Nazira El-Hage, Ph.D. - US grants

DESCRIPTION (provided by applicant): Among injection drug users (IDUs), human immunodeficiency virus (HIV) and hepatitis C virus (HCV) are the two blood-borne pathogens most commonly transmitted. About 25% of HIV infected persons in the United States are also infected with Hepatitis C virus (HCV) while the incidence of HCV infection among persons who acquired HIV from injection drug use approaches 90%. Although the role of HCV in progression of HIV disease remains unclear, co-infection with HIV infection has been associated with accelerated progression of chronic hepatitis C towards cirrhosis and end stage liver disease. The mechanisms responsible for more rapid progression of hepatic disease and increased HCV viremia in individuals co-infected with HIV-1 are not fully understood. Viral infection rapidly triggers intracellular signaling events, leading to an innate cellular antiviral state, and damage to the innate immunity may generate a favorable microenvironment for persistent viral infection. Most liver damage associated with HCV infection is mediated by innate and acquired immune responses. Morphine, the major metabolite of heroin, is the most common opiate drug and preferentially activates <-opioid receptors (MOR). Chronic morphine use and abuse has been shown to impair host innate immune responses, including the production of chemokines and pro-inflammatory cytokines, phagocytosis, and neutrophil migration, which can lead to increased susceptibility to bacterial and viral infections. Morphine, through down- regulation of IFN-1 mediated innate immunity, favors HCV replication in hepatic cells. Chemokines and pro- inflammatory cytokines are important mediators of the immune response and the inflammatory process. More specifically, the pro-inflammatory cytokine, tumor necrosis factor-1 (TNF-1) plays an integral role in hepatocyte injury and cell death in a number of pathophysiological states such as liver injury from toxins, ischemia/ reperfusion, and hepatitis virus. In addition, morphine-induced oxidative damage has been hypothesized to contribute too many of the systemic manifestations of liver disease and hepatotoxicity experimentally shown in mice and in heroin abusers. Activation of MOR can trigger increased production of reactive oxygen species (ROS) and apoptosis. Using a recently established in vitro HCV infection system, we will test the hypothesis that opioids contributes to HCV disease progression by disrupting the response of hepatocytes to HCV and HIV through 1) increase production of cytokines and chemokines and 2) induction of reactive oxidative species(ROS) and nitric oxide (NO). PUBLIC HEALTH RELEVANCE: Studies in this proposal will focus on assaying the extent by which opioids and HIV enhances the susceptibility of HCV infection in human hepatocytes in particular, to proinflammatory cytokine tumor necrosis factor-alpha (TNF-1) and we will investigate the mechanisms by which opiates and HIV affect viral replication and toxicity in HCV infected hepatocytes.

DESCRIPTION (provided by applicant): Approximately one-third of HIV-1 infected individuals develop HIV-1-associated neurocognitive disorders (HAND). Symptoms range from minor difficulties to severe neurodegenerative dementia and even with the advent of highly active antiretroviral therapy (HAART), while severe HIV-associated dementia is less common, minor cognitive motor disorders remains an important part of infection. The deleterious effects of opiates are complex and information regarding the actions of drugs of abuse on glial cell function is very limited though they are known to be critical to opiate response. Moreover, the consequent effects of the actions of drugs of abuse and the manifestation of substance abuse on neuronal function are not well known. The proposed project will investigate the role of autophagy in microglia-induced neuroAIDS in the context of substance abuse. The three correlated specific aims are directed at: 1) identifying a novel mechanism in the biology of microglia that regulates HIV-1 replication and whether morphine converges on HIV-1 infection through this pathway~ 2) characterization at cellular levels the consequences of HIV-1 induced lysosomal dysfunction and whether prior and/ or concurrent substance abuse modifies cell function~ 3) examining the signaling pathways and the significance (protective vs. toxic) of the autophagy process activated by HIV-1 and morphine in microglia cells and neurons. Dr. El-Hage has the expertise, leadership, and motivation necessary to run a successful research program and the purpose of getting this award is to enable Dr. El-Hage to become an independent scientist.

? DESCRIPTION (provided by applicant): Growing evidence suggests that opioid abuse exacerbates neuroAIDS and these effects are mainly mediated through the ?-opioid receptor (MOR). There is also evidence to suggest that MOR-1 may have a direct role in HIV type-1 (HIV-1) replication and pathogenesis. MOR is not a single entity, but instead exists as multiple isoforms that differs in function. Despite significant fundamental differences, most studies examining MOR have focused on the canonical MOR-1, and this is particularly true in studies of opioid drug-HIV interactions. Although MOR-1- HIV-1interactions have been studied extensively, the role of MOR splice variant regulation by HIV and, conversely, MOR variant regulation of HIV cellular binding and entry is an unexplored area. Therefore, given that MOR-1 is thought to interact with HIV co-receptors such as CCR5 via heterodimerization and/or downstream signaling, the goal of this grant is to address the variation of MOR in relation to CCR5 mediation of HIV-associated processes in the CNS in the context of morphine and the HIV-1 entry inhibitor maraviroc. Preliminary studies found that: (i) MOR-1 may not be the predominate form of MOR on all CNS cell types, (ii) the N-terminal variant MOR-1K differs functionally in cellular signaling compared to MOR-1 (iii) although both astrocytes and microglia harbors multiple MOR variants, astrocytes harbors the most (iv) MOR-CCR5 interaction occurs in both microglia and astrocytes, although more so in astrocytes and (iv) morphine the predominant agonist of MOR was able to abolish the antiviral effect of the CCR5-mediated viral entry inhibitor maraviroc in a MOR-dependent manner in both astrocytes and microglia. The present proposal is designed to 1. (i) identify the MOR splice variants that interact with the chemokine and HIV (co-) receptor CCR5 in glia (astroglia and microglia) and (ii) determine how these interactions affect viral entry in opioid using individuals in the context of the clinically relevant viral entry inhibitor maraviroc which targets CCR5-mediated entry. The findings from this project could further define the mechanisms in the progression of HIV-related complications in opioid abusers by targeting specific MOR variants and whether a particular cell type is modulating the vulnerability to these complications in the CNS. These findings may also extend to further develop HIV entry inhibitors targeting this interaction.

PROJECT SUMMARY Develop and Evaluate the Efficacy of Nanoformulated siBeclin1 Delivered Intranasally to Eliminate HIV in the Brain. The overall hypothesis is that small interfering (si) RNAs targeting the autophagy pathway can act as a synergistic therapeutic agent with antiretroviral drugs to eliminate central nervous system (CNS) HIV reservoirs and viral associated inflammatory responses in perivascular macrophages, microglia [1,2] and astrocytes [3]. To this end, we will synthesize a siBeclin1 siRNA- polyethylenimine (PEI) polyplex [4] to facilitate intranasal delivery to the brain [4]. The nanoformulated siBeclin1 which transiently diminishes expression of host protein Beclin1, will be tested for its efficacy in eliminating brain cell HIV reservoirs in humanized mice. Efficient intranasal delivery (Figure 1), deployment of mannose decorated particles (Figure 2) and quantitative measures of viral replication will be employed (Figure 3). In the first two sub aims (a &b) of Aim 1, we will quantitatively measure the pharmacokinetics and bio-distribution of siBeclin1 in brain, lung, heart, liver and kidney by reverse transcription polymerase chain reaction (RT-PCR) and liquid chromatography- tandem mass spectrometry (LC-MS/MS) methodologies. Since cell toxicity is a problem encountered with many antiretroviral therapies, in Aim 1c, morphological changes due to cytotoxicity in the brain will be assessed by histology using Hematoxylin and eosin (H&E) and Nissl staining to detect for neuronal damages. Followed by immunohistochemical labeling of neuronal nuclei (NeuN) or microtubule-associated protein 2 (MAP2) to assess for the surviving neuronal cells in the brain. The cell marker, ionized calcium binding adaptor molecule 1 (Iba-1) or CD68 specific for microglia and glial fibrillary acidic protein (GFAP) specific for astrocytes will be used to detect Glial activation (gliosis). Inflammatory responses will be measured by cytokine and chemokine membrane-based antibody arrays and confirmed by colorimetric sandwich enzyme-linked immunosorbent assay (ELISA). Chemistry analysis on the levels of blood urea nitrogen and alanine transaminase activity, will indicate toxicity of the kidneys and liver, respectively. In Aim 2a, we will determine the efficacy of the nanoformulated siBeclin1 on the different aspects of CNS pathology induced by HIV ± antiretroviral drugs including (1) viral replication (measured by PCR-based assays); (2) secretion of immune responses (measured by ELISA-based assays); (3) glial activation and (4) neuronal health (measured by histological and immunohistochemical based assays). Special emphasis will be placed on the hippocampus and the basal ganglia as these regions are most affected by HIV disease and are critical for brain development, learning and memory processes and sensory motor function. In aim 2b, behavioral changes elicited by HIV infection alone or in combination with antiretroviral drug regimen ± nanoformulated siBeclin1 will be assessed using the novel object recognition test as an indicator of short-term memory and the Morris water maze to measure spatial memory. The sensory motor test will be assessed by using the Rota-rod, grip-strength and horizontal bar.