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Biomedical subjects

M G Macey

Publications and source records attributed to M G Macey.

At least 37 records · Page 2Linked to original sources
PubMed1997

Mitochondrial electron transport chain activity, but not ATP synthesis, is required for drug-induced apoptosis in human leukaemic cells: a possible novel mechanism of regulating drug resistance.

There is increasing evidence for an association between mitochondrial function and susceptibility to apoptosis. It has been shown that the vinblastine-resistant leukaemic cell line CEM/VLB100 has a more active mitochondrial electron transport chain (ETC) than the parental CCRF-CEM cell line. Inhibition of mitochondrial DNA replication by ethidium bromide (EB) depleted the activity of the ETC and reduced cellular respiratory rate. Depletion of mitochondrial DNA was associated with increased resistance to vinblastine-induced apoptosis in both cell lines. In contrast, the highly specific inhibitor of the energy producing mitochondrial enzyme F1Fzero-ATPase, oligomycin, rendered CEM/VLB100 cells more sensitive to vinblastine by inhibiting the energy-dependent P-glycoprotein (Pgp) pump, suggesting that the effect of EB is independent of energy generation and ATPase activity. Both mitochondrial ETC depletion and ATPase inhibition decreased vinblastine-induced cell cycle changes in the CCRF-CEM cell line, suggesting that cell cycle changes are dependent on ATP generation. However, EB-induced ETC depletion in CEM/VLB100 cells inhibited apoptosis in response to high concentration of vinblastine, but not G2M arrest. We suggest that: (1) over-expression of Pgp by drug-resistant cells may up-regulate mitochondrial energy production; (2) mitochondrial ETC activity is required for DNA fragmentation in response to vinblastine, but the mechanism is independent of Pgp activity and ATP generation; (3) down-regulation of mitochondrial ETC activity may confer resistance to vinblastine-induced apoptosis; (4) the mitochondrial ETC is involved in vinblastine-induced apoptosis downstream of microtubule disruption and cell cycle changes.

Adenosine Triphosphatases↗
PubMed1997

Thymic function in adults: evidence derived from immune recovery patterns following myeloablative chemotherapy and stem cell infusion.

We studied 45 patients aged 14-66 years who had undergone stem cell transplantation for a variety of malignant conditions at least 12 months previously. Compared to normal controls, they had significantly reduced absolute numbers of CD4+, CD4+ CD45RA+ and CD4+ CD45RO+ T cells and a reduced CD4+ CD45RA+:CD4+ CD45RO+ ratio. In all subsets T-cell numbers were significantly greater 24 months, compared to 12-24 months, after transplantation and there was a nonsignificant trend towards lower T-cell numbers with increasing age. We conclude that the thymus, or putative thymic-equivalent tissue, remains functional in older adults.

Adolescent↗
PubMed1997

Adhesion molecules are upregulated on dendritic cells isolated from human blood.

This study investigated whether the high expression of adhesion molecules on enriched preparations of circulating dendritic cells (DCs) was an intrinsic property of the cells or whether it was a consequence of the procedure used to isolate them from blood. Expression of the beta 1, beta 2 integrins (CD11/CD18 family) and other adhesion molecules on DCs in whole blood was compared with that on isolated DCs. Dendritic cells were identified by flow cytometry as leucocytes that were positive for human leucocyte antigen (HLA)-DR, but negative for CD3, CD14, CD16, CD19 and CD56. In contrast to a minority of DCs in whole blood, the majority of isolated DCs expressed the beta 2 integrins and there were a greater number of cells bearing CD44, CD54 and some of the beta 1 integrins (notably CD49b, CD49d, CD49e and CD29). An increase in the proportion of DCs bearing adhesion molecules was generally apparent at the isolation stage when mononuclear cells, which had been incubated overnight, were centrifuged on a metrizamide gradient to enrich for cells of low density. Inclusion of an inhibitor of protein glycosylation and exocytosis (brefeldin A) at all stages of separation partially prevented an increase in the percentage of DCs bearing CD18, C29 and C54 whereas the inclusion of cycloheximide (an inhibitor of polypeptide synthesis) interfered with increases in the percentage of cells bearing CD29 and CD54. Neither of these antagonists had an effect on the intensity of adhesion molecule expression. We suggest that some of the adhesion-dependent functions of isolated DCs are caused, in part, by an upregulation of surface adhesion molecules induced by the enrichment procedure.

Brefeldin A↗
PubMed1997

Cutaneous lymphocyte associated antigen (CLA) and alpha e beta 7 integrins are expressed by mononuclear cells in skin and oral lichen planus.

The cutaneous lymphocyte associated- (CLA-) positive subset of lymphocytes appears to migrate preferentially into skin by interacting with E-selectin on vascular endothelium; lymphocytes expressing the alpha e beta 7 integrin accumulate preferentially in the epithelium of the gastrointestinal tract. To determine whether the mononuclear cell population of the oral mucosa resembles that of the skin or intestine, and using lichen planus as a model, the proportions of CLA- and alpha e beta 7-positive cells in the epithelium, lamina propria and peripheral blood were compared by immunostaining and flow cytometry. In both skin and oral lichen planus, selective accumulation of CLA-positive cells was seen in the epithelium but not in the lamina propria. In contrast, large numbers of alpha e beta 7-positive intraepithelial cells were found in oral but not in skin lichen planus. These results show that in terms of CLA and alpha e beta 7 expression there are important differences in the mononuclear cell population of oral mucosa and skin.

Adolescent↗
PubMed1997

Neutrophil adhesion molecule expression during cardiopulmonary bypass: a comparative study of roller and centrifugal pumps.

The purpose of this study was to determine whether adhesion molecules and markers of cell activation were preferentially increased on blood neutrophils during cardiopulmonary bypass (CPB) and whether such effects were influenced by the use of a roller pump or a centrifugal pump. Forty-six patients undergoing open heart surgery were randomly allocated into either the roller or centrifugal groups. Blood (1 ml volumes) was removed from arterial and venous lines immediately before and 1 h after the start of bypass. Whole blood samples were immunolabelled and flow cytometry used to measure the distribution and expression of the adhesion molecules CD11b, CD18, CD62L on neutrophils, monocytes and lymphocytes, in addition to CD64 on neutrophils and monocytes, and CD14 on monocytes. The expression of CD11b was significantly enhanced on neutrophils in arterial and venous samples from both the roller pump (mean 84% and 100% increase, respectively; p < 0.001) and centrifugal pump (mean 74% and 73% increase, respectively; p < 0.001) groups. Neutrophil L-selectin expression increased to a small but significant extent in arterial and venous samples from the centrifugal pump group (mean 16% increase; p < 0.001) and in venous samples from the roller pump group (mean 10% increase; p < 0.01). Neither the percentage of neutrophils bearing CD11b/CD18, CD62L and CD64, nor the expression of adhesion molecules on lymphocytes and monocytes were modified by 1 h of bypass. These results suggest that patients subjected to CPB with roller or centrifugal pumps are equally at risk to neutrophil activation that could lead to increased interaction of these cells with blood vessel walls.

Antigens, CD↗
PubMed1997

Proteinase-activated receptor-2: expression by human neutrophils.

Neutrophils were shown to express the proteinase-activated receptor-2 (PAR-2), a seven transmembrane domain receptor, which is activated by cleavage by trypsin. Granulocytes from 14 donors stained positively for PAR-2 with affinity-purified rabbit antibodies raised against a peptide corresponding to the trypsin cleavage site of human PAR-2. Neutrophil activation in response to a receptor activating peptide (RAP) varied between donors. RAP (Ser-Leu-Ile-Gly-Lys-Val-NH2) alone induced an increase in the forward and side light scatter after 5-10 minutes and a small increase in the expression of the activation molecule CD11b. The increased expression of CD11b induced by RAP was markedly enhanced by priming the neutrophils with a low concentration (1 nM) of formyl-Leu-Met-Phe. Trypsin and RAP also induced an increase in intracellular calcium, but there were large variations in the magnitude of responses between donors also in this assay. The effects of RAP in the different assays were specific; acetylated RAP was completely without activity.

Adult↗
PubMed1996

Novel anticoagulants for flow cytometric analysis of live leucocytes in whole blood.

Enzyme inhibitors have been compared with conventional anticoagulants for the flow cytometric analysis of live leucocytes in whole blood. At 18 to 20 degrees C in vitro, PPACK (60 microM), hirudin (10 units ml-1), leupeptin (20 microg ml-1) and aprotinin (50 microg ml-1) inhibited blood clotting for 4 h or more, whereas PMSF (8 mg ml-1) or AEBSF (5 mg ml-1) inhibited clotting for only 20 or 40 min, respectively. When labelled with CD11b antibodies and analysed immediately ex vivo at 4 degrees C, the percentages of lymphocytes, monocytes, and polymorphs which stained positively and their mean fluorescence intensities were similar, irrespective into which anticoagulant blood was collected. Less than 1.5-fold increases in expression occurred on monocytes and polymorphs when blood anticoagulated with enzyme inhibitors or conventional anticoagulants was kept at 4 degrees C, or when blood anticoagulated with citrate, heparin, or hirudin was kept at 18 to 20 degrees C for 1 h before labelling and analysis, whereas approximately 2-fold increases in expression occurred in blood kept with K3EDTA, leupeptin, or aprotinin and more than 3-fold increases in blood kept with AEBSF or PPACK at 18 to 20 degrees C for 1 h. Further studies showed that leupeptin could be used effectively as the anticoagulant when investigating functional responses of live leucocytes in whole blood samples by flow cytometry and for the isolation of leucocytes with minimal modulation of adhesion molecules.

Adult↗
PubMed1996

The effect of 30 min cycle ergometry on ankylosing spondylitis.

The effect of 30 min cycle ergometry at approximately 100 W (mean 98.8 W; range 34-151 W) in 11 male patients who had no hip involvement were studied. In most patients, exercise produced immediate increases in spinal flexibility and bilateral cervical tilt, and a reduction in pain. However, these improvements steadily waned and all had disappeared by 3-5 h. Exercise induced marked changes in the numbers of circulating leucocytes and platelets, and in the distribution of lymphocytes subsets, similar to those previously reported to occur in individuals without the disease. In a majority of patients there were positive associations (Kendall's tau test) between Schober's index and the platelet count, and negative associations between Schober's index and the percentage of CD4-positive cells over a 5 h period on the exercise day, whereas there were negative associations between the pain score and the leucocyte and neutrophil counts over a comparable period on a control day without exercise. We conclude that exercising those regions of the body unaffected by disease can elicit short-term beneficial effects by a systemically mediated mechanism(s).

Adult↗
PubMed1996

Platelet surface activation antigen expression at baseline and during elective angioplasty in patients with mild to moderate coronary artery disease.

Platelet activation is an important pre-thrombotic event. The elucidation of its pathophysiology could contribute to a reduction in the mortality associated with coronary artery disease-the foremost cause of death in the UK. We examined the platelets of 27 patients with angiographically documented coronary artery disease. All patients had stable angina and were taking their regular medication-including aspirin. We demonstrated significantly increased expression of GP53 and activated GPIIb/IIIa on the platelet surface using a sensitive flow cytometric method of detection. Comparison was made with a control group of 35 patients. Seventeen of the patients had coronary angioplasty carried out. Serial studies of these patients demonstrate an immediate and sustained increase in platelet activation and this has important implications for prevention of restenosis after angioplasty.

Adult↗
PubMed1996

Correlation of GP53 and P-selectin expression in myeloproliferative disorders and normal controls.

Platelet degranulation occurs when platelets are activated. Alpha degranulation releases P-selectin whereas lysosomal degranulation releases GP53. A correlation between these two markers might therefore be expected. We studied the correlation between P-selectin and GP53 in 50 patients with myeloproliferative disorders (MPD), 35 normal controls and 105 disease controls (patients with inflammatory bowel disease [IBD, n = 52], rheumatoid arthritis [RA, n = 26] and coronary artery disease [CAD, n = 27]) by flow cytometry before and after stimulation with thrombin ex vivo. There was no significant correlation between percentage expression of P-selectin and GP53 in unstimulated samples in normal individuals; r = 0.13, P = 0.3, n = 34. Mild thrombin stimulation (10 mU/ml) led to both alpha and lysosomal degranulation with a strong correlation (r = 0.62, P < 0.001, n = 35). Disease controls (IBD, RA and CAD) showed similar trends. In patients with MPD, in contrast, a strong correlation between the expression of these platelet activation markers was demonstrable in unstimulated samples (r = 0.37, P = 0.007, n = 50). P-selection and GP53 expression in stimulated samples also correlated well. The data support the existence of different control pathways for the steady state expression of P-selection and GP53. Heterogeneous steady state responses of P-selectin and GP53 may be physiological and loss of this heterogeneity may be a hitherto unreported and pathologically important feature of MPD. This lack of correlation appears to be specific to MPD and is not simply a function of increased in vivo platelet activation.

Antigens, CD↗
PubMed1996

Analysis of the platelet-type thrombin receptor in 20 cases of large granular lymphocyte proliferations.

The platelet-type thrombin receptor was expressed by large granular lymphocytes (LGLs) in a variety of proliferative diseases. Twenty patients with LGL proliferative disease were examined, including five T cell clones and a variety of polyclonal proliferations, some secondary to rheumatoid arthritis and Felty's syndrome; 17/20 showed high number of CD3+, CD8+, and CD57+ lymphocytes and 9/20 also had high numbers of CD16+ or CD 56+ positive lymphocytes. The thrombin receptor was present on more than 20% of the LGLs in 13/20 patients. The clonal T cell expansions showed the highest receptor expression with greater than 75% cells positive. Regression analysis of all 20 cases showed striking and highly statistically significant positive Spearman rank correlation between the proportion of thrombin receptor and CD57-positive LGLs (r = 0.56, P = 0.009). A negative correlation with CD56 was also found (r = -0.46, P= 0.043). Dual antibody flow cytometry showed the receptor was more often co-expressed with CD57 (64%) than with CD16 (19%) or CD56 (11%). The expression of the platelet-type thrombin receptor by LGLs of this phenotype raises the possibility of a functional role for thrombin in the pathogenesis of LGL proliferative diseases.

Adult↗
PubMed1995

Effects of cell purification methods on CD11b and L-selectin expression as well as the adherence and activation of leucocytes.

This study investigated the effects of commonly used procedures for the isolation of leucocytes from human blood in comparison with cells in whole blood on the surface expression of CD11b and L-selectin (adhesion molecules which are known to be increased and decreased respectively by cell activation). Washing of granulocytes or monocytes with Hanks' buffered salt solution after separation by either dextran sedimentation or density gradient centrifugation, increased surface expression of CD11b (p < 0.05). The number of monocytes bearing CD11b was enhanced (p < 0.05) by dextran sedimentation and two layer density gradient centrifugation (Histopaque). The increase in CD11b expression on granulocytes was associated with enhanced binding of the cells to endothelial monolayers that were either untreated (r = 0.902; p < 0.001) or treated with tumour necrosis factor alpha (TNF-alpha) (r = 0.68; p = 0.004). The expression of L-selectin was reduced on granulocytes that had been isolated by dextran sedimentation followed by hypotonic lysis of contaminating erythrocytes. All isolates of granulocytes demonstrated a loss of L-selectin following activation with fMLP though this effect was less marked with cells subjected to erythrocyte lysis. The various separation methods had little effect on expression or distribution of CD11b or L-selectin on lymphocytes. We conclude that isolation of lymphocytes by density gradient centrifugation and of granulocytes and monocytes by dextran-sedimentation and centrifugation using Histopaque gradients, but avoiding washing and the use of hypotonic erythrocyte lysis, are appropriate techniques for studying the expression and function of adhesion molecules.

Cell Adhesion↗
PubMed1995

Colorectal cell line suppression of lymphokine activated killer cell generation is reversed by suramin.

Progressive tumor growth is associated with a state of immunosuppression. One mode of immunosuppression is thought to be mediated by immunosuppressive factors of tumour origin. We have investigated in vitro the possibility that suramin could be used to blockade tumor derived suppressor factors and enhance the effectiveness of rhIL-2 therapy. These data show that factors derived from cultures of the colorectal carcinoma cell line LoVo suppressed the percentage of cells expressing the natural killer cell antigen CD56 in 92% of individuals and the cytotoxic T cell antigen CD8 in 77% of individuals tested from a panel of 13 normal healthy volunteers. Suramin at 200 micrograms/ml restored the percentage of cells expressing CD56 to levels higher than the control cultures and reduced the suppression in those expressing CD8 to non-significant levels. LoVo produced factors also suppressed the expression of the activation associated antigens CD25, CD71 and HLA-Dr with suramin restoring CD25 expression but not CD71 or HLA-Dr. Functional studies using 51Cr-release assays showed that LoVo produced factors could suppress cytotoxicity in 46% of individuals tested, and of these a reduction in suppression by suramin was demonstrated in 50% of individuals against Daudi target cells and 33% against K562 target cells.

Antigens, CD↗
PubMed1994

Effect of fixation on quantification of the expression of leucocyte function-associated surface antigens.

The surface expression of adhesion molecules and other function-associated antigens on peripheral blood leucocytes may be measured by flow cytometry. However, quantification of these antigens is difficult, because their expression can be rapidly and artefactually modulated if the cells are activated in vitro. Consequently, it is common, when analyzing these antigens either: (1) to label leucocytes in whole blood at 4 degrees C, to lyse erythrocytes and then fix the leucocytes, or (2) to fix the leucocytes in whole blood, to lyse the erythrocytes, and then label the leucocytes. We have compared the mean fluorescence intensity (MFI) values for CD11b, CD18, and L-selectin (Leu-8 and TQ1 epitopes) on human peripheral blood leucocytes, using these two approaches. In addition, we have simultaneously evaluated how anticoagulants (acid citrate, K3EDTA, and heparin) and the presence or absence of divalent metal ions (Ca2+ and Mg2+) affect the expression levels of these antigens. The results for all four epitopes varied markedly depending on the preparation procedure used but were less affected by the choice of anticoagulant and whether divalent cations were or were not present in the media used for cell preparation and labelling. Comparison of the results obtained using these procedures, which involve fixation with formaldehyde, with those obtained by a recently developed procedure in which unfixed leucocytes were labelled with the vital nuclear dye LDS-751 and antibodies together, then analysed in unlysed whole blood at 4 degrees C, showed that formaldehyde-based preparation techniques underestimated the expression (MFI) of CD18, Leu-8, and TQ1. It is recommended that, whenever practicable, measurements are made on unfixed cells stained using the newer procedure.

Ammonium Chloride↗
PubMed1994

Immune thrombocytopenia and Fc receptor-mediated phagocyte function.

The response to intravenous immunoglobulin treatment (IVIG) is thought, in part, to be due to blockade of Fc receptor for IgG in the mononuclear phagocyte system (MPS). We have studied this by measuring splenic clearance of heat-damaged and IgG antibody-coated red cells in immune thrombocytopenic patients receiving IVIG. Clearance of heat-damaged red blood cells (HDRBC) labelled with technetium 99MM was measured in eight patients before and after a 5-day infusion of IVIG. In six of the eight there was an enhanced clearance of HDRBC post IVIG. Clearance studies were performed in eight rhesus-D-positive patients using autologous red cells coated with anti-D and labelled with 99MTc. The time taken for the radioactivity at 3 min to fall to 50% (T1/2) was calculated. The T1/2 increased in seven of the eight patients from a mean of 78.5 min to 137.1 min, a prolongation of 74.6%. In addition, we have examined the effect of IVIG on Fc-mediated phagocytosis by neutrophils. Platelet phagocytosis by patients' neutrophils was measured using opsonized autologous platelets in nine patients. There was a marked reduction in platelet phagocytosis as measured by formazan production in all patients at day 4-5 when compared with the preinfusion value, and in half of these this reduction was still evident at further studies, 1 week after the end of the infusion period. The platelet count rose in parallel with the fall in platelet phagocytic ability of the neutrophils, reaching a peak at 7 days post infusion, and fell with the recovery of phagocytic function in weeks 2 and 3. These findings suggest that IVIG has a marked effect on Fc-mediated phagocytosis.

Antibodies, Anti-Idiotypic↗