Genetic and epitopic analysis of thyroid peroxidase (TPO) autoantibodies : markers of the human thyroid autoimmune response
TL;DR: Monoclonal human TPOautoantibodies will be invaluable for B cell presentation of TPO to determine the T cell epitopes involved in TPO autoantibody production, and evidence for conservation as well as inheritance of the fingerprints in some families may provide insight into the genetic basis of human autoimmune thyroid disease.
Abstract: TPO autoantibodies, the hallmark of human autoimmune thyroid disease, are of IgG class and are associated with thyroid destruction and hypothyroidism. Using the immunoglobulin gene combinatorial library approach, a panel of human monoclonal TPO autoantibodies (expressed as Fab) has been generated from thyroid tissue-infiltrating B cells. TPO-specific Fab closely resemble patients' serum autoantibodies in terms of L chain type, IgG subclass, affinities for TPO as well as epitopes recognized by > 80% of TPO autoantibodies in an individual's serum. TPO autoantibody V region genes are not unique; H chain V genes are usually mutated, while L chain V genes are sometimes in germ-line conformation. The autoantibodies recognize an immunodominant region involving conformational, overlapping epitopes in domains A and B. Finally, TPO autoantibody epitopic fingerprints are distinctive for individual sera, are not associated with hypothyroidism, but are conserved over time (indicating a lack of B cell epitope spreading). Evidence for conservation as well as inheritance of the fingerprints in some families, together with VH gene polymorphisms, may provide insight into the genetic basis of human autoimmune thyroid disease. Furthermore, monoclonal human TPO autoantibodies will be invaluable for B cell presentation of TPO to determine the T cell epitopes involved in TPO autoantibody production.
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TL;DR: This work states that TSHR expression as cell-free translates or as synthetic peptides or ashetic peptides as well as confusion on the terminology and types of T SHR autoantibodies VI is a cause for concern.
Abstract: I. Introduction II. TSHR Ectodomain Structure A. Primary amino acid structure B. Subunit structure C. Disulfide bonds in the TSHR ectodomain D. Carbohydrate moieties in the TSHR ectodomain E. TSHR dimerization III. Recombinant TSHR Expression A. Approaches to TSHR expression B. TSHR expression in prokaryotes C. TSHR expression as cell-free translates or as synthetic peptides D. TSHR expression in eukaryotic insect cells E. TSHR expression in mammalian cells IV. TSH and Autoantibody Binding to the TSHR Ectodomain A. Holoreceptor vs. ectodomain B. Conformational nature of binding sites C. TSHR carbohydrate: part of the binding site? D. TSHR amino acid residues in TSH-binding site E. Autoantibody epitopes V. TSHR Autoantibody Assays A. Historical background B. In vivo and in vitro bioassays C. Indirect TBI assays D. Direct assays for TSHR autoantibodies E. Confusion on the terminology and types of TSHR autoantibodies VI. Monoclonal Autoantibodies to the TSHR Ectodomain A. Introduction B. Mouse TSHR mAb C. Hu...
516 citations
TL;DR: Measuring TPO antibodies in euthyroid subjects can be used to identify subjects with increased risk for hypothyroidism and in women who wish to become pregnant and those with an increased risk per se who are pregnant, patients with other autoimmune diseases, subjects on amiodarone, lithium, or interferon-alpha, and in relatives of patients with autoimmune thyroid diseases.
152 citations
TL;DR: Recent advances in the understanding of the genetic input to the pathogenesis of Hashimoto's thyroiditis are summarized.
Abstract: Hashimoto's thyroiditis (HT) is an organ-specific T-cell mediated disease. It is a complex disease, with a strong genetic component. To date, significant progress has been made towards the identification and functional characterization of HT susceptibility genes. In this review, we will summarize the recent advances in our understanding of the genetic input to the pathogenesis of HT.
143 citations
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...A large proportion of the anti-TPO heavy chain VH domain comes about following a VDJ recombination process that uses inverted D genes [54,55]....
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TL;DR: In this article, a group of 89 subjects undergoing surgery for thyroid carcinomas were compared with a control group for normofunctioning goiter, and a second group of 47 patients undergoing total thyroidectomy for Hashimoto thyroiditis.
Abstract: Several studies report a higher rate of papillary thyroid carcinomas (PTC) in patients with Hashimoto thyroiditis (HT), indicating a possible correlation between the two diseases. We studied a group of 89 subjects undergoing surgery for thyroid carcinomas compared with a control group of 89 subjects operated on for normofunctioning goiter, and a second group of 47 patients undergoing total thyroidectomy for HT. Association with HT was found in 19 of the 71 PTC subjects (26.7%) and in 8 goiter patients (8.9%), which was a significant difference (P < 0.02). Thirteen of the HT patients, mostly with the nodular form, showed coexistent PTC (27.6%). HT and PTC coexisted in several morphological, immunohistochemical, and biomolecular aspects; increased incidence of PTC in HT patients might therefore indicate that HT is a precursor of thyroid cancer. Further studies are required, however, in order to confirm this hypothesis; until then, HT patients should undergo careful clinical and technical follow-up.
143 citations
TL;DR: These models provide unique insight into several aspects of Graves' disease, including manipulating immunity toward Th1 or Th2 cytokines enhances or suppresses hyperthyroidism in different models, and the role of TSHR cleavage and A subunit shedding in immunity leading to thyroid-stimulating antibodies (TSAbs).
Abstract: Graves' hyperthyroidism can be induced in mice or hamsters by novel approaches, namely injecting cells expressing the TSH receptor (TSHR) or vaccination with TSHR-DNA in plasmid or adenoviral vectors. These models provide unique insight into several aspects of Graves' disease: 1) manipulating immunity toward Th1 or Th2 cytokines enhances or suppresses hyperthyroidism in different models, perhaps reflecting human disease heterogeneity; 2) the role of TSHR cleavage and A subunit shedding in immunity leading to thyroid-stimulating antibodies (TSAbs); and 3) epitope spreading away from TSAbs and toward TSH-blocking antibodies in association with increased TSHR antibody titers (as in rare hypothyroid patients). Major developments from the models include the isolation of high-affinity monoclonal TSAbs and analysis of antigen presentation, T cells, and immune tolerance to the TSHR. Studies of inbred mouse strains emphasize the contribution of non-MHC vs. MHC genes, as in humans, supporting the relevance of the models to human disease. Moreover, other findings suggest that the development of Graves' disease is affected by environmental factors, including infectious pathogens, regardless of modifications in the Th1/Th2 balance. Finally, developing immunospecific forms of therapy for Graves' disease will require painstaking dissection of immune recognition and responses to the TSHR.
114 citations
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TL;DR: This review considers recent developments in the understanding of the properties of TRAb, particularly measurement of the antibodies and their sites of action and synthesis and indicates that TSH receptor antibodies nearly always act as TSH agonists in patients with a history of Graves' hyperthyroidism.
Abstract: This review considers recent developments in our understanding of the properties of TRAb, particularly measurement of the antibodies and their sites of action and synthesis. Two new assay methods have allowed considerable improvements in the sensitivity, specificity, precision, and ease of measuring TRAb. In particular: 1) receptor assays based on inhibition of receptor-purified labeled TSH binding to detergent-solubilized TSH receptors and 2) bioassays based on stimulation of cAMP release from monolayer cultures of isolated thyroid cells. Detailed studies with the two assays indicate that TSH receptor antibodies nearly always act as TSH agonists in patients with a history of Graves' hyperthyroidism. Studies in areas of dietary iodine sufficiency suggest that measurement of the antibodies at various stages in the course of treating Graves' disease can be of value in predicting the outcome of therapy. However, in areas of iodine deficiency, difficulties in the ability of patients' thyroid tissue to recover from the effects of antithyroid drugs may prevent the receptor antibodies from causing a relapse of thyrotoxicosis. Consequently, the predictive value of receptor antibody measurements would be expected to be lower in these geographical areas. Although patients with a history of Graves' hyperthyroidism nearly always have TRAb which act as TSH agonists, about 20% of patients with frank hypothyroidism due to autoimmune destruction of the thyroid have TRAb which act as TSH antagonists (blocking antibodies). There is some evidence that these blocking antibodies can cause hypothyroidism particularly in the neonate. With regard to the site of synthesis of TRAb, there is now direct evidence that they are synthesized by thyroid lymphocytes, particularly the lymphocytes in close proximity to thyroid follicular cells. This is consistent with the well established effects of antithyroid treatment (drugs, radioiodine, or surgery) on TRAb levels in addition to their effects on thyroid hormone synthesis. Recent studies using affinity labeling with 125I-labeled TSH have enabled elucidation of the structure of the TSH receptor. TSH receptors in human, porcine, and guinea pig thyroid tissue have a two-chain structure in which the TSH binding site is formed on the outside surface of the cell membrane by a water-soluble A subunit (Mr approximately 50 K). The A subunit is linked by a disulfide bridge and weak noncovalent bonds to the amphiphilic B subunit (Mr approximately 30 K). This subunit, which penetrates the lipid bilayer, probably forms the site for interaction of the receptor with the regulatory subunits of adenylate cyclase.(ABSTRACT TRUNCATED AT 400 WORDS)
556 citations
TL;DR: The new-found ability to generate human antibodies and to evolve their specificities and affinities, ex vivo promises increased the use of this class of molecules in the service of human health.
Abstract: Publisher Summary Combinatorial antibodies will play a significant role in the design of vaccines and new antiviral agents. Antibodies may also provide a way of determining receptor function in vivo and serve as templates for the design of small molecules. Combinatorial antibodies have been shown to provide an accurate functional reflection of the natural response as demonstrated by the ability of cloned antibodies to compete with serum antibodies for binding antigens. Combinatorial antibodies also provide a useful (if somewhat incomplete) guide to the molecular biology of the response. With its ability to provide large numbers of human antibodies directed against a single antigen, the combinatorial approach allows for the rapid assessment of immune dominant as well as neutralizing epitopes in the context of the human response. This information should be utilized in the future to guide the design of more effective vaccines. The new-found ability to generate human antibodies and to evolve their specificities and affinities, ex vivo promises increased the use of this class of molecules in the service of human health.
440 citations
TL;DR: Comparison with 236 rearranged sequences revealed that no more than 24 of these germ‐line sequences could be assigned rearranged counterparts, that some of these were rarely used, and that only about 11 sequences are used frequently, suggesting that the expressed Vχ repertoire is mainly derived from a limited number of segments.
Abstract: From the genomic DNA of a single individual, we have amplified, cloned and sequenced 37 human germ-line V kappa segments. Four of these segments were new. We then compiled a comprehensive directory of all germ-line V kappa segments and identified 50 different sequences with open reading frames. Comparison with 236 rearranged sequences revealed that no more than 24 of these germ-line sequences could be assigned rearranged counterparts, that some of these were rarely used, and that only about 11 sequences are used frequently. This suggests that the expressed V kappa repertoire is mainly derived from a limited number of segments. Most surprisingly, the J kappa-distal region of the locus appears to be rarely used: we could unambiguously assign 162 rearranged sequences to V kappa segments of the J kappa-proximal region, but only 5 to segments of the J kappa-distal region.
405 citations
TL;DR: Eight sequences that have a DN1-like D sequence with two base changes at the same positions were identified, suggesting the likely existence of a new germ line D gene belonging to the DN families.
Abstract: We have examined at the molecular level the CDR3 and adjacent regions in peripheral blood B lymphocytes of normal individuals. A total of 111 sequences (12-28 sequences from six individuals) were obtained after cloning of the polymerase chain reaction-amplified segments into plasmids or phage. The average length of the VDJ joining was 109 nucleotides, with a range from 79 to 151. Approximately 75% of the sequences were in frame when translated into amino acids. Among the JH segments, JH4 was found most frequently (in 52.5% of the sequences), and JH1 and JH2 segments the least frequently (approximately 1% of the clones). A polymorphic JH6 gene with a one-codon deletion accompanied by a base change was present in two of six patients. Preferential breakpoints were found for JH2, JH3, JH4, and JH5, although the breakpoints of JH6 were distributed more heterogenously. In approximately 90% of the cases, significant homology of the D regions with published D sequences was found. Preferential usage of a particular coding frame was observed in in-frame sequences utilizing DA, D21/9, and DM1 segments. However, in general, all coding frames of germline D genes were used to generate CDR3s. Eight sequences that have a DN1-like D sequence with two base changes at the same positions were identified, suggesting the likely existence of a new germ line D gene belonging to the DN families. Using probes specific for a particular CDR3, the frequency of a specific B cell clone in the peripheral blood of normal individuals was estimated to be at most as high as 1/20,000.
377 citations
Journal Article•
TL;DR: Individual H or L chains from a human autoantibody were used to search for other L or H chains that could form antigen-binding fragments, Fab, with the same specificity, raising the possibility that the L chain is critical in defining epitope specificity, even in the presence of completely different D regions and nonidentical VH regions.
Abstract: Individual H or L chains from a human autoantibody were used to search for other L or H chains that could form antigen-binding fragments, Fab, with the same specificity The parent Fab (SP12) exhibits high affinity binding for thyroid peroxidase (TPO), a 107-kDa protein that is the major autoantigen in human autoimmune thyroiditis This autoantibody "roulette," performed by using Ig H and L chain gene libraries expressed in bacteria, increased the frequency of TPO-binding clones in the new libraries However, the frequency was still much lower than would be the case if promiscuous combinations with a variety of H or L chains were compatible with specific Ag binding Nucleotide sequence analysis of the H and L chains of the new TPO-binding clones revealed even more restriction Thus, with the SP12 H chain, all 11 new Fab utilized L chains from the same V kappa 1 family germline gene as SP12 itself Similarly, five of six H chains "captured" by the SP12 L chain were very closely related to the SP12 H chain However, one totally different H chain was isolated: SP46 has a VH region that differs substantially from that of SP12 SP46 also has a distinct D region, uses a different JH, and, unlike SP12, which is an IgG1, belongs to subclass IgG4 The affinities for TPO of SP46 (with the different H chain) and SP120 (which had the least mutated L chain germline gene) were similar to that of SP12 (approximately 10(-10) M) As expected, the SP12 and SP120 Fab, which have the same H chain and closely related L chains, bound to the same domain on TPO However, a similar domain on TPO was recognized by both SP46 and SP12, despite the fact that their V, D, and J regions are quite different This observation raises the possibility that the L chain is critical in defining epitope specificity, even in the presence of completely different D regions and nonidentical VH regions
292 citations