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Biomedical subjects

H E Broxmeyer

Publications and source records attributed to H E Broxmeyer.

At least 271 records · Page 15Linked to original sources
PubMed1990

Enhanced stimulation of human bone marrow macrophage colony formation in vitro by recombinant human macrophage colony-stimulating factor in agarose medium and at low oxygen tension.

Recombinant (r) and natural human (h) macrophage colony-stimulating factor (M-CSF, CSF-1) have been considered poor stimulators of macrophage progenitor cells present in human marrow, although they are potent stimulators of these cells in mouse marrow. We compared the growth characteristics of rhM-CSF-responsive human macrophage progenitor cells placed in semisolid agarose or agar culture medium and incubated for 14 days at ambient (approximately 20%) or lowered (5%) O2 tension. By itself, rhM-CSF was found to be a good stimulator of macrophage colony formation by human bone marrow cells cultured in agarose but not in agar; this growth was enhanced by incubation at 5% O2. Maximal numbers (up to 115/10(5) nonadherent low density cells plated) of macrophage colonies (50 to greater than 500 cells per colony) were stimulated by 500 to 1,000 units rhM-CSF/mL, with 1/2 maximal numbers stimulated by 250 to 500 units/mL. With agarose as the support medium, rhM-CSF was two- to fourfold more active on mouse than on human macrophage colony formation, in contrast to previous reports of 10- to 100-fold greater activity when agar was used as the support medium. Using nonadherent low density T lymphocyte-depleted human bone marrow cells growing in agarose at 5% O2, greater than additive effects on colony formation were observed when 31 to 500 units rhM-CSF were used in combination with either 10 ng rh interleukin-1 alpha (IL-1 alpha), 20, or 200 units rh granulocyte-macrophage (GM)-CSF or rhG-CSF. The agarose assay system should be useful for evaluating factors regulating the proliferation of human macrophage progenitor cells in vitro and during clinical trials with rhM-CSF.

Agar↗
PubMed1990

Gene-expression and release of macrophage-colony stimulating factor in quiescent and proliferating fibroblasts. Effects of serum, fibroblast growth-promoting factors, and IL-1.

Macrophage (M)-CSF induces the proliferation and differentiation of macrophage-precursor cells, and is an important factor in the survival and activation of mature mononuclear phagocytes. Several effector cell populations such as mononuclear phagocytes, endothelial cells, and fibroblasts, have been reported to produce M-CSF at high levels. We investigated the gene-expression and release of M-CSF in fibroblasts. Quiescent, growth-arrested, fibroblasts showed little expression of M-CSF as demonstrated by Northern blot analysis, and little production of M-CSF as measured in the murine bone marrow bioassay. Initiation of proliferation of fibroblasts by fetal bovine serum was followed by an increase in M-CSF transcription and release of the protein. Similarly, single factors (platelet-derived growth factor) or combinations of factors (epidermal growth factor (EGF) + insulin, platelet-derived growth factor + EGF + insulin, or fibroblast growth factor + EGF + insulin) that induced proliferation of the fibroblasts, also induced increased expression of M-CSF. As soon as 1 h after the addition of fetal bovine serum, M-CSF expression was increased, and reached a plateau at 2 to 8 h after induction. IL-1 increased M-CSF expression both in quiescent and proliferating fibroblasts, and induced the expression and release of granulocyte-, and granulocyte/macrophage-CSF. These results show that expression of M-CSF in fibroblasts is not constitutively at a high level, but undergoes regulation depending on the cellular proliferative state and on further activation by acute response proteins such as IL-1.

Animals↗
PubMed1990

Human umbilical cord blood: a clinically useful source of transplantable hematopoietic stem/progenitor cells.

This is a review and discussion of studies leading to the first use of human umbilical cord blood, material usually discarded, for the provision of stem/progenitor cells for clinical hematopoietic reconstitution. This prospect arose as a result of extensive studies of the harvesting and cryopreservation of cord blood and of its numerical content of progenitor cells demonstrable in vitro. A male patient with Fanconi anemia (FA) was conditioned with a modified regimen of cyclophosphamide and irradiation that accommodates the abnormally high sensitivity to these agents that is characteristic of FA. Cryopreserved cord blood had been retrieved at birth from a female sibling known from prenatal testing to be unaffected by FA and to be human leukocyte antigen (HLA)-compatible with the prospective sibling recipient. After conditioning and therapeutic infusion of thawed cord blood, successful hematopoietic reconstitution was indicated by the general health of the patient, who had previously required supportive transfusions, by satisfactory hematological criteria and by counts of hematopoietic progenitor cells of various types in the bone marrow. Complete engraftment of the myeloid system with donor cells was evident from cytogenetics, ABO typing, study of DNA polymorphisms, and normal cellular resistance to cytotoxic agents that reveal the fragility of FA cells; the blood contained a residuum of host lymphocytes exhibiting chromosomal damage, but the trend has been towards eliminating these damaged cells. This implies that cord blood from a single individual should provide sufficient reconstituting cells for effective hematopoietic repopulation of an autologous or an HLA-compatible allogeneic recipient.

Blood Transfusion↗
PubMed1990

New therapeutic strategies in the treatment of murine diseases induced by virus and solid tumors: biology and implications for the potential treatment of human leukemia, AIDS, and solid tumors.

Understanding the biology and treatment of various cancers (including leukemia) and immunodeficiency disorders is still an ongoing and experimental process. Animal models have been and continue to be important to this process. This review will focus in on work by ourselves and others that have used murine models assessing the effects in vivo of the Friend virus complex (FVC, composed of a spleen focus forming virus and a murine leukemia helper virus) and solid tumors with metastatic potential in order to evaluate new and innovative therapies. These therapies include radiation, hyperthermia, and newly recognized naturally occurring biomolecules termed cytokines. These cytokines include, but are not limited to, the interferons, the tumor necrosis factors, the interleukins, the hematopoietic colony stimulating factors, lactoferrin and E-type prostaglandins. For example, it has been found that lactoferrin, when administered early enough, prolongs the survival of mice injected, but not yet infected, with the FVC. Of even greater potential usefulness is that mice already infected with the FVC can be completely rescued from death by treatment with split low dosage (150 cGy) total body irradiation. Irradiation treatment was associated with restoration of the T helper to T suppressor cell ratio, natural killer cell activity and marrow proliferative responses to the mitogens PHA and con A which were compromised by the FVC. More recent studies in our laboratory have demonstrated the potential of the interleukins and colony stimulating factors to decrease the metastatic potential of the B16 melanoma and the Lewis Lung Carcinoma cell lines. The cytokines can act in greater than additive fashion and combinations of therapies are possible. This review is meant to increase the awareness of these investigative animal models and the new types of combination therapies that can then be used as the basis for future clinical trials evaluating therapeutic efficacy.

Acquired Immunodeficiency Syndrome↗
PubMed1990

Prenatal identification of potential donors for umbilical cord blood transplantation for Fanconi anemia.

Reported here are studies of Fanconi anemia fetal cells that led to the first use of umbilical cord blood for hematopoietic reconstitution in a clinical trial. Prenatal diagnosis and HLA typing were performed in fetuses at risk for Fanconi anemia (FA) to identify, prior to birth, those that were unaffected with the syndrome and were HLA-identical to affected siblings. Umbilical cord blood was harvested at the delivery of these infants; assays of progenitor cells indicated the presence of colony-forming units-granulocyte-macrophage (CFU-GM) in numbers similar to those of bone marrow CFU-GM that are associated with successful engraftment in HLA-matched allogeneic bone marrow transplantation. The possibility that umbilical cord blood from a single individual can be used as an alternative to bone marrow for hematopoietic reconstitution has now been demonstrated by the successful engraftment of two patients with FA. Progenitor cell assays of umbilical cord blood collected at the birth of a child affected with FA, who had been misdiagnosed on the basis of chorionic villus sampling (CVS) studies, indicated a profound deficiency in colony formation, consistent with previously reported abnormalities in the growth of FA cells in vitro. These results suggest that the hematopoietic disorder in FA is related to an underlying problem with cell proliferation.

Blood Cell Count↗
PubMed1990

Effect of myogenic and adipogenic differentiation on expression of colony-stimulating factor genes.

The influence of cellular differentiation on colony-stimulating factor gene expression was examined in myogenically and adipogenically determined cell lines derived from 5-azacytidine-treated C3H10T1/2 C18 (10T1/2) mouse embryo fibroblasts. These studies demonstrate that colony-stimulating factor gene expression can be modulated by myogenic and adipogenic determination and terminal differentiation.

Adipose Tissue↗
PubMed1990

Influence of interleukins 3,5, and 6 on the growth of eosinophil progenitors in highly enriched human bone marrow in the absence of serum.

Purified preparations of natural murine interleukin (IL)-5 and recombinant human IL-1 alpha, IL-3, and IL-6 were evaluated alone, and in combination, for effects in vitro on colony formation by eosinophil progenitors (eosinophil colony-forming units, CFU-Eos) in either nonadherent low-density T-lymphocyte depleted (NALT-) or fluorescence-activated cell sorted NALT-HLA-DR+ CD34 (My10) cells from normal human bone marrow, in the absence and presence of serum. Interleukins were assessed on CFU-Eos plated directly in agar, or on CFU-Eos placed in suspension culture for 7 days prior to plating in agar in the presence of IL-5. Eosinophil colonies were identified in situ by staining with Luxol fast blue. IL-3 and IL-5 acted in agar culture as direct stimulators of CFU-Eos, using highly purified cells in the HLA-DR+ My10 fraction of cells, the specificity for CFU-Eos being greatest for IL-5 and most demonstrable in the absence of serum. The IL-3 and IL-5 direct stimulating effects on CFU-Eos were additive. IL-1 alpha and IL-6 had little or no effect, alone or in combination with IL-3 and IL-5; however, IL-3, IL-5, and IL-6 each enhanced the number of IL-5-responsive CFU-Eos found after suspension culture compared to control medium, with the individual effects being additive when the molecules were combined. These results demonstrate that both IL-3 and IL-5, alone and in combination have direct-acting effects on CFU-Eos, with the greatest specificity for stimulation of pure Eos colonies and clusters residing in IL-5, and that IL-3, IL-5, and IL-6, alone and in combination, may play a role in the survival of CFU-Eos.

Antigens, CD↗
PubMed1990

Transplantation of umbilical cord blood in Fanconi's anemia.

It has been shown that human umbilical cord blood contains stem/progenitor cells comparable in number to that of adult bone marrow. We report here the first successful cases of transplantation of umbilical cord blood cells. The patients were suffering from Fanconi's anemia, complicated by severe aplastic anemia. During pregnancy, it was shown that the mother was carrying a sibling unaffected by the disease and with HLA identical to the patient. Cord blood was collected and frozen in liquid nitrogen at birth. After conditioning with low-dose cyclophosphamide (20 mg/kg) and thoraco-abdominal irradiation (5 grays), the patients received a cord blood transplant of thawed cells. Three patients have been transplanted without any immediate side-effect. One has not enough follow-up, but two patients are alive and well with complete donor hematologic reconstitution and no chronic graft versus host disease. Potential developments of this technique are an extension of applicability with regard to other diseases that might be transplanted and whether such transplants can be performed in adults. The relative immaturity of the lymphoid system at birth may be advantageous in decreasing the graft versus host reaction if these cells are used in a mismatched transplantation. Cord blood cell banks may be useful for transplants in patients lacking an HLA-identical donor.

Child↗
PubMed1989

Myelopoietic enhancing effects of murine macrophage inflammatory proteins 1 and 2 on colony formation in vitro by murine and human bone marrow granulocyte/macrophage progenitor cells.

Two recently identified and purified murine macrophage inflammatory proteins MIP-1 and MIP-2 were tested in vitro both alone, and in combination with purified recombinant (r) murine (mu) GM-CSF, natural (n)muCSF-1, or rhuman (hu)G-CSF, for effects on mouse marrow CFU-GM, in combination with erythropoietin for effects on mouse marrow BFU-E, and in combination with rhuGM-CSF or rhuG-CSF for effects on human marrow CFU-GM. MIP-1 and MIP-2 did not stimulate, but did enhance by up to threefold, colony formation of mouse CFU-GM co-stimulated by rmuGM-CSF and nmuCSF-1, but not by rhuG-CSF, in the absence or presence of serum. MIP-1 and MIP-2 were maximally active at concentrations greater than or equal to 100 ng/ml and the actions appeared to be initiated during the DNA synthetic portion of the cell cycle. Neither MIP-1 nor MIP-2 at up to 1 microgram/ml had any effect on mouse BFU-E, in the absence or presence of erythropoietin. Both MIP-1 and MIP-2 had direct acting effects on purified mouse CFU-GM. The cloning efficiency of 200 purified cells plated with 50 U muCSF-1 was 82% with and 43% without MIP; the cloning efficiency with 50 U rmuGM-CSF was 65% with and 35% without MIP. MIP effects were not mimicked by bacterial LPS, rhuIL-1 alpha, rhuIL-6, or rmuIL-4, and were neutralized by their respective specific antibodies. MIP-1 and MIP-2 also enhanced endogenously stimulated and rhuGM-CSF-, but not rhuG-CSF-, stimulated colony formation by human marrow CFU-GM. These results demonstrate a new role for MIP-1 and MIP-2 in vitro as myelopoietic enhancing activities for CFU-GM.

Animals↗
PubMed1989

Effects of recombinant human colony stimulating factors (CSF) (granulocyte-macrophage CSF, granulocyte CSF, and CSF-1) on human monocyte/macrophage differentiation.

Purified recombinant human granulocyte-macrophage (rhuGM)-CSF, rhuG-CSF, and rhuCSF-1 were evaluated for their capacity to influence the differentiation of U-937 cells and normal human monocytes. The human U-937 cell line represents an early stage of monocytic differentiation. It was found that rhuGM-CSF and rhuG-CSF, but not rhuCSF-1, induced phenotypic changes consistent with monocyte/macrophage differentiation in U-937 cells. After 3 days of culture in the presence of either rhuGM-CSF or rhuG-CSF, a small but significant proportion of U-937 cells were able to reduce nitroblue tetrazolium. Nitroblue tetrazolium reduction, however, was maximally induced when rhuGM-CSF and rhuG-CSF were added in combination. These changes were accompanied by increased alpha-naphthyl acetate esterase activity, acquisition of macrophage morphology, Mo-1 Ag expression, and decreased cell proliferation. rhuGM-CSF alone also induced expression of the c-fms proto-oncogene (CSF-1 receptor) in U-937 cells and this expression was enhanced by the combination of rhuGM-CSF and rhuG-CSF. In cultured normal human peripheral blood monocytes, representing a late stage of maturation, rhuGM-CSF and rhuCSF-1 differentially increased Mo-1 and My-4 Ag expression, respectively, whereas rhuG-CSF was without effect. Our results suggest that the interaction of GM-CSF, G-CSF, and CSF-1 may play a fundamental role in the early and late stages of the human monocyte/macrophage differentiation process.

Antigens, Surface↗
PubMed1989

Effect in vivo of multiple injections of purified murine and recombinant human macrophage colony-stimulating factor to mice.

Hematopoietic efficacy in vivo of multiple injections of purified murine L-cell and recombinant human macrophage colony-stimulating factors (M-CSF; specific activity, greater than 2 x 10(7) units/mg) was assessed in mice. Injections i.v. of sterile saline or 20,000 units of M-CSF were administered once (at 0 h), twice (at 0 and 12 h), or three times (at 0, 12, and 24 h) to C57BL/6 x DBA/2 F1 mice. Numbers and cycling rates of marrow and spleen granulocyte-macrophage, erythroid, and multipotential progenitor cells were assessed 32-36 h after the first injection. Marrow, spleen, and peripheral blood cellularity was assessed at intervals of up to 105 h. Progenitor cell cycling rates were significantly increased after one and two injections of M-CSF but were reduced to a slow or noncycling state after three injections. For marrow cells, the third injection resulted in a significant suppression of hematopoietic progenitor cell cycling compared to the control group. No significant changes were noted for number of progenitors per femur or spleen, for marrow, spleen, or peripheral blood cellularity, or for differential cell counts in these organs after any of the M-CSF treatment schedules. Suppression of progenitor cell proliferation noted after three injections of M-CSF may at least partially explain why repeated injections of 20,000 units of M-CSF fails to increase bone marrow, spleen, or blood cellularity even though one injection of M-CSF increases cycling rates of the hematopoietic progenitors.

Animals↗
PubMed1989

Influence of IL-1 alpha and -1 beta on the survival of human bone marrow cells responding to hematopoietic colony-stimulating factors.

Purified recombinant human (rhu) IL-1 alpha and IL-1 beta were evaluated for their effects on the proliferation and survival of granulocyte-macrophage (CFU-GM) and erythroid (BFU-E) progenitor cells from normal human bone marrow (BM). Using nonadherent low density T lymphocyte depleted (NALT-) BM cells cultured in the presence or absence of IL-1, CSF-deprivation studies demonstrated that IL-1 alpha or IL-1 beta by itself did not enhance the proliferation of CFU-GM or BFU-E. They did, however, promote the survival of progenitors responding to the delayed addition of media conditioned by the 5637 cell line (5637 conditioned medium), rhu GM-CSF and erythropoietin. The survival promoting effects of IL-1 alpha on CFU-GM and BFU-E were neutralized by anti-IL-1 alpha mAb added to the cultures. The survival promoting effect of IL-1 alpha did not appear to be mediated by CSF, because neither CSF nor erythroid burst promoting activity were detectable in cultures in which NALT- cells were incubated with rhuIL-1 alpha. In addition, suboptimal concentrations of rhu macrophage CSF (CSF-1), G-CSF, GM-CSF, and IL-3, which were just below the levels that would stimulate colony formation, did not enhance progenitor cell survival. Survival of CFU-GM and BFU-E in low density (LD) bone marrow cells did not decrease as drastically as that in NALT- BM cells, and exogenously added IL-1 did not enhance progenitor cell survival of CFU-GM and BFU-E in LD BM cells. However, addition of anti-IL-1 beta decreased survival of CFU-GM and BFU-E in LD BM cells. These results implicate IL-1 in the prolonged survival of human CFU-GM and BFU-E.

Antibodies, Monoclonal↗
PubMed1989

Differentiation-inducing effect of recombinant human tumor necrosis factor alpha and gamma-interferon in vitro on blast cells from patients with acute myeloid leukemia and myeloid blast crisis of chronic myeloid leukemia.

Tumor necrosis factor alpha (TNF-alpha) and gamma-interferon (IFN-gamma) have been shown to suppress clonogenic growth in cultures containing blast cells obtained from patients with acute myeloid leukemia. We report that recombinant human TNF-alpha and IFN-gamma are also able to induce functional and morphological maturation in fresh myeloid leukemic cells in vitro. Assessing suspension cultures containing cells from patients with acute myeloid leukemia (11 patients) or myeloid blast crisis of chronic myeloid leukemia (5 patients), it was found that recombinant human TNF-alpha and IFN-gamma significantly enhanced the number of cells reducing nitroblue tetrazolium, as compared to control cultures containing no cytokine (P less than 0.001 and P less than 0.001, respectively). Cells from responders showed alterations characteristic of monocyte/macrophage differentiation, adherence to plastic surfaces, development of positive staining for alpha-naphthyl acetate esterase, typical morphology, and expression of cell surface antigens detected by the monoclonal antibodies Mo-1, Mo-2, and My-4. Both cytokines decreased the number of viable cells, the number of blast cells, and the number of cluster-forming units in suspension culture, suggesting inhibitory actions on the growth capacity of leukemic cells. Compared to the maximum effects of either factor alone, the combination of recombinant human TNF-alpha and IFN-gamma significantly increased the extent of growth inhibition and cell adherence but did not result in further increases in nitroblue tetrazolium reduction. The presence of Auer rods in IFN-gamma or TNF-alpha differentiation-induced macrophages with cells from a patient with M5 acute myeloid leukemia demonstrates that these cytokines can induce differentiation of a leukemic clone in primary cells from patients with leukemia.

Adolescent↗
PubMed1989

Low dose total body irradiation: a potent anti-retroviral agent in vivo.

Immunosuppression characterizes many human diseases including leukemia and AIDS. Friend virus (FV)-induced murine leukemia is a useful model for studying both malignancy and immunosuppression. In a previous series of experiments, we have demonstrated that untreated FV-infected mice died within 40 days post-infection, whereas infected mice given 150 cGy total body irradiation (TBI) on days 5 and 12 exhibited long-term survival. In this report, we show that no leukemic cells or type C virus particles are found in the spleens of mice treated with TBI. In addition, both NK activity as well as bone marrow cell's proliferative responses to PHA and Con A were fully restored. This treatment produces long term control of FV-induced murine leukemia, and thus might have relevance for the treatment of a number of immunosuppressive diseases including AIDS.

Animals↗
PubMed1989

Human umbilical cord blood as a potential source of transplantable hematopoietic stem/progenitor cells.

The purpose of this study was to evaluate human umbilical cord blood as an alternative to bone marrow in the provision of transplantable stem/progenitor cells for hematopoietic reconstitution. Although no direct quantitative assay for human hematopoietic repopulating cells is at present available, the granulocyte-macrophage progenitor cell (CFU-GM) assay has been used with success as a valid indicator of engrafting capability. We examined greater than 100 collections of human umbilical cord blood for their content of nucleated cells and granulocyte-macrophage, erythroid (BFU-E), and multipotential (CFU-GEMM) progenitor cells, in many cases both before and after cryopreservation. First it was determined that granulocyte-macrophage, erythroid, and multipotential progenitor cells remained functionally viable in cord blood untreated except for addition of anticoagulant for at least 3 days at 4 degrees C or 25 degrees C (room temperature), though not at 37 degrees C, implying that these cells could be satisfactorily studied and used or cryopreserved for therapy after transport of cord blood by overnight air freight carriage from a remote obstetrical service. Granulocyte-macrophage progenitor cells from cord blood so received responded normally to stimulation by purified recombinant preparations of granulocyte-macrophage, granulocyte, and macrophage colony-stimulating factors and interleukin 3. The salient finding, based on analysis of 101 cord blood collections, is that the numbers of progenitor cells present in the low-density (less than 1.077 gm/ml) fraction after Ficoll/Hypaque separation typically fell within the range that has been reported for successful engraftment by bone marrow cells. Another observation of practical importance is that procedures to remove erythrocytes or granulocytes prior to freezing, and washing of thawed cells before plating, entailed large losses of progenitor cells, the yield of unwashed progenitor cells from unfractionated cord blood being many times greater. The provisional inference is that human umbilical cord blood from a single individual is typically a sufficient source of cells for autologous (syngeneic) and for major histocompatibility complex-matched allogeneic hematopoietic reconstitution.

Blood Preservation↗
PubMed1989

The effects of oxidizing species derived from molecular oxygen on the proliferation in vitro of human granulocyte-macrophage progenitor cells.

In order to better understand the enhancing effects of lowered oxygen (O2) tension on the growth in vitro of granulocyte-macrophage progenitor cells (CFU-GM), the effects of oxidizing species derived from molecular O2 were assessed on CFU-GM. Low density or nonadherent low density normal human bone marrow cells were plated at ambient (20%) or lowered (5%) O2 tension in the presence of a source of colony stimulating factors, and in the absence or presence of superoxide dismutase, catalase, glucose oxidase or horseradish peroxidase, alone or in various combinations. Enhanced colony and cluster formation of CFU-GM was noted when low density cells were grown at 5% O2, or when cells were grown at 20% O2 in the presence of superoxide dismutase or glucose oxidase. Both of these enzymes are capable of generating hydrogen peroxide (H2O2), although by different mechanisms. Low concentrations of glucose oxidase resulted in increased formation of colonies and clusters, but higher concentrations of glucose oxidase were inhibitory. Catalase, which converts H2O2 to H2O, had no effect by itself on cells growing at 20% O2, but it eliminated the superoxide dismutase and glucose oxidase enhancing effects. Catalase decreased colony formation of cells grown at 5% O2. Removal of adherent cells ablated the growth-enhancing effects noted at lowered (5%) O2 tension and also the superoxide dismutase and catalase effects at 20% or 5% O2. Horseradish peroxidase, which converts H2O2 to a more toxic oxidant, hypochlorite, had a suppressive effect on colony and cluster numbers and at 20% O2 converted the glucose oxidase effects from stimulatory to inhibitory. The results suggest that adherent cells and low concentrations of H2O2 may mediate growth-enhancing effects of CFU-GM seen at lowered (5%) O2 tension.

Bone Marrow Cells↗