Farm Exposures and Allergic Disease Among Children Living in a Rural Setting.

OBJECTIVES: The purpose of this study was to examine the association between farm exposures and asthma and allergic disease in children while also highlighting the experiences of non-farm rural children. METHODS: This was a cross-sectional analysis of data collected from across the province of Saskatchewan, Canada in 2014. Surveys were completed by parents of 2275 rural dwelling children (farm and non-farm) aged 0 to 17 years within 46 rural schools. Questionnaires were distributed through schools for parents to complete. RESULTS: Asthma prevalence was 7.6%, of which 29.5% of cases were allergic. After adjustment for potential confounders, home location (farm vs non-farm) and other farm exposures were not associated with asthma and asthma phenotypes. Those who completed farm safety education were more likely to have asthma (11.7% vs. 6.7%; p = .001) compared to children without asthma. In sub-analyses among 6-12-year-old children, boys were more likely to have asthma (non-allergic) and use short-acting beta-agonists compared to girls. Doing farm work in the summer was associated with an increased risk of asthma [adjusted OR (aOR) = 1.71 (1.02-2.88); p = .041]. Doing routine chores with large animals was associated with an increased risk of asthma [aOR = 1.83 (1.07-3.15); p = .027] and allergic asthma [aOR = 2.37 (95%CI = 1.04-5.40); p = .04]. CONCLUSION: The present study showed that the prevalence of asthma and asthma phenotypes were similar between farm and non-farm rural children. There did not appear to be differential involvement in farming activities between those with and without asthma although those with asthma had more training suggesting possible attempts to mitigate harm from farm exposures.

Indoor mold levels and current asthma among school-aged children in Saskatchewan, Canada.

Current knowledge regarding the association between indoor mold exposures and asthma is still limited. The objective of this case-control study was to investigate the relationship between objectively measured indoor mold levels and current asthma among school-aged children. Parents completed a questionnaire survey of health history and home environmental conditions. Asthma cases had a history of doctor-diagnosed asthma or current wheeze without a cold in the past 12 months. Controls were age- and sex-matched to cases. Vacuumed dust samples were collected from the child's indoor play area and mattress. Samples were assessed for mold levels and quantified in colony-forming units (CFU). Sensitization to mold allergens was also determined by skin testing. Being a case was associated with family history of asthma, pet ownership, and mold allergy. Mold levels (CFU/m2 ) in the dust samples of children's mattress and play area floors were moderately correlated (r = 0.56; P < 0.05). High mold levels (≥30 000 CFU/m2 ) in dust samples from play [adjusted odds ratio (aOR) = 2.6; 95% CI: 1.03-6.43] and mattress (aOR) = 3.0; 95% CI: 1.11-8.00) areas were significantly associated with current asthma. In this study high levels of mold are a risk factor for asthma in children.

A dual CysLT1/2 antagonist attenuates allergen-induced airway responses in subjects with mild allergic asthma.

BACKGROUND: The cysteinyl leukotrienes (cysLTs) play a key role in the pathophysiology of asthma. In addition to functioning as potent bronchoconstrictors, cysLTs contribute to airway inflammation through eosinophil and neutrophil chemotaxis, plasma exudation, and mucus secretion. We tested the activity of the dual cysLT1/2 antagonist, ONO-6950, against allergen-induced airway responses. METHODS: Subjects with documented allergen-induced early (EAR) and late asthmatic response (LAR) were randomized in a three-way crossover study to receive ONO-6950 (200 mg) or montelukast (10 mg) or placebo q.d. on days 1-8 of the three treatment periods. Allergen was inhaled on day 7 two hours postdose, and forced expiratory volume in 1 s (FEV1 ) was measured for 7 h following challenge. Sputum eosinophils and airway hyperresponsiveness were measured before and after allergen challenge. The primary outcome was the effect of ONO-6950 vs placebo on the EAR and LAR. RESULTS: Twenty-five nonsmoking subjects with mild allergic asthma were enrolled and 20 subjects completed all three treatment periods per protocol. ONO-6950 was well tolerated. Compared to placebo, ONO-6950 significantly attenuated the maximum % fall in FEV1 and area under the %FEV1 /time curve during the EAR and LAR asthmatic responses (P < 0.05) and allergen-induced sputum eosinophils. There were no significant differences between ONO-6950 and montelukast. CONCLUSIONS: Attenuation of EAR, LAR, and airway inflammation is consistent with cysLT1 blockade. Whether dual cysLT1/2 antagonism offers additional benefit for treatment of asthma requires further study.

Allergen-induced early and late asthmatic responses to inhaled seasonal and perennial allergens.

BACKGROUND: The allergen bronchoprovocation (ABP) test is a validated model to study asthma pathophysiology and response to treatments. The inhibitory effect of agents on the allergen-induced late asthmatic response (LAR) is a predictor of their efficacy in asthma treatment. However, it is difficult to predict the magnitude of a LAR, which may vary according to immune responsiveness and the type of allergen used for ABP. AIM: To determine the relationship between the magnitudes of early asthmatic response (EAR) and LAR in mild asthmatic subjects according to the type of allergen inhaled and its determinants. METHODS: This is a retrospective analysis of a large database of ABPs, all performed with a common standardized methodology. Patients were either challenged with house dust mites (HDMs), animals or pollens allergens. EAR was defined as a ≥ 20% fall in forced expiratory volume in 1 s (FEV1 ) < 3 h following ABP and LAR as a ≥ 15% fall in FEV1 between 3 and 7 h post-ABP. The ratio of EAR % fall in FEV1 /LAR % fall in FEV1 was compared between the groups of subjects according to the allergen used for ABP. RESULTS: Data from 290 subjects were analysed: 87 had an isolated EAR and 203 had a dual response (EAR + LAR). Dual responders had a significantly lower baseline PC20 , a more marked fall in FEV1 at EAR, and a trend towards higher baseline sputum eosinophil percentages. The ratio of EAR over LAR was significantly lower in HDM compared with pollen ABP, indicating a larger LAR for a similar EAR. No correlations were observed between the ratio of EAR over LAR and the various parameters recorded in the different groups analysed. CONCLUSION: Different mechanisms may be involved in modulating the magnitude of the LAR, according to the type of allergen. HDM seems to induce a stronger LAR than pollens, animal allergens being intermediary in this regard.

Agreement between questionnaire report of allergy-related outcomes in school-age children and objective measures of atopy: the Saskatchewan rural health study.

BACKGROUND AND OBJECTIVES: In population-based studies, questionnaires remain the most efficient tool to assess the presence of allergy and atopic conditions, but the quality of the information obtained needs to be validated. We sought to evaluate the agreement and predictive values of a questionnaire to assess atopy in rural children, an understudied population with regard to atopy and allergic disease. METHODS: A total of 480 schoolchildren (grades 1-8) from rural Saskatchewan completed a questionnaire report of allergy and atopic outcomes and participated in skin prick testing (SPT). SPT for 6 common allergens (local grasses, wheat dust, cat dander, house dust mite mixed, Alternaria, and Cladosporium) was completed. Subjects with at least one positive SPT (≥ 3 mm) compared to the negative control were considered to be atopic. We considered per cent concordance, Kappa, sensitivity, specificity, and the positive predictive value and negative predictive value (NPV, PPV) of reported allergies or allergic conditions in comparison with SPT as the gold standard. RESULTS: We found that 25.0% of children reported a history of any allergy and 19.4% were atopic based on SPT. The agreement between questionnaire report of allergic triggers and atopy measured by SPT was high (83.0-89.5%). The agreement between atopy and report of allergic conditions ranged from 67.1% to 79.6%. Individual allergic conditions demonstrated high specificity but low sensitivity. The questionnaire report of any allergy had a low PPV in detecting atopy (47.3%) and high NPV (86.3%). The PPV of reported allergic conditions was low (24.8-43.9%), but the NPV was again high (82.0-82.9%). CONCLUSIONS: We found that the standardized questionnaire report of allergy and atopic conditions was shown not to efficiently and reliably predict atopy. However, given the good specificity and the NPV, the questionnaire may be an efficient tool for epidemiological studies that involve the differential inclusion of subjects without atopy.

Airway responsiveness to mannitol 24 h after allergen challenge in atopic asthmatics.

BACKGROUND: Airway responsiveness to indirect stimuli correlates positively with airway inflammation. In atopic asthmatics, allergen inhalation is associated with an influx of inflammatory cells and increased responsiveness to the direct-acting stimuli methacholine at 3 and 24 h after exposure. We have shown mannitol responsiveness decreases 3 h after allergen inhalation. The current investigation assessed mannitol responsiveness 24 h after allergen challenge. METHODS: Eleven mild atopic asthmatics completed allergen challenges on two separate occasions. In random order, methacholine or mannitol challenges were performed 24 h pre- and post-allergen challenge. Levels of fractional exhaled nitric oxide were also measured. RESULTS: Allergen challenge increased airway responsiveness to methacholine 24 h postchallenge; the geometric mean (95% CI) methacholine PC20 decreased from 5.9 mg/ml (1.8-19.4) to 2.2 mg/ml (0.81-5.89); P = 0.01. This coincided with a significant increase (P = 0.02) in FeNO levels. Conversely, allergen challenge decreased airway responsiveness to mannitol; geometric mean (95% CI) dose-response ratio was significantly higher after allergen exposure (57 mg/% FEV1 fall [27-121] to 147 mg/% FEV1 fall [57-379]; P = 0.03), and FeNO levels were not significantly increased (P = 0.054). CONCLUSION: Allergen-induced changes in airway responsiveness to direct and indirect stimuli are markedly different. The loss in responsiveness to mannitol is likely not explainable by a refractory state. The effect(s) of allergen exposure on airway responsiveness to indirect-acting stimuli require further investigation.

OX40L blockade and allergen-induced airway responses in subjects with mild asthma.

BACKGROUND: The OX40/OX40L interaction contributes to an optimal T cell response following allergic stimuli and plays an important role in the maintenance and reactivation of memory T effector cells. OBJECTIVE: We tested whether treatment with an anti-OX40L monoclonal antibody (MAb) would inhibit allergen-induced responses in subjects with asthma. METHODS: Twenty-eight mild, atopic asthmatic subjects were recruited for a double-blind, randomized, placebo-controlled, parallel-group trial (ClinicalTrials.gov identifier NCT00983658) to compare blockade of OX40L using a humanized anti-OX40L MAb to placebo-administered intravenously in 4 doses over 3 months. Allergen inhalation challenges were carried out 56 and 113 days after the first dose of study drug. The primary outcome variable was the late-phase asthmatic response. Other outcomes included the early-phase asthmatic response, airway hyperresponsiveness, serum IgE levels, blood and sputum eosinophils, safety and tolerability. RESULTS: Treatment with anti-OX40L MAb did not attenuate the early- or late-phase asthmatic responses at days 56 or 113 compared with placebo. In the anti-OX40L MAb treatment group, total IgE was reduced 17% from pre-dosing levels, and sputum eosinophils decreased 75% by day 113 (both P = 0.04). There was no effect of anti-OX40L MAb on airway hyperresponsiveness or blood eosinophils. The frequency of AEs was similar in both groups. CONCLUSION AND CLINICAL RELEVANCE: Pharmacological activity of anti-OX40L MAb was observed by decreases in serum total IgE and airway eosinophils at 16 weeks post-dosing, but there was no effect on allergen-induced airway responses. It is possible that the treatment duration or dose of antibody was insufficient to impact the airway responses.

Single-dose desloratadine and montelukast and allergen-induced late airway responses.

Montelukast and desloratadine synergistically inhibit the allergen-induced early asthmatic response. Montelukast also suppresses the allergen-induced late asthmatic response, but there are no reports on the effect of desloratadine or the combination on the allergen-induced late asthmatic response. Atopic asthmatics (n = 10) completed a multicentric randomised double-blind crossover study comparing single-dose placebo, 5 mg desloratadine, 10 mg montelukast and the combination administered 2 h prior to allergen inhalation challenge. Methacholine challenges were performed 24 h before and after allergen challenge. Exhaled nitric oxide measurements and sputum inflammatory cell counts were also carried out. All active treatments significantly decreased the late asthmatic response area under the curve. Combination therapy provided the greatest inhibition compared to desloratadine and montelukast. Montelukast was nonsignificantly better than desloratadine but not as effective as the combination. There was a trend towards a decrease in airway responsiveness following montelukast and combination. Montelukast, but not desloratadine or the combination, decreased exhaled NO levels 24 h after allergen. The allergen-induced increase in sputum eosinophil numbers was significantly suppressed at 7 h with desloratadine and combination therapy, and at 24 h with montelukast and combination therapy. Single-dose co-administration of desloratadine and montelukast 2 h prior to allergen inhalation clinically abolished the late asthmatic response and eosinophil recruitment.

The effect of IVX-0142, a heparin-derived hypersulfated disaccharide, on the allergic airway responses in asthma.

BACKGROUND: IVX-0142 is a heparin-derived hypersulfated disaccharide devoid of anticoagulant activity while possessing anti-allergic and anti-inflammatory activity in preclinical studies. In a proof-of-concept study, the allergen inhalation challenge model was used to investigate the effect of IVX-0142 in mild atopic asthma. METHODS: Nineteen subjects, not on controller medications, were randomized to an evaluator-blind, placebo-controlled, cross-over study. The effect of a single nebulized dose of IVX-0142 (80 mg) or placebo administered 30 min prior to allergen inhalation was evaluated on the allergic airway responses, airway responsiveness, and airway inflammation. RESULTS: When compared with placebo, 14 and 13 subjects experienced a relatively smaller maximum fall in forced expiratory volume in 1 s (maxFEV(1)%) for the early airway response (EAR) and late airway response (LAR) with IVX-0142, respectively (P < 0.01). The degree of attenuation in the EAR [maxFEV(1)% (mean +/- SE) 26.5 +/- 2.8%vs placebo 31.0 +/- 2.8%, P = 0.059] and LAR (15.6 +/- 2.9%vs placebo 19.0 +/- 2.9%, P = 0.24) with IVX-0142, however, was small and did not reach statistical significance compared with placebo. Similarly, a trend in the attenuation of allergen-induced increase in the absolute sputum cell counts was also observed. No difference in the allergen-induced increase in airway hyper-responsiveness and exhaled nitric oxide was noticed. CONCLUSIONS: The majority of mild atopic asthmatics demonstrated a reduction in the EAR and LAR to IVX-0142. However, the treatment effect observed with a single prechallenge dose of IVX-0142 was small and heterogeneous. The potential anti-allergic and anti-inflammatory effects using multiple higher doses need to be evaluated.

Assessment of endotoxin levels in the home and current asthma and wheeze in school-age children.

UNLABELLED: The relationship between household endotoxin and asthma in children is not clear. To further investigate the relationship between sources of endotoxin and childhood asthma, we conducted a case-control study of children with and without asthma and examined their more frequent household exposures in the home. Children ages 6-13 years with current asthma (n = 70) or wheeze only (n = 19) were sex and age matched (+/-1 year) to 107 controls. Play area and mattress dust were collected for endotoxin analysis. Atopic status was determined by skin prick testing for allergies. A family size of >4 per household was associated with higher endotoxin levels (EU/mg) in the bed dust (P < 0.05). Passive smoking (P < 0.05) and the presence of a cat were associated with higher levels of endotoxin in mattress dust. Endotoxin levels in either the play dust or the bed dust did not differ between cases and controls. Within atopic cases, those with higher endotoxin loads (EU/m2) in bed or play areas were more likely to miss school for chest illness (P < 0.05). In this study, household endotoxin is not a risk factor for current asthma overall but may be associated with increased severity in children with atopic asthma. PRACTICAL IMPLICATIONS: This study did not find that household sources of endotoxin were associated with asthma. However, within atopic asthmatics, asthma severity (as measured by a history of being kept home from school because of a chest illness in the past year) was associated with higher levels of endotoxin in dust from the child's bed. There is a need to further investigate the nature of the relationship between household endotoxin and asthma severity in children which could lead to better management of childhood asthma.

Intracerebral abscess: a complication of severe cystic fibrosis lung disease.

Intracerebral abscess is an uncommon complication of severe cystic fibrosis lung disease. The present report describes a case of fatal multiple intracerebral abscesses in a patient with a severely bronchiectatic, nonfunctioning right lung and chronic low-grade infection. The patient was previously turned down for pneumonectomy. Intracerebral abscess in cystic fibrosis and the potential role of pneumonectomy in the present patient are discussed.

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.

Clinical concerns with inhaled beta2-agonists: adult asthma.

Inhaled beta2-agonists, when used regularly, cause subtle but significant worsening of asthma control. Overuse of inhaled beta2-agonists is associated with increased risk of death from asthma in a dose-response fashion. beta2-Agonists enhance airway responses to allergens, including induced airway hyperresponsiveness and induced airway inflammation. This is a plausible explanation for beta2-agonist-worsened asthma control. These direct effects of inhaled beta2-agonists, including increased airway response to allergen, tolerance, etc., may partially explain the association of overuse with asthma death. However, it is probable that the major reason for the association of beta2-agonists overuse and asthma mortality is an indirect effect. Inhaled beta2-agonists are effective relievers and preventers of bronchoconstriction and asthma symptoms but fail to treat the underlying pathogenesis, namely the airway inflammation. Thus, overuse may mask the true asthma severity and result in both an underappreciation and undertreatment of the disease. This would provide a rational explanation for the relationship of inhaled beta2-agonist use and mortality and also would fit the dose-response pattern. Inhaled beta2-agonists should be used exclusively as needed for relief of symptoms and their requirement should be infrequent: the need for excessive doses of beta2-agonists provides a useful marker of asthma (lack of) control.

Performance of creatinine clearance equations on the original Cockcroft-Gault population.

BACKGROUND: Prediction of endogenous creatinine clearance by mathematical equations such as the Cockcroft-Gault formula is used in clinical practice in spite of the reported concern for their limited predictability. The aim of this study is to determine whether the measured creatinine clearance can be predicted accurately by a number of published equations including the recently modified Cockcroft-Gault formula = Cockcroft-Gault formula x 1.73 m2/body surface area from the original Cockcroft-Gault population. METHODS: The performance of the mathematical equations in patients with different creatinine clearance and body mass indices was assessed by computing accuracy at different percentiles, bias and precision from the original Cockcroft-Gault data. RESULTS: Refitting the modified formula to the Cockcroft-Gault data gave superior results compared to the original Cockcroft-Gault formula with an overall accuracy in the general and subgroup analysis above 70% agreement within 30% estimate of the measured creatinine clearance. On the other hand, analysis of the other equations, including the original Cockcroft-Gault, demonstrated a limited accuracy to predict creatinine clearance particularly in patients with creatinine clearance below 50 ml/min with an overall accuracy in less than 1/3 of the calculated creatinine clearance within 30% range from the measured creatinine clearance. CONCLUSION: The current creatinine clearance equations and even the original Cockcroft-Gault formula did not accurately predict the measured creatinine clearance. Normalization for body surface area in the original Cockcroft-Gault formula demonstrated more accuracy to estimate creatinine clearance, particularly in patients with diminished renal function and is recommended to physicians who wish to use the Cockcroft-Gault formula in their practice until more credible formulas are developed.

The links between allergen skin test sensitivity, airway responsiveness and airway response to allergen.

BACKGROUND: The allergen-induced early asthmatic response [provocation concentration (PC)20, the concentration causing a 20% forced expiratory volume in 1 s (FEV)1 fall] depends on the level of IgE sensitivity and the degree of nonallergic airway hyperresponsiveness (AHR) and can be predicted from histamine PC20 and allergen skin test endpoint. OBJECTIVES: We examined the relationships between allergen PC20, methacholine PC20, and allergen skin test endpoint and assessed the accuracy of both the histamine PC20-based prediction of allergen PC20 (using methacholine) and a new methacholine PC20-based prediction equation. METHODS: From 158 allergen challenges, the allergen PC20, the methacholine PC20, and the skin test endpoint were recorded and relationships between these three were sought. We compared the measured allergen PC20 to that predicted from the previous histamine PC20-based and the new methacholine-based formulae. RESULTS: In single regressions, allergen PC20 correlated with both methacholine PC20 (r=0.25, P=0.0015) and skin test endpoint (r=0.52, P <0.00005). The relationship was improved by multiple regression of log-allergen PC20vs. log-methacholine PC20 and log-endpoint (r=0.61, P <0.00005). The histamine-based formula predicted allergen PC20 to within 2 doubling concentrations in 80% and within 3 in 92%. The new methacholine-based formula to within 2 and 3 concentrations in 81% and 94%, respectively; only nine of 158 subjects were outside the 3 concentrations. CONCLUSIONS: We have confirmed the dependence of the allergen-induced early asthmatic response upon the level of allergic sensitivity and the degree of AHR, the latter as assessed by methacholine challenge. The allergen PC20 can be predicted to within 3 doubling concentrations in 94% of cases.

Mycobacterium saskatchewanense sp. nov., a novel slowly growing scotochromogenic species from human clinical isolates related to Mycobacterium interjectum and Accuprobe-positive for Mycobacterium avium complex.

A pigmented, slowly growing Mycobacterium avium complex AccuProbe-positive organism was isolated from the sputum and pleural fluid of a 72-year-old female with bronchiectasis. The unusual morphology of the organism prompted further identification by 16S rRNA gene sequencing, revealing a perfect identity with previously uncharacterized strain Mycobacterium sp. MCRO 8 (GenBank accession no. X93034), with the closest established species by 16S rDNA analysis being Mycobacterium interjectum. HPLC of the organism corresponded to previously obtained patterns identified as M. interjectum-like and, upon sequence evaluation of a selection of strains with a similar profile, more were subsequently identified as MCRO 8. A total of 16 strains isolated from human respiratory samples were evaluated in the characterization of this novel species, for which the name Mycobacterium saskatchewanense sp. nov. is proposed. The type strain is strain 00-250(T) (=ATCC BAA-544(T)=DSM 44616(T)=CIP 108114(T)).

Regular salbutamol use increases CXCL8 responses in asthma: relationship to the eosinophil response.

Regular salbutamol use can exacerbate allergen-induced airway eosinophilia in asthmatics, but its effect on airway eosinophil chemokine responses is unknown. Asthmatic subjects (n=14) were treated for 10 days with placebo or salbutamol in a double-blind, cross-over study, then given same-dose allergen challenges. Their sputa were then analysed 1 and 7 h later for a panel of eosinophil-related cytokines. Eosinophils from five test and three control subjects were tested for expression of CXCL8/interleukin (IL)-8, and its receptors and responsiveness to CCL11/eotaxin and CXCL8/IL-8. Sputum CXCL8/IL-8, but not IL-5, CCL5/regulated on activation, T-cell expressed and secreted, CCL7/monocyte chemotactic protein-3, CCL11/eotaxin, granulocyte-macrophage colony-stimulating factor or tumour necrosis factor levels, were increased (42%) by the salbutamol treatments. The CXCL8/IL-8 levels correlated with the proportions of sputum eosinophils and these cells, but not other sputum cells, stained strongly for CXCL8/IL-8. The circulating eosinophils of the tested subjects (n=5) expressed CXCL8/IL-8 receptors and secreted high levels of this chemokine. Neutralisation of sputum CXCL8/IL-8 reduced eosinophil chemotactic responses to these samples by 19 +/- 5%. These data suggest that regular use of salbutamol can augment airway CXCL8/interleukin-8 responses to allergen challenge and that this CXCL8/interleukin-8 could contribute to the airway inflammatory response.

Indirect airway challenges.

Indirect challenges act by causing the release of endogenous mediators that cause the airway smooth muscle to contract. This is in contrast to the direct challenges where agonists such as methacholine or histamine cause airflow limitation predominantly via a direct effect on airway smooth muscle. Direct airway challenges have been used widely and are well standardised. They are highly sensitive, but not specific to asthma and can be used to exclude current asthma in a clinic population. Indirect bronchial stimuli, in particular exercise, hyperventilation, hypertonic aerosols, as well as adenosine, may reflect more directly the ongoing airway inflammation and are therefore more specific to identify active asthma. They are increasingly used to evaluate the prevalence of bronchial hyperresponsiveness and to assess specific problems in patients with known asthma, e.g. exercise-induced bronchoconstriction, evaluation before scuba diving. Direct bronchial responsiveness is only slowly and to a modest extent, influenced by repeated administration of inhaled steroids. Indirect challenges may reflect more closely acute changes in airway inflammation and a change in responsiveness to an indirect stimulus may be a clinically relevant marker to assess the clinical course of asthma. Moreover, some of the indirect challenges, e.g. hypertonic saline and mannitol, can be combined with the assessment of inflammatory cells by induction of sputum.