Indirect airway challenges

ERS TASK FORCE

Indirect airway challenges

GF Joos B O9Connor on behalf of the Task ForceCo-authors of the Task Force Report GF Joos (Chairman) B OrsquoConnor (Co-Chairman)

SD Anderson F Chung DW Cockcroft B Dahlen G DiMaria A Foresi FE HargreaveST Holgate M Inman J Lotvall H Magnussen R Polosa DS Postma J Riedler

Indirect airway challenges GF Joos B O9Connor on behalf of the Task ForceCo-authors of the Task Force Report GF Joos (Chairman) B OrsquoConnor(Co-Chairman) SD Anderson F Chung DW Cockcroft B Dahlen G DiMariaA Foresi FE Hargreave ST Holgate M Inman J Lotvall H MagnussenR Polosa DS Postma J Riedler ERS Journals Ltd 2003ABSTRACT Indirect challenges act by causing the release of endogenous mediatorsthat cause the airway smooth muscle to contract This is in contrast to the directchallenges where agonists such as methacholine or histamine cause air ow limitationpredominantly via a direct effect on airway smooth muscle

Direct airway challenges have been used widely and are well standardised They arehighly sensitive but not speci c to asthma and can be used to exclude current asthma ina clinic population Indirect bronchial stimuli in particular exercise hyperventilationhypertonic aerosols as well as adenosine may re ect more directly the ongoing airwayin ammation and are therefore more speci c to identify active asthma They areincreasingly used to evaluate the prevalence of bronchial hyperresponsiveness and toassess speci c problems in patients with known asthma eg exercise-inducedbronchoconstriction evaluation before scuba diving

Direct bronchial responsiveness is only slowly and to a modest extent in uenced byrepeated administration of inhaled steroids Indirect challenges may re ect more closelyacute changes in airway in ammation and a change in responsiveness to an indirectstimulus may be a clinically relevant marker to assess the clinical course of asthmaMoreover some of the indirect challenges eg hypertonic saline and mannitol can becombined with the assessment of in ammatory cells by induction of sputumEur Respir J 2003 21 1050ndash1068

Correspondence GF JoosDept Respiratory DiseasesGhent University HospitalDe Pintelaan 185B-9000 GhentBelgiumFax 32 92402341E-mail guyjoosrugacbe

Keywords Adenosineasthmabronchoprovocationchronic obstructive pulmonary diseaseexercisemethacholine

Received September 24 2002Accepted October 3 2002

CONTENTS

De nition and main properties of an indirectchallenge 1051Mechanisms and receptors involved in indirectchallenges 1052

Mechanisms involved in the airway narrowing tophysical stimuli evidence from studies on exercise-induced bronchoconstriction 1052Mechanisms involved in the airway narrowing causedby pharmacological stimuli evidence on adenosine-tachykinin- and bradykinin-inducedbronchoconstriction 1053

Diagnostic value of the indirect challenges a comparisonwith direct challenges 1053

Diagnostic value of the direct challenges histamine andmethacholine 1053Diagnostic value of indirect challenges 1054

Direct versus indirect airway challenges to monitorasthma 1056Use of indirect airway challenges in epidemiologicalstudies 1058

Safety aspects of indirect airway challenges 1058Conclusions 1059Areas for future research 1059

Mechanisms and receptors 1059Diagnosis 1059Monitoring 1059Epidemiology 1059Safety performance 1059

Appendix 1 Safety issues for exercise challenges inthe lung function laboratory and in eldstudies 1059Appendix 2 Safety issues for airway challenges withhypertonic saline in the lung function laboratory and in eld studies 1060Appendix 3 Performance standards safety issues andprotocol recommendations for airway challenges withadenosine 1060Appendix 4 Performance standards safety issues andprotocol recommendations for airway challenges withlysine-aspirin 1061

Eur Respir J 2003 21 1050ndash1068DOI 101183090319360300008403Printed in UK ndash all rights reserved

Copyright ERS Journals Ltd 2003European Respiratory Journal

ISSN 0903-1936

1Bronchial hyperresponsiveness is an abnormal increase in

air ow limitation following exposure to a nonallergic stimulus[1 2] Bronchial hyperresponsiveness is a characteristic featureof both asthma and chronic obstructive pulmonary disease(COPD) Thus bronchial hyperresponsiveness is frequentlyused to aid in diagnosis and characterisation of individuals withairway disease Although bronchial hyperresponsiveness isnot speci c for asthma nearly all patients with asthma exhibitincreased responsiveness which is more marked during sympto-matic episodes Bronchial hyperresponsiveness to methacholineis also present in a majority of patients with mild to moderateCOPD [3] Moreover the severity of bronchial hyperrespon-siveness predicts the response to inhaled corticosteroids inpatients with asthma [4] and the progression of air ow limita-tion in patients with COPD [5]

Most investigators assess bronchial responsiveness usingmethacholine or histamine as a provocative stimulus Metha-choline and histamine cause air ow limitation predominantlyvia a direct effect on airway smooth muscle By contrastindirect challenges induce air ow limitation by acting on cellsother than smooth muscle cells eg in ammatory cells epithelialcells and nerves which upon stimulation release mediators orneurotransmitters that provoke smooth muscle contractionNearly all the published studies on asthma and COPD haveutilised histamine and methacholine provocation tests forclinical characterisation of patients Furthermore hyper-responsiveness testing is widely used in clinical researchsettings to evaluate potential new therapies For exampledirect challenges with histamine or methacholine are usedto establish a dose response and time course of the acutebronchoprotective effects of b-agonists These challenges havealso been used to assess the potential anti-in ammatoryeffects of prolonged treatment with new agents There arelimitations to this model Inhaled corticosteroids the currentgold standard anti-in ammatory treatment for asthma reducesbronchial responsiveness to histamine or methacholine onlyto a small degree an effect that is both dose and timedependent In recent years an increasing number of studieshave investigated the relative usefulness of indirect airwaychallenges in monitoring anti-in ammatory treatment inasthma but almost none in COPD

In 1998 the European Respiratory Society (ERS) approveda Task Force on Indirect Airway Challenges The objectivesof this Task Force were to develop recommendations concern-ing the role of indirect airway challenges in the assessmentand monitoring of airway diseases The recommendations inthis report are based on a review of the published literatureand were developed during workshops held at the AmericanThoracic Society in San Diego (April 1999) ERS Congress inMadrid (October 1999) ERS Meeting in Ghent (June 2000)and the ERS Congress in Florence (August 2000) Thefollowing topics were included 1) Mechanisms and receptorsinvolved in the airway narrowing caused by indirect airwaychallenges 2) Diagnostic value of indirect challenges 3) Valueof indirect challenges in the monitoring of asthma includingthe use of these challenges as an outcome measure in clinicaltrials 4) Value of indirect challenges in epidemiological studies5) The importance of standardisation of challenge methods6) Areas for further research

De nition and main properties of an indirect challenge

The concept of indirect challenges was developed at the endof the eighties [6] Several publications had con rmed thatmany different nonspeci c stimuli induced airway narrowingin patients with asthma Thus a distinction had to be madebetween direct and indirect stimuli Methacholine and histamine

are direct stimuli because they cause air ow limitation byacting on effector cells predominantly on airway smoothmuscle but also on mucus glands and on airway micro-vasculature without involving intermediate pathways Bycontrast indirect stimuli ie physical stimuli such as exerciseosmotic challenge or pharmacological stimuli such as adeno-sine cause air ow limitation by acting on cells most notablyin ammatory cells and neuronal cells which release mediatorsor cytokines to cause secondary bronchoconstriction

The fact that the pattern of airway narrowing induced byindirect stimuli differs from that provoked by direct stimuli isshown by the following clear evidence 1) Bronchial respon-siveness to direct and indirect challenges are rather poorlycorrelated with each other [6] 2) A wide array of mediatorsincluding histamine leukotrienes prostaglandins acetyl-choline neuropeptides are involved in the airway narrowinginduced by the indirect stimuli [7] 3) The airway narrowingcaused by an indirect but not a direct challenge can beprevented by acute pretreatment with a cromone (cromogly-cate nedocromil) inhaled frusemide andor heparin [7] 4)After the administration of an indirect challenge tachy-phylaxis to a second stimulus with the same or another indirectacting agent (cross refractoriness) is frequently observed [7]The tachyphylaxis observed with the indirect challenges is farmore pronounced than the small changes seen when histamineor methacholine is repeatedly inhaled [8ndash10] 4) In patientswith asthma bronchial responsiveness to an indirect airwaychallenge is more closely associated with airway in ammationthan bronchial responsiveness to a direct stimulus [11] Bron-chial responsiveness to an indirect stimulus may also betterre ect acute changes in airway in ammation induced by allergenavoidance [12] or by treatment with inhaled steroids [13 14]

The authors propose the following practical workingde nition of an indirect challenge Indirect challenges actby causing the release of endogenous mediators that cause theairway smooth muscle to contract with or without effect ininducing microvascular leakage Because the responses tothese challenges are modi ed or even completely inhibited byinhaled steroids the airway response to these challenges maybe a closer re ection of active airway in ammation

Table 1 ndash Overview of direct and indirect stimuli

Indirect stimuli Direct stimuli

Physical stimuli Cholinergic agonistsExercise (acetylcholineNonisotonic aerosols methacholine(hyper- hypotonic carbachol)distilled water Histamineaerosols mannitol) Prostaglandin D2

Eucapnic voluntaryhyperpnoea of dryair

Leukotriene C4D4 E4

Pharmacological stimuliAdenosineTachykininsBradykininMetabisulphiteSO2Propranolol

Endotoxin (LPS)Platelet activating factorOzoneSelective agents

Aspirin and NSAIDAllergen

LPS lipopolysacharides NSAID nonsteroidal anti-in ammatorydrugs SO2 sulphur dioxide

1051INDIRECT AIRWAY CHALLENGES

Mechanisms and receptors involved in indirect challenges

An overview of the different indirect and direct airwaystimuli is given in table 1 In gure 1 the contribution of thedifferent intermediate pathways involved in indirect broncho-constriction are outlined

Mechanisms involved in the airway narrowing to physicalstimuli evidence from studies on exercise-inducedbronchoconstriction

Exercise causes airway narrowing by the loss of watervia evaporation from the airway surface The mechanismwhereby the loss of water causes the airways to narrow isthought to relate to the thermal (cooling and rewarming)[15] and osmotic (increase in airway osmolarity) effects ofdehydration [16] The dehydration results in cell shrinkageand leads to a complex sequence of biochemical events aspart of the homeostatic response producing a restorativeincrease in the cell volume For cells such as the epithelial cellthe mast cell and the sensory nerve cell these biochemicalevents are likely to stimulate the release of mediators [16]In-vitro studies of human lung mast cells show that increasingthe osmolarity of the solution bathing the cells is a potentstimulus to release of histamine [17] The major clinicalevidence to support a role for histamine release is the ndingthat some histamine H1 receptor antagonists have an inhibi-tory effect on exercise-induced bronchoconstriction (EIB)

[18ndash20] Because the inhibitory effect is incomplete histaminecannot be the only mediator involved in EIB

There are other mast cell mediators that are likely to beinvolved in EIB most notably prostaglandin D2 (PGD2) andthe cysteinyl leukotrienes Recent studies have demonstratedthat the PGD2 metabolite 9-a 11-b prostaglandin F2 is signi -cantly increased in the urine 30 60 and 90 min postexercise[21 22] This nding is also supported by the observation that urbiprofen a cyclooxygenase inhibitor also has a partialinhibitory effect on EIB [19]

Leukotrienes are involved in the genesis of EIB and insustaining the bronchoconstriction following exercise Repeatedstudies have reported increases in urinary leukotriene E4following EIB [23 24] Some investigators have also reporteda signi cant increase in leukotrienes in bronchoalveolarlavage following dry air hyperpnoea [25] Also there arenow many studies demonstrating that both 5-lipoxygenaseinhibitors [26ndash28] and leukotriene receptor antagonists [2429] inhibit EIB and enhance recovery of lung function to pre-exercise values The inhibition is incomplete con rming thatmore than one mediator is involved

The epithelial cell is a rich source of mediators One suchmediator is prostaglandin E2 (PGE2) which may act toprotect the airways from narrowing [30] The release of PGE2may in part be dependent on stimulation by leukotrienes [8]Thus PGE2 may play an important role in the refractorinessthat follows exercise In a recently reported study [31] humanepithelial cells in culture when stimulated with hypertonicsolutions rapidly produced interleukin (IL)-8 IL-8 promotes

s

s

s

s

sss

s

s

s

s

s

ssss

s

s

HyperpnoeaHypertonic

Water loss

Water gainDistilled water

Allergen

Adenosine

AMP

Substance PNKA

Bradykinin

Aspirin

AirwayEpithelium Mucus

hypersecretionMicrovascular

leakage

Airwaysmooth muscle

CO LO

AA

P

Leukotriene

NK1NK2

Leukotriene

Histamine

Histamine

Leukotriene

NK1NK2

Histamine

Leukotriene

Sensorynerve

Eosinophil

Activated mast cell

Fig 1 ndash The contribution of different intermediate pathways in airway-narrowing induced by various indirect stimuli NK neurokinin receptorAMP adenosine 59-monophosphate P phosphate group AA arachidonic acid CO cyclooxygenase LO 5-lipoxygenase For details on the differ-ent pathways see the Mechanisms and receptors involved in indirect challenges section of this report and the report by VAN SCHOOR et al [7]

1052 GF JOOS ET AL

neutrophil chemotactic activity which has been reported tobe increased during EIB [32]

Airway sensory nerves may also be affected by alterationsin osmolarity and cell volume There is abundant evidencefrom animal studies that an increase in osmolarity stimulatessensory nerves In addition exercise-induced respiratory waterloss can cause coughing in humans an effect that is blockedby inspiring humid air [33] There is some evidence to supportthe role of tachykinins in EIB the selective tachykinin neurokinin-receptor type-1 (NK1) antagonist FK888 hastened the recoveryin lung function to baseline after exercise [34]

EIB is signi cantly inhibited or even completely blocked bysingle doses of nedocromil sodium sodium cromoglycate [35]frusemide [36] and by repeated dosing with inhaled steroids[37] These drugs have no direct effect on airway smooth musclebut reduce the functional activity of mast cells epithelial cellsand sensory nerves implying a signi cant role for these cellsin EIB

The other physical stimuli nonisotonic aerosols and eucapnicvoluntary hyperpnoea of dry air work through similarmechanisms (table 2)

Mechanisms involved in the airway narrowing caused bypharmacological stimuli evidence on adenosine-tachykinin- and bradykinin-induced bronchoconstriction

Several cells and mediators are involved in the airwaynarrowing due to indirect stimuli these include epithelialcells in ammatory cells (incorporating mast cells) nerve cellsand blood vessels A summary is given in gure 1 and table 2and more details can be found in a recent review on this sub-ject by VAN SCHOOR et al [7] The effect exerted by an indirectacting pharmacological agent on the airways differs fromstimulus to stimulus depending on the targets and receptorsinvolved and by the presence of degrading enzymes [7]

Adenosine Adenosine exerts its effects on human cells throughinteraction with speci c adenosine (P1) receptors of whichfour subtypes (A1 A2A A2B and A3) have been described [104]The A1 A2B and A3 receptors have been shown to be involvedin various animal and human models of in ammationIn particular the potential role of A2B receptors is beingincreasingly recognised [105] The future development ofspeci c and potent adenosine-receptor agonists and antagonistsfor use in vivo in asthma will clarify the relative importance ofthese receptors [106]

Tachykinins The airway effects of the tachykinins aremediated via tachykinin NK1 and NK2 receptors there is

no evidence for the presence of tachykinin NK3 receptors inhuman airways Substance P has the greatest af nity for theNK1 receptor whereas neurokinin A has the greatest af nityfor the NK2 receptor although there is cross-reactivity [107]In vitro tachykinins constrict the smooth muscle of humanairways mainly through tachykinin NK2 receptors [108ndash110]in small and medium sized bronchi tachykinin NK1 receptorsare also involved [50 111] In vivo inhaled neurokinin A causesbronchoconstriction mainly by indirect mechanisms [112]Both tachykinin NK1 and NK2 receptors are involved in thebronchoconstrictor effect of neurokinin A [113 114] Follow-ing their release from sensory cells and immune cells thetachykinins are degraded by at least two enzymes these areneutral endopeptidase (NEP EC 342411) [115 116] andangiotensin converting enzyme (ACE EC 34151) NEP iswidely distributed on a variety of airway cells and in the airwayepithelium NEP appears to be the most important enzyme forthe breakdown of tachykinins in tissues ACE on the otherhand is localised predominantly around the vascular endo-thelium and therefore degrades intravascular peptides [117]

Bradykinin Bradykinin causes contraction of the airwaysby stimulation of B2 receptors [57 58 118] Bradykinin ismetabolised by several peptidases the most important of whichare carboxypeptidase N (kininase I) ACE and NEP [119]Pretreatment with inhaled NG-monomethyl-L-arginine a nitricoxide (NO) synthase inhibitor signi cantly potentiated air owlimitation in response to inhaled bradykinin in asthmatics thissuggests that bradykinin activates the NO synthase pathwayleading to the release of NO which in turn counteracts thebronchoconstrictor response to bradykinin [120] The involve-ment of histamine and prostaglandins in bradykinin-inducedair ow limitation appears to be limited [59 60] The broncho-constrictor effect of bradykinin is at least in part mediated viacholinergic vagal nerves since pretreatment with ipratropiumbromide signi cantly reduced air ow limitation in asthmatics[60]Although bradykinin has been shown to release tachykininsin guinea-pig airways [121ndash123] conclusive evidence for aninvolvement of tachykinins in bradykinin-induced broncho-constriction in man is lacking [51 52 61]

Diagnostic value of the indirect challenges a comparisonwith direct challenges

Diagnostic value of the direct challenges histamine andmethacholine

Physicians need objective measurements such as a bronchialprovocation test to diagnose asthma [124] For historical

Table 2 ndash Mediators and neurotransmitters involved in indirect bronchial responsiveness

Mediator Release Neuronal Stimulation References

Adenosine z(Hi LT PG) z(ACh TK) [27 39ndash49]Tachykinins z(Hi LT PG) z(ACh) [50ndash56]Bradykinin z(Hi PG NO) z(ACh TK) [57ndash63]Propranolol iexcl(Hi) z(ACh) [64ndash68]MetabisulphiteSO2 z(Hi LT PG) z(ACh TK) [47 69ndash75]Exercise z(Hi LT PG) z(AChTK) [19 21 22 24 27ndash30

34 76 77ndash82]Nonisotonic aerosols z(Hi LT PG) z(ACh TK) [17 83ndash88]EVH of dry air z(Hi LT) z(ACh TK) [26 30 89ndash94]PAF z(LT) iexcl [95]Aspirin z(PG LT) [96]Allergen z(Hi PG LT TK) iexcl [97ndash103]

Hi histamine LT leukotriene C4 D4 E4 PG prostaglandins ACh acetylcholine TK tachykinins NO nitric oxide EVH eucapnic voluntaryhyperpnoea PAF platelet activating factor not known for human airways This table was modi ed from [7]


reasons bronchial responsiveness has been most commonlyassessed using the direct stimuli histamine and methacholine[125] Widely used methods include the 2-min tidal breathingmethod [126 127] the counted-breath dosimeter method [128]which produce comparable results with appropriate calibra-tion [129] and the portable counted breath technique [130]The results are usually expressed as the provocation concentra-tion (or dose) producing a 20 fall in forced expiratoryvolume in one second (PC20 PD20 FEV1) Histamine andmethacholine are approximately equivalent on a mg [131] ormmol [132] basis Bronchial responsiveness to histamine andmethacholine (PC20 PD20) is unimodally log-normally dis-tributed within the population this continuous distributionplus the 95 con dence interval (CI) of repeatability in therange of iexcl1ndash16 doubling concentrations [133] leads to asigni cant grey area when trying to de ne a normal response

Inhalation tests have been arbitrarily de ned so that themajority of current asthmatics are identi ed generally by acut-off point that is at the higher end of the borderline rangeBronchial hyperresponsiveness is considered to be presentwhen the histamine or methacholine PC20 is lt8ndash16 mgmL-1

[127] or the PD20 is lt39ndash78 mmol [130] These arbitraryde nitions make the test highly sensitive for the detection ofhyperresponsiveness in a pulmonary function laboratory orhospital clinic population This has been con rmed by anumber of studies documenting sensitivity and closely relatednegative predictive values of histamine and methacholinechallenges approaching 100 for clinically current asthma(symptoms within previous few days) as opposed to epidemio-logically current asthma (symptoms within the past year)[134ndash137] By contrast the speci city and positive predictivevalue of these challenges for asthma symptoms perform lesswell in the eld For example the positive predictive value ofhistamine PC20lt8 mgmL-1 for current symptoms of asthmain a random sample from the general population was shownto be well below 50 [137] When the cut-off point is reducedthe speci city and positive predictive value can approach100 (for example PC20 lt1 mgmL-1 [127]) but the sensi-tivity and negative predictive value perform poorly [137]Thus methacholine and histamine at a cut-off point of PC20of 8ndash16 mgmL-1are highly sensitive tests and are best used toexclude current active disease as opposed to the application ofthe highly speci c cut-off point of PC20 of 1 mgmL-1 whichpermits these tests to con rm disease

Patients with nonasthmatic xed air ow limitation (chronicair ow limitation COPD) also demonstrate bronchial hyper-responsiveness to histamine and methacholine [138ndash141] Thecharacteristics are somewhat different in that there is a stronglinear relationship between bronchial hyperresponsiveness andthe obstructive reduction in FEV1 in subjects with chronicair ow limitation Subjects with COPD also are less hyper-responsive than asthmatics However bronchoprovocationwith direct stimuli lack speci city to be able to detect asthmain the presence of resting air ow obstruction Thus broncho-provocation with the directly acting stimuli histamine andmethacholine is extremely sensitive for current asthma symp-toms but lack speci city both in differentiating asthma fromnormal and asthma from chronic air ow limitation

Diagnostic value of indirect challenges

Physical stimuli Exercise challenge Many comparisons ofexercise challenges (EIB) with histamine and methacholinechallenges have produced somewhat variable results Thereis a weak if statistically signi cant correlation between EIBand log histamine or methacholine PC20 [142 143] Exercisechallenge to a preset threshold is consistently less sensitive

but more speci c than the direct challenges in differentia-ting asthma from normal [142ndash149] There are many asthmaticswith mild bronchial hyperresponsiveness to direct stimuliwho have negative exercise challenges but there are individualswho have positive exercise challenges and negative histamineor methacholine challenges [150] The imperfect relation-ship between EIB and PC20 and the existence of a numberof EIB-positive methacholine-negative individuals are indica-tive of the difference in mechanisms involved

The fewer studies in nonasthmatic lung disease are due inpart to the dif culty such individuals have in performingexercise challenges In children an exercise challenge is betterthan methacholine at distinguishing asthma from chronicairway disorders such as cystic brosis bronchiolitis oblit-erans pulmonary ciliary dyskinesia and bronchiectasis [151152] Additional studies showing that allergen avoidanceresulted in a greater improvement in EIB than in metha-choline PC20 [153] and that EIB correlates better with markersof in ammation than methacholine PC20 [154] would supportthe possibility that EIB may be more clinically relevant thanmethacholine PC20

The investigations described in the previous paragraphscon rm that a positive exercise challenge is highly speci c toidentify clinical asthma but generally is somewhat insensitiveto the presence of clinically relevant mild bronchial hyper-responsiveness In this regard the sensitivity-speci city pro leof exercise challenge resembles that of a histamine or metha-choline PC20 of 1 or 2 mgmL-1 [137] There are two possibleexplanations First as the physical stimulus affects many cellsthat are abnormal in asthma it may more readily identifypatients with this disease than with other airway in ammatorydiseases and therefore has a high speci city Secondly there isa limit to the extent of stimulus that can be achieved due tothe technical and safety constraints of exercise this preventsmaximal airway provocation resulting in low sensitivity

The indications for exercise testing have been summarisedin statements from the ERS [1] and the American ThoracicSociety (ATS) [155] Exercise may be used in the followingways 1) In making a diagnosis of EIB in asthmatic patientswith a history of breathlessness during or after exertion 2) Toevaluate the ability of performing demanding or lifesavingwork (eg military police or re ghting work) in personswith a history suggesting asthma 3) To determine the effective-ness and optimal dosing of medications prescribed to preventEIB 4) To evaluate the effects of anti-in ammatory therapygiven acutely (eg cromones) or chronically (eg steroids andleukotriene antagonists)

The recommendations for conducting an exercise test toidentify those with exercise-induced bronchoconstriction havebeen described in detail in both the ERS [1] and the ATS [155]guidelines The recommendations are similar in both docu-ments In brief the subjects should exercise for 6 (children12 yrs) to 8 (adults) min breathing dry air (lt25 degC andlt50relative humidity or lt10 mg H2OL-1) at an intensity to raisethe minute ventilation 14 times above the FEV1 and pre-ferably to 21 times the FEV1 (60 maximum voluntaryventilation) for the last 4 min of exercise In the absence of ameasure of ventilation the heart rate should achieve 90predicted maximum in the last 4 min of exercise Values forFEV1 are measured before and after exercise Providing theair is dry and the intensity of exercise appropriate it is onlynecessary to increase the time of exercise to increase theseverity of the airway response A reduction in FEV1 of 10of the pre-exercise value is widely accepted as outside theresponse observed in healthy individuals without asthma

Eucapnic voluntary hyperpnoea with dry air Althoughthere are fewer studies available the results are consistent

1054 GF JOOS ET AL

with the ndings for exercise challenge Eucapnic hyperpnoeawith dry air is more speci c and less sensitive than histamineor methacholine challenges [147 156ndash158] Dry air chal-lenge is clearly more able to separate asthmatics from subjectswith chronic air ow limitation than is histamine challenge[139 140] Eucapnic voluntary hyperpnoea (EVH) of dryair containing 5 carbon dioxide (CO2) for 6 min at aventilation equivalent to 30 times the FEV1 mimics theeffects of exercise as described above and has the sameclinical signi cance [156] As with exercise a 10 reductionin FEV1 is outside the range for healthy subjects withoutasthma [156] EVH was recommended to assess winterathletes competing in the Olympic Games in Salt LakeCity as higher levels of ventilation could be more easilyachieved during EVH compared with exercise ergometersFurther with EVH it is possible to simulate the conditionsof exercise (ventilation duration inspired air temperatureetc) in a laboratory setting [156] In contrast to exercisedoseresponse curves can be constructed

Hypertonic aerosols Bronchial responsiveness to hyper-tonic saline challenge correlates better with serum markersof in ammation than bronchial responsiveness to methacholine[159] It improves more than bronchial responsiveness tohistamine after a course of inhaled corticosteroids A challengewith hypertonic saline is easy to perform and allows con-struction of a doseresponse curve [160]

A recently developed highly portable test using mannitolcapsules and a dry-powder inhaler has shown promise asan indirect challenge with good correlation with the otherindirect physical challenges exercise hypertonic saline andhyperventilation [161 162] In one study there was a reason-able correlation between mannitol PD15 and methacholinePC20 [161] There appears to be no published data oncomparative sensitivity and speci city However since somesubjects with positive mannitol tests had mild bronchialresponsiveness to methacholine the mannitol inhalation testmay be more sensitive than other indirect challenges fordetecting mild bronchial responsiveness In a study byBRANNAN et al [162] 22 of the 23 subjects with exerciseasthma were identi ed with mannitol and the only subjectwho did not respond had a 10 fall in FEV1 to exercise

The major indications for using hypertonic aerosols are toidentify bronchial hyperresponsiveness consistent with activeasthma or exercise-induced asthma and to evaluate bronchialresponsiveness that will respond to treatment with anti-in ammatory drugs In a study by RIEDLER et al [163]children with a history of current wheeze were seven timesmore likely to have a positive response to hypertonic salinethan asymptomatic children In an occupational study inpeople responding positively to the question have you everhad an attack of asthma the mean percentage fall in FEV1was 176 compared with 58 for those who respondednegatively [164] From the evidence to date it would appearthat bronchial responsiveness to a hypertonic aerosol isconsistent with an asthma diagnosis

A test using a hypertonic aerosol is an alternative toexercise or hyperventilation to identify patients with EIB [76162 165 166] Although some patients can have EIB and benegative to hypertonic saline or mannitol this is unusual andhas only been found in persons with very mild EIB [162 163]

A challenge with a hypertonic aerosol can be used in theassessment of a patient with a past history of asthma thatwishes to scubadive In a study using 45 saline to assesspotential divers with a past history of asthma (usuallygt5 yrs)17 were found to have an abnormal response consistentwith a diagnosis of current asthma [167]

Another indication for the use of hypertonic aerosols may

be in the identi cation of persons with other airway diseaseseg chronic air ow limitation or cystic brosis who have anasthmatic component to their disease Many patients withcystic brosis are considered to have asthma As some of theinhaled medication used in the treatment of cystic brosis ishyperosmolar it would also seem important to identify thosein whom airway narrowing may occur in response to treat-ment of their primary disease [168] Both hypertonic salineand mannitol increase mucociliary clearance in subjects withasthma bronchiectasis and cystic brosis [169ndash171] Whengiven daily hypertonic saline has been shown to improve lungfunction in patients with cystic brosis [172] Thus a recom-mendation for use of a hypertonic aerosol as a therapeuticagent may need to be preceded by an inhalational challengewith the same hypertonic aerosol [173]

A challenge with a hypertonic aerosol may also be indicatedin persons with cough-variant asthma Hypertonic aerosolscan provoke cough [174 175] so documenting excessivecough in the absence of airway narrowing may indicate thatthe cough is not due to asthma Further the cough normallyprovoked by inhaling hypertonic saline stops very quicklywithin 1ndash2 min suggesting a form of refractoriness to cough inhealthy subjects

Finally a challenge with a hypertonic aerosol may beindicated in pregnancy when a patient chooses not to bechallenged with a pharmacological agent

The inhalation of hypertonic saline has been widely usedto induce sputum and to collect in ammatory cells andcytokines in asthmatics [176ndash179] What is unique to hypertonicchallenge is that it can be used to document bronchialresponsiveness at the same time as collecting sputum [179180] This makes hypertonic challenge attractive for assessingboth acute and chronic treatment with corticosteroids

Distilled water ALLEGRA and BIANCO [181] performed the rst inhalation challenge with ultrasonically nebuliseddistilled water (UNDW) in asthmatic patients The techniquewas later modi ed and standardised by other investigators[182 183] Inhalation of UNDW evokes only a cough insome normal subjects and a cough and bronchoconstrictionin asthmatic patients [184] Bronchial response to UNDWis normally distributed Most asthmatic patients developbronchoconstriction after inhaling lt2 mL of UNDW [185]A positive response to UNDW is more likely when PD20methacholine is lt2 mmol [185 186] Bronchial response toUNDW correlates poorly with methacholine responsiveness[187] The degree of bronchial responsiveness to UNDWis in good concordance with the response to exercise andto eucapnic hyperpnoea [165] A refractory period is evidentafter UNDW in ~50 of patients [185 188] Refractorinessof bronchial airways to UNDW is decreased by histamine-induced bronchoconstriction [189] the UNDW-induced refrac-toriness cross reacts with exercise-induced refractoriness [190]

Pharmacological stimuli Adenosine CUSHLEY et al [191]reported the rst observation that inhaled adenosine butnot related nucleotides caused bronchoconstriction inpatients with asthma Subsequently PHILLIPS et al [38]have shown that atopic subjects when compared to non-atopic controls are relatively more responsive to inhaledadenosine and adenosine 59-monophosphate (AMP) thanthey are to methacholine The airway response to thesepurines may be an index of mast-cell priming probablythrough A2B receptor stimulation linked to mobilisationof intracellular calcium stores Indeed nasal challenge withAMP elicits rhinitic symptoms and an immediate rise inhistamine levels in the lavage uid with the greatest increaseoccurring in atopic compared to nonatopic volunteers [192]


This indicates that atopy and other conditions where mastcells are primed for mediator release are important deter-minants of enhanced adenosine-induced histamine releaseand that this response may be used as an index of mastcell priming in vivo The capacity of adenosine to augmentmediator release from mast cells in vivo indicates thatadenosine-induced bronchoconstriction in asthmatics maydepend on the state of airway mast-cell priming and mightbe useful as an in-vivo test for this

There are limited data available for comparison ofsensitivity and speci city of AMP challenge with the direct-acting stimuli It requires ~30 times as much AMP asmethacholine to induce bronchoconstriction AMP andexercise challenges are better than methacholine challengesfor separating paediatric asthma from paediatric chronicobstructive lung disease ie AMP and exercise challengestended to be negative in the children with cystic brosisbronchiolitis obliterans ciliary dyskinesia and bronchiectasis[151 152] Nonsmoking adults with COPD are signi cantlyless responsive to inhaled adenosine than nonsmokingasthmatics whereas the sensitivity to methacholine is similarin both groups [193] Taken together these ndings indicatethat adenosine challenge may be a useful tool in thedifferential diagnosis of asthma and COPD in patients of allages in whom the diagnosis is clinically uncertain This isespecially the case in nonsmokers since smokers with COPDmay show AMP responsiveness as well [193] In additionthe speci city of adenosine bronchoprovocation for asthmatogether with the high repeatability of this test could be usefulfor epidemiological studies

Propranolol On a molar basis the dose of propranololrequired to induce bronchoconstriction in patients with asthmais ~10ndash15 times larger than methacholine or histamine [194]The limited data supports higher speci city and lower sensi-tivity for propranolol compared to histamine or methacholinePropranolol inhalation tests were negative in the majority ofsubjects with chronic air ow limitation supporting betterspeci city of propranolol challenge for asthma [195] Broncho-constriction induced by propranol is usually less well toleratedby patients compared to that caused by histamine methacholineor adenosine Nevertheless no serious events have ever beenreported following propranolol-induced bronchoconstrictioneither in asthmatics or in patients with COPD In additionpropranolol-induced bronchoconstriction can be weakly reversedby inhaled adrenergic and anticholinergic drugs

Metabisulphite sulphur dioxide In epidemiological studiesairway responsiveness to the indirect stimulus sulphur dioxide(SO2) and the direct stimulus methacholine were compared ina sample of 790 adults aged 20ndash44 yrs In this cohort theprevalence of hyperresponsiveness to SO2 was 34 Amongthe subjects who had hyperresponsiveness to methacholine224 had hyperresponsiveness to SO2 There was no signi -cant correlation between the degrees of hyperresponsiveness tomethacholine and SO2 [196]

Aspirin While there is no in-vitro test available for thedetection of intolerance to aspirin and cross-reacting non-steroidal anti-in ammatory drugs (NSAIDs) in patients withasthma oral provocations with incremental doses of aspirinhave been used to diagnose this syndrome [197] However thechallenge procedure is fairly time consuming potentiallydangerous and should only be performed in a laboratory withconsiderable experience of aspirin elicited reactions Morerecently the lysine-aspirin inhalation challenge introducedby BIANCO et al [198] has proven very useful in identifyingaspirin-intolerant asthmatic subjects [199ndash201] In a prospective

comparative study the lysine-aspirin challenge was found tobe as sensitive as oral provocation with respect to productionof airway obstruction In a study on 22 consecutive patientswith a history andor clinical ndings suggestive of aspirin-intolerance (asthma rhinorrhea nasal polyposis) challenges byboth routes were performed at least two weeks apart A total of10 subjects developed signi cant bronchoconstriction ( 20drop in FEV1) during either challenge with the same absolutesensitivity for both tests (910) Inhalation challenge provokedresponses that developed more promptly (within 20ndash30 min)were limited to the airways caused a lesser degree of airwayobstruction (mean maximal fall in FEV1 29iexcl6 versus38iexcl16 for oral challenge) and were more easily reversed[200] In 19 aspirin-tolerant control subjects with the samebaseline pulmonary function inhalation of lysine-aspirin causedno signi cant changes in FEV1 supporting the speci city ofthe test

Although oral administration is necessary for the detectionand investigation of extrapulmonary reactions inhalationchallenge has the bene t of safety for use in clinical practiceFor research purposes the safety and good repeatability ofinhalation challenge provide a considerable advantage overoral challenge particularly since a signi cant proportion ofaspirin-intolerant asthmatics suffer from moderate-to-severeasthma

Reports on the repeatability of lysine-aspirin challenge [96199] have shown that it is repeatable approximately within asingle doubling concentration or dose difference With themethodology described below the 95 CI for the differencein results between two challenges separated by 10ndash75 days was06ndash18-fold A positive provocation response to inhaled (ororal) aspirin results in a state of refractoriness to furtherdoses of aspirin or other NSAIDs [198] The refractory periodlasts between 2ndash5 days and desensitisation as well as cross-desensitisation may be retained provided aspirin is ingestedwithin a maximum interval of 48 h Complete sensitivity toaspirin and other NSAIDs reappears ~7 days after the lastexposure to these drugs [202] Therefore repeated challengesfor diagnosis or research purpose should be separated by atleast 1 week Another pitfall that may produce false-negativeaspirin provocation is indicated by observations that highdoses of glucocorticosteroids may mask aspirin intolerance[203] Moreover it has been documented that treatment withantileukotrienes [96] and salmeterol [204] blunt the lysine-aspirin induced airway response

The major indication for using lysine-aspirin inhalationchallenge is to identify aspirin-sensitive asthmatic patientsand to study mechanisms involved in bronchoconstrictionelicited by aspirin and other NSAIDs

Direct versus indirect airway challenges to monitor asthma

The monitoring of symptoms air ow obstruction andexacerbations is essential to asthma management Regularmonitoring by physicians improves health outcomes pro-vided it includes monitoring of control of asthma medicationand skills at regular intervals [205] Bronchial responsive-ness can be assessed at regular clinic visits and is related toasthma severity and airway in ammation [205] It has beendemonstrated repeatedly that despite signi cantly improvingsymptoms and decreasing airway in ammation inhaled cortico-steroids produce at best a modest decrease in bronchialhyperresponsiveness as measured by histamine or methacho-line challenges This observation has been made in adults[206] as well as children with asthma [207] Despite theselimitations direct airway challenges may be useful in thetitration of anti-in ammatory therapy [208] Indeed SONTet al [208] have reported that a treatment protocol aimed at

1056 GF JOOS ET AL

improving bronchial hyperresponsiveness to methacholine aswell as symptoms and lung function led to better asthmacontrol fewer exacerbations and reduced chronic airwayin ammation

In view of the clinical and physiological relevance ofindirect challenges it is desirable to design studies thatcompare the improvement in symptoms and markers ofairway in ammation induced by anti-asthmatic therapy withtheir effects on direct and indirect airway challenges The viewthat bronchial responsiveness to adenosine is a more robustmarker of disease activity in relation to allergic airwayin ammation than other nonspeci c stimuli such as hista-mine or methacholine is supported by a number of clinicalstudies In subjects with active allergic rhinitis bronchialresponsiveness to AMP but not methacholine is stronglycorrelated to sputum eosinophilia [209] In a large group ofpatients with asthma PC20 AMP was more closely associatedwith eosinophilic airway in ammation than PC20 metha-choline [11] A series of clinical studies have con rmed thepotential utility of AMP in detecting in ammatory changes inadult and paediatric asthma Regular treatment with inhaledcorticosteroids results in a signi cantly greater reduction inAMP responsiveness compared to that of direct (methacho-line and histamine) and neurally acting stimuli (sodiummetabisulphite and bradykinin) [210 211] In keeping withthis several studies have shown that b-agonists cause greaterbronchoprotection against AMP than against histamine ormethacholine challenge in patients with asthma [212 213]VAN VELZEN et al [12] have shown that improvements inclinical asthma occurred in a group of 16 allergic asthmaticchildren admitted to a high-altitude clinic This was believedto be due to the lower allergen levels encountered and wasaccompanied by a signi cant reduction in bronchial respon-siveness to AMP but interestingly not to methacholine Onthe basis of these observations the authors believe thatadenosine bronchoprovocation may provide an index thatcould be used to survey disease progression monitor therapyand assess prognosis

Osmotic stimuli such as hypertonic (45) saline andmannitol hold promise for monitoring asthma A challengewith hypertonic saline or mannitol can be used to assess theseverity of asthma the effect of treatment and the compliancewith treatment In a recent study in well-controlled asthmaticsLEUPPI et al [214] demonstrated that failure of successfulreduction in steroids could be predicted by responsiveness tomannitol The use of 45 saline as an indication of severityof asthma and need for steroids is supported by the ndingsof RODWELL et al [215] In their study patients with a PD20to 45 saline of 30 mL ie those with moderate-to-mildasthma were most likely to become negative to hypertonicsaline during treatment with steroids and to plateau inresponse to acute administration of nedocromil sodiumBRANNAN et al [216] reported similar ndings for mannitoland nedocromil sodium In the study of ANDERSON et al [217]the increase in PD20 to hypertonic saline in response to8 weeks of treatment with budesonide was predicted by theincrease in PD20 following a single dose of sodium cromo-glycate given 10 min before challenge [217] A negativeresponse to challenge with 45 saline suggests that theperson either does not have asthma or that their asthma iscurrently under control with treatment For example apatient taking budesonide daily for 4ndash8 weeks has a 50likelihood of becoming negative to challenge with hypertonicsaline [160 215] and to mannitol [218] These ndings are inkeeping with 50 of the subjects no longer having EIB aftertreatment with budesonide [37] By contrast it is highly likelythat the same people would remain responsive to inhaledhistamine or methacholine [160 206 219]

A bronchial challenge with hypertonic saline can be combined

with an induction of sputum to assess airway in ammation[176 220] IN9T VEEN et al [179] compared provocation withmethacholine (PC20) hypertonic saline and sputum induc-tion as outcome parameters in patients with severe asthmaduring steroid withdrawal [179] During both induced andspontaneously occurring exacerbations increased bronchialresponsiveness for methacholine was noted However onlythe induced exacerbations were associated with increasedbronchial responsiveness to hypertonic saline and increasedpercentage of sputum eosinophils

Response to indirect challenges can be an interestingoutcome parameter in the evaluation of anti-in ammatorytreatment by inhaled steroids or leukotriene receptor antago-nists In a comparative study on the effects of 4-weektreatment periods with three different doses of budesonide(100 200 and 400 mgday-1) PEDERSEN and HANSEN [221]found a doseresponse effect on lung function and EIB butnot on symptoms or peak expiratory ow rate in the eveningApproximately 53 of the maximum effect against EIB wasachieved by the lowest budesonide dose and ~83 by thehighest dose In a study on the effects of two doses of uticasone propionate (100 and 250 mg bid compared toplacebo) the severity of EIB decreased signi cantly as com-pared to placebo within 3 weeks [13] These reductions in EIBdid not differ between the two doses and were sustainedduring the study period of 6 months In contrast responsive-ness to methacholine improved during the rst 6 weeks of thetreatment with uticasone and steadily increased with timeafter 24 weeks of treatment the difference in improvement ofPD20 methacholine was 16 dose steps for 100 mg uticasonebid and 33 dose steps for 250 mg bid The new inhaledsteroid ciclesonide (50 200 and 800 mgday-1) reduced respon-siveness to AMP and eosinophils in induced sputum Incontrast to sputum eosinophilia the reduction in responsivenessto AMP was dependent on the dose of inhaled steroid [14]

The studies that have compared direct and indirect chal-lenges to monitor asthma during anti-in ammatory therapywith inhaled corticosteroids and leukotriene-receptor antago-nists are summarised in table 3 Inhaled corticosteroids led toan attenuation of bronchial responsiveness to the majority ofdifferent stimuli although to different extents thereby under-lining the antiasthmatic ef ciency of inhaled corticosteroidsAll authors found a signi cant although small reduction inhistamine or methacholine responsiveness Results were lessconsistent for bradykinin responsiveness and inhalation chal-lenges using hyperventilation of air which contained SO2 [211222] It has even been argued that AMP responsiveness atleast in children is a more sensitive predictor of the effect ofanti-in ammatory therapy than bronchial responsiveness tomethacholine or bradykinin [211] In a study on the effect of a2-week treatment with oral or inhaled steroids in adultasthmatic patients PC20 AMP was found to be more sensitiveto changes in acute airway in ammation compared to PC20methacholine [228] This would underline the assertion thatindirect challenges may be better suited to assess therapeuticef cacy than direct challenges Following the same line ofreasoning LEFF et al [29] demonstrated that EIB was signi -cantly attenuated by long-term treatment with a leukotrienereceptor antagonist whereas methacholine responsivenesswas not signi cantly reduced It should be noted however thatthe relatively modest bene t of inhaled steroids on directchallenges should not per se be considered as a disadvantageThis modest bene t may in fact be highly relevant as parts ofbronchial responsiveness to histamine or methacholine maynot be sensitive to steroids or may require very prolongedtherapy The slow response to steroids may actually be moreinformative on eg remodelling aspects which may be moreimportant for the long-term management and prognosis ofthe disease [208]


Use of indirect airway challenges in epidemiologicalstudies

Questionnaires are most frequently used to diagnoseasthma or other respiratory disorders in epidemiologicalstudies They may however be subjective and the level ofawareness of the condition in the community may in uencethe pattern of response Similar problems may occur with adoctor9s diagnosis of asthma These differences in de ningrespiratory diseases often cause problems with comparisonsof epidemiological studies between different populations andover time Thus an objective marker closely associated withdiseases like asthma is desirable

In the past direct-airway challenges using histamine andmethacholine have been considered to be more sensitive for adiagnosis of asthma or asthma symptoms when comparedwith indirect tests However recent laboratory and epide-miological studies have shown that this concept might be inquestion In a laboratory based study of elite summer athletesHOLZER et al [150] found that methacholine PD20 had asensitivity of only 36 to identify the athletes with positiveresponse to EVH a surrogate challenge used to identifyexercise-induced bronchoconstriction For those 16 subjectspositive to EVH and negative to methacholine the meaniexclSD

percentage fall in FEV1 was 178iexcl195 after EVH and thetop dose of methacholine the fall in FEV1 was 76iexcl49 In a eld study by HABY et al [229] in which children were studiedwith histamine and exercise 45 of those positive to astandardised exercise challenge were negative to inhaledhistamine with reduction in FEV1 to the highest cumulativedose of histamine beinglt10 A histamine challenge in 2363Australian schoolchildren aged 8ndash11 yrs yielded a sensitivityof 53 and a speci city of 90 to detect subjects with adiagnosis of asthma [230] Sensitivity and speci city of thehistamine challenge were similar to sensitivity and speci cityof a hypertonic saline challenge and an exercise challenge inanother epidemiological study in children from the samecountry [163]

For many participants in eld studies particularly children

indirect challenges involving more natural stimuli are moreappealing Parents will often not allow their child to inhale apharmacological agent in epidemiological surveys Conse-quently there has been increasing interest in the use ofindirect airway challenges for epidemiological studies Thesetests mainly comprise of the inhalation of nonisotonic solutionssuch as hypertonic saline or distilled water hyperventilationof dry air and various sorts of exercise tests Hypertonic salinechallenge is a relatively inexpensive test that is safe welltolerated and reproducible It can be performed readily in the eld It produces few complaints of dryness or irritation of thethroat In a study on 500 children only 15 of participatingchildren felt that they could not continue the challengebecause of irritation to the throat or cough Similarly 16 of the same subjects were unwilling to complete a free-runningexercise test because of fatigue [163]

The hypertonic saline challenge appears to have somepractical advantages compared to exercise challenge in a eldstudy A challenge with hypertonic saline is not dependent onweather conditions (temperature humidity) nor is it in u-enced by the level of the child9s tness and it allows for doseincrements and measurement of dose response curves makingthe challenge safer The EVH challenge is well standardised[156 157] but needs a special gas mixture source which makesit less suitable for eld studies

Safety aspects of indirect airway challenges

The safety of standardised histamine and methacholinechallenge tests is recognised all over the world Previousguidelines on provocation challenges have stressed the pre-cautions that need to be taken as well as the relative andabsolute contraindications for challenge testing [1] Theseprecautions apply also to indirect airway challenges andinclude laboratory materials personnel training and writtensafety protocols With regard to physical challenges there isgeneral consensus that standardised exercise tests are safe[155] In the literature there is one documented case of a fatal

Table 3 ndash Direct and indirect challenge tests to monitor asthma during anti-in ammatory therapy

First author[ref no]

Year Compound Duration oftreatment

weeks

Dose Challenge

Direct Indirect

Type Reactivity Type Reactivity

WIEBICKE et al 1990 Salbutamolz 3 0205 mg qid Histamine SO2 Oslash[222] BDP Methacholine Hypervent OslashVATHENEN et al 1991 Budesonide 6 800 mg bid Histamine Exercise[223] Cold air hyperventFULLER et al[224] 1991 Budesonide 3 1200 mgday-1 Histamine BradykininGROOT et al [225] 1992 BDP 8 200 mg qid Histamine Dist waterO9CONNOR et al 1992 Budesonide 2 08 mg bid Methacholine Metabisulphite[210] AMPBOOTSMA et al 1995 Fluticasone 6 750 mgday-1 Histamine Dist water[226] BDP 1500 mgday-1 Histamine Dist waterDOULL et al [211] 1997 BDP 12 400 mgday-1 Methacholine Oslash Bradykinin OslashDU TOIT et al[160]

1997 Budesonide 8 1000 mgday-1 Histamine Hypertonic saline

WEERSINCK et al 1997 Salmeterol 6 50 mg bid Methacholine Adenosine[227] Fluticasone 250 mg bid

SalmeterolzFluticasone

50z250 mg bid

LEFF et al [29] 1998 Montelukast 12 10 mgday-1 Methacholine Oslash Exercise

BDP beclomethasone dipropionate Dist water distilled water modest reduction more pronounced reduction Oslash no change

1058 GF JOOS ET AL

asthma attack during inhalation challenge with distilled water[231] Recent studies have reported inhalation of hypertonicsaline eventually in conjunction with sputum induction to besafe [232 233] In the appendices (1ndash4) safety and perfor-mance issues concerning physical challenges with exercise orhypertonic saline and pharmacological challenges with adeno-sine or lysine-aspirin are described in detail

Conclusions

The direct airway challenges methacholine and histaminecause air ow limitation predominantly via a direct effect onairway smooth muscle Indirect airway challenges induceair ow limitation by an action on cells other than smoothmuscle cells which upon stimulation release mediators thatprovoke smooth muscle contraction

A challenge with methacholine or histamine is a highlysensitive measure for the detection of hyperresponsiveness inpatients suspected of having asthma when referred to apulmonary function laboratory or clinic They are useful toexclude current asthma in these populations However directchallenges are not speci c to asthma do not exclude exercise-induced bronchoconstriction and perform less well in theepidemiological setting Indirect bronchial stimuli in parti-cular exercise hyperventilation nonisotonic aerosols as wellas adenosine may re ect more directly the ongoing airwayin ammation and are more speci c but less sensitive toasthma They are increasingly used to evaluate the prevalenceof bronchial hyperresponsiveness and to assess speci c pro-blems in patients with known asthma (eg exercise-inducedbronchoconstriction evaluation before scuba diving)

Bronchial responsiveness can be assessed at regular intervalsand is related to asthma severity and airway in ammation Itis well known that anti-in ammatory therapy with inhaledcorticosteroids results in an improvement of symptoms and adecrease in airway in ammation Direct bronchial respon-siveness is only slowly and to a modest extent in uenced bythe repeated administration of inhaled steroids Indirectchallenges may re ect more closely acute changes in airwayin ammation and be clinically relevant markers to assess theclinical course of asthma Moreover some of the indirectchallenges eg hypertonic saline and mannitol can be com-bined with the assessment of in ammatory cells by inductionof sputum In view of the clinical and physiological relevanceof indirect challenges it is desirable to design studies thatcompare the improvement in symptoms and markers ofairway in ammation induced by antiasthmatic therapy withtheir effects on direct and indirect airway challenges

Areas for future research

Mechanisms and receptors

The following are areas that require further research toimprove the understanding within this eld 1) Furthercharacterisation of receptor(s) involved in bronchoconstrictoreffects of adenosine eg by use of speci c antagonists 2) Theidenti cation of adenosine targets on cells other than mastcells (eg epithelial cells) 3) The relation between mediatorrelease and the response in individual patients combinationof indirect challenges with measurements in breath conden-sate and exhaled air 4) The use of transgenic technologyie knock-outs and knock-ins to de ne in more detail themolecular targets for some of the indirect stimuli

Diagnosis

Further questions that need to be addressed to improve thediagnostic development in this eld include 1) How doindirect challenges relate to mucosal in ammation and tononinvasive measures of airway in ammation such as inducedsputum and exhaled air 2) What is the relationship betweenbronchial responsiveness of different indirect challenges andairway remodelling 3) Can an indirect challenge be used asan index of asthma severity 4) To assess risk for an exacer-bation 5) Are indirect challenges useful in assessing riskseg occupational exposure 6) How can indirect challenges beincorporated in geneticphenotyping studies

Monitoring

To improve knowledge on the value of indirect airwaychallenges for monitoring of asthma the following questionsneed to be investigated further 1) How can indirect challengesbe applied in the short-term and long-term monitoring of anasthma patient 2) How do they compare to the direct stimulihistamine and methacholine 3) Can indirect challenges beused to evaluate the ef cacy of allergen avoidance measures4) Can indirect challenges be used to assess the minimumeffective dose of an inhaled steroid and to monitor complianceto treatment with inhaled steroids 5) Do indirect challengeshave a prognostic value in allergic rhinitis

Epidemiology

A question concerning epidemiology that needs to be addressedis what is the epidemiology of responsiveness to lysine-aspirin or to adenosine Further research into the phenotype-genotype correlation and the standardisation of protocols forindirect challenges in infants and toddlers is also needed

Safety performance

There is a need for better standardisation and also to knowmore about reproducibility in order to improve both safetyand performance of indirect airway challenges

Appendix 1 Safety issues for exercise challenges in thelung function laboratory and in eld studies

A distinction has to be made between challenges applied inthe laboratory and in a eld study General issues includingsafety issues have been discussed in recent documentsEuropean Respiratory Society (ERS) 1993 [1] and AmericanThoracic Society (ATS) 2000 [155]

Safety issues for exercise challenge in the laboratory

Safety issues for the exercise challenge in the laboratoryinclude the following 1) Two experienced people in atten-dance if patient at high risk one should be a physician 2)Adult of gt60 yr to have normal electrocardiogram 3)Ventilation to be measured as this is the stimulus 4) Heartrate measured 5) Bronchodilator plus oxygen (O2) at hand 6)Medical helpresuscitation available within 2 min 7) Forcedexpiratory volume in one second (FEV1) pre-exercise gt70predicted 8) No long-acting b2 agonist for 48 h 9) Cessationof exercise test if patient is distressed the arterial oxygensaturation (SaO2) is falling during exercise ventilation is


reduced or the breathing is laboured 10) The FEV1 indistressed patients must be measured

Safety issues for exercise challenge in the eld

The safety issues for exercise challenge in the eld are asfollows 1) Baseline FEV1gt70 predicted 2) Actual value forFEV1 is to be considered 3) Known asthmatics should beidenti ed 4) Subject exercising observed by one person at alltimes during and especially after exercise 5) Bronchodilatorand O2 are to be at hand 6) Large volume-spacer and pres-surised metered-dose inhalers available 7) Medical or nursinghelp at hand 8) Transport available to nearest accident andemergency facility 9) Oximeter for monitoring O2 saturationand heart rate 10) Bronchodilator given when fall in FEV1gt10

Appendix 2 Safety issues for airway challenges withhypertonic saline in the lung function laboratory and in

eld studies

Safety issues for hypertonic saline challenge in thelaboratory

The safety issues for hypertonic saline challenge in thelaboratory are as follows 1) Baseline FEV1 gt75 pred or65 for some laboratories 2) First exposure 30 s only 3)Patient must be attended at all times 4) Patient must be freeto come off mouthpiece 5) Bronchodilator and oxygen to bein immediate vicinity 6) Medical helpresuscitation availablewithin 2 min 7) Oximeter available for monitoring oxygensaturation 8) No long-acting bronchodilator for 48 h 9)Equipment must be properly cleaned

Safety issues for hypertonic saline challenge in the eld

Safety issues for hypertonic saline challenge in the eld areas follows 1) Baseline FEV1 gt65ndash75 of pred or gt12 L 2)First exposure 30 s only 3) Stop at 15 or 20 fall in FEV1give bronchodilator 4) Subject must be attended at all times5) Subject must be free to come off mouthpiece 6) Broncho-dilator and O2 at hand 7) Medical or nursing help at hand8) Transport available to nearest accident and emergencyfacility 9) Oximeter for monitoring O2 saturation and heartrate 10) Subjects should understand test 11) Bronchodilatorgiven if fall in FEV1 gt10

Appendix 3 Performance standards safety issues andprotocol recommendations for airway challenges with

adenosine

Contraindications and safety

As for more traditional means of bronchial challengecontraindications to adenosine challenge testing are condi-tions that may compromise the quality of the test (eginability to perform acceptable spirometric manoeuvressigni cant airway obstruction) or that may subject the patientto increased risk or discomfort (eg low baselinendashlungfunction recent heart attack or stroke and pregnancy)Moreover to ensure good-quality results and patient safetythe technicianphysician who performs the test should bepro cient in bronchial challenge testing However hundredsof adenosine challenge tests have been performed bylaboratories with no serious side-effects

Patient preparation

Patients undergoing adenosine 59-monophosphate (AMP)challenge testing should be given a list of itemsmedications toavoid before the test Medications such as inhaled broncho-dilators (b2-agonists anticholinergics) [39] theophylline [234]antihistamines [40 234] cromones (sodium cromoglycatenedocromil) [235] nonsteroidal anti-in ammatory drugs(NSAIDs) [41 42] and oral antileukotrienes [27] can reducebronchial responsiveness to adenosine potentially causing afalse-negative response Moreover as adenosine responses areextremely sensitive in detecting changes after inhaled steroidsmuch attention should be dedicated to this confounderespecially when monitoring bronchial responsiveness in thelong term Factors such as allergen exposure recent respira-tory infection and cigarette smoking may temporarilyincrease bronchial responsiveness to AMP and generatefalse-positive results

Making of adenosine 59-monophosphate solution

The sodium salt of AMP (Sigma-Aldrich product noA1752) available as a dry crystalline powder is the agent ofchoice for challenge testing This is preferred to adenosinebecause it is more soluble in sterile normal saline Bulkpowder should be stored with a desiccator in a freezer Sterilenormal saline (09 sodium chloride) may be used as thediluent AMP solutions should be properly mixed labelledand stored (~4 degC) AMP solutions 3125 mgm-1 remainstable for up to 25 weeks at 4 degC

Dosing protocols

Doubling concentrations are widely recommended and aremathematically attractive Many authors favour the ve-breath method (either using a dosimeter at the beginning of adeep inhalation or by continous nebulisation dosing during adeep inhalation) over the others The 2-min tidal breathingmethod is slightly more time consuming but has also beenused successfully in both adults and children [152]

For the ve-breath dosimeter technique the authorsrecommend a dosing schedule using AMP concentrations of3125 625 125 25 50 100 200 and 400 mgmL-1 The ve-breath dosimeter protocol was rst standardised by theNational Institutes of Health (NIH) Institute of Allergic andInfectious Diseases in 1975 [128] and is presented as analternative method by the ERS [1] Dosimeters may improvethe accuracy and repeatability of the dose delivered to theairways but adds additional expense They are widely used inboth clinical and research settings The protocol is as follows1) Set up and check the dosimeter 2) Prepare AMP solutions(3125ndash400 mgmL-1) in sterile vials place them in a holderand store them in a refrigerator 3) Remove the vials from therefrigerator 30 min before testing so that the contents warmto room temperature before use 4) Most current protocolsstart with a diluent step with normal saline 5) The patient isseated throughout the test 6) Perform baseline spirometry 7)Ask the patient to hold the nebuliser upright with themouthpiece in their mouth Watch the patient during thebreathing manoeuvres to ensure that the inhalation andbreathhold are correct 8) Instruct the patient to inhale slowlyand deeply from the nebuliser Trigger the dosimeter soonafter the inhalation begins dosimeters may do this auto-matically 9) Repeat step eight for a total of ve inspiratorycapacity inhalations Take no more than a total of 2 min toperform these ve inhalations 10) Measure the FEV1 at ~60and 180 s after the fth inhalation from the nebuliser Obtain

1060 GF JOOS ET AL

a good-quality FEV1 at each time point This may requirerepeated attempts 11) Report the highest FEV1 fromacceptable manoeuvres The postsaline FEV1 is the referencepoint for comparison and it should not exceed a 10 fall inFEV1 from baseline 12) Pour the rst concentration of AMPsolution into the nebuliser using a sterile syringe and repeatsteps 7ndash9 13) Measure the FEV1 at 60 and 180 s after the fthinhalation from the nebuliser The timing of FEV1 measure-ments at 60 and 180 s after the inhalation is based on theresults of time course studies with AMP 14) At each dosereport the highest FEV1 from acceptable manoeuvres 15) Ifthe FEV1 falls lt20 empty the nebuliser shake it dry andadd 20 mL of the next higher concentration and repeat steps12ndash14 16) If the FEV1 fallsgt20 from baseline (or the highestconcentration has been given) give no further AMP administerinhaled salbutamol wait 10 min and repeat spirometry

Recommendation on nebulisers and dosimeters

The nebuliser must deliver an aerosol with a particle massmedian diameter (MMD) between 10ndash36 mm Avoid the useof nebulisers with MMD lt10 mm Nebulisers for the ve-breath method should deliver 9 mLiexcl10 of solution per 06-sactuation during inhalation [129] A single nebuliser may beused for all concentrations provided it is emptied and thenozzle dried between doses Alternatively six or sevenseparate calibrated nebulisers may be lled before the testIf separate nebulisers are used they must be carefully labelledto avoid dosing errors Inexpensive plastic nebulisers aregenerally not manufactured with tight output tolerances andtheir volume output should be checked before use At least1 mL of solution should remain at the end of nebulisationbecause output decreases below this level

Common end-point measures

Change in FEV1 is the primary outcome measure foradenosine challenge testing Special care should be taken toobtain good quality baseline FEV1 measurements becauseunacceptable manoeuvres may result in false-positive or false-negative results The quality of the owvolume curves shouldbe examined after each manoeuvre

Measures of airway resistance (Raw) usually expressed asspeci c conductance (sGaw) are alternative end-points foradenosine challenge testing but both Raw and sGaw aremore variable and less reproducible than FEV1 Changes inairway resistance may be more sensitive than changes inFEV1 for detecting bronchoconstriction but FEV1 is superiorto other parameters for discriminating relatively healthypersons from those with asthma Changes in peak expiratory ow often parallel changes in FEV1 during bronchocons-triction but have the disadvantages of being more effortdependent and less reproducible [236]

Data presentation

The percentage fall in FEV1 from baseline is plotted on theordinate against the log concentration of AMP on theabscissa and the provocation concentration required toproduce a 20 decrease in FEV1 (PC20) from the postsalinetreatment baseline value is determined by linear interpolationThe PC20 value may be used to summarise the results If theFEV1 does not fall by at least 20 after the highest concentra-tion then the PC20 should be reported as gt 400 mgmL-1The PC20 is recommended as the outcome variable because

it is simple to calculate and avoids the complicated andcontroversial aspects of estimating a provocative dose (PD20)


lysine-aspirin

Challenges must be performed in the hospital under closesupervision of the patients and with emergency resuscita-tive equipment readily available Moreover the responsiblephysician and the technician performing the test should beexperienced with bronchial challenge testing The protocoldescribed below has been used repeatedly at the Dept ofRespiratory Medecine at Karolinska Hospital (StockholmSweden) in cohorts of NSAID-intolerant asthmatic subjectsand in patients with NSAID-tolerant asthma on gt250occasions and with no serious adverse events

Lysine-aspirin is administered by a dosimeter-controlledjet-nebuliser (Spira Elektro 2 Respiratory Care CenterHameenlinna Finland) As indicated in table 4 by the useof two or sometimes three different solutions of lysine-aspirinand by variations in the number of tidal breaths step-wiseincrements in the dose of inhaled aspirin produce the desiredprotocol for cumulative challenge

Nebuliser settings

The settings for the nebuliser are shown in table 5 Thesesettings provide an aerosol with 80 of the particles beinglt58 mm and an MMD of 41 mm [237]

Lysine-aspirin solutions

Crystalline lysine-aspirin (AspisolTM Horby Bayer AGGermany FlectadolTM Maggioni-Winthrop Italy) is providedin vials containing 1 g (AspisolTM) or 2 g (FlectadolTM) oflysine-aspirin This corresponds to 500 and 1000 mg ofacetylsalicylic acid respectively

The lysine-aspirin solutions are prepared fresh just before

Table 4 ndash Concentrations and doses of Aspirin used in thedosimeter-controlled jet-nebuliser protocol

Aspirin M No ofbreaths

Dosemmol

Cumulateddose mmol

log10 log10 unitsincrease

01 1 1 1 0 001 2 2 3 05 0501 7 7 10 10 0510 2 20 30 15 0510 7 70 100 20 0510 8 80 180 226 02610 12 120 300 248 02310 30 300 600 277 029

Table 5 ndash Nebuliser settings

Procedure Parameter

Inspiratory ow rate Ls-1 05Starting volume mL 50Tidal volume L 05ndash06Duration of nebulisation s 08Output mLbreath-1 103


the start of the challenge by dissolving the crystallinelysine-aspirin in saline (09 sodium chloride) Crystallinelysine-aspirin is stable and may be kept at room temperaturefor prolonged periods whereas solutions of lysine-aspirin areonly stable for 2 h in the refrigerator

For most challenges in sensitive subjects it is suf cient tomake up two concentrations of lysine-aspirin (01 and 1 M)Using AspisolTMthe 1 M stock solution (360 mgmL-1 lysine-aspirin 180 mgmL-1 aspirin) is made by dissolving one vialof crystalline lysine-aspirin (1 g lysine-aspirin contains 05 gaspirin) in 28 mL of saline The 01 M solution (36 mgmL-1

lysine-aspirin 18 mgmL-1 aspirin) is produced by adding45 mL of saline to 05 mL of the 1 M stock solution Pleasemake sure that the 10 M stock solution is dissolved beforeperforming the dilution

In subjects who are less sensitive to aspirin it may berequired to prepare also a 20 M solution by dissolving 1 g oflysine-aspirin in 14 mL of saline (720 mgmL-1 lysine-aspirin360 mgmL-1 of aspirin) This more concentrated solutionreduces the number of breaths required to produce the highestdose of lysine-aspirin in the protocol or may be used ifnecessary to increase the dose even further in doubtful cases(table 4) However it should be borne in mind that aspirin isabsorbed through the airways and gives rise to measurableplasma levels

The solutions are kept in the refrigerator during theprovocation but must be brought to room temperaturebefore each administration A minimum of 1 mL of solutionis required in this particular nebuliser

Challenge protocol

Pulmonary function is measured as FEV1 and the baselinede ned as the best of three efforts If baseline FEV1 is gt70of pred the test is started by administration of the diluent(seven breaths of saline) Provided FEV1 at 10 and 20 minafter inhalation of the diluent does not change by gt10 theaspirin challenge is started and the postdiluent FEV1 valueused as baseline

The lysine-aspirin solution is inhaled every 30 min and~05ndash025 log-dose increments are administered according tothe table FEV1 is obtained at 10 20 and 30 min after eachdose The provocation is stopped when FEV1 has fallen 20 from the postdiluent baseline or the maximum dose ofaspirin has been reached (600 mmol cumulative dose)

If the decrease in FEV1 at 30 min after an inhaled dose isbetween 15ndash20 indicating the development of a positivereaction it is advised to wait another 15 min before a furtherdose increment If the drop in FEV1 remains between15ndash20 the responsible physician must make a decision asto whether or not the next dose in the protocol should begiven In subjects with a steep dose-response relation forlysine-aspirin andor moderate to severe asthma it is recom-mended for safety purposes to repeat the previous doserather than giving the next dose in the protocol

After a positive reaction FEV1 is followed every 15 minuntil it returns to within 10 of the post diluent baseline Thepatient should always be observed for at least 1 h after thetermination of provocation Although typical late reactionshave not been documented following aspirin challengethe challenged subject should be advised to record peakexpiratory ow rate (PEFR) in the case of airway symptomsBefore leaving the clinic PEFR should be recorded and aprede ned level marked out on the PEFR-chart to alert forrescue medication andor contact with the hospital

Dose-response relations for aspirin are constructed andused for calculation of the PD20 The lowest FEV1 measure-ment at 10 20 or 30 min after each dose is plotted against the

log cumulated dose of aspirin and the PD20 value is derivedfrom linear interpolation between the two last doses

Acknowledgements The authors would like tothank C Vandeven for expert secretarial assis-tance and V Schelfhout for help in recording thediscussions during the Ghent workshop

Internal referees of the report (andor attendeesof one or more workshop) R Aalbers N BarnesL-P Boulet D Dusser J De Jongste S GodfreyM Ichinose PW Ind KN ParameswaranK Rabe J Solway PJ Sterk

References

1 Sterk PJ Fabbri LM Quanjer PhH et al Airway respon-siveness Standardized challenge testing with pharmacologicalphysical and sensitizing stimuli in adults Report WorkingParty Standardization of Lung Function Tests EuropeanCommunity for Steel and Coal Of cial position of theEuropean Respiratory Society Eur Respir J 1993 6 Suppl16 53ndash83

2 Lotvall J Inman M O9Byrne P Measurement of airwayhyperresponsiveness new considerations Thorax 2000 53419ndash424

3 Tashkin DP Altose MD Bleecker ER et al The lung healthstudy airway responsiveness to inhaled methacholine insmokers with mild to moderate air ow limitation The LungHealth Study Research Group Am Rev Respir Dis 1992 145301ndash310

4 Juniper EF Frith PA Hargreave FE Airway responsivenessto histamine and methacholine - relationship to minimumtreatment to control symptoms of asthma Thorax 1981 36575ndash579

5 Tashkin DP Altose MD Connett JE Kanner RE Lee WWWise RA Methacholine reactivity predicts changes in lungfunction over time in smokers with early chronic obstructivepulmonary disease The Lung Health Study ResearchGroup Am J Respir Crit Care Med 1996 153 1802ndash1811

6 Pauwels R Joos G Van der Straeten M Bronchialhyperresponsiveness is not bronchial hyperresponsiveness isnot bronchial asthma Clin Allergy 1988 18 317ndash321

7 Van Schoor J Joos GF Pauwels RA Indirect bronchialhyperresponsiveness in asthma mechanisms pharmacologyand implications for clinical research Eur Respir J 2000 16514ndash533

8 Manning PJ Watson RM O9Byrne PM Exercise-inducedrefractoriness in asthmatic subjects involves leukotriene andprostaglandin interdependent mechanisms Am Rev RespirDis 1993 148 950ndash954

9 Laprise C Boulet LP Magnitude and determinants ofshort-term tachyphylaxis to methacholine in subjects withsymptomatic or asymptomatic airway hyperresponsivenessand in normal controls Respiration 1996 63 304ndash308

10 Strban M Manning PJ Watson RM O9Byrne PM Effect ofmagnitude of airway responsiveness and therapy with inhaledcorticosteroid on histamine tachyphylaxis in asthma Chest1994 105 1434ndash1438

11 Van Den Berg G Meijer RJ Kerstjens HA et al Pc(20)adenosine 59-monophosphate is more closely associated withairway in ammation in asthma than pc(20) methacholineAm J Respir Crit Care Med 2001 163 1546ndash1550

12 van Velzen E van den Bos JW Benckhuijsen JA van Essel Tde Bruijn R Aalbers R Effect of allergen avoidance at highaltitude on direct and indirect bronchial hyperresponsivenessand markers of in ammation in children with allergicasthma Thorax 1996 51 582ndash584

13 Hofstra WB Neijens HJ Duiverman EJ et al Dose-responsesover time to inhaled uticasone propionate treatment

1062 GF JOOS ET AL

of exercise- and methacholine-induced bronchoconstrictionin children with asthma Pediatr Pulmonol 2000 29 415ndash423

14 Taylor DA Jensen MW Kanabar V et al A dose-dependenteffect of the novel inhaled corticosteroidciclesonide on airwayresponsiveness to adenosine-59-monophosphate in asthmaticpatients Am J Respir Crit Care Med 1999 160 237ndash243

15 McFadden ER Jr Gilbert IA Exercise-induced asthmaN Engl J Med 1994 330 1362ndash1367

16 Anderson SD Daviskas E The mechanism of exercise-induced asthma is hellip J Allergy Clin Immunol 2000 106 453ndash459

17 Eggleston PA Kagey-Sobotka A Lichtenstein LM Acomparison of the osmotic activation of basophils andhuman lung mast cells Am Rev Respir Dis 1987 135 1043ndash1048

18 Patel KR Terfenadine in exercise induced asthma Br MedJ (Clin Res Ed ) 1984 288 1496ndash1497

19 Finnerty JP Holgate ST Evidence for the roles of histamineand prostaglandins as mediators in exercise-induced asthmathe inhibitory effect of terfenadine and urbiprofen aloneand in combination Eur Respir J 1990 3 540ndash547

20 Magnussen H Reuss G Jorres R Aurich R The effect ofazelastine on exercise-induced asthma Chest 1988 93 937ndash940

21 O9Sullivan S Roquet A Dahlen B et al Evidence for mastcell activation during exercise-induced bronchoconstrictionEur Respir J 1998 12 345ndash350

22 Nagakura T Obata T Shichijo K et al GCMS analysis ofurinary excretion of 9alpha 11beta-PGF2 in acute andexercise-induced asthma in children Clin Exp Allergy 199828 181ndash186

23 Kikawa Y Miyanomae T Inoue Y Salto M Makai ASudo M Urinary leukotriene E4 after exercise challenge inchildren with asthma J Allergy Clin Immunol 1992 89 1111ndash1119

24 Reiss TF Hill JB Harman E et al Increased urinaryexcretion of LTE4 after exercise and attenuation of exercise-induced bronchospasm by montelukast a cysteinyl leuko-triene receptor antagonist Thorax 1997 52 1030ndash1035

25 Pliss LB Ingenito EP Ingram RHJ Pichurko B Assessmentof bronchoalveolar cell and mediator response to isocapnichyperpnea in asthma Am Rev Respir Dis 1990 142 73ndash78

26 Israel E Dermarkarian R Rosenberg M et al The effects ofa 5-lipoxygenase inhibitor on asthma induced by cold dryair N Engl J Med 1990 323 1740ndash1744

27 Van Schoor J Joos GF Kips JC Drajesk JF Carpentier PJPauwels RA The effect of ABT-761 a novel 5-lipoxygenaseinhibitor on exercise- and adenosine-induced bronchocon-striction in asthmatic subjects Am J Respir Crit Care Med1997 155 875ndash880

28 Lehnigk B Rabe KF Dent G Herst RS Carpentier PJMagnussen H Effects of a 5-lipoxygenase inhibitor ABT-761 on exercise-induced bronchoconstriction and urinaryLTE4 in asthmatic patients Eur Respir J 1998 11 617ndash623

29 Leff JA Busse WW Pearlman D et al Montelukast aleukotriene-receptor antagonist for the treatment of mildasthma and exercise-induced bronchoconstriction N EnglJ Med 1998 339 147ndash152

30 Ishii Y Kitamura S Hyperventilation stimulates the releaseof prostaglandin I2 and E2 from lung in humansProstaglandins 1990 39 685ndash691

31 Hashimoto S Matsumoto K Yasuhiro G Tomoko NIkuko T Horie T Hyperosmolarity-induced IL-8 expressionin human bronchial epithelial cells through p38 mitogen-activated kinase Am J Respir Crit Care Med 2000 159 634ndash640

32 Lee TH Nagakura T Papageorgiou N Cromwell O Iikura YKay AB Mediators in exercise-induced asthma J AllergyClin Immunol 1984 73 634ndash639

33 Banner AS Green J O9Connor M Relation of respiratorywater loss to coughing after exercise N Engl J Med 1984311 883ndash886

34 Ichinose M Miura M Yamauchi H et al A neurokinin1ndashreceptor antagonist improves exercise-induced airwaynarrowing in asthmatic patients Am J Respir Crit CareMed 1996 153 936ndash941

35 Konig P Hordvik NL Kreutz C The preventive effect andduration of action of nedocromil sodium and cromolynsodium on exercise-inducedasthma (EIA) in adults J AllergyClin Immunol 1987 79 64ndash68

36 Bianco S Vaghi A Robuschi M Pasargiklian M Preventionof exercise-induced bronchoconstriction by inhaled fruse-mide Lancet 1988 2 252ndash255

37 Waalkens HJ van Essen-Zandvliet EE Gerritsen JDuiverman EJ Kerrebijn KF Knol K The effect of aninhaled corticosteroid (budesonide) on exercise- inducedasthma in children Dutch CNSLD Study Group Eur RespirJ 1993 6 652ndash656

38 Phillips GD Ng WH Church MK Holgate ST Theresponse of plasma histamine to bronchoprovocation withmethacholine adenosine 59-monophosphate and allergen inatopic nonasthmatic subjects Am Rev Respir Dis 1990 1419ndash13

39 Polosa R Phillips GD Rajakulasingam K Holgate ST Theeffect of inhaled ipratropium bromide alone and incombination with oral terfenadine on bronchoconstrictionprovoked by adenosine 59-monophosphate and histamine inasthma J Allergy Clin Immunol 1991 87 939ndash947

40 Rafferty P Beasley R Holgate ST The contribution ofhistamine to immediate bronchoconstriction provoked byinhaled allergen and adenosine 59 monophosphate in atopicasthma Am Rev Respir Dis 1987 136 369ndash373

41 Crimi N Palermo F Polosa R et al Effect of indomethacinon adenosine-induced bronchoconstriction J Allergy ClinImmunol 1989 83 921ndash925

42 Phillips GD Holgate ST The effect of oral terfenadine aloneand in combination with urbiprofen on the broncho-constrictor response to inhaled adenosine 59-monophosphatein nonatopic asthma Am Rev Respir Dis 1989 139 463ndash469

43 Hughes PJ Holgate ST Church MK Adenosine inhibits andpotentiates IgE-dependent histamine release from humanlung mast cells by an A2-purinoceptor mediated mechanismBiochem Pharmacol 1984 33 3847ndash3852

44 Polosa R Ng WH Crimi N et al Release of mast-cell-derived mediators after endobronchial adenosine challengein asthma Am J Respir Crit Care Med 1995 151 624ndash629

45 Phillips GD Rafferty P Beasley R Holgate ST Effect oforal terfenadine on the bronchoconstrictor response toinhaled histamine and adenosine 59-monophosphate innon-atopic asthma Thorax 1987 42 939ndash945

46 Crimi N Polosa R Magri S et al Inhaled lysineacetylsalicylate (L-ASA) attenuates the bronchoconstrictorresponse to adenosine 59-monophosphate (AMP) in asth-matic subjects Eur Respir J 1995 8 905ndash912

47 Wang M Wisniewski A Pavord I Knox A Tatters eld AComparison of three inhaled non-steroidal anti-in ammatorydrugs on the airway response to sodium metabisulphite andadenosine 59-monophosphate challenge in asthma Thorax1996 51 799ndash804

48 Crimi N Palermo F Oliveri R Polosa R Settinieri IMistretta A Protective effects of inhaled ipratropiumbromide on bronchoconstriction induced by adenosine andmethacholine in asthma Eur Respir J 1992 5 560ndash565

49 Polosa R Santonocito G Magri S et al Neutral endopepti-dase inhibition with inhaled phosphoramidon no effect onbronchial responsiveness to adenosine 59-monophosphate(AMP) in asthma Eur Respir J 1997 10 2460ndash2464

50 Naline E Molimard M Regoli D Emonds-Alt X Bellamy JFAdvenier C Evidence for functional tachykinin NK1receptors on human isolated small bronchi Am J Physiol1996 271 L763ndashL767

51 Ichinose M Nakajima N Takahashi T Yamauchi HInoue H Takishima T Protection against bradykinin-induced


bronchoconstriction in asthmatic patients by neurokininreceptor antagonist Lancet 1992 340 1248ndash1251

52 Joos GF Van Schoor J Kips JC Pauwels RA The effect ofinhaled FK224 a tachykinin NK-1 and NK-2 receptorantagonist on neurokinin A-induced bronchoconstriction inasthmatics Am J Respir Crit Care Med 1996 153 1781ndash1784

53 Heaney LG Cross LJ Stanford CF Ennis M Substance Pinduces histamine release from human pulmonary mast cellsClin Exp Allergy 1995 25 179ndash186

54 Joos G Van De Velde V Schelfhout V Pauwels RThe leukotriene receptor antagonist za rlukast inhibitsneurokinin-A induced bronchoconstriction in patients withasthma Am J Respir Crit Care Med 2001 163 A418

55 Joos G Pauwels R Van Der Straeten M The effect ofoxitropium bromide on neurokinin A-induced broncho-constriction in asthmatic subjects Pulm Pharmacol 1988 141ndash45

56 Crimi N Palermo F Oliveri R et al In uence of anti-histamine (astemizole) and anticholinergic drugs (ipratropiumbromide) on bronchoconstriction induced by substanceP Ann Allergy 1990 65 115ndash120

57 Molimard M Martin CA Naline E Hirsch A Advenier CContractile effects of bradykinin on the isolated human smallbronchus Am J Respir Crit Care Med 1994 149 123ndash127

58 Hulsmann AR Raatgeep HR Saxena PR Kerrebijn KFde Jongste JC Bradykinin-induced contraction of humanperipheral airways mediated by both bradykinin B-2 andthromboxane prostanoid receptors Am J Respir Crit CareMed 1994 150 1012ndash1018

59 Polosa R Phillips GD Lai CK Holgate ST Contribution ofhistamine and prostanoids to bronchoconstriction provokedby inhaled bradykinin in atopic asthma Allergy 1990 45174ndash182

60 Fuller RW Dixon CM Cuss FM Barnes PJ Bradykinin-induced bronchoconstrictionin humans Mode of action AmRev Respir Dis 1987 135 176ndash180

61 Schmidt D Jorres RA Rabe KF Magnussen H Reproduci-bility of airway response to inhaled bradykinin and effect ofthe neurokinin receptor antagonist FK-224 in asthmaticsubjects Eur J Clin Pharmacol 1996 50 269ndash273

62 Polosa R Milazzo VL Magri S et al Activity of inhaledlysine acetylsalicylate (L-ASA) on bradykinin- inducedbronchoconstriction in asthmatics evidence of contributionof prostaglandins Eur Respir J 1997 10 866ndash871

63 del Bufalo C Fasano L Patalano F Gunella G Inhibitionof fog-induced bronchoconstriction by nedocromil sodiumand sodium cromoglycate in intrinsic asthma a double-blind placebo- controlled study Respiration 1989 55 181ndash185

64 Ind PW Barnes PJ Durham SR Kay AB Propranolol-inducedbronchoconstrictionin asthma beta-receptor blockadeand mediator release Am Rev Respir Dis 1984 129 SupplA10

65 Carpentiere G Castello F Marino S Effect of oralterfenadine on the bronchoconstrictor response to inhaledpropranolol and histamine in asthmatics Curr Ther Res1991 49 507ndash513

66 Ind PW Dixon CM Fuller RW Barnes PJ Anticholinergicblockade of beta-blocker-induced bronchoconstriction AmRev Respir Dis 1989 139 1390ndash1394

67 Okayama M Yafuso N Nogami H et al A new methodof inhalation challenge with propranolol comparison withmethacholine-induced bronchoconstriction and role of vagalnerve activity J Allergy Clin Immunol 1987 80 291ndash299

68 Koeter GH Meurs H Jonkman JH et al Protective effectof oral oxyphenonium bromide terbutaline and theophyl-line against the bronchial obstructive effects of inhaledhistamine acetylcholine and propranolol Eur J ClinPharmacol 1984 26 435ndash441

69 Pavord ID Wisniewski A Tatters eld AE Refractoriness toinhaled sodium metabisulphite in subjects with mild asthmaEur Respir J 1994 7 50ndash54

70 Field PI Simmul R Bell SC Allen DH Berend N Evidencefor opioid modulation and generation of prostaglandins insulphur dioxide (SO)2-induced bronchoconstriction Thorax1996 51 159ndash163

71 Lazarus SC Wong HH Watts MJ Boushey HA Lavins BJMinkwitz MC The leukotriene receptor antagonist za rlu-kast inhibits sulfur dioxide- induced bronchoconstriction inpatients with asthma Am J Respir Crit Care Med 1997 1561725ndash1730

72 Nadel JA Salem H Tamplin B Tokiwa Y Mechanism ofbronchoconstriction during inhalation of sulfur dioxideJ Appl Physiol 1965 20 164ndash167

73 Sheppard D Wong WS Uehara CF Nadel JA Boushey HALower threshold and greater bronchomotor responsivenessof asthmatic subjects to sulfur dioxide Am Rev Respir Dis1980 122 873ndash878

74 Bellingan GJ Dixon CM Ind PW Inhibition of inhaledmetabisulphite-induced bronchoconstriction by inhaled fru-semide and ipratropium bromide Br J Clin Pharmacol 199234 71ndash74

75 Bello ore S Caltagirone F Pennisi A Ciancio N Mistretta ADi Maria GU Neutral endopeptidase inhibitor thiorphanincreases airway narrowing to inhaled sodium metabisul tein normal subjects Am J Respir Crit Care Med 1994 150853ndash856

76 Boulet LP Turcotte H Tennina S Comparative ef cacyof salbutamol ipratropium and cromoglycate in theprevention of bronchospasm induced by exercise andhyperosmolar challenges J Allergy Clin Immunol 1989 83882ndash887

77 Anderson SD Holzer K Exercise-induced asthma Is it theright diagnosis in elite athletes J Allergy Clin Immunol 2000106 419ndash428

78 Shimizu T Mochizuki H Shigeta M Morikawa A Effectof inhaled indomethacin on exercise-induced broncho-constriction in children with asthma Am J Respir CritCare Med 1997 155 170ndash173

79 Finnerty JP Wood-Baker R Thomson H Holgate ST Roleof leukotrienes in exercise-induced asthma Inhibitory effectof ICI 204219 a potent leukotriene D4 receptor antagonistAm Rev Respir Dis 1992 145 746ndash749

80 Robuschi M Riva E Fuccella LM et al Prevention ofexercise-induced bronchoconstriction by a new leukotrieneantagonist (SKampF 104353) A double-blind study versusdisodium cromoglycate and placebo Am Rev Respir Dis1992 145 1285ndash1288

81 Dorward AJ Patel KR A comparison of ketotifen withclemastine ipratropium bromide and sodium cromoglycatein exercise-induced asthma Clin Allergy 1982 12 355ndash361

82 Tullett WM Patel KR Berkin KE Kerr JW Effect oflignocaine sodium cromoglycate and ipratropium bromidein exercise-induced asthma Thorax 1982 37 737ndash740

83 Jongejan RC de Jongste JC Raatgeep RC Stijnen TBonta IL Kerrebijn KF Effects of hyperosmolarity onhuman isolated central airways Br J Pharmacol 1991 102931ndash937

84 Gravelyn TR Pan PM Eschenbacher WL Mediator releasein an isolated airway segment in subjects with asthma AmRev Respir Dis 1988 137 641ndash646

85 Finnerty JP Wilmot C Holgate ST Inhibition of hypertonicsaline-induced bronchoconstriction by terfenadine and ur-biprofen Evidence for the predominant role of histamineAm Rev Respir Dis 1989 140 593ndash597

86 Brannan JD Anderson SD Gomes K King GG Chan H-KSeale JP Fexofenadine decreases sensitivity to and mon-telukast improves recovery from inhaled mannitol Am JRespir Crit Care Med 2001 163 1420ndash1425

87 Makker HK Holgate ST The contribution of neurogenicre exes to hypertonic saline-induced bronchoconstriction inasthma J Allergy Clin Immunol 1993 92 82ndash88

88 Altounyan REC Lee TB Rocchiccioli MS Shaw CL Acomparison of the inhibitory effects of nedocromil sodium

1064 GF JOOS ET AL

and sodium cromoglycate on adenosine monophosphate-induced bronchoconstriction in atopic subjects Eur J RespirDis 1986 69 Suppl 277ndash279

89 Badier M Beaumont D Orehek J Attenuation ofhyperventilation-induced bronchospasm by terfenadine anew antihistamine J Allergy Clin Immunol 1988 81 437ndash440

90 Finnerty JP Harvey A Holgate ST The relative contribu-tions of histamine and prostanoids to bronchoconstrictionprovoked by isocapnic hyperventilation in asthma EurRespir J 1992 5 323ndash330

91 Fischer AR McFadden CA Frantz R et al Effect ofchronic 5-lipoxygenase inhibition on airway hyperrespon-siveness in asthmatic subjects Am J Respir Crit Care Med1995 152 1203ndash1207

92 Sheppard D Epstein J Holtzman MJ Nadel JA Boushey HADose-dependent inhibition of cold-air-induced broncho-constriction by atropine J Appl Physiol 1982 53 169ndash174

93 Wilson N Dixon C Silverman M Bronchial responsivenessto hyperventilation in children with asthma inhibition withipratropium bromide Thorax 1984 39 588ndash593

94 Myers JD Bigby BG Calvayrac P Sheppard D Boushey HAInteraction of cromolyn and a muscarinic antagonist ininhibiting bronchial reactivity to sulfur dioxide and toeucapnic hyperpnea alone Am Rev Respir Dis 1986 1331154ndash1158

95 Schellenberg RR Airway responses to platelet-activatingfactor Am Rev Respir Dis 1987 136 S28ndashS32

96 Dahlen B Kumlin M Margolskee DJ Larsson C BlomqvistHWilliams VC et al The leukotriene-receptor antagonistMK-0679 blocks airway obstruction induced by inhaledlysine-aspirin in aspirin-sensitive asthmatics Eur Respir J1993 6 1018ndash1026

97 Wood-Baker R Finnerty JP Holgate ST Plasma andurinary histamine in allergen-induced early and late phaseasthmatic responses Eur Respir J 1993 6 1138ndash1144

98 Twentyman OP Ollier S Holgate ST The effect ofH1ndashreceptor blockade on the development of early- andlate-phase bronchoconstriction and increased bronchialresponsiveness in allergen-induced asthma J Allergy ClinImmunol 1993 91 1169ndash1178

99 Sladek K Sheller JR FitzGerald GA Morrow JDRoberts LJ Formation of PGD2 after allergen inhalationin atopic asthmatics Adv Prostaglandin Thromboxane LeukotRes 1991 21A 433ndash436

100 Macfarlane AJ Dworski R Sheller JR Pavord ID Kay ABBarnes NC Sputum cysteinyl leukotrienes increase 24 hoursafter allergen inhalation in atopic asthmatics Am J RespirCrit Care Med 2000 161 1553ndash1558

101 Friedman BS Bel EH Buntinx A et al Oral leukotrieneinhibitor (MK-886) blocks allergen-induced airwayresponses Am Rev Respir Dis 1993 147 839ndash844

102 Diamant Z van der Veen H Kuijpers EA Bakker PF SterkPJ The effect of inhaled thiorphan on allergen-inducedairway responses in asthmatic subjects Clin Exp Allergy1996 26 525ndash532

103 Roquet A Dahlen B Kumlin M et al Combined antagon-ism of leukotrienes and histamine produces predominantinhibition of allergen-induced early and late phase airwayobstruction in asthmatics Am J Respir Crit Care Med 1997155 1856ndash1863

104 Fredholm BB Arslan G Kull B Kontny E Svenningsson PAdenosine (P1) receptor signalling Drug Dev Res 1996 39262ndash268

105 Feoktistov I Polosa R Holgate ST Biaggioni I AdenosineA2B receptors a novel therapeutic target in asthma TrendsPharmacol Sci 1998 19 148ndash153

106 Joos G Pauwels RA Adenosine receptors involved in thebronchoconstrictor effect of adenosine Drug Dev Res 199639 330ndash332

107 Maggi CA Tachykinin receptors and airway pathophysiol-ogy Eur Respir J 1993 6 735ndash742

108 Naline E Devillier P Drapeau G et al Characterization ofneurokinin effects and receptor selectivity in human isolatedbronchi Am Rev Respir Dis 1989 140 679ndash686

109 Advenier C Naline E Toty L et al Effects on the isolatedhuman bronchus of SR 48968 a potent and selectivenonpeptide antagonist of the neurokinin A (NK2) receptorsAm Rev Respir Dis 1992 146 1177ndash1181

110 Ellis JL Undem BJ Kays JS Ghanekar SV Barthlow HGBuckner CK Pharmacological examination of receptorsmediating contractile responses to tachykinins in airwaysisolated from human guinea pig and hamster J PharmacolExp Ther 1993 267 95ndash101

111 Amadesi S Moreau J Tognetto M et al NK1 receptorstimulation causes contraction and inositol phosphateincrease in medium-size human isolated bronchi Am JRespir Crit Care Med 2001 163 1206ndash1211

112 Joos GF Pauwels RA Van der Straeten ME The effect ofnedocromil sodium on the bronchoconstrictor effect ofneurokinin A in subjects with asthma J Allergy ClinImmunol 1989 83 663ndash668

113 Joos GF Pauwels RA Pro-in ammatory effects of sub-stance P new perspectives for the treatment of airwaydiseases Trends Pharmacol Sci 2000 21 131ndash133

114 Van Schoor J Joos GF Chasson BL Brouard RJPauwels RA The effect of the NK2 tachykinin receptorantagonist SR 48968 (saredutant) on neurokinin A-inducedbronchoconstrictionin asthmatics Eur Respir J 1998 12 17ndash23

115 Di Maria GU Bello ore S Geppetti P Regulation of airwayneurogenic in ammation by neutral endopeptidase EurRespir J 1998 12 1454ndash1462

116 Cheung D Timmers MC Zwinderman AH den Hartigh JDijkman JH Sterk PJ Neutral endopeptidase activity andairway hyperresponsiveness to neurokinin A in asthmaticsubjects in vivo Am Rev Respir Dis 1993 148 1467ndash1473

117 Nadel JA Peptidase modulation of neurogenic in amma-tion In Geppetti P Holzer P ed Neurogenic in ammationBoca Raton CRC Press 1996 115ndash127

118 Polosa R Holgate ST Comparative airway response toinhaled bradykinin kallidin and [des- Arg9] bradykinin innormal and asthmatic subjects Am Rev Respir Dis 1990 1421367ndash1371

119 Proud D Kinins as mediators of lung disease InCrystal RG West JB Barnes PJ Weibel ER eds TheLung Philadelphia Lippincott Raven 1997 89ndash101

120 Ricciardolo FL Geppetti P Mistretta A et al Randomiseddouble-blind placebo-controlled study of the effect ofinhibition of nitric oxide synthesis in bradykinin-inducedasthma Lancet 1996 348 374ndash377

121 Geppetti P Sensory neuropeptide release by bradykininmechanisms and pathophysiological implications Regul Pept1993 47 1ndash23

122 Ichinose M Belvisi MG Barnes PJ Bradykinin-inducedbronchoconstriction in guinea pig in vivo role of neuralmechanisms J Pharmacol Exp Ther 1990 253 594ndash599

123 Saria A Martling CR Yan Z Theodorsson-Norheim EGamse R Lundberg JM Release of multiple tachykininsfrom capsaicin-sensitive sensory nerves in the lung bybradykinin histamine dimethylphenyl piperazinium andvagal nerve stimulation Am Rev Respir Dis 1988 137 1330ndash1335

124 Adelroth E Hargreave FE Ramsdale EH Do physiciansneed objective measurements to diagnose asthma Am RevRespir Dis 1986 134 704ndash707

125 Woolcock AJ Anderson SD Peat JK et al Characteristicsof bronchial hyperresponsiveness in chronic obstructivepulmonary disease and in asthma Am Rev Respir Dis 1991143 1438ndash1443

126 Cockcroft DW Killian DN Mellon JJ Hargreave FEBronchial reactivity to inhaled histamine a method andclinical survey Clin Allergy 1977 7 235ndash243

127 Juniper EF Cockcroft DW Hargreave FE Histamine and


methacholine inhalatation tests tidal breathing method -laboratory procedure and standardisation Lund SwedenAB Draco 1994

128 Chai H Farr RS Froehlich LA et al Standardization ofbronchial inhalation challenge procedures J Allergy ClinImmunol 1975 56 323ndash327

129 Ryan G Dolovich MB Obminski G et al Standardizationof inhalation provocation tests in uence of nebuliser outputparticle size and method of inhalation J Allergy ClinImmunol 1981 67 156ndash161

130 Yan K Salome C Woolcock AJ Rapid method formeasurement of bronchial responsiveness Thorax 1983 38760ndash765

131 Juniper EF Frith PA Dunnett C Cockcroft DWHargreave FE Reproducibility and comparison of responsesto inhaled histamine and methacholine Thorax 1978 33705ndash710

132 Woolcock AJ Expression of results of airway hyperrespon-siveness In Hargreave FE Woolcock AJ eds AirwayHyperresponsiveness Measurement and InterpretationCanada Astra Pharmaceuticals Canada Ltd 1985 pp 80ndash90

133 Dehaut P Rachiele A Martin RR Malo JL Histaminedose-response curves in asthma reproducibility andsensitivity of different indices to assess response Thorax1983 38 516ndash522

134 Townley RG Bewtra AK Nair NM Brodkey FDWatt GD Burke KM Methacholine inhalation challengestudies J Allergy Clin Immunol 1979 64 569ndash574

135 Hopp RJ Bewtra AK Nair NM Townley RG Speci cityand sensitivity of methacholine inhalation challenge innormal and asthmatic children J Allergy Clin Immunol1984 74 154ndash158

136 Backer V Groth S Dirksen A et al Sensitivity andspeci city of the histamine challenge test for the diagnosisof asthma in an unselected sample of children andadolescents Eur Respir J 1991 4 1093ndash1100

137 Cockcroft DW Murdock KY Berscheid BA Gore BPSensitivity and speci city of histamine PC20 determination ina random selection of young college students J Allergy ClinImmunol 1992 89 23ndash30

138 Ramsdell JW Nachtwey FJ Moser KM Bronchial hyper-reactivity in chronic obstructive bronchitis Am Rev RespirDis 1982 126 829ndash832

139 Ramsdale EH Morris MM Roberts RS Hargreave FEBronchial responsiveness to methacholine in chronic bron-chitis relationship to air ow obstruction and cold airresponsiveness Thorax 1984 39 912ndash918

140 Ramsdale EH Roberts RS Morris MM Hargreave FEDifferences in responsiveness to hyperventilation andmethacholine in asthma and chronic bronchitis Thorax1985 40 422ndash426

141 Verma VK Cockcroft DW Dosman JA Airway respon-siveness to inhaled histamine in chronic obstructive airwaysdisease Chronic bronchitis vs emphysema Chest 1988 94457ndash461

142 Anderton RC Cuff MT Frith PA Cockcroft DW Morse JLJones NL et al Bronchial responsiveness to inhaledhistamine and exercise J Allergy Clin Immunol 1979 63315ndash320

143 Eggleston PA A comparison of the asthmatic response tomethacholine and exercise J Allergy Clin Immunol 1979 63104ndash110

144 Shapiro GG Methacholine challenge ndash relevance for theallergic athlete J Allergy Clin Immunol 1984 73 670ndash675

145 Fourie PR Joubert JR Determination of airway hyper-reactivity in asthmatic children a comparison amongexercise nebulized water and histamine challenge PediatrPulmonol 1988 4 2ndash7

146 Lin CC Wu JL Huang WC Lin CY A bronchial responsecomparison of exercise and methacholine in asthmaticsubjects J Asthma 1991 28 31ndash40

147 Eliasson AH Phillips YY Rajagopal KR Howard RSSensitivity and speci city of bronchial provocation testingAn evaluation of four techniques in exercise-inducedbronchospasm Chest 1992 102 347ndash355

148 Vasar M Braback L Julge K Knutsson A Riikjarv MABjorksten B Prevalence of bronchial hyperreactivity asdetermined by several methods among Estonian school-children Pediatr Allergy Immunol 1996 7 141ndash146

149 Godfrey S Springer C Bar-Yishay E Avital A Cut-offpoints de ning normal and asthmatic bronchial reactivity toexercise and inhalation challenges in children and youngadults Eur Respir J 1999 14 659ndash668

150 Holzer K Anderson SD Douglass J Exercise in elitesummer athletes Challenges for diagnosis J Allergy ClinImmunol 2002 110 374ndash380

151 Godfrey S Springer C Noviski N Maayan C Avital AExercise but not methacholine differentiates asthma fromchronic lung disease in children Thorax 1991 46 488ndash492

152 Avital A Springer C Bar-Yishay E Godfrey S Adenosinemethacholine and exercise challenges in children withasthma or paediatric chronic obstructive pulmonary diseaseThorax 1995 50 511ndash516

153 Benckhuijsen J van den Bos JW van Velzen E de Bruijn RAalbers R Differences in the effect of allergen avoidance onbronchial hyperresponsivenessas measured by methacholineadenosine 59-monophosphate and exercise in asthmaticchildren Pediatr Pulmonol 1996 22 147ndash153

154 Yoshikawa T Shoji S Fujii T et al Severity of exercise-induced bronchoconstrictionis related to airway eosinophilicin ammation in patients with asthma Eur Respir J 1998 12879ndash884

155 Guidelines for Methacholine and Exercise ChallengeTesting-1999 Am J Respir Crit Care Med 2000 161 309ndash329

156 Anderson SD Argyros GJ Magnussen H Holzer KProvocation by eucapnic voluntary hyperpnoea to identifyexercise induced bronchoconstriction Br J Sports Med 200135 344ndash347

157 Steinbrugger B Eber E Modl M Weinhandl E Zach MSA comparison of a single-step cold-dry air challenge and aroutine histamine provocation for the assessment of bron-chial responsiveness in children and adolescents Chest 1995108 741ndash745

158 Nielsen KG Bisgaard H Lung Function Response to ColdAir Challenge in Asthmatic and Healthy Children of 2ndash5Years of Age Am J Respir Crit Care Med 2000 161 1805ndash1809

159 Sont JK Booms P Bel EH Vandenbroucke JP Sterk PJThe determinants of airway hyperresponsiveness to hyper-tonic saline in atopic asthma in vivo Relationship with sub-populations of peripheral blood leucocytes Clin Exp Allergy1993 23 678ndash688

160 Anderson SD Gibson P The use of aerosols of hypertonicsaline and distilled water (fog) for the patient with asthmaIn Barnes PJ Grunstein MM Leff A Woolcock AJ edAsthma New York Raven Press 1997 pp 1135ndash1150

161 Anderson SD Brannan J Spring J et al A new method forbronchial-provocation testing in asthmatic subjects using adry powder of mannitol Am J Respir Crit Care Med 1997156 758ndash765

162 Brannan JD Koskela H Anderson SD Chew N Respon-siveness to mannitol in asthmatic subjects with exercise- andhyperventilation-induced asthma Am J Respir Crit CareMed 1998 158 1120ndash1126

163 Riedler J Reade T Dalton M Holst D Robertson CHypertonic saline challenge in an epidemiologic survey ofasthma in children Am J Respir Crit Care Med 1994 1501632ndash1639

164 Rabone SJ Phoon WO Anderson SD et al Hypertonicsaline challenge in an adult epidemiological survey OccupMed (Lond) 1996 46 177ndash185

165 Smith CM Anderson SD Inhalational challenge using

1066 GF JOOS ET AL

hypertonic saline in asthmatic subjects a comparison withresponses to hyperpnoea methacholine and water EurRespir J 1990 3 144ndash151

166 Belcher NG Lee TH Rees PJ Airway responses tohypertonic saline exercise and histamine challenges inbronchial asthma Eur Respir J 1989 2 44ndash48

167 Anderson SD Brannan J Trevillion L Young I Lungfunction and bronchial provocation tests for intending diverswith a history of asthma SPUMS Journal 1995 25 233ndash248

168 Chua HL Collis GG Le Souef PN Bronchial response tonebulized antibiotics in children with cystic brosis EurRespir J 1990 3 1114ndash1116

169 Daviskas E Anderson SD Gonda I et al Inhalation ofhypertonic saline aerosol enhances mucociliary clearance inasthmatic and healthy subjects Eur Respir J 1996 9 725ndash732

170 Daviskas E Anderson SD Eberl S Chan HK Bautovich GInhalation of dry powder mannitol improves clearance ofmucus in patients with bronchiectasis Am J Respir Crit CareMed 1999 159 1843ndash1848

171 Robinson M Daviskas E Eberl S et al The effect of inhaledmannitol on bronchial mucus clearance in cystic brosispatients a pilot study Eur Respir J 1999 14 678ndash685

172 Eng PA Morton J Douglass JA Riedler J Wilson JRobertson CF Short-term ef cacy of ultrasonicallynebulized hypertonic saline in cystic brosis PediatrPulmonol 1996 21 77ndash83

173 Rodwell LT Anderson SD Airway responsiveness tohyperosmolar saline challenge in cystic brosis a pilotstudy Pediatr Pulmonol 1996 21 282ndash289

174 Eschenbacher WL Boushey HA Sheppard D Alterationin osmolarity of inhaled aerosols cause bronchoconstrictionand cough but absence of a permeant anion causes coughalone Am Rev Respir Dis 1984 129 211ndash215

175 Sheppard D Rizk NW Boushey HA Bethel RAMechanism of cough and bronchoconstriction induced bydistilled water aerosol Am Rev Respir Dis 1983 127 691ndash694

176 Pin I Gibson PG Kolendowicz R et al Use of inducedsputum cell counts to investigate airway in ammation inasthma Thorax 1992 47 25ndash29

177 Fahy JV Wong H Liu J Boushey HA Comparison ofsamples collected by sputum induction and bronchoscopyfrom asthmatic and healthy subjects Am J Respir Crit CareMed 1995 152 53ndash58

178 Gibson PG Saltos N Borgas T Airway mast cells andeosinophils correlate with clinical severity and airway hyper-responsiveness in corticosteroid-treated asthma J AllergyClin Immunol 2000 105 752ndash759

179 In9t Veen JC Smits HH Hiemstra PS Zwinderman AESterk PJ Bel EH Lung function and sputum characteristicsof patients with severe asthma during an induced exacerba-tion by double-blind steroid withdrawal Am J Respir CritCare Med 1999 160 93ndash99

180 Iredale MJ Wanklyn SA Phillips IP Krausz T Ind PWNon-invasive assessment of bronchial in ammation inasthma no correlation between eosinophilia of inducedsputum and bronchial responsiveness to inhaled hypertonicsaline Clin Exp Allergy 1994 24 940ndash945

181 Allegra L Bianco S Non-speci c broncho-reactivityobtained with an ultrasonic aerosol of distilled water EurJ Respir Dis 1980 61 Suppl 106 41ndash49

182 Schoeffel RE Anderson SD Altounyan RE Bronchialhyperreactivity in response to inhalation of ultrasonicallynebulised solutions of distilled water and saline Br Med J1981 283 1285ndash1287

183 Sheppard D Rizk NW Boushey HA Bethel RA Mechanismof cough and bronchoconstriction induced by distilled wateraerosol Am Rev Respir Dis 1983 127 691ndash694

184 Eschenbacher WL Boushey HA Sheppard D Alteration inosmolarity of inhaled aerosols cause bronchoconstriction

and cough but absence of a permeant anion causes coughalone Am Rev Respir Dis 1984 129 211ndash215

185 Anderson SD Bronchial challenge by ultrasonically neb-ulized aerosols Clin Rev Allergy 1985 3 427ndash439

186 Anderson SD Non-isotonic aerosol challenge in theevaluation of bronchial hyperresponsiveness Allergy Proc1991 12 143ndash149

187 Foresi A Mattoli S Corbo GM Polidori G Ciappi GComparison of bronchial responses to ultrasonically neb-ulized distilled water exercise and methacholine in asthmaChest 1986 90 822ndash826

188 Mattoli S Foresi A Corbo GM et al Refractory period toultrasonic mist of distilled water relationship to methacho-line responsiveness atopic status and clinical characteristicsAnn Allergy 1987 58 134ndash140

189 Groot C Lammers JW Festen J van Herwaarden CRefractoriness for ultrasonically nebulized distilled waterand histamine after histamine challenge J Appl Physiol 199170 1011ndash1015

190 Kivity S Shalit Y Greif J Topilsky M Comparison betweenrefractoriness after distilled water-induced asthma andexercise-induced asthma Ann Allergy 1989 62 180ndash183

191 Cushley MJ Tatters eld AE Holgate ST Inhaled adenosineand guanosine on airway resistance in normal and asthmaticsubjects Br J Clin Pharmacol 1983 15 161ndash165

192 Polosa R Pagano C Prosperini G et al Histamine releaseupon adenosine 59-monophosphate (AMP) nasal provoca-tion in allergic subjects Thorax 1999 54 230ndash233

193 Oosterhoff Y de Jong JW Jansen MA Koeter GH Postma DSAirway responsiveness to adenosine 59-monophosphate inchronic obstructive pulmonary disease is determined bysmoking Am Rev Respir Dis 1993 147 553ndash558

194 Chetta A Foresi A Garavaldi G Corbo GM Cuomo AOlivieri D Evaluation of bronchial responsiveness bypharmacological challenges in asthma Inhaled propranololin comparison with histamine and methacholine Respiration1988 54 Suppl 1 84ndash89

195 Woolcock AJ Cheung W Salome C Relationship betweenbronchial responsiveness to propranolol and histamineAm J Respir Crit Care Med 1986 133 A177

196 Nowak D Jorres R Berger J Claussen M Magnussen HAirway responsivenessto sulfur dioxide in an adult populationsample Am J Respir Crit Care Med 1997 156 1151ndash1156

197 Szczeklik A Stevenson DD Aspirin-induced asthmaadvances in pathogenesis and management J Allergy ClinImmunol 1999 104 5ndash13

198 Bianco S Robuschi M Petrini G Aspirin-induced tolerancein aspirin-asthma detected by a new challenge test IRCSJ Med Sci 1997 5 129

199 Phillips GD Foord R Holgate ST Inhaled lysine-aspirin asa bronchoprovocation procedure in aspirin-sensitive asthmaits repeatability absence of a late-phase reaction and therole of histamine J Allergy Clin Immunol 1989 84 232ndash241

200 Dahlen B Zetterstrom O Comparison of bronchial and peroral provocation with aspirin in aspirin-sensitive asthmaticsEur Respir J 1990 3 527ndash534

201 Melillo G Padovano A Cocco G Masi C Dosimeterinhalation test with lysine acetylsalicylate for the detection ofaspirin-induced asthma Ann Allergy 1993 71 61ndash65

202 Pleskow WW Stevenson DD Mathison DA Simon RASchatz M Zeiger RS Aspirin desensitization in aspirin-sensitive asthmatic patients clinical manifestations andcharacterization of the refractory period J Allergy ClinImmunol 1982 69 11ndash19

203 Nizankowska E Szczeklik A Glucocorticosteroidsattenuateaspirin-precipitated adverse reactions in aspirin-intolerantpatients with asthma Ann Allergy 1989 63 159ndash162

204 Szczeklik A Dworski R Mastalerz L et al Salmeterolprevents aspirin-induced attacks of asthma and interfereswith eicosanoid metabolism Am J Respir Crit Care Med1998 158 1168ndash1172

205 Gibson PG Monitoring the patient with asthma an


evidence-based approach J Allergy Clin Immunol 2000 10617ndash26

206 Juniper EF Kline PA Vanzieleghem MA Ramsdale EHO9Byrne PM Hargreave FE Effect of long-term treatmentwith an inhaled corticosteroid (budesonide) on airwayhyperresponsiveness and clinical asthma in nonsteroid-dependent asthmatics Am Rev Respir Dis 1990 142 832ndash836

207 Kerrebijn KF van Essen-Zandvliet EE Neijens HJ Effect oflong-term treatment with inhaled corticosteroids and beta-agonists on the bronchial responsiveness in children withasthma J Allergy Clin Immunol 1987 79 653ndash659

208 Sont JK Willems LN Bel EH van Krieken JHVandenbroucke JP Sterk PJ Clinical control and histo-pathologic outcome of asthma when using airway hyper-responsiveness as an additional guide to long-termtreatment The AMPUL Study Group Am J Respir CritCare Med 1999 159 1043ndash1051

209 Polosa R Ciamarra I Mangano G et al Bronchialhyperresponsiveness and airway in ammation markers innonasthmatics with allergic rhinitis Eur Respir J 2000 1530ndash35

210 O9Connor BJ Ridge SM Barnes PJ Fuller RW Greatereffect of inhaled budesonide on adenosine 59-monophosphate-induced than on sodium-metabisul te-induced bronchocon-striction in asthma Am Rev Respir Dis 1992 146 560ndash564

211 Doull l Sandall D Smith S Schreiber J Freezer NJHolgate ST Differential inhibitory effect of regular inhaledcorticosteroid on airway responsiveness to adenosine 59monophosphate methacholine and bradykinin in sympto-matic children with recurrent wheeze Pediatr Pulmonol 199723 404ndash411

212 O9Connor BJ Fuller RW Barnes PJ Nonbronchodilatoreffects of inhaled beta 2 agonists Greater protection againstadenosine monophosphate- than methacholine-inducedbronchoconstriction in asthma Am J Respir Crit Care Med1994 150 381ndash387

213 O9Connor BJ Aikman SL Barnes PJ Tolerance to thenonbronchodilator effects of inhaled beta 2-agonists inasthma N Engl J Med 1992 327 1204ndash1208

214 Leuppi JD Salome CM Jenkins CR et al Predictivemarkers of asthma exacerbation during stepwise dosereduction of inhaled corticosteroids Am J Respir Crit CareMed 2001 163 406ndash412

215 Rodwell LT Anderson SD Seale JP Inhaled steroidsmodify bronchial responses to hyperosmolar saline EurRespir J 1992 5 953ndash962

216 Brannan JD Anderson SD Freed R Leuppi JD Koskela HChan HK Nedocromil sodium inhibits responsiveness toinhaled mannitol in asthmatic subjects Am J Respir CritCare Med 2000 161 2096ndash2099

217 Anderson SD Du Toit JI Rodwell LT Jenkins CR Acuteeffect of sodium cromoglycate on airway narrowing inducedby 45 percent saline aerosol Outcome before and duringtreatment with aerosol corticosteroids in patients withasthma Chest 1994 105 673ndash680

218 Brannan J Koskela H Anderson SD Chan HK Kim HBudesonide reduces sensitivity and reactivity to inhaledmannitol in asthmatic subjects Respirology 2002 7 37ndash44

219 de Vries K Booij-Noord H Van der Lende RVan Lookeren Campagne JG Orie NGM Reactivity ofthe bronchial tree to different stimuli Les Bronches 1968 18439ndash542

220 Jayaram L Parameswaran K Sears MR Hargreave FEInduced sputum cell counts their usefulness in clinicalpractice Eur Respir J 2000 16 150ndash158

221 Pedersen S Hansen OR Budesonide treatment of moderate

and severe asthma in children a dose-response studyJ Allergy Clin Immunol 1995 95 29ndash33

222 Wiebicke W Jorres R Magnussen H Comparison of theeffects of inhaled corticosteroids on the airway response tohistamine methacholine hyperventilation and sulfur diox-ide in subjects with asthma J Allergy Clin Immunol 1990 86915ndash923

223 Vathenen AS Knox AJ Wisniewski A Tatters eld AEEffect of inhaled budesonide on bronchial reactivity tohistamine exercise and eucapnic dry air hyperventilation inpatients with asthma Thorax 1991 46 811ndash816

224 Fuller RW Choudry NB Eriksson G Action of budesonideon asthmatic bronchial hyperresponsiveness Effects ondirectly and indirectly acting bronchoconstrictors Chest1991 100 670ndash674

225 Groot CAR Lammers JWJ Molema J Festen Jvan Herwaarden CLA Effect of inhaled beclomethasoneand nedocromil sodium on bronchial hyperresponsiveness tohistamine and distilled water Eur Respir J 1992 5 1075ndash1082

226 Bootsma GP Dekhuijzen PN Festen J Mulder PGvan Herwaarden CL Comparison of uticasone propionateand beclomethasone dipropionate on direct and indirectmeasurements of bronchial hyperresponsiveness in patientswith stable asthma Thorax 1995 50 1044ndash1050

227 Weersink EJ Douma RR Postma DS Koeter GHFluticasone propionate salmeterol xinafoate and theircombination in the treatment of nocturnal asthma AmJ Respir Crit Care Med 1997 155 1241ndash1246

228 van den Berge M Kerstjens HA Meijer RJ et alCorticosteroid-induced improvement in the PC(20) ofadenosine monophosphate is more closely associated withreduction in airway in ammation than improvement in thePC(20) of methacholine Am J Respir Crit Care Med 2001164 1127ndash1132

229 Haby MM Peat JK Mellis CM Anderson SD Woolcock AJAn exercise challenge for epidemiological studies of child-hood asthma validity and repeatability Eur Respir J 1995 8729ndash736

230 Salome CM Peat JK Britton WJ Woolcock AJ Bronchialhyperresponsiveness in two populations of Australianschoolchildren I Relation to respiratory symptoms anddiagnosed asthma Clin Allergy 1987 17 271ndash281

231 Saetta M Di Stefano A Turato G et al Fatal asthma attackduring an inhalation challenge with ultrasonically nebulizeddistilled water J Allergy Clin Immunol 1995 95 1285ndash1287

232 Wong HG Fahy JV Safety of one method of sputuminduction in asthmatic subjects Am J Respir Crit Care Med1997 156 299ndash303

233 Jones PD Hankin R Simpson J Gibson PG Henry RLThe tolerability safety and success of sputum induction andcombined hypertonic saline challenge in children Am JRespir Crit Care Med 2001 164 1146ndash1149

234 Mann JS Holgate ST Speci c antagonism of adenosine-induced bronchoconstriction in asthma by oral theophyllineBr J Clin Pharmacol 1985 19 685ndash692

235 Crimi N Palermo F Oliveri R et al Comparative study ofthe effects of nedocromil sodium (4 mg) and sodium cromo-glycate (10 mg) on adenosine-induced bronchoconstrictionin asthmatic subjects Clin Allergy 1988 18 367ndash374

236 Meltzer SS Hasday JD Cohn J Bleecker ER Inhibition ofexercise-inducedbronchospasm by zileuton a 5-lipoxygenaseinhibitor Am J Respir Crit Care Med 1996 153 931ndash935

237 Dahlen B Leukotrienes as mediators of asthma induced byaspirin and allergen Karolinska Instituet StockholmSweden (PhD) 1993

1068 GF JOOS ET AL

1Bronchial hyperresponsiveness is an abnormal increase in

air ow limitation following exposure to a nonallergic stimulus[1 2] Bronchial hyperresponsiveness is a characteristic featureof both asthma and chronic obstructive pulmonary disease(COPD) Thus bronchial hyperresponsiveness is frequentlyused to aid in diagnosis and characterisation of individuals withairway disease Although bronchial hyperresponsiveness isnot speci c for asthma nearly all patients with asthma exhibitincreased responsiveness which is more marked during sympto-matic episodes Bronchial hyperresponsiveness to methacholineis also present in a majority of patients with mild to moderateCOPD [3] Moreover the severity of bronchial hyperrespon-siveness predicts the response to inhaled corticosteroids inpatients with asthma [4] and the progression of air ow limita-tion in patients with COPD [5]

Most investigators assess bronchial responsiveness usingmethacholine or histamine as a provocative stimulus Metha-choline and histamine cause air ow limitation predominantlyvia a direct effect on airway smooth muscle By contrastindirect challenges induce air ow limitation by acting on cellsother than smooth muscle cells eg in ammatory cells epithelialcells and nerves which upon stimulation release mediators orneurotransmitters that provoke smooth muscle contractionNearly all the published studies on asthma and COPD haveutilised histamine and methacholine provocation tests forclinical characterisation of patients Furthermore hyper-responsiveness testing is widely used in clinical researchsettings to evaluate potential new therapies For exampledirect challenges with histamine or methacholine are usedto establish a dose response and time course of the acutebronchoprotective effects of b-agonists These challenges havealso been used to assess the potential anti-in ammatoryeffects of prolonged treatment with new agents There arelimitations to this model Inhaled corticosteroids the currentgold standard anti-in ammatory treatment for asthma reducesbronchial responsiveness to histamine or methacholine onlyto a small degree an effect that is both dose and timedependent In recent years an increasing number of studieshave investigated the relative usefulness of indirect airwaychallenges in monitoring anti-in ammatory treatment inasthma but almost none in COPD

In 1998 the European Respiratory Society (ERS) approveda Task Force on Indirect Airway Challenges The objectivesof this Task Force were to develop recommendations concern-ing the role of indirect airway challenges in the assessmentand monitoring of airway diseases The recommendations inthis report are based on a review of the published literatureand were developed during workshops held at the AmericanThoracic Society in San Diego (April 1999) ERS Congress inMadrid (October 1999) ERS Meeting in Ghent (June 2000)and the ERS Congress in Florence (August 2000) Thefollowing topics were included 1) Mechanisms and receptorsinvolved in the airway narrowing caused by indirect airwaychallenges 2) Diagnostic value of indirect challenges 3) Valueof indirect challenges in the monitoring of asthma includingthe use of these challenges as an outcome measure in clinicaltrials 4) Value of indirect challenges in epidemiological studies5) The importance of standardisation of challenge methods6) Areas for further research

De nition and main properties of an indirect challenge

The concept of indirect challenges was developed at the endof the eighties [6] Several publications had con rmed thatmany different nonspeci c stimuli induced airway narrowingin patients with asthma Thus a distinction had to be madebetween direct and indirect stimuli Methacholine and histamine

are direct stimuli because they cause air ow limitation byacting on effector cells predominantly on airway smoothmuscle but also on mucus glands and on airway micro-vasculature without involving intermediate pathways Bycontrast indirect stimuli ie physical stimuli such as exerciseosmotic challenge or pharmacological stimuli such as adeno-sine cause air ow limitation by acting on cells most notablyin ammatory cells and neuronal cells which release mediatorsor cytokines to cause secondary bronchoconstriction

The fact that the pattern of airway narrowing induced byindirect stimuli differs from that provoked by direct stimuli isshown by the following clear evidence 1) Bronchial respon-siveness to direct and indirect challenges are rather poorlycorrelated with each other [6] 2) A wide array of mediatorsincluding histamine leukotrienes prostaglandins acetyl-choline neuropeptides are involved in the airway narrowinginduced by the indirect stimuli [7] 3) The airway narrowingcaused by an indirect but not a direct challenge can beprevented by acute pretreatment with a cromone (cromogly-cate nedocromil) inhaled frusemide andor heparin [7] 4)After the administration of an indirect challenge tachy-phylaxis to a second stimulus with the same or another indirectacting agent (cross refractoriness) is frequently observed [7]The tachyphylaxis observed with the indirect challenges is farmore pronounced than the small changes seen when histamineor methacholine is repeatedly inhaled [8ndash10] 4) In patientswith asthma bronchial responsiveness to an indirect airwaychallenge is more closely associated with airway in ammationthan bronchial responsiveness to a direct stimulus [11] Bron-chial responsiveness to an indirect stimulus may also betterre ect acute changes in airway in ammation induced by allergenavoidance [12] or by treatment with inhaled steroids [13 14]

The authors propose the following practical workingde nition of an indirect challenge Indirect challenges actby causing the release of endogenous mediators that cause theairway smooth muscle to contract with or without effect ininducing microvascular leakage Because the responses tothese challenges are modi ed or even completely inhibited byinhaled steroids the airway response to these challenges maybe a closer re ection of active airway in ammation

Table 1 ndash Overview of direct and indirect stimuli

Indirect stimuli Direct stimuli

Physical stimuli Cholinergic agonistsExercise (acetylcholineNonisotonic aerosols methacholine(hyper- hypotonic carbachol)distilled water Histamineaerosols mannitol) Prostaglandin D2

Eucapnic voluntaryhyperpnoea of dryair

Leukotriene C4D4 E4

Pharmacological stimuliAdenosineTachykininsBradykininMetabisulphiteSO2Propranolol

Endotoxin (LPS)Platelet activating factorOzoneSelective agents

Aspirin and NSAIDAllergen

LPS lipopolysacharides NSAID nonsteroidal anti-in ammatorydrugs SO2 sulphur dioxide










s

s

s

s

sss

s

s

s

s

s

ssss

s

s


Water loss


Allergen

Adenosine

AMP

Substance PNKA

Bradykinin

Aspirin



leakage

Airwaysmooth muscle

CO LO

AA

P

Leukotriene

NK1NK2

Leukotriene

Histamine

Histamine

Leukotriene

NK1NK2

Histamine

Leukotriene

Sensorynerve

Eosinophil

Activated mast cell


1052 GF JOOS ET AL
































1054 GF JOOS ET AL


























1056 GF JOOS ET AL





















weeks

Dose Challenge

Direct Indirect






50z250 mg bid



1058 GF JOOS ET AL


Conclusions







Diagnosis


Monitoring


Epidemiology


Safety performance











eld studies






adenosine



Patient preparation




Dosing protocols



1060 GF JOOS ET AL







Data presentation




lysine-aspirin



Nebuliser settings







Dosemmol

Cumulateddose mmol


01 1 1 1 0 001 2 2 3 05 0501 7 7 10 10 0510 2 20 30 15 0510 7 70 100 20 0510 8 80 180 226 02610 12 120 300 248 02310 30 300 600 277 029


Procedure Parameter








Challenge protocol









References














1062 GF JOOS ET AL















































































1064 GF JOOS ET AL

















































































1066 GF JOOS ET AL














































































1068 GF JOOS ET AL









s

s

s

s

sss

s

s

s

s

s

ssss

s

s


Water loss


Allergen

Adenosine

AMP

Substance PNKA

Bradykinin

Aspirin



leakage

Airwaysmooth muscle

CO LO

AA

P

Leukotriene

NK1NK2

Leukotriene

Histamine

Histamine

Leukotriene

NK1NK2

Histamine

Leukotriene

Sensorynerve

Eosinophil

Activated mast cell


1052 GF JOOS ET AL
































1054 GF JOOS ET AL


























1056 GF JOOS ET AL





















weeks

Dose Challenge

Direct Indirect






50z250 mg bid



1058 GF JOOS ET AL


Conclusions







Diagnosis


Monitoring


Epidemiology


Safety performance











eld studies






adenosine



Patient preparation




Dosing protocols



1060 GF JOOS ET AL







Data presentation




lysine-aspirin



Nebuliser settings







Dosemmol

Cumulateddose mmol


01 1 1 1 0 001 2 2 3 05 0501 7 7 10 10 0510 2 20 30 15 0510 7 70 100 20 0510 8 80 180 226 02610 12 120 300 248 02310 30 300 600 277 029


Procedure Parameter








Challenge protocol









References














1062 GF JOOS ET AL















































































1064 GF JOOS ET AL

















































































1066 GF JOOS ET AL














































































1068 GF JOOS ET AL
































1054 GF JOOS ET AL


























1056 GF JOOS ET AL





















weeks

Dose Challenge

Direct Indirect






50z250 mg bid



1058 GF JOOS ET AL


Conclusions







Diagnosis


Monitoring


Epidemiology


Safety performance











eld studies






adenosine



Patient preparation




Dosing protocols



1060 GF JOOS ET AL







Data presentation




lysine-aspirin



Nebuliser settings







Dosemmol

Cumulateddose mmol


01 1 1 1 0 001 2 2 3 05 0501 7 7 10 10 0510 2 20 30 15 0510 7 70 100 20 0510 8 80 180 226 02610 12 120 300 248 02310 30 300 600 277 029


Procedure Parameter








Challenge protocol









References














1062 GF JOOS ET AL















































































1064 GF JOOS ET AL

















































































1066 GF JOOS ET AL














































































1068 GF JOOS ET AL













1054 GF JOOS ET AL


























1056 GF JOOS ET AL





















weeks

Dose Challenge

Direct Indirect






50z250 mg bid



1058 GF JOOS ET AL


Conclusions







Diagnosis


Monitoring


Epidemiology


Safety performance











eld studies






adenosine



Patient preparation




Dosing protocols



1060 GF JOOS ET AL







Data presentation




lysine-aspirin



Nebuliser settings







Dosemmol

Cumulateddose mmol


01 1 1 1 0 001 2 2 3 05 0501 7 7 10 10 0510 2 20 30 15 0510 7 70 100 20 0510 8 80 180 226 02610 12 120 300 248 02310 30 300 600 277 029


Procedure Parameter








Challenge protocol









References














1062 GF JOOS ET AL















































































1064 GF JOOS ET AL

















































































1066 GF JOOS ET AL














































































1068 GF JOOS ET AL


























1056 GF JOOS ET AL





















weeks

Dose Challenge

Direct Indirect






50z250 mg bid



1058 GF JOOS ET AL


Conclusions







Diagnosis


Monitoring


Epidemiology


Safety performance











eld studies






adenosine



Patient preparation




Dosing protocols



1060 GF JOOS ET AL







Data presentation




lysine-aspirin



Nebuliser settings







Dosemmol

Cumulateddose mmol


01 1 1 1 0 001 2 2 3 05 0501 7 7 10 10 0510 2 20 30 15 0510 7 70 100 20 0510 8 80 180 226 02610 12 120 300 248 02310 30 300 600 277 029


Procedure Parameter








Challenge protocol









References














1062 GF JOOS ET AL















































































1064 GF JOOS ET AL

















































































1066 GF JOOS ET AL














































































1068 GF JOOS ET AL












1056 GF JOOS ET AL





















weeks

Dose Challenge

Direct Indirect






50z250 mg bid



1058 GF JOOS ET AL


Conclusions







Diagnosis


Monitoring


Epidemiology


Safety performance











eld studies






adenosine



Patient preparation




Dosing protocols



1060 GF JOOS ET AL







Data presentation




lysine-aspirin



Nebuliser settings







Dosemmol

Cumulateddose mmol


01 1 1 1 0 001 2 2 3 05 0501 7 7 10 10 0510 2 20 30 15 0510 7 70 100 20 0510 8 80 180 226 02610 12 120 300 248 02310 30 300 600 277 029


Procedure Parameter








Challenge protocol









References














1062 GF JOOS ET AL















































































1064 GF JOOS ET AL

















































































1066 GF JOOS ET AL














































































1068 GF JOOS ET AL





















weeks

Dose Challenge

Direct Indirect






50z250 mg bid



1058 GF JOOS ET AL


Conclusions







Diagnosis


Monitoring


Epidemiology


Safety performance











eld studies






adenosine



Patient preparation




Dosing protocols



1060 GF JOOS ET AL







Data presentation




lysine-aspirin



Nebuliser settings







Dosemmol

Cumulateddose mmol


01 1 1 1 0 001 2 2 3 05 0501 7 7 10 10 0510 2 20 30 15 0510 7 70 100 20 0510 8 80 180 226 02610 12 120 300 248 02310 30 300 600 277 029


Procedure Parameter








Challenge protocol









References














1062 GF JOOS ET AL















































































1064 GF JOOS ET AL

















































































1066 GF JOOS ET AL














































































1068 GF JOOS ET AL













weeks

Dose Challenge

Direct Indirect






50z250 mg bid



1058 GF JOOS ET AL


Conclusions







Diagnosis


Monitoring


Epidemiology


Safety performance











eld studies






adenosine



Patient preparation




Dosing protocols



1060 GF JOOS ET AL







Data presentation




lysine-aspirin



Nebuliser settings







Dosemmol

Cumulateddose mmol


01 1 1 1 0 001 2 2 3 05 0501 7 7 10 10 0510 2 20 30 15 0510 7 70 100 20 0510 8 80 180 226 02610 12 120 300 248 02310 30 300 600 277 029


Procedure Parameter








Challenge protocol









References














1062 GF JOOS ET AL















































































1064 GF JOOS ET AL

















































































1066 GF JOOS ET AL














































































1068 GF JOOS ET AL


Conclusions







Diagnosis


Monitoring


Epidemiology


Safety performance











eld studies






adenosine



Patient preparation




Dosing protocols



1060 GF JOOS ET AL







Data presentation




lysine-aspirin



Nebuliser settings







Dosemmol

Cumulateddose mmol


01 1 1 1 0 001 2 2 3 05 0501 7 7 10 10 0510 2 20 30 15 0510 7 70 100 20 0510 8 80 180 226 02610 12 120 300 248 02310 30 300 600 277 029


Procedure Parameter








Challenge protocol









References














1062 GF JOOS ET AL















































































1064 GF JOOS ET AL

















































































1066 GF JOOS ET AL














































































1068 GF JOOS ET AL





eld studies






adenosine



Patient preparation




Dosing protocols



1060 GF JOOS ET AL







Data presentation




lysine-aspirin



Nebuliser settings







Dosemmol

Cumulateddose mmol


01 1 1 1 0 001 2 2 3 05 0501 7 7 10 10 0510 2 20 30 15 0510 7 70 100 20 0510 8 80 180 226 02610 12 120 300 248 02310 30 300 600 277 029


Procedure Parameter








Challenge protocol









References














1062 GF JOOS ET AL















































































1064 GF JOOS ET AL

















































































1066 GF JOOS ET AL














































































1068 GF JOOS ET AL







Data presentation




lysine-aspirin



Nebuliser settings







Dosemmol

Cumulateddose mmol


01 1 1 1 0 001 2 2 3 05 0501 7 7 10 10 0510 2 20 30 15 0510 7 70 100 20 0510 8 80 180 226 02610 12 120 300 248 02310 30 300 600 277 029


Procedure Parameter








Challenge protocol









References














1062 GF JOOS ET AL















































































1064 GF JOOS ET AL

















































































1066 GF JOOS ET AL














































































1068 GF JOOS ET AL






Challenge protocol









References














1062 GF JOOS ET AL















































































1064 GF JOOS ET AL

















































































1066 GF JOOS ET AL














































































1068 GF JOOS ET AL















































































1064 GF JOOS ET AL

















































































1066 GF JOOS ET AL














































































1068 GF JOOS ET AL







































1064 GF JOOS ET AL

















































































1066 GF JOOS ET AL














































































1068 GF JOOS ET AL

















































































1066 GF JOOS ET AL














































































1068 GF JOOS ET AL








































1066 GF JOOS ET AL














































































1068 GF JOOS ET AL














































































1068 GF JOOS ET AL



































1068 GF JOOS ET AL

Date post:	21-Nov-2023
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Indirect airway challenges

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