Identified high and low molecular weight RA antigens.

<p>Identification of high molecular weight (HMW) and low molecular weight (LMW) antigens using Q-TOF mass spectrometric (MS) analysis followed by online MASCOT search against the SwissProt and NCBInr protein databases (kDa-kilo Dalton, MW-molecular weight, Obs-observed, Thr- theoretical, pI- isoelectric point, MOWSE- Molecular Weight Search).</p

Identification of Novel Autoantigen in the Synovial Fluid of Rheumatoid Arthritis Patients Using an Immunoproteomics Approach

<div><p>Rheumatoid arthritis (RA) is a chronic, autoimmune and inflammatory joint disease with a poorly understood etiology. Despite widespread diagnostic use of anti-citrullinated protein antibodies and rheumatoid factor proteins there is a strong demand for novel serological biomarkers to improve the diagnosis this disease. The present study was aimed to identify novel autoantigens involved in rheumatoid arthritis (RA) pathogenesis through immune-proteomic strategy. Synovial fluid samples from clinically diagnosed RA patients were separated on two-dimensional gel electrophoresis (2-DE). Samples from patients with non-RA rheumatisms (osteoarthritis and trauma) were used as controls. Immunoreactive proteins were spotted by Western blotting followed by identification through Q-TOF mass spectrometer analysis. Forty Western blots were generated using plasma from ten individual RA patients and 33 reactive spots were identified, 20 from the high molecular weight (HMW) gel and 13 from the low molecular weight (LMW) gel. Among the 33 common immunogenic spots, 18 distinct autoantigens were identified, out of which 14 are novel proteins in this context. Expression analysis of five important proteins, vimentin, gelsolin, alpha 2 HS glycoprotein (AHSG), glial fibrillary acidic protein (GFAP), and α1B-glycoprotein (A1BG) by Western blot analysis using their specific antibodies revealed their higher expression in RA synovial fluid as compared to non-RA samples. Recombinantly expressed GFAP and A1BG protein were used to develop an in-house ELISA to quantify the amount of autoantibodies in the RA patients. RA patients revealed an increase in the expression of GFAP and A1BG in the plasma as compared to osteoarthritis patients. Therefore, GFAP and A1BG can be proposed as potential new autoantigens of diagnostic importance for RA subjects. Further characterization of these proteins in rheumatoid arthritis will be helpful in understanding the role of these proteins in the disease pathogenesis providing new diagnostic tool with better specificity and accurate detection of the disease.</p> </div

Clinical and demographic characteristics of the study subjects.

<p>M-male, F-female, SD-standard deviation, CRP-C-reactive protein, mg-milligram, RF-rheumatoid factor, IU-international units, ml-milliliter, anti CCP- Anti-cyclic citrullinated peptide, EU- ELISA units, ESR-erythrocyte sedimentation rate, mm-millimeter, hr-hour, DAS28- disease activity score (28 joints maximum).</p

Quantification of autoantibodies in RA patient’s plasma by ELISA using recombinant pure protein of GFAP and A1BG.

<p>Confirmation of over-expressed (A) GFAP and (B) A1BG in plasma of RA patients (n = 30) compared to OA (n = 30) and healthy controls (n = 30). Densitometric analysis showed higher expression level of both proteins in the plasma of RA patients compared to OA and healthy controls.</p

Proteome analysis of CNTF knockout mice.

<p>2D-DIGE gel of spinal cord proteins from CNTF knock-out mice with EAE, labeled with Cy3 (shown in green) versus wild type mice with EAE labeled with Cy5 (shown in red). Selected proteins identified by Mass Spectrometry are indicated with roman indices (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007624#pone-0007624-g005" target="_blank">Fig. 5</a>). Proteins were separated on 24 cm IPG Strip pH 3–10 non-linear.</p

Statistical evaluation of regulated proteins in CNTF −/− mice.

<p>2D DIGE analysis of the average spot volume increase ratio of proteins shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007624#pone-0007624-g004" target="_blank">Fig. 4</a>. Using the DeCyder's Biological Variation Analysis module, a paired Student's t-test yielded a p-value within the 99th percentile confidence level. Mean value crosses are connected; IS  =  Internal Standard.</p

Up-regulation of GFAP over the course of MOG-EAE.

<p>(A) Western-Blot analysis of spinal cord protein preparation at the early chronic (day 35 p.i.) and late chronic phase of the disease (day 60 p.i.). Each lane represents the GFAP expression of one single mouse, β-actin serves as a loading control. (B) Quantification of optical densities of the GFAP labeling as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007624#pone-0007624-g002" target="_blank">figure 2A</a>. There was a clear increase in GFAP expression over the course of MOG-EAE (p<0.05 on day 60 p.i.). (C) Immunohistochemistry for GFAP in naïve C57BL/6 mice without EAE (left) and on day 35 p.i. (middle) and 60 p.i. (right). Representative images from spinal cord cross sections are shown, arrows indicate GFAP positive astrocytes which appear elongated in naïve mice and swollen in the chronic phases of MOG-EAE. Bar represents 20 µm.</p

CNTF deficiency leads to destructive EAE lesions with enhanced apoptosis.

<p>EAE lesions in CTNF −/− mice (left) are destructive with vacuolar degeneration (asterisks) and multiple apoptotic cells with pycnotic nuclei (depicted by arrows). These changes are not present in EAE lesions from wild-type control mice (right side). Representative cervical spinal cord cross sections from a CNTF −/− knockout mouse and a wild-type CNTF +/+ control mouse on day 35 p.i. of MOG-EAE are shown. Bar  = 50 µm.</p

Proteome analysis of chronic MOG-EAE.

<p>2D-DIGE gel of brain proteins from wild type mice with EAE, labeled with Cy3 (shown in red) versus healthy control (without EAE) labeled with Cy5 (shown in green). Images of gel region containing the glial fibrillary acidic protein (GFAP; identified by Mass Spectrometry) is shown in detail for the respective CyDye. IS  =  Internal Standard (Mixture of Samples). Proteins were separated on 24 cm IPG Strip pH 3–10 non-linear. CRNP =  Calreticulin Precursor; PDI = Protein Disulfid Isomerase Precursor.</p

Detection of GFAP in CSF samples of secondary progressive MS patients.

<p>(A) Boxplot analysis of a group of unselected MS patients in comparison to control patients without neurological disease. GFAP levels in the CSF are increased in the multiple sclerosis (MS) group (p<0.05). (B) Boxplot analysis comparing secondary progressive MS (SP-MS) patients, relapsing remitting MS (RR-MS) patients and controls. GFAP levels in the CSF are exclusively increased in the secondary progressive MS group.</p