Montelukast treatment may alter the early efficacy of immunotherapy in children with asthma

Asthma and lower airway disease

Montelukast treatment may alter the early efficacy of immunotherapy in children with asthma _ Rychlik, PhD,c qukasz Pu1aski, PhD,c,d Andrzej B1auz, _ PhD,c Brzozowska Agnieszka, MD, Pawe1 Majak, MD, PhD,a B1azej a a b PhD, Monika Bobrowska-Korzeniowska, MD, PhD, Piotr Kuna, MD, PhD, and Iwona Stelmach, MD, PhDa Lodz, Poland Background: Allergen-specific immunotherapy (SIT) is the only available potentially curative approach in the management of allergic diseases. Therapies that boost regulatory T cell induction during SIT might further enhance its effectiveness. Objective: The purpose of this study was to assess the effect of montelukast treatment on early clinical and immunologic effects of allergen-specific immunotherapy in children with asthma. Methods: It was a randomized, double-blind, placebo-controlled trial conducted in 36 children with asthma and allergy to house dust mites who required from 400 to 800 mg of inhaled budesonide per day during the 7-month run-in period. Patients were randomly allocated to receive 5 mg montelukast daily (n 5 18) or placebo (n 5 18) as an addition to inhaled corticosteroid (ICS) treatment during the 3-month build-up phase of SIT, when modification of ICS doses was not allowed. During the 7 months of the maintenance phase of SIT, ICS doses were adjusted to control the asthma symptoms. Results: After 12 months of SIT, a reduction of the median daily ICS dose, necessary to control asthma symptoms, was 16.7% grater in patients from the placebo group than in patients from the montelukast group. Intervention with montelukast significantly impaired the induction of regulatory T lymphocytes. During the build-up phase of SIT, patients in the placebo group frequently experienced an increase in asthma symptoms leading to exclusions from the per protocol population. Conclusion: Our study failed to show a beneficial effect of montelukast on SIT. In fact, quite the opposite occurred: compared with placebo, montelukast intervention led to less effectiveness of SIT. (J Allergy Clin Immunol 2010;125:1220-7.)

From the Departments of aPediatrics and Allergy and bInternal Medicine, Asthma and Allergy, Medical University of Lodz; cthe Department of Molecular Biophysics, University of Lodz; and dthe Laboratory of Transcriptional Regulation, Institute of Medical Biology PAS. Supported by grants 502-12-760 and 503-2056-1 from the Medical University of Lodz, Poland. Montelukast and extracts for allergen-specific immunotherapy were provided by the manufacturers: MSD Polska and Stallergenes Pharmaceutical Co, respectively. Disclosure of potential conflict of interest: P. Kuna has given sponsored lectures for Merck Sharp Dohme. The rest of the authors have declared that they have no conflict of interest. Received for publication October 25, 2009; revised February 12, 2010; accepted for publication February 18, 2010. Available online May 3, 2010. Reprint requests: Iwona Stelmach, MD, PhD, Department of Pediatrics and Allergy, N. Copernicus Hospital, 62 Pabianicka Str, 93-513 Lodz, Poland. E-mail: alergol@ kopernik.lodz.pl. 0091-6749/$36.00 Ó 2010 American Academy of Allergy, Asthma & Immunology doi:10.1016/j.jaci.2010.02.034

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Key words: Immunotherapy, asthma, montelukast

It is widely accepted that the establishment of a tolerant state in peripheral T cells represents an essential step in allergen-specific immunotherapy (SIT).1,2 The mechanism of the development of allergen-specific tolerance is not completely understood, but it might involve the induction of adaptive regulatory T (Treg) cells, such as IL-10–secreting Treg cells,3,4 along with the activation and expansion of natural Treg cells, such as CD41CD251 forkhead box protein 3 (Foxp3)1 cells.5-7 Both populations of Treg cells contribute to the control of allergen-specific immune responses in several ways, including suppression of different effector cells and induction of allergenspecific IgG4.2 Allergen-specific immunotherapy is the only available potentially curative approach in the management of allergic diseases. Therapies that modify immune regulation to boost the Treg cells induction during SIT might further enhance its effectiveness. We have previously shown that the treatment with montelukast sodium significantly upregulated IL-10 production and consequently increased the serum level of IL-10 in children with IgE-dependent asthma.8,9 Taking into account the multifaceted and crucial role of IL-10 in the mechanism of SIT, the treatment able to increase IL-10 concentration in cytokines microenvironment attracts attention, because it may change the immunologic response to allergen administration toward a more effective SIT. To our best knowledge, the direct influence of antileukotriene treatment on the induction of Treg cells has not been studied yet. However, it has been shown that antileukotriene therapies may change the function of dendritic cells and reproduce some regulatory functions played by IL-10 in the inflammatory processes, because cysteinyl leukotriene–induced activation of human dendritic cells may be specifically inhibited by IL-10 as well as by leukotriene receptor antagonists.10 Because premedication with montelukast has been found to reduce the local side-effects of SIT,11 it is possible that the antileukotriene therapy interferes with the inflammation induced by allergen administration. Montelukast’s ability to reduce the serum levels of specific IgE in children with asthma, as indicative of systemic immune response regulation, also seems important.12 To date, the influence of antileukotriene treatment on immunotherapy effectiveness has never been tested. All these findings have encouraged us to conduct this doubleblind, placebo-controlled study assessing the effect of montelukast treatment on the early clinical and immunologic effects of allergen-specific immunotherapy in children with asthma.

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Abbreviations used Foxp3: Forkhead box protein 3 HPRT1: Hypoxanthine phosphoribosyltransferase ICS: Inhaled glucocorticosteroid ITT: Intent-to-treat PP: Per-protocol SIT: Allergen-specific immunotherapy Treg: Regulatory T

METHODS Patients Thirty-six boys and girls age 6 to 12 years were selected from an outpatient population at our Allergy Clinic Center. The main inclusion criteria were moderate IgE-dependent asthma with regular symptoms requiring long-term treatment with inhaled corticosteroids and a disease duration of at least 2 years. Diagnosis of asthma was established by assessing the symptoms of asthma and _12% after administration of salimprovement in the prebronchodilator FEV1 of > butamol (200 mg); asthma severity was calculated according to the daily inhaled glucocorticosteroid (ICS) dose necessary to control the symptoms. To be included in the study, the patients had to be sensitized only to house dust mites (Dermatophagoides farinae and Dermatophagoides pteronyssinus), as evi_3 mm at 15 mindenced by a positive skin prick test (a mean wheal diameter of > utes) and by the presence of serum-specific IgE (a level of >0.7 kU/L). The patients and their parents were required to demonstrate the understanding of a diary used to record daily symptoms. Moreover, the ability to perform reproducible spirometry, exhibiting the resting FEV1 of at least 70% and no contraindications for SIT, was required of the patients. A need for a daily dose below 400 mg or above 800 mg of budesonide calculated during the run-in period and sensitization to allergens other than house dust mites resulted in a patient’s exclusion from the study. Subjects with concomitant allergic rhinitis were excluded. The patients who had previously received immunotherapy were also excluded. Modification of daily ICS dose during the build-up phase of SIT was not allowed. The patients who required modification in antiasthma treatment during the build-up phase of SIT were excluded. The patients were also excluded if SIT was for any reason discontinued, the maintenance dose of allergen extract has not been reached within 3 months of the build-up phase of SIT, or more than 1 maintenance dose of allergen extract was missed. Medications that were prohibited included inhaled long-acting b2-agonists, leukotriene modifiers, b-blockers (including eye drops), and systemic corticosteroids. Asthma was stable in all patients; there had been no exacerbations of a disease nor any need for other treatment for a minimum of 6 months.

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received the first subcutaneous dose of commercial Dermatophagoides pteronyssinus (50%) and Dermatophagoides farinae (50%) extracts (Phostal, Stallergenes, France). Intervention (montelukast or placebo) was discontinued during the second visit (at the end of the SIT build-up phase). The last study visit took place at the end of the first year of SIT, and the double-blind code was revealed. During each study visit, blood samples were obtained from all patients, clinical symptoms were assessed, lung function was measured, and the patients’ diaries were evaluated. The visits were scheduled so that blood sampling and lung function testing in individual patients always took place at the same time and on the same day of each week. During the prestudy visit, antiallergen measures13 were recommended for all patients. In addition, every 3 months visits were scheduled to adjust the corticosteroid doses to the current control of asthma symptoms, according to clinical examination and lung function measurement. All children remained in the same environment during the study. Immunotherapy was continued throughout the study period according to a standard dose schedule (maintenance dose was 0.8 mL [8 IRreactivity index]—59 mcg major allergens). The average cumulative dose for each patient was 93 IR, corresponding to 686 mcg major allergens.

Assessment of the clinical effects of SIT

Montelukast sodium oral tablet once daily at bedtime (Singulair, 5 mg; MSD, Whitehouse Station, NJ)—montelukast group (18 patients) Placebo—control group (18 patients)

The clinical effects of SIT were assessed via each patient’s diary card,8 lung function measurements,14 and analysis of the reduction in the ICS dose. Asthma exacerbations were noted at each doctor’s visits. Because the monitoring of the consumption of inhaled corticosteroids seems to be the best approach for assessing the clinical efficacy of SIT in children with asthma,15 the inhaled steroid-sparing effect of SIT was considered the main outcome of this study. A diary card was completed filled during the run-in period (September through March: the season with the maximum levels of natural allergen exposure16), the build-up phase (April through June: outside the peak exposure season), and the last 7 months of the maintenance phase (September through March: the season with the maximum levels of natural allergen exposure). For these periods, the mean asthma symptom score was calculated. We did not monitor domestic exposure to house dust mites in our study. Diary card. The daily diary card included daytime symptoms (completed at bedtime), incidents of nocturnal awakening (recorded in the morning on awakening), and the number of puffs of as-needed b2-agonist. The daytime asthma symptoms and nocturnal awakenings were scored subjectively as follows: 0, no symptoms during the day/night; 1, symptoms that do not affect any activities during the day/night; 2, symptoms affecting at least 1 daily activity/ disturbing the night; and 3, symptoms affecting 2 or more daily activities/sleep disturbances throughout all night or most of the night. The use of b2-agonists was scored as follows: 0, none; 1, once a day; 2, two to three times a day; and 3, more than 3 times a day. The minimum score for each day was 0 (no symptoms during the daytime, no symptoms at night, and no use of b2-agonists). The maximum score was 9 (severe symptoms during the day and night and the use of the b2-agonists more than 3 times a day). Principles for reducing the ICS dose. All patients were treated with budesonide (dry powder inhaler, Pulmicort turbuhaler; AstraZeneca, Bedfordshire, United Kingdom) twice daily. Before the first visit (the run-in period) and after the second visit (the maintenance phase of SIT), the daily dose of ICS was reduced stepwise every 3 months until asthma was no longer controlled. The treatment was then continued with a dose 1 step higher. The dose steps of budesonide were 100, 200, 400, 600, and 800 mg daily. When the patient was asymptomatic for 3 months with the minimal dose, ICS treatment was discontinued. Uncontrolled asthma was defined according to guidelines17 based on daytime/nighttime asthma symptoms, lung function, and the number of short-acting b2-agonist inhalations. Reducing the ICS dose was not allowed during the build-up phase of SIT. The daily dose of ICS was assessed during the entire study. On the basis of the minimal daily dose capable of maintaining asthma control during the September to March period before SIT and during the SIT period, the reduction in the minimal daily dose of ICS after the first year of SIT was calculated for all patients and compared between the study groups. Reduction in ICS dose was calculated according to following rule:

Active intervention drugs and placebo were blind (prepared in wafers with 0.3 mg lactose) by the hospital pharmacy. During the same visit, all patients

(ICS dose after the first year of SIT– ICS dose at baseline)/ICS dose at baseline 3 100%

Study design This study was a randomized, double-blind, placebo-controlled trial. Fig 1 presents the flow chart of the study. Before beginning the treatment, all patients underwent a run-in period from September 2005 until the end of March 2006. During this period, ICS dose was adjusted stepwise to define the lowest dose necessary to control asthma symptoms. Intervention was initiated in April 2006 and was continued until the end of the build-up phase of SIT (July 2006). The patients attended 1 prestudy and 3 study visits at the clinic. During the prestudy visit (at the beginning of a run-in period), all participants were informed about the purpose of the study, were told how to score asthma symptoms, underwent skin testing, and had peripheral blood samples taken. During the first visit, the patients were classified by age and a minimal daily ICS dose necessary to control asthma symptoms and randomized according to a computer-generated stratified allocation schedule for intervention with the following: d

d

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FIG 1. Flow chart of the study. Two (5.5%) patients were excluded from the ITT population because they withdrew before the second month of intervention (discontinuation of SIT) and therefore had no efficacy assessment. Five patients (13.8%) were excluded from the PP population because of worsening asthma symptoms that required an increase in ICS dose and changes in the SIT schedule.

Assessment of the immunologic effects of SIT Peripheral mononuclear cell isolation and culture. PBMCs isolated from whole blood by density gradient centrifugation on Histopaque-1077 (Sigma-Aldrich, St Louis, Mo) were cultured for 72 hours in standard mammalian cell culture conditions (378C; 5% CO2; 80% relative humidity) in the presence of Der p 2 allergen at a concentration of 10 mg/mL. CD4, CD25, and Foxp3 expression on cultured PBMCs was estimated by flow cytometry (LSRII instrument; BD Biosciences, San Jose, Calif) using appropriate fluorophore-conjugated mAbs. The IL-4, IL-5, IL-10, IL-13, and TGF-b1) levels in cell culture supernatants were determined by using ELISA-based specific tests (R&D, Minneapolis, Minn) according to the manufacturer’s instructions. Foxp3 cDNA quantitation was done by a real-time PCR run on a LightCycler 480 thermocycler (Roche Applied Science, Indianapolis, Ind) using LightCycler 480 SYBR Green I Master (Roche Applied Science, Indianapolis, Ind) according to the manufacturer’s protocol. The number of Foxp3 cDNA molecules in a sample was expressed in relation to the number of hypoxanthine phosphoribosyltransferase (HPRT1) cDNA molecules, HPRT1 being the reference gene of choice for lymphoid cells, using the relative quantitation feature of LightCycler 480 software. The HPRT1-specific primers used were 59-TGACACTGGCAAAACAATGCA-39 and 59-GGTCCT TTTCACCAGCAAGCT-39. The Foxp3-specific primers used were 59AACAG CACATTCCCAGAGTTCCT-39 and 59-CATTGAGTGTCCGCTGCTTCT-39.

Statistical methods The study was designed to achieve at least 85% power (for 15 patients completing the study per treatment arm) in detecting a 25% difference in ICS dose reduction after 12 months of SIT (the primary outcome) between the active

TABLE I. Baseline characteristic of the trial participants according to the study group Control group N 5 18

Age, n (%) 6-8 y 9-10 y 11-12 y Male sex, n (%) Duration of asthma (y) Median Interquartile range ICS dose,* n (%) 400 mg budesonide/d 600 mg budesonide/d 800 mg budesonide/d

6 6 6 11

Montelukast group N 5 18

(33.3) (33.3) (33.3) (61)

6 6 6 12

(33.3) (33.3) (33.3) (67)

3 2-6

2.5 2-5

8 (44.4) 7 (38.9) 3 (16.7)

8 (44.4) 7 (38.9) 3 (16.7)

*Minimal daily ICS dose capable of maintaining asthma control.

and the placebo group. Calculations were performed for a Mann-Whitney test on the basis of the assumption that the variability and distribution of our primary outcome was similar to that observed in a previous study18 and similar to our unpublished data on the steroid-sparing effect of SIT. An allowance was made for a 20% dropout rate. We compared the baseline characteristics of the patients by using x2 tests for discrete variables and the Student t test for continuous variables. To determine differences within and between the groups, the Student t test was used. Variables exhibiting a heavily skewed distribution were compared within and between groups by using Wilcoxon and Mann-Whitney

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FIG 2. Between-group comparisons of reduction of minimal daily dose of inhaled corticosteroid necessary to control the symptoms of asthma (% change; A), asthma symptoms score (points; B), and the resting FEV1 (% of predictive value; C). For between-group comparisons, Mann-Whitney tests were used. Reduction in ICS dose was calculated according to following rule: (ICS dose after the first year of SIT – ICS dose at baseline)/ICS dose at baseline 3 100%. m-s, Months; pred., predictive value.

tests, respectively. All analyses were performed on an intent-to-treat (ITT) basis, with statistical significance set at 5%. The intent-to-treat (ITT) population includes all patients who received intervention for at least 2 months. The perprotocol (PP) population includes all patients who completed the study according to the protocol and had no major protocol violations.

Ethics This study was approved by the Medical Ethics Committee of the Medical University. All parents or guardians provided written consent for the children’s participation in the study.

RESULTS Of 36 children, 29 completed the study according to the protocol. Seven patients were excluded: 5 patients from the

control group (because of changes in immunotherapy schedule and an increase in ICS dose as the reasons of deterioration in asthma symptoms) and 2 patients from montelukast group because of early discontinuation of intervention (no efficacy assessment). The timing and the reasons of discontinuation are indicated in Fig 1. Therefore, the ITT population (n 5 34) included all patients who completed the study according to the protocol (PP population, n 5 29) and patients excluded from the control group but treated more than 2 months (n 5 5). Compared with the montelukast group, the rate of dropouts from the PP population was significantly higher in the control group (P 5 .022). As shown in Table I, the treatment groups were similar in baseline characteristics.

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TABLE II. Measurements at baseline, and after 3 and after 12 months of SIT, premedicated with montelukast sodium (montelukast group) or placebo (control group) in patients with asthma* Visit 1 Baseline Variable

Clinical outcomes ICS dosek (mg/d), median (interquartile range) Asthma symptoms score (points)§ FEV1 (% predicted) Immunologic outcomes % CD41CD251Foxp31 cell Relative expression of Foxp3 IL-10 (in supernatant) (pg/mL) TGF-b1 (in supernatant) (pg/mL) IL-13 (in supernatant) (pg/mL)

Visit 2 After 3 months of SIT

Visit 3 After 12 months of SIT

Montelukast group (n 5 16)

Control group (n 5 13)

Montelukast group (n 5 16)

Control group (n 5 13)

Montelukast group (n 5 16)

Control group (n 5 13)

600 (400-600)

600 (400-600)

NA

NA

400 (300-400)à

200 (200-400)à

0.28 6 0.05

0.28 6 0.03

0.28 6 0.05

0.29 6 0.02

0.18 6 0.03à

0.17 6 0.03à

94.4 6 4.9

93.4 6 6.8

94.1 6 4.7

93.5 6 4.4

95.1 6 3.7

94.8 6 4.2

11.9 6 3.0 43.8 6 23.4

11.3 6 4.8 39.8 6 14.3

12.6 6 6.5 52.3 6 28.3

12.9 6 6.0 76.3 6 23.9à

39.7 6 9.9à 69.2 6 23.4à

51.3 6 8.2 à 78.8 6 22.6à

73.3 6 15.3 2330 6 1047 75.9 6 28.2

68.8 6 17.8 2567 6 1235 72.1 6 15.1

95.0 6 17.4à 3027 6 1309 90.6 6 36.4 

85.8 6 22.9  4625 6 1406à 88.4 6 19.4à

36.7 6 22.9à 1311 6 462à 44.7 6 20.6à

15.7 6 5.5 à 634 6 484à 25.6 6 9.5à

NA, Data not applicable. *Unless otherwise indicated, plus–minus values are means 6 SDs. FEV1 is prebronchodilator forced expiratory volume in one second. ‘‘Relative expression of Foxp3’’ denotes the number of FOXP3 cDNA molecules relative to the number of HPRT1 cDNA molecules. ‘‘% CD41CD251Foxp31’’ presents percentage of Foxp31 cells in the population of CD41CD251.  P < .05. àP < .01 for within-group comparison (vs visit 1). §Mean daily score calculated at baseline (data from September 2005 to the end of March 2006), after 3 months of SIT (data from build-up phase of SIT period) and after 12 months of SIT (data from September 2006 to the end of March 2007). k Minimal daily ICS dose capable of maintaining asthma control.

Clinical outcomes Analyses of clinical outcomes were conducted for the PP population only (n 5 29). We could not conduct analyses of clinical outcomes in whole ITT population (n 5 34) because changes in ICS doses in dropouts (n 5 5) could interfere with the main clinical study outcome: reduction in ICS dose during SIT. After 12 months of SIT, the median daily inhaled corticosteroid dose, necessary to control the symptoms of asthma, was reduced 33% in the group of children from montelukast group. However, a reduction of the median daily ICS dose was 50% (17% greater) in patients from the placebo group. Results of ICS dose reduction after 1 year of SIT were equally distributed between different baseline ICS doses (Fig 2). The main study outcomes at baseline and after 3 and 12 months of SIT in each study group are presented in Table II. We observed a significant reduction in the asthma symptoms score after 12 months of SIT within both study groups. However, differences between the groups were not significant (Fig 2). There were no significant changes in FEV1 in both groups. None of participants had an asthma exacerbation during the study. Immunologic outcomes After 3 months (at the end of the build-up phase of SIT), significant increases in Foxp3 expression and TGF-b level were observed only among patients from the control group (Table II). At the same time, a significant increase in IL-10 and IL-13 production in the PBMC cultures of patients from the control and montelukast groups was measured. In all groups, the percentage of Foxp3-positive cells did not change significantly after 3 months of SIT (Table II). After 12 months of SIT, a significantly smaller increase in the expression of Foxp3 and a smaller increase in the

percentage of Foxp3-positive cells were observed in patients from the montelukast group compared with the control group (Fig 3). After transient changes during the build-up phase of SIT, the levels of IL-10, TGF-b, and IL-13 significantly decreased in all study groups (Table II); however, the reductions were significantly lower in the montelukast group compared with the control group (Fig 3). The analysis of the ITT population (Fig 3) revealed no significant differences between study groups in all immunologic parameters. In patients from the placebo group excluded from the PP population because of the deterioration of asthma symptoms, we observed a diminished immunologic response to SIT in the percentage of Foxp3-positive cells, Foxp3 expression, and TGF-b level (Fig 3). Most of these patients were below the lower 95% CI in the change from baseline: in the percentage of Foxp3-positive cells after 12 months of SIT (4 of 5), in the Foxp3 expression after 3 and 12 months of SIT (5 of 5 and 4 of 5, respectively), and in TGF-b level after 3 and 12 months of SIT (4 of 5). The levels of IL-5 and IL-4 were not analyzed because of very low values that were similar to the manufacturer’s sensitivity cutoff levels. It is possible that conditions of PBMC cultures were not optimal for these cytokines. We used the dose of allergen and time of the PBMC stimulation similar to our previous observation on IL-10 production.9

Safety There were 22 local reactions (9 in the montelukast group vs 13 in the control group; P 5 .239). All these reactions occurred during administration of the maintenance dose. All reactions were immediate, with a diameter smaller than 5 cm. No systemic reactions were reported.

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FIG 3. Between-group comparisons of changes from baseline in the relative expression of Foxp3 (A), in the %CD41CD251Foxp31 cell (B), and in a supernatant concentration of IL-10 (C), TGF-b1 (D), and IL-13 (E). Comparisons were done after 3 and after 12 months (m-s) of SIT in the PP population (left) and in the ITT population (right). Open triangles indicate dropouts from the PP population. The relative expression of Foxp3 indicates the number of Foxp3 cDNA molecules normalized to the number of HPRT1 cDNA molecules. %CD41CD251Foxp31 indicates the percentage of Foxp31 cells in the population of CD41CD251.

DISCUSSION In this study, the addition of montelukast to ICS treatment in patients undergoing allergen-specific immunotherapy was associated with a reduced early clinical and immunologic effectiveness of SIT. However, the patients from the placebo group experienced the deterioration of asthma symptoms in the course of SIT more frequently, which led to exclusions from the PP population. In those cases, an impaired, early immunologic effect of SIT was observed. A direct link between the 5-lipoxygenase proinflammatory pathway and the regulatory mechanisms of immune response has

been previously suggested.10 More recently, it has been shown that leukotriene B4 dose-dependently decreased the percentage of Treg cells and the mRNA expression of Foxp3.19 Unfortunately, so far the immunologic effects and the precise mechanisms of interaction between the leukotriene inflammation pathway and immune regulation processes remain unclear. It has been found that montelukast inhibits TGF-b1 production at the mRNA and the protein levels.20 This phenomenon may have some consequences in the context of allergen-specific immunotherapy, because TGF-b is responsible for Foxp3 induction and maintains a suppressor function of CD41CD251Foxp31

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Treg cells.21 In the current study, after montelukast intervention, we also observed suppressed TGF-b1 production that was induced during SIT. Therefore, we suppose that a nonspecific regulation of TGF-b1 production may at least partially explain the suppressing effect of montelukast treatment on immunologic and consequently clinical effects of SIT in our study. Montelukast treatment was previously found to increase the production of IL-109; however, in vitro model, it failed to regulate directly the IL-10 production.22 In the current study, a simultaneous effect of SIT and montelukast treatment on IL-10 production was assessed. That is probably why the lack of changes in IL-10 concentration after montelukast intervention was noticed. Before immunotherapy, asthma symptoms were controlled in all our patients with inhaled corticosteroids; an additional treatment with montelukast could push the balance between the allergic inflammation and the anti-inflammatory effects of antiasthma treatment toward the suppression of natural regulatory mechanisms that control immunologic responses to allergen. The induction of Treg cells followed by increased production of regulatory cytokines such as IL-10 and TGF-b has been previously observed during effective SIT.6,7 In our study, increase in the production of IL-10 and TGF-b was only transient (first 3 months), and after 12 months of SIT levels of both cytokines significantly decreased. The reason for such discrepancy could be the use of the PBMCs for allergen-specific stimulation in our in vitro model. Because we did not observe any changes in the levels of cytokines produced by unstimulated PBMCs (data not shown) during SIT, a decrease cytokines production of stimulated PBMCs may be the indirect evidence of allergen tolerance induced by SIT. Importantly, the unfavorable effect of montelukast was no longer visible in the analysis of an ITT population. Several patients were excluded from the PP population because of deterioration in asthma symptoms followed by significant changes in SIT schedule. All 5 patients who were excluded because they had worsening asthma symptoms were not on montelukast. These patients had been stable for several months without asthma exacerbation and without requiring any increase in their inhaled steroid. After starting allergen immunotherapy they had asthma deterioration, which did require increases in inhaled steroid. This would seem to imply that the immunotherapy itself caused a worsening in asthma symptoms, and that the montelukast offered some protection from this. This phenomenon can be explained both by the fact that montelukast is able to protect respiratory tract responses induced by allergen administration,23 and by a previously shown positive effect of montelukast on viralinduced asthma exacerbations.24 Although the increase in ICS dose improved asthma control and allowed patients to reach a maintenance dose of allergen, in all those cases, a diminished, early immunologic effect of SIT was observed. Noteworthy is the fact that a diminished Foxp3-positive cell expansion in all drop-outs cases from per-protocol population could be the result of a more severe inflammation per se, because all the patients in our study received during 12 months of SIT almost the same cumulative dose of allergen. Our study has some limitations. Because of an unexpectedly high dropout rate in the control group, the intended sample size for the PP population has not been reached. However, the difference in the main study outcome has been shown, and the conclusion has not changed.

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Based on the asthma severity and treatment schedules, study groups were not homogenous (within-group); however, we did not observe the effect of this feature on the main study outcome (Fig 2). Although the only clinically relevant result was a slightly greater reduction in ICS (about 15%) in the placebo group and the difference between groups in immunologic outcomes was not large, in our opinion, routine use of montelukast during SIT is not justified. The benefit/risk ratio can be more favorable for montelukast in children with seasonal viral-induced asthma exacerbation,24,25 or in children with asthma with exerciseinduced bronchoconstriction14 or with concomitant allergic rhinitis.26 Our study failed to show a beneficial effect of montelukast on SIT. In fact, quite the opposite occurred: compared with placebo, montelukast intervention led to less effectiveness of SIT. We are indebted to Stallergenes Pharmaceutical Co for providing extracts for SIT and Der p 2 for in vitro stimulation. We also thank MSD Polska for providing montelukast.

Clinical implications: When children with asthma are being treated during SIT, routine use of montelukast is not justified. REFERENCES 1. Larche´ M, Akdis CA, Valenta R. Immunological mechanisms of allergen-specific immunotherapy. Nat Rev Immunol 2006;6:761-71. 2. Akdis M, Akdis CA. Mechanisms of allergen-specific immunotherapy. J Allergy Clin Immunol 2007;119:780-91. 3. Bohle B, Kinaciyan T, Gerstmayr M, Radakovics A, Jahn-Schmid B, Ebner C. Sublingual immunotherapy induces IL-10-producing T regulatory cells, allergenspecific T-cell tolerance, and immune deviation. J Allergy Clin Immunol 2007; 120:707-13. 4. Meiler F, Zumkehr J, Klunker S, Ruckert B, Akdis CA, Akdis M. In vivo switch to IL-10-secreting T regulatory cells in high dose allergen exposure. J Exp Med 2008; 205:2887-98. 5. Nagato T, Kobayashi H, Yanai M, Sato K, Aoki N, Oikawa K, et al. Functional analysis of birch pollen allergen Bet v 1-specific regulatory T cells. J Immunol 2007;178:1189-98. 6. Jutel M, Akdis M, Budak F, Aebischer-Casaulta C, Wrzyszcz M, Blaser K, et al. IL- 10 and TGF-beta cooperate in the regulatory T cell response to mucosal allergens in normal immunity and specific immunotherapy. Eur J Immunol 2003;33: 1205-14. 7. Radulovic S, Jacobson MR, Durham SR, Nouri-Aria KT. Grass pollen immunotherapy induces Foxp3-expressing CD41 CD251 cells in the nasal mucosa. J Allergy Clin Immunol 2008;121:1467-72. 8. Stelmach I, Jerzynska J, Kuna P. A randomized, double-blind trial of the effect of glucocorticoid, antileukotriene and beta-agonist treatment on IL-10 serum levels in children with asthma. Clin Exp Allergy 2002;32:264-9. 9. Stelmach I, Majak P, Jerzynska J, Kuna P. The effect of treatment with montelukast on in vitro interleukin-10 production of mononuclear cells of children with asthma. Clin Exp Allergy 2005;35:213-20. 10. Woszczek G, Chen LY, Nagineni S, Shelhamer JH. IL-10 inhibits cysteinyl leukotriene-induced activation of human monocytes and monocyte-derived dendritic cells. J Immunol 2008;180:7597-603. 11. W€ohrl S, Gamper S, Hemmer W, Heinze G, Stingl G, Kinaciyan T. Premedication with montelukast reduces local reactions of allergen immunotherapy. Int Arch Allergy Immunol 2007;144:137-42. 12. Stelmach I, Bobrowska-Korzeniowska M, Majak P, Stelmach W, Kuna P. The effect of montelukast and different doses of budesonide on IgE serum levels and clinical parameters in children with newly diagnosed asthma. Pulm Pharmacol Ther 2005;18:374-80. 13. Maestrelli P, Zanolla L, Pozzan M, Fabbri LM. Regione Veneto Study Group on the ‘‘Effect of immunotherapy in allergic asthma’’. Effect of specific immunotherapy added to pharmacologic treatment and allergen avoidance in asthmatic patients allergic to house dust mite. J Allergy Clin Immunol 2004;113:643-9. 14. Stelmach I, Grzelewski T, Majak P, Jerzynska J, Stelmach W, Kuna P. Effect of different antiasthmatic treatments on exercise-induced bronchoconstriction in children with asthma. J Allergy Clin Immunol 2008;121:383-9.

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