Aryl Hydrocarbon Receptor Agonism Antagonizes the Hypoxia-driven Inflammation in Cystic Fibrosis.

Hypoxia contributes to the exaggerated yet ineffective airway inflammation that fails to oppose infections in cystic fibrosis (CF). However, the potential for impairment of essential immune functions by HIF-1α (hypoxia-inducible factor 1α) inhibition demands a better comprehension of downstream hypoxia-dependent pathways that are amenable for manipulation. We assessed here whether hypoxia may interfere with the activity of AhR (aryl hydrocarbon receptor), a versatile environmental sensor highly expressed in the lungs, where it plays a homeostatic role. We used murine models of Aspergillus fumigatus infection in vivo and human cells in vitro to define the functional role of AhR in CF, evaluate the impact of hypoxia on AhR expression and activity, and assess whether AhR agonism may antagonize hypoxia-driven inflammation. We demonstrated that there is an important interferential cross-talk between the AhR and HIF-1α signaling pathways in murine and human CF, in that HIF-1α induction squelched the normal AhR response through an impaired formation of the AhR:ARNT (aryl hydrocarbon receptor nuclear translocator)/HIF-1ß heterodimer. However, functional studies and analysis of the AhR genetic variability in patients with CF proved that AhR agonism could prevent hypoxia-driven inflammation, restore immune homeostasis, and improve lung function. This study emphasizes the contribution of environmental factors, such as infections, in CF disease progression and suggests the exploitation of hypoxia:xenobiotic receptor cross-talk for antiinflammatory therapy in CF.

Serratiopeptidase Affects the Physiology of Pseudomonas aeruginosa Isolates from Cystic Fibrosis Patients.

Pseudomonas aeruginosa is frequently involved in cystic fibrosis (CF) airway infections. Biofilm, motility, production of toxins and the invasion of host cells are different factors that increase P. aeruginosa's virulence. The sessile phenotype offers protection to bacterial cells and resistance to antimicrobials and host immune attacks. Motility also contributes to bacterial colonization of surfaces and, consequently, to biofilm formation. Furthermore, the ability to adhere is the prelude for the internalization into lung cells, a common immune evasion mechanism used by most intracellular bacteria, such as P. aeruginosa. In previous studies we evaluated the activity of metalloprotease serratiopeptidase (SPEP) in impairing virulence-related properties in Gram-positive bacteria. This work aimed to investigate SPEP's effects on different physiological aspects related to the virulence of P. aeruginosa isolated from CF patients, such as biofilm production, pyoverdine and pyocyanin production and invasion in alveolar epithelial cells. Obtained results showed that SPEP was able to impair the attachment to inert surfaces as well as adhesion/invasion of eukaryotic cells. Conversely, SPEP's effect on pyocyanin and pyoverdine production was strongly strain-dependent, with an increase and/or a decrease of their production. Moreover, SPEP seemed to increase swarming motility and staphylolytic protease production. Our results suggest that a large number of clinical strains should be studied in-depth before drawing definitive conclusions. Why different strains sometimes react in opposing ways to a specific treatment is of great interest and will be the object of future studies. Therefore, SPEP affects P. aeruginosa's physiology by differently acting on several bacterial factors related to its virulence.

Anti-Virulence Potential of a Chionodracine-Derived Peptide against Multidrug-Resistant Pseudomonas aeruginosa Clinical Isolates from Cystic Fibrosis Patients.

Pseudomonas aeruginosa is an opportunistic pathogen causing several chronic infections resistant to currently available antibiotics. Its pathogenicity is related to the production of different virulence factors such as biofilm and protease secretion. Pseudomonas communities can persist in biofilms that protect bacterial cells from antibiotics. Hence, there is a need for innovative approaches that are able to counteract these virulence factors, which play a pivotal role, especially in chronic infections. In this context, antimicrobial peptides are emerging drugs showing a broad spectrum of antibacterial activity. Here, we tested the anti-virulence activity of a chionodracine-derived peptide (KHS-Cnd) on five P. aeruginosa clinical isolates from cystic fibrosis patients. We demonstrated that KHS-Cnd impaired biofilm development and caused biofilm disaggregation without affecting bacterial viability in nearly all of the tested strains. Ultrastructural morphological analysis showed that the effect of KHS-Cnd on biofilm could be related to a different compactness of the matrix. KHS-Cnd was also able to reduce adhesion to pulmonary cell lines and to impair the invasion of host cells by P. aeruginosa. A cytotoxic effect of KHS-Cnd was observed only at the highest tested concentration. This study highlights the potential of KHS-Cnd as an anti-biofilm and anti-virulence molecule against P. aeruginosa clinical strains.

Achromobacter spp. Adaptation in Cystic Fibrosis Infection and Candidate Biomarkers of Antimicrobial Resistance.

Achromobacter spp. can establish occasional or chronic lung infections in patients with cystic fibrosis (CF). Chronic colonization has been associated with worse prognosis highlighting the need to identify markers of bacterial persistence. To this purpose, we analyzed phenotypic features of 95 Achromobacter spp. isolates from 38 patients presenting chronic or occasional infection. Virulence was tested in Galleria mellonella larvae, cytotoxicity was tested in human bronchial epithelial cells, biofilm production in static conditions was measured by crystal violet staining and susceptibility to selected antibiotics was tested by the disk diffusion method. The presence of genetic loci associated to the analyzed phenotypic features was evaluated by a genome-wide association study. Isolates from occasional infection induced significantly higher mortality of G. mellonella larvae and showed a trend for lower cytotoxicity than chronic infection isolates. No significant difference was observed in biofilm production among the two groups. Additionally, antibiotic susceptibility testing showed that isolates from chronically-infected patients were significantly more resistant to sulfonamides and meropenem than occasional isolates. Candidate genetic biomarkers associated with antibiotic resistance or sensitivity were identified. Achromobacter spp. strains isolated from people with chronic and occasional lung infection exhibit different virulence and antibiotic susceptibility features, which could be linked to persistence in CF lungs. This underlines the possibility of identifying predictive biomarkers of persistence that could be useful for clinical purposes.

The Antimalarial Mefloquine Shows Activity against Mycobacterium abscessus, Inhibiting Mycolic Acid Metabolism.

Some nontuberculous mycobacteria (NTM) are considered opportunistic pathogens. Nevertheless, NTM infections are increasing worldwide, becoming a major public health threat. Furthermore, there is no current specific drugs to treat these infections, and the recommended regimens generally lack efficacy, emphasizing the need for novel antibacterial compounds. In this paper, we focused on the essential mycolic acids transporter MmpL3, which is a well-characterized target of several antimycobacterial agents, to identify new compounds active against Mycobacterium abscessus (Mab). From the crystal structure of MmpL3 in complex with known inhibitors, through an in silico approach, we developed a pharmacophore that was used as a three-dimensional filter to identify new putative MmpL3 ligands within databases of known drugs. Among the prioritized compounds, mefloquine showed appreciable activity against Mab (MIC = 16 µg/mL). The compound was confirmed to interfere with mycolic acids biosynthesis, and proved to also be active against other NTMs, including drug-resistant clinical isolates. Importantly, mefloquine is a well-known antimalarial agent, opening the possibility of repurposing an already approved drug, which is a useful strategy to reduce the time and cost of disclosing novel drug candidates.

Essential Oils Biofilm Modulation Activity, Chemical and Machine Learning Analysis. Application on Staphylococcus aureus Isolates from Cystic Fibrosis Patients.

Bacterial biofilm plays a pivotal role in chronic Staphylococcus aureus (S. aureus) infection and its inhibition may represent an important strategy to develop novel therapeutic agents. The scientific community is continuously searching for natural and "green alternatives" to chemotherapeutic drugs, including essential oils (EOs), assuming the latter not able to select resistant strains, likely due to their multicomponent nature and, hence, multitarget action. Here it is reported the biofilm production modulation exerted by 61 EOs, also investigated for their antibacterial activity on S. aureus strains, including reference and cystic fibrosis patients' isolated strains. The EOs biofilm modulation was assessed by Christensen method on five S. aureus strains. Chemical composition, investigated by GC/MS analysis, of the tested EOs allowed a correlation between biofilm modulation potency and putative active components by means of machine learning algorithms application. Some EOs inhibited biofilm growth at 1.00% concentration, although lower concentrations revealed different biological profile. Experimental data led to select antibiofilm EOs based on their ability to inhibit S. aureus biofilm growth, which were characterized for their ability to alter the biofilm organization by means of SEM studies.

Design of a Broad-Range Bacteriophage Cocktail That Reduces Pseudomonas aeruginosa Biofilms and Treats Acute Infections in Two Animal Models.

The alarming diffusion of multidrug-resistant (MDR) bacterial strains requires investigations on nonantibiotic therapies. Among such therapies, the use of bacteriophages (phages) as antimicrobial agents, namely, phage therapy, is a promising treatment strategy supported by the findings of recent successful compassionate treatments in Europe and the United States. In this work, we combined host range and genomic information to design a 6-phage cocktail killing several clinical strains of Pseudomonas aeruginosa, including those collected from Italian cystic fibrosis (CF) patients, and analyzed the cocktail performance. We demonstrated that the cocktail composed of four novel phages (PYO2, DEV, E215 and E217) and two previously characterized phages (PAK_P1 and PAK_P4) was able to lyse P. aeruginosa both in planktonic liquid cultures and in biofilms. In addition, we showed that the phage cocktail could cure acute respiratory infection in mice and treat bacteremia in wax moth (Galleria mellonella) larvae. Furthermore, administration of the cocktail to larvae prior to bacterial infection provided prophylaxis. In this regard, the efficiency of the phage cocktail was found to be unaffected by the MDR or mucoid phenotype of the pseudomonal strain. The cocktail was found to be superior to the individual phages in destroying biofilms and providing a faster treatment in mice. We also found the Galleria larva model to be cost-effective for testing the susceptibility of clinical strains to phages, suggesting that it could be implemented in the frame of developing personalized phage therapies.

Identification of FDA-Approved Drugs as Antivirulence Agents Targeting the pqs Quorum-Sensing System of Pseudomonas aeruginosa.

The long-term use of antibiotics has led to the emergence of multidrug-resistant bacteria. A promising strategy to combat bacterial infections aims at hampering their adaptability to the host environment without affecting growth. In this context, the intercellular communication system quorum sensing (QS), which controls virulence factor production and biofilm formation in diverse human pathogens, is considered an ideal target. Here, we describe the identification of new inhibitors of the pqs QS system of the human pathogen Pseudomonas aeruginosa by screening a library of 1,600 U.S. Food and Drug Administration-approved drugs. Phenotypic characterization of ad hoc engineered strains and in silico molecular docking demonstrated that the antifungal drugs clotrimazole and miconazole, as well as an antibacterial compound active against Gram-positive pathogens, clofoctol, inhibit the pqs system, probably by targeting the transcriptional regulator PqsR. The most active inhibitor, clofoctol, specifically inhibited the expression of pqs-controlled virulence traits in P. aeruginosa, such as pyocyanin production, swarming motility, biofilm formation, and expression of genes involved in siderophore production. Moreover, clofoctol protected Galleria mellonella larvae from P. aeruginosa infection and inhibited the pqs QS system in P. aeruginosa isolates from cystic fibrosis patients. Notably, clofoctol is already approved for clinical treatment of pulmonary infections caused by Gram-positive bacterial pathogens; hence, this drug has considerable clinical potential as an antivirulence agent for the treatment of P. aeruginosa lung infections.

Organization of Patient Management and Fungal Epidemiology in Cystic Fibrosis.

The achievement of a better life for cystic fibrosis (CF) patients is mainly caused by a better management and infection control over the last three decades. Herein, we want to summarize the cornerstones for an effective management of CF patients and to give an overview of the knowledge about the fungal epidemiology in this clinical context in Europe. Data from a retrospective analysis encompassing 66,616 samples from 3235 CF patients followed-up in 9 CF centers from different European countries are shown.

A Different Microbiome Gene Repertoire in the Airways of Cystic Fibrosis Patients with Severe Lung Disease.

In recent years, next-generation sequencing (NGS) was employed to decipher the structure and composition of the microbiota of the airways in cystic fibrosis (CF) patients. However, little is still known about the overall gene functions harbored by the resident microbial populations and which specific genes are associated with various stages of CF lung disease. In the present study, we aimed to identify the microbial gene repertoire of CF microbiota in twelve patients with severe and normal/mild lung disease by performing sputum shotgun metagenome sequencing. The abundance of metabolic pathways encoded by microbes inhabiting CF airways was reconstructed from the metagenome. We identified a set of metabolic pathways differently distributed in patients with different pulmonary function; namely, pathways related to bacterial chemotaxis and flagellar assembly, as well as genes encoding efflux-mediated antibiotic resistance mechanisms and virulence-related genes. The results indicated that the microbiome of CF patients with low pulmonary function is enriched in virulence-related genes and in genes encoding efflux-mediated antibiotic resistance mechanisms. Overall, the microbiome of severely affected adults with CF seems to encode different mechanisms for the facilitation of microbial colonization and persistence in the lung, consistent with the characteristics of multidrug-resistant microbial communities that are commonly observed in patients with severe lung disease.

Hypoxia promotes danger-mediated inflammation via receptor for advanced glycation end products in cystic fibrosis.

RATIONALE: Hypoxia regulates the inflammatory-antiinflammatory balance by the receptor for advanced glycation end products (RAGE), a versatile sensor of damage-associated molecular patterns. The multiligand nature of RAGE places this receptor in the midst of chronic inflammatory diseases. OBJECTIVES: To characterize the impact of the hypoxia-RAGE pathway on pathogenic airway inflammation preventing effective pathogen clearance in cystic fibrosis (CF) and elucidate the potential role of this danger signal in pathogenesis and therapy of lung inflammation. METHODS: We used in vivo and in vitro models to study the impact of hypoxia on RAGE expression and activity in human and murine CF, the nature of the RAGE ligand, and the impact of RAGE on lung inflammation and antimicrobial resistance in fungal and bacterial pneumonia. MEASUREMENTS AND MAIN RESULTS: Sustained expression of RAGE and its ligand S100B was observed in murine lung and human epithelial cells and exerted a proximal role in promoting inflammation in murine and human CF, as revealed by functional studies and analysis of the genetic variability of AGER in patients with CF. Both hypoxia and infections contributed to the sustained activation of the S100B-RAGE pathway, being RAGE up-regulated by hypoxia and S100B by infection by Toll-like receptors. Inhibiting the RAGE pathway in vivo with soluble (s) RAGE reduced pathogen load and inflammation in experimental CF, whereas sRAGE production was defective in patients with CF. CONCLUSIONS: A causal link between hyperactivation of RAGE and inflammation in CF has been observed, such that targeting pathogenic inflammation alleviated inflammation in CF and measurement of sRAGE levels could be a useful biomarker for RAGE-dependent inflammation in patients with CF.

Analysis of gut microbiota in patients with Williams-Beuren Syndrome reveals dysbiosis linked to clinical manifestations.

Williams-Beuren syndrome (WBS) is a multisystem genetic disease caused by the deletion of a region of 1.5-1.8 Mb on chromosome 7q11.23. The elastin gene seems to account for several comorbidities and distinct clinical features such including cardiovascular disease, connective tissue abnormalities, growth retardation, and gastrointestinal (GI) symptoms. Increasing evidence points to alterations in gut microbiota composition as a primary or secondary cause of some GI or extra-intestinal characteristics. In this study, we performed the first exploratory analysis of gut microbiota in WBS patients compared to healthy subjects (CTRLs) using 16S rRNA amplicon sequencing, by investigating the gut dysbiosis in relation to diseases and comorbidities. We found that patients with WBS have significant dysbiosis compared to age-matched CTRLs, characterized by an increase in proinflammatory bacteria such as Pseudomonas, Gluconacetobacter and Eggerthella, and a reduction of anti-inflammatory bacteria including Akkermansia and Bifidobacterium. Microbial biomarkers associated with weight gain, GI symptoms and hypertension were identified. Gut microbiota profiling could represent a new tool that characterise intestinal dysbiosis to complement the clinical management of these patients. In particular, the administration of microbial-based treatments, alongside traditional therapies, could help in reducing or preventing the burden of these symptoms and improve the quality of life of these patients.

Haemophilus influenzae in children with cystic fibrosis: antimicrobial susceptibility, molecular epidemiology, distribution of adhesins and biofilm formation.

Haemophilus influenzae commonly infects the respiratory tract of patients with cystic fibrosis (CF), early in childhood. In this investigation, 79 H. influenzae isolates were recovered from the respiratory secretions of 64 CF patients (median age: 5 years) included in a 5-year follow-up study. Fifteen of the 64 patients contributed two or more H. influenzae isolates overtime. Serotyping, antibiotic susceptibility testing, genotyping, detection of both hmwA and hia adhesin genes and hypermutable strains was carried out. Biofilm formation ability was investigated. Most strains (72/79, 91.2%) were nonencapsulated or nontypeable (NTHi). Resistance to ampicillin (13.9%) and imipenem (17.7%) was the most detected. Few isolates (2.5%) exhibited the hypermutable phenotype. The NTHi strains showed 55 different genotypes, but 19 clusters of closely related strains were identified. Nine clusters included strains that cross-colonised several patients over a long-time period (mean: 3.7 years). Most patients with sequential isolates harboured strains genetically unrelated, but persistent colonisation with the same clone was observed in 37.5% of patients. Over 45% of NTHi strains contained hmwA-related sequences, 26.3%, hia, 8.3% both hmwA and hia, while 19.4% lacked both. A significant association was found between occurrence of an adhesive gene (irrespective of which) and both persistence (P<0.0001) and long-term cross-colonisation (P<0.0001). Mean biofilm level formed by the persistent strains was found significantly increased compared to non-persistent ones (P<0.0001). Hia-positive strains produced significantly more biofilm than hmwA-carrying strains (P<0.01). Although a high turnover of NTHi strains in FC patients was observed, distinct clones with increased capacity of persistence or cross-colonisation occurred.

Potential novel therapeutic strategies in cystic fibrosis: antimicrobial and anti-biofilm activity of natural and designed α-helical peptides against Staphylococcus aureus, Pseudomonas aeruginosa, and Stenotrophomonas maltophilia.

BACKGROUND: Treatment of cystic fibrosis-associated lung infections is hampered by the presence of multi-drug resistant pathogens, many of which are also strong biofilm producers. Antimicrobial peptides, essential components of innate immunity in humans and animals, exhibit relevant in vitro antimicrobial activity although they tend not to select for resistant strains. RESULTS: Three α-helical antimicrobial peptides, BMAP-27 and BMAP-28 of bovine origin, and the artificial P19(9/B) peptide were tested, comparatively to Tobramycin, for their in vitro antibacterial and anti-biofilm activity against 15 Staphylococcus aureus, 25 Pseudomonas aeruginosa, and 27 Stenotrophomonas maltophilia strains from cystic fibrosis patients. All assays were carried out in physical-chemical experimental conditions simulating a cystic fibrosis lung. All peptides showed a potent and rapid bactericidal activity against most P. aeruginosa, S. maltophilia and S. aureus strains tested, at levels generally higher than those exhibited by Tobramycin and significantly reduced biofilm formation of all the bacterial species tested, although less effectively than Tobramycin did. On the contrary, the viability-reducing activity of antimicrobial peptides against preformed P. aeruginosa biofilms was comparable to and, in some cases, higher than that showed by Tobramycin. CONCLUSIONS: The activity shown by α-helical peptides against planktonic and biofilm cells makes them promising "lead compounds" for future development of novel drugs for therapeutic treatment of cystic fibrosis lung disease.

The Fascinating History of Wound Healing Through Fine Arts.

Visual art images narrate the evolution of humankind including different and specific wound managing strategies. Through the observation of some notable art works we explore the empiric historical progress in wound healing and the main reasons they may have been represented. We briefly examine the cultural, symbolic, magical or religious beliefs that have conditioned the approach to a fundamental vital need of humanity: to heal a wound.

In vitro Activity of Antivirulence Drugs Targeting the las or pqs Quorum Sensing Against Cystic Fibrosis Pseudomonas aeruginosa Isolates.

The chronic lung infection caused by Pseudomonas aeruginosa is a major cause of morbidity and mortality in cystic fibrosis (CF) patients. Antivirulence drugs targeting P. aeruginosa quorum sensing (QS) systems are intensively studied as antibiotics substitutes or adjuvants. Previous studies, carried out in non-CF P. aeruginosa reference strains, showed that the old drugs niclosamide and clofoctol could be successfully repurposed as antivirulence drugs targeting the las and pqs QS systems, respectively. However, frequent emergence of QS-defective mutants in the CF lung undermines the use of QS inhibitors in CF therapy. Here, QS signal production and susceptibility to niclosamide and clofoctol have been investigated in 100 P. aeruginosa CF isolates, with the aim of broadening current knowledge on the potential of anti-QS compounds in CF therapy. Results showed that 85, 78, and 69% of the CF isolates from our collection were proficient for the pqs, rhl, and las QS systems, respectively. The ability of both niclosamide and clofoctol to inhibit QS and virulence in vitro was highly variable and strain-dependent. Niclosamide showed an overall low range of activity and its negative effect on las signal production did not correlate with a decreased production of virulence factors. On the other hand, clofoctol displayed a broader QS inhibitory effect in CF isolates, with consequent reduction of the pqs-controlled virulence factor pyocyanin. Overall, this study highlights the importance of testing new antivirulence drugs against large panels of P. aeruginosa CF clinical isolates before proceeding to further pre-clinical studies and corroborates previous evidence that strains naturally resistant to QS inhibitors occur among CF isolates. However, it is also shown that resistance to pqs inhibitors is less frequent than resistance to las inhibitors, thus supporting the development of pqs inhibitors for antivirulence therapy in CF.

Essential Oils Biofilm Modulation Activity and Machine Learning Analysis on Pseudomonas aeruginosa Isolates from Cystic Fibrosis Patients.

The opportunistic pathogen Pseudomonas aeruginosa is often involved in airway infections of cystic fibrosis (CF) patients. It persists in the hostile CF lung environment, inducing chronic infections due to the production of several virulence factors. In this regard, the ability to form a biofilm plays a pivotal role in CF airway colonization by P. aeruginosa. Bacterial virulence mitigation and bacterial cell adhesion hampering and/or biofilm reduced formation could represent a major target for the development of new therapeutic treatments for infection control. Essential oils (EOs) are being considered as a potential alternative in clinical settings for the prevention, treatment, and control of infections sustained by microbial biofilms. EOs are complex mixtures of different classes of organic compounds, usually used for the treatment of upper respiratory tract infections in traditional medicine. Recently, a wide series of EOs were investigated for their ability to modulate biofilm production by different pathogens comprising S. aureus, S. epidermidis, and P. aeruginosa strains. Machine learning (ML) algorithms were applied to develop classification models in order to suggest a possible antibiofilm action for each chemical component of the studied EOs. In the present study, we assessed the biofilm growth modulation exerted by 61 commercial EOs on a selected number of P. aeruginosa strains isolated from CF patients. Furthermore, ML has been used to shed light on the EO chemical components likely responsible for the positive or negative modulation of bacterial biofilm formation.

A Shaving Proteomic Approach to Unveil Surface Proteins Modulation of Multi-Drug Resistant Pseudomonas aeruginosa Strains Isolated From Cystic Fibrosis Patients.

Cystic fibrosis (CF) is the most common rare disease caused by a mutation of the CF transmembrane conductance regulator gene encoding a channel protein of the apical membrane of epithelial cells leading to alteration of Na+ and K+ transport, hence inducing accumulation of dense and sticky mucus and promoting recurrent airway infections. The most detected bacterium in CF patients is Pseudomonas aeruginosa (PA) which causes chronic colonization, requiring stringent antibiotic therapies that, in turn induces multi-drug resistance. Despite eradication attempts at the first infection, the bacterium is able to utilize several adaptation mechanisms to survive in hostile environments such as the CF lung. Its adaptive machinery includes modulation of surface molecules such as efflux pumps, flagellum, pili and other virulence factors. In the present study we compared surface protein expression of PA multi- and pan-drug resistant strains to wild-type antibiotic-sensitive strains, isolated from the airways of CF patients with chronic colonization and recent infection, respectively. After shaving with trypsin, microbial peptides were analyzed by tandem-mass spectrometry on a high-resolution platform that allowed the identification of 174 differentially modulated proteins localized in the region from extracellular space to cytoplasmic membrane. Biofilm assay was performed to characterize all 26 PA strains in term of biofilm production. Among the differentially expressed proteins, 17 were associated to the virulome (e.g., Tse2, Tse5, Tsi1, PilF, FliY, B-type flagellin, FliM, PyoS5), six to the resistome (e.g., OprJ, LptD) and five to the biofilm reservoir (e.g., AlgF, PlsD). The biofilm assay characterized chronic antibiotic-resistant isolates as weaker biofilm producers than wild-type strains. Our results suggest the loss of PA early virulence factors (e.g., pili and flagella) and later expression of virulence traits (e.g., secretion systems proteins) as an indicator of PA adaptation and persistence in the CF lung environment. To our knowledge, this is the first study that, applying a shaving proteomic approach, describes adaptation processes of a large collection of PA clinical strains isolated from CF patients in early and chronic infection phases.

An inflammatory Signature of Glucose Impairment in Cystic Fibrosis.

Objective and Design: Cystic fibrosis-related diabetes (CFRD) is a severe complication associated with increased morbidity and mortality in cystic fibrosis (CF) patients. Extensive inflammatory state in CF leads to pancreas damage and insulin resistance with consequent altered glucose tolerance and CFRD development. The aim of the present study was to identify circulating levels of inflammatory markers specifically associated with impaired glucose tolerance (IGT) and overt CFRD in a sample of young adults with CF. Materials and Methods: Sixty-four CF outpatients, without evident active pulmonary exacerbation, infectious and autoimmune diseases, were enrolled in the study and the levels of 45 inflammatory serum mediators were measured through x magnetic bead panel multiplex technology. Results: Serum levels of PDGF-AA, CCL20/MIP3α, IFNα, CCL11/eotaxin, CXCL1/GROα, GMCSF, B7H1/PDL1, IL13, IL7, VEGF, and TGFα were all significantly (p<0.05) elevated in patients according to glycemic status and directly correlated with glycated hemoglobin and C-reactive protein levels. Conclusion: Our findings suggest that increased levels of specific circulating inflammatory mediators are directly associated with impaired glucose tolerance in CF patients, thus, potentially implicating them in CFRD pathogenesis and warranting larger longitudinal studies to validate their monitoring as predictor of CFRD onset.

Hybrid Lipid/Polymer Nanoparticles to Tackle the Cystic Fibrosis Mucus Barrier in siRNA Delivery to the Lungs: Does PEGylation Make the Difference?

Inhaled siRNA therapy has a unique potential for treatment of severe lung diseases, such as cystic fibrosis (CF). Nevertheless, a drug delivery system tackling lung barriers is mandatory to enhance gene silencing efficacy in the airway epithelium. We recently demonstrated that lipid-polymer hybrid nanoparticles (hNPs), comprising a poly(lactic-co-glycolic) acid (PLGA) core and a lipid shell of dipalmitoyl phosphatidylcholine (DPPC), may assist the transport of the nucleic acid cargo through mucus-covered human airway epithelium. To study in depth the potential of hNPs for siRNA delivery to the lungs and to investigate the hypothesized benefit of PEGylation, here, an siRNA pool against the nuclear factor-κB (siNFκB) was encapsulated inside hNPs, endowed with a non-PEGylated (DPPC) or a PEGylated (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-poly(ethylene glycol) or DSPE-PEG) lipid shell. Resulting hNPs were tested for their stability profiles and transport properties in artificial CF mucus, mucus collected from CF cells, and sputum samples from a heterogeneous and representative set of CF patients. Initial information on hNP properties governing their interaction with airway mucus was acquired by small-angle X-ray scattering (SAXS) studies in artificial and cellular CF mucus. The diffusion profiles of hNPs through CF sputa suggested a crucial role of lung colonization of the corresponding donor patient, affecting the mucin type and content of the sample. Noteworthy, PEGylation did not boost mucus penetration in complex and sticky samples, such as CF sputa from patients with polymicrobial colonization. In parallel, in vitro cell uptake studies performed on mucus-lined Calu-3 cells grown at the air-liquid interface (ALI) confirmed the improved ability of non-PEGylated hNPs to overcome mucus and cellular lung barriers. Furthermore, effective in vitro NFκB gene silencing was achieved in LPS-stimulated 16HBE14o- cells. Overall, the results highlight the potential of non-PEGylated hNPs as carriers for pulmonary delivery of siRNA for local treatment of CF lung disease. Furthermore, this study provides a detailed understanding of how distinct models may provide different information on nanoparticle interaction with the mucus barrier.