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ABSTRACT
The aim of this study was to define, at the molecular level, epitopes of thyroglobulin recognized by heterologous antibodies and autoantisera.
One hundred thousand clones from a λgt11 human thyroid cDNA library were screened using a rabbit antibody to human thyroglobulin at a 1:2000 dilution. Twenty clones were plaque-purified to homogeneity, and characterization and sequencing of their cDNA inserts showed that they represented four distinct regions of the thyroglobulin molecule, one of them being the 22 carboxyl-terminal amino acids. Rescreening of the library with the same rabbit antibody to human thyroglobulin absorbed with peptides encoded by the carboxyl-terminal clone, led to the definition of six further epitope-bearing fragments of thyroglobulin. The ten regions that we have identified were recognized by ten further rabbit antibodies to human thyroglobulin, showing that they are representative of the repertoire of heterologous epitopes.
In contrast, none of the ten heteroepitope-bearing fragments was recognized by sera from ten patients with autoimmune thyroid disease with various titres of thyroglobulin antibodies. Screening of 2 × 106 clones from the library using a pool of ten autoantisera (individual sera diluted to 1:1000), and of 1 × 106 clones using a single autoantiserum of very high antithyroglobulin titre (diluted 1:400) resulted in no thyroglobulin clones being isolated. The significance of these results to the immune process is discussed.
Journal of Endocrinology (1989) 122, 169–176
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ABSTRACT
The effect of excessive tri-iodothyronine (T3) in vivo was assessed using normal human lymphocytes. Cells from normal subjects were frozen in liquid nitrogen before and after oral administration of T3 for 1 week to permit a direct comparison under identical culture conditions. Within the group of individuals studied, some subjects did show changes in B or T cell function but hypertri-iodothyroninaemia produced no consistent effect for the whole group on circulating T cell subsets or T and B cell activation measured by short-term culture or stimulation of lymphocyte cultures with phytohaemagglutinin or pokeweed mitogen. Tri-iodothyronine supplementation of cultures in vitro did not affect pokeweed mitogen stimulation. These findings suggest that the immunological abnormalities in Graves' disease are not the result of increased circulating thyroid hormone levels and that remission following medical treatment is due to an immuno-suppressive effect of the drug rather than the restoration of euthyroidism.
J. Endocr. (1984) 101,81–86
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Iodide concentration by the thyroid gland, an essential step for thyroid hormone synthesis, is mediated by the Na+/I- symporter (NIS). To identify factors that may regulate this process, we have studied NIS gene expression in the Fisher rat thyroid cell line (FRTL-5) by a semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) technique. Increasing concentrations of bovine TSH (0.1, 1, 10, 50 and 100 mU/l), with or without tumour necrosis factor-alpha (TNF alpha), interferon-gamma (IFN gamma) or interleukin-1 alpha (IL-1 alpha) were added to FRTL-5 cells previously deprived of TSH for a minimum of 5 days. RNA was extracted and samples were studied for NIS expression. TSH enhanced NIS mRNA expression in a dose-dependent manner, with induction evident at 0.1 mU/l, reaching a peak at 50 mU/l, an effect detected after 6 h of stimulation, but not in the first 2 h. Both TNF alpha and, to a lesser extent, IL-1 alpha inhibited basal and TSH-induced NIS expression. High concentrations of IFN gamma also downregulated TSH-stimulated NIS mRNA expression. Using the same technique, we also investigated NIS mRNA tissue distribution in two male and one female Wistar rats. High levels of NIS expression were detected in the thyroid, stomach, and mammary gland, lower levels were found in the intestine, adipose tissue and liver, borderline levels were expressed in the salivary gland, and no expression was detected in the kidneys. In summary, we have shown that TSH upregulates rat NIS gene expression in vitro, and this induction can be modulated by cytokines. Analysis of the distribution of rat NIS mRNA ex vivo demonstrated variable levels of NIS transcription in different tissue samples.
