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Thyroid storm is an endocrine emergency which is characterized by multiple organ failure due to severe thyrotoxicosis, often associated with triggering illnesses. Early suspicion, prompt diagnosis and intensive treatment will improve survival in thyroid storm patients. Because of its rarity and high mortality, prospective intervention studies for the treatment of thyroid storm are difficult to carry out. We, the Japan Thyroid Association and Japan Endocrine Society taskforce committee, previously developed new diagnostic criteria and conducted nationwide surveys for thyroid storm in Japan. Detailed analyses of clinical data from 356 patients revealed that the mortality in Japan was still high (∼11%) and that multiple organ failure and acute heart failure were common causes of death. In addition, multimodal treatment with antithyroid drugs, inorganic iodide, corticosteroids and beta-adrenergic antagonists has been suggested to improve mortality of these patients. Based on the evidence obtained by nationwide surveys and additional literature searches, we herein established clinical guidelines for the management of thyroid storm. The present guideline includes 15 recommendations for the treatment of thyrotoxicosis and organ failure in the central nervous system, cardiovascular system, and hepato-gastrointestinal tract, admission criteria for the intensive care unit, and prognostic evaluation. We also proposed preventive approaches to thyroid storm, roles of definitive therapy, and future prospective trial plans for the treatment of thyroid storm. We hope that this guideline will be useful for many physicians all over the world as well as in Japan in the management of thyroid storm and the improvement of its outcome.

SummaryObjective Thyroid storm (TS) is a life-threatening endocrine emergency. This study aimed to achieve a better understanding of the management of TS by analyzing therapeutic modalities and prognoses reported by nationwide surveys performed in Japan. Design, patients and measurements Retrospective analyses were performed on clinical parameters, outcomes, and treatments in 356 TS patients. Results Patient disease severities assessed via Acute Physiology and Chronic Health Evaluation (APACHE) II and Sequential Organ Failure Assessment (SOFA) scores significantly correlated with mortality. Free triiodothyronine (FT3) and the FT3/free thyroxine (FT4) ratio inversely correlated with disease severity. Methimazole (MMI) was used in the majority of patients (78·1%), and there were no significant differences in mortality or disease severity between those treated with MMI and those receiving propylthiouracil (PTU). Patients who received inorganic iodide (KI) demonstrated higher disease severity but no change in mortality compared to those who did not. Patients treated with corticosteroids (CSs) demonstrated significantly higher disease severity and mortality than those who were not. Disease severity in patients treated with intravenous administration of beta-adrenergic antagonists (AAs) was significantly higher than those treated with oral preparations, although no significant difference in mortality was observed between these groups. In addition, mortality was significantly higher in patients treated with non-selective beta-AAs as compared with other types of beta-AAs. Conclusion In Japan, MMI was preferentially used in TS and showed no disadvantages compared to PTU. In severe TS, multimodal treatment, including administration of antithyroid drugs, KI, CSs and selective beta1-AAs may be preferable to improve outcomes.

Primary hyperparathyroidism is the most common hormonal manifestation associated with multiple endocrine neoplasia 1 (MEN1). It is generally caused by parathyroid hyperplasia, and parathyroid carcinoma is rare. Here, we report a case of MEN1 with parathyroid carcinoma in two parathyroid glands causing primary hyperparathyroidism. A 40-year-old man with primary hyperparathyroidism due to MEN1 underwent a total parathyroidectomy. His corrected calcium and intact PTH (i-PTH) serum levels were 10.8 mg/dL and 203 pg/mL, respectively. Although three glands were successfully removed, the left upper parathyroid gland could not be detected. Since the right lower parathyroid lesion had invaded into the thyroid, right lobectomy was performed. A portion of the left lower parathyroid tissue was transplanted into his forearm. The histological findings of the left lower and the right upper parathyroid glands were consistent with hyperplasia while that of the right lower parathyroid gland was parathyroid carcinoma. Since the post-surgical i-PTH levels remained high, the intrathyroidal lesion of the left lobe, which was initally diagnosed as an adenomatous nodule, was suspected to contain parathyroid tumor. A fine needle aspiration of the tumor revealed a high concentration of i-PTH. One week after the first surgery, a left thyroid lobectomy was performed. The pathological diagnosis of the tumor was parathyroid carcinoma. After the surgery, calcium and i-PTH levels were normal. Although it is rare, parathyroid carcinoma should be considered as a cause of hyperparathyroidism in MEN1 patients. Since it is difficult to diagnose parathyroid carcinoma before surgery, intraoperative findings are important for the appropriate treatment.

Temozolomide (TMZ) is an alkylating agent and was a first-line chemotherapeutic agent for malignant gliomas. Recently, TMZ has been documented to be effective against atypical pituitary adenomas (APAs) and pituitary carcinomas (PCs).The clinical and pathological characteristics of APAs and PCs treated with TMZ in Japan were surveyed and analyzed retrospectively.Members of the Japan Society of Hypothalamic and Pituitary Tumors were surveyed regarding the clinical characteristics of APAs and PCs treated with TMZ. Stored tumor samples were gathered from the responders and were assessed by the immunohistochemistry of Ki-67, O(6)-methyl-guanine-DNA methyltransferase, p53, MSH6, and anterior pituitary hormones. Responses to TMZ treatment were defined as complete response (CR), partial response (PR), progressive disease (PD), and stable disease (SD) according to RECIST (Response Evaluation Criteria in Solid Tumors) version 2.0.Three samples from 3 subjects with APA and 11 samples from 10 subjects with PC were available.The 13 subjects had APAs and PCs consisting of 5 prolactin-producing tumors, 5 ACTH-producing tumors, and 3 null cell adenomas. The clinical response to TMZ treatment was as follows: 4 cases of CR and PR (31%), 2 cases of SD (15%), 6 cases of recurrence after CR and PR (46%), and 1 case of PD (8%). However, considerable subjects had recurrent disease after a response to TMZ. The immunohistochemical findings of Ki-67, O(6)-methyl-guanine-DNA methyltransferase, and p53 did not show any significant correlation with the efficacy of TMZ. However, the immunopositivity of MSH6 was positively correlated with TMZ response (P = .015, Fisher's exact test).This study showed that preserving MSH6 function was contributory to the effectiveness of TMZ in malignant pituitary neoplasms. It is necessary to survey more cases and evaluate multifactor analyses.

Painless thyroiditis is characterized by transient thyrotoxicosis with complete recovery. Some patients, however, have repeated episodes of thyrotoxicosis, for which the mechanism of recurrence is not clear. We therefore studied the clinical characteristics in 8 patients with 4 or more episodes of thyrotoxicosis and 40 control patients. The age at onset was significantly earlier and the male-to-female ratio was significantly higher in the case group than the control group. The highest FT4 and FT3 levels were higher in the recurrent type. Multiple regression analysis showed contributions of higher peak FT4 level, male gender, presence of thyroid autoantibody, earlier onset, and smaller size of thyroid to repeated recurrence. Since the peak FT4 value, gender, autoantibody, age of onset and size of thyroid contribute to only 27.5% of the repeated recurrence cases, other factors such as genetic susceptibility or inflammatory tissue changes in thyroid may also be contributory. Further studies are needed to elucidate the mechanism underlying repeated recurrence.

Serum BNP (brain naturiuretic peptide) and ANP (atrial natriuretic peptide) levels are reportedly elevated in patients with thyrotoxicosis. The increases may not be due to thyrotoxicosis itself but to secondary cardiovascular changes such as chronic heart failure (HF) or atrial fibrillation (AF) which frequently accompany thyrotoxicosis. We measured serum ANP and BNP levels in 130 patients with thyrotoxicosis and correlated them with HF severity and thyroid function. Thirty-seven normal subjects served as controls. Serum BNP levels in thyrotoxic patients were significantly higher than those in control subjects and significantly correlated with serum free T4, free T3 and ANP levels. In untreated Graves' disease serum BNP level was significantly elevated in patients with HF or AF. Multiple regression analysis revealed that HF, free T4, female gender and AF are independent contributing factors to the elevated BNP level, and that these four factors contributed about 40%. On the other hand, HF and AF were contributing variables for ANP level but the overall contribution of these factors was only 10%. After normalization of thyroid function, serum BNP levels were normalized in 70.5% of Graves' patients. BNP level in euthyroid state was dependent on the presence of HF and the BNP value before therapy, but not on thyroid hormone levels or AF. These data suggest that the cardiovascular condition is the major factor responsible for the elevated serum BNP and ANP levels in thyrotoxic patients, while thyrotoxicosis itself is an independent but minor contributing factor. Thus, the determination of serum BNP levels in thyrotoxic patients is useful for monitoring cardiovascular conditions of HF.

In TSH-secreting pituitary adenomas (TSHoma), octreotide (OCT) therapy reduces tumor size and TSH secretion in some cases but not in others. As OCT acts through various types of somatostatin receptors (SSTRs), the different responses of TSHoma to OCT might be explained by the differences of SSTR expression. We therefore studied the expression of subtype-specific SSTR mRNA transcripts in tumor tissues by RT-PCR. Type 2 (SSTR2) mRNA transcripts were detected in all 8 tumors but those of SSTR3 and SSTR5 were demonstrated only in 5 of them. Serum TSH levels were decreased by OCT administration test in all patients but OCT therapy was effective in two patients out of three. SSTR5 mRNA was detected in two tumors from the responder, but not in one tumor that was resistant to OCT. These observations suggest that the temporal decrease of TSH by OCT may be mediated by SSTR2, and that the long term response to OCT therapy may be related with the expression of SSTR5. Therefore, the expression of SSTR5 in TSHoma may be a useful marker for predicting the outcome of the therapy, but further studies with larger numbers of patients are necessary.

We have recently used a tissue-engineered cell sheet system with temperature-sensitive culture dishes to regenerate tissues such as corneal epithelial graft sheets, bioartificial tracheas, mucosal epithelial sheets for esophageal ulcers, and pulsatile cardiac tissue grafts, all of which can be used in clinical medicine. With regard to the thyroid gland, the three-dimensional follicular structure consists of polarized thyroid epithelial cells, and this structure is important for the production and secretion of thyroid hormones. In order to regenerate thyroid tissue from isolated cells, we used temperature-responsive culture dishes to harvest thyroid follicular cell sheets in the first step of reconstituting functional thyroid tissue. Initial trials demonstrated that the following conditions improved the recovery of cells sheets: 1) pretreatment of the dishes with serum, 2) use of medium free of thyroid stimulating hormone (TSH), and 3) co-culture with skin fibroblasts.

Background: In a previous study, we found that the amino acid substitution R218H in human serum albumin (HSA) was the cause of familial dysalbuminemic hyperthyroxinemia (FDH) in several Caucasian patients. Subsequently the substitution R218P was shown to be the cause of FDH in several members of a Japanese family. This study attempts to resolve discrepancies in the only other study of R218P HSA and identifies two new Japanese R218P FDH patients unrelated to those described previously.Methods and Results: Recombinant R218H, R218P, and wild-type HSA were synthesized in yeast, and the affinities of these HSA species for l- and d-thyroxine were determined using fluorescence spectroscopy. The dissociation constants for the binding of wild-type, R218P, and R218H HSA to l-thyroxine were 1.44 × 10−6, 2.64 × 10−7, and 2.49 × 10−7 mol/L, respectively. The circular dichroism spectra of thyroxine bound to R218H and R218P HSA were markedly different, indicating that the structure of the thyroxine/HSA complex is different for either protein.Conclusions: The Kd values for l-thyroxine bound to R218P and R218H HSA determined in this study were similar. The extremely high serum total-thyroxine concentrations reported previously for R218P FDH patients (10-fold higher than those reported for R218H FDH patients) are not consistent with the Kd values determined in this study. Possible explanations for these discrepancies are discussed.

In order to identify the mutual interaction between GH and leptin, we studied the effect of GH on fatty Zucker rats. GH administration at a high dose (5.0 IU/kg) reduced % body fat after 7 days. The leptin mRNA level in subcutaneous fat tissue was not changed but that in epididymal fat tissue was decreased by an even lower dose of GH (1.5 IU/kg). IGF-I treatment (200 microg/kg/day) did not change the % body fat or leptin mRNA level. These observations suggest that GH directly interacts with visceral fat and reduces fat mass and leptin expression. We also measured serum leptin levels in patients. The levels in patients with acromegaly were significantly lower than those in normal subjects with the same amount of body fat, but serum IGF-I and urinary C peptide excretion rates were higher in the acromegalic. These observations also suggests that GH directly interacts with adipose tissue and reduces leptin expression. Next we investigated the direct action of leptin on GH release from the pituitary. Leptin pretreatment of pituitary cells in culture or rats in a fasted or fed condition did not change GRH induced GH secretion. As indicated also by other recent studies, leptin may increase GRH or decrease somatostatin secretion by the hypothalamus. Thus GH interacts with fat tissues and leptin may be a good marker of the interaction.

A missense and loss of function mutation of the Na+/I− symporter (NIS) gene, T354P[ Thr354→Pro (ACA→CCA)], was found in the homozygous state in two unrelated Japanese patients with iodide transport defect. In this study we have identified the homozygous T354P NIS germline mutation in seven Japanese patients, including one previously reported, from five unrelated families. No other nucleotide changes were found in the coding regions and the exon-intron boundaries of the NIS gene in these seven patients. These results suggest a common prevalence of the T354P mutation in Japanese patients. Although these seven patients have the identical NIS mutation, T354P, marked heterogeneity in clinical pictures, especially concerning goiter and hypothyroidism, were noted among them. Therefore, another factor(s), but not the nature of the NIS mutation, may account for the clinical heterogeneity among patients with the iodide transport defect. We have previously reported that the NIS messenger ribonucleic acid was markedl...

As leptin, an ob gene product, plays a pivotal role in the regulation of adiposity and energy homeostasis, the level of its expression is likely to fluctuate under various physiological, nutritional, and disease conditions. Reports regarding the effect of GH on serum leptin levels are inconsistent. We have measured serum leptin levels and correlated them with several variables in patients with acromegaly, patients with adult GH deficiency (GHD), and normal controls. In 116 normal subjects, the mean serum concentration of leptin was 5.0 ± 2.8 (mean ± sd) ng/mL in men (n = 42) and 10.7 ± 7.3 ng/mL in women (n = 73), respectively. As reported previously, the leptin levels in women were significantly (P < 0.001) higher than in men, and there was a strong positive correlation between log-transformed serum leptin levels and percent body fat in simple regression analysis (in men: r = 0.606; P < 0.0001; in women: r= 0.707; P < 0.0001). In 36 acromegalic patients, the percent body fat mass was significantly lower ...

The functions of thyroid cells are regulated by a number of cytokines and growth factors in addition to TSH. Recent studies have revealed that several cytokines including interleukin (IL)-6 are involved in thyroid dysfunction. Oncostatin M (OSM) is a glycoprotein belonging to the same family of cytokines as IL-6, to which it is related by sequence and structural homology and the use of the signal-transducing receptor component gp130. We, therefore, studied the effect of OSM on iodide uptake and DNA synthesis by porcine thyroid cells in culture. OSM increased c-fos and c-jun mRNA levels but did not stimulate DNA synthesis. OSM inhibited iodide uptake stimulated by TSH; while IL-6 also inhibited iodide uptake, it was only about one-tenth as potent. IL-6 had about the same potency as OSM when it was added with soluble IL-6 receptor. OSM had no effect on cAMP production but inhibited iodide uptake stimulated by 8-bromo-cAMP and forskolin. These findings suggest that OSM exerts its inhibitory effects at the post-cAMP production step(s). OSM also inhibited thyroid peroxidase mRNA levels but had little effect on thyroglobulin mRNA levels. Investigations of the signal transduction system showed that gp130 and leukemia inhibitory factor (LIF) receptor beta subunit mRNA were detectable in porcine thyroid cells by reverse transcription (RT)-polymerase chain reaction (PCR). Together with the report that serum OSM and IL-6 concentrations are elevated to the same levels in patients with sepsis, these results suggest that OSM may contribute to the thyroid dysfunction in this condition.

Arai M, Tsushima T, Isozaki 0, Demura H, Shizume K, Emoto N, Miyakawa M, Nozoe Y, Murakami H, Ohmura E. Effects of transforming growth factor α (TGF-α) on DNA synthesis and thyrotropin-induced iodine metabolism in cultured porcine thyroid cells. Eur J Endocrinol 1995;132:242–8. ISSN 0804–4643 Transforming growth factor α (TGF-α) is a potent mitogen that is similar structurally to epidermal growth factor (EGF). As EGF is a potent growth stimulator and an inhibitor of iodine metabolism in cultured thyroid cells of several species, we studied whether TGF-α has similar effects using porcine thyroid cells in culture. Recombinant human TGF-α dose-dependently stimulated DNA synthesis of thyroid cells, with maximal stimulation (eight- to ninefold above basal) occurring at 2 nmol/l. The potency was approximately 50% that of mouse EGF and correlated with the ability to compete with EGF for receptor binding, suggesting that the action of TGF-α is mediated by interaction with EGF receptors. When thyroid cells were cultured for 3 days with thyrotropin (TSH) in the presence of TGF-α, TSH-induced iodide uptake was inhibited in a dose-dependent manner. The potency of TGF-α again was approximately 50% that of EGF. Transforming growth factor α did not inhibit TSH-stimulated cAMP production. Moreover, iodide uptake stimulated by either forskolin or 8-bromo-cAMP also was inhibited by TGF-α. Thus, we conclude that TGF-α inhibits TSH-induced iodine metabolism largely by acting at the steps distal to cAMP production. Northern blot analysis revealed expression of TGF-α mRNA in porcine thyroid cells. These observations suggest that TGF-α acts as an autocrine modulator of growth and differentiated functions in porcine thyroid cells. T Tsushima, Department of Medicine 2, Tokyo Women's Medical College, Kawadacho 8–1, Shinjukuku, Tokyo 162, Japan

Thyroid storm (TS) is life threatening. Its incidence is poorly defined, few series are available, and population-based diagnostic criteria have not been established. We surveyed TS in Japan, defined its characteristics, and formulated diagnostic criteria, FINAL-CRITERIA1 and FINAL-CRITERIA2, for two grades of TS, TS1, and TS2 respectively.We first developed diagnostic criteria based on 99 patients in the literature and 7 of our patients (LIT-CRITERIA1 for TS1 and LIT-CRITERIA2 for TS2). Thyrotoxicosis was a prerequisite for TS1 and TS2 as well as for combinations of the central nervous system manifestations, fever, tachycardia, congestive heart failure (CHF), and gastrointestinal (GI)/hepatic disturbances. We then conducted initial and follow-up surveys from 2004 through 2008, targeting all hospitals in Japan, with an eight-layered random extraction selection process to obtain and verify information on patients who met LIT-CRITERIA1 and LIT-CRITERIA2.We identified 282 patients with TS1 and 74 patients with TS2. Based on these data and information from the Ministry of Health, Labor, and Welfare of Japan, we estimated the incidence of TS in hospitalized patients in Japan to be 0.20 per 100,000 per year. Serum-free thyroxine and free triiodothyroine concentrations were similar among patients with TS in the literature, Japanese patients with TS1 or TS2, and a group of patients with thyrotoxicosis without TS (Tox-NoTS). The mortality rate was 11.0% in TS1, 9.5% in TS2, and 0% in Tox-NoTS patients. Multiple organ failure was the most common cause of death in TS1 and TS2, followed by CHF, respiratory failure, arrhythmia, disseminated intravascular coagulation, GI perforation, hypoxic brain syndrome, and sepsis. Glasgow Coma Scale results and blood urea nitrogen (BUN) were associated with irreversible damages in 22 survivors. The only change in our final diagnostic criteria for TS as compared with our initial criteria related to serum bilirubin concentration3 mg/dL.TS is still a life-threatening disorder with more than 10% mortality in Japan. We present newly formulated diagnostic criteria for TS and clarify its clinical features, prognosis, and incidence based on nationwide surveys in Japan. This information will help diagnose TS and in understanding the factors contributing to mortality and irreversible complications.

To investigate the role of basic fibroblast growth factor (FGF) in renal cell carcinoma growth, we have analyzed the expression of mRNA of basic FGF. In 7 of 15 cases, basic FGF mRNA level in renal cell carcinoma tissues was higher than that in corresponding normal tissues. However, the tumor-to-normal ratios of expression levels are chiefly less than 2.0 and, in 5 cases, are even less than 1.0. Furthermore, there was no correlation between the ratio and the clinical stage. In protein analysis, we could not find any difference between basic FGF extracted from renal cell carcinomas and that from normal kidney tissues in bioactivity, immunoreactivity, molecular weight and affinity to heparin. On the other hand, anti-basic FGF monoclonal antibody inhibited the growth of a renal cell carcinoma cell line, VMRC-RCW, and this inhibition was reversed by an extraphysiological amount of exogenous basic FGF (100 ng./ml.). These results suggest that basic FGF itself may have no pivotal role in renal cell carcinoma etiology but is involved in the growth of renal cell carcinomas in an autocrine manner.

Semisynthetic tetracyclines (TCNs) are used for the management of malignant pleural effusions as sclerosing agents. However, their precise mechanism of actions are uncertain. In the present study, the mechanism of inhibitory effects of minocycline (MINO) and doxycycline (DOXY), on the accumulation of ascitic fluid induced by mouse fibrosarcoma (Meth-A) cells were investigated using male mice. Meth-A cells inoculated intraperitoneally elicited 2.5–4 ml of bloody ascites 10 days after implantation. The production of ascitic fluid was suppressed in a dose-related manner by daily intraperitoneal injections of MINO or DOXY, whereas vehicle (normal saline with 0.01N HC1) did not exert a significant effect. The inhibitory activity of these two substances was quite similar; one mg/mouse of MINO or DOXY inhibited the accumulation of fluid by 87% and 84%, respectively. The survival rate of Meth-A-bearing mice treated with MINO or DOXY was higher than that of the controls. Macroscopic examination of the peritoneal cavity did not reveal any obvious effects, such as adhesions, in mice treated with either MINO or DOXY. In vitro studies showed that MINO and DOXY suppressed Meth-A cell growth with IC 5 0 s of 5 μM and 8 μM, respectively. Maximal suppression (95%) was achieved at MINO and DOXY concentrations of 25 μM. The above observations suggest that MINO and DOXY inhibit the accumulation of ascites by a direct effect on Meth-A cell growth. Therefore, it appears that TCNs injected into the pleural cavity to manage malignant effusions in man exert their activity, at least in part, by suppressing malignant cell growth.

Basic fibroblast growth factor (bFGF) increases DNA synthesis in rat FRTL-5 thyroid cells, as measured by increased incorporation of tritiated thymidine into DNA. We show that this action is associated with the ability of bFGF to increase cytosolic Ca2+ levels and transiently increase c-fos mRNA levels. Other agents that increase c-fos mRNA levels and DNA synthesis in FRTL-5 cells include TSH, insulin, insulin-like growth factor-I, phorbol esters, A23187, and alpha 1-adrenergic agents; the last two agents also act by increasing cytosolic Ca2+ levels. Despite its enhancement of DNA synthesis, however, bFGF decreases TSH-induced cAMP-mediated iodide uptake. This action appears to reflect two separate actions of bFGF. First, bFGF decreases TSH receptor mRNA levels and the ability of TSH to acutely increase cAMP levels in FRTL-5 cells. The ability of bFGF to negatively regulate TSH receptor mRNA levels is additive to and independent of the ability of TSH and its cAMP signal to negatively autoregulate TSH receptor mRNA levels. This is consistent with the effect of bFGF on cytosolic Ca2+ levels and the ability of increased cytosolic Ca2+ to decrease TSH receptor mRNA levels. Second, bFGF inhibits cAMP signal expression, as evidenced by its ability to inhibit (Bu)2cAMP-induced iodide uptake in FRTL-5 cells. Both effects are, presumably, associated with the ability of bFGF to counteract TSH/cAMP-induced increases in thyroid peroxidase mRNA levels, which we demonstrate. We suggest, therefore, that bFGF causes opposite effects on DNA synthesis and iodide uptake because of its effect on cytosolic Ca2+ levels and because increases in cytosolic Ca2+ can have opposite effects on gene transcription, particularly in the case of the TSH receptor and c-fos genes.

A 43-year-old woman was admitted to hospital due to loss of consciousness. Her fasting plasma glucose and serum immunoreactive insulin levels were 14 mg/dL and 5 microU/mL, respectively. She needed continuous glucose infusion to avoid hypoglycemia. A dynamic abdominal CT scan revealed two hypervascular tumors in the pancreas. A pancreatectomy was performed and her blood glucose level was normalized. The pathological examination revealed six insulinomas but we could not find either parathyroid tumors or obvious pituitary tumor, which denied the possibility of multiple endocrine neoplasia type I. However, long-term follow-up will be necessary for monitoring the development of other tumors.

The prevalence and titer of glutamic acid decarboxylase antibody (GADAb) in type 1 diabetes mellitus (T1DM) has been reported to be higher in patients with autoimmune thyroid diseases (AITD) than those without them. However, we have no data about the influence of GADAb on AITD. We therefore studied the clinical characteristics of Graves' disease (GD) with GADAb in order to clarify the influence of GADAb on GD. Twelve GD patients with GADAb were enrolled and were compared to 40 GD patients without DM. The male to female ratio and age of onset of GD showed no statistical difference. The titer of TSH receptor antibody (TRAb) at the onset of GD was similar in both groups. Initial treatment with methimazole (MMI) was started in all patients with GADAb but radioactive iodine (RI) therapy was carried out in five patients because of adverse effects of MMI or poor control of hyperthyroidism. The initial titer of TRAb was significantly lower in patients treated with MMI alone compared to that in RI treated patients but none of the patients treated with MMI alone went into remission after more than 3-years of follow up. We also compared these GADAb-positive patients with 14 patients with diabetes mellitus who had matched clinical features. The number of diabetic patients who remained in possible remission was significantly higher than that of GADAb-positive patients (5 in 14 vs 0 in 12). Moreover, the rate of remission in the diabetic patients was no different from that of 21 control patients without diabetes followed for more than 7 years (5 in 14 vs 7 in 21). These data suggested that GADAb-positive patients are unlikely to go into remission with antithyroid agents. Therefore, definitive therapies might be preferable for the initial treatment of GADAb-positive patients.

Effects of retinoids on DNA synthesis, iodine metabolism, and thyroid peroxidase messenger RNA levels were studied in cultured porcine thyroid cells. Retinol (10(-8)-10(-5) M) alone did not affect DNA synthesis but potentiated that induced by epidermal growth factor or insulin-like growth factor-I without changes in the number or affinity of receptors for the growth factors, suggesting that retinol stimulates postreceptor events responsible for DNA synthesis. Retinol was an inhibitor of TSH-stimulated iodine metabolism. Iodide uptake and release of organified iodine stimulated by TSH or forskolin were inhibited dose dependently by treatment with retinol. The inhibition was detected at 10(-8) M and was approximately 50% at 10(-6) M. The potency of retinoic acid was comparable to that of retinol. The inhibitory effect of retinol was detected after treatments of thyroid cells for 24 h, and the maximal effect occurred after 48 h incubation. The cAMP accumulation in cultures treated with TSH plus retinol was lower than that of control cultures treated with TSH alone. However, iodide uptake stimulated by 8-bromo-cAMP was also inhibited by retinoids. Retinol reduced TSH- or 8-bromo-cAMP-stimulated gene expression of thyroid peroxidase. Thus, the data suggest that retinoids inhibit TSH-stimulated iodine metabolism by reducing cAMP accumulation and also by acting on the steps subsequent to cAMP production.

Glucocorticoids are widely used for the treatment of malignancy-associated hypercalcemia to delay the occurrence of an escape phenomenon inherent in calcitonin therapy. Using parathyroid hormone-related protein (PTHrP)-producing squamous carcinoma cells (T3M-1 and EC-GI) established in our laboratory, we investigated the in vitro effects of glucocorticoids and calcitonin on PTHrP mRNA expression in the cells and release of PTHrP into the culture medium. The PTHrP gene was constitutively expressed in the logarithmic growth phase in both squamous carcinoma cell lines. When these cells became superconfluent, PTHrP mRNA expression was greatly diminished in T3M-1 cells but was not distinctly diminished in EC-GI cells. Hydrocortisone inhibited the PTHrP mRNA expression in T3M-1 cells and EC-GI cells in a dose-dependent manner. In accordance with the decreased expression of PTHrP mRNA, the release of immunoreactive as well as bioactive PTHrP also decreased in the conditioned medium of glucocorticoid-treated cells. The minimal effective concentration of prednisolone was about 10(-7) M, which is readily attainable in the serum of patients treated with the agent. Calcitonin and indomethacin did not affect the PTHrP mRNA expression or PTHrP release into the medium. Calcitonin did not modulate the hydrocortisone-induced inhibition of PTHrP production. These in vitro findings suggest that the combined use of glucocorticoids and calcitonin plays a beneficial role in the treatment of malignancy-associated hypercalcemia, since the steroid hormone can suppress PTHrP mRNA expression and release of bioactive PTHrP in certain PTHrP-producing tumors.

Methimazole (MMI) increases thyroglobulin (Tg) mRNA levels in FRTL-5 rat thyroid cells. The increase reflects a transcriptional action of the antithyroid agent and is inhibited by cycloheximide, as is the transcriptional action of TSH. It takes several hours to be apparent, is maximal between 24-48 h, and is specific, in that thyroid peroxidase and beta-actin mRNA levels are not increased simultaneously. The increased mRNA levels are associated with increased recovery of immunoprecipitable Tg in the medium of cells exposed to [35S]methionine. The MMI effect appears to be independent of the action of TSH or its cAMP signal, since the MMI-induced increase in Tg mRNA levels is evident in cells treated with TSH or maintained in its absence and is associated not with increases in cAMP levels but, rather, under some circumstances with a decrease. The effect is evident under conditions in which the ability of insulin or insulin-like growth factor-I to increase Tg mRNA levels is already maximal. The MMI-induced increase is inhibited by concentrations of iodide associated with autoregulation of FRTL-5 rat thyroid cells, is inhibited but not mimicked by propylthiouracil, and is not altered by T3. The increase in Tg mRNA levels does not correlate with increased DNA synthesis as a function of MMI concentration either in cells treated with TSH or in those maintained in its absence. A concentration of MMI (5 mM) that increases Tg mRNA levels can also inhibit 8-bromo-cAMP- or phorbol ester-induced increases in [3H]thymidine incorporation into DNA.

Leptin receptors are distributed throughout the body and leptin has been shown to have various effects. As we have recently demonstrated a positive correlation between serum leptin levels and TSH in euthyroid subjects, we investigated the effect of leptin on the thyroids. It was observed that serum leptin levels were negatively correlated with free thyroxine/TSH ratios in the serum of euthyroid female subjects. This suggests that leptin may modulate TSH effects. RT-PCR for leptin receptor expression revealed that FRTL-5 cells possess the gene transcript to the long cytoplasmic form of the receptor. Leptin actually appeared to induce an increase in c-fos mRNA expression. However, it inhibited iodide uptake typically induced by both TSH and dibutyryl cAMP, while leptin did not inhibit TSH-induced cAMP production or TSH-stimulated DNA synthesis in 4H medium (in the absence of insulin and TSH). Leptin also was observed to inhibit TSH- and dibutyryl cAMP-induced Na+/I– symporter and thyroglobulin mRNA expression. Lastly, leptin was seen to inhibit TSH-stimulated thymidine incorporation in 5H medium. Taken together, these results suggest that leptin suppresses TSH-induced thyroid function. Therefore, we hypothesized that leptin may be one of the regulators of thyroid function in obese patients.

A 50-year-old Japanese man with Grave's disease had been taking propylthiouracil (PTU) for 10 years prior to the diagnosis of pneumonia. He noticed dyspnea on exertion and had a dry cough for at least 2 years and then suddenly developed high fever and dyspnea at rest. Clinical symptoms, chest radiographs, chest computed tomography and lung function revealed interstitial pneumonia. The symptoms were completely resolved after withdrawal of PTU, and consequently he was diagnosed with PTU-induced interstitial pneumonia. He also showed moderate myeloperoxidase (MPO)- antineutrophil cytoplasmic antibody (ANCA) positivity without any signs of vasculitis before as well as after PTU withdrawal. Although PTU-induced interstitial pneumonia is quite rare, with only 3 cases, including the present patient, so far reported, respiratory involvement should be considered in patients treated with PTU.

Although a number of abnormalities in oncogenes have been reported in thyroid neoplasms, little information is available on the signal transduction pathway involved in neoplastic thyroid cell growth. Both p70S6 kinase (p70S6K) and Akt are kinases downstream of phosphatidylinositol 3 kinase (PI3K). These kinases are phosphorylated and activated by growth factors including IGF-1, EGF/TGF-alpha, and HGF in thyroid cells. Since the receptors for these growth factors are reportedly overexpressed in human thyroid cancer, we hypothesized that the PI3K-mediated signalings are overactivated in thyroid cancers. Tumorous and adjacent normal tissues of 20 patients with papillary thyroid cancer were obtained at surgery, and expression of p70S6K and Akt were measured by Western blot. Expression of the protein levels of p70S6K was increased in tumor tissues (T) compared to normal thyroid tissues (N), and expression of phosphorylated p70S6K was also significantly increased in tumor than in surrounding normal tissues. Overexpression of p70S6K in tumor tissues was further confirmed by immunohistochemistry. Strong immunoreactivity in the cytoplasm of thyroid cancer cells was seen in the majority of cases, whereas little immunoreactivity was found in the surrounding normal portion. Expression of phosphorylated Akt (pAkt) was also significantly higher in tumor tissues. Phosphorylation of Bad (pBad), a substrate of Akt, was also increased in the tumor tissues in association with activation of Akt, and the T/N ratio for pAkt positively correlated to the T/N ratio for pBad. The data presented here demonstrate that both p70S6K and Akt are activated in the majority of human papillary cancer cells. Activation of these signalings may be involved in the progression of papillary carcinoma by stimulating cell proliferation and/or preventing apoptosis.

Bioactivity of Insulin-like growth factors (IGEs) are positively or negatively regulated by IGF binding proteins (IGFBPs). Like IGFs, production of IGFBPs are influenced by a number of hormonal factors. We studied the effects of cytokines on production of IGFBPs in human fibroblasts in culture. Both IL-1beta and TNF-alpha inhibited IGFBP-3 (42/38 KDa species) production in a concentration dependent manner judged by Western ligand blot. Expression of IGFBP-3 mRNA was also decreased by these cytokines. Moreover, the treatment with IL-1beta and TNF-alpha resulted in appearance of smaller mol weight (26 KDa) immunoreactive IGFBP-3 fragment(s) which lacked the ability to bind 125I-IGFs, indicating that these cytokines degrade IGFBP-3 via activation of proteases. Both IL-1beta and TNF-alpha decreased production of IGFBP-4, whereas they increased that of IGFBP-6, IL-6 had little effect on production of IGFBPs. Likewise, interferon-gamma failed to affect of production of IGFBPs except at high concentrations. The present data demonstrate that cytokines, especially IL-1beta and TNF-alpha are potent regulators of IGFBPs production and degradation. These cytokines may alter tissue uptake of IGFs and help to counteract the catabolic state induced by them.

We investigated whether thyroid status modulates serum leptin concentrations and body composition as determined by bioelectric impedance analysis (BIA). The percent body fat mass (%FM) in male Graves' disease was significantly lower than that in age- and sex- matched normal subjects, at the levels of 11.4+/-6.4% (mean+/-SD) vs 19.9+/-9.2% for men (n=12, P

The role of cell surface heparan sulfate proteoglycans in the effect of basic fibroblast growth factor (bFGF) on FRTL-5 rat thyroid cells was investigated and compared with that of endothelial cells. FRTL-5 cells were incubated for 2 h with heparitinase (0.5-5.0 mU/mL), which specifically degrades heparan sulfate proteolgycans, and then stimulated by bFGF. The mitogenic effect of bFGF was estimated by measuring [3H]thymidine incorporation. Although cell surface heparan sulfates have been believed to be necessary for bFGF binding to its high affinity receptors, the heparitinase treatment had no significant effect on the DNA synthesis of FRTL-5 cells stimulated by bFGF. The binding study revealed that heparitinase treatment decreased low affinity bindings of [125I]bFGF to FRTL-5 cells by only 50% and did not attenuate the high affinity binding, while the same treatment abolished the high and low affinity binding to bovine pulmonary artery endothelial (CPAE) cells. Analysis of trypsin accessible cell surface 35SO4-labeled materials by Q-sepharose anion-exchange column chromatography showed that heparan sulfate proteoglycans, peaked at 0.55 M NaCl elution, disappeared from the surface of FRTL-5 cells after treatment with 2.0 mU/mL of heparitinase, indicating that the heparitinase resistant low-affinity binding sites are not heparan sulfates. These results demonstrate that cell surface heparan sulfates are not required for the high affinity binding of bFGF to FRTL-5 rat thyroid cells, while proteoglycans are necessary for binding to endothelial cells, and suggest that the mechanism of the action of bFGF is different in rat thyroid cells compared with endothelial cells.

Starting with a previously isolated cDNA for human thyrotropin receptor (TSHR), we established a transformed myeloma cell line, SP56, which expresses human TSHR on its cell surface. Binding analysis showed that SP56 bears 1.1 x 10(5) TSHR per cell with a Kd of 2.2 x 10(-10) M. Using the purified cellular membrane, we established a TSH binding inhibition immunoglobulin (TBII) assay for autoantibodies against TSHR. We compared it with the TBII assay utilizing porcine thyroid membranes expressing porcine TSHR, which has been widely used for TBII assay, by using 96 serum samples from patients with autoimmune thyroid disease and normal individuals. Our TBII assay was more sensitive than the one using porcine TSHR: of 38 sera of patients which were judged negative for autoantibodies to TSHR (TBII value below 10%) by the latter assay, 28 were positive (above 20%) in our assay. By using a perfusion culture system, we obtained as many as 3 x 10(10) SP56 cells, from which 3,450 mg protein of the membrane could be purified; this is sufficient for 15,000 assays. The results indicate that the membrane of the myeloma cell line SP56 is more suitable for use in the TBII assay than the porcine thyroid membrane, in terms of sensitivity to autoantibodies against TSHR in human sera.

In FRTL5 thyroid cells, endothelin(ET)-1 alone had no effect on DNA synthesis but caused a transient increase in c-fos mRNA levels and stimulated IGF-I induced DNA synthesis and cell proliferation. By contrast, ET-1 inhibited the stimulatory effects of TSH actions on DNA synthesis, cell proliferation and c-AMP production. 8-Bromo-cAMP-induced DNA synthesis was also inhibited by ET-1, suggesting that ET-1 exerts its inhibitory effects at step(s) involving cAMP production and post cAMP pathway. ET-1-induced suppression of TSH actions were reversed by a C-kinase inhibitor, H-7. These results suggest that the effect of ET on functions of FRTL5 cells is, at least, in part mediated by C-kinase dependent pathway.

An established cell line (TC-cell, clone 78) derived from human thyroid papillary cancer cells was investigated for production of peptide growth factors. The cells had specific binding sites for insulin-like growth factor-I (IGF-I) and responded to this growth factor with increased proliferation. Culture medium conditioned by TC cells was found to contain insulin-like growth factor (IGF)-I and IGF-binding protein(s). Furthermore, reverse transcription-polymerase chain reaction revealed expression of IGF-I mRNA. When monoclonal antibody to IGF-I receptors (αIR3) was added, the growth of TC cells cultured in serum-free medium was significantly reduced. The growth rate of the cells was restored when the antibody was removed from the medium. These results strongly suggest that TC cells produce IGF-I, which is involved in the regulation of their own growth.

In order to study the mechanism of GH secretion from somatotroph adenoma cells, we have compared the effect of 12-O-tetradecanoyl phorbol-13-acetate (TPA) with that of growth hormone releasing factor (GRF) on GH secretion from human somatotroph adenoma cells cultured in monolayer. Pituitary adenoma cells were obtained from 13 patients with acromegaly undergoing surgery. On the 7th day of culture, the cells were exposed for 2 h to secretagogues. All 13 adenoma cell cultures (100%) responded to TPA (1.6-16.0 nmol/l) with a two- to six-fold increase in GH release (240 +/- 37% increase of control: mean +/- SE). The response was detectable within 10 min, and was maximal at 2 h. Phospholipase C (7.7 mmol/l) also stimulated a two- to ten-fold increase in GH release in all four adenomas examined (100%). GH release was stimulated by GRF (2.0 nmol/l) in eight out of 12 adenoma cells (67%), but the magnitude of the responses to GRF (60 +/- 18% increase of control: mean +/- SE) were much smaller than that of TPA. Five out of 13 adenomas secreted detectable amount of PRL into the medium and these five adenomas (100%) responded to TPA (16.0 nmol/l) with a two- to six-fold increase. These observations indicate that the activation of protein kinase C is the consistent stimulator in GH and PRL secretion in human somatotroph adenoma cells. However, it is not determined whether the protein kinase C is involved in the in-vivo production of GH in patients with acromegaly.

Fibroblast growth factor (FGF) is one of the most potent endothelial cell growth factors and has been found in almost all tissues in the body. However, there is no definitive report showing that FGF exists in the thyroid. In this report, we describe heparin binding endothelial cell growth factor activity in porcine thyroids. The extract from normal adult porcine thyroids was loaded onto a heparin-sepharose affinity column. The mitogenic activity on endothelial cells was found to elute from the column with 0.9 M-2.0 M NaCl. Polyacrylamide gel electrophoresis and an immunoblotting of bioactive fractions showed the basic FGF immunoreactivity in a double band with a molecular weight of 18K and 20K. These results strongly imply that the adult porcine thyroid-derived heparin binding endothelial cell growth factor may be authentic basic FGF and one of its high molecular weight forms. In support of this conclusion, basic FGF mRNA was detected in poly A+ RNA isolated from cultured porcine thyroid cells. On the other hand, recombinant human basic FGF at the concentration of 0.1-10 ng/ml increased the incorporation of 3H-thymidine into FRTL-5 cells and porcine thyroid follicular cells in culture. The stimulatory effects were also observed when the biologically active fractions of heparin-sepharose column of the thyroid extract were added to the culture. In addition, both recombinant human basic FGF at 10 ng/ml and heparin binding fractions from porcine thyroids inhibited TSH-induced iodide uptake by porcine thyroid cells in culture. These results suggest that basic FGF may be one of the important local modulators of thyroid function, cell growth, and angiogenesis.

We investigated the direct effect of lithium on porcine thyroid cells in culture to exclude the secondary regulatory factors. First we have studied the effect of lithium on TSH-induced iodide uptake. Significant suppression was seen at 0.1 mmol/liter, and half-maximal suppression was obtained at the pharmacological concentration reported in patient serum. The suppression was dose dependent and reversible. Besides the suppression of cAMP production stimulated by TSH, lithium also inhibited iodide uptake stimulated by forskolin or 8-bromo-cAMP. These results demonstrated that lithium inhibits TSH-induced iodide uptake not only by reducing cAMP production, but also by acting on the steps of post-cAMP production. Next, we studied the effect of lithium on DNA synthesis of the cultured porcine thyroid cells. Lithium stimulated [3H]thymidine incorporation of the thyroid cells in the basal condition (0.5% fetal calf serum) as well as those stimulated by insulin-like growth factor-I (100 micrograms/liter). The minimal concentrations for the significant increase were 0.5 and 0.1 mmol/liter, respectively. These results suggest that lithium might contribute to the formation of the goiter directly at the cellular levels in patients treated with the agent.