Granulocyte macrophage colony‐stimulating factor promotes regeneration of retinal ganglion cells in vitro through a mammalian target of rapamycin‐dependent mechanism

Lack of regeneration in the adult central nervous system (CNS) is a major hurdle that limits recovery from neurological ailments. Although accumulating research suggests the possibility of axon regeneration by targeting intrinsic signaling mechanisms, it remains a matter of controversy whether functional recovery can be achieved by manipulating aspects of molecular signaling. Recent studies have shown that granulocyte macrophage colony‐stimulating factor (GM‐CSF) may be an effective means of targeting repair following CNS injury; how this molecule is able to produce this effect is not known. Indeed, GM‐CSF has been shown to promote neuronal survival, potentially through activation of as yet unknown cytokine‐dependent signals and potentially through regulation of antiapoptotic mechanisms. It is well established that the loss of intrinsic regenerative ability is highly correlated with development of CNS neurons. We therefore designed experiments, using a well‐established in vitro retinal ganglion cell (RGC) culture system, to evaluate the effect of GM‐CSF on axon growth and cell survival and define possible mechanisms involved in GM‐CSF‐mediated effects in vitro. Several developmental stages were evaluated, with particular focus placed on stages at which axon growth is known to be significantly diminished. Our results reveal that GM‐CSF not only promotes axon growth in postnatal RGCs but also enhances cell survival through a mammalian target of rapamycin (mTOR)‐dependent mechanism. © 2013 Wiley Periodicals, Inc.     

Seeking huntington disease biomarkers by multimodal,cross‐sectional basal ganglia imaging

Neurodegeneration of the striatum in Huntington disease (HD) is characterized by loss of medium‐spiny neurons, huntingtin nuclear inclusions, reactive gliosis, and iron accumulation. Neuroimaging allows in vivo detection of the macro‐ and micro‐structural changes that occur from presymptomatic stages of the disease (preHD). The aim of our study was to evaluate the reliability of multimodal imaging as an in vivo biomarker of vulnerability and development of the disease and to characterize macro‐ and micro‐structural changes in subcortical nuclei in HD. Macrostructure (T₁‐weighted images), microstructure (diffusion tensor imaging), and iron content (R relaxometry) of subcortical nuclei and medial temporal lobe structures were evaluated by a 3 T scanner in 17 preHD carriers, 12 early‐stage patients and 29 matched controls. We observed a volume reduction and microstructural changes in the basal ganglia (caudate, putamen, and globus pallidus) and iron accumulation in the globus pallidus in both preHD and symptomatic subjects; all these features were significantly more pronounced in patients, in whom degeneration extended to the other subcortical nuclei (i.e., thalamus and accumbens). Mean diffusivity (MD) was the most powerful predictor in models explaining more than 50% of the variability in HD development in the caudate, putamen, and thalamus. These findings suggest that the measurement of MD may further enhance the well‐known predictive value of striatal volume to assess disease progression as it is highly sensitive to tissue microimpairment. Multimodal imaging may detect brain changes even in preHD stages. Hum Brain Mapp, 2013. © 2012 Wiley Periodicals, Inc.     

Priming with an adenovirus 35-circumsporozoite protein (CS) vaccine followed by RTS,S/AS01B boosting significantly improves immunogenicity to Plasmodium falciparum CS compared to that with either malaria vaccine alone

The RTS,S/AS02A protein-based vaccine consistently demonstrates significant protection against infection with Plasmodium falciparum malaria and also against clinical malaria and severe disease in children in areas of endemicity. Here we demonstrate with rhesus macaques that priming with a replication-defective human adenovirus serotype 35 (Ad35) vector encoding circumsporozoite protein (CS) (Ad35.CS), followed by boosting with RTS,S in an improved MPL- and QS21-based adjuvant formulation, AS01B, maintains antibody responses and dramatically increases levels of T cells producing gamma interferon and other Th1 cytokines in response to CS peptides. The increased T-cell responses induced by the combination of Ad35.CS and RTS,S/AS01B are sustained for at least 6 months postvaccination and may translate to improved and more durable protection against P. falciparum infection in humans.     

Real-time quantitative PCR for assessment of antiviral drug effects against Epstein-Barr virus replication and EBV late mRNA expression

This study assesses the ability of quantitative real-time PCR to measure the effects of virus DNA polymerase inhibitors on EBV DNA and late mRNAs syntheses in EBV-producing cell lines. In-house real-time quantitative PCRs were used to measure EBV DNA (thymidine kinase) and mRNAs (BLLF1 gene/gp350/220, BVRF2 gene/protease) in P3HR-1 and B95-8 cells induced for EBV production by PMA and exposed to ganciclovir, cidofovir and foscarnet. The calculated 50% effective concentrations (EC(50)) for viral DNA replication inhibition in P3HR-1 cells after 7 days of drug exposure were 0.28+/-0.06, 0.29+/-0.01 and 13.6+/-0.17 microg/mL for ganciclovir, cidofovir and foscarnet, respectively. The EC(50) for B95-8 cells were 0.44+/-0.02, 0.70+/-0.06 and 46.8+/-0.5 microg/mL, respectively. The quantitation of the late viral mRNAs showed a decrease of 79-89% in the mRNA amount after 4 days of antiviral treatment. Nevertheless, a substantial amount of mRNA still remained detectable after drug exposure. The real-time PCR is an improvement in the attempt to simplify EBV DNA-quantitation for antiviral assays. The quantitation of late mRNA does not appear as more informative than DNA quantitation for the assessment of the DNA polymerase inhibitor activity, but it may be useful to assess the antiviral activity of drugs acting by another mechanism.     

CX3CR1-dependent subretinal microglia cell accumulation is associated with cardinal features of age-related macular degeneration

The role of retinal microglial cells (MCs) in age-related macular degeneration (AMD) is unclear. Here we demonstrated that all retinal MCs express CX3C chemokine receptor 1 (CX3CR1) and that homozygosity for the CX3CR1 M280 allele, which is associated with impaired cell migration, increases the risk of AMD. In humans with AMD, MCs accumulated in the subretinal space at sites of retinal degeneration and choroidal neovascularization (CNV). In CX3CR1-deficient mice, MCs accumulated subretinally with age and albino background and after laser impact preceding retinal degeneration. Raising the albino mice in the dark prevented both events. The appearance of lipid-bloated subretinal MCs was drusen-like on funduscopy of senescent mice, and CX3CR1-dependent MC accumulation was associated with an exacerbation of experimental CNV. These results show that CX3CR1-dependent accumulation of subretinal MCs evokes cardinal features of AMD. These findings reveal what we believe to be a novel pathogenic process with important implications for the development of new therapies for AMD.     

In vitro study and semiempirical model for aerosol delivery control during mechanical ventilation

The object of this study was to evaluate in vitro the influence of various ventilatory parameters on the delivery of synchronized nebulization of terbutaline during mechanical ventilation and to determine a semiempirical model to control the quantity of aerosol delivered into the patients lung. An ATOMISOR NL9 M jet nebulizer (La Diffusion Technique Française, France) was filled with terbutaline (Bricanyl, Astra-Zeneca, Sweden) and connected to the inspiratory line of a Horus ventilator (Taema, France). Nebulization was synchronized with the inspiratory phase. We assessed at the end of the endotracheal tube the quantity of terbutaline (terbutaline mass output) and the volume median diameter (VMD) by diffraction-laser method. There was a negative correlation between terbutaline mass output and inspiratory air flow ( r =–0.95, p <0.0001) and between VMD and inspiratory air flow ( r =–0.96, p <0.0001). Moreover, positive end-expiratory pressure levels between 0 cm and 8 cm of water did not significantly change the terbutaline output mass ( p =0.22). Total nebulization time and terbutaline mass output calculated by the mathematical model showed good agreement with experimental data. In conclusion, our semiempirical model allows calculation of the duration of the nebulization required to deliver a given mass of terbutaline into patient lungs.This revised version was published online in May 2005 with a corrected section heading.