Introduction Airway remodelling describes the histopathological changes in tissue architecture observed in obstructive lung diseases such as asthma and may have a negative impact on lung function. These changes do not appear to be treated by current asthma treatments. Changes observed during airway remodelling include increased thickness of airway smooth muscle (ASM) layer and enhanced extracellular matrix (ECM) deposition. Matrix metalloproteinases (MMPs) are a family of proteolytic enzymes, which facilitate tissue remodelling via ECM protein degradation. Matrix metalloproteinase-1 (MMP-1) and mast cells are present in the airways of patients with asthma (but not in healthy people). MMPs expression is highly regulated in lungs and is increased in disease states. My project aimed to assess MMP-1, -2 and -9 expression and activity in asthma airways. Furthermore, the underlying mechanism of MMP-1 activation and subsequently its role in airway remodelling and worsening asthma severity was investigated in the context of asthma exacerbation, which is thought to be an exaggerated lower airway inflammatory response to an environmental exposure such as respiratory virus infection. Methods Patients with stable asthma and healthy controls underwent spirometry, methacholine airway (PC20 ) challenge, exhaled nitric oxide (FeNO) test, bronchoscopy/bronchial washings and primary airway smooth muscle (ASM) cell cultures. A second asthma group (mild to moderate severity) and controls had symptom scores, spirometry and bronchoalveolar lavage (BAL) before and after rhinovirus inoculation. ECM was prepared from decellularised primary ASM cultures. MMP-1 protein levels and activity were assessed in bronchial fluid samples by enzyme-linked immunosorbent assay (ELISA), western blotting and fluorescent activity assay. ASM cell growth was measured by MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) reduction assay and cell counts. Bronchial fluid gelatinase (MMP-2 and -9) expression and activity was assessed by gelatin zymography. Results MMP-1 and MMP-9 expression was enhanced in both stable asthma and during asthma exacerbations, whilst MMP-2 expression was only increased during asthma exacerbations. MMP-1 can be activated by tryptase, which is an inflammatory product of mast cell degranulation. Activated (degranulated) mast cells enhanced proliferation of both control and asthma ASM cells via the production of a pro-proliferative ECM in vitro and the proliferative effect was dependent on MMP-1. In patients with asthma, mast cells numbers within ASM bundles were associated with ASM growth. MMP-1 protein levels were related to bronchial reactivity and MMP-1 activity increased during asthma exacerbations, where its levels were related to exacerbation severity. Conclusion This study suggests that MMP-1 plays an important role in asthma pathophysiology and that ASM/mast cell interactions contribute to asthma severity by transiently increasing MMP-1 activation, ASM growth and airway responsiveness. Moreover, there is increased expression of MMP-2 and -9 during asthma exacerbations compared with stable asthma. As both MMP-2 and -9 act as mediators of inflammation (Okada, S. et al., 1997) (Elkington, P.T.G., 2006) and tissue remodelling (Oshita, Y. et al., 2003), an increase in gelatinolytic activity linked to MMP-2 and MMP-9 is also likely to play a significant role in the pathophysiology of asthma exacerbations.

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No description available.

1. The physiology of mammalian respiration and the pharmacology of the potent, synthetic opioids, the fentanyls, are reviewed. The current knowledge of the opioid receptor sub-type involvements in opioid-induced respiratory depression is summarised, together with the experimental methods which have previously been employed in studies of receptor involvement. 2. The comparative binding characteristics of the mu opioid receptor selective ligand [3H]-[D-Ala2-MePhe4-Glyol5] enkephalin ([3H]- DAGO) and the delta receptor ligand [3H]-[D-Pen2,D-Pen5] enkephalin ([3H]-DPDPE) were studied in homogenates of both whole brain and of pons/medulla regions from the rat. The receptor affinities and site-selectivities of five drugs of the fentanyl series (alfentanil, carfentanil, fentanyl, lofentanil and sufentanil) were determined by inhibition studies, using [3H]-DAGO and [3H]-DPDPE as markers of the mu and delta opioid binding sites, respectively. The concentration of delta opioid sites in pons/medulla was found to be approximately one third that of mu sites. The concentrations of both mu and delta sites in whole brain were similar to that of ? sites in pons/medulla. The rank order of affinities of the unlabelled drugs was dissimilar at the mu and delta sites. The most potent fentanyl derivatives exhibited negligible preference for the mu or delta sites, in contrast to the least potent compound, alfentanil, which showed an extremely high mu-site selectivity. 3. The respiratory depressant properties of the fentanyls were investigated in urethane-anaesthetised rats. Ventilatory parameters were measured using a volumetric pressure transducer connected via a Fleisch tube to a tracheal cannula. Intravenous administration of the fentanyls produced an apnea of immediate onset and dose-related duration, in addition to depressing both tidal volume and respiratory frequency, and thus minute volume, in a dose-related manner. The potency ratios of the fentanyls to produce apnea and to depress minute volume were dissimilar. Studies of the effects of alfentanil on arterial blood gases confirmed the reduction in minute volume to be a respiratory depression. 4. A method was developed for the quantification of antagonism of the respiratory effects of opioids by means of infusion of the opioid antagonist, naloxone, to predicted steady-state blood concentrations, which provided accurate measures of pA2. The basis of the technique rests upon determination of the plasma clearance of the antagonist whence appropriate loading doses and zero order infusion rate constants were calculated. The predicted concentrations of naloxone were verified by direct serum measurements using HPLC. 5. Naloxone pA2 values for antagonism of the apnea produced by the fentanyls were identical and indicated sole mediation by the mu opioid receptor, which was confirmed by intravenous administration of receptor-selective opioids. Apnea was produced by DAGO but not by DPDPE or U69,593. This component of the response was shown to be peripherally-mediated and vagally dependent, being abolished by bilateral vagotomy and the quaternary opioid antagonist, N-methyl levallorphan. 6. Naloxone pA2 values for antagonism of the minute volume depressant effects of the fentanyls showed significant differences through the series, implying both a mu and non-mu receptor involvement in this centrally-mediated response, which could still be elicited after vagotomy. A k receptor contribution to respiratory depression was eliminated by investigation of the respiratory effects of U69,593 and U50,488H. The involvement of the delta receptor was suggested by the finding that intracerebroventricular administration of DPDPE produced respiratory depression which was blocked by high doses of naloxone. 7. A study of the cardiovascular effects of the fentanyls confirmed that the different naloxone pA2 values for depression of respiration could not be attributed to this influence. 8. In conclusion, the binding site selectivities of the drugs of the fentanyl series varied inversely with their affinities at the mu and delta sites. In vivo studies have demonstrated that mu receptors alone in the periphery mediate the apneic responses to intravenously administered opioids. Whilst mu receptors in the brain are the most important in the mediation of the minute volume depressant effects of the fentanyls another receptor, possibly delta, contributes to this response. This non-mu contribution is different for each of the fentanyl drugs, broadly in accord with their binding site selectivity exhibited in vitro.

Genome-wide association studies of human lung function and Chronic Obstructive Pulmonary Disease have identified a highly significant and reproducible signal on 4q24. It remains unclear which of the two candidate genes within this locus may regulate lung function: GSTCD, a gene with unknown function, and/or INTS12, a member of the Integrator Complex which is currently thought to mediate 3’end processing of small nuclear RNAs. An interrogation of bioinformatic datasets showed that in lung tissue, 4q24 polymorphisms associated with lung function correlate with INTS12 but not neighboring GSTCD expression. In contrast to the previous reports in other species, a minor alteration of small nuclear RNA processing was observed following INTS12 depletion. RNA sequencing analysis of knockdown cells instead revealed dysregulation of a core subset of genes relevant to airway biology and a robust downregulation of protein synthesis pathways. Consistent with this, protein translation was decreased in INTS12 knockdown cells. In addition, chromatin immunoprecipitation and sequencing experiments demonstrated INTS12 binding throughout the genome, which was enriched in transcriptionally active regions. Finally, INTS12 regulome was defined which includes genes belonging to the protein synthesis pathways. INTS12 has functions beyond the canonical snRNA processing and evidence is presented showing that it regulates translation by directly controlling the expression of genes belonging to protein synthesis pathways. This thesis provides a detailed analysis of INTS12 activities on a genome-wide scale and contributes to the understanding of biology behind the genetic association for lung function at the 4q24.

Background Evidence from the UK suggests the incidence of idiopathic pulmonary fibrosis is increasing, but there is a lack of data from elsewhere in the World. The cause of the disease remains unknown. New anti-fibrotic therapies may increase the use of surgical lung biopsy for accurate diagnosis, although the risks of this (and other surgery) are not clear. Methods Collated international mortality statistics and a systematic review of the literature were used to assess the incidence and mortality of idiopathic pulmonary fibrosis worldwide. Primary care data from the United Kingdom were used to assess the association between recent major surgery and a new diagnosis of idiopathic pulmonary fibrosis. Secondary care data from the United States and United Kingdom were used to assess the risk of surgical lung biopsy for the diagnosis of interstitial lung disease, and the risk of other major surgery in those with idiopathic pulmonary fibrosis. Results Mortality from idiopathic pulmonary fibrosis in increasing steadily worldwide. Incidence varies worldwide but is in the range of 3-9 per 100,000 in the West. No association was identified between recent major surgery and a new diagnosis of idiopathic pulmonary fibrosis. Surgical lung biopsy for the diagnosis of interstitial lung disease has an in-hospital mortality of under 2% for elective procedures, but this is higher for non-elective surgery, and in those who are older with co-morbidities. In those with idiopathic pulmonary fibrosis undergoing major surgery, in-hospital mortality was higher than the general population. Conclusion Idiopathic pulmonary fibrosis seems to be increasingly common worldwide. Surgery has risks, particularly in unwell older patients, and less invasive diagnostic methods are needed.

Rationale Chronic obstructive pulmonary disease (COPD) is a progressive lung condition with extrapulmonary manifestations- cardiovascular diseases (CVD), impaired physical function, activity and increased frailty. Integrating measures of function into community assessments is hindered by the space and time required. The association of function, activity and CVD has not been extensively investigated in COPD. Objectives Explore the potential utility of Time Up and Go (TUG) as a measure of physical function in COPD Assess association of non-invasive measures of haemodynamics to physical function and self-reported activity Explore ambulatory haemodynamics in COPD and controls Methods Subjects with COPD (n=119) and controls (n=58) were recruited. Ethical and governance approvals were obtained. A medical history including falls, spirometry, peripheral and central haemodynamics, self-reported physical activity questionnaires and functional assessments (TUG and six-minute walk distance (6MWD)) were obtained from all subjects. Ambulatory 24-hour haemodynamics including aortic pulse wave velocity (aPWV) and blood pressure were measured in patients (n=20) and controls (n=19). Results TUG mean(SD) was increased in patients 11.9(3.7)s compared to controls 9.5(1.8)s, p < 0.001. In patients, fallers had longer TUG than non-fallers (p=0.02) and a cut-off time of 12s had the highest sensitivity and specificity to detect fallers and non-fallers. Aortic stiffness was not associated to physical function or physical activity, p > 0.05. In the pilot study, significant nocturnal dip in aPWV was seen in controls, p < 0.01, but not in patients, p=0.07. Conclusion TUG could be a useful measure of function and possibly be incorporated into COPD assessment, particularly where time and space are limited. Finally, ambulatory haemodynamic machine, the Mobil-O-Graph, is feasible and offers opportunity to assess 24-hour haemodynamics profile including aPWV as opposed to a one-off measurement.

Abstract available in the Journal of Investigative Medicine.

This study was performed to compare the delivery of the antimicrobial gentamicin to the respiratory tract of adult horses following aerosol and intravenous administration. Nine adult horses were used in a crossover design. Aerosol administration of gentamicin was performed using a close fitting facemask and an ultrasonic nebulizer. Intravenous gentamicin was administered via a jugular venous catheter. Samples of pulmonary epithelial lining fluid were collected by bronchial lavage performed at 0.5, 4, 8 and 24 hours after gentamicin administration. All samples were analyzed for gentamicin concentration, and cytologic examination was performed on aliquots of bronchial lavage fluid from times 0.5, 8 and 24 hours. Comparisons were made using the Wilcoxon signed-rank test. The bronchial lavage fluid gentamicin concentration after aerosol administration was significantly greater (p<0.05) than after intravenous administration at 0.5, 4, and 8 hours. The bronchial lavage fluid total nucleated cell count increased significantly (p<0.05) from 0.5 to 24 hours following both routes of gentamicin administration, with the increase observed following aerosol administration being significantly greater (p<0.05) than that observed following intravenous administration. A significant increase in neutrophil count was detected between bronchial lavage fluid samples taken at 0.5 hours and 24 hours, regardless of route of gentamicin administration. We conclude that aerosol administration of gentamicin to the equine respiratory tract achieves bronchial lavage fluid gentamicin levels that are significantly higher than levels obtained following intravenous administration for at least the first 8 hours after administration, while inciting a mild inflammatory response. / Master of Science

archives@tulane.edu / The overall goal of this dissertation is to study the effects of environmental exposures on respiratory health and determine if there are more complex relationships between simultaneous exposures that lead to more severe morbidity. Three specific objectives over three manuscripts are proposed to achieve this goal. The first manuscript focuses on assessing the impact of pesticide exposure (i.e., pesticide metabolites in urine) on asthma outcomes in children residing in low-income urban housing. The second manuscript focuses on assessing the dual impact of cockroach allergen exposure and pesticide spray use on asthma morbidity. Finally, the third manuscript utilizes publicly available data from the National Health and Nutrition Examination Survey (NHANES) to conduct an Environment Wide Association Study (EWAS) to assess the factors associated with a marker of eosinophilic inflammation. The proposed dissertation has the potential to increase the understanding of the effect of pesticides on asthma morbidity in a highly vulnerable group and to assess factors associated with eosinophilic inflammation in the general population. / 1 / Derek Werthmann