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Title: Comparative pulmonary fibrosis : imaging fibroproliferation in donkey and man
Author: Miele, Amy Caroline
ISNI:       0000 0004 5991 5918
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2015
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Pulmonary fibrosis is a chronic and debilitating condition that proposes several challenges to both veterinary and medical clinicians. Despite considerable research, many fibrotic lung diseases remain elusive in terms of aetiology, pathogenesis and treatment. Furthermore, progress is hindered by the lack of a translatable animal model with durable and persistent fibrosis. Asinine Pulmonary Fibrosis (APF) is a spontaneous syndrome of aged donkeys with high prevalence (35%). No previous detailed characterisation of APF has been performed and disease diagnosis remains a challenge. APF was studied with regard to clinical, pathological and molecular features and the suitability of this condition as a model for a rare fibrotic lung disease in humans known as pleuroparenchymal fibroelastosis (PPFE) was assessed. In addition, target activatable optical imaging reagents for the real time detection of two key molecular markers of fibrosis: matrix metalloproteinases (MMPs) and lysyl oxidases (LOXF) were evaluated in spontaneous ex vivo models of fibrosis. Such reagents may be used alongside fibred confocal fluorescence microscopy (FCFM), a relatively noninvasive and cutting edge diagnostic tool, to detect and monitor fibroproliferation in animals and man. Whole lungs were collected from 32 aged donkeys at routine necropsy. Gross examination revealed pulmonary fibrosis in 19 donkeys (APF cases), while 13 (controls) had grossly normal lungs. HRCT images and histology sections were reviewed independently and blindly for each of the lungs. Ten of 19 APF lungs were categorised as being ‘consistent with’ PPFE according to previously defined histological and imaging criteria. All 10 PPFE-like lungs had marked pleural and subpleural fibrosis, predominantly within the upper lung zone, with accompanying intra-alveolar fibrosis and elastosis. An activatable Smartprobe for the detection of LOXF, TWB-219, was synthesised by The Bradley Group, Department of Chemistry (UoEDC). The probe was based on a tandem amine oxidation and β-elimination mechanism, resulting in signal amplification detected at the 488nm wavelength. The probe showed increased fluorescence in the presence of diamine oxidase as well as on incubation with aged human lung tissue cell-free homogenate as determined by a fluorescent plate reader. This signal amplification could be inhibited by β-aminopropionitrile, a recognised LOX inhibitor as well as by an in-house inhibitor specific to LOX. An evolutionary family of MMP probes with varying cleavage sequences and structures, synthesised by the UoEDC, was evaluated at each stage of progression with regard to signal to noise ratio, sensitivity and specificity. Probes were tested against recombinant enzymes from the MMP family as well as neutrophil elastase and plasmin. Signal amplification was also assessed on incubation with human and ovine ex vivo lung tissue. The final ‘lead’ MMP probe, SVC-186, was cleaved by MMP-2, -9 and -13. Signal amplification was also seen following incubation with both human and ovine tissue with significant inhibition in the presence of the pan- MMP inhibitor, marimastat. In conclusion, APF is an emerging condition of aged donkeys that shares key pathological and imaging features with human PPFE. Diagnosis of APF and other fibrotic lung conditions across species remains a challenge to veterinary and medical professionals. As such, optical imaging tools may provide dynamic, real time information on the presence and progression of fibroproliferation in the lung. TWB- 219 and SVC-186 produce a detectable increase in fluorescent signal at the 488nm wavelength when activated by LOXF and MMPs respectively. These probes have been shown to function in human ex vivo tissue as assessed by FCFM.
Supervisor: Haslett, Christopher ; McGorum, Bruce ; Dhaliwal, Kanwaldeep Sponsor: Medical Research Council (MRC)
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: pulmonary fibrosis ; fibrosis ; lung ; donkey ; optical ; imaging