Expression of Human Immunodeficiency Virus Type 1 Gp120 from Herpes Simplex Virus Type 1-derived Amplicons Results in Potent, Specific, and Durable Cellular and Humoral Immune Responses

Journal of Virology. Jun, 2002  |  Pubmed ID: 11991985

Herpes simplex virus type 1 (HSV-1) infects a wide range of cells, including dendritic cells. Consequently, HSV-1 vectors may be capable of eliciting strong immune responses to vectored antigens. To test this hypothesis, an HSV-1 amplicon plasmid encoding human immunodeficiency virus type 1 gp120 was constructed, and murine immune responses to helper virus-free amplicon preparations derived from this construct were evaluated. Initial studies revealed that a single intramuscular (i.m.) injection of 10(6) infectious units (i.u.) of HSV:gp120 amplicon particles (HSV:gp120) elicited Env-specific cellular and humoral immune responses. A potent, CD8(+)-T-cell-mediated response to an H-2D(d)-restricted peptide from gp120 (RGPGRAFVTI) was measured by a gamma interferon ELISPOT and was confirmed by standard cytotoxic-T-lymphocyte assays. Immunoglobulin G enzyme-linked immunosorbent assay analysis showed the induction of a strong, Env-specific antibody response. An i.m. or an intradermal administration of HSV:gp120 at the tail base elicited a more potent cellular immune response than did an intraperitoneal (i.p.) inoculation, although an i.p. introduction generated a stronger humoral response. The immune response to HSV:gp120 was durable, with robust cellular and humoral responses persisting at 171 days after a single 10(6)-i.u. inoculation. The immune response to HSV:gp120 was also found to be dose dependent: as few as 10(4) i.u. elicited a strong T-cell response. Finally, HSV:gp120 elicited significant Env-specific cellular immune responses even in animals that had been previously infected with wild-type HSV-1. Taken together, these data strongly support the use of helper-free HSV-1 amplicon particles as vaccine delivery vectors.

Tumor Necrosis Factor-alpha in Normal and Diseased Brain: Conflicting Effects Via Intraneuronal Receptor Crosstalk?

Journal of Neurovirology. Dec, 2002  |  Pubmed ID: 12476354

Tumor necrosis factor-alpha (TNF-alpha) is pleiotropic mediator of a diverse array of physiological and neurological functions, including both normal regulatory functions and immune responses to infectious agents. Its role in the nervous system is prominent but paradoxical. Studies on uninflamed or "normal" brain have generally attributed TNF-alpha a neuromodulatory effect. In contrast, in inflamed or diseased brain, the abundance of evidence suggests that TNF-alpha has an overall neurotoxic effect, which may be particularly pronounced for virally mediated neurological disease. Still others have found TNF-alpha to be protective under some conditions of neurological insult. It is still uncertain exactly how TNF-alpha is able to induce these opposing effects through receptor activation of only a limited set of cell signaling pathways. In this paper, we provide support from the literature to advance our hypothesis that one mechanism by which TNF-alpha can exert its paradoxical effects in the brain is via crosstalk with signaling pathways of growth factors or other cytokines.

Engineered Fibronectin Type III Domain with a RGDWXE Sequence Binds with Enhanced Affinity and Specificity to Human Alphavbeta3 Integrin

Journal of Molecular Biology. Mar, 2003  |  Pubmed ID: 12595259

Fibronectin is an extracellular matrix protein with broad binding specificity to cell surface receptors, integrins. The tenth fibronectin type III domain (FNfn10) is a small, autonomous domain of fibronectin containing the RGE sequence that is directly involved in integrin binding. However, in isolation FNfn10 only weakly bind to integrins. We reasoned that high-affinity and high-specificity variants of FNfn10 to a particular integrin could be engineered by optimizing residues surrounding the integrin-binding RGD sequence in the flexible FG loop. Affinity maturation of FNfn10 to alphavbeta3 integrin, an integrin up-regulated in angiogenic endothelial cells and in some metastatic tumor cells, yielded alphavbeta3-binding FNfn10 mutants with a novel RGDWXE consensus sequence. We characterized one of the RGDWXE-modified clones, FNfn10-3JCLI4, as purified protein. FNfn10-3JCLI4 binds with high affinity and specificity to purified alphavbeta3 integrin. Alanine scanning mutagenesis suggested that both the tryptophan and glutamic acid residues following the RGD sequence are required for maximal affinity and specificity for alphavbeta3. FNfn10-3JCLI4 specifically stained alphavbeta3-positive cells as detected with flow cytometry and it inhibited alphavbeta3-dependent cell adhesion. As with the anti-alphavbeta3 antibody LM609, FNfn10-3JCLI4 can interfere with in vitro capillary formation. Taken together, these data show that FNfn10-3JCL14 is a specific, high-affinity alphavbeta3-binding protein that can inhibit alphavbeta3-dependent cellular processes similar to an anti-alphavbeta3 monoclonal antibody. These properties, combined with the small, monomeric, cysteine-free and highly stable structure of FNfn10-3JCLI4, may make this protein useful in future applications involving detection and targeting of alphavbeta3-positive cells.

Mechanistic Understanding of an Altered Fidelity Simian Immunodeficiency Virus Reverse Transcriptase Mutation, V148I, Identified in a Pig-tailed Macaque

The Journal of Biological Chemistry. Aug, 2003  |  Pubmed ID: 12740369

We have recently reported that the reverse transcriptase (RT) of SIVMNE 170 (170), which is a representative viral clone of the late symptomatic phase of infection with the parental strain, SIVMNE CL8 (CL8), has a largely increased fidelity, compared with the CL8 RT. In the present study, we analyzed the mechanistic alterations of the high fidelity 170 RT variant. First, we found that among several 170 RT mutations, only one, V148I, is solely responsible for the fidelity increase over the CL8 RT. This V148I mutation lies near the Gln-151 residue that we recently found is important to the low fidelity of RT and the binding of incoming dNTPs. Second, we compared dNTP binding affinity (Kd) and catalysis (kpol) of the CL8 RT and the CL8-V148I RT using pre-steady state kinetic analysis. In this experiment, the high fidelity CL8-V148I RT has largely decreased binding to both correct and incorrect dNTP without altering kpol. The fidelity increase imparted by the V148I mutation is likely because of the major reduction seen in RT binding to dNTPs. This parallels our findings with the Q151N mutant. Third, site-directed mutagenesis targeting amino acid residue 148 has revealed that a valine amino acid at this position is essential to RT infidelity. Based on these findings, we discuss possible structural impacts of residue 148 (and mutations at this site) on the interaction of RT with incoming dNTPs and infer how alterations in these properties may relate to viral replication and fitness.

Cellular Immune Responses to Helper-free HSV-1 Amplicon Particles Encoding HIV-1 Gp120 Are Enhanced by DNA Priming

Vaccine. Jun, 2003  |  Pubmed ID: 12744859

A single inoculation of herpes simplex virus type-1 (HSV-1) amplicon vectors encoding human immunodeficiency virus type-1 gp120 (HSV:gp120) results in robust, specific immune responses to gp120. To explore further the utility of this novel vaccine delivery system, we examined the kinetics of the cellular immune response by tetramer staining, following a single intramuscular administration of HSV:gp120 particles, and found that it peaks at 9-28 days post-immunization, before declining to a stable memory response. We also examined the utility of prime-boost regimens using packaged amplicon particles and naked amplicon plasmid DNA (DNA:gp120). These experiments showed that two sequential immunizations with HSV:gp120 resulted in a 5-10-fold increase in gp120-specific cellular immune responses and that plasmid DNA priming, followed by amplicon particle boosting, imparted the strongest acute and memory T cell responses, as determined by tetramer analysis. Collectively, these results demonstrate the utility of HSV amplicon vectors in prime-boost regimens for HIV vaccine development.

A Peptide Containing a Novel FPGN CD40-binding Sequence Enhances Adenoviral Infection of Murine and Human Dendritic Cells

European Journal of Biochemistry / FEBS. May, 2003  |  Pubmed ID: 12752448

CD40 is a receptor with numerous functions in the activation of antigen presenting cells (APCs), particularly dendritic cells (DC). Using phage display technology, we identified linear peptides containing a novel FPGN/S consensus sequence that enhances the binding of phage to a purified murine CD40-immunoglobulin (Ig) fusion protein (CD40-Ig), but not to Ig alone. To examine the ability the FPGN/S peptides to enhance adenoviral infection of CD40-positive cells, we used bifunctional peptides consisting of an FPGN-containing peptide covalently linked to an adenoviral knob-binding peptide (KBP). One of these, FPGN2-KBP, was able to enhance adenoviral infection of both murine and human DCs in a dose-dependent manner. FPGN2-KBP also improved infection of murine B cell blasts, a murine B lymphoma cell line (L10A), and immortalized human B cells. To demonstrate that enhancement of adenoviral infection depended on the presence of CD40, we analyzed infection of the breast cancer line, SKBR3, that does not express CD40 or the adenovirus cellular receptor, CAR. Infection of SKBR3 cells was enhanced by FPGN2-KBP following transient transfection with a plasmid vector that expresses murine CD40, but not when the cells were mock-transfected. In conclusion, we have isolated a peptide that binds to murine CD40, and promotes the uptake of adenoviruses into CD40-expressing cells of both murine and human origin, suggesting that it may have potential applications for antigen delivery to CD40-positive antigen-presenting cells.

Murine Cytomegalovirus Abortively Infects Human Dendritic Cells, Leading to Expression and Presentation of Virally Vectored Genes

Journal of Virology. Jul, 2003  |  Pubmed ID: 12805417

Dendritic cells (DC) are potent antigen-presenting cells that play a crucial role in antigen-specific immune responses. Thus, the targeting of exogenous antigens to DC has become a popular approach for cancer immunotherapy and vaccine development. In this report, we studied the interplay between murine cytomegalovirus (MCMV) and human monocyte-derived DC. The results showed that an enhanced green fluorescent protein (EGFP)-encoding, replication-competent MCMV vector underwent abortive infection in human DC; this was accompanied by the efficient expression of EGFP. Infection of human DC by this vector resulted in a modest increase in the expression of cell surface proteins associated with DC maturation and has no significant effect on the immunostimulatory function of the cells, as reflected by their ability to support T-cell proliferation in a mixed-lymphocyte reaction. Finally, an MCMV vector encoding the human immunodeficiency virus type 1 (HIV-1) gp120 envelope glycoprotein was constructed and used to infect cultured human DC. The infected DC were shown to be capable of stimulating the expansion of autologous, gp120-specific, class I-restricted T lymphocytes from an HIV-1-negative donor, as determined by tetramer staining and enzyme-linked immunospot analysis. Taken together, these results suggest that MCMV may have potential utility as a vector for human vaccine development.

Effects of Codon-optimization on Protein Expression by the Human Herpesvirus 6 and 7 U51 Open Reading Frame

Journal of Virological Methods. Aug, 2003  |  Pubmed ID: 12880930

Codon-optimization refers to the alteration of gene sequences, to make codon usage match the available tRNA pool within the cell/species of interest. Codon-optimization has emerged as a powerful tool to increase protein expression by genes from small RNA and DNA viruses, which commonly contain overlapping reading frames as well as structural elements that are embedded within coding regions; these features are not widespread among large DNA viruses. We therefore examined whether codon-optimization might influence protein expression from a herpesvirus gene. We focused on the U51 gene from human herpesviruses-6 and -7, which was cloned in both native and codon-optimized form, with an N-terminal HA epitope tag to allow protein detection. Codon-optimization was associated with a profound (10-100 fold) increase in U51 expression in human (293A, HSG, K562) or hamster (CHO) cell lines, suggesting this may represent a valuable tool to facilitate functional studies on recalcitrant herpesvirus genes. Finally, it is postulated that the suboptimal expression of native U51 may reflect a regulatory mechanism that controls viral gene expression.

Neuroprotective Activities of Sodium Valproate in a Murine Model of Human Immunodeficiency Virus-1 Encephalitis

The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. Oct, 2003  |  Pubmed ID: 14534250

Human immunodeficiency virus-1 (HIV-1) infection of the nervous system can result in neuroinflammatory events leading first to neuronal dysfunction then to cognitive and behavioral impairments in infected people. The multifaceted nature of the disease process, commonly called HIV-1-associated dementia (HAD), provides a number of adjunctive therapeutic opportunities. One proposed adjunctive therapy is sodium valproate (VPA), an anticonvulsant known to promote neurite outgrowth and increase beta-catenin through inhibiting glycogen synthase kinase 3beta activity and tau phosphorylation. We now show that VPA treatment of rat cortical neurons exposed to HIV-1 gp120 prevents resultant neurotoxic activities. This includes the induction of significant neurite outgrowth and microtubule-associated protein 2 (MAP-2) and neuron-specific nuclear protein (NeuN) antigens in affected neuronal cell bodies and processes. Similarly, VPA protects severe combined immunodeficient (SCID) mice against the neurodegeneration of HIV-1ADA infected monocyte-derived macrophages (MDMs). In SCID mice with HIV-1 MDM-induced encephalitis, VPA treatment significantly reduced neuronal phosphorylatedbeta-catenin and tau without affecting HIV-1 replication or glial activation. We conclude that VPA protects neurons against HIV-1 infected MDM neurotoxicity, possibly through its effects on the phosphorylation of tau and beta-catenin. The use of VPA as an adjuvant in treatment of human HAD is being pursued.

Immunohistochemical Assessment of Fractalkine, Inflammatory Cells, and Human Herpesvirus 7 in Human Salivary Glands

The Journal of Histochemistry and Cytochemistry : Official Journal of the Histochemistry Society. May, 2004  |  Pubmed ID: 15100244

Human fractalkine (CX3CL1), a delta-chemokine, is implicated in the mediation of multiple cell functions. In addition to serving as a chemotactic factor for mononuclear cell subtypes, membrane-bound fractalkine may promote viral infection by interacting with virions that encode putative fractalkine-binding proteins. Fractalkine expression in normal epithelial tissues studied to date is either constitutive or is upregulated with inflammation. In salivary glands, the expression of fractalkine is unknown. Moreover, salivary glands are a major site for the persistent and productive infection by human herpesvirus (HHV)-7, which encodes two putative fractalkine-binding gene products, U12 and U51. Surprisingly, the cellular distribution of HHV-7 in major salivary glands has not been explored. We therefore determined by immunohistochemistry the cellular localization of fractalkine in three different salivary glands: parotid, submandibular, and labial glands. Fractalkine expression was highly variable, ranging from high to undetectable levels. We further examined the association of fractalkine with inflammatory cell infiltration or HHV-7 infection of salivary epithelial cells. Inflammatory cells were always adjacent to epithelial cells expressing fractalkine, consistent with a function of fractalkine in inflammatory cell recruitment and/or retention in salivary glands. In contrast, HHV-7-infected epithelial cells did not always express fractalkine, suggesting that fractalkine may not be an absolute requirement for viral entry.

Transient Overexpression of Kappa and Mu Opioid Receptors Using Recombinant Adenovirus Vectors

Journal of Neuroscience Methods. Jul, 2004  |  Pubmed ID: 15183265

In order to study the trafficking and signal transduction mechanisms of the multiple opioid receptors, these receptors are expressed either transiently or stably in cell lines. Often, it is difficult to express receptors at a sufficiently high density to obtain reproducible results. To achieve a high density of receptors, replication-defective adenovirus (rAd5) vectors encoding the mu (MOR) and kappa (KOR) opioid receptors, both in their native form and as fusion proteins bearing the green fluorescent protein (GFP) at their C-terminus, were constructed. These vectors efficiently and reproducibly infected Chinese hamster ovary (CHO) cells that stably express the human coxsackie-adenovirus receptor (hCAR), with up to 90% of cells becoming infected at a low multiplicity of infection (MOI). Saturation receptor binding studies using mu- and kappa-selective agonists, [3H][D-Ala2, N-Me-Phe4, Gly5-ol]enkephalin (DAMGO) and [3H](5alpha7alpha,8beta)-(-)-N-methyl-N-(7-(1-pyrrolidinyl)-1-oxaspiro(4,5)dec-8-yl)benzeneacetamide (U69,593), respectively, and a nonselective antagonist, [3H]diprenorphine, revealed that rAd5-transduced cells expressed MOR and KOR for at least 3 days, at levels which exceeded those present on widely-used CHO sublines that stably express MOR or KOR. Expression levels were highest for the vectors encoding native MOR or KOR, and slightly reduced for the GFP fusion proteins. These findings demonstrate the feasibility of using rAd5 vectors to express opioid receptors at high densities, which may facilitate opioid receptor studies.

Activation of Adenosine A2A Receptor Protects Sympathetic Neurons Against Nerve Growth Factor Withdrawal

Journal of Neuroscience Research. Jul, 2004  |  Pubmed ID: 15211592

Adenosine mediates a range of effects in the central nervous system (CNS), including the promotion of neuronal survival, but its actions on sympathetic neurons are less well characterized. We therefore sought to understand the role of endogenous adenosine in contributing to the survival of neurotrophin-dependent sympathetic neurons. Rat superior cervical ganglion (SCG) cultures were maintained in the continuous presence of nerve growth factor (NGF) and then exposed to adenosine deaminase (ADA), to deplete endogenous adenosine. This resulted in a marked increase in cellular apoptosis, to a level that approximated the effect of NGF withdrawal. Furthermore, the addition of exogenous adenosine to NGF-deprived SCG neurons resulted in enhanced cell survival. Analysis of adenosine receptor (AR) subtypes on these neurons, using real-time RT-PCR and receptor binding analyses, revealed that the A2A receptor was the major subtype present. Accordingly, the A2A receptor agonist CGS21680 significantly reduced both ADA-induced and NGF-withdrawal-induced neuronal apoptosis, whereas the A1 receptor agonist R-PIA had no such effect. The survival-promoting effect of CGS21680 was eliminated when cells were coincubated with a molar excess of an A2A receptor antagonist. Finally, follow-up experiments revealed that CGS21680 prevented the induction of early apoptotic events, such as changes in mitochondrial integrity and caspase activation, and that it also triggered an increase in ERK activation, which was essential for neurotrophin-independent cell survival. Taken together, these findings provide evidence that endogenous adenosine may be important in mediating protection of sympathetic neurons and that it may act via the A2A receptor subtype.

Human Herpesvirus Type 6 and Human Herpesvirus Type 7 Infections of the Central Nervous System

Herpes : the Journal of the IHMF. Jun, 2004  |  Pubmed ID: 15319097

In developing guidelines for the improved management of herpesvirus infections of the central nervous system (CNS), the International Herpes Management Forum (IHMF) has considered human herpesvirus (HHV) type 6 and type 7 disease. Although HHV-6 is generally asymptomatic, it has been associated with exanthema subitum, febrile convulsions and encephalitis in infants and immunocompromised adults and may play a role in multiple sclerosis, Guillain-Barre syndrome and acute disseminated encephalomyelitis. As HHV-6 is present in the brain tissue of healthy individuals, its role as an aetiological agent in CNS disorders is unclear. While polymerase chain reaction (PCR) is a method useful for diagnosis of other viral CNS infections, it has no value for diagnosing HHV-6. HHV-7 has not been shown to cause a specific disease but is associated with febrile convulsions and has been implicated as a cause of encephalitis. Ganciclovir and foscarnet, either alone or in combination, may be used for the management of HHV-6-related neurological disease. Although ganciclovir is unlikely to be effective against HHV-7-related CNS disease, foscarnet may be useful but prospective trials are needed.

Macrophage Tropism of HIV-1 Depends on Efficient Cellular DNTP Utilization by Reverse Transcriptase

The Journal of Biological Chemistry. Dec, 2004  |  Pubmed ID: 15452123

Retroviruses utilize cellular dNTPs to perform proviral DNA synthesis in infected host cells. Unlike oncoretroviruses, which replicate in dividing cells, lentiviruses, such as human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus, are capable of efficiently replicating in non-dividing cells (terminally differentiated macrophages) as well as dividing cells (i.e. activated CD4+ T cells). In general, non-dividing cells are likely to have low cellular dNTP content compared with dividing cells. Here, by employing a novel assay for cellular dNTP content, we determined the dNTP concentrations in two HIV-1 target cells, macrophages and activated CD4+ T cells. We found that human macrophages contained 130-250-fold lower dNTP concentrations than activated human CD4+ T cells. Biochemical analysis revealed that, unlike oncoretroviral reverse transcriptases (RTs), lentiviral RTs efficiently synthesize DNA even in the presence of the low dNTP concentrations equivalent to those found in macrophages. In keeping with this observation, HIV-1 vectors containing mutant HIV-1 RTs, which kinetically mimic oncoretroviral RTs, failed to transduce human macrophages despite retaining normal infectivity for activated CD4+ T cells and other dividing cells. These results suggest that the ability of HIV-1 to infect macrophages, which is essential to establishing the early pathogenesis of HIV-1 infection, depends, at least in part, on enzymatic adaptation of HIV-1 RT to efficiently catalyze DNA synthesis in limited cellular dNTP substrate environments.

Congenital Infections with Human Herpesvirus 6 (HHV6) and Human Herpesvirus 7 (HHV7)

The Journal of Pediatrics. Oct, 2004  |  Pubmed ID: 15480369

To examine whether: (1) congenital human herpesvirus 6 (HHV6) and human herpesvirus 7 (HHV7) infections occur; whether (2) their manifestations differ from postnatal infections; and whether (3) HHV6 and HHV7 infections differ despite their close relatedness.

Human Herpesvirus 6 (HHV6) DNA Persistence and Reactivation in Healthy Children

The Journal of Pediatrics. Oct, 2004  |  Pubmed ID: 15480370

To determine in healthy children after primary infection the persistence of human herpesvirus 6 (HHV6) DNA, the presence and frequency of HHV6 re-activation or re-infection, and the relationship of both to illness and the presence of human herpesvirus 7 (HHV7) infection.

False Memories in Children. Evidence for a Shift from Phonological to Semantic Associations

Psychological Science. Nov, 2004  |  Pubmed ID: 15482451

Memory illusions in children of three age groups (5-, 8-, and 11-year-olds) were investigated using a modified version of the Deese-Roediger-McDermott (DRM) procedure. Children from each age group falsely recalled nonpresented items related to the study lists. However, the nature of the intrusions varied across the different groups. The 5-year-olds were more likely to falsely recall words that rhymed with the studied items than to recall unrelated items, whereas the 11-year-olds were more likely to falsely recall words that were semantically related to the list theme than to produce either rhyming or unrelated intrusions. Intrusions made by the 8-year-olds were equally likely to be semantic, rhyming, or unrelated to study items. The results are consistent with the notion of a developmental shift from phonological to semantic associations, leading to qualitatively different memory distortions in children of different ages.

Dishevelled Promotes Neurite Outgrowth in Neuronal Differentiating Neuroblastoma 2A Cells, Via a DIX-domain Dependent Pathway

Brain Research. Molecular Brain Research. Dec, 2004  |  Pubmed ID: 15548427

Dishevelled (Dvl) is a cytoplasmic protein involved in the Wnt-Frizzled signaling cascade, which has also been shown to interact with the cytoskeleton in part through inhibition of glycogen synthase kinase 3beta (GSK3beta). Using mouse neuroblastoma 2A (N2A) cells as a model system, we have found that overexpression of Dvl promotes the outgrowth of neurite-like processes, and leads to the induction of a striking, bipolar morphologic phenotype during neuronal differentiation. In contrast, suppression of Dvl expression using isoform-specific siRNAs led to an inhibition of neurite outgrowth in these cells. In order to further elucidate the mechanism(s) responsible for this effect, we overexpressed several mutant forms of Dvl in the N2A cells, including deletions in each of the three major functional subdomains of the protein (DeltaDIX, DeltaPDZ, DeltaDEP) and point mutations in the two well-defined interaction motifs within the DIX domain (the actin-binding and vesicle-association elements; K58A and K68A/E69A, respectively). These experiments revealed that the DIX domain (and its vesicle-binding subregion) was essential for Dvl's effect on neurite extension and morphogenesis in N2A cells. In contrast, direct overexpression of a degradation-resistant form of beta-catenin (S37A), or a dominant negative GSK3beta mutant (K85R), had no effect on neurite outgrowth or morphology in neuronally differentiating N2A cells; exposure of cells to a pharmacologic inhibitor of GSK3beta (lithium) also had no effect. Taken together, these results suggest that Dvl induces cytoskeletal and morphologic rearrangements in neuronal differentiating N2A cells through a mechanism that cannot be attributed exclusively to modulation of GSK3beta or beta-catenin activity, but which does depend upon a DIX-domain/vesicle-association-dependent signaling pathway.

Inhibition of HIV Type 1 Replication Using Lentiviral-mediated Delivery of Mutant TRNA(Lys3)A58U

AIDS Research and Human Retroviruses. Dec, 2004  |  Pubmed ID: 15650425

In previous studies, we showed that residue A58 of cellular tRNALys3 is necessary for appropriate termination of viral plus-strand strong-stop DNA (+SS DNA), and therefore plays a critical role in the life cycle of HIV-1. We also performed proof-of-principle studies that established that a mutant form of this tRNA primer (tRNA(Lys3)A58U, which lacks the M1A58 residue necessary for +SS DNA termination) could inhibit HIV-1 replication. In the present work, we examined whether a third generation lentiviral vector (SIN) could be used to deliver tRNA(Lys3)A58U to CEM cells. Using both viral kinetic studies and limiting dilution assays (LDA), we observed significant impairment of HIV-1 replication, up to 3 logs in the LDA, in CEM sublines expressing mutant tRNA(Lys3)A58U. No inhibition occurred in cells that either expressed wild-type tRNA(Lys3) or were transduced with empty SIN vector. Further, we observed impairment of viral replication using primary isolates of both HIV-1 and HIV-2 in sublines containing tRNA(Lys3)A58U. We also detected "breakthrough" HIV-1 replication in some tRNA(Lys3)A58U-expressing cultures. Interestingly, analyzed breakthrough viruses appeared to be both genetically and phenotypically wild type. One possible explanation for virological breakthrough is that it reflects the gradual accumulation of HIV-1 within the infected cell culture, to a level that ultimately exceeds the containment "threshold" conferred by tRNA(Lys3)A58U. The fact that HIV-1 does not appear to acquire heritable resistance to tRNA(Lys3)A58U-mediated blockade differentiates this antiviral modality from other therapeutic interventions. It also suggests that tRNA-mediated inhibition of viral replication might be a valuable adjunct to other antiviral approaches.

Intention to Learn Influences the Word Frequency Effect in Recall but Not in Recognition Memory

Memory & Cognition. Dec, 2004  |  Pubmed ID: 15900925

Watkins, LeCompte, and Kim (2000) suggested that the recall advantage for rare words in mixed lists is due to a compensatory study strategy that favors the rare words. They found the advantage was reversed when rare and common words were studied under incidental learning conditions. The present study investigated the possibility that the rare-word advantage in recognition memory is also the result of a compensatory study strategy. Experiment 1 replicated the findings of Watkins et al. that the rare-word advantage in recall is eliminated under incidental learning conditions. In contrast, Experiment 2 showed that the rare-word advantage in recognition memory is maintained under both intentional and incidental learning conditions. Experiment 3 replicated the results of Experiments 1 and 2 using different stimuli and a different orienting task. Finally, Experiment 4 showed that the rare-word advantage in recognition is maintained with pure lists. These findings show that the rare-word advantage in recognition memory is not the result of a compensatory study strategy. Instead, rare words are encoded more distinctively than common words, irrespective of participants' intention to remember them.

The Human Herpesvirus 6 G Protein-coupled Receptor Homolog U51 Positively Regulates Virus Replication and Enhances Cell-cell Fusion in Vitro

Journal of Virology. Sep, 2005  |  Pubmed ID: 16140767

Human herpesvirus 6 (HHV-6) is a ubiquitous T-lymphotropic betaherpesvirus that encodes two G protein-coupled receptor homologs, U12 and U51. HHV-6A U51 has been reported to bind to CC chemokines including RANTES, but the biological function of U51 remains uncertain. In this report, we stably expressed short interfering RNAs (siRNAs) specific for U51 in human T cells and then infected these cells with HHV-6. Viral DNA replication was reduced 50-fold by the U51 siRNA, and virally induced cytopathic effects were also inhibited. In contrast, viral replication and syncytium formation were unaltered in cells that expressed a scrambled derivative of the siRNA or an irrelevant siRNA and were restored to normal when a human codon-optimized derivative of U51 was introduced into cells containing the U51 siRNA. To examine the mechanism whereby U51 might contribute to viral replication, we explored the signaling characteristics of U51. None of the chemokines and opioids tested was able to induce G protein coupling by U51, and no evidence for opioid ligand binding by U51 was obtained. The effect of U51 on cell-cell fusion was also evaluated; these studies showed that U51 enhanced cell fusion mediated by the G protein of vesicular stomatitis virus. However, a U51-specific antiserum had no virus-neutralizing activity, suggesting that U51 may not be involved in the initial interaction between the virus particle and host cell. Overall, these studies suggest that HHV-6 U51 is a positive regulator of virus replication in vitro, perhaps because it may promote membrane fusion and facilitates cell-cell spread of this highly cell-associated virus.

Neuroprotective Mechanisms of Lithium in Murine Human Immunodeficiency Virus-1 Encephalitis

The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. Sep, 2005  |  Pubmed ID: 16162919

Lithium (Li) has garnered considerable interest as a neuroprotective drug for a broad range of nervous system disorders. Its neuroprotective activities occur as a consequence of glycogen synthase kinase-3beta (GSK-3beta) inhibition leading to downstream blockade of beta-catenin and Tau phosphorylation. In the present study, we investigated Li-mediated neuroprotective mechanisms in laboratory and murine human immunodeficiency virus-1 (HIV-1) encephalitis (HIVE) models. In laboratory tests, Li protected neurons from neurotoxic secretions of HIV-1-infected monocyte-derived macrophages (MDMs). This neuroprotection was mediated, in part, through the phosphatidyl inositol 3-kinase/Akt and GSK-3beta pathways. To examine the effects of Li treatment in vivo, MDMs were injected into the basal ganglia of severe combined immunodeficient mice and then Li was administered (60 mg/kg/d). Seven days after MDM injection, mice were killed and CNS tissue was collected and subjected to immunocytochemical and Western blot assays for leukocyte and neural antigens, GSK-3beta, and key kinase substrates such as beta-catenin and Tau. Numbers of HIV-1 p24 antigen-positive MDMs were unaltered by Li treatment of HIVE mice. Similarly, the greatly increased extent of astrocyte and microglia activation in HIVE mice (10-fold and 16-fold, respectively, compared with unmanipulated controls) was also unaltered by Li. In contrast, Li restored HIVE-associated loss of microtubule-associated protein-2-positive neurites and synaptic density while reducing levels or activity of phospho-Tau Ser202, phospho-beta-catenin, and GSK-3beta. Electrophysiological recordings showed diminished long-term potentiation in hippocampal slices of HIVE mice that were restored by Li. Based on these data, the use of Li as an adjuvant for HIV-1-associated dementia is now being pursued.

A Simple Method for Displaying Recalcitrant Proteins on the Surface of Bacteriophage Lambda

Nucleic Acids Research. , 2005  |  Pubmed ID: 16224099

Bacteriophage lambda (lambda) permits the display of many foreign peptides and proteins on the gpD major coat protein. However, some recombinant derivatives of gpD are incompatible with the assembly of stable phage particles. This presents a limitation to current lambda display systems. Here we describe a novel, plasmid-based expression system in which gpD deficient lambda lysogens can be co-complemented with both wild-type and recombinant forms of gpD. This dual expression system permits the generation of mosaic phage particles that contain otherwise recalcitrant recombinant gpD fusion proteins. Overall, this improved gpD display system is expected to permit the expression of a wide variety of peptides and proteins on the surface of bacteriophage lambda and to facilitate the use of modified lambda phage vectors in mammalian gene transfer applications.

Selective Interference with the Use of Visual Images in the Symbolic Distance Paradigm

Journal of Experimental Psychology. Learning, Memory, and Cognition. Sep, 2005  |  Pubmed ID: 16248750

Eight experiments investigated the effects of visual, spatial, auditory, and executive interference on the symbolic comparison of animal size and ferocity, semantic goodness of words, and numbers. Dynamic visual noise (DVN) and the reading of visually presented stimulus items were shown to selectively interfere with response times on the animal size comparison task, though the slope of the symbolic distance function remained unchanged. Increased change of DVN significantly increased interference, but interference was reduced by equiluminant DVN. Spatial tracking reduced the slope of the symbolic distance function in contrast to an executive task that only increased mean latency and errors for all comparisons. Results suggest that the generation of an image is necessary for size comparison, but neither imagery nor executive function is responsible for the frequently observed distance-time function.

Human Dendritic Cells Transduced with Herpes Simplex Virus Amplicons Encoding Human Immunodeficiency Virus Type 1 (HIV-1) Gp120 Elicit Adaptive Immune Responses from Human Cells Engrafted into NOD/SCID Mice and Confer Partial Protection Against HIV-1 Challenge

Journal of Virology. Feb, 2005  |  Pubmed ID: 15681415

Small-animal models are needed to test human immunodeficiency virus (HIV) vaccine efficacy following viral challenge. To this end, we examined HIV-1-specific immune responses following immunization of nonobese diabetic-severe combined immunodeficient mice that were repopulated with human peripheral blood lymphocytes (hu-PBL-NOD/SCID mice). Autologous dendritic cells (DC) were transduced ex vivo with replication-defective, helper virus-free, herpes simplex virus type 1 (HSV-1) amplicons that expressed HIV-1 gp120 and were then injected into the hu-PBL-NOD/SCID mice. This resulted in primary HIV-1-specific humoral and cellular immune responses. Serum samples from vaccinated animals contained human immunoglobulin G that reacted with HIV-1 Env proteins by enzyme-linked immunosorbent assay and neutralized the infectivity of HIV-1 LAI and ADA strains. T cells isolated from the mice responded to viral antigens by producing gamma interferon when analyzed by enzyme-linked immunospot assay. Importantly, exposure of the vaccinated animals to infectious HIV-1 demonstrated partial protection against infectious HIV-1 challenge. This was reflected by a reduction in HIV-1(ADA) and by protection of the engrafted human CD4(+) T lymphocytes against HIV-1(LAI)-induced cytotoxicity. These data demonstrate that transduction of DC by HSV amplicon vectors expressing HIV-1 gp120 induce virus-specific immune responses in hu-PBL-NOD/SCID mice. This mouse model may be a useful tool to evaluate human immune responses and protection against viral infection following vaccination.

Valproic Acid Enhances Gene Expression from Viral Gene Transfer Vectors

Journal of Virological Methods. Apr, 2005  |  Pubmed ID: 15737413

Viral vectors represent an efficient delivery method for in vitro and in vivo gene transfer, and their utility may be further enhanced through the use of pharmacologic agents that increase gene expression. Here, we demonstrate that valproic acid (VPA), a drug which is widely used for the treatment of epilepsy and mood disorders, enhances and prolongs expression of exogenous genes in cells transduced with various gene transfer agents, including adenovirus, adeno-associated virus and herpesvirus vectors. This effect occurs in a wide range of cell types, including both primary cells and cell lines, and appears to be associated with VPA's ability to function as a histone deacetylase inhibitor (HDACi). VPA treatment also enhanced adenovirally-vectored expression of a luciferase reporter gene in mice, as demonstrated by in vivo imaging. VPA was also less cytotoxic than a commonly used HDAC inhibitor, TSA, suggesting its use as a safer alternative. Taken together, these results suggest that VPA treatment may represent a useful approach to various gene transfer approaches in which enhanced transgene expression is desirable.

Opposite Effects of Lithium and Valproic Acid on Trophic Factor Deprivation-induced Glycogen Synthase Kinase-3 Activation, C-Jun Expression and Neuronal Cell Death

Neuropharmacology. Mar, 2005  |  Pubmed ID: 15755485

Recent studies demonstrate that lithium and valproic acid (VPA), two commonly used mood-stabilizing drugs, have neuroprotective effects against a variety of insults. Inhibition of the proapoptotic enzyme, glycogen synthase kinase-3 (GSK-3), has been suggested to be the mechanism of action of neuroprotection for both drugs. In this study, we tested if lithium and VPA could protect cultured cerebellar granule neurons (CGNs) from GSK-3-mediated apoptosis induced by trophic factor withdrawal (serum/potassium deprivation). Both lithium and indirubin, a specific GSK-3 inhibitor, protected CGNs in a dose-dependent manner. In contrast, VPA did not provide any neuroprotection and even potentiated cell death. Immunoblot analysis revealed that lithium inhibited the trophic factor deprivation-induced activation of GSK-3 as well as the in vivo phosphorylation of the microtubule-associated protein Tau on Ser199, a specific target site for GSK-3. Under these same experimental conditions, however, VPA neither inhibited GSK-3 activation nor hindered GSK-3 mediated Tau phosphorylation. Furthermore, in accordance with their effects on neuronal survival, lithium prevented the induction of c-Jun expression in trophic factor-deprived CGNs, whereas VPA potentiated it. Collectively, these results show that VPA is not a universal inhibitor of neuronal GSK-3, and that instead of being neuroprotective, VPA can even exacerbate neuronal death under some conditions.

HIV-1 Transactivator of Transcription Protein Induces Mitochondrial Hyperpolarization and Synaptic Stress Leading to Apoptosis

Journal of Immunology (Baltimore, Md. : 1950). Apr, 2005  |  Pubmed ID: 15778398

Despite the efficacy of highly active antiretroviral therapy in reducing viral burden, neurologic disease associated with HIV-1 infection of the CNS has not decreased in prevalence. HIV-1 does not induce disease by direct infection of neurons, although extensive data suggest that intra-CNS viral burden correlates with both the severity of virally induced neurologic disease, and with the generation of neurotoxic metabolites. Many of these molecules are capable of inducing neuronal apoptosis in vitro, but neuronal apoptosis in vivo does not correlate with CNS dysfunction, thus prompting us to investigate cellular and synaptic events occurring before cell death that may contribute to HIV-1-associated neurologic disease. We now report that the HIV-1 regulatory protein transactivator of transcription protein (Tat) increased oxidative stress, ATP levels, and mitochondrial membrane potential in primary rodent cortical neurons. Additionally, a proinflammatory cellular metabolite up-regulated by Tat, platelet-activating factor, also induced oxidative stress and mitochondrial hyperpolarization in neurons, suggesting that this type of metabolic dysfunction may occur on a chronic basis during HIV-1 infection of the CNS. Tat-induced mitochondrial hyperpolarization could be blocked with a low dose of the protonophore FCCP, or the mitochondrial KATP channel antagonist, tolbutamide. Importantly, blocking the mitochondrial hyperpolarization attenuated Tat-induced neuronal apoptosis, suggesting that increased mitochondrial membrane potential may be a causal event in precipitating neuronal apoptosis in cell culture. Finally, Tat and platelet-activating factor also increased neuronal vesicular release, which may be related to increased mitochondrial bioenergetics and serve as a biomarker for early damage to neurons.

Distinctiveness, Typicality, and Recollective Experience in Face Recognition: a Principal Components Analysis

Psychonomic Bulletin & Review. Dec, 2005  |  Pubmed ID: 16615324

In this study, participants rated previously unseen faces on six dimensions: familiarity, distinctiveness, attractiveness, memorability, typicality, and resemblance to a familiar person. The faces were then presented again in a recognition test in which participants assigned their positive recognition decisions to either remember (R), know (K), or guess categories. On all dimensions except typicality, faces that were categorized as R responses were associated with significantly higher ratings than were faces categorized as K responses. Study ratings for R and K responses were then subjected to a principal components analysis. The factor loadings suggested that R responses were influenced primarily by the distinctiveness of faces, but K responses were influenced by moderate ratings on all six dimensions. These findings indicate that the structural features of a face influence the subjective experience of recognition.

Inhibition of Mixed Lineage Kinase 3 Prevents HIV-1 Tat-mediated Neurotoxicity and Monocyte Activation

Journal of Immunology (Baltimore, Md. : 1950). Jul, 2006  |  Pubmed ID: 16785569

The HIV-1 gene products Tat and gp120 are toxic to neurons and can activate cells of myeloid origin, properties that are thought to contribute to the clinical manifestations of HIV-1-associated dementia (HAD). To investigate the intracellular signaling mechanisms involved in these events, the effect of Tat and gp120 on mixed lineage kinase (MLK) 3 activation was examined. Tat and gp120 were shown to induce autophosphorylation of MLK3 in primary rat neurons; this was abolished by the addition of an inhibitor of MLK3 (CEP1347). CEP1347 also enhanced survival of both rat and human neurons and inhibited the activation of human monocytes after exposure to Tat and gp120. Furthermore, overexpression of wild-type MLK3 led to the induction of neuronal death, whereas expression of a dominant negative MLK3 mutant protected neurons from the toxic effects of Tat. MLK3-dependent downstream signaling events were implicated in the neuroprotective and monocyte-deactivating pathways triggered by CEP1347. Thus, the inhibition of p38 MAPK and JNK protected neurons from Tat-induced apoptosis, whereas the inhibition of p38 MAPK, but not of JNK, was sufficient to prevent Tat- and gp120-mediated activation of monocytes. These results suggest that the normal function of MLK3 is compromised by HIV-1 neurotoxins (Tat, gp120), resulting in the activation of downstream signaling events that result in neuronal death and monocyte activation (with release of inflammatory cytokines). In aggregate, our data define MLK3 as a promising therapeutic target for intervention in HAD.

Amplicons As Vaccine Vectors

Current Gene Therapy. Jun, 2006  |  Pubmed ID: 16787189

HSV-1 amplicon vectors efficiently transduce cultured antigen-presenting cells (APC), including both human and murine dendritic cells as well as primary human chronic lymphocytic leukemia (CLL) B cells. Helper-free amplicons have been shown to be especially well-suited for this purpose, since they do not impair the antigen-presenting functions of these target cells. In vivo, amplicon vectors have been used in preclinical studies aimed at the development of therapeutic cancer vaccines, as well as vaccines for Alzheimer's disease, and selected microbial pathogens. Studies in small animal model systems have shown that ex vivo transduction of irradiated tumor cells with amplicon vectors encoding immunomodulatory cytokines such as IL-2 or GM-CSF can elicit protective responses against a tumor challenge. In an experimental model for cancer immunotherapy, direct transduction of preformed tumors with vectors encoding CD40L resulted in slowed tumor growth or tumor eradication. Other studies have examined the ability of amplicons to elicit immune responses against encoded antigens, and have shown that strong cellular immune responses can be generated against amplicon encoded HIV-1 antigens in mice. Thus, amplicon vectors have shown significant promise as vaccine vectors in a range of settings. These promising initial findings highlight the need to perform additional studies, including experiments to evaluate the immunogenicity of amplicon vectors in additional animal models, possibly including nonhuman primates. Overall, amplicon vectors offer compelling advantages when compared to other vaccine-delivery platforms, which include the capacity to incorporate a very large transgene payload and the potential to efficiently transduce mucosal surfaces. It will be important to design future studies to directly test and exploit these features of the amplicon system. The next few years therefore promise to be an exciting and important period in the development of amplicons as vaccine vectors.

Human Immunodeficiency Virus-encoded Tat Activates Glycogen Synthase Kinase-3beta to Antagonize Nuclear Factor-kappaB Survival Pathway in Neurons

The European Journal of Neuroscience. May, 2006  |  Pubmed ID: 16817865

The pathogenesis of human immunodeficiency virus type 1 (HIV-1)-associated dementia is mediated by neuronal dysfunction and death, brought about by the action of soluble neurotoxic factors that are released by virally infected macrophages and microglia. Paradoxically, many candidate HIV-1 neurotoxins also possess the ability to activate nuclear factor-kappa B (NF-kappaB), which has a potent pro-survival effect in primary neurons. The present study explored this conundrum and investigated why NF-kappaB might fail to protect neurons that are exposed to candidate HIV-1 neurotoxins. Here, we evaluated the ability of virus-depleted conditioned medium produced by HIV-1-infected human macrophages (HIV-MCMs) to modulate NF-kappaB activity in neurons. We demonstrated that HIV-MCMs inhibit the normal signaling pathways that lead to NF-kappaB activation in neurons. This inhibitory effect of HIV-MCM is dependent upon the presence of HIV-1 Tat, which activates glycogen synthase kinase (GSK)-3beta in neurons. Activation of GSK-3beta, in turn, results in modification of the NF-kappaB subunit RelA at serine 468, thereby regulating the physical interaction of RelA with histone deacetylase-3 corepressor molecules. Furthermore, neutralization of Tat or inhibition of GSK-3beta activity prevents neuronal apoptosis induced by HIV-MCM. We conclude that HIV-1 Tat may compromise neuronal function and fate by interfering with normal survival pathways subserved by NF-kappaB. These findings may have important therapeutic implications for the management of HIV-1-associated dementia.

Dissociating Word Frequency and Age of Acquisition: The Klein Effect Revived (and Reversed)

Journal of Experimental Psychology. Learning, Memory, and Cognition. Jul, 2006  |  Pubmed ID: 16822157

The Klein effect (G. S. Klein, 1964) refers to the finding that high-frequency words produce greater interference in a color-naming task than low-frequency words. The present study used the Klein effect to investigate the relationship between frequency and age of acquisition (AoA) by measuring their influence on color naming. Two experiments showed reliable effects of frequency (though in the opposite direction to that reported by Klein) but no effects of AoA. Experiment 1 produced a dissociation between frequency and AoA when manipulated orthogonally. Experiment 2 produced the same dissociation using different stimuli. In contrast, both variables reliably influenced word naming. These findings are inconsistent with the view that frequency and AoA are 2 aspects of a single underlying mechanism.

Characteristics and Acquisition of Human Herpesvirus (HHV) 7 Infections in Relation to Infection with HHV-6

The Journal of Infectious Diseases. Apr, 2006  |  Pubmed ID: 16544246

Although both human herpesvirus (HHV) 6 and HHV-7 infections are ubiquitous during childhood, few acute HHV-7 infections are identified. It is unknown whether HHV-7 viremia indicates primary infection, as with HHV-6, or reactivation, and if these differ clinically. We studied, in otherwise healthy children < or =10 years old, HHV-7 and HHV-6 infections and their interaction by serologic assessment, viral isolation, and polymerase chain reaction. In children < or =24 months of age, HHV-7 infections occurred less often than HHV-6 infections (P< or =.002). Of 2806 samples from 2365 children < or =10 years old, 30 (1%) showed evidence of HHV-7 viremia; 23 (77%) of these were primary and 7 (23%) were reactivated HHV-7 infections. Four (13%) showed concurrent HHV-6 viremia, 2 associated with primary HHV-7 infections. The clinical manifestations of primary and reactivated HHV-7 infections were similar, except that seizures occurred more frequently in reactivated infections. These findings, previously unrecognized in otherwise healthy children, suggest that HHV-7 viremia could represent primary or reactivated infection and may be affected by the interaction between HHV-6 and HHV-7.

HIV-1-based Defective Lentiviral Vectors Efficiently Transduce Human Monocytes-derived Macrophages and Suppress Replication of Wild-type HIV-1

The Journal of Gene Medicine. Jan, 2006  |  Pubmed ID: 16142830

Human monocytes play an important role in mediating human immunodeficiency virus type 1 (HIV-1) infection of the central nervous system (CNS), and monocytes-derived macrophages (MDM) represent a major viral reservoir within the brain and other target organs. Current gene transduction of MDM is hindered by a limited efficiency. In this study we established a lentiviral vector-based technique for improved gene transfer into human MDM cultures in vitro and demonstrated significant protection of transduced MDM from super-infection with wild-type HIV-1.

Recombinant Adenovirus Type 5 Vectors That Target DC-SIGN, ChemR23 and Alpha(v)beta3 Integrin Efficiently Transduce Human Dendritic Cells and Enhance Presentation of Vectored Antigens

Vaccine. Jan, 2006  |  Pubmed ID: 16154247

Recombinant adenoviruses (rAds) represent a promising system for vaccine delivery but transduce dendritic cells (DC) relatively poorly. To address this concern, we used a biotin-avidin linkage to conjugate rAd vectors to ligands which bind with high affinity to selected receptors on DC (ChemR23, alpha(v)beta3 integrin, and DC-SIGN). The targeted vectors had an enhanced ability to transduce human monocyte-derived DC compared to untargeted virus. In addition, DC transduced with targeted rAd vectors were more efficient at stimulating cytokine production by autologous memory CD8+ T cells, against a vector-encoded antigen. These results expand the range of cell surface receptors that can be used to target rAd5 vectors to DC, and may facilitate future development of rAd-based vaccines.

Measuring the Speed of the Conscious Components of Recognition Memory: Remembering is Faster Than Knowing

Consciousness and Cognition. Mar, 2006  |  Pubmed ID: 16019226

Three experiments investigated response times (RTs) for remember and know responses in recognition memory. RTs to remember responses were faster than RTs to know responses, regardless of whether the remember-know decision was preceded by an old/new decision (two-step procedure) or was made without a preceding old/new decision (one-step procedure). The finding of faster RTs for R responses was also found when remember-know decisions were made retrospectively. These findings are inconsistent with dual-process models of recognition memory, which predict that recollection is slower and more effortful than familiarity. Word frequency did not influence RTs, but remember responses were faster for words than for nonwords. We argue that the difference in RTs to remember and know responses reflects the time taken to make old/new decisions on the basis of the type of information activated at test.

Enhanced Transduction of Dendritic Cells by FcgammaRI-targeted Adenovirus Vectors

The Journal of Gene Medicine. Dec, 2007  |  Pubmed ID: 17966114

The high affinity Fcgamma receptor I (FcgammaRI; aka CD64) is expressed by dendritic cells (DC) and antigens targeted to this receptor elicit enhanced immune responses. This study was designed to test the hypothesis that targeting an adenoviral (Ad) vector to FcgammaRI would lead to enhanced transduction of DC and an improved immune response to vector-encoded antigens.

Glycogen Synthase Kinase 3 Beta (GSK-3 Beta) As a Therapeutic Target in NeuroAIDS

Journal of Neuroimmune Pharmacology : the Official Journal of the Society on NeuroImmune Pharmacology. Mar, 2007  |  Pubmed ID: 18040831

Highly active antiretroviral therapy (HAART) has made a significant impact on the lives of people living with HIV-1 infection. The incidence of neurologic disease associated with HIV-1 infection of the CNS plummeted between 1996-2000, but unfortunately the number of people currently HIV-1 infected (i.e., prevalence) with associated cognitive impairment has been steadily rising. While the reasons for this may be multifactorial, the implication is clear: there is a pressing need for adjunctive therapy directed at reversing or preventing damage to vulnerable pathways in the central nervous system (CNS) from HIV-1 infection. Using a team of preclinical and clinical investigators, we have focused our efforts on defining how proinflammatory mediators and secretory neurotoxins from HIV-1 disrupt signaling of the survival-regulating enzyme, glycogen synthase kinase 3 beta (GSK-3beta). In a series of studies initiated using in vitro, then in vivo models of HIV-1-associated dementia (HAD), we have demonstrated the ability of the mood stabilizing and anticonvulsant drug, sodium valproate (VPA), that inhibits GSK-3beta activity and other downstream mediators, to reverse HIV-1-induced damage to synaptic pathways in the CNS. Based on these results, we successfully performed pharmacokinetic and safety and tolerability trials with VPA in a cohort of HIV-1-infected patients with neurologic disease. VPA was well tolerated in this population and secondary measures of brain metabolism, as evidenced by an increase in N-acetyl aspartate/creatine (NAA/Cr), further suggested that VPA may improve gray matter integrity in brain regions damaged by HIV-1. These findings highlight the therapeutic potential of GSK-3beta blockade.

The Effects of Divided Attention at Study and Test on False Recognition: a Comparison of DRM and Categorized Lists

Memory & Cognition. Dec, 2007  |  Pubmed ID: 18265611

Three experiments investigated the effects of divided attention at encoding and retrieval on false recognition. In Experiment 1, participants studied word lists in either full or divided attention (random number generation) conditions and then took part in a recognition test with full attention. In Experiment 2, after studying word lists with full attention, participants carried out a recognition test with either full or divided attention. Experiment 3 manipulated attention at both study and test. We also compared Deese/Roediger-McDermott (DRM) and categorized lists, due to recent claims regarding the locus of false memories produced by such lists (Smith, Gerkens, Pierce, & Choi, 2002). With both list types, false "remember" responses were reduced by divided attention at encoding and increased by divided attention at retrieval. The findings suggest that the production of false memories occurs as a result of the generation of associates at encoding and failures of source monitoring retrieval. Crucially, this is true for both DRM and categorized lists.

Effect of Promoter Strength on Protein Expression and Immunogenicity of an HSV-1 Amplicon Vector Encoding HIV-1 Gag

Vaccine. Feb, 2007  |  Pubmed ID: 17145123

Helper-free herpes simplex virus type-1 (HSV-1) amplicon vectors elicit robust immune responses to encoded proteins, including human immunodeficiency virus type-1 (HIV-1) antigens. To improve this vaccine delivery system, seven amplicon vectors were constructed, each encoding HIV-1 Gag under the control of a different promoter. Gag expression levels were analyzed in murine and human cell lines, as well as in biopsied tissue samples from injected mice; these data were then compared with Gag-specific T cell responses in BALB/c mice. The magnitude of the amplicon-induced immune response was found to correlate strongly with the level of Gag production both in vitro and in vivo. Interestingly, the best correlation of the strength of the amplicon-induced immune response was with antigen expression in cultured DC rather than expression at the tissue site of injection or in cultured cell lines. These findings may have implications for the generation of improved HSV-1 amplicon vectors for HIV-1 vaccine delivery.

Infection of Human Immunodeficiency Virus and Intracellular Viral Tat Protein Exert a Pro-survival Effect in a Human Microglial Cell Line

Journal of Molecular Biology. Feb, 2007  |  Pubmed ID: 17157319

The interaction of human immunodeficiency virus type 1 (HIV-1) with CD4+ T lymphocytes is well studied and typically results in virally induced cytolysis. In contrast, relatively little is known concerning the interplay between HIV-1 and microglia. Recent findings suggest that, counter-intuitively, HIV-1 infection may extend the lifespan of microglia. We developed a novel cell line model system to confirm and mechanistically study this phenomenon. We found that transduction of a human microglial cell line with an HIV-1 vector results in a powerful cytoprotective effect following apoptotic challenge. This effect was reproduced by ectopic expression of a single virus-encoded protein, Tat. Subsequent studies showed that the pro-survival effects of intracellular Tat could be attributed to activation of the PI-3-kinase (PI3K)/Akt pathway in the microglial cell line. Furthermore, we found that expression of Tat led to decreased expression of PTEN, a negative regulator of the PI-3-K pathway. Consistent with this, decreased p53 activity and increased E2F activity were observed. Based on these findings, a model of possible regulatory circuits that intracellular Tat and HIV-1 infection engage during the cytoprotective event in microglia has been suggested. We propose that the expression of Tat may enable HIV-1 infected microglia to survive throughout the course of infection, leading to persistent HIV-1 production and infection in the central nervous system.

Human Immunodeficiency Virus Type 1 Pathobiology Studied in Humanized BALB/c-Rag2-/-gammac-/- Mice

Journal of Virology. Mar, 2007  |  Pubmed ID: 17182671

The specificity of human immunodeficiency virus type 1 (HIV-1) for human cells precludes virus infection in most mammalian species and limits the utility of small animal models for studies of disease pathogenesis, therapy, and vaccine development. One way to overcome this limitation is by human cell xenotransplantation in immune-deficient mice. However, this has proved inadequate, as engraftment of human immune cells is limited (both functionally and quantitatively) following transplantation of mature human lymphocytes or fetal thymus/liver. To this end, a human immune system was generated from umbilical cord blood-derived CD34(+) hematopoietic stem cells in BALB/c-Rag2(-/-)gamma(c)(-/-) mice. Intrapartum busulfan administration followed by irradiation of newborn pups resulted in uniform engraftment characterized by human T-cell development in thymus, B-cell maturation in bone marrow, lymph node development, immunoglobulin M (IgM)/IgG production, and humoral immune responses following ActHIB vaccination. Infection of reconstituted mice by CCR5-coreceptor utilizing HIV-1(ADA) and subtype C 1157 viral strains elicited productive viral replication and lymphadenopathy in a dose-dependent fashion. We conclude that humanized BALB/c-Rag2(-/-)gamma(c)(-/-) mice represent a unique and valuable resource for HIV-1 pathobiology studies.

Human Papillomavirus-like Particles Mediate Functional Delivery of Plasmid DNA to Antigen Presenting Cells in Vivo

Vaccine. Apr, 2007  |  Pubmed ID: 17293010

Because recombinant empty viral capsids are potentially attractive vectors for gene therapy, here we examined the ability of human papillomavirus (HPV) virus-like particles (VLPs) to mediate delivery and expression of DNA plasmids in vitro and in vivo. VLP-mediated delivery and expression of a GFP reporter construct in vitro was found to be highly dependent upon the presence of full-length L2 protein within the VLPs. Similarly, expression of GFP and luciferase reporter plasmids in vivo was strongly enhanced by co-administration of L1/L2 VLPs. Interestingly, in these experiments we routinely observed GFP expression in migrating antigen presenting cells (APC) recovered from mice inoculated with GFP plasmid in combination with VLPs, but not in APC recovered from mice inoculated with the plasmid alone. Additional evidence to support this concept was generated in experiments in which co-administration of VLPs with a plasmid designed to express HPV16 E6 oncoprotein was associated with significant enhancement of plasmid-encoded E6-specific cellular immune responses. These findings have implications for the design of vaccines for combined prophylaxis and therapy of HPV-associated diseases, and for other vaccines that rely on the administration of DNA-based immunogens, adjuvants, and/or other factors.

Spatial and Temporal Expression of Herpes Simplex Virus Type 1 Amplicon-encoded Genes: Implications for Their Use As Immunization Vectors

Human Gene Therapy. Feb, 2007  |  Pubmed ID: 17298238

There is great interest in developing new immunization vectors. Helper virus-free herpes amplicons, plasmid-based vectors that encode no viral gene products and have an extremely large coding capacity, are attractive viral vaccine candidates for expressing recombinant proteins in vivo for immunization. Earlier studies in mice, using amplicons encoding the gp120 protein of human immunodeficiency virus (HIV), resulted in strikingly robust cellular immune responses as measured by cytotoxicity and interferon gamma enzyme-linked immunospot assays. To begin to understand how such vectors function in vivo to generate an immune response, we used amplicons encoding reporter constructs including green fluorescent protein (GFP) and luciferase to examine the duration of expression after administration to mice. Luciferase expression, measured with the IVIS system from Xenogen/Caliper Life Sciences (Hopkinton, MA) and by enzymatic assays of tissue extracts, revealed that expression after injection of the HSVluc amplicons peaked earlier than 24 hr after injection into mice. HSVegfp injection resulted in peak accumulation of GFP 24 hr after administration in vivo. Thus, both reporter genes revealed a rather rapid and robust expression pattern of short duration. The short period of expression appears in part to be due to gene silencing. Examination of the cells transduced by amplicons encoding GFP and human B7.1 suggested that the amplicons transduce a variety of cells, including professional antigen-presenting cells. From this and previous work, we conclude that amplicons may engender a potent immune response by directly transducing dendritic cells as well as by cross-priming of antigen produced by other transduced host cells.

Functional Synergy Between CD40 Ligand and HIV-1 Tat Contributes to Inflammation: Implications in HIV Type 1 Dementia

Journal of Immunology (Baltimore, Md. : 1950). Mar, 2007  |  Pubmed ID: 17312171

HIV type 1 (HIV-1)-associated dementia (HAD) is believed to occur due to aberrant activation of monocyte-derived macrophages and brain-resident microglial cells by viral proteins as well as by the proinflammatory mediators released by infected cells. To investigate the inflammatory aspects of the disease, we examined the levels of soluble CD40L (sCD40L) in paired samples of plasma and cerebrospinal fluid obtained from 25 HIV-infected individuals. A significantly higher level of sCD40L was detected in both cerebrospinal fluid and plasma from HIV-infected patients with cognitive impairment, compared with their nonimpaired counterparts. The contribution of sCD40L to the pathogenesis of HAD was then examined by in vitro experiments. rCD40L synergized with HIV-1 Tat to increase TNF-alpha release from primary human monocytes and microglia, in an NF-kappaB-dependent manner. The mechanistic basis for this synergism was attributed to a Tat-mediated up-regulation of CD40 in monocytes and microglia. Finally, the CD40L-mediated increase in TNF-alpha production by monocytes was shown to be biologically important; immunodepletion experiments revealed that TNF-alpha was essential for the neurotoxic effects of conditioned medium recovered from Tat/CD40L-treated monocytes. Taken together, our results show that CD40 signaling in microglia and monocytes can synergize with the effects of Tat, further amplifying inflammatory processes within the CNS and influencing neuronal survival.

A Tractable Method for Simultaneous Modifications to the Head and Tail of Bacteriophage Lambda and Its Application to Enhancing Phage-mediated Gene Delivery

Nucleic Acids Research. , 2007  |  Pubmed ID: 17392341

There is considerable interest in the use of bacteriophage vectors for mammalian cell gene transfer applications, due to their stability, excellent safety profile and inexpensive mass production. However, to date, phage vectors have been plagued by mediocre performance as gene transfer agents. This may reflect the complexity of the viral infection process in mammalian cells and the need to refine each step of this process in order to arrive at an optimal, phage-based gene transfer system. Therefore, a flexible system was designed that alowed for the introduction of multiple modifications on the surface of bacteriophage lambda. Using this novel method, multiple peptides were displayed simultaneously from both the phage head and tail. Surface head display of an ubiquitinylation motif greatly increased the efficiency of phage-mediated gene transfer in a murine macrophage cell line. Gene transfer was further increased when this peptide was displayed in combination with a tail-displayed CD40-binding motif. Overall, this work provides a novel system that can be used to rationally improve bacteriophage gene transfer vectors and shows it may be possible to enhance the efficiency of phage-mediated gene transfer by targeting and optimizing multiple steps within the viral infection pathway.

Story Contexts Increase Susceptibility to the DRM Illusion in 5-year-olds

Developmental Science. May, 2007  |  Pubmed ID: 17444977

False recognition in children aged 5, 8, and 11 years was investigated using the standard version of the Deese-Roediger-McDermott (DRM) procedure and an alternative version in which the DRM stimuli were embedded in stories designed to emphasize their overall theme. Relative to the 8- and 11-year-olds, the 5-year-olds falsely recognized fewer critical lures when the DRM stimuli were presented in lists, but falsely recognized more critical lures when the stimuli were presented in stories. Levels of false recognition in the 8- and 11-year-olds were not affected by study format. We argue that the story context enhanced the ability of the 5-year-olds to make inferences based on the theme of the DRM stimuli. The 5-year-olds then showed higher levels of false recognition than the older children owing to their inability to reject lure words consistent with the stories.

Reinstating Effortful Encoding Operations at Test Enhances Episodic Remembering

Quarterly Journal of Experimental Psychology (2006). Apr, 2007  |  Pubmed ID: 17455065

Two experiments investigated the effects of reinstating encoding operations on remember and know responses in recognition memory. Experiment 1 showed that reinstating an effortful encoding task (generating words from fragments) increased remember responses at test but reinstating an automatic encoding task (reading intact words) did not. This pattern was confirmed in Experiment 2 in which words were either read intact or generated from anagrams. These findings show that repeating effortful (but not automatic) encoding operations at test cues not only the recognition of the information that was acquired via those operations but also the conscious recollection of the encoding episode.

Infectivity of Herpes Simplex Virus Type-1 (HSV-1) Amplicon Vectors in Dendritic Cells is Determined by the Helper Virus Strain Used for Packaging

Journal of Virological Methods. Oct, 2007  |  Pubmed ID: 17606303

Herpes simplex virus type-1 (HSV-1) amplicon vectors are being explored for a wide range of potential applications, including vaccine delivery and immunotherapy of cancer. While extensive effort has been directed towards the improvement of the amplicon "payload" in these vectors, relatively little attention has been paid to the effect of the packaging HSV-1 strains on the biological properties of co-packaged amplicon vectors. We therefore compared the biological properties of amplicon stocks prepared using a panel of primary HSV-1 isolates, a molecularly cloned strain used to package helper-free amplicons (designated here as F5), and two laboratory isolates (KOS and strain 17, which is the parent of the F5 clone). This analysis revealed considerable inter-strain variability in the ability of amplicon stocks packaged by different primary HSV-1 isolates to efficiently transduce established cell lines and primary human dendritic cells (DC). Amplicons packaged by both the F5 molecularly cloned virus and its laboratory-adapted parent (strain 17) were very inefficient at transducing DC, when compared to amplicons packaged by KOS or by several of the primary virus isolates. These finding have important implications for the future development of improved amplicon-based vaccine delivery systems and suggest that DC tropism may be an instrinsic property of some HSV-1 strains, independent of passage history or molecular cloning.

Divided Attention at Retrieval Disrupts Knowing but Not Remembering

Memory (Hove, England). Aug, 2007  |  Pubmed ID: 17654280

The view that remember and know responses can be explained within a dual-process framework has recently been questioned (e.g., Gardiner, Konstantinou, Karayianni, & Gregg, 2005). The aims of the present study were to investigate further discrepancies between remember/know (R/K) studies and dual-process models of recognition memory. In two experiments participants were required to make old/new and R/K decisions under full and divided attention conditions. Experiment 1 used a two-step R/K procedure and showed that attention during retrieval reduced overall recognition performance. Experiment 2 used a one-step R/K procedure and showed that dividing attention at retrieval only affected know responses, suggesting that knowing but not remembering relies on controlled retrieval processes. These findings and findings from recent research provide evidence that is inconsistent with the dual-process explanation for R/K research.

The Effect of Divided Attention on False Memory Depends on How Memory is Tested

Memory & Cognition. Jun, 2007  |  Pubmed ID: 17848024

In three experiments, we investigated the effects of divided attention on false memory, using the Deese/Roediger-McDermott (DRM) paradigm. In Experiments 1 and 2, participants studied six DRM lists with full attention and six in one of two divided-attention conditions (random number generation or digit monitoring). Both divided-attention conditions increased false recall of related words (Experiment 1) but reduced false recognition (Experiment 2). These results were confirmed in Experiment 3, in which the type of secondary task was manipulated within groups. We argue that the increase in false recall with divided attention reflects a change in participants' response criterion, whereas the decrease in false recognition occurs because the secondary tasks prevent participants from generating associates of the words presented at study.

HSV-1 Amplicon Vectors Elicit Polyfunctional T Cell Responses to HIV-1 Env, and Strongly Boost Responses to an Adenovirus Prime

Vaccine. Oct, 2007  |  Pubmed ID: 17868958

HSV-1 amplicon vectors elicit strong T-cell responses to encoded antigens but the qualitative nature of these responses is poorly understood. Antigen-specific CD4(+) and CD8(+) T-cell responses to amplicon and adenovirus (rAd5) vectors encoding HIV-1 gp120 were assessed following immunization of mice, by performing intracellular cytokine staining for IFNgamma, IL2 and TNFalpha, following stimulation of splenocytes with a HIV-1 Env peptide pool. The quality of the primary T-cell response to amplicon and rAd5 vectors was strikingly similar, but there were qualitative differences in responses to amplicon vectors that incorporated different promoters upstream of gp120 - suggesting that promoters can significantly influence immune response quality. When prime-boost combinations were studied, a rAd5 prime and amplicon boost elicited the highest T-cell response. Furthermore, protocols that incorporated a rAd5 prime consistently elicited a greater proportion of polyfunctional CD4(+) T-cells-regardless of boost. This suggests that initial priming can shape immune response quality after a boost. Overall, these findings provide insight into effective vector combinations for HIV-1 vaccine development.

The Effect of Glucose Administration on the Recollection and Familiarity Components of Recognition Memory

Biological Psychology. Jan, 2008  |  Pubmed ID: 17950982

Previous research has demonstrated that glucose administration facilitates long-term memory performance. The aim of the present research was to evaluate the effect of glucose administration on different components of long-term recognition memory. Fifty-six healthy young individuals received (a) a drink containing 25 g of glucose or (b) an inert placebo drink. Recollection and familiarity components of recognition memory were measured using the 'remember-know' paradigm. The results revealed that glucose administration led to significantly increased proportion of recognition responses based on recollection, but had no effect on the proportion of recognition responses made through participants' detection of stimulus familiarity. Consequently, the data suggest that glucose administration appears to facilitate recognition memory that is accompanied by recollection of contextual details and episodic richness. The findings also suggest that memory tasks that result in high levels of hippocampal activity may be more likely to be enhanced by glucose administration than tasks that are less reliant on medial temporal lobe structures.

Dynamic Visual Noise Interferes with Storage in Visual Working Memory

Experimental Psychology. , 2008  |  Pubmed ID: 18683625

Several studies have demonstrated that dynamic visual noise (DVN) does not interfere with memory for random matrices. This has led to suggestions that (a) visual working memory is distinct from imagery, and (b) visual working memory is not a gateway between sensory input and long-term storage. A comparison of the interference effects of DVN with memory for matrices and colored textures shows that DVN can interfere with visual working memory, probably at a level of visual detail not easily supported by long-term memory structures or the recoding of the visual pattern elements. The results support a gateway model of visuospatial working memory and raise questions about the most appropriate ways to measure and model the different levels of representation of information that can be held in visual working memory.

The Human H5N1 Influenza A Virus Polymerase Complex is Active in Vitro over a Broad Range of Temperatures, in Contrast to the WSN Complex, and This Property Can Be Attributed to the PB2 Subunit

The Journal of General Virology. Dec, 2008  |  Pubmed ID: 19008377

Influenza A virus (IAV) replicates in the upper respiratory tract of humans at 33 degrees C and in the intestinal tract of birds at close to 41 degrees C. The viral RNA polymerase complex comprises three subunits (PA, PB1 and PB2) and plays an important role in host adaptation. We therefore developed an in vitro system to examine the temperature sensitivity of IAV RNA polymerase complexes from different origins. Complexes were prepared from human lung epithelial cells (A549) using a novel adenoviral expression system. Affinity-purified complexes were generated that contained either all three subunits (PA/PB1/PB2) from the A/Viet/1203/04 H5N1 virus (H/H/H) or the A/WSN/33 H1N1 strain (W/W/W). We also prepared chimeric complexes in which the PB2 subunit was exchanged (H/H/W, W/W/H) or substituted with an avian PB2 from the A/chicken/Nanchang/3-120/01 H3N2 strain (W/W/N). All complexes were functional in transcription, cap-binding and endonucleolytic activity. Complexes containing the H5N1 or Nanchang PB2 protein retained transcriptional activity over a broad temperature range (30-42 degrees C). In contrast, complexes containing the WSN PB2 protein lost activity at elevated temperatures (39 degrees C or higher). The E627K mutation in the avian PB2 was not required for this effect. Finally, the avian PB2 subunit was shown to confer enhanced stability to the WSN 3P complex. These results show that PB2 plays an important role in regulating the temperature optimum for IAV RNA polymerase activity, possibly due to effects on the functional stability of the 3P complex.

HIV-1 Tat Activates Neuronal Ryanodine Receptors with Rapid Induction of the Unfolded Protein Response and Mitochondrial Hyperpolarization

PloS One. , 2008  |  Pubmed ID: 19009018

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.

Sequential Shrinkage and Swelling Underlie P2X7-stimulated Lymphocyte Phosphatidylserine Exposure and Death

Journal of Immunology (Baltimore, Md. : 1950). Jan, 2008  |  Pubmed ID: 18097031

Patterns of change in cell volume and plasma membrane phospholipid distribution during cell death are regarded as diagnostic means of distinguishing apoptosis from necrosis, the former being associated with cell shrinkage and early phosphatidylserine (PS) exposure, whereas necrosis is associated with cell swelling and consequent lysis. We demonstrate that cell volume regulation during lymphocyte death stimulated via the purinergic receptor P2X7 is distinct from both. Within seconds of stimulation, murine lymphocytes undergo rapid shrinkage concomitant with, but also required for, PS exposure. However, within 2 min shrinkage is reversed and swelling ensues ending in cell rupture. P2X7-induced shrinkage and PS translocation depend upon K+ efflux via KCa3.1, but use a pathway of Cl- efflux distinct from that previously implicated in apoptosis. Thus, P2X7 stimulation activates a novel pathway of cell death that does not conform to those conventionally associated with apoptosis and necrosis. The mixed apoptotic/necrotic phenotype of P2X7-stimulated cells is consistent with a potential role for this death pathway in lupus disease.

Fc Receptor-mediated, Antibody-dependent Enhancement of Bacteriophage Lambda-mediated Gene Transfer in Mammalian Cells

Virology. Apr, 2008  |  Pubmed ID: 18191979

Lambda phage vectors mediate gene transfer in cultured mammalian cells and in live mice, and in vivo phage-mediated gene expression is increased when mice are pre-immunized with bacteriophage lambda. We now show that, like eukaryotic viruses, bacteriophage vectors are subject to Fc receptor-mediated, antibody-dependent enhancement of infection in mammalian cells. Antibody-dependent enhancement of phage gene transfer required FcgammaRI, but not its associated gamma-chain, and was not supported by other FcgammaR family members (FcgammaRIIA, FcgammaRIIB, and FcgammaRIII). Studies using chlorpromazine and latrunculin A revealed an important role for clathrin-mediated endocytosis (chlorpromazine) and actin filaments (latrunculin A) in antibody-enhanced phage gene transfer. This was confirmed by experiments using inhibitors of endosomal acidification (bafilomycin A1, monensin) and by immunocytochemical colocalization of internalized phage particles with early endosome-associated protein-1 (EAA1). In contrast, microtubule-targeting agents (nocodazole, taxol) increased the efficiency of antibody-enhanced phage gene transfer. These results reveal an unexpected antibody-dependent, FcgammaRI-mediated enhancement of phage transduction in mammalian cells, and suggest new approaches to improve bacteriophage-mediated gene transfer.

Akt Inhibitors As an HIV-1 Infected Macrophage-specific Anti-viral Therapy

Retrovirology. , 2008  |  Pubmed ID: 18237430

Unlike CD4+ T cells, HIV-1 infected macrophages exhibit extended life span even upon stress, consistent with their in vivo role as long-lived HIV-1 reservoirs.

False and Veridical Collaborative Recognition

Memory (Hove, England). Jan, 2009  |  Pubmed ID: 19058091

Participants studied DRM words lists (Deese, 1959; Roediger & McDermott, 1995) and then completed a recognition test individually or in a collaborative pair, trio, or quartet. The collaborative groups' responses were compared to those of equivalent sized nominal groups. Non-studied critical lure and studied word recognition increased with group size and these increases were greatest for the collaborative groups. The collaborative groups' critical lure and studied word recognition rates were facilitated as they lowered their response criterion thresholds towards all test words semantically related to those in the DRM lists. Prior collaboration also enhanced later individual critical lure and studied word recognition. The group members believed the critical lures and studied words recognised during collaboration were studied, and they therefore repeated these judgements when tested alone.

Proteasome Inhibitors Enhance Bacteriophage Lambda (lambda) Mediated Gene Transfer in Mammalian Cells

Virology. Feb, 2009  |  Pubmed ID: 19064273

Bacteriophage lambda vectors can transfer their genomes into mammalian cells, resulting in expression of phage-encoded genes. However, this process is inefficient. Experiments were therefore conducted to delineate the rate limiting step(s) involved, using a phage vector that contains a mammalian luciferase reporter gene cassette. The efficiency of phage-mediated gene transfer in mammalian cells was quantitated, in the presence or absence of pharmacologic inhibitors of cell uptake and degradation pathways. Inhibitors of lysosomal proteases and proteasome inhibitors strongly enhanced phage-mediated luciferase expression, suggesting that these pathways contribute to the destruction of intracellular phage particles. In contrast, inhibition of endosome acidification had no effect on phage-mediated gene transfer, presumably because phage lambda is tolerant to extended exposure to low pH. These findings provide insights into the pathways by which phage vectors enter and transduce mammalian cells, and suggest that it may be possible to pharmacologically enhance the efficiency of phage-mediated gene transfer in mammalian cells. Finally, the data also suggest that the proteasome complex may serve as an innate defense mechanism that restricts the infection of mammalian cells by diverse viral agents.

Remembering the Past and Imagining the Future: Differences in Event Specificity of Spontaneously Generated Thought

Memory (Hove, England). May, 2009  |  Pubmed ID: 19235018

A growing interest has emerged in the role that autobiographical memory retrieval plays in simulation of future events. Cognitive explorations in this domain have generally relied on cue word paradigms with instructions to develop specific (relating to one particular day) memories or future events. However, the usefulness of this paradigm has been questioned with respect to its ability to assess habitual patterns of retrieval within autobiographical memory. The current study investigated similarities and differences in how participants spontaneously remember the past and imagine the future when the specificity constraints inherent in the cue word task are removed. A total of 93 undergraduate students completed two sentence-completion tasks, probing for past and future events. A number of differences emerged between past and future thought; in particular, they were less specific when simulating future events compared with past events. This reduction in specificity was the result of participants producing more future thoughts relating to extended lifetime periods and semantic associates. The findings are discussed in relation to the underlying cognitive processes involved in autobiographical memory retrieval and future event simulation.

Lithium Therapy for Human Immunodeficiency Virus Type 1-associated Neurocognitive Impairment

Journal of Neurovirology. Apr, 2009  |  Pubmed ID: 19306230

The objective of this study was to assess lithium safety and tolerability and to explore its impact on cognition, function, and neuroimaging biomarkers in human immunodeficiency virus (HIV)-infected subjects with cognitive impairment. Fifteen cognitively impaired HIV-infected subjects were enrolled in this 10-week open-label study of lithium 300 mg twice daily. Neuroimaging was performed at baseline and following 10 weeks of treatment and included magnetic resonance spectroscopy (MRS), diffusion tensor imaging (DTI), and functional MRI (fMRI). Thirteen of the 14 subjects (93%) that complied with the study visits were able to complete the study on lithium and 11 out of 13 (79%) completed the study at the originally assigned dose of 300 mg twice daily. There were no significant changes in CD4(+) lymphocyte cell count and plasma HIV RNA. Cognitive performance and depressive mood did not improve significantly after the 10-week lithium treatment; however, neuroimaging revealed a decrease in the glutamate+glutamine (Glx) peak in the frontal gray matter, increased fractional anisotropy, and decreased mean diffusivity in several brain areas, and changes in brain activation patterns, suggestive of improvement. These results suggest that lithium can be used safely in HIV-infected individuals with cognitive impairment. Furthermore, the neuroimaging results suggest that lithium may improve HIV-associated central nervous system (CNS) injury; thus, further investigations of lithium as an adjunctive treatment for HIV-associated cognitive impairment are warranted.

Nef Gene Evolution from a Single Transmitted Strain in Acute SIV Infection

Retrovirology. , 2009  |  Pubmed ID: 19505314

The acute phase of immunodeficiency virus infection plays a crucial role in determining steady-state virus load and subsequent progression of disease in both humans and nonhuman primates. The acute period is also the time when vaccine-mediated effects on host immunity are likely to exert their major effects on virus infection. Recently we developed a Monte-Carlo (MC) simulation with mathematical analysis of viral evolution during primary HIV-1 infection that enables classification of new HIV-1 infections originating from multiple versus single transmitted viral strains and the estimation of time elapsed following infection.

The Mechanistic Architecture of Thermostable Pyrococcus Furiosus Family B DNA Polymerase Motif A and Its Interaction with the DNTP Substrate

Biochemistry. Dec, 2009  |  Pubmed ID: 19817489

Thermostable DNA polymerases isolated from archaeal organisms have not been completely characterized kinetically and require further study if we are to understand both their dNTP binding mechanism and their role within the organism. Here, we demonstrate that the thermostable family B DNA polymerase from Pyrococcus furiosus (Pfu Pol) contains sensitive determinants of both dNTP binding and replicational fidelity within the highly conserved motif A. Site-directed mutagenesis of the motif A SYLP region revealed that small shifts in side chain volume result in significant changes in the dNTP binding affinity, steady state kinetics, and fidelity of the enzyme. Mutants of Y410 show high fidelity in both misincorporation assays and forward mutation assays, but display a substantially higher K(m) than the wild type. In contrast, mutations of upstream residue L409 result in a drastically reduced affinity for the correct dNTP, a much higher efficiency of both misincorporation and mismatch extension, and substantially lower fidelity as demonstrated by a PCR-based forward mutation assay. The A408S mutant, however, displayed a significant increase in both dNTP binding affinity and fidelity. In summary, these data show that modulation of motif A can greatly shift both the steady and pre-steady state kinetics of the enzyme as well as the fidelity of Pfu Pol.

Neuroprotective Activities of CEP-1347 in Models of NeuroAIDS

Journal of Immunology (Baltimore, Md. : 1950). Jan, 2010  |  Pubmed ID: 19966207

When the nervous system is infected with HIV-1, it commonly results in neuroinflammation leading to overt neuronal dysfunction and subsequent cognitive and behavioral impairments. The multifaceted disease process, now referred to as HIV-1-associated neurocognitive disorders (HAND), provides a range of molecular targets for adjunctive therapies. One is CEP-1347, an inhibitor of mixed lineage kinases that elicits neuroprotective and anti-inflammatory responses in models of neurodegenerative diseases. Since HAND is associated with inflammatory encephalopathy induced by virus infection and mononuclear phagocytes (perivascular macrophages and microglia) immune activation, we investigated whether CEP-1347 could ameliorate disease in laboratory models of HAND. We now demonstrate that CEP-1347 reduces the levels of secreted proinflammatory cytokines and chemokines in HIV-1-infected human macrophages and attenuates dose-dependent neurotoxicity in rodent cortical neurons. CEP-1347-treated mice readily achieve therapeutic drug levels in peripheral blood. HIV-1 encephalitis (HIVE) mice, where human virus-infected monocyte-derived macrophages are stereotactically injected into the basal ganglia of CB17 severe combined immunodeficient mice, received daily intraperitoneal injections of CEP-1347. Here, CEP-1347 treatment of HIVE mice showed a dose-dependent reduction in microgliosis. Dendritic integrity and neuronal loss were sustained and prevented, respectively. These results demonstrate that CEP-1347 elicits anti-inflammatory and neuroprotective responses in an HIVE model of human disease and as such warrants further study as an adjunctive therapy for human disease.

Mixed Lineage Kinase 3 Deficiency Delays Viral Clearance in the Lung and is Associated with Diminished Influenza-induced Cytopathic Effect in Infected Cells

Virology. May, 2010  |  Pubmed ID: 20185156

Influenza virus leads to acute respiratory disease resulting in seasonal epidemics and periodic pandemics. Little is known about the signaling events that regulate host defense to influenza. One particular pathway, the c-Jun amino-terminal kinase (JNK) cascade is activated following influenza infection and blocking JNK leads to enhanced viral replication. We hypothesize that Mixed Lineage Kinase 3 (MLK3), an upstream regulator of JNK, is involved in the host response to influenza. To test this, wild-type and MLK3-/- mice were infected with pathogenic strain of influenza A virus, A/PR/8/34 (PR8). Although, cellular and humoral immune responses were similar between wild-type and MLK3-/- hosts, the viral load in the lungs was comparatively higher in MLK3-/- mice at day 8 post-infection. Consistent with this, MLK3-/- murine lung fibroblast and epithelial cells had prolonged survival and increased virion production following infection compared to wild-type. These findings support a role for MLK3 in viral production during influenza infection.

Mathematical Modeling of Ultradeep Sequencing Data Reveals That Acute CD8+ T-lymphocyte Responses Exert Strong Selective Pressure in Simian Immunodeficiency Virus-infected Macaques but Still Fail to Clear Founder Epitope Sequences

Journal of Virology. Jun, 2010  |  Pubmed ID: 20335256

The prominent role of antiviral cytotoxic CD8(+) T-lymphocytes (CD8-TL) in containing the acute viremia of human and simian immunodeficiency viruses (HIV-1 and SIV) has rationalized the development of T-cell-based vaccines. However, the presence of escape mutations in the acute stage of infection has raised a concern that accelerated escape from vaccine-induced CD8-TL responses might undermine vaccine efficacy. We reanalyzed previously published data of 101,822 viral genomes of three CD8-TL epitopes, Nef(103-111)RM9 (RM9), Tat(28-35)SL8 (SL8), and Gag(181-189)CM9 (CM9), sampled by ultradeep pyrosequencing from eight macaques. Multiple epitope variants appeared during the resolution of acute viremia, followed by the predominance of a single mutant epitope. By fitting a mathematical model, we estimated the first acute escape rate as 0.36 day(-1) within escape-prone epitopes, RM9 and SL8, and the chronic escape rate as 0.014 day(-1) within the CM9 epitope. Our estimate of SIV acute escape rates was found to be comparable to very early HIV-1 escape rates. The timing of the first escape was more highly correlated with the timing of the peak CD8-TL response than with the magnitude of the CD8-TL response. The transmitted epitope decayed more than 400 times faster during the acute viral decline stage than predicted by a neutral evolution model. However, the founder epitope persisted as a minor population even at the viral set point; in contrast, the majority of acute escape epitopes were completely cleared. Our results suggest that a reservoir of SIV infection is preferentially formed by virus with the transmitted epitope.

Biochemical Characterization of Enzyme Fidelity of Influenza A Virus RNA Polymerase Complex

PloS One. , 2010  |  Pubmed ID: 20454455

It is widely accepted that the highly error prone replication process of influenza A virus (IAV), together with viral genome assortment, facilitates the efficient evolutionary capacity of IAV. Therefore, it has been logically assumed that the enzyme responsible for viral RNA replication process, influenza virus type A RNA polymerase (IAV Pol), is a highly error-prone polymerase which provides the genomic mutations necessary for viral evolution and host adaptation. Importantly, however, the actual enzyme fidelity of IAV RNA polymerase has never been characterized.

CD8+ Cell Depletion Accelerates HIV-1 Immunopathology in Humanized Mice

Journal of Immunology (Baltimore, Md. : 1950). Jun, 2010  |  Pubmed ID: 20495069

Stable engraftment of human lymphoid tissue in NOD/scid-IL-2Rgammacnull mice after CD34+ hematopoietic stem cell reconstitution permits the evaluation of ongoing HIV-1 infection for weeks to months. We demonstrate that HIV-1-infected rodents develop virus-specific cellular immune responses. CD8+ cell depletion, 2 or 5-7 wk after viral infection, resulted in a significant increase of HIV-1 load, robust immune cell activation, and cytopathology in lymphoid tissues but preserved CD4/CD8 double-positive thymic T cell pools. Human CD8+ cells reappeared in circulation as early as 2-3 wk. These data support a role of CD8+ cells in viral surveillance and the relevance of this humanized mouse model for the studies of HIV-1 pathobiology and virus-specific immunity.

Can False Memories Prime Problem Solutions?

Cognition. Nov, 2010  |  Pubmed ID: 20813356

Previous research has suggested that false memories can prime performance on related implicit and explicit memory tasks. The present research examined whether false memories can also be used to prime higher order cognitive processes, namely, insight-based problem solving. Participants were asked to solve a number of compound remote associate task (CRAT) problems, half of which had been primed by the presentation of Deese/Roediger-McDermott (DRM) lists whose critical lure was also the solution to the problem. The results showed that when the critical lure: (a) was falsely recalled, CRAT problems were solved more often and significantly faster than problems that were not primed by a DRM list and (b) was not falsely recalled, CRAT problem solution rates and times were no different than when there was no DRM priming. A second experiment demonstrated that these outcomes were not a simple artifact of the inclusion of a recall test prior to the problem solving task. The implications of these results are discussed with regard to the previous literature on priming and the adaptive function of false memories.

Amyloid-binding Small Molecules Efficiently Block SEVI (semen-derived Enhancer of Virus Infection)- and Semen-mediated Enhancement of HIV-1 Infection

The Journal of Biological Chemistry. Nov, 2010  |  Pubmed ID: 20833717

Semen was recently shown to contain amyloid fibrils formed from a self-assembling peptide fragment of the protein prostatic acid phosphatase. These amyloid fibrils, termed semen-derived enhancer of virus infection, or SEVI, have been shown to strongly enhance HIV infectivity and may play an important role in sexual transmission of HIV, making them a potential microbicide target. One novel approach to target these fibrils is the use of small molecules known to intercalate into the structure of amyloid fibrils, such as derivatives of thioflavin-T. Here, we show that the amyloid-binding small molecule BTA-EG(6) (the hexa(ethylene glycol) derivative of benzothiazole aniline) is able to bind SEVI fibrils and effectively inhibit both SEVI-mediated and semen-mediated enhancement of HIV infection. BTA-EG(6) also blocks the interactions of SEVI with HIV-1 virions and HIV-1 target cells but does not cause any inflammation or toxicity to cervical epithelial cells. These results suggest that an amyloid-binding small molecule may have utility as a microbicide, or microbicidal supplement, for HIV-1.

Rebuilding Synaptic Architecture in HIV-1 Associated Neurocognitive Disease: a Therapeutic Strategy Based on Modulation of Mixed Lineage Kinase

Neurotherapeutics : the Journal of the American Society for Experimental NeuroTherapeutics. Oct, 2010  |  Pubmed ID: 20880503

Work from our laboratories has validated mixed lineage kinase type 3 (MLK3) as an enzyme pathologically activated in the CNS by human immunodeficiency virus 1 (HIV-1) neurotoxins. In this review, we discuss MLK3 activation in the context of the neuropathogenesis of HIV-1 associated neurocognitive deficits (HAND). We use findings from the literature to substantiate the neuropathologic relevance of MLK3 to neurodegenerative disease, with an emphasis on Parkinson's disease that shares a number of important phenotypic and neuropathologic characteristics with HAND. We discuss signal transduction pathways downstream from MLK3 activation, with an emphasis on their involvement in microglia and neurons in preclinical models of HAND. Finally, we make a case for pharmacologic intervention targeted at inhibition of MLK3 as a strategy to reverse HAND, in light of the fact that combination antiretroviral therapy, despite successfully managing systemic infection of HIV-1, has been largely unsuccessful in eradicating HAND.

Ablation of Mixed Lineage Kinase 3 (Mlk3) Does Not Inhibit Ototoxicity Induced by Acoustic Trauma or Aminoglycoside Exposure

Hearing Research. Dec, 2010  |  Pubmed ID: 20971179

Jun N-terminal kinase (JNK) is activated in cochlear hair cells following acoustic trauma or exposure to aminoglycoside antibiotics. Blockade of JNK activation using mixed lineage kinase (MLK) inhibitors prevents hearing loss and hair cell death following these stresses. Since current pharmacologic inhibitors of MLKs block multiple members of this kinase family, we examined the contribution of the major neuronal family member (MLK3) to stress-induced ototoxicity, usingMlk3(-/-) mice. Immunohistochemical staining revealed that MLK3 is expressed in cochlear hair cells of C57/BL6 mice (but not in Mlk3(-/-) animals). After exposure to acoustic trauma there was no significant difference in DPOAE and ABR values betweenMlk3(-/-) and wild-type mice at 48 h following exposure or 2 weeks later. Susceptibility of hair cells to aminoglycoside toxicity was tested by exposing explanted utricles to gentamicin. Gentamicin-induced hair cell death was equivalent in utricles from wild-type and Mlk3(-/-) mice. Blockade of JNK activation with the pharmacologic inhibitor SP600125 attenuated cell death in utricles from both wild-type and Mlk3(-/-) mice. These data show that MLK3 ablation does not protect against hair cell death following acoustic trauma or exposure to aminoglycoside antibiotics, suggesting that MLK3 is not the major upstream regulator of JNK-mediated hair cell death following these stresses. Rather, other MLK family members such as MLK1, which is also expressed in cochlea, may have a previously unappreciated role in noise- and aminoglycoside-induced ototoxicity.

Investigating the Encoding-retrieval Match in Recognition Memory: Effects of Experimental Design, Specificity, and Retention Interval

Memory & Cognition. Dec, 2010  |  Pubmed ID: 21156873

Five experiments investigated the encoding-retrieval match in recognition memory by manipulating read and generate conditions at study and at test. Experiments 1A and 1B confirmed previous findings that reinstating encoding operations at test enhances recognition accuracy in a within-groups design but reduces recognition accuracy in a between-groups design. Experiment 2A showed that generating from anagrams at study and at test enhanced recognition accuracy even when study and test items were generated from different anagrams. Experiment 2B showed that switching from one generation task at study (e.g., anagram solution) to a different generation task at test (e.g., fragment completion) eliminated this recognition advantage. Experiment 3 showed that the recognition advantage found in Experiment 1A is reliably present up to 1 week after study. The findings are consistent with theories of memory that emphasize the importance of the match between encoding and retrieval operations.

Dense Display of HIV-1 Envelope Spikes on the Lambda Phage Scaffold Does Not Result in the Generation of Improved Antibody Responses to HIV-1 Env

Vaccine. Mar, 2011  |  Pubmed ID: 21310193

The generation of strong, virus-neutralizing antibody responses to the HIV-1 envelope spike (Env) is a major goal in HIV-1 vaccine research. To try to enhance the Env-specific response, we displayed oligomeric gp140 on a virus-like scaffold provided by the lambda phage capsid. To do this, an in vitro complementation system was used to "decorate" phage particles with glycosylated, mammalian cell-derived envelope oligomers. We compared the immune response to lambda phage particles displaying HIV-1 Env to that elicited by soluble oligomeric gp140 in rabbits. Env-binding antibody titers were higher in animals that received oligomeric gp140 as compared to Env decorated phage particles, as were virus neutralizing antibody responses. The Env decorated phage particles were, however, able to efficiently boost a protein-primed humoral response to levels equivalent to those elicited by high-dose adjuvanted Env oligomers. These results show that display of HIV-1 envelope spikes on the bacteriophage lambda capsid does not result in an improved, Env-specific humoral immune response.

The Development of Automatic and Controlled Inhibitory Retrieval Processes in True and False Recall

Journal of Experimental Child Psychology. May, 2011  |  Pubmed ID: 21320706

In three experiments, we investigated the role of automatic and controlled inhibitory retrieval processes in true and false memory development in children and adults. Experiment 1 incorporated a directed forgetting task to examine controlled retrieval inhibition. Experiments 2 and 3 used a part-set cue and retrieval practice task to examine automatic retrieval inhibition. In the first experiment, the forget cue had no effect on false recall for adults but reduced false recall for children. In Experiments 2 and 3, both tasks caused retrieval impairments for true and false recall, and this occurred for all age groups. Implicit inhibition, which occurs outside of our conscious control, appears early in childhood. However, because young children do not process false memories as automatically as adults, explicit inhibition can reduce false memory output.

Enhancement of HIV-1 Infectivity by Simple, Self-assembling Modular Peptides

Biophysical Journal. Mar, 2011  |  Pubmed ID: 21354406

Semen-derived enhancer of viral infection (SEVI), an amyloid fibril formed from a cationic peptide fragment of prostatic acidic phosphatase (PAP), dramatically enhances the infectivity of human immunodeficiency virus type 1 (HIV-1). Insoluble, sedimentable fibrils contribute to SEVI-mediated enhancement of virus infection. However, the SEVI-forming PAP(248-286) peptide is able to produce infection-enhancing structures much more quickly than it forms amyloid fibrils. This suggests that soluble supramolecular assemblies may enhance HIV-1 infection. To address this question, non-SEVI amyloid-like fibrils were derived from general amphipathic peptides of sequence Ac-K(n)(XKXE)(2)-NH(2). These cationic peptides efficiently self-assembled to form soluble, fibril-like structures that were, in some cases, able to enhance HIV-1 infection even more efficiently than SEVI. Experiments were also performed to determine whether agents that efficiently shield the charged surface of SEVI fibrils block SEVI-mediated infection-enhancement. To do this, we generated self-assembling anionic peptides of sequence Ac-E(n)(XKXE)(2)-NH(2). One of these peptides completely abrogated SEVI-mediated enhancement of HIV-1 infection, without altering HIV-1 infectivity in the absence of SEVI. Collectively, these data suggest that soluble SEVI assemblies may mediate infection-enhancement, and that anionic peptide supramolecular assemblies have the potential to act as anti-SEVI microbicides.

Methamphetamine Causes Sustained Depression in Cerebral Blood Flow

Brain Research. Feb, 2011  |  Pubmed ID: 21156163

The use prevalence of the highly addictive psychostimulant methamphetamine (MA) has been steadily increasing over the past decade. MA abuse has been associated with both transient and permanent alterations in cerebral blood flow (CBF), hemorrhage, cerebrovascular accidents and death. To understand MA-induced changes in CBF, we exposed C56BL/6 mice to an acute bolus of MA (5mg/kg MA, delivered IP). This elicited a biphasic CBF response, characterized by an initial transient increase (~ 5 minutes) followed by a prolonged decrease (~ 30 minutes) of approximately 25% relative to baseline CBF--as measured by laser Doppler flowmetry over the somatosensory cortex. To assess if this was due to catecholamine derived vasoconstriction, phentolamine, an α-adrenergic antagonist was administered prior to MA treatment. This reduced the initial increase in CBF but failed to prevent the subsequent, sustained decrease in CBF. Consistent with prior reports, MA caused a transient increase in mean arterial blood pressure, body temperature and respiratory rate. Elevated respiratory rate resulted in hypocapnia. When respiratory rate was controlled by artificially ventilating mice, blood PaCO(2) levels after MA exposure remained unchanged from physiologic levels, and the MA-induced decrease in CBF was abolished. In vivo two-photon imaging of cerebral blood vessels revealed sustained MA-induced vasoconstriction of pial arterioles, consistent with laser Doppler flowmetry data. These findings show that even a single, acute exposure to MA can result in profound changes in CBF, with potentially deleterious consequences for brain function.

Test-induced Priming Impairs Source Monitoring Accuracy in the DRM Procedure

Journal of Experimental Psychology. Learning, Memory, and Cognition. Jul, 2011  |  Pubmed ID: 21480753

Three experiments investigated the effects of test-induced priming (TIP) on false recognition in the Deese/Roediger-McDermott procedure (Deese, 1959; Roediger & McDermott, 1995). In Experiment 1, TIP significantly increased false recognition for participants who made old/new decisions at test but not for participants who made remember/know judgments or were given diagnostic information to help them avoid false recognition. In Experiment 2, a TIP effect was observed with old/new recognition but not when participants were required to remember whether study items were spoken by a male or a female speaker. In Experiment 3, false recognition increased when critical lures were preceded by 10 studied items but not when preceded by 5 studied and 5 unstudied items from the same list. These findings suggest that TIP increases false recognition by disrupting source monitoring processes.

PA Residues in the 2009 H1N1 Pandemic Influenza Virus Enhance Avian Influenza Virus Polymerase Activity in Mammalian Cells

Journal of Virology. Jul, 2011  |  Pubmed ID: 21561908

The 2009 pandemic influenza virus (pH1N1) is a swine-origin reassortant containing human, avian, and swine influenza genes. We have previously shown that the polymerase complex of the pH1N1 strain A/California/04/2009 (Cal) is highly active in mammalian 293T cells, despite the avian origin of both its PA and PB2. In this study, we analyzed the polymerase residues that are responsible for high pH1N1 polymerase activity in the mammalian host. Characterization of polymerase complexes containing various combinations of Cal and avian influenza virus A/chicken/Nanchang/3-120/01 (H3N2) (Nan) by reporter gene assay indicates that Cal PA, but not PB2, is a major contributing factor to high Cal polymerase activity in 293T cells. In particular, Cal PA significantly activates the otherwise inactive Nan polymerase at 37 and 39°C but not at the lower temperature of 34°C. Further analysis using site-directed mutagenesis showed that the Cal PA residues 85I, 186S, and 336M contribute to enhanced activity of the Cal polymerase. Recombinant A/WSN/33 (H1N1) (WSN) viruses containing Nan NP and polymerase (PA, PB1, PB2) genes with individual mutations in PA at residues 85, 186, and 336 produced higher levels of viral protein than the virus containing wild-type (WT) Nan PA. Interestingly, compared to the WT, the virus containing the 85I mutation grew faster in human A549 cells and the 336M mutation most significantly enhanced pathogenicity in a mouse model, among the three PA mutations tested. Our results suggest that multiple mutations in PA, which were rarely present in previous influenza isolates, are involved in mammalian adaptation and pathogenicity of the 2009 pH1N1.

Dazed and Confused by HHV-6

Blood. May, 2011  |  Pubmed ID: 21566101

Lentiviral Vector-mediated Stable Expression of STNFR-Fc in Human Macrophage and Neuronal Cells As a Potential Therapy for NeuroAIDS

Journal of Neuroinflammation. , 2011  |  Pubmed ID: 21569583

Human immunodeficiency virus type 1 (HIV-1) infection frequently causes neurologic disease, which is the result of viral replication and activation of macrophages and microglia in the CNS, and subsequent secretion of high levels of neurotoxic products, including tumor necrosis factor-α (TNF-α). We therefore hypothesized that a soluble TNF-α antagonist might have potential utility as a neuroprotective effecter molecule, and conducted proof-of-concept studies to test this hypothesis.

Comprehensive Proteomic Analysis of Influenza Virus Polymerase Complex Reveals a Novel Association with Mitochondrial Proteins and RNA Polymerase Accessory Factors

Journal of Virology. Sep, 2011  |  Pubmed ID: 21715506

The trimeric RNA polymerase complex (3P, for PA-PB1-PB2) of influenza A virus (IAV) is an important viral determinant of pathogenicity and host range restriction. Specific interactions of the polymerase complex with host proteins may be determining factors in both of these characteristics and play important roles in the viral life cycle. To investigate this question, we performed a comprehensive proteomic analysis of human host proteins associated with the polymerase of the well-characterized H5N1 Vietnam/1203/04 isolate. We identified over 400 proteins by liquid chromatography-tandem mass spectrometry (LC-MS/MS), of which over 300 were found to bind to the PA subunit alone. The most intriguing and novel finding was the large number of mitochondrial proteins (∼20%) that associated with the PA subunit. These proteins mediate molecular transport across the mitochondrial membrane or regulate membrane potential and may in concert with the identified mitochondrion-associated apoptosis inducing factor (AIFM1) have roles in the induction of apoptosis upon association with PA. Additionally, we identified host factors that associated with the PA-PB1 (68 proteins) and/or the 3P complex (34 proteins) including proteins that have roles in innate antiviral signaling (e.g., ZAPS or HaxI) or are cellular RNA polymerase accessory factors (e.g., polymerase I transcript release factor [PTRF] or Supt5H). IAV strain-specific host factor binding to the polymerase was not observed in our analysis. Overall, this study has shed light into the complex contributions of the IAV polymerase to host cell pathogenicity and allows for direct investigations into the biological significance of these newly described interactions.

Robust Antigen-specific Humoral Immune Responses to Sublingually Delivered Adenoviral Vectors Encoding HIV-1 Env: Association with Mucoadhesion and Efficient Penetration of the Sublingual Barrier

Vaccine. Sep, 2011  |  Pubmed ID: 21801777

The efficient induction of virus-specific mucosal antibodies is an important unmet objective in Human Immunodeficiency Virus Type-1 (HIV-1) vaccine research. One promising approach is sublingual (SL) immunization. We examined the effectiveness of SL delivery of two different viral vectors: (i) a recombinant adenovirus (rAd5), and (ii) a Herpes Simplex Virus Type-1 amplicon vector (HSV-1). Initial in vitro videomicroscopy experiments showed that rAd5 particles were trapped in saliva (i.e., that Ad5 was mucoadhesive) - unlike HSV-1 virions, which migrated freely in both saliva and water. In vivo imaging studies in mice revealed that only the rAd5 vector efficiently transduced the SL epithelium. Consistent with this, SL delivery of an rAd5 encoding HIV-1 envelope glycoprotein (Env) resulted in robust antigen-specific antibody responses in plasma and in vaginal washes, whereas SL delivery of a HSV-1 amplicon vector encoding HIV-1 Env failed to elicit Env-specific antibodies. In contrast, both vectors elicited equivalent humoral responses following intramuscular (IM) delivery. Finally, SL delivery of the rAd5:Env vector resulted in elevated levels of Env-specific serum IgA, and vaginal IgA and IgG, when compared to IM delivery of the same vector. These results findings shed light on vector properties (mucoadhesion, penetration of the sublingual barrier) which may be important for the induction of potent humoral immune responses following sublingual vector administration. Our data also show that SL delivery of an Env-encoding rAd5 vector can elicit a potent antigen-specific mucosal antibody response in the absence of adjuvant. Overall, these findings support the further exploration of the SL delivery route for HIV-1 vaccine delivery.

Biochemical Impact of the Host Adaptation-associated PB2 E627K Mutation on the Temperature-dependent RNA Synthesis Kinetics of Influenza A Virus Polymerase Complex

The Journal of Biological Chemistry. Oct, 2011  |  Pubmed ID: 21816827

Most avian influenza A viruses, which preferentially replicate at the high temperatures found in the digestive tract of birds, have a glutamic acid at residue 627 of the viral RNA polymerase PB2 subunit (Glu-627), whereas the human viruses, which optimally replicate at the low temperatures observed in the human respiratory tract, have a lysine (Lys-627). The mechanism of action for this mutation is still not understood, although interaction with host factors has been proposed to play a major role. In this study, we explored an alternative, yet related, hypothesis that this PB2 mutation may alter the temperature-dependent enzymatic polymerase activity of the viral polymerase. First, the avian polymerase protein, which was purified from baculovirus expression system, indeed remained significantly active at higher temperatures (i.e. 37 and 42 °C), whereas the human E627K mutant drastically lost activity at these high temperatures. Second, our steady-state kinetics data revealed that the human E627K mutant polymerase is catalytically more active than the avian Glu-627 polymerase at 34 °C. Importantly, the E627K mutation elevates apparent K(cat) at low temperatures with little effect on K(m), suggesting that the E627K mutation alters the biochemical steps involved in enzyme catalysis rather than the interaction with the incoming NTP. Third, this temperature-dependent kinetic impact of the human E627K mutation was also observed with different RNA templates, with different primers and also in the presence of nucleoprotein. In conclusion, our study suggests that the amino acid sequence variations at residue 627 of PB2 subunit can directly alter the enzyme kinetics of influenza polymerase.

What Factors Underlie Associative and Categorical Memory Illusions? The Roles of Backward Associative Strength and Interitem Connectivity

Journal of Experimental Psychology. Learning, Memory, and Cognition. Aug, 2011  |  Pubmed ID: 21875250

Factors that affect categorical and associative false memory illusions were investigated in 2 experiments. In Experiment 1, backward associative strength (BAS) from the list word to the critical lure and interitem connectivity were manipulated in Deese-Roediger-McDermott (DRM) and category list types. For both recall and recognition tasks, the likelihood of producing DRM and category false memories was greater for lists with high BAS and low interitem connectivity. In Experiment 2, DRM and category lists with high BAS showed similar indirect priming effects in a word stem completion task. With low BAS, category lists, unlike DRM lists, showed no priming effect. We discuss the role of BAS, interitem connectivity, and associate-level differences in implicit and explicit measures of false memory production. (PsycINFO Database Record (c) 2011 APA, all rights reserved).

HIV-1 Tat-induced Microgliosis and Synaptic Damage Via Interactions Between Peripheral and Central Myeloid Cells

PloS One. , 2011  |  Pubmed ID: 21912650

Despite the ability of combination antiretroviral treatment (cART) to reduce viral burden to nearly undetectable levels in cerebrospinal fluid and serum, HIV-1 associated neurocognitive disorders (HAND) continue to persist in as many as half the patients living with this disease. There is growing consensus that the actual substrate for HAND is destruction of normal synaptic architecture but the sequence of cellular events that leads to this outcome has never been resolved. To address whether central vs. peripheral myeloid lineage cells contribute to synaptic damage during acute neuroinflammation we injected a single dose of the HIV-1 transactivator of transcription protein (Tat) or control vehicle into hippocampus of wild-type or chimeric C57Bl/6 mice genetically marked to distinguish infiltrating and resident immune cells. Between 8-24 hr after injection of Tat, invading CD11b(+) and/or myeloperoxidase-positive leukocytes with granulocyte characteristics were found to engulf both microglia and synaptic structures, and microglia reciprocally engulfed invading leukocytes. By 24 hr, microglial processes were also seen ensheathing dendrites, followed by inclusion of synaptic elements in microglia 7 d after Tat injection, with a durable microgliosis lasting at least 28 d. Thus, central nervous system (CNS) exposure to Tat induces early activation of peripheral myeloid lineage cells with phagocytosis of synaptic elements and reciprocal microglial engulfment of peripheral leukocytes, and enduring microgliosis. Our data suggest that a single exposure to a foreign antigen such as HIV-1 Tat can lead to long-lasting disruption of normal neuroimmune homeostasis with deleterious consequences for synaptic architecture, and further suggest a possible mechanism for enduring neuroinflammation in the absence of productive viral replication in the CNS.

Shared Cognitive Processes Underlying Past and Future Thinking: The Impact of Imagery and Concurrent Task Demands on Event Specificity

Journal of Experimental Psychology. Learning, Memory, and Cognition. Sep, 2011  |  Pubmed ID: 21928933

Recent literature has argued that whereas remembering the past and imagining the future make use of shared cognitive substrates, simulating future events places heavier demands on executive resources. These propositions were explored in 3 experiments comparing the impact of imagery and concurrent task demands on speed and accuracy of past event retrieval and future event simulation. Results provide support for the suggestion that both past and future episodes can be constructed through 2 mechanisms: a noneffortful "direct" pathway and a controlled, effortful "generative" pathway. However, limited evidence emerged for the suggestion that simulating of future, compared with retrieving past, episodes places heavier demands on executive resources; only under certain conditions did it emerge as a more error prone and lengthier process. The findings are discussed in terms of how retrieval and simulation make use of the same cognitive substrates in subtly different ways. (PsycINFO Database Record (c) 2011 APA, all rights reserved).

Advances in HIV Microbicide Development

Future Medicinal Chemistry. Dec, 2011  |  Pubmed ID: 22098355

There is an urgent need control the spread of the global HIV pandemic. A microbicide, or topical drug applied to the mucosal environment to block transmission, is a promising HIV prevention strategy. The development of a safe and efficacious microbicide requires a thorough understanding of the mucosal environment and its role in HIV transmission. Knowledge of the key events in viral infection identifies points at which the virus might be most effectively targeted by a microbicide. The cervicovaginal and rectal mucosa play an important role in the innate defense against HIV, and microbicides must not interfere with these functions. In this review, we discuss the current research on HIV microbicide development.

Adenoviral Vector Driven by a Minimal Rad51 Promoter is Selective for P53-deficient Tumor Cells

PloS One. , 2011  |  Pubmed ID: 22174876

The full length Rad51 promoter is highly active in cancer cells but not in normal cells. We therefore set out to assess whether we could confer this tumor-selectivity to an adenovirus vector.

Oligovalent Amyloid-Binding Agents Reduce SEVI-Mediated Enhancement of HIV-1 Infection

Journal of the American Chemical Society. Jan, 2012  |  Pubmed ID: 22239120

This paper evaluates the use of oligovalent amyloid-binding molecules as potential agents that can reduce the enhancement of human immunodeficiency virus-1 (HIV-1) infection in cells by semen-derived enhancer of virus infection (SEVI) fibrils. These naturally occurring amyloid fibrils found in semen have been implicated as mediators that can facilitate the attachment and internalization of HIV-1 virions to immune cells. Molecules that are capable of reducing the role of SEVI in HIV-1 infection may, therefore, represent a novel strategy to reduce the rate of sexual transmission of HIV-1 in humans. Here, we evaluated a set of synthetic, oligovalent derivatives of benzothiazole aniline (BTA, a known amyloid-binding molecule) for their capability to bind cooperatively to aggregated amyloid peptides and to neutralize the effects of SEVI in HIV-1 infection. We demonstrate that these BTA derivatives exhibit a general trend of increased binding to aggregated amyloids as a function of increasing valence number of the oligomer. Importantly, we find that oligomers of BTA show improved capability to reduce SEVI-mediated infection of HIV-1 in cells compared to a BTA monomer, with the pentamer exhibiting a 65-fold improvement in efficacy compared to a previously reported monomeric BTA derivative. These results, thus, support the use of amyloid-targeting molecules as potential supplements for microbicides to curb the spread of HIV-1 through sexual contact.

Test-induced Priming Increases False Recognition in Older but Not Younger Children

Journal of Experimental Child Psychology. Jan, 2012  |  Pubmed ID: 21908005

The effect of test-induced priming on false recognition was investigated in children aged 5, 7, 9, and 11 years using lists of semantic associates, category exemplars, and phonological associates. In line with effects previously observed in adults, nine- and eleven-year-olds showed increased levels of false recognition when critical lures were preceded by four studied items. This pattern was present with all three list types. In contrast, no effects of test-induced priming were observed in five- or seven-year-olds with any list type. The results also support those of previous studies in showing a developmental shift from phonological to semantic false memories. The findings are discussed in terms of current theories of children's false memories.

A Gender Difference in the False Recall of Negative Words: Women DRM More Than Men

Cognition & Emotion. , 2012  |  Pubmed ID: 21432635

Gender differences in susceptibility to associative memory illusions in the Deese/Roediger-McDermott paradigm were investigated using negative and neutral word lists. Women (n=50) and men (n=50) studied 20 lists of 12 words that were associates of a non-presented critical lure. Ten lists were associates of negatively valenced lures (e.g., cry, evil) and ten were associates of neutral lures (e.g., chair, slow). When asked to recall the words after each list, women falsely recalled more negative lures than men, but there was no gender difference in the false recall of neutral lures. These findings suggest that women reflect on associations within negative lists to a greater degree than men and are thereby more likely to generate the negative critical lures.