F. De Gregorio

A novel treatment of cystic fibrosis acting on-target: cysteamine plus epigallocatechin gallate for the autophagy-dependent rescue of class II-mutated CFTR

We previously reported that the combination of two safe proteostasis regulators, cysteamine and epigallocatechin gallate (EGCG), can be used to improve deficient expression of the cystic fibrosis transmembrane conductance regulator (CFTR) in patients homozygous for the CFTR Phe508del mutation. Here we provide the proof-of-concept that this combination treatment restored CFTR function and reduced lung inflammation (P<0.001) in Phe508del/Phe508del or Phe508del/null-Cftr (but not in Cftr-null mice), provided that such mice were autophagy-competent. Primary nasal cells from patients bearing different class II CFTR mutations, either in homozygous or compound heterozygous form, responded to the treatment in vitro. We assessed individual responses to cysteamine plus EGCG in a single-centre, open-label phase-2 trial. The combination treatment decreased sweat chloride from baseline, increased both CFTR protein and function in nasal cells, restored autophagy in such cells, decreased CXCL8 and TNF-α in the sputum, and tended to improve respiratory function. These positive effects were particularly strong in patients carrying Phe508del CFTR mutations in homozygosity or heterozygosity. However, a fraction of patients bearing other CFTR mutations failed to respond to therapy. Importantly, the same patients whose primary nasal brushed cells did not respond to cysteamine plus EGCG in vitro also exhibited deficient therapeutic responses in vivo. Altogether, these results suggest that the combination treatment of cysteamine plus EGCG acts ‘on-target’ because it can only rescue CFTR function when autophagy is functional (in mice) and improves CFTR function when a rescuable protein is expressed (in mice and men). These results should spur the further clinical development of the combination treatment.

Prediction of acute pancreatitis risk based on PIP score in children with cystic fibrosis

BACKGROUND Currently no tools to predict risk of acute (AP) and recurrent pancreatitis (ARP) in children with cystic fibrosis (CF) are available. We assessed the prevalence of AP/ARP and tested the potential role of Pancreatic Insufficiency Prevalence (PIP) score in a cohort of children with CF. METHODS We identified two groups of children, on the basis of presence/absence of AP/ARP, who were compared for age at diagnosis, clinical features, genotypes and sweat chloride level. PIP score was calculated for each patient. RESULTS 10/167 (5.9%) experienced at least one episode of AP during follow up; 10/10 were pancreatic sufficient (PS). Patients with AP/ARP showed a PIP score ≤0.25 more frequently (6/10) than patients without AP/ARP. The odds ratio (95% CI) of developing pancreatitis was 4.54 (1.22-16.92) for patients with PIP <0.25 when compared with those who have a PIP score >0.25 (p 0.0151). PIP score was correlated with sweat chloride test (p < 0.01). CONCLUSION PIP score, PS status and normal/borderline sweat chloride levels could be applied to predict pancreatitis development in children with CF. ARP could lead to pancreatic insufficiency.

51 Inhaled GSH tolerability in patients with cystic fibrosis (CF)

Objectives: Oxidative stress biomarkers as reactive oxygen species are induced by the sustained activation of neutrophils and other CF-derived defects in the lung of CF patients. Observed defects include an impaired glutathione (GSH) metabolism. Its supplementation may counterbalance the oxidative stress. A randomized, single blind controlled trial of inhaled GSH versus placebo (NCT01450267) is underway in order to evaluate the effect of GSH in cohort of CF patients. We report preliminary data on tolerability to GSH in a pediatric subset of enrolled patients. Methods: 48 CF patients (F 23, age M±DS 13.53 yrs), in regular follow up at the Regional Pediatric CF Center of Naples, were enrolled for RCT. The main inclusion criteria were: CF diagnosis by sweat test and/or two CF causing mutations, age of patients >6 yrs, FEV1% >40% of the predicted value, negative culture for Burkholderia cepacia. Spirometry was performed before and 10 and 60 minutes after GSH inhalation test (10mg/kg, maximum dosage 600mg/dose) in order to assess tolerability. Conclusions: No patients showed a decrease in FEV1% >15% after GSH inhalation as defined in the study design. A statistically significant increase was observed for FEF25−75% after 10 and 60 minutes from inhalation (FEF25−75 M±DS: T0 71.64±33.35 VS T10 76.37±36.73; p< 0.02 and T0 71.64±33.35 VS T60: 80.26±35.25; p< 0.0001) and for FEV1% after 60 minutes from inhalation (FEV1 M±DS: T0 97.90±21.03 VS T60 100.01±19.42; p< 0.01). No side effects were reported. On the basis of these preliminary results we are currently evaluating the efficacy of inhaled GSH on pulmonary function and inflammatory markers within a 12 months therapy.

10 The role of complex alleles in patients with cystic fibrosis and L997F

Objective: The c.3909C>G (N1303K) is the most frequent mutation after the c.1521_1523delCTT (F508del) in Lebanon. A CFTR gene screening was performed on 10 Lebanese and 5 French CF patients carrying c.3909C>G. Parental studies revealed that all individuals have an association of c.3909C>G (exon 24) with c.869+11C>T (intron 7) in cis, inducing a new complex allele (CA). Since both mutations are located in two distant regions, we studied their combined impact by two plasmid constructions. Method: Firstly, we studied the impact of c.3909C>G on alternative splicing (AS), localization and maturation of the CFTR protein. Secondly, to study the impact of c.869+11C>T on AS, an ex-vivo study will be conducted using a plasmid containing the exon 7 and its flanking introns. After transfection of different type of eukaryotic cells, RNA extraction and RT-PCR, cDNA will be sequenced to verify the possible AS. Results: We showed that c.3909C>G affects the localization and the process. The in-silico study is in favor for a possible role of c.869+11C>T in AS. In fact, the used algorithm human splicing Finder showed that c.869+11C>T might affect the AS since it is located near the donor site. Conclusion: The class II mutation, c.3909C>G, induces mild phenotype with few CFTR on membrane. We suggest that the CA c.[869+11C>T;3909C>G], will act like a class I mutation, explaining the severe phenotype in some c.3909C>G patients. So, the classification of a mutation is not sufficient in a clinical approach, as the possible presence of a CA may alter its the specific effect. Therefore, an individualized approach is required to perform proper diagnostic and treatment when available.

225 PIP score could predict the risk of pancreatitis in patients with cystic fibrosis (CF)

Objective: To assess the level of fecal elastase-1 (FE-1) in relation to CFTR gene mutations in patients with cystic fibrosis (CF). Materials and Methods: This study included 49 patients (23 boys) with CF, mean age 8.43±0.99 years. CF was established on the basis of a positive sweat test (Macroduct, Wescor USA) and the results of molecular genetic analysis. Genetic diagnosis was realized for 36 CFTR mutations in genetic laboratories from Germany and France. To detect pancreatic insufficiency (PI) was defined FE-1 (ScheBo Biotech, Germany). The values of FE-1 in the range 0–100mg/g, are characteristic for severe exocrine PI, 100–200mg/g for moderate exocrine PI, and >200mg/g for pancreatic sufficiency. Results: F508del mutation was revealed in 73.33% cases (40.0% − homozygotes), known genotype with other mutations was found in 18.26% cases. Low values of FE-1 (7.89±3.70mg/g) confirmed exocrine PI in 81.64% patients with CF. PI with FE-1 of 2.90±0.78mg/g was confirmed in 100% children with homozygous state of F508del mutation. F508del mutation in heterozygous state caused decreased levels of FE-1 (33.69±19.34mg/g). Only 5.88% patients with CF F508del heterozygous were pancreatic sufficient and other 11.7% cases were moderate PI. Non-F508del CFTR mutations determined FE-1 levels of 218.64±64.87mg/g, with PI in 57.89% patients. Conclusion: The F508del mutations in homozygous state were associated with severe pancreatic exocrine insufficiency in all patients. Heterozygous state of mutation F508del requires association with another mutation and in most cases led to pancreatic enzyme deficiency. Non-F508del CFTR mutations are not obligatory associated with PI.

COMPARISON AMONG AUXOLOGIC INDEXES TO EVALUATE NUTRITIONAL STATUS OF PEDIATRIC CF PATIENTS AND CREATION OF CF GROWTH CHARTS: A MULTI-CENTRIC ITALIAN STUDY

Chronic airway obstruction may determine early hypoxaemia during exercise and sleep even in patients with normal diurnal blood gases. The aim of our study was to establish the occurrence of sleep-disordered breathing (SDB) in a cohort of patients with cystic fibrosis (CF), followed in our Cystic Fibrosis Unit. We therefore studied 55 patients (49% M; 36% children (mean age 4.3±3.9, SaO2 % awake 97.7±1.5); 64% adults (mean age 20.9±4.9, BMI 21.1±3.6, SaO2 % awake 97.1±1.1). All patients, in stable clinical conditions, underwent a nocturnal standard polysomnography (sleep monitoring system Compumedics S-Series) and respiratory functional evaluation during the day. Scoring of respiratory events was performed according to standard criteria. Polysomnographic monitoring revealed the occurrence of SDB, defined as an apnea-hypopnea index (AHI) >5, in 52% of adults and in 56% of children. In adults we found mean AHI 16.8±7.8, mean nocturnal SaO2 93.9%±2.3, mean SaO2 min 88.2%±2.2; in children we found mean AHI 12.5±5.8, mean nocturnal SaO2 93.7%±1.2, mean SaO2 min 89.2%±3.2. In both adults and children we found a similar prevalence of upper airways obstruction (sinusitis and rhinitis), a major cause of SDB, in groups with SDB and without SDB. In adults we found a lower mean FEV1% in the group with SDB as compared to the group without SDB (50% vs 64%, respectively). These data show a high prevalence of sleep-disordered breathing in patients with cystic fibrosis. In these patients SDB occurs in an early phase of life and in adulthood SDB seems to be associated to a lower respiratory function.