The ability of interferon alpha to induce suicidal depression in patients is of growing concern and unkonwn etiology. I am currently employing several new cutting- edge approaches to dissect the mechanisms of interferon alpha and beta-induced neurotoxicity. The newly evolving field of proteomic biology attempts to define biological events and disease progression in terms of overall protein expression patterns and not gene expression. Afterall, it is the relative levels of the proteins themselves which dictate cell biology. New approaches to define protein expression include IPG (immobilized-pH gradient ) 2D-PAGE electophoresis and SELDI (surface-enhanced laser deapsorption and ionization mass spectroscopy ) protein chip retentate mapping analysis. These techniques allow the rapid-hight-throughput screening of low to high abundance proteins from complex biological systems. Additionally, I have developed a novel technique, through the use of a selective differential display system, to """"""""fingerprint"""""""" any cytokine or growth factor , or diseace state in their ability to induce the tyrosine or serine phosphophorylation of intracellualr proteins which are ultimately responsible for the biological effects of these biologics or diseace processes. I intend to employ each of these techniques to identify speicif toxic pathways that interferon alpha dn beta are using in cell lines and in vivo in mice. Specifically, these approaches include: 2D-PAGE analysis of mouse brain tyrosine and serine mmunoprecipitations, 2D mapping of total whole brain lysates, rapid high-throuhgput multiplexed RNase protection assays for message levels of cytokine, apoptosis and cell-cycle analysis, SELDI protein-chip retentate mapping and protein-protein, protein-DNA analysis, and scanning infra-red and magnetic resonance microscopy analysis of mice brain lysates and fixed tissue sections. New or pre-existing protiens involved in the propogation of toxic events can be isolated and identified via MS-MS sequencing. I am currently the lead investigator-collaborator with Dr. Lance Liotta, Cheif of the Laboratory of Pathology, NCI and am in charge of the cancer proteomics effort underway there, and have at my disposal the Ciphergen SELDI machine and a Finnegan LCQ tandem quadrapole MS-MS ESI mass spectrospoy machine for other research projects. The ability to define toxic pathways and surrogate markers for toxic events, especially in animal models is of extreme importance to the FDA, and it could be argued that this is the most improtant research we could perfrom. Many products, both in drugs and biologics fail to produce obvious toxic events in pre-clinical animal studies. This is especiall true for neurotoxic events, as it is extremely difficult to accurately assess depression, or coginitive behavioral changes in animals. The identification of new surrogate markers will prove invaluable to predict early toxic events which will lead to better safety in phase I trials, saving of time on the part of the manufacturer, and an overall better product.
