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Biomedical subjects

B L Davidson

Publications and source records attributed to B L Davidson.

At least 145 records · Page 8Linked to original sources
PubMed1994

Direct plasmid mediated transfection of adult murine brain cells in vivo using cationic liposomes.

Gene transfer to the central nervous system (CNS) is complicated by the anatomic and physiologic isolation of the brain. Direct injection techniques circumvent this, and allow delivery of transgenes to specific areas of the CNS. Previously, direct transfection of cellular components of the CNS has been achieved using plasmid DNA. We report the use of cationic liposomes as a means of transfecting plasmids into adult mammalian brain. Using the gene for E. coli beta-galactosidase or the cDNA or human beta-glucuronidase as reporters, we demonstrate plasmid mediated gene transfer into the caudate putamen of adult mice with expression of the transgene for at least 21 days post-transfection.

Animals↗
PubMed1994

Expression of Escherichia coli beta-galactosidase and rat HPRT in the CNS of Macaca mulatta following adenoviral mediated gene transfer.

Adenoviral-mediated gene transfer to the caudate nucleus of Macaca mulatta was accomplished using stereotactic injection of two distinct recombinant Ad5 vectors containing the gene for Escherichia coli beta-galactosidase and the cDNA for rat hypoxanthine-guanine phosphoribosylphosphotransferase (HPRT), respectively. Multiple analyses (including immunohistochemistry, histochemistry, transmission electron microscopy, RNA in situ hybridization, nucleotide pool analysis, and enzyme assay) confirmed efficient expression of beta-galactosidase and rat HPRT. Transgene expression was evident in both neurons and glia. Clinically, no evidence of meningitis or cerebritis was observed and no focal neurological deficits were detected in the animal. These preliminary studies indicate that recombinant adenovirus is capable of mediating high level transgene expression to the brains of higher order mammals.

Adenoviridae↗
PubMed1994

A 13 base pair deletion in exon 1 of HPRTIllinois forms a functional GUG initiation codon.

More than 50 mutations in the human hypoxanthine-guanine phosphoribosyltransferase (HPRT) locus have been described, yet only 2 alter the AUG initiation codon. One, variant HPRT1151, results in Lesch-Nyhan syndrome (LNS), and the other, HPRTIllinois, results in partial HPRT deficiency. Although previously undetectable, we used a sensitive gel assay to demonstrate that HPRTIllinois is not only active, but has a native Mr indistinguishable from normal. Confirmatory evidence of activity and native Mr is demonstrated following transfection of HPRT cells with expression plasmids containing cDNA sequences representing HPRTIllinois. These data provide support for the hypothesis that patient RT, or variant HPRTIllinois, is spared manifestations of the LNS as a result of translation at the newly formed GUG initiation codon.

Base Composition↗
PubMed1994

Vaccination of Thai infants with rhesus-human reassortant tetravalent oral rotavirus vaccine.

In a randomized double blind placebo-controlled study, the rhesus-human reassortant tetravalent oral rotavirus vaccine (dose 4 x 10(4) plaque-forming units) was evaluated in Thai infants immunized at ages 2, 4 and 6 months. To investigate dose responses and to compare vaccine-induced and naturally acquired rotavirus immunity in the study population blood specimens were collected before and 1 month after each vaccination and at 12 months of age. No adverse reactions attributable to the vaccine were detected in the vaccinees. Sixty-three of 94 (67%) vaccine recipients showed a seroconversion in rotavirus IgA enzyme-linked immunosorbent assay antibodies after one or more doses, whereas only 15 of 93 (16%) placebo-vaccinated control children showed an IgA enzyme-linked immunosorbent assay antibody response, suggestive of natural rotavirus infection, between 2 and 7 months of age. By measuring rhesus rotavirus-neutralizing antibodies a seroconversion was detected in 49% of the vaccinees and 14% of the controls between 2 and 7 months of age. The geometric mean titers of neutralizing antibodies to human rotavirus serotypes 1, 2, 3 and 4 after the completion of vaccinations and at 12 months of age were higher in the vaccinees than in the controls. It is concluded that, even though maternally acquired rotavirus antibodies are commonly present, the rhesus-human reassortant tetravalent vaccine is immunogenic in many Thai infants ages 2 to 6 months. The immunogenicity of this vaccine is enhanced by multiple doses.

Administration, Oral↗
PubMed1994

Enhanced cytotoxicity of antiviral drugs mediated by adenovirus directed transfer of the herpes simplex virus thymidine kinase gene in rat glioma cells.

The antiviral agents ganciclovir, 1-beta-D-arabinofuranosylthymine (araT), acyclovir, and 5-iodo-5'-amino-2',5'-dideoxyuridine were cytotoxic to rat C6 glioma cells expressing retrovirally transferred herpes simplex virus (HSV) type 1 thymidine kinase (TK) coding sequence, with concentrations that inhibited cell survival by 50% (IC50 values) of 0.06, 3, 13, and 23 mumol/L, respectively. In C6 cells not expressing HSV-TK, the IC50 value for ganciclovir was 140 mumol/L and a concentration of 1 mmol/L killed more than 99% of the cells. The other antiviral agents tested were less toxic in nontransduced cells. Compared with retrovirally transduced cells, transduction of C6BU1 cells with an adenovirus vector containing the coding sequence for HSV-TK (Ad.RSVtk) increased the cellular activity of the viral kinase up to 600-fold with increasing multiplicity of infection (MOl). Cells transduced with Ad.RSVtk exhibited as much as a fivefold and 12-fold decrease in IC50 value for ganciclovir and araT, respectively, compared with retrovirally transduced cells. Sensitivity to antiviral drugs increased with increasing exposure to Ad.RSVtk, with IC50 values of 0.6 and 0.005 mumol/L for araT and ganciclovir, respectively, at an MOl of 1000. These data suggest that adenoviral transfer of HSV-TK will allow the use of less toxic drugs or lower concentrations of toxic drugs such as ganciclovir for directed antitumor therapy in vivo.

Acyclovir↗
PubMed1994

In vivo transfer of a reporter gene to the retina mediated by an adenoviral vector.

PURPOSE: The ability of replication-deficient adenovirus to mediate gene transfer to retinal cells was evaluated. METHODS: A replication-deficient adenoviral vector, AdCMV beta A.ntlacZ, which contains the bacterial beta-galactosidase (lacZ) reporter gene, was injected into the subretinal space of normal, rd, and rds strains of mice at various ages. The efficiency and duration of transgene expression were assessed by histochemical examination and transmission electron microscopy. RESULTS: AdCMV beta A.ntlacZ was effective in mediating gene transfer to the retinal pigment epithelial cells, rod and cone photoreceptor cells, and cells in the inner nuclear layer of the retina for periods of up to 1 month. Gene transfer to retinal pigment epithelial cells occurred at much lower viral titers than was required for gene transfer to photoreceptor cells. The extent to which photoreceptor cells could be transduced varied with the age of the animals and the conditions of the photoreceptor cells: greater numbers of photoreceptor cells were transduced in 5- to 7-day-old pups and in mice at the initial stages of photoreceptor degeneration than in normal adult mice. No evidence of gross pathogenic effects or viremia in recipient mice was observed. CONCLUSIONS: Replication-deficient adenovirus mediates transfer and expression of a foreign gene in retinal pigment epithelial and photoreceptor cells. Gene transfer to photoreceptor cells is enhanced in developing retinas or at the predegenerate stage of photoreceptors in genetically programmed retinal degeneration.

Adenoviruses, Human↗
PubMed1993

Synthesis of normal and variant human hypoxanthine-guanine phosphoribosyltransferase in Escherichia coli.

Naturally occurring mutations in hypoxanthine-guanine phosphoribosyltransferase (HPRT) have been identified by amino acid sequencing, cDNA cloning, and direct nucleotide sequencing of PCR-amplified transcripts. To determine the effect these mutations have on the catalytic properties of the molecule, knowledge of the three-dimensional structure of HPRT is required. A prerequisite for this, however, is the availability of a large amount of purified product for crystallization and x-ray diffraction analysis. For these reasons we have developed an effective means of producing high levels of human HPRT in Escherichia coli using the expression cassette PCR. By taking advantage of a T7 polymerase/promoter system, we have expressed both normal and variant human hprt sequences in E. coli. The proteins synthesized from these sequences are immunologically and enzymatically active, and are physically indistinguishable from the HPRT in B-lymphoblasts derived from normal and three HPRT-deficient subjects.

Base Sequence↗
PubMed1993

A model system for in vivo gene transfer into the central nervous system using an adenoviral vector.

Previous methods of in vivo gene transfer to differentiated neurons of the adult mammalian brain have been inefficient and associated with technical problems. We have therefore developed a model system of direct gene transfer using a replication-defective adenoviral vector containing a beta-galactosidase gene to transduce brain neurons. Following injection of purified high titre recombinant adenovirus into the caudate putamen of seven week old mice, lacZ activity was evident in neural components of the central nervous system (CNS) for at least 8 weeks post infection. The efficiency of adenoviral gene transfer was very high compared to other techniques, suggesting an attractive and efficient alternative for neuronal gene transfer in vivo.

Adenoviridae↗
PubMed1993

Take of rhesus-human reassortant tetravalent rotavirus vaccine in breast-fed infants.

Rhesus-human reassortant tetravalent rotavirus vaccine at a titer of 4 x 10(4) plaque forming units was evaluated for immunogenicity in 194 6-8-week-old breast-fed Turkish infants. The vaccine was administered orally as a single dose following either a meal of breast milk or 30 ml of sodium bicarbonate-buffered soy milk formula. Four-fold or greater responses in rotavirus IgA ELISA antibody were detected in 62% and 65% of the infants in the two groups, respectively (p = 0.62). In a smaller comparison group of non-breast-fed infants, an IgA response was detected in 7 of 11 (64%) cases. In all vaccinees, a serological response was detected in 72% of the initially seronegative and 47% of the initially seropositive infants (p = 0.001). We conclude that the take of rhesus-human reassortant tetravalent rotavirus vaccine in breast-fed infants is not compromised by breast feeding before vaccination. However, a higher titered preparation of the same vaccine may be required to improve overall immunogenicity in young infants, particularly in those with pre-vaccination rotavirus antibody.

Administration, Oral↗
PubMed1993

Adenoviral-mediated gene transfer to rabbit synovium in vivo.

Currently, treatment for rheumatoid arthritis and other inflammatory arthropathies is often ineffective in ameliorating the progression of the disease, particularly the invasive destruction of cartilage and bone by rheumatoid synovium. Multiple aspects of this inflammatory process are mediated by the synovial lining cells (synoviocytes). Genetic modification of these cells in vivo represents a potential method for the treatment of these conditions. In this report, we describe a novel technique for the genetic transduction of synovial lining cells in vivo using recombinant adenoviral vectors and intraarticular injection techniques. Purified high titer suspensions of a recombinant adenoviral vector containing the gene for Escherichia coli beta-galactosidase (AdCMVlacZ) were directly injected into the hind knees of New Zealand white rabbits. Synovial tissues were then examined for transgenic lacZ expression using a combination of in situ staining for beta-galactosidase activity, immunohistochemical staining, and transmission electron microscopy. High efficiency gene transfer and lacZ expression was observed in both type A and type B synoviocytes throughout the articular and periarticular synovium of the rabbit knee, with continued expression of transgenic lacZ detected for > or = 8 wk after infection.

Adenoviridae↗
PubMed1992

Low accuracy of color Doppler ultrasound in the detection of proximal leg vein thrombosis in asymptomatic high-risk patients. The RD Heparin Arthroplasty Group.

OBJECTIVE: To evaluate the ability of color Doppler ultrasound to detect proximal deep venous thrombosis (DVT) in asymptomatic high-risk patients who subsequently underwent contrast venography. DESIGN: Prospective cohort study using blinded observers, with contrast venography as the comparison standard. SETTING: Seven medical centers (university and community hospitals) participating in a clinical trial of low-molecular-weight heparin for prevention of DVT. PATIENTS: A total of 385 consecutive patients undergoing elective unilateral hip or knee replacement. MEASUREMENTS: Ten days after surgery or before hospital discharge (whichever occurred first), patients had bilateral color Doppler ultrasound examinations of the proximal veins of the lower extremities. Subsequently, a contrast venogram of the operated leg was obtained. RESULTS: Color Doppler ultrasound studies and venograms were both evaluable in 319 of the 385 patients. Deep venous thrombosis was identified by contrast venography in 80 patients (prevalence, 25%; 95% CI, 20% to 30%) and involved the proximal veins in 21 patients (prevalence, 7%; CI, 4% to 10%). For proximal DVT, color Doppler ultrasound showed poor sensitivity (38%; CI, 18% to 62%), moderately good specificity (92%; CI, 89% to 95%), and a poor positive predictive value for this population (26%). CONCLUSION: Color Doppler ultrasound examinations are insensitive to proximal DVT in asymptomatic high-risk patients and should not be substituted for venography for identifying proximal DVT in such patients.

Adolescent↗
PubMed1992

Safety and immunogenicity of oral tetravalent human-rhesus reassortant rotavirus vaccine in neonates.

We conducted a prospective randomized double blind study to determine: (1) the safety and immunogenicity of live oral tetravalent human-rhesus rotavirus reassortant vaccine in neonates; and (2) whether a second dose at the age of 6 to 8 weeks enhances the immunogenicity. Two hundred forty healthy neonates were enrolled and received vaccine (183) or placebo (57) on the second day of life. At the age of 6 to 8 weeks 133 received placebo and 88 received a second dose of vaccine. Medical events were noted within 10 days from vaccine administration in 6 of 183 (3.3%) vaccine recipients vs. 0 of 57 placebo recipients (P = 0.34) after the first dose and in 8 of 88 (9%) vs. 4 of 133 (3%) after the second dose (P = 0.069); none was severe and all were of short duration. Seroresponse of any type (detectable IgA or 4-fold increase of titer to rhesus rotavirus was 9% for the placebo, vs. 52 and 46% for those who received one and two doses of vaccine, respectively. However, neutralizing antibodies against human serotypes 1, 2 and 3 were not raised successfully in vaccinated infants when compared with placebo recipients. The same pattern was found when geometric mean titers were compared. Vaccine take was better when cord blood titers were low. At the age of 1 year the vaccinees had more often high titers for antirhesus rotavirus antibodies (> 640) than the placebo recipients (49% vs. 0%; P < 0.001). NO difference was found between the groups in neutralizing antibodies to human serotypes 1, 2 and 3 rotavirus.(ABSTRACT TRUNCATED AT 250 WORDS)

Administration, Oral↗
PubMed1991

Immunogenicity of tetravalent rhesus rotavirus vaccine administered with buffer and oral polio vaccine.

Between January and November 1989, we studied 174 infants aged 6 to 16 weeks in a randomized clinical trial to (1) determine the immunogenicity of a single dose of tetravalent rhesus rotavirus vaccine (RRV-TV) when administered with three different buffer regimens: no antacid buffer and small-volume (2.5-mL) and large-volume (30-mL) antacid buffer; and (2) examine the potential interference of RRV-TV on the immune response to oral polio vaccine. Immunogenicity of RRV-TV, measured as a fourfold rise in antibody titers to rotavirus, was similar in the groups receiving small- and large-dose buffer (45% and 49%, respectively) and significantly less in the group that received RRV-TV alone (23%). Administration of RRV-TV with oral polio vaccine did not significantly interfere with the neutralization response of oral polio vaccine poliovirus serotypes 1, 2, or 3, and overall, 29%, 87%, and 24% of the infants had a fourfold rise in titer to each serotype, respectively.

Antibodies, Viral↗
PubMed1991

Expression of normal and variant human hypoxanthine-guanine phosphoribosyltransferase in E. coli.

1. ECPR is a rapid and effective means for generating recombinant human HPRT. 2. The Bl21 (DE3) T7 polymerase/T7 promoter system provides high level expression of human HPRT constructs after induction of the T7 polymerase gene with IPTG. 3. Human HPRT constructs expressed in E. coli mimic the variant properties originally demonstrated in lymphoblast extracts from affected individuals. 4. Human HPRT expressed in E. coli can be rapidly purified to near homogeneity by a two step purification scheme.

Base Sequence↗
PubMed1991

Determination of the mutations responsible for the Lesch-Nyhan syndrome in 17 subjects.

Hypoxanthine--guanine phosphoribosyltransferase (HPRT) is a purine salvage enzyme that catalyzes the conversion of hypoxanthine to inosine monophosphate and guanine to guanosine monophosphate. Previous studies of mutant HPRT proteins analyzed at the molecular level have shown a significant heterogeneity. This investigation further verifies this heterogeneity and identifies insertions, deletions, and point mutations. The direct sequencing of the polymerase chain reaction-amplified product of reverse-transcribed HPRT mRNA enabled the rapid identification of the mutations found in 17 previously uncharacterized cell lines derived from patients with the Lesch-Nyhan syndrome.

Cell Line↗
PubMed1991

Identification of 17 independent mutations responsible for human hypoxanthine-guanine phosphoribosyltransferase (HPRT) deficiency.

Complete hypoxanthine-guanine phosphoribosyltransferase (HPRT) deficiency causes the Lesch-Nyhan syndrome, an X-linked, purine metabolism disorder manifested by hyperuricemia, hyperuricaciduria, and neurologic dysfunction. Partial HPRT deficiency causes hyperuricemia and gout. One requirement for understanding the molecular basis of HPRT deficiency is the determination of which amino acids in this salvage enzyme are necessary for structural or catalytic competence. In this study we have used the PCR coupled with direct sequencing to determine the nucleotide and subsequent amino acid changes in 22 subjects representing 17 unrelated kindreds from the United Kingdom. These mutations were confirmed by using either RNase mapping or Southern analyses. In addition, experiments were done to determine enzyme activity and electrophoretic mobility, and predictive paradigms were used to study the impact of these amino acid substitutions on secondary structure.

Chromosome Deletion↗