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Osteoarthritis (OA) affects multiple tissues of the synovial joint and is characterised by articular cartilage degeneration and bone remodelling. Interferon-γ (IFN-γ) is implicated in osteoarthritis pathology exerting its biological effects via various mechanisms including activation of protein kinase R (PKR), which has been implicated in inflammation and arthritis. This study investigated whether treatment of articular cartilage chondrocytes and osteoblasts with IFN-γ could induce a degradative phenotype that was mediated through the PKR signalling pathway. IFN-γ treatment of chondrocytes increased transcription of key inflammatory mediators (TNF-α, IL-6), matrix degrading enzymes (MMP-13), the transcription factor STAT1, and PKR. Activation of PKR was involved in the regulation of TNF-α, IL-6, and STAT1. In osteoblasts, IFN-γ increased human and mouse STAT1, and human IL-6 through a mechanism involving PKR. ALP, COL1A1 (human and mouse), RUNX2 (mouse), and PHOSPHO1 (mouse) were decreased by IFN-γ. The number of PKR positive cells were increased in post-traumatic OA (PTOA). This study has revealed that IFN-γ propagates inflammatory and degenerative events in articular chondrocytes and osteoblasts via PKR activation. Since IFN-γ and PKR signalling are both activated in early PTOA, these mechanisms are likely to contribute to joint degeneration after injury and might offer attractive targets for therapeutic intervention., (© 2022 The Authors. Published by Elsevier B.V.)

Objective: Identification of genes differentially expressed in mechano-biological pathways in articular cartilage provides insight into the molecular mechanisms behind initiation and/or progression of osteoarthritis (OA). Quantitative PCR (qPCR) is commonly used to measure gene expression, and is reliant on the use of reference genes for normalisation. Appropriate validation of reference gene stability is imperative for accurate data analysis and interpretation. This study determined in vitro reference gene stability in articular cartilage explants and primary chondrocytes subjected to different compressive loads and tensile strain, respectively., Design: The expression of eight commonly used reference genes (18s, ACTB, GAPDH, HPRT1, PPIA, RPL4, SDHA and YWHAZ) was determined by qPCR and data compared using four software packages (comparative delta-Ct method, geNorm, NormFinder and BestKeeper). Calculation of geometric means of the ranked weightings was carried out using RefFinder., Results: Appropriate reference gene(s) for normalisation of mechanically-regulated transcript levels in articular cartilage tissue or isolated chondrocytes were dependent on experimental set-up. SDHA, YWHAZ and RPL4 were the most stable genes whilst glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and to a lesser extent Hypoxanthine-guanine phosphoribosyltransferase (HPRT), showed variable expression in response to load, demonstrating their unsuitability in such in vitro studies. The effect of using unstable reference genes to normalise the expression of aggrecan (ACAN) and matrix metalloproteinase 3 (MMP3) resulted in inaccurate quantification of these mechano-sensitive genes and erroneous interpretation/conclusions., Conclusion: This study demonstrates that commonly used 'reference genes' may be unsuitable for in vitro cartilage chondrocyte mechanobiology studies, reinforcing the principle that careful validation of reference genes is essential prior to each experiment to obtain robust and reproducible qPCR data for analysis/interpretation., (Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.)

This study investigated whether treatment of articular cartilage chondrocytes with a combination of oncostatin M (OSM) and interleukin-1 (IL-1) could induce a degradative phenotype that was mediated through the protein kinase R (PKR) signalling pathway. High-density monolayer cultures of full depth, bovine chondrocytes were treated with a combination of OSM and IL-1 (OSM+IL-1) for 7 days. To inhibit the activation of PKR, a pharmacological inhibitor of PKR was added to duplicate cultures. Pro- and active matrix metalloproteinase-9 (MMP9) and MMP9 mRNA were significantly upregulated by OSM+IL-1 through a PKR dependent mechanism. ADAMTS4 and ADAMTS5 mRNA were also upregulated by OSM+IL-1. The upregulation of ADAMTS4 and ADAMTS5 were, in part, mediated through PKR. OSM+IL-1 resulted in a loss of type II collagen, which could not be rescued by PKR inhibition. OSM+IL-1 reduced the expression of COL2A1 (type II collagen), COL9A1 (type IX collagen), COL11A1 (type XI collagen), and ACAN (aggrecan) mRNAs. Expression of type II and XI collagen and aggrecan was reduced further when PKR was inhibited. OSM+IL-1 resulted in an 11-fold increase in TNFa mRNA which was, in part, mediated through the PKR pathway. This study demonstrates, for the first time, that a number of catabolic and pro-inflammatory effects known to be important in human arthritis and induced by OSM and IL-1, are mediated by the PKR signalling pathway.

The failure of cartilages to fuse, particularly in the case of articular cartilage under conditions of repair is due to morphological and structural constraints of the tissue. Factors that impede integration include, non-vascularisation, low cellularity, and proteoglycan in the surrounding extracellular matrix acting as a natural barrier to cellular migration. We hypothesised that brief activation of a catabolic cascade by cytokines followed by culture under anabolic conditions would promote tissue fusion in a ring-disk model of cartilage integration. Our results show that transient exposure to 10 ng mL(-1) interleukin-1β, followed by two weeks post-culture under anabolic conditions, enhanced cartilage-cartilage integration compared to untreated explants. Quantitative PCR analysis of catabolism-related genes ADAMTS4 and MMP13 showed both were transiently upregulated and these findings correlated with evidence of extracellular matrix remodelling. At the level of histology, we observed chondrocytes readily populated the interfacial matrix between fused explants in interleukin-1β treated explants, whereas in control explants this region was relatively acellular in comparison. Catabolic cytokine treated explants exhibited 29-fold greater adhesive strength (0.859 MPa versus 0.028 MPa, P 〈 0.05) than untreated counterparts. Collectively, our results demonstrate that a single short catabolic pulse followed by an anabolic response is sufficient to generate mechanically robust, integrative cartilage repair.

Articular cartilage is a challenging tissue to reconstruct or replace principally because of its avascular nature; large chondral lesions in the tissue do not spontaneously heal. Where lesions do penetrate the bony subchondral plate, formation of hematomas and the migration of mesenchymal stem cells provide an inferior and transient fibrocartilagenous replacement for hyaline cartilage. To circumvent the poor intrinsic reparative response of articular cartilage several surgical techniques based on tissue transplantation have emerged. One characteristic shared by intrinsic reparative processes and the new surgical therapies is an apparent lack of lateral integration of repair or graft tissue with the host cartilage that can lead to poor prognosis. Many factors have been cited as impeding cartilage:cartilage integration including; chondrocyte cell death, chondrocyte dedifferentiation, the nature of the collagenous and proteoglycan networks that constitute the extracellular matrix, the type of biomaterial scaffold employed in repair and the origin of the cells used to repopulate the defect or lesion. This review addresses the principal intrinsic and extrinsic factors that impede integration and describe how manipulation of these factors using a host of strategies can positively influence cartilage integration.

Our previous analysis of the genes regulated in cartilage at the onset of spontaneous osteoarthritis in the guinea pig knee revealed up-regulation of the gene for protein kinase R (PKR)-activating protein (PACT), which encodes the cellular activator of the protein kinase, PKR. PACT and PKR are upstream components of a number of signal transduction and gene transcription pathways used by pro-inflammatory cytokines. We have investigated the role of PACT and PKR in articular cartilage degradation using cytokine treatment of bovine primary chondrocytes and cartilage explants. Tumour necrosis factor alpha increased expression of PACT protein after 3 h of treatment. Furthermore, increased phosphorylation of PKR and eukaryotic initiation factor 2-alpha was observed. The known role of PKR in cytokine-induced signalling pathways, together with our data showing cytokine regulation of PACT and PKR in chondrocytes, reveals a novel mechanism of cartilage degradation that may be important in the pathogenesis of arthritic diseases.

We have measured the first state-resolved, absolute cross sections for positron excitation of electronic states of an atom or molecule using a high resolution (Delta E approximately 25 meV FWHM) beam of positrons from a Penning-Malmberg trap. We present cross sections for the excitation of the low-lying levels of Ar, H(2), and N(2) for incident positron energies between threshold and 30 eV. For Ar and H2, comparison can be made with theoretical calculations, and, in the case of H(2), the results resolve a significant discrepancy between the only two available calculations.

Remodelling of the collagen matrix of the myocardium has been implicated in the pathogenesis of dilated cardiomyopathy, a major cause of heart failure in Doberman pinschers. The aim of this study was to characterise the myocardial collagen matrix of Dobermans. In clinically normal Dobermans there was evidence of focal fibrosis. Collagen cross-links were altered in both diseased and clinically normal Doberman myocardium as compared with myocardium from control dogs. Extensive remodelling, in the form of a loss of collagen tethers, increased collagen synthesis and alterations in the collagen cross-links, occurs in diseased Doberman myocardium. Changes in the collagenous matrix are also present in apparently normal Dobermans. These changes are likely to be involved in the progression of the disease and may explain the predisposition of this breed to dilated cardiomyopathy., (Copyright 2000 Harcourt Publishers Ltd.)

Purpose: To identify whether aging of human vitreous is accompanied by an elevation in degradative enzymes within the tissue., Methods: Human vitreous samples from donors aged 10 to 88 years were placed in two groups based on donor age of less than or more than 50 years. Homogenized samples were analyzed by gelatin substrate zymography for matrix metalloproteinases (MMP). Serine proteinases were detected by casein substrate zymography, and a specific antibody was used to confirm the identity of, and to quantify, the serine proteinase, plasmin., Results: Progelatinase A (ProMMP-2) was present in all the vitreous samples but did not show an age-related increase. MMP-2 was also present at low levels. Progelatinase B (ProMMP-9) was found in approximately 80% of samples analyzed, but neither its presence nor level of activity was age dependent. Of the serine proteinase activities detected, an enzyme of approximately 80 kDa was identified by Western blot analysis as plasmin(ogen). Quantitative analysis revealed a significant increase in plasmin(ogen) with age., Conclusions: This study shows there is an age-related increase in potential degradative activity in human vitreous that may be responsible for degenerative changes such as vitreous liquefaction. The data suggest that increased levels of these enzymes precede, or are indicative of, underlying ocular disease in some individuals.

Objectives: The purpose of this study was to evaluate empirically the relevance of animal-bioassay-based models for predicting human risks from exposure to 1,3-butadiene (BD) using epidemiologic data., Methods: Relative-risk results obtained with a regression model in a recent epidemiologic study were used to estimate leukemia risk for occupational and environmental exposures to BD and to compare these estimates with those previously derived from an analysis of animal bioassay data., Results: The estimates of risk were found to be highly dependent on the model used when low levels of exposure were evaluated that are of environmental concern, but not at the levels of occupational concern. For example, at the level (1 part per million) of the recently revised standard of the Occupational Safety and Health Administration in the United States the estimates of lifetime excess risk ranged from 1 to 8 per 1000 workers. The range of the risk estimates derived from the epidemiologic models was remarkably similar to the range of risk estimates for occupational exposures (1 to 9 per thousand) previously developed by Dankovic et al in 1993 from an analysis of a mouse bioassay study for lymphocytic lymphoma., Conclusions: Results for BD seem to provide another example of a high degree of concordance between the risk predictions from models of toxicologic and epidemiologic data, particularly at occupational levels of exposure.

Objective: Canine dilated cardiomyopathy, commonly affecting Doberman pinschers, results in extracellular matrix remodelling within the myocardium. The aim of this study was to examine the proteolytic activity in myocardium from Doberman pinschers with dilated cardiomyopathy., Methods: Samples of myocardium, obtained rapidly post mortem from the left ventricular free wall of Dobermans with dilated cardiomyopathy, clinically normal Dobermans and control dogs (non-Dobermans), were examined for proteolytic activity using substrate gel zymography. Gels were analysed by scanning densitometry., Results: Promatrix metalloproteinase-9 activity was significantly increased in all Doberman myocardium when compared to controls. A significant increase in an enzyme, identified to be neutrophil elastase by inhibition of its activity by Elastatinal and Western blotting, was also detected in all Dobermans when compared to controls., Conclusions: The results indicate that promatrix metalloproteinase-9 and neutrophil elastase, both of which are implicated in inflammatory responses, are present in significantly elevated levels in Doberman dilated cardiomyopathy and are raised in clinically normal Dobermans. Both proteolytic enzymes degrade a wide variety of connective tissue components and thus the increased levels found may play an important role in the structural remodelling seen in the myocardium and subsequent heart failure. Increased proteolytic enzyme levels in clinically normal Dobermans may be indicative of the predisposition of the breed to dilated cardiomyopathy.

This paper describes a new analysis of data from the 1968-72 National Institute for Occupational Safety & Health (NIOSH) Occupational Noise and Hearing Survey (ONHS). The population consisted of 1172 (792 noise-exposed and 380 "controls") predominately white male workers from a cross section of industries within the United States. The analysis focused on how risk estimates vary according to various model assumptions, including shape of the dose-response curve and the amount of noise exposure among low-noise exposed workers (or controls). Logistic regression models were used to describe the risk of hearing handicap in relation to age, occupational noise exposure, and duration exposed. Excess risk estimates were generated for several definitions of hearing handicap. Hearing handicap is usually denoted as an average hearing threshold level (HTL) of greater than 25 dB for both ears at selected frequencies. The frequencies included in the biaural averages were (1) the articulation-weighted average over 1-4 kHz, (2) the unweighted average over 0.5, 1, and 2 kHz, and (3) the unweighted average over 1, 2, and 3 kHz. The results show that excess risk estimates for time-weighted average sound levels below 85 dB were sensitive to statistical model form and assumptions regarding the sound level to which the "control" group was exposed. The choice of frequencies used in the hearing handicap definition affected the magnitude of excess risk estimates, which depended on age and duration of exposure. Although data were limited below 85 dB, an age-stratified analysis provided evidence of excess risks at levels ranging from 80 to 84 dB, 85-89 dB, and 90-102 dB. Due to uncertainty in quantifying risks below 85 dB, new data collection efforts should focus on better characterization of dose-response and longitudinal hearing surveys that include workers exposed to 8-hour time-weighted noise levels below 85 dB. Results are compared to excess risk estimates generated using methods given by ANSI S3.44-1996.