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49 p.-9 fig.-1 tab.-1 graph. abst., Background and purpose: The pathogenesis of osteoarthritis (OA) implicates a low-grade inflammation associated to the activation of the innate immune system. Toll like receptor (TLR) stimulation triggers the release of inflammatory mediators, which aggravate OA severity. The aim was to study the preventive effect of 6-shogaol (6S), a potential TLR4 inhibitor, on the treatment of experimental knee OA., Experimental approach: OA was induced in C57BL6 mice by surgical section of the medial meniscotibial ligament, which received 6S for eight weeks. Cartilage damage, inflammatory mediator presence, and disease markers were assessed in the joint tissues by immunohistochemistry. Computational modelling was used to predict binding modes of 6S into the TLR4/MD2 receptor and its permeability across cellular membranes. Employing LPS-stimulated chondrocytes and MAPK assay, we clarified 6S action mechanisms., Key results: 6S treatment was able to prevent articular cartilage lesions, synovitis, and the presence of pro-inflammatory mediators and disease markers in OA animals. Molecular modelling studies predicted 6S interaction with the TLR4/MD-2 heterodimer in an antagonist conformation through its binding into the MD-2 pocket. In cell culture, we confirmed that 6S reduced LPS-induced TLR4 inflammatory signaling pathways. Besides, MAPK assay demonstrated that 6S directly inhibits the ERK1/2 phosphorylation activity., Conclusion and implications: 6S evoked a preventive action on cartilage and synovial inflammation in OA mice. 6S effect may take place not only by hindering the interaction between TLR4 ligands and the TLR4/MD-2 complex in chondrocytes, but also through inhibition of ERK phosphorylation, implying a pleiotropic effect on different mediators activated during OA, which proposes it as an attractive drug for OA treatment., This work was partially supported by grants from the Instituto de Salud Carlos III (PI16/00991,PI18/00261, PI20/00349) and MICINN (CTQ2017-88353-R). AM’s work was funded by the Instituto de Salud Carlos III through the “Miguel Servet” grant program (CP15/00053). PG and AL were founded by Fundación Conchita Rábago, and AM-R was founded by MINECO through the FPI program (PRE2018-086249). This work is co-funded by Fondo Europeo de Desarrollo Regional (FEDER).

Chondrocytes in osteoarthritic (OA) cartilage acquire a hypertrophic-like phenotype, where Hedgehog (Hh) signaling is pivotal. Hh overexpression causes OA-like cartilage lesions, whereas its downregulation prevents articular destruction in mouse models. Mutations in EVC and EVC2 genes disrupt Hh signaling, and are responsible for the Ellis-van Creveld syndrome skeletal dysplasia. Since Ellis-van Creveld syndrome protein (Evc) deletion is expected to hamper Hh target gene expression we hypothesized that it would also prevent OA progression avoiding chondrocyte hypertrophy. Our aim was to study Evc as a new therapeutic target in OA, and whether Evc deletion restrains chondrocyte hypertrophy and prevents joint damage in an Evc tamoxifen induced knockout (EvccKO) model of OA. For this purpose, OA was induced by surgical knee destabilization in wild-type (WT) and EvccKO adult mice, and healthy WT mice were used as controls (n = 10 knees/group). Hypertrophic markers and Hh genes were measured by qRT-PCR, and metalloproteinases (MMP) levels assessed by western blot. Human OA chondrocytes and cartilage samples were obtained from patients undergoing knee joint replacement surgery. Cyclopamine (CPA) was used for Hh pharmacological inhibition and IL-1 beta as an inflammatory insult. Our results showed that tamoxifen induced inactivation of Evc inhibited Hh overexpression and partially prevented chondrocyte hypertrophy during OA, although it did not ameliorate cartilage damage in DMM-EvccKO mice. Hh pathway inhibition did not modify the expression of proinflammatory mediators induced by IL-1 beta in human OA chondrocytes in culture. We found that hypertrophic—IHH—and inflammatory—COX-2—markers co-localized in OA cartilage samples. We concluded that tamoxifen induced inactivation of Evc partially prevented chondrocyte hypertrophy in DMM-EvccKO mice, but it did not ameliorate cartilage damage. Overall, our results suggest that chondrocyte hypertrophy per se is not a pathogenic event in the progression of OA., This work was financially supported by grants from the Instituto de Salud Carlos III [PI20/00349, PI16/00065, PI18/00261] and Fondo Europeo de Desarrollo Regional (FEDER); and by the Spanish Ministry of Science and Innovatin (MICINN) [SAF-2013-43365-R and SAF2016-75434-R] to VLR-P. AL and PG were funded by Fundación Conchita Rábago.

© Author(s) (or their employer(s)) 2022. No commercial re-use. See rights and permissions. Published by BMJ., Background: Synovial tissue research has become widely developed in several rheumatology centres, however, large discrepancies exist in the way synovial tissue is handled and, more specifically, how data pertaining to biopsy procedure, quality check and experimental results are reported in the literature. This heterogeneity hampers the progress of research in this rapidly expanding field. In that context, under the umbrella of European Alliance of Associations for Rheumatology, we aimed at proposing points to consider (PtC) for minimal reporting requirements in synovial tissue research. Methods: Twenty-five members from 10 countries across Europe and USA met virtually to define the key areas needing evaluation and formulating the research questions to inform a systematic literature review (SLR). The results were presented during a second virtual meeting where PtC were formulated and agreed. Results: Study design, biopsy procedures, tissue handling, tissue quality control and tissue outcomes (imaging, DNA/RNA analysis and disaggregation) were identified as important aspects for the quality of synovial tissue research. The SLR interrogated four databases, retrieved 7654 abstracts and included 26 manuscripts. Three OPs and nine PtC were formulated covering the following areas: description of biopsy procedure, overarching clinical design, patient characteristics, tissue handling and processing, quality control, histopathology, transcriptomic analyses and single-cell technologies. Conclusions: These PtC provide guidance on how research involving synovial tissue should be reported to ensure a better evaluation of results by readers, reviewers and the broader scientific community. We anticipate that these PtC will enable the field to progress in a robust and transparent manner over the coming years., This work was funded by EULAR (SCI020)

BackgroundChondrocytes in osteoarthritic (OA) cartilage acquire a hypertrophic-like phenotype, where Hedgehog (Hh) signaling is pivotal. Hh overexpression causes OA-like cartilage lesions, whereas its downregulation prevents articular destruction in mouse models. Mutations in EVC and EVC2 genes disrupt Hh signaling, and are responsible for the Ellis-van Creveld syndrome skeletal dysplasia. Since Ellis-van Creveld syndrome protein (Evc) deletion is expected to hamper Hh target gene expression we hypothesized that it would also prevent OA progression avoiding chondrocyte hypertrophy. Our aim was to study Evc as a new therapeutic target in OA, and whether Evc deletion restrains chondrocyte hypertrophy and prevents joint damage in an Evc tamoxifen induced knockout (EvccKO) model of OA.MethodsOA was induced by surgical knee destabilization in wild-type (WT) and EvccKO adult mice, and healthy WT mice were used as controls (n=10 knees/group). Hypertrophic markers and Hh genes were measured by qRT-PCR, and metalloproteinases (MMP) levels assessed by western blot. Human OA chondrocytes and cartilage samples were obtained from patients undergoing knee joint replacement surgery. Cyclopamine (CPA) was used for Hh pharmacological inhibition and IL-1β as an inflammatory insult.ResultsTamoxifen induced inactivation of Evc inhibited Hh overexpression and partially prevented chondrocyte hypertrophy during OA, although it did not ameliorate cartilage damage in DMM-EvccKO mice. Hh pathway inhibition did not modify the expression of proinflammatory mediators induced by IL-1 beta in human OA chondrocytes in culture. Hypertrophic – IHH – and inflammatory – COX-2 – markers co-localized in OA cartilage samples.ConclusionsTamoxifen induced inactivation of Evc partially prevented chondrocyte hypertrophy in DMM-EvccKO mice, but it did not ameliorate cartilage damage. Our results suggest that chondrocyte hypertrophy per se is not a pathogenic event in the progression of OA.

Semaphorin 4D (Sema4D) is a neurotrophin that is secreted by osteoclasts and binds to its receptor PlexinB1 on osteoblasts to inhibit their differentiation and function. Adenosine A2A activation inhibits osteoclast Sema4D-mediated secretion, diminishes inflammatory osteolysis and prevents bone loss following tenofovir (one of the most used antivirals in HIV). Therefore, tenofovir might activate Sema4D signaling to alter bone turnover. Female C57Bl/6/A2AKO mice were ovariectomized and treated with saline (control), tenofovir 75 mg/Kg/day, dipyridamole 25 mg/Kg/day or a combination for 5 weeks and long bones were prepared for histology. Primary murine-induced osteoclast/osteoblast were challenged with tenofovir/dipyridamole 1 μM each, and the expression of Sema4D/PlexinB1, RhoA/ROCK/IGF1R was studied by RT-PCR, Western blot and immunostaining. In vivo tenofovir showed an increased expression of Sema4D when compared to control mice, and dipyridamole reverted the expression in an A2A-dependent manner. In vitro, tenofovir increases Sema4D expression and secretion in osteoclast precursors, and pre-treatment with dipyridamole reverted this effect. pRhoA and ROCK1 activation were increased and IRS1/IGF1R expression was diminished by tenofovir in the Vav3/ARHGAP18 mechanism in osteoblast precursors and reverted by dipyridamole in an A2A-dependent manner. This suggests that tenofovir increases bone loss by activation of Sema4D/PlexinB1 signaling, which inhibits osteoblast differentiation. Agents that increase local adenosine concentrations, such as dipyridamole, might prevent bone loss following the inhibition of this pathway.

Copyright © 2021 Mobasheri, Fonseca, Gualillo, Henrotin, Largo, Herrero-Beaumont and Rocha. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms., Osteoarthritis (OA) is the most common form of arthritis affecting more than 500 million people globally. It accounts for more pain and functional disability than any other musculoskeletal disease and is an important source of high societal and economic costs. Although the pathophysiology of OA is poorly understood, the risk factors associated with disease development are well-established. They include age, obesity, sex, previous incidence of joint injuries, meniscal damage, joint instability, malalignment, genetics, bone shape (including anatomical deformities), muscle weakness and sarcopenia, and metabolic disease. Although OA can affect any synovial joint, including joints in the hand, according to studies on the global burden of disease in 2010 and 2017, knee OA represents the greatest societal burden.

Summary Objective We aimed to assess whether an increase in chondrocyte size might be a feature of the articular cartilage (AC) hypertrophic-like phenotype both in experimental and in human osteoarthritis (OA). The anatomical location of these enlarged cells in the cartilage layers was also evaluated. Methods Experimental OA was carried out in female rabbits alone or in combination with osteoporosis (OPOA). The rabbits were subjected to destabilization knee surgery to develop OA. Osteoporosis was induced with ovariectomy and methylprednisolone administration. Human OA samples obtained from knee replacement surgery were also studied. Cartilage lesions and chondrocyte size were assessed in AC sections. Immunostaining of type-X collagen and metalloproteinase-13 were used as markers of the AC hypertrophic transformation. Both the cell size and the gene expression of type-X collagen were further analyzed in primary murine chondrocyte cultures. Results Compared to healthy AC, chondrocyte size was increased both in experimental and in human OA, in correlation with the severity of cartilage damage. No differences in chondrocyte size were found between deeper or more superficial regions of AC. In cell cultures, accretion of hypertrophic markers and cell enlargement were found to occur synchronized. Conclusions We observed an enhancement in the mean size of chondrocytes at the OA cartilage, which showed correlation with cartilage damage, both in human and in experimental OA. The enlarged chondrocytes were homogeneously distributed throughout the AC. Our results suggest that chondrocyte size could be a reliable measure of disease progression, of potential use in the histopathological assessment of OA cartilage.

Summary The major histocompatibility complex (MHC) class I-related chain A (MICA) is induced upon stress, and labels malfunctioning cells for their recognition by cytotoxic lymphocytes. Alterations in this recognition and also abnormal natural killer (NK) functions have been found in systemic lupus erythematosus (SLE). MICA can be shed from cells, subsequently acting as a soluble decoy receptor (sMICA). Our purpose was to study circulating sMICA levels in relationship with the activation of innate pathways in PBMC in a cohort of lupus patients. NK cells were characterized by flow cytometry. Gene expression of Toll-like receptors (TLR), interferon (IFN)-I sensitive genes and MICA were separately analyzed in monocytes, T cells and B cells. Serum sMICA was measured with enzyme-linked immunosorbent assay (ELISA). In our cohort, NK cell counts dropped in relationship with disease activity. sMICA showed an inverse trend with NK cell counts, as well as a significant association with activity indices, but not with complement decrease. Levels of sMICA associated to proteinuria and active nephritis. A multivariate regression model revealed anti-nuclear antibody (ANA) titres, the up-regulation of TLR-4 in T cells and lower vitamin D as predictors of sMICA enhancement. Interestingly, vitamin D showed an inverse association with proteinuria and a strong correlation with T cell MICA mRNA levels. According to our data, circulating sMICA identifies a subgroup of lupus patients with low vitamin D, innate activation of T cells and nephritis. We propose that lymphocyte shedding could account for the enhancement of sMICA and reflect an immune evasion mechanism driving disease activation in lupus.

Osteopenia and fragility fractures have been associated with human immunodeficiency virus (HIV) infection. Tenofovir, a common antiviral in HIV treatment, also leads to increases in bone catabolism markers and decreased BMD in children and young adults. In murine models and human cell lines, tenofovir inhibits adenosine triphosphate release and decreases extracellular adenosine levels. Adenosine and adenosine A2A receptor inhibit osteoclast formation, and increase local adenosine concentration with dipyridamole, an agent that blocks adenosine cellular uptake and stimulates new bone formation as well as bone morphogenic protein 2. We hypothesized that tenofovir regulates bone resorption by diminishing endogenous adenosine levels and questioned whether dipyridamole may be a useful treatment to counteract the deleterous bone effects of tenofovir. Primary murine osteoclasts were induced by M-CSF/RANKL, and the number of TRAP-positive-cells was studied after challenge with tenofovir alone or in combination with dipyridamole. Differentiation markers were studied by RT-PCR and MAPK/NFkB expression by Western blot. Male C57Bl/6 mice were treated as follows: saline 0.9% (control), tenofovir 75 mg/kg/day, dipyridamole 25 mg/kg/day, combination tenofovir/dipyridamole (n = 10, 4 weeks). Calcein/Alizarin Red-labeling of newly formed bone was used, and long bones were prepared for micro-computed tomography (μCT)/histology. Tenofovir produced a dose-dependent increase in osteoclast differentiation (EC50 = 44.5nM) that was reversed by dipyridamole (IC50 = 0.3 μM). Tenofovir increased cathepsin K and NFATc1 mRNA levels and dipyridamole reversed the effect. Dipyridamole reversed the effect of tenofovir on pERK1/2, pp38, and NFkB nuclear translocation. Mice treated with tenofovir lost nearly 10% of their body weight (p < 0.001). μCT revealed decreased BMD and altered trabecular bone in tenofovir-treated mice, reversed by dipyridamole. TRAP-staining showed increased osteoclasts in tenofovir-treated mice (p < 0.005), an effect reversed by dipyridamole. Similar results were obtained for cathepsin K and CD68. RANKL-positive cells were increased in tenofovir-treated mice, whereas osteoprotegerin-positive cells were decreased; both effects were reversed by dipyridamole. These results suggest that treatment with agents that increase local adenosine concentrations, like dipyridamole, might prevent bone loss following tenofovir treatment. © 2019 American Society for Bone and Mineral Research.

Extracellular adenosine triphosphate (ATP) plays a central role in a wide variety of joint diseases. ATP is generated intracellularly, and the concentration of the extracellular ATP pool is determined by the regulation of its transport out of the cell. A variety of ATP transporters have been described, with connexins and pannexins the most commonly cited. Both form intercellular channels, known as gap junctions, that facilitate the transport of various small molecules between cells and mediate cell–cell communication. Connexins and pannexins also form pores, or hemichannels, that are permeable to certain molecules, including ATP. All joint tissues express one or more connexins and pannexins, and their expression is altered in some pathological conditions, such as osteoarthritis (OA) and rheumatoid arthritis (RA), indicating that they may be involved in the onset and progression of these pathologies. The aging of the global population, along with increases in the prevalence of obesity and metabolic dysfunction, is associated with a rising frequency of joint diseases along with the increased costs and burden of related illness. The modulation of connexins and pannexins represents an attractive therapeutic target in joint disease, but their complex regulation, their combination of gap-junction-dependent and -independent functions, and their interplay between gap junction and hemichannel formation are not yet fully elucidated. In this review, we try to shed light on the regulation of these proteins and their roles in ATP transport to the extracellular space in the context of joint disease, and specifically OA and RA.

Background: Bone alterations have been observed in the course of HIV infection, char-acterized by a marked decrease in bone mineral density (BMD) and an increase in the frequency of fractures as a result of fragility. We aim to evaluate early changes in bone metabolic profile and the possible association with tenofovir and other nucleoside and nucleotide reverse transcriptase inhibi-tors (NRTIs) in treatment-naïve HIV patients. Methods: We conducted a prospective study in naïve HIV-infected adults (under 50 years), separat-ed into three groups according to NRTI therapy: tenofovir disoproxil fumarate (TDF); tenofovir alafenamide (TAF) and abacavir (ABC). BMD and epidemiological, immunological and metabolic bone parameters were evaluated. Bone markers were analyzed in plasma at baseline, 12 and 48 weeks after initiating treatment. Results: Average age of patients was 34.8 years (± 9.6). 92.4% of them with CD4 count > 200 cel/μL. At week 12 after starting treatment, both TDF [increase in PN1P (31.7%, p = 0.004), TRAP (11.1%, p = 0.003), OPN (19.3%, p = 0.045) and OC (38.6%, p = 0.001); decrease in OPG (-23.4%, p = 0.003)] and TAF [increase in 42.6% for CTX (p = 0.011), 27.3% for OC (p = 0.001) and 21% for TRAP (p = 0.008); decrease in OPG (-28.8%, p = 0.049)] presented a deep resorption profile compared to ABC, these differences in bone molecular markers, a tendency to equalize at week 48, where no significant differences were observed. Patients treated with TDF showed the greatest decrease in Z-score in both lumbar spine (LS) and femoral neck (FN) at week 48 without statistically significant differences. Conclusion: Treatment-naïve HIV patients have a high prevalence of low bone density. Treatment with TDF is associated with greater bone deterioration at 12 and 48 weeks. TAF seems to present similar early bone deterioration at 12 weeks which disappears at 48 weeks., This research was financially supported by The Carlos III Health Institute through the “Miguel Servet” program (CP15/00053), co-funded by The European Regional Development Fund and research grants from the Spanish Carlos III Health Institute (PI16/00991 and PI19/00744)

ObjectiveTo evaluate the discriminatory ability of ultrasound in calcium pyrophosphate deposition disease (CPPD), using microscopic analysis of menisci and knee hyaline cartilage (HC) as reference standard.MethodsConsecutive patients scheduled for knee replacement surgery, due to osteoarthritis (OA), were enrolled. Each patient underwent ultrasound examination of the menisci and HC of the knee, scoring each site for presence/absence of CPPD. Ultrasound signs of inflammation (effusion, synovial proliferation and power Doppler) were assessed semiquantitatively (0–3). The menisci and condyles, retrieved during surgery, were examined microscopically by optical light microscopy and by compensated polarised microscopy. CPPs were scored as present/absent in six different samples from the surface and from the internal part of menisci and cartilage. Ultrasound and microscopic analysis were performed by different operators, blinded to each other’s findings.Results11 researchers from seven countries participated in the study. Of 101 enrolled patients, 68 were included in the analysis. In 38 patients, the surgical specimens were insufficient. The overall diagnostic accuracy of ultrasound for CPPD was of 75%—sensitivity of 91% (range 71%–87% in single sites) and specificity of 59% (range 68%–92%). The best sensitivity and specificity were obtained by assessing in combination by ultrasound the medial meniscus and the medial condyle HC (88% and 76%, respectively). No differences were found between patients with and without CPPD regarding ultrasound signs of inflammation.ConclusionUltrasound demonstrated to be an accurate tool for discriminating CPPD. No differences were found between patents with OA alone and CPPD plus OA regarding inflammation.

We have read with interest the comment from Angelides and Manolios in which they propose an alternative way to account for the potential mechanism of action of glucosamine in osteoarthritis (OA) and other chronic inflammatory diseases.1 Recent and robust epidemiological data suggest that sustained glucosamine intake could partially prevent cardiovascular disease and cancer.2 Our editorial made some hypothetical considerations about the mechanism of action of this compound.3 The editorial also commented on the difficulty of detecting a beneficial effect of glucosamine in OA, due to the modesty of the therapeutic effect, as well as the weakness of the methodological tools employed in OA clinical trials.3 In some way, the relationship between glucosamine and OA takes us fully into the pathophysiology that revolves around immunometabolic regulation, a driver that can account for tissue deterioration in various chronic diseases and still with many aspects to …

Inflammation triggered by metabolic imbalance, also called metainflammation, is low-grade inflammation caused by the components involved in metabolic syndrome (MetS), including central obesity and impaired glucose tolerance. This phenomenon is mainly due to excess nutrients and energy, and it contributes to the pathogenesis of osteoarthritis (OA). OA is characterized by the progressive degeneration of articular cartilage, which suffers erosion and progressively becomes thinner. Purinergic signaling is involved in several physiological and pathological processes, such as cell proliferation in development and tissue regeneration, neurotransmission and inflammation. Adenosine and ATP receptors, and other members of the signaling pathway, such as AMP-activated protein kinase (AMPK), are involved in obesity, type 2 diabetes (T2D) and OA progression. In this review, we focus on purinergic regulation in osteoarthritic cartilage and how different components of MetS, such as obesity and T2D, modulate the purinergic system in OA. In that regard, we describe the critical role in this disease of receptors, such as adenosine A2A receptor (A2AR) and ATP P2X7 receptor. Finally, we also assess how nucleotides regulate the inflammasome in OA.

Osteoarthritis (OA) is a disease with a very long course and varied clinical expression in the initial stages, when it is really challenging to adequately measure disease outcomes both in clinical trials and in daily life.1 It is also very difficult to accurately study the efficacy of symptomatic slow-acting drugs for OA and that of non-pharmacological treatments.2 Although the efficacy of glucosamine (GlcN) in the treatment of OA is still a controversial issue3 4, recent high-quality epidemiological studies confirm previous data showing that prolonged GlcN intake, regardless of its effect on OA progression, could decrease cardiovascular disease (CVD) events, and the incidence of CVD-associated diseases.5–8 These data should be analysed keeping in mind that CVD is the main cause of death in patients with OA.9 Different authors suggest that this protective effect may be associated with the anti-inflammatory properties of GlcN,5 although the molecular basis has been only partially defined. The imbalance between the mechanical loading and its absorption by the articular cartilage is the origin of joint tissue alteration in OA. While a severe overload can deteriorate any kind of cartilage, a certain load that is physiologically well tolerated by a robust cartilage could be the origin of pathological alterations for a weakened one.9 Cartilage damage begins when the load prevails over the resistance, which in the midterm activates innate immune response in the different joint tissues, and is, at least partially, responsible for joint deterioration. Unbalanced mechanical forces and damage-associated molecular patterns turn on the innate immune system through the activation of the Toll-like receptors. Once this response is activated, a secondary wave of inflammatory mediators is released, with a robust increase in the concentration of cytokines and metalloproteases, the final effectors of cartilage destruction.9 Certainly, these mediators can …

Plasma levels of adenosine deaminase (ADA), an enzyme that deaminates adenosine to inosine, are increased during inflammation. An increase in ADA activity occurs with lower human immunodeficiency virus (HIV) viral load and higher CD4+ T cell counts. We aimed to investigate the role of plasma ADA as a biomarker of inflammation in treatment-na&iuml, ve HIV patients who received tenofovir or another nucleoside analog for comparison. Ninety-two treatment-na&iuml, ve patients were included in the study and grouped by treatment, i.e., tenofovir disoproxil fumarate (TDF), tenofovir alafenamide (TAF) or Triumeq. ADA activity was measured in plasma and cytokines were analyzed by MILLIPLEX&reg, MAP-Luminex&reg, Technology. Plasma concentration of monocytes and neutrophils was measured at 0, 3, and 12 months post-treatment. Treatment-na&iuml, ve HIV patients had increased ADA concentrations (over 15 U/L) that decreased after treatment with TAF and Triumeq, though this did not occur in TDF-treated patients. However, all groups exhibited a pro-inflammatory systemic profile at 12 months of treatment. Plasma GM-CSF levels decreased after 12 months of treatment in the TDF group, with a concomitant decrease in blood monocyte count, and a negative correlation with ADA values was found. In conclusion, ADA levels may be modulated by antiretroviral therapy in HIV patients, possibly affecting inflammatory status.

Osteoarthritis (OA) is a multifactorial joint disease mainly affecting articular cartilage (AC) with a relevant biomechanical component. During endochondral ossification growth plate (GP) chondrocytes arrange in columns. GPs do not ossify in skeletally mature rodents. In neonatal mice, an altered joint loading induces GP chondrocyte disorganization. We aimed to study whether experimental OA involves GP disorganization in adult mice and to assess if it may have additional detrimental effects on AC damage. Knee OA was induced by destabilization of the medial meniscus (DMM) in wild-type (WT) adult mice, and in Tamoxifen-inducible Ellis-van-Creveld syndrome protein (Evc) knockouts (EvccKO), used as a model of GP disorganization due to Hedgehog signalling disruption. Chondrocyte column arrangement was assessed in the tibial GP and expressed as Column Index (CI). Both DMM-operated WT mice and non-operated-EvccKO showed a decreased CI, indicating GP chondrocyte column disarrangement, although in the latter, it was not associated to AC damage. The most severe GP chondrocyte disorganization occurred in DMM-EvccKO mice, in comparison to the other groups. However, this altered GP structure in DMM-EvccKO mice did not exacerbate AC damage. Further studies are needed to confirm the lack of interference of GP alterations on the analysis of AC employing OA mice., This work was financially supported by grants from the Instituto de Salud Carlos III [PI15/00340, PI16/00065, PI18/00261] to RL and GH-B, and Fondo Europeo de Desarrollo Regional (FEDER) by grants SAF-2013–43365-R and SAF2016-75434-R to VL R-P. AL and PG were funded by Fundación Conchita Rábago.

The objective of this study was to validate musculoskeletal ultrasound (US) in a rabbit model of acute gout. Acute gout was induced by intra-articular injection of monosodium urate (MSU) crystals in 10 rabbits; the 3 controls received vehicle. Rabbit knees were assessed by B-mode and power Doppler (PD) US 24 and 72 h after injections. After 72 h, all rabbits were euthanized. US discriminated between the MSU-injected and control groups with respect to the different inflammatory findings at both at 24 and 72 h and for MSU crystal-related findings after 24 h of injection. US synovial thickening, intra-synovial power Doppler signal and global joint distension significantly correlated with the synovial global histopathological score (r = 0.47, p = 0.0188), tissue vascularization measured by CD31 immunohistochemical-positive staining (r = 0.46, p = 0.0172) and tissue levels of interleukin-1β (r = 0.53, p = 0.0078), respectively. US is a valid method for assessment of synovial inflammation in experimental gouty arthritis in rabbits.

BACKGROUND AND PURPOSE Patients with active rheumatoid arthritis (RA) have increased cardiovascular mortality, paradoxically associated with reduced circulating lipid levels. The Janus kinase (Jak) inhibitor tofacitinib ameliorates systemic and joint inflammation in RA with a concomitant increase in serum lipids. We analyzed the effect of tofacitinib on the lipid profile of hyperlipidemic rabbits with chronic arthritis (CA) and on the regulation of reverse cholesterol transport (RCT) during chronic inflammation. EXPERIMENTAL APPROACH CA was induced in previously immunized rabbits fed with a high-fat diet (HFD) by administering four intra-articular injections of ovalbumin. A group of rabbits received tofacitinib (10 mg kg-1 day-1) for two weeks. Systemic and synovial inflammation and lipid content were evaluated. For in vitro studies, THP-1-derived macrophages were exposed to high lipid concentrations, and then stimulated with interferon gamma (IFNγ) in the presence or absence of tofacitinib in order to study RCT mediators. KEY RESULTS Tofacitinib decreased systemic and synovial inflammation and increased circulating lipid levels. Although it did not modify synovial macrophage density, it was able to reduce the lipid content within synovial macrophages. In foam macrophages in culture, IFNγ further stimulated intracellular lipid accumulation, while Jak/STAT inhibition provoked by tofacitinib induced lipid release by increasing cellular liver X receptor alpha (LXRα) and ATP-binding cassette transporter (ABCA1) synthesis. CONCLUSION AND IMPLICATIONS Active inflammation could be associated with lipid accumulation within macrophages of CA rabbits. Jak inhibition induced lipid release though RCT activation, providing a plausible explanation for the effect of tofacitinib on the lipid profile of RA patients.

Background 9The OMERACT Ultrasound (US) in calcium pyrophosphate deposition disease (CPPD) sub-task force has been working on the assessment of the utility of US in CPPD since 2014 first creating definitions for CPPD identification and then demonstrating that US is a reliable tool[1]. Objectives Objective of this study is to assess the diagnostic accuracy of US in CPPD Methods This is a multicentre international diagnostic accuracy study involving 17 centres from 9 countries. We enrolled in this study consecutive patients waiting to undergo knee replacement surgery due to severe osteoarthritis. Each patient underwent US examination of the knee, focusing on the menisci and the hyaline cartilage, the day prior to surgery, scoring each site according to the presence/absence of CPP as defined by OMERACT[1]. After surgery, the menisci and the condyles were collected and examined microscopically. Six samples were collected, both from the surface and from the internal part of menisci and cartilage trying to cover a large part of the structure. All slides were observed under transmitted light microscopy and by compensated polarised microscopy. A dichotomous score was given for the presence/absence of CPP crystals. US and microscopic analysis were performed by blinded operators. Sensitivity and specificity of US were calculated using microscopic findings of the menisci and cartilage as the gold standard. Results These preliminary analyses include 30 patients. The mean age was 71yrs (SD±9.1), 19 (63%) were females. 17 patients were positive at US analysis and 12 at microscopic analysis. Diagnostic accuracy results of US at patient level, are presented in figure 1. Conclusion These preliminary results demonstrate that US is a sensitive exam for identification of CPPD with acceptable specificity. US is the first diagnostic technique with consistent reliability and validity to be applied for non-invasive screening for CPPD in clinical practice. References [1] Filippou G, Scire CA, Adinolfi A, et al. Identification of calcium pyrophosphate deposition disease (CPPD) by ultrasound: reliability of the OMERACT definitions in an extended set of joints—an international multiobserver study by the OMERACT Calcium Pyrophosphate Deposition Disease Ultrasound Subtask Force. Ann Rheum Dis 2018;:annrheumdis-2017-212542. doi:10.1136/annrheumdis-2017-212542 Disclosure of Interests Georgios Filippou Speakers bureau: Laborest, Abbvie, BMS, Sanofi, Anna Scanu: None declared, Antonella Adinolfi: None declared, Carmela Toscano: None declared, Raquel Largo-Carazo: None declared, Esperanza Naredo Consultant for: Abbvie, Speakers bureau: AbbVie, Roche, Bristol-Myers Squibb, Pfizer, UCB, Lilly, Novartis, Janssen, and Celgene GmbH, Emilio Calvo: None declared, Gabriel Herrero-Beaumont: None declared, Pascal Zufferey: None declared, Christel Madelaine-Bonjour: None declared, Daryl MacCarter: None declared, Stanley Makman: None declared, Zachary Weber: None declared, Ingrid Moller: None declared, Marwin Gutierrez: None declared, Carlos Pineda: None declared, Denise Clavijo Cornejo: None declared, Hector Garcia: None declared, Victor Ilizaliturri: None declared, Jaime Mendoza Torres: None declared, Raul Pichardo: None declared, Luis Carlos Rodriguez Delgado: None declared, Emilio Filippucci: None declared, Edoardo Cipolletta: None declared, Teodora Serban: None declared, Florentin Ananu Vreju Consultant for: abbvie and novartis, Speakers bureau: abbvie, novartis, pfizer, sandoz, eli lilly, ucb pharma, Gael Mouterde: None declared, Maria-Antonietta d’Agostino: None declared, Marcello Govoni: None declared, Leonardo Punzi Consultant for: BMS, Fidia, Grunenthal, Menarini, Speakers bureau: BMS, Fidia, Grunenthal, Menarini, Nemanja Damjanov Grant/research support from: AbbVie, Pfizer and Roche, Consultant for: Abbvie, Gedeon Richter, Merck, Novartis, Pfizer and Roche., Speakers bureau: Abbvie, Gedeon Richter, Merck, Novartis, Pfizer and Roche., Lene Terslev Speakers bureau: Speakers fee from : Roche, Novartis, Pfizer, MSD, BMS, Celgene, Irene Azzolin: None declared, Giulio Guerrini: None declared, Carlo Alberto Scire: None declared, Annamaria Iagnocco: None declared

Background: Patients with active rheumatoid arthritis (RA) have increased cardiovascular mortality, paradoxically associated with reduced circulating lipid levels (1-3). Several disease-modifying antirheumatic drugs (DMARDs), such as the JAK inhibitor tofacitinib, ameliorate disease activity along with an increase in serum lipids (4, 5). We previously demonstrated in vitro that tofacitinib favored cholesterol efflux from macrophages through an ABCA1-dependent mechanism (6). Furthermore, tofacitinib-treated chronic arthritic rabbits showed increased circulating lipids and decreased lipid accumulation within the synovium (6). Objectives: Our aim was to explore in vivo whether inflammation impedes cholesterol efflux from macrophages, and whether tofacitinib restores macrophage cholesterol release during chronic arthritis. For that purpose, we assessed the ability of intraperitoneally-injected 3H-cholesterol labelled macrophages to efflux cholesterol to circulating lipoproteins in collagen-induced arthritis (CIA) mice treated with tofacitinib or placebo, as compared to healthy controls. Methods: DBA/1J mice were randomly assigned to healthy controls (Control, n = 9), CIA (CIA, n = 6) and CIA mice receiving 50 mg/kg/day tofacitinib, orally, for three consecutive days, starting on day 39 after CIA induction and coinciding with disease peak (CIA+TOFA, n = 6). The day after, 3H-cholesterol labeled RAW264.7 macrophages were intraperitoneally injected into mice and tracer appearance was monitored in plasma lipoprotein subfractions, synovium, liver, bile and feces. Results: The CIA group showed higher C-Reactive protein levels (CRP, μg/ml; Control: 10.90±0.49, CIA: 13.86±1.05, p=0.03 vs. Control) and lower circulating 3H-cholesterol levels (% of injected disintegrations per minute (dpm)/ml; Control: 1.29±0.11, CIA: 0.92±0.11, p=0.04 vs. Control). Both serum CRP and 3H-cholesterol –particularly the HDL fraction– were restored to baseline after the treatment with the JAK inhibitor (CIA+TOFA: 10.97±0.67 μg/ml and 1.37±0.20 % of injected dpm/ml, respectively, p=0.05 vs. CIA). Concomitantly, we observed an upward trend in 3H-cholesterol accumulation within the synovium of CIA animals as compared to controls, which tended to normalize with the treatment. Conclusion: Systemic inflammation induces cholesterol sequestering within macrophages in vivo by acting on cholesterol efflux transporters (6). Tofacitinib favors cholesterol release to plasma lipoproteins, hence increasing circulating cholesterol. This is not only due to a decrease in inflammation –an effect very likely shared with some other biologic DMARDs–, but also to a direct mechanism on ABCA1 transporters that favors cholesterol efflux. To our knowledge, this is the first report suggesting that cholesterol dynamics in the macrophage may contribute to the overall circulating cholesterol levels, especially considering that this phenomenon may occur in other cell types, such as adipocytes. References [1] Johnsson H. et al. Ann Rheum Dis. 2013;73:1495–9. [2] Myasoedova E. et al. Ann Rheum Dis. 2012;70:482–7. [3] Choy E, Sattar N. Ann Rheum Dis. 2009;68:460–9. [4] Charles-Schoeman C. et al. Semin Arthritis Rheum. 2016;46:71–80. [5] Robertson J. et al. Nat Rev Rheumatol. 2013;9:513–23. [6] Perez-Baos S. et al. Br J Pharmacol. 2017;174:3018–31. Disclosure of Interests: None declared

Background Chondrocytes (CHs) in articular cartilage undergo phenotypic changes and senescence, restricting cartilage regeneration and favoring osteoarthritis (OA) progression. CHs and synovial cells from OA patients show a chronic increase in the channel protein connexin43 (Cx43), which regulates signal transduction. Extracellular vesicles (EVs), including exosomes, have been shown to play important roles in many biological functions and harbour Cx channels that allow the formation of gap junctions (GJs) between the exosome and the target cell, but the role of these EVs and exosomal-Cx43 in OA progression has not been studied yet. Objectives The objective of this study was to investigate the role of EVs released by OA chondrocytes (OACs) in cellular plasticity, inflammation and senescence of surrounding joint tissues. Methods CHs, bone and synovial cells were isolated from healthy and OA donors. EVs were obtained by ultracentrifugation and their protein content was analysed by LC-MS/MS. Protein levels were evaluated by western blot, immunofluorescence and flow cytometry. RNA expression was evaluated by RT-qPCR. Senescence and GJ intercellular communication was studied by flow cytometry and scrape loading assay, respectively. Results OACs showed increased levels of Cx43 within their EVs in comparison to the EVs isolated from healthy donors. Overexpression of Cx43 in CHs increased senescence and exosomal Cx43 levels. Interestingly, the treatment of CHs, bone cells and synoviocytes (target cells) with Cx43-EVs released by OACs, led to a significant increase in both Cx43 mRNA and protein levels in the recipient cells. The increase of Cx43 in target cells acted as a positive regulator of the reversion to a less differentiated state via EMT by activation of Twist-1, associated with increased levels of the mesenchymal markers, as CD105/CD166. The phenotypic changes detected in OACs lead to a decrease in Col2A1 and aggrecan expression in CHs, and increased the levels of cellular senescence and the senescence-associated secretory phenotype (SASP) in the target cells in target cells via p53/p16 and NF-ks. These results were corroborated by analysing the protein cargo of these Cx43 positive EVs by LC-MS/MS, finding enrichment in proteins related with catabolic, senescence and wound-healing pathways. Conclusion Our results indicate that Cx43-positive exosomes released by OACs may be involved in the spread of cellular senescence and inflammation involved in wound healing failure. Further understanding of the role of exosomal Cx43 in OA will help to halt the disease spread and progression. Disclosure of Interests None declared

Background Osteoarthritis (OA) is a degenerative joint disease characterized by cartilage degradation, although both subchondral bone deterioration and synovial inflammation are also hallmarks of the disease. Chiropractic manipulation (CM) is a therapeutic approach focused on the diagnosis, treatment and prevention of musculoskeletal disorders. It is essentially manual, allowing the chiropractor to restore the normal range of motion and function of the joints, muscles, and ligaments. Clinical evidences suggest that CM might exert positive effects in OA patients. Objectives The aim of this study was to evaluate the effects of CM on cartilage, subchondral bone and synovitis state in an OA rabbit model. Methods Ten (4 months old) male New Zealand rabbits underwent knee surgery to induce OA by transection of anterior cruciate ligament. One week after the surgery, CM was performed using the chiropractic adjusting instrument ActivatorV as follows: Force 2 setting was applied onto the tibial tubercle of the right hind limb (true manipulation, TM-OA group), at an angle of approximately 90°, from medial to lateral, whereas the corresponding left hind limb received a false manipulation (FM-OA group) consisting of ActivatorV firing in the air and slightly touching the tibial tubercle. These procedures were repeated 3 times a week for 8 weeks. Three healthy animals were used as control. Following sacrifice, μCT and histological damage evaluation (Mankin score) were done in femur and tibiae. RANKL/OPG protein expressions were studied by immunohistochemistry in tibia samples. Sinovitis was assessed by Krenn score and immunohistochemistry for macrophages (RAM11) and angiogenesis (CD31) were done. Protein expression of VEGF, COX2, TNFa, IL-1b and MMP3 were determinated by Western Blot. Results In the OA rabbits, subchondral BMD decreased in relation to control, been partially reversed in the tibiae of TM-OA group. When subchondral trabecular bone structural parameters were analyzed by microCT, a significant decrease of bone volume/trabecular volume (BV/TV), trabecular number (Tb.N) and trabecular thickness (Tb.Th) was observed in the OA rabbits, while trabecular separation (Tb.S) increased compared to control animals. TM-OA group showed a significant improvement of these parameters compare to FM-OA group. FM-OA joints had higher Mankin score (cartilage damage) than control joints, and TM decreased Mankin score compare to FM-OA (p Conclusion These results suggest that mechanical stimulus induced by CM may retard the pathologic progression of OA. The beneficial effects of CM might be associated with an improvement in bone and cartilage damage and also inflammatory state. Disclosure of Interests None declared

Osteoarthritis (OA) is a chronic joint disease characterized by cartilage degradation, osteophyte formation, subchondral bone sclerosis, and synovitis. Systemic factors such as obesity and the components of the metabolic syndrome seem to contribute to its progression. Breakdown of cartilage ensues from an altered balance between mechanical overload and its absorption by this tissue. There is in this context a status of persistent local inflammation by means of the chronic activation of innate immunity. A broad variety of danger-associated molecular patterns inside OA joint are able to activate pattern recognition receptors, mainly TLR (toll-like receptor) 2 and 4, which are overexpressed in the OA cartilage. Chronic activation of innate immune responses in chondrocytes results in a robust production of pro-inflammatory cytokines and chemokines, as well as of tissue-destructive enzymes, downstream of NF-κB and MAPK (mitogen activated protein kinase) dependent pathways. Besides, the toxic effects of an excess of glucose and/or fatty acids, which share the same pro-inflammatory intracellular signalling pathways, may add fuel to the fire. Not only high concentrations of glucose can render cells prone to inflammation, but also AGEs (advanced glycation end products) are integrated into the TLR signalling network through their own innate immune receptors. Considering these mechanisms, we argue for the control of both primary inflammation and proteolytic catabolism as a preventive strategy in OA, instead of focusing treatment on the enhancement of anabolic responses. Even though this approach would not return to normal already degraded cartilage, it nonetheless might avoid damage extension to the surrounding tissue.

Background In order to gain insight into the early effects drawn by JAK inhibitors on intra-joint JAK/STAT-dependent signaling, we sought synovial activation of STATs and their end-products, along with their modification with tofacitinib (TOFA), at flare-up in antigen induced arthritis (AIA). New Zealand rabbits were randomly assigned to four groups –healthy controls, AIA, TOFA-treated AIA, or TOFA-treated controls–. AIA was induced with 4 weekly intra-articular ovalbumin injections in sensitized animals. TOFA (10 mg·kg− 1·day− 1) was administered for the last 2 weeks. Animals were euthanized 24 h after the last injection. Results AIA animals showed high-grade synovitis, which was partially improved by TOFA. No effects of the treatment were found on serum C-reactive protein or on the synovial macrophage infiltration at this stage. Synovial MMP-1,-3 and -13 expression levels in treated AIA rabbits were found to drop to those of controls, while a downregulation of IL6, IFNγ and TNF was evident in treated versus untreated AIA rabbits. Concurrently, a reduction in pSTAT1 and SOCS1, but not in pSTAT3, SOCS3 or active NFκB-p65, was noted with TOFA. Conclusions Studying the mechanism of action of immunomodulatory drugs represents a major challenge in vivo, since drug-dependent decreases in inflammation very likely mask direct effects on disease mechanisms. This study design allowed us to prevent any confounding effect resulting from reductions in the overall inflammatory status, hence assessing the true pharmacological actions of TOFA in a very severe synovitis. Our findings point to pSTAT1 and MMPs as early molecular readouts of response to this JAK inhibitor. Electronic supplementary material The online version of this article (10.1186/s12950-019-0206-2) contains supplementary material, which is available to authorized users.

Aging is a natural process characterized by the declining ability of the different organs and tissues to respond to stress, increasing homeostatic imbalance and risk of disease. Osteoarthritis (OA) is a multifactorial disease in which cartilage degradation is a central feature. Aging is the main risk factor for OA. In OA cartilage, a decrease in the number of chondrocytes and in their ability to regenerate the extracellular matrix and adequately respond to stress has been described. OA chondrocytes show a senescence secretory phenotype (SSP) consisting on the overproduction of cytokines (interleukins 1 and 6), growth factors (e.g., epidermal growth factor) and matrix metalloproteinases (MMP) (e.g., MMP-3, MMP-13). Reactive Oxygen Species (ROS) play a major role in the induction of the SSP. In chondrocytes, an increase in ROS production leads to hyper-peroxidation, protein carbonylation and DNA damage which alter chondrocyte function. ROS overproduction also induces changes in metabolic pathways such as PI3K-Akt and ERK. Autophagy is a key mechanism for maintaining cell homeostasis by adjusting cell metabolism to nutrient supply and removing damaged organelles. In cartilage, aging-related loss of autophagy leads to cell death and OA, while stimulation of autophagy exerts protective effects on cartilage deterioration. Aging also interferes with epigenetic mechanisms such as activity of histone acetylases that control the pattern of DNA methylation, and induces up- or down-regulation of microRNAs expression. A deeper knowledge of the mechanisms involved in chondrocyte aging could identify potential targets for the treatment of OA, a prevalent and therapeutic-orphan disease.

Objectives Despite the high prevalence of osteoarthritis (OA) in postmenopausal women, a relationship between circulating estrogen levels and the development of OA has not been found. Therefore, the purpose of this study was to evaluate the expression and activity of aromatase, a key enzyme in local production of estrogens, in human OA cultured articular chondrocytes, and to determine the physiological relevance of this enzyme in cartilage. Methods Human OA articular chondrocytes were isolated and cultured. Local production of estradiol was measured after incubation with 100ng/ml testosterone for 8 and 24 h. Furthermore, chondrocytes were culture for 2 h, 48 h, 7 days or 15 days, or in alginate beads for 10 days. Aromatase, type II and X collagen, aggrecan, alkaline phosphatase, and Runx2 expression were evaluated in cartilage, freshly isolated chondrocytes and cultured chondrocytes. Results Aromatase was expressed and active in cultured human chondrocytes. Human cartilage, freshly isolated chondrocytes, and chondrocytes cultured for 2 h expressed an insignificant amount of aromatase; however, expression arose after 48 h of culture and remained increased thereafter. Aromatase expression was not related to estrogen deprivation and was inversely correlated with differentiation. Re-differentiation did not reduce its expression. Conclusions Aromatase presents an almost undetectable expression in human cartilage but is induced in cultured chondrocytes. Therefore, human cartilage might act as a mere target for estrogens rather than a producer, and researchers using cell expansion in culture for latter therapies should consider these changes in estrogen metabolism which may not be reverted after re-differentiation.

Background:The OMERACT Ultrasound (US) in calcium pyrophosphate deposition disease (CPPD) sub-task force has been working on the use of US in CPPD since 2014 first creating definitions for CPPD identification and then assessing the reliability[1].Objectives:Objective of this study is to assess the diagnostic accuracy (truth) of US in CPPD.Methods:Consecutive patients waiting to undergo knee replacement surgery due to osteoarthritis were enrolled in 12 centres from 6 countries. Each patient underwent US examination of the knee, focusing on the menisci and the hyaline cartilage, the day prior to surgery, scoring each site for presence/absence of CPP as defined previously[1]. After surgery, the menisci and the condyles were retrieved and examined microscopically. Six samples were collected, both from the surface and from the internal part of menisci and cartilage trying to cover a large part of it. All slides were observed under transmitted light microscopy and by compensated polarised microscopy. A dichotomous score was given for the presence/absence of CPP. US and microscopic analysis were performed by different operators, blind to each other’s findings. Sensitivity and specificity of US were calculated using microscopic findings as the gold standard.Results:101 patients have been enrolled in the study. 33 patients have been excluded due to loss of anatomical pieces at surgery. The mean age of the remaining 68 pts was 71yo (±8), 44 women, 34 were affected by CPPD according to microscopy. Overall and per site diagnostic US accuracy results are presented in table 1Diagnostic accuracySensitivitySpecificityPositive Predictive valueNegative Predictive valueGlobal0.750.910.590.690.87Medial meniscus0.820.870.770.770.87Lateral meniscus0.750.830.680.680.83Medial cartilage0.860.790.920.880.85Lateral cartilage0.820.710.880.770.84Medial side (combined cartilage and meniscus)0.820.880.760.790.87Lateral side (combined cartilage and meniscus)0.780.880.690.730.86Conclusion:Our results demonstrate that US is an accurate exam for identification of CPPD. The best combination of sensitivity and specificity is achieved by examining the medial aspect of the knee.References:[1]Filippou G, Scirè CA, Adinolfi A,et al.Identification of calcium pyrophosphate deposition disease (CPPD) by ultrasound: reliability of the OMERACT definitions in an extended set of joints—an international multiobserver study by the OMERACT Calcium Pyrophosphate Deposition Disease Ultrasound Subtask Force.Ann Rheum Dis2018;:annrheumdis-2017-212542. doi:10.1136/annrheumdis-2017-212542Disclosure of Interests:Georgios Filippou: None declared, Anna Scanu: None declared, Antonella Adinolfi: None declared, Carmela Toscano: None declared, Dario Gambera: None declared, Raquel Largo: None declared, Esperanza Naredo: None declared, Emilio Calvo: None declared, Gabriel Herrero-Beaumont: None declared, Pascal Zufferey: None declared, Christel Madelaine-Bonjour: None declared, Daryl MacCarter: None declared, Stanley Makman: None declared, Zachary Weber: None declared, Fabiana Figus: None declared, Ingrid Möller: None declared, Marwin Gutierrez: None declared, Carlos Pineda: None declared, Denise Clavijo Cornejo: None declared, Héctor García: None declared, Victor Ilizaliturri: None declared, Jaime Mendoza Torres: None declared, Raul Pichardo: None declared, Luis Carlos Rodriguez Delgado: None declared, Emilio Filippucci Speakers bureau: Dr. Filippucci reports personal fees from AbbVie, personal fees from Bristol-Myers Squibb, personal fees from Celgene, personal fees from Roche, personal fees from Union Chimique Belge Pharma, personal fees from Pfizer, outside the submitted work., Edoardo Cipolletta: None declared, Teodora Serban: None declared, Catalin Cirstoiu: None declared, Florentin Ananu Vreju: None declared, Dun Grecu: None declared, Gael Mouterde: None declared, Marcello Govoni: None declared, Leonardo Punzi: None declared, Nemanja Damjanov Grant/research support from: from AbbVie, Pfizer, and Roche, Consultant of: AbbVie, Gedeon Richter, Merck, Novartis, Pfizer, and Roche, Speakers bureau: AbbVie, Gedeon Richter, Merck, Novartis, Pfizer, and Roche, Lene Terslev Speakers bureau: LT declares speakers fees from Roche, MSD, BMS, Pfizer, AbbVie, Novartis, and Janssen., Carlo Alberto Scirè: None declared, Annamaria Iagnocco Grant/research support from: Abbvie, MSD and Alfasigma, Consultant of: AbbVie, Abiogen, Alfasigma, Biogen, BMS, Celgene, Eli-Lilly, Janssen, MSD, Novartis, Sanofi and Sanofi Genzyme, Speakers bureau: AbbVie, Alfasigma, BMS, Eli-Lilly, Janssen, MSD, Novartis, Sanofi