Search

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.

Plant-derived nanovesicles have been considered interesting in medicine for their breakthrough biological effects, including those relevant to wound healing. However, tomato-derived nanovesicles (TDNVs) have not been studied for their effects on wound closure yet. TDNVs were isolated from Solanum lycopersicum (var. Piccadilly) ripe tomatoes by ultracentrifugation. Extract (collected during the isolation procedure) and NVs (pellet) were characterized by transmission electron microscopy and laser Doppler electrophoresis. Wound healing in the presence of Extract or NVs was analyzed by a scratch assay with monocultures of human keratinocytes (HUKE) or NIH-3T3 mouse fibroblasts. Cell proliferation and migration were studied by MTT and agarose spot assay, respectively. The vesicles in the Extract and NV samples were nanosized with a similar mean diameter of 115 nm and 130 nm, respectively. Both Extract and NVs had already accelerated wound closure of injured HUKE and NIH-3T3 monocultures by 6 h post-injury. Although neither sample exerted a cytotoxic effect on HUKE and NIH-3T3 fibroblasts, they did not augment cell proliferation. NVs and the Extract increased cell migration of both cell types. NVs from tomatoes may accelerate wound healing by increasing keratinocyte and fibroblast migration. These results indicate the potential therapeutic usefulness of TDNVs in the treatment of chronic or hard-to-heal ulcers.

The respiratory mucus, a viscoelastic gel, effectuates a primary line of the airway defense when operated by the mucociliary clearance. In chronic respiratory diseases (CRDs), such as asthma, chronic obstructive pulmonary disease (COPD), and cystic fibrosis (CF), the mucus is overproduced and its solid content augments, changing its structure and viscoelastic properties and determining a derangement of essential defense mechanisms against opportunistic microbial (virus and bacteria) pathogens. This ensues in damaging of the airways, leading to a vicious cycle of obstruction and infection responsible for the harsh clinical evolution of these CRDs. Here, we review the essential features of normal and pathological mucus (i.e., sputum in CF, COPD, and asthma), i.e., mucin content, structure (mesh size), micro/macro-rheology, pH, and osmotic pressure, ending with the awareness that sputum biomarkers (mucins, inflammatory proteins and peptides, and metabolites) might serve to indicate acute exacerbation and response to therapies. There are some indications that old and novel treatments may change the structure, viscoelastic properties, and biomarker content of sputum; however, a wealth of work is still needed to embrace these measures as correlates of disease severity in association with (or even as substitutes of) pulmonary functional tests.

This study focuses on the metrological characterization of a 3D vision system consisting in the fusion of a CMOS camera sensor with a 2D laser scanner for contactless dimensional measurements. The purpose is to obtain an enhanced measurement information as a result of the combination of two different data sources. On one side, we can estimate the pose of the target measurand by solving the well-known Perspective-n-Point (PnP) problem from the calibrated camera. On the other side, the 2D laser scanner generates a discrete point cloud which describes the profile of the intercepted surface of the same target object. This solution allows to estimate the target’s geometrical parameters through the application of fit-to-purpose algorithms that see the data acquired by the overall system as their input. The measurement uncertainty is evaluated by applying the Monte Carlo Method (MCM) to estimate the uncertainty deriving from the Probability Distribution Functions (PDF) of the input variables. Through a Design of Experiments (DOE) model the effects of different influence factors were evaluated.

This research focuses on the characterization of the metrology of Optical 3D Coordinate Measurement Systems (O3DCMS). The focus is set on the identification and execution of the procedure indicated by the currently active technical standards related to industrial O3DCMS, for their metrological assessment, objective comparison, and performance tracking. This work leads to the implementation of an ad hoc software for the execution of the standard tests by the ISO 10360-13 standard. The implemented software application is employed in a real-case scenario for evaluating the performances of an industrial 3D scanner based on structured light. The specific hardware components to be assessed are two light sources of the active stereoscopic vision system, named Digital Light Projectors (DLP). The case study applies the procedures and metrics indicated by the active standards to objectively compare two alternative hardware design of the system under test. This results in the identification of the most performing hardware configuration, allowing the selection of the best system design, basing on objective metrological parameters.

Abstract In this in vitro study, we test our hypothesis that Broccoli-derived vesicles (BDVs), combining the anti-oxidant properties of their components and the advantages of their structure, can influence the metabolic activity of different cancer cell lines. BDVs were isolated from homogenized fresh broccoli (Brassica oleracea L.) using a sucrose gradient ultracentrifugation method and were characterized in terms of physical properties, such as particle size, morphology, and surface charge by transmission electron microscopy (TEM) and laser doppler electrophoresis (LDE). Glucosinolates content was assessed by RPLC–ESI–MS analysis. Three different human cancer cell lines (colorectal adenocarcinoma Caco-2, lung adenocarcinoma NCI-H441 and neuroblastoma SHSY5Y) were evaluated for metabolic activity by the MTT assay, uptake by fluorescence and confocal microscopy, and anti-oxidant activity by a fluorimetric assay detecting intracellular reactive oxygen species (ROS). Three bands were obtained with average size measured by TEM based size distribution analysis of 52 nm (Band 1), 70 nm (Band 2), and 82 nm (Band 3). Glucobrassicin, glucoraphanin and neoglucobrassicin were found mostly concentrated in Band 1. BDVs affected the metabolic activity of different cancer cell lines in a dose dependent manner compared with untreated cells. Overall, Band 2 and 3 were more toxic than Band 1 irrespective of the cell lines. BDVs were taken up by cells in a dose- and time-dependent manner. Pre-incubation of cells with BDVs resulted in a significant decrease in ROS production in Caco-2 and NCI-H441 stimulated with hydrogen peroxide and SHSY5Y treated with 6-hydroxydopamine, with all three Bands. Our findings open to the possibility to find a novel “green” approach for cancer treatment, focused on using vesicles from broccoli, although a more in-depth characterization of bioactive molecules is warranted.

Is it possible to have an available vaccine that eradicates cancer? Starting from this question, this article tries to verify the state of the art, proposing a different approach to the issue. The variety of cancers and different and often unknown causes of cancer impede, except in some cited cases, the creation of a classical vaccine directed at the causative agent. The efforts of the scientific community are oriented toward stimulating the immune systems of patients, thereby preventing immune evasion, and heightening chemotherapeutic agents effects against cancer. However, the results are not decisive, because without any warning signs, metastasis often occurs. The purpose of this paper is to elaborate on a vaccine that must be administered to a patient in order to prevent metastasis; metastasis is an event that leads to death, and thus, preventing it could transform cancer into a chronic disease. We underline the fact that the field has not been studied in depth, and that the complexity of metastatic processes should not be underestimated. Then, with the aim of identifying the target of a cancer vaccine, we draw attention to the presence of the paradoxical actions of different mechanisms, pathways, molecules, and immune and non-immune cells characteristic of the tumor microenvironment at the primary site and pre-metastatic niche in order to exclude possible vaccine candidates that have opposite effects/behaviors; after a meticulous evaluation, we propose possible targets to develop a metastasis-targeting vaccine. We conclude that a change in the current concept of a cancer vaccine is needed, and the efforts of the scientific community should be redirected toward a metastasis-targeting vaccine, with the increasing hope of eradicating cancer.

Cystic fibrosis (CF) is a monogenic syndrome determined by over 2000 mutations in the CF Transmembrane Conductance Regulator (CFTR) gene harbored on chromosome 7. In people with CF (PWCF), lung disease is the major determinant of morbidity and mortality and is characterized by a clinical phenotype which differs in the presence of equal mutational assets, indicating that genetic and environmental modifiers play an important role in this variability. Airway inflammation determines the pathophysiology of CF lung disease (CFLD) both at its onset and progression. In this narrative review, we aim to depict the inflammatory process in CF lung, with a particular emphasis on those genetic polymorphisms that could modify the clinical outcome of the respiratory disease in PWCF. The natural history of CF has been changed since the introduction of CFTR modulator therapies in the clinical arena. However, also in this case, there is a patient-to-patient variable response. We provide an overview on inflammatory/immunity gene variants that affect CFLD severity and an appraisal of the effects of CFTR modulator therapies on the inflammatory process in lung disease and how this knowledge may advance the optimization of the management of PWCF.

Background: The influence of a magnetic field on the activation of bone cells and remodelling of alveolar bone is known to incite bone regeneration. Guided Bone Regeneration (GBR) aims to develop biomimetic scaffolds to allow for the functioning of the barrier and the precise succession of wound healing steps, including haemostasis. The effect of a magnetic field on blood clot dissolution has not been studied yet. Methods: We conducted a methodological study on the clot stability in the presence of a static magnetic field (SMF). Preformed whole blood (WB) clots were treated with either a broad proteolytic enzyme (trypsin) or a specific fibrinolytic agent, i.e., tissue-type plasminogen activator (t-PA). MG63 osteoblast-like cells were added to preformed WB clots to assess cell proliferation. Results: After having experienced a number of clotting and dissolution protocols, we obtained clot stability exerted by SMF when tissue factor (for clotting) and t-PA + plasminogen (for fibrinolysis) were used. WB clots allowed osteoblast-like cells to survive and proliferate, however no obvious effects of the magnetic field were noted. Conclusions: Paramagnetic properties of erythrocytes may have influenced the reduction in clot dissolution. Future studies are warranted to fully exploit the combination of magnetic forces, WB clot and cells in GBR applied to orthodontics and prosthodontics.

Cystic fibrosis (CF) is an autosomal recessive multi-organ disease caused by mutations in the CF Transmembrane Conductance Regulator (CFTR) gene, with morbidity and mortality primacy related to the lung disease. The CFTR protein, a chloride/bicarbonate channel, is expressed at the apical side of airway epithelial cells and is mainly involved in appropriate ion and fluid transport across the epithelium. Although many animal and cellular models have been developed to study the pathophysiological consequences of the lack/dysfunction of CFTR, only the three-dimensional (3D) structures termed “spheroids” and “organoids” can enable the reconstruction of airway mucosa to model organ development, disease pathophysiology, and drug screening. Airway spheroids and organoids can be derived from different sources, including adult lungs and induced pluripotent stem cells (iPSCs), each with its advantages and limits. Here, we review the major features of airway spheroids and organoids, anticipating that their potential in the CF field has not been fully shown. Further work is mandatory to understand whether they can accomplish better outcomes than other culture conditions of airway epithelial cells for CF personalized therapies and tissue engineering aims.

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-α, IL-1β, 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-1β+TNFα 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.

Abstract Background Idiopathic Pulmonary Fibrosis (IPF) is a degenerative interstitial lung disease with both a poor prognosis and quality of life once the diagnosis is made. In the last decade many features of the disease have been investigated to better understand the pathological steps that lead to the onset of the disease and, moreover, different types of biomarkers have been tested to find valid diagnostic, prognostic and therapy response predictive ones. In the complexity of IPF, microRNA (miRNAs) biomarker investigation seems to be promising. Methods We analysed the expression of five exosomal miRNAs supposed to have a role in the pathogenesis of the disease from serum of a group of IPF patients (n = 61) and we compared it with the expression of the same miRNAs in a group of healthy controls (n = 15). Results In the current study what emerged is let-7d down-regulation and, unexpectedly, miR-16 significant down-regulation. Moreover, through a cross-sectional analysis, a clustering of the expression of miR-16, miR-21 and miR-26a was found. Conclusions These findings could help the individuation of previously unknown key players in the pathophysiology of IPF and, most interestingly, more specific targets for the development of effective medications.

Cystic fibrosis (CF) is an autosomal recessive, life-threatening condition affecting many organs and tissues, the lung disease being the chief cause of morbidity and mortality. Mutations affecting the CF Transmembrane Conductance Regulator (CFTR) gene determine the expression of a dysfunctional protein that, in turn, triggers a pathophysiological cascade, leading to airway epithelium injury and remodeling. In vitro and in vivo studies point to a dysregulated regeneration and wound repair in CF airways, to be traced back to epithelial CFTR lack/dysfunction. Subsequent altered ion/fluid fluxes and/or signaling result in reduced cell migration and proliferation. Furthermore, the epithelial-mesenchymal transition appears to be partially triggered in CF, contributing to wound closure alteration. Finally, we pose our attention to diverse approaches to tackle this defect, discussing the therapeutic role of protease inhibitors, CFTR modulators and mesenchymal stem cells. Although the pathophysiology of wound repair in CF has been disclosed in some mechanisms, further studies are warranted to understand the cellular and molecular events in more details and to better address therapeutic interventions.

The lung is an accomplished organ for gas exchanges and directly faces the external environment, consequently exposing its large epithelial surface. It is also the putative determinant organ for inducing potent immune responses, holding both innate and adaptive immune cells. The maintenance of lung homeostasis requires a crucial balance between inflammation and anti-inflammation factors, and perturbations of this stability are frequently associated with progressive and fatal respiratory diseases. Several data demonstrate the involvement of the insulin-like growth factor (IGF) system and their binding proteins (IGFBPs) in pulmonary growth, as they are specifically expressed in different lung compartments. As we will discuss extensively in the text, IGFs and IGFBPs are implicated in normal pulmonary development but also in the pathogenesis of various airway diseases and lung tumors. Among the known IGFBPs, IGFBP-6 shows an emerging role as a mediator of airway inflammation and tumor-suppressing activity in different lung tumors. In this review, we assess the current state of IGFBP-6’s multiple roles in respiratory diseases, focusing on its function in the inflammation and fibrosis in respiratory tissues, together with its role in controlling different types of lung cancer.

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.

Quality inspection in the industrial production field is experiencing a strong technological development that benefits from the combination of vision-based techniques with artificial intelligence algorithms. This paper initially addresses the problem of defect identification for circularly symmetric mechanical components, characterized by the presence of periodic elements. In the specific case of knurled washers, we compare the performances of a standard algorithm for the analysis of grey-scale image with a Deep Learning (DL) approach. The standard algorithm is based on the extraction of pseudo-signals derived from the conversion of the grey scale image of concentric annuli. In the DL approach, the component inspection is shifted from the entire sample to specific areas repeated along the object profile where the defect may occur. The standard algorithm provides better results in terms of accuracy and computational time with respect to the DL approach. Nevertheless, DL reaches accuracy higher than 99% when performance is evaluated targeting the identification of damaged teeth. The possibility of extending the methods and the results to other circularly symmetrical components is analyzed and discussed.

The pathomechanisms of preeclampsia (PE), a complication of late pregnancy characterized by hypertension and proteinuria, and due to improper placentation, are not well known. Mesenchymal stem cells derived from the amniotic membrane (AMSCs) may play a role in PE pathogenesis as placental homeostasis regulators. PLACenta-specific protein 1 (PLAC1) is a transmembrane antigen involved in trophoblast proliferation that is found to be associated with cancer progression. We studied PLAC1 in human AMSCs obtained from control subjects (n = 4) and PE patients (n = 7), measuring the levels of mRNA expression (RT-PCR) and secreted protein (ELISA on conditioned medium). Lower levels of PLAC1 mRNA expression were observed in PE AMSCs as compared with Caco2 cells (positive controls), but not in non-PE AMSCs. PLAC1 antigen was detectable in conditioned medium obtained from PE AMSCs, whereas it was undetectable in that obtained from non-PE AMSCs. Our data suggest that abnormal shedding of PLAC1 from AMSC plasma membranes, likely by metalloproteinases, may contribute to trophoblast proliferation, supporting its role in the oncogenic theory of PE.

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.

Exosomes-like nanoparticles can be released by a variety of plants and vegetables. The relevance of plant-derived nanovesicles (PDNVs) in interspecies communication is derived from their content in biomolecules (lipids, proteins, and miRNAs), absence of toxicity, easy internalization by mammalian cells, as well as for their anti-inflammatory, immunomodulatory, and regenerative properties. Due to these interesting features, we review here their potential application in the treatment of inflammatory bowel disease (IBD), liver diseases, and cancer as well as their potentiality as drug carriers. Current evidence indicate that PDNVs can improve the disease state at the level of intestine in IBD mouse models by affecting inflammation and promoting prohealing effects. While few reports suggest that anticancer effects can be derived from antiproliferative and immunomodulatory properties of PDNVs, other studies have shown that PDNVs can be used as effective delivery systems for small molecule agents and nucleic acids with therapeutic effects (siRNAs, miRNAs, and DNAs). Finally, since PDNVs are characterized by a proven stability in the gastrointestinal tract, they have been considered as promising delivery systems for natural products contained therein and drugs (including nucleic acids) via the oral route.

In keeping with the extraordinary interest and advancement of extracellular vesicles (EVs) in pathogenesis and diagnosis fields, we herein present an update to the knowledge about their role in cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD). Although CF and COPD stem from a different origin, one genetic and the other acquired, they share a similar pathophysiology, being the CF transmembrane conductance regulator (CFTR) protein implied in both disorders. Various subsets of EVs, comprised mainly of microvesicles (MVs) and exosomes (EXOs), are secreted by various cell types that are either resident or attracted in the airways during the onset and progression of CF and COPD lung disease, representing a vehicle for metabolites, proteins and RNAs (especially microRNAs), that in turn lead to events as such neutrophil influx, the overwhelming of proteases (elastase, metalloproteases), oxidative stress, myofibroblast activation and collagen deposition. Eventually, all of these pathomechanisms lead to chronic inflammation, mucus overproduction, remodeling of the airways, and fibrosis, thus operating a complex interplay among cells and tissues. The detection of MVs and EXOs in blood and biological fluids coming from the airways (bronchoalveolar lavage fluid and sputum) allows the consideration of EVs and their cargoes as promising biomarkers for CF and COPD, although clinical expectations have yet to be fulfilled.

Background: Cystic fibrosis (CF) airway epithelium shows alterations in repair following damage. In vitro studies showed that lumacaftor/ivacaftor (Orkambi) may favor airway epithelial integrity in CF patients. Our aim was to evaluate the effect of the novel triple combination elexacaftor/tezacaftor/ivacaftor (ETI) on wound repair in CF airway epithelial cells. Methods: A tip-based scratch assay was employed to study wound repair in monolayers of CFBE14o- cells overexpressing the F508del mutation. ETI was added during wound repair. Results: ETI efficiently rescued CFTR F508del maturation and activity, accelerated wound closure and increased wound healing rates of the injured CF cell monolayers. Conclusions: The triple corrector/potentiator combination ETI shows promise in ameliorating wound healing of the airway epithelium in F508del patients.

Cystic fibrosis (CF) airways are affected by a deranged repair of the damaged epithelium resulting in altered regeneration and differentiation. Previously, we showed that human amniotic mesenchymal stem cells (hAMSCs) corrected base defects of CF airway epithelial cells via connexin (CX)43-intercellular gap junction formation. In this scenario, it is unknown whether hAMSCs, or fibroblasts sharing some common characteristics with MSCs, can operate a faster repair of a damaged airway epithelium. A tip-based scratch assay was employed to study wound repair in monolayers of CFBE14o- cells (CFBE, homozygous for the F508del mutation). hAMSCs were either co-cultured with CFBE cells before the wound or added to the wounded monolayers. NIH-3T3 fibroblasts (CX43+) were added to wounded cells. HeLa cells (CX43-) were used as controls. γ-irradiation was optimized to block CFBE cell proliferation. A specific siRNA was employed to downregulate CX43 expression in CFBE cells. CFBE cells showed a delayed repair as compared with wt-CFTR cells (16HBE41o-). hAMSCs enhanced the wound repair rate of wounded CFBE cell monolayers, especially when added post wounding. hAMSCs and NIH-3T3 fibroblasts, but not HeLa cells, increased wound closure of irradiated CFBE monolayers. CX43 downregulation accelerated CFBE wound repair rate without affecting cell proliferation. We conclude that hAMSCs and fibroblasts enhance the repair of a wounded CF airway epithelium, likely through a CX43-mediated mechanism mainly involving cell migration.

Abstract Introduction The stress response is different in various individuals, however, the mechanisms that could explain these distinct effects are not well known and the molecular correlates have been considered one at the time. Particular harmful conditions occur if the subject, instead to cope the stressful events, succumb to them, in this case, a cascade reaction happens that through different signaling causes a specific reaction named “sickness behaviour.” The aim of this article is to review the complex relations among important molecules belonging to Central nervous system (CNS), immune system (IS), and endocrine system (ES) during the chronic stress response. Methods After having verified the state of art concerning the function of cortisol, norepinephrine (NE), interleukin (IL)‐1β and melatonin, we describe as they work together. Results We propose a speculative hypothesis concerning the complex interplay of these signaling molecules during chronic stress, highlighting the role of IL‐1β as main biomarker of this effects, indeed, during chronic stress its increment transforms this inflammatory signal into a nervous signal (NE), in turn, this uses the ES (melatonin and cortisol) to counterbalance again IL‐1β. During cortisol resistance, a vicious loop occurs that increments all mediators, unbalancing IS, ES, and CNS networks. This IL‐1β increase would occur above all when the individual succumbs to stressful events, showing the Sickness Behaviour Symptoms. IL‐1β might, through melatonin and vice versa, determine sleep disorders too. Conclusion The molecular links here outlined could explain how stress plays a role in etiopathogenesis of several diseases through this complex interplay.

Autoimmune demyelinating diseases—including multiple sclerosis, neuromyelitis optica spectrum disorder, anti-myelin oligodendrocyte glycoprotein-associated disease, acute disseminated encephalomyelitis, and glial fibrillary acidic protein (GFAP)-associated meningoencephalomyelitis—are a heterogeneous group of diseases even though their common pathology is characterized by neuroinflammation, loss of myelin, and reactive astrogliosis. The lack of safe pharmacological therapies has purported the notion that cell-based treatments could be introduced to cure these patients. Among stem cells, mesenchymal stem cells (MSCs), obtained from various sources, are considered to be the ones with more interesting features in the context of demyelinating disorders, given that their secretome is fully equipped with an array of anti-inflammatory and neuroprotective molecules, such as mRNAs, miRNAs, lipids, and proteins with multiple functions. In this review, we discuss the potential of cell-free therapeutics utilizing MSC secretome-derived extracellular vesicles—and in particular exosomes—in the treatment of autoimmune demyelinating diseases, and provide an outlook for studies of their future applications.

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.

A widely investigated approach to bypass the blood brain barrier is represented by the intranasal delivery of therapeutic agents exploiting the olfactory or trigeminal connections nose-brain. As for Parkinson’s disease (PD), characterized by dopaminergic midbrain neurons degeneration, currently there is no disease modifying therapy. Although several bio-nanomaterials have been evaluated for encapsulation of neurotransmitter dopamine (DA) or dopaminergic drugs in order to restore the DA content in parkinsonian patients, the premature leakage of the therapeutic agent limits this approach. To tackle this drawback, we undertook a study where the active was linked to the polymeric backbone by a covalent bond. Thus, novel nanoparticles (NPs) based on N,O-Carboxymethylchitosan-DA amide conjugate (N,O-CMCS-DA) were prepared by the nanoprecipitation method and characterized from a technological view point, cytotoxicity and uptake by Olfactory Ensheating Cells (OECs). Thermogravimetric analysis showed high chemical stability of N,O-CMCS-DA NPs and X-ray photoelectron spectroscopy evidenced the presence of amide linkages on the NPs surface. MTT test indicated their cytocompatibility with OECs, while cytofluorimetry and fluorescent microscopy revealed the internalization of labelled N,O-CMCS-DA NPs by OECs, that was increased by the presence of mucin. Altogether, these findings seem promising for further development of N,O-CMCS-DA NPs for nose-to-brain delivery application in PD.

The continuous improvements in the field of both regenerative medicine and tissue engineering have allowed the design of new and more efficacious strategies for the treatment of chronic or hard-to-heal skin wounds, which represent heavy burden, from a medical and economic point of view. These novel approaches are based on the usage of three key methodologies: stem cells, growth factors, and biomimetic scaffolds. These days, the adipose tissue can be considered the main source of multipotent mesenchymal stem cells, especially adipose-derived stem cells (ASCs). ASCs are easily accessible from various fat depots and show an intrinsic plasticity in giving rise to cell types involved in wound healing and angiogenesis. ASCs can be found in fat grafts, historically used in the treatment of chronic wounds, and have been evaluated as such in both animal models and human trials, to exploit their capability of accelerating wound closure and inducing a correct remodeling of the newly formed fibrovascular tissue. Since survival and fitness of ASCs need to be improved, they are now employed in conjunction with advanced wound dressings, together with dermal regenerative templates and platelet-rich plasma (as a source of growth and healing factors). In this work, we provide an overview of the current knowledge on the topic, based on existing studies and on our own experience.

Abstract Background Chronic respiratory diseases, whose one of the hallmarks is oxidative stress, are still incurable and need novel therapeutic tools and pharmaceutical agents. The phenolic compounds contained in grape are endowed with well-recognized anti-oxidant, anti-inflammatory, anti-cancer, and anti-aging activities. Considering that natural anti-oxidants, such as proanthocyanidins, have poor water solubility and oral bioavailability, we have developed a drug delivery system based on solid lipid nanoparticles (SLN), apt to encapsulate grape seed extract (GSE), containing proanthocyanidins. Methods Plain, 6-coumarin (6-Coum), DiR- and GSE-loaded SLN were produced with the melt-emulsion method. Physicochemical characterization of all prepared SLN was determined by photon correlation spectroscopy and laser Doppler anemometry. MTT assay (spectrophotometry) and propidium iodide (PI) assay (cytofluorimetry) were used to assess cell viability. Flow cytometry coupled with cell imaging was performed for assessing apoptosis and necrosis by Annexin V/7-AAD staining (plain SLE), cell internalization (6-Coum-SLN) and reactive oxygen species (ROS) production (SLN-GSE). NF-κB nuclear translocation was studied by immunofluorescence. In vivo bio-imaging was used to assess lung deposition and persistence of aerosolized DiR-loaded SLN. Results Plain SLN were not cytotoxic when incubated with H441 airway epithelial cells, as judged by both PI and MTT assays as well as by apoptosis/necrosis evaluation. 6-Coum-loaded SLN were taken up by H441 cells in a dose-dependent fashion and persisted into cells at detectable levels up to 16 days. SLN were detected in mice lungs up to 6 days. SLN-GSE possessed 243 nm as mean diameter, were negatively charged, and stable in size at 37 °C in Simulated Lung Fluid up to 48 h and at 4 °C in double distilled water up to 2 months. The content of SLN in proanthocyanidins remained unvaried up to 2 months. GSE-loaded SLN determined a significant reduction in ROS production when added 24–72 h before the stimulation with hydrogen peroxide. Interestingly, while at 24 h free GSE determined a higher decrease of ROS production than SLN-GSE, the contrary was seen at 48 and 72 h. Similar results were observed for NF-κB nuclear translocation. Conclusions SLN are a biocompatible drug delivery system for natural anti-oxidants obtained from grape seed in a model of oxidative stress in airway epithelial cells. They feature stability and long-term persistence inside cells where they release proanthocyanidins. These results could pave the way to novel anti-oxidant and anti-inflammatory therapies for chronic respiratory diseases.

Transfusion (or drinking) of blood or of its components has been thought as a rejuvenation method since ancient times. Parabiosis, the procedure of joining two animals so that they share each others blood circulation, has revitalized the concept of blood as a putative drug. Since 2005, a number of papers have reported the anti-ageing effect of heterochronic parabiosis, which is joining an aged mouse to a young partner. The hallmark of aging is the decline of regenerative properties in most tissues, partially attributed to impaired function of stem and progenitor cells. In the parabiosis experiments, it was elegantly shown that factors derived from the young systemic environment are able to activate molecular signaling pathways in hepatic, muscle or neural stem cells of the old parabiont leading to increased tissue regeneration. Eventually, further studies have brought to identify some soluble factors in part responsible for these rejuvenating effects, including the chemokine CCL11, the growth differentiation factor 11, a member of the TGF-β superfamily, and oxytocin. The question about giving whole blood or specific factors in helping rejuvenation is open, as well as the mechanisms of action of these factors, deserving further studies to be translated into the life of (old) human beings.

Cystic Fibrosis (CF) is caused by mutations on the CF transmembrane conductance regulator (CFTR) gene and is associated with chronic infection and inflammation. Recently, it has been demonstrated that LPS-induced CFTR dysfunction in airway epithelial cells is due to an early oxidative stress. Dimethyl fumarate (DMF) is an approved anti-inflammatory and anti-oxidant drug for auto-immune and inflammatory diseases, but its role in the CF has never been investigated. In this study, we examined the effect of DMF on CF-related cytokines expression, ROS measurements and CFTR channel function. We found that DMF reduced the inflammatory response to LPS stimulation in both CF and non-CF bronchial epithelial cells, both as co-treatment and therapy, and restored LPS-mediated decrease of Trikafta™-mediated CFTR function in CF cells bearing the most common mutation, c.1521_1523delCTT (F508del). DMF also inhibited the inflammatory response induced by IL-1β/H2O2 and IL-1β/TNFα, mimicking the inflammatory status of CF patients. Finally, we also demonstrated that DMF exhibited an anti-oxidant effect on CF cells after different inflammatory stimulations. Since DMF is an approved drug, it could be further investigated as a novel anti-inflammatory molecule to ameliorate lung inflammation in CF and improve the CFTR modulators efficacy.

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, β 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.

Connexin- and pannexin (Panx)-formed hemichannels (HCs) and gap junctions (GJs) operate an interaction with the extracellular matrix and GJ intercellular communication (GJIC), and on account of this they are involved in cancer onset and progression towards invasiveness and metastatization. When we deal with cancer, it is not correct to omit the immune system, as well as neglecting its role in resisting or succumbing to formation and progression of incipient neoplasia until the formation of micrometastasis, nevertheless what really occurs in the tumor microenvironment (TME), which are the main players and which are the tumor or body allies, is still unclear. The goal of this article is to discuss how the pivotal players act, which can enhance or contrast cancer progression during two important process: “Activating Invasion and Metastasis” and the “Avoiding Immune Destruction”, with a particular emphasis on the interplay among GJIC, Panx-HCs, and the purinergic system in the TME without disregarding the inflammasome and cytokines thereof derived. In particular, the complex and contrasting roles of Panx1/P2X7R signalosome in tumor facilitation and/or inhibition is discussed in regard to the early/late phases of the carcinogenesis. Finally, considering this complex interplay in the TME between cancer cells, stromal cells, immune cells, and focusing on their means of communication, we should be capable of revealing harmful messages that help the cancer growth and transform them in body allies, thus designing novel therapeutic strategies to fight cancer in a personalized manner.

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.

Recently, insulin-like growth factor binding protein 6 (IGFBP-6) has been shown to play a putative role in the immune system, as monocyte-derived dendritic cells (Mo-DCs) are stimulated by hyperthermia to express IGFBP-6 at both the mRNA and protein levels. However, the presence of IGFBP-6 in extracellular vesicles (EVs) and whether other pro-inflammatory stimuli can induce IGFBP-6 expression in Mo-DCs are not known yet. In this brief report, we show that hyperthermia (39 °C) induces IGFBP-6 secretion associated with microvesicles and exosomes as early as 3 h. Moreover, free IGFBP-6 is found in conditioned media (CM) of hyperthermia- and H2O2-treated Mo-DCs, but not in CM obtained from monocytes similarly treated. These results show that diverse inflammatory stimuli can induce IGFBP-6 association with EVs and secretion in conditioned medium, indicating a role for IGFBP-6 in communication between immune cells.

The treatment of cystic fibrosis (CF) patients homozygous for the F508del mutation with Orkambi®, a combination of a corrector (lumacaftor) and a potentiator (ivacaftor) of the mutated CFTR protein, resulted in some amelioration of the respiratory function. However, a great variability in the clinical response was also observed. The aim of this study was to evaluate the response to Orkambi® in a small cohort of F508del/F508del patients (n = 14) in terms of clinical and laboratory parameters, including ex vivo CFTR activity in mononuclear cells (MNCs), during a 12-month treatment. Patients responded with an increase in percent predicted forced expiratory volume in 1 s (FEV1%) and body mass index (BMI) as well as with a decrease in white blood cell (WBC) total counts and serum C-reactive protein (CRP) levels, although not significantly. Sweat chloride and CFTR-dependent chloride efflux were found to decrease and increase, respectively, as compared with pre-therapy values. CFTR and BMI showed a statistically significant correlation during Orkambi® treatment. Clustering analysis showed that CFTR, BMI, sweat chloride, FEV1%, and WBC were strongly associated. These data support the notion that CFTR-dependent chloride efflux in MNCs should be investigated as a sensitive outcome measure of Orkambi® treatment in CF patients.

Background: The number of pediatric patients affected by HIV still remains high, mainly in developing countries, where the main cause of infection is vertical transmission from the mother. Even today, a large number of these children do not have access to treatment, and, without proper care, they die in the first few years of life. Objective: The aim of our review was to assess the prevalence of oral hard and soft tissue lesions in HIV-positive pediatric patients by identifying the most common manifestations and the overall impact that they may have on the children’s quality of life. Study design: A systematic review of the articles in the English language in PubMed and Scopus was conducted in March 2019 in order to identify the main epidemiological and cross-sectional studies on the topic. Results: Oral diseases are still one of the most common manifestations in HIV-positive pediatric patients, and they often represent the first form in which immunosuppression shows itself. An analysis of the literature shows that candidiasis is the most common oral lesion found in HIV-positive children. A significant incidence of gingivitis and gingival disease is also evident, though not strictly correlated to HIV infection. However, thanks to the introduction of new antiretroviral therapies, the incidence of HIV-related oral lesions is decreasing. Conclusions: An HIV-positive children care program should also include dental protocols, as oral disease negatively influences the quality of life, affecting both functional and social aspects.

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.

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.