Methods for classification of toxic agents

A method of determining changes in protein expression that result from contacting a cell culture system with a toxic agent, the method comprising:

• contacting a cell culture system with the toxic agent, wherein the cell culture system is selected from the group consisting of:
• (a) primary human endothelial cells cultured in the presence of two or more factors selected from the group consisting of TNF-α, IL-1, IFN-γ, thrombin, oxidized lipids, angiotensin-II, endothelin-1, aldosterone, IL-4, IL-13, TGF-β, histamine, glucose, and insulin;
• and measuring, subsequent to the contacting, the levels of 3 or more proteins selected from the group consisting of:
• E-selectin, VCAM-1, ICAM-1, MCP-1, MIG, IL-8, HLA-DR, uPAR, CD141/thrombomodulin;
• (b) primary human endothelial cells in a co-culture with peripheral blood mononuclear cells, with a factor stimulating the toll-like receptor 4 (TLR4);
• and measuring, subsequent to the contacting, the levels of 3 or more proteins selected from the group consisting of:
• CD14, CD142, CD40, CD69, MCP-1, E-selectin, IL-1a, IL-8, M-CSF, VCAM-1, CD25, thrombomodulin, and tissue factor;
• (c) primary human endothelial cells in a co-culture with peripheral blood mononuclear cells, with a superantigen;
• and measuring, subsequent to the contacting, the levels of 3 or more proteins selected from the group consisting of:
• CD38, CD40, CD69, CD154/CD40L, E-selectin, IL-8, MCP-1, and MIG;
• (d) primary human fibroblasts, early passage primary human keratinocytes, or a combination of the two, cultured with IL-1β, TNF-α, IFN-γ and TGFβ;
• and measuring, subsequent to the contacting, the levels of 3 or more proteins selected from the group consisting of:
• MCP-1 (monocyte chemoattractant protein-1), ICAM-1 (intracellular cell adhesion molecule-1), collagen I, collagen III, IP-10, Mig, M-CSF (macrophage colony stimulating factor), MMP-9, PAI-1, TGFb1, TIMP-1, uPA, uPAR, I-TAC, HLA-DR, IL-1a, keratin 8/18, MMP-1, eotaxin-3, and IL-8;
• (e) macrophages and endothelial cells co-cultured with TLR2/6 ligand zymosan;
• and measuring, subsequent to the contacting, the levels of 3 or more proteins selected from the group consisting of:
• MCP-1, ICAM-1, collagen I, collagen III, IP-10, Mig, M-CSF, MMP-9, PAI-1, TGFβ1, TIMP-1, uPA, uPAR, I-TAC, HLA-DR, IL-1α, keratin 8/18, MMP-1, Eotaxin-3, and IL-8;
• (f) primary human bronchial epithelial cells stimulated with IL-1, TNF and IFNg;
• or TNF, IL4 and IL-13;
• and measuring, subsequent to the contacting, the levels of 3 or more proteins selected from the group consisting of:
• MCP-1, ICAM-1, collagen I, collagen III, IP-10, Mig, M-CSF, MMP-9, PAI-1, TGFβ1, TIMP-1, uPA, uPAR, I-TAC, HLA-DR, IL-1α, keratin 8/18, MMP-1, Eotaxin-3, and IL-8;
• and
• (g) smooth muscle cells, optionally co-cultured with endothelial cells, stimulated with IL-1β, TNF, and IFN-γ;
• and measuring, subsequent to the contacting, the levels of 3 or more proteins selected from the group consisting of:
• MCP-1, ICAM-1, collagen I, collagen III, IP-10, Mig, M-CSF, MMP-9, PAI-1, TGFβ1, TIMP-1, uPA, uPAR, I-TAC, HLA-DR, IL-1α, keratin 8/18, MMP-1, Eotaxin-3, and IL-8;
• determining changes in protein expression that result from the contacting of the toxic agent, wherein the determining is based on differences between the protein levels in the absence of the toxic agent and subsequent to the contacting;
• and
• recording the changes in protein expression.