Laser scanning microscopy combined with image restoration to analyse a 3D model of the human epithelial airway barrier

A laser scanning microscope collects information from a thin, focal plane and ignores out of focus information. During the past few years it has become the standard imaging method to characterise cellular morphology and structures in static as well as in living samples. Laser scanning microscopy combined with digital image restoration is an excellent tool for analysing the cellular cytoarchitecture, expression of specific proteins and interactions of various cell types, thus defining valid criteria for the optimisation of cell culture models. We have used this tool to establish and evaluate a three dimensional model of the human epithelial airway wall.

Macrophages and dendritic cells express tight junction proteins and exchange particles in an in vitro model of the human airway wall

The human airway epithelium serves as structural and functional barrier against inhaled particulate antigen. Previously, we demonstrated in an in vitro epithelial barrier model that monocyte derived dendritic cells (MDDC) and monocyte derived macrophages (MDM) take up particulate antigen by building a trans-epithelial interacting network. Although the epithelial tight junction (TJ) belt was penetrated by processes of MDDC and MDM, the integrity of the epithelium was not affected. These results brought up two main questions: (1) Do MDM and MDDC exchange particles? (2) Are those cells expressing TJ proteins, which are believed to interact with the TJ belt of the epithelium to preserve the epithelial integrity? The expression of TJ and adherens junction (AJ) mRNA and proteins in MDM and MDDC monocultures was determined by RT-PCR, and immunofluorescence, respectively. Particle uptake and exchange was quantified by flow cytometry and laser scanning microscopy in co-cultures of MDM and MDDC exposed to polystyrene particles (1 μm in diameter). MDM and MDDC constantly expressed TJ and AJ mRNA and proteins. Flow cytometry analysis of MDM and MDDC co-cultures showed increased particle uptake in MDDC while MDM lost particles over time. Quantitative analysis revealed significantly higher particle uptake by MDDC in co-cultures of epithelial cells with MDM and MDDC present, compared to co-cultures containing only epithelial cells and MDDC. We conclude from these findings that MDM and MDDC express TJ and AJ proteins which could help to preserve the epithelial integrity during particle uptake and exchange across the lung epithelium.

Biomechanical effects of environmental and engineered particles on human airway smooth muscle cells

The past decade has seen significant increases in combustion-generated ambient particles, which contain a nanosized fraction (less than 100 nm), and even greater increases have occurred in engineered nanoparticles (NPs) propelled by the booming nanotechnology industry. Although inhalation of these particulates has become a public health concern, human health effects and mechanisms of action for NPs are not well understood. Focusing on the human airway smooth muscle cell, here we show that the cellular mechanical function is altered by particulate exposure in a manner that is dependent upon particle material, size and dose. We used Alamar Blue assay to measure cell viability and optical magnetic twisting cytometry to measure cell stiffness and agonist-induced contractility. The eight particle species fell into four categories, based on their respective effect on cell viability and on mechanical function. Cell viability was impaired and cell contractility was decreased by (i) zinc oxide (40-100 nm and less than 44 microm) and copper(II) oxide (less than 50 nm); cell contractility was decreased by (ii) fluorescent polystyrene spheres (40 nm), increased by (iii) welding fumes and unchanged by (iv) diesel exhaust particles, titanium dioxide (25 nm) and copper(II) oxide (less than 5 microm), although in none of these cases was cell viability impaired. Treatment with hydrogen peroxide up to 500 microM did not alter viability or cell mechanics, suggesting that the particle effects are unlikely to be mediated by particle-generated reactive oxygen species. Our results highlight the susceptibility of cellular mechanical function to particulate exposures and suggest that direct exposure of the airway smooth muscle cells to particulates may initiate or aggravate respiratory diseases.

Ultrasound-guided percutaneous tracheal puncture: a computer-tomographic controlled study in cadavers

Ultrasound-guided techniques are increasingly used in anaesthetic practice to identify tissues beneath the skin and to increase the accuracy of placement of needles close to targeted structures. To examine ultrasound's usefulness for dilatational tracheostomy, we performed ultrasound-guided tracheal punctures in human cadavers followed by computer-tomographic (CT) control.

Airway research: the current status and future directions

We highlight the areas we think important for future development of the subspeciality. The ultimate goal is to improve patient care and safety and to do this, we need to identify how and where episodes of harm arise. Simply continuing with current practice does not represent the best path towards our ultimate goal; objective evidence is needed to inform changes in practice.

Performance of supraglottic airway devices and 12 month skill retention: a randomized controlled study with manikins

Airway management for successful ventilation by laypersons and inexperienced healthcare providers is difficult to achieve. Bag-valve mask (BVM) ventilation requires extensive training and is performed poorly. Supraglottic airway devices (SADs) have been successfully introduced to clinical resuscitation practice as an alternative. We evaluated recently introduced (i-gel™ and LMA-Supreme™) and established SADs (LMA-Unique™, LMA-ProSeal™) and BVM used by laypeople in training sessions on manikins.

The airway epithelium: soldier in the fight against respiratory viruses

The airway epithelium acts as a frontline defense against respiratory viruses, not only as a physical barrier and through the mucociliary apparatus but also through its immunological functions. It initiates multiple innate and adaptive immune mechanisms which are crucial for efficient antiviral responses. The interaction between respiratory viruses and airway epithelial cells results in production of antiviral substances, including type I and III interferons, lactoferrin, β-defensins, and nitric oxide, and also in production of cytokines and chemokines, which recruit inflammatory cells and influence adaptive immunity. These defense mechanisms usually result in rapid virus clearance. However, respiratory viruses elaborate strategies to evade antiviral mechanisms and immune responses. They may disrupt epithelial integrity through cytotoxic effects, increasing paracellular permeability and damaging epithelial repair mechanisms. In addition, they can interfere with immune responses by blocking interferon pathways and by subverting protective inflammatory responses toward detrimental ones. Finally, by inducing overt mucus secretion and mucostasis and by paving the way for bacterial infections, they favor lung damage and further impair host antiviral mechanisms.

Impaired type I and type III interferon induction and rhinovirus control in human cystic fibrosis airway epithelial cells

Rhinoviruses are important triggers of pulmonary exacerbations and possible contributors to long-term respiratory morbidity in cystic fibrosis (CF), but mechanisms leading to rhinovirus-induced CF exacerbations are poorly understood. It is hypothesised that there is a deficient innate immune response of the airway epithelium towards rhinovirus infection in CF.

Distinct patterns of inflammation in the airway lumen and bronchial mucosa of children with cystic fibrosis

Studies in cystic fibrosis (CF) generally focus on inflammation present in the airway lumen. Little is known about inflammation occurring in the airway wall, the site ultimately destroyed in end-stage disease.