Human monoclonal thyroglobulin autoantibodies: epitopes and immunoglobulin genes.
01 Oct 2004-The Journal of Clinical Endocrinology and Metabolism (Endocrine Society)-Vol. 89, Iss: 10, pp 5116-5123
TL;DR: The TgAb panel provides novel information regarding the repertoire of H chain genes encoding human TgAbs as well as the relationship between the H chains and the epitopes recognized on this major thyroid autoantigen.
Abstract: Autoantibodies to thyroglobulin (TgAbs) are common markers of thyroid autoimmunity, but relatively few human monoclonal TgAbs have been described. From a panel of 64 human monoclonal TgAbs (isolated from a thyroid-disease derived combinatorial Ig gene library), we selected seven with unique genetic features for detailed characterization. These TgAbs preferentially recognize native (not denatured) Tg, like serum autoantibodies. Most have high affinities for Tg (dissociation constant 10(-10) to 10(-9) m). Their light (L) chain Ig genes are not unusual, but four of the five heavy (H) chain genes are new. Moreover, one H chain belongs to the small VH2 family, not previously reported for autoantibodies to Tg or thyroid peroxidase. The TgAbs inhibit the binding to Tg of the thyroid donor's serum autoantibodies, indicating epitopic overlap. Competition analysis (surface plasmon resonance) shows that the TgAbs recognize overlapping epitopes in an immunodominant region on the Tg dimer ( approximately 660 kDa). Two major and several minor epitopic regions were defined, each associated with a particular H + L chain combination. In conclusion, our TgAb panel provides novel information regarding the repertoire of H chain genes encoding human TgAbs as well as the relationship between the H chains and the epitopes recognized on this major thyroid autoantigen.
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Abstract: In the year 2003 there was a 17% increase in the number of publications citing work performed using optical biosensor technology compared with the previous year. We collated the 962 total papers for 2003, identified the geographical regions where the work was performed, highlighted the instrument types on which it was carried out, and segregated the papers by biological system. In this overview, we spotlight 13 papers that should be on everyone's 'must read' list for 2003 and provide examples of how to identify and interpret high-quality biosensor data. Although we still find that the literature is replete with poorly performed experiments, over-interpreted results and a general lack of understanding of data analysis, we are optimistic that these shortcomings will be addressed as biosensor technology continues to mature.
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TL;DR: The goal of inducing self-tolerance to prevent autoimmune thyroid disease will require accurate prediction of at-risk individuals together with an antigen-specific, not blanket, therapeutic approach.
Abstract: Thyroid autoimmunity involves loss of tolerance to thyroid proteins in genetically susceptible individuals in association with environmental factors. In central tolerance, intrathymic autoantigen presentation deletes immature T cells with high affinity for autoantigen-derived peptides. Regulatory T cells provide an alternative mechanism to silence autoimmune T cells in the periphery. The TSH receptor (TSHR), thyroid peroxidase (TPO), and thyroglobulin (Tg) have unusual properties ("immunogenicity") that contribute to breaking tolerance, including size, abundance, membrane association, glycosylation, and polymorphisms. Insight into loss of tolerance to thyroid proteins comes from spontaneous and induced animal models: 1) intrathymic expression controls self-tolerance to the TSHR, not TPO or Tg; 2) regulatory T cells are not involved in TSHR self-tolerance and instead control the balance between Graves' disease and thyroiditis; 3) breaking TSHR tolerance involves contributions from major histocompatibility complex molecules (humans and induced mouse models), TSHR polymorphism(s) (humans), and alternative splicing (mice); 4) loss of tolerance to Tg before TPO indicates that greater Tg immunogenicity vs TPO dominates central tolerance expectations; 5) tolerance is induced by thyroid autoantigen administration before autoimmunity is established; 6) interferon-α therapy for hepatitis C infection enhances thyroid autoimmunity in patients with intact immunity; Graves' disease developing after T-cell depletion reflects reconstitution autoimmunity; and 7) most environmental factors (including excess iodine) "reveal," but do not induce, thyroid autoimmunity. Micro-organisms likely exert their effects via bystander stimulation. Finally, no single mechanism explains the loss of tolerance to thyroid proteins. The goal of inducing self-tolerance to prevent autoimmune thyroid disease will require accurate prediction of at-risk individuals together with an antigen-specific, not blanket, therapeutic approach.
160 citations
TL;DR: The pattern of Tg recognition is similar when HT patients are compared to GD and NTMG to PTC patients and differs when AITD are compared with non-AITD patients.
Abstract: Context: Thyroglobulin (Tg) epitopes of serum Tg autoantibodies (TgAb) have been characterized using inhibition of Tg binding by human monoclonal TgAb in autoimmune thyroid diseases (AITD) [Hashimoto’s thyroiditis (HT) and Graves’ disease (GD)] but not in non-AITD [nontoxic multinodular goiter (NTMG) and papillary thyroid carcinoma (PTC)]. Objective: Our objective was to compare Tg epitopes of serum TgAb from patients with AITD, non-AITD, and PTC associated with histological thyroiditis (PTC-T) using inhibition of Tg binding by four recombinant human TgAb-Fab (epitopic regions A–D). Design: Inhibition of Tg binding of 24 HT, 25 GD, 19 NTMG, 15 PTC, and 25 PTC-T TgAb-positive sera by each TgAb-Fab was evaluated in ELISA. Inhibition by the pool of the four TgAb-Fab was evaluated using labeled Tg. Results: Levels of inhibition were different for TgAb-Fab regions A (P = 0.001), B (0.007), and D (0.011). Inhibition by region A TgAb-Fab was significantly higher in HT, GD, and PTC-T than in NTMG and PTC patients...
71 citations
TL;DR: Iodine-induced thyroid autoimmunity is related to TgAb and the unmasking of a cryptic epitope on Tg contributes to this relationship in humans.
Abstract: Context: The mechanisms linking thyroid autoimmunity and iodine use in humans are unknown. Objective: Our aim was to correlate iodine intake, thyroid autoimmunity, and recognition of thyroglobulin (Tg) epitopes after implementation of iodine prophylaxis. Setting: The general community living in an Italian village was evaluated. Main Outcome Measures: Thyroglobulin autoantibodies (TgAb), thyroperoxidase autoantibodies (TPOAb), and urinary iodine excretion were assessed in 906 iodized salt users (IS-users) and 389 nonusers (IS-nonusers). Ultrasound (US) was performed to identify thyroid hypoechogenicity, suggestive of Hashimoto thyroiditis (HT). TgAb epitope pattern in 16 IS-users and 17 IS-nonusers was evaluated by an inhibition binding assay to Tg, using human monoclonal TgAb-Fab directed to A, B, C, and D epitopes on Tg. Results: Median urinary iodine excretion was slightly higher in IS-users than in IS-nonusers (112.0 μg/L vs 86.5 μg/L; P < .01). TgAb, and not TPOAb, was more frequent in IS-users (18.9%...
67 citations
TL;DR: In papillary thyroid carcinoma, LT on histology must be carefully searched for because it is frequently associated with TgAb and therefore mistakenly low or undetectable Tg, which can be underestimated in patients with T g autoantibodies (TgAb).
Abstract: Context: Serum thyroglobulin (Tg), the marker of residual tumor in papillary thyroid carcinoma, can be underestimated in patients with Tg autoantibodies (TgAb) TgAb are due to a coexistent lymphocytic thyroiditis (LT) or the papillary thyroid carcinoma per se TgAb assays are highly discordant Design: We evaluated 141 patients with a clinical diagnosis of nodular thyroid disease, 32 of Hashimoto's thyroiditis, and four of Graves' disease, who underwent total thyroidectomy for an associated papillary thyroid carcinoma Patients were classified as papillary thyroid carcinoma-lymphocytic thyroiditis (PTC-T) and papillary thyroid carcinoma (PTC) according to the presence or absence of LT on histology Tg was measured before thyroid remnant ablation, when it is expectedly detectable, by an immunometric assay (IMA) and TgAb by three noncompetitive IMA and three competitive radioimmunoassays (RIA) The number of lymphocytes was compared with TgAb concentration Results: Seventy-two of 177 patients (407%) were
59 citations
References
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TL;DR: It is suggested that in individuals with no overt clinical or biochemical thyroid abnormalities but with Tg Abs, the finding that these TgAbs recognize particular immunodominant clusters may be utilized to predict full-blown thyroid disorders.
Abstract: Thyroglobulin antibodies (TgAbs) are typically found in autoimmune thyroid diseases and, more rarely, in nonautoimmunc thyroid diseases and healthy subjects. To determine whether TgAbs associated with different conditions recognize different epitopes on the thyroglobulin molecule, we studied 28 patients with Hashimoto's thyroiditis, 30 with Graves' disease, 21 with thyroid carcinoma, 18 with nontoxic goiter, and 25 healthy subjects. All patients were selected for the presence of TgAbs; 4/25 healthy subjects also had TgAbs. The sera were assayed for the their ability to inhibit the binding of monoclonal antibodies to thyroglobulin in an EL1SA assay. We found that: 1) TgAbs in Hashimoto's patients preferentially recognized three clusters of epitopes (II, III and typically VI). with no difference between the goitrous and the atrophic variants; 2) TgAbs in Graves' patients were directed toward cluster II, with no difference between the presence or the absence of ophthalmopathy; 3) TgAbs in thyroid carcinoma p...
47 citations
TL;DR: The studies show that TgAAb are directed to two major conformational epitopes on the Tg molecule and that the proportion of T gAAb directed to these epitopes in apparently healthy blood donors and that in patients with AITD appear to be different.
Abstract: A panel of human monoclonal thyroglobulin (Tg) autoantibodies (TgAAb) has been used to analyze autoantigenic determinants on human Tg and to investigate the relationship between variable (V) region gene sequences and epitope specificity Two monoclonal TgAAb bound to the same (or closely related) epitope on Tg, and these were defined as type I TgAAb Three other monoclonals bound to a different site and were defined as type II TgAAb Inhibition studies with mixtures of type I and type II monoclonal TgAAb (Fab)2 preparations indicated that a mixture of the (Fab)2s almost completely inhibited (> 75%) labeled Tg binding to intact TgAAb in the sera of apparently healthy blood donors and patients with autoimmune thyroid disease (AITD) Type I TgAAb predominated in apparently healthy blood donors' sera, whereas type II TgAAb predominated in AITD sera Analysis of V region gene sequences of the TgAAb indicated that a range of light chain and heavy chain genes from different gene families was used Furthermore, the same germline genes that are used by TgAAb are also well represented in the genes coding for other self- and nonself-reactive antibodies No homology in terms of light chain and heavy chain gene families, germline gene usage, or complementarity determining region sequences was observed in TgAAb directed to the same or closely related epitopes Our studies show that TgAAb are directed to two major conformational epitopes on the Tg molecule and that the proportion of TgAAb directed to these epitopes in apparently healthy blood donors and that in patients with AITD appear to be different TgAAb derived from different germline genes and with different complementarity determining region sequences can display similar epitope specificity, and this indicates that AAb directed to the same or a closely related epitope show considerable heterogeneity at the molecular level
46 citations
TL;DR: No difference in TPOAutoantibody epitopes was observed in this cross-sectional study of hypothyroid and euthyroid individuals, demonstrating that the majority of TPO autoantibodies in both groups recognize the TPO immunodominant domain.
Abstract: Human monoclonal immunoglobulin G-class autoantibodies to thyroid peroxidase (TPO), expressed as recombinant F(ab), are powerful tools for analyzing the individual components of polyclonal serum TPO autoantibodies. Four TPO-specific F(ab) interact with epitopes in two closely related domains (A and B) in the immunodominant region on TPO. In the present study, these TPO F(ab) were used to compete for serum autoantibody binding to [125I]TPO to determine the "epitopic fingerprints" in two groups of carefully controlled individuals. All individuals (14 hypothyroid and 32 euthyroid) were elderly women (60-71 yr old) with similar genetic and environmental backgrounds as well as comparable levels of serum TPO autoantibodies. Using the pool of four F(ab), serum TPO autoantibody binding was inhibited to the same extent (approximately 90%) in hypothyroid and euthyroid individuals, demonstrating that the majority of TPO autoantibodies in both groups recognize the TPO immunodominant domain. When tested individually, ...
36 citations
TL;DR: A remarkable lack of spreading of B cell epitopes during a state of perturbation of the immune system over a period of 1 yr is indicates and their constancy over time suggests that TPO autoantibody fingerprints may be inherited.
Abstract: It is not known whether epitopes recognized by autoantibodies in an individual remain constant or change over time, especially during perturbations of the humoral immune response. To address this question, we studied the epitopic profile ("fingerprint") of autoantibodies to thyroid peroxidase (TPO) in the sera of 19 women during the postpartum period. Fingerprints were determined in competition studies using 4 recombinant F(ab). At delivery and at 3 time intervals over the subsequent 9-12 months, the pool of F(ab) inhibited autoantibody binding to TPO by 80-100%, consistent with the definition by these F(ab) of a TPO immunodominant region (A1, A2, B1, and B2 domains). Despite a wide spectrum among individuals, the TPO epitopic fingerprints for all 19 women were relatively unchanged throughout the postpartum period. Fingerprint constancy occurred regardless of fluctuations in serum TPO autoantibody levels. When assessed numerically as a ratio of inhibition by the A domain F(ab) to inhibition by the B domain F(ab), the A/B domain ratios in individual women ranged from 0.2 (predominantly B domain) to more than 3.0 (predominantly A domain). However, for each individual woman, the A/B epitopic ratio was conserved throughout the study interval. Our TPO autoantibody epitopic fingerprint data have potential implications for understanding the humoral autoimmune response in man. First, the present study indicates a remarkable lack of spreading of B cell epitopes during a state of perturbation of the immune system over a period of 1 yr. Second, and perhaps more important, despite marked variations in TPO epitopic profiles among different individuals, their constancy over time suggests that TPO autoantibody fingerprints may be inherited.
35 citations
TL;DR: Serum antiphospholipid antibodies associated with thrombosis may be encoded by either germline or only slightly mutated variable-region genes.
Abstract: Objective. To investigate the origins of antiphospholipid antibodies associated with thrombosis and other disorders that are found in patients with systemic lupus erythematosus and primary antiphospholipid syndrome (APS).
Methods. Characterization, idiotypic study, and nucleotide sequencing of a human monoclonal antiphospholipid antibody generated from a patient with primary APS. Identification of the germline genes from which the antibody is derived.
Results. A human monoclonal antibody, BH1, was generated. This antibody has ligand-binding properties that closely resemble those of the serum antiphospholipid antibodies found in our patient and in other individuals with APS: it recognizes negatively charged phospholipids, and has lupus anticoagulant activity; it does not bind to neutral phospholipids, or to singlestranded or double-stranded DNA. The relevance of BH1 to the patient's serum antibodies is supported by our idiotypic studies. BH1 is encoded by a new germline VH gene, and by a λ light chain gene that displays >99% homology with the VλIII.1 germline gene.
Conclusion. Serum antiphospholipid antibodies associated with thrombosis may be encoded by either germline or only slightly mutated variable-region genes.
34 citations