Increased levels of circulating soluble CD226 in multiple sclerosis.

BACKGROUND: The glycoprotein CD226 plays a key role in regulating immune cell function. Soluble CD226 (sCD226) is increased in sera of patients with several chronic inflammatory diseases but its levels in neuroinflammatory diseases such as multiple sclerosis (MS) are unknown. OBJECTIVE: To investigate the presence and functional implications of sCD226 in persons with multiple sclerosis (pwMS) and other neurological diseases. METHODS: The mechanisms of sCD226 production were first investigated by analyzing CD226 surface expression levels and supernatants of CD3/CD226-coactivated T cells. The role of sCD226 on dendritic cell maturation was evaluated. The concentration of sCD226 in the sera from healthy donors (HD), pwMS, neuromyelitis optica (NMO), and Alzheimer's disease (AD) was measured. RESULTS: CD3/CD226-costimulation induced CD226 shedding. Addition of sCD226 to dendritic cells during their maturation led to an increased production of the pro-inflammatory cytokine interleukin (IL)-23. We observed a significant increase in sCD226 in sera from pwMS and NMO compared to HD and AD. In MS, levels were increased in both relapsing-remitting multiple sclerosis (RRMS) and secondary-progressive multiple sclerosis (SPMS) compared to clinically isolated syndrome (CIS). CONCLUSION: Our data suggest that T-cell activation leads to release of sCD226 that could promote inflammation and raises the possibility of using sCD226 as a biomarker for neuroinflammation.

Clinical reporting following the quantification of cerebrospinal fluid biomarkers in Alzheimer's disease: An international overview.

INTRODUCTION: The current practice of quantifying cerebrospinal fluid (CSF) biomarkers as an aid in the diagnosis of Alzheimer's disease (AD) varies from center to center. For a same biochemical profile, interpretation and reporting of results may differ, which can lead to misunderstandings and raises questions about the commutability of tests. METHODS: We obtained a description of (pre-)analytical protocols and sample reports from 40 centers worldwide. A consensus approach allowed us to propose harmonized comments corresponding to the different CSF biomarker profiles observed in patients. RESULTS: The (pre-)analytical procedures were similar between centers. There was considerable heterogeneity in cutoff definitions and report comments. We therefore identified and selected by consensus the most accurate and informative comments regarding the interpretation of CSF biomarkers in the context of AD diagnosis. DISCUSSION: This is the first time that harmonized reports are proposed across worldwide specialized laboratories involved in the biochemical diagnosis of AD.

Seropositivity and Antibody Profiling of Patients Are Dramatically Impacted by the Features of Peptides Used as Immunosorbents: A Lesson from Anti-Citrullinated Protein/Peptide Antibody.

Quantification of Abs toward a single epitope is critical to understanding immunobiological processes. In autoimmunity, the prognostic value of the serological profiles of patients draws much attention, but the detection of Abs toward a single epitope is not well controlled. Particularly, the rheumatoid arthritis (RA)-specific anti-citrullinated protein/peptide Abs (ACPA) are specific to a two-atom change on arginyl residues and are considered a heterogeneous family of Abs. As a model, we studied ACPA to decipher how peptide features used as immunosorbent impact Ab detection. We synthesized 30 peptides encompassing immunodominant epitopes of citrullinated fibrin differing by their length and biotin location and tested them using ELISA with 120 sera from RA and non-RA rheumatic disease controls, generating over 3000 experimental measurements. We showed that minor molecular changes in peptide chemical structure had dramatic consequences. Even when peptides exhibited the same epitope, measured Ab titers were extremely variable, and patients' seropositivity was discordant in up to 50% of cases. The distance between epitope and biotin was the most critical parameter for efficient Ab detection irrespective of biotin position or peptide length. Finally, we identified a 15-mer peptide bearing a single citrullinated epitope detecting almost all ACPA-positive sera, thus revealing a high degree of homogeneity in RA autoimmune response. This integrative analysis deciphers the dramatic impact of the molecular design of peptide-based technologies for epitope-specific Ab quantification. It provides a model for assay development and highlights that the studies using such technologies can give a wrong perception of biological processes and therefore that medical use of data must be cautious.

Autoantibodies to human citrullinated fibrinogen and their subfamilies to the α36-50Cit and ß60-74Cit fibrin peptides similarly predict radiographic damages: a prospective study in the French ESPOIR cohort of very early arthritides.

OBJECTIVE: To investigate whether subfamilies of the RA-specific autoantibodies to human citrullinated fibrinogen (AhFibA) differentially associate with the RA risk factors, HLA-DRB1 shared epitope containing alleles (SE alleles) and cigarette smoking, and thus help to predict the disease outcome. METHODS: AhFibA and their anti-α36-50Cit and anti-ß60-74Cit subfamilies were assayed by ELISA, at baseline, in the French ESPOIR (Etude et Suivi des Polyarthrites Indifférenciées Récentes) cohort composed of undifferentiated arthritides and RA patients of < 6 months' duration. Cigarette smoking, SE alleles' presence, DAS28, HAQ and modified Sharp-van der Heijde Score data were obtained at baseline, and after follow-up. RESULTS: After 3 years, 701 patients were classified as having RA according to the ACR/EULAR 2010 criteria. Among them, 349 (50%), 203 (29%) and 257 (37%) were AhFibA-, anti-α36-50Cit- and anti-ß60-74Cit-positive, respectively. The presence and titres of AhFibA and their subfamilies similarly associated with SE alleles, irrespective of their fine specificity, without significant effect of smoking. Neither their presence nor their titre was associated with DAS28 or HAQ. The presence of at least one subfamily was associated with a faster Sharp/van der Heijde score progression, albeit without correlation with the titre. CONCLUSION: AhFibA and their main subfamilies are similarly associated with SE alleles without additional effect of smoking. Whatever their fine specificity was, their presence (but not their titre) similarly constituted a marker of faster joint destruction.

In Black Africans with rheumatoid arthritis, ACPA recognize citrullinated fibrinogen and the derived peptides α36-50Cit38,42 and ß60-74Cit60,72,74, like in Caucasians.

Well documented in Caucasians and Asians, the diagnostic value of anti-CCP2 antibodies has been confirmed in Black African populations. However, autoantibodies to other citrullinated peptides/proteins and their fine specificities have not yet been studied. Here, we show that in Cameroonian patients, anti-citrullinated fibrinogen autoantibodies (AhFibA) are sensitive (73%) diagnostic markers for RA. We also determine that autoantibodies directed to α36-50Cit38,42 or ß60-74Cit60,72,74 peptides which bear the immunodominant epitopes of citrullinated fibrin, are present in similar proportions in Black Africans and Caucasians with 25/56 (45%) and 41/56 (73%) positive RA-sera in Cameroonians, respectively. They also account for almost all the AhFibA reactivities since 38/41 (93%) AhFibA-positive sera contain anti-α36-50Cit38,42 and/or anti-ß60-74Cit60,72,74 autoantibodies. Finally, HLA-DRB1 SE alleles were associated with higher titres of AhFibA and anti-ß60-74Cit60,72,74 autoantibodies. In the genetic and environmental backgrounds of Black Africans, AhFibA are a hallmark of RA like in Caucasians, moreover they recognize the same fibrin epitopes.

First Observations of a Potential Association Between Accumulation of Per- and Polyfluoroalkyl Substances in the Central Nervous System and Markers of Alzheimer's Disease.

The entire human population is exposed to persistent organic pollutants throughout their lives. Among them, per- and polyfluoroalkyl substances (PFAS) are synthetic chemicals widely used in industrial and consumer products that are known to exert adverse effects on human health. As they bioaccumulate in the human brain and are known to be neurotoxic in experimental models, they are assumed to be involved in neurodegenerative processes. In this proof-of-concept study, we measured the level of 18 PFAS in cerebrospinal fluid (CSF) from 8 patients hospitalized with suspected normal pressure hydrocephalus. We then analyzed whether PFAS levels could be related to both biological and clinical markers of Alzheimer's disease. We showed that PFAS and perfluorooctanesulfonate were found in all CSF samples from a French region without fluorochemical industries. Moreover, we observed a significant difference between the levels of PFAS and perfluorooctanesulfonate in the CSF of patients with both Alzheimer's disease markers and cognitive impairment compared with those with only 1 or neither. Two previous studies have shown that PFAS levels in human CSF increase with age and are linked to impaired blood-brain barrier integrity. Our results provide the first evidence of a link between PFAS accumulation in the central nervous system and clinical and biological markers of Alzheimer's disease.

Translocator protein (TSPO) genotype does not change cerebrospinal fluid levels of glial activation, axonal and synaptic damage markers in early Alzheimer's disease.

BACKGROUND: PET imaging of the translocator protein (TSPO) is used to assess in vivo brain inflammation. One of the main methodological issues with this method is the allelic dependence of the radiotracer affinity. In Alzheimer's disease (AD), previous studies have shown similar clinical and patho-biological profiles between TSPO genetic subgroups. However, there is no evidence regarding the effect of the TSPO genotype on cerebrospinal-fluid biomarkers of glial activation, and synaptic and axonal damage. METHOD: We performed a trans-sectional study in early AD to compare cerebrospinal-fluid levels of GFAP, YKL-40, sTREM2, IL-6, IL-10, NfL and neurogranin between TSPO genetic subgroups. RESULTS: We recruited 33 patients with early AD including 16 (48%) high affinity binders, 13 (39%) mixed affinity binders, and 4/33 (12%) low affinity binders. No difference was observed in terms of demographics, and cerebrospinal fluid levels of each biomarker for the different subgroups. CONCLUSION: TSPO genotype is not associated with a change in glial activation, synaptic and axonal damage in early AD. Further studies with larger numbers of participants will be needed to confirm that the inclusion of specific TSPO genetic subgroups does not introduce selection bias in studies and trials of AD that combine TSPO imaging with cerebrospinal fluid biomarkers.

Autoantibodies to citrullinated fibrinogen compared with anti-MCV and anti-CCP2 antibodies in diagnosing rheumatoid arthritis at an early stage: data from the French ESPOIR cohort.

OBJECTIVES: To compare the performance of anticitrullinated peptides/protein antibodies (ACPA) detected by three immunoassays in the French ESPOIR cohort of patients with early rheumatoid arthritis (RA) and undifferentiated arthritis (UA) and to study the relationship between ACPA and disease activity. METHODS: A diagnosis of RA (1987 American College of Rheumatology (ACR) criteria) was established at baseline in 497 patients and after a 2-year follow-up in 592 patients. At baseline, antibodies to citrullinated fibrinogen (AhFibA), antimutated citrullinated vimentin (anti-MCV) and anticyclic citrullinated peptide (anti-CCP2) were assayed and the individual and combined diagnostic sensitivities and predictive values of the tests were determined. Relationships between ACPA positivity and the 28-joint disease activity score and Health Assessment Questionnaire scores were analysed. RESULTS: At a diagnostic specificity of at least 98%, the three tests exhibited similar diagnostic sensitivities (47-48.5%). When considering as positive patients with at least one positive test, the sensitivity increased to 53.5% with a probable loss of specificity. Among the patients classified as having UA at baseline, 30% were positive for one ACPA, the positive predictive values for RA of the three tests ranging from 73% to 80% but increasing when two tests were associated. Whatever the test used, the addition of ACPA positivity to the 1987 criteria enhanced their sensitivity by 6%, close to that of the 2010 ACR/European League Against Rheumatism (EULAR) criteria. CONCLUSIONS: In early arthritis, AhFibA, anti-MCV and anti-CCP2 showed similar diagnostic sensitivity with a high diagnostic specificity and a similar high positive predictive value for RA. Adding ACPA to the 1987 ACR criteria significantly increased the number of patients classified as having RA, confirming the validity of the recent inclusion of the serological criterion in the ACR/EULAR criteria.

Clinical heterogeneity of neuro-inflammatory PET profiles in early Alzheimer's disease.

The relationship between neuroinflammation and cognition remains uncertain in early Alzheimer's disease (AD). We performed a cross-sectional study to assess how neuroinflammation is related to cognition using TSPO PET imaging and a multi-domain neuropsychological assessment. A standard uptake value ratio (SUVR) analysis was performed to measure [18F]-DPA-714 binding using the cerebellar cortex or the whole brain as a (pseudo)reference region. Among 29 patients with early AD, the pattern of neuroinflammation was heterogeneous and exhibited no correlation with cognition at voxel-wise, regional or whole-brain level. The distribution of the SUVR values was independent of sex, APOE phenotype, early and late onset of symptoms and the presence of cerebral amyloid angiopathy. However, we were able to demonstrate a complex dissociation as some patients with similar PET pattern had opposed neuropsychological profiles while other patients with opposite PET profiles had similar neuropsychological presentation. Further studies are needed to explore how this heterogeneity impacts disease progression.

Fcγ receptor profile of monocytes and macrophages from rheumatoid arthritis patients and their response to immune complexes formed with autoantibodies to citrullinated proteins.

OBJECTIVE: To analyse Fcγ receptor (FcγR) expression on monocytes and macrophages from rheumatoid arthritis (RA) patients versus healthy controls (HC), and to compare their responses to immune complexes containing RA-specific anti-citrullinated proteins auto antibodies (ACPA). METHODS: Monocytes and monocyte-derived macrophages were obtained from the peripheral blood of 34 RA patients and 69 HC. FcγR expression was studied by flow cytometry. Cells were stimulated with ACPA-containing immune complexes, and tumour necrosis factor alpha (TNFα) was assayed in culture supernatants. RESULTS: Variations distinguished RA from HC monocytes, corresponding to a 5% and 6% decrease in the percentages of monocytes expressing FcγRI and FcγRII, respectively, and a 7% increase in the proportion of FcγRIII-positive monocytes. Although in both HC and RA patients macrophage differentiation was accompanied by a dramatic increase in the percentage of FcγRIII-expressing cells (72% vs 74.5%), the parallel decline in the proportion of FcγRI-positive cells was markedly smaller in RA (7% vs 43%). Monocytes and macrophages from patients were responsive to ACPA-containing immune complexes but TNFα production in both cell types neither differed from that observed with the corresponding cells from HC, nor correlated with FcγR expression or clinical or biological data. In RA as in HC, ACPA-containing immune complexes induced secretions of more TNFα in macrophages than in paired monocytes (ninefold). Finally, the proinflammatory potential of ACPA-containing immune complexes was confirmed in CD14-positive monocyte macrophages from the synovial fluid of four RA patients. CONCLUSIONS: ACPA-containing immune complexes induce TNFα secretion by blood and synovial fluid-derived macrophages from RA patients, fitting with their probable involvement in RA pathophysiology.

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 ß-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 ß60-74Cit(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), ßCit(72) and ßCit(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 ß60-74Cit(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: VECit(42)HQ and α': Cit(38)VVE), and the single 5-mer epitope (ß: 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.

Are antibodies to fine specificities of citrullinated peptides/proteins useful for stratification of rheumatoid arthritis patients?

BACKGROUND: In rheumatoid arthritis (RA), antibodies to citrullinated protein (ACPA) are believed to be heterogeneous and patient stratification by antibody profiling raised clinical interest for patient management. However, heterogeneity might be partially artificial because of the use of heterogeneous methods for ACPA detection. In recent work instead, we found that ACPA were mainly directed towards a single fibrin-derived peptide, ß60-74BiotNt, but a comparative analysis with the presence of other ACPA specificities is still lacking. OBJECTIVES: To present an overview of RA patients' stratification based on the detection of the main ACPA fine specificities with the same method as compared to that of anti-ß60-74BiotNt antibodies. METHODS: Over 4500 measurements were performed with more than 22 standardised ELISAs, sera from 180 RA patients and 200 to 436 non-RA rheumatic disease controls. RESULTS: Four to 81% of RA patients had ACPA towards various targets, confirming the heterogeneity of ACPA specificities. However, the subgroups of patients overlapped up to 97% with ACPA levels of correlation coefficients up to 0.8, showing redundancy of some targets. Multiplexing decreased diagnostic specificity from 95% to 64%. Instead, anti-ß60-74BiotNt detection identified almost all ACPA-positive patients. CONCLUSIONS: Antibodies to citrullinated protein multiplexing shows some degree of redundancy and is not suitable for diagnostic purposes. ACPA fine specificities might be less heterogeneous than perceived by sera testing on multiple peptides. Patient stratification largely depends on detection methods and requires standardisation.

Taking the A Train? Limited Consistency of Aß42 and the Aß42/40 Ratio in the AT(N) Classification.

The consistency of cerebrospinal fluid amyloid-ß (Aß)42/40 ratio and Aß42 has not been assessed in the AT(N) classification system. We analyzed the classification changes of the dichotomized amyloid status (A+/A-) in 363 patients tested for Alzheimer's disease biomarkers after Aß42 was superseded by the Aß42/40 ratio. The consistency of Aß42 and the Aß42/40 ratio was very low. Notably, the proportions of "false" A+T-patients were considerable (74-91%) and corresponded mostly to patients not clinically diagnosed with Alzheimer's disease. Our results suggest that the interchangeability of Aß42/40 ratio and Aß42 is limited for classifying patients in clinical setting using the AT(N) scheme.

Independent Evaluation of the Respective Predictive Values for High-Grade Prostate Cancer of Clinical Information and RNA Biomarkers after Upfront MRI and Image-Guided Biopsies.

Upfront MRI is taking the lead in the diagnosis of clinically significant prostate cancer, while few image-guided biopsies (IGBs) fail to demonstrate clinically significant prostate cancer. The added value of innovative biomarkers is not confirmed in this context. We analysed SelectMDx-v2 (MDx-2) in a cohort of upfront MRI and image-guided biopsy patients. Participants included patients who received a trans-rectal elastic-fusion registration IGB on the basis of DRE, PSA, PCA3, and PCPT-2.0 risk evaluation. Pre-biopsy MRI DICOM archives were reviewed according to PI-RADS-v2. Post-massage first-void urine samples stored in the institutional registered bio-repository were commercially addressed to MDxHealth to obtain MDx-2 scores. Univariate and multivariate analyses were conducted with the detection on IGB of high-grade (ISUP 2 and higher) as the dependent variable. High-grade cancer was demonstrated in 32/117 (27.4%) patients (8/2010-8/2018). Age, prostate volume, biopsy history, MDx-2, and PI-RADS-v2 scores significantly related to the detection of high-grade cancer. MDx-2 scores and the clinical variables embedded into MDx-2 scores were analysed in multivariate analysis to complement PI-RADS-v2 scores. The two combinations outperformed PI-RADS-v2 alone (AUC-ROC 0.67 vs. 0.73 and 0.80, respectively, p < 0.05) and calibration curves confirmed an adequate prediction. Similar discrimination (C-statistics, p = 0.22) was observed in the prediction of high-grade cancer, thereby questioning the respective inputs and added values of biomarkers and clinical predictors in MDx-2 scores. Based on the results of this study, we can conclude that instruments of prediction developed for systematic prostate biopsies, including those that incorporate innovative biomarkers, must be reassessed and eventually confirmed in the context of upfront MRI and IGB.

Association between number and type of different ACPA fine specificities with lung abnormalities in early, untreated rheumatoid arthritis.

BACKGROUND: Rheumatoid arthritis (RA)-associated anticitrullinated protein/peptide antibodies (ACPA) might originate at mucosal sites such as the lungs. We aimed to examine the relationship between the ACPA repertoire and lung abnormalities on high-resolution CT (HRCT) in patients with earlyuntreated RA. METHODS: 106 patients with newly diagnosed untreated RA were examined with HRCT of the lungs. Blood samples were analysed for presence of rheumatoid factor (RF) and ACPA using either a CCP2 detection kit or an immunochip containing 10 different citrullinated peptides. Association between HRCT findings and the antibody repertoire was assessed by logistic regression analysis. RESULTS: The number (%) of patients with HRCT abnormalities was 58 (54.7%) for parenchymal abnormalities and 68 (64.2%) for airway abnormalities. CCP2 IgG, RF IgA and antibodies against citrullinated fibrinogen were associated with the presence of parenchymal lung abnormalities. Interestingly, a high number of ACPA fine specificities gave a high risk of having parenchymal lung abnormalities at the time of RA diagnosis. No significant signals were identified between ACPA specificities and risk for airway abnormalities. CONCLUSIONS: The presence of RF and ACPAs (especially against citrullinated fibrinogen peptides) as well as high number of ACPAs fine specificities are associated with parenchymal lung abnormalities in patients with early, untreated RA. This provides further support for an important pathogenic link between the lung and systemic autoimmunity, contributing to RA development.

The Urinary Transcriptome as a Source of Biomarkers for Prostate Cancer.

Prostate cancer (PCa) is the second most common cancer of men and is typically slow-growing and asymptomatic. The use of blood PSA as a screening method has greatly improved PCa diagnosis, but high levels of false positives has raised much interest in alternative biomarkers. We used next-generation sequencing (NGS) to elucidate the urinary transcriptome of whole urine collected from high-stage and low-stage PCa patients as well as from patients with the confounding diagnosis of benign hyperplasia (BPH). We identified and validated five differentially expressed protein-coding genes (FTH1 BRPF1, OSBP, PHC3, and UACA) in an independent validation cohort of small-volume (1 mL) centrifuged urine (n = 94) and non-centrifuged urine (n = 84) by droplet digital (dd)PCR. These biomarkers were able to discriminate between BPH and PCa patients and healthy controls using either centrifuged or non-centrifuged whole urine samples, suggesting that the urinary transcriptome is a valuable source of non-invasive biomarkers for PCa that warrants further investigation.

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 a- and beta-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.

Predictive value of autoantibodies from anti-CCP2, anti-MCV and anti-human citrullinated fibrinogen tests, in early rheumatoid arthritis patients with rapid radiographic progression at 1†year: results from the ESPOIR cohort.

OBJECTIVES: We compared the ability of antibodies against cyclic citrullinated peptides (anti-CCP2), against mutated citrullinated vimentin (anti-MCV) and against citrullinated fibrinogen (AhFibA) to predict 1 year rapid radiographic progression (RRP; total Sharp score variation ≥5 points), in early rheumatoid arthritis (RA). METHODS: We analysed 566 patients from the ESPOIR cohort with early RA fulfilling the 2010 American College of Rheumatology/European League Against Rheumatism (ACR/EULAR) criteria at year 1. We assayed the 3 anticitrullinated peptide antibodies (ACPA) tests on baseline sera. We compared the performance of these 3 ACPA tests to predict first-year RRP, by comparing areas under the receiver operating characteristic curves (ROCs). We assessed the 1 year RRP risk by ACPA titres. We used a logistic multivariate regression to analyse RRP risk in terms either of ACPA positivity or titre: high (>3 times the N cut-off) and low (1 to 3N). RESULTS: 145 patients displayed RRP. Areas under the ROCs were similar (0.60) for the 3 tests. High ACPA titres were associated with 1 year RRP, whatever the test was, and with similar ORs. Low+ anti-MCV titres were not associated with 1-year RRP, whereas low+ anti-CCP2 titres (p=0.0226) and low+ AhFibA titres (p=0.0332) were significantly associated. In multivariate analysis, 1 year RRP was associated with anti-CCP2 positivity (p<0.0001), AhFibA positivity (p<0.0001) and high anti-MCV titres (p<0.0001). CONCLUSIONS: Anti-CCP2 antibodies and AhFibA were predictive of 1 year RRP in early RA whatever their titre was, whereas only high anti-MCV antibody titres were predictive, potentially making them more discriminant to predict 1 year RRP risk.

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

INTRODUCTION: Recently, 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. METHODS: Using 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 (n = 1013) and the IMPROVED (n = 269) 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. RESULTS: Thirty-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 (27 %). CONCLUSIONS: Additive 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.

Clinical and pathophysiological significance of the autoimmune response to citrullinated proteins in rheumatoid arthritis.

Rheumatoid arthritis (RA) is the most frequent human autoimmune disease, affecting about 1% of the adult population worldwide. A better knowledge of the autoimmune mechanisms involved is essential. We identified the epithelial targets of various autoantibodies specifically associated to RA, as variants of (pro)filaggrin. We also showed that these targets correspond to deiminated ("citrullinated") proteins, of which arginyl residues have been posttranslationally transformed into citrullyl residues by a peptidylarginine deiminase (PAD). Moreover, we and others established that citrullyl residues are indispensable elements of the epitopes recognized by these autoantibodies but only in the context of specific aminoacid sequences. We also demonstrated that these autoantibodies to citrullinated proteins (ACPA) are secreted by plasma cells of the synovial tissue and that their major targets correspond to citrullinated forms of the alpha- and beta-chains of fibrin, abundant in the tissue. These results have allowed the development of new efficient immunochemical methods for the detection of ACPA. Some of them are already commercially available. These new methods have permitted the high diagnostic value of ACPA which are present very early in the course of the disease, and also their prognostic value, to be confirmed. ACPA detection should therefore prove to be also a very valuable tool to guide the choice of therapeutic strategies, from the earliest stages of the disease. The synthesis of ACPA in the rheumatoid synovial tissue and the existence therein of a specific antigenic target constitute a strong argument for the involvement of this specific immunological conflict in the pathophysiology of RA. Indeed, it could lead to activation of effector mechanisms with pro-inflammatory effects, thus to formation in the tissue of new fibrin deposits, secondarily citrullinated. We therefore, propose a new pathophysiological model accounting for the self-maintenance and chronicity of rheumatoid inflammation. Numerous questions about the pathophysiological significance of the autoimmune response to deiminated proteins in RA remain to be answered to confirm this model.