Ated with ACPA pool I. The full residue span within Cit573 may not be necessary for optimal ACPA binding, therefore two truncated linear peptides of 15 and 11 residues (Cit573Lin1 and Cit573Lin2), originating from the full-length Cit573, were tested and found to provide 75 (IC50 51 M ?6, n = 3) and 69 (IC50 123 M ?18, n = 2) ACPA inhibition, respectively (Fig. 3a). The linear truncated peptides were also incubated with ACPA pool II. Here, Cit573Lin1 accomplished 47 inhibition, with an IC50 of 186 M ?67 (n = 2), while Cit573Lin2 achieved 40 inhibition, with an IC50 of 105 M ?34 Staurosporine (n = 2) (Fig. 3b). Interestingly, the cyclic form of the Cit573 peptide performed similarly in ACPA pool I (approximately 90 ) as in ACPA pool II (Fig. 3b).Discussion In the present study we demonstrated extensive autoantibody reactivity against citrullinated fibrinogen epitopes, previously identified in vivo in RA synovial membranes [17]. We also showed that citrullinated peptides, corresponding to these in vivo modified epitopes, can be used as targeting agents, blocking a significant portion of ACPA binding to CCP2, forming the basis of a possible future therapeutic approach for ACPA-positive RA. In the current study we have analyzed the antibody response towards four citrullinated fibrinogen peptides in sera from 927 RA patients, and 47 of the patients were positive for antibodies against at least one of the peptides. The majority of antibody-positive patients were identified in the CCP2-positive subset, but interestingly, between 4 and 8 of CCP2-negative patients also displayed reactivityagainst the citrullinated fibrinogen peptides, supporting previous findings that the CCP2 detection kit leaves out patients who produce antibodies against citrullinated proteins [26, 27]. In addition to the results described here, the same citrullinated fibrinogen peptides were also tested in a pre-RA cohort [28]. In that study, it was shown that antibodies against Cit573 and Cit74 were detected at low frequencies before RA onset, but at significantly increased frequencies after disease onset, suggesting an involvement in RA progression. Antibodies against Cit591 and Cit72, on the other hand, were detected at the earliest time points, pointing to a role in disease onset. It has been suggested that ACPA mediate disease by promoting osteoclastogenesis [13], activation of the complement system [12], induction of TNF- secretion [10, 11], and enhancement of NETosis [14]. In mice, ACPA targeting fibrinogen has been shown to enhance development of arthritis [9]. Since we know that fibrinogen is citrullinated in vivo, and that PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/6833145 the specific epitopes investigated here are expressed in vivo [17], we hypothesize that ACPA of these specificities will be involved in RA pathogenesis. Hence, we suggest that these specific peptide sequences could be used in the development of novel drugs that will target pathogenic ACPA [29]. Additionally, and taking into account the possible association of certain citrullinated peptides (Cit591 and Cit72) with the disease onset, an alternative therapy would be to induce tolerance of Cit591- or Cit72-reactive CCP-positive RA patients with a Cit591- or Cit72-derived compound. The use of self-antigens (or closely-related structures) to induce tolerizing effects on dendritic cells has, for instance, been previously used in cancer. Farkas et al. demonstrated that mucin 1 transgenic mice developed transientaACPA pool I80 60 40 20 0 0 .0 1 C it5 7 3 L.