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Biomedical subjects

M Macey

Publications and source records attributed to M Macey.

16 recordsLinked to original sources
PubMed2002

Differential expression of beta1 and beta2 integrins and L-selectin on CD4+ and CD8+ T lymphocytes in human blood: comparative analysis between isolated cells, whole blood samples and cryopreserved preparations.

Flow cytometric analysis was used to compare the expression of adhesion molecules on human CD4+ and CD8+ T lymphocytes in isolated blood mononuclear cells (MNCs) in whole blood samples and in cryopreserved MNC preparations. Examination of MNCs revealed that the CD11b and CD11c components of the beta2 integrins were preferentially expressed on CD8+ T cells, whereas CD62L was present on more CD4+ T cells. All CD4+ and CD8+ T lymphocytes were positive for CD11a but the CD8+ population had a higher intensity of expression of CD11a and also CD11b. Virtually identical results were obtained with T cells in whole blood samples. In relation to the beta1 integrins, the only difference between isolated CD4+ and CD8+ T cells was that the latter subset had a greater proportion of cells bearing CD49d. The naive cell marker CD45RA was present on the majority of CD8+ T cells whereas CD45RA and the memory marker CD45RO were evenly distributed within the CD4+ T cell subset. Although cryopreservation of lymphocytes did not modify the expression of beta1 and beta2 integrins it produced a marked reduction in the percentage of CD4+ and CD8+ T cells bearing CD62L. With regard to endothelial interactions, it appears that cryopreserved lymphocytes are suitable for inclusion in studies of integrin-mediated adhesion but not for those relating to tethering or recognition of addressins on high endothelial venules. Differences in adhesion molecule expression between CD4+ and CD8+ T lymphocytes could underlie the selective extravasation of these subsets into sites of infection and inflammation.

Adult↗
PubMed2000

Ridogrel, a dual thromboxane synthase inhibitor and receptor antagonist: anti-inflammatory profile in inflammatory bowel disease.

BACKGROUND: Thromboxanes, prostaglandins, reactive oxygen metabolites and pro-inflammatory cytokines are produced in excess in inflammatory bowel disease. Preliminary reports suggest that ridogrel, a thromboxane synthesis inhibitor and receptor blocker, may have therapeutic benefits in ulcerative colitis. AIMS: To investigate the anti-inflammatory profile of ridogrel. METHODS: The effects of ridogrel on the production of eicosanoids, reactive oxygen metabolites and cytokines by cultured inflamed colorectal mucosal biopsies were made using ELISA and chemiluminescence, reactive oxygen metabolite generation in a cell-free system, and platelet activation using flow cytometry. The effects of oral ridogrel on mucosal release of eicosanoids in two patients with active ulcerative colitis were assessed using rectal dialysis. RESULTS: Ridogrel significantly reduced the release of thromboxane B2, but not prostaglandin E2 or tumour necrosis factor-alpha, from biopsies (P < 0.01 for 10 microM ridogrel). Ridogrel showed no direct antioxidant activity but significantly reduced reactive oxygen metabolite production from cultured biopsies (P < 0.01 for 10 microM ridogrel). Platelet activation in vitro was inhibited by ridogrel (P </= 0.05 for >/= 10 microM ridogrel). Mean rectal mucosal thromboxane B2 release was reduced to 86% of pre-treatment levels in two patients treated with oral ridogrel. CONCLUSIONS: Its inhibition of mucosal production of thromboxane B2, reactive oxygen metabolites, and of platelet activation, suggests that ridogrel could have a therapeutic role in inflammatory bowel disease.

Adult↗
PubMed1997

Human blood dendritic cells: binding to vascular endothelium and expression of adhesion molecules.

To investigate the binding properties of dendritic cells (DC) to vascular endothelium, a comparative analysis was undertaken of DC, monocytes and lymphocytes isolated from the blood of 25 healthy subjects using monolayers of human umbilical vein endothelial cells as the adherence substrate. More blood DC (mean 24% adherence) were adherent to endothelial monolayers than monocytes (mean 18%; P < 0.001) and lymphocytes (mean 12%; P < 0.001). When the monolayers were pretreated with tumour necrosis factor-alpha (TNF-alpha) all leucocyte populations exhibited an increased attachment, but there was still greater binding of DC (mean 37% adherence) in comparison with monocytes (mean 23%; P < 0.001) and lymphocytes (mean 18%; P < 0.001). Flow cytometric analysis revealed that in relation to monocytes and lymphocytes the DC had a higher surface expression of the adhesion molecules CD11a (P < 0.05), CD11c (P < 0.005) and CD54 (P < 0.005) but a lower prevalence of cells bearing CD49d (mean 38%; P < 0.05) and the homing receptor CD62L (mean 14%; P < 0.001). CD1a was present on 22% of DC and virtually absent from the surface of monocytes and lymphocytes. The intensity of expression of the beta1-integrins, CD49c, CD49d and CD49e was greater on DC than lymphocytes and monocytes (P < 0.05). Antibody blocking studies demonstrated that DC binding to untreated and TNF-alpha-treated endothelium was dependent upon the expression of CD11a, CD18 and CD49d, and the simultaneous application of anti-CD18 and anti-CD49d antibodies produced an approximate 70% inhibition of adhesion (P < 0.001). Thus, the expression of both beta1- and beta2-integrins contributes to the adhesive interaction between DC and endothelium.

Cell Adhesion↗
PubMed1995

Blood monocytes in rheumatoid arthritis are highly adherent to cultured endothelium.

Monocytes from 17 patients with rheumatoid arthritis (RA) were more adherent than monocytes from 17 control patients to monolayers of pig aortic endothelium irrespective of whether sera was included (median 27-34% increase; p = 0.002) or omitted (median 27% increase; p = 0.022) from the culture media. When human umbilical vein endothelial cells were used as the adherence substrate, rheumatoid monocytes from an additional 21 patients demonstrated a median 31% (p = 0.004) and 20% increase (p = 0.004) in adhesion when compared with monocytes from 21 normal healthy subjects in the absence and presence of autologous sera, respectively. Activation of control monocytes with muramyl dipeptide or treatment with RA sera increased their attachment to endothelium (mean 34 +/- 14% increase; p < 0.001). The expression of the adhesion molecules CD11b (p < 0.005), CD18 (p < 0.005), CD62L (p = 0.01) was enhanced on rheumatoid monocytes, but antibody-blocking studies suggested that CD18 and CD62L were not responsible for the augmented binding of the rheumatoid cells. A subpopulation of rheumatoid monocytes possessed a very low net negative surface charge, a property that favours binding to vessel walls. We propose that many rheumatoid monocytes are predisposed for sheer-resistant adhesion to vascular endothelium.

Adult↗
PubMed1994

Thrombin receptor expression and function in large granular lymphocyte proliferative disorders.

It has recently been shown that peripheral blood NK-cells and a fraction of T-cells which co-express CD16 and either CD56 or CD57 express the platelet type thrombin receptor. Large granular lymphocytes exhibit a T- or NK-cell phenotype, and therefore these results raise the possibility that thrombin and its receptor may be involved in the biology of large granular lymphocytes in health and disease. It is difficult, however, to perform functional studies using normal blood as a source of large granular lymphocytes, because the small fraction of large granular lymphocytes cannot be separated from other lymphocytes in numbers sufficient for most in vitro experiments. Therefore patients with large granular lymphocyte proliferative disorders have been screened in order to identify a population of cells enriched in large granular lymphocytes that express the thrombin receptor. Expression of the receptor was analysed in polyclonal and clonal large granular lymphocyte proliferative disorders. Using flow cytometry, it was found that the proportion of thrombin receptor positive large granular lymphocytes varied from 3% to 86%. Northern analysis indicated a high level of expression of mRNA in a clonal expansion of large granular lymphocytes that stained positively for the receptor by flow cytometry. Thrombin was found to act as a chemotactic stimulus for large granular lymphocytes from a polyclonal expansion with high numbers of thrombin receptor positive cells. At an optimal concentration of 10(-9) M the chemotactic response to thrombin was roughly equivalent to that obtained with the potent chemoattractant 1-oleoyl 2-acetyl glycerol. These findings suggest that thrombin may play a role in the recruitment of large granular lymphocytes in sites of inflammation.

Aged↗
PubMed1993

Platelet activation by Protease I of Porphyromonas gingivalis W83.

Porphyromonas gingivalis produces a trypsin-like enzyme, Protease I, which is thought to be an important virulence determinant of the organism in adult periodontal disease. Protease I is transiently inhibited by physiological inhibitors of human thrombin. The aim of the present work was to establish whether Protease I was able to mimic thrombin by activation of the thrombin receptor on human platelets. Protease I caused true platelet activation at concentrations comparable to thrombin as measured by aggregometry, morphology and fluorescence flow cytometric analysis of CD63 expression. The effect was blocked by protease inhibitors but not by anti-thrombin receptor antibodies which, by contrast, blocked platelet activation by thrombin. We conclude that the activation of platelets by P. gingivalis Protease I involves proteolysis, but not scission of the thrombin cleavage site of the thrombin receptor.

Amino Acid Sequence↗
PubMed1993

Peripheral blood lymphocytes express the platelet-type thrombin receptor.

Northern blot analysis of human mononuclear cells indicated that the platelet thrombin receptor may be expressed by lymphocytes. In order to investigate this, we prepared affinity purified rabbit antibodies against the thrombin receptor which bound platelets and blocked thrombin activation. Using flow cytometry on peripheral blood cells, we found that the vast majority of NK cells (CD16/CD56 positive) and a fraction of CD3/CD4 positive T cells expressed the thrombin receptor. B cells, neutrophils and monocytes were negative. These data suggest that potentially thrombin may play a direct role in regulating NK and T cell function.

Antibodies↗
PubMed1992

Sulphydryl reactivity of the HLA-B27 epitope: accessibility of the free cysteine studied by flow cytometry.

HLA-B27 has an unpaired cysteine on or near its serologically defined spondylitis associated epitope, and it has been argued that its sulphydryl side chain may be chemically reactive. In a previous study it was shown that chemical treatment of HLA-B27 cells with the sulphydryl binding agent p-chloromercuriphenylsulphonic acid (pCMPSA) specifically reduced binding of antibodies to HLA-B27 by up to 80%, as measured in a cellular enzyme linked immunosorbent assay (CELISA). The effect of sulphydryl blockade on intact B27 cells was investigated using flow cytometry. Compared with the CELISA, inhibition required higher concentrations of pCMPSA, and the degree of inhibition produced by a greater than or equal to 30 microM solution of pCMPSA as measured by flow cytometry (median 28.9%) was significantly lower than that measured by CELISA (median 73.6%; p = 1.6 x 10(-6)). Analysis of unfixed, cell surface HLA-B27 by flow cytometry suggests that on most B27 molecules the unpaired sulphydryl site is not available. On the basis of this evidence for modification after translation, a new 'altered self' hypothesis is proposed for the part which HLA-B27 plays in inflammatory disease.

4-Chloromercuribenzenesulfonate↗
PubMed1989

Enhancement of monoclonal antibody dependent cell mediated cytotoxicity by IL2 and GM-CSF.

Rodent monoclonal antibodies (MAb) directed against cells of the immune system may be used in vivo for applications including conditioning prior to marrow transplantation and treatment of lymphoid malignancies. Although some MAb may lyse targets by complement fixation, MAb dependent cell mediated cytotoxicity (MAb-DCC) appears to be an important additional effector mechanism. We have investigated the cellular basis of the phenomenon and the response of the effector cells to recombinant cytokines in an attempt to maximize the efficacy of MAb-DCC and thereby increase the therapeutic potency of MAbs. Blood mononuclear cells (PBM) coated with CAMPATH 1G (pan lymphocyte reactive rat IgG2b) MAb were used as targets (T) and autologous lymphocytes or granulocytes were used as effector (E) cells. We studied function in normal donors as well as patients 1 week before and 3-6 weeks after bone marrow transplantation (BMT). In the absence of CAMPATH 1G, specific 51Cr release from autologous PBM was less than 1% in all groups, even after pre-incubation of the effector cells with granulocyte-macrophage, colony stimulating factor (GM-CSF) or interleukin-2 (IL2). In the presence of 5 micrograms/ml of MAb, and at an E:T ratio of 50:1, lymphocytes from normal donors induced a low level (6.5%) of 51Cr release from auto PBM rising to 9% after pre-incubation of effector cells with IL2 (P = 0.02). Granulocytes had greater activity inducing 10% 51CR release (range 2-23%) which rose to 21.6% with GM-CSF (range 12-48%) (P less than 0.001). Pre-BMT, killing by lymphocytes and granulocytes was not significantly different from normal, and responded to IL2 and GM-CSF. In contrast, granulocyte killing after BMT was significantly impaired (51Cr release 3%) and showed no rise with GM-CSF. Killing by lymphocytes, however, remained normal, as did their IL2 response. Loss of granulocyte mediated MAb-DCC coincided with significant post-BMT impairment of oxidative metabolism; expression of Fc receptors II and III, however, was normal. Optimum therapeutic effect of MAb-DCC is likely to be achieved when MAb are given together with appropriate cytokines, the choice of which will depend upon the clinical circumstances.

Antibodies, Monoclonal↗
PubMed1989

Detection of granulocyte antibodies using flow cytometric analysis of leucocyte immunofluorescence.

Antigranulocyte antibodies are involved in the pathophysiology of a number of clinical disorders, which include: febrile transfusion reactions, severe pulmonary reactions to transfusion, auto-immune neutropenia, drug-induced neutropenia, and iso-immune neonatal neutropenia. Owing to the inherent difficulties of manipulating granulocytes in vitro, many of the serological techniques described for the detection of antigranulocyte antibodies are complex and sometimes difficult to reproduce. We describe the detection of alloreactive granulocyte antibodies using flow cytometric analysis of donor leucocyte suspensions in an indirect immunofluorescent test. The technique provides a semiquantitative detection of granulocyte antibodies in two groups of patients studied and, by providing as a comparison the reactivity on the corresponding mononuclear leucocytes, allows the distinction between granulocyte-specific antibodies and antibodies directed against the histocompatibility antigens.

Blood Grouping and Crossmatching↗
PubMed1986

Allogeneic bone marrow transplantation for adult leukaemia with soy bean lectin fractionated marrow.

A heterogeneous group of 11 consecutive patients with leukaemia have been transplanted successfully with allogeneic marrow depleted of T lymphocytes by soy bean lectin agglutination and neuraminidase-treated sheep erythrocyte rosetting. Effective depletion was achieved, leaving less than 1% of donor T lymphocytes. Despite the small numbers of nucleated cells infused (mean 0.14 X 10(8)/kg) there was only moderately delayed recovery of peripheral blood counts and no graft failures have occurred. Standard methotrexate prophylaxis against graft-versus-host disease (GVHD) was also employed in the first four transplants. Only one case of mild grade I (skin only) acute GVHD has occurred and there has been no chronic GVHD to date. The group of patients show an actuarial cumulative survival of 55% with two early infective deaths (days 42 and 44 post-transplant) and three late deaths, two with leukaemic relapse and the third with probable viral encephalitis. The longest survivor is now 1109 days post-transplant. This series indicates that lectin fractionation of donor marrow, previously employed mainly in children, can also be effective in minimizing GVHD in adults without endangering successful engraftment.

Adolescent↗