Background/Aims: Several recent studies revealed an accumulation of ceramide in bronchial, tracheal and intestinal epithelial cells of mice and patients with cystic fibrosis (CF). Normalization of ceramide concentrations in lungs of CF mice employing the functional acid sphingomyelinase inhibitor amitriptyline also normalized mucociliary clearance, chronic inflammation and infection susceptibility to pulmonary P. aeruginosa in these mice. Methods: To test for a beneficial effect of amitriptyline in vivo, we performed a phase IIb randomised, double-blind, placebo-controlled study. Twenty-one CF patients were treated with 25 mg/d amitriptyline twice daily for 28 days. The placebo consisted of 19 patients and was also treated twice per day. The primary endpoint was the change in lung function in the intention-to-treat (ITT) population. Secondary endpoints were ceramide levels in epithelial cells and safety. Results: After treatment, forced expiratory volume in 1 sec predicted (FEV1) increased 6.3±11.5% (p=0.08) in the ITT population (36 of 40 CF patients) and 8.5±10% (p=0.013) in the per protocol (PP) population (29 of 40 patients). Ceramide levels decreased in nasal epithelial cells after amitriptyline treatment. Amitriptyline had no severe and only mild and mostly transient adverse effects, i.e. xerostomia and tiredness. Conclusion: Amitriptyline is safe in CF-patients, increases FEV1 and reduces ceramide in lung cells of CF patients.
Abstract Pulmonary infections of cystic fibrosis (CF) patients with Staphylococcus aureus ( S. aureus ) occur very early in the disease. The molecular details that cause infection-susceptibility of CF patients to and mediate infection with S. aureus are poorly characterized. Therefore, we aimed to identify the role of α-toxin, a major S. aureus toxin, for pulmonary infection of CF mice. Infection with S. aureus JE2 resulted in severe pneumonia in CF mice, while wildtype mice were almost unaffected. Deficiency of α-toxin in JE2-Δhla reduced the pathogenicity of S. aureus in CF mice. However, CF mice were still more susceptible to the mutant S. aureus strain than wildtype mice. The S. aureus JE2 induced a marked increase of ceramide and a downregulation of sphingosine and acid ceramidase expression in bronchi of CF mice. Deletion of α-toxin reduced these changes after infection of CF mice. Similar changes were observed in wildtype mice, but at much lower levels. Our data indicate that expression of α-toxin is a major factor causing S. aureus infections in CF mice. Wildtype S. aureus induces a marked increase of ceramide and a reduction of sphingosine and acid ceramidase expression in bronchial epithelial cells of wildtype and CF mice, changes that determine infection susceptibility.
Abstract Cystic fibrosis (CF), the most common autosomal recessive disorder, at least in western countries, is caused by mutations of the cystic fibrosis transmembranous conductance regulator (CFTR) molecule and affects approximately 80,000 patients in Europe and the USA. Most, if not all, CF patients develop a chronic pulmonary infection with Pseudomonas aeruginosa . At present it is unknown why CF patients are highly sensitive to P. aeruginosa infections, and most importantly, no curative treatment for CF is available. P. aeruginosa infection results in an activation of the enzyme acid sphingomyelinase which catalyzes the release of ceramide from sphingomyelin in the cell membrane. Ceramide forms large ceramide‐enriched membrane domains that are required for internalization of bacteria, induction of cell death in infected cells and a controlled release of cytokines from infected cells. Ceramide‐enriched membrane platforms seem to serve the reorganization of receptors and intracellular signaling molecules involved in the infection of mammalian cells with P. aeruginosa . The significance of the acid sphingomyelinase and ceramide for the infection of mammalian cells with P. aeruginosa was demonstrated on mice genetically deficient for the acid sphingomyelinase. Further studies with N. gonorrhoeae , S. aureus and rhinoviruses indicate that ceramide‐enriched membrane domains are also important for the infection of mammalian cells with other bacterial and viral pathogens, suggesting a general role of these membrane domains in infectious biology.
Background Staphylococcus aureus is an important pathogen in cystic fibrosis (CF). However, it is not clear which factors are associated with worse lung function in patients with persistent S. aureus airway cultures. Our main hypothesis was that patients with high S. aureus density in their respiratory specimens would more likely experience worsening of their lung disease than patients with low bacterial loads. Methods Therefore, we conducted an observational prospective longitudinal multi-center study and assessed the association between lung function and S. aureus bacterial density in respiratory samples, co-infection with other CF-pathogens, nasal S. aureus carriage, clinical status, antibiotic therapy, IL-6- and IgG-levels against S. aureus virulence factors. Results 195 patients from 17 centers were followed; each patient had an average of 7 visits. Data were analyzed using descriptive statistics and generalized linear mixed models. Our main hypothesis was only supported for patients providing throat specimens indicating that patients with higher density experienced a steeper lung function decline (p<0.001). Patients with exacerbations (n = 60), S. aureus small-colony variants (SCVs, n = 84) and co-infection with Stenotrophomonas maltophilia (n = 44) had worse lung function (p = 0.0068; p = 0.0011; p = 0.0103). Patients with SCVs were older (p = 0.0066) and more often treated with trimethoprim/sulfamethoxazole (p = 0.0078). IL-6 levels positively correlated with decreased lung function (p<0.001), S. aureus density in sputa (p = 0.0016), SCVs (p = 0.0209), exacerbations (p = 0.0041) and co-infections with S. maltophilia (p = 0.0195) or A. fumigatus (p = 0.0496). Conclusions In CF-patients with chronic S. aureus cultures, independent risk factors for worse lung function are high bacterial density in throat cultures, exacerbations, elevated IL-6 levels, presence of S. aureus SCVs and co-infection with S. maltophilia. Trial Registration ClinicalTrials.gov NCT00669760
Background: Liver impairment, ranging from steatosis to cirrhosis, is frequent in cystic fibrosis (CF) patients and is becoming increasingly significant due to their improved life expectancy. One aspect of hepatic alterations is caused by increased fecal loss of the essential nutrient choline, following enterohepatic bile phosphatidylcholine (PC) cycle impairment. Hepatic PC synthesis, both de novo and via phosphatidylethanolamine-N-methyl-transferase (PEMT), is essential for very low-density lipoprotein (VLDL) secretion. VLDL-PC in particular contributes to the organism's supply with polyunsaturated fatty acids (LC-PUFA), namely arachidonic (C20:4) and docosahexaenoic acid (C22:6). Consequently, choline deprivation and altered hepatic PC metabolism may affect plasma PC homeostasis and extrahepatic organ function. Objectives: To investigate relationships between altered plasma choline and PC homeostasis and markers of lung function and inflammation in CF. To assess alterations in hepatic choline and PC metabolism of CF patients. Design: Quantification of plasma/serum choline and PC species in adult CF patients compared to controls. Correlation of PC with forced expiratory vital capacity (FEV1) and interleukin 6 (IL-6) concentrations. Analysis of choline and PC metabolism in CF compared to controls, using deuterated choline ([D9-methyl]-choline) labeling in vivo. Results: Mean choline and PC concentrations in CF patients were lower than in controls. Choline and PC concentrations as well as fractions of C22:6-PC and C20:4-PC correlated directly with FEV1, but inversely with IL-6. Plasma concentrations of deuterated PC were decreased for both pathways, whereas only in PC synthesized via PEMT precursor enrichment was decreased. Conclusion: In CF patients, hepatic and plasma homeostasis of choline and PC correlate with lung function and inflammation. Impaired hepatic PC metabolism, exemplarily shown in three CF patients, provides an explanation for such correlations. Larger studies are required to understand the link between hepatic PC metabolism and overall clinical performance of CF patients, and the perspective of choline substitution of these patients.
