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IntroductionCommercial assays measuring antibodies to citrullinated protein/peptide (ACPA) show poor quantitative agreement. The diagnostic industry has never adopted the International Union of Immunological Societies-Centers for Disease Control and Prevention (IUIS-CDC) ACPA reference standard. Recently, the National Institute for Biological Standards and Control (NIBSC) prepared a new candidate ACPA standard (18/204). We evaluated both reference materials using different commercially available ACPA assays.Materials and methodsThis is an international study in which the NIBSC candidate ACPA standard and the IUIS-CDC ACPA reference material were analysed together with 398 diagnostic samples from individuals with rheumatoid arthritis (RA) and in 1073 individuals who did not have RA using nine commercial ACPA assays.ResultsFor both reference materials and samples from individuals with RA and individuals who did not have RA, there were large differences in quantitative ACPA results between assays. For most assays, values for the IUIS-CDC standard were lower than values for NIBSC 18/204 and the IUIS-CDC/NIBSC ratio was comparable for several, but not all assays. When NIBSC 18/204 was used as a calibrator, an improvement in alignment of ACPA results across several of the evaluated assays was obtained. Moreover, NIBSC 18/204 could align clinical interpretation for some but not all assays.ConclusionAdoption of an international standard for ACPA determination is highly desirable. The candidate NIBSC 18/204 standard improved the standardisation and alignment of most ACPA assays and might therefore be recommended to be used as reference in commercial assays.

Aims: To determine the diagnostic value of anti-acetylated peptide antibodies (AAPA) in patients with rheumatoid arthritis (RA). Methods: Three acetylated peptides (ac-lysine, ac-lysine.inv and ac-ornithine) derived from vimentin were employed to measure AAPA by enzyme-linked immunosorbent assay (ELISA) in sera of 120 patients with early RA (eRA), 195 patients with established RA (est RA), 99 healthy controls (HC), and 216 patients with other inflammatory rheumatic diseases. A carbamylated and a citrullinated version of the vimentin peptide were used additionally. Receiver operating characteristics and logistic regression analyses were used to assess the discriminative capacity of AAPA. Results: AAPA were detected in 60% of eRA and 68.7% of estRA patients, 22.2% of HC, and 7.1– 30.6% of patients with other rheumatic diseases. Importantly, AAPA were also present in 40% of seronegative RA patients, while antibodies to the carbamylated peptide were detected less frequently. Diagnostic sensitivity of individual peptides for eRA was 28.3%, 35.8%, and 34% for ac-lysine, ac-ornithine, and ac-lysine.inv, respectively. Positive likelihood ratios (LR+) for eRA versus HC were 14.0, 7.1, and 2.1. While the presence of a single AAPA showed varying specificity (range: 84–98%), the presence of two AAPA increased specificity considerably since 26.7% of eRA, as compared with 6% of disease controls, were double positive. Thus, double positivity discriminated eRA from axial spondyloarthritis with a LR+ of 18.3. Remarkably, triple positivity was 100% specific for RA, being observed in 10% of eRA and 21.5% of estRA patients, even in the absence of RF and ACPA. Conclusion: AAPA are highly prevalent in early RA and occur also independently of RF and ACPA, thereby reducing the gap of seronegativity. Furthermore, multiple AAPA reactivity increased the specificity for RA, suggesting high diagnostic value of AAPA testing.

Objective: Little is known about the experiences, values and needs of people without arthritis who undergo predictive biomarker testing for the development of rheumatoid arthritis (RA). Our study aimed to explore the perspectives of these individuals and describe their information needs.\ud \ud Methods: A qualitative, multicenter interview study with a thematic analysis was conducted in Austria, Germany and the UK. Individuals who underwent predictive biomarker testing for RA and had a positive test result, but no diagnosis of any inflammatory joint disease, were interviewed. Participants included patients with arthralgia and asymptomatic individuals. Information and education needs were developed from the qualitative codes and themes using the Arthritis Educational Needs Assessment Tool (ENAT) as a frame of reference.\ud \ud Results: Thematic saturation was reached in 34 individuals (76% female; 24 [71%] with arthralgia and 10 [29%] asymptomatic individuals). Thirty‐seven codes were summarized into four themes, namely (i) decision making around whether to undergo initial predictive testing, (ii) willingness to consider further predictive tests and/or (iii) preventive interventions, including medication and (iv) varying reactions after receiving a positive test result. Individuals with arthralgia were more likely to be willing to take preventive action, undergo further testing, and experience psychological distress than asymptomatic individuals. All participants expressed the need for tailored, lay‐understandable information.\ud \ud Conclusion: Individuals at risk of RA are currently the subjects of research aimed at developing better predictive strategies and preventive approaches. Their perceptions and needs should be addressed to inform the future development of interventions combined with education.

Objectives The aims of this study were to investigate the extent of MRI-determined joint disease (erosion and synovitis) in SLE and to link this to autoantibody profiles known to be relevant to SLE, including ACPA, RF and anti-RA33 antibodies. Methods Contrast-enhanced MRI of the hand and wrist was performed in 34 symptomatic SLE patients and in 15 RA patients with similar disease duration. Images were scored by two observers using the OMERACT rheumatoid arthritis MRI scoring (RAMRIS) system. Findings were correlated with clinical examination and autoantibody status. Results Erosions were present at the wrist in 93% of SLE patients and at the MCP joints in 61% of SLE patients. Despite the high prevalence of MRI-determined erosion, only 8.8% of SLE patients were ACPA positive, although these patients had a higher burden of erosive disease. There was no positive correlation with anti-RA33 titres and erosion scores in the SLE patients, but there was a negative correlation with anti-RA33 titres and total bone oedema scores in the SLE patients. Ninety-three per cent of SLE patients had at least grade 1 synovitis at one or more MCP joints, and wrist joint synovitis was present in all the SLE patients. Conclusion An MRI-determined joint erosive phenotype is common in SLE, even in ACPA-negative cases. The conventional radiographic observation that anti-RA33 is not positively associated with erosion in patients with RA was also found to be the case in SLE patients.

Objectives The aim was to explore the function of the T-cell cytokine IFNγ for mesenchymal tissue remodelling in RA and to determine whether IFNγ signalling controls the invasive potential of fibroblast-like synoviocytes (FLS). Methods To assess architectural responses, FLS were cultured in three-dimensional micromasses. FLS motility was analysed in migration and invasion assays. Signalling events relevant to cellular motility were defined by western blots. Baricitinib and small interfering RNA pools were used to suppress Janus kinase (JAK) functions. Results Histological analyses of micromasses revealed unique effects of IFNγ on FLS shape and tissue organization. This was consistent with accelerated migration upon IFNγ stimulation. Given that cell shape and cell motility are under the control of the focal adhesion kinase (FAK), we next analysed its activity. Indeed, IFNγ stimulation induced the phosphorylation of FAK-Y925, a phosphosite implicated in FAK-mediated cell migration. Small interfering RNA knockdown of JAK2, but not JAK1, substantially abrogated FAK activation by IFNγ. Correspondingly, IFNγ-induced FAK activation and invasion of FLS was abrogated by the JAK inhibitor, baricitinib. Conclusion Our study contributes insight into the synovial response to IFNγ and reveals JAK2 as a potential therapeutic target for FLS-mediated joint destruction in arthritis, especially in RA.

Objective Performance of the 2010 American College of Rheumatology (ACR)/European League Against Rheumatism (EULAR) rheumatoid arthritis (RA) criteria was analysed in an internationally recruited early arthritis cohort (≤16 weeks symptom duration) enrolled in the ‘Stop-Arthritis-Very-Early’ trial. This sample includes patients with a variety of diseases diagnosed during follow-up. Methods Two endpoints were defined: Investigators’ diagnosis and disease-modifying antirheumatic drug (DMARD) treatment start during the 12-month follow-up. The 2010 criteria were applied to score Patients’ baseline data. Sensitivity, specificity, predictive values and areas under the receiver operating curves of this scoring with respect to both endpoints were calculated and compared to the 1987 criteria. The optimum level of agreement between the endpoints and the 2010 classification score ways estimated by Cohen’s ϰ coefficients. Results 303 patients had 12-months follow-up. Positive predictive values of the 2010 criteria were 0.68 and 0.71 for RA-diagnosis and DMARD-start, respectively. Sensitivity for RA-diagnosis was 0.85, for DMARD-start 0.8, whereas the 1987 criteria’s sensitivities were 0.65 and 0.55. The areas under the receiver operating curves of the 2010 criteria for RA-diagnosis and DMARD-start were 0.83 and 0.78. Analysis of inter-rater-agreement using Cohen’s ϰ demonstrated the highest ϰ values (0.5 for RA-diagnosis and 0.43 for DMARD-start) for the score of 6. Conclusions In this international very early arthritis cohort predictive and discriminative abilities of the 2010 ACR/EULAR classification criteria were satisfactory and substantially superior to the ’old’ 1987 classification criteria. This easier classification of RA in early stages will allow targeting truly early disease stages with appropriate therapy.

To investigate interferon-gamma (IFNgamma) signaling in peripheral blood mononuclear cells (PBMCs) from patients with systemic lupus erythematosus (SLE) by analyzing IFNgamma receptor (IFNgammaR) expression, STAT-1 expression and phosphorylation, and the regulation of IFNgamma-inducible genes.Fluorocytometry was used to investigate expression of STAT-1, pSTAT-1, CD95, HLA-DR, class I major histocompatibility complex (MHC), IFNgamma-inducible 10-kd protein (IP-10), monokine induced by IFNgamma (Mig), and IFNgammaR in PBMCs from SLE patients and healthy individuals. STAT-1 phosphorylation was determined by fluorocytometry and Western blotting after stimulation with IFNalpha or IFNgamma. Quantitative polymerase chain reaction was used to assess messenger RNA (mRNA) expression of the IFNgamma-inducible genes IP-10 and Mig shortly after preparation or after stimulation with IFNgamma in monocytes.STAT-1 expression was increased in PBMCs from SLE patients and correlated significantly with disease activity and with the IFN-inducible expression of CD95 and HLA-DR. STAT-1 expression also showed a trend toward association with class I MHC expression. In addition, the expression of other IFNgamma-inducible genes, such as IP-10 or Mig, was increased in SLE monocytes. While STAT-1 phosphorylation in SLE PBMCs and PBMCs from healthy individuals was similar after IFNalpha stimulation, incubation with IFNgamma induced STAT-1 phosphorylation only in SLE lymphocytes. Moreover, SLE monocytes showed a considerably higher increase in pSTAT-1 expression upon IFNgamma stimulation than monocytes from healthy individuals. Increased responsiveness of SLE monocytes to IFNgamma was also confirmed on the mRNA level, where expression of the IFN-inducible, STAT-1-dependent genes IP-10 and Mig was more efficiently increased in SLE cells. However, IFNgammaR was similarly expressed on SLE lymphocytes and monocytes and those from healthy individuals.In addition to supporting the role of IFNs in SLE immunopathogenesis in general, the findings of the present study support a role of IFNgamma in this disease.

Objectives:Autoantibody formation and T cell reactivity against the heterogeneous nuclear ribonucleoprotein A2 (hnRNP-A2) has been observed in patients with rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). Since no differences in epitope recognition were reported and the usefulness of anti-hnRNP-A2 antibodies as diagnostic markers of SLE is unknown, it was our objective to characterise linear B cell epitopes of hnRNP-A2 and to relate the anti-hnRNP-A2 antibody responses to disease activity and clinical features of SLE.Methods:Sequential serum samples from 15 patients with SLE and sera from patients with other rheumatic diseases and healthy subjects were investigated by ELISA for autoantibody reactivities against a set of 13 overlapping peptides spanning the RNA-binding region of hnRNP-A2. Antibody reactivity against the complete protein was determined by western immunoblotting and ELISA. SLE disease activity was assessed by European Consensus Lupus Activity Measure scores, by SLE Index scores and the British Isles Lupus Assessment index.Results:Anti-peptide antibody reactivities were found in 60% of SLE sera but in only 5% of control samples, and were mainly directed to four peptides, one of which (p155–175) appeared to be immunodominant. Antibodies to p155–175 were exclusively seen in patients with SLE and correlated with clinical disease activity as well as kidney and skin involvement. No correlations were found for the other anti-peptide antibody responses.Conclusion:Peptide p155–175 encompasses a disease-specific immunodominant epitope of hnRNP-A2. Since antibodies to p155–175 correlate with disease activity and nephritis, they may be useful as markers for active SLE.

Objective T cell intracytoplasmic antigen 1 (TIA-1) and TIA-1–related protein (TIAR) are involved in posttranscriptional regulation of the expression of tumor necrosis factor α (TNFα) and other proteins. Given the pivotal role of TNFα in chronic inflammatory diseases, this study was undertaken to analyze sera from patients with systemic autoimmune diseases for the presence of autoantibodies to TIA proteins and to investigate the expression of these proteins in inflamed tissue. Methods The presence of autoantibodies to TIA proteins in sera from 385 patients with rheumatic diseases and healthy controls was determined by immunoblotting using recombinant antigens. Expression of TIA proteins in skin and kidney tissue was analyzed by immunohistochemistry. Serum levels of TNFα were measured by enzyme-linked immunosorbent assay. Results Autoantibodies to TIA-1 and/or TIAR were detected in 61% of patients with systemic lupus erythematosus (SLE), 42% of patients with systemic sclerosis (SSc), 15–31% of patients with other rheumatic diseases, and 6% of healthy controls. Compared with patients negative for anti-TIA antibody, anti-TIA antibody–positive SLE patients had higher disease activity (P = 0.01), elevated antibodies to double-stranded DNA (P = 0.0003), and increased serum TNFα levels (P = 0.018). In SLE patients, anti-TIAR antibodies were associated with lupus nephritis (P = 0.02), while in patients with SSc, anti–TIA-1 was associated with lung involvement (P = 0.02). Immunohistochemical analysis of skin and kidney tissue revealed aberrant expression of TIA proteins in skin lesions from SLE and SSc patients, as well as in glomerular cells from SLE patients. Conclusion Aberrant expression of TIA proteins in inflammatory tissue may lead to systemic autoantibody responses, particularly in SLE and SSc. Increased occurrence of anti-TIA autoantibodies in patients with severe organ involvement may point to a possible pathogenetic role.

To study the effects of short-term intermediate dose glucocorticoid (GC) therapy in patients with active rheumatoid arthritis (RA) on circulating endothelial progenitor cells (EPC), which are known to influence cardiovascular risk, and to elucidate mechanisms potentially responsible for the reduction of EPCs in patients with active RA.EPCs were quantified in 29 patients with active RA by flow cytometry, colony forming unit (CFU) and circulating angiogenic cell (CAC) assays before and after 7 days of intermediate dose GC therapy. CFU from patients with RA and from healthy referents (HR) were cultured in vitro in the absence or presence of dexamethasone (Dex) and/or TNF.After 1 week of GC therapy, EPC increased from 0.026 (SD 0.003)% to 0.053 (SD 0.010)% (p0.01), and from 12 (SD 4) to 27 (SD 7) CFU/well (p0.02); CAC also increased from 7 (SD 2) to 29 (SD 8) cells/high power field (p0.05). In parallel, disease activity decreased significantly after GC treatment. TNF serum levels also decreased from 36 (SD 10) to 14 (SD 6) pg/ml (p0.0001). Addition of Dex to the RA CFU led to a significant increase of mean CFU counts, whereas addition of TNF induced a decrease of CFU.Our data indicate that TNF may be at least partly responsible for the reduction of EPC seen in patients with RA. Intermediate doses of GCs for a short period of time, apart from reducing disease activity, significantly increase circulating EPC.

OBJECTIVE Abatacept (CTLA-4Ig) blocks CD28-mediated T cell activation by binding to the costimulatory B7 ligands CD80/CD86 on antigen presenting cells. Costimulatory molecules, however, can also be expressed on T cells upon activation. Therefore, the aim of our study was to investigate direct effects of CTLA-4Ig on distinct T cell subsets in RA patients. METHODS Phenotypic and functional analyses of CD4(+) T cells, including CD4(+) FoxP3(+) CD25(+) regulatory T cells (Treg), from RA patients were performed before and during CTLA-4Ig therapy. In addition T cells from healthy volunteers were analysed on in vitro culture with CTLA-4Ig or anti-CD80 and anti-CD86 antibodies. Apoptotic DNA fragmentation in CD4(+) and CD4(+) FoxP3(+) T cells was measured by TUNEL staining. RESULTS We observed an increase in T cells, including Treg cells, after initiation of CTLA-4Ig therapy, which was linked to a downregulation of activation-associated marker molecules and CD95 on CD4(+) T cells and Treg cells. CTLA-4Ig decreased CD95-mediated cell death in vitro in a dose-dependent manner. Functional analysis of isolated Treg cells from RA patients further revealed a diminished suppression of responder T cell proliferation. This was found to be due to CTLA-4Ig-mediated blocking of CD80 and CD86 on responder T cells that led to a diminished susceptibility for Treg cell suppression. CONCLUSION CTLA-4Ig therapy in RA patients exerts effects beyond the suppression of T cell activation, which has to be taken into account as an additional mechanism of CTLA-4Ig treatment.

Josef S Smolen†, Daniel Aletaha, Klaus Machold, Valerie Nell, Kurt Redlich, Georg Schett, Tanja Stamm & Gunter Steiner †Author for correspondence Medical University of Vienna, Department of Rheumatology, Internal Medicine III, Waehringer Guertel 18–20, A-1090 Vienna, Austria Tel.: +43 1 404 004 300; Fax: +43 1 404 004 331; josef.smolen@wienkav.at ‘The time has come to make the topic of pre-arthritis a major item on the research agenda of the current decade.’

Objective To analyze changes in autoantibodies occurring in patients with systemic lupus erythematosus (SLE) treated with 4 infusions of the chimeric anti–tumor necrosis factor α (TNFα) antibody infliximab. Methods In an open-label safety study, 7 patients with SLE were treated with infliximab at weeks 0, 2, 6, and 10 in combination with azathioprine or methotrexate. Antibodies to double-stranded DNA (dsDNA) were determined by radioimmunoassay and the Crithidia luciliae indirect immunofluorescence assay; anticardiolipin antibodies (aCL) and antibodies to histone and chromatin were measured by enzyme-linked immunosorbent assay. Antihistone antibodies were also analyzed by immunoblotting. Peripheral blood mononuclear cells from healthy individuals and SLE patients were incubated for 2 weeks with or without TNFα. TNFα was removed by washing and by the addition of infliximab. Apoptotic cells were stained with annexin V and analyzed by flow cytometry. Results Autoantibodies to dsDNA increased in 5 of 7 patients. Histone, chromatin, and IgM aCL levels were increased in 4 of 7, 6 of 7, and 4 of 7 patients, respectively, peaking 4–10 weeks after the last infliximab infusion, but falling to baseline levels or lower thereafter. In the in vitro experiments, TNF withdrawal after long-term incubation with recombinant human TNF led to increased percentages of apoptotic cells. Conclusion While TNF blockade was clinically effective, the majority of SLE patients treated with infliximab showed an increase in autoantibodies to nuclear antigens and phospholipids. These increases were transient and were not associated with disease flares. Increased availability of apoptotic antigens after TNF blockade may play a role in the autoantibody formation induced by TNF blockade.

For patients who have combined features of rheumatoid arthritis, the limited cutaneous form of systemic sclerosis, and inflammatory myopathies, the concept of mixed connective tissue disease (MCTD) often helps to predict and diagnose organ problems and to educate the patient accordingly. With high titer IgG antibodies to U1 ribonucleoprotein (U1-snRNP), this concept is supported by a specific serologic marker, and autoantibodies to U1-snRNP and to heterogeneous nuclear ribonucleoprotein (hnRNP)-A2 display MCTD specificity with regard to the recognized epitopes. In addition, the association of MCTD with HLA-DR4 distinguishes it from systemic erythematosus lupus and systemic sclerosis, and speaks to its being a disease entity, rather than a mixture of yet undifferentiated collagen vascular diseases. The authors believe that the concept is useful in daily practice and accurate in the idea that MCTD constitutes a disease entity of its own.

Objective To investigate the efficacy of single and combined blockade of tumor necrosis factor (TNF), interleukin-1 (IL-1), and RANKL pathways on synovial inflammation, bone erosion, and cartilage destruction in a TNF-driven arthritis model. Methods Human TNF–transgenic (hTNFtg) mice were treated with anti-TNF (infliximab), IL-1 receptor antagonist (IL-1Ra; anakinra), or osteoprotegerin (OPG; an OPG-Fc fusion protein), either alone or in combinations of 2 agents or all 3 agents. Synovial inflammation, bone erosion, and cartilage damage were evaluated histologically. Results Synovial inflammation was inhibited by anti-TNF (−51%), but not by IL-1Ra or OPG monotherapy. The combination of anti-TNF with either IL-1Ra (−91%) or OPG (−81%) was additive and almost completely blocked inflammation. Bone erosion was effectively blocked by anti-TNF (−79%) and OPG (−60%), but not by IL-1Ra monotherapy. The combination of anti-TNF with IL-1Ra, however, completely blocked bone erosion (−98%). Inhibition of bone erosion was accompanied by a reduction of osteoclast numbers in synovial tissue. Cartilage destruction was inhibited by anti-TNF (−43%) and was weakly, but not significantly, inhibited by IL-1Ra, but was not inhibited by OPG monotherapy. The combination of anti-TNF with IL-1Ra was the most effective double combination therapy in preventing cartilage destruction (−80%). In all analyses, the triple combination of anti-TNF, IL-1Ra, and OPG was not superior to the double combination of anti-TNF and IL-1Ra. Conclusion Articular changes caused by chronic overexpression of TNF are not completely blockable by monotherapies that target TNF, IL-1, or RANKL. However, combined approaches, especially the combined blockade of TNF and IL-1 and, to a lesser extent, TNF and RANKL, lead to almost complete remission of disease. Differences in abilities to block synovial inflammation, bone erosion, and cartilage destruction further strengthen the rationale for using combined blockade of more than one proinflammatory pathway.

Objective To investigate the role of tumor necrosis factor (TNF) in systemic bone loss of chronic inflammatory conditions, such as rheumatoid arthritis (RA), and to address the therapeutic potential of osteoclast blockade. Methods We investigated systemic bone changes in human TNF transgenic (hTNFtg) mice, which spontaneously developed severe inflammatory arthritis. Results Osteodensitometry revealed a significant decrease in trabecular bone mineral density (BMD) (−37%) in hTNFtg mice, and histomorphometry revealed a dramatic loss of bone volume (−85%) compared with wild-type controls. Osteoclast-covered bone surface and serum levels of deoxypyridinoline crosslinks were significantly elevated, suggesting increased osteoclast-mediated bone resorption in hTNFtg mice. Osteoprotegerin (OPG) completely blocked TNF-mediated bone loss by increasing BMD (+89%) and bone volume (+647%). Most strikingly, formation of primary spongiosa was dramatically increased (+563%) in hTNFtg mice after OPG treatment. Osteoclast-covered bone surface and serum levels of deoxypyridinoline crosslinks were significantly decreased by OPG, suggesting effective blockade of osteoclast-mediated bone resorption. OPG did not influence levels of hTNF, TNF receptor I (TNFRI), interleukin-1β (IL-1β), and IL-6. However, OPG decreased bone formation parameters (osteoblast-covered bone surface and serum osteocalcin levels), which were elevated in hTNFtg mice. In contrast to OPG, bisphosphonates and anti-TNF treatment did not affect generalized bone loss in hTNFtg mice. Anti-TNF, however, did not affect levels of TNF and TNFRI at the concentrations tested. These data indicate that generalized bone loss due to increased TNF can be blocked by OPG. Conclusion OPG may represent a potent tool for preventing generalized loss of bone mass in chronic inflammatory disorders, especially RA.

Die Diagnostik der rheumatoiden Arthritis (RA) (bzw. chronischen Polyarthritis) wurde bislang durch das Fehlen krankheitsspezifischer serologischer Marker erschwert. So werden nach wie vor Rheumafaktoren (RF) trotz ihrer begrenzten Spezifitat als einzig anerkannte Markerantikorper verwendet und stellen unter den diagnostischen Kriterien des „American College of Rheumatology” das einzige serologische dar. In den letzten Jahren sind allerdings einige neue Autoantikorper beschrieben worden, denen eine im Vergleich zu RF weit hohere Krankheitsspezifitat zuzukommen scheint. Dies gilt insbesondere fur Autoantikorper gegen citrullinierte Antigene (Anti-Keratin bzw. Anti-Filaggrin-Antikorper sowie Antikorper gegen synthetisch hergestellte citrullinierte Peptide) die weitgehend spezifisch fur die RA zu sein scheinen. Als weitere Antikorper von diagnostischem Interesse sind Anti-RA33-Antikorper sowie Antikorper gegen das Stressprotein BiP zu nennen. Abgesehen von ihrer moglichen Anwendung als diagnostische Marker konnten diese Antikorper bzw. die ihnen zu Grunde liegenden zellularen Autoimmunreaktionen auch eine Rolle im pathologischen Geschehen der RA spielen.

Introduction: Scurfy mice are deficient in regulatory T cells (Tregs), develop a severe, generalized autoimmune disorder that can affect almost every organ and die at an early age. Some of these manifestations resemble those found in systemic lupus erythematosus (SLE). In addition, active SLE is associated with low Treg numbers and reduced Treg function, but direct evidence for a central role of Treg malfunction in the pathophysiology of lupus-like manifestations is still missing. In the present study, we characterize the multiorgan pathology, autoantibody profile and blood count abnormalities in scurfy mice and show their close resemblances to lupus-like disease. Methods: Scurfy mice have dysfunctional Tregs due to a genetic defect in the transcription factor Forkhead box protein 3 (Foxp3). We analyzed skin, joints, lung and kidneys of scurfy mice and wild-type (WT) controls by conventional histology and immunofluorescence (IF) performed hematological workups and tested for autoantibodies by IF, immunoblotting and enzyme-linked immunosorbent assay. We also analyzed the intestines, liver, spleen and heart, but did not analyze all organs known to be affected in scurfy mice (such as the testicle, the accessory reproductive structures, the pancreas or the eyes). We transferred CD4+ T cells of scurfy or WT mice into T cell-deficient B6/nude mice. Results: We confirm previous reports that scurfy mice spontaneously develop severe pneumonitis and hematological abnormalities similar to those in SLE. We show that scurfy mice (but not controls) exhibited additional features of SLE: severe interface dermatitis, arthritis, mesangioproliferative glomerulonephritis and high titers of anti-nuclear antibodies, anti-double-stranded DNA antibodies, anti-histone antibodies and anti-Smith antibodies. Transfer of scurfy CD4+ T cells (but not of WT cells) induced autoantibodies and inflammation of lung, skin and kidneys in T cell-deficient B6/nude mice. Conclusion: Our observations support the hypothesis that lupus-like autoimmune features develop in the absence of functional Tregs.

Objective Rheumatoid arthritis is a prototype of a destructive inflammatory disease. Inflammation triggered by the overexpression of tumor necrosis factor α (TNFα) is a driving force of this disorder and mediates tissue destruction. Since matrix metalloproteinases (MMPs) are among the molecules activated by TNFα, we hypothesized that overexpression of their natural inhibitor, tissue inhibitor of metalloproteinases 1 (TIMP-1), in TNFα transgenic mice could inhibit the development of destructive arthritis. Methods Systemic treatment was carried out by replication-defective adenoviral vectors for TIMP-1, β-galactosidase, or phosphate buffered saline (PBS), which were applied once at the onset of arthritis. Clinical, serologic, radiologic, and histologic outcomes were assessed 18 days after the treatment. Results The AdTIMP-1 group showed significantly reduced paw swelling and increased grip strength compared with the 2 control groups, whereas total body weight, TNFα, and interleukin-6 levels were similar in all 3 groups. Radiographic assessment revealed a significant reduction of joint destruction in the AdTIMP-1 group; this was confirmed by histologic analyses showing reduced formation of pannus and erosions in the AdTIMP-1 group compared with the AdLacZ and PBS control groups. The formation of arthritis-specific autoantibodies to heterogeneous nuclear RNP A2 was not observed in the AdTIMP-1 group but was present in the 2 control groups. Conclusion These results indicate a central role of MMPs in TNFα-mediated tissue damage in vivo and a promising therapeutic role for TIMP-1.

Objective To investigate the expression of the stroma cell product stem cell factor (SCF) in synovial fibroblasts (SFB) in patients with rheumatoid arthritis (RA) and osteoarthritis (OA), and to analyze the capacity of SFB to induce mast cell (MC) chemotaxis. Methods Synovial tissue was obtained from 29 patients with RA and 25 patients with OA. Tissue was dispersed by enzymatic digestion using collagenase. SFB were grown in serial passage and exposed to tumor necrosis factor α (TNFα) or control medium. Expression of SCF in cultured SFB was analyzed by reverse transcription–polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA), and immunostaining. The ability of SFB (supernatants) to induce MC migration was analyzed using a double-chamber chemotaxis assay and the human mast cell line HMC-1. In situ expression of SCF in synovial tissue from patients with RA (n = 6) and OA (n = 6) was examined by double immunohistochemistry using antibodies against SCF and the fibroblast-specific antibody ASO2. Results In both RA and OA, cultured SFB were found to express SCF messenger RNA, as assessed by RT-PCR. In addition, the SCF protein was detectable in cell lysates and supernatants of SFB by ELISA. Incubation of SFB with TNFα resulted in an increased expression and release of SCF. Recombinant human SCF (rHuSCF) and SFB supernatants induced significant migration of HMC-1 cells above control levels. In addition, exposure of SFB to TNFα led to an increased migration of HMC-1, and a blocking anti-SCF antibody inhibited the rHuSCF- and SFB-induced migration of HMC-1. In situ double immunostaining revealed expression of SCF in ASO2-positive SFB in the synovium of patients with RA. Conclusion Our results show that SFB (in RA and OA) express SCF and induce MC chemotaxis. Furthermore, TNFα was found to augment SCF expression in SFB. It is hypothesized that these cellular interactions play an important role in MC accumulation and related events in RA.

To assess the sensitivity and specificity of the recently described anti-Sa autoantibodies in order to determine their potential usefulness for the diagnosis of rheumatoid arthritis (RA).Sera from 67 patients with RA (including 31 patients with early RA of3 months duration), from 180 patients with other rheumatic diseases and from 30 healthy control subjects were investigated by immunoblotting employing partially purified Sa antigen. Additionally, all sera were tested for rheumatoid factor (RF), anti-A2/RA33, antinuclear antibodies (ANA) and ANA subsets.Twenty-one (31%) of the 67 patients with RA, but only four of the 180 control patients, were anti-Sa positive; therefore, anti-Sa was approximately 98% specific for RA. Anti-Sa was not associated with either RF or with anti-A2/RA33. The latter antibody was present in 21 RA sera, only eight of which also contained anti-Sa. Thus, 34 RA sera (51%) showed at least one of these two autoantibodies and, importantly, 18 of these sera were RF negative. Furthermore, of the 31 patients with early RA, 12 (40%) were anti-Sa and/or anti-A2/RA33 positive.Although the numbers studied remain small, taken together, these data suggest that anti-Sa may represent a promising novel serological marker with high specificity for RA.

Objective The characteristics of 3 patients with longstanding rheumatoid arthritis (RA) and consecutive evolution of limited cutaneous systemic sclerosis (lcSSc) were evaluated and compared with those of patients with lcSSc alone (n = 20) or with RA alone (n = 120). Methods Clinical features of the different patient populations were compared. Serologic analyses included tests for antinuclear antibodies (ANA) and ANA subsets, in particular anticentromere antibodies (ACA) and anti-heterogeneous nuclear RNP (hnRNP)-A2/RA33 (anti-A2/RA33). Results The 3 patients with RA developed lcSSc 11, 29, or 50 years after the onset of RA. Features of lcSSc were Raynaud's phenomenon, sclerodactyly, and telangiactasias in all 3 patients, and esophageal dysmotility in 1 patient. Rheumatoid factor (RF) and anti-A2/RA33 were each found in 2 patients, and 1 of these patients was seropositive for both RF and anti-A2/RA33. ACA titers were positive in all cases. However, similar to the development of RA prior to lcSSc, the occurrence of autoantibodies typical of RA preceded the occurrence of ACA, at least in 2 of the patients. Using affinity-purified antibodies, cross-reactivities between anti-centromere protein A (CENP-A) and anti-CENP-B antibodies with anti-A2/RA33 antigens were seen in the 2 anti-A2/RA33-positive patients. Such cross-reactivities were not found in lcSSc patients without concomitant RA. Epitope mapping revealed that both autoantibody specificities recognized the known major epitopes: anti-CENP-B reacted with the C-terminal region and anti-A2/RA33 with the second RNA binding domain in the N-terminal region of hnRNP-A2. Conclusion The RA-lcSSc overlap syndrome in these 3 patients with longstanding RA was characterized by an incomplete CREST (calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, and telangiectasias) syndrome. The study demonstrated the presence of autoantibodies typical of both diseases and cross-reactivity of ACA with hnRNP-A2/RA33 in the sera of these patients.

Objective To elucidate the nature of the antigen reactive with the "lupus erythematosus (LE) cell factor," the autoantibody involved in the LE cell phenomenon. Methods Serum samples from systemic lupus erythematosus (SLE) patients who were positive for the LE cell phenomenon (LEc+) and SLE patients who were negative for the LE cell phenomenon (LEc-) were used to characterize the nuclear antigen bound by the LE cell factor, by immunoblotting and immunoprecipitation techniques. Results All LEc+ sera, but none of the LEc- sera, uniformly reacted with a double band of MW approximately 30 kd in nuclear extracts. Depletion of nuclear protein extracts of antigens bound by pooled LEc- serum allowed precipitation of a low molecular weight protein by pooled LEc+ serum. This protein was able to block LE cell formation by LEc+ serum. Based on its reactivity with antihistone antibody and an electrophoretic mobility identical with that of precipitated and purified histone H1, this protein was identified as histone H1. Moreover, all LEc+ sera, but none of the LEc- sera, reacted with purified histone H1 by immunoblotting, whereas other histones were reactive with both types of sera. In addition, purified histone H1, but none of the other histones, strongly inhibited the induction of LE cells by LEc+ serum. Conclusion Histone H1 represents the major antigenic component recognized by the LE cell factor. Thus, the LE cell phenomenon appears to be due primarily to anti-histone H1 reactivity.

Introduction Systemic lupus erythematosus (SLE) is a heterogenous autoimmune disease, which can affect different organs. Increased proportions of CD4+CD25-Foxp3+ T cells have been described in SLE patients. The exact role of this cell population in SLE patients still remains unclear. We therefore analyzed this T cell subset in a large cohort of SLE patients with different organ manifestations. Methods Phenotypic analyses, proportions and absolute cell numbers of CD4+CD25-Foxp3+ T cells were determined by flow cytometry (FACS) in healthy controls (HC) (n = 36) and SLE patients (n = 61) with different organ manifestations. CD4+CD25-Foxp3+ T cells were correlated with clinical data, the immunosuppressive therapy and different disease activity indices. In patients with active glomerulonephritis, CD4+CD25-Foxp3+ T cells were analyzed in urine sediment samples. Time course analyses of CD4+CD25-Foxp3+ T cells were performed in patients with active disease activity before and after treatment with cyclophosphamide and prednisone. Results CD4+CD25-Foxp3+ T cells were significantly increased in active SLE patients and the majority expressed Helios. Detailed analysis of this patient cohort revealed increased proportions of CD4+CD25-Foxp3+ T cells in SLE patients with renal involvement. CD4+CD25-Foxp3+ T cells were also detected in urine sediment samples of patients with active glomerulonephritis and correlated with the extent of proteinuria. Conclusion CD4+CD25-Foxp3+ T cells resemble regulatory rather than activated T cells. Comparative analysis of CD4+CD25-Foxp3+ T cells in SLE patients revealed a significant association of this newly described cell population with active nephritis. Therefore CD4+CD25-Foxp3+ T cells might serve as an important tool to recognize and monitor SLE patients with renal involvement.

Objective. To assess the significance of autoantibodies to RA33, the A2 protein of the heterogeneous nuclear ribonucleoproteins (hnRNP), and to the related hnRNP proteins A1, B1, and B2 in rheumatic diseases. Methods. Using a partially purified preparation of hnRNP-A and hnRNP-B proteins, sera from 303 patients with various rheumatic diseases were investigated by immunoblotting. For the analysis of crossreactivities, autoantibodies were affinity purified by blot elution. Results. Anti-A2/RA33 was found in 35% of rheumatoid arthritis (RA) patients, 38% of mixed connective tissue disease (MCTD) patients, 23% of systemic lupus erythematosus (SLE) patients, and, apart from single exceptions, not in patients with other rheumatic diseases. All anti-A2/RA33-positive sera were also reactive with B1 and B2, and anti-A2/RA33 antibodies cross-reacted with both proteins. Antibodies to hnRNP-A1 were found less frequently; moreover, the majority of anti-A1-positive sera also contained anti-A2/RA33 antibodies. In anti-A1, anti-A2/RA33 doublepositive sera, cross-reactivity between the 2 antibodies was generally observed. In SLE patients, the presence of anti-A2/RA33 was correlated with the presence of anti-(U1) small nuclear RNP (snRNP) and anti-Sm (P < 0.0001 and P < 0.005, respectively), but there was no evidence for cross-reactivity between antibodies to hnRNP and antibodies to snRNP antigens. Conclusion. Since both hnRNPs and snRNPs are essential components of the spliceosome, the data show that the immune systems of patients with RA, SLE, and MCTD react to this functional complex. However, compared with MCTD and SLE patients, RA patients have a more restricted immune response to the spliceosome: they react to hnRNP proteins, particularly to hnRNP-A2/RA33, but not to snRNPs.

Background To facilitate the development of therapies for rheumatoid arthritis (RA), the Innovative Medicines Initiative BTCure has combined the experience from several laboratories worldwide to establish a series of protocols for different animal models of arthritis that reflect the pathogenesis of RA. Here, we describe chronic pristane-induced arthritis (PIA) model in DA rats, and provide detailed instructions to set up and evaluate the model and for reporting data. Methods We optimized dose of pristane and immunization procedures and determined the effect of age, gender, and housing conditions. We further assessed cage-effects, reproducibility, and frequency of chronic arthritis, disease markers, and efficacy of standard and novel therapies. Results Out of 271 rats, 99.6% developed arthritis after pristane-administration. Mean values for day of onset, day of maximum arthritis severity and maximum clinical scores were 11.8±2.0 days, 20.3±5.1 days and 34.2±11 points on a 60-point scale, respectively. The mean frequency of chronic arthritis was 86% but approached 100% in long-term experiments over 110 days. Pristane was arthritogenic even at 5 microliters dose but needed to be administrated intradermally to induce robust disease with minimal variation. The development of arthritis was age-dependent but independent of gender and whether the rats were housed in conventional or barrier facilities. PIA correlated well with weight loss and acute phase reactants, and was ameliorated by etanercept, dexamethasone, cyclosporine A and fingolimod treatment. Conclusions PIA has high incidence and excellent reproducibility. The chronic relapsing-remitting disease and limited systemic manifestations make it more suitable than adjuvant arthritis for long-term studies of joint-inflammation and screening and validation of new therapeutics.

Objective Both type I interferons (IFNα and IFNβ) and type II IFN (IFNγ) signal via pSTAT-1. Immunohistochemistry and the gene expression signatures of rheumatoid arthritis (RA) synovial tissue suggest an activated IFN/STAT-1 signaling pathway. The aim of this study was to determine the systemic activity of the IFN/STAT-1 signaling pathway in the peripheral blood cells of patients with RA. Methods Fluorocytometry or quantitative polymerase chain reaction was used to measure the expression of STAT-1, pSTAT-1, and IFN-inducible genes (monokine induced by interferon-γ [MIG], interferon-γ–inducible protein 10 [IP-10], and 2′,5′-oligoadenylate synthetase [OAS]) in the peripheral blood mononuclear cells (PBMCs) and purified CD14+ peripheral blood monocytes of patients with RA and healthy control subjects. PBMCs were also incubated for 48 hours with IFNs and several other cytokines to investigate influences on STAT-1 levels. To examine the significance of STAT-1 activation in RA monocytes after stimulation with IFNγ, the expression of pSTAT-1 and of the IFNγ-inducible chemokine MIG was measured using fluorocytometry. Results Levels of STAT-1 were significantly increased in peripheral lymphocytes and monocytes from patients with RA compared with those from healthy control subjects. STAT-1 levels correlated well with RA disease activity, as measured by the Disease Activity Score in 28 joints and the Clinical Disease Activity Index. Furthermore, STAT-1 messenger RNA expression in RA CD14+ monocytes correlated with the expression of other IFN-target genes, such as IP-10, OAS, or MIG. In RA PBMCs, STAT-1 expression was increased not only by IFNs but also by tumor necrosis factor. RA monocytes demonstrated a considerably higher increase in pSTAT-1 and MIG levels upon IFNγ stimulation when compared with monocytes from control subjects, indicating that RA monocytes are more sensitive to IFNγ stimulation. Conclusion In addition to supporting the role of IFNs in systemic proinflammatory activity, the results of this study further suggest preactivation of the IFNγ/STAT-1 signaling pathway, especially in RA monocytes.

Josef S. Smolen and Gu¨nter SteinerAutoimmunity, characterized by autoantibodyexpression, is one of the hallmarks of rheumatoid arthri-tis (RA). This feature distinguishes RA from otherinflammatory and degenerative joint diseases, such aspsoriatic arthritis, reactive arthritis, or osteoarthritis(OA), where autoantibody responses are absent. Theprominence of serologic manifestations of RA suggeststhat, despite the occurrence of joint inflammation anddestruction in the various forms of arthritis, these dis-eases display significant differences in etiopathogenesis.The first autoantibody described in RA morethan 60 years ago was rheumatoid factor (RF) (1). Sincethen, an increasing number of autoimmune responsesfor RA have been detected, some of which appear to bemuch more specific for RA than RF. These autoanti-bodies include the antiperinuclear factor (APF) andantikeratin antibodies (AKA) (2), anticollagen antibod-ies (3), antibodies to nuclear antigens such as Epstein-Barr nuclear antigen and RA33 (4,5), anti-Sa (6), andanti-p68 (7). Many of the autoantigens targeted by theseantibodies have been characterized: RF is directedagainst IgG (8); APF and AKA are directed againstcitrullinated filaggrin and other proteins containing cit-rulline, a posttranslationally modified arginine residue(9); RA33 is identical to the heterogeneous nuclearribonucleoprotein A2 (hnRNP-A2) (10); and p68 hasrecently been identified as the heavy chain bindingprotein BiP (11).Neither the histologic features of the RA synovialmembrane nor the cytokine profile are pathognomonic forthe disease: synovial lining cell hyperplasia and cellularinfiltrates can be found in many disorders (12), and theproinflammatory cytokines tumor necrosis factor (TNF ) and interleukin-1, which are considered hallmarksof RA, can also be found in other disorders, such aspsoriatic arthritis (13), in which TNF blockade can be usedas successfully as in RA (14). Despite these similarities, thetypical bone and cartilage destruction distinguishes RAfrom most other joint disorders. The only other distinguish-ing characteristic of RA is autoimmunity.Is there a link between autoimmunity and RAjoint destruction? Although the role of various auto-immune responses in the pathogenesis of RA is cur-rently unknown, RF, anticollagen, anti-RA33, and anti-citrulline antibodies are all present in synovial fluid. Thisfinding suggests local production (15–18). Therefore, itis tempting to hypothesize that autoimmune responsesin the joint might fuel the inflammatory process andcontribute to destruction by virtue of immune complexformation and cell-mediated reactivities.* Indeed, RAsynovial fluid shows low complement levels (20), andimmune complexes have been found in the fluid as wellas trapped in cartilage of RA patients (21,22). Cellularreactivity against immunoglobulins, collagen, and RA33/hnRNP-A2 has been observed among T cells from RA(and less so from non-RA) patients (23–25). Such auto-reactive T cells appear to be almost exclusively of theTh1 type (25), like those found in the RA synovialmembrane (26–28).One piece of evidence for the pathogenicity inRA of autoimmune responses in general and autoanti-bodies in particular is the passive transfer of disease byautoantibodies or autoreactive cells and/or the inductionof autoimmunity and autoimmune disease by the puta-tive autoantigen (29). Although only collagen amongknown antigens targeted in RA (30) can elicit erosivearthritis in experimental animal models, RF can beobserved in the destructive arthritis of MRL/lpr mice(31) and, interestingly, anti-RA33 has been found in seraof both MRL/

Objectives. To investigate joint involvement in SSc and its relationship with autoantibody to the hnRNP and to anti-cyclic citrullinated peptide (anti-CCP). Methods. Sera from 55 SSc patients were investigated. Joint involvement was determined by clinical, radiological and ultrasonographical evaluation. Anti-hnRNP proteins A1 and A2 (anti-hnRNP-A1/A2) antibodies were determined by immunoblotting. Anti-CCP, ACA, anti-topo I (ATA), Sm, U1-RNP, ribosomal RNP, Ro/SSA, La/SSB autoantibody and RF were determined. Results. Six patients were positive for anti-hnRNP-A2 autoantibody and two were anti-A1 positive. Eight patients had joint erosions: seven of the eight patients positive for anti-hnRNP-A2 or A1 presented articular involvement (P < 0.04) and five of the eight erosive patients were positive for either of the two autoantibodies (P < 0.02). Of the four patients positive for anti-CCP, none had anti-hnRNP but three had erosive aspects. ATAs were found in 10 patients, six of which were also positive for anti-hnRNP (P < 0.05). RF was positive in 16 patients and in seven among those with articular involvement (P < 0.04). RF was significantly associated with anti-hnRNP in patients with erosive arthritis (P < 0.02), but not with the presence of anti-hnRNP alone. Epitope mapping of the three strongest anti-hnRNP-A2-positive sera recognized the same major epitope as patients with RA. SSc patients have higher incidence of erosions and anti-hnRNP-A2/A1 positivity. RF test and anti-hnRNP had a statistically significant diagnostic value for articular involvement. Conclusions. These parameters might suggest that autoantibody to both hnRNP antigens might become a non-specific but useful marker for joint involvement in SSc patients and identify SSc patients prone to develop joint damage.

Objective To investigate which members of the heterogeneous nuclear RNP (hnRNP) family are targeted by autoantibodies from patients with systemic rheumatic diseases. Methods Using a semipurified preparation of natural hnRNP proteins, 365 sera from patients with rheumatic diseases and control subjects were screened by immunoblotting for the presence of autoantibodies. Bacterially expressed recombinant hnRNP D (AUF1) proteins were used for confirming the data obtained. Binding of RNA and autoantibody to AUF1 was investigated by gel retardation assays. Expression of AUF1 in cultivated cells and synovial tissue was analyzed by indirect immunofluorescence and immunohistochemistry. Results Autoantibodies to AUF1 proteins were detected in 33% of patients with systemic lupus erythematosus, 20% of patients with rheumatoid arthritis, 17% of patients with mixed connective tissue disease, and

Objective To investigate the safety of therapeutic tumor necrosis factor α (TNFα) blockade in patients with systemic lupus erythematosus (SLE), in whom this proinflammatory cytokine is significantly increased and may be involved in the disease pathogenesis. Methods In an open-label study, 6 patients with moderately active SLE (4 with nephritis and 3 with arthritis refractory to other therapies) were given 4 300-mg doses of infliximab, a chimeric anti-TNFα antibody, in addition to immunosuppression with azathioprine or methotrexate. Results The only significant adverse events observed were urinary tract infection in 3 patients, 1 of which was accompanied by Escherichia coli bacteremia, and a prolonged febrile episode of putatively viral origin in 1 of them. These patients had similar infectious conditions in the past. In none of the patients was it necessary to terminate the treatment prematurely. Levels of antibodies to double-stranded DNA and cardiolipin increased in 4 patients each, but this was not associated with a decrease in serum complement levels, with vascular events, or with flares. In contrast, disease activity declined during therapy. All 3 patients with joint involvement experienced remission of arthritis, which relapsed 8–11 weeks after the last infliximab infusion. In the 4 patients with lupus nephritis, proteinuria decreased significantly within 1 week after initiation of therapy and was diminished by ≥60% within 8 weeks, remaining at low levels until the end of the observation period (at least several months). Conclusion Infliximab did not lead to adverse events related to an increase in SLE activity, although autoantibodies to double-stranded DNA and cardiolipin increased, as expected. This finding, coupled with the clinical improvement in the inflammatory manifestations of the disease, indicates that further study in larger controlled trials is warranted.

Background Both, type I interferons (IFNα, IFNβ) and the type II IFN IFNγ signal via phosphorylating Stat1. Immunohistochemistry and gene expression signatures of synovial tissues suggest an activated IFN-Stat1-signalling-pathway in rheumatoid arthritis (RA). This study was performed to determine the activity of the IFN-Stat1-signalling-pathway in RA peripheral blood monocytes. Methods Flourocytometry or qPCR was used to measure the expression of Stat1, phospho-Stat1 (pStat1) and IFN-inducible genes, such as IP-10 and OAS in RA and healthy (HC) peripheral blood monocytes. To examine the significance of Stat1 and of the IFNγ-inducible chemokine MIG (monokine induced by IFNγ) were measured using fluorocytometry. Results Levels of Stat1 and pStat1 protein expression were significantly increased in RA monocytes when compared to HC (mfi 14.7±8.1 vs 8.0±3.9, p=0.0002; mfi 5.1±1.3 vs 3.2±0.7, p RA monocytes demonstrated a considerably higher increase in pStat1 and MIG levels upon IFNγ stimulation when compared to monocytes from HC (pStat1: +2.8±1.8 vs +1.5±1.1, p Conclusions Consistent with a systemic proinflammatory activity of RA monocytes, these studies suggest activation of the IFNγ-Stat1-signalling pathway in RA.

Objective To compare and investigate antihistone and antichromatin antibody responses as well as clinical variables in patients with systemic lupus erythematosus (SLE) who were either positive (LEC+) or negative (LEC−) for the lupus erythematosus (LE) cell phenomenon. Methods The binding properties of LEC+ and LEC− SLE sera to chromatin-associated nuclear antigens (histones H1, H2A, H2B, H3, H4; complexes of H2A–H2B, [H2A–H2B]–DNA, H1–DNA; total and H1-stripped chromatin; native and denatured DNA) were investigated. In addition, sera from patients with drug-induced lupus (by procainamide, hydralazine, or quinidine), as well as from patients with rheumatoid arthritis and osteoarthritis, were assessed. Enzyme-linked immunosorbent assay was used to detect specific antibody binding. Results Mirroring the important role of histone H1 in the formation of LE cells, anti–histone H1 reactivity was 8-fold higher in LEC+ sera than in LEC− sera. In addition, reactivities to most of the other antigens tested, i.e., other histones and histone–DNA complexes as well as chromatin and DNA, were significantly higher in LEC+ sera than in LEC− sera. All but 1 serum sample from the patients with drug-induced lupus were negative for LE cell formation as well as for anti–histone H1 reactivity, but displayed high antibody reactivities to histone–DNA complexes, including chromatin. Sera from patients with rheumatoid arthritis and osteoarthritis did not show significant binding to these antigens. When comparing the clinical features of LEC+ and LEC− SLE patients, severe organ involvement, including nephritis and central nervous system involvement, was common in the LEC+ group, but rare in the LEC− group. Conclusion A positive LE cell phenomenon not only correlated with the presence of high anti–histone H1 antibody levels in SLE, but also indicated serologically and clinically active disease with major organ involvement.

An inception cohort of 238 patients having peripheral joint synovitis of less than 12 months duration was evaluated clinically and followed prospectively for 1 year to determine the clinical significance of a number of rheumatoid arthritis (RA) associated autoantibodies. Serum samples collected at the time of the initial evaluation were tested for rheumatoid factor (RF) and antibodies to Sa (anti-Sa), RA-33, (pro)filaggrin [antifilaggrin antibody (AFA)], cyclic citrullinated peptide (anti-CCP), calpastatin, and keratin [antikeratin antibody (AKA)]. RF had a sensitivity of 66% and a specificity of 87% for RA. Anti-Sa, AFA, and anti-CCP all had a specificity of more than 90%, but a sensitivity of less than 50% for this diagnosis. Overall, there was a high degree of correlation between AFA, AKA, anti-Sa or anti-CCP, this being highest between anti-Sa and anti-CCP (odds ratio, 13.3; P < 0.001). Of the 101 patients who were positive for at least one of these four autoantibodies, 57% were positive for only one. Finally, anti-SA identified a subset of predominantly male RA patients with severe, erosive disease. Anti-SA, AFA and anti-CCP are all specific for early RA but, overall, have little additional diagnostic value over RF alone. Although these antibodies may preferentially recognize citrullinated antigens, the modest degree of concordance between them in individual patient sera suggests that it is unlikely a single antigen is involved in generating these responses., Introduction: A spectrum of autoantibodies is now known to be specifically associated with RA. There continues to be uncertainty as to what stage of the disease each of these autoantibodies develop, and whether they are associated with unique clinical features. Aims: To help address these questions, a spectrum of autoantibodies known to be associated with RA in a cohort of patients with early synovitis was evaluated. Methods: An inception cohort of 238 patients having peripheral joint synovitis of less than 12 months duration was evaluated clinicially then followed prospectively for 1 year. Patients were classified as having RA on the basis of fulfilling the 1987 criteria. Serum samples collected at the time of the initial evaluation were tested for anti-Sa and anti-RA-33 using immunoblotting, and to (pro)filaggrin (AFA), anti-CCP, and calpastatin (anti-RA-1) using enzyme-linked immunosorbent assay techniques. AKA were detected using immunoflurescence on human epidermal tissue. RF was tested by nephelometry. HLA-DRB1 alleles were determined using sequence specific primers. Initial and 1 year radiographs were evaluated for the presence of erosions. Results: Of the 238 patients with synovitis of recent onset in the cohort, 106 (45%) met RA criteria, 102 (96%) of whom met the criteria on their initial visit. Diagnoses in the remaining patients included 22 (9%) with reactive arthritis, 14 (6%) with psoriatic arthritis or another form of spondylarthropathy, 11 (5%) with another well-defined rheumatic diagnosis, and 85 (36%) with undifferentiated arthritis. The RA patients were significantly older than the nonRA patients (46 ± 13 versus 39 ± 13; P < 0.001), had higher mean swollen joint count (13.8 ± 9.7 versus 2.3 ± 2.3; P < 0.001), and higher C-reactive protein (CRP) level (1.9 ± 1.9 versus 1.6 ± 2.4; P < 0.01). Table 1 summarizes the prevalence of the various RA associated antibodies in patients diagnosed as having RF-positive (RF+) RA, RF-negative (RF-) RA, and nonRA. Regarding the characteristics of these tests, RF had the highest sensitivity at 66%, and all the other antibodies individually were less than 50% sensitive. AFA, anti-Sa, anti-CCP were greater than 90% specific for RA, while RF and AKA were 80-90% specific, and anti-RA-33 and anti-RA-1 was not specific for this diagnosis. The data further indicate that adding any one of AFA, AKA, anti-Sa, or anti-CCP to RF increases the specificity for RA from 80 to 90%. In the absence of RF, the presence of one or more of these antibodies carried a sensitivity of only 31% for RF- RA, with anti-Sa being the most specific at 98%. Overall, there was a high degree of correlation between AFA, AKA, anti-Sa or anti-CCP, this being highest between anti-Sa and anti-CCP (odds ratio, 13.3; P < 0.001). Despite this high level of correlation, of the 101 patients who were positive for at least one of these four autoantibodies, 57% were positive for only one, suggesting considerable variability in individual reactivity patterns. RA has been shown in multiple populations to be associated with HLA-DRB1 alleles encoding for the shared epitope (SE). In this study, as illustrated in Table 2, the presence of each of these autoantibodies was significantly associated with having two shared epitope alleles, even when only the RA patients were considered. Patients with anti-Sa antibodies were predominantly male (61% versus 28%; P

Autoantibodies have the potential of pathogenicity in several diseases. In rheumatoid arthritis (RA), however, this has not been ultimately proven. RA is characterized by a variety of autoantibodies. Newer insights into characteristics of rheumatoid factors indirectly suggest their pathogenetic involvement. In contrast, antibodies to collagen, despite the availability of an experimental model, do not appear to be pathogenetic in man. Anti-hnRNP antibodies, particularly anti-A2/RA33, are present in RA and experimental models of RA, and therefore, aside from their diagnostic value in established and early RA, could also be involved in the disease process. The nature of Sa, another target antigen in RA, has not yet been elucidated. Filaggrin is the antigen recognized by antikeratin antibodies and antiperinuclear factor; however, citrullin is the target amino acid in filaggrin, and anticitrullin antibodies have a high predictive value. Among a series of cartilage proteins, most have not yet been characterized sufficiently; one, gp39, appears to be of particular interest. Whether or not these antibodies are involved in RA pathogenesis is not yet known. It can be speculated that autoimmunity to some, if not all, of these autoantigens mirrors events important in the development of RA, but further studies on T-cell reactivities and in experimental models are needed to fully understand the involvement.

Background Monocytes (Mo) are among the first hematopoietic cells to migrate into the inflamed synovial tissue of arthritic joints. Monocyte migration appears to be associated with the expanding synovial lining network of fibroblast-like synovial cells (FLS). Whether cognitive interaction between Mo and FLS is required for an orchestrated migratory behaviour has not been analysed so far. We analysed Mo migratory activity under inflammatory conditions in regard to cell-cell interactions with FLS. Methods Human FLS were prepared from synovial tissues obtained as discarded specimens following joint arthroplasty. CD14+ Mo were isolated from peripheral blood by magnetic bead sorting. FLS and Mo were labeled with fluorescent membrane dyes and cultured in spherical extracellular matrix micromasses with an average size of 1.5 mm for up to two weeks. For stimulation experiments, micromasses were cultured in medium containing 10 ng/ml of tumor necrosis factor (TNF). At different time-points cell migration was monitored in individual micromasses by real-time confocal microscopy. Results Cell migration could be subdivided into three successive phases of cell movement. Phase I (day 1-3 of culture) was characterised by the formation of the synovial lining layer. Mo in close contact with FLS appeared sessile. On average 20% of Mo were in no apparent contact with FLS and displayed a mobile and seeking behaviour. During phase II (day 3-7) the majority of Mo remained sessile whereas a fraction of Mo displayed a directed cell movement with an impressive maximum speed of up to 15 mcm/min. In addition the formation of Mo cell clusters was observed. The rapid Mo migration finally ceased during phase III (day 7-14). The addition of TNF i) increased the frequency and size of Mo cell clusters during phase II two and ii) prolonged the mobility of Mo into phase III. Conclusion The 3D synovial tissue culture system allows to monitor and analysed subtle migration patterns of Mo in relation to the organised synovial lining architecture. Ongoing experiments address molecular mechanism(s) of Mo – FLS interaction in order to identify potential targets for future therapeutic intervention in arthritis.

Background and Objectives There is some evidence that release and insufficient removal of endogenous nucleic acids may be involved in triggering harmful autoimmune reactions that might be important in the initiation of RA. Nucleic acid-sensing molecules, such as the endosomal Toll-like receptors (TLRs) 7 and 9, have been linked to pathogenic autoimmune processes, particularly in SLE, but their role in RA is less clear. Data recently obtained in rats with pristane-induced arthritis (PIA) suggested involvement of TLR9, because antagonizing this receptor in the initiation phase of the disease led to an improved clinical outcome of arthritis. To gain more insight into the role of TLR9 in autoimmune arthritis we have extended our studies to the K/BxN serum-transfer arthritis model which is reflecting the effector phase of erosive autoimmune arthritis. Materials and Methods Arthritis was induced in C57BL/6 mice by two subsequent injections of arthritogenic serum. Antagonists for TLR7 and TLR9 or an agonist for TLR9, respectively, a non-inhibitory control sequence or PBS as placebo was applied every third day. The first treatment was given one day before disease induction. To further investigate TLR9 involvement, arthritis was also induced in TLR9 knock-out mice. Furthermore, the effect of TLR9 on osteoclast differentiation and activation was investigated in an in vitro osteoclast formation assay. Results Neither inhibitor affected arthritis onset and severity in the serum transfer model, which is independent of the adaptive immune system, in contrast to PIA. Furthermore, arthritis severity was not changed in mice lacking a functional tlr9 gene in comparison to wild type animals. In contrast, treatment with a TLR9 agonist led to an improved clinical outcome. Remarkably, in vitro formation of osteoclasts appeared to be slightly enhanced in cells isolated from TLR9-/- mice compared to those from wild type animals. Moreover, osteoclastogenesis was reduced by ~60% in cells from wild type animals grown in the presence of the TLR9 agonist whereas, as expected, no effect was seen in cells from TLR9-/- animals. Conclusions These results suggest that TLR9 might play a beneficial regulatory role in the effector phase of erosive autoimmune arthritis by negatively affecting differentiation and activation of bone resorbing osteoclasts. Nevertheless, the precise role of TLR9 in the different stages of arthritis needs to be further elucidated.

Background There is substantial evidence that abundant release and/or insufficient removal of endogenous nucleic acids can trigger autoimmune reactions via activation of nucleic acid recognising Toll-like receptors (TLR) such as TLR7 and TLR9, which may lead to the development of SLE and other systemic autoimmune diseases. However, in RA the involvement of these TLRs is less well understood. Interestingly, in rats with pristane-induced arthritis (PIA) disease can be transferred by T cells together with antigen-presenting-cells pre-activated with TLR7 or TLR9 agonists (Hoffmann et al , J Autoimmunity 2011; 36: 288–300). Objective We aimed to study the role of TLR7 and TLR9 in the pathogenesis of inflammatory erosive arthritis by antagonising them in PIA and the KRN serum transfer model. Methods Arthritis was induced in rats with the mineral oil pristane, and in C57Bl/6 mice by injection of KRN serum. Immunoregulatory oligodeoxynucleotide (ODN) sequences (IRS) antagonising TLR7 or TLR9 were applied either subcutaneously (PIA) or intra-peritoneally (KRN). A non-inhibitory ODN was used as control and PBS served as placebo. Arthritis was scored using established scoring systems, inflammation and bone erosion were quantitatively analysed by histology. Serum and cell culture cytokine levels were measured by ELISA. Results While the TLR7 inhibitor and the control ODN showed no effect on arthritis development and severity, the TLR9 antagonist reduced arthritis severity significantly in PIA. Bone erosion was almost completely abolished, whereas it was moderately aggravated in animals treated with the TLR7 inhibitor. Furthermore, IL-6 serum levels were significantly reduced in animals treated with the TLR9 antagonist. However, these beneficial effects were only observed when the inhibitor was applied before disease onset. Moreover, neither inhibitor affected arthritis onset and severity in the serum transfer model, which is independent of the adaptive immune system. Summary and Conclusions Inhibition of TLR9 significantly reduced inflammation and bone erosion in PIA but not in the KRN serum transfer model that reflects the late effector phase of erosive arthritis. Therefore, these results suggest important involvement of the DNA (CpG) recognising TLR9 in the initiation of autoimmune arthritis whereas nucleic acid binding TLRs do not seem to play a major role in the later phases of the disease. Antagonizing TLR9 in human RA may only act beneficial in the earliest phase of the disease.

Introduction Pristane-induced arthritis (PIA) in the rat has been described as an animal model of inflammatory arthritis which exhibits features similar to rheumatoid arthritis in humans, such as a chronic, destructive, and symmetrical involvement of peripheral joints. However, so far little is known about the earliest inflammatory events and their influence on locomotor behaviour during the course of PIA. To investigate this issue a detailed analysis of the pathologic changes occurring during the prodromal and early stages of PIA was performed. Methods Arthritis was induced in DA.rats by injection of 150 μl 2,6,10,4-tetramethyl-pentadecane (pristane) at the base of the tail and changes in locomotor behaviour of the affected paws were monitored using the CatWalk quantitative gait analysis system. The pathologic events occurring in the joints of pristane-injected animals were studied before onset, at onset, and during acute phase of arthritis by histological methods. Results Gait analysis revealed that changes in locomotion such as reduced paw print areas and stance phase time are already apparent before the onset of clinically discernible arthritis symptoms (erythema, paw swelling) and correlate with PIA scores. In agreement with these findings, inflammatory tenosynovitis could be observed by histology already before the onset of erythema and swelling of the respective paws. In the most heavily affected rats also irregularities in step sequence patterns occurred A kinetic analysis of clinical and histological findings demonstrated that gait changes precede the pathological changes occurring during the acute phase of pristane-induced arthritis. Conclusions Gait analysis allows for pinpointing the initial inflammatory changes in experimental arthritis models such as pristane-induced arthritis. Analysis of early clinically relevant symptoms in arthritis models may facilitate the search for novel therapeutics to interfere with pain, inflammation and joint destruction in patients suffering from inflammatory arthritis.

Rheumatoid arthritis is a heterogeneous disease with respect to clinical manifestations, serologic abnormalities, joint damage and functional impairment. Predicting outcome in a reliable way to allow for strategic therapeutic decision-making as well as for prediction of the response to the various therapeutic modalities available today, especially biological agents, would provide means for optimization of care. In the present article, the current information on biological and clinical markers related to disease activity and joint damage as well as for predictive purposes is reviewed. It will be shown that the relationship of many biomarkers with disease characteristics is confounded by factors unrelated to the disease, and that only few biomarkers exist with some predictive value. Moreover, clinical markers appear of equal value as biomarkers for this purpose, although they likewise have limited capacity in these regards. The analysis suggests the search for better markers to predict outcomes and therapeutic responsiveness in rheumatoid arthritis needs to be intensified.

A hallmark of systemic lupus erythematosus (SLE) is the appearance of autoantibodies to nuclear antigens, including autoantibodies directed to the heterogeneous nuclear ribonucleoprotein A2 (hnRNP-A2), which occur in 20% to 30% of SLE patients as well as in animal models of this disease. To investigate the underlying cellular reactivity and to gain further insight into the nature and potential pathogenic role of this autoimmune response we characterized the T cell reactivity against hnRNP-A2 in patients with SLE in comparison to healthy controls. Cellular proliferation of peripheral blood T cells to hnRNP-A2 was determined by [3H]thymidine incorporation and T cell clones (TCCs) specific for hnRNP-A2 were grown by limiting dilution cloning; IFNγ, IL-4 and IL-10 in culture supernatants were measured by ELISA. Bioactivity of culture supernatants was determined by incubation of anti-CD3/anti-CD28 stimulated peripheral blood CD4+ T cells with supernatants of TCCs. Stimulation assays performed with peripheral blood mononuclear cells of 35 SLE patients and 21 healthy controls revealed pronounced proliferative responses in 66% of SLE patients and in 24% of the controls, which were significantly higher in SLE patients (p < 0.00002). Furthermore, hnRNP-A2 specific TCCs generated from SLE patients (n = 22) contained a relatively high proportion of CD8+ clones and mostly lacked CD28 expression, in contrast to TCCs derived from healthy controls (n = 12). All CD4+ TCCs of patients and all control TCCs secreted IFNγ and no IL-4. In contrast, CD8+ TCCs of patients secreted very little IFNγ, while production of IL-10 did not significantly differ from other T cell subsets. Interestingly, all CD8+ clones producing IL-10 in large excess over IFNγ lacked expression of CD28. Functional assays showed a stimulatory effect of the supernatants derived from these CD8+CD28- hnRNP-A2 specific TCCs that was similar to that of CD4+CD28+ clones. Taken together, the pronounced peripheral T cell reactivity to hnRNP-A2 observed in the majority of SLE patients and the distinct phenotype of patient-derived CD8+ TCCs suggest a role for these T cells in the pathogenesis of SLE.

Tumour necrosis factor (TNF) signalling molecules are considered as promising therapeutic targets of antirheumatic therapy. Among them, mitogen-activated protein kinases are thought to be of central importance. Herein, we investigate the role in vivo of TNF-α signalling through c-Jun N-terminal kinase (JNK)1 in destructive arthritis. Human TNF transgenic (hTNFtg) mice, which develop inflammatory arthritis, were intercrossed with JNK1-deficient (JNK1-/-) mice. Animals (n = 35) of all four genotypes (wild-type, JNK1-/-, hTNFtg, JNK1-/-hTNFtg) were assessed for clinical and histological signs of arthritis. Clinical features of arthritis (swelling and decreased grip strength) developed equally in hTNFtg and JNK1-/-hTNFtg mice. Histological analyses revealed no differences in the quantity of synovial inflammation and bone erosions or in the cellular composition of the synovial infiltrate. Bone destruction and osteoclast formation were observed to a similar degree in hTNFtg and JNK1-/-hTNFtg animals. Moreover, cartilage damage, as indicated by proteoglycan loss in the articular cartilage, was comparable in the two strains. Intact phosphorylation of JNK and c-Jun as well as expression of JNK2 in the synovial tissue of JNK1-/-hTNFtg mice suggests that signalling through JNK2 may compensate for the deficiency in JNK1. Thus, JNK1 activation does not seem to be essential for TNF-mediated arthritis.

Heterogeneity and multifactoriality complicate diagnostics and our understanding of pathogenesis of rheumatoid arthritis (RA). The only accepted serologic parameter (rheumatoid factor [RF]) is not disease specific, nor are any of several novel RA autoantibodies. We aimed at identifying profiles instead of individual autoreactivities allowing for unambiguous prediction of RA. Selected RA autoantigens were tested by ELISA (RF and anti-cyclic citrullinated peptide [anti-CCP]) or Western blot (heavy-chain-binding protein [BiP], heterogeneous ribonucleoprotein particle A2 [RA33/hnRNP A2], calpastatin and calreticulin). Antibody reactivities were assayed from serum samples of 149 RA patients and 132 patients with other rheumatic diseases and from synovial fluids (SF) (58 RA, 65 non-RA). No single autoreactivity was sufficient for unambiguous prediction of RA. Frequencies of multiparameter profiles consisting of 3, 4, 5 and 6 autoreactivites were determined. Fifteen six-parameter serum profiles were exclusively expressed in RA patients, representing a cumulative sensitivity of 59%. Twelve SF profiles were exclusively expressed in 64% of RA patients. The self-learning classification algorithm CLASSIF1 was capable of accurately predicting RA when these profiles were present. Data profile analysis of RF/CCP/BiP/calpastatin/calreticulin/RA33 provided specific discrimination of 64% of RA. Most importantly, RA specific profiles were observed in 64% of patients with early disease (

Local bone erosions and systemic bone loss are hallmarks of rheumatoid arthritis and cause progressive disability. Tumor necrosis factor (TNF) is a key mediator of arthritis and acts catabolically on bone by stimulating bone resorption and inhibiting bone formation. We hypothesized that the concerted action of parathyroid hormone (PTH), which stimulates bone formation, osteoprotegerin (OPG), which blocks bone resorption, and anti-TNF, which reduces inflammation, could lead to repair of local bone erosions and to inhibition of systemic bone loss. To test this, human TNF-transgenic mice with erosive arthritis and established systemic bone loss were treated with PTH, OPG and anti-TNF. Local bone erosions almost fully regressed, suggesting repair of inflammatory skeletal lesions. In contrast, OPG or anti-TNF alone led to arrest of bone erosions but did not achieve repair. Treatment with PTH alone had no influence on the progression of bone erosions. Local bone erosions showed all signs of new bone formation such as the presence of osteoblasts, osteoid formation and mineralization. Systemic bone loss was completely reversed upon combined treatment, and this effect was mediated by osteoblast stimulation and osteoclast blockade. In contrast to local and systemic bone loss, joint inflammation was only affected by anti-TNF. In summary, we conclude that local and systemic inflammatory bone loss due to TNF can regress, and that repair requires a combined approach by reducing inflammation, blocking bone resorption and stimulating bone formation.