IntroductionDiscrimination of rheumatoid arthritis (RA) patients from patients with other inflammatory or degenerative joint diseases or healthy individuals purely on the basis of genes differentially expressed in high-throughput data has proven very difficult. Thus, the present study sought to achieve such discrimination by employing a novel unbiased approach using rule-based classifiers.MethodsThree multi-center genome-wide transcriptomic data sets (Affymetrix HG-U133 A/B) from a total of 79 individuals, including 20 healthy controls (control group - CG), as well as 26 osteoarthritis (OA) and 33 RA patients, were used to infer rule-based classifiers to discriminate the disease groups. The rules were ranked with respect to Kiendl’s statistical relevance index, and the resulting rule set was optimized by pruning. The rule sets were inferred separately from data of one of three centers and applied to the two remaining centers for validation. All rules from the optimized rule sets of all centers were used to analyze their biological relevance applying the software Pathway Studio.ResultsThe optimized rule sets for the three centers contained a total of 29, 20, and 8 rules (including 10, 8, and 4 rules for ‘RA’), respectively. The mean sensitivity for the prediction of RA based on six center-to-center tests was 96% (range 90% to 100%), that for OA 86% (range 40% to 100%). The mean specificity for RA prediction was 94% (range 80% to 100%), that for OA 96% (range 83.3% to 100%). The average overall accuracy of the three different rule-based classifiers was 91% (range 80% to 100%). Unbiased analyses by Pathway Studio of the gene sets obtained by discrimination of RA from OA and CG with rule-based classifiers resulted in the identification of the pathogenetically and/or therapeutically relevant interferon-gamma and GM-CSF pathways.ConclusionFirst-time application of rule-based classifiers for the discrimination of RA resulted in high performance, with means for all assessment parameters close to or higher than 90%. In addition, this unbiased, new approach resulted in the identification not only of pathways known to be critical to RA, but also of novel molecules such as serine/threonine kinase 10.

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Endothelin (ET) is a broncho- and vasoconstrictive cytokine, but it also possesses proinflammatory and mitogenic activity. It is suggested to be involved in the pathogenesis of fibrotic lung diseases. We analyzed the concentration of ET 1 in the bronchoalveolar lavage (BAL) fluid in 95 patients with different lung diseases, among them 41 patients with interstitial lung diseases (13 fibrosing alveolitis in systemic sclerosis (FASS), 9 idiopathic pulmonary fibrosis (IFP), 8 sarcoidosis (S), 6 occupational lung disease (OLD), 5 other alveolitidies A), 27 patients with pneumonia, and 8 patients with chronic obstructive pulmonary disease (COPD). A heterogeneous group of 19 patients served as controls. The median ET concentration was 3.3 pg/ml. Significantly higher concentration was found in patients with FASS (5.8 pg/ml), IPF (5.0 pg/ml), and S (5.1 pg/ml) compared with OLD (2.8 pg/ml), A (1.9 pg/ml), COPD (1.5 pg/ml), and the control group (2.5 pg/ml). In pneumonia, the elevated ET concentration (4.1 pg/ml) was accompanied by a high alveolocapillary leakage. When normalized to BAL albumin concentration, only FASS presented with significantly elevated ET/albumin in the BAL compared with the control group (134.5 vs. 56.l pg/mg, p < 0.05). There were no correlations between ET and BAL differential cell count or pulmonary function tests. In current smokers, ET in BALF was significantly higher compared with non- or ex-smokers (3.9 vs. 2.0 pg/ml, p < 0.01), but not so the ET/albumin ratio (65.0 vs. 62.5 pg/mg). In summary, ET in the BAL is differentially expressed in distinct inflammatory and interstitial lung disease. Consistently high concentrations are found in FASS and elevated ET concentration could be discussed in IPF, sarcoidosis, and pneumonia. ET concentration in BAL is influenced by current smoking habits.

Global scale molecular profiling of diseased tissues is an important first step to unravel candidate target molecules that are involved in the pathogenesis of a disease. We have performed a comparative molecular characterization at the transcriptome (microarray with 12 526 gene specificities) and proteome level (multi-Western blot PowerBlot with 791 antibodies) of synovial tissue from rheumatoid arthritis (RA) compared to osteoarthritis (OA) patients. From the panel of 791 antibodies, 260 (33%) detected their corresponding protein. Out of 58 unambiguous changes at the protein level only 16 coincided at the transcript level (28%). Stat1, p47phox and manganese superoxide dismutase were shown to be reproducibly overexpressed in RA versus OA synovial tissue by Western blots with a panel of 8 RA versus 8 OA samples. Cathepsin D was among the most prominent proteins scored to be underexpressed in RA by the PowerBlot whereas no differences of the respective transcript were observed. The lower abundance of cathepsin D protein in RA compared to OA tissue was also reproduced in other patient samples. Immunohistochemistry assigned the Stat1 protein in RA synovial tissue mainly to macrophages and T lymphocytes and the p47phox protein in particular to macrophages. In conclusion, our approach provided us with new candidate molecules for further analysis of rheumatic diseases and stressed the importance of studies at the protein level.

The synovitis of rheumatoid arthritis (RA) was long regarded merely as an unspecific chronic inflammatory process of minor diagnostic value and therefore did not play a major role in the understanding of the pathogenesis of RA. It is only in recent years, along with the observation that T and B cells are expanded oligoclonally in synovial tissue and that B cells are able to undergo a local germinal center (GC) reaction, that the synovial tissue has come to be regarded as a site of specific immune processes. The analysis of the immunoglobulin (Ig) gene repertoire had great impact on the understanding of B cell response in lymphatic organs and was subsequently applied to B cells from RA patients. The analyses of the variable (V) regions of the Ig heavy (H) and light (lambda) chains suggested that an antigen specific activation and differentiation of B cells into plasma cells (Plc) takes place in the chronically inflamed synovial tissue of patients with RA. It seems that in a subset of RA patients the synovial tissue develops into an ectopic lymphoid tissue that supports a local GC reaction. Ectopic GC are characteristic of RA; however, they are in general absent from synovitis of osteoarthritis (OA). Here the accumulation of Plc follows a different mechanism. Highly mutated VH genes suggest that in OA memory B cells migrate into the synovial tissue with subsequent differentiation into Plc but without further V gene diversification. Therefore in synovitis two patterns of B cell activation can be differentiated: the maturative and the accumulative type. These two patterns are not definitely disease linked. The maturative type is only found in RA whereas the accumulative type occurs in both diseases. Clinically RA is defined via serum antibodies to the constant region of Ig, so-called rheumatoid factor. However, the spectrum of autoreactive B cells in RA patients is wide and is based on the study of antibody specificities in serum, in synovial fluid and B cell lines derived from peripheral blood, bone marrow, synovial fluid and synovial tissue. These analyses defined non-organ-specific and organ-specific antigens. One can reasonably assume that the disease is far too complex to be explained by only a single antigen. There is a whole combination of antigens acting in a multistep manner that is responsible for RA pathogenesis. It can be hypothesized that chronic synovitis, which is the underlying mechanism of joint destruction, follows a three-step process: (a) initiation, (b) destruction, and (c) perpetuation. The characterization of antigens driving the local synovial B cell maturation and accumulation could lead to an understanding of the process perpetuating the disease. Identification of arthritogenic antigens may yield new avenues for diagnostics and immunotherapy but also a new approach for prevention by vaccines with antigens probably defined by synovial B cell reactivity.