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.
518 citations
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...[111–289], antibody–antigen [290–411] and receptor–...
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...research performed using Biacore instruments [111–931]....
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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 demonstrated that the in-cell PCR library generated human anti-TPO scFvs that retained the VH/VL pairing found in vivo and that the different epitope specificities defined by these scFVS overlapped with those found in the sera of patients with autoimmune thyroid disease.
Abstract: In an attempt to explore the natural variable heavy and light chain (V H /V L ) pairing of autoantibodies involved in Graves’ disease, we constructed a phage-displayed Ab library obtained by in-cell PCR of thyroid-infiltrating cells. We report here the molecular cloning and characterization of human single-chain fragment variable regions (scFv) specific for thyroid peroxidase (TPO) generated from this library. On the basis of the nucleotide sequences, three different scFvs were obtained (ICA1, ICB7, and ICA5). All were encoded by genes derived from the VH1 and Vλ1 gene families. Using BIACORE for epitope mapping and kinetic analysis, we showed that these scFvs exhibited high affinity ( K d = 1 nM) for TPO and recognized three different epitopes. The biological relevance of these scFvs as compared with serum anti-TPO autoantibodies was assessed by competition studies. Sera from all the 29 Graves’ disease patients tested were able to strongly inhibit (60–100%) the binding of the 3 scFvs to TPO. These data demonstrate that the in-cell PCR library generated human anti-TPO scFvs that retained the V H /V L pairing found in vivo and that the different epitope specificities defined by these scFvs overlapped with those found in the sera of patients with autoimmune thyroid disease.
27 citations
TL;DR: This study significantly augments the small database on this category of autoantibodies in general and expresses the proteins for three TPO-specific F(ab) for which sufficient protein could be expressed for competition studies and bound less well to denatured TPO as to native TPO.
27 citations
TL;DR: This finding, albeit from a single patient, questions previous observations of serum TgPOAb prepared by affinity chromatography and provides powerful evidence against shared, cross-reactive epitopes on 2 major thyroid autoantigens.
Abstract: Autoantibodies (Ab) to thyroglobulin (Tg) and to thyroid peroxidase (TPO) are reported to share common epitopes, and an assay for bispecific TgPOAb has been developed that may distinguish between different clinical presentations of thyroid autoimmunity. We sought to clone TgPOAb from an Ig gene combinatorial library constructed from B cells infiltrating the thyroid of a patient with TgPOAb. As described for isolating serum TgPOAb, we panned the phage display library by alternating from Tg- to TPO-coated ELISA wells. After panning, the library was enriched for TgPO-binding phage. Of 526 clones tested for expressed Ab, most were negative; 3 clones were specific for Tg, and 5 clones specifically recognized TPO. Antibody from a single clone, encoded by a non-Tg, non-TPO Ig heavy chain gene, bound both Tg and TPO (TgPO activity). However, this antibody also bound equally well to nonthyroid antigens. In conclusion, enrichment for Tg- and TPO-binding phage was largely attributable to phage specific for either Tg or TPO. This finding, albeit from a single patient, questions previous observations of serum TgPOAb prepared by affinity chromatography. Combined with the isolation of a polyreactive monoclonal antibody, our data provide powerful evidence against shared, cross-reactive epitopes on 2 major thyroid autoantigens.
27 citations
TL;DR: Three combinatorial libraries were constructed from unpurified, CD19+, and antithyroid peroxidase (anti-TPO) B cells extracted from thyroid tissue of Graves’ disease patients, demonstrating that in vivo rearrangement can be obtained from a random Combinatorial library.
Abstract: Three combinatorial libraries were constructed from unpurified, CD19+, and antithyroid peroxidase (anti-TPO) B cells extracted from thyroid tissue of Graves’ disease patients. Fifteen of the 41 randomly derived anti-TPO single chain variable region fragments (scFvs), showed VH1–3/Vλ1–51 or VH1–69/Vλ1–40 heavy/light chain pairing similar to that obtained with TPO-specific scFv derived from an in-cell library. One VH1–3/Vλ1–51 scFv, A16, showed exactly the same nucleotide sequence as in-cell scFv ICB7, demonstrating that in vivo rearrangement can be obtained from a random combinatorial library. The majority of the scFvs used a heavy chain gene derived from the VH1–3 gene segment, whereas the light chain gene segments used were more heterogeneous, with dominance of the Vκ1–39 and Vλ1–51 gene segments. The anti-TPO scFvs showed high affinities to TPO, with values between 0.77 and 12.3 nm, and defined seven antigenic regions on the TPO molecule. The anti-TPO fragments, particularly VH1–3/Vλ1–51 randomly associ...
26 citations
"Human monoclonal thyroglobulin auto..." refers result in this paper
...This approach is similar to that described for epitopic mapping of TPOAb (33, 34 )....
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TL;DR: It is suggested that distinct peptides are recognized by Tg autoantibodies from patients with different thyroid diseases.
Abstract: 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.
24 citations