Naotetsu Kanamoto

Human Induced Pluripotent Stem Cells Differentiated into Chondrogenic Lineage Via Generation of Mesenchymal Progenitor Cells

Human induced pluripotent stem cells (hiPSCs) exhibit pluripotency, proliferation capability, and gene expression similar to those of human embryonic stem cells (hESCs). hESCs readily form cartilaginous tissues in teratomas in vivo; despite extensive effort, however, to date no efficient method for inducing mature chondrocytes in vitro has been established. hiPSCs can also differentiate into cartilage in vivo by teratoma formation, but as with hESCs, no reliable system for in vitro chondrogenic differentiation of hiPSCs has yet been reported. Here, we examined the chondrogenic differentiation capability of hiPSCs using a multistep culture method consisting of embryoid body (EB) formation, cell outgrowth from EBs, monolayer culture of sprouted cells from EBs, and 3-dimensional pellet culture. In this culture process, the cell density of monolayer culture was critical for cell viability and subsequent differentiation capability. Monolayer-cultured cells exhibited fibroblast-like morphology and expressed markers for mesenchymal stem cells. After 2-3 weeks of pellet culture, cells in pellets exhibited a spherical morphology typical of chondrocytes and were surrounded by extracellular matrix that contained acidic proteoglycans. The expression of type II collagen and aggrecan in pellets progressively increased. Histological analysis revealed that over 70% of hiPSC-derived pellets successfully underwent chondrogenic differentiation. Using the same culture method, hESCs showed similar histological changes and gene expression, but differentiated slightly faster and more efficiently than hiPSCs. Our study demonstrates that hiPSCs can be efficiently differentiated into the chondrogenic lineage in vitro via generation of mesenchymal progenitor cells, using a simplified, multistep culture method.

Drug-induced neutropenia associated with anti-neutrophil cytoplasmic antibodies (ANCA):possible involvement of complement in granulocyte cytotoxicity

Although antineutrophil antibodies are thought to be involved in drug‐induced neutropenia, neither the precise mechanisms nor the particular antigens on the neutrophil surface have yet been clarified. Recently, we examined a patient with Graves’ disease who developed antineutrophil cytoplasmic antibodies (ANCA) after propylthiouracil treatment and exhibited neutropenia. Because several target antigens of ANCA are expressed on the surface of neutrophils, it was suggested that ANCA might contribute to neutropenia. The patient’s serum bound specifically to neutrophils and HL‐60 cells differentiated into granulocytes, and lysed the HL‐60 cells via a complement‐mediated mechanism. Furthermore, two representative ANCA antigens, proteinase 3 and myeloperoxidase, significantly inhibited both the binding and cytotoxicity of the serum. Finally, tumour necrosis factor‐α, which is known to up‐regulate cell surface expression of several ANCA antigens, enhanced both the binding and cytotoxicity of the serum. These findings suggest that ANCA induced by propylthiouracil contributed to leucopenia through a complement‐mediated mechanism.

Expression of the adrenomedullin gene in adipose tissue.

Adrenomedullin (AM) is a potent vasodilating peptide originally isolated from human pheochromocytoma cells. This report concerns the expression and secretion of AM from adipose tissue. Northern blot analysis demonstrated marked expression of AM mRNA in mouse adipose tissue. Expression levels in adipose tissues were 2.5-3.2 times higher than in the kidney. AM mRNA level in mature adipocytes was 7.3 times higher than in the stroma-vascular fraction of adipose tissue. In mature adipocyte culture, time-dependent increase of AM peptide concentration in the culture medium was detected. AM expression was also detected in human subcutaneous adipose tissue. Adipose AM expression significantly increased in obesity mouse model, high-fat diet fed mice and ob/ob mice. These results suggest that adipose tissue, especially mature adipocytes, is major source of AM in the body, and that adipocyte-derived AM plays a pathophysiological role in obesity.

The Local CNP/GC-B system in growth plate is responsible for physiological endochondral bone growth

Recent studies revealed C-type natriuretic peptide (CNP) and its receptor, guanylyl cyclase-B (GC-B) are potent stimulators of endochondral bone growth. As they exist ubiquitously in body, we investigated the physiological role of the local CNP/GC-B in the growth plate on bone growth using cartilage-specific knockout mice. Bones were severely shorter in cartilage-specific CNP or GC-B knockout mice and the extent was almost the same as that in respective systemic knockout mice. Cartilage-specific GC-B knockout mice were shorter than cartilage-specific CNP knockout mice. Hypertrophic chondrocyte layer of the growth plate was drastically reduced and proliferative chondrocyte layer, along with the proliferation of chondrocytes there, was moderately reduced in either cartilage-specific knockout mice. The survival rate of cartilage-specific CNP knockout mice was comparable to that of systemic CNP knockout mice. The local CNP/GC-B system in growth plate is responsible for physiological endochondral bone growth and might further affect mortality via unknown mechanisms.

2016 Guidelines for the management of thyroid storm from The Japan Thyroid Association and Japan Endocrine Society (First edition)

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.

Increased Bone Turnover and Possible Accelerated Fracture Healing in a Murine Model With an Increased Circulating C-Type Natriuretic Peptide

Recent studies have revealed that C-type natriuretic peptide (CNP) is a potent stimulator of endochondral bone growth. Nevertheless, the effect of CNP on bone turnover has not yet been well studied. To elucidate this issue, we investigated the bone phenotype of a mouse model with elevated plasma CNP concentrations (SAP-CNP-Tg mice) in the present study. Microcomputed tomography (CT) analysis revealed less bone in femurs, but not in lumber vertebrae, of young adult SAP-CNP-Tg mice than that of wild-type mice. Bone histomorphometry of the tibiae from 8-week-old SAP-CNP-Tg mice showed enhanced osteoblastic and osteoclastic activities, in accordance with elevated serum levels of osteocalcin and tartrate-resistant acid phosphatase-5b, respectively. Next we performed an open and stabilized femoral fracture using 8-week-old SAP-CNP-Tg mice and compared the healing process with age-matched wild-type mice. An immunohistochemical study revealed that CNP and its receptors, natriuretic peptide receptor-B and natriuretic peptide clearance receptor, are expressed in hard calluses of wild-type mice, suggesting a possible role of CNP/natriuretic peptide receptor-B signaling in fracture repair, especially in bone remodeling stage. On micro-CT analysis, a rapid decrease in callus volume was observed in SAP-CNP-Tg mice, followed by a generation of significantly higher new bone volume with a tendency of increased bone strength. In addition, a micro-CT analysis also showed that bone remodeling was accelerated in SAP-CNP-Tg mice, which was also evident from increased serum osteocalcin and tartrate-resistant acid phosphatase-5b levels in SAP-CNP-Tg mice at the remodeling stage of fracture repair. These results indicate that CNP activates bone turnover and remodeling in vivo and possibly accelerates fracture healing in our mouse model.

A comparison between 11C-methionine PET/CT and MIBI SPECT/CT for localization of parathyroid adenomas/hyperplasia.

ObjectiveThe purpose of this study was to compare the sensitivity of single-photon emission computed tomography/computed tomography (SPECT/CT) using 99mTc-sestamibi (MIBI) with that of PET/CT using 11C-methionine (MET) for localization of parathyroid adenomas/hyperplasia in primary hyperparathyroidism. Materials and methodsTwenty-three patients with primary hyperparathyroidism were analyzed. Fifteen patients underwent surgery, and the remaining eight did not, but these patients were clinically diagnosed as having primary hyperparathyroidism. Patients underwent both MET PET/CT and MIBI SPECT/CT scanning. The sensitivities of both modalities were evaluated on a per-patient basis, and on a per-lesion basis for parathyroid lesions detected by surgery. The size of the parathyroid adenoma/hyperplasia and serum intact parathyroid hormone levels were compared with the results of each of the two modalities. ResultsPer-patient sensitivities of MET PET/CT and MIBI SPECT/CT were 65 and 61%, respectively. Per-lesion sensitivities of MET PET/CT and MIBI SPECT/CT were 91 and 73% for histologically confirmed adenomas and 30 and 30% for hyperplastic glands, respectively. No significant differences were observed between the two modalities. The size of uptake-positive lesions was significantly larger than that of uptake-negative lesions in both modalities. Intact parathyroid hormone levels showed no significant difference between uptake-positive and uptake-negative patients in both modalities. ConclusionThe sensitivities of MET PET/CT and MIBI SPECT/CT were comparable. MET PET/CT has a complementary role in localizing parathyroid adenomas/hyperplasia when MIBI SPECT/CT is inconclusive.

Recombinant monoclonal thyrotropin-stimulation blocking antibody (TSBAb) established from peripheral lymphocytes of a hypothyroid patient with primary myxedema

Anti-TSH receptor antibodies (TRAbs) have been known to be involved in Graves’ disease and primary hypothyroidism. We previously isolated and reconstituted immunoglobulin (Ig) genes of Epstein-Barr virus-transformed B cell clones producing monoclonal TRAbs obtained from Graves’ patients. In the present study, we performed a similar experiment using a B cell clone, 32A-5, derived from a patient with primary hypothyroidism. The variable region genes of Ig heavy (H) and light (L) chains were isolated and sequenced from the 32A-5 clone. A significant number of somatic mutations were found in variable regions of H and L chain gene segments. Each pair of H and L chain cDNAs was ligated into an expression vector for IgG1 production and stably introduced into myeloma cells. The transfectants were injected ip into BALB/c mice to yield ample volume of the antibody for following applications. Interactions of recombinant 32A-5 with Graves’ sera with varying thyroid-stimulating antibody (TSAb) activities were studied. The recombinant antibody tended to suppress TSAb activities in 10 of 15 Graves’ sera, in which four were significantly inhibited. In summary, this is the first study to analyze human monoclonal TSH-stimulation blocking antibodies (TSBAb) at the molecular level. Use of human recombinant monoclonal TSBAb may be an analytical tool for molecular-basis etiology and an alternative therapeutic path for Graves’ disease.

Treatment and management of thyroid storm: analysis of the nationwide surveys: The taskforce committee of the Japan Thyroid Association and Japan Endocrine Society for the establishment of diagnostic criteria and nationwide surveys for thyroid storm.

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.

Brain-specific natriuretic peptide receptor-B deletion attenuates high-fat diet-induced visceral and hepatic lipid deposition in mice.

C-type natriuretic peptide (CNP) and its receptor, natriuretic peptide receptor-B (NPR-B), are abundantly distributed in the hypothalamus. To explore the role of central CNP/NPR-B signaling in energy regulation, we generated mice with brain-specific NPR-B deletion (BND mice) by crossing Nestin-Cre transgenic mice and mice with a loxP-flanked NPR-B locus. Brain-specific NPR-B deletion prevented body weight gain induced by a high-fat diet (HFD), and the mesenteric fat and liver weights were significantly decreased in BND mice fed an HFD. The decreased liver weight in BND mice was attributed to decreased lipid accumulation in the liver, which was confirmed by histologic findings and lipid content. Gene expression analysis revealed a significant decrease in the mRNA expression levels of CD36, Fsp27, and Mogat1 in the liver of BND mice, and uncoupling protein 2 mRNA expression was significantly lower in the mesenteric fat of BND mice fed an HFD than in that of control mice. This difference was not observed in the epididymal or subcutaneous fat. Although previous studies reported that CNP/NPR-B signaling inhibits SNS activity in rodents, SNS is unlikely to be the underlying mechanism of the metabolic phenotype observed in BND mice. Taken together, CNP/NPR-B signaling in the brain could be a central factor that regulates visceral lipid accumulation and hepatic steatosis under HFD conditions. Further analyses of the precise mechanisms will enhance our understanding of the contribution of the CNP/NPR-B system to energy regulation.