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Biomedical subjects

D M Bodine

Publications and source records attributed to D M Bodine.

At least 55 records · Page 3Linked to original sources
PubMed1996

The level of mRNA encoding the amphotropic retrovirus receptor in mouse and human hematopoietic stem cells is low and correlates with the efficiency of retrovirus transduction.

The low level of amphotropic retrovirus-mediated gene transfer into human hematopoietic stem cells (HSC) has been a major impediment to gene therapy for hematopoietic diseases. In the present study, we have examined amphotropic retrovirus receptor (amphoR) and ecotropic retrovirus receptor mRNA expression in highly purified populations of mouse and human HSC. Murine HSC with low to undetectable levels of amphoR mRNA and relatively high levels of ecotropic retrovirus receptor mRNA were studied. When these HSC were analyzed simultaneously for ecotropic and amphotropic retrovirus transduction, ecotropic provirus sequences were detected in 10 of 13 long-term repopulated animals, while amphotropic proviral sequences were detected in only one recipient. A second distinct population of murine HSC were isolated that express 3-fold higher levels of amphoR mRNA. When these HSC were analyzed simultaneously for ecotropic and amphotropic retrovirus transduction, 11 of 11 repopulated mice contained ecotropic provirus and 6 of 11 contained amphotropic provirus sequences, a significant increase in the amphotropic retrovirus transduction (P = 0.018). These results indicate that, among the heterogeneous populations of HSC present in adult mouse bone marrow, the subpopulation with the highest level of amphoR mRNA is more efficiently transduced by amphotropic retrovirus. In a related study, we found low levels of human amphoR mRNA in purified populations of human HSC (CD34+ CD38-) and higher levels in committed progenitor cells (CD34+ CD38+). We conclude that the amphoR mRNA level in HSC correlates with amphotropic retrovirus transduction efficiency.

Animals↗
PubMed1996

Reversion of monochromosome-mediated suppression of tumorigenicity in malignant melanoma by retroviral transduction.

We have developed a general strategy to reverse monochromosome suppression of the malignant phenotypes by retroviral transduction. Our approach involved the introduction of a retroviral expression vector-carried cDNA library into a chromosome 6-suppressed melanoma subline UACC-903(+6) [J. M. Trent et al., Science (Washington DC), 247: 568-571, 1990]. The cDNA library was constructed from polyadenylated RNA isolated from the suppressed UACC-903(+6) cells, packaged into high-titer amphotropic retrovirus particles, and transduced into UACC-903(+6) cells. Revertant his(R) transductants were selected by isolating colony-forming cells in soft agar. A total of 121 large (> 150 microm) colonies was picked from soft agar culture with 18 of 121 (15%) established as permanent sublines. The revertant sublines demonstrated 7-58% cloning efficiency upon plating in agar, in contrast to <0.05% for the UACC-903(+6) subline. All 18 revertant sublines, termed SRS1-SRS18 (for "selection of revertants for suppression"), displayed a reduced population-doubling time, with 9 of 18 showing focus formation in monolayer similar to the parental (nonsuppressed) cell line. Preliminary evidence for reversion of the suppressed phenotype by injection of cells into athymic nude mice has been completed for one revertant subline. Southern analysis has demonstrated integration of the retroviral vector sequence in all 18 sublines. This approach should facilitate the identification of genes involved in the tumorigenic phenotype of malignant melanoma, and is readily adaptable to other model systems.

Base Sequence↗
PubMed1996

Bone marrow collected 14 days after in vivo administration of granulocyte colony-stimulating factor and stem cell factor to mice has 10-fold more repopulating ability than untreated bone marrow.

We have examined the repopulating ability of bone marrow and peripheral blood cells collected immediately and at intervals after treatment of donor mice with the combination of granulocyte colony-stimulating factor (G-CSF) and stem cell factor (SCF). Using a competitive repopulation assay we showed that the repopulating ability of peripheral blood cells was highest immediately after cytokine treatment and declined to normal levels within 6 weeks of the termination of treatment with G-CSF and SCF. In contrast the repopulating ability of bone marrow cells was low immediately after cytokine treatment and increased to levels that were 10-fold or more greater than marrow from untreated mice by 14 days after termination of treatment with G-CSF and SCF. This high level of repopulating activity declined to normal levels by 6 weeks after termination of treatment with G-CSF and SCF. The high level of repopulating ability was confirmed by injecting cells from G-CSF- and SCF-treated donors into unconditioned recipients. Peripheral blood cells collected immediately after treatment with G-CSF and SCF engrafted into unconditioned mice sevenfold better than an equivalent number of bone marrow cells from untreated mice. Likewise, bone marrow cells collected 14 days after treatment of the donor animal with G-CSF and SCF engrafted at 10-fold higher levels than an equivalent number of bone marrow cells from untreated mice. We conclude that the treatment of donor mice with G-CSF and SCF causes a transient increase in the repopulating ability of peripheral blood and later of bone marrow. These observations may have applications to clinical hematopoietic stem cell transplantation.

Animals↗
PubMed1996

The Dtk receptor tyrosine kinase, which binds protein S, is expressed during hematopoiesis.

Dtk (Tyro 3/Sky/Rse/Brt/Tif) belongs to a recently recognized subfamily of receptor tyrosine kinases that also includes Ufo (Axl/Ark) and Mer (Eyk). Ligands for Dtk and Ufo have been identified as protein S and the related molecule Gas6, respectively. This study examined expression of Dtk during ontogeny of the hematopoietic system and compared the pattern of expression with that of Ufo. Both receptors were abundantly expressed in differentiating embryonic stem cells, yolk sac blood islands, para-aortic splanchnopleural mesoderm, fractionated AA4+ fetal liver cells, and fetal thymus from day 14 until birth. Although Ufo was expressed at moderate levels in adult bone marrow, expression of Dtk in this tissue was barely detectable. In adult bone marrow subpopulations fractionated using counterflow centrifugal elutriation, immunomagnetic bead selection for lineage-depletion and FACS sorting for c-kit expression, very low levels of Dtk and/or Ufo were detected in some cell fractions. These results suggest that Dtk and Ufo are likely to be involved in the regulation of hematopoiesis, particularly during the embryonic stages of blood cell development.

Animals↗
PubMed1995

Improved engraftment of human hematopoietic cells in severe combined immunodeficient (SCID) mice carrying human cytokine transgenes.

We have generated immunodeficient scid-/scid- (SCID)-transgenic mice expressing the genes for human interleukin 3, granulocyte/macrophage-colony stimulating factor, and stem cell factor. We have compared engraftment and differentiation of human hematopoietic cells in transgenic SCID mice with two strains of nontransgenic SCID mice. Human bone marrow cells carrying the CD34 antigen or human umbilical cord blood were injected into sublethally irradiated recipients. Human DNA was detected by polymerase chain reaction in peripheral blood and bone marrow of 14 of 28 transgenic SCID mice after transplantation, but in only 2 of 15 nontransgenic SCID littermates at a 10-fold lower level. Bone marrow cultures 8 wk after transplantation of cord blood gave rise to human burst-forming unit erythroid, colony-forming unit granulocyte/macrophage, or granulocyte/erythroid/macrophage/megakaryocyte colonies. Engraftment was observed for up to 6 mo in transgenic SCID mice, twice as long as nontransgenic littermates or previous studies in which transplanted SCID mice were given daily injections of growth factors. We conclude that the level and duration of engraftment of human cells in SCID mice can be improved by expression of human cytokine transgenes and that transgenic SCID mice are an efficient model system for the study of human hematopoiesis.

Animals↗
PubMed1995

Dysregulated expression of GATA-1 following retrovirus-mediated gene transfer into murine hematopoietic stem cells increases erythropoiesis.

Retrovirus-mediated gene transfer was used to study the effects of dysregulated expression of the zinc-finger transcription factor, GATA-1, which has been shown to be required for erythropoiesis. A retroviral vector (PGK-GATA-1) was constructed with the murine GATA-1 gene linked to the human phosphoglycerate kinase (PGK) promoter. Expression of GATA-1 was demonstrated by super-shift analysis with a monoclonal antibody against murine GATA-1 using extracts of nonerythroid cytotoxic T-lymphocyte line (CTLL) cells transduced with the PGK-GATA-1 virus. Mouse bone marrow cells were transduced in vitro and transplanted into recipient animals. Polymerase chain reaction (PCR) analysis performed on DNA extracted from peripheral blood 12 to 40 weeks posttransplantation demonstrated the presence of the PGK-GATA-1 provirus. Proviral integrity and copy number were demonstrated by Southern blot analysis of DNA from spleen, thymus, and bone marrow tissues from the long-term animals. At 16 weeks posttransplant, animals that received cells transduced by the GATA-1 virus maintained a lower white blood cell (WBC) count and absolute neutrophil count (ANC) and a higher red blood cell (RBC) count than control animals that received cells transduced with a virus containing a neor gene. Erythropoiesis was stimulated in GATA-1 and control animals by phlebotomy. GATA-1 animals required more extensive phlebotomy to reach a hematocrit less than 25 and their hematocrit returned to normal levels sooner than control animals. The effect of twice-daily injections of 10 U recombinant erythropoietin (epo) was also examined. The hematocrit of GATA-1 animals showed a more rapid and elevated response to epo than the hematocrit of control animals. These data suggest that dysregulated expression of GATA-1 in primitive hematopoietic cells enlarges the pool of epo-responsive erythroid progenitor cells.

Anemia↗
PubMed1995

Pluripotent hematopoietic stem cells contain high levels of mRNA for c-kit, GATA-2, p45 NF-E2, and c-myb and low levels or no mRNA for c-fms and the receptors for granulocyte colony-stimulating factor and interleukins 5 and 7.

Pluripotent hematopoietic stem cells (PHSCs) were highly enriched from mouse bone marrow by counterflow centrifugal elutriation, lineage subtraction, and fluorescence-activated cell sorting based on high c-kit receptor expression (c-kitBR). We used reverse transcriptase polymerase chain reaction to assay the c-kitBR subset and the subsets expressing low (c-kitDULL) and no (c-kitNEG) c-kit receptor for expression of mRNA encoding hematopoietic growth factor receptors and transcription factors. The c-kitBR cells had approximately 3.5-fold more c-kit mRNA than unfractionated bone marrow cells. The c-kitDULL cells had 47-58% of the c-kit mRNA found in c-kitBR cells and the c-kitNEG cells had 4-9% of the c-kit mRNA present in c-kitBR cells. By comparing mRNA levels in c-kitBR cells (enriched for PHSCs) with those of unfractionated bone marrow, we demonstrated that c-kitBR cells contained low or undetectable levels of mRNA for c-fms, granulocyte colony-stimulating factor receptor, interleukin 5 receptor (IL-5R), and IL-7R. These same cells had moderate levels of mRNA for erythropoietin receptor, IL-3R subunits IL-3R alpha (SUT-1), AIC-2A, and AIC-2B, IL-6R and its partner gp-130, and the transcription factor GATA-1 and high levels of mRNA for transcription factors GATA-2, p45 NF-E2, and c-myb. We conclude from these findings that PHSCs are programmed to interact with stem cell factor, IL-3, and IL-6 but not with granulocyte or macrophage colony-stimulating factor. These findings also indicate that GATA-2, p45 NF-E2, and c-myb activities may be involved in PHSC maintenance or proliferation.

Animals↗
PubMed1995

Microcytic anemia in mk/mk mice is not corrected by retroviral-mediated gene transfer of wild-type p45 NF-E2.

Mice homozygous for the mk mutation have a severe hypochromic, microcytic anemia that is characterized by a decreased mean-corpuscular hemoglobin concentration and balanced alpha- and beta-globin-chain synthesis. Transplantation studies have shown that the defect in homozygous mk/mk mice is intrinsic to both the hematopoietic system and the gut. The gene for the hematopoietic-specific transcription factor, p45 NF-E2, has been found to cosegregate with the mk phenotype and contain a point mutation in mk/mk mice that results in an amino acid substitution (173V-->A). In order to test the hypothesis that this amino acid substitution is responsible for the mk phenotype, we have used recombinant retroviruses to introduce wild-type p45 NF-E2 into the bone marrow of mk/mk mice. Despite gene transfer and expression of p45 NF-E2 in erythroid cells, we found no evidence for correction of the phenotype in mk/mk mice. These results indicate that the mk mutation cannot be corrected by enforced expression of wild-type p45 NF-E2 and suggest that the 173V-->A mutation of the p45 NF-E2 gene is not the cause of anemia in mk/mk mice.

Actins↗
PubMed1994

Transgenic mice expressing antisense interleukin-3 RNA develop a B-cell lymphoproliferative syndrome or neurologic dysfunction.

Transgenic mice that expressed antisense interleukin-3 (AS-IL-3) RNA were generated and exhibited either a B-cell lymphoproliferative syndrome or progressive neurologic dysfunction. Each syndrome occurred in the founder or progeny mice of three separate transgenic lines. The lymphoproliferative process involved the accumulation, within peripheral lymphoid organs, of B220+/slgM- pre-B cells that had immunoglobulin (Ig) genes predominantly in germline configuration and expressed lambda 5 and Rag-1 transcripts. Transgenic animals that developed neurologic dysfunction exhibited circling behavior that progressed to ataxia and terminal inanition. AS-IL-3 transcripts were detected in mature CD3+ T cells of asymptomatic transgenic animals, as well as in B220+/slgM- pre-B cells, and CD3+ T cells from animals with the lymphoproliferative syndrome. AS-IL-3 transcripts were also detected in the brains of both young asymptomatic transgenic animals and older transgenic animals with neurologic dysfunction. Decreased IL-3 production from ConA-stimulated splenocytes was observed in asymptomatic transgenic animals. These observations suggest that this cytokine may have important roles in B lymphopoiesis and neurologic function.

Animals↗
PubMed1994

Efficient retrovirus transduction of mouse pluripotent hematopoietic stem cells mobilized into the peripheral blood by treatment with granulocyte colony-stimulating factor and stem cell factor.

Cytokine-mobilized peripheral blood cells have been shown to participate in hematopoietic recovery after bone marrow (BM) transplantation, and are proposed to be useful targets for retrovirus-mediated gene transfer protocols. We treated mice with granulocyte colony-stimulating factor (G-CSF) and stem cell factor (SCF) to mobilize hematopoietic progenitor cells into the peripheral blood. These cells were analyzed for the number and frequency of pluripotent hematopoietic stem cells (PHSC). We found that splenectomized animals treated for 5 days with G-CSF and SCF showed a threefold increase in the absolute number of PHSC over normal mice. The number of peripheral-blood PHSC increased 250-fold from 29 per untreated mouse to 7,200 in peripheral-blood PHSC in splenectomized animals treated for 5 days with G-CSF and SCF. Peripheral blood PHSC mobilized by treatment with G-CSF and SCF were analyzed for their ability to be transduced by retroviral vectors. Peripheral-blood PHSC from splenectomized animals G-CSF and SCF were transduced with a recombinant retrovirus containing the human MDR-1 gene. The frequency of gene transfer into peripheral blood PHSC from animals treated for 5 and 7 days was two-fold and threefold higher than gene transfer into PHSC from the BM of 5-fluorouracil-treated mice (P < .01). We conclude that peripheral blood stem cells mobilized by treatment with G-CSF and SCF are excellent targets for retrovirus-mediated gene transfer.

Animals↗
PubMed1994

Biological properties of subpopulations of pluripotent hematopoietic stem cells enriched by elutriation and flow cytometry.

We have studied several features of pluripotent hematopoietic stem cells (PHSCs) and day-12 spleen colony-forming units (CFU-S) obtained from adult murine bone marrow. Single-cell suspensions of C57BL/6J mouse bone marrow were fractionated by counterflow centrifugal elutriation at flow rates (FR) of 15, 25, 30, and 35 ml/min, and with the rotor off (R/O). The fractions FR25 and FR35 contained approximately equal numbers of PHSC that could repopulate W/Wv mice. These PHSCs were further enriched by subtracting lineage-positive cells using monoclonal antibodies (MAb) and magnetic immunobeads. The resulting lineage-negative cells (Lin-) were then stained with a MAb for the c-kit receptor and sorted by flow cytometry. Both subsets were fractionated into cells expressing high (bright) (c-kitBR), low (dull) c-kitDULL and no (negative, c-kitNEG) c-kit receptor. As few as 100 to 200 c-kitBR cells could repopulate the entire thymus and bone marrow in W/Wv mice. No PHSCs were present in the c-kitDULL and c-kitNEG fractions. We assayed fresh bone marrow and elutriation fractions FR25 and FR35 for gene expression by reverse transcriptase polymerase chain reaction. Using a semiquantitative protocol, we detected mRNA for beta-globin and flk-2, a protein tyrosine kinase receptor, in all samples except the FR25 Lin- c-kitBR subset. We consider the cells in FR25 Lin- c-kitBR to be the most primitive set of hematopoietic stem cells.

Animals↗
PubMed1993

Long-term in vivo expression of a murine adenosine deaminase gene in rhesus monkey hematopoietic cells of multiple lineages after retroviral mediated gene transfer into CD34+ bone marrow cells.

Retroviral mediated gene transfer into stem cells has been proposed as therapy for many inherited hematopoietic diseases. Deficiency of the enzyme adenosine deaminase (ADA) results in depletion of T lymphocytes, causing severe combined immunodeficiency syndrome (SCIDS). In this report, we describe retroviral mediated gene transfer of a murine ADA cDNA into Rhesus monkey hematopoietic stem cells. Immunoselected CD34+ bone marrow cells were exposed to medium containing the ADA retrovirus during culture on a stromal cell line engineered to express the transmembrane form of stem cell factor. After infusion of autologous, transduced cells into irradiated recipients, gene transfer was observed in all three monkeys. The ADA provirus was detected in 2% of circulating granulocytes and T cells from 100 days post-transplantation to longer than 1 year and in B cells from 250 days post-transplantation and beyond. Mouse ADA activity was detected in peripheral blood cells at approximately 3% the activity of monkey ADA. Thus, we have shown gene transfer into repopulating cells that contribute to all hematopoietic lineages with persistent gene expression. These data provide support for the use of stem cell targeted gene transfer for therapy of ADA deficiency.

Adenosine Deaminase↗
PubMed1993

Purification and characterization of heterogeneous pluripotent hematopoietic stem cell populations expressing high levels of c-kit receptor.

Mouse pluripotent hematopoietic stem cells (PHSC) were fractionated based on size and density using counterflow centrifugal elutriation (CCE). These heterogeneous PHSC populations were further enriched by subtraction of cells with lineage-specific markers (Lin-) followed by positive sorting for c-kit expression. The cells were characterized for their functional and biochemical properties. We defined a subpopulation of c-kit-positive cells that expressed high numbers of c-kit receptors (c-kitBR). One hundred c-kitBR cells from either low- or higher-density fractions were sufficient to repopulate the lymphohematopoietic system in WBB6F1-W/Wv (W/Wv) recipients, whereas no PHSC were found in cells with low (c-kitDULL) or no (c-kitNEG) c-kit expression. Lin- c-kitBR cells were separated into RhoDULL and RhoBR subsets based on their ability to efflux rhodamine 123 (Rho). The PHSC were concentrated in Lin- c-kitBR RhoDULL cells and the number of Lin- c-kitBR RhoBR cells correlated directly with the number of day 12 colony-forming unit-spleen (CFU-S12) in each fraction. We were not able to enrich further for PHSC using monoclonal antibodies to the cell-surface markers AA4.1 or CD4, which have been used by others to isolate PHSC. The small, low-density Lin- c-kitBR subset contained PHSC and few CFU-S12. This enabled us to assay PHSC for expression of the flk-2 gene, which encodes a tyrosine kinase receptor present on fetal liver PHSC. Purified RNA from the low-density Lin- c-kitBR subset did not contain flk-2 mRNA. We suggest that AA4.1, CD4 and flk-2 are expressed as stage-specific markers on PHSC in cell cycle.

Animals↗
PubMed1993

In vivo administration of stem cell factor to mice increases the absolute number of pluripotent hematopoietic stem cells.

We have examined the effects of administration of stem cell-factor (SCF) on the number and distribution of pluripotent hematopoietic stem cells (PHSC) in normal mice. Using the competitive repopulation assay we found that in vivo administration of SCF increases the absolute number of PHSC per mouse threefold. The increased numbers of PHSC are found in the peripheral blood and spleen of the SCF-treated animals. The spleen and peripheral blood stem cells completely repopulated the erythroid, myeloid, and lymphoid lineages of irradiated or W/Wv hosts, similar to bone marrow PHSC. PHSC from the peripheral blood of SCF-treated mice have a lineage marker-negative, c-kit-positive phenotype that is indistinguishable from that of bone marrow PHSC. The increase in the absolute number of spleen PHSC is associated with efficient gene transfer to these cells without prior treatment with 5-fluorouracil. This is a US government work. There are no restrictions on its use.

Animals↗
PubMed1993

Specific prolongation of skin graft survival following retroviral transduction of bone marrow with an allogeneic major histocompatibility complex gene.

Engrafted allogeneic hematopoietic cells have a unique capacity to induce a state of donor-specific transplantation tolerance across major histocompatibility complex barriers. This state allows permanent acceptance of donor-type organ grafts, with otherwise normal immunocompetence. We hypothesized that introduction of allogeneic MHC genes into autologous bone marrow which is then returned to recipient mice might similarly induce specific tolerance to products of the introduced MHC genes, without the risk of graft-vs-host disease. We demonstrate here that the introduction of MHC class I Kb cDNA by retrovirus-mediated gene transfer into B10.AKM (Kk) hematopoietic cells confers specific hyporesponsiveness to allogeneic skin grafts expressing Kb.

Amino Acid Sequence↗