INTRODUCTION:Visual evaluation of indirect immunofluorescence (IIF) on human epithelial-2 cells is the routine method for screening for antinuclear antibodies (ANA) in connective tissue diseases. Since visual IIF is time-consuming and subjective, automated IIF processors have been developed to offer standardised, valid and cost-efficient IIF assays.OBJECTIVE:The aim of this study was to determine the diagnostic reliability of 2 widely used IIF processors (Aklides, Medipan GmbH and Helios, Aesku Diagnostics) under real-life laboratory working conditions.METHODS:ANA were determined in samples from patients with suspected autoimmune rheumatic disease (n=1008) using both automated IIF processors and compared with the results obtained by visual interpretation. The performance of IIF processors to discriminate positive from negative samples, pattern recognition and end point titre prediction were evaluated.RESULTS:The IIF processors showed moderate agreement with visual interpretation in discriminating positive from negative ANA samples (κ values: Aklides 0.494; Helios 0.415). The sensitivity/specificity was 89%/59% for Aklides and 87%/54% for Helios. However, both processors correctly identified 99% of definitely positive samples (titre ≥1:320). Aklides correctly identified 43% of fluorescence patterns and its light intensity values showed good correlation (Spearman's ρ=0.680) with visually obtained titres.CONCLUSIONS:Automated IIF determination under real-life laboratory working conditions remains a challenge. Owing to their high sensitivity at clinically relevant ANA titres, automated IIF processors can already support but not totally replace visual IIF.

Introduction: Antibodies against mutated citrullinated vimentin (AMCV) represent a useful diagnostic marker with correlation to disease activity in patients with rheumatoid arthritis (RA). Since seropositivity for citrullinated autoantibodies was predictive for response to B-cell depleting therapy (BCDT) with rituximab (RTX), we investigated whether differences in antibody fine reactivity and immunoglobulin (Ig) isotype kinetics among AMCV-positive patients could provide additional information about outcome.Methods: A total of 50 AMCV IgG-positive RA patients (RTX responders (RRs) n = 37 and non-responders (NRRs) n = 13) were analyzed for reactivity against MCV epitopes and co-existent AMCV isotypes IgM and IgA. Antibody titers were determined by enzyme-linked immunosorbent assay at baseline and 24 weeks after the first cycle of RTX, and compared to kinetics of rheumatoid factor (RF) and antibodies against cyclic citrullinated peptide (ACCP).Results: Recognized MCV epitopes by AMCV IgG of RRs and NRRs showed similar baseline patterns, with reducing reactivity in RRs and unchanged or even expanding reactivity in NRRs upon RTX treatment. At baseline, RRs were more frequently negative for AMCV subtypes, especially for IgA (68 %), compared to NRRs (31 %). Being AMCV IgA-negative at baseline indicated a good treatment response to RTX (negative predictive value = 0.86). Co-existence of AMCV IgA and IgG with stable titers upon treatment were associated with poorer responses to RTX. Furthermore, reductions of AMCV IgA levels upon RTX correlated with the improvement of 28-joint Disease Activity Score (DAS28). In comparison, subtypes of RF and ACCP were not of additional value for prediction of RTX response.Conclusions: Restrictive IgG seropositivity against MCV with treatment-associated decline in fine reactivity and titers was predictive for response to RTX. Double-positivity for AMCV IgG and IgA was associated with failure to respond to BCDT, suggesting a pathogenetic and less sensitive IgA-producing B-cell subset in NRRs.

The diagnosis of antiphospholipid syndrome (APS) remains a clinical challenge in particular with regard to the assessment of so called antiphospholipid (aPL) antibodies and their impact on the diagnosis of this autoimmune clinical entity. The recently revised classification criteria for APS recommend, when there is a clinical suspicion, the use of an enzyme-linked immunosorbent assay (ELISA) for the detection of anti-beta 2 glycoprotein I (2GPI) and cardiolipin-dependent anti-2GPI IgG and IgM. In addition, aPL antibodies can be determined as lupus anticoagulant by a functional coagulation assay as laboratory criterion. According to the classification criteria, a diagnosis of APS requires at least one positive laboratory criterion. However, other aPL antibodies are reactive with differing phospholipids and corresponding cofactors have been reported to be useful for the serological diagnosis of APS while the jury is still out concerning the possibility to reliably identify the full range of true APS cases. Antibody profiling of several aPL antibodies appears to pave the way for a new understanding of the risk assessment of thrombotic events in APS patients. The more different aPL antibodies occur in a patient the higher is the risk for the development of clinical APS manifestations such as thrombotic events. Thus, new approaches to multiparametric aPL antibody detection are required to provide the necessary information regarding diagnosis and eventually outcome in APS patients. Multiplex analysis of aPL antibodies candidates as an alternative thereof. In particular, line immunoassays (LIAs) employing membranous solid phases for the immobilisation of phospholipids and their respective cofactors are reported to detect the corresponding antibodies efficiently and represent an alternative to ELISA. Novel developments in the field of aPL antibody profiling and the currently available data on LIAs for APS serology are summarised in this review.

ObjectiveAntibodies against citrullinated antigens (ACPA) represent one rheumatoid arthritis (RA) classification criteria. Recently, mutated and citrullinated vimentin (MCV), containing approx. 45 potentially citrullinated sites, was characterised as another modified autoantigenic RA target. Therefore, we wanted to screen, select and validate predominant MCV autoantigenic epitopes (called here MCE) as possible new diagnostic targets.MethodsMCV-derived peptides with citrullinated sites were screened in healthy controls and patients. Based on this, twelve selected MCE were used for validation of ACPA isotypes (IgA/IgG/IgM) with ELISA in early RA (ERA, <12 months) and established RA (>12 months) Russian patients. Sensitivity of MCE reactivity was compared to commercially available ELISAs for anti-CCP IgG, anti-MCV IgG, and anti-RF IgA/IgM/IgG.ResultsAnti-MCE IgG/IgAl/IgM antibodies were observed in 64.1%, 23.1%, and 15.4% ERA, and 63.9%, 26.7%, and 13.1% established RA patients, respectively. Anti-MCV IgG was present in 64.1% ERA and 55.0% RA patients.Furthermore, anti-CCP IgG and RF IgG/IgA/IgM were detectable in up to 76.9%, 71.8%, 71.8%, and 38.5% ERA, and 80.1%, 72.3%, 67.5%, and 43.0% RA patients. Anti-CCP IgG single positivity was observed in 7.7% ERA and 6.3% RA patients. Only one RA patient was anti-MCE single positive.ConclusionMCV autoantigenic epitopes were emulated by cyclic citrullinated MCV-derived peptides and recognised by all autoantibody-Ig subclasses in RA. Tested MCE were recognized more frequently by IgG as the original MCV antigen. High antibody prevalence against CCP epitopes suggests a strong CCP-linkage to RA pathogenesis in the investigated Russian cohort.

Die Diagnose des Antiphospholipid Syndroms (APS) stellt unverändert eine klinische Herausforderung insbesondere bezüglich der Bestimmung der sogenannten Antiphospholipid Antikörper (aPLAk) und deren Einfluss auf die Diag­nosestellung dieser klinischen Entität dar. Die vor Kurzem revidierten Klassifikationskriterien für APS empfehlen bei klinischem Verdacht die Verwendung von enzyme-linked immunosorbent assays (ELISAs) für die Bestimmung von anti-beta 2 Glykoprotein I (β2GPI) und Cardiolipin-abhängigen anti-β2GPI IgG und IgM. Zusätzlich können aPLAk als Lupusantikoagulanz mittels eines funktionellen Gerinnungstestes als Laborkriterium zur Diagnosestellung ermittelt werden. Entsprechend den Klassifikationskriterien ist ohne positives Laborkriterium keine Diagnose des APS möglich. Andere aPLAk mit Reaktivitäten gegen unterschiedliche Phospholipide und deren Kofaktoren wurden beschrieben und deren potentieller Nutzen für die APS Serologie hervorgehoben. Antikörperprofiling verschiedener aPLAk wird als ein neuer Ansatz für die Risikoabschätzung der Entwicklung von thrombotischen Ereignissen für Patienten mit APS diskutiert. Je mehr aPLAk Spezifitäten gegen unterschiedliche Phospholipid- bzw. Kofaktorepitope vorkommen, desto höher ist das Risiko für das Auftreten von klinischen Komplikationen des APS wie zum Beispiel thrombotische Ereignisse. Daher können neue Wege für die gleichzeitige Bestimmung von mehreren aPLAk in einer Patientenprobe sinnvoll sein, um zusätzliche Information für die Diagnose und letztendlich Risikoabschätzung von APS zu liefern. Multiplextechnologien zur Bestimmung von aPLAk können eine Alternative dafür sein. Insbesondere Line-Immunoassays (LIAs) mit Membranen als Festphase für die Immobilisierung von Phospholipiden und deren Kofaktoren sind als Alternative zu ELISA für die Bestimmung von aPLAk kürzlich beschrieben worden. Diese Übersichtsarbeit diskutiert die neuen Entwicklungen auf dem Gebiet des aPLAk Profilings und die entsprechende Verwendung von LIAs in der APS Serologie.

Background The determination of antibodies (abs) against citrullinated antigens (ACPA) nowadays belongs to the diagnostic tests included in the classification criteria for rheumatoid arthritis (RA). However, the standard cyclic citrullinated peptide antigen (CCP) is artificial and not expressed in the targeted tissues. Recently, mutated and citrullinated vimentin (MCV) was isolated and characterized as another modified autoantigen in RA. It is of interest, whether cyclic peptides from the sequence of MCV are also recognized as antigenic epitopes. Objectives To analyse reactivity against cyclic citrullinated peptides derived from the sequence of MCV in a cohort of patients with early (ERA) and established (RA). Methods Immunglobulin subclass (IgA, IgG and IgM) ab against MCV-cyclic epitopes (MCE, Orgentec) were investigated in a cohort of well characterized Russian patients (n=206) with RA using ELISA. Sensitivity of MCE-reactivity was compared to commercially available ELISAs for anti-CCP IgG (Medipan), anti-RF (IgA, IgG and IgG), and anti-MCV IgG ab (Orgentec). Patients were classified according to the duration of disease as ERA (<12 months; n=34) or established RA (>12 months; n=172). Results Abs isotypes IgG, IgA and IgM against MCE were observed in 64.7%, 23% and 17.6% of patients with ERA, as well as in 68.6%, 27.3% and 13.7% of patients with RA, respectively. Anti-MCV ab IgG were found positive in 61.8% of patients with ERA, and in 55.8% of patients with RA. Thus, the frequency of IgG-ab against MCE was even higher compared to the complete MCV antigen. In comparison, anti-CCP IgG ab and RF IgG, IgA and IgM were detectable in 73.5%, 70.6, 64.7%, and 35.3% of patients with ERA, and in 80.2%, 71.5%, 68.6%, and 41.9% of patients with RA, respectively. Single positive results for CCP IgG ab were observed in 8.8% of ERA and 10.5% of RA patients. By summarizing all MCE subclass abs, single positive results were observed in 11.8% of ERA and 2.3% of RA patients. The highest IgG ACPA titers were measured for the anti-CCP (mean ERA 2274.9 U/ml and RA 1890.1 U/ml) and the anti-MCE IgG ab (mean ERA 516.0 U/ml and RA 591.6 U/ml) followed by anti-MCV ab (mean ERA 338.0 U/ml and RA 443.7 U/ml). Conclusions The antigenic epitopes of MCV are maintained by using cyclic citrullinated peptides. Antibody reactivity against MCE includes all subclasses, with a predominance of IgG-ab. For diagnostic purposes, the standard CCP ELISA provided the highest sensitivity in the analysed cohort. Disclosure of Interest O. Derganowa: None Declared, L. Martinez-Gamboa: None Declared, K. Egerer: None Declared, H. Bang Employee of: Orgentec, D. Roggenbuck Employee of: Medipan, I. Esaulenko: None Declared, G. Burmester: None Declared, T. Chernykh: None Declared, E. Feist: None Declared

Background In patients with rheumatoid arthritis (RA), seropositivity for either rheumatoid factor or anti-CCP antibodies has been identified as a predictive marker for treatment response to rituximab (RTX) in different studies. However, it is unclear, whether analysis of fine-reactivity against certain epitopes of citrullinated antigens or immunoglobulin subclass reactivity can contribute to the prediction of treatment response to RTX. Objectives To investigate whether reactivity patterns against epitopes of the mutated citrullinated vimentin (MCV) antigen and/or anti-MCV subclass reactivities can contribute to differentiation of responders to RTX. Methods Fine-reactivity against MCV was investigated in anti-MCV antibody positive patients with RA (n=34) under treatment with RTX. According to EULAR response criteria, 23 patients were classified as responders by reaching remission as defined by DAS28 <2.6 or low disease activity as defined by DAS28 <3.2. Follow-up analyses of anti-MCV response were performed using 88 synthetic overlapping MCV peptides in ELISA. The peptides used contained all known modifications of vimentin by mutation and citrullination1. In addition, influence of RTX treatment on anti-MCV IgG, IgM and IgA antibody reactivities was investigated by ELISA. Results At baseline or during follow-up, none of the responders showed an anti-MCV IgA antibody response. In contrast, non-responders were positive for anti-MCV IgA in 50% of the cases at baseline and showed no reduction of antibody subclass reactivities in the follow-up. Furthermore, anti-MCV antibody subclass reactivities showed that RTX-responders were characterized by a reduction of anti-MCV IgG antibody titers, and by a normalization of anti-MCV IgM titers in all initially positive cases. At baseline, subsequent responders to RTX treatment were characterized by only a minor different epitope recognition pattern compared to non-responders. Remarkably upon treatment, a reduction of the number of recognized MCV epitopes was observed in 16 out of 23 responders (69.6%) in contrast to 3 out of 11 (27.3%) non-responders in follow-up analyses. Conclusions Responders to RTX were characterized by a restricted immunoglobulin subclass reactivity against MCV lacking IgA anti-MCV antibodies at baseline, and a reduction of anti-MCV antibody titers as well as the epitope recognition pattern under treatment. Fine-reactivity against the MCV antigen can potentially contribute as a useful marker to predict response to RTX in anti-MCV positive RA patients. References Bang H, Egerer K, Gauliard A, Luthke K, Rudolph PE, Fredenhagen G, Berg W, Feist E, Burmester GR. Mutation and citrullination modifies vimentin to a novel autoantigen for rheumatoid arthritis. Arthritis Rheum. 2007 Jul 30;56(8):2503-2511 Disclosure of Interest L. Naumann Grant/Research support from: Roche Pharma AG, H. Bang Employee of: Orgentec, K. Egerer: None Declared, D. Meyer zum Büschenfelde: None Declared, B. Lehmann: None Declared, G. Fredenhagen Employee of: Orgentec, H. Bastian: None Declared, E. Wittenborn Employee of: Roche Pharma AG, G.-R. Burmester: None Declared, E. Feist: None Declared

Background Patients with antiphospholipid syndrome (APS) may develop during the course of disease several antiphospholipid antibodies (aPL). Such aPL antibody profiles are suggested to be associated with particular clinical symptoms. Objectives Assessment of aPL antibody profiles by line immunodot technique employing a novel hydrophobic surface was related to clinical symptoms in follow-up samples of APS patients treated at the Charité Hospital, Universitätsmedizin Berlin. Methods Serum samples of 45 APS patients (41 females, 4 males, median age 45 years) with at least 3 follow-up samples collected over period of up to 10 years were collected. A total of 223 serum samples were tested by line immunodot assay (LIA) for aPL IgG and IgM antibodies to phosphatidylserine (PS), phosphatidylinositol (PI), cardiolipin (CL), and beta2-glycoprotein I (β2GPI) (GA Generic Assays GmbH, Germany). Of the 45 APS patients 3 (6.7%) with a total of 20 follow-up samples had pregnancy morbidity (PM), 28 patients (62.2%) with 128 samples demonstrated deep venous thrombosis (DVT), 10 patients (22.2%) with 57 samples exhibited cerebrovascular events (CE) comprising cerebral transient ischemic attack and/or ischemic stroke, and 4 patients (8.9%) with 18 samples had pulmonary embolism (PE). Results Within these follow-up samples, anti-PI IgM (16/57), anti-PS IgM (38/57), anti-CL IgM (45/57), and anti-β2GPI IgM (35/57) were detected by the LIA and demonstrated a substantially higher prevalence in APS patients suffering from CE compared with the remaining APS patients (P <0.01; respectively). In contrast, anti-PS IgG (18/20) and anti-β2GPI IgG (19/20) showed a significant higher prevalence in APS patients with PM (P <0.001; respectively). The prevalence of IgM to PS (0/20), CL (2/18), and β2GPI (4/16) was significantly diminished in this group (P <0.02; respectively). The detection of at least 1, 2 or 3 aPL IgG antibodies by LIA also revealed a significant higher prevalence in this APS patient cohort (18/20, 18/20, 15/20; P <0.05, respectively). The prevalence of at least 1, 2 or 3 aPL IgM antibodies was significantly reduced (4/20, 3/20, 0/20; P <0.01, respectively). In contrast, APS patients with CE demonstrated a higher prevalence of at least 1, 2, 3 or 4 aPL IgM antibodies 48/57, 40/57, 33/57, 13/57; P <0.00001, respectively). During the course of disease of up to 10 years, aPL antibody profiles may change, but disappeared in just 1 of the 45 patients (2%). Conclusions aPL antibody profiling by LIA candidate to differentiate APS patients with CE or PM from patients venous thrombotic events. The remarkable significant higher prevalence of IgM to PI, PS, CL, and β2GPI in APS patients suffering from CE and of IgG to PI and β2GPI in APS patients with PM warrants further investigation. Disclosure of Interest D. Roggenbuck Shareholder of: GA Generic Assays GmbH, Medipan GmbH, Employee of: GA Generic Assays GmbH, Medipan GmbH, K. Egerer: None Declared, T. Buettner: None Declared, B. Lehmann: None Declared, E. Feist: None Declared, G.-R. Burmester: None Declared, T. Dörner: None Declared

Multi-kinase inhibitors have been established for the treatment of advanced renal cell cancer, but long-term results are still disappointing and immunotherapeutic approaches remain an interesting experimental option particularly in patients with a low tumor burden. DC are crucial for antigen-specific MHC-restricted T cell immunity. Furthermore, allogeneic HLA-molecules pose a strong immunogenic signal and may help to induce tumor-specific T cell responses. In this phase I/II trial, 7 patients with histologically confirmed progressive metastatic RCC were immunized repetitively with 1 × 107 allogeneic partially HLA-matched DC pulsed with autologous tumor lysate following a schedule of 8 vaccinations over 20 weeks. Patients also received 3 Mio IE IL-2 s.c. once daily starting in week 4. Primary endpoints of the study were feasibility and safety. Secondary endpoints were immunological and clinical responses. Vaccination was feasible and safe with no severe toxicity being observed. No objective response could be documented. However, while all patients had documented progress at study entry, 29% of the patients showed SD throughout the study with a mean TTP of 24.6 weeks (range 5 to 96 weeks). In 3/7 patients, TH1-polarized immune responses against RCC-associated antigens were observed. In one patient showing a minimal clinical response and a TTP of 96 weeks, clonally proliferated T cells against yet undefined antigens were induced by the vaccine. Vaccination with tumor antigen loaded DC remains an interesting experimental approach, but should rather be applied in the situation of minimal residual disease after systemic therapy. Additional depletion of regulatory cells might be a promising strategy.

Objective.Dysregulation of proteasome subunit β1i expression has been shown in total blood mononuclear cells (PBMC) from patients with primary Sjögren syndrome (pSS), a B cell-driven systemic autoimmune disorder.Methods.Proteasome activation was investigated in sorted blood cells from patients with pSS and controls by measuring transcript levels of constitutive (β1/β2/β5) and corresponding immunoproteasome catalytic subunits (β1i/β2i/β5i) using real-time PCR. At protein level, β1i protein expression was analyzed by immunoblotting. Functional effects of proteasome inhibition on proteolytic activity and induction of apoptosis were also evaluated in cellular subsets.Results.The proteasome was found to be activated in pSS, with upregulation of gene expression of catalytic proteasome subunits. Western blot analysis revealed decreased β1i protein expression in pSS B lymphocytes, with decreased protein despite increased messenger RNA (mRNA) levels. After proteasome inhibitionin vitro, proteolytic activity was less reduced and resistance to apoptosis was increased in B lymphocytes compared to other cells.Conclusion.In pSS, catalytic subunits of the proteasome are upregulated at the mRNA level, while dysregulation of subunit β1i is attributed to B lymphocytes. B cell resistance after proteasome inhibition differs from the classical concept of increased susceptibility toward inhibition in activated cells, supporting the novel notion that susceptibility depends on cellular intrinsic factors and on proteasome activation.

Objective To investigate the long-term effects of induction therapy with adalimumab (ADA) plus methotrexate (MTX) in comparison with placebo (PBO) plus MTX in DMARD-naive patients with active early rheumatoid arthritis (RA).Methods Patients with active early RA (disease duration of <= 12 months) were randomly assigned to receive 40 mg ADA subcutaneously every other week (eow) plus MTX 15 mg/week subcutaneously or PBO plus MTX subcutaneously at 15 mg/week over 24 weeks. Thereafter, all patients received MTX monotherapy up to week 48. The primary outcome was the Disease Activity Score 28 (DAS28) at week 48. Secondary outcomes included proportions of patients in remission (DAS28<2.6), ACR responses, Health Assessment Questionnaire (HAQ) score and radiographic progression.Results 87 patients were assigned to ADA/MTX and 85 patients to PBO/MTX. At baseline, DAS28 was 6.2 +/- 0.8 in the ADA/MTX and 6.3 +/- 0.9 in the PBO/MTX groups. At week 24, treatment with ADA/MTX compared with PBO/MTX resulted in a greater reduction in DAS28 (3.0 +/- 1.2 vs 3.6 +/- 1.4; p=0.009) and other secondary outcomes such as DAS28 remission rate (47.9% vs 29.5%; p=0.021) and HAQ (0.49 +/- 0.6 vs 0.72 +/- 0.6; p=0.0014). At week 48, the difference in clinical outcomes between groups was not statistically significant (DAS28: 3.2 +/- 1.4 vs 3.4 +/- 1.6; p=0.41). Radiographic progression at week 48 was significantly greater in patients administered PBO/MTX (Sharp/van der Heijde score: ADA/MTX 2.6 vs PBO/MTX 6.4; p=0.03, Ratingen score: 1.7 vs 4.2; p=0.01).Conclusions A greater reduction in radiographic progression after initial combination therapy with ADA and MTX was seen at week 48, even after discontinuation of ADA treatment at week 24. This sustained effect was not found at the primary endpoint (DAS28 reduction).

Detection of anti-phospholipid (aPL) antibodies for state-of-the art diagnosis of antiphospholipid syndrome(APS) still remains a laboratory challenge due to the great diversity of aPL antibodies and their relevance with regard to the diagnostic criteria. According to the recently revised classification criteria for APS, several enzyme-linked immunosorbent assays (ELISAs) should be performed simultaneously in routine laboratories for the detection of aPL antibodies. Therefore, new approaches to aPL profiling have been proposed recently to provide information regarding diagnosis and eventually outcome in APS patients. Multiplex analysis could meet the increasing demand for cost-efficient detection and profiling of aPL antibodies. Multi-line immunodot assays or bead-based multiplex techniques candidate as alternatives to assess several aPL antibodies simultaneously employing different solid-phases for bound/free separation of reactants. Particularly, multi-line immunodot assays present an alternative to ELISA for aPL antibody detection and profiling in APS patients. The use of hydrophobic membranes as solid-surface by this technique appears to offer a distinct solid-phase reaction environment for the assessment of aPL antibodies. This article reviews novel developments in the field of laboratory diagnostics of APS with special emphasis on multiplex assays.

Background Current perception of RA pathogenesis suggests that posttranslational modification (PTM) of either arginine (into citrulline) or lysine (into homocitrulline) residues in antigens are crucial for the generation of specific autoantibodies in RA. Objectives To examine how anti–citrullinated protein antibody (ACPA) and anti-homocitrulline protein antibody (AHPA) arise in the immune response of clinically overt rheumatoid arthritis (RA), and to analyze the influence of B cell directed therapy on antibody reactivity. Methods Homocitrullin residues were inserted into human mutated vimentin by non-enzymatic carbamoylation, in contrast to the enzymatic citrullination. Peptide librariesfrom the identified major RA epitope of mutated vimentin weregenerated with citrulline and homocitrulline residues, respectively. Reactivity to 5 distinct modified peptides was measured by enzymelinked immunosorbent assay and validatedwith sera from human healthy individuals and disease controls (systemic autoimmune diseases such as SLE and Sjögren’s syndrome). The study included 142 patients (mean age at inclusion 54.3±12.6 years) with RA (fulfilled the ACR 1987 RA criteria), who started on treatment with TNF-inhibitor followed by B-cell directed therapy. Multiple consecutive serum samples from RA patients were included for analysis of antibody titers in the follow-up. Results Sensitivities of 86,8% and 69,2% were calculated for the ACPA and AHPA assays, respectively. Sera from healthy persons and disease control groups showed a comparable specificity for both assays (ACPA 97% and AHPA 91%) and none had anti-antibodies against the unmodifed vimentin. With peptide assay distinctive only in citrulline and homocitrulline, more than 45% of RA patients were analyzed to have both ACPA and AHPA, and singular AHPA response was not observed. The lowest concentration of ACPA and AHPA were estimated to be 60 μg/ml and 25 μg/ml, respectively. Remarkably, after one cycle of RTX treatment a bisection of AHPA levels and a 25% reduction of ACPA levels were observed. Patients under TNF blockers without DAS28 remission after a period of 6 months showed a lesser reduction of ACPA and AHPA levels of only 15 – 28%. Conclusions Our findings indicate that the significant serological variation in RA patients may be an indication that RA may encompass a number of distinct serologic entities, of which particularly ACPA and AHPA may play a role in different phases of RA pathogenesis. Disclosure of Interest H. Bang Employee of: Orgentec Diagnostika GmbH, K. Egerer: None Declared, B. Lehmann: None Declared, A. Krämer Employee of: Orgentec Diagnostika GmbH, E. Feist: None Declared, G. R. Burmester: None Declared

INTRODUCTION:Diagnosis of antiphospholipid syndrome (APS) still remains a laboratory challenge due to the great diversity of antiphospholipid antibodies (aPL) and their significance regarding APS-diagnostic criteria.METHODS:A multi-line dot assay (MLDA) employing phosphatidylserine (PS), phosphatidylinositol (PI), cardiolipin (CL), and beta2-glycoprotein I (β2 GPI) was used to detect aPL, immunoglobulin G (IgG) and immunoglobulin M (IgM) in 85 APS patients, 65 disease controls, and 79 blood donors. For comparison, anti-CL and anti-β2 GPI IgG and IgM were detected by enzyme-linked immunosorbent assay (ELISA).RESULTS:The level of agreement of both methods was good for anti-CL IgG, moderate for anti-CL IgM, very good for anti-β2 GPI IgG, and moderate for anti-β2 GPI IgM (kappa = 0.641, 0.507, 0.803 and 0.506, respectively). The frequency of observed discrepancies for anti-CL IgG (1.75%), anti-CL IgM (3.93%), anti-β2 GPI IgG (1.75%), and anti-β2 GPI IgM (0.87%) was low (McNemar test, P < 0.05, not-significant, respectively). Sensitivity, specificity, positive (+LR) and negative (-LR) likelihood ratios for at least one positive aPL antibody assessed by ELISA were 58.8%, 95.8%, 14.1, and 0.4, respectively, and for at least three positive aPl IgM and/or one positive aPL IgG by MLDA were 67.1%, 96.5%, 19.3, and 0.3, respectively. The frequency of IgM to PI, PS and CL, and combination of three or more aPL IgM detected by MLDA was significantly higher in APS patients with cerebral transient ischemia (P < 0.05, respectively).CONCLUSIONS:The novel MLDA is a readily available, single-step, sensitive diagnostic tool for the multiplex detection of aPL antibodies in APS and a potential alternative for single aPL antibody testing by ELISA.

INTRODUCTION:The objective of this study was to compare the clinical usefulness of the new anti-double-stranded DNA nucleosome-complexed enzyme-linked immunosorbent assay (Anti-dsDNA-NcX ELISA), which is based on dsDNA-loaded nucleosomes as antigens, with established test systems based on dsDNA or nucleosomes alone for systemic lupus erythematosus (SLE) diagnostics and determination of disease activity.METHODS:Sera from a cohort of 964 individuals comprising 207 SLE patients, 357 disease controls and 400 healthy donors were investigated using the Anti-dsDNA-NcX ELISA, Farr assay, Anti-dsDNA ELISA, Anti-nucleosome ELISA and Crithidia luciliae immunofluorescence (CLIF) assay, all of which are tests available from EUROIMMUN Medizinische Labordiagnostika AG (Lübeck, Germany). Receiver operating characteristic curve analyses were performed to compare the sensitivity and specificity of each assay. The test results yielded by these assays in a group of 165 fully characterized SLE patients were compared with the corresponding medical records.RESULTS:The Anti-dsDNA-NcX ELISA was found to have a sensitivity of 60.9% and a specificity of 98.9% in all 964 individuals at the manufacturer's cutoff of 100 U/ml. At a comparable specificity of 99%, the sensitivity amounted to 59.9% for the Anti-dsDNA-NcX ELISA, 54.1% for the Farr assay, 53.6% for the antinucleosome ELISA and 35.8% for the anti-dsDNA ELISA. The CLIF assay had a sensitivity of 28.0% and a specificity of 98.2%. The Anti-dsDNA-NcX ELISA correlated mostly with global disease activity in a cross-sectional analysis. In a longitudinal analysis of 20 patients with 69 patient visits, changes in Anti-dsDNA-NcX ELISA and antinucleosome ELISA results correlated highly with changes in disease activity over time.CONCLUSIONS:The use of dsDNA-complexed nucleosomes as antigens in ELISA leads to optimized determination of diagnosis and disease activity in SLE patients and is available for clinical practice.

High-mannose and hybrid-type N-glycans are present in human serum glycoproteins in low abundance but have recently been described to play an important role in immune responses. It is therefore important to find a strategy to selectively analyze their structures in the context of health and disease in order to understand their impact on disease mechanisms. We report here the characterization of high-mannose and hybrid-type N-glycans in total human serum. To this end, N-glycans were released using Endo-beta-N-acetylglucosaminidase H (Endo H) and analyzed by CE-LIF and MALDI-TOF-MS. We found that the high-mannose structures Man59GlcNAc1 represented the majority of the pool. The monoglucosylated structure Glc1Man9GlcNAc1 as well as four hybrid structures could be identified. Then, we compared the Endo H-released serum glycome of patients suffering from rheumatoid arthritis with healthy controls as mannose-binding lectin deficiency (MBL) and modulation of a-mannosidase activity were previously associated with this disease. Interestingly, we observed that both high-mannose and hybrid structures were fairly constant, suggesting that circulating MBL and a-mannosidase may not affect significantly the levels of serum glycoproteins carrying these glycans.