Cystic Fibrosis Sputum Samples Research Articles

Culture-Independent Identification of Nontuberculous Mycobacteria in Cystic Fibrosis Respiratory Samples.

Respiratory tract infections with nontuberculous mycobacteria (NTM) are increasing in prevalence and are a significant cause of lung function decline in individuals with cystic fibrosis (CF). NTM have been detected in culture-independent analyses of CF airway microbiota at lower rates than would be expected based on published prevalence data, likely due to poor lysing of the NTM cell wall during DNA extraction. We compared a standard bacterial lysis protocol with a modified method by measuring NTM DNA extraction by qPCR and NTM detection with bacterial 16S rRNA gene sequencing. The modified method improved NTM DNA recovery from spiked CF sputum samples by a mean of 0.53 log10 copies/mL for M. abscessus complex and by a mean of 0.43 log10 copies/mL for M. avium complex as measured by qPCR targeting the atpE gene. The modified method also improved DNA sequence based NTM detection in NTM culture-positive CF sputum and bronchoalveolar lavage samples; however, both qPCR and 16S rRNA gene sequencing remained less sensitive than culture for NTM detection. We highlight the limitations of culture-independent identification of NTM from CF respiratory samples, and illustrate how alterations in the bacterial lysis and DNA extraction process can be employed to improve NTM detection with both qPCR and 16S rRNA gene sequencing.

A small molecule neutrophil elastase inhibitor, KRP-109, inhibits cystic fibrosis mucin degradation

BackgroundNeutrophil elastase (NE) rapidly degrades gel-forming airway mucins in cystic fibrosis (CF) sputum. We hypothesized that KRP-109, a small molecule NE inhibitor, would inhibit CF mucin degradation in vitro. MethodsSputa were collected from CF patients (n=5) chronically or intermittently infected with Pseudomonas aeruginosa (P.a.). Mucin degradation was analyzed using western blot. Protease inhibitor studies were performed using alpha1-proteinase inhibitor (A1-PI Prolastin®) and KRP-109. Elastase activity assays were performed using spectrophotometry. ResultsThere were significant differences in the amount of active NE in different CF sputum samples. KRP-109 decreased the NE driven mucin degradation in vitro. Pseudomonas elastases appeared to blunt elastase inhibition by A1-PI or KRP-109. ConclusionInhibitors of neutrophil and Pseudomonas-derived elastases might rescue mucus clearance and reverse airway obstruction in CF.

94 Isolation and characterisation of bacteriophage infecting Prevotella spp. recovered from the cystic fibrosis (CF) airways

Objectives Prevotella spp. are the predominant anaerobes detected in CF sputum samples. Whether these organisms contribute to the pathogenicity of chronic lung disease is currently unclear. Bacterial pathogenicity is increasingly being associated with the carriage of temperate bacteriophage. As such, this study aimed to isolate and characterise bacteriophage infecting Prevotella spp. recovered from CF sputum samples. Methods Prevotella spp. (N = 6) were cultivated in basal anaerobic media, under anaerobic conditions, until late stationary/decline phase. Bacteria were pelleted by centrifugation and the supernatant passed through 0.22 µm filters before bacteriophage were concentrated using high speed centrifugation. Phage particles were stained with 2% uranyl acetate and viewed using a transmission electron microscope. Results Bacteriophage like particles with uniformity and icosahedral capsid structures that characterise many bacteriophage species were recovered from all Prevotella cultures. Conclusion CF Prevotella isolates harbour a variety of bacteriophage like particles. Preliminary investigations suggest that the production of these particles may occur in response to increases in bacterial population density. Ongoing genomic analysis may allow further characterisation of these bacteriophage like particles and provide insight into any potential effects they may have on bacterial virulence.

Impact of storage conditions on metabolite profiles of sputum samples from persons with cystic fibrosis

BackgroundAlthough recent studies have begun to elucidate how airway microbial community structure relates to lung disease in cystic fibrosis (CF), microbial community activity and the host's response to changes in this activity are poorly understood. Metabolomic profiling provides a means to investigate microbial activity and human cell activity within diseased airways. However, variables in sample storage and shipping likely affect downstream analyses and standards for sample handling are lacking. MethodsWe assessed the impact of sample storage conditions on liquid chromatography mass spectrometry analysis of CF sputum samples. ResultsSignificant changes in global metabolomic profiles occurred in samples stored at room temperature or at 4°C for longer than one day. Untargeted metabolomic profiles were stable in sputum samples stored at −20°C or −80°C for at least 28days. Quorum sensing molecules and phenazines, both considered important to the in vivo activity of Pseudomonas during airway infection, were detected after sample storage at room temperature for five days. ConclusionsSputum samples can be stored at −20°C or −80°C for weeks with minimal effect on global metabolomic profiles. This observation provides guidance in designing metabolomic studies that have the potential to deepen our understanding of how airway microbial communities impact lung disease progression in CF.

Purifying the impure: sequencing metagenomes and metatranscriptomes from complex animal-associated samples.

The accessibility of high-throughput sequencing has revolutionized many fields of biology. In order to better understand host-associated viral and microbial communities, a comprehensive workflow for DNA and RNA extraction was developed. The workflow concurrently generates viral and microbial metagenomes, as well as metatranscriptomes, from a single sample for next-generation sequencing. The coupling of these approaches provides an overview of both the taxonomical characteristics and the community encoded functions. The presented methods use Cystic Fibrosis (CF) sputum, a problematic sample type, because it is exceptionally viscous and contains high amount of mucins, free neutrophil DNA, and other unknown contaminants. The protocols described here target these problems and successfully recover viral and microbial DNA with minimal human DNA contamination. To complement the metagenomics studies, a metatranscriptomics protocol was optimized to recover both microbial and host mRNA that contains relatively few ribosomal RNA (rRNA) sequences. An overview of the data characteristics is presented to serve as a reference for assessing the success of the methods. Additional CF sputum samples were also collected to (i) evaluate the consistency of the microbiome profiles across seven consecutive days within a single patient, and (ii) compare the consistency of metagenomic approach to a 16S ribosomal RNA gene-based sequencing. The results showed that daily fluctuation of microbial profiles without antibiotic perturbation was minimal and the taxonomy profiles of the common CF-associated bacteria were highly similar between the 16S rDNA libraries and metagenomes generated from the hypotonic lysis (HL)-derived DNA. However, the differences between 16S rDNA taxonomical profiles generated from total DNA and HL-derived DNA suggest that hypotonic lysis and the washing steps benefit in not only removing the human-derived DNA, but also microbial-derived extracellular DNA that may misrepresent the actual microbial profiles.

Purifying the Impure: Sequencing Metagenomes and Metatranscriptomes from Complex Animal-associated Samples

The accessibility of high-throughput sequencing has revolutionized many fields of biology. In order to better understand host-associated viral and microbial communities, a comprehensive workflow for DNA and RNA extraction was developed. The workflow concurrently generates viral and microbial metagenomes, as well as metatranscriptomes, from a single sample for next-generation sequencing. The coupling of these approaches provides an overview of both the taxonomical characteristics and the community encoded functions. The presented methods use Cystic Fibrosis (CF) sputum, a problematic sample type, because it is exceptionally viscous and contains high amount of mucins, free neutrophil DNA, and other unknown contaminants. The protocols described here target these problems and successfully recover viral and microbial DNA with minimal human DNA contamination. To complement the metagenomics studies, a metatranscriptomics protocol was optimized to recover both microbial and host mRNA that contains relatively few ribosomal RNA (rRNA) sequences. An overview of the data characteristics is presented to serve as a reference for assessing the success of the methods. Additional CF sputum samples were also collected to (i) evaluate the consistency of the microbiome profiles across seven consecutive days within a single patient, and (ii) compare the consistency of metagenomic approach to a 16S ribosomal RNA gene-based sequencing. The results showed that daily fluctuation of microbial profiles without antibiotic perturbation was minimal and the taxonomy profiles of the common CF-associated bacteria were highly similar between the 16S rDNA libraries and metagenomes generated from the hypotonic lysis (HL)-derived DNA. However, the differences between 16S rDNA taxonomical profiles generated from total DNA and HL-derived DNA suggest that hypotonic lysis and the washing steps benefit in not only removing the human-derived DNA, but also microbial-derived extracellular DNA that may misrepresent the actual microbial profiles.

Characterization and quantification of the fungal microbiome in serial samples from individuals with cystic fibrosis.

BackgroundHuman-associated microbial communities include fungi, but we understand little about which fungal species are present, their relative and absolute abundances, and how antimicrobial therapy impacts fungal communities. The disease cystic fibrosis (CF) often involves chronic airway colonization by bacteria and fungi, and these infections cause irreversible lung damage. Fungi are detected more frequently in CF sputum samples upon initiation of antimicrobial therapy, and several studies have implicated the detection of fungi in sputum with worse outcomes. Thus, a more complete understanding of fungi in CF is required.ResultsWe characterized the fungi and bacteria in expectorated sputa from six CF subjects. Samples were collected upon admission for systemic antibacterial therapy and upon the completion of treatment and analyzed using a pyrosequencing-based analysis of fungal internal transcribed spacer 1 (ITS1) and bacterial 16S rDNA sequences. A mixture of Candida species and Malassezia dominated the mycobiome in all samples (74%–99% of fungal reads). There was not a striking trend correlating fungal and bacterial richness, and richness showed a decline after antibiotic therapy particularly for the bacteria. The fungal communities within a sputum sample resembled other samples from that subject despite the aggressive antibacterial therapy. Quantitative PCR analysis of fungal 18S rDNA sequences to assess fungal burden showed variation in fungal density in sputum before and after antibacterial therapy but no consistent directional trend. Analysis of Candida ITS1 sequences amplified from sputum or pure culture-derived genomic DNA from individual Candida species found little (<0.5%) or no variation in ITS1 sequences within or between strains, thereby validating this locus for the purpose of Candida species identification. We also report the enhancement of the publically available Visualization and Analysis of Microbial Population Structures (VAMPS) tool for the analysis of fungal communities in clinical samples.ConclusionsFungi are present in CF respiratory sputum. In CF, the use of intravenous antibiotic therapy often does not profoundly impact bacterial community structure, and we observed a similar stability in fungal species composition. Further studies are required to predict the effects of antibacterials on fungal burden in CF and fungal community stability in non-CF populations.

Proposal of a quantitative PCR-based protocol for an optimal Pseudomonas aeruginosa detection in patients with cystic fibrosis

BackgroundThe lung of patients with cystic fibrosis (CF) is particularly sensitive to Pseudomonas aeruginosa. This bacterium plays an important role in the poor outcome of CF patients. During the disease progress, first acquisition of P. aeruginosa is the key-step in the management of CF patients. Quantitative PCR (qPCR) offers an opportunity to detect earlier the first acquisition of P. aeruginosa by CF patients. Given the lack of a validated protocol, our goal was to find an optimal molecular protocol for detection of P. aeruginosa in CF patients.MethodsWe compared two formerly described qPCR formats in early detection of P. aeruginosa in CF sputum samples: a qPCR targeting oprL gene, and a multiplex PCR targeting gyrB and ecfX genes.ResultsTested in vitro on a large panel of P. aeruginosa isolates and others gram-negative bacilli, oprL qPCR exhibited a better sensitivity (threshold of 10 CFU/mL versus 730 CFU/mL), whereas the gyrB/ecfX qPCR exhibited a better specificity (90% versus 73%). These results were validated ex vivo on 46 CF sputum samples positive for P. aeruginosa in culture. Ex vivo assays revealed that qPCR detected 100 times more bacterial cells than culture-based method did.ConclusionBased on these results, we proposed a reference molecular protocol combining the two qPCRs, which offers a sensitivity of 100% with a threshold of 10 CFU/mL and a specificity of 100%. This combined qPCR-based protocol can be adapted and used for other future prospective studies.

P89 The Effect of Sample Handling on Viable Bacterial Community Profiles from Cystic Fibrosis Sputum Samples

IntroductionObtaining accurate information on the composition of Cystic fibrosis (CF) lung infections is essential for the selection of appropriate therapy and maintenance of respiratory function. Whilst significant advances have been...

Contribution by Polymorphonucleate Granulocytes to Elevated Gamma-Glutamyltransferase in Cystic Fibrosis Sputum

BackgroundCystic fibrosis (CF) is an autosomal recessive disorder characterized by a chronic neutrophilic airways inflammation, increasing levels of oxidative stress and reduced levels of antioxidants such as glutathione (GSH). Gamma-glutamyltransferase (GGT), an enzyme induced by oxidative stress and involved in the catabolism of GSH and its derivatives, is increased in the airways of CF patients with inflammation, but the possible implications of its increase have not yet been investigated in detail.Principal FindingsThe present study was aimed to evaluate the origin and the biochemical characteristics of the GGT detectable in CF sputum. We found GGT activity both in neutrophils and in the fluid, the latter significantly correlating with myeloperoxidase expression. In neutrophils, GGT was associated with intracellular granules. In the fluid, gel-filtration chromatography showed the presence of two distinct GGT fractions, the first corresponding to the human plasma b-GGT fraction, the other to the free enzyme. The same fractions were also observed in the supernatant of ionomycin and fMLP-activated neutrophils. Western blot analysis confirmed the presence of a single band of GGT immunoreactive peptide in the CF sputum samples and in isolated neutrophils.ConclusionsIn conclusion, our data indicate that neutrophils are able to transport and release GGT, thus increasing GGT activity in CF sputum. The prompt release of GGT may have consequences on all GGT substrates, including major inflammatory mediators such as S-nitrosoglutathione and leukotrienes, and could participate in early modulation of inflammatory response.

Reverse line blot hybridisation screening of Pseudallescheria/Scedosporium species in patients with cystic fibrosis

The PCR-RLB (reverse line blot hybridisation) was applied as a molecular technique for the detection of members of Pseudallescheria and Scedosporium from sputum of patients with cystic fibrosis (CF). Fifty-nine sputum samples were collected from 52 CF patients, which were analysed by culture and PCR-RLB. Conventional and semi-selective culture yielded five positive samples, but the PCR-RLB hybridisation assay permitted the detection of members of Pseudallescheria/Scedosporium in 32 out of 52 patients (61.5%). In total, PCR-RLB yielded 47 positives. Pseudallescheria apiosperma was detected in 20 samples, while Pseudallescheria boydii and Pseudallescheria aurantiacum were detected in 17 and eight samples, respectively. Six samples gave a positive reaction with two distinct species-specific probes and one sample with three probes. In conclusion, the PCR-RLB assay described in this study allows the detection of Scedosporium spp. in CF sputum samples and the identification of Pseudallescheria apiosperma, P. boydii, S. aurantiacum, Scedosporium prolificans and Pseudallescheria minutispora.

140 Retrospective analysis of isolation of non-tuberculous mycobacteria from cystic fibrosis sputum samples

140 Retrospective analysis of isolation of non-tuberculous mycobacteria from cystic fibrosis sputum samples

Lack of standardization in the procedures for mycological examination of sputum samples from CF patients: a possible cause for variations in the prevalence of filamentous fungi

Filamentous fungi and yeasts are increasingly isolated from respiratory secretions of patients with cystic fibrosis (CF), and persistent fungal colonization of the airways of such patients is thought to exacerbate lung damage. While many independent studies have identified Aspergillus fumigatus complex as the principal colonizing fungus in CF, increased awareness of the role of fungi in CF pathology coupled with improved mycological culture and identification methods have resulted in a number of other fungi being isolated and reported from CF sputum samples, including A. terreus, members of the Pseudallescheria boydii/Scedosporium apiospermum complex, Exophiala dermatitidis, Paecilomyces and Penicillium species. However, the range of fungal pathogens isolated and the relative prevalence of individual species vary widely between reports from different geographical CF centres, and as yet no standardized method for the mycological examination of CF sputum samples has been adopted. Here, we examine the potential contribution of the mycological methods employed to examine CF respiratory secretions relative to the variability in the fungal biota reported. The role of direct microscopic examination of respiratory samples and the impact of the culture conditions used on the detection of specific fungal pathogens are addressed, and the potential significance of isolation of yeast species from CF patient airways is discussed.

Modelling the bacterial communities associated with cystic fibrosis lung infections

In many human diseases that cystic fibrosis (CF) patients suffer from, for example, lung infections, bacteria have been considered to grow as biofilms. The ability of key CF pathogens such as Pseudomonas aeruginosa to resist antibiotic therapies may be due to the poor drug penetration of these biofilms. The overall aim of this study was to develop biofilm models in vitro that resembled the bacterial species composition of CF sputa. Here, this was a step towards a longer term goal of forming multiple bacterial biofilm models in vitro that would serve, in turn, as better assays of antibiotic susceptibilities than conventionally grown cells. Biofilm models were constructed from 31 CF sputum samples, using a modified microtitre plate assay. Three forms of assessment of these biofilms were made, namely, the mass, microscopic analysis and species composition. Species composition in sputa and biofilms, characterised by terminal restriction fragment length polymorphism (T-RFLP) analysis of ribosomal gene polymerase chain reaction (PCR) products amplified from directly extracted nucleic acids, indicated that the bacterial community in sputa was well reproduced in the biofilm models. Typically, fresh sputa contained 4.6 +/- 2.3 bacterial species, with the species number decreasing to 4.0 +/- 1.6 over 5 days-this was not statistically significant (p = 0.29). This study outlines a novel methodology by which to generate and study bacterial biofilms communities. It is also hoped that the versatility of this in vitro approach, combined with its simplicity and high reproducibility, will make it an effective system to study CF sputum biofilm development and, in the longer term, serve as a means of assessing antibiotic susceptibilities.

Assessing the diagnostic importance of nonviable bacterial cells in respiratory infections

Identification of bacteria in clinical samples is fundamental to combating infections. Modern molecular genetic approaches exploit nucleic acids signals from clinical samples. However, DNA-derived signals can originate from nonviable bacterial cells and, therefore, generate data that could be misinterpreted. Terminal restriction fragment length polymorphism profiling of cystic fibrosis sputum samples was combined with propidium monoazide (PMA) photo-induced cross-linking. PMA is highly membrane impermeant and is excluded from viable bacteria but readily penetrates dead cells. Exposure to a light source renders DNA in permeable cells incapable of contributing to polymerase chain reaction. PMA treatment was shown to effectively prevent dead bacteria, spiked into sputum samples, from contributing to profiles. Comparison of treated and untreated clinical samples indicated that dead bacterial cells significantly bias untreated profiles. These findings highlight the significant contribution that nonviable bacteria can make to DNA-based diagnostic analysis of clinical samples while providing a simple and effective means of avoiding such bias.

Investigation of the algT operon sequence in mucoid and non-mucoid Pseudomonas aeruginosa isolates from 115 Scandinavian patients with cystic fibrosis and in 88 in vitro non-mucoid revertants

Pseudomonas aeruginosa is the dominant pathogen causing chronic lung infections in patients with cystic fibrosis (CF). After an initial phase characterized by intermittent colonizations, a chronic infection is established upon conversion of P. aeruginosa from the non-mucoid to the mucoid, alginate-overproducing phenotype. During the chronic infection the isolation of both mucoid and non-mucoid isolates in CF sputum samples is very common. The purpose of the present study was to establish, by sequence analysis, the types of mutations present in the algTmucABD operon in a large number of mucoid and non-mucoid P. aeruginosa isolates from Scandinavian CF patients and in in vitro-derived non-mucoid revertants. Mucoid (83) and non-mucoid isolates (103) from 91 Scandinavian patients with chronic P. aeruginosa infection and 24 non-mucoid isolates from intermittently colonized CF patients were investigated. In addition, 88 spontaneous non-mucoid revertants obtained in vitro from nine mucoid CF isolates were also included in the study. Mutations in mucA were found in 92 % of the mucoid and in up to 70 % of the non-mucoid isolates from chronically infected patients, indicating that the majority of non-mucoid isolates are revertants. None of the non-mucoid isolates from intermittently colonized CF patients harboured mucA mutations. Although algT has been considered an important gene for secondary-site mutations responsible for reversion to non-mucoidy, only 30 % of the mucA-mutated non-mucoid CF isolates had mutations in algT. In contrast, 83 % of the in vitro-derived spontaneous non-mucoid revertants had mutations in algT, showing that in the CF lung there is a selection for non-mucoid revertants with secondary-site mutations in genes other than algT. In addition, we report, to our knowledge for the first time, loss-of-function mutations in the negative regulators mucB and mucD in CF clinical isolates. In some of the CF isolates these mutations are associated with moderate alginate production. In conclusion, most non-mucoid isolates from chronically infected CF patients are revertants and the mechanism of revertance is algT-independent in the CF lung.

Inflammatory Lung Secretions Inhibit Dendritic Cell Maturation and Function via Neutrophil Elastase

Continuous episodes of infection are a feature of lung diseases such as chronic obstructive pulmonary disease (COPD) and cystic fibrosis (CF). Lung antigen-presenting dendritic cells (DCs) sample inhaled antigen to initiate immune responses. Therefore, we hypothesized that inflammatory mediators, such as neutrophil elastase (NE) released into the lung, may be able to modulate their activity. To determine whether sputum (from patients with COPD and those with CF) or NE can alter DC phenotype and function. NE and sputum samples were incubated with immature or mature murine DCs (mDCs). DC phenotype and function were studied by fluorescence-activated cell sorter and Western Blot analysis, assessing their expression of costimulatory molecules and their ability to induce T cell proliferation. COPD/CF sputum samples and human NE downregulated the expression of CD40, CD80, and CD86 (but not major histocompatibility complex II) on DCs and inhibited LPS-induced DC maturation. This effect was partially (sputa) to significantly (NE) reversed by addition of recombinant secretory leukocyte protease inhibitor. Western Blot analysis showed that purified NE degraded CD86 in mDC lysates in a time- and dose-dependent fashion, and caused shedding of CD86 into the supernatants of mDC cultures. NE treatment also inhibited the antigen-presenting ability of mDCs, as measured by their ability to induce ovalbumin-specific D011.10-transgenic T-cell proliferation. Our data indicate that NE in lung inflammatory secretions of patients with COPD/CF may disable DCs and prevent them from mounting an adequate immune response. This may have implications for the infection-driven generation of disease exacerbations in these two pathologies.

202 Cystic Fibrosis sputum samples: too much, too soon?

202 Cystic Fibrosis sputum samples: too much, too soon?

Antioxidant properties of cystic fibrosis sputum

Oxidative stress is a likely contributor to the pathogenesis of cystic fibrosis (CF) lung disease. However, hydrogen peroxide (H(2)O(2)), a physiological oxidant, is not elevated in CF exhalates. H(2)O(2) may be neutralized by antioxidants in CF airway secretions. The H(2)O(2)-detoxifying capacity of CF airway secretions, obtained via sputum induction, was studied in an in vitro H(2)O(2) cytotoxicity model. 16HBE14o- cells were exposed to H(2)O(2) in culture medium containing either 0 or 10% fetal bovine serum (FBS) or 10% CF sputum supernatant (extracted without use of dithiothreitol). The efficiency of H(2)O(2) neutralization was estimated by measuring intracellular oxidant levels (dihydrorhodamine 123) after 2 h and cell viability (propidium iodide) after 24 h of H(2)O(2) exposure. Furthermore, the presence of reduced thiols (DTNB assay) and reduced glutathione (recycling assay) in CF sputum samples was evaluated. CF sputum extracts completely prevented intracellular oxidant accumulation seen in cells incubated with H(2)O(2) in both control media (i.e., 0 or 10% FBS). Furthermore, CF sputum abolished cell death in 16HBE14o- cells exposed to up to 1 mM H(2)O(2). In contrast, there was 100% cytotoxicity in cells exposed to 600 microM H(2)O(2) in both control media. The H(2)O(2)-detoxifying potential of CF sputum was sustained after catalase and heme peroxidases were inactivated by sodium azide, which does not affect glutathione peroxidase. In addition, reduced protein thiols were found in abundance in CF sputum. In conclusion, CF sputum is capable to neutralize H(2)O(2) and abundant reduced thiols and/or glutathione peroxidase are fully sufficient to detoxify H(2)O(2).

Production of the potent neutrophil chemokine, growth-related protein α (GRO α), is not elevated in cystic fibrosis children

Progressive neutrophil-mediated lung damage causes much of the morbidity and mortality in cystic fibrosis (CF). Neutrophil chemoattractants implicated in CF include interleukin (IL-)8, tumour necrosis factor (TNFα ) and leukotriene (LT)B4, but growth-related protein α (GRO α), a highly potent neutrophil chemokine, has not been investigated. Atopic status has been considered to contribute to the marked heterogeneity of pulmonary disease in CF. We hypothesized that GRO α may be produced in biologically-significant amounts in the CF lung, and that enhanced production of GRO α, IL-8 or LTB4may contribute to the poorer lung function seen in atopic CF patients compared to non-atopic CF patients. GRO α, IL-8 and LTB4levels in the sputum of atopic and non-atopic CF patients were assessed by immunoassays, and GRO α and IL-8 levels were also assessed in the plasma of CF patients and normal controls. As expected, there were high levels of IL-8 and LTB4in most CF sputum samples, and IL-8 levels were higher in CF plasma than in control plasma (P=0·02). In contrast, GROα was undetectable (<5 pg ml−1) in the sputum of 21 out of 25 CF patients, with low levels (range 144–825 pg ml−1) in the remainder, and median levels of GRO α in CF plasma (33 pg ml−1, n=24) were not significantly different from controls (34 pg ml−1,n =25). Lung function [forced expiratory volume in 1 sec (FEV1) and forced vital capacity (FVC)] was significantly poorer in atopic CF compared to non-atopic CF patients (P<0·02), but sputum levels of GRO α, IL-8 and LTB4were not different between the subgroups. Our results suggest that unlike LTB4and IL-8, GRO α does not contribute to neutrophilic inflammation in the CF lung, and other factors must determine the impaired lung function observed in atopic CF patients. These results may have important implications in the development of chemokine receptor antagonists as novel anti-inflammatory agents in CF.