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Biomedical subjects

D C Linch

Publications and source records attributed to D C Linch.

At least 181 records · Page 10Linked to original sources
PubMed1991

Role of leucocyte integrins in phagocyte responses to granulocyte-macrophage colony stimulating factor (GM-CSF): in vitro and in vivo studies on leucocyte adhesion deficiency neutrophils.

The role of leucocyte integrins in phagocyte function has been studied by comparing normal neutrophils with those from a patient with partial leucocyte adhesion deficiency (LAD), in whom the levels of CD11b and CD11c were 10% of controls, whereas CD11a levels were normal. Unstimulated LAD neutrophils exhibited defective adhesion to plastic (4.4 +/- 1.5% cf. 14.4 +/- 3.8% in controls), but not to human umbilical vein endothelial cells (HUVECs). The adhesion to HUVECs could be further upregulated by granulocyte-macrophage colony stimulating factor (GM-CSF), but not by 12-O-tetradecanoylphorbol 13-acetate (TPA) which, in normal cells, is a more potent 'pro-adhesive agonist'. The normal neutrophil-endothelial interaction induced by GM-CSF in LAD neutrophils was confirmed in vivo when administration of GM-CSF resulted in rapid phagocyte margination. Neutrophil migration and phagocytosis/killing were defective in LAD neutrophils, and some improvement in phagocytosis/killing was seen following in vivo administration of GM-CSF. These studies illustrate that the degree to which the leucocyte integrins mediate adherence-related phagocyte functions varies not only with the particular function, but also with the conditions of stimulation. High levels of CD11b and CD11c expression appear not to be required for unstimulated or GM-CSF-stimulated neutrophil-endothelial interactions, either in vitro or in vivo. Other neutrophil functions, on the other hand, such as migration and phagocytosis/killing are much more dependent on the leucocyte integrins.

Antigens, CD↗
PubMed1991

The phosphorylation state of the retinoblastoma (RB) protein in G0/G1 is dependent on growth status.

The product of the retinoblastoma gene (RB) is a nuclear phosphoprotein which is thought to regulate the proliferation of cells. Its phosphorylation state changes with passage through the cell cycle and it has been proposed that RB protein in its hypo-phosphorylated form prevents cells proliferating. We have investigated the phosphorylation state of the RB protein in an actively-dividing human B-lymphoblastoid cell line and after cell cycle arrest caused by alpha-Interferon (alpha-IFN). We show that the phosphorylation state of the RB protein in cells with 2N DNA content depends on whether the cells are actively cycling. Our data is compatible with the proposal that dephosphorylation of the RB protein allows cells to enter a quiescent state. This study sheds light on the molecular mechanisms which may mediate the cytostatic effects of alpha-IFN.

Cell Line↗
PubMed1990

Leukaemia complicating treatment for Hodgkin's disease: the experience of the British National Lymphoma Investigation.

OBJECTIVE: To determine the incidence of and risk factors for the development of secondary acute leukaemia and myelodysplasia in patients treated in British National Lymphoma Investigation's studies of Hodgkin's disease since 1970. PATIENTS: 2676 Patients entered into Hodgkin's disease studies between February 1970 and November 1986. Data accrued up to November 1988 were analysed, ensuring a minimum follow up period of two years. DESIGN: Retrospective analysis of multicentre trial data by case-control and life table methods. RESULTS: 17 Cases of secondary leukaemia were recorded in this group of 2676 patients, giving an overall risk at 15 years of 1.7%. The risks of leukaemia after chemotherapy alone and chemotherapy with radiotherapy were not significantly different. The risk of leukaemia increased sharply with the amount of treatment given as measured by the number of attempts at treatment. The 15 year risks of leukaemia were 0.2%, 2.3%, and 8.1% for patients receiving one, two, or three or more attempts at treatment. The highest risk, 22.8% at 15 years, was observed in patients treated with lomustine (CCNU), and a case-control study suggested that this was an independent risk factor. The risk of secondary leukaemia was largely related to the overall quantity of treatment, although exposure to lomustine seemed to be an important risk factor. Treatment with both drugs and radiation was not more leukaemogenic than treatment with drugs alone. The greatest risk of secondary leukaemia was seen in multiply treated patients who were unlikely to be cured of Hodgkin's disease. CONCLUSIONS: Avoidance of secondary leukaemia should be a minor factor in the choice of treatment for Hodgkin's disease.

Acute Disease↗
PubMed1990

Isoquinolinesulfonamide protein kinase inhibitors H7 and H8 enhance the effects of granulocyte-macrophage colony-stimulating factor (GM-CSE) on neutrophil function and inhibit GM-CSF receptor internalization.

Human granulocyte-macrophage colony-stimulating factor (GM-CSF) increases neutrophil surface expression of the cellular adhesion molecule CD11b and primes the respiratory burst stimulated by the bacterial peptide f-met-leuphe (FMLP). We have examined the effects of the isoquinolinesulfonamide protein kinase inhibitors H7 and H8 on these functions of GM-CSF using whole blood assays. Concentrations of H7 and H8 that inhibited the 12-O-tetradecanoyl-phorbol-13-acetate (TPA) stimulated upregulation of CD11b expression and activation of the respiratory burst, both augmented the effects of GM-CSF. H7 and H8 enhanced the GM-CSF-stimulated increase in CD11b expression to 215% +/- 10% (P less than .05) and 233% +/- 45% (P less than .05), respectively, of the value obtained with GM-CSF alone. The GM-CSF priming of the FMLP-stimulated oxidative burst was increased to 190% +/- 44% (P less than .01) by preincubation with H7 and to 172% +/- 25% (P less than .01) with H8. Preincubation with H8 did not affect overall binding of 125I-GM-CSF to neutrophils, but inhibited GM-CSF receptor internalization after ligand binding (P less than .05). These data indicate that the effects of GM-CSF are not mediated by protein kinase C and that a phosphorylation event down-modulates the neutrophil response to GM-CSF. It suggests that internalization of the receptor-ligand complex is not a rate-limiting step in signal transduction, and that regulation of the rate of internalization may be an important level of control of the activity of GM-CSF.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1990

High-dose chemotherapy and autologous bone marrow transplantation in acute myeloid leukemia.

For younger patients with acute myeloid leukemia (AML), an allogeneic transplant from a matched sibling may afford the best chance of cure. In patients who are older or without a matched sibling donor, dose intensification can be achieved with an autologous bone marrow transplant (ABMT). We report here the results of a high-dose chemotherapy regime with nonpurged ABMT in 82 adult patients in first remission of AML with a median follow-up of 31 months. The median age was 40 years (range 16 to 57 years). The median interval between remission and ABMT was 5 months (range 1 to 12 months). Twenty-eight of these patients received a second course of the same high-dose chemotherapy and ABMT. The procedure related mortality rate was 6%. The projected leukemia-free survival (LFS) at 5 years is 48% for all 82 patients and 50% for the 76 patients with no known preceding myelodysplastic syndrome. For those patients with primary AML who received a double ABMT the projected LFS is 67%. The interval between remission and ABMT did not predict for either relapse or LFS. ABMT using a multidrug chemotherapy protocol is less toxic than allogeneic BMT yet results in a similar LFS.

Adolescent↗
PubMed1990

Development of antibodies to unprotected glycosylation sites on recombinant human GM-CSF.

In 4 out of 16 patients receiving recombinant human granulocyte macrophage colony stimulating factor (rhGM-CSF) in phase I/II studies antibodies developed to the recombinant protein. The antibodies react with sites on the native protein backbone which are normally protected by O-linked glycosylation but which are exposed in rhGM-CSF produced in yeast and Escherichia coli. Antigenicity of recombinant human proteins due to non glycosylation may have relevance to the choice of host system for production of factors for clinical use.

Antibodies↗
PubMed1990

Fc gamma RII-mediated superoxide production by phagocytes is augmented by GM-CSF without a change in Fc gamma RII expression.

Freshly purified neutrophils and monocytes respond to multiple cross-linking of Fc gamma RII with the IgG1 monoclonal antibody, CIKM5, with a rapid rise in Ca(2+)i, but not with a respiratory burst, although superoxide is generated by these cells when stimulated with the chemotactic peptide, FMLP, or phorbol ester (TPA). Incubation in vitro for 30-60 min at 37 degrees C in medium + 0.1% FCS had no effect on the neutrophil superoxide response to CIKM5 but induced a weak monocyte response in 11/13 experiments. However, incubation with rhGM-CSF (10 ng/ml) under similar conditions induced a neutrophil respiratory burst in response to cross-linking Fc gamma RII in 12/14 experiments and enhanced the monocyte response by 181%. GM-CSF also enhanced the response of neutrophils and monocytes to FMLP by 308% and 165%, respectively. The response to TPA was not significantly enhanced by GM-CSF. rhIFN-gamma (100 mu/ml) was ineffective as a priming agent for all agonists tested in short-term incubations but augmented the monocyte response to CIKM5 after 5 d exposure in vitro. Whilst GM-CSF induced neutrophil superoxide production in response to cross-linking Fc gamma RII, there was no concomitant change in Fc gamma RII expression either in in vitro studies of neutrophils from healthy individuals or in in vivo studies of patients receiving GM-CSF. Stimulation of unprimed neutrophils with CIKM5 induced a rapid transient increase in intracellular calcium levels to 181% of resting levels. However, incubation with GM-CSF did not further augment the calcium transients above the stimulated level. The mechanism by which GM-CSF induces an enhanced respiratory burst in response to cross-linking of Fc gamma RII remains to be elucidated, but is not related to receptor expression or increases in receptor mediated calcium mobilization.

Antibodies, Monoclonal↗
PubMed1990

Secretion of neutrophil secondary granules occurs during granulocyte-macrophage colony stimulating factor induced margination.

The effects of recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) on neutrophil lactoferrin (LF) and transcobalamin (TC) 1 and 3 secretion were determined in vitro and during in vivo administration in humans. In whole blood, in vitro incubation with GM-CSF reproducibly produced a rise in plasma LF concentration (P less than 0.05) whereas in purified neutrophils the results were variable. Exposure of whole blood to GM-CSF also resulted in a significant rise in plasma TC 1 and 3 (190 +/- 60%, P less than 0.05). The response was dose dependent with maximal effect at GM-CSF concentrations of 10 ng/ml and above. rhGM-CSF was administered on seven occasions to six patients with malignant disease prior to chemotherapy. Plasma LF and unsaturated TC 1 and 3 levels rose significantly in each patient studied and the rise coincided with the initial neutropenia due to margination that occurs during infusions of rhGM-CSF. Patients receiving rhGM-CSF may therefore have hypofunctional neutrophils due to secondary granule depletion.

Cell Adhesion↗
PubMed1990

The effects of rhGM-CSF on the neutrophil respiratory burst when studied in whole blood.

A simple semiquantitative cytofluorometric method has been developed for measuring neutrophil respiratory burst activity in whole blood samples. This technique avoids the introduction of laboratory artefacts which modulate neutrophil function. In addition, flow cytometric analysis allows the response to be studied in individual cells. We show here that neutrophils examined freshly ex vivo, exhibit only weak respiratory burst activity in response to stimulation with the chemotactic peptide FMLP (10(-6) M). Prior incubation with rhGM-CSF results in an increase in the number of responding cells from 13.5 +/- 2.36% (mean +/- SEM) to 46.7 +/- 6.3% (P less than 0.0001) with an increase in total respiratory burst activity of 567% (P = 0.001). The majority of neutrophils in whole blood (67.1 +/- 8.1%) exhibit respiratory burst activity in response to stimulation with phorbol ester (1 micrograms/ml of TPA), and this response is also significantly primed by rhGM-CSF (P = 0.004). The enhancement of respiratory burst activity induced by rhGM-CSF is due to both recruitment of previously unresponsive neutrophils, and to intensification of the response of the responding cells. In vivo administration of rhGM-CSF also results in priming of the respiratory burst in response to FMLP, although the enhancement of activity is not as great as that obtained when pre-infusion blood samples are incubated with rhGM-CSF in vitro.

Blood↗
PubMed1990

Expression of the human carbonic anhydrase I gene is activated late in fetal erythroid development and regulated by stage-specific trans-acting factors.

Using flow cytometric analysis of red cells from different stages of ontogeny with anti-CAI antibody, it was shown that the human carbonic anhydrase I (HCAI) gene product appears in a developmental stage-specific manner. Virtually no CAI protein was detectable in fetal red cells prior to birth. However, at about the time of normal delivery (40 weeks gestation) CAI production is switched on. The proportion of cells containing CAI reaches adult levels during the second half of the first year of life. Northern analysis suggests that the appearance of CAI protein results directly from the activation of the gene and the production of new mRNA. A transient heterokaryon system was set up by fusing the erythroleukaemic cell lines MEL C88 (a mouse cell line in which CAI is expressed) and K562 SAI (a human cell line with an embryonic/fetal phenotype, not expressing CAI). SP6 RNAase mapping of RNA from the fused cells showed activation of the human CAI gene. This shows the developmental stage-specific expression of HCAI to be regulated by trans-acting factors.

Adult↗
PubMed1990

Activation of monocytic cells by monoclonal antibodies to the CD11a/18 (LFA-1) complex: mediation by Fc receptor.

IgG1 antibodies reacting with several monocytic antigens form a bridge between the specific antigen and the Fc receptors also expressed on these cells. This results in calcium mobilization and generation of superoxide. Single IgG1 antibodies reacting with the CD11a/CD18 cellular adhesion molecular complex do not, however, induce monocytic activation. This is not because they induce a negative signal, as the response to formyl-methionyl-leucyl2-phenylalanine (FMLP) or direct cross-linking of FcRII is not inhibited. Furthermore, a combination of an intact CD11a and CD18 antibody does induce a rise in intracellular calcium and production of superoxide. This activation is dependent on the binding of the Fc portion of both antibodies to Fc receptors, as F(ab')2 fragments do not cause activation. This suggests that simultaneous binding of opsonized bacteria to cellular adhesion molecules and to Fc receptors on monocytes would facilitate activation of these cells. Furthermore, it illustrates the importance of using F(ab')2 fragments in the analysis of signal transduction molecules on Fc receptor-bearing cells.

Antibodies, Monoclonal↗
PubMed1989

The regulation of erythropoiesis in man.

The physiological role of erythropoietin (epo) as a "maintenance" and "progression" factor of terminal erythroid maturation is well established. The role of early acting erythroid factors in the maintenance of steady state erythropoiesis has not been ascertained. Primitive erythroid precursor cells (burst forming units) require a biological activity for colony formation in culture, which has been designated burst promoting activity (BPA) before it became clear that granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin 3 (IL-3) are (probably among others) important components of "BPA". Unlike epo, neither GM-CSF nor IL-3 have been detected in the circulation in sufficient amounts to allow recognition of feed back mechanisms between failing erythropoiesis and either of the two factors. Rather, GM-CSF and IL-3 may play a local role in the enhancement of erythropoiesis in the bone marrow, which is resistant to study in vivo. gamma-Interferon and transforming growth factor beta inhibit erythropoiesis in vitro. Their causative role in diseases with depressed erythropoiesis, however, is not proven.

Colony-Stimulating Factors↗
PubMed1989

Enhanced fluorescence in indirect immunophenotyping by the use of fluorescent liposomes.

Small unilamellar liposomes were optimised for cell phenotyping by indirect immunofluorescence. This involved selection and covalent attachment to the liposome of a polyspecific ligand for cell-bound antibody. For this purpose sheep anti-mouse antibody was preferred to protein A because of its ability to attach to cell-bound IgG1 as well as IgG2 at physiological pH. The maximally fluorescent concentration of encapsulated carboxyfluorescein was determined to be 20 mM and liposomes thus comprised gave up to a nine-fold increase in mean cell fluorescence when compared with sheep anti-mouse antibody conjugated to fluorescein isothiocyanate. There was no parallel increase in background fluorescence. Liposomes retained their targeting and fluorescence properties after 3 months storage. They could be sterilised and were as versatile in use as FITC-antibody conjugates.

Animals↗
PubMed1989

Activation of human monocytes occurs on cross-linking monocytic antigens to an Fc receptor.

Murine mAb to CD13, CD14, and class II MHC, are able to mobilize calcium in normal human monocytes and enhance superoxide production in primed cells. Antibodies to CD35 (CR1) also cause a minor calcium response in some individuals. Antibodies to CD11a, CD11b, CD11c, CD15, CD17, CD18, and CD45 do not activate monocytes. The ability of mAb to cause monocyte activation is not only dependent on the Ag with which they react but also on the isotype of the antibodies and the individual from whom the monocytes were obtained. It is shown that this is because the mAb that activate monocytes do so by formation of Ag-antibody-FcR complexes. F(ab')2 fragments of mAb to CD13 and CD14 do not therefore activate monocytes even when cross-linked with F(ab')2 anti-mouse Ig but do so when cross-linked with intact anti-mouse Ig. These data indicate that activation via the FcR requires perturbation of this receptor but does not necessarily require cross-linking of one FcR to another. Antibody-coated particles or cells able to bind to cell surface receptors on monocytes other than the FcR would thus augment FcR-mediated activation.

Antibodies, Monoclonal↗