Anti-filaggrin Antibodies Research Articles

Proteolytic Processing of the Laminin α3 G Domain Mediates Assembly of Hemidesmosomes but Has No Role on Keratinocyte Migration

Laminin-5 (Lm5), the major adhesion ligand of basal epithelial cells, undergoes complex extracellular proteolytic processing that influences cell adhesion and migration. In tumor cell lines, the proteolytic truncation of the C-terminal G domain of the Lm alpha3 chain induces assembly of hemidesmosomes and downregulates cell migration. To define the biological functions of the alpha3 G domain processing in physiological conditions, we have expressed a series of mutant alpha3 complementary DNA in human primary alpha3-null keratinocytes immortalized by human papillomavirus E6E7 (HKalpha3 cells). Using monolayer and organotypic cell cultures we show that: (1) the hinge region between subdomains G3 and G4 carries the proteolytic cleavage sites; (2) nucleation of the hemidesmosomal proteins is independent of the proteolytic maturation of the alpha3 G domain, whereas formation of mature hemidesmosomes relies on proteolytic cleavage of alpha3; and (3) the proteolytic processing plays no role in cell migration, which suggests that nucleation of hemidesmosomal structures in culture does not reflect the migratory potential of the epithelial cells. Our results also demonstrate that HKalpha3 cells are a unique model system, which will be useful to dissect the functions and molecular interactions of Lm5.

Influence of HLA–DR genes on the production of rheumatoid arthritis–specific autoantibodies to citrullinated fibrinogen

Antibodies directed against citrullinated fibrinogen are highly specific for rheumatoid arthritis (RA). This study was undertaken to test whether RA-associated HLA-DR alleles are associated with anti-citrullinated fibrinogen in RA patient sera and whether replacement of arginyl by citrullyl residues on fibrinogen peptides modifies their binding to HLA-DR molecules and their recognition by T cells. Antikeratin, antifilaggrin, and anti-citrullinated fibrinogen antibodies were assayed in RA patients who had undergone HLA-DR typing. Direct assays were performed to investigate binding of citrullinated or native fibrinogen peptides (encompassing the entire alpha- and beta-chains of fibrinogen) to purified HLA-DR molecules. T cell proliferative responses to citrullinated or native fibrinogen peptides were measured in RA patients and controls. HLA-DRB1*0404 was associated with anti-citrullinated fibrinogen in RA sera (P = 0.002). For the RA-associated alleles HLA-DRB1*0401 and HLA-DR1, there was a nonsignificant trend toward association (P = 0.07). Multiple peptides from the alpha- and beta-chains of fibrinogen bound many HLA-DR alleles; DRB1*0404 was the best fibrinogen peptide binder. Citrullination did not influence fibrinogen peptide binding to HLA-DR or fibrinogen peptide recognition by T cells. Peripheral blood T cells that recognized native or citrullinated fibrinogen peptides were common in RA patients but not in healthy controls. The RA-associated HLA-DRB1*0404 allele is also associated with production of antibodies to citrullinated fibrinogen. DRB1*0401 and DRB1*01 tend to be associated with anti-citrullinated fibrinogen, but this is not statistically significant. Citrullination of fibrinogen peptide does not influence peptide-DR-T cell interaction. Finally, T cell proliferation in response to citrullinated or uncitrullinated fibrinogen peptides is frequent in RA patients and very infrequent in controls.

Survey of Literature

Cytokeratin (CK), filaggrin (filament aggregating protein), and p63 expression and cellular distribution during reepithelialization has not been systemically studied in the healing stage of human cutaneous wounds. We examined these proteins by immunohistochemical methods in 12 cases of skin ulcer, using seven anti-keratin antibodies, anti-filaggrin, and anti-p63 antibody. At the edge of the wound in skin ulcers, CK1 and 10 expression was reduced, while CK14, 16, and 17 expression was raised. Beneath the wound bed, all layers of the epidermal tongue, deriving from sweat ducts, were positive for CK14 and 17. Both cytokeratins were also found in basal and luminal cells of the dermal duct. CK expression by epithelia continuous with hair follicles showed that, CK14, 16, and 17 were present, and CK1 and 10 were absent. Filaggrin expression was elevated in reepithelialized epithelium. Expression of p63 expression was verified in the suprabasal layer in reepithelialized epithelia. CK, filaggrin, and p63 expression in the reepithelialization stage at the wound edge and at epidermal appendages remaining in the wound bed is undifferentiated and hyperproliferative. The presence of CK14 and 17 in the remaining epidermal appendages in the pathological wound may be important in epidermal replacement.

Association of Anti‐Cyclic Citrullinated Peptide Antibodies, Anti‐Citrullin Antibodies, and IgM and IgA Rheumatoid Factors with Serological Parameters of Disease Activity in Rheumatoid Arthritis

We evaluated the association of anti-cyclic citrullinated peptide (CCP) antibody titers with serological markers of disease activity. We also compared three different anti-CCP antibody ELISAs with an anti-citrullin ELISA and the IgM and the IgA rheumatoid factor (RF) in their performance of discriminating between rheumatoid arthritis (RA) and other rheumatic diseases. Sera from 333 consecutive patients of the Rheumaeinheit der Medizinischen Poliklinik Munchen, an outpatient clinic for rheumatic diseases, were collected and tested. Anti-CCP antibodies were assayed with three different commercially available ELISAs. Antifilaggrin antibodies were tested with a commercially available ELISA using in vitro deiminated recombinant rat filaggrin. IgA-RF was analyzed with an ELISA, whereas IgM-RF was measured by latex-enhanced turbidimetry. Rheumatoid arthritis (RA) was diagnosed in 87 patients according to the revised classification criteria of the American College of Rheumatology (ACR), probable RA was diagnosed in 23 patients in an early phase not (yet) fulfilling the ACR criteria, and 223 patients had other rheumatic diseases. Differences in sensitivity and specificity were calculated using McNemar's test. A measure of agreement (kappa statistic) was used to examine whether the tests tended to identify the same patients as positive or negative. Correlations between CCP titers and other tests were analyzed by Spearman nonparametric rank correlation. No significant differences in sensitivity and specificity were found between the tested CCP assays (80.0-80.9% and 97.3-98.1%, respectively). All three CCP tests were slightly but not significantly more sensitive and specific than the anti-citrullin assay (77% and 92%, respectively), comparably sensitive but significantly more specific compared with the IgM-RF (86% and 82%, respectively), and significantly more sensitive but comparably specific compared with the IgA-RF (63% and 94.4%, respectively) in detecting the patients with RA. There was no significant correlation between anti-CCP, anti-citrullin, or IgM-RF or IgA-RF antibody titers and C-reactive protein, erythrocyte sedimentation rate, or white blood cell count. A weak but significant linear correlation was found between anti-CCP titers and IgM-RF titers (r = 0.2, P = 0.03). We could not find a significant difference between the three tested anti-CCP assays and the anti-citrullin test in terms of sensitivity and specificity. Compared with the IgM-RF, all the anti-CCP assays were superior in specificity and comparable in sensitivity. Compared with the IgA-RF, they were more sensitive and comparably specific in the discrimination of patients with RA from other rheumatic diseases. No correlation of any tested autoantibody titer with serological parameters of inflammation was found.

Diagnostic Performances of Anti-Cyclic Citrullinated Peptides Antibody and Antifilaggrin Antibody in Korean Patients with Rheumatoid Arthritis

Rheumatoid arthritis (RA) is a systemic autoimmune disease of unknown etiology. We studied the diagnostic performances of anti-cyclic citrullinated peptides antibody (anti-CCP) assay and recombinant anti-citrullinated filaggrin antibody (AFA) assay by enzyme linked immunosorbent assay (ELISA) in patients with RA in Korea. Diagnostic performances of the anti-CCP assay and AFA assay were compared with that of rheumatoid factor (RF) latex fixation test. RF, anti-CCP, and AFA assays were performed in 324 RA patients, 251 control patients, and 286 healthy subjects. The optimal cut off values of each assay were determined at the maximal point of area under the curve by receiver-operator characteristics (ROC) curve. Sensitivity (72.8%) and specificity (92.0%) of anti-CCP were better than those of AFA (70.3%, 70.5%), respectively. The diagnostic performance of RF showed a sensitivity of 80.6% and a specificity of 78.5%. Anti-CCP and AFA showed positivity in 23.8% and 17.3% of seronegative RA patients, respectively. In conclusion, we consider that anti-CCP could be very useful serological assay for the diagnosis of RA, because anti-CCP revealed higher diagnostic specificity than RF and AFA at the optimal cut off values and could be performed by easy, convenient ELISA method.

Differentiation of the epidermis in turtle: an immunocytochemical, autoradiographic and electrophoretic analysis

Proteins involved in the process of cornification of turtle epidermis are not well known. The present immunocytochemical, electrophoretic and autoradiographic study reports on the localization patterns and molecular weights of keratins, which are cornification proteins, and of tritiated histidine in turtle epidermis. Alpha-keratins with a molecular weight of 40-62 kDa are present in the epidermis. Beta-keratin is mainly detectable in the stratum corneum of the carapace and plastron, but is rarely present or even absent in the corneous layer of limb, tail and neck epidermis. After electrophoresis and immunoblotting with an antibody against chicken scale beta-keratin, bands at 15-17, 22-24, and 36-38 kDa appeared. This antibody recognized weaker bands at 38-40 and 58-60 kDa in the soft epidermis. After reduction and carboxymethylation of proteins extracted from carapace and plastron, but not of proteins from the soft epidermis, protein bands at 15-17 and 35-37 kDa were found when using the anti-beta 1-keratin antibody. Loricrin-, filaggrin-, sciellin-, and transglutaminase-like immunostaining was detectable only in the transitional and lowermost corneous layers of the soft epidermis. Vesicular bodies in the transitional layer were immunolabeled by the anti-loricrin antibody, and weakly by the anti-filaggrin and anti-transglutaminase antibodies. In immunoblots, the anti-loricrin antibody reacted with a major band at 50-54 kDa in both carapace-plastron and soft epidermis. The anti-sciellin antibody detected major bands at 38-40 and 50 kDa in hard epidermis, and at 50 and 54-56 kDa in soft epidermis. Filaggrin-like immunostained bands were observed at 50-55 and 62-64 kDa. This immunostaining was probably due to a common epitope in filaggrin and some keratins. Histidine was evenly incorporated in the epidermis, and the ultrastructural study showed random labeling, often associated with keratin bundles of alpha and beta-keratinocytes. Histidine-labeled protein bands were not found in the carapace-plastron. In the soft epidermis, weakly labeled bands at 15-20, 25, and 45-60 kDa were found occasionally. The latter bands probably represented neo-synthesized keratins as was also indicated by the ultrastructural autoradiographic analysis. In conclusion, our study suggests that proteins with epitopes that they have in common with cornification proteins of mammalian epidermis are also present in the epidermis of turtle.

Antifilaggrin antibodies in serum and synovial fluid samples of patients with rheumatoid arthritis show similar reactivity pattern towards citrulline containing peptides

Antifilaggrin antibodies in serum and synovial fluid samples of patients with rheumatoid arthritis show similar reactivity pattern towards citrulline containing peptides

High diagnostic performance of ELISA detection of antibodies to citrullinated antigens in rheumatoid arthritis

Objective: We investigated the rheumatoid arthritis (RA) diagnostic performances of anti‐cyclic citrullinated peptide antibody (anti‐CCP) and antifilaggrin antibody (AFA) in comparison with RF and matrix metalloproteinase‐3 (MMP‐3).Methods: We used a second generation enzyme‐linked immunosorbent assay (ELISA) kit for the detection of anti‐CCP. We constructed recombinant human filaggrin, which was citrullinated in vitro by human peptidylarginine deiminase, and subsequently used it as the coating antigen for AFA‐ELISA. A total of 549 RA patients and 208 other rheumatic disease patients were included in the study.Results: The specificities of anti‐CCP (88.9%) and AFA (94.7%) were superior to those of RF (81.7%) and MMP‐3 (49.5%). The sensitivity of anti‐CCP (87.6%) was superior to all others. However, the sensitivity of AFA (68.7%) was inferior to those of RF (69.8%) and MMP‐3 (75.7%). Furthermore, receiver operating characteristic curves of anti‐CCP and AFA passed closer to the upper left corner than those of RF and MMP‐3, and the areas under the curves (AUC) of AFA and anti‐CCP were significantly larger. In addition, the AUC of anti‐CCP was significantly larger than that of AFA.Conclusion: ELISA detection of antibodies to citrullinated antigens, especially a second generation anti‐CCP, showed higher discriminative ability than other assays, including RF, and would be useful to aid the diagnosis of RA in clinical practice.

Clinical epidemiology: diagnostic and prognostic tests.

Recent studies of diagnostic and prognostic tests have commonly examined serological tests and new imaging techniques. Antifilaggrin antibodies have been found to be highly specific for the diagnosis of rheumatoid arthritis (RA), but uncertainty remains about the sensitivity of this test, particularly in early RA. Magnetic resonance imaging and ultrasound continue to be explored as methods to detect synovitis and erosions in RA. Several recent studies have confirmed the association between the human leukocyte antigen DRB1 shared epitope and worse radiographic outcomes in patients with RA. Interlaboratory variation in detecting autoantibodies remains a concern, as does overuse of tests for antineutrophil cytoplasmic autoantibodies.

Putative histidin-rich proteins in the epidermis of lizards.

In the stratum granulosum of mammalian epidermis, histidin-rich proteins (filaggrins) determine keratin clumping and matrix formation into terminal keratinocytes of the stratum corneum. The nature of matrix, interkeratin proteins in the epidermis of nonmammalian vertebrates, and in particular in that of reptilian, mammalian progenitors are unknown. The present biochemical study is the first to address this problem. During a specific period of the renewal phase of the epidermis of lizards and during epidermal regeneration, keratohyalin-like granules are formed, at which time they take up tritiated histidine. The latter also accumulate in cells of the alpha-keratin layer (soft keratin). This pattern of histidine incorporation resembles that seen in keratohyalin granules of the stratum granulosum of mammalian epidermis. After injection of tritiated histidine, we have analysed the distribution of the radioactivity by histoautoradiography and electrophoretic gel autoradiography of epidermal proteins. Extraction and electrophoretic separation of interfilamentous matrix proteins from regenerating epidermis 3-48 hours post-injection reveals the appearance of protein bands at 65-70, 55-58, 40-43, 30-33, 25-27, and 20-22 kDa. Much weaker bands were seen at 100, 140-160, and 200 kDa. A weak band at 20-22 kDa or no bands at all are seen in the normal epidermis in resting phase and in the dermis. In regenerating epidermis at 22 and 48 hours post-injection, little variation in bands is detectable, but low molecular weight bands tend to increase slightly, suggesting metabolic turnover. Using anti-filaggrin antibodies against rat, human, or mouse filaggrins, some cross-reactivity was seen with more reactive bands at 40-42 and 33 kDa, but it was reduced or absent at 140, 95-100, 65-70, 50-55, and 25 kDa. This suggests that different intermediate degradative proteins of lizard epidermis may share some epitopes with mammalian filaggrins and are different from keratins with molecular weight ranging from 40 to 65-68 kDa. The immunocytochemical observation confirms that a weak filaggrin-like immunoreactivity characterizes differentiating alpha-keratogenic layers in normal and regenerating tail. A weak filaggrin labeling is discernable in small keratohyalin-like granules but is absent from the larger granules and from mature keratinocytes. The present results indicate, for the first time, that histidine-rich proteins are involved in the process of alpha-keratinization in reptilian epidermis. The cationic, interkeratin matrix proteins implicated may be fundamentally similar in both theropsid-derived and sauropsid amniotes.

Investigation of the reactivity patterns of antifilaggrin antibodies in sera and synovial fluids from patients with rheumatoid arthritis using citrullinated synthetic peptides

Antifilaggrin antibodies comprise a heterogeneous population of antibodies directed to citrullinated proteins. Recent studies have shown that their production is highly specific for rheumatoid arthritis (RA) and the initial antigenic trigger for these autoantibodies can be localised to the inflammed synovial tissue. The aim of our study was to compare the reactivity and specificity of antibodies in sera and synovial fluids towards citrullinated epitopes. Peptide sequence corresponding to human profilaggrin (amino acid residues 306–324) and sequences with citrulline substitution at different positions were synthetised by mutipin peptide synthesis on solid supports. Shortened versions of the peptide were also produced by removal of amino acid residues from its N and C terminals. Completely citrullinated variant of the 14-mer peptide was also prepared. Peptide with no citrulline replacement was used as a control antigen. We found significant differences in the sensitivity for RA of 19 individual peptides tested (from 5% to 68%), reflecting previous results that the surrounding amino acids play an important role in creation of an autoantigenic epitope. Further, we tested the reactivities of paired serum and synovial fluids and found very similar peptide recognition patterns in serum and synovial IgG from the same individuals. Studies on larger number of samples are in progress to evaluate the results statistically that may support further evidence of the synovial origin of antifilaggrin autoantibodies.

Novel autoantibodies for the diagnosis of rheumatoid arthritis

The diagnosis of rheumatoid arthritis has been hampered by the lack of a truly disease-specific serologic marker. Thus, despite its moderate specificity rheumatoid factor (RF) is still the only established marker antibody for RA and among the diagnostic criteria of the American College of Rheumatology the only serologic one. However, in recent years, several newly characterized autoantibodies have been described that may have the potential to become diagnostic indicators for RA. In particular, antibodies to citrullinated targets (anti-keratin or anti-filaggrin antibodies, respectively, antibodies to synthetic citrullinated peptides) appear to be highly specific for RA. Other potentially useful antibodies include anti-RA33 autoantibodies and antibodies to the stress protein BiP which seem to have higher specificity for RA than RF. Apart from being promising diagnostic markers these autoantibodies or the underlying cellular autoimmune reactions, respectively, may also play a role in the pathogenesis of RA.

Presence of autoantibodies to the glycolytic enzyme α‐enolase in sera from patients with early rheumatoid arthritis

To identify a new autoantigen/autoantibody population in rheumatoid arthritis (RA) sera. Following a population-based recruitment effort, 255 patients with very early arthritis (median disease duration 4 months) were studied using different clinical, biologic, and radiologic assessments. After a followup period of 1 year, patients were classified as having RA (n = 145), non-RA rheumatic diseases (n = 70), and undifferentiated arthritis (n = 40). Patients' sera were analyzed by one-dimensional (1D) and 2D Western blotting. The recognized 50-kd protein was analyzed by matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry (MS). RA serum reactivities were evaluated against the recombinant protein synthesized by an in vitro coupled transcription-translation system. On 1D Western blots, 36 of the 145 RA sera bound to a 50-kd polypeptide. On 2D Western blots, anti-50-kd+ RA sera recognized a triplet of isoelectric point 6.5-7.0 and a molecular mass of 50 kd. The 3 spots of the triplet were analyzed by MALDI-TOF MS and were shown to correspond to human alpha-enolase. A goat anti-enolase antiserum, which recognized a band comigrating with the 50-kd antigen on 1D Western blots, gave a labeling pattern on 2D Western blots similar to that observed with anti-50-kd+ RA sera. Among the 36 RA sera that identified alpha-enolase in protein maps, only 8 recognized the recombinant (unmodified) alpha-enolase. The specificity of anti-alpha -enolase antibodies for RA was 97.1%. Half of the anti-alpha -enolase-positive RA patients were negative for both rheumatoid factor and antifilaggrin antibodies. The presence of anti-alpha-enolase antibodies was the greatest predictive factor of radiologic progression in the first 66 RA patients included. Autoantibodies to alpha-enolase, an enzyme of the glycolytic pathway, are present in the sera of patients with very early RA and have potential diagnostic and prognostic value for RA.

Specific presence of intracellular citrullinated proteins in rheumatoid arthritis synovium: relevance to antifilaggrin autoantibodies.

To investigate the presence of citrullinated proteins in the synovial membrane of patients with rheumatoid arthritis (RA) and controls, and to analyze a possible relationship with antifilaggrin autoantibody (AFA) reactivity. Synovial biopsy samples were obtained from 88 consecutive patients undergoing needle arthroscopy for knee synovitis associated with RA (n = 36), spondylarthropathy (n = 35), osteoarthritis (n = 9), or other diagnoses (n = 8). Tissue sections were stained with 2 different anticitrulline polyclonal antibodies and an antifilaggrin monoclonal antibody (mAb). The phenotype of citrulline-positive cells and the colocalization with affinity-purified AFA were investigated by double immunofluorescence on frozen sections. Studies with the first antibody showed that citrulline is expressed intracellularly in the lining and sublining layers of RA synovial tissue. Staining with the second antibody, monospecific for proteins containing modified citrulline, and with anti-inducible nitric oxide synthetase confirmed the presence of citrullinated proteins rather than free citrulline in the synovium. Citrulline-positive cells were detected in 50% of the RA patients (18 of 36) but in none of the controls (0 of 52). The anticitrulline reactivity colocalized with affinity-purified AFA reactivity, although stainings with the antifilaggrin mAb indicated the absence of filaggrin in the synovium. Intracellular citrullinated proteins, which are not recognized by an antifilaggrin mAb, are expressed in RA but not in control synovium. The high specificity of this finding and the colocalization with AFA reactivity boost the interest in citrullinated proteins as possible triggers of autoimmune responses in RA. Moreover, this is the first description of a specific histologic marker for RA synovium.

Rheumatoid factors, anti-filaggrin antibodies and low in vitro interleukin-2 and interferon-γ production are useful immunological markers for early diagnosis of community cases of rheumatoid arthritis. A preliminary study

Objective. To determine whether measurements of different autoantibodies (Ab) and cytokines are useful to distinguish very early rheumatoid arthritis (RA) from other inflammatory rheumatisms. Methods. From a population-based recruitment, 32 patients with very early polyarthritis (median duration: 4 months) were studied. Evaluations at entry (M0), and at 6 (M6) and 12 months (M12). Ab tested: rheumatoid factors (RF) by agglutination methods and ELISA, antiperinuclear factor (APF), antikeratin Ab (AKA), anti-Sa and antinuclear Ab. Cytokine production (TNFα, IL2, IFNγ, IL1β, IL10) in whole blood cell culture (WBCC) was determined at M0. At M12, patients were classified as having RA ( N = 15) or other rheumatic diseases. Results. At M0, AKA/APF and anti-Sa Ab frequencies were low, 13% and 7%, respectively. While most Ab detected at MO persisted, others appeared during follow-up, particularly APF, which rose from 13 to 40% at M12. At M6, IgM-RF was detected in two RA patients exclusively by ELISA. AKA/APF were found to be highly specific markers for RA (100% specificity). At some time during follow-up, two RF-negative RA patients were AKA-positive. In two patients, AKA and APF were present at M0 before they satisfied ACR criteria. IL2 and IFNγ production was significantly lower ( P < 0.05) for RA patients. Conclusion . AKA/APF and anti-Sa Ab were detected in community cases of very early RA. AKA/APF and RF detected by ELISA might contribute to an earlier diagnosis of RA. Low production of IFNγ and IL2 in WBCC constituted a distinct immunopathological feature in very early RA patients.

Investigation of the epitopes of human profilaggrin derived peptide targeted by antibodies of patient with rheumatoid arthritis

Anti-filaggrin antibodies (AFA), directed against the 37-40 kD epidermal protein filaggrin are one of the most specific markers of rheumatoid arthritis (RA) and include anti-keratin antibodies (AKA) and anti-perinuclear factor (APF). Although the antigen triggering autoimmune response to filaggrin related proteins has not been identified, recent studies confirmed that citrulline is essential constituent of protein related antigenic determinats recognised by RA specific autoantibody population. The aim of our study was to identify epitopes of filaggrin derived-peptides targeted by RA specific antibodies to provide further information about the nature of the initial autoantigenic substance. Citrullin containing peptides of human profilaggrin region (amio acid residues 306-324) derived from known cDNA and on the basis of the data published by Shellekens were synthetised by the multipin peptide synthesis on solid support and were reacted in situ by patient sera. Two 19-mer peptides were prepared with single cit-rullin substitution at position 312 or 321 respectively and four additional ones with simultanious replacements of two Arg by Cit. Shortened versions of the 306SHQESTCitGRSGRSGRSGR324 peptidewere also produced by removal of 1-6 amino acid residues from its N-terminal and the 14 -mer truncated one was further shortened from its C-terminal as well. The reactivities of these peptides with RA sera and healthy controls were determined. The results showed that substitution of arginine 312 by citrulline plays major role in the anigenicity of filaggrin-derived sequences. Peptides not containing Cit in position 312 almost lost their ability to bind antibodies from RA sera. Replacement of one additional Arg by Cit in different positions did not improved the antigenicity. When the peptide with Cit in position 312 were shortened from its N-terminal, the 14-mer one showed the highest reactivity. Further shortening of this sequence from its C-terminal showed that TXGRS motif seems to be essential to comprise the autoanigenic epitope. In conclusion our results provide further evidence that not simply the presence of citrullin but also the nature of its surronding amino acids have important role in the creation of autoantigenic epitope reactive with anti-filaggrin antibodies.

Detection of rheumatoid arthritis-specific anti-filaggrin antibodies by line immunoassay shows complementarity to RF and corresponds to the AFA-blot using natural antigen

Anti-filaggrin autoantibodies (AFA) are highly specific markers for rheumatoid arthritis (RA) and can be detected by immunoblotting using human epidermal protein extracts. Furthermore, it was demonstrated that citrullination of the filaggrin epitopes is crucial for epitope recognition and that citrullinated peptides are also recognized by these specific autoantibodies. Based on these data, a line immunoassay (LIA) was developed containing as individual markers in vitro citrullinated recombinant filaggrin and two citrullinated synthetic peptides. Firstly, a comparison was made between this prototype LIA and the AFA blot using natural filaggrin. A blind serum panel consisting of 25 early RA, 25 longstanding RA, and 25 disease controls was selected. Results showed a similar performance of both tests at a specificity level of 95%, while the LIA proved significantly better (P = 0.035) than the AFA blot at 99% specificity. At the latter specificity level, 2 out of 17 RF negative samples were retrieved on LIA but not on Western blot. The LIA was further evaluated on sera obtained from 335 RA patients and 254 patients with non-RA rheumatological disorders in a retrospective study. The overall sensitivity of the LIA including three markers (LIA3) was 65.1% versus 61.8% if only the reactivity towards the citrullinated peptides was considered (LIA2). The specificity of LIA3 was 97.6% versus 98.4% for LIA2, which correlates with an estimated positive predictive value (PPV) of 87.3% for LIA3 and 90.7% for LIA2. Thirty-seven percent (30/81) of the RF-negative RA samples proved LIA2-positive, while 52 out of 55 RF positive control samples were negative for anti-filaggrin. Higher specificity and sensitivity was obtained for LIA2 in comparison with anti-RA33 immunoblot, whereas good agreement could be observed with anti-keratin antibody (AKA) testing. In conclusion, anti-filaggrin autoantibodies can be readily detected by a LIA test based on citrullinated peptides, resulting in a high specificity and hence high PPV for RA. The assay can serve as a user-friendly alternative for AKA immunofluorescent and immunoblot techniques. Together with the RF complementarity, this test provides a valuable tool in the differential diagnosis of RA.

Clinical sensitivity of antibodies against cyclic citrulinated peptide in patients with rheumatoid arthritis

The synthetic cyclic citrulinated peptide (CCP) is recognised by rheumatoid arthritis (RA) associated antifilaggrin antibodies, previously determined as antikeratin antibodies or perinuclear factor. Antibodies detected by ELISA using CCP as an antigen (anti-CCP) seem to be of prognostic value in patients with RA. The objective of our study was to determine the clinical sensitivity of anti-CCP in patients with definite RA (according to ARA diagnostic criteria). RF results were considered when measured in the same serum as anti-CCP. Sera from 97 RA patients (15 M, 82 F) were tested for anti-CCP in duplicates by Imunoscan RA ELISA (Euro-diagnostica). RF was tested in the same serum in 69/97 patients. In all 4 assay runs, OD of all calibrators vs. their calculated values completely corresponded to the figure in the manufacturer's analysis certificate. When the units of the lowest calibrator D were calculated by the equation of log calibration curve according to the manufacturer's protocol, they were always about 20% (7-17U) above the defined value (50U). This inconsistency of the curve fitting led to a wrong validation in 26/97 (27%) of the RA samples whith OD below the OD of the calibrator D, but with calculated results above the defined cut-off at 50U. Therefore, we calculated results by the equtation of 3rd degree polynomal curve which fitted perfectly the measured OD values to the defined units. Out of 97 RA patients, 56 were anti-CCP positive (58%). From 69 patients simultaneously tested for RF and anti-CCP, both tests were positive in 24/69 (35%) and both negative in 23/69 (33%). Anti-CCP were positive in 15/38 (40%) patients with negative RF. Despite lower anti-CCP positivity rate, 16% of samples in RF neg. group exhibited high anti-CCP values: The clinical sensitivity of the anti-CCP test in our RA patients was 58%, which is lower than previously reported (68%). The discrepancy might partially be due to different calculation of the results. We believe that the introduction of polynomal standard curve could contribute to a more consistent validation of samples exhibiting OD bellow the lowest calibrator. Our results support the idea that anti-CCP are of diagnostic value especially in RF neg patients. Table 1

Specific presence of intracellular citrullinated proteins in rheumatoid arthritis synovium: Relevance to antifilaggrin autoantibodies

To investigate the presence of citrullinated proteins in the synovial membrane of patients with rheumatoid arthritis (RA) and controls, and to analyze a possible relationship with antifilaggrin autoantibody (AFA) reactivity.Synovial biopsy samples were obtained from 88 consecutive patients undergoing needle arthroscopy for knee synovitis associated with RA (n = 36), spondylarthropathy (n = 35), osteoarthritis (n = 9), or other diagnoses (n = 8). Tissue sections were stained with 2 different anticitrulline polyclonal antibodies and an antifilaggrin monoclonal antibody (mAb). The phenotype of citrulline-positive cells and the colocalization with affinity-purified AFA were investigated by double immunofluorescence on frozen sections.Studies with the first antibody showed that citrulline is expressed intracellularly in the lining and sublining layers of RA synovial tissue. Staining with the second antibody, monospecific for proteins containing modified citrulline, and with anti-inducible nitric oxide synthetase confirmed the presence of citrullinated proteins rather than free citrulline in the synovium. Citrulline-positive cells were detected in 50% of the RA patients (18 of 36) but in none of the controls (0 of 52). The anticitrulline reactivity colocalized with affinity-purified AFA reactivity, although stainings with the antifilaggrin mAb indicated the absence of filaggrin in the synovium.Intracellular citrullinated proteins, which are not recognized by an antifilaggrin mAb, are expressed in RA but not in control synovium. The high specificity of this finding and the colocalization with AFA reactivity boost the interest in citrullinated proteins as possible triggers of autoimmune responses in RA. Moreover, this is the first description of a specific histologic marker for RA synovium.

Association of rheumatoid factors and anti-filaggrin antibodies with severity of erosions in rheumatoid arthritis.

To evaluate and to compare the association of two types of autoantibodies-rheumatoid factors (RF) and anti-filaggrin antibodies (AFA)-with clinical severity and joint damage progression in rheumatoid arthritis (RA) patients. In a cross-sectional study, we determined RF and AFA titres in 199 RA patients and 65 controls. Erosions apparent on X-rays were quantified using the Larsen score in 143 patients, and the distribution of these scores was studied according to disease duration in patients who were positive and negative for RF and AFA. RF were detected in 72% and AFA in 47% of RA patients. AFA were highly specific for RA (100%). RF positivity was correlated with the presence of subcutaneous nodules, sicca syndrome and the severity of erosions for a given disease duration. AFA positivity was correlated only with the presence of the HLA-DRB1 shared epitope. Since no significant correlation was observed between joint damage progression and AFA positivity, the determination of AFA does not appear to be useful in assessing the prognosis of RA. However, AFA, which appear early in RA, could be helpful for the diagnosis of RA in patients who do not fulfil four American College of Rheumatology criteria.