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Biomedical subjects

G F Savidge

Publications and source records attributed to G F Savidge.

At least 37 records · Page 2Linked to original sources
PubMed1999

Immune tolerance therapy for haemophilia A patients with acquired factor VIII alloantibodies: comprehensive analysis of experience at a single institution.

Immune Tolerance Therapy for Haemophilia A Patients with Acquired Factor VIII Alloantibodies: Comprehensive Analysis of Experience at a Single Institution Eleven children with severe haemophilia A associated with the IVS 22 inversion and acquired high titre neutralising antibodies to factor VIII underwent immune tolerance induction. HLA class I and high resolution class II type is detailed for each patient. A three phase approach to immune tolerance induction was used. During phase 1, which lasted a median of six weeks, patients received factor VIII 100 IU/kg twice daily. Phase 2 comprised a factor VIII dose reduction to 100 IU/kg once daily, and continued for a median duration of 14 weeks. Subsequently 10 of the 11 patients satisfied the criteria of absent factor VIII neutralising activity by the Bethesda method, and a factor VIII elimination half life of greater than 5 h, allowing progression to phase 3, a further factor VIII dose reduction to 50 IU/kg three times weekly. A model for dose reduction as factor VIII tolerance evolves, based on pharmacokinetic analysis, is described.

Child, Preschool↗
PubMed1998

Modulation of RGD sequence motifs regulates disintegrin recognition of alphaIIb beta3 and alpha5 beta1 integrin complexes. Replacement of elegantin alanine-50 with proline, N-terminal to the RGD sequence, diminishes recognition of the alpha5 beta1 complex with restoration induced by Mn2+ cation.

Several recent studies have demonstrated that the amino acid residues flanking the RGD sequence of high-affinity ligands modulate their specificity of interaction with integrin complexes. The present study has addressed the role of the residues flanking the RGD sequence in regulating the recognition by disintegrin of the alphaIIb beta3 and alpha5beta1 complexes by construction of a panel of recombinant molecules of Elegantin (the platelet aggregation inhibitor from the venom of Trimerasurus elegans) expressing specific RGD sequence motifs. Wild-type Elegantin (ARGDNP) and several variants including Eleg. AM (ARGDMP), Eleg. PM (PRGDMP) and Eleg. PN (PRGDNP) were expressed as glutathione S-transferase (GST) fusion proteins in Escherichia coli. The inhibitory efficacies of the panel of Elegantin variants were analysed in platelet adhesion assays with substrates immobilized with fibrinogen and fibronectin. Elegantin molecules containing an Ala residue N-terminal to the RGD sequence (wild-type Elegantin and Eleg. AM) showed strong inhibitory activity towards alphaIIbbeta3-dependent platelet adhesion on fibronectin, whereas a Pro residue in this position (Eleg. PM and Kistrin, the inhibitor from the venom of Calloselasma rhodostoma) engendered lower activity. The decreased activity could not be attributed to a decrease in the affinity of the disintegrin for the alphaIIb beta3 complex because both Eleg. AM and Eleg. PM had similar Kd (app) values. In contrast, Elegantin molecules into which a Met residue was introduced in place of the Asn residue C-terminal to the RGD sequence showed 10-13-fold elevated inhibitory activity towards platelet adhesion on fibrinogen and this was maintained with either a Pro or Ala residue N-terminal to the RGD sequence. In experiments with the alpha5 beta1 complex on K562 cells, the inhibitory efficacies of the panel of Elegantin molecules were analysed under two different cation conditions. First, in the presence of Ca2+/Mg2+, K562 cell adhesion on fibronectin was inhibited equally well by Elegantin and Eleg. AM but inhibited poorly by Eleg. PM and Kistrin. In contrast with platelets, the decreased inhibitory efficacy of the PRGDMP disintegrins was due to poor recognition of the alpha5 beta1 complex. In the presence of Mn2+ cation, K562 cell adhesion on fibrinogen was observed in an alpha5 beta1-dependent manner. Under these conditions both PRGD and ARGD containing disintegrins were strong inhibitors of K562 cell adhesion on fibrinogen and this was due to a markedly improved recognition of the alpha5 beta1 complex by the PRGD molecules. These observations demonstrate the pivotal role of the amino acids flanking the RGD sequence for disintegrin recognition of integrin complexes and highlight the subtle nature by which integrin-ligand binding specificity can be modulated by both cation and adhesive motif.

Amino Acid Sequence↗
PubMed1998

Flow cytometric analysis of reticulated platelets: evidence for a large proportion of non-specific labelling of dense granules by fluorescent dyes.

The labelling of platelets with thiazole orange (TO) has been utilized by various laboratories to determine the percentage of reticulated platelets within whole blood or platelet-rich plasma (PRP). A proportion of TO labelling, however, is not entirely mRNA specific and remains to be fully defined. Almost half of the total TO-positive signal within normal platelets (n = 5) was shown to be abrogated upon degranulation with 80 microM thrombin receptor activating peptide (TRAP) (P = 0.006), strongly suggesting that platelet granules are non-specifically labelling with dye. We have confirmed this hypothesis by studying TO labelling of platelets within whole blood from dense granule deficient patients, e.g. Hermansky-Pudlak syndrome (HPS) (n = 5) and storage pool disease (SPD) (n = 4). The levels of TO-positive platelets were found to be significantly lower than normal (P = 0.0003 and P = 0.0002 respectively), but not significantly different from TRAP degranulated platelets. Upon degranulation of HPS and SPD platelets there was very little further reduction in the TO signal. Incubation of normals and SPD whole blood with different concentrations of either TO or coriphosphine-O confirmed that dense granules were non-specifically labelling even at high concentrations of both dyes. These findings suggest that although TO labelling is in part RNA specific, the dense granular pool of nucleotides appears to cause a substantial amount (approximately 50%) of non-specific labelling observed under these conditions of assay. This can easily be controlled for by a degranulation step with a non-enzymatic platelet agonist such as TRAP, and may have important consequences for the eventual standardization. clinical utilization and automation of reticulated platelet assays.

Albinism, Oculocutaneous↗
PubMed1998

Acquired haemophilia A: errors in the diagnosis.

The distinction between a specific factor inactivator and a non-specific inhibitor is important when confronted by a patient with a history of bleeding and abnormal in-vitro coagulation tests. We report on two patients who presented with bleeding and a prolonged activated partial thromboplastin time. Initial factor assays suggested combined deficiency of factors VIII and IX as a result of the presence of inactivators. The use of dilution studies, chromogenic assays, a novel in-house enzyme-linked-immunosorbent-assay-based technique and phospholipid neutralization, demonstrated that Case 1 had a genuine factor VIII inactivator resulting in factor VIII levels of less than 1 IU/dl but no factor IX deficiency. Case 2 had normal levels of factor VIII on further testing and no specific inactivator to either factor VIII or IX but a potent antiphospholipid antibody which had interfered with the phospholipid-dependent in-vitro assays. Care must be taken in the interpretation of laboratory assays in the presence of antiphospholipid antibodies to ensure that the correct diagnosis is made and inappropriate treatment avoided.

Adult↗
PubMed1997

Megakaryocytes and platelets in alpha-granule disorders.

This chapter summarizes research data contributing to current understanding of disorders affecting alpha-granules of megakaryocytes and platelets. Diagnostic features of the gray platelet syndrome are well defined. Combined evidence suggests a defect, specific to the megakaryocyte cell lineage, causing a cytoskeletal abnormality and defective targeting of endogenously synthesized proteins to the alpha-granule. The abnormalities linked by signal transduction pathways. von Willebrand disease and afibrinogenaemia are disorders which highlight the functional importance of platelet storage pools of von Willebrand factor and fibrinogen, essential ligands in the process of adhesion and aggregation. The abnormality in the factor V Quebec disorder leads to a degradation of most proteins contained within the alpha-granule. The familial platelet disorder Paris-Trousseau thrombocytopenia is the only alpha-granule disorder associated with a cytogenetic abnormality, and it presents a useful model for exploring the genetic influence on regulation of thrombopoiesis. Study of these syndromes has elucidated aspects of the physiology of normal megakaryocyte maturation and platelet formation, including storage organelle biosynthesis.

Afibrinogenemia↗
PubMed1997

Continuous infusion therapy with very high purity von Willebrand factor concentrate in patients with severe von Willebrand disease.

Five patients with severe (type III) von Willebrand disease received, by continuous infusion, a solvent-detergent treated von Willebrand factor high purity concentrate to control haemostasis. The clinical indications for treatment included prophylaxis prior to orthopaedic, abdominal and tympanic membrane surgery, and treatment of epistaxis and trauma-related bleeding. Plasma vWf antigen and activity were normalized sooner than factor VIII:C levels after initial bolus followed by infusion of the concentrate. Haemostasis was established in all five patients. The degree of shortening of the bleeding time correlated with concentrate infusion rate and, therefore, with administered dose of high molecular weight multimers. Concentrate clearance decreased over time with continued infusion. The product was shown to be stable and sterile at 24 h after reconstitution with no evidence of neoantigen expression. This report illustrates the effectiveness of high purity vWf concentrate administered by continuous infusion and shows that high-molecular-weight multimers are required to shorten the bleeding time to within the normal range.

Adolescent↗
PubMed1996

NovoSeven (recombinant factor VIIa) in centeral nervous systems bleeds.

Data is presented from two compassionate-use clinical trials using recombinant factor VIIa (rFVIIa) to treat central nervous system haemorrhage in 18 haemophilia A and B patients with inhibitors, and in 3 patients with FVII deficiency. Prior to rFVIIa treatment 78% of the haemophilia patients had inhibitor titres greater than 10 Bethesda units/ml. Sixty-two percent of the bleeding episodes were treated with a mean dose of 80-100 mu g/kg of rFVIIa administered repeatedly until cessation of bleeding. The overall efficacy was 84% with only one fatality and there were no major adverse events or laboratory indicators of disseminated intravascular coagulation.

Adolescent↗
PubMed1996

Continuous infusion of factor VIII for surgery and major bleeding.

In a clinical trial, 24 patients with haemophilia A who needed surgery or had suffered severe bleeding were treated by continuous infusion of Monoclate P, a factor VIII concentrate that is immunopurified by monoclonal antibodies. Continuous infusion of Monoclate P began with a dose of 2 U/kg per h that was adjusted according to the results of factor VIII assays to achieve a factor VIII target level of 100 IU/dl for 2 days and then 80 IU/dl for 5 days. The safety, efficacy, and economics of this approach were assessed. No haemorrhagic episodes were observed. The continuous infusion was convenient and had the advantage of producing steady-state levels of factor VIII. With a single-compartment model, we found median factor VIII clearance values of 3.11 (range 1.79-7.78) x 10(3) litres/kg per h, elimination rates of 5.0-19.4 x 10(-2)/h and a median half-life of 9.9 h (range 4.8-20.0 h). Clearance and the elimination rate appeared to decline over the infusion period, as judged by the decreasing infusion rate required to maintain the target concentration of factor VIII. An economic comparison with bolus therapy, using theoretically derived bolus dosages, indicated that the potential saving was related inversely to the factor VIII half-life. Potential savings of 75% were predicted on the first postoperative day, averaging 35% over the full course of therapy.

Factor VIII↗
PubMed1995

The successful removal of a bleeding intracranial tumour in a severe haemophiliac using an adjusted dose continuous infusion of monoclonal factor VIII.

Ten per cent of patients with haemophilia A develop intracranial haemorrhage (ICH) with a mortality rate of 30% and an incidence of psycho-neurological sequelae in 50% of survivors. ICH may be spontaneous or in association with trauma and other pathology. The generally recommended management is conservative replacement therapy using bolus injections of factor VIII and no neurosurgical intervention. Adjusted dose continuous infusion therapy provides an alternative method of factor VIII administration that is simple, more cost effective and safer through the maintenance of stable plasma VIII:C levels. This method has been successfully used to cover general surgery and the conservative treatment of subarachnoid haemorrhage but is not widely used due to unfamiliarity with the technique. This paper describes the use of continuous infusion of factor VIII concentrates to cover the successful neurosurgical management of a young man with severe haemophilia A who presented with an ICH associated with a bleeding choroid plexus tumour. Surgery was complicated by the development of a factor VIII inhibitor which disappeared following treatment with an immune-tolerance induction programme.

Adolescent↗
PubMed1995

The use of recombinant factor VIIa in a patient with acquired haemophilia A undergoing surgery.

An elderly woman with acquired haemophilia A secondary to a monoclonal gammopathy, required elective surgery for a parotid tumour but was unable to tolerate conventional treatment. Recombinant activated factor VII (rVIIa) was used successfully to cover a biopsy and then subsequent resection of the tumour. The use of rVIIa in the management of acquired inhibitors is discussed.

Aged↗
PubMed1995

Assay of protein S in systemic lupus erythematosus.

Free protein S:Ag and protein S activity determined by clotting assay are often regarded as synonymous; however, in the study group of patients, abnormal protein S activity (PS:Act) results correlated poorly with abnormal free (PS:Ag(r2 = 0.164). Significantly none of the patients in whom lupus anticoagulant and low free protein S:Ag were detected were found to have low PS:Act. These results could not be explained by activated protein C resistance, suggesting that lupus anticoagulant may cause prolongation of the clotting time within the PS:Act assay and thus give falsely high results. As reduced levels of free protein S:Ag and/or protein S activity are considered risk factors of thrombosis in systemic lupus erythematosus, these findings question the suitability of the protein S activity assay for patients who may have phospholipid antibodies.

Antibodies, Anticardiolipin↗
PubMed1994

Do safety issues of plasma products constrain self-sufficiency?

The ultimate responsibility for selecting and administering plasma products to patients rests with the prescribing physician, and it is for him/her to choose the safest product available. However, liability for a product with a full licence rests exclusively with the licence holder. After the problems of HIV and hepatitis C the safety of plasma-derived products has become of paramount importance. Particularly in the public sector, financial, strategic and political obstacles may adversely influence the quantity, quality and safety of plasma collection. The safety of blood products can be substantially enhanced by the harmonization of technical standards across both public and private sectors, thus supporting EC Directive 89/381. Additionally, the goal of European rather than national self-sufficiency should be encouraged.

Blood Banks↗
PubMed1994

Effect of splenectomy on HIV-related thrombocytopenia and progression of HIV infection in patients with severe haemophilia.

Between May 1983 and September 1991 eleven patients with severe haemophilia underwent splenectomy for HIV-related thrombocytopenia. The sustained complete remission rate (platelets > 100 x 10(9)/l) was 82% over a mean follow-up period of 54 months. The group was compared with 22 age-matched non-thrombocytopenic HIV seropositive haemophiliacs who had not undergone splenectomy. Both groups had equivalent use of factor concentrate and there was no significant difference between the groups in terms of anti-retroviral treatment. Analysis of clinical progression of HIV infection and CD4 positive lymphocyte (CD4+) counts, for the time since splenectomy, revealed no significant difference in progression of HIV infection in the splenectomized group compared with the control group. It is concluded that splenectomy is an effective treatment for HIV-related thrombocytopenia and has no adverse effect on the progression of HIV infection.

CD4-Positive T-Lymphocytes↗
PubMed1994

Supramalleolar tibial osteotomy for haemophilic arthropathy of the ankle.

We report the results of supramalleolar varus osteotomy on seven ankles (in six patients) for haemophilic arthropathy and secondary valgus deformity. The operation reduced pain and the frequency of intra-articular bleeding while preserving joint function for a mean of nine years. The procedure is an attractive alternative to the more commonly used surgical option of arthrodesis.

Adolescent↗
PubMed1993

The application of flow cytometry to the study of bacterial responses to antibiotics.

Experiments were performed to determine whether a modern flow cytometer could be used to study bacterial populations in suspension, with particular reference to their morphological characteristics and their responses to antibiotics. The FACScan, a commercial benchtop flow cytometer fitted with an air-cooled laser, designed primarily for the study of eukaryotic peripheral blood mononuclear cells, yielded reproducible data relating to bacterial shape and internal architecture. It was sensitive enough to detect changes in bacterial morphology on entry into the growth cycle and after exposure to antibiotics. Antibiotic-induced morphological changes affecting subpopulations of bacteria were sufficiently specific to allow differentiation between antibiotics with different cell-wall enzyme targets. Simultaneously, the effect of such antibiotics on the integrity of the outer cell membrane of Escherichia coli was assessed by measurement of the association of the nucleic acid-binding dye propidium iodide with the bacteria. These experiments demonstrated complex patterns of probable cell-wall leakage, related to the modes of action of the antibiotics. The FACScan is a useful and sensitive tool for the study of the morphology and physiology of bacterial populations in suspension, and is especially applicable to the study of antibiotic action.

Amdinocillin↗