INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

House-dust mite is said to be the most important allergen in the causation of asthma (1). In some series as many as 85% of asthmatics have had a positive prick test to house-dust mite (2, 3). The importance of house-dust mite, however, is difficult to ascertain from the history, and patients are not usually aware of this relationship. Epidemiological studies support the causative relationship between house-dust mite allergy and asthma (4) but the direct evidence comes mainly from bronchial challenge where incremental doses are given until an immediate response is seen. The relevance of these experimental studies is often questioned as the relatively large doses required to produce immediate responses do not mimic natural exposure (5). The consequence of low-dose subclinical exposure is more relevant to the pathogenesis of chronic allergic asthma. It is likely that significant exposure to mite allergen occurs at night in the bedroom because of the time spent in bed and the proximity to the reservoir (mattress and bedding) during sleep. We report here giving very small quantities of dust-mite allergen extract (equivalent to estimated daily allergen exposure) through a nebulizer, for up to 4 wk and studying its effects on bronchial hyperreactivity and other features of asthma.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Adults (age: 18 to 55 yr) with a history of physician-diagnosed asthma (for at least 1 yr) were screened for eligibility. Each was clinically stable and taking only as-required (pro re nata, prn) inhaled albuterol at the time of entry without a history of upper respiratory infection during the last 3 mo. Other inclusion criteria were airway hyperresponsiveness on methacholine inhalation test (provocative concentration required to produce a 20% reduction in FEV₁ [PC₂₀] < 25 mg/ml) and allergen sensitivity to dust-mite allergens; Dermatophagoides pteronyssinus (Der p) and/or Dermatophagoides farinae (Der f ) as determined by skin prick test (> 5 mm average diameter above saline control) and presence of serum-specific IgE to these dust mites. The same extracts (with the addition of 50% glycerin) were used for prick testing as for bronchial inhalation. Those with more severe asthma (FEV₁ < 70% predicted when off inhaled ₂ agonist for > 4 h) were excluded. Other exclusion criteria were as follows: (1) need for regular bronchodilator; (2) current anti-inflammatory therapy for asthma; (3) inhaled steroids within the last 6 wk, oral steroids within the last 6 mo, or current nasal steroid use; (4) significant sensitivity and exposure to indoor allergens other than house-dust mite; (5) allergen immunotherapy within 30 d; (6) evidence of chronic lung disease including smoking-related lung disease, cystic fibrosis, and bronchiectasis or other chronic illnesses that may impair normal lung function; (7) pregnancy. The study was approved by the local institutional review board and written informed consent was obtained from all subjects.

Baseline Assessments

At enrollment, medical history including asthma history was taken and a brief physical examination was performed. Spirometery and methacholine inhalation test were performed to assess baseline pulmonary function and bronchial responsiveness. Skin prick tests were performed to common aeroallergens including Der p, Der f, cat, dog, cockroach, short ragweed, plantain, grass mix, Alternaria, and Cladosporium. Serum total IgE (IMx Immunoenzymetric assay; Abbott Laboratories, Abbott Park, IL) and specific IgE to Der p and Der f antigens were measured (Pharmacia CAP system; Pharmacia, Columbus, OH). An asthma diary was completed to indicate symptoms; medication used and thrice-daily peak expiratory flow rate (PEFR) were recorded starting 1 wk before the allergen dosing.

Procedure

Once eligibility was established and baseline measurements obtained, the subjects were asked to visit the Asthma and Allergy Center three times a week (usually Monday, Wednesday, and Friday) for 4 wk for inhalation of a small dose of a standardized dust-mite extract. Spirometery was done before and 10 min after the inhalation on each occasion. Subjects kept home diaries for symptoms, medication usage, and three daily home peak flow measurements. PEFR was also recorded during the day or night if the subject developed a cough, wheezing, or shortness of breath and before use of the prn bronchodilator. Symptoms were scored on an ordinal scale from 0 to 3 where 0 = no symptoms and 3 = spells longer than 2 h or those causing the subject to stay home or see a doctor. Subjects were instructed to take two puffs (200 µg) of inhaled albuterol as needed, for their symptoms. Medication scores equaled the number of times inhaled bronchodilator was used during the previous 24 h. At the end of each week, methacholine inhalation test was performed before the dust-mite inhalation dose of that day was given. On these visits inhaled albuterol (200 µg) was used to reverse bronchoconstriction before administering dust-mite extract for the day. A blood sample was obtained once weekly to measure specific IgE to dust-mite allergen. Once the inhalations were stopped, the subjects were followed weekly with spirometry and methacholine inhalation test until PC₂₀ returned to baseline.

The study was terminated early for a particular patient if any of the following occurred: (1) a 10-fold or more decrease from baseline in the PC₂₀ FEV₁ on two consecutive weeks; (2) a persistent increase in medication requirement (regular [three times a day or more] bronchodilator use for more than 2 d); (3) a consistent 15% or more drop in baseline home PEFR for more than 2 d; (4) medically documented respiratory tract infection.

The primary outcome measure was nonspecific bronchial hyperreactivity. This was determined by the FEV₁ response to inhaled methacholine in incremental doses (6). A twofold or more change in PC₂₀ was considered significant. Secondary outcome measures included spirometry, PEFR, symptoms, medication requirement, and serological measurements.

Inhalation Procedures

All inhalation procedures were performed using a DeVilbiss No. 646 nebulizer (DeVilbiss Co., Somerset, PA), with its auxiliary port open, attached to a French-Rosenthal (7) dosimeter that is breath-activated, using a compressed air source (20 psi). The volume of solution in the nebulizer was 1 ml of each dilution. Aerosols were delivered for 0.6 s at the start of slow inspiration from functional residual capacity to inspiratory capacity. Nebulizer output was estimated to be ~ 20 µl per breath.

Dust-mite Extract Inhalations

Dust-mite allergen exposure was estimated from the concentration of Group 1 allergen present in the air. During normal activity conditions, mean airborne levels of dust-mite allergen (as measured by Group 1 allergens of Der p and Der f ) varied widely in various studies (Table 1) but a conservative estimate was approximately 100 pg/m³ (1 m³ = 1,000 L). Amount of air inhaled: 600 min × 7 L/min = 4,200 (assuming time spent indoors is 10 h and minute volume 7 L). Therefore, the estimated amount of Group 1 allergen inhaled per day: 100/1,000 × 4,200 = 420 pg.

Lyophilized and standardized dust-mite extract was obtained (Greers Lab., Greers Laboratories Inc., Lenoir, NC). Der p and f extracts were diluted in phosphate-buffered saline (PBS) diluent to 10,000 AU/ml. To calculate the amount of extract containing 420 pg Group 1 allergen, Der p1 and Der f1 were measured repeatedly in these extracts and means were obtained. Der p and Der f extracts were mixed so that the two extracts contributed equally to the Group 1 allergen content (10,000 ng/ml) of the extract mixture. Aliquots of this mixture were kept at 4° C. On the day of inhalation the extract mixture was further diluted 1,000-fold in PBS to a final Group 1 allergen concentration of 10 ng/ml (0.85 AU/ml in this extract). One ml of this extract was placed in the nebulizer. Twenty microliters (= 0.2 ng Group 1 allergen) of this solution was inhaled with each breath. Amount of allergen equivalent to daily exposure (0.4 ng Group 1 allergen = 0.34 AU) was provided with two breaths. As the subjects attended three times weekly, they inhaled 4 to 6 breaths depending on the time lapsed since their last visit. Subjects were allowed home after 30 min unless there was a significant reduction in postinhalation spirometery or if they were symptomatic, in which case an inhaled bronchodilator was given. Dust-mite inhalation continued for 4 wk unless the criteria from discontinuation were met. Once the endpoint of > 2 fold change in PC₂₀ was reached, dust-mite extract was replaced by PBS inhalations (unknown to the subjects) three times a week for a further week (Week 5). Observations including spirometery, diary card, and methacholine PC₂₀ were continued as during the allergen inhalation period. After that, weekly methacholine challenges continued until the PC₂₀ had returned to baseline. In those with less than 2-fold change in PC₂₀ no further observations were made.

Statistical Analysis

To compare values before and after exposure, a paired t test was used for analyzing PC₂₀ (after log conversion), and pulmonary function test data. A Wilcoxon matched-pairs signed-ranks test was used to analyze symptoms and medication scores. Repeated measure analysis of variance was used to evaluate changes over time in PC₂₀ and pulmonary function test data.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Nine subjects (age range: 19 to 41 yr; 3 M, 6 F) were included in the study (Table 2). Despite a history of mild asthma and near normal FEV₁, there was more than 2 log variation in baseline PC₂₀. Subjects were highly sensitive to house-dust mite with an average wheal diameter of 11 mm on skin prick test. Total IgE was high in all but one subject (W.S.). Most had other evidence of atopy including multiple allergen sensitivity, other allergic diseases such as rhinitis, and a positive family history of one or more of these disorders. Only one subject (A.B.) smoked.

There was a significant (> 2-fold) drop in the PC₂₀ in 7 of 9 subjects with repeated mite-extract inhalation (Table 3). In 2 of 7 subjects (J.R. and L.C.), the drop in PC₂₀ was more than 10-fold at Week 1 which was sustained at Week 2. In these two subjects, dust-mite extract inhalations were replaced with saline inhalation after 2 wk. In 6 of these 7 subjects, the PC₂₀ recovered to near baseline values within 3 wk of cessation of mite-extract inhalation. Using a paired t test, significant differences (p = 0.008) were found between mean PC₂₀ at baseline (6.35 mg/ml) and at the end of mite-extract inhalations (1.22 mg/ml). Similarly mean PC₂₀ at the end of mite-extract inhalations was significantly lower (p = 0.002) than at the end of saline inhalation weeks (8.22 mg/ml). However there was no difference between the mean PC₂₀ at baseline and that at the end of saline inhalation weeks (p = 0.3). Repeated measure analysis of variance indicated that a significant decline in PC₂₀ occurred at Week 1 (p = 0.005).

The mean weekly FEV₁ reduced in 8 of 9 subjects following repeated mite-extract inhalations (Table 4). Compared with baseline, the mean preinhalation FEV₁ was significantly lower at the endpoint (p = 0.001) but baseline was not different from the mean FEV₁ when study was stopped at Week S1 to S3 (p = 0.3). Thus overall reduction in FEV₁ was significant but reversible. A similar effect was seen on the FVC (data not shown). The immediate change in FEV₁ following mite-extract inhalation was < 10% during most visits. However, 7 of 9 subjects did show an early response (drop in FEV₁ of > 15%, 10 min after mite-extract inhalation) on at least one occasion. Overall, 20 of 74 mite-extract inhalations were followed by an early-phase response (EPR). These EPR were more common during the last 2 wk (12 of 32), as compared with the first 2 wk (8 of 42). Among these 8 EPR, 2 were in subjects (J.R. and L.C.) whose first 2 wk were also their last 2 wk as the study was stopped early due to > 10-fold reduction in their PC₂₀. Excluding these, 2 of 7 subjects had 5 EPR during the first 2 wk while 5 of 7 had 12 EPR during the last 2 wk.

There was some reduction in mean weekly home PEFR (Figure 1) between 2 and 4 wk and a trend to rise once the mite-extract inhalations were stopped but these changes were not statistically significant except the bedtime PEFR which rose from 444 to 478 (p = 0.04) in the 2 wk following cessation of dust-mite extract inhalation. In 6 of 9 subjects, a drop of 15% or more in PEFR occurred 12 to 24 h after mite-extract inhalations on one or more occasions (late-phase response). There were a total of 21 late-phase responses (LPR) and in 14 of these there was no preceding early response (isolated LPR). A small and statistically insignificant rise was seen in the symptom score from baseline to study endpoint (Figure 2) which fell to baseline levels during saline inhalation week. Only three subjects were taking rescue medication (inhaled bronchodilator) during baseline observation period, whereas 8 of 9 subjects had to take some rescue medication during their last week of allergen inhalation. The rise in mean medication score from 0.81 prestudy, to 2.53 at Week 4 was statistically significant (p = 0.02). During poststudy follow-up, although 4 of 7 subjects were still using inhaled bronchodilator, the mean medication score (0.82) was not different from the baseline period. We have since made inquiries from some of these subjects, whom we could contact, regarding their symptoms and medication usage and none felt that these were changed as a result of the study. Figure 3 shows the percent changes in PC₂₀, FEV₁, and PEFR in a typical case (T.P.) to illustrate the pattern of changes in the outcome variables during the study. Although, specific IgE to Der p1 and Der f1 was higher in 7 of 9 subjects at the endpoint as compared with baseline values (Figure 4), the difference was not statistically significant (paired t test).

View larger version (11K): [in this window] [in a new window]   Figure 1.   The means of PEFR for each week, including pre- and poststudy, of all subjects are charted to show the reduction during repeated dust-mite inhalation period and partial recovery during saline inhalation week.

View larger version (10K): [in this window] [in a new window]   Figure 2.   Means of symptoms and medication scores of all subjects are shown for each week. Symptoms were increased slightly but medication usage was, on average, increased by more than four times, which fell to baseline values after cessation of dust-mite inhalation.

View larger version (11K): [in this window] [in a new window]   Figure 3.   Percent changes in three main variables; PC20 methacholine, mean FEV1, and mean PEFR, for each week for an index patient (TP) to show the pattern and degree of changes that occurred during and after dust-mite inhalation.

View larger version (20K): [in this window] [in a new window]   Figure 4.   Serum IgE to Der p1 and Der f1 for each patient before the start of the study and at the end of mite-extract inhalation period. There was an increase in specific IgE levels to these dust mites in 7 of 9 subjects.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Asthma is a disease characterized by varying degrees of bronchial obstruction with bronchial hyperreactivity as a predominant feature. An immediate response to allergen challenge either occurring naturally or during experimental conditions resolves rapidly and cannot explain the pathogenesis of a chronic disease such as asthma. Late asthmatic responses with associated inflammatory sequelae and increased nonspecific bronchial reactivity are more relevant in the pathogenesis of bronchial asthma. These late responses are usually studied as part of a dual response following an allergen challenge where incremental doses are given until an immediate response is seen. The relevance of these experimental studies is often questioned as the large doses required to produce immediate responses do not mimic natural exposure. It has been suggested that small doses of allergen are more likely to cause isolated late reactions (14). The likelihood of a late asthmatic response is primarily determined by the degree of sensitization (15). In these highly dust-mite-allergic subjects, isolated late-phase responses were relatively common after repeated small dose inhalations. Bronchial reactivity was increased and other features of asthma developed. Although there were changes in most clinical and functional parameters, an immunological effect, as assessed by the change in specific antibody to Group 1 allergen was not seen. It is possible that the dose of dust-mite allergen was too small to stimulate antibody production or more likely, the duration of inhalation was too short for any change to be significant.

A period of 4 wk was selected arbitrarily as no previous data were available. With repeated measure analysis, we found that most of the drop in the PC₂₀ and FEV₁ occurred at the end of the first week when acute responses were less common. The reduction in FEV₁ could partly explain the subsequent decrease in PC₂₀ FEV₁. As the airways became increasingly hyperractive they responded more often with a drop in FEV₁ to the same dose of allergen. Those initially with the greatest degree of airway responsiveness to methacholine were less likely to show a further increase after dust-mite inhalation. A significant decrease in PC₂₀ was seen in two of four subjects with a high degree of responsiveness (PC₂₀ FEV₁ < 0.5 mg/ml), whereas all the remaining five with a lower degree of airway responsiveness (PC₂₀ FEV₁ > 1.0 mg/ml) at baseline showed a severalfold increase in airway reactivity.

An inhaled bronchodilator was given after the methacholine challenge (to reverse bronchoconstriction) before antigen inhalation on some visits which may have altered the early response. However this is not supported by the data. An early response was seen on 5 of 21 (24%) visits when bronchodilator was given prior to antigen inhalation and on 15 of 52 (23%) visits without such treatment. Administering even smaller doses daily for a week or more might avoid acute responses altogether and still result in increased bronchial reactivity. However, as the sensitivity to dust-mite differs among individuals, it is not possible to avoid an acute response using a fixed dose of allergen. Alternatively the dose of allergen could be titrated for individual subjects if the aim was solely to achieve an increased bronchial reactivity while avoiding acute responses. It is interesting to note that the number and magnitude of EPR did not influence bronchial reactivity. B.F., who developed EPR on 8 of 11 visits, did not show any change in PC₂₀, whereas a significant reduction in PC₂₀ was seen in G.N. who had no EPR.

The dose of house-dust mite allergen was carefully estimated after a literature review and local experience in some Baltimore homes. Group 1 allergen was used to estimate exposure (as is widely used) and we believe the estimated daily dose (0.4 ng) is very modest; according to Carswell and colleagues (16), the average p1 intake of a 1-mo-old infant is 3 ng/ 24 h. Most asthmatics, unless taking stringent dust-mite avoidance measures, have this level of exposure. However, there were differences from natural exposure. First the amount of allergen estimated to be inhaled over 24 to 36 h (time spent indoors in 2 or 3 d) was given over 2 min. This, we believe, was the main reason why some asthmatics had acute responses. Second, in natural circumstances the inhalation is in the dust form with varying particle sizes. In this study the allergen was inhaled as uniform aerosolized dust-mite extract. Technically, it is possible to do bronchial challenge with house dust (17). However, there were concerns regarding safety and nonspecific reactions (due to chemical, toxins, etc.). Moreover, house dust could not be standardized for its allergen content and accurate estimation of the dose would be difficult. Despite these differences, this experimental model with repeated small dose inhalations mimics the natural exposure more closely than the acute bronchial challenge. As the airborne levels of dust-mite allergen are very low in undisturbed conditions, it is possible that the significant exposure is intermittent and occurs only with disturbance of the reservoir. This type of exposure will still be enough for the continuation of allergic inflammation in bronchial airways. From time to time, the exposure will be large enough to cause an acute response as well. This scenario corresponds very closely to our experimental model. Exposure to other allergens during the study was a potential confounding factor. All subjects had multiple allergen sensitivity but there was no known or obvious exposure except for L.C. who was exposed to dog while being sensitive on skin prick test. There could have been other less obvious exposures such as to molds and cockroach but these were unlikely to have changed during the course of the study. The study months were July, August, and the first 2 wk of September. Sensitivity and exposure to grass pollen in S.M., T.P., and L.C., and ragweed in L.C., could have confounded results but reversal of all changes following cessation of mite-extract inhalation argues against it.

These nine subjects had very mild asthma. Their requirement for a bronchodilator was rare and some had not used any treatment for several years. With repeated exposure to mite allergen in small doses, they developed clinically overt asthma. Not only did their bronchial responsiveness increase, but also their pulmonary function was gradually reduced and symptoms and medication requirements increased. These changes were rapidly reversible within 2 to 3 wk. This confirms the importance of IgE and exposure to allergen in the causation of asthma and the usefulness of measures for reducing allergen exposure. Moreover, this experimental model provides a more natural form of controlled (and reversible) induction of bronchial inflammation for detailed study of the pathogenesis of allergic asthma.