Comparison of Replication-Competent, First Generation, and Helper-Dependent Adenoviral Vaccines

All studies using human serotype 5 Adenovirus (Ad) vectors must address two major obstacles: safety and the presence of pre-existing neutralizing antibodies. Helper-Dependent (HD) Ads have been proposed as alternative vectors for gene therapy and vaccine development because they have an improved safety profile. To evaluate the potential of HD-Ad vaccines, we compared replication-competent (RC), first-generation (FG) and HD vectors for their ability to induce immune responses in mice. We show that RC-Ad5 and HD-Ad5 vectors generate stronger immune responses than FG-Ad5 vectors. HD-Ad5 vectors gave lower side effects than RC or FG-Ad, producing lower levels of tissue damage and anti-Ad T cell responses. Also, HD vectors have the benefit of being packaged by all subgroup C serotype helper viruses. We found that HD serotypes 1, 2, 5, and 6 induce anti-HIV responses equivalently. By using these HD serotypes in heterologous succession we showed that HD vectors can be used to significantly boost anti-HIV immune responses in mice and in FG-Ad5-immune macaques. Since HD vectors have been show to have an increased safety profile, do not possess any Ad genes, can be packaged by multiple serotype helper viruses, and elicit strong anti-HIV immune responses, they warrant further investigation as alternatives to FG vectors as gene-based vaccines

T7 RNA Polymerase Functions In Vitro without Clustering

Many nucleic acid polymerases function in clusters known as factories. We investigate whether the RNA polymerase (RNAP) of phage T7 also clusters when active. Using ‘pulldowns’ and fluorescence correlation spectroscopy we find that elongation complexes do not interact in vitro with a Kd<1 µM. Chromosome conformation capture also reveals that genes located 100 kb apart on the E. coli chromosome do not associate more frequently when transcribed by T7 RNAP. We conclude that if clustering does occur in vivo, it must be driven by weak interactions, or mediated by a phage-encoded protein

Pressure-induced amorphous-to-amorphous configuration change in Ca-Al metallic glasses

Pressure-induced amorphous-to-amorphous configuration changes in Ca-Al metallic glasses (MGs) were studied by performing in-situ room-temperature high-pressure x-ray diffraction up to about 40 GPa. Changes in compressibility at about 18 GPa, 15.5 GPa and 7.5 GPa during compression are detected in Ca80Al20, Ca72.7Al27.3, and Ca66.4Al33.6 MGs, respectively, whereas no clear change has been detected in the Ca50Al50 MG. The transfer of s electrons into d orbitals under pressure, reported for the pressure-induced phase transformations in pure polycrystalline Ca, is suggested to explain the observation of an amorphous-to-amorphous configuration change in this Ca-Al MG system. Results presented here show that the pressure induced amorphous-to-amorphous configuration is not limited to f electron-containing MGs

Rebound Discharge in Deep Cerebellar Nuclear Neurons In Vitro

Neurons of the deep cerebellar nuclei (DCN) play a critical role in defining the output of cerebellum in the course of encoding Purkinje cell inhibitory inputs. The earliest work performed with in vitro preparations established that DCN cells have the capacity to translate membrane hyperpolarizations into a rebound increase in firing frequency. The primary means of distinguishing between DCN neurons has been according to cell size and transmitter phenotype, but in some cases, differences in the firing properties of DCN cells maintained in vitro have been reported. In particular, it was shown that large diameter cells in the rat DCN exhibit two phenotypes of rebound discharge in vitro that may eventually help define their functional roles in cerebellar output. A transient burst and weak burst phenotype can be distinguished based on the frequency and pattern of rebound discharge immediately following a hyperpolarizing stimulus. Work to date indicates that the difference in excitability arises from at least the degree of activation of T-type Ca2+ current during the immediate phase of rebound firing and Ca2+-dependent K+ channels that underlie afterhyperpolarizations. Both phenotypes can be detected following stimulation of Purkinje cell inhibitory inputs under conditions that preserve resting membrane potential and natural ionic gradients. In this paper, we review the evidence supporting the existence of different rebound phenotypes in DCN cells and the ion channel expression patterns that underlie their generation

Mechanisms of HIV-associated lymphocyte apoptosis: 2010

The inevitable decline of CD4T cells in untreated infection with the Human immunodeficiency virus (HIV) is due in large part to apoptosis, one type of programmed cell death. There is accumulating evidence that the accelerated apoptosis of CD4T cells in HIV infection is multifactorial, with direct viral cytotoxicity, signaling events triggered by viral proteins and aberrant immune activation adding to normal immune defense mechanisms to contribute to this phenomenon. Current antiviral treatment strategies generally lead to reduced apoptosis, but this approach may come at the cost of preserving latent viral reservoirs. It is the purpose of this review to provide an update on the current understanding of the role and mechanisms of accelerated apoptosis of T cells in the immunopathogenesis of HIV infection, and to highlight potential ways in which this seemingly deleterious process could be harnessed to not just control, but treat HIV infection

Macrophage signaling in HIV-1 infection

The human immunodeficiency virus-1 (HIV-1) is a member of the lentivirus genus. The virus does not rely exclusively on the host cell machinery, but also on viral proteins that act as molecular switches during the viral life cycle which play significant functions in viral pathogenesis, notably by modulating cell signaling. The role of HIV-1 proteins (Nef, Tat, Vpr, and gp120) in modulating macrophage signaling has been recently unveiled. Accessory, regulatory, and structural HIV-1 proteins interact with signaling pathways in infected macrophages. In addition, exogenous Nef, Tat, Vpr, and gp120 proteins have been detected in the serum of HIV-1 infected patients. Possibly, these proteins are released by infected/apoptotic cells. Exogenous accessory regulatory HIV-1 proteins are able to enter macrophages and modulate cellular machineries including those that affect viral transcription. Furthermore HIV-1 proteins, e.g., gp120, may exert their effects by interacting with cell surface membrane receptors, especially chemokine co-receptors. By activating the signaling pathways such as NF-kappaB, MAP kinase (MAPK) and JAK/STAT, HIV-1 proteins promote viral replication by stimulating transcription from the long terminal repeat (LTR) in infected macrophages; they are also involved in macrophage-mediated bystander T cell apoptosis. The role of HIV-1 proteins in the modulation of macrophage signaling will be discussed in regard to the formation of viral reservoirs and macrophage-mediated T cell apoptosis during HIV-1 infection