DNA array analysis of interleukin-2-regulated immediate/early genes

BACKGROUND: Lymphocyte activation culminates in blastogenesis, cell cycle progression, DNA replication and mitosis. These complex cellular changes are programmed almost simultaneously by multiple ligands and receptors that trigger specific signal transduction pathways and transcription factors. Until now, the discovery of the genes regulated by each ligand/receptor pair has been hampered by the technologies available. RESULTS: To identify interleukin-2 (IL-2)-responsive genes, human peripheral blood mononuclear cells (PBMC) were pre-activated with anti-CD3, rested, and restimulated with IL-2 for 4 hr. Gene expression was analyzed using Affymetrix U95Av2 oligonucleotide arrays. To determine the most stringent parameters to score a gene as a bona fide IL-2 target, the expression of 19 known IL-2-regulated genes was examined first. All were induced at least 2-fold, with a difference in fluorescent intensity of ≥ 100 at p < 0.05. An additional 53 unique genes met these criteria. To determine which of these were immediate/early IL-2 targets in T cells, purified T cells were stimulated with IL-2 for 4 hr in the presence of cycloheximide to prevent secondary gene expression. Of the 72 genes identified in PBMCs, 20 were detected as immediate/early IL-2-regulated genes in purified T cells. In addition, 27 unique genes were IL-2-regulated in T cells but not in PBMCs. CONCLUSIONS: For a successful reductionist approach to the analysis of gene expression in lymphocyte activation, it is necessary to examine purified cell populations and immediate/early gene expression regulated by each ligand/receptor pair involved. This approach should allow the discovery of genes regulated by all of the ligand/receptor pairs involved in lymphocyte activation

The Effect of Microseparation on Corrosion Rates of Metal-on-Metal Total Hip Replacements

The poor performance of Metal-on-Metal (MoM) bearings has to date been blamed on “adverse loading” conditions. Studies have focused on the effect of cup inclination and microseparation on gravimetric wear rates and highlighted the importance of surgical technique when implanting such devices. Up to four fold increase in the wear rates of MoM bearings subjected to microseparation has been reported during the bedding-in period. The contribution of corrosive processes to overall material degradation during adverse loading has not previously been investigated. In the present study 28 mm HC CoCrMo alloy Total Hip Replacements were tested to 1 Mcycles under standard gait and severe microseparation conditions in an electrochemically instrumented hip simulator. An order of magnitude increase in material lost as a result of oxidation was noted (0.234 to 2.044 mm3/Mcycle) during microseparation. Corrosive degradation may therefore be a much more significant contribution to poor bearing performance under adverse loading than previously considered

Investigation of the Repassivation Process of CoCrMo in Simulated Biological Fluids

A thorough investigation into the repassivation process of CoCrMo in multiple simulated biological environments has been undertaken, looking in detail at both the kinetics and composition of the reformed oxide film. Specific focus of this research was aimed at determining the effect of bovine serum albumin (BSA) on these features. The kinetics of repassivation were obtained by using a variety of electrochemical techniques. The current transients formed were fitted to a second-order decay curve, which accouts for two separate phases: coverage and growth. The reformation of the passive film was fastest in a phosphate buffered saline environment, with the presence of BSA delaying this process because it inhibits the oxygen-reduction reaction as it obstructs the active sites of the alloy when adsorbed onto the surface. The composition of the newly formed film was analyzed with x-ray photoelectron spectroscopy. As expected, the film was primarily composed of chromium (III) oxide with small contributions from cobalt and molybdenum oxides. In the presence of BSA, the quantity of molybdenum within the film was drastically reduced; it was shown to be extracted into the bulk solution via inductively coupled mass spectroscopy. This is observed because BSA is able to complex preferentially to the molybdenum ions when the alloy is exposed, extracting them into solution and altering the composition and integrity of the film

A Link Between the Tribology and Corrosive Degradation of Metal-on-Metal THRs

The degradation of Metal-on-Metal (MoM) Total Hip Replacements (THRs) is a complex mix of tribological, corrosive phenomena and their synergistic processes. Previous links between the corrosion of these devices and their sliding conditions over a cycle have been observed in simulator studies instrumented with a three-electrode electrochemical cell. This study further quantifies that link; demonstrating clear repeating periodicity in the anodic current transients of a 28 mm diameter MoM bearing under a standard ISO-14242 walking profile. A simplified 2D model and an expression of the Hamrock-Dowson equation was utilised to estimate the Theoretical Minimum Film Thickness (hmin) over a cycle, which was shown to match closely to the measured anodic current in both shape and magnitude

Tribocorrosion of hard-on-hard total hip replacements with metal and ceramic counterfaces under standard and adverse loading conditions

28 mm Metal-on-Metal (MoM) and Metal-on-Ceramic (MoC) Total Hip Replacements were articulated to 1 million cycles under both Standard Gait and Microseparation conditions. The hip simulator was fully instrumented with a three-electrode electrochemical cell to facilitate monitoring of corrosive degradation. The estimated volume loss from corrosion at the bearing surface was seen to increase by nearly an order of magnitude for both devices, representing as much as 17% of total degradation. Anodic current transients also displayed near order of magnitude increases in the peak current for both bearing couples. An adverse loading scenario could cause as much as an order of magnitude increase in the metallic ions released into the joint capsule as well as an increased volume of wear debris

Genetic testing for Familial Hypercholesterolaemia - Past, Present and Future

In the early 1980s, the Nobel Prize winning cellular and molecular work of Mike Brown and Joe Goldstein led to the identification of the Low Density Lipoprotein Receptor (LDLR) gene as the first gene where mutations cause the Familial Hypercholesterolaemia (FH) phenotype. We now know that autosomal dominant monogenic FH can be caused by pathogenic variants of three additional genes (APOB/PCSK9/APOE), and that the plasma LDL-C concentration and risk of premature Coronary Heart Disease (CHD) differs according to the specific locus and associated molecular cause. It is now possible to use Next Generation Sequencing (NGS) to sequence all exons of all four genes, processing 96 patient samples in one sequencing run, increasing the speed of test results and reducing costs. This has resulted in the identification of many novel FH-causing variants, but also some "Variants of Unknown Significance (VUSs)" which require further evidence to classify as pathogenic or benign. The identification of the FH-causing variant in an index case can be used as an unambiguous and rapid test for other family members. An FH-causing variant can be found in 20%-40% of patients with the FH phenotype, and we now appreciate that in the majority of patients without a monogenic cause, a polygenic aetiology for their phenotype is highly likely. Compared to those with a monogenic cause, these patients have significantly lower risk of future CHD. The use of these molecular genetic diagnostic methods in the characterization of FH is a prime example of the utility of precision or personalised medicine

Adverse loading effects on tribocorrosive degradation of 28 mm metal-on-metal hip replacement bearings

Following the high clinical failure rates of metal-on-metal total hip replacements much work has been undertaken to investigate their poor performance. So called adverse loading scenarios such as acetabular inclination and microseparation have been attributed to indicators for failure of the implants. The ISO hip simulation standards (ISO 14242:1) still rely on gravimetric and ex situ analysis, considering only the total wear during articulation. Live in situ sensing can provide valuable insight into the degradation mechanisms of metallic interfaces under such scenarios. Clinical 28 mm diameter metal-on-metal components were articulated in a full-ISO hip simulator. The bearings were subjected to increasing angles of acetabular inclination and retroversion over short-term periods of articulation. Corrosive degradation was monitored during sliding by means of an in situ three-electrode cell. Changing acetabular inclination from 30° to 50° resulted in greater cathodic shifts in OCP upon the initiation of sliding; from −50 mV to as much as −150 mV. Under anodic polarisation (0 mV vs. Ag/AgCl) the resultant currents at the initiation of sliding also increased significantly with inclination; from approximately 4–10 µA to over 120 µA. Increased retroversion of 20° also resulted in increased anodic currents of 55–60 µA. Changing the nature of articulation demonstrated increased corrosive material loss compared to a standard ISO 14242 profile. The sole use of gravimetric assessment to determine a wear rate for hip replacement bearings under simulation can therefore neglect important degradation mechanisms, such as tribocorrosive loss in devices with metal sliding interfaces

Investigation Into the Repassivation Kinetics of CoCrMo for Applications in a Simulated Biological Environment

The re-passivation kinetics and composition of the passive film of CoCrMo alloys in simulated body fluids have been investigated, with key emphasis being to assess the effect that proteins have on these features. The kinetics were analyzed using potentiostatic polarization, applying a second order exponential decay to the current transients obtained, which consists of two phases: coverage and thickening. Repassivation occurred quickest in a phosphate environment with presence of bovine serum albumin (BSA) hindering the process as it inhibits access of the oxidant. By using X-ray photoelectron spectroscopy (XPS) the composition of the re-passivated layer was studied. As expected, the film is mainly composed of chromium (III) oxide with small amounts of cobalt (II) oxide and molybdenum oxides (IV-VI). When exposed to BSA the percentage of molybdenum in the passive film decreases. This is shown to be due to the protein having a high affinity for the element causing it to be lost to solution when the metal was exposed to corrosion

Fretting–corrosion at the modular tapers interface: Inspection of standard ASTM F1875-98

Interest in the degradation mechanisms at the modular tapers interfaces has been renewed due to increased reported cases of adverse reactions to metal debris and the appearance of wear and corrosion at the modular tapers interfaces at revision. Over the past two decades, a lot of research has been expended to understand the degradation mechanisms, with two primary implant loading procedures and orientations used consistently across the literature. ASTM F1875-98 is often used as a guide to understand and benchmark the tribocorrosion processes occurring within the modular tapers interface. This article presents a comparison of the two methods outlined in ASTM F1875-98 as well as a critique of the standard considering the current paradigm in pre-clinical assessment of modular tapers

Importance of surgical assembly technique on the engagement of 12/14 modular tapers

Fretting-corrosion at the modular taper junction in total hip replacements (THR), leading to implant failure, has been identified as a clinical concern and has received increased interest in recent years. There are many parameters thought to affect the performance of the taper junction, with the assembly process being one of the few consistently identified to have a direct impact. Despite this, the assembly process used by surgeons during THR surgery differs from a suggested ‘ideal’ process. For example, taper junctions of cutting tools should be pushed together rather than impacted, while ensuring as much concentricity as possible between the male and female taper and loading axis. This study devised six simple assembly methodologies to investigate how surgical variations affect the success of the compressive fit achieved at the taper interface compared to a controlled assembly method, designed to represent a more ‘ideal’ scenario. Key findings from this study suggest that a more successful and repeatable engagement can be achieved by quasi-statically loading the male and female taper concentrically with the loading axis. This was shown by a greater disassembly to assembly force ratio of 0.626 ± 0.07 when assembled using the more ‘ideal’ process, compared to 0.480 ± 0.05 when using a method closer to that used by a surgeon intraoperatively. Findings from this study can be used to help inform new surgical instrumentation and an improved surgical assembly method