Serena Quattrucci

Ataluren for the treatment of nonsense-mutation cystic fibrosis: a randomised, double-blind, placebo-controlled phase 3 trial

BACKGROUND Ataluren was developed to restore functional protein production in genetic disorders caused by nonsense mutations, which are the cause of cystic fibrosis in 10% of patients. This trial was designed to assess the efficacy and safety of ataluren in patients with nonsense-mutation cystic fibrosis. METHODS This randomised, double-blind, placebo-controlled, phase 3 study enrolled patients from 36 sites in 11 countries in North America and Europe. Eligible patients with nonsense-mutation cystic fibrosis (aged ≥ 6 years; abnormal nasal potential difference; sweat chloride >40 mmol/L; forced expiratory volume in 1 s [FEV1] ≥ 40% and ≤ 90%) were randomly assigned by interactive response technology to receive oral ataluren (10 mg/kg in morning, 10 mg/kg midday, and 20 mg/kg in evening) or matching placebo for 48 weeks. Randomisation used a block size of four, stratified by age, chronic inhaled antibiotic use, and percent-predicted FEV1. The primary endpoint was relative change in percent-predicted FEV1 from baseline to week 48, analysed in all patients with a post-baseline spirometry measurement. This study is registered with ClinicalTrials.gov, number NCT00803205. FINDINGS Between Sept 8, 2009, and Nov 30, 2010, 238 patients were randomly assigned, of whom 116 in each treatment group had a valid post-baseline spirometry measurement. Relative change from baseline in percent-predicted FEV1 did not differ significantly between ataluren and placebo at week 48 (-2.5% vs -5.5%; difference 3.0% [95% CI -0.8 to 6.3]; p=0.12). The number of pulmonary exacerbations did not differ significantly between treatment groups (rate ratio 0.77 [95% CI 0.57-1.05]; p=0.0992). However, post-hoc analysis of the subgroup of patients not using chronic inhaled tobramycin showed a 5.7% difference (95% CI 1.5-10.1) in relative change from baseline in percent-predicted FEV1 between the ataluren and placebo groups at week 48 (-0.7% [-4.0 to 2.1] vs -6.4% [-9.8 to -3.7]; nominal p=0.0082), and fewer pulmonary exacerbations in the ataluern group (1.42 events [0.9-1.9] vs 2.18 events [1.6-2.7]; rate ratio 0.60 [0.42-0.86]; nominal p=0.0061). Safety profiles were generally similar for ataluren and placebo, except for the occurrence of increased creatinine concentrations (ie, acute kidney injury), which occurred in 18 (15%) of 118 patients in the ataluren group compared with one (<1%) of 120 patients in the placebo group. No life-threatening adverse events or deaths were reported in either group. INTERPRETATION Although ataluren did not improve lung function in the overall population of nonsense-mutation cystic fibrosis patients who received this treatment, it might be beneficial for patients not taking chronic inhaled tobramycin. FUNDING PTC Therapeutics, Cystic Fibrosis Foundation, US Food and Drug Administrations Office of Orphan Products Development, and the National Institutes of Health.

Dysfunctional CFTR Alters the Bactericidal Activity of Human Macrophages against Pseudomonas aeruginosa

Chronic inflammation of the lung, as a consequence of persistent bacterial infections by several opportunistic pathogens represents the main cause of mortality and morbidity in cystic fibrosis (CF) patients. Mechanisms leading to increased susceptibility to bacterial infections in CF are not completely known, although the involvement of cystic fibrosis transmembrane conductance regulator (CFTR) in microbicidal functions of macrophages is emerging. Tissue macrophages differentiate in situ from infiltrating monocytes, additionally, mature macrophages from different tissues, although having a number of common activities, exhibit variation in some molecular and cellular functions. In order to highlight possible intrinsic macrophage defects due to CFTR dysfunction, we have focused our attention on in vitro differentiated macrophages from human peripheral blood monocytes. Here we report on the contribution of CFTR in the bactericidal activity against Pseudomonas aeruginosa of monocyte derived human macrophages. At first, by real time PCR, immunofluorescence and patch clamp recordings we demonstrated that CFTR is expressed and is mainly localized to surface plasma membranes of human monocyte derived macrophages (MDM) where it acts as a cAMP-dependent chloride channel. Next, we evaluated the bactericidal activity of P. aeruginosa infected macrophages from healthy donors and CF patients by antibiotic protection assays. Our results demonstrate that control and CF macrophages do not differ in the phagocytic activity when infected with P. aeruginosa. Rather, although a reduction of intracellular live bacteria was detected in both non-CF and CF cells, the percentage of surviving bacteria was significantly higher in CF cells. These findings further support the role of CFTR in the fundamental functions of innate immune cells including eradication of bacterial infections by macrophages.

Synergistic Post-Transcriptional Regulation of the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) by miR-101 and miR-494 Specific Binding

microRNAs (miRNAs) are a class of regulatory small non-coding molecules that control gene expression at post-transcriptional level. Deregulation of miRNA functions affects a variety of biological processes also involved in the etiology of several human mendelian and complex diseases. Recently, aberrant miRNA expression has been observed in Cystic Fibrosis (CF), an autosomal-recessive genetic disorder caused by mutations in the CFTR gene, in which a genotype-phenotype correlation is not always found. In order to determine miRNA role in CFTR post-transcriptional regulation, we searched for miR-responsive elements in the CFTR 3′-UTR. In silico analysis, performed using different computational on-line programs, identified some putative miRNAs. Both miR-101 and miR-494 synthetic mimics significantly inhibited the expression of a reporter construct containing the 3′-UTR of CFTR in luciferase assays. Interestingly, miR-101/miR-494 combination was able to markedly suppress CFTR activity by approximately 80% (p<0.001). This is one of the first in vitro studies implicating microRNAs as negative regulators of the CFTR gene expression. miRNA aberrant expression and function might explain the wide phenotypic variability observed among CF patients.

Genetic history of cystic fibrosis mutations in italy. I. Regional distribution

Earlier analysis of the Italian population showed patterns of genetic differentiation that were interpreted as being the result of population settlements going back to pre‐Roman times. DNA disease mutations may be a powerful tool in further testing this hypothesis since the analysis of diseased individuals can detect variants too rare to be resolved in normal individuals. We present data on the relative frequencies of 60 cystic fibrosis (CF) mutations in Italy and the geographical distribution of the 12 most frequent CF mutations screened in 3492 CF chromosomes originating in 13 Italian regions. The 12 most frequent mutations characterize about 73% of the Italian CF chromosomes. The most common mutation, ΔF508, has an average frequency of 51%, followed by N1303K and G542X, both with average frequencies around 5%. Multivariate analyses show that the relative frequencies of CF mutations are heterogeneous among Italian regions, and that this heterogeneity is weakly correlated with the geographical pattern of non‐DNA ‘classical’ genetic markers. The northern regions are well differentiated from the central‐southern regions and within the former group the western and eastern regions are remarkably distinct. Moreover, Sardinia shows the presence of mutation T338I, which seems absent in any other European CF chromosome. The north‐western regions of Italy, characterized by the mutation 1717‐1G→A, were under Celtic influence, while the north‐east regions, characterized by the mutations R1162X, 2183AA→G and 711 + 5G→A, were under the influence of the Venetic culture.

Cystic fibrosis transmembrane conductance regulator (CFTR) expression in human platelets: impact on mediators and mechanisms of the inflammatory response

Inflammatory lung disease is a primary cause of morbidity and mortality in cystic fibrosis (CF). Mechanisms of unresolved acute inflammation in CF are not completely known, although the involvement of cystic fibrosis transmembrane conductance regulator (CFTR) in nonrespiratory cells is emerging. Here we examined CFTR expression and function in human platelets (PLTs) and found that they express a biologically active CFTR. CFTR blockade gave an ∼50% reduction in lipoxin A4 (LXA4) formation during PLT/polymorphonuclear leukocytes (PMN) coincubations by inhibiting the lipoxin synthase activity of PLT 12‐lipoxygenase. PLTs from CF patients generated ∼40% less LXA4 compared to healthy subject PLTs. CFTR inhibition increased PLT‐dependent PMN viability (33.0±5.7 vs. 61.2±8.2%; P=0.033), suppressed nitric oxide generation (0.23±0.04 vs. 0.11±0.002 pmol/108 PLTs; P= 0.004), while reducing AKT (1.02±0.12 vs. 0.71±0.007 U; P=0.04), and increasing p38 MAPK phosphorylation (0.650±0.09 vs. 1.04±0.24 U; P=0.03). Taken together, these findings indicate that PLTs from CF patients are affected by the molecular defect of CFTR. Moreover, this CF PLT abnormality may explain the failure of resolution in CF.—Mattoscio, D., Evangelista, V., De Cristofaro, R., Recchiuti, A., Pandolfi, A., Di Silvestre, S., Manarini, S., Martelli, N., Rocca, B., Petrucci, B., Angelini, D.F., Battistini, L., Robuffo, I., Pensabene, T., Pieroni, L., Furnari, M.L., Pardo, F., Quattrucci, S., Lancellotti, S., Davi, G., Romano, M. Cystic fibrosis transmembrane conductance regulator (CFTR) expression in human platelets: impact on mediators and mechanisms of the inflammatory response. FASEB J. 24, 3970–3980 (2010). www.fasebj.org

Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Allelic Variants Relate to Shifts in Faecal Microbiota of Cystic Fibrosis Patients

Introduction In this study we investigated the effects of the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) gene variants on the composition of faecal microbiota, in patients affected by Cystic Fibrosis (CF). CFTR mutations (F508del is the most common) lead to a decreased secretion of chloride/water, and to mucus sticky secretions, in pancreas, respiratory and gastrointestinal tracts. Intestinal manifestations are underestimated in CF, leading to ileum meconium at birth, or small bowel bacterial overgrowth in adult age. Methods Thirty-six CF patients, fasting and under no-antibiotic treatment, were CFTR genotyped on both alleles. Faecal samples were subjected to molecular microbial profiling through Temporal Temperature Gradient Electrophoresis and species-specific PCR. Ecological parameters and multivariate algorithms were employed to find out if CFTR variants could be related to the microbiota structure. Results Patients were classified by two different criteria: 1) presence/absence of F508del mutation; 2) disease severity in heterozygous and homozygous F508del patients. We found that homozygous-F508del and severe CF patients exhibited an enhanced dysbiotic faecal microbiota composition, even within the CF cohort itself, with higher biodiversity and evenness. We also found, by species-specific PCR, that potentially harmful species (Escherichia coli and Eubacterium biforme) were abundant in homozygous-F508del and severe CF patients, while beneficial species (Faecalibacterium prausnitzii, Bifidobacterium spp., and Eubacterium limosum) were reduced. Conclusions This is the first report that establishes a link among CFTR variants and shifts in faecal microbiota, opening the way to studies that perceive CF as a ‘systemic disease’, linking the lung and the gut in a joined axis.

Effect of a medium dose of ursodeoxycholic acid with or without taurine supplementation on the nutritional status of patients with cystic fibrosis: a randomized, placebo-controlled, crossover trial

Ursodeoxycholic acid administration has been reported to improve cholestasis and inflammatory activity in primary biliary cirrhosis and, in an uncontrolled study, also in young adults with cystic fibrosis (CF) and chronic cholestasis. As an improvement in nutritional status was also observed in these young adult patients, we investigated whether the administration of a medium dose of ursodeoxycholic acid ameliorates the nutritional status of malnourished young adult CF patients with chronic liver disease. The study included 51 patients (27 male patients and 24 female patients; age range, 8–32 years; median, 14) with body mass percentiles < 90%. Patients were randomly assigned to receive either ursodeoxycholic acid (10–12 mg/kg/day) alone or with taurine (18–22 mg/kg/day). Patients were followed in a crossover fashion within each group; 6 months of treatment was randomly alternated with 6 months of placebo. Nine patients dropped out before concluding the study. Liver function tests, nutritional status, and coefficients of fat absorption were determined at entry and after each 6 months of placebo or treatment. Nutritional status and fat absorption were not significantly modified by either treatment. Liver function tests improved after ursodeoxycholic acid administration only in patients with concomitant chronic liver disease. Our findings indicate that 6 months of therapy with a medium dose of ursodeoxycholic acid, either alone or with taurine, does not improve the nutritional status of young malnourished CF patients. Higher doses given for longer periods might be worth investigating.

A multitask biosensor for micro-volumetric detection of N-3-oxo-dodecanoyl-homoserine lactone quorum sensing signal

N-3-oxo-dodecanoyl-homoserine lactone (3OC(12)-HSL) is the main quorum sensing (QS) signal produced by the human pathogen Pseudomonas aeruginosa, a major cause of hard-to-treat nosocomial infections and years-lasting chronic biofilm infections in the lungs of cystic fibrosis (CF) patients. 3OC(12)-HSL-dependent QS is considered a promising target for novel anti-pseudomonads drugs. However, the screening systems employed to date for the identification of QS inhibitors (QSI) were aimed at the identification of inhibitors of 3OC(12)-HSL signaling rather than of the synthesis or the export of this molecule. Moreover, the low concentration of 3OC(12)-HSL in CF sputum has hampered large scale studies aimed at addressing the role of this molecule in the CF lung infection. Here we describe the construction and characterization of PA14-R3, a new whole-cell biosensor for the quantitative detection of 3OC(12)-HSL. PA14-R3 provides fast and direct quantification of 3OC(12)-HSL over a wide range of concentrations (from pM to μM), and proved to be an easy-to-handle, cost-effective and reliable biosensor for high-throughput screening of 3OC(12)-HSL levels in samples of different origin, including CF sputum. Moreover, the specific features of PA14-R3 made it possible to develop and validate a novel high-throughput screening system for QSI based on the co-cultivation of PA14-R3 with the PA14 wild-type strain. With respect to previous screening systems for QSI, this approach has the advantage of being cost-effective and allowing the identification of compounds targeting, besides 3OC(12)-HSL signaling, any cellular process critical for QS response, including 3OC(12)-HSL synthesis and secretion.

Hyaluronic Acid Improves the Tolerability of Hypertonic Saline in the Chronic Treatment of Cystic Fibrosis Patients: A Multicenter, Randomized, Controlled Clinical Trial

UNLABELLED TRIAL DESIGN AND METHODS: Between December 2009 and July 2011, four cystic fibrosis (CF) centers in Italy participated in a randomized, double-blind, controlled clinical trial to test whether 7% hypertonic saline (HS) administered together with 0.1% hyaluronic acid (HA) was better tolerated by patients who previously did not tolerate HS well on its own. Participants were CF patients at least 8 years old, in clinically stable conditions, with forced expiratory volume in 1 sec (FEV1) at least 50% predicted. Forty patients were recruited and randomized to receive either HS or HS plus HA (5 mL to be inhaled over 15 min, twice daily for 28 days). Primary endpoints were cough, throat irritation, salty taste, and overall acceptability, as assessed by each patient on a semiquantitative scale on a diary card. Secondary endpoint was FEV1 change at the end of treatment. Patients were randomized into randomly permuted blocks. The first and last doses were administered in hospital. In between, patients were treated at home. Patients, all caregivers, and the statistician who conducted the analysis (different from the one who generated the random list) were blinded to group assignment. RESULTS Severity of cough, throat irritation, and saltiness were more severe in patients treated with HS alone, both after the first inhalation and over the entire treatment period. Overall pleasantness was rated higher by patients treated with the combination product. All differences were highly significant. There were no changes in FEV1 between the first and last administrations. Five patients did not complete the study. Four patients (two from each group) withdrew because of cough or throat irritation. One more patient from the HS group withdrew because of a respiratory exacerbation at week 3. CONCLUSIONS HS is currently a cornerstone in the treatment of CF patients. The addition of HA to HS reduces the prevalence and severity of cough, throat irritation, and saltiness and may improve compliance in patients who previously did not tolerate HS well on its own. Longer-term studies could further assess the benefit of chronic treatment.

3‐Min step test and treadmill exercise for evaluating exercise-induced asthma

A simple exercise test would be useful for detecting exercise-induced asthma, a common problem in asthmatic children. The current study compared the 3‐min step test with treadmill exercise for evaluating exercise-induced asthma in asthmatic children and assessed whether responses to both tests are influenced by baseline lung function and habitual physical activity. A series of 154 asthmatic children (84 male children; mean age 12.9±0.9 yrs) underwent a 3‐min step-test and treadmill testing on different days within a week at least 24 h apart. Before both tests each subject did spirometry to obtain the baseline forced expiratory volume in one second (FEV1). After both exercise challenges all subjects did serial spirometry and the lowest FEV1 recorded over time was used to calculate the fall in FEV1 expressed as a percentage of the measured pre-exercise (baseline) value (% fall in FEV1) and the area above the FEV1 curve (AAC0–30 min) expressed as a percentage of the pre-exercise value. Changes in both exercise variables were also analysed in percentile subgroups defined by questionnaire answers on habitual physical activity in hours. The mean % fall in FEV1 was significantly higher for treadmill exercise than for the step test (15.0±7.5 versus 11.7±5.9); and the AAC0–30 min was larger for treadmill than for the step test (−261.6±139.9% versus −197.3±105.0% min). In all subgroups defined by habitual physical activity the mean % fall in FEV1 decreased more after treadmill exercise than after the step test. After step test and treadmill exercise no significant correlation was found between % fall in FEV1 and baseline lung function, or between % fall in FEV1 among groups defined by habitual physical activity. Although the 3‐min step test yields a lower % fall in forced expiratory volume in one second (FEV1) and a lower value of the area above the FEV1 curve than treadmill testing, it is a quick, economical, reproducible and portable alternative procedure for identifying exercise-induced asthma in outpatients and epidemiological studies. Baseline lung function and habitual physical activity have no influence on the amount or duration of exercise-induced asthma.