Objective. To determine whether the plasma levels of a range of inflammatory proteins have utility as biomarkers of disease activity in rheumatoid arthritis (RA) patients.Methods. Plasma proteins (n ‫؍‬ 163) were profiled in 44 patients with RA diagnosed according to the American College of Rheumatology 1987 criteria (22 with active and 22 with quiescent disease) and in 16 ageand sex-matched healthy controls. The utility of a subset of differentially expressed proteins as predictors of RA disease activity was investigated using partial leastsquares discriminant analysis, and their response to therapeutic intervention was evaluated in plasma from an additional cohort of 16 patients with active RA treated with anti-tumor necrosis factor ␣ (anti-TNF␣).Results. The protein profiling study identified 25 proteins that were differentially expressed in plasma samples from patients with active RA (P for the false discovery rate < 0.01) compared with those with quiescent RA, including the previously described interleukin-6 (IL-6), oncostatin M, and IL-2, and the 5 less-established markers macrophage colonystimulating factor (M-CSF), tumor necrosis factor receptor superfamily member 9, CCL23, transforming growth factor ␣, and CXCL13. Systemic levels of these 5 markers correlated with the C-reactive protein level, erythrocyte sedimentation rate, rheumatoid factor level, tender joint count in 68 joints, and Disease Activity Score in 28 joints (DAS28), and their combined plasma levels were shown to be good predictors of disease activity ( ‫؍‬ 0.64). In anti-TNF␣-treated RA patients, plasma levels of CXCL13 were reduced after 1 and 7 days of therapy, and levels of CCL23, M-CSF, and CXCL13 showed a statistically significant positive correlation with the DAS28 score.

SUMMARYBiomarker quantification in disease tissues from animal models of rheumatoid arthritis (RA) can help to provide insights into the mechanisms of action of novel therapeutic agents. In this study we validated the kinetics of IL-1 b , TNF-a and IL-6 mRNA and protein expression levels in joints from DBA/ 1OlaHsd murine collagen-induced arthritis (CIA) and Lewis rat Streptococcal cell wall (SCW)-induced arthritis by real-time polymerase chain reaction (PCR) TaqMan® and Enzyme-linked immunosorbent assay (ELISA). Prednisolone was used as a reference to investigate any correlation between clinical response and cytokine levels at selected time-points. To our knowledge this is the first report showing a close pattern of expression between mRNA and protein for IL-1 b and IL-6, but not for TNF-a , in these two models of RA. The kinetics of expression for these biomarkers suggested that the optimal sampling time-points to study the effect of compounds on both inflammation and cytokine levels were day 4 postonset in CIA and day 3 after i.v challenge in SCW-induced arthritis. Prednisolone reduced joint swelling through a mechanism associated with a reduction in IL-1 b and IL-6 protein and mRNA expression levels. At the investigated time points, protein levels for TNF-a in arthritic joints were lower than the lower limit of detection of the ELISA, whereas mRNA levels for this cytokine were reliably detected. These observations suggest that RT-PCR TaqMan® is a sensitive technique that can be successfully applied to the quantification of mRNA levels in rodent joints from experimental arthritis models providing insights into mechanisms of action of novel anti-inflammatory drugs.Keywords animal models mice rats collagen-induced arthritis SCW-induced arthritis cytokines interleukins prednisolone

Abstract-Hemodynamic forces such as fluid shear stress have been shown to modulate the activity of an expanding family of genes involved in vessel wall homeostasis and the pathogenesis of vascular disease. We have investigated the effect of shear stress on tissue factor (TF) gene expression in human endothelial cells (ECs) and in a rat arterial model of occlusion. As measured by reverse transcriptase polymerase chain reaction, exposure of ECs to 1.5 N/m 2 shear stress resulted in a time-dependent induction of endogenous TF transcripts of over 5-fold. Transient transfection of TF promoter mutants into cultured ECs suggests the involvement of the transcription factor Egr-1 in mediating the response of the TF promoter to shear stress. To address the importance of flow induction of Egr-1 in vivo, we have established a flow-restricted rat arterial model and determined the level of expressed Egr-1 and TF at the site of restricted flow using immunohistochemistry. We report an increase in the level of Egr-1 and TF protein in ECs expressed at the site of restricted flow. Elevated expression of Egr-1 and TF is restricted to a highly localized area, as evidenced by the fact that no significant increase in level can be detected at arterial sites distal to the site of occlusion. These findings suggest a direct role for Egr-1 in flow-mediated induction of TF and further substantiate the importance of shear stress as a modulator of vascular endothelial gene function in vivo. Key Words: fluid shear stress Ⅲ tissue factor promoter Ⅲ Egr-1 transcription factor Ⅲ vascular endothelial cells E ndothelial cells (ECs) lining the inner surface of blood vessels are constantly exposed to blood flow. Locally disturbed flow at arterial curvatures or bifurcations is characterized by both low and high oscillatory wall shear stresses that are conveyed to the cell as both a change in pressure and a change in the stretch capacity of the EC lining. 1-3 As a consequence of the local perturbation in laminar shear stress, a number of genes have been identified whose promoter activity is positively or negatively regulated by shear stress (reviewed in References 4 to 8). Several cis-acting elements have been implicated in mediating shear modulation of gene expression, and the term shear stress responsive element (SSRE) was first proposed after the identification of the sequence GAGACC. 9 This SSRE was demonstrated to confer shear stress responsiveness on the platelet-derived growth factor (PDGF) B promoter, 9 a heterologous SV40 promoter, 10 and has since been located in a number of other promoters that have been shown to be shear stress responsive in ECs. 11 The GAGACC sequence binds nuclear factor B (NF-B) p50 -p65 heterodimers, and consistent with this observation, the wild-type HIV-1 LTR, but not an LTR lacking the NF-B binding site, was also shown to be responsive to shear stress. 5 Other transcription factors that have been shown to mediate shear stress activation of a promoter are AP1, 12,13 Sp1, 14 and Egr-1. 15 Recently, the transcription fa...

The vascular endothelium, lining the blood vessel wall, is constantly exposed to wall shear stresses generated by flowing blood. Gene regulation, critical for endothelial cell function, depends on complex interactions at the promoter level and utilizes overlapping signal transduction cascades to activate the expression of genes involved in many biological processes.

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