Development of a humanised cystic fibrosis mouse model

Animal models are widely used for the study of the pathophysiology of human diseases and the efficacy of new therapies. In the context of cystic fibrosis (CF), several animal (mouse, pig or ferret) models have been developed. However, they show little or no severe respiratory phenotype typically associated with the high morbidity and mortality of the human disease. Based on the observation that the F508del mutation of CFTR leads to a more severe CF phenotype for the human CFTR (hCFTR) than the murine CFTR (mCFTR), this project aims at developing a CF mouse model expressing the hCFTR gene. A KO-CFTR mouse line was generated using the CRISPR/Cas9 genome editing system. In this aim, a guide RNA targeting the exon 1 was used to produce mutations in the mCFTR gene. In parallel, a wild-type (WT) or mutated (F508del mutation) hCFTR overexpressing mouse line was created by transgene addition. For this purpose, a plasmid construct (pcDNA3.1-hCFTR, CMV promoter) was designed, amplified, purified and microinjected into C57BL/6NCrl mouse embryos. Regarding the development of the "knock-out" mCFTR line, 33 mice were generated. After comparing the WT mouse exon 1 sequences with those of the generated mice, 7 mice were shown to have at least one mCFTR mutation. Regarding the humanized mouse model, the constructs of pcDNA3.1-hCFTR for WT and for F508del were injected into 178 and 115 embryos of C57BL/6NCrl mice respectively. Twenty-seven newborns were obtained for the WT group and 14 for the F508del group. Of these, 5 transgenic mice (2 WT-hCFTR and 3 F508del-hCFTR) were obtained. Biochemical studies using immuno-tagging and western blotting are ongoing to check whether the generated founding mice of the KO-CFTR line are indeed devoid of the expression of the CFTR protein. Quantification of the WT or the F508del mutated CFTR is to be analyzed in the founding mice of the humanized lines to check protein overexpression. When these data were verified, the founding lines will be used to generate lineages of the humanized CF mouse model, which will then be characterized from organ and tissue phenotypic point of view. The model will hopefully allow a better understanding of the genotype/phenotype differences between the hCFTR and the mCFTR

Dysfunction of Anoctamin 1 (Ano1) in F508del mouse pancreatic b-cell

Cystic Fibrosis (CF) is complicated by a form of diabetes (CFRD) mainly characterized by a gradual decline in insulin secretion. Although not apparent at birth, its prevalence increases with age, complicating CF in about 50% of the patients > 35 years, worsening their condition and increasing mortality. Yet little is known on the underlying mechanisms and the functional defects of CFRD within the β-cell. Interestingly, the Anoctamin 1 (Ano1) channel, a calcium-activated chloride channel, has been shown to contribute to insulin secretion and its activity appears to be linked to CFTR function. First Ano1 is expressed by mouse pancreatic b-cells as observed in rat and human b-cells. By immunohistochemistry we localized Ano1 in pancreatic islets and by patch clamp analysis we detected a Ca2+-activated anion channel. Increase in extracellular glucose is a pacemaker inducing cyclic oscillations in b-cell membrane potential. After an initial depolarization, due to closure of the KATP channels, alternating phases of depolarization (active) and repolarization (silent) phases can be observed partially explained by the gating of Ano1. During the active phases, spikes then repeated bursts of action potentials (AP) occur witnessing that the threshold for opening voltage-dependent Ca2+ channels has been reached, allowing Ca2+ entry and triggering insulin release. By patch clamp, we studied the fluctuations of the b-cell membrane potential induced by glucose, 11.1 mM. Their pattern was similar in young F508del mice (1 year) when compared to matched WT mice. In these F508del mice, the resting membrane potential was hyperpolarized (-81 vs -74 mV in WT; p=0.0002) and the number of AP/30 min was drastically reduced (811 vs 1394 in WT; p=0.01) with the bursting second phase peak amplitude partially repolarized (-29 vs -15 mV in WT; p=0.009). Moreover the duration of the silent phase separating the first spiking active phase and the second bursting continuous phase was increased (715 vs 275 sec in WT; p=0.025). The Ano1 potentiator bromophenyltetrazolbenzamide (Fact) was used to further study Ano1 contribution, first in WT mice. When Fact was added simultaneously with the increased glucose concentration, no silent phase was anymore observed and the number of AP/30 min was increased from 1394 to 3475 (p=0.0002). When Fact was added after the increase in glucose concentration, the AP events/30 min increased from 1728 to 3240 (p=0.01) while the AP peak was further depolarized (-16 vs -21 mV; p=0.007). Thus in F508del as in WT b-cell, Fact increased AP number and at least partially corrected the decreased electrical activity of F508del b-cells. Insulin secretion by isolated islets is clearly increased by Fact in WT mice. Its effect in aged F508del mice was therefore examined here. In conclusion, b-cell from F508del mice exhibit membrane hyperpolarization and as the mice aged, dysfunctional Ano1 gating appears leading to reduced number of AP/30 min. Fact appears to correct Ano1 dysfunction in F508del mice and thus is of potential interest in CFRD

Immunisation in the current management of cystic fibrosis patients.

Although no special recommendations exist, clearly patients with cystic fibrosis (CF) can benefit from immunisation. We reviewed the literature regarding vaccination in CF and other chronic diseases. CF subjects should follow national immunisation programmes without delay to obtain optimal vaccination coverage. Indeed they may escape normal programmes due to frequent hospital admissions and school absenteeism and may be more at risk to get "vaccine-controlled" diseases at any age. There is no uniform European immunisation schedule for basic infant and childhood vaccines or for vaccines against hepatitis A (HAV) and B (HBV), varicella (VZ) and booster vaccinations. HAV and HBV vaccination is appropriate in CF as recommended in general for patients with chronic liver disease (CLD). Varicella (VZ) vaccination is not recommended in all European countries. There are no recent data about possible worsening of pulmonary status following VZ in CF, but it is known to cause pulmonary damage in non-CF adults and to be potentially fatal post transplantation and during steroid treatment. Therefore it is recommended at least for seronegative adolescents and transplant candidates. Influenza vaccine is recommended annually for CF patients aged > or =6 months. Pneumococcal vaccine is generally indicated for CF patients. RSV infection might play a role in the initial Pseudomonas colonization and the decline in pulmonary function. However no RSV vaccine is available at present. There are no recommendations for palivizumab in CF as an alternative but expensive prophylaxis. Anti-bacterial vaccinations protecting directly against Pseudomonas aeruginosa colonisation are promising for the future, potential candidates are currently being assessed in phase III clinical trials. More studies are needed to complete recommendations especially for CF adults and transplant candidates.Journal ArticleSCOPUS: re.jinfo:eu-repo/semantics/publishe