Cystic Fibrosis Pathology Research Articles

Cystic fibrosis-related diabetes is associated with reduced islet protein expression of GLP-1 receptor and perturbation of cell-specific transcriptional programs

Insulin secretion is impaired in individuals with cystic fibrosis (CF), contributing to high rates of CF-related diabetes (CFRD) and substantially increasing disease burden. To develop improved therapies for CFRD, better knowledge of pancreatic pathology in CF is needed. Glucagon like peptide-1 (GLP-1) from islet α cells potentiates insulin secretion by binding GLP-1 receptors (GLP-1Rs) on β cells. We determined whether expression of GLP-1 and/or its signaling components are reduced in CFRD, thereby contributing to impaired insulin secretion. Immunohistochemistry of pancreas from humans with CFRD versus no-CF/no-diabetes revealed no difference in GLP-1 immunoreactivity per islet area, whereas GLP-1R immunoreactivity per islet area or per insulin-positive islet area was reduced in CFRD. Using spatial transcriptomics, we observed several differentially expressed α- and/or β-cell genes between CFRD and control pancreas. In CFRD, we found upregulation of α-cell PCSK1 which encodes the enzyme (PC1/3) that generates GLP-1, and downregulation of α-cell PCSK1N which inhibits PC1/3. Gene set enrichment analysis also revealed α and β cell-specific pathway dysregulation in CFRD. Together, our data suggest intra-islet GLP-1 is not limiting in CFRD, but its action may be restricted due to reduced GLP-1R protein levels. Thus, restoring β-cell GLP-1R protein expression may improve β-cell function in CFRD.

Cystic Fibrosis: Understanding Cystic Fibrosis Transmembrane Regulator Mutation Classification and Modulator Therapies.

A common life-threatening hereditary disease, Cystic Fibrosis (CF), affects primarily Caucasian infants. High sweat-salt levels are observed as a result of a single autosomal mutation in chromosome 7 that affects the critical function of the cystic fibrosis transmembrane regulator (CFTR). For establishing tailored treatment strategies, it is important to understand the broad range of CFTR mutations and their impacts on disease pathophysiology. This study thoroughly investigates the six main classes of classification of CFTR mutations based on their functional effects. Each class is distinguished by distinct molecular flaws, such as poor protein synthesis, misfolding, gating defects, conduction defects, and decreased CFTR expression at the apical membrane. Furthermore, this paper focuses on the emerging field of CFTR modulators, which intend to restore CFTR function or mitigate its consequences. These modulators, which are characterized by the mode of action and targeted mutation class, have the potential to provide personalized therapy regimens in CF patients. This review provides valuable insights into the genetic basis of CF pathology, and highlights the potential for precision medicine methods in CF therapy by thoroughly investigating CFTR mutation classification and related modulators.

FGF receptors mediate cellular senescence in the cystic fibrosis airway epithelium.

The number of adults living with cystic fibrosis (CF) has already increased significantly because of drastic improvements in life expectancy attributable to advances in treatment, including the development of highly effective modulator therapy. Chronic airway inflammation in CF contributes to morbidity and mortality, and aging processes like inflammaging and cell senescence influence CF pathology. Our results show that single-cell RNA sequencing data, human primary bronchial epithelial cells from non-CF and CF donors, a CF bronchial epithelial cell line, and Cftr-knockout (Cftr-/-) rats all demonstrated increased cell senescence markers in the CF bronchial epithelium. This was associated with upregulation of fibroblast growth factor receptors (FGFRs) and mitogen-activated protein kinase (MAPK) p38. Inhibition of FGFRs, specifically FGFR4 and to some extent FGFR1, attenuated cell senescence and improved mucociliary clearance, which was associated with MAPK p38 signaling. Mucociliary dysfunction could also be improved using a combination of senolytics in a CF ex vivo model. In summary, FGFR/MAPK p38 signaling contributes to cell senescence in CF airways, which is associated with impaired mucociliary clearance. Therefore, attenuation of cell senescence in the CF airways might be a future therapeutic strategy improving mucociliary dysfunction and lung disease in an aging population with CF.

SERS Microsensors for the Study of pH Regulation in Cystic Fibrosis Patient-Derived Airway Cultures.

Acidification of the airway surface liquid in the respiratory system could play a role in the pathology of Cystic Fibrosis, but its low volume and proximity to the airway epithelium make it a challenging biological environment in which to noninvasively collect pH measurements. To address this challenge, we explored surface enhanced Raman scattering microsensors (SERS-MS), with a 4-mercaptobenzoic acid (MBA) pH reporter molecule, as pH sensors for the airway surface liquid of patient-derived in vitro models of the human airway. Using air-liquid interface (ALI) cultures to model the respiratory epithelium, we show that SERS-MS facilitates the optical measurement of trans-epithelial pH gradients between the airway surface liquid and the basolateral culture medium. SERS-MS also enabled the successful quantification of pH changes in the airway surface liquid following stimulation of the Cystic Fibrosis transmembrane conductance regulator (CFTR, the apical ion channel that is dysfunctional in Cystic Fibrosis airways). Finally, the influence of CFTR mutations on baseline airway surface liquid pH was explored by using SERS-MS to measure the pH in ALIs grown from Cystic Fibrosis and non-Cystic Fibrosis donors.

Pancreas and islet morphology in cystic fibrosis: clues to the etiology of cystic fibrosis-related diabetes.

Cystic fibrosis (CF) is a multi-organ disease caused by loss-of-function mutations in CFTR (which encodes the CF transmembrane conductance regulator ion channel). Cystic fibrosis related diabetes (CFRD) occurs in 40-50% of adults with CF and is associated with significantly increased morbidity and mortality. CFRD arises from insufficient insulin release from β cells in the pancreatic islet, but the mechanisms underlying the loss of β cell function remain understudied. Widespread pathological changes in the CF pancreas provide clues to these mechanisms. The exocrine pancreas is the epicenter of pancreas pathology in CF, with ductal pathology being the initiating event. Loss of CFTR function results in ductal plugging and subsequent obliteration. This in turn leads to destruction of acinar cells, fibrosis and fatty replacement. Despite this adverse environment, islets remain relatively well preserved. However, islet composition and arrangement are abnormal, including a modest decrease in β cells and an increase in α, δ and γ cell abundance. The small amount of available data suggest that substantial loss of pancreatic/islet microvasculature, autonomic nerve fibers and intra-islet macrophages occur. Conversely, T-cell infiltration is increased and, in CFRD, islet amyloid deposition is a frequent occurrence. Together, these pathological changes clearly demonstrate that CF is a disease of the pancreas/islet microenvironment. Any or all of these changes are likely to have a dramatic effect on the β cell, which relies on positive signals from all of these neighboring cell types for its normal function and survival. A thorough characterization of the CF pancreas microenvironment is needed to develop better therapies to treat, and ultimately prevent CFRD.

1451-P: Peri-Islet Fibrosis in Cystic Fibrosis Is Associated with Altered Alpha-Cell Phenotype

Cystic fibrosis (CF) is caused by CF transmembrane conductance regulator mutations leading to impaired epithelial cell anion transport and thickened pancreatic exocrine secretions. CF-related diabetes commonly ensues through impact of pancreatic exocrine pathology on islet cell mass and function although specific mechanisms remain unclear. Paraffin-embedded blocks obtained post mortem from pancreatic body in 9 donors with CF (6 (67%) male; age: 0-27 years) were studied. Following haematoxylin and eosin staining, CF changes were scored as early, advanced or end-stage and islet microenvironment was evaluated for presence of surrounding fibrosis and adipocyte replacement. Four donors were categorised as ‘early’ pathology with 0 (1) % (median (IQR)) of islets surrounded by uninterrupted fibrosis and 0 (0) % by adipocyte replacement; 1 donor was categorised as ‘advanced’ with 100% fibrosis, 0% adipocyte replacement; and 4 donors were categorised as ‘end-stage’ with 65 (46) % fibrosis, 12 (32) % adipocyte replacement. Presence of glucagon/vimentin co-expressing cells was demonstrated by immunofluorescence staining / confocal microscopy in 1 (25%) donor with ‘early’ CF pathology and in all with more advanced exocrine changes. All islets with altered alpha-cell phenotype had peri-islet fibrosis. In pancreata with <50% fibrosis (11±17% (mean±SD) ; n=6) altered alpha-cell phenotype was seen in 11±14% of islets vs. pancreata with >50% fibrosis (93±7%; n=3) with altered alpha-cell phenotype in 65±10% islets (p<0.001) . Analysis of a chronic pancreatitis resection with incrementally increasing fibrosis from head to tail showed parallel increase in islet proportion with altered alpha-cell phenotype (head: 28%; body: 80%; tail: 78%) . We have demonstrated that peri-islet fibrosis is associated with aberrant islet glucagon/vimentin co-expression providing further evidence for exocrine-endocrine signalling in human pancreatic pathology. Disclosure N.Kattner: None. Y.Al-selwi: None. D.Tiniakos: None. G.Klöppel: None. J.A.Shaw: None. Funding Cystic Fibrosis Trust (SRC 019)

A Single-Cell Atlas of Large and Small Airways at Birth in a Porcine Model of Cystic Fibrosis.

Lack of CFTR (cystic fibrosis transmembrane conductance regulator) affects the transcriptome, composition, and function of large and small airway epithelia in people with advanced cystic fibrosis (CF); however, whether lack of CFTR causes cell-intrinsic abnormalities present at birth versus inflammation-dependent abnormalities is unclear. We performed a single-cell RNA-sequencing census of microdissected small airways from newborn CF pigs, which recapitulate CF host defense defects and pathology over time. Lack of CFTR minimally affected the transcriptome of large and small airways at birth, suggesting that infection and inflammation drive transcriptomic abnormalities in advanced CF. Importantly, common small airway epithelial cell types expressed a markedly different transcriptome than corresponding large airway cell types. Quantitative immunohistochemistry and electrophysiology of small airway epithelia demonstrated basal cells that reach the apical surface and a water and ion transport advantage. This single cell atlas highlights the archetypal nature of airway epithelial cells with location-dependent gene expression and function.

Differential correlation network analysis of rectal transcriptomes reveals cystic fibrosis-related disturbance.

Background: Intestinal pathology in cystic fibrosis (CF) remains mechanistically underexplored. Aim: We hypothesized that differential correlation network analysis of expression data would reveal hub genes of CF-related disturbance in the large bowel. Materials & methods: Transcriptomes of 29 rectal tissue samples were accessed at ArrayExpress (E-GEOD-15568 by Stanke et al.). Results: We identified 279 transcript pairs differentially correlating in CF and controls, including: ESRRA and RPL18 (rCF=0.55; rcontrols=-0.68; padj=1.60×10-100), SRP14 and FAU (rCF=-0.69; rcontrols=0.48; padj=2.99×10-90), SRP14 and GDI2 (rCF=-0.34; rcontrols=0.60; padj=1.05×10-78). The genes related to membrane protein targeting (q=8.34×10-14) and one cluster was clearly linked to male infertility. Conclusion:FAU, SRP14and GDI2 may be involved in a compensatory protein trafficking mechanism in CF rectum, highlighting their potential therapeutic value.

Defective Glyoxalase 1 Contributes to Pathogenic Inflammation in Cystic Fibrosis.

Cystic fibrosis (CF) is an autosomal recessive disorder that affects multiple organs, although a decline in respiratory function represents the major cause of morbidity and mortality. The airways of CF patients are characterized by a chronic inflammatory state to which the receptor for advanced glycation end-products greatly contributes. Glyoxalase 1 (GLO1) is the major enzyme metabolizing methylglyoxal, a potent precursor of advanced glycation end-products. Its role in CF has never been investigated. We herein resorted to murine and human preclinical models of CF to define the contribution of GLO1 to inflammatory pathology. We found that the expression and activity of GLO1, measured by real-time PCR and Western blot or a specific spectrophotometric assay, respectively, are defective in mice and human bronchial cells from CF patients exposed to Aspergillus fumigatus, a common pathogen in CF, but could be restored upon blockade of interleukin-1 receptor signaling by anakinra in mice. This study suggests that GLO1 contributes to pathology in CF and may be potentially targeted to mitigate inflammation.

Increased Plasma YKL-40 Level and Chitotriosidase Activity in Cystic Fibrosis Patients.

We investigated plasma YKL-40 levels and chitotriosidase (CHIT1) activity in patients with cystic fibrosis (CF) lung disease and evaluated clinically relevant factors that may affect their levels. Plasma samples were obtained from pediatric (n = 19) and adult patients (n = 15) during exacerbation, discharge, and stable period of the disease. YKL-40 levels and chitotriosidase activity were measured by enzyme-linked immunosorbent assay and fluorometric assay, respectively. Data were compared with healthy children and adults of similar age. YKL-40 levels of pediatric and adult CF patients at all periods were significantly higher than controls (p < 0.001 and p < 0.05). CHIT1 activities of adult patients at all periods were significantly higher compared to controls (p < 0.05). On the other hand, CHIT1 activities of pediatric CF patients were similar with controls. YKL-40 levels of exacerbation period of adult CF patients were negatively correlated with forced vital capacity (FVC) (r = - 0.800, p = 0.014) and forced expiratory volume in 1s (FEV1) (r = - 0.735, p = 0.008). YKL-40 levels in the exacerbation period of pediatric CF patients were negatively correlated with FVC (r = - 0.697, p = 0.0082) and FEV1 (r = - 0.720, p = 0.006). CHIT1 activity may be a valuable marker of chronic inflammation in adult CF patients who suffer from CF for a longer period compared to pediatric patients. Increased YKL-40 levels in both pediatric and adult patients compared to controls may point to a role in between CF pathology.

The Multifaceted Roles of MicroRNAs in Cystic Fibrosis.

Cystic fibrosis (CF) is a lifelong disorder affecting 1 in 3500 live births worldwide. It is a monogenetic autosomal recessive disease caused by loss-of-function mutations in the gene encoding the chloride channel cystic fibrosis transmembrane conductance regulator (CFTR), the impairment of which leads to ionic disequilibria in exocrine organs. This translates into a chronic multisystemic disease characterized by airway obstruction, respiratory infections, and pancreatic insufficiency as well as hepatobiliary and gastrointestinal dysfunction. Molecular characterization of the mutational heterogeneity of CFTR (affected by more than 2000 variants) improved the understanding and management of CF. However, these CFTR variants are linked to different clinical manifestations and phenotypes, and they affect response to treatments. Expanding evidence suggests that multisystemic disease affects CF pathology via impairing either CFTR or proteins regulated by CFTR. Thus, altering the expression of miRNAs in vivo could constitute an appealing strategy for developing new CF therapies. In this review, we will first describe the pathophysiology and clinical management of CF. Then, we will summarize the current knowledge on altered miRNAs in CF patients, with a focus on the miRNAs involved in the deregulation of CFTR and in the modulation of inflammation. We will highlight recent findings on the potential utility of measuring circulating miRNAs in CF as diagnostic, prognostic, and predictive biomarkers. Finally, we will provide an overview on potential miRNA-based therapeutic approaches.

Transcriptomic analysis of CFTR-impaired endothelial cells reveals a pro-inflammatory phenotype.

Cystic fibrosis (CF) is a life-threatening disorder characterised by decreased pulmonary mucociliary and pathogen clearance, and an exaggerated inflammatory response leading to progressive lung damage. CF is caused by bi-allelic pathogenic variants of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which encodes a chloride channel. CFTR is expressed in endothelial cells (ECs) and EC dysfunction has been reported in CF patients, but a role for this ion channel in ECs regarding CF disease progression is poorly described.We used an unbiased RNA sequencing approach in complementary models of CFTR silencing and blockade (by the CFTR inhibitor CFTRinh-172) in human ECs to characterise the changes upon CFTR impairment. Key findings were further validated in vitro and in vivo in CFTR-knockout mice and ex vivo in CF patient-derived ECs.Both models of CFTR impairment revealed that EC proliferation, migration and autophagy were downregulated. Remarkably though, defective CFTR function led to EC activation and a persisting pro-inflammatory state of the endothelium with increased leukocyte adhesion. Further validation in CFTR-knockout mice revealed enhanced leukocyte extravasation in lung and liver parenchyma associated with increased levels of EC activation markers. In addition, CF patient-derived ECs displayed increased EC activation markers and leukocyte adhesion, which was partially rescued by the CFTR modulators VX-770 and VX-809.Our integrated analysis thus suggests that ECs are no innocent bystanders in CF pathology, but rather may contribute to the exaggerated inflammatory phenotype, raising the question of whether normalisation of vascular inflammation might be a novel therapeutic strategy to ameliorate the disease severity of CF.

Osteoclastogenesis and sphingosine-1-phosphate secretion from human osteoclast precursor monocytes are modulated by the cystic fibrosis transmembrane conductance regulator

Osteopenia and increased fracture rates are well-recognized in patients with cystic fibrosis (CF) disease. In CF pathology, F508del is the most common CFTR mutation, with more than 85% of patients carrying it on at least one allele. The underlying molecular defect in CFTR caused by the F508del-CFTR mutation in osteoclastogenesis, i.e., on the generation and bone-resorption activity of osteoclasts (OCs) from peripheral blood-derived monocytes (PBMCs) remained unexplored. We therefore investigated whether the F508del mutation could affect the osteoclastogenic capacity of PBMCs collected from 15 adult patients bearing the F508del-CFTR mutation, to modulate their bone-resorptive abilities and the level of sphingosine-1-phosphate (S1P) produced by OCs, a key factor in the bone mineral density and formation. In the present study, a severe, defective differentiation of CF-F508del PBMCs to CF-F508del OCs without any significant difference in nuclei number per OC was found compared to non-CF healthy PBMCs from 13 subjects after 7–14-days culture periods. We observed a reduced number of formed non-CF healthy OCs in the presence of a selective inhibitor of CFTR chloride conductance (CFTR-Inh172). Our data regarding OCs resorptive capabilites revealed that a loss of CFTR chloride activity in OCs led to a marked reduction in their trench-resorption mode. A 7-fold increase of the S1P release by CF-F508del OCs was found compared to non-CF healthy OCs after a 21-days culture period. We hypothesize that defective maturation of F508del-OCs precursor monocytes associated with high S1P production in the bone environment might contribute to low bone mineral density observed in the CF population.

Deletion of cftr Leads to an Excessive Neutrophilic Response and Defective Tissue Repair in a Zebrafish Model of Sterile Inflammation.

Inflammation-related progressive lung destruction is the leading causes of premature death in cystic fibrosis (CF), a genetic disorder caused by a defective cystic fibrosis transmembrane conductance regulator (CFTR). However, therapeutic targeting of inflammation has been hampered by a lack of understanding of the links between a dysfunctional CFTR and the deleterious innate immune response in CF. Herein, we used a CFTR-depleted zebrafish larva, as an innovative in vivo vertebrate model, to understand how CFTR dysfunction leads to abnormal inflammatory status in CF. We show that impaired CFTR-mediated inflammation correlates with an exuberant neutrophilic response after injury: CF zebrafish exhibit enhanced and sustained accumulation of neutrophils at wounds. Excessive epithelial oxidative responses drive enhanced neutrophil recruitment towards wounds. Persistence of neutrophils at inflamed sites is associated with impaired reverse migration of neutrophils and reduction in neutrophil apoptosis. As a consequence, the increased number of neutrophils at wound sites causes tissue damage and abnormal tissue repair. Importantly, the molecule Tanshinone IIA successfully accelerates inflammation resolution and improves tissue repair in CF animal. Our findings bring important new understanding of the mechanisms underlying the inflammatory pathology in CF, which could be addressed therapeutically to prevent inflammatory lung damage in CF patients with potential improvements in disease outcomes.

Genomic, transcriptomic, and protein landscape profile of CFTR and cystic fibrosis.

Cystic Fibrosis (CF) is caused most often by removal of amino acid 508 (Phe508del, deltaF508) within CFTR, yet dozens of additional CFTR variants are known to give rise to CF and many variants in the genome are known to contribute to CF pathology. To address CFTR coding variants, we developed a sequence-to-structure-to-dynamic matrix for all amino acids of CFTR using 233 vertebrate species, CFTR structure within a lipid membrane, and 20ns of molecular dynamic simulation to assess known variants from the CFTR1, CFTR2, ClinVar, TOPmed, gnomAD, and COSMIC databases. Surprisingly, we identify 18 variants of uncertain significance within CFTR from diverse populations that are heritable and a likely cause of CF that have been understudied due to nonexistence in Caucasian populations. In addition, 15 sites within the genome are known to modulate CF pathology, where we have identified one genome region (chr11:34754985-34836401) that contributes to CF through modulation of expression of a noncoding RNA in epithelial cells. These 15 sites are just the beginning of understanding comodifiers of CF, where utilization of eQTLs suggests many additional genomics of CFTR expressing cells that can be influenced by genomic background of CFTR variants. This work highlights that many additional insights of CF genetics are needed, particularly as pharmaceutical interventions increase in the coming years.

Transport properties in CFTR\u2212/\u2212 knockout piglets suggest normal airway surface liquid pH and enhanced amiloride-sensitive Na+ absorption

Previous analysis of CFTR-knockout (CFTR−/−) in piglets has provided important insights into the pathology of cystic fibrosis. However, controversies exist as to the true contribution of CFTR to the pH balance in airways and intestine. We therefore compared ion transport properties in newborn wild-type (CFTR+/+) and CFTR-knockout (CFTR−/− piglets). Tracheas of CFTR−/− piglets demonstrated typical cartilage malformations and muscle cell bundles. CFTR−/− airway epithelial cells showed enhanced lipid peroxidation, suggesting inflammation early in life. CFTR was mainly expressed in airway submucosal glands and was absent in lungs of CFTR−/− piglets, while expression of TMEM16A was uncompromised. mRNA levels for TMEM16A, TMEM16F, and αβγENaC were unchanged in CFTR−/− airways, while mRNA for SLC26A9 appeared reduced. CFTR was undetectable in epithelial cells of CFTR−/− airways and intestine. Small intestinal epithelium of CFTR−/− piglets showed mucus accumulation. Secretion of both electrolytes and mucus was activated by stimulation with prostaglandin E2 and ATP in the intestine of CFTR+/+, but not of CFTR−/− animals. pH was measured inside small bronchi using a pH microelectrode and revealed no difference between CFTR+/+ and CFTR−/− piglets. Intracellular pH in porcine airway epithelial cells revealed only a small contribution of CFTR to bicarbonate secretion, which was absent in cells from CFTR−/− piglets. In contrast to earlier reports, our data suggest a minor impact of CFTR on ASL pH. In contrast, enhanced amiloride-sensitive Na+ absorption may contribute to lung pathology in CFTR−/− piglets, along with a compromised CFTR- and TMEM16A-dependent Cl− transport.

132 Introduction of F508del human mutation into the CFTR gene of sheep fetal fibroblasts using CRISPR/Cas9 ribonucleoprotein

Cystic fibrosis (CF) is an autosomal recessive genetic disease that affects over 30 000 people in the United States and is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The CFTR protein is a cAMP-regulated C− channel responsible for regulation of anion transport, primarily in the epithelial cells. We have previously generated a sheep model of CF by genetically inactivating the CFTR gene (Fan et al. 2018 JCI Insight 3, e123529). The newborn CFTR −/− sheep develops severe disease consistent with CF pathology in humans. The CF model is extremely valuable for understanding the developmental aspects of CF disease, as sheep have been used extensively in the study of human fetal growth and development. Sheep, like humans, typically give birth to only one or two offspring in each pregnancy, which make them more suitable than many other species for testing prenatal gene-editing treatments. Thus, in this new study, we are working on the generation of F508del sheep CF model. The F508del mutation was chosen because it is the most common mutation in the human CFTR gene (~70%). This mutation is characterised by the deletion of the CTT nucleotides, which ultimately deletes the phenylalanine residue at position 508. The F508del mutation causes misfolding of the CFTR protein, which is further degraded by proteases. Even though several CFTR modulators are available, they are not effective in all patients. Additionally, they cannot reverse deleterious prenatal CF manifestations. Hence, this model will be valuable for evaluating both prenatal drug and gene therapies. Here, we used a CRISPR/Cas9 gene-editing approach to introduce the F508del mutation into the sheep genome. We designed an sgRNA targeting exon 11 of the sheep CFTR gene using the Benchling software (https://benchling.com/academic). The sgRNA was synthesised by Synthego and Cas9 purchased from ThermoFisher. Using the Lonza-4D-Nucleofector system, Cas9/sgRNA ribonucleoprotein complex was transfected into sheep fetal fibroblasts (SFFs), along with 100bp single-stranded oligodeoxynucleotide, flanking the F508del mutation, for the homology-directed repair. The transfected cells were subsequently cultured in Dulbecco's modified Eagle's medium, supplemented with 15% fetal bovine serum and 1% penicillin, and incubated at 38.5°C. Two days post-transfection, SFFs were seeded individually into five 96-well plates by limited dilution. After seven days, the individual colonies were expanded into 24-well plates and cultured for three more days. A total of 56 single-cell-derived SFF colonies were isolated. The presence of F508del mutation was confirmed by amplifying the PCR products of the exon 11 flanking the mutation site and subjecting each amplicon to Sanger sequencing. The sequencing results indicated that the indels (insertion/deletion) were introduced in 49 out of 56 (87.5%) of the colonies, and four (7.14%) of them were confirmed to have biallelic F508del mutations based on sequencing peaks. Therefore, we successfully introduced the F508del mutation in SFFs that will be used for the production of F508del CF sheep by somatic cell nuclear transfer.

Growth and Nutrition in Cystic Fibrosis.

Optimal nutrition support has been integral in the management of cystic fibrosis (CF) since the disease was initially described. Nutritional status has a clear relationship with disease outcomes, and malnutrition in CF is typically a result of chronic negative energy balance secondary to malabsorption. As the mechanisms underlying the pathology of CF and its implications on nutrient absorption and energy expenditure have been elucidated, nutrition support has become increasingly sophisticated. Comprehensive nutrition monitoring and treatment guidelines from professional and advocacy organizations have unified the approach to nutrition optimization around the world. Newborn screening allows for early nutrition intervention and improvement in short- and long-term growth and other clinical outcomes. The nutrition support goal in CF care includes achieving optimal nutritional status to support growth and pubertal development in children, maintenance of optimal nutritional status in adult life, and optimizing fat soluble vitamin and essential fatty acid status. The mainstay of this approach is a high calorie, high-fat diet, exceeding age, and sex energy intake recommendations for healthy individuals. For patients with exocrine pancreatic insufficiency, enzyme replacement therapy is required to improve fat and calorie absorption. Enzyme dosing varies by age and dietary fat intake. Multiple potential impediments to absorption, including decreased motility, altered gut luminal bile salt and microbiota composition, and enteric inflammation must be considered. Fat soluble vitamin supplementation is required in patients with pancreatic insufficiency. In this report, nutrition support across the age and disease spectrum is discussed, with a focus on the relationships among nutritional status, growth, and disease outcomes.

Efficacy of model-based iterative reconstruction in cystic fibrosis assessment using CT

To evaluate the efficacy of model-based iterative reconstruction (MBIR) constructed non-enhanced ultra-low dose (ULD) computed tomography (CT) of the chest to evaluate cystic fibrosis (CF) pathology. ULD-CT was compared with chest X-ray and standard adaptive statistical iterative reconstructed (ASIR) non-enhanced low-dose CT (LD-CT). The effective radiation dose was calculated from the recorded dose-length product (DLP) values and compared between the two CT methods. Identification of pathology was compared between ULD-CT and chest X-ray. It was hypothesised that ULD-CT would be superior to chest X-ray in the identification of CF pathology at lower doses than LD-CT. The mean effective radiation dose of ULD-CT was 0.073 mSv, comparable to one chest X-ray, which was a 94% reduction compared to LD-CT. Compared to chest X-ray, ULD-CT detected on average, 2.3 more regions of bronchiectasis per study and better delineated varicose and cystic forms of bronchiectasis (p≤0.0001). ULD-CT identified four-times more mucous plugging than chest X-ray (p<0.000001) and twice the amount of consolidation (p=0.0002). ULD-CT is superior to chest X-ray in quantifying CF disease and achieves remarkable radiation doses significantly lower than LD-CT, comparable to one chest radiograph. The present results suggest that MBIR-constructed ULD-CT is an effective imaging technique for CF surveillance, with potential applications in other disease settings.

Cell-Selective Regulation of CFTR Gene Expression: Relevance to Gene Editing Therapeutics.

The cystic fibrosis transmembrane conductance regulator (CFTR) gene is an attractive target for gene editing approaches, which may yield novel therapeutic approaches for genetic diseases such as cystic fibrosis (CF). However, for gene editing to be effective, aspects of the three-dimensional (3D) structure and cis-regulatory elements governing the dynamic expression of CFTR need to be considered. In this review, we focus on the higher order chromatin organization required for normal CFTR locus function, together with the complex mechanisms controlling expression of the gene in different cell types impaired by CF pathology. Across all cells, the CFTR locus is organized into an invariant topologically associated domain (TAD) established by the architectural proteins CCCTC-binding factor (CTCF) and cohesin complex. Additional insulator elements within the TAD also recruit these factors. Although the CFTR promoter is required for basal levels of expression, cis-regulatory elements (CREs) in intergenic and intronic regions are crucial for cell-specific and temporal coordination of CFTR transcription. These CREs are recruited to the promoter through chromatin looping mechanisms and enhance cell-type-specific expression. These features of the CFTR locus should be considered when designing gene-editing approaches, since failure to recognize their importance may disrupt gene expression and reduce the efficacy of therapies.