Limitations of the semisynthetic library approach for obtaining human monoclonal autoantibodies to the thyrotropin receptor of Graves' disease.

Graves' disease (GD) is characterized by the presence of autoantibodies against the TSH-receptor (TSH-R) which are pathogenic and, upon binding to the receptor, trigger intracellular signal transduction. The autoantibodies are oligoclonal and as they are responsible for disease activity, their characterization would lead to a better understanding of the development of GD. Attempts to isolate anti-TSH-R antibodies from patients have proved to be difficult due to the exceedingly low serum levels due to rarity of these B cells, together with difficulties in obtaining purified TSH-R capable of interacting with patients autoantibodies. We employed phage antibody display technology and performed selection with a previously characterized semisynthetic antibody library on the purified extracellular ectodomain of the TSH-R. We report the isolation of six different anti-TSH-R monoclonal phage antibodies (moPhabs) from this library. All the moPhabs recognized TSH-R and its recombinant fragments by Western blotting, but failed to recognize the native TSH-R by flow cytometry. Consequently, the moPhabs did not lead to TSH-R activation. As these were the first moPhabs to TSH-R, they were analysed in terms of nucleotide and amino acid sequence and epitope specificity on the receptor. The moPhabs used immunoglobulin VH1 and VH3 germ line genes, all associated with Vlambda3 genes. Interestingly, the CDR3 regions of all moPhabs were remarkably similar, though not identical. In light of the common CDR3 usage, the epitopes recognized on TSH-R appeared to be restricted to amino acids residues 405-411 and 357-364. In summary, our results show that semisynthetic libraries may be limited in isolating human monoclonal antibodies that resemble pathogenic antithyrotropin receptor autoantibodies present in patients with GD. It is likely that until preparations of purified TSH-R that can be recognized by patients autoantibodies become available, similar to the recently described glycosylphosphatidylinositol (GPI) anchored TSH-R ectodomain, monoclonal antibodies from phage antibody display to TSH-R will be limited for isolating the rare, pathogenic antibodies of GD.     

Modulation of Fas-mediated apoptosis of human thyroid epithelial cells by IgG from patients with Graves' disease (GD) and idiopathic myxoedema

The expression of two autoimmune thyroid diseases, GD and idiopathic myxoedema, is associated with antibodies to the thyroid-stimulating hormone (TSH) receptor. Thyroid stimulating antibodies (TSAb) in GD are TSH agonists and cause hyperthyroidism as well as goitre, whereas thyroid stimulation blocking antibodies (TSBAb) in idiopathic myxoedema are TSH antagonists and cause hypothyroidism and thyroid atrophy. We investigated the effect of antibodies to TSH receptor on Fas-mediated apoptosis of thyroid epithelial cells (thyrocytes). Human IgG was isolated from healthy donors, patients with GD and idiopathic myxoedema. Human thyrocytes were obtained from surgical specimens. Thyrocytes were cultured in the presence or absence of human IgG with or without interferon-gamma (IFN-γ) or IL-1β for a specified time. After incubation, we examined the level of cAMP in cultured supernatants and both Fas and Bcl-2 expression on thyrocytes. In addition, we examined anti-Fas-mediated apoptosis of thyrocytes. Fas expression on thyrocytes was significantly down-regulated by Graves' IgG and TSH, although idiopathic myxoedema IgG did not affect Fas expression on thyrocytes. Idiopathic myxoedema IgG abrogated the effect of TSH on both cAMP production and inhibition of Fas expression on thyrocytes. Treatment of thyrocytes with IL-1β or IFN-γ caused a marked augmentation of Fas expression on thyrocytes. The increase of Fas expression of thyrocytes induced by IL-1β or IFN-γ was significantly suppressed in the presence of TSH or Graves' IgG. Anti-Fas-induced apoptosis of thyrocytes was observed in thyrocytes treated with IL-1β or IFN-γ, but was markedly inhibited in the presence of TSH or Graves' IgG. Furthermore, idiopathic myxoedema IgG abrogated most of the inhibitory effect of TSH on Fas-mediated apoptosis of thyrocytes treated with IL-1β or IFN-γ. Bcl-2 expression of thyrocytes did not change after stimulation with TSH, Graves' IgG, idiopathic myxoedema IgG, IL-1β or IFN-γ. These results suggest that TSAb found in Graves' patients may be potentially involved in the development of goitre by inhibition of Fas-mediated apoptosis of thyrocytes. In addition, TSBAb inhibit the action of TSH and increase the sensitivity toward Fas-mediated apoptosis of thyrocytes, inducing thyroid atrophy seen in patients with idiopathic myxoedema.     

A novel mouse model of Graves' disease: implications for a role of aberrant MHC class II expression in its pathogenesis

Mice immunized with fibroblasts expressing an MHC class II molecule and human thyrotropin receptor (TSHR), but not either alone, develop major features characteristic of Graves' disease (GD), such as thyroid-stimulating autoantibodies directed against TSHR, increased serum thyroid hormone levels, and enlarged thyroid glands. The results indicate the need for the simultaneous expression of a class II molecule and the TSHR on the surface of the fibroblasts to develop stimulating anti-TSHR antibodies and full-blown GD in our model. A T cell line established from a mouse with hyperthyroidism proliferates in response to fibroblasts expressing a class II molecule and TSHR, but not to the fibroblasts expressing only TSHR, indicating that the class II molecules on the fibroblasts present TSHR-derived peptide(s) to T cells. These results strongly suggest that the acquisition of antigen-presenting ability by thyrocytes can lead to the induction or progression of GD. We identified a T cell epitope of TSHR by the proliferative response of spleen cells from mice immunized with fibroblasts expressing a class II molecule and TSHR to 80 overlapping peptides spanning the extracellular domain of human TSHR. The identification of a major T cell epitope provides an important clue to a novel therapy of GD.     

A Novel Mouse Model of Graves’ Disease: Implications for a Role of Aberrant MHC Class II Expression in its Pathogenesis

Mice immunized with fibroblasts expressing an MHC class II molecule and human thyrotropin receptor (TSHR), but not either alone, develop major features characteristic of Graves’ disease (GD), such as thyroid-stimulating autoantibodies directed against TSHR, increased serum thyroid hormone levels, and enlarged thyroid glands. The results indicate the need for the simultaneous expression of a class II molecule and the TSHR on the surface of the fibroblasts to develop stimulating anti-TSHR antibodies and fullblown GD in our model. A T cell line established from a mouse with hyperthyroidism proliferates in response to fibroblasts expressing a class II molecule and TSHR, but not to the fibroblasts expressing only TSHR, indicating that the class II molecules on the fibroblasts present TSHR-derived peptide(s) to T cells. These results strongly suggest that the acquisition of antigen-presenting ability by thyrocytes can lead to the induction or progression of GD. We identified a T cell epitope of TSHR by the proliferative response of spleen cells from mice immunized with fibroblasts expressing a class II molecule and TSHR to 80 overlapping peptides spanning the extracellular domain of human TSHR. The identification of a major T cell epitope provides an important clue to a novel therapy of GD.     

Generation of a transgenic animal model of hyperthyroid Graves' disease

Graves' disease (GD) is an organ-specific autoimmune disease characterized by hyperthyroidism. Agonistic anti-thyrotropin receptor antibodies (thyroid-stimulating antibodies, TSAb), which mimic the thyrotropin (TSH) action, are thought to cause GD. The precise immunological mechanism of TSAb production, however, remains elusive. Previous immunization approaches using TSH receptor led to transient hyperthyroidism, but did not seem sufficient for comprehensive understanding of the development of autoimmune responses. To create GD-related autoimmunity in mice, we here generated TSAb-transgenic mice in which a patient-derived TSAb is expressed in B cells. Expression of the human TSAb in mice resulted in various manifestations of hyperthyroidism including increased free thyroxine levels with concomitantly decreased TSH levels, increased thyroid uptake of technetium pertechnetate, hyperthermia and thyroid hyperplasia. We found a correlation between the serum levels of human TSAb immunoglobulin and free thyroxine. In addition, conventional B cells expressing the TSAb were partially deleted in the periphery while B1 cells expressing the TSAb persisted and accumulated in the peritoneal cavity, a finding consistent with previous demonstrations that the maintenance of B1 cells plays an important role in the development of autoimmune diseases. Thus, our transgenic mouse may provide a novel and useful animal model for elucidating the pathogenesis and pathophysiology of GD.     

Antibodies to TSH-receptor in thyroid autoimmune disease interact with monoclonal antibodies whose epitopes are broadly distributed on the receptor

The hyperthyroidism of Graves' disease (GD) is caused by TSH-receptor (TSH-R) stimulating autoantibodies (TSAb), leading to overproduction of thyroid hormones. We present evidence for TSAb interaction with three distinct regions of the TSH-R, one in immediate vicinity of the carboxy terminal serpentine. Three murine monoclonal antibodies (MoAbs 28.1, A9 and 31.7) directed to amino acids 36-40, 147-228 and 382-415 were labelled and tested for their binding to human recombinant TSH-R on solid phase. All MoAbs bound to TSH-R with a K(d) of 8-12 nm and showed no competition among themselves. We tested 114 sera from euthyroid controls, 118 TBII positive sera from patients with GD (containing TSAb confirmed by bioassays), 16 TBII positive sera from patients with autoimmune thyroid disease (AIT), who were hypothyroid and had TSH blocking antibodies (TBAb), and 20 patients with AIT, who were hypothyroid but negative for all TRAb. Mid-regional MoAb A9 tracer achieved the highest sensitivity in the GD group (72.0%), whereas C-terminal MoAb 31.7 found most sera positive in the AIT group (87.5%). Surprisingly, the N-terminal MoAb 28.1 had the lowest sensitivity in the GD (10.4%) and AIT group (43.8%). Using a mixture of all three tracer MoAbs did not increase the sensitivity in the GD or AIT group, compared to the best single MoAb alone. Median inhibition of MoAb A9 was significantly (P < 0.001) higher than inhibition of MoAbs 28.1 or 31.7 in the group of GD patients but not in other groups. Almost all patient sera with positive reactivity in the MoAb tracer assays had TBII values in the higher range. However, there were many highly TBII positive sera, which did not show a displacement of the MoAb tracers. We conclude that, contrary to some reports, the binding of TSAb and TBAb to the TSH-R is not restricted to distinct and distant epitopes. The middle part of the TSH-R seems to be more relevant for TSAb binding than the N-terminal part, while a proportion of TSAb autoantibodies also binds to a C-terminal epitope of the TSH-R. The method described here is a TSH independent competitive assay for the detection of TSH-R autoantibodies.     

Characterization of the murine immune response to the murine TSH receptor ectodomain: induction of hypothyroidism and TSH receptor antibodies

The thyrotropin receptor (TSHR) is the major autoantigen of human Graves' disease. In order to define the antigenicity of the TSHR in a defined model, we examined the immune response of BALB/c mice to immunization with a new bioactive, recombinant preparation of the ectodomain of the murine TSHR (mTSHR-ecd). Mice (n = 10) were immunized with 25–50 μg of insect cell expressed, purified and refolded, mTSHR-ecd in alum adjuvant containing pertussis toxin, on days 0, 21, 36, 50 and 70. Control mice received wild-type baculovirus-infected insect cell protein lysate, in a similar way. After 28 days, murine serum contained high titres of antibodies specific to mTSH-ecd and their titres continued to increase over 90 days. Antibody epitope mapping, using 26 peptides spanning the human TSHR-ecd, showed that a variety of regions of the ectodomain were antigenic. The earliest epitope included aa 22–41, but later two regions of reactivity were noted clustered towards the mid portion and carboxyl terminus of the ectodomain. The murine TSHR autoantibodies (TSHR-Abs) inhibited up to 78% of the binding of labelled TSH to native TSHR, demonstrating the presence of antibodies capable of blocking the native TSHR. We showed that these TSHR antibodies acted, in vitro, as TSH blocking antibodies, inhibiting TSH-induced generation of cyclic AMP in chinese hamster ovary (CHO) cells transfected with the hTSHR. Hence, the antibody response to mTSHR-ecd was potentially antagonistic in its influence on the TSHR. Assessment of thyroid function in the immunized mice showed a fall in serum total T3 by 90 days and markedly elevated murine TSH levels (from 64.0 to 239.6 ng/ml), confirming the onset of thyroid failure. However, thyroid histology remained grossly normal. These data demonstrate that mTSHR-ecd is a potent antigen with three major immunogenic regions. The induced mTSHR-Abs blocked TSH action in vivo and reduced murine thyroid function.     

Thyroid-stimulating hormone receptor and its role in Graves' disease

The thyroid-stimulating hormone (TSH, or thyrotropin) receptor (TSHR) mediates the activating action of TSH to the thyroid gland, resulting in the growth and proliferation of thyrocytes and thyroid hormone production. In Graves' disease, thyroid-stimulating autoantibodies can mimic TSH action and stimulate thyroid cells. This leads to hyperthyroidism and abnormal overproduction of thyroid hormone. TSHR-antibodies-binding epitopes on the receptor molecule are well studied. Mechanism of TSHR-autoantibodies production is more or less clear but a susceptibility gene, which is linked to their production, is still unknown. Genetic studies show no linkage between the TSHR gene and Graves' disease. Among three common polymorphisms in the TSHR gene, only the D727E germline polymorphism in the cytoplasmic tail of the receptor showed an association with the disease, and this association is weak. The absence of a strong genetic effect of the TSHR polymorphisms in such a common and complex disorder as Graves' disease may be explained by a high degree of evolutionary conservation in TSHR. This can be shown by naturally existing germline and somatic mutations in the TSHR gene that cause various types of nonautoimmune and hereditary thyroid disease.     

Graves' Patient with Thymic Expression of Thyrotropin Receptors and Dynamic Changes in Thymic Hyperplasia Proportional to Graves' Disease Activity

Thymic hyperplasia is frequently observed in Graves'' disease. However, detectable massive enlargement of the thymus is rare, and the mechanism of its formation has remained elusive. This case showed dynamic changes in thymic hyperplasia on serial computed tomography images consistent with changes in serum thyrotropin receptor (TSH-R) antibodies and thyroid hormone levels. Furthermore, the patient''s thymic tissues underwent immunohistochemical staining for TSH-R, which demonstrated the presence of thymic TSH-R. The correlation between serum TSH-R antibody levels and thymic hyperplasia sizes and the presence of TSH-R in her thymus suggest that TSH-R antibodies could have a pathogenic role in thymic hyperplasia.     

The putative anti-thyrotropin receptor antibodies of Graves' disease. I. Gm allotypes

The hyperthyroidism of Graves' disease is thought to be related to antithyrotropin receptor (TSH-R) antibodies. In order to study the degree of immunogenetic homogeneity of these antibodies, we carried out Gm typing of 'receptor-purified' IgG from patients with active Graves' disease and controls. The results were compared to those of serum, total IgG and IgG which failed to attach to TSH-R. We found that in five out of seven Gm heterozygote patients studied the receptor-purified antibodies were restricted to the products of one haplotype compared to three out of five similar controls. Such eluted antibodies were biologically active. Similar results in terms of immunogenetic restriction and activity were obtained when F(ab)2 preparations were used. An unexpected finding was that sera and IgG from normal persons attached to thyroid membranes and that the attachment occurred via F(ab)2. Normal whole serum and 'receptor-purified' IgG and F(ab)2 inhibited TSH binding in the receptor assay; however, this inhibition showed no specificity for TSH-R.     

Endocrine ophthalmopathy: a re-evaluation of the association with thyroid autoantibodies.

Autoantibodies to the three major thyroid autoantigens, the TSH-receptor (TSH-R), thyroid peroxidase (TPO) and thyroglobulin (TG), have been investigated in 63 Graves' patients with severe endocrine ophthalmopathy. In agreement with other studies, TSH-R antibodies were detectable in 88% of patients and dominated the autoantibody spectrum. TPO antibodies were detectable in 60% of patients and TG antibodies in only 25% of patients. The prevalences, as well as the amounts, of all three thyroid autoantibodies were not significantly different from the values in 51 Graves' patients without clinically significant ophthalmopathy. However, in the subgroup of patients with TG antibodies, the ophthalmopathy patients displayed a shift towards IgG4 TG antibodies. Furthermore, in the same TG antibody positive subgroup, the amount of TSH-R antibody was significantly higher in the ophthalmopathy patients than in Graves' patients without ophthalmopathy. These qualitative differences in thyroid autoantibodies in patients with and without ophthalmopathy raise the possibility that further qualitative differences, such as thyroid autoantibody epitopes, may exist in patients with ophthalmopathy. Our observations, combined with recent evidence for the presence of TSH-R specific mRNA in retro-orbital tissue, suggest that it may be premature to dismiss the potential pathogenetic or diagnostic value of thyroid autoantibodies, particularly TSH-R antibodies, in Graves' ophthalmopathy.     

THE PUTATIVE ANTI-THYROTROPIN RECEPTOR ANTIBODIES OF GRAVES' DISEASE:I. Gm ALLOTYPES *

The hyperthyroidism of Graves' disease is thought to be related to antithyrotropin receptor (TSH-R) antibodies. In order to study the degree of immunogenetic homogeneity of these antibodies, we carried out Gm typing of ‘receptor-purified’ IgG from patients with active Graves' disease and controls. The results were compared to those of serum, total IgG and IgG which failed to attach to TSH-R. We found that in five out of seven Gm heterozygote patients studied the receptor-purified antibodies were restricted to the products of one haplotype compared to three out of five similar controls. Such eluted antibodies were biologically active. Similar results in terms of immunogenetic restriction and activity were obtained when F(ab)₂ preparations were used. An unexpected finding was that sera and IgG from normal persons attached to thyroid membranes and that the attachment occurred via F(ab)₂. Normal whole serum and ‘receptor-purified’ IgG and F(₂ inhibited TSH binding in the receptor assay; however, this inhibition showed no specificity for TSH-R.     

Application of mouse monoclonal antibodies for identification of antigen regions of human thyroid peroxidase in adolescents with Graves' disease and non-toxic multinodular goiter by flow cytometry

Thyroid peroxidase (TPO) is the major thyroid autoantigen recognized by serum autoantibodies from patients with Graves' disease (GD) or Hashimoto's thyroiditis directed to two immunodominant conformational regions termed A and B. The epitopes of human TPO have been defined using a panel of mouse monoclonal antibodies (mAbs).

The aim of this study was to estimate the expression of chosen surface antigen regions of TPO (1, 18, 30, 64 epitopes) on thyroid cells in 15 patients with non-toxic multinodular goiter (NTMG) and 15 patients with GD. The thyrocytes were identified by indirect method: in the first stage we added mouse monoclonal autoantibodies specific for TPO regions and in the second stage we conjugated this complex with rabbit anti-mouse antibodies IgG (Fab')₂ with FITC. All investigations were performed by flow cytometry using Coulter EPICS XL apparatus. The percentages of thyrocytes with expression of epitopes 1, 18, 30, 64 TPO were measured in relation to the respective anti-TPO concentrations: 50–1600 μg/ml.

The analysis of epitopes located in immunodominant regions (IDR) of TPO revealed higher percentages of thyrocytes in cases with GD in comparison to NTNG. The most predominant difference was observed for mAb 64 epitope (48 vs 7%, p < 0.019; 39 vs 5%, p < 0.017) at the concentration of 100–200 μg/ml mAbs. The expression of 18 epitope on thyrocytes was also statistically higher in Graves' patients than in the NTMG (14 vs 6%, p < 0.025) at concentration of 400 μg/ml mAbs. However, this expression was much less pronounced.

In all the cases, the percentages of thyrocytes with epitopes 1 and 30 were in low detection (8–15% of positive cells).

In conclusion, our findings suggest that the elevated expression of TPO epitopes 18 and 64 in young patients with thyroid autoimmune diseases increase stimulation and activation of thyroid cells during inflammatory reaction within the thyroid gland. In addition, predominant expression of 64 TPO epitope that recognizes B domain in GD patients could be a useful marker of the immune process in the thyroid gland.     

A study of human-human hybridomas from patients with autoimmune thyroid disease

Human-human B-cell hybridomas were established using peripheral blood lymphocytes from patients with autoimmune thyroiditis and Graves' disease. Peripheral mononuclear cells (PMC), with or without mitogen prestimulation, were fused with HGPRT-negative human myeloma cell lines (Gm4672 and Gm0462) using 44% polyethylene glycol. Developing hybridomas were screened by enzyme-linked immunosorbent assays (ELISAs) for human IgG and IgM and antibodies to human thyroglobulin (hTg) and microsomal antigen (M-Ag). A¹²⁵I-TSH binding inhibition assay was utilized for detecting antibodies to TSH receptor (TSH-R) protein. Hybridoma formation was observed only after prior mitogen stimulation of PMC. The amount of antibody secreted by the human-human hybridomas was highly variable (10 ng-100 µg/ml IgG/IgM). Nine and six-tenths percent of the hybrids secreted anti-hTg and 8.4% secreted anti-M-Ag. A 5% cloning efficiency was achieved, with detection of specific thyroid autoantibody secretion in one-third of the clones derived from positive hybridomas. Immunoglobulin secretion decreased with time and long-term stable clones were not achieved. Thyroid monoclonal autoantibodies to hTg, M-Ag, and TSH-R (IgG and IgM) detected during these studies were of a low affinity. In addition, antibodies were identified which exhibited marked specificity crossover between hTg, M-Ag, and nonthyroid antigens, suggesting the presence of recurrent epitopes. Such observations may help explain the multiplicity of thyroid autoantibodies in human thyroid disease and indicate a common defect in immunoregulation. We suggest that cross-reacting epitopes may be important in the derivation of thyroid-specific B-cell clones.     

Characterization of a Recombinant Yersinia enterocolitica Lipoprotein; Implications for its Role in Autoimmune Response against Thyrotropin Receptor

Autoimmune Graves' disease (GD), which is characterized by hyperthyroidism, is mediated by autoantibodies to the thyrotropin receptor (TSHR). Yersinia enterocolitica (Y.e.) has been shown to produce a lipoprotein (LP) that can cross-react with the TSHR and thus can act as a potential trigger of thyroid autoimmunity. In this study, to further characterize LP, we cloned the LP gene from Y. enterocolitica and expressed a recombinant LP. This recombinant LP was mitogenic for C3H/HeJ (LPS hyporesponsive) B cells and induced production and secretion of significant levels of IL-6 from splenocytes. A mouse antibody generated against the recombinant LP cross-reacted with TSHR as shown by western blot analysis. FACS analysis of splenocytes from mice immunized with LP revealed that LP could induce increased expression of B7.1 and B7.2. The immunomodulatory effects of LP including up-regulation of B7.1 and B7.2 coupled with its ability to induce antibodies that can cross-react with the TSHR showed several potential mechanisms by which it can cause breakdown of self-tolerance to TSHR.     

Immunological of the Thyrotropin Receptor

Antibodies to the thyrotropin receptor appear to he responsible for hyperthyroidism in Graves disease. The antibodies, described as thyroid-stimulating antibodies (TSAb) mimic the effects of thyrotropin (TSH) by binding to the TSH receptor and activating adenylate cyclase. TSAb consist of an electrophoretically heterogeneous population of IgG and the thyroid-stimulating site is formed by combination of heavy and light chains in the Fab part of the molecule. Binding studies indicate that the TSAb molecule interacts monovalently with membrane bound TSH receptors and that TSAb consists of an antibody population which shows a restricted heterogeneity with regard to TSH receptor affinity. Studies in patients with Graves disease and hyperthyroidism indicate that the levels of TSAb correlate well with thyroidal iodine uptake and the absence of pituitary control of thyroid function. However in some patients with ophthalmic Graves' disease or autoimmune thyroiditis there is evidence of serum antibodies which interact with the TSH receptor but are unable to stimulate thyroid function.     

Application of mouse monoclonal antibodies for identification of antigen regions of human thyroid peroxidase in adolescents with Graves' disease and non-toxic multinodular goiter by flow cytometry

Thyroid peroxidase (TPO) is the major thyroid autoantigen recognized by serum autoantibodies from patients with Graves' disease (GD) or Hashimoto's thyroiditis directed to two immunodominant conformational regions termed A and B. The epitopes of human TPO have been defined using a panel of mouse monoclonal antibodies (mAbs). The aim of this study was to estimate the expression of chosen surface antigen regions of TPO (1, 18, 30, 64 epitopes) on thyroid cells in 15 patients with non-toxic multinodular goiter (NTMG) and 15 patients with GD. The thyrocytes were identified by indirect method: in the first stage we added mouse monoclonal autoantibodies specific for TPO regions and in the second stage we conjugated this complex with rabbit anti-mouse antibodies IgG (Fab')(2) with FITC. All investigations were performed by flow cytometry using Coulter EPICS XL apparatus. The percentages of thyrocytes with expression of epitopes 1, 18, 30, 64 TPO were measured in relation to the respective anti-TPO concentrations: 50-1600 microg/ml. The analysis of epitopes located in immunodominant regions (IDR) of TPO revealed higher percentages of thyrocytes in cases with GD in comparison to NTNG. The most predominant difference was observed for mAb 64 epitope (48 vs 7%, p < 0.019; 39 vs 5%, p < 0.017) at the concentration of 100-200 microg/ml mAbs. The expression of 18 epitope on thyrocytes was also statistically higher in Graves' patients than in the NTMG (14 vs 6%, p < 0.025) at concentration of 400 microg/ml mAbs. However, this expression was much less pronounced. In all the cases, the percentages of thyrocytes with epitopes 1 and 30 were in low detection (8-15% of positive cells). In conclusion, our findings suggest that the elevated expression of TPO epitopes 18 and 64 in young patients with thyroid autoimmune diseases increase stimulation and activation of thyroid cells during inflammatory reaction within the thyroid gland. In addition, predominant expression of 64 TPO epitope that recognizes B domain in GD patients could be a useful marker of the immune process in the thyroid gland.     

Induction of thyroiditis in mice with thyrotropin receptor lacking serologically dominant regions

Grave's disease (GD) is characterized by pathogenic autoantibodies to the human thyrotropin receptor (hTSH-R), and is frequently associated with a lymphocytic infiltrate of the thyroid gland. In attempts to establish a murine model of GD, we and others have previously shown that immunization of mice with recombinant preparations of the hTSH-R ectodomain induces high titres of specific antibodies, which, however, are not pathogenic, nor is the response accompanied by the development of thyroiditis. Since earlier reports identified the serological immunodominant determinants within the N- and C-terminal regions of hTSH-R ectodomain, we reasoned that immunization of mice with truncated fragments of ectodomain lacking these dominant regions might result in skewing of the response to other determinants of the molecule, with consequent induction of immunopathological features present in GD. We show here that multiple challenge of BALB/c mice with an amino acid fragment of residues 43–282 generates antibodies directed at hTSH-R peptides 37–56, 157–176, 217–236 and 232–251. This reactivity pattern is distinct from that induced previously with the whole ectodomain of hTSH-R in BALB/c animals. Thyroid function remained unaffected in these mice, suggesting that pathogenic antibodies were not being induced. Interestingly, some animals developed lymphocytic infiltration of the thyroid gland, clearly indicating the presence of pathogenic T cell determinants within the 43–282 fragment. Challenge with the related fragment 43–316 produced the same pattern of serological response to the synthetic peptides as fragment 43–282, but was not accompanied by thyroiditis. The results demonstrate: (i) the presence of thyroiditogenic determinants within hTSH-R, and (ii) that these pathogenic determinants are likely to be cryptic, as their effect is exhibited only when the hierarchy of immunodominance within hTSH-R is drastically altered.     

Immunological of the Thyrotropin Receptor

Antibodies to the thyrotropin receptor appear to he responsible for hyperthyroidism in Graves disease. The antibodies, described as thyroid-stimulating antibodies (TSAb) mimic the effects of thyrotropin (TSH) by binding to the TSH receptor and activating adenylate cyclase. TSAb consist of an electrophoretically heterogeneous population of IgG and the thyroid-stimulating site is formed by combination of heavy and light chains in the Fab part of the molecule. Binding studies indicate that the TSAb molecule interacts monovalently with membrane bound TSH receptors and that TSAb consists of an antibody population which shows a restricted heterogeneity with regard to TSH receptor affinity. Studies in patients with Graves disease and hyperthyroidism indicate that the levels of TSAb correlate well with thyroidal iodine uptake and the absence of pituitary control of thyroid function. However in some patients with ophthalmic Graves' disease or autoimmune thyroiditis there is evidence of serum antibodies which interact with the TSH receptor but are unable to stimulate thyroid function.     

Immunology of the thyrotropin receptor.

Antibodies to the thyrotropin receptor appear to be responsible for hyperthyroidism in Graves' disease. The antibodies, described as thyroid-stimulating antibodies (TSAb) mimic the effects of thyrotropin (TSH) by binding to the TSH receptor and activating adenylate cyclase. TSAb consist of an electrophoretically heterogeneous population of IgG and the thyroid-stimulating site is formed by combination of heavy and light chains in the Fab part of the molecule. Binding studies indicate that the TSAb molecule interacts monovalently with membrane bound TSH receptors and that TSAb consists of an antibody population which shows a restricted heterogeneity with regard to TSH receptor affinity. Studies in patients with Graves' disease and hyperthyroidism indicate that the levels of TSAb correlate well with thyroidal iodine uptake and the absence of pituitary control of thyroid function. However in some patients with ophthalmic Graves' disease or autoimmune thyroiditis there is evidence of serum antibodies which interact with the TSH receptor but are unable to stimulate thyroid function.