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Nanomedicine modification with ligands directed to receptors on tumor blood vessels has the potential for selectively enhancing nanomedicine accumulation in malignant tissues by overcoming the vascular barrier of tumors. Nevertheless, the development of broadly applicable ligand approaches capable of promoting the transvascular transport of nanomedicines in a wide spectrum of tumors has been elusive so far. By considering the indispensable and persistent glycolytic fueling of tumors, we developed glucose-installed polymeric micelles loading cisplatin (Gluc-CDDP/m) targeting the glucose transporter 1 (GLUT1), which is overexpressed in most tumors and present on vascular endothelial cells, toward improving the delivery efficiency and therapeutic efficacy. The design of the glucose ligands on Gluc-CDDP/m was engineered to control the conjugation via the carbon 6 of the glucose moieties, as well as the ligand density on the poly (ethylene glycol) (PEG) shell of the micelles. The series of micelles was then studied in vitro and in vivo against GLUT1-high human squamous cell carcinoma of the head and neck OSC-19 cells and GLUT1-low human glioblastoma-astrocytoma U87MG cells. Our results showed that precisely tuning the micelles to have glucose ligands on 25% of their PEG chains increased the efficacy against the tumors by significantly enhancing the tumor accumulation, even in GLUT1-low U87MG tumors. The enhancement of the intratumoral levels of these micelles was hindered by concomitant administration of glucose, or the GLUT1 inhibitor STF-31, confirming a GLUT1/glucose-mediated increment of the accumulation. Intravital confocal laser scanning microscopy imaging of tumor tissues further demonstrated the rapid extravasation and penetration of Gluc-CDDP/m in OSC-19 tumors compared to non-targeted CDDP/m. These findings indicate GLUT1-targeting as a promising approach for overcoming the vascular barrier and boosting the delivery of nanomedicine in tumors., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Cancer stem-like cells (CSCs) treatment is a plausible strategy for enhanced cancer therapy. Here we report a glucose-installed sub-50-nm nanocarrier for the targeted delivery of small interfering RNA (siRNA) to CSCs through selective recognition of the glucose ligand to the glucose transporter 1 (GLUT1) overexpressed on the CSC surface. The siRNA nanocarrier was constructed via a two-step assembling process. First, a glucose-installed poly(ethylene glycol)-block-poly(l-lysine) modified with lipoic acid (LA) at the ω-end (Glu-PEG-PLL-LA) was associated with a single siRNA to form a unimer polyion complex (uPIC). Second, a 20 nm gold nanoparticle (AuNP) was decorated with ~65 uPICs through AuS bonding. The glucose-installed targeted nanoparticles (Glu-NPs) exhibited higher cellular uptake of siRNA payloads in a spheroid breast cancer (MBA-MB-231) cell culture compared with glucose-unconjugated control nanoparticles (MeO-NPs). Notably, the Glu-NPs became more efficiently internalized into the CSC fraction, which was defined by aldehyde dehydrogenase (ALDH) activity assay, than the other fractions, probably due to the higher GLUT1 expression level on the CSCs. The Glu-NPs elicited significantly enhanced gene silencing in a CSC-rich orthotopic MDA-MB-231 tumor tissue following systemic administration to tumor-bearing mice. Ultimately, the repeated administrations of polo-like kinase 1 (PLK1) siRNA-loaded Glu-NPs significantly suppressed the growth of orthotopic MDA-MB-231 tumors. These results demonstrate that Glu-NP is a promising nanocarrier design for CSC-targeted cancer treatment., (Copyright © 2019 Elsevier B.V. All rights reserved.)

By a bioinformatics approach, we have identified a novel cysteine knot protein member, VWC2 (von Willebrand factor C domain containing 2) previously known as Brorin. Since Brorin has been proposed to function as a bone morphogenetic protein (BMP) antagonist, we investigated the binding of Brorin/VWC2 to several BMPs; however, none of the BMPs tested were bound to VWC2. Instead, the βA subunit of activin was found as a binding partner among transforming growth factor (TGF)-β superfamily members. Here, we show that Vwc2 gene expression is temporally upregulated early in osteoblast differentiation, VWC2 protein is present in bone matrix, and localized at osteoblasts/osteocytes. Activin A-induced Smad2 phosphorylation was inhibited in the presence of exogenous VWC2 in MC3T3-E1 osteoblast cell line and primary osteoblasts. The effect of VWC2 on ex vivo cranial bone organ cultures treated with activin A was investigated, and bone morphometric parameters decreased by activin A were restored with VWC2. When we further investigated the biological mechanism how VWC2 inhibited the effects of activin A on bone formation, we found that the effects of activin A on osteoblast cell growth, differentiation, and mineralization were reversed by VWC2. Taken together, a novel secretory protein, VWC2 promotes bone formation by inhibiting Activin-Smad2 signaling pathway.

Critical limb ischemia (CLI) is a devastating disease in patients undergoing hemodialysis (HD). Based on the unsatisfactory results of autologous mononuclear cell transplantation for patients with CLI undergoing HD, we conducted a phase II clinical trial to evaluate the safety and efficacy of granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood-derived autologous purified CD34 positive (CD34+) cell transplantation for CLI in patients undergoing HD. Six patients with CLI (two with Rutherford category 4 and four with Rutherford category 5) were enrolled. As for primary endpoint, there were no major adverse events related to this therapy. As for efficacy, the amputation-free survival rate was 100% at 1 year after cell therapy. Both rest pain scale and ulcer size were significantly improved as early as 4 weeks after therapy compared with baseline (p < .01), and three out of five ulcers completely healed within 12 weeks after cell transplantation. Clinical severity, including Fontaine scale and Rutherford category, significantly improved at 24 weeks after cell transplantation (p < .05), and further improved at 52 weeks (p < .01) compared with baseline. The improvement rate from CLI stage to non-CLI stage was 83.3% at 52 weeks. Toe skin perfusion pressure and absolute claudication distance were also significantly improved. In conclusion, G-CSF-mobilized peripheral blood CD34+ cell transplantation was safe, feasible, and effective for patients with CLI undergoing HD. Stem Cells Translational Medicine 2018;7:774-782., (© 2018 The Authors Stem Cells Translational Medicine published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.)

Background: Intravascular large B-cell lymphoma (IVLBCL) is a rare subtype of extranodal diffuse large B-cell lymphoma characterized by proliferation of B cells within small vessels. Herein, we report a case of a 77-year-old man who presented with IVLBCL and massive tumor formation on the aortic wall who was previously diagnosed with sarcoidosis and focal segmental glomerulosclerosis (FSGS). To our knowledge, this is the first reported case of an IVLBCL with aortic tumor formation., Case Presentation: A 77-year-old ambulatory man with sarcoidosis and FSGS had neurological symptoms for nine months. The patient presented to the emergency department with sudden left leg pain, and was diagnosed with acute femoral artery occlusion. Emergency thrombectomy was performed subsequently. Pathological evaluation of the thrombi revealed that its surface was filled with large atypical B cells. Bone marrow biopsy showed infiltration of large atypical B cells within the small vessels. IVLBCL was suspected and further examination was planned, but the patient died due to sudden respiratory and cardiac arrest on hospital day twelve. Autopsy revealed intravascular tumors adherent to the aortic arch, left ventricle, and the abdominal aorta. All enlarged lymph nodes and the ventricular septum of the heart showed hyalinized lesions with granular formation consistent with sarcoidosis. The patient was diagnosed with IVLBCL with aortic tumor formation complicated with sarcoidosis and FSGS., Conclusions: IVLBCL may present with tumor formation on the aortic wall. Although the cause of its affinity to the aortic wall is yet unknown, autopsy findings imply that arteriosclerosis may have contributed to the tumor formation. The literature suggests that T-cell abnormalities could possibly be the common etiology of intravascular lymphoma, sarcoidosis, and FSGS.

Ellis-van Creveld (EvC) syndrome is an autosomal recessive chondrodysplastic disorder. Affected patients present a wide spectrum of symptoms including short stature, postaxial polydactyly, and dental abnormalities. We previously disrupted Evc2 , one of the causative genes for EvC syndrome, in mice using a neural crest-specific, Cre -mediated approach (i.e., P0- Cre , referred to as Evc2 P0 mutants). Despite the fact that P0-Cre predominantly targets the mid-facial region, we reported that many mid-facial defects identified in Evc2 global mutants are not present in Evc2 P0 mutants at postnatal day 8 (P8). In the current study, we used multiple Cre lines ( P0-Cre and Wnt1-Cre , respectively), to specifically delete Evc2 in neural crest-derived tissues and compared the resulting mid-facial defects at multiple time points (P8 and P28, respectively). While both Cre lines indistinguishably targeted the mid-facial region, they differentially targeted the anterior portion of the skull base. By comprehensively analyzing the shapes of conditional mutant skulls, we detected differentially affected mid-facial defects in Evc2 P0 mutants and Evc2 Wnt1 mutants. Micro-CT analysis of the skull base further revealed that the Evc2 mutation leads to a differentially affected skull base, caused by premature closure of the intersphenoid synchondrosis (presphenoidal synchondrosis), which limited the elongation of the anterior skull base during the postnatal development of the skull. Given the importance of the skull base in mid-facial bone development, our results suggest that loss of function of Evc2 within the skull base secondarily leads to many aspects of the mid-facial defects developed by the EvC syndrome.

Background: Understanding disease seasonality is important for improving clinical practice, hospital resource utilization and community-based preventive care. However, no studies have investigated the seasonality of acute kidney injury (AKI)., Methods: In the Tokushukai Medical Database, which includes 38 Japanese community hospitals, we identified hospitalized patients with AKI based on the Kidney Disease: Improving Global Outcomes serum creatinine criteria from January 2012 to December 2014. We plotted the number and proportion of patients with AKI among hospitalized patients by month of hospital admission. Subgroup analyses were conducted by the admission diagnosis category, timing of AKI diagnosis and age. We also examined the association between month of hospital admission and AKI, adjusting for patient characteristics and AKI risk factors. Finally, we assessed seasonal variations in disease severity and 30-day mortality of patients with AKI., Results: We identified 81 279 (14.6%) patients with AKI among 555 940 hospitalized patients. The proportion of patients with AKI was highest in January (16.7%) and lowest in June (13.4%). Subgroup analyses suggested that the seasonality of AKI incidence was driven by community-acquired AKI associated with the admission diagnosis of cardiovascular and pulmonary diseases among older patients. The adjusted odds ratio for AKI (January versus June) was 1.24 (95% confidence interval, 1.17-1.31). Patients with AKI showed a larger number of failing organs in winter, and their 30-day mortality was 16.4% in spring, 14.5% in summer, 15.6% in autumn and 18.4% in winter., Conclusion: AKI is more common among hospitalized patients and patients with AKI are more severely ill in winter.

Polyzwitterions are employed as coating polymers for biomaterials to induce an antifouling property on the surface. Fine-tuning the betaine structure switches the antifouling property to be interactive with anionic tissue constituents in response to a tumorous pH gradient. The ethylenediamine moiety in the carboxybetaine enabled stepwise protonation and initiated the di-protonation process around tumorous pH (6.5). The net charge of the developed polyzwitterion (PGlu(DET-Car)) was thus neutral at pH 7.4 for antifouling, but was cationic at pH 6.5 for interaction with anionic constituents. Quantum dots coated with PGlu(DET-Car) exhibited comparable stealth and enhanced tumor accumulation relative to the PEG system. The present study provides a novel design of smart switchable polyzwitterion based on a precise control of the net charge., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: Bleeding from hemorrhagic shock can be immediately controlled by blocking the proximal part of the hemorrhagic point using either resuscitative thoracotomy for aortic cross-clamping or insertion of a large-caliber (10-14Fr) resuscitative endovascular balloon occlusion of the aorta (REBOA) device via the femoral artery. However, such methods are very invasive and have various complications. With recent progress in endovascular treatment, a low-profile REBOA device (7Fr) has been developed., Objective: The objective of this study was to report our experience of this low-profile REBOA device and to evaluate the usefulness of emergency physician-operated REBOA in life-threatening hemorrhagic shock., Methods: Ten patients with refractory hemorrhagic shock underwent REBOA using this device via the femoral artery. All REBOA procedures were performed by emergency physicians. The success rate of the insertion, vital signs, and REBOA-related complications were evaluated., Results: Median age was 54 years (interquartile range 33-78 years). The causes of hemorrhagic shock were trauma (n = 4; 1 blunt and 3 penetrating), ruptured abdominal aortic aneurysm (n = 3), and obstetric hemorrhage (n = 3). Two patients had cardiopulmonary arrest upon arrival. REBOA procedure was successful in all patients, and all became hemodynamically stable to undergo definitive interventions after REBOA. There were no REBOA-related complications. The mortality rate within 24 h and 30 days was 40%., Conclusions: This REBOA device was useful for emergency physicians in life-threatening hemorrhagic shock because of its ease in handling and low invasiveness., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Rapid localization of injured survivors by rescue teams to prevent death is a major issue. In this paper, a sensor system for human rescue including three different types of sensors, a CO₂ sensor, a thermal camera, and a microphone, is proposed. The performance of this system in detecting living victims under the rubble has been tested in a high-fidelity simulated disaster area. Results show that the CO₂ sensor is useful to effectively reduce the possible concerned area, while the thermal camera can confirm the correct position of the victim. Moreover, it is believed that the use of microphones in connection with other sensors would be of great benefit for the detection of casualties. In this work, an algorithm to recognize voices or suspected human noise under rubble has also been developed and tested., Competing Interests: The authors declare no conflict of interest.

Acute kidney injury (AKI) is a major clinical problem that still has no established treatment. We investigated the efficacy of cultured human peripheral blood mononuclear cells (PBMNCs) for AKI. Ischemia/reperfusion injury (IRI) was used to induce AKI in male nonobese diabetic (NOD/severe combined immunodeficiency) mice aged 7 to 8 wk. PBMNCs were isolated from healthy volunteers and were subjected to quality and quantity controlled (QQc) culture for 7 d in medium containing stem cell factor, thrombopoietin, Flt-3 ligand, vascular endothelial growth factor, and interleukin 6. IRI-induced mice were divided into 3 groups and administered (1) 1 × 10 6 PBMNCs after QQc culture (QQc PBMNCs group), (2) 1 × 10 6 PBMNCs without QQc culture (non-QQc PBMNCs group), or (3) vehicle without PBMNCs (IRI control group). PBMNCs were injected via the tail vein 24 h after induction of IRI, followed by assessment of renal function, histological changes, and homing of injected cells. Blood urea nitrogen and serum creatinine (Cr) 72 h after induction of IRI in the QQc PBMNCs group dramatically improved compared with those in the IRI control and the non-QQc PBMNCs groups, accompanied by the improvement of tubular damages. Interstitial fibrosis 14 d after induction of IRI was also significantly improved in the QQc PBMNCs group compared with the other groups. The renoprotective effect noted in the QQc PBMNCs group was accompanied by reduction of peritubular capillary loss. The change of PBMNCs' population (increase of CD34+ cells, CD133+ cells, and CD206+ cells) and increased endothelial progenitor cell colony-forming potential by QQc culture might be one of the beneficial mechanisms for restoring AKI. In conclusion, an injection of human QQc PBMNCs 24 h after induction of IRI dramatically improved AKI in mice.

We sought to examine if self-reported oral health conditions regarding difficulty eating tough foods, dry mouth, choking, number of teeth and denture use are associated with incident falls. Our study was based on panel data from the Japan Gerontological Evaluation Study conducted in 2010 and 2013 using self-administered questionnaires. Data from 19,995 male and 20,858 female community-dwelling older people aged ≥65 years without a history of falls within the previous year in 2010 were analyzed. Multilevel logistic regression models were used to determine the association between poor oral health in 2010 and multiple incident falls in 2013 after adjusting for possible confounders and considering differences in municipalities. The percentage of males and females who reported falls in 2013 were 2.4% and 2.1%, respectively. After adjusting for age, educational attainment, equivalized income, depression, self-rated health, instrumental activities of daily living, body mass index, present illness related to falls, social participation, walking in min/day, alcohol drinking status, and municipality population density, dry mouth in males (odds ratio [OR] = 1.41; 95% confidence interval [CI]: 1.12-1.77) and choking in females (OR = 1.64; 95% CI: 1.27-2.11) were significantly associated with incident falls. Difficulty eating tough foods in both sexes and choking in males were marginally associated with incident falls (p<0.1). Females having 10-19 teeth without dentures (OR = 1.63; 95% CI: 1.14-2.31), ≤9 teeth with dentures (OR = 1.36; 95% CI: 1.03-1.80), and ≤9 without dentures (OR = 1.46; 95% CI: 1.02-2.08) were significantly associated with incident falls compared with those having ≥20 teeth, respectively. These findings suggest that poor oral function, having fewer teeth, and not using dentures are predictors of incident falls. Further studies are needed to determine whether improving oral health can reduce the risk of falls.

EvC syndrome is a type of autosomal-recessive chondrodysplasia. Previous case studies in patients suggest abnormal craniofacial development, in addition to dwarfism and tooth abnormalities. To investigate how craniofacial development is affected in EvC patients, surface models were generated from micro-CT scans of control mice, Evc2 global mutant mice and Evc2 neural crest-specific mutant mice. The anatomic landmarks were placed on the surface model to assess the morphological abnormalities in the Evc2 mutants. Through analyzing the linear and angular measurements between landmarks, we identified a smaller overall skull, shorter nasal bone, shorter frontal bone, and shorter cranial base in the Evc2 global mutants. By comparing neural crest-specific Evc2 mutants with control mice, we demonstrated that the abnormalities within the mid-facial regions are not accounted for by the Evc2 mutation within these regions. Additionally, we also identified disproportionate length to width ratios in the Evc2 mutants at all levels from anterior to posterior of the skull. Overall, this study demonstrates a more comprehensive analysis on the craniofacial morphological abnormalities in EvC syndrome and provides the developmental insight to appreciate the impact of Evc2 mutation within the neural crest cells on multiple aspects of skull deformities. Anat Rec, 2017. © 2017 Wiley Periodicals, Inc. Anat Rec, 301:46-55, 2018. © 2017 Wiley Periodicals, Inc., (© 2017 Wiley Periodicals, Inc.)

Background: The aim of this study was to examine whether plasma neutrophil gelatinase-associated lipocalin (NGAL) levels predict the outcome of kidney function and correlate with the severity of tubulointerstitial damages in patients with chronic kidney disease (CKD)., Methods: In this prospective 18-month cohort study of 112 patients with CKD between 2010 and 2011, associations between plasma NGAL levels and estimated glomerular filtration ratio (eGFR), further worsening of kidney function and histological lesion on kidney biopsy were investigated., Results: Serum levels of creatinine and eGFR before the study were 1.48 ± 0.65 mg/dl and 42.6 ± 22.0 ml/min/1.73 m 2 . Median plasma NGAL level was 148.5 (83.75-248.25) ng/ml and showed no correlation with eGFR or age. 87 out of 112 patients were able to follow up for 18 months. Patients with higher levels of NGAL (>107.8 ng/ml) showed significantly more decrease in eGFR in CKD stage 1 or 2 than those with lower levels of NGAL (≦107.8 ng/ml), while there was no difference in change in eGFR in CKD stage 3-5 between patients with higher and lower levels of NGAL. In the kidney biopsy of 27 patients out of enrolled patients, plasma NGAL levels correlated significantly with the degree of interstitial cell infiltration and fibrosis, but did not correlate with that of glomerular sclerosis. In ROC analysis, plasma NGAL levels predicted tubulointerstitial cell infiltrations more accurately [AUC = 0.8300 than eGFR (AUC = 0.716)]., Conclusion: Plasma NGAL is a useful marker of interstitial lesions in patients with CKD and a predictor of further kidney worsening in the early CKD stage.

Introduction: Platinum anticancer drugs are effective against a wide range of tumors, though their severe adverse effects and resistance remain unresolved. A breakthrough in these drawbacks could be achieved by using polymeric micelles, i.e. nanoassemblies having a drug loaded core and a protective hydrophilic shell, incorporating platinum drugs for tumor-targeted delivery. Areas covered: The development of cisplatin- (NC-6004) and DACHPt-loaded micelles (NC-4016) has been reviewed, particularly, from the viewpoint of the effect of their structural features on the tumor-targeting efficacy. We have also described new approaches for molecular targeting by installing ligand moieties on the surface of micelles. Moreover, small platinum drugs and platinum drug-loaded nanocarriers are introduced to explain the context for developing platinum drug-loaded polymeric micelles. Expert opinion: NC-6004 and NC-4016 micelles meet critical structural and functional requirements for achieving safe and effective tumor targeting, enhancing efficacy even against drug resistant cancer cells, and have been translated into human studies, aiming to be the first-of-their-kind in the clinic. Due to their flexible design, polymeric micelles could readily integrate new features based on the knowledge arising from the human clinical trials toward the development of innovative formulations with superior performance.

Recalcitrant head and neck squamous cell carcinoma (HNSCC) usually relapses after therapy due to the enrichment of drug resistant cancer stem-like cells (CSCs). Nanomedicines have shown potential for eradicating both cancer cells and CSCs by effective intratumoral navigation for reaching particular cell populations and controlling drug delivery. The installation of ligands on nanomedicines is an attractive approach for improving the delivery to CSCs within tumors, though the development of CSC-selective ligand-receptor systems has been challenging. Herein, we found that the CSC subpopulation in HNSCC cells overexpresses α v β 5 integrins, which is preferentially expressed in tumor neovasculature and cancer cells, and can be effectively targeted by using cyclic Arg-Gly-Asp (cRGD) peptide. Thus, in this study, we propose installing cRGD peptide on micellar nanomedicines incorporating cisplatin for improving their activity against CSCs and enhancing survival. Both cisplatin-loaded micelles (CDDP/m) and cRGD-installed CDDP/m (cRGD-CDDP/m) were effective against HNSCC SAS-L1-Luc cells in vitro, though cRGD-installed CDDP/m was more potent than CDDP/m against the CSC fraction. In vivo, the cRGD-CDDP/m also showed significant antitumor activity against HNSCC orthotopic tumors, i.e. SAS-L1 and HSC-2. Moreover, cRGD-CDDP/m rapidly accumulated into the lymph node metastasis of SAS-L1 tumors, effectively inhibiting their growth, and prolonging mice survival. These findings indicate cRGD-installed nanomedicines as an advantageous strategy for targeting CSCs in HNSCC, and particularly, cRGD-CDDP/m as a significant therapeutic strategy against regionally advanced HNSCC., (Copyright © 2017. Published by Elsevier B.V.)

Background: Cognitive impairment is one of the important critical issues in hemodialysis (HD) patients. However, the associating factors of brain atrophy in HD patients have not been fully elucidated., Purpose and Methods: Brain magnetic resonance imaging (MRI) was performed in 34 of total 72 HD outpatients in our dialysis center. These MRI images were analyzed by an application software; Voxel-based Specific Regional Analysis System for Alzheimer's Disease (VSRAD). VSRAD quantitatively calculates the extent of brain atrophy (percent of volume reduction) comparing with a MRI imaging database of 80 age-matched healthy controls. The extent of both hippocampal and whole-brain atrophy was evaluated with possible contributing factors., Results: In all patients, the mean extent of hippocampal atrophy was 27.3%, and the mean extent of whole-brain atrophy was 11.2%. The extent of hippocampal atrophy was significantly correlated with low body mass index (BMI), total serum homocysteine (tHcy) levels, and brachial-ankle pulse wave velocity (baPWV). The extent of whole-brain atrophy showed significant correlations with age, hypoalbuminemia, and baPWV. Based on the multiple regression analysis, tHcy was an independent determinant of hippocampal atrophy (β = 0.460, R2 = 0.189, P<0.01); while age was an independent determinant of whole-brain atrophy (β = 0.594, R2 = 0.333, P<0.01)., Conclusions: In this exploratory pilot study, hippocampal atrophy was significantly correlated with hyperhomocysteinemia in HD patients.

A 53-year-old woman developed end-stage renal failure during a 15-year clinical course of primary hyperparathyroidism and was referred to our hospital for evaluation of suspected multiple endocrine neoplasia type 1 (MEN1). Genetic testing revealed a novel deletion mutation at codon 467 in exon 10 of the MEN1 gene. Systemic and selective arterial calcium injection (SACI) testing revealed hyperglucagonemia and hypergastrinemia with positive gastrin responses. A pathological examination revealed glucagonoma and a lymph node gastrinoma. The findings in this case indicate the importance of early diagnosis of MEN1 and demonstrate the utility of systemic and SACI testing in renal failure cases.

Ellis-van Creveld (EvC) syndrome is a skeletal dysplasia, characterized by short limbs, postaxial polydactyly, and dental abnormalities. EvC syndrome is also categorized as a ciliopathy because of ciliary localization of proteins encoded by the two causative genes, EVC and EVC2 (aka LIMBIN). While recent studies demonstrated important roles for EVC/EVC2 in Hedgehog signaling, there is still little known about the pathophysiological mechanisms underlying the skeletal dysplasia features of EvC patients, and in particular why limb development is affected, but not other aspects of organogenesis that also require Hedgehog signaling. In this report, we comprehensively analyze limb skeletogenesis in Evc2 mutant mice and in cell and tissue cultures derived from these mice. Both in vivo and in vitro data demonstrate elevated Fibroblast Growth Factor (FGF) signaling in Evc2 mutant growth plates, in addition to compromised but not abrogated Hedgehog-PTHrP feedback loop. Elevation of FGF signaling, mainly due to increased Fgf18 expression upon inactivation of Evc2 in the perichondrium, critically contributes to the pathogenesis of limb dwarfism. The limb dwarfism phenotype is partially rescued by inactivation of one allele of Fgf18 in the Evc2 mutant mice. Taken together, our data uncover a novel pathogenic mechanism to understand limb dwarfism in patients with Ellis-van Creveld syndrome., Competing Interests: The authors have declared that no competing interests exist.

To assess the clinical benefit of combined treatment of below-knee endovascular therapy (BK-EVT) plus low-density lipoprotein apheresis (LDLA) compared with BK-EVT monotherapy, we retrospectively evaluated the clinical outcome of hemodialysis (HD) patients with critical limb ischemia (CLI) due to isolated BK arterial lesions who underwent BK-EVT or BK-EVT plus short-term LDLA. Between October 2011 and September 2014, 62 HD patients underwent isolated BK-EVT monotherapy (BK-EVT group), and 25 HD patients underwent BK-EVT plus LDLA (BK-EVT + LDLA group). LDLA was started within 1 week after BK-EVT and performed four times in total within next 2 weeks. Major adverse limb events (MALE) including major amputation and re-intervention, and all-cause mortality were examined by Kaplan-Meier method and the log-rank test. Baseline characteristics were not different other than low ABI and low dorsal SPP in BK-EVT + LDLA group. Cumulative MALE-free rate was significantly improved in BK-EVT + LDLA group over the BK-EVT group (72.0% and 45.1% respectively at 30 months after treatment, P = 0.04). All-cause mortality did not differ between the two groups. Major causes of death were heart failure and sepsis in both groups. Short-term LDLA hybrid treatment immediately after BK-EVT might improve the outcome of ischemic limbs after re-vascularization therapy., (© 2016 International Society for Apheresis, Japanese Society for Apheresis, and Japanese Society for Dialysis Therapy.)

Background: Neuroendocrine tumors (NETs) of the ampulla of Vater are rare and difficult to diagnose. We report a rare case of a small NET of the ampulla of Vater with metastasis to distant lymph nodes., Case Presentation: The patient was a 54-year-old man complaining of epigastric pain and melena. Upper gastrointestinal endoscopy revealed a bulging papilla with active bleeding, which was diagnosed as a well-differentiated NET of the ampulla of Vater. An approximately 10-mm hypervascular tumor at the ampulla of Vater and a 41-mm cyst adjacent to the wall of the jejunum were revealed by abdominal computed tomography. We performed pylorus-preserving pancreaticoduodenectomy with lymph node dissection. Macroscopic examination revealed a 9-mm tumor of the ampulla of Vater and a 52-mm cyst adjacent to the wall of the jejunum. Histological examination revealed that the cyst was a lymph node metastasis. The final diagnosis was non-functional NET G1 of the ampulla of Vater, designated T1N1M0 stage IIIB. Postoperatively, the patient underwent no treatment and has had no recurrence for 4 years., Conclusions: This case demonstrates that sporadic NETs of Vater's papilla have aggressive metastatic potential even with a small primary lesion, and radical resection with lymphadenectomy is recommended for all cases.

Ellis-van Creveld (EvC) syndrome is a genetic disorder with mutations in either EVC or EVC2 gene. Previous case studies reported that EvC patients underwent orthodontic treatment, suggesting the presence of craniofacial bone phenotypes. To investigate whether a mutation in EVC2 gene causes a craniofacial bone phenotype, Evc2 knockout (KO) mice were generated and cephalometric analysis was performed. The heads of wild type (WT), heterozygous (Het) and homozygous Evc2 KO mice (1-, 3-, and 6-week-old) were prepared and cephalometric analysis based on the selected reference points on lateral X-ray radiographs was performed. The linear and angular bone measurements were then calculated, compared between WT, Het and KO and statistically analyzed at each time point. Our data showed that length of craniofacial bones in KO was significantly lowered by ∼20% to that of WT and Het, the growth of certain bones, including nasal bone, palatal length, and premaxilla was more affected in KO, and the reduction in these bone length was more significantly enhanced at later postnatal time points (3 and 6 weeks) than early time point (1 week). Furthermore, bone-to-bone relationship to cranial base and cranial vault in KO was remarkably changed, i.e. cranial vault and nasal bone were depressed and premaxilla and mandible were developed in a more ventral direction. Our study was the first to show the cause-effect relationship between Evc2 deficiency and craniofacial defects in EvC syndrome, demonstrating that Evc2 is required for craniofacial bone development and its deficiency leads to specific facial bone growth defect. Anat Rec, 299:1110-1120, 2016. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Objective: Our objectives were to determine the expression of EVC2 in craniofacial tissues and investigate the effect of Evc2 deficiency on craniofacial bones using Evc2 knockout (KO) mouse model., Design: Evc2 KO mice were generated by introducing a premature stop codon followed by the Internal Ribosomal Entry Site fused to β-galactosidase (LacZ). Samples from wild-type (WT), heterozygous (Het) and homozygous Evc2 KO mice were prepared. LacZ staining and immunohistochemistry (IHC) with anti-β-galactosidase, anti-EVC2 and anti-SOX9 antibodies were performed. The craniofacial bones were stained with alcian blue and alizarin red., Results: The LacZ activity in KO was mainly observed in the anterior parts of viscerocranium. The Evc2-expressing cells were identified in many cartilageous regions by IHC with anti-β-galactosidase antibody in KO and Het embryos. The endogenous EVC2 protein was observed in these areas in WT embryos. Double labeling with anti-SOX9 antibody showed that these cells were mainly chondrocytes. At adult stages, the expression of EVC2 was found in chondrocytes of nasal bones and spheno-occipital synchondrosis, and osteocytes and endothelial-like cells of the premaxilla and mandible. The skeletal double staining demonstrated that craniofacial bones, where the expression of EVC2 was observed, in KO had the morphological defects as compared to WT., Conclusion: To our knowledge, our study was the first to identify the types of Evc2-expressing cells in craniofacial tissues. Consistent with the expression pattern, abnormal craniofacial bone morphology was found in the Evc2 KO mice, suggesting that EVC2 may be important during craniofacial growth and development., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Mutations in the Family with sequence similarity (FAM) 20 gene family are associated with mineralized tissue phenotypes in humans. Among these genes, FAM20A mutations are associated with Amelogenesis Imperfecta (AI) with gingival hyperplasia and nephrocalcinosis, while FAM20C mutations cause Raine syndrome, exhibiting bone and craniofacial/dental abnormalities. Although it has been demonstrated that Raine syndrome associated-FAM20C mutants prevented FAM20C kinase activity and secretion, overexpression of the catalytically inactive D478A FAM20C mutant was detected in both cell extracts and the media. This suggests that FAM20C secretion doesn't require its kinase activity, and that another molecule(s) may control the secretion. In this study, we found that extracellular FAM20C localization was increased when wild-type (WT), but not AI-forms of FAM20A was co-transfected. On the other hand, extracellular FAM20C was absent in the conditioned media of mouse embryonic fibroblasts (MEFs) derived from Fam20a knock-out (KO) mouse, while it was detected in the media from WT MEFs. We also showed that cells with the conditioned media of Fam20a WT MEFs mineralized, but those with the conditioned media of KO MEFs failed to mineralize in vitro. Our data thus demonstrate that FAM20A controls FAM20C localization that may assist in the extracellular function of FAM20C in mineralized tissues.

Bone and dentin are mineralized extracellular matrices produced by osteoblasts and odontoblasts, respectively, and their major organic portion is type I collagen. Dentinogenesis Imperfecta (DGI) is one of the most common clinically- and genetically-based disturbances of dentin formation, causing irreversible dentin defects. Among several types of DGI, patients with DGI type II exhibit opalescent dentin with partial or complete pulp obliteration. It has been previously reported that the non-sense mutation (c.133C>T) in Dentin Sialophosphoprotein ( DSPP ) was identified in DGI type II patients at glutamine residue 45, resulting in the premature stop codon (p.Q45X). DSPP is known to be synthesized as a single gene product and further processed at Gly 462 -Asp 463 , resulting in the production of Dentin Sialoprotein (DSP) and Dentin Phosphoprotein (DPP). We hypothesized that the shorter form (Q45X) of N-terminal Dentin Sialoprotein (N-DSP) may cause over-production of type I collagen protein as obliterated pulp is occupied by dentin. To test this hypothesis, we generated mouse recombinant Glutathione-S-Transferase (GST)-N-DSP fusion protein, and the effect of GST-N-DSP was investigated in calvarial bone explant culture and MC3T3-E1 osteoblastic culture systems. Here we show that a significant increase in calvarial bone formation is observed by GST-N-DSP. GST-N-DSP accelerates MC3T3-E1 osteoblast cell growth and proliferation and subsequent osteoblast differentiation by inducing the expression of certain osteogenic markers such as type I collagen , Runx2 , Osterix and ATF4 . Interestingly, GST-N-DSP significantly enhances dentinogenesis marker gene expression including Dspp and Dmp1 gene expression in non-odontogenic MC3T3-E1 cells. To rule out any artificial effect of GST-tag, we also used the synthetic peptide of N-DSP and confirmed the results of N-DSP peptide were essentially similar to those of GST-N-DSP. Taken together, our data suggest that N-DSP promotes bone formation by accelerating osteoblast cell proliferation and subsequent osteoblast differentiation accompanied by marked up-regulation of the dentin matrix markers, such as Dspp and Dmp1 genes.

Eventual relapse of tumor growth is commonly observed in head and neck cancer patients, following treatment with platinum-based chemotherapies. This occurrence is believed to be related to the failure to eradicate drug resistant, cancer stem cell (CSC) niches, thereby enriching their population in tumors after treatment. In this study, we show that in contrast to free cisplatin (CDDP), the polymer micelle-based nanomedicine incorporating cisplatin (CDDP/m), can eradicate both the undifferentiated cell and the differentiated cancer cell populations within a head and neck tumor model. Immunohistochemistry of treated tumors showed that opposing to CDDP treatment, CDDP/m could reduce tumor growth without concentrating the CSC-like population. We further showed that CDDP/m, but not CDDP, can localize into hypoxic regions, possibly CSC-rich areas, in the tumors, and can overcome their detoxification mechanism based-on high cellular expression of glutathione to successfully deliver Pt to nuclear DNA. Our data suggests CDDP/m to be a replacement for current platinum therapies, for its ability to eradicate both bulk and CSC-like populations, and in turn to prevent recurrence of tumor growth., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Endovascular stent graft placement has become a major treatment for thoracic and abdominal aneurysms. While endovascular therapy is less invasive than open surgery, it involves the use of a contrast medium. Contrast media can cause renal impairment, a condition termed as contrast-induced nephropathy (CIN). This study sought to evaluate the incidence and risk factors of CIN following endovascular stent graft placement for aortic aneurysm repair. The study included 167 consecutive patients who underwent endovascular stent graft placement in our hospital from October 2013 to June 2014. CIN was diagnosed using the European Society of Urogenital Radiology criteria. Patients with and without CIN were compared. Chi-squared tests, t-tests, and multivariate logistic regression analyses were performed. Thirteen patients (7.8%) developed CIN. Left ventricular dysfunction and intraoperative blood transfusion were significantly more frequent in the CIN group (P = 0.017 and P = 0.032, resp.). Multivariate analysis showed that left ventricular dysfunction had the strongest influence on CIN development (odds ratio 9.34, P = 0.018, and 95% CI = 1.46-59.7). Patients with CIN also experienced longer ICU and hospital stays. Measures to improve renal perfusion flow should be considered for patients with left ventricular dysfunction who are undergoing endovascular stent graft placement.

Lymph node metastasis (LNM) is correlated with decreased survival, indicating high tumor malignancy and being a potential source for subsequent fatal metastases. Targeted therapies inhibiting the formation of LNM, while eliminating established metastatic foci, could provide synergistic effects by reducing the incidence and growth of metastasis. Based on the inhibitory activity of cRGD peptide against the development of metastasis, and the LNM targeting ability of systemically injected drug-loaded polymeric micelles, herein, we studied the capability of cRGD-installed polymeric micelles incorporating the platinum anticancer drug (1,2-diaminocylohexane)platinum(II) (DACHPt) for cooperatively inhibiting the formation and progression of LNM. As cRGD-installed DACHPt-loaded micelles (cRGD-DACHPt/m) presented similar size, drug loading and surface charge to non-conjugated micelles (MeO-DACHPt/m), the differences in the biological performance of the micelles were endorsed to the effect of the ligand. In a syngeneic melanoma model, both MeO-DACHPt/m and cRGD-DACHPt/m showed comparable antitumor activity against the primary tumors and the established metastatic foci in lymph nodes. However, cRGD-DACHPt/m significantly enhanced the efficacy against LNM draining from primary tumors through the effective inhibition of the spreading of cancer cells. This improved inhibition was associated with the ability of cRGD-DACHPt/m to reduce the migration of melanoma cells, which was higher than that of MeO-DACHPt/m, free cRGD and their combination. These results support our strategy of using cRGD-installed micelles for attaining cooperative therapies against LNM exploiting the inhibitory function of the peptide and the cytotoxic effect of the micelles., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Purpose: Surgical site infections (SSI) are a common complication of gastrointestinal tract surgery. In this study, we explored the correlation between the anastomosis method and the incidence of SSI., Methods: A total of 110 patients underwent ileocecal resection or right hemicolectomy for the excision of colon cancer. Two methods (open and closed, 28 and 82 patients, respectively) of functional end-to-end anastomosis were adopted., Results: Increased perioperative blood loss (p = 0.029214), a longer hospital stay (p = 0.026668) and the development of SSI (p = 0.000181) were significantly correlated with the open method. There was no correlation between SSI and the body mass index, or between SSI and the length of the surgery or diabetes mellitus. However, patients that developed SSI tended to be obese., Conclusion: The open method was associated with a higher incidence of SSI. Therefore, it is necessary to consider potential contamination of the surgical field at the time of anastomosis to reduce the incidence of SSI.

Polyion complex (PIC) formation is an attractive method for obtaining molecular assemblies owing to their facile fabrication process in aqueous media, but more insights are required in order to control the higher-dimensional structures of polypeptide-based PICs. Herein, the PIC formation behavior of oppositely charged homochiral polypeptides, poly-L-lysine and poly(ethylene glycol)-b-poly(L-glutamate) (PEG-PLG), and their secondary structures are carefully studied in water. PIC formation takes place in a polymer concentration-dependent manner, and clear β-sheet formation is observed at polymer concentrations ≥0.3 mg mL(-1). The results also confirm that multimolecular aggregation is a prerequisite for β-sheet formation, which indicates that the inner hydrophobic environment of PICs is favorable for β-sheet formation. Furthermore, the PEG weight fraction, stereoregularity of the polypeptide, and ionic strength of the solutions are found to be key factors for generating a secondary structure, presumably because these factors can contribute to the tuning of the inner environment of PICs. This method of producing water-soluble nanoassemblies from oppositely charged polypeptides may expedite self-assembly studies in biological systems and be incorporated into various molecular systems to exploit protein-mimicking features., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

For the functional analysis of insect genes as well as for the production of recombinant proteins for biomedical use, clonal transgenic silkworms are very useful. We examined if they could be produced in the parthenogenetic strain that had been maintained for more than 40years as a female line in which embryogenesis is induced with nearly 100% efficiency by a heat shock treatment of unfertilized eggs. All individuals have identical female genotype. Silkworm transgenesis requires injection of the DNA constructs into the non-diapausing eggs at the preblastodermal stage of embryogenesis. Since our parthenogenetic silkworms produce diapausing eggs, diapause programing was eliminated by incubating ovaries of the parthenogenetic strain in standard male larvae. Chorionated eggs were dissected from the implants, activated by the heat shock treatment and injected with the transgene construct. Several transgenic individuals occurred in the daughter generation. Southern blotting analysis of two randomly chosen transgenic lines VTG1 and VTG14 revealed multiple transgene insertions. Insertions found in the parental females were transferred to the next generation without any changes in their sites and copy numbers, suggesting that transgenic silkworms can be maintained as clonal strains with homozygous transgenes. Cryopreservation was developed for the storage of precious genotypes. As shown for the VTG1 and VTG14 lines, larval ovaries can be stored in DMSO at the temperature of liquid nitrogen, transferred to Grace's medium during defrosting, and then implanted into larvae of either sex of the standard silkworm strains C146 and w1-pnd. Chorionated eggs, which developed in the implants, were dissected and activated by the heat shock to obtain females (nearly 100% efficiency) or by a cold shock to induce development to both sexes in 4% of the eggs. It was then possible to establish bisexual lines homozygous for the transgene., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Ellis-van Creveld (EvC) syndrome (OMIM 225500) is an autosomal recessive disease characterized with chondrodysplastic dwarfism in association with abnormalities in oral cavity. Ciliary proteins EVC and EVC2 have been identified as causative genes and they play an important role on Hedgehog signal transduction. We have also identified a causative gene LIMBIN for bovine chondrodysplastic dwarfism (bcd) that is later identified as the bovine ortholog of EVC2. Here, we report generation of conventional and conditional mutant Evc2/Limbin alleles that mimics mutations found in EvC patients and bcd cattle. Resulted homozygous mice showed no ciliary localization of EVC2 and EVC and displayed reduced Hedgehog signaling activity in association with skeletal and oral defects similar to the EvC patients. Cartilage-specific disruption of Evc2/Limbin resulted in similar but milder skeletal defects, whereas osteoblast-specific disruption did not cause overt changes in skeletal system. Neural crest-specific disruption of Evc2/Limbin resulted in defective incisor growth similar to that seen in conventional knockouts; however, differentiation of amelobolasts was relatively normal in the conditional knockouts. These results showcased functions of EVC2/LIMBIN during formation of mineralized tissues. Availability of the conditional allele for this gene should facilitate further detailed analyses of the role of EVC2/LIMBIN in pathogenesis of EvC syndrome. genesis 53:612-626, 2015. © 2015 Wiley Periodicals, Inc., (© 2015 Wiley Periodicals, Inc.)

A 35-year-old obese diabetic man presented with recurrent primary hyperparathyroidism during a three-year outpatient follow-up. He was clinically diagnosed with multiple endocrine neoplasia type 1 (MEN1) due to the presence of a pituitary adenoma and multiple glucagonomas. The glucagonomas may have affected his glycemic control. However, he did not demonstrate weight loss, suggesting that the patient's obesity could have obscured the early diagnosis of a glucagonoma. Genetic testing revealed a novel missense mutation at codon 561 in exon 10, resulting in an amino acid substitution from methionine to arginine (M561R) in the MEN1 gene. This mutation appeared to be responsible for the MEN1 pathogenicity.

Supramolecular assemblies of amphiphilic block copolymers having polypeptide segments offer significant advantages for tailoring spatial arrangement based on secondary structures in their optically active backbones. Here, we demonstrated the critical effect of α-helix bundles in cisplatin-conjugated poly(L- (or D-)glutamate) [P(L(or D)Glu)-CDDP] segment on the packaging of poly(ethylene glycol) (PEG)-P(L(or D)Glu)-CDDP block copolymers in the core of polymeric micelles (CDDP/m) and enhanced micelle tolerability to harsh in vivo conditions for accomplishing appreciable antitumor efficacy against intractable pancreatic tumor by systemic injection. CDDP/m prepared from optically inactive PEG-poly(D,L-glutamate) (P(D,LGlu)), gradually disintegrated in the bloodstream, resulting in increased accumulation in liver and spleen and reduced antitumor efficacy. Alternatively, CDDP/m from optically active PEG-P(L(or D)Glu) maintained micelle structure during circulation, and eventually attained selective tumor accumulation while reducing nonspecific distribution to liver and spleen. Circular dichroism and small-angle X-ray scattering measurements indicated regular bundled assembly of α-helices in the core of CDDP/m from PEG-P(L(or D)Glu), which is suggested to stabilize the micelle structure against dilution in physiological condition. CDDP/m suffered corrosion by chlorides in medium, yet the optically active micelles with α-helix bundles kept the micelle structure for prolonged time, with slowly releasing unimers and dimers from the surface of the bundled core in an erosion-like process, as verified by ultracentrifugation analysis. This is in sharp contrast with the abrupt disintegration of CDDP/m from PEG-P(D,LGlu) without secondary structures. The tailored assembly in the core of the polymeric micelles through regular arrangement of constituting segments is key to overcome their undesirable disintegration in bloodstream, thereby achieving efficient delivery of loaded drugs into target tissues.

Cognitive dysfunction is prevalent in chronic kidney disease patients. Little is known about the relationship between the regional cerebral blood flow (rCBF) and cognitive function in hemodialysis (HD) patients. We used quantitative single-photon emission-computed tomography (SPECT) to determine whether rCBF decreased in these patients. Fifty-four consecutive HD patients who were able to visit the hospital unassisted and had no history of stroke underwent cognitive assessment based on the Mini Mental State Examination (MMSE). Using quantitative image-analysis software, the SPECT imaging data were used to compare rCBF in HD patients and age-matched healthy controls. Thirty-four patients (63%) had MMSE scores ⩾28 (non-dementia). Regarding the extent of decreased rCBF in HD patients compared with rCBF in normal control patients, SPECT demonstrated significant rCBF decreases in all patients. rCBF in the perfusion area of the middle cerebral artery was significantly more decreased than in other areas. Multiple logistic regression analysis demonstrated that the presence or absence of a previous history of percutaneous coronary intervention, drug therapy with angiotensin II receptor antagonists and diastolic blood pressure (DBP) were independent risk factors for the extent of decreased rCBF. Regarding the severity of decreased rCBF, stepwise multiple regression analysis indicated that HD duration and systolic blood pressure (mm Hg) were chosen. In conclusion, rCBF decreased in all HD patients studied, irrespective of their clinical symptoms or MMSE scores. Blood pressure was an independent risk factor affecting the extent of decreased rCBF.

Cognitive impairment has long been recognized as a complication of chronic kidney disease. However, there is little information available regarding regional cerebral blood flow (rCBF) in patients with peritoneal dialysis (PD). Therefore, we evaluated rCBF using brain single photon emission computed tomography (SPECT). We conducted a cross-sectional study in our hospital. Eighteen consecutive PD patients who could visit the hospital by themselves without any history of stroke were examined by Technetium-99 m-labeled ethylcrysteinate dimer brain SPECT. An easy Z-score imaging system (eZIS) was used to compare rCBF in PD patients with those in age-matched healthy controls. We also evaluated cognitive dysfunction with the mini-mental state examination (MMSE) questionnaire. Only one patient showed an MMSE score of 18 points, and the remaining 14 patients were considered as normal (MMSE ≥ 27), and three patients were considered to have mild cognitive impairment (24 ≤ MMSE ≤ 26). In all patients, rCBF in the posterior cingulated gyri, precunei, and parietal cortices was significantly decreased. The ratio of the reduction of rCBF in each region relative to that of rCBF across the whole brain correlated positively with the PD duration (r = 0.559; P < 0.05). The serum β2-microglobulin level was significantly higher in patients who had a higher ratio of rCBF reduction compared with those with lower ratios. In conclusion, all PD patients in the present study had decreased rCBF irrespective of MMSE scores., (© 2014 The Authors. Therapeutic Apheresis and Dialysis © 2014 International Society for Apheresis.)

We present the first documented case of generalized calciphylaxis that dramatically improved after low-density lipoprotein-apheresis (LA) in a patient undergoing long-term hemodialysis. Calciphylaxis was diagnosed by skin biopsy and was manifest as painful ulcers on the right leg, left buttock, and glans penis. Skin perfusion pressure (SPP), which has recently been used as an indicator of impaired capillary perfusion in distal lesions of the lower extremities, was markedly reduced. The ulcers continued to worsen despite general wound care, correction of levels of calcium × phosphate product, hyperbaric oxygen therapy, and use of bisphosphonate, antiplatelet therapy, and vasodilators. Because LA is known to exert favorable effects on peripheral arterial disease through improved hemorheology, anti-inflammatory action, vasodilation, and angiogenesis, we introduced LA to produce the same effects on calciphylaxis. LA dramatically increased SPP and promoted ulcer healing, demonstrating that LA can be a useful treatment option for calciphylaxis.

Extrahepatic cholangiocarcinoma (EHCC) is a cancer with a poor prognosis, and the postoperative survival of patients depends on the existence of invasion and metastasis. The epithelial-to-mesenchymal transition (EMT) is an important step in EHCC invasion and metastasis. Forkhead box protein C2 (FOXC2) is a transcription factor that has been reported to induce the EMT. Therefore we examined the correlation between FOXC2 expression and clinical pathological factors, and analysed the function of FOXC2. The expression of FOXC2 in 77 EHCC cases was investigated by immunohistochemical staining, and the relationship between FOXC2 expression and clinicopathological factor was assessed. Knockdown by small interfering RNA (siRNA) was performed to determine the roles of FOXC2 in EHCC cell line. FOXC2 expression correlated with lymph node metastasis (P = 0.0205). Patients in the high FOXC2 expression group had a poorer prognosis than the patients in the low FOXC2 expression group. Moreover, FOXC2 knockdown inhibited cell motility and invasion, and decreased the expression of EMT markers (N-cadherin, and matrix metalloproteinase (MMP) -2) and Angiopietin-2 (Ang-2). The EMT inducer FOXC2 contributes to a poor prognosis and cancer progression. FOXC2 may be a promising molecular target for regulating EHCC metastasis., (© 2013 Japanese Cancer Association.)

Background and Objectives: Coronary artery calcification (CAC) is associated with future cardiovascular events and/or death of patients on hemodialysis (HD). We investigated whether progression of CAC in patients on HD could be delayed by switching from a calcium (Ca)-based phosphate (Pi) binder to lanthanum carbonate., Design, Setting, Participants, and Measurements: The CAC scores were evaluated at study enrollment and after 6 months in 52 patients on HD using calcium carbonate (CC) as a Pi binder. Patients were randomly divided into 2 groups assigned to receive either CC or lanthanum carbonate (LC), and the CAC scores were evaluated after a 6-month treatment period. Progression of CAC was assessed, as were serum levels of Ca, Pi, and intact parathyroid hormone (iPTH)., Results: Forty-two patients completed the study (23 receiving CC and 19 receiving LC). In the 6 months prior to randomization, all patients were treated with CC. During this 6-month period, the CAC scores increased significantly in all 42 patients. Once randomized, there was significantly less progression in the group treated with LC than with CC. Changes in CAC scores from 6 to 12 months were significantly smaller in the LC group than the CC group (-288.9 ± 1176.4 vs 107.1 ± 559.6, P = .036), and percentage changes were also significantly different (-6.4% vs 41.2%, P = .024). Serum Ca, Pi, and iPTH levels were similar in both groups during the study period., Conclusions: This pilot study suggested that LC delayed progression of CAC in patients on HD compared with CC.

Aims: Aliskiren inhibits the first step in the renin-angiotensin system (RAS) and recently has been shown to modulate vascular diseases via RAS-dependent and independent pathways. This study aimed to determine the effect of aliskiren-associated direct renin inhibition on endothelial function in patients on hemodialysis via flow-mediated dilatation (FMD) and platelet-derived microparticles (PDMP), as biomarkers of atherosclerosis., Methods: A 12-week prospective study was performed with 24 patients on hemodialysis who were administered 150 mg orally aliskiren once daily for 12 weeks., Results: No significant difference were observed between pre-dialysis, home, and weekly averaged blood pressure at baseline and at 12 weeks (151.5 ± 8.5/80.9 ± 12.9 mmHg vs 150.3 ± 15.3/78.9 ± 21.2 mmHg, 151.4 ± 9.7/82.3 ± 14.7 mmHg vs 151.2 ± 17.7/81.4 ± 10.6 mmHg, and 156.0 ± 18.3/81.9 ± 9.4 mmHg vs 152.5 ± 18.9/81.7 ± 12.3 mmHg, respectively). FMD significantly increased from 2.54% ± 1.45% at baseline to 3.11% ± 1.37% at 12 weeks (P = 0.0267), and PDMP significantly decreased from 13.9 ± 5.8 U/mL at baseline to 10.9 ± 4.5 U/mL at 12 weeks (P = 0.0002)., Conclusion: Aliskiren improved vascular endothelial function and platelet-endothelium activation in patients on hemodialysis independent of antihypertensive effect., (Copyright © 2013 S. Karger AG, Basel.)

Objectives: Porous tantalum is a biomaterial newly applied for artificial joints. We present here 5-years follow-up report of a multicenter clinical trial of total hip arthroplasties (THA) with porous tantalum modular acetabular component (modular PTC)., Methods: Study participants received 82 hips in 79 cases, with 61.2 months follow-up on average. Age at operation was 60.9 years. Clinical results were evaluated using Merle d'Aubigne Postel score. Presence of implant loosening, periacetabular radiolucency, osteolysis, and gap filling were examined for radiographic results., Results: Merle d'Aubigne Postel score improved from 10.0 to 16.4 points. All PTC were radiographically stable, with no evidence of progressive radiolucencies. Average polyethylene wear rate was 0.004 mm/year, with no periacetabular osteolysis. Fifteen hips (18.3%) showed a gap >1 mm; however, all showed bone filling within 12 months. PTC with oversized reaming was significantly less likely to have a gap. No implant failure was noted related to modularity. Resulting survival rate of modular PTC was 100% at 5 years., Conclusions: Modular PTC showed excellent results at 5-years of follow-up. Some hips showed periacetabular gaps, which were filled with bone within 1 year. Further follow-up was needed to determine long-term efficacy.

Background: Peripheral arterial disease (PAD) frequently occurs in patients on hemodialysis (HD); however, little is known about the effectiveness of drugs. We compare the effects of sarpogrelate and cilostazol in HD patients with PAD., Methods: We conducted a prospective, randomized, open-label, and multicenter trial for 24 weeks in HD patients with PAD. Thirty-five patients were divided into two groups: sarpogrelate (n = 17) and cilostazol (n = 18). We analyzed changes in skin perfusion pressure (SPP), levels of oxidative stress biomarkers, and adverse events., Results: At 24 weeks, SPP was increased in both groups (sarpogrelate, 43 ± 17 to 55 ± 15 mmHg; cilostazol, 49 ± 21 to 66 ± 29 mmHg; p < 0.05), and no difference was observed between the groups. Plasma pentosidine levels decreased in both groups (sarpogrelate, 0.65 ± 0.24 to 0.48 ± 0.12 mg/mL; cilostazol, 0.58 ± 0.22 to 0.47 ± 0.17 mg/mL; p < 0.05), and there were no differences between the groups. Serum malondialdehyde-modified low-density lipoprotein (MDA-LDL) levels significantly increased only in cilostazol group (p < 0.05). There were no clinically significant safety concerns linked to the both drugs. Although blood pressure did not differ in both groups, heart rate increased only in cilostazol group from 77 ± 13 to 83 ± 16 beats per minute (p < 0.05)., Conclusion: Sarpogrelate improves SPP in HD patients with PAD without increasing heart rate and serum MDA-LDL levels. We demonstrated that sarpogrelate is an effective and safe drug for the treatment of HD patients with PAD.

Objective: Cells derived from the neural crest (NC) contribute to the development of several adult tissues, including tooth and periodontal tissue. Here, two transgenic lines, Wnt1-Cre/ZEG and P0-Cre/ZEG, were analysed to determine the fate and distribution of neural crest cells (NCCs) in adult mouse periodontal ligament (PDL)., Design: Paraffin-embedded and decalcified histology samples were prepared from Wnt1-Cre/ZEG and P0-Cre/ZEG mice that were 4-, 8-, or 12-weeks old. Expression of GFP (NC-derived cells), NC-markers (Slug, AP-2 alpha, HNK-1, p75NTR and Nestin), and mesenchymal stem cell markers (CD29 and STRO-1) were examined using immunohistochemistry., Results: In four-week-old Wnt1-Cre/ZEG mice, GFP((+)) NC-derived cells were specifically detected in the mid-zone of PDL. The GFP((+)) cells constituted 1.4% of all cells in PDL, and this percentage decreased as the mice aged. The distribution and prevalence of GFP((+)) cells were comparable between Wnt1-Cre/ZEG and P0-Cre/ZEG mice. NC-marker((+)) cells were expressed only in GFP((+)) cells while MSC markers were detected only in GFP((-)) cells., Conclusion: The prevalence and specific distribution of NC-derived cells in adult PDL of Wnt1-Cre/ZEG and P0-Cre/ZEG mouse were examined. Interestingly, various NC markers, including markers for undifferentiated NCCs, were still expressed at high levels in GFP((+)) cells. These observations may indicate that labelled cells in the Wnt1-Cre/ZEG and P0-Cre/ZEG mice did not constituted all NC-derived cells, but rather an interesting subset of NC-derived cells. These findings may be useful in understanding the homeostatic character of the PDL and contribute to establishing successful periodontal tissue maintenance., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

A 78-year-old Japanese female presented with low-grade fever, malaise, and appetite loss lasting for 1 month. Upper and lower gastrointestinal endoscopy and contrast-enhanced whole-body computed tomography (CT) revealed no abnormal findings at a referring hospital. She was referred to our hospital because of bilateral leg edema and 2.5 g/day proteinuria. Serum creatinine was 0.73 mg/dl and the kidneys were not enlarged. Kidney biopsy showed marked endocapillary proliferation with mesangiolysis. Soon after the kidney biopsy, her symptoms improved spontaneously, along with decreases in lactate dehydrogenase (LDH) from 503 to 197 IU/l, C-reactive protein (CRP) from 4.47 to 0.66 mg/dl, and soluble interleukin-2 receptor (sIL-2R) from 1789 to 1001 U/ml. Thus, she was followed carefully as an outpatient. One month later, however, she presented with dysarthria and right-sided hemiparesis, and diffusion-weighted brain magnetic resonance imaging (MRI) showed multiple high-intensity areas. She also had respiratory failure, and lung perfusion scintigraphy showed multiple low blood stream areas. Suspecting some endovascular abnormality, we performed immunohistochemical staining of the kidney biopsy specimen taken previously to find that endocapillary infiltrating cells were CD20-positive B lymphocytes. The infiltrating cells were confined to the endocapillary compartment in glomeruli and peritubular capillaries. Both clinical and pathological findings led us to diagnose intravascular large B cell lymphoma (IVLBCL). Two bone marrow biopsies and random skin biopsies were performed, but no abnormality was found. The present case demonstrates that clinical course and renal biopsy findings of intravascular large B cell lymphoma may mimic other renal conditions and that the identification of cell types with immunohistochemical staining may help establish an accurate diagnosis.

Objective: To assess the mechanism of long-term LDL-C-lowering effect of ezetimibe-plus-statin., Methods: Coronary artery disease patients whose LDL-C ≥ 70 mg/dL after treatment with atorvastatin 10 mg/day or rosuvastatin 2.5 mg/day were randomly assigned to receive ezetimibe 10 mg/day + statin (n = 78) or double-dose statin (n = 72) for 52 weeks., Results: Greater LDL-C reduction was observed and maintained until 52 weeks in ezetimibe-plus-statin, while LDL-C levels re-increased after 12 weeks in double-dose statin. Although lathosterol/TC increased, campesterol/TC decreased more in ezetimibe-plus-statin. In contrast, lathosterol/TC unchanged and campesterol/TC increased, increasing campesterol/lathosterol ratio for 52 weeks in double-dose statin. Plasma PCSK9 levels were higher in double-dose statin than in ezetimibe-plus-statin at 12 weeks, but similar at 52 weeks., Conclusion: Although the difference in PCSK9 between 2 groups was transient, that in both campesterol and lathosterol persisted until 52 weeks. These results demonstrated simultaneous inhibition of cholesterol absorption and synthesis provides stable and greater decrease in LDL-C levels., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Spontaneous formation of polymeric metallosomes with uniform size (~100 nm) was found to occur in aqueous medium through the reaction of an anticancer agent, (1,2-diaminocyclohexane)platinum(II) (DACHPt), with a Y-shaped block copolymer of ω-cholesteroyl-poly(L-glutamic acid) and two-armed poly(ethylene glycol) (PEGasus-PLGA-Chole). Circular dichroism spectrum measurements revealed that the PLGA segment forms an α-helix structure within the metallosomes, suggesting that secondary-structure formation of metallocomplexed PLGA segment may drive the self-assembly of the system into vesicular structure. These metallosomes can encapsulate water-soluble fluorescent macromolecules into their inner aqueous phase and eventually deliver them selectively into tumor tissues in mice, owing to the prolonged blood circulation. Accordingly, fluorescent imaging of the tumor was successfully demonstrated along with an appreciable antitumor activity by DACHPt moieties retained in the vesicular wall of the metallosomes, indicating the potential of metallosomes as multifunctional drug carriers.

Background and Purpose: Synovectomy is an effective procedure for management of the rheumatoid elbow at radiographically early stages (Larsen grades 1 and 2). However, its efficacy for advanced stages (Larsen grades 3-5) is controversial. We investigated the outcome of synovectomy for advanced stages of the rheumatoid elbow., Methods: Between May 1985 and September 1994, synovectomy was performed for 67 rheumatoid elbows in 59 patients (mean age 52 (26-72) years, 54 women). 3 elbows (3 patients) were lost to follow-up after mean 15 (10-23) years. Thus, 64 elbows were evaluated clinically and radiographically., Results: The mean Mayo elbow performance score (MEPS) improved from 42 (15-75) points preoperatively to 78 (45-100) points at the final follow-up examination. In cases of Larsen grade 5, the mean MEPS at final follow-up examination (69 points) was lower than those of Larsen grade 3 and 4 cases (80 and 79 points, respectively) (p < 0.01). Recurrence of synovitis was obvious in 20/67 elbows. 12 cases had a total elbow arthroplasty mean 13 years after the synovectomy. The 10-year, 15-year, and 20-year survival rates were 97%, 75%, and 70%, respectively., Interpretation: Our findings suggest that synovectomy for the rheumatoid elbow gives a good long-term outcome for radiographically judged destroyed joints of Larsen grades 3-4.

The purpose of this study was to evaluate the change in pelvic tilt angle (PA) in the sagittal plane in the standing and supine positions for 2 to 4 years after total hip arthroplasty (THA). Anteroposterior pelvic radiographs of 21 male and 65 female patients were investigated before and after THA yearly over 2 to 4 years. Both the standing and supine PA significantly posteriorly tilted after THA. The difference in PA between the standing and supine positions (dPA) significantly increased after THA. Although the PA in the standing and supine positions plateaued 1 year after THA, the dPA gradually increased. In addition, the percentage of patients who showed a difference of more than 10° in dPA tended to increase yearly. In particular, elderly female patients who showed posterior tilt in PA in the standing or supine positions or a large dPA before THA tended to show a dPA of more than 10° after THA., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Coronary artery calcification (CAC) leads to a significant increase in cardiovascular morbidity and mortality in hemodialysis (HD) patients. Metabolic acidosis, which is common in HD patients, promotes bone resorption in human and animals as a result of buffer function of bone, and calcium and phosphate elute from bone into blood stream. However, the effect of acidosis on CAC in HD patients has never been precisely investigated. This is a cross-sectional observational study performed in a single center. One hundred and seven prevalent HD patients (35 women and 72 men) underwent electron-beam computed tomography (EBCT) to evaluate CAC score (CACS), and then we evaluated associated factors of CACS with clinical and laboratory parameters including pre-HD pH and bicarbonate levels. Pre-HD pH and bicarbonate levels were 7.35 ± 0.04, and 17.6 ± 1.8 mmol/L, respectively. The pre-HD pH had no significant correlation to CACS (r = -0.025, P = 0.81). CACS was significantly negatively correlated with pre-HD bicarbonate levels (r = -0.329, P = 0.0009) and serum albumin levels (r = -0.298, P = 0.0467), while it was positively correlated with age (r = 0.319, P = 0.0008) and HD duration (r = 0.385, P = 0.0004). Serum levels of calcium, phosphorus, intact parathyroid hormone, and use of phosphorus binders were not related to CACS. Multivariate analysis indicated that plasma pre-HD bicarbonate level was independently associated with CACS. The present study showed that blood levels of pre-HD bicarbonate were significantly associated with CAC in HD patients. Further studies are needed to confirm these results and to determine whether correction of metabolic acidosis prevents the development of CAC, one of the features of accelerated atherosclerosis in HD patients., (© 2012 The Authors. Therapeutic Apheresis and Dialysis © 2012 International Society for Apheresis.)