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In the human breast cancer cell line MCF-7, the nucleotides ATPγS and UTP, acting extracellularly through the purinergic receptor P2Y2, lead to elevated intracellular calcium levels and increased proliferation. ATPγS and UTP treatment of MCF-7 cells activated transcription of the immediate early gene c-fos, an important component in the response to proliferative stimulation. c-fos induction was enhanced by co-treatment with ATPγS and a variety of proliferative agents including growth factors, tumour promoters and stress. Stimulation with ATPγS or epidermal growth factor (EGF) led to extracellular signal-regulated kinase (ERK) activation and phosphorylation of the transcription factors CREB and Elk-1. Co-stimulation synergistically activated fos expression and notably led to increased levels of ERK, CREB and EGF receptor phosphorylation, as well as hyperphosphorylation of ternary complex factor. Nevertheless, the ERK pathway does not fully account for this synergy, since fos induction was differentially sensitive to the MEK inhibitor U0126, indicating that these two agonists signal differently to this immediate early gene. Thus, extracellular nucleotides co-operate with growth factors to activate genes linked to the proliferative response in MCF-7 cells through activation of specific purinergic receptors, which thereby represent important potential targets for arresting the neoplastic progression of breast cancer cells. [ABSTRACT FROM PUBLISHER]

Examines the role of thrombospondin-2 in the regulation of bone resorption. Activated platelets' secretion of thrombospondin; Similarities in the mechanisms of resorption.

Characterizes the novel bone antigens using monoclonal antibodies. Methods used in screening conditioned hybridoma supernatants; Proteins associated with skeletal tissues; Factors involved in bone antigen expression at different levels of maturity.

Abstract: The in vivo kinematics of 10 patients after combined anterior cruciate ligament reconstruction (ACLR group) and Oxford unicompartmental knee arthroplasty (UKA) was compared to those of 10 Oxford UKA patients with an intact ACL (ACLI group) and a group of 22 normal knees. The kinematics during a step-up exercise and a deep knee bend exercise was measured using a fluoroscopic technique. The patellar tendon angle (PTA) to knee flexion angle relationship during both exercises was similar for all three groups of subjects. For the UKA groups the pattern of mobile bearing movement during both exercises was similar. This study demonstrates that normal knee kinematics is achieved in the ACL deficient arthritic knee following ACLR and UKA. As a result these patients, who tend to be young and high demand, have excellent outcome and achieve high levels of function. As the relative position of the components and thus component loading are similar to the ACLI UKA, we would expect similar long term survival. [Copyright &y& Elsevier]

The effect of nanotopography on a range of Ti oxide surfaces was determined. Flat Ti, 3%, 19%, 30% and 43% topography densities of 110 nm high hemispherical protrusions were cultured in contact with primary derived human macrophages and osteoblasts in single culture models.Prior to introduction of the test substrate the phenotype and optimum conditions for in vitro cell culture were established. The cellular response was investigated and quantified by assessments of cytoskeletal development and orientation, viable cell adhesion, cytokine production and release and RT-PCR analysis of osteogenic markers.The tested nanotopographies did not have a statistically significant effect on viable cell adhesion and subsequent cytoskeletal formation. Surface chemistry was the dominant factor as established via incorporation of a tissue culture polystyrene, TCPS, control. The topography surfaces induced a release of chemotactic macrophage activation agents at 1 day in conjunction with stress fibre formation and a subsequent fibronectin network formation. Osteoblasts migrated away from the topography surfaces to the exposed TCPS within the wells during the 7-day period. [Copyright &y& Elsevier]

We have assessed the effect of elevated concentrations of hydroxyphenylpyruvic acid (HPPA), hydroxyphenyllactic acid (HPLA) and tyrosine, on a range of chemistry tests in serum and urine to explore the potential for chemical interference on routine laboratory analyses in patients with alkaptonuria (AKU) treated with nitisinone and similarly implications for patients with hereditary tyrosinemia type 1 (HT-1). HPPA, HPLA and tyrosine were added separately to pooled serum from subjects without AKU in a range of assays with Roche Modular chemistries. Effects on urine were assessed by changes in urine strip chemistries after mixing a positive control urine with various amounts of the test compounds and reading on a Siemens urine strip meter. No significant effect (p > 0.1) was observed up to 225 μmol/L of HPPA and HPLA, and up to 5000 μmol/L tyrosine, on any of the serum-based assays including those with peroxidase-coupled reaction systems of enzymatic creatinine, urate, total cholesterol, HDL cholesterol and triglyceride. Both the monohydroxy HPPA, and the dihydroxy homogentisic acid (HGA), at increased urine concentrations typical of nitisinone-treated AKU and non-treated AKU respectively, did however show marked negative interference in strip assays for glucose and leucocytes; i.e. those with peroxide-linked endpoints. The effect of increased HPLA was less marked. In patients with AKU or on nitisinone treatment and HT-1 patients on nitisinone, urine strip chemistry testing should be used sparingly, if at all, to avoid false negative reporting. It is recommended that urine assays should be organised with a suitable specialist laboratory. • Alkaptonuria treated with nitisinone results in reduction of HGA and marked increases in HPPA, HPLA, and tyrosine • The elevated concentrations of HPPA, HPLA and tyrosine did not interfere with a wide range blood/serum assays used in clinical practice • Increased urinary HGA and HPPA interfered with some urine test strip chemistries • The oxidase-based urine assays showed most suppression of response by HGA and HPPA • Urine strip tests should be avoided in patients with alkaptonuria and patients on nitisinone [ABSTRACT FROM AUTHOR]

Highlights: [•] Developed LC–MS/MS method for homogentisic acid and tyrosine in human urine. [•] Validated in both acidified and non-acidified urine matrices. [•] Applicable for diagnosing patients with alkaptonuria. [•] Applicable for monitoring response to therapy in treatment of alkaptonuria. [•] Validated according to European Medicines Agency Guidelines. [ABSTRACT FROM AUTHOR]

Abstract: Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear steroid hormone superfamily and exist in three isoforms: PPARα, β and γ, each with specific functions. In this study, we have investigated the expression of PPARs by human osteoclast precursors and osteoclasts generated in vitro. In addition, the effects of fibrates and isoform-specific PPAR agonists on osteoclast formation and resorption in vitro were determined. Human peripheral blood mononuclear cells (PBMCs) were stimulated with human recombinant RANKL and M-CSF to generate osteoclasts. RNA was extracted at days 0, 7, 14 and 21 and RT-PCR for all three PPAR isoforms demonstrated their expression throughout this culture period. To determine the effect on osteoclast formation, PPAR agonists (10−8 M to 10−5 M) were added from the beginning of the culture until day 14 and the number of multinucleated osteoclasts counted. The effect of PPAR agonists on osteoclast function was similarly determined by treating mature, multinucleated osteoclasts cultured on dentine wafers with PPAR agonists (10−8 M to 10−5 M) for 7 days and quantifying resorption. Bezafibrate and fenofibrate, which non-discriminately activate all PPAR isoforms, significantly inhibited the formation of multinucleated osteoclasts from PBMC in vitro. Bezafibrate treatment of mature osteoclast resulted in 50% inhibition (at 10−8 M and 10−7 M) of resorption, yet fenofibrate had no significant effect. Activation of individual PPARs with isoform-specific agonist (GW9578, L165041 and ciglitizone which preferentially activate PPARα, β and γ respectively) resulted in significant dose-dependent inhibition of multinucleated osteoclast formation. Divergent effects on osteoclast resorption were observed; GW9578 had no significant effect on resorption, whereas ciglitizone and L165041 dose-dependently inhibited and stimulated resorption, respectively. These data show for the first time expression of all three PPAR isoforms throughout the development and maturation period of osteoclasts generated from human PBMCs. In addition, we demonstrate that isoform-specific PPAR agonists have strong effects on multinucleation and highly variable effects on bone resorption. In conclusion, this study highlights the potential of PPARs as therapeutic targets in diseases with accelerated osteoclast formation and resorption. [Copyright &y& Elsevier]

Abstract: Newer designs of total knee arthroplasty (TKA), through the use of added degrees of constraint, attempt to provide a “guided motion” to restore more normal and predictable kinematics. Two such design philosophies are the posterior stabilised (PS) using a cam–post and the medial pivot (MP) concepts. Knee kinematics of 12 patients with a PS TKA, 13 subjects with a MP TKA and 10 normal subjects were compared. For kinematic assessment, patients underwent fluoroscopic assessment of the knee during a step-up exercise and deep knee bend. Fluoroscopic images were corrected for distortion and assessed using 3D model fitting to determine relative 3D motion, and a 2D method to measure the patellar tendon angle (PTA) as function of knee flexion. For the PS design the cam–post mechanism engaged between 70° and 100° flexion. Between extension and 50° there was forward motion of the contact points. Beyond 60° both condyles rolled moved posteriorly. The majority of the external rotation of the femur occurred between 50° and 80°. The PTA was lower than normal in extension and higher than normal in flexion. The MP exhibited no anterior movement throughout the rage of motion. The medial condyle moved minimally. The lateral contact point moved posteriorly from extension to flexion. The femur rotated externally throughout the range of flexion analysed. The PTA was similar to normal from extension to mid flexion and then higher than normal beyond to high flexion. The PS design fails to fully restrain paradoxical anterior movement and although the cam engages, it does not contribute significantly to overall rollback. The MP knee does not show significant anterior movement, the medial pivot concept appears to achieve near normal kinematics from extension to 50° of knee flexion. However, the results show that at high flexion this design does not achieve normal knee kinematics. [Copyright &y& Elsevier]