Project description:In 15 patients with rheumatoid arthritis the baseline expression (t=0) of type I IFN-regulated genes was determined in peripheral blood cells (PAXgene) using cDNA-microarrays and compared to levels one month after (t=1) anti-TNF treatment (infliximab). Therefore, we used 43K cDNA microarrays from the Stanford Functional Genomics Facility (http://microarray.org/sfgf/) printed on aminosilane-coated slides containing ~20.000 unique genes. Only one batch of arrays was used for all experiments. First DNA spots were UV-cross linked to the slide using 150-300 mJoules. Sample preparation and microarray hybridization were performed as described previously (1,2). Data storage and filtering was performed using the Stanford Microarray Database (http://genome-www5.stanford.edu//) as described previously. 1. van der Pouw Kraan TC, Wijbrandts CA, van Baarsen LG, Voskuyl AE, Rustenburg F, Baggen JM et al.: Rheumatoid arthritis subtypes identified by genomic profiling of peripheral blood cells: assignment of a type I interferon signature in a subpopulation of patients. Ann Rheum Dis 2007, 66: 1008-1014. 2. van der Pouw Kraan TC, van Baarsen LG, Rustenburg F, Baltus B, Fero M, Verweij CL: Gene expression profiling in rheumatology. Methods Mol Med 2007, 136: 305-327. Abbreviations: - Bnrs are the different patients analyzed - PAX or Paxgene indicates the total RNA was isolated from peripheral blood obtained in PAXgene tubes - CRS: common reference sample: We made a common reference that consisted of a mixture of mRNAs isolated from 11 different cell lines (Perou CM, Jeffrey SS, van de RM et al. Distinctive gene expression patterns in human mammary epithelial cells and breast cancers. Proc Natl Acad Sci U S A. 1999; 96:9212-9217) supplemented with RNA from rheumatoid synovial tissue, fibroblasts, and activated peripheral blood mononuclear cells (PBMCs). - PMT: photomultiplier tube: the sensitivity/intensity of the scanner can be adjusted to prevent spot saturation. Compound Based Treatment: anti-TNF (infliximab) reference_design

Project description:In 15 patients with rheumatoid arthritis the baseline expression (t=0) of type I IFN-regulated genes was determined in peripheral blood cells (PAXgene) using cDNA-microarrays and compared to levels one month after (t=1) anti-TNF treatment (infliximab). Therefore, we used 43K cDNA microarrays from the Stanford Functional Genomics Facility (http://microarray.org/sfgf/) printed on aminosilane-coated slides containing ~20.000 unique genes. Only one batch of arrays was used for all experiments. First DNA spots were UV-cross linked to the slide using 150-300 mJoules. Sample preparation and microarray hybridization were performed as described previously (1,2). Data storage and filtering was performed using the Stanford Microarray Database (http://genome-www5.stanford.edu//) as described previously. 1. van der Pouw Kraan TC, Wijbrandts CA, van Baarsen LG, Voskuyl AE, Rustenburg F, Baggen JM et al.: Rheumatoid arthritis subtypes identified by genomic profiling of peripheral blood cells: assignment of a type I interferon signature in a subpopulation of patients. Ann Rheum Dis 2007, 66: 1008-1014. 2. van der Pouw Kraan TC, van Baarsen LG, Rustenburg F, Baltus B, Fero M, Verweij CL: Gene expression profiling in rheumatology. Methods Mol Med 2007, 136: 305-327. Abbreviations: - Bnrs are the different patients analyzed - PAX or Paxgene indicates the total RNA was isolated from peripheral blood obtained in PAXgene tubes - CRS: common reference sample: We made a common reference that consisted of a mixture of mRNAs isolated from 11 different cell lines (Perou CM, Jeffrey SS, van de RM et al. Distinctive gene expression patterns in human mammary epithelial cells and breast cancers. Proc Natl Acad Sci U S A. 1999; 96:9212-9217) supplemented with RNA from rheumatoid synovial tissue, fibroblasts, and activated peripheral blood mononuclear cells (PBMCs). - PMT: photomultiplier tube: the sensitivity/intensity of the scanner can be adjusted to prevent spot saturation. Compound Based Treatment: anti-TNF (infliximab)

Project description:The purpose of this experiment was to do investigatge the transcriptional effect of anti-TNF treatment in patients suffering from rheumatoid arthritis. Two series of hybridizations were performed. Each hybridization was done with a technical replicate (the same amount of RNA taken from the same amplified RNA aliquot labeled in two separate reactions and hybridized onto two separate arrays). In the first series of hybridizations RNA exptracted from biopsies taken before treatment was hybridized in a common reference design to enable comparisons between patients. In the second series RNA from biopsies taken after and before treatment were hybridized on the same chip in a direct design to enable investigation of the effect of treatment. 10 patients (8 females and 2 males, median age 54, range 25-69) meeting the American College of Rheumatology (ACR) criteria for RA were recruited for this study. All patients received infliximab (an antibody directed against TNF-alpha) infusions 3mg/kg at week 0 and than after 2 weeks and 6 weeks. Five of these patients were receiving prednisolon and all ten patients were treated with methotrexate to a maximum of 20 mg per week. Synovial biopsies were obtained during arthroscopy from all patients before and after a median of 9 weeks of treatment (with one exception, patient 7 where the second biopsy was obtained during open surgery). Biopsies were taken at the site of inflammation close to cartilage (cc) or not close to cartilage (ncc), defined as less or more than 1.5 cm away from cartilage, respectively. One biopsy was taken before and after treatment from patient 5, 7, 9 and 10. Multiple biopsies were retrieved before and/or after treatment from patient 1, 2, 3, 4, 6 and 7. Due to small amounts of RNA available for patient 9 it was only used in the second series of hybridizations. The average biopsy weight was 22 mg. The ethical committee at the Karolinska Hospital approved all experiments on human cells and tissues. Informed consent was obtained from all study subjects.

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Delayed anti-TNF therapy increases the risk of total knee replacement in patients with severe rheumatoid arthritis.

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