key: cord-0001206-pjbr6yl2 authors: nan title: Abstracts from the 12th International Symposium on NeuroVirology: October 29–November 2, 2013 Washington, D.C., USA date: 2013-10-08 journal: J Neurovirol DOI: 10.1007/s13365-013-0211-9 sha: 6bbd41c340998a90c41cebafe37431b2a13c45b9 doc_id: 1206 cord_uid: pjbr6yl2 nan Case 3: A 32 year old HIV positive male, not on treatment developed fever, dysphagia and somnolence. MRI demonstrated hydrocephalus, confluent T2 hyperintensities along the basal cisterns, slyvian fissures along with diffuse leptomeningeal enhancement. Initial LP showed 9 WBCs which increased to 75, three days later. He later developed a zoster rash, seizures and CSF VZV PCR was positive. Case 4: A 30 year old female with AIDS developed hemiparesis and difficulty with speech. MRI of the brain identified multiple acute infarcts and CTA showed irregularities is multiple large and medium sized vessels. CSF showed 0 WBCs, normal protein and was positive for VZV PCR. Conclusion: VZV reactivation can be complicated by vasculopathy, meningoencephalitis, cranial nerve palsies, myelopathy or myeloradiculitis. These can also occur in seemingly immunocompetent patients, preceded by or even in absence of rash. CSF may or may not show pleocytosis. Characterizing HIV-Infected Monocytes that Transmigrate Across an In-Vitro Blood-Brain-Barrier as a Tool to Study HAND Pathogenesis Melissa Agsalda-Garcia 1 , Dionna W. Williams 2 , Joan W. Berman 2 , Cecilia Shikuma 1 , Lishomwa C. Ndhlovu 1 , Bruce Shiramizu 1 (presenting author: bshirami@hawaii.edu) 1 Hawaii Center for AIDS, John A. Burns School of Medicine, University of Hawaii, Honolulu, HI; 2 Albert Einstein College of Medicine, New York, NY Background: CD14+ monocytes (MO) are an increasingly recognized reservoir for HIV that are implicated in the pathogenesis of HIV-associated neurocognitive disorders (HAND) despite effective combination antiretroviral therapy (cART). The transmigrating characteristics of monocytes that traffic to the central nervous system by MO subset and HIV DNA content may be important in understanding mechanisms leading to HAND. We sought to adapt an established in-vitro blood-brain-barrier (BBB) model for use in a translational approach. Using blood from patients drawn in real time, we set up PBMC co-cultures to establish MO subset transmigration through this BBB model and quantitated HIV DNA from subsets. Materials: Fresh peripheral blood mononuclear cells (PBMC) were obtained from 2 cART-suppressed HIV-seropositive patients. Four distinct MO subsets based on variations in CD14 and CD16 expression were isolated from PMBC by cell sorting and integrated HIV DNA quantified from each MO subset by PCR (pre-BBB). Another aliquot of PBMC was placed in a transwell culture in an in-vitro BBB model established from human vascular endothelial cells and astrocytes and targeted for migration using the MCP-1 chemokine. After 24 hours, the transmigrated cells were re-phenotyped to assess MO subsets representation and cell sorted to re-analyze for HIV DNA copy number (post-BBB). Results: The MO subsets from PBMC that transmigrated across the BBB were characterized by flow cytometry. There was an approximately 1 log higher copy number of HIV DNA in the post-BBB transmigrated MO cells. DrexelMed HIV/AIDS Genetic Analysis cohort, can be classified into X4 and R5 genotypes, with associated specific co-linear long terminal repeat (LTR), Tat, and Vpr, genotypic patterns. Differential amino acid (DAA) signatures in both Tat and Vpr, as well as differential nucleotide (DN) signatures in the LTR were identified between the R5 and X4 virus. Utilizing maximum likelihood tree building strategy, genetic relatedness of Env-V3 between X4 and R5 was readily characterized, whereas, those of Tat, Vpr and LTR were not. On the other hand, genetic diversities of all genes/region of interest determined by the mean genetic distance (MGD) were evaluated between the group of X4 and R5, indicating the differential evolution of viral genetics. It has been theorized that these differential signatures can also be used to define different genotypes and phenotypes of X4 and R5 viruses more accurately in order to develop the next generation of diagnostic and prognostic tools to examine HIV-1 disease and to develop new therapeutics and vaccines to prevent and treat HIV/AIDS and associated neurological complications. This work is supported by NIH/NINDS R01 NS32092, NIDA R01 DA19807, NIMH P30 MH092177, and NIMH T32 MH079785. David Alvarez, Biswajit Das, Jonathan Karn (presenting author: dxa150@case.edu) Whereas the incidence of HIV-associated dementia (HAD) has declined due to successful anti-retrovirals treatment, prevalence of milder forms of HAD, which include asymptomatic neurocognitive impairment (ANI) and minor neurocognitive disorder (MND) has increased. Both ANI and MND are part of what is known as HIV-associated neurocognitive disorders (HAND). However, the molecular mechanisms explaining regulation of HIV activation in the brain remain ill-defined. The Nurr1/CoREST transrepression pathway has been recently described as a regulator of glial cells response to brain inflammation by limiting over-reactivation of NF-kappaBdependent pro-inflammatory genes. We report here that, unlike in latently-infected T-cells, in latently-infected microglial cells (CHME-5/HIV), HIV is induced by pharmacological inhibitors of the CoREST complex chromatin-modifying enzymes LSD1 and G9a/GLP; these inhibitors also sensitized CHME-5/HIV cells for LPS-mediated HIV reactivation. shRNA-mediated knockdown of Nurr1, LSD1, or CoREST yielded similar results. Chromatin immunoprecipitation analysis followed by high throughput next generation sequencing revealed that upon microglia treatment with TNFalpha, the nuclear receptor co-repressor 2 (NCOR2/ SMRT), which strongly interacts with Nurr1 to facilitate transrepression, is present at the HIV promoter before activation, then recruited at the earliest time points, and then its presence fluctuates over time. Likewise, Nurr1, CoREST, LSD1, and G9a are recruited to the HIV promoter, changing the epigenetic signature (lower H3K4Me/higher H3K9Me2). The repressor role of these proteins in regulating HIV emergence from latency has been confirmed by unbiased shRNA screens for factors involved in maintaining HIV silenced in latently-infected microglial cells. Our data indicate that the NCOR2-Nurr1-CoREST axis plays a role in preventing HIV over-reactivation in microglial cells, and studying this mechanism in detail may provide therapeutic targets for the treatment of HAND. David Alvarez, Yoelvis Garcia-Mesa, Biswajit Das, Stephanie Milne, Rojas Roxana, Jonathan Karn (presenting author: dxa150@case.edu) Toll-like receptors (TLRs) recognize molecules derived from microbes and play a key role in mediating innate immune responses. Multiple TLRs are typically expressed in cells of the monocytic lineage, including microglia. Microglial cells constitute the major reservoir for HIV infections in the brain, where inflammatory conditions in the central nervous system (CNS) are believed to induce HIV-associated neurocognitive disorders (HAND). Using HIV-latently infected microglial cell lines, we investigated whether TLR stimulation can induce HIV transcription. Treatment with a panel of TLR ligands, including Mycobacterium tuberculosis (Mtb)-derived molecules, we found that, unlike in monocytic cells, reactivation of HIV by TLR ligands was significantly restricted in microglia. Flagellin (TLR5 agonist) and, to a lesser extent, lipopolysaccharide (LPS; TLR4 agonist) were able to reactivate HIV in hTERT-immortalized glial (hT_Hmicroglia/HIV) and in SV40immortalized (CHME-5/HIV) human fetal microglial cells. By contrast, agonists for TLR1, 2, 4, 5, 6, or 8 (but not for TLR3, 7, or 9), potently reactivated HIV in THP-1/HIV cells and, to a lesser extent, in U937/HIV and SC/HIV monocytic cells in an NF-kappaB-dependent manner. Mtb-derived molecules PIM6 and LprG, which are potent TLR2 agonists, reactivated HIV in THP-1/HIV, but not in U937/HIV or SC/HIV cells which, unlike THP-1/HIV, showed no significant expression of TLR2. We conclude that TLR signaling probably plays only a minor role in activating HIV replication in the CNS, but can potentially drive replication in peripheral monocytic cells. Without Encephalitis Have Higher RNA Expression of Heme Oxygenase-1 (HO-1) than HIV-Brains Dengue virus (DENV) annually affects 100 million individuals worldwide, with approximately 20% of cases developing into dengue hemorrhagic fever. With no known treatments or available vaccines currently on the market and increasing global dispersal of DENV and its mosquito vectors, this emerging pathogen demands further attention. Patients suffering from severe DENV infection often exhibit encephalopathy and encephalitis. This suggests that the virus successfully infiltrates the blood-brain barrier (BBB) and infects local cell populations, thus negatively impacting normal central nervous system (CNS) function. However, the exact mechanism of DENV neuropathogenesis is unknown. Currently, our data shows that DENV infection of human primary PBMC's leads to 2-and 3-fold increases (T = 24 and 48 hrs P.I.) in absolute monocyte populations as compared with the uninfected control. We also detected significant elevation of several adhesion molecules (i.e. CD99, JAM-A and PECAM-1) on the surface of monocytes that are required for monocyte transmigration across the BBB as determined by qRT-PCR, western blot, and confocal microscopy. Furthermore, our results showed that despite infecting only~4% of human primary astrocytes, a~1.4-to 1.7-fold increase in CCL2 was observed in culture supernatants collected from DENV-infected human primary astrocytes. Similarly, our laboratory previously showed, that CCL2 elevation during HIV infection is essential for enhance monocyte transmigration into the CNS. Together, these data suggest that DENV infection increases monocyte populations and, akin to HIV, increase adhesion molecule expression and chemokine production among BBB cells such as astrocytes. We propose that elevation of CCL2 in astrocytes and the increased expression of surface adhesion molecules are key components of DENV neuropathogenesis. 1 The main barrier to HIVeradication continues to be the presence of viral reservoirs in infected patients despite treatment with antiretroviral drugs. Furthermore, as patients are living longer under treatment, the prevalence of HIV-associated neurocognitive disorders (HAND) has increased. Postmortem analyses of CNS tissues suggest that HAND is dependent on the migration of monocytes to the brain. Monocytes have been shown to traffic through the blood brain barrier and populate the perivascular sites where they differentiate into macrophages and contribute to viral persistence and latency. In addition, infected monocytes have been found in the blood of HIV infected patients treated with HAART. Our laboratory has developed and characterized an accelerated, consistent SIV infected pigtailed macaque model for HIVAIDS and neurologic disease. Using SIV infected pigtailed macaques we have characterized by flow cytometry the activation state of monocytes and microglia during infection. In addition, we have recently developed an assay to quantitate the number of infected blood monocytes and tissue macrophages similar to the quantitative outgrowth assay used on HIVand SIV CD4+ T cells. Our assay has allowed us to further investigate the number of SIV infected cells in peripheral blood and tissues of SIV infected pigtailed macaques that could potentially contribute to SIV persistence in the brain. Morphine reduces gp120-mediated cell death in vitro by altering the processing of proBDNF Alessia Bachis, Lee Campbell, Allyssia Boelk, Italo Mocchetti (presenting author: bachisa@georgetown.edu) Opioids have been shown to potentiate glia responses to HIV viral protein Tat and gp120 and to exacerbate gp120 and Tat toxicity. However, morphine protects cortical cultures against the toxic effect of M-tropic gp120 BaL. Using rat primary neurons we have observed that repeated exposure to morphine for 5 days attenuates gp120 IIIB-mediated neurotoxicity. Thus, further research is required to determine whether opioids exhibit neuroprotective or neurotoxic activity in the presence of HIV and HIV viral proteins. HIV and the envelope protein gp120 have been shown to reduce the levels of the trophic factor BDNF by altering the enzymatic processing of its precursor proBDNF. Interestingly, chronic morphine has been shown to increase the levels of tissue plasminogen activator (tPA), the activator of plasmin, that converts proBDNF into BDNF. Thus, opioids may reduce the neurotoxic effect of HIV/ gp120 by augmenting the downstream enzymatic processing of proBDNF. Using rat cortical neurons, we observed that morphine increases the levels and release of BDNF while at the same time decreases proBDNF. Interestingly, a similar imbalance in the ratio proBDNF/mature BDNF was confirmed in rats chronically (5 days) injected with morphine. The effect of morphine on proBDNF is due to an increase in the levels and activity of tPA. These data suggest that morphine's ability to alter proBDNF processing might be crucial to its neuroprotective effect against gp120. Our studies identify a new mechanism of interaction between morphine and HIV proteins. Laboratory of Neurosciences, NIA, NIH, Baltimore, MD. The widespread use of cART in developed countries has dramatically increased the expected lifespan of HIVinfected patients. This extended lifespan is frequently accompanied by premature brain aging, and accelerated deposition of amyloid beta (Abeta) peptides primarily located to lysosomal and autophagic compartments. The molecular mechanism that regulates the intraneuronal deposition pattern of Abeta is not understood. In this study we identified a series of cellular events evoked by neurotoxic HIV-gp120 that promotes the formation and intraneuronal deposition of Abeta. In triple transgenic gp120/APP/PS1 mice, Abeta1-42 containing plaques were deposited at a younger age with an intraneuronal deposition pattern. This Abeta deposition was accompanied by increased levels of sphingomyelin and ceramide. Ultrastructural analysis of brain tissues showed enlarged lysosomes with numerous electron dense and lipid inclusions in gp120/APP/PS1 mice. In neuronal cultures, gp120 accelerated the formation and perturbed the clearance of Abeta through mechanisms that involved an atypical activation of the chemokine receptor CXCR4, with signaling through protein kinase A, and cJun Nterminal kinase (JNK). This mitogen activated protein kinase pathway diverged at the level of JNK to increase transcriptional expression of beta-secretase (BACE1) through inhibition of peroxisome proliferator-activated receptor gamma, and increased amyloid precursor protein (APP) expression through a post-transcriptional mechanism involving enhanced binding of the heterogeneous nuclear ribonucleoprotein C (hnRNPC) to APP mRNA. APP and BACE1 were co-sequestered into membrane microdomains where BACE1 activity was enhanced, and Abeta production increased. This increased Abeta was not effectively cleared, and accumulated in lysosomes in conjunction with sphingomyelin and calcium. Stimulating calcium efflux from lysosomes with an agonist of TRPML1 channels facilitated the clearance of sphingomyelin and Abeta. These data suggest that strategies to preserve or restore lysosomal function in neurodegenerative settings may provide neuronal protection by increasing the cellular clearance of sphingolipids and Abeta peptides. Despite effective viral control through combinational antiretroviral therapies, approximately half of HIVinfected patients will develop some form of cognitive impairment. While we do not fully understand why some individuals infected with HIV will develop cognitive impairments while others do not, persistent lowlevel inflammation is thought to contribute to cognitive decline. Eicosanoids are a family of signaling molecules produced through the oxidation of 20-carbon fatty acids that regulate inflammation and immunity. Although the eicosanoids are comprised of a large number of products, those derived from the omega-3 fatty acids docosahexaenoic acid (DHA), or eicosapentaenoic acid (EPA) tend to be protective, while those obtained through omega-6 fatty acids arachidonic acid (AA), or linoleic acid (LA) are more often pro-inflammatory. In the present study, we measured eicosanoids in plasma from 98 HIV+ patients, and 25 healthy control subjects. We found 8 eicosanoid metabolites that were undetectable in control subjects and elevated in HIV+ patients. These included a mixture of EPA derived metabolites (15-HEPE, 18-HEPE, 8(9)-EpETE), DHA derived metabolite (13-HDoHE), and AA metabolites (LXA4, 8(9)-DiHETrE, HxB3, 5(12)-DiHETE). At baseline, cognitively impaired HIV+ patients who had better cognitive performance at the following visit had higher levels of 26 eicosanoid metabolites compared to HIV+ patients who were either cognitively normal, or cognitively impaired at both visits, or to cognitively normal control subjects. These included 3 EPA metabolites, DHA and 9 DHA metabolites, LA and 4 LA metabolites, AA and 8 AA metabolites. A number of these metabolites have been associated with vascular and cardiac dysfunction, and altered platelet aggregation. As these HIV+ patients cognitive status improved, levels of these metabolites decreased. These preliminary results suggest that alterations in eicosanoid production in HIV-infected patients may contribute to cognitive impairment through mechanisms that involve alterations in vascular and platelet function. Supported by P30-Pilot Award, JHUNIMH Center to Bandaru Srimoye Banerjee, Lindsey Gerngross, Tracy Fischer-Smith (presenting author: srimoye.banerjee@temple.edu) Temple University School of Medicine HIV encephalitis (HIVE) is the neuropathological correlate of the most severe form of HIV-associated neurocognitive disorders (HAND), which affect most HIV infected individuals. Previously, we reported expansion of a monocyte subset (CD14+/CD163+/CD16+) in HIV infected individuals with detectable plasma viremia that is phenotypically similar to macrophages that accumulate in the central nervous system (CNS) in HIVE. We hypothesize that macrophage colony stimulating factor (M-CSF), which is elevated in cerebral spinal fluid in HIVE, drives expansion of this monocyte subset. In peripheral blood mononuclear cells (PBMC), M-CSF increases the frequency of FLT3+ monocytes. Hence, we examined CNS tissue from SIV infected rhesus macaques and patients with HIV for possible alterations in FLT3 expression. Independent of the presence or absence of infection, neuronal expression of FLT3 is observed in all CNS tissues examined but is increased in SIV/HIV, with even greater expression in SIVE/ HIVE. A relationship between FLT3 expression and neuronal injury is implied by the corresponding decrease in MAP2 positivity with increased FLT3. FLT3 ligand (FL) reveals a similar pattern of increased neuronal expression in SIVE. In contrast to neuronal FLT3 and FL, macrophage/microglial expression is only observed in SIV/HIV infection, with greater expression seen in encephalitis. In vitro studies demonstrate altered FLT3 expression in differentiated SK-N-SH cells in response to various concentrations of FL, interleukin-34 (IL-34) and M-CSF and over different time intervals, suggesting multiple factors may contribute to altered FLT3 expression in the CNS. The functional role of FLT3/FL expression in SIV/HIV is unclear; however, interaction of FLT3 with its ligand is associated with neuronal survival in the developing CNS. As such, FLT3 and FL expression in CNS disease may be neuroprotective through promotion of neuronal survival. FLT3 expression by macrophage/microglia, however, may contribute to CNS pathogenesis in HIV/SIV by promoting survival of activated infected and noninfected macrophages/microglia. Victoria Baxter 1 , Michelle Potter 2 , Robert Mathey 2 , Barbara Slusher 2 , Diane Griffin 1 (presenting author: vbaxter1@jhmi.edu) 1 Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD; 2 Brain Science Institute, Department of Neurology, Johns Hopkins University School of Medicine, Baltimore, MD Recent outbreaks of encephalomyelitis caused by arthropodborne alphaviruses reveal their importance as an emerging cause of human disease and disability. Patients that recover from the acute disease, especially infants and children, are often left with life-long debilitating neurological defects, such as cognitive deficits, impaired motor control, and emotional and behavioral disturbances. Sindbis virus (SINV), the prototypic alphavirus, produces rash and arthritis in humans but is neurotropic in mice, providing an excellent model for studying alphavirus-induced encephalomyelitis. Previous studies have shown that infectious virus is cleared within 7-8 days, but viral RNA is cleared more slowly and persists at low levels for the life of the animal. Neuronal damage seen with alphavirus infection is thought to be due to both the immune response and glutamate excitotoxicity. Five-week-old C57BL/ 6 mice were intranasally inoculated with SINV or PBS. A battery of behavioral tests was used to assess motor and neurocognitive function at different phases of infection. Following behavioral tests, brains and spinal cords were collected, and infectious virus titers, SINV RNA levels, and viral antigen presence were assessed. At the height of active virus infection, characterized by peak infectious virus titers and detectable viral antigen in the brain, SINV-infected mice demonstrated increased activity in open field-testing and markedly impaired hippocampaldependent memory in contextual and cued fear conditioning. Following recovery from clinical disease, SINVinfected mice continued to show memory deficits in contextual fear conditioning when only viral RNA was present in the brain. Treatment with a glutamine inhibitor that affects both inflammation and glutamate excitotoxicity, 6diazo-5-oxo-L-norleucine, partially inhibited clinical disease and neurological deficits as measured by contextual fear conditioning. These findings show that SINV induces long-term neurological sequelae in mice that persist beyond active virus infection, and inhibition of both inflammation and glutamate excitotoxicity can partially mitigate development of these deficits. Frank Bearoff 1 , Laure Case 2 , Emma Wall 2 , Naresha Saligrama 2 , Jiri Forejt 3 , Elizabeth Blankenhorn 1 , Cory Teuscher 2 (presenting author: fb99@drexel.edu) 1 Drexel University College of Medicine; 2 University of Vermont; 3 Insitute of Molecular Genetics, Academy of Sciences of the Czech Republic Multiple sclerosis (MS) is a debilitating chronic inflammatory disease of the nervous system which affects approximately one million individuals worldwide with higher occurrences in females. Consomic mouse lines, also known as chromosome (Chr) substitution strains, are unique in that they allow for accelerated detection (compared to traditional backcross or F2 intercross mapping) of binary/quantitative trait loci by direct genome-wide physical mapping from genetically distinct parental strains. We have employed myelin oligodendrocyte glycoprotein-induced experimental allergic encephalomyelitis (MOG-EAE), a mouse model of MS, to evaluate the sex differences in consomic B6-Chr#PWD/PhJ mice (Mus musculus domesticus). We utilized a panel of 26 B6-Chr#PWD/PhJ consomic and sub-consomic strains of mice covering 17 autosomes from the wild derived PWD strain (Mus musculus musculus) to position genes controlling MOG-EAE in this strain combination, fifteen of which exhibited evidence of such loci. Of the twenty linkages found, seven were male-specific, four female-specific, and nine were nonsex specific, suggesting a greater genetic load in males than in females. We then identified common evolutionarily conserved pathways underlying the sexual dimorphisms in EAE and multiple sclerosis (MS) susceptibility. We find that there is a greater overlap of pathways common to MS and male EAE compared to female EAE (33 vs. 7) that are the basis for these sexual dimorphisms. Importantly, our results provide a scheme by which consomic mouse strains can provide a better understanding of polygenic human diseases. Immune pressure exerted by MHC class I-mediated cytotoxic T-lymphocytes (CTLs) drives HIV escape mutations; nonetheless, the relationship between viral escape and HIV CNS disease remains poorly understood. MHC-I allele Mane-A1*084:01:01 confers resistance to SIV-induced CNS disease in pigtailed macaques, and prototypic escape (K165R) develops in the immunodominant SIV Gag KP9 epitope. We hypothesize that 1) escaped virus has reduced fitness in vivo and in vitro, and 2) CTL-mediated immune pressure switches from the immunodominant Gag epitope to a subdominant epitope in animals inoculated with SIV K165R. We used site-directed mutagenesis to insert the K165R Gag escape mutation into the backbone of the neurovirulent molecular clone SIV/17E-Fr, producing the clone SIV/17E-Fr K165R. Replication of SIV/17E-Fr K165R versus SIV/ 17E-Fr was compared in CEMx174 cells and primary macaque microglia, macrophages, and lymphocytes. Although K165R was replication competent in all cell types, viral replication was reduced compared to SIV/17E-Fr. To extend in vitro findings, Mane-A1*084:01:01 expressing macaques were inoculated: three with SIV/17E-Fr K165R, and three with wildtype, parental SIV/17E-Fr. There were no differences in plasma viral loads and slightly lower CSF viral loads in animals inoculated with K165R versus those inoculated with wildtype. In the absence of the immunodominant epitope Gag KP9, CTL-mediated immune pressure redirected to the sub-dominant Mane-A*084:01:01-restricted SIV Tat epitope KVA10 with no viral escape evident. Most surprisingly, in animals inoculated with K165R, the escape mutation K165R was genotypically stable in the plasma, but rapidly reverted to WT Gag KP9 in both CSF and in microglia. These data clearly demonstrate that viral fitness in the CNS is different from the periphery. As therapeutic vaccination strategies to enhance CTL responses against HIV gag could promote HIV escape, it is vital that we understand the consequences of viral escape on CNS disease. 1 The gene ApoE is mapped to chromosome 19, and the E*4 allele is found in approximately 14% of the population. Several recent studies have examined ApoE among individuals with HIV disease, and to date, the data suggest that any links are weak, at best. We describe here the relationships among ApoE genotypes, subject characteristics, incident neuropsychological impairment, and time to death among men enrolled in the Multicenter AIDS Cohort Study (MACS). Genomic DNA was isolated from lysates of either buffy-coat or B-cell immortalized cell lines from 2846 men (median age: 34, range: 17-69) in the MACS. Genomic DNA concentration and quality was assessed using UV and/or by fluorometric quantitation. End-point genotyping of ApoE variants rs429358 [C/T] and rs7412 [C/T] was performed using TaqMan and OpenArray technologies. 1689 of the men were HIV-infected and 1157 were uninfected. 23.2% of the men who identified themselves as white had at least one copy of the E*4 allele, whereas 31.2% of other races had at least one E*4 allele (Odd's Ratio = 0.65 (0.54 -0.79)). In separate models by race (White/Minority), we found no significant associations between the presence of an E*4 allele and age, HIV status, death, or neuropsychological outcome. We used Cox Proportional Hazard Models to investigate time to death and found a significant association between HIV infection and time to death (all cause), as well as older age, race, and education. ApoE status was not significantly associated with time to death. There were significant associations between time to neuropsychological impairment, age, education, and HIV serostatus, but no association with ApoE*4. There were no interactions between ApoE*4, HIV infection, age and the outcomes of interest. These results confirm and extend previous findings that that the ApoE*4 allele is not associated with death or neuropsychological impairment among men in this age group. AIDS Cohort Study (1987 -2012 among the individuals who died during follow-up. Differences in volunteer recruitment are reflected in the cohort characteristics at the baseline visit, and they tended to persist among the active participants in 2012. Loss of data due to death or dropout may affect the estimates of prevalence of neuropsychological abnormalities among the study participants. Human Polyomavirus JC monitoring and noncoding control region analysis in dynamic cohorts of individuals affected by multiple sclerosis under treatment with natalizumab: an observational study Progressive multifocal leukoencephalopathy (PML) onset, caused by Polyomavirus JC (JCPyV) in patients affected by multiple sclerosis (MS) during natalizumab treatment, raised concerns about the safety profile of this agent. Therefore, the aims of this study were the JCPyV reactivation monitoring and the noncoding control region (NCCR) and viral protein 1 (VP1) analysis in patients affected by MS and treated with natalizumab. We performed JCPyV-specific quantitative PCR of biological samples collected at moment of recruitment (t0) and every 4 months (t1, t2, t3) for 1 year. Subsequently, analysis of NCCR and VP1 rearrangements was carried out. Moreover a 2-step virus-like particle-based enzyme-linked immunosorbed assay was performed at t0 and t3, to detect specific anti-JC virus antibodies in serum of the enrolled subjects. Data were analyzed using chi-square test. Results showed a significant association between JC viruria and JCPyVantibodies after 1 year of natalizumab (p=0.04). Moreover, sequences isolated from peripheral blood mononuclear cells (PBMCs) of 2 patients with JCPyV antibody at t0 and t3, showed a NCCR Type IIR with a duplication of a 98bp unit and a 66bp insert, resulting in a boxB deletion and 37 T to G transversion into the Spi-B binding site. In all patients, a prevalence of genotypes 1A and 1B, the predominant JCPyV genotypes in Europe, was observed. In conclusion, it could be important to understand whether the specific inflammatory scenario of multiple sclerosis could affect JCPyV reactivation from latency, in particular from kidneys. Moreover, for a more accurate PML risk stratification, testing JC viruria seems to be useful to identify patients who harbor JCPyV but with an undetectable JCPyV-specific humoral immune response. In these patients, it may also be important to study the JCPyV NCCR rearrangement: in particular, Spi-B expression in PBMCs could play a crucial role in JCPyV replication and NCCR rearrangement. HAND patients exhibit changes in the key brain regions responsible for motivational drive (mesocorticolimbic system). The HIV-1 transgenic rat is chronically exposed to low levels of HIV-1 proteins in the brain, which may occur in humans with cART. The present study used adult female, ovariectomized, HIV-1 transgenic rats (N=29). Initially, a five bottle preference test, using 0%, 1%, 3%, 10%, and 30% (w/v) sucrose solutions, following 22 h of water restriction, was used to evaluate response to novelty as well as potential sensory differences between HIV-1 and control rats. Although there was no difference in overall volume consumed on the initial test day, HIV-1 rats consumed significantly more of the 10% sucrose solution, suggesting a heightened novelty response. Across the following 5 days of testing, HIV-1 and control rats did not significantly differ in their pattern of consumption of sucrose, suggesting that overall taste preference for sucrose was not altered. Subsequently, rats learned to respond for 5% sucrose (w/v) reinforcement in operant conditioning chambers. Animals were briefly water restricted during autoshaping; all animals eventually progressed onto non-restricted training, according to an increasing fixed-ratio requirement (FR1, FR2, FR3). There was a significant retardation in the rate at which the HIV-1 rats progressed to higher FR schedules compared to controls, suggesting a change in the basal internal motivational state. Most critically, the present results suggest alterations in the internal motivational state of HIV-1 animals, as displayed by their inability to meet criterion and progress through the fixed ratio schedules, which were not readily attributable to any alterations in novelty or taste preference. These results suggest that the HIV-1 transgenic rat may have underlying internal motivational deficits for obtaining natural rewards such as sucrose, not unlike their alterations in responding for drugs of abuse. Objective: In endemic areas, malaria and HIV frequently infect the same host but their combined effects on the brain are poorly understood. We hypothesized that the combination of malaria and HIV would amplify monocyte/macrophage activation and contribute to worsening of neurologic HIV disease. Methods: 269 subjects, 174 HIV+ and 95 HIV-, were evaluated using a comprehensive neuropsychological test battery. Global and domain-specific deficit scores (GDS and DDS) were generated and used for determining impairment. All subjects lacked symptoms of acute malaria and AM was diagnosed based on peripheral smear findings. Hemoglobin, hematocrit, CD4+ T-cell counts, HIV RNA levels, soluble CD163, and soluble CD14 were measured. Results: AM was not statistically significantly more common in HIV+ than in HIV-subjects (36% vs. 29%, odds ratio (OR) 1.36, p > 0.10). Overall, AM was not associated with NCI (p > 0.10). Among AM subjects, HIV+ were more likely to have motor impairment than HIV-(35% vs. 42%, p=0.03). AM trended toward an association with executive functioning impairment after accounting for HIV status (p<0.10). An interaction between CD4+ counts and AM was present: AM was associated with NCI among people with CD4+ counts below 200 (35% vs. 22%, OR 1.90) but the opposite was true for subjects who had higher CD4+ counts (21% vs. 30%, OR 0.61). sCD14 levels were significantly higher in the M+/H+ group compared to the M-/H+ group (p=0.015). Higher sCD14 levels were associated with higher odds of impairment (OR=9.4, 95% CI=1.1,88; p=0.046) but the interaction with malaria was not significant. Conclusion: AM is common among HIV+ individuals in Nigeria. Malaria may lead to worse NC functioning, specifically in motor and executive functioning. Individuals with greater immunosuppression are more likely to experience impairment when co-infected with malaria. Malaria also results in higher levels of biomarkers of monocyte/macrophage activation. Human beta-defensins (hBDs) are broad-spectrum antimicrobial peptides, secreted by epithelial cells and astrocytes, which we and others have shown to inhibit HIV-1 in primary CD4+ T cells. In T cells, the intracellular inhibitory activity is mediated by CCR6, a receptor that binds both the chemokine MIP-3alpha and hBDs. Although loss of T cells contributes to mucosal immune dysfunction, macrophages, by virtue of their roles in both innate and adaptive immunity, are a major source of persistence, spread of HIV, and development of complications, especially in the brain. Interestingly, hBDs are expressed in the CNS but very little is known about their role in the brain. We hypothesized that, besides T cells, hBDs protect microglial cells and perivascular macrophages from productive HIV infection. Our data on primary monocytederived macrophages (MDM), which we use as an in vitro model for microglial cells, show that hBD2 and -3 inhibit HIV replication in a dose-dependent manner. We investigated the mechanism(s) involved. We determined that hBD2 neither alters surface expression of HIV receptors nor induces expression of anti-HIV cytokines or beta-chemokines in MDM. Studies using a G-protein signaling antagonist in a single-cycle reporter virus system showed that hBD2 suppresses HIV at an early post-entry stage via GPCR-mediated signaling. Our data also illustrate that hBD2 blocks HIV by enhancing expression of APOBEC3A and 3G. MDM express the shared chemokine-hBD receptors CCR2 and CCR6, albeit at variable levels among donors. However, neither of these receptors is necessary for hBD2-mediated HIV inhibition, suggesting that hBD2 can signal via additional receptor(s). Preliminary data also show that hBD2 is internalized in early endosomes in MDM. These findings suggest that hBD2 inhibits HIV in MDM via more than one mechanism, and may aid the development of new approaches to treat HIV infection systemically and in the CNS. Background: Previous studies suggested a possible role for HHV-6B in mesial temporal lobe epilepsy (MTLE). HHV-6B is the etiologic agent of roseola, while HHV-6A is associated with central nervous system (CNS) disorders and may be more neurotropic than HHV-6B. Methods: Using digital droplet PCR (ddPCR) to examine HHV-6A and HHV-6B viral DNA, we studied a new cohort of 10 MTLE patients and six with focal cortical dysplasia (FCD). DdPCR is a highly sensitive and precise novel PCR technology that enables absolute quantification of target DNA molecules. Results: Viral DNA was detected in 60% of MTLE patients. This positivity frequency is consistent with our previous findings. In addition, we detected HHV-6A as well as HHV-6B. Moreover, HHV-6A and HHV-6B DNA also were detected in a subset of FCD patients. The detected viral copy numbers varied across a wide range between patients and even within a given patient, depending on regional sampling. Conclusions: Our new data suggest viral presence in patients with FCD and extend our findings in patients with MTLE, with detection of HHV-6A not previously reported. Innate immune signalling initiates B cell response in viral induced demyelination Multiple sclerosis (MS) is a demyelinating disorder of the central nervous system (CNS), believed to be autoimmune in nature. Characteristic presence of oligoclonal bands (OCBS) in CSF of almost 95% of MS patients supports the long standing hypothesis of viral etiology. OCBs are found in infectious CNS disorders where their antigenic target represents the causative agent, but a definite target of OCBs in MS patients remains obscure. Mouse hepatitis virus (MHV) induced neuroinflammation in mice is an experimental model used to study mechanisms of demyelination. Neurotropic MHV strains induce meningitis, encephalitis, and myelitis during their acute phase and progressive demyelination in the chronic phase of infection when viral particle is cleared and acute inflammation is resolved. Affymetrix microarray analysis of the mRNA expression from demyelinating MHV strain infected spinal cord tissue revealed that during acute infection there is a robust upregulation of various innate immune genes which are mainly involved in antiviral immune responses, phagolysosome maturation and MHC Class-II expression. During chronic infection, there is a 10-fold upregulation of several Ig mRNAs (Ighg3, IGJ, Igk-V28 andIgkv4-68), with a shift of Th1 and Th2 immune responses in acute and chronic inflammation stages. It has been known for a long time that viral infection can induce Ig molecules, but the mechanism of induction was not clear. Demyelinating MHV infection induced innate immune signalling and the innate immune effector molecules induce B cell activation and expression of several oligoclonal genes which could play a major role in antibody mediated progressive demyelination. This cause effect relationship of viral infection and induction of oligoclonal Ig mRNAs paves a way to understanding B cell activation in demyelination. Findings may help advance our understanding of viral induced and antibody mediated demyelination in MS. The role of resident and infiltrating macrophages in dorsal root ganglia pathology and intraepidermal nerve fiber loss using a rhesus macaque model of AIDS-associated peripheral neuropathy Symptomatic abnormalities of the peripheral nervous system (PNS) are among the most common complications of HIV-1 infection. Although anti-retrovirals have been successful in ameliorating the constitutional manifestations of AIDS and reducing the severity of many neurological complications caused by HIV infection, there has been no significant impact on the incidence or severity of HIV sensory neuropathy (HIV-SN). Here, we used 12 SIV-infected CD8 depleted rhesus macaques as a model of HIV-SN and 5 uninfected controls. 10 of the 12 SIV-infected animals (83%) developed moderateto-severe dorsal root ganglia (DRG) pathology. DRG tissues had extensive activation of satellite cells, as measured by CD68 and CD163 immunoreactivity, some of which were productively SIV-infected via immunohistochemistry (0.1-8.2%). BrdU pulse studies showed an increase in monocyte traffic from bone marrow to the DRGs, which correlated with the severity of pathology; uninfected 0.8%, mild 1.7%, moderate 2.6% and severe 6.7%. Intraepidermal nerve fiber (IENF) densities were determined from biopsies of the central food pad. There was a significant decline in IENF densities in 7 of the 8 animals by 21 days post infection with an average of a 48.7% loss in fibers at necropsy. A positive correlation between the percentage of CD163+ macrophages surrounding the sacral DRG neurons and the percent loss of IENF densities was seen at necropsy (r= 0.94, P= 0.006). We conclude that both resident macrophage activation and monocyte infiltration play significant primary roles in DRG damage during HIV-SN. Our data also suggest a link between macrophage activation in the DRGs, DRG pathology, and IENF loss in the periphery in a rhesus macaque model of AIDS-induced peripheral neuropathy. Thus, future adjunctive therapies that diminish macrophage activation and monocyte infiltration may serve to ameliorate the HIV-SN severity previously observed. Proteomics evaluation of CSF patients with HIV-associated neurocognitive impairment using iTRAQ labeling HIV invasion of the CNS is coincident with acute HIV infection, and persists due to the chronic infection of long-lived microglial cells, astrocytes and perivascular macrophages. Cerebrospinal fluid (CSF) is becoming one of the most frequently used biofluids for physiological studies of neurological disorders since it is an ultra-filtrate of plasma and is the collection point for drainage of the brain parenchyma. In this proteomics study we used a relative quantitation method to study pooled CSF samples from 10 HIV-infected patients with cognitive impairment, 10 HIV-infected patients with normal cognition, and 10 healthy individuals, with the goal to identify potential biomarkers for cognitive impairment. We used advanced two dimensional separation strategies and bioinformatics to identify a total of identification of overall 673 proteins. Using statistical interpretation of the isobaric tags for relative and absolute quantitation (iTRAQ) reporter intensities, 193 proteins with 95% confidence interval and pvalue ≤ 0.05 were selected based on permutation test that were interpretable for quantitation. Using a cut off of 1.5 fold for upregulation and 0.6 fold for downregulation, 16 proteins were differentially expressed in HIV-infected individuals with cognitive impairment (reporter p-value ≤ 0.05). Seven of these proteins have been previously described as HIV-interacting proteins including: endoplasmin, mitochondrial damage mediator-BH3-interacting domanin death agonist (BID), orosomucoid, apolipoprotein E, metalloproteinase inhibitor 2, peroxiredoxin-2 and the nuclear protein, ruvB-like 2. Several proteins with possible neurological implications were also identified including: forming-binding protein 1, C-reactive protein, leukocyte-associated immunoglobulin receptor 1, renin receptor, mediator of RNA polymerase II transcription subunit 14, multimerin-2, alpha-N-acetylglucosaminidase, caldesmon, cadherin EGF LAG G-type receptor. This suggests that not only a few but possibly a combination of biomarkers that are highly correlated can predict neurocognitive status in HIV-infected patients and might be involved in monocyte or macrophage activation. Background: JCV causes progressive multifocal leukoencephalopathy (PML) in immunocompromised patients. While there is no effective anti-viral therapy against JCV, the cellular immune response is crucial in containment of viral replication and is associated with PML survival. Therefore, a preventative and therapeutic vaccine against JCV, which can elicit this cellular immune response is urgently needed. Methods: Replication incompetent adenovirus vaccine vector serotype 5 (rAd5) was generated by insertion of the JCV VP1 gene. Naive C57BL/6 wildtype mice were immunized with a single injection of either the rAd5-JCV or DNA rAd5-JCV. Days 14 to 21 after immunization, mice were sacrificed and splenocytes were isolated, stimulated with overlapping JCV VP1 peptide pools and assayed by intracellular cytokines staining (ICS) for IFN-gamma. Control mice were injected with PBS and JC Mad-4 virions. Results: Mice remained asymptomatic up to 21 days after injections of JC virions, rAd5-JCV, DNA rAd5-JCV or PBS. While JC virions elicited minimal cellular immune responses, rAd5-JCV and DNA rAd5-JCV significantly increased the percentages of JCV-specific CD4+ T-cells compared with those elicited by JC virions (p<0.05). In addition, rAd5-JCV significantly increased JCV-specific CD8+ T-cells compared to DNA rAd5-JCV (p=0.05), and to JC virions (p<0.05). Furthermore, DNA rAd5-JCV significantly increased JCVspecific CD8+ T-cells compared to JC virions (p<0.05). Conclusions: Both recombinant Adenovector and DNA plasmids expressing JCV VP1 proteins elicit strong JCV-specific cellular immune responses in mice. Further development of these vaccines may lead to clinical applications for prevention and treatment of JCV-associated brain diseases. Comprehensive survey of GABAergic neural transmission in HIV infection reveals correlation with anomalies in the neurovascular unit and dopamine receptor expression in frontal neocortex a gap junction-associated transcript expressed selectively in GABAergic neurons (GJD2) were assayed using qPCR. All three GABA-associated mRNAs were significantly decreased in the dorsolateral prefrontal cortex (DLPFC) of HIV-positive subjects. Abnormal expression was distributed widely in the CNS including neocortex, neostriatum and cerebellum. Patients with and without HIV encephalitis had significantly lower GABAergic mRNAs. Patients who died prior to and after the era of HAART both had significantly lower GABAergic mRNAs. Low GABAergic mRNAs were correlated significantly with higher expression of mRNA markers produced by endothelial cells including PECAM1 and VWF, and with high expression of dopamine receptor type 2 mRNA (DRD2L). Low GABAergic mRNA expression was specifically correlated with worse performance on testing for verbal fluency. Immunoblotting and immunohistochemistry showed protein-level changes in the HIV-infected brain specimens including loss of GAD67 and parvalbumin staining intensity, higher DRD2 expression in some GABAergic interneurons, and swelling of endothelial cells. We conclude that abnormal GABAergic tone is prevalent in HIV infected patients, and it has a broad anatomical distribution in the CNS. Novel associations with changes in dopamine transmission and endothelial cell markers suggest roles in synaptic plasticity and connection to the neurovascular unit. Macrophage/microglia lineage-related R5-tropic simian-human immunodeficiency viruses as tools to induce and study HAND Thanks to an increasing panel of antiretroviral drugs, HIV replication can be effectively controlled in infected patients. However, there are limitations to ART, as immune activation is not fully contained and penetration of most drugs into the central nervous system (CNS) is inefficient. Thus, while NeuroAIDS cases have diminished, HIV-associated neurocognitive disorders (HAND) have become a far more prominent problem. HIV neurovirulence has been associated with viral tropism for macrophage/microglia (MG)/monocyte lineages, but there is as yet a paucity of reliable animal models of neurotropic HIV. To address this gap, we generated an MGtropic SHIV, named SHIV-Bo159N4p2. The resulting virus is highly fusogenic and replicates to high levels in monocytederived macrophages (MDM) and MG. We tested its replicative capacity under the cover of depletion of CD8 cells or CD8 and CD20 B cells in rhesus or pigtailed macaques (PMs). These viruses replicated very well in vitro and in vivo (upon intravenous or intrarectal inoculation) and reached peak viral loads in plasma and CSF (10^6 to 10^9 copies/ml in plasma vs 10^5 to 10^8 copies/ml in CSF). Early-stage viruses were sensitive to broadly neutralizing antibodies, soluble CD4 (sCD4) and induced cross-clade neutralizing antibody responses. The latestage viruses differed in their sensitivity to sCD4 and broadly neutralizing antibodies but remained R5 tropic. Notably, under double depletion (CD8+CD20), 8 out of 11 PMs rapidly progressed to disease within 2-4 months, while 2 out of 2 PMs progressed to disease in < 2 years without depletion. Pathological changes in the brain in most PMs included astrogliosis, chronic meningitis, perivascular cuffing or inflammation, gliosis and few infected MG, suggesting that these viruses cause milder forms of neurological diseases. In summary, macrophage/MGtropic SHIVs will be valuable tools to study milder forms of neurological disorders in a biologically relevant primate model. Studies suggest that uninfected and HIV infected CD14+ CD16+ monocytes transmigrate across the blood brain barrier (BBB), mediating viral entry into the CNS and contributing to chronic neuroinflammation that results in HIV associated neurocognitive disorders (HAND) in greater than 50% of HIV infected people. In some HIV infected drug abusers, HAND is reported to be more severe. Increased extracellular CNS dopamine is a common mechanism that occurs acutely in response to intermittent drug use. We report that the number of peripheral CD14+CD16+ monocytes is increased in HIV infected drug abusers. To study the effects of dopamine on chemokine mediated CD14+CD16+ monocyte influx into the CNS, uninfected peripheral blood monocytes were cultured non-adherently with M-CSF to expand the number of CD14+ CD16+ cells. Cultured monocytes expressed CXCR4 and the transmigration of these cells in response to CXCL12 and/or dopamine was characterized using our human BBB model. Unexpectedly, dopamine alone increased CD14+CD16+ monocyte transmigration. In contrast, dopamine had no effect on T cells. Dopamine receptors D1R and D5R are higher, while D4R is lower, in mature monocytes when compared to T cells. The D1R/D5R agonist SKF 38393 increased CD14+ CD16+ monocyte transmigration, indicating the involvement of these receptors in dopamine induced transmigration. Although dopamine does not cross the BBB, CD14+CD16+ monocyte transmigration may be increased by dopamine mediated effects on BBB cells and/or on the transmigration process once monocytes have penetrated the BBB in response to chemokines. Mature monocytes were treated with dopamine to determine the direct effects of dopamine on cellular processes involved in transmigration. Dopamine increased migration and pseudopodia formation, a component of cellular polarization involved in movement. Thus, the combination of higher numbers of circulating CD14+CD16+ monocytes and elevated dopamine in HIV infected drug abusers may result in increased transmigration of these cells into the CNS, contributing to the severity of HAND. Methods: Here we used a VLA-4 blocking antibody, Tysabri (30 mg/kg once a week for three weeks), and treated SIVmac251 infected, CD8+ lymphocyte depleted monkeys with high viral load and CNS injury on 28 days post infection (dpi) to stop monocyte/macrophage traffic (n=4). Additionally, we treated another cohort of animals at the time of infection (day 0, n=6) to stop productive infection of the brain and gut, but not lymph nodes. In animals treated at 28 dpi, metabolite concentrations of n-acetylaspartate over creatine (NAA/Cr) were assessed biweekly by 1H MR spectroscopy (MRS). Results: With Tysabri treatment at 28 dpi we found a stabilization of neuronal injury (NAA/Cr) in frontal and parietal cortex. Tissue analysis revealed significantly lower numbers of activated resident macrophages (CD68+), recently recruited monocytes/macrophages (MAC387+, BrdU+), and productively infected cells (SIV p28+, RNA+) in the brain and small intestine of Tysabri treated macaques when compared to controls. SIV DNA was undetectable in all but one of 24 brain tissue samples analyzed from 0 dpi Tysabri treated animals, whereas similar concentrations of proviral DNA were isolated from gut tissue of Tysabri treated and untreated control macaques. Animals treated with Tysabri had low to no LPS and soluble CD163 in plasma. Ex vivo PBMCs from treated macaques exhibited impaired adhesive and transmigratory capabilities in response to proinflammatory stimulation. Conclusions: Our studies using Tysabri at 28 days post-infection in SIV-infected macaques demonstrate that stopping monocyte traffic stabilizes CNS disease. Treating animals at the time of infection blocked traffic of virus to brain, and reduced the number of productively infected cells in the small intestine, raising the possibility that Tysabri may be an effective agent for preventing the formation of reservoirs during early HIV infection. Multifocal Leukoencephalopathy (PML). Aim of this study was to determine the immunological changes in Natalizumabtreated MS patients over the time, in order to better understand the effects of the treatment on peripheral blood mononuclear cells (PBMCs) subsets. For this purpose, the relative frequency of different leukocyte subsets and their expression of the alpha4 and beta2 integrins were investigated.The analysis were performed by flow cytometry on PBMCs recovered from blood samples of 11 MS patients treated with Natalizumab, followed up to two years. Eleven MS patients treated with conventional therapy and 11 healthy subjects were also enrolled as control groups.The analysis of the relative frequencies showed a decrease of the monocyte population and an increase of the lymphocyte populations, in particular CD19+ and CD34+ cells. Within the monocyte population, we could detect a significant decrease of not classic CD14+CD16+, and an increase of classic CD14+CD16-, associated with the course of the treatment. The integrins expression was assessed by evaluating the Relative Fluorescence Intensity, which allowed to detect significant variations within all the CD14+ subpopulations and the CD34+ population. Additionally, a patients' stratification based on the urinary JCV and BKV DNA excretion was done to define whether the viral status could affect the natalizumab treatment and viceversa: CD3+ cells relative frequency increased in patients excreting the viruses, while CD19+ relative frequency decreased. Human T-cell lymphotropic virus type 1 (HTLV-1) is a retrovirus that is the etiologic agent in a clinical neuromyelopathy known as HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP). Quantification of the amount of virus in a patient samples is critical for monitoring infection, evaluating the efficacy of therapeutic agents, and assisting with clinical diagnosis. Real-time PCR (qPCR) is the standard method for determining HTLV-1 proviral load (PVL). However, qPCR is dependent upon well-defined standards and calibration to a standard curve, precluding precise and accurate quantification particularly at low cell concentrations. In this study, we use a third generation PCR technique, digital droplet PCR (ddPCR), which allows for direct quantification of PVL. DNA samples are partitioned into thousands of nanoliter-sized droplets, amplified on a thermocycler, analyzed for fluorescent signal, and normalized to a housekeeping gene. Poisson distribution statistics are used to determine absolute copy numbers independently of a standard curve. We found that the intra-assay and inter-assay reliability of ddPCR are robust and more consistent than those of qPCR. Moreover, ddPCR yields similar patient PVL by quantifying different viral genes. Our preliminary data suggests ddPCR can also be used to examine viral gene expression, a key player in the pathogenesis of HAM/TSP and an important parameter to monitor during therapeutic interventions in this condition. The reliability and precision of ddPCR for HTLV-1 quantification should be widely acknowledged as it might be an important tool to further understand the clinical correlates of the viral load in the context of HAM/TSP and other HTLV-1 related conditions; however, further investigation is required to determine the efficacy of ddPCR for mRNA analysis. HIV associated neurocognitive disorder (HAND) is a devastating consequence of HIV infection in the current antiretroviral therapy (ART) era. The pathogenesis of HAND is a multifactorial sequence of events including the transmigration of HIV infected monocytes that cross the blood brain barrier (BBB) resulting in infection of the central nervous system (CNS) resident cells such as macrophages, microglia, and a small population of astrocytes. Infection activates inflammation and subsequent cellular dysfunction and apoptosis. In addition, viral reservoirs are generated within the CNS, and upon activation, propagate viral infection and inflammation years after primary infection. Thus, elimination of these cell reservoirs is essential for the eradication of HIV from the brain and its subsequent affects in pathogenesis of HAND. Our preliminary data indicates that HIV infected microglia and astrocytes are protected from cell death despite intracellular pro-apoptotic signaling from mitochondrial dysregulation. Mitochondrial release of cytochrome c into the cytoplasm is normally induced by pro-apoptotic stimuli and is followed by cell death. However, infected microglia and astrocytes resist cell death even when cytochrome c is released from the mitochondria into the cytoplasm. The mechanism by which HIV protects infected cells from cytoplasmic cytochrome c is currently unknown, and understanding these mechanisms may yield novel pharmaceutical targets for the treatment of HAND. Key negative regulators of apoptosis are heat shock proteins (HSP), which inhibit the pathway by which cytochrome c induces cell death. Our hypothesis is that HIV infected cells "hijack" heat shock proteins to avoid apoptosis induced by cytoplasmic cytochrome c. Targeting a pathway utilized specifically by HIV infected cells will generate a potential drug target for treatment of HAND. Background: The risk of JC virus (JCV) reactivation and progressive multifocal encephalopathy (PML), while on natalizumab, may increase with duration of exposure to the drug. We sought to determine the prevalence of JCV reactivation and potential changes in JCV-specific cellular immunity during prolonged treatment. Methods: We enrolled 39 JCV-seropositive multiple sclerosis (MS) patients, including 32 on natalizumab monotherapy for 18-20 months (n=14), 22-25 months (n=7) and > 36 months (n=11), 2 on interferon-beta monotherapy >36 months and 5 untreated MS patients as controls. We performed QPCR in CSF, blood and urine for JCV DNA and we determined JCV specific T-cell responses using enzyme-linked immunospot (ELISpot) and intracellular cytokine staining (ICS) essays, ex vivo and after in vitro stimulation with JCV peptides. Results: JCV DNA was detected in the CSF of 3/35 (8.6%) subjects tested (1 in 18-20 and 2 in >36 months on natalizumab), who had no symptoms or MRI lesions consistent with PML. None of the subjects had JCV DNA in plasma but viruria was detected in 8/39 (20.5%) patients. JCVspecific T cells were detected more frequently by ICS than ELISpot ex vivo [26/39(66.7%) vs 6/39(15.3%);p<0.001] and after in vitro stimulation [39/39 (100%) vs 33/38 tested (86.8%);p=0.03]. Both assays were significantly more frequently positive after in vitro stimulation than ex vivo (p<0.001). JCV-specific CD4+ T-cells were more frequently detected than CD8+ T-cells after in vitro stimulation [39/39(100%) vs 34/39(87.2%);p=0.05]. No differences in TΓÇôcell responses were observed between patients with and without viruria. Testing of JCV DNA in peripheral blood mononuclear cells is in progress. Conclusions: Asymptomatic JCV reactivation may occur in CSF of natalizumab-treated MS patients. In vitro stimulation with JCV peptides allows enhanced detection of JCV-specific cellular immunity which is highly prevalent in MS patients regardless of treatment, and is mediated by CD4+ and CD8+ T-cells. Microglial cells, the resident macrophages of the central nervous system (CNS), play important roles in maintaining homeostasis in the CNS and in the pathogenesis of HIV-associated neurological disorders (HAND). Brain imaging reveals increased microglial activation in patients with chronic HIV-1 infection, and in vitro studies suggest that altered microglial function may affect neuronal health. The role of miRNAs in regulating microglia activities in the context of HAND remains to be defined, although altered miRNA profiles in blood mononuclear cells of patients with chronic HIV-1 infection have been reported. Furthermore, differential expression of selected miRNAs in the cerebrospinal fluid of HIV+ patients with and without encephalitis has been demonstrated. To date, no study has profiled miRNA changes in primary human microglia upon HIV-1 infection and exposure to HIV-1 envelope proteins. To address this, we infected primary human fetal microglia (PHFM) with macrophage-tropic HIV-1 pseudotype (BAL), vesicular stomatitis virus-G pseudotype (VSV-G) or incubated PHFM with HIV-1 envelope glycoprotein gp120. RNAs was extracted from virus/envelope treated cells and mock treated cells at various time points. ABI Openarray assay was performed on the extracted RNAs to profile miRNA changes, and these were also examined using TaqMan quantitative Real-Time PCR (qRT-PCR). BAL infection resulted in 4-fold increase in miR-146a levels at 48hr post infection and gp120 exposure did not have significant effect on miR-146a levels. In future studies, we will elucidate whether observed changes in miRNA levels contribute to altered microglial function during the course of HIV infection in the CNS. Changes to the Genome of Theiler's Virus Can Alter the Pathogenesis Leading to Immunosuppression Matthew Cusick, Jane Libbey, Robert Fujinami (presenting author: matthew.cusick@path.utah.edu) Viruses, such as human immunodeficiency virus, hepatitis A, poliovirus, coxsackievirus B3, and foot-and-mouth disease virus, use a variety of mechanisms to suppress the human immune system in order to evade clearance by the host. Therefore, investigating how a few changes in the viral genome of a nonlethal virus can lead to an alteration in disease, from survivable to immunosuppression and death, would provide valuable information into viral pathogenesis. In this study we provide a model of a murine virus [DA stain of Theiler's murine encephalomyelitis virus (TMEV)] which in its natural host (the mouse) causes, when administered via a peripheral route (intraperitoneal -i.p.), an asymptomatic infection followed by viral clearance with lasting immunity. C57BL/6 mice infected with the DA strain of virus via the intracerebral (i.c.) route develop acute encephalitis; mice survive the acute disease and clear the virus. A mutant of the DA strain of TMEV was inadvertently created as a result of transcription error(s) by the T7 polymerase while using a modified full-length infectious cDNA clone of the DA virus as template. The H101 mutant virus encodes a point mutation (T101I) in VP1 (called H101). In addition, in sequencing the H101 viral genome, there were also several nucleotide substitutions in the 5' untranslated region as well as additional amino acid substitutions in the capsid protein coding region, suggesting that there are a number of perturbations in the viral genome. C57BL/6 mice infected with the H101 mutant virus via a peripheral route become immunosuppressed by killing T cells. This study provides experimental evidence that a virus that is cleared by its natural host can become lethal due to just a few changes in the viral genome. Transcriptional control of the human immunodeficiency virus type 1 (HIV-1) promoter or long terminal repeat (LTR), is achieved by interactions with cis-acting elements present both upstream and downstream of the transcriptional start site. In silico transcription factor binding analysis of the HIV-1 subtype B LAI LTR revealed a potential downstream CCAAT enhancer binding protein (C/EBP) binding site. This element is present immediately downstream of nucleosome 1 in the viral promoter, which suggested that it may have a functional role in transcriptional regulation because remodeling of this nucleosome is a crucial event in transcription in productively infected cells. Analysis across HIV-1 subtypes has indicated that the LTR downstream C/EBP binding site displayed a high degree of conservation in terms of both nucleotide sequence and physical location in the LTR. Interestingly, this element overlaps with a previously identified AP3-like element, which has been shown to bind members of the nuclear factor of activated T cells (NFAT) family of proteins. NFAT c2 exhibited a higher relative affinity for this element as compared with members of the C/EBP family (C/EBP alpha and beta). The downstream C/EBP element was able to compete efficiently with the low-affinity upstream C/EBP binding site I with respect to C/EBP binding, suggesting utilization of both NFAT and C/EBP. Moreover, cyclosporine A treatment, which has been shown to prevent dephosphorylation and nuclear translocation of NFAT isoforms, resulted in enhanced C/EBP beta binding. A downstream C/EBP binding site knockout mutant also demonstrated reduced LTR-driven transcription under both basal and interleukin-6-stimulated conditions, indicating that interactions at this site positively regulate HIV-1 transcription in cells of the monocyte-macrophage lineage. This work is supported by NIH/NINDS R01 NS32092, NIDA R01 DA19807, NIMH P30 MH092177, and NIMH T32 MH079785. The DrexelMed HIV/AIDS Genetic Analysis Cohort in Philadelphia, PA currently follows over 500 HIV-1-infected individuals longitudinally to examine viral genetic variation in conjunction with clinical and neurological disease severity and the impact of comorbidities like drugs of abuse on these parameters. This substantial cohort allows for a unique analysis of neurological decline in the context of substance abuse. Along with standard clinical parameters such as CD4+/CD8+ T-cell count, viral load measurements, and drug testing, a modified version of the Hopkin's Dementia Bedside Test (TMHDS) was performed. Due to the longitudinal nature of the DrexelMed cohort, it has been possible to investigate the complex effects of drug abuse on HIV-1-infected individuals. The complex nature of drug abuse in an urban cohort complicates the traditional method of grouping patients into monouse categories. Instead we abandoned these traditional methods in favor of Markov Chains. These chains model patients as "stateful machines" in which the change in HIVD score at the next visit is a function of parameters measured at the current visit. The current viral load, CD4+/CD8+ T-cell counts, adherence to HAART therapy, and drug testing results were included in the model. Cocaine use was found to have a detrimental effect on the TMHDS score while cannabinoids exhibited a small but seemingly measurable protective effect. As this study progresses, we will work to develop neurological testing protocols with increased sensitivity that will allow us to better understand the effects of poly-abuse on neurocognitive decline. This work is supported by NIH/NINDS R01 NS32092, NIDA R01 DA19807, NIMH P30 MH092177, and NIMH T32 MH079785. Natalizumab-associated JC virus granule cell neuronopathy complicated by immune reconstitution inflammatory syndrome in a patient with multiple sclerosis Background: Natalizumab blocks trafficking of leukocytes to the brain, and has been associated with reactivation of JC virus(JCV) leading to progressive multifocal leukoencephalopathy (PML) in multiple sclerosis (MS) patients. JCV can also infect cerebellar granule cell neurons resulting in JCVgranule cell neuronopathy (JCV GCN). Case report: A 32 year-old JCV-seropositive gentleman with MS who received 63 infusions of natalizumab monotherapy presented with changes in his handwriting, speech and gait. Initial MRI did not identify any new lesions. CSF JCV PCR was positive 3 months after symptoms onset and subsequent MRIs showed progressive cerebellar atrophy. Natalizumab was discontinued and he received plasma exchange and IV steroids, followed by mirtazapine. He developed progressive hyperintensities in the pons and cerebellar peduncles on T2weighted images on MRI, and contrast-enhancement, suggestive of immune reconstitution inflammatory syndrome (IRIS). CSF JC viral load was 16,489 copies/ml. Intracellular cytokine staining showed a robust cellular immune response mediated by JCV-specific CD4+ and CD8+ T-cells. Molecular analysis of CSF JCV strains revealed three different mutations in the C-terminus of the VP1 gene, consistent with previously reported GCN type JCV strains. He was treated with IV steroids, followed by a decrease in contrast enhancement in his cerebellum on MRI. His neurological function stabilized concomitant with negative JCV CSF PCR. Repeat MRI/MR spectroscopy showed stable low grade cerebellar enhancement and a Lip1/Cr ratio <1.0 in cerebellum consistent with subsiding IRIS. However, there were more extensive T2 signal changes in the pons and cerebellar peduncles. Conclusion: This is the second patient with natalizumabassociated JCV GCN IRIS. Contrary to the initial casereport 1, 2, this patient developed contrast enhancement in the cerebellar cortex on MRI and extension of the lesions in the cerebellar white matter and pons. JCV GCN is a novel complication of natalizumab therapy and should be suspected in any MS patient developing cerebellar dysfunction. Innate immunity regulates blood brain barrier function during West Nile virus encephalitis via type-I interferon. Brian Daniels 1 , Lillian Cruz-Orengo 2 , David Holman 2 , Robyn Klein 1-3 (presenting author: brian.daniels@wustl.edu) West Nile virus (WNV) is a mosquito-borne pathogen capable of infecting the central nervous system (CNS) and causing lethal encephalitis in human hosts. However, the mechanisms by which WNV accesses and infects the CNS are mysterious, as pathogens are normally excluded from the CNS by the blood brain barrier (BBB). The BBB is a complex assortment of vascular endothelial cells joined by tight junctions that prevent circulating pathogens from escaping the vasculature in the CNS and accessing parenchymal nervous tissue. Detection of WNV in the circulation by host tissues elicits innate immune responses, including the production of type-I interferon (IFN), which has been shown to promote and preserve BBB integrity in the context of CNS autoimmunity. Here, we utilize an in vitro model of the BBB to demonstrate that pathogen detection of WNV in the vascular endothelia of the BBB promotes barrier integrity via type-I IFN, rescuing barrier dysregulation by inflammatory cytokines and limiting transendothelial trafficking of WNV via modulation of endothelial Rho-GTPase signaling. Similarly, mice with diminished type-I IFN signaling (IFNAR-/-, IRF7-/-) exhibit enhanced BBB permeability after peripheral WNV infection and earlier entry of virus into the CNS. Together, these data are the first to show a functional role for type-I IFN at the BBB in the context of a neurotropic viral infection, suggesting new roles for type-I IFN in the treatment of neuroinflammatory and infectious diseases. Francesca De Simone, Onder Otlu, Ilker Sariyer (presenting author: isariyer@temple.edu) Department of Neuroscience and Center for Neurovirology, Temple University School of Medicine, Philadelphia, PA Immunosuppression caused by pathologic agents such as HIV-AIDS or by regiments used for the treatment of different types of diseases such as multiple sclerosis puts patients in high risk group of developing progressive multifocal leukoencephalopathy (PML), a fatal demyelinating disease of the white matter caused by human neurotropic polyomavirus, JCV. The virus establishes a latent infection and reactivates under immunosuppressive conditions with an unknown mechanism. Immune-dependent reactivation of JCV and the development of the disease suggest that JCV gene expression and replication are tightly controlled by the immune system in latently infected cells potentially mediated by immune mediators at the tissue level. We have developed an in vitro model to study the role of immune-mediators secreted by active immune cells in the viral gene expression and replication. PMBSc from peripheral blood were cultured and activated in tissue culture and conditioned media (CM) was collected to treat glial cells infected with JCV. A series of experiments suggested that CM from induced but not from uninduced PBMCs inhibited the propagation of the virus suggesting immune-mediated control of viral life cycle. Further studies revealed that soluble immune mediators from PBMCs possessed a dual control on T-antigen expression at transcriptional and posttranscriptional level. We recently identified the alternative splicing factor, SF2/ASF, as a potential regulator of JCV as its overexpression in glial cells strongly suppresses viral gene expression and replication. Results from our preliminary studies suggest that immune mediators secreted from PBMCs induce the expression of SF2/ASF, and inhibit the replication of JCV. These observations suggest operation of a novel immune signaling pathway between peripheral immune cells and glial cells that controls the immediate early stage of JCV gene expression during the course of viral reactivation. This work was made possible by grants awarded by NIH to IKS. Lack of correlation between JC Virus urinary shedding and seropositivity in Multiple Sclerosis patients treated with natalizumab Background: The efficacy of natalizumab in the treatment of multiple sclerosis (MS) is high, but 372 cases of progressive multifocal leukoencephalopathy (PML) have been reported among treated patients. The risk of PML is calculated by assessing JC virus (JCV) seropositivity in natalizumab-treated MS patients. Methods: In total, 42 natalizumab-treated MS patients and 45 MS patients treated with conventional therapies (controls) were enrolled in a case-control study. Urine and blood samples were collected monthly for up to 60 months from the natalizumab-treated patients and once from the controls; these samples were used to monitor JCV and BK virus (BKV) replication. Viral loads were assessed using quantitative realtime PCR (qPCR) assays, and serum anti-JCVantibodies were measured with the Stratify and/or Stratify DxSelect tests. Results: JCV DNA was found in 229 of 741 (30%) urine samples overall and at least once in 21 of 42 (50%) urine samples from natalizumab-treated patients. However, JCV DNA was also detected in 11 of 45 (24.4%) control samples (p<0.05). In the natalizumab-treated patients, JCV DNA shedding in the urine significantly increased up to month 24 of treatment (45.2%, R2=0.86). BKV was found in the urine of 59.5% (25/42) natalizumab-treated patients and in a total of 126 of 741 (17.1%) urine samples overall. In contrast, viral genomes were not detected in the blood. Additionally, JCV viruria and seropositivity did not correlate with each other, and three viruric patients were seronegative according to the Stratify and Stratify DxSelect tests. Conclusions: Our findings demonstrate that natalizumab therapy may increase the rate of JCV urinary shedding and that the correct identification of JCV carriers cannot solely rely on serological tests. To correctly stratify patients by the risk of PML, monitoring the presence of JCV DNA in the urine is needed. Satish Deshmane, Paul Pozniak, Kamel Khalili, Prasun Datta (presenting author: sld907@temple.edu) Department of Neuroscience, Comprehensive NeuroAIDS Center, Temple University Neuronal dysfunction and degeneration are the causative mechanisms for the HIV-Associated Neurocognitive Disorders (HAND) in the era of highly active antiretroviral therapy (HAART). In this study we assessed the effects of exosomes derived from PMA activated promonocytic cells U1 that are latently infected with HIV-1 on human fetal neurons. Exosomes secreted by U1 cells were found to be enriched in as many as 20 micro-RNAS, potentially affecting expression of several essential genes in neurons. Neuronal cultures treated with U1 exosomes were found to be severely compromised in their ability to maintain existing neuronal network as well as their ability to form neurites in a scratch-wound assay. Neuronal cultures treated with U1 derived exosomes showed low levels of superoxide dismutase activity indicating heightened oxidative stress. Analysis of the cytokine/chemokine profile in U1 exosomes revealed attenuated levels of MCP-1, MIP-1alpha, MIF, RANTES and IL-4 in comparison to U937 derived exosomes. Similar analysis of medium obtained from exosome-treated neuronal cells revealed increased expression of MCP-2, MCP-3, MIP-3alpha, NAP-2 and uPAR in cultures treated with U937 exosomes. Furthermore, we also analyzed the phosphorylation status of mitogen-activated protein kinases (MAPKs) and other intracellular proteins and kinases, such as Akt, GSK-3, p70S6 Kinase, mTOR, p53, and CREB that are important regulators of signal transduction and cell proliferation, in neurons treated with U1 exosomes using antibody arrays. These studies demonstrated that many signaling pathways that are known to be crucial for cell survival such as Akt, mTOR, PI3K were found to be negatively affected in U1 exosome treated neurons. Collectively, these observations demonstrate that exosomes derived from HIV-1 infected cell can cause neuronal dysfunction and degeneration by targeting multiple pathways. Supported by Comprehensive NeuroAIDS center (CNAC) developmental core grant to PKD. The study also utilized services offered by core facilities of CNAC (NIMH Grant#P30MH092177) to KK. Neuroinflammation mediated by pro-inflammatory cytokines such as interleukin (IL)-1 beta induced by HIV-1 infection of macrophages, microglia, and astrocytes in the CNS play critical role in the pathogenesis of NeuroAIDS. Dysregulation of inflammation-associated microRNAs such as miR-146a within the brain can accelerate the development of HIVAssociated Neurocognitive Disorders (HAND). Thus, we investigated the role of post-translational modification of RelA in miR-146a gene regulation by IL-1 beta in human fetal brain astrocytes and in astroglial cell lines. Our studies demonstrate that IL-1beta-induced phosphorylation of IKKbeta, IkBalpha and p65 at serine 536 regulate NF-kappaB activation in human fetal astrocytes and in astroglial cell lines. Additionally, our results using phosphorylation defective mutants of p65 indicate that phosphorylation status of serine residues 276 and 536 differentially regulate IL-1beta induced transcriptional expression of miR-146a. To demonstrate the role of mitogen-and stress-activated protein kinase1 (MSK1) that phosphorylates S276 in the regulation of mir-146a by IL-1 beta we employed a pharmacological inhibitor of MSK1, H-89. Our studies demonstrate that H-89 (5 and 10 microM) dose dependently inhibited miR-146a expression. Collectively, our observations demonstrate that IL-1beta activates both canonical and noncanonical NF-kappa B pathways to regulate mir-146a expression in astrocytes. Therefore, pharmacological inhibitors that target NF-kappa B pathways may have therapeutic potential for the treatment of neuroinflammation. This work is supported by NIH/NIDA grant to PKD. The study also utilized services offered by core facilities of the Comprehensive NeuroAIDS center (CNAC) NIMH Grant # P30MH092177. A metabolomics approach to the differentiation of cognitive states in HIV-infected patients Currently, there are no defined surrogate measures to determine which HIV+ patients are likely to develop cognitive impairment or to track the effectiveness of potential neurotherapeutics. Here, we analysed CSF samples taken from HIV+ patients with differing neurocognitive states using 1H-NMR spectroscopy and multivariate analysis to identify novel metabolomic biomarkers for cognitive impairment. CSF samples from 100 HIV-infected patients were collected at 2 time points with four distinct neurocognitive states that were defined by temporal changes in cognitive status. These neurocognitive states were stably-normal, stably-abnormal, improving, and worsening. 1D-NOESY 1H-NMR spectroscopy was performed on these samples using a 16.4T NMR system. Multivariate Partial least squares (PLS) regression models showed significant separations between neurocognitive states with q2 values = 0.61 (stably-normal), 0.84 (stably-abnormal), 0.47 (worsened) and 0.52 (improved). The metabolites that underpinned these changes were distinct in each group, but were generally connected to amino acid, and energy metabolism. Using a classification and regression tree analysis we identified changes in 5 energy metabolites (pyroglutamate, citrate, creatine, alanine, and acetate) that were prognostic indicators for declines in cognitive status (sensitivity 100%, specificity 88%), and 5 metabolites that were prognostic indicators for improved cognitive status (glutamate, pyroglutamate, creatine, myoinositol, betaglucose (sensitivity 91%, specificity 92%). Linear regressions showed that increased levels of specific energy metabolites were associated with poor performance on tasks that measure executive, learning, recall and motor functions, but were not related to working memory performance. These findings suggest that declines in cognitive function are accompanied by a compensatory increase in specific energy metabolites. CSF levels of these energy metabolites may be useful surrogate measures for temporal shifts in cognitive status, and reveal new insights into the neuropathogenesis of HAND. Herpes simplex virus type 1 (HSV-1) is a ubiquitous human pathogen which causes recurrent mild orofacial lesions in infected individuals. Acute infection initiates in mucosal surfaces where the virus replicates within epithelial cells. Following propagation of virus at the site of infection, innervating sensory neurons are infected and a latent infection is established which persists for the lifetime of the host. The immune system is critical for controlling initial virus replication in the periphery and limiting infiltration into the nervous system. We investigated the mechanism that immune cells use to detect productive viral replication in the periphery. TLR-2 knockout (TLR2-/-) mice were more vulnerable to HSV-1induced mortality than wild-type (WT) controls. Although natural killer cells were normal, dendritic cell and CD8+ Tcell activation was impaired in TLR2-/-mice. We observed a minor decrease in CD8+ T-cells responding to virus infection in the knockout animals, but the responding cells exhibited impaired production of interferon gamma. Both WT and knockout animals supported comparable HSV-1 replication in the periphery, but the knockout mice had greater infiltration into the nervous system and suffered greater mortality. These results demonstrate the important role of TLR-2 in priming immune cells and control of acute HSV-1 infection. In the future, we will investigate the role of TLR-2 in maintaining immune control of HSV-1 latency by CD8+ T-cells. We will confirm that CD8+ T-cells in the trigeminal ganglia belong to the recently described Tissue Resident Memory (TRM) class of memory T cells. We will explore the homing mechanisms and proliferative capacity of TRM cells for the purpose of enhancing CD8+ T-cell levels in the trigeminal ganglia. Increased presence of CD8+ T-cells is a possible therapeutic treatment option for reducing reactivation events. High frequency of HIV-related neurologic complications in a Romanian cohort of children and young adults infected with subtype F Design: This is a retrospective longitudinal study of children and adolescents with HIV associated neurological complications followed between January 1996 and June 2010. Methods: All patients with HIV encephalopathy (HIVE) or HIV related opportunistic infections were included. We evaluated demographics, viral load markers and brain imaging and hystology, to describe the clinical dynamics in this unique cohort. Results: During the 14.5 year surveillance period a total number of 275 children and young adolescents were diagnosed with neuroAIDS defining diseases representing 49.2% out of 559 subjects. Although the number of HIV associated CNS complications decreased after introduction of cART, the frequency among AIDS defining diseases increased from 44.5% before cART, to 54.1% thereafter. Unique features were observed in PML patients, including cerebellar and brainstem location of lesions and improved survival in cART regimens with good cerebrospinal fluid HIV viral control. Furthermore, we describe a case series of 34 patients diagnosed with a fatal clinical entity: subacute myoclonic measles encephalitis (SMME) that occurred during two consecutive outbreaks in Romania. Conclusions: We found a high frequency of HIV-related neurological complications in a cohort of parenterally infected children that, interestingly, increased in the cART era. We also describe a new AIDS defining neurologic complication of measles in severe immunosuppressed children: SMME. The frequency of almost 50% of neurological AIDS complications suggests a neurotropic potential for HIV clade F. Yoshimi Enose-Akahata, Raya Massoud, Jussi Virtanen, Steven Jacobson (presenting author: akahatay@ninds.nih.gov) Viral Immunology Section, Neuroimmunology Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health Intrathecal antibody synthesis is a well-documented phenomenon in infectious neurological diseases as well as in demyelinating diseases. Intrathecal antibody synthesis against HTLV-1 has been reported in HAM/TSP, but little is known about the role of B cells and humoral immune responses in the central nervous systems (CNS) of HAM/TSP patients. Here we demonstrate profiles of HTLV-1-specific antibodies in cerebrospinal fluid (CSF) of HAM/TSP patients. Of 36 HAM/TSP patients, antibody responses against Gag and Tax were detected in CSF of all the patients. CSF/Serum anti-Gag antibody ratio was elevated more than anti-Tax (anti-Gag: mean 1.20, anti-Tax: mean 0.85), and was significantly correlated with proviral loads in PBMC. More importantly HAM/ TSP patients with lesions or atrophy in spinal cord showed higher CSF/Serum anti-Gag antibody ratio. Antibody response against Env was detected in CSF of 94.4% of patients, but CSF/serum anti-Env ratio was significantly lower than those of anti-Gag and anti-Tax (mean 0.18). Interestingly, CXCL13 (B cell attracting chemokine-1) and IL-21 were increased in CSF of HAM/TSP patients compared to HTLV-1 positive asymptomatic carriers, which was associated with higher HTLV-1-specific antibody responses in CSF. In ex vivo experiments, CD4+ T cells of HAM/TSP patients showed significantly higher IL-21 expression than those of healthy normal donors. In addition, IL-21 stimulated B cells differentiate into plasma cells and produce HTLV-1-specific antibodies in the cultured B cell of HAM/TSP patients. These results highlight the importance of the B cell compartment in HAM/ TSP where production of HTLV-1-specific antibody may be required to control viral persistence and/or may be associated with HAM/TSP disease progression. Background: Despite successful suppression of HIV-1 replication with combination antiretroviral therapy, chronic immune activation persists in some patients, a process that may be mediated through latently infected cells in various compartments, including the central nervous system. Raltegravir, a potent HIV integrase inhibitor was previously shown to inhibit immune activation in peripheral blood. The goal of the present study was to determine how raltegravir influences proinflammatory cytokine production in HIV-infected monocytes, microglia, and assess toxicity in neuron-glia cultures. Methods: Primary human monocyte derived macrophages and microglia from fetal brain tissue were grown in culture and infected with m-tropic HIV (BaL). Infected cultures were treated with 20 nM raltegravir or vehicle (saline). Supernatant was removed periodically for 9 days and pro-inflammatory cytokines were quantified using Mesoscale Discovery 7-plex platform. Primary neuron-glia cultures were exposed to 0, 20, 100 nM raltegravir for 7 days, then stained for beta-III tubulin and glial fibrillary acidic protein, and immunofluorescence quantified. Results: In both microglia and monocyte derived macrophages, the rate of interleukin-8 secretion was significantly lower for HIV-infected cells treated with raltegravir (132 pg/d) compared to Control (245 pg/d), HIV-infected (232 pg/d), and raltegravir alone (268 pg/d), as determined by analysis of covariance and Tukey's Honestly Significant Difference test; p < 0.001. TNF-alpha, interferon-gamma, IL10, IL12,IL1, and IL6 were not significantly different. In the neuron-glia cultures, at 20 nM, there was significantly more beta-III tubulin area. There was a stepwise decrease in GFAP area at 20 and 100 nM raltegravir. Conclusion: Raltegravir, in the context of HIV infection, leads to diminished production of the potent pro-inflammatory chemoactractant, IL-8 in both monocytes and microglia. The data suggest that raltegravir inhibits astrogliosis and may be neuroprotective. Further study on mechanisms, including NF-kappa B nuclear translocation are underway. Eliseo Eugenin (presenting author: eugeniea@umdnj.edu) Cell to cell communication is essential for development of multicellular systems, and is coordinated by soluble factors, membrane associated proteins, gap junction channels and the recently described tunneling nanotubes (TNT). We and others reported that TNT can be used for pathogens, such as HIV between communicated cells. However, the characterization of these processes, especially in primary cells, and their role in HIV infection has not been fully characterized. Our current data indicates that TNT are composed of several smaller TNT. TNT in macrophages are not continuous tubes, rather TNT establish contact with others macrophages by a synaptic type of contact. We identify several blockers of TNT that reduces spread of HIV between HIV infected and uninfected cells by transfer viral genetic material between communicated cells. Blocking formation of TNT using mild actin blockers or siRNA reduced the spread of HIV among connected cells. Thus, we characterize that TNT play a key role in the spread of HIV among connected cells. Viral infection and inflammation in the central nervous system (CNS) can induce significant illness and neuropathology, particularly in the very young. Though neonates are capable of mounting both innate and adaptive immune responses, they are often unable to control viral infections in the brain and suffer extensive neuronal loss and tissue damage. In order to study how the neonatal immune response interacts with infected CNS neurons, our laboratory uses a transgenic mouse model (NSE-CD46) of neuron-restricted measles virus (MV) infection. NSE-CD46 mice express the human isoform of CD46, a MV receptor, under the control of the neuron specific enolase (NSE), allowing for infection only in CNS neurons. Here, we show that the adaptive immune response is detrimental in neonates during a viral infection in CNS neurons, as neonates lacking T-and B-cells (CD46+/RAG2-KO mice) survive longer than immunocompetent pups. CD46+/RAG2-KO neonates also control viral load more effectively than immunocompetent NSE-CD46 neonates. These results are in contrast to adult NSE-CD46 mice, which survive the infection and clear the virus from the brain in a T-cell dependent manner, whereas CD46+/RAG2-KO adult mice succumb to the infection. In addition, neonatal mice that lack interferongamma (IFNg), which is required for viral clearance from the adult brain, lose nestin-positive precursor cells in contrast to NSE-CD46+ neonates despite higher levels of T-cell infiltration in CD46+/ IFNg-KO neonates. These findings suggest that certain pro-inflammatory cytokines can protect neural cells during a neonatal CNS infection, but that the full adaptive response is associated with greater neuropathology. Current work addresses defects in the neonatal Tcell response and the potential role of IFNg-producing natural killer cells in limiting viral spread. Through these studies, we aim to better define the protective and cytotoxic roles of innate and adaptive immune cells during a neonatal CNS infection. Injection and non-injection illicit drug use and abuse remain significant cofactors for HIV infection and transmission. Cocaine is one of the most widely abused drugs in the United States, which both impair the normal functioning of brain cells and also activate human immunodeficiency virus (HIV) expression in central nervous system (CNS). As a result, HIVinfected individuals who abuse cocaine experience more severe and rapid onset of NeuroAIDS than non-abusing individuals. It has been known that cocaine affects the expression of numerous cellular genes by modulating various signaling and epigenetic pathways. Some of those pathways also influence the expression of integrated HIV proviruses and eventually end up enhancing HIV replication and transmission. It is known that cocaine activates NF-kappaB, and given the importance of NF-kappaB during HIV transcription, it is imperative to understand the underlying molecular mechanisms that ultimately result in the enhanced HIV gene expression. Our results establish that cocaine activate specific protein kinases, which in turn induce selective posttranslational modifications in NF-kappaB. To further extend our understanding about the regulation of HIV gene expression via cocaine induced NF-kappaB systems, we have employed a multi-level approach to characterize the effect of cocaine on the chromatin state of provirus by running several ChIP (Chromatin Immunoprecipitation) assays. Overall, our results demonstrate the exchange of various key epigenetic modifications and associated factors at HIV LTR following cocaine treatment. In summary, our data provide direct evidence that cocaine enhances HIV gene expression not only by activating transcription factors such as NF-kappaB, but also by inducing various selective epigenetic modifications. These events eventually support the establishment of transcriptionally active chromatin structure at HIV LTR and facilitate HIV transcription. Cell culture and bioinformatic identification of putative progressive multifocal leukoencephalopathy risk factors Michael Ferenczy 1 , Kory Johnson 2 , Eugene Major 1 (presenting author: michael.ferenczy@nih.gov) 1 Laboratory of Molecular Medicine and Neuroscience, National Institute of Neurological Disorders and Stroke, National Institutes of Health; 2 Bioinformatics Section, National Institute of Neurological Disorders and Stroke, National Institutes of Health JC virus (JCV) is the etiological agent of the human CNS demyelinating disease progressive multifocal leukoencephalopathy (PML). Attempts to identify risk factors for PML are hampered by the lack of an animal model and the limited cell types in culture that are susceptible to JCV infection. To understand JCV pathogenesis, it was necessary to develop a new system to identify transcription regulators important in the JCV lifecycle and development of PML. Traditional techniques of protein over-expression and knockdown are hampered by off-target effects when transcription regulators important in viral gene expression are targeted, limiting the ability to discern direct from indirect effects (overor under-expression of genes regulated by the transcription regulator of interest). Using multiple cell culture models with differing permissiveness to JCV, cellular gene expression in both uninfected and infected cells was characterized. Multiple known and novel transcription regulators with potential JCVregulatory roles were identified by transcriptome profiling of SV40 T antigen immortalized human fetal brain cells displaying characteristics of either CNS stem cells or radial glia. Several of these factors overlapped with factors identified during differentiation of less permissive human fetal neural progenitor (progenitors) cells to highly permissive progenitorderived astrocytes (PDAs). Lymphoid Enhancer Factor 1 (LEF1) was identified as a potential novel negative regulator of JCV transcription, and potential binding sites for LEF1 in the JCV regulatory region were identified using computational promoter analysis. Progenitor-derived neurons (PDNs) and PDAs were used to confirm LEF1 as important for JCV transcription. Chromatin immunoprecipitation revealed that LEF1 bound the JCV promoter to a greater extent in JCVrepressive neurons than in JCV-permissive astrocytes. These results demonstrate that human fetal brain derived CNS stem cell models combined with bioinformatic approaches can facilitate the identification of factors responsible for JCV multiplication and potential development of PML. Proinflammatory cytokines and gp120 may contribute to synaptic injury through upregulation of neuronal ferritin heavy chain in HIV patients The molecular mechanisms involved in HIV-induced synaptodendritic injury, the structural basis for HIVassociated neurocognitive disorders, have yet to be fully elucidated. In this study, we propose that HIV proteins and inflammatory cytokines induce dendritic injury by altering the neuronal expression of the protein ferritin heavy chain (FHC), a known negative regulator of the CXCL12/CXCR4 signaling pair. Previous work from our lab suggests the importance of CXCL12 in increasing dendritic spine density, as well as modulating the expression of specific glutamate receptor subunits (NR2B). Additionally, we have found that opiates, including morphine, can negatively regulate CXCR4 signaling through FHC. In vivo data from brain tissue of HIV+/drug abusers (and SIV-infected/morphine-treated macaques) show an increase in neuronal FHC and a subsequent decrease in CXCR4 activation (Ser339 phosphorylation). Thus, we sought to investigate whether components of HIV infection could modulate FHC. Our results suggest that TNFalpha and IL-1 beta, as well as the HIV envelope glycoprotein gp120, upregulate FHC in neurons. Both of the cytokines altered neuronal FHC in the presence or absence of glia; however, gp120 (X4-or R5-using) only caused significant increases in neuronal/glial bilaminar co-cultures, suggesting that glia are required for the changes in FHC protein levels by gp120. In support of this, the presence of an IL-1 beta neutralizing antibody (or receptor antagonist) in gp120-treated bilaminar cultures abrogated this effect. In two in vivo models of HIV infection (gp120-ICV injected and HIV-Tg rats), we saw a significant reduction in cortical neuron dendritic spine density compared to age-matched controls. Overall, these studies suggest that the effects of gp120, proinflammatory cytokines, and opiates may converge on dendritic spines, through FHC, and augment synaptic injury in HIV/drug abuse patients. Screening for neutral sphingomyelinase inhibitors as a novel target in human immunodeficiency virus-1 associated neurocognitive disorders Perturbations in brain lipid metabolism with accumulation of the bioactive lipid ceramide occur early in the course of human immunodeficiency virus 1 (HIV-1)-associated neurocognitive disorders (HAND). Elevations of ceramide are thought to contribute to neuronal dysfunction through biophysical effects that alter membrane excitability and molecular response to stress. Ceramide can be rapidly generated in response to inflammatory stimuli and by the HIV-1 coat protein gp120 through actions mediated by the sphingomyelin phosphodiesterase neutral sphingomyelinase 2 (nSMase2). Expression and activity of nSMase2 is elevated in brain tissues from HIV-1 infected patients in conjunction with cognitive impairment, suggesting that inhibition of nSMase2 may preserve neuronal function by preventing increases in ceramide. Indeed, both chemical and genetic modulation of nSMase2 expression or activity are neuroprotective, making this enzyme an attractive therapeutic target. There are a few known inhibitors of nSMase2 that include natural products and small molecules, but these compounds lack specificity, are insoluble and/or exhibit low potency. To identify novel inhibitors specific against human nSMase2, we screened pharmacological active compounds, approved drugs, and inhibitors of acid sphingomyelinase in 384-well format. Enzyme activity was monitored as increases in the fluorescence emission of a product generated after three consecutive coupled reactions that involved alkaline phosphatase, choline oxidase, and horseradish peroxidase. Hits were confirmed using a direct assay with 14C-labeled sphingomyelin (SM) as substrate. Several inhibitors with low micromolar potency were identified from distinct chemical series. The identified compounds are being optimized in structure-activity relationship studies. Small molecule inhibitors of nSMase2 may be useful therapeutic agents that could dampen the neuromolecular response to HIV-1-infection and associated inflammation. Development of microglial activation phenotypic assays as a tool to screen therapeutic agents for neuroAIDS and other neuroinflammatory disorders Mariana Figuera Losada, Camilo Rojas, Barbara S. Slusher (presenting author: mfiguer8@jhmi.edu) The brains of human immunodeficiency virus-1 (HIV-1) patients show signs of chronic inflammation including microglia activation which, in part, leads to neurodegeneration and the manifestation of HIV-1-associated neurocognitive disorders (HAND). Activated microglia and macrophages release excess glutamate which causes excitotoxicity that ultimately results in neuronal dysfunction and death. Two proteins, the cystine/glutamate antiporter (xCT), and the phosphateactivated glutaminase, are thought to be partially responsible for the excess glutamate released from activated microglia. xCT is a sodium independent amino acid transporter that imports extracellular cystine and exports intracellular glutamate. Glutaminase is a mitochondrial enzyme that catalyzes the production of glutamate from glutamine. Both have shown increased activity and/or expression levels following infection of microglia or macrophages with HIV-1, or activation with the viral protein Tat. We developed a cell-based phenotypic assay in 96-well format to screen novel xCT and glutaminase compounds for their ability to inhibit glutamate release from lipopolysaccharides (LPS)-activated microglia. Primary microglia obtained from newborn rats cultured for 10-21 days were treated with 1-1000 ng/mL LPS, which induced the production/release of glutamate in a dose-dependent manner (2-20 fold increase in extracellular glutamate). Response was time-(16-24 h) and cell number-dependent (10,000-50,000 cells/well). The prototype xCT inhibitor erastin and the glutaminase inhibitor DON showed dose dependent inhibition of glutamate release/production with EC50 values of 14 ± 3 nM and 1.5 ± 0.4 microM, respectively. Commensurate with its weaker xCT inhibitory capacity, sulfasalazine was significantly less potent than erastin (362 ± 60 nM). Phenotypic assays following excess glutamate release induced by microglia activation represent a novel way to screen for agents to treat neuroAIDS and other neuroinflammatory disorders. Human immunodeficiency virus (HIV)-infection continues to be a major clinical manifestation in the combination antiretroviral therapy era (cART), that is enhanced in opioid-drug abusers. The gastrointestinal (GI) mucosa plays a major role in HIV-infection as HIV is mostly transmitted across mucosal surfaces. The enteric nervous system (ENS) controls several GI processes including motility and secretion which are specifically affected in HIV-infected patients. Morphine and other opioids directly affect the ENS, causing severe constipation. Because the virus does not infect neurons, it is suggested that viral toxins, such as Tat, modulate neuronal function in HIV-1-infected patients. Tat has been shown to increase neuronal excitability but not much is known about effects of Tat on enteric neurons in combination with drug abuse. Recent experimental studies in our lab have shown that Tat increases enteric neuronal excitability by shifting the voltage-dependence of activation of sodium channels to more negative potentials. In contrast, morphine alone decreases the availability of sodium channels in enteric neurons reducing excitability. Voltage gated sodium channels (VGSCs) are responsible for the generation and propagation of action potentials in neurons. In order to determine the effects of Tat (100 nM) in combination with morphine (0.3 uM, 3 uM) on the voltage dependence of steady-state activation/inactivation of sodium channels, we used a double-pulse protocol in which a variable conditioning pulse was applied from -100 mV to +50 mV in 10 mV steps for 50 ms followed by a test pulse. In Tat-treated neurons, the sensitivity to morphine was significantly enhanced. The V0.5 for steady-state inactivation was shifted in Tat-treated neurons at 0.3 uM morphine with 50% decrease in maximal availability at -100 mV. Morphine did not alter the steady-state activation. Taken together, data suggest that Tat increases the sensitivity to morphine in enteric neurons that may exacerbate the deleterious effects of morphine on GI motility. Cari Fritz-French 1 , William Tyor 2 (presenting author: cfritzf@emory.edu) 1 Neurology Department, Emory University; 2 Department of Neurology, Emory University School of Medicine Elevated levels of interferon-alpha (IFNalpha) in the central nervous system (CNS) are linked to cognitive dysfunction in patients with inflammatory CNS diseases such as HIV-associated dementia. Previous studies showed that IFNalpha treatment of neuronal cultures caused a dose dependent decrease in dendritic branching and length, that was prevented after pre-treatment with IFNalpha neutralizing antibodies. Antagonists to NDMA receptor were also found to be partially protective against IFNalpha induced neurotoxicity. To begin to determine the mechanism of IFNalpha induced neurotoxicity, the cell signaling pathway involving IFNalpha receptor (IFNAR) was evaluated. We demonstrate increased interferon stimulating gene 15 (ISG15) expression, an indicator for JAK-STAT cascade activation, after IFNalpha stimulation. Inhibiting IFNAR was found to partially protect neurons from IFNalpha neurotoxicity, but not as efficiently as neutralizing antibodies to IFNalpha. Our preliminary studies suggest that IFNalpha is in part acting through its receptor as well as NMDAR to reduce dendritic arborization in neurons. Further studies are needed to determine what other signaling pathways may be involved in IFNalpha induced neurotoxicity. Determining the mechanism of IFNalpha neurotoxicity could lead to therapies for cognitive dysfunction during neuroinflammation. Macrophages are one of the principle cell types infected with HIV, and the primary source of virus in many tissues, including the CNS. Within the brain, macrophages are exposed to the neurotransmitter dopamine. Dopamine is elevated by the use of drugs of abuse such as cocaine and methamphetamine. We showed previously that macrophages express dopamine receptors and other dopaminergic proteins, and that exposure to dopamine increases HIV replication in macrophages and alters their cytokine production and MAPK signaling. Our current data demonstrate that the dopamine mediated increase in HIV replication is due to an increase in the ability of HIV to enter macrophages. This effect is dose dependent, occurring at greater than 10 nM dopamine, and is not affected by dopamine metabolites. A pan-dopamine receptor antagonist abrogated the effect, indicating that dopamine receptor activation is necessary for increased entry. Infection in the presence of D1-like or D2-like dopamine receptor agonists shows that the increase can be induced by activation of both dopamine receptor subtypes. Antagonizing CCR5 with TAK779 completely blocks entry, indicating that dopamine mediates is effects through the CCR5 entry process. However, the increased susceptibility to entry is not due to an increase in surface CCR5. These data demonstrate that the macrophage dopaminergic system plays an important role in HIV infection of these cells. The development of neuroinflammation and HAND may be accelerated by the effects of dopamine on macrophages, and that these effects maybe exacerbated by drug abuse. In addition, although different drugs of abuse act through distinct pharmacologic mechanisms, dopamine mediates the addictive and reinforcing effects of many drugs of abuse and may be a common mechanism by which drugs of abuse contribute to HIV associated neuropathogenesis. High expression of interleukin 10 and interferon regulatory factor 4 are correlated with integrated HIV DNA in brain neocortex HIV DNA that is integrated into human genomic DNA (HIVint) may become transcriptionally repressed and immunologically inert in infected people in whom virus replication is suppressed with highly active antiretroviral therapy (HAART). The "latent" pool of HIVint represents a key obstacle to curing HIV infection because it re-seeds the body with replicating HIV when HAART is stopped. Little is known about the neuroimmunological factors involved in supporting the latent pool of HIVint in the brain. We measured HIVint in genomic DNA extracted from the frontal neocortex of 40 HIV infected patients using the O'Doherty Alu-Gag two-step assay. Cases were divided into two equal groups having either a high proportion of HIVint (> 3% of the replication rate in brain) versus low (< 3%). The groups had equivalent rates of HIV transcription (HIV RNA load) in the brain specimens. A panel of neuroimmune marker mRNAs was quantified using qPCR in the groups. The group with high HIVint had significantly greater expression of interleukin 10 (IL10) and interferon regulatory factor 4 (IRF4) mRNAs. Several other neuroimmunological marker mRNAs were not significantly different. These two proteins are associated with in vitro polarization of macrophages towards M2 phenotypes. When typical M2 marker mRNAs including CD163 were assayed, they were not expressed at significantly higher rates in the specimens with high HIVint. Conclusion: High HIVint in the brain is associated with IL10 and IRF4 expression, which play key roles in M2 macrophage polarization. We suggest that HIVint accumulates preferentially in a numerically minute subtype of M2 polarized macrophage in the brain. To identify the cellular sources of latent HIV in the brain, we are screening specimens for M2 surface markers that specifically segregate with high HIVint load. Higher Levels of Plasma Soluble Insulin-Like Growth Factor-1 Receptor are Associated with Severity of HAND in HIV-seropositive Women Yamil Gerena 1 , Raissa Menendez-Delmestre 2 , Richard Skolasky 3 , Rosa Hechavarria 4 , Sebastian Perez 5 , Claudia Hilera 6 , Claribel Gonzalez 2 , Avindra Nath 7 , Valerie Wojna 8 (presenting author: yamil.gerena@upr.edu) Background: Insulin resistance is present in HIV-seropositive people using combined antiretroviral treatment (cART) and associated with HAND. However, the mechanisms involved are not well understood. Recently, we reported that higher plasma soluble Insulin Receptor (sIR) levels are associated with the presence and severity of HAND in our cohort of HIVseropositive women on cART. In this study, we investigated if soluble insulin-like growth factor-1 receptor (sIGF1-R) in the plasma of HIV-infected woman is associated with HAND and correlates with plasma sIR levels. Methods: Plasma sIGF1-R levels were assayed in 34 HIV-seropositive women stratified by HAND into normal cognitive (NC; n=11), asymptomatic impairment (AI; n=8), or symptomatic impairment (SI; n=15) and five (5) controls without history of diabetes. Soluble sIGF1-R levels (full-length or intact) were quantified by ELISA. Patients were also characterized for plasma sIR and TNF-alpha levels as determined by ELISA. Nonparametric statistics were used. Results: Higher levels of plasma sIGF1-R were associated with worse cognitive performance (p=0.006) among HIV-seropositive women stratified by HAND. No significant differences were observed in sIGF1-R levels between controls and HIV-seropositive women with NC. However, significant differences were seen between the NC and AI (p=0.001) and between the NC and SI groups (p=0.027). A positive correlation was observed between plasma sIGF1-R and sIR levels (p=0.011). No correlations were observed with age, viral-immune profile, antiretroviral therapy, or TNF-alpha levels. Conclusions: This study provides evidence that sIGF1-R secretion is increased in HIV-infected women and may have a role in the progression of HAND. Our findings also suggest that similar or coordinated cellular mechanisms may be responsible for the secretion of both, sIGF1-R and sIR, to the plasma of these patients and they could represent biomarkers for the presence and severity of HAND. This work was supported by: R21MH095524, S11NS046278, U54NS043011, U54RR026139, U54MD007587, G12RR003051, G12MD007600, and R25MH080661. Chronic neuroinflammation in HIV-1 infection is believed to contribute to the development of HIV-associated neurocognitive disorders (HAND). Previous work from our laboratory and others suggest that the accumulation of activated central nervous system (CNS)-associated macrophages and resident microglia results from the invasion and differentiation of peripheral blood monocytes. This process is significant to the pathogenesis of HAND because these cells constitute the principal cellular reservoirs of HIV-1 in the CNS. Importantly, HIV infection results in altered monocyte/macrophage homeostasis, evidenced by a skewed monocyte phenotype and activation status that may support the development and maintenance of a neuroinvasive monocyte subset. We have previously reported expansion of CD163+CD16+ monocytes in the peripheral blood of HIV+ patients that is phenotypically similar to macrophages/microglia that accumulate in the CNS in HIV infection; however, whether these cells are a source of new infection remains unclear. Here, we test the hypothesis that specific monocyte populations with predilection for invasion are preferentially infected with HIV-1. We compared the cellular location of HIV-1 in the monocyte subsets isolated from the peripheral blood of a cohort of chronically HIV-1 infected donors with a documented history of combination antiretroviral therapy (cART) adherence, with and without detectable plasma viremia, based on CD163 and CD16 expression. Further, we explored a therapeutic strategy targeting the CD163+CD16+ monocyte subset using experimental and FDA-approved tyrosine kinase inhibitors (TKIs) with selectivity for cfms, a type III receptor tyrosine kinase that has been shown to promote this monocyte phenotype in vitro. We anticipate that our studies will provide novel insights into the role of altered monocyte/macrophage homeostasis in HIV disease and identify a novel strategy for targeting long-lived cellular reservoirs of HIV through restored immune homeostasis. Giant cell arteritis (GCA) and multifocal varicella zoster virus (VZV) vasculopathy with temporal artery (TA) infection have overlapping clinical features and laboratory abnormalities. Our analysis of TAs that were pathologically negative for GCA (GCA-) revealed VZV antigen in 39/103 (38%) TAs. Viral antigen was found mostly, but not exclusively in the adventitia. During our continuing search for VZV antigen in GCA-TAs, abundant VZV antigen was found in multiple regions (skip areas) of an artery. This led to additional pathological analysis of sections adjacent to those containing VZV antigen. Reexamination revealed pathological changes with inflammation involving the arterial media and abundant multinucleated giant cells characteristic of GCA. When we analyzed TAs that had been pathologically confirmed to be GCA (GCA+), VZV antigen was found in 17/26 (65%) TAs. Also, despite formalin-fixation, the presence of VZV in TAs was further confirmed by the detection of VZV DNA in 24/31 (77%) of TAs that contained VZV antigen. None of 27 normal TAs from subjects over age 50 contained VZV antigen or VZV DNA. The detection of VZVantigen was noted in 65% of GCA+ TAs in which inflammation with (1) multinucleated giant cells and/ or epithelioid macrophages and (2) damage to the media and/or internal elastic lamina is present. In contrast, little or no inflammation in the adventitia of 38% of GCA-TAs indicates a continuum of disease. Overall, after reactivation from latency in ganglia, VZV travels transaxonally and initially infects the arterial adventitia resulting in clinical features that lead to TA biopsy even though the classic pathological features of GCA described above have not yet developed. By the time GCA pathology develops,~65% of TAs contained VZV. VZV is a major cause of GCA. Although antiretroviral therapy (ART) has significantly improved clinical outcomes in HIV+ patients, the prevalence and associated morbidity of HIV-associated neurocognitive disorders (HAND) remain high (~50%). Thus, adjunctive neuroprotective therapeutic targets that address the pathological processes persisting in ART-treated individuals are needed. Our lab has identified heme oxygenase-1 (HO-1), a sentinel cytoprotective protein, as a host factor protective against HIVmediated neurodegeneration that is deficient in the CNS of HIV+ individuals. In our current study, we quantified the protein expression of HO-1 in the prefrontal cortex of HIV-, HIV+, and HIV encephalitic brains (n=156). HO-1 protein levels were deficient in the prefrontal cortex of HIV+ patients compared to HIV-controls (p<0.01), with more severe HO-1 deficiency observed in HIV encephalitis patients (p<0.001). Furthermore, this HO-1 deficiency correlated significantly with CNS viral load as well as with brain parenchymal markers of macrophage and innate immune activation. These results suggest CNS HIV infection reduces HO-1 protein expression in the prefrontal cortex. Notably, HO-1 deficiency was significantly worse in patients with HAND compared to HIV+ individuals with normal cognition and correlated with neurocognitive impairment in executive and speed of processing domains, suggesting this HO-1 deficiency may play a role in neurocognitive impairment in HAND patients. Using our in vitro model of HIV-mediated neurodegeneration in which HIV-infected monocyte-derived macrophages (HIV-MDM) release neurotoxic levels of glutamate, we have shown that HIV infection of MDM drastically decreased the protein expression of HO-1. Moreover, pharmacologic induction of HO-1 decreased HIV-MDM neurotoxin production and glutamate release; in contrast, inhibition of HO-1 increased neurotoxin production. In summary, these findings report HO-1 deficiency in the brains of HIV+ individuals and identify HO-1 as a potential modulator of macrophagemediated neurodegeneration in HAND. Therefore, pharmacologic inducers of HO-1 should be studied for the potential to reduce the persistent prevalence of HAND in ART-treated individuals. Varicella zoster virus (VZV) is a ubiquitous neurotropic alphaherpesvirus. Primary infection typically causes childhood varicella after which virus becomes latent in ganglionic neurons along the entire neuraxis. As cell-mediated immunity to VZV declines with age or immunosuppression, VZV can reactivate to cause zoster and other serious neurologic and ocular diseases. Unfortunately, few models are available to study VZV latency since the virus infects only humans and latency is established only in neurons. We have successfully maintained normal human neural progenitor cells (NHNP) in tissue-like assemblies (TLAs) in 3-dimenstional (3D) cultures for up to 6 months. NHNP TLAs are a mixture of cells expressing markers for neuronal progenitor cells (CXCR4, CD90 and nestin), terminally differentiated neurons (beta-III-tubulin) as well as non-neuronal cells (GFAP). VZV infected NHNP-TLAs remained viable for 3 months during which time VZV DNA replicates, VZV genes (ORFs 9, 40 and 63) are transcribed and infectious VZV is sporadically released. The ability to maintain VZV infected NHNP cells in culture for an extended time provides a unique opportunity to study molecular interactions between VZV and neurons to an extent previously unattainable. monoclonal antibody therapy, JCV has been association with several human cancers. JCV infection most likely occurs in childhood and it has been hypothesized that the virus is able to persist within the bone marrow. However, identification of the bone marrow cell population that harbor the persistent virus and may therefore facilitate spread of the virus into different organs, including the brain, has been hampered by the heterogeneity of cell populations within the bone marrow. We recently isolated a distinct population of non-hematopoietic bone marrow derived cells from JCV T-antigen transgenic mice. These cells are of neural crest lineage, and we demonstrate that the expression of JCV T-antigen can be activated in this population of bone marrow cells upon exposure to a neural environment. JCV Tantigen positive cells exhibit neural crest characteristics and demonstrate p75, SOX-10 and nestin positivity. JCV T-antigen positive cells could be successfully cultured long-term while maintaining their neural crest characteristics. Furthermore, when these cells are induced to differentiate into neural crest derivatives, JCV T-antigen expression is downregulated in cells differentiating into bone and maintained in glial cells expressing GFAP and S100. We conclude that JCV T-antigen can be stably expressed within a fraction of bone marrow cells differentiating along the neural crest/glial lineage when cells are cultured in vitro. Thus, we identify a previously unrecognized bone marrow-derived neural crest stem cell population permissible for JCV early gene expression suggesting the possibility that these cells could support viral infection and thus provide clues toward understanding the role of the bone marrow in JCV persistence and reactivation. Novel Neuroprotective GSK-3beta Inhibitor Restricts Tat-Mediated HIV-1 Replication The implementation of new antiretroviral therapies targeting transcription of early viral proteins in post-integrated HIV-1 can aid in overcoming current therapy limitations. Using high throughput screening assays, we have previously described a novel Tat-dependent HIV-1 transcriptional inhibitor named 6BIO. The screening of 6BIO derivatives yielded unique compounds that show potent inhibition of HIV-1 transcription. We have identified a second generation derivative called 18BIOder as an inhibitor of HIV-1 Tat-dependent transcription in TZM-bl cells and a potent inhibitor of GSK-3beta kinase in vitro. Structurally, 18BIOder is half the molecular weight and structure compared to its parental compound 6BIO. More importantly, we also have found differential GSK-3beta complex present only in HIV-1 infected cells. In uninfected cells GSK-3beta was present in a complex with a molecular weight of~300 kDa, which is likely to be composed of either homo-or hetero-dimers of GSK-3beta in conjunction with possible chaperone proteins or other bound proteins. However, HIV-1 infected cells displayed an extended set of smaller molecular weight complexes in addition to the dominant~300 kDa complex. 18BIOder preferentially inhibits this novel kinase complex from infected cells at nanomolar concentrations. We observed efficacy of 18BIOder in HIV-1 replication inhibition in humanized mouse model at both 1.0 and 10 mg/kg as measured through reverse transcriptase activity from treated animals. Longitudinal measurements over a span of 29 days showed effective viral suppression at 1.0 mg/kg 18BIOder shortly after treatment. When left untreated the virus would resume replication, a comparable response to cART which require daily dosage for effective plasma control of viral load. Finally, we observed that neuronal cultures treated with Tat protein are protected from Tatmediated cytotoxicity when treated with 18BIOder. Overall, our data suggest that HIV-1 Tat-dependent transcription is sensitive to small molecule inhibition of GSK-3beta. HIV-1 invades the central nervous system (CNS) and establishes infection in brain by infecting monocytes/macrophages and microglia. HIV-1 infected monocytes/ macrophages release cytokine/chemokines that recruit other cells into brain. In this study we focused on the highly expressed chemokine CXCL5, which is a potent chemoattractant of neutrophils. Neutrophils maintain innate immune surveillance under normal conditions, whereas, during inflammation they cause tissue damage. To investigate the role of CXCL5 as well as neutrophils in HIV-1 infected brain monocyte-derived macrophages (MDMs) were infected with HIV-1 that showed a significant upregulation of CXCL5 both at the RNA and protein levels compared to uninfected/mock MDM culture. The significant upregulation of CXCL5 in HIV-1 infected MDMs was partly influenced by overexpression of IL-1beta, as neutralizing antibody against IL-1beta reduced the expression of CXCL5 more in HIV-1 infected MDM compared to mock infected. The increased production of CXCL5 was also in part regulated by the phosphorylation status of ERK1/2 and p38. The association of ERK1/2 and p38 with CXCL5 production was confirmed by blocking these MAPKs that prevented the elevation of CXCL5 in HIV-1 infected MDMs than in uninfected cultures. Functional analyses suggest that increased level of CXCL5 was directly correlated with infiltration of neutrophils in chemotaxis assay. Furthermore, increased level of CXCL5 and enhanced infiltration of activated neutrophils expressing myeloperoxidase (MPO) were also observed in brain tissues from HIV-1 positive subjects. To assess the consequence of MPO on neurons during HIV-1 infection, primary neurons were treated with HIV-1 infected or control MDM supernatants in the presence or absence of MPO. Results indicate that MPO reduced neuronal survival in a dose dependent manner. Collectively, our results suggest that CXCL5 has a profound effect on impairment of innate immune functions in HIV-1 infected brain through neutrophil infiltration that affects the final outcome of HIV-1 infection. IFN activated monocyte-derived exosomes mediate transfer of miRNAs to astrocytes: Implications for neurocognitive impairment in HIV/HCV-infection Archana Gupta 1 , Bing Sun 2 , Hans Rempel 2 , Brian Wigdahl 3 , Lynn Pulliam 2 (presenting author: ag434@drexel.edu) We recently reported that 60% of subjects with HCV and virally-suppressed HIV infection (coinfection) were cognitively impaired. Importantly, we showed that peripheral monocytes in coinfection had a distinct type-I interferon (IFN) activation profile. Since HCV is not considered neurotropic, it suggests that indirect viral mechanisms may be responsible for neural cell dysfunction. Several lines of evidence indicate that exosomes facilitate cell-to-cell communication wherein host RNA, including miRNAs and proteins can be transferred to recipient cells. We hypothesized that IFN-activated monocyte/macrophages mediate neurocognitive impairment in coinfection by the transfer of exosomal miRNAs to neural cells that subsequently dysregulate their function. In support of our hypothesis, we show that monocyte-derived exosomes are internalized by primary human astrocytes. In addition, miRNA profiling of exosomes shed by monocytes from HIV/HCV-infected subjects revealed that the vesicles were highly enriched in miR-223. Further, we developed an in vitro model that recapitulated the activation state of monocytes from coinfected subjects. IFN-activated monocyte-derived exosomes were greatly abundant in miR-223 as well, and when incubated with human astrocytoma cells, resulted in significant elevation of intracellular miR-223 levels. These results suggest that monocytederived exosomes can transfer miRNAs to neural cells, potentially altering the cellular environment of the latter. The transfer of miRNAs from activated monocytes to astrocytes via exosomes may explain, in part, the neurological abnormalities associated with coinfection. Intra-cerebroventricular (ICV) infusions of HIV envelope glycoprotein gp120 induce cognitive decline in a rodent model of HIV-associated neurocognitive disorders (HAND) acquisition phase of the Morris Water Maze task following intra-cerebroventricular (ICV) infusions of gp120 in rats. The present work sought to extend those findings to other domains of cognitive function. Adult male Fischer-344 rats were implanted bilaterally with cannulae into the lateral ventricles. After recovery from the surgery, daily infusions of gp120 (50 ng/microliter) or the bovine serum albumin (BSA) vehicle solution were administered for 7 days. Animals were food restricted the day after their final infusion and were tested in an attention set-shifting task, adapted from Birrell and Brown (2002) , 20 days later. To succeed in this task, the animal must discriminate between sensory cues to determine the location of a food reward. Subjects are also required to ignore cues that were rewarded on previous trials but which are no longer indicative of reward location. Animals exposed to gp120 were significantly impaired in reversal learning (within modality shift) and in their ability to make an extra-dimensional shift (cross-modality switch), suggesting behavioral inflexibility. Establishing a rat model of HAND that mimics the cognitive deficits observed in the clinical population will provide insights regarding mechanisms as well as guide the development of potential therapies. This work is supported by NIH/ NIMH R21 MH097623 and NIMH T32 MH070785. Targeted quantitative proteomics (SWATH-MS) reveals novel insights for reprogramming of transcription regulator proteins in HIV-1-infected macrophages Nicole Haverland, Pawel Ciborowski (presenting author: pciborowski@unmc.edu) Human immunodeficiency virus type 1 (HIV-1) remains a worldwide epidemic and a vaccine or therapy capable of eradicating the virus is not available. Although HIV-1 infection is generally associated with the widespread destruction of T-cells, monocytes and macrophages play an important role in the infection process, serving viral reservoirs capable of harboring latent virus as well as disseminators of the virus throughout the body. A novel therapeutic avenue against HIV-1 would be to target host factors, such as transcription regulators, that are involved in the host response to viral infection but can be bypassed in normal cellular functioning and this approach could circumvent the current limitations of antiretroviral therapy. We hypothesized that transcription regulator proteins -including transcription factors and cofactors, promoters, enhancers, repressors and proteins involved in chromatin structure and modification -were differentially expressed in HIV-1-infected macrophages. To test this hypothesis, we used a novel proteomic platform, SWATH-MS, for the targeted and quantitative proteomic analysis of transcription regulators capturing quantitative proteomic data specifically selected for transcription regulator proteins using an in-house generated database composed of both known and putative transcription regulators. Using this SWATH-MS approach, we identified and quantified 510 transcription regulators in uninfected and HIV-1-infected monocyte-derived macrophages. We then applied novel statistical testing to our proteomics data using methods previously established for intensity-based microarray data analysis; we identified 61 transcription regulators that were significantly (p < 0.05) altered following HIV-1 infection. Bioinformatic analysis of these altered transcription regulators revealed functional enrichment of proteins involved in chromatin structure and epigenetic modifications. Our findings highlight a novel subset of transcription regulator proteins that are involved in HIV-1 infection demanding further investigation. In addition, our study provides a new experimental paradigm that can be readily applied by a broad group of scientists for understanding the host response to microbial infection. Jasmin Herz, Dorian McGavern (presenting author: herzjn@mail.nih.gov) NIH / NINDS Several viruses can infect the mammalian nervous system, some with devastating consequences, and others with little or no overt pathology. Adoptive immunotherapy is an approach that involves administration of anti-viral T cells and has shown promise in clinical studies for the treatment of CMV, EBV, and adenovirus infections. Our laboratory models adoptive immunotherapy by transferring anti-viral memory T cells into mice persistently infected from birth with lymphocytic choriomeningitis virus. Here, we demonstrate that memory T cells can completely purge the brain of persistently infected mice without causing blood brain barrier breakdown or severe tissue damage. This is accomplished by a tailored release of chemoattractants that recruit adaptive immune cells, but few pathogenic innate immune cells such as neutrophils and inflammatory monocytes. Interestingly, memory T cells enlist the support of nearly all brain resident myeloid cells (microglia) by converting them into CD11c-expressing antigen-presenting cells (APCs). Two-photon imaging studies revealed that anti-viral CD8 T cells are more likely to decelerate and form stable interactions with brain-resident APCs than CD4 T cells. Importantly, microglia do not undergo cell death following T cell engagement and appear to protect themselves by upregulating serine protease inhibitors like Spi-6. We propose that non-cytopathic viral clearance from the brain by therapeutic memory T cells results from tailored chemoattractant production and interactions with resident myeloid cells protected by Spi-6. While the introduction of combined antiretroviral therapy, cART, has greatly improved survival rates among AIDS patients, a substantial portion of HIV-1 infected individuals remain at risk for the development of full blown AIDS as a result of reactivation of latently infected cells, partly due to nonadherence to medication and emergence of drug resistant viruses. Moreover, HIV-1 positive long term survivors continue to develop comorbidities including an accelerated aging process, neurocognitive disorders, heart failure, and others. From the virological point of view, as none of the current treatments suppress viral gene transcription, it is suspected that low, yet continuous, levels of viral early proteins with regulatory and pathogenic activities may contribute to the development of these quality of life-threatening illnesses. Sadly, all efforts toward the development of vaccines against HIV-1 have not shown promising outcomes. Thus, curing of AIDS by eradicating the HIV-1 genome in infected subjects requires a novel strategy that is specific, highly effective, irreversible, and sustained. Recently, we have adapted a genetic approach using clustered regulatory interspaced short palindromic repeat-associated system (Cas) and short complementary single-stranded RNA, called guide RNA (gRNA), which specifically targets the U3 region of the HIV-1 long term repeat (LTR) promoter and precisely excises a segment of the viral regulatory sequence required for its expression. In addition, the employment of single and multiplex gRNA in our Cas9 system showed promising results that included eradication of the entire HIV-1 genome in latently infected microglial cells, thus abrogating viral gene expression and reactivation. Based on this observation, we propose a working model for the development of an RNA-guided Cas9 that acts as molecular scissors and, by disrupting various regions of the LTR and/or removing the entire viral genome, abrogates reactivation of the virus in macrophages, microglia and astrocytes which serve as viral reservoirs in the brain. This work was supported by NIH grants R01 MH093271 and P30 MH092177 Comprehensive NeuroAIDS Center Grant awarded to KK. HIV-1 Tat continues to be implicated as a causative agent of HIV-1 associated neurocognitive disorder (HAND). We found previously that Tat elevates endolysosome pH and alters the structure and function of neuronal endolysosomes, a prominent and early pathological feature of HAND. Here, we showed that enlarged neuronal endolysosomes occur in an inducible HIV-1 Tat transgenic model of HAND and determined underlying mechanisms whereby HIV-1 Tat induces endolysosome dysfunction. Our observations that HIV-1 Tat elevates endolysosome pH indicates that HIV-1 Tat escaping from endolysosomes appears to be linked to proton leakage out of endolysosomes and suggests that a proton-dependent peptide transporter might be involved. Of the known protondependent peptide transporters, we demonstrated that protoncoupled oligopeptide transporter 2 (Pept2) is present on neuronal endolysosomes and that siRNA knockdown of Pept2 blocked HIV-1 Tat-induced enlargement of endolysosomes. Thus, Pept2 might be a proton-dependent peptide transporter through which HIV-1 Tat affects endolysosome pH and it might be involved in HIV-1 Tat escape from endolysosomes. Elevation of endolysosome pH could also affect endolysosome calcium homeostasis and we showed that elevation of endolysosome pH not only induces calcium release from endolysosomes but also activates a novel endolysosomedependent calcium influx across plasma membranes, a phenomenon we have termed "acidic store-operated calcium entry." Given our observations that HIV-1 Tat elevates endolysosome pH, increases calcium release from intracellular stores, and enhances calcium influx across the plasma membranes, HIV-1 Tat could activate this novel endolysosome-dependent calcium regulatory mechanism. Pept2 may be involved in the actions of HIV-1 Tat on neuronal endolysosomes and its contribution to the pathogenesis of HAND. (Supported by AG043338, GM103329, NS065957). The highly active antiretroviral therapy reduces HIV RNA in plasma to undetectable level. However, the virus continues persistence in resting T cells and CNS reservoirs, such as perivascular macrophages, microglia and astrocytes. Selective reactivation and eradication of HIV from CNS reservoirs is a critical problem of current HIV therapy. The X-ray irradiation (IR), well-defined stress signal, that is widely used for many therapeutic purposes, has earlier been shown to be capable to activate increased HIV-1 transcription, progeny virion formation and eventual apoptosis of infected cells. Here, using the HIV-1 infected monocyte-derived macrophage (MDM) model cells (PMA-activated U1) and NSG humanized mice infected with dual-tropic HIV-1 89.6 strain, we examined the effect of IR-induced cellular stress on HIV-1 replication and viability of infected cells. Treatment of both PBMCs and MDM with different IR doses led to dramatic increase of HIV-1 transcription, as evidenced by presence of Pol II and reduction of HDAC1 on HIV-1 promoter when using ChIP assay. This coincided with increased level of intracellular HIV-1 RNA. Incubation of IR-treated cells with proteasomal inhibitor ALLN (Calpain inhibitor 1) resulted in the additional increase of HIV-1 transcription probably due to Tat protein stabilization. Interestingly, analysis of infectivity of progeny virions using TZM-bl reporter cells showed decreased infectivity of the virus produced by irradiated cells, suggesting that along with activation of HIV-1 replication, IR increased production of defective viral particles. Treatment of HIV-1 infected humanized mice that did not display viral RNA in the plasma and PBMCs with IR resulted in significant increase of HIV-1 RNA in plasma, lung and especially brain tissues. Taken together, our current data suggest that IRinduced cellular stress activates HIV-1 expression in the infected MDM in different MDM-rich tissues including brain and facilitates the apoptotic death of infected cells possibly via Tat-dependent phosphorylation of p53 protein. Human T-lymphotropic virus type 1 infected cells secrete oncosomes containing Tax Human T-lymphotropic virus type 1 (HTLV-1) is the causative agent of adult T-cell leukemia (ATL) and HTLV-1-associated myelopathy (HAM)/tropical spastic paraparesis (TSP). The HTLV-1 transactivator protein Tax controls many critical cellular pathways including host cell DNA damage response mechanisms, cell cycle progression, and apoptosis. Recently, extracellular vesicles called exosomes have been shown to play critical roles during pathogenic viral infections as delivery vehicles for host and viral components including proteins, mRNA and miRNA. We hypothesized that exosomes derived from HTLV-1 infected cells contain unique host and viral proteins that may contribute to pathogenesis. We found that exosomes derived from infected cells contained the oncogenic Tax protein and pro-inflammatory mediator IL-6, as well as the viral mRNA transcripts, tax, hbz, and env. We also observed drastic reduction of MCP-1 and RANTES in exosomes from infected cells. Exosomes from infected cells deliver functional proteins to naive recipient cells and induce ROS production. Furthermore, we observed that exosomes released from HTLV-1 infected Tax-expressing cells contributed to enhanced survival of target cells when treated with FAS antibody. IL-2 dependent CTLL-2 cells that received Tax containing exosomes were also protected from apoptosis. Similar experiments with PBMCs also showed protection of the cells in 15 day cultures in the absence of PHA/IL-2. Collectively, these results suggest that exosomes may play an important role in extracellular delivery of functional HTLV-1 proteins, especially Tax, and mRNA to recipient cells and contribute to oncogenesis in the recipient cells. The use of nanoparticle technology in capturing HIV-1 virions and viral proteins Ceres Nanosciences HIV-1 infection results in a chronic but incurable illness since long-term HAART can keep the virus to an undetectable level. Discontinuation of therapy rapidly increases virus burden. Moreover, patients under HAART frequently develop various metabolic disorders and also HIV-associated neuronal disease. Today, the main challenge of HIV research is the elimination of the residual virus in infected individuals. The current HIV-1 diagnostics are largely comprised of serological and nucleic acid based technologies. Our goal is to integrate the nanoparticle technology into a standard research tool that will allow for highly sensitive detection of HIV infection. Our study demonstrates that majority of HIV-1 virions and Tat/nef proteins spiked in culture medium can be captured by nanoparticles. Especially at low concentrations, the nanoparticles provided dramatic increase in HIV-1 detection levels over standard assays. To determine the binding specificities of different affinity baits, we incubated target molecules with nanoparticles at room temperature. After short sequestration, materials were either eluted using downstream assay buffer or remained attached to nanoparticles prior to analysis. The unique affinity baits of nanoparticles preferentially bound HIV-1 materials while excluded albumin. The specific capture of full-length Tat or Tat peptide spiked in cell culture medium by nanoparticles NT082 and NT084 was measured by WB. Intracellular Nef protein was captured by NT080, while membrane-associated Nef was captured by NT086. Whole HIV-1 virus capture was enriched by NT073 and NT086, while NT086 nanoparticle captured infectious virus as demonstrated by Tat transactivation in TZM-bl cells. We also demonstrated specific capture of exosomes derived from HIV-1 infected cells and measured TAR-RNA by qRT-PCR. Collectively, our data indicate that some nanoparticles selectively capture specific target molecules of HIV-1 infection, and we propose to use this technology as a platform to enhance HIV-1 detection by concentrating viral proteins and infectious materials from infected samples. Background: While the effects of HIV-Tat protein on neuroglial function has been well studied, the role of Tat in mediating persistent proinflammatory responses in the CNS in virologically controlled individuals on ART is not well known. Persistent, low level inflammation can be demonstrated in the majority of individuals with chronic HIV infection, even when aviremic. Some patients who are well controlled on ART develop an immune reconstitution inflammatory syndrome (IRIS). Methods: We immunostained brain biopsy tissue for Tat and p24 antigen, and T cell markers from two HIV patients on ART who developed CNS-IRIS and compared it to autopsy brain tissue of seven HIV-infected individuals with good virological control on ART but without IRIS and one with HIV encephalitis (HIVE) with no ART. Tat levels were also examined in the CSF of patients on ART (n=31) and in peripheral blood mononuclear cells (PBMC) infected with HIV and treated with darunavir. Results: Monocytic infiltrates strongly stained for Tat in both patients with IRIS, however, p24 immunostaining was negative. In HIV patients without IRIS Tat was present in 2/7 individuals. The patient with HIVE had Tat and p24 expressing macrophages in microglial nodules and perivascular regions. In IRIS patients, infiltrates consisted of CD3 + T-cells which were predominantly CD8+ with few CD4+ cells and occasional IL-17+ cells. Tat was detected in 13/31 CSF samples by ELISA from HIV-infected individuals controlled on ART. Tat was confirmed in a subset of CSF samples by Western blot analysis. Darunavir blocked viral production in HIV-infected PBMC, however Tat production was unimpaired. Conclusions: Robust production of Tat was noted in brain of individuals with CNS-IRIS and in CSF of one third of individuals controlled on ART. While ART can control HIV replication it does not impact Tat production. Therapeutic Background: Iron dysregulation in the brain is a consistent and poorly understood feature of neurocognitive disorders, and functional neuronal iron deficiency has been implicated in certain dementias. We hypothesized that HIV-Associated Neurocognitive Disorder (HAND) pathophysiology involves brain iron deficiency, reflected by increased transferrin receptor (TFR) RNA expression in brain. Methods: Subjects who died of HIV/AIDS and participated in the National NeuroAIDS Tissue Consortium (NNTC) Study underwent uniform autopsy/neuropathology protocols and comprehensive neurocognitive assessments within 6 months antemortem. Total RNA was extracted and purified from frozen brain tissue (frontal neocortex at Brodmann Area 9); TFR messenger RNA was quantified by RT-PCR. Associations of TFR RNA expression with HAND and standardized, neurocognitive domain scores were assessed using multivariable regression to adjust for potential confounders. Results: Among 300 evaluated decedents from the NNTC Brain Bank (mean age 44, median CD4 cell count 109/ul), HAND occurred in 243 (81%), ranging from mild neurocognitive impairment (24%) to dementia (32%). TFR RNA expression levels, available in 274 subjects (91%), were unrelated to brain viral burden. TFR RNA expression in neocortex was associated with HAND (p<0.05; p<0.01 for association with dementia) in unadjusted analyses: TFR RNA levels were 15.4% higher in HAND cases than controls [median (IQR) 0.90 (0.65, 1.20) vs. 0.78 (0.61, 1.0), respectively]. These associations persisted after multivariable adjustment [odds ratio (OR) 5.8 (95% p<0 .05) for all HAND cases; OR 2.3 (95% CI 1.1-4.9, p<0.05) for dementia]. Speed of information processing, attention working memory, and global standardized T-scores were negatively associated with TFR RNA expression [all p≤0.05; beta-coefficient -4.27 (95% CI -8.3 to -0.23) for global T-score]. Conclusions: TFR RNA expression in frontal neocortex is independently associated with both mild and more severe neurocognitive impairment among individuals dying of HIV/AIDS, suggesting a role for brain iron deficiency in HAND etiology and/or progression. Given the association between HIV-induced CNS disease and replication of HIV in macrophages in the brain, CCR5 antagonists could attenuate CNS disease both by limiting HIV replication in macrophages and by downmodulating inflammatory signaling mediated by chemokine-CCR5 interactions. To determine whether CCR5 inhibition altered CNS disease progression independent of treatment with other classes of anti-retroviral drugs, CNS outcomes were compared between SIV-infected animals treated with maraviroc monotherapy versus untreated SIV-infected animals. SIV RNA and SIV DNA levels in brain were markedly lower in maraviroc treated, SIV-infected macaques versus untreated SIV-infected macaques, demonstrating that maraviroc monotherapy limits replication of SIV in the CNS and may reduce the CNS latent viral reservoir. In addition, maraviroc treatment lowered monocyte and macrophage activation, represented by CNS CD68 immunostaining and plasma sCD163 levels, and reduced both TNFalpha and CCL2 RNA expression in brain. Maraviroc treatment also reduced axonal amyloid precursor protein (APP) immunostaining to levels present in uninfected animals, demonstrating protection from development of neuronal dysfunction. Although maraviroc therapy reduced plasma viral load and SIV RNA levels in spleen, relative decreases were less substantial than CNS declines, underscoring the importance of assessing CNS-specific outcomes in evaluating efficacy of CCR5 inhibition. The addition of CCR5 inhibitors to combined anti-retroviral regimens may effectively prevent neurologic disorders in HIV-infected individuals and also may reduce CNS viral reservoirs. In the absence of an effective microbicide that reduces or eliminates the risk of human immunodeficiency type 1 (HIV-1) transmission, the development of new anti-HIV-1 drugs remains a priority. Cell penetrating peptides (CPP), which are short peptides that are capable of crossing the plasma membrane of a living cell, are under development as delivery vehicles for therapeutic agents that cannot themselves enter the cell. One well-studied CPP is the 10-amino acid peptide derived from the human immunodeficiency virus type 1 (HIV-1) Tat protein. In experiments to test the hypothesis that multiple cationic amino acids within Tat peptide confer antiviral activity against HIV-1, introduction of Tat peptide resulted in concentration-dependent inhibition of HIV-1 IIIB (X4) infection yet minimal antiviral activity against BaL (R5). In contrast, Tat peptide variants containing arginine substitutions for two non-ionic residues and two lysine residues, demonstrated a direct relationship between cationic charge and antiviral potency in HIV-1 inhibition experiments. These studies of Tat peptide as an antiviral agent raise new questions about the role of Tat in HIV-1 replication and provide a starting point for the development of CPPs as novel HIV-1 inhibitors. Patients infected with human immunodeficiency virus type 1 (HIV-1) often display neurological complications in late stage disease and increased viral loads directly correlated with higher concentrations of extracellular HIV-1 protein r (Vpr) in the blood serum and cerebrospinal fluid. Additionally, HIV-1-infected patients with a low CD4+ T-lymphocyte count displayed lower concentrations of reduced glutathione (GSH), the main intracellular antioxidant molecule, and lower level of survival. Conditioned media obtained from the human endothelial kidney (HEK) 293T cell line transfected either in the absence or presence of HIV-1 Vpr contained free Vpr. Exposure of U-87 MG cells to this conditioned media decreased intracellular levels of both adenosine triphosphate (ATP) and GSH. These observations were recapitulated using purified recombinant HIV-1 Vpr both in U-87 MG and primary human fetal astrocytes in a dose-and time-dependent manner. Vpr-induced oxidative stress could be partly restored by co-treatment with the antioxidant molecule Nacetyl-cysteine (NAC). In addition, free Vpr augmented production of reactive oxygen species due to an increase in the level of oxidized glutathione (GSSG). This event was almost entirely suppressed by treatment with an anti-Vpr antibody or co-treatment with NAC. These studies confirmed a role for extracellular Vpr in decreasing the levels of intracellular ATP and GSH in astrocytes. Studies are underway to better understand the intricate correlation between reductions in ATP and GSH metabolites and the impact they exert on neuronal survival in end-stage disease. This work is supported by NIH/NINDS R01 NS32092, NIDA R01 DA19807, NIMH P30 MH092177, and NIMH T32 MH079785. Objective: To determine the frequency of hyperintense cortical T1 signal (HCTS) on MRI in progressive multifocal leukoencephalopathy (PML) patients, its association with seizure risk and immune reconstitution inflammatory syndrome (IRIS), and its pathologic correlate. Background: PML, caused by JC virus (JCV) affects predominately white matter. Seizures point to a cortical origin and add to increasing evidence of gray matter involvement in PML. HCTS is a radiologic marker of cortical laminar necrosis and is described in hypoxia, hypoglycemia and status epilepticus. Methods: We reviewed clinical data including seizure history, presence of IRIS, and MRI scans from PML patients evaluated at our institution between 2003 and 2012. Cases that were diagnosed either by CSF JCV PCR, brain biopsy or autopsy, and who had MRI images available were included in the analysis (n=49).We compared pathologic findings in areas of the brain of two patients displaying HCTS with isointense cortex in the same individuals. . Together, HCTS and IRIS predicted seizures with an OR of 9.9 (95% CI of 2.5 -39.3; p=0.001). On histological examination, HCTS areas showed no evidence of ischemia but were associated with striking demyelination of sub-cortical U-fibers, significant macrophage infiltration and a pronounced reactive gliosis in the deep cortical layers. Conclusions: Seizures are a frequent complication in PML. HCTS is associated with seizures as well as IRIS, and correlates histologically with cortical segmental astrogliosis. Hyperperfusion in progressive multifocal leukoencephalopathy is associated with disease progression and absence of immune reconstitution inflammatory syndrome Michael Khoury 1 , Sarah Gheuens 2 , Long Ngo 3 , Xiaoen Wang 4 , David Alsop 4 , Igor Koralnik 1 (presenting author: ikoralni@bidmc.harvard.edu) Objective: We sought to characterize perfusion patterns of progressive multifocal leukoencephalopathy (PML) lesions by arterial spin labeling (ASL) perfusion magnetic resonance imaging and to analyze their association with immune reconstitution inflammatory syndrome, and survival. Background: ASL uses magnetic fields to alter the magnetization of water in the inflowing arterial blood and then measures the effect of the inflow of altered magnetization on the tissue signal. Because the "tracer" used is the endogenous blood water, ASL does not require any injections or other contrast agent. Methods: A total of 22 patients with PML underwent a clinical evaluation as well as magnetic resonance imaging of the brain within 190 days of symptom onset. The presence of immune reconstitution inflammatory syndrome was determined based on clinical and laboratory criteria. Perfusion within PML lesions was determined by arterial spin labeling magnetic resonance imaging. Results: We observed intense hyperperfusion within and at the edge of PML lesions in a subset of subjects. This hyperperfusion was quantified by measuring the fraction of lesion volume showing perfusion in excess of twice normal appearing gray matter. Hyperperfused lesion fraction was significantly greater in PML progressors than in survivors (12.8% vs 3.4% p=0.02) corresponding to a relative risk of progression for individuals with a hyperperfused lesion fraction ≤ 4.0% of 9.1 (95% CI of 1.4-59.5). The presence of hyperperfusion was inversely related to the occurrence of immune reconstitution inflammatory syndrome at the time of scan (p=0.03). Indeed, within three months after symptom onset, hyperperfusion had a positive predictive value of 88% for absence of immune reconstitution inflammatory syndrome. Conclusions: Arterial spin labeling magnetic resonance imaging recognized regions of elevated perfusion within lesions of PML. These regions might represent virologically active areas operating in the absence of an effective adaptive immune response and correspond with a worse prognosis. While long term combined antiretroviral therapy (cART) has greatly improved survival rates among AIDS patients, a substantial proportion of HIV-1 infected individuals continue to develop comorbidities including heart failure (HF) secondary to left ventricular dysfunction at higher rates than non-HIV-1 individuals. The underlying mechanisms whereby HIV-1 increases susceptibility to HF remain poorly understood. In this respect, HIV-1 Tat, which is produced and released by the latent viral reservoir and upon its circulation can be taken up by uninfected cells, has received special attention due to its ability to induce an array of dysregulatory events that perturb cell and organ function. We demonstrate that HIV-1 Tat physically associates with BAG3, a stress induced protein which is involved in protein quality control, and modulates autophagy and apoptosis. BAG3 is critical for normal cardiac development and maintenance as BAG3 knockout mice develop left ventricular (LV) dysfunction and have a shortened lifespan. Furthermore, recent studies have identified a potential role for BAG3 in patients with HF as heterozygous mutations that decrease the level of BAG3 and lead to development of a heritable form of dilated cardiomyopathy. Moreover, ventricular myocardium isolated from failing human hearts examined at the time of heart transplantation exhibit nearly 50% reduction in BAG3 levels in cardiac tissue, all of which supports the importance of BAG3 for healthy heart function. BAG3 is an important regulator of filamin and myopodin and regulates protein turnover by autophagy in cardiomyocytes. We provide evidence suggesting that the interplay between BAG3 and HIV-1 Tat impacts the ability of BAG3 to regulate several pathways involved in the structural and functional integrity of cardiomyocytes. Thus, one can envision a model in which the inactivation of BAG3, upon its association with HIV-1 Tat, recapitulates the clinical manifestations seen in patients with heart failure associated with low/dysfunctional BAG3. HSV-1 is a ubiquitous, neurotropic human pathogen that causes diseases ranging from mild orofacial lesions to potentially fatal encephalitis. Cells present at the infection site express pattern recognition receptors including Toll-like receptors that detect infection, produce antiviral cytokines, and limit viral invasion of the nervous system. We assessed the role of TLR2 in resistance to HSV-1 encephalitis in a natural lip infection model. Multiple cell types express TLR2, which recognizes HSV-1 glycoproteins. We show here that TLR2knockout (Tlr2-/-) mice were highly susceptible to HSV-1induced mortality. Their natural killer cells responded normally, but the mice exhibited impaired dendritic cell and CD8+ Tcell activation. While virus replication within the lips was not different, HSV-1 spread to the trigeminal ganglia more efficiently in the Tlr2-/-group. Additionally, CNS entry was exacerbated in the knockouts and viral loads there positively correlated with disease severity. The elevated CNS viral load precipitated the up-regulation of inducible nitric oxide synthase. Nitric oxide is neurotoxic at high concentrations and presumably contributed to the mortality in the knockout animals. We then assessed the infiltration of leukocytes to the infected CNS to study their impact on disease. Neutrophils, which contribute to irreparable inflammation during viral encephalitis, were observed in the cerebral cortex of the mice exhibiting the most significant signs of disease, irrespective of their TLR2 status. Lastly, we utilized an intracranial infection model to specifically assess the role of TLR2 within the CNS, but the virus strain utilized proved too virulent to reveal a phenotypic difference. In summary, TLR2 is critically important for controlling the level of HSV-1 that spreads to the CNS, and once the virus accesses the brain, TLR2-independent responses contribute to the heightened inflammation observed in the knockout animals. In future studies, we will focus on the identification of the responsible TLR2+ cell type. Despite the widespread use of antiretroviral therapy, HIVassociated neurocognitive disorders (HAND) continue to persist, signifying a need for the development of adjunct therapeutic strategies. We have identified the anti-oxidant and antiinflammatory enzyme heme oxygenase-1 (HO-1) as a potential therapeutic target for HAND. Recently we characterized HO-1 within the prefrontal cortex of HIV+ individuals and found that HO-1 protein was deficient in HIV+ individuals and correlated with neurocognitive impairment; however, HO-1 RNA was increased compared to HIV-controls. We have previously shown that HO-1 protein is also deficient in HIV-infected monocyte derived macrophages (HIV-MDM) and this deficiency is associated with enhanced glutamate release and neurotoxicity. To explain the HO-1 protein deficiency observed in HAND patients and to probe mechanisms by which this deficiency may contribute to neurotoxicity, we examined HO-1 regulation within astrocytes, which are the predominant source of HO-1 within the brain and are critical mediators of glutamate handling. Primary rat and human astrocytes were treated with immune activators relevant to HIV pathogenesis (IFN-gamma, lipopolysachharide (LPS), TNF-alpha) and HO-1 expression was assessed after acute (1, 6, 24 hours) and chronic (15 days) exposure. Chronic, but not acute, treatment significantly reduced HO-1 protein expression in both rat and human astrocyte cultures; HO-1 RNA was not altered throughout treatment. Furthermore, chronic treatment did not alter the expression of other antioxidant proteins (NQO1 and GPX1), suggesting that the reduction of HO-1 protein driven by immune activators is specific and not due to global suppression of the antioxidant response. These results demonstrate that chronic immune activation suppresses HO-1 protein within astrocytes, and suggest a mechanism by which chronic HIV infection leads to a global deficiency of HO-1 within the frontal cortex. Reduced HO-1 within astrocytes may have important functional consequences for glutamate regulation and subsequent neurodegeneration, which will be addressed in future studies. Since the beginning of the AIDS epidemic, HIV infection and substance abuse have been linked. The risk of contracting HIV and transmitting HIV increases significantly with cocaine use. Neurocognitive disorders associated with cocaine abuse or with HIV infection are exacerbated in patients who are HIV+ and use cocaine. Both cocaine and HIV infection induce pro-inflammatory cytokines, such as tumor necrosis factor alpha (TNF-alpha) and ultimately lead to the disruption of numerous neuronal signaling pathways. The retinoic acid (RA) signaling pathway is important in a number of cellular processes and in HIV patients this pathway is disrupted. Our data show for the first time that cocaine and the HIV protein, Tat significantly disrupt this pathway in the CNS. Retinoid X receptors (RXR) can homodimerize or serve as required heterodimeric partners for a number of ligand-activated nuclear receptors, which recognize specific DNA sequences on target genes to regulate transcriptional activation. Our data show that cocaine significantly down-regulates RXR-gamma in brains of cocaine treated-mice, Tat transgenic mice and in neurons in vitro. Additionally, genes regulated by RXR signaling such as neurogranin, which encodes a protein required for adult neuroplasticity, are significantly down regulated. The cocaine-induced degradation of RXR-gamma is proteasome dependent and involves nuclear export of the protein. Additionally, our data show that inhibiting TNF-alpha signaling, or blocking downstream TNF-alpha effectors such as c-jun-NH2-terminal kinase (JNK), can attenuate the cocainemediated decreases in RXR-gamma levels. Data from our studies provide the first evidence that exposure to cocaine or to Tat disrupts RXR-gamma signaling in the CNS, and that TNF-alpha activation likely plays a role. As RXR dysfunction is implicated in a number of neurodegenerative diseases and psychiatric disorders, our findings may extend to other neuropathological conditions characterized by a neuroinflammatory component. While Vpr has been described extensively as an intracellular protein, very little is known about its role as an extracellular protein. In fact, HIV-1 Vpr has been detected in the blood, serum, and cerebrospinal fluid of HIV-1-infected patients, with concentrations increasingly higher in late-stage disease. To determine the role exogenous Vpr plays in HIV-associated central nervous system dysfunction, primary human fetal astrocytes were exposed to recombinant Vpr and a time-and dose-dependent decrease was demonstrated in two fundamental intracellular metabolites (ATP and glutathione (GSH)). Additionally, exposure to exogenous Vpr led to increased caspase activity and secretion of proinflammatory cytokines IL-6 and IL-8 and chemoattractants, monocyte chemotactic protein-1 and migration inhibition factor. Extracellular Vpr also dampened the glycolytic pathway through impairment of GAPDH activity, causing a decline in the levels of ATP. The reduction in intracellular ATP increased reactive oxygen species buildup, decreasing GSH concentrations, which affected several genes in the oxidative stress pathway. In addition, exposure of the SK-N-SH neuroblastoma cell line to conditioned medium from exogenous Vpr-treated astrocytes decreased synthesis of GSH, leading to their apoptosis. These observations point to a role that Vpr plays in altering astrocytic metabolism and indirectly affecting neuronal survival. We propose a model that may explain some of the neurological damage and therefore neurocognitive impairment observed during the course of HIV-1 disease. This work is supported by NIH/NINDS R01 NS32092, NIDA R01 DA19807, NIMH P30 MH092177, and NIMH T32 MH079785. Pur-alpha is a highly conserved sequence specific DNA and RNA binding protein with established roles in DNA replication, RNA translation, cell cycle regulation, and maintenance of neuronal differentiation. Pur-alpha is also an important mediator of HIV-1 gene expression through its strong interaction with the regulatory Tat protein and neurotoxicity associated with Tat is mediated, at least in part, by Pur-alpha. Post-natal expression of Pur-alpha in the brain is essential to the normal development of neurons and synaptic formation in the hippocampus and cerebellum as homozygous deletion of Puralpha causes premature death before adolescence. Animals heterozygous for Pur-alpha demonstrate haploinsufficiency with significantly reduced levels of Pur-alpha in the brain and expire before one year of age suggesting that chronic reduction in Pur-alpha leads to neuropathogenic effects. Here we present for the first time an assessment of the behavioral phenotype of mice heterozygous for the Pur-alpha knockout allele. Due to the observed motor dis-coordination and cerebellar changes in the model, the less physically demanding Barnes maze was used to assess spatial learning and locomotor activity. A reversal protocol on the maze was used to investigate the animals' retention of spatial memory longterm as well as their capacity for new memory formation. A second spatial learning paradigm with low but different motor demands, the novel object location test, was used to further characterize spatial memory function. A third test probed sensory alterations using a free-two-choice thermal gradient to assess peripheral nerve changes. Taken together, our findings from our behavioral and histochemical assays implicated Pur-alpha in the mediation of neurodegeneration in the context of HIV-1 infection. Thus, the Pur-alpha knockout mice may provide a well-defined animal model in which to study mechanisms of HIV-1 associated neurological disorders, including cognitive deficiencies and peripheral neuropathies. Core facility services supported by CNAC at Temple (NIMH P30 MH092177). Adarsh Kumar 1 , Raymond Ownby 2 , Jesus Fernandez 1 , Eridania Valdes 1 , Mahendra Kumar 1 (presenting author: akumar@med.miami.edu) Background: HIV-1 associated neurodegeneration, and dysfunctional neurobiological systems in the CNS in pre-and post-cART era have been investigated in multitudes of studies, including ours. However, studies are scarce on the impact of HIV-1 on the CNS serotonin system, the major neurotransmitter system that regulates behaviors. In an earlier study carried out in the CSF of HIV-1+ individuals in pre-cART era, we reported a dramatic decrease in 5-hydroxytryptamine (5-HT) concentration without any change in 5-HIAA. In the post-cART era, however, the impact of HIV-1 in the CNS serotonin system has remained elusive, although, 30-50% of HIV-1 infected individuals continue to experience spectrum of neuropsychiatric disorders, including depression, affecting quality of life, medication adherence and disease progression. Objectives: To investigate the impact of HIV-1 on CNS serotonin (5-HT) and 5-HIAA status as well as HIV-1 RNA load in different regions (frontal cortex, basal ganglia, hippocampus, hypothalamus, raphe nuclei, amygdale, and substantria nigra) of postmortem brains of HIV-1+ individuals who during life received cART and were evaluated for neuropsychiatric disorders, compared to 5-HT status in the brain regions of non-infected individuals. Results: We found variable changes (%) in 5-HT and 5-HIAA concentrations in different brain regions of HIV-1+ individuals (5-HT, -13.7%, +4.5%, -9.6%, -8.9%, -0.244%, -9.27%, +8.2%; 5-HIAA, +3.84%, -2.1%, -4.91%, -1.7%, +4.6%, -4.3%, -30% respectively), compared to that in non-infected persons. The variable changes found in 5-HT and 5-HIAA concentrations in different brain regions of HIV-1+ individuals in this study are contrary to the dramatic decrease in CSF 5-HT in HIV-1+ individuals in the pre-cART era. Does cART influence CNS 5-HT turnover? Conclusion: Our data suggest that serotonin system in the CNS is vulnerable to HIV-1 infection in the post-cART era. Further studies are warranted on the effect of HIV-1 and cART on multiple indices of CNS serotonergic systems. Dianne Langford 1 , Ahmet Ozdemir 1 , Radhika Adiga 1 , William Yen 1 , Melissa Wasilewski 1 , Alexandra Carides 2 , Pallavi Chitturi 2 (presenting author: tdl@temple.edu) 1 Temple University School of Medicine, Department of Neuroscience; 2 Center for Statistical Analysis, Department of Statistics, Fox School of Business Growing evidence points to significant mechanistic overlap between HIV-associated neurocognitive disorders (HAND) and age-related neurodegeneration. HIV infected individuals diagnosed decades ago are beginning to face age-related neurodegeneration and combined with viral infection and longterm exposure to cART, neurodegeneration is exacerbated. Cellular dysfunction involving accumulation of aberrant proteins, and loss of protein quality control are among the most important causes for disease and age-related neurologic decline. Accumulation of hyperphosphorylated Tau (hpTau) is a neuropathological feature of aging in HIV encephalitis (HIVE) and other neurodegenerative disorders like Alzheimer's disease. We have discovered that an adaptor protein called PINCH is important in the signaling cascade of HIV-related Tauopathy at multiple levels including cell signaling and as a clinical correlate of CNS disease severity. PINCH is nearly undetectable in the healthy CNS, but during HIV infection, it is robustly expressed by neurons. Our in vitro, in vivo and human data show that PINCH binds directly to hpTau and is associated with loss of Tau solubility in disease. Unique isoforms of PINCH are detectable in the CSF of HIV patients and pilot data suggest that CSF levels of PINCH may correspond in part to immune recovery, rather than viral burden. PINCH inhibits the PP1alpha phosphatase and competes with Tat for binding. Tat induces Tau translocation to the somatodendritic compartment of human primary neurons, and decreasing PINCH expression results in the detection of less hpTau. Understanding the contribution of increased PINCH to HIV-associated Tauopathy may provide a new therapeutic avenue for reducing levels of hpTau in the brain. Moreover, characterizing the clinical significance of PINCH in the CSF may warrant including PINCH as a member of biomarker panel to assess severity or progression of HIV-associated neurocognitive alterations. We previously demonstrated the expansion of CD16+ monocytes in HIV and SIV infection. Since the percentage of this monocyte subset correlated with viral load and CD4+ T cell count, we had suggested a role in AIDS and CNS pathogenesis. These cells likely also promote the persistent macrophage reservoir of viral infection in the CNS as well as the periphery. Here, we investigated the dynamics of production and clearance of total (CD14+) and CD16+ monocytes using in vivo BrdU and CFSE labeling in rhesus macaques infected with SIVMac251. Three groups of animals included SIV+ (N=6), SIV+/CD8 depleted (N=5), and SIV-[N=5(BrdU), 3(CFSE)] . BrdU and CFSE were injected intravenously 3 months postinfection in all groups in order to label proliferating (i.e. in bone marrow), and peripheral circulating leukocytes, respectively. Blood specimens were drawn at 30 minutes, 1, 2, 3, 4, 5, and 7 days post-BrdU/CFSE administration and analyzed by flow cytometry. An increase in the production of BrdU+ monocytes was observed at day 1 in the SIV+/CD8 depleted macaques. CD14+ monocytes, emerging as BrdU+, were virtually all CD16-at 24 hours post BrdU administration, despite the increased production of total CD14+ monocytes in SIV+/CD8 depleted animals at this time point. At subsequent time points, however, the percentage of CD16+/BrdU+ monocytes increased, peaking at day 3 in all groups. These results likely suggest an altered distribution of proliferating monocyte precursors within the bone marrow. Monocyte clearance was similar in all groups as determined by CFSE labeling. Taken together, our results suggest the importance of both altered monocyte production (in bone marrow) as well as maturation in peripheral compartments, leading to the overall expansion of CD16+ monocytes in AIDS. Analysis of T cell turnover also provided novel insights regarding CD4+ and CD8+ T-cell dynamics in AIDS. Emily Leibovitch, Giovanna Brunetto, Breanna Caruso, Kaylan Fenton, Joan Ohayon, Daniel Reich, Steven Jacobson (presenting author: emily.leibovitch@nih.gov) The human herpesviruses HHV-6A and HHV-6B are widely distributed in the adult population, though specifically associated with several disorders of the central nervous system (CNS) including multiple sclerosis (MS). The association of these viruses with neurologic diseases has been partly established by the detection of elevated viral DNA levels in patients compared to controls. Despite their high nucleotide homology, HHV-6A and HHV-6B were recently reclassified as separate species, owing to mounting evidence of their biological differences. As it is now especially relevant to investigate each virus separately, we investigated coinfection of HHV-6A and HHV-6B in human biological material using a novel quantitative PCR technology called digital droplet PCR (ddPCR). DdPCR enables the absolute quantification of target DNA molecules and is highly sensitive for lowlevel targets. To investigate coinfection, we designed an assay to PCR amplify both viruses with comparable kinetics, such that small amounts of one could be distinguished in the presence of large amounts of the other. Using this approach, we observed a heretofore-underappreciated frequency of HHV-6A and HHV-6B coinfection in the serum, PBMC and saliva of healthy donors. Interestingly, upon comparing adult MS patients with healthy donors, we detected a significantly elevated frequency of coinfection in MS saliva. We are currently exploring whether there are clinical correlates to coinfection, and assessing saliva samples from pediatric MS patients and controls. Identifying and quantifying both species of HHV-6 may provide clinically relevant information and as we learn more about each virus in health and disease, the observation of coinfection may have profound implications. Transcriptome analysis of HIV-infected peripheral blood monocytes: genes and networks associated with neurocognitive functioning Immunologic dysfunction, mediated via monocyte activity, has been implicated in the development of HIVassociated neurocognitive disorder (HAND). We hypothesized that transcriptome changes in peripheral blood monocytes relate to neurocognitive functioning in HIV+ individuals, and that such alterations could be useful as biomarkers of worsening HAND. METHODS: mRNA was isolated from the monocytes of 86 HIV+ adults and analyzed with the Illumina HT-12 v4 Expression BeadChip. Neurocognitive functioning, HAND diagnosis, and other clinical and virologic variables were determined. Data were analyzed using standard expression analysis and weighted gene co-expression network analysis (WGCNA). RESULTS: Neurocognitive functioning was correlated with multiple gene transcripts in the standard expression analysis. WGCNA identified two nominally significant co-expression modules associated with neurocognitive functioning, which were enriched with genes involved in mitotic processes and translational elongation. CONCLUSIONS: Multiple modified gene transcripts involved in inflammation, cytoprotection, and neurodegeneration were correlated with neurocognitive functioning. The associations were not strong enough to justify their use as biomarkers of HAND; however, the associations of two coexpression modules with neurocognitive functioning warrants further exploration. Wenxue Li, Richa Tyagi, Lisa Henderson, Avindra Nath (presenting author: liw8@mail.nih.gov) Section of Infections of the Nervous System, National Institute of Neurological Diseases and Stroke, National Institutes of Health Amyotrophic Lateral Sclerosis (ALS) is a progressive neurological disease that affects both upper and lower motor neurons. Patients develop gradual loss of voluntary muscle strength and eventually most die of respiratory failure. Most ALS cases are sporadic , while up to 10% may be inherited. Although a number of gene mutations have been identified in the familial form of ALS, the etiology of ALS is still largely unclear. Our group and others have reported that human endogenous retroviruses type K (HERV-K) is activated in ALS patients, indicating the possible involvement of HERV-K in the pathogenesis of ALS. HERVs are fossil sequences of retrovirus infection that were integrated into human germline chromosomes millions of years ago. Most HERVs are defective because of mutations. However, type K HERV has relatively preserved genomic sequence. It may be activated and produce viral particles under certain physiological and pathological conditions. The reverse transcriptase activity of HERVs in serum was detected in some ALS patients. Increased HERV-K pol gene was also found in the brain of ALS patients. In this study, we further studied the expression of HERV-K genes in autopsy brain tissue of ALS patients. Using quantitative real time PCR, we determined the expression levels of HERV-K gag, pol, and env in frontal cortex. The ALS patients had significantly higher expression levels of HERV-K genes compared to controls. The expression of three HERV-K genes correlated with each other. Our results confirmed the activation of HERV-K in the brain of ALS patients. This warrants further investigation of the activation mechanism. Winston Liu, Raya Massoud, Daniel Reich, Govind Nair, Steve Jacobson (presenting author: winston.w.liu@gmail.com) Human T-cell lymphotropic virus-1 associated myelopathy/ tropical spastic paraparesis (HAM/TSP) and multiple sclerosis (MS) can both lead to progressive inflammatory myelopathy. Although spinal cord atrophy can be qualitatively detected on routine clinical MRI, a robust and sensitive method to quantify changes in spinal cord size might improve characterization of disease severity and progression. Such a method could help develop an imaging marker for disease, and serve as a surrogate end point in clinical trials. MRI was performed on 18 HAM/TSP, 9 MS and 10 healthy volunteers on a Siemens 3T Skyra system equipped with head-neck and spine array coils. Cross-sectional area of the spinal cord was measured using a novel algorithm developed in-house that traces axial contours perpendicular to the cord-edge at each point from C1 to T10 vertebral body segments. Average cross-sectional area in both the T-spine and C-spine were significantly lower in HAM/TSP patients (C-spine: 50.75 ± 10.02 mm2; T-spine: 24.76 ± 5.01 mm2) as compared to healthy controls (C-spine: 72.02 ± 5.792 mm2, p<0.0001; T-spine: 38.8 ± 6.048 mm2, p<0.0001). However, cross-sectional area was only significantly different in the T-spine when comparing HAM/TSP with MS patients (C-spine: 61.84 ± 9.54 mm2; T-spine: 34.84 ± 5.72 mm2, p<0.001). In HAM/TSP, the crosssectional areas from multiple cord segments correlated with disease duration (C1-C3, C6, T1-T2), Extended Disability Status Scale (EDSS) (T2), and Hauser Ambulation Index (T2, T6), but no correlation was observed between HAM/TSP cross-sectional area and proviral load. MS cross-sectional area correlated with EDSS (C1-C3, T1-T6). These results suggest that patterns of spinal cord tissue damage are specific to the underlying disease, a finding that has direct implications for the use of average cross-sectional spinal cord area as a surrogate end point for clinical trials. Longitudinal studies are underway to help improve our understanding of the disease process. Structural and functional studies of CCAAT/enhancer binding sites within the human immunodeficiency type 1 subtype C LTR Human immunodeficiency virus type 1 (HIV-1) subtype C, which is the most predominant subtype in sub-Saharan Africa as well as in Asia and India, is also the most prevalent subtype worldwide. A large number of transcription factor families have been shown to be involved in regulating HIV-1 gene expression in T-lymphocytes and cells of the monocyte-macrophage lineage. Among these, proteins of the CCAAT/enhancer binding protein (C/EBP) family are of particular importance in regulating HIV-1 gene expression within cells of the monocytic lineage during the course of hematologic development and cellular activation. Few studies have examined the role of C/EBPs in long terminal repeat (LTR)-directed viral gene expression of HIV-1 subtypes other than subtype B. Within subtype B viruses, two functional C/EBP sites located upstream of the TATA box are required for efficient viral replication in cells of the monocyte-macrophage lineage. We report the identification of three putative subtype C C/ EBP sites, upstream site 1 and 2 (C-US1 and C-US2) and downstream site 1 (C-DS1). C-US1 and C-DS1 were shown to form specific DNA-protein complexes with members of the C/EBP family (C/EBP alpha, beta, and gamma). Functionally, within the U-937 monocytic cell line, subtype B and C LTRs were shown to be equally responsive to C/EBP beta-2, although the basal activity of subtype C LTRs appeared to be higher. Furthermore, the synergistic interaction between C/EBP beta-2 and Tat with the subtype C LTR was also observed in U-937 cells as previously demonstrated with the subtype B LTR. This work is supported by NIH/NINDS R01 NS32092, NIDA R01 DA19807, NIMH P30 MH092177, and NIMH T32 MH079785. Moderate neurocognitive decline can be detected over a four-month period using the HIV-dementia scale Background: The HIV Dementia scale (HDS) has been recommended as a cross-sectional screen to identify HIVassociated neurocognitive disorder (HAND) but its longitudinal usefulness has not been established. Method: 55 HIV+ participants (aged 57.7±8.3, 96% males) underwent baseline and follow-up HDS screening at 3.9±1.1 months, a standard neuropsychological (NP) evaluation, and baseline clinical and laboratory examination. At baseline 49.1% met the gold standard HAND diagnostic definition. Based on longitudinal published normative standards, 12.7% showed statistically significant decline (gold standard decline was based on an 80% confidence interval; 1-tailed to include mild decline). To systematically assess HDS criterion validity against the NP gold standard, we determined HDS baseline impairment status based on the three published HDS cut-offs (≤10, ≤14, and demographically-corrected HDS T-scores<40). To determine statistically significant decline on the HDS (80% confidence interval; 1-tailed), we developed standard HDS regressionbased change scores after arcsine-root transformation to normalize the data distribution. HDS longitudinal criterion validity was determined by comparing HDS-based decline to gold standard NP-based decline using indexes of sensitivity and specificity. Results: Baseline HDS cut-off ≤14 yielded the highest sensitivity and specificity (52% sensitivity, 81% specificity) compared to the other cut-offs ≤10 (26% sensitivity, 96% specificity) and T-score <40 (41% sensitivity, 57% specificity). The magnitude of the HDS reliability was very large r=.76 (p<.0001). We found that 21.8% declined by the HDS. In cases congruently identified, median raw decline=2.75; median HDS-change score decline=1.98. The HDS showed 57% sensitivity and 82% specificity in detecting clinically meaningful decline, signifying that only cases that declined moderately were congruently identified. Having a HAND diagnosis at baseline (p=0.01) and more severe HAND diagnosis were associated with greater chance of decline (p<0.03). No HIV biomarkers were associated with decline. Conclusions: This is the first study to optimally demonstrate that the HDS reliably detects at least moderate neurocognitive decline. Beta-catenin positively regulates key proteins in glutamate cycling in vivo Victoria Lutgen, Srinivas D Narasipura, Stephanie Min, Maureen Richards, Lena Al-Harthi (presenting author: victoria_lutgen@rush.edu) Neurological disorders including HIV-associated neurocognitive disorders (HAND) have been linked to abnormal excitatory neurotransmission. Perturbations in glutamate cycling can have profound impacts on normal activity, lead to excitotoxicity and create or exacerbate impairments in these diseases. Astrocytes play a key role in excitatory signaling as they both clear glutamate from the synaptic cleft and house enzymes responsible for glutamate conversion to glutamine. However, mechanisms responsible for the regulation of glutamate cycling including the main astrocytic glutamate transporter excitatory amino acid transporter 2 (EAAT2) or GLT-1 in rodents and glutamine synthetase (GS) which catalyzes the ATP-dependent reaction of glutamate and ammonia into glutamine, remains largely undefined. We previously demonstrated that betacatenin, a transcriptional co-activator and the central mediator of Wnt/beta-catenin signaling pathway, regulates both EAAT2 and GS expression in astrocytes in vitro. We assessed here whether beta-catenin regulates these two proteins in vivo. Towards this end, we injected vivo-morpholinos (500nM) to knockdown beta-catenin expression or control morpholinos into the prefrontal cortex (co-ordinates, AP+2.0, ML+/-0.3, DV 1.0) of 4-6 week old C57 BL/6 male mice using stereotaxic microinjection technique. Morpholinos, which block the translation initiation of their gene target, were injected at day 0 and day 3. At day six post first injection, a small chunk of brain tissue at the injection site was collected, processed for protein isolation using RIPA buffer and analyzed by western blotting. We demonstrate that knockdown of betacatenin resulted in a significant reduction in GLT-1 and GS protein expression by 99 and 93 percent respectively. These studies confirm that beta-catenin regulates key proteins responsible for excitatory glutamate neurotransmission in vivo and reveal the therapeutic potential of Wnt/ beta-catenin modulation in treating diseases with abnormal glutamatergic neurotransmission and excitotoxicity. This work is supported by R01NS060632 to LA. Shaily Malik, Rinki Saha, Pankaj Seth (presenting author: pseth@nbrc.ac.in) Molecular and Cellular Neuroscience, National Brain Research Centre Co-morbidity of HIV-1 and illicit drugs modulate properties of neural precursor cells (NPCs). In an attempt to gain insights into the pathways that may mediate such comorbidities, human fetal brain derived neural precursor cells (hNPCs) were exposed to HIV Transactivating protein, Tat and illicit opioid, morphine at various doses and time points. Alterations in hNPC proliferation with Tat and morphine exposure were assessed by Ki67 and BrdU immunoreactivity of treated cells. Changes in proliferative genes were analyzed at RNA and protein levels and several molecular mediators were investigated. Using cell biology and molecular approaches, we observed that chronic treatment of HIV protein, Tat and morphine affect hNPC proliferation. DNA content analysis by FACS and decreased BrdU levels in Tat-morphine treated hNPCs indicated perturbations in S-phase of the cell cycle. HIV-Tat and morphine attenuated Sox2 and CyclinD1 and increased the expression of p53 and CDK inhibitor, p21 thereby reducing the actively dividing population of hNPCs. p21 was found to be governed by Extracellular signal-regulated kinase-1/2 (ERK1/2) while p53 was found to be essential for Tat and morphine induced decrease in NPC proliferation. The synergy of Tat and morphine induced changes in hNPC proliferation were mostly observed after chronic exposure as opposed to the acute exposure to these neurotoxic agents. Interferon-gamma (IFN-g) levels were also elevated in NPCs with Tat and morphine exposure, leading to altered Signal Transducer and Activator of Transcription (Stat) levels and derangement of Stat-Sox-2 pathway. Such growth arrest in hNPCs may have far reaching clinical implications as it may lead to impaired ability of these cells to maintain their own pool as well as compromise replenishment of damaged brain cells. Authors acknowledge financial support from Department of Biotechnology, ICMR and NBRC to PS and University Grants Commission, New Delhi for fellowship to SM. The immuno-pathogenic phenomena leading to neurodegeneration of multiple sclerosis (MS) are thought to be triggered by environmental factors operating on predisposing genetic backgrounds. Among proposed co-factors are EBV, and the potentially neuropathogenic HERV-W/MSRV/Syncytin-1 endogenous retroviruses. The ascertained EBV-MS links are late primary infection (possibly with infectious mononucleosis, IM), and high titers of pre-onset anti-EBNA IgG. During MS, there is no MS-specific EBV expression, while continuous expression of HERV-Ws occurs, paralleling MS stages, active/remission phases, and therapy outcome. (A) In vitro data: the expression of HERV-W/MSRV/syncytin-1, with/ without exposure to EBV or to EBVgp350, was studied in PBMC from healthy volunteers and MS patients, and in astrocytes. Expression of HERV-W/MSRV/syncytin-1 is amplified in untreated MS patients, and dramatically reduced during therapy. In EBVgp350-treated PBMC, MSRVenv and syncytin-1 transcription is activated in B cells and monocytes, but not in T cells, nor in the highly expressing NK cells. (B) In vivo data: hospitalized young adults with IM symptoms were analyzed for expression of HERV-W/MSRV transcripts and proteins. Healthy controls were either EBV-negative or latently EBV-infected with/without high titers of anti-EBNA-1 IgG. HERV-W/MSRV is activated in PBMC of IM patients (2Log10 increase with respect to EBV-negative controls). When healthy controls are stratified for high, low, or no anti-EBNA-1 IgG titers, a direct correlation occurs with MSRV expression. Flow cytometry shows increased percentages of cells exposing surface HERV-Wenv protein, that occur differently in specific cell subsets, and in acute disease and past infection. Conclusions: in vitro EBV activates the potentially immunopathogenic and neuropathogenic HERV-W/MSRV/syncytin-1, in cells deriving from blood and brain. In vivo the two main links between EBV and MS (IM and high anti-EBNA-1-IgG titers) are paralleled by HERV-W/ MSRV activation. These novel findings suggest HERV-W/ MSRV activation as the missing link between EBV and MS, and may open new avenues of intervention. Lisa Mangus 1 , Jamie Dorsey 1 , Jonathan Oakley 2 , Joseph Mankowski 1 (presenting author: lmangus1@jhmi.edu) 1 Department of Molecular and Comparative Pathobiology, Retrovirus Laboratory, Johns Hopkins University; 2 Voxeleron Human immunodeficiency virus-associated peripheral neuropathy (HIV-PN) is currently the most frequent neurologic complication of HIV infection, affecting most individuals living with HIV. HIV-PN is a length-dependent small sensory fiber neuropathy typified by bilateral numbness, tingling, and burning sensations in the lower legs. Because electrophysiology is insensitive to changes in small unmeylinated fibers, microscopic evaluation of epidermal nerve fiber density in skin biopsies has become the standard for diagnosis of HIV-PN. However, longitudinal assessment is limited by the invasiveness of this technique. While noninvasive assessment of corneal sensory nerves has proven clinically useful in diagnosing and monitoring other peripheral neuropathic conditions, such as diabetic neuropathy, corneal nerve alterations in HIV have yet to be characterized. In this study based in the SIV/macaque model, we first developed a beta-III tubulin immunostaining protocol for detecting corneal nerves, and then developed both manual and automated counting methods to measure corneal nerve density. These counting methods each demonstrated significantly lower subbasal corneal nerve fiber counts among SIV-infected animals that rapidly progressed to AIDS as compared to slow progressors. Concomitant with decreased corneal nerve fiber density, rapid progressors had increased levels of SIV RNA, CD68-positive macrophages, and GFAP expression by glial satellite cells in the trigeminal ganglia. These findings demonstrate that corneal nerve assessment has great potential to diagnose and monitor HIV-induced peripheral neuropathy and set the stage for introducing noninvasive techniques to evaluate PNS damage in the HIV clinical setting. Sivabalan Manivannan 1 , Barbara Slusher 2 , Diane E. Griffin 1 (presenting author: smanivan@jhsph.edu) 1 Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health,; 2 Department of Neurology and Psychiatry and the Johns Hopkins Brain Sciences Institute, Johns Hopkins School of Medicine Inflammation in the nervous system is a necessary part of the response to CNS infection, but also causes neuronal damage in both infectious and autoimmune diseases of the CNS. Sindbis virus is an enveloped, positive-strand RNA virus that causes acute encephalomyelitis and fatal paralysis in mice. Adult C56BL/6 mice inoculated with the neurovirulent strain of Sindbis virus (NSV) succumb to fatal paralysis despite clearing infectious virus from the central nervous system. We show that low daily doses of the non-reversible glutamine antagonist, DON (6-diazo-5-oxo-l-norleucine), rescues mice from fatal paralysis by inhibiting the inflammatory immune response. DON-treated mice fail to induce an adaptive immune response to the virus in the deep cervical lymph-nodes and show decreased CD45+ and CD3+ lymphocyte infiltration into the brain and delayed viral clearance compared to vehicle-treated controls. DON-treated mice show significantly reduced mRNA expression of neurotoxic and inflammatory cytokines (TNF-alpha, IL-1b, IL-6, and IFN-gamma) during the course of viral infection in the brain compared to vehicle controls. Additionally, in vitro studies using purified CD3+ Tcells show that DON inhibits the growth and proliferation, but not the activation, of primary mouse CD3+ T-cells in response to stimulation with anti-CD3/CD28. Following stimulation, DON-treated lymphocytes show a defect in S6 phosphorylation, a downstream mTOR pathway target suggesting impaired protein translation in primary lymphocytes. Stimulated DON-treated T-cells have lower IL-2 protein production despite having similar levels of IL-2 mRNA compared to vehicle controls. These studies suggest that small molecules that antagonize glutamine metabolism are immunomodulatory and could be beneficial in the treatment of neuroinflammatory diseases. Drug abuse is common among HIV-seropositive persons but questions arise if HIV's neurocognitive effects can be distinguished from more nonspecific effects of drug dependence and associated comorbidities. In a preliminary study, we administered four cognitive neuropsychological tasks to a sample of 77 HIV-and 25 HIV+ men and women with no history of drug dependence that were well-matched on demographic characteristics; and 310 HIV-and 137 HIV+ individuals with lifetime DSM-IV-diagnosed cocaine or opioid dependence. The drug using groups were well matched on demographic, substance use severity, and potentially confounding comorbid disorders including PTSD, depression, ADHD and antisocial traits. Current and nadir CD4 counts and viral suppression did not differ significantly between HIV+ drug users and non-users. All subjects performed motor and cognitive procedural learning tasks dependent on the integrity of neostriatum and two measures of impulsive behavior typically performed poorly by drug users. HIV affects neostriatal systems relatively more than drug dependence, which typically engages ventral/limbic striatal processing; we reasoned that HIV+ groups would perform procedural learning tasks more poorly than HIV-groups, regardless of drug history. In a series of HIV Serostatus x Drug History analyses of variance, we found significant main effects (poorer performance) for Drug Use but not HIV Serostatus on both inhibitory tasks, p < .05 for each task; and significant main effects for HIV Serostatus but not Drug History for each procedural learning task, p = .01 for each test. This demonstration provides proof of concept that theory driven cognitive neuropsychological tasks may have the capacity to detect effects of HIV on neurocognition not attributable solely to drug dependence; however, conclusions that are more definitive will require more sophisticated statistical approaches. Clinical trial of a humanized monoclonal anti-IL15Rbeta (CD122) antibody, in HTLV-1 associated myelopathy/ tropical spastic paraparesis (HAM/TSP) Raya Massoud 1 , Yoshimi Enose-Akahata 1 , Joan Ohayon 1 , Kaylan Fenton 1 , Irene Cortese 1 , Steven Jacobson 1 , Thomas Waldmann 2 (presenting author: raya.massoud@nih.gov) 1 NINDS/NIH; 2 NCI/NIH CD122 is the common beta subunit shared by the receptors of interleukins-2 and -15 (IL-2, IL-15), two cytokines implicated in the immunopathogenesis of HTLV-1 associated myelopathy/ tropical spastic paraparesis (HAM/TSP). Several in vitro findings suggest that CD122 might be a therapeutic target in this condition : HAM/TSP CD8+ T-cells show increased CD122 expression at baseline and the addition of Mik-Beta1, a monoclonal antibody against CD122, to cultures of HAM/TSP peripheral blood mononuclear cells (PBMC) decreases endogenous STAT-5 phosphorylation, spontaneous CD8+T-cell degranulation, spontaneous lymphoproliferation and reduces the frequency and cytotoxicity of Tax-specific CD8+T-cells. Based on these findings, we are currently evaluating the safety, clinical and immunological effects of anti-IL15RBeta monoclonal antibody therapy at 1mg/kg in patients with HAM/TSP. As of today three subjects have been treated at this dose and all showed saturation of the CD122 receptor. The therapy seems to be well tolerated and we have detected a reduction in multiple ex vivo immune activation markers (CD8 spontaneous degranulation, STAT5 phosphorylation and spontaneous lymphocyte proliferation). Notably, one patient reported resolution of neurogenic bladder symptoms and we observed objective clinical improvement in two out of three treated patients. HIV +/-opiate-mediated neurotoxicity: GSK3beta is a potential therapeutic target Ruturaj Masvekar 1 , Nazira El-Hage 2 , Kurt Hauser 2,3 , Pamela Knapp 1,2,3 , Joyce Balinang 1 (presenting author: masvekarrr@vcu.edu) 1 Department of Anatomy and Neurobiology, 2 Department of Pharmacology and Toxicology, 3 Institutes for Drug and Alcohol Studies, Virginia Commonwealth University, Richmond, VA 23298 HIV disrupts normal immune system functioning, and can also induce a wide range of neurological deficits, collectively known as HIV-associated neurocognitive disorder (HAND). Our previous work has shown that neurotoxic effects induced by the HIV-1 proteins Tat and gp120 are variably enhanced by co-exposure to morphine, an opiate that preferentially acts through mu-opioid receptors (MOR). This mimics comorbid neurological effects observed in opiate-abusing HIV patients, which occur even in patients receiving HAART. Previous studies have shown that HIV induces abnormal activation of GSK3beta, leading to neurotoxicity, but little is known about the role of GSK3beta in HIV-opiate interactive effects. As tau-immunoreactive tangles have been found in brains of young opiate abusers, we predict crosstalk between MOR-mediated signaling pathway/s and GSK3beta. To mimic the disease process more closely, the current studies use supernatant from HIV-infected monocytes instead of single HIV proteins. U937 human leukemic monocyte lymphoma cells were infected with HIVSF162, an R5-tropic strain. Supernatants were collected and p24 levels were measured by ELISA to assess infection. Adjusted titers were added to cultured murine striatal neurons, in the presence or absence of morphine; all treatments were carried out either in presence or in absence of GSK3beta-inhibitors (VPA, SB415286 and Calbiochem® GSK3beta Inhibitor XXVI) to determine role of GSK3beta in these processes. Both lethal and sublethal effects on neurons were assessed in populations, and also by time-lapse imaging of individual cells over 72 h. Infective supernatants (p24 = 25 pg/ml) induced neurotoxicity, affecting both survival and neuritic arborization; and for selective neurotoxic measures there were significant interactions with morphine. Importantly, GSK3beta-inhibitors significantly reduced HIV-mediated neurotoxicity, and also negated the interactive effects of morphine. Our results suggest that GSK3beta activation is a point of convergence and therapeutic target for opiate-and HIV-mediated neurocognitive deficits. Altered tight junction protein expression in response to prolonged morphine exposure in an in vitro model of the blood-brain barrier Approximately one-third of human immunodeficiency virus type 1 (HIV-1) cases resulting in acquired immunodeficiency syndrome (AIDS) in the United States have been associated with injection drug use within the HIV-1-positive population. Specifically, opioid abuse within this population exacerbates disease progression, including increased viral replication and peripheral viral load, as well as incidence and severity of neurocognitive impairment, as compared to non-users. The blood-brain barrier (BBB) is altered as a component of the pathology associated with the processes that ultimately result in HIV-associated neurocognitive disorders (HAND). HIV-1 proteins, as well as selected drugs of abuse, have been implicated in compromise of the BBB. Previous studies have suggested that exposure to mu-opioids alters BBB permeability, resulting in increased cellular transmigration, as well as overall barrier leakiness. In this study, a human brain microvascular endothelial cell (hBMEC) line, hCMEC/D3, was utilized to establish an in vitro transwell model of the BBB to investigate the effects of chronic (24, 48, or 72 h) morphine exposure on the tight junction proteins (TJPs) of the BBB. We observed that hCMEC/D3 cells form a confluent monolayer with a basal rate of passage of a 70 kDa tracer molecule comparable to primary hBMECs. Although chronic morphine exposure did not induce overall barrier leakiness, changes in mRNA transcripts of tight junction proteins were observed throughout the course of exposure. At the protein level, TJP localization was analyzed following cell fractionation and western immunoblot analysis. Overall, these studies demonstrated that prolonged morphine exposure induced changes in TJP expression patterns at both the mRNA and protein level. This work is supported by NIH/NINDS R01 NS32092, NIDA R01 DA19807, NIMH P30 MH092177, and NIMH T32 MH079785. Acute phase protein lipocalin-2 and disruption of CCR5 signaling cooperate to prevent microglial activation and neuronal injury by CXCR4-utilizing HIVgp120 HIV-1 infection continues to cause associated neurocognitive disorders (HAND) despite the success of combination antiretroviral therapy (cART). The cellular mechanisms underlying the development of HAND are poorly understood. Virusassociated or soluble viral envelope glycoprotein gp120 interacts with CD4 in conjunction with CCR5 and CXCR4 leading to infection and/or cellular signaling, and we study the role of the viral co-receptors in HIV-associated brain injury. Mice expressing a CXCR4-utilizing HIVgp120 as transgene in the brain (gp120tg) share key neuropathological features with AIDS patients, such as the loss of neurites and synapses, pronounced astrocytosis and activated microglia. However, the genetic knockout of CCR5 in gp120tg mice abrogates neuronal injury and microglial activation, but not astrocytosis. In order to further characterize the protective effect of the CCR5KO we performed a gene expression analysis for brains of CCR5WT and CCR5KO HIVgp120tg mice and non-tg controls. The microarray study revealed that brains of HIVgp120tg mice and HIV patients with neurocognitive impairment, with and without encephalitis (HIVE) share a significant part of differential gene regulation. Furthermore, brains of CCR5WT and CCR5KO HIVgp120tg mice express markers of an innate immune response, and one of the most significantly up-regulated factors is the acute phase protein lipocalin-2 (LCN2). In follow-up in vitro experiments we found that LCN2 is itself neurotoxic in a CCR5-dependent fashion whereas, as expected, blockade of CCR5 alone failed to block neurotoxicity of a CXCR4-utilizing gp120. However, the combination of LCN2 and disruption of CCR5 signaling caused a significant loss of microglial cells and at the same time completely abrogated neurotoxicity induced by the CXCR4-utilizing gp120. Altogether our studies identified LCN2 as a potential novel player in HIV-associated brain injury and provided a mechanism of how CCR5-deficiency could protect against neurotoxicity of a CXCR4-utilizing HIVgp120. Supported by NIH grants R01 MH087332, DA026306 and DA029480 (to M.K.). Apolipoprotein E influences innate immune responses of maturing human neuroepithelial progenitor cells exposed to HIV-1 HIV enters the brain early during infection and induces a chronic inflammatory state that can result in neurological abnormalities ranging from mild cognitive dysfunction to severe encephalitis. To better understand the effects of HIV on neural cells, we have used an in vitro model consisting of human neuroepithelial progenitor (NEP) cells that undergo directed differentiation into astrocytes and neurons. In the presence of HIV-1-containing culture supernatants, maturing neurons having the apolipoprotein E4 allele have lower total neurite lengths per cell and moderately reduced levels of neurofilament protein. To investigate whether apolipoprotein E genotype influences changes in gene expression in HIV-exposed NEP cultures, gene expression was detected using microarrays with the Illumina HT-12 V4_0_R1 platform array. This was performed in 4 independent cultures of the apoE3/E3 genotype and 3 independent cultures of the apoE3/ E4 genotype. Through this approach, we identified 32 genes specifically upregulated with a fold change of ΓëÑ 1.5 during exposure to virus, most related to interferon induced responses and antigen presentation. Interestingly, this innate immune response was more robust in the apolipoprotein E3/E3 genotype cultures than in the apolipoprotein E3/E4 counterparts. Biological processes, as defined by the gene ontology (GO) program, corroborated these observations, and processes related to antigen presentation and the actions of interferons were also different among the apoE cultures. Differences were manifested both in the numbers of genes affected as well as their significance in the GO processes in which they participate, with apoE3/E3 higher and more significant than apoE3/E4. These data suggest that maturing NEP cultures respond to HIV by mounting an innate immune response with a vigor that is influenced by the apolipoprotein E genotype of the cells. Increased expression of CD39 and CD73 in frontal white matter of SIV infected rhesus macaque brain tissue: implications for the immunopathogenesis of AIDS and persistence of infected reservoirs Bioqual Incorporated CD39 and CD73 are ectonucleotidases that hydrolyze extracellular ATP into ADP/AMP and adenosine, respectively. Adenosine exerts suppressive effects on effector T cells via interaction with A2A and A2B receptors and furthermore, promotes M2 polarization of macrophages via A2B receptors. Conversely ATP exerts immune stimulatory effects. The hydrolysis of ATP by extracellular CD39 and CD73 has been investigated in the context of certain tumor microenvironments and has been proposed to provide an important mechanism for evasion of immune surveillance in cancer. HIV, which establishes chronic and persistent infection, resistant to eradication via adaptive immune mechanisms, may also promote immune suppression via the hydrolysis of extracellular ATP. In our initial pilot studies, CD39 and CD73 levels were evaluated qualitatively and quantitatively in the frontal white matter of rhesus macaques by immunohistochemistry. CD39 and CD73 are primarily expressed within microglia, perivascular macrophages, and microglial nodules, the latter in brain tissue from animals with SIV encephalitis. Samples from macaques with SIV infection with and without encephalitis exhibited increased expression of CD39 and CD73 compared to uninfected, control specimens. These results suggest a role for ectonucleotidases in the pathogenesis of AIDS and may provide a mechanism for the persistence of HIV/SIV infected reservoirs within the CNS and potentially other compartments. Acknowledgements: This work was supported by grants from NIH/NIMH 1R01MH090910 and R01MH101010 to JR. We acknowledge the support from the Comprehensive NeuroAIDS Center (Kamel Khalili, Ph.D. Program Director, P30MH092177), Basic Science Core II, for providing Neuropathology consultation services (Yuri Persidsky, M.D., Ph.D.). Jennifer McGuire Background: HIV-associated neurocognitive disorders (HAND) affect up to 50% of HIV-infected adults, independently predict HIV morbidity/mortality, and are pathologically associated with neuronal damage and monocyte activation. CSF neurofilaments (NFL, pNFH) are sensitive surrogate markers of neuronal damage in other diseases. In HIV, CSF NFL is elevated in untreated HAD. However, CSF NFL/ pNFH expression has not been characterized in milder forms of HAND (MND, ANI). In addition, the relationship between neurofilaments and CSF/plasma markers of monocyte activation has not been fully explored. Objectives: 1. Determine CSF NFL/pNFH expression across HAND stages and neurocognitively normal (NCN) HIV+ controls. 2. Determine the relationships between CSF NFL/ pNFH and CSF/plasma markers of monocyte activation (sCD14/sCD163/HO-1). Methods: This retrospective descriptive cross-sectional study included 48 HIV-infected adults (15 each ANI/MND/NCN; 3 HAD) off ART, enrolled in CHARTER. Biomarkers were measured by validated ELISAs of CSF (NFL/pNFH), paired CSF/plasma (sCD14/sCD163), and plasma (HO-1). Intergroup comparisons were performed using Kruskal-Wallis and Wilcoxan rank-sum tests. Spearman's correlation coefficients compared different biomarker profiles. Results: CSF NFL/pNFH were not significantly different across the different sub-groups of HAND in this unadjusted viremic cohort. However, among individuals with CD4 nadirs ≤200, CSF NFL levels were elevated in HAD compared with NCN, ANI, and MND. Plasma sCD163 did not vary significantly across different sub-groups of HAND, in contrast to previous studies of virologically-suppressed individuals on ART. CSF NFL was significantly positively correlated with CSF pNFH/sCD163/sCD14. Conclusion: This study is the first to correlate CSF NFL expression with CSF sCD14/sCD163 in viremic HIVinfected individuals off ART, eliminating unanticipated direct effects of ART on inflammation and neurodegeneration. In addition, this is the first study to examine NFL across different sub-groups of HAND. Our results confirm that systemic HIV replication is strongly correlated with CNS monocyte activation and neurodegeneration in individuals with a history of severe CD4 depletion. Immunodeficiency lentiviruses, including FIV, readily penetrate the host central nervous system (CNS) and establish a protected reservoir of infection in macrophages and microglia. These reservoirs have been difficult to control with combined antiretroviral therapy (CART) due, in part, to limited CNS penetration of current antiretroviral compounds. Using the FIV model, we explored the therapeutic potential of the novel antiretroviral compound, FX101, in an effort to reduce or eliminate CNS infection while minimizing toxicity. FX101 is a proprietary compound that targets a strictly conserved region of the nucleocapsid protein shared by FIV, SIV, and HIV. Binding of the drug appears to disrupt virus particle assembly allowing control of infectious virus production in previously infected cells. FX101 distributes in tissues and has an apparent long plasma and CSF half-life of 4.2 and 3.3 days, respectively. At a dose of 2 mg/kg, plasma concentrations peaked at 21.07 uM. CSF concentrations were variable with peak values ranging from 0.052 to 1.18 uM. Clinical pathology demonstrated no significant renal, liver, or red blood cell/ white blood cell abnormalities. In vitro, neurotoxicity was relatively low with a median toxic concentration of 20.5 uM. FX101 inhibited FIV replication in feline macrophages in vitro to 8% of controls. To establish antiviral efficacy in vivo, six cats, previously intracranially infected with FIV plus 4 sham infected cats were treated with 2 mg/kg FX101 in 2% DMSO intravenously twice per week over a 4 week period. Virus load in plasma and CSF and provirus loads were significantly reduced to 6-23% of baseline and remained at 5-12.5% of original virus burdens even 36 weeks after treatment was completed. These results show that FX101 has significant antiviral activity which is sustained for long periods and may include effects that progressively reduce the proviral burden in cells. Supported by NIH Grant 5R43MH096663 HIV-1 enters the CNS soon after peripheral infection and causes chronic inflammation and CNS damage that leads to cognitive impairment in greater than 50% of HIV infected people even with successful cART treatment. PrPc (protease resistant prion protein) is the non-pathogenic cellular isoform of human prion protein that is constitutively expressed in the CNS and is involved in several physiological processes that are disrupted during HIV neuropathogenesis. PrPc is also expressed on monocytes and brain microvascular endothelial cells (BMVEC) and is essential for the transmigration of monocytes across the blood brain barrier (BBB). The surface expression of PrPc is increased in HIV infected monocytes as detected by flow cytometry analysis. Previously, our laboratory showed that soluble PrPc (sPrPc) levels are increased in the CSF of people specifically with HIV associated neurocognitive disorder (HAND). Our in vitro studies also showed that HIV infection as well as treatment of CNS cells with the chemokine CCL2 caused increased PrPc shedding. All of these data suggest that sPrPc participates in the mechanisms that mediate NeuroAIDS. To determine the effect of this shed PrPc on the transmigration of monocyte across the BBB, we used our in vitro BBB model which consists of astrocytes and endothelial cells co-cultured on opposite sides of 0.3um pore tissue culture insert. In these studies we demonstrated that sPrPc treatment blocked the transmigration of monocytes across the BBB, suggesting that it could be an initial mechanism of protection against the influx of monocytes across the barrier during early stages of HIV infection. Thus, PrPc may be both protective and neuroinflammatory depending upon its temporal and spatial expression during HIV neuropathogenesis. stratified by HAND into normal cognition (NC, n=8), asymptomatic impairment (AI, n=7), and symptomatic impairment (SI, n=9) and 5 controls. Cytokine levels (TNF-alpha, IFNgamma, IL-2, IL-4, IL-6, and IL-10) were quantified by CBA immunoassay kit and flow cytometry. Neuronal cells (SH-SY5Y cells) were cultured in the presence of cytokines for 24 hours using concentrations similar to those quantified from the CSF of the HIV-seropositive women. Secreted sIR levels were assayed by ELISA. Results: CSF levels of TNF-alpha, IFN-gamma, IL-10, and IL-2 were increased in SI and AI when compared to NC (p<0.05). IL-6 levels were significantly increased in SI when compared to NC (p<0.05). No significant differences were observed in IL-4 CSF levels among groups. sIR levels secreted by neurons increased significantly (p=0.02) after 24hrs exposure to TNF-alpha. Although exposure to IL-10 had slight decrease in sIR secretion this was not significant (p= 0.08). IFN-gamma and IL-6 did not induce any change in sIR secretion as compared to control. Conclusion: This study suggests that CSF cytokine levels in HIV-seropositive women alter neuronal sIR secretion, and may contribute to insulin resistance in HAND patients. Partially supported by: R21MH095524, R25MH080661, R25MD007607, U54RR026139, G12MD007600, G12RR003051, U54NS043011, U54MD007587, S11NS046278 Autophagy is an evolutionary conserved and selective degradation pathway of cellular components that is important for the maintenance of cell homeostasis under healthy and pathologic conditions. Here we demonstrate that an increase in the level of BAG3 results in stimulation of autophagy in glioblastoma cells. BAG3 is a member of a co-chaperone family of proteins that associate with Hsp70 through a conserved BAG domain positioned at the C-terminus of the protein. Expression of BAG3 is induced by a variety of environmental changes causing stress to cells. Our results show that BAG3 interacts with Beclin-1, a key regulator of autophagy, through its unique WW domain positioned at the N-terminus of the protein. In addition, similar to Beclin-1, BAG3 associates with Bcl2, which plays an important role in proliferation and apoptosis. Induction of BAG3 and its association with Beclin-1 diminish interaction of Bcl2 and Beclin-1. Interestingly, an elevated level of BAG3 also promotes cleavage of caspase 3 suggesting a dual role for BAG3 in modulating autophagy and apoptosis. These observations place BAG3 in a unique position suggesting that its interplay with Beclin-1 and Bcl2 has a functional consequence on coordinating the stage of autophagy and apoptosis in glioma cells. Landhing M. Moran, Rosemarie M. Booze, Charles F. Mactutus (presenting author: moranl@email.sc.edu) Approximately 50% of HIV-1-positive individuals are afflicted with HIV-1-associated neurocognitive disorders (HAND), despite the effectiveness of combination antiretroviral therapy (CART) in reducing the prevalence of more severe neurocognitive impairment (i.e., dementia). Deficits in executive function are a distinguishing feature of HAND in the CART era, decaying more rapidly during HIV disease progression than other cognitive domains. In the present study, ovariectomized female Fischer HIV-1 Tg (n=41) and control rats (n=43) were tested with a sustained attention task, a component of executive function. Rats were trained to discriminate signals (illumination for 100, 500, or 1000 msec duration) from non-signals (no illumination) and were reinforced with sucrose pellets for pressing the lever corresponding to each event (hits and correct rejections vs. misses and false alarms). A decrease in hits and an increase in misses as a function of decreased signal duration were observed for both groups; however, the HIV-1 Tg group was more adversely affected by the shorter signal durations. In contrast to the performance of controls, the HIV-1 Tg rats did not display differential target detection (hits vs. misses) at the 500 or 100 msec durations. Subsequently, a daily oral dose of Sequol (0.05, 0.1, or 0.2 mg, or vehicle), a metabolite produced via the gut microbiome following ingestion of soy isoflavone daidzein, was administered to determine its potential therapeutic effects. After 45 days of treatment, the HIV-1 Tg animals that received the 0.2 mg dose of Sequol missed significantly fewer signals across all stimulus durations compared to their pre-dosing performance. Their improvement was twofold that of the control group. The phytoestrogen metabolite S-equol may be useful as a therapeutic for attentional deficits in HAND. Funded by NIH grants DA013137 and HD043680. The impact of viral infection on the innate immune response within the brain during chronic neurodegeneration Understanding the mechanism and environmental influences which shape the development of neurodegenerative diseases such as Alzheimer's is of importance given the trend for an aging population. The majority of neurodegenerative diseases involve abnormal protein deposits within the CNS, degeneration of synapses and neurons and the activation of astrocytes and microglia. It has been suggested that microglia in a brain with ongoing chronic neurodegenerative disease are susceptible to systemic inflammatory signals that switch cells from an atypical anti-inflammatory phenotype to one in which they cause neuronal damage. Defining the impact of environmental factors such as infections on the diseased brain will have profound implications for protecting neurones. We are using a well characterised TSE agent, ME7, as a model for neurodegenerative disease. The locus and timing of the initiation of neurodegeneration by ME7 is under precise experimental control and rodent models of these diseases display all the features of a chronic neurodegenerative process. We challenged mice in vivo with a transient neuroinvasive viral infection, Semliki Forest virus, pre-and post-inoculation with ME7 to ask if "priming" of the immune system by a viral infection which is cleared by the host prior to the onset of a neurodegenerative disease would alter the pattern of progression of the neurodegenerative disease. We observe that co-infection with SFV during the early phase of neurodegeneration does not change disease incubation time but does result in a subtle change in clinical disease onset. Interestingly, we see a significant alteration of early stage disease pathology with a change in the pattern of PrPSc deposition and gliosis. In addition, we address if viral infection prior to the onset of chronic neurodegeneration can switch the innate immune response of microglia from a neuroprotective to a tissue damaging phenotype and exacerbate neurodegenerative disease progression. In vivo and in vitro experiments were undertaken to elucidate the role of regulatory B lymphocytes in controlling neuroinflammation following brain infection with murine cytomegalovirus (MCMV). This unique subset of CD19(+)CD1d(hi)CD5(+) Bcells was found to infiltrate the brains of chronically infected animals, reaching highest levels at the latest time point tested (35 d p.i.). B-cell-deficient Jh-/-mice (BKO) displayed exacerbated neuroimmune responses when compared to infected, wild-type (Wt) animals as measured by enhanced accumulation and/or retention of CD8+ T lymphocytes within the brain, as well as increased levels of microglial cell activation (MHC class II). Conversely, levels of regulatory T-cells (Tregs) were found to be significantly lower in infected BKO mice when compared to Wt animals. Further experiments showed that in vitro generated interleukin (IL)-10-secreting Breg cells were able to inhibit cytokine and chemokine responses from microglia following stimulation with viral antigens. In addition, these in vitro generated Bregs were also found to promote conversion of CD4+ Tcells into a regulatory T-cell (Foxp3+) phenotype. Finally, gainof-function experiments demonstrated that reconstitution of Bcells into BKO mice restored these neuroimmune responses to levels exhibited by infected Wt animals. Taken together, these results demonstrate that regulatory B-cells modulate T lymphocyte as well as microglial cell responses within the brain, and promote CD4+ T-cell transition into a Treg phenotype. HIV anti-retroviral therapy nucleoside reverse transcriptase inhibitors induce mitochondrial dysfunction, oxidative stress, and premature senescence in primary human fibroblasts Timothy Nacarelli, Ashley Azar, Elizabeth Crowe, Claudio Torres, Christian Sell (presenting author: tn332@drexel.edu) Drexel University College of Medicine HIV patients are currently treated with highly active antiretroviral therapy (HAART) that employs nucleoside reverse transcriptase inhibitors (NRTIs). Although successful in decreasing the HIV viral-load and rate of infection, NRTIs predispose patients to an increased risk of developing pathologies associated with aging. These age-related pathologies are thought to arise from mitochondrial toxicity that is attributed by NRTI-mediated inhibition of mitochondrial DNA polymerase gamma. The newer generation of NRTIs currently used in HAART, tenofovir and emtricitabine (TDF/FTC), contain a decreased inhibitory affinity for DNA polymerase gamma, but mitochondrial toxicity remains eminent. We postulate that TDF/FTC impair mitochondria in primary human fibroblasts. Primary human fibroblasts are a relevant model to study aging in vitro, as they contain a finite proliferative capacity before entering cellular senescence, an irreversible growth arrest that recapitulates aging at the cellular level. Cellular senescence is activated by oxidative stress, which could be contributed by reactive oxygen species (ROS) produced from impaired mitochondria. Therefore, we also examined whether TDF/FTC increased mitochondrial and cellular ROS and induce premature senescence. Previous studies in the laboratory shown that treatment with rapamycin, an mTORC1 inhibitor, promotes mitochondrial homeostasis, decreases oxidative stress, and delays senescence in aging primary human fibroblasts. We further examined whether rapamycin could alleviate NRTI-induced effects. After treatment with TDF/FTC, we found that mitochondrial integrity is impaired, ROS is increased, and premature senescence is induced. However, these alterations were ameliorated in the presence of rapamycin. Our results demonstrate that the latest generation of NRTIs impair mitochondria and induce senescence in vitro. Clopidogrel, a P2Y12 purinoreceptor antagonist, attenuates VZV infection in human brain vascular adventitial fibroblasts Maria Nagel 1 , Ann Wyborny 1 , Alexander Choe 1 , Igor Traktinskiy 1 , Evgenia Gerasimovskaya 2 (presenting author: maria.nagel@ucdenver.edu) 1 Department of Neurology, University of Colorado School of Medicine; 2 Department of Pediatrics, University of Colorado School of Medicine Varicella zoster virus (VZV) vasculopathy is caused by productive infection of cerebral arteries leading to pathological vascular remodeling and stroke. Purinergic signaling, mediated by ATP and its metabolites, has emerged as important in vascular remodeling. In addition, purinergic signaling is involved in RNA virus infection, but its role in DNA virus infection is unknown. Thus, we hypothesize that specific purinoreceptors are involved in VZV infection of cerebral arteries and vascular remodeling. Because the arterial adventitia is the initial vascular site infected by VZV after virus reactivation, we treated VZV-infected primary human brain vascular adventitial fibroblasts (BRAFs) with antagonists of purinoreceptors P2X1, P2X4, P2X7, P2Y2, P2Y6, P2Y11 and P2Y12. Three days later, viral DNA was quantified by qPCR. Compared to untreated infected controls, the P2Y11 antagonist significantly down-regulated VZV DNA by 37% and the P2Y12 antagonist by 69%. Significant differences were not found with other purinoreceptor antagonists. Because the P2Y12 antagonist (clopidogrel) produced the greatest reduction of VZV DNA, we also studied its effect on HSV-1 and HSV-2-infected BRAFs. Clopidogrel significantly downregulated HSV-1 and HSV-2 by >99% compared to untreated infected controls. Furthermore, clopidogrel reduced the titer of infectious virus for all three alphaherpesviruses. Overall, we demonstrated that the P2Y12 purinoreceptor antagonist decreases VZV, HSV-1 and HSV-2 DNA and infectivity in BRAFs. The clinical effectiveness of the P2Y12 antagonist clopidogrel remains to be determined. Recently, much interest has developed regarding mechanisms of extracellular delivery of nucleic acids and proteins among virally infected and recipient cells. While the role of exosomes in viral pathogenesis and disease states remains largely unknown, it is now widely accepted that exosomes play important roles in intercellular communication, cellular inflammation, antigen presentation, programmed cell death, and pathogenesis. HIV-1 encodes its own miRNAs that regulate viral and host gene expression. The most abundant HIV-1derived miRNA, first reported by us and later by others using deep sequencing, is the TAR (Trans-Activation Response element) miRNA. We have recently found the presence of TAR RNA in exosomes from cell culture supernatants of HIV-1 infected cells and patient sera. We report that transport of TAR RNA from the nucleus into exosomes is a CRM1dependent active process. Prior exposure of naive cells to exosomes from infected cells increased susceptibility of the recipient cells to HIV-1 infection. Exosomal TAR RNA down regulated apoptosis by lowering Bim and Cdk9 proteins in recipient cells. We found 10^4 -10^6 copies/ml of TAR RNA in exosomes derived from infected culture supernatants and 10^3 copies/ml of TAR RNA in the serum exosomes of Highly active antiretroviral therapy (HAART)-treated patients or Long term nonprogressors (LTNPs). Very recently, we have found that TAR is able to activate cytokines in the recipient cells by increasing the nuclear accumulation of both p65 and p50 (component of NFkB complex). This increase may be related to a newly formed IKKb in the TAR treated cells which may be the result of TLR activation by TAR. We will discuss the effect of these biochemical steps in the recipient macrophage cells that result in alteration of cytokines which in part may explain the neuroinflammation observed in AIDS patients who are under HAART treatment. Ferritin Heavy Chain (FHC), a subunit of the ubiquitously expressed iron storage complex Ferritin, has recently been shown to be upregulated in the central nervous system (CNS) in response to HIV/SIV infection and illicit drug abuse. Previous research has suggested that an unexpected consequence of increased CNS FHC lies in its ability to inhibit homeostatic signaling of the chemokine receptor CXCR4, resulting in decreased dendritic spine density in cortical neurons. Interestingly, initial studies indicate that these changes correlate with disease progression and behavioral symptoms observed in HIV associated neurocognitive disorders (HAND). Opiates, including morphine and DAMGO, are able to upregulate FHC in a mu opioid receptor dependent manner, suggesting a novel avenue of CXCR4 regulation via opioid signaling. Previous experiments have shown that chronic morphine treatment (both in vitro and in vivo) caused a general upregulation of FHC in cortical neurons, which corresponded with decreased levels of phosphorylated, or activated CXCR4, and decreases in the activation of downstream signals (i.e. ERK1/2 and Akt). However, this interaction has yet to be studied in other CNS cell types such as astrocytes, which express both CXCR4 and MOR and are critically involved in neuronal function. Additionally, characterization of morphine-induced changes in the subcellular distribution of FHC, in neurons or other CNS cells, may provide clues about the nature of the CXCR4/FHC interaction, and additional roles of FHC in HAND, and other neurodegenerative diseases. Debasis Nayak, Dorian McGavern (presenting author: nayakdn@ninds.nih.gov) NINDS, NIH Tetherin or bone marrow stromal antigen 2 (BST-2) is an interferon-inducible plasma membrane protein that has recently emerged as a key innate mammalian restriction factor which impedes release of enveloped viruses by physically tethering nascent budding virions to the plasma membrane. Several in vitro studies have demonstrated that BST-2 is a potent antiviral that acts upon many virulent enveloped viruses, such as retroviruses (HIV), arenaviruses (Lassa), filoviruses (Ebola), paramyxoviruses (Sendai), orthomyxoviruses (Influenza A), rhabdoviruses (VSV), etc. However, it remains unclear how this important innate immune protein participates in sequestering a persistent viral infection in vivo. In this study, we used the lymphocytic choriomeningitis virus (LCMV) clone 13 (CL13) model of persistent viral infection to evaluate the role of BST-2 in facilitating the transition from an innate to a virus-controlling adaptive immune response. Importantly, BST-2 knockout mice had an impaired ability to control LCMV CL13 in peripheral tissues as well as the central nervous system (CNS). Viral clearance was delayed by one month in the sera and never purged from the CNS. Anatomically, BST-2 expression mapped to plasmacytoid dendritic cells (DCs), myeloid DCs, and brain resident microglia, consistent with the impaired ability of BST-2-/-mice to sequester LCMV antigen in the splenic marginal zone and brain after infection. Analysis of the adaptive immune response to CL13 revealed that BST-2-/-mice generated less polyfunctional virus-specific CD8+ and CD4+ T cells and showed reduced expansion when compared to wild type control mice, demonstrating that BST-2 is an important participant in the bridge between innate and adaptive immunity. Collectively, our data indicate that BST-2 plays an essential role in controlling a persistent viral infection, which explains why so many viruses have developed strategies to thwart the activity of this potent antiviral protein P132 HIV-1 Vpr accessory protein has a negative impact on astrocityc telomerase activity Diego Ojeda, Mauricio Carobene, Jorge Quarleri (presenting author: diegosebastianojeda@gmail.com) HIV-1 Infection leads to a wide spectrum of neurodegenerative diseases. Among central nervous system cells, astrocytes have been shown to be susceptible to HIV-1 infection. Vpr is an HIV-1 accessory protein that negatively affects the highly controlled CNS compartment, inducing astrocytic apoptosis and secretion of neurotoxins that causes neuronal loss. Telomerase is a cellular ribonucleoprotein complex key in controlling cellular senescence, and involved in inhibition of apoptosis and improvement of DNA repair. Considering that HIV-1 has been shown to modify the cellular aging process by interfering with telomerase activity (TA), the aim of our study was to evaluate the impact of Vpr protein expression on TA in astrocytes. Vpr coding sequence from HIV-1 NL4-3 strain was PCR-amplified and cloned into the pEGFP-C3 expression vector. U373 astrocytic cells were transfected with the pEGFP-Vpr vector, and TA was evaluated by real-time PCR (expressed as Relative Telomerase Activity, RTA), 48 h post-transfection. Transfection efficiency, expressed as percentage of GFP-positive cells, was measured by FACS. Statistical differences were calculated by t-student test. Telomerase activity (RTA) was found to be significantly reduced in U373 astrocytic cells transfected with the HIV-1 Vprexpression vector (25.7% RTA, p<0.05), when compared to untransfected (considered as 100% RTA) and pEGFP-C3transfected cells (44.3% RTA). Mean transfection efficiency for pEGFP-C3 and pEGFP-Vpr transfected cells, was 41% and 39.1%, respectively, as determined by the percentage of GFPpositive cells evaluated by FACS. Data presented here indicates that expression of HIV-1 Vpr has a negative impact on astrocityc TA. Given the critical roles this cell type plays in creating a threedimensional framework for the CNS, in maintaining the bloodΓÇôbrain barrier, and in neural metabolism and activity, the observed phenomenon is relevant in the context of understanding the astrocyte biology changes that could be involved neurodegenerative process observed during HIV-1 infection. Resistance to Apoptosis in HIV-Infected astrocyte as a potential mechanism for viral Persistence in CNS Diego Ojeda, Mauricio Carobene, Jorge Quarleri (presenting author: diegosebastianojeda@gmail.com) Apoptosis plays a critical role in HIV-1-associated neuropathogenesis. HIV-1 has developed multiple strategies to modulate apoptosis related-pathways on infected cells thus promoting its survival and favoring its role as viral reservoirs. HIV-1 establishes infection in astrocytes of the CNS causing minimal cytopathology in these long-lived cells. This study aimed to evaluate the HIV infection impact on apoptosis cascade in infected astrocytes and their neighboring uninfected cells. An HIV infectious molecular clone pNL43-derived and tagged with the GFP (named HIV-GFP) was constructed. It was also pseudotyped with the VSV-G glycoprotein by co-transfection of 293T cells. U373 human astrocytic cells were used as in vitro models for astrocyte infection. To detect apoptotic events at 1 to 6 days post infection (dpi), co-staining with 7AAD and annexin-V was performed. Singlecell analysis aimed at simultaneously identifying apoptoticinfected (Ann+/GFP+) and apoptotic-uninfected (Ann+/GFP-) neighbor cells by FACS. The values were expressed as mean ±SD. Statistic analysis was performed by Student T test and p values <0.001 were considered significant. Initially the level of apoptosis (annexin V+) at 1 and 2 dpi depicted no differences (p>0.005) between productively infected astrocytes (13±7.7 and 7.3±9.7 GFP+, respectively) and neighbor uninfected cells (16.5 ±9.5 and 7.65±6.5 GFP-, respectively). In contrast, when comparing the level of apoptosis (annexin-V+) at 3, 4, 5 and 6 dpi between infected vs. neighbor uninfected astrocytes (GFP+/ GFP-) a significant decreased (p<0.0001) (12±4/18±6, 14±3.8/ 26±12.8, 8±2.8/22±10, and 6±3.6/20±10, respectively) was observed. Such differences were independent of the level of viral replication (GFP+ cells). HIV infection of astrocytes results in survival of infected astrocytes and apoptosis of neighbor uninfected cells. Such anti-apoptotic effect was independent of the viral replication magnitude and its kinetic exhibited a biphasic pattern according the infection progress. In advances stages of infection the resistance to apoptosis was more pronounced, which could support the generation of HIV reservoirs. This study evaluated the relationship between illicit drug use and HIV-1 disease severity in HIV-1-infected patients enrolled in the DrexelMed HIV/AIDS Genetic Analysis Cohort. Since cocaine is known to have immunomodulatory effects, the cytokine profiles of preferential non-users (PN), cocaine users (PCo) or multidrug users (MDU) were analyzed to understand the effects of cocaine on cytokine modulation and HIV-1 disease severity. Patients within the cohort are assessed approximately every 6 months for HIV-1 clinical parameters and history of illicit drug, alcohol, and tobacco use. The Luminex human cytokine 30-plex panel was used for cytokine quantification. Analysis was performed using a newly developed biostatistical model. Substance abuse was found to be common within the cohort. Utilizing the drug screens at the time of each visit, it was determined that the cohort could be categorized as PN, PCo, and MDU. The overall health of the PN population was better than that of the PCo population, with peak and current viral loads in PN substantially lower than those in PCo and MDU patients. Among the 30 cytokines investigated, differential cytokine profiles were established within the three populations. The Th2 cytokines, IL-4 and IL-10, known to play a critical role during HIV-1 infection, were positively associated with increasing cocaine use. Clinical parameters such as latest viral load, CD4+ and CD8+ T-cell counts, and CD4:CD8 ratio were also significantly associated with cocaine use. Based on these assessments, cocaine use appears to associate with more severe HIV-1 disease. This work is supported by NIH/NINDS R01 NS32092, NIDA R01 DA19807, NIMH P30 MH092177, and NIMH T32 MH079785. Michael Park 1 , Thuong Tran 1 , Rasha El Baz 1 , Andrea Cuconati 2 , James Arthos 3 , Caitlin Duffy 4 , Irwin Chaiken 4 , Dora Schnur 5 , Fengbin Song 2 , Patrick Lam 6 , Charles Reynolds 2 , Allen Reitz 7 , Zafar Khan 1 , Pooja Jain 1 (presenting author: zafar.khan@drexelmed.edu) DC-SIGN (dendritic cell specific ICAM-3-grabbing nonintegrin), a membrane protein of the C-type lectin family, is found in high levels on monocyte-derived DCs, some macrophages, and activated B cells. In vivo, DC-SIGN-positive cells have been found in lymph nodes, tonsils, skin, and the subepithelial regions of the cervix. With respect to HIV-1 transmission, DC-SIGN has been shown to serve as ligand that efficiently binds, concentrates, and mediates transfer of virus from the mucosal surface to CD4+ T cells. Subepithelial DCs expressing DC-SIGN have been demonstrated to play an important role in cervicovaginal transmission of HIV-1. Additional studies have examined the process of DC-SIGN-mediated transmission of various pathogens, suggesting that targeting this cell surface molecule may serve as a potential therapeutic strategy. DC-SIGN binds to mannose and fucose moieties present on the HIV envelope glycoprotein gp120 with high affinity. Blocking this interaction at the site of primary infection could potentially be prophylactic and/or serve as a potential microbicidal target. We have developed a novel high throughput screening assay (HTS) to identify inhibitors of DC-SIGN:gp120 interaction and validated this assay by using quinoxazoline small molecules that block mannose dimer binding to DC-SIGN demonstrating that HTS of non-carbohydrate libraries can produce micromolar (or lower) non-carbohydrate hits. Further, we completed virtual screen of diverse libraries of small molecule inhibitors by docking into the calcium-binding domain of DC-SIGN crystal structure and subjected virtual hits into the HTS assay. In addition, we began a novel approach of "binding-site directed lipophilic mining" for hypothesis-driven drug discovery. Interplay between macrophage senescence, microRNA expression, and HIV-1 infection Andrea Partridge, Elizabeth Crowe, Claudio Torres, Julio Martin-Garcia (presenting author: atp28@drexel.edu) In the context of HIV-1 infection, senescence is accelerated, facilitating opportunistic infections and increased viral replication. Cellular senescence is irreversible growth arrest in the lifetime of a cell. Although increased macrophage tropism and HIV-1 replication has been shown, the contribution of macrophages to this process has not been clearly established. Changes in microRNA (miRNA) expression during cellular senescence and/or aging have also been described. MiRNAs seem to contribute to the aging process at the level of cellular senescence, tissue aging, and lifespan of whole organism. However, the specific role of miRNAs in macrophages during aging has not yet been established. Alterations in miRNA profiles during cellular senescence may affect HIV-1 infection, and potentially HIV-1 infection could as well affect cellular senescence through miRNA modulation. We have induced senescence in primary human macrophages and THP-1-derived macrophages by oxidative damage (H202 treatment). To confirm the presence of a senescent phenotype, we used a senescence-associated beta-galactosidase (SA betagal) assay, and western blot analysis of the levels of p16, p21, and p53, which have been shown to increase in senescent cells. We determined that aged and senescence-induced macrophages display a senescent-like phenotype that consists of increased expression of p53, p21, and an enlarged flattened morphology with positive staining for SA beta-gal. Further studies will include the analysis of the cytokine and chemokine secretion profile to determine whether or not they exhibit a senescence-associated secretory phenotype, and the investigation of changes in miRNA profiles due to infection and senescence. Yuri Persidsky, Jeremy Hill, Holly Dykstra, Nancy Reichenbach, Slava Rom, Servio Ramirez (presenting author: yuri.persidsky@tuhs.temple.edu) Despite combined antiretroviral therapy achieving efficient HIV replication control, neurocognitive complications defined as HAND continue to be highly prevalent in HIV infection. One of explanations could be constant compromise of blood brain barrier (BBB) function driven by chronic inflammatory responses documented in HIV infected individuals even with well-controlled virus replication yet with HAND progression. Brain endothelial cell injury and its underlying mechanisms have been explored during last years. However, changes in other cells constituting BBB (like pericytes) have not been studied in detail in context of HIV-1 infection.We found partial pericyte loss and down regulation of key receptors on these cells in brain tissue of HIV-1 infected patients (even without detectable HIV-1 in CNS) and HIV-1 infected huNSG mice that paralleled BBB compromise and neuroinflammation. Primary human brain pericytes treated with IL-1 beta or TNF-alpha demonstrated decreased secretion of angiopoetin-1/ transforming growth factor-beta 1, diminished expression of PDGF-R beta 1 (controlling pericyte survival and recruitment to BBB) and connexin 43 (critical for establishing gap junctions between pericytes and brain microvascular endothelial cells, BMVEC). Pericytes exposed to IL-1 beta/TNF alpha showed enhanced expression of numerous pro-inflammatory factors (relevant to HIV-1 neuropathogenesis) and adhesion molecules (ICAM-1, VCAM-1) paralleling increased adhesion of monocytes to pericyte monolayers. Using BBB models (composed of BMVEC-pericytes) we showed increased monocyte migration across BBB constructs in response to relevant chemokine, CCL2. IL-1 beta/TNF alpha treatment of pericytes diminished their ability to migrate providing potential mechanism of their decrease in brain tissue of HIV-1 infected patients. Taken together, these data point to a major decrease in barrier supporting function and enhanced inflammatory responses in brain pericytes after exposure to relevant cytokines and in brain tissues of HIV-1 infected patients. Sudheesh Pilakka-Kanthikeel, Andrea Raymond, Venkata Subba Rao Atluri, Vidya Sagar, Upal Roy, Madhavan Nair (presenting author: spilakka@fiu.edu) Dept of Immunology, Institute of NeuroImmune Pharmacology, Herbert Wertheim College of Medicine, Florida International University, Miami, FL-33199 Human immunodeficiency virus 1 (HIV-1) remains one of the leading causes of death worldwide. HIV penetrates CNS during early infection, establishing a viral reservoir. Even though astrocytes are infected by HIV, unlike microglia and brain macrophages, they are not productively infected. Non-productive infection could be significant to neuropathogenesis. Although the potent antiretroviral therapies have significantly improved the morbidity in HIV patients, the biggest challenge is inability of HAART to eradicate the virus from the reservoirs. The mechanisms governing the establishment of HIV reservoir in vivo are still not fully understood. The brain is also a target organ for cocaine. Cocaine enhances HIV-1 expression in susceptible cells, serving as a co-factor in the susceptibility and progression of infections. Recently identified restriction factor, SAMHD1, which hydrolyses dNTPs has been shown to restrict HIV infection in resting CD4+ T cells. Based on the previous reports from literature, we hypothesized that increased SAMHD1 expression in astrocytes results in suppression of cellular dNTPs subsequently hampering retrovirus reverse transcription, which in turn leads to non-productive infection. We examined constitutive expression of SAMHD1 in astrocytes and microglia at the gene and protein level and its modulation by cocaine. Astrocytes displayed higher expression level of SAMHD1 compared to microglia, suggesting that SAMHD1 plays a role in astrocytes facilitating them to maintain non-productive infection. Cocaine degraded SAMHD1, which in turn increased viral replication in astrocytes. Suppressing the level of SAMHD1 in astrocytes by cocaine reactivates the latent virus from astrocyte. The long terminal repeat (LTR) regulates HIV-1 gene expression by interacting with multiple host and viral factors. Crosssectional studies in the pre-HAART era demonstrated that SNPs in C/EBP site I and Sp site III from peripheral bloodderived LTRs increased in frequency as disease severity increased and correlated with HIV-1-associated dementia. Current studies focus on the identification of LTR signatures derived from peripheral blood virus that can be used as molecular markers to identify HIV-1-infected individuals more prone to developing advanced stage disease and/or neurologic disease. A prospective, longitudinal study was conducted on 504 HIV-1 seropositive patients currently enrolled in the DREXELMED HIV/AIDS Genetic Analysis Cohort in Philadelphia, PA. History of clinical parameters and comorbities were obtained approximately every 6 months. The collection of extensive clinical parameters on these patients have allowed for cross-sectional and longitudinal analyses of the impact of these parameters on the development of SNPs during the course of disease. To date, SNPs have been identified that associated with CD4 T-cell count and viral load. Of the SNPs identified, SNPs at position 108 were the most significant and correlated with a gain in transcription factor binding. These results suggest that the HIV-1 genomic swarm may evolve during the course of disease in response to selective pressures that lead to changes in prevalence of LTR SNPs that may be predictive of more advanced stage HIV disease and that may result in alterations in viral function. This work is supported by NIH/NINDS R01 NS32092, NIDA R01 DA19807, NIMH P30 MH092177, and NIMH T32 MH079785. Alina Popescu Hategan 1 , Joseph Steiner 1 , Emilios Dimitriadis 2 , Avindra Nath 1 (presenting author: alina.popescu@nih.gov) 1 NIH/NINDS, Section of Infections of the Nervous System, Bethesda MD; 3 NIH/NIBIB, Scanning Probe Microscopy Unit, Bethesda MD We show that amyloid beta 1-40 aggregation under physiological conditions in the presence of HIV-Tat protein presents significant structural changes. Atomic force microscopy imaging shows that the predominant typical singular uniform amyloid fibrils formed at a 200 micromolar amyloid concentration turn into a population with more double twisted fibers when 0.08 micromolar HIV-Tat is present and at higher Tat concentrations (0.4 to 1.8 micromolar) turns into thick unstructured filaments and nonspecifically aggregated large patches. At a 1:10 molar ratio of HIV-Tat per amyloid beta at incubation, fibrils are much larger and irregular along the length compared to amyloid fibrils, their dimensions being similar with those of amyloid fibrils formed at extreme concentrations (>1 mM), but without the uniformly striated structure. Importantly, the presence of Tat in the amyloid fibrils significantly increases the neurotoxicity of amyloid fibrils, as shown in neuronal cell culture experiments. Future studies will involve localization of Tat molecules throughout the amyloid filaments and aggregates, for a better understanding of the interaction between HIV-Tat protein and amyloid beta peptide. Maureen H. Richards, Melanie S. Seaton, Stephanie Min, Victoria Lutgen, Lena Al-Harthi (presenting author: maureen_richards@rush.edu) Department of Immunology/Microbiology, Rush University Medical Center HIV invades the CNS shortly after infection and despite antiretroviral therapy leads to a spectrum of neurologic complications termed HIV-Associated Neurocognitive Disorders (HAND). While extensive studies have probed the cellular and molecular mechanisms driving HIV-mediated neuropathogenesis, little is known about the complex interaction between infiltrating immune cells and resident brain cells, especially astrocytes. We evaluated the interaction between peripheral blood mononuclear cells (PBMCs) and astrocytes. We show that conditioned media from primary human progenitor-derived astrocytes (PDAs) induced Beta-catenin expression in PBMCs by 50%, inhibited HIV replication in T cells by 75% and induced CD4 expression on CD8 T cells to generate a double positive (DP) T cell phenotype. Depleting PDAs conditioned media of Wnt ligands (Wnt 1, 2b, 3, 5b and 10b) abrogated these responses, indicating that Wnt ligands secreted from astrocytes mediate these changes in T cells. Conversely, conditioned PBMCs media activated PDAs as indicated by elevated expression of IFNgamma and HLA-DR, reduced Beta-catenin expression in PDAs, and enhanced HIV replication in PDAs. These data indicate a feedback loop whereby activated PBMCs can alter astrocyte phenotype and susceptibility to HIV infection and astrocytes can alter the phenotype and function of T cell. We propose a model in which crosstalk occurs between astrocytes and infiltrating immune cells; shaping the microenvironment of the brain, and perturbing the functional potential of each cell type. This work is supported by R01NS060632-LA and F32 NS08065701-MR. Oregon and Health Sciences University PURPOSE: In the era of combined antiretroviral treatment, HIV-seropositive patients are living longer. With the increase survival, we observe a high prevalence of neurocognitive impairments. Memory Island (MI) is a computer based test useful in identifying susceptibility to cognitive impairments especially in spatial learning and memory with the advantage of being a non-invasive test that can be easily administered. Since MI is not affected by culture, its validation and characterization in neurocognitive disorders could represent a good screening test for HAND. The MAIN OBJECTIVE of this study is to determine the validity and use of MI as a reliable instrument in the characterization of spatial learning and memory in HIV-seropositive women. DESIGN METHODS: 60 women were evaluated, 44 HIV-seropositive and 16 controls HIV-seronegative, with viral-immune profiles, MI, and neuropsychological tests. Parametrics and non-Parametrics statistics were performed. RESULTS: There were no group differences in speed of navigation in the visible or hidden target trials. During the visible target trials, there were no differences in ability of the two groups to locate the target location. However, during the "hidden" trials, HIVseropositive women required more time and moved longer distance than controls. Thus, HIV-seropositive women had less efficient acquisition (learning) and worse performance when compared with HIV-seronegative women. When spatial memory retention was assessed in the probe trial (no target present), HIV-seropositive women spent less time in the part of the island (quadrant) that contained the target previously than the controls. CONCLUSION: The use of MI test is valid in detecting spatial learning and memory deficits in HIVseropositive women. While MI resembles the findings of the Morris Water Maze, serves as a new translational technology and useful valid instrument to spatial and memory screening. Fiorella Rossi, Gokul Swaminathan, Julio Martin-Garcia 3 (presenting author: fpr23@drexel.edu) Human Immunodeficiency Virus type-1 (HIV-1) is transmitted predominantly through the vaginal or rectal mucosa where dendritic cells (DCs) are among the first cells to interact with the virus. In addition to support viral replication, DCs facilitate viral dissemination and contribute to HIV-1 pathogenesis by migrating to the lymph nodes and assisting the infection of CD4+T cells. DCs show less susceptibility to HIV-1 infection depending on the type of stimuli and maturation stage. Upon DCs maturation, various cellular host factors such as and Apolipoprotein B mRNA editing enzyme 3G (APOBEC-3G) are highly up-regulated. However, these upregulation in the context of HIV-1-infection has not been elucidated in DCs. Our goal is to determine the mechanism that explains how maturation of DCs causes suppression of HIV-1 infection in vitro. Immature DCs were activated with different Toll-like receptor ligands (TLR-L) to obtain a mature phenotype and determined the expression levels of miR-155, APOBEC-3G, and the susceptibility of DCs to a HIV-1 recombinant virus (ADA-Fluc). We observed an upregulation on the expression of both factors upon TLR3 stimulation in comparison to non-stimulated cells (p=0.003) and other TLR-L. This up-regulation in TLR3-stimulated DC correlates with increased expression of the maturation markers CD80, CD86, HLA-DR and CD83 (p=0.03), and a complete suppression of HIV-1 infection (p=0.031). By using a miR-155 inhibitor and silencing APOBEC-3G, the phenotype reverses to 60% of wild type infectivity. This suggests that other factors are also involved in suppressing infection upon TLR3 stimulation. Future studies will address other mechanism(s) involved in these processes. M2 macrophages stimulate neural stem/progenitor cell proliferation via a Wnt 5a dependent pathway: Implications for herpes simplex encephalitis Jessica Rotschafer, Erin Roach, Dianna Cheney-Peters, Maxim Cheeran (presenting author: cheeran@umn.edu) Veterinary Population Medicine department, College of Veterinary Medicine, University of Minnesota Activation of macrophages and microglia is a critical component of the host response following brain damage. Previous studies in our laboratory demonstrated that HSV-1 brain infection stimulates neural stem/progenitor cell (NSC) proliferation between 3 and 6 d p.i., concurrent with macrophage infiltration. In the present study, we examined the role of macrophage activation phenotypes on NSC proliferation. Evaluation of infiltrating macrophages [CD45(hi)CD11b(+)] revealed that 68.6±3.3% of the cells were Ly6C(hi) at 5 d p.i. Expression of an M2 marker, CD206, was five-fold higher than CD86, an M1 phenotype marker, in Ly6C(hi) macrophages at 5 d p.i, indicative of an alternative activation phenotype. To determine if macrophage polarization modulated neurogenesis, NSCs were cultured with supernatants from M1 or M2 polarized bone-marrow derived macrophages. NSC cultures treated with M2 conditioned media (M2CM) had 4fold more proliferating cells compared to those cultured in control media. Cells in all treatment groups maintained their stem cell phenotype [≥95% nestin(+)] at collection. M2CM treated NSCs continued to increase in number at 96 h posttreatment with significantly higher numbers of cells in the G2/ M phase of cell division. Transplantation of M2 macrophages into the lateral ventricles of uninfected mice resulted in a 15% increase in Sox2(+) NSC proliferation and the total number of Sox2(+) NSCs doubled compared to saline or heat-killed cell controls, at 5 d post-treatment. Interestingly, Wnt5a expression was significantly increased in M2 polarized macrophages and treatment with either dickkopf-1, a Wnt5a inhibitor, or Wnt5a neutralizing antibody suppressed NSC proliferation to control levels. Finally, Wnt5a gene expression in HSV-1 infected brains increased at 3 d p.i., coinciding with the increase in neurogenesis and M2 macrophage infiltration into the brain. Results from these studies suggest that M2 macrophages promote neurogenesis following herpes virus-induced brain damage and may provide an avenue for therapeutic intervention. Previous studies in the laboratory showed that neural stem/ progenitor cell (NSC) proliferation was significantly impaired during the chronic phase of HSE (15 -30 dp.i.). The current study evaluated the role of CD8(+) T-cells in modulating this anti-proliferative milieu. At 15 dp.i, approximately 50% of CD8 T-cells in HSV-1-infected brains produced interferongamma following virus stimulation. CD8 T-cells were depleted from mice brains at 15 d p.i, using a neutralizing antibody to CD8a. CD8 T-cell depletion resulted in a 5-fold increase in CD45(-) nestin(+) NSCs compared to isotype antibodytreated controls. While CD4(+) T-cell numbers or phenotype did not change significantly following depletion, an increase in the total number of infiltrating macrophages (CD45(hi)CD11b(+)) as well as a down-regulation of MHC-II on microglia was observed. Interestingly, a 3-fold increase in number of CD206(+)infiltrating macrophages, an M2 macrophage phenotype marker, was seen compared to appropriate controls. Further analysis of macrophage phenotypes showed that, in the absence of CD8 T-cells, the ratio of CD206 to CD86 expressing macrophages shifted from 2:1 to approximately 4:1, suggesting that CD8 T-cell depletion promotes an M2 macrophage activation phenotype. Moreover, gene transcription analysis demonstrated a significant reduction of IFN-gamma expression in depleted animals, compared to isotype-treated mice. Previous studies in our laboratory have shown that M2 polarized macrophages enhance NSC proliferation. However, transplantation of M2 polarized macrophages into the lateral ventricles of chronically infected mice showed no significant change in neurogenesis. In addition, cultured NSCs treated with IFN-gamma were refractory to M2 macrophage induced increase in proliferation. Taken together, these data suggest that CD8 T-cells impair endogenous NSC proliferation subsequent to HSE in an IFN-gamma dependent manner. These studies may help identify therapeutic interventions to enhance neurogenesis during viral encephalitis. Walter Royal, III, Joseph Bryant, Ming Guo, Harry Davis, Christina Preminger, Sandra Navas-Reyes (presenting author: wroyal@som.umaryland.edu) Objective: Cigarette smoking has been linked to an increased risk of disease progression in HIV infection, including an increased risk of cognitive impairment. In these preliminary studies were examined effects of cigarette smoke exposure on the induction of gene expression of mediators of inflammation and oxidative stress in brains of HIV-1 transgenic rats. Methods: 3-6 month old wild-type (WT) and transgenic (TG) Fisher 344/NHsd rats were exposed to cigarette smoke (CS) from 3R4F research grade cigarettes, which contain a regular amount of nicotine, in a specially constructed smoking chamber for 5 days per week for 4 weeks. Frontal cortex and subcortical white matter from the CS-exposed and nonexposed animals (n=4 rats group) were examined by realtime PCR for IFN-gamma, TNF-alpha, IL-6, CCL2/MCP-1, CCL-3/MIP-1-alpha, iNOS, the NADPH oxidase isoform dual oxidase 1 (DUOX1), thioredoxin (TXN) and superoxide dismutase (SOD) gene expression. Results: INF-gamma gene expression was higher for WT than for TG rats and increased by CS exposure. TNF-alpha gene expression was increased only in CS+ WT vs CS-WT rats, and there was no difference in IL-6 expression for either TG or WT rats +/-CS exposure. In the absence of CS exposure, MCP-1/CCL2 expression was higher for TG than WT rats and was higher for TG CS+ rats than for all other groups. There was no elevation of MIP-1-alpha/CCL3 expression with CS exposure. Levels of iNOS and TXN expression were similar for all groups. However, SOD was decreased by CS in WT rats but was unchanged in TG rats. Also, CS increased DUOX1 expression in WT rats but did not affect expression in TG rats. Conclusions: Increased pro-inflammatory and decreased oxidative stress marker gene expression resulted from CS exposure in the TG rats. CS effects on oxidative stress responses in the TG rat may reflect altered sensitivity to nicotine. Objective: Neurocognitive impairment (NCI) in HIV infection has been linked to nervous system involvement by virusinfected and activated mononuclear phagocytes. In this study, associations between levels of markers of monocyte/ macrophage activation, levels of circulating monocytes and the presence of NCI were examined in individuals in Nigeria. Methods: 160 antiretroviral-naïve seropositive (SP; 97 women and 63 men) and 56 seronegative (SN; 38 men and 18 women) subjects received a medical assessment and neuropsychological testing in 7 ability domains. An average global deficit score (GDS), with impairment indicated by GDS > 0.5, was derived from demographically adjusted test scores. Percentages of circulating monocytes and HIV RNA measures were performed in a clinical laboratory. Levels of plasma soluble CD163 and soluble CD14 (which are markers of monocyte/macrophage activation) were determined by ELISA. Measures were compared for SP and SN and by gender using t-tests and analysis of variance. Associations between test results were examined using regression analysis, and analyses of categorical data employed chi-square tests. Results: 81.7% of SP had asymptomatic HIV infection. GDS > 0.05 were noted for 9 (16.07%) SN and 34 (21.25%) SP patients (p=0.4). Among impaired SP, measures were higher for sCD163 (p=0.03), sCD14 (p=0.0002) and HIV RNA (p=0.02) than for non-impaired subjects. Higher measures of GDS (p=0.017), sCD163 (p<0.0001), sCD14 (p=0.008) and percent monocytes (p<0.0001) were noted for SP than for SN subjects. GDS correlated with levels of sCD163 (p=0.02) and sCD14 (p<0.0001). HIV RNA measures were higher for women than men (p<0.0001). Among women, sCD163 (p<0.05) and sCD14 (p<0.001) levels were higher for SP than for SN. Among men, sCD14 (p<0.0001) and percent monocytes (p<0.05) were higher for SP than SN. Conclusion: Measures of circulating monocytes and monocyte activation can be useful for detecting evidence of disease activity among clinically stable individuals, particularly women, with HIV infection. Despite the development of combination antiretroviral therapy, HIV-associated neurocognitive disorders remain prevalent. Traditional neuropsychological (NP) approaches commonly use paper and pencil-based psychometric tests for impairment assessment. Although these approaches provide highly standardized control and delivery of performance challenges, the extent to which these tasks predict everyday functioning is not always clear, as these tasks do not simulate real world activities, such as managing finances or remembering appointments. The assessment of neurocognitive ability using tasks to simulate everyday activities may confer an estimate of the patient's functioning more accurate than the one within laboratory conditions. Virtual reality (VR) allows (1) the user to interact in real time with a pseudo immersing 3D environment by a behavioral interface; (2) the presentation of close to real life testing environments. The aim of this study was to design a measure that will detect NP impairment of HIV-infected subjects in a simulated environment and to analyze how the number of errors detected in this environment will correlate with NP dysfunction on traditional tests. To this end, we developed the NP function of an interactive VR supermarket. The facets of NP functions we expect to correlate with the VR test will be episodic memory and executive functions tests. This study was supported by a developmental grant from the Comprehensive NeuroAIDS Center (CNAC NIMH Grant Number P30MH092177) at Temple University School of Medicine. In vivo immunogenicity of Tax 11-19 epitope in HLA-A2/DTR transgenic mice: implication for dendritic cell-based anti-HTLV-1 vaccine Viral oncoprotein Tax plays key roles in transformation of human T-cell leukemia virus type 1 (HTLV-1)-infected T cells leading to adult T-cell leukemia (ATL), and is the key antigen recognized during HTLV-associated myelopathy (HAM). In HLA-A2+ asymptomatic carriers as well as ATL and HAM patients, the Tax(11-19) epitope has exhibited immunodominance. In this regard, the immunotherapeutic potential of this epitope has been evaluated in HLA-A2 transgenic mice in the absence and presence of dendritic cells (DCs) given the recent encouraging observations made with Phase 1 DCbased vaccine trial for ATL. To facilitate these studies, an HLA-A2/DTR hybrid mouse strain carrying the HLA-A2.1 and CD11c-DTR genes was generated. The CD8+ T-cell immune response was generated against the Tax(11-19) epitope delivered in the absence or presence of Freund's adjuvant and/or DCs. Overall, the results demonstrate that naturally presented Tax epitope could initiate an antigen-specific CD8+ T-cell response in vivo but failed to do so upon DC depletion. In addition, the presence of adjuvant potentiated the Tax(11-19)specific response. Elevated serum IL-6 levels coincided with depletion of DCs whereas decreased TGF-beta was associated with adjuvant use. Thus, the Tax(11-19) epitope is a potential candidate for the DC-based anti-HTLV-1 vaccine and the newly hybrid mouse strain could be used for investigating DC involvement in human class-I-restricted immune responses. In the healthy individual, infiltration of dendritic cells (DCs) into the central nervous system (CNS) is tightly controlled by the highly specialized blood-brain barrier (BBB). However, in an inflammatory disease like multiple sclerosis (MS), DCs have potential to transmigrate across the BBB into the CNS due to the presence of chemoattractants, particularly CCL2 or MCP-1. Hence, studying the interaction of circulating DCs with the BBB represents a critical step in cellular trafficking during neuroinflammation. Near-infrared imaging of DC accumulation into CNS correlated with the severity of inflammation in EAE (experimental autoimmune encephalomyelitis) model of MS. Ex vivo histology confirmed the presence of CCL2 in lesions, with DCs emerging from perivascular spaces. Confocal imaging revealed that CCL2 exposure leads to polymerization of actin on DCs suggesting a migratory phenotype. During mechanistic studies in BBB model, DCs exhibited more efficient transmigration than T cells in the presence of CCL2. Additionally, a paracellular versus transcellular pattern of migration by DCs and T cells was seen. The unique enrichment of lectin receptors on DCs led to differential profile for each specific subset of DCs including myeloid (mDC), plasmacytoid (pDC) and monocyte-derived (MDDC). Subsequently, in the presence of CCL2, each DC subset was found to utilize a different lectin receptor in order to adhere and transmigrate across the BBB. At the subcelleular level a cross-talk between actin and lectin pathways was also established. The prospect of selectively regulating DC entry into the CNS will open up new possibilities for disease pathogenesis and propagation in MS. Agnoprotein is a small and multifunctional regulatory protein of JC virus (JCV) and plays essential regulatory roles in the viral replication cycle. In the absence of its expression, JCV is unable to sustain its productive life cycle. We have recently demonstrated that agnoprotein forms highly stable dimers and oligomers in vivo and in vitro and the Leu/Ile/Phe-rich hydrophobic domain of the protein plays a key role in formation of such multimeric structures. Previous mutagenesis analysis of the region resulted in phenotypes that show defects in viral life cycle. In addition, previous attempts to determine the 3D structure of agnoprotein have been unsuccessful. Here, we report the NMR structure of a synthetic agnoprotein peptide encompassing amino acids Thr17-Glu55 demonstrating that amino acids spanning from Lys22 to Phe39 form an amphipathic alpha-helix. We have also performed NMR structure-based mutagenesis studies to identify amino acids critical for agnoprotein multimeric structures and function. Alanine substitution of three leucine residues; Leu29, Leu32 and Leu36 located within alpha-helix region resulted in a mutant that exhibits a substantially reduced level of agnoprotein expression and viral DNA replication. Consistent with these in vivo findings, in vitro studies with this mutant demonstrated considerably low level of protein expression in a bacterial expression system and showed defects in monomer, dimer and oligomer formation. Collectively, these findings suggest that Leu29, Leu32 and Leu36 residues found in the dimer interface of agnoprotein play critical roles in protein stability, dimer/oligomer formation and in viral DNA replication. Thus, the dimerization domain of agnoprotein represents a potential target site for developing novel therapeutics against JCV infections in affected PML patients. JC virus (JCV) agnoprotein harbors a putative nuclear export signal (NES) and interacts with a host RNA export factor, CRM1: Evidence for its involvement in nucleo-cytoplasmic export of JCV transcripts Sami Saribas, Mahmut Safak (presenting author: msafak@temple.edu) Temple University School of Medicine, Department of Neuroscience, Laboratory of Molecular Neurovirology, Philadelphia, PA, 19140, USA Viruses have evolved highly sophisticated mechanisms to exploit the host machinery for their own benefit to produce infectious virions. They achieve this in part by encoding and utilizing multifunctional proteins in multiple steps of the viral infection cycle including RNA export. These types of viral proteins have distinct functional domains used by viruses in such processes. JCV agnoprotein is one of those multifunctional proteins with multiple properties. For example, it localizes mainly to the perinuclear area of the infected cells but a considerable amount of it is also found in the nucleus. This protein is consists of several functional domains, including a bipartite nuclear localization signal (NLS) and a Leu/Ile/Phe-rich multimerization domain and exhibits a highly basic nature containing multiple Arg, and Lys residues found at the N-and C-terminus of the protein. In addition, it specifically interacts with JCV RNA and harbors a putative NES similar to that of HIV-1 Rev; and treatment of the JCV-infected cells with a selective CRM1 inhibitor (Leptomycin B) leads to the accumulation of agnoprotein inside the nucleus, suggesting that it may be involved regulation of the export of the JCV transcripts in a CRM1-dependent manner. To provide evidence for such a possibility, a protein-protein interaction study has been performed between agnoprotein and CRM1 using whole cell extracts from SVG-A cells infected with JCV and purified CRM1. Results showed that agnoprotein strongly interacts with GST-CRM1, but not with GST alone, suggesting that the observed interaction between these two proteins is specific. Collectively, results from these studies support a hypothesis that agnoprotein may play a regulatory role in nucleo-cytoplasmic export of JCV transcripts in a CRM1-dependent manner. Our future studies are aimed at investigating the molecular mechanisms regulated by agnoprotein in export of JCV transcripts. The majority of the human population is latently infected with JC virus, which causes the fatal demyelinating disease of the brain, progressive multifocal leukoencephalopathy, in immunocompromised individuals. The mechanism of viral reactivation and the molecular switch to activate productive replication is not well understood. Previous studies have identified Pur-alpha and SF2/ASF, also known as SRSF1, as positive and negative regulators of JCV gene expression, respectively. Here we studied the functional interplay between Pur-alpha and SRSF1 in regulation of JCV gene expression at the transcriptional and splicing levels. Western blot analysis of brain lysates and embryonic fibroblasts from mice lacking Pur-alpha showed a dramatic increase in SRSF1 levels compared to wild type mice, pointing to a negative correlation in cellular expression of both proteins. In addition, overexpression of Pur-alpha in glial cell lines caused a significant reduction in SRSF1 levels. Next, we tested the effects of Pur-alpha and SRSF1 on JCV transcription by luciferase assay. As expected, while SRSF1 caused a strong inhibition, Pur-alpha showed a moderate increase in transcription levels driven by the JCV-early promoter. Interestingly, we observed that SRSF1 mediated suppression of JCV transcription was significantly restored by expression of Pur-alpha. These data have revealed a novel interaction between SRSF1 and Pur-alpha in the regulation of JCV reactivation. This work was made possible by grants awarded by NIH to IKS and JG. Ilker Sariyer, Onder Otlu, Kasra Houshmand, Kamel Khalili (presenting author: isariyer@temple.edu) Department of Neuroscience and Center for Neurovirology, Temple University School of Medicine, Philadelphia, PA The JC virus (JCV) is one of the abundant polyomaviruses which infects the majority of human population during childhood, and establishes a persistent life-long infection. JCV replicates in glial cells in the brain, and causes the fatal demyelinating disease, progressive multifocal leukoencephalopathy (PML). PML is usually seen in patients with underlying immunocompromised conditions, notably among AIDS patients and those on chronic immunosuppressive regimens. JCV genome is composed of three functional regions, including the viral early and late coding regions and viral noncoding regulatory region. The late leader sequence contains an open reading frame encoding a small regulatory protein called agnoprotein. Previous studies from mutational analysis suggested that agnoprotein is required for the sufficient progression of the infection cycle in glial cells with an unknown mechanism. Here, we have discovered that agnoprotein can be detected in cell-free fractions of glial cultures infected with JCV, transfected with expression plasmids or transduced with adenovirus expression system. Moreover, treatment of cells with Brefeldin A but not Monensin suppressed the secretion of agnoprotein suggesting that conventional secretion pathway was mainly involved in agnoprotein secretion. We further demonstrated that the secreted agnoprotein was uptaken by glial cells. These studies have revealed a novel phenomenon of agnoprotein during the viral life cycle with a potential of developing diagnostic and therapeutic interventions. This work was made possible by grants awarded by NIH to IKS and KK. The activity of HIV-1 Tat and morphine at Toll-like receptor 4: Implications for mechanism of actions leading to HIVE and HAND Opiate drug use by HIV-1 positive individuals is reported to exacerbate HIV encephalitis (HIVE) and HIVassociated neurodegenerative disorders (HAND). It has recently been suggested that, besides mu-opioid receptors, opiate drugs can also activate Toll-like receptor 4 (TLR4). Likewise, this receptor is a plausible target for HIV-1 proteins such as transactivator of transcription (Tat). Therefore, we aimed to clarify the role of morphine and Tat directly at TLR4 using HEK cell lines that express human TLR4, MD2 and CD14 [TLR4(+) cells] or only MD2 and CD14 genes [TLR4(-) cells], as well as an NF-kappaB-inducible reporter which quantifies TLR4 activation. We found that a 16-h exposure to HIV-1 Tat1-86 (100 nM) increased TLR4 reporter activity, but under our current conditions at this time point, exposure to (-)-morphine or (+)-morphine enantiomers (500 nM-5 uM) did not appear to activate the TLR4 reporter; however, higher doses still need to be tested. Additionally, activity of Tat at TLR4 seems to be specific, as co-application of the TLR4-selective antagonist, LPS-RS, significantly decreased Tat-or LPS-EK (TLR4specific agonist)-induced activity similarly. No significant activation by Tat or morphine was noted in TLR4(-) cells. Finally, using an ELISA, we noted that a 24-h exposure to Tat increased MCP-1 production in TLR4(+) cells, though (-)morphine did not. We validated the model by confirming the presence of TLR4 and absence of mu-opioid receptors in the appropriate cell lines using qRT-PCR. Overall, our current results suggest that, in a TLR4 over-expressing HEK cell model designed to purely test TLR4 activity, Tat, but not morphine, activated TLR4 and its signaling pathway. Supported by NIH T32 DA007027, K02 DA027374, and R01 DA034231. A novel model for proviral HIV-1 latency in neural cells reveals similarities between HIV-1 latency in the brain and in the immune system Martha Schneider, Christoph Ziegenhain, Bianca Tigges, Markus Helfer, Ruth Brack-Werner (presenting author: brack@helmholtz-muenchen.de) Helmholtz Center Munich, Institute of Virology Potential carriers of transcriptionally latent HIV-1 proviruses in the immune system and the central nervous system (CNS) include CD4+ memory T-cells and astrocytes and possibly also neural progenitor cells. While powerful cell culture models exist for proviral HIV-1 latency in CD4-positive Tcell reservoirs, comparable models have been lacking for neural cells. To establish a cell culture model for proviral transcriptional HIV-1 latency in neural cells, we subjected cells of the human neural stem cell line HNSC.100 to singleround infections with various Env-defective HIV-1 genomes, including reporter viruses with GFP-encoding sequences. HIV-1 infection and viral expression were monitored by quantitative PCR analysis of proviral DNA and viral transcript levels and by flow cytometry analysis of GFP-expression. Long-term culture led to establishment of cell populations that had ceased HIV-1 expression but retained HIV-1 proviral DNA. HIV-1 expression was reactivated by treatment with the proinflammatory cytokine TNF-alpha and SAHA (suberoylanilide hydroxamic acid), which are potent HIV-1 activators in latently infected T-cells. Inducible re-activation of HIV-1 expression was maintained over numerous passages and in differentiated cells and was retained by single-cell clones. Side-by-side assays with latently infected neural and T-cells (J-Lat) revealed that reactivation of HIV-1 expression could be blocked in both HIV-1 reservoir cell types by pretreatment with various small molecules that inhibit activation of NF-kappaB or the positive transcriptional elongation factor pTEFb. In summary, we report establishment of a potent cell culture model for persisting latent HIV-1 proviruses in neural cells and use this model to compare modulation of proviral HIV-1 latency in brain cells and T-cells. Our results raise the possibility that agents that influence HIV-1 proviral latency in the immune system may also affect proviral latency in the CNS and provide proof-of-concept for blocking of reactivation of proviral HIV-1 latency in different HIV-1 reservoirs by small molecules. Myocyte enhancer factor-2 (MEF-2) plays critical role in HTLV-1 infection and transformation of CD4+ T cells The transactivator protein of human T-cell leukemia virus type 1 (HTLV-1), Tax, is capable of regulating both virus (via transcriptional activation of the viral long terminal repeat, LTR) and the host transcriptional machinery, however there is no clear understanding about the involvement of host transcription factors. Herein, we describe the role of myocyte enhancer factor-2 (MEF-2) in Tax-mediated activation of HTLV-1 LTR in infected primary CD4+ T cells and the virus-producing cell line, MT-2. MEF-2 was confirmed by ChIP assay to be recruited to LTR in MT-2 and primary CD4+ as well as CD4+CD25+ T cells in association with Tax. Furthermore, an increase in MEF-2 expression was observed upon infection and correlated with Tax and HAT complex formation, while MEF2 knockdown through shRNA and inhibition of MEF-2 activity by overexpression of a known repressor, HDAC9, reduced viral mRNA and protein expression in both primary cells and cell lines. Confocal microscopy also confirmed that MEF-2 colocalizes with Tax in virus producing cell lines. Next, in order to understand the regulation of MEF-2, we performed comparative protein-DNA array analyses and observed a global upregulation of many MEF-2 associated transcriptional factors in infected cells. Additional studies demonstrated the expression of key signaling mediators and clearly indicated that MEF-2-integrated signaling pathways are activated during HTLV-1 infection, including PI3K/Akt, NF-kappaB, MAPK, TGF-beta and JAK/STAT signaling. Overall, these studies are the first to describe the involvement and regulation of a novel transcription factor, MEF-2, during the course of HTLV-1 infection and pathogenesis. Quality and miRNA control of dendritic cell responses during interferon/ribavirin therapy in HIV-1/HCV co-infected patients Mohit Sehgal 1 , Zafar Khan 1 , Andrew Talal 2 , Pooja Jain 1 (presenting author: mohit.sehgal@drexel.edu) 1 Drexel Institute for Biotechnology and Virology Research and Department of Microbiology and Immunology, Drexel University College of Medicine; 2 School of Medicine and Biomedical Sciences, Buffalo University HIV-1/HCV co-infection represents a significant burden on global economy and public health. Up to 30% of HIV-infected patients and 60-90% of HIV-infected injection drug users are infected with HCV. It is now widely accepted that HIV accelerates the course of HCV-related chronic liver disease. The current standard treatment for treating HCV infection in HIV-1/HCV co-infected patients is administration of PEGylated interferon alpha-2a/2b (PEG-IFNalpha-2a/2b) and an antiviral drug ribavirin (RBV). Recent breakthroughs in directacting antivirals (DAAs) such as Telaprevir and Boceprevir against HCV genotype 1/2 have provided hope for HCV cure. However, development of viral resistance during DAA treatment, and drug-drug interaction among protease inhibitors are two key concerns in the clinical management of infection. Furthermore, Telaprevir or Boceprevir yield optimal results upon combination with IFN and/or RBV, suggesting IFN/ RBV will still be the backbone of anti-HCV treatment. Plasmacytoid dendritic cells (pDCs) are the natural producers of type 1 IFN in response to many viral infections including HIV-1. However, HCV is known to be a weak inducer of IFNalpha in pDCs and it is not known how this scenario changes in the presence of HIV. Therefore, we investigated the quality of DC responses in co-infected patients during the course of IFN/RBV therapy. Further, to understand the molecular basis of these responses, we analyzed differential expressed miRNAs and their target mRNAs in sustained virological responders (SVRs) and non-responders (NRs), pre-and post-treatment. Collectively, results of these studies led to key immune and molecular correlates of IFN/RBV treatment in HIV-1/HCV co-infected patients. Regulation of HIV-1 transcription by glycolytic enzyme Pyruvate kinase M2 Satarupa Sen 1 , Kamel Khalili 2 , Shohreh Amini 1 , Prasun K. Datta 2 (presenting author: satarupa.sen@temple.edu) 1 Department of Biology, CST Temple University; 2 Department of Neuroscience, Center for Neurovirology, Temple University School of Medicine Identification of cellular proteins in addition to already known transcription factors such as NF-kappaB, SP1, and CEBP that interact with the HIV-1 LTR is critical in understanding the mechanism of HIV-1 replication in monocytes/macrophages. Our studies demonstrate upregulation of pyruvate kinase isoform M2 (PKM2) expression in HIV-1JRFL infected PBMCs and during reactivation of HIV-1 in chronically infected U1 cells. Furthermore, we observed that infection of PBMCs with HIV-1 and reactivation of HIV-1 in U1 cells results in increased levels of nuclear PKM2. Hence we focused on understanding the potential role of PKM2 on HIV-1 LTR transactivation. Our studies demonstrate that over expression of PKM2 in U937 and in TZM-bl cells lead to transactivation of the HIV-1 LTR reporter construct. Using various deletions constructs of HIV-1 LTR, we mapped the region spanning between -203 bp to -80 bp to be essential for PKM2 mediated transactivation. This region contains the NF-kappaB DNA binding site and mutation of NF-kappaB binding site attenuated PKM2 mediated transactivation of HIV-LTR. Chromatin immune-precipitation (ChIP) analysis in PMA activated TZM-bl cells demonstrated interaction of PKM2 with HIV-1 LTR. Our studies suggest that PKM2 is a transcriptional coactivator of HIV-1 LTR. Hence it opens up another possible target to curb HIV-1 replication at transcriptional level. This work was supported by the NIH grants to SA and PKD. The study also utilized services offered by core facilities of the Comprehensive NeuroAIDS center (CNAC) NIMH Grant # P30MH092177 to KK. The role of hexokinase in conferring protection to HIV-1 infected macrophages from apoptosis Satarupa Sen 1 , Kamel Khalili 2 , Prasun K. Datta 2 , Shohreh Amini 1 (presenting author: satarupa.sen@temple.edu) 1 Department of Biology, CST, Temple University; 2 Department of Neuroscience, Center for Neurovirology, Temple University School of Medicine Viruses have evolved various strategies to protect infected cells from apoptosis. Glycolysis is linked closely to the cell fate and it is known that HIV-1 infected macrophages are long lived and considered to be viral reservoirs. We provide evidence that HIV-1 protects infected macrophages from apoptosis by modulating the host glycolytic pathway. Glycolytic enzyme Hexokinase (HK) and product of its enzymatic activity Glucose 6 phosphate (G-6-P) are known to play a non-metabolic role in resisting and supporting apoptosis, respectively. Increased association of HK with voltage dependent anion channel of mitochondrial outer membrane can resist apoptosis by maintaining mitochondrial membrane integrity. On the other hand increased G-6-P pool in cytoplasm can force HK to dissociate from mitochondrial membrane and induce apoptosis by cytochrome c leakage. In this study, we analyzed regulation of HK that converts glucose to G-6-P and Glucose-6-phosphate-dehydrogenase (G6PD) that converts G-6-P in to fructose 6 phosphate, in response to HIV-1 activation in chronically infected U1 cell line. We found that HIV-1 replication in U1 cell induces HK expression followed by translocation of HK from cytoplasm to mitochondria of cell. We also observed that viral replication increases the enzymatic activity of G6PD. As a consequence, HK can associate strongly with mitochondrial membrane and this association is kept under tight control by rapidly turning over G-6-P through increased activity of G6PD enzyme. We also found that treatment with pentose phosphate pathway blocker Dehydroepiandrosterone (DHEA) which also inhibits HK and VDAC interaction; reduced viral replication. Hence our work suggests Hexokinase as a new therapeutic target for intervention to curtail viral persistence in the macrophage. This work was supported by the NIH grants to SA and PKD. The study also utilized services offered by core facilities of the Comprehensive NeuroAIDS center (CNAC) NIMH Grant # P30MH092177 to KK. Impact of SNPs on regulation of the HIV-1 promoter within a chromatin-based microenvironment Selective pressures and the low efficiency of reverse transcriptase lead to genetic alterations within the human immunodeficiency virus type 1 (HIV-1) genome resulting in quasispecies that can be differentially regulated and can potentially form niches within specific cell types and tissues. Previous studies identified a single nucleotide polymorphism (SNP) within C/EBP site I (3T, C-to-T change at position 3 of the consensus B site) that correlated with HIV-1-associated dementia. In addition, our current cohort shows a SNP within Sp site III (5T, C-to-T change at position 5 of the consensus B site) that occurs as frequently as the consensus subtype B sequence. Stably transfected cell lines were developed using bone marrow progenitor, T, and monocytic cell lines to explore the promoter (LTR) phenotype associated with these genotypic changes from an integrated microenvironment. The LAI LTR was coupled to green fluorescent protein (GFP), and polyclonal HIV-1 LTR-GFP stable cell lines were developed. To examine the mechanism of LTR-driven gene expression as well as epigenetic modifications that may control it, clones were derived from each population of cells. The clones were examined with respect to basal transcription, cytokine response, and Tat transactivation. Results have suggested that non-expressing clones containing the 3T5T LTR within TF-1 cell lines can be induced to express. Additionally, the non-expressing LAI 3T and LAI 5T Jurkat clone genotypes could be induced into expression. Results demonstrate that genetic signature, epigenetic modifications to viral and host DNA, and cellular phenotype may determine the overall level of LTR activity. This work is supported by NIH/NINDS R01 NS32092, NIDA R01 DA19807, NIMH P30 MH092177, and NIMH T32 MH079785. The National NeuroAIDS Millions of HIV-1/AIDS patients develop severe chronic pain that drastically deteriorates their life quality, but the pathogenic mechanism is unclear and effective therapies are not available. We are interested in elucidating the host mechanisms in CNS that interact with HIV toxins and contribute to this devastating neurological complication in HIV patients. We are undertaking an interdisciplinary approach in this line of research. In the first approach, we focus on analyzing the postmortem nervous tissues from HIV-1/ AIDS patients. The goal is to identify clinically relevant molecular and cellular abnormalities in the pain-processing pathway. The analyses reveal the critical HIV-1 protein that may cause this pain syndrome. In the second approach, we are interested in generating a rodent model for the HIV-1-associated chronic pain, based on the findings from human specimen analysis. The goal is to establish a clinically relevant animal model. We have created a mouse model that develops extensive similarities to the pathologies seen in the HIV-1 patients with chronic pain. In the third approach, we focus on elucidating the molecular and cellular processes that are critical for the pathogenesis of HIV-1associated chronic pain, using the mouse model that we have generated and characterized. We are currently determining the contribution of synaptic degeneration, glial activation and Wnt signaling dysregulation to the pathogenesis. Genotypic and phenotypic characterization of the Envelope glycoproteins of two highly neurotoxic, CSF-derived, HIV-1 primary isolates HIV-1 enters the CNS early after systemic infection, leading to a variety of neurocognitive and motor impairments referred to as HIV-1 associated neurocognitive disorders (HAND). Macrophages and microglia support productive viral infection in the brain and the virus subsequently adapt to the CNS environment. Our goal is to elucidate the role of the HIV-1 envelope glycoprotein (Env) during HIV-1 infection in the CNS. We obtained viruses derived from the cerebrospinal fluid (CSF) of two HIV-1-infected individuals with HIV-1associated dementia, the most severe form of HAND. These isolates are macrophage tropic (i.e. efficiently infect macrophages, whereas most blood-derived isolates do not) and highly neurotoxic. We amplified by PCR multiple env genes from these isolates and have performed extensive genotypic and phenotypic characterization. Interestingly, a mixture of genotypes and phenotypes has been found. From both viruses, we obtained a mixture of macrophage-tropic and nonmacrophage tropic Env, as well as Env with various degrees of fusogenicity, ability to use low levels of CD4 for infection, sensitivity to entry and fusion inhibitors, and sensitivity to neutralizing antibodies directed against the conserved CD4 binding site. Importantly, some of these Env, in the context of isogenic recombinant viruses, are able to mediate infection of macrophages and lead to the production of conditioned medium which causes neurotoxicity in primary pure neuronal cultures, whereas others do not. Correlations between genetic determinants and the above reported phenotypes are being further investigated. Imaging lentiviral reservoirs in vivo based on molecular imaging reporter genes Jiasheng Song, Won-Bin Young (presenting author: songj2@upmc.edu) Molecular imaging laboratory, Department of Radiology, University of Pittsburgh School of Medicine HIV-1 infections persist in spite of exposure to highly active antiretroviral therapies (HAART) due to the latent stage of the virus. The latently infected cells present a major barrier for curing HIV-1 infections. The identification of viral reservoirs is essential in monitoring the therapeutic response to HAART including during the latent stage. Bioluminance imaging (BLI), positron emission tomography (PET), and magnetic resonance imaging (MRI) are non-invasion imaging techniques that can be employed to identify viral reservoirs eliminating the need for repeat biopsies in vital organs, such as the brain. However, during the latency stage there are no detectable viral proteins for targeted imaging. Our lab has developed a "Trojan Horse" type strategy where we've engineered human, simian, and feline immunodeficiency viruses (HIV, SIV, FIV) to carry a reporter gene for non-invasive imaging macrophages resulted in significantly reduced production of pro-inflammatory cytokines and chemokines. Phosphorylation of the transcription factor IRF-3 (p-IRF3), is an essential step for TLR3 dependent upregulation of type-I IFNs. HIV-1 BAL infection upregulated p-IRF3 3-6hrs post infection, however, inhibition of TLR3, significantly reduced HIV-1 induced p-IRF3 levels. Surprisingly, HIV-1 BAL infection of human macrophages resulted in significant upregulation of TLR3 mRNA levels and moderate increase in TLR3 protein levels. Finally, infection of 293T cells stably expressing the murine TLR3, with VSV-g pseudotyped virions, resulted in significant induction of TNF-alpha and IL-6. Current and future experiments are aimed at investigating the potential for direct interaction between HIV-1 ssRNA and TLR3. Overall, this study provides the first known evidence that TLR3 senses and responds to HIV-1 infection in macrophages. Since TLR3 is a crucial innate immune sensor in the brain, these results have important implications in HIV-1 neuropathogenesis, inflammation and other co-morbidities. Recent studies show that proinflammatory cytokines including osteopontin (OPN, SPP1) remain elevated in the plasma and cerebrospinal fluid (CSF) in individuals suffering from HIV-associated neurocognitive disorders (HAND). Beyond its ability to enhance HIV replication, OPN promotes increased numbers of activated monocytes in the CNS by facilitating chemotaxis and cell survival. However, the molecular mechanisms by which OPN mediates its function in the context of HAND remain unexplored. We used next-generation high-throughput sequencing and transcriptome analyses to identify the genes and pathways that are differentially regulated in primary human macrophages knocked down with siRNA against SPP1 (SPP1 KoD) compared to wildtype macrophages. As predicted by a disruption of SPP1 expression, OPN receptors, the integrins ITGAV and ITGB3, which signal through NF-kappaB were markedly downregulated. Chemokines CXCL10, CXCL9, CCL2, CXCL1, CCL13, and CCL7 were up-or downregulated 3-6 fold. TNFresponsive genes including TRAF1, TANK, TIFAB, TNFSF9, TIFAB, TNFAIP6 and TNFAIP3 and toll-like receptors -1, -4, -5, -6, and -8 were differentially expressed in SPP1 KoD macrophages. Key genes involved in cytokine signaling including STAT1, STAT2, NFKBIZ, NFKBID, were either up-or down modulated in OPN KoD macs. A prominent feature in SPP1 KoD macs was the differential regulation of 13 members of the mitogen-activated protein kinase cascade. Interestingly, several members of the host restriction factors that target HIV including the TRIM and APOBEC proteins were downregulated in SPP1 KoD macrophages. Unexpectedly, CD163, a biomarker strongly associated with HAND was significantly decreased in SPP1 KoD macs suggesting a link between proinflammatory gene regulation by OPN and CD163 expression. Several other host genes implicated in the HIV life cycle were also differentially regulated. Together, these data suggest that OPN is a potent regulator of proinflammatory gene expression impacting macrophage activation and host cell resistance and susceptibility to HIV infection and replication. Host Immune Responses to JC Virus in Hematopoietic Stem Cell Transplantation (HSCT) Background: JCV causes progressive multifocal leukoencephalopathy(PML) in immunocompromised patients. Up to 80% of the general population is seropositive for JCV and JCV-specific cellular immune response is necessary for containment of viral proliferation. The mechanism of JCV reactivation and immunity in a transplanted immune system remains unclear. Methods: We prospectively enrolled 30 patients undergoing allogeneic HSCT, and collected blood and urine samples before and 3, 6, and 12 months after HSCT. We detected JCV DNA by PCR in blood and urine samples, performed ELISA for detection of JCV IgM and IgG, as well as Elispot and ICS for measurement of T-cells responses to JCV. Multivariate analysis accounted for conditioning regimens, cancer diagnosis, concurrent viremia, age, and transplant type. Results: Pre HSCT, JCV DNA was detected in 7/30 urine, 5/ 30 PBMC and 6/30 plasma samples. While viruria remained stable after HSCT, viremia was detected in only 1/22 plasma and none of 22 PBMC samples 12 months after HSCT. Prevalence of anti-JCV IgG was 83% pre HSCT and remained stable at 72% at 12 months. Anti-JCV IgM was rarely detected. A significant increase in JCV-specific T cell responses 12 months after HSCT was mediated by both CD4+ and CD8+ Tcells. While JC viruria correlated directly with detection of anti-JCV IgG, the cellular immune response to JCV measured by ELISPOT was inversely correlated with anti-JCV IgG response. Age of the patients and the diagnosis of leukemia both significantly reduced cellular immune responses to JCV. Conclusions: JC viruria triggers an antibody response in HSCT. However, an increase in JCV-specific cellular immune response 12 months after HSCT leads to suppression of JC viremia, and decrease in JCV humoral immune response. This prospective study in HSCT patients provides a model of interactions between the host immune response and viral activation in multiple compartments over time. Age-dependent susceptibility to La Crosse Virus-induced neurological disease mediated by TLR3/RLR signaling pathways Katherine Taylor, Tyson Woods, Clayton Winkler, Karin Peterson (presenting author: katherine.taylor@nih.gov) Mosquito-borne La Crosse virus (LACV) infections are a leading cause of infectious encephalitis in children, but adults are generally resistant to neuroinvasive disease. Agedependent susceptibility to LACV is also observed in mouse models. Mice younger than 30 days are susceptible to neurological disease while mice older than 30 days are resistant. In the current study, we demonstrate that the resistance of adult animals is dependent on type I IFN responses generated through TLR3 and RIG-I like receptor (RLR) signaling pathways. These pathways are essential for peripheral virus control and prevention of spread to the CNS. In particular, TLR3 and RIG-I receptor signaling in myeloid dendritic cell populations is implicated in virus control. Young mice have significantly lower numbers of myeloid dendritic cells and generate weaker type I IFN responses in peripheral tissues following either LACV infection or direct stimulation with TLR3/RLR ligands compared to older mice. The inability of young mice to generate strong peripheral type I IFN responses in myeloid dendritic cells following TLR3/RLR stimulation may explain their increased susceptibility to viral infection, spread and encephalitis. Neuronal cell death is a hallmark of various neurological diseases including virus infections. The innate immune response in the CNS contributes to neuronal damage through cytokine and neurotoxin production as well as the recruitment of inflammatory cells. However, the direct role of the innate immune response in mediating cell death during virus infection of neurons is not known. In the current studies, we identified sterile alpha and HEAT/ Armadillo motif containing 1 (SARM1), a negative regulator of TLR signaling, as a mediator of bunyavirusinduced neuronal apoptosis. SARM1 was induced in neurons following La Crosse virus (LACV) infection and inhibition of SARM1 suppressed virus-mediated neuronal apoptosis. Mechanistic studies demonstrated that SARM1 upregulation was due to stimulation through MAVS (IPS-1) and that SARM interacted with MAVS on the mitochondria and induced oxidative damage leading to neuronal apoptosis. This data provides a novel mechanism for immune mediated neuronal death during virus infection by activation of pattern-recognition receptors (PRRs) leading to MAVS activation, SARM1 induction and oxidative stress. Since stimulation of neurons through endosomal TLRs can inhibit neuronal growth and induce apoptosis, we also analyzed the role of SARM in TLR-mediated neuronal death. SARM1 was induced by TLR7/TLR9 agonist stimulation of cortical neurons and TLR-induced neuronal apoptosis was SARM1 dependent. These data indicate that activation of pattern-recognition receptors on neurons, in the presence or absence of active virus infection, can lead to neuronal death and that the mechanism of death is through production of SARM which leads to mitochondrial damage. the HIV infected population constitutes a new challenge in the HIVepidemic in the affluent countries. Importantly, older HIV infected patients are more susceptible to neurocognitive impairments associated with the disease. HIV infected brains are characterized by increased deposition of amyloid beta (Abeta) in perivascular space, indicating the importance of brain microvessels in amyloid accumulation. Our research focused on the mechanisms of HIV-1 interaction with Abeta at the blood-brain barrier level. Exposure to HIV resulted in accumulation of Abeta in brain endothelial cells, with the prominent nuclear predisposition. We demonstrated that these effects are dependent on functional caveolae and can be prevented by inhibition of Ras and the receptor for advanced glycation end products (RAGE). In additon, HIV-induced nuclear entry of Abeta involves activation of the dynamin-dependent the EEA1 and TGF-beta/Smad signaling. Using transgenic mice that express a chimeric mouse/human amyloid precursor protein and a mutant human presenilin 1, we next demonstrated that cerebrovascular toxicity of HIV specific protein Tat is enhanced in mice with amyloid deposits in the brain. Indeed, exposure to Tat increased permeability across cerebral capillaries, enhanced disruption of ZO-1 tight junction protein, and elevated brain expression of MMP-9 in transgenic mice as compared to age-matched littermate controls. These changes were associated with increased leukocyte attachment and their transcapillary migration. Supported by MH098891, MH63022, MH072567, DA027569, and NS39254. endogenous retroviruses was reported. HERV-Ws are potentially neuropathogenic, being gliotoxic, superantigenic, fusogenic, immuno-disregolatory, etc. We wondered about JCV/HERV-W interactions, that could contribute to neurodegeneration. To this end human primary fetal astrocytes and U87MG and SVG cell lines were either infected by JCV-Mad1 strain or transfected with expression plasmids carrying early or late JCV genes, to monitor the expression of HERV-W/MSRV/syncytin-1 env, or that of constructs carrying the ERVWE-1/syncytin-1 promoter. Data showed that JCV infection upregulates both MSRV-like and syncytin env transcripts. Transient transfection experiments showed that both JCVearly and late proteins stimulate the transcription and the expression of HERV-W/MSRV/syncytin-1 env genes up to the glycosylated form. These data were obtained by RT-Real time PCR or western blotting analysis. As for the HERV-W promoter activity in presence of JCV proteins, gene reporter experiments with constructs carrying the full-length syncytin-1 promoter, or promoter deletion mutants, co-transfected with JCVearly or late plasmids were carried out. Data indicate that the full-length promoter is upregulated by both JCV early and late proteins. Deletion of the upstream (cellular) regulatory region indicate that the stimulatory effect lies on the 5'LTR (viral) moiety. Due to the identities between the 5'LTR of MSRV and syncytin-1, it is likely that up-regulation by JCV antigens of both MSRV and syncytin-1 requires mainly the promoter's viral moiety. Given the neuropathogenic properties of HERV-W/env proteins, we wonder whether their upregulation by JCV in astrocytes could contribute to neurodegeneration. This novel interaction among JCVand HERV-W may open new avenues in the understanding of neurodegenerative diseases. Victor Valcour, Edgar Busovaca, Lauren Wendelken, Pardis Esmaeili, Katherine P. Rankin, Iryna Lobach, Howard Rosen (presenting author: vvalcour@memory.ucsf.edu) Memory and Aging Center, Neurology Department, University of California San Francisco Brain white matter hyperintensities (WMH) by magnetic resonance imaging (MRI) are known to occur with HIV, typically with late disease and linked to dementia. WMH can also occur with cerebrovascular disease (CVD) in the absence of HIV. Since CVD risk increases with age and as patients living with HIV age, the underlying mechanisms and clinical importance of WMH become less clear. Using a customized WMH quantification pipeline, we measured lesion volume, number of lesions (count) and lobar distribution of WMH in 65 HIV+ compared to 29 age and gender matched healthy HIV-negative controls. HIV+ subjects were all over age 60 years (median, range of 63, 60-76) with median (range) CD4 count of 531 (203-1098) and duration of HIV of 22 (3-31) years. Eightyeight percent had suppression of plasma HIV RNA < 400 copies. WMH volume was tightly linked to WMH count (Spearman's rho=0.89 and 0.75 for HIV+ and HIV-negative, respectively, p<0.005). The highest WMH volumes, adjusted for intracranial volume (ICV), were seen in HIV+ subjects (Wilcoxon test of medians, p=0.006). In HIV+ subjects, higher WMH volume correlated to poorer performance on executive functioning tests (Spearman's rho=-0.35, p=0.006). WMH volume did not correlate to CD4 count, nadir CD4 count, duration of HIV, or plasma HIV RNA. In the HIV+ group, subjects who smoke or have hypertension had higher burden of WMH volume (Wilcoxon test of medians p=0.03 and p=0.04, respectively). Smoking and HIV status have interactive effect on WMH volume adjusted for age (p=0.04). Similarly, hypertension and HIV status tend to have an interactive effect (p=0.09). We conclude that WMH in older HIV+ subjects is more closely related to CVD rather than HIV risk parameters, and that HIVappears to modulate this effect. These lesions are linked to cognitive performance. Lifestyle factors remain important targets for improving cognitive morbidity. HIV enters the CNS in infected monocytes within two weeks of peripheral infection. Once within the CNS, these cells mediate an inflammatory environment that leads to HIV associated neurocognitive disorders in >50% of infected individuals despite successful antiretroviral therapy. Increased amounts of CCL2 and CXCL12 are present in the CNS of individuals with neurocognitive disorders. CCL2 increases the transmigration of HIV-infected CD14+CD16+ monocytes across the blood-brain-barrier (BBB), but the effect of CXCL12 on the transmigration of these cells is largely unknown. CXCL12 engages the HIV co-receptor CXCR4. The finding that CXCL12 also interacts with CXCR7 contributes to a different understanding of CXCL12 mediated processes. To characterize CXCL12 directed transmigration of CD14+ CD16+ monocytes across the BBB, we studied this process in an in vitro BBB model. To examine mature CD14+CD16+ monocytes, we enrich for this population in culture. We now demonstrate that these cultured CD14+CD16+ monocytes transmigrate preferentially across the BBB as effectively to CXCL12 as to CCL2. Moreover, the expression of CXCR4 and CXCR7 on our cultured monocytes is similar as that on monocytes from HIV-positive individuals, validating our culture model. CD14+CD16-and CD14+CD16+ monocytes from HIV-positive individuals express comparable amounts of CXCR4, but CXCR7 expression is specifically increased on the CD14+CD16+ monocyte population. These CD14+CD16+ monocytes transmigrate across the BBB to very low concentrations of CXCL12. CXCL12 is also a potent T cell chemoattractant, yet T cells require higher CXCL12 concentrations for BBB transmigration. Surface CXCR4 on these monocytes is decreased after transmigration, but is greatly increased on T cells, suggesting that CXCR7 may specifically mediate CD14+CD16+ monocyte transmigration across the BBB. We propose that the significant response of CD14+CD16+ monocytes to CXCL12 when transmigrating across the BBB is likely mediated by both CXCR4 and, at least in part, CXCR7, having implications for HIV neuropathogenesis and therapeutics. SDF-1/CXCL12 induces migration of lymphocytes by a mechanism pannexin1 hemichannels dependent In the last few years the role that pannexin hemichannels play in immune cells has received extensive attention. Our previous work showed that HIV infection induced a biphasic opening of these channels, an early opening between 5-30 min and a late opening between 48-120 h after HIV exposure. Our data indicates that opening of panx-1 hemichannels is essential for viral entry and replication, however there function in physiological conditions is unknown. Thus, we proposed that activation of specific chemokine receptors results in the physiological opening of pannexin-1 hemichannels (Panx-1) in T lymphocytes. We determined that treatment of T lymphocytes with SDF-1/ CXCL12, a key chemokine in lymphocyte migration and HIV infection, induces a transient opening of Panx-1 when compared to HIV, but not connnexin43 hemichannels. Blocking Panx-1 blocked the lymphocyte migration induced by SDF-1/ CXCL12, suggesting that opening of Panx-1 is essential for lymphocyte migration by a mechanism that involves local release of ATP. Alterations in migration occur in HIV infected cells but also in other PNS and CNS diseases such as multiple sclerosis (MS). Using a KO mouse model and EAE (an animal model of MS) we demonstrated that Panx-1 hemichannels are essential for migration of immune cells into the CNS and PNS. In conclusion our findings demonstrate that Panx-1 hemichannels play an essential role in HIV infection and in leukocyte migration into the CNS opening potential new therapeutic targets to block the pathogenesis of NeuroAIDS and MS. Jussi Oskari Virtanen 1 , Jeffrey Kowalak 2 , Emily Leibovitch 1 , Naomi Lee 1 , Yoshimi Enose-Akahata 1 , Steve Jacobson 1 (presenting author: virtanenjo@ninds.nih.gov) 1 NIH/NINDS/Neuroimmunology; 2 NIH/NINDS&NIMH/Clinical Proteomics Viral infections have been associated with autoimmune diseases such as multiple sclerosis (MS), but the exact mechanisms how viral infection could induce autoimmunity are poorly characterized. Although molecular mimicry have been suggested as a mechanism, convincing viral and autoantigen targets have not been identified in MS. Another possibility is that viral infection could induce and/or modify cellular proteins that are recognized by the immune system. Human herpesvirus 6 (HHV-6) antigen and nucleic acids have been found in MS brain lesions suggesting that HHV-6 might participate to the lesion development. To identify possible intrathecal humoral immune response targets in human HHV-6 infected cells we used immunoprecipitation based mass spectrometry analysis to identify possible cellular targets present only in HHV-6 infected cells. Furthermore, we identified MS brain endogenous IgG protein targets using similar approach. Cerebrospinal fluid (CSF) IgG pulled 26 unique proteins from infected cells that did not came up with serum or from non-infected cells. Three of these proteins were identified as MS brain lesion associated endogenous IgG targets as well. We are in the process of validating these results with other immunoassays. Our results suggest that HHV-6 infection can induce cellular proteins that are recognized by MS CSF IgG, and thus, might provide targets for the autoimmunity in MS. Human immunodeficiency virus type 1 (HIV-1) infection causes activation of innate immune pathways in the brain, often with ensuing neurovirulence that is evident as neuronal injury and death with accompanying neurobehavioral disorders. The specific innate immune genes that drive early inflammatory responses to HIV-1 remain uncertain in brain cells but activation of inflammasomes in myeloid cells with subsequent cleavage and release of interleukin (IL)-1beta represent important pathogenic components underlying multiple inflammatory disorders. To investigate inflammasome expression and function in the human brain during HIV-1 infection, inflammasome-associated genes were examined showing that IL-1beta, IL-18 and caspase-1 were induced in brains from HIV-infected persons, largely detected in activated brain myeloid cells. Cultured human microglia constitutively expressed inflammasome-associated genes including IL-1beta, caspase-1, NLRP3, NLRP1, AIM2 and ASC, unlike human astrocytes and neurons. HIV-1 induced pro-IL-1beta in human microglia at 4 hr post-infection with appearance of the cleaved form and peak IL-1beta release at 24hr, which was prevented by the caspase inhibitor, YVAD-fmk. Exposure of microglia to soluble HIV-1 gp120 mediated IL-1beta induction and subsequent release by microglia. HIV-dependent release of IL-1beta from a human myeloid cell line, THP-1, was also inhibited by NLRP3 deficiency and high extracellular [K+] . In vivo infection by feline immunodeficiency virus (FIV) caused induction of multiple inflammasome-associated genes in microglia, which was accompanied by neuronal loss and neurobehavioral impairments; multivariate analyses of FIV-infected and uninfected animals disclosed that IL-1beta, NLRP3 and Caspase-1 represented key molecular determinants in the development of neurovirulence. Thus, inflammasome activation was an early and integral aspect of HIV/AIDS neuropathogenesis and might represent a potential target for therapeutic interventions. 70% of HIV infected individuals despite successful combined antiretroviral therapy (cART). Monocytes are critical cells involved in HAND as they bring virus into the CNS upon transmigration across the blood brain barrier (BBB). A subset of circulating mature monocytes that express surface CD14 and CD16 is highly susceptible to HIV infection and accumulates in the brain of those with HAND. The mechanisms by which these mature monocytes transmigrate across the BBB are not fully understood. We previously demonstrated using an in vitro culture system that diapedesis of CD14+CD16+ monocytes across the BBB is facilitated by the junctional proteins ALCAM, JAM-A, CD99, and PECAM-1. We now characterize the CD14+CD16+ monocyte subset in HIV infected individuals from the Manhattan HIV Brain Bank and the Women's Interagency HIV Study cohort. We found that this monocyte population was present in significantly greater numbers irrespective of cART status, viral load, CD4 count, and gender as compared to individuals without HIV. We determined that CD14+CD16+ monocytes from these people preferentially transmigrated across our in vitro model of the human BBB in response to CCL2, a potent monocyte chemoattractant elevated in the CNS of HIV infected people with HAND. CD14+CD16+ monocytes isolated from HIV seropositive individuals had higher surface expression of ALCAM, JAM-A, and PECAM-1 relative to HIV seronegative people. Blocking antibodies to ALCAM and JAM-A inhibited the transmigration of these CD14+CD16+ monocytes to baseline levels, suggesting their importance in facilitating monocyte transmigration across the BBB. Targeting the increased junctional proteins present on CD14+CD16+ monocytes derived from HIV infected people, which preferentially infiltrate the CNS, represents a novel therapeutic strategy which may reduce the ongoing chronic neuroinflammation which occurs during HIV neuropathogenesis. HIV activation of macrophages and microglia cells in the central nervous system (CNS) triggers the secretion of unknown neurotoxins, which are thought to be the cause of neuronal damage. Macrophage phenotype is controlled extensively by extracellular signals but the influence of brain derived signals on macrophage phenotype is poorly understood. We have recently shown that human macrophages and monocytes express p75 and TrkA neurotrophin receptors and respond to the natural ligand, Nerve Growth Factor (NGF). Flow cytometry showed a decrease in receptors p75 and TrkA on human monocytes after stimulation with HIV virions and ProNGF +/-HIV while NGF +/-HIV increased both receptors. Stimulation of macrophages with HIV also caused prolonged calcium accumulation that was blocked by pretreatment with NGF. ProNGF pretreatment however resulted in spikes of calcium activity in macrophages that was blocked with a neutralizing antibody to the HIV Co-Receptor, CXCR4 suggesting interactions between neurotrophin and chemokine receptors . NGF and proNGF also had opposing effects on macrophage neurotoxin secretion as assessed by calcium dysregulation in rat cortical neurons. HIV virions resulted in increased neurotoxic activity of the macrophage conditioned medium. Pretreatment with NGF partially suppressed while ProNGF exacerbated neurotoxicity. Rearrangement of actin cytoskeleton of macrophages, polarity and podosome location were also seen with neurotrophin and HIV stimulation. ProNGF stimulation resulted in an increase in the polarization of podosomes compared to NGF. As with neurons where signaling of the pro and mature forms of neurotrophins results in opposing actions, the pro and mature forms of NGF may also exert opposing regulation of monocyte/macrophage phenotypes. Based on these data we conclude that mature NGF promotes a less destructive macrophage phenotype while proNGF may increase neurotoxicity. Pharmacological control of these interactions may provide new therapeutic avenues for the treatment of HIV-associated cognitive disorders. The current clinical studies have focused on the identification of LTR SNPs derived from peripheral blood (PB) of patients enrolled in the DrexelMed HIV/AIDS Genetic Analysis Cohort in Philadelphia to identify HIV-1-infected individuals more prone to developing advanced disease and/or neurologic dysfunction. Patient demographics and clinical parameters including drug use, CD4/CD8 T-cell count, and viral load are performed at every visit. Drug abuse is common within this cohort with cocaine use being favored. The cohort can be categorized into non-users (PN), preferential cocaine (PC), and multidrug users (MDU). SNPs have been identified that associated with CD4 T-cell count and viral load. SNPs were also identified that are unique to each drug abuse category. Using an in silico sensitivity analysis, we were able to determine significant differences in transcription factor footprints at the Lef-1 site with a variation from an A-to-G at position 321 to be significantly different between the PN and PC and PN and MDU subcohorts. Additionally, an A-to-G alteration at position 286 within C/EBP site II was found to be significant in both the psychomotor and constructive neuropsychologically impaired patients. In previous studies, we have shown this position to have an increased affinity for C/EBP beta, increased HIV-1 basal LTR activity, a decreased prevalence in PB-derived LTRs from patients with increased disease progression, but an increased prevalence in brainderived LTRs. These results suggest that cocaine may be applying selective pressure with respect to the genetic architecture of the LTR within these critical binding sites. This work is supported by NIH/NINDS R01 NS32092, NIDA R01 DA19807, NIMH P30 MH092177, and NIMH T32 MH079785. Clayton Winkler, Tyson Woods, Karin E. Peterson (presenting author: clayton.winkler@nih.gov) Laboratory of Persistent Viral Diseases, Rocky Mountain Labs, NIAID, NIH La Crosse virus (LACV) is a primary cause of pediatric arbovirus encephalitis United States resulting in severe neurologic symptoms and even death. LACV encephalitis is characterized by perivascular infiltration of leukocytes into the central nervous system (CNS) and neuronal death. However, the mechanisms leading to leukocyte infiltration and the role of those cells in LACV pathogenesis remains unclear. Intravenous injection of Evans blue dye demonstrated increased vascular leakage into the CNS at 5 days post infection (dpi), which is prior to the onset of neurological disease. Brain endothelial cells at this time point showed decreased expression of CD31, a primary component of endothelial tight junctions, as well as increased expression of Ccl2, Ccr2 and Cxcl11, which are involved in leukocyte recruitment. These findings suggest CNS blood vessels in LACV infected animals are permissive to cellular infiltration prior to neurological signs. Examination of CNS infiltrates determined that activated monocytes/macrophages, CD4+ and CD8+ T cells were present at 5dpi, with numbers peaking at neurological disease onset. Inflammatory monocytes/macrophages were by far the predominant infiltrating population and were found in close association with areas of neuronal infection suggesting they may be relevant to neurological disease. Concordantly, depletion of T cells, which are present in low numbers, did not alter the kinetics or onset of the disease suggesting their infiltration is not critical for neurological disease development. Interestingly, Rag1-/-mice were more susceptible to LACV CNS infection than wildtype controls suggesting B cells antibody responses have a protective role in disease pathogenesis. However, B cell infiltration was not observed, suggesting their role in LACV-induced neurological disease is distant from the CNS. Collectively, while the role of infiltrating monocytes/ macrophages is still under investigation, we conclude adaptive immune cell infiltration into the CNS does not appear to influence the development of LACV-induced neurological disease. Increased HIV-1 DNA in CSF cells is associated with decreased CSF oxidative stress in HIV-seropositive Hispanic women Background: HIV-associated neurocognitive disorders (HAND) persist in spite of antiretroviral therapy. Recent data demonstrated high HIV-1 DNA in circulating activated monocytes is associated with HAND. These activated monocytes with HIV-1 DNA migrate to the brain and may represent CSF viral reservoirs. Studies have shown a correlation between oxidative stress and HAND. Therefore we proposed to determine the relationship between HIV-1 DNA in CSF cells, oxidative stress, and HAND. Methods: A retrospective review was performed on prospectively collected clinical data and samples from a cohort of 27 HIV-seropositive Hispanic women, stratified by cognitive impairment into normal (n=8), asymptomatic (n=6), mild impairment (n=6), and HAD (n-7). HIV-1 DNA was measured in CSF cells using real time PCR. The CSF oxidative stress markers 3-NT were detected by dot-blot analysis while the protein carbonyls (PC) with oxy-and dot-blot. Cognitive performance was determined by the established criteria. Parametric and non-parametric statistics were used. Results: A positive association was observed between higher HIV-1 DNA copy/cell in CSF cells and use of cART (p=0.007). When controlling for cART use there was a negative correlation between higher HIV-1 DNA in CD14+ blood cells with worse performance in Trail Making B (TMB, p=0.049) and verbal memory (p=0.036) tests. This negative correlation was also observed in the CSF cells with higher HIV-1 DNA copy/ cell and lower CSF levels of 3NT (p=0.015) and PC (p=0.014). Conclusions: Increased HIV-1 DNA in CD14+ correlated with worse cognitive performance while increased HIV-1 DNA in CSF cells correlated with lower CSF oxidative stress levels. Findings may suggest persistence of CSF viral reservoirs containing latent virus. Grant support U54MD008149, U54 RR022762; partially by U54MD007587, S11NS46278, U54NS43011, P20RR11126, G12RR03051, F31AI081450, R01NS053345, U54MD007584 Hassen Wollebo, Kamel Khalili, Mahmut Safak, Martyn White (presenting author: siraj123@temple.edu) Department of Neuroscience and Center for Neurovirology, Temple University School of Medicine, Philadelphia The human polyomavirus JC (JCV) is the etiologic agent of the rare devastating neurologic disease Progressive multifocal leukoencephalopathy. Globally, the prevalence of infection with JCV is high and JCV is believed to persist in a latent state after primary infection. Even though JCV is common in the population, PML is extremely rare which suggest that the virus is very tightly regulated by cell-mediated immunity. Previously our laboratory and others have shown that the JCV promoter and its transcriptional activity are regulated by multiple transcriptional factors. Here we provide evidence that JC virus promoter activity under goes another level of regulation by epigenetic events involving protein acetylation. We have found that histone deacetylase inhibitors profoundly stimulate the transcription of both the early and late JC virus promoters in transient transfection experiments and in stable early and late reporter cell lines. This suggests that the acetylation status of histones may be an important mechanism controlling the latency/ reactivation status of JCV. Statins are a promising candidate adjunctive therapy for prevention or treatment of HIV-1 associated neurocognitive disorders (HAND) Anjana Yadav 1 , Lorraine Kolson 2 , Michael Betts 3 , Dennis Kolson 2 , Ronald Collman 1 (presenting author: ayadav@mail.med.upenn.edu) 1 Department of Medicine, University of Pennsylvania School of Medicine; 2 Department of Neurology, University of Pennsylvania School of Medicine; 3 Department of Microbiology, University of Pennsylvania School of Medicine HIV-1 associated neurocognitive disorders (HAND) result from indirect effects mediated by activated blood monocytes that traffic to the brain, and activated and/or infected brain macrophages that release inflammatory chemokines and cytokines (including MCP-1) and neurotoxic mediators (such as glutamate). HAND remains a major problem even among patients on antiretroviral therapy (ART) with effective viral suppression, and has been linked to persistent immune activation and inflammation. Statins are HMG-CoA reductase inhibitors with pleiotropic immunomodulatory properties mediated by effects on intracellular signal transduction pathways. We first investigated the immunomodulatory effects of lipophilic statins (simvastatin & atorvastatin) on human monocyte/ macrophage activation in vitro. Statin treatment reduced the CD16+ and CD163+ monocyte populations following LPS and MCP-1 stimulation, and strongly suppressed MCP-1, IL-8, MIP-1alpha production by activated PBMC. Statin treatment also strongly inhibited monocyte chemotaxis to MCP-1. Further, we demonstrated that statins inhibited production of the neurotoxin glutamate by HIV-1 infected macrophages, and also had a direct neuroprotective effect on rat neurons exposed to macrophage-HIV supernatants. We then carried out a small open label in vivo pilot study of five HIV+ / ART+ subjects treated with Atorvastatin for 12 weeks followed by a 4-week washout period. In this small number of subjects, atorvastatin treatment was associated with a trend towards decreased total CD14+ monocytes, reduced CD16 mean fluorescent intensity on CD14+ monocytes , and decline in plasma hsCRP during the treatment period. These observations suggest the potential of statins as a candidate adjuvant treatment to prevent HAND Division of Infectious Diseases and HIV Medicine, Department of Medicine, Drexel University College of Medicine The introduction of HAART has resulted in significantly lower mortality rates, culminating in an HIVepidemic that increasingly affects older adults, emphasizing the importance of understanding the risk factors associated with aging and HIV-1 infection. Additionally, immune failure commonly occurs with both aging and HIV infection. Current studies focus on the impact of age on LTR signatures that correlate with HIV-1 clinical parameters including CD4+ and CD8+ T-cell count and viral load (VL). A prospective, longitudinal study was conducted on 458 HIV-1seropositive patients currently enrolled in the DrexelMed HIV/ AIDS Genetic Analysis Cohort in Philadelphia, PA. History of illicit drug, alcohol, and medication use, CD4+ and CD8+ T-cell count, and viral load, along with patient history including age, were collected approximately every 6 months. Using a linear mixed model for longitudinal data, we determined that there were viral SNPs that significantly correlated with increasing age. Additionally, age modified the SNP effect with respect to CD4+ T-cell counts and VL to different degrees. Both HIV-1 infection and age are also known to have immunomodulatory effects. The cytokine profiles of adult and aged individuals were analyzed to understand the impact of age and HIV-1 infection on cytokine modulation and HIV-1 disease severity. Interestingly, among the 30 cytokines investigated, the cytokine profiles appeared different between these two populations. In addition, there appeared to be a number of SNPs that correlated with the age effect on cytokine levels. In conclusion, age, in the context of HIV-1 infection, appears to impact viral gene expression and immune activation. This work is supported by NIH/NINDS R01 NS32092, NIDA R01 DA19807, NIMH P30 MH092177, and NIMH T32 MH079785. The central dopamine (DA) neurotransmission plays a crucial role in the development of neurocognitive dysfunction in patients with HIV-1 associated neurocognitive disorders (HAND). We have demonstrated that HIV-1 Tat protein in vitro inhibits DA transporter (DAT) allosterically and modulates cocaine binding sites on DAT in rat striatal synaptosomes and cells expressing human DAT. To understand how in vivo expression of HIV-1 viral proteins influences DAT function, kinetic analysis of [3H]DA uptake in prefrontal cortex (PFC) and striatum, and [3H]WIN35,428 binding in striatum were determined in HIV-1 transgenic (HIV-1Tg) and Fisher 344 rats. Compared to Fisher 344 rats, the maximal velocity (Vmax) of DAT-mediated [3H]DA uptake into prefrontal and striatal synaptosomes in HIV-1Tg rats was increased by 34% and 32%, respectively, whereas the Km values for DA uptake were reduced in striatum but not in PFC of HIV-1Tg rats. In addition, HIV-1Tg rats exhibit decreased maximal binding sites (Bmax) of [3H]WIN35,428 and increased DA uptake turnover rate (Vmax/Bmax) in striatum, compared to Fisher 344 rats. To explore the potential mechanism(s) underlying alteration of DAT function in HIV-1Tg rats, our ongoing study is to determine whether the increased DA uptake in both regions is due to redistribution of membrane DAT or changes in DAT-associated proteins in membrane microdomains. Collectively, these results suggest that neuroadaptive changes have occurred in the HIV-1Tg rats that might help to compensate for HIV-1 viral protein-induced inhibition of the DAT function. Supported by grant from the NIH to Jun Zhu (R01DA035714) The SIV/Macaque Model: Proof of Concept for HIV Eradication M. Christine Zink 1 , Lucio Gama 1 , Janice Clements 1 , Celina Abreu 2 , Amilcar Tanuri 2 (presenting author: mczink@jhmi.edu) 1 Johns Hopkins University School of Medicine; 2 Division of Genetics -Federal University of Rio de Janeiro, Rio de Janeiro, RJ, Brazil We have developed an SIV/pigtailed macaque model that results in rapid development of CNS disease and AIDS that provides a robust model for testing of antiretroviral and neuroprotective therapeutic strategies. We have also demonstrated that we can obtain suppression of plasma vRNA to below suggesting both NMDA-R and AMPA/KA-R on OLs were activated by HIV-1 Tat. Importantly, both MK801 and CNQX block Tat-induced immature OLs death, but only MK801 reverses Tat-induced mature OL membrane area change. These results add to evidence suggesting that OLs are targets of HIV, and also suggest that viability and phenotypic changes in immature and mature OLs, respectively, may involve different Ca2+-mediated glutamatergic mechanisms. Tabatadze, Nino, P177 Talal, Andrew, P158 Talbot Jr., C. Conover, P170 Tan, Chen Sabrina, P32, P171 Tanaka, Yuetsu, P10 Myelin pallor is frequently reported in HIV patients, and can occur in the CNS prior to other evidence of a disease process On the other hand, mature OLs exposed to 100 nM Tat exhibit reduced myelin-like membrane area, indicating potential injury to cell morphology and function. To investigate if these effects are mediated via Ca2+, Fura-2 AM was used to quantify [Ca2+]i within individual cells. Both mature (O4+, MBP+) and immature OLs (O4+, MBP-) exposed to 1-100 nM Tat showed a dose-dependent, gradual increase in [Ca2+]i up to~600 nM vs.~100 nM in control, and this high [Ca2+]i level was maintained during a 1 h experimental period. This Tat-induced [Ca2+]i increase is completely abolished in Ca2+-free medium, suggesting extracellular Ca2+ as the primary source. The NMDA-R antagonist MK801 reduces Tat-induced modalities, including BLI, PET or MRI. The engineered viruses remain replication competent after infecting target cells for reporter gene expression. The reporter was engineered into the virus either with or without its own promoter to allow detection of the infected cells during the latency stage, while the long terminal repeat (LTR) promoter is inactive. We have demonstrated that the imaging reporter genes are incorporated, expressed and functional in infected cells and the replication kinetics of engineered lentivirus is comparable to that of the wild type virus. Currently, we are working towards the evaluation of the engineered viruses in the individual species: humanized mice (HIV), macaque monkeys (SIV) or felines (FIV) to further evaluate the use of non-invasive imaging to visualize the spatial and temporal nature of viral reservoirs and their response to HAART. The further analyze of these engineered viruses will provide the ability to image viral reservoirs providing a much needed tool for monitoring and evaluating therapeutic efficacy. Virawudh Soontornniyomkij 1 , Anya Umlauf 2 , Sandra Chung 3 , Megan Cochran 4 , Benchawanna Soontornniyomkij 1 , Ben Gouaux 5 , Will Toperoff 2 , Andrea Bucci 6 , David Moore 1 , Eliezer Masliah 7 , Ronald Ellis 8 , Scott Letendre 9 , Dilip Jeste 10 , Igor Grant 2 , Cristian Achim 1,7 (presenting author: cachim@ucsd.edu The benefit of highly active antiretroviral therapy for preventing and treating HIV-associated neurocognitive disorders (HAND), especially HIV-associated dementia, is well documented. Nonetheless, the milder forms of HAND remain prevalent and hence are suspected to be associated with various co-morbid factors including cumulative exposure to antiretroviral (ARV) medications. We propose that chronic toxic effects of ARV drugs on the cell components of blood vessel walls can contribute to brain vascular pathology, particularly cerebral small vessel disease (CSVD, including arteriosclerosis and arteriolosclerosis). Even at its early stages when a decrease in the cerebral blood flow is not severe enough to cause infarcts or white matter lesions, CSVD can lead to disturbance of cerebrovascular autoregulation and deficiency in functional hyperemia. We used multinomial logistic regression to determine associations between the use of ARV drugs and the occurrence of CSVD in 144 autopsy HIV-infected cases in the California NeuroAIDS Tissue Network. Using univariable analysis we found that mild CSVD (vs. absent) was associated with nucleoside reverse transcriptase inhibitors (NRTIs) or protease inhibitors (PIs) [odds ratio (OR) 19.23 and 2.84, P=0.003 and =0.06, respectively], but not with any of the vascular risk factors (i.e., older age [≥50 years], diabetes mellitus, hypertension, hyperlipidemia, cigarette smoking, and lifetime psychostimulant dependence). On the other hand, moderate/severe CSVD (vs. absent) was associated with older age and diabetes (OR 2.56 and 6.55, P=0.035 and =0.01, respectively), but not with any of the ARV-related predictors. Our findings suggest that the use of NRTIs or PIs may increase the risk of mild CSVD and consequently neurocognitive impairment. Of particular interest are further studies of the molecular mechanisms of ARV toxicity to the cell components of brain vessel walls and identifying potential biomarkers for CSVD in HIV-infected persons. study we demonstrate that relatively high concentrations of SP (10 microM) trigger intracellular calcium increase in monocytes, most likely due to the activation of the truncated isoform of neurokinin 1 receptor (NK1R). SP also enhances CD163 expression on monocytes in a dose-and timedependent manner, albeit to a lower extent than IL-10, a cytokine well known to induce the alternative pathway of monocyte differentiation. The SP-induced expression of membrane-bound CD163 is accompanied by increased shedding of sCD163 from human monocyte-derived macrophages (MDM) and this effect is inhibited by the NK1R antagonist, aprepitant. Decreasing the expression of membrane-bound CD163 in MDM using siRNA resulted in a significant decrease of HIV infection. In order to further demonstrate that the level of expression of membrane-bound CD163 is important for HIV infection, we used cell sorting to separate human monocytes expressing high and low levels of CD163. The productivity of HIV infection was higher in cells expressing high levels, vs. cells expressing low levels of CD163. Our study indicates that high levels of membrane-bound CD163 facilitate HIV infection in macrophages. This work was supported by National Institutes of Health Grants U01-MH 090325, R01-MH 049981, R21-AI 108298 (to SDD) Prolonged exposure to morphine induces cell adhesion to an in vitro model of the blood-brain barrier Opioid abuse by human immunodeficiency virus type 1 (HIV-1)-infected individuals leads to more rapid disease progression, increased viral replication and peripheral viral load, and increased incidence and severity of neurocognitive abnormalities compared to non-drug abusers. The blood-brain barrier (BBB) is an obstacle that must be overcome during neuroinvasion with subsequent development of HIVassociated neurocognitive disorders (HAND). Previous studies addressing the role of mu-opioids in altering BBB permeability have suggested that exposure increases cellular transmigration through an uncharacterized mechanism. In this study, a human brain microvascular endothelial cell (hBMEC) line, hCMEC/D3, was used to establish an in vitro transwell model of the BBB to investigate the effects of chronic (24, 48, or 72 h) morphine treatment on barrier structure and function. We observed that hCMEC/D3 cells form a confluent monolayer with a basal rate of passage of a 70 kDa tracer molecule comparable to primary hBMECs. It has also been shown that these cells express mu opioid receptor, and that prolonged morphine treatment induces changes in mRNA levels of cellular adhesion molecules. Functionally, an increase in PBMC transmigration and firm adhesion was observed following prolonged morphine exposure, in the absence of an increase in overall barrier leakiness. These results have suggested that morphine activates hCMEC/D3 cells leading to a cellular environment permissive to transmigration. These studies may uncover a mechanism through which morphine disrupts periphery-CNS homeostasis leading to accelerated HAND. This work is supported by NIH/NINDS R01 NS32092, NIDA R01 DA19807, NIMH P30 MH092177, and NIMH T32 MH079785. Viral particles of HIV-1, the causative agent of AIDS, contain two copies of a ssRNA genome. Toll-Like Receptors (TLRs) such as endosomal TLRs 7 and 8 have been reported to sense HIV-1 RNA sequences. The endosomal TLR3 primarily senses dsRNA, a common viral replication intermediate that is absent during retroviral replication. However, since HIV-1 ssRNAs contain multiple hairpin-like secondary structures that could potentially mimic a dsRNA entity, we hypothesized that TLR3 could sense and respond to HIV-1 infection. No difference in susceptibility to infection with HIV-1 virions pseudotyped with murine leukemia virus envelope glycoprotein or vesicular stomatitis virus glycoprotein (VSV-g) was observed between bone marrow-derived macrophages isolated from wild-type and TLR3 knock-out (TLR3KO) mice. However, production of pro-inflammatory cytokines and chemokines, including type-I IFNs was significantly reduced in BMDMs from TLR3KO mice. Similarly, in the presence of a TLR3-specific inhibitor, HIV-1 BAL pseudotype infection of human monocyte-derived macrophages and THP1-derived Purpose: HIV-associated neurocognitive disorder (HAND) is a complication of HIV, in which HIV-infected individuals suffer a varying degree of neurocognitive impairment. HAND can occur in both treated and untreated HIV patients, making it a large public health problem. The etiology of HAND is generally thought to be at least partly neuroinflammatory, as infected cells in both the periphery and CNS cause immune activation leading to synaptodendritic damage and neuronal apoptosis. Recently, several labs, including ours, have found that peripheral inflammation is correlated with the progress of neurocognitive disease, raising the possibility that peripheral inflammatory mediators may promote neuroinflammation by crossing into the CNS. Since protein expression, lipid synthesis, and small molecule metabolism may all be involved in inflammatory cascades, in this study we determine levels of all three species in the spleen. Methods: In this study, 6 pig-tailed macaques were dual inoculated with a neurovirulent molecular clone and an immunosuppressive viral swarm, in which we have shown pigtailed macaques to develop AIDS and encephalitis within 84 days. Macaques were sacrificed at 7 days post-infection and spleen tissue was harvested, then extracted for protein, lipid and metabolite species. Protein, lipid and metabolite species were then profiled by mass spectrometry. Results: Lipidomic profiling by an MS/MS-ALL method found changes in triacylglycerides. Metabolomic profiling by highflow LC/MS alignment and then targeted MS/MS found changes in the nucleotides guanosine and inosine, the small lipid adipic acid, and the small molecule diethyl oxalopropionate. Jewell Thomas, Matthew Brier, Beau Ances (presenting author: ancesb@neuro.wustl.edu) Background: The question whether HIV+ individuals experience aging-related deficits earlier than healthy individuals or simply experience increased age-associated deficits across the age-range is unresolved. We present graph theory models of brain functional connectivity (FC) that add important evidence that HIV may lead to an advanced brain-age phenotype. Methods: BOLD resting-state FC scans were collected in HIV+ (N=46) and HIV-(N=59) participants on a Siemens 3T scanner. These groups had similar demographics, though the HIV+ group had a higher viral load and lower CD4+ white blood cell count. FC time-series were sampled in 160 ROIs for each subject and graphs were constructed from crosscorrelation matrices. Clustering coefficient (a measure of local efficiency), path length (a measure of global efficiency), and modularity (a measure of sub-network structure) were computed across a range of correlation thresholds for each subject in order to assess the stability of group differences. Effects of HIV and linear and quadratic effects of age as well as age by HIV interactions on graph theory topology were assessed using ANCOVA. Results:Higher clustering coefficient and lower path length were observed for HIV+ (p<.05, FDR). Modularity decreased, suggesting that graph structural changes may explain changes in efficiency measures. Modularity began to decline earlier in HIV+ individuals than HIV-individuals and declined as a quadratic effect of age (p<.01). Immune system strength modulated modularity changes in HIV+ (p<.01). Conclusions: By investigating graph structure (particularly modularity), we showed the first fMRI evidence that agerelated changes may occur earlier in HIV+ individuals. Importantly, we showed that modularity decreases were explained by strength of immune system response (stronger immune activation led to greater decreases). Because immune activation may be an important underlying cause of brain deficits in both HIV and aging, our result suggests HIV & aging may both impair brain function through immune system mechanisms. The role of the blood brain barrier in amyloid beta uptake by HIV-1-infected brain The mechanism by which brain inflammation drives the systemic immune response is currently unknown. In this study we provide evidence that this neuroimmune axis involves microvesicles released from brain that enter into peripheral circulation. IL-1beta, TNFalpha and ATP evoked a rapid and robust efflux of microvesicles from astrocytes. The stimulated release of these microvesicles from astrocytes involved a stabilization of nSmase2 into ceramide-rich membrane caps that were primarily located to filopodia. Microvesicle release was blocked by inhibition of IL-1beta receptor binding, or inhibition of nSMase2. Microvesicles were 65.6 ± 22.2 nm in size, and contained a complex lipid and protein content that was consistent with exosomes. Using an in vitro BBB-model prepared with BmuEC with GFAP-GFP expressing astrocytes we determined that microvesicles carrying GFP as cargo readily crossed endothelial cells with intact tight junctions. Electron microscopy demonstrated that a small fraction of these exosomes were endocytosed into endothelial cells through caveolae, with the majority appearing to readily cross as intact particles, possibly involving local phase transitions in the membrane. We next determined if the release of these exosomes from the injured brain regulated leukocyte trafficking. Intrastriatal injections of IL-1beta induced rapid focal generations of ceramide, and a transmigration of neutrophils to the lesion site within 24h. Genetic mutation of nSMase2, or simultaneous injections of nSMase2 antagonists (GW4869 or altenusin) blocked IL-1beta from inducing ceramide, and blocked neutrophil influx. Purified exosomes isolated from plasma 2h following intrastriatal injections of IL-1beta, but not exosomes isolated from mice receiving intrastriatal injections of saline, re-activated neutrophil influx when infused into the tail vein of acceptor mice who received intrastriatal injections of IL-1beta + altenusin. These data demonstrate that microvesicles released from astrocytes readily cross the BBB and regulate the peripheral immune response to CNS inflammation. Simian varicella virus is present in lymph nodes of rhesus macaques after experimental reactivation Earlier, we demonstrated experimental reactivation of SVV after Xirradiation and treatment with tacrolimus and prednisone of latently infected monkeys. Herein, 5 rhesus macaques were inoculated intrabronchially with 10000 pfu of SVV or SVV-EGFP (SVV expressing green fluorescent protein) and 5 months later, after the establishment of latency, all monkeys were transported to an irradiation facility and 4 of them were exposed to a single dose (200 cGy) of X-irradiation and treated with tacrolimus (80 ug/kg/day) and prednisone (1 mg/kg/day) for the duration of the experiment; one monkey was not immunosuppressed, but was subjected to the stress of transportation. Zoster rash developed 7-12 weeks after immunosuppression in all 5 monkeys which were euthanized 24-48 hrs later, and multiple tissues (skin, lungs, ganglia and lymph nodes) were harvested. In all 5 monkeys, immunohistochemistry revealed SVV antigens in these tissues. Immunofluorescence and confocal analysis of multiple lymph nodes corresponding to the area of rash, showed colocalization of SVV proteins in macrophages and dendritic cells, but not in lymph nodes from a SVV-seronegative monkey. Detection of SVV proteins was confirmed by Western blot in 2 of 2 lymph nodes from one monkey and by qPCR detection of SVV DNA in 3 of 3 lymph nodes from another monkey. These findings suggest that SVV antigens may be transported into draining lymph nodes where macrophages and dendritic cells present them to activate T cells directed against SVV. Richa Tyagi, Wenxue Li, Danelvis Parades, Avindra Nath (presenting author: tyagi.richa@nih.gov) Section of Infections of the Nervous System, National Institute of Neurological Diseases and Stroke, National Institutes of Health Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease, characterized by the progressive loss of motor neurons. Its pathogenesis is unclear, although several mechanisms have been suggested. We previously study showed that Human Endogenous Retrovirus type K (HERV-K) was activated in the brain of ALS patients, indicating that HERV-K may be involved in the pathogenesis of ALS. HERVs are genomic sequences of retroviral origin which were believed to be integrated into germline chromosomes millions of years ago. Although mostly defective and inactive, some of the HERVs may be activated under certain physiological and pathological conditions. While no drugs are designed specifically targeting HERVs, there are a panel of FDA approved HIV antiretroviral drugs. We hypothesized that HIV antiretroviral drugs may also be effective in inhibiting the infection and replication of HERVs and may be used for the treatment of HERV-K activation in ALS. We constructed a plasmid with consensus HERV-K sequence and produced HERV-K virus for testing the effect of antiretroviral drugs on HERV-K replication. We first determined the effects of nucleoside and nonnucleotide reverse transcriptase inhibitors on HERV-K by product enhanced reverse transcription assay (PERT). We found that Abacavir and Zidovudine could significantly inhibit HERV-K reverse transcriptase activity at similar effective dosages for HIV-1. To determine the effects of Abacavir and an integrase inhibitor, Raltegravir on HERV-K viral replication, we pseudotyped HERV-K with VSV-G and used the pseudotyped HERV-K virus to infect HeLa cells. Replication of HERV-K was measured by quantitative real time polymerase chain reaction (qRT-PCR). We found that both Abacavir and Raltegravir blocked the replication of HERV-K at very low concentration. In summary, we identified several FDA approved antiretroviral drugs that can effectively inhibit HERV-K virus. These antiretrovirals may open new prospects for treatment of HERV-K activation in ALS and other neuropsychiatric diseases. Activation of HERV-W human endogenous retroviruses by JC polyomavirus in human astrocytes: a novel effect that could contribute to neurodegeneration Human T-lymphotropic retrovirus (HTLV-1) is the causative agent of the central nervous system disease HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP). Like patients with multiple sclerosis (MS), a related disease of the central nervous system, HAM/TSP patients have increased IgG index and oligoclonal bands (OCBs) as a marker of intrathecal antibody production. In this study, we investigated the specificity of the cerebrospinal fluid (CSF) and serum antibodies to HTLV-1 antigens by luciferase immunoprecipitation system (LIPS) assay and the specificity of the oligoclonal bands to HTLV-1 antigens in HAM/TSP. All of the 22 patients had HTLV-1 gag, env and tax antibodies in CSF and all but one had OCBs specific to at least one HTLV-1 antigen studied. Gagand env-specific OCBs were found more often than tax-specific OCBs in HAM. Our data shows that in HAM/TSP HTLV-1 antibodies are produced intrathecally and most of the OCBs are specific for HTLV-1 antigens. Detection of intrathecal antibody production by LIPS and HTLV-1 antigen specific OCBs by isoelectric focusing and immunoblot might provide tools to differentiate HAM/TSP patients from HTLV-1 seropositive MS cases and might suggest the involvement of humoral immune response in the pathogenesis of HAM/TSP. Natalizumab-associated progressive multifocal leukoencephalopathy in a patient with multiple sclerosis: a post mortem study Background: Natalizumab, a monoclonal antibody directed against alpha4 integrins, has to date been associated with 395 cases of progressive multifocal leukoencephalopathy (PML) worldwide in patients receiving treatment for multiple sclerosis (MS). Due to the limited number of histological studies, the interplay between MS and PML lesions has not been investigated. Objective: We report the clinical, radiological and histological findings of a MS patient who developed PML after 32 months of natalizumab monotherapy. Following withdrawal of natalizumab, she received plasma exchange, mefloquine and mirtazapine, but passed away soon thereafter. Post mortem studies were restricted to examination of the brain and spinal cord. Results: Extensive PML lesions, characterized by the presence of JCV DNA were found in the cerebral white matter and neocortex. Sharply demarcated areas of active PML lesions contained prominent inflammatory infiltrates composed of approximately equal numbers of CD4+ and CD8+ T cells, consistent with an immune reconstitution inflammatory syndrome (IRIS). Conversely, all MS lesions identified were hypocellular, long-standing inactive plaques characterized by myelin loss, relative axonal preservation, and gliosis, and importantly, were devoid of JCV-DNA and active inflammation. Conclusions: This case demonstrates the coexistence of chronic inactive MS and PML lesions harboring active JCV infection.Chronic inactive MS lesions were separate and distinct from nearby PML lesions. Furthermore, IRIS greatly affected the shape and appearance of PML lesions but did not involve MS lesions. Whether PML may alter the nature of active MS lesions remains to be determined. Murine coronavirus (MHV) accessory protein, ns2 is a phosphodiesterase that inhibits activation of the interferon(IFN)induced oligoadenylate synthetase(OAS)-RNase L pathway and facilitates viral replication in macrophage lineage cells and induces hepatitis. However, efficient MHV replication in the brain and subsequent encephalitis are independent of enzymatically active ns2. Compared to the liver and other peripheral organs, the CNS expresses lower basal levels of IFN-stimulated genes including several OASs, which are required for RNase L activation. Thus, we hypothesized that expression of OAS in the CNS was not sufficient to activate RNase L after MHV infection. Indeed, an MHV mutant expressing an inactive ns2 protein (ns2H126R) replicates to a similar extent as wild type (wt) virus in the CNS. We have compared replication of wt and ns2H126R in bone marrow derived macrophages with primary microglia, neurons, astrocytes and oligodendrocytes. In contrast to restriction of ns2H126R replication in macrophages and microglia, wt and ns2H126R replicated with similar kinetics in the other primary cells. In addition, ns2H126R induced RNase L activity, as assessed by ribosomal RNA (rRNA) cleavage, in myeloidderived cells but failed to do so in other cell types. Parenchymal cells pretreated with IFN restricted wt and ns2H126R to similar extents indicating that inhibition is not due to RNase L activity. However, astrocytes and neurons are competent for RNase L activity as they cleave rRNA when transfected with poly I:C. Thus, RNase L activation depends on the virus, kinetics of infection and IFN induction. Furthermore, expression of relatively high levels of OAS genes is necessary but not sufficient for induction of an effective RNase L antiviral response. Activation of RNase L is muted in the CNS perhaps to prevent the potentially destructive effects of unrestricted activity in nonrenewable cells. Although ART can effectively suppress viral suppression, expression of viral proteins such as Tat remains unaffected. This in turn, could lead chronic activation of glia resulting in neuroinflammation. ER stress has recently been implicated to play an important role in the impairment of CNS homeostasis in HIV+ patients. In response to ER stress, cells activate a set of tightly controlled regulatory programs, the unfolded protein response (UPR), to restore normal functioning of the ER. However, if ER stress is sustained and the adaptive UPR fails to eliminate unfolded/misfolded proteins, balance shifts to apoptosis as a mechanism for clearing stressed proteins. We hypothesized that Tat mediated activation/apoptosis of astrocytes involves the ER stress response. Our preliminary data demonstrates that HIV Tat induced ER stress in astrocytes, with activation of the three UPR pathways-PERK, ATF6 & IRE1. This was accompanied by disruption of calcium homeostasis, generation of reactive oxidative stress (ROS) & induction of apoptosis. Of particular interest is the chaperone Sigma-1 Receptor (sigma1-R) that is critical for ER stress. Intriguingly, overexpression of sigma1-R in astrocytes significantly decreased Tat-mediated ROS generation & inhibited the expression of pro-apoptotic CHOP protein. Reciprocally, knocking down sigma1-R resulted in decreased expression of the anti-apoptotic Bcl2 & increased cellular apoptosis. Sigma1-R could be envisioned as a critical modulator of glial cell survival and may be an important target for therapeutic intervention in HAND. NIBP (NIK-and IKK2-Binding Protein) is a novel protein that enhances cytokine-induced NFkB activation and is required for neuronal differentiation. TNFalpha and IL-1beta are well known to stimulate NFkB activation, which contribute to cytokine-induced HIV-1 reactivation. We hypothesize that NIBP may regulate HIV-1 infection and reactivation via NFkB signaling. To test this, we performed HIV-1 long terminal repeat (LTR) driven firefly-luciferase reporter assay using mouse brain neural stem/progenitor cells and human fibroblast with NIBP deletion. To our surprise, NIBP overexpression in mouse neural stem/progenitor cells significantly inhibited constitutive and TNFalpha-induced LTR promoter activation. Such inhibition was independent of NFkB signaling be-cause NFkB site-deletion LTR mutant inhibited TNFalpha-induced LTR activation but did not prevent NIBP-induced inhibition. This was validated in other cells and species including human fibroblast and breast cancer cells as well as rabbit colonic smooth muscle cells. Interestingly, LTR-luciferase activity was increased in NIBP deletion fibroblast cells from patient with mental retardation and overexpression of NIBP in these fibroblasts rescued such inhibition. NIBP is known to interact with bovine viral diarrhea virus non-structural protein 5A (NS5A), which hijacks NIBP-mediated NFkB activation. Coimmunoprecipitation assay in HEK293T cells cotransfected with Flag-tagged NIBP and CFP-tagged TAT or YFP-tagged Vpr vectors showed that both NIBP(1148aa) and NIBP(960aa) interact with TAT but only NIBP(1148aa) interacts with Vpr. These data suggests that NIBP may act as a novel restriction factor to suppress HIV-1 transcription/reactivation through interacting with HIV-1 viral proteins. Such interaction may hijack NIBP-mediated NFkB activation. Since NIBP is highly expressed in HIV-1 non-permissive neurons but not in permissive microglia/macrophage and T cells, NIBP gene therapy may serve as a novel approach to treat NeuroAids. Impact of Aging on markers of HIV-1 disease detectable levels using combinations of three different classes of antiretroviral drugs that are CNS penetrant or nonpenetrant. In the last few years, a number of HIV-infected patients have experienced at least functional cures (the Berlin Patient, the Mississippi Child, the French Cohort), ceasing antiretroviral therapy without viral reactivation or development of disease. This has initiated a search for ways to duplicate these natural experiments. Recently a number of compounds that are able to activate HIV DNA have been suggested as coadjuvant to cART therapy. It is proposed that the antiretroviral drugs would prevent viral spread while host lymphocytes would kill reactivated infected cells expressing viral proteins. We have been investigating a hexanoate derivative of Ingenol (Kyo-IIB), a phorbol ester isolated from Euphorbia tirucalli. Kyo-IIB treatment of resting CD4+ T cells isolated from virus suppressed SIV-infected macaques led to a significant increase in reactivated latent cells and viral RNA transcription. In a pilot trial in SIV-infected macaques, Kyo-IIB was administered in escalating doses orally BID for one week each, alternated with off-treatment weeks. Activation markers in blood leukocytes and plasma viral load increased during treatment and decreased during the washout periods. In contrast to other phorbol esters, Kyo-IIB is less toxic and can be orally administered to animals with no apparent side effects. We plan to treat virus suppressed SIV-infected macaques with Kyo-IIB to determine the ability of the compound to reactivate SIV in vivo and reduce the number of SIV latently infected resting CD4+ T cells, monocytes, and macrophages as proof-ofconcept for a functional HIV cure. Meredith Zoch, Sarah Price, Jeanne Sisk, Lucio Gama, Janice Clements (presenting author: mzoch1@jhmi.edu) Department of Molecular and Comparative Pathobiology, Johns Hopkins University School of Medicine, Baltimore, MD 21205A major barrier to the eradication of HIV is the persistence of virus in latent reservoirs. CD4+ T cells have been widely studied, and resting CD4+ T cells are considered the major latent reservoir in HIV and SIV infections. HIV and SIV replicate in activated CD4+ T cells, but a small percentage of these cells become resting memory cells and, if infected, harbor latent virus. Monocytes, macrophages, and microglia in the brain have been shown to support HIV and SIV infection. In both HIVand SIV infected individuals on combination antiretroviral therapy, viral DNA is found in the brain, and ongoing changes in the brain suggest that the virus is persistent and latent. Because of the recent focus on the eradication of HIV, identification of all latent viral reservoirs has become crucial. However, little is understood about mechanisms of HIVor SIV latency in myeloid cells. In the CNS, the astrocyte, the most abundant cell in brain, is also infected with HIV and SIV but infection is not productive. In order to characterize viral latency in macrophages, microglia, and astrocytes, we are developing in vitro primary cell latency models. In order to recapitulate the environment in the CNS, cytokines are being used to determine their role in establishing viral latency in macrophages, microglia, and astrocytes. Glutamate receptors mediate stage-specific effects of HIV-1 Tat on the viability and phenotype of oligodendroglia