Sputum Of Cystic Fibrosis Patients Research Articles

Characterization of Pseudomonas aeruginosa strains isolated from cystic fibrosis patients by different typing methods

Pseudomonas (P.) aeruginosa strains isolated from the sputum of cystic fibrosis patients (CFP) are frequently difficult to type by conventional typing methods. The purpose of this study was to develop a random amplified polymorphic DNA (RAPD) analysis for the routine typing of these strains. Sixty P. aeruginosa non-repetitive strains recovered from CFP in a teaching hospital were typed. Thirty-five non-serotyped strains were studied by RAPD-PCR analysis with primers 272 and 208. RAPD data were performed to establish the relatedness between bioptype, antibiotic susceptibility and genotype. Fifty-five percent of strains are multiresistant, and no relation was found between antibiotype and biotype. A possible correlation between various phenotypes belonging to a single genotype was observed. RAPD typing characterized 30 distinct genotypes and two small clusters of strains were observed among isolates with each primer. Strains belonging to one cluster were present in two (6%) of the 35 strains. Strains belonging to the other cluster were present in three (8%) of the 35 strains. The occurrence of these clusters indicates that cross-infection may occur. The results indicate also that only the RAPD method can establish a clonal relation whereas the other methods may only reflect phenotypical differences, and thus are inadequate to type these strains.

Atypical Mycobacterial and Fungal Infections in Cystic Fibrosis

Nontuberculous mycobacteria (NTM) and fungi are prevalent in the sputum of cystic fibrosis patients and are increasingly recognized to cause clinically significant disease. In both instances the organisms are ubiquitous within the environment making exposure common, although specific risk factors that contribute to active pulmonary infection have not been identified. A consistent and aggressive approach to screening for NTM and fungi within the cystic fibrosis airway is likely indicated, especially in the setting of clinical deterioration despite standard antipseudomonal therapies.

Pulmonary nocardiosis in cystic fibrosis

Background The treatment of Nocardia species found in the sputum of cystic fibrosis patients is of unknown value. Methods We conducted a retrospective analysis of the impact of directed oral antibiotic therapy against Nocardia spp. isolated from the sputum of 17 cystic fibrosis patients over a 10-year period. Pulmonary Function Tests were used as the clinical indicator of the disease state and the data were analyzed by general linear mixed model statistics with univariate analysis. Results Pulmonary Function Test values of all patients studied showed no significant difference before, during, or after the antibiotic treatment period. Treatment groups did not differ from non-treatment groups. This held true for Forced Expiratory Volume over 1 s and Functional Vital Capacity analysis. In addition, individual patient analysis did not reveal any trends or outliers. Conclusions Oral antibiotic therapy of cystic fibrosis patients colonized with Nocardia does not appear to affect clinical outcome. This suggests that deferring therapy may be an acceptable alternative and justifies conducting a future placebo controlled trial. In addition, this study model may be useful in analyzing the effect of therapy on other rare and difficult organisms, such as fungi and mycobacteria in the cystic fibrosis population.

In vitro antibacterial activity of aminosterols against multidrug-resistant bacteria from patients with cystic fibrosis

Respiratory infections with multidrug-resistant (MDR) bacteria are life-threatening in patients with cystic fibrosis (CF). Squalamine and aminosterol derivatives (ASDs) have previously demonstrated interesting antibacterial activity against bacterial reference strains. This study investigated for the first time their activity against MDR clinical isolates recovered from the sputa of CF patients. Antibacterial activity of squalamine and two ASDs (1 and 2) was evaluated against 135 MDR gram-negative and gram-positive bacteria using the broth microdilution method for MIC determination. For gram-negative bacteria, MICs ranged from 2 to 128 mg/L. Resistance to colistin and mucoidity were significantly associated with higher MICs of squalamine and ASDs 1 and 2. Tested compounds were active against various gram-positive bacteria with MIC values varying from 0.5 to 8 mg/L, with the exception of two capsulated isolates of Streptococcus pneumoniae demonstrating MICs of 32 mg/L. In this study, we present new findings concerning the antibacterial potential of ASDs against MDR bacteria. Colistin-resistant, mucoid and capsulated bacteria were found to exhibit decreased susceptibility to ASDs indicating that these compounds might share some mechanistic aspects with polymyxins towards gram-negative bacteria. However, ASDs were remarkably active against gram-positive species suggesting different mechanisms of action towards gram-positive and gram-negative bacteria. As tested ASDs exhibited elevated MICs in some cases, we believe that these compounds may be developed to be locally administrated as aerosols rather than via systemic administration routes. Further work is warranted to evaluate their in vivo efficacy in aerosol formulations using a lung-infected animal model.

Multiple genotypic changes in hypersusceptible strains of Pseudomonas aeruginosa isolated from cystic fibrosis patients do not always correlate with the phenotype

Although Pseudomonas aeruginosa from cystic fibrosis patients are well known for their antibiotic resistance, isolates that are highly susceptible to multiple drug classes have also been encountered. In this study, hypersusceptible P. aeruginosa isolates were analysed for changes in intrinsic resistance mechanisms to explain the observed phenotype. P. aeruginosa strains PA30 and PA431 were isolated from the sputa of cystic fibrosis patients and susceptibilities were determined by agar dilution. Isolates were genetically unrelated by PFGE analysis. Expression of efflux pumps, porins, a chromosomal cephalosporinase and a gene, glmS, previously implicated in hypersusceptibility were evaluated by real-time RT-PCR, outer membrane protein analysis and beta-lactamase hydrolysis assays. PA30 was hypersusceptible to beta-lactams, fluoroquinolones and antimetabolites, with MICs at least 4-fold lower than those for the prototype strain PAO1, while PA431 was hypersusceptible to beta-lactams and antimetabolites. Both isolates overproduced the porin OprF but showed down-regulation in the production of the carbapenem channel OprD despite carbapenem hypersusceptibility. PA30 had decreased expression of the mexAB-oprM pump involved with intrinsic antibiotic resistance but overexpressed the mexCD-oprJ and mexEF-oprN efflux systems normally associated with acquired resistance. PA431 showed down-regulation of oprM, the last gene in the mexAB-oprM operon, but overexpressed the mexXY pump. The ampC beta-lactamase was weakly inducible in strain PA30, corresponding to cefoxitin hypersusceptibility. The changes in expression of several intrinsic mechanisms in the hypersusceptible strains did not correlate with the observed phenotype. These data highlight the complex interactions of resistance mechanisms in P. aeruginosa and their roles in drug susceptibility.

Persistent elevated tissue‐transglutaminase in cystic fibrosis

Persistent elevated tissue‐transglutaminase in cystic fibrosis

A novel chromogenic medium for isolation of Pseudomonas aeruginosa from the sputa of cystic fibrosis patients

Background A novel chromogenic medium for isolation and identification of Pseudomonas aeruginosa from sputa of cystic fibrosis (CF) patients was evaluated and compared with standard laboratory methods. Methods One hundred sputum samples from distinct CF patients were cultured onto blood agar (BA), Pseudomonas CN selective agar (CN) and a Pseudomonas chromogenic medium (PS-ID). All Gram-negative morphological variants from each medium were subjected to antimicrobial susceptibility testing, and identification using a combination of biochemical and molecular methods. Results P. aeruginosa was isolated from 62 samples after 72 h incubation. Blood agar recovered P. aeruginosa from 56 samples (90.3%) compared with 59 samples (95.2%) using either CN or PS-ID. The positive predictive value of PS-ID (98.3%) was significantly higher than growth on CN (88.5%) for identification of P. aeruginosa ( P < 0.05). Conclusions PS-ID is a promising medium allowing for the isolation and simultaneous identification of P. aeruginosa from sputa of CF patients.

Development of selective media for the isolation of yeasts and filamentous fungi from the sputum of adult patients with cystic fibrosis (CF)

Yeasts and filamentous fungi are beginning to emerge as significant microbial pathogens in patients with cystic fibrosis (CF), particularly in relation to allergic-type responses, as seen in patients with allergic bronchopulmonary aspergillosis (ABPA), Aspergillus bronchitis and in invasive fungal disease in lung transplant patients. Four fungal media were compared in this study, including Sabouraud Dextrose Agar (SDA) and Medium B, with and without the addition of selective antibiotics, where antibiotic-supplemented media were designated with +. These media were compared for their ability to suppress contaminating, mainly Gram−ve pathogens, in CF sputa ( Pseudomonas aeruginosa, Burkholderia cepacia complex [BCC] organisms) and to enhance the growth of fungi present in CF sputum. Medium B consisted of glucose (16.7 g/l), agar (20 g/l), yeast extract (30 g/l) and peptone (6.8 g/l) at pH 6.3 and both SDA + and Medium B + were supplemented with cotrimethoxazole, 128 mg/l; chloramphenicol, 50 mg/l; ceftazidime, 32 mg/l; colistin, 24 mg/l). Employment of SDA + or Medium B + allowed an increase in specificity in the detection of yeasts and moulds, by 42.8% and 39.3%, respectively, over SDA when used solely. SDA + had a greater ability than Medium B + to suppress bacterial growth from predominantly Gram−ve co-colonisers. This is a significant benefit when attempting to detect and isolate fungi from the sputum of CF patients, as it largely suppressed any bacterial growth, with the exception of the BCC organisms, thus allowing for an increased opportunity to detect target fungal organisms in sputum and represented a significant improvement over the commercial medium (SDA), which is currently used. Overall, both novel selective media were superior in their ability to suppress bacteria in comparison with the commercially available SDA medium, which is routinely employed in most clinical microbiology diagnostic laboratories presently. Alternatively, Medium B + had a great ability to grow fungi than SDA + and when employed together, the specificity of combined use was 82%, with a sensitivity for yeasts, filamentous fungi, and combined overall fungi of 96.0%, 92.3% and 96.0%, respectively. Overall, when employing one fungal selective medium for the routine detection of yeasts and filamentous fungi in the sputum of CF patients, we would recommend employment of Medium B +. However, we would recommend the combined employment of SDA + and Medium B +, in order to synergistically isolate and detect the greatest number of fungi present in CF sputa.

Anaerobic bacteria detected in sputum of cystic fibrosis patients by its cellular fatty acid markers

Anaerobic bacteria detected in sputum of cystic fibrosis patients by its cellular fatty acid markers

Cystic fibrosis: ironing out the problem of infection?

the study by moreau-marquis et al. (3) in this issue of AJP-Lung advances our understanding of lung disease in cystic fibrosis (CF). Their findings are important because they may largely explain the peculiar propensity to early Pseudomonas aeruginosa infection that characterizes this most common of lethal genetic diseases. Moreau-Marquis et al. (3) demonstrate that immortalized human CF airway epithelial cells cocultured with P. aeruginosa promote the formation of antibiotic-resistant biofilms. Their series of experiments demonstrate that this is due to abnormal accumulation and loss of iron by CF epithelial cells and that biofilm formation can be prevented by either iron chelation therapy or by increasing normal cell membrane cystic fibrosis transmembrane regulator (CFTR) expression using the novel compound Corr4a. The observations of Moreau-Marquis et al. (3) confirm what has been clinically suspected in CF with respect to iron promotion of infection with P. aeruginosa. Previous studies have demonstrated that the airways of CF patients contain increased amounts of total iron and the iron-binding protein ferritin as well as a relationship between airway iron content and quantitative P. aeruginosa load assessed by colony-forming units per milliliter of sputum (5). This study also found an increase in iron in sputum in CF patients who had not previously been infected with P. aeruginosa, suggesting that elevated airway iron content may predate P. aeruginosa acquisition. Further evidence to support a problem with iron trafficking comes from the CF G551D mouse model. The teeth of these mice exhibit abnormal iron transport in pigment ameloblasts compared with wild-type mice, resulting in unusually white dentition (7). The study by Moreau-Marquis et al. (3) lends biological rationale to these earlier observations and brings two key questions into focus. First, what is the underlying link between abnormal or absent CFTR and cell iron homeostasis? Second, how does this impact on the CF host and P. aeruginosa interaction?

Cif Is Negatively Regulated by the TetR Family Repressor CifR

We previously reported that the novel Pseudomonas aeruginosa toxin Cif is capable of decreasing apical membrane expression of the cystic fibrosis transmembrane conductance regulator (CFTR). We further demonstrated that Cif is capable of degrading the synthetic epoxide hydrolase (EH) substrate S-NEPC [(2S,3S)-trans-3-phenyl-2-oxiranylmethyl 4-nitrophenol carbonate], suggesting that Cif may be reducing apical membrane expression of CFTR via its EH activity. Here we report that Cif is capable of degrading the xenobiotic epoxide epibromohydrin (EBH) to its vicinal diol 3-bromo-1,2-propanediol. We also demonstrate that this epoxide is a potent inducer of cif gene expression. We show that the predicted TetR family transcriptional repressor encoded by the PA2931 gene, which is immediately adjacent to and divergently transcribed from the cif-containing, three-gene operon, negatively regulates cif gene expression by binding to the promoter region immediately upstream of the cif-containing operon. Furthermore, this protein-DNA interaction is disrupted by the epoxide EBH in vitro, suggesting that the binding of EBH by the PA2931 protein product drives the disassociation from its DNA-binding site. Given its role as a repressor of cif gene expression, we have renamed PA2931 as CifR. Finally, we demonstrate that P. aeruginosa strains isolated from cystic fibrosis patient sputum with increased cif gene expression are impaired for the expression of the cifR gene.

Recovery of antimicrobial-resistant Pseudomonas aeruginosa from sputa of cystic fibrosis patients by culture on selective media

To assess the utility of direct plating of whole sputa onto selective media as a means of identifying antimicrobial resistance in strains of Pseudomonas aeruginosa from the sputa of patients with cystic fibrosis (CF). A total of 45 sputum samples from CF patients were cultured onto conventional culture media for isolation of P. aeruginosa and were also cultured directly onto Iso-Sensitest agar plates containing each of 10 antimicrobials incorporated at a 'breakpoint' concentration. A representative of each colonial type (morphotype) recovered from both routine media and selective media was tested for its susceptibility to 10 antimicrobials using a standard agar dilution MIC technique. Of the samples shown to contain resistant strains, the proportion (%) detected using routine media and selective media, respectively, was: 42 and 100 for amikacin, 57 and 100 for gentamicin, 54 and 100 for tobramycin, 88 and 77 for aztreonam, 62 and 90 for ceftazidime, 70 and 97 for meropenem, 61 and 100 for piperacillin/tazobactam, 90 and 86 for temocillin, 66 and 100 for ticarcillin/clavulanic acid, and 80 and 90 for ciprofloxacin resistance. The increased rates of isolation on selective media were statistically significant (P < 0.05) for amikacin, gentamicin, tobramycin, meropenem, piperacillin/tazobactam and ticarcillin/clavulanic acid. For most antimicrobials, selection of colonies from conventional media for antimicrobial susceptibility testing provided a considerable underestimation of resistance in P. aeruginosa. The use of selective media for the culture of whole sputum was effective for the detection of resistant isolates of P. aeruginosa.

The complete genome, comparative and functional analysis of Stenotrophomonas maltophilia reveals an organism heavily shielded by drug resistance determinants

The complete Stenotrophomonas maltophilia genome sequence suggests that it can act as a reservoir of antibiotic resistance determinants.

Neutrophils in Cystic Fibrosis Display a Distinct Gene Expression Pattern

We compared gene expression in blood neutrophils (polymorphonuclear leukocytes, or PMNs) collected from healthy subjects with those of cystic fibrosis (CF) patients devoid of bacterial colonization. Macroarray analysis of 1050 genes revealed upregulation of 62 genes and downregulation expression of 27 genes in CF blood PMNs. Among upregulated genes were those coding for vitronectin, some chemokines (particularly CCL17 and CCL18), some interleukin (IL) receptors (IL-3, IL-8, IL-10, IL-12), all three colony-stimulating factors (G-, M-, GM-CSF), numerous genes coding for molecules involved in signal transduction, and a few genes under the control of gamma-interferon. In contrast, none of the genes coding for adhesion molecules were modulated. The upregulation of six genes in CF PMNs (coding for thrombospondin-1, G-CSF, CXCL10, CCL17, IKKvarepsilon, IL-10Ra) was further confirmed by qPCR. In addition, the increased presence of G-CSF, CCL17, and CXCL10 was confirmed by ELISA in supernatants of neutrophils from CF patients. When comparison was performed between blood and airway PMNs of CF patients, there was a limited difference in terms of gene expression. Only the mRNA expression of amphiregulin and tumor necrosis factor (TNF) receptor p55 were significantly higher in airway PMNs. The presence of amphiregulin was confirmed by ELISA in the sputum of CF patients, suggesting for the first time a role of amphiregulin in cystic fibrosis. Altogether, this study clearly demonstrates that blood PMNs from CF patients display a profound modification of gene expression profile associated with the disease, suggesting a state of activation of these cells.

Is cystic fibrosis a TH17 disease?

Cystic Fibrosis (CF) is the most common lethal genetic disease in the Caucasian population and typically results in the development of bronchial inflammation, bronchiectasis, the progressive loss of lung function and ultimately death. Recently it has been shown that products of the Th(17) subset of T-cells, specifically, IL-17A and IL-17F are elevated in the sputum of CF patients. This review will go over experimental evidence supporting a role for the IL- 23/IL-17 axis in CF lung inflammation.

88* Detection of anaerobic bacteria in high numbers in sputum from Cystic Fibrosis patients with an acute exacerbation of pulmonary infection

Introduction and Aims: We have previously shown by culture that the lungs of clinically stable Cystic Fibrosis (CF) patients are not only colonised by commonly recognised aerobic bacteria, such as Pseudomonas aeruginosa, but also by a range of potentially pathogenic anaerobic species. The aim of this study was to determine whether anaerobes are also present in the sputum of CF patients with an acute exacerbation of pulmonary infection. Methods: Sputum and mouthwash samples were collected, prior to commencing and at the end of antibiotic therapy, from 30 adult CF patients admitted for treatment of an acute exacerbation of pulmonary infection. Bacteria within the samples were detected by plating on selective agars, quantified by total viable count and identified by PCR and sequencing of 16S ribosomal RNA genes. Results: Anaerobes from a range of species including Prevotella, Veillonella, Propionibacterium, Actinomyces and Gemella were detected in high numbers (up to 1×108 cfu/g of sputum) from all patients prior to commencing antibiotic treatment with the predominant aerobes (P. aeruginosa or Burkholderia cepacia complex) detected in similar or smaller numbers. Anaerobes were also detected in all patients at the end of antibiotic treatment but in the majority of patients they were present in lower numbers than detected before antibiotic treatment. Conclusion: These results indicate that anaerobes are present within the CF lung during an acute exacerbation of pulmonary infection. Their presence could be of important clinical relevance to CF patients as they may contribute significantly to the inflammatory process. Supported by: UK Cystic Fibrosis Trust (project grant PJ533).

Validation of an automated sensitive immunoassay for quantitation of cytokines in the sputum of cystic fibrosis patients

There is little information on the reproducibility of measurement of cytokine levels in sputum obtained from cystic fibrosis (CF) patients. Our aim was to investigate whether assay of cytokine levels in CF sputum is reproducible or is hampered by proteolytic degradation. Interleukin-8 (IL-8) and tumor necrosis factor-alpha (TNF-alpha) levels were measured in native and spiked samples (fresh or after freezing for 15 and 23 days at -70 degrees C) obtained from nine CF patients using an immunometric assay with chemiluminescent substrate run on a IMMULITE analyzer. For both cytokines, linearity was >0.98 for dilutions up to 1:32. After storage, cytokine concentrations in native samples varied between -2.9% and -5.6% for IL-8 and between 0.4% and 3.0% for TNF-alpha. In spiked samples, concentrations increased by 5.8%-12.6% for TNF-alpha and decreased by 3.8%-14.3% for IL-8 after 15 and 23 days of storage. In samples spiked with cytokines, the mean recovery rates for IL-8 and TNF-alpha were 109.4% and 106.3%, respectively. Measurement of IL-8 and TNF-alpha in CF sputum is reproducible and is not hampered by freezing and thawing of samples.

Fungal infections in patients with cystic fibrosis

Current US Cystic Fibrosis Foundation (CFF) Registry data indicates an approximate doubling of prevalence of fungi being detected in the sputum of patients with cystic fibrosis (CF), probably due to a better understanding of the disease and the way in which it is managed. The introduction of new antibiotics and the way in which they are used – in double or triple therapy and as well as in antibiotic cycling – has helped address the requirement to control chronic Gram-negative infections in CF. However, this has inadvertently created a niche for the colonization and proliferation of fungi in the CF lung. The ability to detect and isolate fungi in the sputum of CF patients is important due to the emerging significance of these organisms, particularly in relation to: (i) allergic bronchopulmonary aspergillosis (ABPA); (ii) post-transplant fungaemia; and (iii) chronic colonization/infection (bronchitis). Recent data has indicated that ABPA affects approximately 1–15% of CF patients and is associated with an accelerated decline in lung function. Most of the mycological aetiology of this condition is due to Aspergillus fumigatus and other Aspergillus spp, although other fungi have been reported. In addition, patients who have undergone lung transplantation are at increased risk of opportunistic fungal infection, such as invasive pulmonary aspergilloma and other invasive mycoses, due to the immunosuppression of the host. This review summarizes the important fungal pathogens in CF, namely Aspergillus spp., as well as the emerging fungi, including Scedosporium apiospermum and Exophiala dermatitidis and discusses the various conventional and molecular methods of detection and identification.

ADP-ribosyltransferase-specific Modification of Human Neutrophil Peptide-1

Epithelial cells lining human airways and cells recruited to airways participate in the innate immune response in part by releasing human neutrophil peptides (HNP). Arginine-specific ADP-ribosyltransferases (ART) on the surface of these cells can catalyze the transfer of ADP-ribose from NAD to proteins. We reported that ART1, a mammalian ADP-ribosyltransferase, present in epithelial cells lining the human airway, modified HNP-1, altering its function. ADP-ribosylated HNP-1 was identified in bronchoalveolar lavage fluid (BALF) from patients with asthma, idiopathic pulmonary fibrosis, or a history of smoking (and having two common polymorphic forms of ART1 that differ in activity), but not in normal volunteers or patients with lymphangioleiomyomatosis. Modified HNP-1 was not found in the sputum of cystic fibrosis patients or in leukocyte granules of normal volunteers. The finding of ADP-ribosyl-HNP-1 in BALF but not in leukocyte granules suggests that the modification occurred in the airway. Most of the HNP-1 in the BALF from individuals with a history of smoking was, in fact, mono- or di-ADP-ribosylated. ART1 synthesized in Escherichia coli, glycosylphosphatidylinositol-anchored ART1 released with phosphatidylinositol-specific phospholipase C from transfected NMU cells, or ART1 expressed endogenously on C2C12 myotubes modified arginine 14 on HNP-1 with a secondary site on arginine 24. ADP-ribosylation of HNP-1 by ART1 was substantially greater than that by ART3, ART4, ART5, Pseudomonas aeruginosa exoenzyme S, or cholera toxin A subunit. Mouse ART2, which is an NAD:arginine ADP-ribosyltransferase, was able to modify HNP-1, but to a lesser extent than ART1. Although HNP-1 was not modified to a significant degree by ART5, it inhibited ART5 as well as ART1 activities. Human beta-defensin-1 (HBD1) was a poor transferase substrate. Reduction of the cysteine-rich defensins enhanced their ability to serve as ADP-ribose acceptors. We conclude that ADP-ribosylation of HNP-1 appears to be primarily an activity of ART1 and occurs in inflammatory conditions and disease.

EPI-hNE4, a Proteolysis-Resistant Inhibitor of Human Neutrophil Elastase and Potential Anti-Inflammatory Drug for Treating Cystic Fibrosis

EPI-hNE4 (depelstat) is a potent inhibitor of human neutrophil elastase derived from human inter-alpha-trypsin inhibitor and designed to control the excess proteolytic activity in the sputum of cystic fibrosis patients. We analyzed its resistance to the proteolysis it is likely to encounter at inflammatory sites in vivo. EPI-hNE4 resisted hydrolysis by neutrophil matrix metalloproteases (MMPs) and serine proteases that are released from activated neutrophils in inflammatory lung secretions, including MMP-8 and MMP-9, and the elastase-related protease 3 and cathepsin G. It also resisted degradation by epithelial lung cell MMP-7 but was broken down by the Pseudomonas aeruginosa metalloelastase pseudolysin, when used in a purified system, but not when this protease competed with equimolar amounts of neutrophil elastase. We also investigated the inhibitory properties of EPI-hNE4 at the surface of purified blood neutrophils and in the sputum of cystic fibrosis patients where neutrophil elastase is in both a soluble and a gel phase. The elastase at the neutrophil surface was fully inhibited by EPI-hNE4 and formed soluble complexes. The elastase in cystic fibrosis sputum supernatants was inhibited by stoichiometric amounts of EPI-hNE4, allowing titration of the protease. But the percentage of inhibition in whole sputum homogenates varied from 50 to 100%, depending on the sample tested. EPI-hNE4 was rapidly cleaved by the digestive protease pepsin in vitro. Therefore, EPI-hNE4 seems to be an elastase inhibitor suitable for use in aerosols to treat patients with cystic fibrosis.