Correlation between extraocular muscle enlargement and thyroid autoantibodies in thyroid eye disease.

PURPOSE: This study aimed to investigate the factors affecting extraocular muscle enlargement in thyroid eye disease (TED). STUDY DESIGN: Retrospective study. METHODS: The thyroid-stimulating hormone (TSH) receptor antibody (TRAb), thyroid-stimulating antibody (TSAb), antithyroid peroxidase antibody (ATPO), and antithyroglobulin antibody (ATG) levels in patients diagnosed with TED who underwent orbital magnetic resonance imaging were assessed. The control group comprised the contralateral eye of patients who underwent orbital magnetic resonance imaging (MRI) for unilateral eyelid tumors or orbital disease. The thickness of the bilateral rectus muscles and superior oblique muscles was measured on orbital MRI. Muscle enlargement was classified as unilateral/bilateral and symmetric/asymmetric. The effects of age, sex, smoking history, TSH, thyroid hormone, and thyroid autoantibodies on the muscle thickness and number of enlarged muscles were assessed by use of simple and multiple regression analyses. RESULTS: The TED and control groups comprised 41 and 44 cases, respectively. The positivity rate of TSAb in patients with TED was 92.7% higher than that of the other autoantibodies. Muscle enlargement was observed in 29 of the 41 cases (70.7%). Older age and higher TSAb levels were identified as significant factors affecting the total muscle thickness and number of enlarged muscles. Bilateral muscle enlargement and asymmetrical muscle enlargement were observed in 17 (58.6%) and 23 (79.3%) of the 29 cases, respectively. The TSAb levels and age had no significant effect on the type of muscle enlargement. CONCLUSIONS: TSAb showed significant associations with extraocular muscle enlargement. Measurement of TSAb, rather than of TRAb, may be more useful for diagnosing extraocular muscle enlargement in patients with TED.

[Myasthenia--ocular type]. / Miastenia--forma oculara.

The authors present a case of ocular myastenia, suspected by an ophthalmologist and confirmed as diagnosis by a neurologist. There are discussion regarding the latest possibilities of treatment.

Diagnostic yields and clinical features of ocular myasthenia gravis.

ABSTRACT: To investigate clinical features and diagnosis process of ocular myasthenia gravis (OMG) in ophthalmology department.A total of 36 patients with ptosis or diplopia who had follow-up for at least 3 months between March 2016 and December 2019 were included in this study. Clinical symptoms of patients and the test results were analyzed. According to the positivity of serologic test, these patients were divided into 2 groups (confirmed OMG and possible OMG with relief of symptoms after antimyasthenic treatment) for comparison.Ptosis was present in 12 (33.33%) patients, diplopia was present in 14 (38.89%) patients, and both ptosis and diplopia were present in 10 (27.78%) patients. Acetylcholine receptor auto-antibody (AchR Ab) was positive in 14 (38.89%) of 36 patients and ice test was positive in 15 (71.43%) of 21 patients with ptosis. Unequivocal response to pyridostigmine was observed in 31 (86.11%) patients. For seropositive cases, AchR Ab titer was significantly higher in the group with 2 clinical symptoms than that in the 1 clinical symptom (P = .011).This study presents the usefulness and diagnostic validity of antimyasthenic treatment for OMG, especially seronegative OMG, with detailed symptom analysis.

Influence of 4-week or 12-week glucocorticoid treatment on metabolic changes in patients with active moderate-to-severe thyroid-associated ophthalmopathy.

Thyroid-associated ophthalmopathy (TAO) is a serious, progressive, vision-threatening and difficult-to-treat organ-specific autoimmune disease. The course, therapeutic effects and prognosis of moderate to severe TAO vary greatly. High-dose intravenous glucocorticoid (IVGC) therapy is considered a first-line treatment for active moderate-to-severe TAO, but there is still insufficient evidence regarding the treatment duration. Long-term IVGC therapy can influence the metabolism of glucose, lipids, and bone. This study was designed to compare changes in metabolic and immunological indexes as well as the magnetic resonance imaging apparent diffusion coefficient (ADC) of the extraocular muscles after 4 and 12 weeks of IVGC therapy. Forty-eight patients with active moderate-to-severe TAO were included in this retrospective cohort study. Metabolism and immunological indexes were measured before and after therapy. The ADC and clinical activity score (CAS) were used to evaluate the efficacy of treatment in these patients. We found that the patients in the 12-week group had increased fasting plasma glucose (p = 0.004), glycated hemoglobin (p = 0.028), total cholesterol (p < 0.001), and low-density lipoprotein (p < 0.001) after therapy. The patients in both groups had reduced bone metabolism markers after therapy. Thyroid peroxidase antibody and thyrotropin receptor antibody levels decreased after treatment in both groups (p < 0.001). A significant decrease in thyroglobulin antibody levels was found in the 4-week group (p = 0.006). The change in the ADC was higher in the 4-week group than in the 12-week group (p = 0.014). However, there were no significant differences in CAS values between the two groups. Therefore, 4-week IVGC therapy was recommended for patients with TAO with glucose and lipid disorders.

Thyroid autoantibodies in adults with acquired binocular diplopia of unknown origin.

Patients with acquired adult-onset strabismus mainly present with binocular diplopia. Although cranial nerve palsies are reportedly the most common cause of binocular diplopia in adults, thyroid disease can also cause diplopia. In patients with thyroid-associated ophthalmopathy, upper lid retraction and proptosis are the most common initial findings, but diplopia could be the first manifestation. So far, there has been little information on the diagnostic value of thyroid autoantibodies in patients with strabismus. Therefore, we examined adults with acquired binocular diplopia from 2008 to 2016 and evaluated the presence of thyroid autoantibodies and the relationship between thyroid autoantibody status and clinical characteristics in adults with acquired binocular diplopia. Thyroid autoantibody tests were performed for all patients, unless other causes of diplopia were identified. Fifty one (39%) of 132 patients were positive for thyroid autoantibodies. In the thyroid autoantibody-positive (TAb+) group, microsomal autoantibodies, thyroid-stimulating hormone receptor antibodies, thyroglobulin antibodies, and thyroid-stimulating antibodies were observed in 30, 27, 12, and 7 patients, respectively. The vertical deviation and grade of duction limitation were greater in the TAb+ group. The presence of ocular torsion was 15.5% and 39.5% in the TAb- and TAb+ groups, respectively. Thyroid autoantibody evaluation may be helpful in adults with idiopathic acquired binocular diplopia.

Can autoimmunity against calsequestrin explain the eye and eyelid muscle inflammation of thyroid eye disease?

Extra-ocular and upper eyelid (levator) muscle damage in thyroid orbitopathy may be due to autoimmunity against eye muscle auto antigens. The main antigen appears to be the calcium binding protein calsequestrin. In this study we have tested for T lymphocyte sensitization to calsequestrin in patients with Graves' disease, with and without orbitopathy, in standard proliferation assay. We have also tested total RNA prepared from thyroid tissue of patients with Graves' disease with and without orbitopathy for expression across 20,589 genes using micro array analysis technology. We were looking for differences in gene expression between the two groups which might provide information about the early thyroid events that lead to the development of eye muscle autoimmunity. Positive lymphocyte reactivity to calsequestrin was demonstrated in 59% of Graves' patients with orbitopathy, 33% without evident ophthalmopathy and in 43% of patients with Hashimoto's thyroiditis and upper eyelid retraction (UER). Two hundred and ninety six genes were identified to be differentially expressed between in patients with Graves' disease with and without orbitopathy. Of these, the cardiac calsequestrin gene CASQ2 was the most highly up regulated, 2.2-fold. The closely related skeletal muscle calsequestrin gene CASQ1 was also up-regulated, 4.1 fold, but this was not significant, while genes encoding the thyroid antigens thyroglobulin, thyroid peroxidase and the TSH-receptor were not differentially expressed. These findings provide further evidence for a prominent role of autoimmunity against calsequestrin in the pathogenesis of the eye muscle components of thyroid orbitopathy.

Extraocular muscle susceptibility to myasthenia gravis: unique immunological environment?

Extraocular muscle (EOM) is susceptible to neuromuscular junction disorders, in particular, myasthenia gravis (MG). While EOM physiological characteristics and the ocular motor system requirements contribute to the propensity of ocular motor deficits observed among patients with MG, the authors propose that EOM have immunological features that place the muscles at risk for immune attack. Genomic profiling studies have demonstrated that genes associated with the immune response are differentially expressed in EOM, with particular differences in both classical and alternative complement-mediated immune response pathways. Intrinsic complement regulators are expressed at lower levels at rodent EOM neuromuscular junctions, which would put them at risk for the complement-mediated injury that occurs in MG. In fact, systemic C inhibition in experimental autoimmune MG (EAMG) induced by administration of acetylcholine receptor (AChR) antibodies or immunization with AChR will eliminate complement deposition at junctions of other skeletal muscle, but not EOM. Also, EOM junctions have greater injury in active and passive EAMG by several measures, suggesting that the lack of complement inhibition puts the EOM at risk. Among ocular myasthenia patients, serum AChR antibody levels are low, which would support the concept that EOM junctions are more susceptible to antibody injury than are other junctions. These observations suggest that complement inhibitory therapies may prove to be particularly effective in treatment of ocular myasthenia.

Eye signs and serum eye muscle and collagen XIII antibodies in patients with transient and progressive thyroiditis.

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 ophthalmopathy and one with type 2 ophthalmopathy, and in three patients with ST, two with type 1 ophthalmopathy and one with dominant upper eyelid retraction only. Later in the course of their illness, one other patient with ST developed mild type 1 disease, giving an overall prevalence of any eye signs of 50% in patients with TT. Five patients with HT had mild type 1 ophthalmopathy and dominant upper eyelid retraction. One or more eye muscle antibodies were detected in three patients with SAT, four with ST, and seven with HT, of which calsequestrin and Fp antibodies were the most commonly found. TSH-r antibodies were detected in only one patient with ST, at the time when she developed Graves' hyperthyroidism following an episode of ST. CONCLUSION: The development of mild, but definite, ophthalmopathy or dominant upper eyelid retraction in patients with TT and chronic (Hashimoto's) thyroiditis in the absence of TSH-r antibodies or Graves' hyperthyroidism is an interesting observation that should be further addressed in larger groups of patients, including those with postpartum thyroiditis. These preliminary findings also raise questions about the mechanism for the link between ophthalmopathy and thyroid autoimmunity.

Are linear AChR epitopes the real culprit in ocular myasthenia gravis?

Extraocular muscle weakness occurs in most of the myasthenia gravis (MG) patients and it is often the initial complaint. Approximately 10-20% of MG patients with extraocular muscle weakness display only ocular symptoms and rest of the patients subsequently develop generalized muscle weakness. It is not entirely clear why some MG patients develop only ocular symptoms and why extraocular muscle weakness almost always precedes generalized muscle weakness. These facts are often explained by increased susceptibility of extraocular muscles due to their reduced endplate safety factor and lower complement inhibitor expression. Findings of a recently developed animal model of ocular MG suggest that additional factors might be in play. While immunization of HLA transgenic and wild-type (WT) mice with the native acetylcholine receptor (AChR) pentamer carrying conformational epitopes generates severe generalized muscle weakness, immunization of the same mouse strains with recombinant unfolded AChR subunits containing linear epitopes induces ptosis with or without mild generalized muscle weakness. Notably, immunization of mice with deficient T helper cell-mediated antigen presentation with recombinant AChR subunits or whole native AChR pentamer also induces ocular symptoms, AChR-reactive B cells and AChR antibodies. Based on these findings, we hypothesize that ocular symptoms observed in the earlier stages of MG might be triggered by linear and non-conformational AChR epitopes expressed by thymic cells or invading microorganisms. This initial AChR autoimmunity might be managed by T cell-independent and B cell mediated mechanisms yielding low affinity AChR antibodies. These antibodies are putatively capable of inducing muscle weakness only in extraocular muscles which have increased vulnerability due to their inherent biological properties. After this initial attack, as AChR bearing immune complexes form and the immune system gains access to the native AChR expressed by muscle and thymic myoid cells, a more robust anti-AChR autoimmunity develops giving way to high affinity AChR antibodies, thymic germinal center formation and severe generalized muscle weakness. Accurate characterization of chain if events leading to ocular and generalized symptoms in MG might enable development of novel therapeutics that might prevent the transition from mild ocular symptoms to severe generalized weakness in earlier stages of the disease.

99Tcm-octreotide scintigraphy and serum eye muscle antibodies in evaluation of active thyroid-associated ophthalmopathy.

PurposeAs an autoimmune inflammatory disorder, active thyroid-associated ophthalmopathy (TAO) is managed optimally by immunosuppression. In this study, we aimed to evaluate octreotide scintigraphy and the level of serum extraocular muscle antibodies in TAO activity.Patients and methodsThis prospective study comprised 304 patients with active TAO (the clinical activity score; CAS≥3), 73 with inactive TAO (CAS<3), 128 with Graves' disease (GD) without ophthalmopathy, and 100 healthy subjects. Moderate-to-severe active TAO patients (CAS≥5) received intravenous injection of methylprednisolone; mild active patients (3≤CAS≤4) received periocular injection of triamcinolone acetonide. 99Tcm-octreotide scintigraphy and serum levels of calsequestrin, uveal auto-antigen with coiled-coil domains and ankyrin repeats (UACA) and G2s antibodies were detected before and after treatment.Results99Tcm-octreotide scintigraphy was positive in active TAO patients (97%) with elevated uptake ratio (UR) (P<0.05), and showed a significant correlation with CAS (r=0.816, P<0.01). After treatment both UR and CAS decreased significantly (P<0.05). The receiving operator characteristic curve (ROC) showed that the best UR threshold for discriminating active and inactive TAO was 1.34 (sensitivity, 100%; specificity, 89.4%). The level of serum calsequestrin antibody was higher in active TAO (P<0.05), showed a significant correlation with CAS (r=0.738, P<0.05), and also decreased after treatment (P<0.05). The best serum calsequestrin antibody threshold of the ROC curve was 138 ng/l (sensitivity, 88.4%; specificity, 89.2%). The UACA antibody was elevated in both TAO and GD patients (P<0.05), with no significant difference (P>0.05). As to G2s, no significant difference was found between all groups (P>0.05). Moreover, six GD patients (4.69%) with elevated calsequestrin developed active TAO 12 weeks later.Conclusion99Tcm-octreotide scintigraphy played a critical role in the evaluation of the clinical activity and therapeutic efficacy of TAO. Autoimmunity against calsequestrin in the pathogenesis of the eye muscle components may provide further objective evidence of myopathy in active TAO. Furthermore, calsequestrin antibody may predict myopathy in active TAO.

[Current understanding of pathogenesis of Graves ophthalmopathy].

Graves ophthalmopathy most frequently develops in a patient suffering from Graves disease in which autoantibodies to a target antigen, thyrotropin receptor, activate the autoantigen leading to hyperthyroidism. It is well known that in Graves ophthalmopathy inflammatory cells infiltrate and hydrophobic glycosaminoglycan accumulates in the retro -occular tissues. However, in contrast to Graves disease, little has been known as to how this eye disease develops. Here we review recent advance in understanding of pathogenesis of Graves ophthalmopathy.

Ocular myasthenia gravis induced by human acetylcholine receptor ϵ subunit immunization in HLA DR3 transgenic mice.

Extraocular muscles (EOM) are preferentially involved in myasthenia gravis (MG) and acetylcholine receptor (AChR) antibody positive MG patients may occasionally present with isolated ocular symptoms. Although experimental autoimmune myasthenia gravis (EAMG) induced by whole AChR immunization closely mimics clinical and immunopathological aspects of MG, EOM are usually not affected. We have previously developed an EAMG model, which imitates EOM symptoms of MG by immunization of human leukocyte antigen (HLA) transgenic mice with α or γ-subunits of human AChR (H-AChR). To investigate the significance of the ϵ-subunit in ocular MG, we immunized HLA-DR3 and HLA-DQ8 transgenic mice with recombinant H-AChR ϵ-subunit expressed in Escherichia coli. HLA-DR3 transgenic mice showed significantly higher clinical ocular and generalized MG severity scores and lower grip strength values than HLA-DQ8 mice. H-AChR ϵ-subunit-immunized HLA-DR3 transgenic mice had higher serum anti-AChR antibody (IgG, IgG1, IgG2b, IgG2c and IgM) levels, neuromuscular junction IgG and complement deposit percentages than ϵ-subunit-immunized HLA-DQ8 transgenic mice. Control mice immunized with E. coli extract or complete Freund adjuvant (CFA) did not show clinical and immunopathological features of ocular and generalized EAMG. Lymph node cells of ϵ-subunit-immunized HLA-DR3 mice showed significantly higher proliferative responses than those of ϵ-subunit-immunized HLA-DQ8 mice, crude E. coli extract-immunized and CFA-immunized transgenic mice. Our results indicate that the human AChR ϵ-subunit is capable of inducing myasthenic muscle weakness. Diversity of the autoimmune responses displayed by mice expressing different HLA class II molecules suggests that the interplay between HLA class II alleles and AChR subunits might have a profound impact on the clinical course of MG.

The lack of specificity of ophthalmic immunoglobulins in Graves' disease.

Immunoglobulins (Igs) binding to retro-orbital muscle (ROM) antigens, known as ophthalmic Igs (OIg), were measured using a 100,000 X g sediment of porcine ROM as antigen in a solid phase [125I]protein A binding assay. Serum samples from 50 control subjects bound from 0.60-2.42 times the amount of [125I]protein A as did the normal reference serum samples, defined as the OIg ratio. Serum from 95 patients with hyperthyroid Graves' disease had OIg ratios from 0.64-9.99, with 24 (25%) being positive [OIg ratio greater than 2.05 (mean + 2 SD of the normal group)]. Ten patients with euthyroid Graves' ophthalmopathy had OIg ratios from 1.01-6.33, with 6 (60%) being positive. Among those Graves' disease patients with ophthalmopathy (n = 19) and the euthyroid Graves' ophthalmopathy patients there was a good correlation between the severity of eye signs and the OIg ratio. The OIg-positive serum samples cross-reacted with skeletal muscle and thyroid as well as with ROM antigen. This lack of specificity contradicts previous reports, but does not rule out a role for these antibodies in the etiology of Graves' ophthalmopathy.

Immunoglobulin A class fibroblast antibodies in patients with Graves' disease and pretibial myxedema.

The involvement of autoantibodies in the extrathyroidal manifestations of Graves' disease has been the subject of extensive investigation, with fairly inconclusive results to date. We investigated the presence of immunoglobulin A (IgA) and IgG antibodies in patients with Graves' disease and pretibial myxedema (PTM; n = 21) as well as those with Graves' disease with thyroid-associated ophthalmopathy (TAO; n = 10), Graves' disease with no clinical evidence of extrathyroidal manifestations (n = 11), Hashimoto's thyroiditis (n = 9), type 1 diabetes mellitus (n = 10), systemic lupus erythematosus (n = 9) and normal individuals (n = 17). We looked for antibodies to both retroocular muscle and dermal fibroblasts as well as to thyroid peroxidase, thyroid microsomal antigen, thyroglobulin, and human eye muscle membranes. IgA class antibodies to microsomal antigen (30-50% of patients), thyroid peroxidase (5-20%), and human eye muscle membrane (0-26%) antigens were found in the various groups of patients with Graves' disease. With each of these antigens, serum from patients with PTM showed the greatest binding. Highly significant IgA binding was shown by PTM serum to both dermal (P < 0.001) and retroocular muscle (P < 0.001) fibroblasts from 12 different donors. Serum from Graves' patients with and without TAO and that from Hashimoto's thyroiditis patients reacted significantly with 4 of the 12 fibroblasts lines. In contrast, IgG binding was only found for 3 of the 12 fibroblast lines using patient serum. The IgA binding to fibroblasts shown by PTM patients was predominantly of the IgA2 subclass. The activity was absorbed out by both fibroblasts and thyroid cells. In immunoblotting studies, PTM patient serum reacted with a 54-kilodalton dermal fibroblast antigen and a 66-kilodalton retroocular fibroblast antigen. No binding to these antigens was seen with serum from normal controls or patients without PTM. Further elucidation of the nature of this fibroblast antigen will help to determine the role of IgA autoantibodies in the extrathyroidal manifestations of Graves' disease.

Monoclonal antithyroglobulin antibodies derived from immunizations of mice with human eye muscle and thyroid membranes.

A mouse hybridoma clone secreting an immunoglobulin M monoclonal antibody (K-5-4), which reacted with human thyroglobulin (Tg), was obtained from spleen cells of mice immunized with crude membranes of human eye muscle tissues (Em). Its binding to Tg could be inhibited by another monoclonal anti-Tg (F1-11-1) derived from spleen cells of mice immunized with human thyroid cell membranes, but K-5-4 did not inhibit the binding of F1-11-1 to Tg. This finding suggests that K-5-4 may react with a site on the Tg molecule which is susceptible to conformational changes, such as that induced by binding of another anti-Tg antibody at another site on Tg. K-5-4 reacted with human, mouse, rat, guinea pig, bovine, and porcine Tg. Binding and immunohistological staining experiments failed to detect binding of K-5-4 to Em tissue. The very low frequency of one Tg-reacting hybridoma from 6 X 10(8) spleen cells fused after Em immunization contrasts with the relative ease with which monoclonal anti-Tgs were generated from spleen cells of mice immunized with crude human thyroid membranes. In the latter case, 1 anti-Tg hybridoma was generated for every 100,000 spleen cells fused, and an extensive library of monoclonal anti-Tgs was collected. Some of these antibodies were specific for human Tg only, while others cross-reacted with Tg of other animal species. None had the species reativity pattern of K-5-4. The anti-Tgs were used to affinity purify human Tg directly from supernatant of thyroid homogenate; the purified Tg was, in turn, used to affinity purify human polyclonal but monospecific anti-Tg directly from serum of patients in a simple and rapid procedure. We conclude that the monoclonal anti-Tgs are useful reagents in isolating and purifying Tg and anti-Tg.

Immunologically mediated cytotoxicity against human eye muscle cells in Graves' ophthalmopathy.

The possible roles of antibody-mediated complement-dependent cytotoxicity (AMC), antibody-dependent killer (K) cell-mediated cytotoxicity (ADCC), and spontaneous, natural killer (NK) cell-mediated cytotoxicity (NKC) against human eye muscle cells in the pathogenesis of Graves' ophthalmopathy were investigated, using as targets human eye muscle cells, by 51Cr release assays. AMC was not demonstrated in serum from any patient or normal subject. In ADCC assays, eye muscle cell lysis was significantly increased in serum from patients with Graves' ophthalmopathy compared to those with Graves' hyperthyroidism without eye disease and normal subjects. ADCC tests were positive (percent specific lysis greater than the upper limit of normal) in 5 of 13 patients with Graves' ophthalmopathy using serum diluted 1:48 and in 4 of 10 patients using serum diluted 1:6. There was no correlation between the extent of lysis of human eye muscle and that of human (abdominal) skeletal muscle and no difference between patients with Graves' ophthalmopathy and normal subjects in assays in which abdominal muscle cell targets were used. The degree of killing in ADCC tests was independent of the source of K cells, being similar in assays using effector cells from the patient, another patient, or a normal subject. ADCC activity was partially absorbed by thyroid, orbital connective tissue and eye muscle membranes, and eye muscle cells, but not by liver membranes of thyroglobulin. Four of 8 human monoclonal antibodies reactive with eye muscle membrane antigens were cytotoxic in ADCC assays. A noncytotoxic monoclonal antibody blocked the ADCC effect of serum from a patient with Graves' ophthalmopathy, while a cytotoxic monoclonal antibody enhanced killing. NKC against eye muscle cell targets was depressed in cells from hyperthyroid and euthyroid patients with Graves' ophthalmopathy compared to that in normal subjects. Demonstration of ADCC against human eye muscle cells in some patients with Graves' ophthalmopathy suggests that this may be a mechanism for the eye muscle cell damage characteristic of this disorder. Inability to demonstrate cytotoxicity in a greater proportion of patients may reflect the lack of specific criteria to identify patients with active eye muscle inflammation and the unsuitability of currently available tests for the detection of serum antibodies against eye muscle membrane antigens. The mechanism for depressed NK cell-mediated cytotoxicity against eye muscle cells in this disorder is not known.

A thyroid cytotoxic antibody that cross-reacts with an eye muscle cell surface antigen may be the cause of thyroid-associated ophthalmopathy.

A hitherto unrecognized thyroid antibody, which reacts with a thyroid cell surface antigen expressed on passaged thyroid cells, was identified in serum from patients with thyroid-associated ophthalmopathy using antibody-dependent cell-mediated cytotoxicity (ADCC) tests. The antibody was detected in 14 of 23 patients with Graves' hyperthyroidism (Gh) and associated ophthalmopathy, in 3 of 4 patients with Hashimoto's thyroiditis (HT) and ophthalmopathy, but in only 1 of 16 patients with Gh without clinically evident eye disease and 4 of 37 patients with HT without eye disease. The ADCC test also was positive in 2 of 30 patients with thyroid cancer, both of whom had had Gh and ophthalmopathy in the past. There was no correlation, in patients with ophthalmopathy, between the levels of the antibody (expressed as percent specific lysis) and the titers of antithyroid microsomal antibody measured using a hemagglutination assay. Based on the results of blocking experiments using mouse monoclonal antibodies against human thyroid peroxidase, now known to be the thyroid microsomal antigen, the corresponding antigen was not thyroid peroxidase. Moreover, the new antigen was expressed on cultured and passaged thyroid cells which do not express the microsomal antigen. In patients with ophthalmopathy there was a close correlation between the degree of lysis of passaged thyroid cells and that of eye muscle cells, and ADCC activity against passaged thyroid cells was absorbed by preincubation of positive serum samples with eye muscle and thyroid cell, but not other cell, monolayers. The reaction of a newly identified cytotoxic thyroid antibody with a shared epitope on eye muscle cells thus appears to be a possible mechanism for the development of ophthalmopathy in patients with Gh and, less often, HT.

Affinity purification of orbital membrane antigens for the study of the pathogenesis of Graves' ophthalmopathy.

We characterized 22 human monoclonal antibodies (MCAB) reactive with human orbital antigens. Most of these monoclonal antibodies had variable degrees of reactivity with eye muscle, orbital connective tissue, thyroid, and extra-thyroid tissue preparations when tested in an enzyme-linked immunosorbent assay (ELISA). Eight of the MCABs, selected on the basis of their reactivity with human eye muscle or orbital connective tissue antigens, were used to affinity purify orbital membrane antigens. All purified fractions were tested in the ELISA for reactivity with the MCAB used for their purification. Four of the purified antigens with a high reactivity were not proteins. To increase the specificity of the reactivity of serum autoantibodies with affinity-purified antigens, we developed a competitive binding ELISA in which the MCAB-immunoglobulin was directly labeled with alkaline phosphatase. We tested the serum of 24 patients with Graves' ophthalmopathy, 10 patients with a history of Graves' hyperthyroidism and no eye disease, 14 patients with Hashimoto's thyroiditis without eye disease, 8 patients with other nonautoimmune thyroid disorders, and 20 normal subjects for reactivity with an affinity-purified nonprotein orbital antigen. The levels of serum autoantibodies against this antigen in patients with Graves' ophthalmopathy were significantly higher than those in normal subjects or patients with Graves' hyperthyroidism with no eye disease. Although unlikely to be of primary pathogenetic significance, it is possible that levels of this antibody in patients with Graves' ophthalmopathy may reflect the extent of orbital inflammation and, therefore, be useful as a clinical marker.

Beneficial effect of pentoxifylline on thyroid associated ophthalmopathy (TAO)*: a pilot study.

We have previously found that pentoxifylline (Ptx) inhibited cytokine induced HLA-DR expression and glycosaminoglycan (GAG) synthesis by retroorbital fibroblasts. We have now tested the clinical efficacy of Ptx in treating TAO. Ten patients with moderately severe ophthalmopathy were selected for study. All patients were euthyroid before and during the 12 weeks of the Ptx therapy. Serum GAG, TNF-alpha, anti-TSH-receptor, anti-eye muscle, anti-thyroglobulin and anti-thyroid peroxidase antibodies were determined sequentially. At the end of 12 weeks eight of the ten patients showed improvement in soft tissue but not in proptosis or extraocular muscle involvement. At baseline the levels of GAG (5.2+/-0.92 mg/dl v.s. 0.7+/-0.14 mg/dl, p<0.001) and TNF-alpha (33.6+/-6.6 pg/ml v.s. 5.4+/-1.3 pg/ml, p<0.001) were increased in patients compared to controls. They gradually decreased in the eight patients who responded to Ptx: after 4, 8 and 12 weeks of therapy serum GAG was 3.4+/-0.42 mg/dl, 2.5+/-0.77 mg/dl (p<0.01) and 1.1+/-0.2 mg/dl (p<0.001), respectively and serum TNF-alpha was 20.9+/-4.8 pg/ml, 14.9+/-2.2 pg/ml (p<0.05) and 9.7+/-1.8 pg/ml (p<0.01), respectively. Serum GAG and TNF alpha did not fall in the two patients who did not respond. The titre of anti-eye muscle antibodies but not anti-thyroid antibodies were lower at 12 weeks. Ptx has a beneficial effect on inflammatory symptoms of TAO and associated laboratory parameters in the majority of patients.

Orbital tissue-derived T lymphocytes from patients with Graves' ophthalmopathy recognize autologous orbital antigens.

Lymphocytic and other mononuclear cell infiltration of the retro-bulbar space is observed in Graves' ophthalmopathy (GO). We investigated the antigenic character of orbital adipose/connective tissue and muscle from 21 euthyroid patients with severe GO after orbital surgery. Orbital tissue proteins were separated and recovered in soluble form by means of an electroelution technique. Twenty-two protein fractions, identified according to their molecular mass ranges, were used as antigens for orbital tissue-derived and peripheral blood T lymphocytes. Seventeen T cell lines from 6 patients were established from in vivo activated orbital T cells using interleukin-2 and anti-CD3 antibodies. T cell proliferation was measured as [3H] thymidine uptake. When screened for their reactivity to autologous adipose/connective tissue proteins, all T cell lines responded significantly to protein fractions 6-10 kDa [stimulation index (SI) = 32.9 +/- 9.8 (mean +/- SE)] and 19-26 kDa (17 +/- 5), but not to tuberculin, which was used as a control. Phenotypic analysis analysis of 10 orbital T cell lines indicated that 6 lines consisted predominantly of CD4+ cells. Incubation of a representative T cell line with allogeneic orbital protein fraction induced a very low response to protein fraction 19-26 kDa, but not to other fractions. Thyroid protein fraction 6-10 kDa also induced the proliferation of orbital T cell lines. Incubation of peripheral blood mononuclear cells with autologous orbital protein fractions gave similar results; positive responses to 6-10 and 19-26 kDa fractions were observed with orbital tissue from 12 of 14 patients (mean SI = 22 +/- 5.9 and 6.3 +/- 1.7, respectively), and positive responses were observed with orbital tissue from 3 of 4 patients to eye muscle fractions 6-10 and 19-26 kDa (13.8 +/- 6.9 and 6 +/- 2, respectively). When proteins from cultured orbital fibroblasts were used as antigens, autologous peripheral blood mononuclear cells from the 7 of the 9 patients tested responded to these 2 fractions (15.2 +/- 6.9 and 6.8 +/- 2.4, respectively), whereas a response to cultured orbital myoblasts was observed with the 19-26 kDa fraction only (SI = 8). Positive responses to abdominal adipose or muscle proteins, as controls, were not found. The demonstration of sensitized, orbital tissue-specific, T lymphocytes in the peripheral blood and orbit from patients with GO provides evidence for a role of cellular immunity in the pathogenesis of this eye disorder.