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G B Nash

Publications and source records attributed to G B Nash.

At least 73 records · Page 4Linked to original sources
PubMed1993

A simplified method for culture of endothelial cells and analysis of adhesion of blood cells under conditions of flow.

We have developed a simplified technique for culturing human umbilical vein endothelial cells under shear flow conditions, using prefabricated glass microcapillary tubes ("microslides") with a well-defined rectangular cross section and good optical quality. These microslides have been incorporated into a controlled flow system for quantitative video-microscopic analysis of the adhesion of blood cells to endothelial cells. Microslides were pretreated with 3-aminopropyltriethoxy-silane, or gelatin, and then loaded with a suspension of endothelial cells. After the cells had settled and attached to the substrate, the microslides were inserted into a flow-based culture system. Medium was drawn through them at intervals or continuously until confluency was reached (approximately 24 hr). Cells were cultured at wall shear stresses over a range 0.06 to 2.2 Pa. For adhesion assays, the endothelialized microslides were attached to microscope slides, and suspensions of blood cells were drawn through at desired wall shear stresses (0.02-0.5 Pa). Adhesion of malarial-infected red blood cells and of neutrophilic granulocytes was quantitated by direct microscopic observation. The adhesive behavior of both cell types closely resembled that previously described by ourselves and others using flow chambers incorporating endothelial-coated glass coverslips. The use of microslides represents a significant simplification of methodology for endothelial cell growth and adhesion studies under flow conditions.

Blood Cells↗
PubMed1993

Adhesion of parasitized red blood cells to cultured endothelial cells: a flow-based study of isolates from Gambian children with falciparum malaria.

Adhesion of parasitized red blood cells to vascular endothelium is thought to play an important role in the development of the ischaemic complications associated with severe falciparum malaria. Using a novel, flow-based assay, we have investigated the adhesion of parasitized red blood cells to formalin-fixed human umbilical vein endothelial cells (HUVEC), for isolates obtained from 32 Gambian subjects with mild or severe falciparum malaria. Red cells infected with wild strains of Plasmodium falciparum were able to adhere to HUVEC under physiologically relevant flow conditions, but the level of adhesion was highly variable, ranging from 1 to 688 adherent cells per mm2 of HUVEC. Within isolates, some adherent parasitized cells remained stationary, whilst other formed less stable interactions and rolled slowly over the cell surface. There was no significant difference in adhesion of parasitized cells between isolates obtained from mild or severe cases of malaria, although a subset of isolates did show very high levels of adhesion. The results suggest that there is not a simple relationship between the adhesion of parasitized cells to cultured endothelial cells (presumably via the receptor ICAM-1) and the clinical severity of the disease, although variation in microvascular adhesion in vivo may still be a determinant of ischaemic complications.

Cell Adhesion↗
PubMed1993

Effect of activation on adhesion of flowing neutrophils to cultured endothelium: time course and inhibition by a calcium channel blocker (nitrendipine).

1. Adhesion of neutrophils to vascular endothelium plays an important role in inflammation and thrombosis. Modulation of adhesion may be therapeutic in these conditions. 2. A flow model was used to quantify adhesion of neutrophils to human cultured umbilical vein endothelial cells. The time course of the neutrophil response to activation by N-formyl-methionyl-leucylphenylalanine (fMLP, 10(-7) M) was studied and the inhibitory effects of the calcium-channel blockers, nitrendipine and nifedipine, were investigated. 3. Neutrophils adhered firmly to the endothelial cells without rolling, but initial attachment was highly dependent on shear stress; doubling the stress from 0.05 to 0.1Pa decreased the number of neutrophils adhering by over 80%. 4. Adhesion rapidly increased after activation of neutrophils by fMLP, peaking at 1-3 min post-treatment, and then decreased over the next 10-12 min. A monoclonal antibody to the beta 2-integrin component CD18 inhibited adhesion by over 80% for activated or unactivated cells. 5. The Ca-channel blocker, nitrendipine, but not nifedipine, significantly inhibited the fMLP-induced increase of adhesion in a dose-dependent manner (10(-8) to 10(-6) M). Dihydropyridines may be useful agents for modifying neutrophil function.

Adult↗
PubMed1993

Comparative rheology of human and trout red blood cells.

We have studied the comparative rheology of individual red blood cells from humans and rainbow trout (Oncorhynchus mykiss) at their natural body temperatures. Trout red blood cells were large ellipsoids (about 16 microns x 11.5 microns x 2.5 microns) with a mean volume of 250 fl, a surface area of approximately 350 microns 2 and an elongated nucleus of about 9 microns x 5 microns. Although much larger than human red cells (diameter 8 microns, V = 92 fl, A = 136 microns 2), both theoretical calculation and experimental aspiration into micropipettes indicated that the limiting size of a cylindrical vessel that both types of cell could enter was approximately 3 microns. Nevertheless, individual trout red cells had much longer transit times through 5 microns filter pores and were much slower to enter 3-4 microns diameter micropipettes. Interestingly, the relative deformability of the trout cells depended on the pore size and applied pressure, with entry times for trout and human cells converging as pipette diameter increased. The relatively poor overall cellular deformability of the trout cells reflected their membrane rigidity (shear elastic modulus 4-5 times higher than that of human membrane), as well as their large size and the presence of a prominent nucleus. Capillary diameters in trout muscle are similar to those in the human microcirculation (about 3 microns), while systemic driving pressures are much lower. Therefore, either red cell deformability is a less critical circulatory parameter than has previously been thought, or the apparently disadvantageous blood rheology of trout is adequate because of the lower demand for tissue perfusion.

Animals↗
PubMed1993

Membrane rigidity of red blood cells parasitized by different strains of Plasmodium falciparum.

Changes in the structure of parasitized red blood cells may influence their ability to circulate. We have used a micropipette technique to examine the effects of invasion and maturation of Plasmodium falciparum on the membrane rigidity of red blood cells. In the presence of immature, ring form parasites from different laboratory strains, membrane rigidity remained unchanged as compared with uninfected red cells. However, development of more mature pigmented trophozoites caused a marked increase in membrane rigidity. Parasites from knobless strains caused a less-pronounced increase than parasites from knob-positive strains. Using closely synchronized cultures, the dependence of membrane rigidity on parasite maturation was studied in more detail for selected knob-positive and knobless strains. Over a period of 12 hours, while trophozoites developed into schizonts, no further rigidification of the red cell membrane occurred. The increase in membrane rigidity, occurring with the initial development of pigmented trophozoites, may be related to insertion of neoantigens into the red cell surface or modification of native membrane proteins that also occur at this time. In contrast to others, we found no effect of parasite-culture supernatant, harvested at different stages, on the rigidity of uninfected cells exposed to it. Interstrain variation of membrane rigidity could influence pathophysiology in several ways: by promoting margination and cytoadherence of knob-positive strains in the microcirculation, by modulating clearance of parasitized cells by the reticuloendothelial system, and by influencing ischemic complications of severe falciparum malaria.

Animals↗
PubMed1993

Comparative study of the adhesion of sickle cells and malarial-parasitized red cells to cultured endothelium.

Increased adhesion of red cells to vascular endothelium has been implicated in the pathogenesis of falciparum malaria and sickle cell disease. We have carried out a comparative study of the adhesiveness of normal (AA), sickle trait (AS), and homozygous sickle (SS) red cells, with and without parasitization by Plasmodium falciparum, with an in vitro flow system. Adhesion of nonparasitized red cells to cultured human umbilical vein endothelial cells (either glutaraldehyde fixed or untreated) was strongly dependent on the wall shear stress. Many AA and SS cells adhered at low stress (0.02 Pa), but far fewer did so when the stress was increased to a physiologic level (0.1 Pa). Compared with AA cells, SS adhered in greater number (about threefold) and required greater stress (about two-fold) for their subsequent removal. In contrast, the efficiency of adhesion of AA cells parasitized by Plasmodium falciparum was essentially constant up to 0.1 Pa, where it was about 1000 times greater than the efficiency for nonparasitized cells. The stress required to remove parasitized cells was about 6 times that for controls. When parasites were grown in SS cells, fewer cells adhered than when parasites were grown in AA cells. However, the adhesion of malarial-parasitised AS cells was only slightly less than that of parasitized AA cells, so that modulation of adhesion is unlikely to underlie the protective effect of sickle gene in malaria. Adhesion of red cells to endothelium may promote blockage of microvessels, and the interaction of parasitized cells appears strong enough to directly cause ischemic complications in falciparum malaria.

Anemia, Sickle Cell↗
PubMed1992

Basis of unique red cell membrane properties in hereditary ovalocytosis.

Hereditary ovalocytes from a Mauritian subject are extremely rigid, with a shear elastic modulus about three times that of normal cells, and have increased resistance to invasion by the malaria parasite Plasmodium falciparum in vitro. The genetic anomaly resides in band 3; the protein gives rise to chymotryptic fragments with reduced mobility in SDS/polyacrylamide gel electrophoresis, but this is a result of anomalous binding of SDS and not a higher molecular weight. Analysis of the band 3 gene reveals (1) a point mutation (Lys56----Glu), which also occurs in a common asymptomatic band 3 (Memphis) variant and governs the electrophoretic properties, and (2) a deletion of nine amino acid residues, including a proline residue, encompassing the interface between the membrane-associated and the N-terminal cytoplasmic domains. The interaction of the mutant band 3 with ankyrin appears unperturbed. The fraction of band 3 capable of undergoing translation diffusion in the membrane is greatly reduced in the ovalocytes. Cells containing the asymptomatic band 3 variant were normal with respect to all the properties that we have studied. Possible mechanisms by which a structural change in band 3 at the membrane interface could regulate rigidity are examined.

Adult↗
PubMed1992

Rheological analysis of the adhesive interactions of red blood cells parasitized by Plasmodium falciparum.

Adhesion of parasitized red blood cells (RBCs) to vascular endothelium is thought to be a key factor in the pathology of falciparum malaria. However, quantitative analyses of the intercellular forces and of the effects of flow on adhesion have been lacking. We have characterized cytoadhesion of RBCs parasitized by the strains ITO4 (which can bind to receptors ICAM-1 or CD36) and FCR3A2 (which can bind to CD36 only) using micropipette manipulation and flow chamber techniques. Target cells were unfixed or glutaraldehyde-fixed human umbilical vein endothelial cells (HUVEC, bearing ICAM-1 only) or human amelanotic melanoma cells (C32, bearing CD36 and ICAM-1). In the static, micropipette assay, 60% to 70% of parasitized cells would adhere when tested at up to three successive sites. The percentage of cells adhering and the force required for their detachment (approximately 10(-10) N) were similar for each combination of parasite strain and adhesion target (ITO4/HUVEC, ITO4/C32, FCR3A2/C32). In the flow chamber, efficiency of initial adhesion of parasitized cells was essentially constant (at about 1%) up to a stress of 0.1 Pa, and then decreased rapidly with increasing stress. Either receptor (ICAM-1 or CD36) could immobilize flowing cells at a physiologic flow stress (0.1 Pa), but the numbers of cells adhering varied for the different combinations (ITO4/C32 greater than ITO4/HUVEC greater than FCR3A2/C32). When flow was increased in steps, adhered cells were gradually washed off but many could withstand stresses at which they would not initially adhere. The force for detachment estimated in this way was similar to the pipette value, and again, was similar for the different combinations of strains and targets. Adhesion from flow depends on the affinity between surfaces being above a critical level, and once adhesion is established, the fracture energy determines resistance to disruption of adhesion. The results show that the fracture energy is greater than the affinity (ie, that adhesion becomes stabilized after it is initially established) and that the ratio of affinity to fracture energy is different for different receptor/ligand pairs, with ICAM-1 appearing to be the more efficient immobilizing receptor. Also, static and flow-based assays of adhesion clearly differ; the affinity is less critical in the static situation, so that most parasitized cells were capable of adhering in a static assay, but fewer did so under flow. Adhesiveness varied markedly from cell to cell, both for targets and parasitized cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1992

Nitrendipine is a potent inhibitor of the Ca(2+)-activated K+ channel of human erythrocytes.

Nitrendipine, a classical blocker of L-type Ca2+ channels, is shown to be a potent inhibitor of the Ca(2+)-activated K+ channel of human erythrocytes. In erythrocytes suspended in a solution with physiological Na+ and K+ concentrations and in which the channel was activated using the Ca2+ ionophore ionomycin, nitrendipine inhibited K+(86Rb+) influx with an I50 of around 130 nM. Similar results were obtained for K+(86Rb+) efflux, and for K+(86Rb+) influx into cells suspended in a high-K+ medium.

Calcium↗
PubMed1992

Invasion of hereditary ovalocytes by Plasmodium falciparum in vitro and its relation to intracellular ATP concentration.

Hereditary ovalocytes (stomatocytic ovalocytes), when examined within 1-2 days from the time that the blood sample is drawn, are invaded by Plasmodium falciparum in culture to the extent of at least 55% of normal control cells. The ovalocytes have extremely rigid membranes, characterised by a shear elastic modulus some 3-4 times greater than that of normal cells. The extent of invasion falls off very much more rapidly than that into normal cells on storage, and we surmise that this is the reason for earlier reports of resistance of ovalocytes to malarial invasion in vitro. The initial loss of susceptibility to invasion with time is not accompanied by any change in membrane rigidity, but is primarily a consequence of a rapid decline in intracellular ATP concentration: this falls to below the threshold level required for invasion (approx. 0.1 mM) over a period in which the ATP in normal cells remains almost constant. Incubation in a metabolic regenerating medium leads to a rise in the intracellular ATP concentration and invasion by P. falciparum is recovered, though to a much lower extent than in normal cells. The resistance of ovalocytes to invasion becomes irreversible, due possibly to degradative processes in the membrane, on further storage. The developing parasites in ovalocytes have a reduced number of merozoites and show distinct morphological abnormalities.

Adenosine Triphosphate↗
PubMed1992

Rheological changes in the prodromal and established phases of sickle cell vaso-occlusive crisis.

A rheological study has been made in 20 patients with sickle cell anaemia in the steady state and in the prodromal and established phases of 12 vaso-occlusive crises. Rheology of sickle cells was studied by discontinuous density gradient fractionation and by filtration through pores of 5 microns diameter. The prodromal phase of crisis (day 1), when compared with mean steady state values, was associated with the development of a sub-population of poorly deformable dense cells. This sub-population appeared 1 or more days before the acute-phase rise in C-reactive protein, orosomucoid, fibrinogen, plasma viscosity and leucocytes, and before the rise in serum lactate dehydrogenase. As crisis evolved, the sub-population decreased to steady-state values, or below, by days 6-7. Identification of the prodromal phase of sickle cell crisis has allowed the detection of rheological changes of potential aetiological significance.

Acute-Phase Reaction↗
PubMed1992

Rheological properties of rosettes formed by red blood cells parasitized by Plasmodium falciparum.

A proportion of red blood cells parasitized by Plasmodium falciparum form rosettes with non-parasitized red cells. Although these rosettes are thought to impair microcirculatory flow, their rheological characteristics have not been fully described. Using dual-micropipette manipulation to pull apart individual rosettes, we found that the forces binding rosettes together were strong (average force for removal of a cell was 4.4 x 10(-10) N, approximately 5 times that required to detach a parasitized cell adhered to cultured endothelium). If disrupted rosettes were re-formed, cells rosetted immediately on contact, but the strength of attachment increased over minutes, and did not apparently reach its maximal level for hours. All non-parasitized cells tested could adhere to rosette-forming parasitized cells. Rosettes could withstand arterial flow stresses (1.4-1.6 Pa) for minutes without disintegration. To test the effects of rosetting on flow resistance, the time required for entry into a 4.3 microns pipette was measured. Entry times depended strongly on the number of cells in the rosette, and averaged 35 times longer than for non-parasitized cells. Our studies show that the cell-cell attachments within rosettes are strong, and suggest that rosettes might survive both the arterial circulation and passage through microvessels and could contribute to the ischaemic complications of falciparum malaria.

Animals↗
PubMed1992

Substituted benzaldehydes (12C79 and 589C80) that stabilize oxyhaemoglobin also protect sickle cells against calcium-mediated dehydration.

Reversibly sickled cells from patients with homozygous sickle-cell disease were prepared by Percoll-Isopaque density gradient separation and subjected to 15 h of cyclical deoxygenation-reoxygenation in the presence of Ca. After 15 h the sickle cells became dehydrated, losing volume secondary to K efflux via the Ca-activated (Gardos) channel, and showed impaired filterability through 5 microns diameter pores. The substituted benzaldehydes 12C79 and 589C80, which stabilize the oxy-conformation of sickle haemoglobin, showed an additional protective effect at pharmacological concentration by maintaining the K concentration, mean cell volume, and deformability of sickle cells. Drugs that increase the oxygen affinity of sickle haemoglobin may be more effective than specific inhibitors of Ca entry or K efflux in preserving the cation homeostasis and deformability of sickle cells during sickling in vivo.

Anemia, Sickle Cell↗
PubMed1992

Mode of action and comparative efficacy of pharmacological agents that inhibit calcium-dependent dehydration of sickle cells.

1. Selected Ca-channel antagonists were tested at 20 microM as inhibitors of Ca(2+)-uptake in human sickle red cells. Nitrendipine, fendiline, and bepridil (and its stereoisomers), were found to be as effective as methoxyverapamil (D-600) in inhibiting a fraction (25%) of Ca(2+)-uptake. In contrast cetiedil and Org 30701 were ineffective. 2. The drugs were subsequently tested as inhibitors of Ca(2+)-induced K+ efflux (Gardos) from sickle cells. They all showed inhibitory activity, with the order of efficacy nitrendipine greater than fendiline greater than bepridil greater than cetiedil greater than Org 30701. 3. With a 15 h programme of deoxygenation/reoxygenation cycles in a gas exchanger, it was shown that the inhibitors protected against cellular dehydration and loss of filterability in the order nitrendipine greater than fendiline greater than bepridil greater than cetiedil greater than Org 30701. However, significant stomatocytosis occurred at high concentrations of cetiedil, and bepridil (including its stereoisomers and analogues) impairing cell deformability. 4. It is concluded that Ca-antagonists may partially block both Ca(2+)-uptake and Ca(2+)-induced K+ efflux. The latter pathway is significant in contributing to sickle cell dehydration and nitrendipine is the most effective inhibitor of this route.

Anemia, Sickle Cell↗
PubMed1992

Subclinical ischaemic episodes during the steady state of sickle cell anaemia.

AIMS: To determine the clinical, haematological, biochemical and rheological changes that occur in the asymptomatic steady state of sickle cell anaemia. METHODS: Patient self-assessment visual analogue scores (for wellbeing and tiredness), the blood concentration of acute phase proteins (C-reactive protein, orosomucoid, and fibrinogen), and blood rheology (percentage of dense cells and the number of sickled cells that occluded pores 5 microns in diameter) were studied longitudinally on 10 occasions in each of 20 outpatients with sickle cell anaemia. RESULTS: Patients in the steady state showed fluctuation in visual analogue scores, in concentration of acute phase proteins, and in rheological parameters consistent with minor episodes of tissue injury. Significantly more variation in acute phase proteins occurred in the steady state of 14 of the 20 patients who developed one or more vaso-occlusive crises during the 16 month study period. Rheological fluctuation in the steady state simulated rheological change during crisis, namely a transient rise and then fall in the number of dense and poorly filterable cells. CONCLUSIONS: The term "steady state" is a misnomer, being characterised by biochemical and rheological fluctuation consistent with minor episodes of microvascular occlusion that are insufficient to cause the overt tissue infarction of painful crisis.

Acute-Phase Proteins↗
PubMed1991

Decreased rotational diffusion of band 3 in Melanesian ovalocytes from Papua, New Guinea.

Melanesian ovalcytes from Papua New Guinea have an N-terminal extension of the band 3 polypeptide (Jones, G.L., Edmunson, H.M., Wesche, D., Saul, A. 1990. Biochim. Biophys. Acta 1096:33-40). The ovalocytes showed a threefold increase in shear elastic modulus as determined by micropipette aspiration measurements of membrane rigidity. Time-resolved phosphorescence anisotropy has been used to study the rotational freedom of band 3 in membranes prepared from ovalocytes. The ovalocytic polymorphism was found to be associated with a marked decrease in the rotational mobility of band 3. This may indicate participation of band 3 in large homoaggregates or in complexes with other proteins at the cytoplasmic surface. There was no morphological clustering of band 3 detectable by immunofluorescence microscopy.

Anion Exchange Protein 1, Erythrocyte↗
PubMed1991

Blood rheology and ischaemia.

Ischaemia is essentially the failure of tissue to obtain a sufficient oxygen supply for its function. In the context of rheology, which is the study of the deformation and flow of materials, this implies failure to deliver the blood, rather than failure to oxygenate the blood or extract oxygen from it. Resistance to the delivery of blood is generally considered to have vascular and rheological components. Vascular effects on resistance may often be dominant, and there is wide appreciation of the ischaemic consequences of vascular obstruction and narrowing, for example in atherosclerotic disease. However, rheological factors can vary widely between individuals and in disease, and such variations have the potential to influence oxygen supply. Here, the rheological factors which affect blood flow are reviewed and their role in the development of ischaemia is discussed, with particular reference to the eye where possible.

Blood Viscosity↗