Serum antibodies reactive with eye muscle membrane antigens are detected in patients with nonspecific orbital inflammation.

PURPOSE: Nonspecific orbital inflammation, also called "orbital pseudotumor," has many of the features of thyroid-associated ophthalmopathy, especially when localized to the eye muscle. The purpose of this study is to test for circulating autoantibodies against eye muscle antigens and features of possible thyroid autoimmunity in patients with nonspecific orbital inflammation. METHODS: The authors studied eight patients with diffuse or localized nonspecific orbital inflammation. The presence of autoantibodies reactive with pig eye muscle membrane antigens and 1D, a recombinant 64 kilodaltons (kd) thyroid and eye muscle protein, were tested in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. RESULTS: The most frequently detected antibodies were those reactive with eye muscle membrane proteins of 55 and 64 kd, which were demonstrated in 62.5% and 62.5%, respectively, of patients with nonspecific orbital inflammation; antibodies against 95- and 45-kd proteins were each detected in 50% of patients. In health subjects, antibodies reactive with the 55- and 64-kd proteins were detected in 16% and 20% of patients, respectively; those reactive with the 95-kd protein were detected in 24% of patients and with the 45-kd protein in 20% of patients. On the other hand, antibodies to 1D were demonstrated in only one patient with nonspecific orbital inflammation and not at all in healthy subjects. The prevalence of positive tests were significantly greater in patients with nonspecific orbital inflammation than healthy patients only for antibodies reactive with a 55-kd protein. Of the four antigens, only the 55-kd protein was expressed in other (systemic) skeletal muscle. No patient had overt thyroid disease or detectable serum antibodies reactive with the thyroid-stimulating hormone receptor, and only one had antibodies reactive with the thyroid microsomal antigen. CONCLUSION: Serum autoantibodies reactive with eye muscle membrane proteins are demonstrated in the majority of patients with nonspecific orbital inflammation. Although the pathogenesis of this condition is unknown, autoimmunity against eye muscle antigens is a likely mechanism. While antibodies reactive with the thyroid microsomal antigen were detected in only one patient and anti-thyroid-stimulating hormone receptor antibodies in none of the patients, a possible association of nonspecific orbital inflammation with thyroid autoimmunity has not been excluded.

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 1D 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.

Serum antibody production to botulinum A toxin.

PURPOSE: Conflicting data have been reported regarding development of serum antibodies to botulinum A toxin. The purpose of this study is to determine conclusively whether antibody production to this toxin occurs in humans, and, if so, to determine its relationship, if any, to length of treatment, total cumulative dose, and clinical response to treatment. METHODS: Sixty-five sera samples from 42 adults treated with botulinum A toxin for essential blepharospasm, hemifacial spasm, or spasmodic torticollis were analyzed via a sphere-linked immunodiagnostic assay for antibody production. Results were plotted against length of treatment, number of injections, cumulative dose, and treatment effect produced. RESULTS: Twenty-four (57%) of the 42 patients produced antibodies in all three diagnostic groups. No significant differences were found between antibody producers and nonproducers with respect to age (P = 0.216), length of treatment (P = 0.586), number of injections (P = 0.619), or total cumulative dose (P = 0.286). Within the antibody-producing group, there was no significant correlation between amount of antibody and length of treatment (P = 0.081), number of injections (P = 0.134), or cumulative dose (P = 0.250). The presence of demonstrable antibodies in serum did not affect the clinical responsiveness to injection. CONCLUSION: Antibody production is present in a majority of patients treated with botulinum A toxin. The sphere-linked immunodiagnostic assay is a reliable and reproducible method for detecting and quantifying these antibodies. When antibody production occurs, it is likely due to variations in individual immune responsiveness and appears to have no direct effect on the patient's clinical response to treatment.

Pathogenesis of thyroid-associated ophthalmopathy: an autoimmune disorder of the eye muscle associated with Graves' hyperthyroidism and Hashimoto's thyroiditis.

Thyroid-associated ophthalmopathy, the progressive eye disorder which occurs frequently in patients with Graves' hyperthyroidism and, occasionally, in those with Hashimoto's thyroiditis, may be a two-stage disorder of the eye muscle. In the first stage, which may occur in the great majority of patients with Graves' hyperthyroidism and in an unknown, but probably small, proportion of those with Hashimoto's thyroiditis, antibodies and CD4+ (helper) T lymphocytes reactive with eye muscle and thyroid shared antigens, of which 64-kDa membrane proteins are good candidates, may initiate a mild eye muscle inflammation, manifested as eye muscle swelling on orbital imaging. The second stage, which occurs in about 25% of patients with Graves' hyperthyroidism and in 2% of those with Hashimoto's thyroiditis, may be due to reactivity of cytotoxic antibodies against eye muscle-specific membrane antigens, one of which at approximately 35 kDa appears a likely candidate, and, possibly, cytotoxic T cells in the context of the appropriate class I MHC molecule. Orbital connective tissue inflammation, which plays an important role in the development of progressive orbital inflammation, is likely to be secondary to the eye muscle reaction. The recent cloning of a 64-kDa thyroid and eye muscle antigen which shares significant homology with the muscle protein tropomodulin and mapping of its antibody-reactive epitopes provide structural information about one candidate eye muscle autoantigen and promise for a more rational approach to the diagnosis and management of this common, progressive eye disorder.

Re-examination of peripheral blood T cell subsets in dysthyroid orbitopathy.

Patients with dysthyroid orbitopathy (DO) were grouped according to a multifactorial assessment of disease severity and the rate of disease progression. Using this system and flow cytometric measurements of T cell subsets in the peripheral blood, a significant increase in the percentage of CD4+ lymphocytes correlated with disease severity in DO patients with progressive disease. These observations are consistent with the hypothesis that the CD4+ peripheral blood T helper cells play a significant role in the progression of DO.

Correlation of serum immunoglobulin E elevations with clinical stages of dysthyroid orbitopathy.

Total immunoglobulin E (IgE) was measured by an enzyme-linked immunoassay in serum samples from patients with dysthyroid orbitopathy and from a group of healthy volunteers. All the serum donors had no symptoms of allergy or infection and were not given any immunoregulative treatments for at least 6 months before the sampling. One hundred thirty-seven dysthyroid orbitopathy patients were rated clinically as belonging to one of the following groups: (1) stable dysthyroid orbitopathy; (2) active dysthyroid orbitopathy; (3) chronic or recurrent dysthyroid orbitopathy; or (4) dysthyroid orbitopathy characterized by limited myopathy. The serum IgE levels of all these groups were compared with 26 healthy, nonatopic volunteers. The mean IgE levels of groups 3 and 4 were significantly higher than the mean IgE level of the control group as well as that of the group with stable dysthyroid orbitopathy. Furthermore, serial readings on several patients were consistent with the hypothesis that serum IgE is elevated in connection with certain stages of rapid dysthyroid orbitopathy progression and also with two unusual clinical forms of dysthyroid orbitopathy.