Leslie J. DeGroot

CTLA-4 Gene Polymorphism at Position 49 in Exon 1 Reduces the Inhibitory Function of CTLA-4 and Contributes to the Pathogenesis of Graves’ Disease

Activation of T cells requires at least two signals transduced by the Ag-specific TCR and a costimulatory ligand such as CD28. CTLA-4, expressed on activated T cells, binds to B7 present on APCs and functions as a negative regulator of T cell activation. Our laboratory previously reported the association of Graves’ disease (GD) with a specific CTLA-4 gene polymorphism. In theory, reduced expression or function of CTLA-4 might augment autoimmunity. In the present study, we categorized autoimmune thyroid disease patients and normal controls (NC) by genotyping a CTLA-4 exon 1 polymorphism and investigated the function of CTLA-4 in all subjects. PBMCs and DNA were prepared from GD (n = 45), Hashimoto’s thyroiditis (HT) (n = 18), and NC (n = 43). There were more GD patients with the G/G or A/G alleles (82.2% vs 65.1% in NC), and significantly fewer patients with the A/A allele (17.8% vs 34.9% in NC). In the presence of soluble blocking anti-human CTLA-4 mAb, T cell proliferation following incubation with allogeneic EBV-transformed B cells was augmented in a dose-dependent manner. Augmentation induced by CTLA-4 mAb was similar in GD and NC (GD, HT, NC = 156%, 164%, 175%, respectively). We related CTLA-4 polymorphism to mAb augmentation of T cell proliferation in each subgroup (GD, HT, NC). Although PBMC from individuals with the G/G alleles showed 132% augmentation, those with the A/A alleles showed 193% augmentation (p = 0.019). CTLA-4 polymorphism affects the inhibitory function of CTLA-4. The G allele is associated with reduced control of T cell proliferation and thus contributes to the pathogenesis of GD and presumably of other autoimmune diseases.

Comparison of administration of recombinant human thyrotropin with withdrawal of thyroid hormone for radioactive iodine scanning in patients with thyroid carcinoma.

BACKGROUND To detect recurrent disease in patients who have had differentiated thyroid cancer, periodic withdrawal of thyroid hormone therapy may be required to raise serum thyrotropin concentrations to stimulate thyroid tissue so that radioiodine (iodine-131) scanning can be performed. However, withdrawal of thyroid hormone therapy causes hypothyroidism. Administration of recombinant human thyrotropin stimulates thyroid tissue without requiring the discontinuation of thyroid hormone therapy. METHODS One hundred twenty-seven patients with thyroid cancer underwent whole-body radioiodine scanning by two techniques: first after receiving two doses of thyrotropin while thyroid hormone therapy was continued, and second after the withdrawal of thyroid hormone therapy. The scans were evaluated by reviewers unaware of the conditions of scanning. The serum thyroglobulin concentrations and the prevalence of symptoms of hypothyroidism and mood disorders were also determined. RESULTS Sixty-two of the 127 patients had positive whole-body radioiodine scans by one or both techniques. The scans obtained after stimulation with thyrotropin were equivalent to the scans obtained after withdrawal of thyroid hormone in 41 of these patients (66 percent), superior in 3 (5 percent), and inferior in 18 (29 percent). When the 65 patients with concordant negative scans were included, the two scans were equivalent in 106 patients (83 percent). Eight patients (13 percent of those with at least one positive scan) were treated with radioiodine on the basis of superior scans done after withdrawal of thyroid hormone. Serum thyroglobulin concentrations increased in 15 of 35 tested patients: 14 after withdrawal of thyroid hormone and 13 after administration of thyrotropin. Patients had more symptoms of hypothyroidism (P<0.001) and dysphoric mood states (P<0.001) after withdrawal of thyroid hormone than after administration of thyrotropin. CONCLUSIONS Thyrotropin stimulates radioiodine uptake for scanning in patients with thyroid cancer, but the sensitivity of scanning after the administration of thyrotropin is less than that after the withdrawal of thyroid hormone. Thyrotropin scanning is associated with fewer symptoms and dysphoric mood states.

Decreased levels of helper T cells: a possible cause of immunodeficiency in pregnancy.

DECREASED maternal immune responsiveness during pregnancy may partly explain the survival of the fetus as an allograft. It may also account for changes in disease activity and antibody production i...

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 Hashimotos thyroiditis.

Studies of a sibship with apparent hereditary resistance to the intracellular action of thyroid hormone

Abstract Further studies are presented on a previously described sibship 1 with a familial syndrome combining deaf mutism, delayed bone maturition, stippled epiphyses, goiter, and high levels of circulating thyroid hormone in the presence of euthyroid clinical state. Seventy-five to 90% of the circulating iodinated compounds were L-thyroxine and L-triiodothyronine by chromatography, ability to bind to thyroxine-binding globulin, L-amino acid oxidase digestion, and precipitation to constant specific activity. The usual basic laboratory tests were normal, and there was no direct evidence of endocrine disease other than that of the thyroid. Although all standard tests of thyroid function, with the exception of the basal metabolic rate (BMR), were typically those of hyperthyroidism, the patients appeared clinically euthyroid. This diagnosis is supported by the normal BMR, serum lipids, cholesterol, tyrosine, enzymes and albumin metabolism, and by normal caloric intake, dentition, and steady growth. Certain tissues appeared to be resistant to thyroid hormone action. We observed delayed bone age, epiphyseal stippling, and low hydroxyproline excretion. Sensorineural deafness, congenital nystagmus, elevated serum carotene, and possibly metachromasia in the cultured skin fibroblasts were also suggestive of hypothyroidism. Endogenous pituitary thyrotropin secretion was not completely suppresed by the high, blood hormone level. Administration of 1 mg/day L-T 4 or 375 μg/day L-T 3 produced some effect on connective tissues. Thyroid gland activity was only partially suppressed with such treatment. Administration of the acetic acid analogue of triiodothyronine had no effect other than thyroid gland suppression, attributed to the liberation of large amounts of iodine. β-Adrenergic blockage and 45 days of propylthiouracil therapy also failed to produce detectable effects. Labeled thyroxine and triiodothyronine studies indicate that these hormones penetrate the liver and other tissues normally or even at an excess rate and are degrated three- to five fold more rapidly than normal. Progressive amelioration of the syndrome has been noted over the past 6–7 yr. The etiology of this syndrome remains unclear. It appears to date to be a congenital. inherited metabolic and possibly transient defect associated with variable degrees of intracellular resistance to the action of thyroid hormone, which has been partially compensated by excess hormone production.

Reduced nuclear triiodothyronine receptors in starvation-induced hypothyroidism

Abstract Starvation of male rats during 48 hours causes a marked reduction of serum thyroxine, serum triiodothyronine, and liver nuclear triiodothyronine content. Liver nuclear receptors capacity for binding of triiodothyronine was reduced, in contrast to thyro-previc hypothyroidism in which binding capacity is normal. DNA-dependent RNA polymerase activities were reduced. In contrast to typical hypothyroidism, serum thyrotropin was low. This form of pituitary non-responsive hypothyroidism may represent a selective response to caloric and/or amino acid deprivation.

Comparison of 30- and 50-mCi Doses of Iodine-131 for Thyroid Ablation

We compared the utility of lower (30 mCi) and higher (50 to 60 mCi) doses of 131I used to ablate residual thyroid tissue after thyroidectomy for carcinoma. Whole body scans were done using 1 mCi 131I, 3 weeks after withdrawal of triiodothyronine. Patients had received ablation therapy within 3 days after scanning, and one or more subsequent scans were analyzed. Forty-eight patients were treated to ablate residual thyroid tissue that was presumed to be normal. Among 18 patients given the lower dose of 131I as outpatients, 15 had successful ablation and three needed a second administration; all 30 patients treated with the higher dose had successful ablation. Seventeen additional patients, presumed to have residual cancer, received 50 to 150 mCi; and six needed treatment. Although 1 dose of 50 to 60 mCi 131I provides more ablation, use of the usually effective 30-mCi dose for initial ablation is justified by the convenience of outpatient administration, the lower expense, and the lower whole-body radiation dose.

Relation of three polymorphisms of the CTLA-4 gene in patients with Graves’ disease

Graves’ disease is an autoimmune disease believed to be caused by a combination of environmental and genetic factors. One of the candidate genes is CTLA-4, a negative regulator of T cell activation. Three polymorphisms of the gene have been described, in the promoter at position -318, at position 49 in exon 1, and an (AT)n repeat within the 3′-untranslated region of exon 4. Many studies describe the association between a polymorphism of the CTLA-4 gene and autoimmune disease. To investigate the association of these CTLA-4 gene polymorphisms with each other, we analyzed the combined frequencies of each polymorphism and calculated the disequilibrium coefficients. We studied DNA samples from 120 Graves’ disease (GD) patients and 80 healthy donors (NC). The exon 1 position 49 A/G polymorphism and promoter polymorphism at position -318, were typed using a PCR-restriction fragment length polymorphism method (PCR-RFLP). The polymorphic (AT)n repeat in exon 4 was determined by PCR amplification of genomic DNA, resolution of the amplified products on sequencing gels, and detection by autoradiography. There was a significant difference between GD and NC patients and occurrence of the polymorphism in exon 1 and exon 3, but not for the polymorphism in the promoter region. Furthermore, we found that the genotype with both the G allele in exon 1 and the 106 bp allele of the AT repeat in exon 4 occurred with much higher frequency in GD than NC (p<0.01), and that these polymorphisms are in linkage disequilibrium with each other. These results support the concept that CTLA-4 plays a critical role in the autoimmune process in GD, and that GD depends on multiple genetic susceptibility factors. Because the exon 1 and exon 4 polymorphisms are in strong linkage disequilibrium. It is not possible at this time to determine their unique relation to CTLA-4 function. Studies relating each polymorphism to CTLA4 function are required to determine whether one, or both, polymorphism(s) promote autoimmune disease.

Isolation of a complementary DNA clone for thyroid microsomal antigen. Homology with the gene for thyroid peroxidase.

The thyroid microsomal antigen (MSA) in autoimmune thyroid disease is a protein of approximately 107 kD. We screened a human thyroid cDNA library constructed in the expression vector lambda gt11 with anti-107-kD monoclonal antibodies. Of five clones obtained, the recombinant beta-galactosidase fusion protein from one clone (PM-5) was confirmed to react with the monoclonal antiserum. The complementary DNA (cDNA) insert from PM-5 (0.8 kb) was used as a probe on Northern blot analysis to estimate the size of the mRNA coding for the MSA. The 2.9-kb messenger RNA (mRNA) species observed was the same size as that coding for human thyroid peroxidase (TPO). The probe did not bind to human liver mRNA, indicating the thyroid-specific nature of the PM-5-related mRNA. The nucleotide sequence of PM-5 (842 bp) was determined and consisted of a single open reading frame. Comparison of the nucleotide sequence of PM-5 with that presently available for pig TPO indicates 84% homology. In conclusion, a cDNA clone representing part of the microsomal antigen has been isolated. Sequence homology with porcine TPO, as well as identity in the size of the mRNA species for both the microsomal antigen and TPO, indicate that the microsomal antigen is, at least in part, TPO.

Diurnal rhythm in total serum thyroxine levels.

The diurnal variation of serum thyroxine, plasma proteins, and hematocrit were studied in six patients (euthyroid, hypothyroid, and thyrotoxic) and in two obese subjects during total fast. Variations of 7% to 34% were observed in serum total protein, hematocrit, protein-bound iodine, total thyroxine, thyroxine binding globulin, and prealbumin binding capacity, and thyroxine dialyzable fraction, but not in the concentration of serum free thyroxine. Similar and parallel diurnal variation was observed during studies of the disappearance of 131 I-labeled thyroxine and 125 I-labeled albumin. The maximal daily concentration occurred between 10:00 a.m. and 2:00 p.m., and the minimum occurred around 2:00 a.m. These diurnal variations were not related to food intake or adrenocortical function but were influenced by posture. A reversed cyclic change could be produced by reversing the normal sleep-waking pattern. Because of reciprocal changes in the concentration of total serum thyroxine and thyroxine dialyzable fraction, the serum-free thyroxine concentration remained constant. These rhythmic changes are apparently caused by movement of fluid into and out of the vascular compartment. This factor may be an important cause of scatter in the observations recorded during studies of slowly diffusible plasma constituents if repeated blood sampling is not performed under the same conditions.