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Background. Traumatic brain injury (TBI) is the main cause of disabilities over the industrialized countries. Cognitive decline appears in the chronic phase of the pathology consecutively to cellular and molecular processes. Here we described the use of KCC2, a neuronal-specific potassium-chloride transporters as potent biomarker to predict cognitive dysfunctions after TBI. Methods. Using neuronal and total exosomes collection from blood serum in control and TBI subjects we were able to anticipate the decline of cognitive performance. Results. After TBI, we observed a significative and persistant loss of KCC2 expression in the blood exosomes that is correlated to changes in network activity and cellular processes such as secondary neurogenesis. Also we correlated this KCC2 loss in expression to the appearance of the cognitive decline observed in mice and more particularly we correlate the KCC2 loss of expression to the appearance of the depressive-like behavior. Conclusion. According to our protocol, we were able to confirm our previous findings in agreement with the potential therapeutic effect of bumetanide in the prevention of the post traumatic depression after TBI, by restoring the KCC2 expression thus preventing the massive neuronal death of interneurons and the secondary neurogenesis effect observed in such model.

The prevalence of retinal disorders associated with visual impairment and blindness is increasing worldwide, while most of them remain without effective treatment. Pharmacological and molecular therapy development is hampered by the lack of effective drug delivery into the posterior segment of the eye. Among molecular approaches, RNA-interference (RNAi) features strong advantages, yet delivering it to the inner layer of the retina appears extremely challenging. To address this, we developed an original magnetic nanoparticles (MNPs)-based transfection method that allows the efficient delivery of siRNA in all retinal layers of rat adult retinas through magnetic targeting. To establish delivery of RNAi throughout the retina, we have chosen organotypic retinal explants as an ex vivo model and for future high content screening of molecular drugs. Conversely to classic Magnetofection, and similar to conditions in the posterior chamber of the eye, our methods allows attraction of siRNA complexed to MNPs from the culture media into the explant. Our method termed "Reverse Magnetofection" provides a novel and nontoxic strategy for RNAi-based molecular as well as gene therapy in the retina that can be transferred to a wide variety of organ explants.

Advances in RNA technology during the past two decades have led to the construction of replication-competent RNA, termed replicons, RepRNA, or self-amplifying mRNA, with high potential for vaccine applications. Cytosolic delivery is essential for their translation and self-replication, without infectious progeny generation, providing high levels of antigen expression for inducing humoral and cellular immunity. Synthetic nanoparticle-based delivery vehicles can both protect the RNA molecules and facilitate targeting of dendritic cells—critical for immune defense development. Several cationic lipids were assessed, with RepRNA generated from classical swine fever virus encoding nucleoprotein genes of influenza A virus. The non-cytopathogenic nature of the RNA allowed targeting to dendritic cells without destroying the cells—important for prolonged antigen production and presentation. Certain lipids were more effective at delivery and at promoting translation of RepRNA than others. Selection of particular lipids provided delivery to dendritic cells that resulted in translation, demonstrating that delivery efficiency could not guarantee translation. The observed translation in vitro was reproduced in vivo by inducing immune responses against the encoded influenza virus antigens. Cationic lipid-mediated delivery shows potential for promoting RepRNA vaccine delivery to dendritic cells, particularly when combined with additional delivery elements. Keywords: replicon, RepRNA, self-amplifying, cationic lipids, dendritic cells, nanoparticle delivery, influenza vaccines, humoral immunity, cellular immunity

International audience; Background: Gene delivery within the central nervous system at postnatal age is one of the most challenging tasks in neuroscience and currently only a few effective methods are available. Comparison with existing methods: For postnatal central nervous system cells, viral approaches are commonly used for genetic engineering but they face several biosafety requirements for production and use making them less accessible to the community. Conversely, lipid-based methods are widely used in cell culture but face limitation in vivo mainly due to the inflammatory responses they induce. To this aspect, the use of a transgenic mouse line can represent a credible answer to the community working on rat models still requires an effective and successful solution to circumvent these difficulties. New method: We describe a new polymer-based gene delivery system allowing persistent and robust in vivo transfection with low DNA amount, reduced inflammation and high diffusion. The expression profile along the brain, the stability, the diffusion of the DNA together with the quantity of cells transfected were evaluated through in vivo approaches. Results: With a single low-volume injection, we targeted different cell types within the rat brain. We measured the diffusion rate ranging from 1 to 5 mm based on the injected volume, in the three-dimensions axis. Finally, we modified brain susceptibility to epileptic seizures using a specific knock-down of the neuronal specific potassium chloride transporter 2. Conclusions: This safe and easy system opens perspectives for non viral gene delivery in the rat brain with perspectives to study brain function in vivo.

Microglia, the resident phagocytic cells of the brain, have recently been the subject of intense investigation given their role in pathology and normal brain physiology. In general, phagocytic cells are hard to transfect with plasmid DNA. The BV2 cell line is a murine cell line of microglial origin which is often used to study this cell type in vitro. Unfortunately, this microglial cell line is, like other phagocytic cells, resistant to transfection.Magnetofection is a well-established transfection method that combines DNA with magnetic particles which, under the influence of a magnetic field, ensures a high concentration of particles in proximity of cultured cells. Only recently, Glial-Mag was specifically developed for efficient transfection of microglia and microglial cell lines.Magnetofection with Glial-Mag yielded a transfection efficiency of 34.95% in BV2 cells, 24h after transfection with an eGFP-expressing plasmid. Efficient gene delivery caused a modest and short-lived cell activation (as measured by IL6 secretion) that ceased by 24h after transfection.Here we show that Glial-Mag magnetofection of BV2 cells yielded a significantly higher transfection efficiency (34.95%) compared to other chemical transfection methods including calcium-phoshate precipication (0.34%), X-tremeGENE (3.30%) and Lipofectamine 2000 (12.51%).Transfection of BV2 cells using Glial-Mag magnetofection is superior compared to other chemical transfection methods and could be considered as the method of choice to chemically transfect microglial cell lines.

Background: 3D matrices are widely used as cell growth supports in basic research, regenerative medicine or cell-based drug assays. In order to genetically manipulate cells cultured within 3D matrices, two novel non-viral transfection reagents allowing preparation of matrices for in situ cell transfection were evaluated. Results: Two lipidic formulations, 3D-Fect™ and 3D-FectIN™, were assessed for their ability to transfect cells cultured within 3D solid scaffolds and 3D hydrogels, respectively. These reagents showed good compatibility with the most widespread types of matrices and enabled transfection of a wide range of mammalian cells of various origins. Classical cell lines, primary cells and stem cells were thus genetically modified while colonizing their growth support. Importantly, this in situ strategy alleviated the need to manipulate cells before seeding them. Conclusion: Results presented here demonstrated that 3D-Fect and 3D-FectIN reagents for 3D transfection are totally compatible with cells and do not impair matrix properties. 3D-Fect and 3D-FectIN, therefore, provide valuable tools for achieving localized and sustained transgene expression and should find versatile applications in fundamental research, regenerative medicine and cell-based drug assays.

Most current vaccines are either inactivated pathogen-derived or protein/peptide-based, although attenuated and vector vaccines have also been developed. The former induce at best moderate protection, even as multimeric antigen, due to limitations in antigen loads and therefore capacity for inducing robust immune defense. While attenuated and vector vaccines offer advantages through their replicative nature, drawbacks and risks remain with potential reversion to virulence and interference from preexisting immunity. New advances averting these problems are combining self-amplifying replicon RNA (RepRNA) technology with nanotechnology. RepRNA are large self-replicating RNA molecules (12-15 kb) derived from viral genomes defective in at least one structural protein gene. They provide sustained antigen production, effectively increasing vaccine antigen payloads over time, without the risk of producing infectious progeny. The major limitation with RepRNA is RNase-sensitivity and inefficient uptake by dendritic cells (DCs)-absolute requirements for efficacious vaccine design. We employed biodegradable delivery vehicles to protect the RepRNA and promote DC delivery. Encapsulating RepRNA into chitosan nanoparticles, as well as condensing RepRNA with polyethylenimine (PEI), cationic lipids, or chitosans, has proven effective for delivery to DCs and induction of immune responses in vivo.

Gene therapy offers exciting opportunities for the treatment of innate or acquired genetic diseases. However, there is still a need for a safe and efficient strategy to deliver nucleic acids into cells while overcoming the current limitations faced with standard viral vectors. Intensive researches have been carried out over the past decade, focusing both on viral and non-viral (i.e. physical or chemical) strategies. Of these numerous attempts, magnetofection, defined as the combination of nucleic acid vectors with magnetic nanoparticles, holds the promise to achieve high transfection efficiency with reduced toxicity by magnetically focusing the genetic material to be delivered on its cellular target. In vitro as well as in vivo results already demonstrated that this strategy may become a valuable tool towards practical gene therapy.

In recent years, gene therapy has received considerable interest as a potential method for the treatment of numerous inherited and acquired diseases. However, successes have so far been hampered by several limitations, including safety issues of viral-based nucleic acid vectors and poor in vivo efficiency of nonviral vectors. Magnetofection™ has been introduced as a novel and powerful tool to deliver genetic material into cells. This technology is defined as the delivery of nucleic acids, either ‘naked’ or packaged (as complexes with lipids or polymers, and viruses) using magnetic nanoparticles under the guidance of an external magnetic field. This article first discusses the principles of the Magnetofection technology and its benefits as compared with standard transfection methods. A number of relevant examples of its use, both in vitro and in vivo, will then be highlighted. Future trends in the development of new magnetic nanoparticle formulations will also be outlined.

Adenoviruses are among the most powerful gene delivery systems. Even if they present low potential for oncogenesis, there is still a need for minimizing widespread delivery to avoid deleterious reactions. In this study, we investigated Magnetofection efficiency to concentrate and guide vectors for an improved targeted delivery. Magnetic nanoparticles formulations were complexed to a replication defective Adenovirus and were used to transduce cells both in vitro and in vivo. A new integrated magnetic procedure for cell sorting and genetic modification (i-MICST) was also investigated. Magnetic nanoparticles enhanced viral transduction efficiency and protein expression in a dose-dependent manner. They accelerated the transduction kinetics and allowed non-permissive cells infection. Magnetofection greatly improved adenovirus-mediated DNA delivery in vivo and provided a magnetic targeting. The i-MICST results established the efficiency of magnetic nanoparticles assisted viral transduction within cell sorting columns. The results showed that the combination of Magnetofection and Adenoviruses represents a promising strategy for gene therapy. Recently, a new integrated method to combine clinically approved magnetic cell isolation devices and genetic modification was developed. In this study, we validated that magnetic cell separation and adenoviral transduction can be accomplished in one reliable integrated and safe system.

Primary neural stem cells (NSCs) can be cultivated and differentiated in vitro but are difficult to transfect using conventional methods. We describe a simple and rapid magnetofection-based method suitable for the lab bench as well as for high-throughput projects. Our method yields high transfection efficiency and can be used for deciphering the genetic control of neural cell differentiation.

Gene therapy offers exciting opportunities for the treatment of innate or acquired genetic diseases. However, there is still a need for a safe and efficient strategy to deliver nucleic acids into cells while overcoming the current limitations faced with standard viral vectors. Intensive researches have been carried out over the past decade, focusing both on viral and non-viral (i.e. physical or chemical) strategies. Of these numerous attempts, magnetofection, defined as the combination of nucleic acid vectors with magnetic nanoparticles, holds the promise to achieve high transfection efficiency with reduced toxicity by magnetically focusing the genetic material to be delivered on its cellular target. In vitro as well as in vivo results already demonstrated that this strategy may become a valuable tool towards practical gene therapy.

BackgroundDeciphering host responses contributing to dengue shock syndrome (DSS), the life-threatening form of acute viral dengue infections, is required to improve both the differential prognosis and the treatments provided to DSS patients, a challenge for clinicians.Methodology/principal findingsBased on a prospective study, we analyzed the genome-wide expression profiles of whole blood cells from 48 matched Cambodian children: 19 progressed to DSS while 16 and 13 presented respectively classical dengue fever (DF) or dengue hemorrhagic fever grades I/II (DHF). Using multi-way analysis of variance (ANOVA) and adjustment of p-values to control the False Discovery Rate (FDRConclusions/significanceWe provide a global while non exhaustive overview of the molecular mechanisms altered in of DSS children and suggest how they may interact to lead to final vascular homeostasis breakdown. We suggest that some mechanisms identified should be considered putative therapeutic targets or biomarkers of progression to DSS.

Thrombin exerts pleiotropic effects on endothelial cells, including the release of microparticles (EMPs) that disseminate and exchange information with vascular cells. Nevertheless, the mechanisms leading to their generation are not elucidated. We performed microarray analysis to identify genes involved in EMP release by the endothelial cell line HMEC-1 in response to thrombin. We identified a group of genes linked to the cytoskeleton reorganization family. Among these, the Rho-kinase ROCK-II presented a high transcription rate. ROCK-I, another Rho-kinase isoform, was not modulated by thrombin. Pharmacologic inhibition of Rho-kinases or specific depletion of ROCK-II by short interfering (si) RNA inhibited thrombin-induced EMP release. In contrast, ROCK-I mRNA silencing did not modify EMP generation by thrombin. Exposure of HMEC-1 to thrombin in presence of the caspase-2 selective inhibitor Z-VDVAD-FMK prevented ROCK-II cleavage and inhibited the thrombin-induced EMP release. These events were observed in absence of cell death. Our data clearly identified ROCK-II as a target of thrombin in EMP generation. They indicated that the 2 Rho-kinases did not share identical functions. The involvement of caspase-2 in ROCK-II activation independently of cell death points out a novel signaling pathway that emphasizes the proteolytic activity of caspase in EMP generation in response to cell activation.

The association of the presence of anti-phospholipid antibodies (aPL) with thrombosis characterizes the anti-phospholipid syndrome (APS). The activation of the endothelium is a key event in the establishment of the thrombophilic state. However, the intracellular mechanisms leading to endothelial dysfunction are not fully elucidated. We investigated the role of reactive oxygen species (ROS) in the pro-adhesive state elicited by aPL and studied ROS-dependent downstream signaling pathways. Independent incubation of human umbilical vein endothelial cells (HUVEC) with IgG (IgG-APS) from 12 APS patients caused a large and sustained increase in ROS, which was prevented by the antioxidants vitamin C and N-acetyl-L-cysteine. ROS inhibition observed in the presence of diphenylene iodonium and rotenone indicated an involvement of a membrane-bound oxidase and the mitochondrial transport chain as sources of ROS. ROS acted as a second messenger by activating the p38 mitogen-activated protein kinase and its subsequent target, the stress-related transcription factor activating transcription factor-2 (ATF-2). ROS controlled the up-regulation of vascular cell adhesion molecule-1 expression by IgG-APS-stimulated HUVEC and the increase in THP-1 monocytic cells adhesion. The IgG-APS-mediated oxidative stress was observed irrespective of the clinical and biological criterions of the patients studied here. Taken together, these data indicate that the oxidative stress induced by IgG-APS is a key intracellular event that might contribute to the thrombotic complications of APS by controlling the endothelial adhesive phenotype.