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Biomedical subjects

J C Giddings

Publications and source records attributed to J C Giddings.

At least 37 records · Page 2Linked to original sources
PubMed1998

Particle size analysis of dilute environmental colloids by flow field-flow fractionation using an opposed flow sample concentration technique.

An on-line concentration method has been developed for the analysis of dilute suspensions of macromolecules and particles by flow field-flow fractionation (FFF). This method, termed opposed flow sample concentration (OFSC), uses two opposing flowstreams to focus sample into a narrow band near the inlet of the flow FFF channel. Large sample volumes (up to 1 L) have been successfully loaded, concentrated, and separated in a 1 mL volume flow FFF channel. Concentration factors as high as 10(5) have been achieved. The effectiveness of OFSC flow FFF was demonstrated using mixtures of monodispersed standards such as proteins and polystyrene latex beads. Retention times, peak areas, and resolution are comparable for a 10 mL and a typical 10 microL injection volume. The applicability of the method to environmental studies was demonstrated by a separation of river water colloids.

Automation↗
PubMed1998

The antithrombotic effect of potent bifunctional thrombin inhibitors based on hirudin sequence, P551 and P532, on He-Ne laser-induced thrombosis in rat mesenteric microvessels.

The antithrombotic effect of potent synthetic bifunctional non-substrate type thrombin inhibitors based on hirudin sequences, P551 and P532, on Helium-Neon laser-induced thrombosis was investigated in rat mesenteric microvessels and compared with other types of thrombin inhibitors. P551 and P532, when given intravenously, inhibited platelet-rich thrombus formation in both arterioles and venules in a dose-dependent manner. The inhibitory effect was maximal immediately after intravenous administration and persisted for 20-30 minutes in both arterioles and venules. The minimal effective doses of P551 and P532 were 1.0 mg/ kg (intravenously) in both. However, the time course of the antithrombotic effect was not in keeping with the inhibitory effect measured by an activated partial thromboplastin time and was similar to other types of inhibitors in spite of different half-lives. The current findings show that P551 and P532 had significant inhibitory effects on platelet-rich thrombus formation and suggest that these bifunctional thrombin inhibitors could be potent antithrombotic agents.

Animals↗
PubMed1998

The role of platelet von Willebrand factor in the binding of factor VIII to activated platelets.

Factor VIII binds to activated platelets and contributes to the tenase complex assembled on the platelet membrane surface. We have examined the role of platelet von Willebrand factor in the binding of factor VIII to platelets using a platelet captured enzyme-linked immunosorbent assay. Purified factor VIII bound to activated normal platelets in a dose dependent manner. Factor VIII also bound to platelets obtained from a patient with Type 2N von Willebrand disease, although in this case the binding was reduced to approximately 50% of that seen with control platelets. Furthermore, factor VIII bound to Type 3 von Willebrand disease platelets in the absence of detectable von Willebrand factor. In this instance the binding reaction appeared to be approximately 30% of that seen with the same number of normal platelets. An anti-A3 domain monoclonal antibody, NMC-VIII/10, which recognizes the amino-terminal acidic region of the factor VIII light chain, and an anti-C2 domain monoclonal antibody, NMC-VIII/5, which also moderates the binding of factor VIII to phosphatidylserine, inhibited the association between factor VIII and platelets. Inhibition was more remarkable with NMC-VIII/5 than with NMC-VIII/ 10 but not complete. The findings suggest that the binding of factor VIII to activated platelets is not based on a single ligand-receptor relationship, although a predominant role exists for the platelet von Willebrand factor. Furthermore, both the amino-terminal acidic region of the A3 domain and the C2 domain participate in the binding of factor VIII to activated platelets.

Binding Sites↗
PubMed1997

Adjuvant effect of argatroban on staphylokinase induced thrombolysis of platelet rich thrombi in rat mesenteric venules in vivo.

Effective, therapeutic thrombolysis should not only promote dissolution of fibrin but should also regulate continued thrombin-induced fibrin formation and the accumulation of platelets on the thrombotic lesion. The aim of the present study was to assess the use of a synthetic, low molecular weight thrombin inhibitor, argatroban in association with a well defined thrombolytic agent in a reproducible animal model of thrombolysis in vivo. Thrombi were formed in rat mesenteric venules with a helium neon (He-Ne) laser in the presence of Evans blue and were stabilised for 10 minutes. Thrombi formed in this manner were shown by transmission electron microscopy to be composed mainly of platelets. Thrombolysis was induced with recombinant staphylokinase in the presence and absence of argatroban and the process was monitored using computerised image analysis. Co-infusion of argatroban at a dose of 2.0 mg/kg/h with staphylokinase significantly enhanced the rate of thrombolysis. The results suggested that administration of the thrombin inhibitor together with the fibrinolytic agent moderated platelet-dependent mechanisms and led to a more rapid restoration of blood vessel patency.

Animals↗
PubMed1997

The antithrombotic effect of synthetic low molecular weight human factor Xa inhibitor, DX-9065a, on He-Ne laser-induced thrombosis in rat mesenteric microvessels.

The effect of a synthetic low molecular weight factor Xa (FXa) inhibitor, DX9065a, on thrombosis in vivo were examined in a rat animal model using a Helium-Neon (He-Ne) laser method. DX-9065a administered either intravenously or orally promoted anti factor Xa activity in a dose dependent manner. Anti Xa activity was maximal immediately after intravenous injection and persisted for approximately 30 minutes. Inhibitory activity was maximal 15-30 minutes after oral administration and persisted for approximately 90 minutes. Similarly DX-9065a inhibited platelet-rich thrombosis formation in mesenteric arterioles and venules. In these instances inhibition was relatively transient after intravenous injection (10-20 minutes), but persisted for more than 3 hours after oral administration. The minimum effective doses of DX-9065a given intravenously and orally were 3.89 mg/kg and 25.9 mg/kg, respectively. The results confirmed that DX-9065a selectively modulates thrombotic mechanisms, and suggest that development of this synthetic FXa antagonist could constitute an effective intravenous and oral antithrombotic agent.

Animals↗
PubMed1997

Expression of von Willebrand factor, P-selectin (CD62P) and thrombomodulin in human endothelial cells in culture modulated by cyclosporin A.

Endothelial cells were isolated from human umbilical veins and from saphenous veins and were cultured in the presence and absence of cyclosporin A (CSA). Cell morphology and growth characteristics were monitored continuously by video recording microscopy. The cells and culture supernatants were analysed for von Willebrand Factor (VWF), P-Selectin (CD62P) and thrombomodulin (TM) by immunocytochemistry and immunoassay. CSA had little effect on confluent cells in culture. In contrast, the characteristics of both umbilical vein and saphenous vein cells were markedly altered when CSA was added to subconfluent cultures. Under these conditions, CSA did not appear to be directly cytotoxic at the concentrations used but marked changes in cell morphology including the appearance of large multinucleate forms were recorded. Increased amounts of VWF and TM were detected in the culture supernatants of cells grown continuously in the presence of CSA and correspondingly depressed expression of cell associated antigens was observed. Heterogeneous and depressed distribution of CD62P was seen after 5-6 days culture but cellular expression of this ligand did not appear to be affected specifically by the presence of CSA. In addition, mechanically disrupted confluent endothelial cells failed to recover in the presence of CSA as effectively as similar cells grown in the absence of CSA. The results showed that CSA disturbed actively growing cells more severely than quiescent cells and might help to clarify the role of CSA in vascular disturbances in vivo.

Cells, Cultured↗
PubMed1997

Redistribution of von Willebrand factor in porcine carotid arteries after balloon angioplasty.

von Willebrand factor (VWF) is a well-characterized multimeric glycoprotein present in platelets and plasma and synthesized by vascular endothelial cells and megakaryocytes. Its role in platelet-vessel wall interactions has been studied extensively, but its involvement in intravascular events after balloon angioplasty has not been clarified. VWF antigen is not present in porcine arterial endothelium (except for the pulmonary artery) but is readily detected in porcine venous endothelial cells. We have examined the localization of VWF in porcine vessel walls during neointima formation after bilateral carotid balloon-angioplasty. Endothelium was denuded by balloon injury but regenerated by 7 days and was fully confluent by 42 days. VWF was detected at the site of injury in localized, adherent platelet aggregates at 10 minutes after angioplasty that were not present at later time points. A well-demarcated homogeneous layer of VWF was observed on the luminal surface from 30 minutes to day 7, but there was a progressive shift of positive staining from the lumen to the outer media from days 1 to 7. VWF was also strongly detected at sites proximal and distal to the balloon injury from 30 minutes to day 7, although endothelial disruption was minimal and the monolayer remained substantially intact at these sites. Regrowing endothelial cells appeared to contain granular VWF from days 12 to 21, but this was not readily evident at later time points. The results suggest that balloon injury is associated with deposition and medial absorption of plasma or platelet VWF in this porcine model over a time period that precedes and overlaps vascular smooth muscle proliferation and endothelial recoverage. The findings provide evidence to support the concept of a wider role for VWF in tissue injury responses.

Angioplasty, Balloon↗
PubMed1997

The differential involvement of von Willebrand factor, fibrinogen and fibronectin in acute experimental thrombosis in rat cerebral and mesenteric microvessels.

Arterial thrombi are primarily composed of platelets. Platelets are bound to injured endothelial cells, sub-endothelial matrices, and other platelets by a range of adhesive proteins. Some of these reactions are governed by shear forces. The role of adhesive proteins in the pathogenesis of arterial thrombosis is not fully understood. The aim of this study was to examine the involvement of von Willebrand factor (vWF), fibrinogen (Fg), and fibronectin (FN) in the formation of microvascular thrombi in vivo using a helium-neon laser-induced thrombosis method. Transmission electron microscopy demonstrated that laser irradiation resulted in platelet-rich thrombosis in arterioles and venules, and revealed that this occurred in the absence of endothelial denudation. The mean wall shear rates in mesenteric arterioles and venules were 641 +/- 40 and 280 +/- 20 s-1, respectively. Shear rates increased approximately fivefold in arterioles and tenfold in venules during the formation of occlusive thrombi. Antibody to vWF inhibited thrombosis in arterioles and venules. Antibodies to Fg and FN inhibited thrombosis in venules but not in arterioles. These results confirm that vWF, Fg and FN were involved in thrombogenesis in vivo and demonstrated that significantly higher shear rates were required for the reactions involving vWF than those involving either Fg or FN.

Adhesiveness↗
PubMed1996

High-speed particle separation and steric inversion in thin flow field-flow fractionation channels.

The performance of thin (down to 71 microns thick) flow field-flow fractionation (flow FFF) channels was examined at both high channel flow-rates (up to approximately 47 ml/min) and cross flow-rates (up to approximately 11 ml/min). High levels of retention were observed, suggestive of good performance characteristics. Supporting this expectation, four sizes (0.04-0.30 microns) of polystyrene latex microbeads were baseline separated in under 3 min. Nonetheless, a plate height analysis showed that performance was still less than theoretically expected. Fast steric-hyperlayer separations of larger latex beads were observed in the same systems. Furthermore, it was shown that the steric inversion diameter was shifted down to approximately 0.23 micron thus expanding the size range to which this FFF mode is applicable. The steric inversion phenomenon observed using narrow latex standards was shown to be consistent with that found for a polydisperse polyvinylchloride latex.

Chemical Fractionation↗
PubMed1996

Measurement of anti-factor IX IgG subclasses in haemophilia B patients who developed inhibitors with episodes of allergic reactions to factor IX concentrates.

We have established a simple enzyme-linked immunosorbent assay (ELISA) for the detection of anti-factor IX IgG subclasses in haemophilia B patients with inhibitors. The assay was performed using immobilized purified factor IX. Specific IgG subclasses were detected by peroxidase-conjugated anti-human IgG1,2,3 and 4. Ten plasma samples from 6 haemophilia B patients with inhibitors ranging from 1.0 to 253 Bethesda Units/ml were analyzed. All samples were positive for IgG4. Six out of 10 samples were positive only for IgG4. Three samples were positive for IgG2. Five of the 6 patients had previously had allergic reactions to factor IX concentrates. Three patients had allergic episodes within the past month. Three samples from these latter patients taken on the day when the allergy had occurred showed positive also for IgG1. In later samples, however, taken at 4 days and 4 weeks respectively from two of these same patients. IgG1 was not detected. In two of the five patients in whom allergic reactions had occurred more than one month previously IgG1 was not detected. The results suggested that allergic reactions in patients with haemophilia B treated with factor IX concentrates were associated with the development of the specific IgG1 subclass of antibody to factor IX.

Adolescent↗
PubMed1996

Isolation and measurement of colloids in human plasma by membrane-selective flow field-flow fractionation: lipoproteins and pharmaceutical colloids.

Using a modified flow field-flow fractionation (flow FFF) technique termed membrane-selective flow FFF, a capability is developed to isolate various colloidal constituents that are naturally present in or may be introduced into blood plasma. Once isolated, the colloids may be subject to quantitative measurement to provide relative amounts of the different constituents and the size distribution curve of each. The potential of the technique has been demonstrated by isolating and measuring (a) the lipoprotein fractions found in human blood plasma, (b) liposyn II pharmaceutical emulsion added at 2 mg/mL of plasma, and (c) amphotericin B colloidal dispersion (a drug delivery agent) added to plasma at 0.5 mg/mL.

Chemical Fractionation↗
PubMed1996

Effects of NO-donors on thrombus formation and microcirculation in cerebral vessels of the rat.

Sodium nitroprusside (SNP) and 3-morpholinosydnonimine (SIN-1), are known to liberate nitric oxide (NO). In this study the effects of SNP and SIN-1 on thrombus formation in rat cerebral arterioles and venules in vivo were assessed using a helium-neon (He-Ne) laser. SNP infused at doses from 10 micrograms/kg/h significantly inhibited thrombus formation in a dose dependent manner. This inhibition of thrombus formation was suppressed by methylene blue. SIN-1 at a dose of 100 micrograms/kg/h also demonstrated a significant antithrombotic effect. Moreover, treatment with SNP increased vessel diameter in a dose dependent manner and enhanced the mean red cell velocity measured with a fiber-optic laser-Doppler anemometer microscope (FLDAM). Blood flow, calculated from the mean red cell velocity and vessel diameters was increased significantly during infusion. In contrast, mean wall shear rates in the arterioles and venules were not changed by SNP infusion. The results indicated that SNP and SIN-1 possessed potent antithrombotic activities, whilst SNP increased cerebral blood flow without changing wall shear rate. The findings suggest that the NO released by SNP and SIN-1 may be beneficial for the treatment and protection of cerebral infarction.

Animals↗
PubMed1995

Sample dimensionality: a predictor of order-disorder in component peak distribution in multidimensional separation.

While the use of multiple dimensions in separation systems can create very high peak capacities, the effectiveness of the enhanced peak capacity in resolving large numbers of components depends strongly on whether the distribution of component peaks is ordered or disordered. Peak overlap is common in disordered distributions, even with a very high peak capacity. It is therefore of great importance to understand the origin of peak order/disorder in multidimensional separations and to address the question of whether any control can be exerted over observed levels of order and disorder and thus separation efficacy. It is postulated here that the underlying difference between ordered and disordered distributions of component peaks in separation systems is related to sample complexity as measured by a newly defined parameter, the sample dimensionality s, and by the derivative dimensionality s'. It is concluded that the type and degree of order and disorder is determined by the relationship of s (or s') to the dimensionality n of the separation system employed. Thus for some relatively simple samples (defined as having small s values), increased order and a consequent enhancement of resolution can be realized by increasing n. The resolution enhancement is in addition to the normal gain in resolving power resulting from the increased peak capacity of multidimensional systems. However, for other samples (having even smaller s values), an increase in n provides no additional benefit in enhancing component separability.

Chromatography↗
PubMed1994

Theory of field-programmed field-flow fractionation with corrections for steric effects.

This paper deals with the principal perturbation to ideal normal-mode elution of particles in field-flow fractionation (FFF). This perturbation is due to the finite size of particles undergoing migration in the FFF channel. The effects of a first-order correction for particle size are examined. Equations are derived for retention time, fractionating power, and steric inversion diameter for operation at constant field strength, as well as under conditions of both exponential and power programmed field decay. Useful limiting equations for fractionating power are derived and their validity is confirmed for typical experimental conditions. The derived equations are necessary for the future development of a systematic optimization strategy for the selection of operating conditions for particle size analysis by FFF. Calculations confirm our previous conclusion that the fractionating power for exponential field programming varies strongly with particle size; this variation is only slightly reduced by steric perturbations. The uniform fractionating power of power programming is slightly disturbed by steric effects although fractionating power remains much more uniform than for exponential programming. It is shown that a higher uniformity in fractionating power can be gained by manipulating the parameters of power programming but that no improvement is possible with exponential programming. Phenomena giving rise to higher order perturbations and to secondary relaxation are discussed and the conditions identified under which these effects are minimized.

Chemical Fractionation↗