Respiratory Mucus Research Articles

Immobilization of Pseudorabies Virus in Porcine Tracheal Respiratory Mucus Revealed by Single Particle Tracking

Pseudorabies virus (PRV) initially replicates in the porcine upper respiratory tract. It easily invades the mucosae and submucosae for subsequent spread throughout the body via blood vessels and nervous system. In this context, PRV developed ingenious processes to overcome different barriers such as epithelial cells and the basement membrane. Another important but often overlooked barrier is the substantial mucus layer which coats the mucosae. However, little is known about how PRV particles interact with porcine respiratory mucus. We therefore measured the barrier properties of porcine tracheal respiratory mucus, and investigated the mobility of nanoparticles including PRV in this mucus. We developed an in vitro model utilizing single particle tracking microscopy. Firstly, the mucus pore size was evaluated with polyethylene glycol coupled (PEGylated) nanoparticles and atomic force microscope. Secondly, the mobility of PRV in porcine tracheal respiratory mucus was examined and compared with that of negative, positive and PEGylated nanoparticles. The pore size of porcine tracheal respiratory mucus ranged from 80 to 1500 nm, with an average diameter of 455±240 nm. PRV (zeta potential: −31.8±1.5 mV) experienced a severe obstruction in porcine tracheal respiratory mucus, diffusing 59-fold more slowly than in water. Similarly, the highly negatively (−49.8±0.6 mV) and positively (36.7±1.1 mV) charged nanoparticles were significantly trapped. In contrast, the nearly neutral, hydrophilic PEGylated nanoparticles (−9.6±0.8 mV) diffused rapidly, with the majority of particles moving 50-fold faster than PRV. The mobility of the particles measured was found to be related but not correlated to their surface charge. Furthermore, PEGylated PRV (-13.8±0.9 mV) was observed to diffuse 13-fold faster than native PRV. These findings clearly show that the mobility of PRV was significantly hindered in porcine tracheal respiratory mucus, and that the obstruction of PRV was due to complex mucoadhesive interactions including charge interactions rather than size exclusion.

A Novel Depsipeptide Produced by Paenibacillus alvei 32 Isolated from a Cystic fibrosis Patient.

The important viscosity of the respiratory tract mucus of Cystic fibrosis (CF) patients impairs the mucociliary transport system and allows the growth of numerous micro-organisms. Among them, Pseudomonas aeruginosa and Staphylococcus aureus are known to be responsible for pulmonary infections. We imagined that CF microflora could also harbour micro-organisms naturally equipped to compete with these pathogens. A method was developed to recover these antibiotic-producing strains within 20 CF sputum. Using this approach, we have isolated an unusual Gram-positive bacterium identified as Paenibacillus alvei by Api galleries and 16S rRNA gene sequence analysis. This strain has inhibited the growth of P. aeruginosa, S. aureus and Klebsiella pneumoniae, in co-cultures. A liquid mineral medium named MODT50 was designed and optimised for the production and the recovery of the antimicrobial compounds. The supernatant has inhibited the growth of all Gram-positive strains tested, even Methicillin-resistant S. aureus. One antimicrobial compound with a peptide structure (mainly active against S. aureus, Micrococcus luteus, and Pseudomonas stutzeri) has been purified and characterised by liquid chromatography-mass spectrometry. The new active molecule (m/z 786.6) named depsipeptide L possesses a 15-guanidino-3-hydroxypentadecanoic acid side chain (m/z 298) linked on a cyclic part of four amino acids residues (Ser, two Leu/Ile, Arg). This work reports for the first time the production of such a molecule by a P. alvei strain in a mineral medium. The CF lung microflora might represent a valuable source for the discovery of new antimicrobial-producing strains.

Tracheobronchial mycosis in a retrospective case-series study of five status asthmaticus patients

The etiology of status asthmaticus (SA), a complication of severe asthma, is unknown. Fungal exposure, as measured by fungal atopy, is a major risk factor for developing asthma, but the relationship of fungi in SA per se has not previously been reported. In this five patient retrospective case series study, lower respiratory tract cultures were performed on bronchoalveolar lavage or tracheal aspirate fluid, comparing standard clinical laboratory cultures with a specialized technique in which respiratory mucus was removed prior to culture. We show that mucolytic treatment allows an increased detection of fungal growth, especially yeast, from the lower airways of all SA patients. We also demonstrate that inhalation of the yeast Candida albicans readily induces asthma-like disease in mice. Our observations suggest that SA may represent a fungal infectious process, and support additional prospective studies utilizing anti-fungal therapy to supplement conventional therapy, broad-spectrum antibiotics and high-dose glucocorticoids, which can promote fungal overgrowth.

Transcriptional Response of Mucoid Pseudomonas aeruginosa to Human Respiratory Mucus

ABSTRACTAdaptation of bacterial pathogens to a host can lead to the selection and accumulation of specific mutations in their genomes with profound effects on the overall physiology and virulence of the organisms. The opportunistic pathogen Pseudomonas aeruginosa is capable of colonizing the respiratory tract of individuals with cystic fibrosis (CF), where it undergoes evolution to optimize survival as a persistent chronic human colonizer. The transcriptome of a host-adapted, alginate-overproducing isolate from a CF patient was determined following growth of the bacteria in the presence of human respiratory mucus. This stable mucoid strain responded to a number of regulatory inputs from the mucus, resulting in an unexpected repression of alginate production. Mucus in the medium also induced the production of catalases and additional peroxide-detoxifying enzymes and caused reorganization of pathways of energy generation. A specific antibacterial type VI secretion system was also induced in mucus-grown cells. Finally, a group of small regulatory RNAs was identified and a fraction of these were mucus regulated. This report provides a snapshot of responses in a pathogen adapted to a human host through assimilation of regulatory signals from tissues, optimizing its long-term survival potential.

Optical tweezers reveal relationship between microstructure and nanoparticle penetration of pulmonary mucus

In this study, the mobility of nanoparticles in mucus and similar hydrogels as model systems was assessed to elucidate the link between microscopic diffusion behavior and macroscopic penetration of such gels. Differences in particle adhesion to mucus components were strongly dependent on particle coating. Particles coated with 2 kDa PEG exhibited a decreased adhesion to mucus components, whereas chitosan strongly increased the adhesion. Despite such mucoinert properties of PEG, magnetic nanoparticles of both coatings did not penetrate through native respiratory mucus, resisting high magnetic forces (even for several hours). However, model hydrogels were, indeed, penetrated by both particles in dependency of particle coating, obeying the theory of particle mobility in an external force field. Comparison of penetration data with cryogenic scanning EM images of mucus and the applied model systems suggested particularly high rigidity of the mucin scaffold and a broad pore size distribution in mucus as reasons for the observed particle immobilization. Active probing of the rigidity of mucus and model gels with optical tweezers was used in this context to confirm such properties of mucus on the microscale, thus presenting the missing link between micro- and macroscopical observations. Because of high heterogeneity in the size of the voids and pores in mucus, on small scales, particle mobility will depend on adhesive or inert properties. However, particle translocation over distances larger than a few micrometers is restricted by highly rigid structures within the mucus mesh.

P01.09. Effect of Maeg-Moon-Dong-Tang on production and secretion of respiratory mucus

Methods Confluent NCI-H292 cells were pretreated for 30 min in the presence of MMT and treated with ATP (200μM) or PMA (10ng/ml) or EGF (25ng/ml) or TNF-alpha (0.2nM) for 24 hours, to assess the effect of MMT both on ATPor PMAor EGFor TNF-alpha-induced MUC5AC mucin production using enzyme-linked immunosorbent assay (ELISA) and on gene expression by the same inducers using reverse transcription-polymerase chain reaction (RT-PCR). At the same time, hypersecretion of airway mucus was induced by exposure of rats to SO2 during 3 weeks. Effect of orallyadministered MMT during 2 weeks on increase in airway epithelial mucosubstances from tracheal goblet cells of rats was assesed using histopathological analysis after staining the epithelial tissue with PAS-alcian blue. Possible cytotoxicity of MMT was assessed by investigating the potential damage of kidney and liver function by measuring serum GOT/GPT activities and serum BUN concentration of rats and body weight gain during the experiment.

Novel method of detecting H1N1 using microwaves

H1N1 virus is a subtype of influenza A virus and was the most common cause of human influenza flu in 2009. This paper presents a new method of detecting H1N1. The dielectric properties respiratory mucus is studied at microwave frequencies using rectangular cavity perturbation technique at the S-band with the different samples of respiratory mucus obtained from healthy donors as well as from patients suffering from H1N1. It is observed that an appreciably variation is found in the dielectric properties of patient samples as that of normal healthy samples and this measurements were in good agreement with the clinical analysis. This measurement technique is quick, simple and suggests a new novel method of diagnosing H1N1 using microwaves.

Comparison of ozone-specific (OZAC) and oxygen radical (ORAC) antioxidant capacity assays for use with nasal lavage fluid

Antioxidants in respiratory mucus protect the underlying airway epithelium from damage by ozone (O3), a common outdoor air pollutant. To understand O3–antioxidant interactions and the variation of these interactions among individuals, in vitro assays are needed to measure the total antioxidant capacity of airway lavage fluid, a convenient source of (diluted) mucous samples. Here, we compare the oxygen radical absorbance capacity (ORAC), a general method that uses peroxyl radicals as a reactive substance, to the recently developed ozone specific antioxidant capacity (OZAC), a procedure that directly employs O3. For prepared model mucous antioxidant solutions containing uric acid, ascorbic acid or glutathione, the ORAC and OZAC methods yielded comparable antioxidant capacities. The addition of EDTA or DETAPAC, necessary to prevent auto-oxidation of test solutions during the ORAC assay, unpredictably altered ORAC measurements. EDTA did not have a significant effect on OZAC measurements in either prepared uric acid or ascorbic acid solutions. When assessing antioxidant capacities of nasal lavage samples, the ORAC and OZAC assays were no longer comparable. Because the OZAC of nasal lavage samples was positively related to measured uric acid concentrations whereas the ORAC data were not, the OZAC method appears to provide more realistic mucous antioxidant capacities than the ORAC method.

Analysis of the proteome of human airway epithelial secretions

BackgroundAirway surface liquid, often referred to as mucus, is a thin layer of fluid covering the luminal surface that plays an important defensive role against foreign particles and chemicals entering the lungs. Airway mucus contains various macromolecules, the most abundant being mucin glycoproteins, which contribute to its defensive function. Airway epithelial cells cultured in vitro secrete mucins and nonmucin proteins from their apical surface that mimics mucus production in vivo. The current study was undertaken to identify the polypeptide constituents of human airway epithelial cell secretions to gain a better understanding of the protein composition of respiratory mucus.ResultsFifty-five proteins were identified in the high molecular weight fraction of apical secretions collected from in vitro cultures of well-differentiated primary human airway epithelial cells and isolated under physiological conditions. Among these were MUC1, MUC4, MUC5B, and MUC16 mucins. By proteomic analysis, the nonmucin proteins could be classified as inflammatory, anti-inflammatory, anti-oxidative, and/or anti-microbial.ConclusionsBecause the majority of the nonmucin proteins possess molecular weights less than that selected for analysis, it is theoretically possible that they may associate with the high molecular weight and negatively charged mucins to form a highly ordered structural organization that is likely to be important for maintaining the proper defensive function of airway mucus.

Photoacoustic technique: a new method for measuring the rheologic properties of human bronchial mucus

Patients affected by obstructive pulmonary diseases exhibit typical characteristics of airway mucus hypersecretion, including sputum production, increased luminal mucus, goblet cell hyperplasia and submucosal gland hypertrophy. The mucociliary system is a sophisticated defense mechanism to maintain the homeostasis of the respiratory tract by means of the bronchial mucus transport, the process in which airway mucus together with substances trapped within are moved out of the lungs. In the end, the mucus together with the substances trapped within are swallowed or coughed up. It is an important defense mechanism of the human body [1]. Mucociliary clearance is influenced by viscosity and elasticity, that are the fundamental rheologic properties of the respiratory mucus and important determinants of its transportability in the mucociliary system [2]. It has been observed that the rheologic properties of mucus are often abnormal in patients with airways disease [3]. A retardation in mucociliary transport may lead to mucus retention, a condition which predisposes to bacterial colonization and may eventually lead to pneumonia. Drugs may alter the process of mucociliary transport, such that it is necessary to know the effect of the drugs on the rheologic properties of human bronchial mucus. Understanding the interaction of a specific drug with human bronchial mucus may lead to a better therapeutic use of such medication. The photoacoustic technique (PAT) is a new method that permits to monitor indirectly the rheologic properties of any mucus sample [3,4]. PAT enables to study the interaction between therapeutic aerosols and bronchial mucus, making it possible to determine the typical time of interaction between the aerosol and the mucus (T0) and the solubilization period (Δt). According to our knowledge it is important to have an experimental technique that permits the monitoring of the rheologic properties of mucus during the interaction with the specific drug.

Mucus dynamics subject to air and wall motion

This study presents a numerical investigation of basic interactions between respiratory mucus motion, air circulation and epithelium ciliated cells vibration. One focuses on identification of meaningful rheological parameters, physiological and numerical simulation dimensioning. These preliminary results are crucial before the study of more general configurations of respiratory mucus motion. The numerical study presented in this work aims at providing a first numerical tool able to simulate the effects of mucus mobility and its ability to carry out pathogens or to deliver aerosol therapy to membrane wall cells. Momentum diffusion is identified as the dominant effect, as expected in this micrometer scale configuration, and its associate momentum diffusion operator is shown to be extremely stiff. Furthermore, epithelium vibration is shown to be much more efficient than air circulation for mucus propulsion.

Analysis of the Sputum and Inflammatory Alterations of the Airways in Patients with Common Variable Immunodeficiency and Bronchiectasis

INTRODUCTION:Common variable immunodeficiency is characterized by defective antibody production and recurrent pulmonary infections. Intravenous immunoglobulin is the treatment of choice, but the effects of Intravenous immunoglobulin on pulmonary defense mechanisms are poorly understood.OBJECTIVE:The aim of this study was to verify the impact of intravenous immunoglobulin on the physical properties of the sputum and on inflammatory alterations in the airways of patients with Common variable immunodeficiency associated with bronchiectasis.METHOD:The present study analyzed sputum physical properties, exhaled NO, inflammatory cells in the sputum, and IG titers in 7 patients with Common variable immunodeficiency and bronchiectasis with secretion, immediately before and 15 days after Intravenous immunoglobulin. A group of 6 patients with Common variable immunodeficiency and bronchiectasis but no sputum was also studied for comparison of the basal IgG level and blood count. The 13 patients were young (age=36±17 years) and comprised predominantly of females (n=11).RESULTS:Patients with secretion presented significantly decreased IgG and IgM levels. Intravenous immunoglobulin was associated with a significant decrease in exhaled NO (54.7 vs. 40.1 ppb, p<0.05), sputum inflammatory cell counts (28.7 vs. 14.6 cells/mm3, p<0.05), and a significant increase in respiratory mucus transportability by cough (42.5 vs. 65.0 mm, p < 0.05).CONCLUSION:We concluded that immunoglobulin administration in Common variable immunodeficiency patients results in significant improvement in indexes of inflammation of the airways with improvement in the transportability of the respiratory mucus by cough.

DAS181, a sialidase fusion protein, protects human airway epithelium against influenza virus infection: an in vitro pharmacodynamic analysis

The influenza virus (IFV) infection models commonly used to evaluate antiviral agents (e.g. MDCK cell line and mice) are limited by physiological differences from the human respiratory tract in vivo. Here we report the pharmacodynamics of DAS181, a sialidase fusion protein that inhibits influenza infection, in the model systems of well-defined human airway epithelium (HAE) culture and ex vivo culture of fresh human bronchial tissue, both of which are close mimics of the human respiratory tract in vivo. HAE culture and ex vivo human bronchi were used to evaluate the sialic acid removal and regeneration efficiency and IFV inhibition after various DAS181 treatment levels and regimens. DAS181 effectively desialylates HAE cultures and ex vivo bronchi tissues and therefore potently inhibits replication of different IFV strains. The treatment effect of DAS181 occurs immediately upon application to the epithelial surface and is unaffected by the respiratory mucus. In both HAE and human bronchial tissue, the inhibitory effect of DAS181 treatment lasts for at least 2 days. Approximately 80% epithelial surface desialylation and significant anti-IFV efficacy can be achieved at topical concentrations of DAS181 in the range of 5-10 microg/cm(2) when applied once daily. An additional treatment or a higher loading dose of DAS181 on the first day provides significant additional treatment benefit. Comparing the effect of DAS181 versus its two analogues, DAS180 and DAS185, has confirmed that sialidase function is critical for DAS181, and the cell-binding domain (amphiregulin tag) prolongs DAS181 retention and potentiates its function. These results provide valuable insights into DAS181 treatment dose and potential regimens in the clinical setting.

Does milk increase mucus production?

Excessive milk consumption has a long association with increased respiratory tract mucus production and asthma. Such an association cannot be explained using a conventional allergic paradigm and there is limited medical evidence showing causality. In the human colon, beta-casomorphin-7 (beta-CM-7), an exorphin derived from the breakdown of A1 milk, stimulates mucus production from gut MUC5AC glands. In the presence of inflammation similar mucus overproduction from respiratory tract MUC5AC glands characterises many respiratory tract diseases. beta-CM-7 from the blood stream could stimulate the production and secretion of mucus production from these respiratory glands. Such a hypothesis could be tested in vitro using quantitative RT-PCR to show that the addition of beta-CM-7 into an incubation medium of respiratory goblet cells elicits an increase in MUC5AC mRNA and by identifying beta-CM-7 in the blood of asthmatic patients. This association may not necessarily be simply cause and effect as the person has to be consuming A1 milk, beta-CM-7 must pass into the systemic circulation and the tissues have to be actively inflamed. These prerequisites could explain why only a subgroup of the population, who have increased respiratory tract mucus production, find that many of their symptoms, including asthma, improve on a dairy elimination diet.

Guia para prática clínica: fisioterapia em pacientes com Doença Pulmonar Obstrutiva Crônica (DPOC)

INTRODUCAO: No contexto da colaboracao internacional para desenvolvimento de guias praticos (ou guidelines), a Sociedade Real Holandesa de Fisioterapia (Koninklijk Nederlands Genootschap voor Fysiotherapie, KNGF) se propos a desenvolver um guia para esclarecimento sobre a pratica clinica de Fisioterapia em pacientes com Doenca Pulmonar Obstrutiva Cronica (DPOC), assim como tambem optou por estimular a sua traducao para outras linguas, a fim de torna-lo acessivel para publicos internacionais. OBJETIVOS: O presente guia e a versao em lingua portuguesa do Guia para Pratica Clinica de Fisioterapia em pacientes com DPOC desenvolvido pela KNGF, que teve como objetivo descrever a Fisioterapia baseada em evidencias para pacientes com DPOC que apresentam limitacao da funcao pulmonar, da funcao muscular respiratoria e periferica, da capacidade de exercicio, da depuracao mucociliar e da qualidade de vida, alem de limitacoes em relacao a atividade fisica na vida diaria pela dispneia e/ou intolerância ao exercicio. CONCLUSAO: O guia propoe-se principalmente a prover recomendacoes terapeuticas praticas que auxiliem o fisioterapeuta a oferecer o melhor tratamento possivel para pacientes com DPOC, consideradas as evidencias cientificas disponiveis na atualidade.

Guaifenesin in rhinitis

Mucus in the airways is a complex mixture of water, lipids, glycoproteins, sugars, and electrolytes that serves as a lubricant for the epithelium. The efficient flow of respiratory mucus is a first level of immune defense that requires an appropriate viscosity and elasticity for optimal barrier and ciliary functions. Thickening and drying of airway mucus by respiratory tract infections, allergies, and drugs can impair evacuation. Tenacious, bothersome mucus is an annoying and frequent symptom of rhinitis that is difficult to manage. Common remedies include adequate hydration through fluid intake and nasal washes. The use of mucoactive agents is controversial due to limited data and equivocal efficacy in available studies. Nonetheless, some patients benefit. This review examines the use of guaifenesin (glyceryl guaiacolate) on bothersome nasal mucus associated with rhinitis, including the available published data and clinical experience.

Rapidly adapting receptors in acute heart failure and their impact on dyspnea

Acute heart failure is associated with dyspnea that is usually defined as difficulty in breathing that is accompanied by an element of distress. It is commonly associated with an increase in the rate of ventilation, wheezing, an increase in airway secretions and cough. This review examines the reflexes which generate these responses with particular reference to the role of rapidly adapting receptors (RAR) in the airways. The essential feature of acute heart failure is an increase in pulmonary extravascular fluid volume. Small acute increases in extravascular fluid volume in the airways activate the RAR. With larger increases both the RAR and the C-fiber receptors in the airways and the alveoli are activated. Activation of RAR causes a reflex increase in respiratory rate, tracheal tone and mucus secretion from the airways. It is suggested that the RAR play a significant role in monitoring changes in the extravascular fluid volume of the airways and mediate the respiratory reflexes associated with acute heart failure.

Survival of Influenza Virus on Banknotes

Successful control of a viral disease requires knowledge of the different vectors that could promote its transmission among hosts. We assessed the survival of human influenza viruses on banknotes given that billions of these notes are exchanged daily worldwide. Banknotes were experimentally contaminated with representative influenza virus subtypes at various concentrations, and survival was tested after different time periods. Influenza A viruses tested by cell culture survived up to 3 days when they were inoculated at high concentrations. The same inoculum in the presence of respiratory mucus showed a striking increase in survival time (up to 17 days). Similarly, B/Hong Kong/335/2001 virus was still infectious after 1 day when it was mixed with respiratory mucus. When nasopharyngeal secretions of naturally infected children were used, influenza virus survived for at least 48 h in one-third of the cases. The unexpected stability of influenza virus in this nonbiological environment suggests that unusual environmental contamination should be considered in the setting of pandemic preparedness.

Respiratory training with a specific device in cystic fibrosis: A prospective study

Introduction Chest physiotherapy (CP) is used in cystic fibrosis (CF) even if there is no robust scientific evidence of a beneficial effect. We investigated the effects of a training with a specific device (SpiroTiger®) in a group of CF patients. This device, developed for respiratory training through maximal inspirations and espirations without hypocarbia, may improve respiratory function and mucus clearance. Patients where instructed and trained by a physiotherapist with individualized settings of training parameters. Methods Twenty-four patients were enrolled in an open-label 1 year observational study. Baseline and post intervention measurements were determined by lung function (FVC, FEV1, FEF 25-75), patients’ opinions on physiotherapy (questionnaires), need for antibiotic treatment (clinical follow-up and records) and perception of physical fitness (questionnaires) in the year before and in the year of intervention. Adherence to physiotherapy was monitored by means of a specific device software. Results Increased lung function (FEV1 p < 0.01), perception of physical fitness ( p < 0.001) and a reduction in the need for intravenous antibiotic treatment ( p < 0.001) were reported. Adherence to treatment was good/acceptable in 92% of patients. Conclusions This study shows an association between training through a specific device and improved lung function. Further trials are needed to confirm this report.

Mapping of the 45M1 epitope to the C‐terminal cysteine‐rich part of the human MUC5AC mucin

Mucins are large glycoproteins protecting mucosal surfaces throughout the body. Their expressions are tissue-specific, but in disease states such as cystic fibrosis, inflammation and cancer, this specificity can be disturbed. MUC5AC is normally expressed in the mucous cells of the epithelia lining the stomach and the trachea, where it constitutes a major component of the gastric and respiratory mucus. A number of mAbs have been raised against the gastric M1 antigen, an early marker for colonic carcinogenesis. Several of these mAbs recognize epitopes present on MUC5AC, suggesting that MUC5AC is the antigen. However, some of the mAbs raised against the gastric M1 antigen are widely used as antibodies against MUC5AC, despite the fact that their specificity for MUC5AC has not been clearly shown. In this study, we have tested the reactivity of the latter antibodies against a recombinantly expressed C-terminal cysteine-rich part of human MUC5AC. We demonstrate for the first time that the widely used mAb 45M1, as well as 2-12M1 and 166M1, are true antibodies against MUC5AC, with epitopes located in the C-terminal cysteine-rich part of the mucin.