Pro-inflammatory oxidative stress processes in the SIV/macaque model of HIV CNS disease, and the therapeutic potential of minocycline.

摘要

Human immunodeficiency virus (HIV) continues to affect the lives of millions of people worldwide, with an estimated 2.7 million new infections and 33 million people living with infection in 2007 (UNAIDS, 2008). In addition to the development of acquired immune deficiency disorder (AIDS), HIV infection frequently leads to a constellation of neurological symptoms of both the central and peripheral nervous system. HIV CNS disease is characterized by infiltration and activation of macrophages in the brain, activation of microglia, production of inflammatory cytokines, expression of neurotoxic mediators, and neuronal loss. While the incidence of HIV CNS disease is decreasing, its prevalence is on the rise as patients on highly active antiretroviral therapy (HAART) are living longer (Ances and Ellis, 2007; Dore et al., 2003; Robertson et al., 2007; Tozzi et al., 2007).;Minocycline, a tetracyclic antibiotic, has been shown to have neuroprotective activity in many disease models. Our lab has examined minocycline as a potential therapeutic for HIV CNS disease, and demonstrated a decrease in the severity and incidence of encephalitis with minocycline treatment in our rapid, rigorous SIV/macaque model of HIV-associated CNS disease. In this dissertation, we detail studies performed to examine the mechanisms of minocycline's action, particularly its affects on MAPK signaling. Development of SIV encephalitis is accompanied by a shift in the balance of MAPK signaling pathways in the brain, with a net increase in activation of the proapoptotic neurodegenerative MAPKs p38 and JNK at terminal infection. Activation of these kinases is induced by many different stressors, including reactive oxygen and nitrogen species. We performed in vitro studies to examine the mechanism by which minocycline acts to inhibit p38 and JNK activation induced by nitrosative stress. We identified a role for the inhibition of apoptosis signal-regulating kinase 1 (ASK1) by minocycline, and illustrate this inhibition in vivo in the macaque model. Understanding such mechanisms will not only aid in the potential therapeutic use of this promising drug, but also could provide direction for the development of novel therapeutics and a better understanding of the mechanisms of the disease itself.;To further our understanding of disease processes related to oxidative and nitrosative stress, we then extended this work to study the protein thioredoxin. In addition to its key role as a redox cycling enzyme, thioredoxin is a regulatory component in several intracellular signaling processes, including ASK1 activation, and takes on additional properties extracellularly. Under conditions of oxidative stress, including retroviral infection, thioredoxin may be upregulated and secreted. Secreted thioredoxin can be cleaved to a truncated form (TRX80) that possesses unique pro-inflammatory properties. As levels of plasma thioredoxin are known to be increased in HIV infection, and TRX80 has been suggested to promote viral replication, the ability to monitor and quantitate these two forms of the protein may provide important insight into disease mechanisms. In this dissertation, we describe the development of a mass spectrometry based multiple reaction monitoring assay for the sensitive detection and quantitation of TRX and TRX80 from complex sample types. This assay can be used to provide insight into processes linking oxidative stress and inflammation in our SIV macaque model, and the ways in which minocycline may intervene in these processes. Ultimately, this assay also has far reaching application for the study of many other disease conditions.