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Biomedical subjects

M Y Gordon

Publications and source records attributed to M Y Gordon.

At least 73 records · Page 4Linked to original sources
PubMed1994

The binding of acute myeloid leukemia blast cells to human endothelium.

AML blast cell adhesion to endothelium is in all likelihood a prerequisite for blast cell migration across the vascular wall in the periphery and the subsequent establishment of leukemic extravascular disease. A general feature of malignant cells is their acquisition of altered or aberrant adhesive capabilities which appear to be associated with their ability to metastasize. Aberrant expression of integrin adhesion molecules and of membrane oligosaccharide structures is found in AML and various solid tumors. With respect to AML, these alterations in adhesive phenotype may confer a proliferative advantage on the malignant cells in the marrow, may facilitate egress from the bone marrow into the peripheral vasculature and may enable AML blast cells to traverse the vessel wall and so establish extravascular disease. Oncogenes may be directly involved in the acquisition of such aberrant adhesive phenotypes. Neutrophil extravasation is described as a model for leukocyte migration across the vessel wall and brief summaries of experimental work involving aspects of AML blast cell and normal CD34+ bone marrow cell adhesion to endothelium in vitro are described.

Acute Disease↗
PubMed1994

Plastic-adherent cells in human bone marrow generate long-term hematopoiesis in vitro.

This study demonstrates that the human bone marrow mononuclear cell fraction that adheres to plastic during a 2-h incubation period includes stromal precursors and hematopoietic progenitor cells that can sustain hematopoiesis for at least 5 weeks in a long-term culture (LTC) system. Delta (delta) assays were performed on the 2-h plastic-adherent fraction of mononuclear cells, by assaying the numbers of CFU-GM released into the culture supernatant during 1 week of incubation, to investigate the properties of the original progenitor cells. The plastic-adherent delta (P delta) progenitor cells all express CD34, and a variable proportion express Thy-1. The numbers of CFU-GM produced are directly related to the numbers of mononuclear cells used to initiate the cultures, and limiting dilution experiments indicate a frequency for P delta cells of 2.5-10/10(5) mononuclear cells. The numbers of CFU-GM produced by individual P delta cells at limiting dilution vary considerably within experiments but the distributions of CFU-GM per P delta progenitor show little variation between samples. On average, P delta cells generate 6-9 early CFU-GM scored on day 21 of culture; 10-23 CFU-GM scored on day 14, and 18-40 mature CFU-GM scored on day 7. These results show that plastic-adherent cells in human bone marrow are capable of sustained hematopoiesis in vitro and of producing a spectrum of clonogenic progeny. They form the basis of an assay for investigating the kinetics of early human hematopoietic progenitor cells.

Antigens, CD↗
PubMed1994

In vitro proliferation by cells mobilized into the peripheral blood for collection and autologous transplantation.

We have investigated the properties of mobilized, cryopreserved, peripheral blood stem cells (PBSC), collected by leukapheresis over a period of 5 days, from eight myeloma patients in clinical remission. Cells were mobilized by treatment with cyclophosphamide and granulocyte colony-stimulating factor (G-CSF), and each day's collection was evaluated for its content of CD34+ cells, colony-forming units granulocyte/macrophage (CFU-GM), and plastic-adherent pre-CFU-GM. Peak values for these three parameters were observed at different times in different patients. There was no correlation between CD34+ content and CFU-GM, but there was some (r = 0.65) between CD34 numbers and colonies generated from a delta assay initiated using plastic-adherent pre-CFU-GM. In suspension cultures, the cells grew exponentially for 50 days. Thereafter, they did not divide, although they remained viable in culture for up to 1 month longer. Suspension cultures of PBSC grown with interleukin-3 (IL-3) displayed a predominantly myelomonocytic phenotype, but some megakaryocytes and erythroid cells were observed consistently. These results indicate that pre-CFU-GM in PBSC collections are capable of generating large numbers of clonogenic progeny in liquid culture and are capable of producing multiple lineages of differentiation.

Antigens, CD↗
PubMed1994

Routes to repopulation--a unification of the stochastic model and separation of stem-cell subpopulations.

In summary, we propose that the haemopoietic system consists of a hierarchy of clonogenic stem and progenitor cells and that the stochastic effect is superimposed upon these compartments. This means that (i) considerable heterogeneity can exist within major compartments without the need to invoke the existence of numerous subpopulations, and (ii) that the stochastic effect and the separation of stem cells are not totally incompatible. The unifying model offers a reasonable explanation of the situations summarized above. The clear implication of the disappearance of stem cell clones as a result of the stochastic effect is a requirement for multiple stem cells to ensure long-term repopulation in cell replacement or gene therapy. The necessary multiplicity may not be great, possibly 3-fold according to the results of Lemishka, and the scheme shown in Figure 1, but should be considered in the design and interpretation of experimental and therapeutic strategies. Other implications are concerned with the degree to which it is necessary to select 'subpopulations' of haemopoietic stem cells for transplantation or experimental work.

Cell Cycle↗
PubMed1994

Transplantation biology.

A variety of hemopoietic tissues are now being used as a source of stem cells for clinical transplantation. These tissues all have different biological properties, which are not yet fully understood, as well as particular clinical applications, advantages, and disadvantages. The development of protocols for the manipulation of stem cells prior to infusion into patients will, it is hoped, lead to the further improvement of stem cell replacement therapy including the use of stem cells as vectors for expressing transduced genes. Success in these efforts will depend on an improved understanding of the biological principles underlying hemopoietic stem cell transplantation.

Fetal Blood↗
PubMed1993

Human haemopoietic stem cell assays.

Assay systems for human haemopoietic stem cells are necessary for understanding the regulation of haemopoietic cell production. The stem cells are ultimately responsible for the production of mature blood cells but constitute only a tiny minority of the total haemopoietic cell population. The remainder is a heterogeneous mixture of cells at various stages of differentiation and maturation along the myeloid, erythroid and megakaryocytic lineages. Although clonogenic assays for the lineage-committed progeny of human stem cells are well established and have produced a wealth of information about the regulation of the later stages of haemopoiesis, there is no universally accepted assay for self-renewing pluripotent haemopoietic stem cells in human haemopoietic tissue. Nevertheless, several types of in vitro and in vivo assay systems have been devised for very early haemopoietic cells in man. These can be broadly divided into three classes: (1) direct clonogenic assays; (2) assays where non-clonogenic cells are detected by their ability to produce more mature clonogenic progeny and (3) transplantation of human haemopoietic cells into immunocompromised (SCID) mice. The different assay systems are designed to answer important questions about the control of haemopoietic stem cell proliferation and differentiation and to provide qualitative and quantitative information about stem cell numbers and identity.

Animals↗
PubMed1993

Acute myeloid leukaemia blast cells bind to human endothelium in vitro utilizing E-selectin and vascular cell adhesion molecule-1 (VCAM-1).

The adhesion of acute myeloid leukaemia (AML) blast cells to human umbilical vein endothelial cells (HUVECs) was investigated in vitro. Adhesion of blast cells from 10 cases of AML to unstimulated and interleukin-1 beta (IL-1) stimulated HUVECs was similar to or greater than that of control neutrophils. The extent to which endothelial E-selectin and vascular cell adhesion molecule-1 (VCAM-1) were involved in this adhesive process was investigated using blocking monoclonal antibodies to these proteins. In the majority of cases studied (7/8), anti-E-selectin significantly inhibited adhesion to IL-1 stimulated endothelium (26-65% inhibition) and in 5/8 cases so did anti-VCAM-1 (maximum of 31% inhibition). All cases were found to express the sialylated Lewis x antigen and very late activation antigen-4, ligands for E-selectin and VCAM-1 respectively. Our results indicate that leukaemic blast cells adhere to human endothelium and that there are E-selectin and, to a lesser extent, VCAM-1-dependent components to this process. Such adhesive interactions are likely to confer on AML blast cells the ability to migrate across the vascular wall and so to establish extravascular disease.

Acute Disease↗
PubMed1993

Cell biology of CML--a model linking the chronic and terminal phases.

A model for the pathogenesis of chronic myeloid leukaemia (CML) is proposed. It relies on a comparison between normal steady-state and regenerating haemopoiesis and suggests that chronic phase CML stem cells have a finite capacity for self-renewal. According to the model, metamorphosis of the disease occurs once the potential for chronic phase cell production has been exhausted. The model considers also the generation of leukocytosis in the chronic phase and the origin of the terminal phase. Comparison with normal regenerating haemopoiesis allows discrimination between features of CML that are fundamentally abnormal and those which are normally associated with regeneration.

Animals↗
PubMed1993

Regulation of growth in chronic myeloid leukemia.

Chronic myeloid leukemia (CML) is a malignant haemopoietic stem cell disorder which results in excessive production of cells of the myeloid series. It is associated with a consistent molecular abnormality, the BCR/ABL fusion gene. The product of BCR/ABL is a p210 protein tyrosine kinase but it is not known how this dictates the biological features of the disease. This review considers several key processes that can be suggested as candidate targets for the action of p210.

Cell Division↗
PubMed1993

Rapid positive selection of CD34+ cells using magnetic microspheres coated with monoclonal antibody QBEND/10 linked via a cleavable disulphide bond.

Positive selection of CD34+ cells has applications in diagnostic pathology, in peripheral blood and bone marrow transplantation, and in studies on the function and regulation of primitive haemopoietic stem cells. Antibody-coated magnetic microspheres (dynabeads) can be used to isolate these cells by positive selection procedures. However, the advantages of using dynabeads in some positive selection protocols are compromised by the retention of the beads on the cells. We present a protocol which allows the rapid chemical release of the beads from positively sorted cells. The murine immunoglobulin (Ig) G1 CD34 antibody, QBEND/10, was immobilised onto dynabeads as part of a three-layered immune complex: QBEND/10 was attached to F(ab')2 anti-mouse immunoglobulin antibody fragments, which were immunologically bound to a mouse IgG1 myeloma protein. The myeloma protein covalently bonded the triplex to the beads. Thus, disulphide bonds in the hinge region of the F(ab')2 could be reduced with 10 microM dithiothreitol and CD34+ cells released within 20 min. Purified cells can be re-phenotyped by multiple markers and subsets identified. Purity of 97%, recovery of > 50%, and viability over 90% of the CD34+ cells was readily achieved. Furthermore, granulocyte-macrophage colony-forming cells were retained in the positive fraction. This methodology can be used to purify other cell types, including T and B lymphocytes.

Antibodies, Monoclonal↗
PubMed1993

Hyaluronic acid regulates the function and distribution of sulfated glycosaminoglycans in bone marrow stromal cultures.

Treatment with steroid is necessary for adult human bone marrow-derived stroma to support hemopoiesis in vitro. We have investigated the effect of the steroid methylprednisolone on the glycosaminoglycans produced by adult bone marrow stroma. Methylprednisolone did not significantly alter the amount of sulfated glycosaminoglycans produced but the amount of the nonsulfated glycosaminoglycan, hyaluronic acid (HA), was dramatically decreased by the steroid. Culturing methylprednisolone-treated stroma with exogenous HA reduced the proportion of sulfated glycosaminoglycans incorporated into the cell layer and increased the proportion secreted into the medium. It also reduced the capacity of the stroma to bind and stimulate blast colony-forming cells. In fetal liver stroma, methylprednisolone did not decrease the amount of HA or alter the type of heparan sulfate produced. Thus, the ability of stroma cells to respond to steroid and the amount of HA in the extracellular matrix may vary in different marrow microenvironments. This may have functional consequences regarding their abilities to support hemopoiesis.

Adult↗
PubMed1993

An assessment of myeloid colony-forming-cell generation in liquid human bone marrow cultures: influence of accessory cells and cytokines IL-1 alpha, beta and IL-6.

Liquid culture of Ficoll isolated human marrow mononuclear cells followed by secondary clonogenic assays has been used to study the involvement of specific accessory cells and cytokines on the kinetics in vitro of human granulocyte-macrophage-colony forming cells (CFU-GM). Primary cultures produced an oscillation in CFU-GM numbers, typified by increases in CFU-GM after 4 and 9 days and reductions on days 2 and 4. Cultures of marrow cells depleted of mononuclear phagocytes produced a steady decline in CFU-GM number over a similar period. Removal of T cells was without effect. Comparison of colony assays using conditioned medium (CM) from the human bladder carcinoma cell line 5637 and 5637CM plus interleukin (IL)-3 revealed the decline of CFU-GM in mononuclear phagocyte depleted cultures due to lack of maturation of early myeloid progenitors. The cytokines IL-6 and IL-1 beta were detectable in the supernatant from whole marrow cultures and from cultures depleted of T cells but not in cultures depleted of mononuclear phagocytes. Using neutralizing polyclonal antibodies directed against IL-1 and IL-6, singly or in combination, in whole marrow cultures we showed that anti-IL-6 antibody alone had no effect on the number of colonies detected in the secondary clonogenic assay whilst anti-IL-1 and anti-IL-1 plus anti-IL-6 reduced colony counts by 44 and 83% respectively. Thus, endogenously produced IL-1 and IL-6 are involved in the dynamic changes in CFU-GM numbers when marrow mononuclear cells are cultured. Possible synergistic interactions are discussed.

Bone Marrow↗
PubMed1992

Binding of primitive hematopoietic progenitor cells to marrow stromal cells involves heparan sulfate.

Blast colony-forming cells (BI-CFC) and pre-colony-forming unit-granulocyte, monocyte (CFU-GM) in human bone marrow bind to marrow-derived stromal layers grown in the presence of methylprednisolone (MP+), but do not bind to stroma grown without MP (MP-). The BI-CFC bind to stroma and form colonies when overlaid with agar; the pre-CFU-GM bind to stroma and release CFU-GM into the supernatant culture medium (delta assay). These two classes of progenitor may represent similar stages of hematopoietic cell development. Their binding to stroma depends on the presence of heparan sulfate proteoglycan (HS-PG) in the extracellular matrix secreted by the stromal cells. Here, we have analyzed the functional and biochemical properties of HS-PG isolated from MP+ and MP- stromal cultures. HS-PG or isolated HS glycosaminoglycan (GAG) side chains partially blocked progenitor cell binding when they were added to the 2-hour binding phase of the BI-CFC or delta assays. Gel electrophoresis of HS-PG resolved more bands in matrix preparations from MP+ cultures than in preparations from MP- cultures. The blocking activity of the eluted MP+ HS-PG bands depended partly on the amount of GAG attached to the protein core and presumably partly on the structure of the core itself. Time course studies demonstrated that the HS-dependent phase of the binding interaction was limited to the first 30 to 60 minutes of the 2-hour binding phase. The different blocking effects of MP+ and MP- HS indicate that they have different biochemical properties. The HS-GAG in MP+ stroma has a higher degree of sulfation and a greater negative charge to mass ratio compared with MP- HS-GAG. Variations in HS may determine specific binding by hematopoietic progenitor cells and a heparan sulfate receptor is envisaged as acting in concert with further cell adhesion molecules (CAMs) on the progenitor cell surface.

Adult↗
PubMed1992

Circulating stem cells in mice treated with cyclophosphamide.

Chemotherapy has been used clinically to mobilize hematopoietic progenitor cells into the peripheral blood so that they can be harvested for autologous transplantation. In humans, this is demonstrated by the presence of circulating granulocyte-macrophage colony-forming cells (CFU-GM) and CD34-positive cells, but it has not been possible to confirm the presence of marrow-repopulating stem cells. In this study, we treated mice with 200 mg/kg cyclophosphamide (CY) and measured the numbers of white blood cells, day 12 CFU-S (CFU-S12), and CFU-GM in the peripheral blood. There was a peak in the numbers of CFU-S12 and CFU-GM 8 days after treatment with cyclophosphamide. Peripheral blood cells taken at this time rescued lethally irradiated mice and engraftment of donor cells was confirmed after 140 days in sex mismatched recipients using a Y chromosome-specific probe. In vitro culture of the blood cells harvested after cyclophosphamide showed that they proliferated in suspension cultures for at least a year in the presence of interleukin-3. The cultured cells rapidly lost their abilities to rescue irradiated mice and to form colonies in vitro, but they did not become leukemic. Also, CY-treated mice were irradiated with a leukemogenic dose of x-rays to coincide with peak circulating cell numbers but these animals did not develop an excess of leukemias over mice given irradiation alone.

Animals↗
PubMed1992

Synthesis and deposition of glycosaminoglycans in the murine hemopoietic stromal line S17: modulators of the hemopoietic microenvironment.

The murine hemopoietic stromal cell line S17 can support either myelopoiesis or lymphopoiesis depending on the culture conditions (i.e., the presence of steroid or mercaptoethanol). The glycosaminoglycans are important components of the extracellular matrix, which influence hemopoietic cell proliferation. Accordingly, glycosaminoglycans have been compared under different growth conditions. Under myeloid conditions (with steroid) a higher proportion of the sulfated glycosaminoglycans was incorporated into the cell layer and the extracellular matrix was increased, whereas synthesis was reduced under lymphoid conditions (with mercaptoethanol). The inclusion of steroid or mercaptoethanol did not alter the nature of the heparan sulfate synthesized as shown by DEAE anion-exchange chromatography, cleavage with specific enzymes and resolution of the digestion products by gel electrophoresis (oligomapping), and glycosaminoglycan size. The major species of sulfated proteoglycan synthesized under the different growth conditions (200 and 110 kd for the culture supernatant and 110, 71, and 38 kd for the cell layer) were shown to be very similar by polyacrylamide gel electrophoresis. Although no qualitative difference was found biochemically between the major glycosaminoglycans/proteoglycans, scanning electron microscopy revealed major differences in the pattern of deposition of the glycosaminoglycans. Under myeloid conditions, a rich fibrous matrix covered the cell layer, whereas under lymphoid conditions glycosaminoglycan was sparsely deposited. The arrangement of the extracellular matrix may have important consequences for myelo- and lymphopoiesis.

Animals↗
PubMed1992

Physical, phenotypic and cytochemical characterisation of stroma-adherent blast colony-forming cells.

Primitive cells defined as long-term culture initiating cells (LTCIC) and blast colony-forming cells (Bl-CFC) bind to cultured stromal layers, but cells at later stages of maturation [granulocyte-erythroid-macrophage-monocyte colony-forming cells (GEMM-CFC) granulocyte-macrophage (CM-CFC) and erythroid burst-forming units (BFU-E)] do not. The precise relationship between the LTCIC and Bl-CFC is not known and this study was undertaken to determine their relative positions in the haemopoietic hierarchy. We have defined the Bl-CFC population in terms of its density profile and antigenic phenotype and compared these characteristics with GM-CFC and BFU-E. The progenitor cell populations did not differ in density. The major phenotypic difference was seen using the myeloid monoclonal antibody S17-25 which reacted with fewer Bl-CFC than GM-CFC. Also, we have cytochemically analysed the cells in colonies derived from Bl-CFC. Our studies indicate that the Bl-CFC precede BFU-E and GM-CFC but not the LTCIC.

Antigens, Differentiation↗
PubMed1991

The effects of interferon-alpha on the proliferation of CML progenitor cells in vitro are not related to the precise position of the M-BCR breakpoint.

We investigated the effects of brief (2 h) and continuous exposure to recombinant interferon-alpha (2a) (rIFN-alpha) on the proliferation of primitive (blast colony-forming cells, Bl-CFC) and committed myeloid progenitor cells (BFU-E and GM-CFC) derived from blood and bone marrow of patients with chronic myeloid leukaemia (CML) and normal subjects. In all three clonogenic assays, rIFN-alpha suppressed colony formation in a dose-dependent manner. No differences were detected in the proliferation of CML or normal Bl-CFC and GM-CFC exposed to rIFN-alpha. Erythroid colony formation by normal, but not by CML BFU-E, was inhibited by relatively low concentrations (100 U/ml) of rIFN-alpha. However, in patients whose blood or marrow contained a mixture of Philadelphia chromosome (Ph)-positive and Ph-negative BFU-E, cytogenetic analysis of individual erythroid colonies showed no differential inhibition by rIFN-alpha. We found no difference in the sensitivity to rIFN-alpha of GM-CFC from patients whose leukaemic cells expressed BCR/ABL mRNA with the b2a2 junction and that of GM-CFC from patients with the b3a2 mRNA. We conclude that (1) rIFN-alpha does not have a significant leukaemia-specific effect on the progenitor cells detected in these assays, and (2) the sensitivity of CML GM-CFC to rIFN-alpha is independent of the type of BCR/ABL message present in the cells. The clinical efficacy of rIFN-alpha could be due to selective toxicity to cells not assayed in this study, to effects on accessory cells or to alterations induced in progenitor cell/stromal cell interactions.

Blotting, Southern↗