Evidence against the acidification hypothesis in cystic fibrosis

The pleiotropic effects of cystic fibrosis (CF) result from themislocalization or inactivity of an apical membrane chloride channel,the cystic fibrosis transmembrane conductance regulator (CFTR). CFTRmay also modulate intracellular chloride conductances and thus affectorganelle pH. To test the role of CFTR in organelle pH regulation, wedeveloped a model system to selectively perturb the pH of a subset ofacidified compartments in polarized cells and determined the effects onvarious protein trafficking steps. We then tested whether these effectswere observed in cells lacking wild-type CFTR and whetherreintroduction of CFTR affected trafficking in these cells. Our modelsystem involves adenovirus-mediated expression of the influenza virusM2 protein, an acid-activated ion channel. M2 expression selectivelyslows traffic through the trans-Golgi network (TGN) andapical endocytic compartments in polarized Madin-Darby canine kidney(MDCK) cells. Expression of M2 or treatment with other pH perturbantsalso slowed protein traffic in the CF cell line CFPAC, suggesting thatthe TGN in this cell line is normally acidified. Expression offunctional CFTR had no effect on traffic and failed to rescue theeffect of M2. Our results argue against a role for CFTR in theregulation of organelle pH and protein trafficking in epithelial cells.

     

Targeted therapy for cystic fibrosis: cystic fibrosis transmembrane conductance regulator mutation-specific pharmacologic strategies

Cystic fibrosis (CF) results from the absence or dysfunction of a single protein, the CF transmembrane conductance regulator (CFTR). CFTR plays a critical role in the regulation of ion transport in a number of exocrine epithelia. Improvement or restoration of CFTR function, where it is deficient, should improve the CF phenotype. There are >1000 reported disease-causing mutations of the CFTR gene. Recent investigations have afforded a better understanding of the mechanism of dysfunction of many of these mutant CFTRs, and have allowed them to be classified according to their mechanism of dysfunction. These data, as well as an enhanced understanding of the role of CFTR in regulating epithelial ion transport, have led to the development of therapeutic strategies based on pharmacologic enhancement or repair of mutant CFTR dysfunction. The strategy, termed 'protein repair therapy', is aimed at improving the regulation of epithelial ion transport by mutant CFTRs in a mutation-specific fashion. The grouping of CFTR gene mutations, according to mechanism of dysfunction, yields some guidance as to which pharmacologic repair agents may be useful for specific CFTR mutations. Recent data has suggested that combinations of pharmacologic repair agents may be necessary to obtain clinically meaningful CFTR repair. Nevertheless, such strategies to improve mutant CFTR function hold great promise for the development of novel therapies aimed at correcting the underlying pathophysiology of CF.     

Gene therapy of cystic fibrosis: the glycofection approach

Many cells express surface membrane lectins that selectively bind and carry glycoconjugates into intracellular endosomes; in addition, various intracellular membrane and soluble lectins act as shuttles between different compartments. On this basis, we developed glycosylated polycations, now called glycofectins (glycosylated polylysine and polyethyleneimine). Recently, we set up a simple way to transform oligosaccharides into glycosynthons suitable to substitute proteins or polymers. Glycofectins bind plasmid DNA leading to compact glycoplexes. Glycoplexes prepared with glycofectins were found to be much more active than naked plasmid to transfer genes to various types of cells including human airway epithelial and serous cells. The gene transfer efficiency was found to depend on the nature of the sugars borne by glycofectins. It appeared that the sugar-dependent efficiency was not only related to the uptake but also to the intracellular traffic of glycoplexes.     

Evidence for a mitochondrial lesion in cystic fibrosis

Cystic fibrosis (CF) remains a major problem in human genetics and cell pathophysiology. It is a single gene trait caused by a mutation on the long arm of chromosome 7. Among its expressions are abnormal regulation of chloride channels and/or microobstructions in exocrine tissues. Here, evidence is presented that mitochondria are dysfunctional in CF: the major site of increased intracellular Ca in CF is mitochondrial, cells from subjects with CF consume more oxygen than normal, respond differentially to inhibitors of mitochondrial function, express increased electron transport activity and altered kinetics of complex I (NADH dehydrogenase) of the mitochondrial electron transport system. Patients with CF express increased total and resting energy expenditure. Some of these differences from normal occur also in asymptomatic carriers of the CF gene.     

Targets for cystic fibrosis therapy: proteomic analysis and correction of mutant cystic fibrosis transmembrane conductance regulator

Proteomic analysis has proved to be an important tool for understanding the complex nature of genetic disorders, such as cystic fibrosis (CF), by defining the cellular protein environment (proteome) associated with wild-type and mutant proteins. Proteomic screens identified the proteome of CF transmembrane conductance regulator (CFTR), and provided fundamental information to studies designed for understanding the crucial components of physiological CFTR function. Simultaneously, high-throughput screens for small-molecular correctors of CFTR mutants provided promising candidates for therapy. The majority of CF cases are caused by nucleotide deletions (ΔF508 CFTR; >75%), resulting in CFTR misfolding, or insertion of premature termination codons (～10%), leading to unstable mRNA and reduced levels of truncated dysfunctional CFTR. In this article, we review recent results of proteomic screens, developments in identifying correctors for the most frequent CFTR mutants, and comment on how integration of the knowledge gained from these studies may aid in finding a cure for CF and a number of other genetic disorders.     

Antibiotic therapy of cystic fibrosis in children]

It is postulated that P. aeruginosa in monoculture or in association with Staphylococcus aureus keeps its leading position in chronic bacterial inflammatory broncho-pulmonary processes in children with cystic fibrosis. Antibiotic resistant strains of Burkholderia cepacia, Stenotrophomonas maltophila, Alcaligenes xylosoxidans were revealed (7.1% of the strains). P. aeruginosa strains were susceptible to aminoglycosides, ciprofloxacin, and polymixin B. Susceptibility of smooth and mucoid forms of P. aeruginosa to ceftazidime stayed at the level of 49.6-57.1%. Such microbial associations as P. aeruginosa sm. + S. aureus, P. aeruginosa sm. + P. aeruginosa muc. + S. aureus were mainly susceptible to ciprofloxacin, aminoglycosides and resistant to ceftasidime. Meropenem, cefepim and ciprofloxacin are highly effective antibiotics for the treatment of broncho-pulmonary processes exacerbations at children with chronic P. aeruginosa cystic fibrosis. Intravenous use of antibiotics out of hospital for the treatment of the children with cystic fibrosis is clinically effective, and is economically and psychologically reasonable. It should be used more widely in medical practice.     

Oxidative stress and antioxidant therapy in cystic fibrosis

Cystic fibrosis is a lethal autosomal recessive condition caused by a defect of the transmembrane conductance regulator gene that has a key role in cell homeostasis. A dysfunctional cystic fibrosis transmembrane conductance regulator impairs the efflux of cell anions such as chloride and bicarbonate, and also that of other solutes such as reduced glutathione. This defect produces an increased viscosity of secretions together with other metabolic defects of epithelia that ultimately promote the obstruction and fibrosis of organs. Recurrent pulmonary infections and respiratory dysfunction are main clinical consequences of these pathogenetic events, followed by pancreatic and liver insufficiency, diabetes, protein-energy malnutrition, etc. This complex comorbidity is associated with the extensive injury of different biomolecular targets by reactive oxygen species, which is the biochemical hallmark of oxidative stress. These biological lesions are particularly pronounced in the lung, in which the extent of oxidative markers parallels that of inflammatory markers between chronic events and acute exacerbations along the progression of the disease. Herein, an abnormal flux of reactive oxygen species is present by the sustained activation of neutrophils and other cystic fibrosis-derived defects in the homeostatic processes of pulmonary epithelia and lining fluids. A sub-optimal antioxidant protection is believed to represent a main contributor to oxidative stress and to the poor control of immuno-inflammatory pathways in these patients. Observed defects include an impaired reduced glutathione metabolism and lowered intake and absorption of fat-soluble antioxidants (vitamin E, carotenoids, coenzyme Q-10, some polyunsaturated fatty acids, etc.) and oligoelements (such as Se, Cu and Zn) that are involved in reactive oxygen species detoxification by means of enzymatic defenses. Oral supplements and aerosolized formulations of thiols have been used in the antioxidant therapy of this inherited disease with the main aim of reducing the extent of oxidative lesions and the rate of lung deterioration. Despite positive effects on laboratory end points, poor evidence was obtained on the side of clinical outcome so far. These aspects examined in this critical review of the literature clearly suggest that further and more rigorous trials are needed together with new generations of pharmacological tools to a more effective antioxidant and anti-inflammatory therapy of cystic fibrosis patients. This article is part of a Special Issue entitled: Antioxidants and Antioxidant Treatment in Disease.     

Non-viral vectors in cystic fibrosis gene therapy: progress and challenges

Although the viability of cystic fibrosis (CF) gene transfer to airway epithelium has been demonstrated in vitro and in animal models, so far none of the clinical investigations using adenovirus, adeno-associated virus, lentivirus, cationic lipids or polymers has shown a persistent correction of the ion transport defects that occur in CF. Despite disappointing results, these studies have shown that non-viral vectors could represent a viable alternative for gene therapy in CF airway epithelium. The transfer efficiency of non-viral vectors is currently low, however, and thus these systems are not clinically relevant as yet. Before clinical application, several limitations encountered by non-viral delivery systems must be addressed. Recent progress has been made towards overcoming these limitations and towards making non-viral gene therapy a more realistic option for CF.     

Hypertonic saline increases lung epithelial lining fluid glutathione and thiocyanate: two protective CFTR-dependent thiols against oxidative injury

Background

Cystic fibrosis is a debilitating lung disease due to mutations in the cystic fibrosis transmembrane conductance regulator protein (CFTR) and is associated with chronic infections resulting in elevated myeloperoxidase activity and generation of hypochlorous acid (HOCl). CFTR mutations lead to decreased levels of glutathione (GSH) and thiocyanate (SCN) in the epithelial lining fluid (ELF). Hypertonic saline is used to improve lung function however the mechanism is uncertain.

Methods

In the present study, the effect of GSH and SCN on HOCl-mediated cell injury and their changes in the ELF after hypertonic saline nebulization in wild type (WT) and CFTR KO mice was examined. CFTR sufficient and deficient lung cells were assessed for GSH, SCN and corresponding sensitivity towards HOCl-mediated injury, in vitro.

Results

CFTR (-) cells had lower extracellular levels of both GSH and SCN and were more sensitive to HOCl-mediated injury. In vivo, hypertonic saline increased ELF GSH in the WT and to a lesser extent in the CFTR KO mice but only SCN in the WT ELF. Finally, potential protective effects of GSH and SCN at concentrations found in the ELF against HOCl toxicity were examined in vitro.

Conclusions

While the concentrations of GSH and SCN associated with the WT ELF protect against HOCl toxicity, those found in the CFTR KO mice were less sufficient to inhibit cell injury. These data suggests that CFTR has important roles in exporting GSH and SCN which are protective against oxidants and that hypertonic saline treatment may have beneficial effects by increasing their levels in the lung.     

Evidence against the proposed interaction of thionitrobenzoate with protein disulphide bonds

Thionitrobenzoate was prepared from 5,5'-dithiobis-(2-nitrobenzoic acid), and its reaction with several disulphide-containing proteins was examined under various conditions. No evidence for any cleavage of disulphide bonds was obtained. The claim by Robyt et al. [Arch. Biochem. Biophys. (1971) 147, 262-269] that thionitrobenzoate allows the quantitative determination of disulphide bonds in proteins is not substantiated.     

Plant aquaporins: new perspectives on water and nutrient uptake in saline environment

The mechanisms of salt stress and tolerance have been targets for genetic engineering, focusing on ion transport and compartmentation, synthesis of compatible solutes (osmolytes and osmoprotectants) and oxidative protection. In this review, we consider the integrated response to salinity with respect to water uptake, involving aquaporin functionality. Therefore, we have concentrated on how salinity can be alleviated, in part, if a perfect knowledge of water uptake and transport for each particular crop and set of conditions is available.     

Antibiotic therapy and fat digestion and absorption in cystic fibrosis

Antibiotic therapy in the cystic fibrosis (CF) mouse model has been shown to result in reduced bacterial load of the intestine and significant body mass gain. The effect was suggested to be linked to the improvement of intestinal digestion and absorption. Therefore, we aimed to assess the influence of routinely applied antibiotic therapy in CF patients on fat assimilation. Twenty-four CF patients aged 6 to 30 years entered the study. Inclusion criteria comprised confirmed exocrine pancreatic insufficiency and bronchopulmonary exacerbation demanding antibiotic therapy. Exclusion criteria comprised: antibiotic therapy six weeks prior to the test, liver cirrhosis, diabetes mellitus, oxygen dependency, the use of systemic corticosteroids. In all enrolled CF subjects, (13)C-labelled mixed triglyceride breath test ((13)C MTG-BT) was performed to assess lipid digestion and absorption, before and after antibiotic therapy. Sixteen subjects were treated intravenously with ceftazidime and amikacin, eight patients orally with ciprofloxacin. Cumulative percentage dose recovery (CPDR) was considered to reflect digestion and absorption of lipids. The values are expressed as means (medians). The values of CPDR before and after antibiotic therapy did not differ in the whole studied group [4.6(3.3) % vs. 5.7(5.3) %, p = 0.100] as well as in the subgroup receiving them intravenously [4.6(3.2) % vs. 5.7(5.3) %, p = 0.327] or in that with oral drug administration [4.6(3.4) % vs. 5.7(5.4) %, p = 0.167]. In conclusion, antibiotic therapy applied routinely in the course of pulmonary exacerbation in CF patients does not seem to result in an improvement of fat digestion and absorption.     

Hypertonic saline aerosol increases airway reactivity in the canine lung periphery.

Hyperventilation with dry air increases airway surface fluid (ASF) osmolality and causes acute mucosal injury, leukocyte infiltration, and delayed airway obstruction and hyperreactivity in canine peripheral airways. The purpose of this study was to determine whether ASF hypertonicity per se can account for these hyperventilation-associated effects. We first measured ASF osmolality before and after normal (NSC) and hypertonic (HSC) saline aerosol challenges to document the magnitude of hypertonicity produced by these stimuli. We then measured canine peripheral airway resistance and reactivity to hypocapnia and aerosolized histamine before and after NSC and HSC. Cells and eicosanoid mediators recovered in bronchoalveolar lavage fluid at 5 and 24 h after NSC and HSC were examined. We found that HSC but not NSC caused acute ASF hyperosmolality, increased mediator release, and delayed airway hyperreactivity in the absence of mucosal injury and leukocyte infiltration. These observations suggest that ASF hyperosmolality contributes to the development of the late-phase response to hyperventilation and further suggest that hyperventilation-induced mucosal injury independently initiates leukocyte infiltration and late-phase airway obstruction.     

Evidence that mucoid Pseudomonas aeruginosa in the cystic fibrosis lung grows under iron-restricted conditions

Abstract The outer membrane protein composition of mucoid Pseudomonas aeruginosa recovered without subculture from the sputum of a cystic fibrosis patient was studied by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The results indicated that three outer membrane proteins in the range of M _r 80 000–90 000 were induced. The induction of these proteins can be simulated by growing the same isolate under iron-restricted conditions in laboratory media. This initial study gives the first direct biochemical evidence that mucoid P. aeruginosa grows under iron restricted conditions in the lungs of the cystic fibrosis patient.     

Evidence for direct interaction between actin and the cystic fibrosis transmembrane conductance regulator

Previous studies have demonstrated that actin filament organization controls the cystic fibrosis transmembrane conductance regulator (CFTR) ion channel function. The precise molecular nature of the interaction between actin and CFTR, however, remains largely unknown. In this report, interactions between actin and purified human epithelial CFTR were directly assessed by reconstitution of the channel protein in a lipid bilayer system and by atomic force microscopy (AFM). CFTR-containing liposomes in solution were deposited on freshly cleaved mica and imaging was performed in tapping-mode AFM. CFTR function was also determined in identical preparations. Images of single CFTR molecules were obtained, and addition of monomeric actin below its critical concentration showed the formation of actin filaments associated with CFTR. The data indicate a direct interaction between actin and CFTR exists, which may explain the regulatory role of the cytoskeleton in ion channel function. This was confirmed by functional studies of CFTR single-channel currents, which were regulated by addition of various conformations of actin. The present study indicates that CFTR may directly bind actin and that this interaction helps affect the functional properties of this channel protein.     

Cytokine pattern in cystic fibrosis patients during antibiotic therapy and gene therapy using adenoviral vector

Cystic fibrosis (CF) is a genetic disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Despite improvements in treatment, pulmonary disease still remains the primary cause of death among these patients. In order to introduce a normal CFTR gene copy into airway epithelial cells, adenoviral vectors (AV) have been developed. AV are known to induce an inflammatory reaction that limits transgene expression, and can be potentially harmful. No human study has clearly monitored simultaneously, systemic and local inflammatory reaction, during AV administration. We report here the levels of C-reactive protein (CRP), interleukin (IL)-6, IL-8, tumor necrosis factor-alpha (TNF-alpha), and interleukin-1 receptor antagonist (IL-1Ra) in plasma and bronchoalveolar lavage fluid (BALF) from six cystic fibrosis patients receiving AV encoding CFTR (AdCFTR). AdCFTR was administered to three cohorts of two patients into the nose on day 0, at doses ranging from 105 to 4 x 108 plaque-forming units (pfu), followed, on day 1, by aerosolization of 107 to 5.4 x 108 pfu. In order to ensure that patients were in the best clinical condition, and to further attenuate the broncho-pulmonary inflammation secondary to bacterial infection, they received antibiotic therapy, two weeks prior to AdCFTR administration, until 9 to 11 days after. We found that antibiotics markedly decreased CRP, TNF-alpha, IL-6, IL-1Ra levels in blood. In BALF, antibiotics slightly decreased TNF-alpha levels but had no effect on IL-8 and IL-1Ra, while IL-6 levels increased. AdCFTR administration did not induce any systemic or local cytokine release. In both blood and BALF, CRP, IL-8, IL-1Ra, TNF-alpha decreased, while IL-6 levels increased between day -7 and day 3. One patient presented an asymptomatic increase of all parameters in the BALF on day 7. Twenty one days later, he displayed a clinical deterioration suggestive of an exacerbation. In conclusion, this study demonstrates that antibiotic administration tends to attenuate systemic but not local broncho-pulmonary inflammation in CF patients. In the setting of our study, AdCFTR administration did not induce cytokine release. Further studies are necessary to investigate other inflammatory markers and the mechanisms involved during AV-mediated gene transfer for a better understanding of the immune reaction, which continues to hamper the development of gene therapy for CF patients.