The other antibodies, ACarPA and RF, could be specifically associated with HLA-DRB1 alleles that are not included in the shared epitope, but that have not been yet completely defined6668. = 4.4 1016) and revealed that smoking was exclusively associated with Spinorphin the presence of RF in patients with one or two antibodies (RF+1+2vs. RF0+1+2: OR = 1.32, p = 0.0002). In contrast, no specific association with ACPA or ACarPA was found. Therefore, these results showed the need to understand how smoking favors the concordance of RA specific antibodies and RF triggering, perhaps involving smoking-induced epitope spreading and other hypothesized mechanisms. Subject terms:Rheumatoid arthritis, Risk factors == Introduction == Rheumatoid arthritis (RA) is a systemic autoimmune disease that can be divided in two pathogenic subgroups1,2. The largest subgroup comprises the patients Spinorphin presenting RA specific autoantibodies. These antibodies include the rheumatoid factor (RF), which is directed against the Fc of the IgG, and antibodies against some kalinin-140kDa post-translational protein modifications. The best characterized are anti-citrullinated protein antibodies (ACPA), which in the clinic are assayed as anti-cyclic citrullinated peptides or anti-CCP, and the anti-carbamylated protein antibodies (ACarPA), which are not yet analyzed beyond research studies. The antibody positive patients are known as seropositive and they represent more than two thirds of the total, while the remaining are the seronegative patients. The seropositive patients have a clear component of genetic susceptibility and a defined disease story and autoimmune pathogenesis, which are less clear in the seronegative patients15. Also, smoking, which is the major environmental risk factor for RA, is specific of seropositive RA6,7. In the ACPA positive patients, the risk is potentiated by interaction with the HLA-DRB1 shared epitope (SE)810. This interaction together with protein citrullination in the bronchoalveolar lavage (BAL) cells of smokers has been at the basis of an influential pathogenic model11. According to this model, smoking induces protein citrullination in the lung, which predisposes to the production of ACPA and, subsequently, of RA11. This model has been supported by other findings, including the demonstration of increased PAD2 expression in bronchoalveolar lavage (BAL) Spinorphin of smokers12,13. However, not all the findings have been consistent. In particular, no correlation between smoking and protein citrullination12,14,15or between smoking and PAD215have been observed in the lung tissue specimens. Moreover, the production of ACPA in the lung and the presence of lung abnormalities in early RA were directly associated with ACPA+RA without detectable association with smoking in the available studies1619. Also, van Wesemaelet al. reported that smoking was associated with the concurrent presence of RF, ACPA and ACarPA, rather than with ACPA in three patient cohorts20. The novelty and repercussion of these latter results ask for validation through independent replication. Here, we have replicated and extended the findings of van Wesemaelet al. Spinorphin in an independent set of six patient cohorts (n = 2253). Our results confirmed the predominant association of smoking with the concurrent presence of the three RA autoantibodies. Also, thanks to the large power afforded by the combined analysis (n = 4491), we discovered that smoking was exclusively associated with RF positivity in the patients without three concordant positive autoantibodies (RF+1+2). In contrast, we did not find significant associations of smoking with the specific presence of ACPA or ACarPA. These results indicate that smoking promotes pathways leading to the concurrent presence of the three RA autoantibodies and, in its defect, to the production of RF. == Material and Methods == == Patients and samples == Patients from five Spanish (IDIPAZ, PEARL, IDIS, IdISSC, and IDIVAL) and one Italian (Rome) RA cohorts were considered as replication sets (n = 2253 with complete data). The Spanish data were directly available to us, whereas the information corresponding to the Italian patients was extracted from a publication21. Two of the replication collections were early arthritis (EA) prospective.