Lymphocyte blastogenic response to human thyroglobulin in Graves' disease, Hashimoto's thyroiditis, and metastatic thyroid cancer.

In view of the past contradictory reports concerning in vitro lymphocyte transformation induced by human thyroglobulin (Tg) in thyroid diseases, the present study was undertaken to re-examine the response using improved methods in cell separation, culture, and cell harvesting. It has been found that the optimal dose level of Tg for maximal blastogenesis in culture differs from patient to patient. Consequently, it is inappropriate to use a single dose level of Tg for evaluation of the blastogenesis in study groups. By using serial Tg dose levels of 0.5 through 30 micrograms/ml in cultures, it was found that that incidence of positive responders in Graves' disease was 69.2%, in Hashimoto's thyroiditis 71.4%, and in healthy controls 9.1%. Metastatic thyroid cancer patients responded in a 50% incidence. All of the positive responders in the cancer group had elevated Tg levels, but no anti-Tg antibody in their sera.     

A sensitive enzyme immunoassay for anti-thyroglobulin antibody using Fab'-horseradish peroxidase conjugate. Evaluation of in vitro anti-thyroglobulin antibody synthesis by lymphocytes from patients with autoimmune thyroid disease

A sensitive enzyme immunoassay (EIA) for the detection of anti-thyroglobulin (Tg) antibodies was developed using Fab'-horseradish peroxidase (HRP) conjugate. Anti-Tg antibody was assayed by incubation with a thyroglobulin-coated polystyrene ball and then with affinity-purified anti-IgG Fab'-HRP conjugate. The HRP activity was assayed fluorimetrically. The sensitivity was 625 amol/tube and anti-Tg antibody levels between 0.5 and 200 ng/ml could be determined. The recoveries of anti-Tg antibody added to human sera at three different concentrations were 94.2-101.0%. Both within- and between-assay coefficients of variation were below 10%. Significant correlation was observed between values by the EIA and TGHA method (Kendall's rank correlation coefficient = 0.712, P less than 0.001). The present EIA for anti-Tg antibody is sensitive enough to determine anti-Tg antibody synthesized in vitro by the lymphocytes from patients with autoimmune thyroid disease and normal subjects. The amounts of anti-Tg antibody synthesized by peripheral lymphocytes from patients with Hashimoto's disease were significantly greater than those from patients with Graves' disease, although serum levels of anti-Tg antibody were usually elevated in both groups of patients. The results obtained suggest that anti-Tg antibody is synthesized in a different manner in patients with Hashimoto's disease and in patients with Graves' disease.     

Thyroglobulin-treated blood dendritic cells induce IgG anti-thyroglobulin antibody in vitro in Hashimoto's thyroiditis

Nonadherent, low density cells of dendritic morphology from the blood of patients with Hashimoto's thyroiditis were treated with human thyroglobulin (Tg) in vitro and cultured under serum-free conditions with autologous patient B cells and irradiated T cells. The patients were selected for high serum levels of IgG antithyroglobulin antibody (anti-Tg IgG). In 2 out of 12 patients the Tg-treatment induced production of anti-Tg IgG in excess of that secreted spontaneously. The amount of antibody produced in vitro (whether increased by Tg or not) correlated with the serum levels of antibody. In 5 patients (including the 2 who responded to Tg) the ratio of supernatant IgG anti-Tg antibody to total IgG was reduced when polyclonal stimulation was done with BCGF (10%). Antibody production was absent in cultures of cells from 2 patients with Graves disease and 4 normal individuals. Thus, in some patients with Hashimoto's thyroiditis, an extrinsically added autoantigen (Tg) on blood-derived dendritic cells can induce IgG anti-Tg antibody in vitro. These data suggest that "professional" antigen-presenting cells may play a role in autoimmune thyroid disease.     

The detection of antithyroglobulin activity in human serum monoclonal immunoglobulins (monoclonal gammopathies)

The sera of 159 patients with monoclonal gammopathies were examined for the presence of anti-thyroglobulin (Tg) activity. An enzyme-linked immunosorbent assay was employed. Thirty-one (19.5%) sera were found to bind Tg. The activity against Tg was further confirmed by using purified immunoglobulins and employing competition assays. The anti-Tg antibodies were found in the sera of patients with IgG, IgM and IgA gammopathies. Anti-Tg antibodies were more frequent among patients with IgG gammopathy. Autoantibodies to Tg are found in patients with Hashimoto's thyroiditis, Graves' disease and occasionally in patients with thyroid carcinoma. Natural autoantibodies directed against human Tg have been detected, as well, in healthy subjects. None of the patients in the present study whose serum was found to contain high titers of anti-Tg human monoclonal antibodies had any clinical or biochemical evidence of thyroid disease. Our results of a high incidence of anti-Tg activity in the sera of patients with monoclonal gammopathies support previous reports of autoantibody properties characteristic of these immunoglobulins.     

Antibodies to acetylcholinesterase cross-reacting with thyroglobulin in myasthenia gravis and Graves's disease.

In the present study we analysed by ELISA the ability of sera from 50 patients with myasthenia gravis (MG), 20 with Hashimoto's thyroiditis (HT), 53 with Graves' disease (GD) and 36 healthy controls (CR) to react with acetylcholinesterase (AChE) from Electrophorus electricus and human thyroglobulin (Tg). Significantly increased anti-AChE activity was exhibited by a high proportion of MG (IgG 36%) and GD (IgG 21%) sera, while increased anti-Tg activity was detected in all three patient groups (MG, IgG 26% and IgA 26%; HT, IgG 85% and IgA 40%; and GD, IgG 51%). Interestingly, a significant proportion of MG and GD sera exhibited both IgG anti-AChE and anti-Tg activities (MG, 18%; P < 0.001; and GD, 15%; P < 0.001, versus CR, 0%). This bi-reactivity was exhibited by anti-AChE antibodies cross-reacting with Tg (anti-AChE/Tg activity); (i) serum anti-AChE activity was effectively inhibited by soluble Tg, and (ii) affinity-purified anti-Tg antibodies cross-reacted with AChE. Cross-reactivity seems to be a property of pathological (auto)antibodies; induced (rabbit) antibodies to AChE or Tg were highly monospecific. Analysis of clinical data showed that increased IgG anti-AChE/Tg activity was well associated with: (i) overlapping GD in MG (P < 0.02), and (ii) ophthalmopathy in GD (P < 0.01). In contrast, no correlation was noted in MG between anti-AChE activity units and anti-Tg activity units or acetylcholine receptor antibody titres. The clinical significance of anti-AChE/Tg antibodies remains to be elucidated.     

Autoantibodies in autoimmune thyroid disease promote immune complex formation with self antigens and increase B cell and CD4+ T cell proliferation in response to self antigens

B cells are centrally involved as antigen-presenting cells in certain autoimmune diseases. To establish whether autoantibodies form immune complexes (IC) with self-antigens in autoimmune thyroid disease (AITD) and promote B cell uptake of self-antigen, sera from patients with Hashimoto's thyroiditis (HT), Graves' disease (GD) and healthy controls were incubated with human thyroglobulin (Tg) before adding normal peripheral blood mononuclear cells. The deposition of immunoglobulins and C3 fragments on B cells was then assessed. Inclusion of Tg in serum from HT patients promoted B cell capture of IgG and C3 fragments. Furthermore, the binding of Tg to B cells in preparations of normal blood cells was higher in HT serum than in serum from controls and correlated positively with the serum anti-Tg activity, as did the B and CD4+ T cell proliferation. Disruption of the three-dimensional structure of Tg by boiling reduced the proliferative responses. The data indicate that anti-Tg antibodies associated with AITD facilitate the formation of complement-activating Tg/anti-Tg complexes, binding of IC to B cells, and the subsequent proliferation of B and T cell subsets. This represents a novel mechanism for the maintenance of autoimmune processes in AITD and links autoreactive T cell responses with the presence of autoantibodies.     

Anti-thyroglobulin autoantibodies in sera from patients with chronic thyroiditis and from healthy subjects: differences in cross-reactivity with thyroid peroxidase.

A significant portion (about 12.7%) of healthy subjects was found to contain anti-thyroglobulin (anti-Tg) antibodies in their sera. We compared the binding activities of these antibodies and of anti-Tg autoantibodies from sera of patients with chronic thyroiditis with human thyroid peroxidase (TPO). The results obtained by ELISA indicated that out of 10 healthy subjects with anti-Tg antibodies, only four had anti-Tg antibodies capable of binding to TPO, whereas anti-Tg autoantibodies from almost all patients with chronic thyroiditis possessed high binding activities to TPO. By use of the immunoprecipitation method, it was also shown that although all anti-Tg autoantibodies from patients precipitated TPO, a majority of anti-Tg antibodies from healthy subjects could not precipitate TPO. Such findings cannot be ascribed to the differences in levels of anti-Tg autoantibodies and anti-TPO autoantibodies in sera and the differences in avidities of anti-Tg antibodies in sera between healthy subjects and patients with chronic thyroiditis. Thus, it can be concluded that anti-Tg antibodies from healthy subjects differ from those of patients with chronic thyroiditis with respect to TPO binding, probably due to difference in fine specificities of these anti-Tg antibodies.     

Characterization of different types of experimental autoimmune thyroiditis in the Buffalo strain rat

We have compared features of experimental thyroiditis in the Buffalo strain rat induced by neonatal thymectomy, immunization with rat thyroglobulin (Tg) and complete Freund's adjuvant, and subcutaneous administration of trypan blue or 3-methylcholanthrene. The disease produced by neonatal thymectomy resulted in significantly worse thyroiditis and higher antibody levels than the other models and shared other features in common with Hashimoto's thyroiditis. In particular TSH levels were elevated, Tg antibodies had a restricted subclass distribution, the thyroid was infiltrated by both B cells and T cells (comprising equal numbers of W3/25 and Ox8 positive cells) and thyroid follicular cells were Ia antigen-positive in some of the thymectomy group animals. In the other forms of thyroiditis, the thyroid infiltrate was mainly composed of macrophages or dendritic cells and B cells. Different pathogenic mechanisms are probably involved in these models; the disease produced by neonatal thymectomy shows the closest similarity to Hashimoto's thyroiditis.     

Serial studies on the affinity and heterogeneity of human autoantibodies to thyroglobulin

The functional affinity and heterogeneity of autoantibodies to thyroglobulin (Tg) were measured by an IgG subclass-specific solid-phase competition ELISA in patients with autoimmune thyroid disease. High-affinity IgG1 and IgG4 antibodies formed the major anti-Tg response. Both titre and affinity of IgG3 and IgG2 anti-Tg were generally low but in some Hashimoto's disease patients high-affinity IgG2 anti-Tg were found and IgG2 anti-Tg, unlike those of other subclasses, showed very restricted heterogeneity. The affinity of IgG4 anti-Tg was similar in patients with thyroid disease and their clinically euthyroid (normal) relatives. In contrast, a progressive increase in IgG1 anti-Tg affinity was seen in clinically euthyroid individuals compared with their relatives with thyroid disease and high titred Hashimoto's disease patients, suggesting that either rising titres of high affinity IgG1 anti-Tg or affinity maturation of IgG1 anti-Tg may be indicative of impending hypothyroidism.     

Long term spectrotypic and idiotypic stability of thyroglobulin autoantibodies in patients with Hashimoto''s thyroiditis.

Isoelectric focusing of serial bleeds from patients with Hashimoto's thyroiditis was carried out and thyroglobulin (Tg) autoantibodies visualized using 125I-labelled Tg followed by autoradiography. Although the spectrotype of these antibodies was polyclonal and varied from patient to patient, each individual's spectrotype remained constant during the disease. Similar results were obtained if immunoblots were stained with rabbit anti-idiotype (anti-Id) raised to these autoantibodies. Using radioimmunoassay (RIA), it is shown that the levels of Id remain constant over several years whether assayed on crude immunoglobulin (Ig) fractions or affinity-purified anti-Tg. Therefore, once established, the autoimmune disease appears to be stable in terms of autoantibody spectrotype and idiotype in the patients studied.     

Antigen-specific B-cell function in human autoimmune thyroiditis

T-B cells from the peripheral circulation of patients with autoimmune thyroiditis were cocultured with sheep red blood cells (SRBC) or soluble human thyroglobulin (Tg), a self-antigen. The B-cell mitogen, Staphylococcus aureus, combined with macrophage-derived B-cell differentiating factor, induced in vitro lymphoid activation and proliferation in the presence or absence of Tg or SRBC, which was monitored after 6 days by specific anti-Tg and anti-SRBC plaque-forming cell (PFC) responses (expressed as PFC per 10(6) T-B cells). In the presence of SRBC, significantly more anti-SRBC PFC (322 +/- 113, SE) were generated in normals (N = 5) compared with autoimmune thyroiditis patients (58 +/- 36) (N = 8) (P less than or equal to 0.001), data consistent with an antigen-specific T-cell defect. Anti-Tg PFC, not detectable in normal controls, were observed in patients in the absence (111 +/- 41) and presence (171 +/- 64) of Tg (10-1000 ng/ml). However, variable responses were noted after such coculture experiments with Tg. Three patients demonstrated amplification of anti-Tg PFC, while three showed antigen-related inhibition of anti-Tg PFC. These data indicated heterogeneity of responses to Tg antigen in patients with autoimmune thyroid disease compounded by significantly depressed antigen-specific induction mechanisms.     

Ultracentrifugal and Immunological Comparisons of Succinylated Human Thyroglobulin and 27 S Iodoprotein

Human thyroglobulin (Tg) and thyroid 27 S iodoprotein were dissociated with succinic anhydride (SA), to examine similarities in subunit organization and in immunological reactivity of these subunits. In the analytical ultracentrifuge, S-9, S-11, and 5-16 components arose from 19 S Tg dissociation. Four species, including S-7, S-12, S-15, and S-19 subunits, were formed upon dissociation of 27 S protein. More than 80% of the Tg was transformed into the S-9 component at higher SA concentrations. At similar SA levels, all four of the 27 S protein subunits remained. Immunological reactivity of the intact and succinylated iodoproteins was tested with rabbit anti-Tg sera and human autoimmune thyroiditis sera. Products of both Tg and 27 S protein dissociation behaved similarly in immunodiffusion (ID) and in inhibition of tanned cell hemagglutination (TCHI). All succinylated products, even after purification, were active as inhibitors in TCHI against both types of sera. Also, every dissociated sample precipitated with the heteroimmune sera. However, moderately dissociated Tg or 27 S iodoprotein lost the ability to precipitate in gel diffusion with the autoimmune serum.     

IgA class and subclass thyroid auto-antibodies in Graves' disease and Hashimoto's thyroiditis

The reported prevalence of IgA class thyroid antibodies in Hashimoto's thyroiditis is variable and the IgA subclass distribution in unknown, despite recent reports of IgG subclass restriction in the thyroid auto-antibody response. Using an ELISA, IgA class antibodies were found against thyroglobulin (Tg) and microsomes (Mic) in 40-52% of patients with Graves' disease and Hashimoto's thyroiditis, and, against thyroglobulin, they were detected in the absence of IgG antibodies in 10% of the cases. Both IgA1 and IgA2 subclasses were detected in all patients with IgA class antibodies, although a significantly higher proportion of IgA2 relative to IgA1 was found in microsomal compared with thyroglobulin antibodies. In view of the high turnover rate and unique complement-fixing properties of IgA2 antibodies, this class of thyroid auto-antibody may play an important role in determining the response in thyroid auto-immunity.     

Prevalence of Yersinia plasmid-encoded outer protein (Yop) class-specific antibodies in patients with Hashimoto's thyroiditis

Objective   To investigate the prevalence of class-specific antibodies (IgG, IgA) to Yersinia enterocolitica plasmid-encoded outer proteins (Yops) in patients with diagnosed Hashimoto's thyroiditis.
Methods   Seventy-one patients with Hashimoto's disease, 464 healthy blood donors and 250 patients with non-postinfectious rheumatic disorders (matched controls) were tested for class-specific antibodies to Yops. Anti-Yop antibodies were determined by ELISA and Western blot.
Results   The prevalence of class-specific antibodies to Yops as determined by ELISA was 14-fold higher (20 of 71; 28.2%) in people with Hashimoto's thyroiditis than in the two control groups. These results were confirmed by the Western blot, with 16 positive sera, three equivocal and one negative.
Conclusions   There is strong clinical and seroepidemiologic evidence for an immunopathologic causative relationship between Yersinia enterocolitica infection and Hashimoto's thyroiditis. Further investigation concerning the mechanisms involved and the possible effects of antibacterial chemotherapy on the outcome of Hashimoto's disease is warranted.     

Spontaneous secretion of thyroid autoantibodies by cultured peripheral blood lymphocytes from patients with Hashimoto''s thyroiditis detected by micro-ELISA techniques

The spontaneous in vitro production of anti-thyroglobulin (aTg) and anti-microsomal (aM) antibodies by mononuclear cells (MNC) from patients with Hashimoto's thyroiditis (HT) was analysed by an ELISA detection system. MNC from 35 HT patients spontaneously produced detectable levels of both autoantibodies in vitro (i.e., without mitogenic or antigenic stimulation). aTg was quantified using a reference aTg IgG standard and ranged from 55 to 9,000 ng aTg. Specificity of aTg by ELISA was assessed using heterologous Tg antigen (Ag). Microsomal Ag obtained by gel filtration was far less contaminated with Tg than the ultracentrifugation pellet. Specificity of aM ELISA was assessed using insulinoma membrane as unrelated Ag and by blocking aM detection only with microsomal Ag. aM levels in the 35 supernatants ranged from 0.1 to 1.12 OD. A direct correlation was found between aM serum titres detected by haemagglutination and in vitro aM spontaneous production, but not for aTg. This lack of correlation for aTg might have biological relevance. Tg restimulation in vitro enhanced aTg production in only four out of 18 cases, of which only one was significant. This system provides a tool for studies of the immunoregulation of thyroid autoantibody formation in vitro.     

Circulating antibodies to DNA-related antigens in patients with autoimmune thyroid disorders.

A high prevalence of antibodies to double-stranded DNA (AbDNAds) has been recently reported in serum of patients with autoimmune thyroid disorders, but the specificity of this finding has been questioned. For this reason, the prevalence of several antibodies to DNA-related nuclear antigens (AbDRENA) has been evaluated in sera of patients with autoimmune and non-autoimmune thyroid disease. The study group included: 46 Graves' disease patients, 28 Hashimoto's thyroiditis patients, 25 patients with toxic nodular goitre and 11 with non-toxic nodular goitre. Twenty-eight Graves' patients were retested during methimazole (MMI) therapy, and 5 after radioiodine administration. Twenty-two patients with systemic lupus erythematosus and 28 normal subjects served as positive and negative controls, respectively. AbDRENA included: AbDNAds by RIA or immunofluorescence (IF); antibodies to single-stranded DNA (AbDNAss) and antibodies to histone (AbHist) by ELISA methods; antibodies to nuclear antigens (ANA) by immunofluorescence. RIA values were considered to be abnormal when 2 SD above the mean of normal controls. In our study 13% of Graves' patients were positive for AbDNAds by RIA: all of them had negative tests by IF; 11% were positive for AbDNAss, 2% for AbHist and 7% for ANA. A comparable prevalence of positive results for AbDNAds by RIA, with negative IF tests, was found in Hashimoto's thyroiditis patients. No significant changes of antibody levels were observed in Graves' patients during MMI treatment or after radioiodine administration. A positivity for AbDNAds or AbDNAss was found in 8% of patients with toxic nodular goitre, but in none of those with non-toxic goitre.(ABSTRACT TRUNCATED AT 250 WORDS)     

Autoantibodies to p53 in sera of patients with autoimmune thyroid disease

Mutations in the tumor suppressor gene, p53, lead to intracellular accumulation of abnormal p53 protein and are associated with p53 autoantibodies. p53 also accumulates in autoimmune diseases and Hashimoto's thyroiditis, but it is unknown if p53 autoantibodies occur in the latter. We measured p53 autoantibodies in the sera of 93 patients with thyroid disease and 19 patients without thyroid disease. Anti-p53 antibodies were detected in the sera from 4.2% (2/48) of patients with autoimmune thyroid disease, including one patient with Hashimoto's thyroiditis (3.7%, 1/27) and one with Graves' disease (4.8%, 1/21). A third patient with pseudohypoparathyroidism, but without thyroid disease, was also positive (1/19; 5.2%). None of 19 patients with differentiated thyroid cancer had anti-p53 antibodies. We conclude that anti-p53 antibodies can be detected in the sera from approximately 4% of patients with autoimmune thyroid disease. This finding suggests that increased DNA damage and apoptosis may be associated with autoimmune thyroid disease.     

Tryptic peptides of human thyroglobulin: II. Immunoreactivity with sera from patients with thyroid diseases.

Tryptic peptides of human thyroglobulin (Tg) were analysed by Western immunoblot for their reactivity to circulating autoantibodies from patients with Hashimoto's thyroiditis (HT), Graves' disease (GD) and thyroid carcinoma, and from normal human controls. Low molecular weight peptides were released after 4 h incubation of Tg with trypsin. The sera of thyroid disease patients reacted with several peptides, but predominantly bound three peptides with apparent molecular weights (MWap) of 25 kD, 20 kD, and 15 kD; the sera of normal individuals did not bind these fragments of Tg. The pattern of tryptic peptides recognized by the majority of sera from GD patients differed from that recognized by sera from most patients with HT. Autoantibodies from both groups of patients recognized a 15-kD peptide with a high frequency, but the sera from 26/43 (60%) GD patients also recognized a peptide with MWap of 25 kD, whereas the sera from 22/35 (63%) of HT patients recognized a 20-kD peptide. A few sera from patients with thyroid carcinoma reacted with peptides with MWap of 15 and 20-kD, and none bound the 25-kD peptide. The immunoreactivity of autoantibodies in HT sera to the 20-kD peptide paralleled the competitive inhibition of the MoAb 137C1 by these sera. In addition, MoAb 137C1 and Hashimoto's sera showed the same Western immunoblot-binding pattern to Tg tryptic peptides, suggesting that a Hashimoto-associated epitope and the 137C1-binding site are found on the same peptide. These findings suggest that distinct peptides are recognized by Tg autoantibodies from patients with different thyroid diseases.     

Definition, at the molecular level, of a thyroglobulin-acetylcholinesterase shared epitope: study of its pathophysiological significance in patients with Graves' ophthalmopathy

The nature of the putative autoantigen in Graves' ophthalmopathy (Go) remains an enigma but the sequence similarity between thyroglobulin (Tg) and acetylcholinesterase (ACHE) provides a rationale for epitopes which are common to the thyroid gland and the eye orbit. In an attempt to define the shared epitope, we have screened a lambda gt 11 human thyroid cDNA library using a polyclonal antibody to Torpedo ACHE and isolated two clones, which upon sequencing, were shown to contain Tg segments, corresponding to portions of the C terminal part of the molecule which has a high similarity with ACHE. Having demonstrated the existence of an epitope common to Tg and ACHE, the clones have been further tested and found to be positive in lysis plaque assays with 1/10 sera from patients with Hashimoto's thyroiditis (HT), 8/8 from patients with Graves' ophthalmopathy and 0/8 normal sera. We have investigated the physiological significance of this common epitope by in situ immunolocalization studies in which the polyclonal antibody to Torpedo ACHE (which was used for screening the library) and immunoglobulins (Igs) from 6 Go patients tested were shown to bind to end plate regions of human foetal muscle fibres which were concurrently shown to be rich in cholinesterase activity: Igs from 3 normal individuals and 2 patients with Hashimoto's thyroiditis did not bind. The results demonstrate and characterize an epitope which is common to Tg and ACHE and show that Go patients Igs contain antibodies which bind to muscle end plates rich in cholinesterase. The significance of these findings to the pathogenesis of Go is discussed.