Biomedical subjects
M A Howard
Publications and source records attributed to M A Howard.
von Willebrand disease type B: a missense mutation selectively abolishes ristocetin-induced von Willebrand factor binding to platelet glycoprotein Ib.
von Willebrand factor (vWF) is a multimeric glycoprotein that mediates the adhesion of platelets to the subendothelium by binding to platelet glycoprotein Ib. For human vWF, this interaction can be induced in vitro by the antibiotic ristocetin or the snake venom protein botrocetin. A missense mutation, Gly-561-->Ser, was identified within the proposed glycoprotein Ib binding domain of vWF in the proband with von Willebrand disease type B, a unique variant characterized by no ristocetin-induced, but normal botrocetin-induced, binding to glycoprotein Ib. The corresponding mutant recombinant protein, rvWF(G561S), formed normal multimers and exhibited the same functional defect as the patient's plasma vWF, confirming that this mutation causes von Willebrand disease type B. These data show that botrocetin and ristocetin cofactor activities of vWF can be dissociated by a point mutation and confirm that these mediators promote vWF binding to platelets by different mechanisms. The normal botrocetin-induced binding and the defective ristocetin-induced binding of rvWF(G561S) suggest that the primary defect in von Willebrand disease type B may be a failure of normal allosteric regulation of the glycoprotein Ib binding function of vWF.
Lack of sympathetic and cholinergic influences on cerebral vasodilation caused by sciatic nerve stimulation in the rat.
We studied the influences of sympathetic and cholinergic mechanisms on pial arteriolar responses during cortical activation in the rat. Adult male Sprague-Dawley rats were anesthetized with alpha-chloralose and urethane and mechanically ventilated. Pial arterioles on the somatosensory cortex were visualized on a video monitor through a closed cranial window. Changes in arteriolar diameter induced by sciatic nerve stimulation (0.2 V, 5 Hz, 5 ms, for 20 s) were measured before and after (a) ipsilateral superior cervical ganglionectomy (n = 5), (b) intravenous (0.5 mg/kg) administration and topical (10(-5) M) application of atropine (n = 5), and (c) lesion of the nucleus basalis magnocellularis (the major source of intracerebral acetylcholine neurons, n = 7). Unilateral nucleus basalis magnocellularis lesions were performed stereotactically by injection of ibotenic acid (25 nmol/microliter). Sensory cortex cholinergic denervation was confirmed histologically. These treatments had no significant effect on arteriolar responses to sciatic nerve stimulation. Thus, the present results suggest that neither sympathetic nor cholinergic mechanisms play a significant role in somatosensory evoked cerebral vasodilation.
Goniometric motion controller for the superconducting coil in a magnetic stereotaxis system.
For the purpose of positioning the 80 kg, 2 T superconducting coil/cryostat used in the developmental version of a magnetic stereotaxis system, a four degree-of-freedom goniometer has been designed, built, and tested. Computer-controlled, stepping motor actuators enable movement of the coil via either joystick, keyboard, or translator-module keypad commands. An integral arrangement of counterweights and counterbalances minimizes the overall weight and size of the goniometer, while maintaining static and dynamic stability during operation. As much of the structure as possible has been made of nonmagnetic materials (mostly aluminum) to minimize distortion of the superconducting coil's field. In this paper, we present the design principles for the goniometer, describe the essential features of its construction, and discuss its performance characteristics and limitations. We also discuss a strategy for performing precision magnetic stereotaxis procedures with an arrangement of static superconducting coils.
Effect of butyrate and corticosteroids on retinoblastoma in vitro and in vivo.
The effect of sodium butyrate (NaB) alone and in combination with a glucocorticoid was studied on Y-79 retinoblastoma cells both in vitro and in vivo. Both NaB (0.5 and 4 mM) and hydrocortisone (1-100 microM) added separately to cells grown in suspension resulted in a small growth inhibition (less than 15%). A marked synergistic effect (75-77% growth inhibition) was observed in vitro when NaB and hydrocortisone were added in combination at all the concentrations tested. With in vivo experiments using the nude mouse model of retinoblastoma, the combination of NaB and methylprednisolone did not inhibit tumor growth.
Comparison of test methods for the lupus anticoagulant: international survey on lupus anticoagulants-I (ISLA-1).
Six lyophilized plasma samples were sent to 20 "expert" laboratories for assessment of lupus anticoagulant (LA). Four samples contained pooled LA of graded potency mixed with aged normal plasma. One contained LA plus cephalin phospholipid and one contained a nonspecific venom anticoagulant. Sixteen methods were used overall with some participants using up to 8 methods. Results were scored in regard to the known potencies of LA in the samples and other known induced defects. Activated partial thromboplastin time (APTT) tests used by most participants for preliminary screening were relatively sensitive, but non-specific. Platelet or phospholipid neutralization procedures (PNP) appeared to be sensitive and specific but showed a non-linear response to increased LA content. Kaolin clotting time (KCT) tests showed the most sensitive response to increased LA content but the weaker LA were not scored as abnormal by most laboratories as the samples may have contained platelet fragments. Other commonly used tests such as the tissue thromboplastin inhibition (TTI) test and the dilute Russell's viper venom test (DRVVT) were carried out somewhat inconsistently. The variability in performance of tests in different laboratories indicates that standardization of methodology is urgently required. Generally it seemed that most clotting tests were "bypassed" by the addition of phospholipid to a known LA-positive sample in apparently direct proportion to their sensitivity. Sample preparation, especially prevention of contamination with activated platelets is a vital preliminary part in the assay of LA.
Experimental determination of the force required for insertion of a thermoseed into deep brain tissues.
Our laboratories are developing a new technique for delivering localized hyperthermia to deep-seated brain tumors. In this technique, a spherical thermoseed is stereotactically navigated through the brain and tumour tissues via the noncontact application of an external magnetic force. The force required to produce motion of a 3 mm diameter sphere through in vitro brain tissues was measured to be 0.07 +/- 0.03 N. This result was obtained from a series of experiments performed on whole brain specimens extracted from adult canines. Data were also taken with a 3 mm x 3 mm cylinder and a 5 mm sphere. An experimental procedure simulating physiological conditions was developed prior to testing. Evaluations of systematic effects included determinations of the calibration uncertainties, tests of the dependence of the measured force on temperature, and studies of the effects of method of storage of the tissue specimens. The results obtained are compared with (and confirmed by) two different series of experiments performed in vivo on adult canines and with another series of experiments using brain phantom gelatin.
Vancomycin pharmacokinetics in hydrocephalic shunt prophylaxis and relationship to ventricular volume.
Vancomycin pharmacokinetics were determined in 25 patients receiving ventriculoperitoneal shunts for hydrocephalus. Computed tomography scan-derived ventricular-brain ratio as an expression of hydrocephalus varied between 9.3% and 15.4% (12.9% +/- 1.7%). One hour prior to surgery each patient received 1 g of vancomycin infused intravenously over 60 minutes. Samples of cerebrospinal fluid and venous blood were obtained 1 hour later and vancomycin levels assayed by fluorescence polarization immunoassay. There were 11 females and 14 males, with a mean age of 44.5 +/- 10.3 years and a mean weight of 72.0 +/- 11.4 kg. All had normal renal function. Levels of vancomycin in the cerebrospinal fluid at 1 hour ranged from 0.1 to 1.5 micrograms/mL (0.9 +/- 0.3). Weight did not affect these values (p greater than 0.1). Simultaneous blood vancomycin levels varied between 9.1 and 38.7 micrograms/mL (22.3 +/- 8.3). Ventricular volume, expressed as the ventricular-brain ratio, did not correlate with cerebrospinal fluid vancomycin levels (p greater than 0.5). There was no significant increase in concentrations of vancomycin in CSF as cerebrospinal fluid protein concentration increased, nor when blood vancomycin concentration was greater than 20 mg/dL (therapeutic range) (p greater than 0.1). No patient had evidence of infection at 6 months follow up. These results indicate minimal cerebrospinal fluid penetrance of vancomycin when administered systemically 1 hour prior to shunt surgery. In addition concentrations of vancomycin in cerebrospinal fluid bear no relationship to weight, ventricular volume, meningeal inflammation, or blood levels in the therapeutic range. The minimum inhibitory concentration of vancomycin for staphylococci is 1.5 to 3.1, and as bactericidal levels of 5 to 8 minimum inhibitory concentration are needed to kill organisms, a combination of both systemic and intraventricular vancomycin may be needed to ensure adequate cerebrospinal fluid and tissue concentration of antibiotic during shunt prophylaxis.
Studies of natural anticoagulant proteins and anticardiolipin antibodies in patients with the lupus anticoagulant.
Components of the natural anticoagulant system (NAS) and anticardiolipin antibodies were examined in 21 patients with lupus anticoagulant (LA), 13 of whom had past histories of thrombotic episodes. No relationship could be shown between the antigenic levels of protein C and S (PC, PS) and a history of thrombosis. Inhibition of the anticoagulant activity of activated protein C (APC) was observed using plasma from 20/21 patients when phospholipid vesicles were used as the surface for the coagulation reaction. This effect was not affected by the addition of PS. When platelet membranes were employed only 2/21 patients demonstrated inhibition of APC. Under the latter condition, PS functional activity was inhibited in 7/21 patients, six of whom had a past history of thrombosis. Reduced antithrombin III or heparin cofactor II levels were observed in a total of 4/21 patients and may have contributed to the development of thrombosis in three of these patients. Antibodies specifically directed against these proteins were not detected suggesting the possibility of an associated constitutional deficiency. Anticardiolipin antibodies, though elevated in 17/21 patients, did not serve as a useful marker for an increased risk of thrombosis, and the level did not correlate with inhibition of the activity of APC or PS. We conclude that the mechanism of thrombosis in patients with LA is multi-factorial. A subset of patients in whom LA specifically inhibits PS function may represent patients who are at significant risk from thrombosis.
Magnetic stereotaxis: a technique to deliver stereotactic hyperthermia.
Advances in imaging techniques and computer software over the past decade now define brain abnormalities such as tumors in precise, three-dimensional images. We have taken advantage of these technological improvements in designing a system capable of performing magnetic manipulation of an object in a nonlinear trajectory and able to deliver hyperthermia to highly specific targets within the brain. This device relies on external magnets to pull a small metal pellet (thermoceptor) through the brain, and on biplane fluoroscopy to localize the thermoceptor with respect to previously obtained magnetic resonance images. A radiofrequency tuned circuit serves as the hyperthermia applicator and selectively heats the thermoceptor. This paper describes experiments conducted in a series of dogs showing that all three components of the system (magnetic drive, stereotactic real time imaging, and hyperthermia) can be achieved. Integration of the system was accomplished in one animal. These encouraging results need further detailed substantiation in each of the components, yet demonstrate the feasibility of such a device.
Cleavage of human von Willebrand factor by porcine pancreatic elastase.
von Willebrand factor (vWF) was purified from pooled normal plasma, radiolabeled with iodine then cleaved by porcine pancreatic elastase. Cleavage was monitored by sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE), SDS-agarose electrophoresis, crossed immuno-electrophoresis, ristocetin and botrocetin cofactor activities, and ristocetin and botrocetin induced binding to fixed washed platelets. Cleavage of vWF by porcine elastase was dependent on both the concentration of porcine elastase and period of incubation. Incubation of vWF with concentrations of porcine elastase greater than 20 U/mL for 30 minutes resulted in loss of the 240 Kd vWF subunit and a corresponding loss of the high molecular weight multimers and formation of fragments with molecular weights on reduced SDS-PAGE of 150, 125, 115 Kd and minor bands at 44, 28, and 24 Kd and a band at 20 Kd, which increased in intensity as either the concentration of porcine elastase or the period of incubation increased. More than 50% of the ristocetin cofactor activity was lost during a five-minute incubation of vWF with 0.56 U/mL porcine elastase when no detectable structural changes in the vWF molecule had occurred. Botrocetin cofactor activity was more resistant. Similarly botrocetin induced vWF binding to platelets was retained by a fraction produced by digestion of vWF with porcine elastase, which contained predominantly a 20 Kd fragment of vWF. This fragment retained insignificant amounts of ristocetin related functions and therefore represents a useful piece of the vWF molecule for further exploration of the site involved in botrocetin induced binding to platelets.
Identification and significance of multinucleate tumor cells in retinoblastoma.
Multinucleate tumor cells were observed in a case of a retinal tumor suspected to be a retinoma (retinocytoma). On electron microscopy, these cells had characteristics of retinoblastoma. Similar cells were observed in an irradiated model of retinoblastoma grown in a nude mouse. We suggest that the presence of these cells indicates differentiation and/or regression of the tumor.
Comparison of laboratory tests used for identification of the lupus anticoagulant.
A comparison of the sensitivities of the ten most commonly used tests for the identification of the lupus anticoagulant (LA) and the lupus cofactor phenomenon was undertaken on 18 patients. All investigations, except the cardiolipin-antibody ELISA assay, were carried out using patient's plasma alone followed by a 1:1 mix with control plasma. Dilution studies (1:3, 1:6, 1:9--patient:control) were also carried out. The kaolin clotting time (KCT) was the only test positive in all patients at all dilutions, while the dilute activated partial thromboplastin time with kaolin (Dil-APTT) registered 17 of 18 positive at all dilutions. Both the dilute Russell viper venom time (Dil-RVVT) and the tissue thromboplastin inhibition time (TTI) (1/500 thromboplastin) identified the LA in 17 of 18 patients on initial testing but were less sensitive in the dilution studies. The KCT is not a suitable test for routine laboratory use, as it requires an individual filtration step. Therefore a combination of either the Dil-APTT or Dil-RVVT together with the TTI (1/500 dilution thromboplastin) is recommended for routine LA screening, as all patients with LA in this study were identified using these easily automated tests. The lupus cofactor phenomenon was most frequently demonstrated using the Dil-APTT.
Magnetic movement of a brain thermoceptor.
Hyperthermia has significant potential as an adjuvant form of brain tumor therapy. Current intracranial hyperthermia methods, however, are limited in their ability to control spatiotemporal thermal distribution. A stereotaxic magnetic movement system that may be capable of heating discrete regions of brain to a preselected temperature is described. With this system, a ferromagnetic object (referred to as a thermoceptor) is directed through the brain by an external drive magnet. Real time thermoceptor position is monitored with biplanar fluoroscopy and superimposed on a preoperative magnetic resonance imaging scan using a computer. Once in position, the thermoceptor can be inductively heated by externally generated radiofrequency signals. Experiments on the magnetic drive and imaging aspects of this system have been conducted in vitro and in vivo. Mechanical studies of cadaver dog brains revealed that a mean force of 0.07 +/- 0.03 N was required to move a 3-mm diameter sphere through brain at a speed of less than 1 cm/15 s. A cranial phantom with mechanical properties similar to brain was constructed of gelatin and Plexiglas. With the use of a "neck loop" design drive magnet with a maximum magnetic field strength of 0.10 T, a 3 x 3 mm cylindrical neodymium iron boron thermoceptor was smoothly directed through the phantom in two dimensions. Additional experiments were conducted with a larger drive magnet in five anesthetized dogs. Neodymium iron boron and samarium cobalt thermoceptors of various shapes and sizes were placed into the cerebral cortex through a burr hole, then directed with the drive magnet. Fluoroscopy was used to follow the thermoceptor movements.(ABSTRACT TRUNCATED AT 250 WORDS)
Intracerebral drug delivery in rats with lesion-induced memory deficits.
Pharmacological treatments directed at increasing cortical acetylcholine activity in patients with Alzheimer's disease have largely been disappointing, perhaps because denervated areas of brain may not be exposed to adequate amounts of drug. A new method has been developed to enable localized intracerebral delivery of neurotransmitter substances using a polymeric drug delivery system. Microspheres of a polyanhydride sebacic acid copolymer were impregnated with bethanechol, an acetylcholinesterase-resistant cholinomimetic. Twenty rats received bilateral fimbria-fornix lesions, producing cholinergic denervation of the hippocampus and marked impairment in spatial memory. The animals were trained for 2 weeks to run after which they received bilateral intrahippocampal implants of saline (five rats), blank polymer (five rats), or bethanechol-impregnated polymer (10 rats). Following implantation, spatial memory was assessed by radial-maze performance testing for 40 days. Untreated lesioned rats showed persistently poor spatial memory, entering maze arms with near random frequency. Similarly, animals treated with saline and blank polymer did not improve after implantation. Rats treated with bethanechol-impregnated microspheres, however displayed significant improvement within 10 days after implantation; this improvement persisted for the duration of the experiment (p less than 0.05, Student's t-test). Histological analysis of regional acetylcholinesterase staining showed widespread loss of activity throughout the hippocampus bilaterally in all animals. The microsphere implants were visible within the hippocampus, with minimal reactive changes in surrounding brain. It is concluded that intracerebral polymeric drug delivery successfully reversed lesion-induced memory deficits, and has potential as a neurosurgical treatment method for Alzheimer's disease and other neurodegenerative disorders.
Acute subdural hematomas: an age-dependent clinical entity.
Reports prior to 1980 describe overall mortality rates for acute subdural hematomas (SDH's) ranging from 40% to 90% with poor outcomes observed in all age groups. Recently, improved results have been reported with rapid diagnosis and surgical treatment. A relatively large number of older patients (34 patients over 65 years old) were treated recently at Harborview Medical Center, enabling a retrospective comparison with similarly treated younger patients (33 patients aged 18 to 40 years). Clinical information and computerized tomography morphometric data were obtained. Patients in the younger group were most often injured in motor-vehicle accidents (15 cases), whereas falls were most frequent in the older group (19 cases). Patients in both groups were rapidly resuscitated in the field; more than 30% were treated within 1 hour after the time of injury. Injury severity, determined by the admission Glasgow Coma Scale score, was similar for the two groups. Mean acute SDH volume was significantly larger in the older patients than in the younger group (mean +/- standard deviation: 96.2 +/- 117.2 vs. 21.6 + 27.7 cu cm), as was the amount of midline shift (1.2 +/- 1.69 vs. 0.6 +/- 0.75 cm). Surgical treatments were similar, but outcomes were dramatically different for the younger and older patients. Mortality rates were more than four times higher in older patients than in younger ones (74% vs. 18%). Three older patients and 25 younger patients were functional survivors. Old age, a larger SDH volume, and a larger midline shift all correlated with a poor outcome. The results of this study suggest that the pathophysiology of acute SDH varies with age, and that currently employed resuscitation and treatment methods have differentially improved the outcome for younger patients.
Coagulation activities of plasma microparticles.
An evaluation of the effect of plasma microparticles (MP) on in vitro coagulation has been undertaken using platelet rich (PRP), platelet poor (PPP) and platelet free (PFP) plasmas prepared by differential centrifugation. MP provide coagulant material which shortens the activated partial thromboplastin time (APTT) and dilute simplastin time (DSTT) which is different from that contributed by commercial phospholipid preparations. The amount of platelet factor three (PF3) available in plasma is directly correlated with the centrifugal force used in its preparation and is present in large amounts in the MP pellet remaining after preparation of PFP. Factor VIII (F.VIII:C) and von Willebrand factor (vWf) were associated with the MP fraction but could be separated from MP on sucrose gradients. The effect of MP on the APTT was independent of the F.VIII:C/vWf and was not solely due to their PF3 content. Plasma prepared for routine coagulation assays contains MP which contribute to the APTT and DSTT and should be considered in their assessment. High speed centrifugation of plasma reduces the F.VIII:C/vWf:Ag/RCoF levels and this may contribute to losses of these proteins during preparative procedures utilising high speed centrifugation.
Haemophilia A in German shepherd dogs.
Haemophilia A was diagnosed in 14 male German shepherd dogs. Factor VIII: coagulant (FVIII:C) activities ranged from 1.13% of a normal canine plasma pool. von Willebrand's factor antigen values were normal or increased in all 9 of these dogs which were tested. Twelve of these dogs had a common maternal grandsire. Five dogs had exhibited no tendency to bleed (when tested between 2 to 23 months of age). They were tested because of pedigree links with clinically affected animals. Common clinical signs in the latter dogs included: bleeding from the mouth, subcutaneous and intramuscular haematomas and lameness. Since these dogs usually had a mild to moderate deficiency of FVIII:C, they may survive to adulthood without exhibiting clinical signs severe enough to necessitate veterinary attention.