Gene transfer into the central nervous system using Herpes Simplex Virus-1 vectors

Manipulation of gene expression in developing or in mature central nervous systems (CNS) holds a promise for the resolution of many compelling neurobiological questions, including the feasibility of gene therapy to treat diseases of the brain. In this context, a number of viral vectors has been used in recent years to introduce and express genes into the CNS. This article discusses a gene transfer system based on the Herpes Simplex Virus-1 (HSV-1). We describe here the use of non-replicating, non-toxic HSV-1 vector, 8117/43, in a series of studies carried in our joint program. This vector proves further the utility of HSV-1 as a delivery vehicle to a number of distinct sites within the CNS

Polyethyleneimine-mediated transfection of cultured postmitotic neurons from rat sympathetic ganglia and adult human retina

BACKGROUND: Chemical methods of transfection that have proven successful with cell lines often do not work with primary cultures of neurons. Recent data, however, suggest that linear polymers of the cation polyethyleneimine (PEI) can facilitate the uptake of nucleic acids by neurons. Consequently, we examined the ability of a commercial PEI preparation to allow the introduction of foreign genes into postmitotic mammalian neurons. Sympathetic neurons were obtained from perinatal rat pups and maintained for 5 days in vitro in the absence of nonneuronal cells. Cultures were then transfected with varying amounts of a plasmid encoding either E. coli β-galactosidase or enhanced green fluorescence protein (EGFP) using PEI. RESULTS: Optimal transfection efficiency was observed with 1 μg/ml of plasmid DNA and 5 μg/ml PEI. Expression of β-galactosidase was both rapid and stable, beginning within 6 hours and lasting for at least 21 days. A maximum yield was obtained within 72 hours with ∼ 9% of the neurons expressing β-galactosidase, as assessed by both histochemistry and antibody staining. Cotransfection of two plasmids encoding reporter genes was achieved. Postmitotic neurons from adult human retinal cultures also demonstrated an ability to take up and express foreign DNA using PEI as a vector. CONCLUSIONS: These data suggest that PEI is a useful agent for the stable expression of plasmid-encoded genes in neuronal cultures

Systems Genome:Coordinated Gene Activity Networks, Recurring Coordination Modules, and Genome Homeostasis in Developing Neurons

Simple Summary: A synchronized global genome is a flexible, homeostatic system that underwrites ontogenic development and deprograming in disease. Abstract: As human progenitor cells differentiate into neurons, the activities of many genes change; these changes are maintained within a narrow range, referred to as genome homeostasis. This process, which alters the synchronization of the entire expressed genome, is distorted in neurodevelopmental diseases such as schizophrenia. The coordinated gene activity networks formed by altering sets of genes comprise recurring coordination modules, governed by the entropy-controlling action of nuclear FGFR1, known to be associated with DNA topology. These modules can be modeled as energy-transferring circuits, revealing that genome homeostasis is maintained by reducing oscillations (noise) in gene activity while allowing gene activity changes to be transmitted across networks; this occurs more readily in neuronal committed cells than in neural progenitors. These findings advance a model of an “entangled” global genome acting as a flexible, coordinated homeostatic system that responds to developmental signals, is governed by nuclear FGFR1, and is reprogrammed in disease

Assessment of viral and non-viral gene transfer into adult rat brains using HSV-1, calcium phosphate and PEI-based methods

CNS gene transfer could provide new approaches to the modelling of neurodegenerative diseases and devising potential therapies. One such disorder is Parkinson’s disease (PD), in which dysfunction of several different metabolic processes has been implicated. Here we review the literature on gene transfer systems based on herpes simplex virus type 1 (HSV-1) and non-viral polyethyleneimine (PEI) and calcium phosphate nanoparticle methods. We also assess the usefulness of various CNS gene delivery methods and present some of our own data to exemplify such usefulness. Our data result from vectors stereotaxically introduced to the substantia nigra (SN) of adult rats and evaluated 1 week and/or 1 month post injection using histochemical methods to assess recombinant ß-galactosidase enzyme activity. Gene transfer using PEI or calcium phosphate-mediated transfections was observed for both methods and PEI was comparable to that of HSV-1 amplicon. Our data show that the amplicon delivery was markedly increased when packaged with a helper virus and was similar to the expression profile achieved with a full-size replication-defective HSV-1 recombinant (8117/43). We also examine whether PEI or HSV-1 amplicon-mediated gene transfer could facilitate assessment of the biological effects induced by a dominant negative FGF receptor-1 mutant to model the reduced FGF signalling thought to occur in Parkinson’s disease

Effect of three common SNPs in 5′-flanking region of LEP and ADIPOQ genes on their expression in Polish obese children and adolescents

Genes encoding adipokines are considered as candidates for human obesity. In this study we analyzed the expression of leptin (LEP) and adiponectin (ADIPOQ) genes in relation to common 5′-flanking or 5′UTR variants: -2548G>A (LEP), 19A>G (LEP) and -11377C>G (ADIPOQ) in Polish obese children and adolescents. Relative transcription levels in the subcutaneous adipose tissue (real time RT–PCR) and serum protein concentrations (RIA) were measured in 48 obese subjects with known genotypes at three polymorphic sites and in five non-obese controls. None of the studied polymorphisms altered significantly the expression. Significantly elevated relative transcription levels of the LEP gene (P < 0.05) and serum leptin concentrations (P < 0.01) were recorded in obese patients, when compared with the non-obese controls, but such differences were not found for the ADIPOQ gene. Interestingly, the leptin to adiponectin protein concentration ratio (L/A) was approximately sevenfold higher in obese children and adolescents when compared with the non-obese controls (P < 0.001). Taking into consideration the observed relationship between the genotypes and the gene expression level we suggest that these SNPs are not conclusive markers for predisposition to obesity in Polish children and adolescents. On the other hand, we confirmed that the leptin to adiponectin gene expression ratio (L/A) is an informative index characterizing obesity

HMG1A and PPARG are differently expressed in the liver of fat and lean broilers

The expression of nine functional candidates for QT abdominal fat weight and relative abdominal fat content was investigated by real-time polymerase chain reaction (PCR) in the liver, adipose tissue, colon, muscle, pituitary gland and brain of broilers. The high mobility group AT-hook 1 (HMG1A) gene was up-regulated in liver with a ratio of means of 2.90 (P ≤ 0.01) in the «fatty» group (relative abdominal fat content 3.5 ± 0.18%, abdominal fat weight 35.4 ± 6.09 g) relative to the «lean» group (relative abdominal fat content 1.9 ± 0.56%, abdominal fat weight 19.2 ± 5.06 g). Expression of this gene was highly correlated with the relative abdominal fat content (0.70, P ≤ 0.01) and abdominal fat weight (0.70, P ≤ 0.01). The peroxisome proliferator-activated receptor gamma (PPARG) gene was also up-regulated in the liver with a ratio of means of 3.34 (P ≤ 0.01) in the «fatty» group relative to the «lean» group. Correlation of its expression was significant with both the relative abdominal fat content (0.55, P ≤ 0.05) and the abdominal fat weight (0.57, P ≤ 0.01). These data suggest that the HMG1A and PPARG genes were candidate genes for abdominal fat deposition in chickens. Searching of rSNPs in regulatory regions of the HMG1A and PPARG genes could provide a tool for gene-assisted selection

Universe from vacuum in loop-string cosmology

In this paper we study the description of the Universe based on the low energy superstring theory modified by the Loop Quantum Gravity effects.This approach was proposed by De Risi et al. in the Phys. Rev. D {\bf 76} (2007) 103531. We show that in the contrast with the string motivated pre-Big Bang scenario, the cosmological realisation of the $t$-duality transformation is not necessary to avoid an initial singularity. In the model considered the universe starts its evolution in the vacuum phase at time $t\to - \infty$. In this phase the scale factor $a\to 0$, energy density $\rho \to 0$ and coupling of the interactions $g^2_s \to 0$. After this stage the universe evolves to the non-singular hot Big Bang phase $\rho \to \rho_{\text{max}} < \infty$. Then the standard classical universe emerges. During the whole evolution the scale factor increases monotonically. We solve this model analytically. We also propose and solve numerically the model with an additional dilaton potential in which the universe starts the evolution from the asymptotically free vacuum phase $g^2_s \to 0$ and then evolves non-singularly to the emerging dark energy dominated phase with the saturated coupling constant $g^2_s \to \text{const}$.Comment: JHEP3 LaTeX class, 19 pages, 9 figures, v2: added some comments and references, v3: new numerical result added, new figure

Cytotoxicity of Bacterial Metabolic Products, including Listeriolysin O, on Leukocyte Targets

Bacterial toxins can exhibit anticancer activities. Here we investigated the anticancer effects of the listeriolysin O toxin produced by Listeria monocytogenes. We found that supernatants of Listeria monocytogenes strains (wild type, 1189, and 1190) were cytotoxic to the Jurkat cell line and human peripheral blood mononuclear cells (PBMC) in a concentration-dependent manner. The supernatant of strain 1044, not producing listeriolysin O, was inactive. The supernatants of Listeria strains were also cytotoxic toward B cells of chronic leukemia patients, with no significant differences in activities between strains. We also tested supernatants of Bacillus subtilis strains BR1-90, BR1-S, and BR1-89 producing listeriolysin O. BR1-S and BR1-89 were cytotoxic to PBMC and to Jurkat cells, the latter being more sensitive to the supernatants. BR1-90 was not hemolytic or cytotoxic to PBMC, but was cytotoxic to Jurkat cells in the concentration range of 10–30%, suggesting that listeriolysin O is selectively effective against T cells. Overall, the response of human peripheral blood mononuclear and human leukemia cell lines to bacteria supernatants containing listeriolysin O depended on the bacteria strain, target cell type, and supernatant concentration