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.     

Detection of an allergen in dog dander that cross-reacts with the major cat allergen, Fel d 1

BACKGROUND: A considerable proportion of animal-allergic patients are sensitized to both cat and dog allergens but knowledge about cross-reactive allergens in cat and dog dander is limited. OBJECTIVE: To investigate whether dog dander contains an allergen that cross-reacts with the major cat allergen, Fel d 1. METHODS: Recombinant Fel d 1 with the same immunological properties as natural Fel d 1 was used for quantitative (CAP) IgE competition experiments performed with sera obtained from cat-allergic patients (n=36). A Fel d 1 cross-reactive dog allergen was characterized by one- and two-dimensional immunoblotting using rFel d 1 for IgE inhibition experiments and with monospecific, polyclonal rabbit anti-recombinant Fel d 1 antibodies. RESULTS: In 25% of Fel d 1-reactive cat-allergic patients, more than 50% inhibition of IgE reactivity to dog allergens was achieved with recombinant Fel d 1. An Fel d 1 cross-reactive 20 kDa allergen with a pI of approximately 3.4 was detected in dander extracts of several different dog breeds. CONCLUSION: This is the first report demonstrating the presence of an Fel d 1-like allergen in dog dander extracts, which may be responsible for double positivity to cat and dog in serology. However, the clinical relevance of this cross-sensitization needs to be confirmed. These results are important for the diagnostic and therapeutic use of dog dander allergen extracts.     

Nasal histamine release following hyperosmolar and allergen challenge

Allergic rhinitis is characterised by symptoms of sneezing, itching of the nose with watery secretions, and nasal obstruction. We have previously shown that patients can have the diagnosis of allergic rhinitis confirmed by nasal provocation tests and assessment of nasal inspiratory peak flow (NIPF) after specific allergen or hyperosmolar challenge. We now show that histamine is released into the nasal lavage fluid in response to such challenges. Saline lavage alone results in detectable histamine levels in the order of 5 ng/ml, but in the presence of allergen (HDM) there is a significant increase in histamine release in atopics but not in control subjects. With hyperosmolar challenge, atopics showed a biphasic response in that histamine release was increased with 1.8% and 3.6% saline but returned to baseline with 5.4% and 7.2% saline, then showing a further increase with 9.0% saline. This raises the possibility of two populations of responsive mast cells. Hyperosmolar challenge leads to symptoms of nasal itch and sneezing as well as histamine release in atopics but not in controls. This suggests that hyperosmolar challenge can be used as a simple diagnostic test for allergic rhinitis and may provide a model for nasal hyper-reactivity.     

Effects of intranasal fluticasone and salmeterol on allergen-induced nasal responses

BACKGROUND: Combination of inhaled steroid and long-acting beta-agonists has synergistic effects in asthma. OBJECTIVE: To investigate whether nasal corticosteroid and long-acting beta-agonists have synergistic effects on allergen-induced nasal responses. METHODS: The effects of intranasal treatment with fluticasone p-MDI (50 microg bid), salmeterol p-MDI (25 microg bid), their combination, and placebo, on nasal symptoms, eosinophil differential cell count and albumin in nasal lavage fluid (measures of inflammation and leakage respectively) and nasal electrical potential difference (measure of epithelial integrity) were studied in 11 atopic subjects with rhinitis, in a randomized, partially-blinded, 4-period, cross-over study. The measurements were made at baseline, at the end of 1 week of treatment, and immediately after a nasal allergen provocation. RESULTS: Allergen-induced sneeze, postnasal drip and nasal obstruction were significantly reduced by fluticasone, but not by salmeterol. Eosinophil count in postallergen nasal lavage fluid was significantly less after fluticasone (median 1.9%, IQR 4.6) and salmeterol treatment (median 2.5%, IQR 8.5) compared with placebo (median 12.5%, IQR 27.9). Compared with placebo, both fluticasone and salmeterol attenuated allergen-induced change in nasal potential (mean change from baseline -18.5, +0.4 and -7.2% respectively) and the increase in nasal albumin (median 154, 119 and 130 ng/ml respectively). Combination treatment did not have any additional benefits over the individual therapies. CONCLUSIONS: Although salmeterol has anti-inflammatory properties, intranasal salmeterol or its combination with fluticasone do not offer any added benefit over intranasal fluticasone alone for allergen-induced nasal responses.     

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.     

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.     

Modulation of neurotrophin and neurotrophin receptor expression in nasal mucosa after nasal allergen provocation in allergic rhinitis

Background: Patients with allergic rhinitis (AR) feature both allergic airway inflammation and a hyperresponsiveness to nonspecific stimuli which is partly neuronally controlled. Still, it is unclear whether or not neurotrophins are involved in airway pathophysiology of AR and in nasobronchial interaction. Methods: Nine AR patients with mono‐allergy to grass pollen and nine healthy controls underwent nasal allergen provocation (NP). Serum samples, nasal and bronchial biopsies were taken before (T₀) and 24 h after (T₂₄) NP. Pan‐neurotrophin receptor p75^NTR, tyrosine kinase A (trkA), trkB, nerve growth factor (NGF), and brain‐derived neurotrophic factor (BDNF) were assessed with immunohistochemistry, and NGF and BDNF levels with ELISA. Results: At T₂₄, BDNF and NGF were upregulated in nasal mucosa (P < 0.05) and increased in the peripheral blood of AR compared with T₀. The increase in nasal BDNF expression correlated positively with the maximum increase in total nasal symptom score in AR (P = 0.02). p75^NTR was expressed on peripheral nerves and epithelial layer, trkA on endothelial cells, and trkB on mast cells. trkB + mast cells significantly decreased after NP in AR (P < 0.01). NP did not modulate p75^NTR and trkA expression in nasal mucosa and had no effect on the expression of neurotrophins and receptors in bronchial mucosa. Conclusion: This study shows that neurotrophins and their receptors are expressed in human airways. Allergic rhinitis was characterized by a modulation of BDNF, NGF, and trkB in nasal mucosa after NP and a correlation of nasal BDNF with the maximal increase of total nasal symptom score. Therefore, our data suggest that neurotrophins participate in upper‐airway pathophysiology in AR, whereas their role in nasobronchial interaction remains unclear.     

Eosinophilic rhinitis accompanies the development of lower airway inflammation and hyper-reactivity in sensitized mice exposed to aerosolized allergen

BACKGROUND: Allergic rhinitis is a risk factor for the development of asthma. About 80% of asthmatic patients also have rhinitis. However, the pattern of induction of allergic rhinitis and asthma remains unclear. OBJECTIVE: The purpose of this study was to investigate the development of upper airway inflammation in mice during the development of an asthma-like disease and after an acute allergen provocation. METHODS: BALB-c mice were sensitized intraperitoneally (i.p) to ovalbumin (OA, days 1-13) and were challenged with aerosols of either OA or saline on 8 consecutive days (days 33-40). In a second experiment, chronic exposure for 8 days was followed by 10 days of rest and then an acute nebulized allergen provocation was performed (day 50). Inflammatory parameters were investigated at different time-points. RESULTS: Upper and lower eosinophilic airway inflammation were simultaneously induced in the course of repeated inhalations of nebulized OA, as shown by analyses of nasal and broncho-alveolar lavage fluids and histological sections of the nose and bronchi. Mice that developed bronchial hyper-responsiveness also had increased thickness of the nasal mucosa on magnetic resonance image (MRI) scans. When chronic exposure was followed by acute allergen provocation, the latter caused a systemic increase in IL-5 levels, with a concomitant rise in blood and airway eosinophils, primarily in the nose. CONCLUSIONS: Simultaneous induction of eosinophilic inflammation in the nose and lungs was found in a mouse model of respiratory allergy. These findings support the viewpoint that upper and lower airway disease represent a continuum of inflammation involving one common airway and provide evidence for the concept of global airway inflammation after inhalation of allergen.     

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.     

Immediate and dual response to nasal challenge with Dermatophagoides pteronyssinus in local allergic rhinitis

Background Local allergic rhinitis (LAR) is characterized by in situ production of specific IgE (sIgE) antibodies and a positive response to a nasal allergen provocation test (NAPT) in the absence of atopy. Objective The aim of this study was to investigate the immunological mechanisms involved in the immediate and late responses after nasal exposure to Dermatophagoides pteronyssinus (DP) in patients with LAR. Methods A total of 40 subjects with LAR to DP were studied and compared with 50 healthy controls. Immediate and late responses to NAPT‐DP were assessed using a visual analogue scale of nasal symptoms and acoustic rhinometry. Tryptase, ECP, total and sIgE‐DP were measured in the nasal lavage by immunoassay at baseline, 15 min, 1, 6 and 24 h after nasal challenge. Results NAPT‐DP was positive in all patients, with significant increases in tryptase (45%), ECP (65%) and sIgE‐DP (25%) (P<0.05). Sixty percent of the LAR patients presented an immediate response to NAPT‐DP and 40% a dual response. Immediate responders showed a fast release of tryptase with a peak at 15 min after NAPT‐DP, and a progressive increase in nasal ECP and sIgE‐DP from 1 to 24 h after challenge, with a peak at 24 h. Dual responders presented persistently higher levels of tryptase from 15 min to 6 h after challenge, and a similar pattern of nasal release of ECP and sIgE‐DP to immediate responders. There were no isolated late responders. NAPT‐DP was negative in all healthy controls, with no increases in tryptase, ECP, or total and sIgE‐DP in nasal secretions. Conclusions The results demonstrated the existence of immediate and dual responses to a NAPT with DP in LAR patients, with the local presence of sIgE and mast cell/eosinophil activation. Cite this as: S. López, C. Rondón, M. J. Torres, P. Campo, G. Canto, R. Fernandez, R. Garcia, A. Martínez‐Cañavate and M. Blanca, Clinical & Experimental Allergy, 2010 (40) 1007–1014.     

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.     

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.     

Threshold levels of purified natural Bos d 2 for inducing bronchial airway response in asthmatic patients

BACKGROUND: Provocation tests are invaluable in establishing threshold levels and a causal relationship between atopic asthma and a certain allergen source, especially in relation to work-associated exposure. Purified major allergens open possibilities for a more accurate assessment of sensitization. OBJECTIVE: To determine the threshold dose of purified major bovine dander allergen Bos d 2 in bronchial provocation in comparison with the standard allergen and a set of other parameters of allergy. METHOD: Nine consecutive patients referred to hospital for confirming the bovine origin of their occupational asthma were subjected to bronchial provocation tests with purified natural Bos d 2 and a standard bovine dander allergen. Additional tests included bronchial histamine challenge, measurements of total IgE, specific IgE antibody determinations and skin prick tests (SPT) with both allergens. RESULTS: In the provocation tests with Bos d 2, a 15% decrease in the forced expiratory volume in 1 s (FEV1) and/or peak expiratory flow (PEF) values in eight out of nine patients confirmed the predominant role of Bos d 2 in the sensitization. The threshold dose of Bos d 2 varied from 0.1 microg to > 100 microg (median +/- median absolute deviation = 4.5 +/- 3.9 microg). A positive SPT was induced by a median dose of 13.9 +/- 9.8 microg of Bos d 2. Bronchial response to histamine and IgE antibodies against Bos d 2 showed the highest correlations to the provocations results. CONCLUSIONS: The efficacy of Bos d 2 in bronchial provocation in patients with occupational cattle-associated asthma was confirmed and the range of the threshold level was determined. There were individual variations, but the response in provocation remains the reference method for identification of the cause of occupational atopic asthma. SPT and the measurement of specific IgE antibodies, preferably with purified or recombinant major allergens, increase the accuracy of the diagnosis.     

Lower airway inflammatory responses to repeated very-low-dose allergen challenge in allergic rhinitis and asthma

BACKGROUND: Low-dose allergen challenge (LDAC) may be a useful tool for studying the capacity of allergens to induce airway inflammation in atopic subjects. OBJECTIVE: To evaluate lower airway inflammatory changes following repeated inhalation of very low doses of allergen (VLDAC) in non-asthmatic subjects with allergic rhinitis (NAAR) compared with mild allergic asthmatic subjects (AA). METHODS: Fourteen NAAR and 11 AA were seen out of the pollen season and had skin prick tests with common aeroallergens. Baseline spirometry (S) and methacholine challenge (MC) were done and blood and induced sputum (IS) differential cell counts were obtained. Each subject underwent VLDAC on four consecutive mornings with a relevant allergen. S, MC, and blood and IS samplings were repeated 6 h after the second and fourth VLDAC and one week later. RESULTS: Although there were, as expected, no changes in FEV1 or PC20 in either group, mean percentage eosinophils on IS were significantly increased in NAAR on day 2 of VLDAC and decreased in all but one subject on day 4, with a tendency to return to baseline levels one week later. In AA, there was a non-significant trend for sputum eosinophils to increase on day 2; four subjects showed a decrease of eosinophils on day 4 of VLDAC. There was a correlation between eosinophil cationic protein (ECP) levels and eosinophil counts in NAAR throughout the study. There were no variations in other sputum cells or blood inflammatory cells. CONCLUSION: VLDAC can increase the percentage of eosinophils in IS of NAAR subjects without associated respiratory symptoms nor physiological modifications. A reduction in eosinophilic response despite repeated exposure, more common in NAAR subjects, suggests an adaptation process that needs to be further evaluated.