Mune response ( ) against each individual citrullinated peptide in CCP2-positive sera tested. b. Relative frequency ( ) of patient sera reacting to one, two, three, or four citrullinated fibrinogen peptidesFernandes-Cerqueira et al. Arthritis Research Therapy (2015) 17:Page 5 ofWhen incubated with ACPA pool I, soluble linear Cit573 resulted in the highest degree of ACPA blocking, achieving 84 inhibition of reactivity, with an IC50 of 59 M ?8 (n = 6; Fig. 2a, left panel). Fibrinogen linear peptide Cit591 resulted in a similar dose-response curve as Cit573.
Staurosporine A maximum of 63 ACPA blocking was recorded for Cit591, and the IC50 calculated was 194 M ?3 (n = 2; Fig. 2a, right panel). When the two peptides Cit573 and Cit591 were equally mixed and tested in the competition assay, a significantly higher degree of ACPA blocking was achieved, reaching 91 inhibition (data not shown). Using ACPA pool II, Cit573 resulted in a slightly weaker ACPA inhibition in comparison to that observed with ACPA pool I (Fig. 2b, left panel). Approximately 50 of ACPA in the anti-CCP2 plate were blocked (IC50 548 M ?100, n = 4). On the other hand, ACPA pool II targeted by Cit591 showed a similar result to that observed with ACPA pool I (Fig. 2b, right panel; IC50 412 M ?146, n = 4). When equally combined, Cit573 and Cit591 blocked up to 75 of ACPA from pool II (data not shown).No major inhibition was observed for any of the two ACPA pools, when using the unmodified peptide version Arg573 in the same experimental conditions as the citrullinated fibrinogen peptides (Fig. 2a and b, left panels). On the other hand, Arg591 displayed 30 inhibition when incubated with ACPA pool I (Fig. 2a, right panel), but no inhibition was detected when incubated with ACPA pool II (Fig. 2b, right panel). In comparison, a maximum ACPA inhibition of 35 and 26 was observed for the citrullinated fibrinogen chain peptides Cit72 and Cit74, respectively, while no inhibition was registered with the corresponding unmodified peptides (data not shown). Retrieval experiments were performed using the Cit573 peptide in order to evaluate the capability of this peptide to compete with CCP2 for already bound ACPA. Addition of Cit573 reverted up to 80 of ACPA binding to the CCP2 ELISA plate (data not shown). No relevant retrieval capacity was observed for the arginine control peptide. To determine whether cyclization of the peptides improves ACPA targeting, a cyclic version of the fulllength Cit573 peptide was synthesized (Cit573Cyc) andabFig. 2 Dose-response curves representing the percentage of ACPA targeted by fibrinogen chain peptides. ACPA was incubated with the respective peptides at different concentrations for one hour at room temperature, before proceeding with the anti-CCP2 ELISA. a. ACPA (pool I) blocking with citrulline or arginine-containing fibrinogen peptides from the chain, 573 (left panel) and 591 (right panel). b. ACPA (pool II) blocking with citrulline or arginine-containing fibrinogen peptides from the chain, 573 (left panel) and 591 (right panel). X-axes show peptide amount (nmol). Y-axes show ACPA inhibition levels ( ). Circles represent means of two to seven experiments per ACPA pool and
PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/17139194 error bars represent SEM. *P <0.05, **P <0.Fernandes-Cerqueira et al. Arthritis Research Therapy (2015) 17:Page 6 oftested in the ACPA competition assay (Fig. 3). The cyclic version of Cit573 reached 92 of ACPA inhibition (Fig. 3a), with an IC50 of 28 M ?5 (n = 3) when incub.