Poster Presentations

Journal of NeuroVirology, 13(suppl. 1): 59–135, 2007c© 2007 Journal of NeuroVirologyISSN: 1355-0284 print / 1538-2443 onlineDOI: 10.1080/13550280701739265

Poster Presentations

P.1Identification of a cell entry receptor for wild typemeasles virus on human neuronal cellsHaniah Abdullah, Philip Earle, and S Louise CosbyQueens University Belfast, Division of Infection andImmunity, CCRCB, Microbiology Building, Grosvenor Road,Belfast, BT12 6BN, UK

Subacute sclerosing panencephalitis and measles in-clusion body encephalitis are two neurological com-plications of measles virus (MV) infection. Neuronsand oligodendrocytes are infected and to a lesserextent astrocytes in both conditions. However, themolecules which allow virus entry into these cell typesare unknown. The known cell entry receptor for bothvaccine and wild type (WT) MV strains, signallinglymphocyte activation molecule (SLAM or CDw150)is specific to T and B cells, monocytes as well as acti-vated dendritic cells. Although human cofactor CD46has been shown to be expressed at a low level on asubset of neurons, on oligodendrocytes and astrocytesit is not a receptor for wild type (WT) MV strains. Thisindicates that another receptor(s) is present on neu-ral cells and is required for WT virus infection. TheNT2 cell line, derived from a human teratocarcinoma,exhibits properties characteristic of a committed hu-man neuronal precursor at an early stage of devel-opment and can be differentiated to produce mature,CD46 negative neurons (NT2-N) with retinoic acid. Wehave found that these neuronal cells as well as the hu-man epithelial cell derived neuroblastoma cell line,SHSY5Y, which is also CD46 negative, can be infectedwith the WT Dublin strain of MV. Both cell lines ex-press well characterised neuronal markers and there-fore provide suitable systems to investigate cell entryreceptor(s) for WT MV. Using a combined novel ap-proach we have prepared an antibody library againstSHSY5Y cells and have selected a monoclonal anti-body (Mab) which blocks infection of these cells byWT MV. The putative receptor which this antibodyrecognises on SHSY5Y cells is currently being charac-terised. In addition, the effect of the Mab on WT MVinfection in NT2-N cells and other human cell typesas well as on veterinary morbillivirus infection in ap-propriate cell lines is being investigated.

P.2Infection of human neuronal cell lines by wild type,rodent adapted and vaccine strains of measles virus

Haniah Abdullah and S. Louise CosbyQueen’s University Belfast, Division of Infection andImmunity, CCRCB, Microbiology Building, Grosvenor RoadBelfast, UK

Measles virus (MV) is a highly contagious humanpathogen and the etiological agent of subacute scle-rosing panencephalitis (SSPE) and measles inclusionbody encephalitis. Post mortem examination of SSPEbrain tissue demonstrated that MV antigen and RNAwere present in neurons and neuronal processes aswell as oligodendrocytes, microglia and to a lesser de-gree in astrocytes and endothelial cells. Although hu-man cofactor CD46 has been shown to be expressedat a low level on a subset of neurons it is a receptorfor vaccine but not wild type (WT) MV strains. Fur-thermore, signalling lymphocyte activation molecule(SLAM) which has been identified as a receptor forboth vaccine and WT strains of MV is not detected onCNS cells. To examine virus entry in human neuronalcells we used the WT Dublin-3267 MV strain to in-oculate the human neuroblastoma cell line SHSY5Yas well as NT2 cells during different stages of differ-entiation. The latter are derived from a human tera-tocarcinoma and exhibit properties characteristic ofa committed human neuronal precursor at an earlystage of development and can be differentiated usingretinoic acid to mature neurons. WT MV was capa-ble of infecting SHSY5Y cells and fully differentiatedNT2 neuronal cells, both of which were CD46 negativeand MAP-2 positive. However, unlike vaccine and ro-dent brain adapted strains of MV the WT virus did notinfect NT2 cells which were CD46 positive and MAP-2 negative during the early stages of differentiation.The results therefore suggest that an unknown MV re-ceptor(s) is present on mature human neuronal cells,which allows WT MV infection.

P.3Spleen necrosis virus (SNV) based vectors couldefficiently transfer small inhibitory RNA (siRNA)against the chemokine receptor CCR5 into thehuman brain cells to block HIV-1 entryEdward A Acheampong,1,2,3 Elton Kong,1,2,3

Mohammed A Khan,1,2,3 Roger J Pomerantz,4,5,6

Muhammad Mukhtar,7,8,9 and Zahida Parveen1,2,3

1Thomas Jefferson University, 2Dept. of Medicine, Divisionof Infectious Diseases, 3Philadelphia, PA 19107; 4TibotecInc.; 51020 Stony Hill Road, Suite 300; 6Yardley,Pennsylvania 19074; 7Drexel University College

8th International Symposium on NeuroVirology60 Poster Presentations

of Medicine, 8Department of Microbiology andImmunology, Institute for Molecular Medicine andInfectious Disease, 9Philadelphia, Pennsylvania 19102

Human immunodeficiency virus type 1 (HIV-1) as-sociated dementia (HAD) remains a major problemamong HIV-1-infected individuals even in the era ofhighly active antiretroviral therapy (HAART). Lim-ited penetration of antiretrovirals through the blood-brain barrier (BBB), a protective sheath of the brainis considered a major obstacle in overcoming resid-ual HIV-1 in the brain. The BBB constituted by brainmicrovascular endothelial cells (MVECs), besides pro-tecting the brain is also conduit for viral entry intothe central nervous system (CNS). These cells are de-void of CD4, a major receptor for HIV-1, however,they express various chemokine receptors such asCCR5, CXCR4 and CCR3. We hypothesized that down-modulation of CCR5 expression could reduce the pas-sage of viral entry into the brain MVECs. Based on thishypothesis, we used small inhibitory RNA (siRNA)to reduce the expression of CCR5 on human brainMVECs followed by their infectivity with various lab-oratory adapted and primary isolates of HIV-1. Aseries of spleen necrosis virus (SNV)-based retrovi-ral transfer vectors capable of expressing short hair-pin RNA (shRNA) against the chemokine receptorsCCR5 were constructed. For positive controls, lentivi-ral based vector pCMV-GIN-Neo, expressing micro-RNA adapted shRNA against CCR5 (shRNAmir) andPromega Corporation’s GeneClipTM U1 Hairpin Sys-tem expressing shRNA against CCR5 were used. Theability of these constructs to down-modulate the ex-pression of CCR5 was tested in primary isolated hu-man brain MVECS. The results showed that all thethree constructs are capable of reducing CCR5 expres-sion up to 80% of innate levels, while cells trans-fected with control vector maintained normal lev-els of CCR5. To determine if down regulation ofCCR5 co-receptor inhibits viral replication, humanbrain MVECs transfected with siRNA were challengedwith R5-tropic (ADA) strains of HIV-1. Substantialreduction in viral infection as determined by HIVp24 ELISA and Real Time PCR analyses of the HIV-1 gag copy numbers was seen in siRNA transfectedMVECs challenged with R5-tropic HIV-1 when com-pared with control cells. Besides the primary humanbrain MVECS, we also tested these constructs in as-trocytes, CD14+ monocyte derived macrophages andperipheral blood mononuclear cells (PBMCs) fromHIV-1-seronegative donors. Our preliminary data sug-gest that shRNA against CCR5 delivered through SNV-based vectors are highly selective and inhibits the ex-pression of CCR5 co-receptor in these primary cellswhich is further associated with protection from HIV-1 infection. The results of this study suggest thatdelivery of siRNA into the human brain by SNVbased vectors could be used in the design of ther-apeutics against HIV-1-associated neurodegenerativedisorders.

P.4Dual lentivirus infection potentiatesneuroinflammation and neurodegenerationAmir Afkhami-Goli,1 Shu-Hong Liu,2 Yu Zhu,1Joseph M. Antony,1 Hosseinali Arab,3 ChristopherPower1

1Department of Medicine, University of Alberta;2Department of Clinical Neurosciences, University ofCalgary; 3Department of Pharmacology, Faculty ofVeterinary Medicine, University of Tehran

Objective: Herein we investigated the neu-ropathogenic outcomes and underlying mechanismsof dual lentivirus infections with genetically distinctviral strains as co-infections or super-infections.

Design: Experimental Feline ImmunodeficiencyVirus (FIV) infections were performed using culturedfeline cells and an in vivo model of AIDS neuropatho-genesis.

Methods: Dual infections were carried out withtwo FIV strains (FIV-Ch and FIV-PPR) as co-passagedor superinfected viruses ex vivo in feline monocyte-derived-macrophages and in vivo in an establishedfeline model with subsequent analyses of outcomesusing real time RT-PCR, neuropathological features,quantitation of viral load and measures of neurobe-havioral performance.

Results: Dual infections caused greater IL-1beta, TN-Falpha and IDO expression and neurotoxic proper-ties in infected feline macrophages. Co-infection andsequential FIV super-infection in vivo also inducedgreater IL-1beta, TNFalpha and IDO expression in thebasal ganglia (BG) and frontal cortex (CTX), comparedto the mono- and mock-infection groups, althoughviral loads were similar in mono- and dual-infectedanimals. Immunoblot analyses disclosed lower synap-tophysin immunoreactivity resulting from FIV super-infection and co-infection in the CTX. Both choliner-gic and GABAergic neuronal injury were evident inthe in CTX of animals with dual FIV infection. Withincreased astrocytic and microglial activation and neu-ronal loss in dual FIV-infected brains, immunohis-tochemical analysis also revealed elevated detectionof cleaved caspase-3, concurrent with dysmorphicchanges in neurons and associated neurobehaviouralabnormalities.

Conclusions: These findings highlight the adversebiological effects of dual lentivirus infections insuper-infection and co-infection models for whichthe underlying pathogenic mechanisms representan escalation in neuroinflammation and ensuingneurodegeneration.

P.5Induction of proinflammatory cytokines by human Tcell leukemia virus type 1 Tax protein as determinedby multiplexed cytokine protein array analyses ofhuman monocyte-derived dendritic cells

8th International Symposium on NeuroVirologyPoster Presentations 61

Jaya Ahuja, Veronique Lepoutre, Brian Wigdahl,Zafar K. Khan, and Pooja JainDrexel University College of Medicine

Human T cell leukemia virus type 1 (HTLV-1)-associated myelopathy/tropical spastic paraparesis(HAM/TSP) is characterized by a hyperstimulated im-mune response including elevated levels of inflam-matory cytokines/chemokines, and the oligoclonalexpansion of virus-specific CD8+ T cells in the cere-brospinal fluid. Studies have shown that the HTLV-1 transactivator protein Tax is available for immunerecognition by antigen presenting cells (APCs) such asdendritic cells (DCs). DCs are relevant to the pathogen-esis of HAM/TSP since the presentation of Tax pep-tides by activated DCs to naıve CD8+ T cells mayplay an important role in the induction of a Tax-specific immune responses observed in HAM/TSP. Inthis study, a human cytokine protein array was used tostudy the secretion of cytokines by monocyte-derivedDCs (MDDCs) exposed to Tax. Of the 16 cytokinesanalyzed, 6 cytokines were secreted in significantlyhigh amounts (∼2-fold) including Th1 cytokines (IFN-gamma, IL-12, and TNF-alpha) and C-C chemokines(Eotaxin, MCP-1, and MCP-3). Selected cytokines werefurther examined at two concentrations of Tax andtwo time periods. Furthermore, a transient exposureto Tax did not result in any cytokine production whenexamined at three different time points after expo-sure, indicating that the prolonged presence of Tax wasrequired for its activity. Finally, inhibition of the NF-kappaB signaling pathway by specific inhibitors ab-rogated Tax-mediated cytokine secretion. Collectively,these investigations suggest that Tax-induced cytokinesecretion from MDDCs may be an important factorin the cellular activation and tissue damage observedin HAM/TSP.

P.6Analysis of the DrexelMed HIV/AIDS cohort forenvelope markersBenjamas Aiamkitsumrit, Katherine Flaig, Bryan P.Irish, Evelyn M. Kilareski, Michael R. Nonnemacher,Julio Martin-Garcia, and Brian WigdahlDrexel University College of Medicine

Microglial cells and perivascular macrophages sup-port most productive HIV-1 replication within thebrain, although both have low CD4 levels makingmacrophage tropism and preferential CCR5 utilizationfeatures of CNS-derived viruses/envelopes. Sequenceanalysis has demonstrated both viral evolution withinthe CNS and compartmentalization of sequences be-tween brain and peripheral tissues, suggesting thepotential for adaptation to replication in the brain en-vironment. Previous studies of human immunodefi-ciency virus type 1 (HIV-1) long terminal repeat (LTR)have identified the 3T configuration of CCAAT en-hancer binding protein C/EBP site I (C-to-T change atnucleotide position 3) and 5T configuration of Sp site

III (C-to-T change at nucleotide position 5) to correlatewith peripheral blood disease progression and HIV-associated dementia (HAD). The 3T/5T-containingLTR may be representative of an LTR genotype that ispreferentially retained, because of specific functionalproperties involved in disease pathogenesis, in thePB, CNS, and perhaps other compartments as HIV-1-associated disease severity increases suggesting that itmay be useful as a predictive viral marker. In addition,we propose certain envelope sequences are co-selectedwith this LTR, which play a key role in cell types in-fected within the peripheral blood and. Herein, wepresent an analysis of the DrexelMed HIV/AIDS co-hort for the presence of specific configurations of HIV-1 LTR transcription factor binding sites in associationwith specific envelope genotypes. Future studies willaddress functional properties of these viral geneticsignatures.

P.7Phosphorylation of pRb is differentially regulated inneurons in HIV-Associated DementiaCagla Akay, Ying Wang, Rebecca A Chodroff, DennisL Kolson, and Kelly L Jordan-SciuttoUniversity of Pennsylvania

HIV Associated Dementia (HAD) is characterizedby microgliosis, astrogliosis, neuronal loss and den-dritic damage. While the mechanisms of neuronaldeath in HAD remain only partially defined, neu-ronal damage has been linked to soluble factors re-leased by HIV infected, and non-infected, activatedmacrophages/microglia in the brain. The inflammatoryinfiltrate secretes both neurotoxic and neuroprotec-tive factors. Phosphorylation of pRb has been shownto be required in in vitro models of neuronal apopto-sis including trophic factor withdrawal, beta-amyloidtreatment and oxidative stress. To define the differ-ential effects of neurotoxic and neuroprotective fac-tors on pRb phosphorylation in HAD, we used an invitro model where we treated primary rat neuroglialcultures with supernatants from human monocyte-derived-macrophages infected with a neurovirulentstrain of HIV-1 (HIV-M/M). We assessed pRb phospho-rylation on characterized sites and the interaction ofthese phospho-pRb isoforms with cell cycle proteins.Using dilutions of HIV-M/M that produced 25% neu-ronal loss by 4 hours or 50% neuronal loss by 20 hours,we observed an early increase in p-pRbSer249/Thr252,and p-pRbSer795 by western blotting (WB), and a lateincrease in p-pRbSer780 by HIV-M/M by both WB andimmunofluorescence (IF), similar to results obtainedin cultures treated with Rantes. BDNF led to a de-layed increase in these p-pRb epitopes. We observedsimilar increases by WB and IFA in the mid-frontalcortices of autopsied brain tissue of HIV(+) individ-uals. We did not observe an increase in p-pRbSer608or p-pRbThr821 levels with either HIV-M/M, BDNF orRantes by WB in vivo. Interestingly, p- pRbSer807/811,


which was observed in the cytoplasm of untreatedneurons by IF, was detected in the nuclei of neu-rons in HIV M/M treated cultures, while its proteinlevels were not increased, as assessed by WB. p-pRbSer807/811 is a known epitope for cdk5. By co-immunoprecipitation, we observed a loss of interac-tion of p- pRbSer807/811 with cdk5 when cultureswere exposed to HIV-M/M, whereas it was not dis-rupted in response to BDNF, Rantes or NGF treat-ment. Thus, we hypothesize that phosphorylation ofpRb is differentially regulated in neurons in responseto neurotrophic factors and the binding between p-pRbSer807/811 and cdk5 might be a determining fac-tor in neuronal viability.

P.8IL-1beta-induced differentiation of a human bonemarrow promonocytic progenitor cell line andsusceptibility to HIV-1Aikaterini Alexaki, Shendra Passic, Michael R.Nonnemacher, Evelyn M. Kilareski, and BrianWigdahlDrexel University College of Medicine

The role of the bone marrow compartment in thepathologic events associated with AIDS and HIV-1 dementia (HIVD) has not been elucidated. Inter-estingly, CD34+/CD38- progenitor cells within thebone marrow are refractile to HIV-1 infection, pos-sibly due to their low level expression of HIV-1co-receptors, CXCR4 and CCR5, which upon differ-entiation are upregulated, potentially increasing sus-ceptibility to infection. The CD34+/CD38+ TF-1 bonemarrow progenitor cell line was selected as a model tostudy HIV-1 infection during the differentiation pro-cess of hematopoietic progenitor cells. TF-1 cells weretreated with a number of metabolic activators includ-ing PMA, conditioned media from PMA-treated cells,as well as cytokines such as GM-CSF, M-CSF, IL-1beta,TNF-alpha, IL-4 and their maturation was monitoredthrough surface marker expression by flow cytometry.Interestingly, IL-1beta, alone or in combination withTNF-alpha leads to CXCR4 and CCR5 upregulation andpreservation of CD4 expression while differentiatingTF-1 cells, thereby providing a window of opportu-nity for HIV-1 infection to occur. Moreover, transientand stable transfection analysis demonstrated that theHIV-1 LTR activity was significantly increased follow-ing treatment of TF-1 cells with IL-1beta and condi-tioned media. Importantly, IL-beta treatment led to alarge increase in p24 production in TF-1 cells infectedwith the CCR5 using virus BaL and to a modest in-crease in cells infected with the CXCR4 using virusIIIB. These results indicate that progenitor cell differ-entiation leads to upregulation of HIV-1 co-receptorexpression and enhancement of the LTR activity, con-tributing to increased HIV-1 susceptibility and produc-tive replication.

P.9Assessment of leukocyte movement in encephaliticbrains of SIV infected macaques suggestsbidirectional trafficking across the blood brainbarrierXavier Alvarez,1 Heather Vinet-Oliphant,1 EmilySusko,1 Juan T Borda,1 Kenneth Williams,2 andAndrew Lackner1

1Comparative Pathology, Tulane National PrimateResearch Center, Covington, LA, 70433; 2Division of ViralPathogenesis, Harvard Medical School, Beth IsraelDeaconess Medical Center, Boston, MA 02215

Cell movement is essential for immune system func-tion. While much is known about trafficking of leuko-cytes between blood and peripheral tissues, relativelylittle is known about leukocyte trafficking to, and pos-sibly from, the brain. Examination of these issues inthe brain is made more difficult by the relative inabil-ity to safely collect repeated samples. In vitro and im-mediately ex vivo models can be used to address manyissues but the relevance of the observations to the bi-ology in vivo can be difficult to assess. In an effort toaddress this problem we have developed a standard-ized method for analyzing direction of movement ofimmune system cells from static confocal images byapplying simple algorithms based on cell morphology.Immunofluorescence staining for nonmuscle myosinII heavy chain A (MyH9) was performed in suspen-sion. This molecule was examined because of its ac-tivation during cell movement. Specifically, MyH9 isinvolved in cell polarization, cell motility, contractionof the actin-myosin network in the uropods of T-cells.In addition, the actin networks of cells were analyzedby staining with Phalloidin, a molecule that labels F-actin in mobile cells as well as vessels. Visualizing thedistribution of F-actin is important to understand di-rectional cell movement because it is highly localizedin the uropods suggesting involvement in the contrac-tion of actin-myosin networks at the rear of the cell.The staining of these molecules was further examinedby analyzing the morphology of these polarized cellswith the hypothesis that cells in motion have an elon-gated morphology, as compared to static cells withround morphology, from which a mathematical deter-mination of direction of movement can be made inthree dimensions by drawing a vector from the centerof mass of the cytoplasm to the center of mass of thenucleus while implementing simple algorithms of thedistance formula and the point-slope method. The re-sulting mathematical assessment of direction of move-ment of cells in 3 dimensions, was firstly validatedby in vitro live cell microscopy studies, and then ap-plied to CD3+ lymphocytes and CD68+ and CD163+monocytes/macrophages found in brain parenchymaand perivascular space. Such analysis revealed bidi-rectional movement of leukocytes across the BBB incases of SIV encephalitis.

Supported in part by grants RR00164 (AAL, XA, JB,HVO), NS37654, NS50041 (KW)


P.10Differences in cerebral blood flow (CBF) andcerebral metabolic rate of oxygen consumption(CMRO2) in HIV+ patients compared toseronegative controls using quantitative functionalmagnetic resonance imaging (qfMRI)Beau M Ances, Christine Liang, Richard B Buxton,Davey Smith, Susan Little, Scott Letendre, DouglasRichman, Ron Ellis, and the HIV NeurobehavioralResearch CenterUniversity of California San Diego

Introduction: qfMRI has been used to study the basicphysiology of both cortical and subcortical regions. Si-multaneous acquisition of the blood oxygen level de-pendent (BOLD) and CBF responses during functionalactivation and mild hypercapnia experiments allowsfor non-invasive determination of CMRO2. We usedqfMRI to determine if physiological responses withinthe basal ganglia to a simple fingertapping task wereaffected by HIV.

Methods: 16 seronegative healthy controls and16 HIV+ subjects were studied at 3 T usingQUIPSSII/PICORE ASL technique with a dual echospiral k-space acquisition of CBF and BOLD data. Allsubjects initially received two trials of a 5% CO2 gasmixture for 3 minutes with 4 minutes between CO2challenges. Mild hypercapnia was used to manipulateCBF changes independent of CMRO2. During four sub-sequent activation scans, subjects viewed numbers inthe center of the screen flickering at 2Hz correspondingto finger taps on a 4-button box. Each functional scanconsisted of 4 trials using a block-design stimulus (20sec on, 60 sec off). A region-of-interest was drawn foreach subject corresponding to the basal ganglia. Thisregion was chosen as HIV often leads to pathologicalchanges within this subcortical area. From this regiontrial-averaged CBF and BOLD responses were obtainedfor the functional activation paradigm and for mildhypercapnia. CMRO2 values were subsequently cal-culated using a standard equation. Paired t-tests wereperformed between the two groups for both measured(CBF and BOLD) and calculated (CMRO2) values withp values significant if p < 0.05.

Results: Both CBF and BOLD responses were repro-ducibly observed within the basal ganglia for func-tional stimulation and hypercapnia in HIV+ subjectsand seronegative controls. Changes in CBF, BOLD andCMRO2 for functional activation were significantlygreater within HIV+ subjects compared to seronega-tive controls. There was no correlation between nadirCD4, current CD4, or viral load and qfMRI measureswithin HIV+ subjects.

Conclusions: qfMRI may act as a non-invasive surro-gate biomarker for assessing effects of HIV in the brain.

P.11Metabolic syndrome risk factors are elevated inHIV+ patients with previous stroke

Beau M Ances, Archana Bhatt, JenniferMarquie-Beck, Terry Alexander, J Allen McCutchan,and the HNRCUniversity of California San Diego

Introduction: Components of the metabolic syndromeincluding hypertriglycerides (TRG), increased bodymass index (BMI), hypertension (HTN), and elevatedserum glucose (GLU) and uric acid (UA) levels have be-come common in HIV+ patients since introduction ofcombination antiretroviral therapy (cART). These fac-tors can increase the risk of stroke. We compared theprevalence of these risk factors (TRG, HTN, GLU, BMI,and UA) in a confirmed cohort of HIV+ patients withstrokes (cases) to age and sex matched HIV+ subjectswith no history of stroke (controls).

Methods: A retrospective chart and database anal-ysis was performed of over 2,500 HIV+ individualsfollowed at the HIV Neurobehavioral Research Cen-ter (HNRC) at the University of California San Diego(UCSD). Cases were selected based on ICD -9 codesconsistent with stroke (342 and 430–438). From thisgroup 21 cases were identified. Each case was re-viewed by a board certified neurologist (B.A.) to ensurestroke etiology was most likely due to HIV and notother factors (i.e. current stimulant use, progressivemultifocal leukoencephalopathy, toxoplasmosis, etc.).Each case was rated as definite, probable, or unlikelyaccording to focal findings on neurological examina-tion, acute onset of neurological disturbances, labora-tory data, and neuroimaging abnormalities. From thisset, 12 definite cases were identified. For each case fivematched HIV+ controls were identified using the fol-lowing criteria: age (± 4 years), sex, number of HNRCvisits, and year of stroke (± 4 years). Laboratory val-ues and mean arterial pressure (2/3 systolic pressureand 1/3 diastolic pressure) were obtained at the out-patient visit closest to the date of confirmed stroke foreach case. Pair-wise student t-tests with a Sidak cor-rection for multiple comparisons were performed be-tween cases and controls for each risk factors associ-ated with metabolic syndrome.

Results: Mean TRG, random serum GLU, and serumUA were significantly elevated within cases (p = 0.03)compared to controls. However, no significant dif-ferences were seen for other risk factors includingBMI, MAP, CD4 count, plasma HIV RNA, hemoglobin,cholesterol, or current cART regimens between the twogroups.

Conclusions: Three components of the metabolicsyndrome (hyper-triglyceridemia, -glycmeia, and –uricemia) but not CD4 or plasma HIV RNA, appearto be associated with increased stroke risk in HIV in-fected patients, almost all of who were on antiretrovi-ral therapy.

P.12Biomarkers and neuropsychological deficit patternin clinically asymptomatic HIV-positive patients


Gabriele R Arendt and Thorsten NoltingUniversity Hospital of Duesseldorf

Objective: Human immunodeficiency virus (HIV) pro-vokes different forms of brain disease, like virusassociated dementia (HAD) and its precursors. Neu-ropsychological test batteries can identify deficits inthese patients, but to date there is no pattern identified,allowing to determine the prognosis of an individ-ual patient. Biomarkers for disease progression, likeCD4+-cell count and plasma viral load in systemicHIV disease, are lacking as well in virus associatedbrain disease.

Methods: 33 male homo-/bisexual men without clin-ically overt neurological deficits were included ina prospective study. Patients were examined neuro-logically, neuropsychologically as well as immuno-logically by determining cerebrospinal fluid cytokinelevels using a solid-phase protein array in order toevaluate the role of defined cytokines as biomarkersfor HIV-induced brain disease.

Results: Patients were comparable with respect toage, CD4+-cell count and neuropsychological test per-formance. TH2 reaction like interleukin-1, 4 and 10was upregulated in treated AIDS-patients, whereas IL-10 was significantly down-regulated in therapy-naıvenon-AIDS patients (CDC A1+2, B1+2), who showed abad performance in the digit symbol test and in param-eters of fine motor speed. Untreated late stage patients(CDC A3, B3 and C1-3) had a poor performance in theAIDS dementia scale. The longer patients were knownto be infected, the higher were the levels of CXCL16,GM-CSF and TGF-beta1.

Conclusions: The study showed a supportive valueof CSF-cytokine analysis in neuropsychologically im-paired patients. Solid-phase protein arrays are a use-ful method to analyse cytokine patterns in the CSFof HIV-carriers. Thus, neuropsychological testing andCSF-cytokine-analysis may be useful tools for moni-toring HIV associated brain disease.

P.13Cell associated heparan sulfate proteoglycans areinvolved in HIV-1 attachment and entry intoprimary human astrocytesElias G Argyris,1 Jialing Huang,1 EdwardAcheampong,1 Hui Zhang,1 and Kevin Jon Williams2

1Thomas Jefferson University, Dept. of Medicine, Divisionof Infectious Diseases, Center for Human Virology;2Thomas Jefferson University, Dept. of Medicine, Divisionof Endocrinology

Astrocytes represent a major cellular component inthe brain and appear to significantly contribute toHIV-1-mediated neuropathogenesis. Human primaryastrocytes do not express CD4 but still bind HIV-1gp120 through alternative receptor(s). To date sev-eral candidate receptor proteins have been described,including the mannose receptor and the chemokine

receptor D6. We and others have recently demon-strated that cell-associated proteoglycans serve as ma-jor HIV-1 receptors on CD4-negative CNS target cells,namely brain microvascular endothelial cells. In thecurrent study, we examined whether proteoglycansalso act as HIV-1 receptors on normal primary hu-man astrocytes (obtained from ScienCell Research Lab-oratories). First, we characterized the expression ofspecific proteoglycans in astrocytes via RT-PCR andWestern blot analysis. Our preliminary data indicatestrong expression of syndecan-1, a cell-surface trans-membrane heparan-sulfate proteoglycan (HSPG), re-cently shown to efficiently mediate attachment andentry of HIV-1 in human macrophages and in primarygenital epithelial cells through a CD4-independentmechanism. Enzymatic digestion of cell-surfaceheparan-sulfate and chondroitin-sulfate side-chainswith specific lyases (heparitinase III and chondroiti-nase ABC, 10U/ml respectively) significantly inhibitedHIV-1 (X4-tropic strain NL4-3) attachment onto humanastrocytes, as indicated by the amount of p24 in celllysates via ELISA. Moreover, our data from quantita-tive proviral DNA (HIV-1 gag) real-time PCR analysis inastrocytes pretreated with lyases and subsequently in-cubated with NL4-3 indicate that cell-associated pro-teoglycans facilitate efficient entry of HIV-1 into hu-man astrocytes. The molecular mechanisms by whichproteoglycans promote CD4-independent viral entry,infection, and spread in the CNS and its cellularcomponents remain an area of active investigation.Understanding the infection of the human brain byHIV-1 is crucial in targeting this potential viral reser-voir site.

P.14The IFN-induced expression of APOBEC3G inhuman blood brain barrier exerts a potent intrinsicimmunity to block HIV-1 entry to CNSElias G Argyris,1 Edward Acheampong,1 FengxiangWang,1 Jialing Huang,1 Keyang Chen,1 MuhammadMukhtar,2 and Hui Zhang1

1Thomas Jefferson University, Dept. of Medicine, Divisionof Infectious Diseases; 2Drexel University-College ofMedicine, Dept. of Microbiology and Immunology

In the human genome the apolipoprotein B mRNA-editing enzyme catalytic polypeptide (APOBEC)3 genehas expanded into a tandem array of genes termedAPOBEC3A-H. Several members of this family havebeen found to have potent activity against human im-munodeficiency virus type 1 (HIV-1). The expressionand distribution of APOBEC family members in theCentral Nervous System (CNS) is currently unknown.In this study, we have shown that APOBEC-3B/3C/3Fand -3G are expressed in the major component cellsof CNS, namely differentiated post-mitotic matureneurons, primary human astrocytes, and primary hu-man brain microvascular endothelial cells (MVECS).


Moreover, we have demonstrated that both interferon-alpha (IFN-a) and IFN-gamma (IFN-g) significantlyenhance the expression of APOBEC-3G/3F anddrastically inhibit HIV-1 replication in primary hu-man brain MVECS, the major component of bloodbrain barrier (BBB). As the viral inhibition can beneutralized by APOBEC3G-specific short interferingRNA (siRNA), APOBEC3G plays a key role to medi-ate the anti-HIV-1 activity of IFN-alpha and/or IFN-gamma. Our findings suggest that, in addition to therestriction at viral entrance level, the restriction fromAPOBEC3 family could account for the low-levelreplication of HIV-1 in MVECS. Given the markedresponse to IFN stimulation in MVECS, the manipu-lation of IFN-APOBEC3 signaling pathway could bea potent therapeutic strategy to prevent HIV invasionto CNS.

P.15Predictors of progression from HIV-associated minorcognitive-motor disorder to HIV-dementiaLaura E Baldassari,1 Ned Sacktor,1 Karen Marder,2Giovanni Schifitto,3 Derek Adams,3 RichardSkolasky,1 Bruce Cohen,4 Leon G Epstein,5 and JustinC McArthur1

1Johns Hopkins University, Departments of Neurology andEpidemiology, 2Columbia University, Department ofNeurology, 3University of Rochester, 4NorthwesternUniversity, Department of Neurology, 5Childrens MemorialHospital Research Center, Department of Neurology

Background. HIV-associated neurocognitive disorders,such as minor cognitive-motor disorder (MCMD) anddementia, are common and seriously debilitating ill-nesses. The exact pathogenesis of such disorders re-mains unclear, but the use of highly active antiretro-viral therapy (HAART) can improve clinical deficitsand potentially reverse neuronal damage. The objec-tive of this study was to determine the ability of neu-rocognitive tests to predict the progression from HIV-associated minor cognitive-motor disorder (MCMD) toHIV-associated dementia.

Methods. The Northeast AIDS Dementia (NEAD)Consortium cohort study enrolled HIV positive pa-tients (n = 406) in four academic centers with aCD4 cell count <200 cells/uL, or <300 cells/uL withevidence of cognitive impairment. Individuals withMCMD (as defined by the Memorial Sloan Kettering(MSK) stage = 0.5) at baseline and who had com-pleted neurocognitive assessment, and who were seen12 months after baseline were included in this anal-ysis (n = 96). Progression to HIV-associated dementiaat 12 months after baseline, as defined by the MSKscale score greater than or equal to 1. We examinedthe ability of a variety of neurocognitive assessmentz-scores (adjusted for age and education) and other pa-tient characteristics at baseline to predict progressionto HIV-dementia through logistic regression and ROCcurve analysis.

Results. A logistic regression model intended to pre-dict progression to HIV-dementia from MCMD was de-veloped using baseline age (OR = 5.93, 95% CI: 1.42 to24.73, p = 0.015), Rey Delayed Recall Z-Score (OR =0.29, 95% CI: 0.13 to 0.65, p = 0.003), depression asdefined by the Beck Depression Inventory ≥16 (OR =0.21, 95% CI: 0.04 to 1.01, p = 0.052), Karnofsky Per-formance Score (OR = 0.90, 95% CI: 0.84 to 0.97, p =0.004), and the Rey Complex Figure Immediate RecallZ-Score (OR = 0.57, 95% CI: 0.31 to 1.05, p = 0.072). Areceiving operator characteristic (ROC) curve analysiswas performed, and the logistic regression model pre-dicted progression from MCMD to dementia with anarea under the curve (AUC) of 0.87; at a probability cut-point of 0.25, the model had a sensitivity of 80.0%, aspecificity of 80.3%, and a positive predictive value of51.6%.

Conclusions. This logistic regression model mayhave clinical utility in predicting which patients willprogress from MCMD to dementia; the model maytherefore be able to guide HAART regimen modifica-tions to prevent progression to more severe neurocog-nitive impairment.

P.16Biomarkers for temporal change of cognitive statusin patients infected with HIVVeera VR Bandaru, Justin C McArthur, Ned Sacktor,and Norman J HaugheyJohns Hopkins University School of Medicine. Departmentof Neurology. Division of Neuroimmunology

The neurological manifestations of HIV-infection areconsiderably different since the advent of highly ac-tive antiretroviral therapy. In general, the degree ofneurological impairment is less severe, and many peo-ple infected with HIV-1 develop a chronic conditionof minor cognitive motor impairment. With antiretro-viral therapy, there can be bidirectional transitionsin cognitive status, and the onset of dementia is nolonger a predictor of mortality. In this new era ofHIV-dementia, many of the biomarkers that previouslyassociated with dementia are now of limited utility,and new ways of classifying patients, and identify-ing biomarkers are beginning to emerge. One sugges-tion has been to categorize patients according temporalchanges in cognitive status. This approach has the ad-vantage of potentially identifying biomarkers that areassociated with-, and those can predict neurologicaldisease. In this study we sought to identify biomark-ers in CSF that could predict the onset of dementiaand markers that were associated with dementia in pa-tients infected with HIV-1. HIV-infected patients fromthe North Eastern AIDS Dementia (NEAD) cohort weregrouped based on changes in cognitive status over a1-year time period. Sphingolipid, sterol, triglyceride,antioxidant, pro-oxidant, and lipid peroxidation lev-els were quantified from CSF. We found that increased


levels of the antioxidant vitamin E, triglyceride C52,and iron predicted the onset or worsening of demen-tia. Increased levels of sphingomyelin were associ-ated with an inactive dementia. Increased ceramide,with the accumulation of lipid peroxidation productswere associated with an actively progressing demen-tia. These findings suggest that early in the courseof HIV-dementia, there is an up-regulation of endoge-nous antioxidant defenses in brain. The exhaustion ofthese endogenous neuroprotective mechanisms is fol-lowed by increased concentrations of sphingomyelin,and moderate neuronal dysfunction that is clinicallyapparent as an inactive dementia. In an active demen-tia, ceramide, and reactive aldehyde levels are ele-vated, consistent with an ongoing degenerative con-dition. Thus, an increase in endogenous antioxidantdefenses may predict, while disruptions in lipid bal-ance are associated with dementia in patients infectedwith HIV-1.

P.17Critical review on a possible role of Borna diseasevirus infection in neuropsychiatric disordersKarl Bechter1 and Sibylle Herzog2

1Ulm University, Clinic for Psychiatry and PsychotherapyII; 2Giessen University, Institute of Virology

Introduction: After the first description of BDV spe-cific serum antibodies in depressed patients in 1985 alarge number of seroepidemiological studies on BDVantibodies in patients with neuropsychiatric disordershave been published, leading however to controversy.A critical review seems appropriate concerning a pos-sible impact of BDV infections in humans.Method: Published results are discussed regardingmethodological aspects, contradicted and replicatedfindings and compared to standards in other CNS in-fections including our considerations, investigationsand methods - based on the results of extended ownstudies in large neuropsychiatric and in various hu-man control samples, and of investigations of horseswith acute and chronic BDV infections.Results: Considering exclusively certified methodsand confirmed findings and the established goldstandard in neurological medicine, a relatively clearpicture arises. BDV infection seems to share many as-pects with other neurotropic virus infections: 1. Thepathogenicity appears low, probably below 10% of in-fected humans. Nevertheless, epidemiological studieson the prevalence BDV serum antibodies suggest thatBDV infection may be responsible for or contributedto pathogenesis of psychiatric disorders in 1 – 3% ofpsychiatric hospitalized patients. The specificity of thehuman BDV antibodes in serum and CSF was con-firmed by micro array analysis. 2. The sensitivity ofmethods changes with the time of infection: in acuteinfection, only during a short time period of few daysvirus is detectable in CSF but not in blood. Later,

chronic infection or chronic autoimmune reaction candevelop, detected indirectly by demonstration of spe-cific intrathecal immune reactions or directly by inves-tigation of the brain (possible in animals, but rarely inhumans). Such intrathecal BDV specific immune reac-tion was found in few human cases with affective orschizophrenic spectrum disorders, indicating a causalrelationship to BDV infection.Conclusion: Prevalence and specificity of serum andCSF antibodies suggest that BDV or a related agent caninfect humans. But up to now the proof is lacking thatBDV infection can be pathogenic for humans, thoughCSF findings indicate that in rare cases of affective orschizophrenic spectrum psychoses BDV was presum-ably the causal agent. Epidemiological findings maysuggest that BDV infection could be of some relevanceor even causal for psychiatric disorders in 1–3% ofhospitalized psychiatric patients.

P.18Immunophenotypic diversity of brain macrophages:implications for neuroAIDSJuan T Borda, Alvarez Xavier, and Andrew LacknerTulane National Primate Center

Infection of humans with HIV or macaques with SIVoften leads to an encephalitis (HIVE or SIVE) charac-terized by perivascular accumulations of macrophagesand multinucleated giant cells in brain, and many ofwhich are productively infected. It is assumed thatthe development of SIVE/HIVE is associated with in-creased migration (and retention) of circulating mono-cytes into the brain. The immigration of these mono-cytes into the brain would facilitate neuroinvasionand provide additional cells for infection. Direct as-sessment of this hypothesis has been frustrated bythe lack of specific markers that can easily differen-tiate among perivascular macrophages, recently im-migrated monocytes, and parenchymal microglia. Re-cently, expression of CD14 and CD45 in conjunctionwith CD11b has been used to differentiate parenchy-mal microglia (CD11b+CD14–CD45–) from perivascu-lar macrophages (CD11b+CD14+CD45+) and definethe latter as the primary cell type productively in-fected in the CNS. It was clear, however, that the pop-ulation of SIV-infected cells that shared expressionof CD11b, CD14 and CD45 was heterogeneous. Thissuggested that we might be looking at several popula-tions of cells including those that had recently immi-grated from the blood. To assess this further and tryto define the immunophenotype of additional popula-tions of brain macrophages, including those that hasrecently entered the CNS, we performed multilabelconfocal microscopy combined with in situ hybridza-tion (ISH) for SIV for a variety of cell markers whichare known to be expressed by macrophage population.The markers used in this study included CD11b, CD45,myeloid histocyte antigen (clone MAC387), myeloidrelated proteins 8 (MRP8), CD163, LN5, CD68 andHAM56.


Major observations include the following: 1) Com-mon macrophage markers such as HAM56 and CD68colocalized nearly completely and are seen primarilywithin SIVE lesions. Many such cells are also infectedby SIV; 2) CD163 largely colocalized with CD68 andHAM56 within SIVE lesions. Many of these cells wereinfected with SIV. CD163 was also expressed in cellsmorphologically compatible with microglia through-out the CNS of animals with SIVE and others en-cephalitides. No such labeling was seen in normalanimals. None of the CD163+ cells outside of clas-sic SIVE lesions were SIV positive; 3) LN5 are ex-pressed in SIV infected cells with or without colo-calization of CD68 and CD163 in the SIVE lesions,whereas uninfected cells also expressed LN5 aroundSIVE lesions; and 4) MAC387 and MRP8 primarilylabeled cells within vessels or immediately adjacentto vessels, including those in SIVE lesions. However,none of these cells were SIV+ nor CCR5 by ISH or IHC,respectively. This data indicates that the cellular infil-trates of monocyte/macrophages in SIVE are heteroge-neous. The presence of MAC387+ and MRP8+ cellsnot infected by SIV could represent either recentlyrecruited monocyte/macrophages or a population ofmacrophages resistant to infection. Lastly, CD163 ap-pears to label “activated” microglia as no labeling ofparenchymal cells which was seen in normal animals,while in animals with encephalitis CD163 labeling wasextensive.

Support: RR00164, NS030769, MH061192

P.19Search for molecular markers of HIVneuropathogenesis by gene expression profiling ofbrain tissues.Alejandra Borjabad,1,2 Wei Chao,1 Seon-Young Kim,3

Andrew I Brooks,4 Jacinta Murray,5 Susan Morgello,5and David J. Volsky1,2

1Molecular Virology Division, St. Lukes-Roosevelt HospitalCenter, New York, NY 10019; 2Department of Pathology,Columbia University, New York, NY 10032; 3KRIBB, Seoul,Korea; 4EOHSI, Piscatawai, NJ 08854; 5Mount SinaiMedical Center, New York, NY 10029

Neurodegeneration and cognitive impairment causedby HIV-1 infection are common complications of AIDS.Introduction of highly active antiretroviral therapyreduced the incidence of HIV-1-associated dementiawith active viral replication in brain (HAD with en-cephalitis, or HIVE) but so far has had limited effect onthe high frequency of milder neurological disorders.The latter alter include HAD without encephalitis andMinor Cognitive and Motor Disorder (MCMD), both ofwhich can present with no or minimal histopatholog-ical changes in the brain. The prevalence of MCMDcan reach 30-35 percent in patient cohorts followed inthe U.S.A., regardless of their antiviral treatment sta-tus. Clearly there is a need for better understanding

and treatment of these milder CNS manifestations ofHIV.

We are trying to identify molecular markers of HIVinfection in the CNS that can supplement or serve inlieu of brain histopathology for characterization anddesign of better treatments of HIV associated neuro-logical disorders. For the study presented here, weselected archived, snap-frozen brain tissues from 14HIV-positive individuals who were consented partic-ipants in an ongoing prospective organ donation pro-gram run by the Manhattan HIV Brain Bank (MHBB),a member of the National NeuroAIDS Tissue Consor-tium. All the tissues were from the anterior frontal lobeand for each patient they were sub-dissected into sep-arate white (WM) and grey (GM) matter samples whilestill frozen. As controls, we selected similar sets ofbrain tissues as above from 6 HIV-negative individu-als who died without neurological symptoms and hadno pathological changes. Overall, this study included40 brain samples from 20 subjects: 6 controls, 6 HADwith HIVE (HAD/HIVE), and 8 with HIV-associatedcognitive impairment without HIVE: 4 HAD, 1 whohad fluctuated between HAD and MCMD in their life-time, and 3 MCMD. For the purpose of some microar-ray and statistical analyses, MCMD and HAD withoutHIVE were treated as a single brain-symptomatic cat-egory (HAD/MCMD) totaling 8 subjects. The 40 sub-dissected brain samples were further subdivided intosmaller adjoining fragments without thawing; for eachpair of adjoining WM and GM fragments one was testedfor HIV RNA and DNA burdens by real time PCR andthe other was subjected to gene expression profilingon a Affymetrix U133Plus2 oligonucleotide microar-ray platform. We found that HIV burdens in brainsfrom HIVE patients were highly variable. HIV DNAburdens in brains from HAD/MCMD subjects were ei-ther very low or undetectable and none of these pa-tients had detectable viral RNA. Like in HAD/HIVEsamples, there was a limited correlation between brainvirus burdens and peripheral virus. Next we com-pared gene expression profiles obtained from GM andWM of HAD/HIVE and HAD/MCMD groups of pa-tients. Microarray results for the most relevant geneswere confirmed by Real Time PCR and changes insome gene products were confirmed by immunohis-tochemistry in brain sections and Western blottingin tissue extracts. The initial analyses indicate thatboth HAD/HIVE and HAD/MCMD brain tissues ex-hibit extensive, although for the most part different,changes in their cellular gene expression profiles. InHAD/MCMD, molecular changes in the brain were in-dependent of virus burdens in the tissue. Full resultswill be presented. The approach described here mayfacilitate identification of cellular pathways or indi-vidual genes important in HIV mediated neuropatho-genesis, with or without active viral replication inbrain.

Supported by grants NS31492 (DJV) andR24MH59724 (SM).


P.20Varicella zoster virus induced apoptosis in culturedcells proceeds through the intrinsic pathwayElizabeth Brazeau,1 Ravi Mahalingam,1 Don HGilden,1 and Subbiah Pugazhenthi2,3

1Departments of Neurology and Microbiology, University ofColorado Health Sciences Center, Denver, CO 80262;2Veterans Administration Medical Center, Denver,Colorado 80262; 3Departments of Medicine University ofColorado Health Sciences Center, Denver, CO 80262

Primary infection by varicella zoster virus (VZV) usu-ally results in chickenpox (varicella), after which virusbecomes latent in multiple ganglia along the entirehuman neuraxis. Virus reactivation decades later pro-duces shingles (zoster). Previous studies have shownthat VZV induces apoptosis in non-neuronal cells, butnot in neurons. The cascade of apoptosis proceedsthrough intrinsic (mitochondrial) or extrinsic (deathreceptor) pathways. To elucidate the pathway utilizedby VZV, we used Western blot analysis to compareVZV-infected and uninfected MeWo cell lysates for thepresence of various pro-apoptotic proteins (caspases)and anti-apoptotic proteins at 24, 40, 48, 64 and 72hours post infection. Caspases are a group of cysteine-rich proteases that are sequentially cleaved to activeforms that induce apoptosis. Active forms of caspases9, 3, 7 and 6 as well as the cleaved fragments of theDNA degrading protein PARP were increased in VZV-infected cells as compared to uninfected cells begin-ning 40-48 hours post infection. A reduction in theanti-apoptotic protein Bcl-2 was also seen. Levels ofcaspase 8, an extrinsic pathway marker, did not changeduring the course of infection. Apoptosis produced byVZV utilizes the intrinsic (mitochondrial) pathway innon-neuronal cells.

P.21Searching for novel therapeutics with anti-JCVactivity among known bioactive drugs andexperimental compoundsMargot Brickelmaier, Alexey Lugovskoy, KennethSimon, and Leonid GorelikBiogen Idec Inc., Cambridge, MA, USA

Progressive multifocal leukoencephalitis (PML) is arare but frequently fatal disease caused by uncon-trolled replication of JC polyomavirus in the brain ofsome immunocompromised individuals. Currently,no effective anti-viral treatment has been founddespite a number of clinical trials. The best option inthe management of PML is offered by reconstitutionof patients’ own immunity either via HAART therapyin HIV+ individuals or via reduction of an immuno-suppressive treatment regimen in pharmaceuticallyimmunosuppressed individuals. Since effectiverestoration of the immune response is not always pos-sible or timely enough, finding an effective anti-JCVtherapy is highly desirable. As a first step in the iden-tification of such therapy, we undertook screening ofa collection of ∼1500 approved drugs and biologicallyactive molecules, for anti-JCV activity. We looked for

drugs that effectively inhibited active virus produc-tion of a hybrid JC virus Mad1/SVEdelta in SVG-Acells, a JC virus susceptible human glial cell line. Weidentified a number of different drugs and compoundswith significant anti-JCV activity at micromolarconcentrations without any significant toxicity to thecells. Active compounds form several structurallydistinct classes with well-defined structure-activity re-lationship. Reported pharmacokinetic values promiseto achieve comparable plasma concentrations forsome of the drugs we identified. Implications of thesefindings for further steps in identification of humanPML therapy will be discussed.

P.22Redox-based regulation of macrophageinflammation by opiates and HIV TatAnnadora J Bruce-Keller,1 JadwigaTurchan-Cholewo,1 Kurt F Hauser,2 Pamela EKnapp,2 and Jeffrey N Keller1

1University of Kentucky College of Medicine, 2VirginiaCommonwealth University School of Medicine

HIV-1 patients who abuse opiate-based drugs have in-creased rates of HIV dementia, which may be relatedto brain inflammation. To better understand how opi-ates could derange HIV-related brain inflammation,the effects of morphine and HIV-Tat on free radicalproduction and redox-based inflammatory signal-ing was measured in primary macrophages and mi-croglia. Data show that administration of Tat to eithermacrophages or microglia stimulated the productionof free radicals, which were key to the subsequentproduction and release of cytokines. However, co-administration of morphine with Tat significantlyincreased Tat-induced free radical production and in-tracellular oxidation, while significantly decreasingcytokine release. Finally, data show that the abilityof free radicals to control inflammatory signaling maybe based on oxidative alterations to the multicatalyticproteasome and/or on differential recruitment of freeradical–producing enzymes to lipid raft signaling plat-forms. Together, these findings suggest that free radicalproduction in monocytic cells could be a key mecha-nism controling cytokine release, and that this mecha-nism is subject to regulation by opiates. Supported byRO1 NS046267, P20 RR15592, and P01 DA19398.

P.23HIV and cocaine interplay: It is all in the hadShilap J Buch,1 Navneet K Dhillom,1 Fuwang Peng,1

Anil Kumar,2 and Rachel Williams1

1Kansas Univeristy Medical Center, 2University of MissouriKansas City

HIV-1 infection commonly leads to serious HIV-1-associated neurological disorders, such as HIV-1-associated encephalopathy and dementia. Intravenousdrug use (IVDU) and HIV infections are two linkedglobal health crises since needle sharing is a wellrecognized mode of HIV transmission. Cocaine, often


abused by HIV-infected patients, has been suggestedto worsen the HIV-associated dementia via unknownmechanisms. Since brain is the target organ for both co-caine and HIV, the objective of the present study wasto explore the effects of cocaine on virus replicationin macrophages, the target cells for the virus in theCNS. Cocaine markedly enhanced virus productionin Simian Human Immunodeficiency Virus (SHIV)-infected monocyte derived macrophages (MDMs) andin U1 cells, a chronically infected promonocytic cellline as monitored by ELISA and immunocytochem-istry. Cocaine treatment also resulted in the activa-tion of NF-κB and transcriptional activation of theHIV-LTR gag-GFP. Analyses of chemokines in cocaine-treated macrophages by Realtime RT-PCR and Lu-minex assays suggested increased expression of thechemokines, CXCL10 – CCR2 and the cytokine, IL-10,all of which are known to promote HIV replication inMDMs. In addition to enhancing cytokine/chemokineexpression, cocaine also caused an up-regulation ofthe macrophage activation marker, HLA-DR in MDMs.The synergistic effect of cocaine on virus replicationand its enhancement of host activation markers suggestthat cocaine functions at multiple pathways to accel-erate HAD.

P.24PDGF synergistically enhances IFN-? inducedexpression of CXCL10 in blood-derivedmacrophages: implications for HIV-dementiaShilpa J Buch,1 Navneet K Dhillon,1 Fuwang Peng,1

and Richard Ransohoff2

1Kansas Univeristy Medical Center, 2Cleveland ClinicFoundation

There is increasing cumulative evidence that activatedmononuclear phagocytes (macrophages/microglia) re-leasing inflammatory mediators in the CNS are a bettercorrelate of HIV associated dementia (HAD) than theactual viral load in brain. Earlier studies on Simian Hu-man Immunodeficiency Virus (SHIV)/rhesus macaquemodel of NeuroAIDS confirmed that pathologicalchanges in brains of macaques with encephalitiswere associated with upregulation of platelet-derivedgrowth factor and the chemokine, CXCL10. Sincecomplex interplay of inflammatory mediators re-leased by macrophages often leads to the inductionof neurotoxins in HAD, we hypothesized that PDGFcould interact with IFN-? to modulate the expres-sion of CXCL10, in these primary virus target cells.While PDGF alone had no effect on induction ofCXCL10 in human macrophages, in conjunction withIFN-?, it significantly augmented the expression ofCXCL10 RNA & protein through transcriptional andpost-transcriptional mechanisms. Signaling moleculessuch as JAK and STATs, PI-3 kinase (PI3K), MAP ki-nases and NF-κB were found to play a role in the syn-ergistic induction of CXCL10. Furthermore, PDGF viaits activation of p38 MAP kinase was able to increasethe stability of IFN-?-induced CXCL10 mRNA. Under-standing the mechanisms involved in the synergistic

upregulation of CXCL10 could aid in the developmentof therapeutic modalities for HAD.

P.25Inhibition of CXCR4-mediated G protein stimulationin the cortex and the hippocampus ofmorphine-treated animalsSilvia Burbassi,1,2 Rajarshi Sengupta,1,2 Kenny JSimansky,1 and Olimpia Meucci1,2

1Department of Pharmacology and Physiology, DrexelUniversity College of Medicine; 2Institute of MolecularMedicine and Infectious Diseases, Drexel UniversityCollege of Medicine

The chemokine CXCL12/SDF-1a and its specific re-ceptor (CXCR4) have been increasingly studied in theCNS due to their critical roles in neuronal patterningand survival. CXCR4 is also one of the major HIV-1co-receptors and is involved in HIV neuropathogen-esis. Recent evidence suggests that drugs of abuse,including opiates, can facilitate progression to neu-roAIDS. The goal of this study is to establish whetherin vivo morphine treatment alters CXCR4 couplingto G proteins. This hypothesis is based on previousstudies suggesting an interaction between CXCR4 andmu-opioid receptors in cultured neurons and brainslices. The [35S]GTPgammaS incorporation assay wasused to determine CXCL12-induced stimulation ofGTP “binding” in various brain areas. Rat pups (8-14 days old) were used for these experiments sincethey showed similar regional distribution of G pro-tein stimulation in response to CXCL12 and MOR ag-onists. Animals were treated with Morphine for short(1-24 hrs) or relatively long (3-5 days) time periods andGTPgammaS autoradiography was performed at differ-ent times. Brain sections from vehicle and morphine-treated animals were incubated with saline or CXCL12(1-100nM) in the presence or absence of the CXCR4 an-tagonist AMD3100 (100ng/ml). The data show a reduc-tion in CXCR4 coupling to G proteins after morphinepre-treatment in several brain areas, including cortexand hippocampus. These findings support the hypoth-esis that opioids may alter CXCR4 function and aresupported by our studies with cultured cortical neu-rons. We propose that impairment of CXCR4 by opiatescontribute to the neurological deficits associated withHIV infection. (Supported by NIH grants DA15014 andDA19808 to OM, and DK67648 to KJS).

P.26The intrathecal B cell repertoire in subacutesclerosing panencephalitis brain shares mostfeatures with the plasma cell repertoireMark P Burgoon,1 Yupanqui A Caldas,1 Jeffrey LBennett,1,2 Xiaoli Yu,1 Gregory P Owens,1 andDonald H Gilden1,3

1Dept of Neurology, University of Colorado at Denver andHealth Sciences Center; 2Dept of Ophthalmology,University of Colorado at Denver and Health SciencesCenter; 3Dept of Microbiology, University of Colorado atDenver and Health Sciences Center


A prominent immunologic feature of subacute scleros-ing panencephalitis (SSPE), a chronic fatal encephali-tis caused by measles virus, is the presence of in-creased IgG and bands of oligoclonal IgG in brain andCSF that are directed against measles virus. To developstrategies and techniques to analyze oligoclonal IgG inchronic inflammatory CNS diseases of unknown etiol-ogy, we previously demonstrated that antibodies frommost CD38+ plasma cells in SSPE brain are directedagainst measles virus. Herein, we analyzed the reper-toire of CD20+ B cells in the same brain, to comparethe antibody response in the two groups of cells.

B lymphocytes were identified in frozen sections ofpostmortem SSPE brain by immunostaining for CD20,and individual cells were microdissected with a non-destructive infrared laser. cDNA was synthesized fromunfractionated cell lysates, and sequences of specificIgG heavy/light chain pairs expressed by each B cellwere determined by nested RT-PCR amplification. Arepertoire of 51 cells from a single SSPE brain revealedfeatures of antigen-driven selection and affinity matu-ration in both the overrepresented clonal populationsas well as in cells encountered only once in the reper-toire. Seven of the nine clonally expanded sequencesfrom B cells were also found in the plasma cell reper-toire, with virtually identical patterns of mutationfrom germline sequences. Most nonclonal sequencesin the B cell and plasma cell repertoires did not over-lap, and may reflect the limited number of sequencesanalyzed in each repertoire. Overall, IgGs expressed byB-lymphocytes and plasma cells in SSPE share manyclonal features and specificities. These techniques canbe applied to other chronic inflammatory CNS diseasesof unknown cause, such as multiple sclerosis, CNS sar-coid and Behcet’s disease, to determine the disease-relevant targets of the immune response.

P.27Dopamine inhibits claudin 5 and caveolin 1expression in human brain microvascularendothelial cells and HIV induces the novelexpression of D2 dopamine receptors in activatedhuman T lymphocytes: possible role in T lymphocytetransmigration across the blood brain barrierTina M. Calderon, Peter J. Gaskill, and Joan W.BermanAlbert Einstein College of Medicine

Although highly active antiretroviral therapy(HAART) has proven very effective in suppress-ing human immunodeficiency 1 (HIV) replication andviral-mediated dysregulation of the immune system,central nervous system (CNS) complications of HIVinfection persist. The prevalence of cognitive, motorand behavioral abnormalities has increased in theHAART era as infected individuals live longer onantiretroviral therapies. HIV enters the CNS early afterinfection and significant numbers of infected individ-uals develop a wide spectrum of CNS abnormalities.

Although neurons are not productively infected byHIV, neuronal damage results from secondary effectsof HIV infection of macrophages and microglia, andfrom subsequent inflammatory cascades initiated andpropagated within the brain. The entry of HIV into theCNS is thought to occur by the transmigration of in-fected monocytes across the blood brain barrier (BBB).Thus leukocyte transmigration is postulated to playan important role in the neurological pathogenesis ofHIV infection. CNS damage elicited by HIV infectionis compounded by addictive drugs. Evidence suggeststhat in HIV infected individuals who are also drugabusers, accelerated and more severe neurocognitivedysfunction occurs when compared to non drug abus-ing infected populations. In some studies, increasedlocalization of lymphocytes, including CD8+ T lym-phocytes, to perivascular and parenchymal regionsof the CNS has been detected in tissue sections frompresymptomatic HIV infected drug abusers when com-pared to uninfected drug abusers or non drug usingHIV infected individuals. This chronic T lymphocyteinfiltration may contribute to enhanced neurologicaldamage in infected drug abusers. Drugs of abuseshare common stimulatory effects on dopaminergicpathways, which contribute to their rewarding prop-erties. The addictive and reinforcing effects of manydrugs, including cocaine and methamphetamine,have been attributed to elevated synaptic levels ofdopamine. In studies with simian immunodeficiencyvirus (SIV)-infected monkeys, increased CNS levels ofdopamine were associated with enhanced numbers ofperivascular and parenchymal lymphocytes. In orderto determine the effects of dopamine on T lymphocytetransmigration across the BBB, we first treated humanbrain microvascular endothelial cells (BMVEC) withdopamine. Our studies show that BMVEC express D1,D2, D3 and D4 dopamine receptors and that dopaminetreatment (20 umol for 6 hr) decreases the expressionof caveolin 1 and the tight junction protein (TJP)claudin 5 in BMVEC. Activated (PHA and IL-2) hu-man T lymphocytes express D1, D3 and D4 dopaminereceptors and HIV infection induces the unique, addi-tional expression of the D2 dopamine receptor in thiscell type. These data suggest that elevated levels ofdopamine in HIV infected drug abusers may alter theBBB by modulating the expression of TJPs in BMVEC.Dopamine also inhibits caveolin 1, which has beenshown to contribute to TJ disruption in epithelialcells, as well as to changes in transcellular versusparacellular T lymphocyte transmigration across EC.In addition, the unique induction of D2 dopaminereceptor expression in HIV infected T lymphocytesmay contribute to changes in T lymphocyte transmi-gration across the BBB in response to chemokinesexpressed in the brain, in particular CXCL12 (SDF-1).Thus, dopamine may alter the BBB and exacerbateT lymphocyte infiltration into the CNS, resultingin a chronic inflammatory T lymphocyte responsethat contributes to BBB disruption, HIV entry andinfection of parenchymal cells, and neuronal damage.


P.28Efficient expression of sTNFR-Fc in stablytransduced mononuclear phagocytes and neuronalcells: implications for neuroAIDS therapeuticsShengbo Cao,1 Sanjay Maggirwar,2 Huanyu Dou,3Yongbo Yang,1 Stephen Dewhurst,2 Howard EGendelman,3 and Yuanan Lu1

1Dept. Public Health Sciences, University of Hawaii; 2Dept.Microbiology & Immunology, University of Rochester;3University of Nebraska Medical Center

Human immunodeficiency virus type (HIV-1) infectionand immune activation of mononuclear phagocytes(MP; perivascular macrophages and microglia) isdirectly linked to the neuropathogenesis of HIV-1infection. Indeed, viral and cellular products secretedfrom MP affect neuronal viability and cognitive func-tion in disease. Amongst these factors, tumor necrosisfactor-? (TNF-?) stands as one that elicits paracrineand autocrine production of a variety of inflamma-tory factors and neurotoxicity. Our works center onblocking TNF-? through the delivery of a soluble TNFreceptor (sTNFR)-Fc fusion protein as a decoy for cy-tokine binding. To explore this as a potential approachfor neuronal protection, a HIV-1-based lentiviral vec-tor containing an expression cassette for sTNFR-Fcwas constructed. To facilitate detection of transducedcells, cells co-expressed the green fluorescent protein(GFP) using an IRES element. High-titer vector stockswere used to transduce rodent bone marrow derivedmacrophages (BMM), human microglial (CHME-5)and neuroblastoma (HTB-11) cells. At a multiplicityof infection of 10, approximately > 50% of the cellswere GFP positive and increased to nearly 100%when cells were transduced a second time with thevector. Transduced cells showed no morphologicalalteration as compared to control cells, and no lossin viability was detected upon MTT assay. Secre-tion of sTNFR-Fc from these transduced cells wasdemonstrated using Western blotting. High, stablelevel of sTNFR-Fc was detected in the media of allthe transduced cell cultures by ELISA at 116.8 to520.0 ng/mL. The expressed sTNFR-Fc was stablein cell culture medium, and the biological activityof this fusion protein was confirmed by in vitroprotein binding assay using recombinant TNF-? andneuroprotection experiments using primary neuronsexposed to recombinant HIV-1-Tat. BMM were shownto directly penetrate areas of diseased brain withactive HIV-1 encephalitis in a murine model of humandisease. These results establish the feasibility of usinglentiviral vectors to express sTNFR-Fc as a potentialnovel therapeutic intervention for Neuro-AIDS.

P.29JCV infection of human brain microvascularendothelial cells and B lymphocytes: Potentialmechanisms for JCV transmigration across theblood-brain barrier

Moti L Chapagain, Frederic Mercier, Saguna Verma,Richard Yanagihara, and Vivek R. NerurkarDepartment of Tropical Medicine, Medical Microbiologyand Pharmacology, Asia-Pacific Institute of TropicalMedicine and Infectious Diseases, John A. Burns School ofMedicine, University of Hawaii at Manoa, Honolulu, Hawaii

Background: The mechanism(s) of the transmigrationof human polyomavirus JC (JCV) across the blood-brain barrier (BBB) remains unclear. Hypothesis: Wehypothesized that JCV crosses the BBB by infecting hu-man brain microvascular endothelial (HBMVE) cellsand B lymphocytes. Materials and Methods: To testthis hypothesis, we compared the replication kinet-ics of JCV in HBMVE cells and B lymphocytes, andmonitored the transmigration of cell-free JCV acrossan in-vitro BBB model. Results: Our data, based onthe expression of JCV early and late genomes, mRNAtranscripts and protein, conclusively demonstratedthat JCV productively infects HBMVE cells and in-vitro, approximately 4% of JCV crosses the BBB. Onthe other hand, when B cells were infected with JCV,the JCV genome copy number decreased dramaticallyfrom day 0 to day 15 post-inoculation, and early andlate JCV mRNA transcripts and early T antigen pro-tein, were not detected in JCV-infected B lymphocytes.Moreover, immunofluorescence microscopy data indi-cated that the number of JCV-infected B cells decreasedfrom 10% on day 0 to 0% on day 15 post-infectionsuggesting that JCV infection of B lymphocytes is non-productive. However, JCV-infected B cells were capa-ble of transmitting JCV infection to PHFG cells and theDNase protection assay confirmed that the JCV viri-ons remained intact inside the B cells for several days.Conclusions: Efficient replication of JCV in HBMVEcells, the critical component of BBB, suggests that JCVmay cross the BBB by infecting HBMVE cells and non-productively infected B cells may also serve as a vehi-cle to transport JCV across the BBB.

P.30Cytomegalovirus infection and interferon gammamodulate MHC class I expression on neural stemcellsMaxim C-J Cheeran, Shuxian Hu, Zibing Jiang, GenyaGekker, Thomas Bakken, Joseph M Palmquist, andJames R LokensgardCenter for Infectious Diseases and MicrobiologyTranslational Research, University of Minnesota MedicalSchool, Minneapolis, MN 55455

Cytomegalovirus (CMV) is the leading cause of con-genital brain abnormalities in children and ventricu-loencephalitis in AIDS patients. Many of the patholo-gies associated with CMV brain infection are seenpredominantly in the periventricular region, whichis known to harbor neural stem cells (NSCs). In thepresent study, using an adult murine model for CMVbrain infection, we demonstrated that nestin-positiveNSCs in the subventricular zone were positive for the


virus. This infection was further characterized usingmonolayer NSC cultures. NSCs supported productivemurine CMV (MCMV) replication; demonstrating 3-4Log increases in viral titers and peak gB expressionat 3 days postinfection (d p.i). In addition, MCMV in-fection induced a robust infiltration of peripheral im-munocytes into the brain at 5 d p.i. RNA expressionanalysis of FACS sorted brain leukocyte populationsdemonstrated interferon (IFN)-gamma expression inCD45(hi)CD11b(dim) cells. To further characterize theeffects of neuroinflammation and viral infection onNSCs, MHC class I expression and cell proliferationcharacteristics were studied. IFN-gamma and tumornecrosis factor (TNF)-alpha increased MHC I expres-sion on NSCs. On the other hand, viral infection in-hibited IFN-gamma-induced MHC I expression. Fur-thermore, IFN-gamma, but not TNF-alpha, IL-1beta, orIL-10, suppressed NSC proliferation in vitro. Extracel-lular release of lactate dehydrogenase (LDH) was notaltered in IFN-gamma treated cultures suggesting thatthis cytokine may inhibit cell division. It can be con-cluded from these studies that NSCs are susceptibleto MCMV infection and that inflammatory mediators,such as IFN-gamma, alter stem cell function.

P.31Death induction of human immunodeficiencyvirus-infected macrophages using anti-PI3K/Aktpathway agentsPauline Chugh,1 Vicente Planelles,2 SanjayMaggirwar,1 Stephen Dewhurst,1 and Baek Kim1

1University of Rochester Medical Center; 2University ofUtah

The interaction of Human Immunodeficiency VirusType 1 (HIV-1) with CD4+ T lymphocytes is well-studied, and typically results in virally-inducedcytolysis. In contrast, relatively little is knownconcerning the interplay between HIV-1 andmacrophage/microglia in terms of cell fate. A re-cent report suggested that, counter-intuitively, HIV-1infection may extend the lifespan of these cells.Indeed, we have found that expression of a singlevirus-encoded protein, HIV-1 Tat, as well as HIV-1infection, results in a powerful cytoprotective effectin a microglial cell line. Previous mechanistic studiesin our lab showed that the pro-survival effects ofintracellular Tat could be attributed to activationof the PI-3-Kinase (PI-3-K)/Akt pathway. Furtherdissection of this mechanism revealed that bothHIV-1 infection as well as expression of HIV-1 Tatsignificantly reduced levels of PTEN, a negativeregulator of this pathway. As a result, we also ob-served greatly increased Akt kinase activity followingtransduction of macrophage with an HIV-1 vector.Consistent with this, immunoprecipitation studiesrevealed that following binding of intracellular HIV-1Tat to p53, the ability of PTEN to bind to p53 was

drastically reduced. Since the binding of Tat to p53has been shown to occur through the basic domainof Tat, we constructed a Tat basic domain mutant.Interestingly, this mutant had elevated levels ofPTEN and lacked the cytoprotective effect exertedby HIV-1 Tat. Based on these findings, a model ofpossible regulatory circuits that intracellular Tat andHIV-1 infection engage during this cytoprotectiveevent in macrophage and microglia was envisioned.To attempt to antagonize this regulatory circuit, wetested the effect of PI3K and specific Akt inhibitorson viral replication and the survival of HIV-1 infectedmacrophages. Inhibitors of PI3K and Akt kinase,including miltefosine and perifosine which are cur-rently being tested in clinical trials, were able todrastically reduce HIV-1 production in macrophages,due to the increased incidence of cell death in HIV-1infected macrophages exposed to inhibitor. In conclu-sion, we propose that the expression of Tat may enableHIV-1 infected macrophages to serve as long-livingviral reservoirs, leading to persistent infection andHIV-1 production.

P.32Mechanisms of herpes simplex virus type 1 (HSV-1)infection of mice brain organotypic culturesMyetal Cohen,1 Efrat Braun,1 Amos Panet,2 andIsrael Steiner1

1Laboratory of Neurovirology, Neurological Sciences Unit,Hadassah University Hospital, Mount Scopus; 2Departmentof Virology, Hebrew University Medical School

Background: HSV-1 is an important cause of severemorbidity and mortality, including encephalitis. Themechanisms of HSV-1 penetration into the brain andits predilection to infect certain neuronal regions areunknown. We previously reported that HSV-1 has a re-stricted and specific pattern of infection of mice brainin organotypic cultures and that the neonate brain ismore permissive than the mature adult brain for infec-tion (Braun et al, J. Gen. Virol. 2006). We have usedthis system to follow and characterize HSV-1 infec-tion, the role of the extracellular matrix (ECM), and themechanism of viral access into the infected cell. Ma-terials and Methods: Following brain tissue extractionfrom neonate mice, the tissues were treated with ei-ther collagenase, hyaluronidase, heparin, heparinase,acyclovir or 6 different agents that interfere with en-docytosis and infected the mice brain tissue sliceswith HSV-1 mutants expressing reporter genes. Patternand extent of HSV-1 infection was characterized andmeasured. Infection with adenovirus was used as con-trol. Results; Collagenase, an enzyme responsible forcollagen degradation did not affect the pattern andextent of HSV-1 infection of brain tissue slices (com-pared to skin slices infected with HSV-1 that wereaffected), but hyaluronidase did. Heparinase abolishedinfection, as did pre incubation of HSV-1 with heparin.


Pre-treatment of the brain slices with acyclovir didnot prevent infection and reporter gene expression.Using the agents that interfere with endocytosis wewere able to demonstrate that infection takes placemainly via membrane fusion. Conclusions: Brain ECMplays a major role in brain infection in this system.HSV-1 infection is mediated, at least in part via ha-paran sulphate receptors and internalization of HSV-1is achieved mainly by membrane fusion.

P.33Subclinical reactivation and shed of infectious VZVin saliva of astranauntsRandall J Cohrs,1 Satish K Mehta,2 Donald H Gilden,1

D Scott Schmid,3,4 and Duane L Pierson4

1Department of Neurology and Microbiology, University ofColorado Health Sciences Center, Denver, CO; 2EnterpriseAdvisory Services, Inc., Denver, CO; 3National Aeronauticsand Space Administration, Lyndon B. Johnson SpaceCenter, Houston, TX; 4National VZV Laboratory, Centersfor Disease Control, Atlanta, GA

See Page 36

P.34Comparison of different HIV-1 isolates of clades Band C on macrophage-mediated inflammatory factor,glutamate production and neurotoxicityAgnes Constantino,1 Yunlong Huang,1 Alicia Lopez,1

Hong Zhang,2 June He,2 Jianxing Zhao,1 CharlesWood,2 and Jialin Zheng1,2

1CNND and Dept. of Pharmacology and ExperimentalNeuroscience, Univ. NE Med. Ctr., Omaha, NE; 2NebraskaCenter for Virology, University of Nebraska in Lincoln, NE

HIV-1 clade C is currently responsible for more than50% of new infections and is becoming the most com-monly transmitted subtype worldwide. While HIV-1-associated dementia (HAD) continues to be the majorneuropathological manifestation of AIDS among cladeB-infected individuals in the US and Europe, a lowincidence of HAD has been reported in countries likeIndia and sub-Saharan Africa, where clade C is preva-lent. Potential differences between clade B and cladeC regarding neurovirulence and macrophage-mediatedpathogenesis are yet to be investigated. Differen-tial regulation of proinflammatory factors and neuro-toxins (such as glutamate) by macrophages infectedby different clades/strains may differentially induceneuronal injury and dysfunction, consequently al-tering neuropathogenesis. Human monocyte-derivedmacrophages (MDM) were infected for 4, 7, 14, and 21days using the same TCID50 by 3 laboratory clade Bstrains, 4 primary clade B strains derived from HIV-1infected brain, choroid plexus or spleen, and 2 pairs

of clade C isolates from HIV-1 infected mothers (M)and infants (I). Viral infection was monitored by re-verse transcriptase (RT) activity and characterized byproinflammatory factors MIP-1beta, MCP-1 and TNF-alpha production as determined by ELISA. The con-centration of extracellular glutamate was measuredin MDM supernatants by RP-HPLC. Primary rat cor-tical neurons treated with conditioned media fromdifferent viral strain-infected MDM were used to as-sess macrophage-mediated neurotoxicity. Laboratorystrains (clade B) and 3 primary clade B strains (A-00-086, G0048 and D02-2562) reached highest levels ofviral infection 7–14 days after infection, while cladeC strains reached highest levels of viral infection at21 days post-infection. MIP-1beta and MCP-1 produc-tion was associated with HIV-1 infection as determinedby RT. The highest glutamate production and neuro-toxicity induced by different viral strains in infectedMDM ranked as: laboratory strains > primary clade Bstrains> primary clade C strains. Correlation analysiswith all strains tested suggests a correlation betweenRT, glutamate production, and neurotoxicity inducedby HIV-1 infected MDM.

P.35HIV infection inhibits GM-CSF-induced microglialproliferationMelissa Cosenza-Nashat,1 Meng-Liang Zhao,1 SusanMorgello,2 and Sunhee C Lee1

1Department of Pathology, Albert Einstein College ofMedicine, Bronx, NY; 2Departments of Pathology andNeuroscience, Mount Sinai School of Medicine, New York,NY

It is well known that infection by HIV dysregulatescell physiology, but little information is availableon the consequences of HIV infection in primarymacrophages and microglia. We examined the rela-tionship between cell proliferation and HIV infectionin primary cultures of microglia and in human CNS.In cultures infected with HIV (ADA and BaL), gran-ulocyte macrophage colony-stimulating factor (GM-CSF)-mediated cell proliferation was reduced in pro-ductively infected (p24+) cells as compared to p24−cells. The reduction was observed with both Ki67 andBrdU labeling, suggesting a G1/S block. The reduc-tion was insignificant when microglia were infectedwith a Vpr-mutant virus. In human CNS, proliferat-ing (Ki67+) cells were rare but were increased in theHIV+ and HIV encephalitis (HIVE) groups comparedto the HIV− group. We found a positive correlationbetween GM-CSF immunoreactivity and Ki67 counts,implicating GM-CSF as a growth factor in human CNS.The relationship between total macrophage (CD68+)proliferation and infected macrophage (p24+) prolifer-ation was assessed in HIVE by double labeling. While1.2% of total CD68+ cells were Ki67+, only 0.5% of


HIV p24+ cells were Ki67+ (p < 0.05). Furthermore,staining for CD45RB (as opposed to CD68) facilitatedthe identification of Ki67+ microglia, indicating thatCD68 could underestimate proliferating microglia. Weconclude that while there is increased expression ofGM-CSF and increased cell proliferation in the CNSof HIV-seropositive individuals, cell proliferation inthe productively infected population is actually sup-pressed. These data suggest that there might be a viralgain in the suppressed host cell proliferation.

P.36Dynamics of NF-kB activation by HIV-1 Tat andCD40L in microglial cellsMichelle M Crandall and Sanjay B MaggirwarUniversity of Rochester Department of Microbiology andImmunology

HIV-1-associated dementia (HAD) occurs in a signifi-cant number of HIV-1 infected individuals, in part, dueto the inflammatory response to viral proteins in thecentral nervous system. We have shown that host fac-tors, such as soluble CD40 ligand (sCD40L) that arefound to be elevated in the cerebrospinal fluid andplasma of HAD patients, synergize with HIV-1 encodedTat to activate microglial cells in an NF-kB-dependentmanner. Here we demonstrate that, besides proteolysisof the inhibitory molecules IkB-alpha, the activationof NF-kB by Tat in microglial cells is associated withde novo synthesis of RelB, and processing of p100 (asdefined by generation of the processing product p52),followed by translocation of RelB and p52 into the nu-cleus. However, under these conditions, neither RelB,nor p52, exhibited DNA binding activity at a cis-actingNF-kB responsive element. We further show that the si-multaneous exposure to Tat and CD40L promotes DNAbinding activity of p52, but not of RelB. Finally, ec-topic expression of processing-deficient mutant p100,as well as RelB-specific short hairpin RNA (shRNA),revealed that the biogenesis of p52 and RelB in Tat plusCD40L-treated microglia is crucial for the synthesis ofTNF-alpha. Taken together, these studies highlight themechanism by which viral proteins and host factors(like Tat and CD40L) work in concert to activate NF-kB in microglial cells and explain how this process isdynamically controlled.

P.37HIV-1 Tat inhibits NGF-induced Egr-1 transcriptionalactivity and consequent p35 expression in humanneuronal cells, resulting in apoptosisNune Darbinian, Marta Czernik, Kamel Khalili, andShohreh AminiCenter for Neurovirology, Department of Neuroscience,Temple University School of Medicine

In the context of neuroAIDS, the HIV-1 transactiva-tor protein Tat and the host neurotrophin NGF areimportant regulators of neuronal cell survival andare involved in the modulation of the cell cycle, dif-ferentiation, cytoskeleton dysfunction and apoptosis.We reported recently that Tat dysregulates the NGF-signaling pathway, including suppression of Egr-1, akey pleiotropic mediator of the expression of genesinvolved in cell growth. NGF induces the expressionof Egr-1, which in turn induces p35, an activator ofCDK5 by binding to its three Egr-1 binding sites in thepromoter region. Here, we investigate the impact ofHIV-1 Tat on the NGF-induced Egr-1 transcriptionalactivity in SK-N-MC neuroblastoma cells. Our datafrom Western-blot analysis show that Egr-1 and p35protein levels are elevated in NGF-treated SK-N-MCcells, but not in Tat-expressing cells. We have as-sessed functional interactions between Tat and Egr-1transcription factors and demonstrated that whereasTat itself has no strong effect on the p35 promoter,it weakly synergizes with Egr-1 to induce expressionof a p35 promoter-driven reporter in the absence ofNGF. Our results from reporter assays revealed inhi-bition of NGF-induced Egr-1 activity in NGF-treatedcells. NGF treatment of SK-N-MC cells increases thebinding of Egr-1 to the 232-bp element in the p35 genepromoter in ChIP assays, whereas expression of Tatprotein reduces binding to p35 promoter. Tat also in-hibits binding of Egr1 to Egr1-binding oligonucleotidedriven from p35 promoter in electrophoretic mobilityshift assay (EMSA). Our results from GST-pull downassays demonstrated that Tat physically interacts withEgr-1 through its central part and, thus, interferes withits NGF-induced activation of p35. Deletion of the ba-sic amino acid region of Tat results in the loss of itsbinding ability to Egr-1, and can no longer affect Egr-1-mediated transactivation of the p35 promoter. Thesedata suggest that in NGF-treated Tat-expressing cells,Egr-1 loses its ability to up-regulate p35 gene tran-scription and activate Cdk5/p35 complex, and thiscan contribute to the dysregulation of NGF signalingin neuronal cells. Cells expressing Tat produce lessphosphorylated neurofilament NF-H, a substrate of theneuron-specific p35/Cdk5. This reduction was corre-lated with a decreased level of Cdk5 and low kinaseactivity of the p35/Cdk5, complex which plays a crit-ical role in neuronal cell differentiation and survival.Tat had an apoptotic effect in neuronal cells in thepresence of NGF, as shown by the increased level ofthe pro-apoptotic proteins Bax and Bad, Comet as-say and DNA fragmentation assay. Tat also increasedthe level of the mitochondrial porin in NGF-treatedcells, which plays an important role in apoptosis byrecruiting pro-apoptotic proteins of the Bcl2 family tomitochondria. NGF treatment of Tat-expressing cellsresulted in the increase of cleaved active caspase-3 level and induction of caspase-3 activation in thecaspase-3 GLO assay. Altogether these data demon-strate that Tat dysregulates p35/Cdk5 signaling, in-hibits the phosphorylation of the neuroskeletal protein


NF-H, activates pro-apoptotic proteins and caspase-3cleavage, resulting in neuronal cell death.

This work was supported by NINDS grant awardedto SA

P.38Possible role of Puralpha in neurovasculardysfunction in Alzheimer’s disease through theinhibition of Meox2 expressionNune Darbinian,1 Marta Czernik,1 Rafal Kaminski,1Judith Miklossy,2 Armine Darbinyan,1 and KamelKhalili1

1Center for Neurovirology, Department of Neuroscience,Temple University School of Medicine; 2KinsmenLaboratory of Neurological Research, University of BritishColumbia

Alzheimer’s disease is the major cause of demen-tia in the elderly population. The exact causes ofAlzheimer’s disease are unknown, and it is likelythat the etiology of the disease is heterogeneous.Neurovascular dysfunction is an important feature ofAlzheimer’s disease and could have a major impact onthe pathogenesis of a chronic neurodegenerative con-dition. Mesenchyme Homeobox 2 (Meox2) regulatesthe transcription of genes necessary for many vascularcell processes, tube formation and differentiation ofvascular smooth muscle cells and cardiomyocytes.Genes required for the cytoskeleton (SM alpha-actin),cell cycle (Cyclins, p21) and cell adhesion (integrins,uPA/uPAR, and Ephrins/Eph) are all downstreamtargets of Meox2. Meox2 is downregulated in theendothelial cells from blood vessels in Alzheimer’sdisease patient’s brains. Decreased Meox2 levelsreduced neurovascular angiogenesis in the Meox+/-mice. Low levels of expression of Meox2 may havean important role in neurovascular dysfunction inAlzheimer’s disease resulting from the reduction ofbrain capillary density and vessel regression. The linkbetween decreased Meox2 expression and impairedneurovascular functions in Alzheimer’s disease makesmeox2 homeobox gene an attractive target for thetreatment of the disease. To analyze the factors whichmay play a role in the downregulation of Meox2 is im-portant and will help to develop novel treatments fordiseases that result from improper vessel function.Puralpha, a multifunctional protein, implicated ina variety of biological events, including transcrip-tion, replication and cell cycle, negatively regulatessmooth muscle alpha-actin and beta-myosin geneexpression in fibroblast and vascular smooth musclecells, suggesting that it may play an important rolein neurovascular formation. A possible link betweenlow expression of Meox2 and impaired neurovascularfunctions in Alzheimer’s disease, control of smoothmuscle actin gene transcription by Meox2 and in-hibitory regulation of SM alpha-actin by Puralpha,provided a rationale to analyze possible role of Pural-pha in Meox2 expression, neurovascular dysfunction

and Alzheimer’s disease. Microarray assay from thecells stably expressing nuclear Puralpha demonstratedgreater than 9 fold downregulation of meox2 gene.Inhibition of the level of expression of Meox2 proteinwas confirmed by Western blot analysis of cells ex-pressing Puralpha. Expression of Meox2 was also lowin mouse brain extracts, where a high level of Puralphawas present. In fibroblast cells expressing Puralphaa weak staining of Meox2 was observed. Study of themeox2 promoter and gene structure revealed multiplePuralpha GC-rich binding sites in the promoter region,including the 5′-UTR. Interestingly, antisence meox2DNA contains eleven (GGT) repeats, known as aclassical Puralpha-binding domain. Results from invitro transcription/translation studies demonstratedthat Puralpha blocked Meox2 translation. Altogether,in this study we have demonstrated that Puralphainhibited the expression of Meox2. This observation,suggests that Puralpha may play an important rolein neurovascular dysfunction in Alzheimer’s diseasethrough the inhibition of Meox2 expression, pro-viding new insights for possible therapeutic targetsin the treatment of Alzheimer’s disease and a betterundersatanding of this disorder.

This work was supported by NINDS grants awardedto KK and SA

P.39Inflammation and neurogenesis in an experimentalmodel of Japanese EncephalitisSulagna Das and Anirban BasuNational Brain Research Centre, Manesar, India

Most of the CNS infections and the brain disorders thatwe encounter are associated with CNS inflammationand neurodegeneration. In fact, neuroinflammationhas become the hallmark of most of the CNS infectionby neurotropic viruses (e.g. HIV, JEV etc.) Here wedemonstrate how following Japanese encephalitisinfection there is robust microglial activation and amajor upheaval in the level of the pro-inflammatorymediators (the cytokines, chemokines and the reac-tive oxygen and nitrogen species), which representsthe profound neuroinflammatory condition. Thesevarious mediators have been shown to be detrimentalfor the neuronal survival and eventually bring aboutneuronal death. Interestingly, the enhancement in lev-els of these pro-inflammatory mediators was mainlyobserved in the adult neurogenic regions of the brain,the subventricular zone and the dentate gyrus of thehippocampus. Apart from the mature neurons beingthe target of JEV, it was also investigated whethercognitive deficits that have been observed in thesurvivors of JE can be attributed to the deficits in neu-rogenesis and malformation of new neurons from theneural stem/progenitors in these neurogenic regions.In vivo studies have clearly demonstrated that thereis a decrease in the proliferating neuronal progenitorpool as well as in the immature neuron formation.


Neurospheres cultured from P7/P8 mouse pups wereused to assess the impaired neurogenesis pattern in JEinfected animals as compared to control ones. A vividdecrease in the proliferative ability of the progenitorcells were noticed in terms of the generation potentialof the spheres isolated from JE infected animals.Furthermore, neurospheres cultured in presence ofconditioned media obtained in JE-activated microgliashow arrested growth and distorted morphology.Contradictorily, in vitro JE infection to the spheresdid not render a decrease in the progenitor populationas indicated by the percentage of nestin positivepopulation. Moreover, no direct apoptotic death ofthe neuronal progenitor cells was also observed afterin vitro JE infection, probably suggesting that inflam-mation following JEV has a key role in determiningthe fate of the neuronal stem/progenitor proliferationand differentiation.

Acknowledgement: This work was supported bygrants awarded to AB from the Department of Biotech-nology, Government of India: Award #BT/PR/5799/MED/14/698/2005 and BT/PR8682/Med/14/1273/2007

P.40Molecular characterization of JC virus genomicregion encoding Viral Protein 1 outer loops andevaluation of JCV viral load in CSF of patients withdifferent forms of PMLSerena Delbue,1 Sara Tremolada,1 EmanuelaBranchetti,1 Giulia Minnucci,1 Eleonora Tavazzi,2Enrico Marchioni,2 and Pasquale Ferrante1,3

1Dept.of Science and Biomedical technology, University ofMilan, Segrate, Italy; 2Neurology Department, MondinoHospital, IRCCS, Pavia, Italy; 3Center for traslationalmedicine, Saint Joseph Hospital, Milan, Italy

After the introduction of Highly Active Antiretrovi-ral Therapy (HAART), the prognosis of ProgressiveMultifocal Leukoencephalopathy (PML) has changed,becoming variable, and thus reliable and easy-to-usemarkers of PML clinical evolution are needed. The pur-pose of our study was to determine whether or not spe-cific markers, such as amino acid substitutions in thegenomic region encoding Viral Protein 1 (VP1) outerloops and viral load of JC Virus (JCV), isolated fromCerebrospinal Fluid (CSF) of patients with differentforms of PML, could be associated with the progres-sion of the disease.

In the CSF collected from 21, both HIV+ andHIV-, PML patients (9 Fast Progressors-FP- and 12Slow Progressors-SP), JCV viral load was evaluatedby means of specific RealTime PCR. In addition, JCVVP1 outer loops were amplified in CSF collected from8 patients and in urine collected from 10 controls,cloned, sequenced and translated using the ExPASysoftware.

JCV viral load was significantly higher (p < 0.001)in CSF of FP PML patients (5.51 log copies/ml) than

in CSF of SP PML patients (4.14 log copies/ml). Allthe JCV strains isolated from CSF of SP PML patientsshowed specific amino acid mutations at four definedhot spots in the VP1 genomic region encoding the outerloops (amino acid positions 75, 117, 128, 167). No sig-nificant difference from Archetype strains were foundin the VP1 loops isolated from the urine.

Taken together, the results confirm that high CSF JCVviral load is predictive of poor outcome of PML pa-tients, whereas some specific polymorphisms of VP1loops are associated with longer survival of patients.Among slow progressors, mutation at Arg-75, recentlysuggested to be involved in cell recognition and re-sulting in non-viable virus, is highly frequent. Thuswe could hypothesize that the alteration at the de-fined hot spots determines a decrease in the viral ac-tivity with the consequence of a slow progression ofthe disease.

Supported by grant from NIH no. R01MH72528 toPF

P.41Expression of JCV T-antigen in glioblastomamultiforme. Nuclear translocation of IRS-1 andalteration in homologous recombination dependentDNA repairLuis Del Valle, Katarzyna Urbanska, Kamel Khalili,and Krzysztof ReissDepartment of Neuroscience, Center for Neurovirology.Temple University School of Medicine

Despite significant advances in surgical techniquesand development of new chemotherapeutic agents,Glioblastoma Multiforme (GBM), the most aggressiveand frequent of brain tumors, is always associated withpoor prognosis. One of the hallmarks of glioblastomais genomic instability, which inevitably leads to thedevelopment of new cellular adaptations. Therefore,it is possible that basic DNA repair mechanisms re-sponsible for the maintenance of genomic integritycould be impaired in GBMs. We have previously re-ported detection of the early oncoprotein of humanpolyomavirus JC, large T-Antigen, in the nuclei of neo-plastic cells in 25 of 76 glioblastoma cases examined;and the involvement of JCV T-Antigen in the inhibi-tion of homologous recombination DNA repair (HRR),which seem to require the presence of the cellular pro-tein, insulin receptor substrate 1 (IRS-1). Our ongoingstudies demonstrate an active IGF-IR-IRS-1 signalingsystem in both JCV T-Antigen positive and negativeglioblastoma clinical samples. This included a strongmembrane associated immunolabeling of a total, aswell as tyrosine phosphorylated (pY1316) IGF-IR, andthe accumulation of cytosolic IRS-1 in comparison tohealthy brain tissue. Interestingly, we have detecteda strong nuclear IRS-1 in approximately 30% of neo-plastic cells in clinical samples of GBM, which werealso expressing JCV T-Antigen. Double labeling studies


demonstrated nuclear co-localization between IRS-1and JCV T-Antigen. In addition, immunolabeling withthe antibody, which detects the phosphorylated formof histone H2AX (?H2AX, a marker of DNA damage)showed a much stronger immunolabeling in the nucleiof JCV T-Antigen positive glioblastoma cells, as wellas co-localization between ?H2AX and nuclear IRS-1,and between nuclear IRS-1 and the major enzymaticcomponent of HRR, Rad51. In summary, our findingsindicate the involvement of JCV T-Antigen – IRS-1 sig-naling axis in homologous recombination of activelygrowing glioblastomas, a critical mechanism for the fi-delity of DNA repair during DNA replication, whichcould provide a novel target for the treatment of thesehighly aggressive cerebral tumors.

Supported by Grants from the NIH awarded to LDVand KR.

P.42Unique features of measles encephalitis in theromanian HIV cohortDan Duiculescu,1 Luminita Ene,1 EugeniaUngureanu,1 Gratiela Tardei,1 Harry V Vinters,2 RonEllis,3 and Cristian L Achim4

1“Dr. Victor Babes” Hospital for Infectious Diseases,Bucharest, Romania; 2Department of Laboratory Medicine,UCLA, Los Angeles, CA, USA; 3Department ofNeuroscience, UCSD, La Jola, CA, USA; 4Department ofPsychiatry, UCSD, La Jolla, CA, USA

Background: Subacute measles encephalitis was usu-ally described as a rare complication of measles inseverely immuno-supressed patients. During two con-secutive measles outbreaks in Romania, we observeda specific neurologic clinical picture, characterizedby myoclonus, occurring in HIV infected children.We decided to study the new clinical entity, that wecalled SMME, based on epidemiologic, clinical, lab-oratory and postmortem pathologic data in HIV in-fected patients followed at the “Victor Babes” Hospitalin Bucharest, Romania.

Methods: We included in our study 34 HIV infectedchildren and adolescents: 22 from the 1997-1998 and12 from the 2006-2007 measles epidemics. The inclu-sion criteria were: a previous measles episode or pre-sumed contact with measles virus, clinical features ofmyoclonus associated with motor deficits and stablemental status at onset, followed by rapid progress todeath. The definitive diagnosis was based on immuno-cytochemistry for measles virus in paraffin embeddedbrain autopsy tissues.

Results: The average time from exposure to measlesor the onset of clinical disease to SMME was 89 days(range 42-300). Clinical measles diagnosis was madein 12 patients (35%), 5 with atypical features. Themedian CD4 cell count was 44. The onset of SMMEwas sudden in the majority of patients with unilateralcontinuous myoclonus and preserved mental status.

Myoclonic jerks expended then rapidly, followed bysevere motor deficit, altered mental status and coma.The mean survival time from the onset of SMME was19 days (5-66). Serology for measles was positive in 28of 34 patients. Neuroimaging revealed unilateral focalareas of high T2 and FLAIR signal. At autopsy, im-munohistochemistry for measles was positive in allcases studied.

Conclusions: The key clinical feature in our studywas myoclonus and it occurred between 2 and 9months post-exposure to the measles virus, usuallyin the absence of clinical signs of acute measles dis-ease. This is particularly critical in the HIV infectedpopulation in limited resources settings. In the HIVinfected cohort we have studied, 60% of the patientsdid not have protective antibodies to measles, despitethe fact that vaccination is mandatory in Romania. Theabsence of protective levels in immunosupressed pa-tients indicates the need for aggressive prophylactictherapies during measles epidemics. This is particu-larly critical in the HIV infected population in limitedresources settings.

P.43Inhibition of synaptic proteins by HIV-1 Tat involvesthe activity of microRNADavide Eletto,1 Giovanni Passiatore,1 GiuseppeRusso,2 Luis Del Valle,1 and Francesca Peruzzi1

1Temple University School of Medicine and Center forNeurovirology, Philadelphia, PA, and 2Temple UniversitySbarro Institute for Cancer Research, Philadelphia, PA

See page 9

P.44Use of protease inhibitors does not independentlycontribute to distal polyneuropathy in HIV infectionRonald J Ellis,1 Jennifer Marquie-Beck,1 PatrickDelaney,1 Terry Alexander,1 Chris Ake,1 DavidClifford,6 Justin McArthur,2 David Simpson,3 Ann CCollier,4 Benjamin B Gelman,5 J. Allen McCutchan,1Susan Morgello,3 and Igor Grant1

1University of California, San Diego; 2Johns HopkinsUniversity; 3Mt. Sinai School of Medicine; 4University ofWashington, Seattle, Washington; 5University of TexasMedical Branch, Galveston; 6Washington University, St.Louis, Missouri

Background. A recent analysis (Pettersen et al, 2006)found that exposure to HIV protease inhibitors (PI),an important component of modern combinationantiretroviral (ARV) therapy (ART), was associatedwith an increased likelihood of distal polyneuropathy(DPN) in patients with HIV infection. In dorsal rootganglion sensory neurons, PI exposure resulted insignificant neurite retraction and process loss, sug-gesting neuronal toxicity. If confirmed, these findings


could prove to be a substantial limiting factor in thelong-term use of protease inhibitors in HIV-infectedpatients.

Methods. We evaluated current and past exposure toPIs as a risk factor for sensory neuropathy in 1159 HIV-infected individuals enrolled in a large, NIH-funded,multicenter, cohort study. All subjects underwent acomplete neurological exam to determine whethersigns of DPN were present, including diminished abil-ity to sense vibration on bilateral great toes, decreasedability to discriminate between sharp and dull,and absent or weakened ankle reflexes. Subjects weregrouped into 5 categories according to past and currentexposure to any ARV and to PIs into the following cat-egories: Group 1 - past ARV use/no PIs (n = 64), Group2 - past ARV and PI use (n = 107), Group 3 - currentlyon ARVs/never used PIs (n = 170), Group 4 - currentlyon ARVs/past PI use (n = 147), Group 5 - currently onARVs and PIs (n = 507). The odds of having DPN werecalculated for each group relative to a sixth group ofsubjects who were ARV naıve (n = 164). Concomitantrisk factors were evaluated in multivariate models,including age, exposure to dideoxynucleoside reversetranscriptase inhibitors (d-drugs) and markers of HIVdisease progression (CD4 nadir, plasma viral load,and duration of HIV infection).

Results. Relative to individuals never exposed toARVs the odds for having DPN were significantlyhigher for all groups except group 1. Subjects currentlyon PIs or who had been exposed to PIs were more likelyto have DPN relative to ARV naıve subjects (OR [95%CI] Group 1: 1.4 [0.8-2.5], Group 2: 2.2 [1.4-3.7], Group3: 2.8 [1.8-4.4], Group 4: 3.8 [2.4-6.1], Group 5: 4.0 [2.8-5.9]). However, in a multivariate model accounting forother significant concomitant risks, no group was morelikely to have DPN compared to ARV naıve subjects(adjusted OR [95% CI] Group 1: 0.6 [0.2-1.5], Group 2:0.9 [0.4-2.1], Group 3: 1.4 [0.7-2.7], Group 4: 1.1 [0.5-2.3], Group 5: 1.6 [0.9-2.7]).

Conclusions. Evaluation of confounding risk factorsfor neuropathy in HIV infection suggests that the in-dependent risk attributable to PI exposure, if any, issmall. This risk must be weighed against the impor-tant role of PIs in modern ART regimens.

P.45Decreased CSF HIV load in patients with subacutemyoclonic measles encephalitisLuminita Ene,1 Eugenia Ungureanu,1 CristianaOprea,1 Roxana Radoi,1 Gratiela Tardei,1 SimonaTetradof,1 Raluca Cigoianu,1 Raluca Burlacu,1Cristian L Achim,2 and Dan Duiculescu1

1“Dr. Victor Babes” Hospital for Infectious Diseases,Bucharest, Romania; 2Department of Psychiatry, UCSD, LaJolla, CA, USA

Background: It has been previously reported that theplasma HIV load drops significantly during acute

measles infection. However, there are no data on cere-brospinal fluid (CSF) HIV RNA in patients with acutemeasles and neurological complications of measles.We planned to measure HIV RNA levels in the CSFand plasma in a group of Romanian adolescents diag-nosed with subacute myoclonic measles encephalitis(SMME).

Methods: We studied 12 HIV positive, severelyimmunosupressed patients, diagnosed with SMME.The neurologic symptoms consistent with SMMEstarted after a median period of 80 days (range 40-304) post exposure to the measles virus. CD4 cellcounts and HIV plasma and CSF viral load were mea-sured at the time when neurological complicationsstarted.

Results: All 12 patients had severe immune sup-pression; at the time of SMME diagnosis median CD4cell count was 18 (range 1-231). Eleven patients wereon HAART at the debut of neurologic symptoms, 7of them with virological failure. Analysis of pairedplasma-CSF samples at the time of SMME diagnosisshowed that the median HIV RNA load in the CSF was344 copies/ml (range 112-779) while the plasma me-dian level was 34440 copies/ml (range 178-1000000).All patients had preserved blood-brain barrier. Clini-cal diagnosis of measles was established in 6 patients,half of them with uncommon clinical presentations.Dynamic measurements of plasma HIV RNA availablein 4 patients revealed a temporary ten-fold decrease atthe time of measles diagnosis.

Conclusions: The strikingly low CSF HIV RNA lev-els in patients with SMME suggest an inhibitory ef-fect of measles on HIV replication in the central ner-vous system. We propose that a significant drop inthe HIV CSF burden in severely immunosupressed pa-tients may indicate the presence of a serious oppor-tunistic brain pathology, SMME, that can affect a largepopulation in limited resource settings.

P.46Evidence that morphine intensifies oxidative andsynaptic damage in gp120-transgenic miceOsefame Ewaleifoh,1 Ratnam VR Bandaru,1 FangYang,1 Avindra Nath,1 Fang Yang,1 and Joe Steiner1

1Johns Hopkins University School of Medicine. Departmentof Neurology. Division of Neuroimmunology; 2JohnsHopkins University School of Medicine. Department ofNeurology. Division of Neuroimmunology

Nearly 30% of injection drug users are infected withthe human immunodeficiency virus (HIV), and nearlyhalf of the HIV seropositive women in the UnitedStates have contracted the infection via drug use.Although opioids are not generally neurotoxic, opi-oids can synergize with toxic agents, including HIV-proteins, to exacerbate neuronal damage. In this studywe sought to determine if morphine exacerbates neu-ronal damage in mice transgenic for gp120. Morphine,


or placebo tablets were implanted subcutaneously intogp120- and non-transgenic mice. Levels of antioxi-dants, reactive oxygen species, sphingolipids, phos-pholipids and synaptic proteins were measured fromcortical tissue on days 4, and 7 after implant to de-termine the effects of chronic morphine administra-tion, and withdrawal from morphine. On day 4, mor-phine levels in plasma were 2.7–3.2 ng/ml. Morphinelevels were undetectable on day 7, and were not de-tected in animals administered placebo. Gp120-, andnon-transgenic mice administered placebo had sim-ilar levels of cellular oxidation, but sphingomyelinswere decreased and ceramides increased in gp120-compared with non-transgenic mice. Levels of thepre-synaptic protein PSD-95 were lower in gp120-compared with non-transgenic mice. After 4 days ofmorphine, there was evidence of oxidative stress ingp120-, and non-transgenic mice. In non-transgenicmice administered morphine there were trends towarddecreased sphingomyelin, and increased ceramidein rodents. In gp120-transgenic mice administeredmorphine, sphingomyelin levels were increased, andceramide levels decreased compared to placebo.During drug withdrawal, redox, sphingomyelin, andceramide levels normalized to a greater extent innon-transgenic compared with gp120-transgenic mice.PSD95 was decreased in gp120-transgeic mice com-pared to all other groups of animals. These find-ings suggest that the additional perturbation ofgp120 promotes synaptic damage during morphinewithdrawal.

P.47Functional dysregulation of dopamine transmissionand the dopamine transporter associated with theHIV-1 Tat protein and repeated cocaineadministrationMark J Ferris,1 Danielle Frederick-Duus,2 Jim Fadel,2

Charles F Mactutus,1 and Rosemarie M Booze1,2

1Department of Psychology, University of South Carolina;2Department of Pharmacology, Physiology, andNeuroscience, University of South Carolina School ofMedicine

Injection drug use, including the use of cocaine, isa leading risk factor in the transmission of HumanImmunodeficiency Virus (HIV). Injection drug use isresponsible for one-third of all HIV-infections in theUnited States. Furthermore, cocaine abuse and HIVare known to alter brain dopamine (DA) systems. Todate, little research has been aimed at directly investi-gating the interaction of cocaine-associated DA systemneuroplasticity with HIV-induced DA system toxicityin the context of in vivo DA transmission. In vivobrain microdialysis is a technique that is ideally suitedfor direct monitoring of neurotransmitter alterations.

Thus, a series of experiments using traditional, re-verse, and zero-net-flux microdialysis were designed

to elucidate HIV-1 Tat-induced alterations in DA trans-mission and the DA transporter (DAT), both alone andin the presence of repeated cocaine administration. Inexperiment one, a 33% reduction in potassium-evokedDA overflow was observed in Tat-treated rats relativeto vehicle-control. In experiment two, a significant re-duction in DA efflux to cocaine challenge (10 mg/kgi.p.) was demonstrated in chronic cocaine + acute Tattreated animals relative to cocaine + vehicle treatedanimals. Experiment three demonstrated alterations inDAT function in cocaine + vehicle treated animals andcocaine + Tat treated animals relative to saline + ve-hicle treated animals.

Together, these experiments replicate the plasticityassociated with DA transmission and DAT functionupon repeated cocaine administration. Furthermore,these experiments demonstrate that DA transmissionand DAT function in the HIV-positive cocaine abusingpopulation may be more severely compromised thanthe HIV-positive non-drug abusing population.

P.48High affinity binding of Vpr to the HIV-1 LTR atC/EBP site I is correlated with HADAdriano Ferrucci, Evelyn M. Kilareski, Yujie Liu,Sara Webster, Michael R. Nonnemacher, and BrianWigdahlDrexel University College of Medicine

Human immunodeficiency virus type 1 (HIV-1) viralprotein R (Vpr) is a virion-associated protein thattransactivates the HIV-1 long terminal repeat (LTR).The electrophoretic mobility shift assay has been uti-lized to demonstrate the direct binding of purified Vpr(strain pNL4-3) to HIV-1 LTR sequences that span theadjacent CCAAT/enhancer binding protein (C/EBP)site I, NF-kappa B site II and ATF/CREB binding site(nt -95 to -130, relative to the start of transcription).Binding between HIV-1 Vpr and the LTR C/EBP siteII (nt -167 to -175) has also been observed. A totalof 94.7% of LTRs derived from peripheral blooddisplayed high relative Vpr binding affinity at C/EBPsite I, while only 5.3% exhibited a low relative Vprbinding affinity phenotype. All LTRs derived fromperipheral blood exhibited a high relative Vpr bindingphenotype at C/EBP site II. These results suggesta preference for the maintenance of two cis-actingelements with high affinity for Vpr within LTRs de-rived from peripheral blood in late stage disease andduring development of HIVD. Additional studies havealso demonstrated that naturally occurring sequencevariation within C/EBP site I and II that correlates withdisease progression can dramatically alter the relativeaffinity of Vpr for these cis-acting elements. Furtherstudies have also suggested a competitive interactionbetween Vpr and C/EBP factors for binding sites I andII. These studies indicate that Vpr may regulate the


interaction of members of the C/EBP transcriptionfactor family with the viral LTR.

P.49CD163/CD16 co-expression by circulatingmonocytes/macrophages in HIV-1 infection: potentialrole in the pathogenesis of HIV CNS diseaseTracy Fischer-Smith,1 Christie Bell,1 Ellen Tedaldi,2

Sidney Croul,1 Susan Morgello,3 and Jay Rappaport1

1Department of Neuroscience; 2Department of Medicine,Temple University School of Medicine, Philadelphia, PA,USA 19122; 3Mount Sinai School of Medicine, New York,NY, USA

The accumulation of macrophages in the CNSof patients with HIV encephalopathy (HIVE) pro-vides strong evidence for the role of increasedmonocyte/macrophage trafficking in its pathogene-sis. CD163, a monocyte/macrophage specific scav-enger receptor for hemoglobin-haptoglobin complex,is reported to be expressed in the CNS by perivas-cular macrophages but not by resident microglia inhuman brain. Recently, we utilized this disparity inCD163 expression by brain macrophages to furthercharacterize the MPs in the CNS in HIVE. CD163 ex-pression was seen by cells that accumulate withinperivascular cuffs as well as by parenchymal cellswith microglial morphology in HIVE, suggesting thatthe numerous parenchymal microglia seen in HIVEare derived from cells that have recently emigratedfrom the peripheral blood into the CNS compart-ment. Moreover, these cells are largely CD16+andappear to be the sole source of productive HIV-1 in-fection in the CNS. In a separate study, flow cytom-etry analyses of PBMC from HIV infected volunteersshowed a positive correlation between CD163+/CD16+monocytes and viral load that was not observed be-tween CD4+ absolute T cell counts and viral load.Even more significant was our observation of a stronginverse correlation between CD163+/CD16+ mono-cytes and CD4+ absolute number of T cells whenT cell counts drop below 450. Alterations in bloodmonocyte homeostasis may contribute to AIDS patho-genesis and trafficking of monocytes into the CNSand patients who develop HIVE. In these individ-uals, the CD163+/CD16+ subset may contribute tothe development of cognitive dysfunction by bringingvirus into the CNS as well as through secreted fac-tors and by-products associated with their activationstatus.

P.50Developmental neurotoxicity of HIV-1 proteins in therat hippocampal formation: A design-basedstereological study

Sylvia Fitting,1 Rosemarie M Booze,1,2 UllaHasselrot,1 and Charles F Mactutus1

1Program in Behavioral Neuroscience, Department ofPsychology, University of South Carolina; 2Department ofPhysiology, Pharmacology, & Neuroscience, University ofSouth Carolina

Design-based stereology was used to examine the roleof neonatal intrahippocampal injections of HIV-1 pro-teins, gp120 and Tat, on neuroanatomy of the hip-pocampus in adult rats. Postnatal day (P)1-treatedSprague-Dawley rats were bilaterally injected with ve-hicle (VEH) (0.5μl sterile buffer), gp120 100ng, Tat25μg or combined gp120+Tat (100ng+25μg). UsingNissl-stain, estimated total neuron number were quan-tified in five subdivisions of the rat hippocampus[granual layer (GL), hilus of the dentate gyrus (DGH),cornu ammonis fields (CA)2/3, CA1, and subiculum(SUB)], and glial number (astrocytes and oligodendro-cytes) in two subdivisions (DGH and Sub). Overall,results demonstrated a significant reduction of neu-ron number in the CA2/3 subfield by Tat and gp120,[F(1, 16) = 7.6, p < 0.05 and F(1, 16) = 7.2, p <0.05, respectively] and a significant reduction in theDGH by Tat only [F(1, 16) = 5.4, p < 0.05]. Therewas a corresponding significant increase in glial num-bers with gp120 and Tat demonstrating differential ef-fects. For astrocytes a significant gp120xTat interac-tion was noted in the DGH [F(1, 16) = 32.7, p < 0.01]and supported by a significant Tat effect [F(1, 16) =10.4, p < .01] but no effect for gp120. For oligoden-drocytes in the DGH and astrocytes in the SUB a sig-nificant effect was noted only for Tat [F(1, 16) = 12.1,p < .01 and F(1, 16) = 9.1, p < .01]. Collectively, re-sults suggest differential effects of the HIV-1 proteins,gp120 and Tat, on the total number of neurons, aswell as on the expression of glia cells. Potential re-lationships to behavioral observations in the animalswill be examined. (Support by DA013137, DA014401,HD043680).

P.51Use of the DrexelMed HIV/AIDS cohort to identifyviral markers predictive of disease progression andneuroAIDSKatherine Flaig, Bryan P. Irish, Evelyn M. Kilareski,Jeffrey Jacobson, Michael R. Nonnemacher, and BrianWigdahlDrexel University College of Medicine

The long terminal repeat (LTR) regulates humanimmunodeficiency virus type 1 (HIV-1) viral geneexpression via interactions with multiple viral andhost transcriptional control factors. Previous studieshave examined sequence variation at CCAAT enhancerbinding protein (C/EBP) sites I and II, and Sp sitesI, II, and III in peripheral blood (PB)-derived LTRsfrom HIV-1-infected patients representing increasing


degrees of disease severity. The 3T configuration ofC/EBP site I (C-to-T change at position 3) and 5T con-figuration of Sp site III (C-to-T change at position 5)were variants found in low frequencies in PB-derivedLTRs derived from patients at early stages of HIV-1 disease, and at relatively high frequencies in pa-tients in late stage disease. Sequence variation at thesesites was also examined in LTRs derived from vari-ous brain compartments of patients with and withoutHIV-associated dementia. The 3T C/EBP site I and 5TSp site III were identified in brain-derived LTRs frompatients diagnosed with HIVD, but was absent in pa-tients without dementia. To assess whether these orother correlative or predictive viral markers could beidentified in a larger patient population in the era ofhighly active antiretroviral therapy (HAART), HIV-1-positive patients from the Drexel University Collegeof Medicine HIV/AIDS Clinic have been enrolled ina longitudinal study to examine the change in preva-lence of specific viral genetic markers. Herein, an ini-tial analysis of the DrexelMed HIV/AIDS cohort for thepresence of specific configurations of HIV-1 LTR tran-scription factor binding sites is presented.

P.52Macrophages and dopamine: a role in NeuroAIDSPeter J Gaskill, Eliseo A Eugenin, Tina M Calderon,and Joan W BermanDepartment of Pathology, Albert Einstein College ofMedicine

Human Immunodeficiency Virus (HIV) infection ofthe central nervous system (CNS) can result in a va-riety of neuropathological complications, includingminor cognitive motor disorder and dementia, whichaffect a significant number of HIV positive individuals.One of the major pathological hallmarks of these com-plications is trafficking and accumulation of mono-cyte derived macrophages (MDM) in the CNS. HIVinduced neurological complications are compoundedby dopaminergic drugs, such as cocaine and metham-phetamine, that synergistically increase the pathol-ogy of HIV infection in the CNS. Recent studies haveshown that increased extracellular dopamine expres-sion enhances viral expression and neuropathologyin SIV infected macaques, but the mechanisms forthis enhancement remain unclear. As MDM are theprincipal targets for HIV in the CNS, it is impor-tant to examine the role of these cells in this en-hanced neuropathogenesis. We hypothesize that MDMexpress receptors for dopamine and that dopaminetherefore modulates the functions of both infected anduninfected macrophages in the CNS. In addition, wesuggest that HIV infection dysregulates macrophagefunction, resulting in changes in CNS dopamine lev-els, altering the susceptibility of macrophages to HIVinfection and enhancing neuropathogenesis. Our pre-liminary data indicate that MDM express dopaminereceptors 1 and 2, the dopamine transporter and tyro-

sine hydroxylase. We also show that the expression ofthese proteins in MDM is altered by HIV infection, andthat the presence of dopamine during initial infectionenhances HIV infection levels in MDM.

P.53National NeuroAIDS tissue consortiumBenjamin B Gelman,3 Elyse J Singer,4 Igor Grant,5Seth Sherman,1,2 Susan Morgello,6 andDiane K Brandt1,2

1NNTC National Coordinating Office; 2The EMMESCorporation; 3University of Texas Medical Branch atGalveston; 4University of California, Los Angeles;5University of Californai, San Diego; 6Mount Sinai Schoolof Medicine

NIH established the multi-site NNTC in 1998 toprovide neuroAIDS researchers with high qualitybrain and fluid samples from well-characterized HIV-infected patients. In meeting this goal, more than 2873tissues specimens have been shipped for 206 requestssubmitted by 124 different investigators.

The NNTC has become an invaluable resourcethat uniquely expands the field of translational Neu-roAIDS research by making available to both newand established investigators neurologically focusedtissues linked to extensive antemortem clinical data.Nervous and systemic tissue, fluids, and/or associ-ated databases are available to qualified investigatorsworldwide at no charge upon approval of their ap-plication. Consortium-wide protocols are adhered toin the preparation of specimens as well as in thecollection and archiving of associated clinical data.The NNTC collects, stores and distributes specimensfrom the nervous system (brain, spinal cord, peripheralnerve, CSF), muscle, liver, blood and other tissues fromHIV-infected individuals. Linked neuropsychological(e.g., cognitive testing), neuromedical (e.g., neurolog-ical deficits, history of ART use), neuropathologicaland laboratory (e.g., viral loads, CD4 counts) data arealso available.

Since the formation of the NNTC in 1998, signifi-cant changes have occurred in HIV treatment strate-gies and research methods that have an effect on fu-ture NNTC initiatives. Potent antiretroviral treatmentregimens have resulted in the growth of a survivinglongitudinal cohort for which extensive clinical datais available. The NNTC is adapting to these changesby evaluating its methods and resources to ensure itremains an effective ongoing resource to the researchcommunity. The evolving NNTC cohort now presents aunique opportunity more epidemiological studies ex-amining the interactions between aging and HIV andfactors that predict long-term survival. In addition cor-relating neurological progression with CNS pathology,immunology, and virology at autopsy remain a corepotential of our resource. We describe the NNTC’songoing efforts and update of progress made in thecollecting tissues, research usage, longitudinal cohortanalysis, and resource generated publications. The


website for the NNTC, including investigator informa-tion and cohort description, is www.nntc.org.

P.54Dysregulation of TGF-beta signaling invirus-induced neurologic disease: implications forthe function of both regulatory and effector T cellsChristian W Grant,1 Unsong Oh,1 Karen Yao,1Yoshihisa Yamano,2 and Steven Jacobson1

1NIH, NINDS, Viral Immunology Section, Bethesda,Maryland, USA 2Department of Neurology, Kagoshima CityHospital, Kagoshima, Japan

Recent studies from our group have demonstratedexpression of CD4+CD25+ regulatory T cell (Treg)-specific Foxp3 was significantly reduced in patientswith human T lymphotropic virus type I (HTLV-I)-associated myelopathy/tropical spastic paraparesis(HAM/TSP) compared to Tregs from healthy donors.This was associated with an inability of Tregs fromHAM/TSP patients to suppress proliferation of healthydonor CD4+CD25- T cells. Furthermore, expression ofHTLV-I Tax into healthy donor CD4+CD25+ T cellsresulted in down-regulation of Foxp3 expression andloss of suppressive function. The present study wasaimed at determining the mechanisms underlying theloss of Foxp3 expression and lack of suppressor func-tion in Tregs from HAM/TSP patients. Our evidencesuggests that HTLV-I Tax inhibits Foxp3 expressionin human CD4+CD25+ T cells through transactiva-tion of the CREB signaling pathway. As HTLV-I Taxhas been reported to block TGF-beta/Smad signalingthrough its ability to functionally interact with mem-bers of the CREB pathway, and that TGF-beta signalinghas been previously demonstrated to play a criticalrole in the induction and maintenance of Foxp3 ex-pression, we investigated this potential link betweenTax, TGF-beta, and Foxp3. Herein we report a defectin the TGF-beta/Smad signaling pathway in patientswith HAM/TSP, including aberrant expression of thetype II TGF-beta receptor in CD4+CD25+ as well asCD4+CD25- T cells, which was associated with dys-regulation of Treg and effector T cell function. Thisevidence demonstrates a virus-induced breakdown ofimmunological tolerance at the level of both regulatoryand effector T cells.

P.55Characterization of the IGF-I receptor–mediatedgrowth survival and differentiation responses ofneural progenitorsElisa Gualco and Krzysztof ReissCenter for Neurovirology, Department of Neuroscience,Temple University, School of Medicine, Philadelphia, PA

The contribution of insulin-like growth factor Ireceptor (IGF-IR) in neuronal survival is well docu-mented. The question remains however how the sig-

nal from activated IGF-IR affects growth and differ-entiation of neural progenitors. We have addressedthis question by utilizing three-dimensional culturesof neural progenitors (neurospheres) isolated from thebrain of mouse embryos with targeted disruption ofthe IGF-IR gene (IGF-IR(-/-), and from age-matchingnon-transgenic littermates. There are several reasonswhy we have decided to use this model: (i) it will en-able evaluation of the IGF-I -mediated effects on neu-ronal outgrowth in the presence of three-dimensionalinteractions between neurons, astrocytes, and oligo-dendrocytes; (ii) it will allow cell signaling studies inthe context of cell cycle progression, ROS accumula-tion, and cell survival in actively proliferating neuralprogenitors (nestin positive); and in neural progeni-tors stimulated towards astrocytic, oligodendrocytic orneuronal differentiation; and (iii) it will make possi-ble the distinction between insulin receptor-mediatedand IGF-IR-mediated signaling responses. Our resultsindicate that secondary neurospheres kept for 5 daysin the medium supporting cell proliferation, consistof cells that are mostly nestin positive, indicating neu-ral progenitor phenotype. These proliferating neuro-spheres also contain a small population of cells thatare both nestin and GFAP positive, indicating earlyglial differentiation. These double-labeled cells werelocalized at the periphery of the neurosphere. Inter-estingly, we have found also a small number of cells,which were labeled with betaIII tubulin antibody, indi-cating neuronal differentiation. These few cells werenestin negative and showed early stages of neuronalmorphology. Following attachment and differentiationof secondary neurospheres, we have performed sin-gle and double immunocytofluorescent labeling to de-tect cellular markers expressed during differentiationof nestin positive neural progenitors. Five days afterthe induction of differentiation, three morphologicallydistinct cell populations were detected. These cellswere subsequently distinguished by immunolabelingwith anti betaIII tubulin antibody, an early neuronalmarker, anti-GFAP antibody, astrocytic marker, andanti-GalC antibody, oligodendrocytic marker. We havealso started to evaluate potential differences betweenneurospheres from IGF-IR(-/-) embryos and those fromage matched embryos from non-transgenic littermates.Our results indicate that the percentage of cells repli-cating DNA (BrdU incorporation) is significantly lowerin neurospheres from the IGF-IR(-/-) embryos thanin neurospheres isolated from non-transgenic litter-mates. This significantly lower rate of DNA replica-tion was observed despite of the fact that IGF-IR(-/-)neurospheres contained about 20% more nestin pos-itive cells and less betaIII tubulin and GFAP posi-tive cells, again in comparison to neurospheres fromnon-transfenic littermates. These data indicate that theabsence of IGF-IR slows down the rate of cell prolif-eration and decreases the percentage of progenitors,which differentiate towards neuronal and astrocyticlineages.


P.56Molecular bases of JCV T-antigen–mediatedtransformation of neuronalprogenitors—involvement of the IGF-IR – Survivinsignaling axisElisa Gualco, Theresa Sweet, Luis Del Valle, andKrzysztof ReissCenter for Neurovirology, Department of Neuroscience,Temple University, School of Medicine,Philadelphia, PA

The contribution of insulin-like growth factor I recep-tor (IGF-IR) in polyomavirus T-antigen-mediated cel-lular transformation is well documented. In addition,both IGF-IR and JCV T-antigen are suspected to play arole in the transformation of neuronal progenitors fromthe cerebellar granular layer – a critical step in the de-velopment of medulloblastoma. Therefore, we inves-tigated how IGF-IR supports cellular transformationby JCV T-antigen. We have addressed this question byutilizing three-dimensional cultures of neural progeni-tors (neurospheres) isolated from mouse embryos withtargeted disruption of the IGF-IR gene (IGF-IR(-/-), andfrom age-matching non-transgenic littermates. Our re-sults indicate that neurospheres kept in culture con-ditions supporting cell proliferation, maintain nestinpositive neural progenitors phenotype for severalgenerations. Following differentiation of secondaryneurospheres, we have detected three distinct cellpopulations, which were betaIII tubulin (early neu-ronal marker), GFAP (astrocytic marker), and GalC(oligodendrocytic marker) positive, independentlyfrom the presence or absence of the IGF-IR. Nestinpositive neurospheres were subsequently transfectedwith JCV T-antigen, and tested for cell cycle distri-bution, and protein levels. Our results demonstratethat: (i) more than 50% of IGF-IR(+/+) neurosphereswere positive for JCV T-antigen 48 hours after nucle-oporation –mediated gene delivery, while only 7% ofIGF-IR(-/-) neurospheres showed the expression of thisviral oncoprotein. (ii) In IGF-IR(+/+) neurospheres theexpression of JCV T-antigen shifted the cells from G1towards S–G2M phase of the cell cycle, which signif-icantly increased the rate of cell proliferation. In con-trast, vast majority of JCV T-antigen positive IGF-IR(-/-)neuronal progenitors were TUNEL positive. (iii) West-ern blot analyses demonstrated that anti-apoptotic pro-tein, survivin, which expression has been linked withcell proliferation, was downregulated about 10-foldin IGF-IR(-/-) in comparison to IGF-IR(+/+) neuronalprogenitors. Interestingly, JCV T-antigen failed to in-crease survivin protein level in the absence of IGF-IR.In contrast, (iv) JCV T-antigen expressed in differen-tiated IGF-IR(+/+) neurospheres elevated expressionof survivin to the levels detected during cell prolif-eration. Our results indicate that one of the mecha-nisms which could explain necessity of the IGF-IR inJCV T-antigen–mediated cellular transformation is re-

activation of survivin, which according to our resultsrequires IGF-IR.

P.57Bimolecular fluorescence complementation (BiFC) isa sensitive and quantitative method for studying theinteraction between HIV-1 Gag and Annexin 2Alexia V Harrist, Elena V Ryzhova, Thomas Harvey,and Francisco Gonzalez-ScaranoDepartment of Neurology, University of Pennsylvania

Bimolecular fluorescence complementation (BiFC) isa technique for studying protein-protein interac-tions in which fragments of a fluorescent protein,which by themselves do not fluoresce, are fused toproteins of interest. When the fusion proteins arecoexpressed in cells, their binding brings the flu-orescent protein fragments close enough to form acomplex and fluoresce. Because this assay does notrequire cell lysis or antibody staining, BiFC enablesthe visualization of protein-protein interactions in liv-ing cells in their proper intracellular environment(Kerppola, 2006). This assay has thus been used tostudy interactions that would be difficult to inves-tigate with more traditional methods, such as weakand short-lived interactions as well as interactionsthat require certain intracellular components, such asmembranes (e.g. Nyfeler et al., 2005). The interactionbetween the HIV-1 polyprotein Gag and the cellularprotein Annexin 2 occurs near CD63-positive com-partments in macrophages and is important for properviral assembly and budding and the formation of in-fectious virions (Ryzhova et al., 2006). As HIV-infectedmacrophages play an important role in the pathogen-esis of HIV-associated CNS disease, we developed aBiFC assay using YFP as the fluorescent reporter to bet-ter study the interaction between Gag and Anx2. Wecreated constructs with Gag, Anx2, CD63, and p11, thecellular binding partner of Anx2, fused to fragments ofYFP. We confirmed our ability to detect protein-proteininteractions using this assay by cotransfecting Anx2and p11 fusion proteins into 293T cells and detectingYFP fluorescence with confocal microscopy and quan-titatively using flow cytometry. We were then able tosuccessfully detect fluorescence complementation be-tween Anx2 and a Gag protein containing the matrixand capsid regions of the polyprotein (p41), again us-ing both confocal microscopy and flow cytometry. Thespecificity of the assay was confirmed by the findingthat cotransfection of CD63 and p41 fusion proteinsproduced no fluorescence, indicating that mere prox-imity of proteins without direct interaction does notresult in nonspecific fragment complementation. Thisassay provides us with a sensitive tool to study the in-tracellular site of the Gag-Anx2 interaction as well asmap the Anx2 binding site on Gag using mutagenesis.


P.58Antibody blocking of IL-12/23 amelioratesautoimmune but not viral-inducedencephalomyelitisKatherine S Held,1 William G Glass,2 Yevgeniya IOrlovsky,2 Kimberly A Shamberger,2 Ted D Petley,2Patrick J Branigan,2 Mark R Cunningham,2 JacquelineM Benson,2 and Thomas E Lane1,3

1Department of Molecular Biology and Biochemistry,University of California, Irvine; 2Discovery Research,Centocor R&D; 3Center for Immunology, University ofCalifornia, Irvine

The functional role of IL-12 and IL-23 in host de-fense and disease following viral infection of theCNS was determined. Instillation of mouse hepati-tis virus (MHV, a positive-strand RNA virus) into theCNS of mice results in acute encephalitis followed bya chronic immune-mediated demyelinating disease.Antibody-mediated blocking of either IL-23 (anti-IL-23p19) or IL-12 and IL-23 (anti-IL-12/23p40) signal-ing did not alter virus-specific IFN-gamma-secretingT cell generation nor affect viral clearance. Treatmentwith anti-IL-23p19 did not modulate secretion of IFN-gamma or IL-4 in the brains of MHV-infected mice.Moreover, therapeutic administration of anti-IL-23p19to mice with viral-induced demyelination did not at-tenuate disease. In contrast, treatment with anti-IL-12/23p40 or anti-IL-23p19 resulted in inhibition ofexperimental autoimmune encephalomyelitis (EAE).These data indicate that i) IL-12 and IL-23 signaling aredispensable in generating a protective Th1 responsefollowing CNS infection with MHV and ii) IL-23 ampli-fies disease in a model of autoimmune demyelinationyet does not contribute to demyelination in a modelindependent of autoimmune T cell-mediated pathol-ogy. Therapeutic targeting of IL-12 and/or IL-23 for thetreatment of autoimmune diseases may offer uniqueadvantages by reducing disease severity without mut-ing protective responses following viral infection.

P.59Antibody blocking of IL-23 ameliorates autoimmunebut not viral-induced encephalomyelitisKatherine S Held,1 William G Glass,2 Yevgeniya IOrlovsky,2 Kimberley A Shamberger,2 Ted D Petley,2Patrick J Branigan,2 Mark J Cunningham,2 JacquelineM Benson,2 and Thomas E Lane1

1Department of Molecular Biology & Biochemistry,University of California, Irvine; 2Discovery Research,Centocor R&D

The functional role of IL-12 and IL-23 in host de-fense and disease following viral infection of theCNS was determined. Instillation of mouse hepati-tis virus (MHV, a positive-strand RNA virus) into theCNS of mice results in acute encephalitis followed bya chronic immune-mediated demyelinating disease.Antibody-mediated blocking of either IL-23 (anti-IL-23p19) or IL-12 and IL-23 (anti-IL-12/23p40) signal-

ing did not alter virus-specific IFN-gamma-secretingT cells generation nor affect viral clearance. Treat-ment with anti-IL-23p19 did not modulate secretionof IFN-gamma or IL-4 in the brains of MHV-infectedmice. Moreover, therapeutic administration of anti-IL-23p19 to mice with viral-induced demyelination didnot attenuate disease. In contrast, treatment with anti-IL-12/23p40 or anti-IL-23p19 resulted in inhibition ofexperimental autoimmune encephalomyelitis (EAE).These data indicate that i) IL-12 and IL-23 signaling aredispensable in generating a protective T cell responsefollowing CNS infection with MHV and ii) IL-23 ampli-fies disease in a model of autoimmune demyelinationyet does not contribute to demyelination in a modelindependent of autoimmune T cell-mediated pathol-ogy. Therapeutic targeting of IL-12 and/or IL-23 for thetreatment of autoimmune diseases may offer uniqueadvantages by reducing disease severity without mut-ing protective responses following viral infection.

P.60Neuropsychological performance of HIV-infectedindividuals and its association with AntiretroviralResistance, Methamphetamine Use, Hepatitis CVirus, and CSF HIV RNA levels.George K Hightower,1 Scott L Letendre,1 MarianaCherner,1 Eduardo R Cachay,1 Sarah Gibson,1 RonEllis,1,2 Caroline C Ignacio,1 Robert Heaton,1 IgorGrant,1,2 Douglas D Richman,1,2 and Davey MSmith1,2

1University of California, San Diego; 2San Diego VeteransAffairs Healthcare Systems

Background: Before the widespread use of combina-tion antiretroviral therapy (ART), studies indicatedthat for individuals with AIDS, higher HIV RNA levelsin cerebrospinal fluid (CSF) - but not in plasma—wereassociated with neurocognitive impairment. Morerecent studies have reported that HIV RNA levels inCSF are not as strongly associated with neurocognitiveimpairment. It has been suggested that ART lowersCSF HIV RNA levels and therefore lessens the associa-tion between CSF HIV RNA levels and neurocognitivefunction. ART can impact viral dynamics by inhibitingviral replication or by the selection of drug resistantvariants that may be less neuropathogenic. We hy-pothesized that there would be significant differencesin CSF HIV RNA levels and neuropsychological (NP)performance between individuals with drug resistance(DR+) and those without drug resistance (DR-).

Methods: A comprehensive NP battery was admin-istered to 94 HIV-infected participants at the Uni-versity of California, San Diego HIV NeurobehavioralResearch Center. To assess Global Deficit the resultsfrom the NP battery were adjusted for age, educationand ethnicity. Higher Global Deficit Scores indicateworse NP performance. Univariate and multivariateanalyses included: age, ethnicity, duration of HIV in-fection, HIV RNA levels in CSF and blood, current


and nadir CD4 counts, drug resistance, hepatitis Cvirus (HCV) serostatus, past and current ART use, ARTadherence, methamphetamine use and Global Rating.The Viroseq algorithm and Stanford HIV ResistanceDatabase (April 2007) were used to interpret drug re-sistance from genotypic data.

Results: Logistic regression analysis revealed thatthe absence of drug resistance (DR-), history ofmethamphetamine use, lower CD4 count nadir, pos-itive HCV serostatus (HCV+) and no prior ART usewere independently associated with worse NP per-formance. An interaction between HCV status anddrug resistance showed that individuals who wereHCV+/DR- had worse NP performance than the restof the study group who were HCV-/DR+, HCV-/DR- orHCV+/DR+. There was a similar interaction with cur-rent ART use (ART+) and HCV, which showed thatindividuals with ART+/HCV+ demonstrated worseNP performance than all other ART and HCV groups.Additionally, for individuals without drug resistance(DR-), higher CSF HIV RNA levels correlated withworse NP performance. However, in individuals withdrug resistant HIV (DR+), CSF HIV RNA levels did notcorrelate with NP performance.

Discussion: In this study, we found complex mul-tifactorial associations with NP performance, includ-ing CSF HIV RNA levels, HCV serostatus, metham-phetamine use and drug resistance. In this era ofincreasing use of ART, the understanding of each ofthese factors will be important in fully evaluating HIVassociated NP performance.

P.61CXCL12-induced cleavage of CX3CL1: potential rolein neuroprotectionRandi L Hippensteel, Saori Shimizu, and OlimpiaMeucciDepartment of Pharmacology and Physiology, DrexelUniversity College of Medicine, Philadelphia, USA; andInstitute of Molecular Medicine and Infectious Diseases,Drexel University College of Medicine, Philadelphia, USA

Chemokines are involved in various physiologicaland pathological processes in the CNS, including neu-roinflammation/neuroAIDS. CX3CL1 (Fractalkine)and CXCL12 (SDF-1) are two chemokines that are con-stitutively expressed in the brain. CX3CL1 can existas a transmembrane protein, which acts as adhesivemolecule, or can be cleaved by ADAMs (A Disintegrinand Metalloproteinase) and act as a soluble protein.Both soluble and membrane-attached CX3CL1 inter-act with the specific CX3CL1 receptor, CX3CR1. Invitro and in vivo studies suggest that CX3CL1 andCXCL12 may have neuroprotective effects, though themechanisms involved are not completely clear. Theaim of this study was to establish whether neuronalproduction of CX3CL1 is regulated by CXCL12, anddetermine whether this regulation is involved inneuronal protection. Through the use of a pathway-specific DNA microarray, RT-PCR, and qRT-PCR, we

show that the gene for CX3CL1 is up-regulated inneurons treated with CXCL12 (20nM, 18hr). Further-more, treatment with CXCL12 stimulates cleavageof CX3CL1. Addition of different metalloproteinaseinhibitors abolishes CXCL12-induced cleavage. Wealso found that treatment of neuronal cultures withNMDA can affect levels of soluble/cellular CX3CL1in a time-dependent manner. This is important as weand others have previously shown that both CXCL12and CX3CL1 can protect neurons from exitotoxicity.Survival assays will test the hypothesis that solubleCX3CL1 contributes to the action of CXCL12 onneuronal survival. Our findings so far uncover anovel interaction between the two chemokines, whichmay play an important role in the regulation of theirindividual functions in the brain (supported by NIHgrants DA15014 and DA19808 to OM).

P.62CXCR2 signaling protects against oligodendrocyteapoptosis during virus-induced demyelinationMartin P Hosking and Thomas E LaneDepartment of Molecular Biology & Biochemistry,University of California, Irvine

Intracranial infection of susceptible mice with neu-roadapted strains of mouse hepatitis virus (MHV)induces an acute encephalomyelitis followed by achronic demyelinating disease similar to the humandemyelinating disease multiple sclerosis. During thechronic stage of disease, transcripts specific for theCXC chemokine receptor 2 (CXCR2) and it’s bindingligands CXC chemokine ligands (CXCL) 1 and 2 areelevated within the spinal cords of mice suggesting apotential role in either host defense or disease in micepersistently infected with MHV. Immunohistochemi-cal staining revealed that expression of CXCR2 wasrestricted primarily to neurons, astrocytes, and oligo-dendrocytes and was not associated with infiltratingleukocytes. Antibody neutralization of CXCR2 duringthe chronic phases of MHV infection resulted in in-creased mortality compared to mice treated with con-trol antisera. Blocking CXCR2 did not reduce infiltra-tion of virus-specific T cells into the central nervoussystem (CNS) nor result in viral recrudescence. In ad-dition, there was no difference in the severity of de-myelination in mice treated with either anti-CXCR2or control antisera. However, anti-CXCR2 treatmentdid correlate with a three-fold increase in TUNEL-positive cells that were restricted to the white mat-ter of infected mice. Immunofluorescence combinedwith TUNEL staining revealed that a large majority ofthe apoptotic cells in anti-CXCR2 treated mice wereoligodendrocytes (CNPase+) and oligodendrocyte pre-cursor cells (Olig2+). Therefore, in mice persistentlyinfected with MHV and undergoing demyelination,CXCR2 signaling influences oligodendrocyte survivaland protects against apoptosis during chronic neuroin-flammatory disease.


P.63TNF-alpha mediates trail expression in primaryhuman astrocytes: Relevance to HIV-1 associateddementiaYunlong Huang, Angelique Walstrom, AgnesConstantino, and Jialin ZhengThe laboratory of Neurotoxicology, Dept. Pharmacologyand Experimental Neuroscience, University of NebraskaMedical Center

HIV-1 enters the brain within weeks of initial infec-tion establishing a reservoir for viral replication inmononuclear phagocytes (MP, brain macrophage andmicroglia). Behind the blood brain barrier, these HIV-1-infected and/or activated MP drive the pathogenesis ofHIV-1 associated dementia (HAD). Previously, we re-ported that TRAIL, a death ligand of the TNF superfam-ily, participates in MP-mediated neurotoxicity. In thisstudy, we further examined the level of TRAIL in post-mortem samples from HIV-1 encephalitic (HIVE) indi-viduals. TRAIL levels in HIVE samples were found tobe higher than HIV-1 serum-positive individuals with-out encephalitis or HIV-1 serum-negative samples. As-trocytes are the most abundant cell type in the centralnervous system and human fetal astrocyte culture is acommon model to study astrocyte in vitro. Using realtime RT-PCR, we found astrocytes increased TRAILtranscription in response to inflammatory cytokinestimulation. Further, HIV-1-infected and LPS stimu-lated macrophage conditioned media (MCM) inducedsignificantly higher levels of TRAIL as compared toLPS stimulated MCM alone. Upregulation of TRAILexpression was blocked by soluble TNF-alpha recep-tor, indicating a predominant role of TNF-alpha in in-ducing TRAIL expression in astrocytes. Upregulationof TRAIL expression by TNF-alpha was blocked bythe c-Jun N-terminal kinase (JNK) inhibitor SP600125.In conclusion, HIV-1-infected and/or immune acti-vated macrophages mediate TRAIL expression in as-trocytes through the production of TNF-alpha. Defin-ing macrophage-astrocyte interaction in the context ofinflammation and HIV-1 infection may help to betterunderstand the TRAIL-mediated neurotoxicity duringHAD.

P.64Methamphetamine induces increased levels ofinterferon-stimulated protein in human primaryastrocytesBritta J Hult,1,2 Amy Paulino,3 Gursharan Chana,1,2

Anthony Adame,3 Eliezer Masliah,3 and Ian PEverall1,2

1HIV Neurobehavioral Research Center, 2University ofCalifornia, San Diego: Department of Psychiatry;3University of California, San Diego: Department ofNeurosciences

Methamphetamine (MA) has been shown to be toxicto varying neuronal populations in the striatum, hip-

pocampus and frontal cortex and this effect was greaterwith the presence of HIV encephalitis (HIVE). Expo-sure of mesencephalic, striatal and cortical astrocytesto MA has been shown to increase the presence ofreactive oxygen species, suggesting a mechanism forastroglia-dependent MA neurotoxicity. We have re-ported that interferon stimulated gene, 15kD (ISG15)expression is increased in post-mortem tissue from thefrontal cortex of patients with HIVE and HIVE + MAuse, versus HIV alone, using microarray analysis, qRT-PCR, and immunohistochemistry. As astrocytic cellsare immunocompetent and are capable of releasingproinflammatory chemokines and cytokines, the up-regulation of ISG15 may play a role in the neurotoxic-ity of MA and HIV. Confirmation of this effect in an invitro system would allow for further investigation ofthe cause and effect of ISG15 upregulation.

To further investigate the effect of MA on astrocyticcell ISG15 expression, we exposed human primary as-trocytes (HPA) to 5uM and 20uM MA, an ISG15 trans-fection vector, and 1000U/ml interferon-beta, an in-ducer of ISG15 expression, for 24 hours. We assayedfor ISG15 protein product levels using western blot(WB) and immunocytochemistry (ICC).

In response to an exposure of 20uM MA, HPAshowed an increase in ISG15 expression, as measuredqualitatively by ICC and WB. The same effect was alsoseen with interferon-beta and the ISG15 expressionvector.

We have confirmed previous findings of ISG15 ex-pression dysregulation in response to MA exposure atthe protein level. The ability to investigate ISG15 dys-regulation in an in vitro system will allow for moreprofound investigations of the molecular mechanismsinvolved with MA neurotoxicity and how these effectssynergize with HIV infection.

P.65Pre-treatment of mice with nocodazole delays viralentry into the brain following footpad inoculation ofWest Nile virusElizabeth A Hunsperger,1 Manuela Beltran,1 and JohnT Roehrig2

1CDC, Dengue Branch; 2CDC, Arbovirus Diseases Branch

West Nile virus (WNV) infection in humans can causeneurological deficits including flaccid paralysis, en-cephalitis, meningitis, and mental status change. Tobetter understand the neuropathogenesis of WNV inthe peripheral and the central nervous system (PNS& CNS), we use a mouse footpad model to simulate anatural infection. Localization of WNV in the nervoussystem using this model suggested two routes of viralintroduction into the CNS: retrograde transport fromthe PNS and hematogenous diffusion via a breakdownin the blood-choroid-plexus barrier. To test further theretrograde transport hypothesis, C57BL/6J mice weretreated with nocodazole, a microtubule inhibitor that


blocks retrograde transport, and then infected withWNV. Nocodazole-treated WNV-infected mice devel-oped a viremia at least one log greater than untreatedWNV-infected controls at all time points tested (days1-5 post-infection). Although viremia was greater innocodazole treated mice, introduction of virus into thebrain tissue (spinal cord, cortex, brainstem and cere-bellum) as measured by real time RT-PCR occurredmuch later on day 7 instead of day 1. These results sug-gest that nocodazole decreases neuroinvasion duringearly stages of the infection (1-4 days post-infection).However at later time points (7 and 9 days post-infection), the nocodazole- treated animals had viraltiters as high as the untreated WNV-infected controls.These data support a model of biphasic viral infectionof the CNS and suggest that early CNS infection mayalso occur via retrograde transport of virus from thePNS.

P.66Genome-wide screening of host genes involved inHIV-1-induced neurotoxic signaling via lentiviralsiRNA library screeningTsuneya Ikezu,1,2,3 Eva Bouwmans,1,2,3 James LBueshcer,1,2,3 Lindsey B Martinez,1,2,3 and James DEudy3,4

1Center for Neurovirology and NeurodegenerativeDisorders; 2Department of Pharmacology and ExperimentalNeuroscience; 3University of Nebraska Medical Center;4Department of Cell, Genetics, and Anatomy

Viral infection-mediated neurotoxicity is a centralpathologic mechanism for epidemic viral encephali-tis, including HAD [human immunodeficiency virus(HIV)-1 associated dementia]. Survival of neurons dur-ing chronic viral infection is important for maintain-ing the cognitive, motor, and psychiatric functions ofthe brain. Identification of the host genes responsi-ble for neurotoxic signaling will enable us to enhancecell survival and to develop a therapeutic interven-tion. To identify which genes are responsible for viralneurotoxic signaling, we will utilize high-throughputgenome-wide screening of host genes in a human neu-ronal cell line. A lentiviral siRNA library that targets47,400 human mRNA transcripts was utilized to iden-tify which siRNA clones can prevent in vitro humanneuronal cell death induced by pseudotyped HIV-1.The siRNA clones was identified by GeneChip mi-croarray system. The cells protected by siRNA expres-sion showed significant upregulation in the siRNAlevels of 9 genes and downregulation of 5 genes. EachsiRNA clones identified by the library screening wasfurther tested for their neuroprotection efficacy afterHIV-1 infection. This is the first report of such genome-wide functional screening of biologically significantneurotoxic signaling, with a therapeutic potential forNeuroAIDS.

P.67Genetic variants within the HIV-1 LTR may bepredictive of HADBryan P. Irish, Michael R. Nonnemacher, and BrianWigdahlDrexel University College of Medicine

Human immunodeficiency virus type 1 (HIV-1) provi-ral DNA with specific binding site sequence variantswithin the long terminal repeat (LTR), which arise dur-ing the course of infection may have functional impli-cations relative to LTR activation, viral replication, aswell predictive value relative to progressive neurologi-cal disease. The LTR regulates HIV-1 viral gene expres-sion via its interaction with multiple viral and hostfactors, including members of the CCAAT/enhancerbinding protein (C/EBP) and Sp transcription factorfamilies. We have examined sequence variation atC/EBP sites I and II, and Sp sites I, II, and III in pe-ripheral blood (PB)-derived LTRs from HIV-1-infectedpatients representing increasing degrees of diseaseseverity. The 3T configuration of C/EBP site I (C-to-Tchange at nucleotide position 3) and 5T configurationof Sp site III (C-to-T change at nucleotide position 5)were the only variants examined that were found inlow frequencies in PB-derived LTRs derived from pa-tients at early stages of HIV-1 disease, and at relativelyhigh frequencies in patients in late stage disease. Se-quence variation at these sites was also examined inLTRs derived from various brain compartments of pa-tients both with and without HAD (the 3T C/EBP siteI variant was identified in 25% of brain-derived LTRsfrom patients diagnosed with HAD, but was absent inpatients not suffering from dementia). These resultssuggest that 3T C/EBP site I, and possibly 5T Sp siteIII may prove valuable in assessing the likelihood ofHIV-1-infected individuals developing HAD.

P.68Association of focal adhesion kinase (FAK)activation and lentivirus-induced disruption ofblood-brain barrierNathan S Ivey,1,2 Terri M Rasmussen,2 MaheshMohan,2 Reza Izadpanah,3 Dana N Bieniemy,2 NicoleA Renner,2 Andrew A Lackner,2 and Andrew GMacLean2

1Tulane University Neuroscience Graduate Program;2Tulane National Primate Research Center, Pathology;3Tulane National Primate Research Center, Gene Therapy

Disruption of the blood-brain barrier (BBB) occurswith the development of HIV encephalitis (HIVE).While many studies have examined the role of vi-ral strains and macrophages/microglia in the devel-opment of encephalitis, the molecular signaling path-ways within the brain microvascular endothelial cells(BMECs) involved have not been examined. We havepreviously shown that there is activation and disrup-tion of an in vitro BBB model using lentivirus-infectedCEMx174 cells. Others have shown similar disruption


in vivo. Therefore, it was of interest to determine if thepresence of these cells could disrupt intact cerebralmicrovessels immediately ex vivo, and if so, whichsignaling pathways were involved. We find a corre-lation between a transitory 10-fold upregulation ofFAK expression and markedly decreased expressionand altered localization of zo-1 in microvessels. Ourdata suggest that disruption of tight junctions betweenBMECs is mediated through activation of FAK by phos-phorylation at TYR-397. Inhibition of FAK activationis sufficient to prevent tight junction disruption possi-bly via zo-1, a protein thought to recruit and organizestructural elements (fibrils) within the tight junction.Thus, it may be possible to inhibit break down of theBBB associated with the development of HIVE by us-ing inhibitors of FAK.

P.69The clinical disease and outcome are explained bymorphological changes in neurons in experimentalrabies in yellow fluorescent protein transgenic miceAlan C Jackson,1 Courtney A Scott,2 R DavidAndrew,2 and John P Rossiter2

1University of Manitoba; 2Queen’s University

Although human rabies can be effectively prevented,many questions remain unanswered about the patho-genesis of the disease. It is thought that the severeclinical illness and fatal outcome in rabies may re-sult from neuronal dysfunction, rather than neuronalinjury or death, because neuropathological changesare usually relatively mild and degenerative neuronalchanges are not prominent. In this study the effects ofrabies virus infection were evaluated on the structuralintegrity of neurons in experimentally-infected trans-genic mice expressing the yellow fluorescent protein(YFP) in neurons.

Six-week-old transgenic mice (C57BL background)expressing YFP in neurons (H-line) were inoculatedin the right hindlimb footpad with the CVS strain offixed rabies virus or mock-infected with vehicle (PBS).Morphological and immunohistochemical studies us-ing light and fluorescent microscopy were performedon regional brain areas with expression of YFP inneurons, including the cerebral cortex, hippocampus,cerebellum, and brainstem.

Routine histopathogical studies showed some in-flammatory changes, but no significant neurodegener-ative changes in the CVS-infected brains of moribundmice. However, with fluorescent microscopy dendritesand axons of pyramidal neurons in the cerebral cortexand mossy fibers in the cerebellum exhibited markedstructural abnormalities, including beading, whereasthe perikarya showed relatively few changes. In thecerebral cortex, 12% of dendrites and 41% of axonsof pyramidal cortical neurons were affected in CVS-infected mice, whereas 0% of dendrites and 2% of ax-ons were affected in mock-infected mice. Toluidineblue-stained plastic sections showed vacuolation in

perikarya and neuronal processes, which correlatedultrastructurally with swollen mitochondria. Therewas prominent vacuolation in dendrites and in pre-synaptic nerve endings in the neuropil of the cerebralcortex. There was also swelling of Golgi apparati and ofendoplasmic reticula. In contrast, perikarya and pro-cesses of CA1 pyramidal neurons showed only rarechanges.

Thus we have found evidence of marked structuralchanges in neuronal processes in experimental rabiesin mice. These findings indicate that injury promi-nently involving neuronal processes may be sufficientto explain the severe clinical disease with a fatal out-come in rabies rather than neuronal dysfunction with-out morphological changes.

Support from Canadian Institutes of Health Researchgrant MOP – 64376 and the Queen’s University VioletE. Powell Research Fund.

P.70Modulation of dendritic cell maturation andfunction by the Tax protein of human T cellleukemia virus type 1: Implications forHTLV-1-associated neuroinflammatory diseasePooja Jain, Jaya Ahuja, Zafar K. Khan, Saori Shimizu,Olimpia Meucci, Stephen R. Jennings, and BrianWigdahlDrexel University College of Medicine

HTLV-1-associated myelopathy/tropical spastic para-paresis (HAM/TSP) is characterized by the generationof an intense cytotoxic T cell (CTL) response directedagainst the viral transactivator protein Tax. Addition-ally, patients diagnosed with HAM/TSP exhibit rapidactivation and maturation of dendritic cells (DCs)likely contributing to the robust Tax-specific CTL re-sponse. In this study, extracellular Tax has been shownto induce maturation and functional alterations in hu-man monocyte-derived DCs, critical observations be-ing confirmed in freshly isolated myeloid DCs. Tax wasshown to promote the production of proinflammatorycytokines and chemokines involved in DC activationprocess in a dose- and time-dependent manner. Fur-thermore, Tax induced the expression of DC activation(CD40, CD80, and CD86) and maturation (CD83) mark-ers and enhanced the allogenic and antigen-specificT cell proliferation capability of DCs. Heat inactiva-tion of Tax resulted in abrogation of these effects indi-cating a requirement for the native structure of Tax.Tax was found to efficiently bind to the DC mem-brane and was internalized within a few hours sug-gesting that extracellular Tax may possess an intracel-lular mechanism of action subsequent to entry. Finally,inhibitors of cellular signaling pathways NF-kappaB,protein kinase, tyrosine kinase and phospholipase Cwere shown to inhibit Tax-mediated DC activation.This is the first study reporting the immunomodula-tory effects of extracellular Tax in the DC compart-ment. These results suggest that DCs, once exposed to


Tax by uptake from the extracellular environment, canundergo activation providing constant antigen presen-tation and costimulation to T cells leading to the in-tense T cell proliferation and inflammatory responsesunderlying HAM/TSP. Ongoing ex vivo investigationsinclude frequency, activation state, and functions ofvarious DC subsets in HAM/TSP patients.

P.71The secretion of human T cell leukemia virus type 1Tax protein is a regulated process involvingcomponents of cellular secretory pathway andcritical amino acid signals within the viral protein:Implications for HTLV-1 neuropathogenesisPooja Jain,1 Kate Mostoller,2 Katherine E. Flaig,1

Veronique Lepoutre,1 Jaya Ahuja,1 and BrianWigdahl1

1Drexel University College of Medicine, 2The PennsyvlaniaState University College of Medicine

HTLV-1 associated myelopathy/tropical spastic para-paresis (HAM/TSP) is a progressive neurological dis-order. Many pathologic abnormalities associated withHAM/TSP have been attributed to the extracellularactivity of the viral transactivator protein Tax. Cell-free Tax has been detected in the cerebrospinal fluidof HAM/TSP patients indicating its physiologic rele-vance in the neuropathogenesis. We have previouslydemonstrated the presence of a nuclear export signalwithin Tax and its active secretion in vitro. Studies re-ported herein elucidate the process by which Tax issecreted and identify domains of Tax that contributeto its subcellular localization and secretion. Tax wasshown to co-localize with endoplasmic reticulum (ER)and Golgi, the cytoplasmic organelle relevant to pro-tein secretion. An extensive Proteomics analyses fol-lowed by GST pull-down assay revealed the specificinteraction of Tax with several proteins of the cel-lular secretory pathway including COPII, SCAMP1,SCAMP2 and SNAP23 in both the model cell line BHK(baby hamster kidney)-21 and a T cell line C8166 (rel-evant to HTLV-1 infection). The silencing of these pro-teins inhibited Tax secretion further confirming theirinvolvement in this process. Comparison of the Taxprotein sequence with amino acid signals known totarget proteins to the secretory pathway yielded theidentification of a number of putative secretory sig-nals. The mutations in two signals DHE and YTNI re-sulted in aberrant subcellular localization of Tax witha large amount of Tax localized to areas immediatelysurrounding the nucleus. In addition, Tax constructscontaining mutations in both signals significantly al-tered protein secretion. Together, these studies suggestthat secretion of Tax is a regulated event facilitated bythe interaction of Tax with cellular secretory pathwayproteins and the presence of critical secretory signalswithin the carboxy-terminal domain of protein.

P.72Presenilin and the Gamma-secretase Complex inHIV NeuroinflammationSuman Jayadev,1 Amanda Case,1 Huy Nguyen,1 andGwenn A Garden1,2

1Department of Neurology, University of WashingtonMedical Center; 2Center for Neurogenetics andNeurotherapeutics, University of Washington, Seattle

The Presenilin (PS) proteins, PS1 and PS2, form thecatalytic core of the gamma-secretase complex thatcleaves amyloid precursor protein (APP) to release theamyloid beta (Ab) peptide. Ab is deposited in amy-loid plaques found in Alzheimer’s disease (AD) as wellas in HIV+ brain suggesting that pathways involvedin AD neurodegeneration may also contribute to cog-nitive decline in HIV patients. In addition to APP,the gamma-secretase complex cleaves the transmem-brane substrates Notch and ErbB4 through which PS’smay regulate inflammation, development, and cell fatedetermination.

We tested the hypothesis that PS2 participates inHIV neuroinflammation. We found increased PS2 im-munolabeling in microglia from both HAD and ADfrontal cortex autopsy tissue compared to age matchedcontrols. The gamma-secretase substrate ErbB4 is alsoincreased in both the cytoplasm and nuclei of glialcells in HAD. Cultured primary murine microglia,and microglia or macrophage cell lines exposed tothe HIV coat protein gp120 (400pM SF162, NIH AIDSReagent Program) demonstrate increased PS2 protein.Furthermore, we observed an increase in the gamma-secretase cleavage product of Notch-1, Notch intra-cellular domain, in microglia stimulated with gp120and Lipopolysaccharide (LPS). Pharmacological inhi-bition of gamma-secretase activity modulates nitric ox-ide production and release of tumor necrosis factor instimulated primary microglia. Our data suggest thatPS2 and gamma-secretase may participate in the CNSinflammatory response. Further studies into PS andgamma-secretase mediated cellular pathways may pro-vide useful therapeutically relevant insights into im-mune mediated pathophysiology in human dementia.

P.73Neurotropism and neuropathogenesis of a CAEV/SIVchimeric virus in goatsYuhuai Jin, Naomi Holloway, Amel Baya Bouzar,Geraldine Arrode, Opendra Narayan, andYahia CheblouneThe University of Kansas Medical Center, Kansas City

The pathogenesis of AIDS and CNS disease causedby HIV revolves around the tropism of the virus forCD4+ T cells and macrophages, respectively. Themacrophage phase of the disease, exemplified by HIVdementia, is contingent on elimination of CD4+ T cellsby the virus and is thus a late aspect of the patho-genesis. SIV infection in macaques closely modelsHIV pathogenesis. Caprine arthritis encephalitis virus


(CAEV) is an ungulate lentivirus that replicates onlyin macrophages of infected goats and causes in somecircumstances CNS disease in addition to other syn-dromes unique to goats, but there is no loss of T cells.We incorporated the vpr/vpx and nef genes of SIVinto the genome of CAEV (1,2) and hypothesized thatthis chimera would create a caprine model of HIV en-cephalitis. This virus replicated productively in goatmacrophages but not in T cells and caused increasedcythopathic effects. Non infected lymphocytes in co-culture with infected macrophages underwent apop-tosis. We then undertook a study on pathogenesis ofinfection in juvenile and newborn goats using the re-combinant virus and CAEV as control. The chimericvirus caused a persistent infection in the goats andwas present in peripheral blood monocytes more of-ten than CAEV. T cells underwent moderate apopto-sis, but the cell count did not decrease dramaticallyas seen in HIV infections. Examination of tissues fromthe infected juvenile goats 24 weeks after virus inocu-lation and infected new born kids after 12 weeks post-inoculation showed that CAEV caused minimal or nolesions in target tissues. However, the chimeric viruscaused mild, but diffuse encephalitis that was associ-ated with white and gray matter throughout the neu-ropil. All infected new born kids with the recombinantvirus but not with CAEV have virus in most of theexplant cultures derived from different regions of theCNS and encephalitic lesions were observed in someof the sections. This chimeric virus system thereforehas a great promise for providing a simple and repro-ducible model of HIV CNS disease without causingAIDS.

1. Bouzar et al. Virology 2004.2. Bouzar et al. Virology 2007.

P.74Perturbation of the astrocyte integrated stressresponse (ISR) by HIV-1-infected macrophagesKelly L Jordan-Sciutto,1 Michael G White,1 AlisonJ.B. Markowitz,1 Lorraine Kolson,2 PraveenaSabnekar,2 Cagla Akay,1 Ying Wang,1 and Dennis LKolson2

1Department of Pathology, School of Dental Medicine,University of Pennsylvania; 2Department of Neurology,School of Medicine, University of Pennsylvania

Neuronal degeneration in HAD is mediated by solublefactors released by CNS-resident, HIV-infected and/oractivated macrophages and microglia. Although manymolecules have neurotoxic potential, primary ratmixed neuronal/glial cultures treated with HIV-infected, monocyte-derived macrophage supernatants(HIV-MDM) are protected from death by the NMDAantagonist MK801. However, our studies indicate thatonly ∼30% of the toxicity of HIV-MDM supernatants isdue to HIV-MDM glutamate content, suggesting the in-volvement of additional HIV-MDM excitotoxins or themodulation of excitotoxin production by astrocytes in

these mixed cultures. The astrocytic cystine/glutamateantiporter (xC) is a key regulator of glutamate home-ostasis, and can be modulated by the integrated stressresponse (ISR) by transcriptional upregulation of thexCT subunit via Nrf2. xC activity results in stoichio-metric intake of cystine to synthesize the antioxidantglutathione in exchange for glutamate. Exported glu-tamate is scavenged by excitatory amino acid trans-porters (EAATs). We hypothesize that HIV MDM willactivate the astrocytic ISR perturbing glutamate home-ostasis. Consistent with this, HIV-MDM-treated rat as-trocytes exhibited increased protein levels of ISR targetgenes ATF4, BIP, and CHOP, as well as the endogenousantioxidant response protein Nrf2 and its target, xCT,by immunoblotting. Increased xCT levels in astrocytes,by HIV-MDM or by classic ISR inducer thapsigargin, re-sulted in increased antiporter activity thereby increas-ing cystine uptake, and, presumably, glutamate export.HIV-MDM and thapsigargin also inhibited the scaveng-ing of extracellular glutamate by the astrocytic EAATs,similar to effects of TNF-alpha. These findings sug-gest HIV-MDM-released factors may trigger a net effluxof glutamate from astrocytes due to ISR-induced in-creases in xCT-mediated glutamate export and concur-rent decreases in EAAT-mediated glutamate import.Consistent with this mechanism, we have observedincreases in ISR markers, BiP and ATF6B, and xCTin cortices from HIV-infected patients as compared touninfected controls. Taken together, these data suggestthat induction of the ISR in astrocytes by HIV-MDMmay induce neuronal dysfunction and death by alter-ing glutamate homeostasis in HAD.

P.75Purα as a cellular co-factor of Rev/RRE-mediatedexpression of HIV-1 intron-containing mRNARafal Kaminski,1 Nune Darbinian,1 Bassel E Sawaya,1Dorota Slonina,1 Shohreh Amini,1,2 Edward MJohnson,3 Jay Rappaport,1 Kamel Khalili,1 andArmine Darbinyan1

1Department of Neuroscience Center for NeurovirologyTemple University School of Medicine Philadelphia PA19122; 2Department of Biology College of Science andTechnology Temple University Philadelphia, PA 19122;3Department of Microbiology and Molecular Cell BiologyEastern Virginia Medical School Norfolk, VA 23501

To ensure successful replication, HIV-1 has developeda Rev-mediated RNA transport system that promotesthe export of unspliced genomic RNA from nuclei tocytoplasm. This process requires the Rev ResponsiveElement (RRE) that is positioned in the viral transcriptencoding Env protein, as well as in unspliced andsingly-spliced viral transcripts. We identified Pura, asingle-stranded nucleic acid binding protein as a cellu-lar partner for Rev that augments the appearance of un-spliced viral RNAs in the cytoplasm. A decrease in thelevel of Pura expression by siRNA diminishes the levelof Rev-dependent expression of viral RNA. Through itsnucleic acid binding domain, Pura exhibits the ability


to interact with the multimerization and RBD domainsof Rev. Similar to Rev, Pura associates with RRE andin the presence of Rev forms a complex with slowerelectrophoretic mobility than those from Rev:RRE andPura:RRE. The interaction of Pura with RRE occurs inthe cytoplasm where enhanced association of Rev withRRE is observed. Our data indicate that the partnershipof Pura with Rev is beneficial for Rev-mediated expres-sion of the HIV-1 genome.

This work was supported by NINDS grant awardedto SA and KK

P.76The kynurenine pathway metabolite quinolinic acidupregulates CCR5 expressionApsara Kandanearatchi,1 Gilles Guillemin,1 JohnWilkinson,1 Anthony Cunningham,2 and Bruce Brew1

1Centre for Immunology, St Vincent’s Hospital, Sydney,Australia; 2Millenium Institute, Sydney University, Sydney,Australia

Background: Activation of the kynurenine pathway(KP), especially the production of quinolinic acid(QUIN), has been pathogenetically linked to AIDS De-mentia Complex (ADC). HIV isolated from the cen-tral nervous system (CNS) of ADC patients is bothmacrophage tropic and CCR5 dependent. Previously,we have shown that QUIN significantly upregulatesCCR5 expression on astrocytes, and that the abilityto induce the KP is dependent on the strain of HIV –greater in those derived from neurologically impairedpatients. We therefore hypothesized that QUIN upreg-ulates CCR5 expression on macrophages and is straindependent. Methods: PBMCs were isolated from ablood pack by the Lymphoprep method. Macrophageswere cultured in DMEM and 10% fetal bovine serumon coverslips. To observe if QUIN upregulates CCR5expression, the cells were weaned to serum free con-ditions before the addition of QUIN at 150, 350, 500and 1200nM. Negative controls without QUIN andpositive controls with IFNγ were also conducted. Atdays 1 and 3, cells were processed immunohistochem-ically for CCR5 (1:100) expression. 10 images per con-dition were taken and pixel intensities for each cellwere measured. To observe if CCR5 expression is straindependent the neurotropic strains JRFL, C124 (non-demented, primary isolate) and C158 (demented, pri-mary isolate) and the non-neurotropic strain Bal wereall added at the same viral IC50 concentration. Cellswere immunohistochemically processed as before atday3 following. Results: There was no significant dif-ference in CCR5 expression one day following theaddition of QUIN. However, at day 3 there were sig-nificant differences between the control and QUIN(ANOVA, F = 22.9, p = 0.0001). There were also signif-icant differences at day3 between the controls and thetwo neurotropic viruses C158 and C124 (ANOVA, F =121, p = 0.0001). These strains also had a higher CCR5expression compared to the non-neurotropic strain Bal

(ANOVA, F = 121, p = 0.0001). Conclusions: Thesedata add to the significant pathogenetic role of QUINin ADC: not only does QUIN lead to neural damage,it also facilitates macrophage infection through an au-tocrine relationship. Further work will examine theeffect of QUIN on microglia.

Key Words: Kynurenine Pathway, macrophages,neurotropic, QUIN

P.77Astrocytes as potential source of cell-free Tax duringhuman T cell leukemia virus type 1-associatedmyelopathy/tropical spastic paraparesisZafar K. Khan, Pooja Jain, Veronique Lepoutre, KevinQuann, and Brian WigdahlDrexel University College of Medicine

The mechanism of HTLV-1 associated myelopa-thy/tropical spastic paraparesis (HAM/TSP) patho-genesis is heavily reliant on the viral transcriptionaltransactivator protein Tax. The presence of cell-freeTax has been demonstrated in the cerebrospinal fluidof HAM/TSP and the effects of extracellular Tax hasbeen reported in a number of cell types includingmicroglia and neurons. Since astrocytes are theprimary glial cells within the brain and are knownto be infected with HTLV-1, we hypothesized thatHTLV-1-infected astrocytes could play a critical role inHAM/TSP pathogenesis by releasing Tax. In the studyreported herein, astrocytes were transduced withlentivirus vector expressing green fluorescent proteinlabeled Tax (Tax-GFP), and were observed under flu-orescent microscopy to determine localization of theprotein. Tax was found to be present in high concen-trations within cytoplasm, with very little or no Taxbeing found in the nucleus, 48-hr post-transduction.Subsequently, both cell lysate and supernatant werecollected from the transduced cells and analyzed forthe presence of Tax via western immunoblot analysis.Results indicated that Tax is released by astrocyteswithin 48 hr of transduction, in accordance with vi-sual confirmation of Tax presence in the cytoplasm. Acytokine array analyses was also performed to monitorthe changes in selected cytokine levels within astro-cytes in response to Tax. The Tax-mediated inductionof proinflammatory cytokines and chemokines wasobserved indicating activation of astrocytes andalteration in their function. Future experimentationwill determine the effect of Tax-transduced astrocyteson neuronal damage to correlate these observationswith HAM/TSP pathogenesis.

P.78Increased frequency of alpha-synuclein in thesubstantia nigra in HIV infectionNegar Khanlou, G Chana, Mariana Cherner,David J Moore, Deborah Lazzaretto, Sharron Dawes,Igor Grant, Eliezer Masliah, and Ian EverallDepartment of Psychiatry, UCSD


Objective: The frequency of neurodegenerative dis-eases markers among long surviving HIV infectedindividuals is unknown, therefore, the aim of thepresent study was to investigate the frequency ofalpha-synuclein, beta-amyloid and HIV-associatedbrain pathology in the brains of older HIV infectedindividuals.

Design: Cross-sectional analysis of analysis of pre-viously collected brain tissues from HIV infectedpersons enrolled in the National NeuroAIDS TissueConsortium.

Methods: We examined the substantia nigra of 73clinically well-characterized HIV infected individualsaged 50 to 76 years, who died after 1998. Additionally,we examined the frontal and temporal cortical regionson a subset of 36 individuals. The brain regions wereexamined for the presence of alpha-synuclein, beta-amyloid and HIV-associated brain pathology.

Results: Neuritic alpha-synuclein expression wasfound in 16% of the substantia nigra studied, whichwas far higher than previously reported in older con-trols. beta-amyloid was noted in 35 of the 36 casesexamined. The beta-amyloid deposits were primarilyintraneuronal with a few cases having additional stain-ing of the vessel walls. Despite these increases of de-generative pathology, HIV-associated brain pathologywas present in only 10% of cases.

Conclusions: Among older adults with HIV infec-tion there was no increase in the frequency of HIV-associated brain pathology; however, there was asubstantial increase in the frequency of both alpha-synuclein and beta-amyloid staining. The increasedprevalence of alpha-synuclein and beta-amyloid in thebrains of older HIV-infected individuals may predictan increased risk of developing neurodegenerative dis-ease in this cohort.

P.79Dynamics of NF-kB activation by HIV-1 tat andCD40L in microglial cellsMichelle M. Kiebala and Sanjay B. MaggirwarDepartment of Microbiology and Immunology, University ofRochester Medical Center, Rochester, NY 14642

HIV-1-associated dementia (HAD) occurs in a signif-icant number of HIV-1 infected individuals, in part,due to the inflammatory response to viral proteins inthe central nervous system. We have shown that hostfactors, such as soluble CD40 ligand (sCD40L) that arefound to be elevated in the cerebrospinal fluid andplasma of HAD patients, synergize with HIV-1 encodedTat to activate microglial cells in an NF-kB-dependentmanner. Here we demonstrate that, besides proteolysisof the inhibitory molecules IkB-alpha, the activationof NF-kB by Tat in microglial cells is associated withde novo synthesis of RelB, and processing of p100(as defined by generation of the processing productp52), followed by translocation of RelB and p52 intothe nucleus. However, under these conditions, nei-ther RelB, nor p52, exhibited DNA binding activityat a cis-acting NF-kB responsive element. We further

show that the simultaneous exposure to CD40L pro-motes DNA binding activity of p52, but not of RelB.Finally, ectopic expression of processing-deficient mu-tant p100, as well as RelB-specific short hairpin RNA(shRNA), revealed that the biogenesis of p52 and RelBin Tat plus CD40L-treated microglia is crucial for thesynthesis of TNF-alpha. Taken together, these studieshighlight the mechanism by which viral proteins andhost factors (like Tat and CD40L) work in concert to ac-tivate NF-kB in microglial cells and explain how thisprocess is dynamically controlled.

P.80Impact of low affinity C/EBP and Sp binding sites onHIV-1 replication and relevance to commonlyencountered LTR genetic variants observed earlyand late in HIV diseaseEvelyn M. Kilareski, Vanessa Pirrone, ShendraPassic, Michael R. Nonnemacher, and Brian WigdahlDrexel University College of Medicine

Recent studies have identified two genetic variantsin the human immunodeficiency virus type 1 (HIV-1)long terminal repeat (LTR) in the peripheral blood ofinfected patients that correlate with early and late HIVdisease. One incorporates a C-to-T change in C/EBPbinding site I (3T), and the other incorporates a C-to-T change at position 5 of Sp site III (5T). Inter-estingly, in peripheral blood-derived LTRs, 94% ofpatients who had the 3T variant also had the 5T variantwithin the same LTR. In LTRs isolated from the brainsof HIV-infected patients, 3T C/EBP site I and 5T Sp siteIII were only found in patients who had HIV-associateddementia (HIVD), but were not found in patients with-out HIVD. The overall goal of these studies is to de-termine the effect of these particular genetic variantson viral replication. The 3T, 5T, and 3T5T double vari-ant were incorporated into the HIV-1 LAI infectiousmolecular clone, and infectious viral particles weregenerated by transfection of the molecular clone into293T cells. The results of infection of monocytic U-937and lymphocytic Jurkat cell lines, as well as primaryperipheral blood mononuclear cells and monocyte-derived macrophages with genetically altered virusescarrying the 3T, 5T, and 3T/5T C/EBP and/or Sp tran-scription factor binding site variants has suggested thatthe single and double base pair changes in the viralLTR have impact of viral replication capability. In ad-dition, the effect of cytokine stimulation of these HIV-1infected cell populations has also been examined.

P.81Modulation of the NMDA receptor during HIV-tatinduced neuronal apoptosisJessie E King,1 Eliseo A Eugenin,1 Joy E Gibson,1 andJoan W Berman1,2

1Department of Pathology, Albert Einstein College ofMedicine; 2Department of Microbiology and Immunology,Albert Einstein College of Medicine

HIV infection of the CNS can result in neurologic dys-function, including motor impairment and cognitive


deficits, in a significant number of individuals withAIDS. NeuroAIDS is characterized by neuronal injuryand loss, yet there is no evidence of HIV infected neu-rons. Neuronal dropout must therefore be due to theindirect effects of HIV infection of other CNS cells,through elaboration of inflammatory factors and neu-rotoxic viral proteins, including the viral transacti-vating protein tat. Tat induces apoptosis in humanneurons that is dependent on the lipoprotein receptor-related protein (LRP) and the NMDA receptor(NMDAR). We show that NMDAR positive neurons ini-tiate tat induced apoptosis through the rapid forma-tion of a cell surface complex that includes tat, LRP,PSD-95, and NMDAR channels. Neuronal nitric ox-ide synthase (nNOS) is subsequently recruited to thiscomplex and activated. Blocking LRP mediated uptakeof tat, NMDAR activation or nNOS activity with spe-cific inhibitors results in a significant reduction in tat-induced neurotoxicity, suggesting that this complexplays an initial role in generating and amplifying tattoxicity. These data demonstrate the early mechanismsby which tat triggers apoptosis using the synaptic ma-chinery expressed by NMDAR positive neurons.

In addition, tat treatment of neurons results in phos-phorylation of the NMDAR. The NMDAR subunit 2A(NR2A) is rapidly phosphorylated after tat treatment ina Src kinase dependent manner. In vitro kinase assayssuggest that Src can phosphorylate NR2A on at least3 different tyrosine residues, and research is ongoingto determine whether these residues are phosphory-lated in our human neuronal culture model. NMDARphosphorylation is an important mechanism by whichNMDAR activity can be modulated, by altering perme-ability of the channel to specific ions, the kinetics ofchannel opening and closing, and trafficking of NMDAreceptors to and from the cell membrane. Preliminarydata indicate that there may be a reduction in clathrinmediated internalization of surface receptors after tattreatment. These findings may provide further under-standing of the neuropathogenesis of NeuroAIDS, andindicate avenues of treatment for those vulnerable toHIV cognitive impairment.

P.82HIV-1 tat interaction with proteins at the cellularPCNA promoter and at JCV early and late promotersmodulates JCV DNA synthesis and repairYayoi Kinoshita,1 Margaret J Wortman,2 Michelle RStettner,2 Tracy Fischer-Smith,3 Jay Rappaport,3Kamel Khalili,3 Dianne C Daniel,1,2 andEdward M Johnson2

1Department of Pathology, Mount Sinai School ofMedicine, New York, NY; 2Department of Microbiology andMolecular Cell Biology, Eastern Virginia Medical School,Norfolk, VA 23507; 3Department of Neuroscience andCenter for Neurovirology, Temple University School ofMedicine, Philadelphia, PA 19122

JC virus (JCV) us the etiologic agent of progressive mul-tifocal leukoencephalopathy, a defining opportunis-tic infection in AIDS. JCV infects primarily oligoden-droglial cells in the brain. The Tat protein encoded byHIV-1, which also infects the brain, interacts with cel-lular protein Pur-alpha to regulate several aspects ofJCV gene transcription and DNA replication throughbinding of Pur-alpha to DNA regulatory sequences. Re-cent reports help elucidate the Pur-alpha replicativepathway. Pur-alpha plays a role in repairing double-stranded DNA breaks induced by stalled forks in repli-cating chromosomal DNA. Pur-alpha may thus act withcellular replicative proteins at both initiation and elon-gation stages of chromosomal DNA replication. Cellu-lar protein PCNA is known to function as a DNA poly-merase processivity factor during both DNA synthesisand repair, and PCNA is necessary for replication of allpolyoma viral DNAs. We have thus asked whether Tatand Pur-alpha act to regulate expression of the PCNAgene in glial cells. Using chromatin immunoprecipita-tion (ChIP) we find that Tat, at concentrations as lowas 10-12M, strongly enhances binding of Pur-alpha tothe PCNA promoter. This result is highlighted by a po-tential unusual structure in the PCNA promoter. Usinga new double ChIP method we find that Tat and Pur-alpha are bound together to a specific region of thePCNA promoter. Pur-alpha acts with JCV T-antigen andwith Smad effectors of the TGF-Beta pathway at theJCV transcription/replication regulatory region. TheTGF-Beta1 response is modulated by Tat. We find thatTat enhances binding of Smad 2/3, Smad 4 and Fast1to the JCV regulatory region. We also find co-ChIP ofSmad4 and Fast1 at the PCNA promoter. Functionalaspects of Tat and its partner proteins at the PCNApromoter are detailed. Results indicate a mechanismby which Tat and Pur-alpha influence JCV DNA repli-cation by facilitating the processivity of ongoing DNAreplication forks, thus preventing the damage inducedby stalled forks.

P.83Gamma-herpesvirus infection of the CNS isindependent of B-cells and leads to immunecell-triggered cellular damageMichael Kleines,1 Simone Scheithauer,1 BerndSellhaus,2 Klaus Ritter,1 and Martin Hausler3

1Division of Virology, UK Aachen, RWTH Aachen,Germany; 2Department of Neuropathology, UK Aachen,RWTH Aachen, Germany; 3Department of Pediatrics, UKAachen, RWTH Aachen, Germany

We established a model system for neurologic gamma-herpesvirus diseases based on the murine her-pesvirus 68 (MHV-68)/mouse system for investigationof Epstein-Barr virus (EBV)-associated neurologicalcomplications ranging from facial palsy to encephali-tis. All types of inflammation observed in children inresponse to EBV can be observed in MHV-68 infectedmice.


The kinetics of the viral appearance in the CNS andthe related inflammatory response were determinedby PCR, IFA, H.E. staining or immunhistochemistry inwildtype BALB/c-, wildtype C57BL/6-, and immunod-eficient B6-(Rag1) mice and linked to each other.

In BALB/c mice viremia was maximal one week p.i.and decreased rapidly. The viral load in brain tissueexceeded viremia significantly six days post infection(p.i.) indicating intracerebral viral replication. Maxi-mal viral load in brain was gained on day 12 p.i. andremained positive for months. Cellular infiltrates co-localizing with viral activity were initially detected 9days p.i. (T-lymphocytes), their extent increased 16-48 days p.i. and decreased again 100 days p.i. A strongpositive correlation was observed between CNS viralload and severity of inflammation, particularly in thetemporal lobe. The inflammation reaction can be asso-ciated with severe cellular damages.

C57BL/6 mice showed also CNS inflammation, butwith reduced cellular damage suggesting differencesin the susceptibility to the virus in distinct mousestrains. In immunodeficient B6-(Rag1) mice developedfrom the identical genetic background higher CNS vi-ral loads were observed compared to wildtype ani-mals, but no cellular damages, which suggests thatthe viral entry to the CNS is B-cell independent andthat the antiviral immune response contributed to thelesions.

Taken together, MHV-68 leads to delayed, persistent,B-cell independent CNS infection followed by inflam-mation patterns observed in patient with cerebral EBVinfection. The antiviral immune response contributesto CNS lesions.

P.84Targeted siRNA delivery into CNS using in vitroblood brain barrier modelElton Kong,1,2,3 Edward A Acheampong,1,2,3 Roger JPomenrantz,4,5,6 Muhammad Mukhtar,7,8,9 andZahida Parveen1,2,3

1Department of Medicine, Division of Infectious Diseases;2Thomas Jefferson University; 3Philadelphia, PA 19107;4Tibotec Inc.; 51020 Stony Hill Road, Suite 300; 6Yardley,Pennsylvania 19067; 7Drexel University College ofMedicine; 8Department of Microbiology and Immunology,Institute for Molecular Medicine and Infectious Disease;9Philadelphia, Pennsylvania 19102

Background: RNA interference (RNAi) has emerged asa highly effective and conserved mechanism for thedown regulation of gene expression. While gene si-lencing by RNA interference holds promises for newapproaches for the treatment of a wide range of dis-eases, delivery of the RNAi to the right location hasbeen a major obstacle, particularly in central nervoussystem (CNS) by the presence of blood brain barrier(BBB), regulated by a group of tight junction pro-teins (tjs) including ZO-1. Previous methods of deliv-ery through BBB have been poorly effective. Produc-

tion of chimeric peptides represents a physiologicalbased strategy for targeted RNAi delivery into CNSby selective increase in blood brain barrier perme-ability without affecting the other tissues. We havedeveloped a novel system for the targeted deliveryof RNAi into CNS, using spleen necrosis viral (SNV)vectors.

Methods: Phage peptide library was purchased fromNew England Biolabs NEB and used to screen thepeptides specific for the human brain micro vas-cular endothelial cells (hMVECS) using manufac-turer protocol. MVECs were cultured in 60mm platesMVECs and biopanned with12-mer peptide libraries.After three round of successive biopanning and se-quencing a12-mer peptide was selected for MVECs.The selected phage was amplified and the DNA se-quences were cloned into the transmembrane regionof the SNV envelope vector pTC53 using Nae1 site.Different short interfering RNA template sequences(siRNA) were designed using human ZO-I tight junc-tion gene sequences. The 19 base pair hairpin tem-plate sequences were cloned into SNV transfer vec-tor using humanU6 promotor(RNA polymerase III)with a Bbs1 and Hind111cloning sites. Antibodiesagainst ZO-1 were purchased from to select the ef-ficient siRNA by tranfecting the MVECs followed bywestern blot, RT-PCR and immunocytochmistry. Celltype specificity of the12-mer phage peptide clonedinto SNV envelope was tested by generating SNVbased viral particles transcribing siRNA against ZO-1 and displaying MVECs specific peptides on thesurface of the virus, by using quadruple transfec-tion method. Different CNS cells including astrocytesand MVECs were transduced and analyzed for tar-geting the MVECs by immunostaining and westernblot. The ZO-1 inhibition was also tested by samemethods.

Results: The results by of the transfected and trans-duced MVECs using immunocytochemistry,westernblot and RT-PCR indicate that the 12-mer peptidehighly selective for human MVECs and ZO-1 expres-sion could be inhibited 60% by this targeted siRNA.

Conclusions: Targeted siRNA delivery into CNS bySNV based vectors could be ideal system to design thetherapeutics against neurodegenerative disorders. Tomimic the in vivo conditions, in vitro BBB system willbe ideal for further studies.

Key wards: Blood brain barier, Spleen necrosis virusand ZO-1

P.85Astrocyte-IL-8 regulation during HIV-1 infection ofthe CNS: The Yin and Yang of neuroprotection andglial activationWei Kou,1 Kathleen Borgmann,1 Sugato Banerjee,1 LiWu,1 Raisa Persidsky,1 and Anuja Ghorpade1,2,3


1Laboratory of Cellular Neuroimmunology, Center forNeurovirology and Neurodegenerative Disorders,Department of Pharmacology and ExperimentalNeuroscience, University of Nebraska Medical Center,Omaha, Nebraska 68198-5215, USA; 2Department ofPathology/Microbiology, University of Nebraska MedicalCenter, Omaha, Nebraska 68198-5215, USA; 3Departmentof Biochemistry/Molecular Biology, University of NebraskaMedical Center, Omaha, Nebraska 68198-5215, USA

Reactive astrogliosis is a key pathological featureof HIV-1-associated dementia (HAD) and is associ-ated with neural injury. Activated astrocytes pro-duce chemokines including Interleukin-8 (IL-8), a keymediator of neutrophils and monocytes recruitmentand activation in the periphery. Recent research fromour own and other laboratories suggests that IL-8 isinvolved in neuroinflammatory responses, and con-tributes to several neurodegenerative disorders, in-cluding multiple sclerosis, HIV-1-associated dementia(HAD) and Alzheimer’s disease. Functional analysiswith our gene microarray data revealed that IL-8 wasone of the central mediators of the immune responsein IL-1σ -activated astrocytes. The current study is de-signed to investigate the regulation of astrocyte IL-8in the context of brain HIV-1 infection and its impacton neurodegeneration. Primary human astrocytes, mi-croglia, and neurons were cultured and treated withdifferent stimuli including, but not limited to, IL-1σ ,TNF-�, HIV-1ADA, and HIV-1gp120. The cytokineand/or chemokine production from these cells wasmeasured by real-time PCR and ELISA. The effectsof IL-8 on neuronal survival were tested by double-stranded DNA fragmentation ELISA. The IL-8 expres-sion in human brain tissues from HIV-1-infected pa-tients was also examined. Our data show that 24 htreatment with HIV-1ADA, HIV-1gp120 or IL-1σ cause10- to 1000-fold increases in astrocyte IL-8 produc-tion as measured by ELISA. Both real-time PCR andELISA data show that chronic activation of astro-cytes with IL-1σ for 7 days also significantly increasedtheir IL-8 expression. Furthermore, while blockingthe NF-χB pathway completely abrogated the IL-1σ -induced astrocyte IL-8 production, it shows no ef-fect on HIV-1gp120–induced astrocyte IL-8 expression.Although, microglia and neurons may also produceIL-8, immunohistochemical analysis of brain sectionsfrom HIV-1-infected patients reveals that reactive as-trocytes, but not microglia in the microglial noduleswere positive for IL-8. While IL-8 by itself did nothave any significant effects on astrocyte MCP-1 pro-duction, similar levels of IL-8 completely abolishedthe HIV-1ADA–induced neurotoxicity in primary hu-man neurons. In summary, our data indicate that astro-cytes are the major cellular sources of IL-8 in brain inthe context of HIV-1 infection and this likely involvesNF-χB regulation. The impact of IL-8 overexpressionon neuronal survival is probably determined by directeffects on neurons and by microenvironment modula-tion through glial cell activation. How astrocyte-IL-8regulation ultimately contributes to the pathogenesisof HAD is currently under investigation.

P.86Macrophage-mediated dorsal root ganglion damagemediates altered nerve conduction in SIV-infectedmacaquesVictoria A Laast,1 Patrick M Tarwater,2 Robert JAdams,1 Justin C McArthur,1 Matthias Ringkamp,1 MChristine Zink,1 and Joseph L Mankowski1

1Johns Hopkins University; 2University of Texas HealthScience Center, El Paso Regional Campus

Peripheral neuropathy is the most common neurologi-cal complication of HIV-1 infection, affecting over one-third of infected individuals including those treatedwith antiretroviral therapy. To study the pathogenesisof HIV-induced PNS disease, we established a modelin which SIV-infected macaques developed changesclosely resembling alterations reported in sensory gan-glia of HIV-infected individuals including macrophageinfiltration, SIV replication in macrophages, and neu-ronal loss. Significant declines in epidermal nervefiber (ENF) density also developed in SIV-infectedmacaques (P = 0.002) similar to HIV-infected indi-viduals with neuropathy. SIV-infected macaques alsohad significantly lower C-fiber conduction velocityin sural nerves than uninfected animals (P < 0.001)and the magnitude of CV decline correlated stronglywith extent of DRG macrophage infiltration (P =0.006). The finding that injury to neurons in theDRG mediated by activated macrophages was closelyassociated with altered function of unmyelinatednerve fibers in SIV-infected macaques suggests thatmacrophage-mediated DRG damage may be the initi-ating event in HIV-induced sensory neuropathy. Treat-ment of SIV-infected macaques with a combination ofPMPA and minocycline initiated during early asymp-tomatic infection prevented both DRG macrophage ac-tivation and protected against loss of DRG neuronsand epidermal nerve fibers. The SIV/macaque modelwill be valuable for defining mechanisms underlyingHIV-induced PNS disease and for discovering noveltherapies.

P.87Place and Strategy Learning Deficits in the HIV-1Transgenic RatAbigail L LaShomb,1 Michael Vigorito,1 and Sulie LChang2

1Seton Hall University, Department of Psychology; 2SetonHall University, Department of Biology

Infection with HIV-1 has been linked with several cog-nitive deficits. The nature of these cognitive deficitsrange from mild to profound and typically affect mem-ory, attention, and motor function. In humans, these ef-fects are difficult to study due to frequent co-morbiditywith HIV. A new transgenic rat model has been de-veloped that allows for more controlled investiga-tions into the link between HIV and cognitive deficits.The HIV-1 transgenic (HIV-1 Tg) rat has almost thecomplete HIV-1 genome incorporated into its DNAwith only the replication genes, gag and pol, deleted,


making this animal model of HIV non-infectious. Theremaining HIV-1 Tg genes code functional viral pro-teins, including gp120 and Tat, both of which havebeen shown to be linked to cognitive deficits.

The HIV-1 Tg rats, however, have congenitalcataracts that make visual information difficult forthem to utilize during testing. For this reason, the ratswere tested in the dark in a modified Morris watermaze which provided rats with a combination of audi-tory, tactile, and olfactory cues arranged in such a waythat each quadrant had a unique combination of cues.We previously found a learning deficit in HIV Tg ratsthat were tested in this modified water maze. In thepresent study we further characterize the nature of thelearning deficit by making changes in the training pro-cedures to test place, strategy, and reversal learning.

This experiment contained three distinct phases.Phase 1 was designed to test response learning. Ratswere started from a fixed start point and were requiredto swim to a fixed platform location. Place learningand flexibility of behavior was tested following Phase1 by starting the rats from randomized start locationsand requiring them to swim to the same fixed plat-form location (Test 1) and to a moving platform in thesame target quadrant (Test 2). Phase 2 tested the ratsin the same procedure used in Test 2 but in the oppo-site target quadrant, and thus tested reversal learning.A single day (4 trials) of testing with the cues rotated90 degrees from their original orientation tested therats’ reliance on the available cues. Place learning wasmade irrelevant in Phase 3 by requiring the animals tochange their search strategy from a general quadrantsearch to a perimeter search.

The HIV-1 Tg rats showed subtle to strong impair-ments in all phases of the experiment. These resultssuggest that the HIV-1 Tg rat has deficits in place learn-ing, strategy learning, and reversal learning as mea-sured by this paradigm. These deficits are presumablydue to viral interactions within the CNS. These resultsindicate that the HIV-1 Tg rat may be a good model forstudying the cognitive deficits associated with Neu-roAIDS. Future studies will investigate the relation be-tween the behavioral deficits seen here and the levelsof viral proteins within the brain.

P.88The role of type 1 astrocytes in pro-inflammatoryCNS signalingEhud Lavi and Lin CongDep. of Pathology, Weill Medical College of CornellUniversity

Astrocytes are the most abundant cells in the cen-tral nervous system (CNS), but astrocytes constitutea heterogenous population of cells with a variety ofdifferent functions. Astrocytes are divided into threemajor subgroups (type 1,2 and 3) but very little isknown about the function of each subtype. Althoughit is known that astrocytes are capable of produc-ing cytokines in response to a variety of stimulations

including in response to pathogen infections, it iscurrently unknown which subtype is involved inimmune functions in the CNS. To investigate the po-tential role of astrocytic subtypes in immune functionwe investigated the differential response of clones ofthe three subtypes of mouse astrocytes to neurotropicand non-neurotropic coronavirus infections. We stud-ied the differential ability of astrocyte subtypes tomount pro-inflammatory immune reaction by analyz-ing by cDNA arrays cytokines, interferons and TNFprofiles of each subtype of astrocytes and microglia inresponse to these infections. Although both type 1 and2 astrocytes as well as microglia were infected by theviruses, only type 1 astrocytes and microglia cells pro-duced different pro-inflammatory cytokine responsesto neurotropic viral infection. The novel differentialresponse of astrocyte subtypes suggests that only type1 astrocytes serve along with microglia cells as the lo-cal immune system of the CNS.

P.89Brain metabolism changes observed in subjectsduring primary/early HIV infectionMargaret R Lentz,1 Vallent Lee,1 Woong-Ki Kim,2

Kenneth Williams,2 Ramon G Gonzalez,1 and EricRosenberg3

1Massachusetts General Hospital - Division ofNeuroradiology—Harvard Medical School; 2Beth IsraelDeaconess Medical Center - Division of ViralPathogenesis—Harvard Medical School; 3MassachusettsGeneral Hospital - Infectious Disease Unit - Partners AIDSResearch Center—Harvard Medical School

Introduction: HIV infection is believed to infiltrateand affect the brain early during the course of infec-tion. Previously reported results in the SIV/macaquemodel of neuroAIDS have demonstrated neuronal in-jury during primary (acute) infection using both neu-ropathology and proton magnetic resonance spec-troscopy (MRS). Temporal changes of metabolites inthe frontal cortex of these animals led to the conjec-ture that SIV infiltration was occurring at this stage ofinfection. Until now, the translation of these resultshas not been verified in humans. Herein, we reportboth metabolic and immunologic differences betweensubjects deemed to be healthy controls and those withprimary/early HIV infection.

Methods: Six subjects with primary/early HIV in-fection and eight healthy control subjects underwentmagnetic resonance imaging and spectroscopy (1.5TGE Signa scanner). Subjects identified with primaryHIV infection were imaged within 60 days of theirwestern blot indicating indeterminate/positive results,and while they still had detectable viral RNA levels(on average, 105 copies of RNA/mL). Each subject hadperipheral blood drawn within 48 hours of imagingand viral RNA levels were determined by RT PCR.Spectral analyses determining the quantities of N-acetylaspartate (NAA, the sum of NAA+NAAG), myo-inositol (MI), choline (Cho), glutamate + glutamine


(Glx), and creatine (Cr) in the frontal cortex were per-formed using LCModel software. Absolute concentra-tions and ratios were calculated. T-tests were used toconfirm changes between controls and those with pri-mary infection.

Results: Cho/Cr levels in the frontal cortex of sub-jects with primary HIV infection were 15% lower thanthose of healthy controls (p < 0.03). Similarly, abso-lute concentrations of Cho was found to be 11% lowerin those with primary HIV infection (p < 0.03). Crea-tine levels were found to be similar between the twogroups at this stage of infection in this brain region (p =0.14). MI/Cr and absolute concentration of MI of thosewith primary HIV infection were not significantly al-tered from that of healthy controls. Levels of NAA andGlx were each discover to be 14% lower in subjectswith primary HIV infection as well (p = 0.01, p < 0.01respectively).

Conclusion: Within the frontal cortex of subjectswith primary HIV infection, lower levels of both Choand Cho/Cr were observed. These results confirm thepreviously reported decreases of Cho/Cr found withinSIV infected macaques after peak virema. Choline de-creases thus far have rarely been reported in neuro-logic diseases. Interestingly, preliminary results indi-cate that creatine, the central energy marker of neuronsand astrocytes, does not appear to be different at thisstage of infection in the frontal cortex. NAA, a markerof neuronal integrity, and Glx levels are decreasing,suggesting that the virus is capable of causing neu-ronal injury not only in the chronic stage of infection,but soon after HIV infection.

P.90Olfactory nerve axotomy delays the onset of theacute phase response in mice inoculatedintranasally with influenza virusVictor H Leyva-Grado, Joshep W Harding, MarcusUrza, Jeannine A Majde, and James M KruegerDepartment of VCAPP Washington State University

The olfactory nerve is posited to be a possible routefor viruses to reach the brain after intranasal (IN) in-fection. In previous studies with the mouse-adaptedA/PR/8/34 (H1N1) (PR8) influenza virus we detectedviral genomic RNA and viral replication intermediatesas well as increased cytokine mRNA levels and cy-tokine immunoreactive cells in the olfactory bulb (OB)as early as 4 h (15 h for the cytokines) after IN infec-tion. These results suggest that the olfactory nerve islikely being used by this strain of influenza virus toreach the OB. Furthermore, the OB has indirect con-nections with the hypothalamus, suggesting the possi-bility that the OB cytokines communicate with the hy-pothalamus to induce the acute phase response (APR).We hypothesized that surgically severing the olfactorynerves prior to IN infection with influenza virus willreduce or delay the APR. Adult C57BL/6 mice (n =16) maintained at 24oC were surgically implanted with

radio transmitters in the peritoneum to continuouslymonitor body temperature (Tb) and locomotor activ-ity (LA). During the same surgical procedure the pri-mary olfactory nerves were severed (OBA) using a flex-ible teflon transector blade inserted between the OBand the cribiform plate to sever all the olfactory ax-ons projecting from the main olfactory epithelium (n =9). Sham-operated mice (n = 7) received an identicalsurgical exposure of the OB as the experimental miceexcept the blade was not inserted. Following surgerymice were given 7 days to recover. After recovery, base-line temperature and activity were recorded for 3 daysand after that all mice (n = 16) were infected IN atlight onset (8:00 AM) with live PR8 virus. Mice weremonitored for Tb and LA for 48 h after inoculation.Tb baseline values were not different between shamand OBA mice. Hypothermia was first apparent at 15h post-infection (PI) in the sham group with the low-est value of 35oC recorded at 20 h PI. In contrast, inthe OBA mice, hypothermia was first observed 26 hPI. After hypothermia was observed in the OBA mice,Tb behaved similarly in both groups with a declinethat reached 33oC at 48 h PI. Normal diurnal fluctu-ations were observed only during the first 12-14 h PI.LA was diminished in the sham operated mice aroundthe same time hypothermia was observed between 13and 15 h PI. In OBA mice LA was higher until 26 hPI when it reached the same level of that in sham-operated mice; from that time on, both groups behavedsimilarly. These results suggest that the olfactory nerveroute is important for early expression of the APRsince severing the pathway delays the response to theinfection.

NIH Grant No. HD36520 to JMK. Leyva-Grado wasalso supported by the Direccion General de Apoyo alPersonal Academico of the National Autonomous Uni-versity of Mexico.

P.91Altered recruitment of Sp1 and Sp3 isoforms duringmonocytic differentiation influences the activity ofthe HIV-1 LTRLuna Li,1 John J. McAllister,2 Michael R.Nonnemacher,1 Bryan P. Irish,1 Fred C. Krebs,1Evelyn Kilareski,1 and Brian Wigdahl1

1Drexel University College of Medicine; 2The PennsylvaniaState University College of Medicine

Infection of cells of the monocyte-macrophage lineageby human immunodeficiency virus type 1 (HIV-1) hasbeen shown to be important in the pathogenesis of theacquired immunodeficiency syndrome. Viral replica-tion in this cell lineage is, in part, mediated by inter-actions between the HIV-1 long terminal repeat (LTR)and a variety of host cell and viral proteins. Consis-tent with this logic, basal and activated LTR activity isdependent on interactions between the G/C box arraywithin the HIV-1 LTR and the Sp family of transcrip-tion factors. The effect of monocytic differentiation on


Sp factor binding and transactivation has been exam-ined with respect to the HIV-1 LTR. Primary monocyte-derived macrophages (MDM), as well as monoblastic(U-937 and THP-1) and myelomonocytic (HL-60) celllines were utilized in the absence or presence of chem-ical differentiating agents, dimethylsulfoxide (DMSO)or phorbol myristate acetate (PMA), to model selectedaspects of monocytic differentiation. The binding ofSp1, full-length Sp3, and truncated Sp3 to a high affin-ity HIV-1 Sp element was examined utilizing elec-trophoretic mobility shift (EMS) analyses. Sp1 bindingincreased relative to the sum of full-length and trun-cated Sp3 binding following PMA-induced monocyticdifferentiation in U-937, THP-1, and HL-60 cells. Rela-tive Sp factor binding ratios obtained with nuclear ex-tracts from PMA-induced cell lines were also shownto correlate with those derived from studies performedwith extracts derived from primary MDMs. In addi-tion, the altered Sp binding phenotype was shown tobe associated with changes in the transcriptional acti-vation generated by the HIV-1 G/C box array.

P.92Seizures induced by Theiler’s virus: a model forhuman seizure disorderJane E. Libbey,1 Kerry-Ann Stewart,2 Nikki J.Kirkman,1 Karen S. Wilcox,2 H. Steve White,2 andRobert S. Fujinami1

1University of Utah School of Medicine, and 2University ofUtah College of Pharmacy

We have established an infectious murine model forhuman seizure disorder. Infection of C57BL/6 micewith Theiler’s murine encephalomyelitis virus in-duced transient seizures in about 50% of C57BL/6mice. Seizures were not associated with fever and werevery prominent between 3 to 10 days post infection.Coordination and motor function were impaired inthe seized mice. Pyramidal neuronal loss and gliosiswere prominent features of seized mice. Seizure ac-tivity inversely correlated with inflammation in thehippocampus. Due to the timing of the seizures, directviral and innate immune responses contribute to thedevelopment of the seizures. The characterization ofthis model will allow us to study the involvement ofvirus and innate immune response in the central ner-vous system to the development of seizure disordersin humans.

P.93Activation of the integrated stress response inpatients with HIV-associated dementiaKathryn A Lindl, Cagla Akay, Ying Wang, Michael GWhite, and Kelly L Jordan-SciuttoUniversity of Pennsylvania

Despite the introduction of highly active antiretro-viral therapy (HAART), the prevalence of human-immunodeficiency virus associated dementia (HAD)has been steadily increasing. Neuronal injury and

loss in HAD is initiated by HIV-infected, activatedmacrophages/microglia via soluble neurotoxic medi-ators, including reactive oxygen species (ROS). Thesemediators, which induce oxidative stress, are knownto injure neurons directly and to alter astrocytichomeostatic functions such as glutamate homeostasis,which can lead to excitotoxic neuronal injury. A majordownstream response to oxidative stress is inductionof the integrated stress response (ISR), which includesboth the ER stress response and the antioxidant re-sponse. We hypothesize that the ISR is activated inHAD patients. To begin to investigate ISR activation inHAD, we assessed the expression patterns of ISR pro-teins, Binding Protein (BiP) and NF-E2-related factor2 (Nrf2), by immunofluorescence (IFA) and westernblotting in autopsy tissue from HAD patients. BiP isa molecular chaperone protein whose expression iscontrolled by all three independently regulatedbranches of the ISR. Nrf2 is the primary regulator ofthe endogenous cellular antioxidant stress response,and is upregulated by the ISR. We saw a significant in-crease in total BiP levels by both IFA and western blotin HAD cortical tissue as compared with control tissue.Additionally, by phenotypic analysis of IFA, we sawcell-type specific increases in BiP levels in astrocytes.Nrf2 levels increased significantly in neurons, astro-cytes, and overall in cortical tissue of HAD patientsover that of control patients as determined by IFA. Fur-ther, analysis of IFA stained tissue revealed that, whileNrf2 levels increased in HAD tissue, 0 out of 5 HADcases showed strong Nrf2 nuclear translocation. Thistrend suggests that, while Nrf2 protein levels may beupregulated by an activated ISR in HAD patients, theantioxidant response controlled by Nrf2 may not besuccessfully initiated, due to failure of this transcrip-tional regulator to translocate to the nucleus. Conse-quently, ROS released by activated macrophages, mi-croglia, and astrocytes in this disease may overwhelmendogenous protection mechanisms resulting in neu-ronal death. Together these results suggest that the ISRis activated in HAD, although the endogenous antiox-idant response, an important component of the ISR,may be aberrant in these patients.

P.94Structure and function of a highly conserved HIV-1LTR downstream regulatory elementYujie Liu, Michael R. Nonnemacher, Evelyn M.Kilareski, Aikaterini Alexaki, and Brian WigdahlDrexel University College of Medicine

At least one CCAAT enhancer binding protein (C/EBP)site upstream of the TATA box is necessary for hu-man immunodeficiency virus type 1 (HIV-1) long ter-minal repeat (LTR) activity in cells of the monocyte-macrophage lineage. However, no studies have beenperformed concerning C/EBP sites downstream (DS) ofthe start of transcription. TRANSFAC analyses of 100subtype B LTRs have indicated three potential C/EBP


binding sites within the DS LTR. However, EMS analy-ses demonstrated that only one of the three sites (DS3,+158 to +175) was able to form DNA-protein com-plex(es) containing C/EBPbeta. Transient transfectionstudies utilizing the parental HIV-1 LAI-LTR or theLAI-LTR containing a knock-out 9C variant (LTR-DS3-9C, G-to-C change at position 9 of the site) demon-strated loss of binding to this site results in an ap-proximate 50% decrease in basal LTR activity in theTF-1, U-937, and THP-1 monocyte-macrophage celllines. In addition, C/EBP-induced activation of theLAI-LTR was reduced based on the loss of C/EBP bind-ing to DS3. Interestingly, Tat-mediated transactivationof the LAI-LTR was elevated in the presence of the 9Cknockout with co-expression of C/EBPbeta and Tat re-sulted in an even greater level of transactivation. Tat-mediated transactivation of the HIV-1 LAI-LTR wasalso enhanced in the presence IL-6 with the LAI-DS3-9C knockout. These results suggest that sequence alter-ations in the highly conserved DS3 element may influ-ence the interaction of Tat with the HIV-1 LTR therebyaffecting HIV-1 transcription. This interaction may bea critical element required for HIV-1 transcriptionalregulation.

P.95Perturbation of the viral co-receptor CCR5 by NB325inhibits HIV-1 infectionKarissa Lozenski,1 Nina Thakkar,1 Shendra Passic,1Tina Kish-Catalone,1 Brian Wigdahl,1 Robert FRando,2 Mohamed Labib,2 and Fred C Krebs1

1Drexel University College of Medicine; 2NovafluxBiosciences, Inc.

Despite the successes achieved using the latest thera-pies effective against HIV-1-associated immunopatho-genesis, neurological disease associated with HIV-1infection of the central nervous system remains apressing problem. For this reason, the developmentof compounds effective against HIV-1, specifically theCCR5-utilizing strains predominant in the brain, re-mains a high priority. Our efforts in this area focuson compounds that inhibit HIV-1 infection by inter-fering with the role of CCR5 in viral binding and en-try. NB325, which is a biguanide-based compound,is characterized by low toxicity and notable activityagainst HIV-1 BaL, a CCR5-utilizing viral strain. Re-sults from flow cytometric analyses of primary humanperipheral blood mononuclear cells (PBMCs) demon-strated increased CCR5 detection following exposureto NB325 despite effective inhibition of HIV-1 BaL in-fection. Differences in CCR5 detection were also notedbetween quiescent PBMC sub-populations and cellsactivated with PHA-P. We hypothesize that inhibitionof HIV-1 BaL infection by NB325 may involve pertur-bation of CCR5 within the plasma membrane. Thesechanges interfere with interactions with HIV-1, andmake CCR5 more accessible to detection by CCR5-specific antibodies. Our current mechanism of actionstudies are building on the observation that NB325 alsointeracts specifically with CXCR4 extracellular loop

2 (ECL2). Ongoing investigations are exploring simi-lar interactions between NB325 and CCR5 that maycause changes in co-receptor availability, localization,and/or conformation that result in inhibition of HIV-1 infection. These studies are being used to facilitatethe development of novel compounds that can be usedsafely to inhibit HIV-1 CNS infection.

P.96Chromatin immunoprecipitation of the HIV-1 LTRand sequencing of the HIV-ADA LTRAimee J Luers and Joan W BermanAlbert Einstein College of Medicine, Department ofPathology

Forty million people worldwide are infected with HIV,which causes AIDS and NeuroAIDS. NeuroAIDS in-cludes behavioral and cognitive defects as well asmotor abnormailities. Upon autopsy, over 90% of in-fected individuals show the characteristic pathologyof NeuroAIDS, which includes neuronal damage anddeath, inflammation, elevated levels of the chemokineCCL2 in the CNS, and leukocyte infiltration of the CNS.Studies in humans and macaques indicate that cog-nitive impairment correlates with leukocyte presenceand CNS inflammation. The cellular activities of HIVproteins are still being resolved. The viral protein Tathas pleiotropic activities and interacts directly withhost cell chromatin. In astrocytes, Tat can directly in-teract with the CCL2/MCP-1 promoter, upregulatingthe expression of this chemokine. We hypothesize thatTat interacts with cellular proteins and chromatin di-rectly in cells of the immune system, affecting tran-scription of host cell genes responsible for inflamma-tion and chemotaxis of cells of the monocytic lineageinto the CNS. To examine this, we will use ChIP onchip in order to identify host cell genes with whichTat directly interacts and Mass Spectroscopy in or-der to identify the proteins with which Tat interactsat the chromatin. To date we have successfully im-munoprecipitated the HIVADA LTR using Tat antibodyand sequenced this region of the virus, thereby demon-strating that direct immunoprecipitation of this pro-tein while chemically crosslinked to DNA is possible.This project will identify components of the mecha-nism through which Tat mediates inflammation andchemotaxis. A more complete understanding of the ef-fects of Tat on host cell function will contribute to theidentification of therapeutic targets able to limit thedevastating consequences of NeuroAIDS.

P.97Functional synergy between CD40 ligand and HIV-1tat contributes to inflammation: implications inHIV-1 dementiaSanjay B Maggirwar,1,2 Ziye Sui,2 Lynn F Sniderhan,1

Giovanni Schifitto,3 Richard P Phipps,1 Harris AGelbard,1,2,3 and Stephen Dewhurst1

1Department of Microbiology and Immunology;2Interdepartmental Program in Neuroscience; 3Departmentof Neurology, University of Rochester Medical Center,Rochester, NY 14642


HIV-1 associated dementia (HAD) is believed to oc-cur due to aberrant activation of monocyte-derivedmacrophages and brain resident microglial cells by vi-ral proteins as well as by the pro-inflammatory me-diators released by infected cells. To investigate theinflammatory aspects of the disease, we examinedthe levels of soluble CD40 ligand (sCD40L) in pairedsamples of plasma and cerebrospinal fluid (CSF) ob-tained from 25 HIV-infected individuals. A signifi-cantly higher level of sCD40L was detected in bothCSF and plasma from HIV-infected patients with cog-nitive impairment, compared to their non-impairedcounterparts. The contribution of sCD40L to the patho-genesis of HAD was then examined by in vitro experi-ments. Recombinant CD40L synergized with HIV-1 Tatto increase TNF-alpha release from primary humanmonocytes and microglia, in a nuclear factor kappa B(NF-?B)-dependent manner. The mechanistic basis forthis synergism was attributed to a Tat-mediated upreg-ulation of CD40 in monocytes and microglia. Finally,the CD40L-mediated increase in TNF-alpha produc-tion by monocytes was shown to be biologically im-portant; immunodepletion experiments revealed thatTNF-alpha was essential for the neurotoxic effectsof conditioned medium recovered from Tat/CD40Ltreated monocytes. Taken together, our results showthat CD40 signaling in microglia and monocytes cansynergize with the effects of Tat, further amplifying in-flammatory processes within the central nervous sys-tem and influencing neuronal survival.

P.98Visualization of West Nile virus particles forexamination on the cellular entry mechanismYoshinori Makino,1,2 Tadaki Suzuki,1 Rie Hasebe,3Akihiko Maeda,2,3 Hidehiro Takahashi,4 TakashiKimura,1 and Hirofumi Sawa1,2

1Department of Molecular Pathobiology, Center forZoonosis Control, Hokkaido University; 221st Century COEProgram for Zoonosis Control, Hokkaido University;3Department of Prion Diseases, Graduate School ofVeterinary Medicine, Hokkaido University; 4Department ofPathology, National Institute of Infectious Diseases, Japan

West Nile virus (WNV) is a flavivirus transmitted byCulex mosquitoes to vertebrate hosts and causes epi-demics of febrile illness, meningitis, and, encephali-tis. WNV is found throughout Africa, Europe, CentralAsia, and, most recently in North America. Since theoutbreak in New York during 1999, WNV has been sub-sequently spreading across the United States. In 2002,there were 4,156 reported cases of human illness, as-sociated with 284 deaths in United States. Althoughvirus entry into host cells is a crucial step of viral in-fection, the behavior of WNV at the entry step into hostcells is still not clear.

As native WNV is limited to use in the biosafety level3 (BSL-3), we initially attempted to synthesize subviralparticles (SVPs) of WNV, which exclusively consist ofWNV envelope proteins, M and E. We confirmed thatthe SVPs have similar characters as native WNV. To ex-

amine the cellular entry mechanism of WNV, we nexttried to visualize the SVPs. We chose DiD a far-red flu-orescent lipophilic dye to label the SVPs, because thelong wavelength of DiD excitation shows a high S/Nratio, and succeeded in synthesis of the SVPs labelledwith DiD (SVPs-DiD). We confirmed that fluorescentintensity level of the SVPs-DiD was enough high forobservation by usual fluorescent microscopy. This sys-tem is useful for tracking the movement of the SVPs ofWNV in living cells at the entry step using a time-lapsemicroscope.

P.99NFAT4 is required for JCV infection of glial cellsKate Manley and Walter J AtwoodDepartment of Molecular Biology, Cell Biology, andBiochemistry, Brown University, Providence, RI02912

The human polyomavirus, JCV, infects 35-70% ofthe population worldwide. In immunosuppressed pa-tients, JCV infection can lead to Progressive Multifo-cal Leukoencephalopathy (PML), a fatal demyelinat-ing disease of the central nervous system (CNS). JCVtropism is restricted to oligodendrocytes, astrocytesand B lymphocytes. The factors responsible for thislimited tropism are not well understood. Previouslyour lab established a glial cell line (SVGR2) that isresistant to both wild-type JCV and a related poly-omavirus, SV40, by selecting glial cells (SVG-A) thatsurvived repeated rounds of JCV lytic infection. Theblock to viral replication in SVGR2 cells was identifiedto be at stage of viral transcription. Microarray compar-ison of SVG-A and SVGR2 cellular gene expression re-vealed a role for the transcription factor Nuclear Factorof Activated T-cell (NFAT) in both JCV and SV40 tran-scription and infection. In addition, viral activation ofNFAT is important for the maintenance of viral tran-scription throughout the viral lifecycle.

P.100Interplay of dendritic cell surface receptors andadhesion molecules with respect to HTLV-1 binding,entry, and transmissionSharron L. Manuel, Brian Wigdahl, Jaya Ahuja, ZafarK. Khan, and Pooja JainDrexel University College of Medicine

Despite the susceptibility of dendritic cells (DCs) toHTLV-1 infection and a demonstrated role of these cellsin disease pathogenesis, the mechanisms of virus bind-ing and entry have not been delineated. The identifi-cation of HTLV-1 receptor has been hindered due toits ubiquitous nature. Recently, a glucose transporterGLUT-1, heparan sulfate proteoglycans (HSPGs) andNeuropilin-1 (NRP-1) were demonstrated to facilitateHTLV-1 binding and entry, primarily in T cells. DCsexpress their own array of antigen receptors, most no-tably DC-specific ICAM-3-grabbing non-integrin (DC-SIGN), with respect to retrovirus binding. In this study,we analyzed the role of DC-SIGN and other knownHTLV-1 receptors with respect to viral binding, entry,


and transmission using primary human monocyte-derived DCs and B cell lines stably expressing DC-SIGN. A comparative expression of DC-SIGN, GLUT-1,HSPG, and NRP-1 was examined and correlated with anumber of biological endpoints. Inhibition of all thesemolecules dramatically reduced the binding of HTLV-1virions as well as envelope glycoprotein gp46. With re-spect to HTLV-1 entry, GLUT-1 and HSPCs played animportant role while inhibition of DC-SIGN and NP-1 had no apparent effect. Finally, the transmission ofHTLV-1 from infected DCs to target T cells was ana-lyzed and was found to be primarily mediated by DC-SIGN consistent with its known role for other viruses.These observations were also repeated ex vivo withmyeloid and plasmacytoid DCs. This study representsthe first comprehensive analyses of known HTLV-1 re-ceptors on a major immune cell population that playsa critical role in viral binding and transmission

P.101Bone marrow rearrangement of JC virus regulatoryregion in progressive multifocalleukoencephalopathyAngela Marzocchetti,1 Christian Wuthrich,1 Chen STan,1,2 Troy Tompkins,1 Francisco Bernal-Cano,1,3

Parul Bhargava,4 Rivka Sachdev,5 Judy Hinchey,5Alan H Ropper,6 and Igor J Koralnik1,3

1Division of Viral Pathogenesis, BIDMC, Harvard MedicalSchool; 2Division of Infectious Diseases, BIDMC, HarvardMedical School; 3Department of Neurology, BIDMC,Harvard Medical School; 4Department of Pathology,BIDMC, Harvard Medical School; 5Department ofNeurology , Caritas St Elizabeth’s Medical Center;6Department of Neurology, Brigham and Women’s Hospital,Harvard Medical School

Background: JC virus (JCV), the agent of progressivemultifocal leukoencephalopathy (PML), displays a sta-ble archetypal regulatory region (RR) in the kidneys,where it remains quiescent. However, rearrangementsof the RR are found in the CNS of PML patients andare associated with JCV neurotropism and neuroviru-lence.

Methods: JCV DNA viral load was measured in bonemarrow (BM), PBMC, plasma and urine by QPCR us-ing JCV T-specific primers. JCV RR was amplified bynested PCR, cloned and sequenced. Double immuno-histochemical staining (IHC) was performed on BMbiopsy tissue sections using an anti-VP1 and anti-CD138 ab. JCV-specific CD8+ cytotoxic T lympho-cytes (CTL) was analyzed in blood samples by tetramerstaining assay.

Case report and Results: A 70 year old man withrheumatoid arthritis (RA) treated with methotrexatedeveloped PML.

Methotrexate was discontinued and the patient wastreated with cytarabine and mirtazapine, but he passedaway 13 weeks after disease onset. JCV specific CTLresponse was at limit of detection of the assay.

All JCV RR clones from plasma had a tandem re-peat of 98bp elements consistent with the neurotropic

Mad-1 RR, and urine samples contained an RR withpartial truncation and duplications of the 98 bp ele-ment. Interestingly, the major form of BM RR (92%of the clones) contained a partial duplication of the98bp element and truncation of the 23 and 66bp in-serts, while a minor BM species of RR was similar toarchetype (8% of the clones).

IHC on the BM biopsy sample showed that 94%of JCV-infected cells were CD138+ plasma cells, andthese accounted for 9% of all plasma cells in the biopsyspecimen.

Discussion: These results indicate that archetypeJCV RR is present in BM and that rearrangementsof the RR occur in this compartment. It is thereforepossible that JCV gained its neurotropic phenotypein the BM of this patient, and this strain may havereached the CNS via hematogenous spread. Demon-stration of productive JCV infection of BM plasma cellssupport this hypothesis. Finally this case illustratethat RR with partial tandem repeat can also arise fromkidney.

Conclusion: This is the first report of a detailed anal-ysis of JCV RR from BM of a PML pt. and representevidence that rearrangements in the RR that may leadto PML can occur in this body site.

Acknowledgements: This work was made possiblein part by Public Health Service Grants R01 NS/AI041198 and NS 047029, as well as the Ellen R. Cav-allo research fund to IJK.

P.102Dendritic cells as a therapeutic vaccine againstProgressive Multifocal LeukoencephalopathyAngela Marzocchetti,1 Marco Lima,1,2,3 TroyTompkins,1 Daniel G Kavanagh,4 Rajesh T Ghandi,4Nina Bhardwaj,5 and Igor J Koralnik1,2

1Division of Viral Pathogenesis, BIDMC, Harvard MedicalSchool; 2Department of Neurology, BIDMC, HarvardMedical School; 3The Reference Center on neuroinfectionsand HTLV, Fundacao Oswaldo Cruz; 4Partners AIDSResearch Ctr, Massachusetts General Hospital, HarvardMedical School; 5New York University School of Medicine

Background: Progressive multifocal leukoen-cephalopathy (PML) is a demyelinating diseaseof the brain caused by JC virus (JCV) which occurs inimmunosuppressed individuals. There is no effectivetreatment for this disease, but PML patients who haveJCV-specific cytotoxic T lymphocyte (CTL) in theirblood have a better clinical outcome. One of the mostpromising strategies for boosting T cell immunity isthrough autologous dendritic cell (DC) immunization.In this study we sought to expand JCV- specific CTLex-vivo using JCV antigen-loaded dendritic cells orperipheral blood mononuclear cells (PBMC) culturesfrom healthy individuals (HI), HIV+/PML – (HIV) andPML patients (PML).

Patients and methods: Peripheral blood sampleswere obtained from HLA-A*0201+ subjects, includ-ing 10 JCV-seropositive HI, 6 PML and 4 HIV+


patients. Monocyte-derived mature DC pulsed withHLA-A*0201-restricted JCV VP1p36 and/or VP1p100epitopes were mixed with autologous T cells at a ratio1:10. In parallel, JCV - VP1p36 and/or VP1p100 epi-topes stimulated PBMC were cultured in the presenceof IL-2. After 12 days, JCV specific CTL responses werecompared in both cultures using tetramer stainingassay.

Results: JCV-specific CTL responses against VP1p36and/or VP1p100 epitopes, present in 5/10 HI, 3/4HIV+ pts and 4/6 PML pts using peptide stimulationof PBMC, were enhanced when using Ag-loaded DCstimulation. After stimulation with Ag-pulsed DC a re-sponse was detected in 1 HIV+ pt who had no responsewith peptide stimulation of PBMC only. In 4 HI and 1PML pts. no JCV-specific expansion was obtained ei-ther in PBMC or DC cultures. The mean percentage ofJCV-specific CD8+ T cells in peptide-stimulated PBMCcompared to DC cultures was 1.1% (0.2-2.6%) vs 29%(12.3-48.2%) in HI, 2.1%(0-4.2%) vs 5.2% (0.2-8%) inHIV+ pts and 0.5% (0.3-1.1%) vs 7.2% (0.4-26%) inPML pts.

Discussion: DC pulsed with HLA-A201-restrictedJCV peptides were able to enhance the CTL expansionin the 3 study groups. The lack of any detectable JCVCTL expansion in PBMC or DC cultures of 4 HI and 1PML pts. could be explained by an immune responsedirected toward other JCV epitopes.

Conclusion: Monocyte-derived mature DC loadedwith JCV antigens can induce potent specific CTL ex-pansion in vitro and offer a promising approach toPML immunotherapy.

Acknowledgements: This work was supported inpart by Public Health Service Grants R01 NS/AI041198 and NS 047029 to IJK.

P.103Interplay of host and viral factors in the clinicaloutcome of patients with Progressive MultifocalLeukoencephalopathy (PML).Angela Marzocchetti,1 Troy Tompkins,1 David BClifford,2 Rajesh T Ghandi,3 Santosh Kesari,4 JosephR Berger,5 David M Simpson,6 Andrea de Luca,7 andIgor J Koralnik1,8

1Division of Viral Pathogenesis, BIDMC, Harvard MedicalSchool; 2Department of Neurology, Washington UniversitySchool of Medicine; 3Department of Infectious Diseases,MGH, Harvard Medical School; 4Departemnt of Neurology,Brigham and Women’s Hospital, Harvard Medical School;5Department of Neurology, University of Kentucky Collegeof Medicine; 6Division of Neurology, Mount Sinai MedicalCenter; 7Division of Infectious Diseases, CatholicUniversity Rome; 8Department of Neurology, BIDMC,Harvard Medical School

Objectives: To determine the role of immunologicaland virological factors in the clinical outcome of PML

Methods: We enrolled 60 PML pts (75% HIV+, 25%HIV-) in this study, as well as 20 HIV+ pts matchedfor CD4 count and plasma HIV VL and 15 healthy in-dividuals (HI). JCV DNA viral load (VL) was measured

in PBMC, plasma and urine by QPCR using JCV T-specific primers. JCV RR was amplified by nested PCR,cloned and sequenced. JCV-specific CD8+ cytotoxic Tlymphocytes (CTL) were analyzed in blood samplesof HLA A*0201+ subjects by tetramer staining assayfor VP1 p36 and p100 and HLA A*0201- subjects by51Cr release assay using pool of overlapping peptidescovering the VP1 protein.

Results: The mean age of PML and HIV+ pts was 49y and 35y for HI. HIV+/PML and HIV+ pts had a CD4cell count of 263 vs 398 /μL, and a plasma HIV VL of1.5 vs 2.2 log10 copies/ml, respectively. JCV DNA wasdetected in the urine of 48% PML, 61% HIV+ and 26%HI. JCV DNA was detected in plasma and PBMC of 39and 54% PML, 10 and 30% HIV+ and 0 and 0% HI.JCV detection was significantly increased in plasmaof PML vs HIV+ pts. (p = 0.024).The mean JC VL inblood was similar in PML and HIV+ pts and amongthe 3 study groups in urine.

In HLA A*0201+ PML pts there was a positive cor-relation between the magnitude of JCV VP1 p100 CTLresponse and JC VL in urine (r = 0.92 p = 0.03) or blood(plasma r = 0.63 p = 0.05; PBMC r = 0.76 p = 0.04).Among PML pts, survival at one year was 72 vs 42%for those with detectable vs undetectable JCV-specificCTL within one year of diagnosis, respectively (n = 34Hazard Ratio of death (HR) 0.42, p = 0.14). Of 52 PMLwho had an MRI, 13 (25%) had contrast enhancement(CE) of PML lesions. Of these 13 pts, 12 (92%) survivedmore than one year, and 10 (77%) had detectable JCVCTL compared to 18/39 (46%) pts with no CE who hada CTL response (OR 3.9; p = 0.064). The analysis of JCVRR sequences is in progress and will be presented atthe meeting.

Conclusion: JCV detection in plasma is significantlymore frequent in PML vs. HIV+ pts. The presenceof JCV CTL was associated with a higher rate of sur-vival at one year. CE of PML lesions on MRI ap-pears to be a useful prognostic marker of diseaseevolution.

Acknowledgements: This work was supported inpart by Public Health Service Grants R01 NS/AI041198 and NS 047029 to IJK.

P.104JC Virus Excretion Pattern in HIV infectedPortuguese PatientsAna M Matos,1 Vitor M Duque,2 Antonio AMelico-Silvestre,2 and Joao M Poiares da Silva1

1Laboratorio de Microbiologia, Faculdade de Farmacia daUniversidade de Coimbra, Portugal; 2Laboratorio deVirologia do Departamento de Doencas Infecciosas dosHospitais da Universidade de Coimbra, Portugal

Objectives: JC virus (JCV) is ubiquitous in human pop-ulation. After initial infection that normally occursduring childhood, JCV seems to remain latent in renaltissue, and reactivation occurs under immunosupres-sion conditions, with progeny shed in urine.

However, in severe immunosupresion situations asthose resulting from advanced HIV infection, JCV


reactivation could result in PML, a fatal CNS disease.Actually PML is one of the most frequent CNS op-portunistic infections among HIV-infected people, andAIDS represents it’s most frequent underlying cause ofimmunossupression.

In this order, the present study intends to begin theevaluation of prevalence and pattern of JC virus excre-tion in HIV infected Portuguese patients. The resultsobtained will be compared with those described fornon-HIV infected population, in order to evaluate ifHIV infection affects the demographics of JCV urinaryexcretion.

Methods: Urine samples from 49 HIV infected Por-tuguese patients in different stages of the disease, werecollected and screened for the presence of JCV DNA.

This patient group includes 10 women and 33 men,aged between 28 and 75 years old (43 mean age).

The median CD4 cell count was 350 cells/microLand the median viral load was 131062 copies/mL.

After DNA extraction, each sample was subjectedto PCR amplification performed with specific primersfrom the VP1 coding region of JCV genome. Am-plified products were visualized by agarose gelelectrophoresis.

Results and Conclusions: Twenty-five (58%) out of49 urine samples revealed the presence of the expectedfragment from JCV genome. Thirty percent of womenand 67% of men in this study excretes JC virus in urine.

The age groups presenting a greater prevalence ofJCV viruria were 30 to 39 and over 60 years old, with79% and 100%, respectively.

Rate of JCV urinary excretion don’t seems to varywith CD4 cell count as well as with HIV RNA viralload.

Conclusions: The present study, although only at thebeginning, points to a value of 58% of HIV-infectedPortuguese population that excretes JC virus in urine,which is concordant with prevalence rates observed inother countries.

Our preliminary data also reveals and confirms thatimmunodeficiency as well as HIV plasmatic viral loaddon’t seems to affect JC virus urinary excretion.

Although the excretion rate observed in HIV-infected Portuguese populations is similar to that de-scribed in immunocompetent population, age distri-bution doesn’t seems equivalent, which could be dueto the little number of samples evaluated.

P.105CCR5-deficiency prevents injury to neuronaldendrites and synapses in a transgenic mouse modelof HIV-1 neuropathology expressing viral envelopeprotein gp120 in the brainRicky Maung,1 Kathryn E Medders,1 Rossella Russo,1Maya K Desai,1 Natalia Sejbuk,1 Leah Le,1 Gwenn AGarden,2 and Marcus Kaul1,3

1Center for Neuroscience and Aging and Center forInfectious and Inflammatory Disease, Burnham Institute forMedical Research, La Jolla, CA 92037, USA; 2Departmentof Neurology, University of Washington, Seattle, WA 98195,

USA; 3Department of Psychiatry, University of CaliforniaSan Diego, San Diego, CA 92093, USA

We investigated the potential role of coreceptorsfor human immunodeficiency virus-1 (HIV-1) inpromotion and prevention of the neuropathologyassociated with HIV-1 infection. HIV-1 coreceptorsCCR5 and CXCR4 are G protein-coupled chemokinereceptors that interact, in conjunction with CD4, withthe envelope protein gp120 of HIV-1. In earlier in vitroexperiments using mixed neuronal-glial cultures fromeither wild-type or knock-out rodents (genetically de-ficient in one or both HIV-1 coreceptors), we found thatdepending on the viral strain, CCR5, CXCR4, or bothmediated neurotoxicity of HIV-1/gp120. Conversely,physiological CCR5 ligands MIP-1beta or RANTESprotected neurons from gp120-induced toxicity. Forthe present in vivo study we crossed mice expressingan HIV-1 gp120 transgene in brain (gp120tg; ToggasS. et al., Nature 367: 188, 1994) with CCR5KO miceonly, since CXCR4-deficiency is perinatally lethal.One hallmark of HIV-1 neuropathology that brainsof AIDS patients and gp120 transgenic mice shareis the reduced number of neurites and synapses incomparison to wild type (wt) controls. This loss ofneuronal processes and synapses correlates well withneurocognitive impairment in AIDS patients and isreflected by a diminished immunoreactivity of theneuropil for microtubule-associated protein (MAP)-2and Synaptophysin, respectively. We estimated thepercentage of MAP-2 or Synaptophysin positiveneuropil by deconvolution microscopy in sagittalbrain sections of 6 month old gp120tg mice bothexpressing and lacking CCR5 using CCR5KO andwild type animals as control. CCR5wt/gp120+ micedisplayed a significant reduction in the percentage ofMAP-2 positive neuropil in comparison to all of theother three genotypes (MAP-2 positive neuropil in %+/− SEM, n = 3 animals per group; CCR5wt/gp120+:35 +/− 3, p < 0.001 (ANOVA and post hoc test)in comparison to CCR5KO/gp120+: 51 +/−2, orCCR5wt/gp120-: 47 +/− 2, or CCR5KO/gp120-: 50+/− 2). Similarly, a reduction of Synaptophysinreactivity was observed in CCR5wt/gp120+ mice butnot the other three genotypes. We thus propose thatCCR5 is required for the HIV-1 gp120 transgene tocause damage to neuronal processes and synapsesin this in vivo model, a finding consistent with thecoreceptor usage of most HIV-1 strains previouslyisolated from brains of AIDS patients.

Supported by NIH grant R01 NS050621 (to M.K).

P.106Neuroprotective effects of small, non-peptideneurotrophin mimetics in neural cultures challengedwith gp120 or feline immunodeficiency virusRick B Meeker,1 Winona Poulton,1 Wenhai Feng,1Stephen M Massa,2 and Frank M Longo3

1Department of Neurology, University of North Carolina;2Department of Neurology, UCSF/VAMC; 3Department ofNeurology, Stanford University


HIV-associated CNS disease is largely due to the devel-opment of an inflammatory response that subsequentlydamages neurons. Circulating cytokines, chemokinesand viral proteins provoke dysfunctional regulation ofcalcium, mitochondrial reactive oxygen species gen-eration and changes in receptor signaling that dis-rupt neuronal function and ultimately shift the home-ostatic balance toward apoptosis. Signal transductionpathways that favor Akt activation and discourageGSK3β signaling are thought to play an important rolein reducing inflammation, maintaining normal neu-ronal function and discouraging apoptosis. Growthfactors are strong modulators of this pathway buthave had limited therapeutic application due to dif-ficulties in delivery to the CNS. The development ofseveral small, non-peptide mimetics of nerve growthfactor and brain-derived neurotrophic factor havemade it possible to more effectively exploit the po-tential benefits of neurotrophin receptor activation.To assess their therapeutic potential in the context ofHIV neuropathogenesis, we tested three compoundson primary rat neural cultures challenged with gp120or lipopolysaccharide/interferon-gamma. Addition ofthree prototype compounds, LM11A-31, BD2-4 andBD2-10, to the culture medium at concentrations of 10,500 and 1000 nM resulted in a 50-100% reduction incell death for each challenge. For a more sensitive as-sessment of neuronal dysfunction, we evaluated den-dritic damage and neuronal loss in FIV-infected neuralcultures over a period of 3-14 days post-inoculation.The appearance of dendritic beading, pruning andtortuosity, the loss of neurons, microglial prolifera-tion and astrogliosis were all inhibited by these com-pounds. Neuron survival was completely restored byeach compound although subtle differences in the re-versal of dendritic damage and astrogliosis were seen.These compounds offer a novel therapeutic approachto prevent the destructive effects of lentiviral infectionof the nervous system.

Supported by NIH Grants MH079726 andMH063646

P.107Varicella zoster virus in saliva of patients withherpes zosterSatish K. Mehta,1,2,3 Stephen K. Tyring,4,5

Donald H. Gilden,6,7,8 Randall J. Cohrs,9,10,11 MelanieJ. Leal,1,2,5 Victoria A. Castro,1,2,3 Alan H.Feiveson,12,13,14 C. Mark Ott,12,13,14 and Duane L.Pierson12,13,14

1Enterprise Advisory Services, Inc.; 2Lyndon B. JohnsonSpace Center; 3Houston, TX; 4University of Texas HealthScience Center; 5Houston, TX; 6Departments of Neurologyand Microbiology; 7University of Colorado Health SciencesCenter; 8Denver, CO; 9Department of Neurology;10University of Colorado Health Sciences Center; 11Denver,CO; 12National Aeronautics and Space Administration;13Lyndon B. Johnson Space Center; 14Houston, TX

Varicella zoster virus (VZV) DNA has been found insaliva of healthy astronauts without rash during and

after space flight as well as in saliva of patients with theRamsay Hunt syndrome, which results from VZV reac-tivation in geniculate ganglia. It is not known whetherVZV DNA is present in saliva of patients with herpeszoster in other dermatomes. We hypothesized that aprospective analysis of patients with zoster would re-veal VZV in saliva. We treated 54 patients with herpeszoster with oral valacyclovir. On the first day of treat-ment, and 7- and 14-days later, pain levels were scoredand saliva was examined for VZV DNA. Saliva from 6control subjects with chronic non-dermatomal distri-bution pain and from 14 healthy control subjects wassimilarly studied.

For 50 of the 54 zoster patients, follow-up data wereavailable. During the 14-day study period, pain de-creased in 43 (86%), disappeared entirely in 37 pa-tients (74%), recurred after it had disappeared in 3patients (6%), and increased in 4 patients (8%). VZVDNA was found in saliva of every zoster patient on theday treatment was started. In 47 (94%), the salivaryVZV DNA burden decreased. In 41 patients (82%), vi-ral DNA disappeared. No patients exhibited any in-crease in viral DNA after it had begun to decline ordisappear. There was a significantly positive correla-tion between the presence of VZV DNA in saliva andpain levels (P < 0.0005), as well as between the VZVDNA burden and degree of pain (P < 0.0005). Saliva of2 zoster patients was cultured for virus isolation, andinfectious VZV was isolated from 1 patient. VZV DNAwas present in saliva in 1 patient before rash began andin 4 patients after pain resolved. No VZV DNA wasdetected in saliva from any control subjects. Analysisof human saliva is a non-invasive procedure with po-tential usefulness in diagnosing neurological diseaseproduced by VZV in the absence of rash.

P.108Spatiotemporal localization of TLR3 in humanneurons infected with rabies virusPauline Menager,1 Pascal Roux,2,4 Francoise Megret,1

Jean-Pierre Bourgeois,3 Mireille Lafage,1 ChristophePrehaud,1 and Monique Lafon,11Laboratory of Viral Neuroimmunology, Institut PasteurParis France; 2Bioimaging Facility Institut Pasteur ParisFrance; 4Neuroscience Department, Institut Pasteur ParisFrance

Human neurons express the innate immune responsereceptor, Toll-like receptor-3 (TLR-3) as it has beenshown in human post mitotic neurons and in Purkinjeneurons of cerebellar cortex in postmortem human ra-bies cases (Prehaud et al, 2005) (Jackson et al, 2006).Here, we investigated further the expression and func-tion of neuronal TLR3 in the time course of rabies virusinfection in human neurons. In the absence of infec-tion, TLR3 molecules are located in endosomes. Fol-lowing rabies virus infection, TLR3 is not only presentin endosomes but also in detergent resistant inclu-sions bodies located in the nucleus vicinity. Absence


of association with centrioles and absence of ubiqui-tinylated proteins suggested the inclusions bodies arenot aggresomes, structures where proteins produced inexcess or misfolded proteins are stored before elimina-tion. However, by using nocodazole, we observed thatthe structures are tightly associated with the micro-tubule network. Besides TLR3, these inclusions bodiescontain rabies virus proteins (N and P but no G). Detec-tion of nucleocapsids in these structures suggests thatinclusion bodies may correspond to sites involved invirus transcription/replication processes.

The size of the inclusion bodies (2–4 μm), their com-position and the absence of surrounded membrane,suggest they correspond to the previously describedNegri Bodies (NB). Confocal analysis and 3D modeli-sation indicate that NB structure is strictly organizedwith a nuclear core containing TLR3 surrounded by acorona composed with viral N and P proteins. The roleof TLR3 in the course of rabies virus infection by usingRNA silencing is under investigation.

This work was supported by PTR grant 186 fromInstitut Pasteur, Paris France

P.109Agnoprotein of JCV inhibits oligodendrocytedifferentiation and survival of oligodendrocyte-likecellsNana Merabova,1 Dorota Kaniowska,1 RafalKaminski,1 Satish Deshmane,1 Shohreh Amini,1,2

Armine Darbinyan,1 and Kamel Khalili1

1Department of Neuroscience Center for Neurovirology,Temple University School of Medicine; 2Department ofBiology College of Science and Technology TempleUniversity Philadelphia, PA 19122

We utilized a bipotential cell line that can be inducedto differentiate into oligodendrocytes and type 2astrocytes to investigate the biological impact of JCVagnoprotein. Replication of JCV in brain, an event thatoccurs upon immune suppression, leads to the de-struction of oligodendrocytes and the developmentof the fatal demyelinating progressive multifocalleukoencephalopathy (PML). Our results showthat in the absence of productive viral replication,agnoprotein is capable of affecting oligodendro-cyte differentiation as evidenced by morphologicalchanges due to delayed formation of processes and lowcomplexity of outgrowth network. Differentiation ofoligodendrocytes resulted in a high level of DNA dam-age in agnoprotein-positive cells as detected by DNAfragmentation and Comet assays. Analysis of themolecular profiles of differentiated cells revealedinduction of proapoptotic and suppression of pro-survival signaling pathways in the presence ofagnoprotein. These observations and our previousreports indicating involvement of agnoprotein inregulation of the cell cycle and DNA damage repairpathways suggest that, independent of productiveviral infection cycle, agnoprotein may contribute

to the destruction of myelin-forming cells that isobserved upon JCV infection in PML.

This work was supported by a grant awarded byNINDS to KK.

P.110Treatment of acute flaccid paralysis caused by WestNile virus in hamsters with a humanizedneutralizing monoclonal antibodyJohn D. Morrey,1 Venkatraman Siddharthan,1 HongWang,1 Ramona Skirpstunas,1 Jeffery O. Hall,1 ScottKoenig,2 Syd Johnson,2 Jeffery L. Nordstrom,2Melanie A. Samuel,3 and Michael S. Diamond3

1Institute for Antiviral Research, Department of Animal,Dairy, and Veterinary Sciences, Utah State University,Logan, UT, USA; 2MacroGenics, Inc., Rockville, MD;3Departments of Molecular Microbiology, Medicine, andPathology & Immunology, Washington University School ofMedicine, St. Louis, Missouri

Acute flaccid polio-like paralysis occurs during natu-ral WNV infection in a small fraction of cases in an-imals and humans. To evaluate pathogenesis and thepossibility for therapeutic intervention, we developeda uniform model of flaccid hind limb paralysis by in-jecting WNV directly into the sciatic nerve or spinalcord of hamsters. When WNV was injected into thethoracic (T8) or lumbar spinal cord (L1-4), 100% ofhamsters developed paralysis within 6-18 days andthen subsequently died from systemic infection. Whenthe sciatic nerve was injected with WNV and either leftintact or transected below the injection site, 50% and21% of the animals became paralyzed, respectively,on the ipsilateral side of injection. In contrast, if thesciatic nerve was transected proximal to the WNV in-jection site, animals did not developed paralysis onthe ipsilateral side. These data suggested that WNVundergoes axonal transport from the sciatic nerve intothe lumbar spinal cord to cause paralysis. Confocalimmunohistochemical staining of spinal cord sectionsfrom paralyzed hamsters indicated that WNV-infectedneurons rapidly underwent apoptosis. WNV infectionlocalized primarily to the ventral motor horn of thegray matter, consistent with the polio-like clinical pre-sentation. Cell culture studies using a compartmental-ized neuronal culture system established that WNVcan rapidly spread via axonal transport in both retro-grade and anterograde directions, and that this spreadwas disrupted by a neutralizing humanized mono-clonal antibody (mAb), hE16. Accordingly, a singlei.p. injection of hE16 (32 mg/kg) significantly reducedparalysis and mortality of hamsters injected at day 2or 3 after WNV inoculation of the spinal cord at T8vertebrae. Additionally, a single i.p. injection of hE16administered as late as 5 days after WNV inoculationof the sciatic nerve prevented paralysis in all hamsters.Overall, our experiments establish that (a) WNV under-goes axonal transport, which leads to a specific clin-ical phenotype, acute flaccid paralysis; and (b) hE16antibody therapy, prevents hind limb flaccid paralysis


in hamsters, even after WNV infects spinal cordneurons.

Funding: NIH NO1-AI-15435 (JDM) VirologyBranch, NIAID, NIH; 1-U54 AI06357-01 RockyMountain Regional Centers of Excellence (JDM);U01-AI061373 (MSD) HHSN266200600013C (Macro-Genics).

P.111Characterization of recombinant monoclonalantibodies directed against VZV IE63 proteinexpressed in eukaryotic and prokaryotic cellsNiklaus H Mueller,1 Laurie L Graf,1 Donald HGilden,1,2 and Randall J Cohrs1

1Neurology, University of Colorado Health Sciences Center,Denver, CO, USA; 2Microbiology, University of ColoradoHealth Sciences Center, Denver, CO, USA

The varicella zoster virus (VZV) genome encodes twocopies (ORFs 63 and 70) of the tegument-associated,immediate early 63 protein (IE63). During latency,IE63 is expressed in the cytoplasm of neurons, but ispredominantly nuclear during productive infection.Phosphorylation of IE63 may account for proteinlocalization and function, a hypothesis, which couldnot be tested due to lack of antibody that differentiatesnuclear and cytoplasmic forms of IE63. Herein, wereport the construction and characterization of re-combinant monoclonal antibodies that bind differentforms of IE63. To construct these antibodies, VZVORF 63 was inserted into vectors which expressedN-terminally Flag-tagged IE63 either in bacteria (toidentify unmodified forms of IE63) or in mammaliancells (to identify fully processed IE63). After expres-sion of VZV IE63, IE63 was purified by Flag-affinitychromatography and used to immunize mice. Whenmice developed anti-IE63 antibodies (2 months),spleens were harvested, total RNA was extracted,mRNA was isolated and cDNA was synthesized. Bothheavy and light chain IgG variable regions were PCRamplified and inserted into the phage-display vectorpCOMB3H. Phage libraries which displayed recom-binant IgG-Fab were panned against both prokaryoticor eukaryotic expressed Flag-tagged VZV IE63. TheDNA sequence of enriched recombinant phage heavyand light chain variable regions were determinedand shuttled into the mammalian expression vectorpCEP4. Expression of the pCEP4 plasmids in human293 cells permitted affinity purification of two mon-oclonal antibodies. Western blot analysis showedthat the first monoclonal antibody detected VZV IE63expressed in prokaryotic or eukaryotic cells as well asin VZV-infected MeWo cells. The second monoclonalantibody detected IE63 expressed predominately ineukaryotic cells (both transfected or infected); thisantibody produced against eukaryotic expressed IE63detected IE63 predominantly in the nucleus of VZV-infected MeWo cells, while the antibody producedagainst prokaryotic expressed IE63 detected VZV IE63predominantly in the cytoplasm. The epitopes that arebound by these antibodies have been characterized

and will be reported. These antibodies will allow thecharacterization of the post-translational modifiedforms of IE63 in VZV-infected cells.

P.112Identification of the mechanisms used by p53 toinhibit HIV-1 gene expression and replicationRuma Mukerjee,1 Pier Paolo Claudio,2 Luis DelValle,1 Shongshan Fan,1 Satish L Deshmane,1 RogerLeng,3 Sujatha Radhakrishnan,1 Antonio Giordano,2Kamel Khalili,1 Shohreh Amini,1,4 SamuelBenchimol,3 and Bassel E Sawaya1

1Department of Neuroscience & Center for Neurovirology,School of Medicine, Temple University, Philadelphia, PA19122; 2Center for Biotechnology, Sbarro Institute forCancer Research and Molecular Medicine, www.shro.org,College of Science and Technology, Temple University,Philadelphia, PA 19122; 3Department of Biology, YorkUniversity, 4700 Keele St., Toronto, Ontario Canada;4Department of Biology, College of Science and Technology,Temple University, Philadelphia, PA 19122

Increasing evidences point to the involvement of p53in neurodegenerative diseases including HIV-1 asso-ciated dementia (HAD). p53 accumulation and DNAdamage have been reported in neurons from HAD pa-tients, as well as in non-neuronal CNS cells, includingmicroglia and astrocytes. Taken together, these stud-ies demonstrate that the CNS environment in HADleads to a sufficient degree of injury to activate p53in neuronal and non-neuronal cells. The mechanismsleading to p53 induction in HIV-1 infected patients arenot well understood and remain to be identified. Inthis regard, we recently demonstrated that the pro-tein kinase cdk9 phosphorylates p53 on serine 392via their physical interaction (Claudio et al., J. Cell.Physiol. 2006). Therefore, we sought to investigatewhether p53-cdk9 association affects HIV-1 gene ex-pression and replication in CNS cells. By employing anadenovirus expression vector, we demonstrated thatoverexpression of p53 prevents cdk9-induced phos-phorylation of polymerase II. Further, we demon-strated that wild type, but not mutant, p53 decreasesthe levels of Tat-induced activation of HIV-1 LTR. Inaddition, we showed that p53 significantly reducedHIV-1 replication in primary microglial. Using ELISAmeasurement, we demonstrated that p53 decreasedHIV-1-induced cytokines production (e.g. TNFα). Toovercome the effect of p53, we demonstrated that cdk9associate with Pirh2 to promote the ubiquitination anddegradation of p53. These studies present a novel ther-apeutic approach for the inhibition of HIV-1 gene ex-pression and replication and the treatment of AIDS inbrain.

P.113Role of p53 family in neuropathogenesis of AIDSRuma Mukerjee,1 Shohreh Amini,2 Brian Wigdahl,3and Bassel E Sawaya1


1Department of Neuroscience & Center for Neurovirology,School of Medicine, Temple University, Philadelphia, PA19122; 2Department of Biology, College of Science andTechnology, Temple University, Philadelphia, PA 19122;3Department of Microbiology and Immunology, Institute forMolecular Medicine and Infectious Disease and Centers forMolecular Virology and Neuroimmunology and CancerBiology, Drexel University College of Medicine,Philadelphia, PA 19129

Despite the use of highly active antiretroviral therapy(HAART), neuronal cell death remains a problem thatis frequently found in the brains of HIV-1-infected pa-tients. HAART has successfully prevented many ofthe former end-stage complications of AIDS, however,with increased survival times, the prevalence of mi-nor HIV-associated cognitive impairment appears to berising among AIDS patients. Further, HIV encephalitis(HIVE) is still prevalent in treated patients as well as at-tenuated forms of HIVE and CNS opportunistic disor-ders. HIV-associated cognitive impairment correlateswith the increased presence in the CNS of activated,though not necessarily HIV-1-infected, microglia andCNS macrophages. This suggests that indirect mech-anisms of neuronal injury and loss/death occur inHIV/AIDS as a basis for dementia since neurons arenot themselves productively infected by HIV-1. Re-cent evidence indicates that the p53 tumor suppres-sor protein and its related family member, p73, playan essential role in regulating neuronal loss/apoptosis.The mechanisms involved in induction of p53 or p73which lead to dendritic retraction and neuronal lossare not fully elucidated. Increased levels of p53 wereobserved in the neurons of AIDS patients, which maybe connected to the occurrence of HIV-1-associateddementia (HAD) in those patients. Similar to p53 ac-tivation in neurons, p53 activation in microglia andastrocytes may also contribute to alterations in thephysiology of these cells that eventually result in aneurotoxic environment and neuronal loss. Impor-tantly, for p53 to be functional and stable, activatedp73 is required. Based on this finding, we havestudied the relationship between p73 and Tat, andhave demonstrated the ability of HIV-1 in general, andof Tat in particular, to induce the endogenous lev-els of p73. P73 induction prevents acetylation of Taton lysine 28 through their direct physical interaction,which also inhibits Tat’s apoptotic activity. The asso-ciation of Tat with p73 also reduces Tat-activation ofthe HIV-1 LTR, and prevents p73 from causing celldeath in astrocytes. These data suggest a basis forthe restricted replication of HIV-1 in astrocytes. Wealso investigated the interplay between Tat and p73in neuronal cells. Surprisingly, Tat was unable to pro-mote neuronal death in the absence of either p73 orp53, suggesting a strong link between Tat, p73, p53and neuronal cell death. Hence, we now propose tostudy the mechanism(s) whereby Tat induces p73. Thiswill include analysis of control of the p73 promoterand of p73 protein turnover. We will also examinewhether induction of p73 by Tat leads to activation

of p53 and induction of neuronal cell death. Theoutcome from the proposed studies should providenew information regarding mechanisms of neuronalloss in AIDS patients and suggest possible new thera-peutic approaches.

P.114Characterization of HIV-1 binding to peripheralblood mononuclear cells (PBMCs) andmonocytes/macrophages (M/M): relationship toneuropathogenesisSody Munsaka,1,2 Melissa Agsalda,1 DavidTroelstrup,1 Ningjie Hu,1 Qigui Yu,1,2 and BruceShiramizu1,2

1Hawaii AIDS Clinical Research Program, University ofHawaii, Honolulu, Hawaii, USA; 2Department of TropicalMedicine, Medical Microbiology and Pharmacology,University of Hawaii, Honolulu, Hawaii, USA

Background. Individuals with HIV-1-associated de-mentia (HAD) are characterized with increasedpercentages of circulating activated M/M withCD14/CD16 phenotype. Higher levels of HIV-1 DNAare detected in these activated cells, therefore we hy-pothesize that they are more permissive to infectionand thus the magnitude of viral binding will be higher.

Methods. The study was performed following guide-lines established by the local institutional reviewboard. From a non-HIV-1-infected volunteer, periph-eral blood mononuclear cells (PBMCs) and magneticbead-separated monocytes were immediately placedin 96-well polypropylene plates [5x10(5) cells/well]and exposed to 2ng p24 units of LAI (X4 Strain) andp89.6 (dual tropic but preferentially X5 strain) for onehour at 37◦C, 5% CO2 in triplicate. Unbound viruswas washed off using PBS and the cells were lysed forRNA extraction. Viral binding capacity was assayed byRT-PCR using HIV GAG and beta-actin primers withappropriate positive and negative control RNA. Am-plified fragments were resolved on 2.5% agarose gelsand analyzed by densitometry. From the scanned gels,the ratio of HIV GAG light units/beta-actin light unitswas compared between the two cell populations andthe virus strains. Differences in binding capacities be-tween each of the two groups were considered signifi-cant by Student’s t-test if p < 0.05.

Results. As expected, M/M displayed a higher HIV-1 binding to p89.6 than to LAI, 0.497 vs. 0.328 (p <0.05), respectively. In the PBMCs, viral binding capac-ity was increased compared to M/M , for LAI: 0.492vs. 0.328, respectively (p < 0.05); for p89.6: 0.878 vs.0.497, respectively (p < 0.05). Of note was the signif-icantly higher binding found with p89.6 (0.878) com-pared to LAI (0.492) (p < 0.05), since the PBMCs werefrom the same volunteer obtained at the same time.

Conclusion. These results demonstrate that periph-eral M/M preferentially bind R5 virus suggesting thatthe high HIV DNA found in PBMCs represents boundvirus on the M/M subset. The enhanced binding of R5strains to M/M may lead to more permissive infection


of this PBMC subset. The theory that increased traffick-ing of HIV-1-infected M/M to the central nervous sys-tem is consistent with our findings. (Supported by NIHgrants NS053345, U01A134853, and P20RR011091;and the Fulbright Staff Development Fellowship)

P.115The varicella zoster virus vasculopathiesMaria A. Nagel,1 Randall J. Cohrs,1 RaviMahalingam,1 Mary C. Wellish,1 Bagher Forghani,2Andreas Schiller,3 Joseph E. Safdieh,4 ElenaKamenkovich,4 Lyle W. Ostrow,5 Michael Levy,5Benjamin Greenberg,5 Andrew N. Russman,6 IreneKatzan,7 Craig J. Gardner,8 Martin Hausler,9 RolandNau,10 Takeshi Saraya,11 Hiroo Wada,11 HajimeGoto,11 Maurizio de Martino,12 Makoto Ueno,13

William D. Brown,14 Christoph Terborg,15 and DonaldH. Gilden1,16

1Department of Neurology, University of Colorado HealthSciences Center, Denver, CO, USA; 2Viral and RickettsialDisease Laboratory, Division of Communicable DiseaseControl, California Department of Health Services,Richmond, CA, USA; 3Department of Neurology, UniversityHospital, Zurich Switzerland; 4Department of Neurologyand Neuroscience, Weill Medical College of CornellUniversity, New York, NY, USA; 5Department of Neurology,Johns Hopkins Hospital, Baltimore, MD, USA; 6Departmentof Neurology, Henry Ford Hospital, Detroit, MI, USA;7Department of Neurology, Cleveland Clinic Foundation,Cleveland, OH, USA; 8Department of Neurology, ThomasJefferson University School of Medicine, Philadelphia, PA,USA; 9Department of Pediatrics, University Hospital RWTHAachen, Aachen, Germany; 10Department of Neurology,University of Gottingen, Gottingen, Germany; 11Departmentof Respiratory Medicine, Kyorin University School ofMedicine, Tokyo, Japan; 12Department of Pediatrics,University of Florence, Florence, Italy; 13Division of ChildNeurology, Institute of Neurological Sciences, TottoriUniversity, Tottori, Japan; 14Department of Neurology,Children’s National Medical Center, Washington, DC, USA;15Department of Neurology, Friedrich-Schiller-University,Jena, Germany; 16Department of Microbiology, University ofColorado Health Sciences Center, Denver, CO, USA

Varicella zoster virus (VZV) vasculopathy producestransient ischemic attacks or stroke secondary to viralinfection of cerebral arteries. Unfortunately, diagnosisis not always straightforward. Not all patients haverash before cerebral ischemia or stroke. Furthermore,other vasculitides produce similar clinical features,as well as comparable imaging, angiographic and CSFabnormalities. Herein, we review our 23 publishedcases and 7 unpublished cases of VZV vasculopathy.All CSFs were tested for VZV DNA by PCR and foranti-VZV IgG antibody by enzyme immunoassay andfound to be positive for either or both. Among the 30patients, rash occurred in 19 (63%), CSF pleocytosisin 20 (67%), and abnormalities on brain magneticresonance imaging (MRI) or computerized axial to-mography (CAT) scanning in 29 (97%). Conventionalangiography or magnetic resonance angiography(MRA) in 23 patients revealed abnormalities in 16(70%). Both large and small arteries were involved

in 15 (50%), small arteries alone in 11 (37%) andlarge arteries alone in only 4 (13%) of the 30 patients.The average time from rash to onset of neurologicalsymptoms and signs was 4.1 months. Disease wasusually protracted so that the average time from neu-rological symptoms and signs to virological analysisof CSF was 4.2 months. The CSF of 9 (30%) patientscontained VZV DNA while 28 patients (93%) hadanti-VZV IgG antibody in their CSF; in each of these28 patients, a reduced serum/CSF ratio of VZV IgGconfirmed intrathecal synthesis.

Overall, the data reveal that rash or CSF pleocyto-sis is not required for diagnosis of VZV vasculopathy,whereas MRI/CAT abnormalities are seen in almost allpatients. Furthermore, unlike earlier descriptions ofexclusive involvement of large cerebral arteries, mostpatients had mixed large and small artery involve-ment. Detection of anti-VZV IgG antibody in CSF wasa more sensitive indicator of VZV vasculopathy thandetection of VZV DNA (p < 0.001). Most patients weretreated with acyclovir, with or without steroids, andimproved or stabilized. However, because our patientsreceived different treatment regimens at different insti-tutions in an uncontrolled setting, the determination ofoptimal dose, duration of antiviral treatment, and ben-efit of concurrent steroid therapy awaits studies withlarger case numbers.

P.116HIV induces complement C3 in primary humanastrocytes indirectly through a NFkB-dependentpathwayJadwiga B. Nitkiewicz,1,2 Wei Chao,1 Seon-YoungKim,3 Galina Bentsman,1 Luni Emdad,2 Seok-GeunLee,2 Devanand Sarkar,2 Paul B. Fisher,2 and David J.Volsky1,2

1Molecular Virology Division, St. Lukes-Roosevelt HospitalCenter, New York, NY 10019; 2Department of Pathology,Columbia University, New York, NY 10032; 3KRIBB, Seoul,Korea

Innate immunity is believed to play an important rolein protecting brain against HIV infection and develop-ment of HIV-associated dementia (HAD). ComplementC3 is a key component of the innate immunity sys-tem but in models of other neurodegenerative diseasessuch as multiple sclerosis, uncontrolled C3 productioncan lead to tissue injury and inflammation. C3 is se-creted by several cell types in the brain including as-trocytes. We and others have previously shown thatHIV interaction with astrocytes in vitro alters cellulargene expression, induces interleukin-6, and disruptsimportant physiological functions of the cells such asglutamate transport.

Here we investigated the mechanism of C3 induc-tion in human fetal astrocytes (HFA) exposed to HIVin vitro. Using transient transfection assays with a C3promoter-driven indicator gene, real-time PCR, andC3 ELISA we found that exposure of astrocytes to HIVincreased C3 promoter activity, mRNA expression,and protein production, indicating that HIV acts


by transcriptional activation of the C3 gene. Unlikeother effects of HIV in astrocytes, which are inducedrapidly after the viral stimulus, elevated expressionof C3 mRNA and protein peaked at 7-10 days, indi-cating an indirect pathway of induction. Studies withpharmacological inhibitors revealed that activationof C3 by HIV requires, among others, the NFkB andPTK signaling pathways; however, surprisingly, theC3 core promoter lacks NFkB and SP binding sites. Incontrast, activation of the C3 promoter by HIV was ab-solutely dependent on the presence of the interleukin1-B/interleukin 6 responsive element at -109 to -90and its sub-domain -106 to -100. The requirement forfunctional NFkB for HIV effect on C3 was confirmedby overexpression of the NFkB inhibitor IkB-a inastrocytes; adenovirus-mediated transduction of IkB-ainto HFA, but not of control virus, blocked nucleartranslocation of NFkB and prevented induction ofC3 promoter and mRNA. These results indicate thatHIV activates C3 in astrocytes indirectly throughNFkB-dependent induction of an unknown mediatorthat in turn activates C3 transcription via the -109 to-90 element in the promoter. Studies are in progressto identify the factor that mediates HIV activation ofC3 in HFA. Activation and possibly dysregulation ofastrocyte innate immune responses by HIV, as shownby induction of C3 in HFA here, may have deleteriousconsequences to the normal neuroprotective functionsof astrocytes.

Supported by grants NS31492 (to DJV and PBF),DA17618 (to DJV), and CA035675 (to PBF).

P.117Identification and characterization of C/EBP bindingsites within the HIV-1 subtype C LTRMichael R. Nonnemacher, Yujie Liu, Evelyn M.Kilareski, Luna Liu, and Brian WigdahlDrexel University College of Medicine

Human immunodeficiency virus type 1 (HIV-1)has been transmitted worldwide and regional viralsubtypes have been designated as subtype A throughK. Subtype C, which is concentrated in SoutheastAsia and sub-Saharan Africa, is the most prevalentsubtype worldwide. To date, few studies have exam-ined the role of CCAAT/enhancer binding proteins(C/EBP) with respect to long terminal repeat (LTR)-directed viral gene expression from the subtype CLTR. Within subtype B viruses, two functional C/EBPsites upstream of the TATA box have been shown tobe required for efficient viral replication in cells ofmonocyte-macrophage lineage. In order to assess theroles of C/EBP sites within the subtype C viral LTR,211 HIV-1 subtype C LTR sequences were collected andaligned via the Clustal V method. From these analyses,three putative C/EBP binding sites were identified: twoupstream binding sites 1 and 2 (US1 and US2) and onedownstream binding site (DS1). Interestingly, the pu-tative downstream site (DS1) is highly conserved be-tween subtypes B and C, suggesting the presence ofa functionally important cis-acting element that has

yet to be characterized. Electrophoretic mobility shiftanalysis demonstrated that two of three sites withinthe HIV-1 subtype C were able to bind C/EBP factors(US1 and DS1). Additional studies focused on exam-ining relative binding affinities of naturally occurringvariants of these two sites are currently underway. Fu-ture studies will examine the molecular architectureof these sites relative to the functional properties ofthe HIV-1 LTR.

P.118Apelin, an endogenous neuronal peptide, protectshippocampal neurons against HIV-associatedexcitotoxic injuryLauren A O’Donnell, Arpita Agrawal, PraveenaSabnekar, Marc A Dichter, David R Lynch, DeniseCooke, and Dennis L KolsonDept. Neurology, University of Pennsylvania

Several G-protein couple receptors (GPCRs) have beenshown to mediate neuronal cell migration and survivalupon activation by their native peptide ligands, andto activate death-signaling pathways when activatedby certain non-native ligands. We recently described,in cultured neurons, expression of the unique seven-transmembrane (7TM)-GPCR, APJ, which is alsostrongly expressed in neurons in the brain as wellas different cell types in other tissues. We have nowdemonstrated the ability of the endogenous APJpeptide ligand, apelin, to activate signaling in rat hip-pocampal neurons and to modulate neuronal survival.We found that 1) Both APJ and apelin are expressedin hippocampal neurons; 2) apelin peptides inducephosphorylation of the cell survival kinases AKT/PKBand Raf/ERK-1/2 in hippocampal neurons; and 3)apelin peptides protect hippocampal neurons againstN-methyl-D-aspartate receptor (NMDAR)-mediatedexcitotoxicity, including including that induced byhuman immunodeficiency virus type 1 (HIV-1). Thus,apelin/APJ signaling likely represents an endogenoushippocampal neuronal survival response, and our re-sults suggest that apelin should be further investigatedas a potential neuroprotectant against hippocampalinjury.

P.119Pharmacological cdk inhibitor suppresses JC virusproliferationYasuko Orba,1,2,3 Yuji Sunden,4,5 Tadaki Suzuki,1,3

Takashi Kimura,1 Shinya Tanaka,2 KazuoNagashima,6 and Hirofumi Sawa1,5

1Department of Molecular Pathobiology, HokkaidoUniversity Research Center for Zoonosis Control, Sapporo,Japan; 2Laboratory of Molecular & Cellular Pathology,Hokkaido University Graduate School of Medicine,Sapporo, Japan; 3Research Fellow of the Japan Society forthe Promotion of Science; 4Laboratory of ComparativePathology, Hokkaido University School of VeterinaryMedicine, Sapporo, Japan; 521st Century COE Program forZoonosis Control, Hokkaido University, Sapporo, Japan;6Department of Pathology, Sapporo Higashi-TokushukaiHospital, Sapporo, Japan


JC virus (JCV) utilizes cellular proteins for viral repli-cation and transcription in the host cell nucleus. Thesecellular proteins represent potential targets for an-tiviral drugs against the JCV. In this study, we ex-amined the antiviral effects of the pharmacologicalcyclin-dependent kinase (cdk) inhibitor, which hasbeen shown to have antiviral activity against otherviruses. We found that cdk inhibitor significantly sup-pressed the viral production and cytopathic effects ofthe JCV in a JCV-infected cell line. It attenuated thetranscriptional activity of JCV late genes, but not earlygenes, and also prevented viral replication via inhibit-ing phosphorylation of the viral early protein, large Tantigen. These data suggest that the JCV requires cdksto transcribe late genes and to replicate its own DNA.Pharmacological cdk inhibitor exhibited antiviral ac-tivity in JCV-infected cells, suggests that it might havetherapeutic utility in the treatment of progressive mul-tifocal leukoencephalopathy (PML).

P.120Expanded plasma cell clones in multiple sclerosiscerebrospinal fluid do not produce antibody againstabundant myelin proteinsGregory P Owens,1 Alanna M Ritchie,1 AndrewShearer,1 Mark P Burgoon,1 Xiaoli Yu,1 Jeffrey LBennett,1 and Donald H Gilden2

1Department of Neurology, University of Colorado HealthSciences Center; 2Departments of Neurology andMicrobiology, University of Colorado Health SciencesCenter

Multiple sclerosis (MS) is the most common inflam-matory demyelinating disease of humans. A hallmarkof disease is increased intrathecal IgG synthesis andpersistence of oligoclonal IgG in CSF and brain. Tobetter understand the nature of humoral immunity inMS, we used FACS and single-cell RT-PCR to analyzeIgG variable (V) region sequences of CD138+ plasmacells recovered from the CSF of MS patients. Clonal ex-pansion, somatic hypermutation and the preferentialuse of VH4 family heavy chain gene segments werecharacteristic features of the 11 repertoires examined,supporting the notion of a targeted B cell responsewithin the CNS. To identify their antigenic targets,we produced over 40 different monoclonal humanIgG1 recombinant antibodies (rAbs) by co-expressionof heavy- and light-chain V region sequences fromplasma cell clones in mammalian cells. Because MS isconsidered a putative autoimmune disease, rAbs werefirst assayed for immunoreactivity to myelin basic pro-tein, proteolipid protein and myelin oligodendrocyteprotein. Whereas mouse mAbs and rAbs producedfrom anti-myelin hybridomas readily reacted withmyelin antigens in multiple immunoassays, none ofthe MS CSF rAbs displayed clear immunoreactivity.Our findings indicate that common myelin antigensare not the major target of the humoral immune re-sponse in MS. The identification of novel MS-specificantigens is being pursued.

P.121Measles virus-specific plasma cell clones areprominent in subacute sclerosing panencephalitisCSFGregory P Owens,1 Alanna M Ritchie,1 Mark PBurgoon,1 Andrew Shearer,1 Jeffrey L Bennett,1 andDonald H Gilden2

1Department of Neurology, University of Colorado HealthSciences Center; 2Departments of Neurology andMicrobiology, University of Colorado Health SciencesCenter

Chronic infectious and inflammatory diseases of theCNS are often characterized by a robust B cell re-sponse that manifests as increased intrathecal IgG syn-thesis and the presence of oligoclonal bands. Further,the oligoclonal IgG is directed against the agent thatcauses the disease. To demonstrate the specificity ofexpanded CD138+ plasma cell clones recovered fromthe cerebrospinal fluid (CSF) of a patient with subacutesclerosing panencephalitis (SSPE) for measles virus(MV), variable (V) region sequences of CD138+ cellssorted from SSPE CSF were amplified by single-cellPCR and analyzed. Human IgG1 recombinant antibod-ies (rAbs) were produced from four expanded CD138+clones and assayed for immunoreactivity against MVproteins. Clonal expansion was a prominent feature ofthe SSPE plasma cell repertoire, and each of the fourrAbs assayed were specific for either the MV fusionor MV nucleocapsid protein. Thus, expanded plasmacell clones in the CSF of patients with SSPE producedisease-relevant antibodies and provide a link to ourprevious observation of MV-specific plasma cell clonesin the parenchyma of SSPE brain. Because CSF is read-ily accessible, recombinant Abs derived from B cellclones could provide a tool to identify target antigensin idiopathic inflammatory disorders.

P.122Stereology of dorsal root ganglia neurons andperineuronal macrophages in HIV infected tissuesCarlos A Pardo-Villamizar and Christin A LawlerJohns Hopkins University School of Medicine

The most abundant types of HIV-Neuropathies are thedistal sensory polyneuropathy (DSP) and antiretrovi-ral toxic neuropathies (ATN). The pathological fea-tures of DSP and ATN include macrophage infil-tration in peripheral nerves and dorsal root ganglia(DRG). Increased activation of macrophages and proin-flammatory cytokines appear to be the principal im-munopathogenic factors in DSP.

Perineuronal inflammation in the DRG leads todamage of DRG neurons. Our preliminary data havedemonstrated a reduction of DRG neurons in HIV tis-sues with significant infiltration by CD68+ and CD14+macrophages. We suggest that this aberrant inflamma-tory response plays a critical role in damaging or sen-sitizing DRG neurons and axons, and subsequently af-fects the inputs to central pain pathways.


Therefore, we used stereological techniques to quan-tify DRG neurons and perineruonal macrophage cellsin HIV samples in order to determine the virus’s effecton the DRG.

P.123Importance of the second exon of HIV-1 Tat on thesurvival of neuronal progenitorsGiovanni Passiatore, Elisa Gualco, Krzysztof Reiss,Davide Eletto, Kamel Khalili, and Francesca PeruzziTemple University School of Medicine and Center forNeurovirology, Philadelphia, PA

HIV-Encephalopathy (HIVE) is a neurological disorderassociated with HIV-1 infection and AIDS. Amongthe molecular mechanisms thought to participatein neuronal dysfunction, the viral trans-activatingprotein Tat has been shown to be neurotoxic in variouscellular models. Tat gene consists of two exons thatencode for two variants of the protein, which are 72and 101 amino acids in length, respectively. Althoughthe 101 aa Tat is considered the natural full-lengthisoform of Tat, shorter peptides of 72 and 86 aminoacids are often used for in vitro experiments. While itis clear that the first exon (72 aa in length) is requiredfor viral replication, the function of the carboxy-terminal region of Tat is less clear. Here, we utilized arat neuronal progenitor cell line as a model of retinoicacid induced neuronal differentiation and variousplasmids for the expression of C-terminus truncatedmutants of Tat protein: Tat-72, Tat-86, and Tat-101.Our results strongly suggest that: (i) truncation of theC-terminus correlates with higher toxicity in bothproliferating and differentiating cells; and (ii) survivalof Tat-transfected cells is enhanced upon retinoic acidtreatment and induction of cellular differentiation.Altogether, these results indicate that neuronal toxic-ity of Tat is attenuated by the presence of a sequencein the second exon and by the activation of neuronaldifferentiation.

P.124Structure-activity relationships of biguanide-basedcompounds with activity against HIV-1Shendra Passic,1 Vanessa Pirrone,1 Brian Wigdahl,1Tina Kish-Catalone,1 Robert F. Rando,2 MohamedLabib,2 and Fred C. Krebs1


Combination antiretroviral therapy, which is usedas a highly effective treatment of HIV-1-associatedimmunopathogenesis, has also had a positive im-pact on neurologic disease caused by HIV-1 infec-tion of the central nervous system (CNS). How-ever, limits on treating or preventing HIV-1-associatedneuropathogenesis due to reduced CNS drug penetra-tion and drug-associated neurologic side effects under-score the importance of developing new drugs with

greater efficacy within the CNS. In this regard, wehave focused on the development of biguanide-basedHIV-1 inhibitors. Our investigations, which demon-strated that biguanide-based compounds have activityagainst HIV 1, also suggested a relationship betweenbiological activity and the lengths of hydrocarbonlinkers surrounding the positively charged biguanideunit. To better define biguanide structure-activity re-lationships (SAR), biguanide oligomers with selectlinker lengths were evaluated for cytotoxicity, anti-HIV-1 activity, and in vivo toxicity. Results of thein vitro experiments demonstrated that (i) increasesin linker length (and, therefore, increases in com-pound lipophilicity) were generally correlated withincreases in cytotoxicity and antiviral activity againstHIV-1, and (ii) polyethylene hexamethylene biguanide(PEHMB; biguanide units spaced between alternat-ing ethylene and hexamethylene linkers) providedthe greatest in vitro therapeutic indices (CC50/IC50)among the compounds tested. Additionally, the negli-gible toxicity of PEHMB relative to polyhexamethy-lene biguanide (PHMB; biguanide units alternatingwith hexamethylene linkers) in a murine model ofcervicovaginal microbicide toxicity was consistentwith considerable differences in cytotoxicity betweenPEHMB and PHMB observed during in vitro experi-ments. These SAR investigations represent an essen-tial step in the development of biguanide-based in-hibitors effective against HIV-1 CNS disease.

P.125Platelet-derived growth factor protects neuronsagainst Gp120-mediated toxicityFuwang Peng, Shannon Callen, Navneet Dhillon,Xuhui Zhu, and Shilpa BuchKansas University Medical Centre

The human immunodeficiency virus (HIV)-1 envelopeglycoprotein gp120 has been implicated in mediat-ing neuronal apoptosis, a hallmark feature of HIV-associated dementia (HAD). Mitigation of the toxic ef-fects of gp120 could thus be a potential mechanism forreducing HIV toxicity in the brain. In this study we hy-pothesized that neurotrophic factor, such as platelet-derived growth factor (PDGF), could protect the neu-rons against gp120-mediated apoptosis. The in vitromodel system we have used are the neuronal SH-SY5Ycell culture exposed to gp120 with and without pre-treatment with PDGF. Cells treated with gp120 exhib-ited increased cell death when measured by TUNELassay with concomitant loss of neurites and increasedcell rounding. Pre-treatment with PDGF, however, re-duced gp120-associated neurotoxicity and rescued theneurite outgrowth. Additionally, gp120-mediated ac-tivation of caspase-3, was also significantly reducedin cells pretreated with PDGF. Taken together thesestudies suggest that PDGF may be considered as atherapeutic agent to reduce gp120-mediated neurotox-icity in HAD.


P.126Development of a novel animal/human hybrid modelfor multiple sclerosis in SCID-mice with functionalhuman lymphoid system: experimental allergicencephalomyelitis induced by envelope protein fromhuman endogenous retrovirus“W”(MSRV-ENV/Syncytin).Herve Perron,1 Kamel Sanhadji,2 Iuliana Popa,3Corinne Bernard,1 Jean-louis Touraine,2 NicolasKopp,2 and jacques portoukalian3

1Geneuro SA; Plan-Les-Ouates (Geneva) Switzerland;2Universite Lyon1; Lyon, France; 3INSERM, Lyon, France

Multiple Sclerosis associated RetroVirus element(MSRV), which further unraveled the HERV-W mul-ticopy family, was discovered to encode a power-ful imunopathogenic envelope protein (MSRV-ENV orHERV-W Syncytin copy), causing sequential activa-tion of a pro-inflammatory and autoimmune cascadethrough initial interaction with Toll-Like receptor 4on antigen-presenting cells (APC), accompanied bygliotoxicity causing blood brain barrier (BBB) break-down and further triggering superantigen-like dysreg-ulation of T-lymphocytes.

Initial animal studies in severe combined immunod-eficiency mice (SCID) grafted with a functional humanlymphoid system (thus creating a mouse/human hy-brid) resulted in an hyperacute BBB breakdown withlethal brain haemorrhage was associated with peaksof TNF-alpha, but was inhibited by T-cell depletion.This dramatic effect was obtained with live IP-injectedMSRV concentrated viral particles.

Using recombinant ENV protein at much lowerdoses in this hybrid model with human immune sys-tem, we have now been able to cause an “experi-mental allergic encephalomyelitis” (EAE) using hu-man MBP as autoantigen (highly homologous to mouseMBP). Clinical symptoms and neurological deficitsstarted about 10 days post-inoculation and partialremission was observed in the following week. Re-peated injections with ENV caused new aggravationsreaching complete paralysis with, e.g., spastic retrac-tion of paralyzed forelegs(s). This pattern was rela-tively similar to that obtained in positive controls se-ries with complete Freund’s adjuvant (CFA) whereascontrols with Incomplete Freund’s adjuvant (IFA) re-mained normal. Nonetheless weight curves indicateda continuous loss in ENV series, when ACF micerapidly compensated weight loss during the phase ofrecovery.

Histopathological examination of CNS sections fromsequentially sacrificed mice, after perfusion with PBS+ 4% Paraformaldehyde before dissection and tis-sue embedment in paraffin, revealed massive demyeli-nation prominent in the spinal cord of “ENV se-ries”. Numerous lymphoid cells were associated withtissue destruction in the vicinity of blood vessels,whereas very large macrophage cells were seen atdistance surrounded by large plaques of demyeli-

nation. Brain lymphoid infiltrates were also seen,with more restricted demyelination. Such featuresappeared less extended with much fewer activatedmacrophage cells in demyelinated areas in CFA series.Moreover, Magnetic Resonance Imaging (MRI) was per-formed at regular intervals, evidencing apparition anddissemination of characteristic T2-weighted hypersig-nals in white matter of both spinal cord and brain ofENV+ animals. IFA controls remained normal in alltechniques.

Lympocytes recovered from spleen dissection be-fore animal perfusion, confirmed a time- and dose-dependent increase in MBP autoreactive T-cells inENV or CFA, but not IFA, series.

We have thus developed a novel animal model ofMS characterized i) by the induction of EAE withthe autologous immunotoxin as detected post-mortemin MS brain plaques by independent immunohis-tology studies as well as in MS serum by our re-cent multi-center study with immunoassay dosage(Cf. Epidemiology Abstract) in spite of the heterolo-gous and “MS-irrelevant” M. tuberculosis extract cur-rently used in CFA for classical EAE models andii) by the development of an autoimmunity againstmouse CNS mediated by human immune cells acti-vated against homologous human MBP, from whichhuman anti-MBP autoreactive T-cell clones can beisolated.

This novel model caused by an MS “immunotoxin”and mediated by human immune cells, is now usedfor pre-clinical studies with therapeutic monoclonalantibodies (Cf. therapeutic antibody abstract).

P.127Monoclonal antibody against envelope protein fromHuman Endogenous Retrovirus “W” (MSRV-ENV orSyncytin) inhibits TLR4-initated immunotoxicitycascade in human peripheral blood mononuclearcells and displays therapeutic effects in novelpre-clinical models for Multiple SclerosisHerve Perron,1,2,3 K. Sanhadji,4 Corinne Bernard,1jean-louis Touraine,4 and Patrice Marche5

1Geneuro SA; 2Plan-Les-Ouates (Geneva); 3Switzerland;4Univesity Lyon 1 France; 5INSERM-Universite JosephFourier, Grenoble, France

HERV-W endogenous retroviral family encodes a pow-erful imunopathogenic envelope protein (MSRV-ENVor Syncytin), causing sequential activation of a pro-inflammatory and autoimmune cascade through ini-tial interaction with Toll-Like receptor 4 (TLR4) onantigen-presenting cells, accompanied by gliotoxicitycausing blood brain barrier (BBB) breakdown and fur-ther triggering superantigen (Sag)-like dysregulation ofT-lymphocytes. The specific association of this pro-tein with MS brain lesions post-mortem, as evidencedby independent immunohistological studies, its pres-ence in about 75% of MS sera ex-vivo versus none


in healthy controls (Cf. Epidemiology abstract) and itsability to reproduce the “EAE” MS animal model as-sociated with demyelination and autoimmunity, hasconducted us to select anti-ENV specific monoclonalantibodies (mAb) capable to block pathogenic effectsof this protein in vitro. Further confirmation of ther-apeutic efficiency on clinical effects in vivo (animalmodels) was also pursued.

We can now present an anti-ENV mAb blockingENV interaction with TLR4 (preventing IL6 and IL12-P40 release by human blood mononuclear cells), andinhibiting later Sag-like activation of T-lymphocytes(preventing IFN-Gamma release in the same culture).Similar effects are obtained with anti-TLR4 blockingantibody, which also prevent the immune system fromintercepting bacteria (by LPS) and thus may cause se-rious opportunistic infections if used as therapeuticagent. Anti-ENV blocking mAb do not block TLR4 andthus, do not interfere with LPS activation of such in-nate immunity receptors.

Anti-ENV mAb was further tested in two MS ani-mal models (EAE induced by ENV in either C57/Bl6mice with MOG, or in SCID mice grafted with ahuman lymphoid system with MBP-Cf. Abstract onanimal model), with significant inhibition or preven-tion of clinical symptoms compared to untreated con-trols and to treated animals in EAE models inducedby complete Freund’s adjuvant. Therapeutic efficiencyin the human/mouse hybrid SCID model was evenobtained during a third “challenge” after two sepa-rate ENV injections had already triggered previous“relapses”.

We have cloned the variable chains of this mAb andtested resulting activity of recombinant scFv, whichshowed complete preservation of affinity.

Such results in vitro and in vivo, now pave the wayto the molecular humanization of this antibody for pre-clinical and clinical trials.

P.128Activation of anti-apoptotic protein survivin uponJCV infection in glial cells. Implications for thedevelopment of progressive multifocalleukoencephalopathySergio Pina-Oviedo, Katarzyna Urbanska, ThersaSweet, Krzysztof Reiss, Kamel Khalili, andLuis Del ValleDepartment of Neuroscience, Center for Neurovirology,Temple University School of Medicine

Progressive Multifocal Leukoencephalopathy (PML) isa sub-acute and fatal disease of the Central NervousSystem, result of the productive infection of glial cells,particularly oligodendrocytes, by the opportunistic JCVirus. The lytic destruction of oligodendrocytes andthe activation of astrocytes in response to JCV infectionresults in the characteristic histopathological land-

marks of PML; extensive and confluent areas of myelinloss, in which numerous bizarre reactive astrocyteswith atypical and pleomorphic nuclei, and enlargedoligodendrocytes harboring intra-nuclear eosinophilicinclusion bodies, which represent the site of active vi-ral replication.

Apoptosis is a host defence mechanism to disposeof senescent, damaged or potentially harmful cells, in-cluding virally infected cells, however, certain viruseshave the ability to de-regulate apoptotic pathways inorder to complete their vital cycles. Although apop-tosis has been extensively demonstrated in a varietyof viral infections of the brain, including Herpes, Po-lio and HIV-encephalopathy, there is little evidenceto support the occurrence of apoptosis in PML, sug-gesting that the cellular machinery that controls pro-grammed cell death may be disrupted by the presenceof JCV. One of these pathways involves a novel pro-tein, Survivin, a member of the inhibitors of apoptosisfamily, which is abundantly expressed during embry-onic development in proliferating tissues, but is nor-mally completely absent in terminally differentiatedcells. Immunohistochemical experiments performedon a collection of 20 cases of PML, revealed the in-creased expression of Survivin in the intra-nuclear in-clusion bodies of JCV infected oligodendrocytes, andin the cytoplasm of bizarre astrocytes. Corroboratingthese findings, in vitro experiments showed the activa-tion of Survivin expression at the transcriptional andtranslational levels in JCV infected primary cultures ofastrocytes and oligodendrocytes, compared with un-infected cultures, which, as expected, lack expressionof Survivin. Furthermore, in order to identify the JCVprotein responsible for the activation of the Survivinpromoter, we transfected primary astrocytes with theSurvivin promoter driving a Luciferase reporter gene,which was activated in the presence of JCV T-antigen.Cell cycle analysis and TUNEL assay demonstrated asignificantly lower number of cells undergoing apop-tosis upon JCV infection compared with uninfectedcultures. Finally, the specificity of these events wastested by siRNA inhibition of Survivin, which resultedin a dramatic increase of apoptosis in JCV infectedcultures.

This is the first time that expression of the anti-apoptotic protein Survivin has been demonstrated inclinical samples of PML and in JCV infected glial cellcultures. Based on these observations we hypothesizethat upon its reactivation, JCV attempts to prevent cellsfrom entering the apoptosis pathway by activating theSurvivin gene, which results in expression of the nor-mally silent anti-apoptotic protein, which in turn dis-rupts the apoptotic machinery, allowing JCV to suc-cessfully complete its lytic cycle and resulting in thedevelopment of PML. Understanding of this pathwaymay lead to the development of more effective thera-peutic strategies against a thus far incurable and fataldisease.

Supported by Grants from the NIH awarded to LDV.


P.129Induction of brain tumors by human neurotropic JCvirus in a transgenic animal modelSergio Pina-Oviedo, Jennifer Gordon, Kamel Khalili,and Luis Del ValleDepartment of Neuroscience, Center for Neurovirology,Temple University School of Medicine

JC Virus, a member of the Polyomaviridi family ofviruses, is a neurotropic virus with wide distributionamong the human population of the world, accordingto sero-epidemiological studies. JCV is the establishedopportunistic infectious agent of Progressive Multifo-cal Leukoencephalopathy, a fatal demyelinating dis-ease of the brain, result of the cytolytic infection ofoligodendrocytes. Mutations in the regulatory regionof JCV are responsible for the viral strain. The proto-type strain or Mad-1, which has been associated withPML contains a promoter with two 98 base pair re-peats, whereas the archetype strain or CY, isolatedfrom the urine of healthy individuals contains onlyone 98 tandem with two insertions of 62 and 23 basepairs respectively. JC Virus has been shown to possessoncogenic potential, as demonstrated by the develop-ment of a wide variety of brain tumors upon directinoculation of viral particles into rodents and non-human primates. Furthermore, JCV DNA sequencesand expression of viral proteins have been detectedin a variety of brain tumors, including medulloblas-tomas, oligodendrogliomas, astrocytomas, anaplasticglial tumors, glioblastomas, ependymomas and CNSlymphomas.

To further understand the oncogenicity of JC virusand its role in the development of human neoplasiawe have generated a transgenic mouse colony, whichcontains the early region of the archetype strain of JCvirus (CY), under the regulation of its own promoter.These transgenic animals developed a wide variety ofneural tube and neural crest origin tumors, includ-ing: Primitive Neuroectodermal Tumors (PNETs) (173cases), Medulloblastomas (4 tumors), Pituitary Tumors(21 tumors), Malignant Peripheral Nerve Sheath Tu-mors (18 cases), Adrenal Neuroblastomas (6 tumors),and Glioblastoma Multiforme (3 cases). The most fre-quently observed phenotype thus far is the PNET.The neoplasms originate from different areas of thebrain, including in order of frequency, the lower partof the Brainstem and upper portion of the CervicalSpinal Cord, the Basal Ganglia, the Cerebellum, theFrontal Cortex, and the Temporal Cortex (includingthe Hipocampus). Rarely the tumors arise from the ol-factory bulb. The tumors range from small foci of fewneoplastic cells to huge masses, which substitute up to80% of the brain parenchyma. Interestingly, in 5 casesa combination of PNETs and Pituitary tumor was ob-served, and in 7 cases, PNETs and MPNSTs were de-veloped concurrently.

Immunohistochemical studies for the detection ofthe JCV early product, T-antigen demonstrated the nu-clear robust expression in the nuclei of neoplastic cellsin all the different phenotypes of tumors. Furthermore,

the cell cycle regulatory protein p53 was also detectedin the nuclei of neoplastic cells, and double label-ing demonstrated co-localization of both proteins inthe nuclear compartment of neoplastic cells. Expres-sion of the accessory product Agnoprotein has beenalso constantly detected in the cytoplasm of neoplas-tic cells, demonstrating the active transcription of theviral sequences. Interestingly, while some cells withinthe tumors express abundantly T-Antigen, some othercells demonstrate weak expression and other are com-pletely negative. Alterations in several pathways arecurrently investigated including the Wnt signalingpathway, the IGF-1 / IRS-1 axis, and the antiapoptoticSurvivin pathway. Results from these experiments willhelp us further understanding the mechanisms of JCVoncogenicity.

Supported by Grants from the NIH awarded to LDVand KK.

P.130Cooperative inhibition of HIV-1 by drugcombinations containing the biguanide-basedinhibitor NB325Vanessa Pirrone,1 Shendra Passic,1 TinaKish-Catalone,1 Brian Wigdahl,1 Robert F. Rando,2Mohamed Labib,2 and Fred C. Krebs1


Combination antiretroviral therapy (ART), which con-tinues to be used as highly effective treatment of HIV-1-associated immunopathogenesis, has also had a pos-itive impact on neurologic disease caused by HIV-1infection of the central nervous system (CNS). How-ever, limits on treating or preventing HIV-1-associatedneuropathogenesis due to reduced CNS drug pen-etration and drug-associated neurologic side effectsunderscore the importance of developing new drugsand combinations with greater efficacy. Our effortsin this direction have focused on NB325, which isa biguanide-based compound that is an effective in-hibitor of both R5 and X4 strains of HIV-1. Assays ofdrug combinations have demonstrated that NB325 canbe cooperatively paired with several types of HIV-1 in-hibitors, including nonnucleoside and nucleoside re-verse transcriptase inhibitors (efavirenz and tenofovir,respectively), and a fusion inhibitor (cyanovirin).Assays of cytotoxicity and anti-HIV?1 activity demon-strated that these compound combinations were min-imally cytotoxic and highly effective against HIV-1entry and replication. Further analyses demonstratedadditive or synergistic activity against HIV-1, and thatthe nature of the activity was dependent on the part-ner for NB325 and the combination ratio, as confirmedthrough the use of two distinctly different softwareprograms-CalcuSyn and MacSynergy II – that are usedspecifically to analyze activities of combined agents.These results suggest that cooperative antiviral activ-ity between two agents may be influenced by similari-ties or differences between each agent’s mechanism of


action. Furthermore, these results support the furthertesting of combinations that may be used effectively totreat HIV-1 CNS infection.

P.131Sequence variation within the HIV-1 LTR correlateswith brain regionalization and HIV-1-associateddementiaJonathan Pitcher,1 Michael R. Nonnemacher,1

Aikaterini Alexaki,1 Evelyn M. Kilareski,1 SuzanneGartner,2 and Brian Wigdahl1

1Drexel University College of Medicine; 2Johns HopkinsUniversity School of Medicine

The long terminal repeat (LTR) regulates human im-munodeficiency type 1 (HIV-1) viral gene expressionduring the course of infection. The activity of the LTRis regulated by the relative availability of viral andcellular transcriptional control proteins and the ge-netic variation that accumulates during replication.The C/EBP site II consensus B (conB) variant has beenshown to be highly conserved in brain-derived HIV-1LTR populations, and that LTRs containing C/EBP siteII 4C and 6G variants were only found in brain tissueof patients with HIV-1-associated dementia (HIVD). Astatistically significant difference was also found in theregional distribution of LTRs containing the C/EBP siteII conB, 4C, or 6G variant in brain regions derived frompatients with and without HIVD. A low affinity C/EBPsite II 4C variant accumulates in the cerebellum, a re-gion exhibiting little viral gene expression, while thehigh affinity C/EBP site II 6G variant accumulated inthe mid-frontal gyrus, a site of highly productive repli-cation. A 3T C/EBP site I variant was identified in 25%of brain-derived LTRs from patients diagnosed withHIVD, but was absent in patients without dementia.LTRs containing a C/EBP site I 6G variant were foundto accumulate in a highly specific manner in the headof caudate and the C/EBP site I 3T variant accumu-lated in the mid-frontal gyrus. In conclusion, distinctLTR populations with specific C/EBP site I and II con-figurations were found in different neuroanatomicalregions of the brain and correlated with neurologicaldisease.

P.132Increases CD14+/CD16+ monocytes in Hispanicwomen with asymptomatic HIV-associated cognitiveimpairment with HAARTMarines Plaud-Valentin,1 Valerie Wojna,1 Richard L.Skolasky,2 Raul Mayo,1 Rosa Hechavarria,1 ElizabethMaldonado,1 Tania de la Torre,1 and LoydaMelendez1

1University of Puerto Rico, Medical Sciences Campus;2Johns Hopkins University

HIV-associated dementia (HAD) has remained preva-lent despite highly activated antiretroviral treatment(HAART). The advent of HAART has affected the phe-

notype of HAD which now present in a milder formtermed cognitive impairment (CI) and with variableprogression patterns. Prior to HAART, this disease hasbeen associated with an increased subset of mono-cytes (CD14+/CD16+/CD69+). In the post HAART erathis phenotype has been reported to decrease signif-icantly with HAD. In a recent study, similar of ex-pression of CD14+/CD16+ was reported between HIV-seropositive and HIV-seronegative controls. To ourknowledge no similar study has been conducted in acohort of HIV-seropositive women. Therefore, we de-signed this study to compare the expression of thismonocyte subset in HIV-seropositive Hispanic womenon HAART. Sixty-three HIV-seropositive women and15 HIV-seronegative controls stratified for cognitivefunction using the American Academy of NeurologyHAD criteria modified to include an asymptomaticgroup (mAAN) into 17 (32%) women with normal cog-nition , 12 (23%) with asymptomatic cognitive im-pairment, 11 (21%) with MCMD, and 13 (24%) withHAD. Co-infection with HCV was present in 24% casesand drug abuse was present in 6%. For the purpose ofthis study MCMD and HAD were grouped together assymptomatic CI. Monocyte subsets were determinedin whole-blood samples of HIV-seropositive women byusing antibodies against CD14, CD16, and CD69 anti-gens and flow cytometry analysis. We found no signif-icant differences in age, CD4 cell count, plasma or CSFviral load between the three groups. Increase percent-age of CD14+/CD16+ positive monocytes was foundin HIV-seropositive women compared to seronegativecontrols (p < .05). In HIV-seropositives after adjust-ing for age, education, CD4 cell count, and viral loadusing a generalized lineal equation model we foundthat the percentage of CD14+/CD16+ positive mono-cytes significantly increased with the degree of cog-nitive impairment; being higher in the asymptomaticgroup when compared with those with normal cog-nition. Likewise, CD14+/CD16+ expression in mono-cytes from women with symptomatic CI was higherthan those with asymptomatic and those with nor-mal cognition. In conclusion, our result show thatmonocyte activation occurs in early stages of CI an re-main elevated in HIV-seropositive women while usingHAART.

P.133A novel humanized mouse model for HIV-1encephalitisLarisa Y Poluektova, Hannah Sneller, CatherineGebhard, Howard E Gendelman, and Santhi GorantlaUniversity of Nebraska Medical Center, Omaha, NE, USA

A novel rodent model was developed for studiesof HIV-1 neuropathogenesis. Human hematopoieticstem cells derived from umbilical cord blood wereused to reconstitute newborn irradiated NOD/scid-IL-2Rgc-/- mice (hu-NSG). Thirty hu-NSG animals devel-oped human lymphoid tissue and showed migration


of blood-derived macrophages into the brain. HIV-1infection with CCR5 utilizing viruses induced lym-phadenopathy and viral replication with sustainedplasma viral loads. At 3 months after infection, humanHIV-1p24+ macrophages were present in the meningesand perivascular spaces. Depletion of CD8a+ cells oneweek before and 3 weeks after infection by cM-T807antibodies, increased viral replication and induceda profound meningoencephalitis. Brain infiltrationby lymphocytes, formation of perivascular cuffs andthe presence of HIV-1p24+ cells with microglial-like morphology were documented. These neu-rovirological and neuropathological disease featurescorresponded to reduced CD4/CD8 T cell ratios incirculation and lymphoid tissues with concomitanthumoral immune responses against HIV-1 gp120. Weconclude that hu-NSG mice show sustained viral repli-cation, immunosuppression and macrophage ingressacross the blood brain barrier. These are reflective ofwhat can occur early after viral exposure in an infectedhuman host.

P.134Perturbation of glutamate metabolism inHIV-infected macrophages: Implications for the CNSFabrice Porcheray,1,2,3 Cathie Leone,1,2,3 BoubekeurSamah,1,2,3 Nathalie Dereuddre-Bosquet,4 andGabriel Gras1,2,3

1UMR E-01 Service d’ImmunoVirologie, CEA-DSV/iMETI,Fontenay aux Roses, France; 2University Paris-Sud 11,France; 3Institut Paris Sud Cytokines IPSC, IFR13, France;4SPI-BIO, CEA, Fontenay aux Roses, France

Central nervous system disorders in HIV infectionare mostly the consequences of an inflammatorymacrophage activation and relate to glutamate-mediated excitotoxicity. However, recent studies alsosuggest neuroprotective aspects of macrophage activa-tion through the expression of glutamate transportersand glutamine synthetase. We thus aimed to studywhether HIV infection or activation of macrophagescould modulate glutamate metabolism in these cells.We assessed the effect of HIV infection on glutamatetransporter expression as well as on glutamate uptakeby macrophages and showed that glutamate transportwas partially decreased (30%) in the course of virusreplication whereas EAAT-2 gene expression was dra-matically increased (x20). The consequences of HIVinfection on glutamine synthetase was also measuredand for the first time we show the functional expres-sion of this key enzyme in macrophages. This ex-pression was repressed during virus production. Wethen quantified EAAT-1 and EAAT-2 gene expressionas well as glutamate uptake in differentially activatedmacrophages (TNFalpha, IL-1beta, LPS, IFNgamma,GM-CSF, IL-4, PGE2, M-CSF, IL-10, dexamethasone)and show that the effects of HIV are not directly relatedto pro- or anti-inflammatory mediators. Indeed, EAAT-2 gene expression was upregulated by LPS and PGE2

and EAAT-1 one by dexamethasone. Of note, the func-tion of EAATs was not directly correlated to gene ex-pression levels, as both TNFalpha and LPS increasedit, dexamethasone exhibiting a weaker effect.

We finally showed that the glutamate transport bymacrophages is less affected than what has been de-scribed in astrocytes. Macrophages may thus play arole in neuroprotection against glutamate in the in-fected brain, through their expression of both EAATsand glutamine synthetase. As glutamate metabolismby activated macrophages is sensitive to both HIV in-fection and inflammation, it may thus be of potentialinterest as a therapeutic target in HIV encephalitis.

P.135Simian varicella virus induces apoptosis in monkeykidney cells by an intrinsic pathway in which BCL-2expression is downregulatedSubbiah Pugazhenthi,1,2 Donald H Gilden,3,4 AnneMcAdoo,1,2 Mary C Wellish,3 Elizabeth Brazeau,3,4

and Ravi Mahalingam3

1Medicine, University of Colorado at Denver and HealthSciences Center, Denver, CO; 2Veterans AdministrationMedical Center, Denver, CO, USA; 3Neurology, Universityof Colorado at Denver and Health Sciences Center, Denver,CO; 4Microbiology, University of Colorado at Denver andHealth Sciences Center, Denver, CO

Like varicella zoster virus (VZV) infection in humans,SVV causes chickenpox in primates, becomes latent inganglionic neurons and reactivates to produce zoster.Thus, SVV has been a useful model of varicella in-fection. We showed that the cytopathic effect in SVV-infected monkey kidney (Vero) cells in tissue cultureis apoptotic as evidenced by nuclear condensation.Apoptosis is triggered by sequential activation of agroup of cysteine-rich proteases (caspases). To studythis caspase cascade, we searched for markers of apop-tosis by Western blot analysis in virus-infected and un-infected cells at 24 h, 40 h, 48 h and 64 h post infection(p.i). At 40 to 64 h p.i., there was a significant increasein the level of active caspase-3. This increase was ac-companied by a parallel increase in caspase-3 activitymeasured with DEVD-pNA, thus confirming apopto-sis. Apoptosis proceeds through extrinsic or intrinsicpathways. The extrinsic pathway is mediated by deathreceptors, and the intrinsic pathway is caused by animbalance between pro-apoptotic proteins (e.g., Bad,Bax) and anti-apoptotic proteins (Bcl-2 and Bcl-XL) inmitochondria. The active form of caspase -9 (an in-trinsic pathway marker) was elevated in infected cells40-64 h p.i., while active caspase 8 (an extrinsic path-way marker) was not detected at any of the time points.Bcl-2 mRNA levels in SVV-infected cells decreased ina time-dependent manner with a maximum decrease of90% at 64 p.i. compared to uninfected cells. Bcl-2 pro-tein in infected cells was decreased 40-60% at all timepoints while Bcl-XL, Bad and Bax were unchanged.These findings suggest that SVV infection of Vero cells


leads to induction of apoptosis thru an intrinsic (mi-tochondrial) pathway, as evidenced by decreased ex-pression of the anti-apoptotic gene Bcl-2. Because VZVhas been shown to induce apoptosis in cultured hu-man fibroblasts, but not in human sensory neurons,varicella-induced apoptosis requires further analysisin neurons and non-neuronal cells both in vitro as wellas in vivo.

P.136JC virus T-antigen transforms circulating bonemarrow mesenchymal stem cellsSujatha Radhakrishnan, Jennifer Gordon, Luis DelValle, S. Ausim Azizi, Kamel Khalili, andBarbara KrynskaTemple University School of Medicine, Philadelphia, PA

Bone marrow-derived stromal cells (MSCs), also re-ferred to as mesenchymal stem cells, have the abil-ity of trafficking and multilineage differentiation intodifferent tissues and organs. JC virus is a humanpolyomavirus and the etiological agent of progressivemultifocal leukoencephalopathy. The JC virus earlyprotein, T-antigen, has been described to have onco-genic potential in several animal models and has beendetected in the variety of human tumors. In this study,we demonstrate that MSCs can be infected with JCvirus, and can undergo transformation after trans-fection with JC virus T-antigen. Cultured T-antigentransfected MSCs lost their original morphology andcontact inhibition. These cell when transplanted sub-cutaneously in nude mice formed tumors, but T-antigen negative MSCs did not form tumors. This studyshows for the first time the infection and transforma-tion of MSCs, a globally accessible adult stem cell, witha naturally occurring human viral oncoprotein. In lightof earlier reports on the association of JCV with the va-riety of human tumors our data contributes to identify-ing a possible common origin for a variety of cancers.Moreover JCV T-antigen transformed MSCs provide apowerful tool for study of molecular pathways and an-imal models of oncogenesis.

P.137Regulation of HTLV-1 gene expression in primaryversus secondary target cell population in thecontext of chromosomally integrated viral promoterSaifur Rahman, Devanshi Pandya, Brian Wigdahl,Zafar K. Khan, and Pooja JainDrexel University College of Medicine

HTLV-1 exhibits a broad cellular tropism due to theubiquitous nature of its receptor. In addition to theCD4+/CD25+ T cells other cell populations, includ-ing the cells of the monocyte-macrophage lineage, arealso susceptible to HTLV-1 infection. Viral-inducedalterations in these cells and their trafficking to thebrain may play important roles in the progressionof HAM/TSP. However, very little information existsconcerning the regulation of HTLV-1 promoter or long

terminal repeat (LTR) in secondary target cell popu-lations. Moreover, most of the studies of HTLV-1 geneexpression have been performed using transientlytransfected LTRs that differ physically from a chro-mosomally integrated provirus, which like eukaryoticDNA, is wrapped with histone and nonhistoneproteins to form the nucleosome, the basic unit ofchromatin. Hence it is important to understand howthe viral promoter is regulated when formatted in thecontext of chromatin. To this end, we have generateda number of clones of CD4+ T cell line Jurkat andmonocytic line U-937 (representative primary and sec-ondary target cell type, respectively) stably integratedwith an HTLV-1 LTR. The single cell clones fromboth the cell types were propagated and examined forLTR integration by a reporter gene (luciferase) assay.Selected clones with varying luciferase expressionwere analyzed for integrated LTR copy number by realtime PCR with higher levels of LTR expression shownto correlate with higher LTR copy number. An equalnumber of cells from the clones with comparable copynumbers were pooled and transfected with a Tax-expressing plasmid and analyzed for the luciferaseexpression as well as for global microRNA profiling.Tax has demonstrated a clear differential pattern inthe activation and suppression of the cellular miRNApathway indicating that the differential interplay ofviral and cellular factors in primary versus secondarytarget cell populations regulates viral activationpost-integration. Our results also demonstrated, forthe first time, that HTLV-1 Tax protein can interferewith miRNA pathway as shown previously with otherviral transactivator proteins such as HIV-1 Tat. Theseobservations are currently being confirmed in theprimary T cells and monocytes and are being extendedto the B cell (Raji), and bone marrow progenitor cell(TF-1) lines. Future investigations will analyze therole of individual miRNAs and potential transcriptionfactors in regulating Tax-mediated integrated LTRactivation in selected cell types.

P.138Monocyte protein sialoadhesin facilitates HIV-1trans infection and induces inflammatorychemokine expressionHans Rempel,1 Cyrus Calosing,1 Bing Sun,1 Gaik-WeiTew,1 and Lynn Pulliam1,2

1Veterans Affairs Medical Center, San Francisco, CA;2Department of Laboratory Medicine, University ofCalifornia, San Francisco

Monocyte/macrophages are mediators of HIV-1-associated neuronal dysfunction by actively trans-porting HIV-1 to the CNS and through the release ofneurotoxic factors. Using high-density microarrays toexamine how HIV-1 infection dysregulates the im-mune system and alters gene expression in circulatingmonocytes, we found increased expression of siaload-hesin (Sn, CD169) in CD14+ monocytes from subjects


infected with HIV-1 compared to non-infected con-trols. When analyzed by flow cytometry, Sn expressionon freshly isolated monocytes correlated with the sub-ject’s HIV-1 viral load and could be induced in culturedhuman monocytes by interferon. Sialoadhesin is a 190kDa transmembrane protein that binds sialic acid andwhen expressed on monocytes is capable of bindingHIV-1 through interaction with the sialic acid residueson gp120. When evaluated in an infectivity assay, HIV-1 bound to Sn-expressing monocytes, infected targetcells in trans within 48 hr. Infection of the target cellswas dependent on CD4+ receptor and CXCR4+ core-ceptor expression and occurred in the absence of pro-ductive infection of the Sn-expressing monocytes. Inaddition to binding HIV-1, Sn expression triggeredthe elaboration of cytokines from transfected THP-1cells. THP-1 cells either stably or transiently trans-fected with a CMV-SN construct secreted proinflam-matory chemokines IL-8 (CXCL8), MIP-1alpha (CCL3),MIP-1beta (CCL4) and IP-10 (CXCL10) into the cul-ture medium. These findings suggest that monocyteexpression of Sn in the periphery may facilitate trans-port of HIV-1 to the CNS and that transmigration ofSn-expressing monocyte/macrophages could impairneuronal function through secretion of inflammatorychemokines.

P.139Modulation of galectin-1 by drugs of abuseJessica L. Reynolds, Supriya D Mahajan, Donald E.Sykes, and Stanley A. SchwartzUniversity at Buffalo, The State University of New York

It is well established that drug abuse is a signifi-cant risk factor for contracting human immunodefi-ciency virus (HIV-1) infection and its progression toHIV-1 associated encephalopathy (HIVE). Astrocytes,integral components of the central nervous system(CNS), maintain a homeostatic environment and par-ticipate in a bidirectional communication with neu-rons. Upon activation of astrocytes, the expression ofvarious immunoregulatory proteins involved in thedevelopment of HIVE is modulated. Proteomic anal-yses (difference gel electrophoresis) of heroin treatednormal human astrocytes (NHA) demonstrated the dif-ferential expression of several biologically significantproteins, including galectin-1, as identified by highperformance liquid chromatography-tandem massspectrometry (HPLC-MS/MS). Galectins are a familyof β-galactoside binding lectins that regulate cell tocell and cell to matrix interactions, cell adhesion, andcell signaling. We observed that treatment of NHA withheroin, morphine or methamphetamine increased thegene and protein expression of galectin-1 in a timeand dose dependent manner. Gene and protein expres-sion of galectin-1 was assessed by quantitative real-time PCR and western blot analyses respectively. Ad-dition of HIV-1 envelope protein, gp120, to treatmentof NHA with heroin, morphine or methamphetamine

results in a further increase in galectin-1 expression.Recent evidence indicates that galectin-1, acting as asoluble adhesion molecule, enhances HIV-1 infectivityand replication in immune cells. Our studies demon-strate that drugs of abuse enhance HIV-1 replicationin NHA. The mechanism underlying the associationof HIVE with drug abuse may be mediated by the in-creased expression of galectin-1 which, in turn, en-hances HIV binding to target cells. Our findings maylead to novel strategies for preventing HIVE in highrisk, drug abusing populations.

P.140MCP-1 levels and neurocognitive performance inHIV seropositive marijuana usersMary Ricardo-Dukelow,1 Kathy Conant,2 ChristineCloak,1 Eric Miller,3 and Linda Chang1

1University of Hawaii, Department of Medicine, Honolulu,HI; 2Department of Neurology, Johns Hopkins UniversitySchool of Medicine, Baltimore, Maryland, USA; 3NPI,University of California Los Angeles, Los Angeles, CA

Chronic marijuana use among Human immunodefi-ciency virus (HIV) patients is common, however, itseffects on the brain are not fully known. The aim of thisstudy is to evaluate whether marijuana use in HIV in-fection alters neuroinflammation, which in turn mightalter cognitive function. Neuroinflammation is vital fornormal function and protection of the central nervousSystem (CNS); however, excess inflammatory responsemay be neurotoxic and contribute to the pathophys-iology of many neurodegenerative diseases, such asParkinson’s disease, Alzheimer’s disease and HIV en-cephalitis. The inhibitory effect of cannabinoids onreactive oxygen species, glutamate and tumor necro-sis factor suggests that they may be potent neuropro-tective agents. Monocyte chemotactic protein-1 (MCP-1) is a chemokine that attracts monocytes. MCP-1 hasbeen implicated as an important mediator of monocyteand T lymphocyte infiltration of tissues in several in-flammatory diseases. We measured CSF MCP-1 levelsin HIV patients with and without marijuana use anddetermined whether their CSF MCP-1 level correlateswith neuropsychological test performance. Methods:We evaluated 94 subjects (46 HIV without marijuanause, 48 HIV with any recent marijuana use). CSF MCP-1 levels were measured with a commercial ELISA kit(human MCP-1 by R&D Systems) and a battery of neu-ropsychological tests was performed in each subject.

Results: The two subject groups were well-matchedin terms of age (p = 0.63 ), education (p = 0.50) andestimated verbal IQ (p = 0.90), and the two HIV sub-ject groups (marijuana vs non-marijuana) had similarCD4 count ( 241 ± 23 vs.258 ± 25 /mm3, p = 0.61),Log plasma viral load (3.90 ± 0.24 vs. 3.39 ± 0.21 p =0.11), HIV dementia scale (13.6 ± 0.4 vs. 13.0 ± 0.47,p = 0.35) and Karnofsky score (88 ± 1.3 vs.87 ± 1.5,p = 0.55). MCP-1 levels (pg/ml) showed a trend forhigher levels in HIV patients without marijuana use


(487 ± 29 pg/mL) compared to HIV patients with mar-ijuana use (474 ± 30) . MCP-1 levels correlated sig-nificantly with KS (p = 0.015 Spearman Correlation;and grouped as scores of 100, 90, 80, and 70 & 60 forANOVA p = 0.002). MCP-1 also correlated with threetests of psychomotor speed Trails A (r = 0.33, p =0.0016), Trails B (r = 0.27, p = 0.009), Symbol Digit (r= -0.25, p = 0.019), Stroop tasks (r = 0.21 to 0.25, p =0.01 to 0.03) and response reversal on a computerized(CalCAP) reaction time test during working memory (r= 0.2, p = 0.02). Higher MCP-1 consistently associatedwith slower performance, and with trends for poorerexecutive function.

Conclusion: Trends for immunosuppressive effectsof was observed in HIV-infected individuals who hadany marijuana usage compared to those who did notuse the drug. Since higher MCP-1 levels were associ-ated with poorer cognitive performance and generalfunctioning, cannabinoid drugs may have neuropro-tective effects and may have therapeutic values in HIVpatients. Further evaluation of other chemokines andcytokines are underway to better delineate the neu-roinflammatory patterns associated with these indi-viduals. Relationship between drug usage (dose, dura-tion, and recency) and inflammatory changes in thesesubjects also will be evaluated.

P.141Mechanisms of HIV infected monocytetransmigration across the blood brain barrierToni K Roberts and Joan W BermanAlbert Einstein College of Medicine, Department ofPathology

HIV infected individuals are living longer due tothe success of highly active antiretroviral therapy(HAART). Despite control of peripheral viral repli-cation, HAART is relatively ineffective at crossingthe blood brain barrier (BBB). As such, HIV per-sists in the central nervous system (CNS) and viral-mediated damage to the parenchyma accumulates.Consequently, clinical manifestations of CNS dam-age, including motor and neurocognitive impairment,are increasingly presenting as devastating sequelae ofHIV infection. Early during primary infection, HIVgains entry into the CNS, at least in part, by thetransendothelial migration (TEM) of infected mono-cytes across the BBB, and establishes a viral reser-voir in resident macrophages and microglia. CCL2,the predominant monocyte chemoattractant, is signif-icantly elevated in the cerebral spinal fluid (CSF) ofpatients with HIV Associated Dementia (HAD) andHIV Encephalitis (HIVE). Thus, HIV may establish aCNS microenvironment that promotes recruitment ofmonocytes. As a mediator of both inflammation andviral replication, monocytes are thought to be a majorcontributor to the neuropathogensis of HIV infection.In fact, autopsy specimens of seropositive individu-als demonstrate BBB disruption and suggest that HAD

and HIVE more directly correlate with monocyte re-cruitment, macrophage and microglia activation, andneuroinflammation than with CNS viral load.

In non-pathologic states, monocytes traffic acrossthe BBB at low levels without barrier disruption. Thisprocess is regulated by interactions between homotyp-ically interacting adhesion proteins present on boththe monocyte and the endothelial cell (EC) of the BBBincluding PECAM-1, CD99, and PrPC. However, stud-ies from our lab using a human BBB cell culture modeldemonstrate that monocyte infection with HIV and ex-posure to CCL2 work together to disrupt the homeosta-sis of this system, causing both an increase in mono-cyte TEM across the BBB and a loss of barrier integrity.HIV and CCL2 appear to act in concert, as neither vari-able on its own alters base line monocyte recruitmentor disrupts barrier integrity to the same extent. There-fore, we hypothesize that HIV infection and CCL2 stim-ulation result in aberrant interactions between inter-cellular adhesion proteins on the monocyte and ECduring diapedesis, promoting neuroinflammation andviral entry into the brain. The goal of this research in-cludes characterizing the changes to whole cell proteinexpression, cellular localization, membrane dissocia-tion, and phosphorylation of the intercellular adhe-sion molecules PECAM-1, CD99, and PrPC in boththe monocyte and EC during HIV infection and CCL2-mediated TEM. Changes in expression, localization,or structural integrity of these proteins could alter thebalance of maintaining barrier integrity while accom-modating monocyte TEM, while phosphorylation ofcell adhesion proteins is known to affect their local-ization, interactions with adaptor proteins and the cy-toskeleton, and activity as mediators of cell signaling.Preliminary data suggest that monocytes increase theirwhole cell expression of PECAM-1 and CD99 in re-sponse to HIV infection, potentially increasing the mi-gratory capacity of the monocyte. In response to CCL2,EC demonstrate an early increase in PECAM-1 wholecell expression, followed by a decline, while CD99is redistributed away from intercellular junctions.These changes to EC adhesion molecules could dis-rupt the resealing of the BBB behind the transmigratingmonocyte.

P.142Proteomic fingerprints for HIV-1 cognitiveimpairmentsWojciech Rozek,1 Mary Ricardo-Dukelow,1 Howard EGendelman,1 Loyda Melendez,2 andPawel Ciborowski1

1University of Nebraska Medical Center, Omaha, NE;2University of Puerto Rico Medical Sciences Campus,San Juan, PR

The need for suitable biomarkers in cerebrospinal fluid(CSF) and sera to predict the onset and tempo of cog-nitive impairments (CI) linked directly to advancedHIV infection cannot be overstated. To these ends


a “state of the art” proteomics platform integrating2-dimensional electrophoresis (2DE) with DifferenceGel Electrophoresis (DIGE) and Liquid Chromatog-raphy Tandem Mass Spectrometry (LC-MS/MS) wasdeveloped within our laboratories. A bioinformaticsystem was used for identifications of differential ex-pression of proteins retrieval and identification using(DeCyder©R 6.5 software, GE Healthcare, Inc.) and pro-tein identification (BioWorks©R 3.2 utilizing Sequestalgorithm). To facilitate analysis of low abundanceproteins, we removed the 6 most abundant proteins:Albumin, IgG, IgA, α1-Antitrypsin, Haptoglobin, andTransferrin from CSF samples and 12 most abundantproteins from sera samples: 6 proteins listed aboveand additional 6: Fibrinogen, α2-macroglobulin, IgM,Orosomucoid, Apolipoprotein A-I, Apolipoprotein A-II. Subsequently, we used a 2DE DIGE for proteomicprofiling. Analysis was performed using DeCyder©R 6.5software (GE Healthcare, Inc.). Protein spots showingstatistically significant differences (p < 0.05) were sub-jected to protein identification using LC-MS/MS. Al-together 20 CSF samples, 10 from demented and 10from patients with normal cognition were used. Withinthe CSF, proteins with regulatory functions (comple-ment C3 and its fragments, neuronal cell adhesionmolecule (NrCAM), cystatin C, vitamin D binding pro-tein, clusterin, gelsolin, procollagen C-endopeptidaseenhancer) were found to be differentially expressedand have been validated by Western-blot analysis. 14sera samples, 7 from demented and 7 from patientswith normal cognition were used. Several proteinswith regulatory functions have been initially identifiedas differentially expressed and are being validated bywestern blot analysis. In conclusion, these biomarkerswhile preliminary in number of samples tested pro-vide new means to follow disease progression and topinpoint therapeutic needs in an HIV-1-associated cog-nitive impairment.

P.143Novel host defense mechanisms in theneuropathogenesis of HIV infectionJeffrey A Rumbaugh, Muznabanu Bachani, WenxueLi, Guanhan Li, David Galey, Tanya Malpica-Llanos,and Avindra NathJohns Hopkins University

We have discovered two novel host defense mecha-nisms, which prevent excitotoxicity in the setting ofHIV infection. We treated human fetal neuronal cul-tures with recombinant Tat, matrix metalloproteinases(MMPs), antisera to Tat, and NMDA, individually or invarious combinations.

Surprisingly, we found the combination of Tat andMMPs produced significant attenuation of neurotoxi-city, as measured by cell death and mitochondrial po-tential. Using protein electrophoresis techniques, wefound that MMP-1 can degrade Tat, an effect blockedby MMP inhibitors. MMP-2 and -9 also prevented Tat-

toxicity but did not degrade Tat. The degradation byMMP-1 was specific for Tat and not other viral pro-teins.

Interestingly, monoclonal antibodies against the C-or N-terminal of Tat not only prevented Tat-toxicity,but the Tat-antisera complexes also attenuated NMDA-mediated excitotoxicity. This effect was specific forNMDA and was not seen for kainate.

Further characterization of these MMP and anti-body mediated defense mechanisms will be impor-tant in developing new therapeutic strategies for HIVdementia, and may also be operative in other viralencephalitides.

P.144Toll-like receptors play a crucial role in protectionagainst West Nile virus infectionAmir H. Sabouri,1 Maria Cecilia G. Marcondes,2Howard S. Fox,2 and Nora E. Sarvetnick1

1Department of Immunology, The Scripps ResearchInstitute, La Jolla, CA 92037; 2Molecular and IntegrativeNeurosciences Department, The Scripps Research Institute,La Jolla, CA 92037

West Nile virus (WNV) is a neurotropic flavivirus thatappeared as an emerging infectious disease in thenortheastern United States in 1999. WNV, now en-demic throughout North America, has become a signif-icant global cause of viral encephalitis, primarily in theelderly and immunocompromised individuals. Priorstudies have established an essential protective roleof both innate and adaptive immune response compo-nents. Toll-like receptors (TLR) play an essential rolein initiating innate responses by sensing non-self com-ponents, and are shown to decreased in aged animals.It has been shown that TLR3 deficient mice are moreresistant to lethal infection. However, TLR signalingpathways in host defense against WNV infection havenot been fully explored. In the present work, we eval-uated the role of MyD88 and TRIF, two key adaptormolecules of TLR signaling, in WNV susceptibility andspreading into the brain. The absence of TLR signal-ing through MyD88 and TRIF simultaneously resultedin increased vulnerability to WNV infection with arise in mortality from 68% (wild-type mice) to 100%(MyD88/TRIF dKO mice), and a decrease in the av-erage survival time. MyD88/TRIF dKO mice showedincreased virus replication in the brain and in associ-ated lymphoid tissues. Brain-infiltrating macrophageswere more abundant in infected MyD88/TRIF dKOmice, and expressed higher CD86 than in WT mice.However these cells failed to up-regulate MHC ClassII, suggesting a defective activation of selective path-ways. Interestingly, mice lacking only MyD88 (withintact TLR3 signaling pathway) also exhibited highermortality and increased levels of virus replication inthe brain than WT mice. Our observations suggest thedominant protective role of Toll-like receptors againstWNV infection.


P.145Higher relative frequencies of HIV-specific CD8+ Tcells in cerebrospinal fluid (CSF) versus peripheralblood in antiretroviral-naive immune controllersShanmugalakshmi Sadagopal,1 Shelly Lorey,1 LouiseBarnett,1 Rebecca Basham,1 Laurie Lebo,1 HusamettinErdem,1 David W Haas,1,2 and Spyros A Kalams1,2

1Department of Medicine/Infectious Diseases, VanderbiltUniversity Medical Center, Nashville, TN; 2Department ofMicrobiology and Immunology, Vanderbilt UniversityMedical Center, Nashville, TN

Background: HIV-specific CD8+ T cell responses play acentral role in controlling HIV replication. While manystudies have evaluated CD8+ T cell responses in theperipheral blood and tissues, relatively little is knownregarding qualitative and quantitative characteristicsof HIV-specific T cell responses in the central nervoussystem (CNS). It has been speculated that activatedvirus-specific T cells may participate in the pathogene-sis of CNS dysfunction during HIV-associated demen-tia (HAD). Conversely, these responses may be criticalfor controlling HIV replication in the CNS.

Methods: Seven asymptomatic antiretroviral-naiveHIV+ immune controllers were assessed for the fre-quency of HIV-specific (tetramer+) CD8+ T cellsand their maturation phenotypes (CD45RO, CD57and CCR7) in cerebrospinal fluid (CSF) and periph-eral blood using a 9-colour flow cytometry panel.In addition to direct study of unexpanded cells,CSF cells were expanded with phytohaemagglutinin(PHA) and assessed for frequency of HIV-specific Tcells by flow cytometry and for IFN-gamma produc-tion to HLA-class I-restricted optimal peptides byELISpot. Approximately 40 mL of CSF was analyzedper participant.

Results: Plasma HIV-1 RNA ranged from 60 to 16,800copies/mL and CD4 T cells were >500/cubic mm.CSF HIV-1 RNA was <50 copies/mL in 6 of 7 sub-jects, and less than the plasma HIV-1 RNA levels inall participants. HIV tetramer positive T cells couldbe directly enumerated and phenotyped with as fewas 10,000 total CSF mononuclear cells. Frequenciesof tetramer+ CD8+ T cells in CSF, assessed by di-rect ex vivo staining, were higher than in blood (p =0.0004). The expansion potential of tetramer+ CSF Tcells was similar to those from blood, and the relativehigher frequencies of HIV-specific T cells in CSF wasmaintained in expanded cell populations (p = 0.0016).There were also higher frequencies of IFN- producingT cells in expanded cells from CSF compared to theblood in response to HLA-restricted optimal peptides(p = 0.012). By direct ex vivo evaluation of T cell mat-uration markers, both CD8+ and CD4+ T cells in CSFhad a higher frequency of memory CD45RO+ T cellsthan in blood. Among HIV-specific T cells, frequenciesof CD45RO+, CD57+ (a marker of replicative senes-cence) and CCR7+ T cells were similar between CSFand blood.

Conclusions: Asymptomatic HIV+ controllers havehigher relative frequencies of HIV specific T cells in

CSF compared to blood and lower concentrations ofvirus in CSF. These cells are capable of expansionand IFN-gamma production. This pattern is consistentwith homing of HIV-specific T cells to the CNS and/orpreferential expansion within this compartment withresultant control of virus.

P.146Dephosphorylation of JC virus agnoprotein byprotein phosphatase 2A: Inhibition by small tantigenIlker Sariyer, Kamel Khalili, and Mahmut SafakTemple University School of Medicine, Department ofNeuroscience and Center for Neurovirology, 1900 N., 12thStreet, Philadelphia, PA, USA

Previous studies have demonstrated that the JC virus(JCV) late regulatory protein agnoprotein is phos-phorylated by the serine/threonine-specific proteinkinase-C (PKC) and mutants of this protein at thePKC phosphorylation sites exhibit defects in the viralreplication cycle. We have now investigated whetheragnoprotein phosphorylation is regulated by PP2A,a serine/threonine-specific protein phosphatase andwhether JCV small t antigen (Sm t-Ag) is involvedin this regulation. Protein-protein interaction studiesdemonstrated that PP2A associates with agnoproteinand dephosphorylates it at PKC-specific sites. Sm t-Ag was also found to interact with PP2A and this in-teraction inhibited the dephosphorylation of agnopro-tein by PP2A. The interaction domains of Sm t-Ag andagnoprotein with PP2A were mapped, as were the in-teraction domains of Sm t-Ag with agnoprotein. Themiddle portion of Sm t-Ag (aa 82-124) was found to becritical for the interaction with both agnoprotein andPP2A and the N-terminal region of agnoprotein for in-teraction with Sm t-Ag. To further understand the roleof Sm t-Ag in JCV regulation, a stop codon was intro-duced at Phe86 immediately after splice donor site ofthe JCV early gene and the functional consequencesof this mutation were investigated. The ability of thereplication of this mutant virus reduced substantiallycompared to WT. Next, the functional significance ofPP2A in JCV replication was examined by siRNA tar-geting and down-regulation of PP2A caused a signifi-cant reduction in the level of JCV replication. Collec-tively, these results suggest that there is an interplaybetween agnoprotein, Sm t-Ag and PP2A with respectto the regulation of JCV life cycle and this could be im-portant for the progression of the JCV-induced disease,PML.

P.147JC virus small t antigen targets the host translationsystemIlker K Sariyer, Kamel Khalili, and Mahmut SafakDepartment of Neuroscience and Center for Neurovirology,Temple University School of Medicine, 1900 N., 12th Street,Philadelphia, PA, USA


Sm t-Ag is produced by the translation of the alterna-tively spliced transcripts of the JCV early pre-mRNA.Unlike LT-Ag, the function of Sm t-Ag in JCV life cycleis unclear. To understand the role of Sm t-Ag in JCV reg-ulation, a stop codon was introduced at Phe86 imme-diately after splice donor site of the JCV early gene sothat the unique region of Sm t-Ag is not produced andthe functional consequences of this mutation were in-vestigated. Results showed that the ability of the repli-cation of this mutant virus significantly reduced com-pared to WT, confirming the critical importance of thisprotein in JCV life cycle. To investigate the mechanismby which Sm t-Ag exerts its function during the in-fection cycle, we analyzed the protein complexes thatinteract with the unique region of Sm t-Ag by MassSpectroscopy. This analysis revealed the presence ofprotein complexes that are involved in host transla-tion, including, translation elongation factor 1 and 2suggesting that Sm t-Ag might function as a regula-tor of host translation machinery during the infectioncycle. The specificity of the interaction between Smt-Ag and host elongation factor was further confirmedby coimmunoprecipitation assay. To gain more insightinto the functional significance of this targeting, wewill further characterize the role of small t antigen inhost translation during the viral replication cycle.

P.148Kinetics of cytokine expression in brain and spleenfollowing intracerebral and intraperitonealchallenge of JEV in BALB/c miceVandana Saxena,1 Asha Mathur,2 Narendra KKrishnani,3 and Tapan N Dhole1

1Dept. of Microbiology, SGPGIMS, Lucknow, India; 2Dept.of Microbiology, Saraswati Dental college, Lucknow, India;3Dept. of Pathology, SGPGIMS, Lucknow, India

Introduction: Japanese encephalitis is a serious CNSdisease due to its complexity, lack of specific treat-ment and long term neuro-psychiatric disabilities inthe survivors. Immune mechanisms during JEV infec-tion are complex and need further elucidations for thedevelopment of safe and efficacious vaccines.

Methods: We investigated iNOS and cytokine ex-pression in spleen and brain of BALB/c mice chal-lenged intracerebrally (IC) with 3x106 pfu and in-traperitoneally (IP) with 1x108 pfu of JEV at varioustime points during disease development at mRNA andprotein level. Histopathology and viral growth assayswere performed in brain.

Results: In IC group, there is a significant upregu-lation of proinflammatory cytokines like IFN-g (P =0.03) and TNF-α (P = 0.009) while downregulationof anti-inflammatory IL-10 and IL-4 (P = 0.017 and<0.001) both in transcript as well as protein expres-sion with the disease progression, histopathologicalchanges and increased viral load in the brain. iNOSmRNA was downregulated following IC challenge. Inspleen however, TNF-α, IL-4 and iNOS mRNA was up-

regulated till 3 d.p.i. while IL-10 mRNA was upregu-lated from 1 to 5 d.p.i. but IFN-g was not detected. InIP group, spleen had a gradual, significant upregula-tion of all cytokines till 5 d.p.i. followed by decreaseat 7 and 11 d.p.i. IL-10 expression outcompeted bothIFN-g and TNF-α. In the brain, there is no significantdifference in iNOS and cytokines’ transcript as well asprotein between control and infected mice. But at 11d.p.i., all the cytokines showed significant increase.No virus was isolated from brain but histopathol-ogy shows slight meningeal inflammation at 11 d.p.i.and real-time PCR analysis for JEV E protein waspositive.

Conclusion: An insufficient anti- inflammatory cy-tokine response in the brain is associated with in-creased tissue pathology and viral load and regulatesinflammatory responses driven by IFN-g in concertwith TNF-α, to cause brain tissue damage. Deleteriouseffect of IFN-g in brain is independent of NO induc-tion. TNF-α is involved in pathogenesis of JEV androle of IL-10 needs further investigations.

P.149Impact of selegiline transdermal system on measuresof brain metabolism and markers of oxidative stressin hiv infected individuals with cognitiveimpairmentGiovanni Schifitto,1 Constantin T Yiannoutsos,2Thomas Ernst,3 Bradford Navia,4 Ned Sacktor,5Avindra Nath,6 and David B Clifford7

1University of Rochester, Rochester NY; 2Indiana UniversityMedical School, Indianapolis IN; 3John A. Burns School ofMedicine, University of Hawaii, Honolulu, HI;4Departments of Neurology and Psychiatry, Tufts-NewEngland Medical Center, Boston, MA; 5Johns HopkinsUniversity School of Medicine, Baltimore MD; 6JohnsHopkins University School of Medicine, Baltimore MD;7Washington University School of Medicine, St. Louis MO

Objectives: This substudy of A5090, “ Phase II,Placebo-Controlled, Double-Blind Study of the Selegi-line Transdermal System (STS) in the Treatment ofHIV-Associated Cognitive Impairment” examined theeffectiveness of STS in reversing metabolic brain in-jury, and the relationship between the oxidative stressmarker protein carbonyl and brain metabolite changes,as measured by 1H-MRS.

Background: HIV-associated cognitive impairmenthas been associated with oxidative stress induced neu-ronal injury. Oxygen radicals can attack proteins, de-oxynucleic acids, and lipid membranes resulting incellular dysfunction and cell death. Among the prod-ucts of oxidation, elevated CSF levels of protein car-bonyl have been reported in HIV infected subjects withcognitive impairment. Furthermore, brain metabolitesincluding N-acetyl aspartate (NAA), choline (CHO),and myoinositol (MI), measured non invasively bymagnetic resonance spectroscopy (MRS), have been re-ported to be affected in early stages of HIV infectionand cognitive impairment.


Methods: One hundred twenty-eight HIV infectedsubjects with cognitive impairment were enrolled inA5090 and randomized to receive STS 3mg/24h, STS6mg/24h, or matching placebo for 24 weeks. Random-ization was stratified by plasma HIV viral load (un-detectable, <200 copies/ml; versus detectable, ≥200copies/ml) and clinical stage of the AIDS dementiacomplex (ADC; 0.5 versus 1.0 or higher). Subjectswith ADC stage 1 or higher were eligible to be co-enrolled in this 1H-MRS substudy. Cognitive impair-ment was defined as performance at least one standarddeviation below the mean on two or more indepen-dent neuropsychological tests, or at least two standarddeviations below the mean on one neuropsychologi-cal test using a standard neuropsychological battery.Cognitive performance was evaluated using the NPZ-8 (average z-score of 8 neuropsychological tests) andNPZ-6 (average z-score of 6 neuropsychological tests),as well as cognitive domain scores (average z-scoreof neuropsychological tests corresponding to cogni-tive domain). Sixty-two subjects (25 placebo, 19 STS3mg/24h, and 18 STS 6mg/24h) were co-enrolled inthe substudy, and underwent proton MRS at study en-try, and weeks 12 and 24. MRS-measured metaboliteswere obtained from the midline of the frontal lobes,right (or left) mid-frontal centrum semiovale, and right(or left) basal ganglia. Metabolites measured includedNAA, CHO, MI, creatine (Cr) and glutamate/glutamine(Glx), and were expressed as a ratio relative to Cr. CSFwas obtained at baseline and week 24. CSF protein car-bonyls were detected and quantified using a slot blottechnique and an Oxyblot protein detection kit fromChemicon.

Results: The sixty-two subjects enrolled were pre-dominantly male (87%), African American (55%) andCaucasian (39%). Median age at baseline was 46, witha median education level of 12.5 years. The threetreatment groups did not differ significantly at base-line with respect to these demographic characteristics,ADC stage, HIV viral load, CD4+ count, NPZ scores, orcognitive domain scores. No significant differences in1H MRS metabolite ratios were detected between treat-ment arms at baseline, with the exception of Glx/Cr inthe centrum semiovale (STS 3mg/24 h mean = 1.95, SD= 0.29; STS 6mg/24h mean = 1.72, SD = 0.21; placebomean = 1.81, SD = 0.21; p = 0.037). Change in MRSmetabolite ratios from baseline to weeks 12 and 24 wasevaluated using an analysis of covariance adjusted forbaseline metabolite levels and multiple comparisons.There was a slight increase in NAA/Cr (a marker ofneuronal function) in the basal ganglia and centrumsemiovaleof the placebo group compared to the STSgroups (p = 0.023 and p = 0.072 respectively). Theplacebo group also demonstrated a greater increase inCho/Cr (a glia marker) in the mid-frontal cortex (p =0.002), when compared to the combined STS groups.Evaluation of the change in NPZ-8 and NPZ-6 scores,as well as cognitive domain scores from baseline toweeks 12 and 24 revealed no significant differences be-tween treatment arms. Protein carbonyl analysis will

be available and included in the presentation of thiscommunication at the meeting.

Conclusion: The results of this 24-week study showthat STS had no effect on brain metabolites as mea-sured by MRS. The lack of effect on brain metaboliteswas also reflected in the lack of cognitive improvementin the STS groups compared to placebo.

P.150MMP-7 cleaves the NR1 NMDA receptor subunit andmodifies NMDA receptor functionArek Scklarczyk, David Knorr, Norman Haughey, andKatherine ConantJohns Hopkins University Department of Neurology

Matrix metalloproteinases (MMPs) are zinc dependentenzymes that play a role in the inflammatory response.These enzymes may be substantially increased in as-sociation with viral infection of the brain. MMPs areknown to cleave proteins of the blood brain barrierbasement membrane and accumulating evidence sug-gests they can also target proteins critical to synapticstructure and neuronal survival, including integrinsand cadherins. Here we show that one member of theMMP family, MMP-7, which may be released fromcells including microglia, can cleave a protein criti-cal to synaptic function. Through analysis of extractsfrom murine cortical slice preparations, we show thatMMP-7 cleaves the NR1 subunit of the NMDA receptorto generate an N terminal fragment of approximately65 kDa. Moreover, studies with recombinant proteinshow that MMP-7 mediated cleavage of NR1 occurs atamino acid 517, which is extracellular and just distalto the first transmembrane domain. Data suggest thatNR2A, which shares sequence homology with NR1, isalso cleaved following treatment of slices with MMP-7while select AMPA receptor subunits are not. Consis-tent with a potential effect of MMP-7 on ligand bind-ing, additional experiments demonstrate that NMDAmediated calcium flux is significantly diminished byMMP-7 pretreatment of cultures. These data suggestthat synaptic function may be altered in neurologicalconditions associated with increased levels of MMP-7.

P.151Impact of single nucleotide sequence configurationsin stably integrated HIV-1 LTRs on gene expression,under basal and activating cellular phenotypesSonia Shah, Aikaterini Alexaki, Evelyn M. Kilareski,Michael R. Nonnemacher, and Brian WigdahlDrexel University College of Medicine

Human immunodeficiency virus type 1 (HIV-1) geneexpression is driven by the long terminal repeat(LTR), which has a variety of binding sites for theinteraction with multiple viral and host factors, in-cluding members of the CCAAT/enhancer bindingprotein (C/EBP) and Sp transcription factor families.Previous studies have identified specific nucleotide


sequence configurations within C/EBP site I and Spsite III (3T, C-to-T change at position 3 of the bindingsite, and 5T, C-to-T change at position 5 of the bind-ing site, respectively) which correlate with increasedseverity of HIV-1 disease and HIV-1-associated demen-tia. To begin exploring the LTR phenotype, associ-ated with these genotypic changes, from an integratedchromatin-based microenvironment, a series of stablytransfected cell lines have been developed utilizingbone marrow progenitor, T, and monocytic cell lines(TF-1, Jurkat, and U-937, respectively). Macrophage-,T cell-, and dual-tropic LTRs were coupled to the geneencoding green fluorescent protein (GFP) and poly-clonal HIV-1 LTR-GFP stable cell lines were developedand examined under basal, and chemical or cytokineactivation, as well as in the presence of Tat. The resultsdemonstrate that the cell type and LTR backbone havea significant effect on the expression profiles. In addi-tion, TF-1 cells the 3T/5T-containing LTR is associatedwith a lower expressing phenotype, however it can beinduced to similar levels as the parental LTR, follow-ing stimulation and/or in the presence of Tat. Currentstudies are underway to examine the integration pat-tern and proviral DNA level utilizing uncloned andcloned HIV-1 LTR-GFP stably expressing cell lines.

P.152West Nile virus induced flaccid paralysis mediatedby apoptosis in the ventral horn of the spinal cord inhamster modelVenkatraman Siddharthan,1 Hong Wong,1 Ramona TSkirpstunas,2 and John D Morrey1

1Institute for Antiviral Research, Animal, Dairy andVeterinary Sciences, Utah State University, Logan UT84322; 2Veterinary Diagnostic Laboratory, Department ofAnimal, Dairy and Veterinary Sciences, Utah StateUniversity, Logan UT 84322

Symptoms of West Nile Neurological Disease (WNND)in human patients include tremors, myclonus, parkin-sonism, cerebral ataxia, limb atrophy and acute flaccidparalysis. Since a subset of patients with WNND de-velop acute flaccid paralysis caused by poliomyelitis-like lesions, we hypothesize that West Nile virus(WNV) induced paralysis was due to infection anddeath of motor neurons in the ventral horn of spinalcord. Here we developed a hamster model for WestNile Virus (WNV) infection showing the hind limb flac-cid paralysis. To develop the paralysis we injected NYWNV 1) sub-cutaneously, 2) into the spinal cord at the8th thoracic vertebrae by laminectomy or 3) into thesciatic nerve. By 8 or 9 days post-viral infection (dpi)a small proportion of hamsters developed the hindlimb paralysis after sub-cutaneous infection. Hamstersinjected into the thoracic spinal cord developed ip-silateral hind limb paralysis at day 8 – 9 followedby bilateral paralysis or death by injection into thethoracic spinal cord. With the sciatic nerve injection,the hamsters developed paralysis on day 8. Confocal

microscopy was performed using Terminal deoxynu-cleotidyl Transferase Biotin-dUTP Nick End Labeling(TUNEL) and WNV and neuron specific enolase (NSE)immunohistochemistry. The virus predominantly in-fected neurons in the gray matter of the ventral horn,analogous to human infection, most particularly in thethoracic and lumbar spinal cord. Co-localized stainingof specific anti-WNV antibody, NSE and TUNEL assayand the anatomy of the cell indicated that the dyingcells were infected motor neurons. Some infected ani-mals that were not paralyzed had WNV infected cells,but TUNEL staining was not extensive. This suggestedthat motor neurons stained with WNV and TUNEL aremarkers for the progression of paralysis in WNV in-fected hamster. (Supported by PHS NO1-A1-15435 andPHS 1-U54-A1-06357 (JDM)).

P.153West Nile virus infection in central nervous systemof hamsterVenkatraman Siddharthan,1 Hong Wong,1 Ramona TSkirpstunas,2 and John D Morrey1

1Institute for Antiviral Research, Department ofAnimal,Dairy and Veterinary Sciences, Utah StateUniversity, Logan UT 84322; 2Veterinary DiagnosticLaboratory,Department of Animal,Dairy and VeterinarySciences, Utah State University, Logan UT 84322

West Nile virus (WNV) infects the Central NervousSystem (CNS) and induces a variety of neurologi-cal disease symptoms in human. To investigate theneurological disease, hamsters were sub-cutaneously(s.c.) injected using prototypic NY99 WNV strain.CNS tissues were obtained by cardiac perfusion withparaformaldehyde. Histopatholgic analysis of 3-μmthick sections revealed mild gliosis, neuropil vac-uolation, myelin sheath dilatation, axonal swelling,infiltration of lymphocytes around the vessels, andglial cuffing around neurons in brain stem area. Inspinal cord, there was a moderate bilateral ventralhorn gliosis, neuronophagia, satellitosis and hyper-eosinophilic tissues in the cervical and thoracic cord.Particularly neurons in the ventral horns were dif-fusely hypercellular, and were infiltrated by increasednumbers of glial cells and neutrophils. Inflamma-tion was most severe in cervical and thoracic seg-ments, and less severe in lumbar segments and in thebrain stem. Immunohistochemical analysis showedthat WNV focally infected the different nuclei ofbrain stem, such as lateral alpha-gigantocellular nu-clei, para-gigantocellular nuclei and lateral spinal nu-clei. In the medulla and pons of the brain, the fa-cial nuclei, parvicellular reticular nuclei, intermediatereticular nuclei, pontine reticular nucleus, paraolivarynuclei and superior olive were infected. In thoracicand lumbar spinal cord, motor neurons in the ven-tral horn were profoundly infected. In few cases, neu-rons in the dorsal horn were also infected. Apart fromthese areas, the next intensely infected areas were the


hippocampus, cornu ammonis 1, 2, 3 and dentategyrus. Neurons in the cerebral cortex and Purkinje incerebellum were also positive to a lesser extent. In thisWNV animal model, we observed a clear cytoplasmicimmuno-staining on the neuronal cell body, whetherit was a neuron in the brain stem or motor neuronin the spinal cord. Even though, the histoplathologicevidence showed the infiltration and accumulation oflymphocytes and glial cells in brain stem or mid brain,these cells were not stained for WNV. This informationmay be useful to understand the WNV induced neu-rological disease in human and animal models. (Sup-ported by PHS Contract NO1-A1-15435 and PHS 1-U54- A1-06357 (JDM)).

P.154Molecular mimicry in inducing DNA damagebetween HIV-1 Vpr and the anti-cancer agent,cisplatinKhwaja Siddiqui,1 Shongshan Fan,2 Luis Del Valle,2

Nelly Morellet,3 Jianqi Cui,2 Mohammad Ghafouri,2Ruma Mukerjee,2 Katarzyna Urbanska,2,4 ChristopherB Pattillo,5 Satish L Deshmane,2 Mohammad FKiani,5 Ramin Ansari,5 Kamel khalili,2 Bernard PRoques,3 Krzysztof Reiss,2 Serge Bouaziz,3 ShohrehAmini,1,2 Alagarsamy Srinivasan,6 and Bassel ESawaya2

1Department of Biology, College of Science and Technology,Temple University, Philadelphia, PA 19122; 2Department ofNeuroscience & Center for Neurovirology, School ofMedicine, Temple University, Philadelphia, PA 19122;3Unite de Pharmacologie Chimique et Genetique, INSERMU640, CNRS UMR 8151, 4, Avenue de l’Observatoire, 75270Paris Cedex 06, France; 4Department of Cell Biology,Faculty of Biotechnology, Jagiellonian University, Krakow,Poland; 5Department of Mechanical Engineering, TempleUniversity, 1947 North 12th Street, Philadelphia, PA 19122;6Department of Microbiology and Immunology, KimmelCancer Center, Thomas Jefferson University, Philadelphia,PA 19107

The human immunodeficiency virus type 1 (HIV-1) vprgene is an evolutionarily conserved gene among theprimate lentiviruses. Several functions are attributedto Vpr including the ability to cause cell death, cellcycle arrest, apoptosis, and DNA damage. The Vpr do-main responsible for DNA damage as well as the mech-anism(s) through which Vpr induces this damage isunknown. Using site directed mutagenesis, we iden-tified the helical domain II within Vpr (aa 37–50) asthe region responsible for causing DNA damage. Inter-estingly, Vpr ?(37-50) failed to cause cell cycle arrestor apoptosis, to induce Ku70 or Ku80, and to suppresstumor growth, but maintained its capability to activatethe HIV-1 LTR, to localize to the nucleus, and to pro-mote NHEJ. In addition, our cytogenetic data indicatedthat helical domain II induced chromosomal aberra-tions, which mimicked those induced by cisplatin, ananti-cancer agent. This novel molecular mimicry func-tion of Vpr might lead to its potential therapeutic useas a tumor suppressor.

P.155Unique role of ARMS in neurotrophin-mediatedactivation of NF-kB and neuronal protection againstHIV-1 encoded gp120Lynn F Sniderhan,1 Servio H Ramirez,1 AngelaLitzburg,1 Yuanan Lu,2 Moses V Chao,3 and Sanjay BMaggirwar1

1University of Rochester School of Medicine and Dentistry;2University of Hawaii at Manoa; 3New York UniversitySchool of Medicine

Activation of the transcription factor NF-kB is a keyfeature of the signaling triggered by neurotrophins, andthis activity has been shown to be critical for neu-ronal survival examined in in vitro models of neu-roAIDS. However, the precise mechanism by whichneurotrophins activate NF-kB is not well understood.In an effort to dissect this process, we analyzed therole of brain-derived neurotrophic factor (BDNF) andits receptor, TrkB, on NF-kB activity in cortical neu-ronal cultures. Here we show that TrkB expressionis developmentally regulated in primary cortical cul-tures, and expression levels correlate with the abilityof BDNF to activate NF-kB. This action of BDNF re-quires functional Trk since addition of K252a, a potentinhibitor of Trk, completely abrogated NF-kB activa-tion. Additional experiments were performed to un-derstand the exact mechanism of NF-kB activation byBDNF. It has been shown that Trk interaction with anaccessory protein, ankyrin-rich membrane-spanningprotein (ARMS), is necessary for persistent Rap1-dependent MAPK activation (Embo Journal, 2004, 00,1-11). We report that the activation of NF-kB by BDNFrequires normal expression of ARMS and its associ-ation with Trk. Notably, an overexpression of ARMSaugments BDNF-induced NF-kB signaling in corticalneurons, while dissociation of TrkB:ARMS interac-tion by overexpression of the membrane -spanning re-gion of ARMS blocks NF-kB signaling. These analy-ses were further extended through the generation ofan ARMS siRNA expressing lentivirus and essentiallysupported our conclusions. Finally, we examined thepotential role of ARMS in BDNF-mediated neuropro-tection. We found that ARMS is required for BDNFto protect neurons from toxicity induced by HIV-1encoded gp120. Thus, we propose that the interac-tion of TrkB with ARMS is essential for NF-kB acti-vation by neurotrophins and subsequent neuroprotec-tion against candidate HIV-1 neurotoxins.

P.156Cannabinoid rescue of striatal neurogenesis inchronic encephalitis in ratsMarylou V Solbrig, Daniel Y Chang, and NealHermanowiczDept of Neurology, University of CA-Irvine, Irvine CA USA

Although neurotropic viruses and inflammatory dis-eases target neural progenitor cells, neurogenesiscan be induced in the adult brain by various


pharmacological treatments. Cannabinoids conferneuroprotection, promote cell survival in various ex-perimental paradigms, and increase neurogenesis inhippocampus in rat models of neural injury. BornaDisease virus (BDV) infection reduces neurogenesisin hippocampus of adult rats (Solbrig et al. Brain2006), reduces numbers of BrdU labeled cells in sub-ventricular zone (SVZ) of striatum and causes dis-orderly migrations of newly generated SVZ cells toprefrontal cortex (Solbrig unpublished). The hypoth-esis that cannabinoids rescue SVZ neurogenesis dur-ing encephalitis was tested by examining the ability ofcannabinoid treatments to increase number and sur-vival of newly generated cells in the striatal complexof adult rats with BDV, using pharmacologic probes,immunohistochemistry and cell counting techniques.After BrdU labeling of mitotic cells in S phase fol-lowed by cannabinoid treatment of rats (Win-55,212-2 1mg/kg ip twice daily x 1 week), there are signifi-cant increases in survival of newly generated cells inSVZ dorsal striatum in cannabinoid-treated BDV ratscompared to BDV-infected vehicle-treated rats. BothBD groups have fewer BrdU positive cells than age-matched uninfected control rats. There were no appar-ent cannabinoid effects on cell birth in infected rats,as BDV rats pretreated x 1 week with cannabinoid orvehicle prior to BrdU administration showed no differ-ences in SVZ cell labeling the day after BrdU injection.Therefore, cannabinoids have the potential to improvestriatal motor and procedural learning functions com-promised by viral injury, not only as modulators ofneurotransmission in motor and behavior circuits, butalso by neuroprotective effects on new cell survival inthis region.

P.157Paroxetine treatment for HIV-mediatedneurodegenerationJoseph P Steiner, Carlos Pardo-Villamizar, DaniellaAsch, Norman J Haughey, Justin C McArthur, andAvindra NathDepartment of Neurology, Johns Hopkins University Schoolof Medicine

See Page 18

P.158HIV-1 tat enhances localization of smad proteins tothe JCV regulatory region and modulatestranscriptional activity in oligodendroglial cellsMichelle R Stettner,1 Jonas A Nance,1 ClaytonWright,1 Jay Rappaport,2 Kamel Khalili,2 JenniferGordon,2 and Edward M Johnson1

1Department of Microbiology and Molecular Cell Biology,Eastern Virginia Medical School, Norfolk, VA 23507;2Department of Neuroscience and Center forNeurovirology, Temple University School of Medicine,Philadelphia, PA 19122

HIV-1 infection alters pathways of production andsignal transduction of immunomodulators, including

TGF-Beta1, in the CNS. Therefore, we have examinedeffects of HIV- Tat on binding of TGF-Beta1 effectors,Smads, to the JCV transcription and replication con-trol region. Immunoblotting reveals endogenous ex-pression of Smad2/3, Smad4 and Fast1in KG-1 oligo-dendroglioma cells. Fast1 is reportedly a mediator ofDNA binding of Smad4. Of these proteins, Smad4 isdetected at significantly lower levels than the others.Even upon transfection to overexpress Smad4, thatprotein is only detectable at low levels. Yet, Smad4is readily detected on the JCV regulatory region usingchromatin immunoprecipitation (ChIP). Results so farindicate a situation in which Smad4 not bound to chro-matin is rendered less detectable, either through in-stability of the protein, its mRNA, or through maskingof epitopes. Real-time PCR allows detection of 10-16moles of a 0.3 kb segment representing the JCV controlregion in <30 cycles. ChIP demonstrates that Smad2/3,Smad4 and Fast1 each bind in living KG-1 cells to aspecific segment of the JCV control region. Smad4 andFast1 were detected on the control region at 10 to100-fold higher levels than were Smad2/3. Tat stimulatedbinding of Smad2/3 and Smad4 to the JCV control seg-ment each >10-fold and also statistically significantlystimulated binding of Fast1. In the presence of Tat lev-els of binding of Smad2/3, Smad4 and Fast1 to thecontrol region were virtually equal. At picomolar con-centrations Tat stimulates JCV early and late gene tran-scription. At high levels of overexpression, however,Tat inhibits early gene transcription. This inhibition isreversed by expression of Smad2, 3 and 4, which stim-ulate transcription more than 1000-fold. The presentresults reveal a mechanism by which HIV-1, throughinteraction with immunomodulators, can significantlyinfluence the infection of oligodendroglial cells by JCV.

P.159Histone deacetylase inhibitors suppress the astrocyteinnate immune responseHyeon-Sook Suh, Shinyeop Choi, Meng-Liang Zhao,Celia F Brosnan, and Sunhee C LeeAlbert Einstein College of Medicine, Bronx, NY

Astrocytes are important modulators of immune andinflammatory reactions in the CNS participating inboth innate and acquired immune responses. As-trocytes express certain members of the toll-like-receptor (TLR) family, in particular, the receptor fordouble stranded RNA (dsRNA), TLR3. The syntheticTLR3 ligand, polyinosinic-polycytidylic acid (PIC),is a potent activator of human astrocytes resultingin the activation of specific sets of transcription fac-tors (IRF3 and NF-κB) that leads to the induction ofcytokines, chemokines and antiviral proteins. Acety-lation of various histones is a posttranslationalmodification involved in the regulation of gene ex-pression or silencing. Histone acetylation is reg-ulated by histone acetyl transferases (HAT) andhistone deacetylase (HDAC). In general, HAT in-duces gene transcription, whereas HDAC suppresses


the transcription. Surprisingly, recent studies havedemonstrated that type I IFN (IFNalpha)-stimulatedanti-viral gene expression is downregulated by thegeneral HDAC inhibitors such as valproic acid (VA) ortrichostatin A (TSA). Using a well-established modelof primary human glial cell cultures, we investigatedthe effect of TSA and VA on PIC-induced gene ex-pression. We hypothesized that HDAC inhibitors willinhibit transcription of genes involved in innate im-mune response by inhibiting the activation of thecritical transcription factor IRF3. Our results showthat TSA and VA suppress the PIC-induced expres-sion of cytokines, chemokines and antiviral proteinsin astrocytes. TSA and VA also suppressed the ex-pression of IFNbeta as well as IRF7 and TLR3, im-plying IFNbeta as the converging point. These datasuggest that the site of inhibition is in the TLR3signal cascade upstream of IFNbeta production. Re-markably, microarray analysis demonstrated that theproteins belonging to the IFN-inducible gene familyare globally restricted and are among the most severelydownregulated genes. Our results have clinical impli-cations as they predict that the clinical use of HDACinhibitors will lead to a compromised innate im-mune system. (Supported by MH55477, NS07098 andAI051519)

P.160HIV/HCV co-infection has a monocyte phenotypethat induces MIP3alpha and reactivates MCP-1Bing Sun,1 Cyrus Calosing,1 Sue Currie,1 JamesRyan,1,2 and Lynn Pulliam1,2,3

1Veterans Affairs Medical Center, San Francisco, CA;2Department of Medicine, University of California, SanFrancisco; 3Department of Laboratory Medicine, Universityof California, San Francisco

Nearly 24% of HIV-infected veterans on highly activeantiretroviral therapy (HAART) are co-infected withHCV. While neuropsychological (NP) consequences inHIV infection are well established, HCV-infected sub-jects also have NP abnormalities and co-infection maycause a greater impairment of NP functions. Sincemonocytes normally traffic into the brain and activatedmonocyte/macrophages significantly impact cognitionin HIV infection, we explored monocyte gene expres-sion profiles from subjects with HIV/HCV co-infectionand HCV mono-infection.

Four HIV/HCV co-infected (HIV, low viral load,[LVL], <100 copies/mL; HCV >200,000 IU/mL), 4HCV mono-infected (>200,000 IU/mL), 17 HIV mono-infected (LVL) and 7 healthy controls were en-rolled. Total RNA from CD14+ monocytes was hy-bridized to microarrays. Data were analyzed usingR/BioConductor and GeneSpring.

HCV mono-infection induced 550 differentially ex-pressed (DE) genes. Co-infection induced 736 DEgenes, which included MIP-3alpha (CCL20), MCP-1(CCL2), CD83, IL6, and IP10 (CXCL10). CCL20 was

up regulated >100 fold in HCV mono-infected and inHIV/HCV co-infected subjects but not in HIV mono-infected subjects. CCL20 has been implicated in en-hanced inflammatory cell recruitment to the liver inHCV subjects. CCL20 can be produced by astrocytesduring inflammatory conditions in the CNS, where itmay play a role in monocyte and dendritic cell recruit-ment. CCL2 was up-regulated 3 fold in co-infectionbut not in LVL HIV or HCV mono-infected subjects.CCL2 appears to be reactivated by HCV infection inHIV-infected subjects with LVL.

HIV/HCV co-infection altered the monocyte pheno-type compared to HIV or HCV mono-infection. Thissynergistic effect may play a role in NP impairmentseen in HIV/HCV co-infected subjects. CCL20 plusCCL2 induction may cause an increase in monocytechemotaxis to the CNS.

P.161Biological effects of Natalizumab on JC virusinfectivity in its permissive human neural cell linesin vitroTadaki Suzuki,1,2 Satoko Yamanouchi,1 YujiSunden,3,4 Yasuko Orba,2,5 Shinya Tanaka,5 TakashiKimura,1 and Hirofumi Sawa1,4

1Department of Molecular Pathobiology, HokkaidoUniversity Research Center for Zoonosis Control; 2Researchfellows of the Japan Society for the Promotion of Science;3Laboratory of Comparative Pathology, HokkaidoUniversity School of Veterinary Medicine; 421st CenturyCOE Program for Zoonosis Control, Hokkaido University;5Laboratory of Molecular & Cellular Pathology, HokkaidoUniversity School of Medicine

The human Polyomavirus JC virus (JCV) infects the 70-80% of humans and establishes latent infection in thekidney. In immunosuppressed patients JCV can reac-tivate and result in a fatal and progressing neurologi-cal disease known as progressive multifocal leukoen-cephalopathy (PML). Over the past three decades, PMLhas become one of the important neurological com-plications in acquired immunodeficiency syndrome(AIDS) patients. Recently, it has been reported that pa-tients being treated with therapeutics targeted at theintegrin, very late antigen (VLA)-4, are unexpectedlyfound to have an increased risk for development ofPML. However, there have not been interpreted deter-minately the relationship between sideration of PMLand natalizumab. Therefore, we have investigated thebiological effects of Natalizumab on the growth of JCVin its permissive human neural cells, IMR-32 cells withinoculation of the virus. Initially we have determinedthe effective concentration of Natalizumab using thecell adhesion assay in the IMR-32 cells. Natalizumabdid not affect on the expression levels of the viral pro-teins by immunblotting using the specific antibodies,nor hemmaglutination activities of the cellular lysatesfrom the infected cells. These results suggested thatNatalizumab did not have an influence on JCV infec-tivity in IMR-32 cells in vitro.


P.162JC virus agnoprotein modifies plasma membranepermeability and facilitatesTadaki Suzuki,1,2 Yuki Okada,3 Yasuko Orba,2,3 YujiSunden,4,5 Takashi Kimura,1 Kazuo Nagashima,3 andHirofumi Sawa1,5

1Department of Molecular Pathobiology, HokkaidoUniversity Research Center for Zoonosis Control; 2Researchfellows of the Japan Society for the Promotion of Science;3Laboratory of Molecular & Cellular Pathology, HokkaidoUniversity School of Medicine; 4Laboratory of ComparativePathology, Hokkaido University School of VeterinaryMedicine; 521st Century COE Program for ZoonosisControl, Hokkaido University, Sapporo 060-0818, Japan

Viruses inflict a number of injuries as they infect sus-ceptible cells. Some of these injuries affect the plasmaand intracellular membranes, resulting in modifica-tion of their functions. A typical feature observed dur-ing the animal virus infection is enhanced permeabil-ity of infected cells. Viroporins are a group of proteinsthat participate in the promotion of release of viral par-ticles from cells, and interact with cellular membranesmodifying permeability. These proteins are not essen-tial for the replication of viruses, but their presenceenhances virus growth.

Progressive multifocal leukoencephalopathy (PML)is a fatal demyelinating disease resulting from lyticinfection of oligodendrocytes by the polyomavirus JCvirus (JCV). The genome of JCV encodes six major pro-teins including agnoprotein. Previous studies from ourand other laboratories indicated that the JCV smallauxiliary protein, agnoprotein, plays an important,though not fully understood, role in the propagationof JCV.

Agnoprotein encoded in the late coding region ofJCV is a highly basic protein and contains a hydropho-bic domain in the central portion of the protein.

Here, we demonstrate that agnoprotein possessesproperties commonly associated with viroporins. Ourfindings indicate that: (i) Agnoprotein localizes to theER, Golgi apparatus, and plasma membrane in trans-fected cells; (ii) Agnoprotein can homo-oligomerize intransfected cells; (iii) Agnoprotein enhances perme-ability of cells to the translation inhibitor hygromycinB; (iv) Agnoprotein induces the influx of extracellularCa2+; (v) The N-terminal basic amino acid residues ofagnoprotein are critical for intracellular membrane tar-geting and modification of the membrane permeability.These viroporin-like properties which enhances mem-brane permeability, thereby disrupting the intracellu-lar Ca2+ homeostasis and ultimately causing the dys-function of the membrane may contribute to the role ofagnoprotein in virus release and may have importantimplications for pathogenicity of JCV infections.

P.163Anti viral and anti inflammatory effect of rosmarinicacid in an experimental murine model of Japaneseencephalitis

Vivek Swarup and Anirban BasuNational Brain Research Centre, Manesar, India

Flaviviruses are important human pathogens causinga variety of diseases ranging from mild febrile ill-nesses to severe encephalitis and hemorrhagic fever.Among them, Japanese encephalitis virus (JEV) tar-gets the central nervous system (CNS) and is a ma-jor cause of acute encephalopathy in children. Ros-marinic acid (RA) a phenolic compound found in var-ious Labiatae herbs, such as Ocimum basilicum In thepresent study, we investigated the efficacy of RA asa therapy against murine JE. Four to five weeks oldBALB/c mice of either sex were infected with intra-venous (through tail vein) inoculation of lethal doseof 3x105 p.f.u of JE virus strain GP78). After one dayfollowing virus inoculation animals started receivingRA intra-peritoneally, twice daily (25 mg/kg of bodyweight) until the first animal died from the group ofinfected animals, which did not receive any RA treate-ment. Rosmarinic acid (RA) reduced the mortality ofadult BALB/c mice infected with Japanese Encephali-tis virus (JEV). ). RA treatment completely reduced thepresence of viral protein and significantly reduced theviral transcripts as compared to infected animal with-out any treatment. RA also decreased the level of proin-flammatory mediators like TNF-a, IL-12, IFN-g, IL-6and MCP-1 (p < 0.001) in infected animals. Thus RApresents a potential for therapy for JE.

Acknowledgement: This work was supported bygrant no BT/PR/5799/MED/14/698/2005 from the De-partment of Biotechnology (DBT), Government of In-dia to A.B. and a core grant from DBT to National BrainResearch Center.

P.164Molecular mechanism of neuronal apoptosis inJapanese encephalitisVivek Swarup, Sulagna Das, and Anirban BasuNational Brain Research Centre, Manesar, India

Japanese Encephalitis (JE) is an acute viral infectionof the central nervous system caused by a mosquito-borne flavivirus called JE virus. JEV is a neurotropicvirus, which causes apoptosis in immature neurons.The molecular cascades of neuronal death followingJEV infection are not known in literature. In this work,we investigated the molecular events leading to JEVinduced neuronal apoptosis. JEV induced apoptosis isinitiated by activating membrane-bound tumor necro-sis factor receptor-1 (TNFR1/CD120b). The adapterprotein TNFR1-associated death domain (TRADD),which is an essential component of TNFR1 complex, isinvolved in recruiting other members of the complexto the receptor. TNFR-associated factor (TRAF2) is oneon these proteins and is involved in initiating theapoptosis cascade. Overexpression of TRADD can ini-tiate neuronal apoptosis even in the absence of TNF-a.Since TRADD-knockout animals or deficient cell linesare unavailable, it has been difficult to definitivelyaddress the physiological role of TRADD in diseases


pathology following JEV infection. We circumventedthis problem by silencing TRADD expression withsmall-interfering RNA (siRNA). We have found thatfollowing in vitro infection with JE virus, TRADD isrequired for TNFR-1 initiated neuronal apoptosis. In-terestingly, siRNA against TRADD also decrease the vi-ral load in Neuro2a cells. Furthermore, siRNA againstTRADD increased the survival of JEV infected miceby altering the expression of pro apoptotic versus antiapoptotic molecules. These studies show that TNFR-1 TRADD engagement following JEV infection playsa crucial role in neuronal apoptosis. Overexpressionof the TRAF2 stimulates Apoptosis Signaling Kinase-1 (ASK1, which initiates the signaling leading to theactivation of caspase-3, DNA fragmentation and ulti-mately cell death. Increased neuronal death then trig-gers microglial activation and increased microvascu-lar permeability. While Blood Brain Barrier disruptionleads to the entry of leukocytes into brain, our stud-ies indicate that this phenomenon can be preventedin TRADD siRNA mice. TRADD siRNA mice also hadreduced levels of inflammation and reduced astroglio-sis.

Acknowledgement: This work was supportedby grants awarded to AB from the Depart-ment of Biotechnology, Government of India:Award # BT/PR/5799/MED/14/698/2005 andBT/PR8682/Med/14/1273/2007.

P.165A Prospective cohort study in JCV and nonJCV-related leukoencephalopathies in hiv patients:clinical and MRI findingsEleonora Tavazzi,1 Pasquale Ferrante,2 LorenzoMinoli,3 Serena Del Bue,2 Renato Maserati,3 StefanoNovati,3 Poloni Guy,1 Stefano Bastianello,1 andEnrico Marchioni1

1IRCCS Foundation Neurological Institute C. Mondino,Pavia, Italy; 2San Giuseppe Hospital, Milano, Italy; 3IRCCSPoliclinico S.Matteo, Pavia, Italy

Objective: to describe the clinical and radiologicalfeatures at the diagnosis and during the follow-upof a cohort of HIV related Leukoencephalopathies(LE).Background: The spectrum of LE in HIV+ patientshas changed in the recent years due to multiple rea-sons directly or indirectly linked to the introductionof highly active antiretroviral therapy (HAART). Inboth JCV and non-JCV related LE the clinical features,the survival time and the possible role of immune-reconstitution are discussed. Methods: in 2002 westarted the enrolment of HIV+ patients with at least7 years of disease; 185 patients underwent brain MRI.Twentyseven patients (16%) showing non opportunis-tic CNS involvement, underwent the full clinical, neu-ropsychological, immunological and virological CSFand serum analyses, MRI assessment at the diagnosisstage and during the follow-up. Depending on the de-tection of JCV in the CSF, 14 were diagnosed as Not

Determined LE (NDLE), 13 as Progressive MultifocalLeukoencephalopathy (PML). Results: Median follow-up was 27 months. All patients were HAART treated.We classified both NDLE and PML in different swub-groups according to the disease course. Different MRIpatterns, according to the presence and the anatom-ical distribution of lesions, were identified: multifo-cal, inflammatory, diffuse and vascular-like. Moreover,non conventional MRI techniques (NCMT) were ap-plied (spectroscopy MRI and diffusion weighted imag-ing), in order to detect microscopic damage of thebrain tissue, and to possibly identify distinctive fea-tures of PML and NDLE. DWI showed a diffuse whiteand grey matter damage, more severe in HIV patientsthan in healthy subjects (HS), and in HIV patientswith leukoencephalopathy (HIV LE+) than in HIV pa-tients without leuokoencephalopathy (HIV LE-). Con-cerning spectroscopy, all HIV LE+ showed a loweredN-Acetyl Aspartate/Creatine ratio in frontal white mat-ter and thalamus with respect to HIV LE- and HS; adecreasing gradient of severity from PML to HIV LE-was demonstrated. No difference between HS, HIVLE-, HIV LE+ was found regarding Choline/Creatineratio.

Conclusions: In our series, the amount of atypicalPML cases with good outcome is surprisingly highand can occur in every stage of HIV infection. Thesepreliminary data suggest that the slow PML variantshould be distinguished from the PML with benignoutcome, the former being related to an intrinsic fea-ture of the disease, the latter being treatment-related.Finally, PML show a worse phenotype than NDLE.NCMT could be a useful tool in detecting early involve-ment of the brain tissue, and in understanding thepathogenetic mechanisms underlying different formsof LE.

This study is supported by GRANT R01MH72528from NIH to Pasquale Ferrante.

P.166Inhibition of HIV-1 infection by NB325 may beindependent of the neuroglial functions of thechemokine receptor CXCR4Nina Thakkar,1 Shendra Passic,1 TinaKish-Catalone,1 Brian Wigdahl,1 Robert F. Rando,2Mohamed Labib,2 and Fred C. Krebs1


Within the central nervous system (CNS), CXCR4 andits ligand SDF-1alpha (stromal cell derived factor-1alpha) participate in neurogenesis, neuronal survival,and axonal pathfinding; inhibition of HIV-1 infectionby a CXCR4 inhibitor may adversely affect normal neu-ronal functions. For this reason, chemokine receptor(CCR) inhibitors developed for use in the CNS shouldspecifically inhibit HIV-1 infection while permittingnormal CCR functions. Our efforts in this area havefocused on compounds that inhibit HIV-1 infection


by interfering with interactions between the virus andcell surface proteins. NB325 (a biguanide-based com-pound) is characterized by low toxicity and consider-able activity against HIV-1 IIIB, which uses CXCR4 asa viral co-receptor. The particular potency of NB325in the presence of both virus and target cells led usto hypothesize that NB325 may interfere with viralbinding and entry mechanisms. Flow cytometric as-says demonstrated that NB325 altered the detection ofCXCR4 on activated primary human CD4+ T lympho-cytes in an epitope-specific manner. Further mecha-nistic studies demonstrated that NB325 (i) did not in-hibit CXCR4 binding by its sole ligand, SDF-1alpha, (ii)did inhibit SDF-1alpha-induced chemotaxis, (iii) wascompetitively inhibited from binding CXCR4 by a pep-tide derived from CXCR4 extracellular loop 2 (ECL2),and (iv) did not induce CXCR4 internalization. Ongo-ing investigations will further explore the mechanismof NB325 activity and its impact on normal CXCR4functions in both immune and neuroglial cells. Thesestudies will facilitate the development of novel com-pounds that can be used safely in the CNS to inhibitHIV-1 infection.

P.167The HIV-1 infected monocyte-derived macrophageproteome: Analysis following infection with primaryviral isolates from infected women with cognitiveimpairmentDianedis M Toro,1 Juliana Perez Laspiur,1 PawelCiborowski,2 Fenghai Duan,2 Valerie Wojna,1 HowardE Gendelman,2 and Loyda Melendez1

1University of Puerto Rico-Medical Sciences Campus, SanJuan PR; 2University of Nebraska Medical Center, OmahaNE

The signature for HIV-1 neurovirulence remains asubject of intense debate. One hypothesis revolvesaround the idea that specific viral strains can inducerobust neurotoxic secretions from monocyte-derivedmacrophages (MDM) and as such incite progressiveneurodegeneration. We hypothesize that neuroviru-lent HIV-1 variants will influence the macrophageproteome and as such affect the onset and progres-sion of NeuroAIDS. To test this hypothesis, primaryHIV-1 isolated from blood of women with cogni-tive impairment (CI) were compared to isolates withidentical replication properties but from noncognitiveimpaired (NC) individuals. The neurotropic isolate,SF-162 served as a control. HIV-1 isolates were inoc-ulated onto MDM and viral replication tested by lev-els of HIV-1 p24 antigens in culture fluids. Uninfectedand HIV-1 infected MDM were lysed at 14 days post-infection, fractionated, analyzed by surface enhancedlaser desorption ionization time of flight (SELDI-TOF)for protein profiling. Spectral peaks were assessed byGeneralized Estimated Equations statistics. Gel cubeswere cut from 1D-SDS PAGE and proteins identified

by tandem mass spectrometry (LC-MS/MS). Levels ofHIV-1 replication were similar amongst isolates, al-though the highest levels of viral replication were seenfrom isolates obtained from patients with CI. Signif-icant differences were found in protein profiles be-tween MDM and MDM infected with NC, CI and SF-162 (q < 0.10). We identified 6 and 7 proteins uniqueto NC and SF-162. Three proteins were found com-mon to SF-162 and CI strains, and 20 to CI HIV-1.The MDM proteins linked to viral replication with CIstrains were related to redox, apopototic processes, sig-naling, and intracellular trafficking. We conclude thatdifferent viral isolates may influence the macrophagesproteome.

P.168Neutralizing antibody to interferon-alpha treatmentin SCID mice with HIV encephalitisWilliam R Tyor,1,2 Andrew R Sas,1 and HeatherBimonte-Nelson3

1Medical University of South Carolina, Department ofNeurosciences; 2Ralph H. Johnson VAMC, Charleston, SC;3Arizona State University, Department of Psychology

The pathogenesis of HIV associated dementia (HAD)is incompletely understood. Since highly active an-tiretroviral therapy is only partially effective, newtreatments are needed based on delineation of HADpathogenesis. It is hypothesized that HIV infectedcells produce putative neurotoxins that damage neu-rons. Interferon Alpha (IFNa) is a pleomorphic cy-tokine produced by nucleated cells in response toviral infection. Treatment with IFNa in patients hasside effects including cognitive impairment resem-bling subcortical dementia, which is the hallmark ofHAD. It has been shown that IFNa is increased inthe cerebrospinal fluid of HIV dementia patients com-pared to HIV patients without dementia symptoms.We hypothesized that the inhibition of IFNa in thecentral nervous system (CNS) would ameliorate thecognitive dysfunction function and pathological ab-normalities seen in SCID mice with HIV encephali-tis. In addition, we have shown a correlation betweenIFNa in the CNS and cognitive performance in thismodel with higher levels of IFNa correlating with in-creased cognitive deficits during testing. In this study,HIV mice treated with intraperitoneal (i.p) injectionsof IFNa neutralizing antibodies demonstrated signifi-cantly improved cognitive function (p < 0.05) as de-termined in the water radial arm maze compared toHIV mice that received either isotype matched con-trol antibody treatment or a saline injection. Patho-logical analysis showed presence of IFNa neutralizingantibodies in the brain. Anti- IFNa antibody treatedHIV mice exhibited decreased microgliosis (p < 0.05)compared to HIV mice with control antibody or salineinjections. Also, anti- IFNa antibody treated miceshowed improvements in loss of dendritic arborization


surrounding HIV infected cells compared to other HIVinfected mice.

P.169The signaling cooperation between IGF-IR andEGF-R in human glioblastoma cell lines withdifferent PTEN backgroundKatarzyna Urbanska, Paola Pannizzo, Luis Del Valle,Kamel Khalili, and Krzysztof ReissCenter for Neurovirology, Department of Neuroscience,Temple University, School of Medicine, Philadelphia, PA

Glioblastoma is the most aggressive and frequent braintumor, which accounts for approximately 25% of allprimary intracranial neoplasms. Despite recent ad-vances in surgical and clinical sciences, glioblastomasare always associated with poor prognosis. Patientswith glioblastoma have a median survival of one yeardespite of aggressive therapies, and fever than 5%will survive 5 years. The important characteristic ofglioblastoma is its unusual ability to invade surround-ing brain tissues, and its autonomy in respect to growthfactors requirements, the feature which is often asso-ciated with PTEN mutations. In this study we evalu-ate two tyrosine kinase receptors, IGF-IR and EGF-R,and their possible cooperation in supporting growth,survival and invasiveness of glioblastoma cell lines,characterized by different PTEN background. We haveutilized five human glioblastoma cell lines: U-87 MGand U-118 MG lacking PTEN expression; LN-18 andLN229, which express wt PTEN; and T98G, which ex-press mutated PTEN. We have observed that in theabsence of functional PTEN, levels of expression ofIGF-IR, EGFR, as well as the major IGF-IR signalingmolecule, IRS-1, are about 10 fold lower in compar-ison with cells expressing PTEN. Despite that, IGF-I or EGF stimulation mediated similar responses atthe levels of Akt, Erks and GSK3beta phosphoryla-tion in all cell lines examined. Another observationis that independently of the PTEN background EGFis more effective than IGF-I in phosphorylating ERKs,and IGF-I seems to be more efficient in Akt phospho-rylation. Interestingly, all five cell lines have simi-lar growth requirements. They grow exponentially inthe presence of 10%FBS, slow down substantially inserum-free medium, and surprisingly, do not respondwell with increased cell proliferation when stimu-lated singly by IGF-I or EGF. In contrast, the addi-tion of both IGF-I and EGF strongly supported cellproliferation in the absence of serum. In a similarmanner, single inhibition of IGF-IR by NVP-AEW541(Novertis), or inhibition of EGFR by PD153035 (Cal-biochem) had only marginal effects on cell survival,or on the inhibition of cell proliferation. In contrast,combination of NVP-AEW541 and PD153035 appliedat relatively low doses inhibited serum -dependentcell proliferation, and efficiently challenged survival

of glioblastoma cells in anchorage-independence. Insummary, our results indicate that in five humanglioblastoma cell lines examined there is a signalingsynergy between IGF-IR and EGFR, which supportsmalignant growth of glioblastoma, and that the ab-sence of functional PTEN, although decreases growthfactors requirements, does provide growth factorsindependence.

P.170Dysregulation of Cu/Zn superoxide dismutase-1 inthe CSF and monocytes of Hispanic women withcognitive impairmentIxane Velasquez,1 Marines Plaud,1 Juliana PerezLaspiur,1 Valerie Wojna,1 Richard Skolasky,2 PawelCiborowski,4 Howard E Gendelman,4 andLoyda M Melendez1

1University of Puerto Rico Medical Sciences Campus;2Johns Hopkins University; 3University of Nebraska;4University of Nebraska Medical Center

Cognitive impairment (CI) occurs in advanced stagesof HIV-infection and despite the wide spread use ofantiretroviral medicines. Disease is linked to activa-tion of brain mononuclear phagocytes (MP; perivas-cular blood-borne macrophages and microglia) thatresults in neuroinflammation and secretion of toxicsubstances. These include pro-inflammatory cy-tokines, chemokines and immune products that affectoxidative stress and neuronal death. Our prior workshave demonstrated that alterations in the macrophageand CSF proteome are associated with CI. These stud-ies revealed that among other candidates, CSF Cu/ZnSuperoxide Dismutase (SOD-1) in HIV-1 seropositiveHispanic women with CI. Since MP activation is ahallmark of neuronal injury in HIV associated CNSdisease, we tested the hypothesis that SOD-1 is dys-regulated in the CNS, and HIV-infected macrophagescontribute to this effect during CI. For this purposeSOD-1 expression was measured in CSF, monocytes,monocyte-derived macrophages (MDM), and plasmafrom a group of HIV seropositive Hispanic womencharacterized for cognitive function by ELISA andWestern blots. SOD activity was measured in the CSFfrom 32 patients by a colorimetric assay based on inhi-bition of xanthine oxidase by SOD (Calbiochem). Wefound that SOD-1 expression was not significantly dif-ferent in the CSF and macrophages of women with CIcompared with NC. When stratified by the degree of CI,we found that SOD expression in the CSF decreasedin asymptomatics (A) compared to normal cognition(ND) and HIV associated dementia (HAD), while SODactivity in the CSF increased in A compared to ND andHAD (p < 0.5). These results indicate that there is dys-regulation of SOD-1 in the CNS with increased activityin early stages of CI.


P.171Lipopolysaccharide protects against neurologicaldisease in cats infected with felineimmunodeficiency virusSerena Viappiani, Amir Afkhami-Goli, Yu Zhu, andChristopher PowerDepartment of Medicine, University of Alberta

Infection by the human immunodeficiency virus (HIV)results in neurodegeneration caused by aberrant acti-vation of glial cells with ensuing neuronal death. Sincepatients with HIV/AIDS often develop opportunisticinfections, we studied the role of systemic immune ac-tivation on neurological function and viral replicationin the brain. For this purpose, we repeatedly admin-istered lipopolysaccharide (LPS), a component of themembrane of gram-negative bacteria, to cats infectedwith feline immunodeficiency virus (FIV), as an an-imal model of HIV encephalopathy. After behavioralassessment, animals were sacrificed and proteins andmRNA were extracted from cerebral cortex and basalganglia, for immunoblotting of neuronal markers andRT-PCR analyses of genes for inflammatory cytokinesand neurotrophic factors.

FIV-infected cats treated with LPS developed lesssevere neurobehavioral deficits (mean deficit score1.4 ± 0.2 in FIV-LPS group versus 3.6 ± 0.2 in FIVgroup, p < 0.05) and showed lower brain viral loadthan the FIV-infected control group. Moreover, we ob-served reduced loss of the presynaptic protein synap-tophysin and the vesicular acetylcholine transporter,and up-regulation of the mRNA for brain-derived neu-rotrophic factor (BDNF) in the basal ganglia, despitehigh levels of the pro-inflammatory cytokine IL-1beta.Other neurotrophins (IGF-1, GDNF), as well as themannose receptor, were not affected by LPS treatment.

Thus, systemic LPS improved neurological func-tion and synaptic architecture in FIV-infected animalsthrough a mechanism involving increased expressionof BDNF and reduced viral replication. Our findingsemphasize the beneficial aspects of selective activa-tion of innate immunity in neurological disease.

P.172Expression of CXCL10 from the mouse hepatitisvirus genome results in protection fromviral-induced neurological and liver diseaseKevin B Walsh,1 Robert A Edwards,2 Kimberley MRomero,1 Matthew V Kotlajich,1 Stephen AStohlman,3 and Thomas E Lane1

1Department of Molecular Biology & Biochemistry,University of California, Irvine; 2Department of Pathology,University of California Irvine, School of Medicine;3Department of Neurology, University of SouthernCalifornia, Keck School of Medicine

Using a recombinant murine coronavirus (mousehepatitis virus, MHV) expressing the T cell-chemoattractant CXCL10 (MHV-CXCL10), wedemonstrate a potent antiviral role for CXCL10

in host defense. Instillation of MHV-CXCL10 intothe central nervous system (CNS) of CXCL10 de-ficient mice (CXCL10−/− mice) resulted in viralinfection and replication in both brain and liver.Expression of virally-encoded CXCL10 within thebrain protected mice from death and correlated withincreased infiltration of T lymphocytes, enhancedIFN-? secretion, and accelerated viral clearance whencompared to mice infected with an isogenic controlvirus, MHV. Similarly, viral clearance from the liversof MHV-CXCL10-infected mice was accelerated incomparison to MHV-infected mice, yet was indepen-dent of enhanced infiltration of T lymphocytes andnatural killer (NK) cells. Moreover, CXCL10-/- miceinfected with MHV-CXCL10 were protected fromsevere hepatitis as evidenced by reduced pathologyand serum alanine aminotransferase (sALT) levelscompared to MHV-infected mice. CXCL10-mediatedprotection within the liver was not dependent onCXC-chemokine receptor 2 (CXCR2) signaling asanti-CXCR2 treatment of MHV-CXCL10-infected micedid not modulate viral clearance or liver pathology.In contrast, treatment of MHV-CXCL10-infectedCXCL10−/− mice with anti-CXCL10 antibody re-sulted in increased clinical disease correlating withenhanced viral recovery from the brain and liver aswell as increased sALT levels. These studies high-light that CXCL10 expression promotes protectionfrom coronavirus-induced neurological and liverdisease.

P.173E2F1 expression is changed by activation of theNMDA receptor pathway in an in vitro model of HIVAssociated DementiaYing Wang, Cagla Akay, Michael G White, Kathryn ALindl, Rebecca Chodroff, Marc A Dichter, MargaretMaronski, Dennis L Kolson, and Kelly LJordan-SciuttoUniversity of Pennsylvania

In the pre-HARRT era, about 15∼25% of HIV infectedindividuals develop HIV associated dementia (HAD),which is characterized by neuronal and dendritic dam-age, neuronal loss, microgliosis and astrogliosis in thecentral nervous system. Although neurons are dam-aged in HIV infected patients, HIV does not infectneurons directly. Instead neuronal damage is medi-ated by neurotoxic factors from infiltrating, activatedand /or infected macrophage/microglia. Neuroinflam-mation and the associated oxidative stress seen inpatients with HAD is also seen in other neurode-generative diseases including Alzheimer disease andParkinson disease. Interestingly, the pro-apoptotic cellcycle protein, E2F1, exhibits increased immunoreac-tivity and altered subcellular distribution in severalneurodegenerative diseases including Alzheimer’sdisease, Parkinson’s disease, Amyotrophic LateralSclerosis and HIV encephalitis. However, in these


diseases E2F1 localized predominantly to the cyto-plasm, a localization distinct from its well-defined nu-clear role as a transcription factor during apoptosisand cell cycle progression. Based on these observa-tions we hypothesized that cytoplasmic E2F1 mightalso contribute to neuronal death in HAD. We usedan in vitro model of HAD in which we treated pri-mary rat neuroglial cultures with supernatants fromhuman monocyte-derived-macrophages infected witha neurovirulent strain of HIV-1 (HIV M/M). Unexpect-edly, using a concentration of HIV M/M that produced50% neuronal loss by 20 hours, we observed a lowermolecular weight E2F1 expression, accompanied byincreased calpain activation by western blotting. Be-sides, this generation of lower band E2F1 was reversedby NMDA receptor antagonist and Calpain inhibitor.Furthermore, both of the mRNA and protein expres-sion of E2F1 targets didn’t change in the in vitro model.And there is no change of E2F1 targets protein ex-pression in the mid-frontal cortices of patients withHIV and those with HIV associated cognitive impair-ment. Based on our in vitro observations, we proposethat the generation of lower band of E2F1 is medi-ated via the activation of NMDA pathway, probablythrough Calpain, but whether this change of E2F1 hasits functional significance is still needed to be furtherexplored.

P.174CART results in decreased brain virus burden andreversal of neuronal injury in treated SIV-infectedCD8-depleted rhesus macaquesSusan V. Westmoreland,1,2 LakshmananAnnamalai,1,2 Eva Ratai,3,4,5 Margaret R. Lentz,3,4,5

Kenneth C. Williams,6,7 Gilberto Gonzalez3,4,5 and1Harvard Medical School2New England Primate Research Center; 3MassachusettsGeneral Hospital; 4Department of Neuroradiology; 5A.A.Martinos Center for Biomedical Imaging; 6Beth IsraelDeaconess Medical Center; 7Division of Viral Pathogenesis

The clinical entity of HIV-associated dementia is dueto a combination of neuronal injury, dysfunction, andloss, which may be partially reversible. Although an-tiretroviral drugs have been shown to be effective indecreasing the HIV/SIV burden in plasma, the effectof these drugs on HIV/SIV replication in the brain isnot clearly known. Because many antiretroviral agentsdo not cross the blood-brain barrier (BBB), it has beensuggested that the brain may serve as a proviral reser-voir in HIV patients treated with antiretroviral agents.To quantitatively determine the effect of short-term an-tiretroviral therapy on virus replication and neuronalinjury in different regions of brain we used QC realtime RT-PCR to accurately analyze brain virus bur-den in eight rhesus macaques that were inoculated in-travenously with SIVmac251 and CD8-depleted. Fouranimals were treated with CART, comprised of dailysubcutaneous injections of PMPA (30 mg/kg) and RCV

(10 mg/kg), beginning 28 days post-inoculation (dpi)and continuing until the animals were sacrificed at 57dpi. Neither PMPA nor RCV cross the BBB. Untreatedanimals were sacrificed when moribund with AIDS (57to 85 dpi). Parenchymal brain virus burden was quan-tified in the frontal cortex, putamen, hippocampus andbrain stem. Significantly less virus replication was ob-served in frontal cortex of animals that were treatedwith CART compared to those that were untreated (me-dian values of 3.7 vs. 5.1 log10 SIV RNA copies/mg, re-spectively; p < 0.03). The median virus burdens werelower in the CART animals than untreated macaquesin putamen, hippocampus, and brain stem, as well (5.0vs. 5.8, 5.1 vs. 5.4, and 4.3 vs. 5.5, respectively); how-ever, the differences were not statistically significantin these regions. Longitudinal and terminal magneticresonance spectroscopy (MRS) analysis of NAA/Crfrom frontal cortex demonstrated an inverse relationwith increasing NAA/Cr and decreasing brain virusburden in treated animals (mean NAA/Cr untreated0.666 ± 0.137, treated 0.707± 0.103). Taken together,these data suggest that short-term systemic antiretrovi-ral agents are effective in reducing replicating virus inthe brain, and can result in partial reversal of neuronalinjury.

P.175Role of C/EBP-beta isoforms in JC virus latency andreactivationMartyn K White, Luca Romagnoli, Ruma Mukerjee,and Kamel KhaliliCenter for Neurovirology, Department of Neuroscience,Temple University School of Medicine, Philadelphia, PA,USA

The human neurotropic JC virus (JCV) causes the fataldemyelinating disease progressive multifocal leukoen-cephalopathy (PML) when it reactivates from a la-tent state to destroy oligodendrocytes in the brain.It is thought that this reactivation may involve pos-itive regulation through cellular transcription factorsthat become activated by the action of cytokines. TheJCV non-coding control region (NCCR) contains el-ements that regulate transcription including an NF-kappa-B binding site (kappa-B element) that maybe involved in this transcriptional activation. Wenow report that isoforms of the C/EBP-beta transcrip-tion factor, especially the LIP isoform, also bind tothe kB element but that this binding inhibits bothbasal and NF-kappa-B-stimulated JCV transcription.Gel shift analysis showed that C/EBP-beta LIP boundthis region in vitro but not to other regions of theNCCR. ChIP assays confirmed LIP binding in vivo.Co-immunoprecipitation experiments with oligonu-cleotides to this site indicated the formation of ternarycomplexes of NF-kappa-B p65, C/EBP-beta LIP andJCV DNA. Mutagenesis analysis of the JCV kappa-B ele-ment indicated that p65 and LIP bound to adjacent butdistinct sites both of which are required for basal and


p65-stimulated transcription. Finally, immunohisto-chemistry for C/EBP-beta on tissue from a PML lesionand from a normal brain showed that C/EBP-beta isexpressed in the cytoplasm of oligodendrocytes har-boring viral inclusion bodies, which are negative forC/EBP-beta. In contrast, normal uninfected oligoden-drocytes show robust nuclear C/EBP-beta expression.These observations indicate a novel inhibitory role forC/EBP-beta isoforms in the regulation of JCV, whichmay be important in controlling latency and the tran-scriptional reactivation that occurs in PML.

P.176The early growth response-1 protein is induced byJCV infection and binds and regulates the JCVpromoterMartyn K White,1 Luca Romagnoli,1 Ilker K Sariyer,1

Mariha Feliciano,1 Bassel E Sawaya,1 MahmutSafak,1 Luis Del Valle,1 Pasquale Ferrante,2 andKamel Khalili1

1Center for Neurovirology, Department of Neuroscience,Temple University School of Medicine, Philadelphia, USA;2Laboratory of Molecular Medicine and Biotechnology,IRCCS S Maria Nascente, Don C. Gnocchi Foundation,Milan, Italy

JC virus (JCV) is a human neurotropic polyomavirusthat infects many people subclinically but then entersa latent state. Under conditions of immunosuppres-sion, usually AIDS, JCV can emerge from latency tocause the cytolytic destruction of glial cells of the brainin the fatal demyelinating disease, Progressive Mul-tifocal Leukoencephalopathy (PML). Previous studiesdescribed an important cis-acting transcriptional reg-ulatory element in the JCV non-coding control region(NCCR) that is involved in the response of JCV tocytokines and may have a role in viral reactivation.This consists of a 23 base pair GGA/C rich sequence(GRS) near the replication origin (5112 to +4) that con-tains predicted potential binding sites for the cellulartranscription factors Sp1 and Egr-1. Gel shift analysisshowed that Egr-1, but not Sp1, bound to GRS. Evi-dence is presented that the GRS gel shift seen on cellu-lar stimulation is due to Egr-1. Thus, TPA-induced GRSgel shift is blocked by antibody to Egr-1. Furthermore,the TPA-induced GRS DNA/protein complex band wasisolated from non-denaturing gels and found to con-tain Egr-1 by Western blot. No other Egr-1 sites werefound in the JCV NCCR. Functionally, Egr-1 was foundto stimulate transcription of the JCV late promoter butnot the early promoter. JCV infection of primary astro-cytes induced nuclear Egr-1 expression that was max-imal at 5-10 days post-infection. Finally, upregulationof Egr-1 was detected in the nuclei of JCV-infected cellsin clinical PML samples by immunohistochemistry.These data suggest that Egr-1 induction may be im-portant in the life cycle of JCV and in the pathogenesisof PML.

P.177Development of lentiviral encephalitisClayton WileyDivision of Neuropathology, Department of Pathology,University of Pittsburgh School of Medicine

A quarter of terminally ill AIDS patients develop asevere neurological disease term HIV Associated De-mentia (HAD). The neuropathological substrate ofthis disease is termed HIV encephalitis and con-sists of abundant activation and infection of brainmacrophages. Diagnosis of this disease is complicatedby a myriad of minor cognitive impairments that areseen in earlier stages of HIV infection. We examinedthe utility of the peripheral benzodiazepine recep-tor ligand PK11195 in positron-emission tomography(PET) imaging to assess microglial/macrophage acti-vation in the brains of HIV infected subjects withminor neurocognitive impairment. None of the 12HIV-infected subjects nor 5 controls demonstrated in-creased retention of [11C](R)-PK11195 in the brainparenchyma. These results suggest that macrophageactivation is not the pathological substrate of this mi-nor cognitive impairment. To develop better biomark-ers for the development of lentiviral encephalitis, westudied the SIV non-human primate model. We longi-tudinally followed Rhesus and Pigtailed macaques in-fected with the B670 examining blood, CSF and lymphnodes in addition to serial MRI and PET scans. Thesestudies permitted us to identify the onset of encephali-tis and examine the pathogenesis of neurodegenera-tion associate with lentiviral infection.

P.178The role of IGF-IR–p66Shc-FOXO3a signaling axisin high glucose-mediated accumulation of reactiveoxygen species (ROS) and neuronal damageShuo Yang, Jin Ying Wang, Kamel Khalili, ShohrehAmini, and Krzysztof ReissCenter for Neurovirology, Department of Neuroscience,Temple University, School of Medicine, Philadelphia, PA

It has been demonstrated that diabetes mellitus and as-sociated hyperglycemia alter the redox status of cellsthrough the overproduction of ROS by the mitochon-drial electron transport chain and NADPH oxidase.Our results indicate that high glucose (HG) concentra-tion (25mM for 16 hours) elevated the accumulationof ROS in differentiated PC12 neurons in comparisonto the cells kept in normal glucose (NG; 5mM). Theaccumulated ROS was found in neuronal processesand in perinuclear cytoplasm. This abnormal ROSaccumulation in HG was associated with a gradual(over a period of 10 days) loss of neuronal extensions.Interestingly, the addition of IGF-I to PC12 neuronscultured in HG prevented ROS accumulation andsignificantly improved the stability of neuronal pro-cesses. Since, p66Shc – FOXO3a signaling axis is


involved in ROS metabolism, and Shc proteins are di-rect substrates for the IGF-IR kinase activity, it was rea-sonable to speculate that the IGF-IR may affect ROSmetabolism by changing the status of p66Shc phos-phorylation and FOXO3a subcellular localization. Wehave selected several clones of PC12 cells, which sta-bly express a dominant negative mutant of p66Shc(DNp66Shc) in which Ser36 was replaced with ala-nine. We have analyzed protein levels for Shc proteinsincluding p42, p52 and p66; DNp66shc, as well as IRS-1, IGF-IR, and FOXO3a. Importantly, in the presenceof HG, PC12 neurons stably expressing DNp66Shcshowed significantly lower levels of ROS in compari-son to parental PC12 neurons, further confirming theimportance of functional p66Shc in ROS accumula-tion in differentiated neurons. We have evaluated alsoFOXO3a nuclear localization in PC12 cells culturedin NG, HG and HG+IGF-I. The purpose of this exper-iment was to determine whether FOXO3a, which isthe key transcription factor for superoxide dysmutase(SOD), translocates to the nucleus when ROS accu-mulation is attenuated. Digital images collected frommid sections of the nuclei of PC12 neurons, show anapparent immunolabeling for FOXO3a detected in thecytoplasm of examined cells. In normal glucose con-centration (NG) an apparent FOXO3a nuclear foci werealso detected. In high glucose (HG), the number ofnuclear FOXO3a foci decreased nearly 10-fold com-pared to NG. Importantly, an average number of nu-clear foci of FOXO3a in HG was restored when the cellsfrom HG cultures were treated with IGF-I. This corre-sponded well with ROS levels in these three exper-imental conditions and with the detection of nuclearFOXO3a in cortical neurons from HIV encephalopathyclinical samples.

P.179Identification of Epstein Barr virus epitopes inmultiple sclerosis cerebrospinal fluid usingphage-displayed random peptide libraries

Xiaoli Yu, Cecily E. DuPree, Greg P. Owens, andDonald H. GildenUniversity of Colorado Health Sciences Center

Multiple sclerosis (MS) is a chronic inflammatory de-myelinating disease of unknown cause. The most com-mon laboratory abnormality associated with MS is in-creased intrathecal IgG synthesis and the presence ofbands of oligoclonal IgG in brain and cerebrospinalfluid (CSF). However, the major antigenic targets ofthe antibody response are unknown. Recent interesthas focused on a possible role for Epstein Barr virus(EBV) in the development of disease. The risk of MSis increased after infectious mononucleosis, and MSpatients have higher serum titers of EBV antibodiesthan control populations. To identify disease-relevantantibodies, we screened phage-displayed random pep-tide libraries with IgG purified from an acute MSbrain. Two different phage-displayed peptides wereselected that share linear sequence homologies withEBV nuclear antigens 1 and 2 (EBNA-1 and EBNA-2), respectively. The specificity of the EBV epitopesto panning MS brain IgG were confirmed by ELISAand competitive inhibition assays. Using a highly sen-sitive phage mediated immuno-PCR assay, we mea-sured specific binding of each phage-displayed EBVepitope to IgG from 50 MS CSFs and 5 inflamma-tory control (IC) CSFs. IgG binding to the EBNA-1epitope, but not the EBNA-2 epitope, was found in25 of 50 (50%) MS CSFs, and in 1 of 5 IC CSFs. Wealso studied antibody titers directed against the puri-fied EBNA-1 protein and lysates of EBV-infected cells.On average, EBV titers in MS CSF (n = 50) were sig-nificantly greater than in inflammatory controls (n =9). Although we detected increased antibody to anEBNA-1 epitope as well as to purified EBNA-1 pro-tein in MS CSF, it remains to be determined whetherEBV antibodies are synthesized intrathecally and cor-respond to bands of oligoclonal IgG in MS CSF andbrain.

Poster Presentations

Documents

Transcript of Poster Presentations