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T Hotta

Publications and source records attributed to T Hotta.

At least 73 records · Page 4Linked to original sources
PubMed2000

Clonality analysis by methylation-specific PCR for the human androgen-receptor gene (HUMARA-MSP).

The human androgen-receptor gene (HUMARA) has been used for analysis of X chromosome inactivation (XCI) pattern because of a polymorphic short tandem repeat (STR) near the 5'-promoter region correlated with XCI. We introduce a novel method to analyze XCI pattern, named HUMARA methylation-specific PCR (HUMARA-MSP) assay, which analyzes methylation status of the HUMARA gene by bisulfite modification instead of a methylation-sensitive restriction enzyme. Although the original MSP method shows whether there is a methylated band or not, our HUMARA-MSP method identifies the patterns of methylated and unmethylated bands. Because this method identifies either unmethylated or methylated alleles in each PCR tube and shows opposite band patterns dependent on methylation status, we can assess the XCI pattern independently twice. This method can avoid false results by incomplete enzyme digestion and incomplete bisulfite modification will not affect the results. Extremely small quantities of samples, such as hematopoietic colonies, were also available for HUMARA-MSP assay. Because DNA modified by sodium bisulfite is also available for assessment of methylation status of other genes by setting specific primers for them, we performed the simultaneous assessment of clonality and aberrant hypermethylation of p15INK4B gene in myelodysplastic syndromes. These simultaneous assessments were easily possible and provided much information despite requiring only a small volume of DNA. The HUMARA-MSP assay may facilitate the analyses for pathogenesis of hematological disorders because of its simplicity, sensitivity and wide applicability. Leukemia (2000) 14, 207-212.

Base Sequence↗
PubMed2000

Methylation status of the p15INK4B gene in hematopoietic progenitors and peripheral blood cells in myelodysplastic syndromes.

We previously reported that the hypermethylation of the p15INK4B gene promoter was frequently observed in myelodysplastic syndromes (MDS), and that it may be associated with disease progression. An unanswered question is whether p15INK4B gene methylation is restricted to undifferentiated blastic cells, or whether differentiated cells such as granulocytes or erythrocytes of MDS origin also harbor this epigenetic alteration. In this study, we analyzed the methylation status of the p15INK4B gene in MDS by the methylation-specific PCR (MSP) method, which is more sensitive than Southern blotting. The bone marrow mononuclear cells (BM-MNCs) of 23 MDS patients were analyzed, and six of them showed p15INK4B methylation. Progenitor assay with methylcellulose medium was also performed in all patients. In two of the six patients with p15INK4B-methylated BM-MNCs, erythroid and/or non-erythroid colonies formed were subjected to molecular analysis. Colonies with and without p15INK4B methylation were detected in both patients. Furthermore, X-chromosome inactivation (XCI) pattern of each colony was simultaneously determined by MSP-based human androgen receptor gene analysis (HUMARA-MSP), and all p15INK4B-methylated colonies showed the same XCI pattern, which was dominant among the colonies, while p15INK4B-unmethylated colonies showed both patterns of XCI, in each of the two patients. We then examined the methylation status of the p15INK4B gene of granulocyte (PB-PMN) fractions from 10 patients with available peripheral blood cells. In all four patients with p15INK4B-methylated BM-MNCs, their PB-PMNs showed p15INK4B methylation. These results suggest that p15INK4B methylation in hematopoietic cells in MDS patients is restricted to the MDS clone but not necessarily to blast cells.

Acute Disease↗
PubMed2000

Increased circulating skin-homing cutaneous lymphocyte-associated antigen (CLA)+ type 2 cytokine-producing cells, and decreased CLA+ type 1 cytokine-producing cells in atopic dermatitis.

BACKGROUND: Skin-homing T cells are characterized by expression of cutaneous lymphocyte-associated antigen (CLA). Few data are available on the frequency of circulating CLA+ cytokine-producing T cells in atopic dermatitis (AD) patients. OBJECTIVES: We aimed to investigate cytokine synthesis capability vs. CLA expression in phorbol myristate acetate and ionomycin-stimulated, secretion-inhibited peripheral blood T cells of AD patients compared with healthy subjects and psoriatic patients. METHODS: Multiparameter flow cytometry was used. RESULTS: The expression of CLA among CD4+ T cells was significantly elevated in AD patients compared with healthy subjects and psoriatic patients, whereas there was no significant difference between each group in CLA expression among CD8+ T cells. The frequency of interleukin (IL)-4- and IL-13-producing cells in AD patients was significantly higher than in healthy subjects (in both CD4+ and CD8+ T-cell subsets) and psoriatic patients (in CD4+ T cells). In contrast, the frequency of interferon (IFN)-gamma-producing cells was significantly reduced in AD patients, among both CD4+ and CD8+ T cells, compared with healthy subjects and psoriatic patients. Moreover, in AD patients, the frequency of IL-4- and IL-13-producing cells was remarkably increased among the CLA+ subset (in both CD4+ and CD8+ T cells), whereas the frequency of IFN-gamma-producing cells was decreased in the CLA+ subset (in CD4+, but not in CD8+ T cells). CONCLUSIONS: These results provide evidence for the expansion of skin-homing type 2 cytokine-secreting T cells, associated with a reduction in skin-homing type 1 cytokine-producing T cells, in peripheral blood of AD patients.

Adult↗
PubMed2000

Quantitative expression of erythropoietin receptor (EPO-R) on acute leukaemia cells: relationships between the amount of EPO-R and CD phenotypes, in vitro proliferative response, the amount of other cytokine receptors and clinical prognosis. Japan Adult Leukaemia Study Group.

Expression of erythropoietin (EPO) receptor (EPO-R) was analysed in leukaemia cells from 150 patients with acute myeloid leukaemia (AML) or acute lymphoblastic leukaemia (ALL). EPO-R was expressed in 81 (60%) out of 136 AML, and in vitro treatment with EPO led to proliferation of leukaemia cells in 13 (16%) out of 81 AML examined. EPO-R expression and in vitro response to EPO were observed in all subtypes of AML according to the French-American-British (FAB) classification. All eight patients with FAB-M6 expressed EPO-R, and one out of four showed an in vitro response to EPO. Although there was no significant correlation (r = 0.2522) between the amount of EPO-R and the in vitro response to EPO, all of the AML patients who showed in vitro response expressed EPO-R. Stem cell factor significantly enhanced both EPO-R expression and in vitro response to EPO. Interleukin-3 tended to increase in vitro response to EPO. CD phenotypes, the amount of granulocyte colony-stimulating factor (G-CSF) receptors and the amount of TPO receptors had no significant relationship with the amount of EPO-R. Patients with both EPO-R expression and in vitro response to EPO had shorter duration of complete remission than those without EPO-R (P = 0.0053). EPO-R was expressed in four (29%) out of 14 ALL, and none out of five ALL showed in vitro response to EPO.

Acute Disease↗
PubMed2000

Enrichment of lineage-CD34- cells using a newly developed filter system.

Recent studies have demonstrated the presence of human lineage- (lin-)CD34- cells that are capable of differentiating into CD34+ cells in xenogeneic transplantation systems. We developed a new filter system that can enrich lin-CD34- cells, a precursor cell population of CD34+ cells. The filter consists of polyethylene terephthalate non-woven fabrics coated with hydrophilic polymer. The frequency of lin-CD34- cells in the cell population after filtration was 7.45 +/- 4.41%, a 16.8 +/- 8.81-fold enrichment compared with 0. 49 +/- 0.31% in the cell population before filtration. The mean recovery of lin-CD34- cells was 48.57 +/- 13.59% (n = 15). Purified lin-CD34- cells, obtained by sorting the filtrated cell population, acquired CD34 expression and colony-forming activity after 7 d of culture. Our results indicate that this filter system is useful for isolating lin-CD34- cells, including precursors of CD34+ cells, and will help further the study of lin-CD34- cells.

Antigens, CD34↗
PubMed2000

Inhibition of Epstein-Barr virus (EBV)-specific CD8+ cytotoxic T lymphocyte (CTL) activity by soluble HLA class I in vitro.

In the present study, the effects of soluble HLA (sHLA) class I molecules against EBV-specific CTL were examined. Two different sources of sHLA class I, either bioengineered spliced form of HLA-B7 (sB7) or natural production from EBV-transformed B cells (natural sHLA), were added during the induction of CTL or incubated with MHC-restricted CD8+ CTL, which were selected by immunobeads just before testing for their cytotoxic activity. Both sB7 and natural sHLA class I blocked the generation of CD8+ CTL and also inhibited the cytotoxic activity of established CTL in a dose-dependent manner. In both ways, natural sHLA class I was effective in 10-fold lower concentrations compared with sB7. The inhibitory effect did not require a sharing of the HLA allotypes between sHLA and the CTL. CTL, after being treated with sHLA, underwent apoptosis, which was considered here as the main mechanism.

Apoptosis↗
PubMed2000

Membrane-bound form of ADP-ribosyl cyclase in rat cortical astrocytes in culture.

ADP-ribosyl cyclase activities in cultured rat astrocytes were examined by using TLC for separation of enzymatic products. A relatively high rate of [3H]cyclic ADP-ribose production converted from [3H]NAD+ by ADP-ribosyl cyclase (2.015+/-0.554 nmol/min/mg of protein) was detected in the crude membrane fraction of astrocytes, which contained approximately 50% of the total cyclase activity in astrocytes. The formation rate of [3H]ADP-ribose from cyclic ADP-ribose by cyclic ADP-ribose hydrolase and/or from NAD+ by NAD glycohydrolase was low and enriched in the cytosolic fraction. Although NAD+ in the extracellular medium was metabolized to cyclic ADP-ribose by incubating cultures of intact astrocytes, the presence of Triton X-100 in the medium for permeabilizing cells increased cyclic ADP-ribose production three times as much. Isoproterenol and GTP increased [3H]cyclic ADP-ribose formation in crude membrane-associated cyclase activity. This isoproterenol-induced stimulation of membrane-associated ADP-ribosyl cyclase activity was confirmed by cyclic GDP-ribose formation fluorometrically. This stimulatory action was blocked by prior treatment of cells with cholera toxin but not with pertussis toxin. These results suggest that ADP-ribosyl cyclase in astrocytes has both extracellular and intracellular actions and that signals of beta-adrenergic stimulation are transduced to membrane-bound ADP-ribosyl cyclase via G proteins within cell surface membranes of astrocytes.

ADP-ribosyl Cyclase↗
PubMed2000

Repeated dedifferentiation of low-grade intraosseous osteosarcoma.

Low-grade intraosseous osteosarcoma is an uncommon bone tumor that is characterized by minimum cytological atypism and a much better prognosis than conventional osteosarcoma. This report describes a patient who had a low-grade osteosarcoma that mimicked fibrous dysplasia (FD). The tumor had an area of high-grade sarcoma at the initial diagnosis. Ten years after incomplete resection of FD-like tumor, local recurrence with areas of high-grade tumor developed. This case illustrates the potential of dedifferentiation in low-grade intraosseous osteosarcoma.

Bone Neoplasms↗
PubMed2000

Low-grade intraosseous osteosarcoma in northern Japan: advantage of AgNOR and MIB-1 staining in differential diagnosis.

Low-grade intraosseous osteosarcoma is an uncommon form of bone cancer. It is occasionally difficult to recognize as a malignant tumor and is commonly misdiagnosed as a benign fibrous lesion. We retrospectively studied the records of 8 patients with low-grade intraosseous osteosarcoma in the files of the Tohoku Musculoskeletal Tumor Society in Japan. All tumors arose in the lower limb. The most common symptom was pain, with a duration exceeding 2 years in 4 patients. Radiologic findings, including those at magnetic resonance imaging (MRI), suggested malignancy in 5 lesions, whereas 3 were diagnosed as benign. Two patients initially presented with pathological fracture. The initial pathological diagnosis was malignant in 5 patients and benign in 3. All eight tumors were grade 1 in Broders' classification. The tumor showed a permeative pattern in all eight cases, but this pattern could not be confirmed in the multiple tiny fragments obtained as biopsy specimens in 3 cases. The number of silver-staining nucleolar organizer regions (AgNOR) per nucleus and MIB-1-positive rate were significantly higher in low-grade intraosseous osteosarcoma than in fibrous dysplasia, offering an advantage in differential diagnosis. Three patients (38%) developed high-grade sarcoma at the site of local recurrence after multiple intralesional excisions, and one of them died of the disease. The other 5 patients had a good clinical course after surgery with a wide margin. These findings indicate that preoperative diagnosis with radiologic investigation, including magnetic resonance (MR) imaging and histologic examination of biopsy specimens is essential in preparation for surgery with a wide margin, assuring a good clinical course, and the results of AgNOR and immunohistochemical MIB-1 staining might be helpful in differentiating low-grade intraosseous osteosarcoma from fibrous dysplasia.

Adolescent↗
PubMed2000

Competitive reverse transcription-polymerase chain reaction assay for quantification of human multidrug resistance 1 (MDR1) gene expression in fresh leukemic cells.

We have analyzed MDR1 gene expression in 69 clinical samples obtained from 64 patients with leukemic hematologic malignancies by using a competitive reverse transcription-polymerase chain reaction assay with a heterologous competitor RNA. To exclude a false-positive result caused by concomitant normal lymphocytes that physiologically express MDR1, in samples we determined a cut-off value of 8 amol MDR1 transcript per microgram of RNA by simultaneous measurement of rhodamine 123 dye efflux either in lymphocyte or gated leukemic cell populations. Consequently, 23 of 69 samples were concluded to be MDR1-positive in leukemic cells per se. The MDR1 expression rate was significantly correlated with factors such as a history of preceding chemotherapy, elder age of the patient, and certain disease types (eg, leukemia progressed from myelodysplastic syndrome). Moreover, the complete response rate after chemotherapy was significantly higher in MDR1-negative patients than in MDR1-positive patients (52% vs 17%, respectively; P = .01). The assay established will enable the quantification of MDR1 gene expression in blood samples from patients with leukemic hematologic malignancies and will be applicable to clinical laboratories as a routine test.

Adolescent↗
PubMed2000

A case of bilateral heel ulcers associated with hydroxyurea therapy for chronic myelogenous leukemia.

Bilateral heel skin ulcers developed in a 50-year-old male in the chronic phase of chronic myelogenous leukemia who had been receiving hydroxyurea (HU) therapy for 3 years. Histological examination showed perivascular lymphocytic inflammation without vasculitis. After interruption of HU administration, the heel ulcers were completely resolved within 2 months. The clinical course strongly suggested that the heel ulcers were induced by long-term HU therapy.

Antineoplastic Agents↗
PubMed2000

Alternating combination chemotherapy C-MOPP (cyclophosphamide, vincristine, procarbazine, prednisone) and ABVd (adriamycin, bleomycin, vinblastine, dacarbazine) in clinical stage II-IV Hodgkin's disease: a multicenter phase II study (JCOG 8905). The Lymphoma Study Group of the Japan Clinical Oncology Group.

BACKGROUND: The main form of cytotoxic treatment for advanced Hodgkin's disease (HD) is conventional dose multiagents chemotherapy. As HD is not common in Japan, we conducted a phase II study of the commonly used combination chemotherapy (CCT) regimen established in the West for Japanese patients with advanced HD to confirm the efficacy and safety. METHOD: Between October 1989 and February 1993, a multicenter phase II study of alternating CCT C-MOPP (cyclophosphamide, vincristine, procarbazine, prednisone) and ABVd (adriamycin, vinblastine, bleomycin, dacarbazine) to evaluate its clinical usefulness for clinical stage (cS) II-IV HD was conducted by the Lymphoma Study Group of the Japan Clinical Oncology Group. RESULTS: Seventy-nine previously untreated patients were enrolled in the study. For 67 eligible patients, the response rate was 92.5% with 83.6% complete response (CR). For 40 cS II and 27 cS III/IV patients the response rate was 95.0% with 90.0% CR and 88.9% with 74.1% CR, respectively. The overall 5-year survival rate was 84.8%. Those of cS II and cS III/IV were 92.5 and 73.1%, respectively. There was no significant difference between cS II and cS III/IV (p = 0.1025). The progression-free 4-year survival rate was 72.8%. Those of cS II and cS III/IV were 77.5 and 65.7%, respectively. There was no significant difference between cS II and cS III/IV (p = 0.2483). Grade 4 toxicity by the criteria of the World Health Organization consisted of leukocytopenia in 28.4% of patients. There was GPT elevation in 4.5%, nausea/vomiting in 11.9% and CNS in 1.5% of patients, but there was no treatment-related death. CONCLUSION: The C-MOPP/ABVd regimen for Japanese patients with advanced HD is considered to be one of the effective CCTs according to the results of the present phase II study.

Adolescent↗
PubMed2000

Murine stromal cell line HESS-5 maintains reconstituting ability of Ex vivo-generated hematopoietic stem cells from human bone marrow and cytokine-mobilized peripheral blood.

Human bone marrow (BM) or mobilized peripheral blood (mPB) CD34(+) cells have been shown to loose their stem cell quality during culture period more easily than those from cord blood (CB). We previously reported that human umbilical CB stem cells could effectively be expanded in the presence of human recombinant cytokines and a newly established murine bone marrow stromal cell line HESS-5. In this study we assessed the efficacy of this xenogeneic coculture system using human BM and mPB CD34(+) cells as materials. We measured the generation of CD34(+)CD38(-) cells and colony-forming units, and assessed severe-combined immunodeficient mouse-repopulating cell (SRC) activity using cells five days after serum-free cytokine-containing culture in the presence or the absence of a direct contact with HESS-5 cells. As compared with the stroma-free culture, the xenogeneic coculture was significantly superior on expansion of CD34(+)CD38(-) cells and colony-forming cells and on maintenance of SRC activity. The PKH26 study demonstrated that cell division was promoted faster in cells cocultured with HESS-5 cells than in cells cultured without HESS-5 cells. These results indicate that HESS-5 supports rapid generation of primitive progenitor cells (PPC) and maintains reconstituting ability of newly generated stem cells during ex vivo culture irrespective of the source of samples. This xenogeneic coculture system will be useful for ex vivo manipulation such as gene transduction to promote cell division and the generation of PPC and to prevent loss of stem cell quality.

ADP-ribosyl Cyclase↗
PubMed2000

[Efficacy and safety of cefozopran (CZOP) monotherapy and combination therapy with CZOP and amikacin (AMK) for infections accompanying hematological diseases].

We evaluated efficacy and safety of monotherapy with CZOP (1-2 g x 2/day) and combination therapy with CZOP (1-2 g x 2/day) and AMK (200 mg x 2/day) for infections in patients with hematological diseases. Efficacy was evaluated in 71 patients of monotherapy group and 70 patients of combination therapy group. Underlying diseases were mostly leukemia and lymphoma. Infections included sepsis, suspected sepsis, pneumonia and so on. Efficacy in CZOP monotherapy was excellent in 21 patients (31.3%), good in 23 patients (34.3%), fair in 5 patients (7.5%) and the efficacy rate was 65.7%. On the other hand, in combination therapy, each was 14 patients (21.2%), 23 patients (34.8%), 12 patients (18.2%) and the efficacy rate was 56.1%. Side effects such as eruption were noted in 2 patients. Abnormal laboratory findings were noted in 9 patients. All side effects as well as abnormal laboratory findings were minimal. It was concluded that CZOP monotherapy was effective in the treatment of various infections accompanying hematological diseases.

Adolescent↗
PubMed1999

Ex vivo generation of CD34(+) cells from CD34(-) hematopoietic cells.

The human Lin(-)CD34(-) cell population contains a newly defined class of hematopoietic stem cells that reconstitute hematopoiesis in xenogeneic transplantation systems. We therefore developed a culture condition in which these cells were maintained and then acquired CD34 expression and the ability to produce colony-forming cells (CFC) and SCID-repopulating cells (SRCs). A murine bone marrow stromal cell line, HESS-5, supports the survival and proliferation of Lin(-)CD34(-) cells in the presence of fetal calf serum and human cytokines thrombopoietin, Flk-2/Flt-3 ligand, stem cell factor, granulocyte colony-stimulating factor, interleukin-3, and interleukin-6. Although Lin(-)CD34(-) cells do not initially form any hematopoietic colonies in methylcellulose, they do acquire the colony-forming ability during 7 days of culture, which coincides with their conversion to a CD34(+) phenotype. From 2.2% to 12.1% of the cells became positive for CD34 after culture. The long-term multilineage repopulating ability of these cultured cells was also confirmed by transplantation into irradiated NOD/SCID mice. These results represent the first in vitro demonstration of the precursor of CD34(+) cells in the human CD34(-) cell population. Furthermore, the in vitro system we reported here is expected to open the way to the precise characterization and ex vivo manipulation of Lin(-)CD34(-) hematopoietic stem cells.

Animals↗
PubMed1999

Altered expression of CD45 isoforms in differentiation of acute myeloid leukemia.

Specific expression of different CD45 isoforms can be seen in various stages of differentiation of normal nucleated hematopoietic cells. Association of membrane expression of CD45 isoforms and differential levels of leukemia cells was studied in 91 cases with de novo acute myeloid leukemia (AML). Membrane expression of CD45RA and CD45RO was analyzed by flow cytometry and their expression patterns were compared with AML subtypes classified according to the French-American-British (FAB) classification. CD45RA was essentially expressed in all of the FAB myelocytic subtypes (M0-M3). Its expression in percentage was lower in the most differentiated subtype of AML (M3) when compared with other myelocytic subtypes. CD45RO expression was rarely observed in cases with myelocytic subtypes (1/56 cases of M0, M1, M2, and M3) except for the minimally differentiated myelocytic subtype (M0) or those with potential for differentiation to T-cell lineage where three of 12 cases showed CD45RO expression. When leukemia cells of an M3 case were differentiated to mature granulocytes by treatment of all-trans-retinoic acid, they showed increasing expression of CD45RO. In subtypes with a monocytic component (M4 and M5), both of CD45RA and CD45RO expression were observed and mutually exclusive. When 10 cases of M5 were subdivided by the differential level into undifferentiated (M5a) and differentiated monocytic leukemia (M5b), expression of CD45RA and CD45RO was strictly restricted to cases with M5a and M5b, respectively. These results suggest that CD45 isoform expression in AML characterizes differential levels both in myelocytic and monocytic lineages and specifically disturbed in each subtype. The assessment of CD45 isoform expression appears to provide an insight on biological characteristics and a useful supplementary test for differential diagnosis of AML subtypes.

Acute Disease↗