Prevención del vasospasmo

This research is supported by the 7th _{Framework Programme for Research (project} NanoDiaRA, NMP4-LA-2009-228929) and by the Center for Translational Molecular Medicine, project “TRACER”.

Imaging of cathepsin and MMP activity

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References

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5. Ryu JH, Lee A, Chu JU, et al. Early diagnosis of arthritis in mice with collagen-induced arthritis, using a fluorogenic matrix metalloproteinase 3-specific polymeric probe. Arthritis Rheum 2011;63:3824- 32.

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7. Cunnane G, FitzGerald O, Hummel KM, et al. Collagenase, cathepsin B and cathepsin L gene expression in the synovial membrane of patients with early inflammatory arthritis. Rheumatology (Oxford) 1999;38:34-42.

8. Itoh T, Matsuda H, Tanioka M, et al. The role of matrix metalloproteinase-2 and matrix metalloproteinase-9 in antibody-induced arthritis. J Immunol 2002;169:2643-7.

9. Nakagawa TY, Brissette WH, Lira PD, et al. Impaired invariant chain degradation and antigen presentation and diminished collagen-induced arthritis in cathepsin S null mice. Immunity 1999;10:207-17.

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19. Joosten LA, Helsen MM, van de Loo FA, et al. Anticytokine treatment of established type II collagen- induced arthritis in DBA/1 mice. A comparative study using anti-TNF alpha, anti-IL-1 alpha/beta, and IL-1Ra. Arthritis Rheum 1996;39:797-809.

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Assessment of inflammation by the Saa3 promoter reporter

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Abstract

Objective

To evaluate the applicability of a lentiviral (LV) Saa3-promoter luciferase (Luc) reporter for assessing inflammation in experimental arthritis, synovial fibroblasts (SF) from osteoarthritis (OA) and rheumatoid arthritis (RA) patients.

Methods

In mice, synovium was transduced in vivo by cholesterol optimized LV and two flares of acute joint inflammation were induced by injection of streptococcal cell wall (SCW) material into the knee joint cavity. The time course of synovial inflammation was assessed using ex vivo luciferase assays, and histology. Uptake of 99m_technetium (Tc) was used to assess edema. SF (n=12) of RA and OA patients were stratified by hierarchical clustering of whole genome expression profiles. Relative Saa3-promoter responses were determined in cytokine- or toll-like receptor-stimulated SF subgroups. Results

In vivo, the Saa3-promoter reporter activity was strongly upregulated at one and two

days after the first and second SCW challenge. The Saa3-promoter activities during acute inflammation correlated with Tc uptake measurements, but was more sensitive and able to respond to the ongoing synovitis in the chronic phase of SCW arthritis. Molecular stratification defined two inflammatory SF subtypes, unrelated to disease classification. Relative Saa3-promoter responses to IL-1b, TNFa and TLR4 agonist were significantly increased in OA/RA SF with a high compared to a low inflammatory profile subtype. Serum stimulation of the Saa3-promoter reporter cell-line could discriminate between healthy and RA-patients.

Conclusion

The Saa3-promoter reporter demonstrates a robust and feasible tool for assessing the course and severity of experimental arthritis and to distinguish molecularly distinct inflammatory SF subtypes from a heterogeneous patient population.

Assessment of inflammation by the Saa3 promoter reporter

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Introduction

Rheumatoid arthritis (RA) is a systemic chronic autoimmune disease that mainly affects the synovial joints that ultimately leads to joint destruction. Prior to and during joint inflammation the synovial lining tissue, consisting of synovial fibroblasts (SF) and macrophages, becomes activated. In both experimental and human arthritis, SF have been identified as cells that actively drive inflammation and joint destruction [1- 3]. Moreover, transmigration of activated RASFs has been implicated in mediating the spreading of destructive arthritis to unaffected joints[4]. Due to their key role in RA pathogenesis, SF are regarded not only as major target cells for treatment, but also for diagnostic assessment of disease activity or phenotype.

Non-invasive assessment of disease activity is frequently performed using radiographic agents such as 99m_{technetium pertechnetate (}99m_{Tc) [5]. Histological} and immunohistochemical analyses of synovial tissue comprise the classic ex vivo methods for assessing the grade of inflammation and molecular pathology in human and experimental arthritis and have evidently pointed out that RA is a heterogeneous disease [6, 7]. Moreover, these have shown that disease heterogeneity is stably imprinted in SF and molecularly distinct inflammatory subtypes can be discerned. Gene expression is regulated at multiple levels including epigenetic, transcriptional and translational control. Transcriptional control is exerted by the differential activation of transcription factors (TF) and pivotal TFs implied in RA pathogenesis include nuclear factor-kB (NF-kB), activator protein-1 (AP-1) and CCAAT/Enhancer binding protein b (C/EBPb) [14-16]. Activity of TFs can be dynamically assessed using transcriptional assays based on expression of luminescent, fluorescent or enzymatic reporter genes driven by a promoter containing TF-response elements. Until now, the diagnostic potential of transcriptional reporters in RA has not been extensively investigated. Recently, we have designed a number of promoter-luciferase reporters, using gene expression profiling data of inflamed synovium in murine collagen-induced arthritis (CIA), that demonstrated responsive to experimental arthritis and inflammatory stimuli in human SF [17]. These reporters contained composite elements of AP-1, NF-kB and C/EBPb binding sites. Notably, we found an enrichment of C/EBPb-binding sites in the promoters of genes whose expression correlated with disease severity in CIA, in particular the promoter of serum amyloid A3 (Saa3). This led to the hypothesis that a Saa3-promoter reporter might serve as a novel diagnostic tool for assessing inflammatory subtypes in arthritis.

Therefore, we performed kinetic analyses of Saa3-promoter activity in SCW arthritis and compared this novel method with 99m_{Tc uptake and histological assessment of joint} inflammation. In addition, we evaluated whether Saa3-promoter responses in SF would be able to discriminate between SF of low and high inflammatory expression profiles, as deduced by gene expression profiling.

Assessment of inflammation by the Saa3 promoter reporter

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