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1.  Primary adenocarcinoma of the stomach in von Recklinghausen's disease with high serum levels of multiple tumor markers: a case report 
Gastric tumors in patients affected by neurofibromatosis type 1 are usually carcinoids or stromal tumors, and rarely adenocarcinomas.
Case presentation
We report a case of an adenocarcinoma of the stomach in a 53-year-old Japanese man with neurofibromatosis type 1. An abdominal computed tomography scan and ultrasonography showed tumors in his liver. Gastric fibroscopy revealed a Borrmann type III tumor on his cardia that had spread to his esophagus and was highly suspicious for malignancy. Multiple biopsies showed an adenocarcinoma of the stomach, which was evaluated as gastric cancer, stage IV. Chemotherapy with TS-1 was performed. Our patient died four weeks after initial admission. Histological examination of a liver needle biopsy showed metastatic adenocarcinoma in his liver.
To the best of our knowledge, high serum levels of α-fetoprotein, carcinoembryonic antigen, and carbohydrate antigen 72-4, resulting from gastric adenocarcinoma, have not been reported previously in a patient with neurofibromatosis type 1. We report this rare case along with a review of the literature.
PMCID: PMC3212996  PMID: 22018031
2.  Inhibition of Quorum Sensing in Serratia marcescens AS-1 by Synthetic Analogs of N-Acylhomoserine Lactone▿  
Applied and Environmental Microbiology  2007;73(20):6339-6344.
Quorum sensing is a regulatory system for controlling gene expression in response to increasing cell density. N-Acylhomoserine lactone (AHL) is produced by gram-negative bacteria, which use it as a quorum-sensing signal molecule. Serratia marcescens is a gram-negative opportunistic pathogen which is responsible for an increasing number of serious nosocomial infections. S. marcescens AS-1 produces N-hexanoyl homoserine lactone (C6-HSL) and N-(3-oxohexanoyl) homoserine lactone and regulates prodigiosin production, swarming motility, and biofilm formation by AHL-mediated quorum sensing. We synthesized a series of N-acyl cyclopentylamides with acyl chain lengths ranging from 4 to 12 and estimated their inhibitory effects on prodigiosin production in AS-1. One of these molecules, N-nonanoyl-cyclopentylamide (C9-CPA), had a strong inhibitory effect on prodigiosin production. C9-CPA also inhibited the swarming motility and biofilm formation of AS-1. A competition assay revealed that C9-CPA was able to inhibit quorum sensing at four times the concentration of exogenous C6-HSL and was more effective than the previously reported halogenated furanone. Our results demonstrated that C9-CPA was an effective quorum-sensing inhibitor for S. marcescens AS-1.
PMCID: PMC2075062  PMID: 17675425
3.  Recombinant anti-podoplanin (NZ-1) immunotoxin for the treatment of malignant brain tumors 
Current study demonstrates the glioma tumor antigen podoplanin to be present at very high levels (>90%) in both glioblastoma (D2159MG, D08-0308MG, and D08-0493MG) and medulloblastoma (D283MED, D425MED, and DAOY) xenografts and cell line. We constructed a novel recombinant single-chain antibody variable region fragment (scFv), NZ-1, specific for podoplanin from the NZ-1 hybridoma. NZ-1-scFv was then fused to Pseudomonas exotoxin A, carrying a C-terminal KDEL peptide (NZ-1-PE38KDEL). The immunotoxin was further stabilized by a disulfide (ds) bond between the heavy-chain and light-chain variable regions as the construct NZ-1-(scdsFv)-PE38KDEL. NZ-1-(scdsFv)-PE38KDEL exhibited significant reactivity to glioblastoma and medulloblastoma cells. The affinity of NZ-1-(scdsFv), NZ-1-(scdsFv)-PE38KDEL and NZ-1 antibody, for podoplanin peptide was 2.1×10−8 M, 8.0×10−8 M, and 3.9×10−10 M, respectively. In a protein stability assay, NZ-1-(scdsFv)-PE38KDEL retained 33-98% of its activity while that of NZ-1-PE38KDEL declined to 13% of its initial levels after incubation at 37°C for 3 days. In vitro cytotoxicity of the NZ-1-(scdsFv)-PE38KDEL was measured in cells isolated from glioblastoma xenografts, D2159MG, D08-0308MG, D08-0493MG, and in the medulloblastoma D283MED, D425MED, and DOAY xenografts and cell line. The NZ-1-(scdsFv)-PE38KDEL immunotoxin was highly cytotoxic, with an IC50 in the range of 1.6–29 ng/mL. Significantly, NZ-1-(scdsFv)-PE38KDEL demonstrated tumor-growth delay, averaging 24 days (P<0.001) and 21 days (P<0.001) in D2159MG and D283MED in vivo tumor models, respectively. Crucially, in the D425MED intracranial tumor model, NZ-1-(scdsFv)-PE38KDEL caused a 41% increase in survival (P≤0.001). In preclinical studies, NZ-1-(scdsFv)-PE38KDEL exhibited significant potential as a targeting agent for malignant brain tumors.
PMCID: PMC3809846  PMID: 23115013
podoplanin; glioblastoma multiforme; medulloblastoma; recombinant immunotoxin; single-chain disulfide Fv
4.  Dexamethasone Palmitate Ameliorates Macrophages-Rich Graft-versus-Host Disease by Inhibiting Macrophage Functions 
PLoS ONE  2014;9(5):e96252.
Macrophage infiltration of skin GVHD lesions correlates directly with disease severity, but the mechanisms underlying this relationship remain unclear and GVHD with many macrophages is a therapeutic challenge. Here, we characterize the macrophages involved in GVHD and report that dexamethasone palmitate (DP), a liposteroid, can ameliorate such GVHD by inhibiting macrophage functions. We found that host-derived macrophages could exacerbate GVHD in a mouse model through expression of higher levels of pro-inflammatory TNF-α and IFN-γ, and lower levels of anti-inflammatory IL-10 than resident macrophages in mice without GVHD. DP significantly decreased the viability and migration capacity of primary mouse macrophages compared to conventional dexamethasone in vitro. DP treatment on day 7 and day 14 decreased macrophage number, and attenuated GVHD score and subsequent mortality in a murine model. This is the first study to provide evidence that therapy for GVHD should be changed on the basis of infiltrating cell type.
PMCID: PMC4012982  PMID: 24806147
5.  Applying spatial epidemiology to hematological disease using R: a guide for hematologists and oncologists 
“Spatial statistics” is an academic field that deals with the statistical analysis of spatial data, and has been applied to econometrics and various other policy fields. These methods are easily applied by hematologists and oncologists using better and much less expensive software. To encourage physicians to use these methods, this review introduces the methods and demonstrates the analyses using R and FleXScan, which can be freely downloaded from the website, with sample data. It is demonstrated that spatial analysis can be used by physicians to analyze hematological diseases. In addition, applying the technique presented to the investigation of patient prognoses may enable generation of data that are also useful for solving health policy-related problems, such as the optimal distribution of medical resources.
PMCID: PMC3949695  PMID: 24624001
leukemia; malignant lymphoma; Tango’s index; spatial regression model
6.  A Single-Cell and Feeder-Free Culture System for Monkey Embryonic Stem Cells 
PLoS ONE  2014;9(2):e88346.
Primate pluripotent stem cells (PSCs), including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), hold great potential for research and application in regenerative medicine and drug discovery. To maximize primate PSC potential, a practical system is required for generating desired functional cells and reproducible differentiation techniques. Much progress regarding their culture systems has been reported to date; however, better methods would still be required for their practical use, particularly in industrial and clinical fields. Here we report a new single-cell and feeder-free culture system for primate PSCs, the key feature of which is an originally formulated serum-free medium containing FGF and activin. In this culture system, cynomolgus monkey ESCs can be passaged many times by single-cell dissociation with traditional trypsin treatment and can be propagated with a high proliferation rate as a monolayer without any feeder cells; further, typical PSC properties and genomic stability can be retained. In addition, it has been demonstrated that monkey ESCs maintained in the culture system can be used for various experiments such as in vitro differentiation and gene manipulation. Thus, compared with the conventional culture system, monkey ESCs grown in the aforementioned culture system can serve as a cell source with the following practical advantages: simple, stable, and easy cell maintenance; gene manipulation; cryopreservation; and desired differentiation. We propose that this culture system can serve as a reliable platform to prepare primate PSCs useful for future research and application.
PMCID: PMC3915054  PMID: 24505480
7.  Tet3 CXXC Domain and Dioxygenase Activity Cooperatively Regulate Key Genes for Xenopus Eye and Neural Development 
Cell  2012;151(6):1200-1213.
Ten-Eleven Translocation (Tet) family of dioxygenases offers a new mechanism for dynamic regulation of DNA methylation and has been implicated in cell lineage differentiation and oncogenesis. Yet their functional roles and mechanisms of action in gene regulation and embryonic development are largely unknown. Here, we report that Xenopus Tet3 plays an essential role in early eye and neural development by directly regulating a set of key developmental genes. Tet3 is an active 5mC hydroxylase regulating the 5mC/5hmC status at target gene promoters. Biochemical and structural studies further reveal a novel DNA binding mode of the Tet3 CXXC domain that is critical for specific Tet3 targeting. Finally, we show that the enzymatic activity and CXXC domain are crucial for Tet3’s biological function. Together, these findings define Tet3 as a novel transcription factor and reveal a molecular mechanism by which the 5mC hydroxylase and DNA binding activities of Tet3 cooperate to control target gene expression and embryonic development.
PMCID: PMC3705565  PMID: 23217707
8.  Isocitrate dehydrogenase 2 mutation is a frequent event in osteosarcoma detected by a multi-specific monoclonal antibody MsMab-1 
Cancer Medicine  2013;2(6):803-814.
Somatic mutations of isocitrate dehydrogenase (IDH) 1 and IDH2 occur in gliomas, acute myeloid leukemia, and cartilaginous tumors. Somatic mosaic IDH1/2 mutations are also reported in Ollier disease and Maffucci syndrome, which are characterized by multiple central cartilaginous tumors. Although IDH1/2 mutation analysis against osteosarcoma has been performed in several studies, no IDH1/2 mutation has been reported. Herein, we newly report the IDH2-R172S mutation in three of 12 (25%) osteosarcoma patients, which was detected by direct DNA sequencing. No monoclonal antibody (mAb) has been reported against IDH2-R172S mutation. However, we demonstrate that the IDH2-R172S peptide was recognized by our established multi-specific anti-mutated IDH1/2 mAb, MsMab-1, in enzyme-linked immunosorbent assay. Western blot analysis revealed that MsMab-1 reacts with PA tag combined recombinant proteins of IDH2-R172S. Furthermore, MsMab-1 stained IDH2-R172S-expressing osteosarcoma tissues in immunohistochemistry. The MsMab-1 stained nine of 32 (28.1%) osteosarcomas in a tissue microarray. This report is the first describing IDH2 mutations in osteosarcoma, which can be detected by MsMab-1 mAb. Taken together, these results show that MsMab-1 can be anticipated for use in immunohistochemical determination of IDH1/2 mutation-bearing osteosarcoma.
PMCID: PMC3892385  PMID: 24403254
Isocitrate dehydrogenase 1; isocitrate dehydrogenase 2; monoclonal antibody; mutations; osteosarcoma
9.  Effects of KIR ligand incompatibility on clinical outcomes of umbilical cord blood transplantation without ATG for acute leukemia in complete remission 
Blood Cancer Journal  2013;3(11):e164-.
To clarify the effect of killer cell immunoglobulin-like receptor (KIR) ligand incompatibility on outcomes of acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) patients in complete remission after single cord blood transplantation (CBT), we assessed the outcomes of CBT registered in the Japan Society for Hematopoietic Cell Transplantation (JSHCT) database. A total of 643 acute leukemia (357 AML and 286 ALL) patient and donor pairs were categorized according to their KIR ligand incompatibility by determining whether or not they expressed HLA-C, Bw4 or A3/A11 by DNA typing. A total of 128 patient–donor pairs were KIR ligand-incompatible in the graft-versus-host (GVH) direction and 139 patient–donor pairs were incompatible in the host-versus-graft (HVG) direction. Univariate and multivariate analyses showed no significant differences between the KIR ligand-incompatible and compatible groups in the GVH direction for both AML and ALL patients of overall survival, disease-free survival, relapse incidence, non-relapse mortality and acute GVH disease. However, KIR incompatibility in the HVG direction ameliorated engraftment in ALL patients (hazard ratio 0.66, 95% confidence interval 0.47–0.91, P=0.013). Therefore, there were no effects of KIR ligand incompatibility in the GVH direction on single CBT outcomes for acute leukemia patients without anti-thymocyte globulin use. However, it is necessary to pay attention to KIR incompatibility in the HVG direction for engraftment.
PMCID: PMC3880445  PMID: 24292416
10.  Targeted Gene Deletion of miRNAs in Mice by TALEN System 
PLoS ONE  2013;8(10):e76004.
Mice are among the most valuable model animal species with an enormous amount of heritage in genetic modification studies. However, targeting genes in mice is sometimes difficult, especially for small genes, such as microRNAs (miRNAs) and targeting genes in repeat sequences. Here we optimized the application of TALEN system for mice and successfully obtained gene targeting technique in mice for intergenic region and series of microRNAs. Microinjection of synthesized RNA of TALEN targeting each gene in one cell stage of embryo was carried out and injected oocytes were transferred into pseudopregnant ICR female mice, producing a high success rate of the targeted deletion of miRNA genes. In our condition, TALEN RNA without poly(A) tail worked better than that of with poly(A) tail. This mutated allele in miRNA was transmitted to the next generation, suggesting the successful germ line transmission of this targeting method. Consistent with our notion of miRNAs maturation mechanism, in homozygous mutant mice of miR-10a, the non- mutated strand of miRNAs expression was completely diminished. This method will lead us to expand and accelerate our genetic research using mice in a high throughput way.
PMCID: PMC3797721  PMID: 24146809
11.  Detection of Human Herpesviruses (HHVs) in Semen of Human Male Infertile Patients 
Recently we demonstrated an ectopic expression of the human herpesvirus 1 thymidine kinase (HHV1-TK) gene by functioning of an intrinsic endogenous promoter in the transgenic rat (TG-rat), suggesting that HHV1 infection in humans induces expression of the TK gene with the ectopic promoter in the testis and results in accumulation of HHV1-TK protein, triggering male infertility similar to that in the TG-rat. Hence, in this study, we started to investigate a relationship between infection of herpesvirus and human male infertility. Semen was donated by Chinese male infertile patients (153 men, aged 21–49 years) with informed consent, followed by DNA preparation and analysis by PCR and DNA sequencing. Semen volume, sperm number and density, and sperm motility were examined. DNAs of HHV1, HHV4, HHV5 and HHV6 were confirmed by PCR, electrophoresis and DNA sequencing. Finally, virus DNA was identified in 59 patients (39%). The number of carriers was 39 (25%) for HHV1, 6 (4%) for HHV4, 33 (22%) for HHV5 and 3 (2%) for HHV6, respectively. Moreover, double-infection was found in 22 out of 59 specimens (37%), most of which were double-infection of HHV1 and HHV5 (15 out of 22 carriers). Though slight severity was present in some of the carriers, the relationship between virus infection and sperm impairment was not conclusive. Accordingly, it is essential to examine whether the viral HHV1-TK gene is expressed in the testis of the infertile human HHV carrier.
PMCID: PMC3934121  PMID: 23748714
Human herpesvirus (HHV); Human; Male infertility; Spermatogenesis; Thymidine kinase
12.  Clinical efficacy of abatacept compared to adalimumab and tocilizumab in rheumatoid arthritis patients with high disease activity 
Clinical Rheumatology  2013;33:39-47.
Favourable clinical results in rheumatoid arthritis (RA) patients with high disease activity (HDA) are difficult to achieve. This study evaluated the clinical efficacy of abatacept according to baseline disease activity compared to adalimumab and tocilizumab. This study included all patients registered in a Japanese multicenter registry treated with abatacept (n = 214), adalimumab (n = 175), or tocilizumab (n = 143) for 24 weeks. Clinical efficacy of abatacept in patients with HDA (DAS28-CRP > 4.1) and low and moderate disease activity was compared. Clinical efficacy of abatacept, adalimumab, and tocilizumab was compared in patients with HDA at baseline. In patients treated with abatacept, multivariate logistic regression identified HDA at baseline as an independent predictor for achieving low disease activity (LDA; DAS28-CRP < 2.7) [OR 0.26, 95 % CI 0.14–0.50] or remission (DAS28-CRP < 2.3) [OR 0.26, 95 % CI 0.12–0.56] at 24 weeks. In patients with HDA at baseline, logistic regression did not identify treatment with adalimumab or tocilizumab as independent predictors of LDA or remission compared to abatacept. Retention rates based on insufficient efficacy were significantly higher in patients treated with abatacept compared to adalimumab and lower than tocilizumab. Retention rates based on adverse events in patients treated with abatacept were significantly lower compared to tocilizumab. Clinical efficacy of abatacept was affected by baseline disease activity. There were no significant differences between the three different classes of biologics regarding clinical efficacy for treating RA patients with HDA, although definitive conclusions regarding long-term efficacy will require further research.
PMCID: PMC3890049  PMID: 24057092
Abatacept; Adalimumab; High disease activity; Japanese multicenter registry system; Rheumatoid arthritis; Tocilizumab
13.  Structured self‐monitoring of blood glucose reduces glycated hemoglobin in insulin‐treated diabetes 
The aim of the preset study was to investigate the effectiveness of structured self‐monitoring of blood glucose (SMBG) in insufficiently controlled insulin‐treated diabetes. A total of 86 insulin‐treated patients were randomized to a routine testing group (RTG; n = 43) and a structured testing group (STG; n = 43). The STG used a chart to record seven‐point blood glucose (BG) profile on three consecutive days per month. The primary end‐point was the glycated hemoglobin (HbA1c) at 3 months and 6 months. There were no significant differences of HbA1c between the RTG and STG at 3 months. However, the STG had significantly improved HbA1c at 6‐month follow‐up compared with the RTG (P = 0.002). In the STG, HbA1c decreased by 0.5% from 7.9 (SD 0.5) to 7.4 (0.7)%, whereas it decreased by 0.1% in the RTG from 7.9 (0.5) to 7.8 (0.7)%. In the STG, 55% of the patients were willing to continue structured SMBG and they achieved a 0.7% decrease of HbA1c. The present findings suggest that structured SMBG significantly improves glycemic control.
PMCID: PMC4025113  PMID: 24843694
Glycemic control; Insulin‐treated diabetes; Self‐monitoring of blood glucose
14.  Frequency of elevated biomarkers in patients with cryptogenic hepatocellular carcinoma 
The incidence of hepatocellular carcinoma (HCC) continues to increase in Japan, but the clinical characteristics of Japanese patients with HCC have not been well described. The aim of this study was to determine the frequencies and utilities of elevated α-fetoprotein (AFP) and des-gamma-carboxy prothrombin (DCP) levels as biomarkers in cryptogenic HCC.
A total of 2638 patients with HCC diagnosed between 1999 and 2010 in the Nagasaki Association Study of Liver (NASLD) were recruited for this study. The cause of HCC was categorized into 4 groups; HCC-B, HCC-C, HCC-BC, and HCC-nonBC. The significance of factors was examined for HCC-nonBC using logistic regression analysis in all patients.
Multivariate analysis identified age, sex, BMI, alcohol consumption, platelet count, AST, ALT, AFP, DCP, and TNM stage as independent and significant risk factors for HCC-nonBC. According to TNM stage, the median AFP levels in HCC-nonBC with TNM stages I, II, and III were significantly lower than in either HCC-B or HCC-C. In TNM stage IV, the median AFP level in HCC-nonBC was significantly lower than in either HCC-B or HCC-BC. The median DCP levels in HCC-nonBC with TNM stages I and II were significantly higher than those in either HCC-B or HCC-C. In TNM stage III, the median DCP level in HCC-nonBC was significantly higher than that in HCC-C.
DCP was more sensitive than AFP for the diagnosis of early stage cryptogenic HCC. DCP should be used as the main serum test for cryptogenic HCC detection.
PMCID: PMC3775616  PMID: 24008520
15.  Extension of lifespan and protection against oxidative stress by an antioxidant herb mixture complex (KPG-7) in Caenorhabditis elegans 
Excessive generation of reactive oxygen species within cells results in oxidative stress. Furthermore, accumulation of reactive oxygen species has been shown to reduce cell longevity. Many dietary supplements are believed to have anti-aging effects. The herb mixture KPG-7 contains several components with antioxidant activity. We aim to clarify the mechanisms responsible for the antioxidant activity of KPG-7 and to establish whether KPG-7 has an anti-aging effect. We examined whether dietary supplementation with KPG-7 could provide protection against oxidative stress, extend lifespan, and delay aging in Caenorhabditis elegans (C. elegans). We found that KPG-7 extended lifespan and delayed aging in adult C. elegans. The expression of oxidation resistance 1 protein was induced by juglone and this effect was significantly suppressed in KPG-7-treated. In addition, the amount of oxidized protein was significantly lower in KPG-7-treated worms than untreated worms. Furthermore, locomotive activity was increased in C. elegans at 3 days of age following the treatment with KPG-7. On the other hand, the level of cellular ATP was lower at 3 days of age in worms treated with KPG-7 than in untreated worms. KPG-7 increases lifespan and delays aging in C. elegans, well corresponding to its activity to protect against oxidative stress.
PMCID: PMC3774924  PMID: 24062604
oxidative stress; C. elegans; aging; herb mixture; antioxidant
16.  1α,25-Dihydroxyvitamin D3 Modulates the Hair-Inductive Capacity of Dermal Papilla Cells: Therapeutic Potential for Hair Regeneration 
This study examined the effects and signaling pathways associated with the actions of 1α,25-dihydroxyvitamin D3 (VD3) on dermal papilla cells (DPCs). Results suggest that VD3 may promote functional differentiation of DPCs and be useful in preserving the hair follicle-inductive capacity of cultured DPCs for hair regeneration therapies.
Dermal papilla cells (DPCs) have the potential to induce differentiation of epithelial stem cells into hair, and Wnt signaling is deeply involved in the initiation process. The functional limitation of expanded adult DPCs has been a difficult challenge for cell-based hair regrowth therapy. We previously reported that 1α,25-dihydroxyvitamin D3 (VD3) upregulates expression of transforming growth factor (TGF)-β2 and alkaline phosphatase (ALP) activity, both features of hair-inducing human DPCs (hDPCs). In this study, we further examined the effects and signaling pathways associated with VD3 actions on DPCs. VD3 suppressed hDPC proliferation in a dose-dependent, noncytotoxic manner. Among the Wnt-related genes investigated, Wnt10b expression was significantly upregulated by VD3 in hDPCs. Wnt10b upregulation, as well as upregulation of ALPL (ALP, liver/bone/kidney) and TGF-β2, by VD3 was specific in hDPCs and not detected in human dermal fibroblasts. Screening of paracrine or endocrine factors in the skin indicated that all-trans retinoic acid (atRA) upregulated Wnt10b gene expression, although synergistic upregulation (combined atRA and VD3) was not seen. RNA interference with vitamin D receptor (VDR) revealed that VD3 upregulation of Wnt10b, ALPL, and TGF-β2 was mediated through the genomic VDR pathway. In a rat model of de novo hair regeneration by murine DPC transplantation, pretreatment with VD3 significantly enhanced hair folliculogenesis. Specifically, a greater number of outgrowing hair shafts and higher maturation of regenerated follicles were observed. Together, these data suggest that VD3 may promote functional differentiation of DPCs and be useful in preserving the hair follicle-inductive capacity of cultured DPCs for hair regeneration therapies.
PMCID: PMC3659730  PMID: 23197867
Cell culture; Cell signaling; Cellular therapy; Clinical translation; Gene expression; Signal transduction; Stem cell-microenvironment interactions; Tissue regeneration
17.  Use of in-vitro experimental results to model in-situ experiments: bio-denitrification under geological disposal conditions 
SpringerPlus  2013;2:339.
Some of the low level radioactive wastes from reprocessing of spent nuclear fuels contain nitrates. Nitrates can be present in the form of soluble salts and can be reduced by various reactions. Among them, reduction by metal compounds and microorganisms seems to be important in the underground repository. Reduction by microorganism is more important in near field area than inside the repository because high pH and extremely high salt concentration would prevent microorganism activities. In the near field, pH is more moderate (pH is around 8) and salt concentration is lower. However, the electron donor may be limited there and it might be the control factor for microorganism's denitrification activities. In this study, in-vitro experiments of the nitrate reduction reaction were conducted using model organic materials purported to exist in underground conditions relevant to geological disposal. Two kinds of organic materials were selected. A super plasticizer was selected as being representative of the geological disposal system and humic acid was selected as being representative of pre-existing organic materials in the bedrock. Nitrates were reduced almost to N2 gas in the existence of super plasticizer. In the case of humic acids, although nitrates were reduced, the rate was much lower and, in this case, dead organism was used as an electron donor instead of humic acids. A reaction model was developed based on the in-vitro experiments and verified by running simulations against data obtained from in-situ experiments using actual groundwaters and microorganisms. The simulation showed a good correlation with the experimental data and contributes to the understanding of microbially mediated denitrification in geological disposal systems.
PMCID: PMC3755790  PMID: 24010028
Geological disposal; Nitrates; Denitrification; Microorganism; Modeling
18.  Comparison of Barium and Arsenic Concentrations in Well Drinking Water and in Human Body Samples and a Novel Remediation System for These Elements in Well Drinking Water 
PLoS ONE  2013;8(6):e66681.
Health risk for well drinking water is a worldwide problem. Our recent studies showed increased toxicity by exposure to barium alone (≤700 µg/L) and coexposure to barium (137 µg/L) and arsenic (225 µg/L). The present edition of WHO health-based guidelines for drinking water revised in 2011 has maintained the values of arsenic (10 µg/L) and barium (700 µg/L), but not elements such as manganese, iron and zinc. Nevertheless, there have been very few studies on barium in drinking water and human samples. This study showed significant correlations between levels of arsenic and barium, but not its homologous elements (magnesium, calcium and strontium), in urine, toenail and hair samples obtained from residents of Jessore, Bangladesh. Significant correlation between levels of arsenic and barium in well drinking water and levels in human urine, toenail and hair samples were also observed. Based on these results, a high-performance and low-cost adsorbent composed of a hydrotalcite-like compound for barium and arsenic was developed. The adsorbent reduced levels of barium and arsenic from well water in Bangladesh and Vietnam to <7 µg/L within 1 min. Thus, we have showed levels of arsenic and barium in humans and propose a novel remediation system.
PMCID: PMC3689667  PMID: 23805262
19.  Expression of Membrane Complement Regulatory Proteins Crry and CD55 in Normal Rats 
Journal of Toxicologic Pathology  2013;26(2):223-226.
Some anticancer therapeutic antibodies are designed to act through complement-dependent cytotoxicity (CDC). It has been reported that there are many membrane complement regulatory proteins (mCRPs) that inhibit CDC. In the present study, we examined the expression of two mCRPs, the complement receptor 1-related gene/protein Y (Crry) and the decay-accelerating factor CD55, in three normal rats by immunohistochemistry. Crry and CD55 were detected widely in rat organs and tissues. Crry was found mainly in the urinary, digestive, respiratory, immunohematopoietic, circulatory and neuroendocrine systems. CD55 was found in the urinary, digestive and neuroendocrine systems. However, the two molecules were expressed in separate cells within the same organ. These results suggest that the distribution of mCRPs is related to the strict regulation of CDC activation in these organs and tissues and that the two molecules have a nonoverlapping expression pattern, a fact indicating specific roles in CDC regulation.
PMCID: PMC3695346  PMID: 23914066
membrane complement regulatory proteins; Crry; CD55; rat
20.  Novel Cell Culture-Adapted Genotype 2a Hepatitis C Virus Infectious Clone 
Journal of Virology  2012;86(19):10805-10820.
Although the recently developed infectious hepatitis C virus system that uses the JFH-1 clone enables the study of whole HCV viral life cycles, limited particular HCV strains have been available with the system. In this study, we isolated another genotype 2a HCV cDNA, the JFH-2 strain, from a patient with fulminant hepatitis. JFH-2 subgenomic replicons were constructed. HuH-7 cells transfected with in vitro transcribed replicon RNAs were cultured with G418, and selected colonies were isolated and expanded. From sequencing analysis of the replicon genome, several mutations were found. Some of the mutations enhanced JFH-2 replication; the 2217AS mutation in the NS5A interferon sensitivity-determining region exhibited the strongest adaptive effect. Interestingly, a full-length chimeric or wild-type JFH-2 genome with the adaptive mutation could replicate in Huh-7.5.1 cells and produce infectious virus after extensive passages of the virus genome-replicating cells. Virus infection efficiency was sufficient for autonomous virus propagation in cultured cells. Additional mutations were identified in the infectious virus genome. Interestingly, full-length viral RNA synthesized from the cDNA clone with these adaptive mutations was infectious for cultured cells. This approach may be applicable for the establishment of new infectious HCV clones.
PMCID: PMC3457305  PMID: 22787209
21.  Anti-Thy-1 Antibody-mediated Complement-dependent Cytotoxicity is Regulated by the Distribution of Antigen, Antibody and Membrane Complement Regulatory Proteins in Rats 
Some therapeutic antibodies as anticancer agents exert their effects through the host immune system, but the factors that predict their cytotoxicity, including complement-dependent cytotoxicity (CDC), are unclear. In the present study, we attempted to elucidate some of these factors in a preclinical model. CDC-related mesangiolysis caused by administration of the anti-Thy-1.1 antibody can be studied in the rat anti-Thy-1 glomerulonephritis model, so the model was used in this study. Three animals each were sacrificed at 0.5, 1, 8, 24 and 48 hours after i.v. administration of the anti-Thy-1.1 antibody at 1mg/kg. The distribution of the Thy-1.1 antigen and 2 membrane complement regulatory proteins (mCRPs), Crry and CD55, in three non-treated animals and the distribution of the injected antibody and C3 in the model was studied by immunohistochemistry. In the mesangial cells of the kidney, both expression of the antigen and distribution of the antibody with C3 deposition were observed with weak expression of mCRPs. There was also antigen and antibody distribution in the medullary cells of the adrenal gland and in the lymphocytes of the thymus but no C3 deposition, which was thought to be related to high expression of mCRPs. The antigen was observed in several other organs and tissues without distribution of the antibody. Cell death was only observed in the mesangial cells. These results clearly demonstrate that activation of CDC is regulated by several factors, such as distribution of the target molecule, antibody distribution and the balance among the molecules of the CDC cascade and mCRPs.
PMCID: PMC3620213  PMID: 23723567
antibody; complement-dependent cytotoxicity; antigen; membrane complement regulatory protein; rat anti-Thy-1 glomerulonephritis model
22.  Factors Predictive of Adverse Events Associated with Endoscopic Ultrasound-Guided Fine Needle Aspiration of Pancreatic Solid Lesions 
Digestive Diseases and Sciences  2013;58(7):2093-2099.
Endoscopic ultrasound-guided fine needle aspiration (EUS-FNA) provides high diagnostic accuracy with a low incidence of procedural complications. However, it occasionally causes serious complications, and factors that increase the susceptibility to such adverse events remain unknown.
We aimed to examine post-procedural events and determine risk factors associated with EUS-FNA of pancreatic solid lesions.
This single-center retrospective study included 316 consecutive patients with pancreatic solid lesions who underwent 327 EUS-FNA procedures from April 2003 to September 2011. We registered all patients undergoing EUS-FNA in the database and retrospectively ascertained the presence/absence of post-procedural adverse events.
The incidence of post-procedural adverse events, including moderate to mild pancreatitis, mild abdominal pain, and mild bleeding, was 3.4 %. Univariate analysis showed that the incidence of post-procedural events was significantly increased in patients with tumors less than or equal to 20 mm in diameter (P < 0.001), those with pancreatic neuroendocrine tumors (PNET) (P = 0.012), and patients who had intervening normal pancreas for accessing the lesion (P = 0.048). Multivariate analysis identified tumors measuring less than or equal to 20 mm in diameter (OR 18.48; 95 % CI 3.55–96.17) and case of PNETs (OR 36.50; 95 % CI 1.73–771.83) were an independent risk factors.
EUS-FNA of pancreatic solid lesions is a safe procedure. However, pancreatic lesions with small diameters and pancreatic neuroendocrine tumors are important factors associated with adverse events after EUS-FNA.
PMCID: PMC3695684  PMID: 23423501
EUS-FNA; Adverse events; Pancreatitis; Risk factor; Pancreatic cancer; Pancreatic neuroendocrine tumor
23.  Recombinant human soluble thrombomodulin administration improves sepsis-induced disseminated intravascular coagulation and mortality: a retrospective cohort study 
Thrombosis Journal  2013;11:3.
Early treatment of disseminated intravascular coagulation (DIC) can be associated with improved patient outcomes. The Japanese Ministry of Health and Welfare (JMHW) and the International Society on Thrombosis and Haemostasis (ISTH) criteria are the most specific for diagnosis of septic DIC. The revised Japanese Association for Acute Medicine (JAAM) criteria are able to diagnose sepsis-induced DIC in the early stage. Recombinant human soluble thrombomodulin (rhTM) has recently been used for treating DIC. Previous studies have shown a benefit of using rhTM for D,IC diagnosed by the JMHW or ISTH criteria, but not the JAAM criteria. The purpose of this study was to sequentially evaluate coagulation biomarkers and the DIC score after giving rhTM treatment to patients with sepsis-induced DIC diagnosed according to the JAAM criteria.
We performed a retrospective cohort study. Critically ill patients were included if diagnosed with sepsis-induced DIC according to the JAAM criteria. They were either treated without rhTM (control group) or with rhTM (treatment group). The primary outcome was the DIC score on day 7. The secondary outcome was 28-day mortality from the start of DIC treatment. Changes in the results of coagulation tests were assessed over time from the start of treatment to day 7.
Twelve and 23 patients were assigned to the treatment and control groups, respectively. The DIC score on day 7 was significantly higher in the treatment group (3.3 ± 1.4) than in the control group (4.9 ± 1.8, p < 0.05). Estimated survival showed lower in treatment group than control group. There was significant difference between the control group and the treatment group (p < 0.05). The D-dimer level on day 7 was significantly lower in the treatment group (7.5 ± 4.1 μg/mL) than in the control group (30.9 ± 33.6 μg/mL, p < 0.05). Life-threatening bleeding did not occur. Our results indicated that rhTM improved sepsis-induced DIC and mortality.
Recombinant human soluble thrombomodulin may improve sepsis-induced DIC diagnosed according to the JAAM criteria without an increased bleeding risk.
PMCID: PMC3599946  PMID: 23414216
Disseminated intravascular coagulation; Sepsis; Thrombomodulin; Intensive care unit; Critically ill patient; Anticoagulant; Multiple organ failure; DIC score; JAAM
24.  Acute kidney injury and inflammatory immune reconstitution syndrome in mixed genotype (A/E) hepatitis B virus co-infection in HIV-associated lymphoma 
We report a first case of HIV-associated lymphoma (HAL) presenting with acute kidney injury (AKI) and inflammatory immune reconstitution syndrome (IRIS). A 39-year-old male, treated with nonsteroidal anti-inflammatory drugs (NSAIDs) for one month prior to admission, developed AKI, left testicular tumor, and recurrent swelling of the right parotid gland. A resected testicular tumor exhibited features intermediate between diffuse large B-cell lymphoma and Burkitt lymphoma. Renal biopsy showed hydro-degeneration of renal tubules, interstitial inflammatory cells, and a small number of lymphoma cells in the sub-capsule, compatible with acute interstitial nephritis. His renal dysfunction rapidly recovered following chemotherapy and combination antiretroviral therapy (cART). He developed pneumonia concomitantly with a decrease in HIV-RNA level and an increase in CD4+ cells after the first cycle of chemotherapy, which spontaneously resolved after the second cycle of chemotherapy without additional anti-infection drugs; thus, his pneumonia fulfilled the diagnostic criteria for IRIS. We suggest that IRIS may frequently develop during chemotherapy for HAL, but may be overlooked. He was coinfected with hepatitis B virus (HBV), which genotypes known as is associated with liver-related mortality and response to antiviral therapy; recently, an intimate interplay between HIV and HBV in the onset of lymphoma has been reported. Therefore, we addressed the HBV genotype in the patient. The analysis revealed that he exhibited a mixed genotype (A/E) not native to Japan and primarily found in Europe and North America or West Africa. These findings suggest that universal vaccination for juveniles against HBV is warranted in Japan.
PMCID: PMC3563184  PMID: 23411777
Hepatitis B virus; HIV; genotype; immune reconstitution inflammatory syndrome; acute kidney injury; ptosis
25.  CSN5 specifically interacts with CDK2 and controls senescence in a cytoplasmic cyclin E-mediated manner 
Scientific Reports  2013;3:1054.
The fifth component (CSN5) of the mammalian COP9 signalosome complex plays an essential role in cell proliferation and senescence, but its molecular mediator remains to be determined. Here, we searched for interactors among various cell cycle regulators, and found that CSN5, but not the CSN holo-complex, bound to CDK2 in vivo and in vitro. Depletion of CSN5 enhanced phosphorylation of CDK2 by Akt, resulting in cytoplasmic accumulation of CDK2 together with cyclin E in a leptomycin B-resistant manner, and impaired phosphorylation of the retinoblastoma protein. Additional knockdown of CDK2, which reduced the expression of cyclin E to the normal level, did not restore cell proliferation, but significantly suppressed senescence in CSN5-depleted cells. Enforced expression of cytoplasmic cyclin E induced premature senescence in immortalized cell lines. These results show that CSN5 functions through CDK2 to control premature senescence in a novel way, depending on cyclin E in the cytoplasm.
PMCID: PMC3542532  PMID: 23316279

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