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  • 11
    Publication Date: 1990-02-01
    Description: A retroviral vector (N2-SV-GC) was constructed by inserting a normal human glucocerebrosidase (GC) cDNA under control of the SV40 early region promoter into the Moloney murine leukemia virus-derived N2 vector. N2-SV-GC produced human GC in murine 3T3 fibroblasts at levels in the range of the endogenous murine GC as determined by enzymatic assay and Western blot analysis. The N2-SV-GC retroviral vector was used for studies of gene transduction of murine hematopoietic progenitor cells (HPC). Infection of bone marrow cultured for 2 to 10 days in medium containing hematopoietic growth factors was significantly more efficient than infection of freshly isolated marrow cells (24% to 32% G418-resistant CFU-GM v 15%, respectively). The marrow infected by N2-SV-GC was maintained in long-term bone marrow culture (LTBMC) and had a stable level of G418-resistant HPC over 2 months of serial assays. The human GC gene of the vector was persistently expressed in the nonadherent cell fraction of the murine LTBMC as determined by Northern blotting, Western blotting, and immunohistochemical staining using a monoclonal antibody specific for human GC. N2-SV-GC also expressed the human GC gene in day 12 CFU-S. LTBMC represents a novel system for retroviral vector-mediated gene transduction of HPC and may accurately predict the activities of vectors in vivo.
    Print ISSN: 0006-4971
    Electronic ISSN: 1528-0020
    Topics: Biology , Medicine
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  • 12
    Publication Date: 1996-06-15
    Description: NBXFO hybridoma cells produced both the membrane and secreted isoforms of macrophage colony-stimulating factor (M-CSF). Murine bone marrow cells stimulated by the secreted form of M-CSF (sM-CSF) became Mac1+, Mac2+, Mac3+, and F4/80+ macrophages that inhibited the growth of NBXFO cells, but not L1210 or P815 tumor cells. In cytotoxicity studies, M- CSF activated macrophages and freshly isolated macrophages killed NBXFO cells in the presence of polymyxin B, eliminating the possibility that contaminating lipopolysaccharide (LPS) was responsible for the delivery of the cytotoxic signal. Retroviral-mediated transfection of T9 glioma cells with the gene for the membrane isoform of M-CSF (mM-CSF), but not for the secreted isoform of M-CSF, transferred the ability of macrophages to kill these transfected T9 cells in a mM-CSF dose- dependent manner. Macrophage-mediated killing of the mM-CSF transfected clone was blocked by using a 100-fold excess of recombinant M-CSF. Catalase, superoxide dismutase, and the nitric oxide inhibitor, N-omega- nitro-arginine methyl ester (NAME), did not effect macrophage cytotoxicity against the mM-CSF transfectant T9 clones. T9 parental cells when cultured in the presence of an equal number of the mM-CSF transfectant cells were not killed, indicating specific target cell cytotoxicity by the macrophages. Electron microscopy showed that macrophages were capable of phagocytosizing mM-CSF bearing T9 tumor cells and NBXFO hybridoma cells; this suggested a possible mechanism of this cytotoxicity. This study indicates that mM-CSF provides the necessary binding and triggering molecules through which macrophages can initiate direct tumor cell cytotoxicity.
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  • 13
    Publication Date: 1996-04-15
    Description: X-SCID, the most common form of human SCID, is due to mutations in the common gamma chain gene (gamma-c) that encodes an essential component of the cytokine receptors for interleukin-2 (IL-2), IL-4, IL-7, IL-9, and IL-15. Activation of the Janus family tyrosine kinases Jak1 and Jak3 is necessary for appropriate signalling through the IL-2 receptor (IL-2R). Neither Jak1 nor Jak3 was phosphorylated after IL-2 stimulation of an Epstein-Barr virus-transformed cell line (LCL) from an X-SCID patient with a gamma-c null mutation. However, we now show that appropriate IL-2R function can be restored in an X-SCID LCL by transduction of a wild-type gamma-c gene. A retroviral vector, G1gamma- cSvNa, was constructed and produced in the PG13 packaging line. Transduced X-SCID LCL expressed the G1gamma-cSvNa transcript. IL-2 stimulation of the transduced cell line resulted in appropriate tyrosine phosphorylation of both Jak1 and Jak3. Thus, retroviral- mediated transduction of normal gamma-c can reconstitute downstream signalling through the IL-2R in X-SCID cell lines, suggesting that gene therapy may be a treatment for this disease.
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  • 14
    Publication Date: 1994-05-01
    Description: Gaucher disease is an inherited lysosomal storage disease in which the loss in functional activity of glucocerebrosidase (GC) results in the storage of its lipid substrate in cells of the macrophage lineage. A gene therapy approach involving retroviral transduction of autologous bone marrow (BM) followed by transplantation has been recently approved for clinical trial. Amelioration of the disease symptoms may depend on the replacement of diseased macrophages with incoming cells expressing human GC; however, the processes of donor cell engraftment and vector gene expression have not been addressed at the cellular level in relevant tissues. Therefore, we undertook a comprehensive immunohistologic study of macrophage and microglia replacement after murine BM transplantation with retrovirus-marked BM. Serial quantitative PCR analyses were employed to provide an overview of the time course of engraftment of vector-marked cells in a panel of tissues. Following reconstitution of hematopoietic tissues with vector- marked donor cells at early stages, GC+ cells began to infiltrate the liver, lung, brain, and spinal cord by 3 months after transplant. Immunohistochemical analyses of PCR+ tissues using the 8E4 monoclonal antibody specific for human GC revealed that macrophages expressing human GC had partially reconstituted the Mac-1+ population in all tissues in a manner characteristic to each tissue type. In the brain, 20% of the total microglia had been replaced with donor cells expressing GC by 3 to 4 months after transplant. The finding that significant numbers of donor cells expressing a retroviral gene product immigrate to the central nervous system suggests that gene therapy for neuronopathic forms of lysosomal storage diseases as well as antiviral gene therapy for AIDS may be feasible.
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  • 15
    Publication Date: 1993-12-01
    Description: Gene transfer into human cells using murine amphotropic retroviral vectors is the basic technique used in most current gene therapy studies. The identity of the cell surface receptor for the amphotropic envelope remains unknown and thus its importance in gene transfer is poorly understood. We have measured specific retrovirus binding to cells to study amphotropic virus receptor regulation in human CD34+ bone marrow (BM) progenitors and primitive CD34+CD38- human hematopoietic cells. The rat monoclonal antibody 83A25 recognizes an epitope common to the envelope glycoprotein of all classes of Moloney murine leukemia virus. Indirect fluorescent labeling of 83A25 allows flow cytometric analysis of specific virus-cell interactions and is an indirect measure of specific receptors. Using this assay, amphotropic virus binding to fresh CD34+ cells was minimal. However, when CD34+ cells were cultured with or without growth factors for 4 days, specific binding of amphotropic retrovirus was readily shown. Inclusion of interleukin-3 (IL-3), IL-6, and Steel factor in cultures increased the fluorescence associated with amphotropic virus binding by twofold to four-fold (mean fold increase 2.7 +/- 0.84). Virus binding to CD34+CD38- cells was shown only in those cells culture in IL-3, IL-6, and Steel factor. These results suggest that certain cytokines may cause an increase in the number and/or affinity of amphotropic receptors on primitive human hematopoietic cells. Upregulation of viral receptor expression may be one of the mechanisms by which cytokines enhance gene transfer into primitive BM cells.
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  • 16
    Publication Date: 1988-11-01
    Description: The Wiskott-Aldrich syndrome (WAS) is an X-linked recessive disease characterized by immunodeficiency and severe thrombocytopenia in affected males, but no demonstrable clinical abnormalities in carrier females. Through analysis of the methylation patterns of X-linked genes that display restriction fragment length polymorphisms (RFLPs), we studied the pattern of X-chromosome inactivation in various cell populations from female relatives of patients with WAS. The peripheral blood T cells, granulocytes, and B cells of eight obligate WAS carriers were found to display specific patterns of X-chromosome inactivation clearly different from these of normal controls. Thus, carriers of WAS could be accurately identified using this analysis.
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  • 17
    Publication Date: 1990-02-01
    Description: A retroviral vector (N2-SV-GC) was constructed by inserting a normal human glucocerebrosidase (GC) cDNA under control of the SV40 early region promoter into the Moloney murine leukemia virus-derived N2 vector. N2-SV-GC produced human GC in murine 3T3 fibroblasts at levels in the range of the endogenous murine GC as determined by enzymatic assay and Western blot analysis. The N2-SV-GC retroviral vector was used for studies of gene transduction of murine hematopoietic progenitor cells (HPC). Infection of bone marrow cultured for 2 to 10 days in medium containing hematopoietic growth factors was significantly more efficient than infection of freshly isolated marrow cells (24% to 32% G418-resistant CFU-GM v 15%, respectively). The marrow infected by N2-SV-GC was maintained in long-term bone marrow culture (LTBMC) and had a stable level of G418-resistant HPC over 2 months of serial assays. The human GC gene of the vector was persistently expressed in the nonadherent cell fraction of the murine LTBMC as determined by Northern blotting, Western blotting, and immunohistochemical staining using a monoclonal antibody specific for human GC. N2-SV-GC also expressed the human GC gene in day 12 CFU-S. LTBMC represents a novel system for retroviral vector-mediated gene transduction of HPC and may accurately predict the activities of vectors in vivo.
    Print ISSN: 0006-4971
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  • 18
    Publication Date: 1993-12-01
    Description: Gene transfer into human cells using murine amphotropic retroviral vectors is the basic technique used in most current gene therapy studies. The identity of the cell surface receptor for the amphotropic envelope remains unknown and thus its importance in gene transfer is poorly understood. We have measured specific retrovirus binding to cells to study amphotropic virus receptor regulation in human CD34+ bone marrow (BM) progenitors and primitive CD34+CD38- human hematopoietic cells. The rat monoclonal antibody 83A25 recognizes an epitope common to the envelope glycoprotein of all classes of Moloney murine leukemia virus. Indirect fluorescent labeling of 83A25 allows flow cytometric analysis of specific virus-cell interactions and is an indirect measure of specific receptors. Using this assay, amphotropic virus binding to fresh CD34+ cells was minimal. However, when CD34+ cells were cultured with or without growth factors for 4 days, specific binding of amphotropic retrovirus was readily shown. Inclusion of interleukin-3 (IL-3), IL-6, and Steel factor in cultures increased the fluorescence associated with amphotropic virus binding by twofold to four-fold (mean fold increase 2.7 +/- 0.84). Virus binding to CD34+CD38- cells was shown only in those cells culture in IL-3, IL-6, and Steel factor. These results suggest that certain cytokines may cause an increase in the number and/or affinity of amphotropic receptors on primitive human hematopoietic cells. Upregulation of viral receptor expression may be one of the mechanisms by which cytokines enhance gene transfer into primitive BM cells.
    Print ISSN: 0006-4971
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  • 19
    Publication Date: 1993-12-15
    Description: Two human melanoma cell lines were transduced with the human interleukin (IL)-7 and IL-2 genes using retroviral-mediated gene transfer. Stable, high-level cytokine expression was achieved. The in vitro growth of transduced tumors was unaltered. Neither of the IL-2- transduced melanoma cell lines grew in athymic mice, whereas one IL-7- transduced melanoma line showed retarded in vivo growth. This is consistent with animal studies suggesting a predominantly T-cell response to IL-7-transduced tumors and a more nonspecific response to IL-2-transduced tumors. Both IL-7- and IL-2-transduced melanoma cell lines could induce cytotoxic lymphocytes in mixed lymphocyte-tumor cultures. The expression of putative melanoma antigens (MAGE)-1 and MAGE-3 was unaltered by cytokine transduction. In one cell line, IL-7 transduction resulted in a marked inhibition of the immunosuppressive peptide transforming growth factor (TGF)beta 1. The results allow a comparison of immunobiologic properties of IL-7- and IL-2-transduced human melanoma cell lines in consideration of their use in genetically engineered tumor vaccines. IL-7 transduction results in stable cytokine expression and phenotypic alterations that appear to be favorable for enhanced immunogenicity and it deserves clinical testing.
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