A novel DNA/histone H4 peptide complex detects autoantibodies in systemic lupus erythematosus sera.

BACKGROUND: The detection of anti-dsDNA antibodies is critical for the diagnosis and follow-up of systemic lupus erythematosus (SLE) patients. The presently available assays are characterized by a non-optimal specificity (solid phase assays) or sensitivity (Crithidia Luciliae immunofluorescence test (CLIFT)). To overcome the limits of CLIFT and solid phase chromatin assays, we explored the diagnostic potential of an assay based on plasmid DNA containing a highly bent fragment of 211 bp from Crithidia Luciliae minicircles, complexed with histone peptides. METHODS: Electrically neutral complexes of PK201/CAT plasmid (PK) DNA and histone 4 (H4) peptides were evaluated by electromobility shift assay. Complexes of H4 peptides and PK were absorbed to the solid phase to detect specific immunoglobulin G (IgG) in sera. Sera from 109 SLE patients, 100 normal healthy subjects, and 169 disease controls were tested. RESULTS: H4(14-34) containing the consensus sequence for DNA binding interacts with PK, retarding its migration. H4(14-34)/PK complexes were used to test sera by ELISA. Anti-H4-PK antibodies were detected in 56 % of SLE sera (more frequently in patients with skin or joint involvement) versus 5.9 % in disease controls; inhibition assays show that sera react with epitopes present on DNA or on the complex, not on the peptide. Antibody titer is correlated with European Consensus Lupus Activity Measurement (ECLAM) score and anti-complement component 1q (C1q) antibodies, negatively with C3 levels. Anti-H4-PK antibodies compared with CLIFT and solid phase dsDNA assays display moderate concordance. CONCLUSIONS: The H4/PK assay is a simple and reliable test which is useful for the differential diagnosis and evaluation of disease activity in SLE patients.

Surface Plasmon Resonance Method to Evaluate Anti-citrullinated Protein/Peptide Antibody Affinity to Citrullinated Peptides.

Surface plasmon resonance (SPR) technique is extremely interesting in immunology because it has the potential to directly visualize biomolecular interactions in real-time monitoring antibody affinity, one of the parameters affecting pathogenicity in autoimmune diseases. Herein we describe the affinity evaluation of anti-citrullinated peptide antibodies (ACPA) to a peptide-based biosensor by SPR. The method describes the purification of ACPA isolated from rheumatoid arthritis (RA) patients using affinity columns, the strategy employed for the immobilization of citrullinated peptides onto a sensor chip, and the evaluation of the specific binding of purified ACPA to immobilized peptides.

Biosensor analysis of anti-citrullinated protein/peptide antibody affinity.

Anti-citrullinated protein/peptide antibodies (ACPAs) are detected in rheumatoid arthritis (RA) sera and because of their strict association with the disease are considered marker antibodies, probably endowed with pathogenic potential. Antibody affinity is one of the parameters affecting pathogenicity. Three diagnostic citrullinated peptides-viral citrullinated peptide 1 (VCP1) and VCP2 derived from Epstein-Barr virus (EBV)-encoded proteins and histone citrullinated peptide 1 (HCP1) derived from histone H4-were synthesized as tetrameric multiple antigen peptides and immobilized on sensor chips CM5 type in a Biacore T100 instrument. Specific binding of purified antibodies from RA patients to the three peptides was analyzed by surface plasmon resonance using two arginine-containing sequences as controls. Employing a 1:1 binding model for affinity constant calculation, ACPAs interacted with VCP1 and VCP2 with lower apparent affinity (10(-6) M>KD>10(-7) M) and interacted with HCP1 with higher apparent affinity (KD=10(-8) M). The results indicate that the binding to citrullinated peptides is characterized by wide differences in affinity, with slower association and faster dissociation rates in the case of antibodies to viral citrullinated peptides as compared with antibodies specific for the histone peptide. This biosensor analysis shows the high cross-reactivity of purified ACPAs that bind other citrullinated peptides besides the one used for purification.

Antibodies from patients with rheumatoid arthritis target citrullinated histone 4 contained in neutrophils extracellular traps.

BACKGROUND: Histone deimination regulates gene function and contributes to antimicrobial response, allowing the formation of neutrophil extracellular traps (NETs). Deiminated proteins are target of anti-citrullinated peptides antibodies (ACPA) in rheumatoid arthritis (RA). OBJECTIVE: The objective of this paper is to test the hypothesis that RA sera react with deiminated histones contained in NETs. METHODS: Neutrophils from peripheral blood were stimulated with A23187 and acid treated; NETosis was induced by phorbol myristate acetate, and NET proteins were isolated. Sera were tested by immunoblot on acid extracted proteins from neutrophils and from NETs, and by ELISA on deiminated histone H4 or H4-derived peptides. Bands reactive with RA sera were excised from gels, digested with trypsin and subjected to matrix-assisted laser desorption/ionisation time of flight (MALDI-TOF) analysis, before and after derivatisation to detect citrullinated peptides. RESULTS: RA sera reacted with a deiminated antigen of 11 KDa from activated neutrophils, recognised also by anti-H4 and antideiminated H4 antibodies. A similar reactivity was observed with NET proteins. The antigen from neutrophils or NETs was identified as citrullinated H4 by MALDI-TOF analysis. By ELISA, RA sera bound in vitro citrullinated H4. Citrullinated H4 14-34 and 31-50 peptides detected antibodies in 67% and 63% of RA sera and in less than 5% of controls; antibody titre was correlated with anti-CCP2. CONCLUSIONS: Citrullinated H4 from activated neutrophils and NETs is a target of antibodies in RA, and synthetic citrullinated H4-derived peptides are a new substrate for ACPA detection. As NETosis can generate antigens for ACPA, these data suggest a novel connection between innate and adaptive immunity in RA.

Endostatin and Thrombospondin-1 levels are increased in the sera of patients with chronic spontaneous urticaria.

Chronic urticaria (CU) is a common disease characterized by recurrent itchy wheals and/or angioedema for more than 6 weeks. Increased levels of the pro-angiogenic mediator vascular endothelial growth factor (VEGF) have been described in skin disorders, such as chronic urticaria (CU), psoriasis and atopic dermatitis. Up to now, no data on the role of VEGF endogenous inhibitors Endostatin (ES) and Thrombospondin-1 (TSP-1) in CU are available. The aim of our study is to investigate the potential involvement of ES and TSP-1 in patients with chronic spontaneous urticaria (CSU). The levels of ES and TSP-1 were measured in the sera of 106 adult patients with CSU and 98 healthy subjects by enzyme immunoassays. The serum levels of the anti-angiogenic mediators ES and TSP-1 resulted significantly higher in CSU than in control subjects. Analysis of these mediators in CSU sub-groups, defined by the results of the autologous serum skin test (ASST), identified a significant increase of ES and TSP-1 in both ASST-positive and ASST-negative sub-groups as compared to the controls. Levels of ES and TSP-1 do not parallel the disease severity in CSU. Our study suggests that the extracellular matrix (ECM) fragments ES and TSP-1 with anti-angiogenic activity play a potential role in the pathogenesis of CSU but do not parallel disease activity.

CCL5/RANTES, sVCAM-1, and sICAM-1 in chronic spontaneous urticaria.

BACKGROUND: Chronic urticaria (CU) is a common disease characterized by recurrent itchy wheals and/or angioedema for more than 6 weeks. We aimed to investigate the potential involvement of chemotactic mediators and soluble adhesion molecules as markers of endothelial dysfunction in the pathogenesis of chronic spontaneous urticaria (CSU). The potential relevance of these soluble mediators in the evaluation of disease activity was also investigated. METHODS: We measured the levels of CCL5/RANTES, CXCL8/IL-8, sVCAM-1, and sICAM-1 in the sera of 87 patients with CSU and 61 normal healthy subjects (NHS) using ELISA assays. According to the results of autologous serum skin tests (ASST), CSU patients were classified into ASST-positive and ASST-negative subgroups. Furthermore, we investigated in 4 patients whether H1-antihistamine therapy decreases sVCAM-1 and sICAM-1 levels. RESULTS: We detected a significantly higher concentration of CCL5/RANTES (p < 0.0001) but not of CXCL8/IL-8 in CSU patients compared to NHS. The serum levels of sICAM-1 and sVCAM-1 were significantly increased in CSU patients compared to NHS (p = 0.0121 and p = 0.0043, respectively). No difference in chemokine or soluble adhesion molecule levels was detected between the ASST-positive and ASST-negative subgroups. A positive correlation was found between sICAM-1 and sVCAM-1 (p = 0.0022) but not between these and CCL5/RANTES. After H1-antihistamine therapy, sVCAM-1 and sICAM-1 levels did not decrease in the 4 CSU patients tested. CONCLUSIONS: Our study suggests that CCL5/RANTES, sICAM-1, and sVCAM-1 play a potential role in the pathogenesis of CSU but they do not parallel disease activity and are not predictive of the response to H1-antihistamine therapy.