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The mucus clearance system is the dominant mechanical host defense system of the human lung. Mucus is cleared from the lung by cilia and airflow, including both two-phase gas-liquid pumping and cough-dependent mechanisms, and mucus transport rates are heavily dependent on mucus concentration. Importantly, mucus transport rates are accurately predicted by the gel-on-brush model of the mucociliary apparatus from the relative osmotic moduli of the mucus and periciliary-glycocalyceal (PCL-G) layers. The fluid available to hydrate mucus is generated by transepithelial fluid transport. Feedback interactions between mucus concentrations and cilia beating, via purinergic signaling, coordinate Na+absorptive vs Cl−secretory rates to maintain mucus hydration in health. In disease, mucus becomes hyperconcentrated (dehydrated). Multiple mechanisms derange the ion transport pathways that normally hydrate mucus in muco-obstructive lung diseases, e.g., cystic fibrosis (CF), chronic obstructive pulmonary disease (COPD), non-CF bronchiectasis (NCFB), and primary ciliary dyskinesia (PCD). A key step in muco-obstructive disease pathogenesis is the osmotic compression of the mucus layer onto the airway surface with the formation of adherent mucus plaques and plugs, particularly in distal airways. Mucus plaques create locally hypoxic conditions and produce airflow obstruction, inflammation, infection, and, ultimately, airway wall damage. Therapies to clear adherent mucus with hydrating and mucolytic agents are rational, and strategies to develop these agents are reviewed.

Mucus hyperconcentration in cystic fibrosis (CF) lung disease is marked by increases in both mucin and DNA concentration. Additionally, it has been shown that half of the mucins present in bronchial alveolar lavage fluid (BALF) from preschool-aged CF patients are present in as non-swellable mucus flakes. This motivates us to examine the utility of mucus flakes, as well as mucin and DNA concentrations in BALF as markers of infection and inflammation in CF airway disease.In this study, we examined the mucin and DNA concentration, as well as mucus flake abundance, composition, and biophysical properties in BALF from three groups; healthy adult controls, and two CF cohorts, one preschool aged and the other school aged. BALFs were characterized via refractometry, PicoGreen, immunofluorescence microscopy, particle tracking microrheology, and fluorescence image tiling.Mucin and DNA BALF concentrations increased progressively from healthy young adult controls to preschool-aged people and school-aged people with CF. Notably, mucin concentrations were increased in bronchoalveolar lavage fluid (BALF) from preschool-aged patients with CF prior to decreased pulmonary function. Infrequent small mucus flakes were identified in normal subjects. A progressive increase in the abundance of mucus flakes in preschool and school-aged CF patients was observed. Compositionally, MUC5B dominated flakes from normal subjects, whereas an increase in MUC5AC was observed in people with CF, reflected in a reduced flaked MUC5B/MUC5AC mucin ratio.These findings suggest mucus composition and flake properties are useful markers of inflammatory and infection-based changes in CF airways.

Influenza virus is a major cause of death on a global scale. Seasonal vaccines have been developed to combat influenza; however, they are not always highly effective. One strategy to develop a more broadly active influenza vaccine is the use of multiple rounds of layered consensus buildings to generate recombinant antigens, termed computationally optimized broadly reactive antigen (COBRA). Immunization with the COBRA hemagglutinin (HA) can elicit broad protection against multiple strains of a single influenza subtype (e.g., H1N1). We formulated a COBRA H1 HA with a stimulator of interferon genes agonist cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) into a nasal gel for vaccination against influenza. The gel formulation was designed to increase mucoadhesion and nasal retention of the antigen and adjuvant to promote a strong mucosal response. It consisted of a Schiff base-crosslinked hydrogel between branched polyethyleneimine and oxidized dextran. Following a prime-boost-boost schedule, an intranasal gel containing cGAMP and model antigen ovalbumin (OVA) led to the faster generation of serum IgG, IgG1, and IgG2c and significantly greater serum IgG1 levels on day 42 compared to soluble controls. Additionally, OVA-specific IgA was detected in nasal, vaginal, and fecal samples for all groups, except the vehicle control. When the COBRA HA was given intranasally in a prime-boost schedule, the mice receiving the gel containing the COBRA and cGAMP had significantly higher serum IgG and IgG2c at day 41 compared to all groups, and only this group had IgA levels above the background in vaginal, nasal, and fecal samples. Overall, this study indicates the utility of an intranasal gel for the delivery of COBRAs for the generation of serum and mucosal humoral responses.

The last years have shown enormous advancement in the therapeutic potential of RNA-related treatments, specifically for antisense oligonucleotide (ASO)-based drugs, leading to increased numbers of ASO regulatory approvals. In this study we focus on SPL84, an inhaled ASO-based drug, developed for the treatment of the pulmonary disease, Cystic Fibrosis (CF). Pulmonary drug delivery is challenging, due to a variety of biological, physical, chemical, and structural barriers, especially when aiming to target the cell nucleus. The efficient distribution of SPL84 in the lungs, penetration into the cells and nucleus, and stability are critical parameters that will impact drug efficacy in a clinical setting. In this study, we demonstrate the proper distribution and cell and nucleus penetration of SPL84 in mouse and monkey lungs. In vivo and in vitro studies confirmed the stability and mobility of our inhaled ASO drug through CF patient-derived mucus and in lung lysosomal extracts. Our results, supported by a promising pre-clinical pharmacological effect, emphasize the high potential of SPL84 as an effective drug for the treatment of CF patients.In addition, successfully tackling the lung distribution of SPL84 and specific cell targeting offers huge opportunities for further development of SpliSense inhaled ASO-based drugs for unmet pulmonary diseases.

Impaired mucociliary clearance (MCC) is a key feature of many airway diseases, including asthma, bronchiectasis, chronic obstructive pulmonary disease, cystic fibrosis, and primary ciliary dyskinesia. To improve MCC and develop new treatments for these diseases requires a thorough understanding of how mucus concentration, mucus composition, and ciliary activity affect MCC, and how different therapeutics impact this process. Although differentiated cultures of human airway epithelial cells are useful for investigations of MCC, the extent of ciliary coordination in these cultures varies, and the mechanisms controlling ciliary orientation are not completely understood. By introducing a pattern of ridges and grooves into the underlying collagen substrate, we demonstrate for the first time, to our knowledge, that changes in the extracellular matrix can induce ciliary alignment. Remarkably, 90% of human airway epithelial cultures achieved continuous directional mucociliary transport (MCT) when grown on the patterned substrate. These cultures maintain transport for months, allowing carefully controlled investigations of MCC over a wide range of normal and pathological conditions. To characterize the system, we measured the transport of bovine submaxillary gland mucin (BSM) under several conditions. Transport of 5% BSM was significantly reduced compared with that of 2% BSM, and treatment of 5% BSM with the reducing agent tris(2-carboxyethyl)phosphine (TCEP) reduced viscosity and increased the rate of MCT by approximately twofold. Addition of a small amount of high-molecular-weight DNA increased mucus viscosity and reduced MCT by ∼75%, demonstrating that the composition of mucus, as well as the concentration, can have significant effects on MCT. Our results demonstrate that a simple patterning of the collagen substrate results in highly coordinated ciliated cultures that develop directional MCT, and can be used to investigate the mechanisms controlling the regulation of ciliary orientation. Furthermore, the results demonstrate that this method provides an improved system for studying the effects of mucus composition and therapeutic agents on MCC.

Cystic fibrosis (CF) is characterized by abnormal transepithelial ion transport. However, a description of CF lung disease pathophysiology unifying superficial epithelial and submucosal gland (SMG) dysfunctions has remained elusive. We hypothesized that biophysical abnormalities associated with CF mucus hyperconcentration provide a unifying mechanism. Studies of the anion secretion–inhibited pig airway model of CF revealed elevated SMG mucus concentrations, osmotic pressures, and SMG mucus accumulation. Human airway studies revealed hyperconcentrated CF SMG mucus with raised osmotic pressures and cohesive forces predicted to limit SMG mucus secretion/release. Using proline-rich protein 4 (PRR4) as a biomarker of SMG secretion, CF sputum proteomics analyses revealed markedly lower PRR4 levels compared to healthy and bronchiectasis controls, consistent with a failure of CF SMGs to secrete mucus onto airway surfaces. Raised mucus osmotic/cohesive forces, reflecting mucus hyperconcentration, provide a unifying mechanism that describes disease-initiating mucus accumulation on airway surfaces and in SMGs of the CF lung.

Background The combination of antibacterial and mucolytic actions makes nitric oxide (NO) an attractive dual-action cystic fibrosis (CF) therapeutic. The delivery of any therapeutic agent through pathological mucus is difficult, and the use of inhaled NO gas is inherently limited by toxicity concerns. Herein, we directly compare the ability of NO to eradicate infection and decrease mucus viscoelastic moduli as a function of delivery method (i.e., as a gas or water-soluble chitosan donor). Methods To compare bactericidal action in tissue, an ex vivo porcine lung model was infected and treated with either gaseous NO or NO-releasing chitosan for 5 h. In vitro Pseudomonas aeruginosa biofilm viability was quantified after NO treatment. Human bronchial epithelial mucus and CF sputum were exposed to NO and their viscoelastic moduli measured with parallel plate macrorheology. Results Larger NO concentrations were achieved in solution when delivered by chitosan relative to gas exposure. The bactericidal action in tissue of the NO-releasing chitosan was greater compared to NO gas in the infected tissue model. Chitosan delivery also resulted in improved antibiofilm action and reduced biofilm viability (2-log) while gaseous delivery had no impact at an equivalent dose (~0.8 µmol/mL). At equivalent NO doses, mucus and sputum rheology were significantly reduced after treatment with NO-releasing chitosan with NO gas having no significant effect. Conclusions Delivery of NO by chitosan allows for larger in-solution concentrations than achievable via direct gas with superior bactericidal and mucolytic action.

N-glycans on IgG and IgM antibodies (Ab) facilitate Ab-mediated crosslinking of viruses and nanoparticles to the major structural elements of mucus and basement membranes. Nevertheless, the chemical moieties in these biological hydrogel matrices to which Ab can bind remain poorly understood. To gain insights into the chemistries that support Ab-matrix interactions, we systematically evaluated IgG- and IgM-mediated trapping of nanoparticles in different polysaccharide-based biogels with unique chemical features. In agarose, composed of alternating d-galactose and 3,6-anhydro-l-galactopyranose (i.e. hydroxyl groups only), anti-PEG IgM but not anti-PEG IgG trapped PEGylated nanoparticles. In alginate, comprised of homopolymeric blocks of mannuronate and guluronate (i.e. both hydroxyl and carboxyl groups), both IgG and IgM trapped PEGylated nanoparticles. In contrast, chitosan, comprised primarily of glucosamine (i.e. both hydroxyl and primary amine groups), did not facilitate either IgG- or IgM-mediated trapping. IgG-mediated trapping in alginate was abrogated upon removal of IgG N-glycans, whereas IgM-mediated trapping was eliminated in agarose but not alginate upon desialylation. These results led us to propose a model in which hydrogen bonding between carboxyl and hydroxyl groups of glycans on both Ab and matrix facilitates Ab-mediated trapping of pathogens in biogels. Our work here offers a blueprint for designing de novo hydrogels that could harness Ab-matrix interactions for various biomedical and biological applications. STATEMENT OF SIGNIFICANCE: Here, we interrogated the molecular mechanism of antibody-mediated trapping to address what are the chemical moieties on biogels that are essential for facilitating trapping in biogels. We systematically evaluated the potencies of IgG and IgM to trap nanoparticles in different polysaccharide-based biogels with unique and highly defined chemical moieties: hydroxyl groups (agarose), amine groups (chitosan), and carboxyl groups (alginate). We discovered that only hydroxyl/carboxyl hydrogen bonds (and stronger) are sufficiently strong enough to facilitate antibody-mediated trapping; weaker hydroxyl/hydroxyl bonds or hydroxyl/amine bonds fail to adequately slow particles. Our findings presents the first blueprint for how to engineer de novo biogels that are capable of harnessing antibodies to immobilize foreign entities in the biogels, for applications ranging from infectious disease to contraception to purification processes.

Cystic fibrosis (CF) is characterized by abnormal transepithelial ion transport. However, a description of CF lung disease pathophysiology unifying superficial epithelial and submucosal gland (SMG) dysfunctions has remained elusive. We hypothesized that biophysical abnormalities associated with CF mucus hyperconcentration provide a unifying mechanism. Studies of the anion secretion-inhibited pig airway CF model revealed elevated SMG mucus concentrations, osmotic pressures, and SMG mucus accumulation. Human airway studies revealed hyperconcentrated CF SMG mucus with raised osmotic pressures and cohesive forces predicted to limit SMG mucus secretion/release. Utilizing proline-rich protein 4 (PRR4) as a biomarker of SMG secretion, proteomics analyses of CF sputum revealed markedly lower PRR4 levels compared to healthy and bronchiectasis controls, consistent with a failure of CF SMGs to secrete mucus onto airway surfaces. Raised mucus osmotic/cohesive forces, reflecting mucus hyperconcentration, provide a unifying mechanism that describes disease-initiating mucus accumulation on airway surfaces and within SMGs of the CF lung. One Sentence Summary Hyperconcentrated Mucus Unifies Submucosal Gland and Superficial Airway Dysfunction in Cystic Fibrosis

Significance: Imaging biofluid flow under physiologic conditions aids in understanding disease processes and health complications. We present a method employing a microparallel plate strain induction chamber (MPPSIC) amenable to optical coherence tomography to track depth-resolved lateral displacement in fluids in real time while under constant and sinusoidal shear. Aim: Our objective is to track biofluid motion under shearing conditions found in the respiratory epithelium, first validating methods in Newtonian fluids and subsequently assessing the capability of motion-tracking in bronchial mucus. Approach: The motion of polystyrene microspheres in aqueous glycerol is tracked under constant and sinusoidal applied shear rates in the MPPSIC and is compared with theory. Then 1.5 wt. % bronchial mucus samples considered to be in a normal hydrated state are studied under sinusoidal shear rates of amplitudes 0.7 to 3.2 s−1. Results: Newtonian fluids under low Reynolds conditions (Re∼10−4) exhibit velocity decreases directly proportional to the distance from the plate driven at both constant and oscillating velocities, consistent with Navier–Stokes’s first and second problems at finite depths. A 1.5 wt. % mucus sample also exhibits a uniform shear strain profile. Conclusions: The MPPSIC provides a new capability for studying biofluids, such as mucus, to assess potentially non-linear or strain-rate-dependent properties in a regime that is relevant to the mucus layer in the lung epithelium.

The gain-of-function MUC5B promoter variant rs35705950 is the dominant risk factor for developing idiopathic pulmonary fibrosis (IPF). Here we show in humans that MUC5B, a mucin thought to be restricted to conducting airways, is co-expressed with surfactant protein C (SFTPC) in type 2 alveolar epithelia and in epithelial cells lining honeycomb cysts, indicating that cell types involved in lung fibrosis in distal airspace express MUC5B. In mice, we demonstrate that Muc5b concentration in bronchoalveolar epithelia is related to impaired mucociliary clearance (MCC) and to the extent and persistence of bleomycin-induced lung fibrosis. We also establish the ability of the mucolytic agent P-2119 to restore MCC and to suppress bleomycin-induced lung fibrosis in the setting of Muc5b overexpression. Our findings suggest that mucociliary dysfunction might play a causative role in bleomycin-induced pulmonary fibrosis in mice overexpressing Muc5b, and that MUC5B in distal airspaces is a potential therapeutic target in humans with IPF., The promoter variant rs35705950 confers a gain of function to the MUC5B gene and is the dominant risk factor for idiopathic pulmonary fibrosis. Here the authors show that mice overexpressing Muc5b in distal airspaces show impaired mucociliary clearance and increased susceptibility to bleomycin-induced lung fibrosis, and that both characteristics are reduced by treatment with a mucolytic agent.

The goal of this project was to validate the functional relevance and utility of mucus produced by an in vitro intestinal cell culture model. This is facilitated by the need to physiologically replicate both healthy and abnormal mucus conditions from native intestinal tissue, where mucus properties have been connected to intestinal disease models. Mucus harvested from colonic cell cultures derived from healthy donors was compared to mucus collected from surgically resected, noninflamed transverse colon tissue. The rheological and biochemical properties of these mucus samples were compared using oscillational rheometry, particle-tracking microrheology, multiangle laser light scattering, refractometry, and immunohistochemical imaging. An air-liquid interface culture of primary human colonic epithelial cells generated a continuous monolayer with an attached mucus layer that displayed increasing weight percent (wt%) of solids over 1 week (1.3 ± 0.5% at 2 days vs. 2.4 ± 0.3% at 7 days). The full range of mucus concentrations (0.9–3.3%) observed during culture was comparable to that displayed by ex vivo mucus (1.3–1.9%). Bulk rheological measurements displayed similar wt%-based complex viscosities between in vitro and ex vivo mucus, with the complex viscosity of both systems increasing with wt% of solids. Particle-tracking microrheology showed higher complex viscosities for ex vivo mucus samples than in vitro mucus which was explained by a greater fraction of water present in in vitro mucus than ex vivo, i.e., in vitro mucus is more heterogeneous than ex vivo. Refractometry, multiangle laser light scattering, and immunostaining showed increased mucus complex size in ex vivo mucus compared with in vitro mucus, which may have been due to the admixture of mucus and cellular debris during ex vivo mucus collection. The air-liquid interface culture system produced intestinal mucus with similar composition and rheology to native human gut mucus, providing a platform to analyze pathological differences in intestinal mucus.

Airway mucociliary clearance (MCC) is required for host defense and often diminished in chronic lung diseases. Effective clearance depends upon coordinated actions of the airway epithelium and a mobile mucus layer. Dysregulation of the primary secreted airway mucin proteins, MUC5B and MUC5AC, is associated with a reduction in the rate of MCC; however, how other secreted proteins impact the integrity of the mucus layer and MCC remains unclear. We previously identified the gene Bpifb1/Lplunc1 as a regulator of airway MUC5B levels using genetic approaches. Here, we show that BPIFB1 is required for normal mucociliary clearance in vivo using Bpifb1 knockout (KO) mice. Reduced MCC in Bpifb1 KO mice occurred in the absence of defects in sodium or chloride ion transport or reduced ciliary beat frequency. BPIFB1 loss resulted in airway mucus flakes with significantly increased complex viscosity, a key biophysical property of mucus known to impact MCC. Finally, we detected colocalization of BPIFB1 and MUC5B in secretory granules in mice and in the protein mesh of secreted mucus in human airway cultures. Collectively, our findings demonstrate that BPIFB1 is an important component of the mucociliary apparatus in mice and a key component of the mucus protein network.

The excessive production of thick, viscous mucus in severe respiratory diseases leads to obstruction of the airways and provides a suitable environment for the colonization of pathogenic bacteria. The effect of nitric oxide (NO)-releasing alginates with varying NO release kinetics on the viscoelastic properties of human bronchial epithelial (HBE) mucus was evaluated as a function of the NO-release kinetics using parallel plate rheology. Low molecular weight (~5 kDa) alginates with high NO flux (~4000 ppb/mg) and sustained release (half-life ~0.3 h) proved to be most effective in reducing both mucus elasticity and viscosity (≥60% reduction for both). The efficacy of the NO-releasing alginates was shown to be dose-dependent, with high concentrations of NO-releasing alginates (~80 mg•mL(−1)) resulting in greater reduction of the viscosity and elasticity of the mucus samples. Greater reduction in mucus rheology was also achieved with NO-releasing alginates at lower concentrations when compared to both NO-releasing chitosan, a similarly biocompatible cationic polymer, and N-acetyl cysteine (NAC), a conventional mucolytic agent.

We present a micro-parallel plate rheometer amenable to OCT to track depth-resolved lateral displacement in fluids in real-time while under dynamic shear. Newtonian fluid experiments show direct proportionality between lateral velocity of particles and depth.

QuestionCystic fibrosis (CF) is characterised by the accumulation of viscous adherent mucus in the lungs. While several hypotheses invoke a direct relationship with cystic fibrosis transmembrane conductance regulator (CFTR) dysfunction (i.e. acidic airway surface liquid (ASL) pH, low bicarbonate (HCO3−) concentration, airway dehydration), the dominant biochemical alteration of CF mucus remains unknown.Materials/methodsWe characterised a novel cell line (CFTR-KO Calu3 cells) and the responses of human bronchial epithelial (HBE) cells from subjects with G551D or F508del mutations to ivacaftor and elexacaftor-tezacaftor-ivacaftor. A spectrum of assays such as short-circuit currents, quantitative PCR, ASL pH, Western blotting, light scattering/refractometry (size-exclusion chromatography with inline multi-angle light scattering), scanning electron microscopy, percentage solids and particle tracking were performed to determine the impact of CFTR function on mucus properties.ResultsLoss of CFTR function in Calu3 cells resulted in ASL pH acidification and mucus hyperconcentration (dehydration). Modulation of CFTR in CF HBE cells did not affect ASL pH or mucin mRNA expression, but decreased mucus concentration, relaxed mucus network ultrastructure and improved mucus transport. In contrast with modulator-treated cells, a large fraction of airway mucins remained attached to naïve CF cells following short apical washes, as revealed by the use of reducing agents to remove residual mucus from the cell surfaces. Extended hydration, but not buffers alkalised with sodium hydroxide or HCO3−, normalised mucus recovery to modulator-treated cell levels.ConclusionThese results indicate that airway dehydration, not acidic pH and/or low [HCO3−], is responsible for abnormal mucus properties in CF airways and CFTR modulation predominantly restores normal mucin entanglement.

Aerosol delivery of chemotherapeutic nanocarriers represents a promising alternative for lung cancer therapy. This study optimized gemcitabine (Gem)-loaded gelatin nanocarriers (GNCs) cross-linked with genipin (Gem-GNCs) to evaluate their potential for nebulized lung cancer treatment.Gem-GNCs were prepared by two-step desolvation and optimized through Taguchi design and characterized for physicochemical properties. Particle size and morphology were confirmed by scanning and transmission electron microscopy. In vitro release of Gem from Gem-GNCs performed in Dulbecco's phosphate-buffered saline and simulated lung fluid was evaluated to determine release mechanisms. Particle size stability was assessed under varying pH. Differential scanning calorimetry and powder X-ray diffraction were used to determine the presence and stability of Gem-GNC components and amorphization of Gem, respectively. Gem-GNC efficacy within A549 and H460 cells was evaluated using MTT assays. Mucus rheology upon treatment with Gem-GNCs, lactose, and normal saline control was measured. Andersen cascade impaction identified the aerodynamic particle size distribution of the nebulized formulation.Gem-GNCs had particle size, zeta potential, entrapment efficiency, and loading efficiency of 178 ± 7.1 nm, -18.9 mV, 92.5%, and 9.1%, respectively. The Gem and formulation excipients where molecularly dispersed and configured amorphously. Gem-GNCs were stable at pH 5.4-7.4 for 72 hours. Gem release from Gem-GNCs was governed by non-Fickian controlled release due to diffusion/erosion from a matrix-based nanocarrier. Gem-GNCs elicited a 40% reduction of the complex viscosity η*(1 Hz) of human bronchial epithelial cell mucus containing 3 wt% solids to mimic mild airway disease. The nebulized Gem-GNCs had a mass median aerodynamic diameter (MMAD) of 2.0 ± 0.16 μm, geometric standard deviation (GSD) of 2.7 ± 0.16, and fine particle fraction (FPF) of 75.2% ± 2.4%. The Gem-GNC formulation did not outperform the Gem solution in A549 cells. However, in H460, Gem-GNCs outperformed the Gem IC50 reduction by ∼5-fold at 48 and 10-fold 72 hours.Stable, effective, and sustained-release Gem-GNCs were developed. The nebulized Gem-GNCs had satisfactory MMAD, GSD, and FPF and the formulation reduced the dynamic complex viscosity of mucus consistent with increased mobility of nanoparticles.

Nitric oxide (NO)-releasing chitosan oligosaccharides were modified with ester functional groups to examine how the mucoadhesive nature of the scaffold impacts the ability of NO to degrade mucins from human bronchial epithelial cell cultures and clinical sputum samples collected from patients with cystic fibrosis (CF). Agarose gel electrophoresis experiments indicated that the mucoadhesive NO-releasing chitosan oligosaccharides degraded both the purified mucins and sputum, while control scaffolds (without NO release or mucoadhesive ligands) had no effect on mucin structure. Microscopic observations of sputum treated with the mucoadhesive NO-releasing chitosan oligosaccharide confirmed degradation of the mucin and DNA networks. Similarly, the viscosity and elasticity of sputum were reduced upon treatment with the mucoadhesive NO-releasing chitosan, demonstrating the potential utility of these NO-releasing scaffolds as mucolytic agents.

The solid concentration of pulmonary mucus (wt%) is critical to respiratory health. In patients with respiratory disease, such as Cystic Fibrosis (CF) and Chronic Obstructive Pulmonary Disorder (COPD), mucus hydration is impaired, resulting in high wt%. Mucus with high wt% is a hallmark of pulmonary disease that leads to obstructed airways, inflammation, and infection. Methods to measure mucus hydration in situ and in real-time are needed for drug development and personalized therapy. We employed plasmonic gold nanorod (GNR) biosensors that intermittently collide with macromolecules comprising the mucus mesh as they self-diffuse, such that GNR translational diffusion (DT) is sensitive to wt%. GNRs are attractive candidates for bioprobes due to their anisotropic optical scattering that makes them easily distinguishable from native tissue using polarization-sensitive OCT. Using principles of heterodyne dynamic light scattering, we developed diffusion-sensitive optical coherence tomography (DS-OCT) to spatially-resolve changing DT in real-time. DS-OCT enables, for the first time, direct monitoring of changes in nanoparticle diffusion rates that are sensitive to nanoporosity with spatial and temporal resolutions of 4.7 μm and 0.2 s. DS-OCT therefore enables us to measure spatially-resolved changes in mucus wt% over time. In this study, we demonstrate the applicability of DS-OCT on well-differentiated primary human bronchial epithelial cells during a clinical mucus-hydrating therapy, hypertonic saline treatment (HST), to reveal, for the first time, mucus mixing, cellular secretions, and mucus hydration on the micrometer scale that translate to long-term therapeutic effects.

Muco-obstructive lung diseases (MOLDs), like cystic fibrosis and chronic obstructive pulmonary disease, affect a spectrum of subjects globally. In MOLDs, the airway mucus becomes hyperconcentrated, increasing osmotic and viscoelastic moduli and impairing mucus clearance. MOLD research requires relevant sources of healthy airway mucus for experimental manipulation and analysis. Mucus collected from endotracheal tubes (ETT) may represent such a source with benefits, e.g., in vivo production, over canonical sample types such as sputum or human bronchial epithelial (HBE) mucus. Ionic and biochemical compositions of ETT mucus from healthy human subjects were characterized and a stock of pooled ETT samples generated. Pooled ETT mucus exhibited concentration-dependent rheologic properties that agreed across spatial scales with reported individual ETT samples and HBE mucus. We suggest that the practical benefits compared with other sample types make ETT mucus potentially useful for MOLD research.

Building performance simulation [BPS] tools are important in all design stages. However, barriers such as time, resources, and expertise inhibit their use in the early design stages. This study aims to develop, as part of decision-support framework, metamodels to assess the thermal discomfort in a naturally ventilated Brazilian low-cost house during early design. The metamodels predict the degree-hours of discomfort by heat and/or by cold as a function of design parameters for three Brazilian cities: Curitiba, Sao Paulo, and Manaus. The key design parameters, related with passive design strategies, are building orientation, shading devices position and dimensions, thermal properties of the walls and roof, window-to-wall ratio, and effective window ventilation area. The method consists of three main stages: (i) baseline model development; (ii) Monte Carlo simulation; (iii) multivariate regression. Overall, the metamodels showed R2 values higher than 0.95 for all climates, except the ones predicting discomfort by heat for Curitiba (R2 =0.61) and Sao Paulo (R2 =0.75). The proposed metamodels can quickly and accurately assess the thermal performance of naturally ventilated low-cost houses. They can be used to guide professionals during the early design stages, and for educational purposes in building design pedagogy.

Background: While protein therapeutics are invaluable in managing numerous diseases, many require frequent injections to maintain therapeutically effective concentrations, due to their short half-life in circulation. PolyXen™, a platform and patented technology employing biodegradable, non-immunogenic and hydrophilic Polysialic Acids (PSA) for drug delivery, is being utilized to overcome such limitations, thereby potentially enabling the clinical utility of a broad range of protein therapeutics. Here, we report the recent progress on two development candidates, polysialylated deoxyribonuclease I (PSA-DNase) and polysialylated erythropoietin (PSA-EPO). Methods and Results: Chemical polysialylation of DNase I (DNase) using PSA with different chain length at various conjugation sites led to improved stability against proteases and thermal stress, and slightly reduced enzymatic activity. Polysialylation of EPO resulted in retention of protein structure and PSA-EPO remained biologically active. PSA-EPO had a significantly prolonged circulating half-life (e.g. t 1/2 of PSA-EPO = ~400 h in patients after subcutaneous administration, aimed for once monthly administration, vs. t 1/2 of EPO = ~22 h, administered twice or thrice weekly), and retained in vivo efficacy. Conclusion: This approach has been clinically validated in phase I (in healthy volunteers) and II studies of PSA-EPO [for managing anemia in patients with chronic kidney disease (CKD)].

Rationale: Airways obstruction with thick, adherent mucus is a pathophysiologic and clinical feature of muco-obstructive respiratory diseases, including chronic obstructive pulmonary disease, asthma, and cystic fibrosis (CF). Mucins, the dominant biopolymer in mucus, organize into complex polymeric networks via the formation of covalent disulfide bonds, which govern the viscoelastic properties of the mucus gel. For decades, inhaled N-acetylcysteine (NAC) has been used as a mucolytic to reduce mucin disulfide bonds with little, if any, therapeutic effects. Improvement of mucolytic therapy requires the identification of NAC deficiencies and the development of compounds that overcome them. Objectives: Elucidate the pharmacological limitations of NAC and test a novel mucin-reducing agent, P3001, in preclinical settings. Methods: The study used biochemical (e.g., Western blotting, mass spectrometry) and biophysical assays (e.g., microrheology/macrorheology, spinnability, mucus velocity measurements) to test compound efficacy and toxicity in in vitro and in vivo models and patient sputa. Measurements and Main Results: Dithiothreitol and P3001 were directly compared with NAC in vitro and both exhibited superior reducing activities. In vivo, P3001 significantly decreased lung mucus burden in βENaC-overexpressing mice, whereas NAC did not (n = 6–24 mice per group). In NAC-treated CF subjects (n = 5), aerosolized NAC was rapidly cleared from the lungs and did not alter sputum biophysical properties. In contrast, P3001 acted faster and at lower concentrations than did NAC, and it was more effective than DNase in CF sputum ex vivo. Conclusions: These results suggest that reducing the viscoelasticity of airway mucus is an achievable therapeutic goal with P3001 class mucolytic agents.

Mucociliary clearance (MCC) plays an essential role in maintaining airway sterility and health. Conversely, mucociliary dysfunction is implicated across many airway obstructive diseases. Understanding the necessary requirements for successful MCC is imperative to establish the pathology of disease, as well as to develop therapeutic strategies. Although postural, that is, gravitational, drainage is used clinically to aid mucus clearance, it is ignored in both animal and cell culture models of MCC. In this study, we develop a novel mucus clearance assay that enables the first particle image velocimetry of human bronchial epithelial cell cultures tilted relative to the gravitational field. This tilting system makes it possible to observe drainage of the airway surface liquid and, thus, reveals the effect gravity has on mucociliary clearance. First, we use this assay to demonstrate that beating cilia alone cannot transport buffer upward against gravity. Next, we show the same cilia successfully transporting mucus upward. These results indicate that the biophysical and biochemical properties of mucus enable vertical clearance and that current assay systems are not equipped to determine which properties are required for physiologically relevant vertical mucociliary clearance.

Although destructive airway disease is evident in young children with cystic fibrosis (CF), little is known about the nature of the early CF lung environment triggering the disease. To elucidate early CF pulmonary pathophysiology, we performed mucus, inflammation, metabolomic, and microbiome analyses on bronchoalveolar lavage fluid (BALF) from 46 preschool children with CF enrolled in the Australian Respiratory Early Surveillance Team for Cystic Fibrosis (AREST CF) program and 16 non-CF disease controls. Total airway mucins were elevated in CF compared to non-CF BALF irrespective of infection, and higher densities of mucus flakes containing mucin 5B and mucin 5AC were observed in samples from CF patients. Total mucins and mucus flakes correlated with inflammation, hypoxia, and oxidative stress. Many CF BALFs appeared sterile by culture and molecular analyses, whereas other samples exhibiting bacterial taxa associated with the oral cavity. Children without computed tomography–defined structural lung disease exhibited elevated BALF mucus flakes and neutrophils, but little/no bacterial infection. Although CF mucus flakes appeared “permanent” because they did not dissolve in dilute BALF matrix, they could be solubi-lized by a previously unidentified reducing agent (P2062), but not N-acetylcysteine or deoxyribonuclease. These findings indicate that early CF lung disease is characterized by an increased mucus burden and inflammatory markers without infection or structural lung disease and suggest that mucolytic and anti-inflammatory agents should be explored as preventive therapy.

A tracer particle is called anomalously diffusive if its mean squared displacement grows approximately as $\sigma^2 t^{\alpha}$ as a function of time $t$ for some constant $\sigma^2$, where the diffusion exponent satisfies $\alpha \neq 1$. In this article, we use recent results on the asymptotic distribution of the time-averaged mean squared displacement (Didier and Zhang (2017)) to construct statistical tests for detecting physical heterogeneity in viscoelastic fluid samples starting from one or multiple observed anomalously diffusive paths. The methods are asymptotically valid for the range $0 < \alpha < 3/2$ and involve a mathematical characterization of time-averaged mean squared displacement bias and the effect of correlated disturbance errors. The assumptions on particle motion cover a broad family of fractional Gaussian processes, including fractional Brownian motion and many fractional instances of the generalized Langevin equation framework. We apply the proposed methods in experimental data from treated $P.\ aeruginosa$ biofilms generated by the collaboration of the Hill and Schoenfisch Labs at UNC-Chapel Hill.

Modeling historical structures in virtual space poses many challenges, especially when the original physical structure no longer exists. This chapter reconstructs the pavilion’s architectural history as an expression of its time and as part of a longer tradition of design experiments in which garden pavilions play an important role. The chapter then illustrates the research and digital processes used to reconstruct the building. The research team coupled historical documents—original sketches, engravings, color plates, and a lone black-and-white photo—with architectural modeling tools to achieve sufficiently accurate form, space, and material representations of the pavilion. Experienced through various forms of immersive and interactive media, the model becomes a critical element in portraying and understanding the pavilion’s historical narrative.

Fully nonlinear flow-constitutive model simulations are employed to investigate constructive and destructive interference of counter-propagating shear waves and their associated stress profiles within a viscoelastic layer of “intermediate depth”. Linear unidirectional shear waves in sufficiently thick viscoelastic layers, the viscoelastic analog of Stokes’ second problem, were exploited as a rheological tool by Ferry et al. [1] , [2] . The extension to intermediate gap heights for linear and nonlinear driving amplitudes was studied by our group [3] , [4] , [5] , and by Balmforth et al. [6] for viscoplastic fluids. Here we explore the nonlinear quasi-stationary response of the entire viscoelastic layer to an oscillating boundary, greater than the gap-loading limit of typical shear rheometers but less than the depth of effective attenuation. We illustrate how to tune the degree and partitioning of nonlinearity versus driving amplitude and frequency, gap height, and fluid viscoelasticity.

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 in vivo rheological techniques are lacking. Gold nanorods (GNRs) are attractive biological probes whose diffusion through tissue is sensitive to the correlation length of comprising biopolymers. Through employment of dynamic light scattering theory on OCT signals from GNRs, we find that weakly-constrained GNR diffusion predictably decreases with increasing wt% (more disease-like) mucus. Previously, we determined this method is robust against mucus transport on human bronchial epithelial (hBE) air-liquid interface cultures (R2=0.976). Here we introduce diffusion-sensitive OCT (DS-OCT), where we collect M-mode image ensembles, from which we derive depth- and temporally-resolved GNR diffusion rates. DS-OCT allows for real-time monitoring of changing GNR diffusion as a result of topically applied mucus-thinning agents, enabling monitoring of the dynamics of mucus hydration never before seen. Cultured human airway epithelial cells (Calu-3) with a layer of endogenous mucus were doped with topically deposited GNRs (80×22nm), and subsequently treated with hypertonic saline (HS) or isotonic saline (IS). DS-OCT provided imaging of the mucus thinning response up to a depth of 600μm with 4.65μm resolution, over a total of 8 minutes in increments of ≥3 seconds. For both IS and HS conditions, DS-OCT captured changes in the pattern of mucus hydration over time. DS-OCT opens a new window into understanding mechanisms of mucus thinning during treatment, enabling real-time efficacy feedback needed to optimize and tailor treatments for individual patients.

Muco-ciliary transport in the human airway is a crucial defense mechanism for removing inhaled pathogens. Optical coherence tomography (OCT) is well-suited to monitor functional dynamics of cilia and mucus on the airway epithelium. Here we demonstrate several OCT-based methods upon an actively transporting in vitro bronchial epithelial model and ex vivo mouse trachea. We show quantitative flow imaging of optically turbid mucus, semi-quantitative analysis of the ciliary beat frequency, and functional imaging of the periciliary layer. These may translate to clinical methods for endoscopic monitoring of muco-ciliary transport in diseases such as cystic fibrosis and chronic obstructive pulmonary disease (COPD).