EAACI Position paper on the standardization of nasal allergen challenges

Nasal allergen challenge (NAC) is an important tool to diagnose allergic rhinitis. In daily clinical routine, experimentally, or when measuring therapeutic success clinically, nasal allergen challenge is fundamental. It is further one of the key diagnostic tools when initiating specific allergen immunotherapy. So far, national recommendations offered guidance on its execution; however, international divergence left many questions unanswered. These differences in the literature caused EAACI to initiate a task force to answer unmet needs and find a consensus in executing nasal allergen challenge. On the basis of a systematic review containing nasal allergen challenges of the past years, task force members reviewed evidence, discussed open issues, and studied variations of several subjective and objective assessment parameters to propose a standardized way of a nasal allergen challenge procedure in clinical practice. Besides an update on indications, contraindications, and preparations for the test procedure, main recommendations are a bilaterally challenge with standardized allergens, with a spray device offering 0.1 mL per nostril. A systematic catalogue for positivity criteria is given for the variety of established subjective and objective assessment methods as well as a schedule for the challenge procedure. The task force recommends a unified protocol for NAC for daily clinical practice, aiming at eliminating the previous difficulty of comparing NAC results due to unmet needs.     

Effect of levocetirizine on nasal provocation testing with adenosine monophosphate compared with allergen challenge in allergic rhinitis

Background End‐organ hyperreactivity is an important feature of the allergic airway. There are no data directly comparing the responsiveness to treatment of different nasal provocation tests (NPT). Objective We compared the effect of levocetirizine on nasal adenosine 5′‐monophosphate (AMP) with specific allergen challenge in patients with intermittent and persistent allergic rhinitis (AR). Methods Patients with AR were randomized in double‐blind cross‐over fashion to receive single doses of levocetirizine 5 mg or identical placebo, with nasal challenge performed 12 h after dosing. Sixteen participants completed per protocol. Nasal AMP or allergen challenge was conducted on separate days with 1‐ and 2‐week washout periods in between, respectively. Measurements of peak nasal inspiratory flow (PNIF) were made over 60 min after each challenge. The primary end‐point was the provocative concentration of AMP or allergen causing a 20% drop in the PNIF (PC₂₀). Results The time‐profile for PNIF recovery [area under the 60 min time–response curve as % PNIF change (min)] were significantly attenuated for AMP challenge, as mean difference [95% confidence interval (CI)]: 11.57 (3.87, 19.25), P=0.005 and for allergen challenge: 17.82 (0.11, 35.53), P=0.04. A highly significant correlation was shown between methods for the area under the curve: (R=0.86, P<0.001). A statistically significant correlation was also seen for the PC₂₀: (R=0.94, P<0.001). PC₂₀ improvement amounted to a 1.26 (95% CI 0.16, 2.35) and 0.16 (95% CI ?0.41, 0.73) doubling‐dilution shifts for allergen and AMP challenges, respectively. Bland–Altman plots confirmed good agreement between methods. Conclusion A high correlation and statistical agreement has been demonstrated between AMP and allergen challenge for all outcome measures. In particular, the recovery profile after NPT is a sensitive and discriminatory measure of anti‐allergic treatment.     

Evaluation of acoustic rhinometry in a nasal provocation test with allergen

BACKGROUND: The objective was to validate acoustic rhinometry (AR) in a nasal challenge with allergen. METHODS: Nasal response to allergen provocation was based on clinical and symptom scores, cross-sectional changes of the nasal mucosa as measured by AR with the Rhinoklack system, and peak nasal inspiratory flow (PNIF), in atopic and nonatopic volunteers. RESULTS: After allergen challenge, mean variation in minimal cross-sectional area (deltaMCA), as measured by AR, or in peak nasal inspiratory flow (deltaPNIF) in nonatopic volunteers, was -0.4+/-14.3% and 5.2+/-15.7%, respectively, compared to baseline. This allowed the determination of a reaction threshold of -29% for deltaMCA and of -26% for deltaPNIF. All but one of the 30 atopic patients reached the AR reaction threshold, whereas all patients reached the PNIF reaction threshold. AR and PNIF closely correlated with clinical and symptom scores for nasal congestion, since there was no significant difference at reaction threshold between both methods. CONCLUSIONS: In an allergen provocation test, AR appears to be as specific and sensitive as peak nasal inspiratory flow, with the advantage of being independent of the patient's active cooperation. Discrepancies between both methods emphasize the role of nasal cavity anatomy in measuring nasal congestion by AR.     

Epithelial shedding is associated with nasal reactions to cold, dry air

BACKGROUND: Cold, dry air (CDA) can cause symptoms of rhinitis and obstructive airway responses. The pathophysiology of these reactions is not understood. One hypothesis is that the respiratory mucosa of individuals with CDA sensitivity cannot compensate for the loss of water that occurs on exposure to the stimulus, leading to epithelial damage. OBJECTIVE: To test for an association between nasal reactions to CDA and the number of epithelial cells recovered in nasal fluids. METHODS: Ten CDA-sensitive subjects received nasal provocations with CDA and warm, moist air; 10 CDA-insensitive subjects received CDA; and 10 subjects with allergic rhinitis received allergen and diluent challenges. Nasal lavage cytology was performed at baseline and after the challenge. Symptoms were recorded and histamine, [3H]-N-alpha-tosyl-L-arginine methyl ester-esterase activity, tryptase, and albumin were assayed in nasal lavages. RESULTS: A 6-fold increase in nasal lavage epithelial cells was found in the CDA-sensitive group after CDA (P < .01), but not after warm, moist air. No changes were observed in the CDA-insensitive group, or after allergen or diluent in allergic rhinitis. CONCLUSION: Epithelial cell shedding accompanies clinical responses to CDA in the human nose. This supports the hypothesis that the airway mucosa of CDA-sensitive individuals cannot compensate for the water loss that occurs under extreme conditions leading to epithelial damage. CLINICAL IMPLICATIONS: A defect in mucosal water homeostasis may need to be considered in individuals who get excessive nasal symptoms when exposed to cold and dry, windy environment.     

Facial thermography during nasal provocation tests with histamine and allergen

Changes of skin temperature (T°) of the nose area during nasal provocation tests with histamine and allergen were followed by means of an infrared thermography camera. By a colimator system in which temperatures measured on a given surface can be integrated and averaged, thermography allows the continuous and quantitative recording of the temperature during the whole procedure in a completely noninvasive way. In 10 normal subjects, increasing doses of histamine induced a dose-dependent rise of the nose external temperature. No significant change was observed with the vehicle solution. In six subjects allergic to grass pollen, the nebulization of increasing concentrations of a pollen extract induced a dose-dependent rise in T°. The T° rise observed after histamine or allergen corresponded to a marked nasal obstruction. The nebulization of the highest dose of the pollen extract did not induce any T° rise in six nonallergic subjects. The continuous recording of the skin temperature by a noninvasive method might yield additional information on the vascular changes rapidly occurring during nasal challenges.     

Metachromatic cells in nasal mucosa after allergen challenge

Metachromatic cells in the nasal mucosa were studied in relation to symptoms in 16 schoolchildren and 11 adults with hay fever who were challenged with pollen outside the pollen season, using either a gentle scraping-cytocentrifugation method for collection of mucosal specimens or biopsies. There was a temporary redistribution of metachromatic cells towards the mucosal surface appearing 5-24 h after challenge, with a correlation between the quantity of metachromatic cells and symptom scores. Thus, a single exposure to high doses of allergen may contribute to priming in susceptible individuals.     

Bilateral nasal allergen provocation monitored with acoustic rhinometry. Assessment of both nasal passages and the side reacting with greater congestion: relation to the nasal cycle

BACKGROUND: The effect of bilateral nasal provocation on nasal mucosa measured with the use of acoustic rhinometry (AR) can be assessed for both nasal passages or for the side responding with greater congestion. Assessment of changes in nasal congestion during the nasal provocation test (NPT) can be affected by the nasal cycle (NC). The aim of this study was to find out the most accurate method to evaluate changes observed during bilateral nasal provocation. METHODS: Cross-sectional areas (CSA) at the level of inferior nasal turbinate (CSA-2) were recorded by AR in 26 volunteers with allergic rhinitis during the NC for 5-7 h and subsequently during NPT. The risk of spontaneous total and unilateral CSA-2 decrease was established. Sensitivity of the NPT assessment for the total CSA-2 and for the side responding with greater congestion was evaluated at chosen thresholds. These thresholds were selected in a way that the risk levels of spontaneous decrease of unilateral and total CSA-2 were equal. RESULTS: The assessment of the total CSA-2 was found to be more sensitive than the assessment of the side responding with greater congestion. The highest sensitivity and specificity of the test was achieved by using a combination of both assessments. Optimum thresholds of the CSA-2 decrease for assessment at 15 min after provocation, with this method, were 27% and 40% for the side responding with greater congestion and for the total CSA-2, respectively. CONCLUSIONS: Recognition of the risk of spontaneous unilateral and total CSA-2 decreases enables introduction of combined assessment of bilateral NPT. This assessment seems to be the most accurate method for evaluation of the test results.     

An approach to the understanding of the nasal early-phase reaction induced by nasal allergen challenge

Quantitative determinations of the inflammatory mediators in nasal secretions were performed and correlated with the objective nasal symptoms within 1 h after nasal allergen challenge (NAC). Twenty-six patients with seasonal allergic rhinitis were enrolled outside the pollen season. All measurements were performed before (as a baseline control) and at 1, 5, 10, 30, and 60 min after NAC. This study aimed to clarify the pathogenic mechanism of the early-phase reaction (EPR) by monitoring the evolution of early-phase mediators in nasal secretions and the presence of nasal symptoms during this period. The results showed that, after NAC, the maximal mediator concentration was already reached after 1 min for histamine (124 ng/g), 5 min for tryptase (56 μU/g), and 5-10 min for leukotriene C₄ (40 ng/g). Itching and sneezing started as early as 20-30 s, and they were predominant symptoms within 5 min. Rhinorrhea and nasal obstruction started a few minutes after NAC and lasted until more than 1 h after NAC. There was no significant correlation between any single mediator and nasal symptoms during the sampling period. In conclusion, this study demonstrated that during the EPR the presence of nasal symptoms involves a complex mechanism, reflecting the interaction between the mediators released by inflammatory cells, and the receptors on different target organs. When evaluating symptoms during the EPR, one must consider not only the severity of these symptoms but also the time period within which these symptoms occur. For the symptoms of nasal obstruction and rhinorrhea, the early-phase reaction often lasted more than 1 h.     

Efficacy and onset of action of fluticasone propionate aqueous nasal spray on nasal symptoms, eosinophil count, and mediator release after nasal allergen challenge in patients with seasonal allergic rhinitis

We studied the effect and onset of action of fluticasone propionate aqueous nasal spray (FPANS) on mediator release and eosinophil accumulation in nasal secretions and on nasal symptoms of patients with seasonal allergic rhinitis after nasal allergen challenge (NAC). At the end of the pollen season, 28 patients were randomized in a double-blind and crossover design to receive 7 days' treatment with FPANS (200 μg, once daily) and matching placebo. NACs were performed before and at 6 h and 1. 2. 3. and 7 days during treatment with FPANS or placebo. Nasal secretions were collected for a quantitative determination of mediators and eosinophil count before and 5 min after each challenge. Nasal symptoms were assessed by scales grading the severity of symptoms at the same time. Results showed that for mediator concentrations there was a significant decrease of leukotriene C4 (P<0.001) at 7 days after the first administration of FPANS as compared to placebo. Two days after FPANS. both eosinophil counts and eosinophil cationic protein (ECP) concentrations were lower than those of placebo (eosinophils; f=0.032; ECP; F=0.038). The onset became even more important at day 7 (eosinophils; P=0.001; ECP; P=0.009) during the FPANS treatment period. For the subjective nasal symptoms, a significant reduction of symptom scores for nasal obstruction occurred also at day 3 (F=0.017) and for sneezing at day 7 (f=0.003). There was not yet any significant improvement of the objective nasal airway resistance after the different NACs during the study period. In conclusion, this study demonstrated that topical fluticasone propionate is effective in the treatment of mucosal inflammation induced by NAC. For optimal control of nasal symptoms induced by repeated maximal allergen challenges, a treatment period of more than 1 week is required.     

Topical corticosteroid inhibits interleukin-4, -5 and -13 in nasal secretions following allergen challenge

BACKGROUND: Cytokines and chemokines produced by allergen-reactive T-helper type 2 (Th2) cells may be pivotal to the pathophysiology of allergic disorders. OBJECTIVE: This study was performed to assess the effect of 7 days of topical corticosteroid on nasal allergen challenge (NAC) in terms of eosinophils, cytokines and chemokines obtained by nasal lavage and filter paper methods. METHODS: Patients with grass pollen seasonal-allergic rhinitis (n = 13) out of season received nasal challenge following matched placebo (twice daily into each nostril for 7 days) and fluticasone propionate (100 microg twice daily into each nostril for 7 days). Chemokine and cytokine levels were analysed using a sensitive automated bead immunoassay system at intervals up to 8 h after NAC. RESULTS: Levels of cytokines and chemokines from filter paper were generally higher than from nasal lavage. Fluticasone propionate caused a reduction in symptoms, total leukocyte counts and eosinophils, and abrogation of IL-4, IL-5, IL-6 and IL-13 responses in the filter paper taken in the late phase (P < 0.05 for IL-4 and IL-13, P < 0.01 for IL-5 and IL-6). Levels of chemokines (eotaxin, RANTES, MCP-1, MIP-1alpha, IL-8 and IP-10) were also reduced in the late phase (P < 0.01 at 8 h). However, levels of IL-2, IL-3, IL-7, IL-12 (p40 and p70), -15, TNF-alpha, IFN-gamma and GM-CSF were not affected. CONCLUSION: Fluticasone propionate has selective inhibitory effects on Th2 cytokine synthesis following nasal challenge, while also decreasing release of chemokines, but not affecting levels of Th1 cytokines.     

Nasal nitric oxide: longitudinal reproducibility and the effects of a nasal allergen challenge in patients with allergic rhinitis

BACKGROUND: Exhaled nitric oxide (eNO) is a validated noninvasive marker of airway inflammation in asthma. In patients with allergic rhinitis (AR), increased levels of nasal nitric oxide (nNO) have also been measured. However, the applicability of nNO as a marker of upper airway inflammation awaits validation. AIM: To test the longitudinal reproducibility of standardized nNO measurements in patients with AR and the effects of nasal allergen challenge. METHODS: Twenty patients with clinically stable, untreated AR participated in a combined study design. First, reproducibility of nNO was tested over 1, 7, and 14-21 days. Subsequently, the effect of nasal allergen challenge on nNO was studied in a placebo-controlled, parallel design. Nasal NO was measured with a chemoluminescence analyzer. Ten subjects randomly underwent a standardized nasal allergen challenge; 10 subjects received placebo. Response to nasal challenge was monitored by composite symptom scores. RESULTS: There was a good reproducibility of nNO up to 7 days [coefficient of variation (CV) over 1 (16.45%) and 7 days (21.5%)], decreasing over time [CV (14-21 days): 38.3%]. As compared with placebo, allergen challenge caused a significant increase in symptom scores (P < 0.001), accompanied by a decrease in nNO at 20 min postchallenge (P = 0.001). Furthermore, there was a gradual increase in nNO at 7 h, reaching significance at 24-h postallergen (P = 0.04). CONCLUSIONS: Similar to eNO in asthma, nNO is a noninvasive marker, potentially suitable to monitor upper airway inflammation following allergen-induced late response. Present data show a good reproducibility of nNO measurements, decreasing over time, probably because of subclinical seasonal influences.     

Sequence polymorphism of the group 1 allergen of Bermuda grass pollen

BACKGROUND: Cyn d 1, the major allergen of Bermuda grass pollen, consists of a number of isoforms. OBJECTIVE: To examine the extent of sequence variation of Cyn d 1 isoforms at the molecular level. METHODS: A Bermuda grass pollen lambdaZAP II cDNA expression library was immunoscreened with anti-Cyn d 1 monoclonal antibodies. The reactive clones were isolated, subcloned into Escherichia coli, and sequenced. Some of them were expressed in the yeast Pichia pastoris to obtain recombinant Cyn d 1 proteins. RESULTS: Ten cDNA clones were obtained, all these clones encode the full length of Cyn d 1 protein. Their deduced mature proteins can be grouped into: the long ones with 246 amino acids, and the short ones with 244 amino acids. The last two amino acids (AG) of the long Cyn d 1 are deleted in the short Cyn d 1. The remaining amino acid sequences share more than 98% identity; a total of nine amino acid variations were observed. Two recombinant Cyn d 1 proteins (rCyn d 3-2 and rCyn d 5-4) with three amino acid substitutions showed differential IgE-binding profiles. CONCLUSION: The present study extended our understanding of the primary structure of isoforms of Cyn d 1.     

Effect of nasal allergen challenge on serotonin, substance P and vasoactive intestinal peptide in plasma and nasal secretions

We have studied the changes in concentration of serotonin, substance P, and vasoactive intestinal peptide (VIP) in plasma following a nasal allergen provocation in 14 grass pollen-allergic subjects; in five the urinary excretion of serotonin and 5-hydroxy-indolyl-acetic acid (5-HIAA) was also measured. In addition, the concentration of serotonin and substance P was measured in nasal secretions following nasal challenge with allergen and methacholine. The results showed an allergen-induced increase in free plasma serotonin (P less than 0.01) and no change in platelet serotonin, urinary serotonin and urinary 5-HIAA. The plasma substance P level tended to fall (P greater than 0.1), while plasma VIP increased significantly (P less than 0.02). In nasal secretions, there were measurable levels of serotonin in all samples and of substance P in all but one. There was no difference between the concentrations of serotonin and substance P in secretions collected after allergen challenge and after methacholine challenge. For both substances, the secretion median value was comparable to that of plasma. Symptom reduction by topical and systemic pretreatment with a serotonin- and VIP-antagonist before nasal allergen provocation is necessary to define the role of these two agents in allergic rhinitis.