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Abstract Systemic sclerosis (SSc) is a rare autoimmune disease in which interstitial lung disease (ILD) is the leading cause of morbidity and mortality. Clinical management of the lung disease is mainly based on pulmonary function testing (PFT) and their changes over time. Little is known about the reproducibility of PFT testing in SSc patients. The aim of this study was to assess the test–retest reliability and reproducibility of PFTs in SSc patients with or without ILD over 30 days in order determine the potential physiologic variation over the time. We performed prospective observational study of SSc patients. The FVC, FEV1/FVC ratio, DLCO and KCO parameters were assessed in this population at four different timepoints; T0 (time 0) and H3 (T0 + 3 h) defined test–retest reliability, D15 (T0 + 15 days) and D30 (T0 + 30 days) for reproducibility. A mixed linear model was used to test the effect of time (and therefore reproducibility) on patients and we looked for an interaction. We included 25 SSc patients divided in two groups, 14 with ILD and 11 non-ILD. Interactions between time and group were not significant and were not reported. Time and group did not significantly influence the different measures of the PFT: FVC [p values time and group effect respectively (0.33; 0.34)], FEV1/FVC ratio (0.093; 0.056) and DLCO (0.99; 0.13) in the ILD and non ILD group (Table S2). The analyse with interactions between time and group were not significant and are not reported. We also used a Bland Altman test to assess reproducibility for FVC (L) and DLCO (mMKpa/min/L), Figs. 1 and 2 respectively. The measurements were therefore reproducible over time and in each group. PFT parameters are reproducible over time in a clinically stable population of SSc (no significant effect of the time T0, H3, D15 and D30) and there is no significant distinction between patients with ILD and no ILD. These respiratory functional data can further underline their use in clinical practice.

Abstract Systemic sclerosis (SSc) is a potentially serious and disabling connective tissue disease specially in case of interstitial lung disease (SSc-ILD). The aim of our study was to evaluate the potential utility of dosing in the induced sputum (IS) and to compare their levels in SSc-ILD and SSc-nonILD patients, as well as in healthy volunteers (HV). IS and sera values were also compared. In a prospective cross-sectional analysis, we studied the IS and serum provided from 25 SSc patients, 15 SSc-nonILD and 10 SSc-ILD, compared to 25 HV. We analyzed sputum cell composition and quantified in the supernatant and corresponding serum by commercially available immunoassays: IGFBP-1, IGFBP-2, IGFBP-3, TGF-β, IL-8, TNF-α, YKL-40, MMP-7 and MMP-9. Lung function was studied by the determination of FEV-1 (%), FVC (%), DLCO (%) and KCO (%). The IS of SSc patients had a lower weight than HV (p

Background: There is a pressing need to identify early biomarkers of lung involvement in systemic sclerosis (SSc) to start as soon as possible antifibrotic therapy. We aimed to identify extracellular vesicle-derived microRNAs (EV-miRNAs) that are differentially expressed between SSc patients with and without interstitial lung disease (ILD), explore their diagnostic value and investigate their functional properties., Methods: Small EVs (sEVs) derived from plasma were isolated from 91 well-characterised SSc patients with ILD (SSc-ILD, n=45), without ILD (SSc-no ILD, n=46) and 43 matched healthy subjects (HS). Small RNA sequencing followed by quantitative RT-PCR were used to identify and validate sEV-miRNAs associated to SSc-ILD. Correlations between SSc-ILD-associated miRNAs and clinical parameters were assessed, as well as the impact of related miRNAs/sEVs on fibrosis., Results: We identified a 4-miRNA signature associated with ILD in SSc context (miR-584-5p, miR-744-5p, miR-1307-3p and miR-10b-5p) (ROC AUC=0.85, 95% CI 0.76-0.94, p<0.0001). Deeper analysis revealed a correlation of these candidates with pulmonary function tests (DLCO and FVC), highlighting their capacity to monitor lung fibrosis progression in SSc patients. Furthermore, SSc-ILD-associated sEV miRNAs are positively correlated and enriched in circulating lymphocytes, suggesting that these immune cells are their cellular source. Finally, functional studies highlighted an alteration of functional properties of sEVs in SSc-ILD context mainly due to the transfer of profibrotic miR-584-5p in lung fibroblasts., Conclusions: Our sEV-based biomarker approach enabled to identify a promising 4-miRNA signature characteristic of ILD in SSc patients. Furthermore, the profibrotic properties of SSc-ILD-associated sEVs suggest a prominent role of these vesicles on SSc severity., (Copyright ©The authors 2025.)

Senescent cells promote progressive tissue degeneration through the establishment of a combined inflammatory and trophic microenvironment. The cellular senescence state has therefore emerged as a central driving mechanism of numerous age-related diseases, including osteoarthritis (OA), the most common rheumatic disease. Senescence hallmarks are detectable in chondrocytes, synoviocytes and sub-chondral bone cells. This study investigates how the senescence-driven microenvironment could impact the cell fate of resident osteoarticular mesenchymal stromal/stem cells (MSCs) that are hence contributing to OA disease progression. For that purpose, we performed a comparative gene expression analysis of MSCs isolated from healthy donors that were in vitro chronically exposed either to interferon-gamma (IFN-γ) or Transforming Growth Factor beta 1 (TGFβ1), two archetypical factors produced by senescent cells. Both treatments reduced MSC self-renewal capacities by upregulating different senescence-driven cycle-dependent kinase inhibitors. Furthermore, a common set of differentially expressed genes was identified in both treated MSCs that was also found enriched in MSCs isolated from OA patients. These findings highlight an imprinting of OA MSCs by the senescent joint microenvironment that changes their matrisome gene expression. Altogether, this research gives new insights into OA etiology and points to new innovative therapeutic opportunities to treat OA patients.

Osteoarthritis (OA) synovial membrane is mainly characterized by low-grade inflammation, hyperplasia with increased cell proliferation and fibrosis. We previously underscored a critical role for CEMIP in fibrosis of OA cartilage. However, its role in OA synovial membrane remains unknown. An in vitro model with fibroblast-like synoviocytes from OA patients and an in vivo model with collagenase-induced OA mice were used to evaluate CEMIP-silencing effects on inflammation, hyperplasia and fibrosis. Our results showed that i. CEMIP expression was increased in human and mouse inflamed synovial membrane; ii. CEMIP regulated the inflammatory response pathway and inflammatory cytokines production in vitro and in vivo; iii. CEMIP induced epithelial to mesenchymal transition pathway and fibrotic markers in vitro and in vivo; iv. CEMIP increased cell proliferation and synovial hyperplasia; v. CEMIP expression was increased by inflammatory cytokines and by TGF-β signaling; vi. anti-fibrotic drugs decreased CEMIP expression. All these findings highlighted the central role of CEMIP in OA synovial membrane development and underscored that targeting CEMIP could be a new therapeutic approach., (© 2022. The Author(s).)

An inflamed synovial membrane plays a major role in joint destruction and is characterized by immune cells infiltration and fibroblast proliferation. This proteomic study considers the inflammatory process at the molecular level by analyzing synovial biopsies presenting a histological inflammatory continuum throughout different arthritis joint diseases. Knee synovial biopsies were obtained from osteoarthritis (OA; n = 9), chronic pyrophosphate arthropathy (CPPA; n = 7) or rheumatoid arthritis (RA; n = 8) patients. The histological inflammatory score was determined using a semi-quantitative scale based on synovial hyperplasia, lymphocytes, plasmocytes, neutrophils and macrophages infiltration. Proteomic analysis was performed by liquid chromatography-mass spectrometry (LC-MS/MS). Differentially expressed proteins were confirmed by immunohistochemistry. Out of the 1871 proteins identified and quantified by LC-MS/MS, 10 proteins (LAP3, MANF, LCP1, CTSZ, PTPRC, DNAJB11, EML4, SCARA5, EIF3K, C1orf123) were differentially expressed in the synovial membrane of at least one of the three disease groups (RA, OA and CPPA). Significant increased expression of the seven first proteins was detected in RA and correlated to the histological inflammatory score. Proteomics is therefore a powerful tool that provides a molecular pattern to the classical histology usually applied for synovitis characterization. Except for LCP1, CTSZ and PTPRC, all proteins have never been described in human synovitis.

Osteoarthritis (OA) is recognized as being a cellular senescence-linked disease. Intra-articular injections of glucocorticoids (GC) are frequently used in knee OA to treat synovial effusion but face controversies about toxicity. We investigated the influence of GC on cellular senescence hallmarks and senescence induction in fibroblast-like synoviocytes (FLS) from OA patients and mesenchymal stem cells (MSC)., Methods: Cellular senescence was assessed via the proliferation rate, β-galactosidase staining, DNA damage and CKI expression (p21, p16 INK4A ). Experimental senescence was induced by irradiation., Results: The GC prednisolone did not induce an apparent senescence phenotype in FLS, with even higher proliferation, no accumulation of β-galactosidase-positive cells nor DNA damage and reduction in p21mRNA, only showing the enhancement of p16 INK4A . Prednisolone did not modify experimental senescence induction in FLS, with no modulation of any senescence parameters. Moreover, prednisolone did not induce a senescence phenotype in MSC: despite high β-galactosidase-positive cells, no reduction in proliferation, no DNA damage and no CKI enhancement was observed., Conclusions: We provide reassuring in vitro data about the use of GC regarding cellular senescence involvement in OA: the GC prednisolone did not induce a senescent phenotype in OA FLS (the proliferation ratio was even higher) and in MSC and did not worsen cellular senescence establishment.

Autophagy receptor p62/SQSTM1 signals a complex network that links autophagy-lysosomal system to proteasome. Phosphorylation of p62 on Serine 349 (P-Ser349 p62) is involved in a cell protective, antioxidant pathway. We have shown previously that P-Ser349 p62 occurs and is rapidly degraded during human synovial fibroblasts autophagy. In this work we observed that fingolimod (FTY720), used as a medication for multiple sclerosis, induced coordinated expression of p62, P-Ser349 p62 and inhibitory TFEB form, phosphorylated on Serine 211 (P-Ser211 TFEB), in human synovial fibroblasts. These effects were mimicked and potentiated by proteasome inhibitor MG132. In addition, FTY720 induced autophagic flux, LC3B-II up-regulation, Akt phosphorylation inhibition on Serine 473 but down-regulated TFEB, suggesting stalled autophagy. FTY720 decreased cytoplasmic fraction contained TFEB but induced TFEB in nuclear fraction. FTY720-induced P-Ser211 TFEB was mainly found in membrane fraction. Autophagy and VPS34 kinase inhibitor, autophinib, further increased FTY720-induced P-Ser349 p62 but inhibited concomitant expression of P-Ser211 TFEB. These results suggested that P-Ser211 TFEB expression depends on autophagy. Overexpression of GFP tagged TFEB in HEK293 cells showed concomitant expression of its phosphorylated form on Serine 211, that was down-regulated by autophinib. These results suggested that autophagy might be autoregulated through P-Ser211 TFEB as a negative feedback loop. Of interest, overexpression of p62, p62 phosphorylation mimetic (S349E) mutant and phosphorylation deficient mutant (S349A) in HEK293 cells markedly induced P-Ser211 TFEB. These results showed that p62 is involved in regulation of TFEB phosphorylation on Serine 211 but that this involvement does not depend on p62 phosphorylation on Serine 349., (© 2021 The Author(s).)

Nowadays, in the study of rheumatoid arthritis (RA), more and more interest is directed towards an earlier effective therapeutic intervention and the determination of companion markers for predicting response to therapy with the goal to prevent progressive joint damage, deformities, and functional disability. With the present work, we aimed at quantifying in a cohort of early RA (ERA) patients naïve to DMARD therapy, proteins whose increase was previously found associated with RA: serum amyloid A (A-SAA) and alarmins. Five A-SAA variants (SAA1α, SAA1β, SAA1γ, SAA2α, and SAA2β) but also S100A8 and S100A9 proteins were simultaneously quantified in plasma applying a method based on single targeted bottom-up proteomics LC-MS/MS. First, we compared their expression between ERA (n = 100) and healthy subjects (n = 100), then we focused on their trend by monitoring ERA patients naïve to DMARD treatment, 1 year after starting therapy. Only SAA1α and SAA2α levels were increased in ERA patients, and SAA2α appears to mostly mediate the pathological role of A-SAA. Levels of these variants, together with SAA1β, only decreased under biologic DMARD treatment but not under methotrexate monotherapy. This study highlights the importance to better understand the modulation of expression of these variants in ERA in order to subsequently better characterize their biological function. On the other hand, alarmin expression increased in ERA compared to controls but remained elevated after 12 months of methotrexate or biologic treatment. The work overcomes the concept of considering these proteins as biomarkers for diagnosis, demonstrating that SAA1α, SAA1β, and SAA2α variants but also S100A8 and S100A9 do not respond to all early treatment in ERA and should be rather considered as companion markers useful to improve the follow-up of treatment response and remission state. Moreover, it suggests that earlier use of biologics in addition to methotrexate may be worth considering., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Ciregia, Nys, Cobraiville, Badot, Di Romana, Sidiras, Sokolova, Durez, Fillet, Malaise and de Seny.)

Systemic sclerosis (SSc) is a rare connective tissue disease associated with rapid evolving interstitial lung disease (ILD), driving its mortality. Specific biomarkers associated with the progression of this lung disease are highly needed. We aimed to identify specific biomarkers of SSc-ILD to predict the evolution of the disease. For this, we compared prospectively serum levels of several biomarkers associated with lung fibrosis in SSc patients (n = 102), among which SSc-no ILD (n = 63) and SSc-ILD (n = 39), compared to healthy subjects (HS) (n = 39). We also performed a longitudinal study in a subgroup of 28 patients analyzing biomarkers variations and pulmonary function tests over a period of 2 years. Serum level of IGFBP-2 was significantly increased in SSc patients compared to HS, and negatively correlated with pulmonary function (assessed by carbon monoxide transfer coefficient (KCO)) (r = - 0.29, p < 0.01). Two-year longitudinal analysis in a subgroup of 28 SSc patients determined that IGFBP-2 variation was positively correlated with KCO at 2-year follow-up (r = 0.6, p < 0.001). SSc patients with a lower variation of IGFBP-2 (less than 22%) presented significant deterioration of pulmonary function at 2-year follow-up (p < 0.01). ROC curve analysis enabled us to identify that baseline IGFBP-2 > 105 ng/ml was associated with a poor outcome (KCO < 70% predicted) at 2-year follow-up (AUC = 0.75, p < 0.05). We showed for the first time that serum levels of IGFBP-2 might be a prognostic factor of the development of SSc-ILD.

Osteoarthritis is characterized by structural alteration of joints. Fibrosis of the synovial tissue is often detected and considered one of the main causes of joint stiffness and pain. In our earlier proteomic study, increased levels of vitronectin (VTN) fragment (amino acids 381-397) were observed in the serum of osteoarthritis patients. In this work, the affinity of this fragment for integrins and its putative role in TGF-β1 activation were investigated. A competition study determined the interaction of VTN (381-397 a.a.) with α V β 6 integrin. Subsequently, the presence of α V β 6 integrin was substantiated on primary human fibroblast-like synoviocytes (FLSs) by western blot and flow cytometry. By immunohistochemistry, β 6 was detected in synovial membranes, and its expression showed a correlation with tissue fibrosis. Moreover, β 6 expression was increased under TGF-β1 stimulation; hence, a TGF-β bioassay was applied. We observed that α V β 6 could mediate TGF-β1 bioavailability and that VTN (381-397 a.a.) could prevent TGF-β1 activation by interacting with α V β 6 in human FLSs and increased α-SMA. Finally, we analyzed serum samples from healthy controls and patients with osteoarthritis and other rheumatic diseases by nano-LC/Chip MS-MS, confirming the increased expression of VTN (381-397 a.a.) in osteoarthritis as well as in lupus erythematosus and systemic sclerosis. These findings corroborate our previous observations concerning the overexpression of VTN (381-397 a.a.) in osteoarthritis but also in other rheumatic diseases. This fragment interacts with α V β 6 integrin, a receptor whose expression is increased in FLSs from the osteoarthritic synovial membrane and that can mediate the activation of the TGF-β1 precursor in human FLSs.

We previously reported 18 FPRGD 2 uptake by the coxofemoral lining, intervertebral discs and facet joint osteophytes in OA using PET/SCAN imaging. However, the molecular mechanism by which the PRGD 2 tracer interacts with joint tissues and osteophytes in OA remains unclear. As PRGD 2 ligands are expected to belong to the RGD-specific integrin family, the purpose of this study was (i) to determine which integrin complexes display the highest affinity for PRGD2-based ligands, (ii) to analyze integrin expression in relevant tissues, and (iii) to test integrin regulation in chondrocytes using OA-related stimuli to increase the levels of fibrosis and ossification markers. To this end, the affinity of PRGD 2 -based ligands for five heterodimeric integrins was measured by competition with 125 I-echistatin. In situ analyses were performed in human normal vs. OA cartilage and spinal osteophytes. Osteophytes were characterized by (immuno-)histological staining. Integrin subunit expression was tested in chondrocytes undergoing dedifferentiation, osteogenic differentiation, and inflammatory stimulation. The integrins α V β 5 , α V β 3 , and α V β 6 presented the highest affinity for PRGD 2 -based ligands. In situ, the expression of these integrins was significantly increased in OA compared to normal cartilage. Within osteophytes, the mean integrin expression score was significantly higher in blood vessels, fibrous areas, and cells from the bone lining than in osteocytes and cartilaginous zones. In vitro, the levels of integrin subunits were significantly increased during chondrocyte dedifferentiation (except for β 6 ), fibrosis, and osteogenic differentiation as well as under inflammatory stimuli. In conclusion, anatomical zones (such as OA cartilage, intervertebral discs, and facet joint osteophytes) previously reported to show PRGD 2 ligand uptake in vivo expressed increased levels of α V β 5 , α V β 3 , and β 6 integrins, whose subunits are modulated in vitro by OA-associated conditions that increase fibrosis, inflammation, and osteogenic differentiation. These results suggest that the increased levels of integrins in OA compared to normal tissues favor PRGD2 uptake and might explain the molecular mechanism of OA imaging using the PRGD 2 -based ligand PET/CT., Competing Interests: Competing interestsThe authors declare no competing interests., (© The Author(s) 2020.)

It is now well recognized that osteoarthritis (OA) synovial membrane presents inflammatory components. The aim of this work is to provide evidence that similar inflammatory mechanisms exist in synovial membrane (n = 24) obtained from three pathologies presenting altogether an inflammatory gradient: OA, chronic pyrophosphate arthropathy (CPPA) and rheumatoid arthritis (RA). Synovial biopsies were first characterized by a histological score based on synovial hyperplasia and infiltration of lymphocytes, plasma cells, polymorphonuclear and macrophages. All biopsies were also analyzed by 2D-nano-UPLC-ESI-Q-Orbitrap for protein identification and quantification. Protein levels were correlated with the histological score. Histological score was in the range of 3 to 8 for OA, 5 to 13 for CPPA and 12 to 17 for RA. Of the 4,336 proteins identified by mass spectrometry, 51 proteins were selected for their strong correlation (p < 0.001) with the histological score of which 11 proteins (DNAJB11, CALR, ERP29, GANAB, HSP90B1, HSPA1A, HSPA5, HYOU1, LMAN1, PDIA4, and TXNDC5) were involved in the endoplasmic reticulum (ER) stress. Protein levels of S100A8 and S100A9 were significantly higher in RA compared to OA (for both) or to CPPA (for S100A8 only) and also significantly correlated with the histological score. Eighteen complement component proteins were identified, but only C1QB and C1QBP were weakly correlated with the histological score. This study highlights the inflammatory gradient existing between OA, CPPA and RA synovitis either at the protein level or at the histological level. Inflamed synovitis was characterized by the overexpression of ER stress proteins.

Background: Serum protein glycosylation is an area of investigation in inflammatory arthritic disorders such as rheumatoid arthritis (RA). Indeed, some studies highlighted abnormalities of protein glycosylation in RA. Considering the numerous types of enzymes, monosaccharides and glycosidic linkages, glycosylation is one of the most complex post translational modifications. By this work, we started with a preliminary screening of glycoproteins in serum from RA patients and controls., Methods: In order to isolate glycoproteins from serum, lectin wheat germ agglutinin was used and quantitative differences between patients and controls were investigated by LC-MS/MS. Consequently, we focused our attention on two glycoproteins found in this explorative phase: corticosteroid-binding globulin (CBG) and lipopolysaccharide-binding protein (LBP). The subsequent validation with immunoassays was widened to a larger number of early RA (ERA) patients (n = 90) and well-matched healthy controls (n = 90)., Results: We observed a significant reduction of CBG and LBP glycosylation in ERA patients compared with healthy controls. Further, after 12 months of treatment, glycosylated CBG and LBP levels increased both to values comparable to those of controls. In addition, these changes were correlated with clinical parameters., Conclusions: This study enables to observe that glycosylation changes of CBG and LBP are related to RA disease activity and its response to treatment.

Serum amyloid A (SAA) and S100 (S100A8, S100A9 and S100A12) proteins were previously identified as biomarkers of interest for rheumatoid arthritis (RA). Among SAA family, two closely related isoforms (SAA-1 and SAA-2) are linked to the acute-phase of inflammation. They respectively exist under the form of three (α, β, and γ) and two (α and β) allelic variants. We developed a single run quantitative method for these protein variants and investigated their clinical relevance in the context of RA. The method was developed and validated according to regulations before being applied on plasma coming from RA patients (n = 46), other related inflammatory pathologies (n = 116) and controls (n = 62). Depending on the activity score of RA, SAA1 isoforms (mainly of SAA1α and SAA1β subtypes) were found to be differentially present in plasma revealing their dual role during the development of RA. In addition, the weight of SAA1α in the total SAA response varied from 32 to 80% depending on the pathology studied. A negative correlation between SAA1α and SAA1β was also highlighted for RA early-onset (r = -0.41). SAA2 and S100A8/S100A9 proteins were significantly overexpressed compared to control samples regardless of RA stage. The pathophysiological relevance of these quantitative and qualitative characteristics of the SAA response remains unknown. However, the significant negative correlation observed between SAA1α and SAA1β levels in RA early-onset suggests the existence of still unknown regulatory mechanisms in these diseases., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

SSc is a rare disease of unknown origin associated with multiple organ involvement. One of the major complications that drives the mortality of SSc patients is interstitial lung disease. The course of SSc-interstitial lung disease progression has a wide spectrum. Since the treatment is based on aggressive immunosuppression it should not be given to stable or non-progressing disease. The correct identification of disease with high risk of progression remains a challenge for early therapeutic intervention, and biomarkers remain urgently needed. In fact, eight categories of biomarkers have been identified and classified according to the different biological pathways involved. The purpose of this article is to describe the main biomarkers thought to be of interest with clinical value in the diagnosis and prognosis of SSc-interstitial lung disease., (© The Author(s) 2019. Published by Oxford University Press on behalf of the British Society for Rheumatology.)

Background/aims: Synovial fibrosis is a pathological process that is observed in several musculoskeletal disorders and characterized by the excessive deposition of extracellular matrix, as well as cell migration and proliferation. Despite the fact that glucocorticoids are widely employed in the treatment of rheumatic pathologies such as osteoarthritis (OA) and rheumatoid arthritis, the mechanisms by which glucocorticoids act in the joint and their impacts on pro-fibrotic pathways are still unclear., Materials: Human OA synovial fibroblasts were obtained from knee and hip joints. Cells were treated with prednisolone (1 mM) or transforming growth factor-beta 1 (TGF-β1) (10 ng/ml) for 1 and 7 days for quantification of RNA and protein expression (by real-time quantitative reverse transcription-PCR and western blot, respectively), 72 h for immunocytochemistry analysis, and 48 h for proliferation (by BrdU assay) and migration (by wound assay) studies. In addition, cells were preincubated with prednisolone and/or the peroxisome proliferator-activated receptor gamma (PPAR-γ) agonist 15-deoxy-Δ-12,14-prostaglandin J2 (15d-PGJ2) for 6 h before adding TGF-β1. pSmad1/5, pSmad2 and β-catenin levels were analyzed by Western blot. The activin receptor-like kinase-5 (ALK-5) inhibitor (SB-431542) was employed for the mechanistic assays., Results: Prednisolone showed a predominant anti-fibrotic impact on fibroblast-like synoviocytes as it attenuated the spontaneous and TGF-β-induced gene expression of pro-fibrotic markers. Prednisolone also reduced α-sma protein and type III collagen levels, as well as cell proliferation and migration after TGF-β stimulation. However, prednisolone did not downregulate the gene expression of all the pro-fibrotic markers tested and did not restore the reduced PPAR-γ levels after TGF-β stimulation. Interestingly, anti-fibrotic actions of the glucocorticoid were reinforced in the presence of the PPAR-γ agonist 15d-PGJ2. Combined pretreatment modulated Smad2/3 levels and, similar to the ALK-5 inhibitor, blocked β-catenin accumulation elicited by TGF-β., Conclusions: Prednisolone, along with 15d-PGJ2, modulates pro-fibrotic pathways activated by TGF-β in synovial fibroblasts at least partially through the inhibition of ALK5/Smad2 signaling and subsequent β-catenin accumulation. These findings shed light on the potential therapeutic effects of glucocorticoids treatment combined with a PPAR-γ agonist against synovial fibrosis, although future studies are warranted to further evaluate this concern., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Osteoarthritis (OA) is a degenerative joint disease characterized by progressive cartilage degradation but also synovial membrane inflammation, osteophyte formation and subchondral bone sclerosis. Medical care is mainly based on alleviating pain symptoms, but to date, no effective drug can stop the disease progression. Cartilage is a tissue composed of only one cell type, chondrocytes, wrapped in a collagen rich extracellular matrix they synthesize. Chondrocytes can adopt different phenotypes in vivo and in vitro, defined by the collagen type they produce. Isolated from their matrix, chondrocytes present the particularity to dedifferentiate, producing fibroblastic type I and III collagens. With OA onset, chondrocytes undergo multiple changes, in terms of proliferation, viability, but also secretory profile. The acquisition of a hypertrophic phenotype (producing aberrant type X collagen and catabolic MMP-13 protease) by chondrocytes is well documented and contributes to OA development. However, it is increasingly believed that chondrocytes rather acquire a variety of degenerated phenotypes at the onset of OA, including a "dedifferentiated-like" phenotype that might also contribute to OA progression. In this review, we will (i) present molecular knowledge underlying dedifferentiation process, (ii) emphasize connections between dedifferentiation and OA and (iii) consider OA therapeutic strategies aiming at the maintenance of chondrogenic phenotype., (Copyright © 2019 Elsevier Inc. All rights reserved.)

CEMIP (for "Cell migration-inducing protein" also called KIAA1199 and Hybid for "Hyaluronan-binding protein") expression is increased in cancers and described as a regulator of cell survival, growth and invasion. In rheumatoid arthritis, CEMIP is referred to as an angiogenic marker and participates in hyaluronic acid degradation. In this study, CEMIP expression is investigated in healthy and osteoarthritis (OA) cartilage from human and mouse. Its role in OA physiopathology is deciphered, specifically in chondrocytes proliferation and dedifferentiation and in the extracellular matrix remodeling. To this end, CEMIP, αSMA and types I and III collagen expressions were assessed in human OA and non-OA cartilage. CEMIP expression was also investigated in a mouse OA model. CEMIP expression was studied in vitro using a chondrocyte dedifferentiation model. High-throughput RNA sequencing was performed on chondrocytes after CEMIP silencing. Results showed that CEMIP was overexpressed in human and murine OA cartilage and along chondrocytes dedifferentiation. Most of genes deregulated in CEMIP-depleted cells were involved in cartilage turnover (e.g., collagens), mesenchymal transition and fibrosis. CEMIP regulated β-catenin protein level. Moreover, CEMIP was essential for chondrocytes proliferation and promoted αSMA expression, a fibrosis marker, and TGFβ signaling towards the p-Smad2/3 (Alk5/PAI-1) pathway. Interestingly, CEMIP was induced by the pSmad1/5 (Alk1) pathway. αSMA and type III collagen expressions were overexpressed in human OA cartilage and along chondrocytes dedifferentiation. Finally, CEMIP was co-expressed in situ with αSMA in all OA cartilage layers. In conclusion, CEMIP was sharply overexpressed in human and mouse OA cartilage and along chondrocytes dedifferentiation. CEMIP-regulated transdifferentiation of chondrocytes into "chondro-myo-fibroblasts" expressing α-SMA and type III collagen, two fibrosis markers. Moreover, these "chondro-myo-fibroblasts" were found in OA cartilage but not in healthy cartilage.

Phosphorylation of p62/SQSTM1 (p62) on Serine 349 (P-Ser349 p62) as well as proteasome dysfunction have been shown to activate the cell protective Keap1/Nrf2 pathway. We showed previously that BAY 11-7085-induced human synovial fibroblast cell death includes autophagy and p62 downregulation. In this work, we have studied expression of P-Ser349 p62 in human synovial fibroblasts. Results showed that P-Ser349 p62 was not detected in synovial cell extracts unless cells were cultured in the presence of proteasome inhibitor (MG132). MG132 revealed P-Ser349 p62 turnover, that was further increased by concomitant autophagy inhibition and markedly enhanced in serum starved cells. Starvation sensitized synovial fibroblasts to BAY 11-7085 while MG132 protected both non-starved and starved cells from BAY 11-7085-induced cell death. Lentivirus mediated overexpression of phosphorylation-mimetic p62 mutant S349E markedly protected synovial fibroblasts from BAY 11-7085. Inhibitor of Keap1-P-S349 p62 interaction, K67, had synergistic effect with MG132. Starvation increased p62 molecular weight, that was reversed by serum and bovine serum albumin re-feeding. Furthermore, starvation markedly induced RAD23B. Increased endo-β-N-acetylglucosaminidase (ENGase) turnover was detected in starved synovial fibroblasts. PNGase F treatment produced faster migration p62 form in human synovial tissue extracts but starvation-like p62 form of higher molecular weight in synovial cell extracts. Co-transfection of NGLY1, with p62 or p62 mutants S349A and S349E markedly stabilized p62 expressions in HEK293 cells. Tunicamycin upregulated p62 and protected synovial fibroblasts from BAY 11-7085-induced cell death. These results showed that P-Ser349 p62 has pro-survival role in human synovial fibroblasts and that de-glycosylation events are involved in p62 turnover., Competing Interests: CONFLICTS OF INTEREST The authors declare that they have no conflicts of interest with the contents of this article.

Background: 18 F-FDG PET/CT has been proposed in the evaluation of the disease activity in rheumatoid arthritis (RA). The goals of this study were to evaluate the reproducibility of the technique, to compare metabolic parameters to clinical, biological and ultrasonographic parameters before and after treatment and to evaluate whether the early metabolic response was related to the outcome. 18 F- FDG PET/CT of the hands, wrists and knees was obtained in 15 patients with anti-TNFα refractory RA, at baseline and 16 weeks after treatment with rituximab. The number of PET-positive joints (PET+ joints), the cumulative standard uptake value (cSUV) and the composite index (CI) were defined. The composite clinical index DAS 28 , CRP serum levels and the number of joints positive at ultrasonography (US+ joints) and the cumulative synovial thickness (CST) were also assessed at baseline and week 24., Results: High interobserver agreement was observed, both at baseline and after treatment. The number of PET+ joints was not correlated with the number of joints tender or swollen. The 3 metabolic parameters were strongly correlated with US, CRP and DAS 28 at baseline and with US and CRP (CSUV, CI) at week 16, but no longer with the DAS 28 index. The metabolic response based on the change in the visual PET/CT joint analysis predicted the outcome with a high negative predictive value of 91%, with a 91% specificity, and an 86% accuracy., Conclusions: These preliminary data suggest that 18 F- FDG PET/CT is a reproducible and accurate tool for evaluating disease activity in refractory rheumatoid arthritis and its non-response to rituximab. The correlation obtained with US joint assessment gives relevance to objective diseased joints through imaging techniques., Competing Interests: The authors declare that they have no competing interests.Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Background: Osteoarthritis (OA) is the most common chronic joint disease usually diagnosed at relatively advanced stages when there is irreparable damage to the joint(s). Recently, we have identified two novel biomarkers C3f and V65 which appear to be OA-specific and therefore potential markers of early disease. We report the development of immunoassays for quantitative measure of these two novel biomarkers., Method: Monoclonal and polyclonal antibodies were generated by immunising mouse and rabbits respectively with peptide-carrier conjugates of C3f and V65. Affinity purified antibodies were used for immunoassays development and assays validated using serum from OA patients and controls., Results: The ELISAs developed showed spiked recovery of up to 96% for C3f and V65 peptides depending on serum dilutions with a coefficient of variation (CV) <10%. The intra- and inter-assay CVs for C3f and V65 were 1.3-10.8% and 4.2-10.3% respectively. Both assays were insensitive for measurements of the peptides in patients and the use of different signal amplification systems did not increase assay sensitivity., Conclusion: We have developed two immunoassays for measurements of C3f and V65 peptides biomarkers discovered by our earlier proteomic study. These assays could detect the endogenous peptides in serum samples from patients and controls but lacked sensitivity for accurate measurements of the peptides in patients. Our study highlights the difficulties and challenges of validating biomarker from proteomic studies and demonstrates how to overcome some of the technical challenges associated with developing immunoassays for small peptides.

Microfluidic liquid chromatography coupled to a nanoelectrospray source ion trap mass spectrometry was used for the absolute and simultaneous quantitation of C3f and the V65 vitronectin fragment in serum. The method was first carefully optimized and then validated in serum biological matrix. Stable isotopes for the two biomarkers of interest were used as stable isotope labeled peptide standards. A weighted 1/x 2 quadratic regression for C3f and a weighted 1/x quadratic regression for the V65 vitronectin peptide were selected for calibration curves. Trueness (with a relative bias <10%), precision (repeatability and intermediate precision <15%) and accuracy (risk <15%) of the method were successfully demonstrated. The linearity of results was validated in the concentration range of 2.5-200ng/mL for C3f and 2.5-100ng/mL for the V65 vitronectin fragment. Serum samples (n=147) classified in 7 groups [(healthy volunteers, OA with 5 grades of severity and rheumatoid arthritis (RA) patients] were analyzed with our new quantitative method. Our data confirm that C3f and the V65 vitronectin fragment are biomarkers of OA severity, but also that C3f fragment is further related to OA severity whereas the V65 vitronectin fragment is more related to early OA detection., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Osteoarthritis (OA) is a joint pathology characterized by progressive cartilage degradation. Medical care is mainly based on alleviating pain symptoms. Compelling studies report the presence of empty lacunae and hypocellularity in cartilage with aging and OA progression, suggesting that chondrocyte cell death occurs and participates to OA development. However, the relative contribution of apoptosis per se in OA pathogenesis appears complex to evaluate. Indeed, depending on technical approaches, OA stages, cartilage layers, animal models, as well as in vivo or in vitro experiments, the percentage of apoptosis and cell death types can vary. Apoptosis, chondroptosis, necrosis, and autophagic cell death are described in this review. The question of cell death causality in OA progression is also addressed, as well as the molecular pathways leading to cell death in response to the following inducers: Fas, Interleukin-1β (IL-1β), Tumor Necrosis factor-α (TNF-α), leptin, nitric oxide (NO) donors, and mechanical stresses. Furthermore, the protective role of autophagy in chondrocytes is highlighted, as well as its decline during OA progression, enhancing chondrocyte cell death; the transition being mainly controlled by HIF-1α/HIF-2α imbalance. Finally, we have considered whether interfering in chondrocyte apoptosis or promoting autophagy could constitute therapeutic strategies to impede OA progression., Competing Interests: The authors declare no conflict of interest.

Background: Glucocorticoid-induced leucine zipper (GILZ) is a mediator of the anti-inflammatory activities of glucocorticoids. However, GILZ deletion does not impair the anti-inflammatory activities of exogenous glucocorticoids in mice arthritis models and GILZ could also mediate some glucocorticoid-related adverse events. Osteoarthritis (OA) is a metabolic disorder that is partly attributed to adipokines such as leptin, and we previously observed that glucocorticoids induced leptin secretion in OA synovial fibroblasts. The purpose of this study was to position GILZ in OA through its involvement in the anti-inflammatory activities of glucocorticoids and/or in the metabolic pathway of leptin induction. The influences of mineralocorticoids on GILZ and leptin expression were also investigated., Methods: Human synovial fibroblasts were isolated from OA patients during knee replacement surgery. Then, the cells were treated with a glucocorticoid (prednisolone), a mineralocorticoid (aldosterone), a glucocorticoid receptor (GR) antagonist (mifepristone), a selective glucocorticoid receptor agonist (Compound A), mineralocorticoid receptor (MR) antagonists (eplerenone and spironolactone), TNF-α or transforming growth factor (TGF)-β. Cells were transfected with shRNA lentiviruses for the silencing of GILZ and GR. The leptin, IL-6, IL-8 and matrix metalloproteinase (MMP)-1 levels were measured by ELISA. Leptin, the leptin receptor (Ob-R), GR and GILZ expression levels were analyzed by western blotting and/or RT-qPCR., Results: (1) The glucocorticoid prednisolone and the mineralocorticoid aldosterone induced GILZ expression dose-dependently in OA synovial fibroblasts, through GR but not MR. Similar effects on leptin and Ob-R were observed: leptin secretion and Ob-R expression were also induced by prednisolone and aldosterone through GR; (2) GILZ silencing experiments demonstrated that GILZ was involved in the glucocorticoid-induced and mineralocorticoid-induced leptin secretion and Ob-R expression in OA synovial fibroblasts; and (3) GILZ inhibition did not alter the production of pro-inflammatory cytokines by OA synovial fibroblast or the anti-inflammatory properties of glucocorticoids., Conclusions: The absence of GILZ prevents corticoid-induced leptin and Ob-R expression without affecting the anti-inflammatory properties of glucocorticoids in OA synovial fibroblasts. Mineralocorticoids also induce leptin and Ob-R expression through GILZ.

Background: Nonunion is a failure of healing following a bone fracture. Its physiopathology remains partially unclear and the discovery of new mediators could promote the understanding of bone healing., Methods: Thirty-three atrophic nonunion (NU) patients that failed to demonstrate any radiographic improvement for 6 consecutive months were recruited for providing serum samples. Thirty-five healthy volunteers (HV) served as the control group. Proteomics studies were performed using SELDI-TOF-MS and 2D-DIGE approaches, associated or not with Proteominer® preprocessing, to highlight biomarkers specific to atrophic nonunion pathology. Peak intensities were analyzed by two statistical approaches, a nonparametric Mann-Whitney U tests (univariate approach) and a machine-learning algorithm called extra-trees (multivariate approach). Validation of highlighted biomarkers was performed by alternative approaches such as microfluidic LC-MS/MS, nephelometry, western blotting or ELISA assays., Results: From the 35 HV and 33 NU crude serum samples and Proteominer® eluates, 136 spectra were collected by SELDI-TOF-MS using CM10 and IMAC-Cu(2+) ProteinChip arrays, and 665 peaks were integrated for extra-trees multivariate analysis. Accordingly, seven biomarkers and several variants were identified as potential NU biomarkers. Their levels of expression were found to be down- or up-regulated in serum of HV vs NU. These biomarkers are inter-α-trypsin inhibitor H4, hepcidin, S100A8, S100A9, glycated hemoglobin β subunit, PACAP related peptide, complement C3 α-chain. 2D-DIGE experiment allowed to detect 14 biomarkers as being down- or up-regulated in serum of HV vs NU including a cleaved fragment of apolipoprotein A-IV, apolipoprotein E, complement C3 and C6. Several biomarkers such as hepcidin, complement C6, S100A9, apolipoprotein E, complement C3 and C4 were confirmed by an alternative approach as being up-regulated in serum of NU patients compared to HV controls., Conclusion: Two proteomics approaches were used to identify new biomarkers up- or down-regulated in the nonunion pathology, which are involved in bone turn-over, inflammation, innate immunity, glycation and lipid metabolisms. High expression of hepcidin or S100A8/S100A9 by myeloid cells and the presence of advanced glycation end products and complement factors could be the result of a longstanding inflammatory process. Blocking macrophage activation and/or TLR4 receptor could accelerate healing of fractured bone in at-risk patients.

Inhibition of proapoptotic pathways in synovial fibroblasts is one of the major causes of synovial proliferation and hyperplasia in rheumatic diseases. We have shown previously that NF-κB inhibitor BAY 11-7085, through inactivation of PPAR-γ, induces apoptosis in human synovial fibroblasts. In this work we showed that BAY 11-7085 induced autophagy that preceded BAY 11-7085-induced apoptosis. Of interest, BAY 11-7085 induced Serine 211 phosphorylation and degradation of glucocorticoid receptor (GR). Glucocorticoid prednisolone induced both activation and degradation of GR, as well as autophagy in synovial fibroblasts. BAY 11-7085-induced cell death was significantly decreased with glucocorticoid inhibitor mifepristone and with inhibitors of autophagy. Both BAY 11-7085-induced autophagy and GR activation were down regulated with PPAR-γ agonist, 15d-PGJ2 and MEK/ERK inhibitor UO126. Inhibition of autophagy markedly decreased endogenous and BAY 11-7085-induced ERK phosphorylation, suggesting a positive feed back loop between ERK activation and autophagy in synovial fibroblasts. Co-transfection of MEK1 with PPAR-γ1 in HEK293 cells caused known inhibitory phosphorylation of PPAR-γ1 (Serine 112) and enhanced GR degradation, in the absence or presence of prednisolone. Furthermore, GR was both phosphorylated on Serine 211 and down regulated in synovial fibroblasts during serum starvation induced autophagy. These results showed that GR activation and PPAR-γ inactivation mediated BAY 11-7085-induced autophagy., Competing Interests: The authors declare that they have no conflicts of interest with the contents of this article.

Objective: The aetiology of OA is not fully understood although several adipokines such as leptin are known mediators of disease progression. Since leptin levels were increased in synovial fluid compared to serum in OA patients, it was suggested that joint cells themselves could produce leptin. However, exact mechanisms underlying leptin production by chondrocytes are poorly understood. Nevertheless, prednisolone, although displaying powerful anti-inflammatory properties has been recently reported to be potent stimulator of leptin and its receptor in OA synovial fibroblasts. Therefore, we investigated, in vitro, spontaneous and prednisolone-induced leptin production in OA chondrocytes, focusing on transforming growth factor-β (TGFβ) and Wnt/β-catenin pathways., Design: We used an in vitro dedifferentiation model, comparing human freshly isolated hip OA chondrocytes cultivated in monolayer during 1 day (type II, COL2A1 +; type X, COL10A1 + and type I collagen, COL1A1 -) or 14 days (COL2A1 -; COL10A1 - and COL1A1+)., Results: Leptin expression was not detected in day1 OA chondrocytes whereas day14 OA chondrocytes produced leptin, significantly increased with prednisolone. Activin receptor-like kinase 1 (ALK1)/ALK5 ratio was shifted during dedifferentiation, from high ALK5 and phospho (p)-Smad2 expression at day1 to high ALK1, endoglin and p-Smad1/5 expression at day14. Moreover, inactive glycogen synthase kinase 3 (GSK3) and active β-catenin were only found in dedifferentiated OA chondrocytes. Smad1 and β-catenin but not endoglin stable lentiviral silencing led to a significant decrease in leptin production by dedifferentiated OA chondrocytes., Conclusions: Only dedifferentiated OA chondrocytes produced leptin. Prednisolone markedly enhanced leptin production, which involved Smad1 and β-catenin activation., (Copyright © 2015 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.)

Objective: This work reports on musculoskeletal uptake of ¹⁸F-FPRGD₂, targeting the integrin αvβ3, in patients who had undergone ¹⁸F-FPRGD₂ positron emission tomography combined with computed tomography (PET/CT) for oncologic purposes., Methods: Whole-body ¹⁸F-FPRGD₂ PET/CT images of 62 cancer patients were retrospectively reviewed to detect foci of musculoskeletal ¹⁸F-FPRGD₂ uptake. For 37 patients, a FDG PET/CT performed in clinical settings was available. In each joint with an abnormal uptake, the maximum standardized uptake value (SUVmax) was estimated., Results: A total of 260 musculoskeletal foci of ¹⁸F-FPRGD₂ uptake were detected. Most common sites of uptake were joints and discs (n = 160; 61.5%), entheses (osteotendinous and osteoligamentous junctions; n = 55; 21.2%) and recent fractures (n = 18; 6.9%). In addition, 27 (10.4%) miscellaneous foci were detected. Out of the 146 lesions for which a FDG PET was available, 63% showed both ¹⁸F-FPRGD₂ and FDG uptake, 33.6% did not show FDG avidity and 3.4% showed only FDG uptake. The uptake intensity of the 92 lesions positive with ¹⁸F-FPRGD₂ and FDG was similar with both radiopharmaceuticals, but the target-to-background (blood pool or muscle) ratios were significantly higher with ¹⁸F-FPRGD₂ than with FDG (p < 0.0001)., Conclusion: The ¹⁸F-FPRGD₂ uptake in joints, spine degenerative diseases and tendons was highly prevalent in our population. Up to one-third of ¹⁸F-FPRGD₂ foci showed no FDG uptake suggesting that ¹⁸F-FPRGD₂ signal may not be related to inflammatory angiogenesis only.

Objective: Glucocorticoids are powerful anti-inflammatory compounds that also induce the expression of leptin and leptin receptor (Ob-R) in synovial fibroblasts through TGF-βsignalling and Smad1/5 phosphorylation. Compound A (CpdA), a selective glucocorticoid receptor agonist, reduces inflammation in murine arthritis models and does not induce diabetes or osteoporosis, thus offering an improved risk:benefit ratio in comparison with glucocorticoids. Due to the detrimental role of leptin in OA pathogenesis, we sought to determine whether CpdA also induced leptin and Ob-R protein expression as observed with prednisolone., Methods: Human synovial fibroblasts and chondrocytes were isolated from the synovium and cartilage of OA patients after joint surgery. The cells were treated with prednisolone, TGF-β1, TNF-α and/or CpdA. Levels of leptin, IL-6, IL-8, MMP-1 and MMP-3 were measured by ELISA and expression levels of Ob-R phospho-Smad1/5, phospho-Smad2, α-tubulin and glyceraldehyde 3-phosphate dehydrogenase were analysed by western blotting., Results: CpdA, unlike prednisolone, did not induce leptin secretion or Ob-R protein expression in OA synovial fibroblasts. Moreover, CpdA decreased endogenous Ob-R expression and down-regulated prednisolone-induced leptin secretion and Ob-R expression. Mechanistically, CpdA, unlike prednisolone, did not induce Smad1/5 phosphorylation. CpdA, similarly to prednisolone, down-regulated endogenous and TNF-α-induced IL-6, IL-8, MMP-1 and MMP-3 protein secretion. The dissociative effect of CpdA was confirmed using chondrocytes with no induction of leptin secretion, but with a significant decrease in IL-6, IL-8, MMP-1 and MMP-3 protein secretion., Conclusion: CpdA, unlike prednisolone, did not induce leptin or Ob-R in human OA synovial fibroblasts, thereby demonstrating an improved risk:benefit ratio., (© The Author 2014. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Osteoarthritis (OA) is associated with a local inflammatory process. Dyslipidemia is known to be an underlying cause for the development of OA. Therefore, lipid and inflammatory levels were quantified ex vivo in blood and synovial fluid of OA patients (n=29) and compared to those of rheumatoid arthritis (RA) patients (n=27) or healthy volunteers (HV) (n=35). The role of apolipoprotein A-I (ApoA1) was investigated in vitro on inflammatory parameters using human joint cells isolated from cartilage and synovial membrane obtained from OA patients after joint replacement. Cells were stimulated with ApoA1 in the presence or not of serum amyloid A (SAA) protein and/or lipoproteins (LDL and HDL) at physiological concentration observed in OA synovial fluid. In our ex vivo study, ApoA1, LDL-C and total cholesterol levels were strongly correlated to each other inside the OA joint cavity whereas same levels were not or weakly correlated to their corresponding serum levels. In OA synovial fluid, ApoA1 was not as strongly correlated to HDL as observed in OA serum or in RA synovial fluid, suggesting a dissociative level between ApoA1 and HDL in OA synovial fluid. In vitro, ApoA1 induced IL-6, MMP-1 and MMP-3 expression by primary chondrocytes and fibroblast-like synoviocytes through TLR4 receptor. HDL and LDL attenuated joint inflammatory response induced by ApoA1 and SAA in a ratio dependent manner. In conclusion, a dysregulated lipidic profile in the synovial fluid of OA patients was observed and was correlated with inflammatory parameters in the OA joint cavity. Pro-inflammatory properties of ApoA1 were confirmed in vitro.

Acute graft-versus-host disease (aGVHD) remains a life-threatening complication of hematopoietic stem cell transplantation (HSCT) therefore limiting its application. To optimize the management of aGVHD and reduce therapy-related toxicity, early specific markers are needed. The main objective of this study was to uncover diagnostic biomarkers by comparing plasma protein profiles of patients at the time of acute GVHD diagnosis with those of patients undergoing HSCT without aGVHD. Additional analysis of samples taken 15 days before aGVHD diagnosis was also performed to evaluate the potential of our newly discovered biomarkers for early diagnosis. To get complementary information from plasma samples, we used three different proteomic approaches, namely 2D-DIGE, SELDI-TOF-MS and 2D-LC-MS(E). We identified and confirmed by the means of independent techniques, the differential expression of several proteins indicating significantly increased inflammation response and disturbance in the coagulation cascade. The variation of these proteins was already observed 15 days before GVHD diagnosis, suggesting the potential early detection of the disease before symptoms appearance. Finally, logistic regression analysis determined a composite biomarker panel comprising fibrinogen, fragment of fibrinogen beta chain, SAA, prothrombin fragments, apolipoprotein A1 and hepcidin that optimally discriminated patients with and without GVHD. The area under the receiver operating characteristic curve distinguishing these 2 groups was 0.95., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Objective: To determine if serum amyloid A (A-SAA) could be detected in human osteoarthritic (OA) joints and further clarify if high A-SAA level in joints result from a local production or from a diffusion process from abnormally elevated plasma concentration. Regulatory mechanism of A-SAA expression and its pro-inflammatory properties were also investigated., Methods: A-SAA levels in serum and synovial fluid of OA (n = 29) and rheumatoid arthritis (RA) (n = 27) patients were measured and compared to matched-healthy volunteers (HV) (n = 35). In vitro cell cultures were performed on primary joint cells provided from osteoarthritis patients. Regulatory mechanisms were studied using Western-blotting, ELISA and lentiviral transfections., Results: A-SAA was statistically increased in OA plasma patients compared to HV. Moreover, A-SAA level in OA plasma and synovial fluid increased with the Kellgren & Lauwrence grade. For all OA and RA patients, A-SAA plasma level was higher and highly correlated with its corresponding level in the synovial fluid, therefore supporting that A-SAA was mainly due to the passive diffusion process from blood into the joint cavity. However, A-SAA expression was also observed in vitro under corticosteroid treatment and/or under IL-1beta stimuli. A-SAA expression was down-regulated by PPAR-γ agonists (genistein and rosiglitazone) and up-regulated by TGF-β1 through Alk1 (Smad1/5) pathway. RhSAA induced proinflammatory cytokines (IL-6, IL-8, GRO-α and MCP-1) and metalloproteinases (MMP-1, MMP-3 and MMP-13) expression in FLS and chondrocytes, which expression was downregulated by TAK242, a specific TLR4 inhibitor., Conclusion: Systemic or local A-SAA expression inside OA joint cavity may play a key role in inflammatory process seen in osteoarthritis, which could be counteracted by TLR4 inhibition.

Leptin plays a central role in maintaining energy balance, with multiple other systemic effects. Despite leptin importance in peripheral regulation of mesenchymal stem cells (MSC) differentiation, little is known about its expression mechanism. Leptin is often described as adipokine, while it is expressed by other cell types. We have recently shown an in vitro leptin expression, enhanced by glucocorticoids in synovial fibroblasts (SVF). Here, we investigated leptin expression in MSC from bone marrow (BM-MSC) and umbilical cord matrix (UMSC). Results showed that BM-MSC, but not UMSC, expressed leptin that was strongly enhanced by glucocorticoids. Transforming growth factor β1 (TGF-β1) markedly inhibited the endogenous- and glucocorticoid-induced leptin expression in BM-MSC. Since TGF-β1 was shown to signal via ALK-5-Smad2/3 and/or ALK-1-Smad1/5 pathways, we analyzed the expression of proteins from both pathways. In BM-MSC, TGF-β1 increased phosphorylated Smad2 (p-Smad2) expression, while ALK-5 inhibitor (SB431542) induced leptin expression and significantly restored TGF-β1-induced leptin inhibition. In addition, both prednisolone and SB431542 increased p-Smad1/5 expression. These results suggested the ALK-5-Smad2 pathway as an inhibitor of leptin expression, while ALK-1-Smad1/5 as an activator. Indeed, Smad1 expression silencing induced leptin expression inhibition. Furthermore, prednisolone enhanced the expression of TGF-βRII while decreasing p-Smad2 in BM-MSC and SVF but not in UMSC. In vitro differentiation revealed differential osteogenic potential in SVF, BM-MSC, and UMSC that was correlated to their leptin expression potential. Our results suggest that ALK-1/ALK-5 balance regulates leptin expression in MSC. It also underlines UMSC as leptin nonproducer MSC for cell therapy protocols where leptin expression is not suitable.

Objective: Knee osteoarthritis (OA) is a heterogeneous, complex joint pathology of unknown aetiology. Biomarkers have been widely used to investigate OA but currently available biomarkers lack specificity and sensitivity. Therefore, novel biomarkers are needed to better understand the pathophysiological processes of OA initiation and progression., Methods: Surface enhanced laser desorption/ionisation-time of flight-mass spectrometry proteomic technique was used to analyse protein expression levels in 284 serum samples from patients with knee OA classified according to Kellgren and Lawrence (K&L) score (0-4). OA serum samples were also compared to serum samples provided by healthy individuals (negative control subjects; NC; n=36) and rheumatoid arthritis (RA) patients (n=25). Proteins that gave similar signal in all K&L groups of OA patients were ignored, whereas proteins with increased or decreased levels of expression were selected for further studies., Results: Two proteins were found to be expressed at higher levels in sera of OA patients at all four K&L scores compared to NC and RA, and were identified as V65 vitronectin fragment and C3fpeptide. Of the two remaining proteins, one showed increased expression (unknown protein at m/z of 3762) and the other (identified as connective tissue-activating peptide III protein) was decreased in K&L scores >2 subsets compared to NC, RA and K&L scores 0 or 1 subsets., Conclusion: The authors detected four unexpected biomarkers (V65 vitronectin fragment, C3f peptide, CTAP-III and m/z 3762 protein) that could be relevant in the pathophysiological process of OA as having significant correlation with parameters reflecting local inflammation and bone remodelling, as well as decrease in cartilage turnover.

In most diseases, the clinical need for serum/plasma markers has never been so crucial, not only for diagnosis, but also for the selection of the most efficient therapies, as well as exclusion of ineffective or toxic treatment. Due to the high sample complexity, prefractionation is essential for exploring the deep proteome and finding specific markers. In this study, three different sample preparation methods (i.e., highly abundant protein precipitation, restricted access materials (RAM) combined with IMAC chromatography and peptide ligand affinity beads) were investigated in order to select the best fractionation step for further differential proteomic experiments focusing on the LMW proteome (MW inferior to 40,000 Da). Indeed, the aim was not to cover the entire plasma/serum proteome, but to enrich potentially interesting tissue leakage proteins. These three methods were evaluated on their reproducibility, on the SELDI-TOF-MS peptide/protein peaks generated after fractionation and on the information supplied. The studied methods appeared to give complementary information and presented good reproducibility (below 20%). Peptide ligand affinity beads were found to provide efficient depletion of HMW proteins and peak enrichment in protein/peptide profiles., (Copyright 2010 Elsevier B.V. All rights reserved.)

Protein profiling using SELDI-TOF-MS has gained over the past few years an increasing interest in the field of biomarker discovery. The technology presents great potential if some parameters, such as sample handling, SELDI settings, and data analysis, are strictly controlled. Practical considerations to set up a robust and sensitive strategy for biomarker discovery are presented. This paper also reviews biological fluids generally available including a description of their peculiar properties and the preanalytical challenges inherent to sample collection and storage. Finally, some new insights for biomarker identification and validation challenges are provided.

It was shown recently that synovial fibroblast transformation into adipocytes reduced the expression of interleukin-6 (IL-6) and IL-8. However, the synovial fibroblast adipogenesis was inhibited in inflammatory conditions induced by the tumor necrosis factor-alpha (TNF-alpha). Furthermore, adipogenesis is often accompanied by leptin production, a proinflammatory adipokine in rheumatic diseases. In this study, we tested the phytohormone genistein for adipogenic and anti-inflammatory properties on human synovial fibroblasts. Results showed that genistein was able to transform synovial fibroblasts into adipocytes that expressed perilipin-A and produced adiponectin, but not leptin. Furthermore, genistein enhanced glucocorticoid-mediated synovial fibroblast adipogenesis and, in parallel, downregulated glucocorticoid-induced leptin and leptin receptor. Endogenous and TNF-alpha-induced expressions of IL-6, IL-8, p38, p65 and C/EBP-beta were also downregulated by genistein, showing its anti-inflammatory properties. Peroxisome proliferator- activated receptor-gamma (PPAR-gamma) agonist, rosiglitazone, had a synergic effect on genistein-induced adipogenesis, whereas the non-active tyrosine kinase inhibitor, daidzein, had a significantly inferior adipogenic activity than genistein. The Janus kinase-2 tyrosine kinase inhibitor, AG 490, mimicked the anti-leptin effect of genistein. These results showed that genistein-induced adipogenesis involves PPAR-gamma induction and tyrosine kinase inhibition. In conclusion, genistein, alone or coupled with glucocorticoids, have both adipogenic and anti-inflammatory effects on synovial fibroblasts.

Asthma is a complex inflammatory disease of airways. A network of reciprocal interactions between inflammatory cells, peptidic mediators, extracellular matrix components, and proteases is thought to be involved in the installation and maintenance of asthma-related airway inflammation and remodeling. To date, new proteic mediators displaying significant activity in the pathophysiology of asthma are still to be unveiled. The main objective of this study was to uncover potential target proteins by using surface-enhanced laser desorption/ionization-time of flight-mass spectrometry (SELDI-TOF-MS) on lung samples from mouse models of allergen-induced airway inflammation and remodeling. In this model, we pointed out several protein or peptide peaks that were preferentially expressed in diseased mice as compared to controls. We report the identification of different five proteins: found inflammatory zone 1 or RELM alpha (FIZZ-1), calcyclin (S100A6), clara cell secretory protein 10 (CC10), Ubiquitin, and Histone H4.

Clinical proteomics is a technical approach studying the entire proteome expressed by cells, tissues or organs. It describes the dynamics of cell regulation by detecting molecular events related to diseases development. Proteomic techniques focus mainly on identification of new biomarkers or new therapeutic targets. It is a multidisciplinary approach using medical, biological, bioanalytical and bioinformatics knowledges. A strong collaboration between these fields allowed SELDI-TOF-MS proteomics studies to be performed at the CHU and the University of Liège, in GIGA-Research facilities. The aim of these studies was driven along three main axes of research related to the identification of biomarkers specific to a studied pathology, to a common biological pathway and, finally, to a treatment response. This work was presented in the setting of the "Synthèse CHU 2009" meeting.

Objectives: Infliximab is the first anti-TNFalpha accepted by the Food and Drug Administration for use in inflammatory bowel disease treatment. Few clinical, biological and genetic factors tend to predict response in Crohn's disease (CD) patient subcategories, none widely predicting response to infliximab., Design and Methods: Twenty CD patients showing clinical response or non response to infliximab were used for serum proteomic profiling on Surface Enhanced Lazer Desorption Ionisation-Time of Flight-Mass Spectrometry (SELDI-TOF-MS), each before and after treatment. Univariate and multivariate data analysis were performed for prediction and characterization of response to infliximab., Results: We obtained a model of classification predicting response to treatment and selected relevant potential biomarkers, among which platelet aggregation factor 4 (PF4). We quantified PF4, sCD40L and IL-6 by ELISA for correlation studies., Conclusions: This first proteomic pilot study on response to infliximab in CD suggests association between platelet metabolism and response to infliximab and requires validation studies on a larger cohort of patients.

Background: SELDI-TOF mass spectrometry (MS) is a high-throughput proteomic approach with potential for identifying novel forms of serum biomarkers of arthritis., Methods: We used SELDI-TOF MS to analyze serum samples from patients with various forms of inflammatory arthritis. Several protein profiles were collected on different Bio-Rad Laboratories ProteinChip arrays (CM10 and IMAC-Cu(2+)) and were evaluated statistically to select potential biomarkers., Results: SELDI-TOF MS analyses identified several calgranulin proteins [S100A8 (calgranulin A), S100A9 (calgranulin B), S100A9*, and S100A12 (calgranulin C)], serum amyloid A (SAA), SAA des-Arg (SAA-R), and SAA des-Arg/des-Ser (SAA-RS) as biomarkers and confirmed the results with other techniques, such as western blotting, immunoprecipitation, and nano-LC-MS/MS. The S100 proteins were all able to significantly differentiate samples from patients with rheumatoid arthritis (RA), psoriatic arthritis (PsA), and ankylosing spondylitis (AS) from those of patients with inflammatory bowel diseases used as an inflammatory control (IC) group, whereas the SAA, SAA-R, and SAA-RS proteins were not, with the exception of AS. The 4 S100 proteins were coproduced in all of the pathologies and were significantly correlated with the plasma calprotectin concentration; however, these S100 proteins were correlated with the SAA peak intensities only in the RA and IC patient groups. In RA, these S100 proteins (except for S100A12) were significantly correlated with the serum concentrations of C-reactive protein, matrix metalloproteinase 3, and anti-cyclic citrullinated peptide and with the Disease Activity Score (DAS(28))., Conclusions: The SELDI-TOF MS technology is a powerful approach for analyzing the status of monomeric, truncated, or posttranslationally modified forms of arthritis biomarkers, such as the S100A8, S100A9, S100A12, and SAA proteins. The fact that the SELDI-TOF MS data were correlated with results obtained with the classic calprotectin ELISA test supports the reliability of this new proteomic technique.

Crohn's disease and ulcerative colitis known as inflammatory bowel diseases (IBD) are chronic immuno-inflammatory pathologies of the gastrointestinal tract. These diseases are multifactorial, polygenic and of unknown etiology. Clinical presentation is non-specific and diagnosis is based on clinical, endoscopic, radiological and histological criteria. Novel markers are needed to improve early diagnosis and classification of these pathologies. We performed a study with 120 serum samples collected from patients classified in 4 groups (30 Crohn, 30 ulcerative colitis, 30 inflammatory controls and 30 healthy controls) according to accredited criteria. We compared protein sera profiles obtained with a Surface Enhanced Laser Desorption Ionization-Time of Flight-Mass Spectrometer (SELDI-TOF-MS). Data analysis with univariate process and a multivariate statistical method based on multiple decision trees algorithms allowed us to select some potential biomarkers. Four of them were identified by mass spectrometry and antibody based methods. Multivariate analysis generated models that could classify samples with good sensitivity and specificity (minimum 80%) discriminating groups of patients. This analysis was used as a tool to classify peaks according to differences in level on spectra through the four categories of patients. Four biomarkers showing important diagnostic value were purified, identified (PF4, MRP8, FIBA and Hpalpha2) and two of these: PF4 and Hpalpha2 were detected in sera by classical methods. SELDI-TOF-MS technology and use of the multiple decision trees method led to protein biomarker patterns analysis and allowed the selection of potential individual biomarkers. Their downstream identification may reveal to be helpful for IBD classification and etiology understanding.

Objective: To identify serum protein biomarkers specific for rheumatoid arthritis (RA), using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS) technology., Methods: A total of 103 serum samples from patients and healthy controls were analyzed. Thirty-four of the patients had a diagnosis of RA, based on the American College of Rheumatology criteria. The inflammation control group comprised 20 patients with psoriatic arthritis (PsA), 9 with asthma, and 10 with Crohn's disease. The noninflammation control group comprised 14 patients with knee osteoarthritis and 16 healthy control subjects. Serum protein profiles were obtained by SELDI-TOF-MS and compared in order to identify new biomarkers specific for RA. Data were analyzed by a machine learning algorithm called decision tree boosting, according to different preprocessing steps., Results: The most discriminative mass/charge (m/z) values serving as potential biomarkers for RA were identified on arrays for both patients with RA versus controls and patients with RA versus patients with PsA. From among several candidates, the following peaks were highlighted: m/z values of 2,924 (RA versus controls on H4 arrays), 10,832 and 11,632 (RA versus controls on CM10 arrays), 4,824 (RA versus PsA on H4 arrays), and 4,666 (RA versus PsA on CM10 arrays). Positive results of proteomic analysis were associated with positive results of the anti-cyclic citrullinated peptide test. Our observations suggested that the 10,832 peak could represent myeloid-related protein 8., Conclusion: SELDI-TOF-MS technology allows rapid analysis of many serum samples, and use of decision tree boosting analysis as the main statistical method allowed us to propose a pattern of protein peaks specific for RA.

Motivation: Modern mass spectrometry allows the determination of proteomic fingerprints of body fluids like serum, saliva or urine. These measurements can be used in many medical applications in order to diagnose the current state or predict the evolution of a disease. Recent developments in machine learning allow one to exploit such datasets, characterized by small numbers of very high-dimensional samples., Results: We propose a systematic approach based on decision tree ensemble methods, which is used to automatically determine proteomic biomarkers and predictive models. The approach is validated on two datasets of surface-enhanced laser desorption/ionization time of flight measurements, for the diagnosis of rheumatoid arthritis and inflammatory bowel diseases. The results suggest that the methodology can handle a broad class of similar problems.

Increased resistance to beta-lactam antibiotics is mainly due to beta-lactamases. X-ray structures of zinc beta-lactamases unraveled the coordination of the metal ions, but their mode of action remains unclear. Recently, enzymes in which one of the zinc ligands was mutated have been characterized and their catalytic activity against several beta-lactam antibiotics measured. A molecular modeling study of these enzymes was performed here to explain the catalytic activity of the mutants. Coordination around the zinc ions influences the way the tetrahedral intermediate is bound; any modification influences the first recognition of the substrate by the enzyme. For all the studied mutants, at least one of the interactions fails, inducing a loss of catalytic efficiency compared to the wild type. The present studies show that the enzyme cavity is a structure of high plasticity both structurally and mechanistically and that local modifications may propagate its effects far from the mutated amino acid.