20 results on '"Sellers, S E"'
Search Results
2. New Aspects on the Safety of Multidrug-Resistance 1 Gene Transfers: no Indication for Clonal Dominance after Long-Term Follow-Up in a Primate Transplantation Model: O329
- Author
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Bozorgmehr, F., Buss, E. C., Laufs, S., Nagy, K. Z., Sellers, S. E., Zeller, W. J., Dunbar, C. E., and Fruehauf, S.
- Published
- 2004
3. Recombinant adeno-associated virus (rAAV)-mediated expression of a human gamma-globin gene in human progenitor-derived erythroid cells
- Author
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Samulski, R. J., Sellers, S. E., Donahue, R. E., Miller, J. L., Nienhuis, A. W., and Young, N. S.
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viruses ,hemic and lymphatic diseases - Abstract
Effective gene therapy for the severe hemoglobin (Hb) disorders, sickle-cell anemia and thalassemia, will require an efficient method to transfer, integrate, and express a globin gene in primary erythroid cells. To evaluate recombinant adeno-associated virus (rAAV) for this purpose, we constructed a rAAV vector encoding a human gamma-globin gene (pJM24/vHS432A gamma). Its 4725-nucleotide genome consists of two 180-bp AAV inverted terminal repeats flanking the core elements of hypersensitive sites 2, 3, and 4 from the locus control region of the beta-globin gene cluster, linked to a mutationally marked A gamma-globin gene (A gamma) containing native promoter and RNA processing signals. CD34+ human hematopoietic cells were exposed to rAAV particles at a multiplicity of infection of 500-1000 and cultured in semisolid medium containing several cytokines. A reverse transcriptase polymerase chain reaction assay distinguished mRNA signals derived from transduced and endogenous human gamma-globin genes. Twenty to 40% of human erythroid burst-forming unit-derived colonies expressed the rAAV-transduced A gamma-globin gene at levels 4-71% that of the endogenous gamma-globin genes. The HbF content of pooled control colonies was 26%, whereas HbF was 40% of the total in pooled colonies derived from rAAV transduced progenitors. These data establish that rAAV containing elements from the locus control region linked to a gamma-globin gene are capable of transferring and expressing that gene in primary human hematopoietic cells resulting in a substantial increase in HbF content.
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- 1994
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4. Recombinant adeno-associated virus (rAAV)-mediated expression of a human gamma-globin gene in human progenitor-derived erythroid cells.
- Author
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Miller, J L, primary, Donahue, R E, additional, Sellers, S E, additional, Samulski, R J, additional, Young, N S, additional, and Nienhuis, A W, additional
- Published
- 1994
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5. Anti-neutrophil-elastase defenses of the lower respiratory tract in alpha 1-antitrypsin deficiency directly augmented with an aerosol of alpha 1-antitrypsin.
- Author
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Hubbard, Richard C., Brantly, Mark L., Sellers, Stephanie E., Mitchell, Marc E., Crystal, Ronald G., Hubbard, R C, Brantly, M L, Sellers, S E, Mitchell, M E, and Crystal, R G
- Subjects
DIAGNOSTIC use of aerosols ,ALPHA 1-antitrypsin ,AEROSOLS ,ALPHA 1-antitrypsin deficiency ,ANIMAL experimentation ,CLINICAL trials ,LUNGS ,NEUTROPHILS ,PERMEABILITY ,PROTEOLYTIC enzymes ,PULMONARY alveoli ,SHEEP ,CHEMICAL inhibitors - Abstract
Study Objective: To determine if aerosolization of purified human plasma alpha 1-antitrypsin is an effective means for increasing lower respiratory anti-neutrophil-elastase defenses in alpha 1-antitrypsin deficiency.Design: Nonrandomized, before-and-after trial with a 7-day treatment period. Companion studies in animals to determine lung epithelial permeability to alpha 1-antitrypsin.Patients: Twelve patients with homozygous Z-type alpha 1-antitrypsin deficiency and mild to moderate emphysema.Interventions: Aerosol administration of human plasma alpha 1-antitrypsin, 100 mg every 12 hours for 7 days. Single, 100-mg aerosol dose to anesthetized sheep with indwelling thoracic lymph duct catheters for direct assessment of lung permeability.Measurements and Main Results: Treatment resulted in increased alpha 1-antitrypsin levels in the lung epithelial lining fluid (0.28 +/- 0.07 microM before therapy to 5.86 +/- 1.03 microM after therapy) and increased anti-neutrophil-elastase capacity (0.78 +/- 0.38 microM before therapy to 4.16 +/- 0.95 microM after therapy). Aerosolized alpha 1-antitrypsin diffused across the respiratory epithelium and entered lung interstitial lymph (in sheep) and reached the systemic circulation (in sheep and humans). No side effects were noted.Conclusion: Short-term aerosol administration of human plasma alpha 1-antitrypsin to patients with alpha 1-antitrypsin deficiency is safe and feasible, resulting in a return to normal of anti-neutrophil-elastase defenses in the lower respiratory tract. The aerosol approach, therefore, merits serious long-term evaluation as an alternative to other parenteral forms of administering therapeutic proteins. [ABSTRACT FROM AUTHOR]- Published
- 1989
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6. The effects of SCF/G-CSF prestimulation on radiation sensitivity and engraftment in nonmyeloablated murine hosts
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Giri, N., Kaushiva, A., Wu, T., Sellers, S. E., and Tisdale, J. F.
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- 2001
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7. Fate of aerosolized recombinant DNA-produced alpha 1-antitrypsin: use of the epithelial surface of the lower respiratory tract to administer proteins of therapeutic importance.
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Hubbard, R C, Casolaro, M A, Mitchell, M, Sellers, S E, Arabia, F, Matthay, M A, and Crystal, R G
- Abstract
To evaluate the possibility of administering therapeutic proteins via the respiratory route, we administered an aerosol of recombinant DNA-produced human alpha 1-antitrypsin (rAAT) to anesthetized sheep and measured levels of the protein in epithelial lining fluid (ELF), lung lymph, blood, and urine. Using a nebulizer that generated aerosol droplets with a mass median aerodynamic diameter of 2.7 micron (55% of droplets were less than 3 micron, a particle size optimal for deposition on the alveolar epithelium), in vitro studies demonstrated that the aerosolized rAAT remained intact and fully functional as an inhibitor of neutrophil elastase. When aerosolized to sheep, the 45-kDa rAAT molecule diffused across the alveolar epithelium, as evidenced by its presence in lung lymph and in blood. Comparison of ELF, lymph, blood, and urine rAAT levels demonstrated that the process was concentration dependent, with highest levels in ELF and in descending concentrations with approximately 10-fold concentration differences in each consecutive compartment, respectively. Importantly, evaluation with aerosolized 125I-labeled rAAT demonstrated that the rAAT molecules that reached the lung lymph and the systemic circulation remained intact as a 45-kDa protein. These results demonstrate the feasibility of using aerosolization to the pulmonary epithelial surface to administer sizeable proteins of therapeutic interest, thus circumventing the necessity of the traditional parenteral modes of administration of such molecules.
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- 1989
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8. Recombinant DNA-produced alpha 1-antitrypsin administered by aerosol augments lower respiratory tract antineutrophil elastase defenses in individuals with alpha 1-antitrypsin deficiency.
- Author
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Hubbard, R C, primary, McElvaney, N G, additional, Sellers, S E, additional, Healy, J T, additional, Czerski, D B, additional, and Crystal, R G, additional
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- 1989
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9. Analysis of origin and optimization of expansion and transduction of circulating peripheral blood endothelial progenitor cells in the rhesus macaque model.
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Hu J, Takatoku M, Sellers SE, Agricola BA, Metzger ME, Donahue RE, and Dunbar CE
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- 3T3 Cells, Analysis of Variance, Animals, Antigens, CD34 genetics, Bacterial Proteins metabolism, Cell Lineage, Cells, Cultured, Clone Cells, Endothelium, Vascular drug effects, Endothelium, Vascular immunology, Erythroid Precursor Cells metabolism, Genetic Vectors, Granulocyte Colony-Stimulating Factor pharmacology, Green Fluorescent Proteins, Hematopoietic Stem Cells, Leukocytes, Mononuclear cytology, Leukocytes, Mononuclear drug effects, Luminescent Proteins metabolism, Macaca mulatta, Mice, Models, Animal, Retroviridae genetics, Transduction, Genetic, Endothelium, Vascular metabolism
- Abstract
Adult marrow-derived cells have been shown to contribute to various nonhematologic tissues and, conversely, primitive cells isolated from nonhematopoietic tissues have been shown to reconstitute hematopoiesis. Circulating endothelial progenitor cells (EPCs) have been reported to be at least partially donor derived after allogeneic bone marrow transplantation, and shown to contribute to neovascularization in murine ischemia models. However, it is unknown whether these EPCs are actually clonally derived from the same population of stem and progenitor cells that reconstitute hematopoiesis, or from another cell population found in the marrow or mobilized blood that is transferred during transplantation. To approach this question, we characterized circulating EPCs and also endothelial cells from large vessels harvested at autopsy from rhesus macaques previously transplanted with retrovirally transduced autologous CD34-enriched peripheral blood stem cells (PBSCs). Endothelial cells were grown in culture for 21-28 days and were characterized as CD31(+) CD14(-) via flow cytometry, as acLDL(+) UEA-1(+) via immunohistochemistry, and as Flk-1(+) by reverse transcriptase-polymerase chain reaction (RT-PCR). Animals had stable vector marking in hematopoietic lineages of 2-15%. Neither cultured circulating EPCs collected in steady state (n = 3), nor endothelial cells grown from large vessels (n = 2), had detectable retroviral marking. EPCs were CD34(+) and could be mobilized into the circulation with granulocyte colony-stimulating factor. Under ex vivo culture conditions, in which CD34(+) cells were optimized to transduce hematopoietic progenitor and stem cells, there was a marked depletion of EPCs. Transduction of EPCs was much more efficient under conditions supporting endothelial cell growth. Further elucidation of the origin and in vivo behavior of EPCs may be possible, using optimized transduction conditions and a vascular injury model.
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- 2002
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10. The effect of multidrug-resistance 1 gene versus neo transduction on ex vivo and in vivo expansion of rhesus macaque hematopoietic repopulating cells.
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Sellers SE, Tisdale JF, Agricola BA, Metzger ME, Donahue RE, Dunbar CE, and Sorrentino BP
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- Animals, Cell Culture Techniques, Cell Division drug effects, Gene Dosage, Genetic Therapy standards, Genetic Vectors adverse effects, Genetic Vectors standards, Hematopoietic Stem Cell Transplantation standards, Hematopoietic Stem Cells drug effects, Humans, Macaca mulatta, Models, Animal, Neomycin, Transduction, Genetic methods, Transduction, Genetic standards, Drug Resistance, Microbial genetics, Genes, MDR genetics, Genetic Therapy methods, Hematopoietic Stem Cell Transplantation methods
- Abstract
Transduction of murine stem cells with a multidrug-resistance 1 gene (MDR1) retrovirus results in dramatic ex vivo and in vivo expansion of repopulating cells accompanied by a myeloproliferative disorder. Given the use of MDR1-containing vectors in human trials, investigations have been extended to nonhuman primates. Peripheral blood stem cells from 2 rhesus monkeys were collected, CD34-enriched, split into 2 portions, and transduced with either MDR1 vectors or neo vectors and continued in culture for a total of 10 days before reinfusion. At engraftment, the copy number in granulocytes was extremely high from both MDR vectors and neo vectors, but the copy number fell to 0.01 to 0.05 for both. There were no perturbations of the leukocyte count or differential noted. After 3 cycles of stem cell factor/granulocyte colony-stimulating factor, there were no changes in the levels of MDR1 vector- or neo vector-containing cells. There was no evidence for expansion of MDR1 vector-transduced cells. Long-term engraftment with MDR1 vector- and neo vector-transduced cells occurred despite prolonged culture.
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- 2001
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11. Many multipotential gene-marked progenitor or stem cell clones contribute to hematopoiesis in nonhuman primates.
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Kim HJ, Tisdale JF, Wu T, Takatoku M, Sellers SE, Zickler P, Metzger ME, Agricola BA, Malley JD, Kato I, Donahue RE, Brown KE, and Dunbar CE
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- Animals, Antigens, CD34 blood, Cell Differentiation, Colony-Forming Units Assay, Gene Transfer Techniques, Genes, Reporter, Genetic Therapy methods, Genetic Vectors, Hematopoietic Stem Cell Mobilization, Humans, Kanamycin Kinase genetics, Macaca mulatta, Retroviridae, Transfection, Whole-Body Irradiation, B-Lymphocytes cytology, Hematopoiesis, Hematopoietic Stem Cells cytology, Hematopoietic Stem Cells physiology, T-Lymphocytes cytology
- Abstract
Retroviral insertion site analysis was used to track the contribution of retrovirally transduced primitive progenitors to hematopoiesis after autologous transplantation in the rhesus macaque model. CD34-enriched mobilized peripheral blood cells were transduced with retroviral marking vectors containing the neo gene and were reinfused after total body irradiation. High-level gene transfer efficiency allowed insertion site analysis of individual myeloid and erythroid colony-forming units (CFU) and of highly purified B- and T-lymphoid populations in 2 animals. At multiple time points up to 1 year after transplantation, retroviral insertion sites were identified by performing inverse polymerase chain reaction and sequencing vector-containing CFU or more than 99% pure T- and B-cell populations. Forty-eight unique insertion sequences were detected in the first animal and also in the second animal, and multiple clones contributed to hematopoiesis at 2 or more time points. Multipotential clones contributing to myeloid and lymphoid lineages were identified. These results support the concept that hematopoiesis in large animals is polyclonal and that individual multipotential stem or progenitor cells can contribute to hematopoiesis for prolonged periods. Gene transfer to long-lived, multipotent clones is shown and is encouraging for human gene therapy applications.
- Published
- 2000
12. Introduction of a xenogeneic gene via hematopoietic stem cells leads to specific tolerance in a rhesus monkey model.
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Heim DA, Hanazono Y, Giri N, Wu T, Childs R, Sellers SE, Muul L, Agricola BA, Metzger ME, Donahue RE, Tisdale JF, and Dunbar CE
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- Animals, Base Sequence, Blood Transfusion, Autologous, DNA Primers genetics, Gene Expression, Genetic Therapy methods, Genetic Vectors, Hematopoietic Stem Cell Transplantation, Kanamycin Kinase genetics, Kanamycin Kinase immunology, Lymphocyte Transfusion, Lymphocytes immunology, Lymphocytes metabolism, Macaca mulatta, Models, Biological, RNA, Messenger biosynthesis, RNA, Messenger genetics, Transduction, Genetic, Transplantation, Autologous, Hematopoietic Stem Cells immunology, Hematopoietic Stem Cells metabolism, Immune Tolerance
- Abstract
Host immune responses against foreign transgenes may be a major obstacle to successful gene therapy. To clarify the impact of an immune response to foreign transgene products on the survival of genetically modified cells, we studied the in vivo persistence of cells transduced with a vector expressing a foreign transgene compared to cells transduced with a nonexpressing vector in the clinically predictive rhesus macaque model. We constructed retroviral vectors containing the neomycin phosphotransferase gene (neo) sequences modified to prevent protein expression (nonexpressing vectors). Rhesus monkey lymphocytes or hematopoietic stem cells (HSCs) were transduced with nonexpressing and neo-expressing vectors followed by reinfusion, and their in vivo persistence was studied. While lymphocytes transduced with a nonexpressing vector could be detected for more than 1 year, lymphocytes transduced with a neo-expressing vector were no longer detectable within several weeks of infusion. However, five of six animals transplanted with HSCs transduced with nonexpression or neo-expression vectors, and progeny lymphocytes marked with either vector persisted for more than 2 years. Furthermore, in recipients of transduced HSCs, infusion of mature lymphocytes transduced with a second neo-expressing vector did not result in elimination of the transduced lymphocytes. Our data show that introduction of a xenogeneic gene via HSCs induces tolerance to the foreign gene products. HSC gene therapy is therefore suitable for clinical applications where long-term expression of a therapeutic or foreign gene is required.
- Published
- 2000
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13. Prolonged high-level detection of retrovirally marked hematopoietic cells in nonhuman primates after transduction of CD34+ progenitors using clinically feasible methods.
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Wu T, Kim HJ, Sellers SE, Meade KE, Agricola BA, Metzger ME, Kato I, Donahue RE, Dunbar CE, and Tisdale JF
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- Animals, Blotting, Southern, Cells, Cultured, Colony-Forming Units Assay, Fibronectins metabolism, Granulocyte Colony-Stimulating Factor pharmacology, Interleukin-3 pharmacology, Interleukin-6 pharmacology, Macaca mulatta, Membrane Proteins pharmacology, Polymerase Chain Reaction, Stem Cell Factor pharmacology, Stromal Cells metabolism, Thrombopoietin pharmacology, Antigens, CD34 genetics, Gene Transfer Techniques, Hematopoietic Stem Cells metabolism, Retroviridae genetics
- Abstract
Low-level retroviral transduction and engraftment of hematopoietic long-term repopulating cells in large animals and humans remain primary obstacles to the successful application of hematopoietic stem cell (HSC) gene transfer in humans. Recent studies have reported improved efficiency by including stromal cells (STR), or the fibronectin fragment CH-296 (FN), and various cytokines such as flt3 ligand (FLT) during ex vivo culture and transduction in nonhuman primates. In this work, we extend our studies using the rhesus competitive repopulation model to further explore optimal and clinically feasible peripheral blood (PB) progenitor cell transduction methods. First, we compared transduction in the presence of either preformed autologous STR or immobilized FN. Long-term clinically relevant gene marking levels in multiple hematopoietic lineages from both conditions were demonstrated in vivo by semiquantitative PCR, colony PCR, and genomic Southern blotting, suggesting that FN could replace STR in ex vivo transduction protocols. Second, we compared transduction on FN in the presence of IL-3, IL-6, stem cell factor (SCF), and FLT (our best cytokine combination in prior studies) with a combination of megakaryocyte growth and development factor (MGDF), SCF, and FLT. Gene marking levels were equivalent in these animals, with no significant effect on retroviral gene transfer efficiency assessed in vivo by the replacement of IL-3 and IL-6 with MGDF. Our results indicate that SCF/G-CSF-mobilized PB CD34+ cells are transduced with equivalent efficiency in the presence of either STR or FN, with stable long-term marking of multiple lineages at levels of 10-15% and transient marking as high as 54%. These results represent an advance in the field of HSC gene transfer using methods easily applied in the clinical setting.
- Published
- 2000
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14. No discrepancy between in vivo gene marking efficiency assessed in peripheral blood populations compared with bone marrow progenitors or CD34+ cells.
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Sellers SE, Tisdale JF, Bodine DM, Williams DA, Karlsson S, Meztger M, Donahue RE, and Dunbar CE
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- Animals, Base Sequence, DNA Primers, Drug Resistance genetics, Genetic Markers, Humans, Macaca mulatta, Neomycin pharmacology, Transduction, Genetic, Antigens, CD34 immunology, Blood Cells metabolism, Gene Transfer Techniques standards, Hematopoietic Stem Cells metabolism
- Abstract
Reports of 1- to 2-log higher gene transfer levels in purified CD34+ cells or marrow CFU compared with levels in mature circulating blood cells after transplantation of retrovirally transduced primitive human hematopoietic cells have resulted in concern that transduced progenitors do not contribute proportionally to ongoing hematopoiesis (Kohn et al., 1995; Brenner, 1996). To study the issue in a relevant large animal, we analyzed samples of mature blood cells, marrow CD34-enriched cells and marrow CD34-depleted cells, and marrow CFU from a cohort of 11 rhesus transplanted with retrovirally transduced cells and followed for up to 5.5 years. They were transplanted with CD34-enriched bone marrow (BM) or G-CSF/SCF-mobilized peripheral blood (PB) cells transduced with vectors containing either neo, human glucocerebrosidase, or murine adenosine deaminase genes. There were no significant differences between the levels of vector sequences found in BM CD34+ cells, BM CD34- cells, PB granulocytes, or PB mononuclear cells (MNCs) in any animal. In four animals transplanted with SCF/G-CSF-primed BM cells and analyzed 3-6 months posttransplantation, the percentage of CFU containing the neo vector appeared to be 1 log higher than the representation of marked cells in the PB of these animals, but this discrepancy did not persist at time points greater than 6 months posttransplantation. The level of CFU marking was no higher than PB granulocyte or MNC marking at any time points in the other animals. Low levels of mature gene-modified cells probably reflect poor transduction of repopulating stem cells, not a block in differentiation or specific immune rejection of mature cells. This study represents the longest follow-up of primates transplanted with transduced hematopoietic cells, and it is encouraging that the levels of vector-containing cells appear stable for up to 5 years.
- Published
- 1999
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15. Ex vivo expansion of genetically marked rhesus peripheral blood progenitor cells results in diminished long-term repopulating ability.
- Author
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Tisdale JF, Hanazono Y, Sellers SE, Agricola BA, Metzger ME, Donahue RE, and Dunbar CE
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- Animals, Cells, Cultured transplantation, Coculture Techniques, Colony-Forming Units Assay, Filgrastim, Genes, Reporter, Genetic Markers, Genetic Vectors, Graft Survival, Granulocyte Colony-Stimulating Factor pharmacology, Hematopoietic Stem Cell Mobilization, Hematopoietic Stem Cells drug effects, Interleukin-3 pharmacology, Interleukin-6 pharmacology, Kanamycin Kinase genetics, Proto-Oncogene Proteins pharmacology, Radiation Chimera, Receptor Protein-Tyrosine Kinases pharmacology, Recombinant Proteins, Stem Cell Factor pharmacology, Stromal Cells cytology, Vascular Endothelial Growth Factor Receptor-1, Whole-Body Irradiation, Hematopoietic Stem Cell Transplantation, Hematopoietic Stem Cells cytology, Macaca mulatta blood
- Abstract
The possibility of primitive hematopoietic cell ex vivo expansion is of interest for both gene therapy and transplantation applications. The engraftment of autologous rhesus peripheral blood (PB) progenitors expanded 10 to 14 days were tracked in vivo using genetic marking. Stem cell factor (SCF)/granulocyte colony-stimulating factor (G-CSF)-mobilized and CD34-enriched PB cells were divided into two equal aliquots and transduced with one of two retroviral vectors carrying the neomycin-resistance gene (neo) for 4 days in the presence of interleukin-3 (IL-3), IL-6, and SCF in the first 5 animals, IL-3/IL-6/SCF/Flt-3 ligand (FLT) in 2 subsequent animals, or IL-3/IL-6/SCF/FLT plus an autologous stromal monolayer (STR) in the final 2. At the end of transduction period, one aliquot (nonexpanded) from each animal was frozen, whereas the other was expanded under the same conditions but without vector for a total of 14 days before freezing. After total body irradiation, both the nonexpanded and expanded transduced cells were reinfused. Despite 5- to 13-fold higher cell and colony-forming unit (CFU) doses from the expanded fraction of marked cells, there was greater short- and long-term marking from the nonexpanded cells in all animals. In animals receiving cells transduced and expanded in the presence of IL-3/IL-6/SCF/FLT, engraftment by the marked expanded cells was further diminished. This discrepancy was even more pronounced in the animals who received cells transduced and expanded in the presence of FLT and autologous stroma, with no marking detectable from the expanded cells. Despite lack of evidence for expansion of engrafting cells, we found that the addition of FLT and especially STR during the initial brief transduction period increased engraftment with marked cells into a clinically relevant range. Levels of marked progeny cells originating from the nonexpanded aliqouts were significantly higher than that seen in previous 4 animals receiving cells transduced in the presence of IL-3/IL-6/SCF, with levels of 10% to 20% confirmed by Southern blotting from the nonexpanded IL-3/IL-6/SCF/FLT/STR graft compared with 0.01% in the original IL-3/IL-6/SCF cohort. These results suggest that, although expansion of PB progenitors is feasible ex vivo, their contribution towards both short- and long-term engraftment is markedly impaired. However, a brief transduction in the presence of specific cytokines and stromal support allows engraftment with an encouraging number of retrovirally modified cells.
- Published
- 1998
16. Transplantation and gene transfer of the human glucocerebrosidase gene into immunoselected primate CD34+Thy-1+ cells.
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Donahue RE, Byrne ER, Thomas TE, Kirby MR, Agricola BA, Sellers SE, Gaudernack G, Karisson S, and Lansdorp PM
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- Adenoviruses, Human genetics, Adenoviruses, Human isolation & purification, Animals, Antigens, CD34 analysis, CD2 Antigens analysis, CD8 Antigens analysis, DNA, Recombinant analysis, Defective Viruses genetics, Defective Viruses isolation & purification, Genetic Vectors genetics, Genetic Vectors isolation & purification, Graft Survival, Humans, Immunomagnetic Separation, Lymphocyte Subsets chemistry, Lymphocyte Subsets immunology, Lymphocyte Subsets virology, Macaca mulatta, Polymerase Chain Reaction, Proviruses genetics, Proviruses isolation & purification, Transplantation, Autologous, Bone Marrow Cells, Gene Transfer Techniques, Glucosylceramidase genetics, Hematopoietic Stem Cell Transplantation, Hematopoietic Stem Cells metabolism, Thy-1 Antigens analysis
- Abstract
In an attempt to improve our gene transfer efficiency into hematopoietic stem cells and to evaluate the capacity of immunoselected CD34+Thy-1+(CDw90) cells to reconstitute hematopoiesis following myeloablation, bone marrow (BM) transplantation was performed using autologous, immunoselected CD34+Thy-1+ cells in rhesus macaques. BM samples were positively selected for cells that express CD34, further subdivided using high gradient immunomagnetic selection for cells that express Thy-1, and transduced using a 7-day supernatant transduction protocol with a replication-defective retroviral vector that contained the human glucocerebrosidase (GC) gene. Circulating leukocytes were evaluated using a semiquantitative polymerase chain reaction (PCR) assay for the human GC gene, with the longest surviving animal evaluated at day 558. Provirus was detected at all time points in both CD20+ B cells and CD2+ dim T cells, but long-term gene transfer was not observed in the granulocyte population. The CD2+ dim population was phenotypically identified as being CD8+ natural killer cells. By day 302 and day 330, both the CD2+ bright and dim cell populations and sorted CD4+ and CD8+ cells had detectable provirus. Vector-derived GC mRNA was detected by reverse transcriptase (RT)-PCR analysis as far out as day 588. Thus, CD34+Thy-1+ cells isolated using high gradient magnetic separation techniques can engraft, be transduced with a replication-defective retroviral vector, and contribute to CD20+ B lymphocytes, CD8+ T lymphocytes, and CD4+ T lymphocytes; making them a suitable cell population to target for gene therapies involving lymphocytes.
- Published
- 1996
17. Peripheral blood CD34+ cells differ from bone marrow CD34+ cells in Thy-1 expression and cell cycle status in nonhuman primates mobilized or not mobilized with granulocyte colony-stimulating factor and/or stem cell factor.
- Author
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Donahue RE, Kirby MR, Metzger ME, Agricola BA, Sellers SE, and Cullis HM
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- ADP-ribosyl Cyclase, ADP-ribosyl Cyclase 1, Animals, Antigens, CD34 analysis, Antigens, Differentiation analysis, Blood Cells cytology, Bone Marrow Cells, Cell Cycle, Cell Separation, Cells, Cultured, Flow Cytometry, Granulocyte Colony-Stimulating Factor pharmacology, Immunophenotyping, Leukapheresis, Leukocyte Count, Lymphocyte Subsets, Macaca mulatta, N-Glycosyl Hydrolases analysis, Stem Cell Factor pharmacology, Antigens, CD, Blood Cells immunology, Bone Marrow immunology, Thy-1 Antigens metabolism
- Abstract
Granulocyte colony-stimulating factor (G-CSF) and stem cell factor (SCF) have been shown to stimulate the circulation of hematopoietic progenitor cells in both mice and nonhuman primates. We evaluated the immunophenotype and cell cycle status of CD34+ cells isolated from the bone marrow (BM) and leukapheresis product of cytokine-mobilized nonhuman primates. CD34+ cells were isolated from rhesus macaques that had received no cytokine therapy, 100 micrograms/kg/d G-CSF, 200 micrograms/kg/d SCF, or a combination of both 100 micrograms/kg/d G-CSF and 200 micrograms/kg/d SCF as a subcutaneous injection for 5 days. BM was aspirated before (day 0) and on the last day (day 5) of cytokine administration. On days 4 and 5, peripheral blood (PB) mononuclear cells were collected using a novel method of leukapheresis. Threefold more PB mononuclear cells were collected from animals receiving G-CSF alone or G-CSF and SCF than from animals that had received either SCF alone or no cytokine therapy. CD34+ cells were positively selected using an immunoadsorptive system from the BM, PB, and/or leukapheresis product. Threefold and 10-fold more CD34+ cells were isolated from the leukapheresis product of animals receiving G-CSF or G-CSF and SCF, respectively, than from animals receiving no cytokine therapy or SCF alone. The isolated CD34+ cells were immunophenotyped using CD34-allophycocyanin, CD38-fluorescein isothiocyanate, and Thy-1-phycoerythrin. These cells were later stained with 4', 6-diamidino-2-phenylindole for simultaneous DNA analysis and immunophenotyping. BM-derived CD34+ cells did not differ significantly in cell cycle status and Thy-1 or CD38 phenotype before or after G-CSF and/or SCF administration. Similarly, CD34+ cells isolated from the leukapheresis product did not differ significantly in immunophenotype or cell cycle status before or after G-CSF and/or SCF administration. However, there were consistent differences in both immunophenotype and cell cycle status between BM- and PB-derived CD34+ cells. CD34+ cells isolated from the PB consistently had a smaller percentage of cells in the S+G2/M phase of the cell cycle and had a higher percentage of cells expressing Thy-1 than did CD34+ cells isolated from the BM. A greater proportion of PB-derived CD34+ cells were in the S+G2/M phase of the cell cycle after culture in media supplemented with interleukin-6 and SCF, However, culturing decreased the proportion of CD34+ cells expressing Thy-1.
- Published
- 1996
18. Infection of mononucleated phagocytes with human cytomegalovirus.
- Author
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Maciejewski JP, Bruening EE, Donahue RE, Sellers SE, Carter C, Young NS, and St Jeor S
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- Antigens, Viral analysis, Base Sequence, Cell Division, Cell Survival, Cells, Cultured, Cytomegalovirus genetics, DNA, Viral, Humans, Macrophages cytology, Macrophages metabolism, Macrophages microbiology, Molecular Sequence Data, Monocytes cytology, Monocytes metabolism, Monokines biosynthesis, Phenotype, Polymerase Chain Reaction, Promoter Regions, Genetic, Virus Replication, Cytomegalovirus physiology, Monocytes microbiology
- Abstract
Human cytomegalovirus (HCMV) can be isolated from peripheral blood leukocytes; however, in vitro, only abortive infection of monocytes, lymphocytes, and granulocytes has been detected. These studies demonstrate that freshly isolated monocytes can be infected with HCMV. Infection of monocytes was not associated with loss of cell viability. The virus replication cycle in monocytes resembled that observed in fibroblasts but the virus yield was approximately 0.1% of that observed in fibroblasts. Transient phenotypical changes occurred in HCMV-infected monocytes. Virus persists in infected monocytes upon differentiation to macrophages, suggesting that monocytes may serve as a carrier of HCMV and a vector for viral dissemination. Differentiated mononuclear phagocytes appear to support a productive HCMV infection. Using a recombinant HCMV strain to express beta-galactosidase, we were able to transduce the bacterial beta-galactosidase gene into monocytes and macrophages.
- Published
- 1993
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19. Anesthesia with ketaset plus in guinea pigs and hamsters.
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Mulder JB, Johnson HB Jr, McKee GS, and Sellers SE
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- Animals, Drug Combinations, Female, Male, Anesthesia veterinary, Benzhydryl Compounds, Cricetinae, Guinea Pigs, Ketamine, Promazine
- Published
- 1979
20. Replacement therapy for alpha 1-antitrypsin deficiency associated with emphysema.
- Author
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Wewers MD, Casolaro MA, Sellers SE, Swayze SC, McPhaul KM, Wittes JT, and Crystal RG
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- Adult, Evaluation Studies as Topic, Female, Humans, Infusions, Parenteral, Lung enzymology, Male, Middle Aged, Neutrophils enzymology, Pancreatic Elastase antagonists & inhibitors, Pulmonary Emphysema etiology, Smoking, alpha 1-Antitrypsin administration & dosage, alpha 1-Antitrypsin Deficiency, Pulmonary Emphysema drug therapy, alpha 1-Antitrypsin therapeutic use
- Abstract
In patients with alpha 1-antitrypsin deficiency, the development of emphysema is believed to be caused by the unchecked action of proteases on lung tissue. We evaluated the feasibility, safety, and biochemical efficacy of intermittent infusions of alpha 1-antitrypsin in the treatment of patients with alpha 1-antitrypsin deficiency. Twenty-one patients were given 60 mg of active plasma-derived alpha 1-antitrypsin per kilogram of body weight, once a week for up to six months. After a steady state had been reached, the group had trough serum levels of alpha 1-antitrypsin of 126 +/- 1 mg per deciliter as compared with 30 +/- 1 mg per deciliter before treatment, and serum anti-neutrophil elastase capacities of 13.3 +/- 0.1 microM as compared with 5.4 +/- 0.1 microM. The alpha 1-antitrypsin level in the epithelial-lining fluid of the lungs was 0.46 +/- 0.16 microM before treatment, and the anti-neutrophil elastase capacity was 0.81 +/- 0.13 microM. Six days after infusion, alpha 1-antitrypsin levels (1.89 +/- 0.17 microM) and anti-neutrophil elastase capacities (1.65 +/- 0.13 microM) in the lining fluid were significantly increased (P less than 0.0001). Because of the chronicity of the disorder and the lack of sensitive measures of lung destruction, the clinical efficacy of this therapy could not be studied rigorously. No changes in lung function were observed in our patients over six months of treatment. The only important adverse reactions to the 507 infusions were four episodes of self-limited fever. This study demonstrates that infusions of alpha 1-antitrypsin derived from plasma are safe and can reverse the biochemical abnormalities in serum and lung fluid that characterize this disorder. Together with lifetime avoidance of cigarette smoking, replacement therapy with alpha 1-antitrypsin may be a logical approach to long-term medical treatment.
- Published
- 1987
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