Howard S Fox - US grants

Hormones, particularly estrogens and androgens, can cause different immune responses in males and females. Females generally have a more vigorous immune system than males, and while it is clear that differences in sex steroids contribute to immunologic differences between the sexes, the mechanism(s) by which they alter immune responses is unknown. One of the most dramatic sex-related differences in the immune system is the disproportionate prevalence of autoimmune diseases in females. For example, systemic lupus erythematosus (SLE) occurs nine times more frequently in females than males, and rheumatoid arthritis occurs in females at a ratio of three to one over males. Understanding the basis for sex steroid/immune system interactions will greatly elucidate the pathologic states of autoimmune diseases in particular, and immunology in general. Although the reported experimental effects of sex steroids on immune responses vary, estrogens generally increase, and androgens generally decrease, autoimmune reactions. The nature of the interactions between the endocrine and immune system, and how these affect disease, has yet to be determined. In this regard, we intend to address three specific aims: 1.) Define the immune system cell types responsive to estrogen; 2.) identify immune effector molecular regulated by estrogen, and 3.) evaluate the effects of estrogen on an SLE-xenograft mouse model. Different immune cell populations from cultured cell lines and mice will be examined molecularly for the presence of estrogen receptors. Such cells will then be manipulated in cell culture to examine the effect of estrogen treatment on the production of immune effector molecules, such as interferons and interleukins. These studies will allow identification of the sex steroid target cells and well as the molecules these hormones may modulate that could lead to sex-related differences in immune responses. Finally, an animal model of autoimmunity that allowed the in vivo manipulation of human SLE lymphoid cells will be examined for possible sex steroid effects on the transferred disease. To assess these effects, disease parameters will be measured in SCID mice that received parallel lymphocyte transfers of human SLE cells, and the effects of estrogen treatment measured.

Hormones, particularly estrogens and androgens, can cause different immune responses in males and females. Females generally have a more vigorous immune system than males, and while it is clear that differences in sex steroids contribute to immunologic differences between the sexes, the mechanism(s) by which they alter immune responses is unknown. One of the most dramatic sex-related differences in the immune system is the disproportionate prevalence of autoimmune diseases in females. For example, systemic lupus erythematosus (SLE) occurs nine times more frequently in females than males, and rheumatoid arthritis occurs in females at a ratio of three to one over males. Understanding the basis for sex steroid/immune system interactions will greatly elucidate the pathologic states of autoimmune diseases in particular, and immunology in general. Although the reported experimental effects of sex steroids on immune responses vary, estrogens generally increase, and androgens generally decrease, autoimmune reactions. The nature of the interactions between the endocrine and immune system, and how these affect disease, has yet to be determined. In this regard, we intend to address three specific aims: 1.) Define the immune system cell types responsive to estrogen; 2.) identify immune effector molecular regulated by estrogen, and 3.) evaluate the effects of estrogen on an SLE-xenograft mouse model. Different immune cell populations from cultured cell lines and mice will be examined molecularly for the presence of estrogen receptors. Such cells will then be manipulated in cell culture to examine the effect of estrogen treatment on the production of immune effector molecules, such as interferons and interleukins. These studies will allow identification of the sex steroid target cells and well as the molecules these hormones may modulate that could lead to sex-related differences in immune responses. Finally, an animal model of autoimmunity that allowed the in vivo manipulation of human SLE lymphoid cells will be examined for possible sex steroid effects on the transferred disease. To assess these effects, disease parameters will be measured in SCID mice that received parallel lymphocyte transfers of human SLE cells, and the effects of estrogen treatment measured.

This investigator-initiated interactive research project is designed to combine in vitro and in vivo studies to examine the role of the blood- brain barrier in central nervous system disease induced by HIV. The integrity of the blood-brain barrier is essential for normal brain function. However abnormalities in this barrier have been reported in AIDS patients, and has been found at a high frequency in those with HIV-induced dementia. Such a defect may play a role in causing the CNS functional deficits seen in AIDS by a number of mechanisms, including facilitating a high level of infection of the brain by virus or infiltration by virus- carrying cells, attracting inflammatory cells to the brain which themselves can have toxic effects, and/or disturbing the normal homeostasis of the brain by allowing damaging molecules to enter the CNS that are normally excluded. Determining the role of the blood-brain barrier in HIV-induced neuropathogenesis and the etiopathogenesis of such disorders is extremely limited in human disease due to the impossibility of controlling important scientific variables such as viral strains, timing of infection, and the restricted access to CNS-derived samples. The rhesus monkey-SIV model is ideal in this respect, as many of the CNS disease parameters in this model mimic those in humans HIV. In this study, we will first examine the role of blood-brain barrier dysfunction by comparing breakdown of the barrier to virologic, immunologic, pathologic, and functional parameters measured in animals infected with a neurovirulent strain of SIV. Second, we will study the relationship of tropism of the virus to infect the brain capillary endothelial cells, the central components of the blood-brain barrier, with neurovirulence. Possible etiopathogenic mechanisms in CNS infection blood-brain barrier dysfunction induced by this infection will be examined. Finally, a brain capillary endothelial cell-tropic molecular clone of SIV will be derived and studied for its neuropathogenic effects. These studies will allow the further development of an animal model to study the effect of HIV on the CNS, and yield insights into the role of the blood-brain barrier in disease, the mechanisms of CNS infection and dysfunction, and its prevention and treatment.

Simian immunodeficiency virus infection of rhesus macaques (Macaca mulatta) represents one of hte best available experimental models for human AIDS. In SIV infeciton a AIDS patients. In order to develop a reliable experimental model for neuropathological, behavioral, cognitive and pharmacologic studies of AIDS dementia complex we have initiated experiments to derive a neuroinvasive strain of SIV. By selection of virus populations replicating in brain derived microglial cells, we have demonstrated neuropathological changes in four of four infected macaques. Changes include perivascular and infiltrating mononuclear cells and multinucleate giant cells, viral gene expression, and host response. In the next project period we will continue to develop this model to enable us to probe the mechanisms and potential therapy of CNS alterations. Specific aims of this project include development and analysis of the pathologic effects of a neuroinvasive microglial cell derived stock of SIV, establishment of its growth characteristics and disease potential, and use of the neurovirulent stock to assess host and viral factors contributing to disease. LTR sequence control of replication in the macrophage/microglial cell lineage will be assessed as will the correlation between CNS viral titers and local cytokine production, host immune responses and pathological changes. Parameters of viral pathogenesis will be assessed in cohorts of behaviorally trained monkeys to establish correlation with pathology, temporality of measurement, cognitive change and host responses. Finally, a neurovirulent molecular clone of microglial adapted SIV will be derived in order to provide a stable benchmark for future cognitive and pharmacological studies, and to promote detailed studies of the mechanisms of pathogenesis of AIDS dementia.

tight junctions; membrane proteins; blood brain barrier; vascular endothelium permeability; molecular assembly /self assembly; protein protein interaction; embryogenesis; cell adhesion molecules; pancreatic islets; surface antigens; gene expression; angiogenesis; experimental allergic encephalomyelitis; protein localization; cell line; confocal scanning microscopy; laboratory mouse; genetically modified animals; site directed mutagenesis; immunofluorescence technique; embryo /fetus; tissue /cell culture;

The experiments proposed here are designed to address the relationship between the virus, the host's response to virus, and alterations in CNS functions. We will focus on testing the hypothesis that there is a CTL response in the CNS that serves to help control viral load, but at the cost of damage to the CNS. First, we will investigate the nature and extent of the early CTL response to SIV infection in the CNS. We will asses whether there is a regional specificity of SIV infection or CTL response in the brain. Examination of measures of oxidative stress, a common potential mechanism for many dementing conditions, will assess the potential for these CTL and other events occurring in the host/viral interaction to induce damage to the CNS. Additionally, control of CTL migration by chemokines produced by the infected CNS will be investigated. Second, we will assess the nature of the CTL response in the CNS over time, compare it to the CTL response occurring in the periphery, and relate these findings to the development of CNS functional abnormalities. In order to test whether the level of peripheral viral load plays a role in the maintenance of a CNS immune response, one group of animals will receive anti-viral treatment at two weeks post-viral inoculation, at the peak of peripheral viral load and CTL response. The potential for diminution of the CNS CTL response, versus a perpetuation, will be analyzed to determine the relationship of CNS CTL to peripheral viral load, and their joint relationship to CNS dysfunction and pathology. This will aid in the examination of whether the viral-immune interactions in the CNS compartment can occur independently of those in the blood and lymphoid tissue. Third, we will examine the clonality of CTL responses in the CNS during the acute and chronic infection to assess whether the CNS response is representative of the immune response in the peripheral compartment or whether evidence of local, possibly clonal or oligoclonal amplification occurs within the CNS. We will also use anti-viral therapy and its withdrawal to model the development of viral resistance or failure of effective therapy, and determine the vulnerability of the CNS to re-infection in this setting, and the role CNS CTL play in the response to the rebound viremia.

Drugs of abuse, such as the psychostimulant methamphetamine (METH), are frequently used by the population at risk for HIV infection. Studies in experimental animals indicates that METH has the potential to produce selective toxic effects on dopaminergic and serotoninergic neurons in the CNS. The mechanisms of METH-induced damage remains the subject of active investigation. The effects of energy metabolism, endogenous neurotransmitters, temperature, and free radical-induced toxicity have all been proposed to be important in the mechanisms mediating the effects of METH. Many of these mechanisms and sites of damage converge with those in HIV- induced CNS damage. Interactions of METH and HIV, which both affect an increasing population, is unknown. Using SIV-infected rhesus monkeys we have been able to derive a neurovirulent strain of virus that can induce both histopathological and functional abnormalities in the CNS. Nonhuman primates are also appropriate models for the effects of METH on the CNS of humans. Here we propose to examine the interactions of SIV and METH in CNS infection and injury. Our working hypothesis is that the CNS is infected by virus early after SIV inoculation, and can be continuously infected by circulating SIV or SIV-infected cells. The toxicity to the CNS is related to the number and activation state of the brain microglia and macrophages, both of which increase following CNS virus infection. METH may increase this toxicity by a number of nonexclusive mechanisms; specifically we will examine the possibility that functional measures of physiology (temperature and movement), neuronal circuitry (evoked potentials), behavior (bimanual motor skills), viral load (quantitative SIV RNA analysis), histopathological indicators (macrophage and lymphocyte infiltration) are altered by the interaction of METH and SIV.

Description (adapted from applicant's abstract): This is a revised five-year R01 grant application. A research team headed by Dr. Howard Fox with collaborators Steven Henriksen (electrophysiologist) and John Polich (bio-statistician) and research support staff from the Scripps Research Institute, La Jolla, CA has proposed to examine the relationship between virus levels and associated CNS disease in SIV-infected macaques. This group proposes to use an SIV-rhesus macaque model to test their hypothesis that CNS virus-associated pathology can be blocked, limited or reversed by the administration of antiviral drugs. Both mono-therapy (PMPA) and combination-therapy (PMPA and ZDV) will be used at various stages of infection. Further, the proposed research seeks to describe and determine the cause of CNS tissue damage associated with CNS dysfunction. CNS dysfunction will be detected by physiological monitoring during SIV infection. Their three Specific Aims will determine: 1.) Whether early antiviral treatment (two weeks PI) prevents or reduces the CNS pathology and whether termination of the treatment will result in the development of CNS symptoms and pathology, 2.) The histopathological characteristics of CNS pathology in treated and untreated SIV-infected macaques during a serial sacrifice experiment and 3.) Whether CNS abnormalities are reversible by lowering viral load in chronically infected macaques by initiating antiviral therapy at five months PI. A total of 44 Rhesus macaques will be used in the proposed experiments. All animals will be monitored by a well thought out series of assays and physiological assessments followed by detailed tissue analysis at necropsy.

[unreadable] DESCRIPTION (provided by applicant): The Scripps NeuroAIDS Preclinical Studies (SNAPS) Center was organized in 2000 in order to promote the innovative and highly productive cross-disciplinary research program at The Scripps Research Institute (TSRI) that focused on modeling the untoward effects of HIV in the central nervous system (CNS). Significant success has been achieved, with the SNAPS NIMH P30 Center providing leadership and support for multiple investigators from many fields of inquiry through its activities and Cores. This has resulted, to date, in 138 original research publications and numerous interdisciplinary collaborations. Here, we present our plans to renew the Center. [unreadable] [unreadable] Studies from the SNAPS Center, those of others, and the new clinical findings in neuroAIDS lead us to propose new and restructured Cores. These capitalize on state of the art scientific advances, illustrative in vivo and in vitro systems, and now, building on our preclinical basic science success, direct links and projects with clinical researchers to analyze clinical specimens. We will insure a strong focus on the problems of neuroAIDS. [unreadable] [unreadable] Three themes: Virus-Host Interactions, Integrative Neuropathogenesis, and Discovery, provide the basis for the research, which are united under a systems biology approach to understand the current and changing dynamics of neuroAIDS. The four Research Cores, a single Developmental Core, and an Administrative Core will serve key roles in unraveling the complexity of neuroAIDS. [unreadable] [unreadable] The organization and communication among investigators under the auspices of the SNAPS has, and is expected to continue to, foster numerous other spin-off projects/grants, and to provide training, mentoring, and resources for neuroAIDS work. Additional, open access databases and bioinformatic tools will be made available to the scientific community as a whole, aiding in the efforts to prevent and/or treat this devastating condition. [unreadable]

The experiments proposed here are designed to address the relationship between the virus, the host's response to virus, and alterations in CNS functions. We will focus on testing the hypothesis that there is a CTL response in the CNS that serves to help control viral load, but at the cost of damage to the CNS. First, we will investigate the nature and extent of the early CTL response to SIV infection in the CNS. We will asses whether there is a regional specificity of SIV infection or CTL response in the brain. Examination of measures of oxidative stress, a common potential mechanism for many dementing conditions, will assess the potential for these CTL and other events occurring in the host/viral interaction to induce damage to the CNS. Additionally, control of CTL migration by chemokines produced by the infected CNS will be investigated. Second, we will assess the nature of the CTL response in the CNS over time, compare it to the CTL response occurring in the periphery, and relate these findings to the development of CNS functional abnormalities. In order to test whether the level of peripheral viral load plays a role in the maintenance of a CNS immune response, one group of animals will receive anti-viral treatment at two weeks post-viral inoculation, at the peak of peripheral viral load and CTL response. The potential for diminution of the CNS CTL response, versus a perpetuation, will be analyzed to determine the relationship of CNS CTL to peripheral viral load, and their joint relationship to CNS dysfunction and pathology. This will aid in the examination of whether the viral-immune interactions in the CNS compartment can occur independently of those in the blood and lymphoid tissue. Third, we will examine the clonality of CTL responses in the CNS during the acute and chronic infection to assess whether the CNS response is representative of the immune response in the peripheral compartment or whether evidence of local, possibly clonal or oligoclonal amplification occurs within the CNS. We will also use anti-viral therapy and its withdrawal to model the development of viral resistance or failure of effective therapy, and determine the vulnerability of the CNS to re-infection in this setting, and the role CNS CTL play in the response to the rebound viremia.

The experiments below are designed to address the relationship between the SIV and METH in the induction of alterations in CNS function. We hypothesize that METH and HIV induce convergent cellular processes in the CNS to induce neuropathogenesis. During the life and at autopsy, we have chosen measures to examine these processes and the changes they produce. Since viral load has significant consequences on the course of AIDS and likely neuroAIDS, we will assess virus in the periphery and CNS, as well as determining the CTL response, critical in controlling viral load. Since METH is known to affect neurons and monoamine neurotransmitter systems in the striatum, quantification of neurons and neurochemicals will assess our hypothesis that SIV synergizes with METH in inducing damage to the CNS. In Aim 1, we hypothesize that METH administration surrounding the early phase of SIV infection will lead to increased damage to the CNS and effects on the crucial viral-host interaction setting the course of disease. Since the CNS itself is both infected and affected early following inoculation, changes occurring during the acute injection period may dramatically alter CNS disease. METH administration is predicted to lead to more profound, or quicker development of, SIV-induced abnormalities. During life, a longitudinal study of selected electrophysiological, physiological, behavioral, viral, monoamine metabolite and immune parameters will be performed. This aim will model an active METH user who becomes HIV infected. In Aim 2, we hypothesize that METH administration in the setting of chronic injection can work through numerous mechanisms to augment the CNS deficits induced by SIV infection. Among these are making the CNS more susceptible to damage through neurotoxic effects, affecting viral replication kinetics, or altering immune cell trafficking to the CNS. Here we will treat with METH for two periods following stable establishment of SIV infection. In this manner we will model an HIV-infected individual who uses METH. The first period of METH administration will allow us to determine the effects of measures of physiology, CNS function, and viral/host interactions. The second will allow the same determinations for repeat METH use, and CNS tissue analysis at the end of the second METH period will allow direct examination of the pathological consequences of recent METH use, in the setting of SIV injection and a history of METH use.

DESCRIPTION (provided by applicant): In HIV-induced disease, the central nervous system (CNS) is both a target for virus infection as well as for damage induced by infection. Alcohol abuse is frequent among HIV-infected individuals and those at high risk for contracting HIV infection. Here we propose to examine the interactions of alcohol on the factors controlling SIV infection of the CNS of monkeys, as a model for ethanol effects on HIV in humans. Our working hypothesis is that the toxicity of HIV to the brain is initiated by viral infection of the brain, but is related more to the number and activation state of the brain microglia and macrophages, both of which increase following CNS virus infection. Does alcohol have an effect upon HIV entry and macrophage infiltration into the brain? This key question will be directly addressed here by these studies. This will be studied, in vivo, in the rhesus/SIV model. Events occurring during the acute phase after inoculation may greatly influence the disease induced by SIV. Since the CNS itself is both infected and affected early following inoculation, changes occurring during the acute infection period may dramatically alter CNS disease. Alcohol-treated and non-alcohol treated animals will be infected with SIV, and studied to determine the effect of ethanol on the initial course of SIV infection, and the effect of ethanol on SIV and macrophage entry into the CNS. We will examine the hypothesis that the CNS viral load and CNS macrophage infiltration resulting from SIV infection is altered by ethanol administration. There are numerous convergent processes that can be affected by alcohol and HIWSIV, such as effects on monocytic lineage cells, cytokines, chemokines, oxidative stress, viral infection and cell trafficking, that can result in untoward effects on the CNS. The mechanisms controlling CNS infection and macrophage infiltration will be investigated to pursue the nature of alcohol's effect.

DESCRIPTION (provided by applicant): Infection with HIV-1, the causative agent of AIDS, results in central nervous system (CNS) as well as immunological dysfunction. NeuroAIDS, the constellation of symptomatology found in individuals with cognitive and/or motor disorders, can be a devastating consequence of infection. Although it is generally accepted that the products of infected and/or activated cells of the monocytic lineage (monocytes, macrophages, and microglia) lead to CNS dysfunction in neuroAIDS, our understanding of the nature of the alterations resulting in macrophage accumulation in the brain and the nature of their injurious effects on neurons are still limited. The studies proposed here will combine in vivo studies, using the SIV/rhesus monkey model of AIDS and mouse models, to examine key pathogenic factors leading to brain macrophage accumulation, and the injurious neuronal response. In vitro studies on monocytes, macrophages, and neurons will complement these experiments to further test the molecular mechanisms involved in these effects. Three Specific Aims are proposed, in which we hypothesize that bone marrow changes, potentially due to TGFbeta, results in the production of monocytes with properties leading to increased ability to enter and accumulate in the brain. Osteopontin, discovered to be upregulated in brains in SIVE and HIVE, is postulated to play a key role in this accumulation. Macrophage accumulation and microglia activation in the brain leads neurons to respond to this altered environment, and we have identified cyclin D3 as a potential pathogenic initiator of an eventually damaging neuronal response. The effects of this expression in neurons will be investigated. These combined studies will expand our knowledge of the pathogenesis of this disorder, and should identify crucial pathways in which therapeutic or preventative interventions can be directed.

DESCRIPTION (provided by applicant): The CNS remains a target of HIV in the era of HAART, and the effects of CNS dysfunction can be devastating. The SIV-infected monkey model for HlV-related diseases has been invaluable in studies of HIV/AIDS pathogenesis and treatment/vaccine research. We and others have developed systems and techniques to enable the study of CNS disease in SIV-infected monkeys. Using brain RNA from different stages of SIV-induced disease, we have found distinct alterations in the transcriptional profile both during the stable phase of disease, when animals are generally asymptomatic but have measurable CNS functional abnormalities, as well as in end-stage neurological disease. Here, we will first investigate 2 mechanisms for neuronal damage identified from alterations in neuronal gene expression in microarray studies. Second, using a recently developed HAART protocol, we will now study CNS function and changes in CNS gene expression during HAART treatment of SIV-infected monkeys. Furthermore, we have developed methodologies for metabolomic analysis, enabling us to perform a longitudinal study to identify significant biomarkers in plasma and CSF that correlate with CNS disease. These experiments will yield novel data, and lead to the identification of unique molecules that can then be examined in humans, as well as provide the starting point for new studies aimed at the sources of the identified metabolic differences. The identification of molecular markers for neuroAIDS is valuable for both diagnostic and etiopathogenic reasons, and we believe the recent developments in metabolomics give these techniques a distinct advantage in achieving this goal.

[unreadable] DESCRIPTION (provided by applicant): Drugs of abuse are a significant co-factor in HIV infection. Although it has been postulated that drugs of abuse may also represent significant co-factors in disease progression, addressing this hypothesis has been problematic. We have obtained preliminary data that such an interaction indeed occurs between the commonly used psychostimulant methamphetamine (METH) and SIV, and propose here to further investigate this interaction. Both METH and SIV/HIV affect the brain, and many of the mechanisms and sites of damage converge for these two agents. We have found increased neurophysiological deficits in animals receiving both agents. Furthermore, signs of simian AIDS appeared within the study period only in SIV-infected animals receiving METH. Furthermore, METH-treated SIV-infected animals developed a kidney disease not seen in the other animals. We propose here to study the interactions of SIV and METH in systemic and organ-specific damage and dysfunction. Our working hypothesis is that at least three distinct areas of interaction occur between these agents that combine to increase disease: a combined toxic effect in the CNS, an interaction in the immune system, and an induction of renal disease. Specifically, in our three aims, we will examine functional measures of physiology and neuronal circuitry, viral load, and histopathological and neurostructural indicators for interactions of METH and SIV in the CNS. Systemic/peripheral (non-CNS) manifestations of disease will also be examined, in terms of viral load, immune function, and renal pathophysiology. In addition to in vivo and pathological studies, we will perform in vitro studies on CCL15, a proinflammatory chemokine found to be upregulated by METH in astrocytes, and a potential mediator of neuronal injury. Through these appropriately powered, controlled experiments, we will determine the nature of the interaction between METH and SIV, and uncover important mechanisms relevant to human health. [unreadable] [unreadable]

functional /structural genomics; protein quantitation /detection

AIDS /HIV neuropathy; disease /disorder model; laboratory mouse

DEVELOPMENTAL CORE Abstract The Developmental Core is committed to accelerating the growth of neuroAIDS investigators by supporting innovative and collaborative research, particularly between basic and clinical investigators; conducting a robust, peer-reviewed pilot grant program; assisting in the mentoring and establishment of new/junior investigators; supporting the transition of established investigators to HIV, aging, and neuroAIDS work; and promoting formation of multidisciplinary teams involving both early-career and senior investigators in cutting- edge issues emerging in HIV, aging, and CNS research. In this renewal application, we propose an innovative approach - pairing junior investigators with experienced senior investigators to form mentor-mentee teams that will meet regularly, participate in a workshop on career development, and present their work at the annual CHAIN retreat. We realize that for this approach to succeed, having a pool of skilled volunteer mentors is critical. One important aspect will be to leverage this with ongoing mentoring programs at University of Nebraska Medical Center such as the Faculty Mentoring Program for mentees and mentors and the ?Women?s Mentoring Program (WMP)? the mission of which is to promote and nurture the careers of early-stage women scientists. It must be clarified that the focus of this core is not to exclusively foster the careers of only ?women scientists,? but we are cognizant that increased mentoring is necessary in the current climate of research training and career advancement. We will significantly expand our mentoring capacities to take advantage of all opportunities and sources of expertise. While the CHAIN Developmental Core has a broad mission, its primary goal remains to support and entice investigators from other disciplines into the HIV/aging arena. Applications will undergo rigorous peer review and will be prioritized on the basis of scientific merit, translational relevance, emerging concepts that are high risk/high payoff, addressing key scientific problems like HIV and aging, HIV infection in women, and new biomedical approaches to prevention. Our previous successes support our likelihood for success, and the additional funds procured from UNMC will greatly enhance our ability to enable these investigators to successfully enter the field.

The Chem Library Screening Core of the Scripps NeuroAIDS Preclinical Studies (SNAPS) Center will[unreadable] facilitate the screening of diverse chemical libraries in the context of research relevant to neuroAIDS.[unreadable] Numerous investigators at TSRI have generated libraries and are eager to collaborate with[unreadable] biologists/biochemists working on defined biological systems. The major mission of the proposed Core will[unreadable] be to facilitate such interactions by development of a series of high through-put screens and aid in the[unreadable] development of specialized screens as the need arises. Both biochemical and cell-based screening assays[unreadable] will be developed and employed to identify discrete mixtures of compounds that interfere with viral protein[unreadable] function(s) and interrupt the virus life cycle and/or virus penetration of the CNS. The Library Screening Core[unreadable] will interact with a very diverse group of investigators, integrating their expertise and creating additional[unreadable] know-how for the screening of compounds. The ultimate goal will be to aid in identification of specific lead[unreadable] compounds to aid in development of anti-virals effective against Neuro-AIDS.

DESCRIPTION (provided by applicant): Even in the current era of HAART, the central nervous system remains a target for viral infection and disease-induced damage in HIV-infected individuals. HIV is known to infect and persist in the brain in cells of the monocytic lineage. We have recently identified co-expression of the enzyme indoleamine 2,3- dioxygenase (IDO) with SIV in the brains of monkeys with SIV encephalitis, and have found IDO induction in microglia and macrophages in brains at both early and late stages of disease. IDO catalyzes the oxidative breakdown of tryptophan, and this reaction leads to a number of effects on both the immune and nervous system with important implications for HIV neuropathogenesis. We hypothesize that the expression of IDO in virus-infected macrophage creates a safe haven for the virus in the CNS, allowing persistence and spread of the infection. In the three proposed specific aims, we will examine the basis for the induction of IDO in infected cells, assess the factors that modulate IDO expression, and perform functional studies to uncover how this safe haven is created. In these studies, will investigate three hypotheses: 1) SIV infection of macrophages leads to IDO induction and sensitizes the cells to produce IDO at low levels of other stimulating agents; 2) SIV and the local environment influence the production and activity of IDO through specific transcriptional and post- transcriptional mechanisms; and 3) IDO alters the ability of host T cells to inhibit viral replication, with compensatory mechanisms protecting macrophage viability and viral production. These studies will uncover the molecular mechanisms by which IDO is regulated in microglia and macrophages, the means by which it contributes to neuropathogenesis in HIV infection, and lead to discoveries allowing such pathways to be manipulated therapeutically.

The Scripps NeuroAIDS Preclinical Studies (SNAPS) is a multidisciplinary center supporting biomedical[unreadable] research relevant to neuroAIDS.[unreadable] In HIV infection, the HIV-1 -associated cognitive/motor disorder, also known as the AIDS dementia complex[unreadable] or neuroAIDS, occurs in approximately one-third of patients. Symptoms range from a "minor" disorder,[unreadable] affecting 25% of individuals, to dementia, affecting 15 to 20% of those with AIDS. Although recent[unreadable] therapeutic advances have reduced mortality from HIV, most of these agents do not show significant[unreadable] penetration of the blood-brain barrier (BBS). The central nervous system (CNS) may remain a reservoir as[unreadable] well as suffer continued damage over the treatment-prolonged course of infection.[unreadable] The etiology of the HIV-induced CNS alterations is unknown. One of our working hypotheses are that viral[unreadable] infection of the brain, occurring via a "Trojan horse" mechanism of infected macrophages trafficking to the[unreadable] brain initiates a sequence of events leading to neuronal dysfunction. However we believe that neurons, at[unreadable] least early, are not irreversibly damaged by a virus-induced pathological cascade. Thus HIV-induced CNS[unreadable] damage may be amenable to timely therapy to prevent the chronic consequence of CNS dysfunction.[unreadable] Using a multisystem approaches, the SNAPS center research aims to investigate the host and viral factors[unreadable] leading to CNS dysfunction, and investigate ways to prevent or ameliorate these untoward effects. The[unreadable] SNAPS center brings together scientists with different expertise from proteomic, genomic and physiology.[unreadable] The Systems Biology and Integrative Network Core (SBINC) aims to develop a suite of bioinformatics tools[unreadable] for extracting pathway models from molecular interaction networks using a system of complex queries.[unreadable] Because the database query is a well-established means of extracting information from large bioinformatics[unreadable] databases, it may serve as a key concept for bringing complex network operations, such as pathway[unreadable] construction and simulation, to the SNAPS center at large. Thus helping the SNAPS scientists to better[unreadable] query their large highthroughput dataset and to utilize a integrative system approach to better characterize[unreadable] this complex neuropathology.

[unreadable] DESCRIPTION (provided by applicant): Drugs of abuse are known to be a significant co-factor in HIV infection. However addressing whether drugs of abuse are significant co-factors in disease progression has been difficult. Methamphetamine (METH) is a frequently and increasingly used drug among those infected with HIV, and those at high risk for becoming HIV infected. Our Preliminary Data indicates that METH can negatively affect many parameters of simian immunodeficiency virus (SIV)-induced disease in monkeys. Here we proposed a well- powered study to obtain definitive results on the interaction of METH and SIV. Our hypothesis is that there are distinct areas of interaction occur between these agents that combine to increase disease. First, there is a combined toxic effect in the central nervous system (CNS), in which SIV and METH combine to yield functional and biochemical deficits both greater and different than either agent alone. Second, there is an interaction in the immune system that affects disease course and likely secondarily the CNS. Third, both SIV and METH affect autophagy, an essential process in neurons whose disruption leads to neuronal damage, and the effects of SIV and METH together are greater than either alone. Pharmacological agents that affect autophagy will be tested in a preclinical setting to provide both mechanistic insights and provide leads for treatment strategies. Those infected with HIV, and those at high risk for HIV infection, include people that frequently use drugs of abuse, including methamphetamine, the use of which is increasing nationwide. Methamphetamine can damage the brain, as can HIV infection. Here we will examine how these two agents interact to cause brain as well as other problems, and such work will provide insight into not only these two agents, but means to protect neurons from other damaging processes. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Both HIV-infection and drugs of abuse are worldwide health problems and pose significant obstacles for improved disease diagnosis and treatment. We posit that one approach to reach these goals is through proteomics. In this regard, we will use a well developed track record in proteomics research relevant to neurodegeneration and specifically neuroAIDS and apply it to a strategy for useful biomarker discovery that reflect cellular responses to HIV infection and drug abuse, which can be translated to human disease. This aim drives three proposed studies focusing on the theme that methamphetamine (METH) combines with HIV to increase the damaging affect of glial activation on the brain, impairing neurocognitive functions. We will utilize the scientific and technical expertise of three institutions at the University of Nebraska Medical Center (UNMC), the Scripps Research Institute (TSRI), and the University of California San Diego (UCSD) to investigate the interrelationships between drug abuse (with a focus on METH and HIV infection. In project 1, H.E. Gendelman, we will use well-validated in vitro systems to perform mechanistic investigations focusing on the influence of METH on HIV-induced proteomic changes during glial crosstalk and resulting neuronal function. These results will feed directly into the primate studies in projects 2, H. S. Fox, which uses the nonhuman primate model of AIDS and drug abuse, investigating changes in the proteome as well as up-stream gene expression in affected brain tissue for both biomarker discovery and disease pathogenesis. These studies will lead directly into evaluation of clinical specimens proposed in project 3, P. Ciborowski. In this project, biomarker discovery will be performed on plasma from clinical samples of well-characterized individuals, HIV infected, from time points when they are using, or not using, METH. Data from Project 2, examining both the CNS and plasma, will prove essential in selecting candidates. Furthermore, such candidates from these studies, as well as other proteins uncovered from Projects 1 and 2, will be cross-validated in studies of human plasma and CSF analysis. These integrated studies will bring results from cell biology, virology, immunology, and animal pathogenesis to the bedside in attempts to improve care through diagnostic and clinical benefits. There is a high overlap in the population of people who are HIV infected and those who abuse METH. METH use is on the increase in the US. The relevance of this project is that it will result in the discovery of the mechanisms that HIV and METH interact to damage the brain, and identify proteins mark disease processes and serve as targets of therapeutic intervention.

We propose to use quantitative proteomics approaches to study the interactions between simian immunodeficiency virus (SIV) and methamphetamine (METH) in the central nervous system (CNS) of rhesus monkeys. This proposal builds on our previous works for examining the SIV infected brains of rhesus monkeys for biomarker discovery using transcriptomics (gene array) and metabolomics (mass spectrometry) based technologies. For this project, we will perform hypothesis-based studies building on the wealth of data generated in functional proteomics at the University of Nebraska Medical Center (UNMC, Drs. Howard Gendelman, Project 1 and Pawel Ciborowski, Project 3). Our working hypothesis is that in the presence of METH, the harmful effects of SIV on the CNS are increased: when SIV and METH are joined there are dual and interacting untoward effects on CNS function and neuronal viability. Furthermore, since the proteome is broadly affected during disease, we believe that the use proteomics methodologies in the rhesus monkey/SIV/METH model will enable us to explore distinct and synergistic CNS disease mechanisms. We will utilize both unbiased and directed approaches to study how the effects of SIV on the primate brain are affected by METH. These studies are designed to utilize optimal samples for such discovery and follow-up validation, minimizing experimental confounds and directly studying the targets of SIV and METH, as well as accessible biofluids. In combination with the in vitro systems in H. Gendelman's Project ,1 and clinical samples in P. Ciborowski's Project 3, these nonhuman primate studies will form the necessary bridge between the projects as well as allow us to directly assess, in the most accessible and valid system, the target structures in the brain. Given the growing epidemic of METH use, and its relatively frequent abuse in the HIV infected population, this work is relevant to public health in the US. Knowledge about how METH is toxic to the brain in the setting of HIV infection will be useful not only in discouraging METH use, but in obtaining disease-specific markers that can be used for diagnosis and guiding therapeutic interventions.

AIDS; AIDS neuropathy; Acquired Immune Deficiency; Acquired Immune Deficiency Syndrome; Acquired Immuno-Deficiency Syndrome; Acquired Immunodeficiency Syndrome; Address; Adherence; Adherence (attribute); Advisory Committees; Animal Experimental Use; Animal Experimentation; Animal Husbandry; Animal Research; Animal Rights; Animal Welfare; Animals; Articulation; Arts; Attention; Authorship; Awareness; Awarenesses; BSL-3 facility; BSL3 facility; Biological; Biometrics; Biometry; Biometry and Biostatistics; Biostatistics; Biostatistics Core; Caring; Charge; Chemicals; Clinical; Collaborations; Commit; Communication; Communities; Consultations; Data; Decision Making; Development; Dimensions; Drugs; Educational process of instructing; Employee; Ensure; Environment; Equilibrium; Equipment; Ethics; Evaluation; Exposure to; Feedback; Fostering; Foxes; Funding; Future; Genus - Lotus; Goals; Grant; Guidelines; Hazardous Materials; Hazardous Substances; Head; Hearing; Hour; Human; Human Resources; Human Subject Research; Human, General; Husbandries, Animal; IACUC; Immunologic Deficiency Syndrome, Acquired; Individual; Infectious Agent; Infrastructure; Institutional Animal Care and Use Committee; Instruction; International; Investigators; Joints; Laboratories; Laboratory Infection; Leadership; Lotus; Mails; Man (Taxonomy); Man, Modern; Manpower; Manuscripts; Medical; Medication; Mentors; Methods and Techniques; Methods, Other; Monitor; NIDA; Names; National Institute of Drug Abuse; Nebraska; Neurosciences; Other Resources; Outcome; Participant; Personal Satisfaction; Persons; Pharmaceutic Preparations; Pharmaceutical Preparations; Pharmacology; Phone; Policies; Postdoc; Postdoctoral Fellow; Procedures; Productivity; Programs (PT); Programs [Publication Type]; Proteomics; Publications; Puerto Rico; R01 Mechanism; R01 Program; RPG; Reagent; Real-Time Systems; Recommendation; Reliance; Research; Research Associate; Research Grants; Research Infrastructure; Research Personnel; Research Project Grants; Research Projects; Research Projects, R-Series; Research Resources; Research Specimen; Researchers; Resource Allocation; Resources; Risk; SCHED; Safety; Sampling; Schedule; Science; Scientific Publication; Scientist; Series; Services; Site; Specimen; Structure; Students; System; System, LOINC Axis 4; Systems, Real-Time; Task Forces; Teaching; Techniques; Technology; Telephone; Tetragonolobus; Time; Training; Training and Education; Training of Investigators; Universities; Work; abused drugs; balance; balance function; base; biosafety level 3 facility; career; conference; day; drug of abuse; drug/agent; drugs abused; drugs of abuse; experience; hearing perception; human subject; infectious organism; interest; investigator training; member; neuroAIDS; next generation; occupational health/safety; personnel; post-doc; post-doctoral; programs; quality assurance; response; social; sound perception; statistics/biometry; success; symposium; well-being

The new Phenomics Core of the Scripps NeuroAIDS Preclinical Studies (SNAPS) Center will provide SNAPS investigators and others with cutting-edge analytical support and data access, to address the primary challenges in metabolite and protein profiling, and functional genomics. Identification, quantification, and correlation of the broad range of metabolites and proteins with biochemical and transcriptional events relevant to neuroAIDS and the central nervous system. Meeting this challenge is integral to understanding the progression of disease and the development of diagnostic and treatment tools. Applying state-of-thescience approaches in high density gene expression arrays, global metabolite profiling and comparative proteomic analysis will permit the identification of specific metabolites and proteins associated with neurodegenerative processes and facilitate a deeper understanding of basic problems in neuroAIDS. Affymetrix GeneChip expression arrays and custom array technology will be provided that would otherwise be unavailable or prohibitively expensive. A key component of the Phenomics Core support will be the expansion of bioinformatics and statistics resources through active collaboration with the proposed Systems Biology and Integrative Network Core and the Statistics section of the Administrative Core. This collaborative effort will result in data collection, sharing and analysis capabilities that are simultaneously diverse and integrated, generating a unique resource for neuroAIDS research. The Core will continue providing training and assistance with bioinformatics tools for microarray data analysis, protein-ligand interactions, metabolomics and proteomics, and the management and dissemination of datasets. The ultimate goal of the Prvenomics Core is to provide analytical support in the design and implementation of cutting edge transcriptomics, metabolomics and proteomics focused upon the pathoneurological effects of HIV/AIDS.

The major goal of the Physiology Core of the Scripps NeuroAIDS Preclinical Studies (SNAPS), CSPAR, is to continue to provide neurophysiological assessment of AIDS-related functional pathology using a variety of established and new animal models with clinical phenotypes relevant for neuroAIDS. The Core services will include continued evaluation of current primate and feline models infected with the analogous lentivirus (SIV and FIV, respectively) as well as assessment of existing and new mouse models representing molecularly engineered phenotypes relevant for HIV-1 infection. The Core will provide a broad spectrum of analyses for the detection of functional disease progression and identification of underlying mechanisms. In addition, the Core will work to recruit new SNAPS collaborators who will further enrich the assessment potential of the Core and its contribution to the neuroAIDS field. The Core is organized into two interacting units that carry out specific scientific analyses. The analyses provide sensitive measures of disease progression, functional deficits and underlying mechanisms. The in vivo analysis unit will carry out studies in mouse, primate and feline models of NeuroAIDS utilizing in vivo techniques including extracellular single unit electrophysiological recording, sensory potential recording and radio telemetry. The in vitro analysis unit will use electrophysiological recordings of brain slices from animal models of NeuroAIDS or cultures prepared from these models. The in vitro unit will also carry out studies of these models using live cell measurement of cytosolic calcium levels and anatomical studies of receptor expression and localization. Important strengths of this SNAPS Core include the expertise and knowledge of the faculty and staff, their long-term working relationships, and their commitment to neuroAIDS research.

Adherence to antiviral therapy is a significant issue for HIV-infected drug abusers. The combined studies of lenfivirus-infected rodent and rhesus macaques proposed here build upon exciting preliminary data obtained on nanoformulafions of anfiretrovirals (nanoART) and the data that will result from projects 1 and 2 (A. Kabanov and H. Gendelman). Before proceeding to nonhuman primate studies, rodent testing of nanoART in virus-infected EcoHIV and/or humanized mice. These studies will be conducted with D. Volsky and L. Poluektova. Thus, we will begin in Aim 1 performing rodent biodistribufion (including bioimaging) and pharmacokinefic (PK) tesfing (years 1 and 2, using cores B and C, M. Boska and C. Fletcher) in infected animals as a first pass screen for antiretroviral efficacy and tissue toxicities in vivo (oversight and biostafisfician support through core A, H. Gendelman). We will then proceed in specific aim 2 for PK, drug tissue distribufion, and safety studies in uninfected nonhuman primates (year 3), and then in specific aim 3 perform nanoART evaluafions in SHIV-infected monkeys, including PK, imaging, and efficacy studies, with extensive tissue analyses (years 3-5). These studies will predict whether long-spaced dosing protocols would yield acceptable drug plasma levels in monkeys and whether such developed protocols are efficacious against virus in primates. In toto, studies in the SHIV/rhesus monkey model will yield important information immediately applicable to future human clinical trials. The wealth of data, from pharmacokinefics, organ distribufion, effects on the virus, immune and other physiological systems are crucial pre-clinical steps in the development ofthis excifing therapeufic development.

DESCRIPTION (provided by applicant): HIV-1 infection and drug of abuse have devastating effects on function of the entire organism. The macrophage is the prime member of the mononuclear phagocyte class of cells and a key part of innate immunity system. Because the macrophage is also a target of HIV, a reservoir of productive viral infection and a vehicle to spread infection to organs including the brain, its impact on the course of disease is central. The complexity of HIV infection is further complicated and intensified by use of drugs of abuse. Methamphetamine (METH) was chosen since it is a drug with increasing popularity among the drug-abusing population and used by those with, or at risk for, HIV. Treatment of these individuals is a very complex process because it has to target two entities that are quite different in nature. In addition, life-long cART treatment of HIV infection has adverse toxic effects. As two main avenues of Systems Biology, global profiling techniques and computational processing of large data sets, mature, it becomes feasible to start analyzing data from multivariate experiments (HIV/METH/cART). Prior reductionist approaches precluded performing experiments at this level of complexity. Moreover, despite substantive research efforts, the broad picture of molecular mechanisms underlying functions of macrophages in the complex environment of HIV-1 infection METH use and/or cART is far from being understood. Summarizing, we hypothesize that the systems biology approach will provide unique information which will lead to identification of new paradigm how the human macrophage is regulated in the complex environment of HIV infection, cART and METH. We expect that our experimental plan, examining transcription factors and other nuclear proteins through the use of omic techniques, computational biology and bioinformatic analyses, will provide unique information which will lead to identification of new paradigms in how the human macrophage is regulated in the complex environment of HIV infection, cART and METH. PUBLIC HEALTH RELEVANCE: The transformation of HIV to a chronic disease represents a great success of therapy but has opened up a number of new clinical problems due to the long-term infection, effects of the anti-retroviral therapies, and coexisting factors such as drug abuse which affects many systems including the brain and cardiovascular systems. Macrophages, key component of immune system, play many roles in persistence of infection as well as disease causation. We propose to use a global approach to investigate the effects of HIV infection, drug abuse and anti-retroviral therapy on macrophage which should lead us to better understand molecular mechanisms of disease and propose new targets for therapy.

DESCRIPTION (provided by applicant): Our overall goal is to understand the means by which methamphetamine (Meth) induces functional immune system abnormalities, how these changes alter the response to viral infection, and how the unique pattern of T cell dysfunction resulting from Meth leads to defective immunity to HIV with increased systemic and central nervous system (CNS) disease. Our experiments comprise five specific aims, working systematically and progressively through basic mechanisms in rodents to highly translatable experiments in nonhuman primates. We will learn new information regarding the regulation of the immune system by Meth and its modulation of the host response to HIV. In addition to in vivo studies, we will perform quantitative phosphoproteomics of the signaling in T cells to identify the molecular mechanism whereby Meth alters CD8 T cell function. These combined studies will reveal mechanistic crosscurrents in complementary systems modeling Meth abuse and HIV infection in humans, allowing the identification of the networks of cells and signaling events which are perturbed due to the intersection of Meth and HIV. PUBLIC HEALTH RELEVANCE: The pandemics of HIV infection and Methamphetamine abuse continue to devastate individuals, and they are increasingly seen in combination. We have found a combined effect of both HIV and Meth on the immune system as well as the brain. Our studies are designed to uncover the mechanisms of their damaging effects, leading to preventative and therapeutic measures.

DESCRIPTION (provided by applicant): The now chronic nature of HIV has been a major advancement in AIDS care, but as with any chronic condition brings with it the recognition of other sequelae, including the HIV-associated neurocognitive disorder (HAND). In addition, as a natural consequence of the chronicity of infection and reduced new infection, the face of HIV infection is changing to an older population;soon over half of infected individuals will be 50 years of age or greater. As detailed in our Research Plan, we will now take the lessons others and we have learned about HIV and the brain and apply it to the changing epidemic - the graying of HIV, the nature of the effects on the brain of the chronic infection, means to treat HIV in the brain, and potential interaction with conditions linked to aging and other neurodegenerative disorders. We will set about this through the Chronic HIV Infection and Aging in NeuroAIDS (CHAIN) Center as described in this proposal. We will accomplish four Action Items novel Biomarkers, the Role of Macrophages CNS Penetrating Antiviral Therapeutics, and the Effects on Chronic CNS Infection on Neurons, and support our investigators'research efforts through our Cores (Administrative, Biophysical Assessment Cells/Tissues/Animals, Developmental, International, Phenomics, Systems Biology, and Therapeutics). Our goals driven by our framework: researching of the chronicity of HIV infection in the CNS with a systems biology approach.

The mission of the CHAIN Center is to provide leadership and resources to facilitate multidisciplinary, state-of-the-science research on neuroAIDS and mental health. The Administrative Core will continue to improve and expand research efforts by leading, supen/ising and coordinating the actions of the Center; monitoring and evaluating the work of all Cores; facilitating collaborative efforts; and organizing and implementing input and recommendations from the Advisors. The Core will continue its responsibility for maintaining the Center's focus on contemporary issues in neuroAIDS and mental health while sustaining an environment in which innovative and significant work is pert'ormed. Specifically, the Administrative Core will has been successful in achieving our following Aims. Given these successes and our relatively short time of operation many of the aims are still germane, and our modifications of these aims for the renewal are indicated. Space prohibits detailed descriptions, and the majority ofthe documentation of our Accomplishments can be found in the Research Plan and evidenced by our publications.

Project Summary/Abstract The Data Coordinating Center (DCC) supports the National NeuroAIDS Tissue Consortium (NNTC). The consortium is composed of four NNTC Clinical Sites and the DCC that work cooperatively as a resource for the research community, providing HIV/AIDS investigators with clinically annotated datasets of antemortem information and associated postmortem tissue and fluids. The DCC works cooperatively with the NNTC Clinical Sites to provide (1) management and database capabilities to ensure effective clinical and brain banking operations; (2) scientific expertise in biostatistics and HIV epidemiology to support broad analyses of the NNTC clinical database and analyses that support the recruitment and retention goals for the cohort; and (3) identify gaps and provide solutions to neuroAIDS investigators. The DCC serves as the point of contact for users of the resource (tissue and/or data requests) and services the requests from the time they are submitted through fulfillment of the request and follow up with the requestor to provide the results of their research. The DCC expanded to include the CHARTER study bringing in longitudinal participants and ancillary studies. In this next funding period, the Global CERCH will be developed to enable collaborations and the sharing of specimens and data relating to CSF escape, expanding the DCC's ability to support ongoing neuroAIDS research. The DCC websites are used extensively for dissemination of resource information (query tools, reports, request applications) and management tasks such as resource tracking, document libraries, and communication activities. The DCC provides a central inventory of NNTC data and specimens. Along with this inventory, additional research data such as bioinformatics datasets by the users of the resource are available. The DCC promotes the NNTC through public relations materials including a comprehensive website and meeting presentations. In the past funding period, the NNTC supported 356 separate research grants and 157 peer- reviewed publications.

? DESCRIPTION (provided by applicant): Opiate abuse and HIV-1 have been described as two linked global health crises, and despite the advent of anti-retroviral therapy, abuse of opiates has been shown to result in increased neurologic and cognitive deficits. Using the morphine-dependent rhesus macaques (RMs) infected with CCR5-utilizing SIVR71/17E we recapitulated the human syndrome demonstrating augmentation of neuropathology & neuroinflammation and rapid disease progression compared with SIV-infected RMs without opiate dependence. Mortality in these rapid progressors was associated with robust microglial activation and neuronal injury. MicroRNA (miR)- mediated regulation of disease pathogenesis represents an evolving area of research that has ramifications for identification of potential therapeutic target for various neurodegenerative disorders for which currently there exists no cure. Herein we hypothesize that morphine & HIV Tat modulate increased neuropathology via two complementary mechanisms: a) Morphine exposed astrocytes upregulate expression & release of miR-138 in the extracellular vesicles (EVs), which, following uptake by the microglia, results in their activation via the TLR7-dependent pathway and, b) HIV Tat exposed astrocytes upregulate expression & release of miR-9 in the EVs, that are taken up by the microglia, leading in turn, to increased microglial migration. Two experienced PIs will join on this project to investigate this hypothesis. The innovative aspects of this proposal are based on our unique observation that in chronically (SIV)-infected rhesus macaques, morphine mediated potentiation of neuropathogenesis correlates with dysregulation of miRs, with specific upregulation of miR-138 & miR-9 in the post mortem brain tissues from SIV and morphine-dependent macaques, respectively. These experiments will be brought full circle with the examination of functional studies to examine the mechanisms uncovered in a rodent model of HIV Tat/morphine exposure. This proposal is responsive to the RFA, focusing on release of EVs in HIV/AIDS and including a drug of abuse (morphine).

? DESCRIPTION: HIV-associated neurocognitive disorders (HAND), varying in severity from an asymptomatic to mild neurocognitive impairment to in its most serious form a debilitating dementia, develop in a subset of individuals infected with HIV-1. HAND results from an indirect neurotoxicity, as HIV infects macrophages and microglia, but not neurons, in the brain. The molecular mechanisms underlying neurotoxicity by HIV-1 infection in the brain are still largely unknown. Our studies and those of others have discovered a class of regulatory RNAs, microRNAs (miRNA) that are dysregulated in HIV-1 associated neurological disease. Recent findings indicate that miRNAs can be carried in extracellular vesicles such as exosomes, which have lately emerged as important mediators of cell-to-cell communication in the brain. Exosomes can release their cargo into target cells and trigger downstream signaling pathways. We are particularly interested in understanding the effect of such exosome-carried miRNAs on neurons. In particular, we will study miR-21. We have previously identified that miR-21 is significantly upregulated during SIV/HIV infection in the brain. We now find it is present within macrophages in the infected brain, and our in vitro studies reveal both human and mouse macrophages release miR-21 in exosomes. These miRNAs had a G/U rich region, capable of activating TLR7/TLR8. We believe that miR-21 and similar miRNAs are potential neurotoxic factors and are specifically released during HIV-1 induced insult to the brain. Here we hypothesize to study exosomal miR-21 and its effect on neurons in SIV/HIV-1 infection. These studies will be done in two specific aims; (1) In specific aim 1 we will utilize a robust strategy o isolate exosomes form SIV and HIV-1 infected brain tissue, characterize them, and determine SIV/HIV induced alterations in exosomal miR-21. (2) Specific aim 2 is specifically designed identify induction of neuro-injurious molecular signaling pathways during SIV/HIV-1 infection by exosomal miR-21. We will examine whether exosomal miR-21 activates toll like receptors and which downstream signaling pathways can harm neuronal health. These experiments will expand our knowledge on understanding the mechanisms accounting for the exacerbated neuronal damage during HIV-infection of the brain and therefore build a strong ground to build further therapeutic studies for the prevention of long-term neuronal damage in HAND.

Core C Abstract: Long-acting nanoformulated antiretroviral therapy (nanoART) is poised for bench to bedside clinical translation. However, hurdles remain to see nanoART as a significant part of the therapeutic armamentarium. This includes, but is not limited to, evidence for improved dosing, tissue biodistribution and pharmacodynamics (PD). All require support from readily available and affordable rodent models of human immunodeficiency virus (HIV)-infection for PD and pharmacokinetic (PK) and antiretroviral efficacy tests as performed on non- human primates. To these ends, the Core C animal model core will provide humanized mice for efficacy evaluation and rhesus macaques for PK and tissue distribution testing and pilot efficacy studies. The purpose is to assist researchers with animal experiments (Projects 2 and 3 and Core B). These will include assessments of viral and immune parameters, tissue dissections and when relevant, biopsies of lymph nodes and rectal tissues. All will be processed, in collaborative experiments, for molecular, virologic and biochemical studies. The goal of the humanized mice component is to assess direct antiretroviral efficacy and HIV eradication experiments of selected and drug formulation combinations. Based on the program needs specific formulations will contain GSK744 and GSK8232, an integrase and maturation inhibitor, respectively, or mixtures of both. Alternatively, prodrugs of 3TC, abacavir, maraviroc, and ritonavir-boosted atazanavir will be used, in part, as combination therapy. These nanoformulations or their combinations will be tested for their abilities to reduce residual virus or eradicate HIV when administered with indoleamine 2, 3-dioxygenase inhibitors in platform development. Humanized mice will be employed for metabolomics and molecular imaging studies in order to provide rapid assessments of drug success or failures as they can assess tissue homeostasis and predict end organ disease. Non-human primates will be made available, specifically for drug PK, tissue distribution and toxicological studies. Tissue distribution will be assessed in bioimaging experiments (Project 3) by ligand-targeted magnetite nanoformulations and linked back to PK and PD tests as contained in Projects 1, 2 and Core B.

Abstract: HIV-1 infection and drug of abuse have devastating effects on function of the entire organism. The macrophage is the prime member of the mononuclear phagocyte class of cells and a key part of innate immunity system. Because the macrophage is also a target of HIV, a reservoir of productive viral infection and a vehicle to spread infection to many organs, its impact on the course of disease is central. The complexity of HIV infection is further complicated and intensified by use of drugs of abuse. Methamphetamine (Meth) was chosen since it is a drug with increasing popularity among the drug-abusing population and used by those with, or at risk for HIV. Treatment of these individuals is a very complex process because it has to target two entities that are quite different in nature. As two main avenues of Systems Biology, global profiling techniques and computational processing of large data sets, mature, it becomes feasible to start analyzing data from multivariate experiments. Prior reductionist approaches precluded performing experiments at this level of complexity. Moreover, despite substantive research efforts, the broad picture of molecular mechanisms underlying functions of macrophages in the complex environment of HIV-1 infection and/or Meth use is far from being understood. Summarizing, we hypothesize that merging proteomics, metabolomics and computational analyses into a comprehensive systems biology approach to investigate regulatory mechanisms will provide unique information that will lead to the identification of new paradigms on how the MP responds to the complex environment of HIV infection and/or Meth insult. We expect that targeted profiling, computational biology, and bioinformatic analyses will uncover new and unreported mechanisms of MP regulation and will model functional networks that can be validated by focused and targeted experiments as they exist in ex vivo material from non-human primates. Better understanding of the epigenetic regulation of MP will enable us to define weak points of the major target cell under insult of viral infection and Meth. Three specific aims are proposed: 1. To characterize the networks of epigenetic factors in human monocyte derived macrophage (MDM) induced by HIV infection and/or Meth. 2. To characterize changes in intra- and extracellular metabolomes of MDM induced by HIV infection and/or Meth treatment resulting from alterations in regulatory functions of transcription factors and regulators (TF&R) and 3. To validate integrated complex proteomic and metabolomic data in ex vivo human samples to build new paradigms of how HIV and/or Meth regulate the MP.

Pilot Projects Program offers a rapid mechanism to test the most promising ideas and novel clinical and translational research (CTR) projects and help investigators obtain the preliminary data necessary for successful investigator-initiated grants. The overall objective of this KCA is to foster the development of successful CTR investigators; drive the development of new, diverse CTR teams; develop new tools and technologies that will support remote conduct of CTR or improve health outcomes; and address the diverse causes of health disparities to change health outcomes. The four overall aims of the CTR Pilot Projects Program KCA are to 1) administer a Collaborative CTR Pilot Projects Program that spurs the most innovative and collaborative CTR approaches and teams, 2) develop successful extramurally funded CTR investigators with the IDeA-CTR Scholar Grant Program, 3) review and prioritize requests to use clinical research resources through the Clinical Research Resources Grant Program, and 4) educate potential CTR investigators about the grant review process with a Mock Study Section Program in collaboration with the Professional Development KCA.

Abstract Despite effective treatments for HIV, the presence of viral reservoirs has prevented the development of a cure for HIV infection. Although most reservoir work has focused on CD4+ T cells, these are not the primary infected cells in the CNS instead long-lived macrophages and microglia are infected. Furthermore, many of the anti-retroviral treatments do not penetrate the brain. In addition, drug abuse is frequently co-morbid with HIV infection and affects CNS virus and disease. Studying the state of virus in the brain, as well as doing controlled experiments with drugs of abuse, cannot be performed in humans. Here we will use the highly relevant SIV- infected macaque model treated with an effective anti-retroviral treatment regimen in three groups: those receiving the stimulant methamphetamine, those receiving the opiate morphine and those receiving no drugs of abuse. Our experimental design will test the hypothesis that the brain is a reservoir under controlled treatment regimens and determine the effects drugs of abuse have on such a reservoir. In parallel, we will examine a known reservoir (lymph node CD4+ T cells), both as a comparison and to illuminate additional effects of drugs of abuse on reservoirs. Specific Aim 1 will examine the effects of drugs of abuse on modulating the effectiveness of antiretroviral treatment in SIV infected macaques. Innovative aspects include our hypothesis based mechanistic studies examining changes in cellular markers that may impact infection and tissue migration, and our analyses of intracellular antiretroviral concentrations uniquely performed here in brain cells as well as in lymphoid cells in the setting of drugs of abuse. Specific Aim 2 will utilize a series of state of the art assays to examine the hypotheses that under suppressive anti-retroviral treatment, the brain is indeed a viral reservoir for SIV and substance abuse increases the size of this reservoir. We will utilize PCR detection, viral outgrowth in indicator cells and the highly sensitive TILDA assays to examine virus in microglia/macrophages purified from the brains of the animals in Aim 1. To ensure a definite result with high sensitivity and specificity, we will adoptively transfer infection to naïve monkeys as a new platinum standard for reservoir detection to definitely address this question of the CNS as a reservoir and the effects of drugs of abuse. Such knowledge is crucial for devising strategies to cure HIV infection, including in drug abusers.

Administrative Core Abstract: The Administrative Core is committed to carrying out the mission of the CHAIN Center by providing the leadership and organizational framework to coordinate and facilitate multidisciplinary, state-of-the-science research on HIV infection of the CNS. Over the past five years, the CHAIN leadership took heed of the recommendations of the Advisory Committees and feedback from the NIH. This grant successfully propelled exciting new multidisciplinary research in the realm of HIV and aging resulting in the publication of 226 papers (many of high impact) and procurement of over $18.5 million in HIV-related extramural funding from the NIH. The Core has established and maintained open lines of communication and new collaborations both within and outside the Center and has actively contributed to the field by advancing scientific exchange on emerging contemporary topics through its highly anticipated annual colloquium that are broadcast globally. The Administrative Core will provide administration, oversight, coordination and management for the operation of the Center. Core leaders will lead, supervise and coordinate the actions of the Center and also monitor and evaluate the work of the cores and investigators. The Core will facilitate collaborative efforts and implement input and recommendations from the internal and external advisors. The Core will continue its responsibility for maintaining the Center?s focus on HIV, the CNS and aging while sustaining an environment in which innovative and significant work is performed, particularly in light of the NIH priorities. The Core will provide accountability for the research performed and funds used in support of the work. Integrated into the Core is a Biostatistics subcore, providing key expertise in research design and analysis, furthering our rigor and transparency. The Administrative Core is committed to creating and maintaining a stimulating intellectual environment within UNMC and between Center investigators and the field, providing a research and administrative infrastructure to maximize interdisciplinary research in neuroAIDS that is focused on NIH high priority areas of including aging, inflammation, cure/reservoir work, comorbidities and sex/gender differences, and poised to support investigations into new and emerging areas in the field.

Project Summary/Abstract HIV-infected adults in the western world have a life expectancy near that of the general population, but are at a significantly elevated risk of developing cognitive deficits. Such impairments are the most common neurological complication of HIV disease, with prevalence estimates ranging from 35-70% of all HIV-infected individuals, and research targeting such comorbidities has been identified as a top priority by the Office of AIDS Research (NOT-OD-15-137). Substance use disorders (SUD) are also more prevalent in the HIV-infected population, yet their relative contribution to the increased rate of cognitive impairment in this group remains poorly understood. A key barrier to progress in this area has been the historic lack of diagnostic tests and biomarkers that can precisely assess the neurological complications of HIV-infection, which has all but precluded quantification of the additive impact of SUD comorbidity. This barrier is central to RFA-DA-18-023, which requests applications that ?foster biomarker and signature identification that could advance the clinical assessment of the degree of deterioration or damage, of functional reserve, and of resilience of host defense mechanisms, towards HIV- infection and comorbidity of HIV with SUDs.? Specifically, the RFA requests proposals that identify biomarkers derived from blood, plasma, noninvasive neuroimaging modalities, and/or other sources, with an emphasis on biomarkers that can be quantitated, with levels assessed for their ability to predict disease progression. The current project directly targets the scientific gaps identified in this RFA, Identification of Biomarkers of HIV Pathogenesis and Substance Abuse Comorbidity, using a multipronged approach that includes state-of-the-art neurophysiological, structural, and spectroscopic noninvasive imaging, cognitive assessment, and cellular and molecular analyses of blood/plasma in the context of cannabis use disorder (CUD). Specifically, the Signatures of Cannabis Abuse in NeuroHIV (SCAN) Consortium will utilize advanced magnetoencephalographic (MEG) imaging to quantify the neural dynamics serving cognitive processing, 3-Tesla MRI and multimodal parcellation methods to map brain architecture, functional MRI (fMRI) for hemodynamics and intrinsic networks, and 7-Tesla spectroscopic imaging to quantify local GABA levels across the brain. These data will be integrated with a comprehensive molecular screen that includes 35 plasma biomarkers covering the immune, inflammatory, coagulation, endothelial, and neurological systems, a 10-color flow cytometry panel delineating CD4 and CD8 T cells, B cells, NK cells, monocyte subsets, and activation markers, known clinical markers, and mitochondrial function in immune cells using the Seahorse Analyzer method. To enhance rigor, demographically-matched groups of uninfected controls with and without CUD will be enrolled, which will enable the interaction of HIV and CUD to be quantified. In sum, to truly meet the goals of this RFA, we will use a broad range of neuroimaging, molecular, and cellular functional assays to uncover biomarker signatures of HIV and CUD that will enable identification of early dysfunction, progression, and prognosis, enabling future preventative and treatment trials.

Project Summary An innovative approach is proposed to determine the Role of Myeloid Cells in Persistence and Eradication of HIV-1 Reservoirs from the Brain, in response to RFA-MH-20-702. We will perform using the gold-standard nonhuman primate system, in SIV-infected animals with chronic viral suppression using combination antiretroviral therapy. The infected cells in the brain will be uncovered by intra-CNS anti-CD8 treatment, following subsequently by myeloid cell isolation and purification, single-cell RNA sequencing (scRNA-seq) and single-cell Assay for Transposase-Accessible Chromatin sequencing (scATAC-seq), followed by bioinformatic analysis and validation. This will give result into unparalleled insight into the nature of the myeloid cells in which SIV, as a model for HIV, persists in the brain, combining individual cellular transcriptomes with epigenetic information on the whole genome. These will be done in a new paired format, obtaining linked information on the two modalities from the same cell. combined with sensitive assays for virus in the brain. This will take place in the context of a two-year R21 under two Specific Aims. In Aim 1, we will uncover the SIV-infected cells in the brain in the presence of suppressive cART, allowing their identification and, in comparison with the information on the companion uninfected cells, as well as known data on brain myeloid cells from single cell and other studies, the phenotype of brain myeloid cells in which SIV/HIV can persist. In Aim 2, in-depth molecular characterization of these cells will be performed, yielding additional information on their phenotype, as well as upstream control mechanisms, transcriptional potential, as well as relationships to genes and their control regions that affect HIV, CNS disease, and other conditions. We will ensure our data adhere to the Findable, Accessible, Interoperable, and Reusable (FAIR) standards in order that we and others can utilize this information in future strategies to silence or clear virus from the brain. These studies are designed with rigor and reproducibility factored in, thus the results from these studies will provide new insights into the role of myeloid cells in persistence and eradication of HIV-1 reservoirs from the brain.

Project Summary A comprehensive interdisciplinary approach proposed to support the Single Cell Opioid Responses in the Context of HIV (SCORCH) Program Expansion: CNS Data Generation for Chronic Opioid, Methamphetamine, and/or Cocaine Exposures RFA from the National Institute on Drug Abuse (NIDA). We will perform studies on the interplay of HIV pathogenesis and opioid abuse in the gold-standard SIV/nonhuman primate system, using single-cell RNA sequencing (scRNAseq) to assess individual cellular transcriptomes. This will give us obtain unparalleled insight into the viral and drug-induced effects on diverse cell types in brain in regions critical for HIV infection and opioid abuse. In addition, we will also focus on primary targets of HIV and drivers of neuropathogenesis, microglia and brain macrophages, using scRNAseq as well as single-cell Assay for Transposase-Accessible Chromatin sequencing (scATACseq) to gain insight into the epigenetic regulation of gene expression at the single cell level. These will be combined with state-of-the-art sensitive assays for virus in the brain. We will ensure our data adhere to the FAIR (Findable, Accessible, Interoperable, and Reusable) standards, and will work closely with other members of the SCORCH program to share and disseminate data to yield maximal advancement to the field. Our experiments are carefully designed to address the role of virus, drugs, and their interactions, as well as include the clinically relevant scenarios of those taking antiretrovirals to suppress virus, and those who do not. Our rigor and reproducibility are facilitated by our detailed power analysis with proper sample sizes, as well as our experimental design to match the standards of blocking, replication and randomization. The results from these studies will provide novel insights into the CNS effects of HIV and drug abuse.