Connective Tissue Antigens Research Articles

Specific Immunological Investigation in the Management of Patients with Spine and Joint Pathology

Abstract Objective: The immunological investigation is used for the improvement of the quality of the treatment of various diseases. The aim of the study was to estimate the significance of specific immunological methods in the management of inflammatory diseases. Materials and Methods: Determination of the level of autoimmune lymphocytotoxic and granulocytotoxic antibodies (ALA and AGA) and reaction of inhibition of leukocyte migration were used for investigation of persons with orthopedic, metabolic, and lung pathology. Results: The study showed increased serum levels of ALA and AGA, decreased nonspecific LIF production and increased level of LIF in the presence of own serum, increased leukocyte migration to synovial membrane, bone, cartilage, Candida lusitaniae, Staphylococcus epidermidis, Staphylococcus aureus in persons with periprosthetic infection.. Decreased leukocyte migration to the antigens of S. aureus, Streptococcus pyogenes, Escherichia coli, and Pseudomonas aeruginosa and connective tissue antigens was determined in persons with inflammatory diseases of bones. Increased ALA, reduced LIF, and leukocyte migration enhancement to synovial membrane were revealed in impaired glucose metabolism. The persons with anemia were characterized by increased ALA, sensitization to bone and cartilage, Staphylococcus, and humoral sensitization to E. coli. Increased serum levels of ALA, AGA, decreased level of nonspecific LIF production and sensitization to connective tissue, Streptococcus and E. coli were determined in persons with impaired cholesterol level. Chronic obstructive pulmonary disease patients were characterized by sensitivity to S. aureus, P. aeruginosa, and humoral sensitization to the lung tissue. Conclusions: Inflammatory diseases are characterized by specific signs of immunological status and require definition of individual tactics of the treatment.

PMON21 Autoimmune Polyglandular Syndrome, Type 1: A Rare Condition Manifested by Multiple Endocrine and Non-Endocrine Disorders

Abstract Introduction Autoimmune Polyglandular Syndrome Type 1 (APS-1) is a rare autoimmune disorder with a prevalence of 1 in 2-3 million births. It is an autosomal recessive disorder caused by loss of function mutation of the Auto- Immune REgulator gene (AIRE gene) on chromosome 21. It leads to formation of autoantibodies to various hormonal, connective tissue, and protein antigens. It is characterized by the presence of two of the following three cardinal features: chronic muco-cutaneous candidiasis, hypoparathyroidism, and Addison's disease (primary adrenal insufficiency). Recently AIRE gene mutations have been seen in patients who have one of the three classic findings, but have other less commonly associated autoimmune disorders. The other auto-immune conditions associated with APS-1 are: type 1 diabetes mellitus, vitiligo, pernicious anemia, primary hypothyroidism, primary hypogonadism, pituitary failure, autoimmune hepatitis, and interstitial nephritis. The diagnosis is based on the history suggestive of endocrine and autoimmune disorders and the biochemical testing for endocrinopathies. The definitive diagnosis is made by genetic testing for AIRE gene mutation. Treatment is identical as for each isolated condition, i.e., hormone replacement, electrolyte correction and antifungals. Case Presentation A 19-year-old patient with a past medical history of congenital single kidney, oral thrush and vaginal candidiasis, hypothyroidism, intellectual disabilities, seizure disorder (onset two years earlier) and depression, was referred to the Endocrinology clinic for the evaluation of hypocalcemia. Her serum calcium was 7.9 mg/dl (normal: 8.5-10.0 mg/dl). She complained of fatigue, and constipation, but denied carpopedal spasms or perioral paresthesia. She has a family history of thyroid dysfunction and diabetes (mother). Initial workup for hypocalcemia revealed low serum calcium with low ionized calcium. Normal magnesium, creatinine, vitamin D-1,25 dihydroxy and 25-hydroxy vitamin D, and TSH (patient on levothyroxine for hypothyroidism). The parathyroid hormone level was low at 6.4 pg/ml (normal 8.0 to 65.0 pg/ml). She was diagnosed with hypoparathyroidism of unknown etiology. She was started on vitamin D and calcium supplementation. The biochemical testing for adrenal insufficiency was negative. The anti-adrenal and thyroid antibody panel was negative. She had a hormone-releasing birth control implant so ovarian function could not be assessed. Given the history of candidiasis and hypothyroidism with the new diagnosis of hypoparathyroidism, she was diagnosed with autoimmune polyglandular syndrome type 1. Conclusion Autoimmune Polyglandular Syndrome, type 1 is a rare autoimmune disorder and needs a high level of suspicion for diagnosis. The syndromic nature of this rare disorder should be recognized to facilitate early detection of other possible auto-immune conditions which can be associated with it. A history of more than one endocrine disorder and the presence of autoimmune conditions should prompt the physician to consider APS-1 as one of the possible diagnoses. Presentation: Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

Immunological aspects of carbohydrate and lipid metabolism in patients with periprosthetic infection and osteomyelitis

A study of carbohydrate and lipid metabolism and immunological examination of patients with periprosthetic infection after knee and hip joint endoprosthetics (n = 16) and osteomyelitis (n = 20) was conducted. The patients with periprosthetic infection were characterized by anemia, eosinophilia, impaired glucose tolerance, cholestasis, predisposition to elevated atherogenesis, increased circulating immune complexes, autoimmune granulocytotoxic antibodies, immunoglobulins IgM, IgG, IgA and sensitization to synovial membrane and Staphylococcus aureus. The patients with osteomyelitis did not develop increased glucose and cholesterine level, but showed increased aminotransferases, thymol test, chondroitinsulfates, decrease of calcium and production of the leukocyte migration inhibition factor (LIF), both non-specific and to the antigens of cartilage tissue and sensitization to Streptococcus and E. coli. In patients with infections of the musculoskeletal system, glucose metabolism disturbances have been associated with the presence of autoimmune lymphocytotoxic antibodies, reduced production of LiF, both non-specific and to the antigens of synovial membrane. Atherogenesis was associated with increased circulating immune complexes, autoimmune lymphocytotoxic and granulocytotoxic antibodies, decreased LIF and sensitization to connective tissue antigens and pathogenic microorganisms, especially to Streptococcus and Proteus. Anemia was associated with increased autoimmune lymphocytotoxic antibodies, delayed-type sensitization to bone and cartilage tissue, to Staphylococcus and sensitization by accelerated type to E. coli and Proteus. Physicians should analyse immunological data while treating and monitoring the patients with connective tissue infections and disturbances of glucose and cholesterine metabolism.

Pathogenesis of thyroid-associated ophthalmopathy: does autoimmunity against calsequestrin and collagen XIII play a role?

Thyroid-associated ophthalmopathy (TAO), or thyroid eye disease, is a complex inflammatory disorder of the eye that, as its name implies, is associated with thyroid disease. TAO can be divided into three subtypes: ocular myopathy, congestive myopathy and mixed congestive and myopathic ophthalmopathy. Although the precise pathophysiology of TAO remains unclear it is likely to reflect an autoimmune reaction involving sensitized T-cells and autoantibodies directed against a thyroid and orbital tissue shared antigen. One well studied candidate in this immune reaction is the thyroid-stimulating hormone receptor (TSH-r), expressed in the orbital fibroblast and pre adipocyte. In our studies of TAO, we have investigated the nature and significance of antibodies targeting other eye muscle and orbital connective tissue (OCT) antigens. Our findings suggest that autoimmunity against the eye muscle antigen calsequestrin and the OCT antigen collagen XIII plays a role in the pathogenesis of TAO. We propose that ocular myopathy and chronic eyelid retraction are due to autoimmunity against skeletal muscle calsequestrin in the extraocular and eyelid muscles, respectively. This may be initiated in the thyroid where calsequestrin expression is upregulated, possibly due to a stimulatory effect of TSH-r antibodies. We also propose that congestive ophthalmopathy results from a reaction against the TSH-r or collagen XIII in orbital fibroblast cell membranes. Further insight into the role of eye muscle and OCT antigens in the pathogenesis of TAO may allow for the development of new therapies to treat the eye disorder and reduce patient morbidity.

Study of Serum Antibodies Against Three Eye Muscle Antigens and the Connective Tissue Antigen Collagen XIII in Patients with Graves' Disease With and Without Ophthalmopathy: Correlation With Clinical Features

The extraocular muscles are one of the primary tissues implicated in the autoimmune-mediated inflammation of thyroid-associated ophthalmopathy (TAO). Our aim was to determine the prevalence and level of antibodies against three candidate eye muscle antigens and the orbital fibroblast membrane antigen collagen XIII, in well-characterized patient groups. The study cohort consisted of patients with Graves' hyperthyroidism with and without ophthalmopathy, controls patients with other thyroid or other autoimmune disorders and healthy subjects. The presence of eye muscle antibodies was determined using an optimized and standardized enzyme-linked immunosorbent assay. We measured antibodies against (i) the 67-kDa flavoprotein (Fp) subunit of the mitochondrial enzyme succinate dehydrogenase; (ii) G2s, a 141 amino acid fragment of the winged-helix transcription factor FOXP1; (iii) calsequestrin, a 63-kDa calcium-binding protein; and (iv) collagen XIII, a connective tissue protein that is closely linked to the congestive ophthalmopathy subtype of TAO. Eye muscle antibody levels were correlated with clinical diagnosis and presence or not of ophthalmopathy. Prevalences of positive antibody tests to calsequestrin (75.0%) and collagen XIII (43.8%) were significantly greater in Graves' disease (GD) patients with ophthalmopathy than in healthy subjects, whereas modest significance was demonstrated with antibodies against Fp, but not G2s. Significantly greater serum levels of antibodies against calsequestrin, G2s, and collagen XIII, but not Fp, were found in GD patients with ophthalmopathy compared to control patients without eye disease and healthy subjects. Calsequestrin and collagen XIII antibodies are the most specific to TAO, whereas antibodies against G2s, and to a lesser extent Fp, are also markers of ophthalmopathy, but less reliable. These results are unique in that it is the first time the significance of a panel of three candidate eye muscle antibodies and a connective tissue antibody have been evaluated in the same patients with ophthalmopathy.

Immune regulation in adjuvant-induced arthritis: possible implications for innovative therapeutic strategies in arthritis.

Adjuvant-induced arthritis (AIA), the first experimental arthritis model, was described in 1956 (1,2), at a time when experimental autoimmune diseases affecting the central nervous system, peripheral nervous system, thyroid, and eye had been described, and autoimmunity began to be considered a significant mechanism in the pathogenesis of organ-specific inflammatory disease (3,4). AIA, which appeared in rats immunized with Mycobacterium tuberculosis in oil, showed suggestive similarities in clinical onset and course, as well as the pattern of lesions in joints, skin, eye, and mucosae, to reactive arthritis associated with infection by enteric organisms (primarily gram-negative bacilli) (for review, see ref. 5). The intensified, precocious appearance of AIA lesions at joint or skin sites traumatized by various means suggested that the disease might represent a disseminated autoimmune response to a joint or connective tissue antigen (6). Indeed, AIA could be transmitted by transferring sensitized lymph node cells (7,8) or thoracic duct cells (9) from sensitized donors to normal recipients, without administering M tuberculosis to the latter group.

Flow cytometry analysis of the effects of pre-immersion on the biocompatibility of glass-reinforced hydroxyapatite plasma-sprayed coatings

Multilayered coatings composed of mixtures of HA and P 2O 5-based bioactive glasses are of potential clinical benefit in orthopaedic and dental surgery. Pre-immersion of these materials has been reported to further enhance their efficacy in vivo, although the precise biological effects of this treatment are not yet known. In this study we have therefore prepared double-layer plasma-sprayed coatings and evaluated the effects of pre-immersion on the growth and function of human osteosarcoma cells in vitro, using the MTT assay and flow cytometry analysis, respectively. The results showed that the increase in numbers of viable cells was the same or elevated following incubation on the pre-immersed HA and glass-reinforced HA coatings compared with the non-immersed materials. In addition, the expression of bone sialoprotein and fibronectin, two key connective tissue antigens, was up-regulated in cultures grown on the pre-immersed surfaces compared with the non-treated materials. Moreover, cell numbers and antigen expression both improved as the proportion of glass increased, particularly in the pre-immersed samples. Our findings thus suggest that the immersion treatment of these materials appeared to improve the response of these bone-like cells.

Thyroid-associated ophthalmopathy in black South Africans with Graves' disease: relationship to serum antibodies reactive against eye muscle and orbital connective tissue autoantigens.

The prevalence of hyperthyroidism owing to Graves' disease is increasing among urban black South Africans. Thyroid-associated ophthalmopathy is often observed in this context, but its pathogenesis remains unclear. No close relationship has been noted between antiflavoprotein (Fp) antibodies or thyrotropin receptor antibodies and ocular involvement in black patients. We measured serum antibodies against eye muscle and orbital connective tissue antigens in black patients with Graves' disease, correlating them with eye signs. Of 11 patients with clinical ophthalmopathy, 2 (18%) had antibodies against collagen type XIII, 3 (27%) against flavine adenine dinucleotide (FAD), 1 (9%) against Fp, and 4 (35%) against G2s. Antibody prevalences in eight patients without clinical ophthalmopathy were 12.5% for collagen XIII, 12.5% for FAD, 25% for Fp and 0% for G2s. These differences were not statistically significant. None of the individual mean antibody levels were significantly different between the two subgroups of thyrotoxic patients. Serum antibody levels were negative in 10 black South African controls. In summary, eye muscle and orbital connective tissue antibodies were found in small proportions of patients with Graves' disease with no close relationship of any antibody to eye signs. Thus, a substantial proportion of black South Africans with overt clinical ophthalmopathy remains in whom currently availabe serologic tests are unhelpful for screening and laboratory confirmation.

Antibodies against striated muscle, connective tissue and nuclear antigens in patients with thyroid-associated ophthalmopathy: should Graves' disease be considered a collagen disorder?

The identity and subcellular localization of the principal extraocular muscle (EOM) antigens and prevalences of the corresponding serum autoantibodies in thyroid-associated ophthalmopathy (TAO) need to be clarified. We have used porcine eye muscle tissue, which expresses all autoantigens identified in human tissue, as substrate in an indirect immunofluorescence assay. Several different patterns of antibody binding to EOM tissue antigens were observed with sera from patients with TAO namely, membrane, cytoplasmic, interstitial (endomysial) and nuclear. Overall, sera from 75% of patients with TAO contained one or more antibodies reactive with EOM, compared to 32% of patients with Graves' hyperthyroidism, 38% with Hashimoto's thyroiditis, and 16% of normals. All sera which reacted with EOM membrane or cytoplasmic antigens also reacted with the same antigen(s) in other skeletal muscle, but not in the other tissues tested. Sera from 31% of patients with TAO, but only 7% of those with Hashimoto's thyroiditis, and no patient with Graves' hyperthyroidism without evident ophthalmopathy, contained antinuclear antibodies (ANA). The most common nuclear fluorescence pattern was the finely speckled type typically associated with anti-Sm or anti-RNP antibodies. Significant positive correlations in patients with TAO were found between (i) EOM dysfunction and ANA (ii) eye disease of < 1 yr duration and EOM membrane-reactive antibodies and (iii) eye disease of < 1 yr duration and interstitial (endomysial) tissue-reactive antibodies. Although patients with Graves' disease do not usually exhibit other signs or immunologic features of a generalized collagen disorder, the finding of high prevalences of ANA and anti-striated muscle antibodies and, less often, anti-connective tissue antibodies in patients with ophthalmopathy, is consistent with it being a collagen-like disorder of the striated muscle, connective tissue and the thyroid. The reason why the inflammatory process is mainly limited to these tissues is unclear although cross reaction of ANA with tissue specific proteins or increased expression of muscle and connective tissue antigens in the orbit and skin, are possibilities.

Cellular immune reactivities in women with silicone breast implants: a preliminary investigation.

Surgical implantation of silicone breast prostheses has been conducted and considered safe for over 30 years. Some implant recipients, however, complain of a group of symptoms similar to those observed in connective tissue disorders, rheumatoid arthritis, systemic lupus erythematosus, or polymyositis. To date, immunologic sequelae have not been confirmed and remain controversial. To examine an autoimmune-like basis for the "silicone associated disease" reported by some women with silicone breast prostheses. Proliferative responses of peripheral blood mononuclear cells against a panel of control and connective tissue proteins and to compounds common to silicone prostheses were measured in 26 women who received silicone breast implants (with implants in place an average of 166.4 [standard deviation (SD) 58.3] months), and 23 age-matched and sex-matched healthy controls. The frequency and intensity of cellular immune responses against collagen I, collagen III, fibrinogen, and fibronectin were significantly increased in silicone breast implant recipients versus controls. In implant subjects, the highest frequency of immune reactivity was directed against collagen I (11/26, 42%) with collagen III being the most immunostimulatory self-antigen with a mean stimulation index (SI) of 8.2 [95% confidence interval (95% CI) 3.2]. In addition, 10/26 (39%) of the implant recipients responded to more than one of the connective tissue antigens versus 0/23 (0%, P = .0007) healthy controls. Immunologic reactivities to other antigens, including silicone-based compounds, were remarkably similar. The identification of self-reactivity towards these connective tissue antigens may provide important information for attempts at associating silicone breast implants with disease.

T Cell Receptor (VR) Bias in the Response of Rheumatoid Arthritis Synovial Fluid T Cells to Connective Tissue Antigens

T cell receptor (VB) use in the response to type II collagen and cartilage proteoglycans was analysed in peripheral blood and synovial fluid T cells from RA patients. T cells from RA patients with an immune response to connective tissue antigens, and paired PB and SF samples were stimulated in vitro with type II collagen, high density aggrecan proteoglycans (PG), and the Tcell mitogen concanavalin A. After short term culture, mRNA was extracted from cells and a reverse transcription-polymerase chain reaction was performed, using primers specific for eight TCR VB determinants. Blood cells stimulated with ConA generated strong bands with virtually all the V&bT primers tested, but the TCR (V&bT) expression by SF T cells stimulated with mitogen was biased, suggesting a selection process during joint infiltration. The VB phenotypes of cells responding to PG was restricted in individual RA patients, but the pattern of V&bT use in the the RA population was not consistent. In contrast, the V&bT phenotypes of SF cells responding to CII was highly biased in both individual patients and the RA population, with V&bT14, V&bT17, and V&bT8 phenotypes predominant. We conclude that the Tcell response to connective tissue antigens is restricted compared with mitogen stimulation, with the highest degree of TCR bias seen in the response of SF T cells to stimulation with type II collagen.

Graves' hyperthyroidism, ophthalmopathy and pretibial myxedema may be components of a multi-system autoimmune disorder

The authors have used the indirect immunofluorescence test to investigate reactivity of sera from patients with Graves' disease with porcine extraocular muscle and other tissue substrates. Sera from 75% of patients with thyroid-associated ophthalmopathy (TAO) contained one or more antibodies reactive with extraocular muscle, compared to 32% of those with Graves' hyperthyroidism without the eye disorder, 41% of patients with Hashimoto's thyroiditis, and 16% of normals. Antibodies reactive with a non-organ specific connective tissue antigen(s), seen as fluorescence of the interstitium and endomysium, were found in sera from 10% of patients with TAO and 16% of those with Graves' hyperthyroidism, but not from any patient with Hashimoto's thyroiditis or normal subject. Antinuclear antibodies were detected in sera from 31% of patients with TAO, but from only 8% with Hashimoto's thyroiditis, in no patient with Graves' hyperthyroidism and in only 3% of normal subjects. The finding of a high prevalence of antinucl...

Should Graves' disease be considered a collagen disorder of the thyroid, skeletal muscle and connective tissue?

Graves' disease comprises hyperthyroidism, ophthalmopathy, pretibial myxedema and acropachy, which occur separately or in various combinations. We have used the indirect immunofluorescence test to investigate reactivity of sera from patients with autoimmune thyroid disorders with and without ophthalmopathy, with porcine extra ocular muscle (EOM) and control tissue substrates. Sera from 75% of patients with Graves' hyperthyroidism (GH) and ophthalmopathy, which we call thyroid-associated opthalmopathy (TAO), contained one or more antibodies reactive with EOM compared to 32% of those with GH without the eye disorder, 41% of patients with Hashimoto's thyroiditis (HT), and 16% of normals. Antibodies reactive with an EOM connective tissue antigen(s), seen as fluorescence of the interstitium and endomysium, were found in sera from 10% of patients with TAO and 16% of those with GH, but not from any patient with HT or normal subject. Similar patterns of connective tissue reactivity were also found in lacrimal gland, skeletal muscle, kidney and salivary gland. Antinuclear antibodies were detected in sera from 31% of patients with TAO, but from only 8% with HT, in no patient with GH and in only 3% of normal subjects. The most common pattern was a fine speckled fluorescence, found in 45% of sera, consistent with reactivity against the Sm antigen or nuclear RNP. The finding of a high prevalence of ANA and, less often, anti-connective tissue antibodies in patients with thyroid autoimmunity and ophthalmopathy, is consistent with Graves' disease being a "collagen-like disorder". The reason why inflammation and resulting tissue damage is limited to the thyroid, connective tissue of the skin and orbit, skeletal muscle and, possibly, the lacrimal gland, is unclear. One possibility is cross reaction of ANA with tissue specific membrane proteins in these sites. The extent of immunologic abnormalities, and the resulting clinical features, in patients with Graves' disease may reflect the severity of a putative defect in immune regulation.

Oligoclonal T-receptor (V beta) use in the response to connective tissue antigens by synovial fluid T-cells from rheumatoid arthritis patients.

Oligoclonal T-receptor (V beta) use in the response to connective tissue antigens by synovial fluid T-cells from rheumatoid arthritis patients.

Heterophilic antibodies to connective tissue antigens in the blood sera of rheumatic patients do not cross-react with group AStreptococcus antigens

Heterophilic antibodies reacting with antigens of interstitial connective tissue of bovine myocardium were found in the sera of patients with rheumatic fever. These antibodies were referred to class IgG. Immunologic specificity of the reaction with these antigens was confirmed in experiments with F(ab′)2 fragments from IgG isolated from the sera of rheumatic patients. Heterophilic antibodies were not adsorbed by various antigens of group AStreptococcus, nor were they isolated in a column with immunosorbent prepared on the basis of nontype-specific streptococcal antigens. The reaction of patients' sera was not inhibited by monoclonal antibodies to nontype-specific antigens cross-reacting with antigens of myocardial interstitial connective tisSue.

Classification of Eye Changes of Graves' Disease

Classification of Eye Changes of Graves' Disease

The histochemistry of collagens and fibronectin in the gallbladders of the guinea pig and mouse.

In the gallbladders of the guinea pig and mouse, the localizations and histochemical properties of certain connective tissue antigens were studied by means of indirect immunoperoxidase methods in light microscopy. These antigens were types I, III and IV collagens and fibronectin. The results obtained show that the four antigens exhibited such histochemical natures as are characteristic to each of them. The histophysiological significances of these antigens are discussed with special reference to the known physiological functions of the gallbladder to regulate biliary pressure throughout the bile drainage system of mammals.

Immunity to ocular and collagen antigens in childhood arthritis and uveitis.

Humoral and cellular immunity to ocular antigens (S antigen and alpha, beta heavy, beta light, and gamma crystallins) and connective tissue antigens (type I, II, III, and IV collagens) were studied in children with juvenile rheumatoid arthritis (JRA) with or without uveitis and in controls. There was no association between the presence of uveitis and antibody to any collagen type or of lymphocyte transformation to type II collagen. Antibodies to all crystallins were more common in children with JRA than in controls, and antibodies to BH crystallins were more frequent in children with JRA and uveitis than in those with JRA alone. Such reactivity did not appear to represent antibody cross-reactive with collagen antigens. Lymphocyte proliferation to alpha crystallin was increased in JRA with or without uveitis, whereas lymphocyte response to S antigen was associated with the presence of uveitis.

Rheumatoid Arthritis and its Association with HLA-DR Antigens. II. Antibodies to Native Connective Tissue Antigens Detected by Enzyme Linked Immunosorbent Assay

The distribution of frequencies of HLA-DR alloantigens in HLA-DR4 negative subjects was determined in patients with Rheumatoid arthritis (RA) and normal individuals. An increased incidence of HLA-DR1 alloantigen in DR4 negative RA patients (45.9%) compared with DR4 negative healthy controls (23.6%) was found. The difference became significant when the incidence of DR1 was compared between patients with severe disease stages (III-IV) (75%) in contrast to 32% of incidence in patients of the milder stages (I-II) (p less than 0.05). Using Enzyme Linked Immunosorbent Assay we have determined the incidence of serum antibodies to native bovine type I and type II collagens and proteoglycans in patients with RA. Presence of serum antibodies to native type I collagen was detected in 59% of patients with RA, 60% of sera exhibited reactivity to type II collagen and 12% had antibodies to proteoglycans. There was no correlation between the presence of antibodies to type I and II collagens and disease stages, however, the incidence of serum antibodies to proteoglycans was increased in severe disease stages. On the other hand, the presence of high levels of antibodies to type I collagen was associated to HLA-DR1 antigen, (p less than 0.05).

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.