Does protein kinase R mediate TNF-α- and ceramide-induced increases in expression and activation of matrix metalloproteinases in articular cartilage by a novel mechanism?

We investigated the role of the proinflammatory cytokine TNF-α, the second messenger C(2)-ceramide, and protein kinase R (PKR) in bovine articular cartilage degradation. Bovine articular cartilage explants were stimulated with C(2)-ceramide or TNF-α for 24 hours. To inhibit the activation of PKR, 2-aminopurine was added to duplicate cultures. Matrix metalloproteinase (MMP) expression and activation in the medium were analysed by gelatin zymography, proteoglycan release by the dimethylmethylene blue assay, and cell viability by the Cytotox 96(® )assay. C(2)-ceramide treatment of cartilage explants resulted in a significant release of both pro- and active MMP-2 into the medium. Small increases were also seen with TNF-α treatment. Incubation of explants with 2-aminopurine before TNF-α or C(2)-ceramide treatment resulted in a marked reduction in expression and activation of both MMP-2 and MMP-9. TNF-α and C(2)-ceramide significantly increased proteoglycan release into the medium, which was also inhibited by cotreatment with 2-aminopurine. A loss of cell viability was observed when explants were treated with TNF-α and C(2)-ceramide, which was found to be regulated by PKR. We have shown that C(2)-ceramide and TNF-α treatment of articular cartilage result in the increased synthesis and activation of MMPs, increased release of proteoglycan, and increased cell death. These effects are abrogated by treatment with the PKR inhibitor 2-aminopurine. Collectively, these results suggest a novel role for PKR in the synthesis and activation of MMPs and support our hypothesis that PKR and its activator, PACT, are implicated in the cartilage degradation that occurs in arthritic disease

Enhanced Tissue Integration During Cartilage RepairIn VitroCan Be Achieved by Inhibiting Chondrocyte Death at the Wound Edge

Objective: Experimental wounding of articular cartilage results in cell death at the lesion edge. The objective of this study was to investigate whether inhibition of this cell death results in enhanced integrative cartilage repair. Methods: Bovine articular cartilage discs (6mm) were incubated in media containing inhibitors of necrosis (Necrostatin-1, Nec-1) or apoptosis (Z-VAD-FMK, ZVF) before cutting a 3mm inner core. This core was left in situ to create disc/ring composites, cultured for up to 6 weeks with the inhibitors, and analyzed for cell death, sulfated glycosaminoglycan release, and tissue integration. Results: Creating the disc/ring composites resulted in a significant increase in necrosis. ZVF significantly reduced necrosis and apoptosis at the wound edge. Nec-1 reduced necrosis. Both inhibitors reduced the level of wound-induced sulfated glycosaminoglycan loss. Toluidine blue staining and electron microscopy of cartilage revealed significant integration of the wound edges in disc/ring composites treated with ZVF. Nec-1 improved integration, but to a lesser extent. Push-out testing revealed that ZVF increased adhesive strength compared to control composites. Conclusions: This study shows that treatment of articular cartilage with cell death inhibitors during wound repair increases the number of viable cells at the wound edge, prevents matrix loss, and results in a significant improvement in cartilage-cartilage integration

Exogenous sphingomyelinase increases collagen and sulphated glycosaminoglycan production by primary articular chondrocytes: an in vitro study

We previously established a role for the second messenger ceramide in protein kinase R (PKR)-mediated articular cartilage degradation. Ceramide is known to play a dual role in collagen gene regulation, with the effect of ceramide on collagen promoter activity being dependent on its concentration. Treatment of cells with low doses of sphingomyelinase produces small increases in endogenous ceramide. We investigated whether ceramide influences articular chondrocyte matrix homeostasis and, if so, the role of PKR in this process. Bovine articular chondrocytes were stimulated for 7 days with sphingomyelinase to increase endogenous levels of ceramide. To inhibit PKR, 2-aminopurine was added to duplicate cultures. De novo sulphated glycosaminoglycan and collagen synthesis were measured by adding [(35)S]-sulphate and [(3)H]-proline to the media, respectively. Chondrocyte phenotype was investigated using RT-PCR and Western blot analysis. Over 7 days, sphingomyelinase increased the release of newly synthesized sulphated glycosaminoglycan and collagen into the media, whereas inhibition of PKR in sphingomyelinase-treated cells reduced the level of newly synthesized sulphated glycosaminoglycan and collagen. Sphingomyelinase treated chondrocytes expressed col2a1 mRNA, which is indicative of a normal chondrocyte phenotype; however, a significant reduction in type II collagen protein was detected. Therefore, small increments in endogenous ceramide in chondrocytes appear to push the homeostatic balance toward extracellular matrix synthesis but at the expense of the chondrocytic phenotype, which was, in part, mediated by PKR

AMPA/kainate glutamate receptor antagonists prevent posttraumatic osteoarthritis

Musculoskeletal disorders represent the 3rd greatest burden on health in the developed world. Osteoarthritis is the single greatest cause of chronic pain, has no cure, and affects 8.5 and 27 million in the UK and US respectively. Osteoarthritis commonly occurs after joint injury, particularly affecting younger patients. Painful joints are often treated with injections of steroid or hyaluronic acid (HA), but treatments to prevent subsequent joint degeneration remain elusive. In animals, joint injury increases glutamate release into the joint, acting on nerves to cause pain, and joint tissues to cause inflammation and degeneration. This study investigated synovial fluid glutamate concentrations and glutamate receptor (GluR) expression in injured human joints and compared efficacy of GluR antagonists with current treatments in a mouse model of injury-induced osteoarthritis (ACL rupture). GluRs were expressed in ligament and meniscus after knee injury and synovial fluid glutamate concentrations ranged from 19–129 µM. Intra-articular injection of NBQX (GluR antagonist), administered at the time of injury, substantially reduced swelling and degeneration in the mouse ACL rupture model. HA had no effect and depo-medrone reduced swelling for 1 day, but increased degeneration by 50%. Intra-articular administration of NBQX was both symptom and disease modifying to a greater extent than current treatments. There is an opportunity for repurposing related drugs, developed for CNS disorders, with proven safety in man, to prevent injury-induced osteoarthritis. This could quickly reduce the substantial burden associated with osteoarthritis

Structures of monomeric and oligomeric forms of the Toxoplasma gondiiperforin-like protein 1

Toxoplasma and Plasmodium are the parasitic agents of toxoplasmosis and malaria, respectively, and use perforin-like proteins (PLPs) to invade host organisms and complete their life cycles. The Toxoplasma gondii PLP1 (TgPLP1) is required for efficient exit from parasitophorous vacuoles in which proliferation occurs. We report structures of the membrane attack complex/perforin (MACPF) and Apicomplexan PLP C-terminal β-pleated sheet (APCβ) domains of TgPLP1. The MACPF domain forms hexameric assemblies, with ring and helix geometries, and the APCβ domain has a novel β-prism fold joined to the MACPF domain by a short linker. Molecular dynamics simulations suggest that the helical MACPF oligomer preserves a biologically important interface, whereas the APCβ domain binds preferentially through a hydrophobic loop to membrane phosphatidylethanolamine, enhanced by the additional presence of inositol phosphate lipids. This mode of membrane binding is supported by site-directed mutagenesis data from a liposome-based assay. Together, these structural and biophysical findings provide insights into the molecular mechanism of membrane targeting by TgPLP1

Post-exercise management of exertional hyperthermia in dogs participating in dog sport (canicross) events in the UK

Exercise is a common trigger of heat-related illness (HRI) events in dogs, accounting for 74% of canine HRI cases treated under primary veterinary care in the United Kingdom. However, few empirical studies have evaluated the effectiveness of differing cooling methods for dogs with exertional hyperthermia or HRI. This study aimed to prospectively evaluate effects of ambient conditions and post-exercise management practices (cooling methods and vehicular confinement) on the post-exercise temperature change of dogs participating in UK canicross events. Canine temperature was recorded at three intervals post-exercise: as close as possible to 0- (immediately post-exercise), 5-, and 15-min post-exercise. Ambient conditions and post-exercise management were recorded for 115 cooling profiles from 52 dogs. In 28/115 (24.4%) profiles, the dog's temperature increased during the first 5-min post-exercise. Overall, 68/115 (59.1%) profiles included passive cooling (stood or walked outside), 35 (30.4%) active cooling (cold-water immersion or application of a cooling coat), and 12 (10.4%) involved no cooling and were immediately housed in vehicles. No dogs developed hypothermia during the study and no adverse effects were observed from any cooling method. In hyperthermic dogs, overall post-exercise body temperature change was significantly negatively associated (i.e. the dogs cooled more) with 0-min post-exercise body temperature (β = −0.93, p &lt; 0.001), and not being housed in a vehicle (β = −0.43, p = 0.013). This study provides evidence cold-water immersion (in water at 0.1–15.0 °C) can be used to effectively and safely cool dogs with exertional hyperthermia. Progressive temperature increases in many dogs - even after exercise has terminated - supports the message to “cool first, transport second” when managing dogs with HRI. When transporting dogs post-exercise or with HRI even after active cooling, care should be taken to cool the vehicle before entry and promote air movement around the dog during transport to facilitate ongoing cooling and prevent worsening of hyperthermia during travel.</p

Point pattern simulation modelling of extensive and intensive chicken farming in Thailand : accounting for clustering and landscape characteristics

In recent decades, intensification of animal production has been occurring rapidly in transition economies to meet the growing demands of increasingly urban populations. This comes with significant environmental, health and social impacts. To assess these impacts, detailed maps of livestock distributions have been developed by downscaling census data at the pixel level (10 km or 1 km), providing estimates of the density of animals in each pixel. However, these data remain at fairly coarse scale and many epidemiological or environmental science applications would make better use of data where the distribution and size of farms are predicted rather than the number of animals per pixel. Based on detailed 2010 census data, we investigated the spatial point pattern distribution of extensive and intensive chicken farms in Thailand. We parameterized point pattern simulation models for extensive and intensive chicken farms and evaluated these models in different parts of Thailand for their capacity to reproduce the correct level of spatial clustering and the most likely locations of the farm clusters. We found that both the level of clustering and location of clusters could be simulated with reasonable accuracy by our farm distribution models. Furthermore, intensive chicken farms tended to be much more clustered than extensive farms, and their locations less easily predicted using simple spatial factors such as human populations. These point-pattern simulation models could be used to downscale coarse administrative level livestock census data into farm locations. This methodology could be of particular value in countries where farm location data are unavailable

The unequal burden of human-wildlife conflict

The costs of human-wildlife conflict are measurably more impactful in areas of the developing world where the loss of cattle can mean the entire livelihood of a family being erased.Human-wildlife conflict is one of the most pressing sustainable development challenges globally. This is particularly the case where ecologically and economically important wildlife impact the livelihoods of humans. Large carnivores are one such group and their co-occurrence with low-income rural communities often results in real or perceived livestock losses that place increased costs on already impoverished households. Here we show the disparities associated with the vulnerability to conflict arising from large carnivores on cattle (Bos taurus) globally. Across the distribution of 18 large carnivores, we find that the economic vulnerability to predation losses (as measured by impacts to annual per capita income) is between two and eight times higher for households in transitioning and developing economies when compared to developed ones. This potential burden is exacerbated further in developing economies because cattle keepers in these areas produce on average 31% less cattle meat per animal than in developed economies. In the lowest-income areas, our estimates suggest that the loss of a single cow or bull equates to nearly a year and a half of lost calories consumed by a child. Finally, our results show that 82% of carnivore range falls outside protected areas, and five threatened carnivores have over one third of their range located in the most economically sensitive conflict areas. This unequal burden of human-carnivore conflict sheds light on the importance of grappling with multiple and conflicting sustainable development goals: protecting life on land and eliminating poverty and hunger

Inflammatory and degenerative phases resulting from anterior cruciate rupture in a non-invasive murine model of post-traumatic osteoarthritis

Joint injury is the predominant risk factor for post-traumatic osteoarthritis development (PTOA). Several non-invasive mouse models mimicking human PTOA investigate molecular mechanisms of disease development; none have characterised the inflammatory response to this acute traumatic injury. Our aim was to characterise the early inflammatory phase and later degenerative component in our in vivo non-invasive murine model of PTOA induced by anterior cruciate ligament (ACL) rupture. Right knees of 12-week-old C57Bl6 mice were placed in flexion at a 30° offset position and subjected to a single compressive load (12N, 1.4mm/s) to induce ACL rupture with no obvious damage to surrounding tissues. Tissue was harvested 4 hours post-injury and on days 3, 14 and 21; contralateral left knees served as controls. Histological, immunohistochemical and gene analyses were performed to evaluate inflammatory and degenerative changes. Immunohistochemistry revealed time-dependent expression of mature (F4/80 positive) and inflammatory (CD11b positive) macrophage populations within the sub-synovial infiltrate, developing osteophytes and inflammation surrounding the ACL in response to injury. Up-regulation of genes encoding acute pro-inflammatory markers, inducible nitric oxide synthase, interleukin-6 and interleukin-17, and the matrix degrading enzymes, ADAMTS-4 and MMP3 was detected in femoral cartilage, concomitant with extensive cartilage damage and bone remodelling over 21-days post-injury. Our non-invasive model describes pathologically distinct phases of the disease, increasing our understanding of inflammatory episodes, the tissues/cells producing inflammatory mediators and the early molecular changes in the joint, thereby defining the early phenotype of PTOA. This knowledge will guide appropriate interventions to delay or arrest disease progression following joint injury