BRONCHOPROVOCATION TESTING

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BRONCHOPROVOCATION TESTING Andrew H. Zwiebel, BS

A variety of techniques known as bronchial provocation challenges have been developed to determine the presence and severity of airway hyperactivity. These challenges.can be used by the clinician as a valuable tool for the evaluation of the patient with asthma. Measurement of airway hyperactivity can be used in the clinical practice not only to diagnose the presence of asthma but also to help assess current and future levels of treatment? Airway hyperactivity refers to the characteristic constriction of airways after exposure to stimuli that would not typically affect the airways of a normal patient. Often, inhaled chemicals that cause bronchoconstriction can be used to help clarify an impression of airway hyperactivity. Because the most widely used bronchial provocation challenges measure airway reactivity with the chemical agents methacholine and histamine, the primary focus of this article is based on these challenge procedures.

WHY PERFORM BRONCHOPROVOCATION CHALLENGES? The need for a diagnostic test for the assessment and treatment of asthma arose from the poor predictive value of using only a patient’s past medical history and physical Given that the typical symptoms of cough, shortness of breath, and wheeze are not always observed in the initial presentation of a patient, it would not be unusual to have a physician overlook the diagnosis of asthma? To confront this From ASTHMA, Inc., Seattle, Washington IMMUNOLOGY AND ALLERGY CLINICS OF NORTH AMERICA VOLUME 19 a NLTMBER 1 * FEBRUARY 1999

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Table 1. PURPOSES FOR BRONCHOPROVOCATION CHALLENGES IN CHILDREN Clarify diagnosis of asthma suggested by history Quantify severity of disease Determine and compare utility of drugs for asthma control Determine and compare duration of action drugs Understand asthma mechanisms Epidemiologic studies of airway responsiveness Modified from Shapiro GG, Bierman CW Inhalation bronchoprovocation in children. In Spector SL (ed): Provocative Challenge Procedures: Background and Methodology. New York, Futura Publishing, 1989, pp 395-416; with permission.

issue, the use of bronchoprovocation testing to aid in the diagnosis of asthma has now become a widely accepted practice. In the patient with asthma, airway hyperactivity is often characterized by the clinical symptoms of cough, chest tightness, and wheezing. If baseline pulmonary function testing is considered normal in the face of such a patient history, bronchoprovocation challenges should be considered to help clarify whether symptoms suggestive of asthma are indeed caused by this condition? Some of the primary reasons for performing a challenge technique on certain patients are listed in Table 1. PHARMACOLOGIC CHALLENGES

Bronchial provocation challenges can be divided into two broad categories: pharmacologic (e.g., methacholine and histamine) and nonpharmalogic (e.g., hypertonic saline, exercise, and cold air hyperventilation) challenges. A significant portion of data from the last 20 years demonstrate the usefulness of pharamacologic bronchoprovocation testing.l3* 28 Over this period, methacholine and histamine emerged as powerful agents for eliciting bronchial hyperresponsiveness because of their similar dose-response curves and capability to differentiate patients with reactive and nonreactive airways.15,25, 47 Methacholine, a cholinergic agent, works by contracting smooth muscle in hyperreactive airways.18, 23, Histamine is similar in its mechanism of action to methacholine; however, a slight disparity in response from some individuals to both agents suggests that there must be other mechanisms involved as ~e11.41.43~45 Challenges using histamine and methacholine have been well standardized and are easily applied to children and adults.= Two popular bronchoprovocation challenge techniques have evolved to administer methacholine and histamine. The first technique involves tidal volume breathing, and the second relies on a dosimeter device. Both methods involve inhalation of increasing concentration of stimulatory agent, with each period of inhalation followed by pulmonary function testing. The tidal volume method involves the patient’s cooperation in breathing continuously generated aerosol delivered directly into a face

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mask or mouth piece. Commonly, this technique is used with small children because little patient cooperation is required. The tidal volume breathing method uses a Wright nebulizer, which is connected to a HansRudolph valve that contains a mouthpiece, inspiratory air valve, and expiratory air valve. The purpose of the Hans-Rudolph device is to contain the methacholine or histamine aerosol. Nebulizer output is initiated in a direct manner to the patient’s mouthpiece with no impediment from the valves. The patient has a nose clip in place and breathes with tidal volume through the entire challenge. The patient inhales saline followed by nine standard increasing doses of methacholine or histamine. Careful regulation of the agent delivery and nebulizer output must be recorded if accurate results are to be obtained. An increase in output or in the duration of the nebulization may deposit more aerosol in the lung and decrease the patient’s forced expiratory volume at 1 second (FEVJ at a more significant rate. If the nebulizer output is held constant between 0.13 to 0.16 mL per minute and the duration of the nebulization is maintained at 2 minutes, the total dose delivered to the lungs during this period of tidal volume breathing should be between 0.26 and 0.32 mL. Challenges using the dosimeter method are slightly different. The dosimeter method involves intermittent generation of aerosol by a nebulizer connected to a Rosenthal-French dosimeter, a solenoid valve device that allows aerosol to be generated for a specific time interval. The patient initiates nebulization by pressing a button or spontaneously initiating the device with each inspiration. With the dosimeter method, compressed air at 20 psi is connected through a tube to the input value of the dosimeter. The patient inhales from the mouthpiece containing methacholine or histamine solution. Inspiration begins from functional residual capacity, and each inhalation of methacholine or histamine is delivered for approximately 0.6 seconds? For accurate dosimeter results, output per inhalation and the number of inhalations must be standardized. Patients must consistently start at the same lung volume and attempt to inhale at the same rate with each inhalation. When these guidelines have been followed, the tidal volume and intermittent dosimeter methods result in similar levels of sensitivity and show good re~roducibility.~~ The standard dosage schedule often used for methacholine or histamine challenge involves increasing concentrations two fold (Table 2). In addition, a truncated methacholine protocol (Table 3) has been useful clinically for adults and children because it decreases the number of concentrations from nine to five and reduces the overall time for assessment.2I Truncated challenges have become particularly useful in epidemiologic studies when time and feasibility are essential to consistent outcomes. An abbreviated histamine protocol (Table 4), using fewer doses and a shorter time interval for measuring FEVI, also compares favorably with the truncated methacholine According to the Bronchoprovocation Committee of the American Academy of Allergy and Immunology? after an initial baseline establish-

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Table 2. BREATH UNITS FOR METHACHOLINE AND HISTAMINE CHALLENGE ON A rng/rnL BASIS' Methacholine Concentrations

(mglmL)

Cumulative No. Breaths

Unitsl Breath

Unital5 Breaths

Cumulative Units/ 5 Breathst

5

0.375 0.750 1.55 3.10 6.25 12.50 25.00 50.00 125.00

0.375 1.125 2.68 5.78 12.0 24.5 49.5 99.5 225.0

0.075 0.15 0.31 0.62 1.25 2.50 5.00 10.00 25.00

15 20 25 30 35 40 45

0.075 0.15 0.31 0.62 1.25 2.50 5.00 10.00 25.00

Histamine Base Concentrations (mglmL)

Cumulative No. Breaths

Unital Breath

Units6 Breaths

Cumulative Units/ 5 Breathst

0.03 0.06 0.12 0.25 0.50 1.00 2.50 5.00 10.00

5 10 15 20 25 30 35 40 45

0.03 0.06 0.12 0.25 0.50 1.oo 2.50 5.00 10.00

0.15 0.30 0.60 1.25 2.50 5.00 12.00 25.00 50.00

0.15 0.45 1.05 2.30 4.80 9.80 22.30 47.30 97.30

10

*One breath unit = 1 inhalation of mg/mL histamine base tIf final FEV, test is performed at other than a 5-breath interval (e.g., 23 breaths), the cumulative units are calculated by adding 3 X 0.25-0.75 to 2.30-3.05 Modijiedfrom Chai H, Fan RS, Roehlich LA,et al: Standardization of bronchial inhalation challenge procedure. J Allergy Clin Immunol56:32>327,1975; with permission.

Table 3. TRUNCATED METHACHOLINE PROTOCOL Methacholine Concentrations

(mglmL) 0.025 0.25 2.50 10.00 25.00

Cumulative Breaths

Unitd5 Breaths

Cumulative Unitd5 Breaths

5

0.13 1.25 12.50 50.00 125.00

0.13 1.38 13.88 63.88 188.88

10

15 20 25

From Shapiro GG, Bierman CW Inhalation bronchoprovwation in children. In Spector SL (ed): Provocative Challenge Procedures: Background and Methodology. New York, Futura Publishing, 1989, pp 395416; with permission. t

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Table 4. DOSING AND CUMULATIVE BREATH UNITS FOR THE STANDARD HISTAMINE PROTOCOL AND THE ABBREVIATED HISTAMINE PROTOCOL Abbreviated Histamine Protocol

Standard Histamine Protocol

Concentration Cumulative Breath Concentration Cumulative Breath (mghnL) Inhalations Units (mg/mL) (mg/mL) Inhalations Units (mg/mL)

0.03 0.06 0.12 0.25 0.5 1.0 2.5 5.0 10.0

5 5 5 5 5 5

5 5 5

0.15 0.45 1.05 2.3 4.8 9.8 22.3 47.3 97.3

0.06 0.16 2.12 2.12 10.6 10.6

1 3 1 4 3 4

0.16 0.64 2.76 11.24 43.04 85.44

From Schmidt LE, Thome Fs, Watt JL, et a1 Is an abbreviated bronchial challenge with histamine valid? Chest 101:141-145,1992; with permission.

ment, FEVl is measured at 3 minutes after each concentration until all nine concentrations have been delivered. A decrease in FEV, of 20% or more is generally accepted as a discriminating end point to separate the normal and hyperresponsive groups during a bronchoprovocation challenge procedure. Failure to obtain a decline in FEV, of 20% or more makes the diagnosis of hyperactivity unlikely without further evaluation by the physician5 As a safety parameter for patients, it is usually recommended that the FEV, be 70 percent or more of the predicted normal to perform either challenge method. If a patient’s FEV, drops considerably or the patient becomes uncomfortable during the challenge procedure, inhalation of a bronchodilator ( p p agonist) should rapidly return pulmonary function to normal ranges. In general, the standardization of histamine and methacholine protocols, the short duration of bronchoconstriction, the lack of late-phase response, and the ability to control the dose of bronchoconstriction stimuli all add to the popularity and safety of these two pharmacologic challenge^.^ NONPHARMACOLOGIC CHALLENGES

Bronchoprovocation challenges involving nonpharmacologic methods include inhalation of cold air, ultrasonically nebulized distilled water, hypertonic saline, specific antigens, and exercise challenges. The majority of these challenge procedures elicit bronchial hyperresponsiveness by altering internal temperature and fluid homeostasis in the airways?, 14,37 In contrast to pharmacologic challenges, which directly affect smooth muscle, nonpharmacologic challenges,affect the cellular epithelium and may stimulate nervous and inflammatory processes that can lead to bronchocon~triction.~ The following section offers brief descriptions of the various nonpharmacologic challenge procedures.

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Methods

A common method for bronchoprovocation with nonpharmacologic agents employs cold air hyperventilation. The set up required for hyperventilation of cold air uses a source of compressed air delivered to a heat exchanger that can bring the temperature to -15°C or -10°C. The patient is coached to hyperventilate at about 25 times FEV,, which compares with that of moderate to strenuous activity. Hypocapnia is avoided by the use of a compressed air mixture of 5% C02, 21% 0, 74% N2, or by adding a constant stream of 5% C 0 2 at 21 minutes into the inspiratory line?" 46 Hyperventilation occurs for a 4-minute period, with spirometry performed every few minutes after the challenge for 15 to 20 minutes. The best FEV, of several trials is used. A drop in FEV, of 10% or more has been considered a positive test value.= Maximal faIls in FEV, typically occur at 3 to 4 minutes.@ The ultrasonically nebulized distilled water (UNDW) challenge provides another bronchoprovocation method for analysis in the adult and pediatric patient. UNDW challenges are favorable in the clinical setting because they require minimal equipment and patient cooperation.14A dose-response curve can be generated because patients begin by breathing a low dose of water and proceed to gradually higher doses. The rate of UNDW should be held constant for accuracy in the test procedure; the delivered dose should be checked by weighing the canister and tubing before and after each interval of the challenge. Hopp et all9have used a system generating approximately 8 mL of water per minute. Subjects inhale nebulized water for 15 seconds, 30 seconds, and then 1,2, and 4 minutes. For each interval, the subject tidal breathes with nose clip in place through a mouthpiece connected to a Hans-Rudolph valve, which is attached to the nebulizer. Three minutes after each step, patients perform forced expiratory tests and record the highest FEV,. The test is stopped when the patient's FEV, drops to 20% or more compared with ba~eline.~ Bronchoconstriction typically persists for 10 minutes after challenges, and late reactions are not uncommon." Over the past 10 years, bronchial provocation challenges using hypertonic saline of various concentrations have emerged as powerful tools for the diagnosis of asthma. In one study of adults and children, ultrasonically nebulized hypertonic saline was shown to have a sensitivity of 85% in adults and 72% in children with a specificity of 100% in detecting a ~ t h m aChallenges .~ with hypertonic saline require little patient cooperation and have been shown to be safe in laboratory studies in adult and pediatric patients with asthma.3,4o 2To proceed with hypertonic saline challenges, a baseline FEV, of 65% or more of predicted normal must be established. During the testing, the nebulizer output is kept constant, and the dose of saline (4.5% NaC1) is increased successively by doubling the inhalation time stakting with 0.5 and then 1,2,4, and 8 minutes. The patient's FEV, is typically measured

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in duplicate before the initial challenge and at 60-second intervals after each challenge step. A positive challenge is ended when the patient's FEV, falls by 20% or more. Clinical experience with the test suggests that the total volume to be administered can be as little as 15 mL, with only a minor decrease in sensitivity.* There is currently no accepted standardized hypertonic saline protocol. In contrast to UNDW challenges, neither increases in methacholine reactivity nor late response have been observed after hypertonic saline challenge^.^^ Although less commonly used, bronchial provocation using antigen can be a useful means to measure bronchial hyperactivity. Antigen challenges have one advantage over pharmacologic challenges in their ability to document response to environmental antigens; however, the clinician should use caution in performing such challenges because of the potentially dangerous late-phase response that may occur. Antigen provocation can be performed with dust mite, mold, weed, grass, animal, pollen, and other environmental antigens. Initial prick testing followed by serial dilutions of intradermal skin testing is recommended to establish the concentration for challenge; the weakest concentration producing a wheal 5 mm greater than control on intradermal test is the typical starting p ~ i n tDuring .~ the procedure, five inhalations of saline are delivered, and spirometry is performed with the best of three expiratory efforts being taken as a baseline. At 10-minute intervals, five inhalations of increasing concentrations of antigen are delivered unless there is a drop of 15% or more in FEV,. For a decrease of 15%, the same dose is repeated, and fewer inhalations may be given at the physician's discretion? If a decrease of 20% or more in FEV, occurs or the entire sequence of dilutions is administered, the challenge is complete. Patients undergoing antigen challenge require constant observation, especially in view of possible late-phase reactions that may occur. It has been recommended that the patient be studied at hourly intervals, at least with a peak flow meter device after testing has been completed. Finally, given the rising prevalence of exercise-induced asthma, exercise provocation testing has emerged as yet another effective way to quantify bronchial hyperresponsiveness. Exercise challenge testing is believed to provoke bronchial constriction in a similar manner to hyperventilation and ultrasonically nebulized solutions.1, 22 Typically, a standard treadmill is used in the exercise provocation test. Using an initial rate at approximately 3 miles per hour and a slope of lo%, both variables can then be manipulated until the patient's pulse rate reaches 90% of his or her predicted age maximum within 4 minutes.", 38 FEV, measurements are taken before and after the completion of the challenge, with the measurements taken after at intervals of 5, 10, 15, 20, 25, and 30 minutes. Again, a 20% or more drop in FEV, is considered a positive L test result. Together, these various nonpharmacologic challenges offer the clinician a wide variety of diagnostic tools for the evaluation of the patient with asthma; however, a positive challenge response to any procedure

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must not be overinterpreted. Ultimately, a positive challenge may only be consistent with, but not entirely conclusive of, airway hyperreactivity. FUTUREAVENUES

Future research into bronchial provocation testing is on its way. One of the primary focuses has been on pediatric populations, considering there are no acceptable lung function tests to measure hyperactivity in children under age 5. The use of transcutaneous oxygen tension (PtCOJ has been employed as a particularly useful method for measuring bronchial reactivity after bronchial provocation. Stephen et alQ showed that a challenge method using PtC02 was repeatable for clinical use and compared well with the repeated assessment of FEV,. On the pharmacologic side, new agents are being tested and retested to measure bronchial hyperactivity. Since Cushley et all2 reported that adenosine was capable of provoking dose-related bronchoconstriction when administered by inhalation to the patient with asthma but not to normal volunteers, there has been a resurgence of interest in the role of this naturally occurring agent. Bronchial challenges with adenosine have shown that smooth muscle of individuals'with asthma is more responsive than that of individuals who do not have asthma.5,6, 29 Given these results, the airway response to adenosine may add to our knowledge of asthma pathophysiology and add yet another diagnostic tool for the clinician. COMPARISON OF CHALLENGES

Methacholine and histamine challenge testing of bronchial hyperresponsiveness will likely continue to receive significant attention in the clinical and research setting. The safety and clinical utility of methacholine and histamine challenges have been documented,36with the determination that both are considered more sensitive than a history or physical examination in the diagnosis of asthma.3oShapiro et a135evaluated 166 children for a variety of respiratory symptoms. On the basis of a positive pharmacologic bronchoprovocation challenge, 65% were tentatively diagnosed as having asthma. Follow-up using a favorable response to bronchodilator therapy as an outcome later confirmed the initial diagnosis in patients. In 35% of the patients that had a negative bronchoprovocation challenge, the lack of any clinical evidence of asthma symptoms over a 1-year period virtually excluded asthma as a possible explanation of the patient's symptoms. Ultimately, Shapiro et a1 determined that the results from bronchoprovocation bhallenges accurately predicted patients who would prove to have asthma based on long-term follow-up. Bronchoprovocation challenges using methacholine and histamine have been beneficial in the clinical setting for a variety of reasons. First,

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these chemicals can provide a useful method for studying dose-response relationships for the reduction of airway reactivity. For example, the degree to which a patient's airway reactivity is altered can be used as a means to compare the safety and effectiveness of new drug therapies. Second, bronchoprovocation challenges have also been useful in longterm epidemiologic studies of large groups of healthy patients, as well as patients with asthma. The choice of using either methacholine or histamine bronchoprovocation challenges ultimately rests on the clinician's best judgment. Bronchial responsiveness to histamine and methacholine correlates fairly well; however, it must be noted that methacholine and histamine have different pharmacologic properties that may be clinically or scientifically significant. Side effects, half-lives, and cumulative effects differ between the two agents?, 2o Typically, dose-related side effects to either agent are observed with increasing frequency at concentrations of 8 mg/mL or more.8Flushing and headache have been reported more frequently with histamine; however, symptoms of cough and chest tightness occur with similar frequency in both agents.%In making the decision to use either agent, the unique characteristics of both chemicals should be considered, and the results from these challenges should warrant cautious interpretation as an aid to diagnosis. Comparisons of cold air, UNDW, and methacholine inhalation tests in children with and without asthma confirm the increased sensitivity of methacholine: 95% versus 57% for cold air challenge and 71% for UNDW.16 Comparing UNDW challenge with methacholine challenge in children, Gables-Sebaldt et all6 found 100% specificity for the former compared with the latter, but only 71% sensitivity with UNDW compared with 95% sensitivity with methacholine. In regard to the additional nonpharmacologic methods of testing, cold air challenges have been considered advantageous, although a few disadvantages to this technique also exist. Most cold air challenges use a single period of stimulation after which pulmonary function is measured; however, this technique does not allow the clinician to establish a dose-response curve for interpretation. In addition, this method involves considerable patient cooperation and time. When comparing the results of exercise challenge with the pharmacologic challenges, it has been observed that exercise testing is less sensitive and a poorer screening test for bronchial hyperresponsiveness." For example, Chatham et allo noted that 6 of 15 patients with asthma did not respond to maximal exercise challenge with a 10% fall in FEVl, whereas 1 out of 10 normal subjects did. With the use of hypertonic saline, the equipment and experience required to perform this challenge are very similar to methacholine and histamine challenges. Hypertonic saline has the added advantage over exercise testing in that it produces a dose-response curve in addition to identifying patients responsive to exercise and hyperventilation? From these comparisons, methacholine and histamine seem to be the most sensitive indicators of bronchial hyperresponsiveness. It should

be emphasized that a positive pharmacologic challenge must not be overinterpreted by the clinician. Because the future development of symptoms for any particular individual is unpredictable, an initial positive methacholine challenge cannot always be considered a definitive test. The challenge that one chooses largely depends on one’s goal, although for most purposes, chemical challenge with methacholine or histamine are most valuable. These agents promise excellent sensitivity, reasonable specificity, are safe and easy to administer, have a short duration of action, and bronchoprovocation challenges have been well standardized in the clinical and research setting. Ultimately, the likelihood of asthma in patients with a positive bronchoprovocation challenge is influenced by the history, physical examination, and the clinician’s judgment.l7,27

References 1. Anderson SD: Is there a unifying hypothesis for exercise-induced asthma? J Allergy Clin Immunol 73:660-665, 1984 2. Anderson SD Bronchial challenge by ultrasonically nebulized aerosols. Clin Rev Allergy 3427-439, 1985 3. Anderson SD, Smith CM, Rodwell LT, et a 1 The use of nonisotonic aerosols for evaluating bronchial hyperresponsiveness. In Spector SL (ed): Provocative Testing in Clinical Practice. New York, Marcel Dekker, 1994, pp 249-278 4. Belcher N, Murdock R, Dalton N, et a1 A comparison.of mediators and catecholamine release between exercise and hypertonic saline-induced asthma. American Review of Respiratory Disease 1371026-1032,1988 5. Bierman CW, Pearlman DS, Shapiro GG (eds): Jnhalation bronchoprovocation. In Allergy, Asthma, and Immunology from Infancy to Adulthood, ed 3. Philadelphia, WB Saunders 1996, pp 173-186 6. Bjorck T, Gustafsson LE, Dahlen SE: Isolated bronchi from asthmatics are hyperresponsive to adenosine, which apparently acts indirectly by liberation of leukotrienes and histamine. American Review of Respiratory Disease 1451087-1091, 1992 7. Bumey PGJ, Chin S, Britton J, et al: What symptoms predict the bronchial response to histamine? Evaluation in a community survey of the bronchial symptoms questionnaire 1984 of the International Union Against Tuberculosis and Lung Disease. Eur J Resp. Disease 18:165-173, 1989 8. Cartier A, Malo J, Begin P, et a 1 Time course of the bronchoconstriction induced by inhaled histamine and methacholine. J Appl Physiol54821-823, 1983 9. Chai H, Farr RS, Roehlich LA, et a1 Standardization of bronchial inhalation challenge procedures. J Allergy Clin Immunol56323-327, 1975 10. Chatham M, Bleecker ER, Smith PL, et a 1 A comparison of histamine, methacholine, and exercise airway reactivity in normal and asthmatic subjects. American Review of Respiratory Disease 126235-240, 1982 11. Chin-Chi L, Jen-Liang W, et al: A bronchial response comparison of exercise and methacholine in asthmatic subjects. J Asthma 28:31-40, 1991 12. Cushley MJ, Tattersfiled AE, Holgate ST: Inhaled adenosine and guanosine on airway resistance in normal and asthmatic subjects. Br J Clin Pharmacol 15161-165, 1983 13. DeVries k, Booij-Nord H, Goei JT, et al: Hyperreactivity of the bronchial tree to drugs and chemical and physical agents. In Orie NGM, Sluiter HJ (eds): Bronchitis: Second International Symposium. Assen, Royal Vangorium, 1964, pp 167-180 14. Eggleston P, Kagey-Sobotka A, Schleimer R, et al: Interaction between hyperosmolar

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and IgE-mediated histamine release from mast cells. American Review of Respiratory Disease 130:86-91,1984 15. Fish JE, Rosenthal RR, Batra G, et al: Airway responses to methacholine in allergic and nonallergic subjects. American Review of Respiratory Disease 113:579-586, 1976 16. Gables-Sebaldt M, McLaughlin FJ, Levison H: Comparison of cold air ultrasonic mist and methacholine inhalation as tests of bronchial reactivity in normal and asthmatic children. J Pediatr 107526-530,1985 17. Gilbert R, Auchincloss JH: Post-test probability of asthma following methacholine challenge. Chest 97562-565,1990 18. Hargreave FE, Ryan G, Thomson NC, et a1 Bronchial responsiveness to histamine or methacholine in asthma: Measurement and clinical significance. J Allergy Clin Immuno1 68347-355, 1981 19. Hopp RJ, Christy J, Bewtra AK, et a 1 Incorporation and analysis of ultrasonically nebulized distilled water challenges in an epidemiological study of asthma and bronchial reactivity. Ann Allergy 60129-133, 1988 20. Juniper EF, Frith PA, Dunnett C, et al: Reproducibility and comparison of responses to inhaled histamine and methacholine. Thorax 33:705-710, 1978 21. LaBraico JM, Reed CE, Rosenthal RR, et a1 Multicenter evaluation of airways hyperreactivity using a standardized methacholine challenge [abstract]. J Allergy Clin Immuno1 73224, 1984 22. Lee TH, Nagakura T, Papageorgiou N, et al: Mediators in exercise-induced asthma. J Allergy Clin Immunol 73:634-639, 1984 23. Makker HK, Holgate ST: Relation of the hypertonic saline responsiveness of the airways to exercise-induced asthma symptom severity and to histamine or methacholine reactivity. Thorax 48142-147, 1993 24. Mattioli S, Foresi A, Corbo GM, et aE Jncrease in bronchial responsiveness to methachol i e and late response after the inhalation of ultrasonically nebulized distilled water. Chest 90:726-732,1986 25. Muittari A: The value of the methacholine test as a diagnostic method on bronchospastic disorders. Ann Intern Med 57197-203, 1968 26. Onellette JJ, Reed CE: Increase response of asthmatic subjects to methacholine after influenza vaccine. J Allergy 3555G563, 1965 27. Palmerio EM, Hopp RJ, Biven RE, et al: Probability of asthma based on methacholine challenge. Chest 101:630433, 1992 28. Parker CD, Bildo RE, Reed CE: Methacholine aerosol as test for bronchial asthma. Arch Intern Med 115:452-458,1965 29. Phillips GD, Holgate ST: The response of plasma histamine to bronchoprovocation with methacholine, adenosine 5'-monophosphate and allergen in atopic nonasthmatic subjects. American Review of Respiratory Disease 141:9-13, 1990 30. Pratter MR, Hingston DM, Irwin Rs: Diagnosis of bronchial asthma by clinical evaluation.Chest 84:42-47, 1983 31. Pratter MR, Irwin Rs: The clinical value of pharmacological bronchoprovocation challenge. Chest 85260-265,1984 32. Reisman J, Mappa L, de Benedicits F, et al: Cold air challenge in children with asthma. Pediatr Pulmonol3251-254, 1987 33. Ryan G, Dolovich MB, Roberts RS, et al: Standardization of inhalation provocation tests: Two techniques of aerosol generation and inhalation compared. American Review of Respiratory Disease 123195-199,1981 34. Schmidt LE, Thorne PS, Watt JL, et a1 Is an abbreviated bronchial challenge with histamine valid? Chest 101:141-145, 1992 35. Shapiro GG, Furukawa CT, Pierson WE, et al: Methacholine bronchial challenge in children. J Allergy Clin Immunol69:365, 1982 36. Shapiro GG, Simon RA: Bronchoprovocation Committee Report. J Allergy Clin Immuno1 89775778,1992 c 37. Silber G, Proud D, Warner J, et al: In vivo release of inflammatory mediators by hyperosmolar solutions. American Review of Respiratory Disease 127:602412, 1988 38. Silverman M, Anderson SD: Standardization of exercise tests in asthmatic children. Arch Dis Child 478824585, 1972

39. Smith CM, Anderson SD, Black JL: Methacholine responsiveness increases after ultrasonically nebulized water but not after ultrasonically nebulized hypertonic saline in patients with asthma. J Allergy Clin Ipununol7985-92,1987 40. Smith RE, Anderson SD, Altounyan RE: Bronchial hyperreactivity in response to inhalation of ultrasonically nebulized solutions of distilled water and saline. BMJ 28312851287, 1981 41. Spector SL, Farr RS: A comparison of methacholine and histamine inhalations in asthmatics. J Allergy Clin Immunol56308, 1975 42. Stephen BP, Nicola MW, Michael S Repeatability of methacholine challenge in asthmatic children measured by change in transcutaneous oxygen tension. Thorax 47804808, 1992 43. Sterk PJ, Be1 E H Fundamental aspects of airway hyperresponsiveness. In Kerrebijin KF, Sluter HJ (eds): Nocturnal Dyspena, Mammation and Reactivity. Rijswijk, The Netherlands, Astra 1986, pp 73-91 44. Tal A, Pasterkamp H, Serrette C, et al: Response to cold air hyperventilation in normal and in asthmatic children. J Pediatr 104:51&521,1984 45. Tammeling GJ, DeVries K, et al: The circadian pattern of the bronchial reactivity to histamine in healthy subjects and in patients with obstructive lung disease. In McGovem, Smolensky M, Reinberg A (eds): Chronobiology in Allergy and Immunology. Springfield, IL, Thomas, 1976 46. Townley RJ, Hopp RJ: Inhalation methods for the study of airway responsiveness. J Allergy Clin Immunol80111-124,1987 47. Townley RG, Ryo UK, Kolotkin BM, et a1 Bronchial sensitivity to methacholine in current and former asthmatics and allergic rhinitis patients and control subjects. J Allergy Clin Immunol56:429-442, 1975

Address reprint requests to Andrew H. Zwiebel, BS ASTHMA, Inc. 4500 Sand Point Way NE, Suite 222 Seattle, WA 98105