Autoantibodies against complement factor B in rheumatoid arthritis.

Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disorder affecting the joints. Many patients carry anti-citrullinated protein autoantibodies (ACPA). Overactivation of the complement system seems to be part of the pathogenesis of RA, and autoantibodies against the pathway initiators C1q and MBL, and the regulator of the complement alternative pathway, factor H (FH), were previously reported. Our aim was to analyze the presence and role of autoantibodies against complement proteins in a Hungarian RA cohort. To this end, serum samples of 97 ACPA-positive RA patients and 117 healthy controls were analyzed for autoantibodies against FH, factor B (FB), C3b, C3-convertase (C3bBbP), C1q, MBL and factor I. In this cohort, we did not detect any patient with FH autoantibodies but detected C1q autoantibodies in four patients, MBL autoantibodies in two patients and FB autoantibodies in five patients. Since the latter autoantibodies were previously reported in patients with kidney diseases but not in RA, we set out to further characterize such FB autoantibodies. The isotypes of the analyzed autoantibodies were IgG2, IgG3, IgGκ, IgGλ and their binding site was localized in the Bb part of FB. We detected in vivo formed FB-autoanti-FB complexes by Western blot. The effect of the autoantibodies on the formation, activity and FH-mediated decay of the C3 convertase in solid phase convertase assays was determined. In order to investigate the effect of the autoantibodies on complement functions, hemolysis assays and fluid phase complement activation assays were performed. The autoantibodies partially inhibited the complement-mediated hemolysis of rabbit red blood cells, inhibited the activity of the solid phase C3-convertase and C3 and C5b-9 deposition on complement activating surfaces. In summary, in ACPA-positive RA patients we identified FB autoantibodies. The characterized FB autoantibodies did not enhance complement activation, rather, they had inhibitory effect on complement. These results support the involvement of the complement system in the pathomechanism of RA and raise the possibility that protective autoantibodies may be generated in some patients against the alternative pathway C3 convertase. However, further analyses are needed to assess the exact role of such autoantibodies.

The Role of IgG Fc Region N-Glycosylation in the Pathomechanism of Rheumatoid Arthritis.

Anti-citrullinated protein antibodies (ACPAs) are involved in the pathogenesis of rheumatoid arthritis. N-glycosylation pattern of ACPA-IgG and healthy IgG Fc differs. The aim of this study is to determine the relative sialylation and galactosylation level of ACPAs and control IgG to assess their capability of inducing TNFα production, and furthermore, to analyze the correlations between the composition of Fc glycans and inflammatory markers in RA. We isolated IgG from sera of healthy volunteers and RA patients, and purified ACPAs on a citrulline-peptide column. Immunocomplexes (IC) were formed by adding an F(ab)2 fragment of anti-human IgG. U937 cells were used to monitor the binding of IC to FcγR and to trigger TNFα release determined by ELISA. To analyze glycan profiles, control IgG and ACPA-IgG were digested with trypsin and the glycosylation patterns of glycopeptides were analyzed by determining site-specific N-glycosylation using nano-UHPLC-MS/MS. We found that both sialylation and galactosylation levels of ACPA-IgG negatively correlate with inflammation-related parameters such as CRP, ESR, and RF. Functional assays show that dimerized ACPA-IgG significantly enhances TNFα release in an FcγRI-dependent manner, whereas healthy IgG does not. TNFα production inversely correlates with the relative intensities of the G0 glycoform, which lacks galactose and terminal sialic acid moieties.

Biologia Futura: Emerging antigen-specific therapies for autoimmune diseases.

Autoimmune diseases are caused by breaking the central and/or peripheral tolerance against self, leading to uncontrolled immune response to autoantigens. The incidences of autoimmune diseases have increased significantly worldwide over the last decades; nearly 5% of the world's population is affected. The current treatments aim to reduce pain and inflammation to prevent organ damage and have a general immunosuppressive effect, but they cannot cure the disease. There is a huge unmet need for autoantigen-specific therapy, without affecting the immune response against pathogens. This goal can be achieved by targeting autoantigen-specific T or B cells and by restoring self-tolerance by inducing tolerogenic antigen-presenting cells (APC) and the development of regulatory T (Treg) cells, for example, by using autoantigenic peptides bound to nanoparticles. Transferring in vitro manipulated autologous tolerogenic APC or autologous autoantigen-specific Treg cells to patients is the promising approach to develop cellular therapeutics. Most recently, chimeric autoantibody receptor T cells have been designed to specifically deplete autoreactive B cells. Limitations of these novel autoantigen-specific therapies will also be discussed.

Inflammatory signal induced IL-10 production of marginal zone B-cells depends on CREB.

IL-10 is a suppressive cytokine that has been implicated in the pathophysiology of autoimmune disorders and can be produced by different cell types such as regulatory B-cells. Our previous work showed that under inflammatory condition MZ B-cells differentiated into IL-10 producing cells and contributed to the downregulation of collagen-induced arthritis, while follicular B-cells failed to do so. Based on these observations, we aimed to investigate how inflammatory signals mediated through the BCR, TLR9 and IFN-γ receptors trigger IL-10 production in MZ B-cells but leave FO B-cells unresponsive. We particularly focused on the CREB transcription factor as it is involved in all three signalling cascades and analysed its contribution to IL-10 production. Our results demonstrate that the IL-10 production of MZ B-cells induced by the BCR, TLR9 and IFN-γ receptors is mediated by CREB. We showed that the activation of CREB is prolonged in MZ B-cells while the transcription factor only transiently phosphorylated in FO B-cells. The sustained phosphorylation of CREB is clearly associated with its prolonged binding to molecular partner CBP, whereas inhibition of their association decreased IL-10 production. We assume that sustained activation of CREB is required for IL-10 production by B-cells under inflammatory conditions.

The Role of Interleukin-10 in Mediating the Effect of Immune Challenge on Mouse Gonadotropin-Releasing Hormone Neurons In Vivo.

Immune challenge alters neural functioning via cytokine production. Inflammation has profound impact on the central regulation of fertility, but the mechanisms involved are not clearly defined. The anti-inflammatory cytokine interleukin (IL)-10 is responsible for balancing the immune response in the brain. To examine whether IL-10 has an effect on the function of the gonadotropin-releasing hormone (GnRH) neurons, we first examined the effect of immune responses with distinct cytokine profiles, such as the T cell-dependent (TD) and T cell-independent (TI) B-cell response. We investigated the effect of the TD and TI immune responses on ERK1/2 phosphorylation in GnRH neurons by administering fluorescein isothiocyanate/keyhole limpet hemocyanin (KLH-FITC) or dextran-FITC to female mice. Although dextran-FITC had no effect, KLH-FITC induced ERK1/2 phosphorylation in GnRH neurons after 6 d. KLH-FITC treatment increased the levels of IL-10 in the hypothalamus (HYP), but this treatment did not cause lymphocyte infiltration or an increase in the levels of proinflammatory cytokines. In IL-10 knock-out (KO) mice, KLH-FITC-induced ERK1/2 phosphorylation in the GnRH neurons was absent. We also showed that in IL-10 KO mice, the estrous cycle was disrupted. Perforated patch-clamp recordings from GnRH-GFP neurons, IL-10 immunohistochemistry, and in vitro experiments on acute brain slices revealed that IL-10 can directly alter GnRH neuron firing and induce ERK1/2 phosphorylation. These observations demonstrate that IL-10 plays a role in influencing signaling of GnRH neurons in the TD immune response. These results also provide the first evidence that IL-10 can directly alter the function of GnRH neurons and may help the maintenance of the integrity of the estrous cycle.

Affinity Purification and Comparative Biosensor Analysis of Citrulline-Peptide-Specific Antibodies in Rheumatoid Arthritis.

BACKGROUND: In rheumatoid arthritis (RA), anti-citrullinated protein/peptide antibodies (ACPAs) are responsible for disease onset and progression, however, our knowledge is limited on ligand binding affinities of autoantibodies with different citrulline-peptide specificity. METHODS: Citrulline-peptide-specific ACPA IgGs were affinity purified and tested by ELISA. Binding affinities of ACPA IgGs and serum antibodies were compared by surface plasmon resonance (SPR) analysis. Bifunctional nanoparticles harboring a multi-epitope citrulline-peptide and a complement-activating peptide were used to induce selective depletion of ACPA-producing B cells. RESULTS: KD values of affinity-purified ACPA IgGs varied between 10-6 and 10-8 M and inversely correlated with disease activity. Based on their cross-reaction with citrulline-peptides, we designed a novel multi-epitope peptide, containing Cit-Gly and Ala-Cit motifs in two-two copies, separated with a short, neutral spacer. This peptide detected antibodies in RA sera with 66% sensitivity and 98% specificity in ELISA and was recognized by 90% of RA sera, while none of the healthy samples in SPR. When coupled to nanoparticles, the multi-epitope peptide specifically targeted and depleted ACPA-producing B cells ex vivo. CONCLUSIONS: The unique multi-epitope peptide designed based on ACPA cross-reactivity might be suitable to develop better diagnostics and novel therapies for RA.

Antigen-specific immunotherapies in rheumatic diseases.

The main goal of antigen-specific immunotherapy (ASI) in autoimmune and rheumatic diseases is to reprogramme or remove autoreactive cells and/or induce immune tolerance to self-antigens. Current therapies in these diseases either treat symptoms or slow down disease progression but are not yet curative or preventative - disease-specific treatments are urgently needed. In contrast to the nonspecific treatments in current use that induce generalized immune suppression, which is associated with several adverse effects including increased risk of infections, ASIs target a restricted subset of B cells or T cells, and thus do not compromise systemic immunity and host defence. This Review provides a summary of novel approaches for identifying autoepitopes and detecting and targeting autoreactive cells that might help in the development of ASIs. Promising approaches include the use of tolerizing peptides coupled to MHC constructs and/or nanocompounds, tolerizing dendritic cells and antigen-specific vaccines. Following studies in animal models of rheumatoid arthritis and systemic lupus erythematosus, several of these strategies have now entered clinical trials. However, to use these approaches in humans, several important limitations must first be addressed, such as; selecting the proper immunodominant autoantigen; identifying the optimal timing, dosing and route of administration; finding biomarkers for monitoring the therapy; and optimizing methodology.

Regulatory B cells in rheumatoid arthritis: Alterations in patients receiving anti-TNF therapy.

Cytokines, including tumor necrosis factor alpha (TNFα) are involved in Rheumatoid arthritis (RA) pathogenesis by augmenting autoimmunity, sustaining long term inflammation in the synovium, and promoting joint damage. Anti-TNF therapy is one of the most efficient and widely used therapies for RA, although its mechanism is not clarified yet. Earlier we demonstrated that RA patients have a reduced number of IL-10 producing regulatory B cells (B10 cells) as compared to healthy individuals and they are functionally impaired. Our aim was to study the influence of anti-TNF therapy on B10 cells in RA, to follow the alteration of B cell activation markers (CD25, CD69) and to monitor the level of citrullinated peptid-specific antibodies and the secreted IL-10 in patients' sera during the therapy. We have observed that at six month after starting the therapy the frequency of B10 cells remarkably increased, while the expression of the activation marker, CD69 decreased on B cells. In contrast, serum levels of IL-10 and anti-citrullinated peptide antibodies did not change post-treatment. CONCLUSION: The reduced activation state of B cells and the increasing number of regulatory B10. cells might contribute to the therapeutic efficacy of anti-TNF agents in RA.

Induction and Differentiation of IL-10-Producing Regulatory B Cells from Healthy Blood Donors and Rheumatoid Arthritis Patients.

The most important feature of B cells is the production of Abs upon activation; additionally, B cells produce pro- and anti-inflammatory cytokines in response to certain stimuli. IL-10-producing B cells represent a major subset of regulatory B cells (Bregs) that suppress autoimmune and inflammatory responses. B cells play a crucial role in the development and maintenance of the chronic inflammatory autoimmune disease rheumatoid arthritis (RA); however, controversial data are available on IL-10- producing Bregs in RA. Our aim was to identify the optimal conditions that induce IL-10+ Bregs and, furthermore, to shed light on the signaling pathways that are responsible for their expansion. The results show that dual stimulation by CpG and CD40L for 48 h is optimal for IL-10 induction, and this can be synergistically boosted by IL-21. We identified the CD19+CD27+ memory B cell population as the major source of IL-10+ Bregs. We detected significantly fewer CD19+CD27+IL-10+ cells in RA patients compared with healthy controls, and these were functionally defective in suppressing IFN-γ production by CD4+ T cells in coculture. IL-21 drastically increased the number of IL-10+ Bregs within the CD19+CD27+ and CD19+CD27- populations; furthermore, it induced the appearance of IL-10+Blimp-1+ plasmablasts. Monitoring the phosphorylation of key signaling molecules revealed that activation of ERK, p38, and CREB is indispensable for the induction of IL-10 production, whereas phosphorylation of STAT3 further enhances IL-10 expression in human Bregs. We conclude that CREB and STAT3 are the key transcription factors responsible for the expansion and differentiation of human IL-10-producing Bregs.

MZ B cells migrate in a T-bet dependent manner and might contribute to the remission of collagen-induced arthritis by the secretion of IL-10.

In mice, marginal zone (MZ) B cells are found principally in the MZ of the spleen and characterized as CD23-negative cells, primarily express polyreactive BCRs, high levels of complement receptor-2 and TLRs. Collagen-induced arthritis (CIA) is a commonly used animal model of human rheumatoid arthritis, considered as a Th1-mediated disease. Although the importance of MZ B cells in the initiation of CIA is well established, their role in remission is unexplored. Besides, playing a central role in Th1 cell development, T-box transcription factor (T-bet) has important functions in B cells. T-bet is regulated by IFN-γ and through the BCR and TLR9, the signals that have an impact on regulatory IL-10 production. In this work, we aimed to analyze the contribution of T-bet to the function of IL-10-positive MZ B cells. We demonstrate that during the remission phase of CIA, MZ B cells express an elevated level of T-bet and confirm the existence of IL-10/T-bet coexpressing cells. Moreover, we show that T-bet-expressing MZ B cells migrate toward CXCR3 ligand and secrete IL-10 by inflammatory stimuli. Our data suggest that T-bet might contribute to the remission of CIA by facilitating the regulatory potential of IL-10-positive MZ B cells.

In vitro eradication of citrullinated protein specific B-lymphocytes of rheumatoid arthritis patients by targeted bifunctional nanoparticles.

BACKGROUND: Autoreactive B cells are crucial players in the pathogenesis of rheumatoid arthritis (RA). Autoantibodies specific for citrullinated proteins (ACPA), present in the serum of approximately 60-70 % of patients, have a pathogenic role in the disease. B cell depleting therapies may result in a transient immunosuppression, increasing the risk of infections. Our aim was to develop a new therapeutic approach to selectively deplete the ACPA producing autoreactive B cells. METHODS: To target B cells synthetic citrullinated peptide derived from the ß chain of fibrin, ß60-74Cit 60,72,74 (ß60-74Cit), the predominant epitope recognized by ACPA was used. Complement dependent cytotoxicity (CDC) was induced by a modified peptide derived from gp120 of HIV-1. To trigger CDC both the targeting peptide and the complement activating peptide were covalently coupled in multiple copies to the surface of poly (DL-lactic-co-glycolic acid) nanoparticles (NPs). Ex vivo antibody synthesis was examined by ELISA and ELISpot. CDC was tested after dead cell staining by flow cytometry. RESULTS: The ß60-74Cit peptide was selectively recognized by a small subset of B cells from RA patients having high level of peptide specific serum antibody, suggesting that the peptide can target diseased B cells. The modified gp120 peptide covalently coupled to NPs induced the formation of the complement membrane attack complex, C5b-9 in human serum. We show here for the first time that bifunctional NPs coupled to multiple copies of both the targeting peptide and the complement activating effector peptide on their surface significantly reduce ß60-74Cit peptide specific ex vivo ACPA production, by inducing complement dependent lysis of the citrullinated peptide specific B cells of seropositive RA patients. CONCLUSIONS: Bifunctional NPs covalently coupled to autoantigen epitope peptide and to a lytic peptide activating complement may specifically target and deplete the peptide specific autoreactive B-cells.

Synthetic Peptide-Based ELISA and ELISpot Assay for Identifying Autoantibody Epitopes.

Enzyme-linked immunosorbent assay (ELISA) is an invaluable diagnostic tool to detect serum autoantibody binding to target antigen. To map the autoantigenic epitope(s), overlapping synthetic peptides covering the total sequence of a protein antigen are used. A large set of peptides synthesized on the crown of pins can be tested by Multipin ELISA for fast screening. Next, to validate the results, the candidate epitope peptides are resynthesized by solid-phase synthesis, coupled to ELISA plate directly, or in a biotinylated form, bound to neutravidin-coated surface and the binding of autoantibodies from patients' sera is tested by indirect ELISA. Further, selected epitope peptides can be applied in enzyme-linked immunospot assay to distinguish individual, citrullinated peptide-specific autoreactive B cells in a pre-stimulated culture of patients' lymphocytes.

Suppression of innate and adaptive B cell activation pathways by antibody coengagement of FcγRIIb and CD19.

The Fc receptor (FcγRIIb) inhibits B cell responses when coengaged with B cell receptor (BCR), and has become a target for new autoimmune disease therapeutics. For example, BCR and FcγRIIb coengagement via the Fc-engineered anti-CD19 XmAb5871 suppresses humoral immune responses. We now assess effects of XmAb5871 on other activation pathways, including the pathogen-associated molecular pattern receptor, TLR9. Since TLR9 signaling is implicated in autoimmune diseases, we asked if XmAb5871 could inhibit TLR9 costimulation. We show that XmAb5871 decreases ERK and AKT activation, cell proliferation, cytokine, and IgG production induced by BCR and/or TLR9 signals. XmAb5871 also inhibited differentiation of citrullinated peptide-specific plasma cells from rheumatoid arthritis patients. XmAb5871 may therefore have potential to suppress pathogenic B cells in autoimmune diseases.

Bead arrays for antibody and complement profiling reveal joint contribution of antibody isotypes to C3 deposition.

The development of antigen arrays has provided researchers with great tools to identify reactivities against self or foreign antigens from body fluids. Yet, these approaches mostly do not address antibody isotypes and their effector functions even though these are key points for a more detailed understanding of disease processes. Here, we present a bead array-based assay for a multiplexed determination of antigen-specific antibody levels in parallel with their properties for complement activation. We measured the deposition of C3 fragments from serum samples to reflect the degree of complement activation via all three complement activation pathways. We utilized the assay on a bead array containing native and citrullinated peptide antigens to investigate the levels of IgG, IgM and IgA autoantibodies along with their complement activating properties in serum samples of 41 rheumatoid arthritis patients and 40 controls. Our analysis revealed significantly higher IgG reactivity against the citrullinated fibrinogen ß and filaggrin peptides as well as an IgA reactivity that was exclusive for citrullinated fibrinogen ß peptide and C3 deposition in rheumatoid arthritis patients. In addition, we characterized the humoral immune response against the viral EBNA-1 antigen to demonstrate the applicability of this assay beyond autoimmune conditions. We observed that particular buffer compositions were demanded for separate measurement of antibody reactivity and complement activation, as detection of antigen-antibody complexes appeared to be masked due to C3 deposition. We also found that rheumatoid factors of IgM isotype altered C3 deposition and introduced false-positive reactivities against EBNA-1 antigen. In conclusion, the presented bead-based assay setup can be utilized to profile antibody reactivities and immune-complex induced complement activation in a high-throughput manner and could facilitate the understanding and diagnosis of several diseases where complement activation plays role in the pathomechanism.

TGFß activated kinase 1 (TAK1) at the crossroad of B cell receptor and Toll-like receptor 9 signaling pathways in human B cells.

B cell development and activation are regulated by combined signals mediated by the B cell receptor (BCR), receptors for the B-cell activating factor of the tumor necrosis factor family (BAFF-R) and the innate receptor, Toll-like receptor 9 (TLR9). However, the underlying mechanisms by which these signals cooperate in human B cells remain unclear. Our aim was to elucidate the key signaling molecules at the crossroads of BCR, BAFF-R and TLR9 mediated pathways and to follow the functional consequences of costimulation.Therefore we stimulated purified human B cells by combinations of anti-Ig, B-cell activating factor of the tumor necrosis factor family (BAFF) and the TLR9 agonist, CpG oligodeoxynucleotide. Phosphorylation status of various signaling molecules, B cell proliferation, cytokine secretion, plasma blast generation and the frequency of IgG producing cells were investigated. We have found that BCR induced signals cooperate with BAFF-R- and TLR9-mediated signals at different levels of cell activation. BCR and BAFF- as well as TLR9 and BAFF-mediated signals cooperate at NFκB activation, while BCR and TLR9 synergistically costimulate mitogen activated protein kinases (MAPKs), ERK, JNK and p38. We show here for the first time that the MAP3K7 (TGF beta activated kinase, TAK1) is responsible for the synergistic costimulation of B cells by BCR and TLR9, resulting in an enhanced cell proliferation, plasma blast generation, cytokine and antibody production. Specific inhibitor of TAK1 as well as knocking down TAK1 by siRNA abrogates the synergistic signals. We conclude that TAK1 is a key regulator of receptor crosstalk between BCR and TLR9, thus plays a critical role in B cell development and activation.

T-bet is a new synergistic meeting point for the BCR and TLR9 signaling cascades.

The importance of the BCR and TLR9 in autoimmunity and in the production of auto-antibodies is well established but the underlying molecular mechanism still needs to be determined. Here, we aim to characterize the BCR-TLR9 cross-talk by its effect on T-bet, as T-bet is activated and regulated by both receptors and has an important role in class-switching to pathological IgG2a in mice. Using primary mouse B cells, we demonstrate that T-bet expression is synergistically elevated by the cross-talk between the BCR and TLR9. To test the effect of this synergy on IgG2a-switching, the levels of switched B cells were checked by functional tests. We found that BCR costimulation had no additional effect on TLR9-induced IgG2a expression, however the expression of Rad51 was synergistically increased. To check the biological significance of the synergy, we compared T-bet expression in B cells from healthy and collagen-induced arthritis mice but no differences were found. Taken together, we demonstrate here that signaling cascades driven by the BCR and TLR9 have a newly identified meeting point at T-bet. The two cascades act synergistically on T-bet; however additional signals may be needed to induce prolonged functional responses such as class-switch recombination.

Recognition of new citrulline-containing peptide epitopes by autoantibodies produced in vivo and in vitro by B cells of rheumatoid arthritis patients.

Anti-citrullinated peptide/protein antibodies (ACPAs) are highly sensitive and specific markers of rheumatoid arthritis (RA). Identification of peptide epitopes that may detect different subgroups of RA patients might have diagnostic and prognostic significance. We have investigated citrulline- and arginine-containing peptide pairs derived from filaggrin, collagen or vimentin, and compared this citrulline-peptide panel with the serological assays conventionally used to detect ACPAs. Furthermore, we studied if the same citrulline-peptides identify antibody-secreting cells in in vitro cultures of RA B cells. Recognition of citrulline- and arginine-containing filaggrin, vimentin and collagen peptide epitopes were tested by Multipin ELISA system, by indirect ELISA and by a peptide-specific microarray. B cells were purified from blood by negative selection; antibody-producing cells were enumerated by ELISPOT assay. The panel composed of citrulline-peptide epitopes of filaggrin, collagen and vimentin was recognized by RA sera with a sensitivity and specificity comparable with the currently used tests. Moreover, the combined citrulline-peptide panel including the new short epitope peptide of filaggrin, fil311-315, also identified nearly one-third of RA cases that were negative for antibodies against cyclic citrullinated peptides, mutated citrullinated vimentin or for rheumatoid factor. The results with the peptide-specific microarray have shown that although most ACPAs recognizing the four citrulline peptides are IgG, some of them specifically recognizing citrulline-containing filaggrin peptides (fil311-315 and fil306-326) are IgM, and so may be produced either by newly formed activated B cells or by unswitched B memory cells. Furthermore, the citrulline-peptides of filaggrin and vimentin detect ACPA-producing cells, and so could also be applied to study the B cells of RA patients.

Role of N- or C-terminal biotinylation in autoantibody recognition of citrullin containing filaggrin epitope peptides in rheumatoid arthritis.

Here, we report on the synthesis, conformational analysis, and autoantibody binding properties of new sets of rheumatoid arthritis (RA) specific biotin-peptide conjugates derived from filaggrin epitope peptides. The biotin with or without a linker was attached to the Cit or Arg containing epitope core ((311)TXGRS(315)) or epitope region ((306)SHQESTXGXSXGRSGRSGS(324)) peptide (where X = Cit), through an amide bond at the N- or C-terminal of the epitopes. Antibody binding was detected by indirect enzyme-linked immunosorbent assay (ELISA) using sera from RA, Systemic lupus erythematosus (SLE) patients, as well as healthy individuals, and the secondary structure of conjugates was investigated by electronic circular dichroism (ECD). We found that autoantibodies from RA patients recognize specifically both filaggrin epitope region ((306)SHQESTXGXSXGRSGRSGS(324)) and short epitope core ((311)TXGRS(315)) peptides. Our data also indicate that the positioning of the biotin label within a peptide sequence can markedly influence the antibody binding, but the length of the linker incorporated has essentially no effect on the recognition. ECD experiments demonstrate that the Arg/Cit change does not influence the solution conformation of the peptide conjugates. However, the presence and position of the biotin moiety has a pronounced effect on the conformation of the 5-mer epitope core peptides, while it does not alter the secondary structure of the 19-mer epitope region peptides.