The allergen bronchoprovocation model: an important tool for the investigation of new asthma anti-inflammatory therapies

Allergen bronchoprovocation tests have been used for more than two decades in the investigation of respiratory allergic diseases such as asthma and rhinitis. These bronchial challenges are now well standardized and can offer key information on the therapeutic potential of new agents and on their anti-inflammatory effects on the airways. Both standard and low-dose allergen provocations are safe when performed by experienced investigators and do not lead to persistent worsening of asthma or change in airway function. The evaluation of new therapeutic agents by these methods can also provide important information on the mechanisms of development and persistence of airway diseases.     

Comparison of incremental and bolus dose inhaled allergen challenge in asthmatic patients

BACKGROUND: Attenuation of airway responses to inhaled allergen is increasingly used to evaluate anti-asthma drugs. Many studies use different allergen challenge methods and the presence of the late asthmatic response can be identified by a screening challenge with inhalation of incremental doses of allergen. Once defined, subsequent challenges are often administered as a constant dose based on the dose from the screening challenge. Previously, constant dose challenges have been employed but never validated. OBJECTIVE: A comparative study of two methods of delivering inhaled allergen by evaluating the responses of an incremental dose allergen challenge and the same cumulative dose administered as a bolus over a single inhalation. METHODS: Thirty-five male patients with mild allergic asthma underwent incremental dose challenge followed 3-6 weeks later by a bolus dose challenge. Bronchoconstrictor responses were expressed as the maximum percentage fall in FEV1 from baseline during the early (0-2 h) and late (4-10 h) asthmatic responses and area under the percentage change in FEV1-time curve (AUC). RESULTS: There were no significant differences between the challenges. The mean +/- SEM fall in FEV1 following incremental and bolus dose challenge was 33.1 +/- 1.8% and 29.9 +/- 2.2% during the early response, and 36.9 +/- 2.4% and 34.0 +/- 3.1% during the late response, respectively. The mean +/- SEM AUC following incremental and bolus dose challenge was 35 +/- 3 and 33 +/- 3 Delta%FEV1/h for the AUC0-2 h, 147 +/- 12 and 139 +/- 16 Delta%FEV1/h for the AUC4-10 h, and 204 +/- 14 and 190 +/- 19 Delta%FEV1/h for the AUC0-10 h, respectively. CONCLUSION: Bolus dose allergen challenge is a safe method to administer inhaled allergen in clinical trials with a valid response when compared with incremental dose allergen challenge.     

A comparative study of four different bronchial challenge tests

Haugaard L, Iversen M, Dahl R. A comparative study of four different bronchial challenge tests.
The correlation between 4 commonly used tests for assessment of airway hyperresponsiveness was studied in 10 asthmatics by performing bronchial challenges with histamine, ultrasonically nebulised distilled water (UNDW), exercise, and a relevant allergen. All tests were performed within 4 weeks for each individual and at least 48 h apart. A significant correlation was found between histamine challenge and exercise challenge (r=−0.74). Other correlations were substantial (0.4–0.5) but did not reach statistical significance. This suggests that the challenges have different mechanisms or pathways leading to bronchoconstriction, and assessment of the pattern of individual patients' bronchial hyperresponsiveness may require application of a number of different bronchial challenge tests. For routine use histamine challenge seems the most appropriate.     

Early increase in urinary leukotriene E4 (LTE4) is dependent on allergen dose inhaled during bronchial challenge in asthmatic subjects

BACKGROUND: Urinary leukotriene E4 (LTE4) excretion is a good marker of the rate of total body production of sulfidopeptide leukotrienes released during allergen challenge. METHODS: Twenty-three subjects with allergic asthma were challenged with inhaled allergen, and the urinary excretion of LTE4 was determined by immunoenzymatic assay (associated with HPLC separation) at various intervals after challenge. RESULTS: Allergen challenge caused an early airway response (EAR) with a drop in FEV1 of 40.3+/-9.9%. This was associated with an increase in urine LTE4 excretion for 0-3 h after allergen inhalation (296+/-225.25 pg/mg creatinine) in comparison with baseline values obtained during the night before challenge (101.02+/-61.97 pg/mg creatinine). Urinary LTE4 excretion was significantly higher in subjects who inhaled a higher dose of allergen during challenge (LTE4 during EAR: 211+/-192 pg/mg creatinine in subjects with inhaled total dose of allergen <0.1 biologic units; 408+/-223 pg/mg creatinine in subjects with inhaled total dose >0.1 biologic units). All subjects showed a late airway response (LAR) to allergen of different severity, from mild (FEV1 fall: 15-20%) to severe (>30%); no correlation was found between the increase in urine LTE4 excreted during LAR (3-7 h after challenge) and the severity of LAR, but only subjects with severe LAR showed a significant increase in LTE4 during LAR in comparison with baseline value. CONCLUSIONS: A release of sulfidopeptide leukotrienes, as evaluated by urinary LTE4 excretion, can be documented during EAR and LAR to allergen in relation to the dose of inhaled allergen, and it can represent a useful index of the events underlying the airway inflammatory responses during allergen challenge.     

Predictors of early- and late-phase reactions to bronchial allergen challenge

The influence of inhaled steroids and predictive factors on the response to bronchial allergen challenge (BCA) was evaluated in. 80 asthmatics allergic to Dermatophagoides pteronyssinus (Der p). All underwent BCA with Der p and measurement of early (EAR) and late asthmatic reaction (LAR). The cumulative dose of allergen producing 20% fall in FEV₁, in the EAR (PD₂₀) was calculated. Bronchial histamine provocation, conjunctival provocation test (CPT), and skin prick test with Der p extract were performed. Specific IgE to Der p in serum (RAST), blood eosinophil (EOS) count, serum eosinophil cationic protein, and eosinophil protein X were measured. Thirty patients (38%) were treated with inhaled steroids. All patients had at least a 20% fall in FEV₁ in EAR. Some 42% of nonsteroid- and 33% of steroid-treated patients had LAR with fall in peak flow of at least 20%. For patients not treated with steroid, 35% of variation in PD₂₀ was explained by RAST and histamine reactivity, and 53% of variation of observed PD₂₀ could be predicted. The baseline FEV₁, EOS, and EAR explained 28% of variation in LAR, and 28% of variation in observed LAR could be predicted. For patients treated with steroids, 38% of variation in PD₂₀ was explained by EOS and histamine reactivity, and only 18% of variation of observed PD₂₀ could be predicted. For patients treated with steroids, it was impossible to predict LAR. We conclude that to achieve a quantitative estimation of allergen-specific EAR and LAR, BCA cannot be replaced by the tests used in this study. Treatment with inhaled steroids modifies the response to BCA, making quantitative prediction of EAR less accurate and prediction of the magnitude of LAR impossible.     

Influence of bronchial allergen challenge on histamine release by human basophils

BACKGROUND: Basophils can be primed by cytokines such as interleukin (IL) -3, IL-5 or granulocyte macrophage-colony stimulating factor (GM-CSF). It has been described that the concentrations of these cytokines are enhanced at sites of allergic inflammation as well as systemic in allergic asthma. OBJECTIVE: To investigate the priming status of basophils as detected by thapsigargin-induced histamine release during bronchial allergen challenge. METHODS: Ten subjects allergic to house dust mite were challenged via an aerosol delivery system. Spontaneous leucocyte histamine release as well as histamine release induced by various stimuli was measured in vitro at several time points. In addition, lung function parameters, serum IL-5 and blood eosinophil counts were evaluated. RESULTS: We found no effect of bronchial allergen challenge upon spontaneous leucocyte histamine release, nor upon histamine release induced by anti-immunoglobulin (Ig) E, house dust mite extract, C5a, fMLP, IL-3, PMA+ thapsigargin or IL-3+ thapsigargin. However, the priming status of basophils as measured by thapsigargin-induced histamine release was enhanced at 24 h after bronchial allergen challenge. Analysis of the individual data showed a heterogeneous initial response (30 min, 6 h) followed by a predominant increase at 24 h after allergen challenge. This increase in the thapsigargin-induced histamine release correlated with the increase in serum IL-5 levels at 24 h after allergen challenge. CONCLUSION: The priming status of human basophils as measured by thapsigargin-induced histamine release is enhanced 24 h after allergen challenge.     

Plasma protein profiles in early asthmatic responses to inhalation allergen challenge

Although mediators, such as lipids, cytokines, and chemokines, are related to the appearance of an IPR, there has been no reliable indicator to predict conditions for the appearance of an IPR.
In this study, we adopted a proteomic approach to investigate the pathogenesis at the level of the plasma proteins and to develop plasma markers to predict the appearance of an IPR following an inhalation challenge with Dermatophagoides pteronyssinus (D.p.). Sixteen mild asthmatics were recruited. Plasma was obtained before challenge and when a decline in forced expiratory volume in 1 s (FEV₁) values greater than 20% from the phosphate-buffered saline value was achieved during D.p. allergen challenge (positive responders), or at 60 min after the highest concentration of D.p. allergen was inhaled (negative responders). After comparing normalized volumes of the spots in the two groups, differentially expressed spots were identified using intra-gel digestion and mass spectrometric analysis. Before D.p. antigen challenge, four spots of gamma fibrinogen and its isoforms were significantly decreased and two spots of complement C3 fragments were significantly increased in the positive responders compared to the negative responders. After D.p. antigen challenge, complement C3 fragment was persistently higher, while gamma fibrinogen was lower in the positive responders than in the negative responders. A validation study using Western blotting showed that gamma fibrinogen expression in the IPR-positive asthmatics was significantly decreased compared to the average of the IPR-negative asthmatic control group. These results indicate that alterations in the complement cascade and fibrinogen may predispose patients to the appearance of an immediate response to D.p. allergen challenge and may provide plasma markers to predict the appearance of an IPR.     

Eosinophils in bronchial mucosa of asthmatics after allergen challenge: effect of anti-IgE treatment

Background: Anti‐IgE, omalizumab, inhibits the allergen response in patients with asthma. This has not been directly related to changes in inflammatory conditions. We hypothesized that anti‐IgE exerts its effects by reducing airway inflammation. To that end, the effect of anti‐IgE on allergen‐induced inflammation in bronchial biopsies in 25 patients with asthma was investigated in a randomized, double‐blind, placebo‐controlled study. Methods: Allergen challenge followed by a bronchoscopy at 24 h was performed at baseline and after 12 weeks of treatment with anti‐IgE or placebo. Provocative concentration that causes a 20% fall in forced expiratory volume in 1 s (PC₂₀) methacholine and induced sputum was performed at baseline, 8 and 12 weeks of treatment. Changes in the early and late responses to allergen, PC₂₀, inflammatory cells in biopsies and sputum were assessed. Results: Both the early and late asthmatic responses were suppressed to 15.3% and 4.7% following anti‐IgE treatment as compared with placebo (P < 0.002). This was paralleled by a decrease in eosinophil counts in sputum (4–0.5%) and postallergen biopsies (15–2 cells/0.1 mm²) (P < 0.03). Furthermore, biopsy IgE+ cells were significantly reduced between both the groups, whereas high‐affinity IgE receptor and CD4+ cells were decreased within the anti‐IgE group. There were no significant differences for PC₂₀ methacholine. Conclusion: The response to inhaled allergen in asthma is diminished by anti‐IgE, which in bronchial mucosa is paralleled by a reduction in eosinophils and a decline in IgE‐bearing cells postallergen without changing PC₂₀ methacholine. This suggests that the benefits of anti‐IgE in asthma may be explained by a decrease in eosinophilic inflammation and IgE‐bearing cells.     

Bronchial and cutaneous responses in atopic dermatitis patients after allergen inhalation challenge

Background Atopic dermatitis (AD) is often associated with allergic asthma (AA). Inhalation of allergens influences the activity of AA but the effect on the skin in AD is unclear. Objectives We evaluated the degree of bronchial hyperresponsiveness to methacholine in eight AD patients with AA (AD⁺) and eight AD patients without AA (AD^–) and studied bronchial and cutaneous responses after allergen inhalation challenge. Methods All patients were treated in hospital for their eczema with tar ointment (pix liquida) and orally administered antihistamines (mean hospital stay 37 days). After clearing of the skin lesions allergen inhalation challenge was performed. Cutaneous responses were studied by measuring the‘Costa’ score before and 24 h after allergen inhalation challenge. Results The median value of the provocative concentration of methacholine causing a 20% fall (PC₂₀ Mch) in forced expiratory volume in 1 second (FEV₁) was significantly higher in the AD^– group compared to the AD⁺ group with median values of 10.70 and 0.60mg/mL, respectively. These values did not change significantly in both groups during hospital stay. After challenge all AD⁺ patients showed early and late asthmatic responses whereas only four AD^– patients showed early asthmatic responses (mean values of the maximal fall in FEV_l during the EAR 37%/16% and in PEF during the LAR 27%/4% for AD⁺ and AD^–patients, respectively). The‘Costa’ score increased in both groups (mean score before 19.1/ 24.4 and after challenge 26.8/26.9 for AD⁺ and AD⁺ patients, respectively). The increase in the AD^– group was significantly higher compared with the AD^– group (P= 0.016). Conclusion We conclude that allergen inhalation challenge causes a flare up of the skin lesions in atopic dermatitis patients. This was more prominent in atopic dermatitis patients who already suffered from an IgE-mediated allergic inflammation in the lung.     

Changes in serum haptoglobin level after allergen challenge test in asthmatic children

Bronchial asthma is characterized by airway inflammation, which underlies the phenomenon of bronchial hyperresponsiveness. Previous studies have shown that this correlates with the serum concentration of haptoglobin. The occurrence of the late asthmatic response (LAR) after an allergen challenge test is associated with airway inflammation. The objectives of this study were to examine serum levels of haptoglobin during the 24 h after allergen challenge and to compare changes between the subjects with and without LAR. We studied two groups of children with perennial asthma who developed the early asthmatic response (EAR) only (group 1: n = 14), and EAR but also LAR (group II: n = 14) after an allergen (Dermatophagoides pteronyssinus) challenge test. Serum concentrations of haptoglobin were measured at baseline, at EAR, and at 2 h (recovery), 8 h (LAR), and 24 h after the challenge. Baseline levels were similar in the two groups (group I: 128±57 mg/dl: group II: 129±50 mg/dl). In group I, there was no significant change in the level at any time point; in contrast, the subjects in group II showed a relative fall (92±40 mg/dl) at 8 h, and an increase (161±79 mg/dl) at 24 h after the challenge. Our results indicate that the serum concentration of haptoglobin decreases at the time of LAR and is subsequently replenished during the ensuing time. Although further studies are needed, we think that haptoglobin may be inflused into the airways during the inflammatory process associated with LAR, and that this may be followed by "overshooting" production.     

Late airway response increases at repeat allergen challenge

The relationship between the immediate and late responses to repeated inhalations of allergen was studied. Sixteen male atopic asthmatics were challenged twice with an interval of 2 weeks. Forced expiratory volume in 1 s (FEV1) was measured serially over an 8-hour period after challenge. The method of provocation used implied that only slight differences between the immediate responses on the two provocation days were observed. However, differences in the late responses were demonstrated. Thus, the maximum percent change in FEV1 at the first and second provocation differed significantly in the late (P less than 0.01), but not in the immediate phase. The increase in maximal late response was greater than the small change in maximal immediate response (P less than 0.05). Further, the FEV1 values from 4 to 8 h post-challenge were in each recording significantly lower on the second day suggesting a more pronounced late bronchial response to repeat challenge. The results suggest that after one challenge specific airway reactivity, i.e., reactivity to allergen, at a subsequent rechallenge is increased in the late phase. This late phase hyperreactivity seems to persist for at least 2 weeks after allergen provocation.     

Direct and indirect bronchoprovocation tests in dose-response studies of inhaled corticosteroids: Past,present, and future directions

Inhaled corticosteroids (ICS) are a mainstay of treatment in eosinophilic asthma. Many studies have explored the dose-response effect of different formulations of ICS through direct or indirect bronchoprovocation testing. Such studies are important for investigating efficacy and identifying the relative potency between formulations. However, lack of consistency in methods and designs has hindered the comparability of study findings. This review discusses current knowledge of the dose-response, or lack thereof, of different formulations of ICS through direct and indirect bronchoprovocation testing. The strengths and weaknesses of past studies inform recommendations for future methodological considerations in this field, such as utilizing a randomized double-blind crossover design, enrolling participants likely to respond to ICS therapy, and carefully selecting treatment durations and washout periods to assess incremental improvement in airway hyperresponsiveness while reducing the likelihood of a carryover effect.     

Increases in eotaxin‐positive cells in induced sputum from atopic asthmatic subjects after inhalational allergen challenge

BACKGROUND: Eosinophils are believed to be critical proinflammatory cells in airway mucosal damage in asthma. Eotaxin is a C-C chemokine with selective activity for eosinophils and basophils. Previous studies have shown increased expression of eotaxin in the airways of asthmatics at baseline. We aimed to investigate eotaxin expression during the late-phase reaction to allergen inhalation in atopic asthmatics. METHODS: Sputum induction was performed before and 24 h after inhalational allergen challenge in atopic asthmatics, and eotaxin protein was detected immunocytochemically. RESULTS: Thirteen patients with a mean decrease in forced expiratory volume in 1 s of 28% (+/-1.5) during the early asthmatic reaction, and 39% (+/-4.7) during the late asthmatic reaction produced sufficient sputum for study. The percentage of eosinophils in sputum was increased 24 h after allergen challenge (P<0.004), and eosinophil percentages in sputum after challenge correlated with the magnitude of the late-phase reaction (r=0.56, P=0.05). The percentage of eotaxin-positive cells increased from 12.6% (range 2-43.8) to 24.3% (8.1-47.1, P<0.005). Allergen-induced increases in eotaxin-positive cells correlated with increases in eosinophils (r=0.63, P<0.01). CONCLUSIONS: These findings suggest that eotaxin may contribute to allergen-induced recruitment of eosinophils to the airway in asthmatic subjects.     

Oxidative stress and airway inflammation after allergen challenge evaluated by exhaled breath condensate analysis

Background Allergen exposure may increase airway oxidative stress, which causes lipid membrane peroxidation and an increased formation of 8‐isoprostane. Objective The aim of the study was to investigate oxidative stress induced by allergen challenge in mild asthmatics, by measuring 8‐isoprostane in exhaled breath condensate (EBC), and to examine their relationship with mediators derived from arachidonic acid. Methods 8‐isoprostane, cysteinyl leukotrienes (cys‐LTs) and prostaglandin E2 (PGE₂) concentrations in EBC were measured at baseline and after allergen challenge in 12 patients with mild allergic asthma sensitized to cat allergen. Results At 24 h after allergen challenge, compared with baseline values, EBC 8‐isoprostane increased [48.64 pg/mL (44.14–53.61) vs. 21.56 pg/mL (19.92, 23.35), P<0.001], cys‐LTs increased [27.37 pg/mL (24.09–31.10) vs. 13.28 pg/mL (11.32, 15.57), P<0.001] and PGE₂ decreased [18.69 pg/mL (12.26, 28.50) vs. 39.95 pg/mL (34.37, 46.43), P<0.001]. The trend of increasing 8‐isoprostane after allergen challenge was significantly correlated with the trend of increasing cys‐LTs (R²=0.85, P<0.001) whereas the trend of decreasing PGE₂ after allergen challenge was significantly correlated with the trend of increasing cys‐LTs (R²=0.52, P=0.001). Conclusions and Clinical Relevance The increase in EBC 8‐isoprostane observed after allergen challenge indicates that allergen exposure increases airway oxidative stress in allergic asthma. The strict correlation between cys‐LTs and 8‐isoprostane underlines the relationship between allergic inflammation and oxidative stress. A shift of arachidonic acid metabolism towards lipoxygenase pathway is induced by the allergen challenge. Airway oxidative stress occurs after allergen challenge even in patients with mild intermittent allergic asthma. Cite this as: L. Brussino, I. Badiu S. Sciascia, M. Bugiani, E. Heffler G. Guida, A. Malinovschi, C. Bucca and G. Rolla, Clinical & Experimental Allergy, 2010 (40) 1642–1647.     

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.     

Oral and Inhaled Sodium Cromoglycate in Challenge Test with Food Allergens or Acetylsalicylic Acid

The prophylactic effect of oral and inhaled sodium cromoglycate (SCG) in challenge tests of patients with IgE-mediated food allergy or sensitivity towards acetylsalicylic acid (ASA) was investigated. In food allergic patients SCG administered orally protected against an asthmatic reaction whereas inhaled SCG was without effect. In ASA sensitivity neither oral nor inhaled SCG protected the patients against bronchospasms. SCG seems to act on mucosal surfaces and may inhibit uptake of macromolecular antigens but not affect the absorption of ASA.     

Inhaled formaldehyde exposure: effect on bronchial response to mite allergen in sensitized asthma patients

BACKGROUND: Formaldehyde, an indoor air pollutant, is known to be an irritant and an etiologic factor in occupational asthma. An epidemiologic study suggests that it may also increase the risk of childhood asthma for concentrations above 60 microg/m(3). AIM: To evaluate the influence of pre-exposure to low-dose formaldehyde (100 microg/m(3) in 30 min according to the World Health Organization's recommended maximum value for indoor environments) on bronchial response to Dermatophagoides pteronyssinus. METHOD: Nineteen asthmatic subjects were included. Each subject underwent a mite allergen bronchial challenge test immediately after a standardized exposure in a chamber to formaldehyde or air (random order). Induced sputum were collected 24 h before and after mite challenge. RESULTS: After formaldehyde inhalation, patients developed an immediate bronchial response at a significantly lower dose of mite allergen than after air exposure (the geometric mean PD(20) for Der p 1 was 34.3 ng after formaldehyde and 45.4 ng after placebo, P = 0.05). The late-phase reaction, expressed as the maximum fall in forced expiratory volume in 1 s (FEV(1)) from baseline, was significantly higher after formaldehyde (15%vs 11%, P = 0.046). CONCLUSION: Our study demonstrated that exposure to low levels of formaldehyde significantly enhanced bronchial responsiveness to mite allergen in mite-sensitized subjects with asthma.