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Biomedical subjects

M G Macey

Publications and source records attributed to M G Macey.

At least 55 records · Page 3Linked to original sources
PubMed1994

Characterization and modulation of drug transport kinetics in K562 c1.6 daunorubicin-resistant cell line.

The effects of cyclosporin A (CSA) and cellular energy depletion on daunorubicin (DAU) transport kinetics were investigated in a human erythroid leukaemia cell line K562 c1.6 selected for resistance to daunorubicin. K562 c1.6/DAU resistant cells displayed high levels of P-glycoprotein and a high level of multidrug resistance against several antitumour drugs. The resistance factors of K562 c1.6/DAU cells to DAU, doxorubicin, vinblastine and etoposide were 106, 114, 85 and 13 respectively. A 1.6-fold decrease (P < 0.01, n = 8) in DAU accumulation and a 4-fold increase (P < 0.001, n = 8) in DAU efflux were shown in the resistant cells when compared to K562 c1.6 drug-sensitive parental cells. K562 c1.6/DAU cells were also shown to reach a DAU saturation level (SL) 8-fold faster (P < 0.001, n = 8) than the parental cells. Addition of CSA to the resistant cells led to a dose-dependent increase in cellular DAU retention, while no such effect was observed in the sensitive cells by the introduction of CSA. Resistance to the antitumour drugs could be reduced to various extents by CSA. The patterns of changes and modulations of DAU transport kinetics, as well as chemosensitivity in K562 c1.6/DAU cells were found to be similar to a vinblastine-resistant leukaemia cell line CEM/VLB100. However, K562 c1.6/DAU cells were more resistant to DAU, doxorubicin and etoposide than the CEM/VLB100 cells. An increase in DAU accumulation, intracellular SL and the time to reach 90% saturation level (SL90), and a decrease in DAU efflux in the resistant but not the sensitive cells were found in response to ATP depletion by sodium azide. These effects could be completely reversed by addition of glucose. Our results suggest that the presence of an energy-dependent effluxing mechanism responsible for the decreased drug accumulation and enhanced drug efflux may make a major contribution to the mechanism of resistance in K562 c1.6/DAU resistant cells.

Antineoplastic Agents↗
PubMed1994

The ex vivo function and expression of function-associated antigens of peripheral blood neutrophils and monocytes.

The availability of recombinant human granulocyte and granulocyte-macrophage colony-stimulating factors (rhG-CSF and rhGM-CSF) has prompted many studies of the analysis of antigen expression and function of monocytes and neutrophils from patients receiving these factors as therapeutic agents. Preparatory procedures for leukocytes are known to alter antigen expression and so function. We therefore investigated the use of a novel procedure in which live leukocytes are analyzed by flow cytometry without isolation from blood. The expression levels of CD11b, CD13, CD14, CD16, and CD18 antigens and L-selectin (TQ1 and Leu-8 epitopes) on neutrophils and monocytes from 15 normal individuals were determined and compared with a previously used method in which the leukocytes were fixed and the erythrocytes lysed before analysis. Significant differences for the apparent expression of CD11b, CD18, and L-selectin were observed between the two methods. The reasons for this were investigated. Since the new method allowed analysis of live cells, we also investigated whether modulation of antigen expression could be determined following receptor agonist interaction. This was found to be easily achievable, and we advocate using the new procedure where possible for the ex vivo analysis of function and function-associated antigens on monocytes and neutrophils.

Adult↗
PubMed1993

A simple flow cytometric procedure for the determination of surface antigens on unfixed leucocytes in whole blood.

A novel procedure has been developed for the quantitation by flow cytometry of function-associated antigens on neutrophils and monocytes in unlysed, unfixed, peripheral blood samples. Freshly drawn blood anticoagulated with the serine esterase inhibitor, phenylmethylsulphonyl fluoride, is mixed with the vital nucleic acid stain, LDS-751, labelled with monoclonal antibodies for 5 min at 4 degrees C, diluted and analysed in a five-parameter flow cytometer. The three major leucocyte subpopulations (neutrophils, lymphocytes and monocytes) can be resolved in real time on the basis of their side light scattering and staining intensity with LDS-751 in the FL3 channel (erythrocytes and platelets stain very weakly), whilst the fluorescence intensity due to bound fluorescein isothiocyanate- or phycoerythrin-labelled antibody is monitored simultaneously in the FL1 or FL2 channels respectively. This procedure avoids potential artefacts that can occur due to the use of fixatives, erythrocyte lysing agents, or anticoagulants which are also divalent metal ion chelators. It should be widely applicable for the quantitation of those function-associated antigens, such as adhesion molecules and immune complex receptors, whose surface expression can be rapidly upregulated following activation, as well as for the quantitation of those leucocyte surface antigens whose expression is not subject to rapid modulation.

Antigens, Surface↗
PubMed1993

von Willebrand factor release and P-selectin expression is stimulated by thrombin and trypsin but not IL-1 in cultured human endothelial cells.

The stimulated release of von Willebrand factor (vWF) from endothelial cells by secretagogues such as thrombin is associated with the translocation of Weibel-Palade bodies to the cell membrane and the surface expression of P-selectin (also known as GMP 140, PADGEM and CD 62). P-selectin, which is stored in Weibel-Palade bodies, is a neutrophil and monocyte adhesion molecule important in the initiation of inflammation. We have developed a simple assay for the detection of P-selectin on endothelial cells using indirect immunofluorescence and flow cytometry and have confirmed that this is temporally related to vWF release. The assay has been used to demonstrate that IL-1 does not cause Weibel-Palade body degranulation but that trypsin does. This has implications for the use of passaged endothelial cells in the study of vWF release and the assay has numerous possible applications in study of mechanisms of stimulated vWF release.

Cells, Cultured↗
PubMed1993

Quantitation of adhesion molecules and other function-associated antigens on human peripheral blood leucocytes.

Quantitating satisfactorily the expression of adhesion molecules and other functional antigens on the surface of human peripheral blood granulocytes by flow cytometry is difficult. Artefactual (in vitro) changes, which occur in the expression of these molecules during conventional cell preparation and labelling procedures, led to the development of a leucocyte preparation technique that prevents upregulation of the leucocyte integrins and other antigens. The technique, as originally evaluated, involves fixing freshly drawn blood with formaldehyde (final concentration 0.2%) for 4 min at 37 degrees C before adding ammonium chloride to lyse the erythrocytes; procedures which are inconvenient when blood samples are taken in wards or clinics that are some distance from a laboratory. We therefore investigated i) whether blood could be kept with commonly used anticoagulants for up to 1 h before fixation, and ii) whether unanticoagulated blood could be kept with formaldehyde for a similar period before processing was continued. The results showed that when quantitating those granulocyte surface antigens which can be rapidly upregulated: i) that it is preferable to mix freshly drawn unanticoagulated blood directly with formaldehyde; ii) that if blood must be collected into anticoagulants, it should be kept for only the briefest possible time before processing; and iii) that the time for which cells are exposed to 0.2% formaldehyde must be carefully controlled and limited to 4 min. A corollary of the results with anticoagulants is that it is probably impossible to isolate live granulocytes by currently available procedures without artefactually altering the expression of these antigens in vitro.

Anticoagulants↗
PubMed1993

Leukocyte activation and function-associated antigens in inflammatory disease.

Expression of adhesion molecules, CD11a, CD11b and CD18, and of the function-associated molecules CD3, CD4, CD8, CD16, CD19, CD56 and CD57 was assayed on peripheral blood leukocytes from normal control subjects (n = 10), and from patients with adult periodontitis PD (n = 9), ankylosing spondylitis AS (n = 11) and rheumatoid arthritis RA (n = 14). A novel rapid fixation leukocyte preparation technique was used which prevents artefactual up-regulation of surface antigens. In RA patients, the percentage of CD18+ lymphocytes was decreased and that of CD11b+ neutrophils was increased. On lymphocytes the mean fluorescence intensity (MFI) of both CD11b and CD18 was decreased whereas that of CD57 was increased. In AS patients the percentages of CD11b+ lymphocytes and neutrophils were increased and CD18+ lymphocytes and neutrophils were decreased. On lymphocytes the MFIs of CD11b and CD18 were decreased, whilst that of CD16 was increased. On neutrophils the MFI for CD18 was increased. No significant differences (p < 0.01) were seen for the periodontitis patients. It is suggested that the antigen expression on peripheral blood cells from RA and AS patients is consistent with leukocyte activation.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1993

Abnormal binding properties of blood monocytes in rheumatoid arthritis.

Blood monocytes from patients with RA exhibited a greater binding to monolayers of umbilical cord vein endothelium than monocytes from control subjects (mean 42% increase; p < 0.01). When control monocytes were added to TNF or IL-1 treated endothelium their adhesion was enhanced (mean 24% increase; p < 0.05), whereas the number of monocytes from RA patients binding to TNF or IL-1 treated monolayers was less than that adhering to untreated endothelial cells (mean 22% inhibition; p < 0.02). The surface expression of CD11b/CD18 on RA monocytes was increased and pretreatment of normal and RA cells with an anti-CD18 monoclonal antibody inhibited their attachment to untreated and cytokine-treated endothelial cells. Normal blood monocytes activated with LPS demonstrated an enhanced binding to untreated cultures (mean 23% increase; p < 0.05) and an inhibited attachment to cytokine-treated endothelial cells. This study suggests that blood monocytes in RA may be activated and that this property modifies the attachment of these cells to normal and "inflammatory" endothelium.

Arthritis, Rheumatoid↗
PubMed1993

Structure-dependent antitumor activities of novel anthracyclines YM1, YM3, YM4 and YM6: drug transport properties and effects on biomacromolecule synthesis in drug sensitive and resistant leukemia cells.

The antitumor activities of four novel doxorubicin (DOX) analogues, YM1, YM3, YM4 and YM6 in relation to their structure and drug transport properties, have been investigated in U937 monocytic and CCRF-CEM lymphoid drug sensitive leukemia cell lines, as well as in CEM/VLB100, a drug resistant subline displaying high levels of P-glycoprotein. Treatment of all cell lines with YM1, 3, 4 and 6 produced a dose-dependent decrease in DNA, RNA and protein synthesis as measured by [3H]-thymidine, [3H]-uridine and [3H]-leucine uptake respectively. YM1 was more effective than YM3, YM4 or YM6 against the drug sensitive cells. The antitumor effects of all these DOX-analogues on macromolecule synthesis in U937 and CCRF-CEM cells were lower than that of DOX and epirubicin (EDR). A rapid accumulation of the novel anthracyclines was found in all cell lines compared with DOX or EDR. However, the maximal accumulation of the DOX-analogues was lower than that of EDR. There is a greater efflux from CCRF-CEM sensitive cells and less from CEM/VLB100 resistant cells of the DOX-derivatives when compared with EDR and DOX. Drug-induced cytotoxicity significantly correlated (P < 0.05) with drug retention levels in CCRF-CEM and U937 drug sensitive cells as indicated by an inverse correlation curve between anthracycline retention and drug-induced IC50 value. It was demonstrated that an increased level of drug retained within the sensitive cells would therefore produce a more cytotoxic effect of the drug. However, no such correlation was observed in CEM/VLB100 resistant cells. YM3 was shown to have an increased antitumor activity against CEM/VLB100 resistant cells compared with DOX with a lower resistance factor. These results showed that the antitumor effects of four novel DOX-analogues, like DOX or EDR, were associated with inhibition of DNA replication, transcription and translation. The finding that resistant leukemic cells are more susceptible to the cytotoxic effect of YM3 than DOX warrants further investigation to identify the intrinsic mechanism of resistance.

Biological Transport↗
PubMed1993

Fixation with formaldehyde induces expression of activation dependent platelet membrane glycoproteins, P selectin (CD62) and GP53 (CD63).

Platelet activation in vivo is important in the pathogenesis of thrombosis. Accurate measurement is difficult due to artefactual in vitro preparation related activation. This can be overcome by using whole blood techniques, such as flow cytometry. However, there is little consensus on methods of platelet preparation, procedures for use of fixation, or the optimal types/amounts of fixative. The use of unfixed platelets has received little attention. We describe a series of experiments comparing platelet activation antigen expression detected by flow cytometry in fixed and unfixed samples. Formaldehyde increased the expression of both CD62 and CD63. We recommend that fixation with formaldehyde should not be used when studying platelet activation.

Antigens, CD↗
PubMed1993

Modulation of CD4 by suramin.

Suramin is a polysulphonated compound which can selectively bind to, and inhibit the activity of, a wide range of growth factors. There has been renewed interest recently in suramin as an anti-cancer agent and therefore we have studied its effects on lymphocyte subset populations and recombinant human IL-2 (rhIL-2) activation on lymphocytes in vitro. In the presence of rhIL-2 (1000 U/ml), suramin (200 micrograms/ml) caused a decrease in percentage of cells expressing the predominantly T cell antigen CD3; no change in percentage of cells expressing the T suppressor/cytotoxic subset antigen, CD8; a small rise in those expressing the natural killer cell antigen, CD56; and a large significant fall in those expressing the T helper subset antigen CD4 (48.51% versus 27.97%; P < 0.001). CD4 modulation by suramin was also found on the CD4+ cell lines CEM and MOLT-4. The effect of suramin on rhIL-2-induced activation antigen expression remains equivocal, since a small rise in CD25 expression and small falls in CD71 and HLA-Dr expression were recorded. The modulatory effect of suramin on CD4 expression was not reversible over a 96-h culture period in its continued presence. However, on removal of suramin by extensive washing, recovery of CD4 expression was detected within 24 h. Suramin-induced modulation, but not PMA-induced modulation, could be partially inhibited by preincubation with tyrphostin (12 microM), a tyrosine kinase inhibitor.

CD4 Antigens↗
PubMed1993

Pilot study: effects of G-CSF on neutrophil ex-vivo function post bone marrow transplantation.

The effects of human recombinant granulocyte colony stimulating factor (G-CSF) on neutrophil recovery have been studied in patients undergoing bone marrow transplantation for haematological disease. Nine patients (five autografts and four allografts) were studied while receiving daily doses of G-CSF (range 2-20 micrograms/kg body weight) were compared to eight patients (four autografts and four allografts) who did not receive G-CSF as a control group. In both groups flow cytometry was used to determine neutrophil IgG Fc receptor (FcRII, FcRIII) expression. Phagocytosis and metabolic burst was assessed using IgG opsonized bacteria. The patients' neutrophils were studied prior to conditioning in autografts and donor cells were studied in allografts. Studies were repeated at neutrophil recovery (neutrophil count 1.0 x 10(9)/l) and ten days post recovery. At recovery FcRII expression was slightly increased in all groups, whereas the number of cells expressing FcRIII was reduced. This reduction was significant in the patients receiving rG-CSF at recovery and post recovery compared to the pretreatment levels. The ability to phagocytose bacteria in cells from patients receiving G-CSF was also reduced. The associated metabolic burst was significantly reduced in the autograft group but not the allograft group receiving rG-CSF. The reduced function and reduced expression of FCRIII suggests the presence of immature neutrophils in patients receiving rG-CSF post BMT.

Bone Marrow Transplantation↗
PubMed1993

Programmed cell death (apoptosis) in immunity and haematological neoplasia.

Programmed cell death, also commonly referred to as apoptosis, is a genetically controlled sequence of events, often requiring protein synthesis, which results in cell death. Although initially described 20 years ago, it is only recently that its role as a mechanism in controlling cell population size and content has been fully realised. In this review the biochemical events of apoptosis are discussed briefly, followed by a more detailed look at the role of programmed cell death in the control of haemopoiesis and the maintenance of a balanced functional immune system. The role of proto-oncogenes and suppressor genes known to regulate programmed cell death is covered, and also their influence on the induction and maintenance of neoplastic disease. Finally, the potential role of apoptosis in the resistance of haematological malignancies to chemotherapy is commented on.

Animals↗
PubMed1993

Induction of surface tumor necrosis factor (TNF) expression and possible facilitation of surface TNF release from human monocytic cells by granulocyte-macrophage colony-stimulating factor or gamma interferon in combination with 1,25-dihydroxyvitamin D3.

1,25-dihydroxyvitamin D3 (1,25(OH)2D3), gamma interferon (IFN-gamma) and granulocyte-macrophage colony-stimulating factor (GM-CSF) can regulate monocyte maturation and activation. Using the human monocytoid cell line U937, we have shown that these agents increase surface tumor necrosis factor (TNF) expression without directly affecting TNF release. GM-CSF and IFN-gamma combined with 1,25(OH)2D3 increased cellular TNF secretion to levels not seen with these agents alone. Ability to express and secrete TNF in part depended on degree of monocytic maturation. The combination of 1,25(OH)2D3 and GM-CSF, however, facilitated lipopolysaccharide (LPS)-mediated release of surface TNF from U937 cells, an effect that was temporally independent of maximal maturation. 1,25(OH)2D3 plus IFN-gamma was less effective than 1,25(OH)2D3 plus GM-CSF at facilitating TNF secretion. We postulate that 1,25(OH)2D3 and GM-CSF are required together to prime a specific mechanism, probably a protease, which cleaves TNF from the surface of monocytic cells. This protease, once primed, can be activated by a secondary stimulus such as LPS.

Calcitriol↗
PubMed1992

Expression of functional antigens on neutrophils. Effects of preparation.

We have studied how different conditions of cell labelling and isolation affect the expression of five functional antigens on neutrophils from healthy subjects. Fluorescein isothiocyanate conjugated (FITC) antisera specific for the C3bi receptor CR3 (CD11b), aminopeptidase N (CD13), the LPS:LPS binding protein receptor (CD14) and the receptors for human IgG (Fc gamma RII CDw32 and Fc gamma RIII CD16) were incubated with (i) unfixed whole blood at 4 degrees C and at room temperature (RT, approximately 20 degrees C), and the leukocytes prepared for analysis using the Coulter Q-Prep system, (ii) leukocytes which had obtained following the removal of erythrocytes from whole blood by dextran sedimentation and which had been washed or left unwashed at RT, and (iii) leukocytes which had been prepared from whole blood that had been formaldehyde fixed immediately following venesection. The amount of fluorescence associated with the cells was determined by flow cytometry. The expression of CD14 was low under all conditions. However the expression of CD11b, CD16 and CDw32 was significantly higher (p less than 0.05) on neutrophils obtained by dextran sedimentation (n = 4) than on cells which had been fixed with formaldehyde ex vivo; the increase in expression was even greater if the cells had been washed. In contrast, the expression of CD13 on formaldehyde fixed cells was higher than on cells which had been labelled at 4 degrees C or at room temperature and was similar to or slightly lower than that on cells obtained by dextran sedimentation. Increasing the time between 10 and 60 min for which the whole blood was incubated with antisera at RT or at 4 degrees C, resulted in progressive increases in the expression of CD11b and CD13. When neutrophils which had been obtained by dextran sedimentation were incubated with unlabelled antibodies to CD16 or CDw32 and FITC labelled antibodies to CD11b there was a marked increase in the expression of CD11b. Altogether these findings indicate that the analysis of functional molecules on neutrophils (which may be rapidly up-regulated during activation) should be performed under clearly defined and controlled conditions. Dual fluorescence studies may, in some circumstances, produce misleading results.

Analysis of Variance↗
PubMed1992

In vitro modulation of haematoporphyrin derivative photodynamic therapy on colorectal carcinoma multicellular spheroids by verapamil.

Photodynamic therapy has possible applications in the treatment of colorectal carcinoma. The photosensitizer haematoporphyrin derivative (HpD) is selectively retained by tumours. Agents which block p-glycoprotein, an export protein expressed to increased levels in a high proportion of colorectal carcinomas, may modulate photodynamic therapy by reducing HpD efflux from cells. Multicellular spheroids derived from the colorectal cell lines HRT18 and HT29 were incubated for 24 h with 1 microgram ml-1 HpD and 0, 1, 2 and 4 microM verapamil. Bioactivity demonstrated a dose-dependent potentiation of HpD-photodynamic therapy growth retardation. Clonogenic survival of cells disaggregated from spheroids treated with HpD-photodynamic therapy was reduced when spheroids were coincubated with verapamil. The mean(s.e.m.) efflux of HpD from spheroids into fresh medium assessed by fluorimetry was greater in spheroids treated with HpD alone (93.2(18.8) arbitrary units ml-1) than in those treated with verapamil (18.1(2.8) arbitrary units ml-1), P = 0.003. Flow cytometry demonstrated increased HpD fluorescence in cells derived from spheroids coincubated with verapamil over a range of HpD incubation concentrations. Verapamil can potentiate the bioactivity of HpD-photodynamic therapy and HpD may be a substrate for p-glycoprotein.

Colorectal Neoplasms↗
PubMed1992

The anti-leukaemic effects and the mechanism of sodium selenite.

The anti-proliferative effects of selenium were studied both in vivo and in vitro. At a selenium concentration of 0.6 micrograms/ml, cells from patients with ALL-L1, L2 and AML-M1, M3 and M5 were more sensitive than cells from patients with CML. Cells from patients with AML-M2, CLL and leukaemic lymphoma were least sensitive. Normal bone marrow or peripheral blood cells were not sensitive to selenium at this concentration. In the mouse leukaemia models (L797, L615, L7712), the sensitivity of leukaemic cells were: L797 (93% cytotoxicity) greater than L615 (49.7% cytotoxicity) greater than L7712 (4.4% cytotoxicity). Sodium selenite injected i.p. increased the longevity of L797-inoculated mice. Administration of 40 micrograms selenium daily for 7 days resulted in a significant increase in the longevity of mice inoculated with 10(5) L797 cells. However, no remarkable increase of the longevity was observed in either L615- or L7712-inoculated mice after treatment with sodium selenite for 7 days. Treatment of the HL-60 leukaemic cell line with selenium caused a dose- and time-related decrease in DNA, RNA and protein syntheses as measured by [3H]-thymidine, [3H]-uridine and [3H]-leucine uptake respectively. The inhibitory effect of selenium on DNA synthesis was reversed when selenium was removed from the medium, demonstrating that selenium-induced inhibition of DNA synthesis was due to interference with DNA biosynthesis rather than DNA template damage. These results suggest that the anti-leukaemic effect of sodium selenite is associated with inhibition of DNA replication, transcription and translation.

Animals↗