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Biomedical subjects

M A Howard

Publications and source records attributed to M A Howard.

At least 73 records · Page 4Linked to original sources
PubMed1988

Comparison of two dimensional immunoelectrophoresis and multimer analysis in the study of von Willebrand factor.

To assess the validity of the multimeric analysis of von Willebrand factor (vWf), this technique was compared with two dimensional immunoelectrophoresis using samples of purified vWf obtained by gel filtration and plasma samples from a patient with severe von Willebrands disease who was receiving prophylaxis with cryoprecipitate. It is concluded that except for specialised clinical and research purposes, two dimensional immunoelectrophoresis provides a clear picture of the multimeric composition of the vWf molecule, and that this is sufficient for routine research and clinical use, without having to confirm the data by multimeric analysis.

Electrophoresis, Agar Gel↗
PubMed1988

Brain xenografts: the effect of cyclosporin A on graft survival.

Animal models of Parkinson's disease and Alzheimer's disease have shown dramatic functional improvement after transplantation of embryonic neurons into denervated regions of the adult brain. Because of the ethical and logistic problems associated with the use of human embryonic brain tissue, cross-species transplants are an attractive alternative. An experimental model of cross-species brain transplantation was developed to evaluate cell survival in untreated and cyclosporin A (CyA)-treated animals. Cholinergic ventral neurons from embryonic mice were transplanted into the frontal lobes of 18 adult Sprague-Dawley rats using a cell suspension technique. Nine animals were treated for 13 days with CyA (10 mg/kg/day) and nine were not treated. Twelve weeks after transplantation, frozen sections through the transplant volume were obtained. Alternate sections were prepared with hematoxylin and eosin and acetylcholine esterase stains. Cell counts through a 2-cu mm volume incorporating the transplant were compared to a contralateral control volume. Eight of the nine untreated transplants were successful (mean transplant cells +/- standard error of the mean: 90.7 +/- 19.4/2 cu mm). All of the nine CyA-treated transplants survived, with mean transplant count 28.7 cells/2 cu mm greater than untreated transplants (mean increase 28.7: p less than or equal to 0.05, Wilcoxon matched-pairs signed ranks test). It is concluded that: 1) this model is useful for quantitating transplant cell survival; 2) untreated xenografts survive well; and 3) a 13-day course of CyA improved long-term graft survival.

Acetylcholinesterase↗
PubMed1987

Shortening of bleeding time by 1-deamino-8-arginine vasopressin (DDAVP) in the absence of platelet von Willebrand factor in Gray platelet syndrome.

The Gray platelet syndrome is a rare disorder characterised by the absence of platelet alpha-granules and their contents. We describe a new patient and the effects of infusions of 1-deamino-8-arginine vasopressin (DDAVP). The patient had a prolonged skin bleeding time and his platelets had reduced numbers of alpha-granules, increased vacuolation and reduced retention on glass beads. Platelet von Willebrand factor antigen (vWf:Ag) was undetectable and levels of platelet fibrinogen, beta-thromboglobulin, platelet factor 4 and thrombospondin were reduced. All tests of plasma coagulation factors were normal, including Factor VIII (F.VIII:C), vWf:Ag, ristocetin cofactor (R:CoF) and botrocetin cofactor. Platelet ATP, ADP, platelet albumin, surface membrane glycoproteins and 14C-serotonin uptake were also normal. Infusions of DDAVP increased plasma F.VIII:C, vWf:Ag and R:CoF and shortened the bleeding time on two occasions. This suggests that DDAVP shortens the bleeding time by releasing vWf:Ag and/or other proteins from cellular storage sites other than the platelet.

Adult↗
PubMed1987

Lupus anticoagulant in women with multiple spontaneous miscarriage.

A link between the presence of the lupus anticoagulant (LA) and recurrent spontaneous miscarriage exists; however, the incidence of the LA in patients with unexplained recurrent miscarriage is unknown. We have therefore investigated the incidence of the LA in women with recurrent miscarriage but without evidence of systemic lupus erythematosus or other syndromes known to predispose towards spontaneous abortion. Plasma from 29 such women was obtained and tested in the activated partial thromboplastin time (APTT), testing for correction with normal plasma if prolonged and in the dilute simplastin time test (DSTT), again mixing with normal plasma if prolonged. Using a prolongation in either/or both tests which was not corrected by mixing with normal plasma as criteria for the presence of the LA, 14 (48%) patients showed evidence for the presence of the LA. Substitution of platelet-rich plasma for phospholipid and plasma in the APTT corrected the prolonged clotting time in every patient (9/29) in which it was prolonged, further substantiating the presence of the LA. We suggest that the presence of the LA should be investigated in any patient with unexplained recurrent miscarriage.

Abortion, Habitual↗
PubMed1987

Further investigations on the significance of a plasma coagulation-accelerating factor in glomerular disease.

The prevalence of the coagulation acceleration factor (CAF) in patients with renal disease (predominantly glomerulonephritis) has been investigated. The relationship between the activated partial thromboplastin time, F.VIII:C/vWf, prekallikrein, plasminogen and the pathogenesis of glomerulonephritis was also investigated. One hundred and seventeen patients were entered into the study on the basis of admission for renal biopsy. Thirteen percent of the patients showed evidence for CAF and 15% of patients were found to have reduced levels of plasma prekallikrein; however, no correlation was found between any of the variables measured and the type, activity or progression of glomerular disease in these patients.

Adolescent↗
PubMed1987

Analysis of platelet von Willebrand factor antigen.

Platelet lysates were obtained from suspensions of normal washed platelets by freeze-thawing or Triton X-100 lysis. The resultant platelet lysates contained 0.34 +/- 0.15 U/10(9) platelets (n = 8) of von Willebrand factor antigen (vWf:Ag) as determined by radioelectroimmunoassay using a monospecific antibody to vWf:Ag. The vWf:Ag level was higher in platelet lysates prepared from freshly drawn blood than from outdated platelet packs. Platelet lysates from patients with severe von Willebrand's disease type I (n = 2) did not contain detectable vWf:Ag. When normal platelet lysates were analyzed by radiocrossed immunoelectrophoresis in agarose using a monospecific polyclonal antibody to plasma vWf:Ag, two immunochemically identical precipitin peaks were seen. One of the platelet vWf:Ag peaks corresponded in its electrophoretic mobility to plasma vWf:Ag, while the other peak, i.e. platelet vWf:Ag-peak II, migrated to a more anodal position. The presence of the platelet vWf:Ag-peak II suggests structural differences between plasma and platelet vWf:Ag and illustrates previously unrecognized heterogeneity of platelet vWf:Ag.

Antigens↗
PubMed1986

Binding of human thrombin to human factor VIII:RAg.

The binding capacity of radiolabelled human alpha-thrombin (alpha-thrombin), inactivated with phenyl-methyl sulphonyl fluoride (PMSF), to factor VIII related antigen (VIII:RAg) multimers isolated by immune precipitation and resolved by sodium dodecyl sulphate (SDS) agarose gel electrophoresis has been examined. 125I-PMSF alpha-thrombin bound predominantly to four low molecular weight multimers (LMW) of VIII:RAg prepared from either normal or haemophilia A plasma. Two of these VIII:RAg multimers present in serum had lost the capacity to bind thrombin. No bands were observed when either plasma or serum from three patients with severe von Willebrand's disease (vWd) was processed in an identical manner. This binding was not dependent on the presence of an intact catalytic site on the thrombin molecule. A similar pattern of binding to VIII:RAg multimers was observed with 125I-labelled human anti-FVIII antibodies as 125I-alpha-thrombin and appears to involve the same multimers.

Antigens↗
PubMed1986

Use of the Philadelphia collar as an alternative to the halo vest in patients with C-2, C-3 fractures.

We analyzed retrospectively 27 individuals with C-2, C-3 fractures, 8 of whom were treated with a Philadelphia collar rather than the more commonly used halo vest. Successful fusion without neurological deficit occurred in all cases whether treatment was by Philadelphia collar (n = 8), halo apparatus (n = 16), or prolonged bed rest (n = 3). There was an increase in subluxation in 3 patients in both the Philadelphia collar group and the halo apparatus group. However, 1 of the 3 in the collar group was uncooperative and discarded his collar during treatment. If he is excluded, the subluxation rate in the Philadelphia collar group is 27%; the rate in the halo group is 19%. The Philadelphia collar seems to be an acceptable means of stabilizing the neck in C-2, C-3 fractures, but one must consider the degree of cervical movement in the collar (as detected by dynamic x-ray films), patient reliability, and patient age. The presence of subluxation did not preclude successful fusion in either the Philadelphia collar or the halo vest groups.

Adolescent↗
PubMed1986

Pharmacokinetics of intraventricular vancomycin in hydrocephalic rats.

An animal model was developed for studying the pharmacokinetics of antibiotics administered intraventricularly in hydrocephalus. Obstructive hydrocephalus was consistently produced in craniectomized adult rats by injecting kaolin into the cisterna magna. After induction of hydrocephalus, vancomycin was injected into the right lateral ventricle of each rat. Bilateral ventricular cerebrospinal fluid (CSF) and brain parenchymal samples were obtained at 0.5, 1, 2, 4, 8, and 12 hours and the concentration of vancomycin in these samples was determined. Brain tissue was also analyzed histologically. The results show: (a) vancomycin is rapidly distributed within the CSF, including the contralateral ventricle, within 30 minutes; (b) vancomycin concentrations were nearly identical in both ventricles at all time points; (c) mean peak CSF vancomycin concentrations occurred at 2 hours and were 23.8 and 21.3 micrograms/ml for the left and right lateral ventricles, respectively; (d) elimination from CSF was slow (T1/2 beta = 2.22 hours, T1/2 gamma = 19.65 hours); (e) no vancomycin was detected (less than or equal to 2 micrograms/g) in the samples of periventricular white matter; (f) histological changes observed were consistent with untreated obstructive hydrocephalus and did not seem to be related to vancomycin treatment. The clinical significance of these results and the usefulness of the experimental model are discussed.

Animals↗
PubMed1986

Proteolytic cleavage of human von Willebrand factor induced by enzyme(s) released from polymorphonuclear cells.

In vivo fragmentation of the von Willebrand factor antigen (vWF:Ag) molecule has been demonstrated on radiocrossed immunoelectrophoresis (CIE) in the plasma from patients with disseminated intravascular coagulation, in factor VIII concentrates, and in normal serum. Experiments reported here show that polymorphonuclear (PMN) cells contain a non-calcium-dependent protease(s) that when released and incubated with vWF:Ag results in an additional vWF:Ag peak on radio-CIE. Production of fragments of vWF:Ag by incubation with PMN cells occurred in a time-dependent manner. The protease(s) responsible was inhibited by diisopropyl fluorophosphate, soybean trypsin inhibitor, and aprotinin, but not by benzamidine, azide, epicron, or hirudin. Citrate, EDTA, and leupeptin also had no effect on the PMN cell enzyme's activity, indicating that the enzyme(s) is not calcium dependent. The PMN cell enzyme responsible for vWF:Ag fragmentation is located intracellularly and released by freezethaw lysis or cell activation by calcium or the calcium ionophore A23187.

Aprotinin↗
PubMed1985

Investigation of a coagulation accelerating factor (CAF) in glomerulonephritis.

A coagulation accelerating factor was purified from the plasma of two patients with glomerulonephritis (GN) who suffered from thrombotic complications. The factor co-purified with factor VIII/von Willebrand factor complex (FVIII/vWf) and under dissociating conditions remained associated with the factor VIII coagulant activity (FVIII). Control purified FVIII/vWf showed no coagulation accelerating activity under the experimental conditions used. The levels of coagulation accelerating factor, FVIII and von Willebrand factor (vWf) were reduced by incubation with rabbit anti-human FVIII/vWf or human anti-FVIII serum indicating a close association of these three activities. Multimeric analysis of the plasma FVIII/vWf complex from the two patients demonstrated a reduction in the high molecular weight multimers and the presence of an additional band not present on analysis of normal FVIII/vWf. It is suggested that the coagulation accelerating factor represents an active form of FVIII which has different in vitro properties to thrombin activated FVIII.

Adult↗
PubMed1984

The agglutination of human platelets by botrocetin: evidence that botrocetin and ristocetin act at different sites on the factor VIII molecule and platelet membrane.

Botrocetin caused a factor VIII (FVIII) dependent platelet agglutination which was associated with a reduction in the plasma levels of all FVIII parameters as a result of specific binding of FVIII to the platelets. The site of binding of FVIII to the platelet in response to ristocetin or botrocetin involves the glycoprotein I complex. This is suggested by the inability of chymotrypsin treated platelets or platelets from patients with the Bernard-Soulier syndrome to agglutinate in response to ristocetin. These platelets responded to botrocetin , but this was greatly reduced compared to normal. Crossed immunoelectrophoretic analysis indicated that in the presence of botrocetin most multimetric forms of FVIII bound to the platelet, whereas ristocetin caused binding of high and intermediate molecular weight forms. The antibiotic vancomycin inhibited platelet agglutination by ristocetin but had no effect on that caused by botrocetin . Assays of FVIII von Willebrand factor (VIII:vWf) using botrocetin compared well with those obtained using ristocetin in plasmas from normal individuals and from patients with classical von Willebrand disease (vWd). However, a patient with variant vWd demonstrated 100% botrocetin cofactor activity and 0% ristocetin cofactor activity. This suggested that the site of interaction on the FVIII molecule for botrocetin and ristocetin are different. Therefore the diagnosis of some von Willebrand variants cannot be excluded on the basis of a normal botrocetin cofactor assay.

Blood Platelets↗
PubMed1983

Investigations of the lupus-like inhibitor by-passing activity of platelets.

Plasmas from ten patients with a lupus or lupus-like inhibitor were investigated. In each case the partial thromboplastin time with kaolin (PTTK) was prolonged and failed to correct on the addition of an equal volume of normal plasma. Activated control platelets corrected the inhibitory effect in the PTTK or thrombin generation time (TGT) in every instance. Activated autologous platelets were as effective as control platelets and may thus explain why bleeding is rarely associated with the lupus inhibitor. Experiments using platelets or plasma from patients congenitally deficient in a single clotting factor or normal washed platelets resuspended in deficient plasma indicated that the inhibitor by-passing activity is platelet and not plasma derived. Platelet fractionation studies suggested that this activity is localised at the platelet membrane.

Blood Coagulation Disorders↗
PubMed1983

Fast migrating protein, immunochemically related to human factor VIII, studied by crossed immunoelectrophoresis in agarose.

A fast migrating protein (FMP) was detected by agarose radio-crossed immunoelectrophoresis, in addition to factor VIII antigen (VIII:RAg), using antiserum to human factor VIII (FVIII). FMP had partial immunochemical identity with FVIII, migrated as an alpha-protein, and was distinct from alpha-2 macroglobulin, fibronectin or IgM. FMP was precipitated by concanavalin A and was separable from the bulk of VIII:RAg by ammonium sulphate fractionation. A significant amount of FMP was seen in normal serum (n = 12), plasma from patients with: (a) disseminated intravascular coagulation (n = 12) and (b) severe haemophilia A (n = 6). Trace amounts of FMP were observed in plasma from normal donors (n = 12), but neither VIII:ARg nor FMP was detectable in the plasma or serum from patients with severe von Willebrand's disease (n = 3). Freshly prepared cryoprecipitate contained trace amounts of FMP, similar to normal plasma, but increased levels were observed in antihaemophilic factor concentrates prepared for patient use. Significant levels of FMP were also seen in cryoprecipitate after storage at 4 degrees C for 7 d and this generation of FMP was diminished by the addition of protease inhibitors. The presence of significant levels of FMP in situations where proteolytic enzymes may be activated and inhibition of its generation by protease inhibitors, suggest that this protein is produced by proteolytic action of enzyme(s) on the FVIII molecule.

Antigens↗
PubMed1982

Simplified immunoradiometric assay for factor VIII coagulant antigen.

A simplified, non-competitive, solid phase immunoradiometric assay has been developed for the quantitation of factor VIII coagulant antigen (VIII:CAg)--the antigenic counterpart of FVIII coagulant activity (VIII:C). Both homologous and heterologous antibodies to human factor VIII (FVIII) were used in this assay. Initially, FVIII in a test sample was attached to immobilized, human IgG obtained from a polytransfused haemophilia A patient with a high titre antibody to VIII:C. The bound FVIII was then detected using rabbit 125I-IgG specific for human FVIII. The concentration of VIII:CAg correlated well with VIII:C levels in the plasma from normal donors (r = 0.84, n - 15). Homozygote von Willebrand's disease patients had undetectable levels of VIII:CAg in their plasma. Patients with severe haemophilia A (VIII:C less than 0.01 u/ml) could be divided into groups on the basis of the VIII:CAg levels, i.e. those having undetectable VIII:CAg and other with measurable VIII:CAg. VIII:CAg detected in normal serum was less than 0.002 u/ml. In this assay the use of human antibody to FVIII is considerably decreased compared to other methods for VIII:CAg, and the time-consuming steps to immunopurify human anti-FVIII antibody are eliminated.

Antigens↗
PubMed1982

Variant von Willebrand's disease type B--revisited.

Results of investigations of the factor VIII (FVIII) of a patient with an unusual variant form of von Willebrand's disease (vWD) are presented. A two-peak crossed-immunoelectrophoresis (CIE) pattern was seen when fresh plasma was electrophoresed, but the CIE pattern became normal by incubating the plasma at 37 degree C for more than 72 hr. The two peaks on CIE were separated by cryoprecipitation: the slow-moving peak precipitating and the fast-moving forms of FVIII remaining in the cryosupernate. An additional protein band was seen on multimeric analysis of FVIII. The platelet-rich plasma (PRP) from this patient did not respond to ristocetin, but agglutinated normally in response to botrocetin. Multimeric and CIE analysis of the FVIII post agglutination and 125I-FVIII binding studies to normal formalin-fixed platelets indicated that this patient's FVIII interacted normally with botrocetin but failed to interact with ristocetin. These data strongly suggest that the sites on the FVIII molecule or the multimeric forms involved for ristocetin and botrocetin are different and that the ristocetin reaction is more closely aligned to the physiologic function of FVIII.

Animals↗
PubMed1981

Quantitation of binding of factor VIII antigen to concanavalin A.

This study establishes a convenient method for screening plasma samples for abnormalities of the carbohydrate content of the factor VIII (FVIII) molecule. A radioimmuno-electrophoretic technique has been developed to quantitate the percentage binding of FVIII-related antigen (VIII-Ag) to the lectin concanavalin A (Con A). Plasma samples were electrophoresed through a strip of agarose containing Con A into agarose containing a mixture of unlabelled anti-FVIII and 125I-anti-FVIII where precipitant lines formed, the height of which was dependent upon the degree of VIII-Ag binding to Con A in the first gel. Using this system reduced binding of VIII-Ag to Con A was found in the plasma of 12 patients with moderate classical von Willebrand's disease (vWd), while the Con A binding of six haemophilia A patients fell within the normal range. The VIII-Ag in normal cryoprecipitate showed increased % binding to Con A while the VIII-Ag remaining in the cryo-supernate demonstrated reduced Con A % binding.

Antigens↗