Andrew I. Dayton

Identification of a Potential HIV-Induced Source of Bystander-Mediated Apoptosis in T Cells: Upregulation of TRAIL in Primary Human Macrophages by HIV-1 Tat

The induction of apoptosis in T cells by bystander cells has been repeatedly implicated as a mechanism contributing to the T cell depletion seen in HIV infection. It has been shown that apoptosis could be induced in T cells from asymptomatic HIV-infected individuals in a Fas-independent, TNF-related apoptosis-inducing ligand (TRAIL)-dependent manner if the cells were pretreated with anti-CD3. It has also been shown that T cells from HIV-infected patients were even more sensitive to TRAIL induction of apoptosis than they were to Fas induction. Recently, it has been reported that in an HIV-1 SCID-Hu model, the vast majority of the T cell apoptosis is not associated with p24 and is therefore produced by bystander effects. Furthermore, few apoptotic cells were associated with neighboring cells which were positive for either Fas ligand or TNF. However, most of the apoptotic cells were associated with TRAIL-positive cells. The nature of these TRAIL-positive cells was undetermined. Here, we report that HIV infection of primary human macrophages switches on abundant TRAIL production both at the RNA and protein levels. Furthermore, more macrophages produce TRAIL than are infected by HIV, indicating that a bystander mechanism may, at least in part, upregulate TRAIL. Exogenously supplied HIV-1 Tat protein upregulates TRAIL production by primary human macrophages to an extent indistinguishable from infection. The results suggest a model in which HIV-1-infected cells produce extracellular Tat protein, which in turn upregulates TRAIL in macrophages which then can induce apoptosis in bystander T cells.

Development of Dengue virus type 2 replicons capable of prolonged expression in host cells

BackgroundAs part of a program to develop a Dengue virus vaccine which avoids the deleterious effects of antibody dependent enhancement (ADE) of infection mediated by antibodies to Dengue virus structural proteins, we have begun to investigate the possibility of designing Dengue vaccines based on non-structural proteins.ResultsDengue constructs which lack major structural proteins replicate intracellularly in tissue culture. These replicons are capable of prolonged expression of Dengue virus non-structural proteins for at least seven days in culture.ConclusionsDengue virus genomes lacking major structural proteins can, like other flaviviruses, replicate intracellularly and express virus non-structural proteins with minimal toxicity to host cells. These findings pave the way for the development of dengue virus replicons as a form of live, attenuated virus vaccine.

Monocytes-macrophages are a potential target in human infection with West Nile virus through blood transfusion

BACKGROUND:  West Nile virus (WNV) transmission by transfusion was documented in 2002. Approximately 80 percent of WNV infections are asymptomatic and 1 percent develop severe neurological illness. In animals, Langerhans‐dendritic cells support initial viral replication, followed by replication in lymphoid tissues and dissemination to organs and possibly to the CNS. The cellular tropism of WNV infection after transfusion and the particular human blood cells that sustain viral replication remain largely unknown. Whether primary monocyte‐derived macrophages (MDMs) support WNV infection‐replication and produce infectious virions, with an in vitro system, was investigated.

The Rev Axis of HIV-1 and Its Associated Host Cofactors: A Viral Window onto the Workings of Eukaryotic Posttranscriptional RNA Processing.

The Rev axis of HIV is one of two key autoregulatory pathways required for viral replication and pathogenesis. The viral Rev protein interacts with its RNA target sequence, the RRE, to overcome the inhibitory effects of constitutive repressor sequences and promote nucleocytoplasmic transport and expression of viral RNAs. The Rev axis is the subject of intense scrutiny not only because it plays a central role in the viral life cycle, but also because it offers a window onto the workings of key mechanisms of posttranscriptional regulation, including splicing, polyadenylation, degradation, transport, and translation. Recent reports have conclusively demonstrated a central role for transport in the Rev mechanism and have identified cellular factors that are good candidates for mediating the transport phenomena. Other potentially involved cellular factors are being investigated. Much of the apparent heterogeneity in the observed effects of Rev may actually derive from heterogeneity in the constitutive repressor sequences rather than from heterogeneity in the mechanism of action of Rev per se. Copyright 1996 S. Karger AG, Basel

MicroRNA regulation of STAT4 protein expression: rapid and sensitive modulation of IL-12 signaling in human natural killer cells.

IL-12 exerts several regulatory effects on natural killer (NK) cells by activating IL-12 signaling. IL-12 signaling is tightly auto-regulated to control its onset and termination, with prolonged IL-12 treatment resulting in IL-12 hyporesponsiveness. However, the mechanisms underlying IL-12 auto-regulation are still unclear. In this study we report that prolonged IL-12 treatment significantly up-regulates microRNAs (miRNAs), including miR-132, -212, and -200a in primary human NK cells. This up-regulation correlates temporally with gradually decreasing STAT4 levels and decreasing IFN-γ expression, after an initial increase within the first 16 hours of IL-12 treatment. The IL-12 hyporesponsiveness is dependent on IL-12 concentration, and associated up-regulation of miR-132, -212, and -200a. Furthermore, IL-12-hyporesponsive cells regain responsiveness of IFN-γ production 24 hours after IL-12 removal, which correlates with decreases in miR-132, -212, and -200a levels. Overexpression of miR-132, -212, and -200a by transfection into NK cells mimics IL-12 priming, inducing IL-12 hyporesponsiveness, whereas transfection of miR-132, -212, and -200a inhibitors largely abolishes IL-12 induction of IL-12 hyporesponsiveness. These data suggest that miR-132, -212, and -200a up-regulation during prolonged IL-12 treatment, negatively regulates the IL-12 signaling pathway by reducing STAT4 expression in primary human NK cells.

Quantitative estimate of the risks and benefits of possible alternative blood donor deferral strategies for men who have had sex with men

BACKGROUND: Implementation of sensitive screening methods for human immunodeficiency virus (HIV) and hepatitis viruses prompts the question of what quantitative risks may result from altered deferral strategies for donation of blood by men who have had sex with men (MSM).

Bcl-2 Upregulation by HIV-1 Tat during Infection of Primary Human Macrophages in Culture

The ability of cells of the human monocyte/macrophage lineage to host HIV-1 replication while resisting cell death is believed to significantly contribute to their ability to serve as a reservoir for viral replication in the host. Although macrophages are generally resistant to apoptosis, interruption of anti-apoptotic pathways can render them susceptible to apoptosis. Here we report that HIV-1(BAL )infection of primary human monocyte-derived macrophages (MDM) upregulates the mRNA and protein levels of the anti-apoptic gene, Bcl-2. Furthermore, this upregulation can be quantitatively mimicked by treating MDM with soluble HIV-1 Tat-86 protein. These results suggest that in infecting cells of the monocyte/macrophage lineage, HIV-1 may be benefiting from additional protection against apoptosis caused by specific upregulation of cellular anti-apoptotic genes.

Anti-West Nile virus activity of in vitro expanded human primary natural killer cells

BackgroundNatural Killer (NK) cells are a crucial component of the host innate immune system with anti-viral and anti-cancer properties. However, the role of NK cells in West Nile virus (WNV) infection is controversial, with reported effects ranging from active suppression of virus to no effect at all. It was previously shown that K562-mb15-41BBL (K562D2) cells, which express IL-15 and 4-1BBL on the K562 cell surface, were able to expand and activate human primary NK cells of normal peripheral blood mononuclear cells (PBMC). The expanded NK cells were tested for their ability to inhibit WNV infection in vitro.ResultsCo-culture of PBMC with irradiated K562D2 cells expanded the NK cell number by 2-3 logs in 2-3 weeks, with more than 90% purity; upregulated NK cell surface activation receptors; downregulated inhibitory receptors; and boosted interferon gamma (IFN-γ) production by ~33 fold. The expanded NK (D2NK) cell has strong natural killing activity against both K562 and Vero cells, and killed the WNV infected Vero cells through antibody-dependent cellular cytotoxicity (ADCC). The D2NK cell culture supernatants inhibited both WNV replication and WNV induced cytopathic effect (CPE) in Vero cells when added before or after infection. Anti-IFN-γ neutralizing antibody blocked the NK supernatant-mediated anti-WNV effect, demonstrating a noncytolytic activity mediated through IFN-γ.ConclusionsCo-culture of PBMC with K562D2 stimulatory cells is an efficient technique to prepare large quantities of pure and active NK cells, and these expanded NK cells inhibited WNV infection of Vero cells through both cytolytic and noncytolytic activities, which may imply a potential role of NK cells in combating WNV infection.

Development of dengue virus replicons expressing HIV-1 gp120 and other heterologous genes: a potential future tool for dual vaccination against dengue virus and HIV

BackgroundToward the goals of providing an additional vector to add to the armamentarium available to HIV vaccinologists and of creating a bivalent vaccine effective against dengue virus and HIV, we have attempted to create vectors which express dengue virus non-structural proteins and HIV immunogens. Previously we reported the successful construction of dengue virus replicons which lack structural genes necessary for virion release and spreading infection in culture but which can replicate intracellularly and abundantly produce dengue non-structural proteins. Here we attempted to express heterologous genetic material from these replicons.ResultsWe cloned into a Δpre-M/E dengue virus replicon genes for either green fluorescent protein (GFP), HIV gp160 or HIV gp120 and tested the ability of these constructs to express dengue virus proteins as well as the heterologous proteins in tissue culture after transfection of replicon RNA.ConclusionsHeterologous proteins were readily expressed from these constructs. GFP and gp120 demonstrated minimal or no toxicity. Gp160 expressing replicons were found to express proteins abundantly at 36 hours post transfection, but after 50 hrs of transfection, few replicon positive cells could be found despite the presence of cellular debris positive for replicon proteins. This suggested that gp160 expressed from dengue virus replicons is considerably more toxic than either GFP or gp120. The successful expression of heterologous proteins, including HIV gp120 for long periods in culture suggests this vector system may be useful as a vaccine vector, given appropriate delivery methods.

Interleukin-12 treatment down-regulates STAT4 and induces apoptosis with increasing ROS production in human natural killer cells

NK cells are prominent mediators of the immunomodulating and antiangiogenic activity of IL‐12. However, the effect of prolonged IL‐12 treatment on NK cells is unclear. In this study, we observed that IL‐12 initially activates NK cells, but prolonged IL‐12 treatment specifically down‐regulates IL‐12 signaling and induces NK cell apoptosis associated with a significant reduction in cytolytic activity and IFN‐γ production in response to further IL‐12 stimulation. Further results demonstrate that prolonged IL‐12 stimulation of NK cells specifically decreases the level of activated STAT4 protein, a critical IL‐12 signaling component, through decreasing STAT4 mRNA and protein levels rather than inducing STAT4 protein degradation. IL‐12 treatment induces NK cell activation as well as levels of ROS, but prolonged IL‐12 treatment causes ROS accumulation, which in turn, results in the loss of Δψm, the release of cytochrome c, and the activation of caspase‐3, resulting in NK cell apoptosis. These findings provide new insights into IL‐12 regulation in human NK cells, where IL‐12 initially promotes NK cell activation but subsequently limits this response through a negative‐feedback mechanism.