Effects of Etanercept Are Distinct from Infliximab in Modulating Proinflammatory Genes in Activated Human Leukocytes Asifa S. Haider, Irma R. Cardinale,

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Effects of Etanercept Are Distinct from Infliximab in Modulating Proinflammatory Genes in Activated Human Leukocytes Asifa S. Haider, Irma R. Cardinale, Julia A. Whynot, James G. Krueger Journal of Investigative Dermatology Symposium Proceedings Volume 12, Issue 1, Pages 9-15 (May 2007) DOI: 10.1038/sj.jidsymp.5650032 Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Genes downregulated by etanercept in activated PBMCs. mRNA from cultured PBMCs was hybridized to individual oligonucleotide arrays containing ∼12,000 human genes (Affymetrix HG-U95Av2 chips). Heatmaps show unsupervised hierarchical clusters of genes regulated by etanercept in PBMCs using similarity measure: Pearson's correlation. Gene lists were created after significance analysis as defined by a paired Welch test, with a subsequent correction for multiple gene comparisons by the Benjamini and Hochberg method (P<0.05). The genes regulated by >1.2-fold mean expression levels are represented with red lines (upregulated) as compared to green lines (downregulated). Genes in PBMCs activated with anti-CD3/CD28 (ACT) are compared to non-stimulated and activated in the presence of TNF inhibitor etanercept, n=6. (a) Heatmap of 86 genes significantly downregulated by treatment with etanercept. (b) Heatmap representing >1,700 genes upregulated by activation but not affected after treatment with etanercept. The top 36 genes downregulated by etanercept are listed in the Table S1 online is available with a complete list of etanercept-regulated genes, with fold changes and P-values. Journal of Investigative Dermatology Symposium Proceedings 2007 12, 9-15DOI: (10.1038/sj.jidsymp.5650032) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Real-time RT-PCR confirmation of genes regulated by etanercept as compared to infliximab: IL-4, -5, -9 (IL-4, -5, and -9), IL-12-receptor β 2, IFN-γ, LTA, and TARC. mRNA from PBMCs activated with anti-CD3/CD28 antibody (ACT) was compared to non-stimulated PBMCs (NS), activation in the presence of anti-TNF antibodies etanercept or infliximab. Mean of relative mRNA expression normalized to human acidic ribosomal protein is plotted on column bars. Significance of ACT versus treatment is indicated with asterix (*): n=6, P<0.01. Journal of Investigative Dermatology Symposium Proceedings 2007 12, 9-15DOI: (10.1038/sj.jidsymp.5650032) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Comparison of effects of etanercept and infliximab on gene activation. Venn diagram shows comparison of genes regulated by etanercept (left circle) with genes regulated by infliximab (right circle). Fifty-five genes are regulated exclusively by etanercept, 513 genes are regulated by infliximab and both inhibitors commonly regulate 29 genes. Selected list of genes with fold changes are listed in the figure. Journal of Investigative Dermatology Symposium Proceedings 2007 12, 9-15DOI: (10.1038/sj.jidsymp.5650032) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions