Kelly Tilleman

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

IntroductionRheumatoid 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.MethodsIC 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.ResultsCirculating IC in the serum of RA patients and healthy controls contain fibrinogenβ and fibronectin, both in a non-citrullinated form. Additionally, in IC isolated from RA SF, fibrinogenγ and vimentin were identified as well. More importantly, vimentin and a minor portion of fibrinogenβ 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.ConclusionsCitrullinated fibrinogenβ 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.

Synovial detection and autoantibody reactivity of processed citrullinated isoforms of vimentin in inflammatory arthritides

OBJECTIVES To investigate the presence and characteristics of citrullinated vimentin in protein extracts of inflamed synovial tissue. METHODS Cytosolic protein extracts obtained from RA (n = 14) and SpA patients (n = 14) were analysed by gel electrophoresis and western blotting. Citrullinated vimentin isoforms were visualized by a combination of anti-modified citrulline (AMC) staining and anti-vimentin detections (V9, H-84). This was subsequently confirmed by immunoprecipitation. Autoantibody detection was verified using sera obtained form RA (n = 6) and SpA (n = 6) patients. RESULTS A specific cluster of spots displayed on the 2D gel images of cytosolic synovial tissue extracts, was identified by mass spectrometry as vimentin. Interestingly, our results suggested that these isoforms could be the result of caspase cleavage. In addition, these cleaved forms of vimentin were found to be citrullinated in synovial cytosolic protein extracts of inflammatory arthritides, mainly in RA patients. Caspase-3 is able to cleave vimentin at amino acid 85. Western blot analysis with a specific antibody against amino acids 1-84 of vimentin (H-84) confirmed that the citrullinated isoforms of vimentin were lacking this part of the protein. These results were also confirmed by immunoprecipitation of vimentin derived from cytosolic protein extracts of RA and SpA patients. Furthermore, the presence of autoantibodies against these citrullinated processed forms of vimentin was found to be predominantly associated with RA patients. CONCLUSIONS These findings show the presence of processed citrullinated vimentin in inflammatory arthritides, mainly in RA and suggest a possible origin of the ACPA immune response in RA.

The HCV serum proteome: a search for fibrosis protein markers.

Summary.  Liver fibrosis/cirrhosis is a serious health issue in hepatitis C virus (HCV‐) infected patients and is currently diagnosed by the invasive liver biopsy. The aim of this study was to find useful fibrosis markers in HCV‐patients’ sera of different fibrosis degrees (METAVIR F0‐F4) based on proteomics. Serum proteome profiles were created by two‐dimensional gel electrophoresis. Profiles were analysed between different degrees of fibrosis (F0‐F4) and between early (F0F1) and late (F2F3F4) fibrosis by univariate analyses (P ≤ 0.05). Differentially expressed proteins were subsequently identified by mass spectrometry. Mac‐2‐binding protein, α‐2‐macroglobulin and hemopexin were increased in F4 opposite F0/F1. A‐1‐antitrypsin, leucine‐rich α‐2‐glycoprotein and fetuin‐A were decreased in F4 opposite F0/F1. Late fibrosis was characterized by an increase in Mac‐2‐binding protein, α‐2‐macroglobulin and α‐1B‐glycoprotein expression and a decrease in haptoglobin expression. Mac‐2‐binding protein expression was confirmed by dot blot assay and enzyme‐linked immunosorbent assay in a secondary population. In conclusion, serum proteome analysis enabled the detection/identification of existing and new candidate markers in line with fibrosis progression in HCV‐patients.

The relevance of citrullinated vimentin in the production of antibodies against citrullinated proteins and the pathogenesis of rheumatoid arthritis

Antibodies against citrullinated proteins (ACPAs) are highly specific for RA. Since the discovery of these antibodies, several of studies that focused on the presence and identity of citrullinated proteins in the joints of RA patients have been carried out. The best-known antigens that bind ACPAs are citrullinated filaggrin, Type II collagen (CII), α-enolase, fibrinogen and vimentin. This review compares citrullinated filaggrin, CII, α-enolase and fibrinogen with vimentin in their contribution to ACPA triggering, and gives an overview of the literature in which the role of citrullinated and non-citrullinated vimentin in the onset of ACPA production and the pathogenesis of RA is discussed.

Modification of citrulline residues with 2,3-butanedione facilitates their detection by liquid chromatography/mass spectrometry

Citrullination is a post-translational modification (PTM) that results from the deimination of the amino acid arginine into citrulline by Peptidyl Arginine Deiminase enzymes and occurs in a wide range of proteins in health and disease. This modification causes a 1 Da mass shift, which can be used to identify citrullination sites in proteins by the use of mass spectrometry. However, other PTMs, such as deamidation from asparagine to aspartic acid or from glutamine to glutamic acid, can also cause a 1 Da mass shift, making correct interpretation of the data more difficult. We developed a chemical tagging strategy which, combined with an open source search application, allowed us to selectively pinpoint citrullinated peptides in a complex mixture after liquid chromatography/mass spectrometry (LC/MS) analysis. After incubation of a peptide mixture with 2,3 butanedione, citrulline residues were covalently modified which resulted in a 50 Da shift in singly charged mass. By comparison of the peptide mass fingerprint from a modified and an unmodified version of the same sample, our in-house search application was able to identify the citrullinated peptides in the mixture. This strategy was optimized on synthetic peptides and validated on a digest of in vitro citrullinated fibrinogen, where different proteolytic enzymes were used to augment the protein coverage. This new method results in easy detection of citrullinated residues, without the need for complex mass spectrometry equipment.

Galectin-3-binding protein: a serological and histological assessment in accordance with hepatitis C-related liver fibrosis.

Objectives Invasive liver biopsy is the current method for the assessment of liver fibrosis. In search of noninvasive alternatives, galectin-3-binding protein (G3BP) was introduced as a candidate-marker of hepatitis C-related fibrosis based on serum proteomics. We investigated the role of G3BP as a single-marker of significant fibrosis and cirrhosis by serology and histology and studied the effect of glycosylation on antibody-affinity in hepatitis C and alcoholic cirrhosis. Methods Sera and available biopsies of hepatitis C patients with various fibrosis-grades and patients with alcoholic cirrhosis were used for G3BP-measurements by enzyme-linked immunosorbent assay and immunohistochemistry, respectively. Glycosylation-effect was analyzed by western blot. Data was analyzed in accordance to fibrosis. Results G3BP-levels (mean±standard deviation) were increased during cirrhosis (22.7±10.1 μg/ml) compared to mild (11.3±6.4 μg/ml) and moderate fibrosis (13.4±8.3 μg/ml) (P<0.001; P=0.004, respectively). Receiver operator characteristic curves showed areas under the curve of 0.68, 0.75 and 0.81 for detection of significant fibrosis, severe fibrosis, and cirrhosis, respectively. Similar findings in hepatic G3BP expression were obtained, in which cirrhosis was associated with diffuse, parenchymal expression (P=0.002). The observed difference between hepatitis C and alcoholic cirrhosis (13.5±9.0 μg/ml) (P=0.009) could not be explained by glycosylation. Conclusion Our recent findings confirm our initial proteome results on serological and histological level as well as the role of G3BP as a marker of hepatitis C-related fibrosis, especially cirrhosis. Implication of this protein in future multi-marker study should be considered.

Comparative proteome analysis of porcine follicular fluid and serum reveals that excessive α2-macroglobulin in serum hampers successful expansion of cumulus-oocyte complexes

Porcine follicular fluid (pFF) constitutes the micro‐environment of the maturing oocyte. Although pFF is a transudate of serum, in pigs, it is superior to serum in promoting in vitro expansion of the cumulus cells, a specialized cell population surrounding the oocyte. A comparative proteome analysis of autologous serum and pFF was performed to investigate proteins involved in successful cumulus expansion of porcine oocytes. iTRAQ labeling followed by 2‐D LC ESI‐Q‐TOF MS/MS revealed 63 proteins common to both fluids of which the abundance of 13 proteins was significantly different (p<0.05). Seven proteins were more concentrated in serum whereas six proteins were more abundant in pFF. To investigate the importance of these proteins, the cumulus matrices of COCs were collected after in vitro maturation in media supplemented with either of both biologically fluids and then subjected to 2‐D PAGE analysis. α2‐Macroglobulin and CH4 and secrete domains of swine IgM, which were both less abundant in pFF, were absent from cumulus matrix extracts after in vitro maturation in pFF. Although both proteins were incorporated in the matrices of cumulus‐oocyte complexes matured in serum, depletion of α2‐macroglobulin from serum could significantly compensate for the impaired cumulus expansion of oocytes matured in serum.

Usefulness of a novel serum proteome-derived index FI-PRO (fibrosis-protein) in the prediction of fibrosis in chronic hepatitis C.

Background Liver biopsy is an imperfect standard for the assessment of chronic hepatitis C liver fibrosis. In this study, the diagnostic role of proteome-derived protein markers and the usefulness of a protein-based index were assessed. Methods Characteristics, clinical biochemistry, and protein markers of patients with chronic hepatitis C from a study (n=62) and validation group (n=73) were statistically assessed according to fibrosis severity. Multivariate models were built using linear discriminant analysis for the prediction of minor fibrosis (F0–F1), moderate fibrosis (F2–F3), and cirrhosis (F4). The best model was validated and diagnostic performance was compared with the aspartate aminotransferase-to-platelet ratio index based on their receiver operator characteristic curves. Results Statistical analysis resulted in significant outcomes for both clinical and protein markers. The best multivariate model was based on four protein markers: &agr;-2-macroglobulin (A2M), haptoglobin, hemopexin, and galectin-3-binding protein. A2M and hemopexin were the primary predictors according to this model. A novel index A2M/hemopexin [fibrosis-protein (FI-PRO) index] showed a diagnostic performance rate of 0.80–0.92 for the detection of significant fibrosis (F2–F4) and advanced fibrosis (F3–F4) in the validation group, which was better compared with aspartate aminotransferase-to-platelet ratio index. FI-PRO had an overall positive predictive value of 86% for significant fibrosis and a negative predictive value of at least 90% for advanced fibrosis. Conclusion Proteome-derived protein markers were successfully implemented in clinical diagnosis of hepatitis C fibrosis, which resulted in the FI-PRO index. The efficiency and usability of FI-PRO should be validated in large-scale, prospective studies.

Proteomics in liver fibrosis is more than meets the eye

Liver fibrosis is a serious health issue for many liver patients and is currently diagnosed using liver biopsy. The erroneous nature of this technique urges the search for better, noninvasive alternatives. In this regard, proteomics has been described as a useful biomarker discovery tool and has become increasingly applied in the study of liver fibrosis. Experimental and clinical studies have already provided deeper insights in the molecular pathways of liver fibrosis and even confirmed previous findings. Recent advances in proteomic strategies and tools enable multiple fractionation, multiple protein identifications and parallel analyses of multiple samples. Despite its increasing popularity, proteomics still faces certain pitfalls concerning preanalytical variability, protein coverage and statistic reliability. Proteomics is still evolving, but will undoubtedly contribute to a better understanding of the basics of the pathology and certainly offer opportunities in liver fibrosis diagnostics and therapeutics.

Fluoxetine reduces murine graft-versus-host disease by induction of T cell immunosuppression.

Serotonin reuptake inhibitors (SRIs) are widely used drugs in the treatment of depression and anxiety disorders. Although SRIs are generally regarded as safe drugs with relatively few side effects, literature suggests that high concentrations of SRIs may alter immune function. We investigated whether high-dose treatment with fluoxetine was able to suppress acute graft-versus-host disease (GvHD) in a MHC-matched, minor histocompatibility antigen mismatched murine bone marrow transplantation model. We found that high doses fluoxetine induce a significant reduction of clinical symptoms and increase survival of these animals. The amelioration of clinical GvHD was accompanied by a reduced expansion of alloreactive T cells. We further analyzed the direct in vitro effect of six SRIs on the viability and proliferation of human T cells and found an anti-proliferative and pro-apoptotic effect that was significantly larger in activated than in resting T cells. We discuss these results in the light of potential future exploration of SRIs as a novel class of T cell immunosuppressive drugs.