Comparison of bronchosorption with bronchoalveolar lavage

As with nasal sampling, IL-6 serves as a good cytokine to compare the two bronchial techniques as it was measurable in all study subjects at baseline. The Th1 chemokine CXCL10/IP-10 was also measured at levels above the lower limit of detection in all subjects using both sampling techniques.

Correlations between BAL levels and bronchosorption levels for these cytokines revealed close correlations (IL-6, r=0.65, P< 0.001; CXCL10/IP-10, r=0.47, P=0.004). The median level of IL-6 in BAL

82 was 5.4 pg/mL compared to 38.5 pg/mL in bronchosorption, whilst the median level of CXCL10/IP-10 in BAL was 144 pg/mL compared to 1014 pg/mL in bronchosorption. In both cases this is equivalent to a 7-8 fold increase in concentration over what can be measured in BAL. When dealing with cytokines near the lower limits of detection with BAL, the benefits of bronchosorption can be readily appreciated.

Figure 3.4 Comparison of lower airway sampling techniques. Levels of IL-6 and CXCL10/IP-10 in bronchial mucosal lining fluid measured by bronchosorption are significantly greater than bronchoalveolar lavage

83 3.8 Discussion

One of the main novel aspects of this study is the inclusion of a new sampling technique to measure secreted mediators in airway mucosal lining fluid, and in so doing provide an opportunity to measure previously undetectable inflammatory mediators and advance our understanding of disease mechanisms.

However, despite the successes of nasosorption in the three previously published studies^201–203, it appears the use of Accuwick for sampling nasal mucosal lining fluid has never been formally assessed or properly validated. At the time of writing, the results presented in this chapter represent the first such assessment of using these matrices for this purpose.

In view of the fact that Accuwick is no longer being manufactured, it has also been possible to identify an alternative sampling matrix in Leukosorb that shows equivalence in protein binding and release characteristics to Accuwick and is of an appropriate strength for safe sampling of the bronchial mucosa. Furthermore it has been possible to demonstrate that protein recovery can be greatly improved by the addition of a buffer containing both protein (1% BSA) and detergent (1%

Triton) prior to elution, a finding that will greatly aid future projects using these sampling techniques. The additional finding that protein recovery is not significantly affected by the matrix drying out prior to application of the buffer is also a useful finding as there can frequently be a delay between taking a human sample and it arriving in the lab for initial processing.

The finding that some cytokines are more easily recovered than others was unexpected and cannot easily be explained. In view of the near complete recovery of IL-1β and the demonstration of good reproducibility (irrespective of the percentage recovery) it does not appear to be secondary to a sampling error. It also appears not to be related to the molecular weights of the cytokines as both IL-1β and IFN-α are 17KDa yet their initial recoveries on Accuwick varied greatly from one another.

Even TNF-α at 57KDa is still small enough to have easily passed through the spin filter with ease, and

84 the subsequent experiments performed to assess retention by the cellulose acetate filter present in the polypropylene tubes excluded this as a possible cause definitively. However, it is possible that the differences relate to the charge of the proteins as some of the cytokines are known to be more charged than others and therefore may bond to the matrix more robustly. Further work and additional experiments are clearly needed to better understand the reasons for this and assess how other cytokines differ in their protein binding / release properties.

Nasosorption has been shown for the first time to be a reproducible technique that correlates well with nasal lavage but is able to consistently and reliably measure greatly increased concentrations of mediators. This technique can therefore offer insights into pathways that have not been amenable to investigation previously. Moreover, it is well-tolerated and easy to perform which makes it suitable for sampling both children and adults. Similarly, the development of the bronchosorption sampling device is a significant advance for respiratory medicine. It correlates well with BAL and should permit previously undetectable proteins to be accurately measured across a wide range of airway diseases providing valuable insights into mechanisms of disease.

Over the course of this study approximately 100 bronchoscopies and 200 bronchosorptions have been performed. The technique is well-tolerated, quick to complete, does not appear to have the potential to cause bronchospasm in the same way as bronchoalveolar lavage is recognised to, and does not appear to be associated with the fever that sometimes follows lavage. The theoretical danger of SAM detachment whilst sampling never materialised despite often vigorous coughing on the part of exacerbating asthmatics. It seems unlikely that this technique will not become in widespread use in both the research and healthcare setting.

85 Chapter 4

Results chapter 2

Clinical and Virological Outcomes of Experimental Rhinovirus Infection in Asthma

4.1 Introduction

The human model of experimental rhinovirus infection permits the study of a rhinovirus infection under controlled conditions, allows comparison of symptoms, airway physiology, lung function, airway inflammation, and use of asthma medication to name but a few. Its evolution to include the study of asthma has led to a significant increase in our understanding of the processes that underlie virus-induced exacerbations and offers a model with which new medications can be tested. To date, this model has demonstrated that mild asthmatics experience increased rhinovirus-associated morbidity compared to healthy subjects, with increased lower respiratory symptoms of a mild exacerbation, falls in lung function, and airway hyperresponsiveness.⁶⁸ However the model has been limited by the inclusion of only mild asthmatics, adequately-controlled at study entry. This has meant that the findings are not directly relevant to the group of asthmatics that new asthma therapies are most often targeted to. Another consequence has been that the influence of baseline asthma severity and asthma control on the outcome of rhinovirus infections in asthma has remained unknown. This is in the context of an increased appreciation in recent years of the link between 'current control' and 'future risk' in asthma - the idea that good current asthma control can reduce the future risk of adverse outcomes.²²⁷ Reference to respiratory virus infections with regard to this literature has been completely absent, despite their role as the dominant trigger for exacerbations.

Secondly, to date the current model of experimental infection has used a high-dose virus inoculum (1000 to 30,000 TCID50) to inoculate study subjects with. However this may not truly reflect the onset of a naturally-occurring infection, which would start with a low dose natural inoculation,

86 followed (if immune responses do not succeed in preventing) by replication of virus to high virus loads, leading to consequent expression of symptoms and signs.

The aim of the work presented in this chapter has therefore been to firstly test the hypothesis that rhinovirus infection leads to greater morbidity in moderately-severe and inadequately-controlled asthma compared to subjects with milder and well-controlled disease; and secondly to test the hypothesis that a low-dose virus-inoculum (100 TCID50) is as effective in inducing clinical infection in asthmatic and healthy subjects as the higher doses used in previous studies.