Federico Cresta

Calpain Inhibition Promotes the Rescue of F508del-CFTR in PBMC from Cystic Fibrosis Patients

A basal calpain activity promotes the limited proteolysis of wild type (WT) cystic fibrosis conductance regulator (CFTR), inducing the internalization of the split channel. This process contributes to the regulation in the level of the active CFTR at the plasma membranes. In peripheral blood mononuclear cells (PBMC) from 16 healthy donors, the inhibition of calpain activity induces a 3-fold increase in the amount of active WT CFTR at the plasma membranes. Instead, in PBMC from cystic fibrosis (CF) patients, calpain activity is expressed at aberrant levels causing the massive removal of F508del-CFTR from the cell surface. In these patients, the inhibition of such abnormal proteolysis rescues physiological amounts of active mutated CFTR in 90% of the patients (25 over 28). The recovery of functional F508del-CFTR at the physiological location, in cells treated with a synthetic calpain inhibitor, indicates that F508del-CFTR folding, maturation, and trafficking operate in CF-PBMC at significant rate. Thus, an increase in the basal calpain activity seems primarily involved in the CFTR defect observed in various CF cells. Furthermore, in CF-PBMC the recovery of the scaffolding protein Na+/H+ exchanger regulatory factor 1 (NHERF-1), occurring following inhibition of the aberrant calpain activity, can contribute to rescue CFTR-functional clusters.

A long-term follow-up of residual mass neuroblastoma in a patient with cystic fibrosis

Purpose To report a long-term follow-up of a young woman affected by cystic fibrosis (CF) with a residual retroperitoneal mass of neuroblastoma (NBL) after treatment. Case report We reviewed the patients database and analysed a 20-year follow-up by considering pulmonary exacerbation, nutritional condition, pulmonary function (forced expiratory volume in 1 s), microbiological data and residual retroperitoneal mass volume. We observed stable pulmonary and nutritional conditions. No variation was found in the residual retroperitoneal mass volume. Discussion We report this case of a patient with CF with previous NBL because such a long time of follow-up of a NBL with a stable retroperitoneal remaining tumour is uncommon and needs to be reported. Multidisciplinary management has been crucial in this case because of the presence of concomitant diseases and consequently, differential diagnosis challenges.

Macrophage activation syndrome induced by A/H1N1 influenza in cystic fibrosis

Bacterial respiratory infections have an important impact on the development and progression of pulmonary disease in cystic fibrosis (CF). Viral infections are possible triggers of acute deterioration in the clinical status of CF patients. Macrophage activation syndrome (MAS) is a life‐threatening complication of rheumatic disease characterized by pancytopenia, hepatitis, hyperferritinemia, coagulopathy, and neurologic symptoms. This syndrome is thought to be caused by the activation and uncontrolled proliferation of T lymphocytes and well‐differentiated macrophages, leading to widespread hemophagocytosis and cytokine overproduction. Here, we report the case of a boy affected by CF who developed MAS triggered by pandemic H1N1 influenza; good clinical response was obtained through high dose prednisone treatment. Pediatr Pulmonol. 2014; 49:E10–E12.

Cystic Fibrosis and Fertility

In the last 20 years, the prognosis of cystic fibrosis (CF) has slightly increased and nowadays more than 50% of CF patients are adults. An obvious consequence of this deep change is the increasing question about fertility in both males and females. Almost 97% of male CF patients are infertile, having significant anatomical abnor‐ malities of the reproductive tract, in most cases a congenital bilateral absence of the vas deferens (CBAVD); even if anatomical defect plays an important role, cystic fibrosis transmembrane conductance regulator (CFTR) is directly involved in many aspects of male reproduction, with well-known consequences in spermatozoa capacitation and bicarbonate secretion. Actually, male CF patients can become parents with assisted fertilization techniques: intracytoplasmic sperm injection (ICSI), currently the most used fertilization treatment worldwide, has dramatically improved the assisted reproduction outcomes for men with obstructive azoospermia. Most CF women have a normal reproductive tract and may be able to conceive spontaneously, but multifactorial fertility problems can affect them also: the main cause could be the difficult transport of sperm through the female reproductive tract, secondary to thick secretions, but also lung function and nutritional status at the time of conception significantly influence their fertility.

34 Alterations in cystic fibrosis peripheral blood mononuclear cells are induced by an increase in intracellular calcium concentration

Objectives: Mucins are abundant glycoproteins in human lungs. It is wellestablished that airway mucin O-glycosylation is aberrant in cystic fibrosis (CF) and involved directly or indirectly in the pathogenesis. As the first, we here investigate and establish the link between protein N-glycosylation and CF. Methods: N-glycosylation of crudely isolated sputum non-mucin proteins of five CF and five non-CF individuals with and without pulmonary infection was mapped using liquid chromatography and tandem mass spectrometry based glycomics and glycoproteomics. The resulting glycoprofiles were qualitatively and quantitatively compared between the patient groups. Results: Despite covering different patient characteristics including CFTR genotypes, age, gender and microbial flora, the sputum N-glycomes showed little interperson and longitudinal variation within the patient groups. Inter-group comparisons revealed that lung infection, primarily caused by P. aeruginosa, extensively altered the CF sputum N-glycosylation to paucimannoside rich profiles with simultaneous over-sialylation/fucosylation and under-bisecting GlcNAcylation of the complex N-glycans. The CF genotype in itself yielded fewer sputum N-glycome alterations by slightly increasing the abundance of paucimannose N-glycans in CF relative to pathogen-infected non-CF individuals. Conclusion: We have established that the absence of a functional CFTR and more importantly the bacterial infection of the respiratory tract of CF patients affect their sputum N-glycosylation phenotype. This study provides an important platform to further understand the complex cellular and molecular environment of the respiratory tract in CF. 34 Alterations in cystic fibrosis peripheral blood mononuclear cells are induced by an increase in intracellular calcium concentration M. Averna1, M. Pedrazzi1, F. Cresta2, L. Minicucci2, E. Melloni1. 1University of Genoa, Department of Experimental Medicine − Biochemistry Section, Genova, Italy; 2IRCCS Istituto Giannina Gaslini, Cystic Fibrosis Center, Genova, Italy

162 Serum amyloid A as a useful serum marker of lung inflammation in cystic fibrosis

Cystic Fibrosis (CF) is characterized by an aggressive inflammatory response. One important marker of CF lung disease, the pleiotropic cytokine TGF-beta, is negatively regulated by E3-ubiquitin ligases, which have been found to be dysregulated in previous studies of F508del-CFTR-related gene expression. To understand the role of E3-ubiquitin ligases in CF, we studied the effects of: 1. F508del mutation and 2. exposure to TGF-beta and TNF-alpha cytokines on mRNA and protein expression of the E3-ubiquitin ligases SMURF1, SMURF2 and NEDD4L in polarized CF bronchial epithelial cell models. Using real-time quantitative PCR, we demonstrated that the F508del mutation is not sufficient to induce significant differential mRNA expression of E3-ubiquitin ligases. However, the F508del-CFTR genotype altered the responsiveness of E3ubiquitin ligases to both inflammatory cytokines. Our results showed that both TGF-beta and TNF-alpha increased the expression of SMURF2 mRNA in F508delCFTR CFBE cells, suggesting an up-regulation of this E3-ubiquitin ligase under inflammatory status. In addition, this increased expression was consistent with an observed decrease in SMAD2 and SMAD3 mRNA expression. These results suggest that increased expression of E3-ubiquitin ligases in CF under inflammation could be partly responsible for the increased pro-inflammatory mediators that characterize CF disease, via an inhibition of the Smad-dependent anti-inflammatory effects of TGF-beta. Our preliminary data enlighten the potential implications of differential expression of enzymes involved in ubiquitination on modulation of CF inflammatory responses. Supported by PEst-OE/BIA/UI4046/2011 grant BioFIG.