Biomedical subjects

L J Degroot

Publications and source records attributed to L J Degroot.

13 recordsLinked to original sources

PubMed1991

Thyroid microsomal antigen in Graves' thyroid is not different from that in normal thyroid.

Differences from normal in microsomal antigen (M-Ag) may be involved in the development of autoimmune thyroid disease. We compared the M-Ag in Graves' thyroid immunologically and biochemically to that in normal thyroid. The concentration of M-Ag, measured with an enzyme-linked immunosorbent assay, was significantly greater in the Graves' microsomes than in normal microsomes. Binding of a patient's microsomal antibody to Graves' microsomes was completely inhibited when the serum was first incubated with normal thyroid microsomes. Sodium dodecylsulfate-polyacrylamide gel electrophoresis and Western blotting were done with a monoclonal antibody to denatured M-Ag. In both Graves' and normal thyroids, M-Ag existed as 107-, 101-, and 95-kDa peptides. After incubation with V8 protease, the residual antigenic peptide had a molecular weight of less than 60,000 and after incubation with trypsin, 95- and 87-kDa peptides and several smaller antigenic peptides were found. There were no significant differences in the pattern of normal and Graves' microsomes after digestion. Two-dimensional gel electrophoresis of Graves' microsomes showed that the isoelectric point for the 107-kDa peptide was at pH 7.2; that for the 101-kDa peptide was at pH 6.2, and that for the 95-kDa peptide was at 6.5. These values were not different from those observed for normal microsomes. These results indicate that M-Ag in Graves' thyroid does not differ from that in normal thyroid, and that microsomal antibodies in autoimmune thyroid disease probably do no arise from differences in the antigen.

Autoantigens↗

PubMed1990

Negative and positive transcriptional regulation by thyroid hormone receptor isoforms.

Multiple forms of human thyroid hormone (T3) receptor have been identified, including true receptors that bind T3 (alpha 1 and beta) and a splicing variant (alpha 2) that does not bind T3. The alpha 1- and beta-receptors activate transcription through interactions with positive thyroid response elements (TREs). The alpha 2 variant is unable to activate transcription and has been reported to inhibit alpha 1 or beta stimulation of positive TREs, a property referred to as dominant negative activity. In this report we have performed studies to assess the functional properties of different members of the thyroid receptor family with regard to both positive and negative transcriptional regulation. The alpha 1-, alpha 2-, and beta-receptors were each coexpressed in JEG-3 cells with either TreTKCAT (CAT = chloramphenicol acetyltransferase), a reporter gene that contains a positive TRE, or TSH alpha CAT, a negatively regulated reporter gene. The alpha 1 and beta isoforms stimulated transcription of TreTKCAT and inhibited TSH alpha CAT transcription in a T3-dependent manner, whereas the alpha 2 variant was inactive. When coexpressed with alpha 1- or beta-receptors, alpha 2 inhibited regulation of positive TREs, but the effects of alpha 2 were modest and only occurred when relatively high doses of receptor were transfected. The alpha 2-receptor variant did not affect negative regulation by alpha 1- or beta-receptors. Thus, in both positive and negative regulation, thyroid hormone receptor isoforms that bind T3 (alpha 1, beta) are functional, whereas the alpha 2 isoform, which does not bind T3, is not functional.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding Sites↗

PubMed1989

Lack of effect of methimazole on thyrocyte cell-surface antigen expression.

The nature of the immunosuppressive effect of antithyroid drugs has been a subject of controversy. It has been claimed that these agents exert a direct effect on the immune system, although we and others have suggested that the drugs affect the thyroid cells primarily with consequent reduced thyrocyte-immunocyte signalling. This may occur from reduced thyroid hormone production and/or reduced antigen presentation by the thyrocytes to local T lymphocytes. Using a cytotoxicity assay system, with chromium-51 labelling, monoclonal antibodies against thyroperoxidase (TPO) and HLA-DR, and complement, we have measured the expression of TPO and HLA-DR on cultured normal human thyroid cells; we have also measured thyroglobulin (Tg) release by radioimmunoassay into the medium of the cultured cells. The thyroid cells were stimulated with TSH or thyrotropin binding inhibitory immunoglobulin (TBII) for 48 hours before measuring for TPO induction, and with interferon gamma (IFN-gamma) (with or without TSH or TBII) for thyrocyte HLA-DR expression. A dosage of 1.6 milliunits per ml of TSH resulted in a significant increase in TPO expression on thyrocytes when compared with control unstimulated thyroid cells (p less than 0.001). The concentrations of Tg released into the medium with TSH or TBII were also significantly higher than those of the control thyrocytes. IFN-gamma at 200 units per ml induced HLA-DR expression, but did not induce thyrocyte TPO expression, or Tg release. Addition of the antithyroid drug, methimazole (MMI), at different concentrations, in addition to the other stimulators, IFN-gamma, TSH, or TBII, did not result in any inhibition of TPO, Tg release, or HLA-DR expression on the thyroid cells. It would thus appear that the pathways for stimulation for the expression of TPO and HLA-DR appear to be different. Finally, MMI does not cause its immunosuppressive effect by any reduction of thyroid antigen expression or release.

Antigens, Surface↗

PubMed1979

PPD-induced blastogenesis is auto-regulated by suppressor cells generated in vitro.

Suppressor cell induction can be demonstrated during antigen specific blastogenesis by using the same methods which have shown induction of suppressor cells by Con A. Since suppressor cells are rapidly generated during antigen specific blastogenesis, they must regulate the final level of blastogenesis induced during the seven day in vitro incubation.

Concanavalin A↗

PubMed1977

Biosynthesis of thyroid hormone: basic and clinical aspects.

Thyroid hormone formation requires the coincident presence of peroxidase, H2O2, iodide, and acceptor protein at one anatomic locus in the cell. The peroxidase enzyme appears to be a protoporphyrin lX containing heme protein, with binding sites for both iodide and tyrosine. It is probable that both iodide and tyrosine are oxidized to free radical forms which unite to form iodotyrosine. The peroxidase is also involved through an uncertain mechanism in iodotyrosine coupling and probably in oxidation of sulfhydryl bonds in thyroglobulin. H2O2 may be supplied by microsomal NADPH-cytochrome c reductase or NADH-cytochrome b5 reductase. Other possible intracellular H2OI generating systems include monoamine oxidase and xanthine oxidase. The usual acceptor for iodide is thyroglobulin, which is currently believed to be iodinated within apical secretory vesicles at the cell border just prior to liberation into the colloid, or possibly after liberation into the colloid. Other soluble an insoluble proteins are also iodinated within the gland. The peroxidase is present in numerous cellular structures, but iodination activity occurs primarily, if not only, at the apical cell border. The controls of iodination are imperfectly known. Thyrotrophin modulation of iodide uptake, H2O2 generation, thyroglobulin synthesis, and peroxidase enzyme level obviously are the main regulations. Many of these actions are thought to involve mediation of adenyl cyclase and subsequent activation of intracellular phosphokinases. Antithyroid drugs of the thiocarbamide group are competitive inhibitors of iodination under some circumstances, but if much iodide is present, they react with the oxidized iodine intermediate and are irreversibly inactivated themselves. Clinical problems involving defective peroxidase function are among the most frequent hereditary defects of thyroid hormone formation. Recognized abnormalities include deficient peroxidase, abnormality in binding of the peroxidase apoprotein to its prosthetic group, and other less well-identified abnormalities in peroxidase structure and function. Peroxidase is typically elevated in thyroid tissue from patients with hyperthyroidism sometimes deficient in cold thyroid nodules, and frequently diminished in tissue from patients with Hashimoto's thyroiditis.

Animals↗

PubMed1977

Familial goitre with partial iodine organification defect, lack of thyroglobulin, and high levels of thyroid peroxidase.

From a sibship of three sisters having congenital goitre and normal hearing, two had impairment of organification of iodine. S1 (4 years old) had goitre since birth, euthyroidism, and a negative perchlorate test. S2 (15 years old) and S3 (13 years old) were hypothyroid, and had radioiodide discharge after potassium perchlorate administration of 19.8% and 26.1%, respectively. Thyroid tissue was obtained at thyroidectomy. Peroxidase activity, in the thyroidal subcellular particles, was found to be qualitatively normal, but quantitatively increased. In the triiodide assay, the activity was: S1 6912 u, S2 2590 u, and S3 3844 u (normal values 900-1700 u). In the tyrosine-iodinase assay, the activities, expressed as nmoles of iodide incorporation per gram of tissue, were S1 1046, S2 471 (normal values 220-410). The activity of the thyroidal NADPH-cytochrome c reductase, an enzyme possibly involved in hydrogen peroxide generation, was: S1 0.084, S2 0.047, and S3 0.005 (normal values 0.018 muEq/min/mg). No thyroglobulin was detected by analytical ultracentrifugation, polyacrylamide gel electrophoresis, or double immunodiffusion in agar of the supernatant fractions. In patient S2, whose gland was labelled in vivo with 125I, 60% of the total radioactivity of the gland (pooled nodular and paranodular specimens) was in a particulate iodoprotein that was solublilized by trypsin, deoxycholate or digitonin. In the soluble fraction there were two iodoproteins: iodalbumin, and a second iodoprotein similar to the solubilized particulate iodoprotein. It is postulated that absence of the normal thyroidal receptor protein might be in some cases a cause of iodine organification defect.

Adolescent↗

PubMed1977

The effect of thyroid hormone on in vitro rat liver mitochondrial RNA synthesis.

Liver mitochondrial preparations from normal, thyroidectomized, and triiodothyronine-treated thyroidectomized rats were assayed for in vitro RNA synthetic activity. Thyroidectomized rat mitochondrial preparations incorporated UTP into RNA at 70% the rate of normal control preparations. Mitochondrial preparations from triiodothyronine-treated thyroidectomized rats incorporated UTP at rates 35%-45% greater than those of sham-injected thyroidectomized rats. These differences were statistically significant and could not be attributed to inequalities in mitochondrial sampling, dilution of labeled precursor specific activity, nucleotide substrate concentrations, or differences in ribonuclease activities.

Acriflavine↗

PubMed1977

Thyroid hormone receptors: release of receptor to the medium during in vitro incubation of isolated rat liver nuclei.

Isolated rat liver nuclei show a substantial amount of T3 receptor release to the medium during in vitro incubation. This has been shown to be a general feature of nuclei compared after several methods of isolation and incubation. About 50% of nuclear receptors are released to the medium when incubated in sucrose-MgCl2-Tris, pH 7.85, for 2 h at 20 C.DNA, histones, and non-histone proteins (NHP) are also released. CaCl2 inhibits about 90% of the release of DNA and histones, but has less effect on inhibiting leakage of NHP and nuclear T3-binding protein (NTBP). The highest leakage for each fraction was found when incubating nuclei in the presence of EDTA. The receptor released to the medium has an affinity virtually identical to the receptor remaining in the nuclei. At least 1 mM dithiothreitol is needed to avoid degradation of the receptor. The NTBP has a sedimentation constant of 4.5 S when studied in low ionic strength gradients. Increasing KCl concentration decreases progressively its sedimentation constant, and in gradients containing 0.4 M KCl the receptor sediments as a single peak of 3.4 S. Since release of receptor to incubation medium decreases free T3 concentration, it must be taken into account in calculating receptor affinity. Total nuclear capacity in vitro is obviously underestimated, unless receptor released to medium is measured. Receptor exchange between cytosol and nucleus may be of physiologic significance.

Animals↗

PubMed1976

Dexamethasone suppression of serum T3 and T4.

Dexamethasone (2 mg q6h for 48 h) decreased serum T3 in normal and athyreotic subjects, and decreased T4 in normal subjects. Dexamethasone probably alters secretion and peripheral metabolism of thyroid hormones.

Clinical Trials as Topic↗

PubMed1976

Factors influencing triiodothyronine binding properties of liver nuclear receptors.

Triiodothyronine (T3) may bind directly to receptors present in liver cell nuclei, or may be transported into nuclei by receptor protein(s) present in the cytosol. To evaluate these possibilities, T3 binding was studied in vitro using liver cell nuclei isolated from rats exposed in vivo to very low (H), normal (N),or high levels of T3 (H + T3), and using nuclei incubated in vitro with added cytosol proteins. Ka for T3 was 0.075 +/- 0.05 x 10(10) M-1 in N, 0.1 + 0.04 in H, and 0.094 + 0.04 in H + T3, and pg T3 bound/100 mug DNA were 47 +/- 17, 31 +/- 14, and 29 +/- 8 in the three groups. The data indicate no difference in binding capacity between the groups related to prior in vivo exposure to T3, and that T3 may bind directly to empty nuclear receptor sites. Rat liver cytosol proteins added to the in vitro incubation medium always depressed T3 uptake by nuclei. Bovine serum albumin had a similar effect. Large amounts of rat serum proteins depressed uptake, but low levels augmented T3 binding through an unknown mechanism. It is probable that free T3 in serum is in equilibrium with free T3 in the cytosol and nucleus, and binds directly to nuclear receptor proteins without mediation by a cytosol receptor protein.

Animals↗

PubMed1975

Immunologic aspects of human thyroid cancer. Humoral and cell-mediated immunity, and a trial of immunotherapy.

Immunologic studies were performed on 16 patients with thyroid cancer. Circulating leukocyte counts increased, parallel to development of the terminal stage of disease, but total lymphocytes decreased. Serum immunoglobulin and complement were high, even though almost all patients showed negative antithyroid antibodies. Delayed skin hypersensitivity to bacterial and viral antigens and lymphocyte responsivity to PHA were not impaired at the initial stage of disease, but were impaired in terminal illness. Cell-mediated immunity (CMI) to tumor antigens(s) was measured using the assays of lymphotoxin, migration inhibition factor, and peripheral leukocyte migration inhibition. A few patients showed significant response to tumor antigen, but not to homogenates of Graves' thyroid gland. Active immunotherapy was applied to three patients. Two patients, who were in the terminal stage of illness, could not develop generalized CMI; immunization did not alter the patients' rapid downhill course. One patient developed in vitro evidence of CMI against cancer tissue antigens, associated with decrease in tumor size. Four months after immunization, CMI was impaired in autologous plasma culture, but not in cultures in allogenic normal plasma.

Adenocarcinoma↗

PubMed1975

Insoluble particulate antigen(s) in cell-mediated immunity of autoimmune thyroid disease.

Cell-mediated immunity (CMI) in patients with Grave's disease, chronic thyroiditis, and primary hypothyroidism was observed by assay of lymphocyte-mediated cytotoxicity (LMC) and leukocyte migration inhibition (LMC). Lymphocyte responsivity to phytohemagglutinin (PHA) is normal in these disease. In the LMC assay, lymphocytes of patients in each category responded to the antigens of thyroid homogenates, but not purified human thyroglobulin. Cytotoxicity is least in Graves disease and most obvious in primary hypothyroidism. In the LMI assay, patients lymphocytes responded to thyroid microsomal--mitochondrial antigens, but not to thyroid cell sap. Lymphocytes of Graves disease patients also responded to liver microsomal mitochondrial antigens. The particulate antigens lost activity when solubilized by ultrasonication or KCL extraction. There is no correlation between the PHA responsivity of lymphocytes and thyroid function, or between CMI and serum antithyroid antibodies or thyroid size. Treated and untreated patients had similar evidence of CMI. These data indicate that function of thymus-derived lymphocyte in vitro is not disturbed in autommune thyroid disease and that CMI against thyroid antigens can be demonstrated by assay of LMC and LMI. Insoluble particulate antigens appear more important than soluble antigens in CMI. LMC, resumably induced by soluble cytotoxic factor, "lymphotoxin," may play an important role in the progress of the autoimmune thyroid disease to hypothyroidism.

Antigens↗

PubMed1975

Differentiation of two abnormalities in thyroid peroxidase causing organification defect and goitrous hypothyroidism.

Clinical and laboratory evaluations are reported on two patients with congenital goiter and hypothyroidism due to iodide organification defect. In one patient, a 31-year-old white male with severe mental retardation, administration of perchlorate caused discharge of 69% of the radioiodine accumulated in the thyroid gland. Thyroid tissue had negligible peroxidase activity in the tyrosine-iodinase, triliodide, and guaiacol assays. Preincubation of subcellular fractions with hematin restored activity. The restored enzyme was labile to high concentrations of H2O2 (5.6times 10-4 h2o2 produced inhibition in the triiodide assay). Heating of the enzyme for 5 min at 46 degrees C produced 50% inactivation, while higher temperatures were required to half-inactivate normal peroxidases. This case represents a second example of the "peroxidase apoenzyme-prosthetic group defect" causing congenital goiter. The second patient, an example of the "deficient peroxidase defect," was a 10-yr-old girl with 35% discharge of thyroidal radioiodine by perchlorate. Peroxidase activity in the goiter tissue was quantitatively decreased (10%-20% of normal values) but kinetically normal with respect to apparent Km for H2O2. Hematin had little effect on the enzyme. Peroxidase activity had abnormal subcellular distribution, since pellets sedimenting between 39,000 and 105,000 g contained most of the activity. Normal thyroglobulin was observed in the thyroid gland of the patient. Two distinct defects of the peroxidase system can produce congenital goiter by limiting organification of iodide.

Adult↗