Defective epithelial barrier function in asthma.

BACKGROUND: Asthma is a complex disease involving gene and environment interactions. Although atopy is a strong predisposing risk factor for asthma, local tissue susceptibilities are required for disease expression. The bronchial epithelium forms the interface with the external environment and is pivotally involved in controlling tissue homeostasis through provision of a physical barrier controlled by tight junction (TJ) complexes. OBJECTIVES: To explain the link between environment exposures and airway vulnerability, we hypothesized that epithelial TJs are abnormal in asthma, leading to increased susceptibility to environmental agents. METHODS: Localization of TJs in bronchial biopsies and differentiated epithelial cultures was assessed by electron microscopy or immunostaining. Baseline permeability and the effect of cigarette smoke and growth factor were assessed by measurement of transepithelial electrical resistance and passage of fluorescently labeled dextrans. RESULTS: By using immunostaining, we found that bronchial biopsies from asthmatic subjects displayed patchy disruption of TJs. In differentiated bronchial epithelial cultures, TJ formation and transepithelial electrical resistance were significantly lower (P < .05) in cultures from asthmatic donors (n = 43) than from normal controls (n = 40) and inversely correlated with macromolecular permeability. Cultures from asthmatic donors were also more sensitive to disruption by cigarette smoke extract. Epidermal growth factor enhanced basal TJ formation in cultures from asthmatic subjects (P < .01) and protected against cigarette smoke-induced barrier disruption (P < .01). CONCLUSIONS: Our results show that the bronchial epithelial barrier in asthma is compromised. This defect may facilitate the passage of allergens and other agents into the airway tissue, leading to immune activation and may thus contribute to the end organ expression of asthma.

Photoinduced crosslinking of double-helical DNA by psoralen covalently linked to a triple helix-forming oligonucleotide under near-physiological conditions.

Stable triplexes have been generated under near-physiological conditions by the introduction of the C and T base analogues 3-methyl-2-aminopyridine-2'-deoxyriboside and 5-(3-aminoprop-2-ynyl)-'-deoxyuridine into psoralen-conjugated triplex-forming oligonucleotides. After irradiation with UV light at 365 nm, photo-induced cross-linking of the TFO to double-helical DNA was observed by UV-melting analysis and fluorescence measurements.

Combining nucleoside analogues to achieve recognition of oligopurine tracts by triplex-forming oligonucleotides at physiological pH.

We have used DNase I footprinting to examine DNA triple helix formation at a 12 base pair oligopurine.oligopyrimidine sequence, using oligonucleotides that contain combinations of 2'-aminoethoxy-5-(3-aminoprop-1-ynyl)uridine (bis-amino-U, BAU) and 3-methyl-2-aminopyridine (MeP) in place of T and C, respectively. This combination acts cooperatively to enable high affinity triple helix formation at physiological pH. The affinity depends on the number of substitutions and their arrangement; oligonucleotides in which these analogues are evenly distributed throughout the third strand bind much better than those in which they are clustered together.

Kinetic studies on the formation of DNA triplexes containing the nucleoside analogue 2'-O-(2-aminoethyl)-5-(3-amino-1-propynyl)uridine.

We have examined the kinetics of triple helix formation of oligonucleotides that contain the nucleotide analogue 2'-O-(2-aminoethyl)-5-(3-amino-1-propynyl)uridine (bis-amino-U, BAU), which forms very stable base triplets with AT. Triplex stability is determined by both the number and location of the modifications. BAU-containing oligonucleotides generate triplexes with extremely slow kinetics, as evidenced by 14 degrees C hysteresis between annealing and melting profiles even when heated at a rate as slow as 0.2 degrees C min(-1). The association kinetics were measured by analysis of the hysteresis profiles, temperature-jump relaxation and DNase I footprinting. We find that the slow kinetics are largely due to the decreased rate of dissociation; BAU modification has little effect on the association reaction. The sequence selectivity is also due to the slower dissociation of BAU from AT than other base pairs.

Actin limits enhancement of nanoparticle diffusion through cystic fibrosis sputum by mucolytics.

BACKGROUND: The secretions in the cystic fibrosis (CF) airways contains high concentrations of polymers, including the respiratory mucins and varying amounts of DNA and actin, the debris of an aggressive neutrophilic inflammatory response to infection. Physical and chemical interactions between these polymers contribute to the viscoelastic nature of a material that is hard to clear without the use of mucolytics. Secretions retained in the CF airway not only restrict airflow and invite infection, but also act as a barrier to the delivery of inhaled drugs and gene therapy vectors to the underlying airway epithelium. The aim of this investigation was to develop a simple, sensitive, assay to measure the diffusion of nanospheres the size of liposomal gene therapy vectors through CF sputum, and to model the polymer interactions that limit diffusion and the diffusion-enhancing activity of mucolytics. METHODS: The diffusion of 200 nm fluorescent carboxylated nanospheres through CF sputum was investigated using a diffusion assay based on the micro-Boyden chamber. Atomic force microscopy (AFM) was used to visualise and measure the pore diameter in CF sputum. The effect of the mucolytics deoxyribonuclease (DNase), N-acetylcysteine and gelsolin on the diffusion of nanospheres though synthetic biogels comprising mixtures of DNA, mucin and F-actin was also investigated. RESULTS: CF sputum significantly retarded the diffusion of 200 nm nanospheres. Pore diameter in CF sputum was highly variable, with a mean greater than 200 nm. At concentrations found in the CF airway, DNA (1-10 mg/ml) and mucin (25-50 mg/ml) also significantly reduced the diffusion of nanospheres. The barrier effects of DNA and mucin were not additive, and the additional presence of F-actin (5 mg/ml) did not influence diffusion of the nanospheres. However, actin (5mg/ml) completely inhibited the ability of DNase (2.9 microg/ml) and N-acetylcysteine (5 mM) to enhance diffusion. The activity of the mucolytics, DNase and N-acetylcysteine, was not restored by the addition of the actin depolymerising agent gelsolin (250nM). CONCLUSION: Actin does not contribute to the barrier properties of CF sputum, but is a key determinant of the ability of mucolytics to enhance drug diffusion through synthetic and biological mucus.

Footprinting: a method for determining the sequence selectivity, affinity and kinetics of DNA-binding ligands.

Footprinting is a simple method for assessing the sequence selectivity of DNA-binding ligands. The method is based on the ability of the ligand to protect DNA from cleavage at its binding site. This review describes the use of DNase I and hydroxyl radicals, the most commonly used footprinting probes, in footprinting experiments. The success of a footprinting experiment depends on using an appropriate DNA substrate and we describe how these can best be chosen or designed. Although footprinting was originally developed for assessing a ligand's sequence selectivity, it can also be employed to estimate the binding strength (quantitative footprinting) and to assess the association and dissociation rate constants for slow binding reactions.

Unfractionated heparin reduces the elasticity of sputum from patients with cystic fibrosis.

Mucus obstruction of the airway in patients with cystic fibrosis (CF) reduces lung function, invites infection, and limits delivery of inhaled drugs including gene therapy vectors to target cells. Not all patients respond to presently available mucolytics, and new approaches are needed. Our objectives were to investigate the in vitro effects of unfractionated heparin (UFH) on the morphology and rheology of sputum and the effect of UFH on diffusion of 200-nm nanospheres through sputum from adult CF patients. Confocal laser scanning microscopy was used to image fluorescently stained actin and DNA components of CF sputum, and atomic force microscopy was used to image isolated DNA networks. The viscoelasticity of CF sputum was measured using dynamic oscillatory rheometry. Nanosphere diffusion was measured through CF sputum using a Boyden chamber-based assay. Actin-DNA bundles in CF sputum were disaggregated by UFH at concentrations of 0.1-10 mg/ml, and UFH enhanced the endonuclease activity in sputum from patients on dornase alfa therapy. UFH significantly reduced the elasticity and yield stress, but not the viscosity, of CF sputum from patients not receiving dornase alfa therapy. Heparin dose-dependently significantly increased the diffusion of nanospheres through CF sputum from patients not on dornase alfa therapy from 10.5 +/- 2.5% at baseline to 36.9 +/- 4.4% at 10 mg/ml but was more potent, with maximal effect at 0.1 mg/ml, in patients who were on dornase alfa therapy. Thus the mucoactive properties of UFH indicate its potential as a new therapeutic approach in patients with cystic fibrosis.

Triplex staples: DNA double-strand cross-linking at internal and terminal sites using psoralen-containing triplex-forming oligonucleotides.

A method has been developed to attach 4'-(hydroxymethyl)-4,5',8-trimethylpsoralen to the 5 position of thymine bases during solid-phase oligonucleotide synthesis. UV irradiation of triplex-forming oligonucleotides (TFOs) containing internally attached psoralens produces photoadducts at TpA steps within target duplexes, thus relaxing the constraints on selection of psoralen target sequences. Photoreaction of TFOs containing two psoralens, located at the 5'- and 3'-ends, has been used to create double-strand cross-links (triplex staples) at both termini of the TFO. Such complexes have no free single-stranded ends. TFOs containing 4'-(hydroxymethyl)-4,5',8-trimethylpsoralen, 3-methyl-2-aminopyridine, and 5-(3-aminoprop-2-ynyl)deoxyuridine formed photoadducts with target duplexes under near-physiological conditions.