Dopamine and Citicoline-Co-Loaded Solid Lipid Nanoparticles as Multifunctional Nanomedicines for Parkinson's Disease Treatment by Intranasal Administration.

This work aimed to evaluate the potential of the nanosystems constituted by dopamine (DA) and the antioxidant Citicoline (CIT) co-loaded in solid lipid nanoparticles (SLNs) for intranasal administration in the treatment of Parkinson disease (PD). Such nanosystems, denoted as DA-CIT-SLNs, were designed according to the concept of multifunctional nanomedicine where multiple biological roles are combined into a single nanocarrier and prepared by the melt emulsification method employing the self-emulsifying Gelucire® 50/13 as lipid matrix. The resulting DA-CIT-SLNs were characterized regarding particle size, surface charge, encapsulation efficiency, morphology, and physical stability. Differential scanning calorimetry, FT-IR, and X ray diffraction studies were carried out to gain information on solid-state features, and in vitro release tests in simulated nasal fluid (SNF) were performed. Monitoring the particle size at two temperatures (4 °C and 37 °C), the size enlargement observed over the time at 37 °C was lower than that observed at 4 °C, even though at higher temperature, color changes occurred, indicative of possible neurotransmitter decomposition. Solid-state studies indicated a reduction in the crystallinity when DA and CIT are co-encapsulated in DA-CIT-SLNs. Interestingly, in vitro release studies in SNF indicated a sustained release of DA. Furthermore, DA-CIT SLNs displayed high cytocompatibility with both human nasal RPMI 2650 and neuronal SH-SY5Y cells. Furthermore, OxyBlot assay demonstrated considerable potential to assess the protective effect of antioxidant agents against oxidative cellular damage. Thus, such protective effect was shown by DA-CIT-SLNs, which constitute a promising formulation for PD application.

Slightly viscous dispersions of mucoadhesive polymers as vehicles for nasal administration of dopamine and grape seed extract-loaded solid lipid nanoparticles.

With the aim to find an alternative vehicle to the most used thermosensitive hydrogels for efficient nanotechnology-based nose-to-brain delivery approach for Parkinson's disease (PD) treatment, in this work we evaluated the Dopamine (DA) and the antioxidant grape seed-derived pro-anthocyanidins (Grape Seed Extract, GSE) co-loaded solid lipid nanoparticles (SLNs) put in slight viscous dispersions (SVDs). These SVDs were prepared by dispersion in water at low concentrations of mucoadhesive polymers to which SLN pellets were added. For the purpose, we investigated two polymeric blends, namely Poloxamer/Carbopol (PF-127/Carb) and oxidized alginate/Hydroxypropylmethyl cellulose (AlgOX/HPMC). Rheological studies showed that the two fluids possess Newtonian behaviour with a viscosity slightly higher that water. The pH values of the SVDs were mainly within the normal range of nasal fluid as well as almost no osmotic effect was associated to both SVDs. All the SVDs were capable to provide DA permeation through nasal porcine mucosa. Moreover, it was found that PF-127/Carb blend possesses penetration enhancer capability better than the Alg OX/HPMC combination. Flow cytometry studies demonstrated the uptake of viscous liquids incorporating fluorescent SLNs by human nasal RPMI 2650 cell in time-dependent manner. In conclusion, the SVD formulations may be considered promising alternatives to thermosensitive hydrogels strategy. Moreover, in a broader perspective, such SVD formulations may be also hopeful for treating various neurological diseases beyond PD treatment.

Dopamine- and Grape-Seed-Extract-Loaded Solid Lipid Nanoparticles: Interaction Studies between Particles and Differentiated SH-SY5Y Neuronal Cell Model of Parkinson's Disease.

Parkinson's disease (PD) is a prevalent neurodegenerative disorder, primarily associated with dopaminergic neuron depletion in the Substantia Nigra. Current treatment focuses on compensating for dopamine (DA) deficiency, but the blood-brain barrier (BBB) poses challenges for effective drug delivery. Using differentiated SH-SY5Y cells, we investigated the co-administration of DA and the antioxidant Grape Seed Extract (GSE) to study the cytobiocompability, the cytoprotection against the neurotoxin Rotenone, and their antioxidant effects. For this purpose, two solid lipid nanoparticle (SLN) formulations, DA-co-GSE-SLNs and GSE-ads-DA-SLNs, were synthesized. Such SLNs showed mean particle sizes in the range of 187-297 nm, zeta potential values in the range of -4.1--9.7 mV, and DA association efficiencies ranging from 35 to 82%, according to the formulation examined. The results showed that DA/GSE-SLNs did not alter cell viability and had a cytoprotective effect against Rotenone-induced toxicity and oxidative stress. In addition, this study also focused on the evaluation of Alpha-synuclein (aS) levels; SLNs showed the potential to modulate the Rotenone-mediated increase in aS levels. In conclusion, our study investigated the potential of SLNs as a delivery system for addressing PD, also representing a promising approach for enhanced delivery of pharmaceutical and antioxidant molecules across the BBB.

Solid Lipid Nanoparticles Containing Dopamine and Grape Seed Extract: Freeze-Drying with Cryoprotection as a Formulation Strategy to Achieve Nasal Powders.

(1) Background: DA-Gelucire® 50/13-based solid lipid nanoparticles (SLNs) administering the neurotransmitter dopamine (DA) and the antioxidant grape-seed-derived proanthocyanidins (grape seed extract, GSE) have been prepared by us in view of a possible application for Parkinson's disease (PD) treatment. To develop powders constituted by such SLNs for nasal administration, herein, two different agents, namely sucrose and methyl-ß-cyclodextrin (Me-ß-CD), were evaluated as cryoprotectants. (2) Methods: SLNs were prepared following the melt homogenization method, and their physicochemical features were investigated by Raman spectroscopy, Scanning Electron Microscopy (SEM), atomic force microscopy (AFM) and X-ray Photoelectron Spectroscopy (XPS). (3) Results: SLN size and zeta potential values changed according to the type of cryoprotectant and the morphological features investigated by SEM showed that the SLN samples after lyophilization appear as folded sheets with rough surfaces. On the other hand, the AFM visualization of the SLNs showed that their morphology consists of round-shaped particles before and after freeze-drying. XPS showed that when sucrose or Me-ß-CD were not detected on the surface (because they were not allocated on the surface or completely absent in the formulation), then a DA surfacing was observed. In vitro release studies in Simulated Nasal Fluid evidenced that DA release, but not the GSE one, occurred from all the cryoprotected formulations. Finally, sucrose increased the physical stability of SLNs better than Me-ß-CD, whereas RPMI 2650 cell viability was unaffected by SLN-sucrose and slightly reduced by SLN-Me-ß-CD. (4) Conclusions: Sucrose can be considered a promising excipient, eliciting cryoprotection of the investigated SLNs, leading to a powder nasal pharmaceutical dosage form suitable to be handled by PD patients.

Combined Dopamine and Grape Seed Extract-Loaded Solid Lipid Nanoparticles: Nasal Mucosa Permeation, and Uptake by Olfactory Ensheathing Cells and Neuronal SH-SY5Y Cells.

We have already formulated solid lipid nanoparticles (SLNs) in which the combination of the neurotransmitter dopamine (DA) and the antioxidant grape-seed-derived proanthocyanidins (grape seed extract, GSE) was supposed to be favorable for Parkinson's disease (PD) treatment. In fact, GSE supply would reduce the PD-related oxidative stress in a synergic effect with DA. Herein, two different methods of DA/GSE loading were studied, namely, coadministration in the aqueous phase of DA and GSE, and the other approach consisting of a physical adsorption of GSE onto preformed DA containing SLNs. Mean diameter of DA coencapsulating GSE SLNs was 187 ± 4 nm vs. 287 ± 15 nm of GSE adsorbing DA-SLNs. TEM microphotographs evidenced low-contrast spheroidal particles, irrespective of the SLN type. Moreover, Franz diffusion cell experiments confirmed the permeation of DA from both SLNs through the porcine nasal mucosa. Furthermore, fluorescent SLNs also underwent cell-uptake studies by using flow cytometry in olfactory ensheathing cells and neuronal SH-SY5Y cells, evidencing higher uptake when GSE was coencapsulated rather than adsorbed onto the particles.

Mucopenetration study of solid lipid nanoparticles containing magneto sensitive iron oxide.

In most chronic respiratory diseases, excessive viscous airway secretions oppose a formidable permeation barrier to drug delivery systems (DDSs), with a limit to their therapeutic efficacy for the targeting epithelium. Since mucopenetration of DDSs with slippery technology (i.e. PEGylation) has encountered a reduction in the presence of sticky and complex airway secretions, our aim was to evaluate the relevance of magnetic PEGylated Solid Lipid Nanoparticles (mSLNs) for pulling them through chronic obstructive pulmonary disease (COPD) airway secretions. Thus, COPD sputum from outpatient clinic, respiratory secretions aspirated from high (HI) and low (LO) airways of COPD patients in acute respiratory insufficiency, and porcine gastric mucus (PGM) were investigated for their permeability to mSLN particles under a magnetic field. Rheological tests and mSLN adhesion to airway epithelial cells (AECs) were also investigated. The results of mucopenetration show that mSLNs are permeable both in COPD sputum and in PGM, while HI and LO secretions are always impervious. Parallel rheological results show a different elastic property, which can be associated with different mucus mesostructures. Finally, adhesion tests confirm the role of the magnetic field in improving the interaction of SLNs with AECs. Overall, our results reveal that mesostructure is of paramount importance in determining the mucopenetration of magnetic SLNs.

Downregulation of epithelial sodium channel (ENaC) activity in cystic fibrosis cells by epigenetic targeting.

The pathogenic mechanism of cystic fibrosis (CF) includes the functional interaction of the cystic fibrosis transmembrane conductance regulator (CFTR) protein with the epithelial sodium channel (ENaC). The reduction of ENaC activity may constitute a therapeutic option for CF. This hypothesis was evaluated using drugs that target the protease-dependent activation of the ENaC channel and the transcriptional activity of its coding genes. To this aim we used: camostat, a protease inhibitor; S-adenosyl methionine (SAM), showed to induce DNA hypermethylation; curcumin, known to produce chromatin condensation. SAM and camostat are drugs already clinically used in other pathologies, while curcumin is a common dietary compound. The experimental systems used were CF and non-CF immortalized human bronchial epithelial cell lines as well as human bronchial primary epithelial cells. ENaC activity and SCNN1A, SCNN1B and SCNN1G gene expression were analyzed, in addition to SCNN1B promoter methylation. In both immortalized and primary cells, the inhibition of extracellular peptidases and the epigenetic manipulations reduced ENaC activity. Notably, the reduction in primary cells was much more effective. The SCNN1B appeared to be the best target to reduce ENaC activity, in respect to SCNN1A and SCNN1G. Indeed, SAM treatment resulted to be effective in inducing hypermethylation of SCNN1B gene promoter and in lowering its expression. Importantly, CFTR expression was unaffected, or even upregulated, after treatments. These results open the possibility of CF patients' treatment by epigenetic targeting.

Solid Lipid Nanoparticles Administering Antioxidant Grape Seed-Derived Polyphenol Compounds: A Potential Application in Aquaculture.

The supply of nutrients, such as antioxidant agents, to fish cells still represents a challenge in aquaculture. In this context, we investigated solid lipid nanoparticles (SLN) composed of a combination of Gelucire® 50/13 and Precirol® ATO5 to administer a grape seed extract (GSE) mixture containing several antioxidant compounds. The combination of the two lipids for the SLN formation resulted in colloids exhibiting mean particle sizes in the range 139-283 nm and zeta potential values in the range +25.6-43.4 mV. Raman spectra and X-ray diffraction evidenced structural differences between the free GSE and GSE-loaded SLN, leading to the conclusion that GSE alters the structure of the lipid nanocarriers. From a biological viewpoint, cell lines from gilthead seabream and European sea bass were exposed to different concentrations of GSE-SLN for 24 h. In general, at appropriate concentrations, GSE-SLN increased the viability of the fish cells. Furthermore, regarding the gene expression in those cells, the expression of antioxidant genes was upregulated, whereas the expression of hsp70 and other genes related to the cytoskeleton was downregulated. Hence, an SLN formulation containing Gelucire® 50/13/Precirol® ATO5 and GSE may represent a compelling platform for improving the viability and antioxidant properties of fish cells.

Small-molecule drugs for cystic fibrosis: Where are we now?

The cystic fibrosis (CF) lung disease is due to the lack/dysfunction of the CF Transmembrane Conductance Regulator (CFTR), a chloride channel expressed by epithelial cells as the main regulator of ion and fluid homeostasis. More than 2000 genetic variation in the CFTR gene are known, among which those with identified pathomechanism have been divided into six mutation classes. A major advancement in the pharmacotherapy of CF has been the development of small-molecule drugs hitting the root of the disease, i.e. the altered ion and fluid transport through the airway epithelium. These drugs, called CFTR modulators, have been advanced to the clinics to treat nearly 90% of CF patients, including the CFTR potentiator ivacaftor, approved for residual function mutations (Classes III and IV), and combinations of correctors (lumacaftor, tezacaftor, elexacaftor) and ivacaftor for patients bearing at least one the F508del mutation, the most frequent mutation belonging to class II. To cover the 10% of CF patients without etiological therapies, other novel small-molecule CFTR modulators are in evaluation of their effectiveness in all the CFTR mutation classes: read-through agents for Class I, correctors, potentiators and amplifiers from different companies for Class II-V, stabilizers for Class VI. In alternative, other solute carriers, such as SLC26A9 and SLC6A14, are the focus of intensive investigation. Finally, other molecular targets are being evaluated for patients with no approved CFTR modulator therapy or as means of enhancing CFTR modulatory therapy, including small molecules forming ion channels, inhibitors of the ENaC sodium channel and potentiators of the calcium-activated chloride channel TMEM16A. This paper aims to give an up-to-date overview of old and novel CFTR modulators as well as of novel strategies based on small-molecule drugs. Further investigations in in-vivo and cell-based models as well as carrying out large prospective studies will be required to determine if novel CFTR modulators, stabilizers, amplifiers, and the ENaC inhibitors or TMEM16A potentiators will further improve the clinical outcomes in CF management.

DNA Methylation Patterns Correlate with the Expression of SCNN1A, SCNN1B, and SCNN1G (Epithelial Sodium Channel, ENaC) Genes.

The interplay between the cystic fibrosis transmembrane conductance regulator (CFTR) and the epithelial sodium channel (ENaC) in respiratory epithelia has a crucial role in the pathogenesis of cystic fibrosis (CF). The comprehension of the mechanisms of transcriptional regulation of ENaC genes is pivotal to better detail the pathogenic mechanism and the genotype-phenotype relationship in CF, as well as to realize therapeutic approaches based on the transcriptional downregulation of ENaC genes. Since we aimed to study the epigenetic transcriptional control of ENaC genes, an assessment of their expression and DNA methylation patterns in different human cell lines, nasal brushing samples, and leucocytes was performed. The mRNA expression of CFTR and ENaC subunits α, ß and γ (respectively SCNN1A, SCNN1B, and SCNN1G genes) was studied by real time PCR. DNA methylation of 5'-flanking region of SCNN1A, SCNN1B, and SCNN1G genes was studied by HpaII/PCR. The levels of expression and DNA methylation of ENaC genes in the different cell lines, brushing samples, and leukocytes were very variable. The DNA regions studied of each ENaC gene showed different methylation patterns. A general inverse correlation between expression and DNA methylation was evidenced. Leukocytes showed very low expression of all the 3 ENaC genes corresponding to a DNA methylated pattern. The SCNN1A gene resulted to be the most expressed in some cell lines that, accordingly, showed a completely demethylated pattern. Coherently, a heavy and moderate methylated pattern of, respectively, SCNN1B and SCNN1G genes corresponded to low levels of expression. As exceptions, we found that dexamethasone treatment appeared to stimulate the expression of all the 3 ENaC genes, without an evident modulation of the DNA methylation pattern, and that in nasal brushing a considerable expression of all the 3 ENaC genes were found despite an apparent methylated pattern. At least part of the expression modulation of ENaC genes seems to depend on the DNA methylation patterns of specific DNA regions. This points to epigenetics as a controlling mechanism of ENaC function and as a possible therapeutic approach for CF.

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

Background: The 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.

G-CSF and GM-CSF Modify Neutrophil Functions at Concentrations found in Cystic Fibrosis.

The role of colony stimulating factors (CSFs) in cystic fibrosis (CF) circulating neutrophils has not been thoroughly evaluated, considering that the neutrophil burden of lung inflammation in these subjects is very high. The aim of this study was to assess granulocyte-CSF (G-CSF) and granulocyte-macrophage-CSF (GM-CSF) levels in CF patients in various clinical conditions and how these cytokines impact on activation and priming of neutrophils. G-CSF and GM-CSF levels were measured in sputum and serum samples of stable CF patients (n = 21) and in CF patients with acute exacerbation before and after a course of antibiotic therapy (n = 19). CSFs were tested on non CF neutrophils to investigate their effects on reactive oxygen species (ROS) production, degranulation (CD66b, elastase, lactoferrin, MMP-9), and chemotaxis. At very low concentrations found in CF patients (0.005-0.1 ng/ml), both cytokines inhibited ROS production, while higher concentrations (1-5 ng/ml) exerted a stimulatory effect. While either CSF induced elastase and MMP-9 secretion, lactoferrin levels were increased only by G-CSF. Chemotaxis was inhibited by GM-CSF, but was increased by G-CSF. However, when present together at low concentrations, CSFs increased basal and fMLP-stimulated ROS production and chemotaxis. These results suggest the CSF levels that circulating neutrophils face before extravasating into the lungs of CF patients may enhance their function contributing to the airway damage.

Peripheral localization of the epithelial sodium channel in the apical membrane of bronchial epithelial cells.

NEW FINDINGS: What is the central question of this study? What is the precise subcellular localization of the epithelial sodium channel (ENaC) in human airway epithelium? What is the main finding and its importance? ENaC protein has an unexpected localization in the peripheral region of the apical membrane of bronchial epithelial cells, very close to tight junctions. This may be important for the mechanism of Na+ absorption ABSTRACT: The epithelial sodium channel (ENaC) has a key role in absorbing fluid across the human airway epithelium. Altered activity of ENaC may perturb the process of mucociliary clearance, thus impairing the innate defence mechanisms against microbial agents. The proteins forming ENaC are present on the apical membrane of the epithelium. However, their precise localization is unknown. In the present study, we used two antibodies recognizing the α and ß ENaC subunits. Both antibodies revealed a restricted localization of ENaC in the peripheral region of the apical membrane of cultured bronchial epithelial cells, close to but not overlapping with tight junctions. In contrast, the cystic fibrosis transmembrane conductance regulator chloride channel was more diffusely expressed on the whole apical membrane. Modulation of ENaC activity by aprotinin or elastase resulted in a decrease or increase in the peripheral localization, respectively. Our results suggest that sodium absorption is mainly occurring close to tight junctions where this cation may be rapidly expelled by the Na+ /K+ pump present in lateral membranes. This arrangement of channels and pumps may limit Na+ build-up in other regions of the cells.

Human Cellular Models for the Investigation of Lung Inflammation and Mucus Production in Cystic Fibrosis.

Chronic inflammation, oxidative stress, mucus plugging, airway remodeling, and respiratory infections are the hallmarks of the cystic fibrosis (CF) lung disease. The airway epithelium is central in the innate immune responses to pathogens colonizing the airways, since it is involved in mucociliary clearance, senses the presence of pathogens, elicits an inflammatory response, orchestrates adaptive immunity, and activates mesenchymal cells. In this review, we focus on cellular models of the human CF airway epithelium that have been used for studying mucus production, inflammatory response, and airway remodeling, with particular reference to two- and three-dimensional cultures that better recapitulate the native airway epithelium. Cocultures of airway epithelial cells, macrophages, dendritic cells, and fibroblasts are instrumental in disease modeling, drug discovery, and identification of novel therapeutic targets. Nevertheless, they have to be implemented in the CF field yet. Finally, novel systems hijacking on tissue engineering, including three-dimensional cocultures, decellularized lungs, microfluidic devices, and lung organoids formed in bioreactors, will lead the generation of relevant human preclinical respiratory models a step forward.

Assembly and Functional Analysis of an S/MAR Based Episome with the Cystic Fibrosis Transmembrane Conductance Regulator Gene.

Improving the efficacy of gene therapy vectors is still an important goal toward the development of safe and efficient gene therapy treatments. S/MAR (scaffold/matrix attached region)-based vectors are maintained extra-chromosomally in numerous cell types, which is similar to viral-based vectors. Additionally, when established as an episome, they show a very high mitotic stability. In the present study we tested the idea that addition of an S/MAR element to a CFTR (cystic fibrosis transmembrane conductance regulator) expression vector, may allow the establishment of a CFTR episome in bronchial epithelial cells. Starting from the observation that the S/MAR vector pEPI-EGFP (enhanced green fluorescence protein) is maintained as an episome in human bronchial epithelial cells, we assembled the CFTR vector pBQ-S/MAR. This vector, transfected in bronchial epithelial cells with mutated CFTR, supported long term wt CFTR expression and activity, which in turn positively impacted on the assembly of tight junctions in polarized epithelial cells. Additionally, the recovery of intact pBQ-S/MAR, but not the parental vector lacking the S/MAR element, from transfected cells after extensive proliferation, strongly suggested that pBQ-S/MAR was established as an episome. These results add a new element, the S/MAR, that can be considered to improve the persistence and safety of gene therapy vectors for cystic fibrosis pulmonary disease.

Preparation of drug-loaded small unilamellar liposomes and evaluation of their potential for the treatment of chronic respiratory diseases.

The aim of the present investigation was to evaluate the influence of liposome formulation on the ability of vesicles to penetrate a pathological mucus model obtained from COPD affected patients in order to assess the potential of such vesicles for the treatment of chronic respiratory diseases by inhalation. Therefore, Small Unilamellar Liposomes (PLAIN-LIPOSOMEs), Pluronic® F127-surface modified liposomes (PF-LIPOSOMEs) and PEG 2000PE-surface modified liposomes (PEG-LIPOSOMEs) were prepared using the micelle-to-vesicle transition (MVT) method and beclomethasone dipropionate (BDP) as model drug. The obtained liposomes showed diameters in the range of 40-65 nm, PDI values between 0.25 and 0.30 and surface electric charge essentially close to zero. The encapsulation efficiency was found to be dependent on the BDP/lipid ratio used and, furthermore, BDP-loaded liposomes were stable in size both at 37 °C and at 4 °C. All liposomes were not cytotoxic on H441 cell line as assessed by the MTT assay. The liposome uptake was evaluated through a cytofluorimetric assay that showed a non-significant reduction in the internalization of PEG-LIPOSOMEs as compared with PLAIN-LIPOSOMEs. The penetration studies of mucus from COPD patients showed that the PEG-LIPOSOMEs were the most mucus-penetrating vesicles after 27 h. In addition, PEG- and PF-LIPOSOMEs did not cause any effect on bronchoalveolar lavage fluid proteins after aerosol administration in the mouse. The results highlight that PEG-LIPOSOMEs show the most interesting features in terms of penetration through the pathologic sputum, uptake by airway epithelial cells and safety profile.

Gap Junctions Are Involved in the Rescue of CFTR-Dependent Chloride Efflux by Amniotic Mesenchymal Stem Cells in Coculture with Cystic Fibrosis CFBE41o- Cells.

We previously found that human amniotic mesenchymal stem cells (hAMSCs) in coculture with CF immortalised airway epithelial cells (CFBE41o- line, CFBE) on Transwell® filters acquired an epithelial phenotype and led to the expression of a mature and functional CFTR protein. In order to explore the role of gap junction- (GJ-) mediated intercellular communication (GJIC) in this rescue, cocultures (hAMSC : CFBE, 1 : 5 ratio) were studied for the formation of GJIC, before and after silencing connexin 43 (Cx43), a major component of GJs. Functional GJs in cocultures were inhibited when the expression of the Cx43 protein was downregulated. Transfection of cocultures with siRNA against Cx43 resulted in the absence of specific CFTR signal on the apical membrane and reduction in the mature form of CFTR (band C), and in parallel, the CFTR-dependent chloride channel activity was significantly decreased. Cx43 downregulation determined also a decrease in transepithelial resistance and an increase in paracellular permeability as compared with control cocultures, implying that GJIC may regulate CFTR expression and function that in turn modulate airway epithelium tightness. These results indicate that GJIC is involved in the correction of CFTR chloride channel activity upon the acquisition of an epithelial phenotype by hAMSCs in coculture with CF cells.

The long and winding road: stem cells for cystic fibrosis.

INTRODUCTION: Cystic fibrosis (CF) is a genetic syndrome with a high mortality rate due to severe lung disease. Despite having several drugs targeting specific mutated CFTR proteins already in clinical trials, new therapies, based on stem cells, are also emerging to treat those patients. AREAS COVERED: The authors review the main sources of stem cells, including embryonic stem cells (ESCs), induced-pluripotent stem cells (iPSCs), gestational stem cells, and adult stem cells, such as mesenchymal stem cells (MSCs) in the context of CF. Furthermore, they describe the main animal and human models of lung physiology and pathology, involved in the optimization of these stem cell-applied therapies in CF. EXPERT OPINION: ESCs and iPSCs are emerging sources for disease modeling and drug discovery purposes. The allogeneic transplant of healthy MSCs, that acts independently to specific mutations, is under intense scrutiny due to their secretory, immunomodulatory, anti-inflammatory and anti-bacterial properties. The main challenge for future developments will be to get exogenous stem cells into the appropriate lung location, where they can regenerate endogenous stem cells and act as inflammatory modulators. The clinical application of stem cells for the treatment of CF certainly warrants further insight into pre-clinical models, including large animals, organoids, decellularized organs and lung bioengineering.

Insulin-like growth factor-6 (IGFBP-6) stimulates neutrophil oxidative burst, degranulation and chemotaxis.

The aim of this study was to understand whether insulin-like growth factor-binding protein-6 (IGFBP-6) has functional effects on neutrophils, in particular when they cross epithelium during inflammation. We found that IGFBP-6 increased ROS production (cytofluorimetry), degranulation of primary and tertiary granules (ELISA) and transmigration through the epithelial monolayer. No priming by IGFBP-6 on neutrophils stimulated by either PMA or fMLP was observed. IGFBP-6 is an agonist of neutrophils' functions, most likely when these cells have been already activated by other stimuli.

Human monocyte-derived dendritic cells exposed to hyperthermia show a distinct gene expression profile and selective upregulation of IGFBP6.

Fever plays a role in activating innate immunity while its relevance in activating adaptive immunity is less clear. Even brief exposure to elevated temperatures significantly impacts on the immunostimulatory capacity of dendritic cells (DCs), but the consequences on immune response remain unclear. To address this issue, we analyzed the gene expression profiles of normal human monocyte-derived DCs from nine healthy adults subjected either to fever-like thermal conditions (39°C) or to normal temperature (37°C) for 180 minutes. Exposure of DCs to 39°C caused upregulation of 43 genes and downregulation of 24 genes. Functionally, the up/downregulated genes are involved in post-translational modification, protein folding, cell death and survival, and cellular movement. Notably, when compared to monocytes, DCs differentially upregulated transcription of the secreted protein IGFBP-6, not previously known to be specifically linked to hyperthermia. Exposure of DCs to 39°C induced apoptosis/necrosis and resulted in accumulation of IGFBP-6 in the conditioned medium at 48 h. IGFBP-6 may have a functional role in the hyperthermic response as it induced chemotaxis of monocytes and T lymphocytes, but not of B lymphocytes. Thus, temperature regulates complex biological DC functions that most likely contribute to their ability to induce an efficient adaptive immune response.