Biological activity of autoantibodies from patients with thyroid-associated ophthalmopathy: in vitro effects on porcine extraocular myoblasts.

To determine whether antibodies to orbital tissue have a pathogenic role in thyroid-associated ophthalmopathy, the in vitro effects of IgG from patients with thyroid-associated ophthalmopathy on porcine extraocular myoblast growth were studied. Growth of primary extraocular myoblast cultures and extraocular myoblast clones was stimulated by patients' IgG, as measured by [3H]thymidine uptake and bromodeoxyuridine incorporation. Myoblasts responded in a dose-related fashion to increasing concentrations of patients' IgG, and the response was reversed by anti-human IgG. The growth stimulating effect of patients' IgG was relatively specific to extraocular myoblasts, compared to skeletal myoblasts. Individual IgG samples from 36 patients with ophthalmopathy gave rise to higher growth responses of cloned extraocular myoblasts, than either 31 normal controls (p < 0.005), or IgG from 10 patients with Graves' disease without evidence of ophthalmopathy (p < 0.001). Myoblast growth-stimulating antibody did not correlate with thyroid autoantibody levels (thyrotrophin receptor antibody or thyroid microsomal antibody levels), but correlated significantly with binding to extraocular muscle membranes (r = 0.524, p < 0.001). Eye-muscle stimulating antibodies were demonstrable in sera of patients with thyroid-associated ophthalmopathy, and may be responsible for the enlargement of eye muscle in this disease.     

Biological Activity of Autoantibodies From Patients With Thyroid-associated Ophthalmopathy: In Vitro Effects on Porcine Extraocular Myoblasts

To determine whether antibodies to orbital tissue have a pathogenicrole in thyroid-associated ophthalmopathy, the in vitroeffectsof IgG from patients with thyroid-associated ophthalmopathyon porcine extraocular myoblast growth were studied. Growthof primary extraocular myoblast cultures and extraocular myoblastclones was stimulated by patients' IgG, as measured by [³H]thymidineuptake and bromodeoxyuridine incorporation. Myoblasts respondedin a dose-related fashion to increasing concentrations of patients'IgG, and the response was reversed by anti-human IgG. The growthstimulating effect of patients' IgG was relatively specificto extraocular myoblasts, compared to skeletal myoblasts. IndividualIgG samples from 36 patients with ophthalmopathy gave rise tohigher growth responses of cloned extraocular myoblasts, thaneither 31 normal controls (p<0.005), or IgG from 10 patientswith Graves' disease without evidence of ophthalmopathy (p<0.001).Myoblast growth-stimulating antibody did not correlate withthyroid autoantibody levels (thyrotrophin receptor antibodyor thyroid microsomal antibody levels), but correlated significantlywith binding to extraocular muscle membranes (r=0.524, p<0.001).Eye-muscle stimulating antibodies were demonstrable in seraof patients with thyroid-associated ophthalmopathy, and maybe responsible for the enlargement of eye muscle in this disease.     

Anti-muscle antibodies in Graves' ophthalmopathy

The nature of the humoral immune response in patients with Graves' ophthalmopathy has been investigated using a solid phase 125I Protein A binding assay. Retro-orbital muscle (R.O.M.), skeletal muscle (Sk.M.), R.O.M.-membranes, thyroid, kidney, liver, harderian gland, acetylcholine receptors (AchR), actin and myosin were used as target antigens. No significant difference in antibody binding profile to R.O.M. and Sk.M. was found indicating that the ophthalmic immunoglobulins (OIgs) were not recognising a R.O.M. specific antigen(s). Comparison between R.O.M. and R.O.M.-membranes, however, revealed that these antigens were detecting very different antibody populations. Using the former, it appeared that the predominant antibody population being measured was anti-myosin whereas the latter appeared to be detecting primarily anti-AchR antibodies. Anti-actin antibodies were also present in some of the sera. Thus a spectrum of anti-R.O.M. antibodies appears to be present in Graves' ophthalmopathy but the cross-reactivity of these with non-R.O.M. skeletal muscle and their similarity to those found in myasthenia gravis prevent them as yet being used to explain the specific immunopathology observed in this disease.     

免疫印迹检测抗眼肌抗体

用含ＴｒｉｔｏｎＸ－１００及脱氧胆酸钠的ＴＤＯＣ缓冲液自牛眼肌提取了眼肌细胞膜抗原，建立了抗眼肌多肽抗体的免疫印迹检测法。     

TSH binding site structures in human eye muscle fractions identified by using covalent-crosslinking.

125I-labelled human TSH was crosslinked to the human thyroid and extraocular eye muscle membrane and cytosol fractions (which were obtained by centrifugation). Studying crosslinking of 125I-labelled TSH to the thyroid fractions, TSH binding sites' structures were demonstrated on the eye muscle membranes and in the cytosol fractions. The binding of 125I-labelled TSH was inhibited by the addition of 120 mIU/mL of unlabelled TSH (and not with 12 mIU/mL) which confirmed the presence of TSH binding sites structures (MW about 66,000 Da) on the eye muscle membrane and in its cytosol. Adding purified IgG fractions from the sera from controls and Graves' disease (with high titer of antibodies against TSH receptor) to the thyroid and eye muscle membranes and cytosol fractions, the binding of 125I-labelled human TSH was inhibited by molecular weight of about 66,000 Da in the cytosol fractions. The affinity constant of the binding sites in the human eye muscle cytosol and the number of TSH receptors were found to be 146 x 10(9) M-1 and 9.8 x 10(10) molecules/mg/mL by Scatchard analysis, respectively.     

Thyroid-associated ophthalmopathy: clinical features, pathogenesis, and management

Thyroid-associated ophthalmopathy (TAO) is a progressive eye disorder characterized by immune-mediated inflammation of the extraocular muscles and orbital connective tissue. TAO is linked, in a unique way, with thyroid autoimmunity, in particular Graves' hyperthyroidism. Our working hypothesis for the pathogenesis of TAO is that recognition of a thyrotropin receptor (TSHR)-like protein in the orbital preadipocytes by antibodies may be the initial event leading to homing of lymphocytes into the orbital tissues. In the course of thyroid inflammation, antibodies and T cells reactive against G2s expressed in thyroid membranes cross-react with the protein in the eye muscle fiber, leading to eye muscle damage and dysfunction. Those patients with anti-G2s antibodies develop ocular myopathy. Antibodies against flavoprotein, the 64-kDa protein, which are produced in the context of eye muscle fiber damage and mitochondrial rupture, are sensitive markers of immune-mediated fiber necrosis in patients with ophthalmopathy but do not directly damage the eye muscle. Antibodies against type XIII collagen, which is localized in the plasma membranes of orbital fibroblast, may be a new marker for the congestive ophthalmopathy subtype of TAO. The measurement of antibodies against key eye muscle and orbital connective tissue autoantigens may have a role in the management of active ophthalmopathy and its prediction in patients with Graves' hyperthyroidism.     

Increased level of soluble interleukin-2 receptor in sera of patients with Graves' disease.

Soluble interleukin-2 receptor was studied in 20 patients with Graves' disease before and after methimazole treatment. Soluble interleukin-2 receptor level was significantly increased in newly diagnosed Graves' disease compared to controls (667 +/- 270 vs 205 +/- 45 U/ml) (P less than 0.001). In untreated patients' sera the soluble interleukin-2 receptor levels were higher in patients with active ophthalmopathy than in those without eye symptoms. Soluble interleukin-2 receptor levels were normalized in remission induced by methimazole treatment in the majority of patients except those with infiltrative ophthalmopathy. Furthermore, a correlation was found at the hyperthyroid stage of the disease between soluble interleukin-2 receptor level and titre of anti-TSH-receptor antibodies. However, the association with other parameters including anti-eye muscle, anti-thyroid peroxidase, anti-thyroglobulin antibodies was not significant.     

Sub-class of IgG in allergic disease. I. IgG sub-class antibodies in immediate and non-immediate food allergy

Previous studies have suggested that, apart from IgE-mediated reactions, some of the symptoms of food allergy may be caused by IgG antibodies to food proteins. This study was carried out to see if there were any distinctive features of the IgG sub-class antibody response to dietary antigens which occurs in food allergic patients. IgG sub-class antibodies were measured using a quantitative enzyme-linked immuno-sorbent assay (ELISA) to wheat gliadin, ovalbumin and bovine casein in twenty patients who had coeliac disease and in twenty-eight egg allergic patients. These were compared with twenty-one atopic dermatitis patients who did not have food allergy and twenty-six healthy control subjects. Coeliac disease patients tended to have raised IgG antibody levels (especially IgG1) to all three antigens but these overlapped considerably with that seen in egg allergic and atopic dermatitis patients. Coeliacs who avoided gluten had anti-gliadin antibody levels which did not differ from those seen in healthy subjects but nevertheless had raised anti-ovalbumin and casein-specific antibodies. The IgG antibody was largely restricted to IgG1 and IgG4 sub-class although the relative amount of each varied with the antigen. Although gliadin-specific antibodies were mainly IgG1, ovalbumin-specific antibodies were mainly IgG4. The increased antibody levels to all three antigens in coeliacs were caused by a raised IgG1 response, IgG4 antibodies were usually normal. Egg allergic patients also had raised IgG1 but not IgG4 antibodies to ovalbumin. These data show that the response to different dietary antigens can vary with the antigen. The fact that IgG1 and not IgG4 antibodies were raised to all three antigens in patients with coeliac disease suggests that they are a secondary consequence of the disease, perhaps reflecting increased transport of antigens across a damaged gut mucosa rather than a specific immunopathological reaction. However, the observation that antibodies to gliadin, and not ovalbumin or casein, fell following gluten avoidance shows that the response to gliadin, at least, is dependent upon continued exposure to antigen.     

Isotype distribution and serial levels of antibodies reactive with dietary protein antigens in dermatitis herpetiformis

Using a sensitive enzyme-linked immunoassay (ELISA), a significantly increased prevalence (p less than 0.001) of serum antibodies reactive with wheat gliadin, bovine milk or ovalbumin has been demonstrated in 75% (33/44) of adult patients with dermatitis herpetiformis (DH), compared with healthy adults. There was no significant difference in the prevalence of antibodies (79%) in patients on a gluten-free diet or not on a gluten-free diet (72%). These serum antibodies reactive with gliadin, milk and ovalbumin were of the IgG isotype. However, IgA anti-gliadin antibodies were also detected in DH patients, but only in patients who were not on a gluten-free diet. In contrast, IgA anti-milk antibodies were also detected in DH patients irrespective of whether the patient was on a gluten-free diet. In DH patients, antibodies reactive with ovalbumin were often restricted to the IgG4 subclass and antibodies reactive with bovine milk antigens (notably casein) were distributed predominantly in both IgG2 and IgG4 subclasses, a similar IgG isotype distribution to that observed in healthy individuals. However, anti-gliadin antibodies in DH patients showed no predominant IgG4 subclass restriction. IgG4 anti-ovalbumin antibodies and IgG4 and/or IgG2 anti-casein antibodies persisted for up to 4 yr without fluctuation, irrespective of whether DH patients were on a gluten-free diet.     

Immunologic factors in thyroid disease

The role of immunologic reactions against orbital-specific and orbital-thyroid antigens in the pathogenesis of autoimmune thyroid disorders and Graves' ophthalmopathy is discussed. Possible mechanisms for the association of ophthalmopathy and autoimmune thyroid disorders is discussed in relation to the role of autoantibodies against eye muscle antigens.     

Celiac disease‐associated antibodies in patients with psoriasis and correlation with HLA Cw6

Etiopathology of psoriasis is not completely understood. Patients with psoriasis show elevated sensitivity to gluten. The aim of this study was to see the expression of celiac disease (CD)‐associated antibodies gliadin IgA, gliadin IgG, and tissue transglutaminase IgA, and their correlation with HLA Cw6 in patients with psoriasis. The study comprised 56 patients with psoriasis and 60 healthy controls (HC). The levels of antibodies were detected by using ELISA technique and HLA Cw6 typing was carried out by microcytotoxicity method. HLA Cw6 was significantly expressed in psoriasis cases when compared with HC (P<0.05). CD‐associated antibodies gliadin IgA/IgG and tissue transglutaminase IgA were significantly higher in the serum of patient with psoriasis when compared with HC (P<0.05, <0.05, and 0.01, respectively). Serum anti tissue transglutaminase IgA (anti tTG IgA) was significantly higher in females when compared with males and expressed more in elderly patients. There was a significant positive correlation among the antibodies (anti gliadin IgA with anti gliadin IgG: r=0.67, P<0.05; anti gliadin IgA with anti tTG IgA: r=0.45, P<0.05, anti gliadin IgG with anti tTG IgA: r=0.26, P<0.05, respectively), whereas insignificant with HLA Cw6. Our study concludes that latent CD or CD‐associated antibodies were present in patients with psoriasis and also concludes that HLA Cw6 has no association with expression of these antibodies in patients with psoriasis. J. Clin. Lab. Anal. 24:269–272, 2010. © 2010 Wiley‐Liss, Inc.     

Detection of class-specific antibodies against Micropolyspora faeni antigens in farmers' lung

Micropolyspora faeni antigens were used for specific IgA, IgG and IgM determination with an ELISA technique, and for specific IgE antibodies by means of RAST in eighteen patients with farmer's lung, in nineteen farmers with other chest conditions, and in twenty-nine controls. The farmers' lung group had significantly higher IgG antibody levels than the controls, while specific IgA levels were elevated in ten cases. Specific IgE and--except for three cases--IgM levels did not differ from the controls. In the group of farmers with other lung diseases, only three had increased levels of specific IgG antibody. The correlation (0.89) between IgG by ELISA and a complement-fixation test indicated that C activation by M. faeni antigens is mediated by IgG antibodies.     

Natural antibodies in sera of patients with systemic lupus erythematosus

Sera obtained from fifty-five patients with active systemic lupus erythematosus (SLE) and from four patients with mixed connective tissue disease (MCTD) previously shown by immunofluorescence and by double immunodiffusion to possess antinuclear antibodies, were tested for the presence of natural antibodies of IgG, IgA, and IgM isotypes. Antibody activity to actin, myosin, DNA, TNP, albumin, and tubulin was examined, using an enzyme-linked immunosorbent assay (ELISA). It was found that, in comparison with the antibody titers in normal sera, most of the SLE and MCTD sera possessed statistically greater amounts of IgG, IgA, and IgM antibodies directed against all the antigens tested. Furthermore, the IgG, IgA, and IgM antibody activity to DNA and TNP, compared to that found with all the other antigens, was significantly higher. Antibodies reacting with a saline extract of calf thymus (ECT) were studied by ELISA and by immunodiffusion. No correlation was observed between the natural antibody titers and the serum antibody levels to ECT detected either by ELISA or by immunodiffusion.     

Production, isolation, and characterization of rabbit anti-idiotypic antibodies directed against human antithyrotrophin receptor antibodies.

Previous studies have shown that anti-idiotypic antibodies can be developed in vivo through animal immunization with idiotype, and that these antibodies can be isolated from other anti-immunoglobulin antibodies by affinity purification. These techniques have relied on large amounts of idiotype, which were produced either by hyperimmunization or by monoclonal antibodies, to serve as the affinity adsorbent. In the present study, we produced anti-idiotypic antibodies to human anti-thyroid-stimulating hormone (TSH) receptor antibodies by first injecting rabbits with (TSH receptor purified) IgG from Graves' patients. The resulting antiserum was then adsorbed with Sepharose-coupled TSH in an attempt to specifically bind and isolate the anti-idiotype. The antibody obtained from this process was shown to bind specifically to TSH receptor-binding antibodies from Graves' patients, and this binding could be inhibited by 56% with the addition of 10(-4) M TSH but not by HCG (10(-2) M). The anti-idiotype also bound to TSH, and this binding could be specifically inhibited by receptor-purified Graves' IgG (60% inhibition at 10 micrograms/ml IgG), but not by IgG from normal subjects (no inhibition at 50 micrograms/ml IgG). In a TSH receptor binding assay, the anti-idiotype could inhibit TSH receptor binding in Graves' sera at a 1,000-fold lower concentration than could anti-kappa/lambda antiserum; the anti-idiotypic antiserum also inhibited in vitro TSH-mediated adenylate cyclase stimulation at an IgG concentration of 5 micrograms/ml, while heterologous anti-TSH antisera and normal IgG at similar concentrations had no effect. Finally, despite being generated against a single patient's TSH receptor binding antibody, the anti-idiotype was able to block TSH receptor binding in the serum of six other Graves' patients, thus suggesting that there may be conformational conservation in the antigen that is recognized by different individuals' TSH receptor-binding immunoglobulins.     

Follow-up of anti-Aspergillus IgG and IgA antibodies in bone marrow transplanted patients with invasive aspergillosis

A total of 89 patients at risk for, or with invasive aspergillosis (IA) were recruited from bone marrow transplantation (BMT) units in two Lisbon hospitals, and followed for 2(1/2) years to monitor their immune response. Of these patients, six developed probable IA, from which five died. The presence of serum IgG or IgA antibodies against seven Aspergillus recombinant antigens was assessed in patients with IA, using an enzyme-linked immunosorbent assay (ELISA). In parallel, the serum levels of galactomannan (GM) were also monitored, using the Platelia Aspergillus kit (Sanofi Pasteur, Marnes-la-Coquette, France). Superoxide dismutase (Sod) and 94 kDa were the most immunogenic antigens for IgA, while the IgG pattern of recognition changed from patient to patient. From our results we conclude that although follow-up of antibodies against these antigens should not be used as a diagnostic method, patients with IA do produce an immune response that may influence disease outcome.     

Clinical studies of the reactivity of serum IgG from patients with Graves' ophthalmopathy with porcine orbital tissue preparations

The clinical utility of ELISA assays for antibodies reactive with a variety of porcine orbital tissue antigen preparations is described using sera from large numbers of patients. Use of such assays does not allow reliable identification of patients with Graves' ophthalmopathy (GO) due to the overlap between patients with and without eye involvement and normal individuals. In 5/6 patients showing high levels of reactivity with porcine eye muscle membrane antigen, a considerable but variable proportion of the binding was found to be species, rather than tissue, specific. No consistent pattern of change in ELISA reactivity with time was seen in studies of serum samples from patients treated medically for hyperthyroidism and patients who received immunosuppressive therapy with cyclosporin A for active, severe ophthalmopathy. Use of previously published ELISA methods using the crude antigen preparations described is therefore not likely to be of use in the routine clinical management of GO patients.     

Clinical value of specific detection of immune complex-bound antibodies in pulmonary tuberculosis

Two actively secreted (38 and 30 kDa) and 1 cytosolic (16 kDa) antigens were purified from Mycobacterium tuberculosis culture filtrate and cytosol, respectively, using a combination of chromatographic and electrophoretic methods. One recombinant antigen (27 kDa) overexpressed in Escherichia coli was also isolated. The diagnostic test characteristics of circulating immune complex (CIC)-bound antibodies to purified protein antigens, singly and in combination, were evaluated in patients with pulmonary tuberculosis. The individual antigens ranged in their sensitivity from 73% to 88%, while considering the IgG response. Addition of IgA results improved the sensitivity. The combination of IgG results for 38, 30, and 16 kDa offered >95% sensitivity and specificity for the smear- and culture-positive tuberculosis, as well as for the smear-negative, culture-positive group. CIC-bound antibodies promise to be a better diagnostic tool than serum antibodies.     

Serological response to plasmid-encoded antigens in children and adults with shigellosis

The immunological response to plasmid-encoded antigens of virulent Shigella was determined in Thai children less than 4 yr of age and in Thai adults by immunoblot analysis and ELISA. Forty-two percent (8/19) of Thai children and 4% (1/22) of Thai adults with shigellosis developed a greater than or equal to 4-fold rise in IgG antibody titer to water-extracted antigens of Shigella flexneri M90T by ELISA (p = 0.006). Two children and one lactating mother with shigellosis developed a 4-fold rise in serum IgA antibody titers to water-extracted antigens of M90T. The results of the ELISA were confirmed by immunoblot analysis in all of the 41 paired sera examined. Five patients developed IgA, and four developed IgM, antibodies as detected by immunoblot analysis, that were not detected by ELISA. The reciprocal log2 geometric mean titers of antibodies to plasmid-encoded antigens in acute sera was higher in Thai adults than Thai children: IgG 7,265 versus 1,659; IgM 879 versus 480; and IgA 662 versus 60 (p less than 0.001). Thai adults had high titers of antibodies to plasmid-encoded antigens in their acute sera, but were susceptible to Shigella infections, although they were historically less susceptible than Thai children.     

B-cell depletion with rituximab in the treatment of autoimmune diseases. Graves' ophthalmopathy the latest addition to an expanding family

In this review, the authors summarise the clinical results obtained after therapy with rituximab in autoimmune diseases, including Graves' disease and Graves' ophthalmopathy. On the basis of qualitative and quantitative analyses of B- and T-cell subsets, and autoantibody levels obtained in other diseases before and after rituximab therapy, the authors interpret the results of the only two clinical investigations of the efficacy of rituximab in the treatment of Graves' disease and Graves' opthalmopathy reported so far. No significant effect on autoantibody levels was observed. Nonetheless, 4 out of 10 Graves' disease patients remained in remission 400 days after rituximab treatment versus none in the control group, and remarkable improvements in the eye symptoms of patients with Graves' ophthalmopathy were observed. This supports a role for B cells in the pathogenesis of Graves' ophthalmopathy, and the authors suggest that abrogation of antigen presentation by B cells accounts for the effect of rituximab. In the authors' opinion, the use of rituximab in severe Graves' ophthalmopathy could be contemplated.     

Antibodies to cytoskeletal proteins in patients with Crohn''s disease

The immunologic basis of inflammatory bowel disease has been the focus of interest of a series of studies on Crohn's disease and the process of immune sensitization at the gastrointestinal mucosal level is functionally poorly understood. To date only few contradictory reports concerning the incidence of autoantibodies in patients with this disease exist. The aim of this study was to investigate the sera drawn from 60 patients suffering from biopsy-proven Crohn's disease to evaluate the prevalence of autoantibodies against nuclear antigens and cytoskeletal proteins. Using standard methods, no anti-nuclear antibodies or antibodies to extractable nuclear antigens could be detected. All sera were also negative for antibodies to double-stranded DNA, anti-mitochondrial antibodies, and antibodies to gastric parietal cells. Using sensitive enzyme-linke immunosorbent assays with purified antigens and Western blotting with cytoskeletal proteins of human intestinal cells, the following antibodies could be demonstrated: cytokeratin 18 autoantibodies (IgG 20.0%; IgM 6.7%; IgA 13.3%), actin antibodies (IgG 36.7%; IgM 48.3%, IgA 26.7%), desmin antibodies (IgG 6.7%; IgM 15.08%; IgA 5.0%), vimentin antibodies (IgG 3.3%; IgM 16.7%; IgA 10.0%) and tropomyosin antibodies (IgG 3.3%; IgM 3.3%, IgA 5.0%). Statistically significant correlations could be found for levels of cytokeratin 18 antibodies (IgM-type) and the BEST index of activity, and for levels of desmin antibodies (IgM-type) and the van HEES index of activity. Highest levels could be measured for actin antibodies (IgG-type) in patients with isolated disease manifestation in the colon. The mechanism of induction of autoantibodies against cytoskeletal components in Crohn's disease still remains obscure. Unmasking of hidden antigens after cell injury during the inflammatory process of disease might lead to sensitization and antibody production. The pattern of antibodies in patients with Crohn's disease seems to be different compared with that of connective tissue diseases.