Biomedical subjects

C E Evans

Publications and source records attributed to C E Evans.

At least 19 recordsLinked to original sources

PubMed2003

Role of cyclical pressure and particles in the release of M-CSF, chemokines, and PGE2 and their role in loosening of implants.

A septic loosening of orthopaedic implants is usually attributed to the action of wear debris from the prosthesis. Recent studies, however, have also implicated physical pressures in the joint as a further cause of loosening. We have examined the role of both wear debris and pressure on the secretion of two chemokines, MIP-1alpha and MCP-1, together with M-CSF and PGE2, by human macrophages in vitro. The results show that pressure alone stimulated the secretion of more M-CSF and PGE2 when compared with control cultures. Particles alone stimulated the secretion of M-CSF and PGE2, when compared with unstimulated control cultures, but did not stimulate the secretion of the two chemokines. Exposure of macrophages to both stimuli simultaneously had no synergistic effect on the secretion of the chemokines, but both M-CSF and PGE2 were increased in a synergistic manner. Our findings suggest that pressure may be an initiating factor for the recruitment of cells into the periprosthetic tissue.

Chemokine CCL2↗

PubMed2002

Bisphosphonates modulate the effect of macrophage-like cells on osteoblast.

Macrophages (MPs) are present in many tissues and have been implicated in the excessive bone resorption seen in patients with skeletal disorders. Our previous studies showed that macrophage-like cells influenced osteoblasts (OB) in co-culture, as number and activity of osteoblasts were decreased in co-cultures compared with controls. Macrophages are probable precursors of osteoblasts which have been shown to be inhibited by bisphosphonates (BPs). Bisphosphonates also modulate macrophage and osteoblasts activity. This study investigated whether addition of bisphosphonates to co-cultures of osteoblast and macrophages could reduce or block the adverse effects of macrophages on osteoblasts. The results showed that, compared to controls, fewer osteoblasts were present over time in macrophage/osteoblast co-cultures (at day 12, 15.5 x 10(4) and 8.8 x 10(4); P<0.0001) and that addition of bisphosphonates (10(-9)-10(-5)M) to the co-cultures prevented this reduction (P<0.001). Bisphosphonates also elicited an increase in numbers of osteoblast (82%) and restored alkaline phosphatase (ALP) activity, which was reduced by 15% (P approximately equal to 0.05) compared to control levels. The number of macrophages in co-cultures was reduced when bisphosphonates were added (P<0.001) and release of lactate dehydrogenase (LDH) was seen, which was not detectable in control cultures. It therefore, appears that bisphosphonates initiated macrophage death. These results demonstrated that the inhibitory effect of macrophages on osteoblasts in vitro could be overcome by the action of bisphosphonates. These findings have implications for the treatment of skeletal conditions where macrophage-derived cytokines are important, such as arthritis and implant loosening, although it is clearly important to distinguish between those bisphosphonates which enhance synthesis of pro-inflammatory cytokines and those which inhibit such synthesis.

Alkaline Phosphatase↗

PubMed2002

Synergistic effect of particles and cyclic pressure on cytokine production in human monocyte/macrophages: proposed role in periprosthetic osteolysis.

Macrophages, activated by particulate wear debris, are important in the process of osteolysis, which occurs during joint implant loosening. We previously found increased levels of interleukin-1beta (IL-1beta), IL-6, and tumor necrosis factor-alpha in cultured macrophages subjected to cyclical pressure of 0.138 MPa, suggesting that cyclic pressure may be another relevant cause of macrophage activation. The current study first investigated the effects of a range of cyclic pressures on cultured macrophages, including an investigation of the time course of cytokine expression. At 0.138 MPa, supernatant levels of TNF-alpha were maximal at 12 h, whereas IL-6 and IL-1beta were maximal at 24 h. All four cyclic pressure levels tested (without particles) resulted in increased production of all three cytokines relative to control. These increases were most marked at 0.069 and 0.035 MPa, and the increase in cytokine production at 0.017 MPa was not statistically significant. Further studies demonstrated that conditioned media from cyclically pressurized macrophages stimulated bone resorption in a neonatal mouse calvarial assay system. There were increased levels of calcium released from calvaria cultured in conditioned media from pressurised monocytes, and an increase in tartate-resistant acid phosphatase-positive osteoclasts was observed microscopically. As particulate wear debris is important in implant loosening, ultra high molecular weight polyethylene particles were also added to the pressurized cell cultures. The experiments compared the effect of atmospheric pressure, cyclic pressure alone, particles alone, and particles and cyclic pressure combined. A combination of ultra high molecular weight polyethylene particles and cyclic pressure at 0.017 MPa resulted in a dramatic synergistic elevation of levels of all three cytokines compared with the levels found with either pressure or particles alone. We propose that monocyte/macrophage activation by cyclic pressure plays a major role in the osteolysis seen in aseptic loosening of implants. The synergistic effect observed between particles and pressure could accelerate implant loosening, and implies that reduction in either cyclic pressure (by improving implant fixation) or wear debris load would reduce osteolysis.

Animals↗

PubMed2001

In-line valve injection for capillary electrophoresis.

Direct in-line injection is successfully demonstrated for capillary electrophoresis using a commercially available injection valve designed for liquid chromatographic applications. The internal, fluid-contacting materials in this valve injector are composed of ceramics and PEEK (polyetheretherketone). In studies up to 20 kV, this materials design provides a sufficient dielectric interface to insulate the high-voltage buffer from the metal valve body. Partial-loop injections from 6 to > 60 nL are shown to be highly reproducible and generally consistent with direct electrokinetic injections under the same experimental conditions. The small extracolumn variance contributed by the valve injection system is symmetrical, and the measured theoretical plates for 75-microm- and 100-microm-i.d. separation capillaries are 1.6 x 10(5) and 2.5 x 10(5), respectively. As a result, the separation performance is quite good, demonstrating the viability of in-line valve injection for capillary electrophoresis. This development in capillary electrophoretic instrumentation has important implications for the advancement of electrophoretic applications as well as for the design of completely integrated analysis systems.

Journal Article↗

PubMed2001

An in vitro comparison of human flexor and extensor tendon cells.

This study examined the in vitro phenotype of cells cultured from both flexor and extensor tendons. Matrix proteins secreted by tendon cells were examined, together with their response to mechanical strain, using cells from the two types of tendon. Immunocytochemical staining using specific antibodies to matrix proteins demonstrated that flexor tendon cells synthesised only three of the five proteins stained for, whilst extensor tendon cells synthesised all five. Gel electrophoresis (used to separate mixtures of proteins) demonstrated that protein secretion was similar for both cell types. Both cell types showed similar patterns of synthesis for total collagen and total protein over time. No difference was found in the response to cyclical strain of cells from the two types of tendon; both cell proliferation and collagen synthesis were stimulated. The study demonstrated that cells obtained from two different types of tendon behave similarly when exposed to identical environmental conditions in vitro.

Cells, Cultured↗

PubMed2000

Element-specific detection in capillary electrophoresis using X-ray fluorescence spectroscopy.

X-ray fluorescence spectroscopy is demonstrated here as a novel, element-specific detector for capillary electrophoresis. Monochromatic 10 keV X-rays from a synchrotron light source are used to excite core electrons, causing emission of characteristic Kalpha X-ray fluorescence (XRF) lines. Using this technique, XRF energies provide elemental identification, while XRF intensities can be used to quantitate the metal composition of each eluent. An X-ray transparent polymer coupling is used to create a window for the on-line, X-ray detection. This coupling contributes no measurable extra-column variance, and electrophoretic mobilities for the metal complexes used as model solutes are highly reproducible. The combination of XRF detection with capillary electrophoresis (CE-XRF) creates the first on-line detection system that is element-specific, nondestructive, and directly applicable to a broad range of applications including nonelectroactive species. CE-XRF is successfully demonstrated here for high binding-constant complexes of Fe(III), Co(II), Cu(II), and Zn(II). Within a single injection, electropherograms are obtained for each element of interest, with the element identity obtained directly from the emission energy. In contrast with ICPMS, this detection technique is directly on-line and does not require volatilization of the eluent. As a result, element-specific detection is not limited by the sample or the buffer volatility or atomization efficiency. Simultaneous XRF and UV absorbance detection can be used to provide an on-line determination of metal/chelate ratios. Although XRF detection limits are presently only in the 0.1 mM (0.5 ng) range, both collection geometry and incident intensity have yet to be optimized. Further optimization is expected to enhance this detection limit by another 2-3 orders of magnitude. As a result, the advent of XRF detection combined with the separating power of CE presents new possibilities for on-line, element-specific analysis.

Chelating Agents↗

PubMed2000

The effect of cyclic pressure on human monocyte-derived macrophages in vitro.

Aseptic loosening and osteolysis around prosthetic joints are the principal causes of failure and consequent revision. During this process activated macrophages produce cytokines which are thought to promote osteolysis by osteoclasts. Changes in pressure within the space around implants have been proposed as a cause of loosening and osteolysis. We therefore studied the effect of two different regimes of cyclic pressure on the production of interleukin-1beta (IL-1beta), IL-6 and tumour necrosis factor-alpha (TNF-alpha) by cultured human monocyte-derived (M-D) macrophages. There was a wide variation in the expression of cytokines in non-stimulated M-D macrophages from different donors and therefore cells from the same donor were compared under control and pressurised conditions. Both regimes of cyclic pressure were found to increase expression of IL-6 and TNF-alpha. Expression of IL-1beta was increased by a higher-frequency regime only. Our findings suggest that M-D macrophages are activated by cyclic pressure. Further work will be required to understand the relative roles of frequency, amplitude and duration of applied pressure in the cellular effects of cyclic pressure in this system.

Cells, Cultured↗

PubMed1999

Pressure-controlled high-performance liquid chromatographic study on the influence of rim chemistry on partial molar volume differences between free and complexed cyclodextrins.

Pragmatic comparison of pressure dependent retention for differing cyclodextrin rim chemistries is assessed using controlled-pressure HPLC. For pressure differences of <300 bar, systematic shifts in solute capacity factor are observed for both native and methylated beta-cyclodextrin stationary phases. In addition to the importance of this observation for the practice of liquid chromatography, this technique can also be implemented in the fundamental determination of the influence of rim chemistry on the cyclodextrin partial molar volume both with and without solute inclusion. That is, pressure-controlled measurements provide a direct comparison between the partial molar volumes for native cyclodextrin (CD) and methylated cyclodextrin (MCD) in the presence and absence of the complexing solute (comp). Surprisingly, direct comparison of the measured partial molar volumes for the two rim chemistries indicates that the presence of neutral solutes does not contribute significantly to the volumetric component of complexation, V(comp,CD) - V(comp,MCD) approximately V(CD) - V(MCD). In contrast, their ionized counterparts are shown to exhibit marked rim chemistry differences in the partial molar volume of cyclodextrins with and without anion inclusion, V(comp(-),CD) - V(comp(-),MCD) < V(CD) - V(MCD). Not previously demonstrated by direct chromatographic measurement, these results have interesting implications for advancing the fundamental understanding of host-guest solvation properties.

Chemical Phenomena↗

PubMed1999

Validation of mitochondrial DNA minisequencing for forensic casework.

The validation of multiplex solid-phase fluorescent minisequencing of mitochondrial DNA (mtDNA) for use in forensic casework is presented. Validation included testing of the reliability and species specificity of the technique, analysis of mixed body fluid samples, analysis of samples and substrate controls from previous cases and somatic stability of mtDNA. Animal, bacterial and fungal species extracts were examined and the test did not show cross-reactivity with other species. Hair, blood, saliva, faeces and semen or vaginal samples were tested from five male and five female individuals. For all the samples tested, heteroplasmy was observed only at position 302/309.1. Body fluid mixtures (blood:saliva, semen:saliva, faeces:semen, vaginal:semen) and DNA:DNA mixtures were examined. In total, 189 mixtures were analysed of which one resulted in a hybrid profile consisting of peaks from each of the two donors. The semen fraction of the semen:saliva and vaginal:semen mixtures appeared to be concentrated in the supernatant fraction of the extract thus highlighting the need to extract both the pellet and supernatant fractions of a stain. Control samples, crime stains and their substrate controls from previous cases were examined. Of the 12 loci typed by minisequencing, 11 could be verified by comparison to results from the sequencing method currently in use for casework and no discrepancies were observed between the two. MtDNA minisequencing was found to be a reliable and reproducible technique and its rapid and discriminating nature make it particularly suitable as a screening technique.

Animals↗

PubMed1999

Cytotoxicity of cyanoacrylate adhesives to cultured tendon cells.

The in vitro cytotoxicity of four cyanoacrylate adhesives was tested using cultures of cells derived from human tendons. All four were found to be cytotoxic, even at concentrations as low as 1.7%, over the experimental period of up to 18 weeks. This study shows that such adhesives in their present state may not be suitable for re-joining cut tendons as their initial and long-term toxicity may hinder the slow healing process of tendons.

Cells, Cultured↗

PubMed1998

Screening for hypercholesterolaemia in primary care: randomised controlled trial of postal questionnaire appraising risk of coronary heart disease.

OBJECTIVES: To validate a self administered postal questionnaire appraising risk of coronary heart disease. To determine whether use of this questionnaire increased the percentage of people at high risk of coronary heart disease and decreased the percentage of people at low risk who had their cholesterol concentration measured. DESIGN: Validation was by review of medical records and clinical assessment. The questionnaire appraising risk of coronary heart disease encouraged those meeting criteria for cholesterol measurement to have a cholesterol test and was tested in a randomised controlled trial. The intervention group was sent the risk appraisal questionnaire with a health questionnaire that determined risk of coronary heart disease without identifying the risk factors as related to coronary heart disease; the control group was sent the health questionnaire alone. SETTING: One capitation funded primary care practice in Canada with an enrolled patient population of about 12 000. SUBJECTS: Random sample of 100 participants in the intervention and control groups were included in the validation exercise. 5686 contactable patients aged 20 to 69 years who on the basis of practice records had not had a cholesterol test performed during the preceding 5 years were included in the randomised controlled trial. 2837 were in the intervention group and 2849 were in the control group. MAIN OUTCOME MEASURES: Sensitivity and specificity of assessment of risk of coronary heart disease with risk appraisal questionnaire. Rate of cholesterol testing during three months of follow up. RESULTS: Sensitivity of questionnaire appraising coronary risk was 87.5% (95% confidence interval 73.2% to 95.8%) and specificity 91.7% (81.6% to 97.2%). Of the patients without pre-existing coronary heart disease who met predefined screening criteria based on risk, 45 out of 421 in the intervention group (10.7%) and 9 out of 504 in the control group (1.8%) had a cholesterol test performed during follow up (P<0.0001). Of the patients without a history of coronary heart disease who did not meet criteria for cholesterol testing, 30 out of 1128 in the intervention group (2.7%) and 18 out of 1099 in the control group (1.6%) had a cholesterol test (P=0.175). Of the patients with pre-existing coronary heart disease, 1 out of 15 in the intervention group (6.7%) and 1 out of 23 in the control group (4.3%) were tested during follow up (P=0.851, one tailed Fisher's exact test). CONCLUSIONS: Although the questionnaire appraising coronary risk increased the percentage of people at high risk who obtained cholesterol testing, the effect was small. Most patients at risk who received the questionnaire did not respond by having a test.

Adult↗

PubMed1998

Soluble factors secreted by macrophage-like cells in vitro cause osteoprogenitor cell detachment.

Explant cultures of adult rabbit bone were obtained from a variety of skeletal sites, with the intention of using the cells in a new type of bone graft. The explant cultures contained large numbers of esterase-positive, alkaline phosphatase-negative, TRAP-negative macrophage-like cells (MP), as well as osteoblast-like osteoprogenitor (OP) cells (alkaline phosphatase-positive). OP numbers were assessed by cell counts and MTT assay. The presence of the MP cells appeared to give rise to a reduction in OP numbers in culture, through cell detachment and cell lysis. In addition, after passaging, many OP were unable to reattach to the culture vessels in the presence of MP, depending upon the surface area available for reattachment. The presence of tumor necrosis factor alpha (TNFalpha) in culture medium from these cell cultures was demonstrated by a specific enzyme-linked immunosorbent assay (ELISA). A direct relationship was demonstrated between MP numbers and TNFalpha concentration and an inverse relationship between MP numbers and OP numbers in co-cultures. This was also found when OP cells were exposed to different concentrations of rTNFalpha, in place of the MP. Incubation with anti-TNFalpha inhibited the effect of MP and TNFalpha on OP. These results suggest that MP are able to cause detachment and lysis of OP cells, probably by secretion of a soluble factor in vitro which may be TNFalpha.

Animals↗

PubMed1998

Fibroblast-like cells from tendons differ from skin fibroblasts in their ability to form three-dimensional structures in vitro.

Tendon samples cultured in vitro produced cells (TC) with fibroblast-like morphology and confluence occurred within 5 weeks. Histological staining demonstrated proteoglycan and collagen secretion by TC. Immunohistochemical staining revealed type I collagen but no type III. Assay of total collagen demonstrated a rapid increase in synthesis with time in culture. Cultures allowed to become 'superconfluent' spontaneously formed three-dimensional structures after about 4 weeks, which became macroscopic, tendon-like structures (TLS). Cells within TLS seemed under cell-generated tension. Haematoxylin and eosin staining of sections of tendon, of TLS and of TC cultures demonstrated similarities in morphology. These studies were performed using human and rabbit cells and findings were similar for the two species, but with some differences in cell metabolism. Skin fibroblasts were also cultured as a comparison.

Animals↗

PubMed1998

Selective opportunistic screening for hypercholesterolemia in primary care practice.

OBJECTIVES: To assess the performance of selective opportunistic screening in a primary care group practice. DESIGN: Cross-sectional survey of coronary heart disease risk factors and retrospective chart audit of cholesterol testing. SETTING: Capitation-funded primary care group practice in Ontario, Canada. SUBJECTS: 7785 enrolled patients between the ages of 20 and 69 years. INTERVENTION: Protocol-based selective opportunistic screening program for hypercholesterolemia of 45 months duration. MAIN OUTCOME MEASURES: Targeting (proportion of screening tests that were appropriate), coverage (proportion of those meeting screening criteria who had a screening test performed), over-screening (proportion of those not meeting screening criteria who had a screening test performed), and screening ratio (likelihood that a screening test was performed on an individual who met screening criteria rather than one who failed to meet screening criteria). RESULTS: 64.7% of patients tested met the practice criteria for screening. 37.7% of patients who met the practice screening criteria were tested and 24.9% of those not meeting practice screening criteria had a cholesterol test performed. The screening ratio was 1.52. CONCLUSION: Our findings bring into question the effectiveness of opportunistic approaches to preventive care.

Adult↗

PubMed1997

Casting the screening net: separating big fish from little fish.

Screening tests are a rapidly growing part of medical practice. If we are going to make the best use of resources, screening tests need to be considered in terms of effectiveness, efficiency and equity. We present a framework as a way to think about screening programmes. The framework expands on existing literature that recognizes two categories of screening: universal and opportunistic. By adding the dimension of 'selectivity', we identify four categories of screening: active non-selective (universal or mass screening), active selective, opportunistic non-selective and opportunistic selective. We illustrate the framework by categorizing screening recommendations for high serum cholesterol levels. We conclude there is no one ideal strategy for screening that simultaneously satisfies criteria of effectiveness, efficiency and equity. However, our framework allows a systematic consideration and balancing of these objectives in the development and assessment of screening programs. In this way, it may assist decision-makers by making this trade-off more explicit.

Canada↗

PubMed1997

The effect of dietary sodium on calcium metabolism in premenopausal and postmenopausal women.

OBJECTIVE: To investigate the effects of high and low sodium diets on urinary calcium, bone turnover and calcium absorption in pre and postmenopausal women. DESIGN: Experimental, prospective and longitudinal study. SETTING: Samples were taken at the hospital and the diets were followed at home. SUBJECTS: Volunteers were recruited from the hospital and were either hospital staff or post-graduate students. No volunteers failed to complete the study but one was omitted from analysis due to lack of compliance. INTERVENTIONS: Eleven healthy premenopausal women aged 22-47 y and 11 healthy postmenopausal women ages 45-70 y followed a high (300 mmol/d) and a low (50 mmol/d) sodium diet for one week each. On the 7th day of each diet, blood and urine samples were taken. RESULTS: On the high sodium diet 24 h urinary sodium and calcium values relative to creatinine were significantly higher for all subjects (P < 0.05). Postmenopausal women on the high sodium diet had biochemical evidence of increased bone resorption in relation to the low sodium diet. However in premenopausal women there was no such change. Calcium absorption did not change significantly in either group. CONCLUSIONS: It appears that postmenopausal, but not premenopausal, women respond to a high sodium diet by an increase in bone resorption which may lead to reduced bone density. SPONSORSHIP: Arthritis and Rheumatism Council Project Grant R44.

Absorption↗

PubMed1996

A comparison of techniques to assess skin blanching following the topical application of glucocorticoids.

Glucocorticoid-induced dermal blanching provides a useful research tool to study steroid potency and sensitivity. Conventional measurement of the intensity of blanching relies on subjective assessment by a trained observer using a visual score. Several objective techniques have recently been reported to detect skin blanching, but their sensitivity has not been compared previously with subjective visual recordings. In this report we aimed to establish whether objective methods offer sufficient sensitivity to be employed in epidemiological studies of glucocorticoid responsiveness. In healthy subjects we applied beclomethasone dipropionate at three concentrations (1, 10 and 100 micrograms/ml) under an occluded dressing overnight. The following morning we measured blanching using a visual score, laser Doppler velocimetry with the MBF 3D monitor (Moor Instruments Ltd, U.K.) and a perfusion imager (Lisca, Sweden), and reflectance spectrophotometry with the Dia-Stron 'erythemameter'. Using the visual score, blanching was detected at all concentrations of steroid. Neither laser Doppler instrument detected vasoconstriction at any concentration. By contrast, the reflectance spectrophotometer successfully recorded blanching at 10 and 100 micrograms/ml, but not at 1 microgram/ml. We conclude that laser Doppler instruments, including the novel scanning perfusion imager, do not detect glucocorticoid-induced skin blanching, perhaps because it reflects venular rather than arteriolar vasoconstriction. By contrast, the Dia-Stron reflectance spectrophotometer has sufficient sensitivity to be used as an alternative to visual assessment in epidemiological studies of human glucocorticoid-induced dermal blanching.

Administration, Topical↗

PubMed1995

Modulation of cell phenotype in human osteoblast-like cells by the simian virus 40.

At present, the majority of in vitro research into bone metabolism is performed on either primary cultures of bone or osteosarcoma lines. A better model of the behaviour of normal bone cells would be a cell line derived from normal, adult bone that retained osteoblast-like characteristics. We infected a culture of bone cells from adult humans with simian virus 1613, a variant of the simian virus 40, and obtained 12 clones of variable morphology. The clones were maintained in culture for as long as 6 months. Population doubling times, synthesis of alkaline phosphatase and osteocalcin, secretion of mineral, morphology, and ability to withstand freezing were examined. SV/EC cell morphology varied from the polygonal, osteoblast-like to the bipolar, fibroblast-like. Population doubling times ranged from 0.55 to 2.8 days (compared with 3.9 days for the nontransformed human osteoblast-like cells). Synthesis of alkaline phosphatase varied but was less than that by the human osteoblast-like cells. With the exception of clone 11, all of the transformed clones synthesised mineral in vitro under mineralising conditions. Four clones showed increased synthesis of alkaline phosphatase and increased population doubling times after passaging. All of the clones were successfully frozen and thawed, but, unlike normal human osteoblast-like cells, none responded to stimulation with parathyroid hormone.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkaline Phosphatase↗