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Objective.To get a better understanding of inflammatory pathways active in the osteoarthritic (OA) joint, we characterized and compared inflammatory cells in the synovium and the infrapatellar fat pad (IFP) of patients with knee OA.Methods.Infiltrating immune cells were characterized by flow cytometry in 76 patients with knee OA (mean age 63.3, 52% women, median body mass index 28.9) from whom synovial tissue (n = 40) and IFP (n = 68) samples were obtained. Pain was assessed by the visual analog scale (VAS; 0–100 mm). Spearman rank correlations and linear regression analyses adjusted for sex and age were performed.Results.Macrophages and T cells, followed by mast cells, were the most predominant immune cells in the synovium and IFP, and were equally abundant in these tissues. Macrophages and T cells secreted mostly proinflammatory cytokines even without additional stimulation, indicating their activated state. Accordingly, most CD4+ T cells had a memory phenotype and contained a significant population of cells expressing activation markers (CD25+, CD69+). Interestingly, the percent of CD69+ T cells was higher in synovial than IFP CD4+ T cells. Preliminary analyses indicated that the number of synovial CD4+ T cells were associated with VAS pain (β 0.51, 95% CI 0.09–1.02, p = 0.02).Conclusion.Our data suggest that the immune cell composition of the synovium and the IFP is similar, and includes activated cells that could contribute to inflammation through secretion of proinflammatory cytokines. Moreover, preliminary analyses indicate that synovial CD4+ T cells might associate with pain in patients with endstage OA of the knee.

Objective: Human cohort studies have demonstrated a role for systemic metabolic dysfunction in osteoarthritis (OA) pathogenesis in obese patients. To explore the mechanisms underlying this metabolic phenotype of OA, we examined cartilage degradation in the knees of mice from different genetic backgrounds in which a metabolic phenotype was established by various dietary approaches.Design: Wild-type C57BL/6J mice and genetically modified mice (hCRP, LDLr-/-. Leiden and ApoE*3Leiden. CETP mice) based on C57BL/6J background were used to investigate the contribution of inflammation and altered lipoprotein handling on diet-induced cartilage degradation. High-caloric diets of different macronutrient composition (i.e., high-carbohydrate or high-fat) were given in regimens of varying duration to induce a metabolic phenotype with aggravated cartilage degradation relative to controls.Results: Metabolic phenotypes were confirmed in all studies as mice developed obesity, hypercholesteremia, glucose intolerance and/or insulin resistance. Aggravated cartilage degradation was only observed in two out of the twelve experimental setups, specifically in long-term studies in male hCRP and female ApoE*3Leiden. CETP mice. C57BL/6J and LDLr-/-. Leiden mice did not develop HFD-induced OA under the conditions studied. Osteophyte formation and synovitis scores showed variable results between studies, but also between strains and gender.Conclusions: Long-term feeding of high-caloric diets consistently induced a metabolic phenotype in various C57BL/6J (-based) mouse strains. In contrast, the induction of articular cartilage degradation proved variable, which suggests that an additional trigger might be necessary to accelerate diet-induced OA progression. Gender and genetic modifications that result in a humanized pro-inflammatory state (human CRP) or lipoprotein metabolism (human-E3L. CETP) were identified as important contributing factors. (c) 2017 Published by Elsevier Ltd on behalf of Osteoarthritis Research Society International.

Background Obesity is associated with the development and progression of osteoarthritis (OA). Although the infrapatellar fat pad (IFP) could be involved in this association, due to its intracapsular localization in the knee joint, there is currently little known about the effect of obesity on the IFP. Therefore, we investigated cellular and molecular body mass index (BMI)-related features in the IFP of OA patients. Methods Patients with knee OA (N = 155, 68% women, mean age 65 years, mean (SD) BMI 29.9 kg/m2 (5.7)) were recruited: IFP volume was determined by magnetic resonance imaging in 79 patients with knee OA, while IFPs and subcutaneous adipose tissue (SCAT) were obtained from 106 patients undergoing arthroplasty. Crown-like structures (CLS) were determined using immunohistochemical analysis. Adipocyte size was determined by light microscopy and histological analysis. Stromal vascular fraction (SVF) cells were characterized by flow cytometry. Results IFP volume (mean (SD) 23.6 (5.4) mm3) was associated with height, but not with BMI or other obesity-related features. Likewise, volume and size of IFP adipocytes (mean 271 pl, mean 1933 μm) was not correlated with BMI. Few CLS were observed in the IFP, with no differences between overweight/obese and lean individuals. Moreover, high BMI was not associated with higher SVF immune cell numbers in the IFP, nor with changes in their phenotype. No BMI-associated molecular differences were observed, besides an increase in TNFα expression with high BMI. Macrophages in the IFP were mostly pro-inflammatory, producing IL-6 and TNFα, but little IL-10. Interestingly, however, CD206 and CD163 were associated with an anti-inflammatory phenotype, were the most abundantly expressed surface markers on macrophages (81% and 41%, respectively) and CD163+ macrophages had a more activated and pro-inflammatory phenotype than their CD163- counterparts. Conclusions BMI-related features usually observed in SCAT and visceral adipose tissue could not be detected in the IFP of OA patients, a fat depot implicated in OA pathogenesis. Electronic supplementary material The online version of this article (doi:10.1186/s13075-017-1395-9) contains supplementary material, which is available to authorized users.

Previous studies have shown accumulation and an enhanced proinflammatory profile of macrophages in adipose tissue of obese mice, indicating the presence of an interaction between adipocytes and macrophages in this tissue. However, the consequences of this interaction in humans are yet incompletely understood. In this study, we explored the modulating effects of adipocytes on the phenotype of macrophages in humans and studied the possible molecular pathways involved. Adipocyte-conditioned media (ACM) treatment of macrophages for 48 h strongly reduced the LPS-induced IL-12p40 secretion by macrophages, whereas the production of TNF-α and other cytokines remained largely unaffected. This effect was independent of the source of adipocytes. Interestingly, the level of inhibition correlated directly with body mass index (BMI) of the adipocyte donor. Because adipocytes release many different cytokines, adipokines, and lipids, we have separated the protein and lipid fractions of ACM, to obtain insight into the molecular nature of the soluble mediators underlying the observed effect. These experiments revealed that the inhibitory effect resided predominantly in the lipid fraction. Further studies revealed that PGE2 and linoleic and oleic acid were potent inhibitors of IL-12p40 secretion. Interestingly, concentrations of these ACM-derived lipids increased with increase in BMI of the adipocyte donor, suggesting that they could mediate the BMI-dependent effects of ACM. To our knowledge, these results provide first evidence that obesity-related changes in adipose tissue macrophage phenotype could be mediated by adipocyte-derived lipids in humans. Intriguingly, these changes appear to be different from those in murine obesity.

Objective: To investigate the course of synovitis on contrast-enhanced magnetic resonance images (CE-MRI) in osteoarthritic knees over 2 years, and its association with pain and cartilage deterioration. Design: Consecutive patients (n = 39, mean age 61 years, 79% woman, median (range) body mass index (BMI) 29 (24-48) kg/mm2) with clinical osteoarthritis (OA) were included. Baseline and follow-up CE-MRI (3 T) were scored paired in chronological order for synovitis (semi-quantitatively at 11 sites (range 0-22)), cartilage deterioration and bone marrow lesions (BMLs) (semi-quantitatively according to Knee Osteoarthritis Scoring System (KOSS)). Changes in sum scores were calculated. Cartilage deterioration was defined as change of ≥2 above the smallest detectable change (SDC). Pain was assessed by standardized questionnaires. Analysis of covariance (ANCOVA) and linear regression models were used to investigate association between synovitis change and cartilage deterioration and between synovitis change or cartilage deterioration and change in pain. Results: The total synovitis score did not change over 2 years (mean change 0.2 (standard deviation (SD) 3.2)), although changes in individual patients were observed. Cartilage deterioration was observed in 51% of patients. Synovitis change score was lower in patients without compared to patients with cartilage deterioration, taking BML change in account (mean difference -2.1 (-4.1 to -0.1)). Change in synovitis was not associated with change in pain, whereas cartilage deterioration was associated with change in Intermittent and Constant OsteoArthritis Pain (ICOAP) constant pain in adjusted models (unstandardised coefficient (B) (95% confidence interval (CI)) 2.8 (0.4-5.3)). Conclusions: In individual patients synovitis fluctuates during disease course. Synovitis change was not associated with change in pain. Increase in synovitis is associated with cartilage deterioration, suggesting a role for synovitis as a target for disease-modifying treatment.

OBJECTIVES: Obesity is a risk factor for the development of osteoarthritis (OA) in hands and knees. Adipose tissue can secrete different adipokines with powerful immunomodulatory effects. The infrapatellar fat pad (IFP) is an intra-articular organ in the vicinity of the synovium and cartilage. It is hypothesised that IFP-derived soluble factors could contribute to pathological processes in the knee joint. A study was therefore undertaken to compare the release of inflammatory mediators in the IFP and subcutaneous adipose tissue (ScAT) and to characterise the adipocytes and immune cell infiltrate in these tissues.METHODS: Paired IFP and ScAT samples were obtained from 27 patients with primary OA. The stromal vascular cell fraction (SVF) was isolated and characterised by fluorescence activated cell sorting. Cytokine and adipokine release in fat- and adipocyte-conditioned media was measured by luminex.RESULTS: IFP secreted higher levels of inflammatory mediators such as interleukin 6 (IL-6), adipsin, adiponectin and visfatin than ScAT. This could be due to differences in the phenotype of adipocytes and/or in the composition and phenotype of the SVF cells. IFP adipocyte-conditioned media showed a trend towards more IL-6 and adipsin than ScAT. Moreover, the SVF fraction of IFP contained more cells/g tissue, a lower percentage of T cells and a higher percentage of mast cells than ScAT. In addition, T cells had a predominantly pro-inflammatory phenotype while macrophages had a mixed pro- and anti-inflammatory phenotype in the IFP.CONCLUSION: There are profound differences in secreted inflammatory factors and immune cell composition between the IFP and ScAT. These data indicate that IFP-derived soluble mediators could contribute to pathophysiological processes in the OA knee joint.

Here we present an advanced platform for the analysis of specialized pro-resolving mediators (SPM), including a set of pro-inflammatory lipids and respective biochemical pathway markers. The platform is characterized by a largely simplified sample preparation protocol, employing methanol protein-precipitation instead of solid phase extraction. Sample preparation and analysis can be carried out in 96-well format, ensuring higher throughput. In addition, faster analysis times and higher sensitivities have been achieved when compared to other platforms. In total 36 lipid mediators—SPM, their pathway markers and a set of pro-inflammatory lipids including three internal standards—are analyzed within an 11 min LC–MS/MS run. The method was validated using human plasma, including repeatability, linearity, recovery, matrix effects, and accuracy. The presented platform was applied in the analysis of synovial fluid post-mortem samples and cellular extracts from polymorphonuclear cells as well as whole blood treated with ionophore in the presence of the polyunsaturated fatty acid eicosapentaenoic acid. © 2014, Springer-Verlag Berlin Heidelberg.

Objective: To evaluate the association between synovitis on contrast enhanced (CE) MRI with microscopic and macroscopic features of synovial tissue inflammation. Method: Forty-one patients (mean age 60 years, 61% women) with symptomatic radiographic knee OA were studied: twenty underwent arthroscopy (macroscopic features were scored (0-4), synovial biopsies obtained), twenty-one underwent arthroplasty (synovial tissues were collected). After haematoxylin and eosin staining, the lining cell layer, synovial stroma and inflammatory infiltrate of synovial tissues were scored (0-3). T1-weighted CE-MRI's (3 T) were used to semi-quantitatively score synovitis at 11 sites (0-22) according to Guermazi et al. Spearman's rank correlations were calculated. Results: The mean (SD) MRI synovitis score was 8.0 (3.7) and the total histology grade was 2.5 (1.6). Median (range) scores of macroscopic features were 2 (1-3) for neovascularization, 1 (0-3) for hyperplasia, 2 (0-4) for villi and 2 (0-3) for fibrin deposits. The MRI synovitis score was significantly correlated with total histology grade [r = 0.6], as well as with lining cell layer [r = 0.4], stroma [r = 0.3] and inflammatory infiltrate [r = 0.5] grades. Moreover, MRI synovitis score was also significantly correlated with macroscopic neovascularization [r = 0.6], hyperplasia [r = 0.6] and villi [r = 0.6], but not with fibrin [r = 0.3]. Conclusion: Synovitis severity on CE-MRI assessed by a new whole knee scoring system by Guermazi et al. is a valid, non-invasive method to determine synovitis as it is significantly correlated with both macroscopic and microscopic features of synovitis in knee OA patients. (C) 2013 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

Objective: Soluble mediators in synovial fluid (SF) are acknowledged as key players in the pathophysiology of osteoarthritis (OA). However, a wide-spectrum screening of such mediators in SF is currently lacking. In this study, the levels of 47 mediators in the SF of control donors and osteoarthritic (OA) patients were compared. Materials & Methods: SF was collected from control donors (n=16) and end-stage knee OA patients (n=18) and analysed for 47 cytokines, chemokines and growth factors using several multiplex enzyme-linked immunosorbent assays (ELISAs). A Mann-Whitney U test was used to determine differences between OA and control controls. A principal component analysis (PCA) was performed to cluster the 47 mediators. Results: The majority of the mediators could be detected in both control and OA SF. Interleukin (IL)-6, interferon inducible protein (IP)-10, macrophage derived chemokine (MDC), platelet derived growth factor (PDGF)-AA and regulated on activation normal T cell expressed and secreted (RANTES) levels were found to be higher in OA compared to control SF (P

Objective: Recently, computed tomography arthrography (CTa) was introduced as quantitative imaging biomarker to estimate cartilage sulphated glycosaminoglycan (sGAG) content in human cadaveric knees. Our aim was to assess the correlation between in vivo CTa in human osteoarthritis (OA) knees and ex vivo reference standards for sGAG and collagen content. Design: In this prospective observational study 11 knee OA patients underwent CTa before total knee replacement (TKR). Cartilage X-ray attenuation was determined in six cartilage regions. Femoral and tibial cartilage specimens harvested during TKR were re-scanned using equilibrium partitioning of an ionic contrast agent with micro-CT (EPIC-mu CT), which served as reference standard for sGAG. Next, cartilage sGAG and collagen content were determined using dimethylmethylene blue (DMMB) and hydroxyproline assays. The correlation between CTa X-ray attenuation, EPIC-mu CT X-ray attenuation, sGAG content and collagen content was assessed. Results: CTa X-ray attenuation correlated well with EPIC-mu CT (r = 0.76, 95% credibility interval (95% CI) 0.64 to 0.85). CTa correlated moderately with the DMMB assay (sGAG content) (r = -0.66, 95% CI -0.87 to -0.49) and to lesser extent with the hydroxyproline assay (collagen content) (r = -0.56, 95% CI -0.70 to -0.36). Conclusions: Outcomes of in vivo CTa in human OA knees correlate well with sGAG content. Outcomes of CTa also slightly correlate with cartilage collagen content. Since outcomes of CTa are mainly sGAG dependent and despite the fact that further validation using hyaline cartilage of other joints with different biochemical composition should be conducted, CTa may be suitable as quantitative imaging biomarker to estimate cartilage sGAG content in future clinical OA research. (C) 2016 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

Background Infrapatellar fat pad (IPFP) might be involved in osteoarthritis (OA) by production of cytokines. It was hypothesised that production of cytokines is sensitive to environmental conditions. Objectives To evaluate cytokine production by IPFP in response to interleukin (IL)1b and investigate the ability to modulate this response with an agonist for peroxisome proliferator activated receptor alpha (PPAR alpha), which is also activated by lipid-lowering drugs such as fibrates. Methods Cytokine secretion of IPFP was analysed in the medium of explant cultures of 29 osteoarthritic patients. IPFP (five donors) and synovium (six donors) were cultured with IL-1b and PPAR alpha agonist Wy14643. Gene expression of IL-1b, monocyte chemoattractant protein (MCP1), (IL-6, tumour necrosis factor (TNF)alpha, leptin, vascular endothelial growth factor (VEGF), IL-10, prostaglandin-endoperoxide synthase (PTGS)2 and release of TNF alpha, MCP1 and prostaglandin E-2 were compared with unstimulated IPFP and synovium explants. Results IPFP released large amounts of inflammatory cytokines, adipokines and growth factors. IL-1b increased gene expression of PTGS2, TNF alpha, IL-1b, IL-6 and VEGF and increased TNF alpha release in IPFP. MCP1, leptin, IL-10 gene expression and MCP1, leptin and PGE(2) release did not increase significantly. Synovium responded to IL-1b similarly to IPFP, except for VEGF gene expression. Wy14643 decreased gene expression of PTGS2, IL-1b, TNF alpha, MCP1, VEGF and leptin in IPFP explants and IL-1b, TNF alpha, IL-6, IL-10 and VEGF in synovium that responded to IL-1b. Conclusion IPFP is an active tissue within the joint. IPFP cytokine production is increased by IL-1b and decreased by a PPAR alpha agonist. The effects were similar to effects seen in synovium. Fibrates may represent a potential disease-modifying drug for OA by modulating inflammatory properties of IPFP and synovium.

Objective Obesity is associated with systemic inflammation and is a risk factor for osteoarthritis (OA) development. We undertook this study to test the hypothesis that metabolic stress–induced inflammation, and not mechanical overload, is responsible for the development of high-fat diet–induced OA in mice. Methods Human C-reactive protein (CRP)–transgenic mice received a high-fat diet without or with 0.005% (weight/weight) rosuvastatin or 0.018% (w/w) rosiglitazone, 2 different drugs with antiinflammatory properties. Mice fed chow were included as controls. After 42 weeks, mice were killed and histologic OA grading of the knees was performed. To monitor the overall inflammation state, systemic human CRP levels were determined. Results Male mice on a high-fat diet had significantly higher OA grades than mice on chow and showed no correlation between OA severity and body weight. In male mice, high-fat diet–induced OA was significantly inhibited by rosuvastatin or rosiglitazone to OA grades observed in control mice. Both treatments resulted in reduced human CRP levels. Furthermore, a positive correlation was found between the relative individual induction of human CRP evoked by a high-fat diet on day 3 and OA grade at end point. Conclusion High-fat diet–induced OA in mice is due to low-grade inflammation and not to mechanical overload, since no relationship between body weight and OA grade was observed. Moreover, the OA process was inhibited to a great extent by treatment with 2 drugs with antiinflammatory properties. The inflammatory response to a metabolic high-fat challenge may predict individual susceptibility to developing OA later in life. The use of statins or peroxisome proliferator–activated receptor γ agonists (e.g., rosiglitazone) could be a strategy for interfering with the progression of OA.

Background and objectives Obesity is a risk factor for the development of osteoarthritis (OA) in hands and knees. It has become apparent during the last years that adipose tissue can secrete different adipokines with powerful immunomodulatory effects. Because the infrapatellar fat pad (IFP) is an intra-articular organ in the vicinity of synovium and cartilage, the authors hypothesised that IFP-derived soluble factors could contribute to pathological processes in the knee joint. Therefore the authors have extensively compared the release of inflammatory mediators and characterised the adipocytes and immune cell infiltrate in IFP and Sc adipose tissue (ScAT). Materials and methods Paired IFP and ScAT samples were obtained from 27 primary OA patients. The stromal vascular cell fraction (SVF) was isolated and characterised by FACS. Cytokine and adipokine release in fat- and adipocyte-conditioned media was measured by luminex. Results IFP secreted higher levels of inflammatory mediators, like IL-6, adipsin, adiponectin and visfatin than ScAT. This could be due to differences in the phenotype of adipocytes or/and in composition and phenotype of the SVF cells. Indeed, a similar trend was seen for IL-6 and adipsin when adipocyte-conditioned media from IFP and ScAT were compared. Moreover, the SVF fraction of IFP contained more cells per gram tissue, a lower percentage of T cells and a higher percentage of mast cells than ScAT. In addition, IFP-derived T cells displayed a predominantly proinflammatory phenotype, while macrophages in IFP presented a mixed pro- and anti-inflammatory phenotype. Finally, tumour necrosis factor-α release by IFP was correlated with BMI, indicating BMI-related inflammatory changes in IFP. Conclusions The authors show profound differences in secreted inflammatory factors and immune cell composition between IFP and ScAT. These data indicate that IFP is qualitatively different from ScAT and IFP-derived soluble mediators could contribute to pathophysiological processes in the OA knee joint.

Objectives: Age-related changes in articular cartilage are likely to play a role in the aetiology of osteoarthritis (OA). One of the major age-related changes in cartilage is the accumulation of advanced-glycation-endproducts (AGEs). Since, cartilage tissue is not readily available from patients for studying AGE levels, alternative approaches such as analyzing skin and urine are needed to study the role of cartilage AGE levels in OA. Methods: Paired human skin and cartilage samples were obtained post mortem. Paired skin and urine samples were obtained from the CHECK cohort (early OA patients). Pentosidine levels were measured by high-performance liquid chromatography (HPLC). As marker of cumulative cartilage damage X-rays of both knees and hips were scored. Urinary CTXII (uCTXII) levels were measured, to assess current cartilage breakdown. Results: Cartilage and skin pentosidine correlate well (R= 0.473, P= 0.05). Skin pentosidine was higher in mild (summed (Kellgren & Lawrence K&L) over four large joints ≥4) compared to no (summed K&L≤3) radiographic OA (P= 0.007). Urinary pentosidine was not different between these two groups. Skin pentosidine levels were not related to cartilage breakdown (highest vs lowest tertile of uCTXII). Urinary pentosidine, however, was higher in the highest compared to the lowest uCTXII tertile (P= 0.009). Multiple regression analysis showed age to be the only predictor of the summed K&L score and age, creatinine clearance and urinary pentosidine as predictors of uCTXII. Conclusion: The higher skin and urinary pentosidine levels in those with mild compared to no radiographic joint damage and low vs high cartilage breakdown respectively suggest that AGEs may contribute to disease susceptibility and/or progression. However, relations are weak and cannot be used as surrogate markers of severity of OA. © 2010 Osteoarthritis Research Society International.

Fibrosis is characterized by an excessive accumulation of collagen which contains increased levels of pyridinoline cross-links. The occurrence of pyridinolines in the matrix is an important criterion in assessing the irreversibility of fibrosis, which suggests that collagen containing pyridinoline cross-links significantly contributes to the unwanted collagen accumulation. Pyridinoline cross-links are derived from hydroxylated lysine residues located within the collagen telopeptides (hydroxyallysine pathway). Here, we have investigated whether the increase in hydroxyallysine-derived cross-links in fibrotic conditions can be ascribed to an increased expression of one of the lysyl hydroxylases (LH1, LH2 with its splice variants LH2a and LH2b, or LH3) and/or to an increased expression of lysyl oxidase (LOX). In fibroblast cultures of hypertrophic scars, keloid and palmar fascia of Dupuytren's patients, as well as in activated hepatic stellate cells, increased levels of LH2b mRNA expression were observed. Only minor amounts of LH2a were present. In addition, no consistent increase in the mRNA expression levels of LH1, LH3 and LOX could be detected, suggesting that LH2b is responsible for the overhydroxylation of the collagen telopeptides and the concomitant formation of pyridinolines as found in the collagen matrix deposited in long-term cultures by the same fibrotic cells. This is consistent with our previous observation that LH2b is a telopeptide lysyl hydroxylase. We conclude that the increased expression of LH2b, leading to the increased formation of pyridinoline cross-links, is present in a wide variety of fibrotic disorders and thus represents a general fibrotic phenomenon.

Phage exclusion is a form of programmed cell death in prokaryotes in which death is triggered by infection with phage, a seemingly altruistic response that limits multiplication of the phage and its spread through the population. One of the best-characterized examples of phage exclusion is the exclusion of T-even phages such as T4 by the e14-encoded Lit protein in many Escherichia coli K-12 strains. In this exclusion system, transcription and translation of a short region of the major head coat protein gene late in phage infection activates proteolysis of translation elongation factor Tu (EF-Tu), blocking translation and multiplication of the phage. The cleavage occurs between Gly-59 and Ile-60 in the nucleotide-binding domain. In the present work, we show that a 29-residue synthetic peptide spanning the activating region of the major head coat protein can activate the cleavage of GDP-bound EF-Tu in a purified system containing only purified EF-Tu and purified Lit protein. Lit behaves as a bona fide enzyme in this system, cleaving EF-Tu to completion when present at substoichiometric amounts. Two mutant peptides with amino acid changes that reduce the activation of cleavage of EF-Tu in vivo were also greatly reduced in their ability to activate EF-Tu cleavage in vitro but were still able to activate cleavage at a high concentration. Elongation factor G, which has the same sequence at the cleavage site and a nucleotide-binding domain similar to EF-Tu, was not cleaved by this system, and neither was heat-inactivated EF-Tu, suggesting that the structural context of the cleavage site may be important for specificity. This system apparently represents an activation mechanism for proteolysis that targets one of nature’s most evolutionarily conserved proteins for site-specific cleavage.

Background Aging and degeneration of human intervertebral disc (IVD) are associated with biochemical changes, including racemization and glycation. These changes can only be counteracted by protein turnover. Little is known about the longevity of IVD elastin in health or disease. Yet, such knowledge is important for a quantitative understanding of tissue synthesis and degradation. Methods We have measured the accumulation of d-Asp and pentosidine in IVD elastin. Samples representing a broad range of ages (28–82 years) and degeneration grades (1–5) were analyzed. Results d/l-Asp for elastin increased linearly with age from 3.2% (early 30s) to 14.8% (early 80s) for normal tissue (grades 1–2) and from 1.7% (late 20s) to 6.0% (until the mid 50s) for degenerate tissue (grades 3–5) with accumulation rates of 16.2 ± 3.1 × 10− 4 and 11.7 ± 3.8 × 10− 4 year− 1, respectively; no significant difference was found between these values (p < 0.05). Above the mid 50s, a decrease in d-Asp accumulation was recorded in the degenerate tissue. d-Asp accumulation correlated well with pentosidine content for elastin from healthy and degenerate tissues combined. We conclude that IVD elastin is metabolically‐stable and long‐lived in both healthy and degenerate human IVDs, with signs of new synthesis in the latter. The correlation of d‐Asp with pentosidine content suggests that both these agents may be used as markers in the overall aging process of IVD. General significance Accumulation of modified IVD elastin argues for its longevity and may have a negative impact on its role in disc function. Weak signs of newly synthesized molecules may act to counteract this effect in degenerate tissue.

s / Osteoarthritis and Cartilage 23 (2015) A82eA416 A260 remained unclear whether synovitis is also related to cartilage alterations or other features in patients with end-stage knee OA, where articular cartilage is almost disappeared. In addition, magnetic resonance imaging (MRI) measurements are sensitive to detect of OArelated structural changes. The aim of this study was to investigate the MRI-detected OA-structural changes of the knee joint those were associated with histological synovitis in patients with end-stage knee OA who underwent TKA. Methods: The forty patients (female: 88%, 71.1y on average) who fulfilled the American College of Rheumatology criteria for knee OA and required TKA due to the end-stage knee OA of Kellgren and Lawrence grade 4 were enrolled in this study. All participants were performed with 3.0-Tesla MRI. The OA morphological changes, such as (1) cartilage morphology, (2) bone marrow lesions (BMLs), (3) bone cysts, (4) bone attrition, (5) meniscal pathology, (6) osteophyte, (7) synovitis and (8) ligaments were scored using the whole-organ MRI scoring (WORMS) method. The synovial samples were obtained from five regions of interest of the knee joint at the operation. The sections were stained with hematoxylin and eosin. The histological synovitis scores (HSS) were measured by the methods previously described. Next, the sections were stained with TGF-b, COX-2, IL-1b and IL-6. A semi-quantitative analysis of the immune-histochemically stained sections was conducted. Associations between the HSS and WORMS were examined using Spearman's correlation coefficient. The means of each HSS were compared using the Mann-Whitney U test. Results: Among the eight OA-related morphological changes, the total BML scorewere significantly associated with the total HSS (r1⁄4 0.64, p1⁄4 0.02). BMLs were present in all patients in medial femoro-tibial joint (MFTJ), and were present in lateral femoro-tibial joint (LFTJ) in approximately half (53%) of the patients. The HSS in MFTJ and LFTJ were significantly associated with medial and lateral BML scores [r 1⁄4 0.35, p 1⁄4 0.03 (medial), r 1⁄4 0.49, p < 0.01 (lateral), respectively]. The total HSS in patients with BML in LFTJ were significantly increased compared to those without BML in LFTJ (p < 0.001). The synovial TGF-b expression in the LFTJ in patients with BMLs in the LFTJ was significantly higher than that in patients without BMLs in the LFTJ, while these differences were not observed in COX2, IL-1b or IL-6. Conclusions: The BMLs, but not other structural changes detected by MRI, were associated with the histological synovitis and synovial TGF-b expression in patients with end-stage knee OA. 402 HIGH-FAT DIET FEEDING AFFECTS OSTEOARTHRITIS PROGRESSION IN A SURGICAL MOUSE MODEL WITH ESTABLISHED AND DEVELOPING METABOLIC SYNDROME A.E. Kozijn yz, R. Stoop y, F.P. Lafeber z, H. Weinans zx, A.M. Zuurmond y. y TNO Metabolic Hlth.Res., Leiden, Netherlands; zUniv. Med. Ctr. Utrecht, Utrecht, Netherlands; xDelft Univ. of Technology, Delft, Netherlands Purpose: Metabolic syndrome (MS) is a major risk factor for osteoarthritis (OA) development. The MS-associated low-grade systemic inflammation may underlie this link. We investigated whether a systemic metabolic burden due to high-fat feeding in mice would aggravate OA progression, in both established as well as developing MS. Methods:Metabolic syndromewas induced by providing a high-fat diet (HFD, 45% kcal from fat) to 12-week old male C57Bl/6J mice (n1⁄410) for 10 weeks. Control mice of same sex and age received a synthetic low-fat diet (LFD, 10% kcal from fat) during that time period. At 10 weeks the medial meniscotibial ligament (MMTL) of the right knee joint was transected to destabilize the joint and thereby induce OA. The left knee joint served as an internal control and underwent all the surgical procedures except for the MMTL transection (sham). Half of the control mice were switched to HFD at the time of surgery (LFD-to-HFD), to attain a state of developing metabolic syndrome at end-point. Body weights and changes in body composition were monitored during the entire study period. At endpoint, OA severity was graded on Safranin Ostained histological sections of the knee joints according to OARSI recommendations. Results: Bodyweights of the mice on longand short-term HFD were significantly different at end-point (HFD, 41.1 ± 6.9 g; LFD-to-HFD, 38.8 ± 3.1 g) compared to mice of the control group (LFD, 31.8 ± 2.1 g). Body composition analysis showed that the increase in body weight almost completely resulted from an increase in body fat mass; lean mass remained constant between groups. Severity of OA was greater in the mice fed a HFD, for both the short-term and the long-term regimen. Separate assessment of the longand short-term HFD groups showed a statistically significant difference in cartilage degeneration between the HFD and control groups (Figure 1; one-tailed t-test, p 1⁄4 0.026). This effect was mainly observed at the lateral knee compartments, implying that the observed cartilage degeneration at the lateral knee compartments was predominantly diet-induced (Figure 2). Interestingly, analysis of the lateral knee compartments of the LFD-to-HFD group showed a clear trend towards an increase in OA severity compared to control (one-tailed t-test, p1⁄4 0.064). OA severity did not correlatewith body fat mass at end-point. Conclusions: Our results show that high-fat feeding indeed aggravates OA progression in a surgical mouse model for OA. In this model, the diet-induced OA progression was mainly observed at the lateral knee compartments. No correlation between OA severity and body weight or body fat mass was found, suggesting that the MS-associated inflammation may underlie the diet-induced OA progression. 403 CHONDROGENIC PROGENITOR CELLS PROVIDE A NOVEL INSIGHT TO PHAGOCYTOTIC ACTIVITIES WITHIN CARTILAGE C. Zhou, H. Zheng, J.A. Martin. The Univ. of Iowa, Iowa City, IA, USA Purpose: Chondrogenic progenitor cells (CPCs) have been identified on the cartilage surface post injury, as well as in osteoarthritic cartilage. CPCs serve chondroprotective and regenerative functions, but in initial response to cartilage injury, CPCs overexpress markers associated with dendritic cells. Based on these findings we hypothesized that CPCs carry the potential to clear cell and matrix debris through phagocytosis. To test this we compared phagocytotic capacity of CPCs to chondrocytes. Methods: Scratches on bovine knee cartilage were created to stimulate CPCs migration. After culturing for ten days, CPCs and chondrocytes were isolated and co-cultured with DiO-labeled cell debris for various time periods. Confocal microscopy and flow cytometry analyses were performed to determine phagocytosis events in both cell types. Abstracts / Osteoarthritis and Cartilage 23 (2015) A82eA416 A261s / Osteoarthritis and Cartilage 23 (2015) A82eA416 A261 Results: Confocal microscopy images show CPCs migrating toward acartilage injured (Fig 1A). In CPCs, most DiO-labeled cell debris appeared to be engulfed within the cytoplasm, while in chondrocytes, most of the label appeared to be bound to the cell membrane (Fig 1B). Flow cytometry quantitatively confirmed that DiOþ CPC percentage was significantly higher than chondrocytes at each time point (3hrs, 6hrs, 12hrs and 24hrs) (Fig 2). In addition, DiOþ CPCs increased dramatically (12.58% for 3hrs, 28.63% for 6hrs) and peaked at 12hrs (68.10%), while DiOþ chondrocytes increased slightly (4.18%, 6.66%, 8.08%, and 11.56%, respectively). Conclusions: CPCs showed much higher and sustained uptake of cell debris than chondrocytes. Though not conclusive, these findings support the hypothesis that CPCs play a role in clearing cell debris in damaged cartilage. The physiologic significance of this activity is unclear, but it is conceivable that it neutralizes the pro-inflammatory activity of cell debris, an essential step in wound healing. Figure 1. A) Morphological distinction between CPCs and chondrocytes, CPC (long stretched cells indicated by red arrows) residing on the surface of scratch site. CPCs showed dramatic morphological difference compared to the chondrocytes (rounded cells in most upper and lower portion of the figure) B) Visual comparison between CPCs (upper) and chondrocytes (lower) in terms of phagocytosis under microscopy. Cell debris (stained with green-fluorescent DiO label) was vastly ingested into the cytoplasm of CPC. While for chondrocytes, most cell debris was sticking outside the cell membrane. Flow cytometry quantification assay showed DiOþ cells percentage within CPC population is significantly higher than chondrocytes, along with different rates to phagocytose cell debris for CPCs and chondrocytes (12hr shows highest in CPCs, while gradually increase in chondrocytes). 404 SYNOVITIS AND CHANGES IN THE EXPRESSION OF SYNOVIOCYTES MARKERS CD68 AND CD55, DURING THE PROGRESSION OF OSTEOARTHRITIS IN A RAT MODEL R. Cruz, D. Solis-Garcia, M. Miranda-S anchez, J.B. Kouri. Centro de Investigaci on y de Estudios Avanzados del Inst. Polit ecnico Natl., M exico