Clayton Alexander Wiley, MD, University of California San Diego, PhD, University of California San Diego - US grants

Retroviridae disease; nervous system infection; Betaherpesvirinae; virus protein; ubiquitin; nervous system disorder; HIV infections; virus antigen; immunocytochemistry; vesicle /vacuole; blood brain barrier; opportunistic infections; AIDS dementia complex; disease /disorder model; neural degeneration; neurofilament; immunoelectron microscopy; laboratory mouse; electron microscopy; western blottings;

Approximately one third of terminally ill AIDS patients develop severe cognitive deficits called HIV-associated dementi complex (HIV-ADC). As the efficacy of chemoprophylaxis for opportunistic infections and systemic control HIV replication are improved, survival would be expected to increase and thus the incidence of HIV-ADC would also be expected to increase and lead to greater morbidity. It is the consensus of opinion that HIV does not directly mediate neuronal damage by infecting neuroglial cells. Unfortunately, that is where the consensus ends. We are interested in studying the pathogenesis of neurologic damage associated with HIV encephalitis, a known pathologic substrate of ADC. Most hypotheses of the pathogenesis of neurologic damage in HIV encephalitis focus on neurotoxic HIV proteins or immune factors. This grant is examining the latter. There are numerous candidate immune factors in AIDS that could mediate neurologic disease. Given the character of the histopathology, macrophage factors deserve the greatest attention. As a beginning, we have chosen to perform a detailed analysis of 4 cytokines that have the potential to mediate neurotoxicity. Using well characterized autopsy tissues, we will define cytokine abundance and message expression in select regions of the CNS. For cytokines to have an affect, they must bind to specific membrane receptors. We will characterize the distribution of these receptors within the CNS of control and AIDS brains. We will then compare the distribution of the cytokines and their receptors to the presence of HIV infection and associated neurologic damage. Lastly, we will develop an in vitro system that can assess the presence of these cytokines in primary CNS cultures that, in the future, could be used to examine the pathogenesis of HIV encephalitis and the effects of interrupting cytokine pathways in the progression of neurologic damage. For cytokines to have an effect, they must bind to specific membrane receptors. We will characterize the distribution of these receptors within the CNS of control and AIDS brains.

The Neuropathology (NP) Core provides complete brain banking and neuropathology diagnostic services for the Pittsburgh ADRC. To assure harvesting of excellent quality tissues with short post-mortem times, autopsies are performed are performed on a 24 hour basis and the average post-mortem time over the past four years is approximately 4 hours. The brain is bisected sagittally at the time of autopsy. The right half is dissected and extensively sampled (frozen samples banking, the left half of the brain is fixed in 10% buffered formalin. After fixation the left hemi-brain is intensely sampled for NP diagnostic tests including H&E, Congo Red immunocytochemical (beta-A4 amyloid, tau, alpha-synuclein, and ubiquitin) and silver stains (Bielschowsky). Microscopic analysis of the left hemi-brain is performed according to current NIA- recommendations which includes staging of neurofibrillary tangle density (Braak staging) and follows CERAD protocols for sampling and semi- quantitation. Lewy Body pathology is described per the consensus criteria of McKeith et. al. The NP Core has recently initiated use of alpha- synuclein staining to assess for the presence of Lewy bodies. The brain bank is catalogued and correlated with NP diagnoses to make available well characterized tissues to ADRC and other investigators. Quarterly are issued to investigators to alert them to the availability and diagnoses of banked cases. Banked tissue samples are coded with a unique NP Core banking identifier which does not allow investigators to identify the patient, however, provides a link (through the rain bank which maintains the key on a secure pass-work linked file, with access only by NP Core personnel). Investigators are also provided with additional non- identifying information (age, sex, post-mortem time race, etc.) Tissues and data are made available to investigators only after review and approval by the ADRC Executive Committee and Dr. Wiley and requires IRB approval of the research protocol. NP diagnoses and a 2-3 page narrative report are supplied to the ADRC database and families within one month of death. As of 1998 the NP core banks approximately 50 ADRC autopsies per year. Additionally, control brains have been banked and characterized by identical protocols including microscopic neuropathologic evaluation. The core contains reference laboratory (including a Ar/Kr laser confocal microscope and Image 1 workstation) for development of detained and quantitative analyses.

human immunodeficiency virus; pathologic process; tissue /cell culture

DESCRIPTION (Applicant's Abstract): Approximately one quarter of AIDS patients develop severe cognitive deficits called HIV-associated dementia complex. With the advent of "triple drug" therapy, it is unclear whether treatment of systemic viral burden will lead to an increased or decreased incidence of neurologic disease. There is some controversy regarding the importance of viral burden in mediating neurologic disease. With the advent of the RNA assays for viral burden in serum, several groups have examined whether this technique could be used for measuring viral burden in tissues. As reported in the investigators' Preliminary Results section, they believe that their pilot study using their bank of AIDS brain tissues shows substantial promise to quantify brain viral burden with minimal subjective interpretation, using a branched-DNA test. The investigators propose three Specific Aims; # 1: Compare branched-DNA assessment of brain viral burden to previously utilized assays. Results from this study will permit assessment of how the newer detection assay compares to that used over the previous decade. # 2: Expand the viral burden assessment to include the following regions: caudate, putamen, thalamus, hippocampus, substantia nigra, cerebellum and when available, spinal cord. Data from this specific aim will test the hypothesis of whether there is early or more abundant infection of specified regions of the CNS. # 3: The branched DNA test will be used to assess HIV RNA copy number per mL of post-mortem CSF and per milligram of post-mortem spleen tissue to compare these values to overall brain tissue viral burden. These studies aim to show how well viral burden in other body compartments compares to the brain and will test the hypothesis that the CSF may be used as a surrogate marker of brain viral burden. Additionally, these studies aim to begin to assess the relationship between treating systemic viral burden and its relationship to brain viral burden. A proven sensitive and reproducible means of assessing viral load in brain tissue will substantially facilitate clinical (e.g. cross comparison of data between outcome and treatment studies) and basic (e.g. comparison of viral and host message expression within the same RNA sample assessed for viral load) science advances in our understanding of the pathogenesis of neurologic damage associated with AIDS.

AIDS dementia complex; cytokine; neurotrophic factors; growth factor receptors; HIV infections; gene expression; clinical research; human tissue; human subject;

This application requests funds to continue support of a well integrated basic and clinical sciences predoctoral training program. The training program links 17 programs in 6 graduate schools within the University of Pittsburgh and Carnegie Mellon University. The two universities provide well equipped modern biological clinical and basic science research facilities. Alone the University of Pittsburgh ranks 10th in overall NIH research funding, but with the added expertise of CMU, our program is considered one of the top neuroscience and bioengineering training centers. We offer a pioneering and flexible curriculum that integrates Ph.D. and M.D. training throughout the course of the program. Our program focuses primarily on research and clinical training, however, students participate actively in professional development workshops and receive extensive mentoring in developing their careers as combined degree graduates. Ethical training in responsible research conduct is an integral part of the curriculum. Over the 400 training faculty within the affiliated programs, over 80 are currently directly involved in the program. Ph.D. thesis advisors are approved by the career advisory committee. Trainees are provided thorough graduate medical and career advising throughout the program. Upon matriculation, each trainee is assigned a Career Advisor who in conjunction with the Program Director and the entire Career Advisory Committee guide the student throughout their training and beyond and into post-doctoral fellowship and residency positions. Funds are requested to support from 11-15 trainees for a period of five years beginning July 1999. Data are provided to support our belief that we have a proven history of attracting outstanding trainees and placing them in positions for future career development. Trainees are selected from a national pool of well over 100 applicants with superior academic records. The average MCAT (34) and GPA (3.8) for matriculants is quite competitive with current MST Programs. Graduates of this relatively young program continue to achieve premier postdoctoral and residency positions while our earliest graduates have obtained world class faculty and industrial positions.

Approximately one quarter of AIDS patients develop HIV-associated dementia complex (HIVD). We are studying the pathogenesis of neurologic damage associated with HIV encephalitis, a known pathologic substrate of HIVD. Most hypotheses of the pathogenesis of neurologic damage in HIV encephalitis focus on neurotoxic HIV proteins or immune factors. We and others concluded that many cytokines (in particular monokines) are present within CNS tissues showing HIV encephalitis, but it has been difficult to ferret out their relative importance. Using new quantitative techniques to measure tissue viral-load we observed a tight association between HIV DNA and RNA load. Coupling these findings with our previous observations of viral protein expression, HIV encephalitis would appear to be limited to productive infection of CNS macrophages. The absence of significant proviral load prior to the development of HIV encephalitis leads us to hypothesize that neurologic disease results from either, increased trafficking of HIV-infected monocyte elements into the CNS, loss of immune control of macrophage infection within the CNS, or a CNS microenvironment that promotes HIV replication. To test these hypotheses, in the first 2 specific aims we will examine; the relationship between immunologic and neurologic status and peripheral blood monocyte infection and activation in vivo, infectability in vitro, and changes in the capacity of a subject's cell-mediated immune system to control macrophage infection. In addition to viral entry and immune surveillance, there may be some other attributes of the CNS microenvironment that promote HIV infection. In the third specific aim we will examine the role of immunosuppressive and immuno-enhancing chemokines/cytokines in regulation of macrophage viral production in the CNS. In the fourth specific aim we will bank RNA from peripheral blood, CSF and, brain tissue, to decipher whether a neurotropic strain of HIV evolves within an individual or whether HIV encephalitis is the result of macrophage-tropic strains present from early in infection. The proposed experimentation will help explain why HIV homes for, and propagates within, the nervous system. These data will help design means of identifying subjects at risk for the development of CNS disease and help design therapies for arresting HIV encephalitis.

DESCRIPTION (provided by applicant): HIV infected individuals develop a wide spectrum of neurologic disease ranging from minor cognitive and motor deficits (MCMD) to frank dementia (HIV-associated dementia complex, HIVD). While the pathologic substrate of MCMD is unknown, HIV encephalitis (HIVE) is a known pathologic substrate of HIVD. Abundant activation and infection of CNS macrophages characterize HIVE, with surprisingly little evidence of direct neuroglial infection. The relative role of activated and infected CNS macrophages in mediating neurologic damage is not known. We hypothesize that HIV associated MCMD is related to pathologic activation of macrophages within the CNS. Further this activation provides abundant host target-cells for infection and replication of HIV and subsequent development of HIVE and HIVD. Unfortunately there are no current means of documenting activated macrophages within the CNS. We propose to extend our findings from the simian AIDS model, to humans infected with HIV and test the hypothesis that: the radioligand 11C-PK11195 in 3D-PET will detect the presence of activated macrophages in the CNS of HIV infected subjects. By comparing neurocognitive findings, and peripheral and central markers of HIV infection and PK11195 PET we hope to establish a clinical measure of CNS macrophage activation. In Specific Aim 1we will perform a cross sectional study of HIV infected subjects and appropriate controls to determine the relationship between binding of the radioligand 11C-PK11195 in 3D PET imaging and neurologic disease. All subjects will be given a battery of neurocognitive testing: undergo MRI and PK11195-PET imaging and serum testing for HIV titers and CD4 count. We hypothesize that those subjects with MCMD or HIVD will have elevated binding and retention of PK11195 consistent with increased activation of CNS macrophages. In Specific Aim 2we will longitudinally follow HIV infected subjects at risk for developing dementia. At 6-month intervals, subjects will undergo a battery of neurocognitive exams, serum and CSF analysis, head MRI and C-11PK11195 PET. We hypothesize that prior to developing neurologic disease associated with activated CNS macrophages, PET scans will demonstrate elevated binding and retention of PK11195. If neurologic disease symptomatology progresses, this will be accompanied by persistent elevation of PKlll95 on 3D PET, Successful therapeutic intervention would be associated with decreased PET signal. The results of these experiments will help define clinical tests for diagnosing lentiviral encephalitis and help identify infected populations at risk of developing neurologic disease. They will also permit the development of markers to assess efficacy of future therapy to arrest development and progression - of MCMD and HIVD.

DESCRIPTION (provided by applicant): The Pittsburgh Medical Scientist Training Program (MSTP) offers a 10-week research and career development program for underrepresented minority students interested in careers as physician scientists. The program is designed for students in their freshman, sophomore or junior years of college. Students receive intensive research laboratory experience and abundant mentorship. The program is for U.S. citizens and permanent residents, and is limited to minority groups underrepresented in the biomedical sciences including African Americans, Alaskan Natives, Mexican Americans, Native Americans, Other Hispanic Americans, Pacific Islanders and Puerto Rican Mainlanders. Application to the program includes a personal statement, transcript and 2 letters of recommendation each including a coversheet, and is available online at www.mdphd.pitt.edu. Students receive a stipend for the ten-week period plus round trip airfare. The stipend provides students with a comfortable budget to live in Pittsburgh. All students must have medical insurance for the duration of the program. Since they will be working with human materials, they are encouraged to be vaccinated for hepatitis B. Minority undergraduates work with biomedical researchers in a variety of areas including, but not limited to cardiovascular, pulmonary, hematologic, immunology, transplantation or sleep disorders. The undergraduate students are also paired with current MSTP students who are active members of the mentoring team. Students participate in a variety of career development programs, scientific seminars, responsible conduct of research, and specific career enhancement opportunities including preparation for national examinations. By the end of the summer, students have created a research poster for presentation on the last day of the program as well as at a national conference. Social activities are included in the program to help build a network of peers with similar career paths.

DESCRIPTION (provided by applicant): Neuropathologically, Alzheimer's disease (AD) is defined by the presence of tangles and plaques composed of the amyloid-beta (Abeta) protein and activated microglia. Identification of AD pathology in living subjects is an important goal because it could allow refinement of early diagnosis and identification of pre-symptomatic pathology. In addition, the optimal development of new anti-amyloid therapies will require a means to monitor brain amyloid load and its active removal. Our group has developed a novel amyloid-imaging positron emission tomography (PET) radiotracer termed "Pittsburgh compound-B" (PIB) that distinguish AD from control subjects and had a regional distribution consistent with that of the post-mortem distribution of plaques. Additionally we have used radiolabeled PK11195 a peripheral benzodiazepine receptor ligand that permits PET assessment of microglial activation. This R21 will examine the relationship between amyloid deposition and microglial activation in different stages of Alzheimer's Disease. We will recruit 5 control subjects, 5 minor cognitively impaired, 5 mild, and 5 moderate AD patients. All subjects will have been clinically evaluated and diagnosed by the University of Pittsburgh Alzheimer Disease Research Center (ADRC) and further evaluated with a neuropsychological battery through the ADRC. All subjects will be studied cross-sectionally with PIB and PK11195 PET scans. In addition, volumetric MRI will be performed so we can directly compare these current neuroimaging standards. The quantitative PIB and PK11195 PET data will be compared to neuropsychological measures to explore possible correlations between regional amyloid load, activated microglia and performance on function-specific cognitive testing (e.g. frontal or visuospatial tasks). We hypothesize that subjects with more severe cognitive impairment will show greater PIB retention consistent and greater amyloid burden and will show greater PK11195 retention consistent with increased microglial activation. Delineation of the relationship between amyloid deposition and microglial activation will help define the role of microglial activation in the pathogenesis of amyloid deposition and with development of immunotherapies, help discern the role of activated microglia in eliminating amyloid.

DESCRIPTION (provided by applicant): Approximately 1/4 of immunosuppressed AIDS patients develop a neurodegenerative disorder clinically characterized as HIV associated dementia complex. In our experience, autopsies of AIDS patients who had become demented for reasons other than opportunistic infection uniformly demonstrated HIV encephalitis. Why there is such an abundance of activated and infected macrophages in the CNS remains an enigma, however, we theorize that it may be due to increased trafficking of HIV-infected monocytes. Monocytes are activated upon leaving the blood stream and entering the CNS where they transform into macrophages and can initiate viral replication and a neuroinflammatory cascade. Tissue damage begins a cycle of astrocytic and microglial activation, providing susceptible targets for further HIV infection and destruction of synaptic connectivity. We propose to use SIV infection of Macaca nemestrina and Macaco mullata as models of HIV encephalitis to test several hypotheses related to our theory of lentiviral neuropathogenesis. While no animal disease model is perfect, numerous similarities between simian and human nervous systems, between SIV and HIV infection and the capacity to manipulate and monitor CNS damage, make the macaque models optimal for these studies. Our overarching hypothesis is: Progression of SIV infection leads to increased monocyte/macrophage infection and trafficking into the CNS with destruction of the synaptic matrix. For all 3 aims of the current proposal, we will study a group of 36 SIV infected macaques. At 2-week intervals we will measure; absolute CD4 and CD8 T-cell counts and viral loads in the CSF and serum of all animals. In Specific Aim 1 we will examine the relationship between peripheral SIV infection and the development of SIV encephalitis. These experiments will test the hypothesis that: with progression of immune suppression there is increased trafficking of SIV infected monocytes causing increased brain viral burden. In Specific Aim 2 we will assess activation of CNS macrophages using Positron Emission Tomography and the peripheral benzodiazepine receptor radioligand [11C]-DAA1106. We hypothesize that when animals begin to show disease progression (decline in CD4 T-cells, increase in viremia) they will show increased CSF virus and increased binding of DAA1106 consistent with activation of CNS macrophages. In Specific Aim 3 we will measure synaptic and extracellular matrix damage in autopsy brain tissues and compare them to the temporal course of peripheral and central viral loads, DAA1106 binding. In summary, the proposed specific aims will test a set of interconnected hypotheses helping define mechanisms of synaptic damage and therapeutic targets to arrest its development and progression.

DESCRIPTION (provided by applicant): This is a new RO1 entitled Neurodegeneration in aged SIV-infected primates that is a direct extension of our previously investigations in the pathogenesis of SIV encephalitis and neurodegeneration in aged non-human primates. Use of combined active antiretrovirals (CART) in developed countries has led to a near disappearance of severe encephalitis. Unfortunately chronic HIV infection continues to exact a toll on the nervous system with increased prevalence of a spectrum of neurocognitive and motor dysfunctions termed HIV-associated neurocognitive disorders (HAND). CART has also permitted people to survive longer with HIV infection and the CDC projects that by 2015 over half of HIV infected individuals in the US will be over the age of 50. Coupled with the aging process, the extended exposure to both HIV and antiretroviral drugs appears to increase their risk of neurologic and neuropsychiatric complications. In this application we propose to use a well-established non-human primate (NHP) model of chronic lentiviral infection, SIV infection of Macaca mulatta (Rhesus Macaque RM), to model HAND in aged macaques (>20 years old), map the neurological signs to behavioral abnormalities and begin to elucidate the neurological and immunological pathogenesis of this debilitating disease. While no animal disease model is perfect, numerous similarities between simian and human nervous systems, between SIV and HIV infection and the capacity to manipulate and monitor central nervous system (CNS) damage, make the macaque model optimal for these studies. Using 5 groups of SIV infected and control RMs, we will assess the presence of neurocognitive abnormalities and the role of viral suppression in exacerbating age related neurodegeneration. Findings from this grant will have immediate implications on the treatment of aged HIV infected humans. Building upon our previous decade of experience with SIV infection of macaques as a model of chronic HIV infection and our recent studies of neuroinflammation in aged NHPs (Kofler et al 2011 (41) see appendix), we will test our overarching hypothesis that: chronic systemic CART with or without chronic lentiviral infection exacerbates age related damage to the nervous system principally through CNS stress and associated innate immune activation. We will assess whether aggressive suppression of systemic lentiviral infection with CART exacerbates age related cognitive abnormalities and beta amyloid related neuropathology (e.g. plaques, tangles, abnormal tau phosphorylation or beta amyloid oligomeres). Alternatively, HAND may persist even in the presence of viral suppression or as a result of CART alone. In our second specific aim we will use state of the art quantitative neuropathological analysis to elucidate the pathological substrate of HAND. Knowledge of the pathogenesis of neurological dysfunction in the simian model will help define pathways for intervention to mitigate the human disease.