An analysis of the expression/regulation of specific genes in the synovium is critical to an understanding of the events that initiate and propagate chronic inflammatory synovitis in collagen-induced arthritis (CIA) and rheumatoid arthritis. Microarray technology represents a high-throughput approach to study and statistically compare numerous genes The LOU rat CIA system has several advantages over human autoimmune systems since it uses syngeneic rats, can be reproducibly studied at any time point in arthritis development, and allows the capacity to simultaneously harvest control tissue as well as the end organ of interest, the synovium. Because of limitations in microarray quantitation, RT-PCR will be needed for confirmation only of the selected genes that have altered expression. This analysis could define which gene products might be important at each stage of disease and could provide targets for fixture therapeutic interventions. Since not all LOU rats (even though they are syngeneic) develop CIA following immunization with collagen, early differences in gene expression, compared to their arthritic littermates, might explain this observation. Specifically targeted therapeutic interventions, such as sTNF-Rl, IL-lra, or p38 MAP kinase inhibitors, can be evaluated by microarray methods in the CIA system (and potentially in RA) to clarify their proximate (e.g. TNF, TNF-R2, IL-1) and subsequent (e.g. metalloproteinase) effects on synovial gene expression. This could result in a better understanding of the in vivo mechanisms of how these agents work (in rats with arthritis regression) or don't work (in rats with arthritis progression). An initial pilot study with normal LOU rat spleen/lymph node/liver (non-lymphoid control)/synovial tissue will define optimal procedures and conditions for rat microarray methodology. This utilizes tissue that is readily available and establishes a naive background level of gene expression for later comparison. Most of the previous studies on gene expression profiles in arthritis utilized a small panel of genes encoding potentially pro-inflammatory molecules. In contrast, the microarray includes, in addition, genes that encode molecules responsible for anti-inflammatory effects, metallo-proteinases, bmps, differentiation antigens and factors, transcription factors, and signaling molecules. All of these can help to reveal the on-going pathology and also become targets of therapy in the future. After induction of CIA on day 0, synovial and control tissue from rats will be harvested during the course of arthritis development (day 7-subclinical disease, day 10-clinically evident arthritis onset, day 17-established arthritis, day 28-late arthritis). Microarray analysis will provide sequential data on gene expression as it relates to arthritis progression. Candidate genes will be confirmed by RT-PCR. By testing a number of individual rats, a common gene expression profile at each time point will be generated. Subsequent studies will analyze the effects of specific biologic interventions with sTNF-RI , IL-Ira, p38 MAP kinase inhibitors as single agent or combination interventions in rats with early (day 10) CIA. Patterns of gene expression will be compared with the typical expression defined in naive and CIA control rats at day l7 and day 28. If a therapeutic agent does not alter the gene expression profile, it would indicate that the agent is blocking certain final mediators of the disease. If a therapeutic agent alters the gene expression profile drastically, the changes in the downstream molecules are then responsible for the clinical improvement. This is especially useful when testing therapeutic agents that block signaling pathways. These investigations should identify direct and subsequent effects on gene expression using protocols that selectively target an inflammatory element that has already shown potential utility in rheumatoid arthritis therapy.
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