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Purification of the human thyroid peroxidase and its identification as the microsomal antigen involved in autoimmune thyroid diseases.

Human thyroid peroxidase (TPO) has been purified from thyroid microsomes by immunoaffinity chromatography using a monoclonal antibody (mAb) to TPO. The eluted material had a specific activity of 381 U/mg and exhibited a peak in the Soret region. The ratio of A411 to A280 ranged from 0.20 to 0.25. Upon SDS-polyacrylamide gel electrophoresis, the purified enzyme gave two contiguous bands in the 100 kDa region. Further, it has been demonstrated that sera with anti-microsomal autoantibodies from patients presenting Graves' or Hashimoto's thyroiditis diseases were able to bind to purified TPO and to inhibit in a dose-dependent manner the mAb binding to purified TPO. This suggests that TPO is the thyroid antigen termed to date the microsomal antigen.

Antibodies, Monoclonal↗

Relationship of thyroid disease and use of thyroid supplements to breast cancer risk.

An interview study of 1362 breast cancer cases and 1250 controls identified through a multi-center screening program allowed evaluation of effects of thyroid disease and supplementation on breast cancer risk. A previous diagnosis of treated thyroid disease was not associated with an excess risk (RR = 1.0), nor were any specific diagnoses, including hypothyroidism, hyperthyroidism, or goiter. Although based on limited numbers, women with untreated hypothyroidism or goiter had a significantly reduced risk of breast cancer (RR = 0.3, 95% CI 0.1-0.7). Thyroid supplementation for non-disease reasons (primarily weight loss and fertility problems) was associated with a slight elevation in breast cancer risk (RR = 1.2, 95% CI 0.9-1.7), but patterns of risk by duration and latency generally failed to provide evidence of causality. Elevated risks were noted among women who received thyroid medications for fertility problems (RR = 4.2) and among those with a family history of breast cancer (RR = 2.6) or a late age at first childbirth (RR = 2.4), possibly indicating an hormonal interaction.

Adult↗

An improved metaphase index assay for detecting thyroid growth stimulators using FRTL-5 thyroid cells cultured on a microtitre plate.

The metaphase index assay (MIA) for thyroid growth stimulators, as originally described, used FRTL-5 thyroid cells cultured in Bellco culture chambers and glass microscope slides. The metaphases were observed using the nuclear strain aceto-lacto orcein. However the surface properties of the glass proved to be variable and so polystyrene microscope slides were substituted. The aceto-lacto orcein stain was found to be unsuitable for use with polystyrene because of the solvents and mountant used. Therefore combinations of various other nuclear stains and mounting media were tested. The Giemsa stain, which was found to be the most satisfactory, could be applied to FRTL-5 cells maintained on the large variety of plastic supports now available for tissue culture, e.g., 96 well microtitre plates. This permitted the design of an MIA which is much more convenient, robust and economical in its use of clinical samples. The results with seven IgG preparations derived from the sera of patients with a variety of thyroid disorders are presented. In its revised form, the metaphase index assay provides a rapid screening assay for thyroid growth stimulators, such as autoantibodies and TSH.

Azure Stains↗

In vivo stimulation of thyroid hormone release by rabbit antibodies with thyroid stimulating activity raised against the mutated receptor peptide TSH-R-HIS17.

The present study was designed to examine whether thyroid stimulating antibodies (TSAb) against the mutated or non-mutated peptide of human TSH receptor stimulates release of thyroid hormones in mice in vivo. We generated antibodies against mutated TSH receptor peptide TSH-R-HIS17 and non-mutated TSH receptor peptide TSH-R-ASP17 immunizing the peptides to rabbits. We isolated immunoglobulin G (IgG) from these antibodies and injected iv into male ddy mice. Control mice were injected iv with bTSH or TSAb-negative normal rabbit IgG. Serum T3 and T4 concentrations were elevated after the administration of bTSH in a dose-dependent manner. Serum T3 and T4 concentrations were significantly elevated after the injection of IgG from the rabbit antibodies with TSAb activity produced against TSH-R-HIS17. However serum T3 and T4 remained within normal levels after the injection of IgG from the rabbit antibodies without TSAb activity toward TSH-R-ASP17. The present data offer in vivo support of the IgG with TSAb activity interaction with the TSH receptor of the thyroid and raise the possibility that TSAb against mutated human TSH receptor peptide might be contributed to induce thyroid hyperfunction.

Amino Acid Sequence↗

DNA polymerase beta in human thyroid of Graves' disease and thyroid tumors.

We have shown that the level of DNA polymerase beta of rat adrenal cortex is regulated by pituitary trophic hormones and may correlate with their endocrine function. Here we measured DNA polymerase beta activity in human thyroid tissues of various benign and malignant thyroid disorders in order to verify the correlation between DNA polymerase beta activity and endocrine function. In Graves' disease (hyperfunction), the level of DNA polymerase beta per cell was three times higher than in normal thyroid, while in undifferentiated thyroid carcinomas this enzyme level was lower than normal. Furthermore, DNA polymerase beta in the crude extracts of cancer cells showed larger molecular forms, ranging from five to 12S, upon sucrose gradient sedimentation. These observations further support the hypothesis that the activity of DNA polymerase beta correlates, in part, with the functional level of the endocrine organ and with cell differentiation.

Adenoma↗

In situ kinetic analysis of thyroid lymphocyte infiltrate in mice developing experimental autoimmune thyroiditis.

L3T4+ T cells from genetically susceptible mice developing experimental autoimmune thyroiditis (EAT) were shown earlier to proliferate in response to restimulation with mouse thyroglobulin (MTg) in vitro and to mediate the adoptive transfer of EAT, whereas Lyt-2+ cells differentiated in vitro into cells cytotoxic for thyroid monolayers. Leukocyte suspensions from disrupted thyroid glands examined on Days 13-21 after immunization revealed the accumulation of both T cell subsets in the infiltrate at varying ratios. To characterize the in situ kinetics of cellular infiltration in chronic EAT, we extended the observation intervals after immunization to include Days 21 to 42. The leukocytes in thyroid sections were labeled immunohistochemically first with rat monoclonal antibodies to L3T4, Lyt-2, Thy-1, k light chain, or F4/80 macrophage antigen, then with biotinylated anti-rat IgG, utilizing the avidin-biotin-peroxidase technique. Throughout the 21- to 42-day interval, no significant variations were detected in the percentages of L3T4+ subset, but those of Lyt-2+ cells increased and then declined. The shift in the L3T4+:Lyt-2+ ratio, down from 2.4 to 1.6 and then up to 3.0, was directly related to changes in the Lyt-2+ subpopulation. The F4/80+ and B cell populations changed little during this period. These findings illustrate the changing kinetics of T cell subsets in situ in the development and perpetuation of EAT and MTg-immunized mice.

Animals↗

Distribution and functional analysis of B-L/Ia-positive cells in the chicken: expression of B-L/Ia antigens on thyroid epithelial cells in spontaneous autoimmune thyroiditis.

The B-L region of the chicken major histocompatibility complex (MHC), the so-called B-locus, corresponds to the murine H-2 I-region. Using alloantibodies and monoclonal antibodies to B-L we analyzed: (a) the tissue distribution of B-L+ cells, (b) the function of B-L+ cells, and (c) the possible role of B-L+ cells in the development of spontaneous autoimmune thyroiditis (SAT) in Obese strain (OS) chickens. The tissue distribution of B-L+ cells in peripheral blood and various lymphoid and nonlymphoid organs corresponds to what is known for mammals. In the bursa of Fabricius most lymphoid cells and the dendritic cells carry the B-L antigen; B-L+ thymic nurse cells (TNC) first appear on day 17 of embryonic life; chickens possess dendritic B-L+ cells in the skin resembling mammalian Langerhans cells; in addition we found that the microglia is unequivocally B-L+. B-L+ peripheral blood lymphocytes (PBL) were separated with a fluorescence-activated cell sorter. Ten percent of unstimulated PBL and 60% of phytohemagglutinin (PHA) stimulated T-cell blasts are B-L+. In graft-vs-host (GvH) assays B-L- cells were identified as the effector cells. These cells respond to PHA and concanavalin A (Con A), but not to pokeweed mitogen (PWM). B-L+ cells cannot be stimulated by Con A and PHA, but respond to PWM. They possess only a very low activity in GvH assays which can be inhibited by anti-T-cell sera. In OS chickens B-L+/non-B/, non-T and B-L+ T (blasts?) cells are found in the "first line" of mononuclear cell infiltration in the thyroid glands. Most interesting, thyroid epithelial cells--which are normally B-L- -become B-L+ in the neighbourhood of B-L+ infiltrating mononuclear cells. This observation may be of significance for autoantigen presentation and perpetuation in autoimmune thyroiditis. Finally, OS thymuses contain significantly less TNC than normal controls.

Animals↗

Structural features of the autoantigens involved in thyroid autoimmune disease: the thyroid microsomal/microvillar antigen.

The microsomal/microvillar antigen of the human thyroid gland which provokes thyroid autoimmunity was characterised by immunoprecipitation studies. Sera from patients with Hashimoto's thyroiditis, primary myxoedema or Graves' disease containing autoanti-microsomal antibody specifically precipitated a component, which under reducing conditions migrates with a mol. wt of 105,000 on SDS-polyacrylamide gel electrophoresis. This protein was absent in auto- or xeno-anti-thyroglobulin precipitates, which under reducing conditions display four polypeptides of Mr 260,000, 230,000, 180,000 and 142,000. Under non-reducing conditions, the microsomal/microvillar antigen displayed a small shift in mobility to a mol. wt of 117,000 suggesting the presence of intrachain disulphide bonds. In contrast, under these conditions, anti-thyroglobulin precipitated components displaying polypeptides of approx. mol. wts in the region of 240,000-260,000, 170,000-180,000 and 140,000. Absorption of thyroiditis sera on thyroglobulin-Sepharose followed by immunoprecipitation abolished the anti-thyroglobulin components without affecting the binding of the 105,000-dalton polypeptide, if the sera contained antimicrosomal antibody. No comparable material was identified in microsomal membrane preparations prepared from the stomach which is also commonly involved in organ-specific autoimmunity. The 105,000-dalton component does not bind to a Lens culinaris lectin affinity column. We conclude that the epitopes of the microsomal/microvillar antigen are presented on a poorly glycosylated peptide of mol. wt 105,000, which is probably stabilised by intrachain disulphide bonds and which does not share serological reactivity with membrane-bound thyroglobulin.

Antigens↗

Post-partum thyroiditis and goitrous (Hashimoto's) thyroiditis are associated with HLA-DR4.

Using new panels of HLA-DR typing sera, we found an increase in the prevalence of HLA-DR4 in 21 patients with Hashimoto's thyroiditis (57%) and 32 patients with post-partum thyroiditis (53%) compared to controls (21%). Hashimoto's thyroiditis was previously found to be associated with HLA-DR5 and Dw5. We feel that the doubts raised by this study warrant the study of DR antigens in thyroid autoimmune disease using large panels of specific sera.

Diabetes Mellitus, Type 1↗

Increased thyroid volume and frequency of thyroid disorders signs in schoolchildren from nitrate polluted area.

Thyroid volume (ThV) and echogenicity by ultrasound were estimated in 324 schoolchildren (aged between 10 and 13-years) from high nitrate area (HNA) located in agricultural lowland with high nitrate drinking water supply (51-274 mg/l) from shallow wells. The data were compared to children of the same age from low nitrate area (LNA) consisting of 168 children from the neighboring area with very low nitrate (< 2 mg/l) drinking water and of 596 children from the city of Kosice located in a vicinity of LNA and also supplied by low nitrate water. Blood samples were obtained from 315 willing children from HNA and 109 children from LNA and the levels of thyrotropin (TSH), total thyroxine (TT4), free triiodothyronine (FT3) and thyroperoxidase antibodies (anti-TPO) in serum were determined. ThV (mean +/- SE) in 10-year (5.10 +/- 0.14 ml) and 13-year (5.97 +/- 0.11 ml) old children from HNA was significantly higher than that in two groups of respective age from LNA, 4.58 +/- 0.17 (p < 0.02) and 5.23 +/- 0.15 ml (p < 0.05), and from the city of Kosice, 4.77 +/- 0.10 ml (p < 0.05) and 4.87 +/- 0.1 0ml (p < 0.0001). The frequency of hypoechogenicity in HNA was also significantly higher than that in pooled LNA plus Kosice, 13.7% vs. 4.7% (p < 0.01) in 10-year and 10.6% vs. 5.7% (p < 0.03) in 13-year, respectively. The frequency of TSH level in the range of subclinical hypothyroidism (> 4.0 mU/l) in pooled age groups from HNA was 13/324 (4.0%) and that of positive anti-TPO was 8/324 (2.5%), while no case of either increased TSH or positive anti-TPO was found in 109 children from LNA. Finally, no differences in the levels of TT4 and FT3 were found between HNA and LNA. It was concluded that long-term exposure to high nitrate intake by drinking water and home made meals from local products results in increased thyroid volume and increased frequency of signs of subclinical thyroid disorders (thyroid hypoechogenicity by ultrasound, increased TSH level and positive anti-TPO).

Adolescent↗

Isolation of the alligator (Alligator mississippiensis) thyroid hormone receptor alpha and beta transcripts and their responsiveness to thyroid stimulating hormone.

Thyroid hormones (THs) play key regulatory roles in growth, development and metabolism in vertebrates. Modulation of the cellular hormonal response is largely through the activity of two nuclear TH receptors, TRalpha and TRbeta, which act as transcription factors and alter gene expression programs. Little information is available regarding their structure and regulation in reptiles. We have cloned the expressed sequences encoding these two receptors in the American alligator, Alligator mississippiensis. The encoded putative proteins share a high degree of amino acid sequence conservation with other vertebrates, however, both alligator TRs contain putative N-terminal truncations. This phenomenon is shared with the chicken for TRbeta, but not for TRalpha, making this the first demonstration of this type of TRalpha isoform. We measured the steady-state levels of TR transcripts in heart, lung, liver, thyroid, cliterophallus/phallus, and gonad of juvenile alligators 24 and 48 h after injection with thyroid stimulating hormone (TSH). TRalpha transcript levels were increased in the heart, decreased in the lung and cliterophallus/phallus, and unaffected in the liver, thyroid, and gonad. TRbeta transcript levels were increased in the heart, lung, and gonad whereas estrogen receptor alpha transcript levels were elevated by TSH treatment only in the gonad. Modulation of these transcripts in the gonad is consistent with TH playing an important role in this tissue's function since seasonal TH fluctuations coincide with reproductive events. These data demonstrate that alligator tissues are differentially responsive to TSH by regulation of TR expression and provide an important comparative framework among vertebrates.

Alligators and Crocodiles↗

Hashimoto thyroiditis associated with thyroid cancer in adolescent patients.

Three girls had Hashimoto thyroiditis and thyroid cancer. Cervical exploration revealed a follicular carcinoma in one, an oxyphilic cell carcinoma in another, and a papillary carcinoma in the third. These cases add clinical and pathologic data to the previously debated concept that Hashimoto thyroiditis coexists with thyroid carcinoma.

Adenocarcinoma↗

Altered thyroid hormone binding to plasma lipoproteins in the syndrome of resistance to thyroid hormones.

Lipoproteins, especially HDL, are carriers of a small fraction of the thyroid hormones in plasma and participate in the intracellular transport of T4. In previous work we showed that a brief period of hypothyroidism alters the hormone distribution among the lipoproteins, causing a decrease in VLDL and LDL binding despite a relative increase in VLDL and LDL cholesterol, an increase in HDL binding, and a reversal of T4 and T3 binding to the smallest HDL size subgroup. The present study of three patients with thyroid hormone resistance and largely compensated hypothyroidism showed thyroid hormone distribution that differed markedly from both normal and hypothyroid subjects. The most striking difference was a much lower binding of both T4 and T3 to HDL and a much higher binding to LDL. If confirmed in a larger group of patients, this might serve as a marker for thyroid hormone resistance.

Cholesterol↗

The relationship between multiple intrathyroidal involvement in papillary thyroid carcinoma and chronic non-specific thyroiditis.

The relationship between multiple intrathyroidal involvement (MII) and chronic non-specific thyroiditis (CT) was investigated in 69 papillary thyroid carcinoma patients who received a subtotal or total thyroidectomy. The overall incidence of MII in patients with CT and the incidence of MII in the affected lobe of the patients with CT were significantly higher than that without CT (P = 0.0012 and 0.0425, respectively). Because Hashimoto's thyroiditis is believed not to carry the increased risk of associated thyroid malignancy, the high incidence of MII in the affected lobe in the case with CT is postulated to be caused by intraglandular metastases.

Adult↗

Color Doppler measurement of blood flow in the inferior thyroid artery in patients with autoimmune thyroid diseases.

PURPOSE: The aim of the study is to find out whether the measurement of peak systolic velocity in the inferior thyroid artery (ITA) is a valuable parameter to differentiate autoimmune thyroid diseases (hyper-, normo- or hypofunctional) and to evaluate the efficacy of medical treatment. MATERIAL AND METHODS: The ITA of 31 patients (eight with Graves' disease, 23 with subclinical hypothyroidism) was examined with color Doppler and pulsed Doppler. The final diagnosis was obtained by cytology and by hormonal and antibodies assays. The patients were monitorized by ultrasound for a period of 8 months. RESULTS: In all the patients with Graves' disease the peak systolic velocity was always over 150 cm/s, while in other autoimmune thyroiditis the peak systolic velocity was within the normal range, and never exceeding 65 cm/s. In the first group, the measurement taken in the ITA showed also the efficacy of the pharmacological treatment earlier and more reliably than the color Doppler pattern obtained in the parenchyma. CONCLUSIONS: The color Doppler measurement of the ITA seems to be a promising technique with low-cost and easy approach. In our experience, the color Doppler of the ITA could have a clinical role in the differential diagnosis of diffuse thyroid diseases and in the follow-up of the Graves' disease during medical treatment.

Adolescent↗

Computer-assisted image analysis of small cell lymphoma of the thyroid gland. Comparison of nuclear parameters of small lymphocytes in lymphomas and Hashimoto's thyroiditis.

The nuclear parameters of the small lymphocytes in nine cases of small cell lymphomas of the thyroid gland and 17 cases of Hashimoto's thyroiditis were assessed by computer-assisted image analysis. The nuclear area, maximal, minimal and averaged Ferret diameters, perimeter, regularity factor and elongation factor were gauged. Statistically, the nuclear area was ascertained to be the optimum descriptor discriminating between small neoplastic and reactive lymphocytes. Application of a novel variable, combining a nuclear area cut-off value of 14 microm2 with a nuclear averaged Ferret diameter cut-off value of 4.5 micron, allows for the distinction - with a high degree of sensitivity and specificity - between small neoplastic lymphocytes in thyroidal lymphomas and the reactive lymphocytes in Hashimoto's thyroiditis.

Adult↗

Phenothiazine-induced increase in thyroid autoantigens and costimulatory molecules on thyroid cells: a pathophysiological mechanism for drug-induced autoimmunity?

We have previously demonstrated (J Immunol 1995; 154:3593) that MHC class II antigens can be induced on thyroid epithelial cells (TEC) by alimemazine, a member of the phenothiazine group. Although this expression of MHC class II antigens on TEC confers the theoretical ability to behave as antigen-presenting cells (APC), the simultaneous expression of self antigens and co-receptor(s) must also occur for efficient presentation of self antigens. Therefore, we investigated whether alimemazine applied at pharmacologic doses would modify the expression of thyroid antigens, and simultaneously, the expression of intercellular adhesion molecule-1 (ICAM-1), B7, and LFA-1 co-receptors in human TEC in culture. Using polymerase chain reaction (PCR) amplification and Northern blot analysis, we showed that alimemazine induces increases in thyroglobulin (Tg) and thyroid-stimulating hormone receptor (TSH-R) cDNA, within the first 2 h following its addition. This phenomenon is followed 48 h later by an increase of Tg and TSH-R protein expression on the surface of TEC. Furthermore, increases in the expression of ICAM-1 and B7 co-receptors were concomitantly observed. These results suggest that alimemazine, a drug currently used in paediatrics, could play a role in the induction and perpetuation of thyroid autoimmune disorders by transforming TEC into functional APC.

Antigens↗

Galectin-3, a marker of well-differentiated thyroid carcinoma, is expressed in thyroid nodules with cytological atypia.

AIMS: The distribution of galectin-3, a widely recognized marker of well-differentiated thyroid carcinoma, was investigated in 95 thyroid lesions including nodules with foci of cytoarchitectural atypia. METHODS AND RESULTS: Twenty-eight papillary carcinomas, five follicular carcinomas, one Hurthle cell carcinoma, three poorly differentiated carcinomas, one anaplastic carcinoma, 25 nodular hyperplasias and 27 follicular adenomas, including nodules with atypical features, three neoplasms of undetermined malignant potential and two thyroiditis cases were examined. By immunohistochemistry, galectin-3 was consistently found in carcinomas; otherwise benign nodules exhibited galectin-3-positive clusters of cells with poorly developed features of differentiated carcinoma (mainly of papillary type) such as nuclear chromatin clearing, nuclear clefting, pseudoinclusions, which, in each case, were not histologically sufficient to warrant a definitive diagnosis of malignancy. In other nodules galectin-3 staining was negative. The latter were either clearly benign or showed constantly a minor degree of chromatin clearing and of other atypical features when compared with galectin-3-positive cases. CONCLUSIONS: Galectin-3, a reliable marker of differentiated thyroid carcinoma as confirmed in our series of malignant neoplasms, appears expressed in nodules with an overall benign appearance but with focal areas suspicious for malignancy. The significance of such findings needs to be further investigated.

Antigens, Differentiation↗