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Biomedical subjects

E S Ward

Publications and source records attributed to E S Ward.

17 recordsLinked to original sources
PubMed1992

Comparative stabilities in vitro and in vivo of a recombinant mouse antibody FvCys fragment and a bisFvCys conjugate.

A murine antibody FvCys fragment with a single additional cysteine residue at the C terminus of the VH domain was expressed in Escherichia coli from a modified expression plasmid containing the structural genes for the VH and VL domains derived from the anti-lysozyme hybridoma D1.3. Chemical cross-linking between the introduced sulfhydryl groups of two FvCys fragments by means of bis-maleimidohexane was used to generate a bisFvCys conjugate. The stability of the bisFvCys conjugate and an FvCys analogue that had been reacted with N-ethyl-maleimide to block the free sulfhydryl group, FvCys(BL), were compared after 125I-labeling. The bisFvCys conjugate was completely stable to incubation in solution at 37 degrees C for 24 h whereas only 60% of the FvCys(BL) fragment remained soluble. After i.v. administration to normal Wistar rats, both Fv proteins were rapidly cleared from the circulation with biphasic kinetics that were best fitted to a two-compartment open pharmacokinetic model. The alpha-phase half-life of the bisFvCys conjugate, 0.32 h, was significantly longer than that of the FvCys(BL) fragment, 0.15 h (p less than 0.001) whereas there was no significant difference between the beta-phase half-lives, 1.4 to 1.6h. No chain cleavage or covalent attachment to serum protein was detected by SDS-PAGE analysis of serum samples. However, gel permeation HPLC revealed that both Fv proteins associated with serum proteins in vivo and in vitro.

Amino Acid Sequence↗
PubMed1992

Secretion of T cell receptor fragments from recombinant Escherichia coli cells.

This study describes the secretion and purification of T cell receptor (TCR) V alpha, V beta domains and single chain V alpha-V beta fragments (scTCRs) from recombinant Escherichia coli cells. The TCR V alpha and V beta genes are derived from a T cell hybridoma that is associated with disease pathogenesis in murine experimental allergic encephalomyelitis (EAE). Circular dichroism (c.d.) analyses of the single domains and the scTCR indicate that they are folded into beta-pleated sheet structures similar to those of immunoglobulin variable domains. The secreted TCR fragments can be purified in milligram quantities, and could therefore be used in high-resolution structural studies, in immunization to generate anti-clonotypic antibodies or in vaccination.

Animals↗
PubMed1992

Antibody engineering: the use of Escherichia coli as an expression host.

The hypervariable loops of an antibody molecule are supported on the relatively conserved beta-sheeted frameworks of the heavy- and light-chain variable domains (designated VH and VL domains, respectively). Residues within and flanking these loops interact with antigen and confer the specificity and affinity of antigen binding on the immunoglobulin molecule. Thus, the isolation and expression of VH and VL domain genes are of particular interest both for analysis of the determinants of antibody specificity and for generation of fragments with binding affinities for use in therapy and diagnosis. The PCR can now be used to isolate diverse repertoires of antibody VH and VL domain genes from antibody-producing cells from different species, including humans and mice. The genes can be expressed as either secreted or surface-bound Fv or Fab fragments, using Escherichia coli expression systems, and the desired antigen-binding specificity screened for or, preferably, selected. The use of E. coli as an expression host allows the required antigen-binding specificity to be isolated in clonal form in a matter of days. The VH and VL domain genes can also be hypermutated and higher-affinity variants isolated by screening or selection. Thus, the use of this technology should allow the isolation of novel binding specificities or specificities that are difficult to generate by hybridoma technology. It will also facilitate the isolation of human-derived Fv/Fab fragments that may be less immunogenic in therapy. This approach therefore has almost unlimited potential in the generation of therapeutics with binding specificities to order. The fragments can be used either alone or linked to effector functions in the form of antibody-constant domains or toxins. The new technology could prove to be a method of choice for the rapid and convenient production of designer antibodies.

Antibodies↗
PubMed1992

Effect of education on the appropriateness of serum drug concentration determination.

The purpose of this study was to document the effect of education on the appropriateness of serum drug concentration (SDC) collection. This study included a period of education for hospital personnel involved in the acquisition of drug levels. These included nursing staff, lab personnel, house-staff (residents/interns), ward clerks, and pharmacists. This study included patients receiving aminoglycoside and/or vancomycin while on the general medicine service during the specified study periods. Patients receiving less than 72 h of antimicrobial therapy and patients transferred to or from the surgery service were excluded from the study. A retrospective pre- and posteducation comparison using Chi-square analysis was made to determine whether the percentage of appropriate and useful SDCs was improved through education. There was a significant difference (p less than 0.005) between the percentage of appropriate SDCs in the pre- and posteducation period. As a result of the study, a change in procedure for ordering SDCs was initiated, using an educational program, in order to continue the trend of appropriate SDCs at the Veterans Administration.

Aminoglycosides↗
PubMed1992

Use of theophylline in neonates as an aid to ventilator weaning.

Respiratory diseases are major causes of morbidity and mortality in premature neonates. Theophylline has been utilized as an adjunct in facilitating ventilator weaning and in the management of apnea with or without bradycardia. Patient characteristics associated with improved outcome from theophylline have not been determined. The purpose of this study was to evaluate parameters associated with improved outcome in neonates with respiratory diseases receiving theophylline. The study population consisted of premature neonates that were studied retrospectively. Criteria for entry into the study were (1) less than 40 weeks gestation, (2) a diagnosis of respiratory distress syndrome (RDS), apnea of prematurity, hyaline membrane disease (HMD), or bronchopulmonary dysplasia (BPD), (3) dependence on intermittent mandatory ventilation, (4) failure to wean from the ventilator 24 h or more before the study, or (5) receiving theophylline. In this study, we found no correlations between time to wean from the ventilator and postnatal age at the time theophylline was initiated, 5-min APGAR score, and final theophylline serum concentration before complete weaning from the ventilator. However, there were significant negative correlations between birthweight and gestational age with respect to time to wean from the ventilator. The average theophylline serum concentration before weaning from the ventilator for this population of neonates was approximately 5-10 micrograms/ml, indicating that theophylline is not beneficial as an aid to ventilator weaning at serum concentrations < 10 micrograms/ml.

Apgar Score↗
PubMed1991

Expression and secretion of T-cell receptor V alpha and V beta domains using Escherichia coli as a host.

An expression system for the production of recombinant T-cell receptor (TCR) variable domains would, inter alia, allow structural studies to be carried out and provide protein for the generation of anti-clonotypic antibodies. In this report the V alpha and V beta domain genes have been isolated from a T-cell hybridoma which is associated with the pathogenesis of experimental allergic encephalomyelitis (EAE) in the H-2u mouse. These have been expressed as secreted domains in Escherichia coli, using secretion vectors previously used for the production of immunoglobulin fragments. Both V alpha and V beta domains are secreted in milligram quantities into the culture supernatant, although the levels of the V alpha domain are about 10-20 fold higher than those of the V beta domain. This expression system offers a rapid route for the production of recombinant TCRs in soluble form.

Animals↗
PubMed1991

Elevated phenytoin concentration caused by sampling through the drug-administration catheter.

Elevated phenytoin concentrations are a problem due to the toxic effects that can result. When an elevated concentration is reported, it is important to determine the validity of the report because therapeutic action may include withholding further doses. If the concentration is falsely elevated, the potential for seizure activity as it falls is inherent. Assessing validity involves considering laboratory error and assays of blood samples drawn from the wrong patient. The capacity-limited biotransformation of phenytoin complicates the estimation of concentrations, making it more difficult to establish the accuracy of reported values. Our patient had a phenytoin concentration of 80.7 mg/L. It had been 13.4 mg/L the day before, and the patient received only an additional 400 mg prior to the reported high value. The pharmacokinetic team determined that the dose was administered through a triple-lumen catheter from which blood may have been withdrawn for phenytoin analysis the next day. Venous sampling 7 hours after the 80.7-mg/L value yielded a phenytoin concentration of 12.4 mg/L. Clinicians should consider this error potential in the differential evaluation of elevated concentration in apparently nontoxic patients.

Administration, Oral↗
PubMed1990

Crystallization and preliminary X-ray diffraction study of the bacterially expressed Fv from the monoclonal anti-lysozyme antibody D1.3 and of its complex with the antigen, lysozyme.

The associated heavy (VH) and light (VL) chain variable domains (Fv) of the monoclonal anti-lysozyme antibody D1.3, secreted from Escherichia coli, have been crystallized in their antigen-bound and free forms. FvD1.3 gives tetragonal crystals, space group P4(1)2(1)2 (or P4(3)2(1)2), with a = 90.6 A, c = 56.4 A. The FvD1.3-lysozyme complex crystallizes in space group C2, with a = 129.2 A, b = 60.8 A, c = 56.9 A and beta = 119.3 degrees. The crystals contain one molecule of Fv or of the Fv-lysozyme complex in their asymmetric units and diffract X-rays to high resolution, making them suitable for X-ray crystallographic studies.

Amino Acid Sequence↗
PubMed1990

A comparison of venous versus capillary measurements of drug concentration.

Accessing patient's veins for drug level sampling is not always feasible. The use of capillary sampling techniques is often utilized when venous access is hampered. In the therapeutic monitoring of patients, unexpected drug level results often occur that can be caused by a number of different factors. The possibility that differences in assay results might occur if samples were collected by capillary stick vs. venous phlebotomy was examined by simultaneous sampling in 18 patients. Although correlation was very high (0.999) and percentage differences fairly low (range of 0 to 15.4%), a statistical difference was noted in the sampling methods. The precision was 6.5 +/- 6.58% and there was a slight negative bias (-3.76%), with capillary samples less than venous samples. Although there were statistical differences for the drugs studied in the concentration ranges evaluated, capillary samples should provide fairly small errors when compared to venous samples.

Adult↗
PubMed1990

Implementing and maintaining a private pharmacokinetics practice.

The development and maintenance of a private-practice pharmacokinetics service is described. A contracted pharmacokinetics service has been in place at Georgia Baptist Medical Center, a 525-bed hospital, for the past eight years. Physician support, inhouse study results, and literature documentation of the benefits of scheduling drug concentration determinations were used to convince hospital administrators of the value of a pharmacokinetics service. Services were reimbursed by increasing the fee charged for each drug concentration determination, with payment made to the pharmacokinetics service on a monthly basis. The service is associated with the pathology department for administrative purposes. Each member of the pharmacokinetics service is credentialed by the medical staff. The group trains part-time personnel to provide services when needed. Services provided by the group but not required by the contract include collecting quality assurance data, conducting research, serving on hospital committees, writing newsletter articles, conducting inservice-education programs, and providing clinical interventions for nonconsultation patients. This private-practice pharmacokinetics service provides high-quality services and is well accepted within the institution.

Drug Information Services↗
PubMed1990

Multidimensional work sampling to quantify a pharmacokinetics resident's duties.

A multidimensional work-sampling technique was used to quantify a pharmacokinetics resident's duties. Four dimensions were measured: activity, contact, function, and location. A code list of 47 variables was developed to encompass all activities of the resident being observed. A combination of self-reporting and observer reporting was used to test the veracity of the resident's report and to determine whether differences in reporting occurred. Observation was done 20 times a day over a five-week period (24 working days). Random time periods separated the observation points. The observer and the resident recorded 260 and 220 observations, respectively, for a total of 480. The overall reliability measure was 78%, indicating good interobserver agreement. The resident spent about 36% of his time doing work related to pharmacokinetics consultations and 23% of his time attending meetings. He spent a large proportion of time (48.5%) working alone. Much of his remaining time was spent with the Pharm.D. students and the preceptors. Multidimensional work sampling based on a combination of self-reporting and observer reporting is a useful tool for quantifying the work practices of pharmacokinetics residents.

Education, Pharmacy, Graduate↗
PubMed1989

Binding activities of a repertoire of single immunoglobulin variable domains secreted from Escherichia coli.

In antibodies, a heavy and a light chain variable domain, VH and VL, respectively, pack together and the hypervariable loops on each domain contribute to binding antigen. We find, however, that isolated VH domains with good antigen-binding affinities can also be prepared. Using the polymerase chain reaction, diverse libraries of VH genes were cloned from the spleen genomic DNA of mice immunized with either lysozyme or keyhole-limpet haemocyanin. From these libraries, VH domains were expressed and secreted from Escherichia coli. Binding activities were detected against both antigens, and two VH domains were characterized with affinities for lysozyme in the 20 nM range. Isolated variable domains may offer an alternative to monoclonal antibodies and serve as the key to building high-affinity human antibodies. We suggest the name 'single domain antibodies (dAbs)' for these antigen binding demands.

Amino Acid Sequence↗
PubMed1988

Single amino acid changes in the Bacillus thuringiensis var. israelensis delta-endotoxin affect the toxicity and expression of the protein.

Site-directed mutagenesis has been used to change individual amino acids of the larvicidal 27,000 Mr delta-endotoxin of Bacillus thuringiensis var. israelensis. Basic and acidic residues have been systematically replaced by alanine, and the resulting mutant polypeptides analysed for cytolytic and larvicidal activity, and binding to phosphatidyl choline liposomes. Replacement of residues at positions 154, 163, 164, 213 and 225 results in proteins which accumulate as inclusions in recombinant Bacillus subtilis cells similar to the wild-type, but have considerably reduced in-vitro and in-vivo toxicity. One mutant (Glu45 to Ala45) results in a protein that has reduced activity in vitro, but retains wild-type larvicidal toxicity. In addition, seven other mutations of charged residues result in proteins which form small or no inclusions in recombinant cells, despite being produced at levels similar to the wild-type in six out of seven cases. In most instances, the toxicity of these aberrantly expressed proteins is considerably less than the wild-type, although one (Lys124 to Ala124) results in a polypeptide with approximately threefold increased activity in vitro. A secondary structural model is proposed to explain these observations.

Amino Acid Sequence↗
PubMed1988

Cloning and expression of two homologous genes of Bacillus thuringiensis subsp. israelensis which encode 130-kilodalton mosquitocidal proteins.

Two homologous genes encoding 130-kilodalton (kDa) mosquitocidal proteins of Bacillus thuringiensis subsp. israelensis have been cloned and expressed in Escherichia coli or Bacillus subtilis or both. One of these genes, pPC130, was expressed as a lacZ transcriptional fusion in E. coli at a level sufficient to produce phase-bright inclusions, which were purified and shown to be toxic to Aedes aegypti larvae. The second gene, pCH130, was expressed at a low level in recombinant E. coli cells and was therefore cloned in B. subtilis as a transcriptional fusion of the promoter sequences corresponding to a B. thuringiensis subsp. israelensis 27-kDa delta-endotoxin (E. S. Ward, A. R. Ridley, D. J. Ellar, and J. A. Todd, J. Mol. Biol. 191:13-22, 1986) and the structural gene. Recombinant B. subtilis cells produced phase-bright inclusions during late sporulation; these were partially purified and shown to be toxic to A. aegypti larvae at an LC50 (concentration required to cause 50% mortality of larvae after 24 h of assay) which is significantly lower than that of the pPC130 protein. Neither 130-kDa protein was hemolytic under the assay conditions. Comparison of the nucleotide sequences of these two genes indicates that they share a high degree of homology in the C-terminal portions, but relatively little similarity in the N termini. In addition, significant homologies were found between the pCH130 gene and the HD-1 Dipel gene of B. thuringiensis subsp. kurstaki (H. E. Schnepf, H. C. Wong, and H. R. Whiteley, J. Biol. Chem. 260:6264-6272, 1985).

Aedes↗