Identification of rheumatoid arthritis and osteoarthritis patients by transcriptome-based rule set generation.

INTRODUCTION: Discrimination 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. METHODS: Three 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. RESULTS: The 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. CONCLUSION: First-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.

The expression of collagenase 3 (MMP-13) mRNA in the synovial tissue is associated with histopathologic type II synovitis in rheumatoid arthritis.

The histopathologic analysis of the synovial tissue is important to distinguish rheumatoid arthritis (RA) from other forms of synovitis and to provide information about prognosis and therapeutic strategies at early stages of the disease. In this context, the present study was performed to investigate the correlation between immunohistopathological and morphological features of synovitis and the expression of collagenase 3 (MMP-13) known to contribute significantly to cartilage degradation in RA. In the histopathologic scoring system used in this study, type I synovitis is characterized by B lymphocyte infiltration and an intact lining, and is only mild destructive to cartilage and bone. Type II shows marked diffuse infiltrations of macrophages and T lymphocytes, an ulcerated lining, fibrin exudation, and invasive growth into cartilage and bone tissue. Investigating 36 patients with RA, 21 patients (58%) were positive for the expression of collagenase 3 mRNA in the synovial tissue. Among these patients, 19 showed a histopathologic type II synovitis and only 2 patients had undifferentiated synovitis. In contrast, synovial tissue samples from patients without collagenase 3 mRNA expression were characterized in 6 cases by type I, in 5 cases by type II and in 4 cases by undifferentiated synovitis. The analysis of the clinical data revealed that RA patients with a histopathologic type II synovitis and synovial tissue collagenase 3 mRNA expression had elevated levels of systemic markers of inflammation and received stronger therapies. The data suggest, that collagenase 3 expression and the histopathologic type II synovitis are associated with a severe and destructive course of RA.

Identification of the causes of intrauterine death during 310 consecutive autopsies.

OBJECTIVE: Evaluation of causes of death in stillborn infants. METHODS: During a five-year period, 310 consecutive autopsies of stillborn infants were performed using a standardized protocol with systematic examination of all major cranial, thoracic and abdominal organs including microscopic examination. RESULTS: In 71%, the intrauterine death (ID) occurred up to the end of the 37th week of gestation. Thirty-seven percent (115/310) stillbirths represented with maceration and about one-half with minor or major malformations. Thirty-one percent (53/171) of them were responsible for intrauterine death. In 83% (44/53), the intrauterine death of the malformed fetus occurred before the end of 37th week of gestation, most of them (48/53, 90.6%) were small for gestational age infants. In 75.5% (234/310), the placental villous tree and the umbilical cord represented pathologic conditions. In 191 cases (61.1%), utero-placental pathology was responsible for intrauterine death. Intrauterine infections and traumatic lesions were accompanied by intrauterine death in 2.2 and 1.3%, respectively. In 15.2%, unexplained intrauterine death (because of severe maceration, the placenta was not available for autopsy or insufficient clinical data) occurred. CONCLUSIONS: Perinatal autopsy may be valuable in three ways: the confirmation of ante-mortem diagnoses; the identification of unexpected disorders; and exclusion of other (perhaps inheritable) conditions which might be have caused the intrauterine death. Clinically valuable information, obtained from the autopsy, can be improved by high autopsy rate and performing perinatal necropsies by specially trained pathologists.

High CXCR3 expression in synovial mast cells associated with CXCL9 and CXCL10 expression in inflammatory synovial tissues of patients with rheumatoid arthritis.

To improve our knowledge on the pathophysiology of rheumatoid arthritis (RA), we investigated gene expression patterns in synovial tissue from RA and osteoarthritis (OA) patients. DNA oligonucleotide microarray analysis was employed to identify differentially expressed genes in synovial tissue from pathologically classified tissue samples from RA (n = 20) and OA patients (n = 10). From 7131 gene sets displayed on the microarray chip, 101 genes were found to be upregulated and 300 genes to be downregulated in RA as compared with OA. Semiquantitative reverse-transcription polymerase chain reaction, Western blotting and immunohistochemistry were used to validate microarray expression levels. These experiments revealed that Cys-X-Cys receptor (CXCR)1, CXCR2 and CXCR3 mRNAs, as well as Cys-X-Cys ligand (CXCL)9 (monokine induced by IFN-gamma) and CXCL10 (IFN-gamma inducible protein 10) mRNAs, were significantly upregulated in RA as compared with OA disease. Elevated protein levels in RA synovial tissue were detected for CXCR1 and CXCR3 by Western blotting. Using immunohistochemistry, CXCR3 protein was found to be preferentially expressed on mast cells within synovial tissue from RA patients. These findings suggest that substantial expression of CXCR3 protein on mast cells within synovial tissue from RA patients plays a significant role in the pathophysiology of RA, accompanied by elevated levels of the chemokines CXCL9 and CXCL10. Mature mast cells are likely to contribute to and sustain the inflamed state in arthritic lesions (e.g. by production of inflammatory mediators such as histamine, proteinases, arachidonic acid metabolites and cytokines). Thus, the mast cell could become a potential target in therapeutic intervention.

From transcriptome to proteome: differentially expressed proteins identified in synovial tissue of patients suffering from rheumatoid arthritis and osteoarthritis by an initial screen with a panel of 791 antibodies.

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.

Morphological and molecular pathology of the B cell response in synovitis of rheumatoid arthritis.

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.

Shift in Th1 (IL-2 and IFN-gamma) and Th2 (IL-10 and IL-4) cytokine mRNA balance within two new histological main-types of rheumatoid-arthritis (RA).

Cytokines are the main mediators of inflammation in rheumatoid arthritis (RA). Thus, Th2 cytokines--such as IL-4 and IL-10--have protective properties to this disease. In opposite, the Th1 cytokines--such as IL-2 and IFN-gamma--are supporting proinflammatory microenvironment in joints from patients with RA. The imbalance of Th1/Th2 cytokine steady state may play an important role in the pathogenesis of rheumatoid arthritis. The evaluation of this imbalance leads up to the possibility of pathohistological discrimination in this disease. In this context, we investigated Th1- (IFN-gamma, IL-2) and Th2 (IL-10, IL-4)-cell-derived cytokine mRNA expression in two novel pathohistological main-types of RA synovial membrane (SM). These main-types are characterized by different tissue-infiltrating inflammatory cells and different extent of SM destruction. Our findings showed that expression of IL-10 mRNA was an outcome of histological main-type I (p<0.001), whereas expression of IFN-gamma and IL-2 were mainly associated with pathohistological main-type II (p<0.005, p<0.05). Surprisingly, IL4 was not differential expressed and could be associated with another special T cell subset in this disease. These results suggest that Th1/Th2 balance is biased to Th2 cytokines within main-type I and Th1 cytokines in main-type II.