Lungs Of Cystic Fibrosis Patients Research Articles

Role of CFTR,Pseudomonas aeruginosaand Toll-like receptors in cystic fibrosis lung inflammation

CF (cystic fibrosis) is a severe autosomal recessive disease most common in Northwest European populations. Underlying mutations in the CFTR (CF transmembrane conductance regulator) gene cause deregulation of ion transport and subsequent dehydration of the airway surface liquid, producing a viscous mucus layer on the airway surface of CF patients. This layer is readily colonized by bacteria such as Pseudomonas aeruginosa. Owing to the resulting environment and treatment strategies, the bacteria acquire genetic modifications such as antibiotic resistance, biofilm formation, antimicrobial peptide resistance and pro-inflammatory lipid A structures. Lipid A is a component of the lipopolysaccharide cell wall present on bacteria and is recognized by TLR4 (Toll-like receptor 4). Its detection elicits a pro-inflammatory response that is heightened over time due to the addition of fatty acids to the lipid A structure. Eradication of bacteria from the lungs of CF patients becomes increasingly difficult and eventually leads to mortality. In the present review, we describe the role of lipid A as a virulent factor of Ps. aeruginosa; however, it appears that further work is needed to investigate the role of CFTR in the innate immune response and in modifying the pathogen-host interaction.

Molecular analysis of diversity within the genus Pseudomonas in the lungs of cystic fibrosis patients

The genus Pseudomonas contains species that can act as human pathogens and are important in many infections such as those occurring in the lungs of many cystic fibrosis (CF) patients. Current methodologies that rely on the cultivation of bacteria are too cumbersome and often lack sufficient discriminatory power to define the diversity of Pseudomonas spp. As a result, molecular-based approaches have many advantages when attempting to differentiate between species of this genus. This study assessed the ability of terminal restriction fragment length polymorphism (T-RFLP) profiling of the 16S–23S rRNA ITS1 gene region to differentiate species of the genus Pseudomonas. Before application to clinical samples, this approach was validated on a panel of 10 different Pseudomonas spp. T-RFLP profiling of the 16S–23S rRNA ITS1 gene region amplified from these strains differentiated all Pseudomonas spp. tested. The presence of Pseudomonas spp. in CF sputum was assessed through the detection of this ITS1 gene region as amplified from DNA extracted from 40 samples of CF sputum. The ITS1 gene region was detected in 75% of these samples, including 5 from which no Pseudomonas spp. had been identified using culture-based methods. In silico analysis showed that all sequences amplified had a high homology with the ITS1 region of Pseudomonas aeruginosa. T-RFLP gave data that were consistent with the band being generated from P. aeruginosa for each patient. No correlation between Pseudomonas diversity and severity of lung disease was observed in this group of CF patients. This study has however shown that molecular analysis of the ITS1 region is effective in resolving diversity within the genus Pseudomonas. The wider use of this application is discussed.

Iron acquisition by Pseudomonas aeruginosa in the lungs of patients with cystic fibrosis

The bacterium Pseudomonas aeruginosa is commonly isolated from the general environment and also infects the lungs of patients with cystic fibrosis (CF). Iron in mammals is not freely available to infecting pathogens although significant amounts of extracellular iron are available in the sputum that occurs in the lungs of CF patients. P. aeruginosa has a large number of systems to acquire this essential nutrient and many of these systems have been characterised in the laboratory. However, which iron acquisition systems are active in CF is not well understood. Here we review recent research that sheds light on how P. aeruginosa obtains iron in the lungs of CF patients.

Approaches to analyse dynamic microbial communities such as those seen in cystic fibrosis lung

Microbial communities play vital roles in many aspects of our lives, although our understanding of microbial biogeography and community profiles remains unclear. The number of microbes or the diversity of the microbes, even in small environmental niches, is staggering. Current microbiological methods used to analyse these communities are limited, in that many microorganisms cannot be cultured. Even for the isolates that can be cultured, the expense of identifying them definitively is much too high to be practical. Many recent molecular technologies, combined with bioinformatic tools, are raising the bar by improving the sensitivity and reliability of microbial community analysis. These tools and techniques range from those that attempt to understand a microbial community from their length heterogeneity profiles to those that help to identify the strains and species of a random sampling of the microbes in a given sample. These technologies are reviewed here, using the microbial communities present in the lungs of cystic fibrosis patients as a paradigm.

Pyrogallol, an active compound from the medicinal plant Emblica officinalis, regulates expression of pro-inflammatory genes in bronchial epithelial cells

The most relevant cause of morbidity and mortality in cystic fibrosis (CF) patients is the lung pathology characterized by chronic infection and inflammation sustained mainly by Pseudomonas aeruginosa ( P. aeruginosa). Innovative pharmacological approaches to control the excessive inflammatory process in the lung of CF patients are thought to be beneficial to reduce the extensive airway tissue damage. Medicinal plants from the so-called traditional Asian medicine are attracting a growing interest because of their potential efficacy and safety. Due to the presence of different active compounds in each plant extract, understanding the effect of each component is important to pursue selective and reproducible applications. Extracts from Emblica officinalis (EO) were tested in IB3-1 CF bronchial epithelial cells exposed to the P. aeruginosa laboratory strain PAO1. EO strongly inhibited the PAO1-dependent expression of the neutrophil chemokines IL-8, GRO-α, GRO-γ, of the adhesion molecule ICAM-1 and of the pro-inflammatory cytokine IL-6. Pyrogallol, one of the compounds extracted from EO, inhibited the P. aeruginosa-dependent expression of these pro-inflammatory genes similarly to the whole EO extract, whereas a second compound purified from EO, namely 5-hydroxy-isoquinoline, had no effect. These results identify Pyrogallol as an active compound responsible for the anti-inflammatory effect of EO and suggest to extend the investigation in pre-clinical studies in airway animal models in vivo, to test the efficacy and safety of this molecule in CF chronic lung inflammatory disease.

Carotid ultrasound in children: a window to arterial ageing

Inflammation and proteolytic processes play an important role in the progression of cystic fibrosis (CF) lung disease. The goal of this study was to describe bronchoalveolar lavage fluid (BALF) protein...

SELECTIVE USE OF MEMBRANE CD14, BUT NOT SOLUBLE CD14 ANDINHIBITION BY SERUM IN THE INFLAMMATORY CYTOKINE RESPONSE INDUCED BY A NOVELTLR LIGAND (P. AERUGINOSA EXOENZYME S)

Background/Purpose: Soluble andmembrane CD14 (sCD14 and mCD14) bind microbial products, are toll-like receptor(TLR) coreceptors, and induce inflammation. Clinical relevance is demonstrated by a correlation of sCD14 levels with morbidity and mortality in inflammatory diseases. Moreover, P. aeruginosa induces damaging inflammation in the lungs of cysticfibrosis patients and its virulence is often attributed to the TLR2 and TLR4ligand exoenzyme S (ExoS). Because of the importance of the response of CD14 toTLR ligands and its potential for therapeutic manipulation, its role inExoS-induced inflammatory cytokines was investigated. Methods: The ability of ExoS to induce TNFand IL-6 cytokine production in cell lines that express TLR and either CD14 positive or negative was assessed (THP-1 or U373 cells, respectively). Recombinant CD14 or CD14 blocking agents were used to test the contribution ofmCD14 and sCD14. Results: Enhancing expression of mCD14 on THP-1 cells increased TNF production, which was abrogated by blocking or removing mCD14. Transfecting mCD14 into U373 cells demonstrated that mCD14 was required for binding ExoS and subsequent IL-6 production. Unlike TLR ligands that only stimulate one TLR, such as lipopolysaccharide, neither sCD14 nor serum enhanced production of cytokine in response to ExoS. Uniquely, serum inhibited ExoS induced TNF. Conclusion: This work demonstrates a fundamental difference in the requirement of ExoS and other TLR ligands for CD14, which must be considered when designing therapies to block microbe-induced inflammation. The presence of a potential therapeutic molecule in serum could help in the development of an ExoS neutralizing agent.

Immunoproteomics To Examine Cystic Fibrosis Host Interactions with Extracellular Pseudomonas aeruginosa Proteins

The lungs of patients with cystic fibrosis (CF) are typically chronically infected with Pseudomonas aeruginosa. We used an immunoproteomics approach to analyze the responses of patients to secreted P. aeruginosa proteins. Extracellular proteins from P. aeruginosa strain PAO1 that had been grown to stationary phase were separated by two-dimensional polyacrylamide gel electrophoresis and analyzed by Western blotting using sera from four chronically infected patients. Sera from all four patients detected multiple extracellular proteins. The identities of selected proteins recognized by antisera were determined. Production of at least four of these proteins (azurin and three proteases: elastase, PrpL, and PasP) is governed by quorum sensing, consistent with active bacterial quorum sensing in the lungs of CF patients. The CF lung is generally thought to be an iron-deficient environment for infecting bacteria, and growing the bacteria in the presence of an iron-chelating agent, ethylene-diamine-di(o-hydroxyphenylacetic acid), enabled detection of additional proteins that were recognized by patient sera. The sera also detected multiple proteins from cells in the logarithmic growth phase, and protein identification suggested that most of these were the result of cell lysis or secretion in membrane vesicles. Comparison with extracellular proteins from a second P. aeruginosa strain, strain Pa4, showed that many proteins recognized by patient sera are common to both strains, although there are also some strain-specific extracellular proteins. Our data show that while there are some differences in the responses of different patients to P. aeruginosa, there are also many similarities, and that an immunoproteomics approach enables the identification of proteins that are made by P. aeruginosa during infection.

The expression of alpha-haemolysin is required forStaphylococcus aureusphagosomal escape after internalization in CFT-1 cells

Staphylococcus aureus colonizes the lungs of cystic fibrosis patients and treatment with antibiotics usually results in recurrent and relapsing infections. We have shown that S. aureus can invade and replicate within a cystic fibrosis epithelial cell line (CFT-1), and that these internalized bacteria subsequently escape from the endocytic vesicle. The accessory gene regulator, agr, in S. aureus has been shown to control the expression of a large number of secreted toxins involved in virulence. Here we show that an agr mutant of S. aureus strain RN6390 was unable to escape from the endocytic vesicle after invasion of the CFT-1 cells using markers of vesicular trafficking (LAMP-1 and 2, LysoTracker and Vacuolar-ATPase). Trafficking analysis of live S. aureus which did not express alpha-haemolysin, a specific agr regulated toxin, revealed a defect in vesicular escape that was undistinguishable from the trafficking defect exhibited by the agr mutant. Furthermore, overexpression of alpha-haemolysin under an inducible promoter in an agr mutant of S. aureus partially restored the phagosome-escaping phenotype of an agr mutant. These results demonstrate that the expression of agr is required for vesicular escape, and that biologically active alpha-haemolysin is required for S. aureus escape from the endocytic vesicle into the cytosol of CFT-1 cells.

Increased Fitness of Pseudomonas fluorescens Pf0-1 Leucine Auxotrophs in Soil

The annotation process of a newly sequenced bacterial genome is largely based on algorithms derived from databases of previously defined RNA and protein-encoding gene structures. This process generally excludes the possibility that the two strands of a given stretch of DNA can each harbor a gene in an overlapping manner. While the presence of such structures in eukaryotic genomes is considered to be relatively common, their counterparts in prokaryotic genomes are just beginning to be recognized. Application of an in vivo expression technology has previously identified 22 discrete genetic loci in Pseudomonas fluorescens Pf0-1 that were specifically activated in the soil environment, of which 10 were present in an antisense orientation relative to previously annotated genes. This observation led to the hypothesis that the physiological role of overlapping genetic structures may be relevant to growth conditions outside artificial laboratory media. Here, we examined the role of one of the overlapping gene pairs, iiv19 and leuA2, in soil. Although iiv19 was previously demonstrated to be preferentially activated in the soil environment, its absence did not alter the ability of P. fluorescens to colonize or survive in soil. Surprisingly, the absence of the leuA2 gene conferred a fitness advantage in the soil environment when leucine was supplied exogenously. This effect was determined to be independent of the iiv19 gene, and further analyses revealed that amino acid antagonism was the underlying mechanism behind the observed fitness advantage of the bacterium in soil. Our findings provide a potential mechanism for the frequent occurrence of auxotrophic mutants of Pseudomonas spp. in the lungs of cystic fibrosis patients.

The DeltaF508-CFTR mutation results in increased biofilm formation by Pseudomonas aeruginosa by increasing iron availability.

Enhanced antibiotic resistance of Pseudomonas aeruginosa in the cystic fibrosis (CF) lung is thought to be due to the formation of biofilms. However, there is no information on the antibiotic resistance of P. aeruginosa biofilms grown on human airway epithelial cells or on the effects of airway cells on biofilm formation by P. aeruginosa. Thus we developed a coculture model and report that airway cells increase the resistance of P. aeruginosa to tobramycin (Tb) by >25-fold compared with P. aeruginosa grown on abiotic surfaces. Therefore, the concentration of Tb required to kill P. aeruginosa biofilms on airway cells is 10-fold higher than the concentration achievable in the lungs of CF patients. In addition, CF airway cells expressing DeltaF508-CFTR significantly enhanced P. aeruginosa biofilm formation, and DeltaF508 rescue with wild-type CFTR reduced biofilm formation. Iron (Fe) content of the airway in CF is elevated, and Fe is known to enhance P. aeruginosa growth. Thus we investigated whether enhanced biofilm formation on DeltaF508-CFTR cells was due to increased Fe release by airway cells. We found that airway cells expressing DeltaF508-CFTR released more Fe than cells rescued with WT-CFTR. Moreover, Fe chelation reduced biofilm formation on airway cells, whereas Fe supplementation enhanced biofilm formation on airway cells expressing WT-CFTR. These data demonstrate that human airway epithelial cells promote the formation of P. aeruginosa biofilms with a dramatically increased antibiotic resistance. The DeltaF508-CFTR mutation enhances biofilm formation, in part, by increasing Fe release into the apical medium.

Use of the gyrB gene to discriminate among species of the Burkholderia cepacia complex

Bacteria of the Burkholderia cepacia complex (Bcc) are opportunistic pathogens that can cause serious infections in lungs of cystic fibrosis patients. The Bcc comprises at least nine species that have been discriminated by a polyphasic taxonomic approach. In this study, we focused on the gyrB gene, universally distributed among bacteria, as a new target gene to discriminate among the Bcc species. New PCR primers were designed to amplify a gyrB DNA fragment of about 1900 bp from 76 strains representative of all Bcc species. Nucleotide sequences of PCR products were determined and showed more than 400 polymorphic sites with high sequence similarity values from most isolates of the same species. Phylogenetic tree analysis revealed that most of the 76 gyrB sequences grouped, forming clusters, each corresponding to a given Bcc species.

Comparative evaluation of chest radiography, low-field MRI, the Shwachman-Kulczycki score and pulmonary function tests in patients with cystic fibrosis

The aim of this study was to investigate whether the parenchymal lung damage in patients suffering from cystic fibrosis (CF) can be equivalently quantified by the Chrispin-Norman (CN) scores determined with low-field magnetic resonance imaging (MRI) and conventional chest radiography (CXR). Both scores were correlated with pulmonary function tests (PFT) and the Shwachman-Kulczycki method (SKM). To evaluate the comparability of MRI and CXR for different states of the disease, all scores were applied to patients divided into three age groups. Seventy-three CF patients (mean SKM score: 62 +/- 8) with a median age (range) of 14 years (7-32) were included. The mean CN scores determined with both imaging methods were comparable (CXR: 12.1 +/- 4.7; MRI: 12.0 +/- 4.5) and showed high correlation (P < 0.05, R = 0.97). Only weak correlations were found between imaging, PFT, and SKM. Both imaging modalities revealed significantly more severe disease expression with age, while PFT and SKM failed to detect early signs of disease. We conclude that imaging of the lung in CF patients is capable of detecting subtle and early parenchymal destruction before lung function or clinical scoring is affected. Furthermore, low-field MRI revealed high consistency with chest radiography and may be used for a thorough follow-up while avoiding radiation exposure.

Biofilm penetration, triggered release and in vivo activity of inhaled liposomal amikacin in chronic Pseudomonas aeruginosa lung infections

Chronic infections of Pseudomonas aeruginosa in the lungs of cystic fibrosis patients are intractable antibiotic targets because of their biofilm mode of growth. We have investigated the biofilm penetration, mechanism of drug release and in vivo antimicrobial activity of a unique nanoscale liposomal formulation of amikacin designed specifically for nebulization and inhaled delivery. Penetration of fluorescently labelled liposomes into sputum or P. aeruginosa (PA3064) biofilms was monitored by a filter assay and by epifluorescence or confocal scanning laser microscopy. Amikacin release in vitro and rat lung levels after inhalation of nebulized material were measured by fluorescence polarization immunoassay. A 14 day agar bead model of chronic Pseudomonas lung infection in rats was used to assess the efficacy of liposomal amikacin versus free aminoglycosides in the reduction of bacterial count. Fluorescent liposomes penetrated readily into biofilms and infected mucus, whereas larger (1 microm) fluorescent beads did not. Amikacin release from liposomes was mediated by sputum or Pseudomonas biofilm supernatants. Rhamnolipids were implicated as the major releasing factors in these supernatants, active at one rhamnolipid per several hundred lipids within the liposomes. Inhaled liposomal amikacin was released in a slow, sustained manner in normal rat lungs and was orders of magnitude more efficacious than inhaled free amikacin in infected lungs. Penetration of biofilm and targeted, sustained release from liposomes can explain the superior in vivo efficacy of inhaled liposomal amikacin versus free drug observed in a 14 day infection model. Inhaled liposomal amikacin may represent an important therapy for chronic lung infections.

Bilateral sequential lung and simultaneous pancreas transplant: A new approach for the recipient with cystic fibrosis

Cystic fibrosis (CF) is an inherited disorder that presents in childhood as a multisystem disease. Pulmonary failure and pancreatic insufficiency, including CF related diabetes (CFRD) and exocrine insufficiency, are common complications of this disease. In this report we review the first three simultaneous lung and pancreas transplantations in CF patients with diabetes. Methods All three CF patients presented for evaluation for lung transplantation and had pancreatic insufficiency requiring enzyme supplementation and CFRD requiring insulin. All were severely malnourished and required nutritional supplementation. Surgical technique In each case, the allografts were procured from a single cadaveric donor. Bilateral lung transplantation was performed first using two separate thoracic incisions. The pancreas transplant was performed with systemic venous drainage and enteric exocrine drainage. Results The pancreas allografts all functioned normally with normoglycemia independent of insulin. As a result of the enteric drainage of the pancreas allograft, supplemental pancreatic enzymes were no longer required. Despite several complications detailed in the manuscript, all three remain independent of supplemental oxygen, insulin and pancreatic enzyme replacement at 4, 6 and 14 months of follow-up. Conclusion Simultaneous lung and pancreas transplantation in patients with CF can be performed successfully and provides the advantages of normoglycemia and improves nutrition for patients requiring lung transplantation.

Assessment of Morphological MRI for Pulmonary Changes in Cystic Fibrosis (CF) Patients

As pulmonary complications are life limiting in patients with cystic fibrosis (CF), repeated chest imaging [chest x-ray, computed tomography (CT)] is needed for follow up. With the continuously rising life expectancy of CF patients, magnetic resonance imaging (MRI) as a radiation-free imaging modality might become more and more attractive. The goal of this study was to prospectively assess the value of MRI for evaluation of morphologic pulmonary CF-changes in comparison to established imaging modalities. Thirty-one CF patients (19 female, 12 male; mean age 16.7 years) with stable lung disease were examined by MRI: HASTE, coronal/transversal (TR/TE/alpha/TA: infinite/28 ms/180 degrees /18 s), multi-detector computed tomography (MDCT) (30 patients): 120 kV, dose modulated mAs, and chest x-ray (21 patients). Image evaluation: random order, 4 chest radiologists in consensus; chest x-ray: modified Chrispin-Norman score; CT and MRI: modified Helbich score. The maximal attainable score for chest x-ray was 34, for MRI and CT 25. Median scores, Pearson correlation coefficients, Bland-Altman plots, and concordance of MRI to CT on a lobar and segmental basis were calculated. The median MRI and MDCT scores were 13 (min 3, max 20) respectively 13.5 (min 0, max 20). The median chest x-ray score was 14 (min 5, max 32). Pearson correlation coefficients: MRI/CT = 0.80, P < 0.0001; MRI/chest x-ray = 0.63, P < 0.0018; chest x-ray/CT = 0.75, P < 0.0001. The median lobe related concordance was 80% for bronchiectasis, 77% for mucus plugging, 93%, for sacculation/abscesses, and 100% for collapse/consolidation. Morphologic MRI of the lung in CF patients demonstrates comparable results to MDCT and chest x-ray. Because radiation exposure is an issue in CF patients, MRI might have the ability to be used as an appropriate alternative method for pulmonary imaging.

Sputum antibiotic concentrations: Implications for treatment of cystic fibrosis lung infection

The success of antibiotic therapy may be predicted based on the achievement of pharmacodynamic indices (PDIs), which are determined by the susceptibility of the infecting bacteria and the concentrations of antibiotics achieved at the site of infection. The aim of this study was to determine whether PDIs associated with clinical effectiveness for ceftazidime and tobramycin were achieved at the site of infection in the lungs of cystic fibrosis (CF) patients following intravenous administration during treatment of an acute exacerbation. Serum and sputum samples were collected from 14 CF patients and the concentration of both antibiotics in the samples determined. The susceptibility of bacteria cultured from sputum samples to both antibiotics alone and in combination was also determined. A total of 22 Pseudomonas aeruginosa isolates and 4 Burkholderia cepacia complex isolates were cultured from sputum samples with 55% and 4% of isolates susceptible to ceftazidime and tobramycin, respectively. Target PDIs for ceftazidime and tobramycin, an AUC/MIC ratio of 100 and a C(max)/MIC ratio of 10, respectively, were not achieved in serum or sputum simultaneously or even individually for any patient. Although the combination of ceftazidime and tobramycin was synergistic against 20 of the 26 isolates cultured, the concentrations of both antibiotics required for synergy were achieved simultaneously in only 38% of serum and 14% of sputum samples. Key PDIs associated with clinical effectiveness for ceftazidime and tobramycin were not achieved at the site of infection in the lungs of CF patients.

Oxidative stress induces extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase in cystic fibrosis lung epithelial cells: Potential mechanism for excessive IL-8 expression

Cystic fibrosis (CF) is a lethal disease caused by defective function of the cftr gene product, the CF transmembrane conductance regulator (CFTR) that leads to oxidative damage and excessive inflammatory response in lungs of CF patients. We here report the effects of oxidative stress (hyperoxia, 95% O 2) on the expression of pro-inflammatory interleukin (IL)-8 and CXCR1/2 receptors in two human CF lung epithelial cell lines (IB3-1, with the heterozygous F508del/W1282X mutation and CFBE41o- with the homozygous F508del/F508del mutation) and two control non-CF lung epithelial cell lines (S9 cell line derived from IB3-1 after correction with wtCFTR and the normal bronchial cell line 16HBE14o-). Under oxidative stress, the expression of IL-8 and CXCR1/2 receptors was increased in CF, corrected and normal lung cell lines. The effects of oxidative stress were also investigated by measuring the transcription nuclear factor κB (NF-κB) and activator protein-1 (AP-1) activities. Under oxidative stress, no increase of NF-κB activation was observed in CF lung cells in contrast to that observed in normal and corrected CF lung cells. The signalling of mitogen-activated protein (MAP) kinases was further studied. We demonstrated that extracellular signal-regulated kinase (ERK1/2) and AP-1 activity was markedly enhanced in CF but not non-CF lung cells under oxidative stress. Consistently, inhibition of ERK1/2 in oxidative stress-exposed CF lung cells strongly decreased both the IL-8 production and CXCR1/2 expression. Therefore, targeting of ERK1/2 MAP kinase may be critical to reduce oxidative stress-mediated inflammation in lungs of CF patients.

In Vivo Evidence of Pseudomonas aeruginosa Nutrient Acquisition and Pathogenesis in the Lungs of Cystic Fibrosis Patients

One of the hallmarks of Pseudomonas aeruginosa infection in cystic fibrosis (CF) patients is very-high-cell-density (HCD) replication in the lung, allowing this bacterium to induce virulence controlled by the quorum-sensing systems. However, the nutrient sources sustaining HCD replication in this chronic infection are largely unknown. Here, we performed microarray studies of P. aeruginosa directly isolated from the lungs of CF patients to demonstrate its metabolic capability and virulence in vivo. In vivo microarray data, confirmed by real-time reverse transcription-PCR, indicated that the P. aeruginosa population expressed several genes for virulence, drug resistance, and utilization of multiple nutrient sources (lung surfactant lipids and amino acids) contributing to HCD replication. The most abundant lung surfactant lipid molecule, phosphatidylcholine (PC), induces key genes of P. aeruginosa pertinent to PC degradation in vitro as well as in vivo within the lungs of CF patients. The results support recent research indicating that P. aeruginosa exists in the lungs of CF patients as a diverse population with full virulence potential. The data also indicate that there is deregulation of several pathways, suggesting that there is in vivo evolution by deregulation of a large portion of the transcriptome during chronic infection in CF patients. To our knowledge, this is the first in vivo transcriptome analysis of P. aeruginosa in a natural infection in CF patients, and the results indicate several important aspects of P. aeruginosa pathogenesis, drug resistance, nutrient utilization, and general metabolism within the lungs of CF patients.

Burkholderia cenocepacia ET12 strain activates TNFR1 signalling in cystic fibrosis airway epithelial cells

Burkholderia cenocepacia is an important pulmonary pathogen in individuals with cystic fibrosis (CF). Infection is often associated with severe pulmonary inflammation, and some patients develop a fatal necrotizing pneumonia and sepsis ('cepacia syndrome'). The mechanisms by which this species causes severe pulmonary inflammation are poorly understood. Here, we demonstrate that B. cenocepacia BC7, a potentially virulent representative of the epidemic ET12 lineage, binds to tumour necrosis factor receptor 1 (TNFR1) and activates TNFR1-related signalling pathway similar to TNF-alpha, a natural ligand for TNFR1. This interaction participates in stimulating a robust IL-8 production from CF airway epithelial cells. In contrast, BC45, a less virulent ET12 representative, and ATCC 25416, an environmental B. cepacia strain, do not bind to TNFR1 and stimulate only minimal IL-8 production from CF cells. Further, TNFR1 expression is increased in CF airway epithelial cells compared with non-CF cells. We also show that B. cenocepacia ET12 strain colocaizes with TNFR1 in vitro and in the lungs of CF patients who died due to infection with B. cenocepacia, ET12 strain. Together, these results suggest that interaction of B. cenocepacia, ET12 strain with TNFR1 may contribute to robust inflammatory responses elicited by this organism.