Measurement of autoantibodies against human eye muscle plasma membranes in Graves' ophthalmopathy.

Antibodies that reacted with plasma membranes of human eye muscle but showed no binding to plasma membranes of human skeletal muscle were identified in serum of patients with Graves'' ophthalmopathy. Rabbit antithyroglobulin serum at a dilution of 1 X 10(-3) to 20 X 10(-3) had no effect on the binding of these antibodies to eye muscle membrane antigens. There was no correlation between antihuman eye muscle plasma membrane antibodies and antihuman thyroid membrane antibodies or antibodies against thyroglobulin. It is suggested that specific antibodies against eye muscle membranes are present in Graves'' ophthalmopathy and that this disease might represent a distinct autoimmune disorder.     

Graves' ophthalmopathy.

Graves'' ophthalmopathy usually occurs in association with hyperthyroidism. Its occasional occurrence in the absence of thyroid disease suggests, however, that it may be a separate autoimmune disorder. While the evidence supporting an autoimmune pathogenesis is considerable for the ophthalmopathy, it is not so impressive as that for Graves'' hyperthyroidism: orbital antibodies have not been convincingly demonstrated and autoantigens have not been identified. On the other hand, in patients with Graves'' ophthalmopathy the orbital tissues and eye muscle membranes are infiltrated with lymphoid cells and show evidence of cell-mediated immune reactions. Although there is some evidence that binding of thyroid stimulating hormone fragments and thyroglobulin-antithyroglobulin immune complexes to eye muscle membranes may be important in the pathogenesis of the ophthalmopathy, this needs to be confirmed. The mechanism for the association of hyperthyroidism and ophthalmopathy is unknown, but the association likely reflects an influence of thyroid hormones on the immune system. In view of the autoimmune pathogenesis the logical treatment of Graves'' ophthalmopathy appears to be immunosuppression.     

Graves' ophthalmopathy and subclinical hypothyroidism: diagnostic value of the thyrotropin releasing hormone test.

Five patients with Graves'' ophthalmopathy and no previously documented clinical or laboratory evidence of hyperthyroidism were studied. Their serum levels of thyroxine and triiodothyronine (T3) and their T3 uptake were normal. Although the baseline serum level of thyrotropin (TSH) was normal in two patients, it was increased on the other three, and when TSH releasing hormone (TRH) was administered the T3 response was impaired in three patients and the TSH response was exaggerated in all five. These findings facilitated the diagnosis of subclinical hypothyroidism and distinguished the patients from those with Graves'' ophthalmopathy and normal thyroid function or subclinical hyperthyroidism. Thyroid antibodies were detected in the serum of four of the five patients, suggesting the coexistence of chronic autoimmune thyroiditis; this disorder could account in part for the subclinical hypothyroidism, which was even present in the two patients in whom thyroid-stimulating immunoglobulin was found in the serum. These observations indicate the value of a TRH stimulation test in detecting subclinical hypothyroidism in patients with Graves'' ophthalmopathy who appear from clinical and routine laboratory studies to have normal thyroid function but could have normal function or subclinical hyperthyroidism.     

Human orbital tissue and thyroid membranes express a 64 kDa protein which is recognized by autoantibodies in the serum of patients with thyroid-associated ophthalmopathy

A 64 kDa protein has been identified in the membrane fraction of human eye muscle, orbital connective tissue and thyroid, by testing sera of patients with thyroid-associated ophthalmopathy in SDS-polyacrylamide gel electrophoresis and Western blotting. Antibodies to this membrane antigen seem characteristic of the early stage of ophthalmopathy. In the thyroid this newly recognized protein seems different from previously known membrane antigens. A thyroid antibody reactive with a 64 kDa membrane antigen in eye muscle could explain the very frequent association of ophthalmopathy with autoimmune thyroid disease.     

Clinical applications of assays for thyrotropin-receptor antibodies in Graves' disease.

Graves'' disease is characterized by hyperthyroidism, diffuse goitre, infiltrative ophthalmopathy and, rarely, pretibial myxedema. In 1956 a substance capable of prolonged thyroid stimulation was discovered in the serum of some patients with Graves'' disease and termed long-acting thyroid stimulator (LATS). It was shown to be an antibody that could interact with the receptor for thyroid-stimulating hormone (TSH). The term LATS is usually reserved for the activity measured in a laborious in-vivo bioassay in mice. Today the activity of TSH-receptor antibodies (TSH-R Ab) can be measured by in-vitro bioassays or by radioreceptor assays. These assays are now becoming commercially available. TSH-R Ab assays may be useful in predicting the response to therapy for Graves'' disease, investigating euthyroid ophthalmopathy and predicting the likelihood of neonatal hyperthyroidism.     

Long-term follow-up of ophthalmic Graves' disease.

Sixteen patients with ophthalmic Graves'' disease (clinically euthyroid with ophthalmopathy or exophthalmos) were followed up for 4.3 to 14.3 (mean 9.1) years to determine whether thyroid dysfunction developed and whether their ophthalmopathy progressed, regressed or remained stable. Five patients (31%) manifested hyperthyroidism or hypothyroidism, all before the end of the fifth year of follow-up. The ophthalmopathy was mild, and none of the patients required specific treatment. The thyroid function of patients with ophthalmic Graves'' disease should be periodically monitored for at least 5 years.     

Successful treatment of exophthalmos and pretibial myxoedema with plasmapheresis.

A patient with Graves''s disease with acute progressive exophthalmos and pretibial myxoedema was treated twice with plasmapheresis. Two weeks after the first treatment the symptoms recurred, but 20 weeks after the second treatment the exophthalmos was much improved and the pretibial myxoedema had disappeared. Analysis of sequential serum IgG concentrations and the thyroid-stimulating immunoglobulin index suggested that the two conditions were caused by specific IgGs. The results suggest that plasmapheresis has a useful place in the treatment of acute and rapidly progressive ophthalmopathy and pretibial myxoedema in patients with Graves''s disease.     

Mechanisms of immune damage in Graves' ophthalmopathy

We have studied the role of immunologically mediated cytotoxicity in the orbital tissue damage of Graves' ophthalmopathy. Antibody-dependent cell-mediated cytotoxicity (ADCC) against eye muscle (EM) cells and orbital fibroblasts (OF) was demonstrated in a small proportion of patients, all of whom had severe, recent disease. Antibody-mediated (complement-dependent) cytotoxicity against OF was found in only a few patients. No patients showed lysis above background with EM targets. ADCC activity against OF was absorbed by preincubation of serum with thyroid cells, eye muscle cells, and orbital fibroblasts, as well as thyroid, eye muscle and orbital connective tissue membranes. Both EM and OF were able to express class II MHC HLA-DR antigens when stimulated by gamma interferon, phytohemagglutinin or activated T lymphocytes. DR-positive target cells were much more susceptible to lysis, in both ADCC and lymphocyte-mediated cytotoxicity, than DR negative cells. When DR-positive OF and EM were used as targets in ADCC assays, the degree of lysis determined as 51Cr release given by serum from patients with Graves' ophthalmopathy was enhanced, but only in those patients showing positive tests with DR-negative targets. Intrathyroidal T lymphocytes obtained from a patient with Graves' ophthalmopathy were more cytotoxic against DR-positive OF and EM than equal numbers of her peripheral blood T lymphocytes. Antibody-dependent cell-mediated cytotoxicity and lymphocyte-mediated cytotoxicity against orbital fibroblasts and eye muscle cells are thus associated with target cell HLA-DR antigen expression and are likely to be mechanisms for in vivo tissue damage in Graves' ophthalmopathy. The identity of the mononuclear cell subpopulation effecting cell-mediated cytotoxicity against orbital target cells, and the possible significance of reaction of cytotoxic antibodies against orbital, thyroid-shared antigens are unclear.     

Pathogenesis of thyroid eye disease - does autoimmunity against the TSH receptor explain all cases?

Thyroid associated ophthalmopathy, or thyroid eye disease (TED), is a complex inflammatory disorder of the eye that, as its name implies, is usually associated with thyroid disease. Clinical observation supports the existence of three main TED subtypes, namely ocular myopathy, congestive myopathy, and mixed congestive and myopathic ophthalmopathy. Although the precise pathophysiology of TED remains unclear, it is likely to reflect an autoimmune reaction involving sensitised T lymphocytes and autoantibodies directed against a specific orbital or thyroid-and-orbital shared antigen(s). One well-studied candidate in this immune reaction is the thyroid-stimulating hormone receptor (TSHR), which is also expressed in the orbital fibroblast and preadipocyte. Most patients with ophthalmopathy have associated Graves' disease, 10% have Hashimoto's thyroiditis in which the eye changes are often mild and expressed mainly as upper eyelid retraction (UER), and 10% have no apparent associated thyroid disease - so-called "euthyroid Graves' disease". Ophthalmopathy can also occur in some patients with transient thyroiditis, thyroid cancer, and Graves' disease many years after treatment of the hyperthyroidism - situations where TSHR antibodies are not expected to be present, suggesting that the relationship between TSHR antibodies and the eye disorder has not been established for all cases. In our studies of TED we have investigated the nature and significance of antibodies targeting other eye muscle and orbital connective tissue (OCT) antigens, in particular the calcium binding protein calsequestrin (CASQ1) and the orbital fibroblast membrane antigen collagen XIII. Our working hypotheses for the pathogenesis of TED are: i) the initial reaction in the orbit is antibody and T lymphocyte targeting of the TSHR in the OCT compartment, and ii) the associated extra ocular and upper eyelid muscle inflammation reflects either autoimmunity against primary skeletal muscle antigens such as CASQ1 or a secondary, non specific effect of the OCT reactions as proposed by the main proponents of the "TSHR hypothesis". Here, we review the evidence that autoimmunity against the TSHR expressed in the orbit can be implicated in the development of all cases of TED. Although there is a close general correlation between ophthalmopathy and TSHR antibodies there are many exceptions, suggesting that the continued study of the possible role of autoimmunity against calsequestrin and collagen XIII is justified.     

Neonatal Hyperthyroidism and Long-acting Thyroid Stimulator Protector

Neonatal hyperthyroidism has been thought to result from transplacental passage of long-acting thyroid stimulator (L.A.T.S.) from a mother with Graves''s disease. A case is presented here in which no L.A.T.S. was detected in the mother or neonate but another immunoglobulin, L.A.T.S. protector, a specific human thyroid stimulator, was shown to be present in the mother''s serum. This stimulator may have been the cause of the neonatal hyperthyroidism.     

Intravenous methylprednisolone in the treatment of Graves' ophthalmopathy.

Eleven euthyroid patients with severe Graves'' eye disease were treated with intravenous methylprednisolone and followed up for six months or more by ophthalmological assessment, orbital computed tomography (CT), photographs, and antibody measurements. Papilloedema resolved in the single patient in whom it was present; visual acuity was abnormal in seven eyes initially and in only one eye after treatment; the intraocular pressure differential, which reflects muscle dysfunction, was initially abnormal in 18 eyes but showed a progressive and distinct improvement; nine patients showed substantial improvement in inflammatory signs. Exophthalmos improved early after treatment, but this improvement was not maintained. Orbital CT showed a pronounced reduction in the bulk of eye muscles after treatment in eight of nine patients. Autoantibodies to the thyroid stimulating hormone receptor declined. Adverse effects were trivial. Thus eight patients showed a clear response to intravenous methylprednisolone as judged by ophthalmic assessment and CT scan. The two patients who showed little response and one who had none all had a long history (more than a year) of ophthalmopathy. Results were better than those with oral steroids and adverse effects less. Treatment of Graves'' eye disease is more likely to be effective if given early; patients should be referred promptly to specialist centres, where treatment with intravenous methylprednisolone should be considered.     

Oxidation Products and Antioxidant Markers in Plasma of Patients with Graves' Disease and Toxic Multinodular Goiter: Effect of Methimazole Treatment

Oxidative stress plays an important role in hyperthyroidism-induced tissue damage, as well as in development of autoimmune disorders. To clarify influence of thyroid metabolic status and autoimmune factors on blood extracellular indices of reactive oxygen species (ROS) generation and free radical scavenging in hyperthyroidism, we studied patients with newly diagnosed and untreated Graves' disease without infiltrative ophthalmopathy (17 female and 8 male, aged 41.8±8.9) and toxic multinodular goiter (15 female and 9 male, aged 48.4±10.1) under the same antithyroid treatment protocol. Initially and after achievement of stable euthyroidism with methimazole, plasma levels of hydrogen peroxide (H²O²), lipid hydroperoxides (ROOH) and ceruloplasmin (CP) and serum concentrations of thiobarbituric acid-reacting substances (TBARS) were determined. Similarly, activities of plasma superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and glutathione reductase (GR) were assayed. The results were compared to those of age- and sex-matched controls. Average duration of hyperthyroidism and treatment period were similar in both patients groups. H²O², ROOH and TBARS concentrations were significantly higher in hyperthyroid patients compared to controls. Hyperthyroidism caused an evident increase in SOD and CAT activities and CP level, as well as a decrease in GPx and GR activities. Achievement of euthyroidism resulted in normalization of all analyzed parameters in both hyperthyroid patients groups. These findings suggest that the changes in blood extracellular indices of oxidative stress and free radical scavenging in hyperthyroid patients are influenced by thyroid metabolic status, and are not directly dependent on autoimmune factors present in Graves' disease.     

Antigenic relation between thyroid peroxidase and the microsomal antigen implicated in auto-immune diseases of the thyroid

Pools of sera from patients with Graves' disease or Hashimoto's thyroiditis highly inhibit the binding to human thyroid membranes of one of 19 monoclonal antibodies raised against preparations of human thyroid membranes. This monoclonal antibody reacts with human and bovine thyroid peroxidase and bovine lactoperoxidase but not with human hemoglobin, cytochrome c and other related molecules. These results indicate that the thyroid peroxidase and the microsomal antigen are antigenically related. These data taken together with those from other groups, highly suggest that thyroid peroxidase is the microsomal antigen involved in autoimmune thyroid diseases.     

Optimum duration of antithyroid drug treatment determined by assay of thyroid stimulating antibody in patients with Graves' disease.

OBJECTIVE--To determine the optimal duration of antithyroid drug treatment by monitoring serum thyroid stimulating antibody values in patients with Graves'' disease. DESIGN--Prospective longitudinal trial of patients with Graves'' disease followed up for 24 months after withdrawal of treatment. SETTING--Tertiary referral centre. PATIENTS--A total of 64 consecutive patients with untreated Graves'' disease, eight of whom were subsequently excluded. Fifty six patients completed the study. INTERVENTIONS--All patients were treated initially with carbimazole 40 mg, then with decreasing doses that maintained a euthyroid state. Treatment was scheduled to continue for 18 months but was withdrawn earlier if serum thyroid stimulating antibody became undetectable. END POINT--Serum values of thyroid stimulating antibody (assayed by stimulation of human thyroid cells in vitro) and thyroid hormones and thyroid state every three months during treatment and afterwards every six months for 24 months. MEASUREMENTS AND MAIN RESULTS--In 44 patients serum thyroid stimulating antibody became undetectable during treatment and treatment was withdrawn (median duration of treatment nine months, range 3-18 months). In 12 patients the antibody could be detected during 18 months of treatment. Among the first group of 44 patients initial values of the antibody before treatment were significantly lower than in the second group of 12 patients (median 225% (range 138-1236%) v 570% (250-1480%), p less than 0.001); the incidence of relapse was also lower (41% v 92%, p less than 0.001); and among those who did relapse the disease free interval after treatment was longer (median 12 months v 1 month, p less than 0.001). Moreover, the initial median serum values of thyroid stimulating antibodies were not related to the occurrence of relapse or remission as these did not differ between patients who did and did not have a relapse (median 267% (range 139-1480%) v 220% (range 138-1236%). CONCLUSION--Monitoring of serum thyroid stimulating antibody was a good guide to the duration of treatment as it allowed the treatment period to be considerably shortened in a large group of patients with no loss of efficiency.     

Evidence for a primary autoimmune type of diabetes mellitus.

Sixty-eight patients with longstanding diabetes and persistent islet-cell antibody and 35 with coexistent diabetes and Graves''s disease or primary myxoedema were studied with particular reference to the HLA system and autoantibody patterns. A higher incidence of HLA-B8 than normal was observed in the two groups. An additive relative risk exists when type I diabetes and autoimmune thyroid disease coexist, indicating that different HLA-linked genes may confer susceptibility to the pancreatic and thyroid disorders. Other characteristics, including female predominance, a later onset of diabetes, and a strong family history of autoimmune endocrinopathy, provide further evidence that this form of diabetes is aetiologically distinct from that generally seen in children. These results support the hypothesis of a primary autoimmune type of diabetes mellitus.     

Experimental exophthalmos. Binding of thyrotropin and an exophthalmogenic factor derived from thyrotropin to retro-orbital tissue plasma membranes.

Biologically active preparations of 125I-thyrotropin, [3H]thyrotropin, and the [3H]exophthalmogenic factor derived from thyrotropin by partial pepsin digestion have been used to study the binding properties of the thyrotropin receptor on guinea pig retro-orbital tissue plasma membranes. In regard to the optimal conditions of binding, pH, buffer, salt concentrations, and temperature, these properties are the same as those described in any accompanying report concerning thyrotropin binding to bovine thyroid plasma membranes (Tate, R.L., Schwartz, H.I., Holmes, J.M., Kohn, L.D., and Winand, R.J. (1975) J. Biol. Chem. 250, 6509-6515). In addition, thyrotropin receptors on the retro-orbital tissue plasma membranes are similar to thyrotropin receptors on bovine thyroid plasma membranes in their apparent negative cooperativity and in their relative affinities for luteinizing hormone, the beta subunit of thyrotropin, and the alpha subunit of thyrotropin. In contrast, gamma-globulin from patients with malignant exophthalmos enhances binding when added to incubation mixtures containing the retro-orbital tissue plasma membranes but not when added to those containing thyroid plasma membranes. Normal gamma-globulin and gamma-globulin from Graves' disease patients without exophthalmos do not have this property. The gamma-globulin itself does not bind to the membrane except in the presence of thyrotropin or its exophthalmogenic factor derivative. Tryptic digestion of the retro-orbital tissue membranes releases specific thyrotropin and exophthalmogenic factor binding activity into the supernatant phase. Chromatography on Sephadex G-100 indicates that this trypsin-released receptor activity has a molecular weight of 75,000 or greater, rather than 15,000 to 30,000 for the trypsin-released receptor activity from bovine thyroid membranes (Tate, R.L., Schwartz, H.I., Holmes, J.M., Kohn, L.D., and Winand, R.J. (1975) J. Biol. Chem. 250, 6509-6515).     

Grave's disease affecting one thyroid lobe: About 2 cases

Graves’ disease is an autoimmune disorder of thyroid which is characterized by hyperthyroidism, diffuse goiter, ophthalmopathy. Due to the pathophysiological mechanism of the disease, the disease affects both thyroid lobes. Unilateral involvement of the thyroid gland in patients with Graves’ disease is a rare entity, which suggests a difference between the two thyroid lobes. Clarifying this phenomenon could be a line of research for a better understanding of the pathophysiology of Graves's disease. This entity must be known to the clinician in order to take it better and avoid misdiagnosis. Here we present two cases of Graves’ disease which had unilateral involvement of the thyroid gland and discuss the hypotheses explaining this observation.     

Similarity and dissimilarity between clinical and laboratory findings, especially anti-thyrotropin receptor antibody in ophthalmic Graves' disease without persistent hyperthyroidism and hyperthyroid Graves' disease

The aim of this study was to investigate thyroid states, significance of anti-TSH receptor antibodies and the clinical courses of patients with euthyroid Graves' ophthalmopathy. The clinical and laboratory finding of 30 patients with euthyroid Graves' ophthalmopathy were briefly as follows: 1) normal sized thyroid or small goiter; 2) negative or weakly positive thyrotropin binding inhibitor immunoglobulin (TBII); 3) normal thyroid [99 m-Tc] pertechnetate uptake; and 4) frequent observations of low serum TSH values. Besides TBII, thyroid stimulating antibody (TSAb) was measured under low salt and isotonic conditions using FRTL-5 rat thyroid cells. Both TBII and TSAb titers were lower in euthyroid Graves' ophthalmopathy than in hyperthyroid Graves' disease. Serum TSH levels frequently became low in patients considered as euthyroid upon the first examination as well as in Graves' patients in remission, reflecting preceding or mild hyperthyroidism. In follow-up studies, these patients with mildly elevated thyroid hormone levels and low TSH levels seldom reached a state of persistent hyperthyroidism, when TBII was negative or only weakly positive.     

Detection of the novel autoantibody (anti-UACA antibody) in patients with Graves' disease

Uveal autoantigen with coiled coil domains and ankyrin repeats (UACA) is an autoantigen in patients with panuveitis such as Vogt-Koyanagi-Harada disease. The prevalence of IgG anti-UACA antibodies in patients with uveitis is significantly higher than healthy controls, suggesting its potential role as an autoantigen. Originally, UACA was cloned from dog thyroid tissue following TSH stimulation. So, we presumed UACA could be a novel autoantigen in autoimmune thyroid diseases. We measured serum anti-UACA antibody titer using ELISA in patients with autoimmune thyroid diseases (Graves' disease, Hashimoto's thyroiditis, subacute thyroiditis, and silent thyroiditis). The prevalence of anti-UACA antibodies in Graves' disease group was significantly higher than that in healthy group (15% vs. 0%). Moreover, the prevalence of anti-UACA antibodies in Graves' ophthalmopathy was significantly higher than that in Graves' patients without ophthalmopathy (29% vs. 11%). Especially, 75% of severe ocular myopathy cases showed high UACA titer. Immunohistochemical analysis revealed that UACA protein is expressed in eye muscles as well as human thyroid follicular cells. Taken together, UACA is a novel candidate for eye muscle autoantigens in thyroid-associated ophthalmopathy.     

Hypothyroidism after Thyroidectomy for Graves's Disease: A Search for an Explanation

Out of 38 patients who had undergone subtotal thyroidectomy for Graves''s disease seven to 20 years previously 15 developed hypothyroidism. In these 15 patients autoantibodies against thyroid cytoplasm were significantly more frequent than in the 23 euthyroid patients, though there was no difference in the prevalence of autoantibodies against thyroglobulin. Histological examination of the thyroid tissue removed at operation showed that significantly more plasma cells and lymphoid follicles with germinal centres were present in patients who subsequently developed hypothyroidism than in those who remained euthyroid. No differences in the amount of lymphocytic infiltration were seen in hypothyroid and euthyroid patients.The results suggest that B lymphocytes play a part in the development of postoperative hypothyroidism in Graves''s disease. It is proposed that Graves''s disease and Hashimoto''s disease are different aspects of the same basic autoimmune process.