Abstracts from the 14th International Symposium on ...

Abstracts from the 14th International Symposiumon NeuroVirology October 25–28, 2016, Toronto,Ontario, Canada

Published online: 27 September 2016# Journal of NeuroVirology, Inc. 2016

P1Comparative Modeling of Human EndogenousRetrovirus-K Protease Based on HIV Protease PredictsEfficacy of Protease Inhibitors

Rachel Abrams1, Richa Tyagi1,Wenxue Li1, Mario Bianchet2,Avindra Nath1

(corresponding author: [email protected])

1National Institute of Neurological Disorders and Stroke,National Institutes of Health; 2Department of Neurology,Johns Hopkins School of Medicine

Over the course of human evolution, retroviruses haveinfected cells of the germ line, allowing the genome ofthe virus to be passed down from parent to offspring.These human endogenous retroviruses (HERVs) make upabout 8 % of the genome; however, in most cases multiplemutations have made them inactive. One of the most re-cently incorporated (HERV-K); however, has been impli-cated in the development of amyotrophic lateral sclerosis(ALS). Increased expression of HERV-K viral transcriptswas observed in the brains of ALS patients and the ex-pression of HERV-K envelope causes damage to motorneurons in vivo. While there are no treatment optionsfor patients with ALS, in some patients with HIV infec-tion that also display an ALS-like syndrome, antiretroviraldrugs can reverse the ALS symptoms. Both HIV andHERV-K utilize an aspartic acid protease to process theviral polyprotein to its active components. To determine ifHIV protease inhibitors could inhibit HERV-K protease,HIV protease inhibitors were tested in an in vitro HERV-K infection model. The compounds tested had a moderateprotective effect; however, were less effective than againstHIV infection. To investigate the reason for the level of

observed inhibition, similarities and differences betweenHIV and HERV-K protease were examined. There is nocrystal structure of the HERV-K protease available, hencecomparative modeling using the sequence alignment withHIV protease was performed. A homology model wasgenerated and refined using the Prime program in theSchrodinger Suites Software package. This model wasused to compare the active site of HIV protease with thatof HERV-K. It can be seen from the model that while theoverall structures of the proteases appear quite similar,changes in the active pocket caused by the few aminoacid differences may be sufficient to explain the observedreduction in antiviral activity.

P2Role of ATF6b in HIV-Associated NeurocognitiveDisorders

Cagla Akay Espinoza, Ping Lin(corresponding author: [email protected])

University of Pennsylvania, School of Dental Medicine,Department of Pathology

The underlying mechanism of cognitive impairment andb r a i n i n j u r y i n pa t i e n t s w i t h HIV-a s soc i a t edneurocognitive disorder (HAND) on suppressive antiretro-viral therapy are not completely understood. However,synaptic injury, neuronal dysfunction, and damage inthese patients are partially driven by immune activationand chronic inflammation in response to soluble factorsreleased by HIV-infected and/or activated macrophages aswell as a low level of HIV replication in central nervoussystem (CNS) reservoirs. A majority of these mediators as

J. Neurovirol. (2016) 22 (Suppl 1):S1–S89DOI 10.1007/s13365-016-0478-8

well as many of the comorbid conditions were shown toinduce a ubiquitous cellular response, unfolded proteinresponse (UPR) in vitro and in vivo. We have previouslyshown UPR activation in post-mortem tissue from HANDpatients in vivo. One of the three master UPR initiatorproteins, ATF6b, upon cleavage by site-1 and site-2 pro-teases (S1P and S2P), translocates to the nucleus for tran-scriptional induction of ER resident chaperones, apoptoticgenes, and secretory pathway regulatory genes. We hy-pothesized that activation of the ATF6 pathway of theUPR contributed to neuronal damage and death observedin HAND. We found that infection of primary humanmonocyte-derived macrophages (MDMs) with HIV ledto the nuclear translocation of the cleaved, thus active,ATF6b (N-ATF6b), which could be blocked by S1P inhi-bition. We also observed that blocking nuclear accumula-tion of N-ATF6b in macrophages led to attenuation ofdeath of primary rat cortical neuroglial cultures exposedto supernatants from HIV-infected MDMs. Finally, wefound nuclear N-ATF6b accumulation in neurons exposedto HIV-infected MDM supernatants or excitotoxic stimu-lus. These findings suggest that altered function of ATF6bin several cell types within the CNS might be contributingto neuronal dysfunction and damage in patients withHAND.

P3Rift Valley Fever virus-induced encephalitis:characterization of leukocytes in the CNS

Joseph Albe, Michael Kujawa, Tiffany Thompson,Amy L. Hartman(corresponding author: [email protected])

Department of Infectious Disease and Microbiology, Centerfor Vaccine Research, University of Pittsburgh

Background: Rift Valley Fever Virus (RVFV) is a vector-borne infection endemic to the Horne of Africa; Howeverrecent outbreaks in the Arabian Peninsula have expandedits potential range. People can develop encephalitis as aresult of RVFV infection. Our lab uses an immunocompe-tent Lewis rat model to study neuropathogenesis ofRVFV. Lewis rats develop uniformly lethal encephalitiswithin 7–8 days after aerosol exposure. Rats infected sub-cutaneously do not develop disease. The goal of this studyis to characterize the phenotypes, timing, and extent ofimmune cell infiltrate into the CNS of RVFV-infectedLewis rats. Methods: Lewis rats were infected withRVFV ZH501 by aerosol and subcutaneous routes, thenserially sacrificed between days 3 and 6. Rat immunecells were isolated from brains via percoll gradients.

Cell populations were stained with antibodies, run on aBD LSRII flow cytometer, and analyzed with FlowJo7.6.5. Results: In rats infected by aerosol, infiltration ofinflammatory cells into the CNS began at 4 dpi andconsisted of neutrophils, macrophages, and lymphocytes.There was also evidence of microglia activation. Rats in-fected subcutaneously do not develop overt disease yetstill had a detectable infiltrate that was intermediate com-pared to uninfected rats. Neutrophils were a major com-ponent in aerosol but not subcutaneously infected rats.Conclusions: In addition to extensive viral replication inthe brain, leukocyte infiltration and microglia activationare a significant component of disease in the Lewis ratmodel of RVFV. The relative contributions of viral cyto-pathology versus immunopathology remain to be deter-mined, but this study represent the first attempt to char-acterize the leukocytic component.

P4Selection of gRNAs to target the HIV-1 quasispecieswith CRISPR/cas9

Alexander Allen1, Michael Nonnemacher1, WilliamDampier1,2, Matthew Desimone2, Vanessa Pirrone1, KatieKercher1, Shendra Passic1, Jean Williams1, Brian Wigdahl1


1Department of Microbiology and Immunology, Institute forMolecular Medicine and Infectious Disease, DrexelUniversity College of Medicine; 2School of BiomedicalEngineering and Health Systems, Drexel University

HIV-1 viral persistence during long-term antiretroviral thera-py is a major hurdle to a cure. Genomic editing techniques,like the CRISPR/Cas9 system, hold promise to permanentlyexcise integrated virus from a host cell. Targets are defined bya 20 nucleotide guide RNA (gRNA) complementary to thedesired genomic region. However, due to the rapid mutationrate intrinsic to HIV-1 replication, the virus in patients existsas a collection of distinct genomic variants, termedquasispecies. Presented here is a methodology for designinggRNA sequences to cleave a spectrum of HIV-1 quasispeciespresent within individual patients across a population of HIV-1-infected patients. PBMC genomic DNA was isolated frompatients in the Drexel Medicine CNS AIDS Research andEradication Study (CARES) Cohort as well as from brainand spleen tissue from the National NeuroAIDS TissueConsortium (NNTC) and the long terminal repeat (LTR) ofthe HIV-1 quasispecies was sampled using Next GenerationSequencing (NGS). gRNAs were computationally selected byexamining their binding potential across a random training setof CARES patient samples. A package of 4 or 10 gRNAswere

S2 J. Neurovirol. (2016) 22 (Suppl 1):S1–S89

selected based on the training set which in silico cleaved theentire detectable quasispecies of the remaining CARES sam-ples and of the majority of the NNTC samples with an averagenumber of cleavages within each sample group of 3.4+/−1.7or 5.4+/−3 times, respectively. The package was further testedin silico against a national sampling of subtype B NorthAmerican LTRs from the Los Alamos National Databaseand was shown to cleave all sequences. Currently, we arefocused on cloning these packages of gRNAs to initiate func-tional studies to confirm the in silico studies performed todate. These studies represent a step towards understandingthe complex task of using excision therapy to target HIV-1quasispecies in the infected patient population.

P5Functional consequences of HTLV-1 viral antigensdetected in exosomes from HAM/TSP patients

Monique Anderson1, Yoshimi Enose-Akahata1, Maria ChiaraMonaco-Kushner1, Ashley Vellucci1, Yuetsu Tanaka2, BenLepene3, Fatah Kashanchi4, Steven Jacobson5


1National Institutes of Health; 2University of the RyukyusGraduate School of Medicine; 3Ceres Nanosciences;4George Mason University Laboratory of MolecularVirology; 5National Institutes of Health

Human T-cell lymphotropic virus Type I (HTLV-1) is a retro-virus that currently infects an estimated 10–20 million peopleworldwide. The vast majority of those infected will remainasymptomatic, but about 0.1–4 % of patients will developHTLV-1 associated myelopathy/tropical spastic paraparesis(HAM/TSP). HAM/TSP is a neuroinflammatory disease thathas been shown to be immunopathologically mediated.Recent data suggests that viral antigens can be acquired inthe absence of viral infection through the transport ofexosomes. Therefore, the presence of HTLV-1 products inthe exosomes isolated from HAM/TSP patient cerebrospinalfluid (CSF) was investigated. Using a novel nanotrap technol-ogy which utilizes hydrogel particles that trap exosomes,exosomes that were positive for HTLV-1 Tax by Western blotcould be detected from HAM/TSP patient CSF but not fromcontrol HTLV-1 seronegativemultiple sclerosis (MS) patients.Additionally, HAM/TSP PBMCs were shown to secreteexosomes containing HTLV-1 Tax in ex vivo cultures. Tounderstand the source of these exosomes, PBMCs were sortedand found to have increased exosome production by activatedCD4+CD25+ and CD8+CD25+ T cells. Importantly, to de-termine if these exosomes are functionally producing tax pro-teins, exosomes isolated from HAM/TSP PBMCs were ap-plied to recipient target B cells. These exosome-sensitized

target cells were shown to be lysed by HTLV-1 Tax-specificcytotoxic T lymphocytes (CTL). Additionally, initial resultsindicate that Tax can be introduced into exosomes with trans-fection of Tax plasmids into uninfected cells, and this processwas unaltered with Tax mutation. Cumulatively, these find-ings show that there are HTLV-1 proteins present in exosomesfound in virus-free CSF of HAM/TSP patients and that HAM/TSP PBMCs can excrete these exosomes in culture.Furthermore, exosomes containing HTLV-1 Tax can sensitizetarget cells for HTLV-1 specific lysis by CTLs. This suggeststhat exosomes may play a role in perpetuating the activatedimmune response that contributes to HAM/TSP.

P6Extracellular vesicles are amyloid carriers in HIV-infectedbrains

Ibolya E. Andríçs1, Ana Leda1, Marta Garcia-Contreras 2,Luc Bertrand1, Minseon Park1, Marta Skowronska1,Michal Toborek1


1Department of Biochemistry and Molecular Biology,University of Miami School of Medicine; 2DiabetesResearch Institute, University of Miami School of Medicine

HIV-infected brains are characterized by increased amyloidbeta (Abeta) deposition. It is believed that the blood–brainbarrier (BBB) is critical for Abeta homeostasis and contrib-utes to Abeta accumulation in the brain. Extracellular vesi-cles (ECV) gained recently a lot of attention as potentiallyplaying a significant role in Abeta pathology. In addition,HIV-1 hijacks the exosomal pathway for budding and re-lease. Therefore, we investigated the involvement of BBB-derived ECV in the HIV-1-induced Abeta pathology in thebrain. Our results indicate that HIV-1 increases ECV releasefrom brain endothelial cells and elevate their Abeta cargo ascompared to controls. Interestingly, brain endothelial cell-derived ECV transferred Abeta to astrocytes and pericytes.Infusion of brain endothelial ECV carrying fluorescentAbeta into the internal carotid artery of mice resulted inAbeta fluorescence associated with brain microvessels andalso in the brain parenchyma. These data suggest that ECVcarrying Abeta can be successfully transferred across theBBB into the brain. Based on these observations, we con-clude that HIV-1 facilitates shedding of brain endothelialECV carrying Abeta, a process that may increase Abetaexposure of cells of neurovascular unit, such as astrocytesand pericytes. This mechanism may contribute to increasedamyloid deposition in HIV-infected brain. Supported byMH098891, MH072567, DA039576, DA027569,HL126559, and by the Miami CFAR MH063022.

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P7HIV-1 Vpr sequence variants associate specificallywith co-linear genetic determinants of viral co-receptorphenotype

Gregory Ante l l 1 , Wi l l i am Dampie r 2 , Ben jamasAiamkitsumrit2, Michael Nonnemacher2, Vanessa Pirrone2,Wen Zhong2, Katherine Kercher2, Shendra Passic2, JeanWilliams2, Yucheng Liu2, Tony James2, Jeffrey Jacobson3,Zsofia Szep4, Brian Wigdahl2, Fred Krebs2


1School of Biomedical Engineering, Science, and HealthSystems, Drexel University; 2Department of Microbiologyand Immunology, Institute for Molecular Medicine andInfectious Disease, Drexel University College of Medicine;3Department of Medicine, Section of Infectious Disease,Lewis Katz School of Medicine, Temple University;4Division of Infectious Diseases and HIV Medicine,Department of Medicine, Drexel University College ofMedicine

Viral protein R (Vpr) is a 14 kDa HIV-1 accessory proteinthat functions in HIV-1 replication and pathogenesis. Vpris also a likely contributor to the neuropathogenesis thatunderlies deficits collectively referred to as HIV-associated neurocognitive disorders (HAND). HIV-1 in-fection of the brain is predominated by infected macro-phages and microglial cells that harbor viruses character-ized by an R5 (CCR5-utilizing) co-receptor genotype. Instudies that have paralleled our identification of sequencesignatures in HIV-1 Tat and the long terminal repeat(LTR) that associate with co-receptor utilization, we havedemonstrated associations between Vpr variation in HIV-1-infected patients and co-receptor usage. Co-linear HIV-1 Vpr and Env-V3 amino acid sequences were collectedfrom the LANL HIV-1 sequence database, as well as fromwell-suppressed patients enrolled in the Drexel/TempleMedicine CNS AIDS Research and Eradication Study(CARES) Cohort. Vpr sequences were grouped as X4(CXCR4-utilizing) or R5 according to genotypic classifi-cation of the co-linear Env-V3 sequence. The amino aciddiversity of each Vpr population was assessed, as was theJensen-Shannon divergence between groups, in order toidentify sequence signatures associated with co-receptorutilization. Multiple amino acid alphabets were utilizedin order to better assess the impact of amino acid substi-tutions with similar physiochemical properties. Positions35–45 and the C-terminus of Vpr consistently displayedstatistically significant divergence across multiple alpha-bets. Specifically, positions 37 and 41 displayed a consen-sus amino acid switch between R5 and X4 sequence pop-ulations. Overall, the results suggest a functional link

between the evolution of Vpr and gp120 in HIV-1-infected patients, demonstrate the existence of HIV-1 tro-pism beyond the envelope, and suggest associations be-tween Vpr variants and R5 viral genotypes in the brain.

P8Divergent efficacies of antiretroviral therapy in bloodand brain: HIV-1 suppression is dependent on drugand cell type.

Eugene Asahchop1, Wing Chan1, William Branton1, GailRauw2, Manmeet Mamik1, Lothar Resch3, John Gill1, GlenBaker2, Christopher Power4


1Department ofMedicine, University of Alberta; 2Departmentof Psychiatry, University of Alberta; 3Department ofPathology, University of Calgary; 4Department of Medicineand Psychiatry, University of Alberta

Background: Perivascular macrophages and microglia arethe principal productive reservoir for active HIV-1 repli-cation in the brain. In patients receiving suppressive com-bination antiretroviral therapy (cART), HIV-1 replicationin the brain reservoir is assumed to be latent or minimallyproductive although the susceptibility of this reservoir tosuppression by cART is uncertain. We investigated theefficacies and concentrations of established antiretroviraldrugs (ARVs) in HIV-infected microglia and macrophagestogether with evaluating the in vivo brain HIV-1 reservoirduring suppressive cART. Methods: HIV-1 RNA andDNA levels in brain tissues from HIV-infected patients(n = 2) were quantified by digital droplet PCR within12 h of last ARV dosage. The efficacies of individualARVs in HIV-infected primary microglia, bone marrow-derived macrophages and peripheral blood mononuclearcells (PBMCs) were compared. Tissue and cellular con-cen t ra t ions of ARVs were measured by l iqu idchromatography-mass spectrometry (LC-MS) in humanmyeloid cells and mouse brain. Results: In treated patientswith prolonged (>4 years) undetectable plasma viral RNAlevels, HIV-1 RNA and DNA were detected in multiplebrain specimens with associated neuroinflammatory re-sponses. Zidovudine, etravirine and darunavir treatmentof HIV-infected microglia exhibited significantly higherEC50 va lue s than those obse rved in PBMCs .Intracellular and extracellular ARV levels in human mye-loid cells were similar and unaffected by HIV-1 infection.In vivo concentrations of darunavir and raltegravir in micevaried depending on brain region and were >1.0 log lowerthan matched plasma levels at 1 h and undetectable at 8 hpost-treatment. Conclusions: ARVs displayed differential


concentrations and antiviral effects depending on the in-dividual ARV and infected cell type (or tissue), which wassubstantiated by the presence of persistent viral DNA andRNA in brain tissue from patients with undetectable plas-ma viral RNA. These findings underscore considerationsof assessing ARV selection for different viral reservoirs aspart of efforts to eradicate HIV-1.

P9Identification of HIV-1 quasispecies for targeted excisionof latent infection using next generation sequencing

Andrew Atkins1, William Dampier1, Katherine Kercher1,Shendra Passic1, Wen Zhong1, Jean Williams1, SergeyBalashov1, Joshua Mell1, Joshua Earl1, Vanessa Pirrone1,Michael Nonnemacher1, Zsofia Szep2, Jeffrey Jacobson3,Brian Wigdahl1


1Department of Microbiology and Immunology, Institute forMolecular Medicine and Infectious Disease, DrexelUniversity College of Medicine; 2Division of InfectiousDisease, Department of Medicine, Drexel UniversityCollege of Medicine; 3Department of Medicine, Lewis KatzSchool of Medicine, Temple University

Although ART drastically improves the prognosis for HIV-1-infected patients, latent infection due to integrated proviralDNA persists in some tissues (e.g. peripheral blood, lymphoidtissue, and the brain). There is currently no effective strategyfor the removal of persistent proviral DNA from infected pa-tients and the end result of ART interruption is a resurgence ofsystemic viral load. However, recent success has beenachieved in the removal of HIV-1 from infected cell lines aswell as primary cells from infected patients and the results ofthese experiments suggest a new approach to the targetedremoval of latent infection. Bioinformatic analysis of deepsequencing data can potentially be used to design gRNAswhich target persistent HIV DNA using the CRISPR-Cas9system. In order to pursue this strategy, it is necessary to col-lect sequence data from patients with ART regulated viralloads. Furthermore, it is critically important to track geneticvariation over time in HIV-1-infected patients as the geneticsequence of latent HIV infection is not static. To address thiscomplex problem, we have utilized patient samples from theDrexel Medicine CNS AIDS Research and Eradication Study(CARES) Cohort. The CARES Cohort collects longitudinalsamples from patients allowing observation of the progress ofHIV-1 mutation. Presented here are the strategies that we havedeveloped to address the challenge of whole-genome se-quencing of latent HIV infection in ART-regulated patients.We have determined that an effective strategy is to subdivide

the HIV genome into overlapping fragments of 1000 (+/−200)base pairs allowing PCR amplification of each fragment. Nextgeneration sequencing is then used to acquire sequence datawith sufficient depth to identify genetic variants making upless than 1 % of the quasispecies in the sampled genome,allowing for design of potential CRISPR-cas9-based curestrategies.

P10Human neural progenitor cells (hNPCs) are productivelyinfected by R5-tropic HIV-1: Morphine interactionson infection and function involve Cdk5 signaling

Joyce Balinang1, Kurt Hauser2, Pamela Knapp1


1Department of Anatomy and Neurobiology, VirginiaCommonwealth University; 2Department of Pharmacologyand Toxicology, Virginia Commonwealth University

Opiates augment HIV-induced neuropathogenesis in the CNSthrough both direct and indirect mechanisms that disrupt glialand neuronal function. All CNSmacroglia and neurons derivefrom neural progenitor cells (NPCs) during development, andNPCs in the adult brain contribute to repair processes. Sincedisruptions in NPC function are known to impact CNS popu-lations and brain function in a number of disease/injury con-ditions, we examined whether HIV ± opiate exposure affectedthe maturation and fate of human NPCs. As hNPC infectionby HIV has occasionally been reported, we also reexaminedthis question, and parsed between effects due directly to hNPCinfection by HIV, or to hNPC dysfunction caused by the in-fective milieu. Multiple approaches confirmed the infection ofhNPCs by R5 tropic HIVBaL, and demonstrated that activeinfection could be sequentially transferred to naíve hNPCs.Exposure to supernatant from HIVBaL-infected cells(HIVsup) reduced hNPC proliferation and led to prematuredifferentiation into astrocytes and neurons. Morphine co-exposure prolonged hNPC infection and exacerbated func-tional effects of HIVsup. Neither purified virions nor UV-inactivated HIVsup altered proliferation, indicating that thiseffect did not require infection. Gene array analysis and RT-qPCR with immunoblot validation suggested that Cdk5 sig-naling was involved in HIV-morphine interactions. siRNA-mediated knockdown of Cdk5 expression reversed prematureMAP2 differentiation, but also increased hNPC death.Pharmacological inhibition of Cdk5 activity withroscovitine also reversed MAP2 differentiation withoutinducing hNPC death. However, roscovitine also inhibitsCdk2/Cdk4 activity, which may explain its anti-proliferative effects on hNPCs independent of treatments.Overall, dysregulation of hNPC functions by the


infectious environment may create cell population imbal-ances that contribute to CNS deficits in both adult andpediatric patients. Additionally, infected hNPCs may passvirus to their progeny, and serve as an unappreciated viralreservoir. The recent epidemic of opiate/heroin abusehighlights the clinical importance of HIV and opiateinteractions.

P11cAMP signaling enhances HIV-1 long terminal repeat(LTR)-directed transcription and viral replicationin human bone marrow progenitor cells

Anupam Banerjee, Luna Li, Vanessa Pirrone, Fred Krebs,Brian Wigdahl, Michael Nonnemacher(corresponding author: [email protected])

Department of Microbiology and Immunology, Institute forMolecular Medicine and Infectious Disease, DrexelUniversity College of Medicine

CD34+ hematopoietic progenitor cells have been shownto be susceptible to HIV-1 infection, possibly due to a lowlevel expression of CXCR4, a coreceptor for HIV-1 entry.Given these observations, we have explored the impact offorskolin on cell surface expression of CXCR4 in a cellline model (TF-1). The elevation of intracellular cAMP byforskolin through adenyl cyclase resulted in transcription-al upregulation of CXCR4 with a concomitant increase inreplication of the CXCR4-utilizing HIV-1 strain IIIB.Transient expression analyses also demonstrated an in-crease in CXCR4-, CCR5, and CXCR4-/CCR5-utilizingHIV-1 (LAI, YU2, and 89.6, respectively) promoter activ-ity. Studies also implicated the protein kinase A pathwayand the downstream transcription factor CREB-1 in inter-facing with cAMP response elements located in theCXCR4 and viral promoter. These observations suggestthe cAMP signaling pathway may serve as a regulator ofCXCR4 levels and concomitantly of HIV-1 replication inbone marrow progenitor cells.

P12Exosomes from donor and recipient cells: Roleof exosomes in HIV latency in cART treated cells.

Robert Barclay1, Catherine DeMarino1, Angela Schwab1,Michelle Pleet1, Gavin Sampey1, Sergey Iordanskiy1, RaminHakami1, Benjamin Lepene2, Nazira El-Hage3, FatahKashanchi1


1National Center for Biodefense and Infectious Disease -George Mason University; 2Ceres Nanosciences Inc.,Manassas, VA; 3Florida International University, Miami, FL

HIV-1 infection results in a chronic illness since long-termHAART can lower viral titers to an undetectable level.However, discontinuation of therapy rapidly increases vi-rus burden. Moreover, patients under HAART frequentlydevelop various metabolic disorders, neurocognitive ab-normalities, and cardiovascular diseases. We have previ-ously shown that exosomes containing trans-activating re-sponse (TAR) element RNA enhance susceptibility of un-differentiated naíve cells to HIV-1 infection. Up to a mil-lion copies of TAR RNA per microliter were also detectedin the serum from HIV-1 infected humanized mice suggest-ing that TAR RNA may be stable in vivo. We recently havefound another viral non-coding RNA that we termed TAR-gag which does not code for a protein but is present in theexosomes. Incubation of exosomes from HIV-1 infectedcells with primary macrophages resulted in a dramatic in-crease of pro-inflammatory cytokines, IL-6 and TNF-beta,indicating that exosomes containing TAR RNA could playa direct role in control of cytokine gene expression.Furthermore, the single stranded 5’ or 3’ processed stemRNA binding to TLRs activates the NF-kappaB pathwayand regulates cytokine expression. In our most recent data,we find that the number of exosomes from infected cells isincreased when cells are treated with cART. This directlyindicates that HIV viral release and exosome release haveoverlapping biogenesis pathways, including the ESCRTpathway. We will also discuss exosomes from otherneuro-tropic RNA viral infections, including HTLV-1,Ebola, RVFV, and Zika, and how they behave differentlythan exosomes from HIV infected cells. Collectively, ourresults imply that exosomes in viral infection control path-ogenesis and targeting these particles may be a method tolower overall viral burden in infected hosts.

P13Role of DNA damage response pathways in the activationof polyomavirus JC

Anna Bellizzi1,2, Martyn K. White2, Gabriele Ibba2, KamelKhalili2, Hassen S. Wollebo2


1Department of Public Health and Infectious Diseases,Institute Pasteur, Cenci-Bolognetti Foundation, SapienzaUniversity, Rome Italy; 2Center for Neurovirology,Department of Neuroscience, Lewis Katz School ofMedicine at Temple University, Philadelphia PA


Infection of glial cells by the human neurotropic polyomavi-rus JC (JCV), the causative agent of progressive multifocalleukoencephalopathy (PML) rapidly inflicts damage to cellu-lar DNA, which in turn evokes the DNA damage response(DDR). We previously reported that JCV rapidly induces ex-pression of the DNA repair factor Rad51 and Rad51 activatesthe JCVearly promoter in co-operation with the transcriptionfactor NF-kappaB p65. Thus Rad51 induction by JCV infec-tion serves as a positive feedback mechanism for viral activa-tion early in JCV infection. It is known that the DDRmay alsostimulate NF-kappaB activity, a phenomenon known asnucleus to cytoplasm or “inside-out” NF-kappaB signal-ing. This pathway is initiated by Ataxia telangiectasiamutated (ATM) protein, a serine/threonine kinase that isrecruited and activated by DNA double-strand breaks andsubsequently involves a series of post-translational modi-fications of NF-B essential modulator (NEMO), alsoknown as inhibitor of NF-kappaB kinase subunit gamma(IKK-gamma), which activates NF-kappaB. Here weshow that JCV infection causes phosphorylation and acti-vation of ATM while a specific small molecule inhibitorof ATM, KU-55933, inhibits JCV replication. Analysis ofthe effect of JCV infection on the subcellular distributionof NEMO by cell fractionation and immunocytochemistryshowed a redistribution of NEMO from the cytoplasm tothe nucleus. Co-expression of JCV large T-antigen andFLAG-tagged NEMO in cells showed the occurrence ofthe sumoylation of NEMO in the presence of T-antigen,while co-expression of ATM and FLAG-NEMO demon-strated physical association between ATM and NEMO.Taken together, we propose a model where JCV infectioninduces both the expression level of Rad51 protein andnucleus to cytoplasm NF-kappaB signaling, which thenact together to enhance viral gene expression.

P14CRISPR/Cas9 system as an agent for eradicationof polyomavirus JC infection

Anna Bellizzi1,2, Gabriele Ibba2, Rafal Kaminski2, Martyn K.White2, Wenhui Hu2, Jennifer Gordon2, Kamel Khalili2,Hassen S. Wollebo2


1Department of Public Health and Infectious Diseases,Institute Pasteur, Cenci-Bolognetti Foundation, SapienzaUniversity, Rome Italy; 2Center for Neurovirology,Department of Neuroscience, Lewis Katz School ofMedicine at Temple University, Philadelphia, PA

Progressive multifocal leukoencephalopathy (PML) is a fataldemyelinating disease of the CNS caused by lytic infection of

oligodendrocytes, which produce myelin in the brain, withhuman neurotropic polyomavirus JC (JCV). PML lesions areareas of demyelination containing oligodendrocytes with viralnuclear inclusion bodies and bizarre astrocytes, which are alsoproductively infected by JCV. Although JCV was isolatedover 40 years ago and has been extensively studied since,there is still no effective therapy for PML. Recently, a novelgenome-editing method was developed based on clusteredregularly interspaced short palindromic repeat (CRISPR) sys-tems. The CRISPR system uses a nuclease, CRISPR-associated (Cas9), that complexes with small RNAs asguides (gRNAs) to cleave DNA in a sequence-specificmanner upstream of the protospacer adaptor motif(PAM) in any genomic locat ion . Here , we useCRISPER/Cas9 system as a potential tool for JCV elimi-nation by designing guide RNAs (gRNAs) targeting re-gions within the early gene encoding viral protein, T-an-tigen. Our results indicated that Cas9/gRNAs targeted tothe JCV T-antigen gene effectively delete target DNA se-quence, reduce T-antigen expression and late promoteractivity and inhibit JCV DNA replication. No off-targeteffects of the JCV-specific CRISPR/Cas9 editing weredetected using the SURVEYOR assay. These data revealthe promise of CRISPR/Cas9 as a tool for the eliminationof the JCV genome and a potential as a cure for PML.

P15Inhibition of HSV-1 replication by a gene editing strategy

Anna Bellizzi1,3, Pamela C. Roehm2,3, Masoud Shekarabi3,Hassen S. Wollebo3, Gabriele Ibba3, Martyn K. White3,Lifan He3, Julian Salkind3, Kamel Khalili3


1Department of Public Health and Infectious Diseases,Institute Pasteur, Cenci-Bolognetti Foundation, SapienzaUniversity, Rome Italy; 2Department of Otolaryngology/Head and Neck Surgery - Department of Neurosurgery,Lewis Katz School of Medicine at Temple University,Philadelphia PA; 3Center for Neurovirology, Department ofNeuroscience, Lewis Katz School of Medicine at TempleUniversity, Philadelphia PA

HSV-1 induced illness affects more than 85% of adults world-wide and there is no permanent curative therapy. We usedRNA-guided CRISPR/Cas9 gene editing to specifically targetDNA sequences of the HSV-1 genome for deletion that spanthe region directing expression of ICP0, a key viral proteinthat stimulates HSV-1 gene expression and replication. Wefound that CRISPR/Cas9 introduced InDel mutations into ex-on 2 of the ICP0 gene profoundly reduced HSV-1 infectivityin permissive human cell culture models and protected


permissive cells against HSV-1 infection. CRISPR/Cas9mediated targeting ICP0 prevented HSV-1-induced disin-tegration of promonocytic leukemia (PML) nuclear bod-ies, an intracellular event critical to productive HSV-1infection that is initiated by interaction of the ICP0 N-terminus with PML. Combined treatment of cells withCRISPR targeting ICP0 plus the immediate early viralproteins, ICP4 or ICP27, completely abrogated HSV-1infection. We conclude that RNA-guided CRISPR/Cas9can be used to develop a novel, specific and efficacioustherapeutic and prophylactic platform for targeted viralgenomic ablation to treat HSV-1 diseases.

P16Mechanisms associated with differential inflammatoryeffects of Tat proteins derived from HIV-1 subtypes-Band recombinant CRF02_AG on human brainmicrovascular endothelial cells

BIJU BHARGAVAN1, GEORGETTE KANMOGNE1


1Department of Pharmacology and ExperimentalNeuroscience, University of Nebraska Medical Center

Previous studies showed that HIV-1 Tat induce blood–brainbarrier (BBB) dysfunction and is involved in HIV-associatedneurocognitive disorders (HAND). These studies were per-formed mostly with Tat from HIV-1 subtype-B (Tat.B),whereas 70 % of the 37 millions HIV-infected subjects arein Sub-Saharan Africa and are infected with non-B subtypes,including the recombinant HIV-1 CRF02_AG, prevalent inWest-Central Africa. The effects of this recombinant viruson the BBB and HAND are not known. In the current study,we analyzed the effects of Tat.B and Tat from HIV-1CRF02_AG (Tat.AG) on primary human brain microvascularendothelial cells (HBMEC), the major component of theBBB. Exposure of HBMEC to Tat.B increased the expressionand secretion of the proinflammatory cytokine IL6 by 9-fold(p<0.001) and increased IL6 mRNA by 113-fold (p<0.001),whereas Tat.AG increased IL6 expression by 2.7 to 3.8-fold,and increased IL6 mRNA by 35.7-fold (p<0.001) comparedto controls. Mechanistic studies showed that Tat.B inducedBBB inflammation through the IRAK1/4,MKK/JNK path-ways, in an AP1- and NFkB-independent manner, whereasTat.AG effects occurred via the MKK/JNK/AP1/NFkBpathways. We further showed that Tat-induced effectswere associated with activation of c-jun(serine-63) andSAPK/JNK(Thr183/Tyr185), demonstrated increased ex-pression of transcription factors associated with thesepathways (Jun, STAT1, RELB, CEBPA) with higherlevels in Tat.B-treated cells compared Tat.AG-treated

cells. Functional studies showed that both Tat.B andTat.AG decreased the expression of endothelial tight junc-tion proteins claudin-5 and ZO-1, and decreased braintrans-endothelial electric resistance (TEER), but greaterreduction in TEER was observed with Tat.B comparedto Tat.AG. Overall, our data showed increased BBB in-flammation and BBB disruption with Tat.B, compared toTat.G. This may suggests these two HIV-1 subtypes dif-ferentially affect the BBB and the CNS. Our future studieswill validate these findings by analyzing the direct effectsof these viral strains on the BBB and the CNS.

P17Dysregulation of epigenetic control of memory in HIVinduced mild cognitive disease in mice and disease reliefby valproic acid treatment

Alejandra Borjabad1, Wang Hongyin1, Wei Chao1, Boe-HyunKim1, Kelschenbach Jennifer1, Chao-Jiang Gu1, JacintaMurray1, Susan Morgello1, Mary Jane Potash1, David JVolsky1


1Department ofMedicine, Icahn School ofMedicine atMountSinai, New York, USA

HIV infected individuals on suppressive antiretroviral therapydevelop chronic mild neurocognitive impairments (mild NCI)that may affect everyday activities but do not progress to HIVdementia. The pathobiology of mild NCI is unclear and thereis no treatment, but one feature indicative of disease mecha-nism is an apparent discordance between broad neuronal dys-function and limited neuronal death. We investigated NCIbiology and treatment in experimentally induced disease inimmunocompetent mice infected with chimeric HIV,EcoHIV. We first show in behavioral tests that infected ani-mals develop chronic NCI involving at least two cognitivedomains, memory and executive function. The resemblanceof murine disease to human mild NCI was indicated by lowHIV burdens, mild neuroinflammation, and diffuse dendriticpruning in multiple brain regions but absence of overt neuro-pathology and cellular apoptosis. We then applied systemsbiology approach to gain an insight into disease pathobiology.Comparative analysis of brain gene expression profiles fromcognitively impaired mice and patients with HIV cognitivedisease revealed common downregulation of pathways con-trolling neuronal signal transmission and memory.Assessment of epigenetic control of these functions by chro-matin immunoprecipitation and sequencing indicated highcorrelation between histone 3 hypermethylation at lysine9 m3 and transcriptional suppression of genes associated withsynaptodendritic functions. Treatment of cognitively impaired


mice with valproic acid significantly mitigated murine NCI,normalized selected synaptic gene expression, and reversedhistone hypermethylation on their promoters. The results sug-gest dysregulation of epigenetic control of memory in HIVinduced mild cognitive dysfunction and indicate that the dis-ease is treatable.

P18Plasma soluble CD163 is associated with postmortembrain pathology in human immunodeficiency virusinfection.

Alex Bryant1, David Moore1, Tricia Burdo2, Jessica Lakritz3,Ben Gouaux1, Virawudh Soontornniyomkij4, CristianAchim4, Eliezer Masliah5, Igor Grant4, Andrew Levine6,Ronald Ellis5


1HIV Neurobehavioral Research Program, San Diego, CA;2Department of Neuroscience, Temple University, LewisKatz School of Medicine, Philadelphia, PA; 3Department ofBiology, Boston College, Chestnut Hill, MA; 4Department ofPsychiatry, University of California San Diego, La Jolla, CA;5Department of Neurosciences, University of California SanDiego, La Jolla, CA; 6Department of Neurology, University ofCalifornia Los Angeles, Los Angeles, CA

Background: Plasma soluble CD163 (sCD163), shed bymonocy t e s a nd mac r ophage s , c o r r e l a t e s w i t hneurocognitive impairment in human immunodeficiencyvirus (HIV) infection and is a potential biomarker for neu-rologic damage in HIV. We sought to determine the asso-ciation between plasma or cerebrospinal fluid sCD163 andpostmortem brain pathology. Methods: We measuredsCD163 levels in antemortem plasma (n= 54) and cerebro-spinal fluid (n = 32) samples from 74 HIV+ participants(median 5 months before death) who donated their brainsto research at autopsy. We quantified markers ofsynaptodendritic damage (microtubule-associated protein2 [MAP2], synaptophysin [SYP]), microgliosis (HLA-DR, ionized calcium binding adaptor molecule 1),astrocytosis (glial fibrillary acidic protein) and impairedprotein clearance (beta-amyloid) in frontal cortex, hippo-campus, putamen, and internal capsule. Multivariableleast-squares regression was used to evaluate the associa-tion between plasma or cerebrospinal fluid sCD163 andhistological measures, accounting for medical comorbidi-ties and correcting for multiple comparisons. Results:Higher plasma sCD163 was associated with lower MAP2in frontal cortex (B = −0.23, 95 % CI −0.41 to −0.06,p= 0.007), putamen (B = 0.32, 95 % CI −0.52 to −0.12,p= 0.004), and hippocampus (B =−0.23, 95 % CI −0.35

to −0.10, p= 0.007), and with lower SYP in hippocampus(B = −0.25, 95 % CI −0.42 to −0.03, p = 0.003) but notputamen or frontal cortex (p > 0.05). Higher plasmasCD163 was associated with higher HLA-DR in putamen(B= 0.17, 95 % CI 0.08 to 0.26, p< 0.001) and when aver-aged across three brain regions (B= 0.11, 95 % CI 0.05 to0.17, p< 0.001). Cerebrospinal fluid sCD163 was not as-sociated with any histological measure (p > 0.05).Conclusion: Higher plasma sCD163 in life is associatedwith greater synaptodendritic damage (SYP, MAP2) andmicroglial activation (HLA-DR) in cortical and subcorticalbrain regions. Plasma sCD163 warrants additional study asa biomarker of neurologic damage in HIV infection.

P19Extracellular vesicle mediated shuttling of miR-9mediatessynaptodendritic alterations in HAND

Shilpa Buch, Lu Yang, YeonHee Kook, Yu Cai, Guoku Hu(corresponding author: [email protected])

Depar tment of Pharmacology and Exper imenta lNeuroscience, University of Nebraska Medical Center

Reversible synaptodendritic injury and underlying neuroin-flammation are important phenotypes and correlates of HIV-associated neurocognitive disorders (HAND). Similar toHIV+ subjects on antiretroviral therapy (ART), SIV-infectedrhesusmacaques onART have also been shown to exhibit lossof synaptophysin, increased glial activation and dysregulationof various signature microRNAs (miRs). MiR-mediated reg-ulation of disease pathogenesis represents an evolving area ofresearch that has ramifications for identification of potentialtherapeutic targets for various neurodegenerative disorders,for which, currently there exists no cure. Parallel to the ad-vances made in miRNA research has been the advent of thefield of extracellular vesicles (EVs). EVs represent an impor-tant mode of intercellular communication by serving as con-duits for transfer of membrane and cytosolic proteins andRNA (including miRs) between cells. Despite effective sup-pression of virus replication in the presence of ART, viralproteins such as HIV Tat have been found to be present intissues such as the CNS and the lymph nodes, likely contrib-uting to ongoing neuroinflammation. In the current study wedemonstrate that exposure of astrocytes to HIV Tat resulted inincreased induction/release of miR-9 in the EVs. MiR-9-enriched EVs, were in turn, taken up by the neurons resultingin significant synaptodendritic injury. Furthermore, we alsoobserved downregulated expression of miR-9 targets -PDGF-CC, NLGN2 and MCPIP1 (key players in regulatingthe normal synaptic functioning in neurons) in SIV+/HIV+brains compared with the uninfected controls. Validation of


these findings in rat primary neurons infected with lentivirusexpressing miR-9 demonstrated decreased expression of therespective targets. Moreover, we also observed reduced ex-pression of these targets and concomitant synaprodendriticinjury in the neurons exposed to EVs isolated from condi-tioned media collected from HIV Tat-treated astrocytes.Taken together, our findings implicate that HIV-Tat mediatedalteration of synaptodendritic injury is regulated by exosomalmiR-9 via its targets (PDGF-CC/MCPIP1/NLGN2).

P20Neurotoxic lysosomal protease cathepsin B is presentin macrophage-derived exosomes and internalizedby neurons

Yisel Cantres-Rosario1, Karla Negron Roure2, MarinesPlaud1, Loyda Melendez3


1Department of Microbiology and Medical Zoology, Schoolof Medicine, University of Puerto Rico-Medical SciencesCampus; 2Department of Biology, University of Puerto Rico- Bayamon Campus; 3Department of Microbiology andMedical Zoology, School of Medicine & TranslationalProteomics Center, University of Puerto Rico-MedicalSciences Campus

Despite the efficacy of antiretroviral therapy, mild forms ofHIV-associated neurocognitive disorders (HAND) remainprevalent. HIV-infected macrophages infiltrate into thebrain secreting viral and cellular proteins that trigger neu-ronal dysfunction and death. One of the monocyte-derivedmacrophages (MDM) secreted proteins is cathepsin B, alysosomal protease that triggers neuronal apoptosisin vitro, and co-localizes with beta-amyloid peptides inbrain tissue from HAND and Alzheimer’s patients. Pre-treatment of macrophage-conditioned media (MCM) withanti-cathepsin B antibodies or inhibitors reduces neuronalapoptosis and beta-amyloid peptides in vitro. We havedemonstrated that recombinant active cathepsin B addedin MCM is internalized by neurons, and the levels of inter-nalization are proportional to the levels of HIV infection.Therefore, we hypothesize that targeting cathepsin B inMCM represents a viable approach to elucidate its mecha-nism of neuronal dysfunction. To test this hypothesis, weexposed SK-N-SH neuroblastoma cells to histidine-taggedcathepsin B in culture media alone or in presence of anti-cathepsin B antibody, and localized the histidine tag inneurons by intracellular flow cytometry. Histidine-taggedcathepsin B was internalized by neurons (52.0 %), a mech-anism that was partially reduced by the pre-treatment withanti-cathepsin B antibody (34.9 %). The neuroprotective

potential of cathepsin B antibody was confirmed by immu-nofluorescence, demonstrating increased neuronal MAP2staining and recovered morphology. Then, we examinedthe presence of cathepsin B in exosomes released by unin-fected and HIV-infected MDM at 12 days post-infection.Western blots revealed that exosomes isolated from MDMinfected in vitro contain cathepsin B in both pro-peptideand mature forms, and express CD63, an exosome marker.Our results suggest that cathepsin B might be secreted inexosomes by HIV-infected macrophages, proposing a nov-el mechanism by which cathepsin B triggers neuronal dys-function and opening doors for new studies in search forapproaches to decrease the neuronal dysfunction and sub-sequent development of HAND.

P21A novel deep sequencing platform (ViroFind) revealsa complex JC virus population in the brain of PMLpatients

Spyros Chalkias1, Joshua Gorham1, Erica Mazaika1, MichaelParfenov1, Xin Dang1, Steve DePalma1, David McKean1,Jonathan Seidman1, Igor Koralnik2


1Harvard Medical School; 2Rush University Medical Center

Background: Deep sequencing enables the unbiased detectionof viruses in clinical samples but it is limited by low cost-effectiveness. We developed a novel target-enrichment deep-sequencing platform (ViroFind) for the cost-effective se-quencing of viral genomes and we investigated the composi-tion of viral populations in the brain of 5 PML patients and in18 controls with no known neurological disease. Methods:ViroFind comprises 165,433 probes, complementary to theentire genomes of 386 DNA and RNAviruses known to infecthumans. The ViroFind probes are used in a hybridization re-action with nucleic acid libraries from clinical samples to en-rich only viral sequences. The output of this reaction is se-quenced via an Illumina platform and the sequencing data arealigned against a database of all viral genomes. Mapped se-quences are used to analyze viral genomes and identify viralvariants. Results: Compared to direct deep sequencing,ViroFind enriched the JC virus (JCV) sequences detectedper PML brain sample, ranging from 584 viral sequences(each of 75 bp length) per 1,000,280 total sequences up to375,653 viral sequences per 430,842 total sequences. Theenrichment of JCV sequences attributable to ViroFind rangedfrom 33 to 127-fold. Each JCV nucleotide was sequenced atleast 10 times (coverage 10X). We identified 24 viral capsidprotein VP1 variants and 12 of these variants were associatedwith amino acid substitutions. Mutation D66H, likely related


to VP1 conformational changes that could impact tissue tro-pism, was observed in 4 of 5 (80 %) PML brain samples.Lastly, we detected low frequency JCV sequences in 10 of18 (56 %) control brains. Conclusion: Multiple JCV variantsconstitute highly complex viral populations in the brain ofPML patients. JCV genome fragments can also be detectedin the brain of up to 56 % of individuals with no knownneurological disease, which suggests that JCV is present at alatent stage in the human brain.

P22HHV-6A infection induces the expression of HumanEndogenous RetroViral elements (HERV-W-Env)in human cells: link to the neuroinflammation

Benjamin Charvet1, Josephine Reynaud1, Gilda Raguenez2,Hei-Lanne Dougier2, Julie Medina3, Herve Perron3, PatriceMarche2, Branka Horvat1


1International Centre for Infectiology Research, INSERMU1111, CNRS UMR5308, Ecole Normale Superieure deLyon, University of Lyon 1, 69365 Lyon, France; 2INSERMU823, Universite J Fourier, IAPC, 38041 Grenoble, France;3GeNeuro Innovation, Lyon, France

Herpesvirus infections have been associated with the induc-tion of human endogenous retroviruses (HERV), suggestingthey could synergistically participate in the establishment ofdifferent pathologies in humans. Among the HERV-W fam-ily, the envelope protein (HERV-W-ENV) seems to be in-volved in the pathogenesis of multiple sclerosis (MS). Wehave analyzed here the interaction between HHV-6A andHERV-W-ENV and have particularly focused on thetransactivator role of CD46 molecule, acting as the receptorfor several viruses, including HHV-6A and measles. We in-fected several primary and immortalized human cell lines,including T lymphocytes, monocytes, glioblastoma and neu-roblastoma cells with HHV-6A (GS). The mRNA expressionof ENV from different HERVs (syncytin-1, MSRV andHERV-K18) was determined by quantitative RT-PCR andresults were confirmed by immunofluorescence. The in-volvement of CD46 in HERV-W-ENV activation was ana-lyzed using several CD46 ligands, including UV-inactivatedHHV-6A, measles virus and anti-CD46 antibody and recom-binant C3b complement fragment, which binds naturallyCD46. We showed that HERV-W-ENV over-expression inHHV-6A-infected human cells at transcript and protein level.Furthermore, the stimulation of HHV-6A receptor CD46,using UV-inactivated HHV-6A, anti-CD46-SCR3 domainAb or C3b fragment of complement also increases the pro-duction of HERV-W-ENV, in contrast to measles virus,

which binds different region of CD46 (SCR1-2 domains).These results suggested that induction of HERV-W is spe-cific for HHV-6A infection in neural and immune cells andthat engagement of CD46-SCR3 domain might be requiredfor the induction of HERV-W. The reactivation of HERV-W-ENV was shown to induce inflammatory responses throughTLR4. Therefore, these results expand the spectrum ofHHV-6 effects and of its receptor CD46 in the modulationof the immune response and support the hypothesis thatcross-talk between HHV-6A and HERV as well as comple-ment activation and consequent CD46 activation may par-ticipate in the pathogenesis of the neuroinflammation.

P23Neuro-inflammation modulates Fcgamma receptorexpression on microglial cells following viral braininfection

Priyanka Chauhan, Shuxian Hu, Wen Sheng, Sujata Prasad,James Lokensgard(corresponding author: [email protected])

Department of Medicine, University of Minnesota MedicalSchool

Fcgamma receptors (FcgammaRs) for IgG couple innate andadaptive immunity through their ability to activate effectorcells by antigen-antibody complexes. Brain-residentmicroglial cells, which are pivotal to pathogen detectionand initiation of innate neuro-immune responses, co-express activating (FcgammaRI, FcgammaRIII, andFcgammaRIV) as well as inhibitory Fcgamma Receptor(FcgammaRIIB). Thus, the threshold for cellular activationis determined by the relative ratios of these opposingFcgammaRs. In this study, we investigated the relative ex-pression of both activating and inhibitory FcgammaRs onmicroglia using an in vivo model of chronic neuro-inflammation following murine cytomegalovirus (MCMV)-induced encephalitis. Flow cytometric analysis of microglialcells obtained from infected brain tissue demonstrated thatthe activating FcgammaRs were expressed maximally at 5 dpost infection (dpi), while the inhibitory receptor(FcgammaRIIB) remained highly elevated during the chron-ic phase of infection (i.e., 30 and 60 dpi). The highly in-duced expression of activating FcgammaRIV during theacute phase of infection was also noteworthy. While,FcgammaRI was found to be constitutively expressed, therewas minimal expression of FcgammaRIII. Subsequently,when the relative expression of FcgammaRs was analyzedwithin the brains of mice post MCMV-infection by semi-quantitative RT-PCR (semi-qRT-PCR), we observed maxi-mal expression of FcgammaRIV during the acute phase


commensurate with our flow cytometric studies.Furthermore, in vitro analysis of cultured primary murinemicroglial cells using flow cytometry and semi-qRT-PCRdemonstrated maximal expression of FcgammaRIV andFcgammaRIIB following stimulation with either IFN-gamma or IL-4, respectively. We also demonstrated the roleof IFN-gamma and IL-4 in polarizing microglia towards M1or M2 phenotype, respectively. Thus, we conclude that acuteneuro-inflammation following viral infection increases ex-pression of activating FcgammaRs to promote pathogenclearance through increased effector cell activation. In con-trast, preferential expression of the inhibitory receptor duringchronic infection may provide a protective mechanism toprevent hyper-immune responses and subsequent bystanderbrain damage.

P24Computational detection of off-target effectsof CRISPR/Cas9-associated gRNAs

Cheng-Han (James) Chung, Michael Nonnemacher, BrianWigdahl, William Dampier(corresponding author: [email protected])


Despite antiretroviral therapy, HIV-1 infection remains alife-long clinical problem due to reservoirs harboring provi-ral DNA in a latent/persistent form. Recently, gene-editingstrategies utilizing the CRISPR/Cas9 system have been de-veloped to eradicate the HIV-1 genome from infected cells.These results offer a new avenue toward the elimination ofHIV-1 to cure HIV/AIDS. However, due to the promiscuousnature of the gRNA targeting, there is concern over off-target binding sites that may cause unwanted DNA damagewithin the human genome that need to be assessed. This iscomplicated by the non-linear nature of the binding penal-ties associated with gRNA recognition. With the availabilityof unbiased identification of double stranded breaks enabledby sequencing (GUIDE-seq), the off-target cleavage sitescan be precisely observed. In order to measure the off-target effect, we have developed a new database containingall potential cleavage sites within the entire human genomealong with all known single nucleotide polymorphisms indbSNP. Tree construction occurs on the order ofO(N)~Nlog(N) time where N is the size of the databaseand tree search occurs in O(m)~m where m is the lengthof the query. In practice, the entire human genome anddbSNP were indexed within 72 h and a query takes lessthan 10 ms per gRNA. This database will greatly increase

the ability of researchers to design gRNA that can cleaveHIV-1 while avoiding off-target effects. Future studies willbe performed to validate the off-target predictions usingbiomedical research laboratory techniques includingGUIDE-seq.

P25Alteredmitochondrial biogenesis in brains during HAND:Another result of Tat-mediated neurotoxicity?

Hamza Coban1, Jerel A. Fields1, Edward Rockenstein2, SarahGough1, Ilse Flores1, Eliezer Masliah2, Paula Desplats2,Cristian L. Achim1


1Department of Psychiatry, University of California SanDiego; 2Department of Neuroscience, University ofCalifornia San Diego

Background: Despite combined antiretroviral therapy(cART), HIV-associated neurocognitive disorders(HAND) remain a significant problem for the 1.4 millionpeople living with HIV in the USA. The causes of HANDare probably a diverse set of initiating factors, such aslow-level viral expression, drugs of abuse, cART neuro-toxicity, a combination of these factors and others, whichset in to action a cascade of inflammatory events thatculminate in the neurodegeneration associated withHAND. Multiple recent publications implicate alterationsin mitochondrial dynamics in neurons as a mechanism ofneurotoxicity in some HAND patients. Methods: In thecurrent study, we asked whether alterations in mitochon-drial biogenesis activities might also contribute to thepathogenesis of HAND in the cART era. To this end, weanalyzed expression levels and distribution patterns ofmitochondrial biogenesis related genes and proteins inthe frontal cortex of 37 well-studied cART-era HANDdonors from the NNTC cohorts. Results: We found sig-nificant increases in the master regulator of mitochondrialbiogenesis, peroxisome proliferator-activated receptorgamma coactivator (PGC)-1alpha, in brain tissues ofHAND donors; mRNA levels also indicated alteredPGC-1alpha expression. Immunohistochemichal analysesof PGC-1alpha show a distinct pattern in the brains ofHAND donors compared to HIV- control. Importantly,we found similar alterations brains of in inducible Tat tgmice compared to their non-tg counterparts. Discussion:In combination with recent reports, our data suggest thatalterations at both ends of the mitochondrial biogenesis-dynamics axis may contribute to HAND, and Tat may bean important player in some patients. As mitochondrialdysfunction is implicated in many neurodegenerative


disorders, further elucidation of these mechanisms mayprovide a promising therapeutic target.

P26Three Dimensional NMR Structure of the HumanPolyomavirus JC Agnoprotein Revealed That the NH2-and COOH-termini of the Protein Adopt IntrinsicallyUnstructured Conformation While the Center PortionForms an Alpha-helix

Pascale Coric1, A. Sami Saribas2, Jason Saredy2, MartynWhite2, Serge Bouaziz1, Mahmut Safak2


1Universite Paris Descartes, Sorbonne Paris Cite, Laboratoirede Cristallographie et RMN Biologiques, UMR 8015 CNRS,4 av. de l’Observatoire, Paris, France; 2Department ofNeuroscience, Center for NeuroVirology, Laboratory ofMolecular Neurovirology, Lewis Katz School of Medicine atTemple University, Philadelphia, USA

JC virus (JCV) causes a fatal demyelinating disease of thecentral nervous system (CNS), known as progressive multi-focal leukoencephalopathy (PML), in a subset of the patientswith underlying immunosuppression, including lymphoma,leukemia and AIDS due to the lytic infection of the glial cells,including oligodendrocytes, by the virus. The incidence ofPML dramatically increased in the era of the AIDS epidemicand this disease has also been steadily increasing among au-toimmune disorder patients, including multiple sclerosis andCrohn’s disease, who are treated with antibody-based drugs.JCVestablishes a sub-clinical persistant infection in the bodyduring early childhood, but upon reactivation, it causes theneurological manifestations of PML. JCV encodes a limitednumber of structural and regulatory proteins. Among those,agnoprotein has been shown to play essential regulatory rolesduring the viral replication cycle where JCV was demonstrat-ed to be unable to sustain its productive life cycle in theabsence of agnoprotein expression. Previous studies alsodemonstrated that agnoprotein forms highly stable dimersand oligomers through its Leu/Ile/Phe-rich domain.Subsequent NMR structural studies using a partialagnoprotein peptide revealed that the amino acids fromLys23 to Phe39, containing Leu/Ile/Phe-rich domain, adopta alpha-helical conformation. Here we report the complete 3Dstructure of the full-length peptide of agnoprotein by NMR.This 3D structure confirmed the localization of the previouslyreported alpha-helical domain of the protein and further re-vealed the presence of another shorter alpha-helix locatedbetween residues Leu6 and Lys13. The remaining regionsof the agnoprotein exhibit an intrinsically unstructured con-formation, the significance of which has yet to be

demonstrated. We are currently exploring the functions ofthese regions by mutagenesis. This study has been made pos-s ible by grants awarded to M. Safak from NIH(1R01NS090949-01A1) and Temple University DrugDiscovery Initiative (161398).

P27Effects of cocaine and Tat on astrocyte-derived cholesterolsupplied to neurons

Bianca Cotto1, William Yen1, Timothy Luongo2, PrasunDatta1, Satish Deshmane1, John Elrod2, Dianne Langford1


1Department of Neuroscience, Center for Neurovirology;2Department of Pharmacology, Center for TranslationalMedicine, Lewis Katz School of Medicine at TempleUniversity, Philadelphia, PA USA

The human brain requires a balance of cholesterol homeo-stasis to meet the high metabolic requirements of neuronsand maintain myelin integrity. Neurons rely on cholesterolsynthesized by astrocytes for synaptogenesis and normalfunctioning. In the brain, cholesterol synthesis and clear-ance are tightly regulated to maintain cholesterol levels.Liver X receptors (LXRs) are the master regulators ofcholesterol homeostasis in the CNS. LXR activation leadsto the transcription of target genes involved in cholesteroltrafficking and efflux, including apolipoprotein E (ApoE).The disturbance of LXR signaling in the brain can lead tosignificant dysfunction in cholesterol homeostasis and dis-ruptions have been implicated in several neurological dis-eases. Likewise, HIV infection and cocaine use are asso-ciated with myelin loss and synaptodendritic damage sug-gesting that dysregulation in CNS cholesterol metabolismmay be involved in the progression of neurological impair-ment. Therefore, given the importance of astrocytes inLXR-mediated cholesterol regulation and its role in pro-viding metabolic support to other CNS cells, we hypothe-sized that exposure of astrocytes to cocaine and Tat wouldlead to disruptions in LXR signaling and the expression ofLXR regulated genes. Alterations in these pathways mayin turn decrease the bioavailability of cholesterol fromastrocytes to neurons and oligodendrocytes and promotecellular dysfunction. We found that in astrocytes, exposureto cocaine and Tat significantly decreased LXRbeta ex-pression. In addition, we discovered that LXR target genesincluding ApoE were also significantly reduced upon treat-ment with cocaine and Tat. In response, the ABC1a trans-porter responsible for transporting ApoE bound cholesterolout of the cell is increased significantly. Our findingsdemonstrate the combined effects of cocaine and Tat on


the overall bioavailability of cholesterol provided by astro-cytes to neurons and identify a novel role for LXR agonistas a therapeutic intervention in the severity and progres-sion of HIV infection and/or cocaine abusing individuals.

P28Microglial activation and cocaine addiction

Bianca Cotto1, Christina Chambers2, Hongbo Li2, WilliamYen1, Ronald Tuma2, Sara Jane Ward2, Dianne Langford1


1Department of Neuroscience, Center for Neurovirology;2Department of Pharmacology, Center for Substance AbuseResearch, Lewis Katz School of Medicine at TempleUniversity, Philadelphia, PA USA

Recent research shows that glial cells are intimatelyinvolved in synaptic plasticity, and that drugs of abuseaffect glial activity. Specifically, microglia contribute tosynaptic plasticity via direct interactions with dendriticspines, synaptic pruning, and regulation of hippocampalneurogenesis. We have combined a series of in vivo,ex vivo, and in vitro experiments to test the hypothesisthat cocaine exposure can lead to significant alterationsin microglial activation, enhancing cocaine-inducedneuroplasticity. In turn, these cascades contribute to pe-ripheral immune cell invasion, thereby priming theneuroimmune axis for a cycle of neuroinflammation inthe context of cocaine addiction. We found that cocaineself-administration in the rat model increases microglialactivation in reward regions of the brain, and increasesexpression of the transcription factor and synaptic plas-ticity marker MeCP2. We then determined in vitro thatmicroglia express MeCP2 and that this expression in-creases significantly following cocaine exposure and in-ducing release of BDNF, suggesting that cocaine’s ef-fects on MeCP2 expression may participate in cocaine-induced neuroplasticity. In addition, we observed thatchronic cocaine administration increases cerebrovascularleukocyte rolling and adhesion and subsequent BBBweakening that persists during withdrawal, setting upthe likelihood for a persistent dysregulation ofneuroimmune signaling that may mirror the cycle ofcocaine addiction. These findings identify novelneuroimmune targets such as activated microglia fortreating psychostimulant abuse, for which no approvedmedications exist. Our findings also illustrate mecha-nisms in the cocaine abuse paradigm that are likelyimportant in CNS disease progression in HIV-infectedpatients who use cocaine.

P29Agnoprotein interferes with the neuroimmune responseto JC Virus

Michael Craigie, Stephanie Cicalese, Martina Donadoni,Ilker Sariyer(corresponding author: [email protected])

Department of Neuroscience, Center for Neurovirology,Lewis Katz School of Medicine at Temple University,Philadelphia, PA USA

JC virus is a human neurotropic polyomavirus and thee t i o l o g i c a g e n t o f p r o g r e s s i v e m u l t i f o c a lleukoencephalopathy, a demyelinating disease of grayand white matter. PML is primarily observed in immu-nocompromised patients, including patients with AIDSand those undergoing immunomodulatory therapies.During JCV infection, the virus encodes multiple viralproteins including the regulatory agnoprotein. We haverecently discovered that agnoprotein is secreted frominfected cells into the extracellular matrix. Also, previ-ous data demonstrated that JCV infection is suppressedby treatment with conditioned media from peripheralblood mononuclear cells, suggesting that the immunesystem and immunological soluble factors play a majorrole in the regulation of JCV. Here, we have investi-gated the possible role of extracellular agnoprotein inthe neuroimmune response to JCV in glial cells.Following the cellular expression of agnoprotein,agnoprotein is released by glial cells and is able tobe internalized by both astrocytes and microgliain vitro. This internalization of agnoprotein was foundto impact the profile of cytokines released by both celltypes. Moreover, the treatment of astrocytes with me-dia containing agnoprotein resulted in a significant re-duction in GM-CSF secretion. Subsequent reportergene analysis demonstrated that agnoprotein is capableof suppressing the transcription of GM-CSF, implicat-ing a possible mechanism for the reduction of releasedGM-CSF levels. Likewise, the treatment of a humanmonocyte cell line, U937, with agnoprotein resultedin decreased macrophage differentiation, including de-creased attachment and decreased phagocytic ability.Similarly, treatment of peripheral blood mononuclearcells with agnoprotein resulted in decreased overall mi-gration across an in vitro blood brain barrier model.These findings have suggested that extracellularagnoprotein is able to modulate aspects of the immuneresponse to JCV, primarily through the suppression ofGM-CSF release and subsequent impact on monocyte/macrophage function.


P30HIV-1 genetic variation resulting in the developmentof new quasispecies continues to be encounteredin the peripheral blood of well-suppressed patients

William Dampier1, Michael Nonnemacher1, Joshua Mell1,Joshua Earl1, Vanessa Pirrone1, Benjamas Aiamkitsumrit1,Wen Zhong1, Katherine Kercher1, Shendra Passic1, JeanWilliams1, Zsofia Szep2, Jeffrey Jacobson3, Brian Wigdahl1


1Department of Microbiology and Immunology, Institute forMolecular Medicine and Infectious Disease; 2Department ofMedicine, Division of Infectious Diseases and HIVMedicine,Drexel University College of Medicine; 3Department ofMedicine, Section of Infectious Disease, Lewis Katz Schoolof Medicine, Temple University

As a result of antiretroviral therapeutic strategies, humanimmunodeficiency virus type 1 (HIV-1) infection has be-come a long-term clinically manageable chronic diseasefor many infected individuals. However, despite this prog-ress in therapeutic control, including undetectable viralloads and CD4+ T-cell counts in the normal range, viralmutations continue to accumulate in the peripheral bloodcompartment over time, indicating either low level reacti-vation and/or replication. Using patients from the DrexelMedicine CNS AIDS Research and Eradication Study(CARES) Cohort, whom have been sampled longitudinal-ly for more than 7 years, genetic change was modeledagainst the dominant integrated proviral quasispecies withrespect to selection pressures such as therapeutic interven-tions, AIDS-defining illnesses, and other factors.Phylogenetic methods based on the sequences of theLTR and tat exon 1 of the HIV-1 proviral DNAquasispecies were used to obtain an estimate of an aver-age mutation rate of 5.3 nucleotides (nt)/kilobasepair (kb)/year (yr) prior to initiation of antiretroviral therapy(ART). Following ART the baseline mutation rate wasreduced to an average of 1.02 nt/kb/year. The post-ARTbaseline rate of genetic change, however, appears to beunique for each patient. These studies represent our initialsteps in quantifying rates of genetic change among HIV-1quasispecies using longitudinally sampled sequences frompatients at different stages of disease both before and afterinitiation of combination ART. Notably, while long-termART reduced the estimated mutation rates in the vast ma-jority of patients studied, there was still measurable HIV-1mutation even in patients with no detectable virus by stan-dard quantitative assays. Determining the factors that af-fect HIV-1 mutation rates in the peripheral blood may leadto elucidation of the mechanisms associated with changesin HIV-1 disease severity.

P31Defining Vpr sequence variants and associatedmechanisms that enhance HIV CNS pathogenesisand disease

William Dampier1, Gregory Antell2, Michael Cruz3,Benjamas Aiamkitsumrit1, Michael Nonnemacher1, ZsofiaSzep4, Jeffrey Jacobson5, Vanessa Pirrone1, Wen Zhong1,Katherine Kercher1, Shendra Passic1, Jean Williams1, TonyJames1, Kathryn Devlin6, Tania Giovannetti7, David Libon8,Garth Ehrlich1, Brian Wigdahl1, Fred Krebs1


1Department of Microbiology and Immunology, Institute forMolecular Medicine and Infectious Disease, DrexelUniversity College of Medicine; 2School of BiomedicalEngineering, Science, and Health Systems, DrexelUniversity; 3Interdisciplinary Health Sciences (IHS)Program, Graduate School of Biomedical Sciences andProfessional Studies, Drexel University College ofMedicine; 4Department of Medicine, Division of InfectiousDiseases and HIV Medicine, Drexel University College ofMedicine; 5Department of Medicine, Section of InfectiousDisease, Lewis Katz School of Medicine, TempleUn ive r s i t y ; 6Depa r tmen t o f Neu ro sc i ence andComprehensive NeuroAIDS Center, Lewis Katz School ofMedicine, Temple University; 7Department of Psychology,Temple University; 8Department of Geriatrics andGerontology, New Jersey Institute for Successful Aging,School of Osteopathic Medicine, Rowan University

Studies of HIV-associated neurocognitive disorders(HAND) have identified viral protein R (Vpr) as aneuropathogenic factor. Extracellular Vpr, which has beendetected in the sera and cerebrospinal fluids of HIV-1-infected patients, causes adverse changes in brain residentcells, including macrophages, astrocytes, and neurons. Ourrecent studies of Vpr have turned to investigating connec-tions between naturally occurring Vpr sequence variationand neuropathogenesis in HIV-1-infected patients from theDrexel Medicine CNS AIDS Research and EradicationStudy (CARES) Cohort. Analyses of peripheral blood-derived Vpr sequences from Cohort patients revealed threeamino acids in Vpr (positions 37, 41, and 55 in the 96 aaprotein) associated with significant differences in neuro-psychological impairment (niVpr variants) measured bycomprehensive neuropsychological testing. 37-I and 41-Swere linked to reductions in diagnosed neuropathogenesis(with respect to the average Global Deficit Score or GDS),while 41-N and 55-A were associated with increasedneuropathogenesis and higher GDS. The effects of niVprvariants appear to be cumulative, since the combined ef-fect sizes for Vpr sequences with two or three niVpr


variants preliminarily correlate with changes in averagepatient GDS. Deep sequencing demonstrated the appear-ance of niVpr variants in HIV-1-infected patient blood,spleen, and brain samples, and indicated sequence com-partmentalization as well as variations in relative preva-lence. The pathogenic effects of Vpr variation onimmunopathogenesis and neuropathogenesis were sug-gested, respectively, by higher CD4 T-cell counts in pa-tients with the 41-N variant (relative to cell counts inpatients with the 41-S variant) and greater reductions inastrocyte glutathione pools subsequent to transient expres-sion of the 41-N Vpr variant (relative to the 41-S variant).

P32Astrocyte-shed extracellular vesicles regulate synchronousnetwork burst activity

Raha Dastgheyb1, Saja Khuder1, Joelle Dorskind1, ShengWang1, Amrita Datta Chaudhuri1, Seung-Wan Yoo1,Norman Haughey2


1Department of Neurology, Johns Hopkins University Schoolof Medicine; 2Department of Neurology, Johns HopkinsUniversity School of Medicine. Department of Psychiatry,Johns Hopkins University School of Medicine

Synaptic damage and disruption of neuronal networks areprominent features of HIV Associated NeurocognitiveDisorders (HAND). The underlying mechanisms for thesepathological alterations in synaptodendritic networks arelargely unknown, but are thought to involve chronic glialactivation and inflammation. Based on evidence thatastroglia to neuron communication regulates the formationand strength of synaptic connections, we hypothesized thatHIV infection disrupts neural network activity by modifyingthe quality of astrocyte to neuron communication.Extracellular vesicles (EVs) are emerging as a mechanismby which astroglia regulate neuronal communication throughthe delivery of protein, RNA and lipid cargo that regulatethe activity of target cells. In this study we isolatedexosomes shed from astrocytes in response to ATP (a tro-phic stimulus), or media from HIV-infected macrophages(HIV-MDM; HIV and EV depleted), and exposed primaryrodent neurons to a dose response of 5–100 EVs/cell. Theeffects of EVs on dendritic length, branching, and complex-ity were determined using structured fluorescent imaging,and neural connectivity was determined using multichannelelectrode arrays. Spike detection was accomplished usingfiltered voltage measurements with a threshold for detectionof 5 standard deviations above baseline. A MATLAB basedcode was developed to extract spike times that allow for the

creation of 3D animations to visualize spike progression inindividual electrodes over time, burst identification, spikeand burst firing rates, and periodicity detection. We foundthat exosomes released from astrocytes in response to ATPstimulation produced a dose-dependent increase in dendriticcomplexity, increased neural network connectivity, and peri-odic synchronous network burst activity. In contrast,exosomes shed from astrocytes in response to HIV-MDSreduced dendritic complexity. These data suggest that EVsshed from astrocytes strengthen neural network connectivityand in the setting of HIV infection EVs may deconstructneural networks possibly by modifying the composition ofEV cargo.

P33Follicular DCs within deep cerebral lymph nodes arepotential reservoir of HIV-CNS infection

Rajnish S. Dave1, Artinder P. Nehra1, Roshell R. Muir2,Rashida Ginwala1, Claire Deleage3, Divya Sagar1, BrianWigdahl1, Zafar K. Khan1, Elias Haddad4, Jacob D. Estes3,Francois Villinger5, Aftab Ansari5, Siddappa Byrareddy6,Pooja Jain1


1Department of Microbiology and Immunology, and theInstitute for Molecular Medicine and Infectious Disease,Drexel University College of Medicine, Philadelphia, PA;2Division of Infectious Disease and HIV Medicine,Department of Medicine, Drexel University College ofMedicine, Philadelphia, PA; 3AIDS and Cancer VirusProgram, Frederick National Laboratory for CancerResearch, Leidos Biomedical Research, Inc., Frederick, MD;4Division of Infectious Disease and HIV Medicine,Department of Medicine, Drexel University College ofMedicine, Philadelphia, PA; 5Department of Pathology &Laboratory Medicine, School of Medicine and EmoryVaccine Center, Emory Universi ty, Atlanta, GA;6Department of Pharmacology and ExperimentalNeuroscience, University of Nebraska Medical Center,Omaha, NE

Human immunodeficiency Virus (HIV) neuroinvasion leadsto neurodegeneration and HIV-associated neurocognitive dis-order. In the central nervous system (CNS) circulating dendrit-ic cells (cDCs) encounter HIV virions or viral proteins andmigrate along the rostral migratory system to the cervical orcerebral lymph nodes (CxLNs). Subsequently, HIV accumu-lates in the germinal centers (GCs) and there it appears boundto follicular dendritic cells (FDCs). In this study, we sought todissect the mechanisms underlying harboring of HIVon FDCsand their transmission to CD4+ T follicular helper (TFH) cells


in CxLNs of chronically SIV-infected rhesus macaques(RMs). Chronic SIV infection was established in all RMs.CD35+ FDCs in CxLNs were identified to harbor SIV.Furthermore, FcRs (FcgammaRIIB) immune complexes co-localized with the FDCs and B-cells, suggesting SIV presen-tation by FDCs to B-cells. Significantly, B-cell interactionwith TFH cells resulted in the latter getting infected. Hence,FDCs in CxLN not only retained SIV, but they also infectedTFH cells. Based on these observations, we infer a novel rolefor FDCs in viral entrapment and creation of persistent CNSreservoir. Hence, our study highlights the role of FDCs inaccumulation and transmission of HIV with implications forHIV egress from the CNS.

P34Sex Matters: Differential Deficits in Sustained Attentionin the Male and Female HIV-1 Transgenic (Tg) Rat

Iris K. Dayton, Kristen A. McLaurin, Rosemarie M. Booze,Charles F. Mactutus(corresponding author: [email protected])

Department of Psychology, University of South Carolina

Neurocognitive impairment (NCI) is a prevalent conditionof HIV-1 infected individuals because of the remarkablesuccess of combined antiretroviral therapy. Recent studiessuggest an effect of gender on the domains of speed ofinformation processing, verbal fluency, learning, andmemory. Although it has been reported that the risk fordeveloping NCI for HIV-1 men and women differs, theexamination of biological sex differences in NCI, inde-pendent of societal factors, has not been previously con-ducted. Deficits in executive function are a distinctivecharacteristic of NCI. Presently, we examined the poten-tial sex differences in sustained attention deficits, as acore component of executive function using the HIV-1Tg rat. Male Fischer HIV-1 Tg (n= 35) and F344/N con-trol rats (n = 35), and intact female Fischer HIV-1 Tg(n = 35) and F344/N control rats (n = 33) were trained(shaping) and tested (vigilance and manipulation of stim-ulus duration) in a signal detection task. There weremarked sex differences in initial training of the signaldetection task, acquisition of the vigilance task, and per-formance on the duration task. Females acquired the sig-nal detection task at a much slower (4X) rate than males.Females met criterion of five consecutive days or sevennon-consecutive days at 70 % or higher for the vigilancetask at a significantly slower rate than males; there was a14-day lag before any female met the acquisition criteri-on. The factor of biological sex altered the ability to dis-criminate signals (illumination for 100, 500, or 1000 msec

duration) from non-signals (no illumination) during theduration task. Both HIV-1 Tg and control females wereunable to discriminate signals presented at shorter dura-tions than their male counterparts. Collectively, thesefindings suggest that the factor of biological sex has asignificant effect on the development of NCI for HIV-1patients. Funded by NIH grants DA013137, HD043680,and MH106392.

P35IFN-Gamma Inhibits JC Virus Replication in Glial Cellsby Suppressing T-Antigen Expression.

Francesca Isabella De Simone, Rahsan Sariyer, ShadanYarandi, Michael Craigie, Jennifer Gordon, Ilker Sariyer(corresponding author: [email protected])

Temple University Lewis Katz School of Medicine

Patients undergoing immune modulatory therapies for thetreatment of autoimmune diseases such as multiple sclerosis,and individuals with an impaired-immune system, most nota-bly AIDS patients, are in the high risk group of developingprogressive multifocal leukoencephalopathy (PML), an oftenlethal disease of the brain characterized by lytic infection ofoligodendrocytes in the central nervous system (CNS) with JCvirus (JCV). Here, we investigated the impact of soluble im-mune mediators secreted by activated PBMCs on viral repli-cation and gene expression by cell culture models and molec-ular virology techniques. Our data revealed that viral geneexpression and viral replication were suppressed by solubleimmune mediators. Further studies demonstrated that solubleimmune mediators secreted by activated PBMCs inhibit viralreplication induced by T-antigen, the major viral regulatoryprotein, by suppressing its expression in glial cells. This un-expected suppression of T-antigen was mainly associated withthe suppression of translational initiation. Cytokine/chemokine array studies using conditioned media from acti-vated PBMCs revealed several candidate cytokines with pos-sible roles in this regulation. Among them, only IFN-gammashowed a robust inhibition of T-antigen expression. Whilepotential roles for IFN-beta, and to a lesser extent IFN-alphahave been described for JCV, IFN-gamma has not been pre-viously implicated. Further analysis of IFN-gamma signalingpathway revealed a novel role of Jak1 signaling in control ofviral T-antigen expression. Furthermore, IFN-gamma sup-pressed JCV replication and viral propagation in primary hu-man fetal glial cells, and showed a strong anti-JCV activity.Our results suggest a novel role for IFN-gamma in the regu-lation of JCV gene expression via downregulation of the ma-jor viral regulatory protein, T-antigen, and provide a new av-enue of research to understand molecular mechanisms for


downregulation of viral reactivation that may lead to develop-ment of novel strategies for the treatment of PML.

P36Alcohol-Mediated Alternative Splicing of Mcl-1pre-mRNA is Involved in Ethanol Induced Neurotoxicity

Francesca Isabella De Simone1, Rahsan Sariyer1, Sulie L.Chang2, Ilker Sariyer1


1Department of Neuroscience, Center for NeuroVirology,Lewis Katz School of Medicine at Temple University;2Institute of NeuroImmune Pharmacology, Seton HallUniversity

Heavy and chronic ethanol exposure can cause signifi-cant structural and functional damage to the adult brain.The developing nervous system is even more vulnerableto ethanol exposure. Prenatal exposure of ethanol duringpregnancy can lead to fetal alcohol spectrum disorders(FASD), characterized by malformation of the nervoussystem and mental retardation. The most devastatingconsequence of ethanol exposure is the neurotoxicityassociated with the depletion of neurons. It is crucialto elucidate mechanisms of neuroapoptosis in order todevelop effective therapeutic approaches to overcomeethanol-induced neuropathologies. Regulation of splicevariants in the brain can modulate protein functions,which may ultimately affect behaviors associated withalcohol dependence and ethanol-mediated neurotoxicity.Limited number of studies has shown that pre-mRNAsplicing patterns of genes are potentially altered andinvolved in behavior changes associated with alcohol-ism. Since alcohol consumption is associated with neu-rotoxicity, it is possible that altered splicing of survivaland pro-survival factors during the development of al-coholism may contribute to the neurotoxicity. Our re-sults suggest that ethanol exposure can lead to pre-mRNA missplicing of Mcl-1, a pro-survival member ofthe Bcl-2 family, by downregulating the expressionlevels of serine/arginine rich splicing factor 1 (SRSF1).The pre-mRNA of Mcl-1 can be alternatively spliced toremove exon 2, which produces shortened form of Mcl-1, named Mcl-1S. While the longer gene product Mcl-1 L enhances cell survival, the alternatively splicedshorter gene product Mcl-1S promotes apoptosis. Ourpreliminary data has indicated that ethanol exposure toneurons leads to a decrease in the ratio of Mcl-1 L/Mcl-1S by favoring pro-apoptotic Mcl-1S splicing over anti-apoptotic Mcl-1 L isoform suggesting that Mcl-1S may

play a crucial role in neurotoxicity associated with al-cohol consumption.

P37The role of infiltrating macrophages and IL-6 productionon seizure development after viral infection

Ana Beatriz DePaula-Silva, Tyler Hanak, Daniel Doty, JordanSim, Jane Libbey, Robert Fujinami(corresponding author: [email protected])

University of Utah, School of Medice, Department ofPathology

Epilepsy is a chronic neurological disorder characterizedby recurrent seizures. Seizures, which are often associ-ated with viral encephalitis, occur in response to imbal-ances between excitatory and inhibitory inputs withinthe brain. Patients with viral encephalitis are 16 timesmore likely to develop epilepsy. It is estimated thataround 65 million people worldwide suffer from epilep-sy. Although treatments to prevent seizures are avail-able, they are mainly anticonvulsants and around 30 %of the patients do not respond to the medication andnumerous side effects related to the drugs have beenreported. In order to develop new immunomodulatorytreatments that could lead to an eventual cure for sei-zures/epilepsy, a better understanding of the seizure de-velopment mechanism is required. We have developedan experimental model of virus-induced seizures/epilep-sy. In our model, C57BL/6 mice are infected intra-cranially (i.c) with the Daniels strain (DAV) ofTheiler’s murine encephalomyelitis virus (TMEV).Between 3 and 10 days post infection (d.p.i), around50 % of the infected mice will develop spontaneousacute seizures and approximately 50 % of the mice thathave experienced spontaneous acute seizures will devel-op epilepsy. Because seizures begin to develop at 3d.p.i, we believe it happens as a consequence of theactivation of the innate immune response. We found thatseizures are correlated with an increase in myeloid andlymphoid cells infiltrating into the brain. We also foundthat the pro-inflammatory cytokines IL-6 and TNF alphawere elevated at 3 d.p.i. IL-6 knockout mice infectedwith DAV experienced significant fewer seizures.Because infiltrating macrophages are the main producersof IL-6, we depleted macrophages from mice and weinfected them with DAV. We did not observed seizuresin animals lacking macrophages, suggesting macro-phages are playing a central role in the developmentof seizures after viral infection.


P38Macrophage OXPHOS regulation by HIV-1 Vprand cocaine

Satish Deshmane, Shohreh Amini, Prasun Datta(corresponding author: [email protected])

Neuroscience, Comprehensive NeuroAIDS center, LKSOMat Temple University

HIV-1 infected macrophages play a significant role in thepathogenesis of AIDS. HIV-1 viral protein R (Vpr) notonly facilitates HIV-1 infection but also contribute tolong-lived persistence in macrophages. Our previous stud-ies using SILAC-based proteomic analysis showed that theexpression of critical metabolic enzymes in the glycolyticpathway and tricarboxylic acid (TCA) cycle were altered inresponse to Vpr expression in macrophages. Mitochondriaare key players in the generation and regulation of cellularbioenergetics, producing the majority of adenosine triphos-phate molecules by the oxidative phosphorylation system(OXPHOS). We therefore assessed the effects of Vpr aloneand Vpr in combination with cocaine on the relative pro-tein expression of OXPHOS mitochondrial complexes inmacrophages. U937 differentiated macrophages weretransduced with adenoviral vector harboring Vpr gene.Transduced cultures were either left untreated or treatedwith 5‘microM of cocaine for 1 and 3 days. Cell-lysateswere prepared and electron transport complexes were de-tected using an antibody mix that detects key componentproteins from each complex (CI-NDUFB8, CII-SDHB,CIII-UQCRC2, CIV-MTCO1 and CV-APT5A). ComplexIII, IV and V levels stayed constant however, complex Iand II were found to be negatively affected by Vpr.Complex-I was found to be downregulated by 32 % onday 1 and day 3 post-transduction with Vpr. Complex-IIwas found to be downregulated by 33 and 71 % on day 1and day 3. With cocaine, a transient upregulation of com-plex I and II was found on day 1. However, both com-plexes I and II were found to be downregulated by 70 %and 32 % respectively on day 3 post-transduction.Together, our data show that Vpr and cocaine reprogrammacrophage OXPHOS. Funded by NIDA.

P39Critical Role of Human Endogenous Retroviruses(HERVs) in Neurodevelopment and NeurodevelopmentalTumors.

Tara Doucet-O’Hare1, Kory Johnson2, Tongguang Wang3,Marie Medynets3, Winson Ho4, Zhengping Zhuang5,Avindra Nath6


1Section of Infection of the Nervous System; NationalInstitute of Neurological Disease and Stroke (NINDS); NIH;2Bioinformatics Section of Information Technology Program,NINDS, NIH; 3Neural Differentiation Unit, NINDS, NIH;4C ommun i t y N e two r k s P r o g r am , S e c t i o n o fNeurophysiology and Biophysics, NINDS, NIH; 5NationalCancer Institute, NIH; 6Section of Infection of the NervousSystem, NINDS, NIH

Human Endogenous Retroviruses (HERVs) comprise approx-imately 8 % of the human genome and play a critical, highlyregulated spatiotemporal role in cellular differentiation duringdevelopment. We hypothesize that HERVs play a critical rolein neuronal differentiation, and their dysregulation results inneurodevelopmental tumors. Atypical Teratoid RhabdoidTumor (AT/RT) is a rare type of pediatric brain cancer. Thetumor cells have an expression profile similar to embryonicstem cells (ESCs) and induced pluripotent stem cells (iPSCs).We characterized the cell line by immunostaining and foundthe cells strongly express Pax6, a neuro-ectodermal marker,and Oct4, an iPSC marker while having low expression ofBeta tubulin, a neuronal marker, and Nestin, a neuronal stemcell marker. Interestingly, when the AT/RTcell line was grownas an adherent line using matrigel and incubated in differenti-ation media, the cells began to differentiate into neuronal pre-cursors and neurons with increased expression of both MAP2and Nestin proteins. We analyzed each of the cell types anddetermined the stage of differentiation using RT-PCR andqPCR; furthermore, we compared the AT/RT derived neuro-nal cells with neurons generated from iPSCs. Similar toiPSCs, the AT/RT cells expressed HERV-K; however,HERV-K expression decreased dramatically as the cells dif-ferentiated into neuronal precursors and finally neurons.When we blocked HERV-K expression using siRNA iniPSCs we observed neuronal differentiation as determinedby RT-PCR, qPCR, and immunostaining. Our data leads usto conclude that HERV elements likely play a key role inneuronal differentiation; furthermore, it raises the possibilitythat manipulation of HERV expression could be utilized as atreatment modality for neurodevelopmental tumors.

P40Use of a lip scarification mouse model to studythe pathology, immunology, and virology associatedHSV-1 Infection of the periphery and correspondingtrigeminal ganglia

Kevin Egan, Alex Allen, Brian Wigdahl, Stephen Jennings(corresponding author: [email protected])



Herpes simplex virus type 1 (HSV-1) replicates in epithelialcells of mucosal surfaces before establishing a lifelong latentinfection within the trigeminal ganglion. Although there isan established ocular infection model in the laboratorymouse which reproduces primary infection and latency ob-served in human, the majority of primary human infectionsoccur within the lip and latency is established within a dif-ferent branch of the trigeminal ganglion. Based on this, thelip scarification model has been used to study the geneticloci of HSV-1 resistance in the laboratory mouse, howeverthe basic kinetics of and the pathologic and immunologicresponses to viral infection have not been fully defined.Using the McKrae strain of HSV-1, we have proceeded todefine the kinetics of viral infection in the lip. The lower lipof 3-month-old mice were scarified and inoculated withHSV-1 and tissue was collected at 7 time points up to day60 post infection for detection of infectious virus andresponding immune cells. High virus titers were detectedin the lip at early time points that resolved after 8 days ofexposure. Lip pathology peaked 5 days post-infection andresolved 15 days post-infection with residual lymphocytespresent. CD45+ cells including CD4+ and CD8+ T lympho-cytes infiltrated the TG and associated with neurons. CD8+and CD4+ T lymphocytes were retained in the trigeminalganglia and were present at 30 and 60 days post-infection.Analysis of genome copies revealed that genome copiespeaked at 8 days post-infection in the lip. In the trigeminalganglia genome copies established a steady level at 5 dayspost-infection. These results are the first descriptions ofHSV-1 kinetics and pathology in the mouse lip. This modelwill be useful in studying the virologic and immunologicaspects associated with HSV-1 infection of the lower lipwhere most human infections occur.

P41Novel molecular pathway of perturbation of ATP releasein astrocytes contributes to neuronal death in HAND

Mahar Fatima1, Bharat Prajapati2, Kanza Saleem2, ChitraSingal2, Pankaj Seth2


1Cellular and Molecular Neuroscience, National BrainResearch Centre, Manesar, India; 2Cellular and MolecularNeuroscience, National Brain Research Centre, Manesar

Astroglia are indispensable component of the tripartite synap-se that regulate neuronal functions, health and survival

through dynamic neuroglia crosstalk. Susceptibility to HIV-1infection and subsequent latency in astrocytes contribute toneuronal damage that culminates into HIV-1 AssociatedNeurocognitive Disorders (HAND). The neurotoxic viralprotein HIV-1 Transactivator of Transcription (Tat), is re-leased in the brain even in successful cART cases. Tat isreported to be produced in HIV-1 infected astrocytes.Recently, we have demonstrated that astrocytes mediate in-direct neuronal death by releasing excess ATP through acti-vation of purinergic receptor system and experimentallyproved that enhanced ATP levels induce astrocytes to secretemore MCP-1(CCL2), that is critical for monocyte aided viraltrafficking into CNS. Using well characterized model systemof human primary astrocytes and neurons, we further probedinto the molecular mechanism for enhanced ATP release inTat affected astrocytes. It was observed that HIV-1 Tat mod-ulates the miRNA machinery in astrocytes to perturb thelevels of voltage dependent anion channel-1 (VDAC1), achannel present in the outer mitochondrial membrane andplasma membrane which regulates extracellular ATP release.We report a novel molecular cascade of miRNA-mediatedATP release through regulation of VDAC1 and provide ev-idence that HIV-1 Tat dysregulates miR- 320a that in turnperturbs astrocytic ATP release. Down-regulation ofVDAC1 either with miR-320a mimic or VDAC1 siRNAin HIV-1 Tat affected astroglia, could rescue the neuronsfrom glia-mediated indirect death. We corroborated thesein vitro findings using HIV-1 patients suffering from mildcognitive disorder (MCI) and age-matched control; we ob-served dysregulated miR-320a- VDAC1 axis in frontal cor-tex of HIV-MCI patients as compared to control subjects.Our findings reveal a novel upstream therapeutic target thatcould be employed to abolish the astroglia-mediated neuro-toxicity in HIV neuropathogenesis.

P42Expression of mitochondrial biogenesis and innateinflammation genes in brains of HIV+ donors stratifiedby mtDNA haplogroups

Jerel Fields, Hamza Coban, Sarah Gough, Ilse Flores, EliezerMasliah, Paula Desplats, Cristian Achim(corresponding author: [email protected])

University of California, San Diego

Despite combined antiretroviral therapy (cART), HIV-associated neurocognitive disorders (HAND) remain a signif-icant problem for 1.4 million people living with HIV in theUSA, and minority populations are disproportionately affect-ed. The causes of HAND are multifactorial and may includelow-level viral expression, drugs of abuse, cART


neurotoxicity, a combination of these and others, which initi-ate a cascade of inflammatory events that culminate in neuro-degeneration. Recent studies suggest mitochondrial DNA(haplogroups), and hence mitochondrial function. A re-cent study suggests that Hispanics identified as carryinghaplogroup B mtDNA may be less likely to developHAND. Here we sought to investigate mitochondrial bio-genesis and inflammatory gene expression in HAND do-nors from different haplogroups. In the frontal cortex of60 (32 Hispanic) well-studied cART-era HAND donors,we found significant increases in the master regulator ofmitochondrial biogenesis, peroxisome proliferator-activated receptor gamma coactivator (PGC)-1alpha, inbrain tissues of HAND donors; mRNA levels also indi-cated altered PGC-1alpha expression, with more signifi-cant increases in Hispanic donors. Immunohistochemichalanalyses show a distinct pattern in the brains of HANDdonors compared to HIV- control and HIV+ Normal, andbetween haplogroups. Importantly, we found similar alter-ations in complement pathway protein expression, withthe highest gene expression levels detected in Hispanics.In combination with recent reports, these data suggest thatalterations at both ends of the mitochondrial biogenesis-dynamics axis may contribute to HAND. In Hispanic do-nors, other than those of haplogroup B, altered comple-ment pathway protein expression may increase the risk ofdeveloping HAND. As mitochondrial dysfunction is im-plicated in many neurodegenerative disorders, further elu-cidation of these mechanisms may provide a therapeutictarget to improve disparities in HAND outcomes.

P43Development of an inducible cell line screeningfor therapeutic antagonists to HIV Tat protein

Nicholas Geiger1, Wenxue Li1, Richa Tyagi1, MuznaBachani2, Nasir Malik2, Joseph Steiner2, Avindra Nath3


1Section of Infections of the Nervous System, NINDS/NIH,Bethesda, MD; 2Translational Neuroscience Center, NINDS/NIH, Bethesda, MD, USA; 3SINS and TranslationalNeuroscience Center, NINDS/NIH, Bethesda, MD, USA

The Tat protein of HIV is a small, highly basic, regulatoryprotein that is the first protein to be produced during viralreplication. Tat plays a critical role in driving viral replica-tion by transactivation of the Long Terminal Repeat (LTR)promoter region of the virus, and has long been implicatedin the pathophysiology of HIV-Associated NeurocognitiveDisorders (HAND). Although Tat has been extensivelystudied, it remains an elusive target. We have established

a Tat-mediated HIV LTR transactivation assay with whichwe can identify new antiviral agents that target Tat itself.Using the Tet-On 3G regulated transcription system, wehave successfully developed and optimized a stable, induc-ible HeLa cell line that: a) is fast-growing and easy tomaintain in culture, b) exhibits minimal background Tatexpression in the absence of induction, c) enables Tat ex-pression and LTR transactivation to be monitored by afluorescent tag and a luminescent reporter, respectively, d)demonstrates reliable and consistent GFP fluorescence andLuciferase luminescence data reproducibility and e) dis-plays minimal LTR activation in the absence of Tat stimu-lation. Using this cell line in high throughput screeningassays in 384 well plate format, we’ve screened more than2000 compounds from the Spectrum Collection to identifytherapeutic antagonists to Tat-TAR binding and subsequentLTR transactivation. Subsequent studies will confirm theactivity of the “hit” compounds, characterize them in con-centration dependent analyses and determine their directbinding interactions with HIV Tat, in order to provide anovel therapeutic for HIV infection.

P44Whole body mapping shows a relatively small poolof latent HIV in the CNS not strongly related to poolsin other body compartments

Benjamin Gelman1, Joshua Lisinicchia1, Patel Vipulkumar1,Lalita Singh1, Janice Endsley1, Dennis Kolson2


1Department of Pathology, University of Texas MedicalBranch; 2Department of Neurology, University ofPennsylvania

The effectiveness of therapies to eradicate latent HIV DNAmust be assessed primarily by sampling peripheral bloodmononuclear cells (PBMCs). The relationship betweenchanges in the size of the latent pool of HIV DNA inPBMCs versus that of the deep body compartments suchas the central nervous system (CNS) is unknown. A wholebody map of HIV DNA concentration was produced in 16HIV infected people; half of the decedents were virally sup-pressed. 17 tissue types were assayed for HIV RNA andDNA using PCR and the two-step alu-gag assay for integrat-ed HIV DNA. Correction for latent HIV DNA within bloodpools transiting through the tissues was estimated with he-moglobin assays. The largest HIV DNA pool size in thebody was in the gastrointestinal (GI) tract which contained52 % of the total. Latent HIV in PBMCs had substantialpredictive power pertaining to latent pool sizes in many deepbody compartments including the gut, but notably not in


CNS. The CNS pool of HIV DNA contained less than 1 %of the whole body total and was more concentrated in whitematter, which contained 70 % of the CNS pool. Gray mattercontained 30 % of the pool and was about half as concen-trated. In the liver active hepatitis was significantly asso-ciated with a high latent pool size. In the spleen highlatent pool size was associated with low expression ofcandidate biomarker mRNAs including CXCL16,CXCL13 and CCL1. Whole body mapping shows thatthe latent pool of HIV DNA in PBMCs varies proportion-ately with latent pools in many deep body compartments,but not with the relatively small pool sequestered withinthe CNS. The size of the HIV DNA pool in PBMCs alsowas not significantly related to the degree of viral sup-pression in patients with less than 100,000 copies/mlblood plasma.

P45HIV-1 Tat Induces Neuronal Insulin Receptor SecretionThrough the TNF-Alpha Receptor

Yamil Gerena1, Raissa Mení©ndez-Delmestre2, AndreaDelgado-Nieves1, Anibal Gonzíçlez-Escalante1, LuisColí_n-Cruz1, Valerie Marshall-Freites1, Paola Rivera-Morales1, Avindra Nath3, Valerie Wojna4


1Department of Pharmacology, University of Puerto Rico-Medical Sciences Campus; 2NeuroAIDS Research Program,University of Puerto Rico-Medical Sciences Campus; 3NIH,National Institute of Neurological Disorders and Stroke;4Department of Internal Medicine, Neurology Division,University of Puerto Rico-Medical Sciences Campus

Background: Previously we found that high levels of solubleinsulin receptor (sIR) in the cerebrospinal fluid (CSF) of HIV-seropositive women were associated with presence and sever-ity of cognitive impairment. However, the possibility thatcomponents present in the CSF of these patients may influ-ence sIR secretion from neuronal cells remains totally unclear.In this study we investigated if HIV-1 Tat protein and TNF-alpha influence the secretion of sIR from human neuronalcells and then analyzed the effects of R5070, a TNF-alphareceptor blocker, on HIV-1 Tat or TNF-induced neuronal sIRsecretion. Methods: Human neuronal cells (SH-SY5Y;5× 106 cells) were exposed to HIV-1 Tat (100nM), TNF-alpha (5 pg/ml), or a combination of both proteins for 24 h.Culture medium was collected and sIR levels were quantifiedby ELISA. The secretion of sIR was also measured in mediumof cells exposed to either TNF-alpha, HIV-1 Tat, or a combi-nation of both in the presence of R7050 (10-8 M) for 24 h.Results: A significant increase (p<0.0001) was observed in

sIR secreted from cells exposed to the HIV-1 Tat or TNF-alpha when compared to untreated. Incubation of cells witha combination of HIV-1 Tat and TNF-alpha also increasedsignificantly (p<0.0001) sIR secretion, however this combi-nation did not enhanced the effects observed with either pro-tein individually. R5070 was able to partially reverse the in-creased sIR secretion induced by either HIV-1 Tat or TNF-alpha, however the drug completely abolished the receptorsecretion induced by the combination of both proteins.Conclusion: Our data suggests that TNF-alpha andHIV-1 Tat regulate the secretion of sIR from neuronalcells and that the effects of Tat on sIR is dependent onTNF-alpha receptor activation. Further studies will inves-tigate if the effects of Tat on sIR secretion are mediatedby neuronal TNF-alpha being released to the extracellu-l a r m e d i um . S u p p o r t e d b y R 0 1NS 0 9 9 0 3 6 ,R21MH095524, U54MD007587, G12RR003051,G12MD007600.

P46Microglial gene expression is altered in HIV infection,even in the absence of detectable virus in brain

Stephen D. Ginsberg1, Melissa J. Alldred1, Satya M.Gunnam2, Tracy Fischer2


1Center for Dementia Research, Nathan Kline Institute,Orangeburg, NY; Departments of Psychiatry and Physiol. &Neurosci., New York University Langone Medical Center,New York, NY; 2Temple University School of Medicine,Department of Neuroscience, Philadelphia, PA

We have previously reported substantial accumulation ofCD163+/CD16+ macrophages and microglia in brains of pa-tients with HIVencephalitis (HIVE), a neuropathological cor-relate of the most severe form of HIV-associatedneurocognitive disorders (HAND), HIV-associated dementia(HIV-D). More recently, we found neurocognitively impairedHIV+ individuals without encephalitis (HIV/noE) or detect-able virus production in the brain also have increased CD163+and CD16+ brain macrophage’s and microglia. Accordingly,we hypothesize that microglial activation is a common mech-anism between lesser and more severe HIV-associated neuro-degenerative processes, regardless of virus production in thebrain. To begin to explore this premise, we investigated geneexpression changes of select classes of transcripts in paren-chymal macrophage’s/microglia from archival brain tissue ofpatients with HIVE, HIV/noE, and age-matched seronegative(HIV-) controls. Microarray analyses were performed on lasercapture microdissected parenchymal CD163+, CD16+ orCD68+ macrophage’s/microglia, using terminal continuation


(TC) RNA amplification and a custom-designed array plat-form. Here, we report pronounced differences in gene expres-sion between HIV- and HIV infection with and without en-cephalitis. Altered expression of several classes of transcripts,including genes associated with cell death, inflammatorymarkers, and protein kinases, are observed in the context ofHIV infection. Several transcripts suggesting macrophage’s/microglia activation are up regulated in cells recovered fromHIV/noE brain; however, many of these are decreased inHIVE. This may suggest impaired function of macro-phage’s/microglia, resulting from prolonged neuroinflamma-tion. These data indicate the utility of profiling macrophage’s/microglia in HIV infection to identify potential factors fortargeted treatment modalities. In addition, our work providesimportant insights into the diverse roles microglia play inmaintaining brain homeostasis, as well as alterations inmicroglial function that likely contribute to neuronal injuryand cognitive impairment.

P47HTLV-1 infection and neuropathogenesis in the contextof Rag1−/−γc−/− (RAG1) mice

Rashida Ginwala1, Paige Charlins2, Ramesh Akkina2,Breanna Caruso3, Ronak Loonawat1, Steven Jacobson3,Sreesha Nambiar1, Glen M. Chew4, Lishomwa C. Ndhlovu4,Pooja Jain1, Zafar K. Khan1


1Department of Microbiology and Immunology, and theInstitute for Molecular Medicine and Infectious Disease,Drexel University College of Medicine, Philadelphia, PA;2Department of Microbiology, Immunology and Pathology,Colorado State University, Fort Collins, CO;3ViralImmunology Section, Neuroimmunology Branch, NationalInstitutes of Health, Bethesda, MD; 4Department of TropicalMedicine, Medical Microbiology and Pharmacology, John A.Burns School of Medicine, University of Hawaii, Honolulu,HI

HTLV-1-associa ted myelopathy/ t ropica l spas t icparaparesis (HAM/TSP) is a disabling chronic inflamma-tory disease of the central nervous system (CNS) withsimilarities to multiple sclerosis. To date, the lack of asuitable small animal model has hindered our quest tounderstand the immuno- and neuropathogenesis ofHTLV-1 in an in vivo system. Additionally, the host im-mune response that plays a critical role in the outcome ofHTLV-1 infection could be better tested in the context ofhumanized (hu) mice. Thus, we employ here neonatal andadult Balb/c-Rag1−/−gammac−/− or Rag1 as well as Bonemarrow-Liver-Thymic (BLT) mouse models for

engraftment of human CD34+ hematopoietic stem cells.Flow cytometry and histological analyses revealed recon-stitution of mice with human immune cells, includingmacrophages, dendritic cells, T cells and B cells.Proviral load (PVL) was determined in the peripheralblood, spleen, and other organs of Rag1 and BLT miceby droplet digital PCR. Within blood, PVL and viral pro-tein Tax was detected as early as 2 weeks post-infection(wpi) with continued expression until 16 wpi. Both PVLand Tax expression was considerably higher in the adultRag1 mice as compared to the neonates with the lattershowing less than 20 % PVL in the peripheral blood,brain, and liver. Thus far, several members of the CD28:B7family of immunoglobulin superfamily co-signaling mole-cules that include PD-1 and its ligand PDL-1 have been asso-ciated with T-cell dysfunctions in HTLV-infected patients. Wethus assessed and found over expression of PD-1, TIM-3,TIGIT and 2B4 on CD4 and CD8 T cells in HTLV-1 infectedsamples from both hu-mice and human subjects. Moreover,lymphocytic infiltration with concomitant Tax expression andresulting myelin disruption was observed in the spinal cordand brain of the infected mice. This data represents the firstattempt to establish HTLV-1 neuropathogenesis in the contextof RAG1 and BLT mice.

P48Nutraceutical Apigenin regulates DC functionin a RelB-dependent manner during neuroinflammation

Rashida Ginwala1, Emily McTish1, Nikil Revuri1, ChanderRaman2, Narendra Singh3, Mitzi Nagarkatti3, PrakashNagarkatti3, Divya Sagar1, Pooja Jain1, Zafar K. Khan1


1Department of Microbiology and Immunology, and theInstitute for Molecular Medicine and Infectious Disease,Drexel University College of Medicine, Philadelphia, PA;2Division of Clinical Immunology and RheumatologyUniversity of Alabama School of Medicine, Birmingham,AB; 3Department of Pathology, Microbiology andImmunology University of South Carolina, Columbia, SC

Apigenin, a natural flavonoid, found in several plants isknown to have anti-oxidant and anti-inflammatory propertiesindicated by its use for centuries as a medicinal approach totreat inflammatory disorders. However, there is a considerabledearth of information regarding its effect on immune cells,especially dendritic cells (DC) that maintain the critical bal-ance between an immunogenic and tolerogenic immune re-sponse, in an immunospecialized location like the central ner-vous system (CNS). Thus we looked at the anti-inflammatoryproperties of Apigenin in restoration of immune cell function,


especially DCs. In vitro upon LPS stimulation, Apigenininhibited IL-6 and TNF-alpha secretion and the cell surfaceexpression of alpha4 integrin, CD86, CLEC12A and MHC IImolecules on murine DCs. Further, Apigenin treatment ame-liorated severity of disease progression and relapse after onsetof experimental autoimmune encephalomyelitis (EAE) inC57BL/6 and SJL mouse models of multiple sclerosis immu-nized with MOG35-55 and PLP139-151 respectively.Apigenin treated EAE mice showed decreased expression ofalpha4 integrin and CLEC12A on splenic DCs and an in-creased retention of DCs and macrophages in the peripherycompared to untreated EAE mice. This correlated with de-creased immune cell infiltration and reduced demyelinationin the CNS. Mechanistically, we observed Apigenin treat-ment reduces cytoplasmic RelB expression in presence ofLPS in human peripheral blood DCs, which is central toDC maturation, its antigen presentation capabilities andDC-mediated T cell activation. Concomitantly, IFN-gamma a downstream target of RelB was also reducedupon Apigenin treatment in these cells. RelB also playsa role in mitochondrial bioenergetics during inflammation,which explains the metabolic shift away from glycolysisthat we observed upon Apigenin treatment in the inflamedDCs. These results indicate a protective role of Apigeninagainst neurodegenerative effects resulting from the entryof DC stimulated pathogenic T cells into the CNS thusimplicating a potential therapy for neuroinflammatorydisease.

P49Autophagy Facilitates Nanoformulated antiretroviralDrug Depots for Sustained Release and EnhancedAnti-Retroviral Activity

Divya Prakash Gnanadhas1, Prasanta Dash1, Brady Sillman1,Zhiyi Lin1, Aditya Bade1, Harris Gelbard2, JoEllynMcMillan1, Benson Edagwa1, Howard Gendelman1, SanthiGorantla1


1Department of Pharmacology and ExperimentalNeuroscience, College of Medicine, University of NebraskaMedical Center, Omaha, Nebraska; 2Center for NeuralDevelopment and Disease, University of Rochester MedicalCenter, Rochester, NY

Long-acting anti-HIV products can substantively change thestandard of care for HIV/AIDS. To this end, antiretroviraldrugs (ARVs) that can be administered once a month or longerand the ability to transform short acting hydrophilic drugs toextended release prodrugs are positive steps forward towardsrealizing this goal. We now report yet another means to extend

the half-life of ARVs and enhance drug delivery to viral res-ervoirs. This is achieved by co-administration of long-actingnanoformulated ARVs (nanoARV) with URMC-099, a mixedlineage kinase-3 inhibitor. URMC-099 was shown to be neu-roprotective and anti-inflammatory in a rodent model of HIV-associated neurocognitive disorders (HAND). We now showthat URMC-099 activates autophagy through nucleartranslocation of transcription factor EB (TFEB) resultingin improved antiretroviral activities and extended drug de-pots in human monocyte derived macrophages (MDMs).Induction of autophagy also leads to retention ofnanoARV, containing the protease inhibitor atazanavir, inthe autophagosomes of MDMs. This occurs in parallel withan up to 4-fold improvement in mitochondrial activitiesand cell vitality compared to nanoARV alone. In rodents,URMC-099 induced transcriptional activation of autopha-gy led to a 50-fold increase in the half-life of the viralintegrase inhibitor dolutegravir. Activation of autophagylead to decrease in HIV-1 induced IL-1beta secretion ininfected MDMs and humanized mice. Such improvedanti-inflammatory and antiretroviral responses paralleledwhat was shown previously in HIV-1 infected humanizedmice. We conclude that pharmacologic induction of au-tophagy provides a novel means to further extend the ac-tion of long acting nanoformulated antiretroviral therapy.

P50Cognitive Change Trajectories in Virally SuppressedHIV-Infected Individuals Suggest High Prevalenceof Ongoing Disease Activity

Chloe Gott1, Thomas Gates2, Nadene Dermody3, BruceBrew4, Lucette Cysique5


1Psychology Department, Macquarie University, Sydney,NSW, Australia; 2Centre for Applied Medical Research, StVincent’s Hospital Sydney, NSW, Australia; 3NeuroscienceResearch Australia (NeuRA), Sydney, NSW, Australia;4Centre for Applied Medical Research, St Vincent’s Hospital& UNSWAustralia, Sydney, NSW, Australia; 5NeuroscienceResearch Australia (NeuRA), & UNSW Australia, Sydney,NSW, Australia

Background: In persons with stable, treated, and virally-suppressed HIV infection, cognitive decline rate and profileis not established. As such this study aims to quantify the rateof cognitive decline and define clinically meaningful cogni-tive trajectories in a cohort of virally-suppressed HIV+ per-sons. Methods: Ninety-six HIV+ (97 % virally undetectable;median current cART duration: 24 months; median HIV du-ration: 19 years, mean age: 56 years) and 44 demographically


comparable HIV- participants underwent standard neuropsy-chological testing assessing 7 cognitive domains at baselineand 18-months follow-up. We determined clinically relevantcognitive trajectories based on a history of HAND, baselineHAND status and cognitive decline using norms for changecorrected for practice effect. The definition of cognitive de-cline was based on a continuous global change score (GCS)and dichotomous definition of clinically meaningful cognitivedecline (decline/stable; 90 % confidence interval, 1-tailedaround normative GCS). Results: Relative to HIV- controls(4.5 %), 14 % of HIV+ participants declined (p= .11); more-over the HIV+ group scored significantly lower on the GCS(p= .03) and showed greater decline in processing speed(p= .02) and mental flexibility/inhibition (p= .02). BaselineHAND status significantly predicted cognitive decline atfollow up (p= .005). We determined 7 clinically relevantcognitive trajectories which in order of prevalence were: 1)Always neurocognitively-normal (39 %), 2) Stable base-line impairment (35 %), 3) Sustained long-term impair-ment since historical HAND, (9 %) 4) Baseline impairmentand decline (7 %), 5) Historical HAND fully recovered(3 %), 6) Incident decline at 18 months (3 %) 7) Alwaysprogressing (3 %). There was no relationship betweenthese trajectories, and traditional HIV disease biomarkers(nadir, current, standard difference baseline and follow-upCD4+ T cell counts; study period undetectable/detectable;baseline HIV duration, and baseline cART duration).Conclusions: When determining clinically relevant trajec-tories, we found that more than half of virally-suppressedHIV+ individuals (57 %) have ongoing disease activitythat is not related to traditional HIV biomarkers.

P51Preclinical studies of Bardoxolone for the preventionand treatment of HIV-associated neurocognitive disorders

Analise Gruenewald1, Irene Kim2, Dennis Kolson1, DimitriosVatakis2, Andrew Levine3


1Department of Neuroscience, University of Pennsylvania;2Department of Medicine, Division of Hematology-Oncology, UCLA AIDS Institute, David Geffen School ofMedicine at the University of California Los Angeles;3Department of Neurology, David Geffen School ofMedicine at the University of California Los Angeles

Background: HIV-associated neurocognitive disorders(HAND) are the clinical manifestation of inflammation andoxidative stress in the CNS. Nrf-2 is a transcription factorthat promotes expression of a wide variety of antioxidantand anti-inflammatory factors. The KEAP1/nrf-2 pathway

has previously been implicated in HAND via animal andin vitro studies, and in a large gene expression study inhumans. Therefore, the KEAP1/nrf2 pathway is a candidatetarget for HAND treatment/prevention. Here we assess thesafety and efficacy of Bardoxolone, a compound that in-duces nrf2 nuclear translocation, stimulating expression ofseveral protective genes. Methods: Several experiments wereconducted in two independent labs: 1) To assess neuropro-tective effects, monocyte-derived macrophages (MDM) werepre-treated for 24-h prior to infection with Bardoxolone,Tempol (0.5 or 5microM), or vehicle and the following mea-sures were evaluated several times over 15 days: HIV re-verse transcriptase (RT) activity in supernatants, heme-oxygenase-1 (HO-1) expression in lysates, and microtubuleassociated protein-2 (MAP2) expression in primary rodentneurons exposed to culture supernatants 2) HIV-infected U1monocytes were stimulated using LPS (1microg/ml) to de-termine if pretreatment with Bardoxolone (5 or 50nM)inhibited viral replication. Control cells were compared tocells pre-treated with either Tempol or Bardoxolone over-night. Drug efficacy was assessed by measuring levels ofp24 gag expression. Results: 1) Bardoloxone pre-treatmentreduced HIV replication in MDM, demonstrated by reducedHIV RT activity in the culture supernatants. Bardoxoloneenhanced expression of HO-1 in HIV-infected and non-infected MDM. Bardoxolone treatment of HIV-infectedMDM dramatically reduced the neurotoxicity of cell culturesupernatants, demonstrated by maintenance of MAP2 levelsin the neuron cultures as compared to vehicle and Tempol.2) Bardoxolone pre-treatment decreased the amount of p24gag expression following stimulation by LPS. Conclusion:This series of preclinical studies lends support for the safetyand efficacy of Bardoxolone in the prevention and/or treat-ment of HAND.

P52Macrophage-tropic Simian Immunodeficiency VirusMolecular Clone causes NeuroAIDS in Rhesus Macaques

Sanjeev Gumber1, Shawna Woollard2, Praveen KumarAmancha3, Po-Jen Yen4, Dana Gabuzda4, FrancoisVillinger3, Siddappa Byrareddy2


1Division of Pathology, Yerkes National Primate ResearchCenter, Emory University, Atlanta, GA 30329; 2Departmentof Pharmacology and Experimental Neuroscience, Universityof Nebraska Medical Center, Omaha, NE 68198; 3New IberiaResearch Center, University of Louisiana at Lafayette, NewIberia, LA 70560; 4Department of Cancer Immunology andVirology, Dana-Farber Cancer Institute, Boston, MA, USA


Macrophages are a major target of HIV/SIV and are relativelyresistant to the cytopathic effects of infection. These cells playan important role in disease pathogenesis and serve as viralreservoirs in the central nervous system (CNS). Previously, aunique early SIVmac251 Env variant, deSIV147, was isolatedfrom blood of a rhesus macaque with rapid disease progressionand SIV-associated encephalitis (SIV-E). A replication-competent SIV251 molecular clone encoding deSIV147 Envwas shown to be highly fusogenic, replicate efficiently inrhesus macaque PBMC and macrophages, and induce multi-nucleated giant cell formation during macrophage infectionin vitro. Furthermore, deSIV147 Env shared close sequencehomology to sequences in the brains of infected animals withneurological disease. Here, we show that molecularly clonedSIV251 virus expressing deSIV147 Env is neurotropicin vivo. The deSIV147 virus infected rhesus macaques fol-lowing intravenous or intrarectal exposure, causing sys-temic infection with plasma viral loads ranging from10^5 to 10^7 copies/ml. Furthermore, immunohistologicalevidence of macrophage infection was present in brain,lymph nodes, spleen, colon, lung, and liver, and CD4+ Tcells in gut were depleted during acute infection in 3/3animals tested. Next, deSIV147 virus was inoculated intomacaques depleted of CD4+ T cells, creating selectionpressure for the virus to infect macrophages in the CNS.All animals were infected, with plasma and CSF viral loadsof 10^5 to 10^8 and 10^3 to 10^7 copies/ml, respectively,and evidence of SIVp27-positive macrophages in brain.Furthermore, accumulation of perivascular macrophages,formation of multinucleated giant cells, microgliosis, andmultifocal neuronal injury in gray and white matter of thebrain was detected in 1 of 3 animals tested. These findingssuggest the neurotropic deSIV147 clone will be useful tostudy the role of macrophages in HIV/SIV-associatedneurocognitive disorders, gain new insights into the CNSas a viral reservoir, and test strategies for eradication

P53System mapping of CCR5 signaling in HIVneuropathogenesis and viral infection of primarymacrophages

Jig Guo, Yuntao Wu(corresponding author: [email protected])

NCBID, George Mason University

The CCR5-utilizing viruses are the predominant species dur-ing the course of HIV infection. The R5 viruses infectmonocytes/macrophages that are considered as major media-tors of HIV-mediated neuropathogenesis; the activation andtrafficking of infected/uninfected macrophages into the central

nervous system (CNS) lead to neuronal dysfunction.However, how HIV infection leads to accelerated traffickingof macrophages into the CNS is largely unknown. Previously,we demonstrated that during infection, HIV triggers chemo-tactic signaling through gp120 binding to CXCR4, which ac-tivates cofilin and Arp2/3, two of the major regulators of actindynamics. Given that cell migration are driven by actin dy-namics, we investigated whether R5 virus binding to macro-phages may alter their chemotactic responses and mobility.Here, we report the modulation of both the cofilin andArp2/3 signaling pathways in macrophages by R5 viruses.In addition, we also probed the magnitude of R5 gp120-mediated signal transduction in primary macrophages,using a systematic pathway mapping technology, the re-verse phage phospho-protein micro-array (RPPA), we in-terrogated over 200 phospho-proteins for their involve-ment in R5 gp120 signaling. We identified four majorpathways, the actin signaling network (cofilin, LIMK1,PTEN, VASP, FAK, Pyk2), the PI3K-Akt signaling net-work (Akt), the MAPK signaling network (c-Raf,ERK1/2), and the Jak/Stat signaling network (Jak1,Stat3), that are selectively activated. We future demon-strated that targeting some of these signaling networkseffectively block R5 viral infection of primary macro-phages, Our studies suggest that novel therapeutics canbe developed based on targeting R5 HIV-mediated signal-ing pathways. These novel approaches may prevent theestablishment of viral reservoirs in the CNS.

P54Nef, an auxiliary protein produced by HIV-1 inhibitsautophagy in neonatal cardiac myocytes

Manish Gupta1, Rafal Kaminski1, Brian Mullen1, JenniferGordon1, Joseph Cheung2, Arthur Feldman2, MuniswamyMadesh3, Kamel Khalili3


1Department of Neuroscience, Center for Neurovirology,Lewis Katz School of Medicine at Temple University;2Department of Medicine, Lewis Katz School of Medicine atTemple University; 3Center for Translational Medicine,Department of Medical Genet ics and MolecularBiochemistry, Lewis Katz School of Medicine at TempleUniversity

Introduction: Patients with HIV-1 infection develop heart fail-ure with reduced ejection fraction (HFrEF); however, thecausative mechanism is unclear because cardiac dysfunctionoften occurs in HIV infected individuals in the absence ofmyocarditis. Hypothesis: We tested the hypothesis that theexpression of the HIV-1 auxiliary protein Nef inhibits


autophagy leading to diminished protein quality control andenhanced cell death in cardiac myocytes. Methods and result:Using primary cardiomyocytes isolated from neonatal rat ven-tricle we tested the role of Nef protein in cardiomyocyte au-tophagy and cell survival. Our data show that Nef proteincauses inhibition of cardiomyocyte autophagy at the terminalsteps and induces accumulation of p62 and ubiquitin positiveaggregates at the perinuclear space.Mechanistically, we foundthat Nef protein interacts with the autophagy maturation fac-tors, Belin1 and Rab7 and forms aggregate at the perinuclearspace. In addition, Nef dysregulatedmitochondrial turnover asevidenced by accumulation of Tom20, increased generation ofreactive oxygen species and decreased mitochondrial mem-brane potential. Interestingly, pharmacological drugrapamycin-mediated induction of autophagy can neutralizesthe Nef induced cardiomyocyte’s abnormalities. Conclusion:Nef reversibly inhibits cardiomyocyte autophagy through in-hibition of autophagy maturation factors and may thereforeserve as a therapeutic target in patients with HIV-associatedHFrEF.

P55Reduced cingulate structural and functional integrityin HIV and aging

Shiva Hassanzadeh1, Manya Magnus2, Matthew Dawson3,Allison Budzinski4, Rebecca Barasky2, Cuiwei Wang5,Princy Kumar5, Mary Young5, David Moore3, Ronald Ellis4,Xiong Jiang1


1Department of Neuroscience, Georgetown UniversityMedical Center; 2Department of Epidemiology andBiostatistics, George Washington University; 3Department ofPsychiatry, University of California, San Diego; 4Departmentof Neuroscience, University of California, San Diego;5Department of Medicine, Georgetown University MedicalCenter;

Cingulate cortex is one of less understood brain regions,but has been implied in many cognitive functions, includ-ing executive function. A recent study with a task-switching paradigm revealed that HIV+ older adults adaptless quickly to changing task demands than HIV-uninfected controls, and the behavioral impairments cor-related with reduced brain activation in the anterior cin-gulate cortex (ACC) (Jiang et al., AIDS Care 2016), sug-gesting ACC dysfunction might underlie reduced execu-tive function in HIV. In addition, using resting-state func-tional connectivity MRI (rs-fcMRI) technique, we havefound that both HIV and aging led to an isolated cingu-late, and the reduced functional connectivity correlated

with altered fMRI response in the ACC (Jiang et al.,CROI 2016), providing further evidence suggesting cin-gulate might be affected in HIV. Here using a modifiedvoxel-based morphometry (VBM) technique that is sensi-tive to local changes in gray matter (GM) volume, weexamined the impact of aging and HIV on the volume ofcingulate cortex. Forty-two adults (43–63 years old, 27HIV+) without major psychiatric disorders and other con-founding health problems participated in this study. AllHIV+ subjects were on antiretroviral therapy, and didnot have a clinical diagnosis of cognitive impairments.At a whole brain level, there is no difference betweenthe HIV-infected and HIV-uninfected groups. However,the volume of cingulate cortex was reduced in the HIV+group than the age-matched uninfected controls (aftercontrolling for age). In addition, a negative correlationbetween cingulate volume and age was observed.Furthermore, a strong correlation between the reducedfunctional connectivity and the cingulate volume was ob-served. These results provide further evidence suggestingthat the cingulate cortex might be one of brain regionsaffected by HIV early on, and the additive impairmentsdue to aging and HIV might underlie the increased riskof cognitive impairments in HIV+ older adults.

P56Informatic Interrogation of CSF Proteomic Profilesfrom HIV-Infected Subjects Implicates Acute Phaseand Complement Systems in Shifting Cognitive Status

Norman Haughey1, Ceereena Ubaida-Mohien1, BenjaminLamberty2, Alex Dickens1, Michelle Mielke3, ThomasMarcotte4, Scott Letendre4, Donald Franklin4, Ned Sacktor1,Igor Grant4, Pawal Cibrowski2, Ravi Tharakan1, JustinMcArthur1, Howard Fox2


1Johns Hopkins; 2University of Nebraska; 3Mayo Clinic;4UCSD

The prevalence of HIV-Associated Neurocognitive Disorders(HAND) has not changed considerably in the last two de-cades. Potent antiretroviral therapy (ART) has shifted the se-verity of HAND to milder phenotypes, but excess morbidityand mortality continue to be associated with HAND. Changesin numerous markers of immune function, inflammation andcellular stress have been repeatedly associated with HAND,but the underlying systems that drive these changes have notbeen identified. In this study we conducted an untargeted pro-teomic analysis of CSF samples from the Central NervousSystem HIV Anti-Retroviral Therapy Effects Research


(CHARTER) and the HIV Neurobehavioral Research Center(HNRC) studies. Subject selection was based on a case reviewof ~3,500 clinical visits from 430 study participants. Subjectshad CSF available from two consecutive study visits withcomplete clinical and neuropsychological data available andwere on stable ART for at least 3 months before the first visitand for the duration of the observation period. Participantswere demographically matched, and were similar in durationof HIV infection, HCV serostatus, nadir CD4+ T-cell count,and plasma and CSF HIV RNA. Based on changes inneurocognitive (NC) performance, participants were cate-gorized as: Stably normal (n= 25), Improving (n= 20),Worsening (n= 22) and Stably Impaired (n= 22). To en-sure the stability of these categories of NC performance,NC data from a third visit was used to validate the trajec-tory of change in cognitive status. An untargeted proteo-mic analysis was conducted using CSF from the two in-dex visits. Systems informatics was used to interrogate theproteomic content of CSF. The patterns of change in CSFprotein content implicated the induction of acute phaseand complement systems as important regulators of NCstatus. Worsening NC performance was preceded by in-duction of acute phase and complement systems, whileimproving NC performance was preceded by downregu-lation of these systems.

P57Modulation of HIV replication in lymphocytes,macrophages and astrocytes by association of HIVmRNAwith TAR DNA binding protein 43

Lisa Henderson, Wenxue Li, Richa Tyagi, Avindra Nath(corresponding author: [email protected])

Section of Infections of the Nervous System, NationalInstitute of Neurological Disorders and Stroke

Human immunodeficiency virus (HIV) colonizes the brainearly in infection, where it infects macrophages, microgliaand astrocytes. These cells are long-lived and resistant to cy-topathic effects of HIVand can thus harbor virus for extendedperiods of time. It is vital to gain a greater understanding of themechanism(s) that regulate HIV expression in these cells.TAR DNA binding protein 43 (TDP-43) plays critical rolesin mRNA splicing and stability. TDP-43 can also bind directlyto target promoters, including the HIV long terminal repeat(LTR), to modulate transcription. TDP-43 also binds to NFkBsubunit p65, to function as co-activator for NFkB genes andregulates splicing of interleukin-6 and interleukin-10 mRNA.We found that overexpression of TDP-43 enhanced HIV tran-script levels in monocyte-derived macrophages (MDM) andlymphocytes as measured by qRT-PCR and enhanced viral

release as shown by quantification of HIV reverse transcrip-tase (RT) in culture supernatant. Surprisingly, overexpressionof TDP-43 in astrocytes strongly inhibited viral transcription,suggesting that the role of TDP-43 on HIV replication is cell-type specific and may be dependent on TDP-43 post-transla-tional modifications or availability of cellular co-factors.Furthermore, TDP-43-mediated enhancement of HIV didnot require previously identified binding sites on the HIVLTR, as mutation of these regions had no effect on HIVreplication. Similarly, deletion of NFkB elements fromHIV LTR had no impact on the TDP-43 effect, indicatingthat TDP-43 association with p65 was not involved.In vitro binding assays and RNA immunoprecipitation(RIP) revealed that TDP-43 bound to multiple HIV tran-scripts and identified UG-rich sequences in the 5’ untrans-lated region (UTR) of HIV mRNA as the putative bindingregion. These findings strongly suggest that TDP-43 playsa regulatory role in HIV infection, its effects are cell typespecific and that it may exert this effect via altered pro-cessing or stability of HIV transcripts.

P58Detection of HIV-1 Tat protein and TAR RNAin cerebrospinal fluid from patients on antiretroviraltherapy: targeting persistent Tat production usingantisense oligonucleotides

Lisa Henderson1, Tory Johnson2, Robert Barclay3, RichaTyagi1, Muzna Bachani4, Ned Sacktor2, Scott Letendre5,Joseph Steiner4, Fatah Kashanchi6, Avindra Nath1


1Section of Infections of the Nervous System, NationalInstitute of Neurological Disorders and Stroke; 2Departmentof Neurology, Johns Hopkins University School of Medicine;3National Center for Biodefense and Infectious Diseases,School of Systems Biology, George Mason University;4Translational Neuroscience Center, National Institute ofNeurological Disorders and Stroke; 5University of CaliforniaSan Diego; 6Laboratory of Molecular Virology, School ofSystems Biology, George Mason University

Background: Antiretroviral therapy (ART) has greatly dimin-ished the mortality associated with HIV-1 infection and limit-ed its transmission. However, HIV remains a life-long infec-tion with associated health concerns including persistent in-flammation and neurocognitive impairment. HIV-1 Tat is aneurotoxic and pro-inflammatory viral protein not targetedby current therapies. Methods: Tat levels in cerebrospinal fluid(CSF) from patients on ART (n=57) or collected from pa-tients pre- and post-ART initiation (n=5) were evaluated byELISA. Tat and viral RNA were also measured in exosomes


isolated from patient CSF by ultracentrifugation using westernblot and qRT-PCR, respectively. We designed DNA antisenseoligonucleotides (ASO) complementary to exon 1 of tatmRNA or an intronic sequence not present in the mature tattranscript but retained in env. ASO efficacy was evaluated byco-transfection of HeLa or primary human astrocytes withHIV and ASO followed by Tat ELISA or product enhancedreverse transcriptase (PERT) assay to measure viral release.Results: Tat protein was detected in 42 % (24/57) of pa-tient CSF samples. CSF Tat levels increased in 4 out of 5individuals after initiation of therapy, indicating that Tatwas not diminished by ART. Similarly, exosomes isolatedfrom CSF contained Tat protein (3/10) and/or TAR RNA(4/10), with 2 out of 10 samples positive for both Tat andTAR. ASO directed against the first exon of Tat effective-ly reduced Tat protein levels but did not inhibit productiveviral replication in astrocytes. In contrast, ASO thattargeted the intronic sequence repressed Tat translationand reduced viral release. Conclusions: These findingsconfirm that both Tat and viral RNA continue to be pro-duced in individuals otherwise controlled on ART, andhighlight a need for new therapies that target Tat. Tat-directed antisense are effective in reducing Tat protein aswell as inhibiting total viral replication and provide newavenues for therapeutic intervention.

P59A mouse model of enterovirus D68-induced paralysis

AlisonM. Hixon1, Guixia Yu2, J. Smith Leser3, Shigeo Yagi 4,Penny Clarke3, Charles Y. Chiu2, Kenneth L. Tyler5


1Medical Scientist Training Program, Neuroscience Program,University of Colorado; 2Department of Laboratory Medicineand Medicine, Division of Infectious Diseases, Abbott ViralDiagnostics and Discovery Center, University of California,San Fransicso; 3Department of Neurology, University ofColorado; 4California Department of Public Health; 5DenverVAMedical Center; Department of Neurology, Department ofMicrobiology and Immunology, Department of Medicine,University of Colorado

Enterovirus D68 (EV-D68) is a unique enterovirus that sharescharacteristics with rhinoviruses, including respiratory trans-mission. Although previously rare, the prevalence of EV-D68respiratory disease has increased in the last decade. In 2014,the US saw an unusual increase in acute flaccid myelitis(AFM) cases in children coincident with a nationwide out-break of EV-D68 respiratory disease, and up to 50 % ofAFM patients with available samples had EV-D68 RNA de-tected by RT-PCR in their respiratory secretions. EV-D68 has

only been detected in cerebrospinal fluid (CSF) from oneAFM patient to date. The lack of consistent CSF viral isolationor nucleic acid amplification, and the fact that EV-D68 strainswere not generally considered neurotropic, made the causalrole of EV-D68 in AFM uncertain. Here we show that arepresentative EV-D68 strain (US/MO/18947) isolatedfrom a respiratory sample during the 2014 outbreak in-duces paralytic disease in neonatal mice following intrace-rebral, intramuscular, intraperitoneal, and intranasal inocu-lation that shares key features with the human disease. Incontrast, the 1962 prototype strain, Fermon, does not pro-duce disease in mice. In-depth investigation of diseasepathogenesis of US/MO/14-18947 revealed infectious vi-rus, viral RNA corresponding to the full genome, and viralparticles in spinal cords of paralyzed mice. Paralysis wasassociated with infection and subsequent loss of motorneurons in the corresponding spinal cord anterior horn asassessed by immunohistochemistry. On-going investiga-tion of other 2014 EV-D68 strains has shown that many,but not all, genetically distinct isolates have the ability toinduce paralytic disease in mice. These studies establishthe neuronotropic and neurovirulent potential of EV-D68and provide a novel experimental model to further studyEV-D68-induced disease. In addition, preliminary investi-gations of disease modifying treatments, such as passiveantibody transfer, show promise that this model can beused to develop EV-D68 specific therapies.

P60Role of endolysosome de-acidification in antiretroviraldrug-induced amyloidogenesis

Liang Hui, Leo Lakpa, Jonathan Geiger, Xuesong Chen(corresponding author: [email protected])

School of Medicine & Health Science Department ofBiomedical Sciences, University of North Dakota

Combined antiretroviral therapeutic (ART) strategies have ef-fectively increased the long-term survival of HIV-1 infectedindividuals. However, along with increased longevity comesage-related neurological disorders including a very high prev-alence of HIV-1 associated neurocognitive disorders (HAND)including clinical manifestations and pathological features ofAlzheimer’s disease (AD). However, the pathogenesis ofHAND remains unclear, and little is known about how AD-like pathology is developed as a result of HIV-1 infection and/or long-term use of ART drugs. We postulate that neuronalendolysosome de-acidification leads to the co-pathogenesis ofHAND and AD. Here, we used rat primary cultured neuronsto test the effects of 14 separate ART drugs on endolysosomepH and amyloid beta (Abeta) protein generation. The 14 ART


drugs tested included (1) the nucleoside reverse-transcriptaseinhibitors zidovudine, abacavir, lamivudine, deoxythymidineand emtricitabine, (2) the nucleotide reverse-transcriptaseinhibitors tenofovir and disoproxil, (3) the non-nucleosidereverse-transcriptase inhibitors efavirenz and nevirapine,(4) the protease inhibitors ritonavir, nelfinavir anddarunavir, (5) the integrase inhibitors dolutegravir andelvitegravir, and (6) the booster (liver enzyme inhibitor)cobicistat. The ART drugs that de-acidified endolysosomepH also increased Abeta levels, whereas those ART drugsthat acidified endolysosome pH decreased Abeta levels.Furthermore, an agent that acidifies endolysosomes(MLSA-1) blocked ART drug-induced increases inAbeta levels. Collectively, our data suggest endolysosomede-acidification plays an important role in ART drug-induced amyloidogenesis. (This work was supported byP 3 0 GM 1 0 3 3 2 9 , a n d R 0 1 MH 1 0 0 9 7 2 , a n dR01MH105329.)

P61A new approach to “Shock and Kill” for various organsincluding CNS: Therapeutic doses of irradiation activateviral transcription and induce apoptosis in HIV-1 infectedcells.

Sergey Iordanskiy1, Gavin Sampey1, Rachel Van Duyne2,Caitlin Woodson1, Kelsi Fry1, Mohammed Saifuddin1, FabioRomerio3, Fatah Kashanchi4


1National Center for Biodefense and Infectious Disease -George Mason University; 2Center for Cancer Research,National Cancer Institute, National Institues of Health;3Deparment of Medicine, Uvinersity of Maryland School ofMedicine; 4National Center for Biodefense and InfectiousDisease - George Mason University

The highly active antiretroviral therapy reduces HIV-1 RNAin plasma to undetectable levels. However, the virus continuesto persist in the long-lived resting CD4+ Tcells, macrophages,and astrocytes, which form a viral reservoir in infected indi-viduals. Selective activation of viral transcription is criticalsince the host immune response, in combination with antire-troviral therapy, may eradicate the virus following reactivation(“Shock and Kill”). X-ray irradiation (IR), a well-definedstress signal that is widely used for many therapeutic pur-poses, has been shown to be capable of activating HIV-1 tran-scription, progeny virion formation, and eventual apoptosis ofinfected cells. Using chronically HIV-1 infected Tlymphoblastoid and monocytic cell lines, primary restingCD4+ T cells, and humanized mice infected with dual-tropicHIV-1 89.6, we examined the effect of IR-induced cellular

stress on HIV-1 transcription and viability of infected cells.Treatment of both T cells and monocytes with therapeutic IRdoses led to dramatic increase of HIV-1 transcription, as evi-denced by the presence of Pol II and reduction of HDAC1 andmethyl transferase SUV39H1 on the HIV-1 promoter usingChIP assay. Increased HIV-1 replication after IR correlatedwith higher cell death as compared with uninfected cells.Importantly, the level of phosphorylated Ser46 in p53,responsible for apoptosis induction, was markedly higherin HIV-1 infected cells following IR treatment. Exposureof HIV-1 infected humanized mice treated withantiretrovirals, which did not display viral RNA in theplasma and PBMCs, to IR resulted in a significant in-crease of HIV-1 RNA in plasma, lung, and brain tissuesof these animals. IR-induced cellular stress facilitates en-hanced apoptotic death of infected cells, possibly via p53.Collectively, these data point to the use of combination oflow to moderate dose of IR with HIV-1 transcription ac-tivators as a potential application for the “Shock and Kill”strategy for latently infected cells.

P62Use of novel Nano-particles to deliver specific HIVactivator Tat and proteins that kill infected cells

Sergey Iordanskiy1, Benjamin Lepene2, Fatah Kashanchi1


1National Center for Biodefense and Infectious Disease -George Mason University; 2Ceres Nanosciences Inc.,Manassas, VA

HIV-1 infection results in a chronic illness since long-termHAART can lower viral titers to an undetectable level.However, discontinuation of therapy rapidly increases virusburden. Moreover, patients under HAART frequently developvarious metabolic disorders, neurocognitive abnormalities,and cardiovascular diseases. Our understanding of both thedegree of treatment adherence needed to maintain long-termvirological suppression and the pathophysiology of some ofthe major adverse events associated with HIV infection andtherapy has improved. Anti-retroviral drugs targeting HIVencoded enzymes have slowed the death rate from AIDSbut, to date, no effective vaccines have been developed andthere is no practical cure. Thus, the selective activation of thelatently HIV-infected cells resulting in the replication of pro-viral genome is critical, since the virus itself, as well as thehost immune response in combination with antiretroviraldrugs (“Shock and Kill” strategy), may eliminate the virusfollowing reactivation. Additionally, elicitation of the treat-ment that can directly eliminate latently HIV-infected cellswithin the pool of resting Tcells is also important for effective


depletion of the viral reservoirs. A direct means of achiev-ing this is to first specifically activate the latent virus(i.e., with Tat) and then kill infected cells based on dis-play of specific antigens such as the HIV envelope gly-coprotein (Env) on the external surface of productiveinfected cells, where it can be recognized by a specificbinding molecule such as an antibody. The Env-targetingmoiety can be linked to various types of cytotoxicagents, yielding novel molecules that selectively killHIV-infected cells. We will show data on how this ap-proach can be used to target the infected cells in vivousing specific set of nanoparticles. Our approach is noveland unique in the sense that it can specifically activateHIV transcription and specifically destroy infected cellsin vitro and in vivo.

P63Time of Day May Influence Concentrations of Albuminand HIV RNA in Cerebrospinal Fluid (CSF)

Jennifer Iudicello1, Scott Letendre1, J. Allen McCutchan1,Qing Ma2, Igor Grant1, Ronald J. Ellis1


1University of California, San Diego; 2University at Buffalo

Background: Recent research shows that glial aquaporinchannels generate large water fluxes through interstitial andCSF during sleep, flushing solutes from brain tissue intoglymphatic and venous channels. We tested the effect of thiscircadian glymphatic flushing on levels of albumin and HIVRNA in CSF, hypothesizing that levels would be lower insamples collected earlier in the day. Methods: Albumin andHIV RNA were measured in CSF and blood in 165 HIV+adults who were taking antiretroviral therapy (ART) andwho enrolled in the CHARTER project. Concentrations werecompared to the time of collection of either CSF or bloodusing bivariate and multivariate regression. Recursivepartitioning was used to identify potentially informativethreshold values in the time-of-day. Results: CSF was collect-ed between 9:00 am and 5:20 pm (median 1:10 pm). Latercollection times tended to correlate with higher albumin con-centrations (r=0.15, p=0.057). The 35 (21.2 %) participantswho had CSF collected after 2:50 pm had higher albuminconcentrations (mean 21.8 vs. 17.4 mg/dL, Cohen’s d=0.60,p=0.019). Multivariate analysis indicated that higher CSFalbumin was associated with both serum albumin and CSFcollection time after 2:50 pm (Model R2 = 0.106,p=0.0001). Among the 24 participants with HIV RNA inCSF>50 c/mL, CSF collection time did not correlate withHIV RNA (p=0.41). Excluding 2 outliers that fell near thelimit of quantification and farthest from the regression line

strengthened the correlation (r= 0.44, p= 0.038). The 18(75.0 %) participants who had CSF collected after 11:15 amhad higher HIV RNA in CSF (Cohen’s d=0.83, p=0.053).Conclusions: Circadian variation in two CSF constituentsis consistent with experimental observations of theglymphatics system. While the observed differences maynot be clinically meaningful, they are consistent withglymphatic-mediated surges in CSF flow and suggest thatresearch studies should account for the CSF collectiontime in analyses.

P64Myocyte enhancer factor (MEF)-2 plays essential rolesin T-cell transformation associated with HTLV-1 infectionby stabilizing complex between Tax and CREB

Pooja Jain1, Alfonso Lavorgna2, Mohit Sehgal1, LinlinGao3, Divya Sagar1, Rashida Ginwala1, Edward Harhaj2,Zafar Khan1


1Department of Microbiology and Immunology, and theInstitute for Molecular Medicine and Infectious Disease,Drexel University College of Medicine, Philadelphia, PA;2Department of Oncology, Sidney Kimmel ComprehensiveCancer Center, Johns Hopkins School of Medicine,Baltimore, MD;3Graduate Program in Cancer Biology,Sylvester Comprehensive Cancer Center, The University ofMiami, Miller School of Medicine, Miami, FL

The exact molecular mechanisms regarding HTLV-1 Tax-me-diated viral gene expression and CD4 T-cell transformationhave yet to be fully delineated. Herein, utilizing virus-infected primary CD4+ T cells and the virus-producing cellline, MT-2, we describe the involvement and regulation ofMyocyte enhancer factor-2 (specifically MEF-2A) duringthe course of HTLV-1 infection and associated disease syn-drome. Inhibition of MEF-2 expression by shRNA and itsactivity by HDAC9 led to reduced viral replication and T-cell transformation in correlation with a heightened expressionof MEF-2 in ATL patients. Mechanistically, MEF-2 was re-cruited to the viral promoter (LTR, long terminal repeat) in thecontext of chromatin, and constituted Tax/CREB transcrip-tional complex via direct binding to the HTLV-1 LTR.Furthermore, an increase in MEF-2 expression was observedupon infection in an extent similar to CREB (known Tax-interacting transcription factor), and HATs (p300, CBP, andp/CAF). Confocal imaging confirmed MEF-2 co-localizationwith Tax and these proteins were also shown to interact by co-immunoprecipitation. MEF-2 stabilization of Tax/CREBcomplex was confirmed by a novel promoter-binding assaythat highlighted the involvement of NFAT (nuclear factor of


activated T cells) in this process via Tax-mediated activationof calcineurin (a calcium-dependent serine-threonine phos-phatase). MEF-2-integrated signaling pathways (PI3K/Akt,NF-kappaB, MAPK, JAK/STAT, and TGF-beta) were alsoactivated during HTLV-1 infection of primary CD4+ T cells,possibly regulating MEF-2 activity.

P65Effects of Buprenorphine on CCL2 mediatedCD14+CD16+ Monocyte Migration in the contextof NeuroAIDS

Matias Jaureguiberry-Bravo1, Loreto Carvallo 2, ChaojiangGu2, Jennifer Kelschenbach2, David Volsky2, JoanW. Berman1

(co r r e spond ing au tho r : ma t i a s . j au r egu ibe r [email protected])

1Department of Pathology, Albert Einstein College ofMedicine; 2Department of Medicine, Division of InfectiousDiseases, Icahn School of Medicine at Mount Sinai

HIV entry into the CNS mediates viral seeding, chronicneuroinflammation, and CNS damage that lead to HIVassociated neurocognitive disorders (HAND) in more than50 % of infected people despite successful cART. HIVenters the CNS by transmigration of infected monocytesacross the blood brain barrier (BBB). A mature subpopu-lation of CD14+CD16+ monocytes is increased in numberin infected people. Mature monocytes can be productivelyinfected with HIV, and transmigrate preferentially acrossthe BBB to CCL2, a chemokine elevated in the CNS ofinfected people. Thus, CD14+CD16+ monocytes are keymediators of HAND. Drug abuse is a major risk factor forHIV infection and opioids have been shown to alter theprogression and severity of HAND, in part by modulatingimmune cell functions. Buprenorphine is an opiate deri-vate used to treat opioid dependency. It is a partial agonistof MOR, and a full antagonist of KOR, opioid receptors.However, its effects on CCL2 mediated CD14+CD16+monocyte migration and subsequent neuroinflammationhave not been studied. We showed by FACS thatCD14+CD16+ monocytes express MOR and KOR, andthat mature monocytes obtained from HIV infected peopleexpress higher surface levels of both receptors comparedto monocytes from uninfected people. We also demon-strated that treatment of CD14+CD16+ monocytes withbuprenorphine reduced CCL2-mediated adhesion to brainmicrovascular endothelial cells. Additionally we foundthat buprenorphine decreased mature monocyte chemotax-is to CCL2, but that methadone, another opioid derivateused to treat heroin dependency, does not appear to affectthis process. Our findings suggest buprenorphine is

neuroprotective by limiting crucial steps in mature mono-cyte transmigration in response to CCL2 in the context ofHIV neuropathogenesis and opioid abuse. We are testingthis hypothesis using the ECOHIV mice model that reca-pitulates HIV infection and cognitive impairments ob-served in HIV infected people on cART.

P66Inducible zinc finger nuclease (ZFN) by HIV Tat to exciseHIV-1 from host genome for eradication of AIDS

Haiyan Ji1, Xiying Qu2, Won-Bin Young2, Huanzhang Zhu1


1Key Laboratory of Medical Molecular Virology of Ministryof Education/Health, School of Life Sciences, FudanUniversity, Shanghai, China; 2Department of Radiology,University of Pittsburgh School of Medicine

Current combination antiretroviral therapy (cART) cannotclear the infected cells harboring HIV proviral DNA fromthe HIV-infected patients. We previously demonstrated thatZinc-finger nucleases (ZFNs) could specifically and effi-ciently excise HIV-1 proviral DNA from the latently infectedhuman T cells by targeting the long terminal repeats (LTRs),a novel and alternative antiretroviral strategy for eradicatingHIV-1 infection. To prevent the unwanted off-target effectsfrom constantly expressed ZFN, in this study, we engineeredthe expression of ZFN under the control of HIV LTR, bywhich ZFN expression can be activated by the HIV Tatprotein during or after HIV infection. Our results show thatfunctional expression of ZFN induced by Tat excised theintegrated proviral DNA of HIV-NL4-3-GFP at the positionof Ch16p13.3 in at least 30 % population of previouslyestablished cell line. For visualizing the induction of ZFNby HIV infection, an internal ribosome entry site (IRES) wasplaced between ZFN and an optical imaging reporter geneNanoLuc, a Deep-Sea shrimp luciferase, to become a LTR-ZFN-IRES-Nanoluc. The induced expression of NanoLucand ZFN by HIV transduction was first demonstratedin vitro using bioluminescence imaging and Western blottingon the tested HuT-R5 or GHOST(3) X4/R5 cells. To dem-onstrate the proof of principle in vivo, this LTR-ZFN-IRES-Nanoluc was delivered in vivo using Adeno-Associated viral(AAV) vectors for visualizing the induction of ZFN inmouse models for HIV-1 infection. The spatial-temporalbiodistribution of the ZFN-treated cells along with HIV dis-semination in the same mice are currently under evaluationusing dual-reporter bioluminescence imaging. Taken togeth-er, positively regulated expression of ZFN in the presence ofHIV Tat may provide a safer and novel implementation of


the genome-editing technology, including Cas9/CRISPR, foreradicating the HIV-1 proviral DNA from the infected host.

P67TZIP, a synthetic Pur-protein-based peptide, highlightsa pathway linking CNS actions of Pur-alpha in HIV-1infection and in amyotrophic lateral sclerosis

Edward M. Johnson1, Earl W. Godfrey2, Dianne C. Daniel1


1Department ofMicrobiology andMolecular Biology, EasternVirginia Medical School, Norfolk VA 23507; 2Department ofAnatomy, Eastern Virginia Medical School, Norfolk VA23507

Amyotrophic lateral sclerosis (ALS, Lou Gehrig’s disease)involves degeneration of neurons in the spinal cord andbrain leading to loss of neuromuscular control. Effects ofHIV-1 infection on ALS have previously been noted butnot mechanistically dissected. We report here that the pa-thology of ALS can be influenced by the HIV-1 protein,Tat, through its interaction with cellular protein, Pur-alpha.This influence is highlighted by a new synthetic peptide,TZIP, which strongly modulates the action of Tat in HIV-1transcription as well as the interaction of Pur-alpha withpolynucleotides essential for induction of a type of ALS.ALS is of mixed etiology, including a familial form calledC9ORF72 (C9). This chromosome locus contains a repeatof the hexanucleotide GGGGCC, which is greatly expand-ed in the C9 form of ALS. GGGGCC is a Pur-alphabinding element, both as ssDNA and + strand mRNA.Pur-alpha binds a 24-mer of this sequence with a Kd ofnearly 10-8 M. TZIP was engineered to contain a genericPur protein nucleic acid-binding motif and a transportersequence allowing exogenous cell entry. TZIP has acysteine-containing motif essential for Pur-alpha bindingto Tat. TZIP binds the C9 hexanucleotide repeat nearly100-fold tighter than Pur-alpha and causes the repeat tobind Pur-alpha in an altered 3-dimensional structure. In itstranscribed RNA form the C9 repeat is reportedly involvedin the formation of cytoplasmic stress granules (SG) andatypically-translated dipeptide repeats, considered patholog-ical features of ALS. We have found that ectopic Pur-alpha expression influences formation of the SG and ame-liorates certain other aspects of ALS in primary neurons.TZIP also inhibits Tat-dependent transcription of HIV-1 at<10-10 M. HIV-1 does not infect neurons, but Tat frominfected glia can enter neurons exogenously. Elucidation ofthe Tat-Pur-alpha pathway may provide clues to treatmentof non-AIDS-related ALS.

P68Varicella zoster virus downregulates PD-L1 and MHC-1in human brain vascular adventitial fibroblasts,perineurial cells and lung fibroblasts

Dallas Jones, Anna Blackmon, C. Preston Neff, Brent Palmer,Don Gilden, Hussain Badani, Maria Nagel(corresponding author: [email protected])

University of Colorado School of Medicine

Varicella zoster virus (VZV) vasculopathy is a signifi-cant cause of intracerebral vasculopathy (stroke), giantcell arteritis and granulomatous aortitis. All 3 forms ofVZV vasculopathy develop after virus reactivates fromganglia and spreads transaxonally to the adventitia ofarteries where VZV induces persistent inflammation,which in turn leads to pathological vascular remodel-ing. The mechanism(s) by which inflammatory cellspersist to cause vascular damage in VZV-infected ar-teries is unknown. However, downregulation of pro-grammed death ligand 1 (PD-L1) on target cells causespersistent inflammation in some autoimmune diseasesand may play a role in the VZV vasculopathies.Specifically, PD-L1 can be expressed on virtually allnucleated cells and suppresses the immune system byinteracting with the programmed cell death protein re-ceptor 1 (PD-1) found exclusively on immune cells.Inhibition of PD-L1 by autoantibodies is associatedwith disease progression in rheumatoid arthritis, anddeletion of PD-L1 increases neuroinflammation in amurine model of stroke. Thus, we hypothesized thatVZV infection of adventitial cells downregulates PD-L1 and contributes to persistent vascular inflammation.Adventitial cells (brain vascular adventitial fibroblasts(HBVAFs) and perineurial cells (HPNCs)), as well ashuman fetal lung fibroblasts (HFLs), were mock- andVZV-infected and analyzed at 24 and 72 h post-infec-tion. Flow cytometry and immunofluorescence analysesshowed decreased PD-L1 expression in VZV-infectedBRAFs, HPNCs, and HFLs compared to mock-infected cells. Quantitative RT-PCR analyses showedno change in PD-L1 transcript levels between mock-a n d VZV- i n f e c t e d c e l l s , i n d i c a t i n g a po s t -transcriptional mechanism for VZV-mediated inhibitionof PD-L1 expression. Flow cytometry analyses showeddecreased major histocompatibility complex 1 (MHC-I)expression in both VZV-infected cells and adjacent un-infected cells compared to mock-infected cells. Overall,reduced PD-L1 expression in VZV-infected adventitialcells may contribute to persistent vascular inflamma-tion, while downregulation of MHC-1 may prevent vi-ral clearance in the VZV vasculopathies.


P69HIV-1 Subtype G associated with higher impairmentof neurocognitive function compared to CRF02_AGamong treatment naíve patients in Nigeria

Jibreel Jumare1, Nicaise Ndembi1, Samer El-Kamary2,Laurence Magder2, Laura Hungerford2, Tricia Burdo3,Lindsay Eyzaguirre1, Anya Umlauf4, Mariana Cherner4,Alash’le Abimiku1, Man Charurat1, William Blattner1,Walter Royal III5


1Division of Epidemiology, Institute of Human Virology,University of Maryland Baltimore; 2Department ofEpidemiology and Public Health, University of MarylandBaltimore; 3Boston College, Chestnut Hill, MA; 4HIVNeurobehavioral Research Program, University of CaliforniaSan Diego; 5Department of Neurology, University ofMaryland School of Medicine, Baltimore

Introduction: HIV-1 subtype has been shown to be associat-ed with disease progression. We compared cognitive func-tion between individuals infected with HIV-1 CRF02_AGand subtype G in Nigeria. Methods: A total of 146 antire-troviral naïve participants with plasma HIV RNA ≥1000copies/ml were selected at baseline for this study.Genotypic analysis was performed by nested PCR of prote-ase and reverse transcriptase genes. Amplification productswere sequenced, assembled using Sequencer 5.4, andaligned with HIV-1 curated subtype references. Utilizing de-mographically adjusted T scores obtained from a 7-domainneuropsychological test battery, cognitive status was deter-mined by the global deficit score (GDS) approach, with aGDS of ≥0.5 indicating cognitive impairment. Results: Atotal of 76 (52.1 %) participants were infected with the cir-culating recombinant form CRF02_AG, 48 (32.8 %) withsubtype G, and 22 (15.1 %) with other HIV-1 strains (A,C, D, A1G, CRF06_cpx, CRF43_02G). In a multivariablelinear regression adjusting for plasma HIV RNA viral load,CD4 count and depression score, global T score was higheramong CRF02_AG infected participants as compared tothose with subtype G [Mean difference (MD): 2.3; P-value:0.01]. Also, T scores were significantly higher amongCRF02_AG as compared to subtype G infected participantsfor the speed of information processing, executive function,and verbal fluency cognitive ability domains [MD: 4.4, 5.4,2.9; P-values: 0.002, 0.001, and 0.02 respectively].Adjusting for viral load and CD4 count in a multivariablelogistic regression, the odds of cognitive impairmentwere2.4 times higher among subtype G as compared toCRF02_AG infected participants (OR: 2.4 [95 % CI: 0.95,6.1]; P-value: 0.065). Conclusion: We found significantlybetter cognitive performance among antiretroviral naíve

individuals infected with HIV-1 CRF02_AG as comparedto those with subtype G. Further studies are required tocharacterize the mechanistic basis for the observeddifferences.

P70Elevated Serum S100B Levels are Associated WithImpaired Working Memory and Reduced Brain GrayMatter Volumes in ART-Experienced ChronicallyHIV-Infected Adults

Kalpana Kallianpur, Brooks Mitchell, Cecilia Shikuma,Lishomwa Ndhlovu(corresponding author: [email protected])

Hawaii Center for AIDS, John A. Burns School of Medicine,University of Hawaii, Honolulu, HI

Neuropsychological (NP) impairment and brain atrophy per-sist in chronically HIV-infected patients virally suppressed onantiretroviral therapy (ART). Produced by adipocytes, S100Bprotein in serum correlates with body mass index (BMI) inhealthy individuals. High circulating S100B concentration isalso a marker of brain injury and of blood–brain barrier dis-ruption that may precede neuronal damage. We examined se-rum S100B for cross-sectional associations with NP test per-formance, regional brain volumes, and abdominal fat mass ina subset of participants from the HIVAging with HIV Cohort-Cardiovascular Disease study. Serum S100B was assayed byELISA. Composite, domain-specific NP z-scores were obtain-ed, and T1-weighted magnetic resonance imaging at 3 T wasperformed. Visceral, subcutaneous, and total abdominal fatcontent was assessed by computed tomography. SerumS100B data were available for 39 HIV+ adults on ART [me-dian age=50 years; median CD4 count=466 cells/mL; medi-an BMI= 25.9; 35 male; 33 with plasma HIV RNA <50copies/mL]. Twenty-nine had undetectable S100B (<10 pg/mL) and 10 had detectable S100B levels (median 287.4 pg/mL). Detectability of serum S100B was associated with di-minished working memory (z-scores: −0.80 vs. 0.07,p=0.008) and psychomotor speed (z-scores: 0.17 vs. 0.38,p=0.11); lower CD4 count (390 vs. 502 cells/mL, p=0.04);and higher frequencies of activated (CD38 +HLA-DR+)CD8+ T-cells (26.1 % vs. 10.3 %, p=0.008). High S100Bcorrelated with decreased volumes of brain regions involvedin working memory and psychomotor speed; e.g., anteriorcingulate cortex (Spearman rho=−0.89, p=0.001); total cor-tical gray matter (rho=−0.72, p=0.02); and caudate nucleus(rho=−0.75, p=0.01). S100B did not relate to BMI, waist-to-hip ratio, or abdominal fat mass. Elevated serum S100B inART-treated chronic HIV infection appears to reflect an in-creased release from brain tissue and may hold potential as a


biomarker of HIV-related brain atrophy and neurocognitiveimpairment. Furthermore, circulating S100Bmay help to clar-ify mechanisms underlying neurodegeneration in well-controlled HIV disease.

P71Elimination of HIV-1 genomes from human T-lymphoidcells by CRISPR/Cas9 gene editing

Rafal Kaminski1, Yilan Chen1, Tracy Fischer1, Ellen Tedaldi2,Alessandro Napoli1, Yonggang Zhang1, Jonathan Karn3,Wenhui Hu1, Kamel Khalili1


1Department of Neuroscience/Center for Neurovirology,Comprehensive NeuroAIDS Center, Lewis Katz School ofMedicine, Temple University, Philadephia; 2Department ofMedicine, Temple HIV Program, ComprehensiveNeuroAIDS Center, Lewis Katz School of Medicine,Temple University, Philadephia; 3Department of MolecularBiology and Microbiology, Case Western ReserveUniversity, Cleveland

We employed an RNA-guided CRISPR/Cas9 DNAediting system to precisely remove the entire HIV-1 ge-nome spanning between 5’ and 3’ LTRs of integratedHIV-1 proviral DNA copies from latently infected humanCD4+ T-cells. Comprehensive assessment of whole-genome sequencing of HIV-1 eradicated cells ruled outany off-target effects by our CRISPR/Cas9 technologythat might compromise the integrity of the host genomeand further showed no effect on several cell health indicesincluding viability, cell cycle and apoptosis. Persistent co-expression of Cas9 and the specific targeting guide RNAsin HIV-1-eradicated T-cells protected them against newinfection by HIV-1. Lentivirus-delivered CRISPR/Cas9significantly diminished HIV-1 replication in infected pri-mary CD4+ T-cell cultures and drastically reduced viralload in ex vivo culture of CD4+ T-cells obtained fromHIV-1 infected patients. Thus, gene editing usingCRISPR/Cas9 may provide a new therapeutic path foreliminating HIV-1 DNA from CD4+ T-cells and potential-ly serve as a novel and effective platform toward curingAIDS.

P72Excision of HIV-1 DNA by gene editing:a proof-of-concept in vivo study

Rafal Kaminski1, Ramona Bella2, Chaoran Yin1, JessicaOtte1, Pasquale Ferrante2, Howard E. Gendelman3, Hailong

Li4, Rosemarie M. Booze4, Jennifer Gordon1, Wenhui Hu1,Kamel Khalili1


1Department of Neuroscience, Center for Neurovirology,Lewis Katz School of Medicine at Temple University,Philadelphia; 2Microbiology and Clinical Microbiology,Department of Biomedical, Surgical and Dental Sciences,University of Milan, Milan; Department of Pharmacologyand Experimental Neuroscience, University of NebraskaMedical Center, Durham Research Center, Omaha;4Behavioral Neuroscience, Department of Psychology,University of South Carolina, Columbia

A CRISPR/Cas9 gene editing strategy has been remarkablein excising segments of integrated HIV-1 DNA sequencesfrom the genome of latently infected human cell lines andby introducing InDel mutations, suppressing HIV-1 replica-tion in patient-derived CD4+ T-cells, ex vivo. Here, weemployed a short version of the Cas9 endonuclease,saCas9, together with a multiplex of guide RNAs (gRNAs)for targeting the viral DNA sequences within the 5’-LTRand the Gag gene for removing critically important segmentsof the viral DNA in transgenic mice and rats encompassingthe HIV-1 genome. Tail-vein injection of transgenic micewith a recombinant Adeno-associated virus 9 (rAAV9) vec-tor expressing saCas9 and the gRNAs, rAAV:saCas9/gRNA,resulted in the cleavage of integrated HIV-1 DNA and exci-sion of a 978 bp DNA fragment spanning between the LTRand Gag gene in the spleen, liver, heart, lung and kidney aswell as in the circulating lymphocytes. Retro-orbital inocu-lation of rAAV9:saCas9/gRNA in transgenic rats eliminateda targeted segment of viral DNA and substantially decreasedthe level of viral gene expression in circulating blood lym-phocytes. The results from the proof-of-concept studies, forthe first time, demonstrate the in vivo eradication of HIV-1DNA by CRISPR/Cas9 on delivery by an rAAV9 vector in arange of cells and tissues that harbor integrated copies ofviral DNA.

P73Negative feedback regulation of HIV-1 by gene editingstrategy

Rafal Kaminski1, Yilan Chen1, Julian Salkind1, RamonaBella2, Won-bin Young3, Pasquale Ferrante2, JonathanKarn4, Thomas Malcolm5, Wenhui Hu1, Kamel Khalili1


1Department of Neuroscience/Center for Neurovirology,Comprehensive NeuroAIDS Center, Lewis Katz School ofMedicine, Temple University, Philadelphia; 2Microbiology


and Clinical Microbiology, Department of Biomedical,Surgical and Dental Sciences, University of Milan, Milan;3Department of Radiology, University of Pittsburg School ofMedicine, Pittsburg; 4Department of Molecular Biology andMicrobiology, Case Western Reserve University, Cleveland;5Excision Biotherapeutics, Inc.

The CRISPR/Cas9 gene editing method is comprised of theguide RNA (gRNA) to target a specific DNA sequence forcleavage and the Cas9 endonuclease for introducing breaks inthe double-stranded DNA identified by the gRNA. Co-expression of both a multiplex of HIV-1-specific gRNAsand Cas9 in cells results in the modification and/or excisionof the segment of viral DNA, leading to replication-defectivevirus. In this study, we have personalized the activity ofCRISPR/Cas9 by placing the gene encoding Cas9 under thecontrol of a minimal promoter of HIV-1 that is activated by theHIV-1 Tat protein. We demonstrate that functional activationof CRISPR/Cas9 by Tat during the course of viral infectionexcises the designated segment of the integrated viral DNAand consequently suppresses viral expression. This strategywas also used in a latently infected CD4+ T-cell model aftertreatment with a variety of HIV-1 stimulating agents includingPMA and TSA. Controlled expression of Cas9 by Tat offers anew strategy for safe implementation of the Cas9 technologyfor ablation of HIV-1 at a very early stage of HIV-1 replicationduring the course of the acute phase of infection and the reac-tivation of silent proviral DNA in latently infected cells.

P74Depression among HIV-infected and seronegative controlsubjects in Cameroon: effect of age, education, and gender

Georgette Kanmogne1, Fonsah Julius2, Fang Qiu1, ClaudeTagny2, Dora Mbanya2, Dora Njamnshi3, Callixte Kuate2,Roland Doh2, Anne Marie Kengne4, Mariana Cherner5,Robert Heaton5, Alfred Njamnshi2


1University of Nebraska Medical Center; 2University ofYaounde-I; 3Yaounde Day Hospital; 4Yaounde CentralHospital; 5University of California San Diego;

Depression is a leading cause of disease burden; it worsensHIV/AIDS outcome and quality of life. Addressing thisproblem requires accurate quantification of the added burdenof depression to HIV/AIDS in a given population, andknowledge of the baseline depression prevalence in the gen-eral seronegative population. There has been no previousstudy of depression in the general Cameroon population.The current study begins to fill that important gap in oneSub-Saharan African country. We use the Beck Depression

Inventory-II to assess the prevalence and severity of depres-sive symptoms in 270 controls and HIV-infectedCameroonians. Univariable analysis showed a trend towardhigher risk of depressive symptoms among cases, comparedto controls (p=0.055), and among subjects >40 years-old,compared to subjects ≤40 years (p=0.059); but multivari-able analyses showed no effect of age and no significantdifference in the risk of depressive symptoms between casesand controls. Analysis of depression severity showed that33.73 % of cases had moderate-to-severe depressive symp-toms, compared to 19.8 % of controls (p < 0.01). Bothunivariable and multivariable regression analyses showedsignificantly higher risk of depressive symptoms among fe-males compared to males (p=0.02); both among femalecontrols (p=0.04; 95 % CI: 0.02 to 1.04) and female cases(p=0.03; 95 % CI: 0.04 to 1.03), compared to male con-trols. Gender, ART, or the presence of opportunistic infec-tions did not influence the risk of depression among cases.Lower (≤10 years) education was associated with increasedrisk of depression among controls (p=0.04), but educationdid not influence the risk of depression among cases, andthere was no association between education and depressionseverity. This study shows increased presence of depressivesymptoms in both controls and HIV-infected Cameroonians.Integrating care for mental disorders such as depression intoprimary health care and existing HIV/AIDS care programsin Cameroon may enhance the wellbeing of the general pop-ulation and could lower HIV/AIDS burden.

P75Transgenic expression of HIV-1 in Tg26 mice acceleratesatherosclerosis; and implications in vascular dementia

Alison Kearns, Shen Dai, Fengming Liu, Elizabeth Kiernan,Xiao Peng, Jennifer Gordon, Xuebin Qin(corresponding author: [email protected])

Department of Neuroscience and Center for Neurovirology,Lewis Katz School of Medicine at Temple University

HIV-1-associated comorbidities including vascular dementiaand cardiovascular disease are the most frequent cause ofdeath among HIV-1+ patients in the HAART era. Extensiveclinical evidence suggests that HIV-1 accelerates develop-ment of atherosclerosis, an immune and inflammatory dis-ease that is associated with vascular dementia and cardio-vascular diseases including brain vascular disorders andstroke. However, the mechanisms underlying HIV-1-associated atherosclerosis have not been fully elucidatedexperimentally and how atherosclerosis contributes to thedevelopment of vascular dementia, especially in the contextof chronic HIV-1 infection, remains unclear. This is mainly


due to the lack of a suitable in vivo model. Here, wepresent the characterization of a new mouse model forHIV-1-associated atherosclerosis. By crossing Tg26 mice(Tg26+/−) which contain a transgene encoding the HIV-1genome with Apolipoprotein E (ApoE) deficient mice(ApoE−/−) on a C57BL/6 J background, we introducedTg26 mice to hyperlipidemia conditions (Tg26/ApoE−/−),a widely accepted method for inducing atherosclerosis inmice. We demonstrate that 1) Tg26/ApoE−/− mice fed onhigh fat diets for either 8 or 12 weeks developed moresevere atherosclerosis in both the aortic root and aorta thanApoE−/− mice, 2) Tg26/ApoE−/− mice highly expressHIV-1 transcripts in hematopoietic tissues, which are theorigin of inflammatory cells including macrophages andT-cells, 3) Tg26/ApoE−/− did not have significantly higherlevels of cholesterol or triglycerides than ApoE−/− mice, 4)Tg26/ApoE−/− mice did not display signs of nephropathy,5) HIV-1 expression accelerated the production of foamcells, a hallmark for atherogenesis, and 6) HIV-1 expressionincreased the activation of the caspase-1, a signaling path-way critical for inflammasome formation, in Tg26/ApoE−/−‘s macrophages. Taken together, these results indicatethat Tg26/ApoE−/− mice provide a new mouse model forunderstanding the pathogenesis of HIV-1-associated athero-sclerosis. The implications of these findings on potentialmechanisms underlying HIV-associated changes in thebrain microvasculature will be discussed.

P76Chronic morphine administration exacerbatesEcoHIV-induced neurocognitive impairments in mice

Jennifer Kelschenbach1, Alejandra Borjabad1, Boe-HyunKim1, Chao-Jiang Gu1, Hongxia He1, Wei Chao1, OttavioArancio2, Mary Jane Potash1, David Volsky1


1Department of Medicine Icahn School of Medicine at MountSinai; 2Department of Pathology and Cell Biology, ColumbiaUniversity

Mild HIV associated neurocognitive disorders (mild HAND)continue to occur at high frequency despite the success ofcombined antiretroviral therapies (cART) in alleviating immu-nological abnormalities of HIV infection and preventing ordelaying onset of AIDS. Recent data suggest that the molec-ular processes underlying mild HAND are triggered withoutovert symptoms early, possibly during primary/acute infectionwhen absence of adaptive antiviral immunity facilitates virusexpansion, neuroinvasion, and establishment of stable virusreservoirs. Host and environmental factors, such as drugs ofabuse, may facilitate these processes and enhance HAND

pathogenesis. Preliminary data using conventional mice in-fected with a mouse-tropic HIV, EcoHIV, suggests that chron-ic morphine administration prior to primary HIV infectionaccelerates HIV neuroinvasion and induct ion ofneurocognitive impairments (NCI) as assessed by radial armwater maze (RAWM) testing. This finding was accompaniedby a significant increase in bran viral load, suggesting thatchronic morphine administration may facilitate viral transitinto the brain. In addition, both NCI as well as the virologicalchanges were reversed in the presence of mu opioid receptorantagonist, naltrexone, indicating that these findings are opiatedependent. Based on these results we hypothesize thatchronic morphine exposure facilitates induction ofHAND pathogenesis at the early stage of HIV infectionwhen brain reservoirs of virus are being established andmolecular processes that pre-determine NCI are initiated.The importance of the proposed research is underscoredby significant coincidence between illicit substance useand the progression of HIV infection to HAND. The ulti-mate goal of this work is to maximize currently availabletherapeutics to help the individuals at risk for the devas-tating neurocognitive complications associated with HIV-1 infection, with a particular focus on drug abusing HIVpopulations, which are both prevalent and underserved.Supported by DA037611, MH104145, and DA017618.

P77HIV-1 TAT protein expression enhancesmethamphetamine behavioral sensitization and altersdopaminergic function in mice.

James P. Kesby1, Julia A. Najera2, Benedetto Romoli1, YidingFang2, Liana Basova2, Amanda Birmingham3, Maria CeciliaG. Marcondes2, Davide Dulcis1, Svetlana Semenova1


1Department of Psychiatry, School of Medicine, University ofCalifornia San Diego, La Jolla, CA; 2Department ofMolecular and Cellular Neurosciences, The ScrippsResearch Institute, La Jolla, CA; 3Center for ComputationalBiology and Bioinformatics, University of California SanDiego, La Jolla, CA

Methamphetamine abuse is common among humans with im-munodeficiency virus (HIV). The HIV-1 regulatory protein TATis one of several HIV-related proteins associated with HIV-induced neurotoxicity. TAT-induced dysfunction of themesolimbic dopaminergic systems may result in impaired re-ward processes and contribute to methamphetamine abuse.Transgenic mice with doxycycline-induced TAT protein expres-sion in the brain show neuropathology resembling brain abnor-malities observed in humans affected by HIV disease. These


studies investigated the impact of TAT expression onmethamphetamine-induced locomotor sensitization and underly-ing changes in dopamine function/receptors and adenosine re-ceptors in mesolimbic brain areas. Mice were tested for locomo-tor activity in response to repeated methamphetamine injectionsandmethamphetamine challenge after a 7-day abstinence period.Dopamine function in the nucleus accumbens (Acb) was deter-mined using high performance liquid chromatography.Expression of dopamine and adenosine A receptors (ADORA)in the Acb was assessed using RT-PCR. Microarrays with path-way analyses assessed dopamine and adenosine signaling in thecaudate putamen (CPu). Activity-dependent neurotransmitterswitching of reserve pool neurons to a dopaminergic pheno-type in the ventral tegmental area (VTA) was determined usingimmunohistochemistry and stereology. TAT expression en-hanced methamphetamine-induced sensitization. Levels of do-pamine and associated metabolites in the Acb did not differbetween TAT-expressing and control mice regardless of meth-amphetamine exposure. However, TAT expression alone de-creased striatal dopamine (D1, D2, D4, D5) and ADORA1Areceptor expression, while increasing ADORA2A receptorsexpression. Moreover, TAT expression combined with meth-amphetamine exposure was associated with increased adeno-sine A receptors (ADORA1A) expression and increased re-cruitment of dopamine neurons in the VTA. Together thesefindings suggest that TAT-induced enhancement of metham-phetamine sensitization may involve altered dopamine andadenosine receptors expression and enhanced recruitment ofreserve pool neurons to a dopaminergic phenotype.Dopamine-adenosine receptor interactions and reserve poolneuronal recruitment may represent potential targets to developnew treatments for methamphetamine abuse in individualswith HIV.

P78CCR5-inhibitor combinedwith PrEP to potentially reduceHIV-infected monocyte trafficking across the blood brainbarrier

Joanna Kettlewell1, Robert Oda2, Christie Nakamura3


1Department of Tropical Medicine, Medical Microbiology,and Pharmacology; Hawaii Center for AIDS; John A. BurnsSchool of Medicine-University of Hawaii at Manoa;2Department of Molecular Biosciences & Bioengineering;University of Hawaii at Manoa; 3Department of TropicalMedicine, Medical Microbiology, and Pharmacology; JohnA. Burns School of Medicine-University of Hawaii at Manoa

Background: Morbidity from cognitive problems followingHIV infection continues to be significant. Since HIV-1 enters

the brain early following infection, pre-exposure prophylaxis(PrEP) (tenofovir, emtricitabine) could prevent HIV-infectionbut could also decrease virus entry into the brain. However,efficacy drops when patients are non-compliant. We hypoth-esize that combining PrEP with a CCR5-inhibitor, such asmaraviroc (MVC) could reduce HIV-infected monocyte(MO) trafficking into the CNS. We present an in-vitroblood–brain-barrier (BBB) model to test our hypothesis.Objective: Determine the level of HIV-infected MO in thepresence of PrEP compared to PrEP+MVC. Assess the levelof HIV-infected MO trafficking across the BBB with PrEPcompared to PrEP+MVC. Methods: Human brain astrocytes(HBA) and human brain microvascular endothelial cells(HBMVEC) were cultured on opposite sides of a matrix-coated cell-culture insert. In-vitro BBB integrity was assessedby trans-endothelial electronic resistance (TEER) and perme-ability with 0.45 % Evan’s Blue Albumin (EBA). MO HIV-infection will be assessed in the presence of 0, 0.02, 0.06, and0.2 microM emtricitabine and 0, 0.01, 0.03, and 0.1 microMtenofovir; and compared to infection with PrEP+MVC atMVC concentrations of 0, 0.01, 0.05, and 0.2 microM.Transmigration of MO across the BBB will then be mea-sured to assess MVC efficacy. Results: A BBB bilayer mod-el was established. BBB integrity reached its highest on day8 of culture with TEER ranging from lowest 101 Ω /cm2 tohighest 495 Ω/cm2 (4–24 replicates). BBB EBA permeabil-ity on days 6–8 measured mean of 5.3–6.0 % respectivelycompared to cell-free controls 65–68 % (p=0.0001). BBBTEER remained ≥60 Ω /cm2 (3–15 replicates) up to 6 dayspost-media change from growth to basal medium.Conclusions: A reliable BBB model was created which willbe used for MO infection and transmigration studies follow-ing PrEP±MVC. The results could provide a different para-digm and approach in preventing HIV-associated cognitiveimpairment.

P79APOE isoform specific effects on Tat-mediated HIV-1LTR transactivation in astrocytes

Nabab Khan, Jonathan D Geiger, Xuesong Chen(corresponding author: [email protected])

School of Medicine and Health Sciences, University of NorthDakota

Currently 36 million people worldwide are infected with HIV-1, and it is estimated that 50 % of those HIV-1 infected indi-viduals have HIV-1 associated neurocognitive disorders(HAND). Although unclear mechanistically, various formsof apolipoprotein E cholesterol carriers are known to affectHIV-1 replication and the prevalence of HAND: apoE4 allele


especially has been associated with higher steady-state viralloads and accelerated rates of cognitive decline. HIV-1transactivator of transcription (Tat) protein, a protein essentialfor HIV-1 replication, can be secreted from infected ortransfected cells and can be taken up by bystander cells fromthe extracellular environment via receptor-mediated endocy-tosis. Because HIV-1 Tat can bind to lipoprotein receptorprotein-1 (LRP-1) that is a major contributor to the uptake ofapoE-cholesterol in brain, we determined the extent to whichdifferent isoforms of apoE affected HIV-1 Tat-mediated HIV-1 LTR transactivation in U87MG astrocytoma cells express-ing LTR-driven luciferase. Compared to apoE2-cholesteroland apoE3-cholesterol, apoE4-cholesterol (co-incubationor post-incubation) is less effective in preventing Tat-mediated HIV-1 LTR transactivation. Similar effects wereobserved when cells were treated with isoforms of apoEwithout the incorporation of cholesterol. Furthermore, wedemonstrated that a small peptide that contains the apoEreceptor-binding region attenuated HIV-1 Tat-mediatedLTR transactivation. These findings contribute to our un-derstanding of the effects of apoE on HIV-1 infection andassociated neurocognitive disorders and suggest that apoEmimetic peptides might be used as a therapeutic strategyagainst HIV-1 infection and associated neurocognitivedisorders. (This work was supported by P30GM103329,R01MH100972, and R01MH105329.)

P80Role of endolysosmal pH in Tat-mediated HIV-1 LTRtransactivation in astrocytes

Nabab Khan, Jonathan D Geiger, Xuesong Chen(corresponding author: [email protected])

School of Medicine and Health Sciences, University of NorthDakota

Currently 36 million people worldwide are infected with HIV-1, and it is estimated that 50 % of those HIV-1 infected indi-viduals have HIV-1 associated neurocognitive disorders(HAND). Human immunodeficiency virus type 1 (HIV-1)transactivator of transcription (Tat) is essential for HIV-1 viralreplication. HIV-1 Tat can be secreted from infected ortransfected cells and taken up by bystander cells from extra-cellular environment via receptor-mediated endocytosis.Upon internalization, Tat is then accumulated inendolysosomes, from which it can be released into the cyto-plasm and enters the nucleus to facilitate viral replication. It isstill unknown the underlying mechanisms of Tat escapingendolysosomes and subsequent transactivation in the nucleus.We postulate that endolysosome pH plays a key role in Tat-mediated HIV-1 LTR transactivation. In U87MG astrocytoma

cells expressing the LTR-driven luciferase (LTR-Luc), we de-termined the extent to which modulating endolysosome pHaffects Tat-mediated HIV-1 LTR transactivation. We demon-strated that endolysosome de-acidifying agents,Chloroquine and PI5P, enhance Tat-mediated HIV-1 LTRtransactivation; whereas endolysosome acidifying agents,ML-SA1 and PI(3,5) P2, blocks Tat-mediated HIV-1 LTRtransactivation. Our findings suggest that endolysosomepH plays a key role in Tat-mediated HIV-1 LTRt ransac t iva t ion . (Th i s work was suppor t ed byR01MH100972, and R01MH105329.)

P81Pre-insulin resistance predicts worsening cognitivefunction in HIV-infected patients

Saja Khuder1, SooAh Kim1, Scott Letendre2, ThomasMarcotte2, Igor Grant2, Ned Sacktor1, Justin McArthur1,Alex Dickens1, Norman Haughey3


1The Johns Hopkins University School of Medicine; 2HIVNeurobehavorial Research Program and Department ofPsychiatry; 3The Johns Hopkins University School ofMedicine, The Johns Hopkins University School ofMedicine, Department of Psychiatry

HIV-infection and combination antiretroviral therapy (cART)are independently associated with increased risk of develop-ing lipodystrophy and metabolic abnormalities that includealterations in fat metabolism and insulin sensitivity.Reduction in insulin sensitivity is associated with lipolysisof adipocytes, increased release of fatty acid derivatives intocirculation and subsequent storage in liver, skeletal muscleand heart. This redistribution accelerates the development ofinsulin resistance. Although insulin resistance has been firmlyassociated with cognitive impairment in diabetes, the relation-ship of this metabolic syndrome with the development of cog-nitive impairment in HIV is emerging. We have previouslyshown that abnormalities in CNS lipid metabolism are asso-ciated with the development and trajectory of cognitive im-pairment in HIV infected subjects. Here, we sought to deter-mine the associations of metabolic abnormalities with cARTand cognitive function in the setting of HIV infection. Weconducted untargeted lipidomic analysis, targeted clinicaland metabolic measures in longitudinal plasma samples fromHIV+ patients exhibiting temporal changes in cognitive status(stably normal, stably impaired, declining and improving).Increased plasma levels of triacylglycerols (TAG), their etherderivatives monoalkyl-diacylglycerols (MADAG) and elevat-ed insulin were associated with stably impaired and decliningcognitive status compared to stably normal. Subjects with


improving cognitive status exhibited decreased plasma levelsof TAG and MADAG compared to stably impaired subjects.High plasma c-peptide levels in subjects with stably im-paired and declining cognitive function suggested that anincrease in pancreatic insulin production and secretionrather than a decrease in clearance was responsible forelevated plasma insulin. Low plasma adiponectin andHDL in subjects with stably impaired and declining cog-nitive functions further implicated the involvement of ametabolic syndrome in cognitive decline. These resultssuggest that pre-insulin resistance in HIV is associatedwi th impaired or decl in ing cogni t ive funct ion.Restoration of insulin sensitivity in the setting of HIVinfection may improve cognitive function.

P82MALT1 contributes to the pathogenicity of the virulentrabies virus CVS-11 in mice.

Elodie Kip1, Vanessa Suin2, Jens Staal3, Rudi Beyaert3,Steven VanGucht2


1Na t i o n a l Re f e r e n c e Cen t r e o f Rab i e s , Vi r a ldiseases,Communicable and Infectious Diseases, ScientificInstitute of Public Health (WIV-ISP), Brussels and Unit ofMolecular Signal Transduction in Inflammation,Inflammation Research Center, VIB, Ghent and Gh;2National Reference Centre of Rabies, Viral diseases,Communicable and Infectious Diseases, Scientific Instituteof Public Health (WIV-ISP), Brussels; 3Unit of MolecularSignal Transduction in Inflammation, InflammationResearch Center, VIB, Ghent and Ghent University,Department of Biomedical Molecular Biology, Ghent,Belgium

Rabies virus is a neurovirulent RNAvirus, which causes about59000 deaths in humans each year. The paracaspase MALT1is crucial for immune and inflammatory cell activation bydifferent receptors, including antigen receptors, lectin recep-tors, and some GPCRs. MALT1 acts as a scaffold signallingprotein and a cysteine protease that cleaves substrates, pro-motingNF-kB signalling andmRNA stabilization. As a result,MALT1 activation drives the expression of multiple immuno-regulatory genes. Mepazine, an inhibitor of MALT1 proteo-lytic activity, proved to have therapeutic effects in B cell lym-phoma and experimental autoimmune encephalomyelitis inmice. We examined the role of MALT1 in the developmentof rabies disease using MALT1 knock-out (KO) mice ormepazine treatment after infection with the rabies strainCVS-11. The development of disease was significantlyslowed down in MALT1 KO mice compared to wild-type

(WT) mice (mortality at 12 days instead of 8 days post inoc-ulation). Moreover, MALT1 KO mice exhibited reduced ex-pression of IL-1beta IFN-gamma and iNOS compared to WTmice which might explain the delay in the appearance of clin-ical signs. Viral RNA loads were decreased, suggestingthat inhibition of MALT1 somehow reduces virus replica-tion and/or spread of the virus in the brain. Stronger Tlymphocyte and macrophage infiltration and microglia ac-tivation were observed in WT mice compared to KOmice, which suggests that these cells contribute to patho-genicity rather than protection of the host. Daily mepazinetreatment also delayed the onset of disease, showing aninvolvement of the MALT1 proteolytic activity. We arecurrently further examining the impact of MALT1 defi-ciency in mice with conditional KO in specific cell typesto better understand the role of MALT1 in rabies patho-genesis. Our study shows that MALT1 inhibition has asignificant impact on the progression of rabies virus in-fection and disease, which emphasizes the importance ofinflammatory/immunological mechanisms on rabies dis-ease development.

P83Rift Valley fever virus is highly neurotropic and cytotoxicin a rat model of lethal encephalitis

Michael Kujawa1, Aaron Walters1, Tim Oury2, LeonardD’Aiuto3, Amy Hartman1


1Department of Infectious Diseases and Microbiology, Centerfor Vaccine Research, University of Pittsburgh; 2Departmentof Pathology, University of Pittsburgh; 3University ofPittsburgh

Background: Rift Valley Fever (RVF) is a mosquito-borneviral disease found primarily within Africa. In humans, lethalencephalitis is one of the most severe clinical outcomes. Dueto the lack of a reproducible, immunocompetent mouse modelof RVF encephalitis, not much is known about how RVFVcauses neurological disease, despite the importance of thisclinical outcome in human epidemics. Our lab recently devel-oped the Lewis rat model to study the neuropathogenic mech-anisms of RVFV. After inhalational exposure to RVFV, Lewisrats develop a uniformly lethal encephalitic disease with neu-rological symptoms. The goal of this research study is to usein vitro and in vivo methodology to understand virus-CNSinteractions.Methods: For in vivo studies: Lewis rats (infectedwith ZH501 by aerosol exposure) were euthanized and viralinfection was assessed by q-RT-PCR and immunohistochem-istry. For in vitro studies: viral infection and growth wasassessed in human NPCs, SH-SY5Y, HBMEC, Vero E6, and


BHK-21 cells. Results: Viral replication in the CNS begins inthe olfactory bulb (1–2 dpi) and proceeds posteriorly throughthe brain and spinal cord, while peripheral replication is verylimited. Pathologic lesions in the brain include pervasivevasculitis and meningitis. Preliminary studies indicate thatneurons are the primary cells expressing antigen. In vitro,SH-SY5Y and HBMEC cells were both highly permissivefor viral growth compared to Vero E6 and BHK-21 cells.iPSC-derived primary human neurons were extremely per-missive for viral infection, with near-100 % infectionlevels observed by 24 hpi followed by extensive celldeath. Conclusions: Lewis rats are thus far the most ap-propriate rodent model for studying neurotropic RVFV.In vitro and in vivo evidence shows the virus is highlyneurotropic and cytotoxic, a previously unrecognized andunappreciated finding. Our findings pave the way for fu-t u r e s t ud i e s unde r s t and ing the mechan i sm ofneuropathogenesis of RVFV.

P84The antiviral cytokine interferon-gamma restricts neuralstem/progenitor cell proliferation through activationof STAT1 and modulation of retinoblastoma proteinphosphorylation.

Apurva Kulkarni, Kristen N. Fantetti, Taylor J. Scully, LaurenA. O’Donnell(corresponding author: [email protected])

Mylan School of Pharmacy, Duquesne University

Viral infections in the central nervous system (CNS) result inimmune cell infiltration and release of pro-inflammatory cy-tokines. Interferon-gamma (IFN), a key anti-viral cytokine, isrequired for non-cytolytic clearance of many viruses from theCNS. IFN-mediated cell signaling is responsible for alteringcell survival and proliferation in different neural cells. Here,we address the effects of IFN signaling on the proliferationand differentiation of neural stem/progenitor cells (NSPCs).We hypothesized that IFN would inhibit NSPC proliferationand induce glial differentiation via the Janus activated kinase(Jak)/Signal Transducers and Activators of transcription(STAT) signaling pathways. We observed that IFN inhibitedNSPC proliferation and cell cycle progression in a STAT1-dependent manner characterized by inhibition of neurospheregrowth. Pulse-chase experiments with BrdU confirmed thatIFN slowed cell cycle progression as compared to untreatedcells. IFN restricted cell cycle progression at the late G1/Scheckpoint, which was mediated by dephosphorylation ofthe retinoblastoma protein (pRb) at serine 795 and decreasedcyclin E/cdk2 expression. In addition, IFN increased astrocyt-ic differentiation and reduced neurogenesis in vitro. During a

neuron-restricted measles virus infection in neonatal mice,IFN did not affect proliferation of nestin+NSPCs; however,it reduced neurogenesis as measured by BrdU incorporationinto newly-born neurons. These studies identify a role of IFNin modulating NSPC activity, which would identify potentialnew targets that could salvage the developing CNS from dis-ruptive effects of an anti-viral immune response. Funding forthis work is provided by the NIH (1R15NS087606-01A1) andDuquesne University’s Mylan School of Pharmacy.

P85Molecular and structural traits of IRS-1/LC3 nuclearstructures and their role in glioblastoma drug resistance

Adam Lassak1, Dorota Wyczechowska1, Anna Wilk2,Mathew Dean1, Adriana Zapata1, Luis DelValle1, FrancescaPeruzzi1, Krzysztof Reiss1


1Louisiana State University, Stanley S Scott Cancer Center;2University of South Alabama, Mitchell Cancer Institute

Insulin receptor substrate 1 (IRS-1) is a common cytosolicadapter molecule, which transduces the signal from the insulinand insulin-like growth factor I (IGF-I) receptors. We havepreviously reported that IRS-1 translocates to the nucleus ei-ther in the presence of the human polyomavirus JConcoprotein, large T-antigen, or in the presence of estrogenreceptors. Nuclear IRS-1 (nIRS-1) has been also detected incells expressing the SV40 T-antigen, v-Src, and in breast can-cer cells in association with estrogen receptor alpha, which allindicate that nIRS-1 may play a role in tumor cell biology.Here we report that nuclear IRS-1 can form unique ring-likestructures in a limited number of cells found in glioblastomabiopsies and glioblastoma xenografts. In these structures IRS-1 localizes at the periphery and the center harbors a key au-tophagy protein, LC3. They appear more frequently aftergenotoxic stress, and can be reproduced by ectopic expressionof IRS-1 with nuclear localization signal. These IRS-1-containing nuclear structures are highly dynamic, disassembleduring mitosis, rapidly exchange IRS-1 content with nucleo-plasm, and interact with other nuclear suborganelles includingPML and Polycomb bodies. Remarkably, cells engineered toform these ring-like structures acquired several new adapta-tions including drugs resistance and growth advantage in sub-optimal conditions, which correlated with the recruitment ofBmi1 to Polycomb bodies and repression of the canonicalPolycomb Repressor Complex 1 gene target, Ink4a-Arf locus.This is the first demonstration of a functional interplay be-tween IRS-1 and LC3, which are known to have distinct rolesin cytoplasm; however, in the nucleus they form unique


structure, which according to our data could play a role incontrolling autophagy and acquired stress/drug resistance.

P86Herpesvirus trigger accelerates a neuroinflammatorydemyelinating disease in a nonhuman primate modelof multiple sclerosis

Emily Leibovitch, Breanna Caruso, Matthew Schindler, NickLuciano, Seung-Kwon Ha, Nathanael Lee, Bridgette Billioux,Joseph Guy, Xiaozhen Li, Cecil Yen, Pascal Sati, AfonsoSilva, Daniel Reich, Steven Jacobson(corresponding author: [email protected])

NINDS

Viruses are implicated as triggers of disease onset/progression in multiple sclerosis (MS) and similarneuroinflammatory disorders. Human herpesviruses (HHV)in particular are strongly associated, however the time be-tween viral acquisition and disease presentation complicatesthe study of such mechanisms. Using a small nonhumanprimate, we demonstrate that prior intranasal (IN) inocula-tions with the HHV-6 viruses accelerate an experimentalMS-like neuroinflammatory disease (experimental autoim-mune encephalomyelitis (EAE)). In this study, marmosets(n= 12) received intranasal HHV-6A, 6B or mock inocula-tions, and then were induced with EAE 6 months later. PostEAE induction, brain MRIs were performed every 2 weeksuntil predetermined clinical endpoints. Animals receiving in-tranasal virus alone were asymptomatic, and we observedpatterns of viral DNA detection that reflect thecompartment-specific tropism observed in humans.Moreover, in one 6B-inoculated marmoset that met endpointcriteria before EAE induction, low levels of viral antigenwere demonstrated in the brain, suggesting that the olfactorypathway is sufficient for viral entry into the CNS. FollowingEAE induction, virus-inoculated marmosets exhibited re-duced time to inflammatory perivascular brain lesions andsignificantly reduced overall survival compared to uninfect-ed, EAE-induced controls. By intracellular cytokine staining,levels of the most highly proinflammatory CD8 T cell subsetsignificantly correlated with post-EAE survival time in thevirus-inoculated marmosets, suggesting that a (viral?)antigen-driven expansion occurred in the periphery duringthe EAE disease course. By histopathology, viral antigen inthe brain of virus-inoculated EAE marmosets was markedlyelevated and concentrated in lesions relative to the surround-ing parenchyma. The observation of viral antigen localizationto brain lesions mirrors the findings linking HHV-6 with MS.Collectively, we demonstrate that in a nonhuman primatemodel of MS, asymptomatic intranasal viral acquisition can

accelerate and exacerbate subsequent neuroinflammatory dis-ease through direct CNS infection and peripheral activationthat may promote blood brain barrier permeability.

P87HIV-1 Tat causes regional differences in phagocytosisby perivascular macrophages and microglia and causesdisruption of the blood–brain barrier

Crystal Leibrand1, Jason Paris2, Pamela Knapp3, Woong-KiKim4, Kurt Hauser2, MaryPeace McRae1


1Department of Pharmacotherapy and Outcomes Science,School of Pharmacy, Virginia Commonwealth University;2Department of Pharmacology and Toxicology, School ofMedicine, Virginia Commonwealth University; 3Departmentof Anatomy and Neurobiology, School of Medicine, VirginiaCommonwealth University; 4Department of Microbiologyand Molecular Cell Biology, Eastern Virginia Medical School

The mechanisms by which HIV infection compromisesthe blood–brain barrier (BBB) are not completely under-stood. This is important because HIV-infected macro-phages are thought to cross the BBB and infect residentmicroglia and astroglia within the CNS. We utilized atransgenic mouse model that conditionally expresses theHIV-1 regulatory protein, transactivator of transcription(Tat 1–86), in a GFAP-driven, doxycycline-dependentmanner, to examine the role of Tat in disrupting BBBintegrity and triggering activation of perivascular macro-phages and resident microglia within several brain regionsin vivo. Inducible Tat(+) transgenic and control Tat(−)mice received an intracerebroventricular (ICV) injectionof AlexaFluor 488-labeled dextrans 5 days (long-durationexposure), followed by a second ICV injection ofAlexaFluor 594-labeled dextrans 4 h (short-durationexposure) prior to sacrifice to assess potential changes inmacrophage/microglial turnover, and an intracardiac in-jection of 44-kDa horseradish peroxidase (HRP) 5 minprior to sacrifice. CNS HRP labeling was significantlygreater in Tat(+) mice in comparison with Tat(−) controls,consistent with impairment of BBB integrity. Withincaudate/putamen there was a significant increase in alllabeled (short-, long-, and dual-labeled) perivascular mac-rophages and microglia among Tat(+) expressing micecompared to controls. No significant differences were ob-served between the number of short- and long-labeledmacrophages within either intra-striatal or perivascularspaces, indicating no differences in macrophage/microglial turnover. The number of long-labeled phago-cytic monocyte-derived cells was significantly increased


in the caudate/putamen of Tat(+) mice, but significantlydecreased in Tat(+) mice in cortical regions, including thesomatosensory cortex, agranular insular cortex, andpiriform cortex, compared to Tat(−) controls. No signifi-cant differences in phagocytic monocyte-derived cellswere observed within nucleus accumbens, anterior cingu-late cortex, or primary motor cortex. These findings sug-gest that expression of HIV-1 Tat disrupts the BBB andcauses regional differences in the distribution ofperivascular macrophages and microglia in vivo.

P88HIV infection of astrocytes is detected in vivoby RNAscope and occurs in vitro via a non-classicalmechanism

Guan-Han Li1, MyoungHwa Lee1, Eugene Major2,Avindra Nath1


1Section of Infections of the Nervous System, NINDS, NIH;2Laboratory of Molecular Medicine and Neuroscience,NINDS, NIH

Astrocytes are an ideal reservoir for HIV since onceinfected the virus can stay dormant and thus reside in-definitely without perturbing the cell. However, demon-stration of HIV-infected astrocytes in vivo poses techni-cal challenges. Similarly, infecting the cells in vitro withcell free virus has been very difficult. Now, we showthat both hurdles have been overcome. In this study, wedemonstrated unambiguous infection of astrocytes atleast in 2 of 6 brain tissues with HIV encephalitis byRNAscope and GFAP co-immunostaining techniques. Inthe in-vitro experiments, we found that HIV infection inprimary fetal astrocytes was blocked due to lysosomaldegradation after the virus was endocytosed in a non-CD4 receptor-mediated manner. In fact, we could con-sistently detect low levels of CD4 mRNA in astrocytesand small amounts of CD4 protein by immunoprecipita-tion. Interestingly, CD4 mRNA but not protein was sig-nificantly up-regulated by pro-inflammatory cytokines.Therefore, increased infection of HIV was not seen inthe cells pre-treated with pro-inflammatory cytokines.However, productive infection of astrocytes was consis-tently demonstrated in the transwell cultures loaded withHIV-infected lymphocytes. This infection was CD4-independent but CXCR4-dependent since it could beblocked by anti-CXCR4 antibody and AMD3100. Wescreened a panel of laboratory strains and primary iso-lates of HIV including one isolated from the CSF andfound that the infection occurred with X4 or R5X4

viruses but not with R5 viruses. We hypothesize thatengagement of CXCR4 in the absence of CD4 couldoccur by immature virus since the binding site forCXCR4 might be exposed within the gp120 of the vi-rus. Before the virus is fully mature, the binding siteshould be available for interaction with CXCR4, thusmediating viral entry. These observations conclusivelyimplicate astrocytes as a reservoir for HIV and demon-strate that the mechanism of infection requires immaturevirus.

P89Human Endogenous Retrovirus-K envelope proteininduces neurotoxicity by causing p53-dependent nucleolarstress response

Wenxue Li1, Myoung-Hwa Lee1, Richa Tyagi1, MuznaBachani2, Joseph Steiner2, Avindra Nath1


1Section of Infections of the Nervous System, NationalInstitute of Neurological Disorders and Stroke, NationalInstitutes of Health; 2Neurotherapeutics Unit, NationalInstitute of Neurological Disorders and Stroke, NationalInstitutes of Health

About 8 % of the human genome is composed ofretrovirus-like sequences called human endogenous retro-virus (HERVs), yet their function is poorly understood.Recent studies have shown that HERVs may play criticalroles in both pathological and physiological conditions.Our group found that HERV type K (HERV-K) was ac-tivated in the brain of patients with amyotrophic lateralsclerosis (ALS). Overexpression of HERV-K envelopeprotein (Env) caused neurotoxicity in a cell culture sys-tem and motor neuron specific degeneration in a trans-genic mouse model. HERV-K Env caused nucleolar dys-function in the transgenic mouse as determined by mi-croarray analysis. Immunostaining showed translocationof the nucleolar proteins into the cytoplasm. The Envhas a signal peptide (SP) that gets cleaved andtranslocated into the nucleolus. In vitro studies show thatSP directly interacts with nucleolar proteins as deter-mined by immunoprecipitation studies. The binding ofSP to nucleolar proteins may disrupt their interactionwith MDM2, the negative regulator of p53 signal path-way. Indeed, transfection of SP upregulates p53 and itsdownstream target p21 protein, and causes neuronaldeath. In conclusion, HERV-K Env may induce neuro-toxicity by causing p53-dependent nucleolar stressresponse.


P90Phosphorylation of SAMHD1 in Brain MacrophagesIncreases with Viral Loads in the Brains of SIV-InfectedRhesus Macaques

Allison Lindgren1, Adam Filipowicz1, Marcelo Kuroda2,Woong-Ki Kim1


1Department of Microbiology and Molecular Cell Biology,Eastern Virginia Medical School; 2Division of Immunology,Tulane National Primate Research Center

SAMHD1 is a host restriction factor that blocks thereplication of HIV-1 and SIV via its depletion of theintracellular deoxynucleoside triphosphate (dNTP) pool.Recently, in vitro, it has been shown that phosphoryla-tion of SAMHD1 (pSAMHD1) in cycling macrophagesby cyclin-dependent kinase 2 acts to increase the dNTPpool, thereby increasing susceptibility to HIV-1 infec-tion. We therefore investigated, using our rhesus ma-caque SIV model, whether the level of SIV infectionof the brain corresponds to the level of pSAMHD1 ex-pression in situ in brain macrophages. Using paraffin-embedded brain tissues of uninfected macaques (n= 4)and SIV-infected macaques with (n= 5) or without en-cephalitis (n= 5), we performed immunohistochemistryto examine the expression of SAMHD1 or pSAMHD1.Automated cell counting was performed using ImageJ.Viral loads from all of the animals and their correspond-ing brain areas were also measured by qPCR and qRT-PCR. Phenotypic characterization of these SAMHD1- orpSAMHD1-expressing cells was undertaken using im-munofluorescent microscopy for various markers, in-cluding CD68, CD163, Ki-67, and SIV p28. All statis-tical analyses were carried out using GraphPad Prism 5.Both SAMHD1 and pSAMHD1 showed a significantdifference between uninfected controls and SIV-infectedanimals wi th encephal i t i s (P < 0.01) , but onlypSAMHD1 showed a significant difference betweenSIV-infected animals without encephalitis and animalswith encephalitis (P < 0.01). Results from RNA andDNA analysis show a strong correlation (r = 0.79) ofincreasing pSMAHD1 with increasing viral loads, anda mild correlation (r = 0.66) of increasing SMAHD1with increasing viral loads. These cells were confirmedto be perivascular macrophages (PVM) based on theircellular marker expression. Our results demonstrate thatSIV infection increases the number of PVM expressingpSAMHD1 and Ki-67, suggesting that SAMHD1 phos-phorylation during development of encephalitis renderscycling PVM more susceptible to SIV/HIV infection.Supported by NIH grants R01 MH107333 and R21

MH108458 to W-KK and also by R01 AI097059 toMJK.

P91The hCD59 transgenic rodent modelof Intermedilysin-mediated cell ablation

Fengming Liu1, Shen Dai1, Alison Kearns1, Xiao Peng1,Jennifer Gordon1, Sulie L Chang2, Xuebin Qin1


1Department of Neuroscience, Center for Neurovirology,Lewis Katz School of Medicine at Temple University,Philadelphia, PA; 2Institute of NeuroImmune Pharmacologyand Biological Sciences, Seton Hall University, SouthOrange, NJ

Conditional and targeted cell ablation is powerful andwidely used for studying specific cellular functions, aswell as tissue repair and recovery. Current cell ablationmodels have some inherent shortfalls that includeoffäóñtarget effects, narrow pharmacological windows,and relatively slow onset of cell death. Thus their thera-peutic applications are limited. Intermedilysin (ILY), acytolytic pore-forming toxin that is secreted byStreptococcus intermedius, lyses human cells exclusivelythrough the necrotic effect on the cells by binding to thehuman complement regulator CD59 (hCD59). Taking ad-vantage of these features, we previously reported a novelapproach by generating hCD59 transgenic mice and rats.Administering ILY these hCD59 transgenic strains couldachieve conditional, rapid and targeted cell ablation.Recently, we established a Cre-inducible hCD59 transgen-ic mouse line (ihCD59) where hCD59 expression onlyoccurs after Cre-mediated recombination. Administrationof ILY to various lines of double transgenic (Cre +ihCD59+) mice resulted in the rapid and specific ablationof immune, liver, epithelial or neural cells without off-target effects. Importantly, ILY had a large pharmacolog-ical window, which allowed us to perform dose-dependentstudies. Most recently, by crossing ihCD59 with endothe-lial specific Cre transgenic line (Tek-Cre), we haveestablished specific endothelial cell hCD59 expressingmice (Tek-Cre+/ihCD59+). Injection of ILY at the non-lethal doses to the Tek-Cre+/ihCD59+ had specificallysevere damage on the brain blood barrier of which endo-thelial cells are the key component. Together, ILY/hCD59method is an important and versatile tool that can be usedto study the effects of cell ablation in any organ system orcell type and therefore has universal application and valueto investigators in multiple fields. This approach can bealso used to study the functions of individual cell


populations in multiple disease models including infec-tious diseases and to investigate tissue injury and regen-eration including alcohol and drug abuse-associatedorgan/cell damage & recovery.

P92Interclade HIV-1 Gp120 mediates Unfolded ProteinResponse anti-apoptotic effects on glioma cells

Sheila Lopez-Acevedo, Madeline Rodriguez-Valentin,Gabriel Valentin, Mariela Rivera-Serrano, Luis Cubano,Lilia Kucheryavykh, Nawal Boukli(corresponding author: [email protected])

Universidad Central del Caribe

Patients infected with HIV are more prone to developcancers, including glioblastoma brain cancer (GBM).The median survival of GBM patients with HIV issignificantly smaller compared to HIV-negative GBMpatients, receiving same treatment. This indicates thatHIV infection influences GBM tumors’ responsivenessto treatment. The purpose of this study is to reveal themechanism of action of HIV-1 clades B and C shellproteins Gp120 and their effect on glioma resistanceto apoptosis-inducing factors. We hypothesize thatHIV-1 clades B and C glycoproteins Gp120, initiatedifferential protein expression in human astrocytomacells modulating survival abilities of cancer cells. ATandem Mass tag (TMT) isobaric labeling quantitativeproteomic approach followed by oxidative stressH2DCFDA analysis, autophagy assay by AcridineOrange staining, western blot, and apoptosis assaysbased on Trypan Blue and Annexin 5/PropidiumIodide staining were used on HIV-1 clades B and Ctreated U87 human astrocytoma cells. The study re-vealed significant up-regulation of apoptotic, endoplas-mic reticulum, oxidative stress, and chaperone-mediated autophagic markers in U87 cells treated withHIV-1 Gp120 for 24 h. Moreover, the study revealedthat HIV-1 Gp120 C and HIV-1 Gp120 B provideddifferent regulation on survival mechanisms in U87cells. HIV-1 Gp120 C induced nitrite release and pro-duction of reactive oxidative species, causing apopto-sis and tumor inhibition by an autophagy-induced celldeath mechanism. HIV-1 Gp120 clade B protein in-duced the expression of key endoplasmic reticulummolecular response and unfolded protein responsema rk e r s s u ch a s : GRP78 , P r o t e i n D i s u l f i d eIsomerase, Calreticulin, eIF2 alpha, and Bcl-2 leadingto a protective and anti-apoptotic response in gliomacells. Our data suggests that Gp120 B induces a

differential protein expression and activation of keyendoplasmic reticulum response markers in astrocyto-ma cells inducing an anti-apoptotic response, whileGp120 Clade C caused activation of pro-apoptoticmechanisms on astrocytoma cells.

P93Development of a humanized astrocyte/humanperipheral blood mononuclear cells NOD/scid-IL-2Rgcnull mouse model to assess the role of astrocytesin HIV infection

Victoria Lutgen, Srinivasa Narasipura, Maureen Richards,Lena Al-Harthi(corresponding author: [email protected])

Rush University

HIV invades the brain shortly after infection and canbecome latent in cells such as astrocytes. Given theinability for invasive studies to examine the role ofastrocytes in HIV in humans coupled with the inabilityof HIV to infect non-human astrocytes, we developed anovel humanized astrocyte/human peripheral bloodmononuclear cells (PBMCs) NOD/scid-IL-2Rgc null(NSG) mouse models using either neonatal pups oradult mice. Neonatal (PND1) NSG pups were injectedwith normal fetal human astrocytes (NHAs) either un-infected or infected with HIV-GFP (NLENG1-IRES70), with or without VSVG pseudotyping. Rate ofHIV integration in microinjected astrocytes was at 1–3 % or 20–30 % based on GFP expression from wild-type or VSVG pseudotyped virions, respectively. Pupswere weaned at 5 weeks of age and reconstituted withhuPBMCs at 6 weeks. We found that at 10 weeks ofage, NHAs continue to survive, proliferate, and migratethroughout many areas of the forebrain and form pro-cesses. To circumvent the length of time required forthe neonatal studies, we performed these studies inadult (5–6 weeks old) NSG mice. Five days after bi-lateral microinjection of HIV-infected or uninfectedNHAs into the striatum, mice were reconstituted andsacrificed 4 weeks later. NHAs survived and formedprocesses in adult NSG mice, however, there is lessproliferation and little migration at this time point. Inboth models, we have evidence for widespread HIVdissemination from HIV infected astrocytes to bloodand other tissues. Each model has its advantages, neo-nates have widespread engraftment whereas adult ex-periments have a shorter timeframe. Both models sup-port survival of human astrocytes up to 5 weeks afterinjection. These novel models allow for further studies


to determine the role of astrocytes in brain/peripherydynamics and HIV persistence/latency.

P94PROGRESSIVE DEFICITS IN SUSTAINEDATTENTIONWITH ADVANCING AGE IN THE HIV-1TRANSGENIC (Tg) RAT

Charles F. Mactutus, Kristen A. McLaurin, Rosemarie M.Booze(corresponding author: [email protected])

Program in Behavioral Neuroscience, Department ofPsychology, University of South Carolina

Despite the great success of combination antiretroviraltherapy (cART) in diminishing the most severe forms ofneurocognitive impairment, antiretrovirals alone are notsufficient to protect the brain as HAND remains as prev-alent as before cART, afflicting up to 40–70 % of HIV-1infected individuals. Attention and executive function arethe cognit ive domains most affected in HAND.Asymptomatic neurocognitive impairment conveys a 2-6X risk for progression to symptomatic HAND.Fundamental to research progress in this area is a well-characterized model of neurocognitive deficits that per-mits a prospective approach to assess chronic, progres-sive, impairments across the lifespan. Accordingly, weemployed a longitudinal design, with both male and fe-male HIV-1 Tg and Fischer-344 N control rats (back-ground strain), to test the hypothesis that with advancingage, there will be an HIV-1 viral protein-induced progres-sive loss of neurocognitive function as indexed by accu-racy of performance in a sustained attention task.Beginning at 60 days of age, sustained attention wasassessed with a signal detection task (162 trials/session)that were either reinforced (hit or correct rejection) ornon-reinforced (miss or false alarm) with sucrose pelletsuntil a criterion was met (70 % accuracy for 5 consecutiveor 7 total days). The control rats learned and improved onthe task when retested every 60 days, and with this dis-tributed practice, reached asymptotic criterion of ~6 daysby 300 days of age. In contrast, the HIV-1 transgenic ratsdisplayed an initial learning impairment, an asymptoticcriterion performance of ~7.5 days at 300 days of age,and deterioration of performance beginning at 360 daysof age. The neurocognitive impairment was most promi-nent in HIV-1 Tg female rats. The demonstration of pro-gressive neurocognitive deficits in the HIV-1 Tg rat offersa translational model for HAND as well as the testing ofnovel therapeutic approaches. Funded by NIH grantsDA013137, HD043680, & MH106392

P95Simian varicella virus reactivates in rhesus macaquesafter depletion of CD4 T cells

Ravi Mahalingam1, Mary Wellish1, Meredith Hunter2,Amy Frieman1, Lindsay Hacker1, Don Gilden1, VickiTraina-Dorge2


1Department of Neurology, University of Colorado Denver,Aurora, CO; 2Division of Microbiology, Tulane University,Tulane National Primate Research Center, Covington, LA

Simian varicella virus (SVV) infection of primates is thecounterpart of human varicella zoster virus (VZV) infec-tion. Intrabronchial inoculation of 5 rhesus macaques with4X 10 5 PFU of SVV resulted in varicella rash 10–14 dayspost inoculation (p.i.). Eight months later, 4 monkeyswere treated with 50 mg/kg of anti-CD4 antibody. After1 week, the percent of CD4-T cells in blood was reducedfrom 40–60 % to 5–30 % in treated monkeys andremained low for rest of the experiment. Seven days aftertreatment, zoster rash developed in the absence of viremiain the monkey with lowest (5 %) level of CD4-T cells.Zoster also developed in all other treated monkeys 28–55 days post treatment (d.p.t.) and in the untreated mon-key 10 months p.i. Examination of affected skin revealedSVV ORF 63 protein mostly in sweat glands. All mon-keys were euthanized 1 week after the time of their re-spective zoster, and lymph nodes were analyzed by dualcolor immunohistochemistry. SVVORF63 protein wasfound to be colocalized in macrophages. Our findingsindicate that CD4-T cell immunity is important in SVVreactivation.

P96SIV Sequences in the Choroid Plexus Represent CNSViral Reservoirs in Rhesus Macaques that Develop AIDSwith SIVE

Jaclyn Mallard1, Brittany Rife2, Emily Papazian1, AriannaNoggle1, David J Nolan2, Marco Salemi2, Kenneth CWilliams1


1Department of Biology, Boston College, Chestnut Hill, MA;2Emerging Pathogens Institute, University of Florida,Gainesville, FL

HIV-associated neurocognitive disorders (HAND) and SIV-associated encephalitis (SIVE) are characterized bymonocyte/macrophage (Mo/MΦ) accumulation and virus in


the CNS. Mo/MΦ traffic is critical for CNS viral reservoirs. Tcells and Mo/MΦ enter the CNS from cerebrospinal fluid(CSF) via the choroid plexus (CP) or via the blood brainbarrier. CSF is the only route to sample CNS virus. CNSMo/MΦ s are the predominantly infected CNS cells inHAND and SIVE. We hypothesized CP and CSF viral se-quences represent CNS, Mo/MΦ and T cell-derived se-quences, but CP and not CSF virus seed CNS viral reservoirs.We assessed CNS, CP and peripheral (CSF, plasma, T cellsand Mo/MΦ) tissues and cells from 19 SIVmac251-infectedand 2 uninfected rhesus macaques (11 CD8-depleted, 10 non-depleted). SIV+ animals included: 4 without AIDS, 6 AIDSwith SIVE and 9 AIDSwithout SIVE (SIVnoE). Double-labelin situ hybridization and immunohistochemistry was done tocount SIV-infected T cells and Mo/MΦ in CP. SIVE animalshad higher numbers of Mo/MΦ, not Tcells in CP compared toSIVnoE animals. Mo/MΦ and T cells in CP contained SIV-RNA+ subsets. Phylogenetic analysis of SIV gp120cDNA was used to compare CSF, CP and CNS virus withperipheral virus. CSF viral sequences were dispersed be-tween CNS and peripheral sequences. CP and CNS viralsequences clustered together and were highly compart-mentalized in SIVE animals, but not in SIVnoE animals.Detection of SIV-RNA+ T cells and Mo/MΦ underscoresthe CP as a source of CSF virus. Dispersed phylogeny ofCSF viral sequences among peripheral and CNS se-quences indicates that CSF is not a viral reservoir. Mo/MΦ accumulation and compartmentalized CP and CNSvirus suggests infected Mo/MΦ in these tissues are thesource of CNS viral reservoirs. Increased compartmental-ization of CP and CNS virus with SIVE highlights theimportance of viral reservoirs in SIVE pathogenesis.

P97Insulin signaling suppresses virus replicationand neuroinflammation in HIV/AIDS.

Manmeet Mamik1, Eugene Asahchop1, Wing Chan1, YuZhu41 William Branton1, Brienne Mckenzie2, Eric Cohen 3,Christopher Power4


1Department ofMedicine, University of Alberta; 2Departmentof Medical Microbiology & Immunology, University ofAlberta; 3Department of Microbiology, Infectiology andImmunology, Universite de Montreal; 4Departments ofMedicine and Medical Microbiology & Immunology,University of Alberta

HIV-1 infection of the brain results in neuroinflammation andcontributes to the development of HIV-associatedneurocognitive disorder (HAND), for which there is no

specific treatment. Insulin resistance is also a major complica-tion of contemporary HIV/AIDS care. Studies have shownthat insulin treatment suppressed HIV-1 replication in leuko-cytes. Herein, we investigated the actions of insulin usingex vivo and in vivo models of HAND. Primary human mi-croglia (HFM) were infected with HIV-1 and treatedwith/without insulin, followed by mRNA analyses. Primaryhuman neurons were exposed to viral protein R (Vpr) andtreated with/without insulin followed by toxicity assays. Inan in vivo model of HAND, cats infected with a neurovirulentfeline immunodeficiency virus (FIV) strain, were treated dailyfor 6 weeks with intranasal (IN) insulin or PBS. Increasedneuroinflammatory gene expression was observed in brainsfrom patients with HIV/AIDS. The insulin receptor was de-tected on both neurons and glia but its expression was unaf-fected by HIV-1 infection. Insulin treatment of HIV-infectedHFM suppressed supernatant HIV-1 p24 levels, reducedCXCL10 and IL-6 transcript levels while PPAR-gamma ex-pression was induced. Insulin treatment of primary humanneurons prevented HIV-1 Vpräóñmediated cell process re-traction and death. In FIV-infected (FIV[+]) cats, daily INinsulin treatment (20.0 IU/200 μl for 6 weeks) reducedCXCL10, IL-6 and FIV RNA detection in brain althoughPPAR-gamma in glia was increased, compared to PBS-treated FIV[+] control animals. These molecular changeswere accompanied by diminished glial activation in cere-bral cortex and white matter of insulin-treated FIV[+] an-imals, with associated preservation of cortical neurons.Moreover, IN insulin treatment improved neurobehavioralperformance including both memory and motor functionsin FIV[+] animals. Thus, insulin exerted ex vivo andin vivo antiviral, anti-inflammatory and neuroprotectiveeffects in models of HAND, representing a new therapeu-tic option for patients with neurodegenerative disordersincluding HAND.

P98A CNS IRIS-like phenotype in SIV-infected pigtailedmacaques on cART

Lisa Mangus, Sarah Beck, Suzanne Queen, Elizabeth Engle,Joseph Mankowski(corresponding author: [email protected])

Johns Hopkins University

Histologic examination of the central nervous system (CNS)of simian immunodeficiency virus (SIV)-infected pigtailedmacaques treated with combination antiretroviral therapy(cART) revealed that animals with persistent low viral loadin plasma and/or CSF (n=9 macaques) frequently developeda unique moderate to severe lymphocyte-rich encephalitis that


was morphologically distinct from SIV-encephalitis (SIVE).CNS lesions in cART-treated macaques consisted of multifo-cal perivascular infiltrates of lymphocytes and plasma cellswith fewer macrophages and no multinucleated giant cells.Further, while SIV RNA is abundant in the CNS of untreatedanimals with SIVE, brain viral RNA levels in cART-treatedanimals with lymphocytic lesions were typically low or unde-tectable by digital droplet PCR (ddPCR) and in situ hybridi-zation. Immunophenotyping demonstrated that the infiltrateswere comprised of a mixture of CD20+ B cells, CD3+ Tcells,and CD68+ macrophages, and that approximately 30 % ofcells were positive for Ki67, indicating active proliferation.Western blot analysis of CSF from these animals revealedintrathecal antibodies targeting multiple SIV proteins.Immunostaining for Epstein Barr nuclear antigen(EBNA) to detect associated gamma-herpesviral infectionwas negative; no evidence of other opportunistic infec-tions (OI) in the CNS or other organ system was found.Taken together, these findings may represent an enhancedadaptive immune response to residual low level SIV rep-lication or viral protein production in the brain. While themorphologic features resemble those reported in HIV pa-tients with CNS immune reconstitution inflammatory syn-drome (CNS-IRIS) in the absence of OI, we have notidentified a correlation of the syndrome with CD4 nadir.In addition, the preponderance of B lymphocytes in themacaque lesions is unique. Further research into the path-ogenesis of these lesions may aid our understanding of theconsequences of residual CNS HIV replication in the set-ting of an intact immune system.

P99Interaction between HIV-1 Tat and morphine causesdisruption of GABAergic systems within CA1

WilliamDMarks1, Aaron J Barbour2, Jason J Paris3, ChristinaJ Schier3, Melissa D Denton3, Sylvia Fitting4, A RoryMcQuiston2, Pamela E Knapp2, Kurt F Hauser3


1Department of Pharmacology and Toxicology; 2Departmentof Anatomy and Neurobiology, Virginia CommonwealthUniversity; 3Department of Pharmacology and Toxicology,Virginia Commonwealth University; 4Department ofPsychology and Neuroscienc, University of North CarolinaChapel Hill

The spatial memory deficits associatedwith HIV can be tracedback to hippocampal dysfunction caused in part by the actionsof HIV-1 Tat. Previous investigations found that Tat exposuresignificantly decreased LTP in CA1 in HIV transgenic mice;however, the loss in function was accompanied by only

modest reductions in excitatory proteins and structures. Incontrast, Tat markedly decreased synaptotagmin 2 (Syt2)levels, while increasing gephyrin protein levels reflecting adisruption in GABAergic transmission within CA1. Furtherinvestigation of the GABAergic cells within CA1 revealedthat parvalbumin (PV) positive cells of the pyramidal layer(putatively bistratified cells), SST positive cells of the stratumoriens (putatively O-LM cells), and nNOS positive, NPY neg-ative cells of the pyramidal layer and stratum radiatum (IS3sand neurogliaform cells respectively) were preferentially vul-nerable to Tat. These interneurons form a microcircuit withinCA1 that has a demonstrated role in spatial memory forma-tion. Electrophysiological analysis of CA1 pyramidal cellsin slice preparations from Tat-expressing or control trans-genic mice treated with morphine or a placebo showedthat Tat increases the resting membrane potential, andincreases the amount of current needed to depolarize cellsto their firing threshold. Surprisingly, this was unchangedin pyramidal cells from Tat and morphine exposed mice.However, numbers of action potentials were reduced ifmorphine was removed from the recording bath. Theseresults suggest a compensatory upregulation of GABAAreceptors (GABAR) in pyramidal cells, which offsets theeffects of morphine to inhibit mu opiate receptor-express-ing-PV+ basket cells and consistent with the observedincrease in gephyrin. Additional electrophysiologicalstudies will utilize GABAR antagonists to determinewhether Tat and morphine reduce spontaneous inhibitorypost synaptic currents (iPSCs) as predicted by diminishedinterneuronal presynaptic inputs.

P100Sex Differences in HIV Effects on Mechanismsof Executive Dysfunction Among Drug Users

Eileen Martin1, Michael Keutmann2, Raul Gonzalez3, PaulineMaki2, Leah Rubin2


1Department of Psychiatry, Rush University Medical Center;2Department of Psychology, University of Illinois-Chicago;3Department of Psychology, Florida International University

Rationale. Performance on tests of verb (action) fluency (AF)is a robust predictor of functional independence and CSF ab-normalities among persons living with HIV/AIDS. AF perfor-mance is strongly correlated with neurocognitive measures ofexecutive function and is critically dependent on integrity ofprefrontal-striatal circuitry, but has not been compared sys-tematically between HIV+ men and women or among sub-stance dependent individuals (SDIs). We compared AF per-formance of HIV+ and HIV- male and female SDIs and


investigated the relationship between AF and performance onmeasures of two different types of executive function withknown sensitivity to HIV. Methods. We tested 329 HIV- and148 HIV+ substance dependent individuals (SDIs), primarilyusers of cocaine and alcohol, enrolled in a larger study of sexand HIV serostatus effects on neurocognitive function amongSDIs. The sample was approximately 70 % women and 85 %African American. Subjects were free of AIDS-defining andother neurologic illness or injury and HIV+ subjects wereambulatory and stable on cART. All subjects performed atimed measure of verb fluency requiring speeded retrievalof names of human activities. Subjects also completedtwo executive function measures of verbal learning strat-egy and decision making under risk. Subjects were veri-fied abstinent from drugs and alcohol at testing and werewell matched on demographic, substance use, and comor-bid variables. Results. HIV+ men retrieved significantlymore words than HIV- men and HIV+ women, p= .002.Decision making was significantly correlated with AF forHIV+ men only, p< .001, but verbal learning strategy cor-related significantly with AF performance for HIV+ wom-en, p = .003. Conclusions. Different types of executivefunction contribute to verb fluency performance amongHIV+ men and women, suggesting possible sex differ-ences in brain circuitry affected by neuroAIDS.Supported by the National Institute on Drug Abuse.

P101Identifying the Neurocognitive Determinants of HIV-RiskBehaviors

Cherie Marvel1, Brian Anderson2, Jordan Mandel1, NedSacktor1


1Johns Hopkins University; 2Texas A&M University

HIV-risk behavior is often motivated by the possibility ofimmediate reward (e.g., unprotected sex). Moreover, re-ward stimuli co-opt one’s attention. Our previous studyshowed that attention to reward was stronger in HIV+individuals with vs. without a drug history. In this pre-liminary follow-up study, we have tested 10 HIV+ indi-viduals (5 with and 5 without a drug history), usingfMRI combined with a task that measures the ability tolearn a stimulus-reward association and then, subsequent-ly, to ignore that reward when presented in a novel,irrelevant context. The basal ganglia (BG), a key targetfor damage by HIV infection, play a central role in re-ward processing and addiction. We hypothesized that ifreward salience was high and difficult to ignore, BGactivity would increase in response. This effect would

be greater for those with a drug history. HIV+ individ-uals with a drug history showed hyper BG activity whentask demands required them to ignore the reward infor-mation, indicating reward salience was high. HIV+ indi-viduals without a drug history showed no such BG ac-tivity. Thus, HIV+ individuals with a drug history mayface the greatest challenge in ignoring reward informa-tion and, consequently, controlling risky behaviors inpursuit of that reward.

P102Alpha-synuclein restricts West Nile virus in neuronsduring exocytosis and contributes to neuronal subtypesusceptibility to infection

Aaron Massey1, Katherine Shives2, J David Beckham3


1Department of Medicine, Division of Infectious Diseases,University of Colorado; 2Department of Immunology andMicrobiology, University of Colorado; 3Division ofInfectious Diseases

Introduction: West Nile Virus infection can cause sub-cortical encephalitis with injury to specific regions ofthe brain including the basal ganglia, substantia nigra,and cerebellum. Given the injury of dopaminergicpathways, patients can present with Parkinsoniansymptoms. Alpha-synuclein (Asyn) is known to causeParkinson’s disease but the function of native Asynexpression in neurons is not known. Recent evidencesuggests that Asyn interacts with Rab proteins and N-ethylmaleimide-sensitive factor attachment protein re-ceptor (SNARE) proteins in membranes to alter vesicletransport in neurons. Thus, we determined the role ofAsyn in WNV pathogenesis in neurons. Methods &Results: In a primary neurons and a mouse model ofWNV encephalitis, we have shown that Asyn restrictsviral growth and prevents neuronal injury and diseasein the brain. To determine the mechanism of Asyn-induced restriction of WNV, primary cortical andstriatal neurons from E18 mice were inoculated withmock or WNV (MOI 1) and analyzed using immuno-fluorescence analysis. We found that Asyn localizeswith WNV envelope and Rab1-positive membranes.WNV-inoculated primary cortical neurons exhibit sig-nificantly higher levels of Asyn co-localization withRab1 compared to primary striatal neurons. Thus, weexamined primary cortical neurons for sensitivity toWNV infection and injury. Using wild-type and Asynknockout neurons, we found that Asyn expressionprotected primary cortical neurons from WNV growth


and injury compared to primary striatal neurons.Conclus ions: Our f indings suggest that Asyn-dependent resistance to WNV infection is associatedwith Rab1-dependent vesicle trafficking and contributesto neuron subtype susceptibility to infection.

P103In vitro modeling of blood–brain barrier deregulationby HIV-1 Tat and opioids

Monique Maubert1, Katherine Kercher1, Marianne Strazza1,Vanessa Pirrone1, Wei Lin2, Rui Feng2, Brian Wigdahl1,Michael Nonnemacher1


1Department of Microbiology and Immunology, Institute forMolecular Medicine and Infectious Disease, DrexelUniversi ty College of Medicine; 2Department ofBiostatistics and Epidemiology, Center for ClinicalEpidemiology and Biostatistics, University of PennsylvaniaSchool of Medicine

Many of the pathological observations made in humanimmunodeficiency virus type 1 (HIV-1)-associatedneurocognitive disorders (HAND) have been attributedto compromise of blood–brain barrier (BBB) integrity,and selected viral proteins have been implicated in de-regulation of the BBB, including the transactivator oftranscription (Tat). In addition, illicit drug use is aknown comorbidity in exacerbation of disease in HIV-1-infected individuals. Importantly, opioid abuse withinthis population confounds disease progression in multi-ple ways, including increased viral replication and pe-ripheral viral load, and enhanced incidence and severityof neurocognitive impairment including dementia, ascompared to non-users. Studies have suggested that ex-posure to both HIV-1 Tat protein and mu-opioids dis-rupts BBB homeostasis and permeability in primarycells, including an increased pro-inflammatory state, aswell as augmented cellular transmigration, and enhancedbarrier leakiness. In this study, a human brain microvas-cular endothelial cell line, hCMEC/D3, was utilized toestablish an in vitro model of the BBB to investigatethe effects of Tat or morphine exposure on BBB com-promise. Changes in mRNA transcripts of tight junctionproteins (TJP) were observed throughout the course ofexposure. Differences in TJP expression and localizationwere also observed at the protein level following cellu-lar fractionation and western immunoblot analysis.These studies demonstrate that exposure to Tat or mor-phine compromises BBB integri ty by inducing

alterations in molecular expression at both the mRNAand protein levels.

P104Lipocalin-2 contributes via a CCR5-dependentmechanism to neuronal degeneration in an in vivo modelof HIV-associated brain injury

RickY Maung1, Daniel Ojeda1, Ana Sanchez1, Marcus Kaul2


1Infectious & Inflammatory Disease Center, SanfordBurnham Prebys Medical Discovery Institute, La Jolla,CA 92037; 2Infectious & Inflammatory Disease Center,Sanford Burnham Prebys Medical Discovery Institute,La Jol la, CA 92037, Department of Psychiatry,University of California, San Diego, 9500 Gilman Drive,San Diego, CA 92093

Infection with HIV-1 often triggers neurocognitive im-pairment and degenerative brain injury. Viral proteins,such as the envelope protein gp120, trigger neuroinflam-mation and production of neurotoxins by microglia andmacrophages. We recently observed in a transgenicmodel of HIV-associated brain injury induced by aCXCR4-utilizing viral envelope gp120 (gp120tg) thatthe genetic knockout of CCR5 ameliorated microglialactivation and abrogated neuronal damage and behavior-al impairment. A microarray analysis of CNS RNA ex-pression showed that brains of CCR5 wild-type (WT)and CCR5KO gp120tg mice express markers of an in-nate immune response and neuroinflammation. Theacute phase protein lipocalin-2 (LCN2) was one of themost up-regulated factors. In vitro, LCN2 was itselfn eu ro t ox i c i n a CCR5-dependen t f a sh i on incerebrocortical cell cultures comprising neurons, astro-cytes and microglia. As expected, inhibition of CCR5alone failed to abrogate neurotoxicity of the CXCR4-utilizing gp120, but paradoxically it rescued neuronsfrom gp120 toxicity in combination with LCN2 whileconcomitantly reducing activation of microglial cells,thus recapitulating the finding in CCR5-deficientgp120tg brains. Moreover, in vivo, the genetic knockoutof LCN2 in gp120tg mice protected neurons fromgp120-induced injury without reducing astrocytosis.Altogether, our study provided evidence for a combinedand interdependent contribution of the acute phase pro-tein LCN2 and CCR5 to microglial activation and HIVgp120-induced brain injury. Supported by grants fromthe NIH, MH087332, MH104131, MH105330 andDA026306 (to M.K.)


P105Reduced Resting-State Connectivitybetween the Ventromedial Prefrontal Cortex and InsulaCorresponds to Longer QTc Interval in HIV+ and HIV-Individuals

Roger McIntosh1, Dominic Chow2, Corey Lum3,Mel i ssa Hida lgo1 , Cec i l i a Shikuma2 , KalpanaKallianpur2


1Department of Health Psychology, University ofMiami; 2Hawaii Center for AIDS, Department ofMedicine, John A. Burns School of Medicine;3Division of Cardiology, Department of Medicine,University of Hawaii John A. Burns School ofMedicine

Chronic human immunodeficiency virus (HIV) infec-tion is associated with increased risk of cerebrovascu-lar and cardiovascular disease. Prolongation of the QTinterval is a precursor to fatal cardiac arrhythmias ob-served in HIV. Although resting-state functional con-nectivity of neural networks (RSFC) is altered in per-sons with HIV, the RSFC of brain structures that reg-ulate cardio-autonomic function has not been assessed.This study examined the relationship between heartrate-corrected QT interval (QTc) and RSFC of the leftand right ventromedial prefrontal cortex (VMPFC).Eighteen HIV+ patients on stable antiretroviral therapy[median age = 52 years; median CD4 count = 457 cells/μl; 14 with plasma HIV RNA <50 copies/ml; medianQTc = 412 msec] and no history of coronary artery dis-ease were compared to 26 HIV-negative participantswith similar demographic and cardio-metabolic vari-ables. Contrasts of seed-based RSFC of the left andright VMPFC revealed lower anterior-posterior connec-tivity in patients than in comparison subjects. Whole-brain regression analyses of RSFC of the VMPFCseeds showed that longer QTc interval was associatedwith reduced RSFC between (a) left VMPFC and boththe left insula and right dorsal cingulate cortex(p < 0.001), and (b) right VMPFC and both left insulaand left dorsal cingulate (p < 0.001). After controllingfor age, longer QTc interval corresponded to decreasedRSFC between the left VMPFC and (a) right cingulate(R = −0.33, p < 0.05) and (b) left insula (R = −0.40,p < 0.01). Reduced right VMPFC connectivity with theleft insula was also associated with longer QTc(R = −0.37, p < 0.05), and for HIV+ individuals, waslinked to decreased CD4 count (R = 0.45, p = 0.06).RSFC appears sensitive to clinical markers of cardio-autonomic function and may shed light on mechanisms

underlying ventricular arrhythmias and sudden cardiacdeath.

P106fMRI Correlates of Cardioautonomicand Endothelial Dysfunction in PostmenopauseWomen with HIV

Roger McIntosh1, Melissa Hidalgo1, Tali Naibryf2, NehaRajan1, Judith Lobo1


1Department of Psychology, Unversity of Miami;2Department of Psychology, University of Chicago

Endothelial dysfunction is associated with a predomi-nance of sympathetic nervous system activity, particu-larly after menopause. The central nervous system,which is susceptible to HIV infection, has direct influ-ence on parasympathetic and sympathetic balance.Postmenopause is a period concomitant with endothe-lial, cardioautonomic and functional brain connectivityimpairment, however, it is unclear how these markersare related in HIV. Seven postmenopausal women onstable antiretroviral therapy and no history of coronaryartery disease performed a deep breathing test, fromwhich E:I ratio was calculated from largest RR inter-val, during expiration, and smallest RR interval, duringinspiration. Blood was drawn to assess the proportionof classical, intermediate, and non-classical peripheralblood mononuclear cells along with colony-forming ca-pacity of endothelial progenitor cells (EPCs), i.e., pro-portion of the number of tunneling nanotubules to EPCcolonies after 7 and 14 days. A 7 min resting statebrain scan was also recorded on a subsequent visit.There was a significant (p < .001) positive correlationbetween E:I ratio and EPC colony forming capacityat 7 days; a negative (p < .001) correlation betweenduration of HIV infection and colony forming capacityat 14 days; and a trending (p = .089) negative correla-tion between the proportion of intermediate (CD14++CD16+) mononuclear cells and EPC colony formingcapacity at 7 days. The E:I ratio was then regressedon a map of brain areas showing functional connectiv-ity with the left anterior insula - a structure commonlyactivated during parasympathetic response. Greaterconnectivity between the left anterior insula and twostructures implicated in the generation of sympatheticresponse, i.e., right posterior insula and cingulate werefound in patients with higher E:I ratios. This studyprovides preliminary evidence for a neurogenic model


of cardioautonomic and endothelial dysfunction inpostmenopause women with chronic HIV infection.

P107Alterations in hippocampal CA3 synaptodendriticstructure in the HIV-1 Tat transgenic mouse

Virginia McLane, William Marks, Pamela Knapp, KurtHauser(corresponding author: [email protected])

Virginia Commonwealth University

Nearly one-third of human immunodeficiency virus( H I V ) - 1 p a t i e n t s d e v e l o p H I V- a s s o c i a t e dneurocognitive disorders (HAND). HIV-1 rapidly in-fects the central nervous system (CNS), where neuro-toxic HIV-1 proteins such as transactivator of transcrip-tion (Tat) are expressed by infected glia. Tat exertsdirect and indirect toxic effects on sensitive neuronssuch as those in the hippocampus, a region criticalfor learning and memory. Previously, we observed re-duced apical dendritic spines and disrupted synapticintegrity in pyramidal CA1 neurons in an inducibleHIV-1 tat transgenic mouse model. This dendritic pa-thology correlated to deficits in Barnes Maze perfor-mance and alterations in CA1 interneuron microcircuit-ry. In the current work, we investigated the effect ofTat expression on pyramidal neuron dendrite structureand total volume in the CA3 region of the hippocam-pus. Male mice (60–80 days old) expressing GFAP-driven reverse tetracycline transcriptase activator(rtTA) + and Tat transgenes received 14 days of doxy-cycline chow (6 mg/g). GFAP-rtTA+, Tat- mice servedas controls. Tat expression correlated to a 27 % reduc-tion in dendritic spine density in the Golgi-impregnatedneurons of Tat + mice as compared to Tat- controls(9.13 ± 0.53 vs. 12.50 ± 0.91 spines/10 μm, p = 0.012).Using stereological methods, we observed no signifi-cant change in the volume of four major hippocampalsubregions: CA1 (Tat- 10.39 ± 0.47 mm^3 vs. Tat +10.16 ± 0.77 mm^3), CA2 (Tat- 1.24 ± 0.12 mm^3 vs.Tat + 1.43 ± 0.06 mm^3), CA3 (Tat- 7.35 ± 0.33 mm^3vs. Tat + 7.57 ± 0.47 mm^3), and the dentate gyrus(Tat- 6.72 ± 0.41 mm^3 vs. Tat + 6.57 ± 0.33 mm^3).Overall, this work suggests that Tat induction causessynaptodendritic injury and subtle alterations in hippo-campal network connectivity, rather than gross neuro-nal losses, in area CA3. The findings also suggest Tatmediates key aspects of the pathology cause by HIV-1that underlie the cognitive deficits associated with

HAND. Supported by NIH grants R01 DA018633,K02 DA027374, K99 DA039791, and T32 DA007027.

P108INTACT SENSORYAND MOTOR SYSTEMFUNCTION THROUGH ADVANCING AGE IN THEHIV-1 TRANSGENIC RAT

Kristen McLaurin, Rosemarie Booze, Charles Mactutus(corresponding author: [email protected])

Program in Behavioral Neuroscience, Department ofPsychology, University of South Carolina

Understanding the progression of HIV-1 associatedneurocognitive disorders (HAND) is a critical need asthe preva lence of HIV-1 in older ind iv idua ls(>55 years) is markedly increasing due to the adventof combinat ion ant i re t rovi ra l therapy (cART).Longitudinal experimental designs, used to assess age-related disease progression in HIV-1, however, are de-pendent upon intact sensory and motor system functionthrough advancing age. Three experimental paradigmswere employed to assess the integrity of the sensoryand motor systems in the aging HIV-1 transgenic (Tg)rat. The HIV-1 Tg rat, which resembles HIV-1 seropos-itive individuals on cART, expresses 7 of the 9 HIV-1genes, is non-infectious, and has been promoted forinvestigating the effect of long-term HIV-1 viral pro-tein exposure on neurocognitive deficits, as in HAND.HIV-1 Tg and control animals were assessed at ad-vancing ages (i.e., ≥ 6 months of age) in all experi-mental paradigms. First, the integrity of the auditory,visual, and tactile sensory systems was assessed usingcross-modal prepulse inhibition (PPI). HIV-1 Tg andcontrol animals exhibited robust inhibition of the audi-tory startle response to all sensory modalities, indicat-ing the ability to detect stimuli. Second, gustatory sys-tem function was assessed using a sucrose taste pref-erence test, controlling for bottle position and test dayusing a Latin-square experimental design. As sucroseconcentration increased, a linear increase in mean con-sumption was observed regardless of genotype. Third,motor system function was assessed using locomotoractivity. Through advancing age, HIV-1 Tg animalsdisplayed significantly greater ambulatory activity rela-tive to control animals, suggesting no motor systemimpairment. Thus, the present studies provide strongevidence for intact sensory and motor system functionthrough advancing age in the HIV-1 Tg rat indicatingthe utility of the HIV-1 Tg rat for longitudinal studiesinvestigating the progression of neurocognit ive


impai rment in HAND. Funded by NIH grantsDA013137, HD043680, and MH106392

P109HIV-1 Tat variation correlates with neurocognitiveimpairment in the Drexel Medicine CARES cohort

Anthony Mele1, Gregory Antell2, William Dampier1, VanessaPirrone1, Benjamas Aiamkitsumrit1, Jean Williams1, ShendraPassic1, Katie Kercher1, Wen Zhong1, Zsofia Szep3, JeffreyJacobson4, Brian Wigdahl1, Uri Hershberg2, MichaelNonnemacher1


1Department of Microbiology and Immunology, Institute forMolecular Medicine and Infectious Disease, DrexelUniversity College of Medicine; 2School of BiomedicalEngineering, Science and Health Systems, DrexelUniversity; 3Division of Infectious Disease and HIVMedicine, Department of Medicine, Drexel UniversityCollege of Medicine; 4Department of Medicine, Section ofInfectious Disease, Lewis Katz School of Medicine, TempleUniversity

In recent years, HIV-1 mortality has decreased; however,the prevalence of neurocognitive impairment has in-creased. Previous studies have examined the HIV-1 proteinTat and demonstrated that expression continues even inpatients currently on antiretroviral therapy (ART) withlow-to-undetectable viral titers. Tat has also been shownto be neurotoxic and a causative agent of central nervoussystem (CNS) inflammation. The current studies seek toidentify and characterize the predominant variations withinHIV-1 Tat correlated with the neurocognitive impairment.HIV-1 Tat sequences were obtained from the DrexelMedicine CNS AIDS Research and Eradication Study(CARES) Cohort. The sequences were amplified from pe-ripheral blood mononuclear cells, translated, and aligned tothe HIV-1 subtype B consensus reference genome to com-pare amino acid substitutions. Multiple positional hotspotsof high variation in Tat were identified within the cysteine-rich domain, glutamate-rich domain, and second exon.Statistical analyses were applied to amino acid positionsand variants associated with the neurocognitive impair-ment status of each patient. To assess the functional impli-cations of these results, ten patients, five asymptomatic andfive symptomatic for neurocognitive impairment, were se-lected based on their Global Deficit Score (GDS). Tat exonfractions were quantified based on the frequency in eachpatient. The predominant Tat exon 1 and 2 sequences werereconstructed into expression vectors. These will be uti-l ized to identify and characterize functional and

morphological alterations in vitro. Overall, these analysesseek to determine a correlation between GDS and the prev-alence of Tat variants in asymptomatic or symptomaticneurocognitively impaired patients, which may prove use-ful characterizing HIV-1 neuropathogenesis.

P110Higher hypocretin (orexin) levels correlates with bettermotor skills in HIV+ women

Raissa Menendez-Delmestre1, Claribel Gonzalez1, RosaRodrí_guez-Benitez2, Miriam Matos1, Richard Noel3,Valerie Wojna1


1University of Puerto Rico, Medical Sciences Campus;2University of Puerto Rico, Rio Piedras Campus; 3PonceHealth Sciences University

The hypocretin (orexin) system is associated with multiplebrain functions including alertness and central motor con-trol due to the many projections of the hypocretin-secretingneurons. These neurons are located specifically in the lat-eral hypothalamus, an area known to be dysfunctional inHIV-positive individuals suffering from HIV-associatedneurocognitive disorders (HAND). HAND is characterizedby cognitive, behavioral and motor dysfunctions. In thisstudy we investigated the correlation of hypocretin-1(hcrt-1) levels with cognitive performance in HIV-positive women. In a retrospective study, we measuredserum and CSF hcrt-1 levels of HIV-seropositive womenwi thou t a h i s to ry o f drug abuse eva lua ted fo rneurocognitive performance. Cognitive performance wasdetermined using the HAND criteria where eight cognitivedomains were evaluated. Serum and CSF hcrt-1 levelswere determined using the fluorescent immunoassay kit(Phoenix Pharmaceuticals), with an intra- and inter-assayvalidity of 10 and 15 % respectively. Non-Parametric sta-tistics were used to determine correlation with a p valueless than 0.05 to determine significance. A positive corre-lation was found between Hcrt-1 levels and age in the HIV-seropositive group with normal cognition (Serum:p= 0.020; CSF: p= 0.026). Serum and CSF hcrt-1 levelswere positively correlated (p= 0.022). After adjusting forage, a significantly positive correlation was observed be-tween CSF hcrt-1 levels and better performance in the mo-tor skills domain (p= 0.0016), mainly due to the effect ofthe grooved pegboard test when performed with the non-dominant hand (CSF: p= 0.008). This was also true for theCSF/serum hcrt-1 ratio (motor skills domain: p = 0.047;grooved pegboard non-dominant: p= 0.024). No other cor-relations were observed between hcrt-1 levels and other


cognitive domains or individual neuropsychological tests.These results suggest that the hcrt system may have a rolein the motor skill performance of HIV-positive women.Further investigations need to be conducted to determinethe role of the hcrt system in cognitive performance ofHIV-positive individuals.

P111Discriminant analysis of neuropsychological testingdifferentiates HIV-associated Neurocognitive Disorder(HAND) from Mild Cognitive Impairment (MCI) dueto Alzheimer’s disease in HIV over age 60

Benedetta Milanini1, Isabel Allen2, Shireen Javandel1, JoannaHellmuth1, Robert Paul3, Victor Valcour1


1Memory and Aging Center, Department of Neurology,University of California San Francisco, San Francisco;2Department of Epidemiology & Biostatistics, University ofCalifornia San Francisco, San Francisco; 3Missouri Instituteof Mental Health, University of Missouri, St. Louis

Differentiating HIV-associated neurocognitive disorder(HAND) from Alzheimer’s disease (AD) in older HIV-infected individuals has become a major clinical challenge.Here we hypothesized that the clinical expressions ofHAND and Mild Cognitive Impairment (MCI) due to ADare characterized by distinct neuropsychological profiles. Allparticipants underwent a comprehensive neuropsychologicalbattery.We performed a discriminant function analysis includ-ing a set of cognitive measures and demographic factors (i.e.,age, gender, education and premorbid functioning) to identifythe best model to differentiate HAND from MCI. Logisticregression analyses were performed to identify tests associat-ed with group membership; only these tests were includedwhen collinearity was noted. The sample included 95 individ-uals with HAND (79 % cognitively symptomatic according toFrascati criteria) with an average age of 65 (5), average edu-cation of 15 (2.2) and CD4 T-cell count of 545 (245.4), and 72non-HIV individuals with MCI with an average age of 69(10.5) and education of 18 (3.3). Most of the HIV-infectedindividuals were on antiretroviral therapy (97 %) and werevirologically suppressed (83 %). Results revealed a cognitivephenotype of HAND characterized by significantly higherdiscrimination index (difference between hits and false posi-tives on the California Verbal Learning Test), better perfor-mance on the Digit Backward, as well as worse performanceon theMiniMental State Exam, Trails A,Modified Trails, andDigit Symbol (p’s < 0.05). Our model correctly classified86 % of the cases using this combination of measures(Wilks’ Lambda = 0.54, p < 0.001). The classification

accuracy was 90 % for HAND and 82 % for MCI. This workhas clinical relevance as the HIV-infected population ages andhas increased vulnerability to neurodegenerative diseases.Recognizing these distinct profiles might be useful in a clini-cal setting in identifying HIV-infected patients whose patternof impairment is not typical of HAND and who may requirefurther evaluation or monitoring.

P112Neuropsychological Phenotypes Among Men with andWithout HIV Disease in the Multicenter AIDS CohortStudy

Samantha A. Molsberry1, Yu Cheng2, Lawrence Kingsley3,Lisa Jacobson4, Andrew Levine5, Eileen Martin6, CynthiaMunro7, Ann Ragin8, Ned Sacktor9, James T. Becker10, Forthe Neuropsychology Working Group of the MACS(corresponding author: [email protected])

1Population Health Sciences Program, Harvard UniversityGraduate School of Arts and Sciences; 2Department ofStatistics, University of Pittsburgh; 3Departments ofInfectious Diseases and Microbiology and Epidemiology,University of Pittsburgh Graduate School of Public Health;4Department of Epidemiology, Johns Hopkins BloombergSchool of Public Health; 5Department of Neurology,University of California Los Angeles; 6Department ofPsychiatry, Rush University School of Medicine;7Department of Psychiatry, Johns Hopkins UniversitySchool of Medicine; 8Department of Radiology,Northwestern University Feinberg School of Medicine;9Department of Neurology, Johns Hopkins UniversitySchool of Medicine; 10Departments of Psychiatry,Neurology, and Psychology, University of Pittsburgh

Background: Since the beginning of the HIV epidemic, ithas been clear that HIV infection affects brain function.Prior to the development of combination anti-retroviraltherapies, HIV infected individuals performed poorly ontests of cognition and often became severely cognitivelyimpaired. Since the development of these therapies, severeimpairment is rare but milder impairment remains com-mon. The objective of this study was to identifyparticipant-based latent structure of cognitive functionusing cluster analysis to identify subgroups within theMulticenter AIDS Cohort Study and to build a model topredict cluster membership. Methods: We conducted k-means cluster analysis using MACS participantsäó» neu-ropsychological domain scores from the time of their firstcognitive classification. Exploratory analyses were used toidentify factors associated with cluster membership andstepwise regression was performed to select variables for


a multinomial logistic regression model to predict clustermembership. Results: The k = 4 solution was selected, gen-erating clusters characterized as follows: cluster 1 per-formed poorly for every domain, cluster 2 performedabove average on learning and memory and below averagefor all other domains; cluster 3 performed above averagefor executive function, speed, and working memory, andbelow average on all other domains; and cluster 4 per-formed highly for every domain except learning. Afterconducting exploratory analyses and stepwise regression,the variables retained for the final model were age, testlanguage, CESD score, race, and ever having syphilis.When tested on a separate subset of the data, the modelpredicted cluster membership poorly. Conclusions: Thereis an identifiable pattern of neuropsychological domainscores among MACS members but we were unable to de-velop a model that accurately predicts cluster membership.Neither HIV serostatus nor HIV-related biomarkers wererelated to cluster membership, which is consistent withother recent findings that patterns of neuropsychologicaltest performance do not map directly onto HIV serostatus.

P113Transdermal Rivastigmine as Adjuvant Therapyfor HIV-Associated Neurocognitive Disorders

Jose A. Munoz-Moreno1, Anna Prats1, Nuria Perez-Alvarez1,Jose Molto1, Maite Garolera2, Crisanto Diez-Quevedo3,Cristina Miranda1, Carmina R. Fumaz1, Maria J. Ferrer1,Bonaventura Clotet4


1Lluita Contra la SIDA Foundation - Germans Trias i PujolUniversity Hospital; 2Consorci Sanitari Terrassa Hospital;3Germans Trias i Pujol University Hospital; 4Lluita Contrala SIDA Foundation - Germans Trias i Pujol UniversityHospital - IrsiCaixa Foundation - Universitat de Vic

Background: Adjuvant therapies could serve as supportivetreatments for improvement of HIV-associated neurocognitivedisruption. To date, no studies have been reported investigat-ing the effects of transdermal rivastigmine in people with HIVinfection and cognitive impairment. We developed a random-ized controlled pilot trial to study the safety and efficacy oftransdermal rivastigmine as potential adjuvant therapy forHIV-associated neurocognitive disorders. Methods: We en-rolled HIV-infected patients with cognitive impairment onstable antiretroviral therapy in a 48-week follow-up, random-ized, controlled, pilot trial. Participants received transdermalrivastigmine (9.5 mg daily), lithium (400 mg twice daily, ti-trated progressively), or did not start a new treatment (controlgroup). The primary efficacy endpoint was change in a

neuropsychological composite score (NPZ-7). Secondaryendpoints included specific cognitive measures and domains,as well as functional outcomes (daily functioning, emotionaland quality of life variables). Safety analysis covered frequen-cy of adverse events and changes in laboratory test results.Results: Seventy-six subjects were screened and 29 finallyrecruited. All groups showed increased cognitive out-comes, although they failed to reach significant change.The rivastigmine group showed the greatest trend (meanNPZ-7 [SD], baseline vs wk 48): rivastigmine: −0.47(0.22) vs −0.11 (0.29), p= 0.06; lithium: −0.50 (0.40) vs−0.26 (0.21), p = 0.22; control: −0.52 (0.34) vs −0.32(0.52), p= 0.44. Regarding the primary efficacy endpoint,there were no differences among groups (mean NPZ-7change [SD]): 0.35 (0.14), 0.25 (0.40), 0.20 (0.44);p= 0.78. No significant changes were also observed withregard to secondary outcomes. Severe adverse eventswere not recorded and blood tests revealed no relevantchanges in blood count, biochemistry or immunology.Conclusions: In this small randomized trial, transdermalrivastigmine was safe and well-tolerated in HIV-infectedpatients on stable antiretroviral therapy after a 48-weekfollow-up, although no significant benefits were observedin cognitive status or functional parameters. A larger ran-domized trial should contrast these findings.

P114Accuracy of the NEU Screen to Detect CognitiveImpairment in Older People with HIV

Jose A. Munoz-Moreno1, Estela Lopez-Masramon1, AnnaPrats1, Nuria Perez-Alvarez1, Maite Garolera2, Maria J.Ferrer1, Bonaventura Clotet3


1Lluita Contra la SIDA Foundation - Germans Trias i PujolUniversity Hospital; 2Consorci Sanitari Terrassa Hospital;3Lluita Contra la SIDA Foundation - Germans Trias i PujolUniversity Hospital - IrsiCaixa Foundation - Universitat deVic

Background: The NEU Screen is a proposed tool for practicaldetection of HIV-associated cognitive impairment in the clin-ical setting. This method includes 3 paper-based neuropsycho-logical measures and has an expected administration time of≤10 min. We investigated its precision in a sample of agingHIV population. Methods: A total of 368 HIV-infected peoplewith available neurocognitive data were studied. All subjectshad undergone a comprehensive neuropsychological battery(2–3 h, 7 domains) and had accessible demographic and clin-ical information. Presence of cognitive impairment wasestablished as gold standard, and the accuracy of the NEU


Screen was compared according to several age cutoffs (spe-cifically 30, 40, 50 and 60 years). Sensitivity, specificity, andpositive and negative predictive values (PPV, NPV) were cal-culated. Results: Subjects were mostly men (80 %), with amedian (interquartile range) age of 43 (37; 48) years old,undetectable plasma viral load (70 %), median CD4 cellcount of 511 (375; 720) cells/μL, and median nadir CD4cell count of 223 (101; 360) cells/μL. Frequency of cog-nitive impairment was 51 %, with a comorbidity rate of39 %. When age cutoffs were compared, both sensitivityand specificity appeared to be greater as the age washigher, starting at 75.43 % and 73.75 %, respectively.The highest accuracy was found at the 60 years cutoff(≥60 vs <60 years): sensitivity: 90.91 % vs 74.01 %;specificity: 92.31 % vs 71.26 %; PPV: 90.91 % vs73.18 %; NPV: 92.31 % vs 72.12 %. This was confirmedby a significantly greater correct classification at the samecutoff (91.67 % vs 72.67 %; p= 0.022). Conclusions: TheNEU Screen offers a remarkable good sensitivity andspecificity to detect cognitive impairment in people withHIV, particularly those aged 60 years or older. We pro-pose the use of this brief and feasible method to help inthe screening of HIV-associated neurocognitive disorders,considering specially its benefits in the elderly HIVpopulation.

P115Cerebrospinal Fluid Viral Escape in ART-experiencedHIV-1-Infected Adults

Shibani Mukerji1,2, Vikas Misra2, David Lorenz3, AnnaCervantes-Arslanian4, Jennifer Lyons5, Spyridon Chalkias6,Alysse Wurcel7, Deirdre Burke7, Nagagopal Venna2, SusanMorgello8, Igor Koralnik9, Dana Gabuzda1


1Department of Cancer Immunology and Virology, Dana-Farber Cancer Institute, Boston, MA; 2Department ofNeurology, Massachusetts General Hospital, Boston, MA;3Department of Cancer Immunology and Virology, Dana-Farber Cancer Institute, Boston, MA; 4Departments ofNeurology and Neurosurgery, Boston Medical Center,Boston, MA; 5Department of Neurology, Brigham andWomens Hosp i t a l , Bo s t on , MA; 6D iv i s i on o fNeuroImmunology, Beth Israel Deaconess Medical Center,Boston, MA; 7Department of Geographic Medicine andInfectious Diseases, Tufts Medical Center, Boston, MA;8Division of Infectious Diseases, Icahn School of Medicinea t Moun t S in a i , New York , NY; 9D iv i s i on o fNeuroImmunology, Beth Israel Deaconess Medical Center,Boston, MA; Department of Neurological Sciences, RushUniversity Medical Center, Chicago, IL

Among ART-experienced HIV-1-infected adults, cerebrospi-nal fluid (CSF) viral escape has been associated with neuro-inflammation, drug resistance-associated mutations (DRMs),and neurological symptoms. In a retrospective case-seriesfrom Boston hospitals and the National NeuroAIDS TissueConsortium, we identified 41 HIV-1-infected patients onARTwith discordant CSF and plasmaHIV-1 RNA levels from2005 to 2016. Discordance was defined as detectable CSFHIV-1 RNA when plasma levels were <50 copies/ml, orHIV-1 RNA >0.5-log higher in CSF than plasma. The estimat-ed prevalence of CSF viral escape was 4.7 %; median baselineage was 50, and duration of HIV-1 infection was 17 years(IQR 13–22). Median CD4 count and nadir were 329 cells/mm^3 (IQR 217–388), and 21 cells/mm^3 (IQR 2–60), re-spectively; 76 % were on boosted-protease inhibitors.Neurological symptoms were present in 29 cases; 3 caseshad a concurrent diagnosis of PML or lymphoma. The cohortwas classified into subgroups based on plasma HIV RNAlevels in the preceding 24 months: high-level viremia (HLV:>1000 copies/ml), low-level viremia (LLV: 51–1000copies/ml), and plasma suppression with CSF blip or escape(CSF HIV-1 RNA ≤200 or >200 copies/ml, respectively).Subjects classified as HLV reported more substanceabuse, while those classified as LLV or plasma suppres-sion were more frequently neurosymptomatic (81 % vs.53 %). The majority of DRMs in Boston cases and pub-lished reports were detected in both plasma and CSF;M184V/I mutations were more frequent in CSF than plas-ma. Characteristics frequently observed in HIV-1-infectedindividuals with CSF viral escape include HIV-1 infection>15 years, low CD4 nadir, boosted-protease inhibitor use,and LLV in the preceding 6 months. M184V/I genotype isanother factor that may predispose to CSF escape.Classification of CSF escape subtypes based on longitu-dinal plasma HIV RNA levels provides a conceptualframework that will enable better understanding of viraland host factors leading to CSF viral escape.

P116Getting in to the Brain: Potential of Nanotechnologyto Manage Neuro-AIDS and Drug Addictions

Madhavan Nair(corresponding author: [email protected])

Department of Immunology- Herbert Wertheim College ofMedicine- Florida International University

2014 report suggests that more than 36.9 million people areliving with HIV/AIDS in the world today that includes morethan 1.2 million people from US. Current studies also showthat more than 247 million people are affected with substance


abuse in the world that includes more than 24 millionAmericans. Reports also show that more than 3–4 millionpeople are co-affected with HIV and illicit drug use.Although highly active anti-retroviral therapy (HAART) hasresulted in remarkable decline in the morbidity and mortalityin AIDS patients, inadequate delivery of HIV drugs across theblood–brain barrier (BBB) to the brain results in HIV persis-tence. Drugs of abuse such as opiates act synergistically withHIV-1 to potentiate the HIV-related neurotoxicity that leads todevelopment of Neuro-AIDS. In recent years, use of nano-technology has shown exciting prospect for developmentof novel drug delivery systems. We herein report the de-velopment of a Magneto-Electric Nanocarrier (MEN) todeliver and release on demand of HIV drugs and opiateantagonist which are otherwise impenetrable to brain andinhibit HIV and reverse opiate mediated adverse neurolog-ical effects. The proposed nanocarrier is anticipated tosimultaneously reduce Neuro-AIDS and opiate addictionin HIV-1 infected opiate addicts. Further, this invented/patented new technology will have universal applicabilityfor targeting and controlled release of drugs against avariety of other CNS diseases such as Parkinson’s,Alzheimer’s, brain tumors etc.

P117HIV-1 infected astrocytes support HIV disseminationinto blood and other tissues

Srinivas Narasipura, Victoria Lutgen, Maureen Richards,Lena Al-Harthi(corresponding author: [email protected])

Rush University

Ample evidence demonstrates that HIV infects the brain dur-ing acute stage, setting the stage for inflammatory responses,leading to HIV-Associated Neurocognitive Disorders. Thebrain is a reservoir for HIV, yet whether this reservoir is com-partmentalized or HIV can egress from it to re-seed the pe-riphery is not clear. We evaluated here the role of HIV-1 in-fected astrocytes in disseminating virus to other tissues out-side of the brain. We used a chimeric humanized astrocyte/human peripheral blood mononuclear cells (hu-astro/PBMCs)NSG mice to address this question. Primary normal humanastrocytes (NHA) were infected in vitro with full infectiousHIV with GFP under the control of IRES. Rate of infectionwas 20–30 % when pseudotyped with VSVG or 1–3 % whennon-pseudotyped. Neonate and adult NSGmice were injectedwith HIV-1 infected NHAs or free virion. Astrocyte engraft-ment and survival was assessed using flurescent microscopy.Six weeks (neonatal) or 5 days (adult) post engraftment, micewere reconstituted with huPBMCs and 4 weeks post

reconstitution, animals were sacrificed. HIV-1 was measuredin blood, spleen and lymph nodes using Taqman realtime PCRand GFP detection by microscopy or flow cytometry. Wefound HIV in all of the tissues tested in about 80 % of theinfected animals. Viral outgrowth assays of the huPBMCsisolated from spleen demonstrated replication compe-tence of the virus. Injection of free virus into the braindid not lead to HIV detection in or outside of the brain.These data demonstrate that HIV-1 infected astrocytesare capable of harboring replication competent virusin vivo that can egress from the brain to seed the periph-ery. Given that astrocytes make up ~60 % of the braincells and rate of infection is around 1–2 %, astrocytescan be a significant reservoir for HIV that is not com-partmentalized in the brain but can disseminate to othertissues.

P118Brain delivery of the glutamine antagonist6-Diazo-5-oxo-L-norleucine (DON) via prodrugapproach: a potential treatment for HIV-associatedneurocognitive disorders (HAND)

Michael Nedelcovych1, Boe-Hyun Kim2, Rana Rais1, AndrejJancarik3, Lukas Tenora3, Jesse Alt4, Jennifer Kelschenbach5,Pavel Majer3, David Volsky5, Barbara Slusher6


1Department of Neurology, Johns Hopkins Drug Discovery,Johns Hopkins School of Medicine; 2Department ofMedicine, Icahn School of Medicine at Mount Sinai;3Institute of Organic Chemistry and Biochemistry, Academyof Sciences of the Czech Republic v.v.i.; 4Johns Hopkins DrugDiscovery, Johns Hopkins School of Medicine; 5Departmentof Medicine, Icahn School of Medicine at Mount Sinai;6Departments of Neurology, Psychiatry, Neuroscience, andMedicine, Johns Hopkins Drug Discovery, Johns HopkinsSchool of Medicine

Recent studies revealed that cART-treated patients with HIV-associated neurocognitive disorders (HAND) have increasedlevels of cerebrospinal fluid (CSF) glutamate compared tothose without neurocognitive impairment, suggesting that al-terations in glutamate homeostasis may contribute to HANDpathogenesis. The glutamine antagonist 6-Diazo-5-oxo-L-norleucine (DON) attenuates glutamate release from activatedmicroglia/macrophages exposed to HIV, and reverses cogni-tive deficits in preclinical models of CNS viral infection, buthas not yet been tested in a model of HAND. Furthermore, theclinical utility of DON is limited by GI toxicity. We thus testedDON in a murine model of HAND and subsequently rational-ized a targeted DON prodrug design to enhance its brain/


plasma ratio, limiting systemic exposure and GI toxicity.DON was evaluated in the EcoHIV-infected murine modelof HAND. Cognitive performance in radial arm water maze(RAWM) was measured in mice administered DON (1mg/kg,i.p., q.o.d) beginning before (prevention) or after (treatment)EcoHIV inoculation. CSF glutamate was measured in thetreatment paradigm. To enhance DON’s therapeutic indexfor HAND, we then utilized a dual promoeity prodrug strategyto increase lipophilicity and brain delivery. DON preventedand reversed cognitive impairment as measured by RAWMperformance. DON also normalized EcoHIV-induced in-creases in CSF glutamate. However, DON-treated mice suf-fered systemic side effects such as lethargy and weightloss. In an attempt to synthesize DON prodrugs with en-hanced brain/plasma ratio and improved tolerability, alkylesters were added to DON’s carboxylate functionalitywith additional masking of DON’s amine group byN-(acyloxyalkoxycarbonyl) derivatives. Dual promoietyDON prodrugs were metabolically stable in human, mon-key, and pig plasma and provided >10 fold enhancementin CSF/plasma ratio after in vivo administration in mon-key and pig. These studies provide the first evidence thatglutamine antagonism may be useful in the treatment ofHAND. DON prodrugs may provide an innovative andsafe clinical path for this therapeutic strategy.

P119Brain Microstructural Changes in HIV Infection: AMeta-analysis and longitudinal study

Erin O’Connor1, Assia Jaillard 2, Felix Renard2, ThomasZeffiro3


1Lewis Katz School of Medicine at Temple University;2Centre Hospitalier Universitaire Grenoble; 3TempleUniversity

Purpose: Because of strong histopathological evidencedocumenting HIV effects on cerebral white matter, numerousstudies have used diffusion tensor imaging (DTI) to measurehow HIVaffects white-matter microarchitecture. While manyhave reported reduced fractional anisotropy (FA) and in-creased mean diffusivity (MD) in HIV infection, the quantita-tive inconsistencies observed across studies motivated us to:(1) do a cross-sectional literature meta-analysis of DTI mea-sures in HIV infection and then (2) examine white-matter mi-crostructural changes in a small longitudinal study of HIVinfected participants studied before and after beginning com-bination anti-retroviral therapy (cART). Methods: The meta-analysis included 15 cross-sectional studies reporting FA and11 studies reporting MD in the corpus callosum. Random

effects meta-analysis was used to estimate individual studystandardized mean differences along with between and withinstudy sources of variation. Next, cART effects were studiedwith DTI in a separate sample of HIV infected participants andcontrols studied before, and 3 and 6 months after beginningtreatment. Results: Meta-analysis revealed standardized meandifferences of −0.56 for FA (p<0.01) and 0.21 (p=0.06) forMD. Nevertheless, estimates of between study heteroge-neity showed that 62–76 % of the observed variance wasbetween studies. In a separate longitudinal sample thatshowed some evidence of mild neuropsychological im-pairment, but no evidence of AIDS dementia, pre-treatment FA was higher and MD lower in HIV infectedparticipants than controls (p < .001), and measuresremained stable over 6 months. Conclusions: Publishedstudies of white-matter microstructure following HIV in-fection reveal substantial between study variations thatcould result from image acquisition, image processing orcohort selection differences. Low temporal variation in FAand MD in our longitudinal cohort demonstrates repro-ducibility of imaging biomarkers when uniform acquisi-tion and processing methods are used. Use of standardizedacquisition and processing methods and longitudinalstudy designs might better isolate sources of biologicalvariation in measured diffusion parameters.

P120Blood Brain Barrier Integrity Compromised by PBMCfrom HIV Positive Individuals

Robert Oda1, Joanna Kettlewell1, Christie Nakamura1,Melissa Agsalda-Garcia1, Cecilia Shikuma2, Nancy Hanks2,Bruce Shiramizu1


1Department of Molecular Biosciences & Bioengineering,College of Tropical Agriculture and Human Resources,University of Hawaii at Manoa; 2Hawaii Center for AIDS;John A. Burns School of Medicine; University of Hawaii atManoa

Objectives: For HIV infected individuals who are on stableantiretroviral therapy (ART) with maximally suppressed HIVRNA levels, HIV associated cognitive impairment (HACI)continues to persist. Data from our group suggest that additionof entry inhibitor drug Maraviroc (MVC) can improve HACIin patients on stable ART. We assessed the effect of peripheralblood mononuclear cells (PBMC) on blood brain barrier(BBB) integrity before introduction of MVC. Methods: TheBBB was established with astrocytes and endothelial cells onan insert. Inserts were cultured in growth medium for 7 daysprior to trans-migration studies. PBMC isolated from


consented patients were placed on the BBB and allowed totransmigrate for 24 h before the flow through was collected.Confluence of the barriers was analyzed using trans endothe-lial electronic resistance (TEER) and permeability wasassessed using a 0.45 % concentration of Evanäó»s Blue dyein bovine serum albumin (EBA). Results: PBMC from 4HIV-positive (HIV+), cognitively impaired individualsbefore Maraviroc intensification were collected andplaced on the BBB. TEER values decreased from pre-migration (123.1 ± 2.305Ω/cm2, n= 14) to post-migration(90.72 ± 4.57Ω/cm2, n= 14) and EBA permeability valuesincreased from pre-migration (3.538 ± 0.8056 %, n= 13) topost-migration (15.93 ± 2.833 %, n = 14), p-values<0.0001 and 0.0004 respectively. Compared to post trans-migration data from HIV-negative subjects (n = 4,TEER = 112.5 ± 6Ω/cm2, EBA= 3.925 ± 1.153 %), posttransmigration data from HIV+ PBMC showed decreasedconfluence (p = 0.0313) and increased permeability(p= 0.0425). Conclusions: Preliminary results indicated asignificant compromise in BBB integrity upon exposureto HIV+ PBMC. The impact of MVC on PBMC and BBBintegrity will be compared to entry results after partici-pants have reached the study time point. Results of thestudy could provide valuable insight into the impact ofMVC on BBB integrity.

P121Role of peroxisome proliferator activated receptors(PPARs) in the regulation of HIV-1 gp120 associated braininflammation: implications in HIV-1 neuropathogenesisand its treatment

Amila Omeragic, Tozammel Hoque, David Hampson, ReinaBendayan(corresponding author: [email protected])

Leslie Dan Faculty of Pharmacy, University of Toronto

Despite the use of combination antiretroviral therapy for thetreatment of HIV-1 infection, cognitive impairments remainprevalent due to persistent viral replication and associatedbrain inflammation. Primary cellular targets of HIV-1 in thebrain are microglia and astrocytes which in response to infec-tion release inflammatory markers, viral proteins [i.e., glyco-protein 120 (gp120)] and exhibit impaired glutamate uptake.Peroxisome Proliferator-Activated Receptors (PPARs) aremembers of the nuclear receptor superfamily of ligand-activated transcription factors. Compelling evidence suggeststhat PPARs exert anti-inflammatory properties in neurologicaldisorders. The goal of this study was to examine the role ofPPARgamma in the context of HIV-1 gp120 induced inflam-mation in vitro, in primary cultures of rat astrocytes and

in vivo, in acute and chronic rodent models of HIV-1-gp120-associated brain inflammation. Primary cultures of rat astro-cytes were exposed to gp120, and inflammatory and oxidativestress markers (TNFalpha, IL-1beta, iNOS) were measuredusing qPCR. For the acute in vivo model, rats were adminis-tered an icv injection of gp120 and an intraperitoneal (ip)injection of PPARgamma agonist (rosiglitazone) or co-administration with PPARgamma antagonist (GW9662).qPCR and immunoblot analysis were applied to measure in-flammatory markers, glutamate transporter-1 (GLT-1) andPPARgamma. For the in vivo chronic model, rats wereadministered icv injection of adeno-associated viral(AAV) vector expressing GFP (positive control) orgp120, 3 weeks post icv, immunohistochemical andqPCR analysis were used to measure transgenes and in-flammatory markers. In primary cultures of rat astrocytesand the in vivo acute model, gp120 exposure resulted inan elevation of inflammatory markers, and a decrease inGLT-1 which were attenuated with rosiglitazone treat-ment. In the in vivo chronic model, AAV-GFP adminis-tered rodents, showed efficient transduction throughoutthe hippocampus while AAV-gp120 administration result-ed in elevated inflammatory markers. Our data suggestthat targeting PPARgamma may provide a novel therapeu-tic option for preventing/treating HIV-associated braininflammation.

P122Allopregnanolone attenuates combined neurotoxicityassociated with morphine and HIV-1 Tat in human cells

Jason J. Paris, Joyce M. Balinang, ShiPing Zou, Pamela E.Knapp, Kurt F. Hauser(corresponding author: [email protected])

Virginia Commonwealth University

Human immunodeficiency virus (HIV) infection is associatedwith neurocognitive impairment that can be exacerbated byopioid drug use. One virotoxin that may contribute to theseeffects is the HIV-1 regulatory protein, trans-activator of tran-scription (Tat). Opioids, such as morphine, are observed toexert additive or synergistic effects on HIV-1 Tat, promotingneuroinflammation and neurotoxicity. We have observed theprogesterone metabolite and neurosteroid, 5alpha-pregnan-3alpha-ol-20-one (i.e., allopregnanolone), to ameliorate Tat-mediated dysregulation of intracellular calcium homeostasisin microglia and neurons, as well as microgliosis and neuronalcell death, in murine cells in vitro. However, it was not knownwhether these effects would extend to human cells, nor was itknown whether the protective effects of allopregnanolonewould be sustained when Tat-toxicity was catalyzed by an


opioid challenge. Differentiated human neuroblastoma cells(SH-SY5Y) or neurons derived from fetal neural progenitorswere incubated with media that did, or did not, containallopregnanolone (0.1–100 nM) in the absence or presenceof morphine (500 nM) and/or HIV-1 Tat1-86 (100 nM) orHIV-1BaL (25–500 pg/ml p24). Tat significantly increasedintracellular calcium, depolarized mitochondrial inner mem-brane potential, increased reactive oxygen species, and pro-moted loss of neurons. While morphine was not toxic on itsown, the combination of morphine and Tat displayed ad-ditive effects for the observed neurotoxic profile associat-ed with HIV-1 Tat. Allopregnanolone conferred aconcentration-dependent protection against Tat- ormorphine/Tat-mediated toxicity. The addition of morphineshifted the concentration curve for allopregnanolone pro-tection to the left, revealing 10 nM to be the more effica-cious than the previous 100 nM concentration (which wasefficacious in the absence of morphine). These data sup-port the notion that neurosteroid-based therapeutics mayconfer prophylactic advantages for opioid-catalyzed HIV-neurotoxicity.

P123Targeting inflammation at the synapse in neuroAIDS :Role of Synaptic PEBP1

Gurudutt Pendyala1, Steven Lisco1, Rick Bevins2, ShilpaBuch1


1University of Nebraska Medical Center; 2University ofNebraska at Lincoln

A characteristic hallmark associated with HIV and metham-phetamine (meth) synergy induced brain dysfunction is neu-roinflammation. Our focus is on understanding the role ofinflammation especially at the synapse including applicationof novel and efficacious treatment strategies to treat progres-sion of neuroAIDS. One emerging anti-inflammatory drug isibudilast, a phosphodiesterase inhibitor that modulates the ac-tivity of glial cells by suppressing the production of proinflam-matory cytokines. We recently demonstrated for the first timea novel role for ibudilast’s anti-inflammatory effect at the syn-apse and its ability to attenuate meth seeking during absti-nence. Furthermore our proteomics screen on purified synap-tosomes from animals that self-administered meth identifiedthe synaptic signaling protein phosphatidylethanolamine-binding protein 1 (PEBP1) whose decreased expression bymeth was reversed by ibudilast. Extending this initial obser-vation in the context of HIV and meth synergy, we employedan in vitro mixed cerebrocortical cultures comprising of neu-rons and glia. While both Tat and meth led to a significant

decrease in PEBP1 expression, this decrease was further aug-mented by Tat and meth synergy. Interestingly, pretreatmentwith ibudilast prevented this down regulation of PEBP1 byboth Tat and meth. Given the localization of PEBP1 ondendritic spines that are actin rich and regulate synapticfunction, we tested a more causal effect of PEBP1 onactin dynamics at the synapse. While Tat and meth alonedecreased the expression of F-actin in cultures treatedwith siRNA against PEBP1, this effect was significantlyaugmented during Tat and meth synergy. This markeddramatic reduction in F-actin levels was ameliorated bypretreatment with ibudilast thus suggesting a novel role ofPEBP1 in modulating actin dynamics associated with aneuroinflammatory state due to HIV and meth synergy.Ongoing studies are aimed at further dissecting down-stream mechanisms of PEBP1 down regulation on actindynamics during HIV/meth synergy at the synapse.

P124HERV-W envelope expression in microglia from MultipleSclerosis patients: a lifelong role in neuroinflammation,demyelination and axonal lesion.

Herve Perron1, David Kremer2, Ranjan Dutta3, JackVanHorssen4, Sadie Deckard3, Sandra Amor4, Bruce Trapp3,Patrick Kury5


1Geneuro; 2Dusseldorf University Hospital; 3ClevelandClinic; 4VU-University of Amsterdam; 5University ofDusseldorf

Multiple Sclerosis (MS) associates genetic predisposition andenvironmental factors. Human genome studies have investi-gated exons encoding somatic proteins and their regulatorysequences, which only generated variable panels of suscepti-bility genes. Attempts to identify a causative agent of MSfrom the environment have consistently failed. Since the1990’s, a new horizon arose from studies of MS cells fromwhich a human endogenous retrovirus (MSRV) and its relatedmulticopy family (HERV-W) were identified. Endogenousretroviruses and their relatives occupy approximately 8 % ofthe human genome and are part of the broader category of“mobile genetic elements”, which together account for onehalf of the human genome. Cumulated results from numerousstudies over past decades brought converging and consistentobservations and provide a global picture suggesting that en-vironmental infectious agents may trigger the expression ofcertain HERV-Welements inMS, thereby engaging pathogen-ic pathways leading to final pathognomonic features. Thesescientific advances are now providing a new perspective of“gene - e nv i r onmen t i n t e r p l a y ” unde r l y i ng t h e


etiopathogenesis of MS. Within this global understanding, itnow appears that microglia is the lifelong and key pathogenicplayer of neuroinflammation, recruiting T-cells in RRMSphase and causing chronic innate immune activation, as wellas of myelin and axonal lesions. We shall present how thismicroglial activity is strongly associated with the expres-sion of the HERV-W envelope protein (MSRV-Env) (i) inmicroglia within areas of active demyelination, from ear-liest lesions to late progressive plaques, and (ii) in mi-croglia directly involved in axonal lesions. This endoge-nous retroviral protein is likely to play a role in the con-text of pro-inflammatory activation of microglia contrib-uting to myelin damage, oligodendrocyte cell death andultimately axonal degeneration in progressive MS. Alongwith previous and still ongoing studies, these observationssuggest that MSRV-ENV can be an appropriate therapeu-tic target in MS.

P125Utilization of HIV-1 envelope V3 to identify X4-and R5-specific Tat and LTR sequence signatures

Vanessa Pirrone1, Gregory Antell2, William Dampier1,Benjamas Aiamkitsumrit1, Michael Nonnemacher1, JeffreyJacobson3, Wen Zhong1, Katherine Kercher1, ShendraPassic1, Jean Williams1, Gregory Schwartz2, Uri Hershberg2,Fred Krebs1, Brian Wigdahl1


1Department of Microbiology and Immunology, Institute forMolecular Medicine and Infectious Disease, DrexelUniversity College of Medicine; 2School of BiomedicalEngineering, Science, and Health Systems, DrexelUniversity; 3Department of Medicine, Section of InfectiousDisease, Lewis Katz School of Medicine, Temple University

HIV-1 entry is a receptor-mediated process directed by theinteraction of the viral envelope with the host cell CD4 mol-ecule and one of two co-receptors, CCR5 or CXCR4. Theamino acid sequence of the third variable (V3) loop of theHIV-1 envelope is highly predictive of co-receptor utilizationpreference during entry, and machine learning predictive al-gorithms have been developed to characterize viral sequencesas CCR5-utilizing (R5) or CXCR4-utilizing (X4). It was hy-pothesized that while the V3 loop is predominantly responsi-ble for determining co-receptor binding, additional compo-nents of the HIV-1 genome may contribute to overall replica-tive properties following viral entry and display sequence sig-natures associated with co-receptor utilization. The accessoryprotein Tat and the HlV-1 long terminal repeat (LTR) wereanalyzed with respect to genetic diversity and compared byJensen-Shannon divergence which resulted in a correlation

with both mean genetic diversity as well as the absolute dif-ference in genetic diversity between R5- and X4-genome spe-cific trends. As expected, the V3 domain of the gp120 proteinwas enriched with statistically divergent positions.Statistically divergent positions were also identified inTat amino acid sequences within the transactivation andTAR-binding domains, and in nucleotide positionsthroughout the LTR. We further analyzed LTR sequencesfor putative transcription factor binding sites using theJASPAR transcription factor binding profile database andfound several putative differences in transcription factorbinding sites between R5 and X4 HIV-1 genomes, specif-ically identifying the C/EBP sites I and II, and Sp site III,among others, to differ with respect to sequence configu-ration for R5 and X4 LTRs. These observations support thehypothesis that co-receptor utilization coincides with spe-cific genetic signatures in HIV-1 Tat and the LTR, likelydue to differing transcriptional regulatory mechanisms andselective pressures applied within specific cellular targetsduring the course of productive HIV-1 infection.

P126Expression profile of cathepsin B, cystatins B / C,and related proteins in CD14+ monocyte subpopulationsand HAND severity

Marines Plaud/Valentin1, Yamil Gerena1, Valerie Wojna1,Richard Skolasky2, Loyda M Melendez1


1University of Puerto RicoMedical Sciences; 2Johns HopkinsUniversity

HIV-1 infection affects approximately 34 million peopleworldwide and is associated with neurocognitive disorders(HAND). Particularly its milder forms, HAND, remains prev-alent even in patients receiving combined antiretroviral thera-py. There is a need for early diagnosis and establishment oftherapeutic agents that can halt HAND. Previous studies usingour Hispanic women cohort indicated that intracellular cathep-sin B and cystatin B levels are elevated in CD14+ monocytesof HIV+ women with HIV associated dementia (HAD). It isunknown if these proteins increase in monocytes since theasymptomatic stage to help in predicting the development ofHAD. We hypothesize that the expression of cathepsin B,cystatin B, increased in early HAND. Cystatin B, cathepsinB interacting protein partners (APOC2, APP, SAPC) and re-ceptor (IGF2R) were analyzed in CD14+ resting (CD16-) andactivated (CD16low and high+) monocytes from HIV+Hispanic women. Peripheral blood monocytes from 40 pa-tients (14 N, 13 ANI, and 13 HAD) controlled for age, viralload, and therapy were immunolabeled for CD14 and CD16


surface markers. For detection of intracellular protein levels,cells were permeabilized using the BD Cytofix/Cytoperm kit,incubated with antibodies for cystatins B and C, cathepsin B,APOC2, APP, SAPC, and IGF2R followed by fluorescencelabeled secondary antibodies. The mean fluorescence inten-sity of each protein in CD14/CD16 subsets was deter-mined by flow cytometry. We found elevated levels ofcystatins B in CD14highCD16low (p= 0.010) and CD14high CD16 high subpopulations (p= 0.017), and APOC2in CD14high/CD16 low cells (p= 0.005) correlating withHAND severity. No differences were found with cystatinC, cathepsins B, APP, SAPC, and IGF2R. These resultsindicate that cystatin B increases in CD14+ monocytesregardless the activation state, while cathepsin B proteinpartner, APOC2 increased in CD16low monocytes.Evaluation of these two biomarkers for predictive valuewill be performed using the outcome from subsequentvisits.

P127HIV-Tat protein: molecular structure, aggregationand interaction with amyloid beta

Alina Popescu Hategan1, Mario Bianchet2, ElenaKarnaukhova3, Joseph Steiner1, Emilios Dimitriadis4,Avindra Nath1


1NINDS, Section of the Infectious of the Nervous System;2Johns Hopkins School of Medicine, Department ofNeurogogy; 3FDA, CBER; 4NIBIB, Scanning ProbeMicroscopy Unit

Numerous studies show that Tat protein plays a critical role inHIV neuropathogenesis however, little is known about itsstructure, propensity for aggregation and for interaction withother proteins. Highly purified recombinant Tat protein wasstudied by atomic force microscopy. Only 8 % of the proteinwas in monomeric state. The remaining Tat aggregates rangedfrom dimers, trimers, tetramers to large oligomers (50-mersand larger) and all structures presented globular shape.Circular dichroism and force spectroscopy showed that 20–29 % of the sampleäó»s structure was alpha-helical, whichlikely represents the aggregated state. Force spectroscopyshowed that 44 aminoacids long domains could be mechani-cally unfolded. Adhesion forces of 88 pN showed the in-creased adhesion capacity of Tat and its aggregates, as com-pared to adhesion forces between cells. The reducing agentdithiothreitol dissociated the aggregates into smaller aggre-gates, but structures smaller than the monomer were alsofound, indicating that the molecule is prone to rupture. Highsalt reduced the size of aggregates, and storage at room

temperature at pH7.4 for several days led to apparition of longfibrillar and annular structures, with reduced alpha-helicalstructure. We showed that Tat and its aggregates directlyinteracted with amyloid beta peptide. With Tat, uniform am-yloid fibrils turned into double twisted fibrils followed bypopulations with thick unstructured filaments and aggre-gated large patches. The fibers became more rigid andmechanically resistant. Tat attached externally to fibrils,causing their lateral aggregation into thick multifibrilarstructures, that presented enhanced adhesion. As result,the neurotoxic properties of Tat and amyloid beta aggre-gates were strongly synergistic when complexed together.These data suggest that Tat is a multimeric protein withstrong adhesive properties but sensitive to storage condi-tions. When complexed with amyloid beta, the increasedrigidity, mechanical resistance and adhesion of the com-plexes led to neuronal damage, likely through pore for-mation in membranes.

P128The PD- 1:PD-L1 pathway promotes development of CNSTRM cells following acute viral encephalitis

Sujata Prasad, Shuxian Hu, Wen S Sheng, Priyanka Chauhan,James R Lokensgard(corresponding author: [email protected])

Depatment of Medicine, University of Minnesota MedicalSchool

Initial T-cell-mediated immune responses to infection withcytomegalovirus (CMV) trigger generation of a large numberof short-lived effector cells (SLEC), as well as a pool of mem-ory precursor effector cells (MPEC). These MPEC subse-quently develop into tissue resident memory (TRM) cells,which could be critical in controlling viral reactivation, partic-ular in the tissues like the brain. Previously we reported thatduring acute infection microglial cells and astrocytes modu-late antiviral T-cell effector responses through the PD1: PD-L1 pathway, thereby controlling neuroinflammation. Here, weevaluated the role of the PD-1: PD-L1 pathway in the devel-opment of TRM within the murine CMV (MCMV)-infectedbrain. Flow cytometric analysis of brain-infiltrating cells wasperformed at 7, 14, and 30 d post-infection (dpi) to assess thetransition from SLEC to MPEC, as well as generation ofTRMs. In wild-type (WT) animals, we observed a switch inthe phenotype of brain-infiltrating CD8+ T-cells fromKLRG1+CD127- (SLEC) to KLRG1-CD127+ (MPEC) dur-ing transition from acute through chronic phases of infection.The majority of CNS-infiltrating CD8+ T-cells expressedCD127, a marker of memory cells at 14 and 30 dpi. In con-trast, fewer CD8+ T-cells expressed CD127 within the brains


of infected, PD-L1-KO animals. Notably, in WT mice a largepopulation of CD8+ T-cells was phenotyped as CD103+, amarker of TRM and striking differences were observed inthe numbers of these TRM cells when compared to PD-L1-KO animals. Further immunohistochemical studies revealedthat the majority of brain-resident CD103+ TRM cells werelocalized to the brain parenchyma. Interestingly, in explantreactivation studies, reactivated virus was more efficiently re-covered from the brains of latently-infected PD-L1-KO ani-mals, which contained less TRMs, when compared to WTanimals. In conclusion, our results indicate that the PD1:PD-L1 pathway is critical in the generation of brain TRMs,which control spread of reactivated, latent virus.

P129Cerebrospinal Fluid Galectin-9 is a Novel Biomarkerfor Neurocognitive Impairment Among Elderly HIV-1Infected Adults

Thomas Premeaux1, Michelle D’Antoni1, Bruce Shiramizu1,Melissa Agsalda-Garcia1, Cecilia Shikuma2, Mohamed AbdelMohsen3, Satish Pillai4, Lishomwa Ndhlovu1


1Department of Tropical Medicine, Medical Microbiology,and Pharmacology, Hawaii Center for AIDS, John A. BurnsSchool of Medicine, University of Hawaii; 2Hawaii Center forAIDS, John A. Burns School of Medicine; 3Blood SystemsResearch Institute, UCSF School of Medicine; 4BloodSystems Research Institute, UCSF Department ofLaboratory Medicine, UCSF-GIVI Center for AIDS Research

The prevalence of HIV-associated neurocognitive impairment(NCI) remains high in the HIV-infected population despiteviral suppression with combination antiretroviral therapy(cART). Circulating cell-associated HIV DNA and markersof macrophage activation have been suggested as potentialindicators of NCI, however more sensitive and reliable bio-markers are needed.We previously identified Galectin-9 (Gal-9), a soluble lectin with immunomodulatory properties, as acomponent of the first wave of the plasma cytokine stormduring HIV acquisition. Gal-9 is known to be released byastrocytes and elevated in the cerebrospinal fluid (CSF) ofpatients with progressive multiple sclerosis. We assessed therelationship between Gal-9 and HIV-associated NCI in 15elderly HIV-1 infected individuals with detectable CSF viralloads (VL) enrolled in the Hawaii Aging with HIV Cohort(86.7 % male; age range: 50–58 years; mean: log10 plasma/CSF VL=4.33/2.99 copies/mL, CD4 count/nadir = 392/285cells/μl; on cART: 6/15). Plasma and CSF Gal-9 levels weremeasured by ELISA and analyzed with available neuropsy-chological (NP) test assessments and clinical data. CSF Gal-9

was significantly higher in participants with mild cognitivemotor disorder (mean = 4.43 ng/mL, p= 0.008) and HIV-associated dementia (mean=5.73 ng/mL, p=0.0005) com-pared to those with normal cognition (mean=1.2 ng/mL).CSF Gal-9 levels inversely correlated with NP globalcomposite z-scores (r=−0.63,p= 0.015), and subdomainz-scores in executive function (r=−0.70,p= 0.005), work-ing memory and concentration (r=−0.65,p= 0.011), mo-tor skills (r = −0.65,p = 0.010), and psychomotor speed(r=−0.66, p= 0.009). Moreover, Gal-9 in both CSF andplasma directly correlated with HIV DNA copy number inperipheral blood mononuclear cells (r = 0.79,p = 0.001;r = 0.55,p = 0.036 respectively), and CSF levels of them a c r o p h a g e a c t i v a t i o n m a r k e r s C D 1 6 3(r = 0.59,p = 0.024; r = 0.78,p = 0.001 respectively).Collectively, these data suggest that Gal-9 may be in-volved in the neuropathogenesis of NCI and serve as anovel indicator of HIV-associated NCI. The effects of vi-ral suppression on the relationship between circulatingGal-9 and NCI would be of further interest.

P130In vivo visulation of HIV transmission and viremiarebound in humanized mice

Xiying Qu, Won-Bin Young(corresponding author: [email protected])

Department of Radiology, University of Pittsburgh School ofMedicine

Knowing where HIV-infected cells are located allows formonitoring of the infection spread from the infection site toother sites in the body. The whole-body examination ormonitoring dynamics of HIV infection in real time overthe course of infection provides an opportunity for inves-tigating where the viral infection starts, where the sanctu-aries are during suppressive ART and, when and whereviremia rebound will occur following ART withdraw. Wepreviously demonstrated that bioluminescence imaging(BLI) based on an enhanced firefly luciferase is sensitivefor visualizing as few as 5 single cells in a niche (Songet al., 2015). In this study, we engineered replication-competent HIV-BaL (CCR5/macrophage tropism) carryingthis luciferase reporter for infecting humanized Bonemarrow-Liver-Thymus (BLT) mice via vaginal mucosa orintraperitoneal injection. Compared to the intraperitonealroute, penetration of mucosal barrier by HIV vaginal trans-mission requires at least 17 fold more virus. Infection inthe female reproductive tract (FRT) could be visualized inthe vaginal vulva/canal 48 h post inoculation (p.i.). Robustinfection from FRT to initiate a systemic infection was


visualized as early as 21 days, p.i., presumably an eclipsephase during local HIV expansion. Compared to intraperi-toneal inoculation, the spatial-temporal dynamics of HIVmucosal transmission is significantly different, which isunlikely to be observed via conventional measurement ofviral load in the blood. Treating the inoculated hu-BLTmice with combination ART, consisting of Truvada plusRaltegravir, resulted in declined HIV dissemination in only2 weeks. HIV rebound was observed in the neck lymphnode 2 weeks after ARTwithdrawal. We also demonstratedthat HIV can escape from the clinically approved Pre-Exposure Prophylaxis (PrEP) Truvada, if not adhering todaily PrEP schedule, resulting in a systemic HIV infection3 weeks after the PrEP.

P131Peripheral blood monocyte transcriptome at baselineimplicates Wnt-signaling pathway in later neurocognitivechange

Austin Quach1, Stefan Horvath2, Dimitrios Vatakis2, OtonielMartinez-Maza3, Paul Shapshak4, Mallory Witt5, ElyseSinger6, Andrew Levine7


1Departments of Biostatistics and Human Genetics, DavidGeffern School of Medicine at the University of CaliforniaLos Angeles; 2Department of Medicine, Division ofHematology-Oncology UCLA AIDS Institute, David GeffenSchool of Medicine at the University of California LosAngeles; 3Department of Obstetrics & Gynecology andDepartment of Microbiology, Immunology & MolecularGenetics, David Geffen School of Medicine at theUniversity of California Los Angeles; 4Department ofMedicine, Division of Infectious Disease & InternationalMedicine, University of South Florida, Morsani College ofMedicine; 5Los Angeles Biomedical Research Institute atHarbor-UCLA Medical Center; 6Department of Neurology,National Neurological AIDS Bank, David Geffen School ofMedicine at the University of California Los Angeles;7Department of Neurology, David Geffen School ofMedicine at the University of California Los Angeles

Background: Cross-sectional transcriptome studies have im-plicated a variety of genes and biological pathways associatedwith HIV-associated neurocognitive impairment and demen-tia. To further develop such methods towards prognostic bio-markers, we sought to determine if gene expression character-istics of peripheral blood monocytes at a baseline visit arepredictive of neurocognitive changes 2 years later. Methods:Eighty-two HIV+ participants in the Multicenter AIDSCohort Study in Los Angeles completed the study. Baseline

neurocognitive functioning was determined with a battery ofstandardized tests. Global neurocognitive functioning calcu-lated by averaging all scores. Monocytes were isolated fromwhole blood via Rosette separation. Global gene expressiondetermined with the Illumina HT-12 v4 Expression BeadChip.Neurocognitive functioning was re-evaluated 2 years later.Genes showing the greatest associations with change in globalneurocognitive functioning after adjustment for age and racewere subjected to DAVID gene set enrichment analysis.Results were adjusted using FDR for multiple comparisons.Results: The strongest DAVID category positively associ-ated with improved neurocognitive function was the Wntsignaling pathway (enrichment score = 2.24, FDR=0.026),as well as several other Wnt-related pathways. Categoriesenriched in genes negatively associated with improvingneurocognitive functioning were largely related to tran-scription and mRNA processing (enrichment score = 2.83,FDR = 0.0005), as well as NF-kappaB (enrichmentscore = 1.83, FDR = 0.0004). Conclusion: While cross-sectional transcriptome studies of HAND have implicatedoxidative stress and inflammation processes, the most no-table finding from this longitudinal analysis is that Wnt-signaling pathway activity at baseline is prognostic forneurocognitive change 2 years later. These findings lendsupport to in vitro and animal studies demonstrating thatthe Wnt-pathway is a repressor of HIV transcription inmultiple cell types in the periphery and in the CNS.

P132SIV Can Emerge from the CNS Despite SustainedPeripheral Suppression in ART-treated Macaques

Suzanne Queen, Sarah Beck, Lisa Magnus, Lucio Gama,Janice Clements, Joseph Mankowski(corresponding author: [email protected])

Department of Molecular and Comparative Pathobiology,Johns Hopkins University

Despite successful suppression of HIV with antiretroviraltherapy (ART), HIV associated neurocognitive disorders(HAND) remain prevalent. Additionally, the brain remainsan unappreciated reservoir of replication-competent virus.The SIV macaque model of HIV neurologic disease is widelyrecognized as an excellent model of HAND because it reca-pitulates the various stages of HAND development. Using thismodel, we treated 4 SIV-infected pigtailed macaques withCNS penetrant ART consisting of once daily dolutegravir(2.5 mg/kg), PMPA (20 mg/kg), and FTC (40 mg/kg) begin-ning at day 12 post-inoculation. Virus suppression (<50copies/mL) in both CSF and plasma occurred by 44 days afterinitiating treatment. Despite maintenance of undetectable


virus for 2 months, one pigtail (PT4) rebounded only in theCSF then had persistent detectable SIV RNA in CSF untileuthanasia, approximately 3 months later. Examination of vi-ral loads in tissues showed replicating virus in the brain ofPT4, but no detectable virus in peripheral tissues (spleen, lungand mesenteric LN). PT4 also had replication-competent SIVin cultured primary microglia. The remaining 3 pigtailedmacaques in the study had no detectable SIV RNA ineither the CNS or periphery. Four additional macaqueswere SIV-inoculated and treated under the same ART pro-tocol but with addition of twice daily oral maraviroc(200 mg), a CCR5 antagonist. In addition to suppressingvirus in both CSF and plasma 14 days earlier, these 4macaques receiving maraviroc had no detectable SIVRNA in CSF or plasma throughout the experiment; noSIV RNA was detected in CNS or periphery at terminaltime-points. This study demonstrates that 1) SIV canemerge from the CNS despite suppression in the peripheryand 2) addition of the CCR5 antagonist maraviroc poten-tiates suppression of SIV.

P133Modeling brain lentiviral infections during antiretroviraltherapy in AIDS

Weston Roda1, Michael Li1, Michael Akinwumi1, EugeneAsahchop2, Benjamin Gelman3, Kenneth Witwer4,Christopher Power2


1Department of Mathematical and Statistical Sciences,University of Alberta; 2Division of Neurology, Departmentof Medicine, University of Alberta; 3Texas NeuroAIDSResearch Center and Department of Pathology, University ofTexas Medical Branch; 4Department of Molecular andComparative Pathobiology, Johns Hopkins UniversitySchool of Medicine

Objective: Understanding HIV-1 replication and latency indifferent reservoirs is an ongoing challenge in the care ofpatients with HIV/AIDS. A mathematical model was createdthat predicted HIV-1 and SIV infection dynamics within thebrain during effective combination antiretroviral therapy(cART). Design: By developing a two compartment mathe-matical approach, a predictive model was generated fromexisting empiric data. Methods: Based on previous reportsquantifying total viral DNA levels in brain from HIV-1 andSIV infections, estimates of proviral DNA burden were made,which were fit to a mathematical model predicting viral ac-crual in brain macrophages from primary infection. Results:The annual rate at which susceptible brain macrophages be-come HIV-1 infected was estimated to be in the range 6.72–

7.60×10^–7 per year for cART-treated HIV/AIDS patientswithout comorbid neurological disorders. The transmissionrate for SIV infection among untreated macaques was 6.40–9.45 times greater than cART treated HIV-1 patients. An im-provement in cART efficacy (11–21 %) would suppress HIV-1 infection in patients without neurological disorders. Amongpatients with advanced disease, a substantial improvementin cART efficacy (50 %) would eradicate HIV-1 provirusfrom the brain within 9–11 years in patients without neu-rological disorders, whereas 20–26 years of efficaciouscART would be required for HIV/AIDS patients with co-morbid neurological disorders. Conclusions: HIV-1 andSIV provirus burdens in the brain increase over time. Amoderately efficacious antiretroviral therapy regimencould eradicate HIV-1 infection in the brain that was de-pendent on brain macrophage lifespan and the presence ofneurological comorbidity.

P134Sex Differences in the Expression and Activity of HANDNeurotoxic Factor, Cathepsin B.

Lester Rosario1, Krystal Colon1, Loyda Melendez2


1University of Puerto Rico, Medical Sciences Campus;2University of Puerto Rico, Medical Sciences Campus andRCMI Translational Proteomics Center

HIV-associated neurocognitive disorders (HAND) are preva-lent despite combined antiretroviral therapy. Women accountfor more than a half of the 35 million people living with HIVworldwide, and also represent a risk factor for HAND.Cathepsins and cystatins are lysosomal proteins secreted bymacrophages and microglia that play important roles inneuroregulatory responses. Our laboratory, has demonstratedincreased secretion and neurotoxicity of cathepsin B from in-vitro HIV-infected women monocyte-derived macrophages,including increased expression in postmortem brain tissuewith HIV encephalitis and HAND. However, cathepsins andcystatins expression and their neuroregulatory responses havenot being studied in men. We hypothesize that HIV-positivemen express similar levels of cathepsin B and its inhibitors asHIV-positive women. Monocyte-derived macrophages wereisolated from peripheral blood of 7 HIV seronegative womenand 5 HIV seronegative men. Cells were infected with HIV-1-ADA at 0.1 MOI and, at day 11 post-infection, supernatantswere collected, and ELISA pro-cathepsin B and cystatin Cassays were performed, including a cathepsin B activity assay.Our results show that HIV-1-positive men significantlyexpressed lower levels of pro-cathepsin B and cystatin C com-pared to HIV-1-positive women. However, cathepsin B


activity was higher in HIV-1-positive men than HIV-1-positive women, and significantly increased in HIV-1-positive men compared to non-infected men. Moreover,HIV-positive men had significant lower levels of cystatin Cthan HIV-positive women. These results suggest that HIV-positive men express less pro-cathepsin B than HIV-positive women, with increased activity after HIV infec-tion. In conclusion, sex-linked differences were observedin the secretion of pro-cathepsin B, and HIV-1 infectionis mediating cathepsin B activation in men outside of thecell. Future studies on the relationships of sex hormoneswith cathepsin B secretion and activation, along withneurotoxicity assays will lay the groundwork for the de-velopment of treatments for men and women againstHAND.

P135Increased oral self-administration of methylphenidateand decreased dopamine release in the nucleus accumbenscore region of HIV-1 transgenic rats

Robert F. Roscoe Jr.1, Srimal Samaranayake2, ParastooHashemi2, Steven B. Harrod1, Hailong Li1, Charles F.Mactutus1, Rosemarie M. Booze3


1Dept. of Psychology, University of South Carolina; 2Dept. ofChemistry, University of South Carolina; 3Dept. ofPsychology, Bicentennial Endowed Chair for BehavioralNeuroscience, University of South Carolina

HIV+ youths have an increased risk for mental health disor-ders, including ADHD. As a result, HIV+ children have twicethe odds of receiving psychostimulant medications, such asmethylphenidate (MPH, Ritalin®). Unfortunately, little isknown regarding the etiology of this psychopathology andpotential for psychostimulant abuse in HIV-1+ adolescents.First, we examined oral self-administration of methylpheni-date (MPH) in female OVX F344 (n= 20) and female OVXHIV-1Tg rats (n= 19). Animals were given access for 14 days,on a FR1 schedule, to MPH/sucrose (max dose 4 mg/kg/day).A significant increase in MPH self-administration was foundduring the first week for HIV-1 Tg animals, relative to F344controls (p<0.05), indicating escalation in MPH dosing dur-ing drug initiation in HIV-1 Tg animals. Second, we evaluateddopamine release from the nucleus accumbens core (NAcc)region using an electroanalytical technique, fast-scan cyclicvoltammetry. Animals (F344 n= 8 males/6 females; HIV-1Tg n= 7 males/7 females) were fully anesthetized and thestimulating electrode was positioned in the medial forebrainbundle. HIV-1 Tg rats of both sexes exhibited a significantdecrease in dopamine release in the NAcc, (p<0.05), relative

to controls. Proper electrode placement was later confirmedvia Nissl stain. Together, these experiments establish the roleof the dopamine system in contributing to, if not mediating,potential abuse liability of MPD in the treatment of ADHD inHIV-1+ youths. Funded by: DA013137, MH106563,MH106392, HD043680

P136Low dose hydrocortisone has acute enhancing effectson verbal learning in HIV-infected men

Leah Rubin1, Luan Phan1, Sheila Keating2, Kathleen Weber3,Pauline Maki1


1Department of Psychiatry, University of Illinois at Chicago,Chicago, IL; 2Department of LaboratoryMedicine, Universityof California San Francisco, San Francisco, CA; BloodSystems Research Institute, San Francisco, CA; 3The CoreCenter, Cook County Health and Hospitals System andHektoen Institute of Medicine, Chicago, IL

Background: Glucocorticoids are released in response tostress and alter cognitive performance and brain functionthrough both rapid, nongenomic mechanisms and slow,genomic mechanisms. Administration of glucocorticoidsin the form of low dose hydrocortisone generally enhancescognition in individuals with trauma, but impairs cognitionin healthy individuals. Here we target the time-dependenteffects of glucocorticoids on learning and memory in HIV-infected men, a group that generally reports higher thanaverage levels of stress and trauma. Methods: In a dou-ble-blind, placebo-controlled, cross-over study, we exam-ined the time-dependent effects of a single low dose ofhydrocortisone (10 mg; LDH) on learning and memory in45 HIV-infected men between the ages of 18 and 45.Participants were randomized to receive either LDH orplacebo, and 1 month later were given the opposite treat-ment. At each intervention session, cognition was assessedboth 30 min (assessing nongenomic effects) and 4 h(assessing genomic effects) after pill administration (LDHvs. placebo). Verbal learning and memory, attention/con-centration, executive functioning, and visuospatial abilities(only at 30 min) were assessed. Self-reported stress andanxiety and cortisol and cytokines in saliva were measuredrepeatedly throughout each session. Results: Compared toplacebo, LDH increased salivary cortisol levels by 201 %.Cortisol levels returned to baseline 4 h post administration.At the 30-min assessment, LDH enhanced verbal learningcompared to placebo; the greater the increase in cortisol,the greater the enhancement in verbal learning. LDH didnot affect subjective stress, anxiety, or any other cognitive


outcomes at the 30-min or 4-h time point. Conclusion: Therapid effects of LDH on verbal learning in HIV-infectedmen suggest a nongenomic mechanism by which glucocor-ticoids can enhance cognition. The non-enduring nature ofthis enhancement may limit its clinical utility, but providesnovel insights into mechanisms underlying the effects ofacute glucocorticoids on learning.

P137Antibody blockade of CLEC12A delays EAE onsetand attenuates disease severity by impairing myeloid cellCNS infiltration and restoring positive immunity

Divya Sagar1, Narendra P. Singh2, Rashida Ginwala1,Xiaofang Huang3, Ramila Philip3, Mitzi Nagarkatti4,Prakash Nagarkatti5, Konstantin Neumann6, Jurgen Ruland6,Allison Andrews7, Servio Remirez7, Zafar K. Khan1, PoojaJain1


1Department of Microbiology and Immunology, DrexelUniversity College of Medicine, Philadelphia, PA;2Department of Pathology, Microbiology and Immunology,School of Medicine, University of South Carolina,Columbia , SC; 3Immunotope Inc. , PennsylvaniaBiotechnology Center, Doylestown, PA; 4Department ofPathology, Microbiology and Immunology, School ofMedicine, University of South Carolina and WilliamJennings Bryan Dorn VA Medical Center, Columbia, SC;5Department of Pathology, Microbiology and Immunology,School of Medicine, University of South Carolina,Columbia, SC; 6Institut fur Klinische Chemie undPathobiochemie, Klinikum rechts der Isar, TechnischeUniversitat Munchen, Munich; 7Department of Pathologyand Laboratory Medicine, Lewis Katz School of Medicine,Temple University, Philadelphia, PA

The mechanism of dendritic cells (DCs) recruitmentacross the blood brain barrier (BBB) during neuroinflam-mation has been the least explored amongst all leuko-cytes. For cells of myeloid origin, while integrins functionat the level of adhesion, the importance of lectins remainsunknown. Here, we identified functions of one C-typelectin receptor, CLEC12A, in facilitating DC bindingand transmigration across the BBB in response to CCL2chemotaxis. This process involved Src homology region 2domain-containing phosphatase (SHP)1/2-mediated sig-naling in coordinating actin polymerization in DCs thatexpress the WASP Interacting Protein (WIP). To test func-tion of CLEC12A in an animal model of multiple sclero-sis (MS), we administered blocking antibody toCLEC12A that significantly ameliorated disease scores

in MOG35-55-induced progressive, as well as PLP138-151-induced relapsing-remitting experimental autoim-mune encephalomyelitis (EAE) mice. The decline in bothprogression and relapse of EAE occurred as a result ofreduced demyelination and myeloid cell infiltration intothe CNS tissue. DC numbers were restored in the spleenof C57BL/6 and peripheral blood of SJL/J mice alongwith a decreased TH17 phenotype within CD4+ T-cells.The effects of CLEC12A blocking were further validatedusing CLEC12A knockout (KO) animals wherein EAEdisease induction was delayed and reduced disease sever-ity was observed. These studies reveal the utility of aDC-specific mechanism in designing new therapeuticsfor MS.

P138Cocaine promotes both the initiation and the elongationphases of HIV-1 transcription by activating NF-kappaB,MSK1 and P-TEFb, besides inducing selective epigeneticmodifications at HIV-1 LTR

Geetaram Sahu1, Kalamo Farley1, Nazira El-Hage2, BenjamasAiamkitsumrit1, Ryan Fassnacht1, Fatah Kashanchi3,Jonathan Karn4, Gary Simon1, Kurt Hauser2, Mudit Tyagi1


1Division of Infectious Diseases, Department of Medicine,George Washington Univers i ty ; 2Depar tment ofPharmacology and Toxicology, Virginia CommonwelthUnivers i ty; 3NCBID, George Mason Universi ty;4Department of Molecular Biology and Microbiology, CaseWestern Reserve University

Illicit drug users are a high risk population for infection withthe Human Immunodeficiency Virus (HIV). A strong correla-tion exists between prohibited drugs use and an increase rateof HIV transmission. Cocaine is one of the most widelyabused drugs in the United States, which both impairs thenormal functioning of brain cells and also activate HIV ex-pression in central nervous system (CNS). Cocaine acceleratesHIV replication by altering specific cell-signaling and epige-netic pathways. We have elucidated the underlying molecularmechanisms through which cocaine exerts its effect in mye-loid cells, a major target of HIV-1 in CNS. We demonstratethat cocaine treatment promotes HIV gene expression by ac-tivating both nuclear factor-kappa B (NF-kappaB) andmitogen- and stress-activated kinase 1 (MSK1). MSK1 sub-sequently catalyzes the phosphorylation of histone H3 at ser-ine 10, and p65 subunit of NF-kappaB at 276th serine residue.We demonstrate that a short-term (acute) cocaine treatmentpromotes HIV-1 transcription by activating both nuclearfactor-kappa B (NF-kappaB) and MSK1. However, during


longer-term or chronic cocaine treatment MSK1 is the mainfacilitator of HIV1 transcription. These activation events en-hance the interaction of NF-kappaB with histone acetyl-transferases (HATs) and promote the recruitment of thepositive transcription elongation factor b (P-TEFb) to theHIV-1 LTR, suppor t ing the development of anopen/relaxed chromatin configuration, and facilitatingboth the initiation and elongation phases of HIV-1 tran-scription. Results are also confirmed in primary monocytederived macrophages (MDM). Overall, our study providesdetailed insights into cocaine-driven HIV-1 transcriptionand replication.

P139HIV-1/gp120 protein and methamphetamine inducealterations in dopaminergic and serotonergicneurotransmission systems

Ana Sanchez1, Ricky Maung1, Marcus Kaul1, TMARCGroup1


1Sanford Burnham Prebys Medical Discovery Institute,Infectious and Inflammatory Disease Center; 2UCSD,Translational Methamphetamine AIDS Res. Ctr.

Individuals infected with human immunodeficiency virustype-1 (HIV-1) frequently use methamphetamine (METH).The incidence of both HIV-1 gp120 viral protein and METHin the central nervous system (CNS) is suspected to exacerbateHIV-associated neurocognitive disorders (HAND). In addi-tion METH is a psychostimulant drug that compromises sev-eral neurotransmitter systems, including the dopaminergic andserotonergic network. However, the combined effects of HIV-1 and METH on the brain are incompletely understood at themolecular level. We recently treated 3–4 months old HIV-1/gp120 transgenic (gp120tg) and wild type (wt) mice with anescalating METH binge regimen for 25 days. At 10–12 months of age, HIV-1/gp120tg and METH-exposed ani-mals showed significant impairment in spatial learning andmemoryand neuropathology. METH-exposed andHIVgp120tg animals also displayed changes components ofthe glutamatergic and GABAergic neurotransmission sys-tems. METH-treated HIVgp120tg mice were the most severe-ly affected. In order to further investigate underlying mecha-nisms in the brain, we used in the present study RT-qPCRarrays to assess expression of genes related to the dopaminer-gic and serotonergic neurotransmission systems. Six compar-isons between the four experimental groups revealed signifi-cant gene regulation due toMETH exposure and chronic HIV-1/gp120 expression: 1) WT Saline (SAL) versus (vs.) WTMETH: e.g. BDNF, AKT1, VMAT2, SERT1; 2) WT SAL

vs. gp120 SAL: e.g. GFAP, HTR6, SYNPHILIN, CDK5;3) WT SAL vs. gp120 METH: e.g. GFAP, BDNF, DRD5,CASP3, HTR1D/7; 4) WT METH vs. gp120 SAL: e.g.GFAP, HTR6, CDK5; 5) WT METH vs. gp120 METH:e.g. GFAP, B2M, HTR1A/1D/1 F/2C/4/7, VMAT1; 6)gp120 SAL vs. gp120 METH: e.g. GFAP, DRD5,HTR1A/1D/2C/4/7. In summary, histopathology and im-paired spatial learning and memory due to METH expo-sure and HIV-1 gp120 expression are associated with sig-nificant alterations in the four major neurotransmissionsystems of the brain.

P140Role of autophagy and extracellular vesiclesin neurotoxicity associated with HIV-1 Nef

Sami Saribas, Stephanie Cicalese, Taha Ahooye, KamelKhalili, Shohreh Amini, Ilker Sariyer(corresponding author: [email protected])

Department of Neuroscience, Center for Neurovirology,Lewis Katz School of Medicine at Temple University,Philadelphia, PA

HIV-associated neurological disorders (HAND) affect the ma-jority of AIDS patients and are a significant problem amongHIV-1-infected individuals who live longer due to combinedanti-retroviral therapies (cART). The virus utilizes viral pro-teins and subsequent cytokine inductions to unleash its toxic-ity on neurons. Among these viral proteins, Nef is a smallHIV-1 protein expressed abundantly in astrocytes of HIV-1-infected brains and has been suggested to have a role in thepathogenesis of HAND. In order to explore its effect in theCNS, Nef was expressed in primary human fetal astrocytes(PHFA) using an adenovirus. Our results revealed that Nef isreleased in extracellular vesicles (EVs) derived from PHFAcells expressing the protein. Interestingly, Nef release in EVswas enriched significantly when the cells were treated withperifosine, MG-132, LY294002, and wortmannin suggestinga novel role of autophagic signaling in Nef release from astro-cytes. Next, Nef-carrying exosomes were purified from astro-cyte cultures and neurotoxic effects on neurons were ana-lyzed. We observed that Nef containing extracellular vesicleswere readily taken up by neurons as evidenced by immuno-cytochemistry and immunoblotting. Furthermore, treatment ofneurons with Nef -carrying EVs induced oxidative stress asevidenced by a decrease in glutathione levels. To further in-vestigate its neurotoxic effects, we expressed Nef in primaryneurons by adenoviral transduction. Intracellular expressionof Nef caused axonal and neurite degeneration of neurons.Furthermore, expression of Nef decreased the levels ofphospho-tau while enhancing total tau in primary neurons.


In addition, treatment of primary neurons with Nef-carrying exosomes suppressed functional neuronal firingactivity assessed by multi-electrode array studies (MEA).Collectively, these data suggested that HIV-1 Nef can be aformidable contributor to neurotoxicity along with otherfactors which leads to HAND in HIV-1 infected AIDSpatients.

P141Discovery and Characterization of the Novel ORFsResulting from Trans-Splicing of the HumanPolyomavirus JC Late Transcripts

A. Sami Saribas, Jason Saredy, Martyn White, Mahmut Safak(corresponding author: [email protected])

Department of Neuroscience, Center for Neurovirology,Lewis Katz School of Medicine at Temple University,Philadelphia, USA

RNA splicing is a highly regulated nuclear processingevent where various combinations of exons from pre-mRNA molecules are jointed together to generate multi-ple protein isoforms by cis-and trans-splicing. The JCvirus genome is known to transcribe two primary tran-scripts from its early and late coding regions and pro-duces several predicted alternatively spliced products bycis-splicing, capable of encoding regulatory and structur-al proteins. Our recent NMR-based mutational analysisof the agnoprotein dimerization domain transcripts byRT-PCR has led us discover two new open readingframes (ORF1 and ORF2) by trans-splicing. These unex-pected ORFs results from (i) a trans-splicing of the 5’-short coding region of VP1 between the coding regionsof both agnoprotein and VP2 after replacing the intron 1and (ii) a further frame-shift occurring within the 5’-shortcoding sequences of VP2. ORF1 and ORF2 have thecapacity to generate 58 and 72 aa long proteins respec-tively. Initial characterization of these ORFs by RT-PCRutilizing total RNA isolated from both the infected pri-mary tissue culture cells and the PML brain tissue sam-ples has confirmed that both ORFs are transcribed notonly in vitro but also in vivo. Mutagenesis analysis ofthe ORF1 in the viral background suggests that it mayplay a role in transport of viral capsids into nucleus.Subsequent analysis of the possible protein coding ca-pacity of the ORF1 by immunoblotting studies usingnewly raised polyclonal antibodies against its predictedamino acid coding sequence has revealed that it doesindeed encode a small protein. Immunocytochemistrystudies revealed that both ORFs mostly localize to thecytoplasmic compartment of the cells, but a noted

localization of ORF2 into nucleus also occurs implicatingregulatory roles in the several aspects of the viral lifecycle including, replication, transcription or in virionbiogenesis. We are currently further investigating theirroles in viral life cycle.

P142Agnoprotein of JC Virus, BK Virus and Simian virus 40 isReleased from Cells: Importance of the DimerizationDomain for Release

A. Sami Saribas, Jason Saredy, Martyn White, Mahmut Safak(corresponding author: [email protected])


JC virus (JCV) is the etiological agent of the fatal dis-ease, progressive multifocal leukoencephalopathy(PML), in the central nervous system (CNS), seen in asubset of patients with underlying immunosuppression,including lymphoma and AIDS. JCV specifically infectsoligodendrocytes and astrocytes and causes an extensivemyelin loss in CNS leading to the neurological manifes-tations of PML. PML is a rare disease, but the inci-dence of PML dramatically increased in the era of theAIDS epidemic (5–7 %). PML is also steadily increas-ing among a group of patients with autoimmune disor-ders, including multiple sclerosis (MS) and Crohn’s dis-ease, who are treated with immunomodulatory antibody-based therapies. Agnoprotein is an important regulatoryprotein of several polyomaviruses including JCV, BKVand SV40 and these viruses are unable to replicate ef-ficiently in the absence of this protein. Agnoproteinforms highly stable dimers/oligomers through its Leu/Ile/Phe-rich alpha-helix domain, which plays a criticalrole in stability of the protein. In this report, we haveinvestigated whether agnoprotein of BKV and SV40 ex-hibits similar characteristics with respect to its releasefrom cells as was previously reported for JCV. Our datashowed that agnoprotein of BKV and SV40 is also re-leased from cells. We further investigated the regionsand amino acid residues responsible for this release byemploying mapping and site-directed mutagenesis stud-ies. These studies revealed that amino acid residuesPhe31 and Asp38 located within the dimerization do-main of JCV agnoprotein play important roles in re-lease. In addition, treatment of cultured cells with arecombinant agnoprotein demonstrated a strong interac-tion of agnoprotein with the cell surface, suggesting anovel role for this protein during initiation of the next


round of the viral infection cycle. This study has beenmade possible by grants awarded to M. Safak from NIH(1R01NS090949-01A1) and Temple University DrugDiscovery Initiative (161398).

P143Pur-Alpha Induces JCV Gene Expression and ViralReplication by Suppressing SRSF1 in Glial Cells.

Ilker Sariyer, Rahsan Sariyer, Jessica Otte, Jennifer Gordon(corresponding author: [email protected])


PML is a rare and fatal demyelinating disease of the CNScaused by the human polyomavirus, JC virus (JCV), whichoccurs in AIDS patients and those on immunosuppressivemonoclonal antibody therapies (mAbs). We sought to iden-tify mechanisms that could stimulate reactivation of JCV ina cell culture model system and targeted pathways whichcould affect early gene transcription and JCV T-antigen pro-duction, which are key steps of the viral life cycle forblocking reactivation of JCV. Two important regulatorypartners we have previously identified for T-antigen includePur-alpha and SRSF1 (SF2/ASF). SRSF1, an alternativesplicing factor, is a potential regulator of JCV whose over-expression in glial cells strongly suppresses viral gene ex-pression and replication. Pur-alpha has been most extensive-ly characterized as a sequence-specific DNA- and RNA-binding protein which directs both viral gene transcriptionand mRNA translation, and is a potent inducer of the JCVearly promoter through binding to T-antigen. Pur-alpha andSRSF1 both act directly as transcriptional regulators of theJCV promoter and here we have observed that Pur-alpha iscapable of ameliorating SRSF1-mediated suppression ofJCV gene expression and viral replication. Interestingly,Pur-alpha exerted its effect by suppressing SRSF1 at boththe protein and mRNA levels in glial cells suggesting thiseffect can occur independent of T-antigen. Pur-alpha andSRSF1 were both localized to oligodendrocyte inclusionbodies by immunohistochemistry in brain sections from pa-tients with HIV-1 associated PML. Interestingly, inclusionbodies were typically positive for either Pur-alpha orSRSF1, though some cells appeared to be positive for bothproteins. Taken together, these results indicate the presenceof an antagonistic interaction between these two proteins inregulating of JCV gene expression and viral replication andsuggests that they play an important role during viral reac-tivation leading to development of PML.

P144Serum Inflammatory Markers and Risk of Dementiaand Neuropathy among HIV+ adults in Rakai, Uganda

Deanna Saylor1, Anupama Kumar1, Gertrude Nakigozi2,Noeline Nakasujja3, Kevin Robertson4, Carlos Pardo-Villamizar1, Ronald Gray5, Maria Wawer6


1Department of Neurology, Johns Hopkins University Schoolof Medicine; 2Rakai Health Sciences Program; 3Departmentof Psychiatry, University of Makarere; 4Department ofNeurology, University of North Carolina - Chapel Hill;5Department of Epidemiology, Johns Hopkins BloombergSchool of Public Health; 6Department of Epidemiology,Johns Hopkins University Bloomberg School of Public Health

Background: Systemic inflammation has been linked to risk ofHIV-associated neurocognitive disorder (HAND) westernHIV+ cohorts. Its relationship to HIV-associated peripheralneuropathy has not been investigated. Objective: We investi-gated the association between serum interleukin-6 (IL-6) andD-dimer with HAND and neuropathy among HIV+ adults inrural Uganda. Methods: 400 HIV+ antiretroviral therapy naíveadults were included. All participants completed asociodemographic survey, neurological examination,neurocognitive assessment, and venous blood draw.Neuropathy was defined as the presence of >1 subjective neu-ropathy symptom and >1 objective sign of neuropathy on ex-amination. Presence of dementia was determined using locallyderived normative data and Frascati criteria. Serum D-dimerlevels were determined using ELISA, and serum IL-6 levelswere determined using singleplex assays. Means of log-transformed IL-6 and D-dimer were compared using t-tests.Quartiles of IL-6 and D-dimer levels were compared usingWilcoxon rank sum tests. Univariate and multivariate logisticregression analyses were used to compare odds of dementia byserum markers. Results: Participants were 53 % male, meanage 35+8 years, and mean education 5+3 years. Half of par-ticipants had CD4 counts <200 and half had CD4 counts 350–500. Neuropathy was present in 19 % (n= 76), and 16 %(n= 66) had dementia. Participants with CD4 <200 hadhigher levels of IL-6 (1.77 vs. 1.01; p< 0.001) and a trendtoward higher D-dimer levels (13.16 vs. 12.15; p= 0.06).IL-6 was higher among participants with dementia (1.83vs. 1.32; p= 0.03) but not those with neuropathy. D-dimerdid not vary by dementia or neuropathy status. Odds ofdementia increased by 39 % for every quartile increase inIL-6 (OR 1.39; 95 % CI [1.04, 1.85]; p= 0.02) after con-trolling for CD4 count and D-dimer. Conclusions:Systemic inflammation as measured by IL-6 is associatedwith an increased risk of dementia but not neuropathy inHIV+ adults in rural Uganda.


P145Activation of SLC1A2 promoter using CRISPR/Cas9 geneediting technology

Masoud Shekarabi, Prasun Datta(corresponding author: [email protected])


Solute carrier family 1, member 2 (SLC1A2) encodes theglutamate transporter 2 (EAAT2) protein primarilyexpressed in astrocytes that reuptakes excess glutamatefrom the synaptic cleft to prevent excitotoxicity. EAAT2plays an essential role in cognitive functions and decreasedexpression of EAAT2 protein is observed in NeuroAIDS.In the current study, we investigated whether engineeredtranscriptional activation systems based on CRISPR/Cas9can be harnessed to activate HIV-1 Tat mediated dysregu-lation of EAAT2 expression in astrocytes. We have devel-oped a stable astrocytic cell line that expresses thedeactivated Cas9 (dCas9) protein which includes dCas9cassette in frame with the catalytic domain of p300 pro-tein. We designed guide RNAs to target the promoter andinduce the expression of EAAT2 protein. Using multipletechniques, we show that the SLC1A2 promoter is inducedby the gRNAs in presence dCas9-p300 in the human gli-oma cell line. Induction of SLC1A2 promoter led to in-crease in EAAT2 mRNA and protein expression. In addi-tion, we show that co-transfection of gRNAs with dCas9-p300 can mitigate HIV-1 Tat induced downregulation ofEAAT2 protein expression in primary human fetal brainastrocytes. Collectively, these results demonstrate thatCRISPR/Cas9 system can be used for potential inductionof EAAT2 expression not only in NeuroAIDS but also inother neurodegenerative diseases such as ALS andAlzheimer’s disease.

P146Epileptogenesis in Patients with Progressive MultifocalLeukoencephalopathy

Fabian Sierra Morales 1, Susan Herman2, Mary-AnnDobrota2, Dhanashri P. Miskin2, Igor J. Koralnik 1


1Rush University Medical Center; 2Beth Israel DeaconessMedical Center

Although up to 44 % of progress ive mult i focalleukoencephalopathy (PML) patients will develop seizures,

the mechanisms of epileptogenesis in PML remain unclear.We aimed to determine the incidence and risk factors for sei-zures in PML. In a prospective observational study of PMLpatients at BIDMC, we reviewed demographics, symptoms,etiology, CSF studies, JCV PCR, brain biopsy results, andCD4+T-cell counts. We measured JCV-specific T cell re-sponse in their blood by intracellular cytokine staining(ICS). Patients underwent neurologic exam, MRI of the brain(including arterial spin labeling and MR spectroscopy) and256-channel EEG at enrollment and at 3, 6, and 12 months.EEG was acquired on a Geodesics EEG system 400 with 256channels Hydrocel Geodesic sensor net. EEG findings wereco-registered to the 3D rendition of the brain on MRI. Of 29PML patients, 48 % of patients had only one enrollment visit,and 52 % were followed for 3–12 months. These included10 % of patients with clinical seizures prior to enrollmentand 38 % during the study. EEG showed focal or multi-focal slowing in 21 patients (72 %), both focal and diffuseslowing in 7 (24 %), and no abnormalities in 1 (3 %).Focal slowing was concordant with location of PML de-myelinating lesions on MRI in 24 patients (83 %).Epileptiform discharges were present on at least oneEEG in 13 patients (45 %), most commonly in temporal(10/13, 77 %) and frontal (3/13, 23 %) regions.Epileptiform discharges were concordant with PML lesionlocation in 12/13 patients (92 %) and were not associatedwith a history of IRIS. (p= .70). Focal EEG slowing andepileptiform activity was concordant with demyelinatinglesions of PML on MRI in almost al l pat ients .Demyelinating lesions appear to be associated with corti-cal irritability and epileptogenesis. Correlation with perfu-sion MRI, MR spectroscopy and results of the cellularimmune response to JCV will be presented.

P147Protective Role of Smoothened Agonist in HIV-associatedNeuro-cognitive Disorder.

Meera V Singh1, Vir B Singh1, Santhi Gorantla2, Larisa YPoluektova1, Sanjay B Maggirwar1


1Department of Microbiology and Immunology, University ofRochester Medical Center; 2Department of Pharmacology andExperimental Neuroscience, University of Nebraska MedicalCenter

Sonic Hedgehog (Shh) signaling is critical duringneurogenesis, however; recently, it has also been identifiedto play an important role in the maintenance of blood brainbarrier (BBB) homeostasis in adult CNS. Previous work fromour lab suggests that Human Immunodeficiency Virus (HIV)


infection causes impaired BBB. Excessive migration ofinflammatory/infected leukocytes across the compromisedBBB in to the CNS can exacerbate HIV-associated neuro-cog-nitive disorder (HAND), a chronic neuro-inflammatory con-dition, which afflicts nearly 50 % of HIV-infected individualson antiretroviral therapy (ART). In this report, humanizedmice were used as a model for HIV infection, to demonstratethe involvement of impaired Shh signaling in HAND. Micewere chronically infected for 10 weeks and were treated withSmoothened Agonist (SAG) for the last week. Results showedreduced expression of Shh and Gli-1 in the brains of infectedmice along with astrogliosis, microglial activation, and re-duced dendritic arbor. However upon SAG administration,mice showed increased expression of tight-junction pro-teins (Claudin5, Occludin) and reduced astrogliosis, indic-ative of better BBB integrity and decreased inflammation.Further the ability of SAG to prevent leukocyte infiltra-tion into the brain during acute infection was assessed.Mice were pre treated with SAG followed by infectionwith HIV for 2–3 days. Interestingly, SAG significantlyreduced the number of leukocytes extravasating into thebrain indicating that these mice may eventually have re-duced viral burden. Our results emphasize a neuroprotec-tive role for Shh signaling, while revealing SAG as apotential therapeutic agent. These results are also consis-tent with the outcome of clinical trial (RV254/SEARCH010), which showed HIV entry into the CNS as early as8 days of infection. Reduced viremia during this phase,possibly via SAG treatment, might subsequently result ina better prognosis for HAND.

P148Assessing the correlation between the grey and whitematter damage and HHV-6 involvement in the frontaland temporal lobes of the brain of the elderly

Sandra Skuja1, Anete Zieda1, Kristine Ravina2, SvetlanaChapenko3, Silvija Roga4, Ojars Teteris5, Valerija Groma1,Modra Murovska3


1Institute of Anatomy and Anthropology, Riga StradinsUniversity; 2Department of Neurosurgery, StanfordUniversity School of Medicine; 3A. Kirchenstein Institute ofMicrobiology and Virology, Riga Stradins University;4Department of Pathology, Riga 1st Hospital; 5Latvian StateCentre for Forensic Medical Examination

Aging is an important risk factor for developing neurode-generative disorders. High percentage of world’s populationis seropositive for human herpesvirus 6 (HHV-6), most com-monly the elderly. The immune responses the host can

mount against infectious agents undergo age-dependentchanges. Microglial cells are the main immune-competentcells of the CNS forming the first line of defense againstinvading pathogens in case of injury or disease of the brain.Human brain tissue autopsy samples from the frontal andtemporal lobes of 25 elderly subjects and 25 controls wereused in this study. The inc lus ion cr i te r ia werepathomorphological signs of an unspecified encephalopa-thy on tissue examination. Whole slide scanning, conven-tional immunohistochemistry using anti-HHV-6 and anti-CD68 antibodies and confocal microscopy were applied.Immunostaining intensity was assessed with an additionalquantitative estimation of immunopositive cells. Brain tis-sue samples were assayed for HHV-6 using nested andreal-time PCR. SPSS 23.0 program was used for statisticalanalysis. The HHV-6 DNA was detected in the frontal andtemporal lobes respectively of the following number ofcases - encephalopathy group - 36 % (9/25), 44 % (11/25); and control group - 16 % (4/25), 38.8 % (7/25). Inboth regions studied, there were significantly higher(p=<0.001) number of HHV-6 positive gray matter astro-cytes, oligodendrocytes, microglial cells and neurons inthe encephalopathy group when compared to controls.By contrast, there were significantly (p = <0.001) moreHHV-6 positive oligodendrocytes and microglial cellsfound in the white matter when compared to the graymatter. An increased number of activated microglial cellswere detected in the white matter of the frontal and tem-poral lobes of the encephalopathy group. More neuronaland oligodendroglial HHV-6 positivity was detected in thegray and the white matter, respectively, demonstratingheterogeneity of damage to the cortex and subcorticalwhite matter.

P149In vivo visualization and quantificationof Spatial-Temporal kinetics of Chimeric HIV infectionand the efficacy of Truvada as PrEP

Jiasheng Song1, Alexander G. White1, Yonggang Zhang2, FeiYu1, Wenhui Hu2, Won-Bin Young1


1Department of Radiology, University of Pittsburgh School ofMedicine; 2Department of Neuroscience, Temple UniversitySchool of Medicine, Philadelphia, PA, USA

Visualizing primary sites of HIV-1 infection and viral reser-voirs longitudinally would be extremely useful for the devel-opment of antiretroviral drugs and vaccines. Currently, surro-gate animal models are the major platform for testing newlydeveloped antiretroviral therapy (ART) and vaccines against


HIV infection. To establish a lower-cost high-throughput drugscreening platform in conjunction with studying how viralreservoirs are formed and maintained, we have employed achimeric HIV-1 virus which contains a replacement of theHIV gp120 protein with gp80 from the ecotropic murine leu-kemia virus. This allows for infection of conventional micewithout the need of expensive humanized mice. Insertion ofthe enhanced firefly luciferase gene in this chimeric HIV en-ables the infected cells to be visualized via in vivo biolumi-nescence imaging. We systemically imaged the spatial-temporal biodistribution of the infected cells to revealthe viral infection kinetics during the first 24 h andfollowed the infection for 107 days. The biodistributionof infected cells, mainly neutrophils, was visualized andquantified longitudinally using bioluminescence imagingto illustrate the migration of infected cells in vivo. Exvivo imaging revealed the infected cells reside in liver,gastrointestinal (GI) tract, spleen, peripheral lymph nodes,and nasal mucosa. The viral RNA in the spleen was line-arly correlated to the photon flux output measured fromthe light emission from the spleen during whole animalimaging. We also showed that this imaging/chimeric HIVplatform can be used directly to evaluate the efficacy ofTruvada as Pre-Exposure Prophylaxis against HIV infec-tion in living animals.

P150Substance P Mediated Chemokine Production PromotesMigration of Human Monocytes

Sergei Spitsin1, Steven D. Douglas2


1Division of Allergy and Immunology, The Children’sHospital of Philadelphia Research Institute, Philadelphia,PA; 2Division of Allergy and Immunology, The Children’sHospital of Philadelphia Research Institute, Department ofPediatrics, Perelman School of Medicine, University ofPennsylvania, Philadelphia, PA

Substance P (SP) is a neuropeptide which plays a role inneurotransmission in the central and peripheral nervous sys-tem and has immunomodulatory properties. SP is a member ofthe tachykinin family of neuropeptides and acts through inter-action with neurokinin-1 receptor (NK1R) which is expressedby a variety of cells including lymphocytes and monocyte/macrophages. SP has physiological and pathophysiologicalroles in both CNS and peripheral tissues and is involved incross talk between nervous and immune systems in variousconditions including HIV and SIV infection. Increased SPlevels were demonstrated in plasma of HIV positive individ-uals as well as in the CNS of SIV infected non-human

primates. SP increases HIV infection in macrophages throughinteraction with NK1R. The SP effect on immune system isboth pro- and anti-inflammatory and includes upregulationof a number of cytokines and cell receptors. We determinedwhether there is interplay between monocyte exposure toSP and their recruitment into CNS. We demonstrated thatexposure of both human monocytes/macrophages andPBMC to SP leads to increased production of chemokinesincluding MCP-1 which is expressed only by cells of my-eloid lineage. This effect was inhibited by the NK1R an-tagonist, aprepitant. Exposure to conditioned media fromSP treated PBMC resulted in increased monocyte migra-tion through semipermeable membrane and in vitro humanBBB model. Cell migration was blocked by anti-MCP-1antibodies. Conclusions: The exposure of monocyte/macrophages to a greater levels of SP during HIV infectionmay lead to increased MCP-1 production in the CNS andpromote further monocyte migration into CNS thus con-tributing to HIV infection of the brain. The transmigratedmonocytes may be a major source of the CNS reservoir inHIV infected individuals and factor contributing to CNSinflammation. Supported by NIH PO1 MH-105303, UO1MH-090325, R21 AI-108296, P30 MH-097488 to SDD

P151A role for BACE1 in HIV-associated neurotoxicity

Anna Stern1, Patrick Gannon1, Benjamin Gelman2, DennisKolson1, Kelly Jordan-Sciutto1


1The University of Pennsylvania; 2University of TexasMedical Branch

HIV-associated neurocognitive disorder (HAND) persists in30–50 % of HIV+ patients despite effective viral suppres-sion by combined antiretroviral therapy. Though a distinctdisorder, HAND has symptoms and neuropathological fea-tures in common with Alzheimer’s Disease (AD). A hall-mark of AD pathology is accumulation of amyloid-beta(Abeta), which is generated by cleavage of amyloid precur-sor protein (APP) by the beta-secretase BACE1. BACE1 isincreased in AD brain, and BACE1 inhibitors reverse neu-ronal loss and cognitive decline in animal models of ADwith human trials underway. Although some studies havefound evidence of altered APP processing in HIV+ patients,it is unknown whether increased BACE1 or accumulation ofoligomeric Abeta are a feature of HAND. As HIV does notinfect neurons, neurotoxicity is attributed to factors releasedfrom HIV-infected macrophages including glutamate, andNMDA receptors mediate HIV-associated neurotoxicityin vitro. Herein, we hypothesize that HIV-associated


neurotoxicity is mediated by NMDA-dependent elevation ofBACE1 and subsequent altered processing of APP. In sup-port of this hypothesis, we have observed elevated levelsof BACE1 and Abeta oligomers in CNS of HIV+ patients.As a model of HIV-induced neurotoxicity, we treated pri-mary rat neurons with supernatants from HIV infectedmonocyte derived macrophages (HIV/MDMs). We ob-served elevation of BACE1 protein levels in bothHIVMDM- and NMDA-treated neurons, and HIVMDM-induced BACE1 increases were blocked by NMDA recep-tor inhibition. Furthermore, blocking BACE1 activitywith a BACE1 inhibitor abrogated HIV/MDM-inducedand NMDA-induced neurotoxicity. These findings suggestthat increased BACE1 and resultant Abeta productionmay contribute to HAND neuropathology, and inhibitionof BACE1 may have therapeutic potential for HANDpatients.

P152Research Training in Neurovirology: SupportingPathways to Success

David Stoff(corresponding author: [email protected])

National Institute of Mental Health, Division of AIDSResearch

This session represents our annual efforts at the currentISNV meet ing to support research tra ining inneurovirology. The session is designed to provide newand early stage research investigators with the tools nec-essary to continue along the path of competitive re-search support and transition to independence. As inprevious meetings, active T32 and other predoctoraland/or postdoctoral trainees will briefly present dataand research directions for their current mentored re-search projects and will be offered feedback for im-provements. Preceding and subsequent to the traineepresentations, the symposium content will include: (1)Introductory presentation on neurovirology research tra-jectories, with special attention to barriers faced byeme rg i ng young i nve s t i g a t o r s t o suc c e s s i nneurovirology at various levels (individual, institutional,organizational, systemic), and (2) Panel discussion in-cluding predoctoral, post-doctoral and faculty investiga-tors on strategies for successfully navigating obstaclesand developing potential solutions on the journey to asuccessful research career. One of the intentions of thisroundtable discussion will be to facilitate mentoring re-lationships at multiple levels.

P153EVALUATION OF COMBINATION LONG-ACTINGNANOFORMULATED ANTIRETROVIRALTHERAPY IN EARLY HIV-1 INFECTED huPBLNOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ MICE

Hang Su, Prasanta Dash, Mariluz Arainga, Benson Edagwa,JoEllyn McMillan, Larisa Poluektova, Santhi Gorantla,Howard Gendelman(corresponding author: [email protected])

Depar tment of Pharmacology and Exper imenta lNeuroscience, University of Nebraska Medical Center

Early clinical intervention with combination antiretroviraltherapy (ART) in human immunodeficiency virus type one(HIV-1) infected people was demonstrated to havesustained benefits by lowering viral set points, protectingCD4+ T lymphocytes and limiting viral reservoir poolsthat include central nervous system (CNS), which holdslimited ART excess, persistent viral replication and cellularactivation, leading to continued neuropsychological impair-ment, causing HIV-associated dementia (HAD). Notablyand as shown in our prior studies cell-targetednanoformulated ART (nanoART) improves pharmacokinet-ic and pharmacodynamics profiles and reduces local andsystemic drug toxicities in experimental models of HIV/AIDS. In addition, with optimized size, shape and proteinand lipid coatings, nanoART can circumvent the blood–brain barrier (BBB) to facilitate CNS-directed drug deliv-ery. These drugs are being considered in a step-wise drugregimen for viral eradication. In the present study, weadministrated long-acting nanoformulated combinations offolic acid-decorated cabotegravir, lamivudine and abacavirto human peripheral blood lymphocyte (PBL) reconstitutedNOD. Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice 24 or72 h after HIV-1 infection. Replicate mice received freedrugs at equivalent doses. Animals were sacrificed 2 weeksafter HIV-1ADA infection with blood and tissues harvest-ed for viral measurements. NanoART-treated animals dem-onstrated 75 % recovery of CD4+ T lymphocytes calcu-lated from a total CD3+ cell pool. Immunohistochemicaltests showed HIV-1p24+ cells (~80/1000HLA-DR+ cells)only in untreated infected animals. NanoART suppressedHIV-1gag RNA expression to <10copies as assessed byreal-time RT-PCR in brain, spleen, live, lung and kidneyin 90 % animals while total viral DNA remained detect-able. Limited tissue proviral DNA was seen in nanoARTtreated animals with significant reduction, up to or exceed-ing 85 %, in plasma viral RNA levels was observed in24 h nanoART-treated group (<20 copies/ml). Early treat-ment of combination nanoART in infected PBL mice caneffectively suppress HIV-1 growth in both central and


peripheral viral sanctuaries while preventing CD4+ T lym-phocyte loss.

P154Clinical and functional characterization of viral singlenucleotide polymorphisms (SNPs) within the HIV-1 LTRassociate with increased virus persistence in the DrexelMedicine CARES Cohort

Neil Sullivan1, Michael Nonnemacher1, Vanessa Pirrone1, RuiFeng2, Brian Moldover3, William Dampier1, Shendra Passic1,Jean Williams1, Benjamas Aiamkitsumrit1, Wen Zhong1,Brandon Blakey1, Sonia Shah1, Zsofia Szep4, JeffreyJacobson5, Brian Wigdahl1


1Department of Microbiology and Immunology, Institute forMolecular Medicine and Infectious Disease, DrexelUniversi ty College of Medicine; 2Department ofBiostatistics and Epidemiology, Center for ClinicalEpidemiology and Biostatistics, University of PennsylvaniaSchool of Medicine; 3B-Tech Consulting, Ltd; 4Division ofInfectious Diseases and HIV Medicine, Department ofMedicine, Drexel University College of Medicine;5Department of Medicine, Section of Infectious Disease,Lewis Katz School of Medicine, Temple University

The HIV-1 LTR is continuously under selective pressureand LTR SNPs can alter viral transcription in a cell-typedependent manner. To elucidate the clinical and functionalimpact of HIV-1 SNPs, the Drexel Medicine CNS AIDSResearch and Eradication Study (CARES) Cohort con-ducted a prospective, longitudinal study on >500 HIV-1-infected patients. Numerous SNPs were strongly correlat-ed with clinical disease parameters, such as CD4+ T-cellcount and viral load. Of interest, LTR position 108, aCOUP/AP1 binding site, increased in frequency in pa-tients with high viral loads and low CD4+ T-cell counts.Electrophoretic mobility shift assays (EMSAs) functional-ly demonstrated differential transcription factor (TF)/DNAbinding profiles with Jurkat T-cell and monocytic U-937nuclear extract (NE) when the nucleotide at position 108is changed. The binding site with an A at position 108(108A) formed 3 complexes while a G at this position(108G) formed 4 complexes with Jurkat NE. Three com-plexes were formed with both constructs when U-937 NEwere used in the EMSAs. JASPER and supershift EMSAssupport the presence of GATA-2, ETS-1, AP-1, andCOUP binding to the sequences surrounding 108.Transient expression analyses with an LAI LTR contain-ing a 108G showed increased transcription as compared to108A in monocytic U-937 cells but not in Jurkat T cells.

This observation correlates with the increased viral loadand persistence associated with the 108G change in spe-cific HIV-1-infected individuals. These results demon-strate that mutations are occurring in individuals on anti-retroviral therapy that are likely clinically and functional-ly important.

P155IFNbeta PROTECTS NEURONS IN ACCL4-DEPENDENT FASHION AGAINST HIV-1GP120-INDUCED INJURY

Victoria Thaney1, Alan O’Neill1, Melanie Hoefer1, RickyMaung1, Marcus Kaul1


1Sanford Burnham Prebys Medical Discovery Institute

Human immunodeficiency virus-1 (HIV-1) invades thecentral nervous system (CNS) soon after peripheral infec-tion and can cause HIV-associated neurocognitive disor-ders (HAND). Type I interferons are critical mediators ofanti-viral immune responses and interferon-beta (IFNbeta)has been implicated in the control of HIV and SIV infec-tion in the brain. However, whether IFNbeta has neuropro-tective activity in the context of HIV-mediated neuronalinjury is unknown. In the present study, we usedHIVgp120 transgenic mice (HIVgp120tg) as a model forHIV-induced brain injury that may underlie the develop-ment of HAND. Analysis by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) detectedan IFN response in gp120 transgenic brains with IFNbetamRNA significantly increased at 1.5 months, but not at 3and 6 months. Therefore, we examined whether recombi-nant IFNbeta treatment can prevent neuronal injury and/orglial activation in vivo and in mixed neuronal-glialcerebrocortical cell cultures. In vivo, we administeredIFNbeta intranasally in order to maximize IFNbeta deliv-ery to the brain and to minimize side effects in peripheraltissues. We found that a 4-week IFNbeta treatment trig-gered an IFN response that included increased CCL4 ex-pression. Neurohistological analysis revealed that IFNbetatreatment prevented loss of neuronal dendrites and pre-synaptic terminals in the frontal cortex and hippocampusin gp120tg brains. Similarly, treatment of mixed neuronal-glial cerebrocortical cells with IFNbeta provided completeneuronal protection against recombinant gp120 toxicity.Neuroprotection was dependent on IFNalpha receptor 1(IFNAR1) and CCL4, as IFNAR1 deficiency and neutral-izing antibodies against CCL4, respectively, abolished theneuroprotective effects of recombinant IFNbeta. Taken to-gether, these results identify IFNbeta as a neuroprotective


factor that can ameliorate HIV gp120-induced brain injuryvia induction of CCL4 and be delivered in vivo usingintranasal appl icat ion. Supported by NIH R01MH087332, MH104131 and MH105330

P156HIV-1 Tat protein activates microglia through defectivemitophagy

Annadurai Thangaraj, Palsamy Periyasamy, Shilpa Buch(corresponding author: [email protected])

Depar tment of Pharmacology and Exper imenta lNeuroscience, University of Nebraska Medical Center,Omaha, NE, ZIP: 68198

HIV-1 invades CNS early in the course of infectionand primes a cascade of neuroinflammatory responsesby activating immune cells. Since HIV-1 transactivatorprotein, Tat has been well-documented to activate glialcells, the objective of this study was to explore wheth-er Tat-mediated activation of microglia also involvedmitophagy. Our results demonstrated that exposure ofmicroglia to Tat resulted in cellular activation by alter-ing the mitochondrial membrane potential and by in-creasing the expression of mitophagy markers, Pink1and Parkin in both BV-2 cells and mouse primarymicroglial cells. Increased Pink1 accumulation on thedefective mitochondria has been shown to recruit cyto-solic Parkin to the site of damaged mitochondria fortheir clearance via mitophagy. We also showed thatexposure of microglia to Tat resulted in increased ex-pression of DRP1, thereby escalating the fission ofdamaged mitochondria. Tat exposure also upregulatedthe expression of autophagosome markers, Beclin1 andLC3-II, indicating thereby that increased levels ofPink1, Parkin, and DRP-1 proteins likely tether thedamaged mitochondria to the LC3-positive phagophorefor mitophagosome formation. These findings were val-idated by imaging studies which demonstrated in-creased co-localization of damaged mitochondria withthe LC3 puncta. Intriguingly, Tat exposure also in-creased the expression of p62, signifying thereby a possi-ble blockade of mitophagy flux, that in turn, results inaccumulation of mitophagosomes. This phenomenon wasfurther validated by mRFP-EGFP-LC3 transfectionwherein the results confirmed Tat-mediated defectivemitophagy. Interestingly, Tat-mediated activation of mi-croglia correlated with increased expression of TNF-al-pha, IL-1beta, and IL-6. Using both pharmacologicaland gene-silencing approaches to block either autopha-gy/mitophagy, our findings demonstrated that Tat-

induced activation of microglia involved sequential acti-vation of mitophagy with defective clearance. In summa-ry, Tat activates microglia by both increasing mitochon-drial damage and defective mitophagy. Interventionsaimed at blocking mitochondrial dynamics could thus pro-vide promising therapeutic targets for abrogating Tat-mediated neuroinflammation. (Supported by DA040397;DA035203)

P157Reduced cellular energy as a potential markerof HIV brain latency related neuropathogenesis:a pilot study

Michael Tobia1, Simon Jones2, Michael Lovelace2, CarolineRae1, Lucette Cysique1, Bruce Brew1


1UNSWAustralia; Neuroscience Research Australia; 2UNSWAustralia; Applied Neurosciences Program, Peter DuncanNeuroscience Research Unit, St. Vincent’s Centre forApplied Medical Research

Background: In virally suppressed HIV-infected (HIV+)the cause of brain injury is not known. One hypothesisis that the brain acts as a reservoir for latent HIV in-fection, and that the burden of HIV brain latencycauses a cascade of neuropathological events. In thecurrent study we explore whether major brain metabo-lites may serve as markers of brain HIV latency, de-fined by CSF BCL11b (a microglia cell transcriptionfactor that inhibits HIV transcription). Methods: Thestudy included 26 HIV+ men (mean age 57 years old;stable on cART ≥6 months, nadir CD4+ lymphocytecount ≤350 cells/mm3, HIV infection ≥5 years; 96 %undetectable <50 cp/mL; 100 % <100cp/mL). All un-dertook lumbar puncture; a 1H Magnetic ResonanceSpectroscopy (MRS) scan. CSF samples were analyzedfor BCL11b, neopterin, NFL, and tat. All were hadundetectable HIVRNA at <50 cp/mL. 1H MRS includ-ed measurements of N-acetyl Aspartate (NAA), choline,creatine, Myo-inositol, glutamine/glutamate in the fron-tal white matter, posterior cingulate cortex, and caudatenucleus area. MRS spectra were measured with refer-ence to the unsuppressed water signal and quantifiedusing JMRUI V.03. Results: In a series of adjusted(for neopterin, NFL and tat) regression models; wefound that a higher CSF BCL11b was consistently as-sociated with lower frontal white matter creatine (ad-justed for neopterin, Std beta =−.30; p = .15; adjustedfor NFL, Std beta =−.51; p= .03; adjusted for tat, Stdbeta =−.47; p= .02). Conclusions: Frontal white matter


reduced cellular energy may indicate ongoing HIVbrain latency related neuropathogenesis. These pilot da-ta need to be further tested in a larger sample.

P158Neutralization of interferon-alpha by B18Rin a mouse model of HIV associated neurocognitivedisorders

William Tyor1, Rajeth Koneru2, Heather Bimonte-Nelson3,Woldeab Haile4, Vincent Ciavatta5, Jennifer Ward6, LeonardMaroun6


1Emory University School of Medicine and Atlanta VAMedical Center; 2Atlanta VA Medical Center; 3Arizona StateUniversity; 4Emory University; 5Emory University and theAtlanta VA Medical Center; 6Meiogen

Infection with HIV commonly causes HIV associatedneurocognit ive disorders (HAND) despi te l i fe-prolonging combined antiretroviral therapy (cART).There is substantial evidence that interferon-alphaplays a role in cognitive impairment in HAND. Wehave previously shown that a decoy protein that se-questers type I interferons, B18R, ameliorates HIV in-duced histopathology. We tested if co-administration ofB18R with cART provides enhanced protection fromHIV encephalitis in comparison to B18R or cARTtreatments alone in a mouse model of HAND. After10 days of treatment mice were sacrificed and brainsextracted and frozen for tissue sectioning. The datasuggests that B18R simultaneously administered withcART reduced encephalitis markers more than eitherof the two treatments administered alone. In a separateset of experiments, B18R was tested for its ability toreverse behavioral abnormalities of HAND mice usingan Object Recognition Test (ORT). The ORT was ad-ministered to HAND mice and controls before and af-ter treatment with B18R. Prior to B18R treatmentHAND mice were behaviorally impaired on the ORT.Then HAND mice received either B18R or saline sub-cutaneously twice a day for 5 days prior to repeatORT. Performance was significantly improved inB18R treated HAND mice compared to saline treatedmice. Pathological studies in these ORT, B18R treatedand untreated HAND mouse groups are ongoing.Because of its ability to act in concert with cART aswell as reverse behavioral abnormalities in HANDmice, B18R is a potential therapeutic agent for aPhase I trial in HAND patients.

P159The epidermal growth factor counteracts the effectsof HIVtat on the expression of endogenous retrovirusesof the W family in human astrocytes

Elena Uleri, Gabriele Ibba, Claudia Piu, Maurizio Caocci,Caterina Serra, Antonina Dolei(corresponding author: [email protected])

Department of Biomedical Sciences, University of Sassari

The pathogenic mechanisms of HIV-related neurodegen-eration are not fully elucidated, and include both host andviral mechanisms. The epidermal growth factor (EGF) hasbeen proposed to control the phenotypic features of adultastrocytes. Activation of EGFR, the EGF receptor, wasproposed as a master pathway of astrocyte activation inresponse to different neural injuries. We showed previous-ly that HIVtat activates the neuropathogenic andimmunopathogenic MSRV and Syncytin-1 elements ofthe HERV-W family of endogenous retroviruses, in hu-man macrophages and in astrocytes. We hypothesized thatthese elements could contribute to the pathogenic phe-nomena leading to HIV-related neurodegeneration. Themechanism of HERV-Ws stimulation by Tat is an indirectone, through interaction with Toll-like Receptor-4(TLR4), without internalization, and induction of TNF-alpha. It was shown that HIVtat interacts with the 1–6EGF-like repeats of Notch proteins, with a possible linkwith AIDS pathogenesis. Therefore, we exposed thePHFA, SVG and U87MG human astrocyte cells toHIVtat, EGF and other stimuli, and the expression ofMSRV and Syncytin-1 HERV-W transcripts was evaluatedby real time RT-PCR. The results indicate that EGF coun-teracts the effects of HIVtat on the HERV-Ws, by inter-fering with the induction of TNF-alpha by HIVtat. TheEGF-mediated counteraction of HIVtat effects isprevented by pre-exposure to antibody against the EGF-R.

P160JC polyomavirus expression and bell-shaped regulationof its SF2/ASF suppressor in multiple sclerosis patientsunder therapy with Natalizumab

Elena Uleri, Gabriele Ibba, Claudia Piu, Maurizio Caocci,Caterina Serra, Antonina Dolei(corresponding author: [email protected])

Department of Biomedical Sciences, University of Sassari

Natalizumab is effective against multiple sclerosis (MS), butr a r e l y a s soc i a t ed w i t h p rog r e s s i v e mu l t i f o c a l


leukoencephalopathy (PML), fatal brain disease due to theJCV polyomavirus. The SF2/ASF (splicing factor2/alternative splicing factor) inhibits JCV in glial cells.We wondered about SF2/ASF modulation in the bloodof Natalizumab-treated patients, and if this could influ-ence JCV reactivation. A 2-year study was performed onblood cells from Natalizumab-treated and Fingolimod-treated MS patients. A bell-shaped SF2/ASF regulationwas observed only under Natalizumab: SF2/ASF was up-regulated, during the first year, only in JCV DNA-positive patients, or with high anti-JCV antibody, withenrichment of SF2/ASF-negative B-cells. The expressionof the JCV T-Ag protein in circulating B cells was in-versely related to SF2/ASF protein expression. The in-crease of SF2/ASF-negative B-cells, parallel to JCV ac-tivation, during the second year of therapy withNatalizumab, but not with Fingolimod, may helpexplaining the increased risk of PML with Natalizumab,and not with Fingolimod.

P161JAM-A and ALCAM Mediate PreferentialTransmigration Across the BBB of HIV-infected HIV +CD14+CD16+ Monocytes: Continued Viral Seedingof the CNS and HAND.

Mike Veenstra1, Bruce Shiramizu2, Lucio Gama3, KathrynAnastos4, Matilde Inglese5, Desiree Byrd5, Susan Morgello5,Joan Berman1


1Department of Pathology, Albert Einstein College ofMedicine; 2John A Burns School of Medicine, University ofHawaii at Manoa; 3Department of Molecular andComparative Pathobiology, Johns Hopkins University;4Department of Medicine, Albert Einstein College ofMedicine; 5Department of Neurology, Mount Sinai Schoolof Medicine

HIV associated neurocognitive disorders (HAND) affect>50 % of HIV-infected individuals. HAND is mediated byentry of infected CD14+CD16+ monocytes into the CNS thatestablish and reseed CNS viral reservoirs, even when peoplehave undetectable viral loads. CD14+CD16+ monocytestransmigrate across the blood–brain barrier (BBB) into theCNS in response to chemokines, including CCL2. Viral res-ervoirs release early viral proteins and cytokines, even duringactive cART, that mediate persistent neuroinflammation andneuronal damage leading to HIV neuropathogenesis. To de-velop therapeutics that can prevent viral (re)seeding of theCNS and subsequent HAND, we examined the mechanismof entry of these HIV-infected cells into the CNS. We show

for the first time that HIV-infected HIV+CD14+CD16+monocytes preferentially transmigrate across the BBB in com-parison to uninfected but HIV-exposed HIVexpCD14+CD16+ monocytes, both by p24 FACS and HIV DNA. Wealso show that this is mediated, in part, due to increasedjunctional proteins JAM-A and ALCAM on HIV+CD14+CD16+ monocytes. We also showed that JAM-A andALCAM blocking antibodies inhibit transmigration acrossthe BBB of HIV+CD14+CD16+ monocytes. In addition,CCR2, the only known CCL2 receptor on monocytes, isincreased on CD14+CD16+ monocytes from HIV-infectedindividuals with HAND, but not on those with impairmentdue to other causes. Using neuroimaging, we showed thatincreased CCR2 on CD14+CD16+ monocytes correlateswith decreased neuronal activity in the basal ganglia.Additionally, using HIV DNA analysis we found that in-creased CCR2 correlates with increased peripheral PBMCHIV DNA. This provides strong evidence that CCR2 iden-tifies individuals with cognitive impairment due to HIV,and that CCR2 may be a HAND biomarker. To examineCCR2 in addition to JAM-A and ALCAM as a therapeutictarget, we will use Cenicriviroc to prevent transmigrationof HIV+CD14+CD16+ monocytes into the CNS. It ishoped that these strategies will result in decreased neuronaldamage and inflammation, and, HAND.

P162Characterization of Zika virus infection in HumanAstrocytes

Courtney Veilleux, Eliseo Eugenin(corresponding author: [email protected])

Public Health Research Institute (PHRI), Rutgers the StateUniversity of New Jersey, Newark, NJ, USA; Department ofMicrobiology, Biochemistry, and Molecular Genetics,Rutgers the State University of New Jersey, Newark, NJ, USA

Zika viral infection is correlated with an increased rate ofmicrocephaly in children born of mothers infected duringpregnancy and neurocognitive disorders in adults. Althoughdocumented as a mild febrile illness in less than two dozencases prior to 2003, the Brazilian Zika outbreak strain spreadto over 40 counties and territories since its suspected origin inBrazil in 2015 and has infected over 7300 in the US and USterritories since June 2015. Additionally, 479 pregnancieswithin the continental US and 493 pregnancies in US terri-tories have had evidence of Zika infection during pregnancy.Microcephaly as a result of Zika has been identified in morethan a dozen US births. The pathogenesis of Zika and itseffects on the developing brain are unknown. Previous re-search in our laboratory has demonstrated that astrocytes play


a significant role in perpetuating viral infection within thebrain. Our laboratory has identified astrocytes as key viralreservoirs and mediators of apoptosis in CNS cells duringviral infection. Thus, our objective is to characterizewhether Zika infection in human fetal astrocytes alterssurvival or glial and neuronal cells. We hypothesize thatZika infection of human fetal astrocytes causes apoptosisthat contributes to the devastating CNS consequences ofthis virus. Our results indicate that Zika induces apoptosisof glial and neuronal cells. Altogether, these results illus-trate a distinct mechanism of Zika infection in humanfetal astrocytes. Understanding infection in primary CNSresident cells is critical in understanding the pathogenesisof Zika infection in the developing brain.

P163Modulation of Tryptophan degradation viathe kynurenine pathway during HIV and SIV infection

Stephani Velasquez1, Wendeline Wagner2, Mark G. Lewis2,Jay Rappaport1


1Department of Neuroscience, Lewis Katz School ofMedicine Temple University; 2Bioqual

Tryptophan (TRP) is an essential amino acid and precursor forthe de novo synthesis of nicotinamide adenine dinucleotide(NAD), as well as serotonin and melatonin, by two differentpathways. TRP is catabolized by the kynurenine (KYN) path-way to produce NAD and the methoxyindole pathway to gen-erate serotonin and melatonin. Increased KYN/TRP ratioshave been associated with the progression of acquired im-mune deficiency syndrome (AIDS) as determined by associ-ation with lower CD4/CD8 ratios, reduced T cell recoveryafter initiation or cART, and increased mortality risk.Activation of the KYN pathway as well as the neurotoxicmetabolite quinolinic acid has been implicated in the patho-genesis of HIVassociated neurocognitive disorders (HAND).We identified an increase in the KYN/TRP ratios in the SIVmacaque model with a positive correlation with viral load andsoluble CD163 in plasma, with the latter suggesting the role ofmacrophage activation in this process. In an effort to modulatethe K/T pathway, and decreased tryptophan catabolism, weperformed a dose escalation study with the NAD salvage path-way precursor, nicotinamde riboside (NR), in SIV infectedmacaques. NR treatment significantly reduced the mean fluo-rescence intensity and percent frequency of CD14+/CD16+monocytes as determined by flow cytometry, in both SIVinfected and uninfected animals. This monocyte subset hasbeen implicated in the pathogenesis of HIV infection,HAND, as well as other HIV associated comorbidities.

In uninfected animals NR treatment had no significantimpact on the KTR relative to pretreatment values. Ininfected animals, however, NR treatment appeared to in-crease KTR at 100 mg/kg dose, and decrease CD4/CD8ratio at the highest dose (400 mg/kg). The regulation ofthe kynurenine pathway appears to be complex, yet anattractive target for HIV therapeutics. Understanding themechanism by which HIV modulates the KYN/TRP path-way could provide new therapeutic targets for the treat-ment of HIV and HAND.

P164A novel murine model of enterovirus-71 (EV-A71)-induced neurogenic pulmonary edema

Carla Bianca Luena Victorio1, Yishi Xu1, Qimei Ng1, BengHooi Chua2, Sylvie Alonso3, Vincent Chow3, Kaw BingChua1


1Temasek Lifesciences Laboratory; 2Curtin University;3Department of Microbiology and Immunology, Yong LooLin School of Medicine, National University of Singapore

Enterovirus 71 (EV-A71) is now the most common neuro-trophic enterovirus following the near eradication of poliovi-ruses. It causes Hand, Foot, and Mouth Disease (HFMD) - aself-limiting febrile illness - and occasional fatal neurologicaldisease in children. Current animal models of EV-A71 infec-tion are not clinically authentic. They fail to replicate the fullspectrum of human diseaseäóîspecifically that of neurogenicpulmonary edema (NPE), the major cause of EV-A71 infec-tion-related child mortalityäóîand lack key pathological fea-tures observed in fatal human cases. In order to more fullyunderstand the neuro-pathogenesis of severe EV-A71 infec-tion, especially with respect to NPE, our group generated nov-el mouse-cell-adapted (MCA-EV-A71) viral strains that pro-ductively infected both primate and rodent cell lines (Victorioet al., 2014) and utilize the physiologically expressed viralreceptor expressed in rodents (mSCARB2) to infect murinecells (Victorio et al., 2016a). We subsequently used thesestrains to infect 1-week-old BALB/c mice. The infected micenot only exhibited severe, acute lethal disease, but also in-duced a spectrum of neurological deficits, including that ofNPE. We noted signs of respiratory distress and upon necrop-sy, gross pathological observations confirmed pulmonary ede-ma. Pathological findings in the lung tissues include focalhaemorrhagic lesions and accumulation of proteinaceous fluidin the alveoli; while CNS tissue sections revealed massiveneuronal destruction and accumulation of viral antigens inthe brainstemäóîmost notably in the medulla oblongata(Victorio et al., 2016b). We also observed high levels of serum


catecholamines, confirmingNPE. Thus, we have developedan authentic mouse model of EV-A71 infection that re-capitulated the wide spectrum of clinical signs observedin patients as well as the underlying pathological fea-tures observed in fatal cases (Wong et al., 2008). Thismodel could aid in the further understanding of thepathogenesis of EV71-induced neurological diseasesand in the development of novel therapies andprophylactics.

P165Detection of HIV-1 DNA in brain tissue of virallysuppressed patients supports the existence of a persistentCNS viral reservoir

Emma L. Wanicek1, Lachlan R. Gray2, Wan-Jung Cheng1,Jacob D. Estes3, Sharon R. Lewin4, Paul R. Gorry5, MelissaJ. Churchill6


1Centre for Biomedical Research, Burnet Institute,Melbourne, VIC; 2Centre for Biomedical Research, BurnetInstitute, Melbourne, VIC 2. Department of InfectiousDiseases, Alfred Hospital and Monash University,Melbourne, VIC; 3AIDS and Cancer Virus Program,Frederick National Laboratory for Cancer Research, LeidosBiomedical Research, Inc., Frederick, MD; 4Department ofInfectious Diseases, Alfred Hospital and Monash University,Melbourne, VIC 2. The Peter Doherty Institute for Infectionand Immunity, The University of Melbourne and RoyalMelbourne Hospital, Melbourne, VIC; 5Centre forBiomedical Research, Burnet Institute, Melbourne, VIC 2.Department of Infectious Diseases, Alfred Hospital andMonash University, Melbourne, VIC 3. School of Healthand Biomedical Sciences, RMIT University, Melbourne,VIC 4. Department o; 6Centre for Biomedical Research,Burnet Institute, Melbourne, VIC 2. Department ofMedicine, Monash University, Melbourne, VIC 3.Department of Microbiology, Monash University, Clayton,VIC

Combination antiretroviral therapy (cART) has transformedpatient care and disease outcomes for millions of HIV-1 pa-tients globally. However, cART alone is unable to eradicateHIV-1 due to the persistence of numerous viral reservoirsthroughout the body. The central nervous system (CNS) is apotential viral reservoir but is difficult to study due to limita-tions in access to brain tissue. Here, we have applied a noveland highly sensitive (single copy sensitivity) in situ hybridi-zation technique called DNAScope, to detect and visualizeHIV-1 DNA in the brain tissue from HIV-infected individualson suppressive cART. Using a comprehensive bank of tissue

samples from clinically well-defined HIV+ patients (n=45),including both symptomatic and asymptomatic individuals,and representing a range of CNS disease severities, HIV-1infection was measured by DNAScope. We observed an in-crease in HIV-1 staining that correlated with increaseddisease severity. Significantly, HIV-1 DNA was detectedin both macrophages/microglia (CD68) and astrocytes(GFAP) in virally suppressed patients. Further, HIV-1Envelope V3 sequences isolated by laser capture micro-dissection from macrophages/microglia of 1 patient, dem-onstrated compartmentalization relative to V3 sequencesisolated from PBMCs from the same patient. DNAScopecoupled with laser capture micro-dissection, is a powerfultechnique that allows for the detection, quantification andcharacterisation of HIV-1 infected cells within the CNSand allows for the characterization of the CNS viral res-ervoir. This study provides strong evidence that HIV-1DNA can persist within the CNS of virally suppressedpatients and supports the existence of a CNS viral reser-voir with important implications for cure research and thedevelopment of cure strategies.

P166Investigating the role of Tetherin-dependentpro-inflammatory microparticle release

Emily Weber, Meera Singh, Vir Singh, Joe Jackson, NicoleStirpe, Sanjay Maggirwar(corresponding author: [email protected])

Department of Microbiology and Immunology, University ofRochester School of Medicine and Dentistry

Despite the emergence of combined antiretroviral therapy(cART) that is able to suppress the human immunodeficiencyvirus (HIV) to undetectable levels in plasma, HIV-positiveindividuals still face an array of co-morbidities, including anincreased risk of neurocognitive disorders. Studies have sug-gested that chronic inflammation resulting from HIV infectionmay be due to cytokines/chemokines and viral proteins secret-ed by activated/infected cells as well as cART administration.In particular, small vesicles known as microparticles (MPs)secreted by various activated cell types, such as monocytes,often contain pro-inflammatory signals, which can contributeto neurodegenerative diseases. Increased inflammatory MPlevels have been reported in cART treated HIV-infected indi-viduals. Recent data from our lab suggests that the host re-striction factor Tetherin (BST-2/CD317) can sequester andinhibit the release of MPs from monocytes. Further, mono-cytes from HIV-infected individuals have reduced levels ofTetherin, and plasma samples from these individuals show


increased levels of monocyte-derived MPs. Exposure to theHIV viral protein, Tat, can lead to the down regulation ofsurface levels of Tetherin on monocytes and an increasedrelease of monocyte-derived MPs from healthy individ-uals. Further, MP sequestration is observed on the surfaceof monocytes that over express a degradation-resistantmutant of Tetherin, in which the sequence that is usedfor Vpu-mediated ubiquation is mutated. Additional datafrom mice harboring a degradation-resistant form ofTetherin shows decreased levels of MPs and increasedblood brain barrier function. Information gleaned fromthese studies will reveal the underlying mechanism ofTetherin-mediated MP release and its role in neurologicaldisease along with other co-morbidities evident in HIV-infected individuals. Furthermore, this work is importantin enhancing our understanding of how this novel molec-ular mechanism of Tetherin-dependent MP release relatesto HIV infected individuals, as well as other MP associ-ated diseases.

P167Effects of flaxseed lignin, Secoisolariciresinol diglucose,on endogenous antioxidant pathway in HIV-infectedmacrophages

Kimberly Williams, Andrea Guerrero, Shelley Pu, KellyJordan-Sciutto(corresponding author: [email protected])

University of Pennsylvania

Macrophages and microglia play pivotal roles in pathogen-esis HIV associated neurocognitive disorders. During acuteinfection, activated and/or infected monocytes infiltrate thecentral nervous system (CNS) and differentiate intoperivascular macrophages. HIV can then spread to microg-lia, creating an active, protected reservoir in the CNS. Theensuing inflammatory macrophage/microglia activation andsecretion of a myriad of toxic factors cause damage to neu-rons, which are not infected. Thus, current work strives todevelop strategies to suppress M/M-driven inflammation andoxidative stress. Numerous studies utilizing exogenous anti-inflammatory and antioxidants to mitigate disease progres-sion have been unsuccessful; however, targeting endogenousantioxidant pathways may be more advantageous. A cellularmechanism that mitigates oxidative stress and inflammationis the endogenous antioxidant response (EAR) pathway,which upregulates key antioxidant enzymes, including hemeoxygenase 1 (HO-1), to reduce free radicals and suppresstissue damage. In this study, we investigated the role of aflaxseed lignin, Secoisolariciresinol Diglucose (SDG), onviral replication and oxidative stress in HIV-infected

macrophages. To evaluate EAR, human monocyte derivedmacrophages were infected with HIV. By immunoblot, HIVsuppressed HO-1 levels, in macrophages, while concur-rent treatment with SDG increased HO-1 levels. Thiswas paralleled by increased translocation of nrf2 proteinfrom the cytoplasm to the nucleus and decreased nuclearexpression of HO-1’s negative regulator, Bach1.Prolonged HO-1 deficiency in macrophages has been neg-atively correlated with increased HIV replication in hu-man macrophages. HO-1 levels were decreased in macro-phages 12-day post infection. To determine if SDG canreverse prolonged HO-1 deficits, macrophages werepretreated with SDG for 1 h prior to infection andreplenished every 3 days. SDG treatment increased HO-1 protein levels after 12 days post infection. Given thesedata, SDG increases endogenous antioxidants pathwaysand may be a possible adjunctive therapeutic for HIVassociated neurocognitive disorders.

P168Insulin Receptor Secretion is not associatedwith abnormal glucose metabolism in HIV-seropositivewomen

Valerie Wojna1, Miriam Matos2, Rosa J. Rodriguez-Benitez2,Raissa Menendez-Delmestre2, Avindra Nath3, Yamil Gerena4


1Neurology Division, NeuroAIDS Program, University ofPuerto Rico, Medical Sciences Campus; 2NeuroAIDSProgram, University of Puerto Rico, Medical SciencesCampus; 3Section of Infections of the Nervous System,NINDS, NIH; 4Department of Pharmacology, NeuroAIDSProgram, University of Puerto Rico, Medical SciencesCampus

Background: Previously we found that high levels of solubleinsulin receptor (sIR) in the plasma of HIV-seropositive wom-en were associated with HIV-associated neurocogntivie disor-ders (HAND). Although we found evidence of cellular insulinresistance it is not clear if sIR is associated with peripheralinsulin resistance as determined by the homeostasis modelassessment-estimated insulin resistance (HOMA-IR) index.In this study we investigated if sIR is associated withHOMA-IR. Methods: 50 HIV-seropositive women and 18controls without history of diabetes were tested for oral glu-cose tolerance test (including fasting blood sugar [FBS] andinsulin levels), glycosylated hemoglobin (HgA1c), HOMA-IR, and plasma sIR full-length. Participants were also evalu-ated for metabolic syndrome with blood pressure, anthropo-metric measures, and lipid profile. HAND was determinedfollowing the HAND nosology criteria. Results: No


significant differences were observed between controls andHIV-seropositive women stratified by HAND (normal cogni-tion [18], asymptomatic neurocognitive impairment[ANI,13], and symptomatic neurocognitive impairment[SNI, 19]) regarding age, BMI, HgA1c, insulin andFBS, and HOMA-IR. An association was observed be-tween plasma sIR levels and HAND (p= 0.003), wherewomen with ANI presented with higher levels. NoSpearman’s correlation were observed between plasmasIR levels and age, BMI, HIV RNA copies/mL, CD4 cellcount, use of protease inhibitors, HGA1c, FBS, insulinlevels, and HOMA-IR. Conclusion: Plasma sIR is not as-sociated with standard measures of peripheral insulin re-sistance such as HOMA-IR. It is possible that the pres-ence of plasma sIR levels and its association with HANDis related to a chronic subclinical cellular insulin resis-tance. Supported by R21MH095524, R01NS099036,U54MD007587, G12MD007600

P169In vivo excision of HIV provirus by SaCas9 and multiplexsgRNAs in pre-clinical animal models

Chaoran Yin1, Ting Zhang1, Xiying Qu1, Yonggang Zhang1,Raj Putatunda1, Fang Li1, Huaqing Zhao1, Shen Dai1, XuebinQin1, Xianming Mo2, Jennifer Gordon1, Won-bin Young3,Kamel Khalili1, Wenhui Hu1


1Department of Neuroscience, Center for Neurovirology andThe Comprehensive NeuroAIDS Center, Temple UniversityLewis Katz School of Medicine; 2Laboratory of Stem CellBiology, State Key Laboratory of Biotherapy, West ChinaHospital, West China Medical School, Sichuan University;3Department of Radiology, University of Pittsburgh Schoolof Medicine, Pittsburgh

Cas9/gRNA-mediated genome editing provides a promisingcure for HIV-1/AIDS by excising the proviral DNA from hostgenome. Here, we demonstrate the feasibility of excising theHIV-1 proviral genome in experimental animal models usingsaCas9 along with 2–4 multiplex sgRNAs in a single AAVvector. Both duplex and quadruplex sgRNAs/saCas9 can beefficiently packaged into AAV-DJ or AAV-DJ/8 with high titerat 10e13-14 genome copy (GC)/ml. The quadruplex sgRNA/saCas9 vector outperformed the duplex one in cleaving theintegrated HIV-1 genome in cultured neural stem/progenitorcells and various organs of HIV Tg26 transgenic mice.Excision of HIV-1 proviral DNA by quadruplex AAV-DJ/8(i.v.) was demonstrated by PCR genotyping in liver, lung,brain and spleen of the mice inoculated (i.v.) with chimericEcoHIV carrying a firefly luciferase reporter (eLuc). Live

bioluminescence imaging showed a significant reduction ofEcoHIV infection in the neck lymph nodes and whole body.To demonstrate if Cas9/sgRNAs excise HIV provirus in la-tently infected cells in vivo, we administrated quadruplexsgRNA/saCas9 AAV-DJ/8 (2x10e12 GC/mouse, i.v.) inhumanized Bone marrow-Liver-Thymus (BLT) mice withintravaginal inoculation of HIV-1NL-BaL-eLuc. TheseHIV-infected BLT mice showed significant vaginal infec-tion during the first 3 weeks of inoculation but no observedsystemic HIV dissemination for at least 60 days prior toAAV/saCas9 treatment. Successful excision of HIV-1 pro-virus was detected by PCR genotyping in spleen, lung,heart, colon and brain as well as human thymic organoidon the left kidney as early as 2 weeks after AAV injection.In conclusion, in vivo excision of HIV proviral DNA insolid tissues/organs can be achieved via a single intrave-nous injection of AAV-DJ/8 carrying quadruplex sgRNA/saCas9. This is an important step moving forward to theclinical application of the CRISPR/Cas9 gene editing strat-egy. Supported by NIH grants to KK/WH (R01NS087971),KK (P30MH092177) and WY (R21MH100949).

P170Evidence that Dysfunctional Endolysosomal Biogenesis isAssociated with Cognitive Impairment in HIV-InfectedIndividuals; Agonists of TRPML1 May RestoreLysosomal Function Through Luminal Acidification

Seung-Wan Yoo1, Joshua Lisinicchia2, Jacqueline Lovett1,Benjamin B Gelman2, Norman J Haughey3


1Department of Neurology, Richard T Johnson Division ofNeuroimmunology and Neurological Infections, JohnsHopkins University School of Medicine, Baltimore,Maryland; 2Department of Pathology, Neuroscience and CellBiology, University of Texas Medical Branch Galveston,Texas; 3Department of Neurology, Richard T JohnsonDivision of Neuroimmunology and Neurological Infections,Johns Hopkins University School of Medicine, Baltimore,Maryland

Dysfunction of endolysosomal systems in HIV-AssociatedNeurocognitive Disorders (HAND) has been implicated by dis-turbances in transluminal ion gradients, and intraluminal depo-sition of proteins and lipids. Here, we determined if theCoordinated Lysosomal Expression And Regulation(CLEAR) gene network is impaired by HIV infection usingtissues from the National NeuroAIDS Tissue Consortium(NNTC; mini gauntlet sample set). Frontal neocortices wereobtained from HIV+ cognitively normal (CN; n = 41),subsyndromic (SS; n= 9), Minor Cognitive Motor Disorder


(MCMD; n= 36), HIV Associated Dementia (HAD; n= 29),and HIV- controls (n=68). All HIV infected subjects showedalterations in the expression of multiple genes coding for hy-drolytic enzymes and luminal acidification compared to HIV-.A master regulator of lysosomal biogenesis, transcription factorEB, was increased, and genes related to luminal acidification(chloride channel 7 and transient receptor potential mucolipin-1; TRPML1) were decreased inMCMD and HAD compared toCN. These data suggest that HIV infection is associated with adysregulation of the CLEAR network in neocortex, and cogni-tive impairment is specifically associated with the induction oflysosomal biogenesis and a reduced ability for luminal acidifi-cation. Based on these human data we next used a rodent modelof HIV-associated endolysosomal dysfunction (gp120/APP/PS1 mice) in which the CLEAR network is impaired and lyso-somes are engorged with Abeta and sphingomyelin to deter-mine if luminal acidification could rescue lysosomal function.A 26 day intraventricular infusion of the TRPML1 agonist ML-SA1 by mini osmotic pump reduced cortical Abeta,sphingomyelin, and restored CLEAR network gene expressiontoWt levels. These findings suggest that therapeutic approachesto enhance lysosomal function may protect the CNS in thesetting of HIV infection.

P171Drug Use and Age Effects on White MatterMicrostructure and Behavior in Treated HIV Infection

Thomas Zeffiro1, Erin O’Connor2, Melissa Murray3,Lawrence Kingsley3, James Becker3


1Temple University; 2Lewis Katz School of Medicine atTemple University; 3University of Pittsburgh

Purpose: Although diffusion tensor imaging (DTI) studieshave documented white matter (WM) microarchitecturechanges following HIV infection, WM properties are also

known to change with age and drug use. As the HIVinfected population is steadily aging, the effects of ageand past drug use may confound attempts to identifyspecific effects of serostatus on WM microstructure. Ina cross-sectional study, we examined the effects ofserostatus, age and past drug use on WM microstruc-ture. Methods: Participants included 15 seropositive and20 seronegative men, ages 54–77, with 12 reportingpast drug use. Seropositive participants were all treatedwith anti-retroviral therapies. Mixed effects models wereused to explore effects of serostatus, race, age, and druguse on executive function, learning/memory, attention/working memory, verbal fluency, processing speed, andmotor performance domains. WM microstructure mea-sures included fractional anisotropy (FA) and apparentdiffusion coefficient (ADC). Results: Seropositive partic-ipants exhibited bihemispheric increases in ADC(p < 0.05 FWE-corrected) and FA (p < 0.05 FWE-corrected). Independent effects of age and past druguse on FA and ADC were seen (p < 0.05 FWE-corrected). Processing speed decreased with age(p < 0.01). Motor performance decreased with age,showing effects of race and an interaction betweenserostatus and age (all p < 0.01). Executive function,verbal fluency, attention/working memory, and learning/memory did not show effects of serostatus, age, race ordrug use. Conclusions: In a sample of cognitively intactolder participants, we observed separate effects ofserostatus, age and past drug use on two measures ofWM microstructure and associated slowing of move-ment execution. The relatively wide variation in pub-lished studies of HIV infection effects on white-mattermicrostructure might reflect differences among experi-mental samples related to age or past drug use.Moreover, WM microstructural changes following HIVinfection, while associated with slow motor perfor-mance, are not invariably associated with cognitivechanges assessed across multiple domains.


Author Index

Abrams, Rachel, P1Abimiku, Alash"le, P69Achim L., Cristian, P25Achim, Cristian, P18, P42Agsalda-Garcia, Melissa, P120, P129Ahooye, Taha, P140Aiamkitsumrit, Benjamas, P154, P7,

P30, P31, P138, P125, P109Akay Espinoza, Cagla, P2Akinwumi, Michael, P133Akkina, Ramesh, P47Al-Harthi, Lena, P93, P117Albe, Joseph, P3Alldred, J., Melissa, P46Allen, Alex, P40Allen, Alexander, P4Allen, Isabel, P111Alonso, Sylvie, P164Alt, Jesse, P118Amancha, Praveen Kumar, P52Amini, Shohreh, P38, P140Amor, Sandra, P124Anastos, Kathryn, P161Anderson, Monique, P5Anderson, Brian, P101Andrews, Allison, P137Andríçs E., Ibolya, P6Ansari, Aftab, P33Antell, Gregory, P7, P31, P125, P109Arainga, Mariluz, P153Arancio, Ottavio, P76Asahchop, Eugene, P8, P133, P97Atkins, Andrew, P9

Bachani, Muzna, P43, P58, P89Badani, Hussain, P68Bade, Aditya, P49Baker, Glen , P8Balashov, Sergey, P9Balinang, Joyce, P10Balinang, M., Joyce, P122Banerjee, Anupam , P11Barasky, Rebecca, P55Barbour, J., Aaron, P99Barclay, Robert, P12, P58Basova, Liana, P77Beck, Sarah, P132, P98Becker, James, P171Becker, T., James, P112Beckham, J David, P102

Bella, Ramona, P72Bella, Ramona, P73Bellizzi, Anna, P13, P14, P15Bendayan, Reina, P121Berman, Joan, P161Berman, W., Joan, P65Bertrand, Luc, P6Bevins, Rick, P123Beyaert, Rudi, P82BHARGAVAN, BIJU, P16Bianchet, Mario, P1, P127Billioux, Bridgette, P86Bimonte-Nelson, Heather, P158Birmingham, Amanda, P77Blackmon, Anna, P68Blakey, Brandon, P154Blattner, William, P69Booze, Rosemarie, P108Booze, M., Rosemarie, P34, P72, P94,

P135Borjabad, Alejandra, P76, P17Bouaziz, Serge, P26Boukli, Nawal, P92Branton, William, P8, P97Brew, Bruce, P50, P157Bryant, Alex, P18Buch, Shilpa, P19, P156, P123Budzinski, Allison, P55Burdo, Tricia, P18, P69Burke, Deirdre, P115Byrareddy, Siddappa, P33, P52Byrd, Desiree, P161

Cai, Yu, P19Cantres-Rosario, Yisel, P20Caocci, Maurizio, P160, P159Caruso, Breanna, P47, P86Carvallo, Loreto, P65Cervantes-Arslanian, Anna, P115Chalkias, Spyros, P21Chalkias, Spyridon, P115Chambers, Christina, P28Chan, Wing, P8, P97Chang, L., Sulie, P36, P91Chao, Wei, P17, P76Chapenko, Svetlana, P148Charlins, Paige, P47Charurat, Man, P69Charvet, Benjamin, P22Chaudhuri, Amrita Datta, P32

Chauhan, Priyanka, P23Chauhan, Priyanka, P128Chen, Xuesong, P60Chen, Yilan, P71, P73Chen, Xuesong, P79, P80Cheng, Wan-Jung, P165Cheng, Yu, P112Cherner, Mariana, P69, P74Cheung, Joseph, P54Chew, M., Glen, P47Chiu, Y., Charles, P59Chow, Vincent, P164,Chow, Dominic, P105Chua, Beng Hooi, P164Chua, Kaw Bing, P164Chung, Cheng-Han (James), P24Churchill, J., Melissa, P165Ciavatta, Vincent, P158Cibrowski, Pawal, P56Cicalese, Stephanie, P29, P140Clarke, Penny, P59Clements, Janice, P132Clotet, Bonaventura, P114, P113Coban, Hamza, P25, P42Cohen, Eric, P97Colí_n-Cruz, Luis, P45Colon, Krystal, P134Coric, Pascale, P26Cotto, Bianca, P27, P28Craigie, Michael, P28, P35Cruz, Michael, P31Cubano, Luis, P92Cysique, Lucette, P50, P157

D'Aiuto, Leonard, P83D'Antoni, Michelle, P129Dai, Shen, P75, P91, P169Dampier, William, P4, P154, P7, P9,

P24, P30, P31, P125, P109Dang, Xin, P21Daniel, C., Dianne, P67Dash, Prasanta, P49, P153Dastgheyb, Raha, P32Datta, Prasun, P27, P38, P145Dave, S., Rajnish, P33Dawson, Matthew, P55Dayton, K., Iris, P34De Simone, Francesca Isabella, P35, P36Dean, Mathew, P85Deckard, Sadie, P124


Deleage, Claire, P33Delgado-Nieves, Andrea, P45DelValle, Luis, P85DeMarino, Catherine, P12Denton, D., Melissa, P99DePalma, Steve, P21DePaula-Silva, Ana Beatriz, P37Dermody, Nadene, P50Deshmane, Satish, P27, P38Desimone, Matthew, P4Desplats, Paula, P25, P42Devlin, Kathryn, P31Dickens, Alex, P56, P81Diez-Quevedo, Crisanto, P113Dimitriadis, Emilios, P127Dobrota, Mary-Ann, P146Doh, Roland, P74Dolei, Antonina, P160, P159Donadoni, Martina, P29Dorskind, Joelle, P32Doty, Daniel, P37Doucet-O'Hare, Tara, P39Dougier, Hei-Lanne , P22Douglas, D., Steven, P150Dulcis, Davide, P77Dutta, Ranjan, P124

Earl, Joshua, P9, P30Edagwa, Benson, P49, P153Egan, Kevin, P40Ehrlich, Garth, P31El-Hage, Nazira, P12El-Hage, Nazira, P138El-Kamary, Samer, P69Ellis, Ronald, P55, P18Ellis, J., Ronald, P63Elrod, John, P27Endsley, Janice, P44Engle, Elizabeth, P98Enose-Akahata, Yoshimi, P5Estes, D., Jacob, P33, P165Eugenin, Eliseo, P162Eyzaguirre, Lindsay, P69

Fang, Yiding, P77Fantetti, N., Kristen, P84Farley, Kalamo, P138Fassnacht, Ryan, P138Fatima, Mahar, P41Feldman, Arthur, P54Feng, Rui, P154, P103Ferrante, Pasquale, P72, P73Ferrer, J., Maria, P114, P113

Fields A., Jerel, P25Fields, Jerel, P42Filipowicz, Adam, P90Fischer, Tracy, P46, P71Fitting, Sylvia, P99Flores, Ilse, P25, P42For the Neuropsychology Working

Group of the MACS, P112Fox, Howard, P56Franklin, Donald, P56Frieman, Amy, P95Fry, Kelsi, P61Fujinami, Robert, P37Fumaz, R., Carmina, P113

Gabuzda, Dana, P52, P115Gama, Lucio, P161, P132Gannon, Patrick, P151Gao, Linlin, P64Garcia-Contreras, Marta, P6Garolera, Maite, P114, P113Gates, Thomas, P50Geiger, Nicholas, P43Geiger, Jonathan, P60Geiger, D., Jonathan, P79, P80Gelbard, Harris, P49Gelman, Benjamin, P44, P151, P133Gelman, B., Benjamin, P170Gendelman, Howard, P49, P153Gendelman, E., Howard, P72Gerena, Yamil, P45, P168, P126Gilden, Don, P68, P95Gill, John, P8Ginsberg, D., Stephen, P46Ginwala, Rashida, P33, P47, P48, P64,

P137Giovannetti, Tania, P31Gnanadhas, Divya Prakash, P49Godfrey, W., Earl, P67Gonzalez, Claribel, P110Gonzalez, Raul, P100Gonzíçlez-Escalante, Anibal, P45Gorantla, Santhi, P49, P153, P147Gordon, Jennifer, P14, P35, P54, P72,

P75, P91, P169, P143Gorham, Joshua, P21Gorry, R., Paul, P165Gott, Chloe, P50Gouaux, Ben, P18Gough, Sarah, P25, P42Grant, Igor, P18, P56, P63, P81Gray, R., Lachlan, P165Gray, RonaldWawer, Maria, P144

Groma, Valerija, P148Gruenewald, Analise, P51Gu, Chao-Jiang, P17, P76Gu, Chaojiang, P65Guerrero, Andrea, P167Gumber, Sanjeev, P52Gunnam, M., Satya, P46Guo, Jig, P53Gupta, Manish, P54Guy, Joseph, P86

Ha, Seung-Kwon, P86Hacker, Lindsay, P95Haddad, Elias, P33Haile, Woldeab, P158Hakami, Ramin, P12Hampson, David, P121Hanak, Tyler, P37Hanks, Nancy, P120Harhaj, Edward, P64Harrod, B., Steven, P135Hartman L., Amy, P3Hartman, Amy, P83Hashemi, Parastoo, P135Hassanzadeh, Shiva, P55Hategan, Alina Popescu, P127Haughey, Norman, P32, P56, P81Haughey, J., Norman, P170Hauser, Kurt, P10, P87, P138, P107Hauser, F., Kurt, P122, P99He, Lifan, P15He, Hongxia, P76Heaton, Robert, P74Hellmuth, Joanna, P111Henderson, Lisa, P57, P58Herman, Susan, P146Hershberg, Uri, P125, P109Hidalgo, Melissa, P106, P105Hixon M., Alison, P59Ho, Winson, P39Hoefer, Melanie, P155Hongyin, Wang, P17Hoque, Tozammel, P121Horvat, Branka, P22Horvath, Stefan, P131Hu, Wenhui, P14, P149, P169, P71,

P72, P73Hu, Guoku, P19Hu, Shuxian, P128, P23Huang, Xiaofang, P137Hui, Liang, P60Hungerford, Laura, P69Hunter, Meredith, P95


Ibba, Gabriele, P13, P14, P15, P160, P159Inglese, Matilde, P161Iordanskiy, Sergey, P12, P61, P62Iudicello, Jennifer, P63

Jackson, Joe, P166Jacobson, Steven, P86, P5Jacobson, Jeffrey, P7, P9, P30, P31,

P125, P105Jacobson, Steven, P47Jacobson, Lisa, P112Jaillard, Assia, P119Jain, Pooja, P33, P47, P48, P64, P137James, Tony, P7, P31Jancarik, Andrej, P118Jaureguiberry-Bravo, Matias, P65Javandel, Shireen, P111Jeffrey, Jacobson, P154Jennifer, Kelschenbach, P17Jennings, Stephen, P40Ji, Haiyan, P66Jiang, Xiong, P55Johnson, Kory, P39Johnson, Tory, P58Johnson, M., Edward, P67Jones, Dallas, P68Jones, Simon, P157Jordan-Sciutto, Kelly, P167, P151Julius, Fonsah, P74Jumare, Jibreel, P69

Kallianpur, Kalpana, P70Kallianpur, Kalpana, P105Kaminski, Rafal, P14, P54, P71, P72,

P73Kanmogne, Georgette, P74KANMOGNE, GEORGETTE, P16Karn, Jonathan, P71, P73, P138Karnaukhova, Elena, P127Kashanchi, Fatah, P5, P12, P58, P61,

P62, P138Kaul, Marcus, P155, P139, P104Kearns, Alison, P75, P91Keating, Sheila, P136Kelschenbach, Jennifer, P65, P76, P118Kengne, Anne Marie, P74Kercher, Katie, P4, P109Kercher, Katherine, P7, P9, P30, P31,

P125, P103 ,Kesby, P., James, P77Kettlewell, Joanna, P78Kettlewell, Robert, P120Keutmann, Michael, P100

Khalili, Kamel, P13, P14, P54, P71,P72, P73, P15, P169, P140

Khan, Nabab, P79, P80Khan, Zafar, P64Khan, K., Zafar, P33, P47, P48, P137Khuder, Saja, P32, P81Kiernan, Elizabeth, P75Kim, Boe-Hyun, P17, P118, P76Kim, Irene, P51Kim, SooAh, P81Kim, Woong-Ki, P87, P90Kingsley, Lawrence, P171, P112Kip, Elodie, P82Knapp, Pamela, P10, P87, P107Knapp, E., Pamela, P122, P99Kolson, Dennis, P44, P51, P151Koneru, Rajeth, P158Kook, YeonHee, P19Koralnik, Igor, P21, P115Koralnik, J., Igor, P146Krebs, Fred, P7, P11, P31, P125Kremer, David, P124Kuate, Callixte, P74Kucheryavykh, Lilia, P92Kujawa, Michael, P3, P83Kulkarni, Apurva, P84Kumar, Princy, P55Kumar, Anupama, P144Kuroda, Marcelo, P90Kury, Patrick, P124

Lakpa, Leo, P60Lakritz, Jessica, P18Lamberty, Benjamin, P56Langford, Dianne, P27, P28Lassak, Adam, P85Lavorgna, Alfonso, P64Leda, Ana, P6Lee, Nathanael, P86Lee, MyoungHwa, P88, P89Leibovitch, Emily, P86Leibrand, Crystal, P87Lepene, Ben, P5Lepene, Benjamin, P12, P62Leser, J. Smith, P59Letendre, Scott, P81, P56, P58, P63Levine, Andrew, P18, P51, P131, P112Lewin, R., Sharon, P165Lewis, G., Mark, P163Li, Fang, P169Li, Guan-Han, P88Li, Hailong, P135, P72Li, Hongbo, P28

Li, Luna, P11Li, Michael, P133Li, Wenxue, P43, P1, P57, P89Li, Xiaozhen, P86Libbey, Jane, P37Libon, David, P31Lin, Ping, P2Lin, Zhiyi, P49Lin, Wei, P103Lindgren, Allison, P90Lisco, Steven, P123Lisinicchia, Joshua, P44, P170Liu, Yucheng, P7Liu, Fengming, P75, P91Lobo, Judith, P106Lokensgard, James, P23Lokensgard, James R, P128Loonawat, Ronak, P47Lopez-Acevedo, Sheila, P92Lopez-Masramon, Estela, P114Lorenz, David, P115Lovelace, Michael, P157Lovett, Jacqueline, P170Luciano, Nick, P86Lum, Corey, P105Luongo, Timothy, P27Lutgen, Victoria, P93, P117Lyons, Jennifer, P115

Ma, Qing, P63Mactutus, Charles, P108Mactutus, F., Charles, P34, P94, P135Madesh, Muniswamy, P54Magder, Laurence, P69Maggirwar, Sanjay, P166Maggirwar, B., Sanjay, P147Magnus, Manya, P55Magnus, Lisa, P132Mahalingam, Ravi, P95Majer, Pavel, P118Major, Eugene, P88Maki, Pauline, P136, P100Malcolm, Thomas, P73Malik, Nasir, P43Mallard, Jaclyn, P96Mamik, Manmeet, P8, P97Mandel, Jordan, P101Mangus, Lisa, P98Mankowski, Joseph, P132, P98Marche, Patrice, P22Marcondes, G., Maria Cecilia, P77Marcotte, Thomas, P56, P81Marks, William, P107


Marks, D., William, P99Maroun, Leonard, P158Marshall-Freites, Valerie, P45Martin, Eileen, P112, P100Martinez-Maza, Otoniel, P131Marvel, Cherie, P101Masliah, Eliezer, P18, P25, P42Massey, Aaron, P102Matos, Miriam, P168, P110Maubert, Monique, P103Maung, Ricky, P155, P139, P104Mazaika, Erica, P21Mbanya, Dora, P74McArthur, Justin, P56, P81McCutchan, J. Allen , P63McIntosh, Roger, P106, P105McKean, David, P21Mckenzie, Brienne, P97McLane, Virginia, P107McLaurin, Kristen, P108McLaurin, A., Kristen, P34, P94McMillan, JoEllyn, P49, P153McQuiston, A Rory, P99McRae, MaryPeace, P87McTish, Emily, P48Medina, Julie, P22Medynets, Marie, P39Mele, Anthony, P109Melendez, Loyda, P20, P134Melendez, Loyda, P134Mell, Joshua, P9, P30Menendez-Delmestre, Raissa, P168, P110Mení©ndez-Delmestre, Raissa, P45Mielke, Michelle, P56Milanini, Benedetta, P111Miranda, Cristina, P113Miskin, P., Dhanashri, P146Misra, Vikas, P115Mitchell, Brooks, P70Mo, Xianming, P169Mohsen, Mohamed Abdel, P129Moldover, Brian, P154Molsberry, A., Samantha, P112Molto, Jose, P113Monaco-Kushner, Maria Chiara, P5Moore, David, P18, P55Morales, Fabian Sierra, P146Morgello, Susan, P17, P161, P115Muir, R., Roshell, P33Mukerji, Shibani, P115Mullen, Brian, P54Munoz-Moreno, A., Jose, P114, P113Munro, Cynthia, P112

Murovska, Modra, P148Murray, Jacinta, P17Murray, Melissa, P171

Nagarkatti, Mitzi, P48, P137Nagarkatti, Prakash, P137, P48Nagel, Maria, P68Naibryf, Tali, P106Nair, Madhavan, P116Najera, A., Julia, P77Nakamura, Christie, P78, P120Nakasujja, Noeline, P144Nakigozi, Gertrude, P144Nambiar, Sreesha, P47Napoli, Alessandro, P71Narasipura, Srinivasa, P93, P117Nath, Avindra, P1, P39, P43, P45, P57,

P58, P88, P89, P168, P127Ndembi, Nicaise, P69Ndhlovu, Lishomwa, P70, P129Ndhlovu, C., Lishomwa, P47Nedelcovych, Michael, P118Neff, C. Preston, P68Nehra, P., Artinder, P33Neumann, Konstantin, P137Ng, Qimei, P164Njamnshi, Dora, P74Njamnshi, Alfred, P74Noel, Richard, P110Noggle, Arianna, P96Nolan, J., David, P96Nonnemacher,Michael, P154, P4, P7, P9,

P11, P24, P30, P31, P125, P109, P103

O'Connor, Erin, P171, P119O'Donnell, A., Lauren, P84O’Neill, Alan, P155Oda, Robert, P78, P120Ojeda, Daniel, P104Omeragic, Amila, P121Otte, Jessica, P72, P143Oury, Tim, P83

Palmer, Brent, P68Papazian, Emily, P96Pardo-Villamizar, Carlos, P144Parfenov, Michael, P21Paris, Jason, P87Paris, J., Jason, P122, P99Park, Minseon, P6Passic, Shendra, P4, P7, P9, P30, P31,P154, P125, P109Paul, Robert, P111

Pendyala, Gurudutt, P123Peng, Xiao, P75, P91Perez-Alvarez, Nuria, P114, P113Periyasamy, Palsamy, P156Perron, Herve, P22, P124Peruzzi, Francesca, P85Phan, Luan, P136Philip, Ramila, P137Pillai, Satish, P129Pirrone Vanessa, P154, P4, P7, P9, P11,

P30, P31, P125, P109, P103Piu, Claudia, P160, P159Plaud, Marines, P20Plaud/Valentin, Marines, P126Pleet, Michelle, P12Poluektova, Larisa, P153Poluektova, Y., Larisa, P147Potash, Mary Jane, P17, P76Power, Christopher, P8, P133, P97Prajapati, Bharat, P41Prasad, Sujata, P23, P128Prats, Anna, P114, P113Premeaux, Thomas, P129Pu, Shelley, P167Putatunda, Raj, P169

Qin, Xuebin, P75, P91, P169Qiu, Fang, P74Qu, Xiying, P66, P169, P130Quach, Austin, P131Queen, Suzanne, P132, P98

Rae, Caroline, P157Ragin, Ann, P112Raguenez, Gilda, P22Rais, Rana, P118Rajan, Neha, P106Raman, Chander, P48Rappaport, Jay, P163Rauw, Gail, P8Ravina, Kristine, P148Reich, Daniel, P86Reiss, Krzysztof, P85Remirez, Servio, P137Renard, Felix, P119Resch, Lothar, P8Revuri, Nikil, P48Reynaud, Josephine, P22Richards, Maureen, P93Richards, Maureen, P117Rife, Brittany, P96Rivera-Morales, Paola, P45Rivera-Serrano, Mariela, P92


Robertson, Kevin, P144Rockenstein, Edward, P25Roda, Weston, P133Rodrí_guez-Benitez, Rosa, P110Rodriguez-Benitez, J., Rosa P168Rodriguez-Valentin, Madeline, P92Roehm C., Pamela, P15Roga, Silvija, P148Romerio, Fabio, P61Romoli, Benedetto, P77Rosario, Lester, P134Roscoe Jr., F., Robert, P135Roure, Karla Negron, P20Royal III, Walter, P69Rubin, Leah, P136, P100Ruland, Jurgen, P137

Sacktor, Ned, P56, P58, P81, P112, P101Safak, Mahmut, P26, P142, P141Sagar, Divya, P33, P48, P64, P137Sahu, Geetaram, P138Saifuddin, Mohammed, P61Saleem, Kanza, P41Salemi, Marco, P96Salkind, Julian, P15, P73Samaranayake, Srimal, P135Sampey, Gavin, P12, P61Sanchez, Ana, P139, P104Saredy, Jason, P26, P142, P141Saribas, A. Sami, P26, P142, P141Saribas, Sami, P140Sariyer, Ilker, P29, P140, P143, P35, P36Sariyer, Rahsan, P143, P35, P36Sati, Pascal, P86Saylor, Deanna, P144Schier, J., Christina, P99Schindler, Matthew, P86Schwab, Angela, P12Schwartz, Gregory, P125Scully, J., Taylor, P84Sehgal, Mohit, P64Seidman, Jonathan, P21Semenova, Svetlana, P77Serra, Caterina, P160, P159Serra, Caterina, P159Seth, Pankaj, P41Shah, Sonia, P154Shapshak, Paul, P131Shekarabi, Masoud, P15, P145Sheng, Wen, P23, P128Shikuma, Cecilia, P70, P129, P120, P105Shiramizu, Bruce, P161, P129, P120Shives, Katherine, P102

Sillman, Brady, P49Silva, Afonso, P86Sim, Jordan, P37Simon, Gary, P138Singal, Chitra, P41Singer, Elyse, P131Singh, Lalita, P44Singh, Narendra, P48Singh, Meera, P166Singh, Vir, P166Singh, B., Vir, P147Singh, P., Narendra, P137Singh, V., Meera, P147Skolasky, Richard, P126Skowronska, Marta, P6Skuja, Sandra, P148Slusher, Barbara, P118Song, Jiasheng, P149Soontornniyomkij, Virawudh, P18Spitsin, Sergei, P150Staal, Jens, P82Steiner, Joseph, P43, P58, P89, P127Stern, Anna, P151Stirpe, Nicole, P166Stoff, David, P152Strazza, Marianne, P103Su, Hang, P153Suin, Vanessa, P82Sullivan, Neil, P154Szep, Zsofia, P7, P9, P30, P31, P154, P109

Tagny, Claude, P74Tanaka, Yuetsu, P5Tedaldi, Ellen, P71Tenora, Lukas, P118Teteris, Ojars, P148Thaney, Victoria, P155Thangaraj, Annadurai, P156Tharakan, Ravi, P56Thompson, Tiffany , P3TMARC Group, P139Tobia, Michael, P157Toborek, Michal, P6Traina-Dorge, Vicki, P95Trapp, Bruce, P124Tuma, Ronald, P28Tyagi, Richa, P43, P57, P58, P89, P1Tyagi, Mudit, P138Tyler, L., Kenneth, P59Tyor, William, P158

Ubaida-Mohien, Ceereena, P56Uleri, Elena, P160, P159

Umlauf, Anya, P69

Valcour, Victor, P111Valentin, Gabriel, P92Van Duyne, Rachel, P61Van Horssen, Jack, P124VanGucht, Steven, P82Vatakis, Dimitrios, P51, P131Veenstra, Mike, P161Veilleux, Courtney, P162Velasquez, Stephani, P163Vellucci, Ashley, P5Venna, Nagagopal, P115Victorio, Carla Bianca Luena, P164Villinger, Francois, P33, P52Vipulkumar, Patel, P44Volsky J., David, P17Volsky, David, P65, P76, P118

Wagner, Wendeline, P163Walters, Aaron, P83Wang, Sheng, P32Wang, Tongguang, P39Wang, Cuiwei, P55Wanicek, L., Emma, P165Ward, Sara Jane, P28Ward, Jennifer, P158Weber, Emily, P166Weber, Kathleen, P136Wellish, Mary, P95White K., Martyn, P13, P14, P15White S., Martyn, P26White, Martyn, P142, P141White, G., Alexander, P149Wigdahl, Brian, P4, P7, P9, P11, P24,

P30, P154, P31, P33, P40, P125, P109,P103

Wilk, Anna, P85Williams, Jean, P4, P109, P125, P154,

P30, P31, P7, P9Williams, Kimberly, P167Williams, C., Kenneth, P96Witt, Mallory, P131Witwer, Kenneth, P133Wojna, Valerie, P45, P168, P126, P110Wollebo S., Hassen, P13, P14, P15Woodson, Caitlin, P61Woollard, Shawna, P52Wu, Yuntao, P53Wurcel, Alysse, P115Wyczechowska, Dorota, P85

Xu, Yishi, P164


Yagi , Shigeo, P59Yang, Lu, P19Yarandi, Shadan, P35Yen, William, P27, P28Yen, Cecil, P86Yen, Po-Jen, P52Yin, Chaoran, P72, P169Yoo, Seung-Wan, P32, P170Young, Mary, P55

Young, Won-Bin, P66, P73, P169,P149, P130

Yu, Guixia, P59Yu, Fei, P149

Zapata, Adriana, P85Zeffiro, Thomas, P171, P119Zhang, Yonggang, P71, P149, P169Zhang, Ting, P169

Zhao, Huaqing, P169Zhong, Wen, P7, P9, P30, P31, P154,

P125, P109Zhu, Huanzhang, P66Zhu, Yu, P97Zhuang, Zhengping, P39Zieda, Anete, P148Zou, ShiPing, P122


Abstracts from the 14th International Symposium on ...

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