A germline mutation, 1001delC, of the multiple endocrine neoplasia type 1 (MEN 1) gene in a Japanese family.

Multiple endocrine neoplasia type 1 (MEN 1) is an autosomal dominant inherited disorder characterized by tumors of the enteropancreas, parathyroid and anterior pituitary. The MEN 1 gene was recently cloned, and germline mutations of the gene have been demonstrated in cases of MEN 1. Here, we report a Japanese family with a germline mutation of the MEN 1 gene. The proband (44 y.o., male) had primary hyperparathyroidism (PHP) and pancreatic carcinoid, and his older sister (50 y.o.) had a history of parathyroidectomy for primary hyperparathyroidism at the age of 40. Clinical examination revealed no evidence of PHP or other MEN 1-related tumors in his son. Direct sequencing analysis revealed a heterozygous germline mutation (1001delC) at codon 297 in exon 6 of the MEN 1 gene in the proband and his son. Loss of heterozygosity (LOH) was also found in the resected parathyroid tissue of the proband. The deletion of cytosine 1001 observed in this case induces a frame shift, which causes the appearance of a stop codon (TAG) at codon 367. This mutation appears to be associated with tumors of the endocrine tissues in the cases studied.

Regulation of calcitonin receptor by glucocorticoid in human osteoclast-like cells prepared in vitro using receptor activator of nuclear factor-kappaB ligand and macrophage colony-stimulating factor.

Using mouse osteoclast-like cells (OCs), we have shown that treatment with glucocorticoids (GCs) resulted in an increase in calcitonin (CT) binding by enhancing CT receptor (CTR) gene transcription. Additionally, treatment with GCs demonstrated increased sensitivity to CT. There is, however, scant information on the effects of GC or CTR regulation by GCs in human osteoclasts. In this study we examined CTR regulation by GCs and the effects of GCs and CT together in human OCs. OCs were prepared by treatment of peripheral blood mononuclear cells in vitro with soluble receptor activator of nuclear factor-kappaB ligand and macrophage colony-stimulating factor. Treatment of mature OCs with dexamethasone (Dex) resulted in a dose- and time-dependent increase in [(125)I]salmon CT (sCT) binding capacity. Treatment with Dex enhanced CTR messenger RNA (mRNA) expression, suggesting that CTR up-regulation is at least partly due to an increase in de novo CTR synthesis. Triamcinolone and prednisolone reproduced the Dex effect on [(125)I]sCT-specific binding and CTR mRNA expression, but 17beta-estradiol, progesterone, dehydroepiandrosterone, and aldosterone did not. A Scatchard plot analysis showed that Dex enhanced CTR number with a minimal change in the affinity to sCT. Autoradiographic studies using [(125)I]sCT showed that Dex enhanced the CTR density on individual multinuclear OCs. Up-regulation of [(125)I]sCT-specific binding and CTR mRNA expression was seen even in the presence of sCT, but the enhancement diminished subsequently at later times (36-48 h after sCT removal), which was consistent with our previous observation in mouse OCs. This suggests that GCs and CTs act on CTR expression differently, consistent with our previous work using mouse OCs, in which we found that GCs increased transcription of CTR gene expression, whereas CT reduced CTR mRNA stability. The results obtained in this study show that GC increased CTR expression and sensitivity to CT in cells of the human osteoclast lineage and provide the basis for understanding the beneficial effects of combination treatment with GCs and CTs in malignancy-associated hypercalcemia.

Expression of fibrillins and other microfibril-associated proteins in human bone and osteoblast-like cells.

Fibrillin-containing microfibrils are structural components of extracellular matrices of a diverse range of tissues, including bone. Their importance in bone biology is illustrated by the skeletal abnormalities manifest in the congenital disorder, Marfan syndrome, which results from mutations in the fibrillin-1 gene. We investigated the expression of fibrillins and other microfibril-associated proteins in human bone and bone-derived osteoblasts. Analysis of RNA extracted from cancellous bone showed expression of mRNAs encoding fibrillin-1 and -2, MAGP-1 and -2, LTBP-2, and MP78/70 (Big-h3). In demineralized normal mature bone, fibrillin-1 was immunolocalized to fibrils within the bone matrix and pericellularly to cells lining the endosteal surfaces of trabecular bone, some osteocytes, and cells associated with blood vessels. LTBP-2 was also identified at the endosteal surface and within the bone matrix in a lamellar fashion. In addition, primary osteoblast-like cells cultured from human trabecular bone (obtained from patients at joint replacement surgery) were found to express abundant mRNA for fibrillins and associated glycoproteins. Moreover, using western blot analysis, fibrillin-1 protein was shown to be secreted into the medium and to be deposited into the cell layer. Immunofluorescence staining of the cell layer visualized fibrillin-1 in the matrix as a three-dimensional network of fine filaments. Expression of fibrillin-1 by osteoblast-like cells was constitutive, and a number of skeletally active agents had little effect on mRNA or protein levels. These results show that human osteoblasts from mature bone express fibrillins and other microfibril-associated proteins, and suggest a role for these molecules in adult human bone.

Hypercalcemia accompanied by hypothalamic hypopituitarism, central diabetes inspidus and hyperthyroidism.

We present here a case of prominent hypercalcemia accompanied by hypothalamic tumor and Graves' disease. A 24-year-old man with hypothalamic tumor showed hypopituitarism, central diabetes inspidus (DI) and hyperthyroidism. Nausea, loss of thirst and appetite, and general fatigue were found with the unveiling of hypercalcemia and hypernatremia. Parathyroid hormone (PTH) and 1alpha-dihydroxyvitamin D levels were suppressed with a normal range of PTH-related protein values. One-desamino-(8-D-arginine)-vasopressin (DDAVP) and half-saline administration normalized hypernatremia, while hypercalcemia was still sustained. Administration of cortisone acetate and thiamazole reduced the elevated serum Ca level. In the present case, concurrent hyperthyroidism was assumed to accelerate skeletal mobilization of calcium into the circulation. Hypocortisolism and central DI was also considered to contribute, to some extent, to the hypercalcemia through renal handling of Ca.

Increased serum vascular endothelial growth factor levels and intrathyroidal vascular area in patients with Graves' disease and Hashimoto's thyroiditis.

Vascular endothelial growth factor (VEGF) is one of the angiogenic factors. We examined both thyroid volume and intrathyroidal vascular area by color flow Doppler ultrasonography in patients with Graves' disease (GD), Hashimoto's thyroiditis (HT), and subacute thyroiditis. The serum concentrations of thyroid hormones, TSH, TSH receptor antibodies, and VEGF were also examined. There was a significant increase in serum VEGF levels in patients with untreated GD and goitrous HT compared with those in healthy subjects. The serum VEGF levels in untreated patients with subacute thyroiditis were significantly higher than those in patients with untreated GD or HT. There was a significant correlation between serum VEGF levels and the ratio of intrathyroidal vascular area and thyroid area in untreated patients with GD who had a goiter larger than or equal to 40 cm3. There was also a significant correlation between serum VEGF and TSH levels in patients with HT who were hypothyroid and had a goiter. Serum VEGF levels decreased significantly in these patients after treatment; this was accompanied by a significant decrease in intrathyroidal vascular area and thyroid volume. Our study demonstrates that VEGF appears to play an important role in intrathyroidal angiogenesis in patients with GD and goitrous HT.

Serum substances that interfere with thyroid hormone assays in patients with chronic renal failure.

OBJECTIVE: Serum thyroid hormone concentrations in patients with chronic renal failure are usually low, despite normal serum TSH levels. We investigated the effect on thyroid hormone assays of serum dialysable organic acids that are elevated in uraemic patients. PATIENTS: Serum samples from 42 patients with chronic renal failure who were receiving haemodialysis and 37 sex- and age-matched healthy subjects were examined. DESIGN AND MEASUREMENTS: Serum thyroid hormone concentrations were measured with an analogue radioimmunoassay (RIA), a labelled antibody assay, and an equilibrium dialysis/RIA method. Serum concentrations of organic acids were determined with high performance liquid chromatography. RESULTS: Serum thyroid hormone levels determined by an analogue RIA and a labelled antibody assay in uraemic patients increased, and serum concentrations of organic acids decreased following haemodialysis. A significant association existed between serum free T3 (FT3) levels determined by an analogue RIA and serum concentrations of indoxyl sulphate (IS) prior to dialysis. There was also a significant association between serum free T4 (FT4) levels determined by an analogue RIA and serum concentration of IS and hippuric acid (HA) prior to dialysis. There was a significant association between the changes of serum concentrations of indole acetic acid (IAA) and FT4 concentrations prior to and following haemodialysis when determined by an analogue RIA. Serum FT3 and FT4 levels significantly decreased after the addition of IS to serum from healthy subjects when determined by an analogue RIA but not by a labelled antibody assay. Serum FT4 levels, but not FT3 levels, decreased after addition of IAA when determined by an analogue RIA. Serum FT4 concentrations determined by an equilibrium dialysis/RIA were significantly higher than those determined by the other two methods. The addition of IS, IAA, and HA to serum samples from healthy subjects significantly increased FT4 concentrations when determined by an equilibrium dialysis/RIA method. CONCLUSIONS: Increased serum levels of indoxyl sulphate, indole acetic acid and hippuric acid in sera of uraemic patients may interfere with thyroid hormone measurements when an analogue radioimmunoassay is used. In contrast, there was little Interference with a labelled antibody assay. Dialysable organic acids may also interfere with thyroid hormone assays determined by an equilibrium dialysis/radioimmunoassay method.

Elevated serum granulocyte colony-stimulating factor levels in patients with Graves' disease.

OBJECTIVE: Agranulocytosis is a serious side effect of anti-thyroid drugs (ATD). Granulocyte colony-stimulating factor (G-CSF) is one of the cytokines that increase granulocyte number. The aim of this study was to examine the sequential variation of serum G-CSF levels in patients with Graves' disease before and after ATD therapy. PATIENTS: Sixty-three patients with Graves' disease were studied before, during and after treatment with methimazole (MMI). Serum samples from 71 healthy subjects were used as controls. DESIGN AND MEASUREMENTS: Serum levels of G-CSF were measured by a novel chemiluminescent enzyme immunoassay, which was sensitive enough to determine G-CSF levels in healthy subjects. Blood granulocyte counts, serum, thyroid hormone and TSH levels, and titres of thyroid autoantibodies were also measured. RESULTS: Serum G-CSF levels in Graves' patients before and 2 weeks after MMI were significantly higher than in healthy subjects. There was a significant correlation between serum G-CSF levels and granulocyte counts in untreated patients with Graves' disease. Untreated patients with granulocyte counts less than 2 x 10(9)/I had significantly lower serum G-CSF levels as compared with other untreated patients. Serum G-CSF levels gradually decreased thereafter. No correlation was observed between serum G-CSF levels and serum thyroid hormone levels or titres of thyroid autoantibodies. After ATD treatment, no correlation was found between serum G-CSF levels and granulocytes counts. There was no significant correlation between the change of serum G-CSF levels and that of granulocyte counts before and after MMI treatment. Graves' patients with mild agranulocytosis had normal or elevated serum G-CSF levels. CONCLUSIONS: Significantly elevated serum G-CSF levels were observed in patients with Graves' hyperthyroidism. During ATD therapy, deficiency of G-CSF was not identified as a cause of agranulocytosis in this study.

[A study of gastroesophageal reflux by 24-hour esophageal pH monitoring in patients with bronchial asthma].

To evaluate the incidence of abnormal gastroesophageal reflux in patients with bronchial asthma and the influence of drug therapy on the gastroesophageal reflux, we investigated the gastroesophageal reflux patterns using an ambulatory 24-hour esophageal monitoring in 25 healthy volunteers and 58 asthmatics. All the patients were stable conditions at the time of the study. Bronchodilator therapy was continued, if necessary along with steroid inhalation and xanthines. Compared with healthy volunteers, the asthmatics had significantly greater esophageal acid exposure time, more frequent reflux episode, and longer single reflux time. About 70% of asthmatics had abnormal gastroesophageal reflux. Asthma medications were not associated with the incidence of abnormal gastroesophageal reflux. However, asthmatics receiving beta(2)-stimulants therapy (n = 27) had significantly greater esophageal reflux exposure time than those not receiving (n = 31). Our study suggested that most asthmatics have abnormal gastroesophageal reflux unrelated to asthma attack or asthma medications and that beta(2)-stimulants used in asthmatics may worsen gastroesophageal reflux.

Transplantable rat thyroid cancer cell line FRTC transformed with muramyl dipeptide.

A rat thyroid cancer cell line, FRTC, was established from the normal rat thyroid cell line, FRTL-5. FRTL-5 cells were cultured in vitro with N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP) for 4 days and were transplanted intraperitoneally into Fisher rats. Disseminated tumour formation in the peritoneum was found in ten out of ten rats in which MDP-treated FRTL-5 cells were transplanted. Colloid-like structures stained with anti-thyroglobulin (Tg) antibodies were observed in the tumours. On the other hand, no tumour was found in any of the rats in which untreated FRTL-5 cells were transplanted. No morphological changes were observed in FRTL-5 cells after long-term in vitro culture in the presence of MDP. MDP had no effect on thymidine incorporation, the production of cAMP or the expression of c-myc in FRTL-5 cells in vitro. Cells from the tumour (FRTC) secreted Tg in vitro and expressed Tg, thyroid peroxidase (TPO) and thyrotropin (TSH) receptor mRNA. The expression of TSH receptor mRNA increased in FRTC cells after TSH stimulation. FRTC cells produced cAMP in response to TSH stimulation in a dose-dependent manner. However, the growth of FRTC cells was TSH independent. Expression of c-myc and c-fos was observed in FRTC cells in vivo as well as in vitro. FRTC cells formed tumours in Fisher rats when transplanted subcutaneously. FRTC cells have several characteristics of differentiated thyroid cancer cells and may provide a good model for the study of human differentiated thyroid cancers.

Decrease in serum levels of thyroid hormone in patients with coronary heart disease.

The aim of this study was to evaluate the relationship between subclinical hypothyroidism and/or autoimmune thyroid disease and coronary heart disease (CHD). Ninety seven patients diagnosed as having CHD by a coronary angiography (CHD group) and 103 healthy subjects matched for age, sex and body mass index (control group) were included in the study. Thyroid function, thyroid autoantibodies and serum lipid concentrations were measured in the CHD and control groups. The CHD group exhibited significantly decreased serum free T3 (FT3) and free T4 (FT4) levels, and significantly increased serum TSH levels as compared with the control group, indicating a significant decrease in thyroid function in the CHD patients. Serum high density lipoprotein cholesterol (HDL-C) levels were significantly decreased in the CHD group. The incidence of subclinical hypothyroidism and thyroid autoantibodies was similar in both two groups. These observations were also true of women even after those who had diabetes mellitus (DM), hypertension (HT) and a smoking habit were excluded. This was not the case, however, in men without DM, HT, or a smoking habit. Patients with CHD had significantly lower serum levels of HDL-C than the control subjects, regardless of gender (P < 0.01). In the group with CHD, there was no difference between the serum lipid levels in patients with subclinical hypothyroidism and those with normal thyroid function. Female patients with CHD had significantly lower serum levels of thyroid hormone and HDL-C, but their subclinical hypothyroidism or thyroid autoimmunity did not seem to be related to the development of CHD.

Thyroid involvement by malignant histiocytosis of Langerhans' cell type.

Involvement of the thyroid gland by Langerhans' cell histiocytosis is quite rare. We describe the case of a 58-year-old man referred for treatment of a progressively enlarging goitre. The trachea was severely stenotic and adjacent structures such as the left carotid vein and the thyroid cartilage were also involved. Central diabetes insipidus and severe combined immunodeficiency were associated. Although fine needle aspiration biopsy of the thyroid was initially interpreted as papillary carcinoma, anaplastic thyroid cancer was suspected. Treatment with prednisolone, doxorubicin and irradiation controlled the tracheal compression. A diagnosis of thyroid Langerhans' cell histiocytosis was finally made on the basis of the presence of Birbeck granules and CD1a and CD4 antigen in the thyroid tumour cells. Furthermore, positive staining for CD68 and lysozyme suggested that the tumour cells may have had the character of phagocytic cells in addition to their dendritic cell nature. This is the first case of thyroid involvement by malignant histiocytosis of Langerhans' cell type with unusual phagocytic markers.

[A case of Tsutsugamushi disease infected by mountain climbing in the Republic of Korea].

A 47-year-old male was infected with Tsutsugamushi disease showing typical findings after mountain climbing in the Republic of Korea (ROK). Immunoserological examinations suggested that he had been infected in ROK. In this case, detection of the characteristic sting was useful for early diagnosis and treatment. Until now, Tsusugamushi disease has not been reported in Hokkaido. It is the first case registered in Hokkaido. With extensive international intercourse these days, this case suggests that Tsutsugamushi disease can occur potentially all over Japan.

Disturbed lipid metabolism in patients with subclinical hypothyroidism: effect of L-thyroxine therapy.

To evaluate whether patients with subclinical hypothyroidism have a disturbance in lipid metabolism, and whether supplemental L-thyroxine (L-T4) therapy would improve their lipid parameters, we measured serum levels of thyroid hormones, TSH and lipid parameters in 34 patients with subclinical hypothyroidism before and 2 months after treatment with L-T4. Before treatment, patients with subclinical hypothyroidism had elevated serum low density lipoprotein cholesterol (LDL-C) concentrations compared with control subjects (P < 0.05). Overall, L-T4 therapy significantly decreased the serum level of TSH (P < 0.01), total cholesterol (TC; P < 0.02), high density lipoprotein cholesterol (P < 0.02), LDL-C (P < 0.05), and the ratio of apolipoprotein B to apolipoprotein A1 (P < 0.05). Lipid values in patients with basal serum TSH levels below 10 mU/l were not affected by L-T4 therapy, whereas serum levels of TC and LDL-C decreases significantly (P < 0.01) in patients with serum TSH levels above 10 mU/l. Thus, the L-T4 treatment appears to have a preventive effect on the disturbance of lipid metabolism in patients with subclinical hypothyroidism, especially in patients with serum TSH levels above 10 mU/l.

Involvement of protein kinase A and C in the production of interleukin-1 alpha-induced prostaglandin E2 from mouse osteoblast-like cell line, MC3T3-E1.

Human recombinant interleukin-1 alpha (IL-1) stimulated the mouse osteoblast-like cell line, MC3T3-E1, to produce prostaglandin E2 (PGE2). This was inhibited by 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7) in a dose-dependent manner. The protein kinase A (PKA)-specific inhibitor, KT5720, also inhibited the IL-1-induced PGE2 production in MC3T3-E1 cells, as did staurosporine, a potent inhibitor of protein kinase C (PKC). The PKA activator, 8-bromoadenosine 3',5'-cyclic monophosphate (8-Br-cAMP), weakly stimulated MC3T3-E1 cells to produce PGE2, as did the PKC activator, 12-O-tetradecanoylphorbol 13-acetate (TPA). However, 8-Br-cAMP and TPA acted synergistically to induce PGE2 production equal to that of IL-1. These observations suggest that activation of both PKA and PKC are involved in IL-1-induced PGE2 production in MC3T3-E1 cells.

[Intracellular mechanism of interleukin-1 alpha-induced prostaglandin E2 (PGE2) production in a mouse osteoblast-like cell line and the possible involvement of protein kinase A and protein kinase C].

Interleukin-1 (IL-1) is known to be a potent stimulator of bone resorption. The effect of IL-1 has been shown to be mediated, at least in part, by IL-1-induced prostaglandin (PG) E2 production in osteoblasts. The intracellular signal transduction mechanism of IL-1 in the PG production, however, is unknown. In the present study using a mouse osteoblastic cell line, MC3T3-E1 (MC), the possible involvement of two representative signal transduction pathways, protein kinase A (PKA) or protein kinase C (PKC)-dependent pathway in the IL-1 alpha stimulated PGE2 production, was investigated. MC produced PGE2 30 min after the IL-1 stimulation. This was inhibited with cycloheximide, suggesting the involvement of de novo protein synthesis. The IL-1-induced PGE2 production was inhibited by H-7 in a dose dependent manner. Since H-7 is known to inhibit PKA as well as PKC, a more specific inhibitor of PKA KT5720 or staurosporin was used to determine the respective role of PKA or PKC in the production of PGE2. KT5720 inhibited the IL-1-induced PGE2 production in MC in a dose dependent fashion. Similarly, staurosporin inhibited the IL-1-induced PGE2 production in MC in a dose dependent manner. The effect of the activity of PKA or PKC on the production of PGE2 was also investigated. A stimulator of PKA, 8-bromoadenosine 3'5'-cyclicmonophosphate (8-Br-cAMP), as well as a stimulator of PKC phorbol 12-myristate 13-acetate (PMA) induced PGE2 production in MC. The effect of these agents on the PGE2 production was additive.(ABSTRACT TRUNCATED AT 250 WORDS)

[The effect of arotinolol on the thyroid function and the autonomic nerve systems].

beta-blockers have been accepted as a reasonable adjunct therapy for the treatment of hyperthyroidism. They lessen the sympathetic symptoms such as tachycardia and finger tremor. On the other hand, many studies have demonstrated a decrease in 3, 3', 5-triiodothyronine (T3) during treatment with beta-blockers (especially propranolol). The purpose of this study is to clarify the effect of arotinolol (alpha 1, beta-blocker) on the thyroid functions and autonomic nerve systems (ANS) of patients with Graves' disease. Arotinolol 20mg a day p.o. was given to untreated patients with Graves' disease (n = 16) for 2 weeks. Blood sampling and the ANS function-tests were done before and after the treatment. In addition, the in vitro effects of arotinolol on the cAMP production and the radioactive iodine uptake (RAIU) using rat thyroid cell line FRTL5 were evaluated to examine the direct influence on thyroid cells. Arotinolol improved hyperthyroid symptoms including tachycardia, but had no effect on ANS function-tests. It is of interest that not only T3 but also T4 decreased after the arotinolol treatment. We therefore suspected the direct suppressive effects of arotinolol on the thyroid. There were, however, no in vitro inhibitory effects on the cAMP production and the RAIU in TSH-stimulated FRTL5 cells. The reason why serum T4 levels in patients with untreated Graves' disease have decreased after the treatment of arotinolol could not be clarified. In conclusion, arotinolol is a very useful drug for the initial therapy of patients with Graves' disease to reduce the serum thyroid hormone levels and symptoms of hyperthyroidism when combined with antithyroid drugs.

Incidence of anti-mouse IgG in normal subjects and patients with autoimmune thyroid disease.

Serum antibodies to mouse IgG occasionally interfere with two-site immunometric assays in which mouse monoclonal antibodies are used. We examined the titers of antibodies to mouse IgG in serum samples from normal subjects and patients with autoimmune thyroid disease (AITD) using an enzyme-linked immunosorbent assay. Anti-mouse IgG antibodies were positive in 7/119 patients with Graves' disease (5.9%) and 3/60 patients with Hashimoto's thyroiditis (5.0%). One of the 15 patients with a thyroid neoplasm (6.7%) also had antibodies to mouse IgG, as did 5/60 healthy subjects (8.3%). These antibodies were either of the IgG or IgM class. There was no significant difference in the incidence of positive anti-mouse IgG antibody between normal subjects and patients with AITD. It is important to note this high incidence of antibodies to mouse IgG due to the potential of interference with immunometric assays employing mouse monoclonal antibodies.