Emmanuel Koumoundouros

Chronic airway disease: deteriorating pulmonary function in sheep associated with repeated challenges of house dust mite.

The aim of this work was to characterize lung function and cellular responses in a large animal model for chronic asthma. All sheep were sensitized to house dust mite (HDM) by subcutaneous injection of HDM before lung challenges. Groups of sheep were given weekly lung challenges with either HDM (n = 12) or saline (control, n = 5) for 3 months. Post challenge, there were significant increases in lung resistance in 7 out of 12 HDM-challenged sheep, compared to control sheep. In HDM-responding sheep, there was a progressive increase in the magnitude of HDM-induced resistance throughout the trial. All HDM-challenged sheep developed BAL eosinophilia and bronchial hyperresponsiveness. In conclusion, sheep chronically challenged intralung with HDM consistently develop airway hyperresponsiveness and eosinophilia, whereas allergen-specific bronchoconstriction is observed in just over half of these sheep.

KCa3.1 Channel-Blockade Attenuates Airway Pathophysiology in a Sheep Model of Chronic Asthma

Background The Ca2+-activated K+ channel KCa3.1 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 KCa3.1 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 KCa3.1-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 KCa3.1-activity contributes to allergen-induced airway responses, inflammation and vascular remodelling in a sheep model of asthma, and that inhibition of KCa3.1 may be an effective strategy for blocking allergen-induced airway inflammation and hyperresponsiveness in humans.

Increased vascular density is a persistent feature of airway remodeling in a sheep model of chronic asthma

ABSTRACT Background: Increases in blood vessel density and vascular area are now recognized as important features of remodeled airways in asthma. However, the time sequence for these vascular changes and whether they resolve in the absence of continued antigenic exposure is not well elucidated. The aim of the present study was to correlate progressive changes in airway vascularity with changes in functional airway responses in sheep chronically challenged with house dust mite (HDM) allergen, and to examine the resolution of vascular remodeling following allergen withdrawal. Methods: Progressive changes in vascular indices were examined in four spatially separate lung segments that received weekly challenges with HDM allergen for 0, 8, 16, or 24 weeks. Reversibility of these changes was assessed in a separate experiment in which two lung segments received 24 weeks of HDM challenges and either no rest or 12 weeks rest. Lung tissue was collected from each segment 7 days following the final challenge and vascular changes assessed by a morphometric analysis of airways immunohistochemically stained with an antibody against type IV collagen. Results: Blood vessel density and percent airway vascularity were significantly increased in bronchi following 24 weeks of HDM challenges compared to untreated controls (P < .05), but not at any of the other time-points. There was no significant correlation between vascular indices and airway responses to allergic or nonspecific stimuli. The increase in blood vessel density induced by repeated allergen exposures did not return to baseline levels following a 12-week withdrawal period from allergen. Conclusions: Our results show for the first time that the airways of sheep chronically exposed to HDM allergen undergo vascular remodeling. These findings show the potential of this large animal model for investigating airway angiogenesis in asthma.

Assessment of Peripheral Airway Function following Chronic Allergen Challenge in a Sheep Model of Asthma

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 (Rp) 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 Rp 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 (rs = 0.095, P<0.01). There was no correlation between resting Rp 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.

Lung function in developing lambs: is it affected by preterm birth?

Children born before term often have reduced lung function, but the effects of preterm birth alone are difficult to determine owing to iatrogenic factors such as mechanical ventilation. Our objective was to determine the effects of preterm birth alone on airway resistance, airway reactivity, and ventilatory heterogeneity as an index of intrapulmonary gas mixing. Preterm birth was induced in sheep 12 days before term; controls were born at term ( approximately 147 days). Lung function was assessed at 8 wk postterm. To assess medium-large airway function we measured airway resistance and reactivity to carbachol. Multiple breath N(2) washout (MBW) was used to assess ventilatory heterogeneity in conducting (S(cond)) and acinar (S(acin)) airways. Baseline airway resistance and responsiveness to carbachol were similar in preterm and term lambs. Airway responsiveness to carbachol was greater in females than males (P < 0.05), and baseline airway resistance tended to be higher in females than males (P = 0.06). There were no significant differences in ventilatory heterogeneity between preterm and term lambs; for all animals combined, mean S(acin) was 0.29 +/- 0.05 liter(-1) and S(cond) was 0.26 +/- 0.03 liter(-1). Males had significantly higher S(cond) than females, indicating poorer gas mixing in small conducting airways; there was no sex difference in S(acin). We conclude that preterm birth per se in lambs does not affect baseline airway resistance, airway responsiveness, or ventilatory heterogeneity as measured by MBW. The observed sex-related differences in airway responsiveness and ventilatory heterogeneity in the conducting airways could help explain sex differences in lung function observed in humans.

Altered airway responsiveness in adult sheep born prematurely: effects of allergen exposure.

The aim of this study was to determine the effects of preterm birth per se on airway function in adult sheep. Preterm birth was induced at ∼0.89 of term. At ∼1 year of age the authors measured pulmonary resistance (RL) and airway responsiveness before and after house dust mite (HDM) challenge. Mature preterm sheep tended to have greater baseline RL than controls (P = .12): the smaller preterm sheep showed significantly greater RL than controls following bronchoconstrictor challenge. Preterm animals tended to have greater baseline total blood leukocyte count (P = .06). It was concluded that preterm sheep, especially with low postnatal growth, have greater airway responsiveness to bronchoconstrictor and higher baseline RL.

A novel segmental challenge model for bleomycin-induced pulmonary fibrosis in sheep

ABSTRACT Background: Idiopathic Pulmonary fibrosis (IPF) is a fatal respiratory disease, characterized by a progressive fibrosis and worsening lung function. While the outcomes of recent clinical trials have resulted in therapies to slow the progression of the disease, there is still a need to develop alternative therapies, which are able to prevent fibrosis. Aim: This study uses a segmental lung infusion of bleomycin (BLM) to investigate pulmonary fibrosis in a physiologically relevant large animal species. Methods: Two separate lung segments in eight sheep received two fortnightly challenges of either 3U or 30U BLM per segment, and a third segment received saline (control). Lung function was assessed using a wedged-bronchoscope procedure. Bronchoalveolar lavage fluid and lung tissue were assessed for inflammation, fibrosis and collagen content two weeks after the final dose of BLM. Results: Instillation of both BLM doses resulted in prominent fibrosis in the treated lobes. More diffuse fibrosis and loss of alveolar airspace was observed in high-dose BLM-treated segments, while multifocal fibrosis was seen in low-dose BLM-treated segments. Extensive and disorganised collagen deposition occurred in the BLM-treated lobes, compared to controls. Significant loss of lung compliance was also observed in the BLM-treated lobes, which did not occur in controls. Conclusions: Fibrosis comparable to IPF was induced into isolated lung segments, without compromising the respiratory functioning of the animal. This model may have potential for investigating novel therapies for IPF by allowing direct comparison of multiple treatments with internal controls, and sampling and drug delivery that are clinically relevant.

Nebulized perflubron and carbon dioxide rapidly dilate constricted airways in an ovine model of allergic asthma

BackgroundThe 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.MethodsSheep 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.In additional naïve sheep, a segmental bronchus was pre-contracted with methacholine (MCh) and treated with nebulized S-1226 administered via a bronchoscope catheter. Subsequent bronchodilatation was monitored by real time digital video recording.ResultsTreatment 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.ConclusionsS-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.

Inhibition of the KCa3.1 Channel Alleviates Established Pulmonary Fibrosis in a Large Animal Model

&NA; Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive disease of increasing prevalence marked by poor prognosis and limited treatment options. Ca2+‐activated KCa3.1 potassium channels have been shown to play a key role in the aberrant activation and responses to injury in both epithelial cells and fibroblasts, both considered key drivers in the fibrotic process of IPF. Pharmacological inhibition of IPF‐derived fibroblasts is able to somewhat prevent TGF‐&bgr;‐ and basic fibroblast growth factor‐dependent profibrotic responses. In the current study, we investigated whether blockade of the KCa3.1 ion channel in vivo with a selective inhibitor, Senicapoc, was able to attenuate both histological and physiological outcomes of early fibrosis in our large animal (sheep) model for pulmonary fibrosis. We also determined whether treatment was targeting the profibrotic activity of sheep lung fibroblasts. Senicapoc was administered in established fibrosis, at 2 weeks after bleomycin instillation, and drug efficacy was assessed 4 weeks after treatment. Treatment with Senicapoc improved pre‐established bleomycin‐induced changes compared with vehicle control, leading to improved lung compliance, reduced extracellular matrix and collagen deposition, and a reduction in both &agr;‐smooth muscle actin expression and proliferating cells, both in vivo and in vitro. These studies show that inhibiting the KCa3.1 ion channel is able to attenuate the early fibrogenic phase of bleomycin‐dependent fibrosis and inhibits profibrotic behavior of primary sheep lung fibroblasts. This supports the previous research conducted in human IPF‐derived fibroblasts and suggests that inhibiting KCa3.1 signaling may provide a novel therapeutic approach for IPF.

The Effects of Tumstatin on Vascularity, Airway Inflammation and Lung Function in an Experimental Sheep Model of Chronic Asthma

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.