Thyroid hormones affect plasma ghrelin and obestatin levels.

Using radioimmunoassay, the effects of thyroid hormones on plasma total ghrelin (Gh) and obestatin (Ob) concentrations were evaluated in thyrotoxic patients with an excess of thyroid hormones and in hypothyroid patients lacking endogenous thyroid hormones. 24 patients with thyrotoxicosis, 25 hypothyroid patents after total thyreoidectomy performed due to thyroid cancer, and 17 control subjects were examined. Compared with the controls, the ghrelin and obestatin were elevated in hypothyroidism, while they were decreased in thyrotoxicosis. The plasma Gh and Ob levels differ depending on the thyroid function. In thyroid hormones deficiency, plasma Gh and Ob are increased, while in patients with excess of thyroid hormones, the levels of both Gh and Ob are definitely lower. Gh/Ob ratio is higher in hypothyroidism than in control subjects and thyrotoxic patients.

Naturally occurring mutations in human steroid 21-hydroxylase influence adrenal autoantibody binding.

Human 21-hydroxylase (21-OH) genes containing various mutations, truncations, and deletions were expressed in yeast, and autoantibody binding was studied by Western blotting using patient sera and rabbit antibodies to 21-OH. 21-OH autoantibodies in 13 Addisonian sera showed a marked reduction in their ability to recognize 21-OH mutated at Pro453-->Ser (mean +/- SD, 31 +/- 9% of binding to wild type), whereas the effect on rabbit antibody binding was small (88 +/- 11% of binding to wild type; n = 7). Mutation at Arg339-->His had a less pronounced effect on autoantibody binding (85 +/- 11% of binding to wild type; n = 13) and caused a small enhancement of rabbit antibody binding (124 +/- 16% of binding to wild type; n = 7). These studies indicate that Pro453 has a key role in forming an autoantigenic epitope on 21-OH. It is important to note, however, that the Pro453 mutation caused only partial loss of autoantibody binding, i.e. all Addisonian sera studied still reacted with the mutated protein. This may indicate that each serum sample contains at least two different populations of 21-OH autoantibodies, only one of which recognizes a site dependent on Pro453. A series of more extensive modifications of the 21-OH sequence, including truncations (amino acids 460-494, 448-494, and 418-494) and deletions (amino acids 165-379, 142-240, and 142-280) indicated that most of the sequence of amino acids from 241-494 is important for autoantibody binding. The involvement of such an extensive region of the molecule suggests that the binding sites are generated by three-dimensional folding, with Pro453 having a critical role in forming at least one major autoantigenic epitope.

Autoimmune Addison's disease. Analysis of autoantibody binding sites on human steroid 21-hydroxylase.

Human steroid 21-hydroxylase (21-OH) expressed in an in vitro translation system was found to react specifically with adrenal autoantibodies from patients with Addison's disease. The epitopes on 21-OH which reacted with autoantibodies were studied by incorporating a series of terminal and internal deletions into the 21-OH gene and analysing the expressed proteins by Western blotting. N-Terminal deletions up to amino acid 280 had no effect on autoantibody binding whereas a series of C-terminal deletions and truncations (amino acids 281-494) showed marked effects. Our results indicate that a central segment (281-379) and a C-terminal segment (380-494) of 21-OH interact to form at least one major autoantibody binding site.

Steroid 21-hydroxylase is a major autoantigen involved in adult onset autoimmune Addison's disease.

An adrenal-specific protein reacting with autoantibodies in the sera of patients with adult onset Addison's disease has been purified from human adrenal glands. The protein, mol.wt. 55K, has the biochemical characteristics of steroid 21-hydroxylase and reacts on Western blots with rabbit antibodies to recombinant 21-hydroxylase. Absorption of the native human 55K adrenal protein with human adrenal autoantibodies prevented the subsequent reaction of the 55K protein with rabbit antibodies to 21-hydroxylase in Western blot analysis. In addition, human adrenal autoantibodies reacted with recombinant 21-hydroxylase expressed in yeast. These data indicate that the adrenal specific enzyme steroid 21-hydroxylase is a major autoantigen involved in adult onset autoimmune Addison's disease.

Expression of human thyroid peroxidase in the yeasts Saccharomyces cerevisiae and Hansenula polymorpha.

Saccharomyces cerevisiae and the methylotrophic yeast Hansenula polymorpha have been used to express both full-length and a large hydrophilic domain of human thyroid peroxidase (TPO). Expression of TPO in S. cerevisiae, using the natural signal sequence or the yeast alpha-mating factor (MF alpha) signal sequence, resulted in undetectable or very low levels of recombinant TPO production. However, TPO was expressed when the natural TPO leader sequence was replaced by the yeast STE2 signal sequence. This recombinant TPO reacted with both rabbit anti-human TPO polyclonal and mouse anti-human TPO monoclonal antibodies on Western blots. In the case of H. polymorpha, TPO expression was achieved when the natural TPO leader sequence was replaced by the MF alpha leader and the construct placed under the control of the methanol-regulated promoter from the methanol oxidase gene. The recombinant TPO produced in H. polymorpha reacted with both TPO polyclonal and TPO monoclonal antibodies. No TPO was produced when the signal sequence of SUC2 (invertase) or the TPO natural signal sequence was used to direct expression.

[Method for detection of antitubulin antibodies. Results of assays in autoimmune thyroid diseases]. / Metoda wykrywania autoprzeciwcial antytubulinowych. Wyniki oznaczen w autoimmunologicznych chorobach tarczycy.

A cytoskeletal protein--tubulin was isolated from the cerebral tissue by the sequential microtubule polymerization and depolymerization technique described by Schelansky. The purity of the isolated protein was verified by SDS polyacrylamide gel electrophoresis. The binding reaction with monoclonal antibodies against various cytoskeletal proteins confirmed the presence of pure tubulin. The isolated tubulin was used as reactant in a solid phase radioimmunoassay for the detection of antitubulin antibodies in tubulin-coated plastic tubes. Antitubulin antibody assays were performed in 20 cases of hypothyroidism and 56 cases of Graves' disease. In 70 percent of cases of hypothyroidism and 50 percent of cases of Graves' disease the levels of antitubulin autoantibodies were elevated. The highest titres of antibodies had some patients with Graves' disease. The antitubulin autoantibodies were of the IgM class.

Polyendocrine autoimmunity in thyroid diseases.

Anti-adrenal and anti-pituitary autoantibodies have been determined in 45 patients with autoimmune diseases of the thyroid, including 25 patients with Graves' disease and 20 patients with hypothyroidism of autoimmune origin. The determinations were carried out with the use of solid-phase RIA methods previously developed by us, involving polyethylene tubes coated with the solubilized microsomal fractions obtained from human adrenal and pituitary glands. In the majority of patients with autoimmune diseases of the thyroid the presence of both anti-adrenal and anti-pituitary autoantibodies was detected. In 13 among 20 patients with hypothyroidism of autoimmune origin, the presence of anti-adrenal autoantibodies, and in 12 the presence of anti-pituitary autoantibodies was found. Among 25 patients with Graves' disease, 19 had both anti-adrenal and anti-pituitary autoantibodies. In the majority of patients with autoimmune diseases of the thyroid the titers of autoantibodies of both types were high but in no case were the clinical symptoms of adrenal or pituitary hypofunction observed. Our studies indicate that the thyroid diseases of autoimmune origin can be regarded as manifestation of some more generalized autoimmunization process.

[Detection of pituitary autoantibody in patients with pituitary tumors and hypopituitarism]. / Wykrywanie autoprzeciwcial przysadkowych u chorych z gruczolakami przysadki i w niedoczynnosci przysadki.

UNLABELLED: In 80 patients, 73 cases of pituitary tumours and 7 cases of hypopituitarism, we performed pituitary autoantibodies assays in serum samples because in our previous studies we had found a high prevalence of pituitary autoantibodies in several autoimmune endocrine disorders. To detect the presence of pituitary autoantibodies we applied 2 methods, radioimmunoassay (RIA) and immunoblotting. The RIA was performed by solid phase technique in human pituitary microsome-coated polyethylene tubes. Following incubation with diluted sera of the patients labelled 125I-protein A was added to the tubes to detect the retained antibodies. In the sera of 33 patients we detected the presence of antibodies; in the other 47 patients no antibodies were found. The majority of the patients with positive antibody results were previously treated by pituitary irradiation. To evaluate the molecular weights of pituitary autoantigens the microsomal proteins were separated on SDS PAGE, then electrophoretically transferred to nitrocellulose membranes and reacted with diluted sera of 30 antibody-positive patients. The nitrocellulose strips were incubated with labelled 125I-protein A and autoradiographed. Using immunoblotting, 13 out of these 30 patients we found autoantibodies reacting with pituitary microsomal antigens of different molecular weights, most frequently reacting with a 68 kDa autoantigen. CONCLUSIONS: The prevalence of pituitary autoantibodies in patients with pituitary diseases is 41% lower than in autoimmune endocrine diseases. Pituitary autoantibodies usually appear in patients after pituitary irradiation or after neurosurgery followed by irradiation, but occur rarely in untreated patients with pituitary adenomas.

[Bone mineral density in primary hyperparathyroidism]. / Gestosc mineralna kosci w pierwotnej nadczynnosci przytarczyc.

UNLABELLED: Skeletal complications of advanced hyperparathyroidism include clinically bone pains, muscle weakness, bone deformities and fractures. X-ray studies reveal subperiosteal bone resorption, atrophy of the cortex of long bones, cysts, brown tumours and calcifications of soft tissues; these changes appear in the late period of the disease. In recent onset of hyperparathyroidism bone changes may be detected by X-ray absorptiometry. Thus the aim of our study was to evaluate bone mineral density with the use of dual energy X-ray absorptiometry (DEXA) at two sites: in lumbar vertebral bodies consisting mainly of the trabecular bone and in 1/3 distal part of the radius composed predominantly of the cortical bone. Twenty-three patients with primary hyperparathyroidism were included in our study. Hypercalcemia (ionized calcium above 5.4 mg/100 ml, total calcium above 10.6 mg/100 ml) and increased serum PTH, above 100 pg/ml, were detected in all patients. Isotope scintigraphy using 99mTc-MIBI revealed the presence of a parathyroid adenoma; this was confirmed at surgery and histopathologically. In bone densitometry we found greatly reduced bone mineral density (BMD) in 1/3 distal part of the radius amounting to 66.8 +/- 12.0% of the age-matched range and markedly smaller bone loss in lumbar spine, BMD was 91.7 +/- 14.6%. In 10 patients control densitometry, performed 6-24 months after parathyroid adenomectomy, revealed a marked 10 to 22% increase in bone density of lumbar vertebral bodies in the first year. BMD of the 1/3 distal part of the radius increased to a smaller degree 6.3% per year. CONCLUSIONS: 1. Bone densitometry in primary hyperparathyroidism reveals pronounced decrease in bone mineral density in the 1/3 distal part of the radius and much smaller decrease of the lumbar spine density. 2. Parathyroid adenomectomy leads to a rapid increase in density of the trabecular bone L1-L4 vertebral bodies and much smaller increase in the cortical bone of the radius. 3. Pronounced differences in bone mineral density of cortical bone and trabecular bone surpassing 20% are characteristic of hyperparathyroidism as they do not occur in other types of osteoporosis.