Quantification of IFNγ- and IL17-producing cells after stimulation with citrullinated proteins in healthy subjects and RA patients

Antibodies against citrullinated proteins are highly specific for rheumatoid arthritis (RA) and are currently used as a diagnostic marker. In this study, we wanted to quantify the numbers of T cells that react to a wide range of citrullinated proteins in a wide range of HLA-DR subtypes in order to investigate whether citrullination might create T-cell neo-epitopes and could initiate a universal T-cell response. Therefore, PBMCs from healthy volunteers and RA patients were stimulated with a citrullinated and non-citrullinated cell extract on IFN gamma-ELISpot. We found a significantly higher number of IFN gamma-secreting cells after stimulation with citrullinated proteins compared to non-citrullinated proteins in RA patients (1:14,441 cells vs. 1:32,880 cells) as well as in healthy subjects (1:6,261 reactive cells compared to 1:16,212 cells). Additionally, a higher number of IL17-secreting cells were found after stimulation with citrullinated proteins compared to their non-citrullinated counterparts. Our data indicate that citrulline-dependent T-cell response is not restricted to RA patients but that citrullination as such gives rise to a universal break in tolerance

Citrullinated vimentin as an important antigen in immune complexes from synovial fluid of rheumatoid arthritis patients with antibodies against citrullinated proteins

Introduction: Rheumatoid arthritis (RA) is an inflammatory disease, which results in destruction of the joint. The presence of immune complexes (IC) in serum and synovial fluid of RA patients might contribute to this articular damage through different mechanisms, such as complement activation. Therefore, identification of the antigens from these IC is important to gain more insight into the pathogenesis of RA. Since RA patients have antibodies against citrullinated proteins (ACPA) in their serum and synovial fluid (SF) and since elevated levels of citrullinated proteins are detected in the joints of RA patients, citrullinated antigens are possibly present in IC from RA patients. Methods: IC from serum of healthy persons, serum of RA patients and IC from synovial fluid of RA patients and Spondyloarthropathy (SpA) patients were isolated by immunoprecipitation. Identification of the antigens was performed by SDS-PAGE, mass spectrometry and immunodetection. The presence of citrullinated proteins was evaluated by anti-modified citrulline (AMC) staining. Results: Circulating IC in the serum of RA patients and healthy controls contain fibrinogen beta and fibronectin, both in a non-citrullinated form. Additionally, in IC isolated from RA SF, fibrinogen. and vimentin were identified as well. More importantly, vimentin and a minor portion of fibrinogen beta were found to be citrullinated in the isolated complexes. Moreover these citrullinated antigens were only found in ACPA+ patients. No citrullinated antigens were found in IC from SF of SpA patients. Conclusions: Citrullinated fibrinogen beta and citrullinated vimentin were found in IC from SF of ACPA+ RA patients, while no citrullinated antigens were found in IC from SF of ACPA-RA patients or SpA patients or in IC from serum of RA patients or healthy volunteers. The identification of citrullinated vimentin as a prominent citrullinated antigen in IC from SF of ACPA+ RA patients strengthens the hypothesis that citrullinated vimentin plays an important role in the pathogenesis of RA

Mass spectrometry-based proteomics as a tool to identify biological matrices in forensic science

In forensic casework analysis, identification of the biological matrix and the species of a forensic trace, preferably without loss of DNA, is of major importance. The biological matrices that can be encountered in a forensic context are blood (human or non-human), saliva, semen, vaginal fluid, and to a lesser extent nasal secretions, feces, and urine. All these matrices were applied on swabs and digested with trypsin in order to obtain peptides. These peptides were injected on a mass spectrometer (ESI Q-TOF) resulting in the detection of several biomarkers that were used to build a decision tree for matrix identification. Saliva and blood were characterized by the presence of alpha-amylase 1 and hemoglobin, respectively. In vaginal fluid, cornulin, cornifin, and/or involucrin were found as biomarkers while semenogelin, prostate-specific antigen, and/or acid phosphatase were characteristic proteins for semen. Uromodulin or AMBP protein imply the presence of urine, while plunc protein is present in nasal secretions. Feces could be determined by the presence of immunoglobulins without hemoglobin. The biomarkers for the most frequently encountered biological matrices (saliva, blood, vaginal fluid, and semen) were validated in blind experiments and on real forensic samples. Additionally, by means of this proteomic approach, species identification was possible. This approach has the advantage that the analysis is performed on the first "washing" step of the chelex DNA extraction, a solution which is normally discarded, and that one single test is sufficient to determine the identity and the species of the biological matrix, while the conventional methods require cascade testing. This technique can be considered as a useful additional tool for biological matrix identification in forensic science and holds the promise of further automation