Human Thyroglobulin Research Articles

Fine-needle biopsy of parathyroid adenomas.

High-resolution real-time sonography was performed in 15 cases of clinically and chemically suspected primary hyperparathyroidism and in 20 patients with different thyroid nodules. The suspected enlarged parathyroid glands and the thyroid nodules were percutaneously punctured under sonographic control. Concentrations of parathyroid hormone, human thyroglobulin, and human calcitonin were measured in the aspirate, and immunocytology was performed. The mean concentration of the aspirated parathyroid hormone in the parathyroid glands was 4,013.6 pmol/l +/- 4,519 (SD) as compared with 14.9 pmol/l +/- 8.7 in the thyroid nodules. Thyroglobulin was present in the aspirated fluid of parathyroid adenomas located behind the thyroid (mean +/- SD, 398.1 ng/ml +/- 317). In comparison, the aspirated thyroglobulin from the thyroid nodules averaged 9,689.7 ng/ml +/- 3,732. Immunocytology for parathyroid hormone was positive in 14 of the 15 biopsied specimens. Of 15 patients who were scanned for suspected hyperparathyroidism, six had concomitant thyroid nodules. It is concluded that the measurement of high concentrations of parathyroid hormone in the aspirate from a cervical mass, with sonographic control of needle position and/or positive immunocytology provides absolute localization of parathyroid tissue.

Characterization of a hormonogenic domain from human thyroglobulin

A polypeptide domain of molecular mass near 22 kDa was purified from CNBr-digest of iodine poor human thyroglobulin (hTgb). This fragment represents the N-terminal part of the hTgb molecule and consequently contains the preferential hormonogenic tyrosine ‘acceptor’ of the protein. This fragment could correspond to the non-iodinated and unreduced form of the thyroxinyl-containing 26 kDa peptide previously purified from reduced and iodinated hTgb. This 22 kDa fragment is capable by itself, i.e. independently of the remaining hTgb molecule, of synthesizing thyroxine with a high efficiency after in vitro iodination. Its study should constitute a valuable way to identify at least one of the hormonogenic tyrosine ‘donor’ residues of hTgb.

Human Monoclonals from Antigen-Specific Selection of B Lymphocytes and Transformation by EBV

Hybridoma technology has made it possible to prepare monoclonal antibodies with the use of murine lymphocytes. Attempts to extend this technology to the human level, however, have met with difficulties. A method has been developed for making human monoclonal antibodies of predetermined specificity. Biotinylated antigens (human thyroglobulin or tetanus toxoid) were incubated with human B lymphocytes from peripheral blood. The lymphocytes to which the antigens bound were selected by fluorescence-activated cell sorting. Positively selected (high fluorescence) and negatively selected (low fluorescence) cells were then transformed with Epstein-Barr virus (EBV) and grown in microculture wells. All wells from the positively selected fraction produced antigen-specific antibody (95 to 1800 nanogram-equivalents per milliliter), whereas fewer than 6% of the wells from negatively selected fraction made any detectable antibody (less than 10 nanogram-equivalents per milliliter). When the positively selected EBV-transformed cells were cultured in limiting dilution, clones were obtained that made antigen-specific monoclonal antibodies. By this method, monoclonal antibodies to both foreign antigens and autoantigens can be prepared from the normal human B-cell repertoire.

Glycosylation of human thyroglobulin and characterization by lectin affinity electrophoresis

Thyrolgobulin, a 660 kDa glycoprotein, is the major product of protein synthesis in the thyroid gland. It has been suggested that modifications of thyroglobulin glycosylation occur in various thyroid disorders. In order to study possible changes in glycosylation of tissue thyroglobulin associated with thyroid disease, we have developed a lectin affinity electrophoresis system which allows characterization of small (less than 1 μg) quantities of thyroglobulin. Human thyroglobulin was extracted and purified. Agarose gels were cast containing concanavalin A, Ricinus communis agglutinin, L-phytohaemagglutinin and pokeweed mitogen at various concentrations. Purified human thyroglobulin was serially diluted, loaded onto lectin gels and electrophoresed. Concanavalin A, R. communis agglutinin and phytohaemagglutinin all bound thyroglobulin in a concentration-dependent manner. Pokeweed mitogen did not bind thyroglobulin. Purified thyroglobulin was treated with neuraminidase and endoglycosidase H. Two-dimensional immunoelectrophoresis revealed the migration of thyroglobulin to be modified by neuraminidase but not by endoglycosidase H. Lectin affinity electrophoresis of purified human thyroglobulin with and without enzyme treatment indicated the presence of: (a) oligomannose structures as shown by concanavalin a reactivity and modification by endoglycosidase H, and (b) complex oligosaccharides as shown by affinity for R. communis agglutinin and modification by neuraminidase. These structures are in keeping with the proposed patterns of glycosylation of human thyroglobulin and indicate suitability of the method for characterizing the glycosylation of small quantities of thyroglobulin

Monoclonal antithyroglobulin antibodies derived from immunizations of mice with human eye muscle and thyroid membranes.

A mouse hybridoma clone secreting an immunoglobulin M monoclonal antibody (K-5-4), which reacted with human thyroglobulin (Tg), was obtained from spleen cells of mice immunized with crude membranes of human eye muscle tissues (Em). Its binding to Tg could be inhibited by another monoclonal anti-Tg (F1-11-1) derived from spleen cells of mice immunized with human thyroid cell membranes, but K-5-4 did not inhibit the binding of F1-11-1 to Tg. This finding suggests that K-5-4 may react with a site on the Tg molecule which is susceptible to conformational changes, such as that induced by binding of another anti-Tg antibody at another site on Tg. K-5-4 reacted with human, mouse, rat, guinea pig, bovine, and porcine Tg. Binding and immunohistological staining experiments failed to detect binding of K-5-4 to Em tissue. The very low frequency of one Tg-reacting hybridoma from 6 X 10(8) spleen cells fused after Em immunization contrasts with the relative ease with which monoclonal anti-Tgs were generated from spleen cells of mice immunized with crude human thyroid membranes. In the latter case, 1 anti-Tg hybridoma was generated for every 100,000 spleen cells fused, and an extensive library of monoclonal anti-Tgs was collected. Some of these antibodies were specific for human Tg only, while others cross-reacted with Tg of other animal species. None had the species reativity pattern of K-5-4. The anti-Tgs were used to affinity purify human Tg directly from supernatant of thyroid homogenate; the purified Tg was, in turn, used to affinity purify human polyclonal but monospecific anti-Tg directly from serum of patients in a simple and rapid procedure. We conclude that the monoclonal anti-Tgs are useful reagents in isolating and purifying Tg and anti-Tg.

Idiotypic analysis of five xenogeneic antisera to anti-human thyroglobulin monoclonal antibodies

Five xenogeneic anti-idiotypic antisera (anti-id) were produced against individual BALB/c-derived monoclonal antibodies (mAb) which bound to different peptidic determinants on the human thyroglobulin molecule (Tg). Idiotypic analysis performed using sensitive radioimmunoassays revealed that: (1) the anti-id highly precipitated their homologous ligands; (2) two anti-id displayed minor cross-reactivities with one or two heterologous mAb; (3) each unlabelled homologous mAb was able to inhibit the idiotype binding of the corresponding anti-id; (4) no significant inhibition of homologous idiotype binding was observed with large excess of heterologous mAb; (5) efficient inhibition of mAb binding to Tg was observed only when homologous anti-id served as inhibitor. The data support the conclusion that xenogeneic anti-id may detect on their corresponding ligands individual idiotypic specificities that can be located at the mAb-combining site. Such reagents may constitute appropriate probes for further studies on anti-Tg autoimmunity.

Production of murine monoclonal antibodies against human thyroglobulin using an in vitro immunization procedure in serum-free medium

A serum-free in vitro immunization method for the generation of hybridomas producing specific antibodies to an antigen is described. The method was tested with human thyroglobulin as antigen. The serum-free medium used (Yssel et al., 1984) consisted of Iscove's modification of Dulbecco's modified Eagle's medium, supplemented with albumin, transferrin, insulin, ethanolamine and linoleic, oleic and palmitic acids. An optimal response was obtained when splenocytes from BALB/c mice were cultured for 3 days in the presence of 1.5 nM thyroglobulin and thymocyte-conditioned medium prior to fusion with SP2/0 myeloma cells and seeding of the fused cells in microtitre plates. The frequency of positive wells, defined as the number of wells secreting anti-(thyroglobulin) antibodies/number of viable cells used for the fusion, was 1.6 × 10 −6 ± 0.25 × 10 −6 (mean ± SD; n = 4). Eight stable clones producing anti-(thyroglobulin) antibodies were isolated. One clone (3D12) produced antibodies reacting only with human thyroglobulin. The antibodies produced by the other clones reacted with human, murine and porcine thyroglobulins. Seven of the clones produced antibodies of the IgM class and one clone produced IgG. The specificity of 3D12 (IgM) for human thyroglobulin and the absence of any reactivity with murine thyroglobulin provides evidence for a primary response of splenocytes in culture to the presence of an antigen.

Activation of cytotoxic T cells and effector cells in experimental autoimmune thyroiditis by shared determinants of mouse and human thyroglobulins

Previous studies have shown that T cells from genetically susceptible mice developing experimental autoimmune thyroiditis (EAT) proliferate in response to restimulation with mouse thyroglobulin (MTg) in vitro and differentiate into cells cytotoxic for syngeneic thyroid monolayers. To examine further the effector cells involved in pathogenesis and the determinants on MTg responsible for their activation, spleen cells (SC) and lymph node cells (LNC) from mice immunized with MTg or human (H) Tg. and adjuvant (complete Freund's adjuvant (CFA) or lipopolysaccharide (LPS)) were cultured in vitro with MTg or HTg. Control cultures were incubated with concanavalin A (Con A) or purified protein derivative (PPD). The in vitro-activated cells which proliferated in response to MTg, HTg, or Con A adoptively transferred thyroiditis to normal recipients, whereas cells transferred directly without in vitro culture were very ineffective. The capacity to transfer EAT was abrogated by irradiation (1500 R), and SC from CFA-immunized control mice which responded in vitro to PPD stimulation did not transfer thyroiditis. The serum titers of MTg autoantibodies were uniformly low and were not correlated with severity of disease. The localization of EAT-effector (precursor) cells depended upon the site of immunization; they were found in the spleens after inguinal (subcutaneous) or systemic (intravenous) immunizations, but were present in the popliteal lymph nodes after hind footpad injections. Both homologous MTg and heterologous HTg functioned as in vivo sensitizing antigen and in vitro activating antigen for each other; such cultured cells transferred thyroiditis in vivo and became cytotoxic for thyroid monolayers in vitro. These findings show that shared determinants are autoantigenic and thyroiditogenic, and support the hypothesis that EAT-effector cells responsible for initiating thyroid damage include cytotoxic cells.

Suppression of in vitro human antithyroglobulin antibody secretion by private and cross-reactive anti-idiotypic antibodies

This is a report on suppression of in vitro human antithyroglobulin antibody secretion in Epstein-Barr (EB) virus-transformed B lymphocytes by private and cross-reactive anti-idiotypic antibodies. Two polyclonal anti-idiotypic antibodies, private anti-Yo-Id and cross-reactive anti-Uc-Id antibodies, were raised in rabbits. Human IgG, λ antithyroglobulin antibody-producing cell line (Yo3) and human IgG-producing cell line (Yo5), which did not contain the antithyroglobulin activity were established by EB virus transformation of peripheral lymphocytes of patient Yo with chronic thyroiditis. Binding of anti-Yo-Id and anti-Uc-Id antibodies to IgG F(ab′) 2 antithyroglobulin derived from patient Yo was significantly inhibited by human thyroglobulin and affinity-purified IgG antithyroglobulin of Yo3 cell line. Both private anti-Yo-Id and cross-reactive anti-Uc-Id antibodies suppressed the antithyroglobulin antibody secretion in Yo3 cell line. But these anti-idiotypic antibodies did not suppress the IgG secretion in Yo5 cell line. These results suggested that interactions between idiotype and anti-idiotype may play a role in the immune system of human chronic thyroiditis.

Serum Thyroglobulin and Thyroid Cancer

Thyroglobulin is a large glycosylated protein (MW 660000 daltons) which is synthesised and secreted by the thyroid cell into the follicular lumen. In the thyroglobulin molecule the iodotyrosines undergo oxidative coupling to form the iodothyronines thyroxine (T4) and triiodothyronine (T3). Production of thyroid hormones results from micropinocytosis of iodinated thyroglobulin from the colloid into the thyroid cell and its hydrolysis by lysosymal enzymes. The resulting T4 and T3 diffuse across the basal membrane into the blood stream whilst iodotyrosines are de-iodinated and the iodine recycled. Thyroglobulin thus acts as the template for thyroid hormone synthesis and a storage form of inactive thyroid hormones and iodine. Thyroid function and activity are under the positive control of thyrotrophin (TSH) which stimulates many steps of thyroid cell metabolism including protein synthesis, iodothyronine synthesis, thyroglobulin secretion into the follicular lumen, and replacement of micropinocytosis of thyroglobulin by massive colloid macropinocytosis [1]. The action of TSH on the thyroid cell is mediated via adenylate cyclase activation and recent work has shown that transcription of the thyroglobulin gene is regulated by cyclic AMP [2]. For many years thyroglobulin was thought to be confined to the thyroid gland but the sensitive radioimmunoassays now available have demonstrated that the protein is unequivocally present in the peripheral circulation of man. It is assumed that some of the protein escapes the potent lysosymal mechanism of the thyroid gland and crosses the basal membrane with the thyroid hormones to appear intact in the circulation. The first radioimmunoassays for thyroglobulin in human, monkey and rat serum were reported in 1967 [3]. A wide variation in the normal range of human serum thyroglobulin levels has been reported but a recent international co-operative study evaluating serum thyroglobulin standards has shown this to be due to remarkable interlaboratory variations [4]. No age-related variation of thyroglobulin levels has been reported (except in children) although several studies have reported higher values in women than men, higher values in pregnant women at delivery than non-pregnant controls, and elevated thyroglobulin levels in cord blood from newborn infants. Serum concentrations in man appear to be closely regulated within a narrow range suggesting a mechanism for controlling thyroglobulin release. Thyroid hormone administration suppresses thyroglobulin levels and after injection of thyrotrophin releasing hormone, TSH or thyroid stimulating immunoglobulins, the levels of serum thyroglobulin rise. The data support the concept that thyroglobulin is a normal secretory product of the thyroid gland under the control of TSH, but the mechanism of this 'secretion1, rather than simple diffusion, remains unknown [3].

Methylation and expression of the human thyroglobulin gene

The DNA methylation pattern at the 5′ end of the human thyroglobulin gene has been determined in different tissues. Out of the four HpaII MspI sites (5′-CCGG-3′) present in this region, three were found to be non-methylated in thyroid DNA, while full methylation was observed in liver, salivary gland and sperm DNA. This demethylation therefore correlates with expression of the thyroglobulin gene. However, all four sites were found to be non-methylated in placental DNA, regardless of the activity of the gene.

Genetic control of the production of anti-thyroid hormone antibodies in mice immunized with human thyroglobulin.

Various strains of mice with different H-2 and Igh-I allotypes were immunized with human thyroglobulin (HTg) and genetic control of the production of anti-thyroid hormone antibodies was examined. Anti-HTg antisera obtained from different strains of mice were examined for the presence of anti-thyroid hormone antibodies. At least one Ir-gene which controls the production of anti-T4 antibodies by immunization with HTg was identified in the I-A subregion. The presence of Ir-genes outside the H-2 was also suspected. Concerning the production of anti-T3 antibodies, only three strains (B10.A, C3H.SW, BALB/cJ) were high responders and therefore we were unable to identify the Ir-gene for them. These results indicate the presence of Ir-gene which controls the immune response in mice against thyroid hormone and suggest that the anti-thyroid hormone antibodies observed in various thyroidal and non-thyroidal disorders could be anti-HTg antibodies. The significance of genetic control of the production of anti-thyroid hormone antibodies in mice immunized with HTg is discussed.

A murine monoclonal antibody derived from the immunization of human thyroglobulin reacts equally with L-thyroxine and reverse triiodo-L-thyronine and has a unique idiotype.

Murine monoclonal antibody (mAb 16.3.2) to human thyroglobulin which bound equally to various thyroglobulins derived from nine species was obtained from the fusion of C3H/He spleen cells sensitized with normal human thyroglobulin. Characterization of mAb 16.3.2 revealed that both L-thyroxine (T4) and reverse triiodo-L-thyroxine (rT3) were very efficient in the competitive binding inhibition test and that a molar ratio between T4 and rT3 needed for 50% inhibition of binding to immunized thyroglobulin was about 1:1. Further studies on the idiotype of mAb 16.3.2 using both binding and competitive binding inhibition tests showed that mAb 16.3.2 had a unique idiotype not cross-reacting with other monoclonal antibodies to thyroglobulins. Therefore, a possible explanation offered was that mAb 16.3.2 was endowed with a unique idiotype to be regulated by a distinct idiotype network from those of other mAbs.

Concentrations of free thyroxine, total thyroxine, and total triiodothyronine in rabbits immunized with human thyroglobulin.

Levels of serum free thyroxine, total triiodothyronine and total thyroxine were measured in two rabbits (TG-1, TG-2) which had been immunized with human thyroglobulin and produced both anti-thyroglobulin and anti-thyroid hormone antibodies. Despite the presence of both anti-triiodothyronine and -thyroxine antibodies, levels of serum free thyroxine in serial sera obtained from a rabbit (TG-1) stayed within normal range up to 249 days after the first immunization, whereas in the other rabbit (TG-2), serum free thyroxine concentrations of serial sera declined after three immunizations and stayed at lower levels than normal range up to 249 days. Extraction of serial sera from both rabbits disclosed the presence of larger amounts of triiodothyronine and thyroxine in immune sera than preimmune sera. From these results, it is concluded that the presence of anti-thyroid hormone antibodies per se can reduce serum level of free thyroxine in rabbits.

Antigen Presentation in Human Autoimmune Thyroid Disease

Monocyte/macrophage function in patients with autoimmune thyroiditis was investigated by their presentation of two distinct antigens; sheep red blood cells (SRBC) and human thyroglobulin (hTg) using in vitro systems designed for antibody induction. Purified peripheral blood monocyte/macrophages were primed by prefeeding with antigen for 60 min at 37 degrees C, washed, and co-cultured with autologous lymphocytes under a variety of incubation conditions. The most successful system employed 5% monocyte/macrophages with autologous T-B cells in the presence of the mitogen Staphylococcus aureus and B-cell differentiating factors. Under such conditions the anti-SRBC plaque-forming cell (PFC) response was amplified equally (approximately 10-fold) by SRBC-fed monocyte/macrophages in normal controls and patients with autoimmune thyroiditis rendered euthyroid with thyroxine replacement. hTg-fed monocyte/macrophages induced a 4-fold increase in anti-hTg PFC in selected patients with autoimmune thyroiditis examined under similar conditions (mean 36 +/- 3 PFC per 10(6) T-B cells). These data indicated that antigen processing by monocyte/macrophages was normal in patients with autoimmune thyroid disease.

Radioimmunoscintigraphy with Anti-Thyroglobulin Monoclonal Antibodies

Monoclonal mouse antibodies to human thyroglobulin were conjugated to the cyclic dianhydride of DTPA. After radiolabelling with 111In this compound was injected into nude mice bearing various human thyroid carcinomas. Repeated imaging studies were carried out 15 min to 50 h after tracer administration. In both papillary and undifferentiated thyroid carcinoma no significant uptake of radiolabelled anti-hTG-MAb was observed.

The human thyroglobulin gene is over 300 kb long and contains introns of up to 64 kb.

Thyroglobulin (Tg), the precursor of thyroid hormones, is a 660.000 Da dimeric glycoprotein synthesized exclusively in the thyroid gland. We have cloned the human thyroglobulin gene from cosmid and phage libraries and constructed a complete restriction map. The gene encodes an 8.7 kb mRNA, covers at least 300 kb DNA and contains at least 37 exons separated by large introns of up to 64 kb. A striking difference in structure between the 5' and 3' part of the gene suggests that it is composed of two evolutionarily different regions. The first 30 kb DNA encode 3 kb of the mRNA, yielding an exon:intron ratio of 1:10, whereas the remaining 270 kb encodes 5.7 kb of the mRNA with an exon:intron ratio of 1:47. In thyroid cells, the Tg gene is not rearranged and nuclear RNA homologous with sequences internal to the 64 kb intron is present, suggesting that the Tg gene is transcribed as a 300 kb RNA.

Immunohistochemical localization of a melanin concentrating hormone-like peptide in the rat brain

Using antisera generated in rabbits against salmon melanin concentrating hormone (MCH) coupled to human thyroglobulin, the distribution of MCH-like immunoreactivity was mapped throughout the rat central nervous system. The distribution of MCH-like immunoreactivity in rat brain is unique and different from the distribution of other neuropeptides. MCH-like immunoreactive perikarya and fibers are predominant in the posterior hypothalamic area, mostly in the medial forebrain bundle—lateral hypothalamic area subzona incerta and the perifornical area. Cell bodies are located mainly in the medial forebrain bundle and in proximity to well defined hypothalamic nuclei. Fibers are seen throughout the rat brain in all neocortical areas, the neostriatum and the amygdala, in the diencephalon in most hypothalamic nuclei, the habenula, the mamillary body and very dense in the medial forebrain bundle and just ventral to the zona incerta (“subzona incerta”). In the mesencephalon there are fibers in the central gray; in the pons-medulla fibers are contained in the dorsal and ventral parabrachial nuclei; in the tegmental area ventral to the fourth ventricle; in the spinal trigeminal area, the substantia gelatinosa and the reticular nuclei. In the spinal cord there are more fibers in the dorsal than in the ventral horn. The posterior pituitary also contained few MCH-like fibers. It is suggested that a peptide similar, but not identical, to salmon MCH is present in the rat central nervous system.

Mapping of human thyroglobulin gene on the long arm of chromosome 8 by in situ hybridization.

We report the structural organization of a segment of the human thyroglobulin gene, located 70kb from the 3' end of the gene, containing the exons 8 and 9 starting from the 3' end. Selected probes from this region have been used for the chromosomal mapping of the thyroglobulin gene by in situ hybridization techniques. Only one site in the human haploid karyotype is labeled with the genomic DNA probes. Twenty percent of the grains are localized on the long arm of chromosome 8, mostly in the subregion q-2-23 q-2-24 of the long arm of chromosome 8. The localization of the autoradiographic grains suggests a subregional assignment of the human thyroglobulin gene locus to 8q 2-23 or 8q 2-24.

Heterogeneity of 125I-labeled human thyroglobulin preparations

This study was undertaken to evaluate the heterogeneity and stability of 125I-labeled human thyroglobulin (Tg) tracers. Tg, labeled with 125I by either a Chloramine T (CT) or a Glucose Oxidase/Lactoperoxidase (GO) method, showed considerable heterogeneity of labeled components, the relative proportions of which were a function of the Tg preparation and the iodination method used. The four largest components had apparent molecular weights as follows: A = 1200000 Da; B = 670000 Da; C = 530000 Da and D = 290000 Da. Both B and C were immunoactive. B was considered to be 19S Tg. A non-specific binding difference, (NSBΔ) between nonhuman matrices used for diluting standards and human sera was found for the partly immunoactive aggregate component A, (5–20%) and the nonimmunoactive component D, (20–50%), but was minimally present for components B and C (<5%). The [ 125I]19S Tg(B), stored at -70°C, showed rapid spontaneous decomposition with time (50% lost by 8 days), with generation of C, D and iodide. The loss of B was related to specific activity and was least in GO labels. 125I labeling of Tg by GO produced tracers with better immunoactivity, stability and lower NSBΔ than comparative CT tracers. Definitive purification and repurification of [ 125I]Tg tracers before use is necessary in order to remove degradation products with the potential to compromise the accuracy and specificity of serum Tg radioimmunoassay (RIA) measurement.