Overview

The cells that predominate in the early phase of classical inflammation include platelets, endothelial cells and neutrophils, which are followed in later phases by macrophages, lymphocytes and fibroblasts. Inflammation plays a key role in the pathophysiology of many diseases including atherosclerosis, diabetes and Alzheimer’s disease. A number of inflammatory markers that are measurable in blood have been investigated for their ability to predict an inflammation response. These markers include IL-1β, IL-6, IL-8, IL-10, TNF-α, TGFb, EGF, PDGF and VEGF. The most popular treatment to control the inflammatory responses is use of non-steroidal inflammatory drugs that inhibit cyclooxygenase.

In response to environmental or cellular stresses, cells initiate a program of gene expression either to cope with the stress by inducing repair mechanisms or to mark the cell for apoptosis leading to cell death. This cellular stress response is a universal mechanism of extraordinary physiological/pathophysiological significance. Compromising the integrity of lipid, protein, DNA, redox status, cell cycle control, protein folding, or any one of many other events can elicit a new program of gene expression that can induce a repair process. Common stress-inducible genes include p53, JNK, AP-1, NF-kappaB, MAPK. The stressed endoplasmic reticulum (ER) responds to misfolded proteins via the unfolded protein response pathway involving ER resident transmembrane kinases and chaperones. High content is ideally suited to measuring this cascade of responses in this key area of disease research, learn more about the key targets in inflammation and cell stress below.

Oxidative Stress

Oxidative stress is a consequence of normal aerobic respiration. It can also occur from exposure to UV, environmental stress or toxins, and xenobiotics. When the cellular balance shifts from an antioxidant to pro-oxidant state, reactive oxygen species (ROS) are produced that damage many cellular proteins, enzymes and DNA. ROS are difficult to monitor because they have a short life in the cell. Ideally, to study the effects of oxidative stress a method that can correlate the production of reactive oxygen species to the intracellular damage and cellular response to repair that damage is needed.

Heat Shock Proteins

The major function of heat shock proteins is to facilitate protein folding and protein synthesis as molecular chaperones. However, heat shock proteins also play crucial roles in cell signaling, cell
survival, protein degradation, protein targeting and trafficking, and the regulation of transcription factors. The altered expression level of small heat shock proteins (HSPs) is associated with many human diseases including cataracts, cancer, neurodegenerative disorders and cardiovascular disease, making them ideal targets for drug development.

mTOR Signaling

Cancer cells manipulate many intracellular pathways to enhance cancer cell growth and metastasis. These pathways often converge at AKT and mTOR to determine cell fate. Phospho-mTOR regulates protein translation machinery. Signaling through the PI-3 kinase pathway is critical for cell growth and proliferation, and responding via growth factor stimulation through receptor tyrosine kinases can activate both AKT and mTOR. AKT phosphorylates proteins for both positive and negative regulation of cell growth, and is also coupled to translational and metabolic machinery through its ability to phosphorylate and inactivate TSC, a negative regulator of mTOR. mTOR is responsive to growth factor signaling as well as energy metabolism and nutrients to upregulate protein translation. Proteins in the PI-3 kinase pathway are frequently mutated in cancer cells to promote constitutive growth and proliferation. However, measuring changes in these pathways is challenging because of the constitutive activation of key targets that require multiple parameters to be assessed simultaneously and the rapid turnover of targets that provides only a narrow time window for conducting the assay. Quantitative cell-based imaging assays, such as high-content imaging, provide an advantage over other assay methods because both the activation state of the target (e.g., phosphorylation) and its cellular location can be simultaneously monitored in individual cells. With high-content imaging, AKT and mTOR can be visualized and quantified, as well as subsequent positive and negative downstream effectors (FOXO1A, FOXO3A, GSK3b for AKT, and S6 and 4E-BP-1 for mTOR).

Inflammation Response due to iNOS and COX-2

An inflammatory response is elicited after a harmful stimulus, such as bacterial or viral exposure or a disease state such as cancer, diabetes, rheumatoid arthritis or cardiovascular disease. The inflammatory response involves activation of numerous transcription factors including NF-kB, NFAT, c-Jun, ATF-2 and CREB that can serve as markers for inflammation. Additionally, several upstream and downstream elements to this transcription factor activation can be assayed. Of these, cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) are important. COX-2 increases synthesis of the prostaglandin E2, involved in signaling events in the inflammatory response. Induction of iNOS increases nitric oxide production, a radical important in the oxidative burst of macrophages as well as in signaling. Traditionally, these enzymes are measured by Western blotting or ELISAs that detect their products, NO (iNOS), and PGE-2 (COX-2). Thermo Scientific High-content platforms and Cellomics HCS Reagents are ideally suited to not only measuring the numerous transcriptiofactors involved in the inflammation response, but also can quantitatively measure COX-2 and iNOS activation using indirect immunofluorescence antibody staining and allow for multiplexing experiments.