Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Allelic Variants Relate to Shifts in Faecal Microbiota of Cystic Fibrosis Patients

Introduction

In this study we investigated the effects of the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) gene variants on the composition of faecal microbiota, in patients affected by Cystic Fibrosis (CF). CFTR mutations (F508del is the most common) lead to a decreased secretion of chloride/water, and to mucus sticky secretions, in pancreas, respiratory and gastrointestinal tracts. Intestinal manifestations are underestimated in CF, leading to ileum meconium at birth, or small bowel bacterial overgrowth in adult age.

Methods

Thirty-six CF patients, fasting and under no-antibiotic treatment, were CFTR genotyped on both alleles. Faecal samples were subjected to molecular microbial profiling through Temporal Temperature Gradient Electrophoresis and species-specific PCR. Ecological parameters and multivariate algorithms were employed to find out if CFTR variants could be related to the microbiota structure.

Results

Patients were classified by two different criteria: 1) presence/absence of F508del mutation; 2) disease severity in heterozygous and homozygous F508del patients. We found that homozygous-F508del and severe CF patients exhibited an enhanced dysbiotic faecal microbiota composition, even within the CF cohort itself, with higher biodiversity and evenness. We also found, by species-specific PCR, that potentially harmful species (Escherichia coli and Eubacterium biforme) were abundant in homozygous-F508del and severe CF patients, while beneficial species (Faecalibacterium prausnitzii, Bifidobacterium spp., and Eubacterium limosum) were reduced.

Conclusions

This is the first report that establishes a link among CFTR variants and shifts in faecal microbiota, opening the way to studies that perceive CF as a ‘systemic disease’, linking the lung and the gut in a joined axis.     

Correction of Chloride Transport and Mislocalization of CFTR Protein by Vardenafil in the Gastrointestinal Tract of Cystic Fibrosis Mice

Although lung disease is the major cause of mortality in cystic fibrosis (CF), gastrointestinal (GI) manifestations are the first hallmarks in 15–20% of affected newborns presenting with meconium ileus, and remain major causes of morbidity throughout life. We have previously shown that cGMP-dependent phosphodiesterase type 5 (PDE5) inhibitors rescue defective CF Transmembrane conductance Regulator (CFTR)-dependent chloride transport across the mouse CF nasal mucosa. Using F508del-CF mice, we examined the transrectal potential difference 1 hour after intraperitoneal injection of the PDE5 inhibitor vardenafil or saline to assess the amiloride-sensitive sodium transport and the chloride gradient and forskolin-dependent chloride transport across the GI tract. In the same conditions, we performed immunohistostaining studies in distal colon to investigate CFTR expression and localization. F508del-CF mice displayed increased sodium transport and reduced chloride transport compared to their wild-type littermates. Vardenafil, applied at a human therapeutic dose (0.14 mg/kg) used to treat erectile dysfunction, increased chloride transport in F508del-CF mice. No effect on sodium transport was detected. In crypt colonocytes of wild-type mice, the immunofluorescence CFTR signal was mostly detected in the apical cell compartment. In F508del-CF mice, a 25% reduced signal was observed, located mostly in the subapical region. Vardenafil increased the peak of intensity of the fluorescence CFTR signal in F508del-CF mice and displaced it towards the apical cell compartment. Our findings point out the intestinal mucosa as a valuable tissue to study CFTR transport function and localization and to evaluate efficacy of therapeutic strategies in CF. From our data we conclude that vardenafil mediates potentiation of the CFTR chloride channel and corrects mislocalization of the mutant protein. The study provides compelling support for targeting the cGMP signaling pathway in CF pharmacotherapy.     

Voluntary Exercise Prevents Cisplatin-Induced Muscle Wasting during Chemotherapy in Mice

Loss of muscle mass related to anti-cancer therapy is a major concern in cancer patients, being associated with important clinical endpoints including survival, treatment toxicity and patient-related outcomes. We investigated effects of voluntary exercise during cisplatin treatment on body weight, food intake as well as muscle mass, strength and signalling. Mice were treated weekly with 4 mg/kg cisplatin or saline for 6 weeks, and randomized to voluntary wheel running or not. Cisplatin treatment induced loss of body weight (29.8%, P<0.001), lean body mass (20.6%, P = 0.001), as well as anorexia, impaired muscle strength (22.5% decrease, P<0.001) and decreased glucose tolerance. In addition, cisplatin impaired Akt-signalling, induced genes related to protein degradation and inflammation, and reduced muscle glycogen content. Voluntary wheel running during treatment attenuated body weight loss by 50% (P<0.001), maintained lean body mass (P<0.001) and muscle strength (P<0.001), reversed anorexia and impairments in Akt and protein degradation signalling. Cisplatin-induced muscular inflammation was not prevented by voluntary wheel running, nor was glucose tolerance improved. Exercise training may preserve muscle mass in cancer patients receiving cisplatin treatment, potentially improving physical capacity, quality of life and overall survival.     

CFTR Regulation of Intracellular Calcium in Normal and Cystic Fibrosis Human Airway Epithelia

In cystic fibrosis airway epithelia, mutation of the CFTR protein causes a reduced response of Cl⁻ secretion to secretagogues acting via cAMP. Using a Ca²⁺ imaging system, the hypothesis that CFTR activation may permit ATP release and regulate [Ca²⁺] _i via a receptor-mediated mechanism, is tested in this study. Application of external nucleotides produced a significant increase in [Ca²⁺] _i in normal (16HBE14o⁻ cell line and primary lung culture) and in cystic fibrosis (CFTE29o⁻ cell line) human airway epithelia. The potency order of nucleotides on [Ca²⁺] _i variation was UTP ≫ ATP > UDP > ADP > AMP > adenosine in both cell types. The nucleotide [Ca²⁺] _i response could be mimicked by activation of CFTR with forskolin (20 μm) in a temperature-dependent manner. In 16HBE14o⁻ cells, the forskolin-induced [Ca²⁺] _i response increased with increasing temperature. In CFTE29o⁻ cells, forskolin had no effect on [Ca²⁺] _i at body temperature-forskolin-induced [Ca²⁺] _i response in CF cells could only be observed at low experimental temperature (14°C) or when cells were cultured at 26°C instead of 37°C. Pretreatment with CFTR channel blockers glibenclamide (100 μm) and DPC (100 μm), with hexokinase (0.5 U/mg), and with the purinoceptor antagonist suramin (100 μm), inhibited the forskolin [Ca²⁺] _i response. Together, these results demonstrate that once activated, CFTR regulates [Ca²⁺] _i by mediating nucleotide release and activating cell surface purinoceptors in normal and CF human airway epithelia. Received: 3 April 2000/Revised: 30 June 2000     

The Mutation Spectrum of the CFTR Gene in Cystic Fibrosis Patients from Bashkortostan

Mutations of CFTR were studied in patients with cystic fibrosis (CF) from Bashkortostan. In total, 15 mutations were observed and 51% of all mutant alleles identified. The most diagnostically significant mutations were delF508 (33.8%), 394delTT (3.52%), CFTRdele2,3(21kb) (1.41%), R334W (1.41%), 3849 + 10kbC T (1.41%), and N1303K (1.41%). Mutations G542X, 2184insA, S1196X, and W1282X were each found in less than 1% patients. Five new mutations and two neutral substitutions were revealed. These were I488M (exon 10), 1811 + 12A C (intron 11), T663S (exon 13), I1226R (exon 19), 4005 + 9A C (intron 20), 2097A C (A655A, exon 13), and 3996G C (V1288V, exon 20). Bashkortostan was shown to differ in the CFTR mutation spectrum from other regions of Russia. The results will allow direct DNA diagnostics of CF in far more families. Molecular screening of probands" relatives will contribute to identification and medical genetic counseling of heterozygous carriers, which is essential for CF prevention.     

The Lung Inflammation and Skeletal Muscle Wasting Induced by Subchronic Cigarette Smoke Exposure Are Not Altered by a High-Fat Diet in Mice

Obesity and cigarette smoking independently constitute major preventable causes of morbidity and mortality and obesity is known to worsen lung inflammation in asthma. Paradoxically, higher body mass index (BMI) is associated with reduced mortality in smoking induced COPD whereas low BMI increases mortality risk. To date, no study has investigated the effect of a dietary-induced obesity and cigarette smoke exposure on the lung inflammation and loss of skeletal muscle mass in mice. Male BALB/c mice were exposed to 4 cigarettes/day, 6 days/week for 7 weeks, or sham handled. Mice consumed either standard laboratory chow (3.5 kcal/g, 12% fat) or a high fat diet (HFD, 4.3 kcal/g, 32% fat). Mice exposed to cigarette smoke for 7 weeks had significantly more inflammatory cells in the BALF (P<0.05) and the mRNA expression of pro-inflammatory cytokines and chemokines was significantly increased (P<0.05); HFD had no effect on these parameters. Sham- and smoke-exposed mice consuming the HFD were significantly heavier than chow fed animals (12 and 13%, respectively; P<0.05). Conversely, chow and HFD fed mice exposed to cigarette smoke weighed 16 and 15% less, respectively, compared to sham animals (P<0.05). The skeletal muscles (soleus, tibialis anterior and gastrocnemius) of cigarette smoke-exposed mice weighed significantly less than sham-exposed mice (P<0.05) and the HFD had no protective effect. For the first time we report that cigarette smoke exposure significantly decreased insulin-like growth factor-1 (IGF-1) mRNA expression in the gastrocnemius and tibialis anterior and IGF-1 protein in the gastrocnemius (P<0.05). We have also shown that cigarette smoke exposure reduced circulating IGF-1 levels. IL-6 mRNA expression was significantly elevated in all three skeletal muscles of chow fed smoke-exposed mice (P<0.05). In conclusion, these findings suggest that a down-regulation in local IGF-1 may be responsible for the loss of skeletal muscle mass following cigarette smoke exposure in mice.     

Cystic Fibrosis: Channel, Catalytic, and Folding Properties of the CFTR Protein

The identification and characterization of the CFTR gene and protein have provided not only a major impetus to the dissection of the molecular pathophysiology of cystic fibrosis (CF) but also a new perspective on the structure and function of the large supeifamily of membrane transport proteins to which it belongs. While the mechanism of the active vectorial translocation of many hydrophobic substrates by several of these transporters remains nearly as perplexing as it has for several decades, considerable insight has been gained into the control of the bi-directional permeation of chloride ions through a single CFTR channel by the phosphorylation of the R-domain and ATP interactions at the two nucleotide binding domains. However, details of these catalytic and allosteric mechanisms remain to be elucidated and await the replacement of two-dimensional conceptualizations with three dimensional structure information. Secondary and tertiary structure determination is required both for the understanding of the mechanism of action of the molecule and to enable a more complete appreciation of the misfolding and misprocessing of mutant CFTR molecules. This is the primary cause of the disease in the majority of the patients and hence understanding the details of the cotranslational interactions with multiple molecular chaperones, the ubiquitin-proteasome pathway and other components of the quality control machinery at the endoplasmic reticulum could provide a basis for the development of new therapeutic interventions.     

Exogenous Melatonin Alleviates Skeletal Muscle Wasting by Regulating Hypothalamic Neuropeptides Expression in Endotoxemia Rats

To investigate whether exogenous melatonin (MLT) could alleviate skeletal muscle wasting by regulating hypothalamic neuropeptides expression. Adult male Sprague Dawley rats were intraperitoneally injected with lipopolysaccharide (LPS) (10 mg/kg), followed by MLT (30 mg/kg/day) or saline for 3 days. Hypothalamic tissues and skeletal muscle were obtained on day 3. Skeletal muscle wasting was measured by the mRNA expression of two E3 ubiquitin ligases, muscle atrophy F-box and muscle ring finger 1 as well as 3-methylhistidine (3-MH) and tyrosine release. Three hypothalamic neuropeptides (POMC, AgRP, CART) expression were detected in all groups. POMC expression knockdown was achieved by ARC injection of lentiviruses containing shRNA against POMC. Two weeks after ARC viruses injection, rats were i.p. injected with LPS (10 mg/kg) followed by MLT (30 mg/kg/day) or saline for 3 days. Brain tissues were harvested for immunostaining. In septic rats, 3-MH, tyrosine release and muscle atrophic gene expression were significantly decreased in MLT treated group. POMC and CART expression were lower while AgRP expression was higher in MLT treated group. Furthermore, in septic rats treated with MLT, muscle wasting in those with lower expression of neuropeptide POMC did not differ from those with normal POMC expression. Exogenous MLT could alleviate skeletal muscle wasting in septic rats by regulating hypothalamic neuropeptides.

     

Apical CFTR Expression in Human Nasal Epithelium Correlates with Lung Disease in Cystic Fibrosis

Introduction

Although most individuals with cystic fibrosis (CF) develop progressive obstructive lung disease, disease severity is highly variable, even for individuals with similar CFTR mutations. Measurements of chloride transport as expression of CFTR function in nasal epithelial cells correlate with pulmonary function and suggest that F508del-CFTR is expressed at the apical membrane. However, an association between quantitative apical CFTR expression in nasal epithelium and CF disease severity is still missing.

Methods and Materials

Nasal epithelial cells from healthy individuals and individuals with CF between 12–18 years were obtained by nasal brushing. Apical CFTR expression was measured by confocal microscopy using CFTR mAb 596. Expression was compared between both groups and expression in CF nasal epithelial cells was associated with standardized pulmonary function (FEV₁%).

Results

The proportion of cells expressing apical CFTR in columnar epithelium is lower in CF compared to non-CF. The apical CFTR expression level was significantly correlated with FEV₁% in F508del homozygous subjects (r = 0.63, p = 0.012).

Conclusion

CFTR expression in nasal epithelial cells is lower in subjects with CF compared to healthy subjects. The proportion of cells expressing F508del-CFTR at the apical membrane is variable between subjects and is positively correlated with FEV₁% in F508del-CFTR homozygous subjects.     

Electrophysiological evaluation of Cystic Fibrosis Conductance Transmembrane Regulator (CFTR) expression in human monocytes

Background

Cystic fibrosis is caused by mutations of CFTR gene, a protein kinase A-activated anion channel, and is associated to a persistent and excessive chronic lung inflammation, suggesting functional alterations of immune cells. Leukocytes express detectable levels of CFTR but the molecule has not been fully characterized in these cells.

Methods

Freshly isolated monocytes from healthy individuals and CF patients were assessed by protein expression, single cell electrophysiological and membrane depolarization assays.

Results

We recorded chloride currents by patch clamp in healthy monocytes, after the administration of a CFTR stimulus. Currents were sensitive to a specific blocker of the CFTR channel, CFTR_inh-172 and were absent in CF monocytes. Next, we evaluated the effects of ex vivo exposure of monocytes from cystic fibrosis patients carrying the F508del mutation to a chemical corrector, Vertex-325. We found an increase in CFTR expression by confocal microscopy and a recovery of CFTR function by both patch clamp and single cell fluorescence analysis.

Conclusions

We confirm the expression of functional CFTR in human monocytes and demonstrate that blood monocytes can represent an adequate source of primary cells to assess new therapies and define diagnosis of CF.

General significance

Tests to evaluate CFTR functional abnormalities in CF disease might greatly benefit from the availability of a convenient source of primary cells. This electrophysiological study promotes the use of monocytes as a minimally invasive tool to study and monitor CFTR function in individual patients.     

Lack of Differences in Serum Binding of Calcium in Cystic Fibrosis,Carriers and Controls

Attempts have been made to detect carriers for cystic fibrosis (CF) by some means other than genetic inference from affected children to their otherwise normal parents¹. The ability to detect CF carriers would result in substantial advances in genetic counselling. Moreover, an assay by which heterozygotes are identifiable must involve a parameter close to the elusive primary gene abnormality. Accordingly the paper by Fitz-patrick et al.² in which serum binding of tracer amounts of calcium (determined by equilibrium dialysis) of CF > heterozygotes > controls was a potentially significant report. We were, however, sceptical about data in which healthy carrier individuals demonstrated so great a difference from normal (an increase in serum binding of ⁴⁵Ca in heterozygotes over controls of 65 ± 11%) for a clearly nonspecific characteristic. As we have substantial serum and salivary electrophoretic data³ in which no consistent protein differences were apparent in carriers and because we wondered about the biological explanation of the findings of Fitzpatrick et al.², we decided to repeat their serum binding of calcium experiments. We repeated the reported procedures as closely as possible. We also assayed calcium binding with the additional buffer and salt concentration conditions described below. Sera from different individuals [N (controls = 30; N (heterozygotes) = 31; N (CF) = 33] were used in 4 experiments.     

Four Novel Cystic Fibrosis Mutations in Splice Junction Sequences Affecting the CFTR Nucleotide Binding Folds

Single cases of the four novel splice site mutations 1525-1G & rarr; A (intron 9), 3601 2A → G (intron 18), 3850 3T → G (intron 19), and 4374 → 1G → T (intron 23) were detected in the CFTR gene of cystic fibrosis patients of Indo-Iranian, Turkish, Polish, and German descent. The nucleotide substitutions at the +1, -1, and -2 positions all destroy splice sites and lead to severe disease alleles associated with features typical of gastrointestinal and pulmonary cystic fibrosis disease. The 3850 -- 3 T-to-G change was discovered in a very mildly affected 33-year-old ΔF508 compound heterozygote, suggesting that the T-to-G transversion at the less conserved -3 position of the acceptor splice site may retain some wildtype function.     

Cigarette Smoke-induced Ca2+ Release Leads to Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Dysfunction

Chronic obstructive pulmonary disease affects 64 million people and is currently the fourth leading cause of death worldwide. Chronic obstructive pulmonary disease includes both emphysema and chronic bronchitis, and in the case of chronic bronchitis represents an inflammatory response of the airways that is associated with mucus hypersecretion and obstruction of small airways. Recently, it has emerged that exposure to cigarette smoke (CS) leads to an inhibition of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl⁻ channel, causing airway surface liquid dehydration, which may play a role in the development of chronic bronchitis. CS rapidly clears CFTR from the plasma membrane and causes it to be deposited into aggresome-like compartments. However, little is known about the mechanism(s) responsible for the internalization of CFTR following CS exposure. Our studies revealed that CS triggered a rise in cytoplasmic Ca²⁺ that may have emanated from lysosomes. Furthermore, chelation of cytoplasmic Ca²⁺, but not inhibition of protein kinases/phosphatases, prevented CS-induced CFTR internalization. The macrolide antibiotic bafilomycin A1 inhibited CS-induced Ca²⁺ release and prevented CFTR clearance from the plasma membrane, further linking cytoplasmic Ca²⁺ and CFTR internalization. We hypothesize that CS-induced Ca²⁺ release prevents normal sorting/degradation of CFTR and causes internalized CFTR to reroute to aggresomes. Our data provide mechanistic insight into the potentially deleterious effects of CS on airway epithelia and outline a hitherto unrecognized signaling event triggered by CS that may affect the long term transition of the lung into a hyper-inflammatory/dehydrated environment.     

Skeletal Muscle Cellularity and Glycogen Distribution in the Hypermuscular Compact Mice

The TGF-beta member myostatin acts as a negative regulator of skeletal muscle mass. The Compact mice were selected for high protein content and hypermuscularity, and carry a naturally occurring 12-bp deletion in the propeptide region of the myostatin precursor. We aimed to investigate the cellular characteristics and the glycogen distribution of the Compact tibialis anterior (TA) muscle by quantitative histochemistry and spectrophotometry. We have found that the deficiency in myostatin resulted in significantly increased weight of the investigated hindlimb muscles compared to wild type. Although the average glycogen content of the individual fibers kept unchanged, the total amount of glycogen in the Compact TA muscle increased two-fold, which can be explained by the presence of more fibers in Compact compared to wild type muscle. Moreover, the ratio of the most glycolytic IIB fibers significantly increased in the Compact TA muscle, of which glycogen content was the highest among the fast fibers. In summary, myostatin deficiency caused elevated amount of glycogen in the TA muscle but did not increase the glycogen content of the individual fibers despite the marked glycolytic shift observed in Compact mice.Key words: Compact mice, fiber-type, GDF-8, glycogen, muscle, myostatin     

Wnt Signaling in Skeletal Muscle Dynamics: Myogenesis,Neuromuscular Synapse and Fibrosis

The signaling pathways activated by Wnt ligands are related to a wide range of critical cell functions, such as cell division, migration, and synaptogenesis. Here, we summarize compelling evidence on the role of Wnt signaling on several features of skeletal muscle physiology. We briefly review the role of Wnt pathways on the formation of muscle fibers during prenatal and postnatal myogenesis, highlighting its role on the activation of stem cells of the adult muscles. We also discuss how Wnt signaling regulates the precise formation of neuromuscular synapses, by modulating the differentiation of presynaptic and postsynaptic components, particularly regarding the clustering of acetylcholine receptors on the muscle membrane. In addition, based on previous evidence showing that Wnt pathways are linked to several diseases, such as Alzheimer's and cancer, we address recent studies indicating that Wnt signaling plays a key role in skeletal muscle fibrosis, a disease characterized by an increase in the extracellular matrix components leading to failure in muscle regeneration, tissue disorganization and loss of muscle activity. In this context, we also discuss the possible cross-talk between the Wnt/β-catenin pathway with two other critical profibrotic pathways, transforming growth factor β and connective tissue growth factor, which are potent stimulators of the accumulation of connective tissue, an effect characteristic of the fibrotic condition. As it has emerged in other pathological conditions, we suggests that muscle fibrosis may be a consequence of alterations of Wnt signaling activity.     

Dystonin-Deficient Mice Exhibit an Intrinsic Muscle Weakness and an Instability of Skeletal Muscle Cytoarchitecture

Dystonia musculorum (dt) was originally described as a hereditary sensory neurodegeneration syndrome of the mouse. The gene defective in dt encodes a cytoskeletal linker protein, dystonin, that is essential for maintaining neuronal cytoskeletal integrity. In addition to the nervous system, dystonin is expressed in a variety of other tissues, including muscle. We now show that dystonin cross-links actin and desmin filaments and that its levels are increased during myogenesis, coinciding with the progressive reorganization of the intermediate filament network. A disorganization of cytoarchitecture in skeletal muscle from dt/dt mice was observed in ultrastructural studies. Myoblasts from dt/dt mice fused to form myotubes in culture; however, terminally differentiated myotubes contained incompletely assembled myofibrils. Another feature observed in dt/dt myotubes in culture and in skeletal muscle in situ was an accumulation and abnormal distribution of mitochondria. The diaphragm muscle from dt/dt mice was weak in isometric contractility measurements in vitro and was susceptible to contraction-induced sarcolemmal damage. Altogether, our data indicate that dystonin is a cross-linker of actin and desmin filaments in muscle and that it is essential for establishing and maintaining proper cytoarchitecture in mature muscle.     

Tissue Depletion of Taurine Accelerates Skeletal Muscle Senescence and Leads to Early Death in Mice

Taurine (2-aminoethanesulfonic acid) is found in milimolar concentrations in mammalian tissues. One of its main functions is osmoregulation; however, it also exhibits cytoprotective activity by diminishing injury caused by stress and disease. Taurine depletion is associated with several defects, many of which are found in the aging animal, suggesting that taurine might exert anti-aging actions. Therefore, in the present study, we examined the hypothesis that taurine depletion accelerates aging by reducing longevity and accelerating aging-associated tissue damage. Tissue taurine depletion in taurine transporter knockout (TauTKO) mouse was found to shorten lifespan and accelerate skeletal muscle histological and functional defects, including an increase in central nuclei containing myotubes, a reduction in mitochondrial complex 1 activity and an induction in an aging biomarker, Cyclin-dependent kinase 4 inhibitor A (p16INK4a). Tissue taurine depletion also enhances unfolded protein response (UPR), which may be associated with an improvement in protein folding by taurine. Our data reveal that tissue taurine depletion affects longevity and cellular senescence; an effect possibly linked to a disturbance in protein folding.