Leonor Nogueira

Diagnostic value of anti-human citrullinated fibrinogen ELISA and comparison with four other anti-citrullinated protein assays

We studied the diagnostic performance of the anti-human citrullinated fibrinogen antibody (AhFibA) ELISA for rheumatoid arthritis (RA) in a consecutive cohort (population 1) and evaluated the agreement between the AhFibA ELISA and four other assays for anti-citrullinated protein/peptide antibodies (ACPAs) as well as rheumatoid factor in patients with longstanding RA (population 2). Population 1 consisted of 1024 patients with rheumatic symptoms; serum samples from these patients were sent to our laboratory for ACPA testing within the context of a diagnostic investigation for RA. Ninety-two of these patients were classified as having RA according to the American College of Rheumatology criteria and 463 were classified as non-RA patients. Population 2 consisted of 180 patients with longstanding RA and was used to assess agreement and correlations between five ACPA assays: anti-cyclic citrullinated peptide (CCP)1 and anti-CCP2 antibodies were detected using a commercially available ELISA, AhFibA using ELISA, and anti-PepA and anti-PepB antibodies using line immunoassay. Applying previously proposed cut-offs for AhFibA, we obtained a sensitivity of 60.9% and a specificity of 98.7% in population 1. Receiver operating characteristic curve analysis could not detect a significant difference in diagnostic performance between the AhFibA ELISA and anti-CCP2 assay. Performing a hierarchical nearest neighborhood cluster analysis of the five different ACPA assays in population 2, we identified two clusters: a cluster of anti-pepA, anti-pepB and anti-CCP1, and a cluster of AhFibA and anti-CCP2. In conclusion, we found that AhFibA and anti-CCP2 antibodies had similar diagnostic performance. However, disagreement between ACPA tests may occur.

IgG subclass distribution of the rheumatoid arthritis-specific autoantibodies to citrullinated fibrin

In the rheumatoid synovium, deiminated (‘citrullinated’) forms of fibrin are the major targets of IgG autoantibodies to citrullinated proteins (ACPA), the most specific serological markers of rheumatoid arthritis (RA). To further the characterization of ACPA, we determined their subclass distribution. From a previously validated highly sensitive and specific enzyme‐linked immunosorbent assay (ELISA) onto in vitro deiminated human fibrinogenu2003−u2003antihuman fibrin(ogen) autoantibodies (AhFibA)‐ELISAu2003−u2003we derived and calibrated four ELISAs, using monoclonal antibodies to each of the four IgG subclasses, to determine the proportions of AhFibA subclasses in the sera. A series of 186 serum samples from RA patients was analysed. All AhFibA‐positive sera contained IgG1‐AhFibA, which reached the highest titres and accounted for more than 80% of AhFibA in three‐quarters of the sera. One or two other subclasses were associated with IgG1 in 39% of the sera, IgG4‐AhFibA being observed much more frequently and at higher titres than IgG3‐ or IgG2‐AhFibA. IgG1 alone or IgG(1u2003+u20034)‐AhFibA were the AhFibA subclass profiles found in more than 80% of patients. AhFibA are mainly IgG1 and, to a lesser extent, IgG4. Such IgG subclass profiles may influence the effector phases of the immunological conflict between ACPA and deiminated fibrin that takes place specifically in the rheumatoid synovium and therefore may play a critical role in the self‐maintenance of rheumatoid inflammation.

Tumor necrosis factor polymorphisms in multiple sclerosis: No additional association independent of HLA

In order to investigate whether genes coding for tumor necrosis factors (TNF) contribute to the pathogenesis of multiple sclerosis (MS) and also whether they have a non-random association with the MS associated HLA-DRB1*1501-DQA1*0102-DQB1*0602 haplotype, 40 MS patients and their parents were characterized at four polymorphic loci in the region of the TNF genes: a NcoI RFLP and three microsatellites. We were able to determine the parental haplotypes and used those which were not transmitted to the proband as controls. Fifty percent of the HLA-DRB1*1501-DQA1*0102-DQB1*0602 haplotypes carried the TNFc1-n2-a11-b4 allelic combination in both the patient and the control groups. However, there was no association of any of these TNF polymorphisms with MS, independent of that already described for the class II region. This, with the lack of association of DP alleles with MS, effectively marks the boundaries of the MS associated haplotype.

Autoantibodies to citrullinated proteins: ACPA.

Anti-perinuclear factor and anti-keratin antibodies have long been known to be specifically associated with rheumatoid arthritis (RA). They were first demonstrated to target various forms of (pro)filaggrin, a protein of stratified epithelia. Then, they were found to belong to a single family of autoantibodies targeting proteins that bear peptidic epitopes centered by a citrullyl residue: the anti-citrullinated protein autoantibodies (ACPA). The main targets of ACPA in the synovial tissue were demonstrated to be citrullinated forms of the α- and β-chains of fibrin. A chronic conflict between locally produced ACPA and deposits of citrullinated fibrin is probably responsible for self-maintaining of RA synovial inflammation. Various tests for the detection of ACPA have been developed: recent ELISAs confirm their high diagnostic specificity and improve their diagnostic sensitivity. Since ACPA appear very early in the course of the disease, their detection is of major interest to identify RA among recent arthritides. Moreover, their prognostic value may lead to start early ‘aggressive’ treatments to prevent irreversible joint damage.

Validation of a multiplex chip-based assay for the detection of autoantibodies against citrullinated peptides

IntroductionAutoantibodies directed against citrullinated proteins/peptides (ACPAs) are highly specific and predictive for the development of rheumatoid arthritis (RA). Different subgroups of RA patients, which have different prognoses and may require different treatments, are characterized by different autoantibody profiles. The objective of this study was to develop a microarray for the detection of multiple RA-associated autoantibodies, initially focusing on responses against citrullinated epitopes on candidate autoantigens in RA.MethodsThe microarray is based on Phadias ImmunoCAP ISAC system, with which reactivity to more than 100 antigens can be analyzed simultaneously, by using minute serum volumes (< 10 μl). Twelve citrullinated peptides, and the corresponding native arginine-containing control peptides, were immobilized in an arrayed fashion onto a chemically modified glass slide, allowing a three-dimensional layer with high binding capacity. The assay was optimized concerning serum dilution and glass surface, whereas each individual antigen was optimized concerning coupling chemistry, antigen concentration, and selection of spotting buffer. The performance of each peptide in the ImmunoCAP ISAC system was compared with the performance in enzyme-linked immunosorbent assays (ELISAs). Serum from 927 RA patients and 461 healthy controls from a matched case-control study were applied onto reaction sites on glass slides, followed by fluorescent-labeled anti-human immunoglobulin G (IgG) antibody. Fluorescence intensities were detected with a laser scanner, and the results analyzed by using image-analysis software.ResultsStrong correlations between the ImmunoCAP ISAC system and ELISA results were found for individual citrullinated peptides (Spearman ρ typically between 0.75 and 0.90). Reactivity of RA sera with the peptides was seen mainly in the anticyclic citrullinated peptide 2 (CCP2)-positive subset, but some additional reactivity with single citrullinated peptides was seen in the anti-CCP2-negative subset. Adjusting for reactivity against arginine-containing control peptides did not uniformly change the diagnostic performance for antibodies against the individual citrullinated peptides.ConclusionsThe multiplexed array, for detection of autoantibodies against multiple citrullinated epitopes on candidate RA autoantigens, will be of benefit in studies of RA pathogenesis, diagnosis, and potentially as a guide to individualized treatment.

A new classification of HLA-DRB1 alleles differentiates predisposing and protective alleles for autoantibody production in rheumatoid arthritis.

The HLA-DRB1 gene was reported to be associated with anticitrullinated protein/peptide autoantibody (ACPA) production in rheumatoid arthritis (RA) patients. A new classification of HLA-DRB1 alleles, reshaping the shared epitope (SE) hypothesis, was recently found relevant in terms of RA susceptibility and structural severity.We investigated the relevance of this new classification of HLA-DRB1 SE+ alleles in terms of rheumatoid factor (RF) and ACPA production in a sample of French RA patients.We studied 160 early RA patients included in a prospective longitudinal cohort of French Caucasian patients with recent-onset arthritis. RF, anticyclic citrullinated peptide 2 (anti-CCP2) and antideiminated human fibrinogen autoantibodies (AhFibA) were assessed in all patients at inclusion. The HLA-DRB1 gene was typed by PCR-sequence specific oligonucleotides probes (PCR-SSOP), and SE+ alleles were classified into four groups (S1, S2, S3P, S3D) according to the new classification.The new classification of HLA-DRB1 SE+ alleles distinguishes predisposing and protective alleles for RF, anti-CCP2 or AhFibA production. The presence of S2 or S3P alleles is associated with both RF, anti-CCP2 or AhFibA positivity, whereas the presence of S3D or S1 alleles appears to be protective for RF, anti-CCP2 or AhFibA positivity.The new classification of HLA-DRB1 SE+ alleles is relevant in terms of autoantibody production in early RA patients by differentiating predisposing and protective alleles for RF or ACPA production.

The antigen specificity of the rheumatoid arthritis-associated ACPA directed to citrullinated fibrin is very closely restricted

The major targets of the disease-specific autoantibodies to citrullinated proteins (ACPA) in synovium of rheumatoid arthritis (RA) patients are borne by the citrullinated α- and β-chains of fibrin. We demonstrated that ACPA target a limited set of citrullinated fibrin peptides and particularly four multicitrullinated peptides which present the major epitopes. In this study, we established the clear immunodominance of the peptides α36-50Cit(38,42) and β60-74Cit(60,72,74) which were recognised by 51/81 (63%) and 61/81 (75%) of ACPA-positive patients, respectively, more than 90% recognising one, the other or both peptides. We also identified the citrullyl residues αCit(42), βCit(72) and βCit(74) as essential for antigenicity, and at a lesser degree αCit(38). Then, we assayed on overlapping 7-mer peptides encompassing the sequences of the two peptides, 3 series of sera recognising either α36-50Cit(38,42) or β60-74Cit(60,72,74) or both peptides. In each series, the reactivity profiles of the sera, largely superimposable, allowed identification of the two 4/5-mer overlapping epitopes (α:xa0VECit(42)HQ and α: Cit(38)VVE), and the single 5-mer epitope (β: GYCit(72)ACit(74)), all located to a flexible globular domain of fibrin on a topological 3D model. In conclusion, we demonstrated that only 3 immunodominant epitopes are targeted by ACPA on citrullinated fibrin stressing their actual oligoclonality. However, the reactivity to the 3 epitopes distinguishes three subgroups of patients. The closely restricted antigen specificity suggests that the autoimmune reaction to citrullinated fibrin is antigen-driven. The accessibility of the epitopes reinforces the hypothesis of a pathogenic role for ACPA via immune complexe formation in the synovial tissue.

Sphingosine Kinase-1 Is Central to Androgen-Regulated Prostate Cancer Growth and Survival

Background Sphingosine kinase-1 (SphK1) is an oncogenic lipid kinase notably involved in response to anticancer therapies in prostate cancer. Androgens regulate prostate cancer cell proliferation, and androgen deprivation therapy is the standard of care in the management of patients with advanced disease. Here, we explored the role of SphK1 in the regulation of androgen-dependent prostate cancer cell growth and survival. Methodology/Principal Findings Short-term androgen removal induced a rapid and transient SphK1 inhibition associated with a reduced cell growth in vitro and in vivo, an event that was not observed in the hormono-insensitive PC-3 cells. Supporting the critical role of SphK1 inhibition in the rapid effect of androgen depletion, its overexpression could impair the cell growth decrease. Similarly, the addition of dihydrotestosterone (DHT) to androgen-deprived LNCaP cells re-established cell proliferation, through an androgen receptor/PI3K/Akt dependent stimulation of SphK1, and inhibition of SphK1 could markedly impede the effects of DHT. Conversely, long-term removal of androgen support in LNCaP and C4-2B cells resulted in a progressive increase in SphK1 expression and activity throughout the progression to androgen-independence state, which was characterized by the acquisition of a neuroendocrine (NE)-like cell phenotype. Importantly, inhibition of the PI3K/Akt pathway—by negatively impacting SphK1 activity—could prevent NE differentiation in both cell models, an event that could be mimicked by SphK1 inhibitors. Fascinatingly, the reversability of the NE phenotype by exposure to normal medium was linked with a pronounced inhibition of SphK1 activity. Conclusions/Significance We report the first evidence that androgen deprivation induces a differential effect on SphK1 activity in hormone-sensitive prostate cancer cell models. These results also suggest that SphK1 activation upon chronic androgen deprivation may serve as a compensatory mechanism allowing prostate cancer cells to survive in androgen-depleted environment, giving support to its inhibition as a potential therapeutic strategy to delay/prevent the transition to androgen-independent prostate cancer.

An investigation of the added value of an ACPA multiplex assay in an early rheumatoid arthritis setting

IntroductionRecently, arrays have become available that allow the simultaneous analysis of several anti-citrullinated protein antibody (ACPA) reactivities using distinct citrullinated peptides. Such assays are designed for exploratory studies. The interpretation of positive antibody reactivities can best be made if the diagnostic and prognostic value of a multiplex array in an early arthritis setting is known and if the multiplex-positive patients who are negative according to three commonly used commercial ACPA assays are characterized.MethodsUsing Thermo Scientific’s ImmunoCap ISAC (Immuno Solid-phase Allergen Chip) system, a multiplexed array that determines reactivities to 11 citrullinated peptides, we analysed serum/plasma of 195 healthy controls and 1282 early arthritis patients from two independent cohorts: the Leiden Early Arthritis Clinic (nu2009=u20091013) and the IMPROVED (nu2009=u2009269) cohort. Findings were compared with results primarily of the anti-citrullinated cyclic peptide 2 (anti-CCP-2) assay but also with anti- CCP-3 and anti-mutated citrullinated vimentin (anti-MCV) assays. The associations between ACPA reactivities and patient characteristics, risk factors (shared epitope, smoking) and disease outcomes (progression of undifferentiated arthritis to rheumatoid arthritis (RA) and severity of joint destruction) were assessed.ResultsThirty-one percent of anti-CCP-2-negative RA patients displayed reactivity toward citrullinated peptides in the multiplex assay. These patients had a positive signal toward a more restricted peptide repertoire than anti-CCP-2-positive RA patients (median of 1 versus 5). Within anti-CCP-2-negative patients, ACPA reactivity as detected by multiplex array was not significantly associated with known risk factors or clinical or prognostic parameters. The frequency of sera from anti-CCP-2-negative RA patients who were positive for the multiplexed peptides was comparable to the frequency in non-RA arthritic patients (27xa0%).ConclusionsAdditive citrulline peptide reactivities detected by the current multiplex system did not reach significant power to be RA-specific. The presence of residual citrulline reactivities detected by this multiplex system in arthritis patients who are negative in commercial ACPA assays needs to be interpreted with caution.

VIDAS-EDRA fully automated testing of autoantibodies to citrullinated proteins for the diagnosis of rheumatoid arthritis

Antiperinuclear factor and antikeratin antibodies (AKA) were shown to belong to the same family of rheumatoid arthritis (RA)-specific autoantibodies directed at citrullinated peptidic epitopes borne by (pro)filaggrins. We showed that their major target in the synovial tissue of RA patients is deiminated (citrullinated) fibrin. Although (pro)filaggrins are probably only cross-reactive proteins, their in vitro detection allowed the development of several highly efficient tests for the diagnosis of RA. Among those, the CCP ELISAs (CCP1 and CCP2), based on a cyclic citrullinated peptide derived from human filaggrin, and the ArFA-ELISA that we developed using a citrullinated recombinant rat filaggrin appear to be the most promising. The rapidly growing interest of rheumatologists in autoantibodies to citrullinated proteins rendered the development of a convenient, fully automated test the next challenge. Based on the ArFA-ELISA, we developed an automated test on the VIDAS machine (bioMerieux), called VIDAS-EDRA (early diagnosis rheumatoid arthritis). Thresholds were defined on a large series of 617 patients with well defined, established rheumatic diseases, including 181 patients with RA. Antibodies to CCP1 and CCP2 (Immunoscan, Eurodiagnostica) were sought following the manufacturer procedures. Rheumatoid factor (RF) and AKA were also analyzed in the series. The diagnostic performances of the tests were compared. In established diseases the diagnostic sensitivity of the VIDAS-EDRA was found to be identical to that of CCP2, and was significantly higher than tests for AKA, CCP1 and RF. n nAutoantibodies to citrullinated proteins can be efficiently detected with the highly specific and sensitive automated test VIDAS-EDRA. n n n nTable 1 n nDiagnostic sensitivity (%) computed at thresholds allowing 95.2% and 98.6% diagnostic specificity to be achieved