Ronald Ellis - US grants

AIDS /HIV neuropathy; methamphetamine; cerebrospinal fluid; drug abuse; biomarker; HIV infections; neurochemistry; macrophage inflammatory proteins; neurotoxins; cytokine receptors; tumor necrosis factor alpha; clinical research; human subject;

The biological, medical and social consequences of HIV infection of the central nervous system (CNS) include neurodegeneration, disability from neurocognitive disorders, increased mortality, and decreased quality of life. The CNS may serve as reservoir and sanctuary for HIV because the blood-brain barrier shields infection from certain immunologic and pharmacologic defenses. Recently, quantitative assays for HIV RNA and chemokines have been developed that promise to enhance our understanding of HIV neuropathogenesis and to facilitate the diagnosis and treatment of HIV CNS infection. This proposal outlines an integrated series of clinical and laboratory investigations to assess the role of the CNS in the development of antiretroviral (ARV) resistance and to evaluate strategies for therapeutic intervention in humans through the study of cerebrospinal fluid (CSF). Three clinical studies form the core of this 5-year proposal. Study 1A is an open-label trial of individualized combination ARV therapy (ART) for HIV neurocognitive disorder. The principal objective is to correlate improvements in neurocognitive function with CSF virologic (HIV RNA) suppression and CSF chemokine changes during ART. Eighty participants with neurocognitive disorders will be enrolled for a total of 155 treatment-observation periods of 12-weeks each. Study 1B is a controlled comparison with virologic responses in CSF and plasma with ART in individuals without neurocognitive disorders. Its main goal is to compare rates of change in CSF and plasma HIV RNA levels (HIV dynamics) and chemokines during ART, and to evaluate determinants of virologic efficacy and failure in the CSF. Fifty neurocognitively normal participants will be enrolled for 80 treatment-observation periods of 12- weeks each. Studies 1A and 1B have a parallel design in which participants are randomized to one of two treatment strategies (plasma versus plasma+CSF) based on the use of phenotypic ARV resistance testing. Study 2 compares virologic and immunologic (chemokine) responses in CSF and plasma during the withdrawal of antiretroviral treatment ("structured treatment interruption") in 2o subjects. Its goal is to determine the nature and extent of compartmentalization of CSF responses. Intensive laboratory investigations of HIV genetics , chemokines and chemokine receptors genotypes will be applied to the fluid samples derived from these studies. Performance of the work proposed here will benefit from this grant's relationship to the HIV Neurobehavioral Research Center and to the infrastructure that the HNRC provides in neurocognitive assessment, participant recruitment, data management, statistics and tissue archiving.

[unreadable] DESCRIPTION (provided by applicant): HIV-1 causes neurocognitive and neurological disorders that represent a public health problem as they result in a substantial burden to patients living with HIV infection, their caregivers, and the healthcare system. A recent report suggests that clade C viruses may possess a reduced capacity for neuroinvasion because clade C isolates bear tat sequences coding for a variant Tat protein shown in vitro to be defective in monocyte chemotaxis as compared to clade B Tat. The defective Tat chemokine motif is hypothesized to be clade C-specific based on isolates from several countries and could explain the lower prevalence of HIV brain involvement reported in some of these countries. However, no study has directly compared neurocognitive function among individuals infected with HIV-1 clades C and B. Thus, lower reported rates of dementia in regions where clade C is prevalent may result from ascertainment bias, differential prevalence of co-infections or other factors. This application proposes to perform a cross-sectional comparison of the influence of HIV-1 clade on neurocognitive and neurological disorders in southern Brazil. UCSD investigators, who have extensive experience in NeuroAIDS clinical research, will support investigators in Curitiba, Parana, Brazil, who care for large numbers of patients infected with either clade C or B. In this setting, clade-specific effects can be compared while controlling for potential confounding factors such as ethnicity, sociodemographic characteristics, risk factors for HIV infection and co-infections. UCSD investigators will train and guide the administration and interpretation of the clinical assessment instruments, transfer technology to the Brazilian site. [unreadable] [unreadable]

In the past twenty years, the fields of Evolutionary and Developmental Biology have been revolutionized by the discovery of regulatory genes that are conserved in all animals. These discoveries established the universal nature of many aspects of development, but also revealed profound differences in the developmental machinery. Thus, two crucial questions today are: (1) what aspects of these regulatory pathways can change to create new traits during evolution, and (2) how does the structure of these pathways influence the process of evolution?
Intellectual Merit: The best way to address these questions is by comparing regulatory pathways from closely related species. Sexual characteristics make ideal subjects, since they are among the most rapidly evolving traits, and of interest to many biologists. One of the most dramatic differences in sexual development occurs in the elegans group of nematodes, a clade within the genus Caenorhabditis that contains some species with self-fertile hermaphrodites, and others with normal females. Because C. elegans is a leading model for the study of sex-determination, a wealth of data exists that can aid in the study of mating systems in this group. Recent results suggest that hermaphroditism evolved independently in C. elegans and C. briggsae. To test this hypothesis, the regulation of hermaphroditism in these two species must be compared at the level of gene regulatory pathways. In addition, these regulatory pathways have to be compared with those operating in a closely related male/ female species.
Aim #1. In a pilot screen for mutations that prevent hermaphrodite development in C. briggsae, Dr. Ellis found a single allele of the glf-1 gene. These mutants reproduce as a male/ female population, so glf-1 is required for XX animals to become self-fertile hermaphrodites. Mutations like this one provide the key to understanding hermaphrodite development in C. briggsae, and comparing it with C. elegans. Thus, he will continue this screen to saturation, to find additional alleles of glf-1, and alleles of other genes that control hermaphrodite development. He will backcross each mutation, assign recessive alleles to complementation groups, map each gene, and analyze double mutants to see where each gene acts in the regulatory pathway.
Aim #2. The ancestor of these nematodes was probably a male/ female species. To learn how C. elegans and C. briggsae evolved self-fertile hermaphroditism, it is necessary to learn how sex-determination worked in this ancestor. Thus, this aim involves the genetic characterization of how tra-1, the key sex-determination gene in C. elegans, regulates germ cell fates in the male/ female species C. remanei. In addition, Dr. Ellis has produced XX C. remanei that make sperm and oocytes. Surprisingly, their sperm do not activate in vivo. He will study the requirements for sperm activation in these animals, to test the model that hermaphroditism required two steps to evolve: the production of XX sperm, and the activation of these sperm to allow self-fertilization.
Broader Impacts: Recruiting minority students into the Biological Sciences, and providing them with the background and training needed to excel, are as important in South Jersey as in any part of the country. However, despite the existence of the Rutgers-Camden and Rowan campuses, there are few local opportunities to expose talented Undergraduates to cutting-edge research. This proposal describes an innovative Summer Research program for Undergraduates that should address this problem. It features an intensive week of full-time lectures and computer exercises designed to bring the students up to speed in Classical, Molecular and Population genetics, with examples that focus on the interplay between human medicine and basic research on model organisms. The next ten weeks are devoted to research projects in one of the basic science or medical laboratories at the UMDNJ. Finally, the experience closes with workshops on presenting scientific research, and talks by each of the students.

Advances in antiretroviral therapy have reduced but not eliminated mortality and neurologic morbidity related to HIV infection. The epidemic has been transformed into a chronic disease in which there is a dynamic equilibrium between mechanisms maintaining viral persistence and host immune strategies that, in combination with antiretroviral therapy, hold viral activity in check. Prospects for eradication or cure are many years or decades away. Looking toward the future, the HNRC perceives four general areas in which research needs are greatest. In the renewal period, the Neuromedical Core (NMC) will therefore develop and mature its research infrastructure to support studies addressing the themes of aging, acute and early HIV infection and clinical relevance. A common thread linking these three themes is genetics-both host and viral. The newly-organized NMC is made up of two cooperative units comprising common resources (equipment, supplies) and staff that interact on a daily basis: a Clinical Research Support Unit (CRSU); and a Laboratory, Pharmacology & Biomarker Unit (LPBU). Together these units will perform evaluations on all HIV+ subjects and HIV- controls, including those enrolled in pilot studies of the Developmental Core. The over-arching objectives of the Neuromedical Core (NMC) are to provide comprehensive clinical and laboratory evaluations, scientific consultation, and mentorship, training, and community education in NeuroAIDS. In the current funding period the NMC facilitated 70 research projects including observational cohort studies, clinical-pathological investigations, imaging studies, clinical trials and international research performed by collaborating investigators at UCSD and many other institutions. By assisting investigators in widely diverging disciplines at all levels of project development, from conceptualization of hypotheses to study implementation, analysis, interpretation and dissemination, the NMC encourages the application of multiple scientific perspectives and stimulates interdisciplinary collaboration and coordination. Through a variety of flexible mechanisms including face-to-face meetings and video teleconferences we will collaborate closely with the Neuroimaging, Neurobehavioral, Neurovirology, Neurobiology, Developmental and International Cores to further the HNRC's transdisciplinary aims.

DESCRIPTION (provided by applicant): HIV creates complex genetic populations within a host, and remains latent in a variety of cell types and tissues under antiretroviral therapy, representing a barrier to therapeutic cure. Yet little is known about the relative sizes of latent viral reservoirs in different tissues such as the central nervous system (CNS), and even less about exchange between viral subpopulations in different tissues over time. Deep sequencing technologies allow extensive sampling of these populations. Previous studies have been hampered by limited sampling of viral diversity in the CNS and because they studied only single or occasionally multiple, sparse time points. Designing workable viral eradication strategies will require a characterizing the CNS viral reservoir and its population dynamics with higher resolution. To address these gaps in knowledge, we will apply recently developed techniques, digital PCR (dPCR) and ultra deep sequencing (UDS), to quantify tissue reservoir size and the dynamics of viral subpopulations in archived, longitudinal samples of cerebrospinal fluid cells and supernatant and peripheral blood mononuclear cells and plasma. Here we will evaluate viral subpopulation interactions occurring after antiretroviral treatment interruption, which provides a substantial perturbation of steady state viral and cellular dynamics to anchor the observations. We will apply phylogenetic methods to viral population sequences from CNS and blood over time to assess population dynamics. The study sample comprises a unique, densely serially sampled group of 10 HIV+ subjects undergoing antiretroviral (ART) treatment interruption or viral rebound in the face of continued ART. The project will generate approximately 1.2 million viral sequences. These will be analyzed to delineate how the CNS viral reservoir interacts dynamically with other viral subpopulations and contributes to viral diversification and pathogenicity. The project also will measure the size of CNS viral reservoirs. After de-identification of human subjects, viral sequences will be posted as appropriate on public scientific data resource utilities and software generated for this project will be made publicly available.

NEUROASSESSMENT CORE PROJECT SUMMARY/ABSTRACT: The newly reorganized, streamlined NeuroAssessment Core (NAC) will have the overall goal of supporting comprehensive neuromedical, neurobehavioral and neuroimaging assessments for the characterization of HIV+ individuals and HIV- controls in HNRC-affiliated studies. The Core will comprise 3 integrated subunits: Neuromedical (NM), Neurobehavioral (NBh) and Neuroimaging (NI). We aim to facilitate and enhance multidisciplinary research in neuroAIDS by providing a range of services, training and scientific consultation to projects such as investigator-driven R01s and R21s, international neuroAIDS studies, and a variety of intra- and extramural investigations. Historically the Core and its investigators have been leaders in the field, spurring the development of new methods and adapting to the continually evolving scientific landscape. Consistent with this, the Core will provide resources to support the Center's new themes of HIV persistence and cure, and how the human gut microbiome interacts with HIV and its treatment to affect the CNS. Specific services relevant to HIV persistence and cure include serial lumbar punctures to evaluate viral rebound following HIV reservoir interventions, and high volume CSF collection and neurocognitive (NC) testing to assess effects of such interventions on brain functioning. The gut microbiome will be characterized by collecting and processing samples of stool. Standard evaluations will continue to include a structured neuromedical history, clinical and laboratory examinations, and establishment of diagnoses of central and peripheral neurological complications of HIV. The NAC's NI Unit will provide resources, including training, consultation, and technical support, for the detection and measurement of HIV- related brain injury using a broad array of techniques, including structural, functional, and metabolite MRI and PET imaging. This work will support investigations of the cognitive consequences and neuromedical correlates of these effects; the underlying pathological mechanisms that give rise to this damage; and potential biomarkers for treatment success. The NBh Unit will perform a comprehensive assessment of all participants in the HNRC cohort that will include well-validated measures of NC functioning, psychiatric status, and everyday functioning, in accordance with the HAND diagnostic guidelines. The NBh Unit's specific Resource Objectives are to provide: 1) demographically-adjusted individual, domain, and global summary scores of NC functioning; 2) Determinations of NC change in individual participants using reliable and validated actuarial methods; 3) Self-assessment and laboratory-based measures of everyday functioning; 4) Current and lifetime history of mood and substance use diagnoses; and 5) Technical assistance, training, certification, consultation, and career development mentoring. Data generated by the NAC range from NC performance and diagnostic formulations to ART information, disease staging and viral loads (VLs), to clinical laboratory measurements, medical histories, and physical examinations.

Central nervous system (CNS) complications are common among people with HIV (PWH), even those who are taking antiretroviral therapy (ART). The spectrum of CNS complications is broad, ranging from mild cognitive deficits that do not affect daily functioning to life-threatening encephalitis. Cognitive and mood disorders are among the most common CNS diseases that affect PWH and share a common risk factor, inflammation. Inflammation persists in effectively treated PWH for multiple reasons, including production of HIV RNA and proteins and gut dysbiosis and microbial translocation. CHARTER is a multisite, U.S.-based, neuroHIV cohort study that is funded by NIH and that has investigated CNS disorders in PWH for nearly two decades, during which it has completed more than 6,000 assessments generating more than 10 million data points. CHARTER has made important contributions to understanding the frequency, risk factors, and pathogenesis of these disorders. In recent years, new questions have arisen about the heterogeneity, biological basis, clinical impact, and management of CNS disorders. This debate has highlighted the need to create new classifications of CNS disorders in PWH that are more biology-based. We propose to use methods such as machine learning and an agnostic approach to categorize CHARTER?s high-dimensional neurobehavioral, neuromedical, psychiatric, substance use, and imaging data. Such analyses would yield not just cognitive phenotypes but biopsychosocial (BPS) phenotypes that could identify new mechanisms that lead to clinically useful diagnostic tests, new therapies, and better management of CNS disorders in PWH. Our overarching goal is to leverage prior investment in CHARTER by using its data and stored specimens to a) better understand cognitive and BPS phenotypes in PWH and b) relate them to biological mechanisms. To accomplish this, we will use unsupervised and supervised machine learning methods to analyze data from CHARTER?s comprehensive assessments with the goal of identifying new cognitive and BPS phenotypes (Aim 1). We will then compare these new phenotypes to high-dimensional data from CHARTER?s completed genomewide association study as well as new data we will generate on inflammation (45-plex bead-based array) and highly novel assays of the microbiome and the metabolome in blood and CSF (Aim 2). Our analyses will include a specific focus how sex affects the observed relationships. To determine if this novel approach relates more strongly to biology than prior methods, we will also compare the performance of the new phenotypes to those defined by the 2007 HAND criteria. This highly innovative application is supported by strong preliminary data, responds well to Office of AIDS Research priorities, and will address key gaps in the field, including the need to better understand the pathogenesis of comorbid disease.