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Biomedical subjects

Steven Reid

Publications and source records attributed to Steven Reid.

At least 19 recordsLinked to original sources
PubMed2007

Efficient removal of immunoglobulin free light chains by hemodialysis for multiple myeloma: in vitro and in vivo studies.

Of patients with newly diagnosed multiple myeloma, approximately 10% have dialysis-dependent acute renal failure, with cast nephropathy, caused by monoclonal free light chains (FLC). Of these, 80 to 90% require long-term renal replacement therapy. Early treatment by plasma exchange reduces serum FLC concentrations, but randomized, controlled trials have shown no evidence of renal recovery. This outcome can be explained by the low efficiency of the procedure. A model of FLC production, distribution, and metabolism in patients with myeloma indicated that plasma exchange might remove only 25% of the total amount during a 3-wk period. For increasing FLC removal, extended hemodialysis with a protein-leaking dialyzer was used. In vitro studies indicated that the Gambro HCO 1100 dialyzer was the most efficient of seven tested. Model calculations suggested that it might remove 90% of FLC during 3 wk. This dialyzer then was evaluated in eight patients with myeloma and renal failure. Serum FLC reduced by 35 to 70% within 2 hr, but reduction rates slowed as extravascular re-equilibration occurred. FLC concentrations rebounded on successive days unless chemotherapy was effective. Five additional patients with acute renal failure that was caused by cast nephropathy then were treated aggressively, and three became dialysis independent. A total of 1.7 kg of FLC was removed from one patient during 6 wk. Extended hemodialysis with the Gambro HCO 1100 dialyzer allowed continuous, safe removal of FLC in large amounts. Proof of clinical value now will require larger studies.

Acute Kidney Injury↗
PubMed2006

Importance of virus-medium interactions on the biological activity of wild-type Heliothine nucleopolyhedroviruses propagated via suspension insect cell cultures.

In vitro serial passaging of nucleopolyhedroviruses often results in virus instability, leading to reduction of both yield and biological activity of polyhedra (virus occlusion bodies). In this study, uncloned Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus (HaSNPV) and cloned Helicoverpa zea single-nucleocapsid nucleopolyhedrovirus (HzSNPV) were each serially passaged five times in both low cost (VPM) and commercial (Excell 401) media using H. zea cell cultures. When the experimental data was analysed as a 2(3) full factorial design (testing two levels of virus, medium and passage number), the passage number was shown to have the most significant effect on polyhedra yield, while the virus-medium interaction had the most important effect on polyhedra biological activity. This interaction was most pronounced for HzSNPV, which experienced a dramatic decline in biological activity when switched from its original Excell 401 medium to the in-house VPM medium. This suggests that genetically homogeneous viruses are less able to adapt to perturbations in the nutrient environment, hence medium changes should be avoided. Therefore, it is important to standardise the scale-up process of nucleopolyhedrovirus biopesticides at the earliest stage of development, especially when it requires both a low cost medium and a plaque purified virus.

Animals↗
PubMed2005

Synthesis and kinetic analysis of the N-acetylhexosaminidase inhibitor XylNAc-isofagomine.

[reaction: see text] An efficient 10-step preparation from 4-methoxypyridine of (2R,3R,4R)-2-acetamido-3,4-dihydroxypiperidine ("XylNAc-isofagomine") in optically active form is described. Key steps include an enantioselective reduction with catecholborane/(S)-2-methyl-CBS-oxazaborolidine, and a stereoselective pseudo-glycosylation of lithium azide by a cyclic sulfite ester. The title compound showed a Ki = 21 microM when evaluated against the N-acetyl-beta-hexosaminidase from Streptomyces plicatus.

Enzyme Inhibitors↗
PubMed2005

A cognitive-behavioural approach to the management of idiopathic cervical dystonia.

A case is described in which a patient with idiopathic cervical dystonia is treated successfully with cognitive-behavioural therapy. It was hypothesised that although the dystonia was organic in origin, it was exacerbated and perpetuated by catastrophic thoughts and abnormal illness beliefs, and that modification of maintaining cognitive and behavioural responses would lead to improvement. Self-report outcome measures were consistent with this hypothesis and improvements were maintained at 6-month follow-up.

Activities of Daily Living↗
PubMed2003

Purification and characterisation of functional early pregnancy factor expressed in Sf9 insect cells and in Escherichia coli.

Early pregnancy factor (EPF) is a secreted protein with growth regulatory and immunomodulatory properties. It is an extracellular form of the mitochondrial matrix protein chaperonin 10 (Cpn10), a molecular chaperone. An understanding of the mechanism of action of EPF and an exploration of therapeutic potential has been limited by availability of purified material. The present study was undertaken to develop a simple high-yielding procedure for preparation of material for structure/function studies, which could be scaled up for therapeutic application. Human EPF was expressed in Sf9 insect cells by baculovirus infection and in Escherichia coli using a heat inducible vector. A modified molecule with an additional N-terminal alanine was also expressed in E. coli. The soluble protein was purified from cell lysates via anion exchange (negative-binding mode), cation exchange, and hydrophobic interaction chromatography, yielding approximately 42 and 36mg EPF from 300ml bacterial and 1L Sf9 cultures, respectively. The preparations were highly purified (#10878;99% purity on SDS-PAGE for the bacterial products and #10878;97% for that of insect cells) and had the expected mass and heptameric structure under native conditions, as determined by mass spectrometry and gel permeation chromatography, respectively. All recombinant preparations exhibited activity in the EPF bioassay, the rosette inhibition test, with similar potency both to each other and to the native molecule. In two in vivo assays of immunosuppressive activity, the delayed-type hypersensitivity reaction and experimental autoimmune encephalomyelitis, the insect cell and modified bacterial products, both with N-terminal additions (acetylation or amino acid), exhibited similar levels of suppressive activity, but the bacterial product with no N-terminal modification had no effect in either assay. Studies by others have shown that N-terminal addition is not necessary for Cpn10 activity. By defining techniques for facile production of molecules with and without immunosuppressive properties, the present studies make it possible to explore mechanisms underlying the distinction between EPF and Cpn10 activity.

Animals↗
PubMed2002

Kinetics of baculovirus replication and release using real-time quantitative polymerase chain reaction.

The study of viral-based processes is hampered by (a) their complex, transient nature, (b) the instability of products, and (c) the lack of accurate diagnostic assays. Here, we describe the use of real-time quantitative polymerase chain reaction to characterize baculoviral infection. Baculovirus DNA content doubles every 1.7 h from 6 h post-infection until replication is halted at the onset of budding. No dynamic equilibrium exists between replication and release, and the kinetics are independent of the cell density at the time of infection. No more than 16% of the intracellular virus copies bud from the cell.

Animals↗
PubMed2002

Production of the baculovirus-expressed dengue virus glycoprotein NS1 can be improved dramatically with optimised regimes for fed-batch cultures and the addition of the insect moulting hormone, 20-Hydroxyecdysone.

A perennial problem in recombinant protein expression is low yield of the product of interest. A strategy which has been shown to increase the production of baculovirus-expressed proteins is to utilise fed-batch cultures. One disadvantage of this approach is the time-consuming task of optimising the feeding strategy. Previously, a statistical optimisation routine was applied to develop a feeding strategy that increased the yield of beta-Galactosidase (beta-Gal) by 2.4-fold (Biotechnol. Bioeng. 59 (1998) 178). This involves the single addition of nutrient concentrates (amino acids, lipids, glucose and yeastolate ultrafiltrate) into Sf 9 cell cultures grown in SF 900II medium. In this study, it is demonstrated that this optimised fed-batch strategy developed for a high-yielding intracellular product beta-Gal could be applied successfully to a relatively low-yielding glycosylated and secreted product such as the dengue virus glycoprotein NS1. Optimised batch infections yielded 4 microg/ml of NS1 at a peak cell density of 4.2 x 10 (6) cells/ml. In contrast, optimised fed-batch infections exhibited a 3-fold improvement in yield, with 12 microg/ml of NS1 produced at a peak cell density of 11.3 x 10 (6) cells/ml. No further improvements in yield were recorded when the feed volumes were doubled and the peak cell density was increased to 23 x 10 (6) cells/ml, unless the cultures were stimulated by the addition of 4 microg/ml of 20-Hydroxyecdysone (an insect moulting hormone). In this case, the NS1 yield was increased to 20 microg/ml, which was nearly 5-fold higher than optimised batch cultures.

Animals↗
PubMed2002

Phenotypic and genotypic analysis of Helicoverpa armigera nucleopolyhedrovirus serially passaged in cell culture.

Rapid accumulation of few polyhedra (FP) mutants was detected during serial passaging of Helicoverpa armigera nucleopolyhedrovirus (HaSNPV) in cell culture. 100% FP infected cells were observed by passage 6. The specific yield decreased from 178 polyhedra per cell at passage 2 to two polyhedra per cell at passage 6. The polyhedra at passage 6 were not biologically active, with a 28-fold reduction in potency compared to passage 3. Electron microscopy studies revealed that very few polyhedra were produced in an FP infected cell (<10 polyhedra per section) and in most cases these polyhedra contained no virions. A specific failure in the intranuclear nucleocapsid envelopment process in the FP infected cells, leading to the accumulation of naked nucleocapsids, was observed. Genomic restriction endonuclease digestion profiles of budded virus DNA from all passages did not indicate any large DNA insertions or deletions that are often associated with such FP phenotypes for the extensively studied Autographa californica nucleopolyhedrovirus and Galleria mellonella nucleopolyhedrovirus. Within an HaSNPV 25K FP gene homologue, a single base-pair insertion (an adenine residue) within a region of repetitive sequences (seven adenine residues) was identified in one plaque-purified HaSNPV FP mutant. Furthermore, the sequences obtained from individual clones of the 25K FP gene PCR products of a late passage revealed point mutations or single base-pair insertions occurring throughout the gene. The mechanism of FP mutation in HaSNPV is likely similar to that seen for Lymantria dispar nucleopolyhedrovirus, involving point mutations or small insertions/deletions of the 25K FP gene.

Amino Acid Sequence↗
PubMed2002

Frequent attenders with medically unexplained symptoms: service use and costs in secondary care.

BACKGROUND: Frequent attenders in medical settings account for a disproportionate amount of health-care resources. Little is known about service use and costs of secondary care in those frequent attenders presenting with medically unexplained symptoms. AIMS: To compare health-care use and costs of patients with medically unexplained symptoms with other frequent attenders in secondary care. METHOD: In a sample of 400 frequent attenders of secondary care services, those presenting with medically unexplained symptoms were identified by a review of medical records. Their use of health-care resources was compared with that of other frequent attenders. RESULTS: Of the frequent attenders 17% had at least two medically unexplained consultation episodes. These patients had a greater number of referrals to secondary care and were more likely to undergo particular investigations. CONCLUSIONS: Frequent attenders with medically unexplained symptoms account for levels of service use and expenditure that are comparable with other frequent attenders, but the use and cost of medical investigations in this group are significantly greater.

Adolescent↗
PubMed2000

Virus morphogenesis of Helicoverpa armigera nucleopolyhedrovirus in Helicoverpa zea serum-free suspension culture.

Helicoverpa armigera single nucleopolyhedrovirus (HaSNPV) replication in Helicoverpa zea serum-free suspension culture was studied in detail and the sequence of virus morphogenesis was determined by transmission electron microscopy. By 16 h post-infection (p.i.), virus replication was observed in the virogenic stroma by the appearance of nucleocapsids. Polyhedron formation was detected by 24 h p.i. and the polyhedron envelope (PE) was completely formed by 72 h p.i. PE morphogenesis of HaSNPV is significantly different compared to the extensively studied Autograph californica (Ac)MNPV. In AcMNPV-infected cells, fibrillar structures are found in both cytoplasm and nuclei, and the fibrillar structures in nuclei are in close association with maturing polyhedra during PE formation. Fibrillar structures that resemble the AcMNPV fibrillar structures were detected only in the cytoplasm of HaSNPV-infected cells and appeared to interact with calyx precursors there, but their role in PE formation is unclear. However, prominent calyx precursor structures of various shapes and sizes were observed in the nuclei of HaSNPV-infected cells as well, and they appeared to interact with polyhedra during PE formation. Both the calyx precursor structure and the cytoplasmic fibrillar structure were detected only after HaSNPV virion occlusion had started, indicating that they might have a role in formation of PE. Similar calyx precursor structures and cytoplasmic fibrillar structures were observed in both serum-supplemented and serum-free suspension cultures, as well as in HaSNPV-infected larval tissues, indicating that the structures observed are not cell culture artefacts.

Animals↗
PubMedDate not supplied

Ganser's syndrome.

Explore the source record for details and available documents.

Cognition Disorders↗
PubMedDate not supplied

Effect of time of harvest of budded virus on the selection of baculovirus FP mutants in cell culture.

Rapid formation and selection of FP (few polyhedra) mutants occurs during serial passaging of Helicoverpa armigera nucleopolyhedrovirus (HaSNPV) in insect cell culture. The production of HaSNPV for use as biopesticides requires the passaging of the virus over a number of passages to produce enough virus inoculum for large-scale fermentation. During serial passaging in cell culture, FP mutants were rapidly selected, resulting in declined productivity and reduced potency of virus. Budded virus (BV) is usually harvested between 72 and 96 h postinfection (hpi) in order to obtain a high titer virus stock. In this study, the effect of time of harvest (TOH) for BV on the selection rate of HaSNPV FP mutants during serial passaging was investigated. BV were harvested at different times postinfection, and each series was serially passaged for six passages. The productivity and percentage of FP mutants at each passage were determined. It was found that the selection of FP mutants can be reduced by employing an earlier TOH for BV. Serial passaging with BV harvested at 48 hpi showed a slower accumulation of FP mutants compared to that of BV harvested after 48 hpi. Higher cell specific yields were also maintained when BV were harvested at 48 hpi. When BV that were formed between 48 and 96 hpi were harvested and serially passaged, FP mutants quickly dominated the virus population. This suggests that the BV formed and released between 48 and 96 hpi are most likely from FP mutant infected cells.

Animals↗