Search

Cyclophilins play a key role in the life cycle of coronaviruses. Alisporivir (Debio 025) is a nonimmunosuppressive analogue of cyclosporine with potent cyclophilin inhibition properties. Alisporivir reduced SARS-CoV-2 RNA production in a dose-dependent manner in Vero E6 cells, with a 50% effective concentration (EC50) of 0.46 ± 0.04 μM. Alisporivir inhibited a postentry step of the SARS-CoV-2 life cycle. These results justify rapidly conducting a proof-of-concept phase 2 trial with alisporivir in patients with SARS-CoV-2 infection.

International audience; Background: In hepatitis C virus (HCV) infection, treatment failure is generally associated with the selection of resistance-associated substitutions (RAS) conferring reduced susceptibility to direct-acting antiviral (DAA) drugs. Resistant variants continue to replicate after the end of treatment with potential for transmission. This may result from the selection of "fitness-associated substitutions".Aim: To characterise potential "fitness-associated substitutions" in patients infected with genotype 3a failing DAA drugs METHODS: By means of shotgun metagenomics, we sequenced full-length HCV genomes at treatment initiation and at virological relapse in eight patients infected with genotype 3a with cirrhosis failing sofosbuvir and an NS5A inhibitor. The impact of amino acid changes occurring outside of DAA target regions selected in at least two patients were assessed on the in vitro susceptibility to an NS5A inhibitor and replication capacity.Results: At treatment failure, besides selection of known NS5A RASs, especially Y93H, a large number of amino acid changes was observed outside of DAA target regions. We identified four amino acid positions at which observed changes substantially improved in vitro replication capacity without affecting NS5A inhibitor susceptibility.Conclusions: This is the first in vivo observation combined with in vitro confirmation of selection of phenotypically characterised "fitness-associated substitutions" together with RASs at the time of sofosbuvir-NS5A inhibitor treatment failure in patients infected with genotype 3a with cirrhosis. Our findings may explain the persistence of resistant HCV variants after treatment in patients who did not achieve sustained virological remission.

Purified microglial cells in culture are frequently used to model brain inflammatory responses but obtaining large yields of these cells on a routine basis can be quite challenging. Here, we demonstrate that it is possible to achieve high-yield isolation of pure microglial (MAC-1(+) /Fcrls(+) /Ccr2(-) ) cells from postnatal brain tissue through a simple culture procedure that mainly relies on the adhesion preference of these cells to the polycation polyethyleneimine (PEI) in serum-supplemented DMEM medium. Accordingly, other synthetic or biological substrates failed to mimic PEI effects under the same culture conditions. Replacement of DMEM by DMEM/F12 nutrient mixture did not permit microglial cell isolation on PEI coating, indicating that PEI effects were context-dependent. Remarkably, the lack of culture feeding during progression of microglial cell isolation strongly improved cell yield, suggesting that nutritional deprivation was required to optimize this process. When generated in large culture flasks coated with PEI, cultures of microglial cells were easily recovered by trypsin proteolysis to produce subcultures for functional studies. These cultures responded to lipopolysaccharide (LPS, 1-10 ng/ml) treatment by secreting pro-inflammatory cytokines such as TNF-α, IL-6, IL-1β and by generating nitric oxide and reactive oxygen species. Most interestingly, this response was curtailed by appropriate reference drugs. Microglial cells were also strongly responsive to the mitogenic cytokine GM-CSF, which confirms that the functional repertoire of these cells was well preserved. Because of its high yield and simplicity, we believe that the present method will prove to be especially convenient for mechanistic studies or screening assays. GLIA 2016;64:1912-1924.

International audience; When activated, microglial cells have the potential not only to secrete typical proinflammatory mediators but also to release the neurotransmitter glutamate in amounts that may promote excitotoxicity. Here, we wished to determine the potential of the Parkinson's disease (PD) protein α-Synuclein (αS) to stimulate glutamate release using cultures of purified microglial cells. We established that glutamate release was robustly increased when microglial cultures were treated with fibrillary aggregates of αS but not with the native monomeric protein. Promotion of microglial glutamate release by αS aggregates (αSa) required concomitant engagement of TLR2 and P2X7 receptors. Downstream to cell surface receptors, the release process was mediated by activation of a signaling cascade sequentially involving phosphoinositide 3-kinase (PI3K) and NADPH oxidase, a superoxide-producing enzyme. Inhibition of the Xc- antiporter, a plasma membrane exchange system that imports extracellular l-cystine and exports intracellular glutamate, prevented the release of glutamate induced by αSa, indicating that system Xc- was the final effector element in the release process downstream to NADPH oxidase activation. Of interest, the stimulation of glutamate release by αSa was abrogated by dopamine through an antioxidant effect requiring D1 dopamine receptor activation and PI3K inhibition. Altogether, present data suggest that the activation of microglial cells by αSa may possibly result in a toxic build-up of extracellular glutamate contributing to excitotoxic stress in PD. The deficit in dopamine that characterizes this disorder may further aggravate this process in a vicious circle mechanism.

Purified microglial cells in culture are frequently used to model brain inflammatory responses but obtaining large yields of these cells on a routine basis can be quite challenging. Here, we demonstrate that it is possible to achieve high-yield isolation of pure microglial (MAC-1(+) /Fcrls(+) /Ccr2(-) ) cells from postnatal brain tissue through a simple culture procedure that mainly relies on the adhesion preference of these cells to the polycation polyethyleneimine (PEI) in serum-supplemented DMEM medium. Accordingly, other synthetic or biological substrates failed to mimic PEI effects under the same culture conditions. Replacement of DMEM by DMEM/F12 nutrient mixture did not permit microglial cell isolation on PEI coating, indicating that PEI effects were context-dependent. Remarkably, the lack of culture feeding during progression of microglial cell isolation strongly improved cell yield, suggesting that nutritional deprivation was required to optimize this process. When generated in large culture flasks coated with PEI, cultures of microglial cells were easily recovered by trypsin proteolysis to produce subcultures for functional studies. These cultures responded to lipopolysaccharide (LPS, 1-10 ng/ml) treatment by secreting pro-inflammatory cytokines such as TNF-α, IL-6, IL-1β and by generating nitric oxide and reactive oxygen species. Most interestingly, this response was curtailed by appropriate reference drugs. Microglial cells were also strongly responsive to the mitogenic cytokine GM-CSF, which confirms that the functional repertoire of these cells was well preserved. Because of its high yield and simplicity, we believe that the present method will prove to be especially convenient for mechanistic studies or screening assays. GLIA 2016;64:1912-1924.

To determine whether orexinergic hypothalamic peptides can influence the survival of brainstem dopamine (DA) neurons, we used a model system of rat midbrain cultures in which DA neurons degenerate spontaneously and progressively as they mature. We established that orexin (OX)-B provides partial but significant protection to spontaneously dying DA neurons, whereas the homologous peptide OXA has only marginal effects. Importantly, DA neurons rescued by OXB accumulated DA efficiently by active transport, suggesting that they were functional. G-protein-coupled OX1 and OX2 receptors were both present on DA neurons, but the protective effect of OXB was attributable solely to OX2 receptors; a selective inhibitor of this receptor subtype, N-ethyl-2-[(6-methoxy-3-pyridinyl)[(2-methylphenyl)sulfonyl]amino]-N-(3-pyridinylmethyl)-acetamide (EMPA), suppressed this effect, whereas a selective agonist, [Ala(11), d-Leu(15)]OXB, reproduced it. Survival promotion by OXB required intracellular calcium mobilization via inositol-1,4,5-triphosphate and ryanodine receptors. Nicotine, a well known neuroprotective molecule for DA neurons, improved OXB-mediated rescue through the activation of α-bungarotoxin-sensitive (presumably α7) nicotinic receptors, although nicotine had no effect on its own. Altogether, our data suggest that the loss of hypothalamic orexinergic neurons that occurs in Parkinson's disease might confer an increased vulnerability to midbrain DA neurons in this disorder.