A phosphorothioate oligonucleotide blocks reverse transcription via an antisense mechanism

AbstractWe have studied the inhibition by a phosphorothioate oligodeoxynucleotide (17PScap) of cDNA synthesis performed by either avian or murine reverse transcriptase. Three different mechanisms of inhibition were identified: at low concentrations (⊃100 nM), the cleavage of the RNA template by the retroviral RNase H at the level of the RNA/17PScap duplex accounted for most of the effect, whereas hybrid-arrested cDNA synthesis by an RNase H-independent mechanism marginally contributed to the inhibition. Both mechanisms were sequence-specific. Above 100 nM, the overall cDNA synthesis was reduced in a non-specific manner

Differential aquaporin 4 expression during edema build-up and resolution phases of brain inflammation

<p>Abstract</p> <p>Background</p> <p>Vasogenic edema dynamically accumulates in many brain disorders associated with brain inflammation, with the critical step of edema exacerbation feared in patient care. Water entrance through blood-brain barrier (BBB) opening is thought to have a role in edema formation. Nevertheless, the mechanisms of edema resolution remain poorly understood. Because the water channel aquaporin 4 (AQP4) provides an important route for vasogenic edema resolution, we studied the time course of AQP4 expression to better understand its potential effect in countering the exacerbation of vasogenic edema.</p> <p>Methods</p> <p>Focal inflammation was induced in the rat brain by a lysolecithin injection and was evaluated at 1, 3, 7, 14 and 20 days using a combination of in vivo MRI with apparent diffusion coefficient (ADC) measurements used as a marker of water content, and molecular and histological approaches for the quantification of AQP4 expression. Markers of active inflammation (macrophages, BBB permeability, and interleukin-1β) and markers of scarring (gliosis) were also quantified.</p> <p>Results</p> <p>This animal model of brain inflammation demonstrated two phases of edema development: an initial edema build-up phase during active inflammation that peaked after 3 days (ADC increase) was followed by an edema resolution phase that lasted from 7 to 20 days post injection (ADC decrease) and was accompanied by glial scar formation. A moderate upregulation in AQP4 was observed during the build-up phase, but a much stronger transcriptional and translational level of AQP4 expression was observed during the secondary edema resolution phase.</p> <p>Conclusions</p> <p>We conclude that a time lag in AQP4 expression occurs such that the more significant upregulation was achieved only after a delay period. This change in AQP4 expression appears to act as an important determinant in the exacerbation of edema, considering that AQP4 expression is insufficient to counter the water influx during the build-up phase, while the second more pronounced but delayed upregulation is involved in the resolution phase. A better pathophysiological understanding of edema exacerbation, which is observed in many clinical situations, is crucial in pursuing new therapeutic strategies.</p

Cells

Stem cells isolated from the apical papilla of wisdom teeth (SCAPs) are an attractive model for tissue repair due to their availability, high proliferation rate and potential to differentiate in vitro towards mesodermal and neurogenic lineages. Adult stem cells, such as SCAPs, develop in stem cell niches in which the oxygen concentration [O] is low (3-8% compared with 21% of ambient air). In this work, we evaluate the impact of low [O] on the physiology of SCAPs isolated and processed in parallel at 21% or 3% O without any hyperoxic shock in ambient air during the experiment performed at 3% O. We demonstrate that SCAPs display a higher proliferation capacity at 3% O than in ambient air with elevated expression levels of two cell surface antigens: the alpha-6 integrin subunit (CD49f) and the embryonic stem cell marker (SSEA4). We show that the mesodermal differentiation potential of SCAPs is conserved at early passage in both [O], but is partly lost at late passage and low [O], conditions in which SCAPs proliferate efficiently without any sign of apoptosis. Unexpectedly, we show that autophagic flux is active in SCAPs irrespective of [O] and that this process remains high in cells even after prolonged exposure to 3% O

The Journey of SCAPs (Stem Cells from Apical Papilla), from Their Native Tissue to Grafting: Impact of Oxygen Concentration

Tissue engineering strategies aim at characterizing and at optimizing the cellular component that is combined with biomaterials, for improved tissue regeneration. Here, we present the immunoMap of apical papilla, the native tissue from which SCAPs are derived. We characterized stem cell niches that correspond to a minority population of cells expressing Mesenchymal stromal/Stem Cell (CD90, CD105, CD146) and stemness (SSEA4 and CD49f) markers as well as endothelial cell markers (VWF, CD31). Based on the colocalization of TKS5 and cortactin markers, we detected migration-associated organelles, podosomes-like structures, in specific regions and, for the first time, in association with stem cell niches in normal tissue. From six healthy teenager volunteers, each with two teeth, we derived twelve cell banks, isolated and amplified under 21 or 3% O2. We confirmed a proliferative advantage of all banks when cultured under 3% versus 21% O2. Interestingly, telomerase activity was similar to that of the highly proliferative hiPSC cell line, but unrelated to O2 concentration. Finally, SCAPs embedded in a thixotropic hydrogel and implanted subcutaneously in immunodeficient mice were protected from cell death with a slightly greater advantage for cells preconditioned at 3% O2

Enrichment of an in vivo phage display repertoire by subtraction for easy identification of pathology biomarkers <br /> <br />

Titre de la communication : Enrichment of an in vivo phage display repertoire by subtraction for the easy identification of biomarkers of pathology. Vargas-Sanchez J.K., Vekris A., Mordelet E., Dubourdieu-Cassagno N., Ottones F., Petry K.G and Boiziau C. INSERM U1049, Bordeaux University, Neuroinflammation, Imaging and Therapy of Multiple Sclerosis, 146 rue Leo Saignat, 33 076 Bordeaux cedex, France. Abstract Aim. To develop a method that enriches, after in vivo phage display selection, the repertoire with clones specific for a pathological state. Materials and Methods. In a neuroinflammation model, we performed a subtraction based on DNA hybridization and restriction enzyme digestion of the common clones recovered from the central nervous system (CNS) of healthy and experimental autoimmune encephalomyelitis (EAE) rats. The subtraction efficiency was monitored by massive sequencing and clones were examined for binding specificity onto EAE CNS rat tissues and human blood brain barrier cells (hCMEC/D3) under inflammatory conditions. Results. 95% of the clones common to EAE and HEALTHY repertoires were subtracted, allowing clone enrichment. Conclusion. This physical subtraction discarded from a complex repertoire the non-specific selected ligands. STRATEGY 1) Three rounds of in vivo phage peptide selection in EAE female Lewis rats ("EAE repertoire") vs controls ("HEALTHY repertoire"). 2) DNA subtraction of the most common sequences between «HEALTHY» and «EAE» phage repertoires to obtain a third EAE specific «SUBTRACTION » phage repertoire. 3) Massive sequencing of the three repertoires and bioinformatic analysis to identify the peptides sequences with high EAE specificity. 4) Biological tests of potential EAE specific phage clones with CNS tissues from EAE and Healthy control rats. 5) Biological tests of the EAE specific peptide and phage clones on the BBB in vitro model (hCMEC/D3 cells) under inflammatory conditions (IL-1β stimulation). 6) Target separation and identification by cross-link between the selected phage clones and hMEC/D3 endothelial cells targets under IL-1β stimulation vs controls

Type 3 deiodinase expression in inflammatory spinal cord lesions in rat experimental autoimmune encephalomyelitis

BACKGROUND: We have shown substantial expression of type 3 deiodinase (D3, a major enzyme involved in the inactivation of thyroid hormone) in infiltrating leukocytes in several models of inflammation. Recently, thyroid hormone has been shown to improve remyelination in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. As induction of D3 may play an important role in decreasing local bioavailability of thyroid hormone at inflammation sites, we hypothesized that D3 is induced in spinal cord inflammatory lesions in EAE. METHODS: The aim of the study was to evaluate D3 expression in spinal cord inflammatory lesions of EAE Dark Agouti rats and to investigate D3 induction in activated monocytes. RESULTS: Here, we show marked expression of D3 by granulocytes and macrophages in spinal cord inflammatory lesions of EAE rats. We further confirm induction of D3 expression in vitro in monocytes that were activated toward proinflammatory or immunomodulatory phenotypes. CONCLUSIONS: We observed increased D3 expression both in spinal cord inflammatory lesions during EAE and in activated monocytes. Although increased D3 expression theoretically results in decreased triiodothyronine availability, it is unknown at present whether reduced local triiodothyronine concentrations are involved in impaired remyelination as observed during EA

A disulfide based low molecular weight gel for the selective sustained release of biomolecules

Constructing biocompatible soft materials via supramolecular approaches remains an important challenge for in vivo applications. Substantial efforts have been made to develop biocompatible non-polymeric materials allowing sustained release of biomolecules and/or drugs in vivo. Herein, we introduce disulfide based low molecular weight gels (LMWGs) allowing the in vitro selective sustained release of proteins containing thiol residues. The novel glycosylated nucleoside based bola-amphiphile (GNBA), which features a disulfide bond inserted in the hydrophobic segment, self-assembles to stabilize the resulting hydrogel. Rheological studies show the dominant elastic character and thixotropic properties of the disulfide LMWG demonstrating its injectability. In vitro and in vivo biodegradation investigations reveal that the disulfide LMWG is stable for several weeks. Importantly, disulfide bonds can be cleaved through the thiol-disulfide exchange reactions with small redox molecules such as dithiothreitol (DTT). The disulfide LMWG loaded with a thiol-containing protein (bovine serum albumin) features sustained release in vitro, whereas a dextran of the same molecular weight, lacking a thiol biomolecule, shows quick release. The disulfide GNBA is the first example of a LMWG allowing selective long term sustained release in vitro via a disulfide reshuffling mechanism