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Proteasome inhibitors (PIs) can preferentially restore bone in bone-defective lesions of patients with multiple myeloma (MM) who respond favorably to these drugs. Most prior in vitro studies on PIs used continuous exposure to low PI concentrations, although pharmacokinetic analysis in patients has shown that serum concentrations of PIs change in a pulsatile manner. In the present study, we explored the effects of pulsatile treatment with PIs on bone metabolism to simulate in vivo PI pharmacokinetics. Pulsatile treatment with bortezomib, carfilzomib, or ixazomib induced MM cell death but only marginally affected the viability of osteoclasts (OCs) with F-actin ring formation. Pulsatile PI treatment suppressed osteoclastogenesis in OC precursors and bone resorption by mature OCs. OCs robustly enhanced osteoblastogenesis in cocultures with OCs and MC3T3-E1 pre-osteoblastic cells, indicating OC-mediated coupling to osteoblastogenesis. Importantly, pulsatile PI treatment did not impair robust OC-mediated osteoblastogenesis. These results suggest that PIs might sufficiently reduce MM cell-derived osteoblastogenesis inhibitors to permit OC-driven bone formation coupling while suppressing OC differentiation and activity in good responders to PIs. OC-mediated coupling to osteoblastogenesis appears to be a predominant mechanism for preferential occurrence of bone regeneration at sites of osteoclastic bone destruction in good responders.

Objective : To examine diagnostic performance of corticotropin-releasing hormone (CRH) test combined with baseline dehydroepiandrosterone sulfate (DHEA-S) in patients with a suspect of central adrenal insufficiency. Methods : Patients (n=215) requiring daily or intermittent hydrocortisone replacement, or no replacement were retrospectively checked with their peak cortisol after CRH test and baseline DHEA-S. Results : None of 106 patients with the peak cortisol ≥ 17.5 µg / dL after CRH test required replacement, and all 64 patients with the peak cortisol 10.0 µg / dL required daily replacement. Among 8 patients with 10.0 µg / dL ≤ the peak cortisol 17.5 µg / dL and baseline DHEA-S below the reference range, 6 patients required daily replacement and 1 patient was under intermittent replacement. Among 37 patients with 10.0 µg / dL ≤ the peak cortisol 17.5 µg / dL and baseline DHEA-S within the reference range, 10 and 6 patients were under intermittent and daily replacement, respectively. Conclusions : No patients with the peak cortisol ≥ 17.5 µg / dL required hydrocortisone replacement, and all patients with the peak cortisol below 10.0 µg / dL required daily replacement. Careful clinical evaluation was required to determine requirement for replacement in patients with 10.0 µg / dL ≤ the peak cortisol 17.5 µg / dL even in combination with baseline DHEA-S. J. Med. Invest. 69 : 287-293, August, 2022.

Background: Advanced glycation end product (AGE) accumulation is thought to be an independent predictor for cardiovascular disease in people with diabetes and renal failure. Since it has remained unclear whether AGE has a clinical impact on diabetic kidney disease (DKD) , the aim of this study was to determine the association between AGE accumulation, measured as skin autofluorescence (AF) , and the progression of DKD. Methods: Skin AF was measured by AGE reader® and spot urine biomarkers, including albumin and liver-type fatty acid-binding protein (L-FABP) , were determined as the urine albumin-to-creatinine ratio (uACR) and the urine L-FABP-to-creatinine ratio (uL-FABPCR) in 300 Japanese people with diabetes (173 males and 127 females, mean age, 67.8 ± 10.7 years) . The relationships between skin AF and those urine biomarkers of DKD were statistically evaluated. Results: Simple regression analysis showed that skin AF was associated with uACR (p Conclusion: Skin AF was associated with urinary L-FABP but not with albuminuria in people with diabetes. The results suggest that skin AF can serve as a novel predictive factor for the development of diabetic tubular injury. Disclosure H.Yamagami: None. T.Yuasa: None. S.Yoshida: None. A.Kuroda: None. M.Matsuhisa: Research Support; Nissui , Sysmex Corp., Speaker's Bureau; Astellas Pharma Inc., Eli Lilly and Company, Merck Sharp & Dohme Corp., Mitsubishi Tanabe Pharma Corporation, Novo Nordisk, Sanofi, Sumitomo Dainippon Pharma Co., Ltd., Takeda Pharmaceutical Company Limited. I.Endo: None. K.Aihara: None. M.Abe: None. S.Yasui: None. M.Hosoki: None. T.Hori: None. T.Hara: None. Y.Mitsui: None. K.Kurahashi: None. S.Nakamura: None. T.Otoda: None.

Aims/Introduction: Anemia is one of the common complications of diabetes mellitus (DM) and is associated with an increased risk of death. In recent years, phase angle (PhA) and the ratio of extracellular water to total body water (ECW/TBW) measured by bioelectrical impedance analysis (BIA) have been commonly used for estimating body composition. Since PhA and ECW/TBW have been reported to be useful as prognostic indicators for various chronic disease, we aimed to clarify the clinical significance of PhA and ECW/TBW in anemia of patients with DM. Materials and Methods: The values of PhA and ECW/TBW were measured by a portable BIA device (Inbody S10, Inbody Japan, Tokyo, Japan) and blood samples were collected in 403 Japanese patients with DM (217 males and 186 females) atAnan Medical Center, Tokushima Japan. The relationships of PhA and ECW/TBW with hematocrit (Ht) and hemoglobin (Hb) were statistically evaluated. Results: The level of PhA was decreased with age, whereas the value of ECW/TBW was increased with age. There was close inverse relationship between PhA and ECW/TBW (R2=0.7540, p< 0.001) . PhA was positively correlated with Hb and Ht levels (p Conclusions: PhA and ECW/TBW were associated with levels of Hb and Ht in patients with diabetes. The results suggest that BIA parameters reflect red blood cell lifespan and erythropoiesis. Disclosure T. Hori: None. H. Yamagami: None. S. Yasui: None. M. Hosoki: None. T. Hara: None. K. Kurahashi: None. S. Yoshida: None. M. Abe: None.

Aims and Introduction: The prevalence and incidence of nonalcoholic fatty liver disease (NAFLD) have been increasing worldwide. Thrombin exerts various pathophysiological functions via activation of protease-activated receptors (PARs) , and activation of PARs promotes the development of NAFLD. Since heparin cofactor II (HCII) specifically inhibits thrombin action by binding to dermatan sulfate proteoglycans in various tissue matrices, we hypothesized that plasma HCII activity is associated with the development of NAFLD. Materials and Methods: Plasma HCII activity and clinical markers for assessment of hepatic fibrosis including Fibrosis 4 (FIB-4) index, NAFLD fibrosis score (NFS) , and aspartate aminotransferase to platelet ratio index (APRI) were determined in 322 Japanese patients with diabetes mellitus (183 males and 139 females; mean age, 66.0±11.3 years) . The relationships between plasma HCII activities and the clinical markers were statistically evaluated. Results: The mean plasma HCII activity in all participants was 94.0 ± 17.7%. Multivariate regression analysis with adjustments for confounding factors including age, sex, and serum albumin showed that plasma HCII activity independently contributed to reduction of the FIB-4 index (p < 0.0001) , NFS (p < 0.0001) , and APRI (p < 0.05) . Conclusions: Plasma HCII activity was independently and inversely associated with the clinical markers of hepatic fibrosis. The results suggest that HCII can serve as a novel therapeutic target for the development of NAFLD in patients with diabetes. Disclosure T.Hara: None. I.Endo: None. M.Matsuhisa: Research Support; Nissui , Sysmex Corp., Speaker’s Bureau; Astellas Pharma Inc., Eli Lilly and Company, Merck Sharp & Dohme Corp., Mitsubishi Tanabe Pharma Corporation, Novo Nordisk, Sanofi, Sumitomo Dainippon Pharma Co., Ltd., Takeda Pharmaceutical Company Limited. M.Abe: None. K.Aihara: None. H.Yamagami: None. S.Masuda: None. Y.Mitsui: None. K.Kurahashi: None. S.Yoshida: None. T.Otoda: None. T.Yuasa: None. A.Kuroda: None.

A 70-year-old woman was hospitalized for diarrhea, vomiting, loss of appetite, fatigue, and dyspnea on exertion for the past 3 weeks and treated with intravenous fluid for dehydration. She was receiving prednisolone for polymyositis. She did not have a history of thyroid disease. On day 4 of hospitalization, the patient was diagnosed with congestive heart failure and tachycardiac atrial fibrillation, and treatment with a diuretic agent was initiated. On day 7 of hospitalization, a clinical laboratory evaluation revealed that the level of free thyroxine was 9.95 ng/dL, free triiodothyronine was >30 pg/mL, and thyroid-stimulating hormone was

Thrombin exerts various pathophysiological functions by activating protease-activated receptors (PARs), and thrombin-induced activation of PARs promotes the development of non-alcoholic fatty liver disease (NAFLD). Since heparin cofactor II (HCII) specifically inactivates thrombin action, we hypothesized that plasma HCII activity correlates with the severity of NAFLD.A cross-sectional study was conducted. Plasma HCII activity and noninvasive clinical markers of hepatic fibrosis including fibrosis-4 (FIB-4) index, NAFLD fibrosis score (NFS) and aspartate aminotransferase-to-platelet ratio index (APRI) were determined in 305 Japanese patients with type 2 diabetes mellitus (T2DM). The relationships between plasma HCII activity and the clinical markers were statistically evaluated.Multiple regression analysis including confounding factors showed that plasma HCII activity independently contributed to decreases in FIB-4 index (p＜0.001), NFS (p＜0.001) and APRI (p=0.004). In addition, logistic regression analysis for the prevalence of advanced hepatic fibrosis defined by the cutoff points of the clinical scores showed that plasma HCII activity was the sole and common negative factor for prevalence of advanced hepatic fibrosis (FIB-4 index: p=0.002, NFS: p=0.026 and APRI: p=0.012).Plasma HCII activity was inversely associated with clinical hepatic fibrosis indices including FIB-4 index, NFS and APRI and with the prevalence of advanced hepatic fibrosis in patients with T2DM. The results suggest that HCII can serve as a novel biomarker for assessment of hepatic fibrosis of NAFLD in patients with T2DM.

Exercise offers mechanical loading to the bone, while it stimulates energy expenditure in the adipose tissue. Thus, bone may secrete a factor to communicate with adipose tissue in response to mechanical loading. Interleukin (IL)-11 is expressed in the bone, upregulated by mechanical loading, enhances osteogenesis and suppresses adipogenesis. Systemic IL-11 deletion (IL-11−/−) exhibited reduced bone mass, suppressed bone formation response to mechanical loading, enhanced expression of Wnt inhibitors, and suppressed Wnt signaling. Enhancement of bone resorption under mechanical unloading was unaffected. Unexpectedly, IL-11−/− mice showed increased systemic adiposity and glucose intolerance. Osteoblast/osteocyte-specific IL-11 deletion in osteocalcin-Cre;IL-11fl/fl mice showed reduced serum IL-11, blunted bone formation under mechanical loading, and increased systemic adiposity similar to IL-11−/− mice. Adipocyte-specific IL-11 deletion in adiponectin-Cre; IL-11fl/fl mice exhibited no abnormality. Thus, IL-11 from osteoblast/osteocyte controls both osteogenesis and systemic adiposity in response to mechanical loading. These findings may bring new therapeutic approaches to osteoporosis and metabolic syndrome.

Exercise results in mechanical loading of the bone and stimulates energy expenditure in the adipose tissue. It is therefore likely that the bone secretes factors to communicate with adipose tissue in response to mechanical loading. Interleukin (IL)−11 is known to be expressed in the bone, it is upregulated by mechanical loading, enhances osteogenesis and suppresses adipogenesis. Here, we show that systemic IL-11 deletion (IL-11−/−) results in reduced bone mass, suppressed bone formation response to mechanical loading, enhanced expression of Wnt inhibitors, and suppressed Wnt signaling. At the same time, the enhancement of bone resorption by mechanical unloading was unaffected. Unexpectedly, IL-11−/− mice have increased systemic adiposity and glucose intolerance. Osteoblast/osteocyte-specific IL-11 deletion in osteocalcin-Cre;IL-11fl/fl mice have reduced serum IL-11 levels, blunted bone formation under mechanical loading, and increased systemic adiposity similar to IL-11−/− mice. Adipocyte-specific IL-11 deletion in adiponectin-Cre;IL-11fl/fl did not exhibit any abnormalities. We demonstrate that osteoblast/osteocyte-derived IL-11 controls both osteogenesis and systemic adiposity in response to mechanical loading, an important insight for our understanding of osteoporosis and metabolic syndromes.

Receptor activator of NF-&kappa, B ligand (RANKL), a critical mediator of osteoclastogenesis, is upregulated in multiple myeloma (MM). The xanthine oxidase inhibitor febuxostat, clinically used for prevention of tumor lysis syndrome, has been demonstrated to effectively inhibit not only the generation of uric acid but also the formation of reactive oxygen species (ROS). ROS has been demonstrated to mediate RANKL-mediated osteoclastogenesis. In the present study, we therefore explored the role of cancer-treatment-induced ROS in RANKL-mediated osteoclastogenesis and the suppressive effects of febuxostat on ROS generation and osteoclastogenesis. RANKL dose-dependently induced ROS production in RAW264.7 preosteoclastic cells, however, febuxostat inhibited the RANKL-induced ROS production and osteoclast (OC) formation. Interestingly, doxorubicin (Dox) further enhanced RANKL-induced osteoclastogenesis through upregulation of ROS production, which was mostly abolished by addition of febuxostat. Febuxostat also inhibited osteoclastogenesis enhanced in cocultures of bone marrow cells with MM cells. Importantly, febuxostat rather suppressed MM cell viability and did not compromise Dox&rsquo, s anti-MM activity. In addition, febuxostat was able to alleviate pathological osteoclastic activity and bone loss in ovariectomized mice. Collectively, these results suggest that excessive ROS production by aberrant RANKL overexpression and/or anticancer treatment disadvantageously impacts bone, and that febuxostat can prevent the ROS-mediated osteoclastic bone damage.

Immunoglobulin A vasculitis (IgAV), formerly known as Henoch-Schönlein purpura, primarily occurs during childhood between the ages of 3 and 15 years and is the most common form of systemic vasculitis in children ; its occurrence in adults has been rarely reported. Such low incidence could be attributable to either under-diagnosis or misdiagnosis. Thus, not only pediatricians but also physicians should be able to diagnose IgAV accurately to manage the patients appropriately and avoid its associated complications. In addition, treatment of adult onset IgAV with renal involvement has not been fully established yet. We describe here a case of adult onset IgAV complicated by proteinuria and pharyngitis, which was cured by no specific treatment. J. Med. Invest. 66 : 344-346, August, 2019.

Acromegaly is caused by excessive growth hormone secretion, usually from pituitary adenomas. Somoatostatin analogues are widely used as primary or adjunctive therapy in the management of acromegaly. In this report, we present a case with remarkable shrinkage of a tumor after relatively short-term octreotide long-acting release (LAR) administration. During the 30-month follow-up after starting octreotide LAR, there was no recurrence of acromegaly with remarkable shrinkage of the tumor on pituitary magnetic resonance imaging. A literature review of the predictors for tumor shrinkage after the administration of somatostatin analogues in patients with acromegaly is also discussed in relation to this case.

Fibroblast growth factor (FGF) 23 produced by the bone is the principal hormone to regulate serum phosphate level. Serum FGF23 needs to be tightly regulated to maintain serum phosphate in a narrow range. Thus, we hypothesized that the bone has some phosphate-sensing mechanism to regulate the production of FGF23. Previously we showed that extracellular phosphate induces the phosphorylation of FGF receptor 1 (FGFR1) and FGFR1 signaling regulates the expression of Galnt3, whose product works to increase FGF23 production in vitro. In this study, we show the significance of FGFR1 in the regulated FGF23 production and serum phosphate level in vivo. We generated late-osteoblast/osteocyte-specific Fgfr1-knockout mice (Fgfr1fl/fl; OcnCre/+) by crossing the Ocn-Cre and the floxed Fgfr1 mouse lines. We evaluated serum phosphate and FGF23 levels, the expression of Galnt3 in the bone, the body weight and life span. A selective ablation of Fgfr1 aborted the increase of serum active full-length FGF23 and the enhanced expression of Galnt3 in the bone by a high phosphate diet. These mice showed more pronounced hyperphosphatemia compared with control mice. In addition, these mice fed with a control diet showed body weight loss after 23 weeks of age and shorter life span. These results reveal a novel significance of FGFR1 signaling in the phosphate metabolism and normal life span.

Background : Serum albumin concentration (SAC) is a prognostic factor that is affected by many factors such as postural change, liver function and food intake. Chronic kidney disease (CKD) patients excrete proteinuria, have low-protein diet, and receive glucocorticoid therapy. No one has evaluated the most influential factors on SAC in CKD patients. Methods : A retrospective study. Hospitalized CKD patients with less than 1 g/ gCreatinine proteinuria receiving glucocorticoid therapy (n=28), with 1 or more g/gCreatinine proteinuria not receiving glucocorticoid therapy (n=36), and with 1 or more g/gCreatinine proteinuria receiving glucocorticoid therapy (n=39) were enrolled. SAC, hemoglobin, proteinuria and blood pressure at the last outpatient check-up before hospitalization, on the second day of hospitalization, at the last laboratory examination before discharge, as well as at the first outpatient follow-up after discharge were analyzed. Results : SAC decreased on the second day of hospitalization and increased at the first outpatient follow-up significantly in all groups. Unexpectedly, the change of SAC was irrelevant to the amount of proteinuria. Conclusions : SAC was affected by not only proteinuria, but also postural change, physical activity, and food in CKD patients. SAC should be analyzed by standardizing a patient’s condition during phlebotomy.

Aim: Accelerated thrombin action is associated with insulin resistance. It is known that upon activation by binding to dermatan sulfate proteoglycans, heparin cofactor II (HCII) inactivates thrombin in tissues. Because HCII may be involved in glucose metabolism, we investigated the relationship between plasma HCII activity and insulin resistance. Methods and Results: In a clinical study, statistical analysis was performed to examine the relationships between plasma HCII activity, glycosylated hemoglobin (HbA1c), fasting plasma glucose (FPG), and homeostasis model assessment-insulin resistance (HOMA-IR) in elderly Japanese individuals with lifestyle-related diseases. Multiple regression analysis showed significant inverse relationships between plasma HCII activity and HbA1c (p = 0.014), FPG (p = 0.007), and HOMA-IR (p = 0.041) in elderly Japanese subjects. In an animal study, HCII+/+ mice and HCII+/− mice were fed with a normal diet or high-fat diet (HFD) until 25 weeks of age. HFD-fed HCII+/− mice exhibited larger adipocyte size, higher FPG level, hyperinsulinemia, compared to HFD-fed HCII+/+ mice. In addition, HFD-fed HCII+/− mice exhibited augmented expression of monocyte chemoattractant protein-1 and tumor necrosis factor, and impaired phosphorylation of the serine/threonine kinase Akt and AMP-activated protein kinase in adipose tissue compared to HFD-fed HCII+/+ mice. The expression of phosphoenolpyruvate carboxykinase and glucose-6-phosphatase was also enhanced in the hepatic tissues of HFD-fed HCII+/− mice. Conclusions: The present studies provide evidence to support the idea that HCII plays an important role in the maintenance of glucose homeostasis by regulating insulin sensitivity in both humans and mice. Stimulators of HCII production may serve as novel therapeutic tools for the treatment of type 2 diabetes.

Paraneoplastic syndromes are generally defined as clinical disorders associated with malignant diseases, and hypocalcemia associated with cancer is a rare condition. A woman in her 60s was referred to our hospital for the further examination of massive ascites due to carcinoma of unknown primary origin. She complained of numbness around her lips, and marked hypocalcemia of 5.0 mg/dL was noted. After two courses of chemotherapy, computed tomography showed a decrease in the ascites, and her serum calcium level increased. Although hypocalcemia is a very rare condition in patients with gastric cancer, serum calcium values should be evaluated when neurological symptoms are observed.

// Ariunzaya Bat-Erdene 1 , Hirokazu Miki 2 , Asuko Oda 1 , Shingen Nakamura 1 , Jumpei Teramachi 1, 4 , Ryota Amachi 1, 3 , Hirofumi Tenshin 1, 3 , Masahiro Hiasa 1, 3 , Masami Iwasa 1 , Takeshi Harada 1 , Shiro Fujii 1 , Kimiko Sogabe 1 , Kumiko Kagawa 1 , Sumiko Yoshida 1 , Itsuro Endo 1 , Kenichi Aihara 1 , Masahiro Abe 1 1 Department of Hematology, Endocrinology and Metabolism, Institute of Biomedical Sciences, Tokushima University Graduate School of Medicine, Tokushima, Japan 2 Division of Transfusion Medicine and Cell Therapy, Tokushima University Hospital, Tokushima, Japan 3 Department of Orthodontics and Dentofacial Orthopedics, Tokushima University Graduate School of Oral Sciences, Tokushima, Japan 4 Department of Histology and Oral Histology, Tokushima University Graduate School of Oral Sciences, Tokushima, Japan Correspondence to: Masahiro Abe, email: masabe@tokushima-u.ac.jp Keywords: multiple myeloma, panobinostat, proteasome inhibitors, caspase-8, Sp1 Received: July 16, 2016 Accepted: September 29, 2016 Published: October 12, 2016 ABSTRACT Panobinostat, a pan-deacetylase inhibitor, synergistically elicits cytotoxic activity against myeloma (MM) cells in combination with the proteasome inhibitor bortezomib. Because precise mechanisms for panobinostat’s anti-MM action still remain elusive, we aimed to clarify the mechanisms of anti-MM effects of panobinostat and its synergism with proteasome inhibitors. Although the transcription factor Sp1 was overexpressed in MM cells, the Sp1 inhibitor terameprocol induced MM cell death in parallel with reduction of IRF4 and cMyc. Panobinostat induced activation of caspase-8, which was inversely correlated with reduction of Sp1 protein levels in MM cells. The panobinostat-mediated effects were further potentiated to effectively induce MM cell death in combination with bortezomib or carfilzomib even at suboptimal concentrations as a single agent. Addition of the caspase-8 inhibitor z-IETD-FMK abolished the Sp1 reduction not only by panobinostat alone but also by its combination with bortezomib, suggesting caspase-8-mediated Sp1 degradation. The synergistic Sp1 reduction markedly suppressed Sp1-driven prosurvival factors, IRF4 and cMyc. Besides, the combinatory treatment reduced HDAC1, another Sp1 target, in MM cells, which may potentiate HDAC inhibition. Collectively, caspase-8-mediated post-translational Sp1 degradation appears to be among major mechanisms for synergistic anti-MM effects of panobinostat and proteasome inhibitors in combination.

Expansion of Th 1 -like Vγ 9 Vδ 2 T cells by new-generation IMiDs, lenalidomide and pomalidomide, in combination with zoledronic acid

Background: To prevent the end-stage renal failure, it is important to identify the high-risk patients with rapid decline of renal function (RDRF). Aim: We intended to clarify the risk factors for RDRF in Japanese patients with diabetes. Methods: One hundred sixteen out-patients with diabetes (type 1/2, 38/78; M/F, 60/56; age 60.1±12.3 years; BMI, 25.3±4.8; HbA1c, 7.3±1.0%; eGFR, 68±22mL/min/1.73m2) were enrolled in this study, and were followed for one year. We defined RDRF as more than 20% of annual decline of eGFR. The sodium intake was estimated from the urine sodium excretion from early morning urine specimen according to previous report (J Hum Hypertens. 2002). The visceral fat area (VFA) was determined by the impedance method (HDS-2000, Omron Healthcare, Japan) Results: 8.6% of participants were matched to the criteria of RDRF. In subjects with RDRF, the energy intake (45.6±10.6 kcal/kg BW/day), sodium intake (12.5±1.8 g/day), VFA (157±51 cm2) and prevalence of hypertension (100%) were higher than those without RDRF. Annual decline of eGFR was significantly correlated with the stage of diabetic nephropathy, VFA, systolic and diastolic blood pressure, urinary albumin excretion rate, triglyceride, and daily intake of energy, sodium and protein (p Conclusion: Visceral obesity, and high intake of energy and sodium contributed to rapid decline of renal function in Japanese patients with diabetes. Body weight management with adequate energy and sodium intake, therefore, may contribute to prevent progression of diabetic kidney disease. Disclosure M. Matsuhisa: None. H. Mori: None. M. Ohishi: None. M. Ishizu: None. R. Suzuki: None. I. Takikawa: None. S. Taniguchi: None. M. Tamaki: None. Y. Akehi: None. K. Kurahashi: None. S. Yoshida: None. I. Endo: None. K. Aihara: None. M. Funaki: Research Support; Self; Otsuka Holdings Co., Ltd., The Knowledge Cluster from the Ministry of Education, Science, and Culture of Japan. Board Member; Self; Mechanogenic C.C.. A. Kuroda: None.

Background: CpG cytosine in the human insulin gene (INS) is uniquely unmethylated in pancreatic β-cells. It has been reported that a few unmethylated CpG specific PCR in circulating cell-free DNA could detect injury of pancreatic β-cells. However, the same CpG methylation pattern with these CpG sites are also existing in non-β-cells, thus the false positive results might be involved in these reports. Aim: To develop a precise method for detecting pancreatic β-cell DNA in the circulation. Methods: We have developed the dual Amplification Refractory Mutation System (ARMS) PCR, which amplifies only if 4 CpG sites were all unmethylated. The first ARMS PCR amplifies if 2 CpG sites (+331, 404bp of transcription start sites (TSS) of INS) are unmethylated simultaneously, and followed by the second ARMS PCR, which amplifies if 2 CpG sites (+367, 374bp of TSS of INS) are unmethylated simultaneously. This method was applied to 52 patients with type 1 diabetes (T1D) (duration 12.6 +/- 10.1 years). We confirmed the positive samples using DNA sequence analysis. Results: Pancreatic β-cell DNA were detected in 3 T1D (11.8, 2.5, 912.7 copies in 0.1mL of serum) who are diagnosed as slowly progressive T1D, and 4 among 16 healthy control subjects (1.2, 2.2, 1.9, 3.7 copies in 0.1mL of serum). Conclusion: We have developed the dual ARMS PCR which is a precise method to detect circulating pancreatic β-cell DNA. Almost no β-cell DNA was circulating in the long standing T1D, however, β-cell DNA was detectable in slowly progressive T1D. Also, there were not so many, but a few β-cell DNA in the circulation of healthy control subjects. Therefore, this method is useful to evaluate pathogenesis of type 1 diabetes. Disclosure A. Kuroda: None. M.Y. Yamada: None. Y. Tominaga: None. R. Suzuki: None. M. Tamaki: None. Y. Akehi: None. Y. Takashi: None. D. Koga: Employee; Self; Otsuka Pharmaceutical Co.,Ltd.. E. Shimokita: None. F. Tanihara: None. K. Kurahashi: None. S. Yoshida: None. I. Endo: None. K. Aihara: None. M. Abe: None. K. Ferreri: None. M. Matsuhisa: None.

A 78-year-old Japanese man showed suppression of the hypothalamic-pituitary-adrenal axis during maximum androgen blockade (MAB) therapy including chlormadinone acetate (CMA) for prostate cancer. After stopping the MAB therapy, both the basal ACTH level and the response to CRH recovered. While no reports have indicated that CMA suppresses the hypothalamic-pituitary-adrenal axis in patients with prostate cancer, CMA has been shown to inhibit this axis in animals. These observations suggest that we must monitor the hypothalamic-pituitary-adrenal axis in patients treated with CMA, especially under stressful conditions.

Activating mutations of calcium-sensing receptor (CaSR) cause autosomal dominant hypocalcemia (ADH). ADH patients develop hypocalcemia, hyperphosphatemia, and hypercalciuria, similar to the clinical features of hypoparathyroidism. The current treatment of ADH is similar to the other forms of hypoparathyroidism, using active vitamin D3 or parathyroid hormone (PTH). However, these treatments aggravate hypercalciuria and renal calcification. Thus, new therapeutic strategies for ADH are needed. Calcilytics are allosteric antagonists of CaSR, and may be effective for the treatment of ADH caused by activating mutations of CaSR. In order to examine the effect of calcilytic JTT-305/MK-5442 on CaSR harboring activating mutations in the extracellular and transmembrane domains in vitro, we first transfected a mutated CaSR gene into HEK cells. JTT-305/MK-5442 suppressed the hypersensitivity to extracellular Ca(2+) of HEK cells transfected with the CaSR gene with activating mutations in the extracellular and transmembrane domains. We then selected two activating mutations locating in the extracellular (C129S) and transmembrane (A843E) domains, and generated two strains of CaSR knock-in mice to build an ADH mouse model. Both mutant mice mimicked almost all the clinical features of human ADH. JTT-305/MK-5442 treatment in vivo increased urinary cAMP excretion, improved serum and urinary calcium and phosphate levels by stimulating endogenous PTH secretion, and prevented renal calcification. In contrast, PTH(1-34) treatment normalized serum calcium and phosphate but could not reduce hypercalciuria or renal calcification. CaSR knock-in mice exhibited low bone turnover due to the deficiency of PTH, and JTT-305/MK-5442 as well as PTH(1-34) increased bone turnover and bone mineral density (BMD) in these mice. These results demonstrate that calcilytics can reverse almost all the phenotypes of ADH including hypercalciuria and renal calcification, and suggest that calcilytics can become a novel therapeutic agent for ADH.

Proviral Integrations of Moloney virus 2 (PIM2) is overexpressed in multiple myeloma (MM) cells, and regarded as an important therapeutic target. Here, we aimed to validate the therapeutic efficacy of different types of PIM inhibitors against MM cells for their possible clinical application. Intriguingly, the thiazolidine-2,4-dione-family compounds SMI-16a and SMI-4a reduced PIM2 protein levels and impaired MM cell survival preferentially in acidic conditions, in contrast to other types of PIM inhibitors, including AZD1208, CX-6258 and PIM447. SMI-16a also suppressed the drug efflux function of breast cancer resistance protein, minimized the sizes of side populations and reduced in vitro colony-forming capacity and in vivo tumourigenic activity in MM cells, suggesting impairment of their clonogenic capacity. PIM2 is known to be subject to ubiquitination-independent proteasomal degradation. Consistent with this, the proteasome inhibitors bortezomib and carfilzomib increased PIM2 protein levels in MM cells without affecting its mRNA levels. However, SMI-16a mitigated the PIM2 protein increase and cooperatively enhanced anti-MM effects in combination with carfilzomib. Collectively, the thiazolidine-2,4-dione-family compounds SMI-16a and SMI-4a uniquely reduce PIM2 protein in MM cells, which may contribute to their profound efficacy in addition to their immediate kinase inhibition. Their combination with proteasome inhibitors is envisioned.

Tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) agonists induce tumor-specific apoptosis indicating that they may be an attractive therapeutic strategy against cancers, including multiple myeloma (MM). Osteoclastogenesis is highly induced in MM, which in turn enhances MM growth, thereby forming a vicious cycle between MM tumor expansion and bone destruction. However, the effects of TRAIL on MM-enhanced osteoclastogenesis remain largely unknown. Here, we show that TRAIL induced apoptosis in MM cells, but not in osteoclasts (OCs), and that it rather facilitated receptor activator of NF-kB ligand–induced osteoclastogenesis along with upregulation of cellular FLICE inhibitory protein (c-FLIP). TRAIL did not induce death-inducing signaling complex formation in OCs, but formed secondary complex (complex II) with the phosphorylation of transforming growth factor b–activated kinase-1 (TAK1), and thus activated NF-kB signaling. c-FLIP knockdown abolished complex II formation, thus permitting TRAIL induction of OC cell death. The TAK1 inhibitor LLZ1640-2 abrogated the TRAIL-induced c-FLIP upregulation and NF-kB activation, and triggered TRAIL-induced caspase-8 activation and cell death in OCs. Interestingly, the TRAIL-induced caspase-8 activation caused enzymatic degradation of the transcription factor Sp1 to noticeably reduce c-FLIP expression, which further sensitized OCs to TRAIL-induced apoptosis. Furthermore, the TAK1 inhibition induced antiosteoclastogenic activity by TRAIL even in cocultures with MM cells while potentiating TRAIL’s anti-MM effects. These results demonstrated that osteoclastic lineage cells use TRAIL for their differentiation and activation through tilting caspase-8–dependent apoptosis toward NF-kB activation, and that TAK1 inhibition subverts TRAIL-mediated NF-kB activation to resume TRAIL-induced apoptosis in OCs while further enhancing MM cell death in combination with TRAIL.