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One of the clinical feature of cystic fibrosis (CF) is a deep inflammatory process, which is characterized by production and release of cytokines and chemokines, among which interleukin 8 (IL-8) represents one of the most important. Accordingly, there is a growing interest in developing therapies against CF to reduce the excessive inflammatory response in the airways of CF patients. Since transcription factor NFkappaB plays a critical role in IL-8 expression, the transcription factor decoy (TFD) strategy might be of interest. In order to demonstrate that TFD against NFkappaB interferes with the NFkappaB pathway we proved, by chromatin immunoprecipitation (ChIP) that treatment with TFD oligodeoxyribonucleotides of cystic fibrosis IB3-1 cells infected with Pseudomonas aeruginosa leads to a decrease occupancy of the Il-8 gene promoter by NFkappaB factors. In order to develop more stable therapeutic molecules, peptide nucleic acids (PNAs) based agents were considered. In this respect PNA-DNA-PNA (PDP) chimeras are molecules of great interest from several points of view: (1) they can be complexed with liposomes and microspheres; (2) they are resistant to DNases, serum and cytoplasmic extracts; (3) they are potent decoy molecules. By using electrophoretic mobility shift assay and RT-PCR analysis we have demonstrated that (1) the effects of PDP/PDP NFkappaB decoy chimera on accumulation of pro-inflammatory mRNAs in P. aeruginosa infected IB3-1 cells reproduce that of decoy oligonucleotides; in particular (2) the PDP/PDP chimera is a strong inhibitor of IL-8 gene expression; (3) the effect of PDP/PDP chimeras, unlike those of ODN-based decoys, are observed even in the absence of protection with lipofectamine. These informations are of great impact, in our opinion, for the development of stable molecules to be used in non-viral gene therapy of cystic fibrosis. [ABSTRACT FROM AUTHOR]

The most common mutation of the cystic fibrosis (CF) gene, the deletion of Phe508, encodes a protein (ΔF508-CFTR) that fails to fold properly, thus mutated ΔF508-cystic fibrosis transmembrane conductance regulator (CFTR) is recognized and degraded via the ubiquitin-proteasome endoplasmic reticulum-associated degradation pathway. Chemical and pharmacological chaperones and ligand-induced transport open options for designing specific drugs to control protein (mis)folding or transport. A class of compounds that has been proposed as having potential utility in ΔF508-CFTR is that which targets the molecular chaperone and proteasome systems. In this study, we have selected deoxyspergualin (DSG) as a reference molecule for this class of compounds and for ease of cross-linking to human serum albumin (HSA) as a protein transporter. Chemical cross-linking of DSG to HSA via a disulfide- based cross-linker and its administration to cells carrying ΔF508- CFTR resulted in a greater enhancement of ΔF508-CFTR function than when free DSG was used. Function of the selenium-dependent oxidoreductase system was required to allow intracellular activation of HSA-DSG conjugates. The principle that carrier proteins can deliver pharmacological chaperones to cells leading to correction of defective CFTR functions is therefore proven and warrants further investigations. [ABSTRACT FROM AUTHOR]

Neonatal hypertrypsinaemia with normal sweat chloride detected during CF screening may be related to trypsin activation. We have looked for mutations of the cationic trypsinogen (PRSS1) and pancreatic secretory trypsin inhibitor (PSTI) genes in 50 hypertrypsinaemic neonates with known CFTR genotypes and negative sweat test. No mutations were found in either gene. Two silent polymorphisms were detected in the PRSS1 gene. A polymorphism in the promoter region and an intronic polymorphism of the PSTI gene were found. No difference was observed in the frequency of PRSS1 or PSTI polymorphisms in neonates carrying or not carrying CF mutations. These results do not provide an indication for an increased frequency of mutations in the PRSS1 and PSTI genes in this group of neonates with transient hypertrypsinaemia. [ABSTRACT FROM AUTHOR]

(1) Background: Up-regulation of the Cystic Fibrosis Transmembrane Conductance Regulator gene (CFTR) might be of great relevance for the development of therapeutic protocols for cystic fibrosis (CF). MicroRNAs are deeply involved in the regulation of CFTR and scaffolding proteins (such as NHERF1, NHERF2 and Ezrin). (2) Methods: Content of miRNAs and mRNAs was analyzed by RT-qPCR, while the CFTR and NHERF1 production was analyzed by Western blotting. (3) Results: The results here described show that the CFTR scaffolding protein NHERF1 can be up-regulated in bronchial epithelial Calu-3 cells by a peptide-nucleic acid (PNA) targeting miR-335-5p, predicted to bind to the 3′-UTR sequence of the NHERF1 mRNA. Treatment of Calu-3 cells with this PNA (R8-PNA-a335) causes also up-regulation of CFTR. (4) Conclusions: We propose miR-335-5p targeting as a strategy to increase CFTR. While the efficiency of PNA-based targeting of miR-335-5p should be verified as a therapeutic strategy in CF caused by stop-codon mutation of the CFTR gene, this approach might give appreciable results in CF cells carrying other mutations impairing the processing or stability of CFTR protein, supporting its application in personalized therapy for precision medicine. [ABSTRACT FROM AUTHOR]

Current anti-inflammatory strategies for the treatment of pulmonary disease in cystic fibrosis (CF) are limited; thus, there is continued interest in identifying additional molecular targets for therapeutic intervention. Given the emerging role of sphingolipids (SLs) in various respiratory disorders, including CF, drugs that selectively target the enzymes associated with SL metabolism are under development. Miglustat, a well-characterized iminosugar-based inhibitor of β-glucosidase 2 (GBA2), has shown promise in CF treatment because it reduces the inflammatory response to infection by P. aeruginosa and restores F508del-CFTR chloride channel activity. This study aimed to probe the molecular basis for the anti-inflammatory activity of miglustat by examining specifically the role of GBA2 following the infection of CF bronchial epithelial cells by P. aeruginosa. We also report the anti-inflammatory activity of another potent inhibitor of GBA2 activity, namely N-(5-adamantane-1-yl-methoxy)pentyl)-deoxynojirimycin (Genz-529648). In CF bronchial cells, inhibition of GBA2 by miglustat or Genz-529648 significantly reduced the induction of IL-8 mRNA levels and protein release following infection by P. aeruginosa. Hence, the present data demonstrate that the anti-inflammatory effects of miglustat and Genz-529648 are likely exerted through inhibition of GBA2. [ABSTRACT FROM AUTHOR]

Malignant gliomas, the most frequent primary brain tumors, are characterized by a dismal prognosis. Reliable biomarkers complementary to neuroradiology in the differential diagnosis of gliomas and monitoring for post-surgical progression are unmet needs. Altered expression of several microRNAs in tumour tissues from patients with gliomas compared to normal brain tissue have been described, thus supporting the rationale of using microRNA-based biomarkers. Although different circulating microRNAs were proposed in association with gliomas, they have not been introduced into clinical practice so far. Blood samples were collected from patients with high and low grade gliomas, both before and after surgical resection, and the expression of miR-21, miR-222 and miR-124-3p was measured in exosomes isolated from serum. The expression levels of miR-21, miR-222 and miR-124-3p in serum exosomes of patients with high grade gliomas were significantly higher than those of low grade gliomas and healthy controls and were sharply decreased in samples obtained after surgery. The analysis of miR-21, miR-222 and miR-124-3p in serum exosomes of patients affected by gliomas can provide a minimally invasive and innovative tool to help the differential diagnosis of gliomas at their onset in the brain and predict glioma grading and non glial metastases before surgery.

Peptide nucleic acids (PNAs) are very useful tools for gene regulation at different levels, but in particular in the last years their use for targeting microRNA (anti-miR PNAs) has provided impressive advancements. In this respect, microRNAs related to the repression of cystic fibrosis transmembrane conductance regulator (CFTR) gene, which is defective in cystic fibrosis, are of great importance in the development of new type of treatments. In this paper we propose the use of an anti-miR PNA for targeting miR-145, a microRNA reported to suppress CFTR expression. Octaarginine-anti-miR PNA conjugates were delivered to Calu-3 cells, exerting sequence dependent targeting of miR-145-5p. This allowed to enhance expression of the miR-145 regulated CFTR gene, analyzed at mRNA (RT-qPCR, Reverse Transcription quantitative Polymerase Chain Reaction) and CFTR protein (Western blotting) level., Competing Interests: The authors declare no conflict of interest. The founding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.

The angelicin analogue 4,6,4'-trimethylangelicin (TMA) was recently reported as a strong inhibitor of nuclear factor- κ B (NF- κ B) activity and of the expression of the interleukin-8 (IL-8) gene in bronchial epithelial cells in which the inflammatory response has been challenged with P. aeruginosa , the most common bacterium found in the airways of patients affected by cystic fibrosis (CF). These findings encouraged us to analyze new synthetic analogues of TMA in order to evaluate their biological activities on human bronchial epithelial CF IB3-1 cells and to find more potent anti-NF- κ B agents exhibiting only minor antiproliferative effects. Analogues able to inhibit NF- κ B/DNA interaction at lower concentration than TMA were found and selected to investigate their biological activity on IB3-1 cells induced with TNF- α . In this biological system, NF- κ B-mediated IL-8 gene expression was investigated. Some analogues showed similar activity to the lead compound TMA. Other analogues displayed higher activities; in particular, the most interesting compounds showing relevant anti-inflammatory effects were found to cause 56-83% reduction of IL-8 mRNA expression at low concentrations (1-10  μ M), without changes in cell proliferation pattern, demonstrating their potential interest for a possible development of anti-inflammatory therapy of cystic fibrosis.

The biological activity of a combined treatment of U251, U373 and T98G glioma cell lines with two anti-miR PNAs, directed against miR‑221 and miR‑222 and conjugated with an ocataarginine tail (R8-PNA-a221 and R8-PNA-a222) for efficient cellular delivery, was determined. Apoptosis was analyzed, and the effect of the combined treatment of glioma cells with either or both PNAs on the reversion of drug-resistance phenotype was assessed in the temozolomide-resistant T98G glioma cell line. Selectivity of PNA/miRNA interactions was studied by surface plasmon resonance (SPR)-based Biacore analysis. Specificity of the PNA effects at the cellular level was analyzed by RT-qPCR. These experiments support the concept that the effects of R8-PNA-a221 and R8-PNA-a222 are specific. The studies on apoptosis confirmed that the R8-PNA-a221 induces apoptosis and demonstrated the pro-apoptotic effects of R8-PNA-a222. Remarkably, increased pro-apoptotic effects were obtained with the co-administration of both anti-miR‑221 and anti-miR‑222 PNAs. In addition, co-administration of R8-PNA-a221 and R8-PNA-a222 induced apoptosis of TMZ-treated T98G cells at a level higher than that obtained following singular administration of R8-PNA-a221 or R8-PNA-a222.

The most common mutation of the cystic fibrosis (CF) gene, the deletion of Phe508, encodes a protein (DeltaF508-CFTR) that fails to fold properly, thus mutated DeltaF508-cystic fibrosis transmembrane conductance regulator (CFTR) is recognized and degraded via the ubiquitin-proteasome endoplasmic reticulum-associated degradation pathway. Chemical and pharmacological chaperones and ligand-induced transport open options for designing specific drugs to control protein (mis)folding or transport. A class of compounds that has been proposed as having potential utility in DeltaF508-CFTR is that which targets the molecular chaperone and proteasome systems. In this study, we have selected deoxyspergualin (DSG) as a reference molecule for this class of compounds and for ease of cross-linking to human serum albumin (HSA) as a protein transporter. Chemical cross-linking of DSG to HSA via a disulfide-based cross-linker and its administration to cells carrying DeltaF508-CFTR resulted in a greater enhancement of DeltaF508-CFTR function than when free DSG was used. Function of the selenium-dependent oxidoreductase system was required to allow intracellular activation of HSA-DSG conjugates. The principle that carrier proteins can deliver pharmacological chaperones to cells leading to correction of defective CFTR functions is therefore proven and warrants further investigations.

Chronic pulmonary inflammation in patients affected by cystic fibrosis (CF) is characterized by massive bronchial infiltrates of neutrophils, which is sustained by the interaction of pathogens (e.g., Pseudomonas aeruginosa) with surface bronchial cells. To explore new treatment options focused on the reduction of neutrophil chemotaxis, we applied the transcription factor (TF) decoy approach, based on the intracellular delivery of double-stranded oligodeoxynucleotides (ODNs) causing inhibition of the binding of TF-related proteins to the different consensus sequences in the promoter of specific genes. In CF bronchial IB3-1 cells, P. aeruginosa induced transcription of the neutrophil chemokines IL-8 and GRO-gamma, of the adhesion molecule intercellular adhesion molecule (ICAM)-1, and of the cytokines IL-1beta and IL-6. Since consensus sequences for the TF, NF-kappaB, are contained in the promoters of all these genes, IB3-1, CuFi-1, Beas-2B, and CaLu-3 cells were transfected with double-stranded TF "decoy" ODNs mimicking different NF-kappaB consensus sequences. IL-8 NF-kappaB decoy ODN partially inhibited the P. aeruginosa-dependent transcription of IL-8, GRO-gamma, and IL-6, whereas decoy ODNs to both HIV-1 long terminal repeat and Igk produced a strong, 80 to 85% inhibition of transcription of IL-8, without reducing that of GRO-gamma, ICAM-1, IL-1beta, and IL-6. In conclusion, intracellular delivery of "decoy" molecules aimed to compete with the TF, NF-kappaB, is a promising strategy to obtain inhibition of IL-8 gene transcription.