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Biomedical subjects

Y Ebihara

Publications and source records attributed to Y Ebihara.

At least 55 records · Page 3Linked to original sources
PubMed2000

Natural killer cell depletion by anti-asialo GM1 antiserum treatment enhances human hematopoietic stem cell engraftment in NOD/Shi-scid mice.

The scid mutation was backcrossed on to the NOD/Shi mouse background, resulting in the development of NOD/Shi-scid mice, which showed lack of mature lymphocytes, macrophage dysfunction and absence of circulating complement, but were not as impaired in natural killer (NK) cell activity as NOD/LtSz-scid mice. We then examined the effect of recipient NK cell depletion by anti-asialo GM1 antiserum on the repopulation of human cord blood (CB) hematopoietic stem cells (HSC) in NOD/Shi-scid mice to clarify the role of recipient NK cells in human HSC engraftment. The anti-asialo GM1 antiserum treatment significantly enhanced the engraftment of CB CD34+ cells, but did not affect the differentiation of the engrafted HSC into each hematopoietic lineage. The NK cell depletion was effective at early stages of the engraftment, but not 3 weeks after the transplantation. The anti-asialo GM1 antiserum treatment did not improve the engraftment by human HSC in scid mice which lack mature lymphocytes, but show neither macrophage dysfunction nor a reduction in circulating complement, indicating that macrophages and/or complement also have roles in HSC graft rejection. The present study indicates that the preconditioning targeting of recipient NK cells in addition to T cell suppression and myeloablation might prevent HSC graft failure, and that NOD/Shi-scid mice treated with anti-asialo GM1 antiserum could provide a useful tool for evaluating the repopulating ability of transplantable human HSC.

Animals↗
PubMed2000

Exclusive expression of G-CSF receptor on myeloid progenitors in bone marrow CD34+ cells.

Although granulocyte colony-stimulating factor (G-CSF) has been reported to act on cells of neutrophilic lineage, the administration of G-CSF to induce the mobilization of various haematopoietic progenitors into the circulation. We analysed the expression of receptors for G-CSF (G-CSFR) on human bone marrow and G-CSF-mobilized peripheral blood CD34+ cells, and examined the proliferation and differentiation capabilities of sorted CD34+G-CSFR+ and CD34+G-CSFR- cells using methylcellulose clonal culture. Flow cytometric analysis showed that G-CSFR was expressed on 14.9 +/- 4.9% of bone marrow CD34+ cells, most of which were included in CD34+CD33+ and CD34+CD38+ cell fractions. In clonal cultures, CD34+G-CSFR+ cells produced only myeloid colonies, whereas CD34+G-CSFR- cells produced erythroid bursts, megakaryocyte and multilineage colonies. When incubated with the cytokine cocktail for 5 d, CD34+G-CSFR- cells generated CD34+G-CSFR+ myeloid progenitors. In G-CSF-mobilized peripheral blood, CD34+ cells contained 10.8 +/- 5.8% of G-CSFR+ cells, most of which were also myeloid progenitors, although CD34+G-CSFR- cells contained a substantial number of myeloid progenitors. These results indicated that the expression of G-CSFR on CD34+ cells is restricted to myeloid progenitors, suggesting that the specific activity of G-CSF on myelopoiesis depends on the exclusive expression of its receptor on myeloid progenitors, and that the mobilization of various haematopoietic progenitors is not a direct effect of G-CSF in humans.

Adult↗
PubMed2000

Detection of telomerase activity in biopsy specimens for diagnosis of biliary tract cancers.

BACKGROUND: Telomerase activity is detectable in more than 85% of primary cancers. We determined telomerase activity in biopsy specimens obtained from biliary tract cancers to evaluate the clinical application of telomerase activity detection in combination with p53 immunostaining and routine histologic examination. METHODS: Biopsy specimens obtained during percutaneous transhepatic cholangioscopy from 13 patients with cholangiocarcinoma, 3 patients with gallbladder carcinoma, and 4 patients with intrahepatic bile duct stones were evaluated by routine histologic examination, p53 immunostaining, and telomerase activity. Semiquantitative determination of telomerase activity was performed using a fluorescence-based telomeric repeat amplification protocol. RESULTS: Thirteen of 16 specimens of malignant tissue had detectable telomerase activity, whereas no specimen of nonmalignant tissue had detectable telomerase activity. A p53 overexpression was recognized by immunostaining in 9 of 16 samples with cancers. Combining both telomerase activity and p53 overexpression resulted in the detection of all cancer with a sensitivity of 100%. There were no false-positive results by either modality (specificity 100%). CONCLUSIONS: The detection of telomerase activity in biopsy specimens and p53 overexpression in combination with routine histologic examination may improve the diagnosis of biliary tract cancers. (Gastrointest Endosc 2000;52:380-6).

Aged↗
PubMed2000

Intraperitoneal injection of dextran sulfate as an anti-adherent drug for the prevention of peritoneal metastasis of cancer shows low toxicity in animals.

Intraperitoneal dextran sulfate with a mean molecular weight of 5 x 10(5) has been developed for use in an anti-adherent therapy against peritoneal carcinomatosis. The present study examined acute toxicity of i.p. injection of dextran sulfate in mice and rabbits. The 10, 50 and 90% lethal dose values are 0.213 (0.146-0.252), 0.336 (0.291-0.405) and 0.530 mg/g (0.431-0.873 mg/g: 95% confidence interval) in mice, respectively. These are markedly larger than the efficacious dose of 0.005-0.01 mg/g obtained previously. Death or symptoms of intoxication were seen within 3 days after administration of toxic doses. Rabbits received i.p. injection of dextran sulfate at 0.02 mg/g, which was close to the efficacious dose. At 2, 4, 6, 8 and 13 days after administration, blood was taken for biochemical and hematological analyses. Dextran sulfate at 0.02 mg/g induced no remarkable abnormal findings. These results suggest that the i.p. dextran sulfate is safe as an anti-adherent agent against peritoneal metastasis of cancer.

Animals↗
PubMed2000

Helicobacter pylori may have only a transient presence in the oral cavity and on the surface of oral cancer.

We used the reverse transcription polymerase chain reaction (RT-PCR) and culture methods to study the presence of Helicobacter pylori in the gastric and oral samples from a total of 116 gastritis and peptic ulcer patients, including 58 with oral cancer. Detection rates of H. pylori were 46.6% in stomach samples and 12.1% in oral swab samples. All of the oral cancer surface swab samples were positive for H. pylori, as were their gastric samples suggesting that oral H. pylori derived from the stomach. The culture supernatants of Streptococcus mutans and Prevotella intermedia inhibited the growth of the H. pylori strain and caused the formation of the coccal form. In cases where H. pylori was detected in the oral cavity samples, including the oral cancer surface samples, it was believed that this species had colonized the stomach and were present in the oral cavity only as a transient organism.

Bacterial Proteins↗
PubMed2000

Human tissue distribution of TA02, which is homologous with a new type of aspartic proteinase, napsin A.

The N-terminal amino acid sequence of TA02 (molecular weight 35.0 kDa, isoelectric point 5.29), which is associated with primary lung adenocarcinoma, was determined and a fragment peptide was used to generate mouse monoclonal antibodies (mAbs) against TA02. The amino acid sequence suggested that TA02 might be homologous with napsin A, a new type of aspartic proteinase. In this context, we confirmed the expression of napsin A in primary lung adenocarcinoma using reverse-transcription polymerare chain reaction (RT-PCR) and showed that the TA02 mAbs reacted with glutathione-S-transferase (GST)-napsin A fusion protein. We concluded that TA02 is the same molecule as napsin A, and showed immunohistochemically that it is distributed mainly in type II pneumocytes, alveolar macrophages, renal tubules and exocrine glands and ducts in the pancreas. In particular, type II pneumocytes and alveolar macrophages showed high expression of TA02 among human normal tissues. In primary lung adenocarcinoma, 47 out of 58 (81.0%) primary lesions were positive. All well-differentiated adenocarcinomas except those of goblet cell type showed high expression of TA02. In addition, two out of seven (28.6%) large cell carcinomas showed low expression of TA02. The other histopathological types of primary lung cancer did not express TA02 at all. A few cases of renal cell cancer, pancreatic cancer, breast cancer, thyroid cancer, colon cancer and ovarian cancer showed low expression, but the staining patterns were completely different from that of primary lung adenocarcinoma, which showed a granular staining pattern. Our novel mAbs should be valuable for immunochemical detection of TA02/napsin A.

Adenocarcinoma↗
PubMed2000

Expansion of human NOD/SCID-repopulating cells by stem cell factor, Flk2/Flt3 ligand, thrombopoietin, IL-6, and soluble IL-6 receptor.

Here, we demonstrate a significant ex vivo expansion of human hematopoietic stem cells capable of repopulating in NOD/SCID mice. Using a combination of stem cell factor (SCF), Flk2/Flt3 ligand (FL), thrombopoietin (TPO), and a complex of IL-6 and soluble IL-6 receptor (IL-6/sIL-6R), we cultured cord blood CD34(+) cells for 7 days and transplanted these cells into NOD/SCID mice. Bone marrow engraftment was judged successful when recipient animals contained measurable numbers of human CD45(+) cells 10-12 weeks after transplantation. When cells were cultured with SCF+FL+TPO+IL-6/sIL-6R, 13 of 16 recipients were successfully engrafted, and CD45(+) cells represented 11.5% of bone marrow cells in engrafted recipients. Cells cultured with a subset of these factors were less efficiently engrafted, both as measured by frequency of successful transplantations and prevalence of CD45(+) cells. In animals receiving cells cultured with all 4 factors, human CD45(+) cells represented various lineages, including a large number of CD34(+) cells. The proportion of CD45(+) cells in recipient marrow was 10 times higher in animals receiving these cultured cells than in those receiving comparable numbers of fresh CD34(+) cells, and the expansion rate was estimated at 4.2-fold by a limiting dilution method. Addition of IL-3 to the cytokine combination abrogated the repopulating ability of the expanded cells. The present study may provide a novel culture method for the expansion of human transplantable hematopoietic stem cells suitable for clinical applications.

Animals↗
PubMed2000

Hematopoietic repopulating ability of cord blood CD34(+) cells in NOD/Shi-scid mice.

Although umbilical cord blood (CB) is increasingly being used as an alternative to bone marrow (BM) as a source of transplantable hematopoietic stem cells (HSC), information on the hematopoietic repopulating ability of CB HSC is still limited. We recently established a xenotransplantation system in NOD/Shi-scid mice to evaluate human stem cell activity. In the present study, we transplanted 5 to 10 x 10(4) CB CD34(+) cells into six NOD/Shi-scid mice treated with anti-asialo GM1 antiserum to investigate the hematopoietic repopulating ability of CB. The BM of all recipients contained human CD45(+) cells 10 to 12 weeks after the transplantation (43.8 +/- 17.7%). Clonal culture of the recipient BM cells revealed the formation of various types of human hematopoietic colonies, including myelocytic, erythroid, megakaryocytic, and multilineage colonies, indicating that CB HSC can differentiate into hematopoietic progenitors of various lineages. However, the extent of the differentiation and maturation differed with each lineage. CD13(+)/CD14(+)/CD33(+) myelocytic cells were mainly repopulated in BM and peripheral blood (PB). While CD41(+) megakaryocytic cells and platelets were present, few glycophorin A(+)CD71(+) or hemoglobin alpha-containing erythroid cells were detected. CD19(+) B cells were the most abundantly repopulated in NOD/Shi-scid mice, but their maturational stage differed among the hematopoietic organs. Most of the BM CD19(+) cells were immature B cells expressing CD10 but not surface immunoglobulin (Ig) M, whereas more mature CD19(+)CD10(-) surface IgM(+) B cells were predominantly present in spleen and PB. CD3(+) T cells were not detected even in the recipient thymus. The transplantation to the NOD/Shi-scid mouse may provide a useful tool for evaluating the repopulating ability of transplantable human HSC.

Animals↗
PubMed2000

Massive pericardial and pleural effusion with anasarca following allogeneic bone marrow transplantation.

A 10-year-old girl presented with massive pericardial/pleural effusion with anasarca 216 days after an allogeneic bone marrow transplantation from her HLA-matched sibling for relapsed acute lymphoblastic leukemia. She did not show any other symptoms of chronic graft-versus-host disease (GVHD). The antinucleolar antibody was elevated in the blood and the pleural fluid. The lymphocytes in the fluid were mostly CD8+/HLA-DR+, and a majority of CD8+ cells in the blood expressed CD57. These data suggested that she had chronic GVHD. Immunosuppressive therapy including prednisolone, cyclosporin A, high-dose methylprednisolone, tacrolimus (FK506), and methotrexate had no effect, and the patient died of Aspergillus pneumonia 183 days after the presentation of the disease. Although it has not been described before, isolated serositis with edema should be recognized as a clinical feature of chronic GVHD.

Antigens, CD↗
PubMed2000

[Diffuse large B-cell lymphoma showing CNS invasion by CD30-positive multinuclear giant cells mimicking the clinical features of progressive multifocal leukoencephalopathy].

We report a 55-year-old woman with diffuse large B-cell lymphoma showing central nervous system (CNS) infiltration by CD30-positive lymphoma cells. The patient was admitted with pleural effusion, ascites and a large mass in the abdominal cavity. Southern blot analysis of DNA extracted from the ascites revealed IgJH rearrangement, and therefore she was initially diagnosed as B-cell neoplasia. She received combined chemotherapy (DICE and CHOP regimens), and achieved a transient clinical response. Three months later, she developed various neurological abnormalities, and brain magnetic resonance imaging revealed diffuse infiltration of the cerebral white matter. We considered the possibility of CNS involvement by the lymphoma or progressive multifocal leukoencephalopathy (PML), and began a course of anti-virus therapy and radiation therapy. Because multiple lumbar punctures demonstrated large multinuclear lymphoma cells in the cerebrospinal fluid, a diagnosis of metastatic CNS lymphoma was made. Immunohistochemistry revealed that these lymphoma cells were reactive with anti-CD30 antibody. Although the radiation therapy was temporarily effective against the CNS involvement, the patient died of systemic invasion of the lymphoma cells. The final diagnosis was diffuse large B-cell lymphoma on the basis of pathologic findings, immunohistochemistry, and Southern blot analysis using a mesenteric lymph node obtained at autopsy. Cytospin preparations and immunohistochemistry of specimens obtained from frequent lumbar punctures were useful for differentiating CNS lymphoma from PML.

Brain Neoplasms↗
PubMed1999

Expression of stromal cell-derived factor-1/pre-B cell growth-stimulating factor receptor, CXC chemokine receptor 4, on CD34+ human bone marrow cells is a phenotypic alteration for committed lymphoid progenitors.

We found that the stromal cell-derived factor-1/pre-B cell growth-stimulating factor receptor, CXC chemokine receptor 4 (CXCR4), is expressed on human CD34+ bone marrow (BM) cells. Stringently FACS-sorted CD34+CXCR4+ BM cells completely lack myeloid, erythroid, megakaryocytic, and mixed colony-forming potential (myeloid progenitors), but give rise to B and T lymphoid progenitors, whereas CD34+CXCR4- BM cells can generate colonies formed by myeloid progenitors and can also develop into these lymphoid progenitors. Therefore, expression of CXCR4 on CD34+ BM cells can allow lymphoid progenitors to be discriminated from myeloid progenitors. Because CD34+CXCR4+ cells are differentiated from CD34+CXCR4- cells, multipotential progenitors located in the BM are likely to be negative for CXCR4 expression. CXCR4 seems to be expressed earlier than the IL-7R and terminal deoxynucleotidyl transferase during early lymphohemopoiesis. These results suggest that the expression of CXCR4 on CD34+ BM cells is one of the phenotypic alterations for committed lymphoid progenitors.

Adult↗
PubMed1999

Differential effects of human granulocyte colony-stimulating factor (hG-CSF) and thrombopoietin on megakaryopoiesis and platelet function in hG-CSF receptor-transgenic mice.

Granulocyte-colony stimulating factor (G-CSF) has been found to act on the neutrophilic lineage. We recently showed that human G-CSF (hG-CSF) has effects similar to early-acting cytokines such as interleukin-3 (IL-3) in the development of multipotential hematopoietic progenitors in transgenic (Tg) mice expressing receptors (R) for hG-CSF. In the present study, we examined the effects of hG-CSF on more mature hematopoietic cells committed to megakaryocytic lineage in these Tg mice. The administration of hG-CSF to the Tg mice increased the numbers of both platelets in peripheral blood and megakaryocytes in the spleen, indicating that hG-CSF stimulates megakaryopoiesis in the Tg mice in vivo. The stimulatory effect of hG-CSF was also supported by the results of studies in vitro. hG-CSF supported megakaryocyte colony formation in a dose-dependent fashion in clonal cultures of bone marrow cells derived from the Tg mice. Direct effects of hG-CSF on megakaryocytic progenitors in the Tg mice were confirmed by culture of single-cell sorted from bone marrow cells. hG-CSF showed a stronger effect on maturation of megakaryocytes in the Tg mice than that of IL-3 alone, but weaker than that of TPO alone. In addition, hG-CSF induced phosphorylation of STAT3 but not Jak2 or STAT5, while TPO induced phosphorylation of both. In contrast to TPO, hG-CSF did not enhance ADP-induced aggregation. Thus, hG-CSF has a wide variety of functions in megakaryopoiesis of hG-CSFR-Tg mice, as compared with other megakaryopoietic cytokines, but the activity of hG-CSF in megakaryocytes and platelets does not stand up to a comparison with that of TPO. Specific signals may be required for the full maturation and activation of platelets.

Animals↗
PubMed1999

Expression of E2A-HLF chimeric protein induced T-cell apoptosis, B-cell maturation arrest, and development of acute lymphoblastic leukemia.

The E2A-HLF fusion gene, generated by t(17;19)(q22;p13) in acute lymphoblastic leukemia (ALL), encodes a chimeric transcription factor in which the trans-activating domains of E2A are fused to the DNA-binding and dimerization domains of hepatic leukemic factor (HLF). To investigate its biological role, we generated transgenic mice expressing E2A-HLF using Ig enhancer and promoter, which direct transgene expression in cells committed to the lymphoid lineage. The transgenic mice exhibited abnormal development in the thymus and spleen and were susceptible to infection. The thymus contained small numbers of thymocytes, and TUNEL staining showed that higher population of thymocytes were undergoing apoptosis. The spleen exhibited a marked reduction in splenic lymphocytes and the flow cytometric analyses and the in vitro colony formation assays showed that the B-cell maturation was blocked at a very early developmental stage. These findings indicated that the expression of E2A-HLF induced T-cell apoptosis and B-cell maturation arrest in vivo and that the susceptibility of the transgenic mice to infection was due to immunodeficiency. Moreover, several transgenic mice developed acute leukemia, classified as T-ALL based on the surface marker analysis and DNA rearrangements, suggesting that an additional event is required for malignant transformation of lymphoid cells expressing E2A-HLF. Our findings provide insight into the biological function of E2A-HLF in lymphoid development and also its role in leukemogenesis.

Adenovirus E2 Proteins↗
PubMed1999

Soluble interleukin-6 (IL-6) receptor with IL-6 stimulates megakaryopoiesis from human CD34(+) cells through glycoprotein (gp)130 signaling.

We have recently shown that stimulation of glycoprotein (gp) 130, the membrane-anchored signal transducing receptor component of IL-6, by a complex of human soluble interleukin-6 receptor (sIL-6R) and IL-6 (sIL-6R/IL-6), potently stimulates the ex vivo expansion as well as erythropoiesis of human stem/progenitor cells in the presence of stem cell factor (SCF). Here we show that sIL-6R dose-dependently enhanced the generation of megakaryocytes (Mks) (IIbIIIa-positive cells) from human CD34(+) cells in serum-free suspension culture supplemented with IL-6 and SCF. The sIL-6R/IL-6 complex also synergistically acted with IL-3 and thrombopoietin (TPO) on the generation of Mks from CD34(+) cells, whereas the synergy of IL-6 alone with TPO was barely detectable. Accordingly, the addition of sIL-6R to the combination of SCF + IL-6 also supported a substantial number of Mk colonies from CD34(+) cells in serum-free methylcellulose culture, whereas SCF + IL-6 in the absence of sIL-6R rarely induced Mk colonies. The addition of monoclonal antibodies against gp130 to the suspension and clonal cultures completely abrogated the megakaryopoiesis induced by sIL-6R/IL-6 in the presence of SCF, whereas an anti-TPO antibody did not, indicating that the observed megakaryopoiesis by sIL-6R/IL-6 is a response to gp130 signaling and independent of TPO. Furthermore, human CD34(+) cells were subfractionated into two populations of IL-6R-negative (CD34(+) IL-6R-) and IL-6R-positive (CD34(+) IL-6R+) cells by fluorescence-activated cell sorting. The CD34(+) IL-6R- cells produced a number of Mks as well as Mk colonies in cultures supplemented with sIL-6R/IL-6 or TPO in the presence of SCF. In contrast, CD34(+) IL-6R+ cells generated much less Mks and lacked Mk colony forming activity under the same conditions. Collectively, the present results indicate that most of the human Mk progenitors do not express IL-6R, and that sIL-6R confers the responsiveness of human Mk progenitors to IL-6. Together with the presence of functional sIL-6R in human serum and relative unresponsiveness of human Mk progenitors to IL-6 in vitro, current results suggest that the role of IL-6 may be mainly mediated by sIL-6R, and that the gp130 signaling initiated by the sIL-6R/ IL-6 complex is involved in human megakaryopoiesis in vivo.

Antigens, CD↗
PubMed1999

Loss of protection by hypoxic preconditioning in aging Fischer 344 rat hearts related to myocardial glycogen content and Na+ imbalance.

OBJECTIVES: The objective of this study was to determine whether hypoxic preconditioning (HP) could lessen the myocardial increase in [Na+]i, thus protecting the aging myocardium against ischemia. BACKGROUND: A decrease in ischemic tolerance with aging is associated with an accelerated increase in [Na+]i during ischemia. Ischemic preconditioning fails to protect the middle-aged and senescent myocardium against ischemia. METHODS: Isolated hearts of young adult (12-week-old), middle-aged (50-week-old) and senescent (100-week-old) Fischer 344 rats were subjected to 25 min of ischemia with or without HP followed by 30 min of reperfusion. Left ventricular (LV) function, myocardial energy metabolites and [Na+]i were measured. RESULTS: In the older groups, the recovery of LV function and high-energy phosphates (HEPs) was lower with an increased release of creatine kinase (CK) during reperfusion than in the young group. The increased [Na+]i at the end of ischemia was greater in the former groups than in the young group. HP decreased myocardial glycogen and lessened the increased [Na+]i in the young group, resulting in an improved recovery of LV function and HEPs, as well as decreased CK release. However, the levels of glycogen before HP in the older groups were higher than in the young group and its levels after HP were similar to that before HP in the young group. HP did not affect the [Na+]i, exacerbated CK release and inhibited the recovery of LV function and HEPs in the older groups. CONCLUSIONS: HP failed to lessen the increased [Na+]i or to protect the aging hearts, probably due to the preexistence of increased glycogen level.

Aging↗
PubMed1999

Amyloid deposition in renal angiomyolipoma.

An irregular tumor shadow, seen on the left kidney in a 48-year-old woman by computed tomography, was pathologically diagnosed as 'angiomyolipoma'. HMB-45 immunoreactivity distinguished angiomyolipoma from renal cell carcinoma of sarcomatoid type. The amyloid deposition was limited to the tumor. M-protein and Bence-Jones protein were negative. For amyloid protein characterization, immunohistochemical studies were performed with antiamyloid A, anti-kappa, anti-lambda, anti-prealbumin and anti-beta-2 microglobulin, but none reacted with the amyloid. This is the first documented case of amyloid deposition in angiomyolipoma and may represent a novel precursor protein of amyloid.

Amyloid↗
PubMed1999

A retroperitoneal bronchogenic cyst with malignant change.

A unique case of adenocarcinoma arising in a retroperitoneal bronchogenic cyst is presented. A 55-year-old woman presented with lower abdominal discomfort. Computed tomography revealed a retroperitoneal cystic mass attached to the ascending colon. The resected cyst was unilocular and filled with milky white mucus and hemorrhagic debris. Histologically, most of the cyst wall was of well-differentiated papillary adenocarcinoma with no cyst wall invasion. Other small areas of the cyst were lined with variably atypical dysplastic/metaplastic cuboidal to pseudostratified columnar epithelium. The cyst wall was mostly hyalinized, but there was apparent thickened subepithelial basement membrane, elastosis, and a single layer of smooth muscle that suggested bronchial wall structures. A mucin staining study with O-acylated sialic acid, which is used for the demonstration of gastrointestinal, cholecystic and uterine cervical mucins, was negative for the mucin-producing epithelial cells of the cyst. Thus, to our knowledge, this is the first reported case of adenocarcinoma arising in a retroperitoneal bronchogenic cyst.

Adenocarcinoma↗