Sandra Lechner

Expression of the thioredoxin-thioredoxin reductase system in the inflamed joints of patients with rheumatoid arthritis

OBJECTIVE To examine the expression of the thioredoxin (TRX)-thioredoxin reductase (TR) system in patients with rheumatoid arthritis (RA) and patients with other rheumatic diseases. METHODS Levels of TRX in plasma and synovial fluid (SF) were measured using enzyme-linked immunosorbent assay. Cellular distribution of TRX was determined by flow cytometry and histochemistry. Cellular expression of TR was studied by in situ messenger RNA (mRNA) hybridization. The effect of oxidative stress and tumor necrosis factor alpha (TNF alpha) on TRX expression by cultured rheumatoid fibroblast-like synoviocytes was studied. RESULTS Significantly increased TRX levels were found in the SF from 22 patients with RA, when compared with plasma levels in the same patients (P < 0.001) and compared with SF TRX levels in 15 patients with osteoarthritis (P < 0.001), 13 patients with gout (P < 0.05), and 9 patients with reactive arthritis (P < 0.0001). The presence of TRX could be demonstrated within the SF-derived mononuclear cells and synovial tissue (ST) of RA patients. Concordantly, expression of TR mRNA was observed in the ST of these patients. Stimulation of synovial fibroblast-like synoviocytes with either H2O2 or TNF alpha induced an increase in the production of TRX. CONCLUSION The data demonstrate significantly increased concentrations of TRX in the SF and ST of RA patients when compared with the levels in patients with other joint diseases. Evidence is presented that the local environment in the rheumatic joint contributes to increased TRX production. Based on its growth-promoting and cytokine-like properties, it is proposed that increased expression of TRX contributes to the disease activity in RA.

Use of Simplified Transcriptors for the Analysis of Gene Expression Profiles in Laser-Microdissected Cell Populations

Because colorectal epithelia are prone to malignant transformation, it is important to understand their normal regulation and then to identify differences between the normal cells and the transformed cells. We investigated the gene expression pattern along colonic crypts using a novel gene expression analysis strategy. We combined laser-mediated microdissection of distinct areas within colonic crypts and used modified RNA arbitrarily primed PCR to generate probes for cDNA array hybridization. In the basal part of the crypt, proliferation-related and cell cycle-related genes such as the multifunctional transcription factor e2f-1 or the mismatch-related gene p58/HHR23B were predominantly expressed. In the lumenal part of the crypt, up-regulations of the cysteine protease mch4 and the proto-oncogene c-jun were found. Our findings indicate that e2f1, p58/HHR23B, and mch4 may be involved in key mechanisms leading to malignant transformation in the colonic crypt. Our results also suggest that the technique elucidated here allows identification of gene expression patterns in distinct areas of intestinal tissue samples.

Thioredoxin Reductase 1 Expression in Colon Cancer: Discrepancy between In Vitro and In Vivo Findings

Thioredoxin and thioredoxin reductase 1 (TR1) are redox proteins that have been implicated in cellular events such as proliferation, transformation, and apoptosis. Analysis of the expression and localization of TR1 in different normal and cancer cell lines and in colon tissues (normal, neoplastic, or inflamed) was performed using reverse transcription–PCR and in situ hybridization. TR1 mRNA was expressed in all analyzed tissues with TR mRNA-positive cells restricted to the stroma of colon crypts, partly being CD3 or CD56 positive. In neoplastic areas of colonic cancer tissue, a loss of TR was obvious. None of the epithelial cells in colonic mucosa expressed TR mRNA, whereas more than 70% of HT-29 cells grown in monolayer were positive for TR. In contrast, HT-29 cells, grown as spheroids or as tumors in SCID mice, were negative for TR. In contrast to these in vitro findings and previous studies, there is no evidence that TR plays a significant role in vivo in normal cell growth in colonic epithelial cells. The mechanism underlying the loss of TR1-positive/CD3-positive/CD56-positive cells or the biologic consequence of this phenomenon observed in neoplastic colonic tissue remains to be clarified.

Evaluation of differentially expressed genes by a combination of cDNA array and RAP–PCR using the AtlasImage™ 2.0 software

Evaluation of genes regulated differentially is essential for the development of therapeutic approaches in multifactorial diseases. To characterize gene expression profiles in multifactorial inflammatory and malignant diseases such as rheumatoid arthritis (RA) or colon adenoma (CA), RNA arbitrarily primed PCR (RAP-PCR) combined with cDNA array hybridization were performed and evaluated using an array-specific software.RNA of synovial fibroblasts from patients with RA and osteoarthritis (OA), and laser microdissected normal and colon adenoma tissue was used. RAP-PCR reactions were hybridized to cDNA array membranes. Arrays were analyzed by phosphor imaging, and the AtlasImage 2.0 software with different normalization settings. The AtlasImage 2.0 software was a useful tool to evaluate differentially expressed genes. However, software settings were needed to be optimized for every experimental approach and should be used without changes for all experiments. To compare RA vs. OA synovial fibroblasts and normal vs. CA expression patterns, global normalization using the sum method is recommended.