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-Delta 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-DeltaERM, 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.

The aim of this work was to evaluate the efficiency and duration of gene expression mediated by a VSV-G pseudotyped last generation lentiviral (LV) vector. We studied LV efficiency in ex-vivo models of respiratory epithelial cells, obtained from bronchial biopsies and nasal polyps, by GFP epifluorescence and cytofluorimetry. In vivo efficiency and persistence of gene expression was investigated by GFP immunohistochemistry and luciferase activity in lung cryosections and homogenates, respectively, upon intranasal and intratracheal administration protocols in C57Bl/6 mice. Both primary bronchial and nasal epithelial cells were transduced up to 70-80% 72 hr after the LV infection. In vivo nasal luciferase expression was increased by lysophosphatidylcholine pre-treatment of the nose. Conversely, the bronchial epithelium was transduced in the absence of any pre-conditioning treatment and luciferase expression lasted for at least 6 months without any decline. We conclude that a last generation LV vector is a promising gene transfer agent in the target organ of genetic and acquired lung diseases, as in the case of cystic fibrosis.

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.

Reactive oxygen species (ROS) are small oxygen-derived molecules that are used to control infections by phagocytic cells. In macrophages, the oxidative burst produced by the NOX2 NADPH-oxidase is essential to eradicate engulfed pathogens by both oxidative and non-oxidative killing. Indeed, while the superoxide anion ( O2- ) produced by NOX2, and the other ROS derived from its transformation, can directly target pathogens, ROS also contribute to activation of non-oxidative microbicidal effectors. The response of pathogens to the phagocytic oxidative burst includes the expression of different enzymes that target ROS to reduce their toxicity. Superoxide dismutases (SODs) are the primary scavengers of O2- , which is transformed into H2O2. In the Gram-negative Salmonella typhimurium, periplasmic SODCI has a major role in bacterial resistance to NOX-mediated oxidative stress. In Pseudomonas aeruginosa, the two periplasmic SODs, SODB, and SODM, appear to contribute to bacterial virulence in small-animal models. Furthermore, NOX2 oxidative stress is essential to restrict P. aeruginosa survival in macrophages early after infection. Here, we focused on the role of P. aeruginosa SODs in the counteracting of the lethal effects of the macrophage oxidative burst. Through this study of the survival of sod mutants in macrophages and the measurement of ROS in infected macrophages, we have identified a dual, antagonistic, role for SODB in P. aeruginosa survival. Indeed, the survival of the sodB mutants, but not of the sodM mutants, was greater than that of the wild-type (WT) bacteria early after infection, and sodB-infected macrophages showed higher levels of O2- and lower levels of H2O2. This suggests that SODB contributes to the production of lethal doses of H2O2 within the phagosome. However, later on following infection, the sodB mutants survived less that the WT bacteria, which highlights the pro-survival role of SODB. We have explained this defensive role through an investigation of the activation of autophagy, which was greater in the sodB-infected macrophages.

Chronic lung diseases, such as cystic fibrosis (CF), asthma, and chronic obstructive pulmonary disease (COPD) are incurable and represent a very high social burden. Stem cell-based treatment may represent a hope for the cure of these diseases. In this paper, we revise the overall knowledge about the plasticity and engraftment of exogenous marrow-derived stem cells into the lung, as well as their usefulness in lung repair and therapy of chronic lung diseases. The lung is easily accessible and the pathophysiology of these diseases is characterized by injury, inflammation, and eventually by remodeling of the airways. Bone marrow-derived stem cells, including hematopoietic stem/progenitor cells (HSPCs) and mesenchymal stromal (stem) cells (MSCs), encompass a wide array of cell subsets with different capacities of engraftment and injured tissue regenerating potential. Proof-of-principle that marrow cells administered locally may engraft and give rise to specialized epithelial cells has been given, but the efficiency of this conversion is too limited to give a therapeutic effect. Besides the identification of plasticity mechanisms, the characterization/isolation of the stem cell subpopulations represents a major challenge to improving the efficacy of transplantation protocols used in regenerative medicine for lung diseases.

Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, with most of the mortality given by the lung disease. Human amniotic mesenchymal stromal (stem) cells (hAMSCs) hold great promise for regenerative medicine in the field of lung disease; however, their potential as therapeutics for CF lung disease has not been fully explored. In the present study, hAMSCs were analysed in co-cultures on Transwell filters with CF immortalized airway epithelial cells (CFBE41o- line) at different ratios to exploit their potency to resume basic defects associated with CF. The results show that F-actin content was increased in co-cultures as compared with CF cells and actin was reorganized to form stress fibres. Confocal microscopy studies revealed that co-cultures had a tendency of increased expression of occludin and ZO-1 at the intercellular borders, paralleled by a decrease in dextran permeability, suggestive of more organized tight junctions (TJs). Spectrofluorometric analysis of CFTR function demonstrated that hAMSC-CFBE co-cultures resumed chloride transport, in line with the appearance of the mature Band C of CFTR protein by Western blotting. Moreover, hAMSC-CFBE co-cultures, at a 1:5 ratio, showed a decrease in fluid absorption, as opposed to CFBE cell monolayers that displayed a great rate of fluid resorption from the apical side. Our data show that human amniotic MSCs can be used in co-culture with CF respiratory epithelial cells to model their engraftment into the airways and have the potential to resume a tight epithelium with partial correction of the CF phenotype.

Human papillomavirus (HPV) is the most common sexually transmitted virus. The high-risk HPV types (i.e., HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59) are considered to be the main etiological agents of genital tract cancers, such as cervical, vulvar, vaginal, penile, and anal cancers, and of a subset of head and neck cancers. Three prophylactic HPV vaccines are available that are bivalent (vs. HPV16, 18), tetravalent (vs. HPV6, 11, 16, 18), and non-avalent (vs. HPV6, 11, 16, 18, 31, 33,45, 52, 58). All of these vaccines are based on recombinant DNA technology, and they are prepared from the purified L1 protein that self-assembles to form the HPV type-specific empty shells (i.e., virus-like particles). These vaccines are highly immunogenic and induce specific antibodies. Therapeutic vaccines differ from prophylactic vaccines, as they are designed to generate cell-mediated immunity against transformed cells, rather than neutralizing antibodies. Among the HPV proteins, the E6 and E7 oncoproteins are considered almost ideal as targets for immunotherapy of cervical cancer, as they are essential for the onset and evolution of malignancy and are constitutively expressed in both premalignant and invasive lesions. Several strategies have been investigated for HPV therapeutic vaccines designed to enhance CD4+ and CD8+ T-cell responses, including genetic vaccines (i.e., DNA/ RNA/virus/ bacterial), and protein-based, peptide-based or dendritic-cell-based vaccines. However, no vaccine has yet been licensed for therapeutic use. Several studies have suggested that administration of prophylactic vaccines immediately after surgical treatment of CIN2 cervical lesions can be considered as an adjuvant to prevent reactivation or reinfection, and other studies have described the relevance of prophylactic vaccines in the management of genital warts. This review summarizes the leading features of therapeutic vaccines, which mainly target the early oncoproteins E6 and E7, and prophylactic vaccines, which are based on the L1 capsid protein. Through an analysis of the specific immunogenic properties of these two types of vaccines, we discuss why and how prophylactic vaccines can be effective in the treatment of HPV-related lesions and relapse.

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.

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.

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.

Cystic fibrosis (CF), which is caused by mutations in the CF transmembrane conductance regulator (CFTR), is characterised by chronic bacterial lung infection and inflammation. In CF, monocytes and monocyte-derived macrophages have been shown to display defective phagocytosis and antimicrobial activity against relevant lung pathogens, including Pseudomonas aeruginosa. Thus, we addressed the effect of CFTR triple modulator therapy (elexacaftor/tezacaftor/ivacaftor (ETI)) on the activity of CF monocytes against P. aeruginosa.

Gene transfer to airway epithelial cells is hampered by extracellular (mainly mucus) and cellular (tight junctions) barriers. Magnetofection has been used to increase retention time of lentiviral vectors (LV) on the cellular surface. In this study, magnetofection was investigated in airway epithelial cell models mimicking extracellular and cellular barriers. Bronchiolar epithelial cells (H441 line) were evaluated for LV-mediated transduction after polarization onto filters and dexamethasone (dex) treatment, which induced hemicyst formation, with or without magnetofection. Sputum from cystic fibrosis (CF) patients was overlaid onto cells, and LV-mediated transduction was evaluated in the absence or presence of magnetofection. Magnetofection of unpolarized H441 cells increased the transduction with 50 MOI (multiplicity of infection, i.e., transducing units/cell) up to the transduction obtained with 500 MOI in the absence of magnetofection. Magnetofection well-enhanced LV-mediated transduction in mucus-layered cells by 20.3-fold. LV-mediated transduction efficiency decreased in dex-induced hemicysts in a time-dependent fashion. In dome-forming cells, zonula occludens-1 (ZO-1) localization at the cell borders was increased by dex treatment. Under these experimental conditions, magnetofection significantly increased LV transduction by 5.3-fold. In conclusion, these results show that magnetofection can enhance LV-mediated gene transfer into airway epithelial cells in the presence of extracellular (sputum) and cellular (tight junctions) barriers, representing CF-like conditions.

The pathophysiology of cystic fibrosis (CF) airway disease stems from mutations in the CF Transmembrane Conductance Regulator (CFTR) gene, leading to a chronic respiratory disease. Actin cytoskeleton is disorganized in CF airway epithelial cells, likely contributing to the CF-associated basic defects, i.e. defective chloride secretion and sodium/fluid hypersorption. In this work, we aimed to find whether this alteration could be pointed out by means of Atomic Force Microscopy (AFM) investigation, as roughness and Young's elastic module. Moreover, we also sought to determine whether disorganization of actin cytoskeleton is linked to hypersoption of apical fluid. Not only CFBE41o- (CFBE) cells, immortalized airway epithelial cells homozygous for the F508del CFTR allele, showed a different morphology in comparison with 16HBE14o- (16HBE) epithelial cells, wild-type for CFTR, but also they displayed a lack of stress fibers, suggestive of a disorganized actin cytoskeleton. AFM measurements showed that CFBE cells presented a higher membrane roughness and decreased rigidity as compared with 16HBE cells. CFBE overexpressing wtCFTR became more elongated than the parental CFBE cell line and presented actin stress fibers. CFBE cells absorbed more fluid from the apical compartment. Study of fluid absorption with the F-actin-depolymerizing agent Latrunculin B demonstrated that actin cytoskeletal disorganization increased fluid absorption, an effect observed at higher magnitude in 16HBE than in CFBE cells. For the first time, we demonstrate that actin cytoskeleton disorganization is reflected by AFM parameters in CF airway epithelial cells. Our data also strongly suggest that the lack of stress fibers is involved in at least one of the early step in CF pathophysiology at the levels of the airways, i.e. fluid hypersorption.

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.

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.

Non-nonavalent vaccine (9v) Human papillomavirus (HPV) types have been shown to have high prevalence among HIV-positive women. Here, 1444 cervical samples were tested for HPV DNA positivity. Co-infections of the 9v HPV types with other HPV types were evaluated. The HPV81 L1 and L2 genes were used to investigate the genetic variability of antigenic epitopes. HPV-positive samples were genotyped using the HPVCLART2 assay. The L1 and L2 protein sequences were analyzed using a self-optimized prediction method to predict their secondary structure. Co-occurrence probabilities of the 9v HPV types were calculated. Non9v types represented 49% of the HPV infections; 31.2% of the non9v HPV types were among the low-grade squamous intraepithelial lesion samples, and 27.3% among the high-grade squamous intraepithelial lesion samples, and several genotypes were low risk. The co-occurrence of 9v HPV types with the other genotypes was not correlated with the filogenetic distance. HPV81 showed an amino-acid substitution within the BC loop (N75Q) and the FGb loop (T315N). In the L2 protein, all of the mutations were located outside antigenic sites. The weak cross-protection of the 9v types suggests the relevance of a sustainable and effective screening program, which should be implemented by HPV DNA testing that does not include only high-risk types.

Considerable evidence supports the prediction that CD25 is directly regulated by the forkhead transcription factor FOXP3. However, given that CD25 is normally upregulated in activated T cells, regardless of whether they express FOXP3, this issue has still to be definitively demonstrated. Here we describe that FOXP3, induced by CD28 signals in human CD4(+)CD25(-) T lymphocytes, synergizes with RelA on a regulatory region of Cd25 promoter to mediate the transcriptional activation of Cd25 gene. We found that a striking feature of this regulatory region is the presence of a κB site and of two tandem copies of a non-consensus FOXP3 binding site separated at 5' ends by 19 nucleotides that allow FOXP3 and RelA binding to DNA and their physical interaction. The occupancy of the two FOXP3 binding sites in conjunction with RelA binding site occupancy allows FOXP3 to function as a positive activator of Cd25 gene. Indeed mutations of both FOXP3 binding sites such as mutation of κB site on Cd25 promoter abolished FOXP3 activatory functions. Moreover, FOXP3 mutation ΔE251, that compromises FOXP3 homotypic interactions, failed to trans activate Cd25 promoter, suggesting that both FOXP3 DNA binding and dimerization are required to trans activate Cd25 promoter. These findings identify a novel mechanism by which RelA and FOXP3 cooperate to mediate transcriptional regulation of target genes and characterize a region on Cd25 promoter where FOXP3 dimer could bridge intramolecularly two DNA sites and trans activate Cd25 gene.

Cystic fibrosis (CF) is an inherited disease that is characterised by susceptibility to bacterial infections and chronic lung inflammation. Recently, it was suggested that macrophages contribute to impaired host defence and excessive inflammatory responses in CF. Indeed, dysfunction attributed to CF macrophages includes decreased bacterial killing and exaggerated inflammatory responses. However, the mechanisms behind such defects have only been partially defined. MicroRNAs (miRNAs) have emerged as key regulators of several macrophage functions, including their activation, differentiation and polarisation. The goal of this study was to investigate whether miRNA dysregulation underlies the functional abnormalities of CF macrophages. MiRNA profiling of macrophages was performed, with 22 miRNAs identified as differentially expressed between CF and non-CF individuals. Among these, miR-146a was associated with significant enrichment of validated target genes involved in responses to microorganisms and inflammation. As miR-146a dysregulation has been reported in several human inflammatory diseases, we analysed the impact of increased miR-146a expression on inflammatory responses of CF macrophages. These data show that inhibition of miR-146a in lipopolysaccharide-stimulated CF macrophages results in increased interleukin-6 production, which suggests that miR-146a overexpression in CF is functional, to restrict inflammatory responses.

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.

Torque Teno virus (TTV) is a ubiquitous virus that causes chronic infection in humans with unknown clinical consequences. Here, we investigated the influence of TTV infection on HCV direct-acting antiviral (DAA) efficacy in HIV/HCV coinfected and HCV monoinfected patients as controls. Of 92 study patients, 79.3% were TTV DNA positive; untreated patients exhibited a significantly higher proportion of TTV DNA-positivity vs. sustained virological response (SVR) patients (100.0% vs. 65.2%, p < 0.001), while TTV positivity was not significant in DAA failure patients vs. SVR patients despite HIV/HCV coinfection. TTV DNA viral load was higher among HCV monoinfected patients vs. HIV/HCV coinfected, although marginally significant (p = 0.074) and no significant viral load difference was detected between DAA failures and SVR patients, while untreated vs. SVR patients had a significantly higher viral load (19,884, IQR 5977-333,534, vs. 469, IQR 10-4124, p = 0.004). Alpha-genogroup 3 TTV was the most prevalent genetic group, and no specific strain or genogroup was observed in relapser patients. Among HIV/HCV patients with HCV RNA detectable at end of treatment (EOT), TTV DNA was detected in 9/17 treatment responder patients and 3/5 relapser patients, thus, TTV infection does not appear to influence the control HCV viremia after EOT. Levels of IL-6 IL-4, and CD14 were not significantly different between TTV PCR-positive and -negative patients. These results suggest no association between TTV DNA positivity or viral load and HCV DAA failure whether patients were HIV/HCV coinfected or HCV monoinfected.