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M Y Gordon

Publications and source records attributed to M Y Gordon.

At least 37 records · Page 2Linked to original sources
PubMed1998

Interleukin 3 (IL-3), but not stem cell factor (SCF) increases self-renewal by human erythroid burst-forming units (BFU-E) in vitro.

Interleukin 3 (IL-3) and stem cell factor (SCF) are both important regulators of early haemopoietic cell development. Here, we have compared their effects or the kinetics of erythroid burst formation by BFU-E in normal adult bone marrow. We grew the BFU-E in the presence of erythropoietin (Epo) alone, Epo + IL-3 or Epo + SCF and scored the numbers of subcolonies in individual bursts after 14 days. The data were plotted as the cumulative distribution of the numbers of subcolonies per erythroid burst then linearised by logarithmic transformation. Analysis of the data revealed that IL-3 increases the numbers of subcolonies in BFU-E whilst SCF increases the size of the subcolonies themselves. Experiments involving combinations of Epo + IL-3 + SCF and the delayed addition of IL-3 or SCF indicated that the actions of IL-3 and SCF are largely independent of one another. We conclude that: (1) IL-3 acts at an earlier stage of erythroid differentiation than SCF, and (2) it may be possible to classify haemopoietic growth factors according to their effects on cell kinetics in vitro.

Cells, Cultured↗
PubMed1998

BCR-ABL-positive progenitors in chronic myeloid leukaemia patients in complete cytogenetic remission after treatment with interferon-alpha.

To determine the source of residual disease detected in patients with chronic myeloid leukaemia (CML) in complete cytogenetic remission (n=8) after treatment with interferon-alpha (IFN-alpha), we have tested CFU-GM colonies grown from bone marrow mononuclear cells or from plastic-adherent (Pdelta) cells for BCR-ABL mRNA using a nested multiplex RT-PCR. We compared our results with those obtained by analysis of colonies from newly diagnosed patients (n=4) and patients achieving no cytogenetic response (n=1) or incomplete cytogenetic response to treatment with IFN-alpha (n=5). A total of 1239 informative colonies were analysed. A small proportion of BCR-ABL-positive colonies was detected in all eight patients in complete cytogenetic remission, suggesting the persistence of leukaemia that could potentially lead to relapse. The overall proportion of BCR-ABL-positive colonies in patients achieving a cytogenetic response to IFN-alpha correlated with the levels of BCR-ABL transcripts detected in the peripheral blood by competitive RT-PCR (P=0.004). We conclude that residual disease detected in the peripheral blood of complete cytogenetic responders to IFN-alpha is at least partly derived from clonogenic myeloid cells. It is probable that the leukaemia clone in CML is only very rarely or never entirely eradicated by treatment with IFN-alpha.

Adult↗
PubMed1998

The impact of antenatal and perinatal variables on cord blood haemopoietic stem/progenitor cell yield available for transplantation.

We investigated the impact of maternal and fetal variables on cord blood (CB) haemopoietic stem/progenitor cell content. These included maternal age, ethnic origin, parity, ABO and Rhesus D blood group, antenatal haemoglobin, alcohol and cigarette consumption at time of registration, mode of delivery, duration of the first and second stages of labour, gestational age, birth weight, cord pH and cord erythrocyte mean cell volume (MCV). Cord volumes and total nucleated cellularities (TNC) were recorded, the colony assay for granulocyte-macrophage colony-forming-cells (CFU-GM) was used to quantify the progenitor cells and the potential of CFU-GM to produce secondary colonies on replating was used as a measure of progenitor cell quality. We found: (1) significantly greater (P=0.04) volumes were collected from babies who weighed > or = 2.5kg versus babies with a birth weight <2.5kg; (2) significantly greater numbers of mononuclear cells (MNC) from mothers who drank 0-3 units versus those who drank > or = 4 units of alcohol weekly (P=0.03), and in babies with a cord pH < or = 7.1 v > 7.1 (P=0.02); (3) Significantly greater numbers of cord CFU-GM in mothers who drank 0-3 v > or = 4 units weekly (P=0.004) and smokers of > or = 10 v 0-9 cigarettes daily (P=0.02) and in spontaneous vaginal deliveries than assisted vaginal and caesarean deliveries (P=0.04), and (4) the potential of CFU-GM to produce secondary colonies was significantly greater in CB derived from Caucasians than from non-Caucasians ( P=0.02); in assisted vaginal delivery v spontaneous vaginal (P=0.02) and in deliveries with prolonged first stage of labour v short first stage of labour (P=0.04). We conclude that antenatal and perinatal variables may influence the CB stem/progenitor cell yield available for transplantation.

Alcohol Drinking↗
PubMed1998

A two-color BCR-ABL probe that greatly reduces the false positive and false negative rates for fluorescence in situ hybridization in chronic myeloid leukemia.

The t(9;22) translocation resulting in the fusion of BCR and ABL genes is pathognomonic in chronic myeloid leukemia (CML) and may be investigated at the molecular level using fluorescence in situ hybridization (FISH). Two-color BCR-ABL probes visualizing one fusion signal (1F FISH) have high false positive rates (FPR) and false negative rates (FNR). The FPR is a result of the random spatial association of probe signals within normal interphase cells so that some cells appear to contain the BCR-ABL fusion gene. The FNR of 1F FISH probes depends on the distance between the BCR and ABL probes hybridized to the BCR-ABL fusion gene (< or =368 kb); the "gap" between the signals causing the cell to be interpreted as normal. To overcome these difficulties, a two-color probe was used, employing four yeast artificial chromosome (YAC) sequences that span the breakpoint regions of the BCR and ABL genes and that visualize the two fusion signals BCR-ABL and ABL-BCR in CML cells (2F FISH). The FNR for the 2F FISH probes was assessed on clonal Ph+ granulocyte-macrophage-colony-forming cell (CFU-GM) derived colonies and was reduced to 0.4% (2/450), compared with an FNR of 13.5% (111/823) with 1F FISH. The FPR in normal mononuclear cells for the 2F FISH was 0. 19 +/- 0.12% (3/1,700), whereas the FPR using 1F FISH was 4.5 +/- 2.3% (63/1,294). The 2F FISH can thus be used to evaluate very small frequencies of BCR-ABL-positive and -negative interphase cells and may be of use in the clinical monitoring of CML.

Adult↗
PubMed1997

Characterization of human recombinant interleukin 2 binding to heparin and heparan sulfate using an ELISA approach.

We have developed an enzyme-linked immunosorbent assay (ELISA) approach for the study of interactions between cytokines and glycosaminoglycans. This involves, as solid phase, a synthetic heparin-bovine serum albumin (BSA) complex in which the heparin is coupled via its reducing terminus to the protein using sodium cyanoborohydride. We have investigated the sensitivity and specificity of this experimental technique, employing antithrombin (AT III) and fibroblast growth factor 2 (FGF-2) as well-characterized heparin binding proteins. Using this ELISA method, we have established that human recombinant interleukin (IL-2) binds to heparin in a concentration-dependent manner. Soluble heparin competes for the binding of IL-2 to the complex with 50% inhibition at 5 microg/ml. This IC50 value provides an estimate of the binding constant of around 0.5 microM. This value is at least two orders of magnitude larger than that for the binding of IL-2 to its dimeric and trimeric cell surface receptors, but similar to that for binding to the IL-2 receptor beta polypeptide acting alone. Our ELISA shows that in addition to soluble heparin, fuciodan also competes for IL-2 binding, but chondroitin sulfate and dermatan sulfate are inactive. Of six heparan sulfates tested, only one highly sulfated preparation competed for IL-2. The interaction between IL-2 and heparin-like glycosaminoglycans is likely to be an important mechanism for retaining IL-2 close to its sites of secretion, thus giving rise to localized concentration gradients in the tissues.

Antithrombin III↗
PubMed1997

CD34+ cell selection in chronic phase chronic myeloid leukaemia: a comparison of laboratory grade columns.

CD34 positive (CD34+) cell selection is increasingly used for a number of important applications including gene therapy studies, ex vivo expansion and purging. However there are no data regarding the use of different technologies for CD34+ cell selection in chronic myeloid leukaemia (CML). We therefore compared the performance of three laboratory grade CD34+ selection columns (MiniMACS, Cellpro Ceprate LC and Baxter Isolex 50), using CML chronic phase peripheral blood (PB) and bone marrow (BM). With different CML samples the CD34+ purity from the three columns was equivalent, but comparing five paired samples the Ceprate purity was greater than MiniMACS, at 92.5 and 80.9%, respectively, P = 0.04. Combining results from paired and unpaired CML samples, MiniMACS (n = 7) gave a higher CD34+ yield than Ceprate LC (n = 8) or Isolex 50 (n = 4) with a mean of 51.1%, 24.3% and 13.2% respectively, (P = 0.04 and 0.01). Cell losses with all columns were similar. Attempts to improve the yield from the Ceprate LC columns by modifying the method were unsuccessful. Following MiniMACS and Ceprate LC separation the clonogenic potentials of CD34+ cells in the pre- and positive cell fractions were the same. The proportion of CD34+ 38- or CD34+ DR- cells was unchanged following column separation. These data suggest that the MiniMACS column may be the best column for CD34+ cell selection in CML but these results must be confirmed using large scale clinical columns once the MiniMACS column is licensed. It is possible that variations in CD34+ cell yields between the different columns reflect differences in antibody binding affinity to CML cells, or differences in column technologies.

Antigens, CD↗
PubMed1997

Expression of interferon regulatory factor (IRF) genes and response to interferon-alpha in chronic myeloid leukaemia.

Interferon regulatory factors (IRF) 1 and 2 are DNA-binding proteins which control interferon (IFN) gene expression. IRF1 functions as an activator for IFN and IFN-inducible genes, whereas IRF2 represses the action of IRF1. Expression of the two regulatory genes is itself IFN-inducible. Because therapeutic responses of chronic myeloid leukaemia (CML) patients to IFN-alpha may be determined by intracellular levels of these two mutually antagonistic transcription factors, we have devised a competitive reverse-transcription polymerase chain reaction (RT-PCR) assay which provides an estimate of the ratio of IRF1 to IRF2 expression in a given cell population. Analysis of peripheral blood leucocytes from 25 normal individuals showed that the IRF1:IRF2 ratio varied between 1.13 and 2.30 (mean +/- s.d. 1.49 +/- 0.33). Similar values were obtained for normal bone marrow specimens, with no significant difference between CD34+ and CD34- cells. In contrast, the IRF1:IRF2 ratio in leucocytes from CML patients showed a much wider variation (0.53-5.11). Eleven out of 130 patients in chronic phase had ratios above the normal range, whereas none of the 33 blast crisis samples had a ratio >2.5. Analysis of diagnostic specimens in 59 CML patients treated subsequently with IFN-alpha showed a high IRF1:IRF2 ratio of 5.11 in one of two patients who became complete responders; all the 53 patients with minimal or no cytogenetic response had ratios below 2.5. In a separate series of 97 CML patients studied after IFN-alpha therapy a highly significant correlation was found between the IRF1:IRF2 ratio and both the cytogenetic and the molecular response (ie low concentration of BCR-ABL transcripts) to treatment: 53 out of 115 prospectively analysed samples of good cytogenetic responders had ratios above 2.0, as opposed to only 13 out of 91 samples from poor responders (P < 0.0001; chi2 test). We conclude that a high ratio of IRF1/IRF2 expression may be associated with good cytogenetic and molecular response to IFN-alpha in CML.

Adult↗
PubMed1997

BCR/ABL-negative progenitors are enriched in the adherent fraction of CD34+ cells circulating in the blood of chronic phase chronic myeloid leukemia patients.

Philadelphia chromosome-positive (Ph+) hemopoietic cells predominate in patients with chronic myeloid leukemia (CML) in chronic phase, but some Ph presumably normal stem cells persist in most patients. Ph cells are relatively frequent, compared to mature cell populations, in primitive hemopoietic cell populations from CML patients. We have purified CD34+ cells from chronic phase CML blood and separated them into two fractions on the basis of adherence or non-adherence to tissue culture plastic. We also separated CD34+ CML cell populations into HLA-DR(hi) and HLA-DR(lo) fractions and CD38(hi) and CD38(lo) fractions by flow cytometry. The CD34+ cells that adhered to plastic were predominantly CD33-, CD38- and HLA(-)-DR; cells with these phenotypic properties were significantly rarer in the CD34+ non-adherent cell population (P = 0.008-0.02). Expression of p210 BCR/ABL mRNA by adherent, non-adherent, HLA-DR(hi) and HLA-DR(lo)CD34+ cell subpopulations was demonstrated by RT-PCR. Using fluorescence in situ hybridization (FISH) in conjunction with BCR and ABL probes we detected Ph+ and Ph- cells in both adherent and non-adherent CD34+ cell fractions of 15/15 patients studied and in the HLA-DR(lo) or CD38(lo) sorted CD34+ cell fractions. The concentration of Ph- cells in the adherent CD34+ cell fraction was three-fold higher than in the non-adherent fraction (P = 0.001). Ph- adherent cells were detected in untreated CML patients and as late as 6 years after diagnosis of CML in patients treated with hydroxyurea (HU) or interferon-alpha (IFN-alpha). We conclude that whilst appreciable numbers of Ph- primitive hemopoietic progenitors are present in the circulation in untreated patients and also in treated patients in late chronic phase, the majority of cells expressing CD34 but not CD33, CD38 or HLA-DR antigens, are part of the CML clone.

Adult↗
PubMed1997

Biological properties of peripheral blood progenitor cells mobilized by cyclophosphamide and granulocyte colony-stimulating factor.

Patients transplanted with mobilized blood progenitor cells (PBPC) recover their neutrophil counts more rapidly than patients transplanted with bone marrow even when they receive the same dose/kg of granulocyte-macrophage colony-forming cells (CFU-GM). Here we have sought a biological explanation for this phenomenon. Most CD34-positive PBPC are quiescent (<1% in S phase) when they are collected from the bloodstream of patients treated with cyclophosphamide and granulocyte colony-stimulating factor (G-CSF), but we have shown that they are able to resume proliferation rapidly in vitro by measuring the kinetics of CFU-GM production by primitive plastic-adherent (Pdelta) cells. Also, Pdelta cells in PBPC harvests, unlike normal marrow Pdelta cells, were insensitive to cell-cycle restraint imposed by contact with marrow-derived stromal cells. We found that Pdelta cells in PBPC collections produce relatively more CFU-GM and relatively fewer BFU-E than Pdelta cells in bone marrow, indicating that granulopoiesis might occur at the expense of erythropoiesis, but we were unable to find any differences in the kinetics of granulocytic maturation between PBPC and bone marrow. Our interpretation of these findings is that transplanted PBPC rapidly enter the cell cycle and contact with stromal cells in the marrow does not reduce the proportion of progenitors participating in neutrophil production. Consequently. neutrophil recovery after PBPC infusion is more rapid than neutrophil recovery after marrow infusion. Granulopoiesis at the expense of erythropoiesis may also contribute to this effect.

Cell Cycle↗
PubMed1997

Stromal cells negatively regulate primitive haemopoietic progenitor cell activation via a phosphatidylinositol-anchored cell adhesion/signalling mechanism.

We have tested the effect of stromal cells on the proliferation in long- and short-term cultures of primitive (Thy-1+, CD34+, CD33-, CD38- , HLA-DR , adherent in vitro and quiescent in vivo) progenitors in normal human bone marrow. These primitive cells produce granulocyte-macrophage colony-forming cells (CFU-GM) that are measured in secondary clonogenic assays. Addition of stromal cells to normal adherent haemopoietic progenitor cells reduced CFU-GM production by 80% (P =0.0002) after 1 week of incubation. In long-term culture (LTC), in the presence of stroma. the normal adherent cells did not produce significant numbers of CFU-GM until 3-4 weeks later which suggests that stromal cells reduce the probability of quiescent cell activation. This effect could not be attributed to soluble inhibitory factors and was specific to stroma grown with, rather than without, methylprednisolone. It was blocked by heparanase (H'ase) II treatment of stromal cells, by phosphatidylinositol-specific phospholipase C (PI-PLC) treatment of progenitor cells, by antibody blocking of beta1 integrin molecules or by exposure to glucose/N-acetyl-D-glucosamine/alpha-methyl-D-mannoside, but not by exposure to galactose or fructose. Moreover, these interventions enabled the progenitor cells to respond to stimulatory factors in the culture supernatant. We interpret these results as support for a model involving primitive progenitor cell binding to stroma by PI-CAM/HS, beta1 integrin activation via lectin-like interactions and the transduction of signals which reduce the ability of primitive cells to respond to ambient stimulators. This model provides a mechanism for the maintenance of the quiescent state of stem cells by adhesion to stromal cells.

Cell Communication↗
PubMed1996

Effects of interleukin 6 administration on platelets and haemopoietic progenitor cells in peripheral blood.

Platelet numbers and circulating haemopoietic progenitor cells were examined in 12 patients with advanced malignancies who were receiving recombinant human interleukin-6 (rhIL-6) as part of an investigation of its thrombopoietic effects. Patients received recombinant glycosylated IL-6 by daily subcutaneous injection for 7 consecutive days in doses of 1, 3 or 10 micrograms/kg/day. Platelet numbers increased reaching a peak on days 12-15 with a mean on day 15 of 198.1% of pre-treatment values. This was accompanied by a significant fall in the mean platelet volume (mean decrease of 10.6%, P = 0.0044). No significant correlation was seen between the IL-6 dose and the change in platelet number. No significant differences were observed between pre- and post-treatment levels of circulating erythroid burst-forming units (E-BFU) and granulocyte macrophage colony-forming units (GM-CFU) but a small significant increase was seen in circulating primitive progenitor cells measured in a plastic-adherent (P delta) assay (P = 0.025). As positive controls, a group of patients treated with cyclophosphamide/G-CSF showed significant increases in GM-CFU (P = 0.018), E-BFU (P = 0.018) and P delta progenitors (P = 0.028). These data suggest that the thrombopoietic effects of IL-6 are mediated at a relatively late stage via effects on megakaryocyte differentiation, with a relatively small effect on circulating haemopoietic progenitors.

Adult↗
PubMed1996

Kinetics of colony formation by BFU-E grown under different culture conditions in vitro.

Colony formation by erythroid burst-forming units (BFU-E) involves a variable number of cell divisions before individual 'subcolonies' begin to appear. Consequently the numbers of subcolonies vary amongst individual bursts. If this observation is interpreted as a reflection of a stochastic process, the number of subcolonies in each individual burst represents the number of divisions by the BFU-E prior to commitment to terminal differentiation. This provides a means for quantitating the probability of erythroid differentiation (pD) and the probability of renewal (1 - pD). In order to determine whether these kinetics of burst formation can be influenced by exogenous factors we used three commercially available media designed for the growth of BFU-E. We found that subcolony numbers per burst ranged from one to 64 and that the cumulative distributions of subcolonies per burst followed a logarithmic curve (r > 0.90). Differences were observed in the distribution of subcolonies per burst when BFU-E were grown in different media (P=0.03; Kruskall-Wallis test). The probability of immediate terminal differentiation (i.e. committment to form a subcolony) was 0.25 for two of the media and 0.7 for the third. The corresponding renewal probabilities were O.75 and O.3. These data indicate that the proliferation kinetics of BFU-E are susceptible to regulation by exogenous factors.

Cell Culture Techniques↗
PubMed1996

Abnormal kinetics of colony formation by erythroid burst-forming units (BFU-E) in chronic myeloid leukaemia.

We have investigated the kinetics of colony formation by progenitor cells in chronic myeloid leukaemia (CML) using erythroid burst-forming units (BFU-E) as a model system. For this, we scored the numbers of subcolonies produced by individual BFU-E in cultures of normal marrow and blood cells and in cultures of CML blood cells. The formation of an erythroid burst consisting of a single subcolony was taken as evidence for immediate terminal differentiation; the formation of multiple subcolonies was taken as evidence for commitment to terminal differentiation only after several cell generations. Therefore the probability of differentiation can be obtained by scoring the numbers of subcolonies in individual erythroid bursts. We found that the probability of differentiation is decreased (P = 0.0004) and the number of subcolonies increased (P = 0.01) in CML BFU-E compared with normal BFU-E. The cellularity of the BFU-E was also increased in CML. Using the probabilities of differentiation and renewal obtained from the BFU-E cultures the results fitted the predictions of a stochastic branching model. These results indicate that (a) commitment to terminal erythroid differentiation occurs over several cell generations in populations of BFU-E, (b) the probability of commitment to terminal differentiation (PD) within a particular population of BFU-E, remains a constant independent of the number of cell generations involved, (c) PD is lower during burst formation by CML BFU-E than by normal BFU-E, and (d) commitment to terminal differentiation occurs over more cell generations in CML burst formation than in normal burst formation. Therefore a reduced probability of differentiation may be a primary defect and could explain the expansion of the erythroid progenitor cell compartment in CML.

Bone Marrow↗
PubMed1996

Evaluation of "discordant maturation' in chronic myeloid leukaemia using cultures of primitive progenitor cells and their production of clonogenic progeny (CFU-GM).

The 'discordant maturation hypothesis' proposes that the most mature proliferating cells in chronic-phase chronic myeloid leukaemia (CML) are responsible for the expansion of the Ph-positive population. To evaluate this hypothesis we used a delta assay for primitive haemopoietic cells (P delta assay for P delta cells) which allows investigation of the kinetics of granulocyte-macrophage progenitor (CFU-GM) production. The frequencies of these primitive (P delta) cells were similar in CML blood (14.5/10(5) mononuclear cells), CML marrow (17.3/10(5)) and normal marrow (11.6/10(5)) The average frequency in normal blood is only 0.58/10(6). The absolute numbers of P delta cells in CML patients are therefore greatly increased. The average numbers of CFU-GM produced by individual P delta cells were reduced in CML blood (8.1) and marrow (11.6) compared with normal marrow (28.5). This is consistent with a reduced probability of differentiation at the single cell level in CML. Although the absolute number of CFU-GM produced by individual CML P delta cells was subnormal there was a relative increase in the number of day 7 CFU-GM compared with the number of day 14 and 21 CFU-GM, which agrees with the 'discordant maturation hypothesis'. This bias towards day 7 colony formation could reflect accelerated maturation by the CFU-GM produced by P delta cells or, alternatively, the production of CFU-GM with shorter than normal maturation pathways. Overall, these results suggest that discordant maturation does not by itself account for myeloid expansion in CML. It is more likely that myeloid expansion in CML is due mainly to an increase in the number of primitive haemopoietic progenitor cells.

Bone Marrow↗