Melbourne Veterinary School - Research Publications
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ItemKCa3.1 Channel-Blockade Attenuates Airway Pathophysiology in a Sheep Model of Chronic Asthma(PUBLIC LIBRARY SCIENCE, 2013-06-24)BACKGROUND: The Ca(2+)-activated K(+) channel K(Ca)3.1 is expressed in several structural and inflammatory airway cell types and is proposed to play an important role in the pathophysiology of asthma. The aim of the current study was to determine whether inhibition of K(Ca)3.1 modifies experimental asthma in sheep. METHODOLOGY AND PRINCIPAL FINDINGS: Atopic sheep were administered either 30 mg/kg Senicapoc (ICA-17073), a selective inhibitor of the K(Ca)3.1-channel, or vehicle alone (0.5% methylcellulose) twice daily (orally). Both groups received fortnightly aerosol challenges with house dust mite allergen for fourteen weeks. A separate sheep group received no allergen challenges or drug treatment. In the vehicle-control group, twelve weeks of allergen challenges resulted in a 60±19% increase in resting airway resistance, and this was completely attenuated by treatment with Senicapoc (0.25±12%; n = 10, P = 0.0147). The vehicle-control group had a peak-early phase increase in lung resistance of 82±21%, and this was reduced by 58% with Senicapoc treatment (24±14%; n = 10, P = 0.0288). Senicapoc-treated sheep also demonstrated reduced airway hyperresponsiveness, requiring a significantly higher dose of carbachol to increase resistance by 100% compared to allergen-challenged vehicle-control sheep (20±5 vs. 52±18 breath-units of carbachol; n = 10, P = 0.0340). Senicapoc also significantly reduced eosinophil numbers in bronchoalveolar lavage taken 48 hours post-allergen challenge, and reduced vascular remodelling. CONCLUSIONS: These findings suggest that K(Ca)3.1-activity contributes to allergen-induced airway responses, inflammation and vascular remodelling in a sheep model of asthma, and that inhibition of K(Ca)3.1 may be an effective strategy for blocking allergen-induced airway inflammation and hyperresponsiveness in humans.
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ItemIncreased Mast Cell Density and Airway Responses to Allergic and Non-Allergic Stimuli in a Sheep Model of Chronic Asthma(PUBLIC LIBRARY SCIENCE, 2012-05-14)BACKGROUND: Increased mast cell (MC) density and changes in their distribution in airway tissues is thought to contribute significantly to the pathophysiology of asthma. However, the time sequence for these changes and how they impact small airway function in asthma is not fully understood. The aim of the current study was to characterise temporal changes in airway MC density and correlate these changes with functional airway responses in sheep chronically challenged with house dust mite (HDM) allergen. METHODOLOGY/PRINCIPAL FINDINGS: MC density was examined on lung tissue from four spatially separate lung segments of allergic sheep which received weekly challenges with HDM allergen for 0, 8, 16 or 24 weeks. Lung tissue was collected from each segment 7 days following the final challenge. The density of tryptase-positive and chymase-positive MCs (MC(T) and MC(TC) respectively) was assessed by morphometric analysis of airway sections immunohistochemically stained with antibodies against MC tryptase and chymase. MC(T) and MC(TC) density was increased in small bronchi following 24 weeks of HDM challenges compared with controls (P<0.05). The MC(TC)/MC(T) ratio was significantly increased in HDM challenged sheep compared to controls (P<0.05). MC(T) and MC(TC) density was inversely correlated with allergen-induced increases in peripheral airway resistance after 24 weeks of allergen exposure (P<0.05). MC(T) density was also negatively correlated with airway responsiveness after 24 challenges (P<0.01). CONCLUSIONS: MC(T) and MC(TC) density in the small airways correlates with better lung function in this sheep model of chronic asthma. Whether this finding indicates that under some conditions mast cells have protective activities in asthma, or that other explanations are to be considered requires further investigation.
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ItemAssessment of Peripheral Airway Function following Chronic Allergen Challenge in a Sheep Model of Asthma(PUBLIC LIBRARY SCIENCE, 2011-12-12)BACKGROUND: There is increasing evidence that the small airways contribute significantly to the pathophysiology of asthma. However, due to the difficulty in accessing distal lung regions in clinical settings, functional changes in the peripheral airways are often overlooked in studies of asthmatic patients. The aim of the current study was to characterize progressive changes in small airway function in sheep repeatedly challenged with house dust mite (HDM) allergen. METHODOLOGY/PRINCIPAL FINDINGS: Four spatially separate lung segments were utilized for HDM challenges. The right apical, right medial, right caudal and left caudal lung segments received 0, 8, 16 and 24 weekly challenges with HDM respectively. A wedged-bronchoscope technique was used to assess changes in peripheral resistance (R(p)) at rest, and in response to specific and non-specific stimuli throughout the trial. Allergen induced inflammatory cell infiltration into bronchoalveolar lavage and increases in R(p) in response to HDM and methacholine were localized to treated lung segments, with no changes observed in adjacent lung segments. The acute response to HDM was variable between sheep, and was significantly correlated to airway responsiveness to methacholine (r(s) = 0.095, P<0.01). There was no correlation between resting R(p) and the number of weeks of HDM exposure. Nor was there a correlation between the magnitude of early-phase airway response and the number of HDM-challenges. CONCLUSIONS: Our findings indicate that airway responses to allergic and non-allergic stimuli are localized to specific treated areas of the lung. Furthermore, while there was a decline in peripheral airway function with HDM exposure, this decrease was not correlated with the length of allergen challenge.
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ItemNebulized perflubron and carbon dioxide rapidly dilate constricted airways in an ovine model of allergic asthma(BIOMED CENTRAL LTD, 2014-09-16)BACKGROUND: The low toxicity of perfluorocarbons (PFCs), their high affinity for respiratory gases and their compatibility with lung surfactant have made them useful candidates for treating respiratory diseases such as adult respiratory distress syndrome. We report results for treating acute allergic and non-allergic bronchoconstriction in sheep using S-1226 (a gas mixture containing carbon dioxide and small volumes of nebulized perflubron). The carbon dioxide, which is highly soluble in perflubron, was used to relax airway smooth muscle. METHODS: Sheep previously sensitized to house dust mite (HDM) were challenged with HDM aerosols to induce early asthmatic responses. At the maximal responses (characterised by an increase in lung resistance), the sheep were either not treated or treated with one of the following; nebulized S-1226 (perflubron + 12% CO2), nebulized perflubron + medical air, 12% CO2, salbutamol or medical air. Lung resistance was monitored for up to 20 minutes after cessation of treatment. RESULTS: Treatment with S-1226 for 2 minutes following HDM challenge resulted in a more rapid, more profound and more prolonged decline in lung resistance compared with the other treatment interventions. Video bronchoscopy showed an immediate and complete (within 5 seconds) re-opening of MCh-constricted airways following treatment with S-1226. CONCLUSIONS: S-1226 is a potent and rapid formulation for re-opening constricted airways. Its mechanism(s) of action are unknown. The formulation has potential as a rescue treatment for acute severe asthma.
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ItemThe Effects of Tumstatin on Vascularity, Airway Inflammation and Lung Function in an Experimental Sheep Model of Chronic Asthma(NATURE PORTFOLIO, 2016-05-20)Tumstatin, a protein fragment of the alpha-3 chain of Collagen IV, is known to be significantly reduced in the airways of asthmatics. Further, there is evidence that suggests a link between the relatively low level of tumstatin and the induction of angiogenesis and inflammation in allergic airway disease. Here, we show that the intra-segmental administration of tumstatin can impede the development of vascular remodelling and allergic inflammatory responses that are induced in a segmental challenge model of experimental asthma in sheep. In particular, the administration of tumstatin to lung segments chronically exposed to house dust mite (HDM) resulted in a significant reduction of airway small blood vessels in the diameter range 10(+)-20 μm compared to controls. In tumstatin treated lung segments after HDM challenge, the number of eosinophils was significantly reduced in parenchymal and airway wall tissues, as well as in the bronchoalveolar lavage fluid. The expression of VEGF in airway smooth muscle was also significantly reduced in tumstatin-treated segments compared to control saline-treated segments. Allergic lung function responses were not attenuated by tumstatin administration in this model. The data are consistent with the concept that tumstatin can act to suppress vascular remodelling and inflammation in allergic airway disease.
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ItemUnique mechanisms of connective tissue growth factor regulation in airway smooth muscle in asthma: Relationship with airway remodelling(WILEY, 2018-05)Neovascularization, increased basal membrane thickness and increased airway smooth muscle (ASM) bulk are hallmarks of airway remodelling in asthma. In this study, we examined connective tissue growth factor (CTGF) dysregulation in human lung tissue and animal models of allergic airway disease. Immunohistochemistry revealed that ASM cells from patients with severe asthma (A) exhibited high expression of CTGF, compared to mild and non-asthmatic (NA) tissues. This finding was replicated in a sheep model of allergic airways disease. In vitro, transforming growth factor (TGF)-β increased CTGF expression both in NA- and A-ASM cells but the expression was higher in A-ASM at both the mRNA and protein level as assessed by PCR and Western blot. Transfection of CTGF promoter-luciferase reporter constructs into NA- and A-ASM cells indicated that no region of the CTGF promoter (-1500 to +200 bp) displayed enhanced activity in the presence of TGF-β. However, in silico analysis of the CTGF promoter suggested that distant transcription factor binding sites may influence CTGF promoter activation by TGF-β in ASM cells. The discord between promoter activity and mRNA expression was also explained, in part, by differential post-transcriptional regulation in A-ASM cells due to enhanced mRNA stability for CTGF. In patients, higher CTGF gene expression in bronchial biopsies was correlated with increased basement membrane thickness indicating that the enhanced CTGF expression in A-ASM may contribute to airway remodelling in asthma.
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ItemImmune response to allergens in sheep sensitized to house dust mite(BMC, 2008-10-20)BACKGROUND: House dust mite (HDM) allergens are a major cause of allergic asthma. Most studies using animal models of allergic asthma have used rodents sensitized with the 'un-natural' allergen ovalbumin. It has only recently been recognized that the use of animal models based on HDM provide a more relevant insight into the allergen-induced mechanisms that underpin human allergic disease. We have previously described a sheep model of human allergic asthma that uses Dermatophagoides pteronyssinus HDM. The present study extends our understanding of the immune effects of HDM and the allergens Der p 1 and Der p 2 in the sheep model of asthma. METHODS: Peripheral blood sera from non-sensitized (control) sheep and sheep sensitized to HDM was collected to determine immunoglobulin (Ig) reactivities to HDM, Der p 1 and Der p 2 by ELISA. Bronchoalveolar lavage (BAL) fluid collected following allergen challenge was also assessed for the presence of HDM-specific antibodies. To examine the cellular immune response to HDM allergens, T cell proliferation and cutaneous responses were assessed in sensitized and control sheep. RESULTS: Strong HDM- and Der p 1-specific IgE, IgG1, IgG2 and IgA serum responses were observed in sensitized sheep, while detectable levels of HDM-specific IgG1 and IgA were seen in BAL fluid of allergen-challenged lungs. In contrast, minimal antibody reactivity was observed to Der p 2. Marked T cell proliferation and late phase cutaneous responses, accompanied by the recruitment of eosinophils, indicates the induction of a cellular and delayed-type hypersensitivity (DTH) type II response by HDM and Der p 1 allergen, but not Der p 2. CONCLUSION: This work characterizes the humoral and cellular immune effects of HDM extract and its major constituent allergens in sheep sensitized to HDM. The effects of allergen in HDM-sensitized sheep were detectable both locally and systemically, and probably mediated via enzymatic and immune actions of the major HDM allergen Der p 1. This study extends our understanding of the actions of this important allergen relevant to human allergic asthma and its effects in sheep experimentally sensitized to HDM allergens.