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Background Idiopathic pulmonary fibrosis (IPF) is a devastating form of chronic lung injury of unknown aetiology characterised by progressive lung scarring. A diagnosis of definite IPF requires High Resolution Computed Tomography (HRCT) appearances indicative of usual interstitial pneumonia (UIP), or in patients with 'possible UIP' CT appearances, histological confirmation of UIP. However the proportion of such patients that undergo SLB varies, perhaps due to a perception of risk of biopsy and additive diagnostic value of biopsy in individual patients. We hypothesised that an underlying UIP pathological pattern may result in increased risk of death and aimed to explore this by comparing the risk of SLB in suspected idiopathic interstitial pneumonia, stratified according to HRCT appearance. Additionally we sought to determine the positive-predictive value of biopsy to diagnose IPF in patients with 'possible UIP HRCT' in our population. In patients with possible UIP who are not biopsied, the clinical value of bronchoalveolar lavage (BAL) is uncertain. We aimed to prospectively study the diagnostic and prognostic value of BAL differential cell count (DCC) in suspected IPF and determine the feasibility of repeat BAL and the relationship between DCC and disease progression in two successive BALs. We hypothesised that BAL DCC between definite and possible IPF was different and that baseline DCC and change in BAL DCC predicted disease progression. Alveolar macrophages (AMs) are an integral part of the lung's reparative mechanism following injury, however in IPF they contribute to pathogenesis by releasing pro-fibrotic mediators promoting fibroblast proliferation and collagen deposition. Expansion of novel subpopulations of pulmonary monocyte-like cells (PMLCs) has been reported in inflammatory lung disease. We hypothesised that a distinct AM polarisation phenotype would be associated with disease progression. We aimed to perform detailed phenotyping of AM and PMLCs in BAL in IPF patients. Several prognostic scoring systems and biomarkers have been described to predict disease progression in IPF but most were derived from clinical trial patients or tertiary referral centres and none have been validated in separate cohorts. We aimed to identify a predictive tool for disease progression utilising physiological, HRCT and serum biomarkers in a unique population of incident treatment naïve IPF patients. Methods Between 01/01/07 and 31/12/13, 611 consecutive incident patients with suspected idiopathic interstitial pneumonia (IIP) presented to the Edinburgh lung fibrosis clinic. Of these patients 222 underwent video-assisted thoracoscopic lung biopsy and histological pattern was determined according to ATS/ERS criteria. Post-operative mortality and complication rates were examined. Fewer than 2% received IPF-directed therapy and less than 1% of the cohort were lost to follow-up. Disease progression was defined as death or ≥10% decline in VC within 12 months of BAL. Cells were obtained by BAL and a panel of monoclonal antibodies; CD14, CD16, CD206, CD71, CD163, CD3, CD4, CD8 and HLA-DR were used to quantify and selectively characterise AMs, resident PMLCs, inducible PMLCs, neutrophils and CD4+/CD8+ T-cells using flow cytometry. Classical, intermediate and non-classical monocyte subsets were also quantified in peripheral blood. Potential biomarkers (n=16) were pre-selected from either previously published studies of IPF biomarkers or our hypothesis-driven profiling. Linear logistic regression was used on each predictor separately to assess its importance in terms of p-value of the associated weight, and the top two variables were used to learn a decision tree. Results Based on the 2011 ATS/ERS criteria, 87 patients were categorised as 'definite UIP', of whom 3 underwent SLB for clinical indications. IPF was confirmed in all 3 patients based on 2013 ATS/ERS/JRS/ALAT diagnostic criteria. 222 patients were diagnosed with 'possible UIP'; 55 underwent SLB, IPF was subsequently diagnosed in 37 patients, 4 were diagnosed with 'probable IPF' and 14 were considered 'not IPF'. In this group, 30 patients were aged 65 years or over and 25/30 (83%) had UIP on biopsy. 306 patients had HRCTs deemed 'inconsistent with UIP', SLB was performed in 168 patients. Post6 operative 30-day mortality was 2.2% overall, and 7.3% in the 'possible UIP' HRCT group. Patients with 'definite IPF' based on HRCT and SLB appearances had significantly better outcomes than patients with 'definite UIP' on HRCT alone (P=0.008, HR 0.44 (95% CI 0.240 to 0.812)). BAL DCC was not different between definite and possible UIP groups, but there were significant differences with the inconsistent with UIP group. In the 12 months following BAL, 33.3% (n=7/21) of patients in the definite UIP group and 29.5% (n=18/61) in the possible UIP group had progressed. There were no significant differences in BAL DCC between progressor and non-progressor groups. Mortality in patients with suspected IPF and a BAL DCC consistent with IPF was no different to those with a DCC inconsistent with IPF (P=0.425, HR 1.590 (95% CI 0.502 to 4.967)). There was no difference in disease progression in either group (P=0.885, HR 1.081 (95% CI 0.376 to 3.106)). There was no statistically significant difference in BAL DCC at 0 and 12 months in either group. There was no significant change in DCC between 0 and 12 month BALs between progressors and non-progressors. Repeat BAL was well tolerated in almost all patients. There was 1 death within 1 month of a first BAL and 1 death within 1 month of a second BAL; both were considered 'probably procedure-related'. AM CD163 and CD71 (transferrin receptor) expression were significantly different between groups (P < 0.0001), with significant increases in the IPF group vs non fibrotic ILD (P < 0.0001) and controls (P < 0.0001 and P < 0.001 respectively). CD71 expression was also significantly increased in the IPF progressor vs non-progressor group (P < 0.0001) and patients with high CD71 expression had significantly poorer survival than the CD71low group (P=0.040, median survival 40.5 and 75.6 months respectively). CD206 (mannose receptor) expression was also significantly higher in the IPF progressor vs non-progressor group (P=0.034). There were no differences in baseline BAL neutrophil, eosinophil or lymphocyte percentages between IPF progressor or non-progressor groups. The percentage of rPMLCs was significantly increased in BAL fluid cells of IPF patients compared to those with non-fibrotic ILD (P < 0.0001) and healthy controls (P < 0.05). Baseline rPMLC percentage was significantly higher in IPF progressors vs IPF non-progressors (P=0.011). Baseline BAL iPMLC:rPMLC ratio was also significantly different between IPF progressor and non-progressor groups (P=0.011). Disease progression was confidently predicted by a combination of clinical and serological variables. In our cohort we identified a predictive tool based on two key parameters, one a measure of lung function and one a single serum biomarker. Both parameters were entered into a decision tree, and when applied to our cohort yielded a sensitivity of 86.4%, specificity of 92.3%, positive predictive value of 90.5% and negative predictive value of 88.9%. We also applied previously reported predictive tools such as the GAP Index, du Bois score and CPI Index to the Edinburgh IPF cohort. Conclusions SLB can be of value in the diagnosis of ILD, however perhaps due to the perceived risks associated with the procedure, only a small percentage of patients undergo SLB despite recommendations that patients have histological confirmation of the diagnosis. Advanced age is a strong predictor for IPF, and in our cohort 83% of patients aged over 65 years with 'possible UIP' HRCT appearances, had UIP on biopsy. BAL and repeat BAL in IPF is feasible and safe (< 1.5% mortality). Of those that underwent repeat BAL, disease progression was not associated with a change in DCC. However, 22% of lavaged patients died or were deemed too frail to undergo a second procedure at 12 months. These data emphasise the importance of BAL in identifying a novel human AM polarisation phenotype in IPF. Our data suggests there is a distinct relationship between AM subtypes, cell-surface expression markers, PMLC subpopulations and disease progression in IPF. This may be utilised to investigate new targets for future therapeutic strategies. Disease progression in IPF can be predicted by a combination of clinical variables and serum biomarker profiling. We have identified a unique prediction model, when applied to our locally referred, incident, treatment naïve cohort can confidently predict disease progression in IPF. IPF is a heterogeneous disease and there is a definite clinical need to identify 'personalised' prognostic biomarkers which may in turn lead to novel targets and the advent of personalised medicines.

The objective of this study was to determine if R. equi IgG purified from commmercially available hyperimmune R. equi plasma and delivered to the lungs of adult ponies would cause a local inflammatory response and if increases in total and R. equi specific IgG occurred post administration. IgG was purified and concentrated from plasma via protein G affinity chromatography. A cross over study was performed. Eight healthy adult ponies were randomly assigned to two groups of four; each pony acted as its own control. Either the IgG product or 0.9% Saline was delivered via a vibrating mesh nebulizer during the first treatment phase. During the second treatment phase ponies recieved the oppostie treatment. A 4 week washout period was allowed between phases. Bronchoalveolar fluid was recovered using a low volume endoscopic technique prior to aerosolization (time 0), and at 1 hr, 6 hrs, and 24 hours post administration. The BAL fluid total IgG concentration and R. equi specific IgG titers were determined via ELISA and cytologic analysis was performed. No clinically significant local inflammatory response was identified in response to IgG treatment. While total IgG concentrations were increased at T1 compared to T0, no significant effects of time were found (P=0.19). However, overall significantly higher concentrations of total IgG were found after administration of saline when compared to IgG administration (P=0.023).  While the R. equi specific titer increased at T1 after IgG administration, no significant difference was identified between treatment or time (P=0.261). Overall the individual response to IgG was variable. It is possible that the protein rich IgG acted as a relatively hypertonic solution and caused fluid influx from the pulmonary parenchyma after treatment thereby diluting the total IgG present when compared to saline administration. This conclusion cannot be verified as BAL dilution correction was not performed. However, it is unknown what titer or level of increased IgG is nessecary to assist with prevention of disease. Future research should focus on the effect of R. equi specific IgG on pulmonary cells to determine if administration of local R. equi specific IgG would alter intrapulmonary immune responses to R. equi.

Background Cetuximab, an antibody targeting the epidermal growth factor receptor (EGFR), increases survival in patients with advanced EGFR-positive non-small cell lung cancer when administrated in combination with chemotherapy. In this study, we investigated the role of complement activation in the antitumor mechanism of this therapeutic drug. Results EGFR-expressing lung cancer cell lines were able to bind cetuximab and initiate complement activation by the classical pathway, irrespective of the mutational status of EGFR. This activation led to deposition of complement components and increase in complement-mediated cell death. The influence of complement activation on the activity of cetuximab in vivo was evaluated in xenografts of A549 lung cancer cells on nude mice. A549 cells express wild-type EGFR and have a KRAS mutation. Cetuximab activity against A549 xenografts was highly dependent on complement activation, since complement depletion completely abrogated the antitumor efficacy of cetuximab. Moreover, cetuximab activity was significantly higher on A549 cells in which a complement inhibitor, factor H, was genetically downregulated. Conclusions We demonstrate for the first time that the in vivo antitumor activity of cetuximab can be associated with a complement-mediated immune response. These results may have important implications for the development of new cetuximab-based therapeutic strategies and for the identification of markers that predict clinical response.

Unrecognised airway infection and inflammation may begin early in life in children with cystic fibrosis (CF). This may lead to lung function decline and irreversible lung disease before conventional investigations such as chest x-rays and spirometry become abnormal. Bronchoalveolar lavage (BAL), the current standard for assessing airway infection and inflammation in young children, is invasive, resource-intensive and not easily repeated. Sensitive, non-invasive measures to detect and monitor early CF lung disease are required. The main aim of this thesis was to determine whether the lung clearance index (LCI), a measure of ventilation inhomogeneity reflecting peripheral airway disease, the neutrophilic S100 proteins, S100A8/A9 (calprotectin) and S100A12 measured in serum and urinary desmosine, a measure of (lung) elastin breakdown, were sensitive, non-invasive markers of airway infection and inflammation in newborn-screened infants and young children with CF. A secondary aim was to determine whether distal gastro-oesophageal reflux and BAL pepsin were associated with early lung disease. The LCI was measured in sedated infants and young children with CF and healthy children of the same age using a multiple breath washout method and a portable, commercially available ultrasonic flowmeter. S100A8/A9 and S100A12 were measured by a commercially available and an “in-house” assay respectively. Urinary desmosine was measured by a modified high performance liquid chromatography technique and pepsin activity by fluorometric assay. This thesis has shown for the first time that the LCI is elevated in CF infants and young children especially in the presence of P. aeruginosa infection and airway inflammation. Further, the LCI was assessed to have good within and between test repeatability. The S100 proteins were elevated in BAL fluid and serum in the presence of infection and inflammation but serum levels were insufficiently sensitive to be useful non-invasive markers of early lung disease. Urinary desmosine was not elevated in clinically stable CF infants and young children. BAL Pepsin was associated with airway infection and inflammation. These findings suggest that the LCI but not the S100 proteins or urinary desmosine are useful, sensitive markers of early disease in minimally symptomatic young children with CF and that microaspiration may contribute to airway inflammation.

Multiple biomarkers of oxidative stress have been measured and used in human medicine to diagnose and monitor airway disease. The purpose of the study was to determine if similar relationships existed between inflammatory and oxidative stress biomarkers in exhaled breath condensate (EBC), bronchoalveolar lavage fluid (BALF), red blood cells, white blood cells, and plasma; and cytokine expression in airway inflammatory cells and mucosal biopsies of RAO-affected horses and their controls. Sixteen horses in pairs were used: 8 non-RAO-affected (controls) and 8 RAO-affected horses. Samples from all horses were collected at remission (S1), during environmental challenge (S2) and at recovery (S3). RAO-affected horses had significant alterations in cellular glutathione peroxidase (cGPx) activity, ascorbic acid and pH in a number of biological samples. Concentrations of 8-isoprostanes, isofurans, amino acids and mRNA expression of interleukin 4 (IL4), gamma interferon (INFγ), inducible nitric oxide synthase (iNOS), extracellular glutathione peroxidase (GPx-3), and cytosolic superoxide dismutase (SOD-1) were not significantly different or were at the limits of detection. Conductivity was measured and assessed as a potential correctional factor for respiratory fluid dilution. The alterations in biomarker concentrations demonstrate that oxidative stress is an important component of airway inflammation in RAO-affected horses. Further research is warranted in the use of biomarkers and the effects of dietary interventions.