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Nasal AMP and histamine challenge within and outside the pollen season in patients with seasonal allergic rhinitis
Rhinitis, sinusitis, and upper airway disease
Nasal AMP and histamine challenge within and outside the pollen season in patients with seasonal allergic rhinitis Sriram Vaidyanathan, MBBS, Peter Williamson, MBChB, Karine Clearie, MBChB, Ashley Morrison, MSc, and Brian Lipworth, MD Dundee, United Kingdom Background: Nasal hyperreactivity is a prominent feature of allergic rhinitis. Variation in nasal hyperreactivity with different challenge agents in and out of the pollen season has not been examined. Objective: We sought to compare nasal hyperreactivity with different challenge agents before, during, and after the pollen season. Methods: Grass pollen–monosensitized patients performed cumulative-dose challenges with nasal AMP (25-800 mg $ mL21) and histamine (0.25-8 mg $ mL21) before, during, and after the grass pollen season. Outcomes included the provocative concentration of agent causing a 30% decrease in the peak nasal inspiratory flow (PNIF) (PC30), recovery profile, and diary cards. Results: Nineteen participants completed per protocol. AMP PC30 values for PNIF worsened by 1.33 (95% CI, 0.20-2.44; P 5 .02) doubling dilutions during the season but recovered after the season. The AMP recovery curve showed a 214.39% difference (95% CI, 221.11% to 27.66%; P < .001) during the season and remained abnormal after the season (28.05% [95% CI, 214.78% to 21.33%; P < .05). Histamine PC30 values did not change during the season, but recovery was prolonged by 214.47% (95% CI, 222.19% to 26.76%, P < .001), returning to baseline values after the season. Nasal symptoms, domiciliary PNIF, and serum eosinophil-derived neurotoxin levels returned to baseline values after the season. Conclusions: There is a reduction in AMP PC threshold but not histamine PC threshold during the pollen season, indicating that AMP is a more sensitive indicator of allergic inflammation. The residual hyperreactivity to nasal AMP, but not histamine, outside of the pollen season, seen as a persistently prolonged recovery curve, suggests the presence of primed airway mucosal mast cells, even in asymptomatic patients, and persistent activation of mediator pathways, such as cysteinyl leukotrienes. (J Allergy Clin Immunol 2011;127:173-8.)
From the Asthma and Allergy Research Group, Centre for Cardiovascular and Lung Biology, University of Dundee. Disclosure of potential conflict of interest: The authors have declared that they have no conflict of interest. Received for publication May 11, 2010; revised August 25, 2010; accepted for publication September 13, 2010. Reprint requests: Brian Lipworth, MD, Asthma and Allergy Research Group, Centre for Cardiovascular and Lung Biology, University of Dundee, Dundee DD1 9SY, Scotland, United Kingdom. E-mail: [email protected]. 0091-6749/$36.00 Ó 2010 American Academy of Allergy, Asthma & Immunology doi:10.1016/j.jaci.2010.09.006
Key words: Nasal hyperreactivity, nasal provocation, adenosine monophosphate, histamine, peak nasal inspiratory flow rate, nasal airway resistance
Nasal hyperreactivity is a cardinal feature of allergic rhinitis and is defined as an abnormal responsiveness of the airway to a variety of provocative agents.1 Nasal provocation testing is widely used to assess the onset, effectiveness, and tolerability of antiallergic therapy to ascertain specific diagnoses in cases of discrepancy between skin prick testing and symptom history or before commencement of immunotherapy.2 Different provocation agents reflect different aspects of the nasal inflammatory process. Histamine, a commonly used challenge agent, is a nonspecific marker of nasal vasomotor and neuronal response.1 By contrast, AMP challenge reflects underlying eosinophilic inflammation and has a high correlation with the nasal allergen response.3,4 AMP acts on the primed airway mucosal mast cells through the adenosine A2b receptors, causing degranulation and release of proinflammatory mediators, including cysteinyl leukotrienes, prostaglandins, and IL-8, as well as histamine.3,5 Studies comparing bronchial challenge with histamine or AMP in patients with allergic asthma have shown that AMP is a better marker of mast cell and eosinophil activity.6 Moreover, although histamine challenge can discriminate between patients with rhinitis and healthy control subjects, it cannot discriminate patients with allergic rhinitis from patients with nonallergic rhinitis.7 Furthermore, we have previously shown that nasal AMP, but not histamine, provocation testing is a good predictor of short-term corticosteroid treatment response in patients with allergic rhinitis and correlates significantly with the nasal early-phase allergen challenge response.4,8 How nasal hyperreactivity varies within and outside the pollen season in patients with seasonal rhinitis is crucial to understanding the pathophysiology of the disease because it is apparent that a priming effect can occur even in patients who are asymptomatic outside the pollen season, with persistence of low levels of inflammation outside of the pollen season.9 Indeed, continuous therapy in patients with seasonal rhinitis offers better symptom control than on-demand treatment and, more importantly, a greater reduction in the underlying mucosal inflammation.10 It has also been shown that commencing treatment a few weeks before the start of the pollen season and continuing it beyond this period results in significantly greater reduction in nasal symptom scores and nasal lavage eosinophil counts.11 However, the presence and characteristics of nasal hyperreactivity outside of the pollen season have not yet been determined. 173
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Measurements Abbreviations used DD: Doubling dilution EDN: Eosinophil-derived neurotoxin NAR: Nasal airway resistance PC30/PC60: Provocative concentration of challenge agent causing a 30% or 60% reduction in outcome variable from postdiluent baseline PNIF: Peak nasal inspiratory flow TNSS: Total nasal symptoms score
We conducted a prospective study evaluating and comparing nasal hyperreactivity to AMP and histamine before, during, and after the pollen season in a cohort of grass pollen2monosensitized patients. We hypothesized that nasal hyperreactivity to AMP would be more closely related to the underlying allergic priming processes than that to histamine. We also measured the outcomes of nasal patency, airway and systemic inflammation, and lower airway outcomes.
METHODS The Tayside committee for medical research ethics provided institutional approval to the study protocol before commencement of the study, and written informed consent was obtained from each participant.
Participants Invited participants were male or female, were aged 18 to 70 years, had seasonal allergic rhinitis, were monosensitized to grass pollen (Phleum pratense), and had nasal symptoms only during the grass pollen season, which in Tayside, Scotland, is from June until August according to national monitoring data collated by the Scottish Crop Research Institute, Invergowrie, Scotland. Patients treated with oral prednisolone within 3 months with a recent upper respiratory tract infection, with septal deviation of greater than 50% or grade 2 nasal polyposis,4 with an FEV1 of 60% or less of predicted value (if they had concomitant asthma), with asthma requiring more than 800 mg/d inhaled corticosteroid (chloroflurocarbon–beclomethasone equivalent), who were pregnant, or who were lactating were excluded. Participants withheld antihistamines, leukotriene receptor antagonists, intranasal corticosteroids, and decongestants for 2 weeks before each visit. For subjects prone to symptoms of rhinoconjunctivitis, rescue medication in the form of nasal cromoglycate spray and ocular cromoglycate drops was given, but these were avoided 48 hours and 6 hours before the scheduled visit, respectively.
Study design In this prospective study participants attended the research unit 6 times between February and November 2008. At screening, inclusion and exclusion status were determined. Routine blood tests (full blood count, urea and electrolytes, and liver function tests), rigid nasal endoscopy (2.7 mm, 308 Karl Storz-Endoskope; Karl Storz, Tuttlingen, Germany), spirometry (SuperSpiro; VIASYS, Hampshire, United Kingdom), and skin prick testing to a panel of aeroallergens (Diagenics Ltd, Milton Keynes, United Kingdom) were performed. During each occasion (ie, before, during, and after the grass pollen season), the patients underwent a nasal AMP challenge and a nasal histamine challenge in a randomized assignment order at the same time of day at least 48 hours apart to prevent any carryover effect.8 The preseason visits were held in February and March, in-season visits were held in July and August, and postseason visits were held in October and November. Participants were given a diary for recording concomitant medication, total nasal symptom scores (TNSSs),12 and peak nasal inspiratory flow (PNIF)13 rates taken each morning for a week before each visit.
Each visit started at the same time between 8 and 10 AM, and measurements were taken at a constant room temperature of 218C to 238C and constant relative humidity after a 20-minute acclimatization period. No caffeinecontaining drinks were permitted in the preceding 2 hours, and alcohol was not allowed for 24 hours. Before nasal provocation testing, all other efficacy outcomes were measured. These included nasal and tidal nitric oxide measurements,14 spirometry,15 nasal impulse oscillometry to estimate nasal resistance at 5 Hz (total airway resistance), and serum eosinophil-derived neurotoxin (EDN) measurement (intra-assay coefficient of variation, 9.1%; interassay coefficient of variation, 20%; ELISA, Immunodiagnostik AG, Bensheim, Germany). PNIF rate measurements were taken as the best of 3 measures from an In-check flowmeter (Clement Clarke International Ltd, Harlow, England). The technique was evaluated to ensure a seated posture, horizontal positioning of the meter, correct restoration of the reading to zero, a closed mouth, and an adequate mask seal during a maximal nasal inspiration. Nasal airway resistance (NAR) with active anterior rhinomanometry was measured at 150 Pa by using an NR6 rhinomanometer (GM instruments, Kilwinning, United Kingdom), according to the recommendations of the Standardisation Committee on Objective Assessment of the Nasal Airway.16 Nasal AMP and histamine challenges were performed as described previously,4,8 with PNIF rate and NAR as outcomes. The provocative concentration of AMP or histamine causing a 30% reduction in PNIF rate or a 60% reduction in NAR was estimated by using a standard algebraic formula based on logarithmic extrapolation of the dose-response curve.17 When a greater than 20% decrease was reached with the very first concentration of AMP or histamine, a single-point formula was used instead.18 A 60-minute recovery profile after each challenge with percentage change in PNIF rate measured at 5, 10, 20, 40, and 60 minutes was recorded. Domiciliary PNIF rates and TNSSs were measured as the mean of the last 5 days. All pollen counts were obtained courtesy of the Scottish Crop Research Institute, a designated national pollen aerobiology monitoring site, which collects data from more than 27 species and subspecies of grass and tree pollen in this region.
Statistical analysis Based on previous studies,12,19 we anticipated a sample size requirement of 16 patients to detect a 1 doubling-dilution difference in the AMP provocative concentration causing a 30% reduction in outcome variable from postdiluent baseline (PC30; primary outcome), with an a error of .05 (2-tailed) and overall power of greater than 80%. Each outcome was assessed for normality by using the Shapiro-Wilk test and by means of visual inspection of histograms and Q-Q plots, with consideration given to previous datasets and literature. Nonnormal data were logarithmically transformed. An overall repeatedmeasures ANOVA was performed with subject, treatment, and sequence as cofactors, followed by Bonferroni-corrected pairwise comparisons with a 2-tailed a error set at .05. All analyses were performed on a per-protocol basis with SPSS software (version 17; SPSS, Inc, Chicago, Ill).
RESULTS Participants Of 33 patients screened, 19 were eligible to participate. Twelve female and 7 male participants with a mean age of 44 years (range, 19-68 years) completed per protocol. Demographic data are presented in Table I. Effects of pollen season on all outcomes are shown in Tables II and III. The median pollen (P pratense) count during the season was 33 m23 (interquartile range, 10-55 m23), and pollen was undetectable during preseason and postseason visits. Prechallenge outcomes at each visit before, within and after the season, irrespective of sequence are shown in Table E1 (see Table E1 in this article’s Online Repository at www. jacionline.org). The sequence of nasal challenge testing analyzed as a covariate did not significantly alter any outcome. Asthmatic subjects (n 5 7) were not significantly different from nonasthmatic subjects (data not shown) for any outcome.
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TABLE I. Demographics Patient no./sex/age (y)*
1/F/48 2/M/20 3/F/22 4/M/45 5/F/58 6/F/61 7/M/46 8/F/26 9/F/45 10/F/49 11/F/37 12/M/68 13/F/19 14/M/33 15/F/47 16/F/39 17/M/57 18/F/58 19/M/63 Mean (SEM)
Rhinitis duration Rhinitis (y) medications Asthma
15 4 4 13 26 32 25 6 17 5 29 36 5 5 15 5 6 12 17 14 (2)
A A, S A A A, S S A A S S A A A, S, C A S A A A, S A, C
Y N N N N Y Y N N N Y Y N N N Y Y N N
ICS (mg)
PNIF (L $ min21)
400 80 2 90 2 180 2 220 2 150 800 100 400 170 2 150 2 150 2 170 800 110 800 180 2 170 2 200 2 310 400 140 2 110 2 100 2 130 400 (400-800) 153.1 (12.8)
NAR (Pa $ s21 $ cm23)
2.02 3.15 1.65 0.79 2.82 2.76 0.96 1.80 3.92 3.84 3.98 2.65 1.12 1.79 2.24 1.29 2.07 3.08 0.71 2.24 (0.25)
FEV1 (%)
FVC (%)
FEF25-75 (%)
86 86 74 97 95 89 100 105 82 102 97 111 105 104 86 95 101 62 86 95 56 110 119 84 112 126 80 76 72 86 85 92 60 86 91 59 94 102 69 100 105 85 106 101 101 101 108 80 105 120 77 92 98 59 123 124 100 97.9 (2.7) 102.1 (3.2) 78.9 (3.6) 769.3
Nasal NO (ppb)
512.6 1,110.0 1,295.7 988.3 605.6 1,045.0 421.3 559.3 905.6 402.2 744.7 696.0 1,119.5 1,115.8 613.4 775.2 992.9 999.6 566.9 (689.7-938.7)à
A, Antihistamine; C, sodium cromoglycate nasal spray; FEF25-75, forced expiratory flow between 25% and 75% of forced vital capacity; FVC, forced vital capacity (% predicted); ICS, Inhaled corticosteroids (micrograms of chloroflurocarbon–beclomethasone equivalent units; NAR, total nasal inspiratory resistance; NO, nitric oxide; S, intranasal corticosteroids. *The mean age was 44 years (range, 19-68 years). Median (interquartile range). àGeometric mean (standard error of geometric mean).
Nasal provocation testing Nasal AMP challenge. The PNIF PC30 threshold shifted by 1.33 (95% CI, 0.21 to 2.45; P 5 .02) doubling dilutions (DDs; ie, showing a worsening [reduction] during pollen exposure compared with the preseason baseline value; see Fig E1 in this article’s Online Repository at www.jacionline.org). There was no significant difference between in-season and postseason values, with a 20.68 DD shift (95% CI, 21.81 to 0.43; P 5 .29). The NAR provocative concentration of challenge agent causing a 60% reduction in outcome variable from postdiluent baseline (PC60) threshold showed a 3.45-DD worsening (reduction; 95% CI, 1.34-5.55; P < .001) for in-season exposure versus preseason baseline values, as well as a 22.31-DD improvement (95% CI, 24.42 to 20.21; P 5 .02) for in-season versus postseason values. The time-profile recovery curve showed a 214.39% difference (95% CI, 221.11% to 27.66%; P < .001) in recovery during the season compared with preseason values, which remained abnormal after the pollen season (ie, a 28.05% reduction [95% CI, 214.78% to 21.33%]; Tables II and III and Fig 1). Nasal histamine challenge. There was no significant change in the histamine PNIF PC30 or NAR PC60 threshold values during the season versus those before the season (Tables II and III and see Fig E1). The time profile for recovery was prolonged by 214.47% (95% CI, 222.19% to 26.76%; P < .001) during the season versus before the season but was restored to baseline values after the season (Tables II and III and Fig 1). Upper airway outcomes There was no difference in preseason and in-season values for visit-based PNIF rates (P 5 .47), NAR (P 5 .67), and airway resistance at 5 Hz (P 5 .49).
Lower airway outcomes There was no significant difference in FEV1 percent predicted (P 5 .50), FEV1/forced vital capacity ratio (P 5 .72), and forced expiratory flow between 25% and 75% of forced vital capacity (P 5 .96) when comparing preseason and in-season visits. Inflammation Nasal and tidal nitric oxide levels did not change when comparing preseason versus in-season values as the geometric mean change (0.94 [95% CI, 0.75-1.18; P 5 .60] and 0.81 [95% CI, 0.60-1.08; P 5 .29], respectively). Serum EDN levels showed a geometric mean fold change of 1.47 (95% CI, 1.36-1.92; P 5 .007) during the pollen season. Domiciliary measures There was a significant reduction in domiciliary morning PNIF rates (Tables II and III and see Fig E2 in this article’s Online Repository at www.jacionline.org) when comparing preseason versus in-season values as the mean difference (21.7 L $ min21 [95% CI, 1.6-41.7 L $ min21]; P 5 .04) and domiciliary TNSSs (22.64 [95% CI, 24.68 to 20.60 L $ min21]; P 5 .01). Postseason values for morning PNIF rates and TNSSs were not different from preseason values (27.5 [95% CI, 227.5 to 12.6; P 5 .63] and 1.08 [95% CI, 20.96 to 3.12; P 5 .20], respectively). DISCUSSION The present study evaluated nasal hyperreactivity to histamine and AMP by using monosensitized seasonal allergic rhinitis as a model of seasonal exposure. Our results demonstrated that there is residual hyperreactivity to nasal AMP, but not histamine, after the pollen season, indicating a mast cell memory or priming effect,
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TABLE II. Effect of pollen season on outcomes* Variable
Nasal AMP challenge AMP PNIF PC30 (mg $ mL21) AMP NAR PC60 (mg $ mL21) AMP PNIF recovery (% $ h) Nasal histamine challenge Histamine PNIF PC30 (mg $ mL21) Histamine NAR PC60 (mg $ mL21) Histamine PNIF recovery (% $ h) Visit-based PNIF (L $ min21) NAR (Pa $ s21 $ cm23) Nasal impulse oscillometry R5 Nasal nitric oxide (ppb) Spirometry FEV1 (% predicted) FEV1/FVC ratio (%) FEF25-75 (% predicted) Tidal NO (ppb) Domiciliary measures PNIF (L $ min21) TNSS (U) Serum EDN (nmol $ L21)
Before the season
During the season
After the season
P value
258.85 (1.32) 381.36 (1.54) 27.54 (6.15)
103.25 (1.28) 34.92 (1.47) 41.92 (9.26)
165.99 (1.30) 174.25 (1.67) 35.59 (7.76)
.01 .001 <.0001
0.99 0.74 28.32 152.3 2.66
(1.41) (1.58) (6.43) (11.2) (0.33)
1.36 (1.09) 753.35 (1.09) 97.2 81.6 78.4 17.34
(2.1) (1.0) (3.0) (1.14)
136.7 (9.3) 1.42 (0.43) 3.91 (1.41)
0.82 0.37 42.79 141.1 2.85
(1.36) (1.43) (9.69) (10.8) (0.31)
1.51 (1.14) 799.83 (1.10) 98.9 80.8 77.0 21.53
(2.1) (1.0) (2.6) (1.11)
115.0 (6.6) 4.06 (0.90) 6.66 (1.36)
0.59 0.53 27.82 154.6 3.20
(1.33) (1.33) (6.64) (12.1) (0.24)
1.36 (1.09) 767.36 (1.09) 99.8 80.6 77.1 16.90
(2.1) (0.89) (2.9) (1.11)
144.2 (9.05) 0.34 (0.14) 3.47 (1.21)
.16 .17 <.001 .12 .08 .40 .79 .03 .10 .67 .09 .002 .0001 .005
FEF25-75, Forced expiratory flow between 25% and 75% of forced vital capacity; FVC, forced vital capacity; NAR, total nasal inspiratory resistance; NO, nitric oxide; R5, nasal resistance at 5 Hz. *Data are presented as arithmetic means (SEMs) unless otherwise indicated. Geometric mean (geometric SEM).
even in those patients who do not have symptoms or systemic eosinophilic activation. We showed that both AMP PC30 and PC60 threshold values worsened during pollen exposure, whereas PC60 values significantly improved after the season. AMP recovery was prolonged during pollen exposure and remained so even after the pollen season when compared with baseline values. In contrast, histamine PC30 and PC60 values were not altered by pollen exposure. Although histamine recovery was prolonged during the season, it returned to preseason baseline values afterward. Moreover, the difference in recovery was virtually identical for both challenges (14.4% for AMP vs 14.5% for histamine) when comparing within-season values versus preseason baseline values. This shows a clear difference between AMP and histamine recovery after the pollen season, suggesting that the former takes longer to return to normal after allergen exposure has ceased. This might point to a mast cell priming effect driving the delayed recovery of AMP challenge after the pollen season. Interestingly, this difference between AMP and histamine seems independent of systemic eosinophilic activation or histamine secretion, as evidenced by the complete recovery of histamine challenge and serum EDN levels after the season. We hypothesize that this residual effect could be driven by mediators, such as cysteinyl leukotrienes. This is in keeping with the findings of Lee et al,20 in which antihistamines and leukotriene modifiers had similar effects on attenuating AMP recovery, demonstrating that the nasal action of AMP is partly mediated by cysteinyl leukotrienes. The implications of this are that nasal AMP challenge might be a better indicator of allergic inflammation and more sensitive at testing clinical efficacy, especially outside of the pollen season, compared with histamine challenge. For AMP challenge, the difference in findings between the recovery curve and not the PC threshold for within versus after the season suggest that the
recovery curve is the more sensitive outcome when looking at allergen priming in patients with allergic rhinitis. Furthermore, the fact that there is a difference in the recovery curve but not the threshold for histamine challenge when comparing preseason versus in-season exposure once again indicates greater sensitivity for the recovery curve. The finding of a prolonged recovery curve for AMP, but not histamine, challenge after the season infers a possible persistent effect on priming of airway mast cells. Histamine is a direct stimulant and as such is representative of a general milieu of nonspecific hyperreactivity. The return of the histamine recovery curve after the season shows that mast cell priming might be less important for this particular challenge compared with AMP challenge in the absence of allergen exposure. It might also indicate the role of arachidonic acid metabolites, such as cysteinyl leukotrienes, or other TH2 cytokines known to affect mast cells, such as IL-4, IL-9, and IL-13, in the maintenance of this priming through interaction with adenosine receptors on mast cells and basophils.21 It is known that histamine correlates less well with the symptoms of allergic rhinitis than arachidonic acid metabolites, particularly nasal obstruction.22,23 Also, in patients with perennial rhinitis, no increase in nasal histamine release has been demonstrated, indicating a short-term role for histamine only during acute seasonal exposures to pollen.24 There was a reduction in domiciliary morning PNIF rates during the season but not in the visit-based measurements, such as PNIF rate or NAR. This is likely a function of the increased variability of snapshot measurements as opposed to using serial measures from a 5-day average of repeated measurements. We have shown previously that single visit-based measures are more variable and have a lower responsiveness than domiciliary outcomes.19 PNIF measurement is simple, portable, and cheap, and yet previously, we have shown it to be more reproducible when compared with acoustic rhinometry or rhinomanometry.12 Although we did not
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TABLE III. Within-subject changes in outcomes Variable
Nasal AMP challenge AMP PNIF PC30 (DD shift)§ AMP NAR PC60 (DD shift)§ AMP PNIF recovery (% $ h) Nasal histamine challenge Histamine PNIF PC30 (DD shift)§ Histamine NAR PC60 (DD shift)§ Histamine PNIF recovery (% $ h) Visit-based PNIF (L $ min21) NAR (Pa $ s21 $ cm23) Nasal impulse oscillometry R5k Nasal NO (ppb)k Spirometry FEV1 (% predicted) FEV1/FVC ratio (%) FEF25-75 (% predicted) Tidal NO (ppb)k Domiciliary measures PNIF (L $ min21) TNSS (U) Serum EDN (nmol $ L21)k
Before vs during the season
During vs after the season
Before vs after the season
1.33 (0.21 to 2.45)* 3.45 (1.34 to 5.55) 214.39 (221.11 to 27.66)à
20.68 (21.81 to 0.43) 22.32 (24.42 to 20.21)* 6.33 (20.39 to 13.06)
0.64 (20.48 to 1.76) 1.13 (20.98 to 3.23) 28.05 (214.78 to 21.33)*
0.27 0.98 214.47 11.2 20.19
0.48 20.51 14.97 213.6 20.35
0.75 0.47 0.50 22.3 20.54
(20.71 to 1.25) (20.31 to 2.27) (222.19 to 26.76)à (25.8 to 28.3) (20.79 to 0.40)
0.90 (0.72 to 1.12) 0.94 (0.75 to 1.18) 21.7 0.8 1.3 0.81
(24.1 to 0.6) (25.1 to 6.8) (22.8 to 5.5) (0.60 to 1.08)
21.7 (1.6 to 41.7)* 22.64 (24.68 to 20.60) 0.68 (0.52 to 0.88)
(20.50 to 1.46) (21.80 to 0.78) (7.26 to 22.69)à (230.6 to 3.5) (20.94 to 0.24)
1.11 (0.89 to 1.38) 1.04 (0.83 to 1.31) 20.9 0.61 20.1 1.27
(23.2 to 1.5) (21.1 to 2.3) (24.2 to 4.1) (0.95 to 1.70)
229.2 (249.2 to 29.1) 3.72 (1.68 to 5.76)à 1.46 (1.13 to 1.89)
(20.23 (20.82 (27.21 (219.4 (21.14
to to to to to
1.73) 1.76) 8.22) 14.7) 0.05)
1.00 (0.80 to 1.24) 0.98 (0.78 to 1.24) 22.6 1.46 1.3 1.02
(24.9 to 20.2) (20.2 to 3.1) (22.9 to 5.5) (0.77 to 1.37)
27.5 (227.5 to 12.6) 1.08 (20.96 to 3.12) 0.99 (0.77 to 1.29)
FEF25-75, forced expiratory flow between 25% and 75% of forced vital capacity; FVC, forced vital capacity; NAR, total nasal inspiratory resistance; NO, nitric oxide; R5, nasal resistance at 5 Hz. *P < .05. P < .01. àP < .001. §DD shifts in PC threshold. kGeometric mean fold change (95% CI): a CI that excludes unity denotes a significant difference. The rest of the data are presented as arithmetic mean differences (95% CIs).
explicitly compare rhinomanometry and PNIF rate as study end points, our results suggest a potentially greater sensitivity of the former versus the latter with respect to seasonal priming. However, unlike AMP recovery, it should be noted that PNIF rates and TNSSs recover to preseason values after the pollen season. This is another example of a difference between underlying inflammation and self-reported patient symptoms. This validates the in vivo findings that persistent mucosal inflammation might extend beyond the pollen season and remain independent of overt symptoms.9 We also showed a significant increase in serum EDN levels, a systemic marker of eosinophilic activation, during the pollen season, which did not persist beyond the season.25 Measurement of EDN is a less-expensive alternative to measurement of eosinophil cationic protein, another marker of eosinophil turnover, and does not require strict standardization of the bloodsampling procedure and the handling of the blood sample. It is possible that persistent local eosinophil activation might play a role in the prolonged AMP recovery after the season. We did not measure local tissue eosinophil counts in nasal lavage samples or local markers of activation during these challenges; however, it is nasal obstruction and its measures that have the greatest relevance in a clinical setting. Nasal obstruction constitutes the principal symptom of allergic rhinitis and is the end result of allergic inflammation, mucosal edema, and mucosal hypersecretion.23 Similarly, authors have looked at parameters, such as nasal symptoms, during challenges or surrogates, such as the number of tissues and the amount of secretions. We have shown previously, in a clinical trial using nasal AMP challenge, that for the purposes of monitoring during the challenge, nasal symptoms are soft measures with low responsiveness and a high coefficient of variation.19 PNIF measurement has been shown to be more sensitive than acoustic
rhinometry or rhinomanometry in evaluating nasal responses to provocation testing and correlates well with mucosal changes.2,26 Future application of our study results could be in the form of a randomized controlled trial to look at histamine antagonists alone or in combination with leukotriene antagonists or cromones on nasal AMP and histamine thresholds and recovery in and out of the season, which is similar to what was done previously by Currie et al27 in the lower airway. Likewise, Kurowski et al9 have shown that taking the combination of cetirizine and montelukast 6 weeks in advance of the pollen season had a significant effect on symptoms and nasal lavage eosinophil counts and eosinophil activation when measured during subsequent pollen exposure. Further work needs to be done on the mechanisms underpinning the upregulation of the adenosine A2a and A2b receptors within and outside the pollen season and how various TH2 cytokines or arachidonic acid metabolites might influence this upregulation. Such a mechanistic study conducted after the season in patients with seasonal allergic rhinitis might shed further light on why many patients with seasonal allergic rhinitis have persistent symptoms or whether continued treatment after the conclusion of the pollen season despite the absence of symptoms might reduce mast cell priming and influence the level of symptomatology the following year. There might also be value in a study in which markers of mast cell degranulation (ie, histamine or tryptase) are measured in nasal lavage samples before and after provocation with AMP. Although there are studies that have looked at this in the lung, research within the nasal mucosa is lacking.3,5,6 There are also gaps in the literature regarding seasonal variation in the nasal expression of histamine receptors in the nasal mucosa of allergic subjects that require further exploration. In summary, there is residual hyperreactivity to nasal AMP, but not histamine, outside of the pollen season, which is seen as a
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FIG 1. Recovery profile over 60 minutes for percentage PNIF change after nasal AMP and nasal histamine challenge. The asterisk (*) denotes a significant difference (P < .05) between preseason and in-season timeprofile averaged values. The postseason time-profile curve for recovery for nasal AMP did not improve to preseason values (P < .05) and is denoted by a dagger ( ).
persistently prolonged recovery curve. After the pollen season, unlike symptoms and eosinophilic activation, the AMP recovery curve for PNIF rate does not return to preseason levels. This in turn suggests the presence of primed airway mucosal mast cells, even in asymptomatic patients, and persistent activation of mediator pathways, such as cysteinyl leukotrienes. Although antihistamines are first-line therapy in patients with allergic rhinitis, our data suggest that other inflammatory pathways are of equal importance in the underlying pathophysiology of residual priming outside of the pollen season. Clinical implications: There is residual nasal hyperreactivity to AMP, but not histamine, after the pollen season in patients with asymptomatic seasonal allergic rhinitis. Prophylaxis with cromones or antileukotrienes before the season requires evaluation. REFERENCES 1. Bonini S, Rasi G, Brusasco V, Carlsen KH, Crimi E, Popov T, et al. Nonspecific provocation of target organs in allergic diseases: EAACI-GA(2)LEN consensus report. Allergy 2007;62:683-94. 2. Litvyakova LI, Baraniuk JN. Nasal provocation testing: a review. Ann Allergy Asthma Immunol 2001;86:355-65, 86. 3. Polosa R, Ng WH, Crimi N, Vancheri C, Holgate ST, Church MK, et al. Release of mast-cell-derived mediators after endobronchial adenosine challenge in asthma. Am J Respir Crit Care Med 1995;151:624-9. 4. Vaidyanathan S, Nair A, Barnes ML, Meldrum K, Lipworth BJ. Effect of levocetirizine on nasal provocation testing with adenosine monophosphate compared with allergen challenge in allergic rhinitis. Clin Exp Allergy 2009;39:409-16. 5. Church MK, Holgate ST, Hughes PJ. Adenosine inhibits and potentiates IgEdependent histamine release from human basophils by an A2-receptor mediated mechanism. Br J Pharmacol 1983;80:719-26. 6. van den Berge M, Kerstjens HA, Postma DS. Provocation with adenosine 5’-monophosphate as a marker of inflammation in asthma, allergic rhinitis and chronic obstructive pulmonary disease. Clin Exp Allergy 2002;32:824-30. 7. Wuestenberg EG, Hauswald B, Huettenbrink KB. Thresholds in nasal histamine challenge in patients with allergic rhinitis, patients with hyperreflectory rhinopathy, and healthy volunteers. Am J Rhinol 2004;18:371-5. 8. Wilson AM, Sims EJ, Orr LC, Robb F, Lipworth BJ. An evaluation of short-term corticosteroid response in perennial allergic rhinitis using histamine and adenosine monophosphate nasal challenge. Br J Clin Pharmacol 2003;55:354-9. 9. Ricca V, Landi M, Ferrero P, Bairo A, Tazzer C, Canonica G, et al. Minimal persistent inflammation is also present in patients with seasonal allergic rhinitis. J Allergy Clin Immunol 2000;105:54-7.
10. Ciprandi G, Passalacqua G, Mincarini M, Ricca V, Canonica GW. Continuous versus on demand treatment with cetirizine for allergic rhinitis. Ann Allergy Asthma Immunol 1997;79:507-11. 11. Kurowski M, Kuna P, Gorski P. Montelukast plus cetirizine in the prophylactic treatment of seasonal allergic rhinitis: influence on clinical symptoms and nasal allergic inflammation. Allergy 2004;59:280-8. 12. Sims EJ, Wilson AM, White PS, Gardiner Q, Lipworth BJ. Short term repeatability and correlates of laboratory measures of nasal function in patients with seasonal allergic rhinitis. Rhinology 2002;40:66-8. 13. Vaidyanathan S, Barnes M, Lipworth BJ. Comparative safety and efficacy of 2 formulations of fluticasone aqueous nasal spray in persistent allergic rhinitis. Ann Allergy Asthma Immunol 2009;102:76-83. 14. ATS/ERS recommendations for standardized procedures for the online and offline measurement of exhaled lower respiratory nitric oxide and nasal nitric oxide, 2005. Am J Respir Crit Care Med 2005;171:912-30. 15. Miller MR, Hankinson J, Brusasco V, Burgos F, Casaburi R, Coates A, et al. Standardisation of spirometry. Eur Respir J 2005;26:319-38. 16. Clement PAR, Gordts F. Standardisation Committee on Objective Assessment of the Nasal Airway IRSaERS. Consensus report on acoustic rhinometry and rhinomanometry. Rhinology 2005;43:169-79. 17. Cockcroft DW, Murdock KY, Mink JT. Determination of histamine PC20. Comparison of linear and logarithmic interpolation. Chest 1983;84:505-6. 18. Cockcroft DW, Davis BE. Methacholine PC20: 1-point formula. Ann Allergy Asthma Immunol 2007;98:498-9. 19. Barnes ML, Biallosterski BT, Fujihara S, Gray RD, Fardon TC, Lipworth BJ. Effects of intranasal corticosteroid on nasal adenosine monophosphate challenge in persistent allergic rhinitis. Allergy 2006;61:1319-25. 20. Lee DK, Jackson CM, Soutar PC, Fardon TC, Lipworth BJ. Effects of single or combined histamine H1-receptor and leukotriene CysLT1-receptor antagonism on nasal adenosine monophosphate challenge in persistent allergic rhinitis. Br J Clin Pharmacol 2004;57:714-9. 21. Holgate ST. Adenosine provocation: a new test for allergic type airway inflammation. Am J Respir Crit Care Med 2002;165:317-8. 22. Miadonna A, Tedeschi A, Leggieri E, Lorini M, Folco G, Sala A, et al. Behavior and clinical relevance of histamine and leukotrienes C4 and B4 in grass polleninduced rhinitis. Am Rev Respir Dis 1987;136:357-62. 23. Howarth PH. Mediators of nasal blockage in allergic rhinitis. Allergy 1997;52: 12-8. 24. Wilson J, Reilly K, Salter D, Yap PL, Dawes J, Barnetson R, et al. Nasal histamine and heparin in chronic rhinitis. Ann Otol Rhinol Laryngol 1988;97:389-92. 25. Venge P. Monitoring the allergic inflammation. Allergy 2004;59:26-32. 26. Wilson AM, Sims EJ, Robb F, Cockburn W, Lipworth BJ. Peak inspiratory flow rate is more sensitive than acoustic rhinometry or rhinomanometry in detecting corticosteroid response with nasal histamine challenge. Rhinology 2003;41: 16-20. 27. Currie GP, Haggart K, Brannan JD, Lee DKC, Anderson SD, Lipworth B. Relationship between airway hyperresponsiveness to mannitol and adenosine monophosphate. Allergy 2003;58:762-6.
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FIG E1. Scatter plots showing PC30 and PC60 values during AMP and histamine nasal challenges. The y-axis is a logarithmic scale, and geometric means with 95% CIs are displayed. There was a significant decrease in AMP PC30, but not histamine PC30, during the pollen season.
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FIG E2. Scatter plots showing individual values for domiciliary PNIF rates and TNSSs before, during, and after the pollen season. Horizontal lines and error bars represent arithmetic means and SEMs, respectively. There is a decrease (P < .05) in PNIF rate and an increase (P < .05) in nasal symptoms during the pollen season.
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TABLE E1. Prechallenge outcomes at each visit before the season, within the season and after the season, irrespective of sequence* Before the season Variable
Before AMP 21
Before histamine
Visit-based PNIF (L $ min ) 157.7 (12.4) 146.9 (11.4) 2.78 (0.31) 2.54 (0.40) NAR (Pa $ s21 $ cm23) Nasal impulse oscillometry R5 1.33 (1.09) 1.41 (1.10) Nasal NO (ppb) 714.12 (1.10) 795.83 (1.08) Spirometry 97.7 (2.3) 96.6 (2.1) FEV1 (%predicted) 80.9 (1.1) 82.3 (1.0) FEV1/FVC ratio (%) 79.4 (3.9) 77.3 (4.4) FEF25-75 (% predicted) Tidal NO (ppb) 17.77 (1.14) 18.26 (1.16)
During the season P value
Before AMP
Before histamine
After the season P value
Before AMP
Before histamine
P value
.96 .39
144.8 (13.0) 137.29 (12.0) 2.57 (0.40) 3.13 (0.33)
.95 .49
150.7 (13.4) 158.52 (12.0) 3.37 (0.30) 3.03 (0.34)
.53 .13
.92 .54
1.56 (1.13) 1.47 (1.20) 847.37 (1.09) 752.29 (1.12)
.56 .91
1.22 (1.09) 1.50 (1.14) 703.28 (1.08) 821.20 (1.15)
.48 .36
.84 .73 .96 .74
100.9 81 76.7 21.10
(2.1) (1.5) (2.5) (1.12)
96.8 80.8 77.3 23.25
(2.1) (1.3) (2.4) (1.14)
.82 .57 .91 .96
101.8 80.9 76.5 15.94
(2.0) (1.3) (3.2) (1.14)
97.8 80.2 77.4 17.60
(2.1) (1.0) (2.9) (1.15)
.66 .93 .76 .75
FEF25-75, forced expiratory flow between 25% and 75%; FVC, forced vital capacity; NAR, total nasal inspiratory resistance; NO, nitric oxide; R5, nasal resistance at 5 Hz. *Data are presented as arithmetic means (SEMs), unless otherwise indicated. Each column represents data collected before either AMP or histamine nasal challenge, irrespective of sequence. There was no significant difference in sequence-based values (data not shown). Geometric mean (geometric SEM).
Nasal AMP and histamine challenge within and outside the pollen season in patients with seasonal allergic rhinitis Sriram Vaidyanathan, MBBS, Peter Williamson, MBChB, Karine Clearie, MBChB, Ashley Morrison, MSc, and Brian Lipworth, MD Dundee, United Kingdom Background: Nasal hyperreactivity is a prominent feature of allergic rhinitis. Variation in nasal hyperreactivity with different challenge agents in and out of the pollen season has not been examined. Objective: We sought to compare nasal hyperreactivity with different challenge agents before, during, and after the pollen season. Methods: Grass pollen–monosensitized patients performed cumulative-dose challenges with nasal AMP (25-800 mg $ mL21) and histamine (0.25-8 mg $ mL21) before, during, and after the grass pollen season. Outcomes included the provocative concentration of agent causing a 30% decrease in the peak nasal inspiratory flow (PNIF) (PC30), recovery profile, and diary cards. Results: Nineteen participants completed per protocol. AMP PC30 values for PNIF worsened by 1.33 (95% CI, 0.20-2.44; P 5 .02) doubling dilutions during the season but recovered after the season. The AMP recovery curve showed a 214.39% difference (95% CI, 221.11% to 27.66%; P < .001) during the season and remained abnormal after the season (28.05% [95% CI, 214.78% to 21.33%; P < .05). Histamine PC30 values did not change during the season, but recovery was prolonged by 214.47% (95% CI, 222.19% to 26.76%, P < .001), returning to baseline values after the season. Nasal symptoms, domiciliary PNIF, and serum eosinophil-derived neurotoxin levels returned to baseline values after the season. Conclusions: There is a reduction in AMP PC threshold but not histamine PC threshold during the pollen season, indicating that AMP is a more sensitive indicator of allergic inflammation. The residual hyperreactivity to nasal AMP, but not histamine, outside of the pollen season, seen as a persistently prolonged recovery curve, suggests the presence of primed airway mucosal mast cells, even in asymptomatic patients, and persistent activation of mediator pathways, such as cysteinyl leukotrienes. (J Allergy Clin Immunol 2011;127:173-8.)
From the Asthma and Allergy Research Group, Centre for Cardiovascular and Lung Biology, University of Dundee. Disclosure of potential conflict of interest: The authors have declared that they have no conflict of interest. Received for publication May 11, 2010; revised August 25, 2010; accepted for publication September 13, 2010. Reprint requests: Brian Lipworth, MD, Asthma and Allergy Research Group, Centre for Cardiovascular and Lung Biology, University of Dundee, Dundee DD1 9SY, Scotland, United Kingdom. E-mail: [email protected]. 0091-6749/$36.00 Ó 2010 American Academy of Allergy, Asthma & Immunology doi:10.1016/j.jaci.2010.09.006
Key words: Nasal hyperreactivity, nasal provocation, adenosine monophosphate, histamine, peak nasal inspiratory flow rate, nasal airway resistance
Nasal hyperreactivity is a cardinal feature of allergic rhinitis and is defined as an abnormal responsiveness of the airway to a variety of provocative agents.1 Nasal provocation testing is widely used to assess the onset, effectiveness, and tolerability of antiallergic therapy to ascertain specific diagnoses in cases of discrepancy between skin prick testing and symptom history or before commencement of immunotherapy.2 Different provocation agents reflect different aspects of the nasal inflammatory process. Histamine, a commonly used challenge agent, is a nonspecific marker of nasal vasomotor and neuronal response.1 By contrast, AMP challenge reflects underlying eosinophilic inflammation and has a high correlation with the nasal allergen response.3,4 AMP acts on the primed airway mucosal mast cells through the adenosine A2b receptors, causing degranulation and release of proinflammatory mediators, including cysteinyl leukotrienes, prostaglandins, and IL-8, as well as histamine.3,5 Studies comparing bronchial challenge with histamine or AMP in patients with allergic asthma have shown that AMP is a better marker of mast cell and eosinophil activity.6 Moreover, although histamine challenge can discriminate between patients with rhinitis and healthy control subjects, it cannot discriminate patients with allergic rhinitis from patients with nonallergic rhinitis.7 Furthermore, we have previously shown that nasal AMP, but not histamine, provocation testing is a good predictor of short-term corticosteroid treatment response in patients with allergic rhinitis and correlates significantly with the nasal early-phase allergen challenge response.4,8 How nasal hyperreactivity varies within and outside the pollen season in patients with seasonal rhinitis is crucial to understanding the pathophysiology of the disease because it is apparent that a priming effect can occur even in patients who are asymptomatic outside the pollen season, with persistence of low levels of inflammation outside of the pollen season.9 Indeed, continuous therapy in patients with seasonal rhinitis offers better symptom control than on-demand treatment and, more importantly, a greater reduction in the underlying mucosal inflammation.10 It has also been shown that commencing treatment a few weeks before the start of the pollen season and continuing it beyond this period results in significantly greater reduction in nasal symptom scores and nasal lavage eosinophil counts.11 However, the presence and characteristics of nasal hyperreactivity outside of the pollen season have not yet been determined. 173
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Measurements Abbreviations used DD: Doubling dilution EDN: Eosinophil-derived neurotoxin NAR: Nasal airway resistance PC30/PC60: Provocative concentration of challenge agent causing a 30% or 60% reduction in outcome variable from postdiluent baseline PNIF: Peak nasal inspiratory flow TNSS: Total nasal symptoms score
We conducted a prospective study evaluating and comparing nasal hyperreactivity to AMP and histamine before, during, and after the pollen season in a cohort of grass pollen2monosensitized patients. We hypothesized that nasal hyperreactivity to AMP would be more closely related to the underlying allergic priming processes than that to histamine. We also measured the outcomes of nasal patency, airway and systemic inflammation, and lower airway outcomes.
METHODS The Tayside committee for medical research ethics provided institutional approval to the study protocol before commencement of the study, and written informed consent was obtained from each participant.
Participants Invited participants were male or female, were aged 18 to 70 years, had seasonal allergic rhinitis, were monosensitized to grass pollen (Phleum pratense), and had nasal symptoms only during the grass pollen season, which in Tayside, Scotland, is from June until August according to national monitoring data collated by the Scottish Crop Research Institute, Invergowrie, Scotland. Patients treated with oral prednisolone within 3 months with a recent upper respiratory tract infection, with septal deviation of greater than 50% or grade 2 nasal polyposis,4 with an FEV1 of 60% or less of predicted value (if they had concomitant asthma), with asthma requiring more than 800 mg/d inhaled corticosteroid (chloroflurocarbon–beclomethasone equivalent), who were pregnant, or who were lactating were excluded. Participants withheld antihistamines, leukotriene receptor antagonists, intranasal corticosteroids, and decongestants for 2 weeks before each visit. For subjects prone to symptoms of rhinoconjunctivitis, rescue medication in the form of nasal cromoglycate spray and ocular cromoglycate drops was given, but these were avoided 48 hours and 6 hours before the scheduled visit, respectively.
Study design In this prospective study participants attended the research unit 6 times between February and November 2008. At screening, inclusion and exclusion status were determined. Routine blood tests (full blood count, urea and electrolytes, and liver function tests), rigid nasal endoscopy (2.7 mm, 308 Karl Storz-Endoskope; Karl Storz, Tuttlingen, Germany), spirometry (SuperSpiro; VIASYS, Hampshire, United Kingdom), and skin prick testing to a panel of aeroallergens (Diagenics Ltd, Milton Keynes, United Kingdom) were performed. During each occasion (ie, before, during, and after the grass pollen season), the patients underwent a nasal AMP challenge and a nasal histamine challenge in a randomized assignment order at the same time of day at least 48 hours apart to prevent any carryover effect.8 The preseason visits were held in February and March, in-season visits were held in July and August, and postseason visits were held in October and November. Participants were given a diary for recording concomitant medication, total nasal symptom scores (TNSSs),12 and peak nasal inspiratory flow (PNIF)13 rates taken each morning for a week before each visit.
Each visit started at the same time between 8 and 10 AM, and measurements were taken at a constant room temperature of 218C to 238C and constant relative humidity after a 20-minute acclimatization period. No caffeinecontaining drinks were permitted in the preceding 2 hours, and alcohol was not allowed for 24 hours. Before nasal provocation testing, all other efficacy outcomes were measured. These included nasal and tidal nitric oxide measurements,14 spirometry,15 nasal impulse oscillometry to estimate nasal resistance at 5 Hz (total airway resistance), and serum eosinophil-derived neurotoxin (EDN) measurement (intra-assay coefficient of variation, 9.1%; interassay coefficient of variation, 20%; ELISA, Immunodiagnostik AG, Bensheim, Germany). PNIF rate measurements were taken as the best of 3 measures from an In-check flowmeter (Clement Clarke International Ltd, Harlow, England). The technique was evaluated to ensure a seated posture, horizontal positioning of the meter, correct restoration of the reading to zero, a closed mouth, and an adequate mask seal during a maximal nasal inspiration. Nasal airway resistance (NAR) with active anterior rhinomanometry was measured at 150 Pa by using an NR6 rhinomanometer (GM instruments, Kilwinning, United Kingdom), according to the recommendations of the Standardisation Committee on Objective Assessment of the Nasal Airway.16 Nasal AMP and histamine challenges were performed as described previously,4,8 with PNIF rate and NAR as outcomes. The provocative concentration of AMP or histamine causing a 30% reduction in PNIF rate or a 60% reduction in NAR was estimated by using a standard algebraic formula based on logarithmic extrapolation of the dose-response curve.17 When a greater than 20% decrease was reached with the very first concentration of AMP or histamine, a single-point formula was used instead.18 A 60-minute recovery profile after each challenge with percentage change in PNIF rate measured at 5, 10, 20, 40, and 60 minutes was recorded. Domiciliary PNIF rates and TNSSs were measured as the mean of the last 5 days. All pollen counts were obtained courtesy of the Scottish Crop Research Institute, a designated national pollen aerobiology monitoring site, which collects data from more than 27 species and subspecies of grass and tree pollen in this region.
Statistical analysis Based on previous studies,12,19 we anticipated a sample size requirement of 16 patients to detect a 1 doubling-dilution difference in the AMP provocative concentration causing a 30% reduction in outcome variable from postdiluent baseline (PC30; primary outcome), with an a error of .05 (2-tailed) and overall power of greater than 80%. Each outcome was assessed for normality by using the Shapiro-Wilk test and by means of visual inspection of histograms and Q-Q plots, with consideration given to previous datasets and literature. Nonnormal data were logarithmically transformed. An overall repeatedmeasures ANOVA was performed with subject, treatment, and sequence as cofactors, followed by Bonferroni-corrected pairwise comparisons with a 2-tailed a error set at .05. All analyses were performed on a per-protocol basis with SPSS software (version 17; SPSS, Inc, Chicago, Ill).
RESULTS Participants Of 33 patients screened, 19 were eligible to participate. Twelve female and 7 male participants with a mean age of 44 years (range, 19-68 years) completed per protocol. Demographic data are presented in Table I. Effects of pollen season on all outcomes are shown in Tables II and III. The median pollen (P pratense) count during the season was 33 m23 (interquartile range, 10-55 m23), and pollen was undetectable during preseason and postseason visits. Prechallenge outcomes at each visit before, within and after the season, irrespective of sequence are shown in Table E1 (see Table E1 in this article’s Online Repository at www. jacionline.org). The sequence of nasal challenge testing analyzed as a covariate did not significantly alter any outcome. Asthmatic subjects (n 5 7) were not significantly different from nonasthmatic subjects (data not shown) for any outcome.
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TABLE I. Demographics Patient no./sex/age (y)*
1/F/48 2/M/20 3/F/22 4/M/45 5/F/58 6/F/61 7/M/46 8/F/26 9/F/45 10/F/49 11/F/37 12/M/68 13/F/19 14/M/33 15/F/47 16/F/39 17/M/57 18/F/58 19/M/63 Mean (SEM)
Rhinitis duration Rhinitis (y) medications Asthma
15 4 4 13 26 32 25 6 17 5 29 36 5 5 15 5 6 12 17 14 (2)
A A, S A A A, S S A A S S A A A, S, C A S A A A, S A, C
Y N N N N Y Y N N N Y Y N N N Y Y N N
ICS (mg)
PNIF (L $ min21)
400 80 2 90 2 180 2 220 2 150 800 100 400 170 2 150 2 150 2 170 800 110 800 180 2 170 2 200 2 310 400 140 2 110 2 100 2 130 400 (400-800) 153.1 (12.8)
NAR (Pa $ s21 $ cm23)
2.02 3.15 1.65 0.79 2.82 2.76 0.96 1.80 3.92 3.84 3.98 2.65 1.12 1.79 2.24 1.29 2.07 3.08 0.71 2.24 (0.25)
FEV1 (%)
FVC (%)
FEF25-75 (%)
86 86 74 97 95 89 100 105 82 102 97 111 105 104 86 95 101 62 86 95 56 110 119 84 112 126 80 76 72 86 85 92 60 86 91 59 94 102 69 100 105 85 106 101 101 101 108 80 105 120 77 92 98 59 123 124 100 97.9 (2.7) 102.1 (3.2) 78.9 (3.6) 769.3
Nasal NO (ppb)
512.6 1,110.0 1,295.7 988.3 605.6 1,045.0 421.3 559.3 905.6 402.2 744.7 696.0 1,119.5 1,115.8 613.4 775.2 992.9 999.6 566.9 (689.7-938.7)à
A, Antihistamine; C, sodium cromoglycate nasal spray; FEF25-75, forced expiratory flow between 25% and 75% of forced vital capacity; FVC, forced vital capacity (% predicted); ICS, Inhaled corticosteroids (micrograms of chloroflurocarbon–beclomethasone equivalent units; NAR, total nasal inspiratory resistance; NO, nitric oxide; S, intranasal corticosteroids. *The mean age was 44 years (range, 19-68 years). Median (interquartile range). àGeometric mean (standard error of geometric mean).
Nasal provocation testing Nasal AMP challenge. The PNIF PC30 threshold shifted by 1.33 (95% CI, 0.21 to 2.45; P 5 .02) doubling dilutions (DDs; ie, showing a worsening [reduction] during pollen exposure compared with the preseason baseline value; see Fig E1 in this article’s Online Repository at www.jacionline.org). There was no significant difference between in-season and postseason values, with a 20.68 DD shift (95% CI, 21.81 to 0.43; P 5 .29). The NAR provocative concentration of challenge agent causing a 60% reduction in outcome variable from postdiluent baseline (PC60) threshold showed a 3.45-DD worsening (reduction; 95% CI, 1.34-5.55; P < .001) for in-season exposure versus preseason baseline values, as well as a 22.31-DD improvement (95% CI, 24.42 to 20.21; P 5 .02) for in-season versus postseason values. The time-profile recovery curve showed a 214.39% difference (95% CI, 221.11% to 27.66%; P < .001) in recovery during the season compared with preseason values, which remained abnormal after the pollen season (ie, a 28.05% reduction [95% CI, 214.78% to 21.33%]; Tables II and III and Fig 1). Nasal histamine challenge. There was no significant change in the histamine PNIF PC30 or NAR PC60 threshold values during the season versus those before the season (Tables II and III and see Fig E1). The time profile for recovery was prolonged by 214.47% (95% CI, 222.19% to 26.76%; P < .001) during the season versus before the season but was restored to baseline values after the season (Tables II and III and Fig 1). Upper airway outcomes There was no difference in preseason and in-season values for visit-based PNIF rates (P 5 .47), NAR (P 5 .67), and airway resistance at 5 Hz (P 5 .49).
Lower airway outcomes There was no significant difference in FEV1 percent predicted (P 5 .50), FEV1/forced vital capacity ratio (P 5 .72), and forced expiratory flow between 25% and 75% of forced vital capacity (P 5 .96) when comparing preseason and in-season visits. Inflammation Nasal and tidal nitric oxide levels did not change when comparing preseason versus in-season values as the geometric mean change (0.94 [95% CI, 0.75-1.18; P 5 .60] and 0.81 [95% CI, 0.60-1.08; P 5 .29], respectively). Serum EDN levels showed a geometric mean fold change of 1.47 (95% CI, 1.36-1.92; P 5 .007) during the pollen season. Domiciliary measures There was a significant reduction in domiciliary morning PNIF rates (Tables II and III and see Fig E2 in this article’s Online Repository at www.jacionline.org) when comparing preseason versus in-season values as the mean difference (21.7 L $ min21 [95% CI, 1.6-41.7 L $ min21]; P 5 .04) and domiciliary TNSSs (22.64 [95% CI, 24.68 to 20.60 L $ min21]; P 5 .01). Postseason values for morning PNIF rates and TNSSs were not different from preseason values (27.5 [95% CI, 227.5 to 12.6; P 5 .63] and 1.08 [95% CI, 20.96 to 3.12; P 5 .20], respectively). DISCUSSION The present study evaluated nasal hyperreactivity to histamine and AMP by using monosensitized seasonal allergic rhinitis as a model of seasonal exposure. Our results demonstrated that there is residual hyperreactivity to nasal AMP, but not histamine, after the pollen season, indicating a mast cell memory or priming effect,
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TABLE II. Effect of pollen season on outcomes* Variable
Nasal AMP challenge AMP PNIF PC30 (mg $ mL21) AMP NAR PC60 (mg $ mL21) AMP PNIF recovery (% $ h) Nasal histamine challenge Histamine PNIF PC30 (mg $ mL21) Histamine NAR PC60 (mg $ mL21) Histamine PNIF recovery (% $ h) Visit-based PNIF (L $ min21) NAR (Pa $ s21 $ cm23) Nasal impulse oscillometry R5 Nasal nitric oxide (ppb) Spirometry FEV1 (% predicted) FEV1/FVC ratio (%) FEF25-75 (% predicted) Tidal NO (ppb) Domiciliary measures PNIF (L $ min21) TNSS (U) Serum EDN (nmol $ L21)
Before the season
During the season
After the season
P value
258.85 (1.32) 381.36 (1.54) 27.54 (6.15)
103.25 (1.28) 34.92 (1.47) 41.92 (9.26)
165.99 (1.30) 174.25 (1.67) 35.59 (7.76)
.01 .001 <.0001
0.99 0.74 28.32 152.3 2.66
(1.41) (1.58) (6.43) (11.2) (0.33)
1.36 (1.09) 753.35 (1.09) 97.2 81.6 78.4 17.34
(2.1) (1.0) (3.0) (1.14)
136.7 (9.3) 1.42 (0.43) 3.91 (1.41)
0.82 0.37 42.79 141.1 2.85
(1.36) (1.43) (9.69) (10.8) (0.31)
1.51 (1.14) 799.83 (1.10) 98.9 80.8 77.0 21.53
(2.1) (1.0) (2.6) (1.11)
115.0 (6.6) 4.06 (0.90) 6.66 (1.36)
0.59 0.53 27.82 154.6 3.20
(1.33) (1.33) (6.64) (12.1) (0.24)
1.36 (1.09) 767.36 (1.09) 99.8 80.6 77.1 16.90
(2.1) (0.89) (2.9) (1.11)
144.2 (9.05) 0.34 (0.14) 3.47 (1.21)
.16 .17 <.001 .12 .08 .40 .79 .03 .10 .67 .09 .002 .0001 .005
FEF25-75, Forced expiratory flow between 25% and 75% of forced vital capacity; FVC, forced vital capacity; NAR, total nasal inspiratory resistance; NO, nitric oxide; R5, nasal resistance at 5 Hz. *Data are presented as arithmetic means (SEMs) unless otherwise indicated. Geometric mean (geometric SEM).
even in those patients who do not have symptoms or systemic eosinophilic activation. We showed that both AMP PC30 and PC60 threshold values worsened during pollen exposure, whereas PC60 values significantly improved after the season. AMP recovery was prolonged during pollen exposure and remained so even after the pollen season when compared with baseline values. In contrast, histamine PC30 and PC60 values were not altered by pollen exposure. Although histamine recovery was prolonged during the season, it returned to preseason baseline values afterward. Moreover, the difference in recovery was virtually identical for both challenges (14.4% for AMP vs 14.5% for histamine) when comparing within-season values versus preseason baseline values. This shows a clear difference between AMP and histamine recovery after the pollen season, suggesting that the former takes longer to return to normal after allergen exposure has ceased. This might point to a mast cell priming effect driving the delayed recovery of AMP challenge after the pollen season. Interestingly, this difference between AMP and histamine seems independent of systemic eosinophilic activation or histamine secretion, as evidenced by the complete recovery of histamine challenge and serum EDN levels after the season. We hypothesize that this residual effect could be driven by mediators, such as cysteinyl leukotrienes. This is in keeping with the findings of Lee et al,20 in which antihistamines and leukotriene modifiers had similar effects on attenuating AMP recovery, demonstrating that the nasal action of AMP is partly mediated by cysteinyl leukotrienes. The implications of this are that nasal AMP challenge might be a better indicator of allergic inflammation and more sensitive at testing clinical efficacy, especially outside of the pollen season, compared with histamine challenge. For AMP challenge, the difference in findings between the recovery curve and not the PC threshold for within versus after the season suggest that the
recovery curve is the more sensitive outcome when looking at allergen priming in patients with allergic rhinitis. Furthermore, the fact that there is a difference in the recovery curve but not the threshold for histamine challenge when comparing preseason versus in-season exposure once again indicates greater sensitivity for the recovery curve. The finding of a prolonged recovery curve for AMP, but not histamine, challenge after the season infers a possible persistent effect on priming of airway mast cells. Histamine is a direct stimulant and as such is representative of a general milieu of nonspecific hyperreactivity. The return of the histamine recovery curve after the season shows that mast cell priming might be less important for this particular challenge compared with AMP challenge in the absence of allergen exposure. It might also indicate the role of arachidonic acid metabolites, such as cysteinyl leukotrienes, or other TH2 cytokines known to affect mast cells, such as IL-4, IL-9, and IL-13, in the maintenance of this priming through interaction with adenosine receptors on mast cells and basophils.21 It is known that histamine correlates less well with the symptoms of allergic rhinitis than arachidonic acid metabolites, particularly nasal obstruction.22,23 Also, in patients with perennial rhinitis, no increase in nasal histamine release has been demonstrated, indicating a short-term role for histamine only during acute seasonal exposures to pollen.24 There was a reduction in domiciliary morning PNIF rates during the season but not in the visit-based measurements, such as PNIF rate or NAR. This is likely a function of the increased variability of snapshot measurements as opposed to using serial measures from a 5-day average of repeated measurements. We have shown previously that single visit-based measures are more variable and have a lower responsiveness than domiciliary outcomes.19 PNIF measurement is simple, portable, and cheap, and yet previously, we have shown it to be more reproducible when compared with acoustic rhinometry or rhinomanometry.12 Although we did not
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TABLE III. Within-subject changes in outcomes Variable
Nasal AMP challenge AMP PNIF PC30 (DD shift)§ AMP NAR PC60 (DD shift)§ AMP PNIF recovery (% $ h) Nasal histamine challenge Histamine PNIF PC30 (DD shift)§ Histamine NAR PC60 (DD shift)§ Histamine PNIF recovery (% $ h) Visit-based PNIF (L $ min21) NAR (Pa $ s21 $ cm23) Nasal impulse oscillometry R5k Nasal NO (ppb)k Spirometry FEV1 (% predicted) FEV1/FVC ratio (%) FEF25-75 (% predicted) Tidal NO (ppb)k Domiciliary measures PNIF (L $ min21) TNSS (U) Serum EDN (nmol $ L21)k
Before vs during the season
During vs after the season
Before vs after the season
1.33 (0.21 to 2.45)* 3.45 (1.34 to 5.55) 214.39 (221.11 to 27.66)à
20.68 (21.81 to 0.43) 22.32 (24.42 to 20.21)* 6.33 (20.39 to 13.06)
0.64 (20.48 to 1.76) 1.13 (20.98 to 3.23) 28.05 (214.78 to 21.33)*
0.27 0.98 214.47 11.2 20.19
0.48 20.51 14.97 213.6 20.35
0.75 0.47 0.50 22.3 20.54
(20.71 to 1.25) (20.31 to 2.27) (222.19 to 26.76)à (25.8 to 28.3) (20.79 to 0.40)
0.90 (0.72 to 1.12) 0.94 (0.75 to 1.18) 21.7 0.8 1.3 0.81
(24.1 to 0.6) (25.1 to 6.8) (22.8 to 5.5) (0.60 to 1.08)
21.7 (1.6 to 41.7)* 22.64 (24.68 to 20.60) 0.68 (0.52 to 0.88)
(20.50 to 1.46) (21.80 to 0.78) (7.26 to 22.69)à (230.6 to 3.5) (20.94 to 0.24)
1.11 (0.89 to 1.38) 1.04 (0.83 to 1.31) 20.9 0.61 20.1 1.27
(23.2 to 1.5) (21.1 to 2.3) (24.2 to 4.1) (0.95 to 1.70)
229.2 (249.2 to 29.1) 3.72 (1.68 to 5.76)à 1.46 (1.13 to 1.89)
(20.23 (20.82 (27.21 (219.4 (21.14
to to to to to
1.73) 1.76) 8.22) 14.7) 0.05)
1.00 (0.80 to 1.24) 0.98 (0.78 to 1.24) 22.6 1.46 1.3 1.02
(24.9 to 20.2) (20.2 to 3.1) (22.9 to 5.5) (0.77 to 1.37)
27.5 (227.5 to 12.6) 1.08 (20.96 to 3.12) 0.99 (0.77 to 1.29)
FEF25-75, forced expiratory flow between 25% and 75% of forced vital capacity; FVC, forced vital capacity; NAR, total nasal inspiratory resistance; NO, nitric oxide; R5, nasal resistance at 5 Hz. *P < .05. P < .01. àP < .001. §DD shifts in PC threshold. kGeometric mean fold change (95% CI): a CI that excludes unity denotes a significant difference. The rest of the data are presented as arithmetic mean differences (95% CIs).
explicitly compare rhinomanometry and PNIF rate as study end points, our results suggest a potentially greater sensitivity of the former versus the latter with respect to seasonal priming. However, unlike AMP recovery, it should be noted that PNIF rates and TNSSs recover to preseason values after the pollen season. This is another example of a difference between underlying inflammation and self-reported patient symptoms. This validates the in vivo findings that persistent mucosal inflammation might extend beyond the pollen season and remain independent of overt symptoms.9 We also showed a significant increase in serum EDN levels, a systemic marker of eosinophilic activation, during the pollen season, which did not persist beyond the season.25 Measurement of EDN is a less-expensive alternative to measurement of eosinophil cationic protein, another marker of eosinophil turnover, and does not require strict standardization of the bloodsampling procedure and the handling of the blood sample. It is possible that persistent local eosinophil activation might play a role in the prolonged AMP recovery after the season. We did not measure local tissue eosinophil counts in nasal lavage samples or local markers of activation during these challenges; however, it is nasal obstruction and its measures that have the greatest relevance in a clinical setting. Nasal obstruction constitutes the principal symptom of allergic rhinitis and is the end result of allergic inflammation, mucosal edema, and mucosal hypersecretion.23 Similarly, authors have looked at parameters, such as nasal symptoms, during challenges or surrogates, such as the number of tissues and the amount of secretions. We have shown previously, in a clinical trial using nasal AMP challenge, that for the purposes of monitoring during the challenge, nasal symptoms are soft measures with low responsiveness and a high coefficient of variation.19 PNIF measurement has been shown to be more sensitive than acoustic
rhinometry or rhinomanometry in evaluating nasal responses to provocation testing and correlates well with mucosal changes.2,26 Future application of our study results could be in the form of a randomized controlled trial to look at histamine antagonists alone or in combination with leukotriene antagonists or cromones on nasal AMP and histamine thresholds and recovery in and out of the season, which is similar to what was done previously by Currie et al27 in the lower airway. Likewise, Kurowski et al9 have shown that taking the combination of cetirizine and montelukast 6 weeks in advance of the pollen season had a significant effect on symptoms and nasal lavage eosinophil counts and eosinophil activation when measured during subsequent pollen exposure. Further work needs to be done on the mechanisms underpinning the upregulation of the adenosine A2a and A2b receptors within and outside the pollen season and how various TH2 cytokines or arachidonic acid metabolites might influence this upregulation. Such a mechanistic study conducted after the season in patients with seasonal allergic rhinitis might shed further light on why many patients with seasonal allergic rhinitis have persistent symptoms or whether continued treatment after the conclusion of the pollen season despite the absence of symptoms might reduce mast cell priming and influence the level of symptomatology the following year. There might also be value in a study in which markers of mast cell degranulation (ie, histamine or tryptase) are measured in nasal lavage samples before and after provocation with AMP. Although there are studies that have looked at this in the lung, research within the nasal mucosa is lacking.3,5,6 There are also gaps in the literature regarding seasonal variation in the nasal expression of histamine receptors in the nasal mucosa of allergic subjects that require further exploration. In summary, there is residual hyperreactivity to nasal AMP, but not histamine, outside of the pollen season, which is seen as a
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FIG 1. Recovery profile over 60 minutes for percentage PNIF change after nasal AMP and nasal histamine challenge. The asterisk (*) denotes a significant difference (P < .05) between preseason and in-season timeprofile averaged values. The postseason time-profile curve for recovery for nasal AMP did not improve to preseason values (P < .05) and is denoted by a dagger ( ).
persistently prolonged recovery curve. After the pollen season, unlike symptoms and eosinophilic activation, the AMP recovery curve for PNIF rate does not return to preseason levels. This in turn suggests the presence of primed airway mucosal mast cells, even in asymptomatic patients, and persistent activation of mediator pathways, such as cysteinyl leukotrienes. Although antihistamines are first-line therapy in patients with allergic rhinitis, our data suggest that other inflammatory pathways are of equal importance in the underlying pathophysiology of residual priming outside of the pollen season. Clinical implications: There is residual nasal hyperreactivity to AMP, but not histamine, after the pollen season in patients with asymptomatic seasonal allergic rhinitis. Prophylaxis with cromones or antileukotrienes before the season requires evaluation. REFERENCES 1. Bonini S, Rasi G, Brusasco V, Carlsen KH, Crimi E, Popov T, et al. Nonspecific provocation of target organs in allergic diseases: EAACI-GA(2)LEN consensus report. Allergy 2007;62:683-94. 2. Litvyakova LI, Baraniuk JN. Nasal provocation testing: a review. Ann Allergy Asthma Immunol 2001;86:355-65, 86. 3. Polosa R, Ng WH, Crimi N, Vancheri C, Holgate ST, Church MK, et al. Release of mast-cell-derived mediators after endobronchial adenosine challenge in asthma. Am J Respir Crit Care Med 1995;151:624-9. 4. Vaidyanathan S, Nair A, Barnes ML, Meldrum K, Lipworth BJ. Effect of levocetirizine on nasal provocation testing with adenosine monophosphate compared with allergen challenge in allergic rhinitis. Clin Exp Allergy 2009;39:409-16. 5. Church MK, Holgate ST, Hughes PJ. Adenosine inhibits and potentiates IgEdependent histamine release from human basophils by an A2-receptor mediated mechanism. Br J Pharmacol 1983;80:719-26. 6. van den Berge M, Kerstjens HA, Postma DS. Provocation with adenosine 5’-monophosphate as a marker of inflammation in asthma, allergic rhinitis and chronic obstructive pulmonary disease. Clin Exp Allergy 2002;32:824-30. 7. Wuestenberg EG, Hauswald B, Huettenbrink KB. Thresholds in nasal histamine challenge in patients with allergic rhinitis, patients with hyperreflectory rhinopathy, and healthy volunteers. Am J Rhinol 2004;18:371-5. 8. Wilson AM, Sims EJ, Orr LC, Robb F, Lipworth BJ. An evaluation of short-term corticosteroid response in perennial allergic rhinitis using histamine and adenosine monophosphate nasal challenge. Br J Clin Pharmacol 2003;55:354-9. 9. Ricca V, Landi M, Ferrero P, Bairo A, Tazzer C, Canonica G, et al. Minimal persistent inflammation is also present in patients with seasonal allergic rhinitis. J Allergy Clin Immunol 2000;105:54-7.
10. Ciprandi G, Passalacqua G, Mincarini M, Ricca V, Canonica GW. Continuous versus on demand treatment with cetirizine for allergic rhinitis. Ann Allergy Asthma Immunol 1997;79:507-11. 11. Kurowski M, Kuna P, Gorski P. Montelukast plus cetirizine in the prophylactic treatment of seasonal allergic rhinitis: influence on clinical symptoms and nasal allergic inflammation. Allergy 2004;59:280-8. 12. Sims EJ, Wilson AM, White PS, Gardiner Q, Lipworth BJ. Short term repeatability and correlates of laboratory measures of nasal function in patients with seasonal allergic rhinitis. Rhinology 2002;40:66-8. 13. Vaidyanathan S, Barnes M, Lipworth BJ. Comparative safety and efficacy of 2 formulations of fluticasone aqueous nasal spray in persistent allergic rhinitis. Ann Allergy Asthma Immunol 2009;102:76-83. 14. ATS/ERS recommendations for standardized procedures for the online and offline measurement of exhaled lower respiratory nitric oxide and nasal nitric oxide, 2005. Am J Respir Crit Care Med 2005;171:912-30. 15. Miller MR, Hankinson J, Brusasco V, Burgos F, Casaburi R, Coates A, et al. Standardisation of spirometry. Eur Respir J 2005;26:319-38. 16. Clement PAR, Gordts F. Standardisation Committee on Objective Assessment of the Nasal Airway IRSaERS. Consensus report on acoustic rhinometry and rhinomanometry. Rhinology 2005;43:169-79. 17. Cockcroft DW, Murdock KY, Mink JT. Determination of histamine PC20. Comparison of linear and logarithmic interpolation. Chest 1983;84:505-6. 18. Cockcroft DW, Davis BE. Methacholine PC20: 1-point formula. Ann Allergy Asthma Immunol 2007;98:498-9. 19. Barnes ML, Biallosterski BT, Fujihara S, Gray RD, Fardon TC, Lipworth BJ. Effects of intranasal corticosteroid on nasal adenosine monophosphate challenge in persistent allergic rhinitis. Allergy 2006;61:1319-25. 20. Lee DK, Jackson CM, Soutar PC, Fardon TC, Lipworth BJ. Effects of single or combined histamine H1-receptor and leukotriene CysLT1-receptor antagonism on nasal adenosine monophosphate challenge in persistent allergic rhinitis. Br J Clin Pharmacol 2004;57:714-9. 21. Holgate ST. Adenosine provocation: a new test for allergic type airway inflammation. Am J Respir Crit Care Med 2002;165:317-8. 22. Miadonna A, Tedeschi A, Leggieri E, Lorini M, Folco G, Sala A, et al. Behavior and clinical relevance of histamine and leukotrienes C4 and B4 in grass polleninduced rhinitis. Am Rev Respir Dis 1987;136:357-62. 23. Howarth PH. Mediators of nasal blockage in allergic rhinitis. Allergy 1997;52: 12-8. 24. Wilson J, Reilly K, Salter D, Yap PL, Dawes J, Barnetson R, et al. Nasal histamine and heparin in chronic rhinitis. Ann Otol Rhinol Laryngol 1988;97:389-92. 25. Venge P. Monitoring the allergic inflammation. Allergy 2004;59:26-32. 26. Wilson AM, Sims EJ, Robb F, Cockburn W, Lipworth BJ. Peak inspiratory flow rate is more sensitive than acoustic rhinometry or rhinomanometry in detecting corticosteroid response with nasal histamine challenge. Rhinology 2003;41: 16-20. 27. Currie GP, Haggart K, Brannan JD, Lee DKC, Anderson SD, Lipworth B. Relationship between airway hyperresponsiveness to mannitol and adenosine monophosphate. Allergy 2003;58:762-6.
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FIG E1. Scatter plots showing PC30 and PC60 values during AMP and histamine nasal challenges. The y-axis is a logarithmic scale, and geometric means with 95% CIs are displayed. There was a significant decrease in AMP PC30, but not histamine PC30, during the pollen season.
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FIG E2. Scatter plots showing individual values for domiciliary PNIF rates and TNSSs before, during, and after the pollen season. Horizontal lines and error bars represent arithmetic means and SEMs, respectively. There is a decrease (P < .05) in PNIF rate and an increase (P < .05) in nasal symptoms during the pollen season.
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TABLE E1. Prechallenge outcomes at each visit before the season, within the season and after the season, irrespective of sequence* Before the season Variable
Before AMP 21
Before histamine
Visit-based PNIF (L $ min ) 157.7 (12.4) 146.9 (11.4) 2.78 (0.31) 2.54 (0.40) NAR (Pa $ s21 $ cm23) Nasal impulse oscillometry R5 1.33 (1.09) 1.41 (1.10) Nasal NO (ppb) 714.12 (1.10) 795.83 (1.08) Spirometry 97.7 (2.3) 96.6 (2.1) FEV1 (%predicted) 80.9 (1.1) 82.3 (1.0) FEV1/FVC ratio (%) 79.4 (3.9) 77.3 (4.4) FEF25-75 (% predicted) Tidal NO (ppb) 17.77 (1.14) 18.26 (1.16)
During the season P value
Before AMP
Before histamine
After the season P value
Before AMP
Before histamine
P value
.96 .39
144.8 (13.0) 137.29 (12.0) 2.57 (0.40) 3.13 (0.33)
.95 .49
150.7 (13.4) 158.52 (12.0) 3.37 (0.30) 3.03 (0.34)
.53 .13
.92 .54
1.56 (1.13) 1.47 (1.20) 847.37 (1.09) 752.29 (1.12)
.56 .91
1.22 (1.09) 1.50 (1.14) 703.28 (1.08) 821.20 (1.15)
.48 .36
.84 .73 .96 .74
100.9 81 76.7 21.10
(2.1) (1.5) (2.5) (1.12)
96.8 80.8 77.3 23.25
(2.1) (1.3) (2.4) (1.14)
.82 .57 .91 .96
101.8 80.9 76.5 15.94
(2.0) (1.3) (3.2) (1.14)
97.8 80.2 77.4 17.60
(2.1) (1.0) (2.9) (1.15)
.66 .93 .76 .75
FEF25-75, forced expiratory flow between 25% and 75%; FVC, forced vital capacity; NAR, total nasal inspiratory resistance; NO, nitric oxide; R5, nasal resistance at 5 Hz. *Data are presented as arithmetic means (SEMs), unless otherwise indicated. Each column represents data collected before either AMP or histamine nasal challenge, irrespective of sequence. There was no significant difference in sequence-based values (data not shown). Geometric mean (geometric SEM).