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Biomedical subjects

E Ingham

Publications and source records attributed to E Ingham.

At least 37 records · Page 2Linked to original sources
PubMed2001

In Staphylococcus aureus, fur is an interactive regulator with PerR, contributes to virulence, and Is necessary for oxidative stress resistance through positive regulation of catalase and iron homeostasis.

The Staphylococcus aureus genome encodes three ferric uptake repressor (Fur) homologues: Fur, PerR, and Zur. To determine the exact role of Fur in S. aureus, we inactivated the fur gene by allelic replacement using a tetracycline resistance cassette, creating strain MJH010 (fur). The mutant had a growth defect in rich medium, and this defect was exacerbated in metal-depleted CL medium. This growth defect was partially suppressed by manganous ion, a metal ion with known antioxidant properties. This suggests that the fur mutation leads to an oxidative stress condition. Indeed, MJH010 (fur) has reduced levels of catalase activity resulting from decreased katA transcription. Using a katA-lacZ fusion we have determined that Fur functions, either directly or indirectly, as an iron-dependent positive regulator of katA expression. Transcription of katA is coregulated by Fur and PerR, since in MJH010 (fur) transcription was still repressed by manganese while transcription in MJH201 (fur perR) was unresponsive to the presence of iron or manganese. Siderophore biosynthesis was repressed by iron in 8325-4 (wild-type) but in MJH010 (fur) was constitutive. A number of putative Fur-regulated genes were identified in the incomplete genome databases using known S. aureus Fur box sequences. Of those tested, the sstABCD and sirABC operons and the fhuD2 and orf4 genes were found to have Fur-regulated expression. MJH010 (fur) was attenuated (P<0.04) in a murine skin abscess model of infection, as was double-mutant MJH201 (fur perR) (P<0.03). This demonstrates the importance in vivo of iron homeostasis and oxidative stress resistance regulation in S. aureus.

Animals↗
PubMed2001

Influence of simulator kinematics on the wear of metal-on-metal hip prostheses.

There is now considerable interest in metal-on-metal bearings for hip prostheses. Extremely low wear rates (0.1 mm3/10(6) cycles) have been reported in some simulator studies, while in vivo studies, although still very low, have shown wear rates of the order of 1 mm3/10(6) cycles. The aim of this study was to compare wear rates of metal-on-metal bearings in two hip simulators with different kinematic inputs. In the simulator with three independent input motions which produced an open elliptical wear path with a low level of eccentricity, the wear rates were very low as recorded previously in other simulators. In the simulator with two input motions which produced an open elliptical wear path with greater eccentricity the wear rate was at least ten times higher and closer to clinical values. The motion and kinematic conditions in the contact are critical determinants of wear in metal-on-metal bearings.

Biomechanical Phenomena↗
PubMed2001

A novel method for the prediction of functional biological activity of polyethylene wear debris.

The comparative performance of artificial hip joints has been extensively investigated in vitro through measurements of wear volumes. In vivo a major cause of long-term failure is wear-debris-induced osteolysis. These adverse biological reactions are not simply dependent on wear volume, but are also controlled by the size and volumetric concentration of the debris. A novel model is presented which predicts functional biological activity; this is determined by integrating the product of the biological activity function and the volumetric concentration function with the wear volume over the whole particle size range. This model combines conventional wear volume measurements with particle analysis and the output from in vitro cell culture studies to provide a new indicator of osteolytic potential. The application of the model is demonstrated through comparison of the functional biological activity of wear debris from polyethylene acetabular cups articulating under three different conditions in a hip joint simulator.

Biocompatible Materials↗
PubMed2001

The influence of phospholipid concentration in protein-containing lubricants on the wear of ultra-high molecular weight polyethylene in artificial hip joints.

There is considerable interest in the wear of polyethylene and the resulting wear-debris-induced osteolysis in artificial hip joints. Proteins play an important role as boundary lubricants in vivo in the pseudosynovial fluid, and these are reproduced in in vitro tests through the use of bovine serum. Little is known, however, about the effect of phospholipid concentrations within proteinaceous solutions on the wear of ultra-high molecular weight polyethylene (UHMWPE). The effects of protein-containing lubricants with 0.05, 0.5 and 5 per cent (w/v) phosphatidyl choline concentrations on the wear of ultra-high molecular weight polyethylene (UHMWPE) were compared with 25 per cent (v/v) bovine serum which had 0.01 per cent (w/v) lipid; the effects were compared in a hip joint simulator with smooth (n = 4) and scratched (n = 3) femoral heads. The control bovine serum lubricant produced UHWMPE wear of 55 and 115 mm3/10(6) cycles on the smooth and rough heads respectively. The increased phospholipid concentration significantly reduced the wear rate. At the higher concentration (5% w/v phosphatidyl choline) the average wear was reduced to less than 2 mm3/10(6) cycles. Even with the relatively low concentrations of 0.05% w/v phosphatidyl choline the wear was reduced by at least threefold compared with the bovine serum tests for both the smooth and rough femoral heads. There may be considerable differences in the phospholipid concentrations in patients' synovial fluid and this is highly likely to produce considerable variation in wear rates. In vitro, differences in the phospholipid concentration of lubricants may also cause variation in wear rates between different simulator tests.

Hip Prosthesis↗
PubMed2001

A novel three-dimensional tissue equivalent model to study the combined effects of cyclic mechanical strain and wear particles on the osteolytic potential of primary human macrophages in vitro.

The effects of cyclic mechanical strain and challenge with physiologically relevant doses of submicrometre-size polyethylene (PE) particles on the osteolytic potential of primary human mononuclear phagocytes were investigated. Cells were seeded into a three-dimensional tissue matrix and co-cultured with particles (mean size 0.21 microm) at particle volume to cell number ratios of 7.5, 15, 30 and 100 microm3/cell. Matrices were then either cultured statically or subjected to 20 per cent cyclic compressional strain in the 'ComCell' for 16 h prior to the assessment of cell viability and quantification of the pro-inflammatory cytokine tumour necrosis factor alpha (TNFalpha). The MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazdium bromide) assay was shown to be too insensitive to detect changes in cell viability. However, when quantified by the adenosine triphosphate (ATP) assay, cell viability was demonstrated to be reduced following exposure to cyclic strain. Macrophages cultured in the static three-dimensional tissue equivalent model produced very high levels of TNFalpha in response to submicrometre PE particles at a ratio of 100 microm3/cell. Cyclic strain in the absence of particles gave only a small increase in TNFa production. However, the combined effects of strain and particle stimulation at a ratio of 30 microm3/cell resulted in the secretion of significantly more TNFalpha than was produced by macrophages subjected to strain alone, or the cells-only control. This synergy between cyclic strain and PE particle stimulation was only evident when the volume of particles was reduced below the volume that maximally stimulated cells. These results suggest that while cyclic strain may not be the primary factor responsible for macrophage activation and periprosthetic osteolysis, at low particle load, it may contribute significantly to the osteolytic potential of macrophages in vitro or in vivo.

Coculture Techniques↗
PubMed2001

A retrieval study of capital hip prostheses with titanium alloy femoral stems.

We have examined 26 retrieved, failed titanium-alloy femoral stems. The clinical details, radiological appearances and the histology of the surrounding soft tissues in each patient were also investigated. The stems were predominantly of the flanged design and had a characteristic pattern of wear. A review of the radiographs showed a series of changes, progressive with time. The first was lateral debonding with subsidence of the stem. This was followed by calcar resorption and fragmentation or fracture of the cement. Finally, osteolysis was seen, starting with a radiolucency at the cement-bone interface and progressing to endosteal cavitation. Three histological appearances were noted: granulomatous, necrobiotic and necrotic. We suggest that an unknown factor, possibly related to the design of the stem, caused it to move early. After this, micromovement at the cement-stem interface led to the generation of particulate debris and fracture of the cement. A soft-tissue reaction to the debris resulted in osteolysis and failure of fixation of the prostheses.

Aged↗
PubMed2001

Comparative wear and wear debris under three different counterface conditions of crosslinked and non-crosslinked ultra high molecular weight polyethylene.

The wear debris generated from ultra high molecular weight polyethylene (UHMWPE) have been recognised as one of the major causes of failure in total hip replacements (THR). It is essential to reduce the wear debris generated from UHMWPE acetabular cups in order to minimise this problem. Debris in the submicron size range is believed to have greater osteolytic potential. It is now known that crosslinked UHMWPE acetabular cups have reduced volumetric wear rates but little is known about the influence of crosslinking on the size and morphology of the wear debris. In this study, the wear of grade GUR 1020 crosslinked (vacuum gamma irradiated), GUR 1120 crosslinked (acetylene enhanced irradiated) and non cross linked (ethylene oxide sterilised) GUR 1020 UHMWPE was compared in multidirectional pin-on-plate wear tests under three different counterface conditions (smooth, isotropically rough and scratched counterfaces). Multidirectional motion was chosen because this motion was closer to the relative motion in the natural hip. From this study, better wear resistance of crosslinked UHMWPE compared with non-crosslinked UHMWPE was demonstrated for the smooth counterface conditions. However, in the rough and scratched counterface conditions, the vacuum gamma irradiated crosslinked material produced significantly higher wear rates than the non-crosslinked material. The analysis of the wear debris showed that the majority of the volume of the acetylene enhanced crosslinked UHMWPE wear debris was in the most biologically active size range (0.1 to 0.5 microm). In contrast, the non-crosslinked material and the vacuum gamma irradiated crosslinked material had a greater proportion of the volume of the debris in the larger size ranges which are less biologically active. This has important implications for its osteolytic potential.

Acetylene↗
PubMed2001

Quantitative analysis of wear and wear debris from metal-on-metal hip prostheses tested in a physiological hip joint simulator.

Osteolysis and loosening of artificial joints caused by UHMWPE wear debris has prompted renewed interest in metal-on-metal (MOM) hip prostheses. This study investigated the wear and wear debris morphology generated by MOM prostheses in a physiological anatomical hip simulator for different carbon content cobalt chrome alloys. The low carbon pairings demonstrated significantly higher "bedding in" and steady state wear rates than the mixed and high carbon pairings. The in vitro wear rates reported here were up to one or two orders of magnitude lower than the clinical wear rates for first-generation MOM hip prostheses. Two methods for characterising the metal wear debris were developed, involving digestion, scanning electron microscopy and transmission electron microscopy. The metal wear particles characterised by the two methods were similar in size, 25-36 nm, and comparable to particles isolated from periprosthetic tissues from first and second-generation MOM hip prostheses. Due to the small size of the metal particles, the number of particles generated per year for MOM prostheses in vitro was estimated to be up to 100 times higher than the number of polyethylene particles generated per year in vivo. The volumetric wear rates were affected by the carbon content of the cobalt chrome alloy and the material combinations used. However, particle size and morphology was not affected by method of particle characterisation, the carbon content of the alloy or material combination.

Biomechanical Phenomena↗
PubMed2001

Comparative analysis of two different types of alumina-alumina hip prosthesis retrieved for aseptic loosening.

We compared and quantified the modes of failure and patterns of wear of 11 Mittelmeier and 11 Ceraver-Ostal retrieved alumina-alumina hip prostheses with reference to the corresponding clinical and radiological histories. Macroscopic wear was assessed using a three-dimensional co-ordinate measuring machine. Talysurf contacting profilometry was used to measure surface roughness on a microscopic scale and SEM to determine mechanisms of wear at the submicron level. The components were classified into one of three categories of wear: low (no visible/measurable wear), stripe (elliptical wear stripe on the heads and larger worn areas on the cups) and severe (macroscopic wear, large volumes of material lost). Overall, the volumetric wear of the alumina-alumina prostheses was substantially less than the widely used metal and ceramic-on-polyethylene combinations. By identifying and eliminating the factors which accelerate wear, it is expected that the lifetime of these devices can be further increased.

Adolescent↗
PubMed2000

Heat shock proteins and inflammatory acne vulgaris: molecular cloning, overexpression and purification of a propionibacterium acnes GroEL and DnaK homologue.

Propionibacterium acnes is associated with inflammatory acne. The genes encoding two putative mediators of inflammation, the heat shock proteins GroEL and DnaK, were cloned from this organism and sequenced. groEL and dnaK encode proteins of 56.8 and 66.4 kDa, respectively, which show a high degree of homology (>75% similarity) to the GroEL and DnaK proteins of mycobacteria and streptomycetes. The promoter regions of both genes contain inverted repeat sequences believed to be involved in the transcriptional regulation of heat shock genes. Recombinant P. acnes GroEL and DnaK were overexpressed in Escherichia coli with C-terminal histidine tags. The recombinant proteins were purified from E. coli by metal affinity chromatography. These proteins will now be used in immunological investigations to determine their role in inflammatory acne.

Acne Vulgaris↗
PubMed2000

Effect of size and dose on bone resorption activity of macrophages by in vitro clinically relevant ultra high molecular weight polyethylene particles.

Polyethylene wear debris generated at the bearing surfaces of total artificial hip joints is thought to play an important role in the periprosthetic osteolysis and ultimately the aseptic loosening of these prostheses. The macrophage is believed to be central to this process by releasing various cytokines and other mediators of osteolysis upon phagocytosis of the polyethylene wear debris. This study evaluated the in vitro bone resorption response of C3H murine peritoneal macrophages to clinically relevant GUR 1120 polyethylene particles. Macrophages were co-cultured in vitro with GUR 1120 particles with a mean size of 0.24, 0.45, 1.71, and 7.62, and GUR 1120 polyethylene resin with a mean size of 88 microm at various particle volume (microm)(3): macrophage ratios (0.1:1; 1:1; 10:1; and 100:1). The conditioned supernatants were incubated with (45)calcium radio-labeled mouse calvariae, and bone resorption was measured as (45)calcium release. The results showed that the 0.24 microm particles stimulated the macrophages to generate bone resorbing activity at a ratio of 10(microm)(3) per macrophage. The 0.45 and 1.71 microm particles were active at a ratio of 100( microm)(3) per macrophage, and the 7.62 and 88 microm particles were inactive at all the doses tested. The co-culture supernatants were also assayed for TNF-alpha, IL-1beta, IL-6, and PGE(2). The results followed the same trend for particle size and volume dose to that observed for the bone resorbing activity. This study has demonstrated, for the first time, the importance of size and dose of clinically relevant polyethylene particles on the osteolytic response of macrophages in vitro.

Animals↗
PubMed2000

Evaluation of the response of primary human peripheral blood mononuclear phagocytes to challenge with in vitro generated clinically relevant UHMWPE particles of known size and dose.

The response of primary human peripheral blood mononuclear phagocytes to challenge with clinically relevant ultra-high molecular weight polyethylene (UHMWPE) wear debris of known particle size and dose was evaluated. Particles with a mean size of 0.24, 0. 45, 1.7, 7.6, and 88 microm were cocultured with cells for 24 h before assessment of cell viability and production of the osteolytic cytokines interleukin (IL)-1 beta, IL-6, tumor necrosis factor-alpha, and granulocyte macrophage colony-stimulating factor, and prostaglandin E(2). All particle fractions were evaluated at particle volume (microm(3)) to cell number ratios of 10:1 and 100:1, which had been previously identified as being the most stimulatory and clinically relevant. None of the test fractions had an effect on cell viability. Whereas the heterogeneity of human individuals was clearly evident in the responses of the donors evaluated in this study (the response of donor 3 was between 5 and 20 times greater than the other donors), the most biologically active particles were found to be submicrometer in size. Stimulation with phagocytosable particles (0.24, 0.45, and 1.7 microm) resulted in enhanced levels of cytokine secretion. Macrophages stimulated with particles outside this size range produced considerably less cytokines at the volumes tested. These results confirm earlier findings and suggest that the size and volume of UHMWPE particles are critical factors in macrophage activation. Furthermore, they suggest that the heterogeneity of human individuals may be another important factor in determining implant life.

Biocompatible Materials↗
PubMed2000

Subcutaneous administration of recombinant glycosylated interleukin 6 in patients with cancer: pharmacokinetics, pharmacodynamics and immunomodulatory effects.

This is the first report of the serum profile of a glycosylated recombinant form of human IL-6 (rhIL-6) administered subcutaneously (1-10 microg/kg/day) in a phase I/II trial as a thrombopoietic agent in patients with advanced cancer. The pharmacodynamic effects of IL-6 were also examined. Detailed pharmacokinetic measurements were made in four patients. Peak concentrations at 5-8 h and a median t0.5 of ca. 5 h were similar to those previously reported for non-glycosylated IL-6. However, higher peak concentrations and apparent differences in effective dose levels to those previously reported with the non-glycosylated form were seen. Indications of an apparent attenuation in circulating IL-6 concentrations with continuing injections were seen in eight of 10 patients examined but anti-IL-6 antibody generation was seen in only two patients. Soluble interleukin 6 receptor concentrations generally decreased. No major changes in T cell subsets were seen but expression of CD25 and CD54 by T lymphocytes significantly increased, accompanied by marked increases in soluble CD25 (sIL-2R) and CD54 (sICAM-1). No consistent change in B cells, monocytes or NK cells were seen. No evidence for induction of TNF-alpha was found. This study demonstrates similar biological effects of glycosylated rhIL-6 to those reported for the non-glycosylated form but illustrates several apparent differences which are discussed further.

Adjuvants, Immunologic↗
PubMed2000

Survival and growth of Burkholderia cepacia within the free-living amoeba Acanthamoeba polyphaga.

The aim of this study was to investigate whether Burkholderia cepacia is capable of survival and growth within the free-living amoeba Acanthamoeba polyphaga using a differential immunofluorescence assay of bacterial-amoebal cocultures and viable counts of bacteria determined after amoebal lysis. The numbers of intra-amoebal bacteria and the numbers of infected amoebae increased over time; although, when heat-killed bacteria were used, no intracellular bacteria were observed. These findings should be taken into account in future studies of environmental reservoirs of Burkholderia cepacia.

Acanthamoeba↗
PubMed2000

Comparison of the response of primary human peripheral blood mononuclear phagocytes from different donors to challenge with model polyethylene particles of known size and dose.

The response of primary human peripheral blood mononuclear phagocytes to challenge with polyethylene particles of known size and dose was evaluated. Particles with mean sizes of 0.21, 0.49, 4.3, 7.2, and 88 microm were co-cultured with cells for 24 h prior to the assessment of cell viability and production of the osteolytic mediators IL-1beta, IL-6, TNFalpha, GM-CSF and PGE2. All particle fractions were evaluated at particle volume (microm3) to cell number ratios of 10:1 and 100:1 which were previously identified as being the most biologically active and clinically relevant. The heterogeneity of human individuals was clearly evident both in the profile and the magnitude of the response of the donors evaluated in this study (the response of donor 5 being 2- to 15-fold lower than that of the other donors). Only the sub-micrometre particles stimulated significantly enhanced cytokine secretion at the ratios tested: mean particle sizes of 0.49 and 0.21 microm being the most biologically active. Macrophages stimulated with particles outside this size range produced considerably lower levels of mediator. These results compared favourably with the results of earlier studies, which demonstrated that particles within the phagocytosable size range (0.1-10 microm) were the most biologically active. These results, therefore, confirm earlier findings and suggest that the size and volume of polyethylene particles are critical factors in macrophage activation. Furthermore, they suggest that the heterogeneity of human individuals may be another important factor in determining implant life and could provide the basis for a valuable diagnostic tool to identify those patients most at risk of implant loosening.

Blood Donors↗
PubMed2000

Production of TNF-alpha and bone resorbing activity by macrophages in response to different types of bone cement particles.

We have compared the capacity of clinically relevant wear debris from seven different cement types to activate macrophages to produce TNF-alpha, IL-1beta, IL-6 and bone resorbing activity in vitro. The bone cements were: CMW 1 original (PMMA only); CMW 1RO (1 microm BaSO4; 9.2%); CMW copolymer bone cement 1 (10 microm BaSO4; 10%); CMW copolymer bone cement 2 (1 microm BaSO4; 10%); Palacos R (10 microm ZrO2; 15.6%); CMW Calcium phosphate cement 20% (10 microm tri-calcium phosphate; 20%) and CMW calcium phosphate cement 30% (10 microm tri-calcium phosphate; 30%). Cement debris was produced aseptically using a simple configuration wear test. The majority of particles were in the size range 0.1-0.5 microm for each cement type. The cement particles were co-cultured with the U937 macrophage cell line at ratios of 10 and 100 microm3 particle volumes to macrophage cell numbers for 24 h. At the 10:1 ratio the particles had no effect on the cells. At the 100:1 ratio, the major cytokine produced was TNF-alpha and there were no statistical differences between the different types of cement debris. The bone resorption activity of the co-culture supernatants was significantly greater than the control (U937 cells without particles) for particles of CMW 1RO, CMW copolymer bone cement 1, CMW copolymer bone cement 2 and Palacos R (P < 0.05, ANOVA). However there were no statistical differences between the levels of bone resoprtion evoked by these four cement types. The CMW1 original and CMW calcium phosphate containing cements failed to induce the macrophages to elaborate bone resorption activity at the 100:1 ratio. These data suggest that the addition of radio-opaque additives to bone cement may increase the capacity of the debris to induce osteolysis.

Acrylic Resins↗
PubMed2000

The effects of lipid extraction on the immunomodulatory activity of Malassezia species in vitro.

Malassezia spp. are believed to be the causative agents of pityriasis versicolor and are strongly implicated in seborrhoeic dermatitis. The yeast also forms part of the normal human cutaneous microflora. We have previously shown that when Malassezia yeast cells are incubated with human peripheral blood mononuclear cells (PBMCs), they are capable of reducing the levels of pro-inflammatory cytokines produced. In order to test the hypothesis that this immunoevasive phenomenon may be related to the unusually high level of lipid in the Malassezia yeast cell wall, we have compared the immunomodulatory capacity of normal and lipid-depleted yeast cells. Stationary phase yeast cells of Malassezia sympodialis, M. globosa and M. restricta were treated with chloroform/methanol to extract the surface lipids. The lipid-depleted and non-depleted yeast cells were then co-cultured with human PBMCs from three different human donors at a ratio of 20 yeasts per leukocyte for 24 h. The levels of interleukin (IL)-1beta, IL-6 and tumour necrosis factor (TNF)-alpha were then determined by enzyme-linked immunosorbent assay (ELISA). The results demonstrated that extraction of lipid reversed the yeast cell capacity to reduce the levels of pro-inflammatory cytokines. The levels of IL-1beta, IL-6 and TNF-alpha produced in response to lipid-extracted Malassezia of all three species were either no different from or significantly greater (P < 0.05; ANOVA) than the constitutive control levels. These results suggest that the lipid microfibrillar layer of Malassezia may prevent the yeast cells from inducing inflammation and provide an explanation for the normal commensal status of the organism on human skin. The hypothesis that the lipid layer is absent or altered in seborrhoeic dermatitis may provide an explanation for the inflammatory nature of this dermatosis.

Cells, Cultured↗