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Demyelination is a key pathogenic feature of multiple sclerosis (MS). Here, we evaluated the astrocyte contribution to myelin loss and focused on the neurotrophin receptor TrkB, whose up-regulation on the astrocyte finely demarcated chronic demyelinated areas in MS and was paralleled by neurotrophin loss. Mice lacking astrocyte TrkB were resistant to demyelination induced by autoimmune or toxic insults, demonstrating that TrkB signaling in astrocytes fostered oligodendrocyte damage. In vitro and ex vivo approaches highlighted that astrocyte TrkB supported scar formation and glia proliferation even in the absence of neurotrophin binding, indicating TrkB transactivation in response to inflammatory or toxic mediators. Notably, our neuropathological studies demonstrated copper dysregulation in MS and model lesions and TrkB-dependent expression of copper transporter (CTR1) on glia cells during neuroinflammation. In vitro experiments evidenced that TrkB was critical for the generation of glial intracellular calcium flux and CTR1 up-regulation induced by stimuli distinct from neurotrophins. These events led to copper uptake and release by the astrocyte, and in turn resulted in oligodendrocyte loss. Collectively, these data demonstrate a pathogenic demyelination mechanism via the astrocyte release of copper and open up the possibility of restoring copper homeostasis in the white matter as a therapeutic target in MS. [ABSTRACT FROM AUTHOR]

Novel amphiphilic copolymers havebeen synthesized based on a biocompatiblepoly(hydroxyethylaspartamide) (PHEA) backbone, bearing both anchoringgroups for gold nanoparticles, such as thiols and disulfide, and conjugablemoieties, such as amino groups, the latter as points suitable forappending further functional agents. The strategy was to functionalizeα,β-poly[(N-2-hydroxyethyl)-d,l-aspartamide] (PHEA) with PEG2000-NH2and with ethylenediamine (EDA) obtaining a partially pegylated copolymerwith a large number of pendant primary amino groups. A fraction ofthe latter was conjugated with molecules bearing terminal thiol moietiessuch as 12-mercaptododecanoic acid (MDA) and disulfide groups suchas lipoic acid (LA), obtaining the two amphiphilic derivatives PHEA–PEG2000–EDA–MDA (PPE–MDA) and PHEA–PEG2000–EDA–LA (PPE–LA), which also provedintrinsically able to self-assemble in polymeric micelles. The twocopolymers efficiently coated gold nanostars (GNSs, size ∼40nm), wrapping around the surface increasing only slightly the hydrodynamicdiameter (reaching ∼45 nm), imparting them stability and apH-switchable surface charge, due to the unreacted amino groups. Remarkably,the poor solvation and the huge steric hindrance experienced by theamino groups lowers the observed logarithmic protonation constantsto 5.6–5.7. In vitroexperiments demonstratedthat PPE–MDA and PPE–LA copolymers have an intrinsicexcellent biocompatibility in both the human brain neuroblastoma (SH-SY5Y)and human bronchial epithelial (16-HBE) cell lines. Interaction ofthe same cell lines with “nude” GNS and GNS coated withPPE–LA was also studied, disclosing a completely satisfactorybiocompatibility of the latter. [ABSTRACT FROM AUTHOR]

Polymeric micelles potentially ableto carry to hepatocytes a ribavirin(RBV) prodrug, exploiting the presence of carbohydrate receptors,that is, ASGPR, were prepared starting from a galactosylated polylactide-polyaminoacidconjugate. This latter was obtained by chemical reaction of α,β-poly(N-2-hydroxyethyl) (2-aminoethylcarbamate)-dl-aspartamide(PHEA-EDA) with polylactic acid (PLA), and subsequent reaction withlactose, obtaining PHEA-EDA-PLA-GAL copolymer. To enhance the entrapmentinto obtained nanostructures, a hydrophobic RBV prodrug, that is,RBV tripalmitate, was synthesized and its capability to release RBVin the presence of an adequate enzymatic activity was demonstrated.Liver-targeted RBV tripalmitate-loaded micelles were obtained in aqueousmedia at low PHEA-EDA-PLA-GAL copolymer concentration value with nanometricsize. By in vitro experiments, the specificity of RBV tripalmitate-loadedPHEA-EDA-PLA-GAL micelles toward HepG2 was demonstrated by using acompetitive inhibition assay in the presence of free GAL. This findingraises hope in terms of future micelles-based liver-targeted drugdelivery strategy for the hepatitis C treatment. [ABSTRACT FROM AUTHOR]

Myelination is a complex process that requires coordinated Schwann cell-axon interactions during development and regeneration. Positive and negative regulators of myelination have been recently described, and can belong either to Schwann cells or neurons. Vimentin is a fibrous component present in both Schwann cell and neuron cytoskeleton, the expression of which is timely and spatially regulated during development and regeneration. We now report that vimentin negatively regulates myelination, as loss of vimentin results in peripheral nerve hypermyelination, owing to increased myelin thickness in vivo, in transgenic mice and in vitro in a myelinating co-culture system. We also show that this is due to a neuron-autonomous increase in the levels of axonal neuregulin 1 (NRG1) type III. Accordingly, genetic reduction of NRG1 type III in vimentin-null mice rescues hypermyelination. Finally, we demonstrate that vimentin acts synergistically with TACE, a negative regulator of NRG1 type III activity, as shown by hypermyelination of double Vim/Tace heterozygous mice. Our results reveal a novel role for the intermediate filament vimentin in myelination, and indicate vimentin as a regulator of NRG1 type III function. [ABSTRACT FROM AUTHOR]

Axonal loss causes disabling and permanent deficits in many peripheral neuropathies, and may result from inefficient nerve regeneration due to a defective relationship between Schwann cells, axons and the extracellular matrix. These interactions are mediated by surface receptors and transduced by cytoskeletal molecules. We investigated whether peripheral nerve regeneration is perturbed in mice that lack glial fibrillary acidic protein (GFAP), a Schwann-cell-specific cytoskeleton constituent upregulated after damage. Peripheral nerves develop and function normally in GFAP-null mice. However, axonal regeneration after damage was delayed. Mutant Schwann cells maintained the ability to dedifferentiate but showed defective proliferation, a key event for successful nerve regeneration. We also showed that GFAP and the other Schwann-cell-intermediate filament vimentin physically interact in two distinct signaling pathways involved in proliferation and nerve regeneration. GFAP binds integrin α vβ8, which initiates mitotic signals soon after damage by interacting with fibrin. Consistently, ERK phosphorylation was reduced in crushed GFAP-null nerves. Vimentin instead binds integrin α 5β1, which regulates proliferation and differentiation later in regeneration, and may compensate for the absence of GFAP in mutant mice. GFAP might contribute to form macro-complexes to initiate mitogenic and differentiating signaling for efficient nerve regeneration. [ABSTRACT FROM AUTHOR]

Inthis work we wish to report on the covalent functionalizationof polylactide (PLA) surfaces by photoradical thiol–yne toyield antibacterial surfaces. At first, hydrophilic and hydrophobicthiol fluorescent probes are synthesized and used to study and optimizethe conditions of ligation on alkyne-PLA surfaces. In a second part,a new antibacterial polyaspartamide copolymer is covalently grafted.The covalent surface modification and the density of surface functionalizationare evaluated by SEC and XPS analyses. No degradation of PLA chainsis observed, whereas covalent grafting is confirmed by the presenceof S2p and N1s signals. Antiadherence and antibiofilm activities areassessed against four bacterial strains, including Gram-negative andGram-positive bacteria. A strong activity is observed with adherencereduction factors superior to 99.98% and biofilm formation decreasedby 80%. Finally, in vitro cytocompatibility tests of the antibacterialsurfaces are performed with L929 murine fibroblasts and show cellviability without promoting proliferation. [ABSTRACT FROM AUTHOR]

Abstract: In the present study the rheological properties of hydrogels obtained through UV irradiation of an aqueous solution of methacrylated inulin (INUMA) alone or in the presence of four different crosslinkers, have been recorded as a function of irradiation time, at two different wavelengths (320 and 365nm) and fixed frequency and amplitude by oscillatory experiments. Each hydrogel has been analyzed by measurements of the frequency-dependency of the elastic modulus, G′, in the linear viscoelastic region. The amount of polymeric chains giving an elastic response has been correlated to the chemical nature of the crosslinker used together with INUMA. Finally the release properties of the hydrogel exhibiting the highest elastic modulus have been tested by using 5-Fluorouracil, as a model drug, and two different loading procedures. The rheological characterization allowed us to determine the most effective condition (appropriate crosslinker, wavelength and irradiation time) to obtain a strong hydrogel that was able to give a modified drug release in simulated gastrointestinal fluids. [Copyright &y& Elsevier]

Astrocytes greatly participate to inflammatory and neurotoxic reactions occurring in neurodegenerative diseases and are valuable pharmacological targets to support neuroprotection. Here we used human astrocytes generated from reprogrammed fibroblasts as a cellular model to study the effect of the compound Laquinimod and its active metabolite de-Laquinimod on astrocyte functions and the astrocyte–neuron interaction. We show that human iAstrocytes expressed the receptor for the inflammatory mediator IL1 and responded to it via nuclear translocation of NFκB, an event that did not occur if cells were treated with Laquinimod, indicating a direct anti-inflammatory activity of the drug on the human astrocyte. Similarly, while exposure to IL1 downregulated glial glutamate transporters GLAST and GLT1, treatment with Laquinimod supported maintenance of physiological levels of these proteins despite the inflammatory milieu. Laquinimod also induced nuclear translocation of the aryl hydrocarbon receptor (AHR), suggesting that drug action was mediated by activation of the AHR pathway. However, the drug was effective despite AHR inhibition via CH223191, indicating that AHR signaling in the astrocyte is dispensable for drug responses. Finally, in vitro experiments with rat spinal neurons showed that laquinimod did not exert neuroprotection directly on the neuron but dampened astrocyte-induced neurodegeneration. Our findings indicate that fibroblast-derived human astrocytes represent a suitable model to study astrocyte–neuron crosstalk and demonstrate indirect, partial neuroprotective efficacy for laquinimod. [ABSTRACT FROM AUTHOR]

High-mobility group box 1 (HMGB1) is a nonhistone protein secreted by airway epithelial cells in hyperinflammatory diseases such as asthma. In order to down-regulate HMGB1 expression in airway epithelial cells, siRNA directed against HMGB1 was delivered through nanocomplexes based on a cationic copolymer of poly( N -2-hydroxyethyl)- d,l -aspartamide (PHEA) by using H441 cells. Two copolymers were used in these experiments bearing respectively spermine side chains (PHEA-Spm) and both spermine and PEG 2000 chains (PHEA-PEG-Spm). PHEA-Spm and PHEA-PEG-Spm derivatives complexed dsDNA oligonucleotides with a w/w ratio of 1 and higher as shown by a gel retardation assay. PHEA-Spm and PHEA-PEG-Spm siRNA polyplexes were sized 350–650 nm and 100–400 nm respectively and ranged from negativity/neutrality (at 0.5 ratio) to positivity (at 5 ratio) as ζ potential. Polyplexes formed either at a ratio of 0.5 (partially complexing) or at the ratio of 5 (fully complexing) were tested in subsequent experiments. Epifluorescence revealed that nanocomplexes favored siRNA entry into H441 cells in comparison with naked siRNA. As determined by flow cytometry and a trypan blue assay, PHEA-Spm and PHEA-PEG-Spm allowed siRNA uptake in 42–47% and 30% of cells respectively, however only with PHEA-Spm at w/w ratio of 5 these percentages were significantly higher than those obtained with naked siRNA (20%). Naked siRNA or complexed scrambled siRNA did not exert any effect on HMGB1mRNA levels, whereas PHEA-Spm/siRNA at the w/w ratio of 5 down-regulated HMGB1 mRNA up to 58% of control levels (untransfected cells). PEGylated PHEA-Spm/siRNA nanocomplexes were able to down-regulate HMGB1 mRNA levels up to 61% of control cells. MTT assay revealed excellent biocompatibility of copolymer/siRNA polyplexes with cells. In conclusion, we have found optimal conditions for down-regulation of HMGB1 by siRNA delivery mediated by polyaminoacidic polymers in airway epithelial cells in the absence of cytotoxicity. Functional and in-vivo studies are warranted. [ABSTRACT FROM AUTHOR]

New fluorinated amphiphilic copolymers based on a biocompatible polyaspartamide have been prepared in order to obtain polymeric micelles useful for delivering anticancer drugs. In particular, α,β-poly( N-2-hydroxyethyl)- d, l-aspartamide (PHEA) has been derivatized with polyethylene glycol (PEG2000) and ethylendiamine (EDA). Both these portions form the hydrophilic part of the copolymer, while the hydrophobic moiety is given by 1,2,4-oxadiazoles: 5-pentafluorophenyl-3-perfluoroheptyl-1,2,4-oxadiazole (PPOX) or 3-carboxyethyl-5-pentadecafluoroheptyl-1,2,4-oxadiazole (CPOX). Copolymers named PHEA-PEG2000-EDA-PPOX and PHEA-PEG2000-EDA-CPOX have been prepared with various degrees of derivatization and characterized by spectroscopic analyses. Size exclusion chromatography, pyrene colorimetric assay, light scattering analysis and scanning electron microscopy have evidenced the occurrence of a self-association process in aqueous medium. The ability of these aggregates to incorporate a hydrophobic drug and increase its solubility has been evaluated by using Flutamide, a fluorinated anticancer agent. Moreover, the activity of Flutamide-loaded micelles on proliferation of dihydrotestosterone stimulated LNCaP cells has been determined and compared to that of free drug. [ABSTRACT FROM AUTHOR]

Abstract: The study investigates if alpha-lipoic acid is neuroprotective against chemotherapy induced neurotoxicity, if mitochondrial damage plays a critical role in toxic neurodegenerative cascade, and if neuroprotective effects of alpha-lipoic acid depend on mitochondria protection. We used an in vitro model of chemotherapy induced peripheral neuropathy that closely mimic the in vivo condition by exposing primary cultures of dorsal root ganglion (DRG) sensory neurons to paclitaxel and cisplatin, two widely used and highly effective chemotherapeutic drugs. This approach allowed investigating the efficacy of alpha-lipoic acid in preventing axonal damage and apoptosis and the function and ultrastructural morphology of mitochondria after exposure to toxic agents and alpha-lipoic acid. Our results demonstrate that both cisplatin and paclitaxel cause early mitochondrial impairment with loss of membrane potential and induction of autophagic vacuoles in neurons. Alpha-lipoic acid exerts neuroprotective effects against chemotherapy induced neurotoxicity in sensory neurons: it rescues the mitochondrial toxicity and induces the expression of frataxin, an essential mitochondrial protein with anti-oxidant and chaperone properties. In conclusion mitochondrial toxicity is an early common event both in paclitaxel and cisplatin induced neurotoxicity. Alpha-lipoic acid protects sensory neurons through its anti-oxidant and mitochondrial regulatory functions, possibly inducing the expression of frataxin. These findings suggest that alpha-lipoic acid might reduce the risk of developing peripheral nerve toxicity in patients undergoing chemotherapy and encourage further confirmatory clinical trials. [Copyright &y& Elsevier]