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Iodine is an essential element required for diverse physiological functions, including thyroid hormone production. Beside thyroid, other organs take up iodide via the sodium/iodide symporter (NIS), such as stomach and salivary and mammary glands. In addition to its secretion into milk, iodide has been suggested to offer an antioxidant protection in the three mentioned tissues, along with a recycling role via salivary glands and stomach secretion of iodide into their respective fluids to avoid kidney clearance. Several studies have linked iodine deficiency (ID), which is still a global problem, to functional alterations and pathologies of those organs. In thyroid, ID is known to activate a TSH-independent microvascular response. We have thus hypothesized that those organs could also activate a vascular response when facing ID. In this work, we show that acute ID increases vascular endothelial factor (VEGF) expression in salivary glands, stomach, and lactating and virgin mammary glands, transiently leading to increased blood perfusion in salivary and mammary glands. This VEGF up-regulation was shown to depend on non-mitochondrial ROS and the mechanistic target of rapamycin (mTOR) in two breast cell lines: MCF7 and MCF12A. Because X-rays are able to activate angiogenic processes, we hypothesized that they could further increase ID-induced response. The combined exposure to ID and to 3 Gy of X-rays indeed led to additive effects on ROS production and VEGF mRNA expression, but in MCF12A cells only. Our results suggest that ROS induced by radiations with or without iodide come at least partly from mitochondria in this cell line. In conclusion, ID induces a transient microvascular response in all three organs studied as it does in thyroid. This microvascular response might be a common reaction of NIS-expressing organs to try to improve iodide supply, but, in the case of stomach and salivary glands, might also be a mechanism to increase iodide recycling and spare it for the, (BIFA - Sciences biomédicales et pharmaceutiques) -- UCL, 2018

CONTEXT: Polyalanine tract variations in transcription factors have been identified for a wide spectrum of developmental disorders. The thyroid transcription factor forkhead factor E1 (FOXE1) contains a polymorphic polyalanine tract with 12-22 alanines. Single-nucleotide polymorphisms (SNP) close to this locus are associated with papillary thyroid cancer (PTC), and a strong linkage disequilibrium block extends across this region. OBJECTIVE: The objective of the study was to assess whether the FOXE1 polyalanine repeat region was associated with PTC and to assess the effect of polyalanine repeat region variants on protein expression, DNA binding, and transcriptional function on FOXE1-responsive promoters. DESIGN: This was a case-control study. SETTING: The study was conducted at a tertiary referral hospital. PATIENTS AND METHODS: The FOXE1 polyalanine repeat region and tag SNP were genotyped in 70 PTC, with a replication in a further 92 PTC, and compared with genotypes in 5767 healthy controls (including 5667 samples from the Wellcome Trust Case Control Consortium). In vitro studies were performed to examine the protein expression, DNA binding, and transcriptional function for FOXE1 variants of different polyalanine tract lengths. RESULTS: All the genotyped SNP were in tight linkage disequilibrium, including the FOXE1 polyalanine repeat region. We confirmed the strong association of rs1867277 with PTC (overall P = 1 × 10(-7), odds ratio 1.84, confidence interval 1.31-2.57). rs1867277 was in tight linkage disequilibrium with the FOXE1 polyalanine repeat region (r(2) = 0.95). FOXE1(16Ala) was associated with PTC with an odds ratio of 2.23 (confidence interval 1.42-3.50; P = 0.0005). Functional studies in vitro showed that FOXE1(16Ala) was transcriptionally impaired compared with FOXE1(14Ala), which was not due to differences in protein expression or DNA binding. CONCLUSIONS: We have confirmed the previous association of FOXE1 with PTC. Our data suggest that the coding p

BACKGROUND/AIM: Thyroid associated orbitopathy (TAO) and Graves' disease (GD) have an autoimmune pathogenesis, possibly related to the thyrotropin receptor (TSHR). The aim of this study was to determine whether TSHR immunoreactivity is correlated with disease severity or serum TSHR antibody (TRAB) levels. METHODS: Orbital tissues from 30 patients with TAO were compared with those of 20 patients with strabismus and four with non-thyroid orbital inflammation. TSHR was detected by immunohistochemistry and TRAB were measured by radioreceptor assay. RESULTS: No TSHR immunoreactivity was detected in the 24 control orbital tissues, whereas in all TAO biopsies elongated fibroblast-like cells, expressing TSHR, were present. These cells were located between the muscle cells, which were separated by oedema in the acute phase but fibrous tissue in the chronic phase of disease. Semi-thin sections showed numerous mast cells present in the chronic phase and in close contact with adipocytes. The number of TSHR immunostained cells was high in early disease, decreased with disease duration, and was positively correlated with TRAB levels at the onset of TAO. CONCLUSION: TSHR immunoreactivity was demonstrated specifically in TAO orbits which highlights the importance of TRAB early in the pathogenesis., Journal Article, info:eu-repo/semantics/published

Pretibial myxedema (PTM), mainly characterized by the accumulation of glycosaminoglycans in the dermis and subcutaneous tissue, is an extrathyroidal manifestation of autoimmune Graves' disease (GD), almost always associated with Graves' ophthalmopathy (GO). The thyrotropin receptor (TSH-R) has been proposed as the common target antigen in GD, GO and PTM, with evidence for receptor transcripts and/or protein in these locations. The aim of this study has been to investigate whether receptor protein is present in the pretibial tissues. Skin biopsies were obtained from two patients with PTM and two normal subjects without thyroid disease. A portion of each sample was fixed to produce semi-thin sections for Toluidine Blue or Periodic Acid Schiff (PAS) staining. The remainder was snap frozen to generate cryostat sections for immunohistochemical analysis using three monoclonal antibodies against TSH-R. In the skin from the two patients suffering from PTM, the dermis was infiltrated by inflammatory cells (lymphocytes, B cells, macrophages, mast cells) and adipocytes. The collagen fibers were dissociated by edema and by the accumulation of a PAS-positive material. Immunodetection of TSH-R produced positive staining on cells localized in the dermis, beneath the epidermis or close to the hypodermis. These cells were elongated and resembled fibroblasts. No immunoreactivity was observed in the dermis from control patients without thyroid disease. In conclusion, we have evidence for TSH-R immunoreactivity in the pretibium of patients with GD, GO and PTM. Further studies are needed to unambiguously identify the positive cells and determine whether the reactivity is due to the receptor itself or to a cross-reacting protein., Case Reports, Journal Article, info:eu-repo/semantics/published

An infant girl was born at 37 weeks gestation and found to be clinically thyrotoxic at 9 months of age. Thyroid autoantibodies were negative, and thyroid function failed to normalize with medical treatment. The patient underwent a total thyroidectomy. DNA obtained from her thyroid gland and leukocytes was analyzed for thyrotropin receptor (TSHR) mutations using single strand conformation polymorphism and direct sequencing. A mobility shift of polymerase chain reaction (PCR)-amplified DNA was detected on single strand conformation polymorphism gel. Direct sequencing identified a novel point mutation in the fifth transmembrane domain of the TSH receptor at codon 597 (GTC to CTC), resulting in the amino acid substitution of leucine for valine. The mutation was heterozygous and germline, and was not identified in DNA from either of her parents. Expression of the V597L mutant is transiently transfected COS 7 cells displayed increased constitutive cyclic adenosine monophosphate (cAMP) production compared with the wild-type receptor. The mutant is expressed at very low levels on the surface of COS cells, and its response to TSH is marginal., Case Reports, Journal Article, Research Support, Non-U.S. Gov't, info:eu-repo/semantics/published

In previous studies we have transferred thyroiditis to naive BALB/c and NOD mice with human thyrotropin (TSH) receptor (TSHR)-primed splenocytes. Because the TSHR has been implicated in the pathogenesis of thyroid eye disease (TED) we have examined the orbits of recipients of TSHR-primed T cells, generated using a TSHR fusion protein or by genetic immunization. In the NOD mice, 25 of 26 animals treated with TSHR-primed T cells developed thyroiditis with considerable follicular destruction, numerous activated and CD8+ T cells, and immunoreactivity for IFN-gamma. Thyroxine levels were reduced. Thyroiditis was not induced in controls. None of the NOD animals developed any orbital pathology. Thirty-five BALB/c mice received TSHR-primed spleen cells. Thyroiditis was induced in 60-100% and comprised activated T cells, B cells, and immunoreactivity for IL-4 and IL-10. Autoantibodies to the receptor were induced, including TSH binding inhibiting Igs. A total of 17 of 25 BALB/c orbits displayed changes consisting of accumulation of adipose tissue, edema caused by periodic acid Schiff-positive material, dissociation of the muscle fibers, the presence of TSHR immunoreactivity, and infiltration by lymphocytes and mast cells. No orbital changes or thyroiditis were observed in control BALB/c mice. We have induced orbital pathology having many parallels with human TED, only in BALB/c mice, suggesting that a Th2 autoimmune response to the TSHR may be a prerequisite for the development of TED., Journal Article, Research Support, Non-U.S. Gov't, info:eu-repo/semantics/published

Thyroid eye disease (TED) has an autoimmune etiology, but the nature of the autoantigen that is the target of the initiating event remains unknown. A number of candidates have been proposed based on Western blotting, library screening, and deduction from sequence similarity. A strong favorite is the thyrotropin receptor (TSHR), which is the target of the thyroid stimulating antibodies (TSAB) of Graves' disease (GD). We have recently demonstrated TSHR transcripts in orbital adipose tissue from a patient with TED by Northern blot, transcripts in normal adipose tissue being at the limit of detection. We have shown that the transcripts are translated into protein by immunohistochemical analysis using two monoclonal antibodies to the TSHR generated by genetic immunization. TSHR immunoreactivity is associated with elongated cells with the appearance of a fibroblast, often adjacent to clusters of adipocytes, in orbital biopsies from patients with TED but not in strabismus or pseudotumor biopsies. In animal studies, we have transferred thyroiditis to naive BALBc and NOD mice, using T cells primed to the human TSHR, either using the receptor expressed as a bacterial fusion protein or by genetic immunization. The BALBc develop a Th2-type response to the receptor, but the NOD a Th1-type with thyrocyte destruction. Orbital pathology, edema, infiltration by mast cells and lymphocytes, and adipose accumulation was also induced in 68% of the BALBc but none of the NOD mice. Together these data indicate that the preadipocyte expresses the TSHR and that a Th2 autoimmune response to the receptor may be an initiating event in TED., Journal Article, Review, info:eu-repo/semantics/published

The generation of Abs recognizing the native structure of the human thyrotropin receptor (hTSHR) has been difficult because there is currently no method allowing the purification of correctly folded Ag in the amounts required by classical immunization protocols. The majority of Abs made against the hTSHR react preferentially with denatured molecules. We report that a humoral response against the native hTSHR, compatible with mAb production, is elicited in mice by immunization with a DNA construct encoding the receptor. BALB/c mice were inoculated in the anterior tibialis muscle with 100 microg of plasmid DNA harboring the hTSHR cDNA. Eleven weeks after the first injection, 10 mice of 14 showed by FACS analysis a strong IgG response against the hTSHR expressed at the surface of Chinese hamster ovary cells. A clear TSH-binding inhibiting Ig and thyrotropin-blocking Ab activity (competition with TSH binding and TSH activity, respectively) was demonstrated in the majority of sera tested. One serum exhibited a clear stimulating activity. Despite the maintenance of normal circulating free T4 levels in all mice, these bioactivities persisted until 18 wk, in which mice were sacrificed, their thyroids were examined histologically, and spleens from two animals were used for mAb production. All mice displayed a severe lymphocytic infiltration of their thyroids, composed mostly of activated B cells. Three mAbs were produced against conformational epitopes of the hTSHR. We conclude that genetic immunization is an efficient method of generating Abs recognizing the native structure of the hTSHR and a new way of inducing thyroiditis in mice murine., Journal Article, Research Support, Non-U.S. Gov't, Communication lors de l'European thyroid association à Athens en 1997, Communication lors de l'Endocrine society à New Orleans en 1998, info:eu-repo/semantics/published

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A number of proteins, many of them enzymes, i.e. glutamic acid decarboxylase (GAD), carboxypeptidase H, 37-40 K tyrosine phosphatase (ICA512, IA2/IA2 beta), have been proposed as islet autoantigens involved in the pathogenesis of IDDM. Until recently, progress in their characterization has been impeded by the inaccessibility of the human pancreas, resulting in many of them being cloned from animal or non-islet sources. Carboxypeptidase H, one of these enzymes, has been cloned and sequenced from human brain and from rat islets but not from human islets. In this study, we describe the production of a human islet cDNA library and the cloning of islet CPH from it. Since CPH clones were also detected in a human thyroid library, we have sequenced CPH from these two endocrine tissue libraries and compared them to the known brain sequence. The sequences from islets and thyroid were identical and differed from brain only in the absence of a second ATG in the predicted 5'non-coding region. Northern blot analysis revealed the presence of an identical 2.5 kb transcript in human islets, thyroid and brain. The confirmation of the existence of a single isoform of CPH expressed in brain and endocrine tissues simplifies future experiments to elucidate the role of CPH as autoantigen., Journal Article, Research Support, Non-U.S. Gov't, info:eu-repo/semantics/published

In this paper we report the isolation of a self-reactive cytotoxic gamma delta T cell line, 158RE.2, that originates from the T lymphocyte population infiltrating the thyroid gland of a patient with Graves' disease. Functional data using this cell line demonstrate that gamma delta T cells expanded in the thyroid tissue specifically recognize a ligand expressed by thyroid epithelial cells and cell lines of endocrine epithelial origin. The TCR expressed by these gamma delta T cells--V gamma I/V delta 5--is unusual in peripheral blood lymphocytes, and its specificity is clearly different from that observed in a high percentage of gamma delta T cells from PBL, which express the common TCR V gamma 9/V delta 2. The V gamma I/V delta 5 receptor is involved in the recognition of the ligand expressed by the thyroid cells, but not in the NK-like activity also displayed by 158RE.2. These cells express CD8 alpha alpha dimers, which participate in the thyroid ligand recognition but not in the NK-like activity. The epithelial cell recognition is not restricted by classical MHC class I or class II molecules, although the CD8 alpha alpha participation in the recognition suggests the involvement of nonclassical MHC molecules. These are the first data to be presented on self-reacting gamma delta T cells in human epithelium., Journal Article, Research Support, Non-U.S. Gov't, info:eu-repo/semantics/published

In previous studies we have induced TSH binding-inhibiting Igs and thyroiditis in BALB/c mice and thyroiditis alone in NOD mice immunized with the extracellular domain of the human TSH receptor produced as a maltose-binding protein fusion in bacteria (MBP-ECD). In this study, our aim was to determine whether thyroiditis can be transferred to syngeneic naive recipients with in vivo and in vitro primed spleen cells. Groups of 6-week-old female BALB/c and NOD mice were immunized ip with MBP-ECD in an adjuvant of alum plus attenuated Bordetella pertussis toxin, on days 0 (100 micrograms), 14, 28, and 35 (50 micrograms). These mice (in vivo primed) and nontreated age- and sex-matched controls were killed on day 43, and their spleens and thyroids were removed, the latter to verify the induction of thyroiditis in the antigen-treated mice. Splenocytes were disrupted mechanically and cultured at 3 x 10(6)/ml in RPMI supplemented with 20 micrograms/ml MBP-ECD for 48-64 h. After this in vitro priming, some of the splenocytes received no further treatment, but a portion was fractionated into a CD4+-enriched population. Groups of 6-week-old female BALB/c and NOD mice were immunized into the tail vein with 100-200 microliters PBS containing approximately 10(5)-10(7) unfractionated T cells (both in vivo primed and not) and CD4+-enriched (in vivo primed) splenocyte populations. The animals were killed 16 days later, and their thyroids were examined histologically and by immunohistochemistry. In addition, levels of antibody to the MBP-ECD priming antigen were assessed by enzyme-linked immunosorbent assay in the antigen- and spleen-treated mice. In the donor animals, in vivo priming resulted in an extensive lymphocytic infiltration of the thyroids in both BALB/c and NOD mice and follicular destruction in the latter. There was no evidence of thyroiditis in all 9 BALB/c mice and all 4 NOD mice who received unfractionated T cells from mice that had not been primed in vivo. In contrast, Journal Article, Research Support, Non-U.S. Gov't, info:eu-repo/semantics/published

We describe a patient with monoclonal gammopathy who subsequently developed thyrotoxicosis, pretibial myxedema and thyroid-associated ophthalmopathy. The pathogenesis of thyrotoxicosis in Graves' disease is due to the presence of autoantibodies that mimic the action of thyrotropin (TSH), called thyroid-stimulating antibodies (TS-ab); these antibodies may or may not inhibit the binding of TSH to the receptor (thyroid-binding inhibiting immunoglobulin, TBII). The patient's immunoglobulins were TS-ab positive and TBII negative when measured on CHO cells expressing the human TSH receptor. The pathogenetic link between the thyroid, orbit and skin is yet to be established but several candidate shared antigens have been proposed, including the TSH receptor itself. The monoclonal immunoglobulins were in evidence before the symptoms of pretibial myxedema, thyrotoxicosis and ophthalmopathy. In addition, the patient had no autoantibodies to thyroglobulin or thyroperoxidase, which are classic markers of thyroid autoimmunity. This combination led us to postulate that the monoclonal gammopathy could be the cause of all the observed pathology. One method to test this hypothesis would be to show that the monoclonal immunoglobulin is a TS-ab. Various methods were used to separate the monoclonal from the polyclonal components of the patient's serum. Preparative isoelectric focusing enabled us to obtain fractions containing only the monoclonal (as revealed by polyacrylamide gel electrophoresis), which were devoid of TS-ab activity (measured as cAMP accumulation in CHO cells expressing the human TSH receptor in a hypotonic bioassay). Subsequently, different oligoclonal fractions were shown to have varying degrees of TS-ab activity, with one fraction having faint biological activity and able to recognize a recombinant TSH receptor preparation in a Western blot. In conclusion, the monoclonal antibody does not seem to be responsible for the thyrotoxicosis, pretibial myxedema and ophthalmo, Case Reports, Journal Article, Research Support, Non-U.S. Gov't, info:eu-repo/semantics/published

OBJECTIVES: Metastatic thyroid carcinoma rarely provokes hyperthyroidism. We describe here the main characteristics of this association observed in a 48-year-old woman. CASE REPORT: The patient had presented signs of hyperthyroidism for one year. Clinical examination revealed severe Graves' disease with nodular goiter. Presurgery investigations demonstrated thyroid carcinoma and lung metastasis. Thyroid stimulating immunoglobulins were elevated. After total thyroidectomy, hyperthyroidism persisted, indicating the functional nature of the lung metastases. DISCUSSION: The effect of thyroid stimulating immunoglobulins on the clinical course of thyroid carcinoma and distant metastases remains unknown., Case Reports, English Abstract, Journal Article, Review, SCOPUS: ar.j, info:eu-repo/semantics/published

Thyroperoxidase is a membrane-bound, heme-containing enzyme which catalyses iodination of thyroglobulin and coupling of resulting iodotyrosines to produce thyroid hormone. In addition to the full length molecule of 933 amino acids (TPO1), Northern blotting and sequencing have revealed several shorter transcripts. The most abundant is a species lacking 171 nucleotides in which the alternative splicing results in the deletion of codons 533-590 in exon 10 (TPO2). Evidence for TPO2 transcripts being translated into a protein is lacking, but in Western blots TPO invariably appears as a doublet of 110 and 105 kDa. In the present study we have produced two recombinant fusion proteins for: (i) the 57 amino acids which are spliced out in TPO2 and (ii) for the 20 amino acids which bridge the splice site (10 amino acids on both sides). Both recombinant fragments have been produced in the pMAL-cRI vector as a maltose-binding protein (MBP) fusion, permitting their purification from a bacterial lysate on an amylose column. Rabbits have been immunized by intradermal injection of 500 micrograms of fusion protein, initially in complete Freund's adjuvant followed by two boosts, at 2-week intervals, in incomplete Freund's adjuvant. The resulting high titre immune sera (IS) were reactive with the relevant immunising antigens, when tested by ELISA. Depletion of each serum by passage through an MBP-CNBr Sepharose column allowed purification of antibodies against the relevant peptides, as demonstrated by ELISA with the appropriate fusion protein and MBP. This demonstrates that we have produced specific polyclonal antibodies for the 57 amino acids unique to TPO1 and for the amino acid segment bridging the splice site, found in TPO2. These polyclonal antibodies were used in Western blotting experiments with normal and Graves' thyroid membranes, in reducing and non-reducing conditions. Monoclonal 47/C21 which recognises a linear epitope (amino acids residues 710-722) common to TPO1 and TPO2, Journal Article, Research Support, Non-U.S. Gov't, info:eu-repo/semantics/published

The thyrotropin receptor (TSHR) has been used as an example to illustrate how disease may be the consequence of: 1. Modifications or inappropriate production of the natural ligand. 2. Production of abnormal agonists or antagonists such as autoantibodies. 3. Modifications in receptor structure resulting in constitutive activation or the absence of activation following ligand binding. 4. Changes in the cellular machinery which transduces the signal from the receptor to the cytoplasmic or nuclear endpoint target. This chapter concentrates on mechanisms (2) and (3). Since the cloning of the TSHR it has been shown that approximately 50% of cases of toxic adenoma can be explained by somatic point mutations in the nucleotide sequence of the receptor gene which causes single amino acid substitutions. The resulting modified TSHR structure constitutively activates adenylate cyclase (via Gs), intracellular cAMP levels are increased and, since cAMP controls both growth and function of the human thyrocyte clonal expansion of the mutated cell ensues. Similarly, activating mutations of the TSH receptor gene in the germline are responsible for hereditary hyperthyroidism with goitre, which is transmitted in the autosomal dominant mode. Changes in receptor primary structure, i.e. a modified autoantigen, do not seem to be responsible for the escape from tolerance which must precede production of thyroid stimulating antibodies (TSAB) which cause hyperthyroid Graves' disease and thyroid blocking antibodies (TBAB) which are responsible for some cases of hypothyroid idiopathic myxoedema. The eukaryotic expression of wild-type, experimentally mutated and chimeric TSHR has enabled some progress in delineating the residues involved in binding TSH, TSAB and TBAB. All three ligands bind numerous discontinuous residues in the extracellular domain of the receptor. The difference between the bioactivity of TSAB and TBAB cannot be explained completely by different binding sites on the receptor. Su, Journal Article, Review, info:eu-repo/semantics/published

Journal Article, Research Support, Non-U.S. Gov't, Review, FLWIN, info:eu-repo/semantics/published

The thyroid gland is the target of several autoimmune diseases. Specific thyroid proteins have been identified as autoantigens associated with these diseases (e.g. thyroperoxidase, thyroglobulin and the thyrotrophin (TSH) receptor). In this paper, we report that the serum of a patient suffering from Hashimoto's thyroiditis, autoimmune gastritis and rheumatoid arthritis was able to inhibit the chronic TSH-induced I- uptake of dog thyrocytes in culture, even at a 1:1000-fold dilution, without affecting their 86Rb+ uptake. This blocking activity is rare as 147 sera (from patients positive for antibodies to the thyroid microsomes and the gastric parietal cell antigen, patients with Sjögren's syndrome, patients with a high titre of microsomal antibodies and low or negative for antibodies to thyroperoxidase, and patients with a high titre of microsomal antibodies and frank hypothyroidism) were negative when tested for their ability to inhibit I- uptake. Subsequently we tested 20 murine monoclonal antibodies previously obtained by immunizing mice with a crude human thyroid membrane preparation, which were all negative when tested against thyroglobulin and thyroperoxidase. One of the monoclonal antibodies displayed a 50% inhibition of the chronic TSH-induced 125I- uptake of dog thyrocytes without affecting the 86Rb+ uptake of the cells. Immunoglobulins purified from the ascite fluid by affinity chromatography on a protein A cellulose column had the same characteristics. Taken together, the data suggest that thyroidal 125I- uptake can be inhibited by antibodies, that autoantibodies in the patient's serum are most probably responsible for the observed inhibition and therefore that the Na+/I- cotransporter is probably an autoantigen., Journal Article, Research Support, Non-U.S. Gov't, info:eu-repo/semantics/published

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The identification of autoantibody epitopes is important to the understanding of autoimmune thyroid diseases. In the case of thyroid peroxidase antibodies (TPO-ab), recent reports have disagreed about the number and type of autoantibody epitopes found in human TPO. In order to clarify the nature of these epitopes, we used an approach that provides recombinant human TPO produced by bacterial cells. The cDNA of four slightly overlapping fragments of human TPO-TPO 1(Glu 17-Ser 227), TPO 2(Tyr 226-Thr 476), TPO 3(Glu 471-Ser 720) and TPO 4(Phe 709-Leu 993)--were amplified by polymerase chain reaction and subcloned into the expression vector pMAL. In addition, a TPO 3 species for an alternatively spliced form of TPO of 876 amino acids was constructed (TPO 3M). Each of these constructs encodes a fusion protein, in which the amino terminal portion is maltose-binding protein, followed by the sequence of the fragment of human TPO. The plasmid constructs were transformed in Escherichia coli and, after growth, bacterial cells were harvested, lysed and the lysate was passed over an amylose affinity column and eluted with maltose. Western blots were performed using 33 sera from patients with autoimmune thyroid disease (group 1) and 17 sera from patients with nodular goiter and focal thyroiditis (group 2), all positive for TPO-ab measured by radio-immunoassay; sera from 10 healthy people with no clinical evidence of thyroiditis and positive for TPO-ab measured by radioimmunoassay (group 3) and sera from 30 patients with antigastric parietal cell antibodies without signs or symptoms of thyroiditis, 16 negative for TPO-ab (group 4a) and 14 positive for TPO-ab (group 4b), were included in the study.(ABSTRACT TRUNCATED AT 250 WORDS), Journal Article, Research Support, Non-U.S. Gov't, info:eu-repo/semantics/published

In a previous study, we have described the induction of thyroid blocking (TBAB) and thyrotropin binding inhibiting antibodies accompanied by thyroiditis in female BALBc mice (H2d) immunised with the extra-cellular domain (ECD) of the human thyrotropin receptor (TSHR) expressed as a maltose binding protein (MBP) fusion. In the present study we have investigated the response induced in mice of varying MHC haplotype. Two groups of female NOD (H2g), CBA (H2k) and C57 (H2b) mice were immunised intra-peritoneally with MBP-ECD or MBP on days 0 (100 micrograms), 15, 30 and 43 (50 micrograms). Blood samples from individual mice were obtained on days 0, 22, 36 and 50 and assessed for thyroid binding inhibiting immunoglobulins (TBII), thyroid stimulating (TSAB) and TBAB. On day 50 the treated mice and five age/sex matched NOD mice were sacrificed, their thyroids removed, examined histologically and any infiltrate characterised. Induction of antibodies to the ECD was tested by ELISA in which plates had been coated with either MBP-ECD or an ECD-protein A fusion. All of the mice developed a strong antibody response to the relevant immunogen but none of them contained TBII, TSAB or TBAB activities. No lymphocytic infiltration of the thyroid glands of the CBA or C57 mice was observed. In contrast, all of the NOD mice displayed severe thyroiditis, whilst one of seven MBP-treated mice had moderate infiltration and none of five untreated controls. Immunohistochemical analysis revealed that the infiltrate was predominantly activated T helper cells with little evidence of B cells or the cytokines IL-10 or IL-4, indicating that a Th1 response had been induced, contrary to our findings in BALBc mice which mount a Th2 response. In conclusion we have shown that the type and extent of response induced by immunising with the TSHR varies in mice of differing genetic background. H2d mice develop thyroiditis and TBAB/TBII, H2g mice develop thyroiditis in the absence of functional TSHR antibodies, Journal Article, Research Support, Non-U.S. Gov't, info:eu-repo/semantics/published

The TSH receptor (TSHR) has been proposed as an antigenic link between the thyroid and the orbit; TSHR transcripts have been demonstrated by other groups, one in orbital tissue and the other in orbital and dermal fibroblasts. In a previous study we were unable to demonstrate transcripts for the complete TSHR in retroocular muscle containing also fibroblasts. We now confirm this finding. A 1.3-kilobase variant of the TSHR messenger ribonucleic acid (mRNA) has been described in normal and Graves' thyroids; it contains exons 1-8 of the major mRNA species and a unique 3'-sequence predicted to encode further amino acids and a polyadenylated tail. Lacking the membrane-spanning region, the corresponding variant protein, if expressed, is not expected to couple to G-proteins. Using primers specific for this variant in reverse polymerase chain reaction experiments, Southern blotting and frequencies, we demonstrate the presence of this transcript in normal and Graves' thyroid, extraocular muscle, peripheral blood mononuclear cells, and, to a lesser extent, in fat and fibroblasts. TSH-mediated protein synthesis, cAMP, and glycosaminoglycan production have been measured in cultured fibroblasts. At 5 mU/mL, bovine TSH stimulated glycosaminoglycan production, but recombinant TSH did not, even at higher concentrations, suggesting that contaminating factors are responsible. Together the data do not support the presence of a functional complete TSHR in orbital tissue. However, they are compatible with a role for the extracellular portion of the receptor as a nonfunctional autoantigen and provide some explanation for the conflicting results with regard to the relevance of the TSHR in the pathophysiology of thyroid-associated ophthalmopathy., Journal Article, Research Support, Non-U.S. Gov't, SCOPUS: ar.j, info:eu-repo/semantics/published

A soluble inositol 1,4,5-trisphosphate 3-kinase (InsP3 3-kinase) has been characterized from extracts of rat thymus. The enzyme was shown to have a molecular weight within the range 98,000-114,000 M(r) as determined by regeneration of enzyme activity from sodium dodecyl sulphate polyacrylamide gels. The enzyme phosphorylates inositol 1,4,5-trisphosphate (InsP3) to inositol 1,3,4,5-tetrakisphosphate (InsP4) with an apparent Km of 3.1 +/- 0.4 microM. The enzyme is stimulated 4-6-fold by Ca2+/calmodulin and is not recognised by polyclonal antisera raised against rat brain InsP3 3-kinase A. High levels of InsP3 3-kinase activity were also detected in soluble extracts of human lymphocyte preparations. The human lymphocyte enzyme was shown to have a molecular weight between 61,000 and 70,000 M(r) as judged by SDS-PAGE, and was stimulated approximately 10-fold in the presence of Ca2+/calmodulin. These results establish that InsP3 3-kinase from rat thymus and human lymphocyte preparations represent high molecular weight isoenzymes of the InsP3 3-kinase family., Journal Article, Research Support, Non-U.S. Gov't, info:eu-repo/semantics/published

Expression of thyrotropin receptor (TSHr) was analyzed in normal human thyroids and differentiated thyroid tumours by Northern blotting of total RNA and by Western blotting of detergent-solubilized membrane proteins with an antibody specific for the extracellular domain of TSHr. Under nonreducing conditions TSHr stained as a 90-kDa protein in normal thyroid tissue. Expression varied in differentiated carcinomas from normal to not detectable in parallel to steady state TSHr mRNA levels and TSH binding in a TSH binding assay. The negative expression of TSHr in some patients with differentiated carcinomas may have consequences for the TSH suppressive treatments of these patients., Journal Article, info:eu-repo/semantics/published

The availability of high affinity antibodies to the human TSH receptor (TSHR) would help in defining its functional domains, but this requires the production of pure receptor as immunogen. We have expressed the extracellular domain (ECD) of the TSHR (residues 21-414) as a fusion protein with maltose-binding protein (MBP) in Escherichia coli, using the pMAL-cR1 vector. The major protein in an electrophoretically separated, crude bacterial lysate had a molecular mass of 89 kDa, in agreement with the size predicted for the MBP-ECD fusion product. Its identity was confirmed by Western blotting in which it was recognized by two polyclonal antibodies to synthetic peptides of the TSHR and an anti-MBP. Following purification on an amylose column, 15 mg pure MBP-ECD per litre of culture were produced, which was 5% of the total bacterial protein. Following extensive dialysis in a buffer which produces slight denaturation, MBP-ECD was cleaved with factor Xa. The identity of each protein was confirmed by Western blotting. To investigate the possibility of using the fusion protein as an immunogen we produced rabbit polyclonal antibodies to the ECD which were able to produce immunofluorescent staining of Chinese hamster ovary cells that expressed the TSHR, and revealed a protein of 95 kDa in Western blots of the same cells, in addition to a protein of 55 kDa. Only the protein of 55 kDa was detected in Western blots of human thyroid membranes.(ABSTRACT TRUNCATED AT 250 WORDS), Journal Article, info:eu-repo/semantics/published

Two groups of 10 Balbc by Jico male mice were immunised on days 0, 15 and 35, with the extra cellular domain (ECD) of the human thyrotropin receptor (TSH-R) expressed as a fusion protein in bacteria (group 1) or with the maltose binding protein (MBP) fusion partner alone (group 2). Blood was obtained on days 0, 22, 32, 42 and 49 and samples from the individual animals pooled for each group. Serum and immunoglobulin (IgG) preparations were tested, using CHO cells expressing the human TSH-R (JP26 and JP09) for thyroid stimulating (TSAb); thyroid blocking (TBAb) and thyrotropin binding inhibiting (TBII) activities. Neither serum nor IgGs were found to contain TSAb at any time point. TBII activity was present in the serum of both groups on day 32 and in group 1 only on day 49; when the test was performed on IgGs, only the MBP-ECD day 49 preparation remained significantly positive for TBII (p < 0.005). Significant TBAb activity was present in both the serum and IgG of group 1 day 49 (p < 0.005) and to a lesser extent on 42 (p < 0.02). Following the second immunisation (day 15) both groups and had decreased circulating T4 levels (p < 0.05) when compared with day 0 in each case. Group 2 were unaffected by the third immunisation on day 35 but the MBP-ECD group again had significantly decreased T4 levels (p < 0.02) compared with MBP day 49 and (p < 0.03) when compared with MBP-ECD day 0. Histological examination of thyroids from group 1 animals revealed extensive vascularisation and an atypical lymphoblastoid infiltration which was not observed in control mice. These preliminary results indicate that care is required in interpreting data since a non-receptor antigen was shown to decrease circulating thyroxine and serum from these animals had apparent TBII like activity. However, the results obtained with the IgGs suggest that receptor autoantibodies can be induced by immunising with the human TSH-R, in addition, the immunised mice show histological evidence for the developme, Journal Article, Research Support, Non-U.S. Gov't, info:eu-repo/semantics/published

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In a preliminary study, we observed the production of TSH binding-inhibiting (TBII) and thyroid-blocking (TBAb) antibodies accompanied by lymphocytic infiltration of the thyroid in a pool of male BALB/c mice immunized with the extracellular domain (ECD) of the human TSH receptor (TSHR) expressed as a maltose-binding protein (MBP) fusion in bacteria. In the present study we evaluated the humoral response to the same antigenic preparation in a new series of individual male and female BALB/c mice immunized ip on day 0 with 100 micrograms MBP-ECD and days 25, 39, and 53 with 50 micrograms MBP-ECD in an adjuvant composed of aluminum oxide, magnesium hydroxide, and Bordetella pertussis vaccine. Mice immunized with MBP served as control. Individual sera and immunoglobulins were tested for TBII, TBAb, and thyroid-stimulating antibodies (TSAb) on days 0, 32, 46, and 60, and total circulating T4 levels were measured by RIA. Animals were killed on day 120, their thyroids were examined histologically, the infiltrates were characterized using monoclonal antibodies specific for T-cells (total, activated, helper, and suppressor), B-cells, and macrophages. Sera and immunoglobulins G of the MBP-treated control group were all negative for TSAb, TBAb, and TBII activity. The receptor-immunized mice, despite having high titers of antibodies to the immunogen in an enzyme-linked immunosorbent assay, displayed a heterogeneous response in terms of biological activity, with 3 of 7 female and 4 of 8 male mice having TBAb/TBII activities that persisted and whose activity increased throughout the experiment. No significant TSAb antibody activity was observed. Total T4 levels were also heterogeneous even before immunization, but 9 of 15 MBP-ECD-treated mice had levels below the normal range after immunization, and 7 of these also had TBII/TBAb activities. At the end of the experiment, only 4 of the MBP-ECD-treated female mice survived, but all of them had a severe lymphocytic infiltration of the, Comparative Study, Journal Article, Research Support, Non-U.S. Gov't, SCOPUS: ar.j, info:eu-repo/semantics/published, Communication lors de l'European thyroid association à Wien en 1994

Chinese hamster ovary (CHO) cells transfected with the cloned human TSH receptor (CHO-R) were used to develop an assay to detect thyroid autoantibodies blocking the TSH-dependent cAMP production (TSHBAb). The study group included 38 patients with goitrous Hashimoto’s thyroiditis (HT) and 47 subjects with atrophic thyroiditis (AT). In the HT group, 8 patients had subclinical hypothyroidism (HT-SH) and 30 had overt hypothyroidism (HT-H). Thirty normal subjects served as controls. Immunoglobulin G (IgG) was prepared from serum by double chromatography on DEAE-Sephadex. CHO-R cells were seeded in 96-well plates and were cultured for 48 h before the assay in RPMI-1640 medium plus 1 mmol/L glutamine, 10% fetal calf serum, and 0.4 g/L geneticin. In the assay for TSHBAb, CHO-R cells were incubated with IgG alone (0.5–2 mg/ml), TSH alone (0.2–625 mU/L), or IgG plus TSH; all samples were diluted in hypotonic medium containing 0.5 mmol/L isobutylmethylxanthine (IBMX). After 2 h of incubation at 37°C in 5% CO2 −95% air atmosphere, TSH-stimulation was quantified by measuring extracellular cAMP by a RIA. IgGs from normal subjects did not significantly modify the stimulation of adenylate cyclase produced by TSH, the results obtained ranging between −30% and +18% (mean±SD=−3±14%). All IgGs producing an inhibition greater than 2SD from the mean of controls (>25%) were considered positive for blocking antibodies. TSHBAb were detected in 1/8 (12.5%) patients with HT-SH, in 7/30 (23.3%) with HT-H and in 16/47 (34.0%) patients with AT. When the same IgGs were tested in FRTL-5 cells, TSHBAb were detected in 1/8 (12.5%) patients with HT-SH, in 5/30 (16.6%) with HT-H and in 15/47 (31.9%) with AT. TSHBAb results in CHO-R cells showed a good correlation with those in FRTL-5 cells (r=0.74, p<0.0001), but 3/24 IgGs were positive for TSHBAb in CHO-R cells and negative in FRTL-5 cells. Using the radioreceptor assay, TSH-binding inhibiting antibodies were detected in 17/24 (70.8%) sera that conta, SCOPUS: ar.j, info:eu-repo/semantics/published

The search for autoantigenic targets in thyroid associated ophthalmopathy (TAO) and a possible link with the thyroid continues. Several authors have presented data suggesting that extrathyroidal tissues, e.g. extraocular muscle (EOM), may contain a functional thyrotropin receptor (TSH-R). We have applied Northern blotting and PCR amplification in an attempt to demonstrate the mRNA for the TSH-R in EOM, which although predominantly composed of muscle, may contain a significant proportion of fibroblasts. In Northern blots of poly(A) mRNA of normal human thyroid, EOM and skeletal muscle (SM) probed with a p32 labeled hTSH-R cDNA, transcripts of 4.6 and 4.4 kb were observed only in the thyroid. PCR amplification was performed on thyroid and EOM cDNAs using pairs of primers designed to amplify exons 1-->9, the extracellular domain (ECD), and exon 10, the intracellular domain (ICD). Both the ECD and ICD were amplified from a thyroidal cDNA library but only the ICD in the EOM cDNA library, this probably being due to the inevitable contamination with genomic DNA. These results do not support the hypothesis that the TSH receptor, as such, would be an autoantigen of EOM involved in TAO., Journal Article, Research Support, Non-U.S. Gov't, info:eu-repo/semantics/published

Unlike autoimmune thyroid disease (AITD) in which a number of autoantigens have been identified and characterized, the situation in thyroid associated ophthalmopathy (TAO) is far from clear. A number of candidate antigens have been identified by probing Western blots of orbital tissue (OT) with sera from TAO patients, the most frequently cited being proteins of molecular weight 23, 28, 55, 64, 78 and 120 kilodaltons. In an attempt to identify autoantigens in TAO we have produced a lambda gt11 human eye muscle expression library. This has been screened with sera from four patients with severe TAO whose antibodies bind to one or more of the aforementioned candidate antigens or to a thyroglobulin/acetylcholinesterase (Tg/Ache) shared epitope. Four clones were isolated and characterized; clone R14 encodes the carboxyl terminal 193 amino acids of an IgE binding protein, clones R10 and R13 encode unknown proteins having significant similarity with heat shock protein 27 and the U1 small nuclear ribonucleoprotein respectively. Clone R1 encodes an unknown peptide of 347 amino acids having no similarity with proteins in available data banks. R1 clone affinity purified autoantibodies bind to a protein of Mr 78 kD in a Western blot of porcine eye muscle tissue. Autoantibodies to the R1 recombinant lysogen were clearly demonstrated in 5 of 20 sera from Graves disease patients, its role merits further investigation. The possible relevance of these clones to the pathogenesis of TAO is discussed as well as the limitations of this type of approach in the identification of unknown autoantigens., Journal Article, info:eu-repo/semantics/published

A clone of Chinese hamster ovary (CHO) cells transfected with the cloned human TSH receptor (CHO-R) was used to optimize an assay for thyroid-stimulating antibody (TSAb), measuring adenylate cyclase stimulation by purified immunoglobulin G from patients with Graves' disease. Optimal sensitivity to bovine TSH (1 mU/L) and TSAb was obtained using hypotonic buffer and measuring extracellular cAMP. In time-response experiments, TSAb stimulation was maximal after 2 h of incubation in hypotonic buffer. Under these conditions, a significant stimulation by Graves' immunoglobulin G was obtained with 33 of 35 (94%) samples from patients with untreated Graves' disease and with 21 of 23 (91%) from patients who relapsed after a course of antithyroid drugs. On the other hand, TSAb was detected in only 12 of 20 (60%) patients who were euthyroid during methimazole treatment and in 4 of 11 (36%) who were euthyroid after a course of antithyroid drugs. All samples from Graves' patients were also tested for TSAb activity on FRTL-5 cells. The results of cAMP stimulation in FRTL-5 and CHO-R showed a fairly good correlation (r = 0.60; P < 0.0001). In particular, of the 58 patients with active Graves' disease (35 with untreated hyperthyroidism and 23 relapsed after methimazole), 43 (74%) were positive in both assays, 3 (5%) were negative in both, 11 (19%) were negative in FRTL-5 and positive in CHO-R, and 1 (1.7%) was negative in CHO-R and positive in FRTL-5. In conclusion, CHO cells transfected with the cloned human TSH receptor are suitable for the clinical assay of TSAb. The sensitivity of this assay is higher than that obtained using FRTL-5 cells, having the additional advantages of expressing the human TSH receptor and requiring less cumbersome procedures for cell culture., Journal Article, Research Support, Non-U.S. Gov't, SCOPUS: ar.j, info:eu-repo/semantics/published

Circulating TSH bioactivity may vary in several clinical and experimental conditions. Since the reliability of the current methods for the measurement of TSH bioactivity is limited, a new bioassay based on cAMP accumulation in Chinese Hamster Ovary cells transfected with recombinant human TSH receptor (CHO-R) was set up. The sensitivity was 0.3 +/- 0.1, 0.4 +/- 0.1 and 0.01 +/- 0.01 micrograms/L for TSH IRP 80/558, recombinant human TSH and bovine TSH, respectively. Standard curves were parallel, and the intra- and inter-assay coefficients of variation were 13 +/- 1.1% and 22 +/- 1.9%, respectively. LH, FSH, CG and TSH subunits did not stimulate cAMP accumulation up to high concentrations. Circulating TSH was partially purified by immunoaffinity separation and concentrated before being bioassayed. However, plain sera with high TSH levels, such as those from primary hypothyroid patients (PH), could be directly tested in CHO-R bioassay, provided that sera were added at concentrations lower than 10%. TSH from 6 normal subjects had biological to immunological ratio (B/I) ranging from 0.6 to 2.1 (mean +/- SD = 1.4 +/- 0.5). TSH from 6 patients with PH showed bioactivity significantly lower than in normals (B/I = 0.6 +/- 0.3; p < 0.001; range = 0.3-1.1). TSH from 5 patients with central hypothyroidism of hypothalamic origin (CH) had undetectable basal bioactivity (B/I < 0.2), which normalized in only one patient after acute TRH and in all patients after chronic TRH administration. In conclusion, CHO-R cells provide an excellent tool for evaluating TSH bioactivity, owing to high sensitivity, specificity, reproducibility and feasibility of the assay.(ABSTRACT TRUNCATED AT 250 WORDS), Journal Article, Research Support, Non-U.S. Gov't, SCOPUS: ar.j, info:eu-repo/semantics/published

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We have tested sera from patients with autoimmune thyroid disorders with or without ophthalmopathy for immunoreactivity, in a dot blot assay, against a recombinant 98 amino acid fragment of a cloned 64 kDa protein, D1, which is expressed in human eye muscle and thyroid, in the form of a Lac Z fusion protein. Tests were positive in 19 out of 40 patients with established thyroid-associated ophthalmopathy (TAO), in 12 out of 21 patients with Graves' hyperthyroidism (GH) without clinically evident ophthalmopathy, in 5 out of 10 patients with thyroid autoimmunity and lid retraction but no other signs of ophthalmopathy, in 4 out of 23 patients with Hashimoto's thyroiditis (HT) without evident ophthalmopathy and in 2 out of 18 patients with benign adenoma or multinodular goitre, but in only 2 out of 37 normal subjects tested. SDS-polyacrylamide gel electrophoresis and Western blotting for an antibody reactive with a 64 kDa antigen in pig eye muscle membranes was also carried out on sera from patients with TAO and GH. While immunoblotting for antibodies reactive with a 64 kDa protein was more often positive in patients with TAO, in whom 58% had serum antibodies which reacted with a 64 kDa protein, this was not the case in patients with GH without eye signs in whom the prevalence of positive immunoblot tests was 35%. Overall there was a fairly close correlation between the two tests although there were many exceptions.(ABSTRACT TRUNCATED AT 250 WORDS), Journal Article, Research Support, Non-U.S. Gov't, Research Support, U.S. Gov't, P.H.S., SCOPUS: ar.j, info:eu-repo/semantics/published

In human thyrocytes and in a permanent CHO cell line expressing the human thyroid stimulating hormone (TSH) receptor cDNA (JP09 cells), TSH activates both the cyclic AMP and the phosphatidylinositol 4,5-bisphosphate (PIP2) cascade, although the latter effect requires higher TSH concentrations. Thyroid stimulating autoantibodies (TSAb) activate also the human thyroid leading to the hyperthyroidism of Graves' disease. They bind to the TSH receptor and mimic the TSH stimulation of the gland by increasing intracellular cyclic AMP, but they do not enhance PIP2 hydrolysis in human thyroid slices. We show in this study that TSAb are able to activate the PIP2 cascade in JP09 cells, a cell line expressing high levels of TSH receptor. This suggests that the mechanism of action of TSAb on the TSH receptor is qualitatively similar to that of TSH., Journal Article, SCOPUS: ar.j, info:eu-repo/semantics/published

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We have characterized a transfected Chinese hamster ovary cell line, JP09, which expresses high levels of the human TSH receptor (TSH-R). Based on a theoretical biological activity for TSH of 40 IU/mg, JP09 has approximately 90,000 receptors per cell, having a dissociation constant of 1.64 x 10(3) mU/L or 1.47 x 10(-9) mol/L. We have used JP09 to prepare solubilized TSH-Rs which have formed the basis of a binding assay for thyroid-binding inhibiting immunoglobulins in unfractionated sera. We have compared the JP09 assay with the TRAK assay (which is based on solubilized porcine TSH-R) and found a highly positive correlation between the two assays, r = 0.83 P < 0.0001, in 55 sera from patients with autoimmune thyroid disease. JP09 can be adapted to growth in suspension culture, permitting large scale production. The tracer in the assay is bovine [125I]TSH; surprisingly, despite the use of a hTSH-R, hTSH had no effect on the binding of the tracer up to 10(3) mU/L and only a minor effect at 10(4) mU/L., Comparative Study, Journal Article, info:eu-repo/semantics/published

We have expressed the complete coding sequence of the human TSH-R in eucaryotic cells. Limiting dilution enabled us to select two clones, JP09 and JP26, which have formed the basis of binding and bioassays respectively, for TSH-R antibodies. Results obtained in the binding assay correlated well with those obtained in the TRAK (Henning Berlin) assay, while the bioassay was at least as sensitive as measurements made with FRTL5 or human thyroid cells. These cell lines provide a reliable source of human TSH-R which will be useful in routine diagnosis., Journal Article, info:eu-repo/semantics/published

We have investigated whether the alternatively spliced form of thyroid peroxidase (TPO), which has been shown clearly to occur in Graves' thyroid, is present in normal thyroid. We have performed a polymerase chain reaction on cDNAs prepared from normal thyroid mRNAs using primers 100 bases up-stream and down-stream from the 171 nucleotides that have been shown to be spliced out. Size analysis of the polymerase chain reaction products by agarose gel electrophoresis revealed bands at 0.2 and 0.37 kilobases (kb), sizes predicted to be obtained when the two forms of TPO cDNA are present. This was confirmed by hybridization with a 32P-labeled probe corresponding to the 20 nucleotides of the junction site. After stringent washing, the 0.2-kb band gave a clear positive signal, which was stronger than that of the 0.37-kb band despite the latter cDNA being more abundant. This demonstrates clearly that the alternatively spliced form of TPO exists in normal thyroid. Its significance is discussed., Journal Article, Research Support, Non-U.S. Gov't, info:eu-repo/semantics/published

Screening 10(6) plaque-forming units from a lambda gt11 human thyroid cDNA library with a pool of 10 sera from patients with Hashimoto's thyroiditis resulted in the isolation of a clone, D1, of 292 basepairs with an open reading frame of 97 amino acids which did not share significant similarity with any known protein. Rescreening the library using D1 as a probe led to the full-length cDNA being cloned and sequenced which has the potential to encode a 572-amino acid polypeptide. This would correspond to a 63-kDa nonglycosylated protein which is probably membrane bound. The recombinant fusion protein from the original D1 clone was recognized in dot blot or Western blot assays by 4 of 19 patients with Hashimoto's thyroiditis, 3 of 9 patients with Graves' disease, and 1 of 6 patients with idiopathic myxedema; all other sera (12), including that from normal subjects, were negative. Clone affinity-purified autoantibodies bound to a protein of 64 kDa in a Western blot of human thyroid tissue. Using D1 as a probe in a Northern blot revealed 3.9-kilobase (kb) transcripts in poly(A)+ RNA from normal human thyroid and extraocular muscle, but not skeletal muscle. The dog equivalent of D1 cDNA was isolated by screening 10(6) plaque-forming units from a dog thyroid cDNA library using clone D1. A fragment of dog cDNA was, in turn, used as a probe in a Northern blot of dog poly(A)+ RNAs from thyroid, brain, lung, heart, liver, kidney, spleen, and stomach; a faint signal at 3.9 kb was observed in all tissues except the thyroid, which displayed a strong transcript of 2.6 kb. It is concluded that the D1 cDNA clone encodes an autoantigen shared by the thyroid and eye muscle. Its possible relevance to autoimmune ophthalmopathy is discussed., Journal Article, Research Support, Non-U.S. Gov't, info:eu-repo/semantics/published

The sequential epitopes on the human thyroperoxidase (TPO) recognized by antibodies in the sera of patients with autoimmune thyroid disease were investigated using a recombinant DNA technique. Previous studies led to the isolation of two overlapping cDNA clones that encode polypeptides of TPO (85 residues, C2; 100 residues, C21) recognized by sera from several patients with autoimmune disease that contained antimicrosomal autoantibodies. In this report the vector pUEX1 was used to clone and express small random fragments of TPO cDNA in Escherichia coli as a beta-galactosidase fusion protein. Colonies were screened with a serum from a patient with Hashimoto's thyroiditis, and immunoreactive peptides were identified by sequencing the corresponding DNA inserts. Two linear epitopes of human TPO (amino acids 590-622 and 710-722) were recognized by the autoantibodies. This confirmed our previous results and provide a more precise localization of the antigenic determinants involved. The same approach has been applied in an attempt to identify the binding site(s) for autoantibodies on the human TSH receptor. In contrast to the data obtained with TPO, sera from patients with blocking (from idiopathic myxoedema) or stimulating (from Graves' disease) activity did not recognize the linear TSH receptor peptide fragments generated in our libraries., Journal Article, SCOPUS: ar.j, info:eu-repo/semantics/published

The effects of thyroid-stimulating antibodies (TSAb) and of thyrotropin (TSH) were compared, on the generation of cyclic AMP and inositol phosphates (InsP), in human thyroid slices incubated in vitro, and on the Rapoport cyclic AMP bioassay. The TSAb positive sera were obtained from 19 patients with Graves' disease. In 14 experiments with the slices system, TSH significantly increased cyclic AMP accumulation (TSH, 0.03-10 mU/ml) as well as the cyclic AMP-independent inositol trisphosphate (InsP3) generation (TSH, 1-10 mU/ml). In the same 14 experiments, TSAb (0.10-28 mg/ml) enhanced cyclic AMP intracellular levels as expected while they did not induce any InsP accumulation. Even when TSAb increased cyclic AMP levels to the same or higher values as those obtained with TSH concentrations allowing InsP3 generation. TSAb were still unable to activate the phosphatidylinositol-Ca2+ cascade. The patterns of the response curves of TSAb and TSH on cyclic AMP accumulation were different, suggesting that different mechanisms may be involved. In addition, unlike TSH, TSAb were not able to stimulate H2O2 generation, which in human tissue mainly depends on the activation of the phosphatidylinositol-Ca2+ cascade. Immunoglobulins from six additional Graves' patients lacking measurable cyclic AMP-stimulating activity in both slices and cells systems did not activate phospholipase C either. In conclusion, our results show that TSAb do not share all the metabolic actions of TSH on human thyroid tissue. The data provide support for the concept that the pathogenesis of Graves' disease can be fully accounted for by the ability of TSAb to stimulate adenylate cyclase. This work also confirms that TSH activates the cyclic AMP and the phosphatidylinositol cascade by independent pathways in the human thyroid., In Vitro, Journal Article, Research Support, Non-U.S. Gov't, info:eu-repo/semantics/published

The human thyrotropin receptor cDNA was transfected in CHO cells and individual clones were isolated. They were tested for their response to thyrotropin, forskolin and antibodies from a patient with high levels of thyroid stimulating antibodies. Several clones were characterized extensively with respect to membrane binding of labeled thyrotropin, cAMP accumulation in response to thyrotropin and kinetics of cAMP production. Data for three representative clones are presented. Receptor number as assessed by membrane binding of labeled thyrotropin, and cAMP production, measured in a thyrotropin response bioassay, are correlated. The Kd value for the human thyrotropin receptor expressed in CHO was estimated to be 50 pM., Journal Article, Research Support, Non-U.S. Gov't, info:eu-repo/semantics/published

We report on two assays for autoantibodies to the TSH-R which have been developed using materials from mammalian cells transfected with the cDNA for the human TSH-R. In the first, a particulate fraction has been prepared from COS cells, transiently expressing the human TSH-R and used in a radioreceptor assay in conjunction with bovine 125I-TSH. Immunoglobulins (IgGs) from patients with Graves' disease (n = 11) and idiopathic myxoedema (n = 2) have been used as competitors of 125I-TSH binding to the COS TSH-R membranes and the results have been compared with those obtained with a commercially available kit for measuring TSH-R autoantibodies, which uses solubilised porcine TSH-R. Both assays showed similar performance, being particularly sensitive to antibodies from patients with idiopathic myxoedema. In the second assay system we have used a CHO cloned cell line (JP26) stably transfected with the human TSH-R. A selection of IgG preparations from patients with Graves' disease and of six normal controls was used to test the ability of this cell line to detect thyroid stimulating immunoglobulins (TSAb) by increasing its cAMP production. The assay was performed under two conditions: in standard (isotonic) medium or in hypotonic medium. Freshly thawed human thyrocytes incubated in hypotonic medium served as a reference method. Only five patients scored positive when tested in the JP26 cell line under isotonic conditions. When the assay was performed in a hypotonic medium, a significant positive correlation was observed between the results given by JP26 cells and human thyrocytes.(ABSTRACT TRUNCATED AT 250 WORDS), Comparative Study, Journal Article, info:eu-repo/semantics/published

Journal Article, Research Support, Non-U.S. Gov't, Research Support, U.S. Gov't, P.H.S., Review, info:eu-repo/semantics/published

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In a previous work we characterized a major epitope of thyroid peroxidase (TPO), which was recognised by 66% of 157 patients with autoimmune thyroid disease (AITD) and 9 out of 50 patients with non-thyroidal autoimmune disease (NTAID) 6 of whom had antibodies to the gastric parietal cell antigen (PCA). In the present study we have affinity purified C2 antibodies and demonstrated that they bind to the native TPO enzyme in a radioimmunoassay (RIA). We have measured antibodies to the C2 peptide and a second TPO peptide, C21, in enzyme-linked immunosorbent assays (ELISA) in 30 patients with NTAID all of whom have antibodies to the gastric PCA, having first determined the incidence of antibodies to C21 in 98 patients with AITD who do not have antibodies to the gastric PCA. 58% of patients with AITD have antibodies to C21, a peptide of TPO which overlaps C2 by 21 amino acids in the region containing an 11 residue fragment which is very similar to a fragment of the H+ K+ ATPase, which has recently been shown to be a major component of the gastric PCA. In patients having NTAID and antibodies to the gastric PCA, 60% are positive for C2 Ab and 100% for C21 Ab, which is suggestive of an epitope shared by TPO and H+ K+ ATPase, corresponding to TPO 659----669 and H+ K+ ATPase 177----187. We conclude that C2 antibodies are heterogenous and comprise activities which bind to the intact enzyme and activities binding to an epitope which may be common to TPO and H+ K+ ATPase., Comparative Study, Journal Article, Research Support, Non-U.S. Gov't, info:eu-repo/semantics/published

The homology between thyroglobulin and acetylcholinesterase (1) has been analyzed in detail. It contains 28.3% identical amino acids and extends over 544 residues, involving more than 90% of the acetylcholinesterase molecule and the C-terminal portion of thyroglobulin. The hydropathy profiles of the homologous regions have been determined and compared. Their striking resemblance suggests that both proteins adopt a similar three dimensional structure and militates for some common property. As thyroglobulin and acetylcholinesterase are known to interact with cell membranes, we suggest that the acetylcholinesterase-like domain of thyroglobulin is involved in the binding. These observations demonstrate that thyroglobulin has evolved from the condensation of a duplicated copy of the acetylcholinesterase gene with an archaic thyroglobulin gene encoding the major hormonogenic domain. The extensive homology in hydropathy profiles suggests that the two proteins may share antigenic determinants. If this were the case, it would provide a rationale for the demonstration of immunoreactive thyroglobulin in neurons (2) and the pathogenesis of Grave's ophthalmopathy., Comparative Study, Journal Article, Research Support, Non-U.S. Gov't, Research Support, U.S. Gov't, P.H.S., SCOPUS: ar.j, info:eu-repo/semantics/published

Previous studies carried out by screening a lambda gt11 human thyroid cDNA library with serum samples from selected patients with Hashimoto's thyroiditis and a polyclonal antibody to porcine thyroid peroxidase (TPO) confirmed, at the molecular level, that TPO is a major component of the thyroid microsomal antigen (M). That investigation led to the isolation of a clone (C2) which encodes an 85-amino acid segment of TPO and harbors a major epitope recognized by serum from several patients with autoimmune thyroid disease that contained anti-M autoantibodies (MAb). In this study, C2 antigen that was produced as a beta-galactosidase fusion protein was used to establish an enzyme-linked immunoabsorbent assay for the detection of anti-C2 autoantibodies (C2Ab). C2Ab then were assayed in 191 patients with different autoimmune and nonautoimmune thyroid disorders, and 50 patients with nonthyroidal autoimmune diseases. The results were compared with the titers of anti-TPO antibodies (TPOAb; as detected by monoclonal antibody-assisted RIA) and MAb (as detected by passive hemagglutination). Positive C2Ab was found in the serum of 85 of 136 (63%) patients whose serum contained TPOAb and/or MAb. A significant positive correlation was found between the levels of C2Ab and those of TPOAb (r = 0.76; P less than 0.001) or MAb (r = 0.69; P less than 0.001), which was independent of the type of underlying autoimmune thyroid disorder. Low levels of C2Ab also were found in 10 of 105 (9%) serum samples that did not contain TPOAb. Western blot analysis carried out on the latter samples showed that in 2 samples the apparent C2Ab reactivity was due to the presence of antibodies reacting with beta-galactosidase. In conclusion, we confirmed the validity of screening lambda gt11 cDNA human thyroid libraries to better characterize thyroid autoantigens and demonstrated the feasibility of using recombinant proteins to establish diagnostic assays for autoantibodies., Journal Article, Research Support, Non-U.S. Gov't, info:eu-repo/semantics/published