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PEGylation is currently the most widespread strategy for the stabilization of nanocarriers for biomedical applications. However, the recognized stealth properties of PEG collide with accelerated blood clearance phenomena and frequently induced side effects. In this work, polydopamine (PDA), a bioinspired polymer, is proposed and evaluated as an alternative to PEG for liposome coating and stabilization. The PDA polymerization conditions are optimized, and the PDA-coated vesicles (Lipo@PDA) are fully characterized from a physicochemical point of view. Protein Corona (PC) formed after vesicle incubation in Fetal Bovine Serum (FBS) is characterized, given that PC determines the behavior of nanocarriers in biological fluids. Proteomic analysis reveals high homology between the most abundant recovered proteins in the PC of Lipo@PDA and PEG-coated liposomes (Lipo@PEG). From a quantitative standpoint, no significant differences were highlighted between the two systems. As for the hemolytic response, it is possible to stay within the 5% red blood cell lysis safety threshold by modulating the concentration and thickness of the PDA shell. Cyto-biocompatibility of Lipo@PDA and Lipo@PEG vesicles towards human respiratory cells NCI–H441 is evaluated. Both kinds of liposomes show a similar behavior: cytotoxicity slightly increases with their concentration. However, biocompatible Lipo@PDA vesicles are obtainable at all tested concentrations by reducing the thickness of the PDA coating. Gathered data suggest that the PDA coating can give the liposomes the same behavior in FBS as the PEG coating, thus offering an opportunity to overcome the drawbacks associated with the use of PEG.

In the context of biointeractive dressings used for enhancing wound healing, the use of stromal vascular fraction (SVF) or adipose-derived stem cells (ASCs) hereof derived has not been fully exploited yet. Noncultured SVF, a heterogeneous mesenchymal population of cells, is attractive in the field of dermal regeneration because it can be instantaneously obtained, avoids genomic alterations, and is comparatively safer than cultured ASCs. Integra®Dermal Regeneration Template (DRT) was sprinkled with ASCs in complete medium supplemented with 10% fetal bovine serum (FBS), or SVF, obtained from emulsified or nonemulsified fat, in medium supplemented with 2% platelet-rich plasma (PRP). The presence and differentiation of cells were evaluated by standard histochemistry and immunohistochemistry, whereas conditioned media were analyzed for vascular endothelial growth factors (VEGF) by ELISA. In vitroexperiments were conducted to analyze ASC proliferation in the presence of either FBS or PRP. Deposition of ASCs in medium supplemented with FBS caused their integration into Integra DRT as early as 1 h. ASCs were found as aggregates until 6–10 days without forming organized structures. When seeded onto Integra DRT, SVF cells in medium supplemented with PRP formed aggregates at early times, which at 7 and 10 days organized into vascular-like structures, lined by CD31+and smooth muscle actin-positive cells. With nonemulsified fat, the lacunar structures did not show an organized distribution of SVF cells. PRP induced ASC proliferation although at lower level than FBS. VEGF secretion was enhanced when fat emulsification was introduced into the protocol. In conclusion, the combination of SVF cells obtained from emulsified fat, PRP, and Integra DRT exhibit synergistic effect on the formation of vessel-like structures indicating a step forward aimed at regenerative surgery for chronic wound healing.

ABSTRACTIntroductionCystic Fibrosis (CF) is caused by mutations in the Cystic Fibrosis Transmembrane Conductance Regulator(CFTR) gene. The most common mutation, F508del, induces protein misprocessing and loss of CFTR function. The discovery through in vitrostudies of the CFTR correctors (i.e. lumacaftor, tezacaftor) that partially rescue the misprocessing of F508del-CFTR with the potentiator ivacaftor is promising in giving an unprecedented clinical benefit in affected patients.Areas coveredOnline databases were searched using key phrases for CF and CFTR modulators. Tezacaftor–ivacaftor treatment has proved to be safer than lumacaftor–ivacaftor, although clinical efficacy is similar. Further clinical efficacy has ensued with the introduction of triple therapy, i.e. applying second-generation correctors, such as VX-569 and VX-445 (elexacaftor) to tezacaftor–ivacaftor. The triple combinations will herald the availability of etiologic therapies for patients for whom no CFTR modulators are currently applied (i.e. F508del/minimal function mutations) and enhance CFTR modulator therapy for patients homozygous for F508del.Expert opinionCF patient-derived tissue models are being explored to determine donor-specific response to current approved and future novel CFTR modulators for F508deland other rare mutations. The discovery and validation of biomarkers of CFTR modulation will complement these studies in the long term and in real-life world.

Parkinson’s disease (PD) is characterized by loss of dopaminergic neurons, oxidative stress and neuroinflammation. The classical therapeutic approach with L-DOPA is not able to control motor symptoms in the long term, thus new disease-modifying or neuroprotective treatments are urgently required in order to match such yet unmet clinical needs. Success in cell-based therapy has been accomplished at a clinical level with human fetal mesencephalic tissue, but ethical issues and a shortage of organs clearly underline the need for novel sources of dopaminergic neurons. Mesenchymal stem cells (MSCs) can be obtained from different adult and fetal tissues that are normally discarded as waste, including adipose tissue, placenta, umbilical cord, and dental tissues. Their neuroregenerative, anti-inflammatory and immunomodulatory properties are mainly mediated by the secretion of an array of bioactive molecules and are heightened when MSCs form tri-dimensional structures called spheroids. Not only can MSCs spontaneously produce neurotrophic factors (NFs) but they can be engineered to synthetize and secrete them in vivo. The aim of this review is to provide a picture of results gained with MSC secretome and spheroids in PD, as well as the possibility of harnessing MSC-based therapy with the use of nano- and micro-structured materials for NF delivery.

ABSTRACTIntroduction: 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.

Few mutations in cishave been annotated for F508del homozygous patients. Southern Italy patients who at a first analysis appeared homozygous for the F508del mutation (n=63) or compound heterozygous for the F508del and another mutation in the cystic fibrosis transmembrane conductance regulator gene (n=155) were searched for the A238V mutation in exon 6. The allelic frequency of the complex allele [A238V;F508del] was 0.04. When the whole data set was used (comprised also of 56 F508del/F508del and 34 F508del/other mutation controls), no differences reached the statistical significance in the clinical parameters, except chloride concentrations which were lower in [A238V;F508del]/other mutation compared with F508del/other mutation (P=0.03). The two study groups presented less complications than the control groups. Within the minimal data set (34 F508del/F508del, 27 F508del/other mutation, 4 [A238V;F508del]/F508del cases and 5 [A238V;F508del]/other mutation cases); that is, presenting all the variables in each patient, forced expiratory volume in 1?s and forced vital capacity presented a trend to lower levels in the study groups in comparison with the F508del/F508del group, and C-reactive protein approximated statistically significant higher levels in the [A238V;F508del]/other mutation as compared with F508del/F508del patients (P=0.09). The analysis of statistical dependence among the variables showed a significant anticorrelation between chloride and body mass index in the [A238V;F508del]/other mutation group. In conclusion, the complex allele [A238V;F508del] seems to be associated with less general complications than in the control groups, on the other hand possibly giving a worse pulmonary phenotype and higher systemic/local inflammatory response. These findings have implications for the correct recruitment and clinical response of F508del patients in the clinical trials testing the new etiological drugs for cystic fibrosis.

Lung diseases represent a significant burden of morbidity and mortality worldwide. Current therapies have not proven adequate in the long term and are often associated with significant side effects. There has been recent interest in the regenerative/reparative potential of cell-based therapies, including cells derived from the placental tissues. Amnionderived cells are fetal-derived and characterized by expression profile and differentiative capacity of pluripotent cells. Moreover, because placenta is discarded after delivery, they represent an ethical source for the purposes of regenerative medicine. Amnion-derived cells are endowed with immunomodulatory, anti-inflammatory, anti-scarring and antibacterial properties, which may explain many of the beneficial effects observed with administration of the cells in animal models for a large number of inflammatory diseases. Both human amniotic epithelial cells (hAEC) and mesenchymal stromal cells (hAMSC) have been shown to acquire in vitro and in vivo some characteristics of epithelial cells, i.e. CFTR (cystic fibrosis transmembrane conductance regulator) and surfactant proteins. Administration of hAEC or hAMSC in vivo in the bleomycin-induced lung injury model has proven their therapeutic effects in term of reduction of pulmonary fibrosis and inflammation, as well as recovery of lung mechanical function. Many biological and clinical information have to be gathered before proposing amnion-derived cells in the clinic for the treatment of acute and chronic lung diseases.

Pulmonary delivery of locally-acting drugs encapsulated in nanocarriers provides several advantages for the treatment of respiratory diseases such as asthma, chronic obstructive pulmonary diseases, cystic fibrosis, tuberculosis and lung cancer. These advantages include, among others, sustained drug delivery to the lungs, reduced therapeutic dose and improved patient compliance. The aim of this review is to give an updated overview on recent advances recorded in the last few years in this field as well as on the major challenges still existing and that remain to be overcome before any clinical application. After an outline on the cellular and extracellular barriers affecting drug delivery to the airways both in physiological and pathological conditions, the significant developments recorded using inhaled polymeric- and lipid-based nanocarriers for drug and gene delivery to the lung are presented. In this discussion, the major challenges existing in the field are evidenced including the understanding of the factors governing the mucus penetration capability of these nanocarriers and the identification of new technologies for delivering drugs to specific regions or cell types of the lungs. In this regard, the recognition of receptor expressed only at lung level may facilitate drug targeting to this organ and it should improve the therapeutic efficacy of nanocarrier-based treatments for respiratory diseases.

Introduction/Purpose:Sleep is critical for health and plays a role in motor learning, which is an important component of post stroke rehabilitation. There is limited research on the impact of sleep on functional outcomes during the early stages of rehabilitation after stroke. The purpose of this study was to explore the associations between sleep parameters and functional recovery during inpatient rehabilitation in people with stroke.Methods:13 participants undergoing inpatient rehabilitation after stroke were recruited to participate. Function was measured at admission and discharge using GG Self Care and Mobility scales. Sleep parameters; sleep efficiency and wake after sleep onset (WASO); were determined from an ActiGraph wGT3X-BT activity monitor (AGAM), which participants wore for 5 days continuously upon admission. Associations between sleep parameters and function were determined using Pearson correlation coefficients.Results:Mean time since stroke was 15.5 (9.0) days, mean National Institutes of Stroke Scale score was 6.2 (4.1), mean age was 65.1 (12.1) years old, and 54% were women. There was a moderate association between sleep efficiency and change in GG mobility score; r=0.67 (p=0.012); and a moderate inverse association between WASO and change in GG mobility scores: r=-0.66 (p=0.013). There was not a significant association between change in GG self-care score and sleep parameters (p>=0.05).Discussion:Quality of sleep is associated with improvement in functional mobility during inpatient rehabilitation after stroke. Participants with better sleep efficiency and less time awake during the night after falling asleep during the first 5 days of their inpatient rehabilitation stay had greater improvement in functional mobility at discharge. Further research is needed to examine the complex relationships between sleep and recovery after stroke and to investigate possible interventions to promote better sleep during inpatient rehabilitation after stroke.

Cystic Fibrosis (CF) lung disease is characterized by high levels of cytokines and chemokines in the airways, producing chronic inflammation. Non-invasive biomarkers, which are also specific for the inflammatory and immune responses, are urgently needed to identify exacerbations and evaluate therapeutic efficacy. The aim of this study is to evaluate the association of sputum and exhaled breath condensate (EBC) biomarker changes with clinical exacerbation and response to therapy. We studied the simultaneous presence and concentration of twelve cytokines and growth factors (EGF, IL-1α, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IFN-γ, MCP-1, TNF-α and VEGF) by a multi-parametric biochip array in sputum and EBC of 24 CF patients before, after 6 and 15 days of therapy, and 15 days after the end of treatment for an acute exacerbation. Correlations with functional respiratory tests (FEV1, FVC) and the systemic marker C-reactive protein (CRP) were looked for. In sputum, before therapy, VEGF and IL-1β levels positively correlated with the respiratory function and CRP. Sputum IL-1α, IL-1β IL-4, IL-10, TNF-α, and VEGF significantly decreased, while EGF increased, during therapy. IL-8 and IL-4 levels negatively correlated with the respiratory function at 15 and 30 days from the start of therapy, respectively. IL-4, IL-6, IL-10 and TNF-α positively correlated with CRP during therapy. Although some EBC biomarkers correlated with respiratory function and CRP, no significant associations with these clinical parameters were found. Sputum IL-1β and VEGF might be considered biomarkers of an acute exacerbation in CF patients. A panel of sputum cytokines and growth factors may better describe the response to intravenous antibiotic treatment of CF than one single systemic marker.

Cystic Fibrosis (CF), the most common autosomal lethal disorder in Caucasians, is caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. Although CF is multi-organ disease, the lung pathology is the chief cause of morbidity and mortality of CF patients. The hallmarks of CF lung disease are respiratory infection by opportunistic pathogens and a deranged inflammatory response. However, clinical and experimental data suggest that CF is a hyperinflammatory disorder which can arise in the absence of infection. Laboratory and animal studies suggest that CFTR is involved in regulating some neutrophil and macrophage functions and indicate that altered properties of immune cells may contribute to the dysregulated inflammation in the CF lung. Moreover, recent investigations point out to the involvement of lymphocyte subpopulations in the onset of an altered immune response to pathogens. The development of novel therapies aimed to reduce the inflammatory and regulate immune responses, including stem cell-based treatment, will be presented.

We have demonstrated that Na+/H+exchanger regulatory factor 1 (NHERF1) overexpression in CFBE41o- cells induces a significant redistribution of F508del cystic fibrosis transmembrane conductance regulator (CFTR) from the cytoplasm to the apical membrane and rescues CFTR-dependent chloride secretion. Here, we observe that CFBE41o- monolayers displayed substantial disassembly of actin filaments and that overexpression of wild-type (wt) NHERF1 but not NHERF1-Δ Ezrin-Radixin-Moesin (ERM) increased F-actin assembly and organization. Furthermore, the dominant-negative band Four-point one, Ezrin, Radixin, Moesin homology (FERM) domain of ezrin reversed the wt NHERF1 overexpression-induced increase in both F-actin and CFTR-dependent chloride secretion. wt NHERF1 overexpression enhanced the interaction between NHERF1 and both CFTR and ezrin and between ezrin and actin and the overexpression of wt NHERF1, but not NHERF1-ΔERM, also increased the phosphorylation of ezrin in the apical region of the cell monolayers. Furthermore, wt NHERF1 increased RhoA activity and transfection of constitutively active RhoA in CFBE41o- cells was sufficient to redistribute phospho-ezrin to the membrane fraction and rescue both the F-actin content and the CFTR-dependent chloride efflux. Rho kinase (ROCK) inhibition, in contrast, reversed the wt NHERF1 overexpression-induced increase of membrane phospho-ezrin, F-actin content, and CFTR-dependent secretion. We conclude that NHERF1 overexpression in CFBE41o- rescues CFTR-dependent chloride secretion by forming the multiprotein complex RhoA-ROCK-ezrin-actin that, via actin cytoskeleton reorganization, tethers F508del CFTR to the cytoskeleton stabilizing it on the apical membrane.

Cystic Fibrosis (CF) is a still life-treathening disease, although therapies have augmented the life span of CF individuals. Isolation of the CF gene, named CFTR (CF Transmembrane Conductance Regulator), led to the discovery that it encodes for a protein kinase A-regulated chloride channel, expressed by epithelial cells mainly in mucosal tissues. Mutated or absent CFTR brings about altered muco-ciliary clearance, by a yet disputed mechanism (likely involving overfunction of the epithelial sodium channel [ENaC]), and ultimately to colonization and infection of the airways by a few opportunistic bacteria species, including Pseudomonas aeruginosa (P.a.). An exuberant and persistent acute inflammatory reaction is commonly observed in the lung from CF patients, representing a key pathogenetic event of lung damage and respiratory insufficiency. Lung disease is the chief cause of morbidity and mortality in CF patients and current therapies are aimed at controlling the respiratory symptoms by antibiotic and anti-inflammatory treatments. Major improvements in the strategy to fight pulmonary P.a. infection are based on treatment of first colonization with P.a., of chronic infection and of multidrug resistant bacteria. Controlling inflammation is a challenging, however crucial, task in CF therapy. Since classical steroidal and non-steroidal anti-inflammatory drugs are endowed with serious side-effects, alternative antiinflammatory strategies are being developed, including drugs which modulate cytokine expression, nitric oxide production and the oxidation unbalance in the CF airways. Soon after gene identification, gene transfer vectors to replace the defective gene were developed and underwent a number of human trials which have not yet produced a viable clinical gene therapy strategy. Novel gene therapy vectors and strategies for delivering them more efficiently and safely to the lung are being studied. In the last few years various in vitro studies have demonstrated the possibility of pharmacological intervention to correct the primary defect in CF. This may be obtained by directly addressing the CFTR protein (by means of so-called correctors and potentiators) or by modulating the activity of other types of ion transport in epithelial cells (such as with Moli1901 or denufosol). Novel interventions aimed to improve the mucociliary clearance, including hypertonic saline, mannitol and ENaC inhibitors, will be finally discussed.

Airway inflammation plays a crucial role in lung damage in cystic fibrosis (CF) and is characterized by a persistent influx of neutrophils into the airways. We hypothesized that the high levels of inflammatory products that accumulate in the microenvironment of the CF lung contribute to induce the persistent neutrophil recruitment and the airway epithelial damage. Thus, we evaluated the in vitroeffect of sputum sol phase (SSP) from CF patients on a) adhesion molecule expression by human microvascular endothelial cells (HMECs) and b) apoptosis of human bronchial epithelial cells (HBECs), both wild-type and CFTR-defective. SSP was obtained from 7 clinically stable adult CF patients and 8 patients with an acute exacerbation. HMECs and HBECs were cultured in the absence or presence of SSP. Cell adhesion molecule expression was assessed by flow cytometry and cell death by the detection of histone-associated DNA fragments, caspase activation, and cytochrome c release. SSP obtained from CF patients, especially at the time of an acute exacerbation, induced a) an upregulation of endothelial adhesion molecules on cultured HMECs that was associated with an increase of neutrophil adhesion to these cells, and was mediated at least in part by TNF-α and IL-1 and b) apoptosis of airway epithelial cells, mainly activated by TNF-α pathway. These results suggest that the high concentrations of inflammatory mediators in CF airways contribute both to the chronic neutrophil influx and the airway damage, and support the crucial role of early anti-inflammatory treatment in the disease.

Background: Although cystic fibrosis (CF) seems a good candidate for gene therapy (GT), 15 years of intense investigations and a number of clinical trials have not yet produced a viable clinical GT strategy. Objective: To review the strategies used to optimize the current GT vectors, to overcome the extra- and cellular barriers to efficient gene delivery to the CF airways, and the poor long-term persistence of gene expression. Methods: We describe a selected set of patents, which were published in the last 5 years (2003 – 2008), with the aim of covering the issues related to GT and the airways; however, in some cases, they are intended for a broader use than CFGT. World Intellectual Property Organization (WIPO), the European network of patent databases, and the US Patent Office were queried. Results/conclusions: Novel biodegradable and biocompatible non-viral GT vectors have been characterized and used for gene transfer into the airways, along with physical methods to enhance their concentration at the level of airway epithelium. Less immunogenic and more efficient adeno-associated viral vectors are being studied, whereas integrating lentiviral viruses are currently investigated for their potential use to give prolonged gene expression. Methods for overcoming the mucus barrier and for increasing accessibility to the basolateral receptors have been described. Although a wealth of GT vectors has been produced, their fabrication, storage and administration are still critical issues, and these shortcomings are tackling translation of laboratory non-viral and viral vectors to the clinic. Thus, in the near future, we expect new developments in GT vector technology, above all of delivery, expression and safety. These improvements, along with better comprenhension of CF lung disease pathophysiology, will aid CFGT to achieve clinical benefits in CF patients.

Cystic fibrosis (CF) is caused by mutations of the CF transmembrane conductance regulator (CFTR) gene, which encodes a cAMP dependent chloride channel whose expression is finely tuned in space and time. Gene therapy approaches to CF lung disease have demonstrated partial efficacy and short-lived CFTR expression in the airways. Drawbacks in the use of classical gene transfer vectors include immune response to viral proteins or to unmethylated CpG motifs contained in bacterially-derived vector DNA, and shut-off of viral promoters. These limitations could be overcome by providing stable maintenance and expression of the CFTR gene inside the defective cells. This strategy makes use of large fragments of DNA of various sizes containing the CFTR transgene and its relevant regulatory regions, (genomic context vectors [GCVs], reaching ultimate complexity in the form of an artificial chromosome [AC]) as vector for the transgene. Appropriate regulation in space and time would be achieved by the presence of the endogenous promoter and other control elements, while retention in daughter cells could be ensured by the presence of sequences which guarantee episomal replication. In this review, we describe recent advances in GCVs and ACs and the technology underlying their construction. These vectors have been shown to be suitable for delivery and expression of therapeutically relevant genes, including CFTR. The major issue which now limits their routine use is delivery inefficiency. Once this issue is resolved, we will be closer to achieving the goal of regulated gene therapy for CF.

Since Cystic Fibrosis (CF) is an autosomal recessive disorder due to mutations in the CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) gene, studies towards a gene therapy approach to its treatment followed immediately upon the cloning of the gene. It was demonstrated that the insertion of a single copy of the wild-type gene restored the normal phenotype in CF cells in vitro. Encouraging results were obtained in many in vivo model systems (CF transgenic mice) involving viral as well as non-viral vectors, which demonstrated the recovery of CFTR function in the airways. These results constituted the basis for human studies. Of those with a non-viral approach, a total of seven clinical trials using cationic lipids have reported data on efficiency, efficacy and safety. An effective gene transfer approach for the treatment of CF lung disease is not however imminent: low transfection efficiency and poor maintenance of gene expression are so far the main obstacles on this therapeutic path. On the other hand, no important adverse effects have been documented and repeated administration in humans is possible.The understanding of tissue and cellular barriers is a prerequisite for the development of more efficient non-viral gene therapy protocols for CF patients. While cationic lipids have been shown to be blocked by the mucous airway barrier and not be able to transfect differentiated respiratory epithelial cells, a new class of non-viral vectors, cationic polymers, are endowed with chemical and biological properties that make them more efficient in mediating gene transfer than lipids. Cationic polymers, such as polyethylenimine, are promising vectors for CF lung gene therapy.

PAI‐1 (plasminogen activator inhibitor‐1) binds the urokinase‐type plasminogen activator (uPA) and causes its degradation via its receptor uPAR and low‐density lipoprotein receptor‐related protein (LRP). While both uPA and PAI‐1 are chemoattractants, we find that a preformed uPA–PAI‐1 complex has no chemotactic activity and that PAI‐1 inhibits uPA‐induced chemotaxis. The inhibitory effect of PAI‐1 on uPA‐dependent chemotaxis is reversed when uPAR internalization is inhibited by the 39 kDa receptor‐associated protein or by anti‐LRP antibodies. Under the same conditions, the uPA–PAI‐1 complex is turned into a chemoattractant causing cytoskeleton reorganization and extracellular‐regulated kinase/mitogen‐activated protein kinases activation. Thus, uPAR internalization by PAI‐1 regulates cell migration.