Kevin Looi

Matrix metalloproteinase activation by free neutrophil elastase contributes to bronchiectasis progression in early cystic fibrosis

Neutrophil elastase is the most significant predictor of bronchiectasis in early-life cystic fibrosis; however, the causal link between neutrophil elastase and airway damage is not well understood. Matrix metalloproteinases (MMPs) play a crucial role in extracellular matrix modelling and are activated by neutrophil elastase. The aim of this study was to assess if MMP activation positively correlates with neutrophil elastase activity, disease severity and bronchiectasis in young children with cystic fibrosis. Total MMP-1, MMP-2, MMP-7, MMP-9, tissue inhibitor of metalloproteinase (TIMP)-2 and TIMP-1 levels were measured in bronchoalveolar lavage fluid collected from young children with cystic fibrosis during annual clinical assessment. Active/pro-enzyme ratio of MMP-9 was determined by gelatin zymography. Annual chest computed tomography imaging was scored for bronchiectasis. A higher MMP-9/TIMP-1 ratio was associated with free neutrophil elastase activity. In contrast, MMP-2/TIMP-2 ratio decreased and MMP-1 and MMP-7 were not detected in the majority of samples. Ratio of active/pro-enzyme MMP-9 was also higher in the presence of free neutrophil elastase activity, but not infection. Across the study cohort, both MMP-9/TIMP-1 and active MMP-9 were associated with progression of bronchiectasis. Both MMP-9/TIMP-1 and active MMP-9 increased with free neutrophil elastase and were associated with bronchiectasis, further demonstrating that free neutrophil elastase activity should be considered an important precursor to cystic fibrosis structural disease. In young children with CF, activation of MMP-9 by free NE may provide one mechanism driving structural lung disease http://ow.ly/KArB5

Impaired airway epithelial cell responses from children with asthma to rhinoviral infection

The airway epithelium forms an effective immune and physical barrier that is essential for protecting the lung from potentially harmful inhaled stimuli including viruses. Human rhinovirus (HRV) infection is a known trigger of asthma exacerbations, although the mechanism by which this occurs is not fully understood.

Alpha-1 Antitrypsin Mitigates the Inhibition of Airway Epithelial Cell Repair by Neutrophil Elastase.

Neutrophil elastase (NE) activity is associated with many destructive lung diseases and is a predictor for structural lung damage in early cystic fibrosis (CF), which suggests normal maintenance of airway epithelium is prevented by uninhibited NE. However, limited data exist on how the NE activity in airways of very young children with CF affects function of the epithelia. The aim of this study was to determine if NE activity could inhibit epithelial homeostasis and repair and whether any functional effect was reversible by antiprotease alpha-1 antitrypsin (α1AT) treatment. Viability, inflammation, apoptosis, and proliferation were assessed in healthy non-CF and CF pediatric primary airway epithelial cells (pAECnon-CF and pAECCF, respectively) during exposure to physiologically relevant NE. The effect of NE activity on pAECCF wound repair was also assessed. We report that viability after 48 hours was significantly decreased by 100 nM NE in pAECnon-CF and pAECCF owing to rapid cellular detachment that was accompanied by inflammatory cytokine release. Furthermore, both phenotypes initiated an apoptotic response to 100 nM NE, whereas ≥ 50 nM NE activity significantly inhibited the proliferative capacity of cultures. Similar concentrations of NE also significantly inhibited wound repair of pAECCF, but this effect was reversed by the addition of α1AT. Collectively, our results demonstrate free NE activity is deleterious for epithelial homeostasis and support the hypothesis that proteases in the airway contribute directly to CF structural lung disease. Our results also highlight the need to investigate antiprotease therapies in early CF disease in more detail.

Bronchial brushings for investigating airway inflammation and remodelling

Asthma is the commonest medical cause for hospital admission for children in Australia, affects more than 300 million people worldwide, and is incurable, severe in large number and refractory to treatment in many. However, there have been no new significant treatments despite intense research and billions of dollars. The advancement in our understanding in this disease has been limited due to its heterogeneity, genetic complexity and has severely been hampered particularly in children by the difficulty in obtaining relevant target organ tissue. This review attempts to provide an overview of the currently used and recently developed/adapted techniques used to obtain lung tissue with specific reference to the airway epithelium.

Reduced transforming growth factor β1 (TGF-β1) in the repair of airway epithelial cells of children with asthma.

Evidence into the role of TGF‐β1 in airway epithelial repair in asthma is still controversial. This study tested the hypothesis that the reduced TGF‐β1 levels previously observed in paediatric asthmatic airway epithelial cells directly contribute to the dysregulated repair seen in these cells.

Effect of human rhinovirus infection on airway epithelium tight junction protein disassembly and transepithelial permeability

ABSTRACT Rationale: No studies have assessed the effects of human rhinovirus (HRV) infection on epithelial tight junctions (TJs) and resultant barrier function. Aim of the Study: To correlate viral infection with TJ disassembly, epithelial barrier integrity, and function. Materials and Methods: Human airway epithelial cells were infected with HRV minor serotype 1B (HRV-1B) at various 50% tissue culture infectivity doses (TCID50) over 72 hours. HRV replication was assessed by quantitative-polymerase chain reaction (qPCR) while cell viability and apoptosis were assessed by proliferation and apoptotic assays, respectively. Protein expression of claudin-1, occludin, and zonula occludens protein-1 (ZO-1) was assessed using In-Cell™ Western assays. Transepithelial permeability assays were performed to assess effects on barrier functionality. RT2 Profiler focused qPCR arrays and pathway analysis evaluating associations between human TJ and antiviral response were performed to identify potential interactions and pathways between genes of interests. Results: HRV-1B infection affected viability that was both time and TCID50 dependent. Significant increases in apoptosis and viral replication post-infection correlated with viral titer. Viral infection significantly decreased claudin-1 protein expression at the lower TCID50, while a significant decrease in all three TJ protein expressions occurred at higher TCID50. Decrease in protein expression was concomitant with significant increases in epithelial permeability of fluorescein isothiocynate labeled-dextran 4 and 20 kDa. Analysis of focused qPCR arrays demonstrated a significant decrease in ZO-1 gene expression. Furthermore, network analysis between human TJ and antiviral response genes revealed possible interactions and regulation of TJ genes via interleukin (IL)-15 in response to HRV-1B infection. Conclusion: HRV-1B infection directly alters human airway epithelial TJ expression leading to increased epithelial permeability potentially via an antiviral response of IL-15.

Determinants of culture success in an airway epithelium sampling program of young children with cystic fibrosis

ABSTRACT Aim of the study: The bronchial brushing technique presents an opportunity to establish a gold standard in vitro model of Cystic Fibrosis (CF) airway disease. However, unique obstacles exist when establishing CF airway epithelial cells (pAECCF). We aimed to identify determinants of culture success through retrospective analysis of a program of routinely brushing children with CF. Materials and methods: Anaesthetised children (CF and non-CF) had airway samples taken which were immediately processed for cell culture. Airway data for the CF cohort was obtained from clinical records and the AREST CF database. Results: Of 260 brushings processed for culture, 114 (43.8%) pAECCF successfully cultured to passage one (P1) and 63 (24.2% of total) progressed to passage two (P2). However, >80% of non-CF specimens (pAECnon-CF) cultured to P2 from similar cell numbers. Within the CF cohort, specimens successfully cultured to P2 had a higher initial cell count and lower proportion of severe CF mutation phenotype than those that did not proliferate beyond initial seeding. Elevated airway IL-8 concentration was also negatively associated with culture establishment. Contamination by opportunistic pathogens was observed in 81 (31.2% of total) cultures and brushings from children with lower respiratory tract infections were more likely to co-culture contaminating flora. Conclusions: Lower passage rates of pAECCF cultures uniquely contrasts with pAECnon-CF despite similar cell numbers. An equivalent establishment rate of CF nasal epithelium reported elsewhere, significant associations to CFTR mutation phenotype, elevated airway IL-8 and opportunistic pathogens all suggest this is likely related to the CF disease milieu.

Usage of spacers in respiratory laboratories and the delivered salbutamol dose of spacers available in Australia and New Zealand

Background and objective:  Purchase and disinfection costs together with medication delivery factors may influence the choice of drug delivery options. This study assessed salbutamol delivery habits used in respiratory laboratories and quantified the delivered salbutamol dose of locally available spacers.

Effects of human rhinovirus on epithelial barrier integrity and function in children with asthma

Bronchial epithelial tight junctions (TJ) have been extensively assessed in healthy airway epithelium. However, no studies have yet assessed the effect of human rhinovirus (HRV) infection on the expression and resultant barrier function in epithelial tight junctions (TJ) in childhood asthma.

Visualisation of Multiple Tight Junctional Complexes in Human Airway Epithelial Cells

BackgroundApically located tight junctions in airway epithelium perform a fundamental role in controlling macromolecule migration through paracellular spaces. Alterations in their expression may lead to disruptions in barrier integrity, which subsequently facilitates entry of potential bacterial and other pathogens into the host. Furthermore, there is emerging evidence that the barrier integrity of the airway in certain airway inflammatory diseases may be altered. However, there is little consensus on the way this is assessed and measured and the type of cells used to achieve this.MethodsHere, we assessed four fixation methods including; (i) 4% (v/v) paraformaldehyde; (ii) 100% methanol; (iii) acetone or; (iv) 1:1 methanol: acetone. Pre-extraction with Triton X-100 was also performed and assessed on cells prior to fixation with either methanol or paraformaldehyde. Cells were also permeabilized with 0.1% (v/v) Saponin in 1× TBS following fixation and subsequently stained for tight junction proteins. Confocal microscopy was then used to visualise, compare and evaluate staining intensity of the tight junctional complexes in order to determine a standardised workflow of reproducible staining.ResultsPositive staining was observed following methanol fixation for claudin-1 and ZO-1 tight junction proteins but no staining was detected for occludin in 16HBE14o- cells. Combinatorial fixation with methanol and acetone also produced consistent positive staining for both occludin and ZO-1 tight junction proteins in these cells. When assessed using primary cells cultured at air-liquid interface, similar positive staining for claudin-1 and ZO-1 was observed following methanol fixation, while similar positive staining for occludin and ZO-1 was observed following the same combinatorial fixation with methanol and acetone.ConclusionsThe present study demonstrates the importance of a personalised approach to optimise staining for the visualisation of different tight junction proteins. Of significance, the workflow, once optimised, can readily be translated into primary airway epithelial cell air-liquid interface cultures where it can be used to assess barrier integrity in chronic lung diseases.