Darryl Knight
The University of Newcastle, Biomedical Science &pharmacy, Faculty Member
The use of cytology brushes for the purpose of obtaining respiratory cells from adults for clinical and research purposes is well established. However, the safety and utility of non-bronchoscopic brushings to study the paediatric airway... more
The use of cytology brushes for the purpose of obtaining respiratory cells from adults for clinical and research purposes is well established. However, the safety and utility of non-bronchoscopic brushings to study the paediatric airway has not been assessed. The purpose of this study was to assess the practicality of using non-bronchoscopic brushing to sample epithelial cells from children for investigation of epithelial function in health and disease using a wide range of molecular and cellular techniques. Non-bronchoscopic brushing was investigated in a non-selected cohort of healthy, and mildly asthmatic children presenting for surgery unrelated to respiratory conditions, at the major children's hospital in Perth. Safety and side-effects of the procedure were assessed. Cell number, phenotype and viability were measured for all samples. The potential of these cells for use in long-term cell culture, immunohistochemistry, western blotting, quantitative PCR and gene arraying wa...
Research Interests: Asthma, Immunohistochemistry, Cell Culture, Quantitative PCR, real time PCR, and 17 moreHumans, Child, Respiratory Disease, Female, Male, Clinical Sciences, Research Purpose, Microarray Analysis, Epithelial cells, Respiratory, Western blot, Bronchoscopy, Protein Expression, Side Effect, Cough, Cell Survival, and Cohort Studies
The fractional concentration of nitric oxide (NO) in exhaled breath (FeNO) is increased in asthma. There is a general assumption that NO synthase (NOS) 2 in epithelium is the main source of NO in exhaled breath. However, there is no... more
The fractional concentration of nitric oxide (NO) in exhaled breath (FeNO) is increased in asthma. There is a general assumption that NO synthase (NOS) 2 in epithelium is the main source of NO in exhaled breath. However, there is no direct evidence to support the assumption and data from animal models suggest that non-inducible NOS systems have important roles in determining airway reactivity, regulating inflammation, and might contribute significantly to NO measured in exhaled breath. Bronchial epithelial cells were obtained from healthy, atopic, and asthmatic children by non-bronchoscopic brushing. Exhaled NO (FeNO) was measured directly using a fast response chemiluminescence NO analyser. RNA was extracted from the epithelial cells and real time polymerase chain reaction was used to determine the expression of NOS isoenzymes. NOS2 was examined in macrophages and epithelial cells by immunohistochemistry. NOS1 mRNA was not detectable. NOS3 mRNA was detected in 36 of 43 samples at l...
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This is the first study to measure inducible nitric oxide synthase (iNOS) gene and protein expression quantitatively in primary epithelial cells from very young children with cystic fibrosis (CF). Low levels of exhaled nitric oxide (NO)... more
This is the first study to measure inducible nitric oxide synthase (iNOS) gene and protein expression quantitatively in primary epithelial cells from very young children with cystic fibrosis (CF). Low levels of exhaled nitric oxide (NO) in CF suggest dysregulation of NO production in the airway. Due to the importance of NO in cell homeostasis and innate immunity, any defect in the pathway associated with CF would be a potential target for treatment. Cells were obtained by tracheobronchial brushing from 40 children with CF of mean (SD) age 2.1 (1.5) years and from 12 healthy non-atopic children aged 3.4 (1.2) years. Expression of iNOS mRNA was measured using quantitative PCR and iNOS protein by immunofluorescence and Western blot analysis. Inducible NOS mRNA expression was significantly lower in CF patients with and without bacterial infection than in healthy children (0.22 and 0.23 v 0.76; p=0.002 and p=0.01, respectively). Low levels of iNOS gene expression were accompanied by low ...
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Asthma exacerbations represent a significant disease burden and are commonly caused by rhinovirus (RV), which is sensed by Toll-like receptors (TLR) such as TLR7. Some asthmatics have impaired interferon (IFN) responses to RV, but the... more
Asthma exacerbations represent a significant disease burden and are commonly caused by rhinovirus (RV), which is sensed by Toll-like receptors (TLR) such as TLR7. Some asthmatics have impaired interferon (IFN) responses to RV, but the underlying mechanisms of this clinically relevant observation are poorly understood. To investigate the importance of intact TLR7 signalling in vivo during RV exacerbation using mouse models of house dust mite (HDM)-induced allergic airways disease exacerbated by a superimposed RV infection. Wild-type and TLR7-deficient (Tlr7(-/-)) BALB/c mice were intranasally sensitised and challenged with HDM prior to infection with RV1B. In some experiments, mice were administered recombinant IFN or adoptively transferred with plasmacytoid dendritic cells (pDC). Allergic Tlr7(-/-) mice displayed impaired IFN release upon RV1B infection, increased virus replication and exaggerated eosinophilic inflammation and airways hyper reactivity. Treatment with exogenous IFN o...
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Rationale: Convincing evidence of epithelial damage and aberrant repair exists in adult asthmatic airways, even in the absence of inflammation. However, comparable studies in children have been limited by access and availability of... more
Rationale: Convincing evidence of epithelial damage and aberrant repair exists in adult asthmatic airways, even in the absence of inflammation. However, comparable studies in children have been limited by access and availability of clinical samples. Objectives: To determine whether bronchial epithelial cells from children with asthma are inherently distinct from those obtained from children without asthma. Methods: Epithelial cells were
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Chronic obstructive pulmonary disease (COPD) is a worldwide epidemic affecting over 200 million people and accounting for more than three million deaths annually. The disease is characterized by chronic inflammation of the airways and... more
Chronic obstructive pulmonary disease (COPD) is a worldwide epidemic affecting over 200 million people and accounting for more than three million deaths annually. The disease is characterized by chronic inflammation of the airways and progressive destruction of lung parenchyma, a process that in most cases is initiated by cigarette smoking. Unfortunately, there are no interventions that have been unequivocally shown to prolong survival in patients with COPD. Regeneration of lung tissue by stem cells from endogenous and exogenous sources is a promising therapeutic strategy. Herein we review the current literature on the characterization of resident stem and progenitor cell niches within the lung, the contribution of mesenchymal stem cells to lung regeneration, and advances in bioengineering of lung tissue.
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The respiratory epithelium produces a variety of inflammatory mediators which may be influencing the bronchial hyperreactivity observed in patients with asthma. Animal studies have demonstrated that removal of the epithelium from tracheal... more
The respiratory epithelium produces a variety of inflammatory mediators which may be influencing the bronchial hyperreactivity observed in patients with asthma. Animal studies have demonstrated that removal of the epithelium from tracheal and bronchial smooth muscle causes enhanced responses to cholinergic agonists and histamine (Hist). In this study the effect of epithelium removal on human bronchial smooth muscle response to acetylcholine (ACh) and Hist was assessed. Bronchial smooth muscle was obtained fresh from the operating theatre from 12 patients undergoing thoracotomy. Cumulative concentration effect curves (CCEC) for Hist and ACh were generated for epithelium intact and epithelium denuded muscle strips. All CCEC's were performed in duplicate and all denuded strips were obtained from the same airway immediately adjacent to the intact strip. The mean (+/- SEM) maximum response for Hist for the intact strip was 8.6 +/- 1.1 (grams/gram wet weight) and 12.0 +/- 1.4 (grams/g...
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The interaction of histamine (Hist) and acetylcholine (ACh) on human isolated bronchial smooth muscle (HIBSM) contraction, and the influence of the epithelium, was assessed using HIBSM obtained from 15 patients undergoing thoracotomy.... more
The interaction of histamine (Hist) and acetylcholine (ACh) on human isolated bronchial smooth muscle (HIBSM) contraction, and the influence of the epithelium, was assessed using HIBSM obtained from 15 patients undergoing thoracotomy. Cumulative concentration effect curves for ACh and Hist, together with combinations of equipotent concentrations of both agonists, were generated using both epithelium-intact and epithelium-denuded HIBSM. In epithelium-denuded HIBSM both ACh (p less than 0.05) and Hist (p less than 0.005) produced a significantly enhanced maximal response and a 2.1 fold increase in the potency of ACh (p less than 0.02, n = 13). When ACh and Hist were added simultaneously, in equipotent concentrations, to epithelium-intact HIBSM, a significantly less (p less than 0.0005, n = 13) than additive response occurred with only 60% of the predicted maximum response being observed. However, following epithelium removal, an additive interaction between the two agonists (n = 8) oc...
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Airway smooth muscle cell contraction is regulated by changes in intracellular Ca2+ concentration ([Ca2+]i) and the responsiveness of the airway smooth muscle cell to this Ca2+. The mechanism controlling [Ca2+]i primarily involves... more
Airway smooth muscle cell contraction is regulated by changes in intracellular Ca2+ concentration ([Ca2+]i) and the responsiveness of the airway smooth muscle cell to this Ca2+. The mechanism controlling [Ca2+]i primarily involves agonist-induced release of Ca2+ from internal stores to generate Ca2+ oscillations. The extent of contraction correlates with the persistence and frequency of these Ca2+ oscillations. The maintenance of the Ca2+ oscillations requires Ca2+ influx, but membrane depolarization appears to have a minor role in initiating or sustaining contraction. Contraction also requires agonist-induced Ca2+ sensitization, which is mediated mainly by decreases in myosin light-chain phosphatase activity. Although it is not clear if airway hyperresponsiveness associated with asthma results from the specific modulation of these Ca2+-based regulatory mechanisms, bronchodilators relax airways by both attenuating the Ca2+ oscillations and by decreasing the Ca2+ sensitivity.
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The fractional concentration of nitric oxide (NO) in exhaled breath (FeNO) is increased in asthma. There is a general assumption that NO synthase (NOS) 2 in epithelium is the main source of NO in exhaled breath. However, there is no... more
The fractional concentration of nitric oxide (NO) in exhaled breath (FeNO) is increased in asthma. There is a general assumption that NO synthase (NOS) 2 in epithelium is the main source of NO in exhaled breath. However, there is no direct evidence to support the assumption and data from animal models suggest that non-inducible NOS systems have important roles in determining airway reactivity, regulating inflammation, and might contribute significantly to NO measured in exhaled breath. Bronchial epithelial cells were obtained from healthy, atopic, and asthmatic children by non-bronchoscopic brushing. Exhaled NO (FeNO) was measured directly using a fast response chemiluminescence NO analyser. RNA was extracted from the epithelial cells and real time polymerase chain reaction was used to determine the expression of NOS isoenzymes. NOS2 was examined in macrophages and epithelial cells by immunohistochemistry. NOS1 mRNA was not detectable. NOS3 mRNA was detected in 36 of 43 samples at lower levels than NOS2 mRNA which was detectable in all samples. The median FeNO was 15.5 ppb (95% CI 10 to 18.1). There was a significant correlation between FeNO and NOS2 expression (R = 0.672, p<0.001). All epithelial cells exhibited NOS2 staining, whereas staining in the macrophages was variable and not related to phenotype. Only NOS2 expression was associated with FeNO in respiratory epithelial cells obtained from children (R = 0.672; p<0.001). This suggests that FeNO variability is largely determined by epithelial NOS2 expression with little contribution from other isoforms.
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The major function of the respiratory epithelium was once thought to be that of a physical barrier. However, it constitutes the interface between the internal milieu and the external environment as well as being a primary target for... more
The major function of the respiratory epithelium was once thought to be that of a physical barrier. However, it constitutes the interface between the internal milieu and the external environment as well as being a primary target for inhaled respiratory drugs. It also responds to changes in the external environment by secreting a large number of molecules and mediators that signal to cells of the immune system and underlying mesenchyme. Thus, the epithelium is in a unique position to translate gene-environment interactions. Normally, the epithelium has a tremendous capacity to repair itself following injury. However, evidence is rapidly accumulating to show that the airway epithelium of asthmatics is abnormal and has increased susceptibility to injury compared to normal epithelium. Areas of detachment and fragility are a characteristic feature not observed in other inflammatory diseases such as COPD. In addition to being more susceptible to damage, normal repair processes are also compromised. Failure of appropriate growth and differentiation of airway epithelial cells will cause persistent mucosal injury. The response to traditional therapy such as glucocorticoids may also be compromised. However, whether the differences observed in asthmatic epithelium are a cause of or secondary to the development of the disease remains unanswered. Strategies to address this question include careful examination of the ontogeny of the disease in children and use of gene array technology should provide some important answers, as well as allow a better understanding of the critical role that the epithelium plays under normal conditions and in diseases such as asthma.
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Research Interests: Microscopy, Multiphoton Microscopy, Asthma, Extracellular Matrix, Humans, and 13 moreMice, Animals, Lung Diseases, Three Dimensional Imaging, Second Harmonic Generation, Clinical Sciences, Fine Structure Constant, Sensitivity and Specificity, Cellular Structure, D structure, Minimally Invasive, Pulmonary Pharmacology, and Harmonic Generation
Granzyme K (GrK) is a trypsin-like serine protease that is elevated in patients with sepsis and acute lung inflammation. While GrK was originally believed to function exclusively as a pro-apoptotic protease, recent studies now suggest... more
Granzyme K (GrK) is a trypsin-like serine protease that is elevated in patients with sepsis and acute lung inflammation. While GrK was originally believed to function exclusively as a pro-apoptotic protease, recent studies now suggest that GrK may possess other non-cytotoxic functions. In the context of acute lung inflammation, we hypothesized that GrK induces pro-inflammatory cytokine release through the activation of protease-activated receptors. The direct effect of extracellular GrK on PAR activation, intracellular signaling and cytokine was assessed using cultured human lung fibroblasts. Extracellular GrK induced secretion of IL-6, IL-8 and MCP-1 in a dose- and time-dependent manner in lung fibroblasts. Heat-inactivated GrK did not induce cytokine release indicating that protease activity is required. Furthermore, GrK induced activation of both the ERK1/2 and p38 MAP kinase signaling pathways, and significantly increased fibroblast proliferation. Inhibition of ERK1/2 abrogated the GrK-mediated cytokine release. Through the use of PAR-1 and PAR-2 neutralizing antibodies, it was determined that PAR-1 is essential for GrK-induced IL-6, IL-8 and MCP-1 release. In summary, extracellular GrK is capable of activating PAR-1 and inducing fibroblast cytokine secretion and proliferation.
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Research Interests: Engineering, Physics, Chemistry, Biology, Medicine, and 17 moreMultidisciplinary, Epithelial-Mesenchymal Transition, Protein Structure and Function, Humans, Mice, Female, Animals, Skin, Fibrosis, Lung, PLoS one, Epithelial cells, Bleomycin, Growth Factor, Human Fibroblasts, Renal Fibrosis, and Gene Expression Regulation
Research Interests: Engineering, Physics, Chemistry, M Rna Processing, Biology, and 16 moreMedicine, Multidisciplinary, Epithelial-Mesenchymal Transition, Humans, Female, Male, Polymerase Chain Reaction, Young Adult, Lung, PLoS one, Aged, Middle Aged, microRNAs, Retrospective Studies, Disease Progression, and Idiopathic Pulmonary Fibrosis
Research Interests: Engineering, Physics, Chemistry, Kinetics, Biology, and 20 moreStructural Integrity, Cardiovascular disease, Medicine, Multidisciplinary, Extracellular Matrix, Biocatalysis, Western blotting, Humans, Phosphorylation, Solubility, PLoS one, Coumarins, Proteoglycans, Substrate Specificity, Transforming Growth Factor Beta, Decorin, Serine Protease, Extracellular Space, Transforming Growth Factor, and Coronary artery(Structural Integrity, Cardiovascular disease, Medicine, Multidisciplinary, Extracellular Matrix, Biocatalysis, Western blotting, Humans, Phosphorylation, Solubility, PLoS one, Coumarins, Proteoglycans, Substrate Specificity, Transforming Growth Factor Beta, Decorin, Serine Protease, Extracellular Space, Transforming Growth Factor, and Coronary artery)
(Structural Integrity, Cardiovascular disease, Medicine, Multidisciplinary, Extracellular Matrix, Biocatalysis, Western blotting, Humans, Phosphorylation, Solubility, PLoS one, Coumarins, Proteoglycans, Substrate Specificity, Transforming Growth Factor Beta, Decorin, Serine Protease, Extracellular Space, Transforming Growth Factor, and Coronary artery)
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Research Interests: Physiology, Immunology, Cell Migration, Calcium, Immunology of the Gut, and 17 moreMolecular Mechanics, Cell line, Thrombin, Molecular and cellular biology, Humans, Platelet aggregation, Sequence alignment, Animals, Anura, Peptides, The, Trypsin, Platelet activation mechanism, Clinical Sciences, Amino Acid Sequence, Biochemistry and cell biology, and Interleukin
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The airway epithelium is the first line of defense against inhaled insults and therefore must be capable of coordinating appropriate inflammatory and immune responses. We sought to test the hypothesis that the nucleotide-binding domain... more
The airway epithelium is the first line of defense against inhaled insults and therefore must be capable of coordinating appropriate inflammatory and immune responses. We sought to test the hypothesis that the nucleotide-binding domain and leucine-rich repeat protein 3 (NLRP3) inflammasome, an intracellular danger-sensing complex, plays a critical role in airway epithelium-mediated immune responses to urban particulate matter (PM) exposure. In this study we (1) identified NLRP3 and caspase-1 expression in human airway epithelium bronchus and primary cells, (2) characterized NLRP3 inflammasome-mediated IL-1β production from human airway epithelium in response to PM, and (3) performed in vivo PM exposure experiments with wild-type and Nlrp3(-/-) mice. Our results demonstrate that human airway epithelium contains a functional NLRP3 inflammasome that responds to PM exposure with caspase-1 cleavage and production of IL-1β. Exposure of Nlrp3(-/-) and wild-type mice to PM in vivo demonstrates NLRP3-dependent production of IL-1β in the lung, airway neutrophilia, and increases in CD11c(+hi)/MHC class II(+hi) cell numbers in intrathoracic lymph nodes. Our study is the first to characterize airway epithelial NLRP3 inflammasome-mediated immune responses to PM exposure, which might have implications in patients with asthma and other lung diseases.
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... Chang Xiao, Sarah M. Puddicombe, Sarah Field, Joel Haywood, Victoria Broughton-Head, Ilaria Puxeddu, Hans Michael Haitchi, Elizabeth Vernon-Wilson, David Sammut, Nicole Bedke, Catherine Cremin, Jody Sones, Ratko Djukanović, Peter H.... more
... Chang Xiao, Sarah M. Puddicombe, Sarah Field, Joel Haywood, Victoria Broughton-Head, Ilaria Puxeddu, Hans Michael Haitchi, Elizabeth Vernon-Wilson, David Sammut, Nicole Bedke, Catherine Cremin, Jody Sones, Ratko Djukanović, Peter H. Howarth, Jane E. Collins ...
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BACKGROUND: The use of cytology brushes for the purpose of obtaining respiratory cells from adults for clinical and research purposes is well established. However, the safety and utility of non-bronchoscopic brushings to study the... more
BACKGROUND: The use of cytology brushes for the purpose of obtaining respiratory cells from adults for clinical and research purposes is well established. However, the safety and utility of non-bronchoscopic brushings to study the paediatric airway has not been assessed. The purpose of this study was to assess the practicality of using non-bronchoscopic brushing to sample epithelial cells from children