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Biomedical subjects

E G Tuddenham

Publications and source records attributed to E G Tuddenham.

At least 145 records · Page 8Linked to original sources
PubMed1984

A clinically useful DNA probe closely linked to haemophilia A.

Genetic linkage studies were done in families segregating for haemophilia A with an X-chromosome-specific probe, DX13, which has been localised to band Xq28. When DNA is digested with the restriction enzyme Bg1 II, this probe recognises a restriction fragment length polymorphism, for which 50% of females are heterozygous. No crossovers were seen between the haemophilia A locus and the DX13 locus (lod score = 5.4 at recombination fraction [theta] 0.0; 95% confidence limits of theta, 0-12%). This indicates that the DX13 probe is closely linked to the haemophilia A locus and is likely to prove useful in carrier detection and prenatal diagnosis.

Alleles↗
PubMed1984

Localization of factor VIIIC: antigen in guinea-pig tissues and isolated liver cell fractions.

Factor VIIIC:antigen (VIII:CAg) was estimated in guinea-pig tissues by an immunoradiometric assay using a human inhibitor antibody. In homogenized guinea-pig tissues, VIII:CAg was shown to be stable and to be predominantly located in the liver (9 +/- 1.2 units; mean +/- SEM, n = 8). Lesser amounts were detected in spleen (1.3 +/- 0.02 units), lung (0.6 +/- 0.07) and kidney (0.4 +/- 0.06). In isolated liver cell fractions separated by centrifugal elutriation VIII:CAg was mainly detected in the hepatocyte fraction (0.3 +/- 0.07 units/10(8) cells;mean +/- SEM, n = 5) and in lesser amounts in the endothelial (0.02 +/- 0.01 units/10(8) cells) and the Kupffer cell fractions (0.05 +/- 0.02 units/10(8) cells). The liver concentration of VIII:CAg was (0.17 +/- 0.02 units/g) which was 20% of the plasma concentration (0.96 +/- 0.01 units/ml, n = 8) suggesting that VIII:CAg may not be stored in the liver but is rapidly exported following synthesis.

Animals↗
PubMed1984

Clinical experience with polyelectrolyte-fractionated porcine factor VIII concentrate in the treatment of hemophiliacs with antibodies to factor VIII.

Circulating antibodies to factor VIII (anti-VIII, "inhibitors") occurring in patients with hemophilia neutralize porcine factor VIII less readily than human factor VIII in vitro. Over an 18-mo period, 8 patients with anti-VIII were treated with 45 courses (297 infusions) of polyelectrolyte-fractionated porcine factor VIII concentrate (PE porcine VIII). Where no anti-PE porcine VIII was detectable, mean post-infusion rise in plasma factor VIII was 1.29 U/dl/units infused/kg. Above 13 Old Oxford units of anti-PE porcine VIII and 48 Bethesda units of anti-human VIII, there were no postinfusion rises in plasma factor VIII. Where postinfusion rises were detected, clinical responses were good and conventional methods could be used to guide dosage. Ten percent of infusions were followed by febrile reactions, but these were usually mild and decreased in frequency and severity with increasing exposure. Multiple and prolonged courses of therapy were given to some patients without evidence of loss of clinical or laboratory efficacy. PE porcine VIII could provoke anamnestic rises of anti-VIII in susceptible patients, but appeared to have a lower immunogenic potential than human VIII. PE porcine VIII is a rational and effective therapeutic alternative for patients with anti-VIII, particularly those with intermediate level inhibitors who cannot be managed effectively using human factor VIII.

Adolescent↗
PubMed1983

Von Willebrand factor multimer patterns in von Willebrand's disease.

The von Willebrand factor antigen (factor VIII-related antigen, VIIIR:Ag) multimer pattern has been analysed by SDS-agarose electrophoresis of plasmas from 116 patients (47 families) with von Willebrand's disease. In addition to previously recognized patterns, a subclassification was established between plasmas that had a type Ia pattern (VIIIR:Ag multimer pattern like that of normal plasma) and those that had a type Ib pattern in which there was a relative reduction in the concentration of the larger VIIIR:Ag multimers even though all multimeric forms were present. The different patterns were consistent within families and were inherited by autosomal dominant transmission. Von Willebrand's disease heterogeneity was apparent in the distribution of these plasmas: type Ia, 43 patients in 18 families; type Ib, 39 patients in 15 families; type II, 22 patients in 10 families, one of which was further classified as type IIB, one of which was type IIC, and three were IIA. Seven patients with severe von Willebrand's disease were also studied. In general, the interpretation of SDS-agarose multimer patterns corresponded to those previously obtained by crossed immunoelectrophoresis, but the former technique was more sensitive and could identify differences that were not apparent by crossed immunoelectrophoresis.

Antigens↗
PubMed1983

Monoclonal antibodies to human procoagulant factor VIII.

VIII:C was purified from intermediate-purity concentrate by adsorption on polyelectrolyte E5 and affinity chromatography on Sepharose/anti-VIIIR:Ag. The highly purified VIII:C preparation (sp. act. 1598 U/mg) was used to immunize Balb-C mice. Spleen cells from a mouse with a serum antibody titer of 963 U/ml were fused with P3 NSI mouse myeloma cells. Hybrid clones were screened by a coagulation inhibition assay and by a four-layer antibody adsorption procedure. Nine monoclonal antibodies specific to VIII:C were produced. Five of these antibodies have been cloned and grown in mouse ascitic fluid. Antibody titers from ascitic fluid ranged from 35 to 82,000 BU/ml. The antibodies, when radiolabeled, form a high-molecular-weight complex with antigens present in normal plasma and factor VIII concentrate, but not when incubated with CRM-negative hemophilic plasma. A two-site assay using a combination of monoclonal antibodies is able to detect VIII:CAg in normal plasma and in factor VIII concentrate. Sensitive two-site immunoradiometric assays using monoclonal antibodies as the solid phase have been set up.

Animals↗
PubMed1982

Immunologic studies of factor VIII coagulant activity (VIII:C). 2. Factor VIII in selected vertebrates.

Cross reactive antigens to factor VIII have been measured in a range of vertebrate phyla. VIII coagulant antigen (VIII:CAg) measured using a human antibody was in general, much lower than reported values of VIII coagulant activity (VIII:C) except in simians and the guinea pig. The dose response curve for the assay was parallel in all cases. Factor VIII-related antigen (VIIIR:Ag) measured using rabbit antibody to human VIIIR:Ag was low or absent in most species except simians and the dose response curve was non-parallel to the human standard except with goat plasma. Avians lacked cross-reactive material.

Animals↗
PubMed1982

Response to infusions of polyelectrolyte fractionated human factor VIII concentrate in human haemophilia A and von Willebrand's disease.

Factor VIII was purified from cryoprecipitate by ion exchange chromatography on solid phase polyelectrolyte E-5 (PE-E5). The product was highly purified (3.5 u VIII:C/mg protein) compared to conventional concentrate (0.3 u VIII:C/mg protein) with low fibrinogen, low isoagglutinin titre, and a ratio of factor VIII coagulant activity (VIII:C) to factor VII related antigen (VIIIR:Ag) of 16:1. Trial infusions of this material (PE VIII) were given to three patients with severe haemophilia A and one patient with homozygous von Willebrand's disease. These patients also each received separate infusions of intermediate purity concentrate (IPC) for comparison. There were no adverse effects. The mean half life of VIII:C after PE VIII infusion in the haemophiliacs was 10.9 h and after IPC was 12.1 h, a statistically insignificant difference. The survival of factor VIII coagulant antigen (VIII:CAg) was similar to that of VIII:C. In contrast, the half life of VIII:C and of VIII:CAg was very short after infusion of PE VIII in the patient wih von Willebrand's disease (2.4 h). IPC when infused in this patient produced a typical secondary rise of VIII:C. Two bleeding episodes in severe haemophiliacs were satisfactorily treated with PE VIII. PE-E5 deserves further study as a means of preparing clinical concentrates of factor VIII.

Antigens↗
PubMed1982

Morphological and functional disturbances of platelets induced by cryopreservation.

In vitro morphological and functional studies were carried out on platelets which had been cryopreserved in the presence of 5% dimethyl sulphoxide (DMSO). Overall loss of platelets was around 50%. Those which survived freezing and reconstitution showed marked morphological deterioration, increase of procoagulant activity (PF3a) and a decrease in their aggregability and adenine nucleotide content. We conclude that if transfused, cryopreserved platelets are likely to be less effective than fresh platelets and may activate coagulation in vivo and that they should only be used when suitable fresh platelets are not available.

Blood Platelets↗
PubMed1982

Preparation of factor IX deficient human plasma by immunoaffinity chromatography using a monoclonal antibody.

A murine hybridoma clone is described that grows continuously in culture and produces a monoclonal antibody we have called Royal Free Monoclonal Antibody to factor IX No. 1 (RFF-IX/1). This has high affinity for a coagulation site on factor IX. RFF-IX/1 immobilised on sepharose can be used to deplete factor IX from normal human plasma. This immunoaffinity depleted plasma is indistinguishable from severe Christmas disease plasma and can be used as the substrate in a one stage coagulation assay for factor IX. The affinity column has high capacity and can be regenerated so that large scale production from normal plasma of factor IX deficient plasma as a diagnostic reagent is now feasible.

Animals↗
PubMed1981

Synthesis and release of factor VIII by cultured human endothelial cells.

Endothelial cells (ECs) derived from human umbilical veins were cultured in order to study the physiological control of factor VIII synthesis and release. The culture media were studied from multiple replicate cultures at confluence. Factor VIII related antigen (VIIIR:Ag) and factor VIII coagulant antigen (VIII:CAg) were measured by sensitive immunoradiometric assays. De novo synthesis of factor VIII related protein (VIII:R) was quantitated by incorporation of labelled amino acids into specific protein subunits. The following agents were added to the culture medium in a range of concentrations from physiological to pharmacological: adrenaline, 5 hydroxytryptamine, 2,3-DPG, cyclic AMP, thyroxine, hydrocortisone, and human growth hormone. None of them had any effect at any concentration on the rate of accumulation of VIIIR:Ag in the culture medium. Addition of exogenous factor VIII had no effect on do novo synthesis of VIII:R. VIII:CAg was found to be stable under the conditions of culture but none was released from the ECs. Long-term monocyte cultures also failed to release VIII:CAg. It appears that VIII:R is a constitutive gene product of umbilical vein endothelial cells and that VIII:CAg is not made by these cells.

Antigens↗
PubMed1981

Detachment of endothelial cells by polymorphonuclear leukocytes in vitro: potentiation by antibody coating and prevention by inflammatory exudate.

A widely advocated hypothesis suggests that polymorphonuclear leukocytes release enzymes at sites of inflammation which in turn cause tissue damage, especially vasculitis. To test this we isolated and grew human endothelial cells and labelled them with chromium-51. Polymorphonuclear leukocytes were separated from whole blood and added to the labelled endothelial cells. Upon incubation with the polymorphonuclear leukocytes the endothelial cells rapidly detached but were not lysed. Detachment was prevented by the addition of small amounts of inflammatory exudate. Prior coating of endothelial cells with a specific antibody accelerated detachment of the cells by polymorphonuclear leukocytes but this was still prevented by addition of inflammatory exudate in low concentration. In a separate experiment extracellular basement membrane synthesized in vitro by endothelial cells was labelled by incubating the cells with 14C-proline. Polymorphonuclear leukocytes caused release of 14C but this was again prevented by addition of either inflammatory exudate or serum. We conclude that polymorphonuclear leukocytes alone could cause vascular intimal damage in vivo if inhibitors, which are present in serum and inflammatory exudate, are excluded or overwhelmed.

Antibodies↗
PubMed1976

Familial pulmonary fibrosis associated with oculocutaneous albinism and platelet function defect. A new syndrome.

A family was studied in which four siblings had oculocutaneous albinism. In three of these a platelet function defect characterized by poor response to collagen was found. The fourth had previously died from cryptogenic fibrosing alveolitis. Two of the survivors had cryptogenic fibrosing alveolitis and the third had physiological lung function disturbance. Bone marrow examination of one showed pigment laden macrophages (Hermansky-Pudlak Syndrome). Three other normally pigmented family members were found to have normal platelets and no evidence of cryptogenic fibrosing alveolitis, although one had pulmonary disease attributable to occupational dust exposure. To elucidate the aetiological factors involved, three unrelated normally pigmented patients with known platelet function defect were studied. One proved to have cryptogenic fibrosing alveolitis. Four unrelated albinos were also studied and had normal lungs and platelets. It is suggested that, in addition to the known association between platelet function defect and albinism, there is an association between a platelet function defect and cryptogenic fibrosing alveolitis.

Adenosine Diphosphate↗