Suppression of MMP activity in bovine cartilage explants cultures has little if any effect on the release of aggrecanase-derived aggrecan fragments

<p>Abstract</p> <p>Background</p> <p>Progressive loss of articular cartilage is a central hallmark in many joint disease, however, the relative importance of individual proteolytic pathways leading to cartilage erosion is at present unknown. We therefore investigated the time-dependant release <it>ex vivo </it>of MMP- and aggrecanase-derived fragments of aggrecan and type II collagen into the supernatant of bovine cartilage explants cultures using neo-epitope specific immunoassays, and to associate the release of these fragments with the activity of proteolytic enzymes using inhibitors.</p> <p>Findings</p> <p>Bovine cartilage explants were cultured in the presence or absence of the catabolic cytokines oncostatin M (OSM) and tumor necrosis factor alpha (TNFα). In parallel, explants were co-cultured with protease inhibitors such as GM6001, TIMP1, TIMP2 and TIMP3. Fragments released into the supernatant were determined using a range of neo-epitope specific immunoassays; (1) sandwich ³⁴²FFGVG-G2 ELISA, (2) competition NITEGE³⁷³ELISA (3) sandwich G1-NITEGE³⁷³ELISA (4) competition ³⁷⁴ARGSV ELISA, and (5) sandwich ³⁷⁴ARGSV-G2 ELISA all detecting aggrecan fragments, and (6) sandwich CTX-II ELISA, detecting C-telopeptides of type II collagen. We found that (1) aggrecanase-derived aggrecan fragments are released in the early (day 2-7) and mid phase (day 9-14) into the supernatant from bovine explants cultures stimulated with catabolic cytokines, (2) the release of NITEGE³⁷³neo-epitopes are delayed compared to the corresponding ³⁷⁴ARGSV fragments, (3) the MMP inhibitor GM6001 did not reduce the release of aggrecanase-derived fragment, but induced a further delay in the release of these fragments, and finally (4) the MMP-derived aggrecan and type II collagen fragments were released in the late phase (day 16-21) only.</p> <p>Conclusion</p> <p>Our data support the model, that aggrecanases and MMPs act independently in the processing of the aggrecan molecules, and furthermore that suppression of MMP-activity had little if any effect on the quantity of aggrecanase-derived fragments released from explants cultures.</p

Forearm fractures–are we counting them all? An attempt to identify and include the missing fractures treated in primary care

Objective: Norway has a high incidence of forearm fractures, however, the incidence rates based on secondary care registers can be underestimated, as some fractures are treated exclusively in primary care. We estimated the proportion of forearm fracture diagnoses registered exclusively in primary care and assessed the agreement between diagnosis for forearm fractures in primary and secondary care. Design: Quality assurance study combining nationwide data from 2008 to 2019 on forearm fractures registered in primary care (Norwegian Control and Payment of Health Reimbursement) and secondary care (the Norwegian Patient Registry). Setting and patients: Forearm fracture diagnoses in patients aged ≥20 treated in primary care (n = 83,357) were combined with injury diagnoses for in- and outpatients in secondary care (n = 3,294,336). Main outcome measures: Proportion of forearm fractures registered exclusively in primary care, and corresponding injury diagnoses for those registered in both primary and secondary care. Results: Of 189,105 forearm fracture registrations in primary and secondary care, 13,948 (7.4%) were registered exclusively in primary care. The proportion ranged from 4.9% to 13.5% on average between counties, but was higher in some municipalities (>30%). Of 66,747 primary care forearm fractures registered with a diagnosis in secondary care, 62% were incident forearm fractures, 28% follow-up controls, and 10% other fractures or non-fracture injuries. Conclusion: An overall small proportion of forearm fractures were registered only in primary care, but it was larger in some areas of Norway. Failing to include fractures exclusively treated in primary care could underestimate the incidence rates in these areas

The effect of farming environment on asthma; time dependent or universal?

Funding Information: MJA holds investigator-initiated grants from Pfizer and Boehringer-Ingelheim for unrelated research. He has undertaken an unrelated consultancy for and received assistance with conference attendance from Sanofi. He has also received a speaker’s fee from GSK. The other authors have no conflicts of interest to declare that are relevant for the content of this article. Funding Information: The ECRHS/RHINE/RHINESSA study was supported by grants from The Faculty of Health, Aarhus University, Denmark (Project No. 240008), The Wood Dust Foundation (Project No. 444508795), The Danish Lung Association, the Swedish Heart and Lung Foundation, the Swedish Association Against Asthma and Allergy, the Swedish Association against Heart and Lung Disease, the Swedish Council for Working Life and Social Research, The Bror Hjerpstedt Foundation, The Vårdal Foundation for Health Care and Allergic Research, The Norwegian Research Council (Grant Nos. 214123, 230827/F20, 228174 and 135773/330), The Norwegian Asthma and Allergy Association, HelseVest Norway (Grant No. 911 631), The Icelandic Research Council, The University of Iceland Research Fund, The Icelandic GP’s Research Fund, The Estonian Science Foundation (Grant No. 4350), The Estonian Research Council (Grant No. PUT562), Melbourne University, National Health & Medical Research Council of Australia, SEPAR Spain, Sociedad Española de Neumologia y Cirugía Toracica Spain and Horizon2020 PHC1 (Grant No. 633212). For further information about funding sources, please consult www.rhinessa.net . Vivi Schlünssen and Cecilie Svanes are members of the COST BM1201 network. Publisher Copyright: © 2022, Springer Nature B.V.The increasing prevalence of asthma is linked to westernization and urbanization. Farm environments have been associated with a lower risk of asthma development. However, this may not be universal, as the association differs across birth cohorts and farming methods. The aim of this study was to investigate the associations of farm upbringing with asthma in different generations and at different times in history. The study population consisted of three generations: 13,868 subjects participating in the ECRHS in 2010, their 9,638 parents, and their 8,885 offspring participating in RHINESSA in 2013. Information on place of upbringing and self-reported ever asthma was provided via questionnaires. Logistic regression was performed including subgroup analysis stratified by generation and birthyear into ten-year-intervals. The prevalence of asthma increased from 8% among grandparents to 13% among parents and to 18% among offspring. An overall analysis showed an inverse association of farm upbringing on the risk of asthma (OR = 0.64; 95%CI 0.55–0.74). Subgroup analysis stratified into ten-year-intervals showed a tendency towards a more pronounced inverse association between growing up on a farm and asthma among subjects born in the 1940s (0.74; 0.48–1.12), 1950s (0.70; 0.54–0.90) and 1960s (0.70; 0.52–0.93). For subjects born in 1970 and thereafter this association appeared less consistent. While growing up on a farm was associated with a reduced risk of developing asthma in participants born between 1945–1999, this was mainly driven by generations born from 1945 to 1973.Peer reviewe

Investigation of the direct effects of salmon calcitonin on human osteoarthritic chondrocytes

<p>Abstract</p> <p>Background</p> <p>Calcitonin has been demonstrated to have chondroprotective effects under pre-clinical settings. It is debated whether this effect is mediated through subchondral-bone, directly on cartilage or both in combination. We investigated possible direct effects of salmon calcitonin on proteoglycans and collagen-type-II synthesis in osteoarthritic (OA) cartilage.</p> <p>Methods</p> <p>Human OA cartilage explants were cultured with salmon calcitonin [100 pM-100 nM]. Direct effects of calcitonin on articular cartilage were evaluated by 1) measurement of proteoglycan synthesis by incorporation of radioactive labeled ³⁵SO₄[5 μCi] 2) quantification of collagen-type-II formation by pro-peptides of collagen type II (PIINP) ELISA, 3) QPCR expression of the calcitonin receptor in OA chondrocytes using four individual primer pairs, 4) activation of the cAMP signaling pathway by EIA and, 5) investigations of metabolic activity by AlamarBlue.</p> <p>Results</p> <p>QPCR analysis and subsequent sequencing confirmed expression of the calcitonin receptor in human chondrocytes. All doses of salmon calcitonin significantly elevated cAMP levels (P < 0.01 and P < 0.001). Calcitonin significantly and concentration-dependently [100 pM-100 nM] induced proteoglycan synthesis measured by radioactive ³⁵SO₄incorporation, with a 96% maximal induction at 10 nM (P < 0.001) corresponding to an 80% induction of 100 ng/ml IGF, (P < 0.05). In alignment with calcitonin treatments [100 pM-100 nM] resulted in 35% (P < 0.01) increased PIINP levels.</p> <p>Conclusion</p> <p>Calcitonin treatment increased proteoglycan and collagen synthesis in human OA cartilage. In addition to its well-established effect on subchondral bone, calcitonin may prove beneficial to the management of joint diseases through direct effects on chondrocytes.</p