Cystic Fibrosis Transmembrane Conductance Receptor Research Articles

Pharmacological inhibition of CFTR attenuates nonalcoholic steatohepatitis (NASH) progression in mice

Nonalcoholic steatohepatitis (NASH) is considered a pivotal stage in nonalcoholic fatty liver disease (NAFLD) progression and increases the risk of end-stage liver diseases such as fibrosis, cirrhosis, and hepatocellular carcinoma (HCC). The etiology of NASH is multifactorial and identifying reliable molecular players has proven difficult. Presently, there are no approved drugs for NASH treatment, which has become a leading cause of liver transplants worldwide. Here, using public human transcriptomic NAFLD dataset, we uncover Cystic fibrosis transmembrane conductance receptor (CFTR) as a differentially expressed gene in the livers of human NASH patients. Similarly, murine Cftr expression was also found to be upregulated in two mouse models of diet-induced NASH. Furthermore, the pharmacological inhibition of CFTR significantly reduced NASH progression in mice and its overexpression aggravated lipotoxicity in human hepatic cells. These results, thus, underscore the involvement of murine Cftr in the pathogenesis of NASH and raise the intriguing possibility of its pharmacological inhibition in human NASH.

Nontuberculous Mycobacterial Lung Disease in the Patients with Cystic Fibrosis-A Challenging Diagnostic Problem.

Background: Cystic fibrosis (CF) is an autosomal, recessive genetic disorder, caused by a mutation in the cystic fibrosis transmembrane conductance receptor regulator (CFTR) gene. Dysregulated mucous production, and decreased bronchial mucociliary clearance, results in increased susceptibility to bacterial and fungal infections. Recently, nontuberculous mycobacteria (NTM) infections were identified as an emerging clinical problem in CF patients. Aim: The aim of the present study was to assess the frequency of NTM isolations in CF patients hospitalized in the pulmonary department, serving as a hospital CF center, and to describe challenges concerning the recognition of NTMLD (nontuberculous mycobacterial lung disease) in those patients. Methods: Consecutive CF patients, who were hospitalized due to pulmonary exacerbations (PEX), in a single CF center, between 2010 and 2020, were retrospectively assessed for the presence of NTM in respiratory specimens. Clinical and radiological data were retrospectively reviewed. Results: Positive respiratory specimen cultures for NTM were obtained in 11 out of 151 patients (7%), mean age—35.7 years, mean BMI—20.2 kg/m2, mean FEV1—58.6% pred. Cultures and phenotyping revealed the presence of Mycobacterium avium (M. avium)—in six patients, Mycobacterium chimaera (M. chimaera) in two, Mycobacterium kansasii (M. kansasii)—in one, Mycobacterium abscessus (M. abscessus)—in one, Mycobacterium lentifavum (M. lentiflavum)—in one. Simultaneously, respiratory cultures were positive for fungi in 91% of patients: Candida albicans (C. albicans)—in 82%, Aspergillus fumigatus (A. fumigatus)—in 45%. Clinical signs of NTMLD were non—specific, chest CT indicated NTMLD in five patients only. Conclusion: Due to non-specific clinical presentation, frequent sputum cultures for NTM and analysis of serial chest CT examinations are crucial for NTMLD recognition in CF patients. Further studies concerning the predictive role of fungal pathogens for NTMLD development in CF patients are needed.

Distinctive metabolic profiles between Cystic Fibrosis mutational subclasses and lung function.

Cystic fibrosis (CF) is a lethal multisystemic disease of a monogenic origin with numerous mutations. Functional defects in the cystic fibrosis transmembrane conductance receptor (CFTR) protein based on these mutations are categorised into distinct classes having different clinical presentations and disease severity. The present study aimed to create a comprehensive metabolomic profile of altered metabolites in patients with CF, among different classes and in relation to lung function. A chemical isotope labeling liquid chromatography-mass spectrometry metabolomics was used to study the serum metabolic profiles of young and adult CF (n = 39) patients and healthy controls (n = 30). Comparisons were made at three levels, CF vs. controls, among mutational classes of CF, between CF class III and IV, and correlated the lung function findings. A distinctive metabolic profile was observed in the three analyses. 78, 20, and 13 significantly differentially dysregulated metabolites were identified in the patients with CF, among the different classes and between class III and IV, respectively. The significantly identified metabolites included amino acids, di-, and tri-peptides, glutathione, glutamine, glutamate, and arginine metabolism. The top significant metabolites include 1-Aminopropan-2-ol, ophthalmate, serotonin, cystathionine, and gamma-glutamylglutamic acid. Lung function represented by an above-average FEV1% level was associated with decreased glutamic acid and increased guanosine levels. Metabolomic profiling identified alterations in different amino acids and dipeptides, involved in regulating glutathione metabolism. Two metabolites, 3,4-dihydroxymandelate-3-O-sulfate and 5-Aminopentanoic acid, were identified in common between the three anlayses and may represent as highly sensitive biomarkers for CF.

Cystic fibrosis transmembrane conductance receptor modulator therapy in cystic fibrosis, an update.

Cystic fibrosis transmembrane conductance receptor (CFTR) modulators are a new class of drugs that treat the underlying cause of cystic fibrosis. To date, there are four approved medications, which are mutation-specific. Although the number of mutations that respond to these agents is expanding, effective CFTR modulators are not available to all cystic fibrosis patients. The purpose of this article is to review the approved CFTR modulators and discuss the mutations that can be treated with these agents, as well as, review the long-term benefits of modulator therapy. More people with cystic fibrosis can be effectively treated with CFTR modulators. The new, highly effective triple therapy, elexacaftor/tezacaftor/ivacaftor is indicated for more than 90% of patients with cystic fibrosis and ivacaftor is now approved for children as young as 6 months of age with 1 of 30 CFTR mutations. Long-term use of modulator therapy is associated with fewer pulmonary exacerbations, maintenance of lung function, improved weight gain, and quality of life. CFTR modulators are the first therapies developed to treat the underlying defect in cystic fibrosis. Their use is associated with preserved lung function and improved health in patients with cystic fibrosis.

Clinical characteristics and genetic analysis of cystic fibrosis transmembrane conductancereseptor-related disease.

Cystic fibrosis (CF) transmembrane conductance receptor (CFTR)-related disease is diagnosed in patients affected by CFTR dysfunction who do not fully meet the CF diagnostic criteria. Only 2% of all CF patients have CFTR-related disease. We define the demographic characteristics of such patients, described the performance of mutational analyses, and describe the clinical findings. Twenty-four patients were followed-up for CFTR-related disease. Patients with CF symptoms but who did not completely fulfil the CF diagnostic criteria were enrolled. Age, body mass index at the times of diagnosis and admission, symptoms, pulmonary function and fecal elastase test results, gene analyses, and clinical findings during follow-up were evaluated. Ten patients (42%) were female and 14 (58%) male. Their mean age was 15.3years (minimum-maximum 6-20years). The mean age at diagnosis was 8.5years (minimum-maximum 3-14years) and the most common presenting complaint was a cough (n = 19). During follow up, chronic sinusitis developed in 15 patients, bronchiectasis in 13, nasal polyposis in six, failure to thrive in three, recurrent pancreatitis in two, asthma in one, and congenital bilateral absence of the vas deferens in one. Fecal elastase levels were low in only one of the three patients who failed to thrive. In terms of CFTR gene mutations, two were found in 10 patients, one in eight patients, and none in six. Cystic fibrosis transmembrane conductance receptor-related disease presents with various clinical findings. Serious symptoms may develop later in life. Late diagnosis significantly compromises the quality and duration of life in such patients.

Gallbladder and bile duct disease in Cystic Fibrosis

Cystic fibrosis (CF) is a multi-organ, clinically diverse disorder caused by mutations in the cystic fibrosis transmembrane conductance receptor (CFTR). Awareness of extra-pulmonary manifestations, including gastrointestinal and hepatobiliary disturbances, is an increasingly important part of providing high-quality care to patients with CF. Furthermore, biliary disorders, including gallbladder and bile duct disease, are common complications of CF. Therefore, a thorough understanding and efficient clinical evaluation of the gallbladder and biliary tree is an important aspect of integrated care for the patient with CF in order to prevent progression of undetected pathology. This best practice article summarizes the basis for gallbladder and bile duct pathology, describes the context and clinical presentation of biliary disease, and provides recommended approaches to delivery of high-quality care for patients with CF.

Preparation for a first-in-man lentivirus trial in patients with cystic fibrosis

We have recently shown that non-viral gene therapy can stabilise the decline of lung function in patients with cystic fibrosis (CF). However, the effect was modest, and more potent gene...

Combined Bilateral Lung-Liver Transplantation Complicated by Intraoperative Right Ventricular Dysfunction and Postoperative Hepatic Artery Thrombosis

COMBINED LUNG AND LIVER transplantation is performed infrequently because simultaneous transplantation of the 2 organs rarely is indicated. Patients selected for this procedure are not expected to survive transplantation of either organ alone. Cystic fibrosis (CF) is a genetic disease caused by dysfunction of the cystic fibrosis transmembrane conductance receptor on epithelial cells leading to abnormally viscous secretions. These secretions cause obstruction of the airways, the intestinal tract, and the biliary and pancreatic ducts.

NF-E2-related factor 2, a key inducer of antioxidant defenses, negatively regulates the CFTR transcription.

A few studies have clearly indicated that oxidative stress suppresses the cystic fibrosis transmembrane conductance receptor (CFTR) function and expression. However, the mechanisms by which this occurs are still poorly understood. To clarify this effect, we investigated the role of NF-E2-related factor 2 (Nrf2) transcription factor, a key cellular sensor of oxidative stress. A conserved antioxidant response element (ARE) in the CFTR minimal promoter, which binds Nrf2, has been identified. Surprisingly, Nrf2 exerts an unexpected repressive role on the CFTR gene promoter activity. To decipher the molecular mechanisms involved, we evaluated the role of YY1 in the Nrf2-mediated transcriptional activity and showed cooperation between these two factors. We demonstrated that Nrf2 promotes YY1 nuclear localization and increases its binding to the CFTR promoter. To our knowledge, this study is the first to report a repressor role of Nrf2 through the cooperation with YY1 and contributes to clarify the cascade events leading to the oxidative stress-suppressed CFTR expression.

Production of CFTR-null and CFTR-ΔF508 heterozygous pigs by adeno-associated virus–mediated gene targeting and somatic cell nuclear transfer

Progress toward understanding the pathogenesis of cystic fibrosis (CF) and developing effective therapies has been hampered by lack of a relevant animal model. CF mice fail to develop the lung and pancreatic disease that cause most of the morbidity and mortality in patients with CF. Pigs may be better animals than mice in which to model human genetic diseases because their anatomy, biochemistry, physiology, size, and genetics are more similar to those of humans. However, to date, gene-targeted mammalian models of human genetic disease have not been reported for any species other than mice. Here we describe the first steps toward the generation of a pig model of CF. We used recombinant adeno-associated virus (rAAV) vectors to deliver genetic constructs targeting the CF transmembrane conductance receptor (CFTR) gene to pig fetal fibroblasts. We generated cells with the CFTR gene either disrupted or containing the most common CF-associated mutation (DeltaF508). These cells were used as nuclear donors for somatic cell nuclear transfer to porcine oocytes. We thereby generated heterozygote male piglets with each mutation. These pigs should be of value in producing new models of CF. In addition, because gene-modified mice often fail to replicate human diseases, this approach could be used to generate models of other human genetic diseases in species other than mice.

A pure population of lung alveolar epithelial type II cells derived from human embryonic stem cells

Alveolar epithelial type II (ATII) cells are small, cuboidal cells that constitute approximately 60% of the pulmonary alveolar epithelium. These cells are crucial for repair of the injured alveolus by differentiating into alveolar epithelial type I cells. ATII cells derived from human ES (hES) cells are a promising source of cells that could be used therapeutically to treat distal lung diseases. We have developed a reliable transfection and culture procedure, which facilitates, via genetic selection, the differentiation of hES cells into an essentially pure (>99%) population of ATII cells (hES-ATII). Purity, as well as biological features and morphological characteristics of normal ATII cells, was demonstrated for the hES-ATII cells, including lamellar body formation, expression of surfactant proteins A, B, and C, alpha-1-antitrypsin, and the cystic fibrosis transmembrane conductance receptor, as well as the synthesis and secretion of complement proteins C3 and C5. Collectively, these data document the successful generation of a pure population of ATII cells derived from hES cells, providing a practical source of ATII cells to explore in disease models their potential in the regeneration and repair of the injured alveolus and in the therapeutic treatment of genetic diseases affecting the lung.

PAR2 activation interrupts E-cadherin adhesion and compromises the airway epithelial barrier: protective effect of β-agonists

The airway epithelium is an important barrier between the environment and subepithelial tissues. The epithelium is also divided into functionally restricted apical and basolateral domains, and this restriction is dependent on the elements of the barrier. The protease-activated receptor-2 (PAR2) receptor is expressed in airway epithelium, and its activation initiates multiple effects including enhanced airway inflammation and reactivity. We hypothesized that activation of PAR2 would interrupt E-cadherin adhesion and compromise the airway epithelial barrier. The PAR2-activating peptide (PAR2-AP, SLIGRL) caused an immediate approximately 50% decrease in the transepithelial resistance of primary human airway epithelium that persisted for 6-10 min. The decrease in resistance was accompanied by an increase in mannitol flux across the epithelium and occurred in cystic fibrosis transmembrane conductance receptor (CFTR) epithelium pretreated with amiloride to block Na and Cl conductances, confirming that the decrease in resistance represented an increase in paracellular conductance. In parallel experiments, activation of PAR2 interrupted the adhesion of E-cadherin-expressing L cells and of primary airway epithelial cells to an immobilized E-cadherin extracellular domain, confirming the hypothesis that activation of PAR2 interrupts E-cadherin adhesion. Selective interruption of E-cadherin adhesion with antibody to E-cadherin decreased the transepithelial resistance of primary airway epithelium by >80%. Pretreatment of airway epithelium or the E-cadherin-expressing L cells with the long-acting beta-agonist salmeterol prevented PAR2 activation from interrupting E-cadherin adhesion and compromising the airway epithelial barrier. Activation of PAR2 interrupts E-cadherin adhesion and compromises the airway epithelial barrier.

A Novel Method for Creating Artificial Mutant Samples for Performance Evaluation and Quality Control in Clinical Molecular Genetics

The lack of readily available, patient-derived materials for molecular genetic testing of many heterozygous or rare disorders creates a major impediment for laboratory proficiency and quality control procedures. The paucity of clinically derived mutation-positive samples could be surmounted if it were possible to construct artificial samples containing mutations of interest that would sufficiently resemble natural human samples. Such samples could then function as acceptable and realistic performance evaluation challenges and quality control reagents for recipient laboratories. Using the cystic fibrosis gene (CFTR) as a prototype, we have devised and executed experiments designed to generate unique DNA samples that could be used for these purposes. We used site-directed mutagenesis to generate mutations of interest in plasmid DNA derived from common bacterial artificial chromosome sources containing the cystic fibrosis transmembrane conductance receptor gene. CFTR mutations G85E and 1078delT were chosen to represent mutations in the original American College of Medical Genetics-recommended population-screening panel of 25 mutations. DNA samples containing predetermined concentrations and ratios of wild-type and mutated plasmids, bacterial artificial chromosomes of interest, and nonhuman genomic carrier DNA were characterized and tested in-house and in a group of nine pilot testing laboratories using a variety of technical platforms. The results indicate that these constructs, containing CFTR mutations in heterozygous and homozygous states, can serve as valid and accessible materials for quality assurance, including performance evaluation, proficiency testing, and assay quality control.

Identification of Differentially Expressed Genes in Normal and Tumor Human Gastric Tissue

The search for differentially expressed genes in gastric cancer may help define molecular alterations and molecular diagnosis of gastric cancer. Using the differential display PCR technique, we identified 18 genes that are differentially expressed between normal and tumor human gastric tissues. Their expressions were verified with reverse Northern blot analysis and Northern blot analysis. Oxidative phosphorylation-related genes, antizyme inhibitor of ornithine decarboxylase, protein phosphatase-1β, 35-kDa peroxisomal membrane protein, and cystic fibrosis transmembrane conductance receptor were highly expressed in tumor tissue, whereas pepsinogen A, Na-K ATPase α subunit, nerve growth factor receptor, and α-tropomyosin were highly expressed in normal tissue. In addition, 3 unknown genes were found to be differentially expressed in paired gastric tissues. These differentially expressed genes may provide significant opportunities for further understanding of gastric carcinogenesis and the molecular diagnosis of gastric cancer.

Capital Appreciation of World Equity Markets 1971–1994

We have recently shown that non-viral gene therapy can stabilise the decline of lung function in patients with cystic fibrosis (CF). However, the effect was modest, and more potent gene transfer agents are still required. Fuson protein (F)/Hemagglutinin/Neuraminidase protein (HN)-pseudotyped lentiviral vectors are more efficient for lung gene transfer than non-viral vectors in preclinical models. In preparation for a first-in-man CF trial using the lentiviral vector, we have undertaken key translational preclinical studies. Regulatory-compliant vectors carrying a range of promoter/enhancer elements were assessed in mice and human air–liquid interface (ALI) cultures to select the lead candidate; cystic fibrosis transmembrane conductance receptor (CFTR) expression and function were assessed in CF models using this lead candidate vector. Toxicity was assessed and ‘benchmarked’ against the leading non-viral formulation recently used in a Phase IIb clinical trial. Integration site profiles were mapped and transduction efficiency determined to inform clinical trial dose-ranging. The impact of pre-existing and acquired immunity against the vector and vector stability in several clinically relevant delivery devices was assessed. A hybrid promoter hybrid cytosine guanine dinucleotide (CpG)- free CMV enhancer/elongation factor 1 alpha promoter (hCEF) consisting of the elongation factor 1α promoter and the cytomegalovirus enhancer was most efficacious in both murine lungs and human ALI cultures (both at least 2-log orders above background). The efficacy (at least 14% of airway cells transduced), toxicity and integration site profile supports further progression towards clinical trial and pre-existing and acquired immune responses do not interfere with vector efficacy. The lead rSIV.F/HN candidate expresses functional CFTR and the vector retains 90–100% transduction efficiency in clinically relevant delivery devices. The data support the progression of the F/HN-pseudotyped lentiviral vector into a first-in-man CF trial in 2017.