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Introduction: The ability to engineer mammalian genomes in a quick and cost-effective way has led to rapid adaptation of CRISPR technology in biomedical research. CRISPR-based engineering has the p...

Obesity is a well-known risk factor for osteoarthritis, but it is unknown what it does on cartilage repair. Here we investigated whether a high fat diet (HFD) influences cartilage repair in a mouse model of cartilage repair. We fed DBA/1 mice control or HFD (60% energy from fat). After 2 weeks, a full thickness cartilage defect was made in the trochlear groove. Mice were sacrificed, 1, 8, and 24 weeks after operation. Cartilage repair was evaluated on histology. Serum glucose, insulin and amyloid A were measured 24 h before operation and at endpoints. Immunohistochemical staining was performed on synovium and adipose tissue to evaluate macrophage infiltration and phenotype. One week after operation, mice on HFD had defect filling with fibroblast-like cells and more cartilage repair as indicated by a lower Pineda score. After 8 weeks, mice on a HFD still had a lower Pineda score. After 24 weeks, no mice had complete cartilage repair and we did not detect a significant difference in cartilage repair between diets. Bodyweight was increased by HFD, whereas serum glucose, amyloid A and insulin were not influenced. Macrophage infiltration and phenotype in adipose tissue and synovium were not influenced by HFD. In contrast to common wisdom, HFD accelerated intrinsic cartilage repair in DBA/1 mice on the short term. Resistance to HFD induced inflammatory and metabolic changes could be associated with accelerated cartilage repair. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

Background In this study we investigated the contribution of high-fat diet-induced metabolic overload to osteoarthritis (OA) progression originally caused by mild mechanical trauma to the mouse knee joint. We hypothesized that metabolic stress would induce a proinflammatory environment by altering systemic lipid levels and immune cell populations. Methods Twelve-week-old male C57BL/6J mice (n=20) were given a low-fat diet (LFD, 10%kcal from fat) or high-fat diet (HFD, 45%kcal from fat) for 18 weeks. OA was initiated by transecting the medial meniscotibial ligament of the right knee joint at t=10 weeks. OA severity and changes in M1/M2 polarization of synovial macrophage populations were determined in serial coronal FFPE-mounted sections. Eicosanoid levels and monocyte populations were evaluated before and after ligament transection. Results Diet-induced metabolic stress assessed by body weight, systemic cholesterol levels, and insulin resistance index was significantly higher in HFD mice. This group also showed increased cartilage damage, synovitis, and osteophyte formation compared with LFD controls. High-fat feeding elevated systemic arachidonic acid levels, which mainly resulted in increased levels of its cytochrome P450-catalysed diol metabolites 5,6-dihydroxyeicosatrienoic acid (DHET) and 8,9-DHET. High-fat feeding also triggered an increase in pro-inflammatory intermediate CD43++Ly6Cint monocytes after ligament resection. Ligament resection in addition to high-fat feeding induced increased expression of the activation marker CD11c selectively on non-classical CD43++Ly6Clow monocytes. No significant changes in synovial macrophage polarization were observed. Conclusions Metabolic stress resulted in a proinflammatory environment and aggravated injury-induced OA progression. Our results suggest that a CYP450-focused eicosanoid metabolism and activated circulating monocytes may be drivers of this metabolic stress-induced OA progression, contributing to the mechanistic understanding and potentially serving as future diagnostic and prognostic biomarkers for metabolic OA.

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.

To investigate the presence of mast cells in the osteoarthritic (OA) synovium and their association with clinical parameters in comparison with rheumatoid arthritis (RA) samples.Synovial tissues of 56 symptomatic OA and 49 RA patients were obtained. Two to three paraffin slides were used to quantify inflammation using haematoxylin and eosin (HE) staining (synovitis score 0-9), and numbers of mast cells (per 10 high-power fields) using double immunofluorescence for CD117 and tryptase. Average scores per patient were used for analysis. Knee radiographs of OA patients were scored according to the Kellgren and Lawrence (KL) system and pain was determined in OA patients at baseline by visual analogue scale (VAS).Median (range) of mast cells was significantly higher in OA samples 45 (1-168) compared to RA samples 4 (1-47) (P-value 0.001), despite a lower median (range) synovitis score in OA (2.5 (0-6.0)) compared to 4.6 (0-8.0) in RA samples. The synovitis score was significantly correlated with the number of mast cells (in OA Spearman's rho (P-value) 0.3 (0.023) and RA 0.5 (P-value 0.001)). Interestingly, we observed a trend towards an association between the number of mast cells and an increased KL-grade (P-value 0.05) in OA patients, independently of synovitis. No associations were found with self-reported pain.Prevalence of mast cells in OA synovial tissue is relatively high and associates with structural damage in OA patients, suggesting a role of mast cells in this disease.

Objective: C-reactive protein (CRP) levels can be elevated in osteoarthritis (OA) patients. In addition to indicating systemic inflammation, it is suggested that CRP itself can play a role in OA development. Obesity and metabolic syndrome are important risk factors for OA and also induce elevated CRP levels. Here we evaluated in a human CRP (hCRP)-transgenic mouse model whether CRP itself contributes to the development of ‘metabolic’ OA. Design: Metabolic OA was induced by feeding 12-week-old hCRP-transgenic males (hCRP-tg, n = 30) and wild-type littermates (n = 15) a 45 kcal% high-fat diet (HFD) for 38 weeks. Cartilage degradation, osteophytes and synovitis were graded on Safranin O-stained histological knee joint sections. Inflammatory status was assessed by plasma lipid profiling, flow cytometric analyses of blood immune cell populations and immunohistochemical staining of synovial macrophage subsets. Results: Male hCRP-tg mice showed aggravated OA severity and increased osteophytosis compared with their wild-type littermates. Both classical and non-classical monocytes showed increased expression of CCR2 and CD86 in hCRP-tg males. HFD-induced effects were evident for nearly all lipids measured and indicated a similar low-grade systemic inflammation for both genotypes. Synovitis scores and synovial macrophage subsets were similar in the two groups. Conclusions: Human CRP expression in a background of HFD-induced metabolic dysfunction resulted in the aggravation of OA through increased cartilage degeneration and osteophytosis. Increased recruitment of classical and non-classical monocytes might be a mechanism of action through which CRP is involved in aggravating this process. These findings suggest interventions selectively directed against CRP activity could ameliorate metabolic OA development.

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.

Objective To determine possible patterns of synovitis on contrast-enhanced magnetic resonance imaging (CE-MRI) and its relation to pain and severity in patients with radiographic knee osteoarthritis (OA). Methods In total, 86 patients (mean age 62 years, 66% women, median body mass index 29 kg/m2) with symptomatic knee OA (Kellgren/Lawrence radiographic score 3) were included. T1-weighted, gadolinium-chelate-enhanced MRI with fat suppression was used to semiquantitatively score the extent of synovitis at 11 knee sites (total score range 0-22). Self-reported pain was assessed with 3 standardized questionnaires. Principal components analysis (PCA) was used to investigate patterns (the location and severity) of synovitis. Subsequently, these patterns were assessed for associations with pain measures and radiographic severity in adjusted logistic regression models. Results Synovitis was observed in 86 patients and was found to be generally mild on CE-MRI (median total synovitis score 7, range 0-16). The median pain scores were 53 (range 0-96) on the visual analog scale for pain, 51.4 (range 2.8-97.2) on the Knee Injury and Osteoarthritis Outcome Score (KOOS) for pain, 35 (range 0-75) on the Intermittent and Constant Osteoarthritis Pain (ICOAP) score for constant pain, and 40.6 (range 0-87.5) on the ICOAP score for intermittent pain. PCA resulted in extraction of 3 components, explaining 53.4% of the variance. Component 1 was characterized by synovitis at 7 sites (mainly medial parapatellar involvement) and was associated with scores on the KOOS pain subscale and the ICOAP constant pain subscale. Component 2 was characterized by synovitis at 4 sites (mainly the site adjacent to the anterior cruciate ligament), but was not associated with pain measures or with radiographic severity. Component 3, characterized by synovitis at 3 sites (mainly at the loose body site), was associated with radiographic severity. Conclusion Different patterns of synovitis in knee OA were observed. The pattern that included several patellar sites was associated with pain, whereas other patterns showed no association, suggesting that pain perception in patients with knee OA is a localized response. Copyright © 2015 by the American College of Rheumatology.

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.

Background Obesity is associated with the development and progression of osteoarthritis (OA). The infrapatellar fat pad (IFP) could contribute to this association due to its localization in the knee joint and secretion of inflammatory mediators. However, little is known about the effects of obesity on the IFP. Therefore, the aim of this study was to investigate the presence of obesity-related features in adipocytes and infiltrating immune cells in the IFP of OA patients. Materials and methods IFP volume was determined by MRI in 79 knee OA patients. IFP and subcutaneous adipose tissue (SCAT) were obtained from 106 knee OA patients (total n=155: 68% women, mean age 65 years, mean (SD) body mass index (BMI) 29.9 kg/m 2 (5.7)) undergoing joint replacement surgery. Crown-like structures (CLS) were determined using immunohistochemistry. Adipocyte size was determined by light microscopy and histology. Stromal vascular fraction (SVF) cells were characterised by flow cytometry. Results IFP volume (mean(SD) 23.6 (5.4) mm 3 ) associated with gender and height, but not with BMI. Likewise, volume and size of IFP adipocytes (mean 271 pl, mean 1933 μm) was not correlated with BMI. Few CLS were observed in IFP and the number did not correlate with BMI. Moreover, high BMI was not associated with higher SVF immune cell numbers in IFP, nor with changes in their phenotype. No molecular differences were observed with BMI, besides an increase in TNFα expression. Extensive characterisation of IFP macrophages revealed that CD206 and CD163, usually associated with an anti-inflammatory phenotype were the most abundantly expressed surface markers on macrophages (81% and 41% respectively), while macrophages produced predominantly IL-6 and TNFα, but little IL-10. Interestingly, surface marker and cytokine expression revealed that CD163 + macrophages had an activated and pro-inflammatory phenotype. Conclusions Obesity-related differences usually observed in SCAT and visceral adipose tissue could not be detected in IFP of OA patients, a fat depot implicated in OA pathogenesis.

New targets for cancer treatment frequently emerge in literature, but the thorough target validation required to consider these targets for a drug discovery program is often lacking. In pharmacological or genetic perturbation studies using complex biological assays, undesired off-target effects cannot be easily distinguished from the intended mode of action at the desired target. This is especially evident in cancer drug development where it is important to discriminate on-target effects on cell viability from off-target effects resulting in non-specific loss of cellular fitness. Neglecting the possibility of being deceived by off-target effects can have tremendous scientific and financial impact on a drug discovery program. Ideally, confidence in a preclinical drug target and a modulating compound is boosted in an early stage by more extensive analysis and validation of the actual drug-target relationship. Rescue of a disease-relevant phenotype by genetic restoration of a target mutation is a gold standard approach in drug discovery by which target validation can be achieved. We aim to follow this approach targeting the BRAF V600E mutation in a number of well described melanoma lines as well as the MAP2K1 Q56P mutation in the non-small cell lung cancer cell line H1437. Target validation for both BRAF and MAP2K1 will be addressed by assessing viability, phenotypic changes and sensitivity to compound modulation upon CRISPR/Cas9 repair of the target mutation or by exogenous re-expression of the wild type variant. Compounds tested will be Vemurafenib for BRAF and Trametinib for MAP2K1. Further investigation into target validity will be done using a physiologically relevant 3D spheroid based co-culture system. Mimicking the tumor microenvironment increases the knowledge about “drug-ability” of a target and sustainability of the target modulation at an early time point in the development process. Such early in-depth validation of the relationship between a compound and the drug target is vital to mitigate the risk of failure at later steps of drug development. Citation Format: Laure Grandmoursel, Lieke Geerts, Geraldine Servant, Miranda van der Ham, Armin Maier, Jamil Aarbiou, Marijn Vlaming, Jeroen DeGroot, Julia Schuler, Ian Waddell, Anne-Marie Zuurmond. Validation of the interaction between a candidate compound and the intended drug target by a phenotypic rescue approach [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C021. doi:10.1158/1535-7163.TARG-19-C021

Targeted therapy of BRCA-deficient cancers has been achieved using poly(ADP-ribose) polymerase (PARP) inhibitors, which block BRCA-independent DNA repair. With first approval in 2014 of Olaparib the concept of tumor-specific synthetic lethality was added to the treatment portfolio of cancer patients. Although the effects of PARPi have shown promising results in multiple cancer types, how and whether patients might benefit from combination with compounds modulating the immune landscape of a tumor is largely unknown. In the current study, we investigate the cross-talk between PARPi and immune checkpoint inhibition, in particular, anti PD-1 and anti CTLA-4, as the most advanced targets in the field. PARP inhibitors Niraparib, Rucaparib, Talazoparib and Olaparib were investigated in vivo using the murine EMT6/BRCA1-/- model in monotherapy as well as in combination with anti-CTLA-4 or anti-PD1 treatment. The four PARPi showed distinct activity profiles in the two breast cancer models. Talazoparib was the most active compound in the BRCA1-/- model (optimal T/C (test/control) of 60%), followed by Niraparib and Rucaparib (65% and 67%, respectively). Olaparib was considered inactive with a T/C value of 80% in monotherapy. The EMT6/BRCA1-/- turned out to be sensitive towards anti CTLA-4 treatment (optimal T/C of 35% - 38% in three independent experiments). Anti PD-1 treatment in monotherapy induced no significant reduction in tumor growth (optimal T/C of 80% - 82%, in two independent experiments). The combination of PARPi and anti CTLA-4 induced a transient but significant reduction of tumor load early in the treatment phase (p< 0.002, one-way ANOVA, day 8). However, anti PD-1 treatment significantly prolonged overall survival in combination with Talazoparib (p< 0.011 log-rank test). The analysis of tumor infiltrating lymphocytes (TILs) by flow cytometry revealed that Talazoparib as monotherapy and in combination with checkpoint (CP) inhibitors enhanced the number of gMDSC as well as the number of CD3/CD11b double positive T cells. Weekly cytokine analysis in the serum of tumor bearing mice will elucidate possible interactions between PARP and CP inhibitors and give guidance to optimal combination and schedules. First results indicate that PARPi as well as CPi induce unique cytokine profiles correlating well with the modified TIL composition of the respective treatment groups. Further mechanistic studies as well as comparative studies with the EMT6/BRCAwt model will elucidate the tumor biology behind these observations and might lead to beneficial combination strategies in patients suffering from triple negative and BRCA1-/- breast cancer. Citation Format: Anya Avrutskaya, Cordula Tschuch, Astrid Jensen, William Durham, Maycee Robinson, Charles Krause, Emily O’Koren, Gerhard Kelter, Anne-Lise Peille, Armin Maier, Anne-Marie Zuurmond, Julia Schüler. Modulation of the tumor-infiltrating lymphocyte population by PARP inhibitor talazoparib in combination with anti-PD1 treatment significantly enhances overall survival in a murine BRCA1-/- breast cancer model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2709.

Objective: Since statins and fibrates are capable of improving the metabolic profile of patients as well as decreasing inflammation, they are considered as potential drugs for preventing osteoarthritis (OA). The goal of the present study was to investigate the effect of these drugs in the STR/Ort spontaneous OA mouse model. Design: Male STR/Ort mice received control diet or control diet containing two different dosages of simvastatin or fenofibrate or a combination of both. Mice were euthanized after 16 weeks of treatment at the age of 24 weeks. Serum analysis for metabolic and inflammatory markers, histologic OA grading and micro computed tomography (IICT) analysis of subchondral bone plate were performed. Results: Simvastatin treatment did not have a statistically significant effect on any of the measured parameters. Fenofibrate treated mice gained less body weight (BW) and had lower serum amyloid A (SAA) levels, but higher Interleukin (1)-1a and MIPla than other mice. Mice treated with 200 mg/kg BW/ day fenofibrate had less subchondral bone plate volume than control, but no statistically significant reduction in cartilage damage. In the combination treatment group, BW and SAA were lower than control. Overall, bodyweight, synovium membrane cell layers and SAA levels correlated to subchondral bone plate changes and subchondral bone plate changes correlated to cartilage damage. Conclusions: Statins and fibrates did not affect development of cartilage damage in the STR/Ort spontaneous OA mouse model. Fenofibrates however, had an effect on BW, serum inflammation markers and subchondral bone plate morphology. 2013 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

Objective: Stiffening of the joint is a feature of knee osteoarthritis (OA) that can be caused by fibrosis of the synovium. The infrapatellar fat pad (IPFP) present in the knee joint produces immune-modulatory and angiogenic factors. The goal of the present study was to investigate whether the IPFP can influence fibrotic processes in synovial fibroblasts, and to determine the role of transforming growth factor β (TGFβ) and prostaglandin F2α (PGF2α ) in these processes. Methods: Batches of fat-conditioned medium (FCM) were made by culturing pieces of IPFP obtained from the knees of 13 patients with OA. Human OA fibroblast-like synoviocytes (FLS) (from passage 3) were cultured in FCM with or without inhibitors of TGFβ/activin receptor-like kinase 5 or PGF2α for 4 days. The FLS were analyzed for production of collagen and expression of the gene for procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 (PLOD2; encoding lysyl hydroxylase 2b, an enzyme involved in collagen crosslinking) as well as the genes encoding α-smooth muscle actin and type I collagen α1 chain. In parallel, proliferation and migration of the synoviocytes were analyzed. Results: Collagen production and PLOD2 gene expression by the FLS were increased 1.8-fold (P < 0.05) and 6.0-fold (P < 0.01), respectively, in the presence of FCM, relative to control cultures without FCM. Moreover, the migration and proliferation of synoviocytes were stimulated by FCM. Collagen production was positively associated with PGF2α levels in the FCM (R = 0.89, P < 0.05), and inhibition of PGF2α levels reduced the extent of FCM-induced collagen production and PLOD2 expression. Inhibition of TGFβ signaling had no effect on the profibrotic changes. Conclusion: These results indicate that the IPFP can contribute to the development of synovial fibrosis in the knee joint by increasing collagen production, PLOD2 expression, cell proliferation, and cell migration. In addition, whereas the findings showed that TGFβ is not involved, the more recently discovered profibrotic factor PGF2α appears to be partially involved in the regulation of profibrotic changes.

Purpose: Osteoarthritis (OA) is associated with obesity in which altered fatty acid levels have been observed. We investigated whether the most common fatty acids in synovial fluid influence cartilage deterioration in OA. Design: Cartilage was obtained from OA patients undergoing total knee arthroplasty. Chondrocytes or cartilage explants were cultured with linoleic (n-6 polyunsaturated), oleic (monounsaturated), or palmitic (saturated) acid. After preculture, media were renewed and inflammation was simulated in half of the samples by addition of 10 ng/mL tumor necrosis factor-α (TNFα) with or without the fatty acids. Effects on lipid uptake (Oil-Red-O), cell toxicity (lactate dehydrogenase), prostaglandin-E2 (PGE2) release and gene expression for prostaglandin-endoperoxide synthase-2 (PTGS2), matrix metalloproteinase-1 (MMP1), and MMP13, and a disintegrin and metalloproteinase with thrombospondin motifs 4 were determined on chondrocytes in monolayer. Effects on glycosaminoglycan (GAG) release were evaluated on cartilage explants. Results: None of the fatty acids were cytotoxic and all were taken up by the cells, resulting in a higher amount of intracellular lipid in chondrocytes. Linoleic acid increased PGE2 production in the presence of TNFα. Oleic acid and palmitic acid inhibited MMP1 gene expression in chondrocytes stimulated with TNFα. In cartilage explants, GAG release was also inhibited by oleic acid and palmitic acid, and oleic acid decreased PTGS2 gene expression in stimulated chondrocytes. Conclusions: Linoleic acid has a pro-inflammatory effect on cartilage whereas oleic acid and palmitic acid seem to inhibit cartilage destruction. These results indicate that altered fatty acid levels may influence loss of cartilage structure in OA. © The Author(s) 2013.Chemicals/CAS: collagenase 3, 175449-82-8; glyceraldehyde 3 phosphate dehydrogenase, 9001-50-7; interstitial collagenase, 9001-12-1; lactate dehydrogenase, 9001-60-9; linoleic acid, 1509-85-9, 2197-37-7, 60-33-3, 822-17-3; oleic acid, 112-80-1, 115-06-0; palmitic acid, 57-10-3; prostaglandin E2, 363-24-6; prostaglandin synthase, 39391-18-9, 59763-19-8, 9055-65-6

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.

Objective: Although osteoarthritis (OA) is considered a non-inflammatory condition, it is widely accepted that synovial inflammation is a feature of OA. However, the role of immune cells and their cytokines in OA is largely unknown. This narrative systematic review summarizes the knowledge of inflammatory properties, immune cells and their cytokines in synovial tissues (STs) of OA patients. Design: Broad literature search in different databases was performed which resulted in 100 articles. Results: Of 100 articles 33 solely investigated inflammation in OA ST with or without comparison with normal samples; the remaining primarily focussed on rheumatoid arthritis (RA) ST. Studies investigating different severity stages or cellular source of cytokines were sparse. OA ST displayed mild/moderate grade inflammation when investigated by means of haematoxylin and eosin (H&E) staining. Most frequently found cells types were macrophages, T cells and mast cells (MCs). Overall the number of cells was lower than in RA, although the number of MCs was as high as or sometimes even higher than in RA ST. Cytokines related to T cell or macrophage function were found in OA ST. Their expression was overall higher than in normal ST, but lower than in RA ST. Their cellular source remains largely unknown in OA ST. Conclusion: Inflammation is common in OA ST and characterized by immune cell infiltration and cytokine secretion. This inflammation seems quantitatively and qualitatively different from inflammation in RA. Further research is needed to clarify the role of inflammation, immune cells and their cytokines in the pathogenesis of OA. © 2012 Osteoarthritis Research Society International.

Objective Adipose tissue is known to release inflammatory cytokines and growth factors. In this exploratory study, the authors examined whether the infrapatellar fat pad (IPFP) closely located to cartilage in the knee joint can affect cartilage metabolism. In addition, the authors analysed whether the macrophage types present in IPFP could explain the effect on cartilage. Methods IPFP explants obtained during total knee replacement of 29 patients with osteoarthritis (OA) were used to make fat-conditioned medium (FCM). Explants of bovine cartilage were cultured with or without FCM. Nitric oxide (NO) and glycosaminoglycan release and gene expression of matrix-degrading enzymes in cartilage were analysed. To stimulate catabolic processes in the cartilage, the authors added interleukin 1β, and the effect of six FCMs was evaluated. The presence of different types of macrophages (CD68+, CD86+ and CD206+) in OA IPFPs was compared with subcutaneous adipose tissue samples and IPFP samples from patients with an anterior cruciate ligament rupture. Results FCM alone reduced NO and glycosaminoglycan release and matrix metalloproteinase (MMP)1 gene expression by the cartilage. Moreover, when catabolic conditions were enhanced with interleukin 1β, FCM inhibited NO production as well as MMP1 and MMP3 gene expression and increased collagen type II gene expression. Significantly more CD206+ cells were present in OA IPFP samples than in subcutaneous fat or anterior cruciate ligament IPFP samples. Conclusion In contrast to the authors' expectations, medium conditioned by end-stage OA IPFP inhibited catabolic processes in cartilage. CD206+ cells present in the IPFPs used for making the FCM might have contributed to the inhibition of catabolic processes in the cartilage.

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.

Pharmacokinetic properties and safety profile of a drug are likely influenced by the disease state of a patient. In this study, we investigated the influence of arthritic processes on pharmacokinetics and immunotoxicity of interleukin-1 receptor antagonist (Anakinra) in the rat adjuvant arthritis model. Anakinra dose-dependently suppressed joint inflammation and degradation as demonstrated by reduced clinical arthritis score, paw thickness, synovial infiltration and bone degradation. In addition, plasma levels of chemokines MCP-1 and GRO/KC were reduced. Pharmacokinetic behaviour of Anakinra was influenced by disease state of the rats as judged from a decrease in Cmax and an increase of the MRT as the disease progressed at a dose of 24 and 72mgAnakinra/kgbody weight. The pharmacokinetic parameters increased dose-dependently, but non-proportionally with increasing dose. Low level anti-Anakinra antibody formation was observed at prolonged exposure to the biologic. Safety parameters, including haematology, splenic lymphocyte subset analysis, ex vivo stimulation of spleen cells and histopathology of immune system organs were affected by the disease itself to such extent that no additional effects of Anakinra could be observed. In conclusion, we demonstrated that pharmacokinetic behaviour of Anakinra was influenced by the arthritis background of the rats resulting in decreased internal exposure. © 2011 Elsevier Inc.

In studies aimed at local treatment of experimental osteoarthritis (OA) it is optimal to have an internal (untreated) OA control. Such an approach excludes interanimal variation, and allows paired statistical evaluation of treatment efficacy. For this purpose, we developed and characterized a bilateral version of the canine Groove model. We hypothesized that the bilateral version of the canine Groove model would show consistent and clear development of features of OA similar to those found in the unilateral version. In six Beagle dogs, grooves were surgically made in the articular cartilage of the femoral condyles of both knee joints. Six additional dogs underwent bilateral sham surgery. The degree of OA was quantified 20 weeks after surgery and was compared in retrospect to 23 animals that undergone the same procedure in a single knee joint with the contralateral knee serving as a non-OA control. Bilateral groove surgery resulted in OA. This was based on the observed ineffective repair response in which an increase in proteoglycan synthesis, a diminished retention of these newly formed proteoglycans, and an enhanced loss of resident proteoglycans resulted in a decreased cartilage proteoglycan content. These biochemical effects were corroborated by clear histological features of OA. All these effects were found in femor as well as in the (surgically untouched) tibia. Interestingly, features of OA were slightly more severe in the bilateral model than in the unilateral variant. The bilateral canine Groove model showed consistent and clear development of features of OA, comparable to the unilateral model. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

Transforming growth factor beta (TGF beta) is often used in cartilage tissue engineering to increase matrix formation by cells with various phenotypes. However, adverse effects of TGF beta, such as extensive cross-linking in cultured fibroblasts, have also been reported. Our goal was to study effects of TGF beta on collagen cross-linking and evaluating the role of cellular phenotype and physical environment. We therefore used four different cell populations in two very different physical environments: primary and expanded chondrocytes and fibroblasts embedded in alginate gel and attached to tissue culture plastic. Matrix production, collagen cross-linking, and alpha-smooth muscle actin (alpha SMA) were analyzed during 4 weeks with or without 2.5 ng/mL TGF beta 2. TGF beta 2 did not affect collagen deposition by primary cells. In expanded cells, TGF beta 2 increased collagen deposition. Chondrocytes and fibroblasts in monolayer produced more collagen cross-links with TGF beta 2. In alginate, primary and expanded cells displayed an unexpected decrease in collagen cross-linking with TGF beta 2. aSMA was not present in alginate cultures and barelay upregulated by TGF beta 2. Organized alpha SMA fibers were present in all monolayer cultures and became more pronounced with TGF beta 2. This study demonstrates that the physical environment determined by the substrate used co-determines the response of cells to TGF beta. The presence of mechanical stress, determined with alpha SMA-staining, is probably responsible for the increase in collagen cross-linking upon addition of TGF beta.

Obesity is a well-known risk factor for osteoarthritis, but it is unknown what it does on cartilage repair. Here we investigated whether a high fat diet (HFD) influences cartilage repair in a mouse model of cartilage repair. We fed DBA/1 mice control or HFD (60% energy from fat). After 2 weeks, a full thickness cartilage defect was made in the trochlear groove. Mice were sacrificed, 1, 8, and 24 weeks after operation. Cartilage repair was evaluated on histology. Serum glucose, insulin and amyloid A were measured 24 h before operation and at endpoints. Immunohistochemical staining was performed on synovium and adipose tissue to evaluate macrophage infiltration and phenotype. One week after operation, mice on HFD had defect filling with fibroblast-like cells and more cartilage repair as indicated by a lower Pineda score. After 8 weeks, mice on a HFD still had a lower Pineda score. After 24 weeks, no mice had complete cartilage repair and we did not detect a significant difference in cartilage repair between diets. Bodyweight was increased by HFD, whereas serum glucose, amyloid A and insulin were not influenced. Macrophage infiltration and phenotype in adipose tissue and synovium were not influenced by HFD. In contrast to common wisdom, HFD accelerated intrinsic cartilage repair in DBA/1 mice on the short term. Resistance to HFD induced inflammatory and metabolic changes could be associated with accelerated cartilage repair. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:1258-1264, 2017.

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.

The hallmark of fibrosis is the excessive accumulation of collagen. The deposited collagen contains increased pyridinoline cross-link levels due to an overhydroxylation of lysine residues within the collagen telopeptides. Lysyl hydroxylase 2b (LH2b) is the only lysyl hydroxylase consistently up-regulated in several forms of fibrosis, suggesting that an enhanced LH2b level is responsible for the overhydroxylation of collagen telopeptides. The present paper reports the effect of profibrotic cytokines on the expression of collagen, lysyl hydroxylases and lysyl oxidase in normal human skin fibroblasts, as well as the effect on pyridinoline formation in the deposited matrix. All three isoforms of TGF-β induce a substantial increase in LH2b mRNA levels, also when expressed relatively to the mRNA levels of collagen type I α2 (COL1A2). The TGF-β isoforms also clearly influence the collagen cross-linking pathway, since higher levels of pyridinoline cross-links were measured. Similar stimulatory effects on LH2b/COL1A2 mRNA expression and pyridinoline formation were observed for IL-4, activin A, and TNF-α. An exception was BMP-2, which has no effect on LH2b/COL1A2 mRNA levels nor on pyridinoline formation. Our data show for the first time that two processes, i.e., up-regulation of LH2b mRNA levels and increased formation of pyridinoline cross-links, previously recognized to be inherent to fibrotic processes, are induced by various profibrotic cytokines. © 2004 Elsevier B.V. All rights reserved. Chemicals / CAS: activin A, 104625-48-1; collagen, 9007-34-5; procollagen lysine 2 oxoglutarate 5 dioxygenase, 9059-25-0; protein lysine 6 oxidase, 99676-44-5; activin A; Activins, 104625-48-1; Amino Acids; Collagen Type I; Cross-Linking Reagents; Cytokines; Inhibin-beta Subunits, 93443-12-0; Interleukin-4, 207137-56-2; PLOD2 protein, human, EC 1.14.11.4; Procollagen-Lysine, 2-Oxoglutarate 5-Dioxygenase, EC 1.14.11.4; Protein Isoforms; pyridinoline, 63800-01-1; RNA, Messenger; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Fibrotic processes in humans are characterised by an excessive accumulation of collagen containing increased levels of hydroxyallysine-derived cross-links. The occurrence of these cross-links appears to be an important criterion in assessing the irreversibility of fibrosis. We hypothesise that increased hydroxyallysine cross-linking results in a collagenous matrix that is less susceptible to proteolytic degradation and therefore the collagen deposition is no longer reversible. In this report, we show that collagen matrices with increased hydroxyallysine cross-link levels were less susceptible to matrix metalloproteinase 1 degradation than are collagen matrices containing low hydroxyallysine levels. These data indicate that the type of collagen cross-link influences collagen catabolism. In vivo evidence for the importance of the cross-linking type in determining the reversibility of the fibrotic process was found using the bleomycin-induced skin fibrosis mouse model. The analysis of the accumulated collagen in the fibrotic skin of bleomycin-treated mice did not reveal an increase in hydroxyallysine cross-link levels. In concurrence with our hypothesis, the collagen accumulation resolved in time when the mice were no longer receiving bleomycin treatment, showing the reversibility of the fibrosis. In conclusion, our data indicate that the type of collagen cross-linking is an important factor in determining whether the outcome of the fibrotic process is reversible or not.

The hallmark of fibrotic processes is an excessive accumulation of collagen. The deposited collagen shows an increase in pyridinoline cross-links, which are derived from hydroxylated lysine residues within the telopeptides. This change in cross-linking is related to irreversible accumulation of collagen in fibrotic tissues. The increase in pyridinoline cross-links is likely to be the result of increased activity of the enzyme responsible for the hydroxylation of the telopeptides (telopeptide lysyl hydroxylase, or TLH). Although the existence of TLH has been postulated, the gene encoding TLH has not been identified. By analyzing the genetic defect of Bruck syndrome, which is characterized by a pyridinoline deficiency in bone collagen, we found two missense mutations in exon 17 of PLOD2, thereby identifying PLOD2 as a putative TLH gene. Subsequently, we investigated fibroblasts derived from fibrotic skin of systemic sclerosis (SSc) patients and found that PLOD2 mRNA is highly increased indeed. Furthermore, increased pyridinoline cross-link levels were found in the matrix deposited by SSc fibroblasts, demonstrating a clear link between mRNA levels of the putative TLH gene (PLOD2) and the hydroxylation of lysine residues within the telopeptides. These data underscore the significance of PLOD2 in fibrotic processes.

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.

For clarification of the action of a new antibiotic, the analysis of resistant mutants is often indispensable. For enacyloxin IIa we discovered four resistant elongation factor Tu (EF-Tu) species in Escherichia coli with the mutations Q124K, G316D, Q329H, and A375T, respectively. They revealed that enacyloxin IIa sensitivity is dominant in a mixed population of resistant and wild-type EF-Tus. This points to an inhibition mechanism in which EF-Tu is the dominant target of enacyloxin IIa and in which a ribosome with a sensitive EF-Tu blocks mRNA translation for upstream ribosomes with resistant EF-Tus, a mechanism similar to that of the unrelated antibiotic kirromycin. Remarkably, the same mutations are also linked to kirromycin resistance, though the order of their levels of resistance is different from that for enacyloxin IIa. Among the mutant EF-Tus, three different resistance mechanisms can be distinguished: (i) by obstructing enacyloxin IIa binding to EF-Tu. GTP; (ii) by enabling the release of enacyloxin IIa after GTP hydrolysis; and (iii) by reducing the affinity of EF-Tu.GDP. enacyloxin IIa for aminoacyl-tRNA at the ribosomal A-site, which then allows the release of EF-Tu.GDP.enacyloxin IIa. Ala375 seems to contribute directly to enacyloxin IIa binding at the domain 1-3 interface of EF-Tu.GTP, a location that would easily explain the pleiotropic effects of enacyloxin IIa on the functioning of EF-Tu.

A method of lambda-mediated gene replacement was used to disrupt tufA or tufB on the chromosome of the E. coli K-12 strain MG1655. Both tuf genes, which are almost identical but map in different chromosomal contexts, encode the essential peptide chain elongation factor EF-Tu, one of the most abundant cytoplasmic proteins. Southern analysis confirmed replacement of the chromosomal tufA or tufB gene by a chloramphenicol resistance marker, demonstrating that both tuf genes are individually dispensable for growth. Under conditions of rapid growth, deletion of tufB had no significant effect on growth rate, but deletion of tufA resulted in a 35% increase in generation time. In minimal medium we observed no negative effects of tufA deletion on growth rate. Strains with a single tuf gene are useful for the expression of mutant forms of EF-Tu as the sole species in cells; this was demonstrated by introducing the hybrid tufAhis gene, encoding EF-TuA extended with a C-terminal (His)6 tag, into the chromosome of a strain lacking tufB.

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 The infrapatellar fat pad (IPFP) in the knee joint is hypothesized to contribute to osteoarthritis (OA) development by the IFPF possibly by influencing inflammatory processes. Oxylipins are essential mediators in the inflammatory process. We undertook this study to investigate secretion by the IFPF of fatty acids and oxylipins derived from those fatty acids. Methods IPFP explants from 13 OA donors undergoing joint replacement surgery and from 10 normal donors postmortem were cultured for 24 hours, and supernatants (fat-conditioned medium [FCM]) were collected. Liquid chromatography tandem mass spectrometry detected fatty acids and oxylipins in FCM samples. Univariate and multivariate (partial least-squares discriminant analysis [PLS-DA]) analyses were performed, followed by pathway analysis. To validate these outcomes, a second set of OA FCM samples was measured (n = 23). Results Twenty-nine oxylipins and fatty acids could be detected in FCM. Univariate analysis showed no differences between normal donor and OA donor FCM; however, PLS-DA revealed an oxylipin/fatty acid profile consisting of 14 mediators associated with OA (accuracy rate 72%). The most important contributors to the model were lipoxin A4 (decreased), thromboxane B2 (increased), and arachidonic acid (increased). The statistical model predicted 64% of the second set of OA FCM samples correctly. Pathway analysis indicated differences in individual mediators rather than in complete pathways. Conclusion The IPFP secretes multiple and different oxylipins, and a subset of these oxylipins provides a distinctive profile for OA donors. It is likely that the observed changes are regulated by the OA process rather than being a consequence of basal metabolism changes, as an increase in fatty acid levels was not necessarily associated with an increase in oxylipins derived from that fatty acid. Copyright © 2013 by the American College of Rheumatology.

Objective Hypercholesterolaemia, a risk factor for atherosclerosis (ATH), has been suggested to have a role in the development of osteoarthritis (OA). To test this hypothesis, the effect of cholesterol and different cholesterol-lowering treatments on OA was investigated in a mouse model resembling human lipoprotein metabolism. Methods Female ApolipoproteinE*3Leiden.human Cholesteryl Ester Transfer Protein mice received a westerntype diet with 0.1% (w/w) cholesterol (LC), 0.3% (w/w) cholesterol alone (HC) or treated with 3 mg/kg/day atorvastatin or 0.3 mg/kg/day ezetimibe. One group remained on chow (control). After 39 weeks, OA grades of the knees and the extent of ATH were determined. Plasma cholesterol levels were measured throughout the study. Results LC and HC groups developed significantly more OA at the medial side than the control group in a dosedependent manner. Atorvastatin but not ezetimibe treatment significantly suppressed OA development. As expected, features of ATH were significantly increased in the LC and HC groups compared with the control group and suppressed by atorvastatin (48%) and ezetimibe (55%) treatment. There were significant correlations between the development of OA on the medial side of the joint and cholesterol exposure (r=0.4) or ATH features (r=0.3). Conclusions Dietary cholesterol and accordingly increased plasma levels play a role in the development of OA. The correlation found between OA, cholesterol and ATH demonstrates that these variables are connected, but indicates the contribution of other ongoing processes in the development of OA. The suppressive effect on OA development of atorvastatin but not of ezetimibe, which had similar cholesterol exposure levels, corroborates these findings.

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.

Stiffening of the joint is a feature of knee osteoarthritis (OA) that can be caused by fibrosis of the synovium. The infrapatellar fat pad (IPFP) present in the knee joint produces immune-modulatory and angiogenic factors. The goal of the present study was to investigate whether the IPFP can influence fibrotic processes in synovial fibroblasts, and to determine the role of transforming growth factor β (TGFβ) and prostaglandin F2α (PGF2α ) in these processes.Batches of fat-conditioned medium (FCM) were made by culturing pieces of IPFP obtained from the knees of 13 patients with OA. Human OA fibroblast-like synoviocytes (FLS) (from passage 3) were cultured in FCM with or without inhibitors of TGFβ/activin receptor-like kinase 5 or PGF2α for 4 days. The FLS were analyzed for production of collagen and expression of the gene for procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 (PLOD2; encoding lysyl hydroxylase 2b, an enzyme involved in collagen crosslinking) as well as the genes encoding α-smooth muscle actin and type I collagen α1 chain. In parallel, proliferation and migration of the synoviocytes were analyzed.Collagen production and PLOD2 gene expression by the FLS were increased 1.8-fold (P0.05) and 6.0-fold (P0.01), respectively, in the presence of FCM, relative to control cultures without FCM. Moreover, the migration and proliferation of synoviocytes were stimulated by FCM. Collagen production was positively associated with PGF2α levels in the FCM (R = 0.89, P0.05), and inhibition of PGF2α levels reduced the extent of FCM-induced collagen production and PLOD2 expression. Inhibition of TGFβ signaling had no effect on the profibrotic changes.These results indicate that the IPFP can contribute to the development of synovial fibrosis in the knee joint by increasing collagen production, PLOD2 expression, cell proliferation, and cell migration. In addition, whereas the findings showed that TGFβ is not involved, the more recently discovered profibrotic factor PGF2α appears to be partially involved in the regulation of profibrotic changes.