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Objectives To evaluate the proportions of rheumatoid arthritis (RA) sera containing anticitrullinated proteins autoantibodies (ACPA) reactive to α36-50Cit38,42 and/or β60-74Cit60,72,74, two peptides identified as bearing the immunodominant epitopes of their major target, citrullinated fibrin. To analyse the relationships of anti-α36--50Cit38,42 and anti-β60-74Cit60,72,74 autoantibodies with autoantibodies reactive to the complete citrullinated human fibrinogen molecule (AhFibA) and with anti-CCP2 antibodies. Methods 617 sera from 181 patients with established RA and 436 with non-RA rheumatic diseases were tested by ELISA for AhFibA, anti-CCP2, anti-α36- 50Cit38,42, anti-β60-74Cit60,72,74 autoantibodies, and by nephelometry for rheumatoid factor (RF). Diagnostic indexes, correlations and concordances between tests were analysed. Crossreactivity of anti-α36-50Cit38,42 and anti-β60-74Cit60,72,74 autoantibodies was assessed in competition experiments. Results At a diagnostic specificity of 95%, the diagnostic sensitivity of AhFibA (83%) was significantly higher than that of all other tests. The diagnostic sensitivity of anti-β60-74Cit60,72,74 (71%) was significantly higher than that of anti-α36-50Cit38,42 autoantibodies (51%) but similar to that of anti-CCP2 (74%). Titres of RF, anti-α36-50Cit38,42 and anti-β60-74Cit60,72,74 autoantibodies were weakly correlated with each other, whereas titres of anti-β60-74Cit60,72,74 were strongly correlated with those of AhFibA (r=0.633) and anti-CCP2 (r=0.634). Anti-α36-50Cit38,42 and anti-β60-74Cit60,72,74 mainly corresponded to two noncrossreactive subfamilies of ACPA. More than 90% of AhFibA-positive or anti-CCP2-positive sera recognised the α36-50Cit38,42 and/or the β60-74Cit60,72,74 peptide. Conclusions Autoantibodies reactive to α36-50Cit38,42 and β60-74Cit60,72,74 form two distinct, non-overlapping subfamilies of ACPA that, together, cover practically all the ACPA reactivity to citrullinated fibrinogen and to CCP2 antigens. In established RA, anti-β60-74Cit60,72,74 autoantibodies show diagnostic indexes similar to those of anti-CCP2. [ABSTRACT FROM AUTHOR]

OBJECTIVE: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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. [ABSTRACT FROM AUTHOR]

Serum IgG, labelling the stratum corneum of the rat oesophagus epithelium, so-called anti-keratin antibodies (AKA) constitute the most specific marker for the diagnosis of rheumatoid arthritis. In this study, we investigated 31 IgG AKA-positive rheumatoid sera and 21 control sera from patients with non-rheumatoid inflammatory rheumatic discuses. The serum level of IgG 1,2,3 and 4 was determined by radial immunodiffusion and the subclass distribution of IgG AKA by a three-step semi-quantitative immunofluorescence assay using standard monoclonal antibodies specific for each of the four human IgG subclasses. In the rhetimatoid sera, the serum level of IgG 1 was found to be significantly increased and the level of IgG2 significantly decreased with regard to the control sera, while the levels of IgG3 and 4 as well as total IgG were in the normal range. IgG1,2,3 and 4 AKA were detected in 27(87%),6(19%),4(13%)and 11 (35%)of the 31 rheumatoid sera, respectively, and were found to be independent of the clinical and biological indices of the disease. In spite of interindividual heterogeneity. Two predominant profiles were distinguished: IgG1 (alone) and IgG(1 + 4,. which together represented 18 sera (58%). The large predominance of IgGl AKA and the quasiabsence of IgG2 AKA suggest that the recognized antigen may be partly comprised of protein. Moreover, the high frequency of occurrence of IgG4 AKA might result from chronic exposure to the eliciting antigen, which could be a genuine autoantigen since we demonstrated that it is also present in the stratum corneum of human epidermis. [ABSTRACT FROM AUTHOR]

Background and Purpose: Chymotrypsin is a pancreatic protease secreted into the lumen of the small intestine to digest food proteins. We hypothesized that chymotrypsin activity may be found close to epithelial cells and that chymotrypsin signals to them via protease-activated receptors (PARs). We deciphered molecular pharmacological mechanisms and gene expression regulation for chymotrypsin signalling in intestinal epithelial cells., Experimental Approach: The presence and activity of chymotrypsin were evaluated by Western blot and enzymatic activity tests in the luminal and mucosal compartments of murine and human gut samples. The ability of chymotrypsin to cleave the extracellular domain of PAR1 or PAR2 was assessed using cell lines expressing N-terminally tagged receptors. The cleavage site of chymotrypsin on PAR1 and PAR2 was determined by HPLC-MS analysis. The chymotrypsin signalling mechanism was investigated in CMT93 intestinal epithelial cells by calcium mobilization assays and Western blot analyses of (ERK1/2) phosphorylation. The transcriptional consequences of chymotrypsin signalling were analysed on colonic organoids., Key Results: We found that chymotrypsin was present and active in the vicinity of the colonic epithelium. Molecular pharmacological studies have shown that chymotrypsin cleaves both PAR1 and PAR2 receptors. Chymotrypsin activated calcium and ERK1/2 signalling pathways through PAR2, and this pathway promoted interleukin-10 (IL-10) up-regulation in colonic organoids. In contrast, chymotrypsin disarmed PAR1, preventing further activation by its canonical agonist, thrombin., Conclusion and Implications: Our results highlight the ability of chymotrypsin to signal to intestinal epithelial cells via PARs, which may have important physiological consequences in gut homeostasis., (© 2024 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

Proteolytic homeostasis is important at mucosal surfaces, but its actors and their precise role in physiology are poorly understood. Here we report that healthy human and mouse colon epithelia are a major source of active thrombin. We show that mucosal thrombin is directly regulated by the presence of commensal microbiota. Specific inhibition of luminal thrombin activity causes macroscopic and microscopic damage as well as transcriptomic alterations of genes involved in host-microbiota interactions. Further, luminal thrombin inhibition impairs the spatial segregation of microbiota biofilms, allowing bacteria to invade the mucus layer and to translocate across the epithelium. Thrombin cleaves the biofilm matrix of reconstituted mucosa-associated human microbiota. Our results indicate that thrombin constrains biofilms at the intestinal mucosa. Further work is needed to test whether thrombin plays similar roles in other mucosal surfaces, given that lung, bladder and skin epithelia also express thrombin.