Biomedical subjects

N Emi

Publications and source records attributed to N Emi.

At least 55 records · Page 3Linked to original sources

PubMed1997

Gene transfer mediated by polyarginine requires a formation of big carrier-complex of DNA aggregate.

Cationic peptide, poly-arginine, promotes transfection efficiency under a proper concentration. Using fluorescence microscopy, we observed the structure of the DNA carrier-complex in solution directly. Under low concentration of poly-arginine, small complexes were observed with fluorescence images like a blight light spot. This small complex (sizes are c.a. 1 approximately 2 microns order) had no transfection ability. As the concentration of poly-arginine became high, the complex grew into a big one whose size reached to a degree of 10 microns. Transfection was succeeded under that condition. The relationship between transfection efficiency and higher-order structure of the DNA carrier-complex was discussed.

Amino Acid Sequence↗

PubMed1997

Effect of gene therapy with the herpes simplex virus-thymidine kinase gene on hepatic metastasis in murine colon cancer.

Hepatic metastasis of colon cancer is an important prognostic factor for survival. In this study, we examined the effect of gene therapy using the herpes simplex virus-thymidine kinase (HS-tk) gene with short-course ganciclovir (GCV) treatment for multiple hepatic metastases of murine colon cancer. Colon26 cells transfected with the HS-tk gene were found to be sensitive to GCV in a concentration-dependent way. On the other hand, induction of the HS-tk gene in the cells had no influence on cell growth in vitro. However, multiple hepatic metastases of Colon26 cells transfected with HS-tk gene were significantly suppressed by the GCV treatment. These results thus suggest that HS-tk gene therapy is useful for the treatment of hepatic metastasis in colon cancer.

Animals↗

PubMed1997

Phase I/II trial of cure-oriented high-dose chemoradiotherapy with transplantation of CD34+ peripheral blood stem cells purified by the immunomagnetic bead method for refractory hematological malignancies. Nagoya CD34+ PBSCT Study Group.

A multicenter phase I/II clinical trial was conducted to evaluate the safety of a device (Isolex System; Baxter Health Corporation, Irvine, Calif., USA) using the immunomagnetic bead method to purify CD34+ stem cells from peripheral blood and to assess the efficacy and toxicity of high-dose chemoradiotherapy with peripheral blood stem-cell transplantation (PBSCT) using purified CD34+ stem cells in patients with refractory hematological malignancies. Patients eligible for the study included those who had T-cell acute lymphoblastic leukemia (T-ALL), lymphoblastic lymphoma (LBL), mantle-cell lymphoma (MCL), high-risk aggressive non-Hodgkin's lymphoma (NHL), and adult T-cell leukemia/lymphoma (ATLL) in first complete remission (CR) and those who had standard-risk aggressive NHL, indolent lymphoma, Hodgkin's disease, or acute promyelocytic leukemia (APL) in second CR or first partial remission (PR) after the completion of first-line chemotherapy and were chemosensitive to salvage chemotherapy, in whom tumor contamination of harvested peripheral blood stem cells (PBSCs) was possible due to bone marrow or peripheral blood involvement. Lack of CD34 expression by tumor cells was an important selection factor. Eight patients with hematological malignancies (six NHL patients, one ATLL patients, and one APL patient) were enrolled; their median age was 41 years (range 26-49 years). After consolidation and mobilization chemotherapy, two or three courses of apheresis were performed in each patient. After high-dose chemo(radio)therapy, in each patient a median of 1.8 x 10(6) cells/kg (range 8.2 x 10(5)-5.1 x 10(6) cells/kg) purified CD34+ PBSCs were infused; granulocyte colony-stimulating factor was given from day 1. Median times to hematopoietic recovery were as follows: WBC of > or = 1,000/microliter, day 11; platelet count of > or = 50,000/microliter, day 19; and reticulocyte count of > or = 10/1000, day 15. Two NHL patients relapsed at 23 and 9 months after PBSCT, respectively; the remaining six patients are alive and in CR. No severe toxicity was observed in any patient. Tumor contamination as measured using a polymerase chain reaction-mediated RNase protection assay at the 10-4 level was detected in the CD34(+)-purified fractions of 2 of the 5 samples analyzed; however, a reduction in contaminating lymphoma cells from the autograft of at least 1,000 to 10,000 orders of magnitude was achieved by CD34+ selection using the immunomagnetic bead method. High-dose chemoradiotherapy with transplantation of CD34+ PBSCs purified by the immunomagnetic bead method was thus shown to be an active and safe therapy for refractory hematological malignancies with bone marrow or peripheral blood involvement. However, it is too early for evaluation of the long-term survival benefit.

Adolescent↗

PubMed1997

Morphological subtyping of acute myeloid leukemia with maturation (AML-M2): homogeneous pink-colored cytoplasm of mature neutrophils is most characteristic of AML-M2 with t(8;21).

Morphologic and cytochemical features of 30 acute myeloid leukemia subtype M2 (AML-M2) patients with t(8;21) were compared with those of 50 AML-M2 patients without t(8;21). It was disclosed that irregular nuclear shape, Auer bodies, and at least 90% myeloperoxidase positivity in blast cells, and pseudo-Pelger-Huët anomaly of the nuclei and homogeneous pink-colored cytoplasm of mature neutrophils were observed in 90-100% of the t(8;21)+ patients. The percentages of patients showing these features were significantly (P < 0.01) lower in the t(8;21)- group. Among these morphological features, homogeneous pink-colored cytoplasm of mature neutrophils is most characteristic of t(8;21)+ AML-M2, because it was seen in 90% of the t(8;21)+ patients but in only 2% of the t(8;21)- patients. Conversely, pale-colored cytoplasm without any granules in mature neutrophils or dyserythropoietic features was observed in 84% of the t(8;21)- patients, but in none of the t(8;21)+ patients. These data suggest that it is possible to subtype AML-M2 patients morphologically by the recognition of homogeneous pink-colored or pale-colored cytoplasm of mature neutrophils and dyserythropoietic features. Thus, the morphologic subtyping of AML-M2 can be utilized alone or in combination with chromosomal or molecular subtyping for biological and clinical studies of AML with maturation.

Adolescent↗

PubMed1996

Folding and aggregation of DNA chains induced by complexation with lipospermine: formation of a nucleosome-like structure and network assembly.

Dioctadecylamidoglycylspermine (DOGS) is a cationic lipid vector capable of efficiently introducing DNA into various eukaryotic cells. We investigated the higher-order structure of the DNA/DOGS complex using fluorescence and electron microscopy. Our results show that the DNA/DOGS complex exhibits a nucleosome-like structure in which DNA wraps around an aggregate of DOGS molecules. In addition, DNA/DOGS complexes tend to associate with each other to form network structures. The resulting network assembly may play a role in effective gene transfection.

Animals↗

PubMed1996

Inactivation of multiple tumor-suppressor genes involved in negative regulation of the cell cycle, MTS1/p16INK4A/CDKN2, MTS2/p15INK4B, p53, and Rb genes in primary lymphoid malignancies.

It is now evident that the cell cycle machinery has a variety of elements negatively regulating cell cycle progression. However, among these negative regulators in cell cycle control, only 4 have been shown to be consistently involved in the development of human cancers as tumor suppressors: Rb (Retinoblastoma susceptibility protein), p53, and two recently identified cyclin-dependent kinase inhibitors, p16INK4A/MTS1 and p15INK4B/MTS2. Because there are functional interrelations among these negative regulators in the cell cycle machinery, it is particularly interesting to investigate the multiplicity of inactivations of these tumor suppressors in human cancers, including leukemias/lymphomas. To address this point, we examined inactivations of these four genes in primary lymphoid malignancies by Southern blot and polymerase chain reaction-single-strand conformation polymorphism analyses. We also analyzed Rb protein expression by Western blot analysis. The p16INK4A and p15INK4B genes were homozygously deleted in 45 and 42 of 230 lymphoid tumor specimens, respectively. Inactivations of the Rb and p53 genes were 27 of 91 and 9 of 173 specimens, respectively. Forty-one (45.1%) of 91 samples examined for inactivations of all four tumor suppressors had one or more abnormalities of these four tumor-suppressor genes, indicating that dysregulation of cell cycle control is important for tumor development. Statistical analysis of interrelations among impairments of these four genes indicated that inactivations of the individual tumor-suppressor genes might occur almost independently. In some patients, disruptions of multiple tumor-suppressor genes occurred; 4 cases with p16INK4A, p15INK4B, and Rb inactivations; 2 cases with p16INK4A, p15INK4B, and p53 inactivations; and 1 case with Rb and p53 inactivations. It is suggested that disruptions of multiple tumor suppressors in a tumor cell confer an additional growth advantage on the tumor.

Adolescent↗

PubMed1996

Inhibitory effect of double transfection to xenoendothelial cells using both decay accelerating factor and homologous restriction factor 20 genes on complement dependent cytolysis.

Hyperacute rejection in discordant xenotransplantation occurs due to the complement activation via classical and/or alternative pathway. Regulator of complement activation (RCA) molecules inhibit species-specific complement dependent cytolysis. To confirm the effect of gene engineering using double RCA molecules on xenogeneic cells, the inhibitory effect on complement dependent cytolysis was compared between bovine aortic endothelial cells (BAEC) doubly transfected with both decay accelerating factor (DAF) and homologous restriction factor (HRF) 20 cDNA and BAEC singly transfected with DAF alone using retroviral vector. The positive percent expression of DAF and HRF20 antigen on double transfectant (BAEC/D+H) was 97.0% and 95.0%, respectively, whereas that of DAF antigen on single transfectant (BAEC/D) was 97.0%. After incubated with 25% human serum and anti-BAEC antibody, the viability of double transfectant (BAEC/ D+H) was significantly preserved, compared with that of single transfectant (BAEC/D). These findings demonstrated that xenoendothelial cells doubly transfected with both DAF and HRF20 cDNA could be protected from complement dependent cytolysis more effectively than those singly transfected with DAF alone in the presence of antixenoendothelial antibody.

Animals↗

PubMed1996

Human superoxide dismutase cDNA transfection and its in vitro effect on cold preservation.

In order to elucidate the role of superoxide dismutase (SOD on ischemia-reperfusion injury, bovine and porcine aortic endothelial cells were transfected with retroviral vector LSODRNL containing human Cu,Zn-SOD cDNA and their SOD activities were evaluated by measuring superoxide scavenging capability. Successful augmentation of Cu,Zn-SOD up to 2.7-fold in bovine and 1.9-fold in porcine cells was obtained. After 24 h cold preservation followed by 4 h rewarming, cell viability compared with that of pre-preservation was significantly increased in both types of cells (P < 0.01). Moreover, after exposure to superoxide, viability of the porcine cells with high SOD activity was significantly improved compared to control cells. We consider that transfection of human Cu,Zn-SOD cDNA to arterial endothelial cells is useful for preventing from cold preservation-rewarming or ischemia-reperfusion injury.

Animals↗

PubMed1996

Inhibition of human complement-dependent cell lysis by bovine aortic endothelial cells transfected with membrane-bound complement-regulatory factor (DAF and HRF20) gene using a retroviral vector.

The endothelial cells (EC) of xenografts are the target of hyperacute rejection induced by complement activation via the classical and/or the alternative pathway. To protect these cells from the attack of human complement, decay-accelerating factor (DAF, CD55) and homologous restriction factor 20 (HRF20, CD59), which belong to human complement regulatory factors, were transfected into bovine aortic EC (BAEC) using retroviral vector. Cell surface expression of DAF and HRF20 on BAEC transfectants (BAEC/DAF, BAEC/HRF20) is comparable to that on human umbilical vein EC. Phosphatidyl inositol-phospholipase C treatment diminished or abolished cell surface expression of DAF and HRF20 on BAEC. The addition of human serum to BAEC led to complement-dependent cell lysis, whereas practically no lysis was observed after addition of human serum to BAEC/DAF and BAEC/HRF20. The addition of human serum plus rabbit complement to BAEC/DAF and BAEC/HRF20 caused complement-dependent cell lysis that was comparable to that observed for BAEC. These data demonstrate that xenograft EC transfected with DAF or HRF20 cDNA using retroviral vector are protected from complement-dependent cell lysis.

Animals↗

PubMed1996

Randomized trials between behenoyl cytarabine and cytarabine in combination induction and consolidation therapy, and with or without ubenimex after maintenance/intensification therapy in adult acute myeloid leukemia. The Japan Leukemia Study Group.

PURPOSE: We analyzed complete remission (CR), disease-free survival (DFS), and event-free survival (EFS) rates in two groups of patients treated with either N4-behenoyl-1-beta-D-arabinosylcytosine (BHAC) or cytarabine, and analyzed DFS with or without ubenimex, a biologic response modifier. PATIENTS AND METHODS: Newly diagnosed patients with acute myeloid leukemia (AML) were randomized to receive either BHAC or cytarabine as remission-induction combination chemotherapy and two courses of consolidation therapy. After maintenance/intensification therapy, patients in CR were randomized to receive either ubenimex and no drug. RESULTS: Of 341 patients registered, 326 were assessable. The age of assessable patients ranged from 15 to 82 years (median, 48). The overall CR rate was 77%: 72% in the BHAC group and 81% in the cytarabine group, and there was a significant difference between the two groups (P = .035, chi 2 test). The predicted 55-month EFS rate of all patients was 30%: 23% in the BHAC group and 35% in the cytarabine group, with a significant difference between groups (P = .0253). The predicted 55-month DFS rate of all CR patients was 38% and that of CR patients less than 50 years of age was 47%. There was no significant difference in DFS between the ubenimex group and the group that did not receive ubenimex. CONCLUSION: Analyses of our clinical trial showed that the use of BHAC in remission-induction therapy and in consolidation therapy resulted in poorer CR and EFS rates in adult AML patients compared with the use of cytarabine at the doses and schedules tested. Immunotherapy with ubenimex after the end of all chemotherapy did not improve DFS.

Acute Disease↗

PubMed1996

Establishment and characterization of a novel human bone marrow stromal cell line, FS-1.

We have established a human stromal cell line derived from the bone marrow of a patient with chronic myelogenous leukemia in blast crisis. This cell line, designated FS-1, exhibits a fibroblastoid morphology and does not express any hematopoietic cell marker tested. FS-1 is negative for alpha-naphthyl acetate esterase, acetylated LDL, von Willebrand factor, and shows no phagocytosis. This cell line is positive for acid phosphatase, alkaline phosphatase, collagen types I, III, IV, and fibronectin. cDNA from FS-1 cells was subjected to amplification by the polymerase chain reaction to assess the constitutive expression of several cytokine genes. Transcripts for interleukin (IL)-6, IL-7, macrophage colony-stimulating factor (M-CSF), and stem cell factor (SCF) were detected in FS-1 cells. IL-6 and SCF also were detected in the culture supernatants of FS-1 at a concentration of 95 pg/ml and 21.2 pg/ml, respectively. These data show that FS-1, established from a human bone marrow, is a stromal cell line which was not generated using transfection with SV40 T antigen. FS-1 cells may be useful in supporting human hematopoietic cells for experimental manipulation.

Antigens, CD↗

PubMed1996

Expression of costimulatory molecules in human leukemias.

In order to determine the indication of B7 (B7-1 and B7-2) molecules-mediated immuno-gene therapy for human leukemias, we investigated 94 human leukemic samples for the expression of MHC molecules required for tumor antigen-specific signals and of B7-1, B7-2, and ICAM-1 molecules required for non-specific costimulatory signals. All samples were strongly positive for MHC class I and 84% for class II antigen. B7-1, B7-2 and ICAM-1 were expressed in 5%, 22% and 16% of the total cases, respectively. Especially in 54 AML samples, B7-1 was only expressed in one case, while B7-2 was detected in as many as 15 cases (28%). We have also examined 13 human myelo/monocytic cell lines for the expression of class II and costimulatory molecules and found that significant expression of costimulatory molecules was induced in human leukemic cells by some suitable drugs, among which interferon-gamma (IFN-gamma) was the most potent inducer. Our results indicate that when the B7-mediated immuno-gene therapy was applied to human leukemias, especially to AML, B7-1 was rather preferable to B7-2 in that the latter was more widely expressed on human leukemic cells. Furthermore, since gene-transfer systems occasionally accompany serious problems, it should be taken into account that costimulatory molecules on human myelo/monocytic leukemic cells could be induced ex vivo without the introduction of exogenous genes.

Antigens, CD↗

PubMed1996

[Synthetic vectors for gene transfection].

Although viruses can be efficient gene transfer vehicles developed by natural evolution, progress also has been made toward developing nonviral gene delivery systems. Various methods for nonviral gene transfer have been proposed and shown to be generally safe. Basically, synthetic vectors have to be capable of binding to DNAs and carrying them inside into target cells. Synthetic vectors are classified into three groups with respect to the chemical structure: cationic lipids, ligand-polycation conjugates and polycationic polymers. In this review, we summarize their ability to mediate gene transfer in relation to their structural characteristics. Although the molecular mechanisms including internalization via cell membrane and nuclear transport are still poorly understood, in the future systematic studies on the structure-activity relationship will be helpful for the design of more efficient artificial vectors.

Cations↗

PubMed1996

Induction of humoral and cellular anti-idiotypic immunity by intradermal injection of naked DNA encoding a human variable region gene sequence of an immunoglobulin heavy chain in a B cell malignancy.

The idiotypic determinants of a B cell neoplasia could provide a tumor-specific target for vaccinating patients against their B cell tumors. Several approaches for inducing idiotype-specific immunization have been reported, but their major theoretical focus was related to the induction of humoral immunity. We have investigated an immunization procedure with naked DNA encoding human variable region gene sequences of an immunoglobulin heavy chain (VH), which may generate both humoral and cellular immune responses against the idiotype of a B cell acute lymphoblastic leukemia (ALL). Using polymerase chain reaction (PCR) amplification, we investigated heavy chain variable region genes obtained from an ALL patient and whether they could induce humoral and cellular immune responses in DBA/2 mice. The VH sequence was cloned into a mammalian expression vector for intradermal DNA vaccination and into a bacterial expression vector to obtain VH protein for Western blotting. The mammalian expression vector encoding the VH gene was injected into mice three times at 7-day intervals and was transduced into P1HTR which is a syngeneic tumor of DBA/2, to serve as target cells to detect cellular immunity. Spleen cells from the inoculated mice exhibited a significant cytotoxic T lymphocyte response to the target cells. By Western blotting, we could also detect trace levels of anti-VH antibodies in all mice serum. This approach for the induction of cellular immunity by intradermal injection is easy and variable region-specific, and may be used as vaccines after chemotherapy treatment of B cell malignancies.

Amino Acid Sequence↗

PubMed1995

Effects of gadolinium chloride on the rat lung following intratracheal instillation.

The metabolic behavior, clearance, and pulmonary effects of gadolinium (Gd), one of the rare earth elements, were investigated after single intratracheal instillation of gadolinium chloride (GdCl3) in male Wistar rats. There was a dose-related increase in Gd content of lung tissue. Gd content in the supernatant of bronchoalveolar lavage fluid (BALF) did not exceed 5 micrograms Gd/BALF even at a dose of 100 micrograms Gd/rat. Gd in the lung tissue decreased very slowly with a biological half-life of 136 days at a dose of 50 micrograms Gd/rat. On the other hand, Gd content in the supernatant of BALF was not detectable after 31 days. These results suggest that intratracheally instilled Gd can be retained in epithelial lining fluid only to a limited extent as soluble forms and is deposited in the lung tissue probably in insoluble forms which are metabolized very slowly. Calcium (Ca) content in BALF increased more rapidly than other toxicological indices such as lactate dehydrogenase activity, protein concentration, and inflammatory cell counts. In the lung tissue, levels of Ca in Gd-instilled groups did not differ from the control value. Although these data suggest that the origin of Ca may be blood plasma, biological and/or toxicological significance of increased Ca is not known. The number of neutrophils reached the maximum at 12 hr after instillation, indicating that Gd has the potency to cause acute lung toxicity. Summarizing the observation, Gd instilled intratracheally into rats was deposited in the lung tissue in nonsoluble forms with an extremely long half-life, while the metal caused a rapid and selective infiltration of serum Ca before acute lung toxicity.

Animals↗

PubMed1995

Retroviral transfer of HSV1-TK gene into human lung cancer cell line.

We used a recombinant retrovirus as one of the potential vectors for human gene therapy to transfer a drug sensitivity gene into human lung cancer cells. The gene encoding the thymidine kinase (TK) of herpes simplex virus type 1 (HSV1) was used as the drug sensitivity gene. The antiherpes drugs acyclovir (ACV) and ganciclovir (GCV) were chosen to test the HSV1-TK activity transferred into the human lung cancer cell lines. The rationale for this approach was that ACV and GCV are nucleoside analogs specifically converted by HSV1-TK to a toxic form capable of inhibiting DNA synthesis or disrupting cellular DNA replication. The results obtained from our experiments demonstrate that the retroviral vector-mediated HSV1-TK gene transfer leads to ACV- and GCV-dependent cytotoxicity in human lung cancer cell lines, including both small-cell carcinoma and non-small-cell carcinoma. Although the gene transfer of HSV1-TK gene into tumor cells would be one model for gene therapy to control lung cancer, further investigations are necessary for the proper choice of the therapeutic gene and vector targeting such as tumor cell specific delivery of the gene or tumor cell specific expression of the transduced gene.

Acyclovir↗

PubMed1995

Acute myeloblastic leukemia (M2) with translocation (7;11) followed by marked eosinophilia and additional abnormalities of chromosome 5.

We present an 18-year-old woman who was diagnosed with acute myeloblastic leukemia (AML M2), and in whom chromosome analysis of bone marrow cells revealed t(7;11), an abnormality rarely found in leukemias with a differentiation potency. She relapsed 1 year after complete remission was achieved by chemotherapy. Bone marrow examination then revealed a t(7;11) abnormality in 48 of 50 metaphases examined, even when there were less than 7.5% leukemic blasts in the marrow, indicating that the morphologically normal cells were derived from leukemic blasts. The number of leukemia clones with the additional abnormalities in chromosome 5 increased, with concurrent development of eosinophilia, fever, asthma-like symptoms, erythema, itching, and hepatosplenomegaly. Elevation of interleukin 5 (IL-5) in serum and an enhanced expression of IL-5 mRNA were also detected. The increase in IL-5 may have been produced by an abnormality on chromosome 5.

Adolescent↗

PubMed1995

Suppressive effect of molybdenum on hepatotoxicity of N-nitrosodiethylamine in rats.

In order to elucidate the mechanism by which molybdenum prevents the carcinogenesis of N-nitroso compounds, the effects of Na2MoO4-pretreatment on N-nitrosodiethylamine (NDEA)-induced DNA strand breaks, fluctuation in cation contents and lipid peroxidation levels in rat liver were examined. Male Wistar rats weighing 170-190 g were pretreated with Na2MoO4 (1.24 mmol/kg body weight, i.p., once a day) for 3 d and on day 4, they were exposed to NDEA (50 mg/kg body weight, once, i.p.). Three days after exposure to NDEA, the nitroso compound caused DNA strand breaks and disrupted potassium (K) and calcium (Ca) metabolism in the liver but did not affect lipid peroxidation levels. Na2MoO4-pretreatment prevented both NDEA-induced DNA damage and disruption of the metabolism of those cations but rather enhanced lipid peroxidation. These results suggest that Mo prevented NDEA-induced DNA damage by preventing disruption of intracellular Ca metabolism while stimulating the metabolism of the nitroso compound via a nontoxic pathway.

Animals↗