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Biomedical subjects

R W Ellis

Publications and source records attributed to R W Ellis.

At least 55 records · Page 3Linked to original sources

A simple method of estimating cerebrospinal fluid pressure during lumbar puncture.

It is often difficult to measure cerebrospinal fluid (CSF) pressure in children. CSF flow through a spinal needle is described by the equation: Flow = pressure/(needle constant x relative viscosity). Thus, CSF flow rate during lumbar puncture can be used to estimate CSF pressure. Because the viscosity of CSF is approximately the same as that of normal saline, 0.9% NaCl was used to model CSF flow in vitro. Flow of saline through various spinal needles was measured as pressure and temperature were varied to determine needle constants and variation in viscosity with temperature. Counting periods for which the number of drops counted equals the pressure (in centimeters of H2O) then were determined for each needle size. At patient temperatures less than 40 degrees C, counting periods were calculated at 21, 39, and 12 seconds, for 22-gauge 1.5-inch, 22-gauge 3.5-inch, and 20-gauge 3.5-inch spinal needles, respectively. Viscosity decreased slightly above 40 degrees C, and counting periods became 20, 37, and 11 seconds. Finally, the method was tested prospectively in 12 patients by comparing drop count (over the calculated counting period) to manometric pressure measurement. Drop counts were within 15% of manometric pressure in all patients. This method allows simple and rapid estimation of CSF pressure during lumbar puncture.

Cerebrospinal Fluid Pressure↗

Protein disulfide isomerase is essential for viability in Saccharomyces cerevisiae.

Protein disulfide isomerase (PDI) is an enzyme involved in the catalysis of disulfide bond formation in secretory and cell-surface proteins. Using an oligodeoxyribonucleotide designed to detect the conserved 'thioredoxin-like' active site of vertebrate PDIs, we have isolated a gene encoding PDI from the lower eukaryote, Saccharomyces cerevisiae. The nucleotide sequence and deduced open reading frame of the cloned gene predict a 530-amino-acid (aa) protein of Mr 59,082 and a pI of 4.1, physical properties characteristic of mammalian PDIs. Furthermore, the aa sequence shows 30-32% identity with mammalian and avian PDI sequences and has a very similar overall organisation, namely the presence of two approx. 100-aa segments, each of which is repeated, with the most significant homologies to mammalian and avian PDIs being in the regions (a, a') that contain the conserved 'thioredoxin-like' active site. The N-terminal region has the characteristics of a cleavable secretory signal sequence and the C-terminal four aa (-His-Asp-Glu-Leu) are consistent with the protein being a component of the S. cerevisiae endoplasmic reticulum. Transformants carrying multiple copies of this gene (designated PDI1) have tenfold higher levels of PDI activity and overproduce a protein of the predicted Mr. The PDI1 gene is unique in the yeast genome and encodes a single 1.8-kb transcript that is not found in stationary phase cells. Disruption of the PDI1 gene is haplo-lethal indicating that the product of this gene is essential for viability.

Amino Acid Sequence↗

Production of hepatitis B surface antigen (preS2 + S) by high-cell density cultivations of a recombinant yeast.

A high-cell density bioprocess has been developed for the production of hepatitis B surface protein (preS2 + S) by recombinant yeast. This fed-batch process utilizes a growth medium containing yeast extract, soy peptone and glucose which was fed at a constant rate to maintain cells in a respiratory state. Cell densities of up to 60 g l-1 dry weight were achieved, which represented a 6-fold increase over those from batch bioprocesses. This increase in cell mass was attained without compromising specific activity; therefore, volumetric productivities of six times those of batch bioprocesses were achieved.

Hepatitis B Surface Antigens↗

Biological activity of Hib conjugates.

Haemophilus influenzae type b (Hib) conjugate vaccines dramatically improve the immunogenicity against the capsular polysaccharide (PRP) of Hib. A new Hib conjugate, PedvaxHIB, is shown to be immunogenic in infant rhesus monkeys. The monkey model appears to correlate well with the immunogenicity of PedvaxHIB in human clinical studies. Not all commercial Hib conjugates are immunogenic in the monkey model. The data from the priming study indicate that HibTITER is not immunogenic in an immune system naive to diphtheria toxoid, such as the infant rhesus monkey. The role of diphtheria toxoid in the immunogenicity of HibTITER in human infants should be studied.

Animals↗

Antibody assays suitable for assessing immune responses to live varicella vaccine.

An enzyme-linked immunosorbent assay for antibodies to varicella-zoster virus (VZV), using purified viral glycoproteins as antigen (gpELISA), was compared with other assays for measuring vaccine-induced antibody responses. The gpELISA was more sensitive than conventional assays, proved highly specific for VZV and agreed well with an assay for neutralizing antibody activity. It was successfully applied to large-scale testing of live varicella vaccine in humans.

Antibodies, Viral↗

An enzyme-linked immunosorbent assay for quantitation of Haemophilus Influenzae type b polysaccharide-specific IgG1 and IgG2 in human and infant rhesus monkey sera.

An enzyme-linked immunosorbent assay (ELISA) has been developed and validated to quantitate IgG1 and IgG2 antibody to polyribosyl-ribitol phosphate (PRP), the capsular polysaccharide of Haemophilus influenzae type b (Hib). The sera of children and infant Rhesus monkeys immunized with an Hib conjugate vaccine composed of Hib PRP covalently linked to an outer membrane protein complex (OMPC) from Neisseria meningitidis serogroup B (PedvaxHIB, PRP-OMPC, Merck, Sharp and Dohme Research Laboratories). The solid-phase antigen employed in the ELISA is a conjugate of PRP to human serum albumin. The enzyme-labeled antibody is alkaline phosphatase-conjugated mouse monoclonal (mAb) anti-human IgG1 or IgG2. A human serum standard was calibrated using parallel titrations with a known antibody standard. The geometric mean titer (GMT) of the anti-PRP IgG1 response to one dose of PedvaxHIB was 3.87 micrograms/ml (n = 82), 11.80 micrograms/ml (n = 62) and 14.57 micrograms/ml (n = 74) in infants and children 12 to 17 months, 18 to 23 months and greater than or equal to 24 months old, respectively. Infants 2 to 11 months old responded with an IgG1 anti-PRP response of 7.10 micrograms/ml while infant monkeys responded with a GMT of 150.65 (n = 9) after two doses of vaccine. The anti-PRP IgG2 GMT responses in all groups were less than 0.25 micrograms/ml, except for humans greater than or equal to 18-months old who exhibited a GMT of greater than or equal to 0.40 micrograms/ml (n = 75). PedvaxHIB, immunization of human infants and children and infant Rhesus monkeys elicits primarily an IgG1 response to PRP. The monkey model appears to be a reliable indicator of the human immune response.

Animals↗

Immunogenicity of Haemophilus influenzae type b conjugate vaccines in infant rhesus monkeys.

Two Haemophilus influenzae type b (Hib) polysaccharide-protein conjugate vaccines were evaluated for immunogenicity in eliciting anti-polyribosyl ribitol phosphate (PRP) antibodies in infant rhesus monkeys. Animals received intramuscular injections of either Hib polysaccharide (PRP)-meningococcal outer membrane protein complex or Hib oligosaccharide-CRM197 (HbOC) conjugate vaccines on d 0, 28, and 56. Because HbOC contains the CRM197 mutant diphtheria toxin from Corynebacterium diphtheriae as its protein carrier, the effect of simultaneous injection of diphtheria toxoid on the immunogenicity of HbOC also was evaluated by dividing monkeys vaccinated with HbOC into three groups: HbOC/saline, HbOC/diphtheria and tetanus toxoids, and HbOC/tetanus toxoid (coadministration of HbOC and other vaccine or placebo injected into the flank muscle of different legs). Infant monkeys vaccinated with the PRP-outer membrane protein complex conjugate responded with anti-PRP antibody after the first dose and showed booster responses after the second and third injections. In contrast, infant monkeys vaccinated with HbOC did not respond after three doses of HbOC/saline or HbOC/tetanus toxoid. However, two of three monkeys given concurrent injections of HbOC and diphtheria and tetanus toxoids did respond. The nonresponder monkey to three doses of HbOC and diphtheria and tetanus toxoids did respond to a subsequent injection with PRP-outer membrane protein complex. Thus, concomitant administration of diphtheria toxoid, a common vaccine for human infants, is necessary to elicit an anti-PRP antibody response to HbOC.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Varicella vaccine (VARIVAX) in healthy children and adolescents: results from clinical trials, 1987 to 1989.

A total of 3303 healthy children and adolescents, aged 12 months to 17 years, were vaccinated with one of five production lots of a live attenuated varicella vaccine (VARIVAX) containing 1000 to 1625 plaque-forming units per dose. The vaccine was generally well tolerated. Ninety-six percent (2381/2475) of vaccinees responded to vaccination by producing antibody as measured by a glycoprotein-based enzyme-linked immunosorbent assay; 99% (569/576) of those tested maintained antibody at 1 year following vaccination. The incidence of varicella following household exposure in vaccinees was approximately 12%; household contact historically results in 87% infection. Nearly all of the vaccinees who had varicella after vaccination had a clinically modified disease.

Adolescent↗

Characterization of recombinant tick anticoagulant peptide. A highly selective inhibitor of blood coagulation factor Xa.

Tick anticoagulant peptide (TAP) is a potent, highly selective inhibitor of blood coagulation factor Xa (Waxman, L., Smith, D. E., Arcuri, K. E., and Vlasuk, G. P. (1990) Science, 248, 593-596). Further detailed studies pertaining to the in vitro and in vivo evaluation of TAP require quantities of the inhibitor which cannot be isolated from ticks. To overcome this limitation we describe here the characterization of recombinant TAP (rTAP) secreted by Saccharomyces cerevisiae. Expression of rTAP was obtained using a chimeric gene containing a fusion between sequences encoding the secretory preproleader of the yeast mating pheromone alpha-factor and a synthetic sequence encoding the 60-amino acid inhibitor under the transcriptional control of a galactose-inducible promoter. Recombinant S. cerevisiae were found to secrete biologically active rTAP into the extracellular medium at levels of 0.1-0.15 g/liter. The secreted inhibitor was purified to homogeneity and found to be indistinguishable from the native inhibitor with respect to several criteria, including primary structure, amino acid composition, and electrophoretic mobility. In addition, purified rTAP and native TAP exhibited similar stoichiometric inhibition of factor Xa in vitro. The in vivo efficacy of rTAP was demonstrated using a model of low grade disseminated intravascular coagulation where the purified inhibitor was shown to significantly inhibit thromboplastin-induced fibrinopeptide A generation following an infusion into conscious rhesus monkeys. The availability of rTAP will allow a detailed evaluation of the in vitro and in vivo properties of this highly specific and potent factor Xa inhibitor.

Amino Acid Sequence↗

Purification and characterization of human plasminogen activator inhibitor type I expressed in Saccharomyces cerevisiae.

The rapidly acting inhibitor of plasminogen activators, PAI-1, was produced intracellularly in Saccharomyces cerevisiae by using the ADH2 promoter to drive the expression of the human PAI-1 cDNA. Approximately 8 mg of human PAI-1 was produced per liter of confluent yeast culture. A purification scheme which resulted in 20% recovery of isolated PAI-1 from the broken yeast cell homogenate was devised. Yeast-derived human PAI-1 differs from endothelial-type PAI-1 isolated from HT1080 fibrosarcoma cells in that the recombinant inhibitor does not contain carbohydrate side chains. Nevertheless, the activity and other functional attributes of yeast-derived PAI-1 are similar to those exhibited by HT1080 fibrosarcoma cell-derived PAI-1. Hence, this study demonstrates that expression of human PAI-1 in yeast is a viable strategy for the production of ample quantities of this key modulator of plasminogen activator-mediated proteolysis.

Antigens↗

Key issues in the selection of an expression system for vaccine antigens.

Three criteria by which the appropriate host cell is chosen for the expression of a recombinant-derived vaccine antigen are efficacy, safety, and scale-up. Efficacy for a vaccine antigen refers to the ability of the host cell to produce a vaccine antigen capable of eliciting a protective immune response. A concern for safety of a vaccine antigen relates to residual DNA in the final product, especially when derived from continuous mammalian cell lines as opposed to microbial cells. Since tens (or hundreds) of millions of doses of a widely used vaccine might be injected into healthy infants and young children during the lifetime of the product, safety is a critical issue, such that the use of a microbial expression system might be preferable to the use of a continuous cell line in certain circumstances.

Animals↗

Vaccination of infants and children against hepatitis B.

Hepatitis B infection and its sequelae constitute a significant public health problem in the United States. It is estimated that 300,000 acute hepatitis B infections occur each year, with about 25% accompanied by both clinical illness and jaundice. Some infections become chronic and ultimately may cause development of liver cirrhosis or primary hepatocellular carcinoma. The risk of chronicity is especially great with infections that occur in infancy. Highly effective vaccines comprised of purified hepatitis B surface antigen (HBsAg) particles are now available for the prevention of hepatitis B infection. A first-generation vaccine utilized HBsAg derived from the plasma of infected persons; this has now been replaced by two similar vaccines that incorporate HBsAg produced by genetically engineered strains of the common bakers' yeast, Saccharomyces cerevisiae. Improved use of vaccine is needed to reduce and ultimately eliminate hepatitis B infection. Vaccination already is recommended for persons recognized to be at increased risk of exposure to virus-containing blood or other body fluids (e.g., infants born to carrier mothers, household or sexual contacts of carriers); however, mass vaccination of adolescents and infants is needed to interdict effectively a majority of all exposures to the hepatitis B virus.

Adolescent↗

In vitro immune responses to hepatitis B surface antigens S and preS2 during acute infection by hepatitis B virus in humans.

This study evaluated the in vitro immune responses to different components of the hepatitis B surface antigen (HBsAg) over the course of acute hepatitis B virus (HBV) infection. Early in the convalescent phase of infection, while HBsAg was present in the serum, peripheral blood mononuclear cells (PBMCs) were stimulated with preS2 peptide or hepatitis B surface protein. Specific IgG directed to different components of HBsAg was produced without a polyclonal increase in total IgG production. Stimulation with preS2 peptide produced IgG to the preS2 peptide (anti-preS2) and to the S antigen (anti-HBs). Hepatitis B surface protein stimulation produced anti-HBs but not anti-preS2. After this initial reactive phase, the PBMCs did not produce specific antibody when stimulated with either component of HBsAg; this effect lasted greater than 1 year. At some time 1-2 years after acute infection, the pattern of in vitro S antigen- and preS2 antigen-stimulated anti-HBs response reemerged in the PBMCs. Reemergence of sustained preS2 peptide-stimulated anti-preS2 response was not observed.

Cells, Cultured↗

Cell-mediated immune responses after immunization of healthy seronegative children with varicella vaccine: kinetics and specificity.

Humoral and cell-mediated immune responses were determined in seronegative children immunized with live attenuated Oka strain varicella vaccine. At 2 weeks after immunization, 80% of children had detectable lymphocyte proliferation to varicella-zoster virus (VZV) antigens, while only 40% had antibodies to VZV as detected by ELISA. By 6 weeks after immunization, 97% of children seroconverted, and 95% of these responded to VZV antigens in the proliferation assay. A high proportion of immunized children also responded in the proliferation assay to purified glycoproteins I, II, and III of VZV. These results indicate that most children develop a broad cell-mediated immune response to VZV antigens within weeks after immunization with varicella vaccine.

Antibodies, Viral↗

Haemophilus influenzae type b conjugate vaccine (meningococcal protein conjugate) (PedvaxHIB): clinical evaluation.

Although systemic infections caused by Haemophilus influenzae type b occur worldwide, detailed epidemiologic data are available in but a few countries. The public health impact of morbidity, mortality, and serious sequelae from disease caused by H influenzae type b has stimulated the search for control strategies. In the United States now, active immunoprophylaxis is largely favored over treatment of prophylaxis with antibiotics. This preference stems from three major observations: that high mortality and morbidity persist despite the availability of potent antimicrobial agents, that antibiotic-resistant strains of H influenzae type b have emerged, and that implementation of antimicrobial prophylaxis on a large scale has been unsatisfactory. Moreover, universal vaccination has been projected as offering a higher economic benefit than other control strategies. A matter of more proximate importance, however, is the search for H influenzae type b vaccines that will confer protection to all age groups, including infants younger than 18 months of age and subpopulations specifically at risk for invasive disease caused by H influenzae type b. Haemophilus b conjugate vaccine (meningococcal protein conjugate), PedvaxHIB (PRP-OMPC), is a conjugate H influenzae type b vaccine developed at Merck Sharp & Dohme Research Laboratories that now is undergoing extensive clinical evaluation to assess its prospects for disease control when first administered in early infancy. This is an interim report of results obtained in studies conducted in diverse locations throughout the United States.

Antibodies, Bacterial↗

The Minnesota project: a focused approach to ambulatory quality assessment.

With national HMO quality assurance requirements pending for Medicare risk contracts, three HMOs in Minnesota established a working group with the state Department of Health to develop and test a new methodology proposed for quality of care review. A two-tiered system was developed for ambulatory chart review based on 15 hospitalization diagnoses having a potential for inadequate prehospital care. This system was applied to 796 cases from the HMOs (2% of admissions). Technical problems limited actual review to 673 of these cases. Although 304 (45%) of reviewed cases failed initial screening, physician review found only 22% of such failures (10% of reviewed cases) to represent probable quality of care problems. The approach appears to be feasible and unusually efficient. Although there is considerable variability that limits its potential use for interhealth plan comparison, the approach holds promise for quality assurance within an individual health plan.

Ambulatory Care↗