To elucidate the associations of the HLA class II alleles with Graves' disease (GD), we examined DRB1, DQA1, DQB1 alleles in 62 Japanese GD patients and 142 control subjects by the PCR-SSOP (polymerase chain reaction-sequence specific oligonucleotide probes) method. We found that DRB1*0803 (P<0.02), DRB1*1403 (P<0.03), DQA1*0103 (P<0.02) alleles and DRB1*0803-DQA1*0103-DQB1*0601 (P<0.01), DRB1*1403-DQA1*0501-DQB1*0301 (P<0.02) haplotypes were significantly increased in GD patients. No DQB1 allele revealed a significant association with GD in Japanese. These weak associations may reflect either the heterogeneity of GD in Japanese or the importance of non-HLA factors in the development of the disease.

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In order to clarify the mechanism of antiatherogenic action of several steroids such as estrogens, dehydroepiandrosterone (DHEA) and dexamethasone, we investigated the effects of various steroids on the copper (Cu²⁺)-catalyzed oxidation of low density lipoprotein (LDL) or high density lipoprotein (HDL) in 0.15 M NaCl by measuring thiobarbituric acid-reactive substances (TBARS). At a concentration of 10^-5 M, estrogens strongly protected against LDL oxidation by 0.5 μM Cu²⁺ in the following order of inhibition: estradiol (E₂)(75%), estrone (E₁)(35%) and estriol (E₃)(30%). However, the corresponding metabolites of these estrogens, the catechol estrogens, had an even more protective effect on LDL oxidation by 0.5 iM Cu2+ in the following order of inhibition: 2-hydroxyestradiol (2-OHE₂)(98%), 2-OHE1 (97%) and 2-OHE₃ (96%). E₂ and 2-OHE2 from 10-7 M to 10-5 M inhibited LDL oxidation in a dose-dependent manner, with a more marked effect for oxidation by 0.1μM Cu²⁺ than by 0.5μM Cu²⁺. 10^-5 M dexamethasone produced a slight (10%) but significant inhibition of LDL oxidation by 0.5 μM Cu²⁺. In addition, the estrogens and catechol estrogens were also effective in protecting against HDL oxidation by 0.5μM Cu²⁺. Other steroids including DHEA and DHEA-sulfate had no antioxidative effects on either LDL or HDL in this system. These results indicate that estrogens and their metabolites, the catechol estrogens, exert antioxidative effects on both LDL and HDL. The catechol estrogens may be more important antioxidants than estrogens for both LDL and HDL. Dexamethasone may exert its antiatherogenic effect partly by inhibiting the oxidation of lipoproteins, but this may not be the case for DHEA and DHEA-sulfate.

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Four cases of polyglandular endocrine disorders associated with pituitary hormone secretion failure are reported. Three of them had both insulin dependent diabetes mellitus (IDDM) and Hashimoto's disease. Each of these patients (cases 1-3) showed isolated deficiency of ACTH, TSH or gonadotropin, respectively. Another patient (case 4) had both Hashimoto's disease and isolated ACTH deficiency. Anti-pituitary antibody to AtT-20 cells was detected in case 1. Serum gamma-globulins from patients 1 and 4 attenuated corticotropin releasing hormone-induced ACTH release in monolayer cultured rat anterior pituitary cells. Gamma-globulins from patients 1 and 2 decreased baseline TSH release but stimulated baseline prolactin release in pituitary cell cultures. It is possible that pituitary hormone deficiency in these patients may be caused by autoimmune disorders.

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One of the determinants of cellular responsiveness to glucocorticoid hormone is the concentration of the receptor protein. It is well-known that cellular receptor levels are down-regulated by the cognate ligands, but the biological significance of this homologous down-regulation of the receptor has not yet been completely understood. We showed that in human histiocytic lymphoma cell line U937 the cellular glucocorticoid receptor was homologously down-regulated by means of both ligand binding and Western immunoblot experiments. Reduction of the receptor was saturable, and the receptor levels tended to return to the levels before treatment after 3-day culture in the presence of the hormone. Next, using the cells which were pretreated with the hormone for 0 to 3 days, hormonal inducibility of the transiently-transfected reporter gene and the inhibitory effect of the hormone on cellular 3-O-methyl glucose uptake were determined. Hormonal inducibility of the reporter gene was progressively reduced, and thereafter tended to be restored, apparently in accordance with the cyclic change in amount of the receptor. The glucose uptake inhibiting effect of the hormone also revealed this cyclic pattern. In summary, in U937 cells glucocorticoid receptor was homologously down-regulated and may play a pivotal role in attenuating hormone responsiveness.

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This paper reports a rare case of isolated ACTH deficiency associated with anti-pituitary antibodies, pituitary cyst, sphenoidal cyst and pineal tumor. A 68-year-old man consulted our clinic for general fatigue. Laboratory data showed low plasma adrenocorticotrophin (ACTH) and cortisol levels with blunted responses to insulin-induced hypoglycemia and corticotrophin releasing factor (CRF). Urinary 17-OHCS was low but responded to ACTH-Z administration. No other pituitary functions were impaired. Antibodies to the cytoplasm of rat pituitary and the surface of GH3 cells were detected in the serum. The magnetic resonance imaging (MRI) showed a high signal intensity mass in the anterior pituitary and in the sphenoidal sinus in both T1 and T2 weighted images as well as a low signal intensity mass in a Ti weighted image of the pineal region. Transsphenoidal surgery was performed to resect the mass in the sphenoid sinus and in the pituitary. Pathological studies showed a benign cyst in the sphenoid sinus, and fibrous degeneration and decreased basophils in the pituitary. No infiltrative mononuclear cells were detected in the pituitary. Immunohistochemical studies revealed a decrease in the number of ACTH-producing cells in the pituitary. The patient was well maintained by glucocorticoid replacement without any growth of a possibly benign pineal tumor.

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We herein report familial defects in the molecular structure of luteinizing hormone (LH). The propositus was a 29-year-old woman with repeated abortion in whom the serum LH concentration was extremely low determined by an immunoradiometric assay utilizing two monoclonal antibodies for the intact LH dimer and β-subunit of LH (SPAC-S kit). Further studies on the serum LH concentration in this propositus by five different assay systems gave various results ranging from low to normal values. Bioassay of the propositus's LH using C57 black mice showed normal in biological activity. These data suggest that LH in the propositus is abnormal in terms of its molecular structure located in the bond region between α and β subunits.
A family study showed that the bioactivity of LH was normal in all family members. The serum LH was not detected in either the propositus or her brother with the SPAC-S kit even after the administration of LHRH, while the serum LH concentrations in the father, mother and sister were approximately half the normal range, indicating that the defect is hereditary and the mode of inheritance is autosomal dominant: the propositus and brother were homozygously affected, and the parents and sister heterozygously affected.

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Deposition of glycosaminoglycan is one of the histological features of Graves' ophthalmopathy. Although retroocular tissue fibroblasts are considered to be responsible for glycosaminoglycan accumulation, it is not known what is stimulating the fibroblasts. There are studies which are in support of and against the role of and-TSH receptor antibodies in the pathogenesis of Graves' ophthalmopathy. TSH-receptor antibodies increase cAMP as a second messenger in thyroid cells. We studied the effects of dibutyryl cyclic AMP (Bt₂ cAMP) on glycosaminoglycan synthesis by retroocular tissue fibroblasts in order to know whether cAMP can modulate glycosaminoglycan synthesis. Retroocular tissue fibroblasts mainly synthesize hyaluronan, the large chondroitin sulfate proteoglycan and the small chondroitin sulfate proteoglycan as glycosaminoglycan in cell culture. The amount of hyaluronan synthesis was measured as [³H] glucosamine incorporation into macromolecule susceptible to hyaluronidase digestion (from Streptomyces hyaluronlyticus). The amount of proteoglycan synthesis was measured as [³⁵S] sulfate incorporation into macromolecules in medium and cell layer fraction. Proteoglycans in medium were further separated into the large proteoglycan and the small proteoglycan on a Superose 6 column. Bt₂ cAMP increased both hyaluronan and proteoglycan synthesis by retroocular tissue fibroblasts, especially stimulating the secretion of the large proteoglycan. Effects of Bt₂ cAMP on glycosaminoglycan synthesis were then compared with those in adult skin fibroblasts. Although the magnitude of response between the two was indistinct, the stimulation of the large proteoglycan synthesis by Bt₂ cAMP was more prominent in retroocular tissue fibroblasts. The results suggest that the regulation of glycosaminoglycan synthesis by retroocular tissue fibroblasts is different from that by adult skin fibroblasts. Although further studies are required to determine its actual role, cAMP stimulates glycosaminoglycan synthesis by retroocular tissue fibroblasts and underlies the mechanism in Graves' ophthalmopathy.

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The purpose of this study was to investigate the effects of pamidronate, a second generation bisphosphonate, on the change in calcium homeostasis in patients with tumor-associated hypercalcemia. Eight patients with tumor-associated hypercalcemia received intravenous infusion of pamidronate (45 mg) and their high mean serum calcium concentration significantly decreased from 3.56 mmol/L to 2.62 mmol/L 7 days after treatment. Serum intact PTH before treatment had been suppressed to below normal in all patients but returned to normal range in six patients within 7 days after treatment. Urinary PTH related peptide (PTHrP) excretion before treatment had been elevated in seven patients and then significantly increased further after pamidronate therapy. The serum bone Gla protein concentration was not apparently changed by the treatment. Pamidronate in serum was rapidly eliminated after the treatment and urinary excretion reached a plateau on the second day (13.8% of the administered dose), suggesting that the major portion of the infused dose had been distributed to the bone and other tissues. These findings suggest that pamidronate has a potent hypocalcemic effect and that PTHrP production in malignant tumors could be affected by pamidronate therapy.

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In order to investigate the immunological aspects of silent thyroiditis, T lymphocyte subsets were analyzed by two-color flow cytometry in 10 patients with silent thyroiditis in the thyrotoxic phase, 12 patients with silent thyroiditis in the recovery phase, and 11 healthy volunteers as a control. The percentages of CD3⁺ CD4⁺, and CD8⁺ cells were similar in all three groups. In contrast, activated matured T (HLA-DR⁺CD3⁺), activated helper/inducer T (HLA-DR⁺CD4⁺) and activated suppressor/cytotoxic T (HLA-DR⁺CD8⁺) cells were more numerous in both the thyrotoxic and the recovery phases of patients with silent thyroiditis when compared with healthy controls. In a serial study of 6 patients with silent thyroiditis, the percentage of activated helper/inducer T (HLA-DR⁺CD4⁺) cells was higher in the thyrotoxic phase than in the recovery phase. These data indicate that the activation of T cells, especially of helper/inducer T cells, might be important for the induction of silent thyroiditis.

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In order to clarify the insulin-release mechanism of β₃-adrenoceptor agonist, we examined the effect of CL 316, 243, a highly specific β₃-adrenoceptor agonist having a relative potency of β₁:β₂:β₃ = 0: 1: 100, 000, on insulin secretion in perfused mouse pancreas. The application of 0.2 mM and 0.5 mM CL 316, 243 produced significant insulin secretion from within 1 min, which lasted until 2 min after its withdrawal. DL-propranolol and ICI 118551 at 0.2 mM partially inhibited the insulin secretion induced by. the 0.2 mM CL 316, 243, but 0.2 mM metoprolol had no effect on the insulin release produced by 0.2 mM CL 316, 243. We conclude that β₃-adrenoceptor agonist stimulates insulin secretion via β₃-action in the perfused mouse pancreas.

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Synergistic insulin release is observed in combination with muscarinic agonist and glucose. In order to define the role of changes in the cytoplasmic free Ca2+level ([Ca²⁺] i), the effect of combination with carbachol and glucose on both phosphoinositide breakdown in rat pancreatic islets and [Ca²⁺] i dynamics in single fura 2-loaded B-cells was investigated. When the islets were prelabeled with myo-[2-³H] inositol, phosphoinositide breakdown was obtained and inositol trisphosphate levels were increased in the presence of carbachol (0.5 mM). Inositol trisphosphate levels induced by carbachol were further increased by co-stimulation with 16.7 mM glucose.[Ca²⁺] i in single-B-cells was increased by 16.7 mM glucose. A prompt, transient rise in [Ca²⁺] i was induced by carbachol. However, carbachol failed to augment the [Ca²⁺] i response in the presence of 16.7 mM glucose. These data suggest that a synergistic release of insulin occurs without an amplification of [Ca²⁺] i regardless of an increase in inositol trisphosphate.

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Previously we found intragranular colocalization of immunoreactive endothelin-1 and neurohypophysial hormones in the axon terminals of the rat neural lobe. To investigate the function of endothelin-1 in the rat neural lobe, immunoreactive endothelin-1 in plasma and the neural lobe was measured by enzyme immunoassay in rats subjected to hemorrhage and in other rats who were deprived of water for 2 days to induce dehydration. Changes in plasma arginine vasopressin were determined by radioimmunoassay. In addition, morphometric analysis was performed in the neural lobe of rats exposed to these stresses. Plasma concentrations of immunoreactive endothelin-1 were unchanged following hemorrhage or dehydration, whereas those of immunoreactive vasopressin were remarkably increased. In the neural lobe, immunoreactive endothelin-1 content and the number of neurosecretory granules decreased significantly after dehydration. However, immunoreactive endothelin-1 in tissue increased nearly three-fold in hemorrhaged rats, whereas the endothelin-1-immunolabeling in the axon terminals was unchanged. These results suggest that endothelin-1 in the hypothalamo-hypophysial system may be involved in the local modulation of vasopressin secretion when an animal is exposed to hypovolemic and/or osmotic stress.

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The cytologic localization and cellular levels of TGFα in the human ovary during follicular growth and regression were examined by avidin/biotin immunoperoxidase techniques with a monoclonal antibody to TGFα. In primordial follicles, only the oocyte showed intense immunostaining for TGFα, whereas the flattened pregranulosa cells were negative for the immunostaining. The earliest stage of follicular growth at which immunostaining for TGFα in granulosa cells and theca interna cells became apparent was the preantral stage. With the increase in the size of follicles, the intensity of TGFα immunostaining in the oocyte decreased, whereas the staining intensity of the granulosa and theca cells increased. The immunostaining for TGFα in granulosa and theca interna cells persisted in the corpus luteum, and further intensified during the midluteal phase. In the regressing corpus luteum, the immunostaining was present only in the peripheral theca lutein cells adjacent to the central scar tissue. The corpus albicans was negative for the immunostaining. Stromal cells exhibited weak staining for TGFα.In atretic follicles, the theca interna cells exhibited intense staining for TGFα without appreciable staining in the scattered granulosa cells. This is the first report to demonstrate that TGFα expression in the oocyte is maximal in primordial follicles, whereas TGFα expression in granulosa and theca cells increases with the progress of follicles and reaches its maximum in the lutein cells during the mid luteal phase. These features of developmental stage-dependent expression of TGFα in the human ovary suggest the possible participation of TGFα in the regulation of initial growth of the oocyte in early folliculogenesis and steroidogenic function of growing follicles. Furthermore, the increased expression of TGFα in the theca interna cells of atretic follicles and theca lutein cells peripheral to the regressing corpus luteum implies that TGFα may participate in the transformation of theca cells into stromal cells to remodel the local tissue following atresia and luteolysis in the human ovary.

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Changes in serum immunoreactive (IR)-inhibin were measured by RIA in two studies, in order to elucidate, firstly whether the pattern of IR-inhibin secretion is similar to that of estradiol (E2), and secondly, whether inhibin suppresses endogenous FSH release. Study 1: Purified urinary FSH (pFSH) or human menopausal gonadotropin (hMG) were daily injected intramuscularly into women with hypogonadotropic amenorrhea at 12 to 14 week intervals. PFSH and hMG stimulated IR-inhibin release in a similar fashion in the ovulatory cycles, but the increase in estradiol (E2) during pFSH administration was delayed and lower than that during the hMG cycles. This suggests that E2 and IRinhibin are secreted independently from the granulosa cells. Study 2: Ovulation induction was performed in 18 cycles of 9 women with polycystic ovarian disease (PCOD) by the step-down administration of pFSH. The serum FSH concentration in cycles with premature LH release increased even after the dose of pFSH was reduced, and were significantly higher than those of cycles without premature LH release. It was also found that the serum IR-inhibin concentration in cycles with the premature LH release was 2 to 4 times as high as in cycles without premature LH release. This suggests that IR-inhibin does not suppress endogenous FSH release associated with premature LH release.

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To examine the androgen receptor in androgen-dependent and-independent tumor immunohistochemically, an indirect immunofluorescence study with an antibody to human androgen receptor was performed. Shionogi Carcinoma 115 (SC 115) cells are an androgen-dependent mouse tumor, but the growth is sustained without androgen when fetal bovine serum is added to serum-free medium. Cells obtained from successive culture (A (-) X cells; X is generations after removal of androgen) were androgen-independent but showed binding to androgen. SC 115 cells, A (-) cells and CS 2 cells which are the other androgen-independent cells derived from SC 115, were used in the study. The androgen receptor (AR) in SC115 cells was stained as small-sized oval granules localized in the nucleus, and the number of the granules was 10-20 per cell. Removal of testosterone for one day as well as one week did not change the size of the AR, but some of the AR in A (-) 10 cells and in generations thereafter appeared to be large. Other small ones were similar to that in SC 115 cells. The nuclear location of the AR did not change in A (-) cells. The ratio of cells containing large AR to the total number of cells increased with each generation after the removal of testosterone from the culture. The addition of testosterone to the culture changed the AR in A (-) 40 cells to small ones, but did not influence the form of the AR in A (-) 60 cells. The AR in CS 2 cells had a similar appearance to that in A (-) 60 cells and did not change the form by addition of testosterone. These results suggest that AR in the functional state is small and oval in shape, and long term removal of testosterone gradually change the AR to a large one. The return of the large form of AR to a small one occurs within a limited period in the absence of androgen, but thereafter no reversibility is noticed.

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We evaluated the effect of the β-adrenergic receptor blocking agent nadolol on serum levels of free thyroid hormones in 20 untreated patients with Graves' disease, aged between 17 and 57 years (mean±SD, 32.9±10.5). All the patients were treated with 30mg of nadolol alone once-daily for 2 weeks, and clinical and laboratory parameters before and after the treatment were compared. Systolic blood pressure was depressed significantly (P<0.001) from 132.7±11.1 mmHg to 122.8±10.9mmHg. The resting pulse rate was also reduced significantly (from 110.3±9.2/min to 86.0±13.2/min, P<0.001). Serum free thyroxine levels were reduced significantly from 7.1±3.1ng/dl to 5.7±3.6 ng/dl (P<0.05) and serum free triiodothyronine levels were reduced from 21.9±5.6pg/ml to 17.2±6.9pg/ml (P<0.01). In the present study, the β-adrenergic receptor blocking agent nadolol was newly found to have a reducing effect on serum free thyroxine, as well as free triiodothyronine concentrations.

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Information about the plasma IGF-I concentrations in domestic animals in a cold environment is still limited. And mechanisms to change plasma IGF-I concentrations in cold environments are not fully elucidated. In this study, plasma insulin-like growth factor-I (IGF-I) in relation to plasma growth hormone (GH) and metabolite concentrations was investigated in pigs living at 20°C and at 4°C. Six pigs (Landrase breed; barrows, 118 days old, 51.0±3.5 kg body weight) were maintained for 2 weeks at 20°C in a climatic room. Then a placebo or recombinant bovine GH (100μg/kg body weight) was injected subcutaneously. Blood samples were taken through a catheter at -2, 0, 1, 2, 3, 4, 5, 6, 9, 12, 22, and 24 h after the injections. The same experiments were conducted on days 5 and 6 after the room temperature was changed to 4°C. Mean (±SD) basal plasma GH concentrations in pigs without bovine GH administration living at 20°C and at 4°C were 4.8±1.7 ng/ml and 4.6±2.8ng/ml, respectively. There were no significant differences between GH concentrations. On the other hand, the mean plasma IGF-I concentrations were 80.8±25.1ng/ml and 57.3±14.3ng/ml respectively. Plasma IGF-I concentrations in pigs living at 4°C were significantly lower than in pigs living at 20°C (P<0.05). Plasma glucose and non-esterified fatty acid (NEFA) concentrations in pigs at 4°C were significantly higher than in pigs at 20°C (P<0.05). Blood urea nitrogen (BUN) concentrations in pigs at 4°C were also higher than in pigs at 20°C. In an experiment on GH administration, the plasma GH concentrations in pigs at both 20°C and 4°C were increased to the peak (49.1±2.5ng/ml and 43.0±23.6 ng/ml, respectively) 2h after the GH injection. They then gradually decreased to the basal level within 22 h. Plasma IGF-I concentrations were significantly increased 3 or 4h after the GH injection, and they reached to the maximum 9 or 12h after GH injection. No statistical significance was observed in the increase in the plasma IGF-I concentrations between pigs living at 20°C and at 4°C after GH injection. These results indicate that basal plasma IGF-I concentrations in pigs living at 4°C were lower than at 20°C. And the increase in the plasma IGF-I after the bovine GH injection were not different at the two environmental temperatures.

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We treated a diabetic patient with familial multiple endocrine neoplasia type 1 (MEN 1) who had undergone total pancreatoduodenectomy. The patient received insulin and showed signs of symptomatic primary hyperparathyroidism (PHPT). The insulin requirement to control blood glucose before and after parathyroidectomy was compared by using an artificial pancreas. The insulin infusion rate during the day and at night was reduced to about one-third and half, respectively, after parathyroidectomy with autotransplantation of parathyroid tissues into the forearm. The daily insulin dose was reduced from 36 units to 14 units 2 weeks after surgery, and glycemic control showed further improvement 2 months after surgery with the same dose of insulin for up to 6 months. These observations suggest that insulin sensitivity increases after surgical correction of PHPT.

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The relative concentrations of total insulin receptor (IR) mRNA were measured in the epididymal adipose tissue and liver of intact untreated rats, dexamethasone-treated rats, adrenalectomized rats and adrenalectomized rats treated with dexamethasone, using RNA blot assays and a specific IR cDNA probe. Northern blot assays revealed two IR mRNA species of approximately 9.5 and 7.5 kb, in both tissues. Dot-Blot assays followed by densitometry indicated that dexamethasone induced an approximately three-fold increase in IR mRNA in liver, but not in epididymal adipose tissue. By contrast, neither adrenalectomy alone nor the combination of adrenalectomy plus dexamethasone treatment altered the IR mRNA concentrations in liver nor in adipose tissue, which indicates that adrenalectomy was able to prevent the stimulation of IR gene expression caused by dexamethasone in rat liver. These results provide evidence for an “in vivo” tissue-specific regulation of IR gene expression, at the mRNA level, in rats under experimental conditions of an excess or insufficiency of glucocorticoids.

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A 49-year-old Japanese woman was referred to our department for evaluation of a thyroid nodule. She underwent subtotal thyroidectomy with modified neck dissection for a follicular thyroid carcinoma, suspected on preoperative diagnosis. The histological diagnosis was tuberculous thyroiditis. She made an uneventful recovery and received antituberculous agents. At follow-up she remains well and is euthyroid. Reports on forty-four patients in the Japanese literature were read. Tuberculous thyroiditis must be differentiated from thyroid cancer and subacute thyroiditis. Surgery plus administration of antituberculous drugs is considered the treatment of choice.

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