Background. Cystic Fibrosis patients with nonsense-mutation in the CFTR gene generally make virtually no CFTR protein and thus often have a more severe form of CF. Recently, Ataluren (formerly PTC124) was suggested to induce the read-through of premature termination codons (PTCs) mainly the UGA codon. However, despite promising results there is not a general consensus of Ataluren efficacy and mechanism of action.
Hypothesis and objectives. The design of new small molecules (PTC124 related) together with the understanding of their mechanism of action could lead to new pharmacologic approaches for the cure of CF caused by nonsense mutations in the CFTR gene. This project was aimed to evaluate the activity of some PTC124 analogues identified by a virtual screening, by different orthogonal assays with vectors containing PTCs in reporter genes and in CF bronchial epithelial cells.
Background. This is a project aimed at the pharmaceutical development of a new antimicrobial peptide (M33) discovered at the University of Siena. M33 showed a strong activity in vitro and in vivo against a panel of bacteria generally involved in CF infections.
Hypothesis and objectives. Before arriving to human experimentation, a new drug must be developed preclinically and this includes the study of pharmacokinetic, bio-distribution and toxicity profiles in animals. This project was exactly designed for the evaluations of these issues. At the end of preclinical procedures of development, regulatory documents for the request of a Clinical Trial Authorization will be submitted to competent authorities.
Background. The diffusion of multidrug resistant (MDR) bacteria has highlighted the need of new antibiotics, but, so far, progress in developing them has been slow. Cationic Antimicrobial Peptides (CAMPs) are the first line of defense of multicellular eukaryotics against microbial invasions. Even if the protein nature of CAMPs makes difficult their use as systemic antimicrobials they are ideally suited for direct delivery to airways and lung.
Hypothesis and objectives. The main aim of this project is to develop inhalable dry powders for lung-delivery of CAMPs and CAMP-releasing proteins (CAMP-RPs) carrying simple chemical modifications which improve their antimicrobial activity.
Background. Cystic fibrosis (CF) patients must deal with pulmonary bacterial infections by antibiotic-resistant pathogens. A weapon that could be used against pulmonary infections are antimicrobial peptides (AMPs), natural molecules with antibiotic activity. The BMAP18 peptide has a potent antibacterial in vitro activity, however it could not protect mice with P. aeruginosa infection and showed quite toxic side effects.
Hypothesis & Objectives. Because of these problems which are common to many others AMPs, BMAP18 could not be directly used in clinic. Our aim was to investigate the reasons of toxicity and scarce activity of BMAP18 in mice model of infection to optimize the molecule for its usage in the particular lung environment of CF patients. Tobramycin and colistin antibiotics have been used for comparison.
Background. Early aggressive and maintenance antibiotic therapies prolong cystic fibrosis (CF) patient life, but are not able to eradicate Pseudomonas aeruginosa lung infection. Anti-virulence drugs represent a promising therapeutic option in CF. These drugs could alleviate the severity of the infection, reduce lung inflammation, and help antibiotics in eradicating the P. aeruginosa infection. The long times and high costs required for the development of “brand new” anti-virulence drugs can be saved by repurposing “old” drugs already used in humans for different diseases. We have recently shown that the antimycotic drug flucytosine and the anthelmintic drug niclosamide can be repurposed to suppress P. aeruginosa virulence in vitro and in animal models of infection. However, the old drugs need to be re-formulated for the new application in CF therapy.
Hypothesis and objectives. 1) To develop and validate inhalable formulations of flucytosine and niclosamide for CF therapy. 2) To discovery additional drugs with anti-virulence activity against P. aeruginosa.
Background. Cystic fibrosis (CF) is characterized by a progressive decline in lung function. Despite antibiotic treatment, patients with CF may show a rapid and severe decline in lung function. In the previous project (FFC#8/2012), we found changes in CF airway microbiota associated with a severe decline in lung function (Paganin, Fiscarelli et al. PLoS ONE 10(4): e0124348). Moreover a large set of metagenomic and metabarcoding data were produced allowing to identify additional novel biomarkers for factors responsible for the seriously decline in lung function.
Hypothesis and objectives. The starting hypothesis is that a number of hidden pathogens/biomarkers from non-culturable bacteria may be present in patient’s airways of cystic fibrosis and could be used as predictive markers of severe decline in lung function, allowing earlier intervention and improving health care treatment of CF patients......
Background. In vivo and ex vivo measurements of CFTR function in human cells and tissues is required for screening and monitoring new therapies and phenotyping controversial CFTR genotypes. We set up a technique enabling intestinal stem cells to expand into closed organoids containing crypt-like structures and an internal lumen lined by differentiated cells (Sato et al Gastroenterology 2011) for measuring CFTR function. 2011).
Hypothesis and objectives. This quantitative method could be useful to detect the effects of CFTR genetic variants/rare mutations as well as of drugs targeting specific CFTR. The combination with other functional tests could support controversial diagnosis and drug development.
Background. PTX3 is a component of innate immunity involved in resistance to infections. In previous research projects funded by Fondazione Ricerca Fibrosi Cistica, our group demonstrated the therapeutic potential of PTX3 in Pseudomonas aeruginosa and Aspergillus fumigatus infections and showed that specific PTX3 genetic variants are associated to P. aeruginosa infection in cystic fibrosis patients and in A. fumigatus infection. Both in infectious and inflammatory conditions, PTX3 behaves as a potential diagnostic and prognostic biomarker. PTX3 is under development for the transfer to the clinic as therapeutic tool for opportunistic infections and as diagnostic/prognostic marker in inflammatory conditions.
Hypothesis and objectives. The main objectives were to investigate whether PTX3 plasma levels and PTX3 production by leukocytes could represent a novel diagnostic and prognostic marker of inflammation and infection in CF patients and/or a marker of defective functional activity of leukocytes.
Background. CFTR gene sequencing enhances the detection rate of molecular analysis but it frequently identifies mutations of uncertain significance for which it is difficult to define the pathogenic role without complex functional studies that require in vitro expression of the mutation.
Hypothesis and objectives. Set up and validate the sampling, culture and analysis of nasal epithelial cells using a series of techniques to study the effect of mutations in a novel “ex-vivo” model specifically obtained from the patient bearing the novel mutation.
Background. P. aeruginosa chronic colonization of CF airways is associated to physical changes, characterized by mucus hypersecretion, degradation of extracellular matrix, and high levels of sulphated glycosaminoglycans (sGAG).
Hypothesis and objectives. We hypothesized that during P. aeruginosa chronic lung infection there is an increasing concentration and sulphation of different sGAG, contributing to inflammation and tissue damage. Thus the objectives of this project were to establish the role of sGAG during P. aeruginosa chronic infection and to modulate the vicious inflammation-damage cycle using modified polysaccarides (PS) derived from heparin.
Background. CF airway inflammation is related to genetic CFTR defect and to dysregulation of iron homeostasis and it can precede bacterial infection. Bacterial infections are favored by iron overload thus worsening inflammation and cell damage. A dangerous vicious circle involving inflammation damage, bacterial infection, and dysregulation of iron homeostasis, is established in CF airways.
Hypothesis and objectives. We hypothesize that lactoferrin (Lf), an iron-chelating glycoprotein of the innate immunity of human secretions, could be a key molecule exerting anti-inflammatory and anti-microbial activities interrupting the vicious circle. Our results show that Lf administered by aerosol reduces inflammation and infection in pre-clinical mouse models of acute and chronic lung infection. Since in airways secretions Lf activity can be reduced by proteolytic enzymes, Lf-liposomes have been prepared to protect Lf against proteolytic activity.
Background. The possibility of monitoring the inflammatory response in a IL-8 transgenic WT and CF non invasive animal model has been demonstrated in this project. Experimental support has been provided for the proposal that azithromycin (AZM) acts by inhibiting the synthesis of bacterial metalloproteases (MPs) thus causing a mitigation of the lung inflammation. The possibility of further take advantage of the CF mouse model to analyze the possible anti-inflammatory activity of other antibiotics with mechanism of action similar to that of AZM and that of inhibitors of human metallo-proteases appears of high interest.
Objectives and Methods. The objectives of this new part of the project are i) to use the IL-8 transgenic CF mouse model to monitoring the expected significant reduction of the pro-inflammatory response induced by Pseudomonas MPs by using protease inhibitors such as Galardin and other approved drugs for human use, namely Marimastat and the antibiotic Doxycycline and ii) to analyze the possible anti-inflammatory activity of other antibiotics with mechanism of action similar to that of AZM and used in CF such as claritromycin (CLAR) and tobramycin (TOB).
Background. The sphingolipid mediator ceramide accumulates in chronic inflammatory diseases, such as Cystic Fibrosis and COPD, promoting inflammation and favoring infection. Sphingolipids (SPLs) are essential components of fungal membranes and signaling. Myriocin (Myr) inhibits SPLs synthesis in both human and fungi. We previously demonstrated that Myr loaded nanocarriers (SLN) reduces CF mice lung inflammation, ceramide content and P. aeruginosa infection (Caretti et al BBA 2014).
Hypothesis. We hypothesize that Myr modulation of SPLs synthesis, by lowering ceramide expression level, could reduce A. fumigatus infection and inflammation in both in vitro and in vivo CF models.
Background. The underlying cause of cystic fibrosis (CF) is a mutation in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR), a cyclic AMP (cAMP)-stimulated chloride channel. The ensuing CFTR hypofunction primarily affects the respiratory system, in which the reduced activity of the channel ultimately leads to respiratory failure and death in 80% of CF patients. A number of CFTR correctors and potentiators, restoring membrane expression and cAMP-mediated gating of the channel, have been developed, but their efficacy appears to be unsatisfactory and to strictly rely on elevated concentrations of intracellular cAMP.
Hypothesis and objectives. Given the well-established role of phosphoinositide 3-kinase γ (PI3Kγ) as negative regulator of cAMP, we intend to explore the therapeutic potential of PI3Kγ inhibition in cell-based and pre-clinical models of CF.
Background. Cystic Fibrosis Related Diabetes, the most prevalent comorbidity in older patients, is often anticipated by pulmonary and nutritional decay. Insulin secretory (IS) and resistance (IR) defects are involved but their progression rates, determinants and implications for nutritional, respiratory and clinical outcomes are unclear. We reported that IS and IR are related to respiratory and nutritional status (Project FFC #16/2005)
Hypothesis and objectives. 1) To provide CF population-specific estimates of the time course of IS and IR defects quantified by a model previously developed; 2) To identify risk factors associated to their progression rates and patients negative outcomes
Background. CF carrier screening is a genetic test to identify carriers among reproductive adults, namely the so-called heterozygotes who have one copy of the mutated gene and therefore are not sick, but can transmit the disease to a child if the partner is carrying a similar mutation. Over the past 10-15 years in the western part of the Veneto and Trentino-Alto Adige, the test has been used with caution and offered to those who already had a case of cystic fibrosis in the family. For the past ten years the University of Padua took a different policy and launched a campaign of active offer of CF testing to the general population. As a consequence, in the eastern region thousands of tests have been performed, identifying CF carriers. In the eastern Veneto region, the number of newborns with cystic fibrosis has fallen year after year until almost to zero, while this decrease was lower in the western part of the Veneto and Trentino-Alto Adige.
Background. Mucins are mucus glycoproteins that are kept compact inside mucous cells because their poly-anionic part are shielded by counter-ions. When mucus is secreted towards the airways, mucins expand to permit an efficient mucociliary clearance. This function, that requires rapid removal of cations, seems to depend on the presence of bicarbonate. In CF patients, the defective ion transport across mutated protein causes a simultaneous reduction of fluid, Cl- and bicarbonate secretion leading to a viscous mucus phenotype with frequent infections and bacterial colonization of the airways.
Hypothesis and objectives. We have hypothesized, and aimed to demonstrate, that the consequence of the defective fluid and bicarbonate secretion in CF is an inappropriate structure of the mucus network with deficient rheological properties, and that pharmacological correction of the mutated CFTR may lead to recover the properties of mucus. In addition, we intended to determine whether inhalation of bicarbonate by CF patients can improve the properties of sputum.
Background. Cystic Fibrosis Transmembrane conductance Regulator (CFTR) carrying the F508del mutation is retained in endoplasmic reticulum and fails to traffic to the cell surface where it functions as a protein kinase A (PKA)-activated chloride channel. Pharmacological correctors that rescue the trafficking of F508del CFTR could partially overcome this defect. However, the rescued F508del CFTR still displays reduced chloride permeability. Therefore, a combined administration of correctors and potentiators of the gating defect is considered to be ideal. We previously found that while a short treatment with 4,6,4'-trimethylangelicin (TMA) potentiates the cAMP/PKA-dependent activation of wild-type CFTR, a long preincubation with nanomolar concentrations of TMA significantly rescues the functional expression of F508del CFTR in primary airway cell monolayers homozygous for F508del mutation.
Objectives. The main objectives of this project were 1) identify the mechanism of action by which TMA rescues F508del CFTR activity; 2) analyze the effect of TMA treatment on actin organization and F508del CFTR stabilization on the apical membrane, 3) analyze TMA effect on cAMP/PKA compartmentalization in the membrane region.
Background. Our research is designed to increase the number of useful substances for treating CF patients with ΔF508 mutation. In particular, our efforts are aimed at expanding the sector of "correctors."
Hypothesis and objectives. 1) Finding, through rational systems, molecules interacting with the mutant CFTR or those proteins responsible for its degradation in order to rescue the ΔF508-CFTR. 2) Discovering new potential correctors between natural substances already present in foodstuff or herbal medicines. Such substances could reach the market in less time than the synthetic drugs.
Background. Cystic fibrosis (CF) is due to dysfunctions of CFTR whose most common mutation, F508Δ, localized in NBD1 domain, causes an abnormal conformation of F508Δ-CFTR that is withhold in the endoplasmic reticulum. The small amount of receptor that reaches the plasma membrane exhibits low activity (gating defect).
Hypothesis & objectives. Current CF therapies are aimed at symptoms alleviation, calling for new drugs to rescue F508Δ-CFTR trafficking (correctors) or gating (potentiators), to the identification of which this study has sought to make an original contribution.