Analysis of Developmental Changes in Avian DNA Methylation Using a Novel Method for Quantifying Genome-wide DNA Methylation

Individual differentiated somatic cells and undifferentiated stem cells have common genome, although their functions or morphological characters are very different. These differences are derived from difference of gene expression pattern. DNA methylation is generally key factor of Suppression of gene and its level is globally change during mammalian early development. But, in birds, whether genome-wide changes in DNA methylation occur during embryonic development is still unknown. Here, we show that genome-wide DNA methylation to assess occurrence during early chick embryonic development. We found that the methylation status at stage 1 was approximately 57%, after which it gradually decreases, reaching a minimum at stage 10 (33%). After stage 10, DNA methylation gradually increased. These results should contribute to clarify the epigenetic mechanisms in birds.ArticleJOURNAL OF POULTRY SCIENCE. 46(4): 286-290(2009)journal articl

A Novel Concentrating System of Chicken Stem Cells by Bone Marrow Side Population Cells

Numerous studies in mammalian species have recently been reported that many stem cells have an ability to efficiently efflux the vital DNA-binding dye Hoechst 33342, and it is called side population (SP) cells. However, few study have been reported on the avian SP cells. It could be possible that concentration of hematopoietic stem cells (HSCs) in birds since the characteristic of SIP cells should be shared in various tissues and species. In this study, we first attempted the isolation of SP cells from chicken bone marrow and the assessment by gene expression and morphologic analyses. Bone marrow cells (BMCs) were flushed from the femurs and tibias of chicks aged at 10 days with PBS. The BMCs were layered on lymphocyte separation medium and centrifuged for excluding the erythrocytes. The separated cells were adjusted to 10(6)/ml in HBSS. Hoechst 33342 were added (1.25 mu g/ml) and incubated 60 to 90 minutes at 37 degrees C. Propidium iodide was added (2 mu g/ml) to exclude dead cells. The SP cells were isolated with flow cytometer. The sorted cells were stained with May-Gruenwald Giemsa (MG) for morphological analysis and RNA was extracted for gene expression analysis. The avian SP cells which was vanished by addition verapamil counld be separated. The percentage of SP cells in chicken bone marrow was about 2.6%. The morphological analysis by MG staining indicated that the SP cells had a larger nuclear and little cytoplasm which were typical characterisation of mouse HSCs. The pattern of gene expressions (CD34, c-Kit, CD4 and CD8) in SP cells also resembled that of the mouse HSCs. These results suggested that the HSCs could be enriched from avian bone marrow cells. Together with these results, it was concluded that SP is one of powerful tools for concentration of avian stem cells.ArticleJOURNAL OF POULTRY SCIENCE. 47(1): 53-56(2010)journal articl

Depletion of Primordial Germ Cells (PGCs) by X-irradiation to Extraembryonic Region of Chicken Embryos and Expression of Xenotransplanted Quail PGCs

The generation of germline chimeras by the transfer of primordial germ cells (PGCs) requires incorporation of the PGCs of the donor into the gonadal tissue of the recipient embryo. We investigated the utility of soft x-irradiation with application of a lead (12-3 x 0.25 mm, similar to 0.1 g) shield to the embryo proper for the production of chicken-quail germline chimeras. Chicken embryos shielded during irradiation for 120 s (similar to 7.2 Gy) at stages 13 to 17 showed a hatchability of 35% (106/301). whereas the hatchability of unshielded embryos was 26% (27/105). The relative Population of gonadal PGCs Lit stage 30 for embryos irradiated at stage 13 with or Without shielding was 13 and 5%. respectively, of the value for nonirradiated controls. Chicken embryos irradiated at stages 13 or 14 with or Without shielding and transfused with quail embryonic blood containing PGCs each exhibited similar to 130 relative population of donor PGCs in the left gonad at stage 30. Xenotransplanted hatchlings exhibited donor-derived PGCs as detected by Southern hybridization and PCR. Exposure of chicken embryos to similar to 7.2 Gy of x-radiation at stage 13 with the application of a lead shield to the embryo proper is thus a feasible approach to depletion of endogenous germ cells and the production of chicken-quail germline chimeras.ArticleJOURNAL OF POULTRY SCIENCE. 46(2): 136-143(2009)journal articl

Clinical trials with artificial CO(2) bathings

パブ浴（人工炭酸浴剤浴）は，組織に酸素を供給し，また組織循環の改善により組織に賦活力を与え，さらに末梢毛細血管の拡張作用を有する。外来患者を対象に，末梢循環障害5例，高血圧症2例，不定愁訴症例2例を選び，半年より1年にわたって家庭でパブ浴を連浴させた。全例に，併用薬の減量，あるいは中止をみた。とくに蛋白尿を伴った高血圧症例では，血圧の正常化に伴って蛋白尿の減少をみた。炭酸泉の効果は連浴によっても変化を認めず，人工炭酸泉浴は家庭においても療養泉としての効果がえられ，疾病の自然予後に有為に作用する。Clinical trials for 5 cases of peripheral circulation insufficiency, 2 cases of hypertension, 2 cases of general malaise and 2 cases of liver function insufficiency were performed with artificial CO(2) baths using BUB-KAO, a 50 g sodium hydogencarbonate and succinic acid tablet, KAO Co. Ltd., Japan, in 150-litre-tub at 40℃ for 10-20 minutes in a period of 6 to 12 months. By virtue of an increase of tissue PO(2) and an improvement of tissue perfusion with artificial CO(2) baths, favorable effects were obtained in all cases but in 2 patients with liver function insufficiency, leading to reduce or discontinue the regular medications. Remarkable effect was observed in 2 cases of hypertension, being brought under control in 2 months, keeping normal range thereafter due possibly to lowering the peripheral resistance caused by capillary dilation effect. A greate favor of hypotensive effect was granted in reducing the excretion of urinary protein. It is very advantageous in maintaining homeostasis to ameliorate natural course of diseases with artificial C (2) baths which are effective by repeated routine use for a long period of time, leading to assist modern way of medical treatment

Roles of Macrophages in Advanced Liver Fibrosis, Identified Using a Newly Established Mouse Model of Diet-Induced Non-Alcoholic Steatohepatitis

Macrophages play critical roles in the pathogenesis of non-alcoholic steatohepatitis (NASH). However, it is unclear which macrophage subsets are critically involved in the development of inflammation and fibrosis in NASH. In TSNO mice fed a high-fat/cholesterol/cholate-based diet, which exhibit advanced liver fibrosis that mimics human NASH, we found that Kupffer cells (KCs) were less abundant and recruited macrophages were more abundant, forming hepatic crown-like structures (hCLS) in the liver. The recruited macrophages comprised two subsets: CD11c+/Ly6C−and CD11c− /Ly6C+ cells. CD11c+ cells were present in a mesh-like pattern around the lipid droplets, constituting the hCLS. In addition, CD11c+ cells colocalized with collagen fibers, suggesting that this subset of recruited macrophages might promote advanced liver fibrosis. In contrast, Ly6C+cells were present in doughnut-like inflammatory lesions, with a lipid droplet in the center. Finally, RNA sequence analysis indicates that CD11c+/Ly6C− cells promote liver fibrosis and hepatic stellate cell (HSC) activation, whereas CD11c−/Ly6C+ cells are a macrophage subset that play an anti-inflammatory role and promote tissue repair in NASH. Taken together, our data revealed changes in liver macrophage subsets during the development of NASH and shed light on the roles of the recruited macrophages in the pathogenesis of advanced fibrosis in NASH

Polarization-Sensitive Optical Coherence Tomographic Documentation of Choroidal Melanin Loss in Chronic Vogt–Koyanagi–Harada Disease

Purpose: Vogt–Koyanagi–Harada (VKH) disease is a systemic autoimmune disorder that affects organs with melanocytes. The sunset glow fundus (SGF) in VKH disease was evaluated with polarization-sensitive optical coherence tomography (PS-OCT).Methods: The study involved 28 eyes from 14 patients with chronic VKH disease, 21 eyes from 21 age-matched controls, and 22 eyes from 22 high-myopic patients with a tessellated fundus. VKH eyes were grouped into sunset or non-sunset groups on the basis of color fundus images. The presence of melanin in the choroid was determined by using the degree of polarization uniformity (DOPU) obtained by PS-OCT. The sunset glow index (SGI) was calculated by using color fundus images. Presence of an SGF was evaluated by using DOPU, SGI, subfoveal choroidal thicknesses, near-infrared images, and autofluorescence images at 488 nm (SW-AF) and 785 nm (NIR-AF).Results: There were 16 eyes in the sunset group and 12 eyes in the non-sunset group. For all eyes in the sunset group, the disappearance of choroidal melanin was clearly detected with PS-OCT. Percentage areas of low DOPU in the choroidal interstitial stroma of the sunset group were significantly lower than those of other groups and showed no overlap with other groups. The distribution of choroidal thicknesses and SGI in the sunset group substantially overlapped with other groups. The subjective analyses of the sunset and non-sunset groups, using near infrared, SW-AF, or NIR-AF, showed substantial inconsistencies with the PS-OCT results.Conclusions: PS-OCT provides an in vivo objective evaluation of choroidal melanin loss of the SGF in chronic VKH disease

Salvage Haploidentical Transplantation Using Low-dose ATG for Early Disease Relapse after First Allogeneic Transplantation: A Retrospective Single-center Review

Second allogeneic stem cell transplantation (allo-SCT) is a potentially curative therapy for patients who relapse after first allo-SCT. Human leukocyte antigen (HLA)-haploidentical related donors provide the broad opportunity to conduct second SCT at the appropriate time, but the efficacy of second SCT from haploidentical donors after relapse has not been established. We retrospectively analyzed the records of 33 patients who underwent second SCT. Twenty patients underwent haplo-SCT with low-dose antithymocyte globulin (ATG), and the other 13 patients underwent conventional- SCTs, including HLA-matched related peripheral blood, unrelated bone marrow or cord blood. Three years after the second SCT, the overall survival (OS) and progression-free survival (PFS) of all patients were 32.5% and 23.9%. Multivariate analyses indicated that non-complete response at second SCT, less than 1-year interval to relapse after first- SCT, and total score ≥ 3 on the hematopoietic cell transplantation-specific comorbidity index were significantly associated with a lower PFS rate. The haplo- and conventional- SCT groups showed equivalent results regarding OS, PFS, cumulative incidences of relapse, non-relapse mortality and graft-versus-host disease. The neutropenic period after transplantation was significantly shorter in haplo- SCT than conventional- SCT (10.5 days vs. 16 days, p=0.001). Our analysis revealed that haplo-SCT could be an alternative therapeutic option for relapsed patients after first SCT

Association Between PSCA Variants and Duodenal Ulcer Risk

Background: While duodenal ulcer (DU) and gastric cancer (GC) are both H. pylori infection-related diseases, individuals with DU are known to have lower risk for GC. Many epidemiological studies have identified the PSCA rs2294008 T-allele as a risk factor of GC, while others have found an association between the rs2294008 C-allele and risk of DU and gastric ulcer (GU). Following these initial reports, however, few studies have since validated these associations. Here, we aimed to validate the association between variations in PSCA and the risk of DU/GU and evaluate its interaction with environmental factors in a Japanese population. Methods: Six PSCA SNPs were genotyped in 584 DU cases, 925 GU cases, and 8,105 controls from the Japan Multi-Institutional Collaborative Cohort (J-MICC). Unconditional logistic regression models were applied to estimate odds ratios (ORs) and 95% confidence intervals (CIs) for the association between the SNPs and risk of DU/GU. Results: PSCA rs2294008 C-allele was associated with per allele OR of 1.34 (95% CI, 1.18–1.51; P = 2.28 × 10−6) for the risk of DU. This association was independent of age, sex, study site, smoking habit, drinking habit, and H. pylori status. On the other hand, we did not observe an association between the risk of GU and PSCA SNPs. Conclusions: Our study confirms an association between the PSCA rs2294008 C-allele and the risk of DU in a Japanese population

放送大学体育実技を受講した身体障害者の実態と体育・スポーツに対する意識

　本研究では、「放送大学身障者体育実技」を受講した全履修生を対象として、身障者のための体育実技に対する印象、現在行っている運動の実施状況、スポーツと運動に対する意識を明らかにすることを目的として意識調査を行った。　研究の対象は、放送大学で実施している身障者のための体育実技の受講生200名とし、調査用紙は、郵送にて2003年6月に配布し、同年7月31日までに回収した。回収できた114名（回収率57.0％）の回答結果をDr.SPSS Windows Ver 11.0を使用して統計処理した。　調査の結果から、以下の結論が得られた。1．日常的に運動を行っている者は少なかった。このことを考察した結果、地域で行われ、障害者が参加できる運動とスポーツについての詳細な情報を提供する必要性が明らかになった。2．放送大学の身障者のための体育実技を選択した理由には、講師である私たちのコンセプトが支持されていた。3．授業回数を増やすこと、および個人にあった運動量を設定することが求められていることが明らかになった。4．講義を受けた結果、運動やスポーツに対する、気持ち、考え方、行動は前向きに変化していた。このことから、これまでの体育やスポーツに対して抱いていた劣等感が減少した可能性が示唆された