Verso un approccio personalizzato per il trattamento del difetto di base in Fibrosi Cistica

Introduction Cystic fibrosis (FC) is a disease caused by mutations that compromise the function of CFTR, a membrane protein with ion channel function permeable to chloride and other anions. FC mutations have been grouped into five different classes depending on the mechanism of action. In the last 15 years, the possibility of a pharmacological approach for the functional recovery of the mutated CFTR protein has become increasingly concrete. However, this approach must be adapted to the different types of mutations. Currently, different types of enhancers (Ivacaftor) and correctors (Lumacaftor, Tezacaftor) of CFTR active on particular types of mutations are already available for use on patients. The introduction of next gen molecules such as Vx445, in association with the aforementioned modulators, has further expanded the population of FC patients eligible for modulatory therapies, as these molecules are active on the most common CF-causing mutation, the F508del, also in single copy. However, a rather large percentage of CF patients with rare genotypes remain (10-25% of the total FC population, given very variable depending on the geographical area), not carriers of the F508del variant, for which at the moment no molecules are available corrective measures on the CFTR function. Objective of the study Our project has proposed to develop an in vitro analysis protocol for the definition of the teratype (in vitro characterization of the response to treatments aimed at modulating CFTR) belonging to Italian FC patients, a particularly important objective given the large number of mutations rare whose class and pharmacological sensitivity are unknown. This analysis was performed on cells collected using the nasal brushing technique. Materials and methods Human nasal cell collection was performed from both nostrils and without local anesthesia, with prior informed consent from the patient. A soft sterile toothbrush was used for the collection, making antero-posterior rotational movements. The toothbrush was then transferred to a 15 mL conical tube containing 6 mL of Ca2 + Mg2 + D-PBS and centrifuged at 1500 rpm for 5 minutes. The pellet was resuspended in 6 ml of medium consisting of LHC9 and RPMI1640 (1: 1) and then pretreated with a collagen solution. To improve growth and increase cell duplication in vitro, the culture medium was supplemented with a mixture of SMAD and ROCK inhibitors, necessary for the reprogramming and immortalization of human epithelial cells. (Palechor-Ceron et al., 2013; Mouet al., 2016). In order to eradicate the contamination, antibiotics and antifungals have been added to the proliferation medium. The cells were then detached by trypsin and re-plated to continue the culture. The cells were cultured in flasks until 90% of the confluence was reached and then sown on Snapwell permeable supports. After 2 hours the LHC / RPMI 1640 medium was replaced with a differentiation medium containing DMEM / Ham's F12 (1: 1), fetal bovine serum (2%), penicillin (100 U / ml), streptomycin (100 \u3bcg / ml) and various hormones and supplements (Galietta et al., 1998). The degree of differentiation of the epithelium was assessed by measuring the difference in potential and transepithelial resistance with an epithelial voltmeter. Subsequently, the soil was added only in the basolateral part; this condition, which lasts for a further 7 days and, favors a more complete differentiation of the epithelium. The effect of individual compounds or combinations of compounds was tested with short circuit current records. In the case of correctors, the epithelia were incubated for 24 hours with the compounds to be tested. The Snapwell porous supports were mounted in the Ussing chamber at 37 \ub0 C. The response to the sequential addition of amiloride, cAMP agonists, VX-770 and inh-17 was measured. Study population 27 patients (F 16, M11) were selected with the diagnosis of Cystic Fibrosis in regular follow-up at our Center with a known CFTR genotype. All 27 selected patients joined the project and provided informed consent. In all cases the sampling procedure was well tolerated (in 3/27 minimal epistaxis with spontaneous resolution) and in 24/27 the cells were isolated in the laboratory without problems. In 2/27 a retest was carried out, in 1 case with success

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

Evidence for alteration of calpain/calpastatin system in PBMC of cystic fibrosis patients

We are here reporting that in peripheral blood mononuclear cells (PBMC) of patients homozygous for F508del-CFTR the calpain\u2013calpastatin system undergoes a profound alteration. In fact, calpain basal activity, almost undetectable in control PBMC, becomes measurable at a significant extent in cells from cystic fibrosis (CF) patients, also due to a 40\u201360% decrease in both calpastatin protein and inhibitory activity. Constitutive protease activation in CF patients' cells induces a large accumulation of the mutated cystic fibrosis transmembrane conductance regulator (CFTR) in the 100 kD + 70 kD split forms as well as a degradation of proteins associated to the CFTR complex. Specifically, the scaffolding protein Na+/H+ exchanger 3 regulatory factor-1 (NHERF-1) is converted in two distinct fragments showing masses of 35 kD and 20 kD, being however the latter form the most represented one, thereby indicating that in CF-PBMC the CFTR complex undergoes a large disorganization. In conclusion, our observations are providing new information on the role of calpain in the regulation of plasma membrane ion conductance and provide additional evidence on the transition of this protease activity from a physiological to a pathological function. \ua9 2011 Elsevier B.V. All rights reserve

Smart Grid and Microgrid Cooperation in a Real Distribution Network under Emergency Conditions

The paper presents a feasibility study regarding the ability of an existing rural microgrid to supply an islanded portion of the MV distribution network. The microgrid is currently off-grid, but in the near future will be connected to the MV distribution grid. Based on load and generation measurements in a six-month period, a statistical analysis is carried out with the aim to check the ability of the microgrid to guarantee such a service whenever requested. Steady state simulations, also taking into account a proposed control strategy of the microgrid energy resources during islanding operation, show that for a significant share of cases the islanding operation is feasible

Calpain inhibition promotes the rescue of F(508)del-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 F(508)del-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 F(508)del-CFTR at the physiological location, in cells treated with a synthetic calpain inhibitor, indicates that F(508)del-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

Targeting the E1 ubiquitin-activating enzyme (UBA1) improves elexacaftor/tezacaftor/ivacaftor efficacy towards F508del and rare misfolded CFTR mutants

The advent of Trikafta (Kaftrio in Europe) (a triple-combination therapy based on two correctors-elexacaftor/tezacaftor-and the potentiator ivacaftor) has represented a revolution for the treatment of patients with cystic fibrosis (CF) carrying the most common misfolding mutation, F508del-CFTR. This therapy has proved to be of great efficacy in people homozygous for F508del-CFTR and is also useful in individuals with a single F508del allele. Nevertheless, the efficacy of this therapy needs to be improved, especially in light of the extent of its use in patients with rare class II CFTR mutations. Using CFBE41o- cells expressing F508del-CFTR, we provide mechanistic evidence that targeting the E1 ubiquitin-activating enzyme (UBA1) by TAK-243, a small molecule in clinical trials for other diseases, boosts the rescue of F508del-CFTR induced by CFTR correctors. Moreover, TAK-243 significantly increases the F508del-CFTR short-circuit current induced by elexacaftor/tezacaftor/ivacaftor in differentiated human primary airway epithelial cells, a gold standard for the pre-clinical evaluation of patients' responsiveness to pharmacological treatments. This new combinatory approach also leads to an improvement in CFTR conductance on cells expressing other rare CF-causing mutations, including N1303K, for which Trikafta is not approved. These findings show that Trikafta therapy can be improved by the addition of a drug targeting the misfolding detection machinery at the beginning of the ubiquitination cascade and may pave the way for an extension of Trikafta to low/non-responding rare misfolded CFTR mutants

Relazione sul Workshop «Paleontologia e biostratigrafia dei sedimenti ammonitiferi toarciano-bajociani in successioni di alto morfostrutturale dell' Appennino umbro-marchigiano: l'area di Monte Nerone. Correlazioni con altre regioni della Tetide occidentale». (Piobbico, 2-14 agosto 1993)

This report is the synthesis of the field work and discussions held during the workshop «PaIaeontology and biostratigraphy of Toarcian-Bajocian Pelagic Carbonate PIatforms ammonitiferous sediments of umbro-marchean Apennines: the Monte Nerone area. Correlations with other western Tethys regions» (Piobbico, CentraI ItaIy, august 2-14, 1993). It presents a preliminary proposition of biohorizons for rock correlation in the investigated area, following the proposais of Italian Commission on Stratigraphy on definition of biostratigraphic units. The sampied «body of rock strata» consists on 113 beds, 25,60 metres thick, where 30 biostratigraphic surfaces (biohorizons) has been recognized

The L467F-F508del Complex Allele Hampers Pharmacological Rescue of Mutant CFTR by Elexacaftor/Tezacaftor/Ivacaftor in Cystic Fibrosis Patients: The Value of the Ex Vivo Nasal Epithelial Model to Address Non-Responders to CFTR-Modulating Drugs

Loss-of-function mutations of the CFTR gene cause cystic fibrosis (CF) through a variety of molecular mechanisms involving altered expression, trafficking, and/or activity of the CFTR chloride channel. The most frequent mutation among CF patients, F508del, causes multiple defects that can be, however, overcome by a combination of three pharmacological agents that improve CFTR channel trafficking and gating, namely, elexacaftor, tezacaftor, and ivacaftor. This study was prompted by the evidence of two CF patients, compound heterozygous for F508del and a minimal function variant, who failed to obtain any beneficial effects following treatment with the triple drug combination. Functional studies on nasal epithelia generated in vitro from these patients confirmed the lack of response to pharmacological treatment. Molecular characterization highlighted the presence of an additional amino acid substitution, L467F, in cis with the F508del variant, demonstrating that both patients were carriers of a complex allele. Functional and biochemical assays in heterologous expression systems demonstrated that the double mutant L467F-F508del has a severely reduced activity, with negligible rescue by CFTR modulators. While further studies are needed to investigate the actual prevalence of the L467F-F508del allele, our results suggest that this complex allele should be taken into consideration as plausible cause in CF patients not responding to CFTR modulators

The role of small intestinal bacterial overgrowth in cystic fibrosis: a randomized case-controlled clinical trial with rifaximin

Background: Scientific literature shows a high prevalence of Small Intestinal Bacterial Overgrowth (SIBO) in patients with Cystic Fibrosis (CF). The role of SIBO in nutritional status and gastrointestinal symptoms in CF is not known. Our aim was to study epidemiology and clinical impact of SIBO while assessing the efficacy of rifaximin in eradicating SIBO in CF patients. Methods: Symptoms questionnaire and Glucose Breath Test (GBT) were given to 79 CF patients (median age 19.6 years; 9.2\u201336.9). Subjects with a positive GBT were enrolled in a randomized controlled trial and received rifaximin 1200 mg for 14 days or no treatment. Questionnaire and GBT were repeated 1 month after the end of treatment or 45 days after the first negative GBT. Results: Out of 79 patients, 25 were affected by SIBO (31.6%) with a significant correlation with lower BMI, SDS-BMI (p < 0.05) and serum albumin levels (p < 0.05), independently from pancreas insufficiency. Twenty-three patients took part in the randomized trial, 13 patients (56.5%) in rifaximin group and 10 patients (43.5%) in control group. Eradication rate of SIBO was 9/10 (90%) in rifaximin group and 2/6 (33.3%) in control group (p < 0.05). In the rifaximin group, gastrointestinal symptom improvement was observed in 4/5 patients aged 64 14 years and in 0/5 patients aged > 14 years (p < 0.05); in 2/6 patients in the control group. Conclusions: CF patients show a high prevalence of SIBO, related to a poorer nutritional status. Rifaximin therapy is well tolerated and the results are promising in terms of efficacy in eradicating small intestinal bacterial overgrowth in C

Rescue by elexacaftor-tezacaftor-ivacaftor of the G1244E cystic fibrosis mutation's stability and gating defects are dependent on cell background

Background: Cystic fibrosis is caused by mutations impairing expression, trafficking, stability and/or activity of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel. The G1244E mutation causes a severe gating defect that it is not completely rescued by ivacaftor but requires the use of a second compound (a co-potentiator). Recently, it has been proposed that the corrector elexacaftor may act also as a co-potentiator. Methods: By using molecular, biochemical and functional analyses we performed an in-depth characterization of the G1244E-CFTR mutant in heterologous and native cell models. Results: Our studies demonstrate that processing and function of the mutant protein, as well as its pharmacological sensitivity, are markedly dependent on cell background. In heterologous expression systems, elexacaftor mainly acted on G1244E-CFTR as a co-potentiator, thus ameliorating the gating defect. On the contrary, in the native nasal epithelial cell model, elexacaftor did not act as a co-potentiator, but it increased mature CFTR expression possibly by improving mutant's defective stability at the plasma membrane. Conclusions: Our study highlights the importance of the cell background in the evaluation of CFTR modulator effects. Further, our results draw attention to the need for the development of novel potentiators having different mechanisms with respect to ivacaftor to improve channel activity for mutants with severe gating defect