The burden of musculoskeletal disease - a global perspective

Musculoskeletal diseases are one of the major causes of disability around the world and have been a significant reason for the development of the Bone and Joint Decade. Rheumatoid arthritis, osteoarthritis and back pain are important causes of disability-adjusted-life years in both the developed and developing world. COPCORD studies in over 17 countries around the world have identified back and knee pain as common in the community and are likely to increase with the ageing population. Musculoskeletal conditions are an enormous cost to the community in economic terms, and these figures emphasise how governments need to invest in the future and look at ways of reducing the burden of musculoskeletal diseases by encouraging exercise and obesity prevention campaigns.

Recent developments in C5/C5a inhibitors

Complement factor 5a (C5a) is formed upon complement system activation in response to infection, injury or disease. Whilst C5a is a potent mediator of immune and inflammatory processes, excessive production or inadequate regulation of C5a has been implicated in the pathogenesis of numerous immuno-inflammatory diseases, predominantly through experimental studies utilising animal models of disease. Both acute and chronic conditions may benefit from C5a inhibition, including rheumatoid arthritis, inflammatory bowel disease, asthma, psoriasis, haemorrhagic shock and neurodegenerative conditions. The potentially broad clinical application for treatments that inhibit the activity of C5a at C5a receptors and the large global market for anti-inflammatory therapeutics have made C5a and the C5a receptor attractive targets for academic and commercial drug development programmes. in the past 5 years, interest in C5a as a drug target has grown substantially, and this activity has resulted in a collection of patents and scientific papers reporting novel C5a and C5a receptor inhibitors and antagonists, and generated a secondary stream of patent applications broadly claiming the use of C5/C5a inhibitors as a method of treating various immune and inflammatory conditions. This paper will review the physiology and pathophysiology of C5a and discuss the development of C5a and C5a receptor inhibitors in light of the recent scientific and patent literature.

The human genome and gene expression profiling

The mapping and sequencing of the human genome has generated a large resource for answering questions about human disease. This achievement is akin in scientific importance to developing the periodic table of elements. Plastic surgery has always been at the frontier medical research. This resource will help us to improve our understanding on the many unknown physiological and pathogical conditions we deal with daily, such as wound heating keloid scar formation, Dupuytren's disease, rheumatoid arthritis, vascular malformation and carcinogenesis. We are primed in obtaining both disease and normal tissues to use this resource and applying it to clinical use. This review is about the human genome, the basis of gene expression profiling and how it will affect our clinical and research practices in the future and for those embarking on the use of this new technology as a research tool, we provide a brief insight on its limitations and pitfalls. (C) 2006 The British Association of Plastic Surgeons. Published by Elsevier Ltd. All rights reserved.

Risk of serious NSAID-related gastrointestinal events during long-term exposure: a systematic review

Objective: Exposure to non-steroidal anti-inflammatory drugs (NSAIDs) is associated wit increased risk of serious gastrointestinal (GI) events compared with non-exposure. We investigated whether that risk is sustained over time. Data sources: Cochrane Controlled Trials Register (to 2002); MEDLINE, EMBASE, Derwent Drug File and Current Contents (1999-2002); manual searching of reviews (1999-2002). Study selection: From 479 search results reviewed and 221 articles retrieved, seven studies of patients exposed to prescription non-selective NSAIDs for more than 6 months and reporting time-dependent serious GI event rates were selected for quantitative data synthesis. These were stratified into two groups by study design. Data extraction: Incidence of GI events and number of patients at specific time points were extracted. Data synthesis: Meta-regression analyses were performed. Change in risk was evaluated by testing whether the slope of the regression line declined over time. Four randomised controlled trials (RCTs) provided evaluable data from five NSAID arms (aspirin, naproxen, two ibuprofen arms, and diclofenac). When the RCT data were combined, a small significant decline in annualised risk was seen: -0.005% (95% Cl, -0.008% to -0.001%) per month. Sensitivity analyses were conducted because there was disparity within the RCT data. The pooled estimate from three cohort studies showed no significant decline in annualised risk over periods up to 2 years: -0.003% (95% Cl, -0.008% to 0.003%) per month. Conclusions: Small decreases in risk over time were observed; these were of negligible clinical importance. For patients who need long-term (> 6 months) treatment, precautionary measures should be considered to reduce the net probability of serious GI events over the anticipated treatment duration. The effect of intermittent versus regular daily therapy on long-term risk needs further investigation.

The v-MFG test: Investigating maternal, offspring and maternal-fetal genetic incompatibility effects on disease and viability

The MFG test is a family-based association test that detects genetic effects contributing to disease in offspring, including offspring allelic effects, maternal allelic effects and MFG incompatibility effects. Like many other family-based association tests, it assumes that the offspring survival and the offspring-parent genotypes are conditionally independent provided the offspring is affected. However, when the putative disease-increasing locus can affect another competing phenotype, for example, offspring viability, the conditional independence assumption fails and these tests could lead to incorrect conclusions regarding the role of the gene in disease. We propose the v-MFG test to adjust for the genetic effects on one phenotype, e.g., viability, when testing the effects of that locus on another phenotype, e.g., disease. Using genotype data from nuclear families containing parents and at least one affected offspring, the v-MFG test models the distribution of family genotypes conditional on offspring phenotypes. It simultaneously estimates genetic effects on two phenotypes, viability and disease. Simulations show that the v-MFG test produces accurate genetic effect estimates on disease as well as on viability under several different scenarios. It generates accurate type-I error rates and provides adequate power with moderate sample sizes to detect genetic effects on disease risk when viability is reduced. We demonstrate the v-MFG test with HLA-DRB1 data from study participants with rheumatoid arthritis (RA) and their parents, we show that the v-MFG test successfully detects an MFG incompatibility effect on RA while simultaneously adjusting for a possible viability loss.

Oxygen radical induced alterations in polyclonal IgG

In the sera and synovial fluid of patients with rheumatoid arthritis, part of the IgG fraction is found in an aggregated and fluorescent form. Oxygen-free radicals have been implicated in this denaturation, although the precise radical species responsible is unknown. In this work, oxygen-free radicals generated radiolytically were allowed to attack polyclonal IgG in solution. OH radicals induced aggregation of the monomer and a new fluorescence appeared in the visible region (Ex 360 nm, Em 454 nm). The superoxide radical anion was found to be inert in both these respects, whilst peroxy radicals induced autofluorescence without concomitant aggregation. The results suggest that OH.and/or peroxy radical attack may be an in vivo mechanism for IgG denaturation.

Free radicals and redox signalling in T-cells during chronic inflammation and ageing

During chronic inflammation and ageing, the increase in oxidative stress in both intracellular and extracellular compartments is likely to influence local cell functions. Redox changes alter the T-cell proteome in a quantitative and qualitative manner, and post-translational modifications to surface and cytoplasmic proteins by increased reactive species can influence T-cell function. Previously, we have shown that RA (rheumatoid arthritis) T-cells exhibit reduced ROS (reactive oxygen species) production in response to extracellular stimulation compared with age-matched controls, and basal ROS levels [measured as DCF (2',7'-dichlorofluorescein) fluorescence] are lower in RA T-cells. In contrast, exposing T-cells in vitro to different extracellular redox environments modulates intracellular signalling and enhances cytokine secretion. Together, these data suggest that a complex relationship exists between intra- and extra-cellular redox compartments which contribute to the T-cell phenotype.

The targeting of corticosteroids to inflamed tissues

In recent years, much interest has focused on the beneficial effects of administering potentially harmful therapeutic agents in drug carriers so as to reduce their toxic side effects. Rheumatoid arthritis is a chronic systemic disease with progressive destruction of the Joints and long term patient disability, Corticosteroids have been shown to retard the progression of Joint destruction but are limited in their use due to adverse side effects,This project, following the line of investigation started by other workers, was designed to study the use of microspheres to deliver corticosteroids to inflamed tissues by both the oral and intravenous routes. Hydrocortisone (HC)-loaded albumin microspheres were prepared by three different methods, by direct incorporation of HC within the particles, by indirect incorporation of HC by the enzymatic conversion of hydrocortisone-21-phosphate (H-21-P) to HC within the particles, and by the adsorption of HC onto the surface. HC was also loaded with PLA microspheres. The level of corticosteriod loading and in vitro release from microspheres was determined by HPLC analysis. A reversed-phase, ion-pairing HPLC method was developed to simultaneously measure both HC and H-21-P. The highest level of corticosteroid loading was achieved using the incorporation of H-21-P with enzymatic conversion to HC method. However, HPLC analysis showed only 5% of the incorporated steroid was HC. In vitro release rates of steroid from albumin microspheres showed >95% of incorporated steroid was released within 2 hours of dissolution. Increasing the protein:steroid ratio, and the temperature and duration of microsphere stabilization, had little effect on prolonging drug release. In vivo studies, using the carrageenan-induced rat hind-paw model of inflammation, indicated steroid-incorporated microspheres administered both orally and intraperitoneally were not therapeutically advantageous when compared to equivalent free steroid doses. The ability of orally and intravenously dosed [125I]~albumin microspheres (2.67 μm mean diameter) to accumulate in acutely and chronically inflamed tissues was investigated, The subcutaneous air-pouch was the model of inflammation used, with carrageenan as the inflammatory stimulus. Acute and chronic inflammation was shown to be consistently formed  in pouch tissues in terms of cell infiltration and fluid exudate formation in the pouch cavity. Albumin microspheres were shown to accumulate in the inflamed tissues and pouch fluids after both oral and intravenous administration. Preliminary, confirmatory studies using latex microspheres and quantitation by GPC analysis, also indicated microsphere accumulation in both acutely and chronically inflamed air-pouch tissues. tntl lUr"'poucbtis,sues; The results indicate the uptake and transfer of microspheres across the gastrointestinal tract into the circulation and their migration through disrupted endothelium and basement membranes at the inflamed sites. , .

Ceramide and reactive oxygen species (ROS) as signal transduction molecules in inflammation

Reactive oxygen species (ROS) and the sphingolipid ceramide are each partly responsible for the intracellular signal transduction of a variety of physiological, pharmacological or environmental agents. Furthermore, the enhanced production of many of these agents, that utilise ROS and ceramide as signalling intermediates, is associated with the aetiologies of several vascular diseases (e.g. atherosclerosis) or disorders of inflammatory origin (e.g. rheumatoid arthritis; RA). Excessive monocyte recruitment and uncontrolled T cell activation are both strongly implicated in the chronic inflammatory responses that are associated with these pathologies. Therefore the aims of this thesis are (1) to further elucidate the cellular responses to modulations in intracellular ceramide/ROS levels in monocytes and T cells, in order to help resolve the mechanisms of progression of these diseases and (2) to examine both existing agents (methotrexate) and novel targets for possible therapeutic manipulation. Utilising synthetic, short chain ceramide to mimic the cellular responses to fluctuations in natural endogenous ceramide or, stimulation of CD95 to induce ceramide formation, it is described here that ceramide targets and manipulates two discrete sites responsible for ROS generation, preceding the cellular responses of growth arrest in U937 monocytes and apoptosis in Jurkat T-cells. In both cell types, transient elevations in mitochondrial ROS generation were observed. However, the prominent redox altering effects appear to be the ceramide-mediated reduction in cytosolic peroxide, the magnitude of which dictates in part the cellular response in U937 monocytes, Jurkat T-cells and primary human peripheral blood resting or PHA-activated T-cells in vitro. The application of synthetic ceramides to U937 monocytes for short (2 hours) or long (16 hours) treatment periods reduced the membrane expression of proteins associated with cell-cell interaction. Furthermore, ceramide treated U937 monocytes demonstrated reduced adhesion to 5 or 24 hour LPS activated human umbilical vein endothelial cells (HUVEC) but not resting HUVEC. Consequently it is hypothesised that the targeted treatment of monocytes from patients with cardiovascular diseases with short chain synthetic ceramide may reduce disease progression. Herein, the anti-inflammatory and immunosuppressant drug, methotrexate, is described to require ROS production for the induction of cytostasis or cytotoxicity in U937 monocytes and Jurkat T-cells respectively. Further, ROS are critical for methotrexate to abrogate monocyte interaction with activated HUVEC in vitro. The histological feature of RA of enhanced infiltration, survivability and hyporesponsiveness of T-cells within the diseased synovium has been suggested to arise from aberrant signalling. No difference in the concentrations of endogenous T-cell ceramide, the related lipid diacylglycerol (DAG) and cytosolic peroxide ex vivo was observed. TCR activation following PHA exposure in vitro for 72 hours did not induced maintained perturbations in DAG or ceramide in T-cells from RA patients or healthy individuals. However, T-cells from RA patients failed to upregulate cytosolic peroxide in response to PHA, unlike those from normals, despite expressing identical levels of the activation marker CD25. This inability to upregulate cytosolic peroxide may contribute to the T-cell pathology associated with RA by affecting the signalling capacity of redox sensitive biomolecules. These data highlight the importance of two distinctive cellular pools of ROS in mediating complex biological events associated with inflammatory disease and suggest that modulation of cellular ceramides represents a novel therapeutic strategy to minimise monocyte recruitment.

Targeting the glycoprotein 130 receptor subunit to control pain and inflammation

The glycoprotein 130 (gp130) is a shared signal-transducing-membrane-associated receptor for several hematopoietic cytokines. Its activation is implicated in pain and in a variety of diseases via signaling of proinflammatory cytokines. These include interleukin-6 (IL-6) subfamily cytokines, many of which play important roles in the pathogenesis of diseases such as rheumatoid arthritis, Castleman's disease, and Kaposi's sarcoma. Several strategies have been developed to block gp130-receptor-mediated signaling. These include the application of monoclonal antibodies, the creation of mutant form(s) of the gp130 with increased binding affinity for such ligands as IL-6/sIL-6R complex, and the generation of antagonists by selective mutagenesis of the specific cytokine/gp130 receptor binding site(s). Other strategies include targeting gp130-mediated signaling pathways such as that involving signal transducer and activator of transcription-3. This review provides a summary of the latest research pertaining to the role of gp130 in the pathogenesis of inflammatory and other diseases in which the gp130 receptor is implicated. An overview of antagonists targeting the gp130 receptor is included with particular emphasis on their mechanism of action and their limitations and potential for therapeutic application.

Dipeptidyl peptidase IV (DPP IV) and related molecules in type 2 diabetes

Dipeptidyl peptidase IV (DPP IV) is a widely distributed physiological enzyme that can be found solubilized in blood, or membrane-anchored in tissues. DPP IV and related dipeptidase enzymes cleave a wide range of physiological peptides and have been associated with several disease processes including Crohn's disease, chronic liver disease, osteoporosis, multiple sclerosis, eating disorders, rheumatoid arthritis, cancer, and of direct relevance to this review, type 2 diabetes. Here, we place particular emphasis on two peptide substrates of DPP IV with insulin-releasing and antidiabetic actions namely, glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP). The rationale for inhibiting DPP IV activity in type 2 diabetes is that it decreases peptide cleavage and thereby enhances endogenous incretin hormone activity. A multitude of novel DPP IV inhibitor compounds have now been developed and tested. Here we examine the information available on DPP IV and related enzymes, review recent preclinical and clinical data for DPP IV inhibitors, and assess their clinical significance.

Cyclin-dependent kinase 9 activity regulates neutrophil spontaneous apoptosis

Neutrophils are the most abundant leukocyte and play a central role in the immune defense against rapidly dividing bacteria. However, they are also the shortest lived cell in the blood with a lifespan in the circulation of 5.4 days. The mechanisms underlying their short lifespan and spontaneous entry into apoptosis are poorly understood. Recently, the broad range cyclin-dependent kinase (CDK) inhibitor R-roscovitine was shown to increase neutrophil apoptosis, implicating CDKs in the regulation of neutrophil lifespan. To determine which CDKs were involved in regulating neutrophil lifespan we first examined CDK expression in human neutrophils and found that only three CDKs: CDK5, CDK7 and CDK9 were expressed in these cells. The use of CDK inhibitors with differing selectivity towards the various CDKs suggested that CDK9 activity regulates neutrophil lifespan. Furthermore CDK9 activity and the expression of its activating partner cyclin T1 both declined as neutrophils aged and entered apoptosis spontaneously. CDK9 is a component of the P-TEFb complex involved in transcriptional regulation and its inhibition will preferentially affect proteins with short half-lives. Treatment of neutrophils with flavopiridol, a potent CDK9 inhibitor, increased apoptosis and caused a rapid decline in the level of the anti-apoptotic protein Mcl-1, whilst Bcl2A was unaffected. We propose that CDK9 activity is a key regulator of neutrophil lifespan, preventing apoptosis by maintaining levels of short lived anti-apoptotic proteins such as Mcl-1. Furthermore, as inappropriate inhibition of neutrophil apoptosis contributes to chronic inflammatory diseases such as Rheumatoid Arthritis, CDK9 represents a novel therapeutic target in such diseases.

The role of apoptotic cell clearance in a high-lipid environment:links to ageing

Individuals within the aged population show an increased susceptibility to infection, implying a decline in immune function, a phenomenon known as immunosenescence. Paradoxically, an increase in autoimmune disease, such as rheumatoid arthritis, is also associated with ageing, therefore some aspects of the immune system appear to be inappropriately active in the elderly. The above evidence suggests inappropriate control of the immune system as we age. Macrophages, and their precursors monocytes, play a key role in control of the immune system. They play an important role in host defence in the form of phagocytosis, and also link the innate and adaptive immune system via antigen presentation. Macrophages also have a reparative role, as professional phagocytes of dead and dying cells. Clearance of apoptotic cells by macrophages has also been shown to directly influence immune responses in an anti-inflammatory manner. Inappropriate control of macrophage function with regards to dead cell clearance may contribute to pathology as we age. The aims of this study were to assess the impact of lipid treatment, as a model of the aged environment, on the ability of macrophages to interact with, and respond to, apoptotic cells. Using a series of in vitro cell models, responses of macrophages (normal and lipid-loaded) to apoptotic macrophages (normal and lipid-loaded) were investigated. Monocyte recruitment to apoptotic cells, a key process in resolving inflammation, was assessed in addition to cytokine responses. Data here shows, for the first time, that apoptotic macrophages (normal and lipid-loaded) induce inflammation in human monocyte-derived macrophages, a response that could drive inflammation in age-associated pathology e.g. atherosclerosis. Monoclonal antibody inhibition studies suggest the classical chemokine CX3CL1 may be involved in monocyte recruitment to apoptotic macrophages, but not apoptotic foam cells, therefore differential clearance strategies may be employed following lipid-loading. CD14, an important apoptotic cell tethering receptor, was not found to have a prominent role in this process, whilst the role for ICAM-3 remains unclear. Additionally, a small pilot study using macrophages from young (<25) and mid-life (>40) donors was undertaken. Preliminary data was gathered to assess the ability of primary human monocyte-derived macrophages, from young and mid-life donors, to interact with, and respond to, apoptotic cells. MØ from mid-life individuals showed no significant differences in their ability to respond to immune modulation by apoptotic cells compared to MØ from young donors. Larger cohorts would be required to investigate whether immune modulation of MØ by apoptotic cells contribute to inflammatory pathology throughout ageing.

Modulation of interleukin-6 and matrix metalloproteinase 2 expression in human fibroblast-like synoviocytes by functional ionotropic glutamate receptors

Objective. Patients with rheumatoid arthritis (RA) have increased concentrations of the amino acid glutamate in synovial fluid. This study was undertaken to determine whether glutamate receptors are expressed in the synovial joint, and to determine whether activation of glutamate receptors on human synoviocytes contributes to RA disease pathology. Methods. Glutamate receptor expression was examined in tissue samples from rat knee joints and in human fibroblast-like synoviocytes (FLS). FLS from 5 RA patients and 1 normal control were used to determine whether a range of glutamate receptor antagonists influenced expression of the proinflammatory cytokine interleukin-6 (IL-6), enzymes involved in matrix degradation and cytokine processing (matrix metalloproteinase 2 [MMP-2] and MMP-9), and the inhibitors of these enzymes (tissue inhibitor of metalloproteinases 1 [TIMP-1] and TIMP-2). IL-6 concentrations were determined by enzyme-linked immunosorbent assay, MMP activity was measured by gelatin zymography, and TIMP activity was determined by reverse zymography. Fluorescence imaging of intracellular calcium concentrations in live RA FLS stimulated with specific antagonists was used to reveal functional activation of glutamate receptors that modulated IL-6 or MMP-2. Results. Ionotropic and metabotropic glutamate receptor subunit mRNA were expressed in the patella, fat pad, and meniscus of the rat knee and in human articular cartilage. Inhibition of N-methyl-D-aspartate (NMDA) receptors in RA FLS increased proMMP-2 release, whereas non-NMDA ionotropic glutamate receptor antagonists reduced IL-6 production by these cells. Stimulation with glutamate, NMDA, or kainate (KA) increased intracellular calcium concentrations in RA FLS, demonstrating functional activation of specific ionotropic glutamate receptors. Conclusion. Our findings indicate that activation of NMDA and KA glutamate receptors on human synoviocytes may contribute to joint destruction by increasing IL-6 expression. © 2007, American College of Rheumatology.