Search PubMed⌕ Search

Biomedical subjects

D B Kohn

Publications and source records attributed to D B Kohn.

At least 91 records · Page 5Linked to original sources
PubMed1993

Transduction of human melanoma cell lines with the human interleukin-7 gene using retroviral-mediated gene transfer: comparison of immunologic properties with interleukin-2.

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.

Animals↗
PubMed1993

Growth factors increase amphotropic retrovirus binding to human CD34+ bone marrow progenitor cells.

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.

ADP-ribosyl Cyclase↗
PubMed1993

Expression of coagulation factor IX (Christmas factor) in human hepatoma (HepG2) cell cultures after retroviral vector-mediated transfer.

PURPOSE: In this study, we compared production of recombinant human factor IX by HepG2 cells transduced with a cytomegalovirus (CMV) promoter-controlled factor IX vector to endogenous production of factor IX by non-transduced primary rat hepatocytes. METHODS AND RESULTS: Northern analysis showed 2.8 kb transcripts corresponding to the known size of factor IX mRNA in primary hepatocyte preparations and vector factor IX transcripts of the expected sizes in transduced HepG2 cell preparations. Factor IX produced by transduced HepG2 cells was completely inhibited by a monospecific antibody against human factor IX. Western analysis showed that recombinant factor IX migrated to the region of native plasma factor IX at 56 Kd. Production of biologically active factor IX by transduced HepG2 cells was 20-fold greater than that by nontransduced primary hepatocytes. CONCLUSION: These data indicate that transduced HepG2 cells transcribe, synthesize, and secrete authentic factor IX, and that these genetically engineered cells secrete significantly greater amounts of factor IX than do nontransduced primary hepatocytes. Studies are in progress to determine the effect of hepatocyte mitogens on production of factor IX in transduced HepG2 cells and primary hepatocytes.

Animals↗
PubMed1993

Comparison of trans-dominant inhibitory mutant human immunodeficiency virus type 1 genes expressed by retroviral vectors in human T lymphocytes.

trans-Dominant inhibitory mutant versions of the human immunodeficiency virus type 1 (HIV-1) regulatory genes tat and rev have previously been described. We have constructed a series of retroviral vectors to transduce these genes and compare their inhibitory activities. The inhibitory activities were measured with transient transfection assays by using a reporter which expresses an HIV-1 gag-Escherichia coli lacZ fusion protein with strict dependence on coexpression of both tat and rev. Additionally, the vectors were packaged as amphotropic virions and used to stably transduce human CEM T lymphocytes. The transduced CEM cells were challenged with HIV-1, and the effects of the mutant HIV-1 genes were determined by measuring the levels of HIV-1 p24gag produced. A tat gene substituted at amino acid 41 (tatk41a) retained partial trans-activating activity and lacked inhibitory activity. A tat gene with a premature stop codon at amino acid 54 (tat54ter) showed moderate trans-dominant inhibition of the reporter plasmid but failed to significantly inhibit HIV-1 replication. The M10 rev mutant, with a 2-amino-acid substitution, showed strong trans-dominant inhibitory activity both in the reporter plasmid and in the HIV-1 infection assay. The greatest inhibition of HIV-1 growth was seen when M10 was expressed under the transcriptional control of a human cytomegalovirus promoter; slightly less inhibition was achieved when expression of M10 was controlled by the Moloney murine leukemia virus long terminal repeat, and minimal inhibition was seen when the HIV-1 long terminal repeat controlled the M10 gene. These results demonstrate the potential utility of retroviral vectors expressing trans-dominant inhibitory mutant HIV-1 genes for gene therapy approaches to AIDS.

Acquired Immunodeficiency Syndrome↗
PubMed1993

Granulocyte recovery in pediatric marrow transplant recipients treated with ganciclovir for cytomegalovirus infection.

PURPOSE: Cytomegalovirus (CMV) infection remains a major cause of morbidity and mortality after bone marrow transplantation (BMT). Ganciclovir is a guanosine analogue that selectively inhibits CMV DNA polymerase and appears to be a useful agent for BMT patients with CMV pneumonia. One major side effect of ganciclovir is neutropenia, and there may be reluctance to administer ganciclovir to neutropenic patients. However, there is evidence that CMV infection may directly or indirectly cause marrow suppression. In this situation, the potential benefit of ganciclovir may outweigh the risk. PATIENTS AND METHODS: We retrospectively reviewed the charts of 11 consecutive patients receiving ganciclovir for CMV infection posttransplant. A total of 13 courses of ganciclovir were administered. RESULTS: In 10 of 13 cases, the absolute neutrophil count was higher at the completion of ganciclovir therapy than at the start of treatment, including five cases where the absolute neutrophil count was < 500/mm3 at the time ganciclovir was started. In only one of 13 cases was ganciclovir discontinued due to neutropenia. CONCLUSIONS: We conclude that the administration of ganciclovir is not often associated with the development of severe neutropenia in pediatric marrow transplant recipients and that ganciclovir can be safely administered to patients with CMV disease, who are severely neutropenic.

Adolescent↗
PubMed1993

Unrelated donor BMT for Wiskott-Aldrich syndrome.

The role of allogeneic sibling BMT for children with Wiskott-Aldrich syndrome is established. Mismatched T cell-depleted BMT has been successful, although significant problems with graft rejection, GVHD, and post-transplant lymphoproliferative disorders have been reported. We have performed four BMTs for children with Wiskott-Aldrich syndrome utilizing phenotypically HLA-identical unrelated donors. A non-TBI (total body irradiation) conditioning regimen was utilized, and BM was not T cell-depleted. All patients engrafted and developed significant, although manageable, GVHD. All patients are alive 3+ to 17+ months post-transplant. These results suggest that matched unrelated donor BMT has a definite role in the treatment of Wiskott-Aldrich syndrome.

Acute Disease↗
PubMed1992

Simultaneous use of two retroviral vectors in human gene marking trials: feasibility and potential applications.

Two Moloney murine leukemia virus (Mo-MLV)-based neoR retroviral vectors--LNL6 and G1Na--were used to transduce various human tumor-infiltrating lymphocytes (TIL) populations. These groups included bulk CD(8+)- and CD(4+)-enriched TIL from human renal cell carcinomas and melanomas. Transduction efficiencies averaged 5% for single 4-hr supernatant infections. Integrated provirus could be detected for up to 4 weeks of in vitro culture. LNL6 provirus could be distinguished from G1Na provirus using specific polymerase chain reaction (PCR) primers. A single neomycin phosphotransferase (neoR) gene copy could be detected in 10(5) TIL. Using quantitative PCR, the relative ratio of LNL6 to G1Na copies in the same sample could be determined even at low copy numbers. These preclinical studies demonstrate the feasibility of using two retroviral marking vectors in human gene therapy efforts.

Base Sequence↗
PubMed1992

Retroviral-mediated transfer of the human glucocerebrosidase gene into cultured Gaucher bone marrow.

Gaucher disease, a lysosomal glycolipid storage disorder, results from the genetic deficiency of an acidic glucosidase, glucocerebrosidase (GC). The beneficial effects of allogeneic bone marrow transplantation (BMT) for Gaucher disease suggest that GC gene transduction and the transplantation of autologous hematopoietic stem cells (gene therapy) may similarly alleviate symptoms. We have constructed a retroviral vector, L-GC, produced by a clone of the amphotropic packaging cell line PA317, which transduces the normal human GC cDNA with high efficiency. Whole-marrow mononuclear cells and CD34-enriched cells from a 4-yr-old female with type 3 Gaucher disease were transduced by the L-GC vector and studied in long-term bone marrow culture (LTBMC). Prestimulation of marrow with IL-3 and IL-6, followed by co-cultivation with vector-producing fibroblasts, produced gene transfer into 40-45% of the hematopoietic progenitor cells. The levels of GC expression in progeny cells (primarily mature myelomonocytic) produced by the LTBMC were quantitatively analyzed by Northern blot, Western blot, and glucocerebrosidase enzyme assay. Normal levels of GC RNA, immunoreactive protein, and enzymatic activity were detected throughout the duration of culture. These studies demonstrate that retroviral vectors can efficiently transfer the GC gene into long-lived hematopoietic progenitor cells from the bone marrow of patients with Gaucher disease and express physiologically relevant levels of GC enzyme activity.

Antigens, CD↗
PubMed1992

Two different genes coding for processable and nonprocessable forms of a viral envelope protein can account for the apparent hormonal stimulation of protein processing in W7MG1 lymphoma cells.

In the mouse mammary tumor virus (MMTV)-infected mouse T-lymphoma cell line W7MG1, glucocorticoid hormone regulates two aspects of MMTV gene expression: hormone stimulates MMTV gene transcription and increases the ratio of mature envelope proteins to envelope precursor protein produced. To separate these two effects and determine the mechanism by which hormone regulates the conversion of the envelope precursor Pr74 to the mature cleaved products gp52 and gp33, we constructed expression vectors in which the envelope gene is constitutively transcribed. Surprisingly, the envelope precursor protein Pr74 encoded by two independently isolated, allelic envelope genes behaved differently. Pr74-P (encoded by the ENV/P gene) was processed efficiently to the mature products gp52 and gp33, independently of the level of expression, hormonal induction of cellular genes, or the presence of other MMTV proteins. In contrast, under the same conditions, Pr74-N (encoded by the ENV/N gene) was not processed further despite being relatively stable. In sucrose gradient analyses, Pr74-P sedimented as monomers, whereas Pr74-N was found in high mol wt aggregates of heterogeneous size. Coimmunoprecipitation analysis determined that Pr74-N associated with BiP, whereas Pr74-P did not. This is indicative of improper folding of Pr74-N in the endoplasmic reticulum.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Retroviral vector-mediated gene transfer into primitive human hematopoietic progenitor cells: effects of mast cell growth factor (MGF) combined with other cytokines.

Retroviral vector-mediated gene transfer into human hematopoietic stem cells may permit gene therapy of numerous genetic diseases. Stimulation of marrow with hematopoietic growth factors (HGFs) has been shown to increase the level of retroviral transduction. We have examined the effects of recombinant human mast cell growth factor (MGF), alone and in combination with other HGFs, on the efficiency of gene transfer into human hematopoietic progenitor cells. MGF acts in concert with interleukin 3 (IL-3) and interleukin 6 (IL-6) to increase the percentage of CD34+ progenitors transduced with a retroviral vector expressing the neo gene. The most potent combination of growth factors that we examined, interleukin 1 (IL-1)/IL-3/IL-6/MGF, resulted in the conferral of G418 resistance to 45% of progenitors and long-term culture-initiating cells. Extending the time of cocultivation of the marrow cells with the vector-producing cells did not further increase gene transfer frequency, suggesting that the amount of available vector is not limiting. To analyze the effects of the HGF on gene transfer into more primitive hematopoietic progenitors, CD34+ cells were isolated from marrow samples that were purged of committed progenitor cells by treatment with 4-hydroperoxycyclophosphamide (4-HC). Preculturing the CD34+ 4-HC-treated cells with the combination of four HGF (IL-1/IL-3/IL-6/MGF) permitted transduction of 20%-28% of the progenitors that formed colonies after 30 days in culture. These results demonstrate that MGF in combination with other HGFs enhances gene transduction of human hematopoietic progenitor cells.

Antigens, CD↗
PubMed1991

Toward gene therapy for Gaucher disease.

We are studying the transfer and expression by retroviral vectors of the human glucocerebrosidase (GC) gene into bone marrow cells as a model of gene therapy for genetic diseases of hematopoietic cells. A simple retroviral vector (G2) was developed that contains a normal human GC cDNA under the control of the Moloney murine leukemia virus long-terminal repeat (LTR) enhancer/promoter. Murine bone marrow was transduced with the G2 vector and maintained in long-term bone marrow culture (LTBMC). Expression of the human GC gene in the transduced murine LTBMC cells exceeded the level of endogenous murine GC mRNA. Murine bone marrow cells were also transduced with G2 and transplanted into irradiated syngeneic recipients. High levels of GC gene transfer and expression were seen in day-12 CFU-S foci, and to a lesser extent in the hematopoietic organs 4 months after gene transfer/bone marrow transplant (BMT). Human bone marrow, from a patient with Gaucher disease, was also used in studies of GC gene transduction. Gene transfer into 35-40% of the Gaucher hematopoietic progenitor cells was achieved, following prestimulation of the marrow with recombinant hematopoietic growth factors. Equal rates of gene transfer were obtained using either total marrow mononuclear cells or progenitor cells enriched 100-fold by immunomagnetic bead separation. GC gene transduction corrected the enzymatic deficiency of the Gaucher marrow. Our results demonstrate the potential utility of retroviral vector-mediated gene transfer for gene therapy of Gaucher disease. Current efforts are aimed at achieving more consistent in vivo GC expression in the murine BMT model and demonstrating transduction of pluripotent human hematopoietic stem cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Expression of human glucocerebrosidase following retroviral vector-mediated transduction of murine hematopoietic stem cells.

The human glucocerebrosidase (GC) gene has been expressed in the progeny of murine hematopoietic stem cells following transduction of marrow with a retroviral vector (G2) containing the human GC cDNA. Murine marrow was transduced via co-cultivation following prestimulation in the presence or absence of recombinant IL-3 and IL-6. A high rate of gene transfer and expression (95%) was demonstrated in primary day 12 CFU-S foci following bone marrow transplantation (BMT) of G2-transduced marrow into lethally irradiated syngeneic recipient mice. Immunoreactive human GC protein was also documented in the CFU-S foci. Primary recipient mice were examined 4-6 months following BMT. A higher rate of gene transfer (87%) was seen in hematopoietic organs of recipients of prestimulated donor marrow compared with organs from initially unstimulated marrow (25%). A high rate of expression of human GC was also documented in the prestimulated organs (50%) when compared with the unstimulated group (25%). Secondary BMT was performed using marrow from the long-lived primary recipients. The human GC gene was present in 88% of secondary day 12 CFU-S foci examined in the prestimulated group versus 23% in the unstimulated group. Expression of the human GC gene was documented in secondary day 12 CFU-S foci, providing strong evidence of initial hematopoietic stem cell transduction.

Animals↗
PubMed1990

Expression of human glucocerebrosidase in murine long-term bone marrow cultures after retroviral vector-mediated transfer.

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.

Animals↗
PubMed1990

Comparison of the effects of growth factors on retroviral vector-mediated gene transfer and the proliferative status of human hematopoietic progenitor cells.

We have examined the ability of the recombinant hematopoietic growth factors (HGF) interleukin-3 (IL-3), IL-6, and granulocyte-macrophage colony-stimulating factor (GM-CSF) to increase retroviral vector-mediated gene transfer into human hematopoietic progenitor cells (HPC). The efficiency of neo gene transfer by the N2 vector into human HPC was enhanced by preculture with either GM-CSF or IL-3 (but not IL-6) and with each combination of the three factors. The combination of IL-3 plus IL-6 consistently produced significantly higher levels of G418-resistant colonies (50-60%) than any of the other combinations of HGF tested. Following preculture with HGF and transduction by N2, marrow was maintained in long-term bone marrow culture (LTBMC) for 2 months. The levels of G418-resistant HPC remained stable, and no apparent depletion of total HPC content resulted from the prior exposure to highly stimulatory doses of factors. The proliferative status of the HPC, following exposure to the HGF, was measured as the percentage of HPC that were inhibited from forming colonies by exposure to the S-phase-specific drug, hydroxyurea. The ability of the different HGF to increase the rate of gene transfer by N2 correlated significantly with the extent to which they stimulated HPC proliferation. These results suggest that the mechanism by which HGF increase rates of gene transfer into HPC is by stimulating cell proliferation. Techniques that produce high rates of gene transfer into long-lived human HPC will facilitate studies to quantitate expression of exogenous genes in hematopoietic cells and may be applicable to clinical gene therapy.

Cell Division↗
PubMed1990

Bone marrow transplantation for genetic diseases.

Bone marrow transplantation is the treatment of choice for a number of genetic diseases. Recently, bone marrow transplantation has been increasingly used for erythroid disorders, such as thalassemia and sickle cell anemia. A number of inherited metabolic disorders (i.e., storage diseases, leukodystrophies, and the like) may be corrected with a marrow transplant. Successful correction of genetic diseases with allogeneic bone marrow transplantation lays the groundwork for the use of specific gene therapy.

Bone Marrow Transplantation↗
PubMed1989

Establishment and characterization of adenosine deaminase-deficient human T cell lines.

We have established long term cell lines from a patient with adenosine deaminase (ADA)-deficient severe combined immunodeficiency by stimulation of blood and bone marrow cells with PHA and IL-2 followed by transformation of the activated cells with the human retrovirus HTLV-I. Despite the absence of detectable T cells in the patients blood, cell lines grew that carried the phenotype of mature activated T cells. TJF-2, the line established from blood, was characterized in detail. The concentration of ADA in TJF-2 cells was less than 1% of normal (3.2 U vs 413.0 U). Studies with pharmacologic inhibitors of ADA suggest that the residual adenosine deaminating activity of TJF-2 is from an enzyme distinct from true ADA, a nonspecific aminohydrolyase. Growth of TJF-2 cells was hypersensitive to inhibition by 2'-deoxyadenosine compared to normal T cells (ID50, 55 microM vs greater than 1000 microM). Analysis of 2'-deoxyadenosine-challenged cells showed that TJF-2 cells accumulated significant levels of deoxyadenosine triphosphate, whereas normal T cells did not unless they were also incubated with the ADA inhibitor deoxycoformycin. Southern and Northern blot analysis of these cells revealed a grossly intact ADA gene that produced a normal size ADA mRNA. Yet, despite ADA deficiency, cells of the TJF-2 line were otherwise indistinguishable from HTLV-I-transformed T cells derived from normal donors with respect to dependence on exogenous IL-2 for growth, clonal rearrangement patterns of TCR beta-chain genes, response to PHA, and rapid restoration of cellular volume after hypotonic challenge. The TJF-2 line thus represents a unique HTLV-I-transformed human T cell line exhibiting ADA deficiency and its expected metabolic consequences.

Adenosine Deaminase↗
PubMed1988

Infection of human hematopoietic progenitor cells using a retroviral vector with a xenotropic pseudotype.

In an effort to determine if viral envelope type influences the infectivity of human hematopoietic progenitor cells with retroviral vectors, we have pseudotyped the retroviral vector N2, which confers G418-resistance, in either an amphotropic or xenotropic envelope. Vector titres obtained by the pseudotype procedure were nearly two orders of magnitude lower than the titer obtained when N2 was packaged using the amphotropic PA317 packaging cell line. Despite its low titer, xenotropically pseudotyped N2 generated G418-resistant hematopoietic colonies at levels approaching those observed after bone marrow was infected using vector packaged using PA317 cells. These results suggest that manipulations of vector envelope may lead to improvements in the level of infection of human hematopoietic stem cells.

Animals↗