Resistencia calefactora

+P<0.05; ++P<0.01.

b a

93

Figure 4.4 Peak and total upper respiratory symptom scores according to baseline asthma control. +P<0.05; ++P<0.01.

a

b

94 4.6 Lower Respiratory Symptom Scores

Asthmatics experienced substantially greater lower respiratory symptom scores compared to healthy subjects following inoculation with RV-16. Compared to healthy subjects, daily lower respiratory symptom scores were significantly greater on days 3-6 for the mild asthmatics and on days 3-13 for the moderate asthmatics (all P<0.05) (figure 4.5a). In addition, symptom scores were significantly greater for the moderate asthmatics compared to the mild asthmatics on days 8, 10, 11 and 13 highlighting a more prolonged, severe exacerbation in these subjects (all P<0.05).

Defined instead by asthma control category, the poorly-controlled sub-group experienced increased daily lower respiratory symptoms following inoculation compared to the well- and partially-controlled asthmatics, with an earlier symptom peak (day 4) and more sustained duration of symptoms (days 7-13) (figure 4.5b). Indeed the peak symptom score for poorly-controlled subjects was significantly greater than observed in asthmatics with better baseline control (all P<0.05). The difference in peak lower respiratory symptom score was not statistically significant between moderate and mild asthmatics (figure 4.6).

In contrast, total lower respiratory symptoms scores (summation of daily scores over the 14 day period post inoculation) were significantly greater in moderate asthmatics than in both healthy subjects (45.2±6.2 vs 1.45±3.5, P<0.001) and mild asthmatic subjects (45.2±6.2 vs 20.3±4.9, P=0.004) and in mild asthma vs healthy subjects (20.3±4.9 vs 1.45±3.5, P=0.005) (figure 4.7a).

Total lower respiratory scores in the poorly-controlled sub-group (59.8±8.6) were almost three times the scores of the well-controlled (21.3±5.5, P=0.001), and almost twice those of the partially-controlled (32.5±6.3, P=0.02) groups (figure 4.7b). It is also noteworthy that the mean total lower respiratory score for poorly-controlled asthmatics was substantially higher than for the larger moderate asthmatic group (from which they were also part) suggesting that these subjects were at the most severe end of the moderate group.

95

Figure 4.5 Daily lower respiratory symptom scores in healthy and asthmatic subjects defined by baseline asthma severity (a) and baseline asthma control category (b).

Panel a: *P<0.05;**P<0.01;***P<0.001 asthma v’s healthy subjects; ⁺P<0.05 moderate v’s mild asthma. Panel b: †P<0.05, ††P<0.01 poorly-controlled v’s well-controlled asthma; ^#P<0.05; ^##P<0.01 poorly-controlled v’s partially-controlled asthma.

a

b

96

Figure 4.6 Peak lower respiratory symptom scores in healthy and asthmatic subjects defined by baseline asthma severity (a) and baseline asthma control category (b).

+P<0.05; ++P<0.01; +++P<0.001.

b

97

Figure 4.7 Total 14 day lower respiratory symptom scores in healthy and asthmatic subjects defined by baseline asthma severity (a) and baseline asthma control category (b).

+P<0.05; ++P<0.01; +++P<0.001.

a

b

98 4.7 Changes in lung function with infection

A similar relationship between asthma severity, control and exacerbation severity was seen for changes in lung function during rhinovirus infection. Asthmatics experienced greater virus-induced falls in morning PEF and FEV1 than healthy subjects which were significantly greater for moderate-severe asthmatics than healthy subjects on days 3-9, 11 and 13 (all P<0.05). The greatest fall in PEF in the moderate asthmatic group occurred on day 5 and this was significantly greater than the fall in mild asthmatics on that day (17.4 ± 3.1% v 7.2 ± 3.5%, P = 0.045) (figure 4.8). The maximal fall in FEV1 during the infection period was also greatest for moderately-severe asthmatics compared to mild asthmatics (20.8 ± 2.04% v's 14.3 ± 1.3%, P = 0.032) and healthy subjects; (6.2 ± 1.2%, P<0.001) figure 4.9a).

The poorly-controlled asthmatics also had the greatest maximal fall in FEV1 (24.6 +/- 3.1% compared to 16.3 ± 1.7%, P = 0.025 for well-controlled and 14.9 +/- 2.0%, P = 0.021 for partially-controlled asthmatics) (figure 4.9b). This is despite the fact the partially-controlled and poorly-controlled asthmatics had similar baseline FEV1 at study entry (figure 4.1 and Table 4.1).

Unsurprisingly, a strong correlation was observed between maximal falls in PEF and FEV1 in asthma (r = 0.614, P = 0.001).

99

Figure 4.8 Change in morning peak expiratory flow from baseline during the 14 day period following rhinovirus inoculation. Asthmatic subjects defined by baseline asthma severity (a) and baseline asthma control category (b)

Panel a: *P<0.05;**P<0.01;***P<0.001 asthma v’s healthy subjects; ⁺P<0.05 moderate v’s mild asthma. Panel b: †P<0.05 poorly-controlled v’s well-controlled asthma; ^#P<0.05 poorly-controlled v’s partially-controlled asthma.

a

b

100

Figure 4.9 Maximal fall in morning FEV

from baseline during the 14 day period following rhinovirus inoculation. Asthmatic subjects defined by baseline asthma severity (a) and baseline asthma control category (b).

+P<0.05; ++P<0.01; +++P<0.001.

a

b

101 4.8 Effects of Virus Infection on Airway Hyperresponsiveness in Asthma

38/39 subjects performed a baseline histamine challenge however due to low FEV1 levels (<70%

predicted) on day 7 post-inoculation the test was not attempted in 6 asthmatics (1 mild, 5 moderate). None of the 11 healthy subjects achieved a 20% drop in FEV1 following the top histamine dose of 16mg/mL at either time-point. Of the 22 asthmatics that completed baseline and day 7 tests, 16 (73%) had evidence of increased BHR on day 7 compared to baseline (P = 0.003). This increase was statistically significant in both mild (P = 0.045) and moderate (P = 0.022) asthma groups. However no significant differences between mild and moderate asthmatics (or between asthma control groups) were seen despite 88% of the moderate asthmatics using regular inhaled corticosteroid therapy throughout the infection. In view of the range of baseline asthma control of these subjects and the fact that it was not possible to perform a day 7 test in several of the most severely affected asthmatics, it is difficult to draw any firm conclusions from these results other than the general finding of virus-induced AHR in asthma.

Figure 4.10 Bronchial hyperresponsiveness at baseline and during RV infection. +,P<0.05.

102 4.9 Assessment of Virus load

Virus load was measured in both bronchoalveolar lavage (BAL) on day 4 and in nasal lavage on days 2, 3, 4, 5, 7, and 10 post-inoculation. Unfortunately, levels in BAL were in general very low with only 3/11 healthy and 11/28 asthmatics measuring over 10,000 copies / mL. Consequently identifying any differences between asthma and healthy subjects and between the individual asthma sub-groups has not been possible.

Virus load in nasal lavage was seen to peak on day 3 in asthmatic subjects but not until day 4 in healthy subjects. On day 3, levels in asthma were significantly greater (nearly 250-fold) compared to healthy subjects (median day 3 copies/mL: 1.68x10⁶ [1.60 x 10⁴ - 1.28 x 10⁷] v's 6.92 x 10³ [1.50 x 10³ -3.21x10⁶], P=0.042). A trend for greater (~10-fold) day 2 levels was also seen in asthma (P=0.058) (figure 4.11a). The peak level of virus load was greater in asthma but again this was not statistically significant (figure 4.11b).

No statistically significant differences in virus load were observed when comparing asthma severity or control categories however greater median levels were seen in the moderately severe and poorly-controlled groups compared to milder and well-poorly-controlled subjects (figure 4.12).

103

Figure 4.11 Virus Load in nasal lavage. +, P<0.05 compared to healthy subjects; NS = non-significant.

a

b

104

Figure 4.12 Peak virus Load according to asthma sub-group. NS = non-significant

b a

105 4.10 Relationships between virus load and clinical outcome

Relationships were observed between day 2 virus load and both upper and lower respiratory symptoms between days 2 - 4. These included relationships with upper respiratory symptoms on day 2 (all subjects, r=0.550, P<0.001; asthma only, r=0.623, P=0.001), and day 3 (all subjects, r=0.485, P=0.002; asthma only, r=0.548, P=0.003), whilst relationships with lower respiratory symptom scores in asthma were most significant on day 3 (r=0.640, P<0.001) and day 4 (r=0.547, P=0.003). No relationships between day 4 BAL virus load and symptom scores were observed.

In asthma, significant relationships were also observed between virus load and falls in lung function.

Specifically, day 3 virus load correlated with maximal fall in PEF (r=-0.586, P=0.001), and day 7 PEF (r=-0.405, P=0.036), whilst peak virus load correlated with maximal fall in PEF (r=-0.501, P=0.008), as well as PEF on days 4 (r=-0.461, P=0.016), and 7 (r=-0.397, P=0.040). Trends towards significance were seen at several other time-points.

There were no relationships between virus load and other baseline clinical features including baseline FEV1 (% predicted), eosinophil count, total IgE or SPT results, and no correlation with virus load and the degree of AHR in asthma was observed.

Relationships between virus load and inflammatory mediators are discussed in chapter 5 (type 1 immune mediators) and chapter 6 (type 2 immune mediators).

4.11 Inflammatory cell measurements

Lower airway inflammation (measured in BAL) was increased during infection compared to baseline in asthma, and in asthma relative to healthy subjects, however, this was only statistically significant for eosinophils (figure 4.13). Median BAL eosinophil percentages: baseline asthma (0.5 [0.0-1.7]) v’s day 4 (1.2 [0.0-3.8]), P=0.025; day 4 asthma v’s day 4 healthy subjects (0.0 [0.0-0.7]), P=0.046. There were no significant differences in inflammatory cell number according to baseline severity, asthma control or treatment. Inflammatory cell proportions can be seen in full in the appendix.

106 The rhinovirus-induced eosinophilia is in keeping with significant inductions observed in both nasal and bronchial IL-5 in the asthmatic subjects in this study (chapter 6) and highlights the potential benefits of anti-IL-5 therapies such as Mepolizumab¹⁴³ in reducing this element of virus-induced inflammation.

It is noteworthy that nine asthmatics (35%) had no eosinophils present at either time point (which included six subjects on maintenance inhaled corticosteroid therapy) stressing the need to target Mepolizumab at selected asthmatics only.