Publications by authors named "Ryo Takagi"

154 Publications

Long-term outcomes after carbon-ion radiotherapy for oral mucosal malignant melanoma.

J Radiat Res 2017 Jul;58(4):517-522

Department of Otolaryngology, Chiba University Graduate School of Medicine, 1-8-1 Inohara, Chuo-ku, Chiba 260-8677, Japan.

Oral mucosal malignant melanoma (OMM) is extremely rare and has a poor prognosis. Owing to its rarity, it has not yet been possible to establish an optimal treatment modality. The objective of this study was to evaluate the long-term efficacy of carbon-ion radiotherapy (C-ion RT) for OMM. Between 1997 and 2013, 19 patients with OMM were treated with C-ion RT alone. Patient ages ranged from 44 to 84 years (median, 69 years). Nine men and 10 women were included. OMMs were restaged in accordance with the seventh edition of the tumour/node/metastasis (TNM) Staging System of the International Union Against Cancer. Before treatment, 14 patients had T3 disease and 5 had T4a disease. Three patients were classified as having N1 disease. All patients were classified as having M0. The hard palate was the most frequently involved oral subsite. All patients were treated with 57.6 Gy (relative biological effectiveness) in 16 fractions. The median follow-up period was 61 months (range, 8-190 months). The 5-year local control, overall survival and progression-free survival rates were 89.5%, 57.4% and 51.6%, respectively. For local control and overall survival, T classification was found to be a significant prognostic factor. Grade 2 and 3 osteoradionecrosis was observed in three and four patients, respectively. The presence of teeth within the planning target volume was a significant risk factor for developing osteoradionecrosis. C-ion RT was an effective treatment option with acceptable toxicity for OMM.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/jrr/rrw117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5570020PMC
July 2017

Definitive carbon-ion radiotherapy for locally advanced parotid gland carcinomas.

Head Neck 2017 04 22;39(4):724-729. Epub 2016 Dec 22.

Department of Otolaryngology, Chiba University School of Medicine, Chiba, Japan.

Background: The purpose of this study was to present our evaluation of the safety and efficacy of carbon-ion radiotherapy (C-ion RT) for locally advanced parotid gland carcinomas.

Methods: Clinicopathological features and outcomes were evaluated in 46 patients receiving C-ion RT for parotid gland carcinomas.

Results: Sixteen patients had adenoid cystic carcinoma; 8 had adenocarcinoma, 8 had mucoepidermoid carcinoma, and 14 had other carcinomas. T2, T3, T4a, and T4b diseases were diagnosed in 3, 18, 8, and 17 patients, respectively. C-ion RT was provided to 25 patients as the primary treatment, to 20 patients for local recurrences after surgery, and to 1 patient for residual tumor after surgery. During follow-up (median duration, 62 months), 5-year local control and overall survival (OS) rates were 74.5% and 70.1%, respectively. Of the 30 patients without facial nerve palsy before C-ion RT, 25 showed no radiation-induced facial nerve palsy.

Conclusion: C-ion RT is effective and has acceptable toxicity levels for locally advanced parotid gland carcinomas. © 2016 Wiley Periodicals, Inc. Head Neck 39: 724-729, 2017.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/hed.24671DOI Listing
April 2017

Radiation-induced Parotid Gland Atrophy in Patients with Head and Neck Cancer After Carbon-ion Radiotherapy.

Anticancer Res 2016 10;36(10):5403-5407

National Institute of Radiological Science Hospital, National Institutes for Quantum and Radiological Sciences and Technology, Chiba, Japan.

Background/aim: This study aimed to clarify the relationship between dosimetric factors and parotid gland (PG) atrophy after carbon ion radiotherapy (C-ion RT).

Patients And Methods: Fifty-four patients with head and neck tumours were enrolled and 93 irradiated PGs were analyzed. Thirty and 24 patients were treated with total doses [relative biological effectiveness (RBE)] of 57.6 Gy and 64.0 Gy, respectively, in 16 fractions. PG volumes were measured using computed tomographic images obtained before C-ion RT and every 3-6 months thereafter.

Results: The median follow-up period was 46.4 months (range=24.0-123.0 months). Univariate analysis showed that PG volumes receiving more than 5, 10, 15, and 20 Gy RBE (V5, V10, V15 and V20, respectively), mean dose, and maximum dose were significantly associated with PG atrophy. Multivariate analysis indicated that only V5 was significantly associated with atrophy.

Conclusion: Increasing V5 was a significant risk factor for PG atrophy after C-ion RT.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.21873/anticanres.11116DOI Listing
October 2016

Analysis of molecular alterations in laterally spreading tumors of the colorectum.

J Gastroenterol 2017 Jun 4;52(6):715-723. Epub 2016 Oct 4.

Division of Gastroenterology, Department of Internal Medicine, Iwate Medical University, 19-1, Morioka, 020-8505, Japan.

Background: Colorectal laterally spreading tumors (LSTs) are classified into LST-Gs and LST-NGs, according to macroscopic findings. In the present study, we determined the genetic and epigenetic alterations within colorectal LSTs and protruding adenomas.

Methods: A crypt isolation method was used to isolate DNA from tumors and normal glands of 73 macroscopically verified colorectal LSTs (histologically defined adenomas; 38 LST-Gs and 35 LST-NGs) and 36 protruding adenomas. The DNA was processed using polymerase chain reaction (PCR) microsatellite assays, single-strand conformation polymorphism (SSCP) assays, and pyrosequencing to detect chromosomal allelic imbalance (AI), mutations in APC, KRAS, and TP53, and the methylation of MLH1, MGMT, CDKN2A, HPP1, RASSF2A, SFRP1, DKK1, ZFP64, and SALL4 genes. In addition, methylation status was examined using the following set of markers: MIN1, MINT2, MINT31, MLH1, and CDKN2A (with classification of negative/low and high). Microsatellite instability (MSI) was also examined.

Results: 5q AI and methylation of the SFRP1 and SALL4 genes were common molecular events in both LST-Gs and LST-NGs. Neither MSI nor mutations in BRAF ware observed in the LSTs. TP53 mutations were rarely found in LSTs. The frequencies of KRAS and APC mutations and the methylation levels of ZFP64, RASSF2A, and HPP1 genes were significantly higher in LST-Gs than in LST-NGs. Protruding adenomas showed alterations common to LST-Gs. Negative/low methylation status was common among the three types of tumors.

Conclusion: Combined genetic and epigenetic data suggested that the molecular mechanisms of tumorigenesis were different between LST-Gs and LST-NGs.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00535-016-1269-yDOI Listing
June 2017

Randomised clinical study comparing the effectiveness and physiological effects of hypertonic and isotonic polyethylene glycol solutions for bowel cleansing.

BMJ Open Gastroenterol 2016 27;3(1):e000101. Epub 2016 Jul 27.

Digestive Disease Center, Akita Red Cross Hospital , Akita , Japan.

Objectives: Bowel cleansing is necessary before colonoscopy, but is a burden to patients because of the long cleansing time and large dose volume. A low-volume (2 L) hypertonic polyethylene glycol-ascorbic acid solution (PEG-Asc) has been introduced, but its possible dehydration effects have not been quantitatively studied. We compared the efficacy and safety including the dehydration risk between hypertonic PEG-Asc and isotonic PEG regimens.

Design: This was an observer-blinded randomised study. Participants (n=310) were allocated to receive 1 of 3 regimens on the day of colonoscopy: PEG-Asc (1.5 L) and water (0.75 L) dosed with 1 split (PEG-Asc-S) or 4 splits (PEG-Asc-M), or PEG-electrolyte solution (PEG-ES; 2.25 L) dosed with no split. Dehydration was analysed by measuring haematocrit (Ht).

Results: The cleansing time using the hypertonic PEG-Asc-S (3.33±0.48 hours) was significantly longer than that with isotonic PEG-ES (3.05±0.56 hours; p<0.001). PEG-Asc-M (3.00±0.53 hours) did not have this same disadvantage. Successful cleansing was achieved in more than 94% of participants using each of the 3 regimens. The percentage changes in Ht from baseline (before dosing) to the end of dosing with PEG-Asc-S (3.53±3.32%) and PEG-Asc-M (4.11±3.07%) were significantly greater than that with PEG-ES (1.31±3.01%).

Conclusions: These 3 lower volume regimens were efficacious and had no serious adverse effects. Even patients cleansed with isotonic PEG-ES showed significant physiological dehydration at the end of dosing. The four-split PEG-Asc-M regimen is recommended because of its shorter cleansing time without causing serious nausea.

Trial Registration Number: UMIN000013103; Results.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1136/bmjgast-2016-000101DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4985908PMC
August 2016

Regional adaptation of collagen in skeletal muscle to repeated bouts of strenuous eccentric exercise.

Pflugers Arch 2016 09 28;468(9):1565-72. Epub 2016 Jul 28.

Department of Life Sciences, The University of Tokyo, 3-8-1 Komaba, Meguro-ku, 153-8902, Japan.

This study investigated the injured region-specific alterations of factors related to the "repeated bout effect" (RBE), i.e., when the first bout of eccentric exercise generates resistance to injuries from the second bout of the same exercise. Wistar rats were divided into single injury (SI) and repeated injury (RI) groups. The right gastrocnemius muscle was subjected to a bout of eccentric contractions (ECs) at the age of 14 weeks in the SI group and 10 and 14 weeks in the RI group. The number of injured fibers after the last bout of ECs was lower in RI than in SI. In the SI group, injured fibers after ECs were mainly located in the superficial region of muscle and expressed myosin heavy chain (MHC) IIx and IIb. Prior to the second bout of ECs, the fiber-type composition in the RI group showed decreased MHC IIx and IIb fibers and increased MHC IIa fibers compared with those in the SI group. However, most regenerating fibers showed either MHC IIx or IIb expression. Heat shock protein 72 and total collagen contents in whole muscle were higher in the RI group than in the SI group; however, only the collagen expression in the RI group was more intense than that in the SI group in the superficial region of muscle. These findings suggest that increased collagen may play a more important role in the injured region of muscle than the other factors in RBE.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00424-016-1860-3DOI Listing
September 2016

Detection of tissue coagulation by decorrelation of ultrasonic echo signals in cavitation-enhanced high-intensity focused ultrasound treatment.

J Ther Ultrasound 2016 14;4:15. Epub 2016 Apr 14.

Graduate School of Biomedical Engineering, Tohoku University, Sendai, 980-8579 Japan.

Background: A noninvasive technique to monitor thermal lesion formation is necessary to ensure the accuracy and safety of high-intensity focused ultrasound (HIFU) treatment. The purpose of this study is to ultrasonically detect the tissue change due to thermal coagulation in the HIFU treatment enhanced by cavitation microbubbles.

Methods: An ultrasound imaging probe transmitted plane waves at a center frequency of 4.5 MHz. Ultrasonic radio-frequency (RF) echo signals during HIFU exposure at a frequency of 1.2 MHz were acquired. Cross-correlation coefficients were calculated between in-phase and quadrature (IQ) data of two B-mode images with an interval time of 50 and 500 ms for the estimation of the region of cavitation and coagulation, respectively. Pathological examination of the coagulated tissue was also performed to compare with the corresponding ultrasonically detected coagulation region.

Results: The distribution of minimum hold cross-correlation coefficient between two sets of IQ data with 50-ms intervals was compared with a pulse inversion (PI) image. The regions with low cross-correlation coefficients approximately corresponded to those with high brightness in the PI image. The regions with low cross-correlation coefficients in 500-ms intervals showed a good agreement with those with significant change in histology.

Conclusions: The results show that the regions of coagulation and cavitation could be ultrasonically detected as those with low cross-correlation coefficients between RF frames with certain intervals. This method will contribute to improve the safety and accuracy of the HIFU treatment enhanced by cavitation microbubbles.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s40349-016-0060-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4831115PMC
April 2016

Brush biopsy of human oral mucosal epithelial cells as a quality control of the cell source for fabrication of transplantable epithelial cell sheets for regenerative medicine.

Regen Ther 2016 Jun 24;4:71-77. Epub 2016 Mar 24.

Institute of Advanced Biomedical Engineering and Science, Tokyo Women's Medical University, 8-1 Kawada-Cho, Shinjuku-ku, Tokyo 162-8666, Japan.

Autologous oral mucosal epithelial cell sheets have been used for treating epithelial defects such as cornea and esophagus. The cell source of patients' oral mucosal epithelial cell sheet should be examined in normality because it has individual difference. In this study, oral mucosal epithelial cells were less invasively collected by brush biopsy from the buccal, gingival, labial, and palate mucosa of four healthy volunteer donors without anesthesia, and analyzed the keratin expressions by western blotting and the obtained results were compared with those by immunohistochemistry of each of the native tissues. All of the oral mucosal epithelial cells expressed keratin 4 (K4) and K13, which were mucosal stratified squamous epithelial cell markers. K1 and K10, keratinized epithelial cell markers, were also detected in keratinized tissues such as gingival and palate mucosa. The markers of epithelial basal cells such as p63 and K15 were not detected by brush biopsy-western blotting. Although this method does not include basal layers of oral mucosa, protein expressions of upper layer of lesion area are different from normal. Therefore, brush biopsy-western blotting was extremely less invasive and would contribute to quality control of the fabrication of autologous oral mucosal epithelial cell sheets.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.reth.2016.02.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6581830PMC
June 2016

Allogeneic Transplantation of Periodontal Ligament-Derived Multipotent Mesenchymal Stromal Cell Sheets in Canine Critical-Size Supra-Alveolar Periodontal Defect Model.

Biores Open Access 2016 1;5(1):22-36. Epub 2016 Jan 1.

Department of Periodontology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University , Tokyo, Japan .

Periodontitis is a chronic inflammatory disease that induces the destruction of tooth-supporting tissues, followed by tooth loss. Although several approaches have been applied to periodontal regeneration, complete periodontal regeneration has not been accomplished. Tissue engineering using a combination of cells and scaffolds is considered to be a viable alternative strategy. We have shown that autologous transplantation of periodontal ligament-derived multipotent mesenchymal stromal cell (PDL-MSC) sheets regenerates periodontal tissue in canine models. However, the indications for autologous cell transplantation in clinical situations are limited. Therefore, this study evaluated the safety and efficacy of allogeneic transplantation of PDL-MSC sheets using a canine horizontal periodontal defect model. Canine PDL-MSCs were labeled with enhanced green fluorescent protein (EGFP) and were cultured on temperature-responsive dishes. Three-layered cell sheets were transplanted around denuded root surfaces either autologously or allogeneically. A mixture of β-tricalcium phosphate and collagen gel was placed on the bone defects. Eight weeks after transplantation, dogs were euthanized and subjected to microcomputed tomography and histological analyses. RNA and DNA were extracted from the paraffin sections to verify the presence of EGFP at the transplantation site. Inflammatory markers from peripheral blood sera were quantified using an enzyme-linked immunosorbent assay. Periodontal regeneration was observed in both the autologous and the allogeneic transplantation groups. The allogeneic transplantation group showed particularly significant regeneration of newly formed cementum, which is critical for the periodontal regeneration. Serum levels of inflammatory markers from peripheral blood sera showed little difference between the autologous and allogeneic groups. EGFP amplicons were detectable in the paraffin sections of the allogeneic group. These results suggest that allogeneic PDL-MSC sheets promoted periodontal tissue regeneration without side effects. Therefore, allogeneic transplantation of PDL-MSC sheets has a potential to become an alternative strategy for periodontal regeneration.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1089/biores.2015.0043DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4744877PMC
February 2016

Evaluation of the safety and efficacy of carbon ion radiotherapy for locally advanced adenoid cystic carcinoma of the tongue base.

Head Neck 2016 04 8;38 Suppl 1:E2122-6. Epub 2016 Feb 8.

Department of Otolaryngology, Chiba University School of Medicine, Chiba, Japan.

Background: Most cases of adenoid cystic carcinoma (ACC) of the tongue base are radioresistant, and are diagnosed in the advanced stage. Therefore, we evaluated the safety and efficacy of carbon ion radiotherapy (C-ion RT) for locally advanced ACC of the tongue base.

Methods: Eighteen patients with ACC of the tongue base were treated with C-ion RT between May 2002 and April 2014. Seventeen patients had T4a disease and 1 patient had T2 disease before C-ion RT.

Results: The median follow-up period was 57 months (range, 10-132 months). The 5-year local control rate was 92%. The 5-year overall survival (OS) and disease-free survival (DFS) rates were 72% and 44%, respectively. Regarding late reactions, 2 patients developed grade 3 mandible osteoradionecrosis, and 1 had grade 3 hemorrhage of the tongue base.

Conclusion: C-ion RT was effective with acceptable toxicities for locally advanced ACC of the tongue base. © 2016 Wiley Periodicals, Inc. Head Neck 38: E2122-E2126, 2016.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/hed.24397DOI Listing
April 2016

Safe use of iodinated and gadolinium-based contrast media in current practice in Japan: a questionnaire survey.

Jpn J Radiol 2016 Feb 10;34(2):130-9. Epub 2015 Dec 10.

Department of Radiology, Nippon Medical School, Tokyo, Japan.

Purpose: To help establish consensus on the safe use of contrast media in Japan.

Materials And Methods: Questionnaires were sent to accredited teaching hospitals with radiology residency programs.

Results: The reply rate was 45.4% (329/724). For contrast-induced nephropathy (CIN), chronic and acute kidney diseases were considered a risk factor in 96.7 and 93.6%, respectively, and dehydration in 73.9%. As preventive actions, intravenous hydration (89.1%) and reduction of iodinated contrast media dose (86.9%) were commonly performed. For nephrogenic systemic fibrosis (NSF), chronic and acute kidney diseases were considered risk factors in 98.5 and 90.6%, respectively, but use of unstable gadolinium-based contrast media was considered a risk factor in only 55.6%. A renal function test was always (63.5% in iodinated; 65.7% in gadolinium) or almost always (23.1; 19.8%) performed, and estimated glomerular filtration rate (eGFR) was the parameter most frequently used (80.8; 82.6%). For the patients with risk factors for acute adverse reaction (AAR), steroid premedication or/and change of contrast medium were frequent preventive actions, but intravenous steroid administration immediately before contrast media use was still performed.

Conclusion: Our questionnaire survey revealed that preventive actions against CIN were properly performed based on patients' eGFR. Preventive actions against NSF and AAR still lacked consensus.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s11604-015-0505-3DOI Listing
February 2016

4D flow MRI assessment of extracranial-intracranial bypass: qualitative and quantitative evaluation of the hemodynamics.

Neuroradiology 2016 Mar 2;58(3):237-44. Epub 2015 Dec 2.

Department of Radiology, Nippon Medical School, Tokyo, Japan.

Introduction: Our aim was to assess the feasibility of using time-resolved 3D phase-contrast (4D flow) MRI to characterize extracranial-intracranial (EC-IC) bypass.

Methods: We enrolled 32 patients who underwent EC-IC bypass (15 men, 17 women; mean age 66.4 years). In all, 16 underwent radial artery graft (RAG) bypass and 16 underwent superficial temporal artery (STA) bypass. 4D flow MRI, time-of-flight (TOF) magnetic resonance angiography (MRA), and computed tomography angiography (CTA) were performed. Bypass patency, flow direction, and blood flow volume (BFV) of each artery were determined by 4D flow MRI. Arterial diameters were measured by TOF-MRA and CTA. We compared RAG and STA bypasses by evaluating the flow direction and BFV of each artery. We evaluated the correlation between arterial diameters (measured by CTA or MRA) and the BFV and the detectability of flow direction (measured by 4D flow MRI) of each artery.

Results: 4D flow MRI confirmed the patency of each bypass artery. Flow direction of the M1 segment of the middle cerebral artery and BFV in the bypass artery differed between RAG and STA groups (p < 0.01). BFV in the bypass slightly correlated with the diameters on CTA (p < 0.05, R (2) = 0.287). Of the 29 arteries in the circle of Willis, nine were not depicted on 4D flow MRI. Cutoff values for arterial diameters on CTA and TOF-MRA for detecting the artery on 4D flow MRI were 2.4 and 1.8 mm, respectively.

Conclusion: 4D flow MRI provided unique information for characterizing EC-IC bypasses, although this detectability is limited when addressing small arteries with slow flow.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00234-015-1626-1DOI Listing
March 2016

Glucocorticoid metabolism in the bovine cumulus-oocyte complex matured in vitro.

Reproduction 2016 Jan 30;151(1):73-82. Epub 2015 Oct 30.

Department of Life Science and AgricultureObihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido 080-8555, Japan.

Glucocorticoid action in target organs is regulated by relative activities of 11β-HSD type 1 (HSD11B1) that mainly converts cortisone to active cortisol and type 2 (HSD11B2) that inactivates cortisol to cortisone. HSD11Bs have been shown to be expressed in the ovary of various species. However, little is known about the expression and activity of HSD11Bs in the bovine cumulus-oocyte complex (COC). In the present study, we investigated the expression and activities of HSD11Bs in in vitro-matured (IVM) bovine COCs. Bovine COCs were matured in M199 supplemented with or without FSH and FCS. The expression of HSD11B1 and HSD11B2 was measured by using quantitative RT-PCR in denuded oocytes (DO) and cumulus cells (CC). Reductive and oxidative activities of HSD11Bs were determined by radiometric conversion assay using labeled cortisol, cortisone or dexamethasone in intact COCs, DO or CC in the presence or absence of 11-keto-progesterone (11kP), a selective inhibitor of HSD11B2. The presence of HSD11Bs in the oocyte was examined by immunofluorescence microscopy. Oocytes exclusively expressed HSD11B2 and its expression and activity were largely unchanged during IVM. CC, on the other hand, exclusively expressed HSD11B1 and its expression and activity were upregulated as IVM progressed. As a result, the net glucocorticoid metabolism shifted from inactivation to activation towards the end of IVM. These results indicate that the bovine COC is capable of modulating local glucocorticoid concentration and, by doing so, may create an environment that is favorable to ovulating oocyte for maturation, fertilization and subsequent development.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1530/REP-15-0363DOI Listing
January 2016

Epigenetic silencing of NTSR1 is associated with lateral and noninvasive growth of colorectal tumors.

Oncotarget 2015 Oct;6(30):29975-90

Department of Molecular Biology, Sapporo Medical University School of Medicine, Sapporo, Japan.

Our aim was to identify DNA methylation changes associated with the growth pattern and invasiveness of colorectal cancers (CRCs). Comparison of the methylation statuses of large (≥ 20 mm in diameter along the colonic surface) noninvasive tumors (NTs) and small (<20 mm in diameter along the colonic surface) invasive tumors (ITs) using CpG island microarray analysis showed neurotensin receptor 1 (NTSR1) to be hypermethylated in large NTs. Quantitative bisulfite pyrosequencing revealed that NTSR1 is frequently methylated in colorectal tumors, with large NTs exhibiting the highest methylation levels. The higher NTSR1 methylation levels were associated with better prognoses. By contrast, NTSR1 copy number gains were most frequent among small ITs. Methylation of NTSR1 was associated with the gene's silencing in CRC cell lines, whereas ectopic expression of NTSR1 promoted proliferation and invasion by CRC cells. Analysis of primary tumors composed of adenomatous and malignant portions revealed that NTSR1 is frequently methylated in the adenomatous portion, while methylation levels are generally lower in the cancerous portions. These results suggest that NTSR1 methylation is associated with lateral and noninvasive growth of colorectal tumors, while low levels of methylation may contribute to the malignant potential through activation of NTSR1. Our data also indicate that NTSR1 methylation may be a prognostic biomarker in CRC.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.18632/oncotarget.5034DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4745776PMC
October 2015

Membrane-Permeable Calpain Inhibitors Promote Rat Oral Mucosal Epithelial Cell Proliferation by Inhibiting IL-1α Signaling.

PLoS One 2015 31;10(7):e0134240. Epub 2015 Jul 31.

Institute of Advanced Biomedical Engineering and Science, Tokyo Women's Medical University, TWIns, 8-1 Kawada-cho, Shinjuku-ku, Tokyo, 162-8666, Japan.

To standardise regenerative medicine using cultured cells, the use of serum-free, chemically defined media will be necessary. We have reported that IL-1α inhibits the growth of epithelial cells in culture and that recombinant IL-1 receptor antagonist (IL-1RA) significantly promotes epithelial cell growth in no feeder layer condition. In this study, we examined inhibitors of calpain, a cysteine proteinase that plays crucial roles in various cellular functions, including IL-1α maturation and secretion. The culturing of epithelial cells in serum-free media supplemented with a membrane-permeable calpain inhibitor significantly promoted growth while suppressing IL-1α maturation and secretion. By contrast, non-membrane-permeable calpain inhibitor treatment did not have these effects. Interestingly, immunoblotting analysis revealed that immature, untruncated, IL-1α expression was also downregulated by cell-permeable calpain inhibitor treatment, and the difference in IL-1α gene expression increased from day 2 to day 6. Although IL-1RA has been reported to promote epithelial cell growth, we detected no synergistic promotion of epithelial cell growth using a calpain inhibitor and IL-1RA. These findings indicate that calpain inhibitors promote epithelial cell proliferation by inhibiting IL-1α maturation at an early phase of epithelial cell culture and by suppressing the positive feedback-mediated amplification of IL-1α signalling.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0134240PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4521813PMC
May 2016

Endoscopic cell sheet transplantation device developed by using a 3-dimensional printer and its feasibility evaluation in a porcine model.

Gastrointest Endosc 2015 Jul 17;82(1):147-52. Epub 2015 Apr 17.

Institute of Advanced Biomedical Engineering and Science, Tokyo Women's Medical University, Tokyo, Japan.

Background: To prevent severe esophageal stenosis after aggressive endoscopic submucosal dissection (ESD), our group previously reported an efficient treatment using cell sheets that had been fabricated from patient cells. However, this transplantation procedure had not been easy for every endoscopist and needed to be improved to derive the full effect of epithelial cell sheets.

Objective: To develop an endoscopic device that enables easy and effective cell sheet transplantation and to evaluate its performance and clinical feasibility.

Design: Animal study.

Setting: Animal experimentation laboratory.

Intervention: Three pigs underwent circumferential esophageal ESD while under general anesthesia. A total of 12 cell sheets were endoscopically transplanted to the ESD site; 6 cell sheets were transplanted by using an endoscopic device that we developed, and 6 cell sheets were transplanted by using the conventional method.

Main Outcome Measurements: Procedure time, transplanted area on the ESD site, transplantation success rate, and monitoring of adverse events or incidents.

Results: The device allowed successful transplantation of all cell sheets with a shorter procedure time than with the conventional method (4.8 ± 0.8 minutes vs 13.3 ± 5.7 minutes, respectively) (P = .005) and onto a larger area (111.3 ± 56.3 mm(2) vs 41.8 ± 4.2 mm(2), respectively) (P = .023) with a higher success rate (100% vs 83%, respectively). No adverse incidents were monitored in each method.

Limitations: Animal study, small sample.

Conclusion: A newly designed endoscopic cell sheet transplantation device would be useful.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.gie.2015.01.062DOI Listing
July 2015

A chemically defined culture medium containing Rho kinase inhibitor Y-27632 for the fabrication of stratified squamous epithelial cell grafts.

Biochem Biophys Res Commun 2015 May 28;460(2):123-9. Epub 2015 Feb 28.

Department of Surgery, Institute of Gastroenterology, Tokyo Women's Medical University, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666, Japan. Electronic address:

With the development of a culture method for stratified squamous epithelial cells, tissue-engineered epithelial cell sheets have been successfully applied as clinical cell grafts. However, the implementation of these cell sheets without the use of any animal-derived materials is highly desirable. In this study, Rho-associated protein kinase inhibitor Y-27632 was used to develop a chemically defined culture medium for the fabrication of stratified epithelial cell grafts consisting of human epidermal and oral keratinocytes, and the proliferation activity, cell morphology, and gene expressions of the keratinocytes were analyzed. The results of a colorimetric assay indicated that Y-27632 significantly promoted the proliferation of the keratinocytes in culture media both with and without fetal bovine serum (FBS), although there were no indications of Y-27632 efficacy on cell morphology and stratification of the keratinocytes in culture medium without any animal-derived materials. The results of quantitative RT-PCR revealed that gene expressions correlated with cell adhesion, cell-cell junction, proliferation markers, and stem/progenitor markers in cultured keratinocytes were not strongly affected by the addition of Y-27632 to the culture medium. Moreover, gene expressions of differentiation markers in stratified keratinocytes cultured in medium without FBS were nearly identical to those of keratinocytes co-cultured with 3T3 feeder cells. Interestingly, the expressions of differentiation markers in cultured stratified keratinocytes were suppressed by FBS, whereas they were reconstructed by either co-culture of a 3T3 feeder layer or addition of Y-27632 into the culture medium containing FBS. These findings indicate that Y-27632 is a useful supplement for the development of a chemically defined culture medium for fabrication of stratified epithelial cell grafts for clinical applications for the purpose of developing the culture medium with a lower risk of pathogen transmission that might arise from animal-derived materials.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bbrc.2015.02.120DOI Listing
May 2015

How to prevent contamination with during the fabrication of transplantable oral mucosal epithelial cell sheets.

Regen Ther 2015 Jun 18;1:1-4. Epub 2015 Feb 18.

Institute of Advanced Biomedical Engineering and Science, Tokyo Women's Medical University, TWIns, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666, Japan.

We have utilized patients' own oral mucosa as a cell source for the fabrication of transplantable epithelial cell sheets to treat limbal stem cell deficiency and mucosal defects after endoscopic submucosal dissection of esophageal cancer. Because there are abundant microbiotas in the human oral cavity, the oral mucosa was sterilized and 40 μg/mL gentamicin and 0.27 μg/mL amphotericin B were added to the culture medium in our protocol. Although an oral surgeon carefully checked each patient's oral cavity and although candidiasis was not observed before taking the biopsy, contamination with () was detected in the conditioned medium during cell sheet fabrication. After adding 1 μg/mL amphotericin B to the transportation medium during transport from Nagasaki University Hospital to Tokyo Women's Medical University, which are 1200 km apart, no proliferation of was observed. These results indicated that the supplementation of transportation medium with antimycotics would be useful for preventing contamination with derived from the oral mucosa without hampering cell proliferation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.reth.2014.12.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6581869PMC
June 2015

The effect of transplantation of nasal mucosal epithelial cell sheets after middle ear surgery in a rabbit model.

Biomaterials 2015 Feb 15;42:87-93. Epub 2014 Dec 15.

Department of Oto-Rhino-Laryngology, Jikei University School of Medicine, Tokyo, Japan.

Postoperative regeneration of the middle ear mucosa and pneumatization of the middle ear cavity are of great importance after middle ear surgery. This study developed a new method to transplant autologous nasal mucosal epithelial cell sheets into the damaged middle ear cavity. The aim of this study was to evaluate postoperative healing after the transplantation of the cell sheets. Rabbit nasal mucosal epithelial cell sheets were fabricated on a temperature-responsive culture dish, and transplanted into the damaged middle ear of rabbit, which was surgically created. The healing of middle ears was evaluated by histology and X-ray computed tomography after transplantation. Functional evaluation was performed by measuring the maximum middle ear total pressure reflecting a trans-mucosal gas exchange function. Two control groups were used: the normal control group and the mucosa-eliminated control group. Transplantation of cell sheets suppressed the bone hyperplasia and the narrowing of pneumatic space in the middle ear cavity compared with the mucosa-eliminated control group. The mucosal gas exchange function was also better in the cell sheet-transplanted group. Nasal mucosal epithelial cell sheet was confirmed to be useful as an effective graft material after middle ear surgery and hopefully become a novel therapy in the future.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.biomaterials.2014.11.037DOI Listing
February 2015

[Regenerative medicine by cell sheet technology: the regeneration for stratified epithelial tissues].

Arerugi 2014 Nov;63(9):1223-8

Institute of Advanced Biomedical Engineering and Science Tokyo Women's Medical University.

View Article and Find Full Text PDF

Download full-text PDF

Source
November 2014

Prevention of esophageal strictures after endoscopic submucosal dissection.

World J Gastroenterol 2014 Nov;20(41):15098-109

Shinichiro Kobayashi, Nobuo Kanai, Takeshi Ohki, Ryo Takagi, Naoyuki Yamaguchi, Yoshiyuki Kasai, Takahiro Hosoi, Masayuki Yamato, Teruo Okano, Institute of Advanced Biomedical Engineering and Science, Tokyo Women's Medical University, Tokyo 162-8666, Japan.

Endoscopic mucosal resection (EMR) and endoscopic submucosal dissection (ESD) have recently been accepted as less invasive methods for treating patients with early esophageal cancers such as squamous cell carcinoma and dysplasia of Barrett's esophagus. However, the large defects in the esophageal mucosa often cause severe esophageal strictures, which dramatically reduce the patient's quality of life. Although preventive endoscopic balloon dilatation can reduce dysphagia and the frequency of dilatation, other approaches are necessary to prevent esophageal strictures after ESD. This review describes several strategies for preventing esophageal strictures after ESD, with a particular focus on anti-inflammatory and tissue engineering approaches. The local injection of triamcinolone acetonide and other systemic steroid therapies are frequently used to prevent esophageal strictures after ESD. Tissue engineering approaches for preventing esophageal strictures have recently been applied in basic research studies. Scaffolds with temporary stents have been applied in five cases, and this technique has been shown to be safe and is anticipated to prevent esophageal strictures. Fabricated autologous oral mucosal epithelial cell sheets to cover the defective mucosa similarly to how commercially available skin products fabricated from epidermal cells are used for skin defects or in cases of intractable ulcers. Fabricated autologous oral-mucosal-epithelial cell sheets have already been shown to be safe.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3748/wjg.v20.i41.15098DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4223243PMC
November 2014

4D Flow Preliminary Investigation for Anterior Fossa Dural Arteriovenous Fistula.

Can J Neurol Sci 2014 Sep;41(5):656-8

Department of Neurosurgery,Nippon Medical School,Tokyo,Japan.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1017/cjn.2014.6DOI Listing
September 2014

Carbon ion radiotherapy for locally advanced squamous cell carcinoma of the external auditory canal and middle ear.

Head Neck 2016 Apr 26;38(4):512-6. Epub 2015 May 26.

Department of Otolaryngology, Chiba University School of Medicine, Chiba, Japan.

Background: The prognosis of advanced squamous cell carcinoma (SCC) of the external auditory canal and middle ear remains poor. Carbon ion radiotherapy (C-ion RT) has shown promise for locally advanced head and neck cancer. Therefore, we evaluated the safety and efficacy of C-ion RT for locally advanced SCC of the external auditory canal and middle ear.

Methods: The cases of 13 patients with advanced (T3 and T4) SCC of the external auditory canal and middle ear who received C-ion RT as the primary treatment were reviewed.

Results: The median follow-up for all patients and the 7 surviving patients was 12 and 32 months, respectively. The 1-year and 3-year local control and overall survival (OS) rates were 72% and 54% and 70% and 40%, respectively. Severe temporal bone necrosis was observed in 2 patients.

Conclusion: C-ion RT is effective and generally safe for locally advanced SCC of the external auditory canal and middle ear.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/hed.23905DOI Listing
April 2016

Feasibility of carbon ion radiotherapy for locally advanced sinonasal adenocarcinoma.

Radiother Oncol 2014 Oct 3;113(1):60-5. Epub 2014 Oct 3.

Department of Otolaryngology, Chiba University School of Medicine, Japan.

Background And Purpose: To evaluate the safety and efficacy of carbon ion radiotherapy (CIRT) for locally advanced sinonasal adenocarcinoma.

Material And Methods: Twenty-two patients with sinonasal adenocarcinoma were treated with CIRT. CIRT was the primary treatment for 16 patients. Four patients received CIRT for local recurrence after surgery and two for residual tumour after surgery or chemotherapy. At the start of CIRT, 1 patient had T-classification (T) 2 disease, 2 had T3 disease, 5 had T4a disease, and 14 had T4b disease. Fourteen patients were treated with 57.6 Gy equivalent (GyE)/16 fractions, and 8, with 64.0 GyE/16 fractions.

Results: The median follow-up period was 43 months for all patients. The 3-year local control and loco-regional control rates for all patients were 76.9% (95% confidence interval [CI]=56.7-97.1%) and 61.3% (95% CI=38.5-84.1%), respectively. The 3-year overall survival and disease-specific survival rates were 59.1% (95% CI=38.6-79.6%) and 65.6% (95% CI=44.9-86.3%), respectively. Acute reactions of grade 3 of the skin and mucosa were observed in 2 and 4 patients, respectively. Late reactions included lateral visual loss (5 patients), mucosal ulceration (1 patient), and brain necrosis with clinical symptoms (1 patient). In the 5 patients who developed visual loss, the optic nerve was close to the tumour.

Conclusions: CIRT was effective and generally safe for locally advanced sinonasal adenocarcinoma.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.radonc.2014.09.009DOI Listing
October 2014

Atypical radiological and intraoperative findings of acute cerebral hemorrhage caused by ruptured cerebral aneurysm in a patient with severe chronic anemia.

J Nippon Med Sch 2014 ;81(4):264-8

Department of Neurosurgery, Nippon Medical School.

Acute intracerebral hemorrhage (ICH) associated with mild anemia is commonly observed on radiological examination, and there are several reports of ruptured aneurysms occurring with ICH but without accompanying subarachnoid hemorrhage. However, the relationship among computed tomography (CT), magnetic resonance imaging (MRI), and intraoperative findings of ICH caused by ruptured cerebral aneurysm in patients with severe chronic anemia has been rarely reported and is poorly understood. Here, we report atypical radiological and intraoperative findings of acute ICH caused by ruptured cerebral aneurysm in a patient with severe chronic anemia. A 64-year-old man with anemia was admitted to our hospital after he experienced left hemiparesis and a disturbance of consciousness. At a referring institution, he showed evidence of macrocytic anemia (white blood cell count, 9,000/μL; red blood cell count, 104×10(4)/μL; hemoglobin, 4.0 g/dL; hematocrit, 12.2%; and platelet count, 26.6×10(4)/μL). Both CT and MRI showed a right frontal ICH. The outer ring of the hematoma appeared as low-density area on CT, a low-intensity area on T1-weighted MRI, and a high-intensity area on T2-weighted MRI with a serous component. The patient received a blood transfusion and underwent surgical removal of the hematoma the following day. The white serous effusion visualized with CT and MRI was identified as a blood clot in the hematoma cavity. The blood that leaks from blood vessels appears as a high-intensity area on CT because it undergoes plasma absorption in a solidification shrinkage process, and is, therefore, concentrated. Although we did not examine the white effusion to determine if serous components were present, we speculated that the effusion may have contained serous components. Therefore, we removed the part of the effusion that appeared as a low-density area on CT. The presence of ICH without subarachnoid hemorrhage suggested the possible adhesion and rupture of a previous aneurysm. Therefore, ICH appeared as a mixed density area on CT because bleeding may have occurred several times. Because radiological findings of ICH caused by ruptured cerebral aneurysm in patients with severe chronic anemia are similar to those of ICH and cerebral edema, we suggest that the atypical radiological findings of ICH caused by ruptured cerebral aneurysm in patients with severe chronic anemia should be carefully evaluated, especially when surgery is indicated.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1272/jnms.81.264DOI Listing
April 2015

Application of regenerative medical technology using tissue-engineered cell sheets for endoscopic submucosal dissection of esophageal neoplasms.

Dig Endosc 2015 Jan 28;27(2):182-8. Epub 2014 Oct 28.

Department of Surgery, Institute of Gastroenterology, Tokyo Women's Medical University, Tokyo, Japan; Institute of Advanced Biomedical Engineering and Science, Tokyo Women's Medical University, Tokyo, Japan.

We have developed a technique for endoscopic transplantation of cultured autologous oral mucosal epithelial cell sheets to an esophageal ulcer following endoscopic submucosal dissection (ESD). The epithelial cell sheets successfully prevented esophageal stricture after ESD. Key technology is that epithelial cell sheets cultured from oral mucosal tissue and attached proteins can be harvested using cell sheet technology and can be transplanted to a wound site without the use of adhesive material. This regenerative procedure can promote the epithelialization of ulceration safely and effectively. In the near future, the development of advanced endoscopic treatment of regenerative medicine shows promise.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/den.12354DOI Listing
January 2015

Analysis of DNA methylation in bowel lavage fluid for detection of colorectal cancer.

Cancer Prev Res (Phila) 2014 Oct 19;7(10):1002-10. Epub 2014 Aug 19.

Department of Molecular Biology, Sapporo Medical University, Sapporo, Japan.

Aberrant DNA methylation could potentially serve as a biomarker for colorectal neoplasms. In this study, we assessed the feasibility of using DNA methylation detected in bowel lavage fluid (BLF) for colorectal cancer screening. A total of 508 BLF specimens were collected from patients with colorectal cancer (n = 56), advanced adenoma (n = 53), minor polyp (n = 209), and healthy individuals (n = 190) undergoing colonoscopy. Methylation of 15 genes (miR-1-1, miR-9-1, miR-9-3, miR-34b/c, miR-124-1, miR-124-2, miR-124-3, miR-137, SFRP1, SFRP2, APC, DKK2, WIF1, LOC386758, and ZNF582) was then analyzed in MethyLight assays, after which receiver operating characteristic (ROC) curves were analyzed to assess the diagnostic performance of BLF methylation. Through analyzing BLF specimens in a training set (n = 345), we selected the three genes showing the greatest sensitivity for colorectal cancer detection (miR-124-3, 71.8%; LOC386758, 79.5%; and SFRP1, 74.4%). A scoring system based on the methylation of those three genes (M-score) achieved 82% sensitivity and 79% specificity, and the area under the ROC curve (AUC) was 0.834. The strong performance of this system was then validated in an independent test set (n = 153; AUC = 0.808). No significant correlation was found between M-score and the clinicopathologic features of the colorectal cancers. Our results demonstrate that DNA methylation in BLF specimens may be a useful biomarker for the detection of colorectal cancer.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1158/1940-6207.CAPR-14-0162DOI Listing
October 2014

Effects of the dose-volume relationship on and risk factors for maxillary osteoradionecrosis after carbon ion radiotherapy.

Radiat Oncol 2014 Apr 3;9(1):92. Epub 2014 Apr 3.

Research Center for Charged Particle Therapy Hospital, National Institute of Radiological Sciences, Chiba, Japan.

Background: Osteoradionecrosis (ORN) is a critical complication after carbon ion (C-ion) or photon radiotherapy (RT) for head and neck tumors. However, the risk factors for ORN after C-ion RT remain unclear. Therefore, the present study aimed to investigate the effects of the dose-volume relationship on and risk factors for ORN development after C-ion RT. We, however, focused on the maxillary bone because most tumors treated with C-ion RT were primarily located in the sinonasal cavity.

Methods: The patients enrolled in this study received more than 10% of the prescribed total dose of 57.6 Gy equivalent (GyE) in 16 fractions to their maxilla. All patients were followed up for more than 2 years after C-ion RT. Those with tumor invasion to the maxilla before C-ion RT or local recurrence after the treatment were excluded from the study to accurately evaluate the effects of irradiation on the bone. Sixty-three patients were finally selected. The severity of ORN was assessed according to the Common Terminology Criteria for Adverse Events version 4.0. The correlation between clinical and dosimetric parameters and ORN incidence was retrospectively analyzed.

Results: The median follow-up period was 79 months. Of the 63 enrolled patients, 26 developed ORN of grade ≥1. Multivariate analysis revealed that the maxilla volume receiving more than 50 GyE (V50) and the presence of teeth within the planning target volume were significant risk factors for ORN. Dose-volume histogram analysis revealed that V10 to V50 parameters were significantly higher in patients with ORN than in those without ORN.

Conclusions: V50 and the presence of teeth within the planning target volume were independent risk factors for the development of ORN after C-ion RT using a 16-fraction protocol.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/1748-717X-9-92DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3992144PMC
April 2014

Feasibility of 4D flow MR imaging of the brain with either Cartesian y-z radial sampling or k-t SENSE: comparison with 4D Flow MR imaging using SENSE.

Magn Reson Med Sci 2014 31;13(1):15-24. Epub 2014 Jan 31.

Department of Radiology, Nippon Medical School.

Purpose: A drawback of time-resolved 3-dimensional phase contrast magnetic resonance (4D Flow MR) imaging is its lengthy scan time for clinical application in the brain. We assessed the feasibility for flow measurement and visualization of 4D Flow MR imaging using Cartesian y-z radial sampling and that using k-t sensitivity encoding (k-t SENSE) by comparison with the standard scan using SENSE.

Materials And Methods: Sixteen volunteers underwent 3 types of 4D Flow MR imaging of the brain using a 3.0-tesla scanner. As the standard scan, 4D Flow MR imaging with SENSE was performed first and then followed by 2 types of acceleration scan-with Cartesian y-z radial sampling and with k-t SENSE. We measured peak systolic velocity (PSV) and blood flow volume (BFV) in 9 arteries, and the percentage of particles arriving from the emitter plane at the target plane in 3 arteries, visually graded image quality in 9 arteries, and compared these quantitative and visual data between the standard scan and each acceleration scan.

Results: 4D Flow MR imaging examinations were completed in all but one volunteer, who did not undergo the last examination because of headache. Each acceleration scan reduced scan time by 50% compared with the standard scan. The k-t SENSE imaging underestimated PSV and BFV (P < 0.05). There were significant correlations for PSV and BFV between the standard scan and each acceleration scan (P < 0.01). The percentage of particles reaching the target plane did not differ between the standard scan and each acceleration scan. For visual assessment, y-z radial sampling deteriorated the image quality of the 3 arteries.

Conclusion: Cartesian y-z radial sampling is feasible for measuring flow, and k-t SENSE offers sufficient flow visualization; both allow acquisition of 4D Flow MR imaging with shorter scan time.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.2463/mrms.2013-0008DOI Listing
November 2014
-->