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D M Bodine

Publications and source records attributed to D M Bodine.

At least 73 records · Page 4Linked to original sources
PubMed1992

Stem cell factor increases colony-forming unit-spleen number in vitro in synergy with interleukin-6, and in vivo in Sl/Sld mice as a single factor.

Hematopoiesis is thought to be modulated by interactions of progenitor cells with hematopoietic growth factors. We have shown that colony-forming units-spleen (CFU-S) and repopulating stem cells require interleukin-3 (IL-3) to survive in vitro, and that CFU-S number and long-term repopulating ability can be increased by culture in the combination of IL-3 and IL-6. In this report, we describe the effects of stem cell factor (SCF) on CFU-S and repopulating stem cells. Injection of SCF into anemic Sl/Sld mice caused a twofold and 20-fold increase in CFU-S number in the bone marrow and spleen of treated animals, respectively. After 6 days in suspension culture, CFU-S number increased threefold in cultures supplemented with SCF and IL-6, or SCF, IL-3, and IL-6 relative to the number at day 0. The long-term repopulating ability of cells cultured in SCF, IL-3, and IL-6 was approximately sevenfold better than that of cells cultured in IL-3 or SCF. Similar experiments were performed on populations of bone marrow cells enriched for, or depleted of, CFU-S by elutriation and lineage subtraction. The combination of SCF and IL-6 increased CFU-S number approximately fourfold to eightfold in the CFU-S-enriched fraction, but had no effect on the CFU-S-depleted cells. These results show that SCF alone can increase CFU-S number in vivo, and in combination with other growth factors increases CFU-S numbers in vitro.

Animals↗
PubMed1992

Mechanism of DNase I hypersensitive site formation within the human globin locus control region.

The human beta-like globin gene locus contains embryonic, fetal, and adult globin genes that are regulated in a developmentally timed, as well as a tissue-specific, manner. The locus control region (LCR), located 5' of the globin genes, is characterized by four erythroid-specific nuclease-hypersensitive sites within native chromatin. These sites contain the active elements of the LCR. The LCR establishes an active chromatin conformation across the globin locus and enhances globin gene expression in transfected erythroleukemia cells and transgenic mice. We have used 5' DNase I hypersensitive site (HS) 4 as a model to define the minimum elements necessary for site formation. We have identified a 101-base-pair fragment within 5' HS4 that is the active site-forming element. DNase I footprint and gel-mobility shift assays have identified binding sites for transcription factors AP-1/NF-E2, Sp-1, and GATA-1 within the HS-forming element. We conclude that HS formation, the characteristic feature of the LCR in nuclear chromatin, requires interaction between erythroid-specific and ubiquitous nuclear proteins.

Animals↗
PubMed1992

Pluripotent hematopoietic stem cells of low and high density can repopulate W/Wv mice.

We have studied several features of pluripotent hematopoietic stem cells (PHSC) and day-12 spleen colony-forming units (CFU-S12) in murine bone marrow. C57BL/6J marrow cell suspensions were separated by elutriation and fractions were obtained at flow rates (FR) of 25 ml/min, 29/30 ml/min, 35 ml/min, and with the rotor off. All four fractions contained PHSC that could repopulate W/Wv mice, but significant numbers of CFU-S12 were found only in the three higher FR fractions. Cells in the FR29/30 fraction were shown to have almost three-fold more repopulating activity than fresh marrow in a competitive repopulation assay. The PHSC in fractions separated by elutriation were enriched by depleting cells expressing specific lineage markers with monoclonal antibodies and magnetic immunobeads. As few as 10(4) lineage negative (lin-) cells from FR35 or 10(5) lin--cells from FR25 conferred long-term multilineage repopulation in W/Wv mice, as demonstrated by Southern blot analysis of DNA from recipient thymus and bone marrow. We conclude that PHSC are heterogeneous for cell size and density and that the highest concentration of PHSC resides in the subset of intermediate density present in the FR29/30 fraction.

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PubMed1991

Effects of hematopoietic growth factors on the survival of primitive stem cells in liquid suspension culture.

We have examined the effects of 10 different growth factors either alone or in combination on colony-forming unit-spleen (CFU-S) and repopulating stem cell survival in vitro. Either interleukin-3 (IL-3), granulocyte-colony-stimulating factor (G-CSF), or IL-4 alone support CFU-S in vitro. The effects of IL-3 or G-CSF could be neutralized by adding antibodies against IL-3 or G-CSF, respectively. However, the effects of IL-4 could be neutralized with antibodies to IL-4 as well as with antibodies to IL-3 and G-CSF. The combinations of IL-3 and IL-6, IL-3 and G-CSF, IL-3 and IL-1 alpha, IL-3 and granulocyte-macrophage CSF (GM-CSF), and IL-4 and IL-6 acted synergistically to increase CFU-S number. Addition of macrophage inflammatory protein-1 alpha (MIP-1 alpha) to IL-3 and IL-6 inhibited the increase in CFU-S number. Repopulating stem cell function was measured in a competitive repopulation assay. Either IL-3 or IL-4 alone could preserve stem cell function in vitro. The combinations of IL-3 and IL-6, and IL-3 and G-CSF increased stem cell function approximately twofold. The combinations of IL-3 + G-CSF + IL-6, and IL-4 and IL-6 (both of which increased CFU-S number fivefold to 10-fold) decreased stem cell function approximately fourfold. These results demonstrate that certain combinations of growth factors can increase CFU-S number at the expense of stem cell function.

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PubMed1991

The upstream region of the human gamma-globin gene promoter. Identification and functional analysis of nuclear protein binding sites.

The promoter of the human gamma-globin gene confers tissue specificity as well as developmental stage specificity to gamma gene expression. Earlier work in our laboratory suggested that a fragment of the gamma-globin promoter between -300 and -137 base pairs upstream of the transcription start site contributed to the developmental specificity of the promoter. In this paper, we have mapped potential regulatory elements within this upstream region of the gamma promoter by a combination of in vitro DNA-protein binding assays and functional determinations of promoter strength in transient expression studies. Four sites between -300 and -130 bind proteins present in nuclear extracts of erythroid and non-erythroid cell lines. Mutation of these binding sites by internal base substitution determined that three of the four influence overall promoter strength in transient assays. We have focused on two protein binding sites, -246 to -212 and -195 to -170, that have been reported to bind erythroid-specific factors. The erythroid binding protein NF-E1 and a ubiquitous octamer protein footprint the -195 to -170 site. While internal mutation of this site did not significantly alter promoter strength, a point mutation at position -175 that is associated with hereditary persistence of fetal hemoglobin increased the activity of a promoter construct 20-fold in erythroid cells. A detailed mutational analysis of this site suggests that NF-E1 binding is necessary but not sufficient for activation of the promoter by the -175 mutation, and we propose that a second protein or co-activator is required. The nucleotides between -246 and -212 appear to bind a complex of at least three proteins, at the core of which is a protein binding to a string of dA:dT residues. This complex also appears to form on the 3' A gamma-globin enhancer, and homologous sites have been identified within the locus activating region of the beta-globin cluster, suggesting that this element may mediate long range interactions with distant regulatory elements.

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PubMed1991

Gene transfer into hematopoietic stem cells.

The ability to reliably transfer genes into hematopoietic stem cells with long-term repopulating potential and to selectively express such genes would allow genetic therapy for diseases such as sickle cell anemia and immunologic deficiencies due to T-cell defects, including acquired immune deficiency syndrome (AIDS). Understanding the biology of the hematopoietic stem cell is a key element in realizing the full therapeutic potential of gene insertion strategies. Current techniques have efficiency rates of gene insertion of approximately 10% to 20% into murine stem cells and 1% to 5% into primate stem cells. Many challenges, some biologic and some logistic, remain before gene transfer protocols that are successful in the mouse model can be extended to humans.

Animals↗
PubMed1991

Survival and retrovirus infection of murine hematopoietic stem cells in vitro: effects of 5-FU and method of infection.

Gene replacement therapy for diseases of the hematopoietic system requires efficient gene transfer to pluripotent hematopoietic stem cells. We have systematically compared a number of protocols for retrovirus-mediated gene transfer into murine repopulating hematopoietic stem cells. Recipients of infected bone marrow cells were analyzed for the presence of the transduced provirus 4 months after transplantation. Our results show that 5-fluorouracil (5-FU) pretreatment of donor animals was required for efficient gene transfer and that 5-FU-treated bone marrow retained more repopulating activity in culture than untreated bone marrow. A comparison of retrovirus-mediated gene transfer by co-cultivation of bone marrow cells with retrovirus producer cells as opposed to gene transfer by culturing bone marrow cells in retrovirus-containing supernatant revealed that gene transfer by cocultivation was more efficient than supernatant infection. However, the repopulating ability of bone marrow cells cocultured with retrovirus producer cells was reduced compared to bone marrow cells cultured in virus-containing medium.

Animals↗
PubMed1990

Development of a high-titer retrovirus producer cell line capable of gene transfer into rhesus monkey hematopoietic stem cells.

Retroviral-mediated gene transfer into primitive hematopoietic cells has been difficult to achieve in large-animal models. We have developed an amphotropic producer clone that generates greater than 10(10) recombinant retroviral particles (colony-forming units) per ml of culture medium. Autologous rhesus monkey bone-marrow cells were cocultured with either high (2 x 10(10) colony-forming units/ml) or low (5 x 10(6) colony-forming units/ml) titer producer clones for 4-6 days and reinfused into sublethally irradiated animals. The proviral genome was detected in blood and bone-marrow cells from all three animals reconstituted with cells cocultured with the high-titer producer cells. In contrast, three animals reconstituted with bone marrow cocultured with the low-titer producer clone exhibited no evidence of gene transfer.

Animals↗
PubMed1990

Development of a high-titer retrovirus producer cell line and strategies for retrovirus-mediated gene transfer into rhesus monkey hematopoietic stem cells.

Retroviral-mediated gene transfer into pluripotent hematopoietic stem cells has been difficult to achieve in large animal models. We have compared several infection protocols in a murine model system and concluded that bone marrow can be maintained and infected in vitro for 2-6 days. We have also developed an amphotropic producer clone that generates greater than 10(10) recombinant retroviral particles (CFU) per milliliter of culture medium. Autologous rhesus monkey bone marrow cells were co-cultured with either high- (2 x 10(10) CFU/ml) or low- (5 x 10(6) CFU/ml) titer producer clones for 4-6 days and reinfused into sublethally irradiated animals. The proviral genome was detected in blood and bone marrow cells from all three animals reconstituted with cells co-cultured with the high-titer producer cells. In contrast, three animals reconstituted with bone marrow co-cultured with the low-titer producer clone exhibited no evidence of gene transfer.

Animals↗
PubMed1990

Dysregulated interleukin 6 expression produces a syndrome resembling Castleman's disease in mice.

Interleukin 6 (IL-6) is an important regulator of the acute phase response, T cell function, and terminal B cell differentiation. Excessive or inappropriate production of this cytokine may be involved in a variety of autoimmune and neoplastic disorders. To investigate the consequences of dysregulated synthesis of IL-6 in vivo, a high-titer recombinant retroviral vector produced in psi-2 packaging cells was used to introduce the coding sequences of murine IL-6 into mouse hematopoietic cells. Congenitally anemic W/Wv mice reconstituted with bone marrow cells transduced with the retroviral vector developed a syndrome characterized by anemia, transient granulocytosis, hypoalbuminemia, and polyclonal hypergammaglobulinemia, with marked splenomegaly and peripheral lymphadenopathy. Extensive plasma cell infiltration of lymph nodes, spleen, liver, and lung was noted. The similarity of these findings to those of multicentric Castleman's disease, taken together with the observation that lymph nodes from these patients elaborate large amounts of this cytokine, suggest that the inappropriate synthesis of IL-6 has a primary role in the pathogenesis of this systemic lymphoproliferative disorder.

Animals↗
PubMed1989

Combination of interleukins 3 and 6 preserves stem cell function in culture and enhances retrovirus-mediated gene transfer into hematopoietic stem cells.

The effects of several hematopoietic growth factors on primitive murine bone marrow progenitor cells [colony-forming unit(s)-spleen (CFU-S)] have been investigated during culture for 2-6 days. Interleukin 3 (IL-3) was required for CFU-S survival in culture, and the combination of IL-3 and interleukin 6 (IL-6) increased the number of CFU-S in culture 10-fold over the number obtained with IL-3 alone. Stem cell function was measured by competitive repopulation; IL-3 was required, and IL-3 and IL-6 appear to act synergistically to enhance stem cell recovery from these cultures. These data appear to be relevant for retroviral-mediated gene transfer into stem and progenitor cells. Murine bone marrow cells were infected with a retrovirus containing the human beta-globin gene in the presence of various growth factors. Only 2 of 17 mice reconstituted with cells infected in the presence of IL-3 alone showed long-term expression of the human beta-globin gene (12 months), as opposed to 6 of 11 mice reconstituted with cells infected in the presence of IL-3 and IL-6. Medium conditioned by 5637 bladder carcinoma cells, a source of several hematopoietic growth factors, increased the frequency of infection of CFU-S but did not enhance stem cell infection or the repopulating potential of cultured bone marrow cells. Stem cells containing the human beta-globin provirus from these animals were shown to be capable of reconstituting secondary recipients in which the human beta-globin gene was expressed.

Animals↗
PubMed1989

Retrovirus-mediated transfer and expression of the interleukin-3 gene in mouse hematopoietic cells result in a myeloproliferative disorder.

A high-titer, recombinant retroviral vector produced in psi 2 packaging cells has been used to introduce the murine interleukin-3 (IL-3) gene into mouse hematopoietic cells. Integration and expression of the IL-3 gene was observed in spleen foci from which could be derived factor-independent, continuously proliferating cell lines. Irradiated or genetically anemic W/Wv recipients of infected hematopoietic cells developed a myeloproliferative syndrome characterized by a marked elevation in leukocyte count, bone marrow hyperplasia, and enlargement of the liver and spleen. The syndrome reflected proliferation of one or more stem cell clones, the progeny of which were capable of repopulating secondary recipients. One animal developed the syndrome primarily by a paracrine mechanism. Endogenous IL-3 production caused amplification of hematopoietic cells but did not appear to alter the maturational or self-renewal potential of these cells.

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