Current literature suggests that increased levels of citrullinated proteins are found in several if not all inflammatory diseases. In RA measurement of anti-citrullinated protein antibodies (ACPA) against citrullinated protein fragments are widely used as a prognostic biomarker. More recently, it has been indicated that levels of selected citrullinated proteins carries additional potential as biomarkers. This includes citrullinated vimentin which provide prognostic information in diseases as fibrosis and ankylosing spondylitis. In addition, recent studies suggest that inhibition of PAD is a target for treatment of diseases such as RA and cancer where proteins that are citrullinated are believed to influence the disease activity.
BackgroundProtein citrullination, i.e. conversion of arginine residues into citrulline residues, is a post-translational modification catalyzed by PAD, and is an important pathophysiological determinant in conditions such as Rheumatoid arthritis (RA). Identification of citrullination sites on putative autoantigens is likely to enhance our understanding of PAD's substrate specificity.ObjectivesCitrullinated fibrinogen is an autoantigen linked to the pathophysiology of RA.We have applied a novel MS-based proteomics approach to estimate the degree of citrullination in synovial fluid (SF) from RA patients, and compare to fibrinogen citrullinated in vitro by PAD2/4, the most important PAD isoforms involved in RA. The estimated degree of citrullination induced by the two isoforms is also compared to evaluate their relative impact.MethodsFibrinogen was citrullinated in vitro by PAD2/4 and citrullination sites were identified by LC-MS/MS on a Q-exactive orbitrap following proteolytic digestion with Lys-C. These in vitro citrullination profiles were compared to those observed in SF fibrinogen of four RA patients with varying DAS28 scores, CRP levels and leukocyte counts. DAS28 scores >5.1 and ≤2.4 correspond to moderate to severe and low activity of disease respectively. Patients with high inflammatory activity gave high CRP level and leukocyte count values.ResultsA total of 52 citrullination sites were identified. Overall, PAD2 generated higher number of identified sites and higher degree of citrulline occupancy at given sites than PAD4. In fibrinogen from SF, 38 citrullination sites were identified, of which 23 have not been previously reported. Several of these sites were identified in more than one patient, and were regarded as hotspots. Fibrinogen from patients with high DAS28 levels contained markedly more citrullination sites and higher citrulline occupancy.ConclusionsStudy suggests that PAD2 citrullinates fibrinogen more efficiently than PAD4 and citrullination of certain sites in fibrinogen from SF reflects disease activity. Identification of such sites may have diagnostic or prognostic value in RA and other inflammatory disorders.References D Makrygiannakis, E af Klint, I E Lundberg, R Lofberg, A-K Ulfgren, L Klareskog, A.I.C. Citrullination is an inflammation-dependent process. Ann. Rheum. Dis. 2006, 65 (9), 1219–1222.Foulquier,C.; Sebbag, M.; Clavel, C.; Chapuy-Regaud, S.; Al Badine, R.; Mechin, M. C.; Vincent, C.; Nachat, R.; Yamada, M.; Takahara, H. Peptidyl arginine deiminase type 2 (PAD-2) and PAD-4 but not PAD-1, PAD-3, and PAD-6 are expressed in rheumatoid arthritis synovium in close association with tissue inflammation. Arthritis Rheum. 2007, 56 (11), 3541–3553.Gestel, A.; Haagsma, C.; Riel, P. Validation of rheumatoid arthritis improvement criteria that include simplified joint counts. 1998, 41 (10), 1845–1850. AcknowledgementsThe Danish Council for Independent Research Natural Sciences (grant number 11–106246) and the Velux foundation. The Danish Research Foundation, Nordic Bioscience A/S and DTU are a...
Purpose: C-reactive protein (CRP) is an acute phase reactant, involved in both acute and chronic inflammatory diseases. CRP is produced mainly by the liver upon elevated levels of cytokines such as IL-6. From the liver CRP is transported back to the inflamed tissue where it binds its receptors and thereby accumulated by the tissue. We have recently identified the presence fragments of CRP, generated by matrix metalloproteinases (MMPs). These fragments are released from the tissue as MMPs are upregulated in response to pro-inflammatory induction. Recently, an assay was developed, CRPM, which can measure this tissue specific inflammation. The aim of the study was to investigate whether the level of the protein fingerprint CRPM together with the systemic inflammatory marker hsCRP could segregate OA patients with and without inflammatory arthritis. Methods: High sensitive CRP (hsCRP) and CRPM were measured in the serum of patients from three OA cohorts; the CCBR (n¼150), the C4P-003 (n¼250) and the HTF-synovitis (n¼50) studies. The CCBR study consisted of control subjects and patients with various degrees of asymptomatic OA (n¼88, KL 1 to 4). The C4P-003 study consisted of patients with different degree of symptomatic OA as well as controls. All patients in the HTF-Synovitis study were eligible and referred to total joint replacement (TJR) and were described as having end stage OA. KL and VAS pain was monitored for each of the patients. The biomarkers were plotted against each other (figure) and cutoffs were set based on reference value. Results: Patients were separated into 4 groups; 1) patient with low CRPM (tissue inflammation) and low hsCRP (systemic inflammation), 2) patients with high CRPM and low hsCRP, 3) patients with low CRPM and high hsCRP, and 4) patients with high CRPM and high hsCRP. 33% of the patients with end stage OA were found in group 4, whereas only 22% of the patients with symptomatic OA segregated to this group. In comparison, 8% of the asymptomatic OA patients and only 4% of the controls went in to group 4. More than 70% of the controls segregated to group 1, whereas more than 60% the asymptomatic OA patients showed up in group 2. The rest of the patients with symptomatic and end stage OA were equally distributed to group 2 and 3. Conclusions: We found that different of the two inflammatory markers could facilitate patient segregation. This may have many implications, however most importantly in the identification of more OA patients with the right diagnosis, inflammatory vs. non-inflammatory disease, which may benefit from a given treatment.
complete. Furthermore, about 30% of OA samples (hip and knee) showed a fragment of approximately 20KDa that it's not present in none of the 18 control samples analyzed. This fragment seems not to be specific of MMP13 since it was present before the incubation with the protease, and the intensity of the band did not increase with the in vitro MMP-13 digestion. Conclusions: We found three ECM proteins susceptible of MMP13 digestion that could be potential OA biomarkers. Interestingly, results showed that 30% of OA patients had a MMP13 not specific fragment from OGN. The presence or absence of this fragment could be useful as a tool to identify a group of patients with similar characteristics
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