Role of Neutrophils in Cystic Fibrosis Lung Disease

Cystic fibrosis (CF) is a genetic syndrome caused by mutations in the CF Transmembrane Conductance Regulator (CFTR) gene. In CF patients, chief morbidity and mortality are due to pulmonary manifestations. CFTR lack/dysfunction brings an altered ion flux through the airway epithelium and ablation of mucociliary clearance, which in turn ensues in colonization and infection by opportunistic bacterial pathogens and subsequent neutrophil‐dominated inflammation. This response eventually leads to the damage of the lung tissue. A host of inflammatory mediators attract, activate, and reprogramme neutrophils to survive (avoiding apoptosis) and produce a wealth of proteases and radical oxygen species. The protease/antiprotease imbalance and oxidative stress have multiple downstream effects, including impaired mucus clearance, increased and self‐perpetuating inflammation, and impaired immune responses, thus facilitating and fostering bacterial infections. On the other hand, CFTR lack or dysfunction is likely responsible for alterations in neutrophils concerning chemotaxis, phagocytosis, oxidative burst, degranulation, and neutrophil extracellular trap (NET) formation. A good opportunity to reveal new and non‐invasive biomarkers of CF lung disease is the evaluation of circulating neutrophils. Indeed, neutrophil responses are now investigated as outcomes of the aetiological therapies in CF, such as hypertonic saline, antiproteases, CFTR correctors and potentiators

Impact of Lentiviral Vector-Mediated Transduction on the Tightness of a Polarized Model of Airway Epithelium and Effect of Cationic Polymer Polyethylenimine

Lentiviral (LV) vectors are promising agents for efficient and long-lasting gene transfer into the lung and for gene therapy of genetically determined pulmonary diseases, such as cystic fibrosis, however, they have not been evaluated for cytotoxicity and impact on the tightness of the airway epithelium. In this study, we evaluated the transduction efficiency of a last-generation LV vector bearing Green Fluorescent Protein (GFP) gene as well as cytotoxicity and tight junction (TJ) integrity in a polarized model of airway epithelial cells. High multiplicities of infection (MOI) showed to be cytotoxic, as assessed by increase in propidium iodide staining and decrease in cell viability, and harmful for the epithelial tightness, as demonstrated by the decrease of transepithelial resistance (TER) and delocalization of occludin from the TJs. To increase LV efficiency at low LV:cell ratio, we employed noncovalent association with the polycation branched 25 kDa polyethylenimine (PEI). Transduction of cells with PEI/LV particles resulted in 2.5–3.6-fold increase of percentage of GFP-positive cells only at the highest PEI:LV ratios (1×107 PEI molecules/transducing units with 50 MOI LV) as compared to plain LV. At this dose PEI/LV transduction resulted in 6.5 ± 2.4% of propidium iodide-positive cells. On the other hand, PEI/LV particles did not determine any alteration of TER and occludin localization. We conclude that PEI may be useful for improving the efficiency of gene transfer mediated by LV vectors in airway epithelial cells, in the absence of high acute cytotoxicity and alteration in epithelial tightness

Insulin-Like Growth Factor Binding Protein (IGFBP-6) as a Novel Regulator of Inflammatory Response in Cystic Fibrosis Airway Cells

Cystic Fibrosis (CF) patients are prone to contracting bacterial lung infections with opportunistic pathogens, especially Pseudomonas aeruginosa. Prolonged P. aeruginosa infections have been linked to chronic inflammation in the CF lung, whose hallmarks are increased levels of cytokines (i.e., TNF-alpha, IL-1 beta, IL-6) and neutrophil attraction by chemokines, like IL-8. Recently, insulin-like growth factor binding protein 6 (IGFBP-6) has been shown to play a putative role in the immune system and was found at higher levels in the sera and synovial tissue of rheumatoid arthritis patients. Moreover, it has been demonstrated that IGFBP-6 has chemoattractant properties towards cells of the innate (neutrophils, monocytes) and adaptive (T cells) immunity. However, it is not known whether IGFBP-6 expression is dysregulated in airway epithelial cells under infection/inflammatory conditions. Therefore, we first measured the basal IGFBP-6 mRNA and protein levels in bronchial epithelial cells lines (Wt and F508del-CFTR CFBE), finding they both are upregulated in F508del-CFTR CFBE cells. Interestingly, LPS and IL-1 beta+TNF alpha treatments increased the IGFBP-6 mRNA level, that was reduced after treatment with an anti-inflammatory (Dimethyl Fumarate) in CFBE cell line and in patient-derived nasal epithelial cultures. Lastly, we demonstrated that IGFBP-6 reduced the level of pro-inflammatory cytokines in both CFBE and primary nasal epithelial cells, without affecting rescued CFTR expression and function. The addition of a neutralizing antibody to IGFBP-6 increased pro-inflammatory cytokines expression under challenge with LPS. Together, these data suggest that IGFBP-6 may play a direct role in the CF-associated inflammation

Eudragit s100 entrapped liposome for curcumin delivery: Anti-oxidative effect in Caco-2 cells

Curcumin is a natural polyphenol with strong antioxidant activity. However, this molecule shows a very poor bioavailability, instability, and rapid metabolism in vivo. In this work curcumin was loaded in Eudragit-coated liposomes to create a gastroresistant carrier, able to protect its load from degradation and free it at the site of absorption in the colon region. Small unilamellar vesicles were prepared and coated with Eudragit by a pH-driven method. The physico-chemical properties of the prepared systems were assessed by light scattering, transmission electron microscopy, infrared spectroscopy, and differential scanning calorimetry. The uptake of vesicles by Caco-2 cells and the anti-oxidant activity in cells were evaluated. The produced vesicles showed dimensions of about forty nanometers that after covering with Eudragit resulted to have micrometric dimensions at acid pH. The experiments showed that at pH > 7.0 the polymeric coating dissolves, releasing the nanometric liposomes and allowing them to enter Caco-2 cells. Delivered curcumin loaded vesicles were then able to decrease significantly ROS levels as induced by H2O2 in Caco-2 cells. The proposed work showed the possibility of realizing effective gastroresistant curcumin liposome formulations for the delivery of antioxidant molecules to Caco-2 cells, potentially applicable to the treatment of pathological conditions related to intestinal oxidative stress. View Full-Tex

Polymer Encapsulated Liposomes for Oral Co-Delivery of Curcumin and Hydroxytyrosol

Curcumin (Cur) is a hydrophobic polyphenol from the rhizome of Curcuma spp., while hydroxytyrosol (HT) is a water-soluble polyphenol from Olea europaea. Both show outstanding antioxidant properties but suffer from scarce bioavailability and low stability in biological fluids. In this work, the co-encapsulation of Cur and HT into liposomes was realized, and the liposomal formulation was improved using polymers to increase their survival in the gastrointestinal tract. Liposomes with different compositions were formulated: Type 1, composed of phospholipids and cholesterol; Type 2, also with a PEG coating; and Type 3 providing an additional shell of Eudragit® S100, a gastro-resistant polymer. Samples were characterized in terms of size, morphology, ζ-potential, encapsulation efficiency, and loading capacity. All samples were subjected to a simulated in vitro digestion and their stability was investigated. The Eudragit®S100 coating demonstrated prevention of early releases of HT in the mouth and gastric phases, while the PEG shell reduced bile salts and pancreatin effects during the intestinal digestion. In vitro antioxidant activity showed a cumulative effect for Cur and HT loaded in vesicles. Finally, liposomes with HT concentrations up to 40 μM and Cur up to 4.7 μM, alone or in combination, did not show cytotoxicity against Caco-2 cells

Amniotic Mesenchymal Stem Cells: A New Source for Hepatocyte-Like Cells and Induction of CFTR Expression by Coculture with Cystic Fibrosis Airway Epithelial Cells

Cystic fibrosis (CF) is a monogenic disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, with lung and liver manifestations. Because of pitfalls of gene therapy, novel approaches for reconstitution of the airway epithelium and CFTR expression should be explored. In the present study, human amniotic mesenchymal stem cells (hAMSCs) were isolated from term placentas and characterized for expression of phenotypic and pluripotency markers, and for differentiation potential towards mesoderm (osteogenic and adipogenic) lineages. Moreover, hAMSCs were induced to differentiate into hepatocyte-like cells, as demonstrated by mixed function oxidase activity and expression of albumin, alpha1-antitrypsin, and CK19. We also investigated the CFTR expression in hAMSCs upon isolation and in coculture with CF airway epithelial cells. Freshly isolated hAMSCs displayed low levels of CFTR mRNA, which even decreased with culture passages. Following staining with the vital dye CM-DiI, hAMSCs were mixed with CFBE41o- respiratory epithelial cells and seeded onto permeable filters. Flow cytometry demonstrated that 33–50% of hAMSCs acquired a detectable CFTR expression on the apical membrane, a result confirmed by confocal microscopy. Our data show that amniotic MSCs have the potential to differentiate into epithelial cells of organs relevant in CF pathogenesis and may contribute to partial correction of the CF phenotype

Cyto/Biocompatibility of Dopamine Combined with the Antioxidant Grape Seed-Derived Polyphenol Compounds in Solid Lipid Nanoparticles

none10The loss of nigrostriatal neurons containing dopamine (DA) together with the “mitochondrial dysfunction” in midbrain represent the two main causes related to the symptoms of Parkinson’s disease (PD). Hence, the aim of this investigation is to co-administer the missing DA and the antioxidant grape seed-derived proanthocyanidins (grape seed extract, GSE) in order to increase the levels of the neurotransmitter (which is unable to cross the Blood Brain Barrier) and reducing the oxidative stress (OS) related to PD, respectively. Methods: For this purpose, we chose Solid Lipid Nanoparticles (SLN), because they have been already proven to increase DA uptake in the brain. DA-SLN adsorbing GSE (GSE/DA-SLN) were formulated and subjected to physico-chemical characterization, and their cytocompatibility and protection against OS were examined. Results: GSE was found on SLN surface and release studies evidenced the efficiency of GSE in preventing DA autoxidation. Furthermore, SLN showed high mucoadhesive strength and were found not cytotoxic to both primary Olfactory Ensheathing and neuroblastoma SH-SY5Y cells by MTT test. Co-administration of GSE/DA-SLN and the OS-inducing neurotoxin 6-hydroxydopamine (100 μM) resulted in an increase of SH-SY5Y cell viability. Conclusions: Hence, SLN formulations containing DA and GSE may constitute interesting candidates for non-invasive nose-to-brain delivery.openAdriana Trapani, Lorenzo Guerra, Filomena Corbo, Stefano Castellani, Enrico Sanna, Loredana Capobianco, Anna Grazia Monteduro, Daniela Erminia Manno, Delia Mandracchia, Sante Di Gioia and Massimo ConeseTrapani, Adriana; Guerra, Lorenzo; Corbo, Filomena; Castellani, Stefano; Sanna, Enrico; Capobianco, Loredana; Monteduro, ANNA GRAZIA; Manno, Daniela Erminia; Mandracchia, Delia; Di Gioia and Massimo Conese, Sant