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Biomedical subjects

N Crawford

Publications and source records attributed to N Crawford.

At least 19 recordsLinked to original sources
PubMed1997

Local delivery of platelets with encapsulated iloprost to balloon injured pig carotid arteries: effect on platelet deposition and neointima formation.

Local delivery of a drug to the arterial wall during angioplasty is an approach which might reduce the incidence of occlusive events such as thrombosis and restenosis, without the risk of systemic side effects. By exploiting their natural primary haemostatic properties, platelets, with encapsulated drugs, can be targeted to a vessel wall injury site and act as a depot for sustained release. The platelet plasma membrane can be reversibly permeabilised by high voltage, short duration electrical pulses (electroporation). Drugs will diffuse into porated platelets and become trapped on resealing. We have studied the effects of autologous platelets, electroloaded with the stable prostacyclin analogue, iloprost on platelet deposition and neointima formation in a pig carotid angioplasty model. Iloprost loaded or control platelets were delivered locally and immediately to the balloon injured site using a double balloon delivery catheter. Acute platelet deposition was measured using 111-Indium, and neointima formation at 21 days post angioplasty was assessed by morphometric analysis. In pigs treated with iloprost loaded platelets, platelet deposition on the artery at 2 hours post injury was dramatically reduced (to approximately monolayer coverage), when compared with arteries from pigs treated with control platelets. In pigs with deeply injured arteries, i.e. with extensively ruptured internal elastic lamina (IEL), platelet deposition was reduced by 88% compared with control arteries (118 +/- 20 x 10(6)/cm vs. 14 +/- 2 x 10(6)/cm, means +/- SI, 2P < 0.001). In minimally injured arteries (IEL intact) a 65% reduction in platelet deposition was observed (55 +/- 24 x 10(6)/cm vs. 19 +/- 3 x 10(6)/cm. 2P < 0.002). A high concentration of free iloprost, delivered to the angioplasty site, with control platelets, had far less effect on platelet deposition, substantiating the advantage of platelet encapsulation. At 21 days post injury, morphometry of the carotid arteries after treatment with iloprost loaded platelets showed significant reductions in intimal area and intimal/medial ratios in minimally injured vessels (P < 0.05) as compared with vessels from pigs treated with control platelets. With deeply injured vessels, the mean differences (control vs. treated) for the same morphometric parameters were not significant. This novel approach of electro-encapsulating drugs within autologous platelets, and using them as highly biocompatible and biodegradable drug targeting vehicles might, with the appropriate choice of encapsulated agent, have potential for reducing the incidence of occlusion after angioplasty and thrombolysis procedures.

Animals↗
PubMed1995

Differences in oxidative response of subpopulations of neutrophils from healthy subjects and patients with rheumatoid arthritis.

OBJECTIVES: To determine whether blood neutrophils from healthy individuals and blood and synovial fluid neutrophils from patients with rheumatoid arthritis (RA) responded differently to priming agonists and stimuli of the oxidative burst and, if so, whether this was a property of a subpopulation of neutrophils. METHODS: Continuous flow electrophoresis was used to separate neutrophils into subpopulations based upon quantitative differences in net negative surface charge. The generation of superoxide anion (O2-) was used as a measure of oxidative activity using 10(-7) mol/l N-formyl-methionylleucyl-phenylalanine (FMLP) as the stimulating agonist and 10(-8) mol/l platelet activating factor (PAF) as the priming agent. RESULTS: The production of O2- by blood and synovial fluid neutrophils from RA patients in response to FMLP was greater than that observed with control blood neutrophils (p < 0.001). Priming of normal blood neutrophils with PAF increased their FMLP induced oxidative burst (p < 0.001), but PAF treatment had no effect on rheumatoid neutrophils. Neutrophils from synovial fluid of RA patients were less electronegative than paired blood samples and exposure of blood neutrophils to FMLP but not PAF reduced their surface charge. Continuous flow electrophoresis isolated three neutrophil subpopulations: cells of least surface electronegativity were ascribed to pool P1 and cells of greatest surface electro-negativity to P3. Normal blood neutrophils from P3, but not P1, showed increased oxidative activity after PAF priming (twofold increase; p < 0.01), whereas the responsiveness of rheumatoid blood and synovial fluid neutrophils from P1 and P3 was not modified by PAF treatment under the same conditions. CONCLUSION: It is suggested that most of the circulating neutrophils in RA are already in a state of readiness to generate O2- upon activation by an inflammatory stimulus. This is in contrast to normal blood neutrophils, which have both responsive and non-responsive subpopulations with respect to priming agonists.

Arthritis, Rheumatoid↗
PubMed1995

Targeting platelets containing electro-encapsulated iloprost to balloon injured aorta in rats.

Drugs can be electro-encapsulated within platelets and targeted to damaged blood vessels by exploiting the platelet's natural haemostatic properties to adhere to collagen and other vessel wall constituents revealed by injury. A rat aorta balloon angioplasty model has been used to study the effect on platelet deposition of giving iloprost loaded platelets i.v. during the balloon injury. After labelling the circulating platelets with 111-Indium before balloon injury, time course studies showed maximum platelet deposition on the injured aorta occurred at about 1 h post-injury and the deposition remained stable over the next 2-3 h. When iloprost-loaded platelets were given i.v. during injury and the circulating platelet pool labelled with 111-Indium 30 min later, platelet deposition, measured at 2 h postinjury, was substantially and significantly reduced compared with control platelet treatment. Some anti-proliferative effects of iloprost-loaded platelets given i.v. during injury have also been observed. Whereas the incorporation of [3H]-thymidine into aorta intima-media DNA at 3 days post injury was 62-fold higher in balloon injured rats than in control sham operated rats, thymidine incorporation into intima/media of rats which had received iloprost loaded platelets during injury was reduced as compared with rats subjected only to the injury procedure. The reduction was only of near significance, however, but at 14 days after injury the total DNA content of the aorta intima/media of rats given iloprost loaded platelets during injury was significantly reduced. Although iloprost loaded platelets can clearly inhibit excessive platelet deposition, other encapsulated agents may have greater anti-proliferative effects.(ABSTRACT TRUNCATED AT 250 WORDS)

Angioplasty, Balloon↗
PubMed1994

Characterization of the distal alpha-fetoprotein enhancer, a strong, long distance, liver-specific activator.

High level expression of the alpha-fetoprotein (AFP) gene is controlled by three upstream enhancers which function even in hepatic cell lines that repress the AFP gene promoter. The most distal ("Complex 3," at -6 kilobases) is the strongest in HepG2 cells. We mapped the main activity of Complex 3 to a 170-base pair (BP) region from -6069 to -5900; progressive deletion of the 5'- and 3'-ends identified an 84-bp segment which accounted for 90% of enhancer activity. Expression studies, which combined the deleted Complex 3 with an AFP or tk promoter chloramphenicol acetyltransferase gene fusion, resolved five regions in the enhancer (Ia, Ib, II, III, and IV). Deletion of Regions Ia or II strongly reduced stimulation of the AFP promoter, while Regions Ia and Ib were essential for stimulation of the tk promoter. Footprinting indicated multiple binding sites in regions Ia, Ib, and II. Gel shift and oligonucleotide competition demonstrated that Regions Ia and II had high affinity HNF3- and C/EBP-binding sites, respectively, while additional unidentified factors bound throughout Regions I-III. Complex 3 is a powerful liver-specific transcriptional regulator and an important model of long distance gene activation.

Base Sequence↗
PubMed1994

Electrokinetic properties of human cryopreserved platelets.

A method for the cryopreservation of human platelets with glycerol/glucose is described which was a simplified modification of the method of Dayian and Pert (1979). The effect of cryoinjury of the platelet surface membrane was investigated by studying the surface electrokinetic properties of the platelet. A significant increase in platelet electrophoretic mobility was found after cryopreservation. The fresh platelets had a mean electrophoretic mobility of 1.04 +/- 0.05 microns s-1 V-1 cm-1 and cryopreserved platelets 1.18 +/- 0.05 microns s-1 V-1 cm-1, P < 0.05. However, the total platelet sialic acid of fresh platelets was 62.5 +/- 5.6 nmol 10(-9) platelets compared to 47.2 +/- 4.6 nmol 10(-9) platelets after cryopreservation, P < 0.001. Similarly, the neuraminidase-labile sialic acid was 26.4 +/- 4.3 nmol 10(-9) platelets for fresh platelets and 17.6 +/- 4.0 nmol 10(-9) platelets after cryopreservation, P < 0.001. Using polyacrylamide gel electrophoresis with Western blotting, we showed a reduction in the platelet glycoprotein Gp Ib after cryopreservation, this was confirmed by using crossed immunoelectrophoresis. Electron microscopy revealed a significant change in platelet morphology after the cryopreservation procedure with disruption of the platelet membrane and also platelet shape change. These features may explain the changes in platelet electrokinetic properties.

Blood Platelets↗
PubMed1994

Inhibition of platelet recruitment to arterial lesions by predeposition of platelets containing encapsulated iloprost.

Drugs can be encapsulated within blood platelets by reversible electroporation and can be haemostatically targeted to vessel wall injury sites. Initial studies with iloprost-loaded pig platelets and pig aorta tunica media in perfusion circuits are presented. After autologous reconstitution into blood, no significant difference was observed in the deposition of 111Indium labelled sham-loaded and untreated platelets onto the tunica media during perfusion under low and high shear conditions. In paired experiments (n = 10 pairs), the deposition of iloprost-loaded platelets was significantly lower (mean 61%) after 5 min perfusion than the deposition from blood containing sham-loaded (control) platelets. A similar significant reduction (mean 54%) was seen after 10 min perfusion. Pre-perfusion of iloprost-loaded platelets for 10 min under low shear conditions (212/s), followed by 5 min perfusion of 111Indium labelled normal platelets, significantly reduced the secondary platelet deposition (p < 0.01) when compared with the deposition seen when control untreated platelets were preperfused. Significant differences (p < 0.001) in secondary deposition were also observed when primary and secondary platelet perfusions were made under high shear (1690/s). Histology of the tunica media segments post perfusion, supported the inhibitory effect of predeposited iloprost-loaded platelets on secondary platelet recruitment. By exploiting their natural haemostatic propensity, drug-loaded platelets can be targeted to vessel wall injury sites. Appropriate drugs could be packaged that may passivate the carrier platelets at the lesion inhibiting thrombus formation or they may act as a depot for sustained drug release.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

A promoter-linked coupling region required for stimulation of alpha-fetoprotein transcription by distant enhancers.

We previously demonstrated that the rat alpha-fetoprotein (AFP) gene has three upstream enhancers that stimulate the AFP promoter additively in HepG2 cells (1). In this paper, deletion analysis demonstrated that a promoter-linked segment from -178 to -155 was required for full activity when the enhancers were distant from the promoter, even at less than their normal genomic distances, but dispensable when the enhancers were moved close to the promoter. This 'promoter-coupling element' appears to interact simultaneously with all three enhancers. Deletion analysis also localized a transcription stimulatory and a negative region in the promoter. Though these latter regions controlled the strength of the isolated promoter, they did not affect 'coupling' to the distant enhancers, and transcription stimulation by these distal promoter elements was small compared to the distant enhancers. Overall, the distant enhancers, acting through the promoter-coupling element, accounted for 70% of the activity of the transfected AFP gene. Footprint analysis with HepG2 nuclear extracts demonstrated protein binding at two sites near the promoter-coupling element. The data indicate a positive transcription control mechanism by which distant enhancers stimulate the AFP promoter through a specific promoter-linked element.

Animals↗
PubMed1993

Alpha 2-adrenoreceptor status of human platelet subpopulations separated by continuous flow electrophoresis.

Human platelets were separated into subpopulations using continuous flow electrophoresis, which uses electrophoretic mobility as the separative parameter. The platelets with the greatest electrophoretic mobility showed higher amounts of total sialic acid than the less electrophoretically mobile subpopulation (74.3 +/- 18.2 nmol/10(9) platelets vs. 49.2 +/- 20.1 nmol/10(9) platelets, p < 0.05 paired Student's t-test). Furthermore, neuraminidase-labile sialic acid was also elevated in the more electrophoretically mobile platelet subpopulation (29.1 +/- 6.0 nmol/10(9) platelets vs. 21.8 +/- 10.4 nmol/10(9) platelets, p < 0.05 paired Student's t-test). We also found that the sialic acid enriched platelet subpopulation had more alpha 2-adrenoreceptors than the less electrophoretically mobile platelets (457 +/- 104 vs. 302 +/- 164 receptors per cell, p < 0.05 paired Student's t-test).

Blood Platelets↗
PubMed1993

Cytoskeletal interactions of Rap1b in platelets.

We have presented evidence that rap1b, a 22 kDa low molecular weight GTP binding protein, becomes associated with the cytoskeleton in thrombin-activated platelets. The initial incorporation is very rapid and occurs as fast as we can measure it. Thus, some rap1b is associated with the cytoskeleton as fast as it is formed. The remainder of the rap1b is incorporated more slowly. This biphasic incorporation of rap1b is similar to the incorporation of GPIIb/IIIa into the cytoskeleton, but no interaction between GPIIb/IIIa and rap1b could be demonstrated. Phosphorylation of rap1b by cAMP-dependent protein kinase did not inhibit its association with the cytoskeleton. We conclude that rap1b is one of an increasing number of proteins that associate with the cytoskeleton during cell activation. The function of rap1b in the cytoskeleton is unclear at this time. However, it is possible to speculate on potential roles. There is growing evidence that low molecular weight G proteins participate in the formation of multi-molecular aggregates. For example, p21rac promotes the assembly of a membrane-associated complex composed of NADPH oxidase, p47, and p67 and this complex is important for activation of NADPH oxidase in neutrophils. Similarly, in yeast, BUD1, a homolog of rap1, forms a complex with BUD5 (a homolog of GDI), BEMI, CDC24, and CDC42 (a homolog of G25K). This multi-protein aggregate may be important in cytoskeletal structure in yeast. In platelets, rad1b, which is membrane associated, may promote the assembly of a complex of proteins during cell activation and may localize this complex to the plasma membrane.(ABSTRACT TRUNCATED AT 250 WORDS)

Blood Platelets↗
PubMed1992

Effect of platelet encapsulated Iloprost on platelet aggregation and adhesion to collagen and injured blood vessels in vitro.

A novel approach to site-directed delivery of drugs in vivo using blood platelets as carrier vehicles is being investigated. In this context some initial studies are reported on the effect of platelet encapsulated anti-platelet drugs on platelet aggregation and adhesion to fibrillar collagen and injured arteries in vitro. The stable prostacyclin analogue Iloprost has been encapsulated within human and pig platelets by high voltage electroporation (Hughes and Crawford 1989 and 1990). After resealing the platelets, the packaged drug has a negligible effect upon platelet adhesion to a surface of fibrillar collagen or to damaged aorta (stripped to the tunica media to simulate deep injury). The rate of platelet recruitment to the collagen shows no dose dependency with respect to intracellular Iloprost concentrations. After high Iloprost loading, as few as 2% drug loaded platelets in a mixture with control (sham encapsulated) platelets, inhibit agonist-induced platelet aggregation > 50%. The prior deposition of a "lawn" of Iloprost-loaded platelets onto fibrillar collagen or damaged aorta has a substantial inhibitory effect (50-70%) upon the secondary recruitment of normal platelets compared with recruitment to a "lawn" of normal platelets. This inhibition of secondary recruitment occurs even in the presence of a platelet activator. If reduction of platelet recruitment to a vessel wall lesion results in a decrease in the local concentration of platelet granule-derived smooth muscle cell chemotactic and proliferative factors, this site-directed drug delivery may well have application for the prevention of restenosis following balloon angioplasty procedures.

Animals↗
PubMed1992

Heterogeneity in the circulating neutrophil pool: studies on subpopulations separated by continuous flow electrophoresis.

Isolated blood neutrophils from healthy individuals have been separated by continuous flow electrophoresis (CFE) as a Gaussian-shaped profile extending over 12-15 fractions, on the basis of differences in cell surface electrical charge. The fractions were pooled into three or four subpopulations; the mean electrophoretic mobilities of the least and most electronegative cells were 0.96 and 1.22 microns/sec/V/cm, respectively. Each pool of neutrophils was analyzed for functional and biochemical differences. Expression of respiratory burst in terms of the rate of superoxide production by the least and most electronegative cells to fixed concentrations of N-formyl-methionyl-leucyl-phenylalanine (fMLP, 10(-7) M) or phorbol myristate acetate (PMA, 1 microgram/ml) revealed that the least electronegative cells generated superoxide anion (O2-) at approximately twice the rate of the most electronegative cells. However, when lower concentrations of fMLP were used (1-5 x 10(-9) M), the most electronegative cells were most active. The least electronegative cells were also the most active in terms of phagocytosis and chemotaxis. In accordance with these differences in motile function, the basal F-actin content of the least electronegative cell pool was greater than the F-actin levels found in the most electronegative cells and remained so upon stimulation with fMLP. Such neutrophil heterogeneity as detected by CFE may be important in selective margination and recruitment of cells to inflammatory foci and sites of infection and may in part represent subsets of cells within the circulation that are primed in vivo to respond to inflammatory stimuli.

Actins↗
PubMed1992

Electrokinetic behaviour and surface sialic acid status of blood platelets in essential thrombocythaemia (ET).

We have previously reported, for platelets from patients with idiopathic thrombocytopenic purpura, that there was in increased electrophoretic mobility that was related to their increased sialic acid content. Heterogeneity in the circulating platelet pool of patients with essential thrombocythaemia (platelet counts 600-1000 x 10(9)/l) has also been investigated by preparative continuous flow electrophoresis (CFE). The surface charge-dependent separation profiles of all the patients' platelets showed marked anodal shifts compared with the profiles of platelets from age- and sex-matched control subjects separated under identical conditions. The increase above normal in the net electronegativity of the patients' platelets could not be accounted for by differences in surface neuraminidase-labile or total platelet sialic acid content.

Blood Platelets↗
PubMed1992

Fractionation of human neutrophils into subpopulations by countercurrent distribution: surface charge and functional heterogeneity.

Isolated blood neutrophils from normal healthy subjects were separated into fractions by sequential countercurrent distribution (CCD) in a charge-sensitive dextran/polyethylene glycol aqueous phase system. The neutrophils separated as a broad profile, and in a charged phase procedure the separation was based upon differences in cell surface electrokinetic properties, as confirmed by electrophoretic mobility measurements of fractions across the profile using analytical cytopherometry. The CCD cell fractions were generally pooled as three or four major subfractions for analysis of functional and metabolic differences. These included measurements of chemotaxis, phagocytosis, and respiratory burst. An inverse relationship was found between the electrophoretic mobility (EPM) of the subfraction pools and their functional competence, with the less electronegative cell fraction pools often as much as 2 to 3-fold more active than the more electronegative pools. This demonstration of electrokinetic and functional heterogeneity in 'resting' neutrophil subpopulations separated by CCD may reflect changes during their sojourn in the circulation that determine selective margination and recruitment of cells to inflammatory foci and sites of infection.

Cell Separation↗
PubMed1991

Priming action of inositol hexakisphosphate (InsP6) on the stimulated respiratory burst in human neutrophils.

After priming by a number of different host, bacterial and chemical agents, human neutrophils may be stimulated to produce a greater respiratory burst than would be elicited by the stimulus alone. Other neutrophil functions may be similarly enhanced by pre-exposure to a priming agent. We describe here a new extracellular role for inositol hexakisphosphate (InsP6) as a priming agent for a variety of human neutrophil functional responses. Preincubation of the cells with InsP6 alone (up to 250 microM) has no stimulatory effect upon the basal production of reactive oxygen intermediates but the response to a subsequent stimulus (FMLP, PMA or phagocytic particles) is substantially enhanced. Levels 100-200% higher than 'stimulus only' controls have been recorded. Peak enhancement of the FMLP-induced oxidative response occurs after 1-2 min preincubation with InsP6 and the effect is dose-dependent (maximum at approx. 100 microM InsP6). As others have shown FMLP stimulation of superoxide anion production has no external Ca2+ dependence but the presence of low levels of Ca2+ and Mg2+ (0.1 mM) during priming appears to be an essential requirement for full expression. Reports of intracellular concentrations of InsP6 in mammalian cells in the 30-100 microM range suggest that the local release of this inositol polyphosphate from damaged or effect cells could have a physiologically important modulatory role on neutrophil functions.

Calcium↗
PubMed1991

Dynamic changes in neutrophil cytoskeleton during priming and subsequent surface stimulated functions.

The naturally occurring inositol polyphosphate, inositol hexakisphosphate (InsP6, phytic acid) primes human neutrophils for enhanced responses to stimulatory agonists. Unlike other primers InsP6 has no effect at high doses (up to 500 microM) on the neutrophil's basal rate of superoxide production (O2-). Pretreatment of the cells for 2 min with InsP6 (100 microM) substantially enhances fMet-Leu-Phe (FMLP) (10(-7) M)-induced respiratory burst. In investigations of the dynamic aspect of neutrophil actin during priming and stimulation with FMLP the content of F-actin has been measured with and NBD (nitrobenz-2-oxa-1,3-diazole)-phallacidin assay. The induced assembly of F-actin is rapid (peaks at approximately 45 s) followed by depolymerization. In contrast, priming with InsP6, with or without FMLP stimulation, results in a sustained assembly of F-actin as visualized with a rhodamine-phalloidin staining technique. These changes in macromolecular status and distribution of F-actin during InsP6 priming occur in the absence of any other demonstrable functional responses and thus InsP6 may be a useful tool to follow early events in neutrophil functions or to monitor the presence of unprimed and preprimed subpopulations in the circulation. Since InsP6 is present in high concentrations in neutrophils (up to 100 microM), its release from damaged or effete cells may have a modulatory role on neutrophil functions.

Actins↗