2 do not share major epitopes. Because antibodies of the IgG1 and IgG3 subclasses are cytotoxic, CASQ1 antibodies may contribute to the eye muscle damage in patients with TED. Because CASQ1 antibodies were positive in only a third of patients with active TED we are unable to draw conclusions about their role in its pathogenesis. On the other hand, a possible role of CASQ2 antibodies in the aetiology of the cardiac complications of Graves' disease is a new avenue for research and appears worthy of further investigation.     

Isotype and immunoglobulin subclass distribution of eye muscle membrane reactive antibodies in the serum of patients with thyroid-associated ophthalmopathy as detected in Western blotting.

We have determined the immunoglobulin (Ig) class (isotype) and IgG subclass of autoantibodies in the serum of patients with thyroid-associated ophthalmopathy (TAO) or autoimmune thyroid disorders without evident ophthalmopathy reactive in Western blotting with antigens of 55, 64, 75 and 95 kDa in pig eye muscle membrane (PEMM). The 22 sera studied were shown, previously, to contain IgG antibodies reactive with one or more of the four antigens. The majority of sera antibodies reactive with PEMM antigens were of two or more IgG subclasses. Of the IgG subclass specificities IgG3 and IgG4 subclass antibodies were, overall, the most common. We were unable to demonstrate IgG subclass restriction for antibodies reactive with the 95 or 55 kDa antigens in PEMM, antibody activity being equally distributed in all four subclasses tested. While most of the sera which recognized a 64 kDa antigen did so with an IgG4 antibody, all other subclasses were also represented. On the other hand all 13 sera reactive with a 75 kDa antigen did so using Ig of the IgG3 subclass and 12 of these used the IgG4 subclass as well, IgG1 and IgG2 subclasses being represented in only 3 and 4 sera, respectively. There were no differences, in respect to Ig class or IgG subclass distribution of eye muscle reactive antibodies between patients with Graves' hyperthyroidism with ophthalmopathy and those with Hashimoto's thyroiditis, and eye disease. Control sera from five normal subjects and three patients with nonautoimmune thyroid disorders did not contain antibodies reactive with these PEMM antigens of any Ig class or IgG subclass.(ABSTRACT TRUNCATED AT 250 WORDS)     

Serum immunoglobulin levels in thyroid disease

Serum levels of IgG, IgA and IgM were assayed by radial immunodiffusion in 261 patients with eight categories of thyroid disease. These composed eighty-three patients with a first episode of untreated active Graves' disease (toxic diffuse goitre), ten with relapsed Graves' disease, seventeen with thyrotoxicosis due to a multinodular goitre, forty-nine with Hashimoto's thyroiditis, twenty-eight with primary (non-goitrous) myxoedema, forty with non-toxic goitre, eighteen with an adenoma and sixteen with euthyroid ophthalmopathy.

Eighteen (21·7%) patients with a first episode of Graves' disease had abnormally high IgG levels whereas eight (80%) of those who had relapsed after a course of Carbimazole had high levels. Those Graves' disease patients with raised IgG levels had a significantly higher 24-hr radioiodine uptake than those with normal levels. Eight (16·3%) patients with Hashimoto's thyroiditis had abnormally high levels of IgG associated with a higher incidence of thyroglobulin autoantibodies. Very few (<6%) patients with primary myxoedema, non-toxic goitre and adenoma had abnormal levels. Euthyroid patients with ophthalmopathy had a significantly lower mean IgG level than the corresponding mean level found in the group with active Graves' disease.

However, despite the differences between groups described above, there were no significant differences of mean IgG, IgA and IgM levels in seven of the eight groups when compared with normal subjects. Only the group with relapsed Graves' disease had a significantly higher mean IgG. None of the patients studied had abnormal IgM or IgA levels.

     

Peripheral Blood Lymphocyte Transformation to G2s in Patients with Autoimmune Thyroid Disease with and without Ophthalmopathy

Thyroid associated ophthalmopathy (TAO) is an autoimmune disease involving the extra ocular muscles and surrounding orbital connective and adipose tissues. The mechanism for the link between ophthalmopathy and thyroid autoimmunity is unknown but current evidence favors an immune reaction against a thyroid and orbital tissue shared antigen such as the novel protein G2s, which is highly expressed in both eye muscles and thyroid, or the TSH receptor (TSHR). Earlier, we showed that serum antibodies against G2s were closely linked to ophthalmopathy. Although lymphocytic infiltration of the eye muscles is a pathologic feature of TAO, it is unclear whether the reaction is in the eye muscle fiber or the surrounding connective tissue. We tested for peripheral blood mononuclear cell sensitization to G2s fusion protein in patients with TAO, Graves' hyperthyroidism or Hashimoto's thyroiditis without evident ophthalmopathy and normal subjects. Results were expressed as counts per min (cpm) and as stimulation indices (SI). Although proliferation tests were positive in 23% of patients with TAO, overall, there were no significant differences between the four groups. Tests were also positive in four out of seven patients with Hashimoto's thyroiditis, suggesting that immune reactivity against G2s could be a marker of this progressive thyroid disorder. There was no significant correlation between T cell reactivity to G2s and serum antibodies against the same protein, measured in enzyme linked immunosorbent assay. Failure to demonstrate significant T lymphocyte sensitization to G2s in the majority of patients with TAO may reflect the small number of sensitized T cells expected to be circulating in the peripheral blood which could be overcome by testing cloned orbital T cells, as available. Another possibility is that the T cell epitope(s) is not present on the 141 amino acid fragment of G2s that we have so far cloned. The finding of positive T cell tests in a small proportion of patients with ophthalmopathy suggests that future studies using cloned orbital T cells and full length G2s, or its dominant epitope, are indicated.     

Significance of anti-eye muscle antibody in patients with thyroid-associated ophthalmopathy by quantitative western blot.

To investigate the prevalence of antibody against rat eye muscle membrane antigen, as determined from SDS-polyacrylamide gel electrophoresis and western blotting, in sera from patients with thyroid-associated ophthalmopathy (TAO), we quantitatively analyzed the binding activity with a rat eye muscle membrane 64 kDa protein using chromato-scanner. Eye muscle antibody activity was expressed as ratio of density of the 64 kDa band to that at 66 kDa found with all normal sera and phosphate buffered saline. The mean (+/- SD) eye muscle antibody activity was 2.7 +/- 2.7 in TAO (P < 0.01 v.s. normal), 1.5 +/- 1.7 in Graves' disease without evident eye disease, 1.6 +/- 2.5 in Hashimoto's thyroiditis and 0.45 +/- 0.26 in normal subjects. A positive band at 64 kDa was found in 71% of patients with TAO, 36% of those of Graves' disease without evident eye disease and in 35% of patients with Hashimoto's thyroiditis without eye disease. The prevalence of this antibody activity tended to correlate to the severity of ophthalmopathy. Furthermore, the level of eye muscle antibody activity decreased in parallel with the improvement of eye signs in two patients. Sera reactive with rat eye muscle membrane 64 kDa protein reacted also with a human eye muscle membrane 64 kDa protein but not with human thyroid, liver, spleen or pancreas membrane preparations. In conclusion, antibody to rat eye muscle membrane 64 kDa protein is present in TAO and may be a useful clinical marker of ophthalmopathy.     

Thyroid size and goitre frequency in hyperthyroidism

Thyroid function, the clinical occurrence of goitre and thyroid gland volume, ultrasonically determined, were investigated in 310 consecutive untreated patients with hyperthyroidism. Of 173 patients with Graves' disease (39%), 67 had no goitre, while 53 patients (31%) had a normal thyroid volume. Twenty-three of 91 patients with multinodular glands (25%) had no goitre and ten patients (11%) had a normal thyroid volume. Of 46 patients with solitary autonomous nodules (7%), three had no goitre while six (13%) had a normal thyroid volume. Out of all the hyperthyroid patients (30%), 93 had no clinically detectable goitre, and 69 (22%) had a normal thyroid volume. The lack of a goitre in a large portion of patients with hyperthyroidism could be responsible for delayed diagnosis and subsequent treatment of these patients.     

Identification of autoantigen recognized by autoimmune ophthalmopathy sera with immunoblotting correlated with orbital computed tomography.

We have demonstrated that there is an antibody related to extraocular muscle enlargement in autoimmune ophthalmopathy (Graves' ophthalmopathy, thyroid-associated correlated with orbital computed tomography (CT). This study was designed to identify the autoantigen and to determine whether there are common antigens among the extraocular muscle, the lacrimal gland, and the thyroid. We prepared a 100,000g sediment fraction of porcine extraocular muscle, lacrimal gland, thyroid, and human thyroid, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting with sera from patients with Graves' disease, with or without ophthalmopathy, classified by symptoms and signs combined with orbital CT and normal controls. The results showed there was an approximately 55-kDa protein band which was recognized by the sera in 32.1% (9/28) of patients with autoimmune ophthalmopathy and in 47.3% (9/19) of patients with extraocular muscle enlargement demonstrated by orbital CT. It was significantly higher than the positive rates in patients without autoimmune ophthalmopathy and normal controls (15.8 and 11.1%, respectively, P < 0.025). However, there was no common antigen among the extraocular muscles, the lacrimal gland, and the thyroid. To further confirm this eye muscle-specific antigen, the approximately 55-kDa protein band was cut and solubilized from the nitrocellulose paper after SDS-PAGE, and electrophoretically transferred and used as an antigen in enzyme-linked immunosorbent assay. The absorbance was significantly higher in patients with autoimmune ophthalmopathy than patients without ophthalmopathy (P < 0.005), and normal controls (P < 0.01). Our findings suggest that an approximately 55-kDa protein may be a possible antigen in the eye muscle related to autoimmune ophthalmopathy.     

Soluble interleukin-2 receptor in sera of patients with Graves' disease.

Activation of T lymphocytes has been found to be associated with an increase in soluble interleukin-2 receptor (sIL-2R) levels. The aim of this study was to investigate serum levels of sIL-2R in 20 untreated patients with Graves' disease and to relate these levels to disease activity and to TSH-receptor, anti-thyroglobulin, anti-microsomal and anti-eye muscle antibodies. sIL-2R levels were significantly increased in newly diagnosed Graves' patients compared with controls (667 +/- 270 vs 205 +/- 45 U/ml) (P less than 0.001). The sIL-2R levels were higher in patients with active infiltrative ophthalmology than in those without eye symptoms (810 +/- 313 vs 525 +/- 180 U/ml). All patients were treated with methimazole for at least 12 months. sIL-2R levels were normalized by methimazole treatment in the majority of patients without ophthalmopathy but not in those with ophthalmopathy. In five patients sIL-2R serum levels were studied after interruption of thyrostatic therapy. An increase was observed in three patients and hyperthyroidism subsequently relapsed in two of these. Furthermore, a correlation was found between soluble interleukin-2 receptor levels and TSH-receptor antibodies but not with other immune parameters examined. Serum sIL-2R represents a useful marker of immunological activity in Graves' disease.