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Wnt-1 inducible signaling pathway protein 2 (WISP-2/CCN5) is a recently identified adipokine that has been described as an important mediator of canonical Wnt activation in adipogenic precursor cells. In osteoarthritis (OA), the most common form of arthritis, chondrocytes exhibit aberrant and increased production of pro-inflammatory mediators and matrix degrading enzymes such as IL-1β and MMP-13. Although recent evidence suggests a role for Wnt signaling in OA physiopathology, little is known about the involvement of WISP-2 in cartilage degradation. In the present study, we determined the expression of WISP-2 in healthy and OA human chondrocytes. WISP-2 expression is modulated along chondrocyte differentiation and downregulated at the onset of hypertrophy by inflammatory mediators. We also investigated the effect of WISP-2 on cartilage catabolism and performed WISP-2 loss-of-function experiments using RNA interference technology in human T/C-28a2 immortalized chondrocytes. We demonstrated that recombinant human WISP-2 protein reduced IL-1β-mediated chondrocyte catabolism, that IL-1β and WNT/b-catenin signaling pathways are involved in rhWISP-2 protein and IL-1β effects in human chondrocytes, and that WISP-2 has a regulatory role in attenuating the catabolic effects of IL-1β in chondrocytes. Gene silencing of WISP-2 increased the induction of the catabolic markers MMP-13 and ADAMTS-5 and the inflammatory mediators IL-6 and IL-8 triggered by IL-1β in human primary OA chondrocytes in a Wnt/β-catenin dependent manner. In conclusion, here we have shown for the first time that WISP-2 may have relevant roles in modulating the turnover of extracellular matrix in the cartilage and that its downregulation may detrimentally alter the inflammatory environment in OA cartilage. We also proved the participation of Wnt/β-catenin signaling pathway in these processes. Thus, targeting WISP-2 might represent a potential therapeutical approach for degenerative and/or inflammatory diseases of musculoskeletal system, such as osteoarthritis.

Despite the benefits of current therapeutic options for treating inflammatory bowel disease (IBD), there are still patients who are refractory to these therapies. Moreover, the relapses caused by incomplete intestinal mucosa healing are frequent. Therefore, there is a need for novel pharmacological targets that can improve the existing IBD therapeutic armamentarium. Chemokine and chemokine receptors have emerged as appealing options to this end. As well as controlling leukocyte trafficking to inflamed tissues, these proteins regulate many other processes related to the development of intestinal inflammation. In this review, we summarise the most recent preclinical studies, along with the putative application of chemokine-based therapies in patients with IBD.

C-reactive protein (CRP) is an acute-phase protein that is used as an established biomarker to follow disease severity and progression in a plethora of inflammatory diseases. However, its pathophysiologic mechanisms of action are still poorly defined and remain elusive. CRP, in its pentameric form, exhibits weak anti-inflammatory activity. On the contrary, the monomeric isoform (mCRP) exhibits potent pro-inflammatory properties in endothelial cells, leukocytes, and platelets. So far, no data exists regarding mCRP effects in human or mouse chondrocytes. This work aimed to verify the pathophysiological relevance of mCRP in the etiology and/or progression of osteoarthritis (OA). We investigated the effects of mCRP in cultured human primary chondrocytes and in the chondrogenic ATDC5 mouse cell line. We determined mRNA and protein levels of relevant factors involved in inflammatory responses and the modulation of nitric oxide synthase type II (NOS2), an early inflammatory molecular target. We demonstrate, for the first time, that monomeric C reactive protein increases NOS2, COX2, MMP13, VCAM1, IL-6, IL-8, and LCN2 expression in human and murine chondrocytes. We also demonstrated that NF-kB is a key factor in the intracellular signaling of mCRP-driven induction of pro-inflammatory and catabolic mediators in chondrocytes. We concluded that mCRP exerts a sustained catabolic effect on human and murine chondrocytes, increasing the expression of inflammatory mediators and proteolytic enzymes, which can promote extracellular matrix (ECM) breakdown in healthy and OA cartilage. In addition, our results implicate the NF-kB signaling pathway in catabolic effects mediated by mCRP.

Injectable and biocompatible novel hybrid hydrogels based on physically crosslinked natural biopolymers and green graphene for potential use in tissue engineering are reported. Kappa and iota carrageenan, locust bean gum and gelatin are used as biopolymeric matrix. The effect of green graphene content on the swelling behavior, mechanical properties and biocompatibility of the hybrid hydrogels is investigated. The hybrid hydrogels present a porous network with three-dimensionally interconnected microstructures, with lower pore size than that of the hydrogel without graphene. The addition of graphene into the biopolymeric network improves the stability and the mechanical properties of the hydrogels in phosphate buffer saline solution at 37 °C without noticeable change in the injectability. The mechanical properties of the hybrid hydrogels were enhanced by varying the dosage of graphene between 0.025 and 0.075 w/v%. In this range, the hybrid hydrogels preserve their integrity during mechanical test and recover the initial shape after removing the applied stress. Meanwhile, hybrid hydrogels with graphene content of up to 0.05 w/v% exhibit good biocompatibility for 3T3-L1 fibroblasts; the cells proliferate inside the gel structure and show higher spreading after 48 h. These injectable hybrid hydrogels with graphene have promising future as materials for tissue repair., The research leading to these results received funding from the Xunta de Galicia Government: program of consolidation and structuring competitive research units [grant number: ED431C 2019/17]. Y.F. is a ‘Sara Borrell’ researcher funded by Instituto de Salud Carlos III (ISCIII) and co-funded by Fondo Europeo de Desarrollo Regional (FEDER) [CD21/00042]. Thanks to Corfo 22CVID-206836, CIPA, ANID Regional, GORE BIO BIO, R17A10003, ACE210016, ACE210012. Funding for open access charge was provided by Universidade da Coruna/CISUG

Objective To investigate the human leukocyte antigen (HLA) association with anti-synthetase syndrome (ASSD). Methods We conducted the largest immunogenetic HLA-DRB1 and HLA-B study to date in a homogeneous cohort of 168 Caucasian patients with ASSD and 486 ethnically matched healthy controls by sequencing-based-typing. Results A statistically significant increase of HLA-DRB1*03:01 and HLA-B*08:01 alleles in patients with ASSD compared to healthy controls was disclosed (26.2% versus 12.2%, P = 1.56E–09, odds ratio–OR [95% confidence interval–CI] = 2.54 [1.84–3.50] and 21.4% versus 5.5%, P = 18.95E–18, OR [95% CI] = 4.73 [3.18–7.05]; respectively). Additionally, HLA-DRB1*07:01 allele was significantly decreased in patients with ASSD compared to controls (9.2% versus 17.5%, P = 0.0003, OR [95% CI] = 0.48 [0.31–0.72]). Moreover, a statistically significant increase of HLA-DRB1*03:01 allele in anti-Jo-1 positive compared to anti-Jo-1 negative patients with ASSD was observed (31.8% versus 15.5%, P = 0.001, OR [95% CI] = 2.54 [1.39–4.81]). Similar findings were observed when HLA carrier frequencies were assessed. The HLA-DRB1*03:01 association with anti-Jo-1 was unrelated to smoking history. No HLA differences in patients with ASSD stratified according to the presence/absence of the most representative non-anti-Jo-1 anti-synthetase autoantibodies (anti-PL-12 and anti-PL-7), arthritis, myositis or interstitial lung disease were observed. Conclusions Our results support the association of the HLA complex with the susceptibility to ASSD.

Osteoarthritis (OA) is the most common form of joint disease. This presents the OA research community and pharmaceutical companies developing disease-modifying osteoarthritis drugs (DMOADs) with great opportunities.. The different OA subtypes complicates the traditional approaches for developing new treatments. If we can identify new markers that can distinguish different subtypes this can greatly facilitate drug development from early discovery to late clinical development. Nevertheless, the current approaches result in poorly targeted treatments and the inability to recruit the right patients for clinical trials. Thus, there is an urgent medical need for objective biomarker tools for patient phenotyping.

Summary Objective Lipocalin-2 (LCN2) is an adipokine that was first identified in neutrophil granules. In the last years it was recognized as a factor that could impair chondrocyte phenotype, cartilage homeostasis as well as growth plate development. Both pro-inflammatory cytokines and glucocorticoids (GCs) modulate LCN2 expression. Actually, GCs were found to be LCN2 inducers, suggesting that part of the negative actions exerted by these anti-inflammatory drugs at cartilage level could be mediated by this adipokine. So, in this study we wanted to investigate whether corticoids were able to act in synergy with IL-1 in the induction of LCN2 and the signaling pathway involved in this process. Materials and methods For the realization of this work, ATDC5 mouse chondrogenic cell line was used. We determined the mRNA and protein expression of LCN2 by real-time reverse transcription-polymerase chain reaction (RT-qPCR) and western blot respectively, after GC or mineralcorticoid treatment. Different signaling pathways inhibitors were also used. Results GC and mineralcorticoid were able to induce the expression of LCN2 in ATDC5 cells. Interestingly, both corticoids synergized with IL-1 in the induction of LCN2. The effect of these corticoids on the expression of LCN2 occurred through GC or mineralcorticoid receptors and the kinases PI3K, ERK1/2 and JAK2. Conclusions Prolonged use of corticoids may have detrimental effects on cartilage homeostasis. Based on our results, we conclude that corticoids could increase the negative actions exerted by IL-1 by increasing the expression of LCN2.

The EMPA-REG OUTCOME (Empagliflozin, Cardiovascular Outcome Event Trial in patients with Type 2 Diabetes Mellitus (T2DM)) trial made evident the potentiality of pharmacological sodium-glucose cotransporter 2 (SGLT2) inhibition for treating patients with diabetes and cardiovascular disease. Since the effect of empagliflozin or other SGLT2 inhibitors on the whole cardiac metabolic profile was never analysed before, and with the purpose to contribute to elucidate the benefits at cardiac level of the use of empagliflozin, we explored the effect of the treatment with empagliflozin for six weeks on the cardiac metabolomic profile of Zucker diabetic fatty rats, a model of early stage T2DM, using untargeted metabolomics approach. Empagliflozin reduced significantly the cardiac content of sphingolipids (ceramides and sphingomyelins) and glycerophospholipids (major bioactive contributing factors linking insulin resistance to cardiac damage) and decreased the cardiac content of the fatty acid transporter cluster of differentiation 36 (CD36); induced significant decreases of the cardiac levels of essential glycolysis intermediaries 2,3-bisphosphoglycerate and phosphoenolpyruvate, and regulated the abundance of several amino acids of relevance as tricarboxylic acid suppliers and/or in the metabolic control of the cardiac function as glutamic acid, gamma-aminobutyric acid and sarcosine. Empagliflozin treatment activated the cardioprotective master regulator of cellular energyhomeostasis AMP-activatedproteinkinase (AMPK) and enhanced autophagy at cardiac level, while it decreased significantly the cardiac mRNA levels of the pro-inflammatory cytokines interleukin-6 (IL-6), chemerin, TNF-α and MCP-1, reinforcing the hypothesis of a direct role for empagliflozin in attenuating cardiac inflammation. Our results provide an advancement on the knowledge of the mechanisms linking the therapy with empagliflozin with protective effects on the development of cardiometabolic diseases whose course is associated with remarkable cardiac bioenergetics dysregulation and disarrangement in cardiac metabolome and lipidome.

Extra-virgin olive oil (EVOO), a principal component of the Mediterranean diet (Med diet), is one of the most ancient known foods and has long been associated with health benefits. Many phenolic compounds extracted from Olea europea L. have attracted attention since their discovery. Among these phenolic constituents, oleocanthal has recently emerged as a potential therapeutic molecule for different diseases, showing relevant pharmacological properties in various pathogenic processes, including inflammation, cancers and neurodegenerative diseases. Here, we discuss and summarize the most recent pharmacological evidence for the medical relevance of oleocanthal, focusing our attention on its anti-inflammatory and chemotherapeutic roles.

Ghrelin is a gastric peptide, discovered by Kojima et al. (1999) [55] as a result of the search for an endogenous ligand interacting with the “orphan receptor” GHS-R1a (growth hormone secretagogue receptor type 1a). Ghrelin is composed of 28 aminoacids and is produced mostly by specific cells of the stomach, by the hypothalamus and hypophysis, even if its presence, as well as that of its receptors, has been demonstrated in many other tissues, not least in gonads. Ghrelin potently stimulates GH release and participates in the regulation of energy homeostasis, increasing food intake, decreasing energy output and exerting a lipogenetic effect. Furthermore, ghrelin influences the secretion and motility of the gastrointestinal tract, especially of the stomach, and, above all, profoundly affects pancreatic functions. Despite of these previously envisaged activities, it has recently been hypothesized that ghrelin regulates several aspects of reproductive physiology and pathology. In conclusion, ghrelin not only cooperates with other neuroendocrine factors, such as leptin, in the modulation of energy homeostasis, but also has a crucial role in the regulation of the hypothalamic–pituitary gonadal axis. In the current review we summarize the main targets of this gastric peptide, especially focusing on the reproductive system.

Scientific interest in the biology of white adipose tissue (WAT) has increased since the discovery of leptin in 1994. The description of the effects of leptin, the product of the ob gene, has started to clarify the role of adipose tissue in the physiopathology of obesity related diseases and has helped in the identification of a great number of other molecules (named adipocytokines), some of them with a proinflammatory nature. In rheumatic diseases, adipocytokines derived from the WAT are among the most important factors related to obesity and promote inflammatory and/or autoimmune conditions. In this review we will present the most recent advances in adipocytokine research, with special attention to the role of leptin, adiponectin, resistin and visfatin and inflammatory, autoimmune and rheumatic diseases.

Ghrelin, an acylated 28 amino acid gastric peptide, was isolated from the stomach as an endogenous ligand for growth hormone (GH) secretagogue receptor in 1999. Circulating ghrelin is mainly produced by specific cells in the stomach's oxyntic glands. Ghrelin potently stimulates GH release and food intake and exhibits diverse effects, including ones on glucose metabolism and on secretion and motility of the gastrointestinal tract. Besides these effects on food intake and energy homeostasis, ghrelin is also involved in controlling reproductive functions, and a role for it as a novel regulator of the hypothalamic-pituitary gonadal axis is clearly emerging. We review recent ghrelin research with emphasis on its roles in the reproductive axis.

Interest in the biology of white adipose tissue has increased dramatically since the discovery of leptin in 1994. The identification of the product of the gene obese ( ob ) threw light on the role of adipose tissue in the physiopathology of obesity-related diseases and spurred the identification of numerous other adipokines, many of a proinflammatory nature. It has become increasingly evident that white adipose tissue–derived cytokines mediate between obesity-related exogenous factors (nutrition and lifestyle) and the molecular events that lead to metabolic syndrome, inflammation, and cardiovascular diseases. Here we review recent adipokine research, with particular attention to the roles of adiponectin, leptin, resistin, visfatin, apelin, omentin, and chemerin in such conditions.

In a previous study, we established that leptin acts synergistically with interferon-gamma in inducing nitric oxide synthase type II in cultured chondrocytes via Janus kinase-2 activation. However, the exact molecular mechanism that accounts for this synergism is not completely understood. The aim of the present study was to further delineate the signalling pathway used by leptin/interferon-gamma in the nitric oxide synthase type II induction in chondrocytes. Consequently, the roles of PI-3 kinase, MEK1 and p38 kinase were investigated using specific pharmacological inhibitors (Wortmannin, LY 294002, PD 098,059 and SB 203580). For this purpose, the amount of stable nitrite, the end product of NO generation by activated chondrocytes, has been evaluated by Griess colorimetric reaction in culture medium of human primary chondrocytes and in the murine ATDC5 cell line stimulated with leptin (400 nM) and interferon-gamma (1 ng/ml), alone or in combination. Specific inhibitors for PI-3K, MEK1 and p38 were added 1 h before stimulation. Nitric oxide synthase type II mRNA was investigated by real-time RT-PCR and NOS type II protein expression has been evaluated by western blot analysis. Our results showed that, as expected, leptin synergizes with IFN-gamma in inducing NO accumulation in the supernatant of co-stimulated cells. Pre-treatment with Wortmannin, LY 294002, PD 098,059 and SB 203580 caused a significant decrease in nitrite production, NOS type II protein expression and NOS type II mRNA expression induced by leptin and interferon-gamma co-stimulation. These findings were confirmed in 15 and 21-day differentiated ATDC5 cells, and in normal human primary chondrocytes. This is the first report showing that NOS type II induction triggered by co-stimulation with leptin and interferon-gamma is mediated by a signaling pathway involving PI-3K, MEK1 and p38.

Preproghrelin is the polypeptide precursor of ghrelin. First discovered in gastric extract as a growth hormone releasing peptide and food intake modulator, it has more recently been revealed to have other physiological aspects. The fine molecular mechanisms of ghrelin biosynthesis show that this peptide is but one piece of a puzzle which contains many other peptides obtained from alternative splicing of the same gene or from extensive post-translational modifications. Recent developments have shown that pro-ghrelin cleavage generates another active peptide named obestatin with an intriguingly subtle but opposite physiological action to ghrelin. Noteworthy, and similar to ghrelin, which requires post-translational processing close to its amino terminus by acylation, the biological activity of the ghrelin-associated peptide obestatin also depends on modification, but by amidation at its carboxyl terminus. In this review we will summarize the steps which led to the identification of pre-proghrelin gene products and will examine the significance and perspectives of the different peptides generated from the same ancestor gene.

s / Osteoarthritis and Cartilage 22 (2014) S57–S489 S173 used in experiments up to the 2nd passage. Two experimental protocols were used: a membrane water permeability assay and measurement of intracellular calcium using Fura-2AM. Results: Using an established permeability assay, whereby a hypotonic solution is applied to the cells and cell volume recorded, aquaporin permeability was measured. In healthy cells permeability was found to be 31 3x104cm.s (n 1⁄4 5). Stimulated cells did not show a significant change in aquaporin permeability (n 1⁄4 12). Calcium measurements showed that healthy cells responded to the same hypotonic challenge with an intracellular calcium increase of 115 15nM (n 1⁄4 16). This calcium increase was inhibited by the TRP channel antagonist, PYR3 (n 1⁄4 69). When applied to cells from the in vitro model of arthritis the same osmotic challenge caused a significantly greater calcium increase of 328 45nM (n 1⁄4 11; p 0.01). Conclusions: We have investigated changes to two ion channels in healthy chondrocytes and those from an in vitro model of arthritis. Aquaporin function appeared unchanged in our cytokine arthritis model, possibly suggesting that this change in gene expression occurs as a result of OA, rather than contributing to OA development. Previous work has shown that intracellular calcium increases greater than :300nM can lead to cell apoptosis and therefore the changes we observe here could implicate important pathological changes in cytokinestimulated chondrocytes. Further work is necessary to identify the exact mechanism by which this calcium increase occurs. 291 ENVIRONMENTAL POLLUTANTS AND OSTEOARTHRITIS: EFFECTS OF NON-DIOXIN-LIKE POLYCHLORINATED BIPHENYLS ON CULTURED CHONDROCYTES V. Abella yz, M. Scotece y, J. Conde y, V. Lopez y, V. Lazzaro yx, J. Pino y, J.J. Gomez-Reino y, R. Meli k, O. Gualillo y. y SERGAS-IDIS Santiago Univ. Clinical Hosp., Santiago de Compostela, SPAIN; zUniv. of A Coru~ na, A Coru~ na, Spain; xUniversit a della Magna Grecia, Catanzaro, Italy; kUniv. of Naples Federico II, Napoli, Italy a. Purpose: Non-dioxin-like polychlorinated biphenyls (PCBs) are persistent organic pollutants that accumulate in fatty tissues causing immune suppression and endocrine disruption. Several studies suggest that PCBs may be involved in pathogenesis of osteoarthritis (OA). Alterations in the mechanisms of programmed cell death (apoptosis) are strongly related to the degradation of extracellular matrix (ECM) in the cartilage of OA subjects. Identification of apoptosis inducers is of paramount relevance to understand the pathogenesis and/or progression of OA. Thus, the aim of the present study was to assess the effect of several PCBs on chondrocytes viability and apoptosis induction. b. Methods: The murine chondrogenic cell line ATDC5 and human juvenile costal chondrocyte cell line T/C-28a2 were treated with several doses of PCB 101, 153 and 180, alone and in combination. Cell viability was examined using a colorimetric assay based on the MTT labeling reagent. Apoptosis was evaluated by Annexin V flow cytometric assay and by the involvement of apoptotic related proteins, such as caspase-3 and Bcl-2/Bax ratio, using western blot analysis. Finally, to evaluate whether PCBs exert necrotic effect, apart from apoptosis pathways, we have also assessed lactate dehydrogenase (LDH) levels in culture supernatants. c. Results: ATDC5 and T/C-28a2 cell lines treated with PCBs, alone and in combination, showed a significant reduction of cell viability rate in a concentration-dependent manner. Neither synergisms nor additive effects were observed on cell viability with the combined treatment. Data from annexin V assays suggested that PCBs clearly induced apoptotic pathways, as well as, a certain rate of necrosis. Actually, this effect was confirmed by evaluating LDH levels that were strongly increased in supernatants of PCBs-treated cells, suggesting that necrotic mechanisms are at play too. PCBs also induced caspase-3 activation by increasing its proteolytic cleavage in a concentration-dependent manner. Finally, the Bcl2/Bax ratio was also altered. d. Conclusions: The viability of murine and human chondrocytes was reduced in presence of PCBs. The activity of PCBs on cell viability is likely to be mediated by alterations in the mechanisms of regulation of apoptosis and necrosis. Overall, this work highlights a novel role of environmental pollutants in the pathophysiology of chondrocytes. 292 IL-1b MEDIATES MMP SECRETION AND IL-1B NEOSYNTHESIS VIA UPREGULATION OF P22PHOX AND NOX4 ACTIVITY IN HUMAN ARTICULAR CHONDROCYTES F. Rousset y, F. Hazane Puch z, L. Grange yz, M. Nguyen y, C. Pinosa y, C. Combaz Lair z, B. Burroni z, B. Rubens-Duval z, F. Morel y, B. Lardy yz. yUniversit e Joseph Fourier, Grenoble, France; zCHU Grenoble, Grenoble

Leptin is a pleiotropic hormone that regulates body weight and energy expenditure. Recent findings suggest that leptin may be involved in acute and/or chronic inflammation, however only limited results are available describing the effects of in vivo models of acute inflammation on leptin secretion. The aim of this study was to evaluate serum leptin levels in response to two well-established models of acute inflammation in rats: carrageenan rat paw induced oedema and carrageenan induced pleurisy. Our results clearly show that leptin levels rise in rats in which both oedema and pleurisy were induced. Serum leptin levels in carrageenan induced paw oedema were 3.86+/-0.16 microg/L in comparison to 1.83+/-0.17 microg/L of control animals (p0.001). A similar result was observed in carrageenan induced pleurisy animals in which leptin levels were 4.87+/-0.27 microg/L in comparison to 2.19+/-0.16 microg/L of control animals (p0.001). The increase in leptin levels induced following carrageenan-induced pleurisy appears to be dependent on adrenal function and it is markedly blunted in adrenalectomized rats. Leptin levels in carrageenan induced pleurisy, carried out on adrenalectomized rats, were lower than intact inflamed animals, suggesting a possible involvement of endogenous glucocorticoids. In summary the results here presented show that: a) an elevated plasma leptin concentration was induced during experimental models of inflammation b) this increase is mediated to a large extent by glucocorticoids. In conclusion, acute experimental models of inflammation are associated with changes in circulating leptin suggesting a possible involvement of this hormone in the anorexia/cachexia that is frequently associated with inflammatory processes. Furthermore, our data indicate the existence of a feedback loop among glucocorticoids and leptin which might contribute to the immune response to lace the inflammatory process.

The methods normally used for the detection of enteroviruses in environmental samples involve the use of cell cultures, which are expensive and time consuming. The Polymerase Chain Reaction (PCR) is a useful tool for the detection of enteroviruses in several matrixes because primary cell culture is not needed and the increased sensitivity of PCR allows detection of the low numbers of target nucleic acids usually found in environmental samples. A 5-h, user-friendly PCR assay was used to detect enteroviruses in bivalves molluscs (clams) and sewage. Reverse transcription and amplification were performed in a one-step reaction using rTth polymerase. Carryover contamination was prevented with dUTP and uracil N-glycosylase. Detection was performed colorimetrically in a microwell titer plate. This method has greater advantages over conventional methodologies for routinely screening a large number of samples, namely, the rapid acquisition of results and cost effectiveness.