INTRODUCTION

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The course of occupational asthma after removal from exposure is variable. Some subjects show persistent asthma even after total removal from exposure whereas others recover completely, with normalization of their nonspecific bronchial reactivity (NSBR) and loss of their asthmatic symptoms (1). However, the loss of asthmatic symptoms does not ensure that these subjects will not experience any further asthmatic reactions upon reexposure to the offending agent. Indeed, we have previously shown that most workers with occupational asthma retain specific bronchial reactivity (SBR) to their occupational allergens even 2 or more years after removal from exposure (2). Some of these workers experience an asthmatic reaction to occupational agents despite normalization of NSBR; this SBR might be due to a persistence of immunologic sensitization to the offending agent. The proportion of subjects who retain a measurable SBR to occupational allergens despite normal NSBR is unknown. This issue has significant practical implications because some subjects considered to be cured from their occupational asthma might consider going back to their previous work. It is therefore important to know whether these subjects risk experiencing an asthmatic reaction when reexposed to the offending agent.

In the present study, we examined subjects with previously diagnosed occupational asthma caused by high-molecular-weight agents to determine if they retain a measurable SBR to these agents despite normalization of NSBR at the time of the study. We also investigated whether the persistence of SBR was related to a persistence of IgE-dependent immunologic reactivity at the time of the study.

	METHODS

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Study Design

Subjects with previously diagnosed occupational asthma were investigated for 2 d at Sacré-Coeur Hospital in Montreal. On the first day, they were asked to complete a questionnaire regarding their respiratory symptoms, smoking habits, and treatment. At the time of diagnosis, skin tests to 22 common inhalant allergens and to the relevant occupational agent were performed, blood samples were taken from all subjects, and sera were kept frozen at 20° C for further analysis. At the time of the study, a skin-prick test to the occupational agent and venesection for specific IgE measurement were again performed. Specific inhalation challenges (SIC) to the offending agent were performed over the 2 d of the study (Day 1, control day; Day 2, exposure to the specific agent). A methacholine inhalation challenge was performed at the end of the 2 d as at the time of the diagnosis. The provocative concentration of methacholine that caused a 20% reduction in FEV₁ (mPC₂₀) obtained at the end of Day 1 was considered as a prechallenge value whereas the mPC₂₀ obtained at the end of Day 2 was considered as a postchallenge value.

Subjects

All subjects were adult workers who (1) had shown an asthmatic reaction after an SIC with high-molecular-weight agents at our center some time during the past 15 yr, (2) had not experienced a severe asthmatic reaction ( 50% decrease in FEV₁) after the earlier exposure, (3) had a normal NSBR (mPC₂₀ > 16 mg/ml) at the time of the study, and (4) were willing to participate in the study. Subjects were no longer exposed to the offending agent in their work or daily lives, did not complain of asthmatic symptoms, and were not receiving any treatment for asthma. The study was approved by the hospital research ethics committee and all subjects gave written consent.

Protocol

Questionnaire. We administered a questionnaire regarding asthma symptoms, need for medication, and smoking habits.

Specific inhalation challenges. SIC were performed in the same way as at the time of diagnosis, as previously described (3). Briefly, on the control day, spirometric tests were performed with a Collins spirometer (W.E. Collins Inc., Braintree, MA). FEV₁ was measured every 10 min for the first hour, every 30 min for the second hour, then hourly for a total of 7 h to ensure that there was no significant fluctuation. FVC was recorded hourly. On Day 2, each subject was exposed to the agent that had caused his or her asthma by the same method that was used at the time of the diagnosis: Eight subjects were exposed by means of a closed-circuit apparatus (4) that allowed accurate generation of the concentration of particles; seven were exposed in a realistic way, with the subject tipping the powder from one tray to another and using approximately the same amount of allergen; and one subject was exposed by using nebulized water-soluble allergen with an increasing concentration of allergen (5). SIC was considered positive if there was a decrease in FEV₁  20%. A methacholine challenge was performed at the end of the 2 d as described previously (6).

Immunologic assessmentskin testing. Skin tests were performed with commercial extracts from cereals (Omega, Montreal, PQ, Canada), and with extracts of pure guar gum and psyllium in saline solution (0.01, 0.1, 1 mg/ml). A positive reaction was set at a weal diameter of  3 mm in the absence of reaction to a control diluent and with a positive reaction to histamine phosphate (1 mg/ml).

Immunologic assessmentradioallergosorbent test (RAST) for specific IgE antibodies to extracts. Antigen extracts for RAST discs were prepared as follows: Thirty grams of unflavored psyllium (Personnelle brand; The Jean Coutu Group, Inc., Longueuil, PQ, Canada) was extracted in 500 ml phosphate-buffered saline (PBS), pH 7.2 (0.01 M) by mixing on a magnetic stirrer for 30 min at 24° C; centrifuged (16,000 × g) for 30 min at 4° C; concentrated on Amicon YM1 filter (Millipore, Danbury, CT; 1,000 molecular weight cutoff, MWCO), and recentrifuged (100,000 × g) for 30 min at 4° C. Ten grams of guar gum (Sigma, St. Louis, MO) was extracted in 700 ml of PBS, pH 7.2 (0.01 M) by mixing for 30 min at 24° C on a magnetic stirrer; centrifuged (16,000 × g) for 30 min at 4° C, and recentrifuged (80,000 × g) for 30 min at 24° C. Fifty grams of all-purpose white flour (Five Roses brand) was extracted in 100 ml PBS, pH 7.2 (0.01 M) by mixing on a magnetic stirrer for 30 min at 24° C; centrifuged (12,000 × g) for 30 min at 4° C; concentrated on an Amicon YM1 filter (1,000 MWCO); and recentrifuged (100,000 × g) for 30 min at 4° C. All extracts were aliquoted, had their dry weights determined, and were stored frozen at 20° C.

Workers' sera were tested by RAST to determine concentrations of specific IgE antibodies to psyllium, guar gum, and flour, as described previously (7). For coupling to RAST disks, extracts were adjusted to 1 mg/ml in pH 8.0 borate buffer and coupled to cyanogen bromide (CNBr) activated filter paper disks. For the test, 100 µl of patient serum was incubated overnight with a test disk. All sera were tested in duplicate. After three washes with 2.5 ml of physiologic saline to remove unreacted serum, disks were incubated overnight with 100 µl of ¹²⁵I-labeled anti-IgE antiserum (15,000 counts per minute [cpm]), washed three times with saline to remove unreacted material, and counted in a gamma counter to determine radioactivity in counts per minute bound to the disks. A RAST binding more than 3% of total activity added was considered positive, a RAST binding between 5% and 15% of total activity added was considered intermediate, and a RAST binding above 15% of total activity added was considered high, based on previous experience (8, 9).

Outcomes

The primary outcome was the proportion of subjects who still experienced an asthmatic reaction after SIC despite normal NSBR.

The secondary outcomes were the level of specific IgE to the offending agent measured in subjects who did and did not experience an asthmatic reaction at the time of the study (second SIC), and the correlation between the difference in specific IgE level and difference in SBR at the time of the diagnosis (first SIC) and at the time of the study.

Data Analysis

Results were expressed as mean and standard deviation, except for data with non-normal distribution, which were expressed as median and interquartile range. We calculated SBR as the dose of high-molecular-weight agent that induced a 20% decrease in FEV₁. Because the concentration of agent was kept constant from one challenge to the other, SBR was estimated as being the duration of exposure that induces a 20% decrease in FEV₁. Two-sided paired t test was used to compare data at the time of first and second SIC. Data with non-normal distribution that could not be normalized using usual transformations were compared using a Wilcoxon signed rank test. Correlations were examined using a Pearson correlation test. Mann-Whitney test was used to compare IgE concentrations between different groups. Significance was accepted at a level of 95%. The analysis was performed using the SPSS 7.5 statistical package (Chicago, IL).

	RESULTS

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Clinical and Functional Features

We investigated 16 subjects (five women, 11 men) with occupational asthma caused by flour (seven cases), psyllium (five cases), and guar gum (four cases). Workers had been removed from exposure for 5.7 ± 4.0 yr (mean ± SD) and none complained of asthmatic symptoms. None of the subjects was being treated with inhaled steroids. Some relevant clinical and functional characteristics are listed in Tables 1 and 2. Baseline FEV₁ was not significantly different at the time of the diagnosis (99.0 ± 14.4% predicted) and at the time of the study (94.8 ± 13.0% predicted). All subjects had normalized their NSBR at the time of the study. Nine subjects already had normal NSBR at the time of the diagnosis before the first SIC; they had already been removed from exposure at the time of the first SIC. Four of them had a significant increase in their NSBR after the first SIC, whereas only two subjects had a change in their NSBR at the time of the second SIC.

Specific Inhalation Challenges

Eight subjects were exposed using a closed-circuit apparatus. The concentration of agent delivered at the time of the diagnosis and at the time of the study was similar (3.0 ± 0.7 mg/m³ versus 3.6 ± 0.9 mg/m³). Eleven of 16 workers experienced an asthmatic reaction during the second SIC. Of the five subjects who did not experience an asthmatic reaction, three were exposed to psyllium and two to flour. One of the three subjects exposed to psyllium had significantly increased airway responsiveness after challenge without any significant decrease in FEV₁, suggesting that an increase in exposure would have induced a significant decrease in FEV₁ (10).

In the whole group of subjects, SBR was 4.62 ± 3.43 min at the time of the diagnosis compared with 14.9 ± 13.4 min at the time of study (p = 0.09). When considering only those subjects who experienced an asthmatic reaction at the time of the second SIC, SBR remained the same (3.5 ± 0.5 min versus 4.2 ± 0.7 min, p = 0.8). Although all the subjects had a normal NSBR and had been removed from exposure for a long time, the duration of exposure required to induce an asthmatic reaction remained the same at the time of the study as it had been at the time of diagnosis. Although not significant, there was a trend toward a shorter period of occupational exposure to the causal agents in subjects who did not experience an asthmatic reaction at the time of the second SIC (5.7 ± 4.4) yr compared with 13.1 ± 10.4; yr, p = 0.15).

Immunologic Assessment

There was a significant decrease in the level of specific IgE at the time of the second SIC (13.6 ± 34.9% binding) compared with the first SIC (31.0 ± 24.7% binding), p = 0.005. Four of the five workers who did not experience asthmatic reactions had a significant decrease in their specific IgE (Table 2). The level of specific IgE was significantly lower in the group of subjects who did not experience an asthmatic reaction compared with the subjects who still had a positive SIC at the time of the study: median 3.0 (interquartile range 16.9) compared with those who did [24.2 (37.5)] (p = 0.05) (Figure 1). There was a significant decrease in the level of specific IgE in the group of subjects exposed to psyllium at the time of the second SIC, whereas the decrease was less in the group exposed to flour and guar gum (Figure 2).

View larger version (18K): [in this window] [in a new window]   Figure 1.   Changes in concentrations of specific IgE in subjects who did and did not experience an asthmatic reaction after the second SIC. Medians are represented by horizontal lines.

View larger version (16K): [in this window] [in a new window]   Figure 2.   Concentration of specific IgE in the groups of subjects sensitized to flour, psyllium, or guar gum at the time of the first and second SIC. Medians are represented by horizontal lines. *p < 0.05.

A significant correlation was found between the difference in SBR at the time of the first and second SIC and the difference in specific IgE at the time of the first and second SIC (r = 0.56, p = 0.03)(Figure 3).There was a negative correlation between the difference in specific IgE at the time of the first and second SIC and the duration of exposure to the causal agent, suggesting that the longer the occupational exposure, the less the decrease in specific IgE (r = 0.62, p = 0.01) (Figure 3).

View larger version (14K): [in this window] [in a new window]   Figure 3.   Positive correlation between the difference in SBR at the time of the first and second SIC and the difference in specific IgE concentration at the same time of the first and second SIC, and negative correlation between the difference in specific IgE at the time of the first and second SIC and the duration of occupational exposure to the causal agent.

	DISCUSSION

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Most subjects in this study still experienced an asthmatic reaction after SIC in spite of a normalization of their NSBR. Most of the subjects who did not show an asthmatic reaction at the time of the second challenge had a significant decrease in their specific IgE level and a lower level of IgE than workers who experienced an asthmatic reaction.

In the late 1950s, Tiffeneau (11) hypothesized that SBR to common inhalants was related to both NSBR and specific immunologic reactivity, and other researchers subsequently confirmed this hypothesis (12). Some investigators set an equation to predict the concentration of allergen needed to induce an asthmatic reaction based on the PC₂₀ value and on wheal diameter of the skin-prick test (5). Those studies were performed in asthmatic subjects with mild to moderate bronchial hyperreactivity. In our study, subjects who experienced an asthmatic reaction did not have an improved SBR, despite improvement and normalization of their NSBR. NSBR might modulate the severity of SBR, but it does not appear to be a prerequisite for the occurrence of an asthmatic reaction after exposure to a sensitizer. After the immediate reactions in asymptomatic subjects, we did not observe any change in NSBR that stayed within the normal range whereas there was an increase in NSBR to the asthmatic range after occurrence of the late reaction. An increase in NSBR after asthmatic reactions caused by occupational agents has been reported to occur in 40% of cases after immediate reaction and in 60% of cases after late reactions, as reported by Malo and coworkers (15). However, when immediate reactions occur in subjects with normal NSBR, which was not the case in subjects investigated by Malo and coworkers (15), the reaction does not seem to increase the degree of NSBR in most cases.

Some subjects experienced dual asthmatic reactions at the time of diagnosis, whereas they developed isolated immediate asthmatic reactions or isolated late reaction on reexposure to the same agent. Reproducibility in the pattern and in the severity of asthmatic reactions after challenges to common inhalants has been reported to be good (16). Although reproducibility of the temporal pattern of asthmatic reactions to occupational agents several years after the initial diagnosis has never, to our knowledge, been examined specifically, some studies have reevaluated SBR several years after diagnosis (2, 17, 18). The occurrence of a different type of asthmatic reaction on reexposure is not uncommon, ranging from 11% (17) to 27% (2) of reactions. This change in the pattern of the asthmatic reaction might depend on the level of sensitization to the allergen and on the dose administered during the challenge. Indeed, we do know that exposure to low concentrations of common allergens leads to an increase in airway responsiveness as well as an increase in eosinophils and interleukin-5 (19). Therefore, a low dose of agent or a substantial decrease in sensitization might lead to a late reaction, whereas a high dose of allergen with a marked degree of sensitization to the allergen might lead to an immediate reaction caused by a massive mast cell degranulation.

SBR to isocyanates (2, 17), platinum salts (18), or high-molecular-weight agents (2) has been assessed several years after removal of subjects from exposure. In those studies, 20 to 40% of subjects did not have asthmatic reactions on reexposure to isocyanates, whereas only 12% did not have asthmatic reactions on reexposure to high-molecular-weight agents. Subjects kept the same SBR to platinum salts 19 mo after removal from exposure as at the time of diagnosis. In the current study, 30% of subjects did not experience an asthmatic reaction. Loss of SBR seems to occur in similar proportions after removal from exposure to either high- or low-molecular-weight agents and does not seem to be associated with the normalization of NSBR.

It seems that a certain amount of specific IgE is mandatory for demonstrating SBR. Bryant and coworkers clearly demonstrated the strong correlation between SBR to common inhalants and the concentration of specific IgE to the same allergens in asthmatic subjects (20). An association between the severity of asthmatic reaction after allergen challenge and the level of sensitization to the allergen has also recently been shown with common allergens (21). High amount of specific IgE might indicate a high risk of developing an asthmatic reaction in case of reexposure to the allergen previously causing occupational asthma, even in subjects who no longer complain of asthmatic symptoms and have normalized their NSBR. Indeed, despite a complete disappearance of asthmatic symptoms, absence of treatment, and a normal NSBR, subjects with a high amount of specific IgE reacted to the offending agent within a few minutes of exposure.

All subjects in the present study were removed from exposure after diagnosis of occupational asthma and had not been exposed to their agent for a mean of 5.7 yr at the time of the study. Despite this, 56% of subjects retained the same concentration of specific IgE.The time-course of specific sensitization to occupational agents seems to vary according to the offending agent. We previously reported a significant reduction in the concentration of specific IgE to snow crab at 1 and 5 yr after removal from exposure (22). In our current study, subjects initially exposed to psyllium decreased their level of sensitization compared with subjects exposed to flour or guar gum (Figure 2). The decreased level of sensitization might be due to a total removal from exposure to psyllium, whereas the subjects with asthma resulting from flour or guar gum may continue to be indirectly exposed to those agents. Subjects are likely to stay exposed to flour in their daily lives and there is a cross-reactivity between grass and flour (23). Guar gum is widely used in the food industry and can be found in ice creams and yogurt. Although psyllium (Plantago ovata) and English plantain (Plantago lanceolata) have a phylogenetic relationship, there is only little cross-allergenicity between the two (24).The time-course of specific sensitization to occupational agents also seems to vary according to the total duration of exposure to the offending agent. Subjects who stayed exposed to the agent for only a short period of time are more likely to have a significant decrease in their specific IgE concentrations after removal from exposure (Figure 3).

In conclusion, most subjects who developed occupational asthma caused by high-molecular-weight agents showed a persistent SBR despite normal NSBR. Persistent immunologic sensitization seems to be a major factor for persistence of SBR. The results of this study emphasize the importance for subjects with occupational asthma caused by high-molecular-weight agents to stay away from work, even if they no longer complain of asthmatic symptoms and have normalized their NSBR. Indeed, these subjects could develop a severe asthmatic reaction within a few minutes of reexposure, even a long time after removal from exposure to the agent.