The ability to directly instill allergens in the lower airway, and evaluate the cellular response thereafter, provides a unique approach to evaluating the pathophysiology of allergic airway inflammation. While the technique of SBP-AG is accepted research tool, there are limited published data regarding its safety, and no publications comparing BAL cellular response or safety profile among different allergens. As with prior reports from other groups 
there were no major complications associated with bronchoscopy or SBP-AG in our study. However, in our study there were significantly fewer adverse events and a smaller degree of spirometry changes post SBP. There was a modest decline in SpO2
and an increase in heart rate during the procedure that returned to normal by the time of discharge. Post-SBP-AG symptoms were mild and transient. Thus, the procedure was well tolerated. The SBP-AG-associated modest decline in FVC and increase in FEV1
/FVC suggest that the allergen administration led to a restrictive defect probably related to the intense inflammatory response in the challenged segment. Interestingly, the study by Moore et al. evaluating the effects of fixed-dose SBP on subjects with mild-to-moderate asthma showed a greater decline in FEV1
and a trend toward reduction in FEV1
. The inverse correlation between the change in FEV1
and the baseline FEV1
percent predicted () was also observed in the bronchoscopy study from the Severe Asthma Research Program evaluating subjects with a wider range of baseline lung function 
. Nonetheless, our safety data for SBP-AG pertain exclusively to subjects with mild disease not requiring inhaled corticosteroids, and these findings cannot be directly translated to subjects with more severe asthma.
Our study provides a unique addition to the limited existing literature on safety of, and response to, SBP-AG especially comparing different allergens. We evaluated the safety of SBP-AG using a protocol with subject-individualized allergen dosing and compared the safety of bronchoprovocation among three different allergens. There were no differences in either symptoms or objective measures among subjects who received RW, HDM, or CAT allergens. In addition, while there was a marked inflammatory response in BAL fluid on D2 with a predominance of eosinophils, there were no differences in the elicited responses among the three allergens. While there were no differences in the safety profile or cellular responses among subjects challenged with the three allergens utilized in our protocol, it is unclear if these results can be generalized to other allergens that could be used in such procedures.
The low incidence of side effects in our study might be, in part, due to the use of individualized allergen dosing for SBP-AG, which is consistent with the report by Julius and colleagues. In their study, when the allergen dose was individualized based on inhaled allergen testing, only 19% required bronchodilator therapy in the immediate post-challenge period; in contrast, when the allergen dose was not subject-specific, 43% of the subjects required β-agonist treatment. It is important to note that in our study we did not use a fixed dose approach, thus direct extrapolation as to the importance of this approach is not feasible. Compared to other methods used for dose selection such as skin prick titration, the utilization of whole-lung allergen inhalation challenge to determine individual dose for SBP-AG allows for monitoring of the subject’s airway response to allergen challenge with direct relevance to their specific response to SBP-AG. In addition, subjects with highly reactive airways to methacholine were also very reactive to inhaled allergen and as such received a smaller dose of allergen during SBP-AG by this dosing method. Since allergen dose is potentially an important factor in the fall in FEV1 after SBP-AG bronchoscopy, this may explain the observed negative correlation between the change in FEV1 post bronchoscopy and methacholine PC20 (). Of note is that subjects who did not tolerate whole-lung allergen inhalation challenge well during screening were not considered for subsequent SBP-AG.
All of our subjects received β-agonists by protocol prior to bronchoscopy and none of the bronchoscopies were discontinued due to wheezing or other acute symptoms during the SBP-AG. While there was a statistically significant reduction in pre-bronchoscopy FEV1
on D2 compared to baseline FEV1
on D0, the absolute difference between these study visits was less than 200 mL, a change that is not considered clinically significant. The modest reductions in spirometry noted in our study stands in contrast to those reported by Krug et al. 
; 29% of their subjects developed significant wheezing during the procedure and showed a significant decrease in FEV1
following the procedure. Additionally, the drop in lung function was severe enough in half of those subjects to warrant direct instillation of β-agonist bronchoscopically and termination of the procedure. Other studies have also reported larger reductions in FEV1
compared to those observed in our study 
. The reasons that may explain the differences between our findings and those in previously published reports are not clear. Subject selection, recruitment of a relatively younger subject cohort with mild asthma, consistent premedication with β-agonist, use of minimal sedation, differences in total allergen dose, or tailoring allergen dose to each subject based on their individual response to whole-lung allergen inhalation are all among the possible explanations. We should note that in our study, three subjects did not undergo the D2 bronchoscopy. One subject developed sore throat, nasal congestion, and chest tightness approximately 36 hours after the procedure, which improved after β-agonist therapy. The second subject developed fevers, myalgias, and sore throat 24 hours after the procedure that resolved without further intervention and were consistent with acquisition of a naturally occurring viral infection, although these symptoms also could have been related to post-bronchoscopy fever. The third subject developed anxiety accompanied by hyperventilation during the initial procedure, but no changes in objective parameters were seen. This complication was also noted by Julius and colleagues 
. Finally, there have been safety concerns raised regarding repeated allergen exposures 
. We did not observe any increase in adverse events in subjects who underwent 2 or 3 SBP-AG studies. This is very consistent with the reports by Julius and Moore 
. However, this point was not specifically investigated in our study.
In conclusion, our results support the safety of research bronchoscopy with SBP-AG in subjects with mild asthma. Furthermore, we demonstrated similar BAL cellular responses to three different allergens. These data support the continued application of this research tool to the investigation of the mechanisms of allergic airway inflammation in asthma.