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Biomedical subjects

J C Lewis

Publications and source records attributed to J C Lewis.

At least 19 recordsLinked to original sources
PubMed2005

Hydroxyapatite-ceramic-coated femoral stems in revision hip surgery.

We describe the clinical and radiological results of 120 consecutive revision hip replacements in 107 patients, using the JRI Furlong hydroxyapatite-ceramic-coated femoral component. The mean age of the patients at operation was 71 years (36 to 92) and the mean length of follow-up 8.0 years (5.0 to 12.4). We included patients on whom previous revision hip surgery had taken place. The patients were independently reviewed and scored using the Harris hip score, the Western Ontario and McMaster Universities osteoarthritis index (WOMAC) and the Charnley modification of the Merle d'Aubigné and Postel score. Radiographs were assessed by three reviewers for the formation of new bone, osteolysis, osseointegration and radiolucent lines in each Gruen zone. The mean Harris hip score was 85.8 (42 to 100) at the latest post-operative review. The mean WOMAC and Merle d'Aubigné and Postel scores were 34.5 and 14.8, respectively. The mean visual analogue score for pain (possible range 0 to 10) was 1.2 overall, but 0.5 specifically for mid-thigh pain. There were no revisions of the femoral component for aseptic loosening. There were four re-revisions, three for infection and one for recurrent dislocation. Radiological review of all the femoral components, including the four re-revisions showed stable bony ingrowth and no new radiolucent lines in any zone. Using revision or impending revision for aseptic loosening as an end-point, the cumulative survival of the femoral component at ten years was 100% (95% confidence interval 94 to 100). We present excellent medium- to long-term clinical, radiological and survivorship results with the fully hydroxyapatite-ceramic-coated femoral component in revision hip surgery.

Adult↗
PubMed2001

Using epitope-aequorin conjugate recognition in immunoassays for complex proteins.

Recombinant techniques are used to fuse biologically important molecules or peptides to the N-terminus of the photoprotein aequorin such that the binding characteristics of the molecule and the bioluminescent activity of aequorin are retained. This work demonstrates that the peptide region of a bulky protein can be used to develop an assay for the protein. A heterogeneous competitive binding assay was first developed for HPC4 epitope, the binding region of protein C, using HPC4-apoaequorin conjugate. It was observed that the binding of HPC4 epitope to its monoclonal antibody and the bioluminescence properties of aequorin were retained in the fusion protein. The same strategy and the same fusion protein were used to develop the assay for protein C. This project could potentially be a model for large biomolecules utilizing only the binding region of the protein in the labeled analyte. Also, this assay can be used in clinical diagnostics for the quantitative detection of protein C.

Aequorin↗
PubMed2001

Bioluminescence immunoassay for thyroxine employing genetically engineered mutant aequorins containing unique cysteine residues.

Genetically engineered one-to-one conjugates between an analyte and a protein label have been demonstrated to yield assays with better detection limits and performance characteristics than those prepared by conventional chemical conjugation methods. To date, the preparation of these conjugates has been limited to fusion techniques where a peptide analyte is fused in frame to the protein label. To further expand the range of analytes that can be detected by using genetic engineering techniques coupled with bioanalytical methods, we have employed site-directed mutagenesis to prepare one-to-one analyte-label conjugates that include nonpeptidic analytes such as drugs, vitamins, and hormones. Specifically, we have prepared mutants of the photoprotein aequorin containing single cysteine residues suitable for site-specific conjugation. Aequorin is a photoprotein that emits light at 469 nm and has been employed as a highly sensitive bioluminescent label in the development of binding assays for important biomolecules. We have performed polymerase chain reaction-based site-directed mutagenesis on apoaequorin to yield four mutant aequorins containing unique cysteine residues at positions 5, 53, 71, and 84 in the polypeptide chain for the purpose of site-specific conjugation to a model analyte. A maleimide-activated thyroxine was selected as the model analyte and site-specifically conjugated to the mutants through their unique cysteine residues. A heterogeneous assay for thyroxine was then developed by employing the genetically engineered aequorin mutants.

Aequorin↗
PubMed2001

Bisphosphonates inhibit the growth of Trypanosoma brucei, Trypanosoma cruzi, Leishmania donovani, Toxoplasma gondii, and Plasmodium falciparum: a potential route to chemotherapy.

We have investigated the effects in vitro of a series of bisphosphonates on the proliferation of Trypanosoma cruzi, Trypanosoma brucei rhodesiense, Leishmania donovani, Toxoplasma gondii, and Plasmodium falciparum. The results show that nitrogen-containing bisphosphonates of the type used in bone resorption therapy have significant activity against parasites, with the aromatic species having in some cases nanomolar or low-micromolar IC(50) activity values against parasite replication (e.g. o-risedronate, IC(50) = 220 nM for T. brucei rhodesiense; risedronate, IC(50) = 490 nM for T. gondii). In T. cruzi, the nitrogen-containing bisphosphonate risedronate is shown to inhibit sterol biosynthesis at a pre-squalene level, most likely by inhibiting farnesylpyrophosphate synthase. Bisphosphonates therefore appear to have potential in treating parasitic protozoan diseases.

Animals↗
PubMed2001

Ethical approval for research involving geographically dispersed subjects: unsuitability of the UK MREC/LREC system and relevance to uncommon genetic disorders.

OBJECTIVES: To assess the process involved in obtaining ethical approval for a single-centre study involving geographically dispersed subjects with an uncommon genetic disorder. DESIGN: Observational data of the application process to 53 local research ethics committees (LRECs) throughout Wales, England and Scotland. The Multicentre Research Ethics Committee (MREC) for Wales had already granted approval. RESULTS: Application to the 53 LRECs required 24,552 sheets of paper and took two months of the researcher's time. The median time taken for approval was 39 days with only seven (13%) of committees responding within the recommended 21 days. In at least nineteen cases (36%) a subcommittee considered the application. Thirty-three committees (62%) accepted the proposal without amendments but, of the remainder, four (8%) requested changes outside of the remit of LRECs. DISCUSSION: Difficulties still exist with the system for obtaining ethical approval for studies involving a single centre but with patients at multiple sites, as is often required for genetic observational research. As such studies differ from true multicentre studies, it may be advantageous to develop a separate and specific process of application to ensure that resources are not unnecessarily expended in the quest for ethical approval.

Cohort Studies↗
PubMed2000

Bioluminescence detection of proteolytic bond cleavage by using recombinant aequorin.

Detection of proteolytic bond cleavage was achieved by taking advantage of the bioluminescence emission generated by the photoprotein aequorin. A genetically engineered HIV-1 protease substrate was coupled with a cysteine-free mutant of aequorin by employing the polymerase chain reaction to produce a fusion protein that incorporates an optimum natural protease cleavage site. The fusion protein was immobilized on a solid phase and employed as the substrate for the HIV-1 protease. Proteolytic bond cleavage was detected by a decrease in the bioluminescence generated by the aequorin fusion protein on the solid phase. A dose-response curve for HIV-1 protease was constructed by relating the decrease in bioluminescence signal with varying amounts of the protease. The system was also used to evaluate two competitive and one noncompetitive inhibitor of the HIV-1 protease. Among the advantages of this assay is that by using recombinant methods a complete bioluminescently labeled protease recognition site can be designed and produced. The assay yields very sensitive detection limits, which are inherent to bioluminescence-based methods. An application of this system may be in the high-throughput screening of biopharmaceutical drugs that are potential inhibitors of a target protease.

Aequorin↗
PubMed2000

Reduction in pain associated with open carpal tunnel decompression.

Patients treated by open carpal tunnel decompression under local anaesthetic experience significant pain with the introduction of local anaesthetic before operation. A prospective double-blind randomized placebo controlled trial was carried out to assess whether this pain could be reduced with the pre-application of a topical lignocaine-prilocaine anaesthetic cream. Nineteen patients undergoing simultaneous bilateral operations were studied. Pain, measured with a visual analogue scale, was significantly reduced on the sides treated with anaesthetic cream. On a four point verbal scale the rating on the placebo side was "moderate" or "severe", compared to "mild" on the anaesthetic cream side. The reduction in pain was greater in women, possibly due to a relatively thinner dermis. There were no adverse effects.

Anesthetics, Local↗
PubMed2000

Detection of hepatitis B virus infection in wild-born chimpanzees (Pan troglodytes verus): phylogenetic relationships with human and other primate genotypes.

Infection with hepatitis B virus (HBV) was detected by serological testing for HBV surface antigen and by PCR assay for HBV DNA in serum samples from two common chimpanzees (Pan troglodytes subsp. verus) born in West Africa. The complete genome sequences obtained by nucleotide sequencing of overlapping DNA fragments amplified by PCR were compared with HBV variants recovered from other primates and with human genotypes A to F. Both chimpanzee sequences were 3, 182 nucleotides in length, and the surface gene sequence predicted the existence of a, d, and w serological determinants. Neither sequence contained stop codons in the precore region. On phylogenetic analysis, the HBV variants infecting the chimpanzees clustered together with a third chimpanzee HBV isolate independently obtained from an infected captive animal (A. J. Zuckerman, A. Thornton, C. R. Howard, K. N. Tsiquaye, D. M. Jones, and M. R. Brambell, Lancet ii:652-654, 1978), with an overall sequence similarity of >94%. This provides strong evidence for a chimpanzee-specific genotype of HBV which circulates in nature. These findings add to the recent evidence for infection in the wild of other Old and New World primates (gibbon, orangutan, and woolly monkey) with species-specific variants of HBV. There is no evidence for close phylogenetic clustering of variants found so far in primates with any of the established HBV genotypes from humans. With the new evidence for the widespread distribution of HBV in primates, hypotheses for the origins of human infection are reviewed.

Animals↗
PubMed2000

Who gets priority? Waiting list assessment using a scoring system.

This study examines the relationship between clinical priority and duration on the waiting list. Patients' priority is also compared from three different perspectives: the consultant at out-patients (clinical urgency), the general practitioner who later sends a letter requesting expedition of admission (clinical urgency) and the 18-month waiters. Clinical priority in 222 patients awaiting primary hip or knee arthroplasty was assessed using a modification of the New Zealand priority criteria scoring system, administered by postal questionnaire. There was no correlation between time on waiting list and clinical score (r = 0.0). The hip and knee patient scores were not significantly different. The mean scores in the consultant and GP groups were higher (greater pain and disability) than in the 18-month waiters. The Kappa inter-rater agreement method demonstrated that both groups of clinician's assessment of clinical urgency had a 'fair' strength of agreement with scoring system, but the agreement of the 18-month waiter group was 'very poor'. Time on a waiting list should not be a decisive factor in establishing priority for primary hip or knee arthroplasty. Scoring systems can aid in assigning clinical priority for operation and indeed for the initial out-patient referral.

Arthroplasty, Replacement, Hip↗
PubMed2000

Mapping international challenges for managed care.

The author in this presentation discusses the factors which he believes constitute the 5 mega-challenges confronting the global managed care industry; namely: Challenge One: Contest the bogus falsehoods and factual errors about managed care. Challenge Two: Engage the debate on the rightful place of private health care in the global economy. Challenge Three: Make the healthcare industry an integral part of every nation's economic development plan. Challenge Four: Lead the public policy debate on the side of safe, health, peaceful, caring communities. Challenge Five: Join with colleagues from pharmaceutical, medical device and information technology companies to promote a robust private health sector.

Global Health↗
PubMed1999

Dual detection of peptides in a fluorescence binding assay by employing genetically fused GFP and BFP mutants.

A competitive fluorescence microplate assay based on a red-shifted green fluorescent protein (rsGFP) and a blue fluorescent protein (BFP) was developed for the detection of two model peptides in the same sample. The assay employed gene fusion to prepare the fluorescently labeled peptide conjugates. Specifically, plasmids were constructed in which the genes encoding for the two small peptides (less than 12 amino acids in length) were fused to either the gene of the rsGFP or the BFP, as desired. The newly constructed plasmids were transformed into E. coli for expression of the fusion proteins. By employing the technique of gene fusion, one-to-one homogeneous populations of peptide-rsGFP or -BFP conjugates were produced. These peptide-GFP mutant conjugates exhibited the same excitation and emission spectral characteristics as the unmodified proteins. The naturally fluorescent proteins act as labels to provide sensitive dual detection of the two selected small peptides in a competitive assay format. To our knowledge, this is the first time that mutants of GFP, such as the rsGFP and BFP, have been used as quantitative labels for the development of a dual-analyte fluorescence immunoassay.

Bacteriophage T7↗
PubMed1999

Genetically engineered obelin as a bioluminescent label in an assay for a peptide.

The marine polyp Obelia longissima produces a protein, obelin, which emits light in a calcium-dependent manner. This photoprotein consists of a stable complex of its apoprotein, a chromophore, and oxygen. In the presence of calcium ions, the protein undergoes a change in conformation that allows it to catalyze the oxidation of the chromophore, coelenterazine, to coelenteramide with the release of light and CO2. Photoproteins are attractive as labels in analytical applications because the bioluminescent signal that they produce is the result of a chemical reaction and, therefore, has virtually no background. Thus, bioluminescence allows for extremely sensitive detection. In that regard, the feasibility of using obelin as a label has been explored with the development of a competitive immunoassay for the determination of a small peptide analyte. To attach the obelin label in a controlled manner to the octapeptide, a fusion protein was produced using recombinant DNA techniques. The protein consisted of the C-terminus of the peptide fused to the N-terminus of obelin. The octapeptide-obelin fusion protein retained the bioluminescence properties of the native protein, and was subsequently used to generate dose-response curves for the free octapeptide.

Animals↗
PubMed1999

Modified LDLs induce and bind to membrane ruffles on macrophages.

Macrophage foam cell formation in vitro requires uptake of modified low density lipoproteins (LDL) such as acetylated LDL (AcLDL) and moderately oxidized LDL (OxLDL), or beta-migrating very low density lipoprotein (betaVLDL), a naturally occurring lipoprotein. Incubation ofmacrophages with AcLDL and OxLDL resulted in stimulation of membrane ruffle formation, while betaVLDL primarily resulted in increased numbers of microvilli. Time-lapse Allen video enhanced contrast differential interference contrast (AVEC-DIC) light microscopy and correlative whole mount intermediate-voltage transmission electron microscopy (IVEM) was used to examine the dynamics ofAcLDL stimulated membrane ruffling and membrane ruffle ultrastructure. Stereo 3D surface replicas confirmed that AcLDL bound to these AcLDL-induced membrane ruffles. Quantification of the plasma membrane surface area after incubation with AcLDL, betaVLDL or LDL confirmed that AcLDL stimulated membrane ruffling, while betaVLDL and LDL stimulated microvilli formation. These studies suggest that modified LDLs induce circular membrane ruffles and modified LDLs bind to these ligand-induced membrane ruffles.

Animals↗