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Objectives: The aim of this work was to evaluate the activation of oxidative stress and inflammation in mice exposed to ultrafine particles (UFPs), and to elucidate putative physiopathological correlations with neurodegeneration. Materials: UFPs derived from two different anthropogenic sources were used: DEP, from EURO 4 diesel engine emission, and BC, from pellet boiler biomass emission (ENEA). Method: Male BALB/c mice were submitted to single or repeated intratracheal instillation of DEP and BC (50µg); in parallel, control mice were always considered (sham). Cortex, cerebellum and hippocampus from sham and treated mice were screened for markers of inflammation and oxidative stress (iNOS, HO-1), endoplasmic reticulum stress (Hsp70), a putative pro-carcinogenic marker (Cyp1B1) and markers related to the onset of Alzheimer Disease (AD) (P-Tau, Tau, P-APP, APP, BACE1). Results: After both single and repeated DEP and BC instillation, we observed an increase in iNOS, HO-1 and Hsp70 levels in all districts, while Cyp1B1 and markers of neurodegeneration changed only after repeated treatment. Discussion: The single DEP and BC instillation induced oxidative stress, endoplasmic reticulum stress and inflammation, while typical AD-related proteins seemed not affected. The repeated instillation was able to sustain oxidative stress and inflammation; moreover, it induced increase of BACE1 levels and changes in Tau and APP total levels and phosphorylation. Finally, BC seemed to be less effective than DEP. Conclusions: Our results suggest UFPs or inflammatory mediators translocation from lungs, and their effects on the brain, after both acute and repeated DEP and BC treatment. Grant by Fondazione Cariplo. Genc S., Zadeoglulari Z., Fuss S.H., Genc K. The adverse effects of air pollution on the nervous system. J Toxicol. 2012. 2012:782462. Levesque S., Surace M.J., McDonald J., Block M.L. Air pollution & the brain: Subchronic diesel exhaust exposure causes neuroinflammation and elevates early markers of neurodegenerative disease. J Neuroinflammation. 2011. 8:105. Block M.L. and Calderón-Garcidueñas L. Air pollution: mechanisms of neuroinflammation and CNS disease. Trends Neurosci. 2009. 9: 506–516.

Objectives: Understanding the ischemic injury contribution to Alzheimer’s disease (AD) onset, pinpointing to post-translational modifications, turnover alterations, secretion of proteins and networks identified as AD hallmarks. Materials: Neuronal cultures prepared from hippocampi of E18-E19 embryos from pregnant Sprague Dawley rats. 5%CO2: 95%N2 gaseous mixtures (Sapio); hypoxia chamber (Billups–Rothenberg). Methods: DIV8 neurons were subjected to oxygen and glucose deprivation (OGD). After medium replacement with a glucose-free balanced salt solution (ogR), cells were incubated for 3 hours in the chamber saturated with the gaseous mixture. The restoration solution (glucose and B27) was administrated to neurons (ogR) for one hour (R1h) or overnight (R16h). Results: We observed Tau and amyloid precursor protein (APP) post-translational and protein level modifications after OGD/ogR. Tau cleavage and secretion occurs at R1h through microvesicles (MVs) population, including LC3 and/or LAMP1 positive vesicles, marker of autophagy-mediated secretion (exophagy). In MVs extract we also identified α- and β- C-terminal fragments (CTFs) of APP, and the peptidyl prolyl cis/trans isomerase Pin1. Discussion: MVs represent an intercellular communication delivering multiple cargo with beneficial or harmful messages [1]. Thus, differences in MVs contents after OGD/ogR suggest that ischemia leads to a robust upheaval with the autophagic mechanisms activation resultant in neuron-to-neuron transfer of material with neurodegenerative potential. Conclusions: OGD treatment leads to mis-processed protein unconventional secretion also including exophagy pathway. These proteins represent seeds for protein aggregation according to the “prion-like” propagation mechanism [2] that may represent an early event in AD and proteinopathies.

In recent years particulate matter (PM) has been found to trigger several mechanisms responsible for neuroinflammation, and accumulating evidence points to neuroinflammation as an active participant in the progression of neurodegenerative diseases. This project aimed to assess whether, in a model of neuronal cells, PM, specifically ultrafine particles (UFP), induces oxidative stress and inflammation. We performed in-vitro analyses using HT22 cells, an immortalized mouse hippocampus cell line. HT22 cells have been treated for 3h and 24h with DEP SRM1650b (Standard Reference Material, National Institute of Standards and Technology, USA), a diesel particulate with aerodynamic diameter of 0,18 µm, suspended at different concentrations (10 µg/ml, 25 µg/ml, 50 µg/ml, 100 µg/ml) in culture medium. The following parameters have been measured: cell viability, oxidative stress related toxicity and activation of inflammation pathways. MTT viability assay demonstrated that DEP does not induce a significant cell death neither at 3h nor at 24h of treatment, for all the doses used. After 3h of DEP treatment, protein analysis revealed the presence of oxidative stress; in fact DEP induced a significant dose-dependent increase of HO-1 levels, a stress related protein which catalysed heme degradation. The increase of iNOS protein levels suggests the activation of inflammation pathway, although we need further studies to confirm it. Moreover, we observed an increase in Cyp1B1 levels, a cytochrome of the P450 superfamily involved in the activation of many xenobiotics and in polycyclic aromatic hydrocarbons metabolism, and in HSP70 levels, a protein activated in cellular stress conditions. After 24h of DEP treatment, analysis showed that HO-1 and HSP70 protein levels are still high, although the dose function increase is more pronounced at 3h, whereas Cyp1B1 and iNOS return almost to control levels. Based on the data so far available, we can hypothesize that exposure of neuronal cells to DEP results in the induction of oxidative stress and in the activation of inflammation pathway.

Objectives. To understand brain ischemia injury contribution to Alzheimer’s disease (AD) onset, looking for post-translational modifications, turnover alterations and AD hallmarks secretion such as tau and amyloid precursor protein (APP), associated to autophagy activation. Methods. Rat cultured hippocampal neurons are subjected to oxygen glucose deprivation (OGD) followed by normal culture condition restoration (R), mimicking an ischemia/reperfusion (I/R) event. Afterwards AD hallmarks and autophagy markers expression and secretion are analyzed in the cells lysate and in microvescicles (MVs) isolated from culture medium. Confocal microscopy is employed to evaluate the potential re- and co-localization of tau and autophagic markers after treatment. Results. In our cellular model subjected to OGD/R, we observed tau and APP post-translational and protein level modifications in parallel to the autophagic markers (LC3II and beclin-1) expression alterations. Moreover, OGD/R treatment elicits a time-dependent increase in tau and APP C-terminal fragments (CTF) secretion by means of a heterogeneous MV population, including a group of LC3II positive vescicles. Preliminary data obtained by confocal microscopy suggest that LC3 might undergo re-localization after OGD/R treatment. Conclusions. The obtained results suggest that OGD treatment leads to multiple unconventional secretion mechanisms for tau and APP CTFs, in which autophagy activation seems to partially outcome in the exophagy secretory pathway. Although exophagy might be a defensive mechanism to prevent intracellular accumulation of misfolded proteins under conventional autophagy alteration, the secretion of misprocessed proteins has been recently associated to dendritic degeneration and neuronal death becoming a possible link to Alzheimer’s disease.

Background: MiBIECT is a new oxadiazolyl-oxazolidinone that has been developed by means of chemoinformatic tools, synthesis and biological evaluation. This agent demonstrates potent activity against Gram-positive pathogens, including linezolid-resistant (LZDR) isolates. The aims of this study were to determine the in vitro activity of MiBIECT against a greater number of Gram-positive cocci with various resistant phenotypes, and to evaluate MiBIECT for in vitro cytotoxicity. Methods: A total of 340 strains of linezolid-susceptible (LZD-S) S. aureus, S. epidermidis, enterococci, S. pneumoniae, S. pyogenes and 20 LZDR strains were analyzed. MiBIECT was evaluated for its in vitro antibacterial activity by determining the minimum inhibitory concentrations (MIC) by the CLSI broth microdilution methods. In vitro cytotoxicity assays in cell lines representative of excretory organs were used to evaluate for potential safety concerns. Results: MiBIECT exhibited potent activity against S. aureus, S. epidermidis, S. pneumoniae and S. pyogenes with MICs in range of

Alzheimer's disease (AD) is a neurodegenerative illness affecting the elderly population, characterized by plaques of Aβ42 aggregates, neurofibrillary tangles and neuronal loss (Allsop, 2000). In AD vascular factors could precede the neurodegenerative process (de la Torre, 2002, 2008); Aβ42 accumulation in the cerebral capillary may be a consequence of a local production in the vascular domain (Natté et al., 1999). Air pollution has been associated with central nervous system (CNS) diseases. Inhaled UFPs (< 100 nm) could easily translocate cross the air-blood barrier, reach the bloodstream and be distributed to the cardiovascular system or the CNS (Oberdorster et al., 2002), thus affecting systemic microvasculature (Nurkiewicz et al., 2011). hCMEC/D3 and HT22 cells have been exposed to different DEP concentrations for different times. The following parameters have been measured: cell viability, oxidative stress and inflammation markers (HO-1, iNOS, Cyp1b1, COX-2, TNFalpha, IL1beta, IL8, VEGF), tight junction proteins (claudin-5, occludin), an amyloidogenic processing marker (BACE-1), besides an AD marker (Tau). In both cell lines, none of the concentrations induced cytotoxicity. In hCMEC/D3, DEP caused increases in HO-1, COX-2 and BACE-1 levels; moreover, the lower dose elicit a significant VEGF release. In HT22, after 3h all the concentrations caused an increase in HO-1, iNOS, HSP70 and Cyp1b1, whereas after 24h iNOS and Cyp1B1 return almost to control levels. Finally, after 24h a decrease in Tau levels has been found. In conclusion, all the parameters, except cytotoxicity, were differently affected in hCMEC/D3 and HT22, confirming the major susceptibility of neurons to toxic insults. Supported by Fondazione Cariplo.

In recent years particulate matter (PM) has been found to trigger several mechanisms responsible for neuroinflammation, an active participant in the progression of neurodegenerative diseases (Block and Calderón-Garcidueñas, 2009; Levesque et al., 2011). This project aimed to assess whether, in a model of neuronal cells, PM, specifically ultrafine particles (UFP), induces oxidative stress and inflammation. HT22 cells, an immortalized mouse hippocampus cell line, have been treated for 3h and 24h with DEP SRM1650b (Standard Reference Material, National Institute of Standards and Technology, USA), a diesel particulate with mean aerodynamic diameter of 0,18 µm, suspended at different concentrations (10 µg/ml, 25 µg/ml, 50 µg/ml, 100 µg/ml) in culture medium. The following parameters have been measured: cell viability, oxidative stress related toxicity and activation of inflammation pathways. MTT viability assay demonstrated that DEP does not induce a significant cell death neither at 3h nor at 24h of treatment, for all the doses used. After 3h of DEP treatment, protein analysis revealed the presence of oxidative stress; in fact DEP induced a significant dose-dependent increase of HO-1 levels, a stress related protein which catalyse heme degradation. The increase of iNOS protein levels suggests the activation of inflammation pathway. Moreover, we observed an increase in Cyp1B1 levels, a cytochrome of the P450 superfamily involved in polycyclic aromatic hydrocarbons metabolism, and in HSP70 levels, a protein activated in cellular stress conditions. After 24h of DEP treatment, analysis showed that HO-1 and HSP70 protein levels are still high, whereas Cyp1B1 and iNOS return almost to control levels. Indeed, we can hypothesize that exposure of neuronal cells to DEP results in oxidative stress and inflammation induction. In in-progress experiments, BALB/c mice will be submitted to intratracheal instillation of single or repeated doses of DEP, and effects on the SNC system (in particular on hippocampus, cortex and cerebellum) will be studied. Figure 1. Protein analyses after 3h of DEP treatment. * p

Introduction Staphylococcus aureus is one of the first pathogens to colonize and infect the lungs of cystic fibrosis (CF) patients, causing recurrent and relapsing infections, particularly in children and adolescents (1,2). Moreover, recent data have demonstrated an increase in S. aureus infections in the CF population being reported both in USA and Europe, with methicillin resistant S. aureus (MRSA) strains being on the rise (3). Emergence of multidrug-resistant bacteria, such as MRSA, therefore poses a special challenge in the treatment of infections in CF and new therapeutic agents are required to eradicate these pathogens. Some persistent lung infections often have low response rates to conventional chemotherapy, in part due to the lack of selectivity of many of antimicrobial drugs and their low bioavailability, especially when administered by a systemic route. Thus, the design of new formulations, that allow dose-intensification with reduced or no systemic side effects, have emerged to provide higher antimicrobial activity. Prolonging plasma circulation time, lowering renal drug exposure, and increasing lung tissue deposition, Solid Lipid Nanoparticles (SLN) are good candidate to facilitate antimicrobial delivery to microbial infection sites. Aim of the work With the ongoing efforts in the nanomedicine field, here we evaluate the biodistribution and “in vivo” safety of SLN specifically designed to encapsulate a new lead antimicrobial agent, exploring the possibility that nanoparticle-based drug delivery systems could improve treatments of bacterial infections. Methods SLN have been produced by Nanovector s.r.l. and specifically designed to encapsulate antimicrobial agents. Their preparation is based on an original warm microemulsion technique allowing the production of nanocarriers in an aqueous solution with a size ranging from approximately 20 nm to 200 nm. The new lead antimicrobial compound SR155 loaded by SLN has been administered in healthy mice via pulmonary route by means of intratracheal aerosolization. The biodistribution of unloaded SLN was evaluated by means of radiochemical technique and in vivo Fluorescent Microscopy Tomography (FMT 1500, Perkin Elmer). Results and discussion Extensive studies have demonstrated that the use of an appropriate nanocarrier for the delivery of therapeutics together with pulmonary route of administration can help to biodistribute the biologically active agents to the lungs and extend their retention period. This can enhance the efficacy of the treatment, reducing possible side effects. Our preliminary results confirmed that [3H]-SLN developed by Nanovector represent a suitable drug delivery system to improve drug bioavailability within the lungs. Inhaled [3H]-SLN ensure greater localization in the lung and lower retention in the liver with a reduced first pass effect. Moreover using a fluorescence microscopy tomography system we were able to evaluate the biodistribution profile of indocarbocyanine iodide(DiR)-labeled SLN in adult mice after single intratracheal instillation (50 µL, 9-18 mg/mL as total lipids). We have shown that DiR-SLN are retained within the lungs for hours after IT aerosolization, and 24 hours after the administration the 20% of the administered dose is still present within the lungs. Biodistribution studies of the SR155 antimicrobial compound are currently running. SR155 loaded by this new SLN formulation IT administered in 4 mice up to the final dose 0.129 mg/mice did not exert any signs of acute toxicity being preserved respiratory functionalities and behavior. Conclusions Many antimicrobial drugs are difficult to administer because of their low water-solubility, cytotoxicity to healthy tissues, rapid degradation and clearance in the blood stream. Their antimicrobial activities against intracellular microbes are also severely limited by poor membrane transport ability. Extensive studies have demonstrated that nanoparticles such as liposomes and solid lipid nanoparticles (SLN) are able to overcome these issues and to facilitate antimicrobial delivery to microbial infection sites. References 1. Cystic Fibrosis Foundation 2012. Patient registry 2011 annual data report. Cystic Fibrosis Foundation, Bethesda, MD, USA. 2. International Journal of Medical Microbiology, 2010, 300: 514-519 3. Med Princ Pract, 2014, DOI:10.1159/000357646