Barbara Pompili

Reactive-Oxygen-Species-Mediated P. aeruginosa Killing Is Functional in Human Cystic Fibrosis Macrophages

Pseudomonas aeruginosa is the most common pathogen for chronic lung infection in cystic fibrosis (CF) patients. About 80% of adult CF patients have chronic P. aeruginosa infection, which accounts for much of the morbidity and most of the mortality. Both bacterial genetic adaptations and defective innate immune responses contribute to the bacteria persistence. It is well accepted that CF transmembrane conductance regulator (CFTR) dysfunction impairs the airways-epithelium-mediated lung defence; however, other innate immune cells also appear to be affected, such as neutrophils and macrophages, which thus contribute to this infectious pathology in the CF lung. In macrophages, the absence of CFTR has been linked to defective P. aeruginosa killing, increased pro-inflammatory cytokine secretion, and reduced reactive oxygen species (ROS) production. To learn more about macrophage dysfunction in CF patients, we investigated the generation of the oxidative burst and its impact on bacterial killing in CF macrophages isolated from peripheral blood or lung parenchyma of CF patients, after P. aeruginosa infection. Our data demonstrate that CF macrophages show an oxidative response of similar intensity to that of non-CF macrophages. Intracellular ROS are recognized as one of the earliest microbicidal mechanisms against engulfed pathogens that are activated by macrophages. Accordingly, NADPH inhibition resulted in a significant increase in the intracellular bacteria survival in CF and non-CF macrophages, both as monocyte-derived macrophages and as lung macrophages. These data strongly suggest that the contribution of ROS to P. aeruginosa killing is not affected by CFTR mutations.

Toward Repositioning Niclosamide for Antivirulence Therapy of Pseudomonas aeruginosa Lung Infections: Development of Inhalable Formulations through Nanosuspension Technology

Inhaled antivirulence drugs are currently considered a promising therapeutic option to treat Pseudomonas aeruginosa lung infections in cystic fibrosis (CF). We have recently shown that the anthelmintic drug niclosamide (NCL) has strong quorum sensing (QS) inhibiting activity against P. aeruginosa and could be repurposed as an antivirulence drug. In this work, we developed dry powders containing NCL nanoparticles that can be reconstituted in saline solution to produce inhalable nanosuspensions. NCL nanoparticles were produced by high-pressure homogenization (HPH) using polysorbate 20 or polysorbate 80 as stabilizers. After 20 cycles of HPH, all formulations showed similar properties in the form of needle-shape nanocrystals with a hydrodynamic diameter of approximately 450 nm and a zeta potential of -20 mV. Nanosuspensions stabilized with polysorbate 80 at 10% w/w to NCL (T80_10) showed an optimal solubility profile in simulated interstitial lung fluid. T80_10 was successfully dried into mannitol-based dry powder by spray drying. Dry powder (T80_10 DP) was reconstituted in saline solution and showed optimal in vitro aerosol performance. Both T80_10 and T80_10 DP were able to inhibit P. aeruginosa QS at NCL concentrations of 2.5-10 μM. NCL, and these formulations did not significantly affect the viability of CF bronchial epithelial cells in vitro at microbiologically active concentrations (i.e., ≤10 μM). In vivo acute toxicity studies in rats confirmed no observable toxicity of the NCL T80_10 DP formulation upon intratracheal administration at a concentration 100-fold higher than the anti-QS activity concentration. These preliminary results suggest that NCL repurposed in the form of inhalable nanosuspensions has great potential for the local treatment of P. aeruginosa lung infections as in the case of CF patients.

Development of inhalable hyaluronan/mannitol composite dry powders for flucytosine repositioning in local therapy of lung infections.

Flucytosine (5-fluorocytosine, 5-FC) is a fluorinated analogue of cytosine currently approved for the systemic treatment of fungal infections, which has recently demonstrated a very promising antivirulence activity against the bacterial pathogen Pseudomonas aeruginosa. In this work, we propose novel inhalable hyaluronic acid (HA)/mannitol composite dry powders for repositioning 5-FC in the local treatment of lung infections, including those affecting cystic fibrosis (CF) patients. Different dry powders were produced in one-step by spray-drying. Powder composition and process conditions were selected after in depth formulation studies aimed at selecting the 5-FC/HA/mannitol formulation with convenient aerosolization properties and drug release profile in simulated lung fluids. The optimized 5-FC/HA/mannitol powder for inhalation (HyaMan_FC#3) was effectively delivered from different breath-activated dry powder inhalers (DPI) already available to CF patients. Nevertheless, the aerodynamic assessment of fine particles suggested that the developed formulation well fit with a low-resistance DPI. HyaMan_FC#3 inhibited the growth of the fungus Candida albicans and the production of the virulence factor pyoverdine by P. aeruginosa at 5-FC concentrations that did not affect the viability of both wild type (16HBE14o-) and CF (CFBE41o-) human bronchial epithelial cells. Finally, pharmacokinetics of HyaMan_FC#3 inhalation powder and 5-FC solution after intratracheal administration in rats were compared. In vivo results clearly demonstrated that, when formulated as dry powder, 5-FC levels in both bronchoalveolar lavage fluid and lung tissue were significantly higher and sustained over time as compared to those obtained with the 5-FC solution. Of note, when the same 5-FC amount was administered intravenously, no significant drug amount was found in the lung at each time point from the injection. To realize a 5-FC lung concentration similar to that obtained by using HyaMan_FC#3, a 6-fold higher dose of 5-FC should be administered intravenously. Taken together, our data demonstrate the feasibility to deliver 5-FC by the pulmonary route likely avoiding/reducing the well-known side effects associated to the high systemic 5-FC doses currently used in humans. Furthermore, our results highlight that an appropriate formulation design can improve the persistence of the drug at lungs, where microorganisms causing severe infections are located.

Assembly and Functional Analysis of an S/MAR Based Episome with the Cystic Fibrosis Transmembrane Conductance Regulator Gene

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.

142 Development of an S/MAR based episomal vector for the CFTR gene delivery

Objectives The recent use of mutation-specific correctors in Cystic Fibrosis, reinforces the concept of gene therapy as a therapeutic treatment for all CF patients. Non-viral vectors are preferable to viral ones, particularly in treatment of chronic diseases, for their simpler composition, lack of immunogenicity and genotoxicity. Episomal vectors based on the S/MAR (scaffold/matrix attachment region) sequence have self-replicating capability and episomal persistence in different cell types, both in vitro and in vivo. To obtain long-term and episomal maintenance of the CFTR gene in target cells, we constructed a vector containing the S/MAR, GFP and the CFTR cDNA (pBQ-S/MAR). Methods pBQ-S/MAR was transfected in CF(DF508/DF508) airway epithelial cells and the cells were grown in non-selective conditions up to 14 days after transfection. The cells were evaluated for the episome maintenance and persistence as well as for CFTR gene expression (RT-PCR, immunofluorescence, western blot). Results The resulting data demonstrate that pBQ-S/MAR is maintained as an episome, with unchanged structure respect to the input vector, for at least 14 days after transfection, corresponding to about six cell duplications. CFTR expression analyses confirmed the presence of the wt transcript and the mature form of the protein. Additionally, the expression of a functional CFTR in pBQ-S/MAR-transfected cells was associated with an increase of Trans Epithelial Resistance respect to parental cells. Conclusion Our results suggest that the pBQ-S/MAR is stably maintained as an episome in airway epithelial cells and support expression of a functional CFTR.

56 Pseudomonas aeruginosa end-stage isolates: analysis of virulence traits and interaction with macrophages

56 Pseudomonas aeruginosa end-stage isolates: analysis of virulence traits and interaction with macrophages N. Cifani1,2, B. Pompili1, V. Ferri1, P. Fradiani3, E.G. Di Domenico1, F. Venuta4, M. Anile4, P. Del Porto1, F. Ascenzioni1. 1Sapienza University of Rome, Department of Biology and Biotechnology ‘C. Darwin’, Rome, Italy; 2Sapienza University of Rome, Department of Clinical and Molecular Medicine, Rome, Italy; 3Sapienza University of Rome, U.O.D. Microbiology, Sant’Andrea Hospital, Rome, Italy; 4Sapienza University of Rome, Department of Thoracic Surgery, Rome, Italy

109 Increased IL-8 production in human CFTR-deficient macrophages

108 Reduced effect of intravenous antibiotic treatment on sinonasal compared to pulmonary inflammatory markers J.G. Mainz1, J. Hentschel1, N. Fischer1, U.R. Markert2, K. Boer3, W. Pfister4, F. Doht1. 1Jena University Hospital, CF-Centre, Jena, Germany; 2Jena University Hospital, Department of Obstetrics, Placenta Laboratory, Jena, Germany; 3Jena University Hospital, Institute for Clinical Chemistry and Laboratory Diagnostics, Jena, Germany; 4Jena University Hospital, Institute of Medical Microbiology, Jena, Germany