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Biomedical subjects

G B Nash

Publications and source records attributed to G B Nash.

At least 55 records · Page 3Linked to original sources
PubMed1996

Effect of temperature on the resistance of individual red blood cells to flow through capillary-sized apertures.

Low temperature can be expected to increase the resistance to deformation of red blood cells, but the effect of such changes on microcirculatory perfusion are unknown. We therefore analysed resistance to flow through capillary-sized apertures for individual human red blood cells, by micropipette aspiration (approximately 3 microm aperture) and pore transit analysis (approximately 5 microm), as well as average resistance to flow of red cell suspension through multipore filters (5-microm pores). Over a range decreasing from 37 to 0 degrees C, rates of flow of single cells through the 3- and 5-microm apertures decreased monotonically by 2.5- to 3-fold. The changes were similar in magnitude to that expected for the viscosity of aqueous fluid (2.5-fold increase). Average flow resistance measured by bulk filtration also increased in line with viscosity of water, while tendency to block pores was not increased. Micropipette aspiration of small membrane tongues showed that membrane rigidity increased as temperature was lowered, but by a factor rather less than the viscosity. Cell volume also responded rapidly to change in temperature, with lower temperature being associated with swelling, although this effect was much reduced in plasma compared with saline buffer. We conclude that, although increased resistance to deformation of red cells may impair microcirculation at low temperature, there is no structural change likely to induce more dramatic occlusion of flow. Moreover, the effect is comparable in magnitude to the increase predicted for changes in plasma and blood viscosity.

Adult↗
PubMed1996

Changes in neutrophil rheology after acute ischemia and reperfusion in the rat hindlimb.

Neutrophils may contribute to the pathogenesis of ischemia-reperfusion injury by impairing microcirculatory flow and causing tissue damage. In this study neutrophil deformability and activation were studied after acute ischemia and reperfusion of the rat hindlimb. A cell transit analyzer with 8 microm pore filters was used to measure the flow resistance of individual neutrophils isolated from arterial blood drawn 30 seconds and 5 minutes after reperfusion, after 2 hours of femoral or iliac artery occlusion, in anesthetized rats. Femoral occlusion followed by 30 seconds of reperfusion resulted in a 16% rise in mean pore transit time, and iliac occlusion caused a 31% increase. Neutrophil activation (percentage of cells with nonspherical shape) rose from 6% to 23% (femoral occlusion) or from 7% to 29% (iliac occlusion). By 5 minutes of reperfusion, transit times, but not shape change, had almost returned to preischemic levels. Sham-occluded rats had no significant change in neutrophil behavior. Plasma isolated from rats after 30 seconds of reperfusion after 2 hours of iliac occlusion caused a 27% increase in mean transit time when incubated with nonautologous neutrophils, while activation rose from 32% to 55%. Thus plasma factor(s) released after ischemia and reperfusion of rat hindlimb caused transient rheologic alteration of circulating neutrophils, which may contribute to local or disseminated vascular occlusion and tissue injury.

Animals↗
PubMed1996

Prolonged E-selectin induction by monocytes potentiates the adhesion of flowing neutrophils to cultured endothelial cells.

We investigated the hypothesis that the infiltration of monocytes into inflamed tissue or damaged vessels would induce a secondary accumulation of neutrophils. Confluent human umbilical vein endothelial cells (HUVEC) and blood monocytes (0.5 or 0.05 monocytes/endothelial cell) were co-incubated for 4 or 24 h. The adhesion of neutrophils flowing over HUVEC was then analysed by video microscopy. Co-incubation caused up to a 40-fold increase in neutrophil adhesion, dependent upon monocyte/HUVEC ratio and duration of incubation. At the lower monocyte/HUVEC ratio, rolling adhesion alone was induced after 4 h co-incubation; however, the full repertoire of rolling, immobilization and migration of neutrophils was observed at all other combinations of co-culture ratio and exposure time. After maximal stimulation by monocytes, antibody blockade of the neutrophil integrin CD18 inhibited neutrophil arrest and migration and revealed underlying rolling adhesion. Rolling was supported by endothelial E-selectin as demonstrated by the almost total abolition of adhesion by a blocking antibody. In a direct comparison, monocytes, tumour necrosis factor alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta) were assessed for their ability to induce endothelial expression of E-selectin. E selectin was significantly increased by all agents at 4 h, but monocytes alone were able to maintain high levels of E-selectin expression for 24 h. We conclude that monocytes can induce prolonged neutrophil adhesion and migration by activating endothelial cells and causing expression of E-selectin.

Cell Communication↗
PubMed1995

Perturbation of red blood cell membrane rigidity by extracellular ligands.

It is known that binding of extracellular antibodies against the major sialoglycoprotein, glycophorin A, reduced the deformability of the red blood cell membrane. This has been taken to result from new or altered interactions between the glycophorin A and the membrane skeleton. We have shown by means of the micropipette aspiration technique that antibodies against the preponderant transmembrane protein, band 3, induce similar effects. A definite but much smaller reduction in elasticity of the membrane is engendered by univalent Fab fragments of the anti-band 3 antibodies. By examining cells genetically devoid of glycophorin A or containing a variant of this constituent, truncated at the inner membrane surface, we have shown that the anti-band 3 antibodies do not act through the band 3-associated glycophorin A. We examined the effect of anti-glycophorin A antibodies on homozygous Wr(a+b-) cells, in which an amino acid replacement in band 3 annihilates the Wright b (Wrb) epitope (comprising sequence elements of glycophorin A and band 3) and thus, by implication disrupts or perturbs the band 3-glycophorin A interaction; these cells show a much smaller response to an anti-glycophorin A antibody than do normal controls. We infer that in this case anti-glycophorin A antibodies exert their rigidifying effect through the associated band 3. Another anti-glycophorin A antibody, directed against an epitope remote from the membrane surface, however, increases the rigidity of both Wr(a+b-) and normal cells. This implies that not all antibodies act in the same manner in modifying the membrane mechanical properties. The effect exerted by anti-band 3 antibodies appears not to be transmitted through the band 3-ankyrin-spectrin pathway because the rigidifying effect of the intact antibody persists at alkaline pH, at which there is evidence that the ankyrin-band 3 link is largely dissociated. The large difference between the effects of saturating concentrations of the divalent and univalent anti-band 3 antibodies implies the existence of an overriding effect on rigidity, resulting from the bifunctionality of the intact antigen. Freeze-fracture electron microscopy shows that the anti-band 3 promotes the formation of small clusters of intra-membrane proteins. Extracellular ligands may in general act by promoting strong or transient interactions between integral membrane proteins, thereby impeding local distortion of the membrane skeletal network in response to shear.

Anion Exchange Protein 1, Erythrocyte↗
PubMed1995

Plasmodium falciparum: characterization of adhesion of flowing parasitized red blood cells to platelets.

Adhesion of parasitized red blood cells to vascular endothelium contributes to the ischaemic pathology of severe falciparum malaria. One of the endothelial cytoadhesion receptors, CD36, is also expressed by platelets. We have studied adhesion of flowing parasitized cells to a surface coated with immobilized, activated platelets, both as a model for CD36-mediated adhesion and because interaction with platelets might play a direct role in thrombotic complications of malaria. Parasitized cells were able to bind firmly to platelets over a range of shear stress (up to 0.3 Pa) close to those found in the microcirculation. The binding was largely abolished by treatment of platelets with antibody to CD36, with only a small effect by antibody to ICAM-1. Binding showed pH sensitivity consistent with previous reports of CD36-mediated cytoadhesion. Fixation of the platelet surface with formaldehyde preserved adhesion and its antibody sensitivity, while fixation with glutaraldehyde greatly reduced adhesion and increased the sensitivity to antibody against ICAM-1. Thus CD36-mediated binding is inhibited by glutaraldehyde--but not formaldehyde--fixation, while ICAM-1 can mediate adhesion after either form of fixation. We conclude that platelet-coated surfaces (with or without fixation) represent a practically simple model for studying malarial cytoadhesion and that platelets are likely to be able to bind parasitized cells in vivo and could thus promote vascular occlusion.

Animals↗
PubMed1995

Mechanism of regulation of malarial invasion by extraerythrocytic ligands.

Invasion of red cells by Plasmodium falciparum in vitro was inhibited by a range of extracellular ligands, none of which block the major receptors for merozoites. Most effective, in terms of dose response, were two monoclonal antibodies against the Wrb antigen on glycophorin A; wheat germ agglutinin which also binds to glycophorin, and an anti-band 3 monoclonal antibody, caused inhibition of invasion at higher levels of saturation, while concanavalin A, which binds to band 3, was without effect. All the ligands except concanavalin A, increased the rigidity of the host cell membrane. The anti-Wrb antibodies generated the highest dose response effect, but no correlation between invasion and shear elastic modulus of the membrane could be established. All ligands, with the exception of concanavalin A, caused a reduction in the translationally mobile fractions of band 3 and glycophorin, as revealed by fluorescence recovery after photobleaching (FRAP). Invasion diminished with loss of mobile band 3, engendered by bound wheat germ agglutinin or anti-band 3, falling precipitately when the mobile fraction fell below 40% of that in unperturbed membranes. Both anti-Wrb antibodies suppressed invasion completely at concentrations insufficient to affect significantly either membrane rigidity or intramembrane protein diffusion. A univalent anti-glycophorin A (Fab) fragment, the parent antibody of which was previously shown to inhibit invasion strongly, had only a modest effect on invasion and induced a correspondingly small change in the mobile fraction of band 3.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Thromboxane and neutrophil changes following intermittent claudication suggest ischaemia-reperfusion injury.

OBJECTIVES: It has been postulated that ischaemia-reperfusion occurs in intermittent claudication resulting in neutrophil activation and release of soluble mediators, increasing systemic vascular permeability and enhancing atherogenesis. METHODS: We measured neutrophil deformability, plasma thromboxane levels, and urinary microalbumin excretion in 30 male claudicants, and 10 age- and sex-matched controls, before and after exercise to maximum walking distance. Blood was taken from an antecubital vein. RESULTS: There was an increase in urinary microalbumin excretion after exercise in claudicants. Statistically significant increases in the median and 90th percentile transit times (markers of neutrophil deformability) for isolated neutrophils from blood drawn 5 min after exercise in the claudicants were observed with no change in control subjects. Plasma thromboxane concentrations in claudicants increased within 10 min post-exercise. Plasma concentrations in controls were significantly lower throughout the study period. In the claudicant group, a positive correlation between the percentage change in the median transit time for neutrophils, and the percentage change in plasma thromboxane at 60 min post-exercise was found. CONCLUSIONS: The results lend further support to the concept of ischaemia-reperfusion events in patients with intermittent claudication, leading to a systemic increase in vascular permeability as a result of endothelial injury or dysfunction (a crucial step in atherogenesis), associated with thromboxane production and neutrophil activation. We suggest that the above changes may contribute to the increased mortality seen in such patients.

Adult↗
PubMed1995

Adhesion of flowing leucocytes to immobilized platelets.

Adhesion of neutrophils, lymphocytes and promyelocytic HL60 cells was compared in a flow-based model in which a monolayer of activated platelets formed the adhesive substrate. Each type of leucocyte formed P-selectin-mediated rolling attachments on the platelet surface under physiologically relevant flow conditions. Lymphocytes adhered less, and HL60 in similar numbers, compared to neutrophils, whereas the lymphocytes and HL60 cells rolled much more rapidly. Sulphated, sialylated saccharide(s) were implicated as ligand(s) for P-selectin for all leucocytes, but L-selectin (borne by neutrophils and lymphocytes, but not HL60 cells) appears to be a major presenter of ligand for neutrophils alone. T cells enriched from peripheral blood lymphocytes adhered in greater numbers than B cells. Differentiation of HL60 cells to neutrophil-like cells (induced by DMSO) caused cell volume to decrease and surface expression of integrin adhesion molecules to increase, but only a small percentage of cells were converted to an L-selectin-bearing phenotype. Differentiated cells showed evidence of stabilization of adhesion with increasing stress and a marked reduction in rolling velocity. These studies indicate that cell differentiation may be accompanied by alteration of adhesive behaviour, resulting from changes in physical characteristics as well as surface properties. Moreover, results suggest that P-selectin could promote lymphocyte attachment to endothelium in acute inflammatory conditions and possibly mediate lymphocyte-platelet interaction during thrombosis.

B-Lymphocytes↗
PubMed1995

Adhesion of flowing neutrophils to cultured endothelial cells after hypoxia and reoxygenation in vitro.

Using a novel in-line deoxygenating system linked to an in vitro flow-based adhesion assay and video microscopy, we have studied neutrophil recruitment and migration after hypoxia and reoxygenation of cultured human umbilical vein endothelial cells (HUVEC). Unstimulated purified neutrophils were perfused over reoxygenating HUVEC immediately after various periods of endothelial hypoxia. Adhesion to HUVEC was dependent on the duration of hypoxia, with 30, 60, and 100 min of exposure causing graded increments in neutrophil recruitment. The degree of hypoxia also markedly influenced the endothelial response. Severe hypoxia (O2 < 2.5%) induced stationary attachment and then migration of neutrophils, in contrast to rolling adhesion alone under a less intense regime (O2 = 2.5-4.0%). Judged from studies with monoclonal antibodies, P-selectin was essential for adhesion after severe hypoxia, and neutrophil immobilization was attributable to the activation of neutrophil beta 2-integrin. Perfusion of neutrophils with an antibody against interleukin-8 or a platelet-activating factor antagonist reduced levels of adhesion. However, IL-8 appeared to be the dominant agent involved in the immobilization from flow, whereas platelet-activating factor was the more potent agent involved in initiating subendothelial migration. Thus endothelial cells alone can initiate all stages of adhesion and migration of flowing neutrophils after hypoxia and reperfusion.

Cell Adhesion↗
PubMed1995

Mechanisms of cytoadhesion of flowing, parasitized red blood cells from Gambian children with falciparum malaria.

Adhesion of parasitized red blood cells to vascular endothelium is considered to be a major factor in the pathophysiology of falciparum malaria, and so the molecular mechanisms and rheologic characteristics of this interaction are of profound importance. We have investigated the adhesive behavior of wild-type parasite isolates cultured from the blood of Gambian children with falciparum malaria and allowed to flow over surfaces coated with formaldehyde-fixed human umbilical vein endothelial cells (HUVEC) or platelets. Parasitized cells were able to attach to HUVEC and/or to platelets, and studies with monoclonal antibodies showed that intercellular adhesion molecule-1 (ICAM-1) and CD36 antigen were the major mediators of adhesion for the two surfaces, respectively. The levels of adhesion to HUVEC and to platelets were highly variable but did not correlate with each other, so that different isolates express independently variable capacities to bind to the two receptors. Adhesion was stationary for platelets and generally at a higher level compared with binding to HUVEC, which was predominantly (about 60%) of a rolling type. The stationary component of adhesion to HUVEC represented a greater proportion of adhesion for the wild isolates than for laboratory-adapted strains, and this form of adhesion was relatively insensitive to antibody to ICAM-1. This suggests the existence of an additional endothelial cell-expressed receptor for the wild isolates. These studies show wide variation in the ability of wild isolates of Plasmodium falciparum to adhere to ICAM-1, CD36 antigen, and possibly other receptors in the presence of physiologically relevant flow.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Natural protection against severe Plasmodium falciparum malaria due to impaired rosette formation.

Genes for two lethal diseases, thalassemia and sickle cell anemia, are favored by evolution because, in their heterozygous form, they protect against cerebral malaria. Rosette formation, the binding of uninfected red cells (RBCs) to Plasmodium falciparum-infected RBCs (PRBCs), has previously been found to be associated with cerebral malaria, the most important severe manifestation of P falciparum malaria. We show here that thalassemic RBCs and, under certain conditions, even hemoglobin S (HbS)-containing RBCs possess an impaired ability to bind to PRBCs, forming small and weak erythrocyte rosettes compared with rosettes formed by normal RBCs. This decreased rosetting ability is associated with the small size of the thalassemic RBCs and with distortion of the mechanical properties of HbS-containing RBCs. The impairment of rosette formation may hinder the development of cerebral malaria by abatement of sequestration.

Anemia, Sickle Cell↗
PubMed1994

Adhesion between neutrophils and platelets: a modulator of thrombotic and inflammatory events?

Recent studies have demonstrated that neutrophilic granulocytes and platelets have the capability to interact with each other. Either type of cell may release soluble factors, which either activate the other or increase its response to separate stimulating agents. Direct contact between the two types of cell generally increases the response, and it may be predicted that neutrophil-platelet adhesion will augment the potential for biochemical interaction in vivo. Adhesion may also promote immobilisation of neutrophils or platelets in thrombotic or inflamed tissue, while circulation aggregates could cause vascular blockage. Adhesion between neutrophils and platelets is dependent on P-selectin on activated platelets and sialyated carbohydrate moieties on the neutrophil, although other molecular mechanisms could stabilise the interaction. The adhesive interaction is strong enough to allow flowing neutrophils to adhere to immobilised platelets, and induce formation of aggregates in vivo. Thus, inhibition of neutrophil-platelet adhesion and modulation of their biochemical interactions could be a novel approach to treatment of thrombotic, ischaemic or inflammatory disorders.

Amino Acid Sequence↗
PubMed1994

Rolling and stationary cytoadhesion of red blood cells parasitized by Plasmodium falciparum: separate roles for ICAM-1, CD36 and thrombospondin.

Adhesion of parasitized erythrocytes to microvascular endothelium is a central event in the pathogenesis of severe falciparum malaria. We have characterized the adhesion of flowing parasitized red blood cells to three of the known endothelial receptors coated on plastic surfaces (CD36, intercellular adhesion molecule-1 (ICAM-1) and thrombospondin (TSP)), and also to cells bearing these receptors (human umbilical vein endothelial cells (HUVEC) and platelets). All of the surfaces could mediate adhesion at wall shear stress within the physiological range. The great majority of adherent parasitized cells formed rolling rather than static attachments to HUVEC and ICAM-1, whereas static attachments predominated for platelets, CD36 and TSP. Studies with monoclonal antibodies verified that binding the HUVEC was mainly via ICAM-1, and to platelets via CD36. Adhesion via ICAM-1 was least sensitive to increasing wall shear stress, but absolute efficiency of adhesion was greatest for CD36, followed by ICAM-1, and least for TSP. TSP did not give long-lasting adhesion under flow, whereas cells remained adherent to CD36 or ICAM-1. We propose that the different receptors may have complementary roles in modulating adhesion in microvessels. Initial interaction at high wall shear stress may be of a rolling type, mediated by ICAM-1 or other receptors, with immobilization and stabilization occurring via CD36 and/or TSP.

Animals↗
PubMed1994

Rheological response of neutrophils to different types of stimulation.

The potential for neutrophils to obstruct microvessels was evaluated by measuring transit of individual neutrophils through 8-microns pores in an automated cell transit analyzer (CTA) or into micropipettes (4-8 microns ID). Stimulation in vitro by the chemotactic agent N-formyl-methionyl-leucyl-phenylalanine. (fMLP), cigarette smoke, or purified antineutrophil cytoplasm antibodies greatly increased flow resistance, but the response varied in its dependence on time and pore diameter. Cigarette smoke or fMLP caused rapid loss of cellular deformability, although observations were complicated by changes in cell shape: progressive bipolar shape formation (after treatment with fMLP) could facilitate entry into larger pores (approximately 8 microns), whereas blebs induced by cigarette smoke caused bridging of these pores with cell immobilization. These processes led to an underestimation of the changes in deformability by the CTA. Neutrophils responded slowly to the antineutrophil cytoplasm antibodies (approximately 30 min), with a greater increase in flow resistance evaluated by a micro-pipette (4-6 microns ID) than by the CTA. We conclude that the effect of neutrophil stimulation on flow through capillary-sized vessels is potentially great (with resistance typically increased 10-fold or even complete blockage) but may depend on the vascular and cellular geometry and may be local or disseminated, depending on the rate of the rheological response.

Adult↗
PubMed1993

Selectin-mediated rolling of neutrophils on immobilized platelets.

Interaction between neutrophils and platelets at the site of vascular damage or in ischaemic tissue may promote thrombosis and/or vascular occlusion. To study this interaction, we have developed a novel technique that allows visualization of adhesion of flowing neutrophils to immobilized, activated platelets. The total number of adherent neutrophils decreased with increasing wall shear stress in the range 0.05 to 0.4 Pa. Although a proportion of the adherent neutrophils were stationary, most were rolling with a velocity greater than 0.4 micron/s. The percentage of rolling cells increased with increasing wall shear stress, but the mean rolling cell velocity was nearly independent of shear stress. Adhesion of neutrophils was nearly abolished by treatment of the platelets with antibody to P-selectin, or by treatment of neutrophils with either neuraminidase, dextran sulfate, or EDTA. Studies with a series of antibodies to L-selectin (TQ-1, Dreg-56, LAM1-3, and LAM1-10) suggested that this molecule was one neutrophil ligand for rolling adhesion. Thus, sialylated carbohydrate on neutrophils appears essential for P-selectin-mediated adhesion, and a proportion of this ligand may be presented by L-selectin. Treatment of the neutrophils with N-formyl-methionyl-leucyl-phenylalanine decreased the number of rolling cells, and increased the rolling velocity, possibly due to shedding of neutrophil ligand(s) and/or cell shape change. In vivo, immobilized platelets could play an important role in promoting attachment of neutrophils to vessel walls, eg, by slowing neutrophils so that integrin-mediated immobilization could occur.

Antibodies, Monoclonal↗
PubMed1993

Neutrophil integrin assay for clinical studies.

The level of expression of neutrophil adhesion molecules may be a useful marker for neutrophil activation in clinical studies. We therefore determined neutrophil integrin expression under various experimental conditions using a Fluorescence Activated Cell Sorter (FACS) after the cells had been labelled with fluorescent conjugated antibodies to the integrin subunits CD11a, CD11b and CD18. Levels of labelled CD11b and CD18 increased after activation with the chemotactic peptide formyl-methionyl-leucyl phenylalanine (fMLP) in a dose- and time-dependent manner, but CD11a did not, indicating that CD11a would not be a useful marker of neutrophil activation. The baseline expression of CD11b and CD18 on unstimulated neutrophils was similar in heparin and EDTA anti-coagulated blood but the response to activation with fMLP was significantly less for the EDTA anti-coagulated samples (p < 0.01 in paired t-test). The labelling of integrins was significantly higher in unfixed whole blood samples compared to samples fixed with 1 per cent paraformaldehyde. However, the increase in labelling induced by fMLP was similar whether or not the samples were fixed after activation. Labelling of CD11b and CD18 was greater for preparations of isolated neutrophils than for neutrophils in whole blood, and the response to fMLP stimulation tended to be lower for the isolated cells. Our results indicate that heparin should be used as anti-coagulant in clinical studies utilizing whole blood if subsequent activation of neutrophils is planned (e.g. to detect in vivo priming), although EDTA may be used if baseline expression alone is to be measured. Fixation of blood samples should not affect the ability to detect neutrophil activation.

Antigens, CD↗