L. Minicucci

Liver disease as risk factor for cystic fibrosis‐related diabetes development

Aim: To evaluate clinical and genetic factors, besides pancreatic insufficiency, associated with increased risk of cystic fibrosis‐related diabetes.

Impact of inhaled corticosteroids on the risk of early Pseudomonas aeruginosa acquisition in cystic fibrosis.

Aim: To investigate the role of inhaled corticosteroids (IC) on the risk of Pseudomonas aeruginosa acquisition before the age of 10 y in cystic fibrosis (CF) patients. Methods: For each subject the cumulative dose kg‐1 of IC received for each year of age was calculated until the end of follow‐up. The age at CF diagnosis, the nutritional status (NS) and the number of respiratory exacerbations (RE) were used as surrogate measures for the severity of CF. Results: A total of 83 patients (40 M, 43 F) entered the study. Their median length of follow‐up was 4.4 y, for a total of 386 person‐years at risk. Twenty‐three patients acquired P. aeruginosa at a median age of 4.6 y (range 0.4‐9.9 y). The estimated survival without P. aeruginosa acquisition was 65% at 10 y of age. The effect of different risk factors (IC, NS, RE and age at CF diagnosis) on the probability of P. aeruginosa acquisition was evaluated: none of them was significantly associated with the risk of P. aeruginosa acquisition. In particular, patients receiving very high cumulative doses of IC (4th quartile) had a non‐significantly increased risk of P. aeruginosa acquisition compared with those receiving low doses of IC (1st quartile) (hazard ratio = 1.73, 95% confidence limits 0.40‐7.38).

Diabetes mellitus after kidney transplantation in pediatric recipients

We read with great interest the paper by Greenspan et al. [1] on the increased incidence of post-transplant diabetes mellitus (PTDM) in kidney allograft recipients. The authors emphasize the central role of a firstand second-degree family history of type 2 diabetes mellitus (DM), use of tacrolimus in immunosuppressive therapy, and hyperglycemia in the first 2 weeks after transplantation as significant risk factors for PTDM. Eleven children (4 males, 7 females) were identified with PTDM in a retrospective review of 101 pediatric kidney allograft recipients (10.9%), referred to the G. Gaslini Institute, Genoa, Italy in the last 5 years. As in the study of Greenspan et al. [1] PTDM was clinically defined by a serum glucose level higher than 200 mg/dl on more than one occasion and the need for subcutaneous insulin therapy. The presentation of PTDM was asymptomatic hyperglycemia during routine laboratory evaluation in all patients. None presented diabetic ketoacidosis. No patient was on human growth hormone after transplantation. The mean age at the time of transplant for the 11 patients with PTDM was 16.78 years (range 8.34– 22.79 years). The mean age of controls was 15.00 years (range 2.8–23.1 years). The mean time from transplantation to diagnosis of PTDM was 5.6 months (range 0– 33 months). All subjects received glucocorticoids, with either cyclosporin (CsA) or tacrolimus, as part of their immunosuppressive regimen. Only 1 of the 11 patients with PTDM had parameters consistent with obesity (body mass index of 30.2 kg/m2 compared with a 50th percentile normal value of 21.38 kg/m2). Islet cell autoantibodies, such as insulin and anti-glutamic acid decarboxylase autoantibodies, were negative in all patients tested. A family history of type 2 diabetes was present in 8 of 11 PTDM patients (72.7%); 2 of 11 (18%) had a first-degree relative and 6 of 11 (54.5%) a second-degree relative with type 2 DM. The 11 PTDM patients were treated with three or four daily insulin injections; 6 of 11 patients remained on insulin therapy at 24 months post PTDM diagnosis (range 11–45 months), while in 5 of 11, insulin therapy was withdrawn after a mean of 4.6 months (range 2– 10 months). We confirmed that the immunosuppressive regimen had an effect on the risk for PTDM. In our cohort, the frequency of PTDM was higher in the group receiving tacrolimus compared with the group receiving CsA [8/30 (26.7%) vs. 3/71 (4.2%), respectively, P<0.001]. At the time of PTDM diagnosis, the patients in the tacrolimus group were receiving a mean dose of 0.22€0.09 mg/kg per day (range 0.08–0.35), with mean trough levels of 12.06€2.8 ng/ml (range 9.1–16.2). No difference was observed in the PTDM frequency according to the steroid load. According to present knowledge, the dose should not exceed 0.3 mg/kg per day and trough levels after the acute post-transplant phase should not exceed 8 ng/ml to prevent the development of diabetes. On the basis of the evidence regarding the role of tacrolimus in inducing PTDM, we agree with Greenspan et al. [1] that it would be advisable to routinely screen for glucose tolerance all children awaiting renal transplantaM. Cotellessa · L. Cresta · L. Minicucci · R. Lorini Department of Pediatrics, University of Genoa, Genoa, Italy

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.

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.