pulmonary mucus hydration assay using rotational and translational diffusion of gold nanorods with polarization-sensitive optical coherence tomography.

Significance: Assessing the nanostructure of polymer solutions and biofluids is broadly useful for understanding drug delivery and disease progression and for monitoring therapy.

Aim: Our objective is to quantify bronchial mucus solids concentration (wt. %) during hypertonic saline (HTS) treatment via nanostructurally constrained diffusion of gold nanorods (GNRs) monitored by polarization-sensitive optical coherence tomography (PS-OCT).

Oligonucleotide-based therapies for cystic fibrosis.

In the clinically successful era of CFTR modulators and Theratyping, 10-20% of individuals with cystic fibrosis (CF) may develop disease due to CFTR mutations that remain undruggable. These individuals produce low levels of CFTR mRNA and/or not enough protein to be rescued with modulator drugs. Alternative therapeutic approaches to correct the CFTR defect at the mRNA level using nucleic acid technologies are currently feasible; e.

Enhanced delivery of peptide-morpholino oligonucleotides with a small molecule to correct splicing defects in the lung.

Pulmonary diseases offer many targets for oligonucleotide therapeutics. However, effective delivery of oligonucleotides to the lung is challenging. For example, splicing mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) affect a significant cohort of Cystic Fibrosis (CF) patients.

Retro-1-Oligonucleotide Conjugates. Synthesis and Biological Evaluation.

Addition of small molecule Retro-1 has been described to enhance antisense and splice switching oligonucleotides. With the aim of assessing the effect of covalently linking Retro-1 to the biologically active oligonucleotide, three different derivatives of Retro-1 were prepared that incorporated a phosphoramidite group, a thiol or a 1,3-diene, respectively. Retro-1⁻oligonucleotide conjugates were assembled both on-resin (coupling of the phosphoramidite) and from reactions in solution (Michael-type thiol-maleimide reaction and Diels-Alder cycloaddition).

Different Munc18 proteins mediate baseline and stimulated airway mucin secretion.

Airway mucin secretion is necessary for ciliary clearance of inhaled particles and pathogens but can be detrimental in pathologies such as asthma and cystic fibrosis. Exocytosis in mammals requires a Munc18 scaffolding protein, and airway secretory cells express all 3 Munc18 isoforms. Using conditional airway epithelial cell-deletant mice, we found that Munc18a has the major role in baseline mucin secretion, Munc18b has the major role in stimulated mucin secretion, and Munc18c does not function in mucin secretion.

Airway surface liquid pH is not acidic in children with cystic fibrosis.

Modulation of airway surface liquid (ASL) pH has been proposed as a therapy for cystic fibrosis (CF). However, evidence that ASL pH is reduced in CF is limited and conflicting. The technical challenges associated with measuring ASL pH in vivo have precluded accurate measurements in humans.

A Novel Family of Small Molecules that Enhance the Intracellular Delivery and Pharmacological Effectiveness of Antisense and Splice Switching Oligonucleotides.

The pharmacological effectiveness of oligonucleotides has been hampered by their tendency to remain entrapped in endosomes, thus limiting their access to cytosolic or nuclear targets. We have previously reported a group of small molecules that enhance the effects of oligonucleotides by causing their release from endosomes. Here, we describe a second novel family of oligonucleotide enhancing compounds (OECs) that is chemically distinct from the compounds reported previously.

Diffusion-sensitive optical coherence tomography for real-time monitoring of mucus thinning treatments.

Mucus hydration (wt%) has become an increasingly useful metric in real-time assessment of respiratory health in diseases like cystic fibrosis and COPD, with higher wt% indicative of diseased states. However, available rheological techniques are lacking. Gold nanorods (GNRs) are attractive biological probes whose diffusion through tissue is sensitive to the correlation length of comprising biopolymers.

Vesicular nucleotide transporter regulates the nucleotide content in airway epithelial mucin granules.

Nucleotides within the airway surface liquid promote fluid secretion via activation of airway epithelial purinergic receptors. ATP is stored within and released from mucin granules as co-cargo with mucins, but the mechanism by which ATP, and potentially other nucleotides, enter the lumen of mucin granules is not known. We assessed the contribution of the recently identified SLC17A9 vesicle nucleotide transporter (VNUT) to the nucleotide availability within isolated mucin granules and further examined the involvement of VNUT in mucin granule secretion-associated nucleotide release.

CFTR, mucins, and mucus obstruction in cystic fibrosis.

Mucus pathology in cystic fibrosis (CF) has been known for as long as the disease has been recognized and is sometimes called mucoviscidosis. The disease is marked by mucus hyperproduction and plugging in many organs, which are usually most fatal in the airways of CF patients, once the problem of meconium ileus at birth is resolved. After the CF gene, CFTR, was cloned and its protein product identified as a cAMP-regulated Cl(-) channel, causal mechanisms underlying the strong mucus phenotype of the disease became obscure.

The UDP-sugar-sensing P2Y(14) receptor promotes Rho-mediated signaling and chemotaxis in human neutrophils.

The G(i)-coupled P2Y(14) receptor (P2Y(14)-R) is potently activated by UDP-sugars and UDP. Although P2Y(14)-R mRNA is prominently expressed in circulating neutrophils, the signaling pathways and functional responses associated with this receptor are undefined. In this study, we illustrate that incubation of isolated human neutrophils with UDP-glucose resulted in cytoskeleton rearrangement, change of cell shape, and enhanced cell migration.

VAMP8 is a vesicle SNARE that regulates mucin secretion in airway goblet cells.

Mucin secretion is an innate defence mechanism, which is noxiously upregulated in obstructive lung diseases (e.g. chronic obstructive pulmonary disease (COPD), cystic fibrosis and asthma).

Cigarette smoke exposure induces CFTR internalization and insolubility, leading to airway surface liquid dehydration.

Cigarette smoke (CS) exposure induces mucus obstruction and the development of chronic bronchitis (CB). While many of these responses are determined genetically, little is known about the effects CS can exert on pulmonary epithelia at the protein level. We, therefore, tested the hypothesis that CS exerts direct effects on the CFTR protein, which could impair airway hydration, leading to the mucus stasis characteristic of both cystic fibrosis and CB.

Rho signaling regulates pannexin 1-mediated ATP release from airway epithelia.

ATP released from airway epithelial cells promotes purinergic receptor-regulated mucociliary clearance activities necessary for innate lung defense. Cell swelling-induced membrane stretch/strain is a common stimulus that promotes airway epithelial ATP release, but the mechanisms transducing cell swelling into ATP release are incompletely understood. Using knockdown and knockout approaches, we tested the hypothesis that pannexin 1 mediates ATP release from hypotonically swollen airway epithelia and investigated mechanisms regulating this activity.

Molecular mechanisms of purine and pyrimidine nucleotide release.

Given the widespread importance of purinergic receptor-evoked signaling, understanding how ATP and other nucleotides are released from cells in a regulated manner is an essential physiological question. Nonlytic release of ATP, UTP, UDP-glucose, and other nucleotides occurs in all cell types and tissues via both constitutive mechanisms, that is, in the absence of external stimuli, and to a greater extent in response to biochemical or mechanical/physical stimuli. However, a molecular understanding of the processes regulating nucleotide release has only recently begun to emerge.

Nucleotide release by airway epithelia.

The purinergic events regulating the airways' innate defenses are initiated by the release of purines from the epithelium, which occurs constitutively and is enhanced by chemical or mechanical stimulation. While the external triggers have been reviewed exhaustively, this chapter focuses on current knowledge of the receptors and signaling cascades mediating nucleotide release. The list of secreted purines now includes ATP, ADP, AMP and nucleotide sugars, and involves at least three distinct mechanisms reflecting the complexity of airway epithelia.

Imaging CFTR protein localization in cultured cells and tissues.

CFTR functions as a chloride channel at the apical membrane of airway, gastrointestinal, and other epithelial cells. Immunofluorescence microscopy is commonly used to assess the subcellular localization and relative abundance of CFTR. Visualization of heterologously overexpressed CFTR is typically unproblematic and straightforward, whereas detection of small quantities of endogenous CFTR in tissues can be challenging and requires highly specific antibodies and optimized staining protocols.

Coupled nucleotide and mucin hypersecretion from goblet-cell metaplastic human airway epithelium.

Adenosine triphosphate (ATP) and its metabolite adenosine regulate airway mucociliary clearance via activation of purinoceptors. In this study, we investigated the contribution of goblet cells to airway epithelial ATP release. Primary human bronchial epithelial (HBE) cultures, typically dominated by ciliated cells, were induced to develop goblet cell metaplasia by infection with respiratory syncytial virus (RSV) or treatment with IL-13.

Receptor-promoted exocytosis of airway epithelial mucin granules containing a spectrum of adenine nucleotides.

Purinergic regulation of airway innate defence activities is in part achieved by the release of nucleotides from epithelial cells. However, the mechanisms of airway epithelial nucleotide release are poorly understood. We have previously demonstrated that ATP is released from ionomycin-stimulated airway epithelial goblet cells coordinately with mucin exocytosis, suggesting that ATP is released as a co-cargo molecule from mucin-containing granules.

Endoplasmic reticulum/golgi nucleotide sugar transporters contribute to the cellular release of UDP-sugar signaling molecules.

Extracellular UDP-sugars promote cellular responses by interacting with widely distributed P2Y(14) receptors, but the mechanisms by which these molecules are released from cells are poorly understood. Given the active role of UDP-sugars in glycosylation reactions within the secretory pathway, we hypothesized that UDP-sugar release includes an exocytotic component. This hypothesis was tested by assessing the contribution of endoplasmic reticulum (ER)/Golgi-resident UDP-GlcNAc transporters to the cellular release of their cognate substrates.

Development of chronic bronchitis and emphysema in beta-epithelial Na+ channel-overexpressing mice.

Rationale: Chronic obstructive pulmonary disease is a leading cause of death worldwide, but its pathogenesis is not well understood. Previous studies have shown that airway surface dehydration in beta-epithelial Na(+) channel (betaENaC)-overexpressing mice caused a chronic lung disease with high neonatal pulmonary mortality and chronic bronchitis in adult survivors.

Objectives: The aim of this study was to identify the initiating lesions and investigate the natural progression of lung disease caused by airway surface dehydration.

Coordinated release of nucleotides and mucin from human airway epithelial Calu-3 cells.

The efficiency of the mucociliary clearance (MCC) process that removes noxious materials from airway surfaces depends on the balance between mucin secretion, airway surface liquid (ASL) volume, and ciliary beating. Effective mucin dispersion into ASL requires salt and water secretion onto the mucosal surface, but how mucin secretion rate is coordinated with ion and, ultimately, water transport rates is poorly understood. Several components of MCC, including electrolyte and water transport, are regulated by nucleotides in the ASL interacting with purinergic receptors.

Direct interaction with filamins modulates the stability and plasma membrane expression of CFTR.

The role of the cystic fibrosis transmembrane conductance regulator (CFTR) as a cAMP-dependent chloride channel on the apical membrane of epithelia is well established. However, the processes by which CFTR is regulated on the cell surface are not clear. Here we report the identification of a protein-protein interaction between CFTR and the cytoskeletal filamin proteins.

Physiological regulation of ATP release at the apical surface of human airway epithelia.

Extracellular ATP and its metabolite adenosine regulate mucociliary clearance in airway epithelia. Little has been known, however, regarding the actual ATP and adenosine concentrations in the thin ( approximately 7 microm) liquid layer lining native airway surfaces and the link between ATP release/metabolism and autocrine/paracrine regulation of epithelial function. In this study, chimeric Staphylococcus aureus protein A-luciferase (SPA-luc) was bound to endogenous antigens on primary human bronchial epithelial (HBE) cell surface and ATP concentrations assessed in real-time in the thin airway surface liquid (ASL).

The cystic fibrosis transmembrane conductance regulator is regulated by a direct interaction with the protein phosphatase 2A.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-activated chloride channel expressed at the apical surface of epithelia. Although the regulation of CFTR by protein kinases is well documented, channel deactivation by phosphatases is not well understood. We find that the serine/threonine phosphatase PP2A can physically associate with the CFTR COOH terminus.