Peripheral blood T lymphocyte sensitisation against calsequestrin and flavoprotein in patients with Graves' ophthalmopathy

Thyroid-associated ophthalmopathy (TAO) is an orbital autoimmune disorder of the extraocular and eyelid muscles and surrounding connective and adipose tissue. Although mononuclear cell infiltration of orbital tissue is a characteristic feature of TAO the likely role of T lymphocyte reactivity against eye muscle antigens in the initiation of eye muscle damage in TAO has not been explored in detail. Therefore, we tested for T lymphocyte sensitisation to three eye muscle antigens namely, calsequestrin, G2s and flavoprotein (Fp), in patients with Graves' ophthalmopathy (GO), Graves' hyperthyroidism (GH) without ophthalmopathy and age and sex matched normal subjects. T lymphocyte reactivity was determined in a proliferation assay, results being expressed as stimulation index (SI). Mean ( +/- SE) SI for patients with GO, but not GH without ophthalmopathy, were significantly greater than that for normal subjects for calsequestrin and Fp, but not G2s. Mean ( +/- SE) SI was also significantly increased in patients with active ophthalmopathy, but not chronic ophthalmopathy, compared to normal subjects, for calsequestrin and Fp, but not G2s. Overall, positive lymphocyte proliferation to calsequestrin was demonstrated in 59% of patients with GO and 33% of patients with GH, which was significantly greater than in normals for both groups. In conclusion, we have demonstrated significant T lymphocyte reactivity to calsequestrin and, to a lesser extent, Fp in patients with GO. Because calsequestrin is located in the cell membranes of the eye muscle cell during the myotubular stage of the cell cycle, its targeting might be the primary reaction which leads to extraocular muscle inflammation in patients with GH.     

Peripheral Blood Lymphocyte Transformation to G2s in Patients with Autoimmune Thyroid Disease with and without Ophthalmopathy

Thyroid associated ophthalmopathy (TAO) is an autoimmune disease involving the extra ocular muscles and surrounding orbital connective and adipose tissues. The mechanism for the link between ophthalmopathy and thyroid autoimmunity is unknown but current evidence favors an immune reaction against a thyroid and orbital tissue shared antigen such as the novel protein G2s, which is highly expressed in both eye muscles and thyroid, or the TSH receptor (TSHR). Earlier, we showed that serum antibodies against G2s were closely linked to ophthalmopathy. Although lymphocytic infiltration of the eye muscles is a pathologic feature of TAO, it is unclear whether the reaction is in the eye muscle fiber or the surrounding connective tissue. We tested for peripheral blood mononuclear cell sensitization to G2s fusion protein in patients with TAO, Graves' hyperthyroidism or Hashimoto's thyroiditis without evident ophthalmopathy and normal subjects. Results were expressed as counts per min (cpm) and as stimulation indices (SI). Although proliferation tests were positive in 23% of patients with TAO, overall, there were no significant differences between the four groups. Tests were also positive in four out of seven patients with Hashimoto's thyroiditis, suggesting that immune reactivity against G2s could be a marker of this progressive thyroid disorder. There was no significant correlation between T cell reactivity to G2s and serum antibodies against the same protein, measured in enzyme linked immunosorbent assay. Failure to demonstrate significant T lymphocyte sensitization to G2s in the majority of patients with TAO may reflect the small number of sensitized T cells expected to be circulating in the peripheral blood which could be overcome by testing cloned orbital T cells, as available. Another possibility is that the T cell epitope(s) is not present on the 141 amino acid fragment of G2s that we have so far cloned. The finding of positive T cell tests in a small proportion of patients with ophthalmopathy suggests that future studies using cloned orbital T cells and full length G2s, or its dominant epitope, are indicated.     

Peripheral blood lymphocyte transformation to G2s in patients with autoimmune thyroid disease with and without ophthalmopathy

Thyroid associated ophthalmopathy (TAO) is an autoimmune disease involving the extra ocular muscles and surrounding orbital connective and adipose tissues. The mechanism for the link between ophthalmopathy and thyroid autoimmunity is unknown but current evidence favors an immune reaction against a thyroid and orbital tissue shared antigen such as the novel protein G2s, which is highly expressed in both eye muscles and thyroid, or the TSH receptor (TSHR). Earlier, we showed that serum antibodies against G2s were closely linked to ophthalmopathy. Although lymphocytic infiltration of the eye muscles is a pathologic feature of TAO, it is unclear whether the reaction is in the eye muscle fiber or the surrounding connective tissue. We tested for peripheral blood mononuclear cell sensitization to G2s fusion protein in patients with TAO, Graves' hyperthyroidism or Hashimoto's thyroiditis without evident ophthalmopathy and normal subjects. Results were expressed as counts per min (cpm) and as stimulation indices (SI). Although proliferation tests were positive in 23% of patients with TAO, overall, there were no significant differences between the four groups. Tests were also positive in four out of seven patients with Hashimoto's thyroiditis, suggesting that immune reactivity against G2s could be a marker of this progressive thyroid disorder. There was no significant correlation between T cell reactivity to G2s and serum antibodies against the same protein, measured in enzyme linked immunosorbent assay. Failure to demonstrate significant T lymphocyte sensitization to G2s in the majority of patients with TAO may reflect the small number of sensitized T cells expected to be circulating in the peripheral blood which could be overcome by testing cloned orbital T cells, as available. Another possibility is that the T cell epitope(s) is not present on the 141 amino acid fragment of G2s that we have so far cloned. The finding of positive T cell tests in a small proportion of patients with ophthalmopathy suggests that future studies using cloned orbital T cells and full length G2s, or its dominant epitope, are indicated.     

A 63 kDa skeletal muscle protein associated with eye muscle inflammation in Graves' disease is identified as the calcium binding protein calsequestrin

It is generally accepted that thyroid-associated ophthalmopathy (TAO) is an autoimmune disease of the eye muscle (EM) and the surrounding orbital connective tissue in which circulating antibodies play an important role. Antibodies against EM membrane proteins of 63-67kDa mol. wt. seem to be the best markers of ophthalmopathy in patients with autoimmune thyroid disease. We purified a 63 kDa EM protein using SDS-polyacrylamide gel electrophoresis technology and TAO patients' sera as probes, digested the protein with cyanogen bromide and sequenced immunoreactive peptides. We also screened a human EM library with a rabbit antiserum against 63-65 kDa proteins and affinity purified antibodies from a TAO patient's serum that reacted with a 55 kDa EM membrane protein. From partial sequence information and from DNA sequencing of positive cDNA clones, the protein was identified as calsequestrin, a 63 kDa calcium binding protein localized in the sarcoplasmic reticulum of the muscle fiber. As determined by Northern blotting, calsequestrin was expressed in EM and other skeletal muscle but not thyroid or fibroblasts. Calsequestrin is different from the "64 kDa protein", which has been identified as succinate dehydrogenase flavoprotein subunit, which has a corrected mol. wt. of 67 kDa. Serum antibodies against calsequestrin were found in 40% of patients with clinically active TAO, but in only 4% of those with stable eye disease, and in 5% of normal subjects, by immunoblotting. Although it is possible that autoimmunity against calsequestrin plays a role in the progressive EM damage that characterizes ophthalmopathy it is more likely that the antibodies are secondary to a reaction against some other cell membrane protein, such as the novel thyroid and eye muscle shared protein G2s or the TSH receptor.     

Cell-mediated immunity to orbital tissue antigens in thyroid-associated ophthalmopathy determined using the leukocyte procoagulant activity assay.

While it is generally accepted that thyroid-associated ophthalmopathy (TAO), a progressive inflammatory disorder of the extraocular muscle and orbital connective tissue (OCT), is immunologically mediated the nature of the underlying abnormalities is poorly understood. Although there is considerable evidence for antibody-mediated immunity against both eye muscle (EM) and OCT antigens in TAO a role of cell-mediated immunity (CMI) has not been studied in detail. We have used a new sensitive test for CMI, the leukocyte procoagulant activity (LPCA) assay and tested blood leukocytes from patients with TAO and autoimmune thyroid disease (ATD) without evident ophthalmopathy for reactivity against pig eye muscle (PEM) and human thyroid and OCT membrane and soluble fractions. In some cases human EM fractions were also tested. Preparations of PEM membrane (PEMM) and human thyroid membrane induced a significant LPCA response in both groups of patients. PEM cytosol, human OCT membrane and cytosol and human spleen membrane did not evoke a significant response in either group of patients. There were significant positive correlations in patients with TAO between (i) LPCA in response to PEMM and that to human thyroid membrane and (ii) LPCA in response to human thyroid membrane and that to human EM membrane. In patients with TAO there were no significant associations between LPCA response to PEMM and the detection of serum antibodies to a 64 kDa EM membrane protein in immunoblotting, or between LPCA response to PEMM and the duration or severity of the ophthalmopathy or clinical evidence for eye muscle involvement. These findings confirm a role of cell-mediated immunity against eye muscle antigens in thyroid-associated ophthalmopathy.(ABSTRACT TRUNCATED AT 250 WORDS)     

Antibodies in the serum of patients with autoimmune thyroid disorders react with a recombinant 98 amino acid fragment of a full length 64 kDa eye muscle membrane protein which is also expressed in the thyroid.

We have tested sera from patients with autoimmune thyroid disorders with or without ophthalmopathy for immunoreactivity, in a dot blot assay, against a recombinant 98 amino acid fragment of a cloned 64 kDa protein, D1, which is expressed in human eye muscle and thyroid, in the form of a Lac Z fusion protein. Tests were positive in 19 out of 40 patients with established thyroid-associated ophthalmopathy (TAO), in 12 out of 21 patients with Graves' hyperthyroidism (GH) without clinically evident ophthalmopathy, in 5 out of 10 patients with thyroid autoimmunity and lid retraction but no other signs of ophthalmopathy, in 4 out of 23 patients with Hashimoto's thyroiditis (HT) without evident ophthalmopathy and in 2 out of 18 patients with benign adenoma or multinodular goitre, but in only 2 out of 37 normal subjects tested. SDS-polyacrylamide gel electrophoresis and Western blotting for an antibody reactive with a 64 kDa antigen in pig eye muscle membranes was also carried out on sera from patients with TAO and GH. While immunoblotting for antibodies reactive with a 64 kDa protein was more often positive in patients with TAO, in whom 58% had serum antibodies which reacted with a 64 kDa protein, this was not the case in patients with GH without eye signs in whom the prevalence of positive immunoblot tests was 35%. Overall there was a fairly close correlation between the two tests although there were many exceptions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Significance of cytotoxic eye muscle antibodies in patients with thyroid-associated ophthalmopathy

We have studied the clinical significance of cytotoxic antibodies against human eye muscle cells in patients with thyroid-associated ophthalmopathy (TAO). Eye muscle reactive antibodies were measured in an antibody-dependent cell-mediated cytotoxicity (ADCC) assay. A positive test was defined as % specific lysis greater than the upper limit of normal, taken as the mean plus two standard deviations for normal subjects tested concurrently. As parameters of the severity of the ophthalmopathy we measured the degree of proptosis (mm), level of intraocular pressure (IOP) (mmHg) and American Thyroid Association classes (0-6). ADCC tests were positive in 21 out of 42 patients with TAO and in 8 out of 14 patients with Graves' disease without evident eye disease but in none of 12 normal subjects tested. In patients with TAO mean (+/- SE) IOP was significantly greater than that in patients with Graves' disease without apparent eye involvement for the primary position and for all gaze positions. There were significant positive correlations between levels of eye muscle reactive cytotoxic antibodies and the severity of the eye disease quantitated as American Thyroid Association classes 0-6, the IOP in the primary position and on downgaze, but not with the degree of proptosis. These results suggest that cytotoxic antibodies, as detected in ADCC, may play a role in the eye muscle damage of TAO and that their measurement may provide a useful clinical test.     

Restricted tissue reactivity of autoantibodies to a 64-kDa eye muscle membrane antigen in thyroid-associated ophthalmopathy.

We studied the tissue specificity of eye muscle (EM) membrane-reactive autoantibodies detected in the serum of patients with thyroid-associated ophthalmopathy (TAO). In preliminary studies, such antibodies were shown to react, in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting, with human thyroid (THY) and other human skeletal muscle (HSM) membrane antigens. We carried out absorption with human EM (HEM), THY, and HSM membranes of sera from patients with TAO and autoimmune thyroid disease without ophthalmopathy which reacted with one or more of 55-, 64-, and 95-kDa antigens in pig eye muscle (PEM) membrane in immunoblotting, the majority of which were also cytotoxic to HEM cells in an antibody-dependent cell-mediated cytotoxicity assay. In Western blotting, serum antibodies reactive with PEM membrane antigens of 55, 64, and 95 kDa were cross-absorbed by HEM, THY, and HSM but not by spleen or brain membranes and showed some species specificity, being absorbed by pig and human, but not bovine, EM membranes. When incubated with cultured HEM, THY, and HSM cells in vitro, autoantibodies in TAO sera immunoprecipitated a 64-kDa antigen from the first two tissues, but not from HSM, suggesting a specific binding to autoantigenic epitopes in HEM and THY. Sera from patients with TAO as well as those from patients with thyroid autoimmunity without ophthalmopathy immunoprecipitated a approximately 66-kDa protein, shown to be distinct from the 64-kDa antigen. The restricted immunological cross-reactivity of antibodies to a THY and HEM 64-kDa membrane antigen is discussed in the context of the association of ophthalmopathy with thyroid autoimmunity. Further experiments are needed to show whether autoantibodies to the 64-kDa eye muscle and thyroid shared antigen are cytotoxic, and thus likely to play a major role in the pathogenesis of the eye disease, or just markers of the orbital autoimmune process.     

Significance of anti-eye muscle antibody in patients with thyroid-associated ophthalmopathy by quantitative western blot.

To investigate the prevalence of antibody against rat eye muscle membrane antigen, as determined from SDS-polyacrylamide gel electrophoresis and western blotting, in sera from patients with thyroid-associated ophthalmopathy (TAO), we quantitatively analyzed the binding activity with a rat eye muscle membrane 64 kDa protein using chromato-scanner. Eye muscle antibody activity was expressed as ratio of density of the 64 kDa band to that at 66 kDa found with all normal sera and phosphate buffered saline. The mean (+/- SD) eye muscle antibody activity was 2.7 +/- 2.7 in TAO (P < 0.01 v.s. normal), 1.5 +/- 1.7 in Graves' disease without evident eye disease, 1.6 +/- 2.5 in Hashimoto's thyroiditis and 0.45 +/- 0.26 in normal subjects. A positive band at 64 kDa was found in 71% of patients with TAO, 36% of those of Graves' disease without evident eye disease and in 35% of patients with Hashimoto's thyroiditis without eye disease. The prevalence of this antibody activity tended to correlate to the severity of ophthalmopathy. Furthermore, the level of eye muscle antibody activity decreased in parallel with the improvement of eye signs in two patients. Sera reactive with rat eye muscle membrane 64 kDa protein reacted also with a human eye muscle membrane 64 kDa protein but not with human thyroid, liver, spleen or pancreas membrane preparations. In conclusion, antibody to rat eye muscle membrane 64 kDa protein is present in TAO and may be a useful clinical marker of ophthalmopathy.     

Antibodies against 1D,a Recombinant 64-kDa Membrane Protein,Are Associated with Ophthalmopathy in Patients with Thyroid Autoimmunity

We have tested for serum antibodies reactive with ₁D, a recombinant 65-kDa human thyroid protein which is also expressed in eye muscle, in patients with thyroid autoimmunity and ophthalmopathy by immunofluorescence and SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. We also measured antibodies to a 64-kDa pig eye muscle membrane protein which is identified by SDS-PAGE and Western blotting, correlating the two reactivities. While antibodies to ₁D, expressed in Chinese hamster ovary (CHO) cell membrane, were detected in approximately 40% of patients with ophthalmopathy, in both tests the greatest prevalence, by immunofluorescence, 73%, was demonstrated in patients with Graves' hyperthyroidism without clinically evident eye disease, although only 50% of these patients were positive in immunoblotting. When the two tests for anti-₁D antibodies were compared, immunofluorescence appeared to be the more specific and immunoblotting appeared to be the more sensitive. The greatest prevalence of antibodies reactive with a 64-kDa pig eye muscle protein, 71%, was in patients with TAO of less than 1 year duration; tests were positive in 49% of patients with more chronic ophthalmopathy and in 50% of patients with Graves' hyperthyroidism without evident eye disease. Antibodies reactive with ₁ D were detected in 17% of normals by immunofluorescence and 24% by immunoblots, while antibodies reactive with the 64-kDa pig eye muscle protein were detected in only 10% of the normal subjects tested. Lesser prevalences of antibodies to the two 64-kDa proteins in patients with established eye disease suggest that such antibodies may be an early abnormality in patients with Graves' hyperthyroidism who are predisposed to develop ophthalmopathy. Although the association was not close, reactivity against ₁D by immunoblotting, but not immunofluorescence, was significantly correlated with reactivity to a 64-kDa eye muscle membrane protein by immunoblotting. On the other hand, when sera containing antibodies reactive with both ₁D and the 64-kDa eye muscle protein were incubated with CHO (₁D) cell membrane, reactivity against ₁D was absorbed while that against the eye muscle protein was not. The precise relationship between the two 64-kDa proteins can only be clarified by cloning the 64-kDa protein from an eye muscle expression library and comparing the sequences with those of ₁D.     

Failure to Detect Complement-Mediated Antibody-Dependent Cytotoxicity Against Human Orbital Tissue Cells in the Serum of Patients with Thyroid-Associated Ophthalmopathy

Antibodies which are cytotoxic to human eye muscle cells and orbital fibroblasts in antibody dependent cell-mediated cytotoxicity (ADCC) are routinely detected in the serum of patients with thyroid-associated ophthalmopathy (TAO). In the present study sera from patients with TAO, most of which were shown to be positive in ADCC against eye muscle cell targets, were tested for complement-mediated antibody-dependent cytotoxicity (CMAC) against human and pig eye muscle cells, human (abdominal) skeletal muscle cells and human orbital fibroblasts, in ⁵¹ Cr release assays. Several different assay protocols and complement sources were used and patient and age, sex matched normal sera compared. In preliminary studies tests were always negative when eye muscle cells, other skeletal muscle cells, or orbital fibroblasts were used as targets, regardless of the complement source, or concentration, or assay conditions used. When larger numbers of patients and normals were tested in a single assay mean (± SE) % specific lysis for patients with TAO was not significantly different from that for normals for either eye muscle cells or other skeletal muscle cells and taking the upper limit of normal as mean + 2 SD for the normals, tests were positive in no patient with either target. On the other hand ADCC tests were positive in 57 % of the same sera tested with the same eye muscle cell targets. When human thyroid cells were used as targets, tests were positive in 10 of the 14 patients tested and monoclonal antibodies, in enzyme-linked immunosorbent assay, reactive with eye muscle antigens gave positive lysis of eye muscle cells.

Although eye muscle reactive autoantibodies have been well described in TAO, particularly IgG₃ subclass antibodies against a 64 kDa membrane antigen, their significance in the pathogenesis of the eye muscle component of this disorder is unclear. In contrast to thyroid cells where microsomal, and perhaps other membrane reactive antibodies, are cytotoxic in both ADCC and CMAC, antibodies associated with TAO appear to be cytotoxic only in ADCC. Although the reason for this discrepancy is unknown one possible mechanism is failure of IgG₃ subclass antibodies, which are usually cytotoxic, to activate complement when the target antigen is in too low a density on the cell surface.     

Failure to detect complement-mediated antibody-dependent cytotoxicity against human orbital tissue cells in the serum of patients with thyroid-associated ophthalmopathy

Antibodies which are cytotoxic to human eye muscle cells and orbital fibroblasts in antibody dependent cell-mediated cytotoxicity (ADCC) are routinely detected in the serum of patients with thyroid-associated ophthalmopathy (TAO). In the present study sera from patients with TAO, most of which were shown to be positive in ADCC against eye muscle cell targets, were tested for complement-mediated antibody-dependent cytotoxicity (CMAC) against human and pig eye muscle cells, human (abdominal) skeletal muscle cells and human orbital fibroblasts, in 51Cr release assays. Several different assay protocols and complement sources were used and patient and age, sex matched normal sera compared. In preliminary studies tests were always negative when eye muscle cells, other skeletal muscle cells, or orbital fibroblasts were used as targets, regardless of the complement source, or concentration, or assay conditions used. When larger numbers of patients and normals were tested in a single assay mean (+/- SE) % specific lysis for patients with TAO was not significantly different from that for normals for either eye muscle cells or other skeletal muscle cells and taking the upper limit of normal as mean + 2 SD for the normals, tests were positive in no patient with either target. On the other hand ADCC tests were positive in 57% of the same sera tested with the same eye muscle cell targets. When human thyroid cells were used as targets, tests were positive in 10 of the 14 patients tested and monoclonal antibodies, in enzyme-linked immunosorbent assay, reactive with eye muscle antigens gave positive lysis of eye muscle cells.2+ t     

Isotype and immunoglobulin subclass distribution of eye muscle membrane reactive antibodies in the serum of patients with thyroid-associated ophthalmopathy as detected in Western blotting.

We have determined the immunoglobulin (Ig) class (isotype) and IgG subclass of autoantibodies in the serum of patients with thyroid-associated ophthalmopathy (TAO) or autoimmune thyroid disorders without evident ophthalmopathy reactive in Western blotting with antigens of 55, 64, 75 and 95 kDa in pig eye muscle membrane (PEMM). The 22 sera studied were shown, previously, to contain IgG antibodies reactive with one or more of the four antigens. The majority of sera antibodies reactive with PEMM antigens were of two or more IgG subclasses. Of the IgG subclass specificities IgG3 and IgG4 subclass antibodies were, overall, the most common. We were unable to demonstrate IgG subclass restriction for antibodies reactive with the 95 or 55 kDa antigens in PEMM, antibody activity being equally distributed in all four subclasses tested. While most of the sera which recognized a 64 kDa antigen did so with an IgG4 antibody, all other subclasses were also represented. On the other hand all 13 sera reactive with a 75 kDa antigen did so using Ig of the IgG3 subclass and 12 of these used the IgG4 subclass as well, IgG1 and IgG2 subclasses being represented in only 3 and 4 sera, respectively. There were no differences, in respect to Ig class or IgG subclass distribution of eye muscle reactive antibodies between patients with Graves' hyperthyroidism with ophthalmopathy and those with Hashimoto's thyroiditis, and eye disease. Control sera from five normal subjects and three patients with nonautoimmune thyroid disorders did not contain antibodies reactive with these PEMM antigens of any Ig class or IgG subclass.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunohistochemical Studies Using Immunized Guinea Pig Sera with Features of Anti-Human Thyroid,Eye and Skeletal Antibody and Graves’ Sera

Type 2 5′ deiodinase enzyme was observed in both thyroid and eye muscle tissues, highlighting its possible role as a common antigen in thyroid-associated ophthalmopathy. Sera of 105 Graves’ patients and 40 controls, and immunized guinea pig sera against TCSS peptide, showing homology to the amino acid sequence from 132 to 152 of type 2 5′ deiodinase, were investigated to demonstrate the binding effects to human thyroid, eye and skeletal muscle tissues. Twenty-two Graves’ patients were positive for anti-TCSS peptide antibodies, of whom 18 cases had ophthalmopathy. The levels of anti-TCSS peptide antibodies were higher not only in Graves’ patients with (P<0.0001) and without (P<0.036) eye symptoms compared to controls but also the difference was significant between patients with and without ophthalmopathy (P<0.049). In Western blot, immunized sera showed binding reactions to the supernatant fractions of human thyroid, eye and skeletal muscle tissues at the range of 29 kDa. Patient sera with Graves’ ophthalmopathy resulted in positive reactions directed to membrane areas in thyroid follicular cells, and to fibers in eye and skeletal muscles using immunohistochemical method, while no positive staining was present after adding control sera. The binding features of immunized guinea pig sera exhibited similar staining in all human tissues but could be blocked with Graves’ sera. Our results suggest that type 2 5′ deiodinase enzyme protein could play a role as an antigen in Graves’ disease. Immunized guinea pig sera against TCSS peptide exhibited similar binding reactions and stainings to human thyroid, eye and skeletal muscle tissues as patient sera with Graves’ ophthalmopathy.     

Nature and significance of orbital autoantigens and their corresponding autoantibodies in thyroid-associated ophthalmopathy.

There is now considerable evidence that the pathogenesis of thyroid-associated ophthalmopathy is closely linked to the presence of a shared autoantigen(s) in the thyroid and the eye muscle, against which cytotoxic mechanisms are directed. Although the orbital connective tissue is certainly involved in the orbital inflammatory process, a 64 kDa membrane protein expressed by both the eye muscle and the thyroid and recognized consistently by antibodies in the sera of TAO patients, seems to be the most likely target candidate. While its presence in non ocular skeletal muscle is not as well established, more recent data tend to suggest the existence of a 64 kDa molecule in the three tissues. The availability of a cDNA encoding a 572 amino acid protein corresponding to a MW of 63-64 kDa, which may be the same molecule, will allow us to determine more clearly the structural characteristics of the different molecules proposed as targets. The role of the corresponding autoantibodies in the pathogenesis of the eye disease is far less well defined. Whether they play a role in the induction of the ophthalmopathy or only represent helpful markers remains to be clarified.     

Protective efficacy of rotavirus 2/6-virus-like particles combined with CT-E29H, a detoxified cholera toxin adjuvant

Identifying a safe and efficacious mucosal adjuvant is crucial for the development of subunit vaccines against rotavirus and other mucosal pathogens. Moreover, recognition of determinants of protective immunity to rotavirus infection is essential to the design of the means to prevent or control this viral gastrointestinal disease. We have studied the kinetics of systemic and mucosal antibody responses elicited upon mucosal immunization of mice with rotavirus recombinant virus-like particles (rVLPs) alone or combined with a detoxified version of cholera toxin, CT-E29H. CT-E29H has been shown to maintain the adjuvant effect of parental cholera holotoxin. Both inbred BALB/c and outbred CD-1 mice were immunized with rotavirus VP2/6-rVLPs (2/6-VLPs) combined with CT-E29H, orally or intranasally (i.n.), and the comparative efficacy of different formulations was then determined. Rotavirus-specific serum and fecal IgA, IgM, and IgG antibodies were determined by enzyme-linked immunoadsorbent assay (ELISA) weekly (or every other week) following vaccination. Animals then were challenged with a murine rotavirus strain, EDIM. The degree to which vaccinated animals were protected from the wild-type rotavirus challenge was reflected in the levels of viral antigen shed in stools (percent reduction in antigen shedding, PRAS). BALB/c mice immunized by either route produced rotavirus-specific serum IgA, IgM and IgG, as well as fecal IgA and IgG, but not IgM; however, the intranasal immunization induced stronger systemic IgG and IgM responses than did oral immunization. Similar levels of prechallenge rotavirus-specific fecal and serum IgA were detected in both the orally and the i.n. immunized groups. Two immunizations with 2-6VLPs and CT-E29H were sufficient to protect BALB/c mice, regardless of the route of administration. PRAS was 99.6, 98.8, and 98.8% for oral, i.n. and the oral + i.n. groups, respectively; in contrast vaccination with 2/6-VLPs alone was not protective (PRAS = 39%), indicating the critical role of CT-E29H in inducing protective levels of immune responses. Two of four outbred CD-1 mice that were immunized orally with 2/6-VLPs-CT-E29H showed no humoral responses (PRAS, 65%), but four of four i.n. immunized CD-1 mice displayed humoral responses (PRAS, 97.9%). Serum anti-VP6 and VP2 antibodies were detected in all immunoresponsive mice. The combined results in two strains of mice indicate that CTE29H is an effective mucosal adjuvant capable of inducing protective immune responses and suggest that intranasal administration is the preferred route of immunization.     

活化淋巴细胞及其染色质诱导抗核抗体生成

目的诱导抗双链ＤＮＡ抗体和其它抗核抗体生成的免疫原是什么？迄今不明。本研究试图阐明不同原因活化的淋巴细胞是诱导抗核抗体生成的免疫原。方法用丝裂原（ＣｏｎＡ／ＬＰＳ）或ＩＬ－２活化淋巴细胞及其染色质免疫同系ＢＡＬＢ／ｃ小鼠，用ＥＬＩＳＡ测定ＩｇＧ抗ｄｓＤＮＡ抗体，用免疫荧光法检测抗核抗体核型和免疫复合物沉积，用免疫印迹法测定抗Ｓｍ和抗ＲＮＰ等抗核抗体。结果上述原因活化的淋巴细胞及其活性染色质均能诱导抗双链ＤＮＡ抗体和其它抗核抗体生成，免疫小鼠的肾脏有显著的免疫复合物沉着。结论据本文结果推测活化淋巴细胞及其细胞核和染色质是诱发抗ｄｓＤＮＡ抗体和其它抗核抗体生成的免疫原。     

T cell responses to orbital antigens in thyroid-associated ophthalmopathy.

Thyroid-associated ophthalmopathy (TAO) is most likely to be a T cell-mediated disease, in which cytokines released in the extraocular muscles activate fibroblasts, increasing glycosaminoglycan production. The nature of the orbital antigen recognized by the infiltrating T cells is unclear, although it is possible that there is cross-reactivity between this and a thyroid autoantigen to explain the close association with thyroid autoimmunity. We have tested the ability of human and porcine eye muscle antigen preparations to stimulate proliferation of circulating T cells from healthy subjects and patients with TAO or Graves' disease without clinical TAO. Occasional responses were seen, particularly after depletion of CD8+ T cells, and two out of 10 TAO patients responded to eye muscle proteins of 25-50 kD after fractionation of antigens on gels and subsequent elution. There was no disease-specific response of T cells to R1, R14, D1 and 1D3, recombinant proteins identified from screening an eye muscle cDNA library with sera from patients with autoimmune thyroid disease. We have also found that interferon-gamma (IFN-gamma) production by T cells from TAO patients was not stimulated by eye muscle membrane antigens or by 1D3. These results suggest that the frequency of circulating T cells responding to eye muscle antigens in TAO is low, and that several candidate orbital antigens, including the 64-kD protein 1D3, are unlikely to be important T cell autoantigens in this condition.     

Rituximab Induces Distinct Intraorbital and Intrathyroidal Effects in One Patient Satisfactorily Treated for Graves’ Ophthalmopathy

Hyperthyroid Graves’ disease (GD) is a B-cell-mediated disease caused by antibodies stimulating the thyroid stimulating hormone (TSH) receptor (TRAb). A proportion of patients (40–60%) present with an associated ophthalmopathy (TAO), a progressive inflammatory autoimmune disease of the retroorbital tissue. We thought that the anti-CD20 monoclonal antibody rituximab (RTX), by inducing transient B-cell depletion, may potentially modify the active inflammatory phase of TAO. One patient with GD and TAO in its active phase and unresponsive to steroid, was treated with RTX. Whereas the ophthalmopathy responded to RTX therapy and a decrease in the clinical activity score from 5 to 2 was observed during the B-cell depletion, serum antithyroid antibodies, and in particular serum TRAb, were not affected by therapy. When the patient underwent total thyroidectomy, we found B-cells in the thyroid tissue specimens. The eye disease remained stable (clinical activity score = 2) and the patient subsequently underwent orbital decompression to correct proptosis of the eye. At that time we did not find lymphocytes in any of the orbital tissue specimens. We believe that RTX therapy in GD may cause amelioration of ophthalmopathy by depleting total lymphocyte population in the orbit, but not lymphocyte depletion in thyroid tissue with consequent unchanged serum TRAb levels.