Recombinant human DNase I reduces the viscosity of cystic fibrosis sputum.
ABSTRACT: Respiratory distress and progressive lung destruction in cystic fibrosis can be attributed to bacterial persistence and the accumulation of viscous purulent secretions in the airways. More than 30 yr ago it was suggested that the large amounts of DNA in purulent secretions contribute to its viscosity and that bovine pancreatic DNase I could reduce the viscosity. To evaluate the potential clinical utility of recombinant human DNase I (rhDNase) in the treatment of cystic fibrosis, we have cloned, sequenced, and expressed rhDNase. Catalytic amounts of rhDNase greatly reduce the viscosity of purulent cystic fibrosis sputum, transforming it within minutes from a nonflowing viscous gel to a flowing liquid. The reduction in viscosity is associated with a decrease in size of DNA in the sputum. Inhalation of a rhDNase aerosol may be a simple direct approach that will help individuals with cystic fibrosis and other patients with pneumonia or bronchitis to clear their airways of purulent secretions.
Project description:Human deoxyribonuclease I (DNase I) is an endonuclease that catalyzes the hydrolysis of extracellular DNA and is just one of the numerous types of nucleases found in nature. The enzymatic mechanism for a single turnover is reasonably well understood based on biochemical and structural studies that are consistent with divalent metal ion dependent nonspecific nicking of a phosphodiester bond in one of the strands of double stranded DNA. Recombinant human DNase I (rhDNase I, rhDNase, Pulmozyme®, dornase alfa) has been expressed in mammalian cell culture in Chinese hamster ovary cells and developed clinically where it is aerosolized into the airways for treatment of pulmonary disease in patients with cystic fibrosis (CF). rhDNase I hydrolyzes the DNA in purulent sputum of CF patients and reduces sputum viscoelasticity. Reduction of high molecular weight DNA into smaller fragments by treatment with aerosolized rhDNase I has been proposed as the mechanism to reduce the mucus viscosity and improve mucus clearability from obstructed airways in patients. The improved clearance of the purulent mucus enhances pulmonary function and reduces recurrent exacerbations of respiratory symptoms. rhDNase I was approved for clinical use in 1993 and has been widely used as a safe and effective therapy for CF patients. The use of rhDNase I has also been investigated in other diseases where exogenous DNA has been implicated in the disease pathology.
Project description:For effective airway gene therapy of cystic fibrosis (CF), inhaled gene carriers must first penetrate the hyperviscoelastic sputum covering the epithelium. Whether clinically studied gene carriers can penetrate CF sputum remains unknown. Here, we measured the diffusion of a clinically tested nonviral gene carrier, composed of poly-l-lysine conjugated with a 10 kDa polyethylene glycol segment (CK(30)PEG(10k)). We found that CK(30)PEG(10k)/DNA nanoparticles were trapped in CF sputum. To improve gene carrier diffusion across sputum, we tested adjuvant regimens consisting of N-acetylcysteine (NAC), recombinant human DNase (rhDNase) or NAC together with rhDNase. While rhDNase alone did not enhance gene carrier diffusion, NAC and NAC + rhDNase increased average effective diffusivities by 6-fold and 13-fold, respectively, leading to markedly greater fractions of gene carriers that may penetrate sputum layers. We further tested the adjuvant effects of NAC in the airways of mice with Pseudomonas aeruginosa lipopolysaccharide (LPS)-induced mucus hypersecretion. Intranasal dosing of NAC prior to CK(30)PEG(10k)/DNA nanoparticles enhanced gene expression by up to ~12-fold compared to saline control, reaching levels observed in the lungs of mice without LPS challenge. Our findings suggest that a promising synthetic nanoparticle gene carrier may transfer genes substantially more effectively to lungs of CF patients if administered following adjuvant mucolytic therapy with NAC or NAC + rhDNase.
Project description:The stasis of mucus secretions in the lungs of cystic fibrosis (CF) patients leads to recurrent infections and pulmonary exacerbations, resulting in decreased survival. Prior studies have assessed the biochemical and biophysical features of airway mucus in individuals with CF. However, these measurements are unable to probe mucus structure on microscopic length scales relevant to key players in the progression of CF-related lung disease, namely, viruses, bacteria, and neutrophils. In this study, we quantitatively determined sputum microstructure based on the diffusion of muco-inert nanoparticle probes in CF sputum and found that a reduction in sputum mesh pore size is characteristic of CF patients with reduced lung function, as indicated by measured FEV1. We also discovered that the effect of ex vivo treatment of CF sputum with rhDNase I (Pulmozyme) on microstructure is dependent upon the time interval between the most recent inhaled rhDNase I treatment and the sample collection. Microstructure of mucus may serve as a marker for the extent of CF lung disease and as a parameter for assessing the effectiveness of mucus-altering agents.
Project description:Highly compacted DNA nanoparticles, composed of single molecules of plasmid DNA compacted with block copolymers of poly-l-lysine and 10kDa polyethylene glycol (CK(30)PEG(10k)), mediate effective gene delivery to the brain, eyes and lungs in vivo. Nevertheless, we found that CK(30)PEG(10k) DNA nanoparticles are immobilized by mucoadhesive interactions in sputum that lines the lung airways of patients with cystic fibrosis (CF), which would presumably preclude the efficient delivery of cargo DNA to the underlying epithelium. We previously found that nanoparticles can rapidly penetrate human mucus secretions if they are densely coated with low MW PEG (2-5kDa), whereas nanoparticles with 10kDa PEG coatings were immobilized. We thus sought to reduce mucoadhesion of DNA nanoparticles by producing CK(30)PEG DNA nanoparticles with low MW PEG coatings. We examined the morphology, colloidal stability, nuclease resistance, diffusion in human sputum and in vivo gene transfer of CK(30)PEG DNA nanoparticles prepared using various PEG MWs. CK(30)PEG(10k) and CK(30)PEG(5k) formulations did not aggregate in saline, provided partial protection against DNase I digestion and exhibited the highest gene transfer to lung airways following inhalation in BALB/c mice. However, all DNA nanoparticle formulations were immobilized in freshly expectorated human CF sputum, likely due to inadequate PEG surface coverage.
Project description:Cystic fibrosis patients suffer from chronic lung infection and inflammation due to the secretion of viscous sputum. Sputum viscosity is caused by extracellular DNA, some of which originates from the release of neutrophil extracellular traps (NETs). During NET formation neutrophil elastase (NE) partially processes histones to decondense chromatin. NE is abundant in CF sputum and is thought to contribute to tissue damage. Exogenous nucleases are a palliative treatment in CF as they promote sputum solubilization. We show that in a process reminiscent of NET formation, NE enhances sputum solubilization by cleaving histones to enhance the access of exogenous nucleases to DNA. In addition, we find that in Cf sputum NE is predominantly bound to DNA, which is known to downregulate its proteolytic activity and may restrict host tissue damage. The beneficial role of NE in CF sputum solubilization may have important implications for the development of CF therapies targeting NE.
Project description:Non-cystic fibrosis bronchiectasis (NCFB) is a chronic, progressive respiratory disorder characterised by irreversibly and abnormally dilated airways, persistent cough, excessive sputum production and recurrent pulmonary infections. In the last several decades, its prevalence has increased, making it likely to be encountered in the primary care setting. The aim was to review the clinical presentation and diagnosis of NCFB, with an emphasis on the role of computed tomography (CT).For this review, trials and reports were identified from PubMed/Medline and ClinicalTrials.gov from the US NIH and the Cochrane Register of Controlled Trials. The search used keywords: bronchiectasis, non-cystic fibrosis bronchiectasis, chronic pulmonary infection and computed tomography. No date/language restrictions were used.Non-cystic fibrosis bronchiectasis often coexists with other respiratory conditions, such as chronic obstructive pulmonary disease. The prevalence of NCFB is increasing, particularly in women and older individuals, possibly as a result of increased physician awareness and widespread use of CT, which is the gold standard for the diagnosis of NCFB. CT can assist in identifying an underlying cause of NCFB and determining the extent and severity of the disease.Non-cystic fibrosis bronchiectasis should be suspected in the primary care setting in patients with chronic cough, purulent sputum and frequent respiratory infections that tend to resolve slowly or partially. Early diagnosis and determination of the extent and severity of the disease by CT and other tests are critical to establish therapy to improve quality of life and potentially slow progressive decline of lung function in patients with NCFB.
Project description:Cystic fibrosis (CF) is a life-threatening genetic disorder, characterized by chronic microbial lung infections due to abnormally viscous mucus secretions within airways. The clinical management of CF typically involves regular respiratory-tract cultures in order to identify pathogens and to guide treatment. However, culture-based methods can miss atypical or slow-growing microbes. Furthermore, the isolated microbes are often not classified at the strain level due to limited taxonomic resolution.Here, we show that untargeted metagenomic sequencing of sputum DNA can provide valuable information beyond the possibilities of culture-based diagnosis. We sequenced the sputum of six CF patients and eleven control samples (including healthy subjects and chronic obstructive pulmonary disease patients) without prior depletion of human DNA or cell size selection, thus obtaining the most unbiased and comprehensive characterization of CF respiratory tract microbes to date. We present detailed descriptions of the CF and healthy lung microbiome, reconstruct near complete pathogen genomes, and confirm that the CF lungs consistently exhibit reduced microbial diversity. Crucially, the obtained genomic sequences enabled a detailed identification of the exact pathogen strain types, when analyzed in conjunction with existing multi-locus sequence typing databases. We also detected putative pathogenicity islands and indicators of antibiotic resistance, in good agreement with independent clinical tests.Unbiased sputum metagenomics provides an in-depth profile of the lung pathogen microbiome, which is complementary to and more detailed than standard culture-based reporting. Furthermore, functional and taxonomic features of the dominant pathogens, including antibiotics resistances, can be deduced-supporting accurate and non-invasive clinical diagnosis.
Project description:Changes in the airway microbiome may be important in the pathophysiology of chronic lung disease in patients with cystic fibrosis. However, little is known about the microbiome in early cystic fibrosis lung disease and the relationship between the microbiomes from different niches in the upper and lower airways. Therefore, in this cross-sectional study, we examined the relationship between the microbiome in the upper (nose and throat) and lower (sputum) airways from children with cystic fibrosis using next generation sequencing. Our results demonstrate a significant difference in both ? and ?-diversity between the nose and the two other sampling sites. The nasal microbiome was characterized by a polymicrobial community while the throat and sputum communities were less diverse and dominated by a few operational taxonomic units. Moreover, sputum and throat microbiomes were closely related especially in patients with clinically stable lung disease. There was a high inter-individual variability in sputum samples primarily due to a decrease in evenness linked to increased abundance of potential respiratory pathogens such as Pseudomonas aeruginosa. Patients with chronic Pseudomonas aeruginosa infection exhibited a less diverse sputum microbiome. A high concordance was found between pediatric and adult sputum microbiomes except that Burkholderia was only observed in the adult cohort. These results indicate that an adult-like lower airways microbiome is established early in life and that throat swabs may be a good surrogate in clinically stable children with cystic fibrosis without chronic Pseudomonas aeruginosa infection in whom sputum sampling is often not feasible.
Project description:Hallmarks of cystic fibrosis (CF) are increased viscosity of mucus and impaired mucociliary clearance within the airways due to mutations of the cystic fibrosis conductance regulator gene. This paves the way for the colonization by microbial pathogens and the concomitant establishment of chronic infections leading to lung tissue damage, reduced lung function, and to decreased life expectancy. Although microbial infections play a key role during disease progression, only a few studies investigated the pathophysiology of the microbial community in vivo so far. Moreover, no CF study so far applied metaproteomics, a powerful approach to unravel molecular mechanisms of microbial infection, mainly reasoned due to (I) the challenging processability of inhomogeneous, viscous, slimy sputum, and (II) the high number of human proteins masking comparably low abundant microbial proteins. Consequently, we developed a reliable, reproducible and widely applicable protocol for sputum processing, microbial enrichment, and subsequent metaproteomics analyses with a focus on microbial pathogens overcoming the aforementioned challenges. Metaproteomics data were complemented and validated by 16S sequencing, metabolomic as well as microscopic analyses. In total, we processed 21 CF sputum samples and selected three for detailed metaproteome analysis. The number of bacterial proteins/protein groups increased from 199-425 to 392-868. Moreover, our data suggest that the arginine deiminase pathway and multiple proteases and peptidases identified from various bacterial genera are so far underappreciated in their contribution to the CF pathophysiology. By providing a standardized and effective protocol for sputum processing and microbial enrichment, our study represents an important basis for future studies investigating the physiology of microbial pathogens in CF in vivo – an important prerequisite for the development of novel antimicrobial therapies against mucoviscidosis.
Project description:Biofilms-communities of bacteria encased in a polymer-rich matrix-confer bacteria with the ability to persist in pathologic host contexts, such as the cystic fibrosis (CF) airways. How bacteria assemble polymers into biofilms is largely unknown. We find that the extracellular matrix produced by Pseudomonas aeruginosa self-assembles into a liquid crystal through entropic interactions between polymers and filamentous Pf bacteriophages, which are long, negatively charged filaments. This liquid crystalline structure enhances biofilm function by increasing adhesion and tolerance to desiccation and antibiotics. Pf bacteriophages are prevalent among P. aeruginosa clinical isolates and were detected in CF sputum. The addition of Pf bacteriophage to sputum polymers or serum was sufficient to drive their rapid assembly into viscous liquid crystals. Fd, a related bacteriophage of Escherichia coli, has similar biofilm-building capabilities. Targeting filamentous bacteriophage or the liquid crystalline organization of the biofilm matrix may represent antibacterial strategies.