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Objective To investigate type II collagen (CII) as a joint-specific target of the anti-citrullinated protein antibody (ACPA) response in rheumatoid arthritis (RA). Methods Potential citrullinated neoepitopes were identified by high-resolution tandem mass spectrometry (MS/MS) of in vitro peptidylarginine deiminase 2 (PAD-2)-treated CII, and the relationship between citrullination and CII conformation was investigated by circular dichroism and conformation-dependent antibodies. Based on the MS analyses, synthetic peptides were designed and analyzed for serum IgG reactivity in the Epidemiological Investigation of RA (EIRA) case-control cohort of 1,949 RA patients and 278 healthy controls. Peptide-specific antibodies were purified from RA patient serum and used to stain RA cartilage specimens. Results We described the conformation-dependent citrullination pattern of CII after PAD-2 treatment at room temperature and 37°C and showed that CII could be citrullinated in its native triple-helical conformation. Screening of Arg and Cit pairs of synthetic peptides revealed new citrullinated B cell epitopes on CII. Antibodies directed to 2 proximal epitopes close to the C-terminus of the CII triple helix were recognized by autoantibodies in 21% and 17% of RA patients, respectively. Affinity-purified antibodies from RA sera directed to these 2 epitopes, but not antibodies directed to citrullinated α-enolase peptide 1, bound to RA cartilage. Conclusion These findings suggest that cartilage-directed anticitrulline immunity contributes to the induction of joint inflammation in RA. [ABSTRACT FROM AUTHOR]

The German Society of Rheumatology approved new German guidelines for the sequential medical treatment of rheumatoid arthritis (RA) based on the European League Against Rheumatism (EULAR) recommendations for the management of RA published in 2010. An update of the EULAR systematic literature research was performed in Medline, Embase, and Cochrane databases. Meta-analyses, controlled trials, cohort studies, and registry data addressing traditional and biologic disease-modifying antirheumatic drugs, glucocorticoids, and treatment strategies published between January 2009 and August 2011 were included. Two reviewers independently evaluated and compared the additional data that had been published after the time limit set by the EULAR recommendations. A national guideline working group developed an adapted set of recommendations. The new German guidelines were accepted by vote using an informal Delphi approach. Twelve recommendations and the resulting updated treatment algorithm were developed and approved as a practical orientation for rheumatologists. These recommendations are based on a successive treatment with traditional and biologic disease-modifying drugs depending on the individual progress of the disease and distinct patient characteristics. The German guidelines have been developed on the basis of the internationally well-recognized EULAR recommendations. In addition, more recent evidence from a systematic literature research was considered. They have been developed and approved by a group of national experts aiming at guidance for rheumatologists to reach best medical practice. [ABSTRACT FROM AUTHOR]

Objective To study the contribution of ADAM15, a disintegrin metalloproteinase that is up-regulated in the rheumatoid arthritis (RA) synovial membrane, to the characteristic resistance of RA synovial fibroblasts (RASFs) to apoptosis induction by genotoxic stress or stimulation with proapoptotic FasL, which is present at high concentrations in RA synovial fluid. Methods Caspase 3/7 activity and the total apoptosis rate in RASFs upon exposure to the DNA-damaging agent camptothecin or FasL were determined using enzyme assays and annexin V staining. Phosphorylated signaling proteins were analyzed by immunoblotting. RNA interference was used to silence ADAM15 expression. NF-κB activity was determined by enzyme-linked immunosorbent assay. Results RASFs displayed significantly higher caspase 3/7 activity upon camptothecin and FasL exposure when ADAM15 had been down-regulated by specific small interfering RNAs. Upon FasL stimulation, RASFs phosphorylated focal adhesion kinase (FAK) and c-Src (Src), and activated phosphatidylinositol 3-kinase as well as the transcription factor NF-κB. This ADAM15-dependent, FasL-induced activation of antiapoptotic kinases and NF-κB was demonstrated by a marked reduction of apoptosis upon knockdown of ADAM15 protein expression. Inhibitors specifically interfering with FAK and Src signaling, such as FAK inhibitor 14 and dasatinib, potently induce apoptosis in RASFs, with significant enhancement by the silencing of ADAM15. Conclusion ADAM15 contributes to apoptosis resistance in RASFs by activating the Src/FAK pathway upon FasL exposure, rendering the FAK/Src signaling pathway an interesting target for potential therapeutic intervention in RA. [ABSTRACT FROM AUTHOR]

Activation of the nuclear transcription factor κB (NF-κB) regulates the expression of inflammatory genes crucially involved in the pathogenesis of inflammatory diseases. NF-κB governs the expression of adhesion molecules that play a pivotal role in leukocyte–endothelium interactions. We uncovered the crucial role of NF-κB activation within endothelial cells in models of immune-mediated diseases using a "sneaking ligand construct" (SLC) selectively inhibiting NF-κB in the activated endothelium. The recombinant SLC1 consists of three modules: (i) an E-selectin targeting domain, (ii) a Pseudomonas exotoxin A translocation domain, and (iii) a NF-κB Essential Modifier-binding effector domain interfering with NF-κB activation. The E-selectin–specific SLC1 inhibited NF-κB by interfering with endothelial IκB kinase 2 activity in vitro and in vivo. In murine experimental peritonitis, the application of SLC1 drastically reduced the extravasation of inflammatory cells. Furthermore, SLC1 treatment significantly ameliorated the disease course in murine models of rheumatoid arthritis. Our data establish that endothelial NF-κB activation is critically involved in the pathogenesis of arthritis and can be selectively inhibited in a cell type- and activation stage-dependent manner by the SLC approach. Moreover, our strategy is applicable to delineating other pathogenic signaling pathways in a cell type-specific manner and enables selective targeting of distinct cell populations to improve effectiveness and risk–benefit ratios of therapeutic interventions. [ABSTRACT FROM AUTHOR]

Objective Chronic inflammation of the peripheral joints is a hallmark of rheumatoid arthritis (RA). The autoantibody response in RA has been shown to be directed mainly to ubiquitous antigens, whereas the response to cartilage proteins has been less extensively investigated. This study was undertaken to characterize the immune response in pristane-induced arthritis (PIA) in the rat to the cartilage-specific proteins type II collagen (CII) and type XI collagen (CXI) and to genetically fine-map their underlying major histocompatibility complex (MHC) associations. Methods The genetic control of CII and CXI immunity was mapped using intra-MHC-recombinant inbred strains immunized with the respective collagens. Reactivity with CII and CXI was tested in acute and chronic PIA and in 356 HLA-typed patients with recently diagnosed RA. Results Mapping of arthritis susceptibility within the MHC region revealed a 144-223-kb locus containing <12 genes, including paralogs for HLA-DQ and HLA-DR. Susceptibility to CII and CXI was linked to haplotypes RT1 av1 (DA) and RT1 f (DA.1F), respectively. After injection of pristane, rats of both strains developed weak T cell and IgG responses to CII, but not to CXI. In chronic arthritis, however, collagen reactivity was stronger, specific for CXI, and restricted to rats with RT1 f MHC. Among RA patients, 12% exhibited a specific IgG response to CXI, 6% to CII, and 6% to both collagens. Conclusion These findings demonstrate a shift in cartilage recognition in early and chronic arthritis in the rat, suggesting that CXI autoreactivity contributes to the perpetuation of chronic disease. The results provide evidence of the importance of joint antigens in arthritis development. [ABSTRACT FROM AUTHOR]

Objective The strongest susceptibility locus of psoriatic arthritis (PsA) is within the major histocompatibility complex (MHC) region (psoriasis susceptibility region 1, or PSORS1), and HLA-Cw*06:02 has been reported as the PSORS1 susceptibility allele. Non-HLA genes within the MHC region have also been implicated in PsA, but because of the strong linkage disequilibrium at chromosome 6p21, it is difficult to make a distinction between susceptibility alleles and linked markers. Recent studies have demonstrated that the association between PsA and the tumor necrosis factor (TNF) promoter polymorphism TNF*-857 is independent of PSORS1. The aim of this study was to replicate the independent association of TNF*-857 in patients with PsA. Methods A total of 909 patients with PsA and 1,315 healthy controls originating from the UK, Germany, and Italy were typed for TNF*-857 and for the estimated risk alleles of HLA-Cw*06:02. Results Overall, the results of genotyping in these 3 case-control cohorts replicated the finding that the frequency of carriers of TNF*-857 TT/CT who were negative for the PSORS1 risk allele was significantly higher among patients with PsA compared with control subjects (30% versus 21%; P = 9.17 × 10−5). Conclusion The results of this collaborative study indicate that TNF*-857T is a susceptibility allele for PsA independent of the PSORS1 allele. [ABSTRACT FROM AUTHOR]

Objective In rheumatoid arthritis, joint inflammation and cartilage destruction are mediated by autoantibodies directed to various self antigens. Type II collagen (CII)-specific antibodies are likely to play a role in this process and have been shown to induce experimental arthritis in susceptible animals. The purpose of this study was to reveal how arthritogenic autoantibodies recognize native CII in its triple-helical conformation. Methods Site-directed mutagenesis and crystallographic studies were performed to reveal crucial contact points between the CII antibody and the triple-helical CII peptide. Results The crystal structure of a pathogenic autoantibody bound to a major triple-helical epitope present on CII was determined, allowing a first and detailed description of the interactions within an arthritogenic complex that is frequently occurring in both mice and humans with autoimmune arthritis. The crystal structure emphasizes the role of arginine residues located in a commonly recognized motif on CII and reveals that germline-encoded elements are involved in the interaction with the epitope. Conclusion The crystal structure of an arthritogenic antibody binding a triple-helical epitope on CII indicates a crucial role of germline-encoded and arginine residues as the target structures. [ABSTRACT FROM AUTHOR]

Objectives. To investigate the presence of autoantibodies against mammalian chaperones of the endoplasmic reticulum (ER) in patients with RA and other immune-mediated diseases.Methods. Sera from healthy donors, from early RA patients with two follow-up samples, patients with SLE, SSc and IBD were collected and analysed for anti-ER chaperone antibodies. Detection of serum IgG antibodies against immunoglobulin heavy chain binding protein (BiP), glucose-regulated protein 94 (Grp94) and calnexin was carried out using ELISA. The specificity of sera positive for individual ER chaperones was confirmed by immunoblotting. Statistical analysis was performed using Welch’s t-test, Mann–Whitney U-test, partial correlation and Pearson’s correlation.Results. In patients with RA and SLE, autoantibody titres against BiP, Grp94 and calnexin were significantly higher than those in healthy controls. These autoantibodies were detectable in patients with early RA and titres remained stable for at least 6–12 months. Also several SSc and IBD patients exhibited autoantibodies against these ER chaperones; however, titres and frequencies were lower than in RA or SLE patients. Furthermore, anti-calnexin antibodies correlated significantly with the presence of BiP and Grp94 autoantibodies in patients with RA and SLE.Conclusion. Calnexin and Grp94 were identified as novel autoantigens in RA and calnexin in SLE. Since calnexin, Grp94 and BiP are ER-resident proteins of eukaryotic cells, our data suggest that autoantibody generation against ER chaperones is independent of initial exposure to the corresponding bacterial chaperones; rather, ER chaperones may represent genuine autoantigens. [ABSTRACT FROM PUBLISHER]

The article presents a study on disintegrin metallorproteinase ADAM15 capacity to defend chondrocytes against apoptosis caused by genotoxic stress and growth factor deprivation. It states that cellular adenosine triphosphate (ATP) was measured to determine the camptothecininduced cytotoxicity while real-time polymerase chain reaction was used to determine X-linked inhibitor apoptosis (XIAP). The results show significant caspase 3/7 activity reduction due to ADAM15-transfected chondrocytes.

The discovery of antibodies specific for citrullinated protein epitopes [anti-citrullinated protein antibodies (ACPAs)] is a hallmark for the diagnosis and prognosis of rheumatoid arthritis (RA) and will also be a useful tool for understanding the fundamental pathologic processes. There are several essential questions pertaining to ACPA that remain to be explored, such as understanding the early specificity of the underlying T-cell recognition, whether the production of ACPA is a primary or secondary process, and in the event of such antibodies being arthritogenic, whether they could possibly regulate the disease development. To answer these questions, animal models are needed, but unfortunately ACPA is not a prominent feature of any of the classical animal models of RA. However, we showed recently that ACPA can be isolated from animals susceptible to collagen-induced arthritis that are specific for citrullinated type II collagen (CII). The citrulline specificity could be visualized, and the specificity is determined primarily by a direct interaction with citrulline. We also demonstrated that these antibodies are specific for the citrullinated epitopes and are pathogenic in vivo. A new hypothesis to explain how inflammation in RA can be directed to cartilaginous joints and be self-perpetuating is suggested, which involves recognition of post-translational modifications (glycosylation and citrullination) on CII by T and B cells that can have both arthritogenic and regulatory consequences. [ABSTRACT FROM AUTHOR]

ADAM15 belongs to a family of transmembrane multi-domain proteins implicated in proteolysis, cell–cell and cell–matrix interactions in various disease conditions. In osteoarthritis (OA), ADAM15 is up-regulated in the chondrocytes already at early stages of cartilage degeneration where it seems to exert homeostatic effects likely associated with its ability to enhance integrin-mediated chondrocyte adhesion to the surrounding collagen matrix. The aim of our present study was, therefore, to characterize functional domains of ADAM15 involved in collagen II (CII) interaction and to analyse associated outside-in signalling events. Accordingly, ADAM15 and respective deletion mutants were stably transfected into the chondrocyte cell line T/C28a4. Transfected cells were adhered to CII and phosphoproteins analysed by Western blotting. Co-immunoprecipitation served to identify protein binding to ADAM15. Our results elucidate the prodomain as critical for the capacity of ADAM15 to enhance CII adhesion, thereby identifying for the first time a cell-adhesive role of a metalloproteinase prodomain. Moreover, the cytoplasmic tail of ADAM15 confers a modulatory effect on the autophosphorylation site Y397 of the focal adhesion kinase (FAK) during chondrocyte–collagen interaction. In conclusion, the newly uncovered impact of ADAM15 on signalling events that arise from chondrocyte interactions with its collagen matrix might contribute to the elucidation of the mechanism underlying its proposed chondroprotective role in degenerative cartilage disease. [ABSTRACT FROM AUTHOR]

The article presents a study which investigated the significance and pathogenic potential of a highly conserved major type II collagen triple-helical epitope-specific antibody in vivo and in vitro in patients with early rheumatoid arthritis (RA) and in experimental animal models of collagen-induced arthritis (CIA). The study compared disease progression in the CIA models in mice and rats with anti-U1 antibodies. The study observed increased levels of U1 antibodies in patients with early RA, especially in association with joint erosions.