Non-invasive monitoring of changes in exhaled markers of airway inflammation in Thoroughbred racehorses

Exhaled breath (EB) and exhaled breath condensate (EBC) contain numerous volatile gases and a wide-array of non-volatile compounds, several of which have been investigated as markers of lower airway inflammation in human and veterinary medicine and have been used to diagnose and monitor diseases associated with pulmonary inflammation. The identification of reliable biomarkers within EB and EBC is an active research focus with the common goal of establishing non-invasive and repeatable assessment of respiratory health and disease in mammals. The application of EB and EBC analysis holds considerable appeal in the investigation of respiratory disease in Thoroughbred racehorses, as inflammatory airway disease (IAD) is a common cause for poor performance in this population of animals. This study documented that EB and EBC samples can be safely collected from Thoroughbred racehorses in their own environment, without adverse effect or interference with the horse’s training regimen. The use of off-line collection and analysis of exhaled gases via chemiluminescence is suitable for the measurement of exhaled carbon monoxide, but is not appropriate for analyzing exhaled nitric oxide in horses. Significant changes in the concentration of exhaled CO and the pH of EBC occurred in response to strenuous exercise and when exercising in different environmental temperatures. Exhaled CO was associated with tracheal mucus score (and the number of neutrophils in the mucus) and EBC pH was significantly different in horses with evidence of neutrophilic IAD compared to horses without IAD. Numerous physiological and environmental variables were identified as confounding factors in the assessment of both exhaled CO and EBC pH, with respiratory rate prior to EB collection, and during EBC collection, consistently identified as an explanatory variable influencing the concentration of exhaled biomarkers. Further studies in EB and EBC analysis in horses need to focus on objectively accounting for key respiratory dynamics during sample collection.

Oxidative stress and its haemodynamic consequences in chronic kidney disease

Chronic kidney disease (CKD) is associated with increased cardiovascular risk in comparison with the general population. This can be observed even in the early stages of CKD, and rises in proportion to the degree of renal impairment. Not only is cardiovascular disease (CVD) more prevalent in CKD, but its nature differs too, with an excess of morbidity and mortality associated with congestive cardiac failure, arrhythmia and sudden death, as well as the accelerated atherosclerosis which is also observed. Conventional cardiovascular risk factors such as hypertension, dyslipidaemia, obesity, glycaemia and smoking, are highly prevalent amongst patients with CKD, although in many of these examples the interaction between risk factor and disease differs from that which exists in normal renal function. Nevertheless, the extent of CVD cannot be fully explained by these conventional risk factors, and non-conventional factors specific to CKD are now recognised to contribute to the burden of CVD. Oxidative stress is a state characterised by excessive production of reactive oxygen species (ROS) and other radical species, a reduction in the capacity of antioxidant systems, and disturbance in normal redox homeostasis with depletion of protective vascular signalling molecules such as nitric oxide (NO). This results in oxidative damage to macromolecules such as lipids, proteins and DNA which can alter their functionality. Moreover, many enzymes are sensitive to redox regulation such that oxidative modification to cysteine thiol groups results in activation of signalling cascades which result in adverse cardiovascular effects such as vascular and endothelial dysfunction. Endothelial dysfunction and oxidative stress are present in association with many conventional cardiovascular risk factors, and can be observed even prior to the development of overt, clinical, vascular pathology, suggesting that these phenomena represent the earliest stages of CVD. In the presence of CKD, there is increased ROS production due to upregulated NADPH oxidase (NOX), increase in a circulating asymmetric dimethylarginine (ADMA), uncoupling of endothelial nitric oxide synthase (eNOS) as well as other mechanisms. There is also depletion in exogenous antioxidants such as ascorbic acid and tocopherol, and a reduction in activity of endogenous antioxidant systems regulated by the master gene regulator Nrf-2. In previous studies, circulating markers of oxidative stress have been shown to be increased in CKD, together with a reduction in endothelial function in a stepwise fashion relating to the severity of renal impairment. Not only is CVD linked to oxidative stress, but the progression of CKD itself is also in part dependent on redox sensitive mechanisms. For example, administration of the ROS scavenger tempol attenuates renal injury and reduces renal fibrosis seen on biopsy in a mouse model of CKD, whilst conversely, supplementation with the NOS inhibitor L-NAME causes proteinuria and renal impairment. Previous human studies examining the effect of antioxidant administration on vascular and renal function have been conflicting however. The work contained in this thesis therefore examines the effect of antioxidant administration on vascular and endothelial function in CKD. Firstly, 30 patients with CKD stages 3 – 5, and 20 matched hypertensive controls were recruited. Participants with CKD had lower ascorbic acid, higher TAP and ADMA, together with higher augmentation index and pulse wave velocity. There was no difference in baseline flow mediated dilatation (FMD) between groups. Intravenous ascorbic acid increased TAP and O2-, and reduced central BP and augmentation index in both groups, and lowered ADMA in the CKD group only. No effect on FMD was observed. The effects of ascorbic acid on kidney function was then investigated, however this was hindered by the inherent drawbacks of existing methods of non-invasively measuring kidney function. Arterial spin labelling MRI is an emerging imaging technique which allows measurement of renal perfusion without administration of an exogenous contrast agent. The technique relies upon application of an inversion pulse to blood within the vasculature proximal to the kidneys, which magnetically labels protons allowing measurement upon transit to the kidney. At the outset of this project local experience using ASL MRI was limited and there ensued a prolonged pre-clinical phase of testing with the aim of optimising imaging strategy. A study was then designed to investigate the repeatability of ASL MRI in a group of 12 healthy volunteers with normal renal function. The measured T1 longitudinal relaxation times and ASL MRI perfusion values were in keeping with those found in the literature; T1 time was 1376 ms in the cortex and 1491 ms in the whole kidney ROI, whilst perfusion was 321 mL/min/100g in the cortex, and 228 mL/min/100g in the whole kidney ROI. There was good reproducibility demonstrated on Bland Altman analysis, with a CVws was 9.2% for cortical perfusion and 7.1% for whole kidney perfusion. Subsequently, in a study of 17 patients with CKD and 24 healthy volunteers, the effects of ascorbic acid on renal perfusion was investigated. Although no change in renal perfusion was found following ascorbic acid, it was found that ASL MRI demonstrated significant differences between those with normal renal function and participants with CKD stages 3 – 5, with increased cortical and whole kidney T1, and reduced cortical and whole kidney perfusion. Interestingly, absolute perfusion showed a weak but significant correlation with progression of kidney disease over the preceding year. Ascorbic acid was therefore shown to have a significant effect on vascular biology both in CKD and in those with normal renal function, and to reduce ADMA only in patients with CKD. ASL MRI has shown promise as a non-invasive investigation of renal function and as a biomarker to identify individuals at high risk of progressive renal impairment.

Utility and safety of invasive (fractional flow reserve) and non-invasive (cardiac magnetic resonance imaging) diagnostic tests in patients with NSTEMI

A prospective randomised controlled clinical trial of treatment decisions informed by invasive functional testing of coronary artery disease severity compared with standard angiography-guided management was implemented in 350 patients with a recent non-ST elevation myocardial infarction (NSTEMI) admitted to 6 hospitals in the National Health Service. The main aims of this study were to examine the utility of both invasive fractional flow reserve (FFR) and non-invasive cardiac magnetic resonance imaging (MRI) amongst patients with a recent diagnosis of NSTEMI. In summary, the findings of this thesis are: (1) the use of FFR combined with intravenous adenosine was feasible and safe amongst patients with NSTEMI and has clinical utility; (2) there was discordance between the visual, angiographic estimation of lesion significance and FFR; (3). The use of FFR led to changes in treatment strategy and an increase in prescription of medical therapy in the short term compared with an angiographically guided strategy; (4) in the incidence of major adverse cardiac events (MACE) at 12 months follow up was similar in the two groups. Cardiac MRI was used in a subset of patients enrolled in two hospitals in the West of Scotland. T1 and T2 mapping methods were used to delineate territories of acute myocardial injury. T1 and T2 mapping were superior when compared with conventional T2-weighted dark blood imaging for estimation of the ischaemic area-at-risk (AAR) with less artifact in NSTEMI. There was poor correlation between the angiographic AAR and MRI methods of AAR estimation in patients with NSTEMI. FFR had a high accuracy at predicting inducible perfusion defects demonstrated on stress perfusion MRI. This thesis describes the largest randomized trial published to date specifically looking at the clinical utility of FFR in the NSTEMI population. We have provided evidence of the diagnostic and clinical utility of FFR in this group of patients and provide evidence to inform larger studies. This thesis also describes the largest ever MRI cohort, including with myocardial stress perfusion assessments, specifically looking at the NSTEMI population. We have demonstrated the diagnostic accuracy of FFR to predict reversible ischaemia as referenced to a non-invasive gold standard with MRI. This thesis has also shown the futility of using dark blood oedema imaging amongst all comer NSTEMI patients when compared to novel T1 and T2 mapping methods.

The impact of denture related disease on the oral microbiome of denture wearers

Advances in healthcare over the last 100 years has resulted in an ever increasing elderly population. This presents greater challenges for adequate systemic and oral healthcare delivery. With increasing age there is a natural decline in oral health, leading to the loss of teeth and ultimately for some having to wear denture prosthesis. It is currently estimated that approximately one fifth of the UK and US populations have some form of removable prosthesis. The microbiology of denture induced mucosal inflammation is a pivotal factor to consider in denture care management, similar to many other oral diseases of microbial influence, such as caries, gingivitis and periodontitis. Dentures support the growth of microbial biofilms, structures commonly known as denture plaque. Microbiologically, denture stomatitis (DS) is a disease primarily considered to be of yeast aetiology, with the literature disproportionately focussed on Candida spp. However, the denture surface is capable of carrying up to 1011 microbes per milligram, the majority of which are bacteria. Thus it is apparent that denture plaque is more diverse than we assume. There is a fundamental gap in our understanding of the bacterial composition of denture plaque and the role that they may play in denture related disease such as DS. This is categorised as inflammation of the oral mucosa, a disease affecting around half of all denture wearers. It has been proposed that bacteria and fungi interact on the denture surface and that these polymicrobial interactions lead to synergism and increased DS pathogenesis. Therefore, understanding the denture microbiome composition is the key step to beginning to understand disease pathogenesis, and ultimately help improve treatments and identify novel targets for therapeutic and preventative strategies. A group of 131 patients were included within this study in which they provided samples from their dentures, palatal mucosa, saliva and dental plaque. Microbes residing on the denture surface were quantified using standard Miles and Misra culture technique which investigated the presence of Candida, aerobes and anaerobes. These clinical samples also underwent next generation sequencing using the Miseq Illumina platform to give a more global representation of the microbes present at each of these sites in the oral cavity of these denture wearers. This data was then used to compare the composition and diversity of denture, mucosal and dental plaque between one another, as well as between healthy and diseased individuals. Additional comparisons included denture type and the presence or absence of natural teeth. Furthermore, microbiome data was used to assess differences between patients with varying levels of oral hygiene. The host response to the denture microbiome was investigated by screening the patients saliva for the presence and quantification of a range of antimicrobial peptides that are associated with the oral cavity. Based on the microbiome data an in vitro biofilm model was developed that reflected the composition of denture plaque. These biofilms were then used to assess quantitative and compositional changes over time and in response to denture cleansing treatments. Finally, the systemic implications of denture plaque were assessed by screening denture plaque samples for the presence of nine well known respiratory pathogens using quantitative PCR. The results from this study have shown that the bacterial microbiome composition of denture wearers is not consistent throughout the mouth and varies depending on sample site. Moreover, the presence of natural dentition has a significant impact on the microbiome composition. As for healthy and diseased patients the data suggests that compositional changes responsible for disease progression are occurring at the mucosa, and that dentures may in fact be a reservoir for these microbes. In terms of denture hygiene practices, sleeping with a denture in situ was found to be a common occurrence. Furthermore, significant shifts in denture microbiome composition were found in these individuals when compared to the denture microbiome of those that removed their denture at night. As for the host response, some antimicrobial peptides were found to be significantly reduced in the absence of natural dentition, indicating that the oral immune response is gradually impaired with the loss of teeth. This study also identified potentially serious systemic implications in terms of respiratory infection, as 64.6% of patients carried respiratory pathogens on their denture. In conclusion, this is the first study to provide a detailed understanding of the oral microbiome of denture wearers, and has provided evidence that DS development is more complex than simply a candidal infection. Both fungal and bacterial kingdoms clearly play a role in defining the progression of DS. The biofilm model created in this study demonstrated its potential as a platform to test novel actives. Future use of this model will aid in greater understanding of host: biofilm interactions. Such findings are applicable to oral health and beyond, and may help to identify novel therapeutic targets for the treatment of DS and other biofilm associated diseases.