Analysis of clusterin expression changes as a biomarker of osteoarthritis

Purpose: The discovery and validation of arthritis-related biomarkers and establishment of methodology for proteomic studies in osteoarthritis (OA) are needed. Proteomics strategies have identified many proteins that may relate to pathological mechanisms of OA, however targeted approaches are required to validate the roles of these proteins. This study aimed to use mass spectrometry and western blotting to identify peptides from several proteins in the secretome of chondrocytes, cartilage explants and osteochondral biopsies treated with inflammatory cytokines over a 2-week period, to evaluate their potential as biomarkers of OA progression. Methods: Healthy cartilage was obtained from fetlock joints of skeletally mature horses, euthanized for unrelated veterinary reasons. Cartilage explants were isolated using a 6 mm biopsy, with discs placed into wells (3 discs per 1 ml DMEM + 1% Pen/Strep) before incubation for 24 hours (37 °C, 5% CO2). After this equilibration period, the media was removed and replaced with either fresh DMEM + 1% Pen/Strep or DMEM supplemented with 1% Pen/Strep containing TNFα and IL-1β both at 10ng/ml. Explants were culture for 7–14 days with the cytokines replaced every 4th day. For cell based assays chondrocytes were isolated from tissue using 70U pronase for 1hr at 37 °C and overnight digestion at 37°C using a 0.2% collagenase II solution. The cell suspension was filtered and washed before being seeded into culture flasks and cultured until confluence was reached (37°C, 5% CO2). Once cultures were established cells were split into two groups: healthy control (DMEM supplemented with 1% Pen/Strep and 10% foetal calf serum) or stimulated cells (DMEM as above plus TNFα and IL-1β both at 10ng/ml). Chondroyctes were cytokine-stimulated for up to one week. Cells were used in experiments up to the 2nd passage. Results: Mass spectrometry data showed that peptides representative of clusterin were found to decrease following 7 days of inflammatory stimulation. Western blotting of secreted proteins in media of cartilage explants or chondrocyte showed that clusterin expression was reduced following 7 days of cytokine treatment. Catabolic matrix metalloproteinase enzymes MMP1, MMP3 and MMP13, as well the matrix component cartilage oligomeric protein (COMP) were all found to have an increased abundance in the media of the cytokine treated samples. This data was supported by qPCR for clusterin gene expression which showed initially mRNA levels increased 3 day after inflammatory stimulation but expression was lost after 7 days. Western blotting of media from the osteochondral biopsies showed an increase in clusterin expression after 7 days of inflammatory stimulation however clusterin protein expression could not be detected after 14 days of treatment, indicating a delayed response compared to cartilage tissue alone. Conclusions: The equine chondrocytes, cartilage explant and osteochondral biopsy models exhibited highest clusterin secretion in untreated cultures. IL-1β and TNFα treatment caused a reduction in clusterin secretion. Clusterin acts as a chaperone to aid protein refolding in situations of stress and is constitutively secreted by mammalian cells. IL-1β and TNFα appear to interrupt clusterin secretion and therefore the protection it may offer healthy functioning cells. Previous studies have reported variable data, with some studies indicating a decrease in clusterin in OA, while others indicate an increase in clusterin expression. Our results suggest the clusterin increases immediately after inflammatory stimulation but is lost after prolonged exposure. Therefore, levels of secreted clusterin may be a candidate biomarker for OA progression

Interventions based on PRECEDE-PROCEED for promoting safety behaviors in Primary school boys

Introduction & Objective: Pedestrian injuries are a significant source of morbidity, mortality and disability among children. Prevention of these injuries is thus a major priority for public health and requires a comprehensive approach and educational strategies. The purpose of this study was designing and evaluation of an educational program, based on the PRECEED-PROCEED model for improvement of safety behavior in primary school boys. Materials & Methods: This study is a field trial carried out in 6th areas of Tehran in 2007. Participants (n=88) were 10 – 11 year old who followed by items measuring the PRECEED-PROCEED model before and after two months of intervention. The collected data were analyzed with T test, X2 and pair T test using SPSS software. Results: After participating in educational program, significant improvements were found in knowledge, attitude, behavior, enabling and reinforcing factors of the components of PRECEEDPROCEED model. Conclusion: The findings suggest the beneficial effects of educational intervention based on the PRECEDE-PROCEED model on improving the safety behavior. It can be recommended that health education plan be used on educational models

Design and development of poly-L/D-lactide copolymer and barium titanate nanoparticle 3D composite scaffolds using breath figure method for tissue engineering applications

In tissue engineering, the scaffold topography influences the adhesion, proliferation, and function of cells. Specifically, the interconnected porosity is crucial for cell migration and nutrient delivery in 3D scaffolds. The objective of this study was to develop a 3D porous composite scaffold for musculoskeletal tissue engineering applications by incorporating barium titanate nanoparticles (BTNPs) into a poly-L/D-lactide copolymer (PLDLA) scaffold using the breath figure method. The porous scaffold fabrication utilised 96/04 PLDLA, dioleoyl phosphatidylethanolamine (DOPE), and different types of BTNPs, including uncoated BTNPs, Al2O3-coated BTNPs, and SiO2-coated BTNPs. The BTNPs were incorporated into the polymer scaffold, which was subsequently analysed using field emission scanning electron microscopy (FE-SEM). The biocompatibility of each scaffold was tested using ovine bone marrow stromal stem cells. The cell morphology, viability, and proliferation were evaluated using FE-SEM, LIVE/DEAD staining, and Prestoblue assay. Porous 3D composite scaffolds were successfully produced, and it was observed that the incorporation of uncoated BTNPs increased the average pore size from 1.6 mu m (PLDLA) to 16.2 mu m (PLDLA/BTNP). The increased pore size in the PLDLA/BTNP scaffolds provided a suitable porosity for the cells to migrate inside the scaffold, while in the pure PLDLA scaffolds with their much smaller pore size, cells elongated on the surface. To conclude, the breath figure method was successfully used to develop a PLDLA/BTNP scaffold. The use of uncoated BTNPs resulted in a composite scaffold with an optimal pore size while maintaining the honeycomb-like structure. The composite scaffolds were biocompatible and yielded promising structures for future tissue engineering applications.Peer reviewe

The cytotoxic domain of colicin E9 is a channel-forming endonuclease

Bacterial toxins commonly translocate cytotoxic enzymes into cells using dedicated channelforming subunits or domains as conduits. We demonstrate that the small cytotoxic endonuclease domain from the bacterial toxin colicin E9 (the E9 DNase) exhibits nonvoltage- gated, channel-forming activity in planar lipid bilayers and that this activity is linked to toxin translocation into cells. A disulfide bond engineered into the DNase abolished channel activity and colicin toxicity but left endonuclease activity unaffected, with NMR experiments suggesting decreased conformational flexibility as the likely reason for these alterations. Concomitant with the reduction of the disulfide bond was the restoration of conformational flexibility, DNase channel activity and colicin toxicity. Our data suggest that endonuclease domains of colicins may mediate their own translocation across the bacterial inner membrane through an intrinsic channel activity that is dependent on structural plasticity in the protein

Physiological effects of oral glucosamine on joint health: Current status and consensus on future research priorities

The aim of this paper was to provide an overview of the current knowledge and understanding of the potential beneficial physiological effects of glucosamine (GlcN) on joint health. The objective was to reach a consensus on four critical questions and to provide recommendations for future research priorities. To this end, nine scientists from Europe and the United States were selected according to their expertise in this particular field and were invited to participate in the Hohenheim conference held in Aug

A machine learning heuristic to identify biologically relevant and minimal biomarker panels from omics data

Background: Investigations into novel biomarkers using omics techniques generate large amounts of data. Due to their size and numbers of attributes, these data are suitable for analysis with machine learning methods. A key component of typical machine learning pipelines for omics data is feature selection, which is used to reduce the raw high-dimensional data into a tractable number of features. Feature selection needs to balance the objective of using as few features as possible, while maintaining high predictive power. This balance is crucial when the goal of data analysis is the identification of highly accurate but small panels of biomarkers with potential clinical utility. In this paper we propose a heuristic for the selection of very small feature subsets, via an iterative feature elimination process that is guided by rule-based machine learning, called RGIFE (Rule-guided Iterative Feature Elimination). We use this heuristic to identify putative biomarkers of osteoarthritis (OA), articular cartilage degradation and synovial inflammation, using both proteomic and transcriptomic datasets.Results and discussion: Our RGIFE heuristic increased the classification accuracies achieved for all datasets when no feature selection is used, and performed well in a comparison with other feature selection methods. Using this method the datasets were reduced to a smaller number of genes or proteins, including those known to be relevant to OA, cartilage degradation and joint inflammation. The results have shown the RGIFE feature reduction method to be suitable for analysing both proteomic and transcriptomics data. Methods that generate large ‘omics’ datasets are increasingly being used in the area of rheumatology.Conclusions: Feature reduction methods are advantageous for the analysis of omics data in the field of rheumatology, as the applications of such techniques are likely to result in improvements in diagnosis, treatment and drug discovery

Communication aid requirements of intensive care unit patients with transient speech loss

Alert and transiently nonvocal intensive care unit (ICU) patients are dependent on augmentative and alternative communication (AAC). Unfortunately, the literature demonstrates that existent AAC devices have not been widely adopted, and unaided methods are often the primary modalities used despite being insufficient, and frustrating. We present the results of a qualitative semi-structured interview study with 8 ex-ICU patients, 4 ICU patient relatives, and 6 ICU staff, exploring their AAC needs and requirements. Participants identified important AAC hardware, software, and content requirements. Salient factors impacting on AAC adoption in the ICU setting were also highlighted and included the need for staff training and bedside patient assessment. Based on the study results, we propose a series of recommendations regarding the design and implementation of future AAC tools specifically targeted at this group

Chondrocyte secretome: a source of novel insights and exploratory biomarkers of osteoarthritis

The extracellular matrix (ECM) of articular cartilage is comprised of complex networks of proteins and glycoproteins, all of which are expressed by its resident cell, the chondrocyte. Cartilage is a unique tissue given its complexity and ability to resist repeated load and deformation. The mechanisms by which articular cartilage maintains its integrity throughout our lifetime is not fully understood, however there are numerous regulatory pathways known to govern ECM turnover in response to mechanical stimuli. To further our understanding of this field, we envision that proteomic analysis of the secretome will provide information on how the chondrocyte remodels the surrounding ECM in response to load, in addition to providing information on the metabolic state of the cell. In this review, we attempt to summarize the recent mass spectrometry-based proteomic discoveries in healthy and diseased cartilage and chondrocytes, to facilitate the discovery of novel biomarkers linked to degenerative pathologies, such as osteoarthritis (OA)

Neuropathic Pain in the IMI-APPROACH Knee Osteoarthritis Cohort: Prevalence and Phenotyping

The study is registered under clinicaltrials.gov nr: NCT03883568.[Abstract] Objectives: Osteoarthritis (OA) patients with a neuropathic pain (NP) component may represent a specific phenotype. This study compares joint damage, pain and functional disability between knee OA patients with a likely NP component, and those without a likely NP component. Methods: Baseline data from the Innovative Medicines Initiative Applied Public-Private Research enabling OsteoArthritis Clinical Headway knee OA cohort study were used. Patients with a painDETECT score ≥19 (with likely NP component, n=24) were matched on a 1:2 ratio to patients with a painDETECT score ≤12 (without likely NP component), and similar knee and general pain (Knee Injury and Osteoarthritis Outcome Score pain and Short Form 36 pain). Pain, physical function and radiographic joint damage of multiple joints were determined and compared between OA patients with and without a likely NP component. Results: OA patients with painDETECT scores ≥19 had statistically significant less radiographic joint damage (p≤0.04 for Knee Images Digital Analysis parameters and Kellgren and Lawrence grade), but an impaired physical function (p<0.003 for all tests) compared with patients with a painDETECT score ≤12. In addition, more severe pain was found in joints other than the index knee (p≤0.001 for hips and hands), while joint damage throughout the body was not different. Conclusions: OA patients with a likely NP component, as determined with the painDETECT questionnaire, may represent a specific OA phenotype, where local and overall joint damage is not the main cause of pain and disability. Patients with this NP component will likely not benefit from general pain medication and/or disease-modifying OA drug (DMOAD) therapy. Reserved inclusion of these patients in DMOAD trials is advised in the quest for successful OA treatments