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Biomedical subjects

R Milne

Publications and source records attributed to R Milne.

At least 55 records · Page 3Linked to original sources
PubMed1999

Structure of a monoclonal 2E8 Fab antibody fragment specific for the low-density lipoprotein-receptor binding region of apolipoprotein E refined at 1.9 A.

The crystal structure of the Fab fragment of 2E8, the monoclonal IgG1,kappa antibody specific for the low-density lipoprotein (LDL) receptor-binding region of apolipoprotein E (apoE), has been solved by molecular replacement and refined at 1.9 A resolution (PDB entry 12E8). Two 2E8 Fab molecules in the asymmetric unit are related by noncrystallographic symmetry and are hydrogen bonded through a beta-sheet-like intermolecular contact between the heavy-chain complementarity-determining regions 3 (CDRH3) of each molecule. The structure has been refined to an R value of 0.22 (Rfree = 0.27). The initially ill-defined heavy-chain constant domain (CH1) of 2E8 has been retraced with the aid of automatic refinement, confirming the beta-sheet tracing independently of any starting models. As a resolution better than 2 A is not common for Fab fragments, this model represents a well defined Fab structure and should prove useful in MR solution of other Fab fragments. Furthermore, in the absence of an LDL-receptor structure, the homology of the 2E8 CDRH2 to the ligand-binding domain of the LDL receptor has been exploited to model the apoE-LDL-receptor interaction.

Animals↗
PubMed1999

Bacterial expression and purification of the Fab fragment of a monoclonal antibody specific for the low-density lipoprotein receptor-binding site of human apolipoprotein E.

We report the bacterial expression and the purification of a monoclonal antibody (mAb) specific for an epitope that coincides with the LDL receptor (LDLr)-binding domain of human apolipoprotein E (apoE). This antibody resembles the LDLr in its primary structure and its specificity for apoE variants. The recombinant Fab (rFab) fragment of mAb 2E8, consisting of the entire light chain and the Fd portion of the heavy chain, was expressed in Escherichia coli and purified to homogeneity. Purification was facilitated by including a five-histidine carboxyl-terminal extension on the Fd chain. A 5- to 10-fold difference in yield of the antibody was observed when the plasmid was expressed in two different strains of E. coli. Typically 2-6 mg of rFab per liter of culture medium was recovered in the periplasm of the TG1 strain and less than 1 mg was recovered in the periplasm of the XL1-Blue strain. Culture temperatures above 35 degrees C or inclusion of sucrose in the medium reduced rFab yields. The 2E8 rFab was indistinguishable from Fab prepared from 2E8 hybridoma-generated IgG with respect to its affinity and fine specificity. We are using this system to express a panel of 2E8 variant Fabs that will be used as probes to establish the structural features responsible for the binding of apoE to the LDLr.

Animals↗
PubMed1999

A Delphi study to establish national cost-effectiveness research priorities for positron emission tomography.

OBJECTIVE: This study aimed to determine the key cost-effectiveness research questions relating to positron emission tomography (PET) in the UK. METHODS: A systematic literature review was conducted to establish the existing knowledge base relating to the cost-effectiveness of PET in the various conditions for which it has been proposed. A three-round postal Delphi study of relevant individuals was used to determine the key cost-effectiveness research questions relating to PET in the UK. The content and structure of the Delphi study was informed by the results of the literature review. RESULTS: The most important cost-effectiveness research priorities for the National Health Service (NHS) relating to PET were in the clinical areas of lung cancer, breast cancer and the assessment of myocardial viability. Gamma camera PET using coincidence imaging was highlighted as a modality whose, clinical role needed to be determined urgently. CONCLUSION: Underlying the cost-effectiveness research priorities which were established is the need for evidence that the use of the various PET modalities as a diagnostic technique will alter patient management as compared to existing diagnostic strategies. The findings of the project provide a contemporary overview of the potential role for PET in the NHS and will be relevant to other countries.

Cost-Benefit Analysis↗
PubMed1999

rhDNase therapy for the treatment of cystic fibrosis patients with mild to moderate lung disease.

OBJECTIVE: To assess the cost-effectiveness of rhDNase (Pulmozyme(R)) for patients with cystic fibrosis (CF) aged 5 years or more, with mild to moderate lung disease. The review addresses four questions: a) does rhDNase therapy work in the short term?, b) does rhDNase therapy work more effectively in certain groups of patients?, c) does rhDNase therapy work in the long term? and d) what is the cost-effectiveness of rhDNase therapy? METHODS: A structured rapid review with modelling and cost-effectiveness calculations. Electronic searches were carried out to identify randomised controlled trials (RCTs), systematic reviews, epidemiological and economic information. Databases searched included Cochrane Library, Medline, Healthstar, Embase, PreMedline and NHS Economic Evaluation Database (NHS EED). Exclusion criteria were trials of very short duration (14 days or less) and those which looked at CF patients with severe lung disease. Open label extensions providing information on longer term outcomes were included. RESULTS: Nine published RCTs were identified, although only one met the inclusion criteria. This large RCT was of good methodological quality, and shows that treatment with rhDNase over a 6-month period improves lung function, and decreases the risk of respiratory exacerbations. Expert opinion suggests that there are identifiable subgroups of patients showing improvement, little or no change, and deterioration after treatment with rhDNase. However, the best supporting evidence for this comes from a retrospective case series, showing that response to rhDNase is highly variable, and that early improvement was a good predictive marker for long-term benefit. Evidence for the long-term impact of rhDNase is not yet available from any RCTs. A simplified model was therefore developed to estimate the decline in lung function for patients treated with rhDNase, compared with those who were not treated. From this model it appears that the continued use of rhDNase over the lifetime of a CF patient might extend their life expectancy by 2 years. If treatment is limited to a subgroup of patients with moderate lung disease who respond to treatment, the continued use of rhDNase might extend their life expectancy by 7 years. Using the model, the discounted cost per life year gained for all patients is estimated at approximately pound52 500, with a range of between approximately pound25 000-57 000 from sensitivity analysis. For the subgroup of patients, the discounted cost per life year gained is estimated at approximately pound16 000, with a range of between approximately pound18 000-36 600 from sensitivity analysis. CONCLUSIONS: Although there is short-term evidence that the use of rhDNase improves lung function and decreases the risk of respiratory exacerbations, at present there is no evidence from RCTs to indicate whether this effect is sustained over a longer time period, or whether rhDNase is associated with a reduction in mortality. RCTs to date have been of insufficient duration to answer important questions about long term outcomes, particularly the effects of rhDNase on lung function, respiratory exacerbations and mortality. Further long-term research is needed, with economic analysis to evaluate the long term cost-effectiveness of rhDNase. Research is also needed to identify, in advance, which patients would benefit most from this expensive treatment.

Adolescent↗
PubMed1999

Volunteer assistance in the treatment of chronic alcoholism.

Chronic alcoholics often have great difficulty in adjusting their lifestyle to accommodate the goals agreed upon in treatment. A 16-week behavioral self-management program, which emphasized creating lifestyle changes in the community, was offered to 193 clients. To assist in the process of treatment delivery, half of the clients were offered the support of trained community volunteers during the treatment program. To give volunteers a time period in which to to meet and start working with their clients, only those clients who completed at least the first 4 weeks of the 16-week program (N = 106) were included in the evaluation. Independent follow-up was conducted over a 12-month period. A significant reduction in alcohol consumption was found for clients of both the volunteer-support (VS; N = 52) and the office-based (OB; N = 54) groups; this reduction was maintained over the 12 months of review, with no major differences found between groups. Volunteers rated high on particular characteristics assisted the client more effectively during treatment than those rated low. The variability in hours spent between volunteer-client pairs appeared to mask any main effects, and also the length of the contact period with the volunteer was too short. It is recommended that the use of volunteers be evaluated further by extending the client contact time into the follow-up period.

Adult↗
PubMed1999

Positron emission tomography: establishing priorities for health technology assessment.

BACKGROUND: Positron emission tomography (PET) is an expensive diagnostic imaging technology. Despite the long history of PET development, the costs and effectiveness of its use in routine clinical practice remain unknown. Against this background of uncertainty regarding the clinical role of PET, the UK Standing Group on Health Technology requested a review of its current and potential role which would enable research priorities in this area to be established. OBJECTIVES: This 3-month project had two explicit objectives: (1) to review the state of knowledge regarding the clinical applications of PET; (2) to determine the key health technology assessment (HTA) research questions relating to the use of PET in the UK. METHODS: A literature review to ascertain the state of knowledge regarding the clinical applications of PET and a three-round Delphi study to inform the key HTA research questions relating to the use of PET in the UK were undertaken. The results of an earlier systematic review, published by the Veteran's Health Administration (VHA) in the USA in 1996, were used as the starting point for the literature review. The VHA review was updated and extended by means of MEDLINE and Cochrane Library database searches. Participants in the Delphi study were selected by discussion with five individuals in the UK with an interest in, and awareness of, developments in PET. As a result of their suggestions, 43 individuals were initially invited to participate, of whom two did not feel appropriately qualified. Questionnaires were sent by facsimile to all invited participants, who were asked to return the completed forms by facsimile within a week. The content and structure of the Delphi study was informed by the results of the literature review. The responses and comments of the participants were a major source of information for this report. RESULTS: Clinical applications for PET have been advocated in three broad disease groups: oncology, cardiology and neuropsychiatric disorders. There are currently four PET modalities that need to be considered when assessing its potential clinical role in the UK: full ring PET scanners operating in two or three dimensions (available at five sites); partial ring rotating PET scanners (one currently operating in the UK); coincidence imaging with modified gamma camera technology; and high-energy collimator imaging of 511 keV photons with modified gamma camera technology. There is a paucity of available evidence relating to the cost-effectiveness of the various PET modalities in all of the clinical indications for which the technology is currently being advocated. In addition, many existing reports on the diagnostic accuracy of PET are limited because they are liable to bias and often relate only to very small patient numbers. The results of the Delphi study indicated that the four most important research priorities for the NHS, in descending order of their importance, are: (1) the relative cost-effectiveness of (a) full ring PET, (b) gamma camera PET using coincidence imaging and (c) existing diagnostic strategies to determine staging prior to operative intervention for lung cancer; (2) partial ring PET compared with full ring PET in oncology (3) the relative cost-effectiveness of (a) full ring PET, (b) gamma camera PET using coincidence imaging and (c) existing diagnostic strategies to stage and monitor treatment response in breast cancer; (4) the relative cost-effectiveness of (a) gamma camera PET using coincidence imaging and (b) 511 keV collimated positron imaging for assessing myocardial viability when selecting patients for revascularisation surgery. Vignettes describing each of the research priorities are provided in the main report. CONCLUSIONS: The findings of this project, which was undertaken rapidly in order to inform HTA research prioritization in the UK, provide a contemporary overview of the potential clinical role for PET in the NHS. Evidence is needed that using PET as a diagnostic

Female↗
PubMed1998

Epitopes close to the apolipoprotein B low density lipoprotein receptor-binding site are modified by advanced glycation end products.

Advanced glycation end products (AGEs) are thought to contribute to the abnormal lipoprotein profiles and increased risk of cardiovascular disease of patients with diabetes and renal failure, in part by preventing apolipoprotein B (apoB)-mediated cellular uptake of low density lipoproteins (LDL) by LDL receptors (LDLr). It has been proposed that AGE modification at one site in apoB, almost 1,800 residues from the putative apoB LDLr-binding domain, may be sufficient to induce an apoB conformational change that prevents binding to the LDLr. To further explore this hypothesis, we used 29 anti-human apoB mAbs to identify other potential sites on apoB that may be modified by in vitro advanced glycation of LDL. Glycation of LDL caused a time-dependent decrease in its ability to bind to the LDLr and in the immunoreactivity of six distinct apoB epitopes, including two that flank the apoB LDLr-binding domain. ApoB appears to be modified at multiple sites by these criteria, as the loss of glycation-sensitive epitopes was detected on both native glycated LDL and denatured, delipidated glycated apoB. Moreover, residues directly within the putative apoB LDLr-binding site are not apparently modified in glycated LDL. We propose that the inability of LDL modified by AGEs to bind to the LDLr is caused by modification of residues adjacent to the putative LDLr-binding site that were undetected by previous immunochemical studies. AGE modification either eliminates the direct participation of the residues in LDLr binding or indirectly alters the conformation of the apoB LDLr-binding site.

Antibodies, Monoclonal↗
PubMed1998

Spruce budworm elastase precipitates Bacillus thuringiensis delta-endotoxin by specifically recognizing the C-terminal region.

A gut juice protein from Choristoneura fumiferana (spruce budworm) larvae that precipitates certain delta-endotoxins shows a unique specificity for the C-terminal amino acid sequence. Using homolog scanning mutants, we have identified a contiguous region of the Cry1Aa toxin which interacts with the 75-kDa toxin precipitating protein (TPP-75)' resulting in precipitation. The contiguous region from Cry1Aa can be transferred to Cry1Ac and results in an identical precipitation reaction. The precipitation reaction occurs rapidly and is unique in that the ratio of precipitating protein to toxin is low (estimated at 0.01), unlike antibody-antigen reactions which exhibit mole ratios close to 1. TPP-75 has been characterized as an elastase-like serine protease. We have taken advantage of this serine protease character and incorporated a radiolabel using an irreversible inhibitor. The radiolabel has allowed us to show the coincidence of the catalytically-inhibited TPP-75 with the toxin in a blotting assay and to follow the degradation of TPP-75 during storage. TPP-75 represents the first evidence that gut juice proteins may selectively attenuate the activity of delta-endotoxins, prior to binding to putative receptors on susceptible cells. TPP-75 should be evaluated as a possible resistance mechanism for those larvae that do not exhibit a receptor-based resistance.

Animals↗
PubMed1998

Increased levels of apolipoprotein D in cerebrospinal fluid and hippocampus of Alzheimer's patients.

Apolipoprotein D (apoD) is a member of the lipocalin family of proteins. Most members of this family are transporters of small hydrophobic ligands, although in the case of apoD, neither its physiological function(s) nor its putative ligand(s) have been unequivocally identified. In humans, apoD is expressed in several tissues, including the CNS, and its synthesis is greatly increased during regeneration of rat peripheral nerves. As apoD may have an important function in the nervous system and, particularly, in nerve regeneration, we measured immunoreactive apoD levels in the hippocampus and in CSF of patients with either Alzheimer's disease (AD) or other neuropathologies. In parallel, we determined the concentrations of apolipoprotein E (apoE), another apolipoprotein also implicated in nerve regeneration and in the etiology of AD. Levels of apoD but not apoE were increased in the hippocampus of AD patients compared with controls. ApoD concentrations, as determined by radioimmunoassay, were significantly increased in the CSF of AD patients (4.23 +/- 1.58 microg/ml) and patients with other pathologies (3.29 +/- 1.35 microg/ml) compared with those in the CSF of normal subjects (1.15 +/- 0.71 microg/ml). Although the differences were smaller than for apoD, the mean apoE concentrations in the CSF of both groups of patients were also significantly higher than those of controls. In AD patients, apoD, but not apoE, levels in CSF and hippocampus increased as a function of inheritance of the epsilon4 apoE allele. This study therefore demonstrates that increased apoD levels in the hippocampus and in CSF are a marker of neuropathology, including that associated with AD, and are independent of apoE concentrations.

Aged↗
PubMed1998

Police officers' perceptions of eyewitness performance in forensic investigations.

Few contemporary data support the assertion that eyewitnesses are important in police investigations. In the present study, 159 UK police officers were surveyed regarding their perceptions of eyewitnesses and eyewitness performance. The respondents indicated that eyewitnesses usually provide the central leads in criminal investigations; however, the police officers also believed that eyewitnesses rarely provide sufficient information, especially descriptive details as opposed to action details. Nevertheless, the officers believed that eyewitnesses are rarely incorrect. A sizable minority reported that witnesses rarely come forward to the police and that those who do are often reluctant to testify in court. Many officers indicated that they do not have enough time to conduct good eyewitness interviews.

Adult↗
PubMed1998

The effects on mortality of the use of combined oral contraceptives.

OBJECTIVE: To assess, using a computerised model, the effects on mortality of the use of combined oral contraceptives (COCs). DESIGN: Two hypothetical cohorts of one million women each, identical except for their use of contraception. The impact of COC use was explored by applying, to each cohort, death rates which were adjusted according to a series of assumptions about the risks associated with COC use. The model also explored the effects of a number of different patterns of COC use. SETTINGS AND SUBJECTS: Women aged 16, followed through to ages 50 and 75, exposed to 1994 UK death rates. MAIN OUTCOME MEASURES: Numbers of deaths from various cancers and cardiovascular diseases attributable to COC use. RESULTS: Based on the standard pattern of use, there were 1.7 per cent more deaths in the COC cohort to age 50. The important effects on mortality of different patterns of use and of different assumptions about risks in ex-users were illustrated. CONCLUSIONS: The results confirm the findings of earlier work and provide some reassurance about the likely adverse effects of COC use.

Adolescent↗
PubMed1997

Impact of the NHS reforms on English hospital productivity: an analysis of the first three years.

OBJECTIVES: To evaluate the effect of purchaser mix, market competition, and trust status on hospital productivity within the NHS internal market. METHODS: Hospital cost and activity data were taken from routinely collected data for acute NHS hospitals in England for 1991-2 to 1993-4. Cross sectional and longitudinal regression methods were used to estimate the effect of trust status, competition, and purchaser mix on average hospital costs per inpatient, after adjusting for outpatient activity levels, casemix, teaching activity, regional salary variation, hospital size, scale of activity, and scope of cases treated. RESULTS: Real productivity gains were apparent across the study period for NHS hospitals on average. Casemix adjustment drastically improved cross sectional comparisons between hospitals. Gaining trust status and increasing host district purchaser share were associated with productivity increases after adjustment for casemix, regional salary differences, and hospital size and scope. Hospitals that became trusts during the study period were on average less productive at the beginning of the period than those that did not, and there were no significant productivity differences between trust waves at the end of the period in 1993-4. Market concentration was not associated with productivity differences. CONCLUSION: Further analysis is needed to determine whether overall and trust associated productivity gains are transient effects, one off shifts, or self perpetuating reorientations of organisational behaviour. Hospitals may have chosen to become trusts because they anticipated being able to increase productivity. Increases in the proportions of small purchasers were associated with increasing costs. Importantly, this study could not adjust for changes in the quality of care.

Cross-Sectional Studies↗
PubMed1997

Cholesteryl ester transfer protein mediates selective uptake of high density lipoprotein cholesteryl esters by human adipose tissue.

We have determined the role of cholesteryl ester transfer protein (CETP) in selective uptake of high density lipoprotein (HDL)-derived cholesteryl esters (CE) by human adipose tissue, using organ culture or collagenase-digested adipocytes. Incubation of the fresh tissue fragments with HDL containing [3H]CE or 125I-apoprotein (apo) A-I resulted in significant uptake of HDL-CE-derived label. Addition of recombinant CETP (rCETP) increased CE uptake in a dose-response fashion. In contrast, little association of 125I-apoA-I with adipose tissue was noted, and addition of rCETP did not alter apoA-I uptake or degradation. Incubation of adipose tissue with TP2, an anti-CETP monoclonal antibody, which inhibits neutral lipid transfer, markedly reduced selective uptake of HDL-CE. Studies using human adipocytes isolated by collagenase digestion also demonstrated selective uptake of HDL-CE and enhancement of this process by rCETP. To confirm that the association of HDL-CE-derived radioactivity with adipose tissue was not due to neutral lipid exchange between adipocytes and HDL, we measured changes in HDL composition following incubation of HDL and rCETP with isolated adipocytes. A decrease in HDL-CE concentration in the medium was observed, an effect which was markedly attenuated when incubations were carried out in the presence of monoclonal antibody TP2. Furthermore, the decrease in HDL-CE was accompanied by an increase in HDL free cholesterol, likely representing efflux of adipocyte cholesterol to HDL. There were no significant changes in phospholipid, apoA-I, or apoA-II in the medium following incubation with adipocytes. These data demonstrate a novel and important role for CETP in selective uptake of HDL-cholesteryl esters by human adipocytes and suggest that this pathway may be of quantitative physiological significance in HDL remodeling and adipocyte cholesterol accumulation.

Adipocytes↗
PubMed1997

Protein intake and urinary albumin excretion rates in the EURODIAB IDDM Complications Study.

For people with insulin-dependent diabetes mellitus (IDDM) renal disease represents a life-threatening and costly complication. The EURODIAB IDDM Complications Study, a cross-sectional, clinic-based study, was designed to determine the prevalence of renal complications and putative risk factors in stratified samples of European individuals with IDDM. The present study examined the relationship between dietary protein intake and urinary albumin excretion rate (AER). Food intake was assessed centrally by a standardized 3-day dietary record. Urinary AER was determined in a central laboratory from a timed 24-h urine collection. Complete data were available from 2696 persons with IDDM from 30 centres in 16 European countries. In individuals who reported protein consumption less than 20% of total food energy intake, mean AER was below 20 microg/min. In those in whom protein intake constituted more than 20%, mean AER increased, a trend particularly pronounced in individuals with hypertension and/or poor metabolic control. Trends reached statistical significance for intakes of total protein (% of energy, p = 0.01) and animal protein (% of energy, p = 0.02), while no association was seen for vegetable protein (p = 0.83). These findings support the current recommendation for people with diabetes not to exceed a protein intake of 20% of total energy. Monitoring and adjustment of dietary protein appears particularly desirable for individuals with AER exceeding 20 microg/min (approximately 30 mg/24h), especially when arterial pressure is raised and/or diabetic control is poor.

Adolescent↗