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.

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.

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.

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.

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.

[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]

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.

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.

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.

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.

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 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.

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.

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).

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.

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.

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 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.