The purpose of this cross-sectional study was to characterize the age-related change in bone metabolism during the pre- and postmenopausal periods, and to define the standard levels of three serum markers of bone metabolism, pyridinoline cross-linked carboxyterminal telopeptide of type I collagen (ICTP), carboxyterminal propeptide of type I procollagen (PICP), and bone gla protein (BGP), in Japanese adult women. The bone mineral density (BMD) of the lumbar spine (L₂-L₄) and the serum levels of ICTP, PICP and BGP were determined in a total of 207 healthy Japanese women (108 premenopausal and 99 postmenopausal). The lumbar BMD decreased sig-nificantly with increasing age not only in postmenopausal women (P<0.001) but also in premenopausal women (P=0.014). There was a clear gap in the serum levels of ICTP, PICP and BGP between the premenopausal and postmenopausal group (P<0.001), but those were absolutely the same within each group except for ICTP in the postmenopausal women. These findings and the values of serum ICTP, PICP and BGP in pre- and postmenopausal women obtained in this study are expected to be very useful for treatment of postmenopausal osteoporosis.

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The modulatory effect of GH on basal, LH and T₃ mediated secretion of testosterone and oestradiol by purified rat (60 day old) Leydig cells was studied in vitro. Percoll gradient purified Leydig cells (1×10³) were cultured for 48 hours at 34°C in a medium containing different concentrations of rat GH (5-400ng/mL), after an initial culture for 24 hours at 37°C. GH increased testosterone and oestradiol secretions in a dose dependent manner. While testosterone secretion reached the saturation point with 50ng GH, oestradiol secretion reached the saturation point with 150ng GH, followed by diminished secretions. Co-administration of minimum (10ng) effective does of GH with minimum (25ng) or maximum (100ng) effective doses of oLH significantly decreased the testosterone secretion. However, an increased secretion of testosterone was observed when maximum effective doses of rGH (50ng) and oLH (100ng) were co-administered. Minimum effective (25ng) or maximum effective (50ng) doses of T₃ inhibited GH mediated secretion of testosterone in vitro. Oestradiol concentration in the culture medium increased when either dose of rGH was co-administered with the minimum or maximum effective doses of oLH. T₃ 50ng augmented the secretion of oestradiol by Leydig cells in the presence of GH. These results indicate that GH acts as a gonadotrophin to stimulate testosterone and oestradiol secretions by Leydig cells, and that it modulates LH or T₃ induced secretion of these steroids, depending on the intensity of their stimulation.

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Here we report a case of a renin-producing adrenocortical carcinoma. A 57-year-old woman was referred to our hospital complaining of thirst and generalized muscle weakness. She was diagnosed as being hypertensive and diabetic with associated hypokalemia and she had a hard elastic mass with a diameter of 10cm on the left side of her neck. An abdominal computed tomography scan revealed a suprarenal mass on the left side (8.5×8×6.5cm). Endocrinological examination demonstrated a marked elevation in the patient's serum glucocorticoid and sex steroid hormones as well as plasma renin activity. Histological examination of a sample taken from the neck mass revealed a metastasis from an adrenal carcinoma, which was stained positively with antibodies against cytochrome P450 and renin, establishing the diagnosis of a renin-producing adrenocortical carcinoma. Trilostane was effective in reducing serum cortisol levels, but mitotane was ineffective.

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To elucidate the involvement of intrathyroidal T cells in the thyroid antigen-specific immune response in Graves' disease (GD), we investigated whether identical T cell clonotypes accumulate clonally in the right and left lobes of thyroid glands of GD patients. mRNAs extracted from thyroid glands of five females patients with GD were reverse-transcribed to cDNA and then the genes coding the T cell receptor B chain variable (V-NDN-J) region were amplified using polymerase chain reaction. Single strand conformation polymorphism analysis and subsequently nucleotide sequencing were also performed to determine the clonotype of accumulating T cells. T cells infiltrating the thyroid glands showed oligoclonal expansion. The expanded T cell clonotypes were not detected in peripheral blood of the same patients. Importantly, the majority of expanding T cell clonotypes in the two lobes of the thyroid glands were identical. Our findings suggest that the clonal expansion of identical T cell clonotypes in the two lobes is driven by factors common to both lobes, such as thyroid-specific antigens, in patients with Graves' disease.

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During the pen-implantation period significant production of ovine interferon-τ (oIFNτ) by the trophectoderm is detected in day 13-16 conceptuses, but its level rapidly declines thereafter. To understand molecular mechanisms by which oIFNτ gene expression is down-regulated, a variety of deletion constructs were prepared from upstream sequences of the oIFNτ gene and examined for possible silencer regions by using transient transfection into human choriocarcinoma, JEG3, cells. Two regions between -700 to -654 bases (distal region) and from -503 to -453 bases (proximal region) were found to be the possible negative regulatory regions. With probes prepared from these regions, gel mobility shift assay (GMSA) was then conducted. DNA-protein complexes were observed, but the gel shift pattern was different between nuclear extracts from days 14 (active oIFNτ production) and 20 (minute oIFNτ production) ovine trophoblasts. Day 20 nuclear extracts exhibited more band patterns than those of day 14; most notably the distal region between -692 and -668 bases exhibited the distinct band with nuclear extracts from day 20, but not from day 14 trophoblasts. In addition, the band patterns from day 20 trophoblast nuclear proteins were similar to those detected with JEG3 and HeLa cell nuclear extracts. Taken together, these observations suggest that the upstream sequences identified could serve as negative regulatory regions to which various nuclear factors bind, resulting in reduction of oIFNτ gene transcription.

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Sandwich cell Immunoblot assay(sandwich CIBA) was used to identify somatotrophs (GH cells), mammosomatotrophs, and mammotrophs (PRL cells) in pituitary tumors obtained from patients with GH-secreting adenomas and prolactinomas. The mean serum GH level was 177.6ng/ml in 19 patients with GH-secreting adenomas and the mean PRL level was 2, 129ng/ml in 9 patients with prolactinomas. GH-secreting adenomas could be divided into 3 groups according to the proportions of the cell types. The GH cell dominant type had more than 70% GH cells. The mammosomatotroph cell dominant type had more than 80% mammosomatotrophs. The nondominant type had no dominant cell type. There was a good correlation (r²=0.804) between serum GH levels and tumor size in patients with the GH cell dominant type (n=10). The nondominant type (n=8) had a low serum level of GH except for one tumor. The mammosomatotroph cell dominant type (n=1) showed high serum levels of both GH and PRL. All prolactinomas had a predominance of PRL cells. Sandwich CIBA is a simple method for detecting GH cells, mammosomatotrophs, and PRL cells, and useful for classification for GH-secreting adenomas.

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A multicenter study was carried out to investigate the prevalence of growth hormone (GH-1) gene deletions among patients with isolated growth hormone deficiency (IGHD) and extremely short stature in Japan, using PCR method. Genomic DNA was extracted from the whole blood samples of 48 patients (34 males and 14 females) at 20 hospitals. All the patients fulfilled the inclusion criteria as follows: (1) IGHD patients whose every peak serum GH level in more than two tests <5ng/ml and (2) pretreatment height <-3SD, regardless of family history and facial feature characteristic of GH-1 gene deletion. The subjects were screened for deletions in GH-1 gene, using a PCR method that could identify deletions of 6.7, 7.0 and 7.6kbp. Three (6.25%) out of 48 subjects were found to have such deletion fragments. The first case was a boy homozygous for deletion of 6.7kbp fragments. The second case was a girl heterozygous for 6.7kbp deletion. A direct sequence analysis revealed a 2-bp deletion in exon 3 on the remaining allele that created a stop codon in exon 4. The third case was a boy also heterozygous for 6.7kbp deletion. By direct sequencing analysis, three point mutations were detected in the promoter region on the opposite allele together with a four-base addition at base 250. One of the mutations was in the area of Pit-1 binding site (at base -123). The latter two cases apparently represent new types of compound heterozygote of GH-1 gene deletion. Our results suggest that GH-1 gene mutation is not so rare in extremely short IGHD children in Japan.

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X-linked hypophosphatemic rickets (XLH) is one of the most common causes of rickets in infancy and childhood. Combination therapy of vitamin D and phosphate is generally used for patients with XLH. Effect of treatment of vitamin D and phosphate during childhood on final height of XLH has to be elucidated in Japanese. There have been only three Caucasian studies on final height of XLH with treatment since childhood. Purpose of this study is to report adult height and therapeutic effect of 22 Japanese participants (5 males, 17 females) with XLH who were treated with phosphate (33-200mg/kg/day as phosphorus divided into 3 or 4 doses) and vitamin D (vitamin D₂ or 1α-hydroxyvitamin D₃) for more than five years and evaluate effect of the treatment on the final height retrospectively. Final height (FHt) for all participants was -1.69±1.11 SD. FHt (-1.69±1.11 SD) was significantly higher than height at the initiation of treatment (-2.38±0.88 SD) for all participants (P<0.01). In conclusion, combination therapy of vitamin D and phosphate improved final height of Japanese patients with XLH as is similar to previous Caucasian studies.

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Parathyroid hormone-related protein (PTHrP), a multi-functional protein, is produced by many tissues in fetus. PTHrP concentration in amniotic fluid is reported to be significantly higher than in either fetal or maternal plasma. Other investigators have reported that PTHrP in amniotic fluid is derived mainly from amnion. The aim of this study was to investigate the contribution of fetus to PTHrP in amniotic fluid and the role of PTHrP in human fetal lung tissue. Samples of amniotic fluid, neonatal intra-tracheal fluid, gastric fluid, and the first urine of neonates were obtained at the time of elective cesarean section (n=11), and the concentrations of PTHrP were measured. The PTHrP level in intra-tracheal fluid (41.0±19.6pmol/l, mean±SD) was significantly higher than the levels in amniotic fluid (22.1±0.8), neonatal gastric fluid (13.5±2.5), first urine (0.95±0.6), umbilical cord venous and arterial plasma (1.35±0.2, 1.63±0.3) and maternal plasma (1.05±0.1). PTHrP and PTH/PTHrP receptor mRNA were detected in human lung tissue obtained from a fetus stillborn at 36 weeks of gestation. The effects of PTHrP on fetal lung maturation were studied in H441 cells from a human lung epithelial cell line. PTHrP (10^-7M) significantly suppressed cell proliferation (p<0.05) to approximately 80% of the control level, while administration of PTHrP significantly increased surfactant protein A production (p<0.01). We first demonstrated the high concentration of PTHrP in intra-tracheal fluid that may suggest the positive production of this protein from the fetal lung. The results obtained by in vitro study using a human lung epithelial cell line suggest that PTHrP derived from the fetal lung might modulate its own maturation.

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The effects of Matrigel, a reconstituted basement membrane, on human granulosa cells were investigated. Cells were obtained from follicular aspirate in the course of oocyte retrieval for in vitro fertilization and were cultured on either a surface coated with Matrigel or uncoated plastic. Light and electron microscopy showed that granulosa cells cultured on Matrigel demonstrated three-dimensional aggregated cells with well differentiated morphology: numerous lipid droplets, microvilli, junctional complexes and lumen-like structures were seen. In contrast, cells cultured on plastic were flattened, poorly differentiated and showed apoptotic cells. Immunocytochemistry showed that the proportion of immunopositive cells for 3β-hydroxysteroid dehydrogenase was increased in cultures on Matrigel. The results of the present study suggest that culture on Matrigel promotes the differentiation of human granulosa cells and provides a useful tool which may improve the efficiency of in vitro fertilization.

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We reported the preliminary outcomes of CT-guided percutaneous injection therapy for aldosteroneproducing adrenocortical adenoma (APA). Five sessions of injection therapy, 4 percutaneous acetic acid injections (PAI) and 1 percutaneous ethanol injection (PEI) were performed in 3 patients with APA. A small amount of acetic acid or ethanol solution was injected via a needle placed precisely inside the tumor. The procedure was frequently monitored by repetitive CT scanning. The follow-up period ranged from 5 to 27 months. After the treatment, hypertension was normalized or controlled by a low dose of conventional anti-hypertensive drug. In 2 of 3 cases the plasma aldosterone levels were normalized. Although temporary symptoms of alcoholic intoxication were observed in the single session of PEI, the 4 sessions of PAI were associated with no adverse symptoms or complications. Although this study covers only short-term results in 3 patients, CT-guided PAI appears to be a safe and effective treatment and may be a promising alternative as a simple and far less invasive therapy for APA.

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Here we report three cases of hyperthyroid Graves' disease that occurred after partial thyroidectomy for papillary carcinoma. In Case 1, the patient first developed hyperthyroidism 2 years after resection of left thyroid lobe, was treated for 2 years with antithyroid drug which was then discontinued, and relapsed with periodic paralysis after 8 years of remission. In Case 2, a hyperfunctioning remnant thyroid was noted 22 years after right hemithyroidectomy. In Case 3, where thyrotoxic symptoms became evident 7 weeks after right hemithyroidectomy, autoantibodies to thyroglobulin and thyroid microsome were positive in preoperative serum, in line with a report by others detecting these antibodies in 2 out of 3 such cases examined. Later bioassay revealed activity of thyroid stimulating antibodies in that serum, with further increase in titer in the sample taken at the clinical manifestation. Hence in Case 3, surgical stress may have altered immunological homeostasis, promoting a preclinical Graves' disease to full-blown hyperthyroidism.

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We report an 11-year-old girl with euthyroid Graves' disease. She was referred to our clinic because of left exophthalmos without other symptoms suggestive of hyperthyroidism. Her serum concentration of free thyroxine (FT₄) and free triiodothyronine (FT₃) were normal, but thyroid-stimulating hormone (TSH) was below normal and impaired TSH response to TSH releasing hormone (TRH) was found. Although the sera were positive
for anti-TSH receptor antibody (TRAb) and thyroid-stimulating antibody (TSAb), both titers were not as high as usually observed in Graves' disease. Three months later, she developed hyperthyroidism and was treated with propylthiouracil. Within 2 weeks of the initiation of therapy, all symptoms except exophthalmos disappeared, and after 2 months of treatment TRAb was negative though TSAb remained positive. TSAb is therefore a good indicator to use in the diagnosis and follow-up of euthyroid Graves' disease and should be measured in patients with exophthalmos of unknown origin, even in children.

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