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Jack R. Wall - One of the best experts on this subject based on the ideXlab platform.
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Cell mediated Immunity Against Three Eye Muscle Antigens and Correlation with Eye Signs in Patients with Transient and Chronic Thyroiditis
The Open Autoimmunity Journal, 2012Co-Authors: Bao Nguyen, Bamini Gopinath, Bernard Champion, Jack R. WallAbstract:Background: Mild Eye signs, especially itchiness and grittiness of the Eyelids and upper Eyelid retraction (UER), are found in about 25% of patients with Hashimoto's thyroiditis (HT) in whom antibodies against calsequestrin and flavoprotein (Fp) are often detected. The role of T lymphocyte reactivity against Eye Muscle antigens in patients with thyroiditis has not been investigated. Methods: We studied peripheral blood T lymphocyte reactivity against calsequestrin, Flavoprotein (Fp) and G2s in pa- tients with transient (sub acute and silent) thyroiditis (TT) and HT, determined in a standard proliferation assay. Reactivity was expressed as stimulation index (SI) and correlated with signs of ophthalmopathy and upper Eyelid disease, assessed as; NOSPECS classes, clinical activity score (CAS) and upper Eyelid retraction (UER). Results: Positive lymphocyte proliferation to calsequestrin was demonstrated in 71% of TT patients all of whom had mild ophthalmopathy and this was significantly increased compared to normal control subjects. The prevalences of positive T cell reactivity to calsequestrin was also significantly increased in HT patients (38%) compared to the controls, all of whom had upper Eyelid disease. Three out of 7 patients with upper Eyelid disease (6 of the patients with TT or HT and one other patient with Graves' disease) taken as a separate group, demonstrated significant T lymphocyte sensitisation to cal- sequestrin. TSH-receptor antibodies were detected in only one TT patient and one patient with upper Eyelid disease. Interpretation: The development of ophthalmopathy and/or Eyelid retraction (in the absence of TSH-r antibodies and Graves' hyperthyroidism) in TT patients is closely associated with an autoimmune reaction against calsequestrin. These findings support the notion that ophthalmopathy and upper Eyelid inflammation and dysfunction are independent autoim- mune disorders not necessarily linked with thyroid autoimmunity and that T cell reactivity plays a role.
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Eye signs and serum Eye Muscle and collagen xiii antibodies in patients with transient and progressive thyroiditis
Thyroid, 2007Co-Authors: Bamini Gopinath, Jack R. WallAbstract:Background: There have been reports of the development of ophthalmopathy in patients with subacute thyroiditis (SAT) in the absence of Graves' disease and thyroid-stimulating hormone receptor (TSH-r) antibodies. Objective: The aim of the study was to determine the prevalences of Eye and Eyelid signs and positive Eye Muscle and collagen XIII antibody tests in patients with SAT and silent thyroiditis (ST) and in patients with Hashimoto's thyroiditis (HT) as chronic thyroiditis controls. Design: Ophthalmopathy was classified as Nunery type 1 (orbital inflammation, proptosis, without restrictive myopathy) or Nunery type 2 (with restrictive myopathy). We tested for antibodies against calsequestrin, flavoprotein (Fp), G2s, and collagen XIII in 5 patients with SAT, 6 with ST, and 11 with HT, and in 12 age- and sex-matched healthy subjects, using an optimized and standardized enzyme-linked immunosorbent assay (ELISA). Main outcome: At the first visit, Eye signs were found in two patients with SAT, one with type 1...
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autoimmunity against Eye Muscle antigens may explain thyroid associated ophthalmopathy
Canadian Medical Association Journal, 2006Co-Authors: Junichi Tani, Jack R. WallAbstract:The ophthalmopathy associated with Graves' hyperthyroidism and Hashimoto's thyroiditis is an autoimmune disorder of the extraocular Muscle and the surrounding orbital connective tissue (OCT) and fat ([Box 1][1]). Its most appropriate name is thyroid-associated ophthalmopathy (TAO). ![Figure][2]
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Eye Muscle antibodies in Graves’ ophthalmopathy: Pathogenic or secondary epiphenomenon?
Journal of endocrinological investigation, 2004Co-Authors: T. Mizokami, Mario Salvi, Jack R. WallAbstract:The extra ocular (Eye) Muscles are one of the principal tissues involved in the autoimmune-mediated inflammation of Graves’ ophthalmopathy (GO). Several Eye Muscle proteins are targeted by autoantibodies or sensitized T lymphocytes, or both, and include: G2s, which is now identified as the terminal 141 amino acids of the winged-helix transcription factor FOXP1, the flavoprotein (Fp) subunit of the mitochondrial enzyme succinate dehydrogenase, the so-called “64kDa protein”, a non-tissue specific membrane protein called 1D and the calcium binding protein calsequestrin. Of these, antibodies against G2s and Fp are the most sensitive markers of Eye Muscle damage in patients with thyroid autoimmunity even though neither antigen is specific to Eye Muscle and neither antibody is specific to GO. However, the recent finding that the calsequestrin gene is 4.7 times more expressed in Eye Muscles than other skeletal Muscles suggests that we should reconsider the possible role of anti-calsequestrin autoantibodies in ophthalmopathy. GO may comprise two main subtypes with different pathogenetic mechanisms, namely ocular myopathy in which Eye Muscle inflammation predominates and congestive ophthalmopathy where inflammatory changes occur in the periorbital connective tissues in the absence of Eye Muscle dysfunction. Anti-G2s and anti-Fp antibodies are closely associated with the ocular myopathy subtype of GO while antibodies targeting type XIII collagen, the only member of the collagen family to have a transmembrane domain, are closely linked to congestive ophthalmopathy. Since both G2s and Fp are intracellular antigens it is unlikely that either antibody causes Eye Muscle fiber damage in GO, although a role in the later stages of the disease when the fiber has released its cellular contents has not been excluded. Eye Muscle antibodies that are cytotoxic to Eye Muscle cells in antibody-dependent cell-mediated cytotoxicity (ADCC) are more likely to play a role in Eye Muscle fiber damage since they target a putative Eye Muscle cell membrane antigen, the identity of which is currently being investigated. While anti-G2s and anti-Fp antibodies are probably secondary to an underlying reaction, such as cytotoxic T lymphocyte targeting of an Eye Muscle membrane antigen that has yet to be identified, they are reliable markers of immunologically mediated Eye Muscle fiber damage in patients with Graves’ hyperthyroidism. In conclusion, while a pathogenic role for Eye Muscle antibodies has not been excluded, they are most likely secondary to cytotoxic T cell reactions in GO and, as such, good markers of this autoimmune disease.
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Eye Muscle antibodies and subtype of thyroid-associated ophthalmopathy.
Thyroid : official journal of the American Thyroid Association, 2002Co-Authors: Matthias Kaspar, George J. Kahaly, Curtis Archibald, Anna Maria De Bellis, Masayo Yamada, Cheng-hsien Chang, Jack R. WallAbstract:The Eye changes associated with Graves' hyperthyroidism can be classified into two subtypes, congestive ophthalmopathy (CO), in which inflammatory changes in the periorbital tissues predominate, and ocular myopathy (OM), in which Eye Muscle damage is the main feature. Antibodies against the flavoprotein (Fp) subunit of succinate dehydrogenase (SDH), the 64-kd protein, and G2s, a thyroid and Eye Muscle shared protein of unknown function, are good markers of Eye Muscle cell damage in patients with OM. Another antigen associated with ophthalmopathy is the flavine adenine nucleotide (FAD) cofactor of several mitochondrial enzymes, including SDH. We tested for serum antibodies against purified human recombinant Fp, FAD, and a G2s fusion protein, in patients with thyroid-associated ophthalmopathy (TAO) and control patients and subjects, in enzyme-linked immunosorbent assay. Antibodies against Fp were detected in 32% of patients with TAO, 30% with Graves' hyperthyroidism (GH), 16% with Hashimoto's thyroiditis (H...
J. R. Wall - One of the best experts on this subject based on the ideXlab platform.
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cloning and characterization of the novel thyroid and Eye Muscle shared protein g2s autoantibodies against g2s are closely associated with ophthalmopathy in patients with graves hyperthyroidism
The Journal of Clinical Endocrinology and Metabolism, 2000Co-Authors: Kazuaki Gunji, S. Kubota, Anna Maria De Bellis, Masayo Yamada, Brian A. C. Ackrell, Sylvia Wengrowicz, Antonio Bellastella, Antonio Bizzarro, Antonio Agostino Sinisi, J. R. WallAbstract:Serum autoantibodies against Eye Muscle antigens are closely linked with thyroid-associated ophthalmopathy (TAO), although their significance is unclear. The two antigens that are most often recognized are Eye Muscle membrane proteins with molecular masses of 55 and 64 kDa, as determined from immunoblotting with crude human or porcine Eye Muscle membranes. We cloned a fragment of the 55-kDa protein by screening an Eye Muscle expression library with affinity-purified anti-55 kDa protein antibody prepared from a TAO patient's serum. A complementary DNA (cDNA) encoding a novel protein, which we have called G2s, was sequenced on both strands, and its size was 411 bp. The open reading frame of G2s corresponded to a 121-amino acid peptide with a size of 1.4 kb. Using the rapid amplification of 5'-cDNA ends technique we were able to clone an additional 0.3 kb of the protein. G2s did not share significant homologies with any other entered protein in computer databases and had one putative transmembrane domain. Using the 1.4 kb cDNA as probe in Northern blotting of a panel of messenger ribonucleic acids prepared from human tissues, the parent protein was shown to correspond to a large molecule of about 5.8 kb with a calculated molecular mass of approximately 220 kDa, consistent with earlier immunoblot studies performed in the absence of reducing agents. G2s was strongly expressed in Eye Muscle, thyroid, and other skeletal Muscle and to a lesser extent in pancreas, liver, lung, and heart Muscle, but not in kidney or orbital fibroblasts. We tested sera from patients with Graves' hyperthyroidism with and without ophthalmopathy and from control patients and subjects for antibodies against a G2s fusion protein by immunoblotting and enzyme-linked immunosorbent assay. In immunoblotting, antibodies reactive with G2s were identified in 70% of patients with TAO of less than 3 yr duration, 53% with TAO of more than 3 yr duration, 36% with Graves' hyperthyroidism without evident ophthalmopathy, 17% with Hashimoto's thyroiditis, 3% with type 1 diabetes, 23% with nonimmunological thyroid disorders, and 16% of normal subjects. The prevalences, compared to normal values, were significant for the two groups of patients with TAO, but not for the other groups. Tests were positive in 54% of patients with active TAO, 33% with chronic ophthalmopathy, 36% with Graves' hyperthyroidism, 54% with Hashimoto's thyroiditis, 23% with type 1 diabetes, and in 11% of normal subjects using enzyme-linked immunosorbent assay. The antibodies predicted the development of the ocular myopathy subtype of TAO in six of seven patients and the congestive ophthalmopathy subtype in seven of eight patients, respectively, with Graves' hyperthyroidism studied prospectively during and after antithyroid drug therapy. Antibodies reactive with G2s may be early markers of ophthalmopathy in patients with Graves' hyperthyroidism. Because G2s is expressed in both thyroid and Eye Muscle, immunoreactivity against a shared epitope in the two tissues may explain the well known link between thyroid autoimmunity and ophthalmopathy.
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The 64-Kilodalton Eye Muscle Protein Is the Flavoprotein Subunit of Mitochondrial Succinate Dehydrogenase: The Corresponding Serum Antibodies Are Good Markers of an Immune-Mediated Damage to the Eye Muscle in Patients with Graves’ Hyperthyroidism
The Journal of clinical endocrinology and metabolism, 1998Co-Authors: S. Kubota, John S Kennerdell, Yuji Hiromatsu, Kazuaki Gunji, C. Stolarski, Sylvia Wengrowicz, B. A. C. Ackrell, B. Cochran, J. R. WallAbstract:Thyroid-associated ophthalmopathy (TAO) is a progressive Eye disorder associated with thyroid autoimmunity, particularly Graves' hyperthyroidism, which is generally considered to have an autoimmune etiology. Eye Muscle membrane proteins reportedly of 55 and 64 kDa are the best markers of the ophthalmopathy. The main focus of our recent studies has been to purify the pertinent proteins from porcine Eye Muscle membranes and characterize them. The 64-kDa protein is now shown from a partial sequence and by Western blotting using specific antibody probes to be the flavoprotein (Fp) subunit of succinate dehydrogenase and to have a correct molecular mass of 67 kDa. The protein was purified and cleaved with cyanogen bromide, and the N-terminal region of an immunoreactive partial peptide was determined. The 20-amino acid porcine sequence so obtained matched one within the Fp subunits of human and bovine succinate dehydrogenases in 20 and 18 of these positions, respectively. Succinate dehydrogenase is both a citric acid cycle enzyme and a component (complex II) of the mitochondrial respiratory chain. It is thus essential for aerobic energy production and is highly conserved. The mature human and bovine Fp subunits are 92% homologous and have a molecular mass of approximately 67 kDa, the same as our redetermined value for the 64-kDa marker protein. Sera from patients with TAO and from those with Graves' hyperthyroidism without evident ophthalmopathy highlighted the 64-kDa marker protein in crude porcine Eye Muscle membranes and the Fp subunit of highly purified bovine succinate dehydrogenase at the identical position on Western blots. Anti-beef Fp antibodies were detected in sera from 67% of patients with active TAO of more than 1-yr duration, in 30% with stable TAO of more than 3-yr duration, and in 30% of patients with Graves' hyperthyroidism without ophthalmopathy, but in only 7% of age- and sex-matched normal subjects. As succinate dehydrogenase is bound to the matrix (inside) surface of the mitochondrial inner membrane, it is unlikely to be accessible to circulating autoantibodies. We would postulate that Eye Muscle damage in ophthalmopathy is probably caused by cytotoxic antibodies or CD+ T lymphocytes targeting a cell membrane antigen, such as the thyroid and Eye Muscle shared protein G2s, and that presentation of succinate dehydrogenase is secondary. On the other hand, an autoantibody response to succinate dehydrogenase may be a good marker of immune-mediated damage to the Eye Muscle fiber and may support the idea that the extraocular Muscles are targets of the autoimmune reactions of TAO.
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Role of Eye Muscle antibody measurement in diagnosis of thyroid-associated ophthalmopathy: a laboratory update.
Endocrine practice : official journal of the American College of Endocrinology and the American Association of Clinical Endocrinologists, 1998Co-Authors: S. Kubota, Carol Stolarski, John S Kennerdell, Kazuaki Gunji, J. R. WallAbstract:ABSTRACT Objective: To review the current role of measurement of serum Eye Muscle antibodies in thyroid-associated ophthalmopathy (TAO). Methods: We conducted laboratory studies to determine the prevalences of serum autoantibodies reactive with Eye Muscle antigens in patients with active and inactive TAO, Graves’ hyperthyroidism, and Hashimoto’s thyroiditis as well as in normal subjects. Results: The two antigens most often recognized in immunoblotting with crude human or porcine Eye Muscle membranes by serum autoantibodies in patients with TAO are Eye Muscle membrane proteins of 55 and 64 kd. One 64-kd Eye Muscle protein has recently been cloned by screening a human Eye Muscle expression library with two different antibody probes and identified from a computer gene bank search as the calcium-binding protein calsequestrin. A fragment of a 220-kd Eye Muscle protein, called G2s, has also been cloned by screening the Eye Muscle library with affinity-purified antibodies reactive with a 55-kd Eye Muscle membrane protein. The prevalences of autoantibodies reactive with these two antigens in our study groups were as follows. Antibodies against calsequestrin were detected in 38% of patients with TAO for 3 years, in 17% of patients with Graves’ hyperthyroidism without ophthalmopathy, in 12% of patients with Hashimoto’s thyroiditis without ophthalmopathy, and in 21% of normal subjects. Antibodies reactive with the 64-kd protein were demonstrated in 62% of patients with recent-onset active TAO, in 33% with Eye disease for > 3 years, in 39% of patients with Graves’ hyperthyroidism without ophthalmopathy, in 25% of patients with Hashimoto’s thyroiditis, and in 16% of normal control subjects. Antibodies reactive with G2s fusion protein were detected in 67% of patients with recent-onset active TAO, in 46% of patients with Graves’ hyperthyroidism, and in 20% of normal subjects. Antibodies reactive with the parent protein, of which G2s is a fragment, may be markers of early Eye Muscle swelling and inflammation, whereas those reactive with the 64-kd protein and, less often, calsequestrin are associated with established Eye disease. Conclusion: Measurement of serum Eye Muscle antibodies is recommended as an aid to the early diagnosis of ophthalmopathy in predisposed patients and first-degree relatives of patients with TAO as well as to monitor active or progressive Eye disease. (Endocr Pract. 1998;4:127-132)
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Eye Muscle Antibodies in Patients with Ocular Myasthenia Gravis: Possible Mechanism for Eye Muscle Inflammation in Acetylcholine-Receptor Antibody-Negative Patients
Clinical immunology and immunopathology, 1998Co-Authors: K. Gunji, John S Kennerdell, C. Skolnick, T. Bednarczuk, S. Benes, Brian A. C. Ackrell, Bruce Cochran, J. R. WallAbstract:Myasthenia gravis is an organ-specific autoimmune disorder generally thought to be caused by an antibody-mediated attack against the skeletal Muscle nicotinic acetylcholine (Ach) receptor (AchR) at the neuromuscular junction. Extraocular Muscle weakness and double vision are present in about 90% of patients with myasthenia gravis and are the predominant complaints in about 20% of patients, when the condition is called ocular myasthenia gravis (OMG). While serum antibodies against the AchR are detected in most patients with generalized myasthenia gravis (GMG), they are not found in about one-third of patients with the ocular variety, and epidemiological, clinical, and serological studies suggest that OMG and GMG are two separate diseases. Both forms of myasthenia gravis are sometimes associated with thyroid autoimmunity or thyroid-associated ophthalmopathy (TAO). We have therefore tested the sera of patients with GMG and OMG by Western blotting for antibodies against porcine Eye Muscle membrane proteins in general, and by enzyme-linked immunosorbent assays (ELISA) specifically for reaction with two skeletal Muscle antigens which are prominent marker antigens for TAO, namely, the calcium-binding protein calsequestrin and the so-called "64-kDa protein." The 64-kDa protein has recently been identified as the flavoprotein subunit of mitochondrial succinate dehydrogenase. Patients with ophthalmopathy and myasthenia were excluded. Nine of the patients had associated Graves' hyperthyroidism without evident ophthalmopathy and one had Hashimoto's thyroiditis. Antibodies against porcine Eye Muscle membrane antigens of M(r) 15-110 kDa were detected in patients with GMG or OMG, one or more antibodies being detected in 100% of patients with GMG and in 88% of those with OMG. The most frequently found antibodies were those targeting Eye Muscle membrane proteins of 15, 67, and 110 kDa. Antibodies reactive with purified calsequestrin (63 kDa) were detected in 21% of patients with OMG but in no patient with GMG. Antibodies recognizing purified succinate dehydrogenase (67 kDa) were found in 42% of patients with OMG, in 100% (5 of 5) of patients with GMG, and in 48% of all patients with myasthenia gravis not associated with Graves' hyperthyroidism. There was no close correlation between any Eye Muscle-reactive antibody and antibodies against the AchR in either group of myasthenic patients. The findings support the notion that immunoreactivity against skeletal Muscle proteins other than the AchR may play a role in the development of the Muscle weakness in AchR antibody-negative patients with OMG and GMG, although it is unlikely that any of the antibodies demonstrated in this study are directly implicated. Similarly, while the demonstration of antibodies reactive with Eye Muscle antigens associated with TAO in patients with OMG raises the possibility that the link between the ocular lesions of myasthenia gravis and Graves' disease may be autoimmunity against a common antigen(s), it is more likely that both disorders are mediated by cytotoxic T cells recognizing another cell membrane antigen, such as the novel thyroid and Eye Muscle shared protein G2s, and that serum antibodies reactive with succinate dehydrogenase Fp subunit and calsequestrin are markers of an immune-mediated Eye Muscle reaction.
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Antibody-dependent cell-mediated cytotoxicity against orbital target cells in thyroid-associated ophthalmopathy and related disorders; close relationship between serum cytotoxic antibodies and parameters of Eye Muscle dysfunction
Journal of Endocrinological Investigation, 1996Co-Authors: A. Barsouk, John S Kennerdell, J. Kiljanski, C. Stolarski, V. Nebes, K. A. Peele, R. Volpe, J. R. WallAbstract:We have carried out tests for antibody-dependent cell-mediated cytotoxicity (ADCC) against extra ocular Muscle (EOM), Müller’s Muscle, orbital fibroblasts and skeletal Muscle in patients with thyroid-associated ophthalmopathy (TAO) and related Eye disorders. Cytotoxicity was measured as lactate dehydrogenase (LDH) release and results expressed as % cytotoxicity. Tests were positive, with EOM cells, in 65% of patients with TAO, 75% with ocular myopathy, a variant of TAO in which periorbital inflammation is minimal, 50% with euthyroid Graves’ disease defined as ophthalmopathy associated with subclinical thyroiditis and in 50% of patients with stable lid lag and retraction but no other signs of progressive ophthalmopathy, but in only 13% of patients with Graves’ hyperthyroidism without ophthalmopathy, 10% with Hashimoto’s thyroiditis and 14% of patients with other thyroid disorders. Tests were positive, with Müller’s Muscle cells, in 40% of patients with TAO, 25% with ocular myopathy, 40% with euthyroid Graves’ disease, 44% with lid lag, 19% with Graves’ hyperthyroidism, 50% with Hashimoto’s thyroiditis and in 37.5% of patients with other thyroid disorders. When skeletal Muscle cells were used as target, tests were positive in 13% of patients with TAO, 31% with lid lag, 25% with Graves’ hyperthyroidism and in 29% of patients with Hashimoto’s thyroiditis, but in no patient with euthyroid Graves’ disease or other thyroid disorders. Tests were negative in all patients and normals tested when EOM-derived fibroblasts were used as targets in ADCC. A significant positive correlation between % cytotoxicity against EOM cells and the severity of the Eye Muscle dysfunction expressed as an Eye Muscle index, was observed in patients with TAO. There was a significant negative correlation between the duration of Eye disease and % cytotoxicity against EOM cells, suggesting higher titers of cytotoxic antibodies in the early stages of TAO. There was no correlation between % cytotoxicity and serum level of anti-TSH receptor antibodies, measured in a radioreceptor assay. These findings suggest that autoimmunity against Müller’s Muscle may play a role in the pathogenesis of persistent lid lag and retraction. The nature of the EOM and Müller’s Muscle autoantigens recognized by cytotoxic antibodies in the serum of patients with TAO and related Eye disorders is unknown.
John S Kennerdell - One of the best experts on this subject based on the ideXlab platform.
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The 64-Kilodalton Eye Muscle Protein Is the Flavoprotein Subunit of Mitochondrial Succinate Dehydrogenase: The Corresponding Serum Antibodies Are Good Markers of an Immune-Mediated Damage to the Eye Muscle in Patients with Graves’ Hyperthyroidism
The Journal of clinical endocrinology and metabolism, 1998Co-Authors: S. Kubota, John S Kennerdell, Yuji Hiromatsu, Kazuaki Gunji, C. Stolarski, Sylvia Wengrowicz, B. A. C. Ackrell, B. Cochran, J. R. WallAbstract:Thyroid-associated ophthalmopathy (TAO) is a progressive Eye disorder associated with thyroid autoimmunity, particularly Graves' hyperthyroidism, which is generally considered to have an autoimmune etiology. Eye Muscle membrane proteins reportedly of 55 and 64 kDa are the best markers of the ophthalmopathy. The main focus of our recent studies has been to purify the pertinent proteins from porcine Eye Muscle membranes and characterize them. The 64-kDa protein is now shown from a partial sequence and by Western blotting using specific antibody probes to be the flavoprotein (Fp) subunit of succinate dehydrogenase and to have a correct molecular mass of 67 kDa. The protein was purified and cleaved with cyanogen bromide, and the N-terminal region of an immunoreactive partial peptide was determined. The 20-amino acid porcine sequence so obtained matched one within the Fp subunits of human and bovine succinate dehydrogenases in 20 and 18 of these positions, respectively. Succinate dehydrogenase is both a citric acid cycle enzyme and a component (complex II) of the mitochondrial respiratory chain. It is thus essential for aerobic energy production and is highly conserved. The mature human and bovine Fp subunits are 92% homologous and have a molecular mass of approximately 67 kDa, the same as our redetermined value for the 64-kDa marker protein. Sera from patients with TAO and from those with Graves' hyperthyroidism without evident ophthalmopathy highlighted the 64-kDa marker protein in crude porcine Eye Muscle membranes and the Fp subunit of highly purified bovine succinate dehydrogenase at the identical position on Western blots. Anti-beef Fp antibodies were detected in sera from 67% of patients with active TAO of more than 1-yr duration, in 30% with stable TAO of more than 3-yr duration, and in 30% of patients with Graves' hyperthyroidism without ophthalmopathy, but in only 7% of age- and sex-matched normal subjects. As succinate dehydrogenase is bound to the matrix (inside) surface of the mitochondrial inner membrane, it is unlikely to be accessible to circulating autoantibodies. We would postulate that Eye Muscle damage in ophthalmopathy is probably caused by cytotoxic antibodies or CD+ T lymphocytes targeting a cell membrane antigen, such as the thyroid and Eye Muscle shared protein G2s, and that presentation of succinate dehydrogenase is secondary. On the other hand, an autoantibody response to succinate dehydrogenase may be a good marker of immune-mediated damage to the Eye Muscle fiber and may support the idea that the extraocular Muscles are targets of the autoimmune reactions of TAO.
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Role of Eye Muscle antibody measurement in diagnosis of thyroid-associated ophthalmopathy: a laboratory update.
Endocrine practice : official journal of the American College of Endocrinology and the American Association of Clinical Endocrinologists, 1998Co-Authors: S. Kubota, Carol Stolarski, John S Kennerdell, Kazuaki Gunji, J. R. WallAbstract:ABSTRACT Objective: To review the current role of measurement of serum Eye Muscle antibodies in thyroid-associated ophthalmopathy (TAO). Methods: We conducted laboratory studies to determine the prevalences of serum autoantibodies reactive with Eye Muscle antigens in patients with active and inactive TAO, Graves’ hyperthyroidism, and Hashimoto’s thyroiditis as well as in normal subjects. Results: The two antigens most often recognized in immunoblotting with crude human or porcine Eye Muscle membranes by serum autoantibodies in patients with TAO are Eye Muscle membrane proteins of 55 and 64 kd. One 64-kd Eye Muscle protein has recently been cloned by screening a human Eye Muscle expression library with two different antibody probes and identified from a computer gene bank search as the calcium-binding protein calsequestrin. A fragment of a 220-kd Eye Muscle protein, called G2s, has also been cloned by screening the Eye Muscle library with affinity-purified antibodies reactive with a 55-kd Eye Muscle membrane protein. The prevalences of autoantibodies reactive with these two antigens in our study groups were as follows. Antibodies against calsequestrin were detected in 38% of patients with TAO for 3 years, in 17% of patients with Graves’ hyperthyroidism without ophthalmopathy, in 12% of patients with Hashimoto’s thyroiditis without ophthalmopathy, and in 21% of normal subjects. Antibodies reactive with the 64-kd protein were demonstrated in 62% of patients with recent-onset active TAO, in 33% with Eye disease for > 3 years, in 39% of patients with Graves’ hyperthyroidism without ophthalmopathy, in 25% of patients with Hashimoto’s thyroiditis, and in 16% of normal control subjects. Antibodies reactive with G2s fusion protein were detected in 67% of patients with recent-onset active TAO, in 46% of patients with Graves’ hyperthyroidism, and in 20% of normal subjects. Antibodies reactive with the parent protein, of which G2s is a fragment, may be markers of early Eye Muscle swelling and inflammation, whereas those reactive with the 64-kd protein and, less often, calsequestrin are associated with established Eye disease. Conclusion: Measurement of serum Eye Muscle antibodies is recommended as an aid to the early diagnosis of ophthalmopathy in predisposed patients and first-degree relatives of patients with TAO as well as to monitor active or progressive Eye disease. (Endocr Pract. 1998;4:127-132)
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Eye Muscle Antibodies in Patients with Ocular Myasthenia Gravis: Possible Mechanism for Eye Muscle Inflammation in Acetylcholine-Receptor Antibody-Negative Patients
Clinical immunology and immunopathology, 1998Co-Authors: K. Gunji, John S Kennerdell, C. Skolnick, T. Bednarczuk, S. Benes, Brian A. C. Ackrell, Bruce Cochran, J. R. WallAbstract:Myasthenia gravis is an organ-specific autoimmune disorder generally thought to be caused by an antibody-mediated attack against the skeletal Muscle nicotinic acetylcholine (Ach) receptor (AchR) at the neuromuscular junction. Extraocular Muscle weakness and double vision are present in about 90% of patients with myasthenia gravis and are the predominant complaints in about 20% of patients, when the condition is called ocular myasthenia gravis (OMG). While serum antibodies against the AchR are detected in most patients with generalized myasthenia gravis (GMG), they are not found in about one-third of patients with the ocular variety, and epidemiological, clinical, and serological studies suggest that OMG and GMG are two separate diseases. Both forms of myasthenia gravis are sometimes associated with thyroid autoimmunity or thyroid-associated ophthalmopathy (TAO). We have therefore tested the sera of patients with GMG and OMG by Western blotting for antibodies against porcine Eye Muscle membrane proteins in general, and by enzyme-linked immunosorbent assays (ELISA) specifically for reaction with two skeletal Muscle antigens which are prominent marker antigens for TAO, namely, the calcium-binding protein calsequestrin and the so-called "64-kDa protein." The 64-kDa protein has recently been identified as the flavoprotein subunit of mitochondrial succinate dehydrogenase. Patients with ophthalmopathy and myasthenia were excluded. Nine of the patients had associated Graves' hyperthyroidism without evident ophthalmopathy and one had Hashimoto's thyroiditis. Antibodies against porcine Eye Muscle membrane antigens of M(r) 15-110 kDa were detected in patients with GMG or OMG, one or more antibodies being detected in 100% of patients with GMG and in 88% of those with OMG. The most frequently found antibodies were those targeting Eye Muscle membrane proteins of 15, 67, and 110 kDa. Antibodies reactive with purified calsequestrin (63 kDa) were detected in 21% of patients with OMG but in no patient with GMG. Antibodies recognizing purified succinate dehydrogenase (67 kDa) were found in 42% of patients with OMG, in 100% (5 of 5) of patients with GMG, and in 48% of all patients with myasthenia gravis not associated with Graves' hyperthyroidism. There was no close correlation between any Eye Muscle-reactive antibody and antibodies against the AchR in either group of myasthenic patients. The findings support the notion that immunoreactivity against skeletal Muscle proteins other than the AchR may play a role in the development of the Muscle weakness in AchR antibody-negative patients with OMG and GMG, although it is unlikely that any of the antibodies demonstrated in this study are directly implicated. Similarly, while the demonstration of antibodies reactive with Eye Muscle antigens associated with TAO in patients with OMG raises the possibility that the link between the ocular lesions of myasthenia gravis and Graves' disease may be autoimmunity against a common antigen(s), it is more likely that both disorders are mediated by cytotoxic T cells recognizing another cell membrane antigen, such as the novel thyroid and Eye Muscle shared protein G2s, and that serum antibodies reactive with succinate dehydrogenase Fp subunit and calsequestrin are markers of an immune-mediated Eye Muscle reaction.
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Antibody-dependent cell-mediated cytotoxicity against orbital target cells in thyroid-associated ophthalmopathy and related disorders; close relationship between serum cytotoxic antibodies and parameters of Eye Muscle dysfunction
Journal of Endocrinological Investigation, 1996Co-Authors: A. Barsouk, John S Kennerdell, J. Kiljanski, C. Stolarski, V. Nebes, K. A. Peele, R. Volpe, J. R. WallAbstract:We have carried out tests for antibody-dependent cell-mediated cytotoxicity (ADCC) against extra ocular Muscle (EOM), Müller’s Muscle, orbital fibroblasts and skeletal Muscle in patients with thyroid-associated ophthalmopathy (TAO) and related Eye disorders. Cytotoxicity was measured as lactate dehydrogenase (LDH) release and results expressed as % cytotoxicity. Tests were positive, with EOM cells, in 65% of patients with TAO, 75% with ocular myopathy, a variant of TAO in which periorbital inflammation is minimal, 50% with euthyroid Graves’ disease defined as ophthalmopathy associated with subclinical thyroiditis and in 50% of patients with stable lid lag and retraction but no other signs of progressive ophthalmopathy, but in only 13% of patients with Graves’ hyperthyroidism without ophthalmopathy, 10% with Hashimoto’s thyroiditis and 14% of patients with other thyroid disorders. Tests were positive, with Müller’s Muscle cells, in 40% of patients with TAO, 25% with ocular myopathy, 40% with euthyroid Graves’ disease, 44% with lid lag, 19% with Graves’ hyperthyroidism, 50% with Hashimoto’s thyroiditis and in 37.5% of patients with other thyroid disorders. When skeletal Muscle cells were used as target, tests were positive in 13% of patients with TAO, 31% with lid lag, 25% with Graves’ hyperthyroidism and in 29% of patients with Hashimoto’s thyroiditis, but in no patient with euthyroid Graves’ disease or other thyroid disorders. Tests were negative in all patients and normals tested when EOM-derived fibroblasts were used as targets in ADCC. A significant positive correlation between % cytotoxicity against EOM cells and the severity of the Eye Muscle dysfunction expressed as an Eye Muscle index, was observed in patients with TAO. There was a significant negative correlation between the duration of Eye disease and % cytotoxicity against EOM cells, suggesting higher titers of cytotoxic antibodies in the early stages of TAO. There was no correlation between % cytotoxicity and serum level of anti-TSH receptor antibodies, measured in a radioreceptor assay. These findings suggest that autoimmunity against Müller’s Muscle may play a role in the pathogenesis of persistent lid lag and retraction. The nature of the EOM and Müller’s Muscle autoantigens recognized by cytotoxic antibodies in the serum of patients with TAO and related Eye disorders is unknown.
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Serum antibodies reactive with Eye Muscle antigens and the TSH receptor in a euthyroid subject who developed ophthalmopathy and Graves' hyperthyroidism.
Thyroid : official journal of the American Thyroid Association, 1996Co-Authors: Jack R. Wall, Carol Stolarski, J. Kiljanski, A. Barsouk, V. Nebes, I. Stachura, K. Peele, Robert Volpé, John S KennerdellAbstract:Serum antibodies reactive with Eye Muscle autoantigens, in particular a 64-kDa protein that is also expressed in the thyroid, and the TSH receptor, are associated with the ophthalmopathy that occurs in about 50% of patients with Graves' hyperthyroidism. We have had the opportunity to study a euthyroid, apparently normal, 35-year-old woman with a family history of thyroid autoimmunity and "colitis" but no clinical or biochemical evidence for thyroid disease or ophthalmopathy, who developed Graves' hyperthyroidism and ophthalmopathy together 18 months later. Serum taken when the patient was first seen was positive for antibodies reactive with (i) 9 different Eye Muscle proteins ranging in size from 15 to 130 kDa, notably those of 64, 55, and 50 kDa, by immunoblotting with Eye Muscle membranes, (ii) Eye Muscle and Muller's Muscle cell membrane antigens in antibody-dependent cell-mediated cytotoxicity (ADCC), (iii) an Eye Muscle cytoplasmic antigen in indirect immunofluorescence, and (iv) the TSH receptor as ...
Kazuaki Gunji - One of the best experts on this subject based on the ideXlab platform.
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cloning and characterization of the novel thyroid and Eye Muscle shared protein g2s autoantibodies against g2s are closely associated with ophthalmopathy in patients with graves hyperthyroidism
The Journal of Clinical Endocrinology and Metabolism, 2000Co-Authors: Kazuaki Gunji, S. Kubota, Anna Maria De Bellis, Masayo Yamada, Brian A. C. Ackrell, Sylvia Wengrowicz, Antonio Bellastella, Antonio Bizzarro, Antonio Agostino Sinisi, J. R. WallAbstract:Serum autoantibodies against Eye Muscle antigens are closely linked with thyroid-associated ophthalmopathy (TAO), although their significance is unclear. The two antigens that are most often recognized are Eye Muscle membrane proteins with molecular masses of 55 and 64 kDa, as determined from immunoblotting with crude human or porcine Eye Muscle membranes. We cloned a fragment of the 55-kDa protein by screening an Eye Muscle expression library with affinity-purified anti-55 kDa protein antibody prepared from a TAO patient's serum. A complementary DNA (cDNA) encoding a novel protein, which we have called G2s, was sequenced on both strands, and its size was 411 bp. The open reading frame of G2s corresponded to a 121-amino acid peptide with a size of 1.4 kb. Using the rapid amplification of 5'-cDNA ends technique we were able to clone an additional 0.3 kb of the protein. G2s did not share significant homologies with any other entered protein in computer databases and had one putative transmembrane domain. Using the 1.4 kb cDNA as probe in Northern blotting of a panel of messenger ribonucleic acids prepared from human tissues, the parent protein was shown to correspond to a large molecule of about 5.8 kb with a calculated molecular mass of approximately 220 kDa, consistent with earlier immunoblot studies performed in the absence of reducing agents. G2s was strongly expressed in Eye Muscle, thyroid, and other skeletal Muscle and to a lesser extent in pancreas, liver, lung, and heart Muscle, but not in kidney or orbital fibroblasts. We tested sera from patients with Graves' hyperthyroidism with and without ophthalmopathy and from control patients and subjects for antibodies against a G2s fusion protein by immunoblotting and enzyme-linked immunosorbent assay. In immunoblotting, antibodies reactive with G2s were identified in 70% of patients with TAO of less than 3 yr duration, 53% with TAO of more than 3 yr duration, 36% with Graves' hyperthyroidism without evident ophthalmopathy, 17% with Hashimoto's thyroiditis, 3% with type 1 diabetes, 23% with nonimmunological thyroid disorders, and 16% of normal subjects. The prevalences, compared to normal values, were significant for the two groups of patients with TAO, but not for the other groups. Tests were positive in 54% of patients with active TAO, 33% with chronic ophthalmopathy, 36% with Graves' hyperthyroidism, 54% with Hashimoto's thyroiditis, 23% with type 1 diabetes, and in 11% of normal subjects using enzyme-linked immunosorbent assay. The antibodies predicted the development of the ocular myopathy subtype of TAO in six of seven patients and the congestive ophthalmopathy subtype in seven of eight patients, respectively, with Graves' hyperthyroidism studied prospectively during and after antithyroid drug therapy. Antibodies reactive with G2s may be early markers of ophthalmopathy in patients with Graves' hyperthyroidism. Because G2s is expressed in both thyroid and Eye Muscle, immunoreactivity against a shared epitope in the two tissues may explain the well known link between thyroid autoimmunity and ophthalmopathy.
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Serum antibodies against the flavoprotein subunit of succinate dehydrogenase are sensitive markers of Eye Muscle autoimmunity in patients with Graves' hyperthyroidism.
The Journal of clinical endocrinology and metabolism, 1999Co-Authors: Kazuaki Gunji, Mario Salvi, Anna Maria De Bellis, Brian A. C. Ackrell, Bruce Cochran, Sumihisa Kubota, Jil Swanson, Sylvia Wengrowicz, Antonio Bellastella, A. BizzarroAbstract:Thyroid-associated ophthalmopathy is an autoimmune disorder of the extraocular Muscles and orbital connective tissue, which is usually associated with Graves' hyperthyroidism. Well-studied markers of ophthalmopathy are Eye Muscle membrane antigens, reportedly of approximately 64-kDa molecular mass. One, originally identified only as the 64-kDa protein, has recently been shown to be the flavoprotein (Fp) subunit of mitochondrial succinate dehydrogenase, which has a correct molecular mass of 67 kDa. We have used purified beef heart Fp as antigen in an enzyme-linked immunosorbent assay for cross-reactive human autoantibodies. Sera have been screened from patients with thyroid-associated ophthalmopathy classified according to activity and presence or not of Eye Muscle disease, and from those with Graves' hyperthyroidism without Eye involvement. Also examined were serum samples taken periodically from 20 patients with Graves' hyperthyroidism during 24 months of treatment of their hyperthyroidism with antithyroid drugs. Four of these patients had ophthalmopathy at the onset, 12 developed ophthalmopathy, and 4 did not develop any Eye signs during treatment. Anti-Fp subunit antibodies were detected in 73% of patients with active ophthalmopathy and evidence of Eye Muscle involvement but only in 25% if there was only congestive ophthalmopathy. These values were 0% and 11% for patients with chronic ophthalmopathy, with or without Eye Muscle dysfunction, respectively. The antibodies were also detected in 14% of patients with Graves' hyperthyroidism without evident ophthalmopathy, 11% of patients with nonimmunologic thyroid disorders, 12% of type I diabetics, and 12% of age- and sex-matched normal subjects. Significantly, appearance of anti-Fp antibodies predicted the development of ophthalmopathy in 5 of the 6 patients with Graves' hyperthyroidism, who developed Eye Muscle dysfunction after treatment of the hyperthyroidism, and coincided with the onset of Eye Muscle signs in the other patient. Antibodies were not detected in any of 6 patients who developed congestive ophthalmopathy without evidence of Eye Muscle damage or in 4 patients who did not develop any Eye signs. In conclusion, we have shown a close relationship between Eye Muscle disease and serum antibodies against the Fp subunit of succinate dehydrogenase in patients with Graves' hyperthyroidism.
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The 64-Kilodalton Eye Muscle Protein Is the Flavoprotein Subunit of Mitochondrial Succinate Dehydrogenase: The Corresponding Serum Antibodies Are Good Markers of an Immune-Mediated Damage to the Eye Muscle in Patients with Graves’ Hyperthyroidism
The Journal of clinical endocrinology and metabolism, 1998Co-Authors: S. Kubota, John S Kennerdell, Yuji Hiromatsu, Kazuaki Gunji, C. Stolarski, Sylvia Wengrowicz, B. A. C. Ackrell, B. Cochran, J. R. WallAbstract:Thyroid-associated ophthalmopathy (TAO) is a progressive Eye disorder associated with thyroid autoimmunity, particularly Graves' hyperthyroidism, which is generally considered to have an autoimmune etiology. Eye Muscle membrane proteins reportedly of 55 and 64 kDa are the best markers of the ophthalmopathy. The main focus of our recent studies has been to purify the pertinent proteins from porcine Eye Muscle membranes and characterize them. The 64-kDa protein is now shown from a partial sequence and by Western blotting using specific antibody probes to be the flavoprotein (Fp) subunit of succinate dehydrogenase and to have a correct molecular mass of 67 kDa. The protein was purified and cleaved with cyanogen bromide, and the N-terminal region of an immunoreactive partial peptide was determined. The 20-amino acid porcine sequence so obtained matched one within the Fp subunits of human and bovine succinate dehydrogenases in 20 and 18 of these positions, respectively. Succinate dehydrogenase is both a citric acid cycle enzyme and a component (complex II) of the mitochondrial respiratory chain. It is thus essential for aerobic energy production and is highly conserved. The mature human and bovine Fp subunits are 92% homologous and have a molecular mass of approximately 67 kDa, the same as our redetermined value for the 64-kDa marker protein. Sera from patients with TAO and from those with Graves' hyperthyroidism without evident ophthalmopathy highlighted the 64-kDa marker protein in crude porcine Eye Muscle membranes and the Fp subunit of highly purified bovine succinate dehydrogenase at the identical position on Western blots. Anti-beef Fp antibodies were detected in sera from 67% of patients with active TAO of more than 1-yr duration, in 30% with stable TAO of more than 3-yr duration, and in 30% of patients with Graves' hyperthyroidism without ophthalmopathy, but in only 7% of age- and sex-matched normal subjects. As succinate dehydrogenase is bound to the matrix (inside) surface of the mitochondrial inner membrane, it is unlikely to be accessible to circulating autoantibodies. We would postulate that Eye Muscle damage in ophthalmopathy is probably caused by cytotoxic antibodies or CD+ T lymphocytes targeting a cell membrane antigen, such as the thyroid and Eye Muscle shared protein G2s, and that presentation of succinate dehydrogenase is secondary. On the other hand, an autoantibody response to succinate dehydrogenase may be a good marker of immune-mediated damage to the Eye Muscle fiber and may support the idea that the extraocular Muscles are targets of the autoimmune reactions of TAO.
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Role of Eye Muscle antibody measurement in diagnosis of thyroid-associated ophthalmopathy: a laboratory update.
Endocrine practice : official journal of the American College of Endocrinology and the American Association of Clinical Endocrinologists, 1998Co-Authors: S. Kubota, Carol Stolarski, John S Kennerdell, Kazuaki Gunji, J. R. WallAbstract:ABSTRACT Objective: To review the current role of measurement of serum Eye Muscle antibodies in thyroid-associated ophthalmopathy (TAO). Methods: We conducted laboratory studies to determine the prevalences of serum autoantibodies reactive with Eye Muscle antigens in patients with active and inactive TAO, Graves’ hyperthyroidism, and Hashimoto’s thyroiditis as well as in normal subjects. Results: The two antigens most often recognized in immunoblotting with crude human or porcine Eye Muscle membranes by serum autoantibodies in patients with TAO are Eye Muscle membrane proteins of 55 and 64 kd. One 64-kd Eye Muscle protein has recently been cloned by screening a human Eye Muscle expression library with two different antibody probes and identified from a computer gene bank search as the calcium-binding protein calsequestrin. A fragment of a 220-kd Eye Muscle protein, called G2s, has also been cloned by screening the Eye Muscle library with affinity-purified antibodies reactive with a 55-kd Eye Muscle membrane protein. The prevalences of autoantibodies reactive with these two antigens in our study groups were as follows. Antibodies against calsequestrin were detected in 38% of patients with TAO for 3 years, in 17% of patients with Graves’ hyperthyroidism without ophthalmopathy, in 12% of patients with Hashimoto’s thyroiditis without ophthalmopathy, and in 21% of normal subjects. Antibodies reactive with the 64-kd protein were demonstrated in 62% of patients with recent-onset active TAO, in 33% with Eye disease for > 3 years, in 39% of patients with Graves’ hyperthyroidism without ophthalmopathy, in 25% of patients with Hashimoto’s thyroiditis, and in 16% of normal control subjects. Antibodies reactive with G2s fusion protein were detected in 67% of patients with recent-onset active TAO, in 46% of patients with Graves’ hyperthyroidism, and in 20% of normal subjects. Antibodies reactive with the parent protein, of which G2s is a fragment, may be markers of early Eye Muscle swelling and inflammation, whereas those reactive with the 64-kd protein and, less often, calsequestrin are associated with established Eye disease. Conclusion: Measurement of serum Eye Muscle antibodies is recommended as an aid to the early diagnosis of ophthalmopathy in predisposed patients and first-degree relatives of patients with TAO as well as to monitor active or progressive Eye disease. (Endocr Pract. 1998;4:127-132)
Yuji Hiromatsu - One of the best experts on this subject based on the ideXlab platform.
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cytokine profiles in Eye Muscle tissue and orbital fat tissue from patients with thyroid associated ophthalmopathy
The Journal of Clinical Endocrinology and Metabolism, 2000Co-Authors: Yuji Hiromatsu, Kyohei Nonaka, Dame Yang, Tomasz Bednarczuk, Ikuyo Miyake, Yoichi InoueAbstract:Eye Muscle (EM) and retroorbital fat tissue are two major sites of involvement in thyroid-associated ophthalmopathy (TAO). Lymphocytic infiltration in these tissues is a prominent histological feature of TAO. We have investigated the cytokine gene profiles in EM and orbital fat (OF) tissues from patients with TAO. Total RNA was isolated from EM tissue of 14 patients and from OF tissues of 29 patients with TAO. Cytokine gene expression was assessed by RT-PCR using paired primers for interferon γ (IFNγ), tumor necrosis factor α (TNFα), interleukin (IL)-1β, IL-2, IL-4, IL-6, IL-10, CD4, CD8, and glyceraldehyde-3-phosphate dehydrogenase. IFNγ, TNFα, IL-1β, and IL-6 messenger RNA (mRNA) were mainly detected in EM tissue, whereas IL-4 and IL-10 mRNA were detected in only one patient. On the other hand, in OF tissue, IL-4 and IL-10 mRNA were detected in 24% and 38% of the patients, respectively, and IFNγ, IL-1β, and IL-6 mRNA were less often detected compared with EM tissue. The enlargement of EM tissue as asses...
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The 64-Kilodalton Eye Muscle Protein Is the Flavoprotein Subunit of Mitochondrial Succinate Dehydrogenase: The Corresponding Serum Antibodies Are Good Markers of an Immune-Mediated Damage to the Eye Muscle in Patients with Graves’ Hyperthyroidism
The Journal of clinical endocrinology and metabolism, 1998Co-Authors: S. Kubota, John S Kennerdell, Yuji Hiromatsu, Kazuaki Gunji, C. Stolarski, Sylvia Wengrowicz, B. A. C. Ackrell, B. Cochran, J. R. WallAbstract:Thyroid-associated ophthalmopathy (TAO) is a progressive Eye disorder associated with thyroid autoimmunity, particularly Graves' hyperthyroidism, which is generally considered to have an autoimmune etiology. Eye Muscle membrane proteins reportedly of 55 and 64 kDa are the best markers of the ophthalmopathy. The main focus of our recent studies has been to purify the pertinent proteins from porcine Eye Muscle membranes and characterize them. The 64-kDa protein is now shown from a partial sequence and by Western blotting using specific antibody probes to be the flavoprotein (Fp) subunit of succinate dehydrogenase and to have a correct molecular mass of 67 kDa. The protein was purified and cleaved with cyanogen bromide, and the N-terminal region of an immunoreactive partial peptide was determined. The 20-amino acid porcine sequence so obtained matched one within the Fp subunits of human and bovine succinate dehydrogenases in 20 and 18 of these positions, respectively. Succinate dehydrogenase is both a citric acid cycle enzyme and a component (complex II) of the mitochondrial respiratory chain. It is thus essential for aerobic energy production and is highly conserved. The mature human and bovine Fp subunits are 92% homologous and have a molecular mass of approximately 67 kDa, the same as our redetermined value for the 64-kDa marker protein. Sera from patients with TAO and from those with Graves' hyperthyroidism without evident ophthalmopathy highlighted the 64-kDa marker protein in crude porcine Eye Muscle membranes and the Fp subunit of highly purified bovine succinate dehydrogenase at the identical position on Western blots. Anti-beef Fp antibodies were detected in sera from 67% of patients with active TAO of more than 1-yr duration, in 30% with stable TAO of more than 3-yr duration, and in 30% of patients with Graves' hyperthyroidism without ophthalmopathy, but in only 7% of age- and sex-matched normal subjects. As succinate dehydrogenase is bound to the matrix (inside) surface of the mitochondrial inner membrane, it is unlikely to be accessible to circulating autoantibodies. We would postulate that Eye Muscle damage in ophthalmopathy is probably caused by cytotoxic antibodies or CD+ T lymphocytes targeting a cell membrane antigen, such as the thyroid and Eye Muscle shared protein G2s, and that presentation of succinate dehydrogenase is secondary. On the other hand, an autoantibody response to succinate dehydrogenase may be a good marker of immune-mediated damage to the Eye Muscle fiber and may support the idea that the extraocular Muscles are targets of the autoimmune reactions of TAO.
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Possible Involvement of Fas-Mediated Apoptosis in Eye Muscle Tissue from Patients with Thyroid-Associated Ophthalmopathy
Thyroid : official journal of the American Thyroid Association, 1998Co-Authors: Mari Koga, Yuji Hiromatsu, Atsuo Jimi, Yoichi Inoue, Kyohei NonakaAbstract:In order to investigate the possible involvement of apoptosis in the pathogenesis of thyroid-associated ophthalmopathy (TAO), we studied the expression of Fas, Fas ligand (FasL), and Bcl-2 in extraocular Muscle tissues from patients with TAO by immunohistochemistry using monoclonal antibodies against Fas, FasL, and Bcl-2. Apoptosis was detected by in situ end-labeling of fragmented DNA. Apoptosis was detected in extraocular Muscle tissues from 17 of 19 patients with TAO and, to a smaller degree in 4 of 7 normal control subjects. The mean percentages of apoptotic nuclei were 12.1% in TAO Eye Muscle fibers and 16.4% in TAO interstitial cells, compared with 2.2% and 0.4% in normal Eye Muscle fibers and interstitial cells, respectively. The percentage of apoptotic nuclei in Eye Muscle fibers correlated with the enlargement of Eye Muscle tissue (r = 0.47, p < 0.05). Fas was demonstrated on the surfaces of extraocular Muscle fibers in 11 of 18 patients with TAO, but not in normal extraocular Muscle tissues. Nei...
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Human histocompatibility leukocyte antigen-DR and heat shock protein-70 expression in Eye Muscle tissue in thyroid-associated ophthalmopathy
The Journal of clinical endocrinology and metabolism, 1995Co-Authors: Yuji Hiromatsu, Kiyoko Tanaka, J. R. Wall, Tomoaki Hoshino, Yoichi Inoue, N Ishisaka, J Kamachi, Toshitaka Kuroki, Kyohei NonakaAbstract:To investigate the expression and localization of human leukocyte antigen (HLA)-DR and heat shock protein-70 (HSP-70) in orbital tissue from patients with thyroid-associated ophthalmopathy (TAO), we carried out an immunohistochemical study using anti-HLA-DR and anti-HSP-70 monoclonal antibodies and a streptavidin-biotinperoxidase detection system. Eye Muscle tissues were obtained at surgery from 38 patients with TAO and 8 control subjects. HLA-DR expression on Eye Muscle cells was demonstrated in orbital tissue from 2 of 3 untreated patients and 2 of 35 patients who had been treated with orbital irradiation or corticosteroids, in all of whom lymphocytic infiltration was also demonstrated. HLA-DR was not detected on Eye Muscle cells from 8 normal controls studied. HLA-DR was expressed on endothelial cells and interstitial cells from almost all patients with TAO and all 8 control subjects. HSP-70 was detected in Eye Muscle cells from 31 of the patients with TAO, including all 3 untreated patients, and 3 of the controls. Although the degree of HSP-70 expression did not correlate with the severity of the ophthalmopathy, significant expression of HSP-70 in Eye Muscle cells was more often demonstrated in patients with Eye disease of short duration (83%) than in those with disease of longer duration (33%). These results support the notion that Eye Muscle fiber is an important target of the orbital autoimmune reactions that characterize TAO.
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Significance of anti-Eye Muscle antibody in patients with thyroid-associated ophthalmopathy by quantitative western blot.
Autoimmunity, 1993Co-Authors: Yuji Hiromatsu, Masayuki Sato, Kiyoko Tanaka, Shingo Shoji, Kyohei Nonaka, Masanobu Chinami, Hiroshi FukazawaAbstract: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
