Mucus Clearance Research Articles

Oral Dantrolene Causing a Decrease in Vital Capacity, Recurrent Lung Collapse, and Respiratory Failure in a Spinal Cord Injury Patient: A Case Report

Introduction: Dantrolene is a muscle relaxant used to treat malignant hyperthermia as well as muscle spasms in spinal cord injury patients. It is known to cause a decrease in skeletal muscle strength in humans, and inhibit diaphragm muscle strength in hamsters. There is at least one other case report of dantrolene leading to respiratory failure in a spinal cord injury patient. Here was present a case of a patient developing poor mucus clearance, lobar collapse and respiratoy failure after being started on dantrolene, and trend vital capacity and negative inspiratory force while titrating off dantrolene.Case: 47 y/o male with history of motor vehicle accident resulting in tetraplegia 19 months prior to admission, presented with pneumonia for the first time since his accident. He also experienced recurrently episodes of bilateral lung collapse and respiratory failure and was found to have significant respiratory muscle weakness. He had been started on dantrolene approximately six weeks prior to admission. After stopping dantrolene his clinical status and his vital capacity and negative inspiratory force dramatically improved. He was then able to clear his secretions, and had no further episodes of lung collapse.Discussion: Our case is only the second in literature of dantrolene causing respiratory failure in a spinal cord patient; and is the first to trend daily vital capacity and NIF while the patient is being tapered off the dantrolene. Our case documented a dramatic improvement NIV and VC after stopping dantrolene. Our case demonstrates the potential and avoidable harm of using dantrolene in group of patients for which is commonly prescribed.

Paranasal sinus size is decreased in CFTR heterozygotes with chronic rhinosinusitis.

Cystic fibrosis (CF) heterozygotes with a single mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene are at significantly higher risk to develop chronic rhinosinusitis (CRS). However the reasons why remain unknown. We tested the hypothesis that CFTR heterozygotes would have smaller sinus volumes than healthy controls. To exclude sinus disease as a confounding factor we also assessed paranasal sinus volume in those with CRS, but without known CFTR mutations. A total of 131 adults of white Northern European and Latino origin were recruited: 81 diagnosed with CRS and 50 healthy controls. Subjects were genotyped for 9 common CFTR mutations covering >80% of mutation prevalence. Those with CRS were separated by CFTR mutational status and matched demographically to healthy controls. Three-dimensional sinus volume, mucosal opacification, and skull volume were quantified to obtain the percentage of pneumatization and extent of mucosal disease in each sinus. Twenty-item Sino-Nasal Outcome Test (SNOT-20) and endoscopy scores were also analyzed. In individuals diagnosed with CRS we identified 7 CFTR heterozygotes (8.64%); no CFTR mutations were identified in our healthy controls. There were no significant differences between the 3 matched groups other than sinus pneumatization. The frontal and maxillary sinuses were significantly smaller in CFTR heterozygotes with CRS compared to CFTR wild-type subjects with or without disease. CFTR heterozygotes with CRS have significantly smaller frontal and maxillary sinus size compared to those without mutations, irrespective of disease state. This sinus hypoplasia may contribute to impaired mucus clearance and chronic sinus disease development.

Impaired mucus clearance exacerbates allergen-induced type 2 airway inflammation in juvenile mice

Type 2 airway inflammation plays a central role in the pathogenesis of allergen-induced asthma, but the underlying mechanisms remain poorly understood. Recently, we demonstrated that reduced mucociliary clearance, a characteristic feature of asthma, produces spontaneous type 2 airway inflammation in juvenile β-epithelial Na+ channel (Scnn1b)-transgenic (Tg) mice. We sought to determine the role of impaired mucus clearance in the pathogenesis of allergen-induced type 2 airway inflammation and identify cellular sources of the signature cytokine IL-13. We challenged juvenile Scnn1b-Tg and wild-type mice with Aspergillus fumigatus and house dust mite allergen and compared the effects on airway eosinophilia, type 2 cytokine levels, goblet cell metaplasia, and airway hyperresponsiveness. Furthermore, we determined cellular sources of IL-13 and effects of genetic deletion of the key type 2 signal-transducing molecule signal transducer and activator of transcription 6 (STAT6) and evaluated the effects of therapeutic improvement of mucus clearance. Reduced mucociliary allergen clearance exacerbated Stat6-dependent secretion of type 2 cytokines, airway eosinophilia, and airway hyperresponsiveness in juvenile Scnn1b-Tg mice. IL-13 levels were increased in airway epithelial cells, macrophages, type 2 innate lymphoid cells, and TH2 cells along with increased Il33 expression in the airway epithelium of Scnn1b-Tg mice. Treatment with the epithelial Na+ channel blocker amiloride, improving airway surface hydration and mucus clearance, reduced allergen-induced inflammation in Scnn1b-Tg mice. Our data support that impaired clearance of inhaled allergens triggering IL-13 production by multiple cell types in the airways plays an important role in the pathogenesis of type 2 airway inflammation and suggests therapeutic improvement of mucociliary clearance as a novel treatment strategy for children with allergen-induced asthma.

Goblet Cell Hyperplasia Requires High Bicarbonate Transport To Support Mucin Release.

Goblet cell hyperplasia, a feature of asthma and other respiratory diseases, is driven by the Th-2 cytokines IL-4 and IL-13. In human bronchial epithelial cells, we find that IL-4 induces the expression of many genes coding for ion channels and transporters, including TMEM16A, SLC26A4, SLC12A2, and ATP12A. At the functional level, we find that IL-4 enhances calcium- and cAMP-activated chloride/bicarbonate secretion, resulting in high bicarbonate concentration and alkaline pH in the fluid covering the apical surface of epithelia. Importantly, mucin release, elicited by purinergic stimulation, requires the presence of bicarbonate in the basolateral solution and is defective in cells derived from cystic fibrosis patients. In conclusion, our results suggest that Th-2 cytokines induce a profound change in expression and function in multiple ion channels and transporters that results in enhanced bicarbonate transport ability. This change is required as an important mechanism to favor release and clearance of mucus.

Direct numerical simulation of particle laden flow in a human airway bifurcation model

During the delivery of inhaled medicines, and depending on the size distribution of the particles in the formulation, airway bifurcations are areas of preferential deposition. Previous studies of laminar flow through airway bifurcations point to an interplay of inertial and centrifugal forces that leads to rich flow phenomena and controls particle deposition patterns. However, recent computational studies have shown that the airflow in the upper human airways is turbulent during much of the respiratory cycle. The question of how the presence of turbulence modifies these effects remains open. In this study, we perform for the first time Direct Numerical Simulations (DNS) of fully developed turbulent flow through a single human airway bifurcation model, emulating steady prolonged inspiration and expiration. We use the rich information obtained from the DNS in order to identify key structures in the flow field and scrutinize their role in determining deposition patterns in the bifurcation. We find that the vortical structures present in the bifurcation during expiration differ from those identified during inspiration. While Dean vortices are present in both cases, a set of three dimensional “carinal vortices” are identified only during expiration. A set of laminar simulations in the same geometries, but at lower Reynolds numbers, allow us to identify key differences in aerosol deposition patterns between laminar and turbulent respiration. We also report deposition fractions for representative Stokes numbers for both laminar and turbulent conditions. Given the suspected role of external mechanical stress on the airway epithelium in determining mucus clearance and chronic disease development, here we report wall shear stress distributions for both the turbulent and laminar cases. Finally, we also perform Large Eddy Simulations (LES) and Reynolds-Averaged Navier-Stokes (RANS) simulations for the same configuration in order to asses their performance as compared to DNS. We find that LES and RANS perform well and that they are able to capture the key characteristics of the flow field. The agreement between DNS and RANS holds true only for the mean flow field, which is primarily influenced by curvature effects.

Multiciliated Cells in Animals.

Many animal cells assemble single cilia involved in motile and/or sensory functions. In contrast, multiciliated cells (MCCs) assemble up to 300 motile cilia that beat in a coordinate fashion to generate a directional fluid flow. In the human airways, the brain, and the oviduct, MCCs allow mucus clearance, cerebrospinal fluid circulation, and egg transportation, respectively. Impairment of MCC function leads to chronic respiratory infections and increased risks of hydrocephalus and female infertility. MCC differentiation during development or repair involves the activation of a regulatory cascade triggered by the inhibition of Notch activity in MCC progenitors. The downstream events include the simultaneous assembly of a large number of basal bodies (BBs)-from which cilia are nucleated-in the cytoplasm of the differentiating MCCs, their migration and docking at the plasma membrane associated to an important remodeling of the actin cytoskeleton, and the assembly and polarization of motile cilia. The direction of ciliary beating is coordinated both within cells and at the tissue level by a combination of planar polarity cues affecting BB position and hydrodynamic forces that are both generated and sensed by the cilia. Herein, we review the mechanisms controlling the specification and differentiation of MCCs and BB assembly and organization at the apical surface, as well as ciliary assembly and coordination in MCCs.

Intérêt de la PPC en aide au désencombrement chez le patient présentant une trachéo-bronchomalacie

The patients suffering of trachea bronchial dyskinesia in a context of trachea bronchomalacia present an important decrease of the expiratory flow during the Forced Expiration Techniques, and during the cough, returning difficult mucus clearance. The use of CPAP for these patients as a pneumatic prothesis can bring a considerable help during the chest physiotherapy techniques, avoiding the tracheobronchial collapse. The objectivation of the efficiency of this technique through a clinical case, its ease of application and control, allows us to propose the attempt of this additive help when the indication and the contraindications are clearly defined.

Endotracheal Tubes Cleaned With a Novel Mechanism for Secretion Removal: A Randomized Controlled Clinical Study.

Intubation compromises mucus clearance, allowing secretions to accumulate inside the endotracheal tube (ETT). The purpose of this trial was to evaluate a novel device for ETT cleaning. We hypothesized that its routine use would reduce tube occlusion due to mucus accumulation, while decreasing airway bacterial colonization. Subjects were randomized to either the use of the device every 8 h, or the institutional standard of care (blind tracheal suction) only. ETTs were collected at extubation and analyzed with high-resolution computed tomography (HRCT) for quantification of mucus volume. Microbiological testing was performed on biofilm samples. Vital signs and ventilatory settings were collected at the bedside. In-hospital follow-up was conducted, and a final evaluation survey was completed by respiratory therapists. Seventy-four subjects expected to remain intubated for longer than 48 h were enrolled (77 ETTs, 37 treatment vs 40 controls). Treated tubes showed reduced mucus accumulation (0.56 ± 0.12 vs 0.71 ± 0.28 mL; P = .004) and reduced occlusion (6.3 ± 1.7 vs 8.9 ± 7.6%; P = .039). The HRCT slice showing the narrowest lumen within each ETT exhibited less occlusion in cleaned tubes (10.6 ± 8.0 vs 17.7 ± 13.4%, 95% CI: 2-12.1; P = .007). Data on microbial colonization showed a trend in the treatment group toward a reduced ETT-based biomass of bacteria known to cause ventilator-associated pneumonia. No adverse events were reported. The staff was satisfied by the overall safety and feasibility of the device. The endOclear is a safe and effective device. It prevents luminal occlusion, thereby better preserving ETT nominal function.

Active cycle of breathing technique for cystic fibrosis.

People with cystic fibrosis experience chronic airway infections as a result of mucus build up within the lungs. Repeated infections often cause lung damage and disease. Airway clearance therapies aim to improve mucus clearance, increase sputum production, and improve airway function. The active cycle of breathing technique (also known as ACBT) is an airway clearance method that uses a cycle of techniques to loosen airway secretions including breathing control, thoracic expansion exercises, and the forced expiration technique. This is an update of a previously published review. To compare the clinical effectiveness of the active cycle of breathing technique with other airway clearance therapies in cystic fibrosis. We searched the Cochrane Cystic Fibrosis Trials Register, compiled from electronic database searches and handsearching of journals and conference abstract books. We also searched the reference lists of relevant articles and reviews.Date of last search: 25 April 2016. Randomised or quasi-randomised controlled clinical studies, including cross-over studies, comparing the active cycle of breathing technique with other airway clearance therapies in cystic fibrosis. Two review authors independently screened each article, abstracted data and assessed the risk of bias of each study. Our search identified 62 studies, of which 19 (440 participants) met the inclusion criteria. Five randomised controlled studies (192 participants) were included in the meta-analysis; three were of cross-over design. The 14 remaining studies were cross-over studies with inadequate reports for complete assessment. The study size ranged from seven to 65 participants. The age of the participants ranged from six to 63 years (mean age 22.33 years). In 13 studies, follow up lasted a single day. However, there were two long-term randomised controlled studies with follow up of one to three years. Most of the studies did not report on key quality items, and therefore, have an unclear risk of bias in terms of random sequence generation, allocation concealment, and outcome assessor blinding. Due to the nature of the intervention, none of the studies blinded participants or the personnel applying the interventions. However, most of the studies reported on all planned outcomes, had adequate follow up, assessed compliance, and used an intention-to-treat analysis.Included studies compared the active cycle of breathing technique with autogenic drainage, airway oscillating devices, high frequency chest compression devices, conventional chest physiotherapy, and positive expiratory pressure. Preference of technique varied: more participants preferred autogenic drainage over the active cycle of breathing technique; more preferred the active cycle of breathing technique over airway oscillating devices; and more were comfortable with the active cycle of breathing technique versus high frequency chest compression. No significant difference was seen in quality of life, sputum weight, exercise tolerance, lung function, or oxygen saturation between the active cycle of breathing technique and autogenic drainage or between the active cycle of breathing technique and airway oscillating devices. There was no significant difference in lung function and the number of pulmonary exacerbations between the active cycle of breathing technique alone or in conjunction with conventional chest physiotherapy. All other outcomes were either not measured or had insufficient data for analysis. There is insufficient evidence to support or reject the use of the active cycle of breathing technique over any other airway clearance therapy. Five studies, with data from eight different comparators, found that the active cycle of breathing technique was comparable with other therapies in outcomes such as participant preference, quality of life, exercise tolerance, lung function, sputum weight, oxygen saturation, and number of pulmonary exacerbations. Longer-term studies are needed to more adequately assess the effects of the active cycle of breathing technique on outcomes important for people with cystic fibrosis such as quality of life and preference.

In vivo imaging of airway cilia and mucus clearance with micro-optical coherence tomography.

We have designed and fabricated a 4 mm diameter rigid endoscopic probe to obtain high resolution micro-optical coherence tomography (µOCT) images from the tracheal epithelium of living swine. Our common-path fiber-optic probe used gradient-index focusing optics, a selectively coated prism reflector to implement a circular-obscuration apodization for depth-of-focus enhancement, and a common-path reference arm and an ultra-broadbrand supercontinuum laser to achieve high axial resolution. Benchtop characterization demonstrated lateral and axial resolutions of 3.4 μm and 1.7 μm, respectively (in tissue). Mechanical standoff rails flanking the imaging window allowed the epithelial surface to be maintained in focus without disrupting mucus flow. During in vivo imaging, relative motion was mitigated by inflating an airway balloon to hold the standoff rails on the epithelium. Software implemented image stabilization was also implemented during post-processing. The resulting image sequences yielded co-registered quantitative outputs of airway surface liquid and periciliary liquid layer thicknesses, ciliary beat frequency, and mucociliary transport rate, metrics that directly indicate airway epithelial function that have dominated in vitro research in diseases such as cystic fibrosis, but have not been available in vivo.

Repurposing tromethamine as inhaled therapy to treat CF airway disease.

In cystic fibrosis (CF), loss of CF transmembrane conductance regulator (CFTR) anion channel activity causes airway surface liquid (ASL) pH to become acidic, which impairs airway host defenses. One potential therapeutic approach is to correct the acidic pH in CF airways by aerosolizing HCO3- and/or nonbicarbonate pH buffers. Here, we show that raising ASL pH with inhaled HCO3- increased pH. However, the effect was transient, and pH returned to baseline values within 30 minutes. Tromethamine (Tham) is a buffer with a long serum half-life used as an i.v. formulation to treat metabolic acidosis. We found that Tham aerosols increased ASL pH in vivo for at least 2 hours and enhanced bacterial killing. Inhaled hypertonic saline (7% NaCl) is delivered to people with CF in an attempt to promote mucus clearance. Because an increased ionic strength inhibits ASL antimicrobial factors, we added Tham to hypertonic saline and applied it to CF sputum. We found that Tham alone and in combination with hypertonic saline increased pH and enhanced bacterial killing. These findings suggest that aerosolizing the HCO3--independent buffer Tham, either alone or in combination with hypertonic saline, might be of therapeutic benefit in CF airway disease.

WS04.1 Impaired mucus clearance promotes Aspergillus fumigatus-induced type 2 airway inflammation in cystic fibrosis-like lung disease in mice

Objectives: The polymicrobial nature in pulmonary infections in patients with CF is one the causes of the lack of efficacy of existing antibiotic treatment. The aim of the present study was to evaluate clinical efficacy of nebulized solution of novel antimicrobial drug candidate Mul-1867 (Poly-N,N′-hexamethyleneguanidine, modified by hydrazine) in murine pneumonia model caused by mixed infection. Methods: 8-weeks old C57BL/6 mice were intranasally infected with P. aeruginosa + S. aureus (each 1×105 cfu/mouse) or P. aeruginosa + C. albicans (each 1×105 cfu/mouse) mix. Treatment was started 12h after infection with Mul-1867 or Amikacin (taken as representative of aminoglycosides that are used for the treatment of lung infections in CF patients), both administered by intranasal inhalation at 32×MIC. Results: Mul-1867 exhibited a high level of antimicrobial activity with the MIC 0.25mg/L against S. aureus; 0.5mg/L against C. albicans and 4.0mg/L against P. aeruginosa. Amikacin was less active; the MICs 128mg/L for both S. aureus and P. aeruginosa and was not active against C. albicans. Induced pneumonia displayed a total death of control animals without of treatment within 72h post-infection in both groups with mixed infections. For the group P. aeruginosa + S. aureus treated with Mul-1867 to the end of observation period (72h) 20% of mice died. Only 10% animals died in P. aeruginosa + C. albicans group treated with Mul-1867. Animals treated with Amikacin displayed 50% mortality in P. aeruginosa + S. aureus group and 70% mortality in P. aeruginosa + C. albicans. Conclusion: Our study demonstrated that Mul-1867 possesses great activity against mixed infection, including bacterial–fungal mix.

The innate immune properties of airway mucosal surfaces are regulated by dynamic interactions between mucins and interacting proteins: the mucin interactome.

SummaryChronic lung diseases such as cystic fibrosis, chronic bronchitis and asthma, are characterized by hypersecretion and poor clearance of mucus, which are associated with poor prognosis and mortality. Little is known about the relationship between the biophysical properties of mucus and its molecular composition. The mucins MUC5B and MUC5AC are traditionally believed to generate the characteristic biophysical properties of airway mucus. However, the contribution of hundreds of globular proteins to the biophysical properties of mucus is not clear. Approximately one-third of the total mucus proteome comprises distinct, multi-protein complexes centered around airway mucins. These complexes constitute a discrete entity we call the “mucin interactome”. The data suggest that while the majority of these proteins interact with mucins via electrostatic and weak interactions, some interact through very strong hydrophobic and/or covalent interactions. Using reagents that interfere with protein-protein interactions, the complexes can be disassembled, and mucus rheology can be dramatically altered. Using MUC5B-glutathione S-transferase (GST) and MUC5B-galectin-3 as a representative of these interactions, we provide evidence that individual mucin protein interactions can alter the biophysical properties of mucus and modulate the biological function of the protein. We propose that the key mechano- and bio-active functions of mucus depend on the dynamic interactions between mucins and globular proteins. These observations challenge the paradigm that mucins are the only molecules that confer biophysical properties of mucus. These observations may ultimately lead to a greater understanding of the system and guide the development of strategies for more effective interventions using better therapeutic agents.

Neonatal Pulmonary Macrophage Depletion Coupled to Defective Mucus Clearance Increases Susceptibility to Pneumonia and Alters Pulmonary Immune Responses.

Resident immune cells (e.g., macrophages [MΦs]) and airway mucus clearance both contribute to a healthy lung environment. To investigate interactions between pulmonary MΦ function and defective mucus clearance, a genetic model of lysozyme M (LysM) promoter-mediated MΦ depletion was generated, characterized, and crossed with the sodium channel β subunit transgenic (Scnn1b-Tg) mouse model of defective mucus clearance. Diphtheria toxin A-mediated depletion of LysM(+) pulmonary MΦs in wild-type mice with normal mucus clearance resulted in lethal pneumonia in 24% of neonates. The pneumonias were dominated by Pasteurella pneumotropica and accompanied by emaciation, neutrophilic inflammation, and elevated Th1 cytokines. The incidence of emaciation and pneumonia reached 51% when LysM(+) MΦ depletion was superimposed on the airway mucus clearance defect of Scnn1b-Tg mice. In LysM(+) MΦ-depleted Scnn1b-Tg mice, pneumonias were associated with a broader spectrum of bacterial species and a significant reduction in airway mucus plugging. Bacterial burden (CFUs) was comparable between Scnn1b-Tg and nonpneumonic LysM(+) MΦ-depleted Scnn1b-Tg mice. However, the nonpneumonic LysM(+) MΦ-depleted Scnn1b-Tg mice exhibited increased airway inflammation, the presence of neutrophilic infiltration, and increased levels of inflammatory cytokines in bronchoalveolar lavage fluid compared with Scnn1b-Tg mice. Collectively, these data identify key MΦ-mucus clearance interactions with respect to both infectious and inflammatory components of muco-obstructive lung disease.

Inhibition of airway surface fluid absorption by cholinergic stimulation.

In upper airways airway surface liquid (ASL) depth and clearance rates are both increased by fluid secretion. Secretion is opposed by fluid absorption, mainly via the epithelial sodium channel, ENaC. In static systems, increased fluid depth activates ENaC and decreased depth inhibits it, suggesting that secretion indirectly activates ENaC to reduce ASL depth. We propose an alternate mechanism in which cholinergic input, which causes copious airway gland secretion, also inhibits ENaC-mediated absorption. The conjoint action accelerates clearance, and the increased transport of mucus out of the airways restores ASL depth while cleansing the airways. We were intrigued by early reports of cholinergic inhibition of absorption by airways in some species. To reinvestigate this phenomenon, we studied inward short-circuit currents (Isc) in tracheal mucosa from human, sheep, pig, ferret, and rabbit and in two types of cultured cells. Basal Isc was inhibited 20–70% by the ENaC inhibitor, benzamil. Long-lasting inhibition of ENaC-dependent Isc was also produced by basolateral carbachol in all preparations except rabbit and the H441 cell line. Atropine inhibition produced a slow recovery or prevented inhibition if added before carbachol. The mechanism for inhibition was not determined and is most likely multi-factorial. However, its physiological significance is expected to be increased mucus clearance rates in cholinergically stimulated airways.

Pharmacokinetics of guaifenesin following administration of multiple doses to exercised Thoroughbred horses.

Guaifenesin is an expectorant commonly used in performance horses to aid in the clearance of mucus from the airways. Guaifenesin is also a centrally acting skeletal muscle relaxant and as such is a prohibited drug with withdrawal necessary prior to competition. To the authors' knowledge, there are no reports in the literature describing single or multiple oral administrations of guaifenesin in the horse to determine a regulatory threshold and related withdrawal time. Therefore, the objective of the current study was to describe the pharmacokinetics of guaifenesin following oral administration in order to provide data upon which appropriate regulatory recommendations can be established. Nine exercised Thoroughbred horses were administered 2g of guaifenesin orally BID for a total of five doses. Blood samples were collected immediately prior to drug administration and at various times postadministration. Serum guaifenesin concentrations were determined and pharmacokinetic parameters calculated. Guaifenesin was rapidly absorbed (Tmax of 15min) following oral administration. The Cmax was 681.3±323.8ng/mL and 1080±732.8 following the first and last dose, respectively. The serum elimination half-life was 2.62±1.24h. Average serum guaifenesin concentrations remained above the LOQ of the assay (0.5ng/mL) by 48h postadministration of the final dose in 3 of 9 horses.

Rheological Properties of Cystic Fibrosis Bronchial Secretion and in Vitro Drug Permeation Study: The Effect of Sodium Bicarbonate.

Cystic fibrosis (CF) is characterized by a thick, sticky mucus responsible for both airway obstruction and resistance to drug diffusion, reducing the effectiveness of drug delivery to the lung. Studies of drug-mucus interaction may be a crucial step in therapeutic management of CF. In the present research, the effect of a saline solution of sodium bicarbonate (100 mM) on sputum viscosity and the permeation properties of ketoprofen lysinate (Klys) from a previously developed dry powder inhaler were evaluated. Rheological measurements were performed using an ARES rotational rheometer (Rheometrics, Inc.) with a parallel plate geometry. The gel fraction, separated from the liquid phase of various sputum samples from CF patients was loaded onto the plate. The elastic (G') and the viscous (G") moduli, tan δ (ratio of G" to G') and η* (complex viscosity) were evaluated as frequency-dependent parameters. Drug permeation across CF sputum from dry powders was studied by means of Franz-type vertical diffusion cells. The experiments were conducted on untreated sputum and on sputum treated with bicarbonate. Rheological studies showed that the elastic modulus (G') was always greater than the viscous modulus (G") and the viscosity decreased with increasing frequency, as for pseudo-plastic fluids. Bicarbonate caused a downward shift of both the elastic and viscous moduli, with a reduction in complex viscosity. As to drug permeation, the untreated sputum slowed down drug dissolution and permeation compared to buffer permeability (control). Permeation studies across CF sputum treated with bicarbonate showed higher Klys dissolution/permeation than untreated sputum. The interesting results confirm the previously reported bicarbonate. effectiveness in CF; this weak base seems to act by decreasing high viscosity of the CF bronchial secretion and, potentially, resulting in better mucus clearance and in fighting pulmonary infections.

Recombinant human deoxyribonuclease attenuates oxidative stress in a model of eosinophilic pulmonary response in mice.

The inflammatory cells infiltrating the airways produce several mediators, such as reactive oxygen species (ROS). ROS and the oxidant-antioxidant imbalance might play an important role in the modulation of airways inflammation. In order to avoid the undesirable effects of ROS, various endogenous antioxidant strategies have evolved, incorporating both enzymatic and non-enzymatic mechanisms. Recombinant human deoxyribonuclease (rhDNase) in clinical studies demonstrated a reduction in sputum viscosity, cleaving extracellular DNA in the airways, and facilitating mucus clearance, but an antioxidant effect was not studied so far. Therefore, we evaluated whether the administration of rhDNase improves oxidative stress in a murine model of asthma. Mice were sensitized by two subcutaneous injections of ovalbumin (OVA), on days 0 and 7, followed by three lung challenges with OVA on days 14, 15, and 16. On days 15 and 16, after 2h of the challenge with OVA, mice received 1mg/mL of rhDNase in the lungs. Bronchoalveolar lavage fluid and lung tissue were obtained on day 17, for inflammatory and oxidative stress analysis. We showed that rhDNase did not alter the population of inflammatory cells, such as eosinophil cells, in OVA-treated rhDNase group but significantly improved oxidative stress in lung tissue, by decreasing oxygen reactive species and increasing superoxide dismutase/catalase ratio, glutathione peroxidase activity, and thiol content. Our data provide the first evidence that rhDNase decreases some measures of oxidative stress and antioxidant status in a murine model of asthma, with a potential antioxidant effect to be further studied in human asthma.

Controlling the mechanical microenvironment of human airway epithelial cells for tracheal tissue engineering

Event Abstract Back to Event Controlling the mechanical microenvironment of human airway epithelial cells for tracheal tissue engineering James Poon1, Thomas K. Waddell1, 2, 3 and Alison P. Mcguigan1, 4 1 University of Toronto, Institute of Biomaterials & Biomedical Engineering, Canada 2 University of Toronto, Institute of Medical Science, Canada 3 University Health Network, Division of Thoracic Surgery, Canada 4 University of Toronto, Department of Chemical Engineering & Applied Chemistry, Canada Many major organs are lined by sheets of epithelial tissue comprised of various cell types; therefore engineering epithelial constructs has emerged as an important field in tissue engineering. We are developing cell alignment tools to engineer functional epithelium for incorporation into engineered tracheas and other artificial organs. Airway epithelium rests on a basement membrane of aligned collagen fibres and significant local variation in mechanical stiffness exists at the scale of the individual cells [1]. However, most tissue engineering tools have been developed for endothelial, muscle, and nerve cells. These cell types are grown on solid culture substrates and do not polarize in a specialized apical-basal fashion. Currently, transwell membrane cultures are used to generate artificial adult airway epithelium in vitro. This air-liquid-interface (ALI) culture system allows for apical-basal polarization of the cells over a four-week maturation period; differentiation is indicated by the formation of motile cilia at the cell apical surface [2]. However, ALI culture does not create planar polarized epithelium. Planar polarity is required for correct alignment of motile cilia, which beat in coordinated waves in order to produce a transport function important for the clearance of mucus and foreign particles from the airway. The mechanical properties of the extracellular environment have been demonstrated to guide cell behaviour. Variations in substrate topography and stiffness influence focal adhesion formation and cytoskeleton organization in epithelial cells in vitro [3],[4]. We are particularly interested in using sub-cellular scale mechanical cues, in the form of groove topography and anisotropic substrate stiffness, to align motile cilia in primary human tracheal epithelial cells (HTECs). We hypothesize that primary HTECs will align and polarize in the direction of our mechanical signals, and produce more organized cilia beating than HTECs on unpatterned substrates. Preliminary studies indicate that HTECs grown on polydimethylsiloxane (PDMS) substrates with deep groove topography (3 µm depth) display a cell alignment response. We are currently fabricating, using photolithography and chemical etching techniques, groove arrays of various dimensions (1-50 µm spacing and 0.2-3 µm depth) to identify the optimal alignment response (whole cell, F-actin and nuclei) of primary cells on these features. These dimensions will be used to mould grooved gelatin inserts onto which HTECs will undergo ALI culture. In parallel, we are developing a simple spin-coating protocol to produce micron-scale anisotropic stiffness patterns, whereby a thin layer (<20μm) of 5% gelatin is spun onto a grooved 15% gelatin substrate to produce a flat surface with alternating regions of soft and stiff substrate. Mucociliary clearance, as assessed by video tracking of fluorescent bead movement on the surface of ALI cultures, is disorganized in control transwell cultures and expected to be organized or enhanced in patterned gel substrates. Engineering specific physical forces during tissue development and regeneration provides a novel strategy for recapitulating large airway epithelium. Understanding mechanical requirements will improve clinical impact by providing the basis for generating gel coatings with specific properties for lining the lumens of engineered airway replacements to guide epithelium organization and maturation.

COUGH IN CHILDREN: RATIONAL CHOICE OF THERAPY

Cough is one of the most frequent reasons for seeking pediatrician’s aid. The fact that, generally, cough has the function of airway clearance is not always taken into account. Short-term, non-persistent cough may be a manifestation of the normal protective reaction. Coughing is beneficial if the sputum has certain rheological properties and there are no obstacles for its evacuation. However, more often coughing indicates the presence of a disease and may be inefficient because of impaired mucus clearance due to various reasons. Therefore, the physician should be able to manage cough and optimize the process of sputum clearance using rational methods of treatment.