Inhalation methods for the study of airway responsiveness

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Continuing Medical Education This continuing medical education self-assessment program is sponsored by The American Academy of Allergy and Immunology. Support for this program has been made possible by a grant from Glaxo, Inc.

Inhalation methods for the study of airway responsiveness Robert J. Townley,

M.D., and Russell J. Hopp, D.O. Omaha, Neb.

Clinically, asthma is recognized by reversible airway obstruction and airway hyperresponsiveness. The hyperresponsiveness of the airways is a universal characteristic of current symptomatic asthma.lm4 The severity of asthma is correlated with the degree of hyperresponsiveness. I4 Airway hyperresponsiveness is essential to the definition of asthma, and an understanding of its mechanism is crucial to the elucidation of the pathogenesis of asthma.’ Airway hyperresponsiveness appears to arise from both genetic and acquired mechanisms, but the increased airway responsiveness can be temporally associated with exposure to environmental stimuli such as allergens, infection, or ozone.5 These stimuli have been found to cause an inflammatory reaction, which, along with the release of mast cell mediators, appears to increase airway, responsiveness. These mediators include histamine, LTB4, LTC,, LTD4, and LTE,, prostaglandins D2 and Fz,, thromboxane, PAF, NCF, and eosinophil chemotactic factor of anaphylaxis. Mediators have a profound contribution to the pathologic derangements observed in asthma; these include constriction of airway smooth muscle, stim-

Abbreviations used PD,,: Provocative dose producing a 20% decrease in FEV, FEV, PD,,: Provocative dose producing a 35% decrease in FEV, PD,,: Provocative dose producing a 35% decrease in. SG,, SG.,: Specific airway conductance FRC: Functional residual capacity CAHC: Cold-air hyperventilation challenge UNDW: Ultrasonic nebulized distilled water LT: Leukotriene PAF: Platelet-activating factor NCF: Neutrophil chemotactic factor R,,: Airway resistance bu: Breath unit V,: Minute ventilation Water content expired air WC,: WC,: Water content inspired air MVV: Maximal voluntary ventilation PEFR: Peak expiratory flow rate MMD: Mass median diameter

of mucus from goblet cells and mucus glands, increased vascular permeability and the ensuing edema of the airways, parasympathetic nervous system activation, denudation of airway epithelial lining

ulation From Creighton University, Omaha, Neb. Reprint requests: Robert J. Townley, M.D., California Omaha, NE 68178.

at 24th St.,

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112 Townley and Hopp

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cells, and influx of inflammatory cells, including neutrophils and eosinophils.‘-’ It appears that a variety of mediators work in concert to induce these pathologic changes. It is likely that no single mediator contributes to the overall pathology or airway hyperresponsiveness. Physical stimuli. as well as pharmacologic stimuli such as histamine, methacholine. prostaglandin D2 or F,,. LTC, or LTD,, are all capable of inducing bronchoconstriction in subjects with asthma3. “. ’ However, the overriding consideration is that the subject with asthma is hyperresponsive to all of the above mediators and not any one specific mediator. Challenges with both physiologic and pharmacologic methods have provided an important tool to advance the understanding of airway reactivity and, hence, the pathogenesis of bronchial asthma. The purpose of such testing includes diagnostic testing, occupational screening, research, and epidemiology and evaluation of the efficacy of various pharmacologic agents for the treatment of asthma.‘ ’ I3 Precise inhalation methods for the study of airway hyperresponsiveness are essential, regardless of the purpose of such testing. Although the individual with asthma demonstrates hyperresponsiveness to a wide variety of physiologic and pharmacologic stimuli, this is not to imply that there are not important differences in the mechanism whereby these stimuli induce bronchoconstriction. Methacholine or histamine induces direct smooth muscle constriction; exercise, inhalation of cold dry air or inhalation of UNDW appears to do this by more indirect mechanisms, including mast cell-mediator release. Furthermore. the response to the pharmacologic stimuli provides a more sensitive parameter than the response to the physiologic stimuli. However, the latter provides a higher degree of specificity. Thus, all current subjects with asthma respond to methacholine inhalation, whereas only 75% to 80% will respond to the various physiologic stimuli to the point of resulting in a PD,,.‘- ’ “I In any population of subjects with asthma, the level of responsiveness to methacholine or histamine tends to parallel the responsiveness to exercise and hyperventilation. In contrast, patients with chronic airflow limitations secondary to emphysema are hyperresponsive to methacholine but not to hyperventilation. ‘” These differences may merely reflect the fact that methacholine challenges are more sensitive tests than CAHC or exercise. Alternatively, the fact that subjects with asthma respond to exercise or UNDW may be because of the differences in mast cell “releasability” in the airways of the subjects with asthma. Since these latter tests involve mediator release, it is entirely conceivable that the differences in

1987

the response between patients with asthma versus emphysema may reflect differences in airway mast cciis and/or basophils and their “releasability.-“. The differences between the pharmacologic und physiologic bronchial challenges are both qualitative and quantitative. Tests with exercise, inhaled water, or hyperventilation of cold dry air will not produce II PD?,, in subjects without asthma even with the masimum stimulus. In contrast. pharmacologic challenges with methacholine or histamine can produce signihcant airway narrowing in some subjects without asthma. Subjects include those with allergic rhinitis. siblings of subjects with asthma or other immediate family members, and individuals with chronic bronchitis or those who have had an upper respimtory infection in the last 4 to 6 weeks.’ ‘. ‘. ’ However. the degree of response in these subjects is quantitatively much less than that observed in subjects with asthma. For this reason it is essential to measure accurately the oral dose of the agent delivered and to quantitate as carefully as possible different aspects of the bronchial challenge when it is used for clinical_ or research purposes.

Indications

for testing

The primary clinical indication for inhalation challenge with methacholine, histamine, UNDW. or CAHC is to identify the presence of bronchial reactivity, an essentia1 component of the asthmatic state. ‘, ‘. ‘). I’-” Most patients presenting with asthma have classic symptoms, and bronchial chalhznges are not necessary for the diagnosis. However. patients may present with cough as their only symptom or atypical dyspnea without physical findings and wGth normal spirometry. In these situations a bronchial challenge may be useful as a clinical test. A negative methachoiine challenge test rules out current bronchial asthma and would guide the clinician to consider.other causes of bronchial disease such as tumor, bronchiecY tasis, or possibly chronic bronchitis. The degree of methachdine and histamine sensitivity also correlate with the severity of asthma in terms of symptomS.?.q. 7 17The indications for antigen challenge and for pharmacologic challenge are listed in Tabk 1. Various manufacturing and mining operarions. exposure to agricultural environments. and animal handling may be associated with a high rate of~occupational asthma. In these situations inhalation challenge may be valuable to determine and identify those workers who are at risk for occupational asthma because of preexisting bronchial responsiveness. Bronchial provocation tests may provide a valuable test of drug efficacy. However, they are not a.-substitute

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TABLE I. Clinical

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challenge

Antigen challenge Clarification of the role of specific allergens in asthma,especially when other diagnostic criteria are inadequate.It should be recognizedthat a false positive reaction is possible, that is, allergic patients may have bronchoreactivity to antigens that are clinically unrelated to their asthma. Conversely, antigen challenge may be useful to clarify the relevance of cutaneousor serum tests when the history is equivocal. Provocation with antigen should have someclinical relevance, such as contingency of intervention by immunotherapy or specific avoidance. Identification of specific antigens suspectedof causing acute or delayed airway obstruction8.‘. I5 Evaluation of new drugs for asthma Almost all the antiasthmamedication and many others were testedagainst allergen challenges. The following drugs are found to prevent the early response:E-adrenergicagonist, theophylline, cromolyn sodium, lodoxamide, and thymoxamine (an cu-adrenergicblocking agent). Corticosteroids, cromolyn sodium, and possibly cyclooxygenaseinhibitors block the late reaction. Atropine and antihistamines do not block the antigen reaction significantly. Clarification of the mechanismof asthma Antigen challenges have been used extensively to study mediatorsof allergic asthma. Histamine, LTC,, LTD,, NCF, and PAF releasehave been documentedafter antigen challenge.’

The antigen challenge helps us to understandthe different reactions: the immediate reaction that begins within minutes, reachesa peak within 10 to 20 minutes, and clears within 1.5 to 3 hours. The immediate reaction is causedby smooth ,nuscle constriction as a result of endogenousmediator release.The late responseusually begins between 3 to 4 hours, peaksbetween 8 and 12 hours, and may last 24 to 36 hours. The late responsesare associatedwith prolonged increases in airway responsivenessto histamine and methacholine.’ The early responseis determinedby the level of airway responsivenessto histamine or methacholine, the dose of antigen, and the level of specific IgE antibodies. To define, when it is appropriate, the natural history of antigen sensitivity when there is no inununologic intervention or, conversely, for evaluation of the therapeutic effect of inununotherapy.‘5 For the evaluation of new or unrecognizedallergens or provocative agentsin pulmonary disease,such as for the assessmentof occupational inhalants in susceptiblepatients. Methacholine, carbachol, or histamine challenge To identify the patient with hyperactive airways regardlessof cause, as well as to measurethe extent of such hyperactivity when it is appropfiak.“3 9,Is

for clinical trials. In testing the effect of various nonbronchodilating and bronchoclilating drugs, the provocation must be standardized and regulated in order that the environmental factors as well as the stimulus and airway measurements are the same for each treatment.

airway hyperresponsiveness is not equivalent to a disease. This is exemplified by the fact that many siblings of subjects with asthma have no history of asthma but may demonstrate positive responses to methacholine.‘* 3

Comparieon of different bronchoprovocetion procedures

The FBV, has remained the most important measurement of airway responsiveness. The virtues of FBV, are simplicity, reproducibility, and the fact that measurement is readily available. FBV, also provides the best separation between patients who have asthma and those who do not. other tests, however, may be used, but it is important that a common parameter such as the FBV, also be included for comparison between various laboratories. other tests also commonly used are R,,, SG,, and partial expiratory flow volume curves. These measurements provide sensitive measures of response and avoid the full inspiratory maneuver of the FEV, that

Bronchoprovocation tests tend to correlate in the same individual; however, certain tests are more closely related than others. For example, histamine and methacholine have a very high correlation coefficient,‘, ‘*, I9 whereas methacholme and exercise or methacholine and CAHC or UNDW correlate to a lesser degree. However, there is a strong correlation between the response to UNDW and with exercise challenges.” Bronchoprovocation tests are more sensitive than bronchodilator responses and in most cases are more sensitive than a careful history. However,

Measurement of response

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114 Townley and Hopp

TABLE II. Recommended between last medication bronchial challenge

avoidab4e, take41kto corksideration in evduating bron-

ideal time interval and

chial challenge results. Factorsinclude acute viraf respiratory infections, bacterial respiratory ~1fe~tion.s.

Time interval (hrl

Inhaled bronchodilators Isoproterenol lsoetharine Metaproterenol Terbutaline Salbutamol Atropine and its analogs Injected bronchodilators Epinephrine Terbutaline Oral bronchodilators Liquid theophylline preparations Short-acting theophylline preparations Aminophylline preparations Intermediate-acting theophylline preparations Terbutaline Long-acting theophytline preparirtions Cromolyn sodium Lodoxamide Long-acting antihistamines Hydroxyzine Terfenadine

CLIN. WvlUi’OL. nuGUST 1987

4

6 8 12 12 10 4 12 12 18 18 24

and pollutants such as NO, and SOZthat are known to increase reactivity. Seasonal exposure to naturally occurring antigen or recent antigen challenge may increase airway hyperresponsiveness. Similarly, power of suggestioncan also influence airway reactivity. Drugs. A variety of drugs that can influence bron-

chial challenge and the recommended withdrawaf times before challenge are listed in Table 11. Aerosol generation. In order for airway respon-

siveness studies to be valid, an aerosol with appropriate and reproducible properties must be delivered to the subject. lmportant properties of an aerosol include the electric charge, surface area, particle density, composition, volatility, shape, and mass distri-

bution. Aerosols may change their properties rapidly becauseof the inherent instability. Changesoccur as a result of evaporation, absoqtion,

con$ensation,

sedimentation, inertial deposition, or diffusion.” 24 For methacholine, histamine, and other pharma48 cologic agents, as well as antigen, the DeVilbiss jet 48 nebulizer model 646 (DeVilbiss Co., Somerset;Pa.) 48 is used by most investigators. The nebulized particle 48 size has an MMD of 4.4 + 2.2 pm. This nebuulizer 96 produces an aerosol with a mass concentration of 72 37 mg/L. The coefficient of variation of different Guidelinesfor bronchialinhalationchaiiengewith pharmacologic DeVilbiss 646 nebulizers is about 25%. and for theandantigenicagents.‘Patients with anFEV, < 70%of predicted same nebulizer used repeatedly, it is 10%. For any shouldnot be tested. nebulizer used, the dose delivered to a subject is the volume of aerosol inhaled times the aerosol mass con-

may have a minimal bronchodilator effect. For this reason they are useful for measuring responsesto a smaller stimulus. Changesin thesepammeterscan be obtained evenwhen no& individuals am challenged with agents that would fail to produce a PDZO.They have the disadvantageof being more variable and less reproducible than the FEV,. Obstruction in the large airways can be detectedby changesin Rw, SG*,, and PEFR, whereasobstruction in smaller ahways can be detected by changes in forced expiratory !Iow rate -between25% and 75%, partial flow volume, and FRC and residual volume. Changesin FEV, reflect the ckmges in the caliber of both large and small airways. EEV,, PBFR, forced expiratory flow rate between 25% and 75%, and flow volume loops can be meas& by a we& spirometer. The measof o&e: parameters~quires the use of a body player that is more expensive and incanveuient. Factorsthat can in@uencethe v to bronchial chdlenge should be avoided or, if factors are un-

centration times the deposition percentage.” Other commonly used nebulizers are the Wright nebulizer and the Wiesbadener Doppelinhalator (Wiesbadener Inhalatoren-Vertrieb, Wiesbaden, W. Germany). The Wright nebulizer has a smaller MMD than the DeVilbiss 646. The MMD is 1.4 km + 2.0. The nebulizer has a massconcentration of aerosol of 29 mg/L. Similar histamine bronchoconstricting effectsand dose-response. curves were produced with a DeVilbiss 646 (with do&meter) and a Wright nebulizer with 2 minutes of tidal breathing.= Ultrasonic nebulization is produced when highfrequency sound waves are focused onto an air-liquid interface. The initial average particle diameter is a function of the crystal frequencymused to generatethe sound waves. Considerable variation occurs in the effectivenessof different types of ultrasonic r&&zers in inducing bronchoconstriction with UNDW. Tubing, mouthpieces, and other ~devicesbetween the aerosol generator and the patient’s mouth should remain the same for all measurebecause the particle size will vary causedby evaporation and impaction of the larger droplets in the tubing.

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Inhalation

Technical factors. The response to the inhalation of a pharmacologicaerosol is determinedby the dose and distribution of the aerosol depositedin the bronchi. The amount deposited in the bronchi and the lower respiratory tract is influenced by the inspiratory flow rate. The two methodsthat are used include the method of continuous aerosol generation and tidal breathing and the dose-delivery methodwith a dosimeter and a specific number of inhalations of a given concentration (see Histamine and methacholine). In the dosimeter method the effect of the volume delivered per puff, lung volume at delivery (residual or FRC), the speedof inhalation, and how long the breath is held at the end of inspiration need to be specified in describing the method used.‘*‘, IS,23 The stability of the various concentrations of the inhaled agentsin diluent is an important consideration. Methacholine chloride and histamine acid phosphate should be preparedin buffered saline to pH of 7.4 to avoid the possible potent bronchoconstricting effect of hypotonic solutions. These agentsare stable for at least 3 months when they are stored at 4” C.‘, 23Preservatives such as benzyl alcohol or 0.4% phenol are added to maintain sterility. As an additional precaution, it is recommended that the final solution be passedthrough a bacterial filter before use. However, prostaglandinsand LTC, and LTD, are very unstable, and therefore, should be prepared fresh each day. Before obtaining the doseresponseto an agent, the effect of inhalation of normal saline needsto be determined. If a fall in FEV, of 10% to 20% occurs after saline solution, the validity of any subsequent results is questionable, and further testing is inappropriate, at least for that subject for that day. The time between inhalations and the time of spirometry measurement should be constant. The responsesshould then be comparedto the postsalinemeasurement.The inhaled agents are delivered in stepwise increasing doses. The duration of the test can be shortenedby adjusting the starting concentration or by increasing the dosesfourfold until there is definite changefrom baseline. Subsequently,twofold increasesin doseare used. Concentrationsof the bronchoprovocationagent are increaseduntil PD, is reached, the R,,, SG,, or expiratory flow volume curves have changedby 35% to 40%, or the final dose is reached. The results are expressedas the PDzoor the PD,, and are discussed below (Expression of results). The dose is expressedin breath units for methacholineor histamine. One breath unit is defined as one inhalation of a concentration of 1 mg/ml. 7,8 It is also appropriate to expressthe dose in milligrams or micromoles and to specify whether the dose is cumulative or noncumulative. The results should be expressedas the dose delivered to the mouth by the method of aerosol gen-

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eration and inhalation, rather than the concentration” (see Histamine and methacholine section). Subject-related factors. In order to assure reproducible results, subject-relatedfactors needto be controlled. These factors include reproducible baseline lung function, no recent exposureto occupationalsensitizers or irritants, no exposure to allergens, and no respiratory tract infections, asthma exacerbation, or recent vaccinations. If all of these technical and subject factors are controlled, the reproducibility of the test is excellent (r = 0.98).** Expression of results The factors that most affect interpretation of tests of airway responsivenessare the dose delivered, how the dose-responsecurve is plotted, the values usedto express its position, slope and shape of the doseresponsecurve, and the lung function test used. The use of different lung function testsresults in a different range of values for airway responsiveness. For example, the PD,, may be one log less than the PD,,. Although the FEV, is less sensitive than some of the other testsused, it remains the most commonly used test, especially in clinical and epidemiologic studies of airway responsiveness.It is the least variable of the tests. The FEVJFVC percent is not a suitable measurementfor challenge testing because the changesin FVC are often similar to changesfor the FEV,, which indicate that during the challenge considerableairway closure occurs. Fig. 1 illustrates typical dose-responsecurves to histamine in subjectswith severe,moderate,and mild asthma, a subject with past asthma, and a normal subject. Resultsare plotted illustrating the cumulative dose and expressedas micromoles of histamine on a log scale against the fall in FEV,. The degreeof airway responsivenessto methacholine can be expressedas milligrams per milliliter for concentration (noncumulative) and micromoles and breath units (cumulative) for individuals with severe, moderate, and mild asthma, as well as normal subjects, e.g., 1 mg/ml = 0.5 pmol = 10 bu. There is a gray zonebetweenmild asthmaandnormal subjects. Although the PD,, is the usual methodof expressing results, some subjects without asthma, particularly subjects with allergic rhinitis, may have a positive reaction, as evidencedby attaining a PDm, and then demonstratea plateau phenomena.*,3*I73~4In these subjects, further administration of methacholine or histamine will fail to produce progressiveairway narrowing. In contrast, patients with asthma, when they are administeredincreaseddosesof inhaled histamine or methacholine, have a progressive airway narrowing. Woolcock,24Townley et al.,*, I7 and Reed and Townley3have demonstratedthat, when an FEV, PD3*

116 Townley and Hopp

J. ALLERGY

FIG. 1. Dose-response curves to histamine in subjects with severe, maderate, and mitd tima, in a subject with past asthma end in a subject without asthma.

is used, individuals with this plateau phenomenaversus individuals with asthma can clearly be differentiated. However, a concern for the severity of airway narrowing beyond the FEV, PD,, makes further administration of the bronchoconstrictive agent too uncomfortable or dnngerous.17.24 An iqwtant advantage of determining the area under the dosec~se curve is that it allows quantitative t3xasumrnentsin subjects who never achieve a PDzoto either histamine or methacholine.*~17,24In many sub&& with allergic rhinitis, a plateau occurs even after achieving a PDm. If only the PD, is compared, these subjects with allergic rhinitis may have results co-e to some subjects with asthma. However, if the areaunder the parabolic dose-response curve is determined (equation in Fig. Z), the representation of results in this manner allows for the differentiation of subjects with asthma and responder subhts with allergic rhinitis (Fig. 2). When the concentration of methacholine is increasedto 60 mg/ml and the number of cumulative breath unitsis %Kl, the vast majority of normal subjects and most subjects with allergic rhinitis do not achieve a-PD,. Expressing the results of the methaC

ural history of airway sensitivity. The sen&&y of a the ability of the test it is present, whemas ape+3

m#%hacholinewitl%the we found thet 2fxlbu opt specificity at 90%.2*3 From others,wecanco~ludeth

area under the dose-response

allows for the results to be expressed as quant&&ive vale: rather than a nonresponse. i&out asthma that do achieve a concentrations (>200 bu) that C

cussedin folio*

s6?&ons.

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bXmax pAREA, =

n 1.5

, 4.5

. ax2 +bx+c dx where xmax = PD,, or 804.5 bu I

,

I

14.5 44.5

144.5

I

I

444.5

804.5

CUMULATIVE DOSE (Breath Units) FIG. 2. Methacholine challenge. A least-squares is the right-hand limit of the-integral.

fidence limits of the PD, value. The effect of an intervention such as an aerosolizedagent on the level of responsivenesscan only be determined if the variability of the baseline is known. It is preferable to assessthe airway response to mediators at the same time of the day as circadian variation has beenobservedwith histamine. The 95% confidence limits for the reproducibility of the PC, for methacholine and histamine challenges done on different days with an interval of 1 to 2 weeks was plus or minus one single twofold concentration.22This samedegree of reproducibility was demonstratedfor both the dosimeter and continuous tidal breathing methods.22 Pharmacologic challenges Histamine and methacholine. Since Dautrebande and Philippot’” introduced inhalation challenge in 1941, various specific and nonspecific agentsand different methods have been used to induce bronchial hyperreactivity. Inhalation challenges with different pharmacologic agents like histamine, methacholine, acetylcholine, carbachol, pilocarpine, serotonin, propranolol, methoxamine, adenosine,prostaglandin D2 and L LTs, and PAF have been used. Of these agents,methacholineand histamine are the most commonly used and have been well standardized.Methacholine is a parasympathomimeticagent that appears to stimulate the muscarinic receptors on bronchial smoothmusclesdirectly, increasingthe bronchomotor activity. Methacholine is inhibited by atropine and its analogues.2,5*27Histamine induces bronchoconstric-

fitted, parabolic

dose-response

curve. FEV, PD,,

tion primarily by a direct bronchoconstrictive effect and partly by reflex vagal stimulation.27 Methacholine and histamine are both extensively used in researchas well as in diagnostic testing. Selection of the agent to be used generally dependson the investigator’s experience and familiarity with the agent. Histamine has a shorter duration of action and may cause headache, flushing, and hoarsenessat higher doses.2g*29 PREPARATION. Patients should have the procedure fully explained. An informed consent should be obtained wheneverit is possible. A physician and emergency equipment must be available at the site of the challenge testing. A general physical examination should be carried out before the challenge, and the findings should be recorded. METHODS.Methacholine and histamine are available as dry powders. Methacholine has beenrecently approved by the Food and Drug Administration and made available through Hoffmann-La Roche Inc., Nutley, N.J. The preparation and preservation has been described in the Technical factors section. Two methods of challenge with methacholine and histamine are widely used, and both methodsrequire nebulization of the solution. A DeVilbiss nebulizer 646 is used with the Rosenthal-French dosimeter (Laboratory for Applied Immunology, Baltimore, Md.),9*I5 and a Wright nebulizer is used for the continuous aerosol method.4Baseline spirometry should be performed before proceeding to any challenge, and FEV, should be at least 70% of the predicted value.

118 Townley and Hopp

Serial dilutions of methacholine or histamine are prepared. The standardprocedure used the following concentrations and sequence of five inhalations of 0.075, 0.15, 0.31, 0.62, 1.25, 2.5, 5.0, 10, and 25 mg/ml for a total of 225 bu.‘, 9An abbreviatedversion of the 1975 dose scheduleof the American Academy of Allergy’ has been suggested.” Two milliliters of the solution is put in the DeVilbiss No 646 nebulizer, and the plumbing tube is attachedto the compressed air reservoir. The aerosol is generatedby the compressedair delivered at 20 psi through the nebulizer. The input is controlled by a solenoid valve that is triggered by the inspiration and is kept open for 0.6 seconds. A noseclip is used. The subjects are instructed to inhale slowly from the FRC to total lung capacity. During the inhalation, the vent of the nebulizer should be kept open. Three minutes after the aerosol inhalation of each dilution, spirometric measurementsare taken. The test is generally terminated if a 20% fall or more from the control FEV, is reached or if the final dilution is reached. Failure to reach a PD, is considered to be a negative challenge. The results are evaluated as discussed in the section on Expression of results. For the continuous tidal breathing method, serial dilutions of methacholineor histamine are used. Three milliliters of the solution is put in the Wright nebulizer. The plumbing tube is attached to the compressedair reservoir. The flow of the air is regulated between 7 and 8 L/min that delivers 0.135 ml/mitt of the aerosol. The aerosolis administeredcontinuously for 2 minutes by the nebulizer directly into the mask that is held over the mouth with a noseclip in place. The spirometric measurementsare taken 1% to 3 minutes after the inhalation of each dilution. The test is terminated if a PD,, or the final concentration is reached. Throughout the challenge, spirometric measurements are taken in duplicate. The consistency should be defined as FEV, within 5% of each other, and the best of the two values is taken for the challenge evaluation at each step. Both the methods require compressedair for the generationof the aerosol. Constancyof the length and diameter of the tubing carrying the compressedair to the nebulizer is maintained. If the challenge is to be repeated in the same subject, the same nebulizer should be used each time to avoid the internebulizer variation9 The nebulizers should be thoroughly cleaned between patients. Other piuwmasologk: mrkts The previously discussedbronchial challenge procedures have widespread diagnostic applications. However, researchin asthmaandbronchial reactivity

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OLIN. IMMUNOL. AUGUST ?987

includes bronchial provocation testing with various other chemicals. These, to date, have included prostaglandins, LTs, adenosine,PAF, propranolol. and serotonin. Many of these agents are known to be rcleased during mast cell-dependent events and may duplicate natur&y occurring asthma. Theseagents are likely to be more widely used in the coming years, especially astools for the investigation of basic mechanisms in asthma. An overview of the pertinent findings to date is, therefore, important. The technical aspects, as they relate to inhalation challenge tests with these agents, are quite variable when these are comparedto more standardizedchallenge protocols. It is important for investigators to clearly outline the inhalation technique used to allow for comparison between studies. Dose concentrations and nebulizer outputs should be detailed in published articles. Since the action of theseagentsis not always well understood, it is not uncommonthat inhaled dose and pulmonary function changesare deliberately and appropriately limited. ProstaglandinD2 is releasedin significant amounts after IgE-mediated stimulation of human lung mast cells. A recent studyMdemonstratedthat,inhaled prostaglandin D, has a 10 times ~morepotent effect on airways than does histamine and is 3% times more potent than prostaglandin FZa.ProstaglandinD, had a minimal effect in normal subjects, whereasFZademonstrated no effect at the doses used. Other studies3’,32have indicated that F, can induce bronchoconstriction in normal subjects, although doses 500 to 1000 times are needed in normal subjects, compared to dosesneededin subjects with asthma. LTC4, LTD,, and LIE4 have been demonstratedto have potent bronchoconstrictive properties in humans.33,34Although this fact is well recognized, conflicting data have arisen concerning (I) the site of action of LTs,~~(2) the relative sensitivity of subjects with asthmaand normal subjects to LTs,%and (3) the relative sensitivity of LTs in subjectswith asthmaand normal subjects when compared to the degree of methacholine sensitivity in these subjects.37Although these questions are important, the significance of LT sensitivity in subjects with asthma has been established and will have a major impact on research in asthmain the coming years. LT challenges may also play an important role in the testing of potential LT-receptor antagonists. PAF, a mediator released from mast cells, has recently been implicated to be an active bronchoconstrictive agent. It also has potent in&nmatory generating properties in experimental animals.R A single study39has been made on the effect of inhaled PAF on normal control subjects. Although small incre-

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Inhalation methods for study of airway responsiveness

ments of inhaled PAF were used and only minimal decreasesof the FEV, were observed, methacholine hyperresponsivenessincreasedin five of the six subjects studied. In several instances, PAF-induced hyperreactivity lastedfor severalweeks. The role of PAF in the induction or accentuationof nonspecific bronchial reactivity will likely come under more intensive investigation. Adenosine inhalation challenges have gained limited use, predominately in the United Kingdom. Adenosine, and its precursor nucleotide, adenosine-5monophosphate, induces bronchial constriction in subjects with asthma but not in normal subjects.40 Adenosine is four times less potent than histamine or methacholine. The action of adenosine is not well understood but probably does not act through vagal stimulation.41Since adenosinereleasecan be detected after antigen-induced bronchial constriction, it appearsreasonableto speculatethat adenosinemay play a role asan inflammatory componentof asthma.Adenosine receptors are blocked by theophylline compounds, and this may in part explain the role of methylxanthines in asthma management. At the present time, however, adenosinechallengesare not used for diagnostic testing in the United Statesbut could have a role in elucidating the pathophysiology of asthma. The well-recognized clinical observation that B-blockerscan induce bronchoconstriction in subjects with asthmahas resulted in the use of propranolol as an inhalation challenge procedure. Increasing concentrations of propranolol are inhaled with a modification of the method of Chai et al.’ or by the method of Yan et al.42Propranalol sensitivity in subjectswith asthmais less than that observedwith histarnine43and methacholine.44Although normal subjectsdo not respond to inhaled propranolol, subjectswith hay fever have been demonstrated to develop sensitivity to methacholine and asthma-like symptomsafter the inhalation of propranolol.45Propranolol challenges are a useful tool in research and could provide further understanding of why B-adrenergic blocking agents induce bronchoconstriction in subjectswith asthma. Physiologic challenges As with inhalation studies of methacholine, histamine, and antigen, graded exercise and CACH can also produce bronchoconstriction in susceptible individuals. Thesetestsare usedto assesslatent asthma, to evaluate the severity of known exercise-induced bronchoconstriction, and to evaluatethe effectsof certain medications.‘, I43I5 The specificity approaches 100%; however, the sensitivity varies from 50% to lOO%, depending on the criteria for a positive response.Their reproducibility is high if tests are per-

119

formed more than 2 hours apart.46Controversy exists, however, in their correlation with histamine and methacholine challenge.474g Exercise. Exercise studies entail free running, treadmill running, cycloergometertesting, kayaking, walking, or swimming with the order of responsiveness as stated.” Free running, however, has many uncontrollable factors such as temperature,humidity, environmental pollutants, and allergensthat make the test less than optimal. ‘OAs a result, treadmill running and cycle studiesin a controlled environment are more popular. With both challenges, the energy of each subject should be quantitatedby a measurementof work rate5’ neededto increasethe oxygen consumption by 30 to 40 ml/min/kg52~53or increaseof the heart rate by 80% to 90% of maximum heart rate basedon the subject’s age5’,53;this is approximately 180 bpm in children and 160 bpm in adults.48(Values may be obtained from tables53or are approximately 210 minus age.) For subjectsolder than 25 years, a three-stepgraded increment approachfor treadmill testing is advisedfor safety reasons.50,5’ With subjects younger than 25 years of age, the target heart rate can be reached in the first 1 to 2 minutes of exercise.” With both treadmill running and cycling, the target heart rate should be sustained for 6 to 8 minutes for a maximum response;longer bouts of exercise will not increasethe bronchoconstriction and may even promote bronchodilation.50q52-55 The mechanics of each challenge has been described elsewhere.5’ Within the first 4 minutes of exercise, bronchodilation occurs and is believed to be due to increased sympatheticdrive.5oThis is followed by a progressive airway constriction that peaks 3 to 4 minutes after exerciseis completedin children and up to 15 minutes later in adults.50+ ” Baseline function is attained on an average20 to 30 minutes later.50.5’ As with other bronchoprovocation studies, baseline pulmonary functions are obtained immediately before the study, and FEV, and PEFR should be at least 65% to 70% of predicted and within 80% of the subject’s usual leve1.54The responseshould be measuredevery 2 minutes after exercise up to 10 minutes and then every 5 minutes thereafteruntil baselineis achieved.54 Disagreementexists regarding what degreeof change in the indices representsa positive response.The percent change from baseline or percent fall index: Value immediately before exercise lowest value after exercise Value immediately before exercise for PEFR and FEV, varies from 310% to 20%. A 230% decreasewith SG,, and ~25% decrease

120 Townley and Hopp

HG. 3. Diagram of equipment used to perform a bronchial challenge with iaezxzapnichyperventilation of subfreezing, dry air.

in maximal midexpiratory flow are generally acC+4?d.M-54

changer, it travels through-an witha to the

It must also be rememberedthat these tests, especially the treadmill test, ate not benign pmcedmes. Far subjects with suspe&ed cardiovascufar disease, obesity, diabetes, and subjectsolder than 35 years, a stress test should be done before testing.53Subjects with a history of clinically significatntheartshould not be tested. In subjectsat risk, a resting EK;Gand blood pressure should be checked before testing, and the bIwd pressureshould be mot&on& inmy and EKG continuously throughout the test.s2~ S, 56If hypoxia or hypercapnia is suspected,then a test for arteriatblwd gas is essentialbefore exercise. The chalbronchoeonstrietion.“’ T&is can be lenge test should be termmatedif any of the .following of 5% 2I% 0, occur: angina with or without ST segmentchanges, at 2 L/mWinto the ectopic supraventrieular tachyeardiaa,ectopic ventricular contractions >IO/min if contractions are unifocal, or any multifocal ve&c&r tachycardia, any type of heart block, signs of peripheral circulatory insuffIciency, or if blood pressure decreasesor fails to itlcrease more than 10 mm IIg during the test.*6 cardiac maaitaring should fixxhue for applC+ximately 3 minutes after the test, and the p&em should be observed for 30 rnirtute~.~~During the test, end tidal CO2 and oximeter measurementsmay be help&l but can be -assumedto be not essential52

between the two. ‘6*51,55 The setup consists of compressed air entrained through a heat exchan nalteqXua#ueat -10” Cf (Fig. 3).

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MVV.57.M,Regardlessof the method, each is maintained for 3 to 4 minutes.I6957,6oAltbough a single V, of 20 to 35 times the FEV, may produce good results, MVV alone or with a cumulative dose elicits more consistent results.@’A cumulative-dose method also allows better observation of the effect of a drug and decreasesthe chanceof marked bronchoconstriction. As with exercise tests, the maximal bronchoconstrictive responsepeaks4 to 8 minutesafter challenge, starts resolving within the next 5 minutes, and approachesbaseline after 15 to 60 minutes.“. 57Here, too, controversy exists as to what is considered a positive response;most agreethat a positive response is at leasta 10%decreasein FEV,. A positive response for SG,, is 330% change.15z “* 6oMeasurementsare done at either 2 to 3-minute intervals until maximal responseis achieved and then at 5-minute intervals until recovery begins.5v,6’ The resultscanbe expressed in terms of VE, the respiratory heat exchange,51, 61or the maximum drop in FEV, obtained. During both exercisetesting and CAHC, a thermal burden is placed on the airways, resulting in heat and water loss.“’ ~4.55The expired air transfersonly some of the heat and water back to the mucosa;therefore, there is a net loss from the mucosa.55How this heat and water loss that occurs during CAHC produces bronchoconstriction is not precisely known. Current theory suggeststhat aswater evaporatesfrom the bronchial mucosa,a hypersmolarenvironment is produced that is responsible for the bronchoconstriction.55In support of this theory are studiesdemonstratingbronchoconstriction produced by inhalation of hypo- and hyperosmolar solutions,58and a good correlation exists in the responseto CAHC, exercise, and hypoand hyperosmolar challenges.55It has also been demonstrated that an osmolarity change itself does not directly stimulate human airway smooth muscle but causesrelease of mediators from mast cells, eosinophils, and basophils.55s 62 Ultrasonic

nebulized distilled

water challenge.

Anesthesiologistswere first to observe an increasein airway resistanceafter inhalation of ultrasonic water. Allegra and Bianco63usedUNDW as bronchial provocation in 1974. Anderson@ introduced a doseresponsecurve and calculated the PD, of UNDW. The test appearsto be very specific, but the sensitivity ranges between 30% to 100%.20The correlation of UNDW challenge to methacholine is variable; a better correlation to exercise and CAHC is found.*’ A possible mechanism by which UNDW causes bronchial constriction is the releaseof mediatorsfrom the mast cells. Shaw et al.‘j5demonstratedan increase in NCF activity and an increasein the plasma hista-

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121

mine level after UNDW challenges. Bascom and Bleecke?’ have demonstrated parallel activity of UNDW and exercise, indicating a possible similar mechanism induced by these two challenges. It is possible that UNDW could induce a change in epithelial permeability with a resultant inflammatory response. These changes could activate subepithelial receptors and induce vagally mediated bronchoconstriction. Inhalation of UNDW can briefly induce an increase in nonspecific bronchial responsivenessto metbacholine,66and a late asthmaticresponsehasbeen reported after UNDW. 66Beta-agonist and cromolyn sodium are effective in inhibiting the response to UNDW.63,65Anticholinergic drugs have not consistently displayed attenuationof UNDW-induced bronchospasm.63* @ Various methods of UNDW challenge are being used, and a standardizedprotocol has yet to be established. The basic principles in doing UNDW challengeinclude (1) starting with a low-dose, (2) increase the dose gradually, and (3) obtain a dose-response curve. UNDW challenge is a safe, specific, moderately sensitive test for bronchial asthma, and it can be usedasa valuable researchtool in testing the patbophysiology of asthma. Hypertonic aerosols with osmolarity up to 1280 have been used as a bronchial challenge. The bronchoconstriction induced by inhalation of 3.6% normal saline was equal to that induced by UNDW inhalation.67 Allergen Antigen provocation was first describedby Lowell and Schiller.68Although it is widely used by many investigators, its use as a clinical diagnostic tool is restricted becauseit addslimited information over the skin test. Investigators have found considerableoverlap in the antigenprovocation responseamongpatients who have atopic asthmaand patients who suffer from allergic rhinitis.‘, 3,69 There is a good correlation between skin tests and in vitro testsand bronchial provocation. The stronger the skin reaction the greater the chance of positive bronchial provocation.1,69With few exceptions,a negative antigen provocation test to a certain antigenrules out the possibility that the antigen is responsible for asthma.In contrast, a positive challengeonly indicates sensitivity to the antigen. Pollens, housedust, mites, molds, and animal danders have beenusedin antigen provocation tests. Pollens are more likely to causepositive responsesthan house dust in comparableskin reactive patients, possibly becauseof the lack of homogeneityof housedust extracts (section 2, Table I).

V?

Towniey

Method’,

and Hopp

’

The aqueous antigenic extracts are diluted with a diluent containing 0.5% sodium chloride, 0.275% sodium bicarbonate, and 0.40% phenol (pW 7.0). Lyophilized extracts are preferable if they are available. The degradation can be reduced by storage between 4” and - 20” C and by addition of stabilizer such as serum albumin. Limited data indicate that concentrations up to 1: 20 can be stored at 4” C for 1 year.* Concentrationsmore diluted than 1: 20 should be used within 7 days of preparation. When studies are performed of drug efficacy against allergen-induced responses,the extractsshould be reconstitutedor thawed and diluted shortly before use, preferably on the same day.**9,‘OIt is important that the potency and stability of the anitgenic extracts are assured. The likelihood of achieving this goal is maximized by the use of lyophilized extracts of a lot that hasbeenstandardized by an approved method for assessingpotency of allergenic extracts. Antigen should be labeled on a weight per volume basis, but the protein nitrogen units and/or micrograms of protein nitrogen per milliliter determination should also be indicated.8 One inhalation unit equals one inhalation of I:5000 w/v or one inhalation of solution with 1 p.g of protein nitrogen per milliliter or 100 PNU/ml. The generalprecautions and safety measuresfor bronchial challenge also apply. Additional precautionspertain to the greatervariability, severity, and duration of the responseto allergens, especially the late response.“. 72 A properly performedskin test should be performed with both skin prick and intradermal tests. Serially diluted antigen extracts are administeredfrom 10e5to 10m3w/v dilution. The diluent control is used as well as histamine. The initial antigen concentration used for the first challenge should be the one that produces a 2 + reaction on intracutaneous injection, i.e., >5 mm wheal (minus diluent control). The antigen should be delivered from a nebulizer connectedto a dosimeter as discussedpreviously. Ten minutes after inhalation of five breaths of diluent, pulmonary function studies are performed. This diluent value is the control, and if the FEY, is not reduced 10% from the baseline, the subjectenters the study. The challenge begins with five breaths of the antigen concentration that was required to elicit the 2 + skin test.*.’ If < 15% reduction from the control FEZV,value occurs 10 minutes after antigen, the next dilution is administered. If the reduction is 15% to 19%, an additional 5- to lo-minute wait is indicated. When the end point is approached, which is sPDZo from the diluent control t&t, less than five breaths may be obtained to reduce the possibility of a precipitous fall in the FEV,.

J. ALLERGY

CLIN. IMMUNOL. AUGUST 1987

The cumukative inhalation units of antigen appear logarithmically on the abscissaand the percentageof FEV, on the ordinate. The PDzocan be calculated in breath units.*. 9 A P-agonist by inhalation should be administered and postbronchodilator spirometry performed to be sure that the FEV, returns to or near the baseline value. A potential late reaction could be expected to begin within 3 to 8 hours. If it is feasible, the patient should be studied at hourly intervals, or a peak Aow meter can be used and results recorded.Is,‘O If another antigen challenge is necessary,it is wise to wait 1 week or longer. The increasein nonspecific bronchial reactivity may take 1 week or more to return to baseline.7@72 The subsequentchallenge should be startedat least two concentrationslower than the previous concentration that induced a significant drop in the FEV,. Wethank Drs. AgaindraK. Bewtra,Nicki Nair, RajendrakumarTrivedi, Fawzi Suliaman,and Shull Lemire for their contribution,andRosemaryBat&andLaurieWilliams for their skillful assistancein prep&ing this manuscript. RFERMCES 1. Townley RG, Dennis M, J&in JM. Camparative action of acetyl-beta-meth, histamine, and pollen antigens in subjects with hay fever and patients with bronchial asthma. J ALLERGY 1%5;36:121.

2. Townley RG, Bewtra AK, Nair NM, Brodkey FD, Watt Gp. Burke KM. Me.mne it&d& chalhge studies. J ALLERGY CLIN IMMUNOL 1979;64@2:569. 3. Reed C, Townley R. Asthma: ciaa@iEcation and pathogem+sis. In: Middteton E Jr, ReedCE, Ellis E%,eds, A&rg$ principles and practice. 2nd ed. St. Louis: The CV Mosby Co. 1983~811. 4. Cockcroft DW, Killian DN, Me&n J.JA,Hargreave FE. Bronchial reactivity to inhaled h&amine: a method and &i&al survey. Clin Allergy 1977;7:235-43. 5. Boushey HA, Holtzman MJ, Shellar JR, Net JA. State of the art: bronchial hyperreactivity. Am Rev- Respir Dis 1980;121:389. 6. Chung KF. Role of inflammation in the hypermactivity of the airways in asthma. Thorax 1986;41:657. 7. Townley RG, Weiss S, Lang W, McCall M, Hopp R. Mechanisms and management of bronchial asthma, chap. 7. In: Spittel, ed. Clinical Medicine. F’hiktdelphia:Hat$er & Row, 1986. 8. Chai H, Farr RS, Froehlich LA, Mat&son IX, McLean JA, RosenthalRR, Sbeffer AL U, Spector St, ‘&w&y RG. Standardization of bronc@aiinhal&- &a&~ pmce&tes. J ALLERGY CLIN lklMuNot 1975;56:322. 9. Cropp GJ, Berm&n IL, BouaheyHA Jr, Hyde RW,-Rosenthal RR, Spector SL, Tow&q RG. G&d&nes for bhitd inhalation challenges with ph&aco&& and a&g&c agents. ATS News 198O;spring:11. 10. RamsdaleEH, Morris MM, RobertaRS, HargreavesFE. Branchial responsivenessto me&a&&e in cl&i& brunchhis: relationship to airflow obstictionand cold air reqonaiveness. Thorax 1984,39:912. 11. Findlay SR, L @~ LM. it3$?aIseQts with asthma. Am Rev &apirDis 1580;122353.

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12. Felarca AB, Itkin I. Studies with the quantitative inhalation challenge technique. I. Curve of dose-responseto acetyl-betamethacholinein patients with asthmaof known and unknown origin, hay fever subjects,andnonatopicvolunteers.J ALLERGY 1966;37:223. 13. Itkin IH. Bronchial hyperreactivity to mecholyl and histamine in asthmasubjects. J ALLERGY 1967;40:245. 14. Bewtra A, Townley RG. Bronchoprovocative tests-clinical usefulness and limitations [Editorial]. Arch Intern Med 1984;144:925. 15. RosenthalRR. Inhalation challenge in asthma, Chap. 14. In: Kaplan AP, ed. Allergy. New York: Churchill Livingstone, 1985. 16. Deal EC, McFaddenER, Ingram RH, et al. Airway responsivenessto cold air and hyperpnea in normal subjectsand in those with hay fever and asthma. Am Rev Respir Dis 1980;121:621. 17. Townley RG, Ryo UY, Kilotkin BM, Kang B. Bronchial sensitivity to methacholine in current and former asthmatic and allergic rhinitis patients and control subjects.J ALLERGYCLIN IMMLJNOL 1975;56:429. 18. Curry JJ. Comparative action of acetyl-beta-methacholineand histamineon the respiratory tract in normals, patients with hay fever, and subjects with asthma. J Clin Invest 1947;26:430. 19. Spector SL, Farr RS. A comparisonof methacholineand histamine inhalations in asthmatics. 3 ALLERGYCLIN IMMUNOL 1975;56:308. 20. BascomB, Bleecker ER. Bmnchoconstrictioninducedby distilled water. Am Rev Respir Dis 1986;134:248. 21. Swift DL. Aerosol generation for inhalation challenge. In: Hargreaves FE, Woolcock AJ, eds. Airway responsiveness: measurementand interpretation. Mississauga, Ontario: Astra PharmaceuticalsCanadaLtd, 1985:l. 22. Ryan Cl, Dolovich MB, Roberts RS, et al. Standardizationof inhalation provocation tests: two techniques of aerosol generation and inhalation compared. Am Rev Respir Dis 1981;123:195. 23. Dolovich MB. Technical factors influencing responseto challenge aerosolsin airway responsiveness.HargreaveFE, Woolcock AJ, eds. Airway responsiveness:measurementand interpretation. Mississauga,Ontario: Astra PharmaceuticalLtd, 198.5:9. 24. Woolcock AJ. Expression of results of airway hyperresponsivenessin airway responsiveness.In: Hargreave FE, Woolcock AJ, eds. Airway responsiveness:measurementand interpretation.Mississauga,Ontario: Astra PharmaceuticalsCanada Ltd, 1985:80. 25. Townley RG, Bewtra A. Airway reactivity to methacholinein asthma family members and twins, chap. 9. In: Spector SL, ed. Provocative challenge procedures, vol. 1. Boca Raton, Fla.: CRC Press, 1983:177. 26. DautrebandeL, Philippot E. Ctise d’asthme experimental par aerosolsde carbaminoylcholine chez l’homme traitee par dispersat de phenylaminopmpane. Etude de l’action sur la respiration de ces substancespar la determination du volume respiratoire utile. PresseMed 1941;49:942. 27. Simoqsson BG, Jacobs FM, Nadel JA. Role of autonomic nervou’ssystem and the cough reflex in the increasedresponsivenessof airways in patients with obstructive airway disease. J Clin Invest 1967;46:1812. 28. Hargreave FE, Ryan G, Thomson NC, O’Byme PM, Latimer K, Juniper EF, Dolovich J. Bronchial responsivenessto histamineor methacholmein asthma:measurementandclinical significance. J ALLERGYCLINJMMUNOL 1981;68:347. 29. Kang B, Townley RG, Lee CK, Kolotkin BM. Bronchial reac-

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tivity to histamine before and after sodium cmmoglycate in bronchial asthma. Br Med J 1976;1:867. 30. Hardy CC, Robinson C, Tattersfield AE, Holgate ST. The bmnchoconstrictoreffect of inhaled prostaglandinD, in normal and asthmatic men. N Engl J Med 1984;311:209. 31. Lewis RA, Hardy C, Tattersfield AE. Low and high dose response to prostaglandin F2 in normal subjects. Clin Sci 1982;623:77. 32. Smith AP, Cuthbert MF, Dunlop LS. Effects of inhaled prostaglandins E,, &, and F2 on the airway resistanceof healthy and asthmatic men. Clin Sci Mel Med 1975;48:421. 33. Holmyde MC, Altounyan REC, Cole M, Dickson M, Elliott EV Bronchoconstrictionproduced in man by leukotrienes C and D. Lancet 1981;2:17. 34. Weiss JW, Drazen JM, Cole M, et al. Bmnchoconstrictoreffects of leukotriene C in humans. Science 1982;216:1%. 35. Gtiffen M, Weiss JW, Leitch AF, McFaddenER, Corey EJ, Austen KF, Drazen JM. Effects of leukotrienesD on the airways in asthma. N Engl J Med 1983;308:436. 36. Smith LJ, GreenbergerPA, PattersonR, Krell RD, Bernstein PR. The effect of inhaled leukotriene D4 in humans. Am Rev Respir Dis 1985;131:368. 37. Adelroth E, Morris MM, HargreaveFE, O’Byme PM. Airway responsivenessto leukotrienesC, and Da and to methacholine in patients with asthma and normal controls. N Engl J Med 1986;315:480. 38. Vargaftig BB, Lefort J, Chignard M, Benveniste J. Plateletactivating factor inducesa platelet-dependentbronchoconstriction unrelated to the formation of prostaglandin derivatives. Eur J Pharmacol 1980;65:185. 39. CussFM, Dickson CMS, BarnesPJ. Effects of inhaledplatelet activating factor in pulmonary function and bronchial responsivenessin man. Lancet 1986;2:189. 40. Cushley MJ, Tattersfield AE, Holgate ST. Adenosine antagonism as an alternative mechanismof action of methylxanthines in asthma. Agents Actions [Suppl] 1983;13:109. 41. Mann JS, Cushley JM, Holgate ST. Adenosine-inducedbmnchoconstrictionin asthma. Am Rev Respir Dis 1985;132:1. 42. Yan K, Salome C, Woolcock AJ. Rapid method for measurement of bronchial responsiveness.Thorax 1983;38:760. 43. Woolcock AJ, Cheung W, Salome C. Relationship between bronchial responsivenessto propranolol and histamine. Am Rev Respir Dis 1986;133:A177. 44. De Vries K, GokemeyerJDM, Koeter GH, De Monchy JGR, Van Bork LE, Cauffman HL, Meurs H. Cholinergic and adrenergic mechanismsin bronchial reactivity. In: Morley J, ed. Bronchial hyperreactivity. London: Academic Press, 1982: 107. 45. Townley RG, McGeady S, Bewtra A. The effect of betaadrenergic blockade on bronchial sensitivity to acetyl-betamethacholine in normal and allergic rhinitis subjects. J ALLERGYCLINIMMUNGL1976;57:358. 46. AndersonSD, SchoeffelRE. Standardizationof exercisetraining in the asthmaticpatient: a challengein itself. In: Hargreave FE, Woolcock AJ, eds. Airway responsiveness:measurement and interpretation. Mississauga, Ontario: Astra Pharmaceuticals Ltd, 1985:51. 47. HeatonRW, HendersonAF, Costello JF. Cold air asa bronchial provocation technique. Clin Invest 1984;86(6):810. 48. HodgsonWC, Cotton DJ, WernerRT, Cockcmft DW, Dosman JA. Relationship between bronchial response to respiratory heat exchange and nonspecific airways reactivity in asthmatic patients. Chest 1984;85(4):465. 49. Nair N, Hopp R, Alper B, Bewtra A, Townley R. Correlation of methacholine-inducednonspecific bronchial reactivity and

124

50. 51. 52. 53. 54.

55. 56. 57. 58. 59.

60.

61.

Townley

and Hopp

cold air hyperventilation challenge. Ann Allergy 1986;56(3): 226. Godfrey S. Exercise-inducedasthma-clinical, psychological, and therapeutic implications. J ALLERGYCL~ I~%~JNOL 1975;56:1. SouhradaJF, Kivity S. Exercise testing. In: Spector SL, ed. Provocative challenge procedures:bronchial, oral, nasal, and exercise, vol. II. Boca Raton, Fla.: CRC Press, 1983:75. Cropp GJA. The exercise bronchopmvocation test: standardization of proceduresand evaluation of response.J ALLERGY CLIN IMMUNOL 1979;64:642. Eggleston PA, RosenthalRR, Anderson SD, et al. Guidelines for the methodology of exercise testing of asthmatics. J ALLERGYCLIN IMMUNOL1979;64:642. Anderson SD, SchoeffelRE. Standardizationof exercise training in the asthmaticpatient: a challenge in itself. In: Hargreave FE, Woolcock AJ. eds. Airway responsiveness:measurement and interpretation. Mississauga, Ontario: Astra Pharmaceuticals CanadaLtd, 1985:51. AndersonSD. Issuesin exercise-inducedasthma[Postgraduate course]. J ALLERGYCLIN IMMUNOL1985;76:763. Trautlein JJ. Risk factors in exercise training. J ALLERGYCL~ IMMUNOL1979;64:625. McLaughlin FJ, Dozor AJ. Cold air inhalation challenge in the diagnosis of asthma in children. Pediatrics 1983;72:503. EschenbacherWL, SheppardD. Respiratory heat loss is not the sole stimulus for bronchoconstrictioninduced by isocapnic hyperpnea with dry air. Am Rev Respir Dis 1985;131:894. Tessier P, Corner A, L’Archeveque J, et al. Within- and between-day reproducibility of isocapnic cold air challenges in subjects with asthma. J ALLERGYCL~ I~UNOL 1986; 781379. Malo JL, Cartier A, L’Archeveque J. Cold air inhalation has a cumulative bronchospasticeffect when inhaled in consecutive dosesfor progressively increasing degreesof ventilation. Am Rev Respir Dis 1986;134:990. O’Byrne PM. Airway challenge using isocapnic hyperventilation. In: Hargreave FE, Woolcock AJ, eds. Airway responsiveness: measurementand interpretation. Mississauga, Gntario: Astra PharmaceuticalsLtd, 198560.

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62. Rimmer J, Bryant DH. Effect of hypo- and hyperosmolatity on basophil histamine release. Clin Allergy 1986;16:221. 63. Allegra L, Bianco S. Nonspecific bmnchoreactivity obtained with an ultrasoaic aerosol of distilled water. Em J Respir Dis [Suppl] 1980;61(suppl106):41. 64. Anderson S. Bronchial challenge by ultrasonically nebulircd aerosol. Clin Rev Allergy 1985;3:427. 65. Shaw RJ, Anderson DS, Durham SR, Taylor KM, Schoeffel RE, Green W, Torzillo R, Kay AB. Mediators of hypersensitivity and “fog’‘-induced asthma. Allergy 1985:40:48. 66. Mattoli S, Foresi A, Corbo G, Valente S. Patalano F, Ciappi A. Increasein bronchial responsivenessto methacholine andlate asthmaticresponseafter the inhalation of ultrasonically nebulized distilled water. Chest 1986;90(5):726. 67. Schoeffel RE, Anderson SD, Altounyan REC. Bronchial hyperreactivity in responseto inhalation of ultrasonically nebulized solutions of distilled water and saline. Br Med J 1981;283:1285. 68. Lowell FC, Schiller IW. Measurementof change in vital capacity as a meansof detecting pulmonary reactionsto inhaled aerosolizedallergenic extractsin asthmaticsubjects.J ALLERGY 1948;19:1OO. 69. Bruce CA, RosenthalRR, Lichtenstein LM, et al. Quantitative inhalation bronchial challenge in ragweed hay fever patients: a comparison with ragweed-allergic asthmatics. J ALLERGY CL~ IMMUNOL1975;56:331. 70. Hargreave FE, Fink JN. Report of the AAAI task force on guidelines for clinical investigutian of nonbronchodilatorantiasthmatic drugs: the role of bronchopmvocation[Wvrkshop]. J ALLERGYCm IMMUN~L1986;78(2):517. 71. Hargreave FE, Dolovich J. Bronctriat responsivenessand late asthmaticresponse.In: Kay AB, LichtensteinLM, Austen KF, eds. Asthma: physiology, immunophannacolcgy, and treatment. London: Academic Press, 1984263. 72. Hargreave FE, Frith PA, Dolovich M, et al. Allergen-induced airway responsesand relations&s with nonspecificreactivity. In: HargreaveFE, ed. Airway reactivity: mechanismsand clinical relevance. Mississauga, Ontario: Astra Pharmaceuticals Ltd. 1980:145.

AVAILABLE NOW! The F%XBEDINGS OF THE INTEBNATKTNAL CONGRESS OF ~R~~Y~~~-~I~~YC~~~~ This collmtiun of “state-of-the-art” present&on6 from the XII CongressheId t%t&er 2025, 1985, in Washiagton, D.C., brings together the current advanc& in basic a& al@kd aspectsof allergy and allergic d&eases.It inch&s 528 pagescove& It@, roles of tk different cell types and their products, clinical lx&ems, *and reactiuns to foods and drugs and occupathal agents, collected and revieuwl by Eklitor Cbarla E. Reed, MD S.A.), Joseph& . (U.S.A.) M.D. Robert J. Davies, M.D. (Spain), and Raymond To purchase, call or write: V. Mosby Company, 118 St. Louis, MO 631445,or te R2E.E 14X%325-4177, J (in Missouri caI1 collect at 314-872-8370, Journal Fulgl quired. Make checkspayable to The C. V. Mosby Company. ( fundadrawnonatJ. . $36.50 in &beU.S., $40. itktanaw (surfw included).