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1.  HIV-1 neuropathogenesis: Glial mechanisms revealed through substance abuse 
Journal of neurochemistry  2006;100(3):567-586.
Neuronal dysfunction and degeneration are ultimately responsible for the neurocognitive impairment and dementia manifest in neuroAIDS. Despite overt neuronal pathology, HIV-1 does not directly infect neurons; rather, neuronal dysfunction or death is largely an indirect consequence of disrupted glial function and the cellular and viral toxins released by infected glia. A role for glia in HIV-1 neuropathogenesis is revealed in experimental and clinical studies examining substance abuse-HIV-1 interactions. Current evidence suggests that glia are direct targets of substance abuse and that glia contribute markedly to the accelerated neurodegeneration seen with substance abuse in HIV-1 infected individuals. Moreover, maladaptive neuroplastic responses to chronic drug abuse might create a latent susceptibility to CNS disorders such as HIV-1. In this review, we consider astroglial and microglial interactions and dysfunction in the pathogenesis of HIV-1 infection and examine how drug actions in glia contribute to neuroAIDS.
PMCID: PMC4305441  PMID: 17173547
Opiates; Cocaine; Methamphetamine; Alcohol; HIV encephalopathy; Neuroimmunology; AIDS
2.  T cells induced by recombinant chimpanzee adenovirus alone and in prime-boost regimens decrease chimeric EcoHIV/NDK challenge virus load 
European journal of immunology  2012;42(12):3243-3255.
The popularity of nonreplicating adenoviruses of chimpanzee origin (ChAdVs) as vectors for subunit vaccines is on the rise. This is mainly for their excellent safety and impressive immunogenicity observed in human studies to date. Here, we recloned the chimpanzee adenovirus sero type 68 (ChAdV-68), also designated SAdV-25 and AdC68, genome and demonstrated its straightforward genetic manipulation facilitated by the use of bacterial artificial chromosome recombineering. To generate the ChAdV68.GagB vaccine, the HIV-1 consensus clade B Gag-derived Tg was inserted into the E1 region. In part confirming previous observations, the ChAdV68.GagB vaccine alone and in heterologous prime-boost regimens with plasmid DNA- and modified vaccinia virus Ankara (MVA)-vectored vaccines induced robust polyfunctional HIV-1-specific CD8+ and CD4+ T-cell responses with a gut-homing phenotype. Importantly, we showed that when a single epitope is expressed as an immunodominant CD8+ T-cell determinant, responses elicited by ChAdV68.GagB alone and in combination lowered surrogate challenge EcoHIV/NDK (where EcoHIV is chimeric ecotropic HIV) virus load in mice both at the peak T-cell frequencies 2 weeks after vaccination and 16 weeks later indicating development of protective effector memory. These results parallel the immunogenicity of similar vaccine regimens in macaques and an ongoing phase I/IIa trial in humans, and support further development of vaccines vectored by ChAdVs.
PMCID: PMC3980942  PMID: 22930183
Chimpanzee adenovirus; EcoHIV challenge; Heterologous prime-boost regimen; T-cell vaccines
4.  Transmission of chimeric HIV by mating in conventional mice: prevention by pre-exposure antiretroviral therapy and reduced susceptibility during estrus 
Disease Models & Mechanisms  2013;6(5):1292-1298.
Heterosexual transmission accounts for the majority of new human immunodeficiency virus (HIV) cases worldwide. The current approach to investigate HIV heterosexual transmission in animals involves application of virus stock to the vaginal surface, a method that does not reproduce the physiological conditions of vaginal intercourse that influence the rate of transmission. We have previously described efficient infection of conventional mice using EcoHIV/NL4-3 and EcoHIV/NDK, chimeric HIV molecular clones constructed to express all HIV structural and regulatory genes except envelope, which is replaced by a rodent-tropic envelope gene. Here we investigated whether EcoHIV/NDK-infected male mice transmit virus to females during coitus, and the sensitivity of this transmission to HIV pre-exposure prophylaxis and the estrus state. Our general approach was to allow mating between EcoHIV/NDK-infected male mice and uninfected females for 1–7 nights. At 1–6 weeks after mating, mice were euthanized and virus burdens were measured by quantitative PCR (qPCR) amplification of HIV RNA or DNA in peritoneal macrophages, inguinal lymph node cells, spleen cells or vas deferens, or by ELISA for antibodies to HIV Gag. We found that 70–100% of female mice mated to EcoHIV/NDK-infected males acquired infection. Pericoital treatment of females with either 2′,3′-dideoxcytidine (ddC) or tenofovir largely prevented their EcoHIV/NDK infection by mating (P<0.05 and P<0.003, respectively). In males, T cells were dispensable for virus transmission. The rate of EcoHIV/NDK sexual transmission to females in estrus declined sharply (P=0.003) but their infection by injection was unaffected, indicating that the local environment in the female reproductive tract influences susceptibility to HIV. We conclude that this system of EcoHIV/NDK transmission during mouse mating reproduces key features of heterosexual transmission of HIV in humans and can be used to investigate its biology and control.
PMCID: PMC3759349  PMID: 23886803
5.  Mice Chronically Infected with Chimeric HIV Resist Peripheral and Brain Superinfection: A Model of Protective Immunity to HIV 
Infection by some viruses induces immunity to reinfection, providing a means to identify protective epitopes. To investigate resistance to reinfection in an animal model of HIV disease and its control, we employed infection of mice with chimeric HIV, EcoHIV. When immunocompetent mice were infected by intraperitoneal (IP) injection of EcoHIV, they resisted subsequent secondary infection by IP injection, consistent with a systemic antiviral immune response. To investigate the potential role of these responses in restricting neurotropic HIV infection, we established a protocol for efficient EcoHIVexpression in the brain following intracranial (IC) inoculation of virus. When mice were inoculated by IP injection and secondarily by IC injection, they also controlled EcoHIV replication in the brain. To investigate their role in EcoHIV antiviral responses, CD8+ T lymphocytes were isolated from spleens of EcoHIV infected and uninfected mice and adoptively transferred to isogenic recipients. Recipients of EcoHIV primed CD8+ cells resisted subsequent EcoHIV infection compared to recipients of cells from uninfected donors. CD8+ spleen cells from EcoHIV-infected mice also mounted modest but significant interferon-γ responses to two HIV Gag peptide pools. These findings suggest EcoHIV-infected mice may serve as a useful system to investigate the induction of anti-HIV protective immunity for eventual translation to human beings.
PMCID: PMC3487595  PMID: 21987348
Mouse model; Chimeric HIV; Superinfection; Adaptive immune responses; HIV neuropathogenesis; Brain
6.  Common Transcriptional Signatures in Brain Tissue from Patients with HIV-Associated Neurocognitive Disorders, Alzheimer’s Disease, and Multiple Sclerosis 
HIV-Associated Neurocognitive Disorders (HAND) is a common manifestation of HIV infection that afflicts about 50 % of HIV-positive individuals. As people with access to antiretroviral treatments live longer, HAND can be found in increasing segments of populations at risk for other chronic, neurodegenerative conditions such as Alzheimer’s disease (AD) and Multiple Sclerosis (MS). If brain diseases of diverse etiologies utilize similar biological pathways in the brain, they may coexist in a patient and possibly exacerbate neuropathogenesis and morbidity. To test this proposition, we conducted comparative meta-analysis of selected publicly available microarray datasets from brain tissues of patients with HAND, AD, and MS. In pair-wise and three-way analyses, we found a large number of dysregulated genes and biological processes common to either HAND and AD or HAND and MS, or to all three diseases. The common characteristic of all three diseases was up-regulation of broadly ranging immune responses in the brain. In addition, HAND and AD share down-modulation of processes involved, among others, in synaptic transmission and cell-cell signaling while HAND and MS share defective processes of neurogenesis and calcium/calmodulin-dependent protein kinase activity. Our approach could provide insight into the identification of common disease mechanisms and better intervention strategies for complex neurocognitive disorders.
Electronic supplementary material
The online version of this article (doi:10.1007/s11481-012-9409-5) contains supplementary material, which is available to authorized users.
PMCID: PMC3515772  PMID: 23065460
Transcriptome meta-analysis; Microarrays; HIV; HAND; Alzheimer’s disease; Multiple sclerosis
7.  Significant Effects of Antiretroviral Therapy on Global Gene Expression in Brain Tissues of Patients with HIV-1-Associated Neurocognitive Disorders 
PLoS Pathogens  2011;7(9):e1002213.
Antiretroviral therapy (ART) has reduced morbidity and mortality in HIV-1 infection; however HIV-1-associated neurocognitive disorders (HAND) persist despite treatment. The reasons for the limited efficacy of ART in the brain are unknown. Here we used functional genomics to determine ART effectiveness in the brain and to identify molecular signatures of HAND under ART. We performed genome-wide microarray analysis using Affymetrix U133 Plus 2.0 Arrays, real-time PCR, and immunohistochemistry in brain tissues from seven treated and eight untreated HAND patients and six uninfected controls. We also determined brain virus burdens by real-time PCR. Treated and untreated HAND brains had distinct gene expression profiles with ART transcriptomes clustering with HIV-1-negative controls. The molecular disease profile of untreated HAND showed dysregulated expression of 1470 genes at p<0.05, with activation of antiviral and immune responses and suppression of synaptic transmission and neurogenesis. The overall brain transcriptome changes in these patients were independent of histological manifestation of HIV-1 encephalitis and brain virus burdens. Depending on treatment compliance, brain transcriptomes from patients on ART had 83% to 93% fewer dysregulated genes and significantly lower dysregulation of biological pathways compared to untreated patients, with particular improvement indicated for nervous system functions. However a core of about 100 genes remained similarly dysregulated in both treated and untreated patient brain tissues. These genes participate in adaptive immune responses, and in interferon, cell cycle, and myelin pathways. Fluctuations of cellular gene expression in the brain correlated in Pearson's formula analysis with plasma but not brain virus burden. Our results define for the first time an aberrant genome-wide brain transcriptome of untreated HAND and they suggest that antiretroviral treatment can be broadly effective in reducing pathophysiological changes in the brain associated with HAND. Aberrantly expressed transcripts common to untreated and treated HAND may contribute to neurocognitive changes defying ART.
Author Summary
HAND is a common complication of HIV-1 infection in the nervous system presenting a varied spectrum of clinical manifestations with cognitive, motor and behavioral symptoms. Introduction of ART has greatly reduced morbidity and mortality in HIV-1 infection; however HAND persists and its overall prevalence appears to have increased despite treatment. The effects of the treatment on neurological disease are not well understood. Here, we used genomic analysis to compare gene expression profiles in brain tissues from treated and untreated patients who died with HAND. We identified a large number of genes and biological pathways dysregulated in untreated HAND compared with uninfected controls. ART appears to be effective in mitigating aberrant gene expression in brain tissues of patients with HAND but a fraction of genes remained dysregulated under ART and the patients continued to manifest HAND in the last evaluation prior to death. Our study provides new insights into the molecular changes in brain tissues of patients with HAND and the effect of the treatment on brain transcriptome. The identification of aberrantly expressed genes common to untreated and treated HAND may contribute to understand the neurocognitive impairment observed in patients under ART.
PMCID: PMC3164642  PMID: 21909266
8.  Gene Expression Profiles of HIV-1-Infected Glia and Brain: Toward Better Understanding of the Role of Astrocytes in HIV-1-Associated Neurocognitive Disorders 
Astrocytes are the major cellular component of the central nervous system (CNS), and they play multiple roles in brain development, normal brain function, and CNS responses to pathogens and injury. The functional versatility of astrocytes is linked to their ability to respond to a wide array of biological stimuli through finely orchestrated changes in cellular gene expression. Dysregulation of gene expression programs, generally by chronic exposure to pathogenic stimuli, may lead to dysfunction of astrocytes and contribute to neuropathogenesis. Here, we review studies that employ functional genomics to characterize the effects of HIV-1 and viral pathogenic proteins on cellular gene expression in astrocytes in vitro. We also present the first microarray analysis of primary mouse astrocytes exposed to HIV-1 in culture. In spite of different experimental conditions and microarray platforms used, comparison of the astrocyte array data sets reveals several common gene-regulatory changes that may underlie responses of these cells to HIV-1 and its proteins. We also compared the transcriptional profiles of astrocytes with those obtained in analyses of brain tissues of patients with HIV-1 dementia and macaques infected with simian immunodeficiency virus (SIV). Notably, many of the gene characteristics of responses to HIV-1 in cultured astrocytes were also altered in HIV-1 or SIV-infected brains. Functional genomics, in conjunction with other approaches, may help clarify the role of astrocytes in HIV-1 neuropathogenesis.
PMCID: PMC3107560  PMID: 19697136
astrocytes; microarrays; gene expression profiling; HIV-1; HAD; brain; neurobiology; neurodegeneration; innate immunity
9.  Protective Efficacy of Serially Up-Ranked Subdominant CD8+ T Cell Epitopes against Virus Challenges 
PLoS Pathogens  2011;7(5):e1002041.
Immunodominance in T cell responses to complex antigens like viruses is still incompletely understood. Some data indicate that the dominant responses to viruses are not necessarily the most protective, while other data imply that dominant responses are the most important. The issue is of considerable importance to the rational design of vaccines, particularly against variable escaping viruses like human immunodeficiency virus type 1 and hepatitis C virus. Here, we showed that sequential inactivation of dominant epitopes up-ranks the remaining subdominant determinants. Importantly, we demonstrated that subdominant epitopes can induce robust responses and protect against whole viruses if they are allowed at least once in the vaccination regimen to locally or temporally dominate T cell induction. Therefore, refocusing T cell immune responses away from highly variable determinants recognized during natural virus infection towards subdominant, but conserved regions is possible and merits evaluation in humans.
Author Summary
The body's reaction to infections is dominated by strong immune responses focused on a small number of protein regions, while ignoring the other. These strong responses may be protective against invariant viruses, but fail to control viruses that can change quickly. In particular for the AIDS virus, it happens that the few targeted regions during natural infection are highly variable, and rapidly change and become unrecognized by the mounted responses, i.e. escape. Here, we demonstrated that even the naturally under-recognized protein regions can induce robust responses and confer protection against whole viruses, and therefore should be made use of by vaccination strategies.
PMCID: PMC3098219  PMID: 21625575
10.  Novel regulation of HIV-1 replication and pathogenicity: Rev inhibition of integration 
Following fusion of the human immunodeficiency virus type-1 (HIV-1) with host cells' membrane and reverse transcription of the viral RNA, the resulted cDNA is integrated into the host genome by the viral integrase enzyme (IN). Quantitative estimations have revealed that only 1–2 copies are integrated per infected cell, although many copies of the viral RNA are reverse-transcribed. The molecular mechanism that restricts the integration degree has not, so far, been elucidated. Following integration, expressed partially spliced and unspliced transcripts are exported from the nuclei by the viral Rev protein. Here, we show that in virally infected cells, the Rev interacts with the IN forming a Rev–IN complex and consequently limits the number of integration events. Disruption of the Rev–IN complex by selected IN-derived peptides or infection by a Rev-deficient virus stimulate integration resulting in large numbers of integration event/cell. Conversely, infection of Rev-expression cells blocks integration and inhibits virus production. Increased integration appears to correlate with increased cell death of infected cultures. Our results thus demonstrate a new regulatory function of Rev and probably establish a link between Rev restriction of HIV-1 integration and protection of HIV-1-infected cells from premature cell death.
PMCID: PMC2777023  PMID: 19875511
cell death; HIV-1; integrase; integration regulation; Rev
11.  HIV-1 infected astrocytes and the microglial proteome 
The human immunodeficiency virus (HIV) invades the central nervous system early after viral exposure but causes progressive cognitive, behavior, and motor impairments years later with the onset of immune deficiency. Although in the brain, HIV preferentially replicates productively in cells of mononuclear phagocyte (MP; blood borne macrophage and microglia), astrocytes also can be infected, at low and variable frequency, particularly in patients with encephalitis. Among their many functions, astrocytes network with microglia to provide the first line of defense against microbial infection; however, very little is known about its consequences on MP. Here, we addressed this question using co-culture systems of HIV infected mouse astrocytes and microglia. Pseudotyped vesicular stomatis virus/HIV was used to circumvent absence of viral receptors and ensure cell genotypic uniformity for studies of intercellular communication. The study demonstrated that infected astrocytes show modest changes in protein elements as compared to uninfected cells. In contrast, infected astrocytes induce robust changes in the proteome of HIV-1 infected microglia. Accelerated cell death and redox proteins, amongst others, were produced in abundance. The observations confirmed the potential of astrocytes to influence the neuropathogenesis of HIV-1 infection by specifically altering the neurotoxic potential of infected microglia and in this manner, disease progression.
PMCID: PMC2579774  PMID: 18587649
astrocytes; microgial; human immunodeficiency virus; pseudotyped viral infection; proteomics; cell mobility; neurotoxicity
12.  Monocyte Chemotactic Protein-1 Regulates Voltage-Gated K+ Channels and Macrophage Transmigration 
Progressive human immunodeficiency virus (HIV)-1 infection and virus-induced neuroinflammatory responses effectuates monocyte-macrophage transmigration across the blood-brain barrier (BBB). A key factor in mediating these events is monocyte chemotactic protein-1 (MCP-1). Upregulated glial-derived MCP-1 in HIV-1 infected brain tissues generates a gradient for monocyte recruitment into the nervous system. We posit that the inter-relationships between MCP-1, voltage gated ion channels, cell shape and volume, and cell mobility underlie monocyte transmigration across the BBB. In this regard, MCP-1 serves both as a chemoattractant and an inducer of monocyte-macrophage ion flux affecting cell shape and mobility. To address this hypothesis, MCP-1 treated bone marrow derived macrophages (BMM) were analyzed for gene and protein expression, electrophysiology, and capacity to migrate across a laboratory constructed BBB. MCP-1 enhanced K+ channel gene (KCNA3) and channel protein expression. Electrophysiological studies revealed that MCP-1 increased outward K+ currents in a dose dependent manner. In vitro studies demonstrated that MCP-1 increased BMM migration across an artificial BBB and the MCP-1-induced BMM migration was blocked by tetraethylammonium, a voltage-gated K+ channel blocker. Together these data demonstrated that MCP-1 affects macrophage migratory movement through regulation of voltage-gated K+ channels and as such, provides a novel therapeutic strategy for neuroAIDS
PMCID: PMC2657224  PMID: 19034671
Monocyte; Monocyte Chemotactic Protein-1; K+ Channels; Blood-Brain Barrier
13.  Vaccine induced protection from infection of mice by chimeric human immunodeficiency virus type 1, EcoHIV/NL4-3 
Vaccine  2007;25(52):8660-8663.
EcoHIV/NL4-3 is a chimeric human immunodeficiency virus type 1 (HIV-1) that can productively infect mice. This study tests the utility of EcoHIV/NL4-3 infection to reveal protective immune responses to an HIV-1 vaccine. Immunocompetent mice were first immunized with VRC 4306 which encodes subtype B consensus sequences of gag, pol, and nef and then were infected by EcoHIV/NL4-3. Anti-Gag antibodies were sampled during immunization and infection. The extent of EcoHIV/NL4-3 infection in spleen cells and peritoneal macrophages was determined by quantitative real-time PCR (QPCR). Although antibody titres were not significantly different in control and vaccinated groups, VRC 4306 immunization induced protective responses that significantly reduced virus burden in both lymphocyte and macrophage compartments. These results indicate that EcoHIV/NL4-3 infection can be controlled by HIV-1 vaccine induced responses, introducing a small animal model to test vaccine efficacy against HIV-1 infection.
PMCID: PMC2219693  PMID: 18023943
HIV-1; mouse model; DNA vaccine
14.  HIV-1 Tat Activates Neuronal Ryanodine Receptors with Rapid Induction of the Unfolded Protein Response and Mitochondrial Hyperpolarization 
PLoS ONE  2008;3(11):e3731.
Neurologic disease caused by human immunodeficiency virus type 1 (HIV-1) is ultimately refractory to highly active antiretroviral therapy (HAART) because of failure of complete virus eradication in the central nervous system (CNS), and disruption of normal neural signaling events by virally induced chronic neuroinflammation. We have previously reported that HIV-1 Tat can induce mitochondrial hyperpolarization in cortical neurons, thus compromising the ability of the neuron to buffer calcium and sustain energy production for normal synaptic communication. In this report, we demonstrate that Tat induces rapid loss of ER calcium mediated by the ryanodine receptor (RyR), followed by the unfolded protein response (UPR) and pathologic dilatation of the ER in cortical neurons in vitro. RyR antagonism attenuated both Tat-mediated mitochondrial hyperpolarization and UPR induction. Delivery of Tat to murine CNS in vivo also leads to long-lasting pathologic ER dilatation and mitochondrial morphologic abnormalities. Finally, we performed ultrastructural studies that demonstrated mitochondria with abnormal morphology and dilated endoplasmic reticulum (ER) in brain tissue of patients with HIV-1 inflammation and neurodegeneration. Collectively, these data suggest that abnormal RyR signaling mediates the neuronal UPR with failure of mitochondrial energy metabolism, and is a critical locus for the neuropathogenesis of HIV-1 in the CNS.
PMCID: PMC2579580  PMID: 19009018
15.  Proteomic Modeling for HIV-1 Infected Microglia-Astrocyte Crosstalk 
PLoS ONE  2008;3(6):e2507.
HIV-1-infected and immune competent brain mononuclear phagocytes (MP; macrophages and microglia) secrete cellular and viral toxins that affect neuronal damage during advanced disease. In contrast, astrocytes can affect disease by modulating the nervous system's microenvironment. Interestingly, little is known how astrocytes communicate with MP to influence disease.
Methods and Findings
MP-astrocyte crosstalk was investigated by a proteomic platform analysis using vesicular stomatitis virus pseudotyped HIV infected murine microglia. The microglial-astrocyte dialogue was significant and affected microglial cytoskeleton by modulation of cell death and migratory pathways. These were mediated, in part, through F-actin polymerization and filament formation. Astrocyte secretions attenuated HIV-1 infected microglia neurotoxicity and viral growth linked to the regulation of reactive oxygen species.
These observations provide unique insights into glial crosstalk during disease by supporting astrocyte-mediated regulation of microglial function and its influence on the onset and progression of neuroAIDS. The results open new insights into previously undisclosed pathogenic mechanisms and open the potential for biomarker discovery and therapeutics that may influence the course of HIV-1-mediated neurodegeneration.
PMCID: PMC2429966  PMID: 18575609
16.  Gene Expression Profiling of Human Primary Astrocytes Exposed to Manganese Chloride Indicates Selective Effects on Several Functions of the Cells 
Neurotoxicology  2006;28(3):478-489.
Exposure of adult humans to manganese (Mn) has long been known to cause neurotoxicity. Recent evidence also suggests that exposure of children to Mn is associated with developmental neurotoxicity. Astrocytes are critical for the proper functioning of the nervous system, and they play active roles in neurogenesis, synaptogenesis and synaptic neurotransmission. In this report, to help elucidate the molecular events underlying Mn neurotoxicity, we systematically identified the molecular targets of Mn in primary human astrocytes at a genome-wide level, by using microarray gene expression profiling and computational data analysis algorithms. We found that Mn altered the expression of diverse genes ranging from those encoding cytokines and transporters to signal transducers and transcriptional regulators. Particularly, 28 genes encoding proinflammatory chemokines, cytokines and related functions were up-regulated whereas 15 genes encoding functions involved in DNA replication and repair and cell cycle checkpoint control were down-regulated. Consistent with the increased expression of proinflammatory factors, analysis of common regulators revealed that 16 targets known to be positively affected by the interferon-γ signaling pathway were up-regulated by Mn2+. In addition, 68 genes were found to be similarly up- or down-regulated by both Mn2+ and hypoxia. These results from genomic analysis are further supported by data from real-time RT-PCR, Western blotting, flow cytometric and toxicological analyses. Together, these analyses show that Mn2+ selectively affects cell cycle progression, the expression of hypoxia-responsive genes, and the expression of proinflammatory factors in primary human astrocytes. These results provide important insights into the molecular mechanisms underlying Mn neurotoxicity.
PMCID: PMC2041834  PMID: 17175027
Manganese; Inflammatory factors; Human Astrocytes; Microarray; Gene expression
17.  Astrocyte Elevated Gene (AEG)-1: recent insights into a novel gene involved in tumor progression, metastasis and neurodegeneration 
Pharmacology & therapeutics  2007;114(2):155-170.
Tumor progression and metastasis are complex processes involving intricate interplay among multiple gene products. Astrocyte Elevated Gene (AEG)-1 was cloned as an HIV-1- and tumor necrosis factor α (TNF-α)-inducible transcript in primary human fetal astrocytes by a rapid subtraction hybridization approach. AEG-1 downregulates the expression of the glutamate transporter EAAT2, thus it is implicated in glutamate-induced excitotoxic damage to neurons as evident in HIV-associated neurodegeneration. Interestingly, AEG-1 expression is elevated in subsets of breast cancer, glioblastoma multiforme and melanoma cells and AEG-1 cooperates with Ha-ras to augment the transformed phenotype of normal immortal cells. Moreover, AEG-1 is overexpressed in >95% of human malignant glioma samples when compared with normal human brain. Overexpression of AEG-1 increases and siRNA inhibition of AEG-1 decreases migration and invasion of human glioma cells, respectively. AEG-1 contains a lung-homing domain facilitating breast tumor metastasis to lungs. These findings indicate that AEG-1 might play a pivotal role in the pathogenesis, progression and metastasis of diverse cancers. Our recent observations indicate that AEG-1 exerts its effects by activating the NF-κB pathway and AEG-1 is a downstream target of Ha-ras and plays an important role in Ha-ras-mediated tumorigenesis. These provocative findings are intensifying interest in AEG-1 as a crucial regulator of tumor progression and metastasis and as a potential mediator of neurodegeneration. In this review, we discuss the cloning, structure and function(s) of AEG-1 and provide recent insights into the diverse actions and intriguing properties of this molecule.
PMCID: PMC2039930  PMID: 17397930
AEG-1; Progression; Metastasis; Ha-ras oncogene; Glutamate excitotoxicity; AEG-1 promoter
18.  Human immunodeficiency virus type 1 efficiently binds to human fetal astrocytes and induces neuroinflammatory responses independent of infection 
BMC Neuroscience  2007;8:31.
HIV-1 infects human astrocytes in vitro and in vivo but the frequency of infected cells is low and its biological significance is unknown. In studies in vitro, recombinant gp120 alone can induce profound effects on astrocyte biology, suggesting that HIV-1 interaction with astrocytes and its functional consequences extend beyond the limited levels of infection in these cells. Here we determined the relative efficiencies of HIV-1 binding and infection in human fetal astrocytes (HFA), mainly at the single cell level, using HIV-1 tagged with green fluorescence protein (GFP)-Vpr fusion proteins, termed HIV-GFP, to detect virus binding and HIV-1 expressing Rev and NefGFP fusion proteins to detect productive infection.
Essentially all HFA in a population bound HIV-GFP specifically and independently of CCR5 and CXCR4. The dynamics of this binding at 37°C resembled binding of an HIV fusion mutant to CD4-positive cells, indicating that most of HIV-GFP arrested infection of HFA at the stage of virus-cell fusion. Despite extensive binding, only about 1% of HFA were detectably infected by HIV-RevGFP or HIV-NefGFP, but this proportion increased to the majority of HFA when the viruses were pseudotyped with vesicular stomatitis virus envelope glycoprotein G, confirming that HFA impose a restriction upon HIV-1 entry. Exposure of HFA to HIV-1 through its native proteins rapidly induced synthesis of interleukin-6 and interleukin-8 with increased mRNA detected within 3 h and increased protein detected within 18 h of exposure.
Our results indicate that HIV-1 binding to human astrocytes, although extensive, is not generally followed by virus entry and replication. Astrocytes respond to HIV-1 binding by rapidly increased cytokine production suggesting a role of this virus-brain cell interaction in HIV-1 neuropathogenesis.
PMCID: PMC1884168  PMID: 17498309
19.  Generalization of DNA microarray dispersion properties: microarray equivalent of t-distribution 
Biology Direct  2006;1:27.
DNA microarrays are a powerful technology that can provide a wealth of gene expression data for disease studies, drug development, and a wide scope of other investigations. Because of the large volume and inherent variability of DNA microarray data, many new statistical methods have been developed for evaluating the significance of the observed differences in gene expression. However, until now little attention has been given to the characterization of dispersion of DNA microarray data.
Here we examine the expression data obtained from 682 Affymetrix GeneChips® with 22 different types and we demonstrate that the Gaussian (normal) frequency distribution is characteristic for the variability of gene expression values. However, typically 5 to 15% of the samples deviate from normality. Furthermore, it is shown that the frequency distributions of the difference of expression in subsets of ordered, consecutive pairs of genes (consecutive samples) in pair-wise comparisons of replicate experiments are also normal. We describe a consecutive sampling method, which is employed to calculate the characteristic function approximating standard deviation and show that the standard deviation derived from the consecutive samples is equivalent to the standard deviation obtained from individual genes. Finally, we determine the boundaries of probability intervals and demonstrate that the coefficients defining the intervals are independent of sample characteristics, variability of data, laboratory conditions and type of chips. These coefficients are very closely correlated with Student's t-distribution.
In this study we ascertained that the non-systematic variations possess Gaussian distribution, determined the probability intervals and demonstrated that the Kα coefficients defining these intervals are invariant; these coefficients offer a convenient universal measure of dispersion of data. The fact that the Kα distributions are so close to t-distribution and independent of conditions and type of arrays suggests that the quantitative data provided by Affymetrix technology give "true" representation of physical processes, involved in measurement of RNA abundance.
This article was reviewed by Yoav Gilad (nominated by Doron Lancet), Sach Mukherjee (nominated by Sandrine Dudoit) and Amir Niknejad and Shmuel Friedland (nominated by Neil Smalheiser).
PMCID: PMC1586001  PMID: 16959036
20.  PAGE: Parametric Analysis of Gene Set Enrichment 
BMC Bioinformatics  2005;6:144.
Gene set enrichment analysis (GSEA) is a microarray data analysis method that uses predefined gene sets and ranks of genes to identify significant biological changes in microarray data sets. GSEA is especially useful when gene expression changes in a given microarray data set is minimal or moderate.
We developed a modified gene set enrichment analysis method based on a parametric statistical analysis model. Compared with GSEA, the parametric analysis of gene set enrichment (PAGE) detected a larger number of significantly altered gene sets and their p-values were lower than the corresponding p-values calculated by GSEA. Because PAGE uses normal distribution for statistical inference, it requires less computation than GSEA, which needs repeated computation of the permutated data set. PAGE was able to detect significantly changed gene sets from microarray data irrespective of different Affymetrix probe level analysis methods or different microarray platforms. Comparison of two aged muscle microarray data sets at gene set level using PAGE revealed common biological themes better than comparison at individual gene level.
PAGE was statistically more sensitive and required much less computational effort than GSEA, it could identify significantly changed biological themes from microarray data irrespective of analysis methods or microarray platforms, and it was useful in comparison of multiple microarray data sets. We offer PAGE as a useful microarray analysis method.
PMCID: PMC1183189  PMID: 15941488
22.  Activation of NF-κB by R5 and X4 Human Immunodeficiency Virus Type 1 Induces Macrophage Inflammatory Protein 1α and Tumor Necrosis Factor Alpha in Macrophages 
Journal of Virology  2002;76(10):5274-5277.
NF-κB is a transcriptional activator that often regulates inflammatory responses. We demonstrate that human immunodeficiency virus type 1 activates nuclear localization of NF-κB in macrophages in a manner dependent upon virus strain but independent of virus replication. Through the use of an inhibitor, NF-κB activation was found to be responsible for the cytokine and chemokine induction that we recently reported.
PMCID: PMC136147  PMID: 11967342
23.  Induction of Rapid and Extensive β-Chemokine Synthesis in Macrophages by Human Immunodeficiency Virus Type 1 and gp120, Independently of Their Coreceptor Phenotype 
Journal of Virology  2001;75(22):10738-10745.
Human immunodeficiency virus type 1 (HIV-1) interacts with its target cells through CD4 and a coreceptor, generally CCR5 or CXCR4. Macrophages display CD4, CCR5, and CXCR4 that are competent for binding and entry of virus. Virus binding also induces several responses by lymphocytes and macrophages that can be dissociated from productive infection. We investigated the responses of macrophages to exposure to a series of HIV-1 species, R5 species that productively infect and X4 species that do not infect macrophages. We chose to monitor production of several physiologically relevant factors within hours of treatment to resolve virally induced effects that may be unlinked to HIV-1 production. Our novel findings indicate that independently of their coreceptor phenotype and independently of virus replication, exposure to certain R5 and X4 HIV-1 species induced secretion of high levels of macrophage inflammatory protein 1α (MIP-1α), MIP-1β, RANTES, and tumor necrosis factor alpha. However two of the six R5 species tested, despite efficient infection, were unable to induce rapid chemokine production. The acute effects of virus on macrophages could be mimicked by exposure to purified R5 or the X4 HIV-1 envelope glycoprotein gp120. Depletion of intracellular Ca2+ or inhibition of protein synthesis blocked the chemokine induction, implicating Ca2+-mediated signal transduction and new protein synthesis in the response. The group of viruses able to induce this chemokine response was not consistent with coreceptor usage. We conclude that human macrophages respond rapidly to R5 and X4 envelope binding by production of high levels of physiologically active proteins that are implicated in HIV-1 pathogenesis.
PMCID: PMC114655  PMID: 11602715
24.  Highly Productive Infection with Pseudotyped Human Immunodeficiency Virus Type 1 (HIV-1) Indicates No Intracellular Restrictions to HIV-1 Replication in Primary Human Astrocytes 
Journal of Virology  2001;75(17):7925-7933.
Human astrocytes can be infected with human immunodeficiency virus type 1 (HIV-1) in vitro and in vivo, but, in contrast to T lymphocytes and macrophages, virus expression is inefficient. To investigate the HIV-1 life cycle in human fetal astrocytes, we infected cells with HIV-1 pseudotyped with envelope glycoproteins of either amphotropic murine leukemia virus or vesicular stomatitis virus. Infection by both pseudotypes was productive and long lasting and reached a peak of 68% infected cells and 1.7 μg of viral p24 per ml of culture supernatant 7 days after virus inoculation and then continued with gradually declining levels of virus expression through 7 weeks of follow-up. This contrasted with less than 0.1% HIV-1 antigen-positive cells and 400 pg of extracellular p24 per ml at the peak of astrocyte infection with native HIV-1. Cell viability and growth kinetics were similar in infected and control cells. Northern blot analysis revealed the presence of major HIV-1 RNA species of 9, 4, and 2 kb in astrocytes exposed to pseudotyped (but not wild-type) HIV-1 at 2, 14, and 28 days after infection. Consistent with productive infection, the 9- and 4-kb viral transcripts in astrocytes infected by pseudotyped HIV-1 were as abundant as the 2-kb mRNA during 4 weeks of follow-up, and both structural and regulatory viral proteins were detected in infected cells by immunoblotting or cell staining. The progeny virus released by these cells was infectious. These results indicate that the major barrier to HIV-1 infection of primary astrocytes is at virus entry and that astrocytes have no intrinsic intracellular restriction to efficient HIV-1 replication.
PMCID: PMC115036  PMID: 11483737
25.  Vif Is Largely Absent from Human Immunodeficiency Virus Type 1 Mature Virions and Associates Mainly with Viral Particles Containing Unprocessed Gag 
Journal of Virology  2001;75(12):5504-5517.
Vif is a human immunodeficiency virus type 1 (HIV-1) protein that is essential for the production of infectious virus. Most of Vif synthesized during HIV infection localizes within cells, and the extent of Vif packaging into virions and its function there remain controversial. Here we show that a small but detectable amount of Vif remains associated with purified virions even after their treatment with the protease subtilisin. However, treatment of these virions with 1% Triton X-100 revealed that most of the virion-associated Vif segregated with detergent-resistant virus particles consisting of unprocessed Gag, indicating that detergent-soluble, mature virions contain very little Vif. To investigate the control of Vif packaging in immature virus particles, we tested its association with Gag-containing virus-like particles (VLPs) in a Vif and Gag coexpression system in human cells. Only a small proportion of Vif molecules synthesized in this system became packaged into VLPs, and the VLP-associated Vif was protected from exogenous protease and detergent treatment, indicating that it is stably incorporated into immature virion-like cores. About 10-fold more Vpr than Vif was packaged into VLPs but most of the VLP-associated Vpr was removed by treatment with detergent. Mutagenesis of the C-terminal sequences in Gag previously shown to be responsible for interaction with Vif did not reduce the extent of Vif packaging into Gag VLPs. Surprisingly, short deletions in the capsid domain (CA) of Gag (amino acid residues 284 to 304 and 350 to 362) increased Vif packaging over 10-fold. The 350 to 363 deletion introduced into CA in HIV provirus also increased Vif incorporation into purified virions. Our results show that Vif can be packaged at low levels into aberrant virus particles or immature virions and that Vif is not present significantly in mature virions. Overall, these results indicate that the Vif content in virions is tightly regulated and also argue against a function of virion-associated Vif.
PMCID: PMC114263  PMID: 11356958

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