- Email: [email protected]
Sublingual immunotherapy for hazelnut food allergy: A randomized, double-blind, placebo-controlled study with a standardized hazelnut extract
Sublingual immunotherapy for hazelnut food allergy: A randomized, double-blind, placebo-controlled study with a standardized hazelnut extract Ernesto Enrique, MD, PhD,a Fernando Pineda, PhD,b Tamim Malek, MD,a Joan Bartra, MD, PhD,c Marı´a Basagan˜a, MD,d Raquel Tella, MD,c Jose´ Vicente Castello´, MD,a Rosario Alonso, MD,d Jose´ Antonio de Mateo, MD,a Teresa Cerda´-Trias, MD,c Marı´a del Mar San Miguel-Moncı´n, MD,d Susana Monzo´n, MD,d Marı´a Garcı´a, PhD,e Ricardo Palacios, PhD,b and Anna Cistero´-Bahı´ma, MD, PhDd Castello´n, Madrid, Girona, and Barcelona, Spain
From athe Allergy Department, Hospital General de Castello´n; bthe Investigacio´n y Desarrollo Department, Laboratorios Diater, Madrid; cthe Allergy Department, Hospital General de Girona Dr Joseph Trueta, Girona; dthe Allergy Department, Institut Universitari Dexeus, Universitat Auto`noma de Barcelona; and ethe Institut d’Investigacio´ Biome´dica, Girona. Disclosure of potential conflict of interest: F. Pineda and R. Palacios are employed by Research and Development, a private laboratory. The other authors have none to disclose. Received for publication June 22, 2005; revised August 16, 2005; accepted for publication August 16, 2005. Available online October 3, 2005. Reprint requests: Ernesto Enrique, MD, PhD, Allergy Department, Hospital General de Castello´n, Avda, Benicasim s/n, 12004 Castello´n, Spain. E-mail: [email protected]. 0091-6749/$30.00 Ó 2005 American Academy of Allergy, Asthma and Immunology doi:10.1016/j.jaci.2005.08.027
Conclusion: Our data confirm significant increases in tolerance to hazelnut after sublingual immunotherapy as assessed by double-blind, placebo-controlled food challenge, and good tolerance to this treatment. (J Allergy Clin Immunol 2005;116:1073-9.) Key words: Sublingual immunotherapy, food immunotherapy, hazelnut allergy, biological standardization in mass units, doubleblind, placebo-controlled food challenge, Cor a 1, Cor a 8
Food allergy, like other atopic disorders, appears to be on the increase. Moreover, food allergy remains a leading cause of anaphylaxis treated in emergency departments in several countries, and the general public has become increasingly aware of the problem.1 Peanut and tree nuts are some of the most allergenic foods worldwide.2 In the United States, peanut and tree nut allergy affects approximately 1.1% of the population.3 In the tree nut group, hazelnut (Corylus avellana) is frequently implicated in allergic reactions.4 In a recent report, Pastorello et al5 analyzed the allergen profile of patients with hazelnut allergic reactions and positive double-blind, placebo-controlled food challenge (DBPCFC). All sera from patients recognized an 18-kd allergen; other major allergens recognized were at molecular weights of 47, 32, and 35 kd. The 18-kd allergen, Cor a 1, is a protein homologous to Bet v 1 allergen. Patients with severe allergic reactions to hazelnut showed IgE reactivity to a 9-kd allergen.5 This allergen was shown to be a hazelnut lipid transfer protein, registered as Cor a 8, and it is a major allergen for Spanish patients with allergy to hazelnut without birch pollen allergy.6 In Europe, it has been estimated that hazelnut allergy affects between 0.1% and 0.5% of the population.7,8 Hazelnut is largely used in prepackaged foods, particularly in the production of pastry and ice creams. The wide use of hazelnut in the food industry represents a considerable risk for subjects with hazelnut allergy, because trace quantities can induce systemic allergic reactions in highly sensitized individuals. Moreover, some food labels may not list a small quantity of a foodstuff, or this may be included as a contaminant, with accidental ingestion 1073
Food allergy, dermatologic diseases, and anaphylaxis
Background: Food allergy may be life-threatening, and patients affected need to receive accurate diagnoses and treatment. Hazelnut has often been implicated as responsible for allergic reactions, and trace quantities can induce systemic reactions. Objective: The aim of this study was to evaluate the efficacy and tolerance of sublingual immunotherapy with a standardized hazelnut extract in patients allergic to hazelnut. Methods: This was a randomized, double-blind, placebocontrolled study. Inclusion criteria were a history of hazelnut allergy and positive skin prick test and double-blind placebocontrolled food challenge results. Patients were then randomly assigned into 2 treatment groups (hazelnut immunotherapy or placebo). Efficacy was assessed by double-blind, placebocontrolled food challenge after 8 to 12 weeks of treatment. Blood samples were drawn for measurement of specific IgE, IgG4, and serum cytokines before and after treatment. Results: Twenty-three patients were enrolled and divided into 2 treatment groups. Twenty-two patients reached the planned maximum dose at 4 days. Systemic reactions were observed in only 0.2% of the total doses administered. Mean hazelnut quantity provoking objective symptoms increased from 2.29 g to 11.56 g (P 5 .02; active group) versus 3.49 g to 4.14 g (placebo; NS). Moreover, almost 50% of patients who underwent active treatment reached the highest dose (20 g), but only 9% in the placebo. Laboratory data showed an increase in IgG4 and IL-10 levels after immunotherapy in only the active group.
1074 Enrique et al
Abbreviations used AU: Allergen unit DBPCFC: Double-blind, placebo-controlled food challenge n: Native r: Recombinant SLIT: Sublingual immunotherapy SPT: Skin prick test
possible despite efforts at avoidance in unexpected food products.9 The management of food allergies continues to consist of educating patients on how to avoid relevant allergens, recognize early symptoms of an allergic reaction in cases of accidental ingestion, and initiate the appropriate emergency therapy. Nevertheless, because of the high risk of life-threatening allergic reactions and the difficulty in avoidance of the culprit food, allergen-specific immunotherapy has been studied as a treatment option. However, subcutaneous immunotherapy was accompanied by a high rate of systemic reactions.10,11 The aim of this study was to evaluate the response to sublingual immunotherapy (SLIT) with hazelnut extract standardized in unit masses of major allergens, Cor a 1 and Cor a 8, in a large group of patients allergic to hazelnut. Efficacy was assessed by double-blind, placebocontrolled food challenges with hazelnut before SLIT and after 2 months of immunotherapy maintenance.
METHODS Patients and study design
J ALLERGY CLIN IMMUNOL NOVEMBER 2005
equal to or larger than the wheal obtained with histamine 10 mg/mL 15 minutes after testing was judged as positive. Each patient was also skin prick tested in duplicate on the volar surface of the forearm with four 10-fold serial dilutions (10, 1, 0.1, and 0.01 mg/mL) of a raw hazelnut extract (Diater Laboratorios). Wheal areas were marked with a fine-tipped ball-point pen and transferred by transparent adhesive tape onto paper for subsequent planimetric evaluation and statistical analysis. Skin wheal areas were determined by computer scanning (AutoCAD; Autodesk, Inc, San Rafael, Calif) in all patients.
DBPCFC Double-blind, placebo-controlled food challenges to hazelnut were performed in all patients. A single common protocol, based on a previous European multicenter study, was adhered to in the 3 centers.4 Patients who had systemic reactions or anaphylaxis were also challenged, but they were started with a modified schedule proposed by Alonso et al,12 after which they continued with the normal protocol, always within the double-blind, placebo-controlled schedule.
Allergen sources and extracts Raw hazelnuts were used for oral challenges and SPT and as source material for production of allergen extracts. The extract was prepared as follows: nuts were defatted with diethyl ether in Soxhlet system and proteins extracted with PBS with 4% Tween 20 at a 50% (wt/vol) ratio by stirring for 1 hour at 4°C. The soluble fraction was separated by centrifugation at 22,000g for 20 minutes at 4°C. The hazelnut extract was then dialyzed against distilled water, filtered, and lyophilized. The hazelnut extract was used for SPT, IgE, and IgG4 measurements. Recombinant hazelnut allergens recombinant (r) Cor a 1 (Biomay, Vienna) and rCor a 8 (Paul Ehrlich Institut, Langen, Germany) were used to obtain rabbit polyclonal antibodies a-rCor a 1 and a-rCor a 8, and biotinylated antibodies against native (n) Cor a 1 and nCor a 8 by affinity purification.13,14 Protein concentration was determined by the Bradford15 method.
Food allergy, dermatologic diseases, and anaphylaxis
This was a randomized, double-blind, placebo-controlled, multicenter study. Patients provided written informed consent. The study was approved by the ethics committees of the participating hospitals. Potential subjects were preselected on the basis of a clear history of hazelnut food allergy and positive skin prick test to hazelnut. Definitive inclusion criteria then also included a positive DBPCFC with hazelnuts. Eligible patients could not be pregnant, and any asthma had to be under control. Systemic corticosteroids and b-blockers were prohibited before screening and throughout the study, and antihistamines and antidepressants were prohibited for 1 week and 2 weeks, respectively, before skin testing and oral food challenge. Patients with systemic diseases and/or oral infection or inflammation not compatible with the correct, easy, and safe administration of the treatment were excluded from the study. Before the onset of treatment, a titrated skin prick test (SPT) with hazelnut extract was performed, and blood samples were drawn for in vitro standardization of a hazelnut extract in mass units of the hazelnut major allergens Cor a 1 and Cor a 8.5,6 Patients were later randomly assigned to 2 treatment groups (hazelnut extract or placebo). They then underwent a final DBPCFC 8 to 12 weeks later. At that time, a titrated SPT with hazelnut extract was repeated, and blood samples were again drawn for in vitro studies.
nCor a 1 and nCor a 8–specific IgE antibodies were measured by an ELISA experiment similar to that described previously16,17 with some modification. In brief, plates were coated with 0.02 mg/mL of nCor a 1 and nCor a 8. A 50-mL serum sample from each patient (before and after immunotherapy) was incubated for 2 hours at room temperature. The bound IgE were detected by biotinylated mouse monoclonal antihuman IgE antibody (1:1000, 50 mL/well; Operon, Cuarte de Huerva, Zaragoza, Spain) followed by streptavidin–horseradish peroxidase—labeled antimouse IgG antibody (1:5000, 50 mL/ well; Sigma-Aldrich, St Louis, Mo); IgE binding was detected by using a solution of 3,3#,5#-tetramethyl-benzidine (50 mL/well; Sigma), and optical densities were read at 450 nm.
SPT
Human serum cytokines
All patients underwent SPT performed according to standard procedure with timothy, parietaria, mugwort, olive, plane tree, birch, and hazel extracts (Diater Laboratorios, Madrid, Spain). A wheal size
Human serum IL-4, IL-5, IL-10, TGF-b, and IFN-g concentrations were measured by ELISA using reagent kits of BLK (Biolink 2000, Barcelona, Spain), according to the manufacturer’s instructions.
Specific IgE Specific IgE against hazelnut was measured by an enzyme allergosorbent test (Hycor Biomedical Inc, Garden Grove, Calif) according to the manufacturer’s instructions. Levels higher than 0.35 IU/mL were considered positive (Hytec-specific IgE enzyme immunoassay, Hycor Biomedical Inc).
ELISA for specific IgE
Enrique et al 1075
J ALLERGY CLIN IMMUNOL VOLUME 116, NUMBER 5
TABLE I. Concentration of vials and build-up treatment schedule* Daily dose (mg) Day
1
Vial concentration (mg/mL)
Drops
211
(F0) 66.25 3 10
(F1) 66.25 3 1028 2
25
(F2) 66.25 3 10
(F3) 66.25 3 1022 3
(FA) 66.25
4
(FA) 66.25
1 10 1 10 1 10 1 10 1 2 3 4 10 25
Administered dose (mg)
Cumulative dose (mg)
211
Cor a 1
Cor a 8
5.68 3 1027
3.68 3 1027
211
2 3 10 2 3 10210 2 3 1028 2 3 1027 2 3 1025 2 3 1024 0.02 0.26 2.65 5.30 7.95 10.60 26.50 66.25
2 3 10 2 3 10210 2 3 1028 2 3 1027 2 3 1025 2 3 1024 0.02 0.26 2.91 8.21 16.16 26.76 53.26 119.51
0.99
0.48
150.5 188.15
97.5 121.9
*Doses were administered at 15-minute intervals.
The active specific immunotherapy consisted of a biologically standardized hazelnut extract, graded in 5 strengths (F0, F1, F2, F3, FA) in glycerosaline solution. The protein concentration of each vial in milligrams per milliliter and the treatment schedule are detailed in Table I. Placebo was prepared as saline solution in vials with exactly the same appearance, color, and taste, but without allergens, to guarantee the double-blind design of the trial. All vials contained 50% glycerin (vol/vol) and 0.3% phenol (wt/vol). All doses of the build-up phase were administered in a hospital setting with the availability of complete resuscitation equipment and trained personnel; the patient was kept under constant observation after each administration and for at least 60 minutes after each day’s last administration. Patients were instructed to keep the allergen solution in the mouth for at least 3 minutes and then to discharge (sublingual-discharge technique). The build-up phase was completed in 4 days according to a rush schedule. During the build-up phase, doses were administered at 15 minutes intervals.18 After the build-up phase, all patients followed the same daily maintenance schedule consisting of 5 drops of the maximum concentration performed at home. SLIT was administered from November 2004 to March 2005 before the birch and hazel pollen season. During the maintenance phase, patients attended the clinic every 21 days.
TABLE II. Demographic characteristics and allergy history of patients randomized in the study (n 5 23)* Characteristics
Sex Male Female Age, y, mean (range) Hazelnut specific IgE, kU/L, mean (quartiles) Associated pollen allergy Grass Mugwort Plane tree Olea Parietaria Birch Hazel Hazelnut allergy symptoms Oral allergy syndrome Anaphylaxis Urticaria-angioedema
Active treatment (n 5 12)
3 9 29.2 5.34 8 5 6 5 5 0 2 1
(25%) (75%) (19-53) (0.45-9.10) (66.6%) (41.6%) (50%) (41.6%) (41.6%) (0%) (16.6%) (8.3%)
6 (54.5%) 5 (45.5%) 0
Placebo (n 5 11)
7 4 29.6 6.41 10 5 5 4 7 4 3 3
(63.3%) (36.7%) (19-42) (4.02-9.91) (81.8%) (44.4%) (44.4%) (36.3%) (63.6%) (36.3%) (27.2%) (27.2%)
6 (54.5%) 1 (9%) 4 (36.5%)
*No statistical differences were found between placebo and active group for all of the parameters.
Statistics Results were expressed as percentages for categorical variables and as mean or median (quartiles) for continuous variables. Proportions were compared by using the x2 test, and the t test and Mann-Whitney test were used to compare continuous variables between the 2 treatment groups. The effect of treatment on interleukin levels, TGF-b, IFN-g, IgG4, and IgE was analyzed by generalized linear models (ANOVA) for repeated measurements. Significance level was 5%, and the statistical program used was SPSS 10.0 (1999; SPSS Inc, Chicago, Ill).
RESULTS Study population Forty-one adult patients age 18 to 60 years were selected on the basis of a history of hazelnut allergy and
positive SPT. DBPCFCs to hazelnut were performed in all of the patients and were positive in 29. All of these 29 patients (M/F, 17/12; mean age, 29.4 years; range, 19-53 years) were included to be randomized. Six of the 29 withdrew their consent for personal reasons, and 23 were divided into 2 groups of treatment: active group (n 5 12) and placebo (n 5 11). One patient of the treatment group withdrew consent for professional reasons the first day of starting the immunotherapy. The main demographic characteristics and allergy history of the randomized patients are summarized in Table II and in Fig 1. No statistical differences were found between placebo and active groups for sex; age; associated pollen sensitizations; clinical symptoms related to
Food allergy, dermatologic diseases, and anaphylaxis
SLIT
1076 Enrique et al
J ALLERGY CLIN IMMUNOL NOVEMBER 2005
FIG 2. Oral challenge tests: mean food amounts tolerated in patients with hazelnut SLIT and placebo SLIT before and after immunotherapy.
FIG 1. Oral challenge test results (quantity of hazelnut provoking objective symptoms). *Cor a 8–specific IgE sensitive patients. dCor a 1–specific IgE sensitive patients. A, Anaphylaxis; OAS, oral allergy syndrome; U, urticaria; U-AE, urticaria-angioedema.
hazelnut ingestion; and levels of serum specific IgE to hazelnut, Cor a 1, or2 Cor a 8.
Food allergy, dermatologic diseases, and anaphylaxis
SPT and hazelnut extract standardization Each patient was also skin prick tested with a raw hazelnut extract to determine the in vivo reactivity of the extract. A concentration of 2.69 mg/mL hazelnut extract was calculated to cause a wheal size equal to that obtained with histamine. For this raw hazelnut extract, 1 mg corresponded to 1.84 mg of the allergen Cor a 8 and 2.88 mg of the allergen Cor a 1 (data not shown). Oral food challenges: efficacy assessment Allergic reactions to hazelnut observed during DBPCFC were generally well controlled by stopping the oral food challenge and treatment with epinephrine, antihistamines, and corticosteroids, as appropriate. No patient required overnight hospitalization, and none of the patients who had anaphylaxis presented severe symptoms or hypotension. The mean quantity of hazelnut provoking objective symptoms increased from 2.29 g to 11.56 g (P 5 .02) in the active group and from 3.49 g to 4.14 g in the placebo group (without statistical significance; Fig 2). Furthermore, almost 50% of patients (5/11) who underwent hazelnut SLIT reached the highest level (20 g) compared with 9% (1/11) of the placebo group (Fig 1). This beneficial effect has been observed in patiens with local reactions and also in patients with systemic reactions after hazelnut ingestion.
Tolerance and adverse reactions: safety assessment All patients reached the planned maximum dose of 25 drops from the most concentrated vial in 4 days. A total of 1466 doses were administered: 309 during the build-up phase and 1157 during maintenance. Systemic reactions were observed in 0.2% (3 reactions/ 1466 doses). They appeared only during the build-up phase, and only antihistamines were required for their control. One facial urticaria occurred in the placebo group and 2 reactions in 1 patient of the active group: skin pruritus a few minutes after the last dose of the second day and 1 delayed urticaria several hours after the last dose of the third day, probably associated with exercise (the patient had urticaria after 1 hour of exercise). Local reactions, mainly in the form of immediate oral itching, were observed in 7.4% (109 reactions/1466 doses). Four patients in the active group reported abdominal pain several hours after the ingestion on 1 occasion each and only during the build-up phase. All local reactions during the maintenance phase were also oral itching, and all were in the same patient. Laboratory parameters Hazelnut-specific IgE levels did not differ between groups before treatment but were lower in both groups after treatment, with no statistical significance both between both groups and within the same group (data not shown). As expected, because patients selected came from birch-free areas, prevalence to Cor a 1–specific IgE was low (22.14%), whereas Cor a 8–specific IgE was present in 50% of the patients selected (Fig 1). Regarding Cor a 1 and Cor a 8 specific IgE, there were no differences between groups before treatment; however, lower rates were observed in both groups after treatment, with no statistical significance within and between both groups (data not shown). Regarding hazelnut-specific IgG4 levels, no statistical significance was found between the studied groups; however, in the active group, an increase in mean IgG4 levels from 7.34 allergen units (AU)/mL to 9.84 AU/mL (P < .05) was observed (Fig 3).
Enrique et al 1077
J ALLERGY CLIN IMMUNOL VOLUME 116, NUMBER 5
An increase in IL-10 levels after treatment was found only in the active group, rising from 1.62 pg/mL to 2.24 pg/mL (P < .05). No differences were observed between the placebo and active groups (Fig 3). We were unable to obtain results of the other cytokines because of the low sensitivity of the tests used.
DISCUSSION Food allergy is potentially life-threatening, and there is no known curative treatment to date. The recommended treatment for individuals with known anaphylactic reactions to foods is avoidance and use of injected epinephrine as soon as they become aware of an inadvertent ingestion. However, avoidance of accidental ingestion is difficult when eating away from home, and many people with known food sensitivity do not routinely carry epinephrine with them.19 Therefore, an improved treatment is needed for anaphylactic reactions to foods. Previous studies have used subcutaneous peanutspecific immunotherapy and have showed a decrease in SPT reactions and decreased sensitivity to peanut ingestion. However, this therapy was accompanied by a high rate of systemic reactions.10,11 Oral desensitization to foods has been reported in numerous limited series but with varying results.20,21 Although oral desensitization is an attractive procedure, well-designed studies are required. Other novel immunotherapeutic approaches have been reported with good results: anti-IgE therapy,22 Chinese herbal medicines,23 the uses of subcutaneous birch pollen immunotherapy in patients with oral allergy syndrome,24,25 and other laboratory studies with animal models.26 Food immunotherapy has recently been suggested again as a food allergy treatment in 2 case report publications: one, oral immunotherapy in patients allergic to peanut,27 and the other, sublingual immunotherapy in a case of anaphylactic reaction to kiwi.28 Nevertheless, double-blind, placebo-controlled randomized studies are required to confirm these results.
The aim of this study was to evaluate a large group of patients allergic to hazelnut for their response to a sublingual immunotherapy with a biological extract standardized in unit mass of major allergens. In the current study, efficacy was assessed by DBPCFC with hazelnut performed before and after 2 to 3 months of maintenance immunotherapy. Our clinical data confirm significant increases in the threshold of sensitivity to hazelnut allergen to a level that should translate into at least partial protection against most unintended ingestion of hazelnuts. Moreover, almost 50% of the patients who underwent active treatment tolerated the highest dose of 20 g, about 15 to 20 hazelnuts, which constitutes a high amount of hazelnuts that should translate into enough protection against most unintended ingestion of hazelnuts. Surprisingly, 1 patient (Cor a 1 sensitive) who received placebo tolerated the same highest dose of 20 g, and another patient who received placebo, with the same characteristic allergen profile, increased his tolerance from 2 to 10 g hazelnut (Fig 1). These patients presented oral allergy syndrome in the DBPCFC and had hazel pollinosis. Although the second challenge test was performed before hazel pollen season, it is tempting to speculate on the possibility of a seasonal variation in hazelnut pollen– dependent allergy.29 Mean provocative dose in a previous study ranged from 1.4 and 2.7 g in patients with hazelnut allergy from Zurich and Copenhagen to 15.3 g in patients from Milan.4 Nevertheless, the current study is conducted in a birch-free area, contrary to Zurich, Copenhagen, and Milan which are in an area of birch pollen exposure. We found a mean provocative hazelnut dose similar to data observed in Zurich and Copenhagen; however, Cor a 8 was described as a major allergen in Milan and also in the geographical area of the current study.4,6 Regarding safety aspects, in the current study, we observed very good tolerance because the rate of side effects was very low and appeared in both groups of treatment. Although the sublingual-discharge technique seems to lead to better tolerance than the sublingual-swallow
Food allergy, dermatologic diseases, and anaphylaxis
FIG 3. A, IgG4 levels before and after treatment. B, IL-10 levels before and after treatment.
1078 Enrique et al
Food allergy, dermatologic diseases, and anaphylaxis
technique, mainly for gastrointestinal symptoms, 4 patients reported abdominal pain. Nevertheless, a relative high rate of gastrointestinal symptoms has been reported with sublingual-swallow immunotherapy with latex,18 and also with a high dose of parietaria extract.30 Our data also confirm regional differences in sensitization pattern reported for other birch pollen–related foods.5,6 In a previous study conducted from Germany in a group of patients with hazelnut allergy and birch pollen allergy, the majority were sensitized to the pollen-related hazelnut allergen Cor a 1.31 In other studies, Cor a 8 (hazelnut lipid transfer protein) was significant for a majority of patients recruited in Spain or Northern Italy.5,6 In this study, we partially repeat the same results. Nevertheless, some patients of the current study with systemic reactions did not react to Cor a 1 or to Cor a 8, suggesting the possible presence of other important pollen-related or nonpollenrelated hazelnut allergens, such as Cor a 9, an 11S globulin related to hazelnut-induced systemic reactions.32 Immunotherapy is capable of modifying the immunologic response to the offending allergen in the earliest stages. The subcutaneous route of administration has been well established, and its mechanisms of action have been extensively investigated.33 However, the clinical use of SLIT is recent. Therefore, some aspects have not been fully investigated, and data on the mechanisms of action are few and contradictory. A recent prospective study of SLIT in children with allergic rhinitis found positive effects on rescue and medication use but observed no significant effects on in vitro T-cell immune responses or immunoglobulins34; such effects were demonstrated in another double-blind trial35 and in a case report of a patient with severe anaphylaxis caused by kiwi allergy treated with sublingual kiwi immunotherapy with good tolerance and efficacy.27 In the current study, we observed, on the one hand, a decrease in specific IgE levels in both treatment groups and on the other hand, an increase in specific IgG4 and IL-10 levels only in the active group, suggesting the possibility of immunoregulation through regulatory T cells producing IL-10 and the oral mucosa Langerhans cells.36-38 In conclusion, this randomized and controlled clinical trial of sublingual immunotherapy performed in patients allergic to hazelnut has found significant increases in the threshold of sensitivity to hazelnut allergen, as assessed by DBPCFC after hazelnut SLIT, and good tolerance to this treatment. This beneficial effect has been observed both in local reactions and in systemic reactions after hazelnut ingestion. Nevertheless, further studies and others on the long-term efficacy of SLIT with food extracts are required. We thank Dr Stephan Scheurer from the Paul-Ehrlich Institut (Langen, Germany) for providing Cor a 8. We acknowledge Teresa Pin˜o´n, Dolores Sa´nchis, Ine´s Saz, Marihor Aguilar, Josefa Fuentes, and Magdalena Sa´nchez for technical assistance. We also thank Ms Christine O’Hara for English review of the manuscript. REFERENCES 1. Sampson HA. Update on food allergy. J Allergy Clin Immunol 2004; 113:805-9.
J ALLERGY CLIN IMMUNOL NOVEMBER 2005
2. Bjo¨rkste´n B. How allergenic is food? Clin Exp Allergy 2004;34:673-5. 3. Sicherer SH, Mun˜oz-Furlong A, Burks W, Sampson HA. Prevalence of peanut and tree nut allergy in the US determined by a random digit dial telephone survey. J Allergy Clin Immunol 1999;103:559-62. 4. Ortolani C, Ballmer-Weber BK, Skamstrup Hansen K, Ispano M, Wu¨trich B, Bindslev-Jensen C, et al. Hazelnut allergy: a double-blind, placebo-controlled food challenge multicenter study. J Allergy Clin Immunol 2000;105:577-81. 5. Pastorello EA, Vieths S, Pravettoni V, Farioli L, Trambaioli C, Fortunato D, et al. Identification of hazelnut major allergens in sensitive patients with positive double-blind, placebo-controlled food challenge results. J Allergy Clin Immunol 2002;109:563-70. 6. Schocker F, Lu¨ttkopf D, Scheurer S, Petersen A, Cistero´-Bahı´ma A, Enrique E, et al. Recombinant lipid transfer protein Cor a 8 from hazelnut: a new tool for in vitro diagnosis of potentially severe hazelnut allergy. J Allergy Clin Immunol 2004;113:141-7. 7. Tariq SM, Stevens M, Rideout S, Twinselton R, Hide RW. Cohort study of peanut and tree nut sensitization by age of 4 years. BMJ 1996;313:514-7. 8. Groot H, Jong NW, Vuijk MH, Gerth van Wijk R. Birch pollinosis and atopy caused by apple, peach, and hazelnut; comparison of three extraction procedures with two apple strains. Allergy 1996;51:712-8. 9. Steinman H. ‘‘Hidden’’ allergens in foods. J Allergy Clin Immunol 1996; 98:241-50. 10. Oppenheimer JJ, Nelson HS, Bock SA, Christensen F, Leung DYM. Treatment of peanut allergy with rush immunotherapy. J Allergy Clin Immunol 1992;90:256-62. 11. Nelson HS, Lahr J, Rule R, Bock A, Leung D. Treatment of anaphylactic sensitivity to peanuts by immunotherapy with injections of aqueous peanut extract. J Allergy Clin Immunol 1997;99:744-51. 12. Alonso R, Enrique E, San Miguel-Moncı´n MM, Tella R, Ferna´ndezParra B, Bartra J, et al. An alternative challenge in oral allergy syndrome. J Allergy Clin Immunol 2004;113:S152. 13. Chapman MD, Heymann PW, Wilkins SR, Brown MJ, Platts-Mills TA. Monoclonal immunoassays for major dust mite (Dermatophagoides) allergens, Der p 1 and Der f 1, and quantitative analysis of the allergen content of mite and house dust extracts. J Allergy Clin Immunol 1987;80:184-94. 14. O’Shannessy DJ, Dobersen MJ, Quarles RH. A novel procedure for labeling immunoglobulins by conjugation to oligosaccharide moieties. Immunol Lett 1984;8:273-7. 15. Bradford M. A rapid and rensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Ann Biochem 1976;72:248-54. 16. Boluda L, Alonso C, Ferna´ndez Caldas E. Purification, characterization and partial sequencing of 2 new allergens of Olea europaea. J Allergy Clin Immunol 1998;101:210-6. 17. Boluda L, Sastre J, Casanovas M, Fernandez Caldas E. Determination of Ole e 1 by enzyme immunoassay and scanning densitometry: validation by skin-prick testing. J Immunol Methods 1999;223:17-26. 18. Cistero´-Bahı´ma A, Sastre J, Enrique E, Ferna´ndez M, Alonso R, Quirce S, et al. Tolerance and effects on skin reactivity to latex of sublingual rush immunotherapy with latex extract. J Invest Allergol Clin Immunol 2004;14:17-25. 19. Bock SA, Mun˜oz-Furlong A, Sampson HA. Fatalities due to anaphylactic reactions to foods. J Allergy Clin Immunol 2001;107:191-3. 20. Bauer A, Ekanayake MS, Wigger-Alberti W, Elsner P. Oral rush desensitization to milk. Allergy 1999;54:894-5. 21. Patriarca G, Nucera E, Roncallo C, Pollastrini E, Bartolozzi F, De Pasquale T, et al. Oral desensitizing treatment in food allergy: clinical and immunological results. Aliment Pharmacol Ther 2003;17:459-65. 22. Leung DYM, Sampson HA, Yunginger JW, Burks AW, Schneider LC, Wortel CH, et al. Effect of anti-IgE therapy in patients with peanut allergy. N Engl J Med 2003;348:986-93. 23. Srivastava KD, Kattan JD, Zou ZM, Li JH, Zhang L, Wallenstein S, et al. The Chinese herbal medicine formula FAHF-2 completely blocks anaphylactic reactions in a murine model of peanut allergy. J Allergy Clin Immunol 2005;115:171-8. 24. Bucher X, Pichler WJ, Dahinden CA, Helbing A. Effect of tree pollen specific, subcutaneous immunotherapy on the oral allergy syndrome to apple and hazelnut. Allergy 2004;59:1272-6. 25. Blotter ST, Tiemessen MM, Zuidmeer L, van Leeuwen A, HoffmanSommergruber K, Bruijnzeel-Koomen CAFM, et al. Efficacy of birchpollen immunotherapy on cross-reactive food allergy confirmed by
26. 27.
28.
29.
30.
31.
skin tests and double-blind food challenges. Clin Exp Allergy 2004;34: 761-9. Eigenmann PA. Future therapeutic options in food allergy. Allergy 2003; 58:1217-23. Scurlock AM, Steele PH, Andrzejewski SS, Pons LA, Althage KA, Christie L, et al. Safety of oral peanut immunotherapy for peanut allergic patients. J Allergy Clin Immunol 2005;115:S246. Mempel M, Rakoski J, Ring J, Ollert M. Severe anaphylaxis to kiwi fruit: Immunologic changes related to successful sublingual allergen immunotherapy. J Allergy Clin Immunol 2003;111:1406-9. Skamstrup Hansen K, Vieths S, Vestergaard H, Skov PS, BindslevJensen C, Poulsen JH. Seasonal variation in food allergy to apple. J Chromatogr B Biomed Sci Appl 2001;756:19-32. La Rosa M, Ranno C, Andre´ C, Carat F, Tosca MA, Canonica GW. Double-blind, placebo-controlled evaluation of sublingual-swallow immunotherapy with standardized Parietaria judaica extract in children with allergic rhinoconjunctivitis. J Allergy Clin Immunol 1999;104: 425-32. Schocker F, Lu¨ttkopf D, Mu¨ller U, Thomas P, Vieths S, Becker W-M. IgE binding to unique hazelnut allergens: identification of non-pollenrelated and heat-stable hazelnut allergens eliciting severe allergic reactions. Eur J Nutr 2000;39:172-80.
Enrique et al 1079
32. Beyer K, Grishina G, Bardina L, Grishin A, Sampson HA. Identification of an 11 S globulin as a major hazelnut food allergen in hazelnut-induced systemic reactions. J Allergy Clin Immunol 2002;110:517-23. 33. Till SJ, Francis JN, Nouri Aria K, Durham SR. Mechanisms of immunotherapy. J Allergy Clin Immunol 2004;113:1025-34. 34. Rollinck-Werninghaus C, Kopp M, Liebke C, Lange J, Whan U, Niggemann B. Lack of detectable alterations in immune responses during sublingual immunotherapy in children with seasonal allergic rhinoconjunctivitis to grass pollen. Int Arch Allergy Immunol 2005;136:134-41. 35. Lima MT, Wilson D, Pitkin L, Roberts A, Nouri Aria K, Jacobson M, et al. Grass pollen sublingual immunotherapy for seasonal rhinoconjunctivitis: a randomized controlled trial. Clin Exp Allergy 2002;32:507-14. 36. Sicherer SH, Leung DYM. Advances in allergic skin disease, anaphylaxis, and hypersensitivity to foods, drugs, and insects. J Allergy Clin Immunol 2005;116:153-63. 37. Francis JN, Till SJ, Durham SR. Induction of IL-101CD41CD251 T cells by grass pollen immunotherapy. J Allergy Clin Immunol 2003; 111:1256-61. 38. Allam JP, Novak N, Fuchs C, Asen S, Berge´ S, Appel T, et al. Characterization of dendritic cells from human oral mucosa: a new Langerhans’ cell type with high constitutive FceRI expression. J Allergy Clin Immunol 2003;112:141-8.
Food allergy, dermatologic diseases, and anaphylaxis
J ALLERGY CLIN IMMUNOL VOLUME 116, NUMBER 5
From athe Allergy Department, Hospital General de Castello´n; bthe Investigacio´n y Desarrollo Department, Laboratorios Diater, Madrid; cthe Allergy Department, Hospital General de Girona Dr Joseph Trueta, Girona; dthe Allergy Department, Institut Universitari Dexeus, Universitat Auto`noma de Barcelona; and ethe Institut d’Investigacio´ Biome´dica, Girona. Disclosure of potential conflict of interest: F. Pineda and R. Palacios are employed by Research and Development, a private laboratory. The other authors have none to disclose. Received for publication June 22, 2005; revised August 16, 2005; accepted for publication August 16, 2005. Available online October 3, 2005. Reprint requests: Ernesto Enrique, MD, PhD, Allergy Department, Hospital General de Castello´n, Avda, Benicasim s/n, 12004 Castello´n, Spain. E-mail: [email protected]. 0091-6749/$30.00 Ó 2005 American Academy of Allergy, Asthma and Immunology doi:10.1016/j.jaci.2005.08.027
Conclusion: Our data confirm significant increases in tolerance to hazelnut after sublingual immunotherapy as assessed by double-blind, placebo-controlled food challenge, and good tolerance to this treatment. (J Allergy Clin Immunol 2005;116:1073-9.) Key words: Sublingual immunotherapy, food immunotherapy, hazelnut allergy, biological standardization in mass units, doubleblind, placebo-controlled food challenge, Cor a 1, Cor a 8
Food allergy, like other atopic disorders, appears to be on the increase. Moreover, food allergy remains a leading cause of anaphylaxis treated in emergency departments in several countries, and the general public has become increasingly aware of the problem.1 Peanut and tree nuts are some of the most allergenic foods worldwide.2 In the United States, peanut and tree nut allergy affects approximately 1.1% of the population.3 In the tree nut group, hazelnut (Corylus avellana) is frequently implicated in allergic reactions.4 In a recent report, Pastorello et al5 analyzed the allergen profile of patients with hazelnut allergic reactions and positive double-blind, placebo-controlled food challenge (DBPCFC). All sera from patients recognized an 18-kd allergen; other major allergens recognized were at molecular weights of 47, 32, and 35 kd. The 18-kd allergen, Cor a 1, is a protein homologous to Bet v 1 allergen. Patients with severe allergic reactions to hazelnut showed IgE reactivity to a 9-kd allergen.5 This allergen was shown to be a hazelnut lipid transfer protein, registered as Cor a 8, and it is a major allergen for Spanish patients with allergy to hazelnut without birch pollen allergy.6 In Europe, it has been estimated that hazelnut allergy affects between 0.1% and 0.5% of the population.7,8 Hazelnut is largely used in prepackaged foods, particularly in the production of pastry and ice creams. The wide use of hazelnut in the food industry represents a considerable risk for subjects with hazelnut allergy, because trace quantities can induce systemic allergic reactions in highly sensitized individuals. Moreover, some food labels may not list a small quantity of a foodstuff, or this may be included as a contaminant, with accidental ingestion 1073
Food allergy, dermatologic diseases, and anaphylaxis
Background: Food allergy may be life-threatening, and patients affected need to receive accurate diagnoses and treatment. Hazelnut has often been implicated as responsible for allergic reactions, and trace quantities can induce systemic reactions. Objective: The aim of this study was to evaluate the efficacy and tolerance of sublingual immunotherapy with a standardized hazelnut extract in patients allergic to hazelnut. Methods: This was a randomized, double-blind, placebocontrolled study. Inclusion criteria were a history of hazelnut allergy and positive skin prick test and double-blind placebocontrolled food challenge results. Patients were then randomly assigned into 2 treatment groups (hazelnut immunotherapy or placebo). Efficacy was assessed by double-blind, placebocontrolled food challenge after 8 to 12 weeks of treatment. Blood samples were drawn for measurement of specific IgE, IgG4, and serum cytokines before and after treatment. Results: Twenty-three patients were enrolled and divided into 2 treatment groups. Twenty-two patients reached the planned maximum dose at 4 days. Systemic reactions were observed in only 0.2% of the total doses administered. Mean hazelnut quantity provoking objective symptoms increased from 2.29 g to 11.56 g (P 5 .02; active group) versus 3.49 g to 4.14 g (placebo; NS). Moreover, almost 50% of patients who underwent active treatment reached the highest dose (20 g), but only 9% in the placebo. Laboratory data showed an increase in IgG4 and IL-10 levels after immunotherapy in only the active group.
1074 Enrique et al
Abbreviations used AU: Allergen unit DBPCFC: Double-blind, placebo-controlled food challenge n: Native r: Recombinant SLIT: Sublingual immunotherapy SPT: Skin prick test
possible despite efforts at avoidance in unexpected food products.9 The management of food allergies continues to consist of educating patients on how to avoid relevant allergens, recognize early symptoms of an allergic reaction in cases of accidental ingestion, and initiate the appropriate emergency therapy. Nevertheless, because of the high risk of life-threatening allergic reactions and the difficulty in avoidance of the culprit food, allergen-specific immunotherapy has been studied as a treatment option. However, subcutaneous immunotherapy was accompanied by a high rate of systemic reactions.10,11 The aim of this study was to evaluate the response to sublingual immunotherapy (SLIT) with hazelnut extract standardized in unit masses of major allergens, Cor a 1 and Cor a 8, in a large group of patients allergic to hazelnut. Efficacy was assessed by double-blind, placebocontrolled food challenges with hazelnut before SLIT and after 2 months of immunotherapy maintenance.
METHODS Patients and study design
J ALLERGY CLIN IMMUNOL NOVEMBER 2005
equal to or larger than the wheal obtained with histamine 10 mg/mL 15 minutes after testing was judged as positive. Each patient was also skin prick tested in duplicate on the volar surface of the forearm with four 10-fold serial dilutions (10, 1, 0.1, and 0.01 mg/mL) of a raw hazelnut extract (Diater Laboratorios). Wheal areas were marked with a fine-tipped ball-point pen and transferred by transparent adhesive tape onto paper for subsequent planimetric evaluation and statistical analysis. Skin wheal areas were determined by computer scanning (AutoCAD; Autodesk, Inc, San Rafael, Calif) in all patients.
DBPCFC Double-blind, placebo-controlled food challenges to hazelnut were performed in all patients. A single common protocol, based on a previous European multicenter study, was adhered to in the 3 centers.4 Patients who had systemic reactions or anaphylaxis were also challenged, but they were started with a modified schedule proposed by Alonso et al,12 after which they continued with the normal protocol, always within the double-blind, placebo-controlled schedule.
Allergen sources and extracts Raw hazelnuts were used for oral challenges and SPT and as source material for production of allergen extracts. The extract was prepared as follows: nuts were defatted with diethyl ether in Soxhlet system and proteins extracted with PBS with 4% Tween 20 at a 50% (wt/vol) ratio by stirring for 1 hour at 4°C. The soluble fraction was separated by centrifugation at 22,000g for 20 minutes at 4°C. The hazelnut extract was then dialyzed against distilled water, filtered, and lyophilized. The hazelnut extract was used for SPT, IgE, and IgG4 measurements. Recombinant hazelnut allergens recombinant (r) Cor a 1 (Biomay, Vienna) and rCor a 8 (Paul Ehrlich Institut, Langen, Germany) were used to obtain rabbit polyclonal antibodies a-rCor a 1 and a-rCor a 8, and biotinylated antibodies against native (n) Cor a 1 and nCor a 8 by affinity purification.13,14 Protein concentration was determined by the Bradford15 method.
Food allergy, dermatologic diseases, and anaphylaxis
This was a randomized, double-blind, placebo-controlled, multicenter study. Patients provided written informed consent. The study was approved by the ethics committees of the participating hospitals. Potential subjects were preselected on the basis of a clear history of hazelnut food allergy and positive skin prick test to hazelnut. Definitive inclusion criteria then also included a positive DBPCFC with hazelnuts. Eligible patients could not be pregnant, and any asthma had to be under control. Systemic corticosteroids and b-blockers were prohibited before screening and throughout the study, and antihistamines and antidepressants were prohibited for 1 week and 2 weeks, respectively, before skin testing and oral food challenge. Patients with systemic diseases and/or oral infection or inflammation not compatible with the correct, easy, and safe administration of the treatment were excluded from the study. Before the onset of treatment, a titrated skin prick test (SPT) with hazelnut extract was performed, and blood samples were drawn for in vitro standardization of a hazelnut extract in mass units of the hazelnut major allergens Cor a 1 and Cor a 8.5,6 Patients were later randomly assigned to 2 treatment groups (hazelnut extract or placebo). They then underwent a final DBPCFC 8 to 12 weeks later. At that time, a titrated SPT with hazelnut extract was repeated, and blood samples were again drawn for in vitro studies.
nCor a 1 and nCor a 8–specific IgE antibodies were measured by an ELISA experiment similar to that described previously16,17 with some modification. In brief, plates were coated with 0.02 mg/mL of nCor a 1 and nCor a 8. A 50-mL serum sample from each patient (before and after immunotherapy) was incubated for 2 hours at room temperature. The bound IgE were detected by biotinylated mouse monoclonal antihuman IgE antibody (1:1000, 50 mL/well; Operon, Cuarte de Huerva, Zaragoza, Spain) followed by streptavidin–horseradish peroxidase—labeled antimouse IgG antibody (1:5000, 50 mL/ well; Sigma-Aldrich, St Louis, Mo); IgE binding was detected by using a solution of 3,3#,5#-tetramethyl-benzidine (50 mL/well; Sigma), and optical densities were read at 450 nm.
SPT
Human serum cytokines
All patients underwent SPT performed according to standard procedure with timothy, parietaria, mugwort, olive, plane tree, birch, and hazel extracts (Diater Laboratorios, Madrid, Spain). A wheal size
Human serum IL-4, IL-5, IL-10, TGF-b, and IFN-g concentrations were measured by ELISA using reagent kits of BLK (Biolink 2000, Barcelona, Spain), according to the manufacturer’s instructions.
Specific IgE Specific IgE against hazelnut was measured by an enzyme allergosorbent test (Hycor Biomedical Inc, Garden Grove, Calif) according to the manufacturer’s instructions. Levels higher than 0.35 IU/mL were considered positive (Hytec-specific IgE enzyme immunoassay, Hycor Biomedical Inc).
ELISA for specific IgE
Enrique et al 1075
J ALLERGY CLIN IMMUNOL VOLUME 116, NUMBER 5
TABLE I. Concentration of vials and build-up treatment schedule* Daily dose (mg) Day
1
Vial concentration (mg/mL)
Drops
211
(F0) 66.25 3 10
(F1) 66.25 3 1028 2
25
(F2) 66.25 3 10
(F3) 66.25 3 1022 3
(FA) 66.25
4
(FA) 66.25
1 10 1 10 1 10 1 10 1 2 3 4 10 25
Administered dose (mg)
Cumulative dose (mg)
211
Cor a 1
Cor a 8
5.68 3 1027
3.68 3 1027
211
2 3 10 2 3 10210 2 3 1028 2 3 1027 2 3 1025 2 3 1024 0.02 0.26 2.65 5.30 7.95 10.60 26.50 66.25
2 3 10 2 3 10210 2 3 1028 2 3 1027 2 3 1025 2 3 1024 0.02 0.26 2.91 8.21 16.16 26.76 53.26 119.51
0.99
0.48
150.5 188.15
97.5 121.9
*Doses were administered at 15-minute intervals.
The active specific immunotherapy consisted of a biologically standardized hazelnut extract, graded in 5 strengths (F0, F1, F2, F3, FA) in glycerosaline solution. The protein concentration of each vial in milligrams per milliliter and the treatment schedule are detailed in Table I. Placebo was prepared as saline solution in vials with exactly the same appearance, color, and taste, but without allergens, to guarantee the double-blind design of the trial. All vials contained 50% glycerin (vol/vol) and 0.3% phenol (wt/vol). All doses of the build-up phase were administered in a hospital setting with the availability of complete resuscitation equipment and trained personnel; the patient was kept under constant observation after each administration and for at least 60 minutes after each day’s last administration. Patients were instructed to keep the allergen solution in the mouth for at least 3 minutes and then to discharge (sublingual-discharge technique). The build-up phase was completed in 4 days according to a rush schedule. During the build-up phase, doses were administered at 15 minutes intervals.18 After the build-up phase, all patients followed the same daily maintenance schedule consisting of 5 drops of the maximum concentration performed at home. SLIT was administered from November 2004 to March 2005 before the birch and hazel pollen season. During the maintenance phase, patients attended the clinic every 21 days.
TABLE II. Demographic characteristics and allergy history of patients randomized in the study (n 5 23)* Characteristics
Sex Male Female Age, y, mean (range) Hazelnut specific IgE, kU/L, mean (quartiles) Associated pollen allergy Grass Mugwort Plane tree Olea Parietaria Birch Hazel Hazelnut allergy symptoms Oral allergy syndrome Anaphylaxis Urticaria-angioedema
Active treatment (n 5 12)
3 9 29.2 5.34 8 5 6 5 5 0 2 1
(25%) (75%) (19-53) (0.45-9.10) (66.6%) (41.6%) (50%) (41.6%) (41.6%) (0%) (16.6%) (8.3%)
6 (54.5%) 5 (45.5%) 0
Placebo (n 5 11)
7 4 29.6 6.41 10 5 5 4 7 4 3 3
(63.3%) (36.7%) (19-42) (4.02-9.91) (81.8%) (44.4%) (44.4%) (36.3%) (63.6%) (36.3%) (27.2%) (27.2%)
6 (54.5%) 1 (9%) 4 (36.5%)
*No statistical differences were found between placebo and active group for all of the parameters.
Statistics Results were expressed as percentages for categorical variables and as mean or median (quartiles) for continuous variables. Proportions were compared by using the x2 test, and the t test and Mann-Whitney test were used to compare continuous variables between the 2 treatment groups. The effect of treatment on interleukin levels, TGF-b, IFN-g, IgG4, and IgE was analyzed by generalized linear models (ANOVA) for repeated measurements. Significance level was 5%, and the statistical program used was SPSS 10.0 (1999; SPSS Inc, Chicago, Ill).
RESULTS Study population Forty-one adult patients age 18 to 60 years were selected on the basis of a history of hazelnut allergy and
positive SPT. DBPCFCs to hazelnut were performed in all of the patients and were positive in 29. All of these 29 patients (M/F, 17/12; mean age, 29.4 years; range, 19-53 years) were included to be randomized. Six of the 29 withdrew their consent for personal reasons, and 23 were divided into 2 groups of treatment: active group (n 5 12) and placebo (n 5 11). One patient of the treatment group withdrew consent for professional reasons the first day of starting the immunotherapy. The main demographic characteristics and allergy history of the randomized patients are summarized in Table II and in Fig 1. No statistical differences were found between placebo and active groups for sex; age; associated pollen sensitizations; clinical symptoms related to
Food allergy, dermatologic diseases, and anaphylaxis
SLIT
1076 Enrique et al
J ALLERGY CLIN IMMUNOL NOVEMBER 2005
FIG 2. Oral challenge tests: mean food amounts tolerated in patients with hazelnut SLIT and placebo SLIT before and after immunotherapy.
FIG 1. Oral challenge test results (quantity of hazelnut provoking objective symptoms). *Cor a 8–specific IgE sensitive patients. dCor a 1–specific IgE sensitive patients. A, Anaphylaxis; OAS, oral allergy syndrome; U, urticaria; U-AE, urticaria-angioedema.
hazelnut ingestion; and levels of serum specific IgE to hazelnut, Cor a 1, or2 Cor a 8.
Food allergy, dermatologic diseases, and anaphylaxis
SPT and hazelnut extract standardization Each patient was also skin prick tested with a raw hazelnut extract to determine the in vivo reactivity of the extract. A concentration of 2.69 mg/mL hazelnut extract was calculated to cause a wheal size equal to that obtained with histamine. For this raw hazelnut extract, 1 mg corresponded to 1.84 mg of the allergen Cor a 8 and 2.88 mg of the allergen Cor a 1 (data not shown). Oral food challenges: efficacy assessment Allergic reactions to hazelnut observed during DBPCFC were generally well controlled by stopping the oral food challenge and treatment with epinephrine, antihistamines, and corticosteroids, as appropriate. No patient required overnight hospitalization, and none of the patients who had anaphylaxis presented severe symptoms or hypotension. The mean quantity of hazelnut provoking objective symptoms increased from 2.29 g to 11.56 g (P 5 .02) in the active group and from 3.49 g to 4.14 g in the placebo group (without statistical significance; Fig 2). Furthermore, almost 50% of patients (5/11) who underwent hazelnut SLIT reached the highest level (20 g) compared with 9% (1/11) of the placebo group (Fig 1). This beneficial effect has been observed in patiens with local reactions and also in patients with systemic reactions after hazelnut ingestion.
Tolerance and adverse reactions: safety assessment All patients reached the planned maximum dose of 25 drops from the most concentrated vial in 4 days. A total of 1466 doses were administered: 309 during the build-up phase and 1157 during maintenance. Systemic reactions were observed in 0.2% (3 reactions/ 1466 doses). They appeared only during the build-up phase, and only antihistamines were required for their control. One facial urticaria occurred in the placebo group and 2 reactions in 1 patient of the active group: skin pruritus a few minutes after the last dose of the second day and 1 delayed urticaria several hours after the last dose of the third day, probably associated with exercise (the patient had urticaria after 1 hour of exercise). Local reactions, mainly in the form of immediate oral itching, were observed in 7.4% (109 reactions/1466 doses). Four patients in the active group reported abdominal pain several hours after the ingestion on 1 occasion each and only during the build-up phase. All local reactions during the maintenance phase were also oral itching, and all were in the same patient. Laboratory parameters Hazelnut-specific IgE levels did not differ between groups before treatment but were lower in both groups after treatment, with no statistical significance both between both groups and within the same group (data not shown). As expected, because patients selected came from birch-free areas, prevalence to Cor a 1–specific IgE was low (22.14%), whereas Cor a 8–specific IgE was present in 50% of the patients selected (Fig 1). Regarding Cor a 1 and Cor a 8 specific IgE, there were no differences between groups before treatment; however, lower rates were observed in both groups after treatment, with no statistical significance within and between both groups (data not shown). Regarding hazelnut-specific IgG4 levels, no statistical significance was found between the studied groups; however, in the active group, an increase in mean IgG4 levels from 7.34 allergen units (AU)/mL to 9.84 AU/mL (P < .05) was observed (Fig 3).
Enrique et al 1077
J ALLERGY CLIN IMMUNOL VOLUME 116, NUMBER 5
An increase in IL-10 levels after treatment was found only in the active group, rising from 1.62 pg/mL to 2.24 pg/mL (P < .05). No differences were observed between the placebo and active groups (Fig 3). We were unable to obtain results of the other cytokines because of the low sensitivity of the tests used.
DISCUSSION Food allergy is potentially life-threatening, and there is no known curative treatment to date. The recommended treatment for individuals with known anaphylactic reactions to foods is avoidance and use of injected epinephrine as soon as they become aware of an inadvertent ingestion. However, avoidance of accidental ingestion is difficult when eating away from home, and many people with known food sensitivity do not routinely carry epinephrine with them.19 Therefore, an improved treatment is needed for anaphylactic reactions to foods. Previous studies have used subcutaneous peanutspecific immunotherapy and have showed a decrease in SPT reactions and decreased sensitivity to peanut ingestion. However, this therapy was accompanied by a high rate of systemic reactions.10,11 Oral desensitization to foods has been reported in numerous limited series but with varying results.20,21 Although oral desensitization is an attractive procedure, well-designed studies are required. Other novel immunotherapeutic approaches have been reported with good results: anti-IgE therapy,22 Chinese herbal medicines,23 the uses of subcutaneous birch pollen immunotherapy in patients with oral allergy syndrome,24,25 and other laboratory studies with animal models.26 Food immunotherapy has recently been suggested again as a food allergy treatment in 2 case report publications: one, oral immunotherapy in patients allergic to peanut,27 and the other, sublingual immunotherapy in a case of anaphylactic reaction to kiwi.28 Nevertheless, double-blind, placebo-controlled randomized studies are required to confirm these results.
The aim of this study was to evaluate a large group of patients allergic to hazelnut for their response to a sublingual immunotherapy with a biological extract standardized in unit mass of major allergens. In the current study, efficacy was assessed by DBPCFC with hazelnut performed before and after 2 to 3 months of maintenance immunotherapy. Our clinical data confirm significant increases in the threshold of sensitivity to hazelnut allergen to a level that should translate into at least partial protection against most unintended ingestion of hazelnuts. Moreover, almost 50% of the patients who underwent active treatment tolerated the highest dose of 20 g, about 15 to 20 hazelnuts, which constitutes a high amount of hazelnuts that should translate into enough protection against most unintended ingestion of hazelnuts. Surprisingly, 1 patient (Cor a 1 sensitive) who received placebo tolerated the same highest dose of 20 g, and another patient who received placebo, with the same characteristic allergen profile, increased his tolerance from 2 to 10 g hazelnut (Fig 1). These patients presented oral allergy syndrome in the DBPCFC and had hazel pollinosis. Although the second challenge test was performed before hazel pollen season, it is tempting to speculate on the possibility of a seasonal variation in hazelnut pollen– dependent allergy.29 Mean provocative dose in a previous study ranged from 1.4 and 2.7 g in patients with hazelnut allergy from Zurich and Copenhagen to 15.3 g in patients from Milan.4 Nevertheless, the current study is conducted in a birch-free area, contrary to Zurich, Copenhagen, and Milan which are in an area of birch pollen exposure. We found a mean provocative hazelnut dose similar to data observed in Zurich and Copenhagen; however, Cor a 8 was described as a major allergen in Milan and also in the geographical area of the current study.4,6 Regarding safety aspects, in the current study, we observed very good tolerance because the rate of side effects was very low and appeared in both groups of treatment. Although the sublingual-discharge technique seems to lead to better tolerance than the sublingual-swallow
Food allergy, dermatologic diseases, and anaphylaxis
FIG 3. A, IgG4 levels before and after treatment. B, IL-10 levels before and after treatment.
1078 Enrique et al
Food allergy, dermatologic diseases, and anaphylaxis
technique, mainly for gastrointestinal symptoms, 4 patients reported abdominal pain. Nevertheless, a relative high rate of gastrointestinal symptoms has been reported with sublingual-swallow immunotherapy with latex,18 and also with a high dose of parietaria extract.30 Our data also confirm regional differences in sensitization pattern reported for other birch pollen–related foods.5,6 In a previous study conducted from Germany in a group of patients with hazelnut allergy and birch pollen allergy, the majority were sensitized to the pollen-related hazelnut allergen Cor a 1.31 In other studies, Cor a 8 (hazelnut lipid transfer protein) was significant for a majority of patients recruited in Spain or Northern Italy.5,6 In this study, we partially repeat the same results. Nevertheless, some patients of the current study with systemic reactions did not react to Cor a 1 or to Cor a 8, suggesting the possible presence of other important pollen-related or nonpollenrelated hazelnut allergens, such as Cor a 9, an 11S globulin related to hazelnut-induced systemic reactions.32 Immunotherapy is capable of modifying the immunologic response to the offending allergen in the earliest stages. The subcutaneous route of administration has been well established, and its mechanisms of action have been extensively investigated.33 However, the clinical use of SLIT is recent. Therefore, some aspects have not been fully investigated, and data on the mechanisms of action are few and contradictory. A recent prospective study of SLIT in children with allergic rhinitis found positive effects on rescue and medication use but observed no significant effects on in vitro T-cell immune responses or immunoglobulins34; such effects were demonstrated in another double-blind trial35 and in a case report of a patient with severe anaphylaxis caused by kiwi allergy treated with sublingual kiwi immunotherapy with good tolerance and efficacy.27 In the current study, we observed, on the one hand, a decrease in specific IgE levels in both treatment groups and on the other hand, an increase in specific IgG4 and IL-10 levels only in the active group, suggesting the possibility of immunoregulation through regulatory T cells producing IL-10 and the oral mucosa Langerhans cells.36-38 In conclusion, this randomized and controlled clinical trial of sublingual immunotherapy performed in patients allergic to hazelnut has found significant increases in the threshold of sensitivity to hazelnut allergen, as assessed by DBPCFC after hazelnut SLIT, and good tolerance to this treatment. This beneficial effect has been observed both in local reactions and in systemic reactions after hazelnut ingestion. Nevertheless, further studies and others on the long-term efficacy of SLIT with food extracts are required. We thank Dr Stephan Scheurer from the Paul-Ehrlich Institut (Langen, Germany) for providing Cor a 8. We acknowledge Teresa Pin˜o´n, Dolores Sa´nchis, Ine´s Saz, Marihor Aguilar, Josefa Fuentes, and Magdalena Sa´nchez for technical assistance. We also thank Ms Christine O’Hara for English review of the manuscript. REFERENCES 1. Sampson HA. Update on food allergy. J Allergy Clin Immunol 2004; 113:805-9.
J ALLERGY CLIN IMMUNOL NOVEMBER 2005
2. Bjo¨rkste´n B. How allergenic is food? Clin Exp Allergy 2004;34:673-5. 3. Sicherer SH, Mun˜oz-Furlong A, Burks W, Sampson HA. Prevalence of peanut and tree nut allergy in the US determined by a random digit dial telephone survey. J Allergy Clin Immunol 1999;103:559-62. 4. Ortolani C, Ballmer-Weber BK, Skamstrup Hansen K, Ispano M, Wu¨trich B, Bindslev-Jensen C, et al. Hazelnut allergy: a double-blind, placebo-controlled food challenge multicenter study. J Allergy Clin Immunol 2000;105:577-81. 5. Pastorello EA, Vieths S, Pravettoni V, Farioli L, Trambaioli C, Fortunato D, et al. Identification of hazelnut major allergens in sensitive patients with positive double-blind, placebo-controlled food challenge results. J Allergy Clin Immunol 2002;109:563-70. 6. Schocker F, Lu¨ttkopf D, Scheurer S, Petersen A, Cistero´-Bahı´ma A, Enrique E, et al. Recombinant lipid transfer protein Cor a 8 from hazelnut: a new tool for in vitro diagnosis of potentially severe hazelnut allergy. J Allergy Clin Immunol 2004;113:141-7. 7. Tariq SM, Stevens M, Rideout S, Twinselton R, Hide RW. Cohort study of peanut and tree nut sensitization by age of 4 years. BMJ 1996;313:514-7. 8. Groot H, Jong NW, Vuijk MH, Gerth van Wijk R. Birch pollinosis and atopy caused by apple, peach, and hazelnut; comparison of three extraction procedures with two apple strains. Allergy 1996;51:712-8. 9. Steinman H. ‘‘Hidden’’ allergens in foods. J Allergy Clin Immunol 1996; 98:241-50. 10. Oppenheimer JJ, Nelson HS, Bock SA, Christensen F, Leung DYM. Treatment of peanut allergy with rush immunotherapy. J Allergy Clin Immunol 1992;90:256-62. 11. Nelson HS, Lahr J, Rule R, Bock A, Leung D. Treatment of anaphylactic sensitivity to peanuts by immunotherapy with injections of aqueous peanut extract. J Allergy Clin Immunol 1997;99:744-51. 12. Alonso R, Enrique E, San Miguel-Moncı´n MM, Tella R, Ferna´ndezParra B, Bartra J, et al. An alternative challenge in oral allergy syndrome. J Allergy Clin Immunol 2004;113:S152. 13. Chapman MD, Heymann PW, Wilkins SR, Brown MJ, Platts-Mills TA. Monoclonal immunoassays for major dust mite (Dermatophagoides) allergens, Der p 1 and Der f 1, and quantitative analysis of the allergen content of mite and house dust extracts. J Allergy Clin Immunol 1987;80:184-94. 14. O’Shannessy DJ, Dobersen MJ, Quarles RH. A novel procedure for labeling immunoglobulins by conjugation to oligosaccharide moieties. Immunol Lett 1984;8:273-7. 15. Bradford M. A rapid and rensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Ann Biochem 1976;72:248-54. 16. Boluda L, Alonso C, Ferna´ndez Caldas E. Purification, characterization and partial sequencing of 2 new allergens of Olea europaea. J Allergy Clin Immunol 1998;101:210-6. 17. Boluda L, Sastre J, Casanovas M, Fernandez Caldas E. Determination of Ole e 1 by enzyme immunoassay and scanning densitometry: validation by skin-prick testing. J Immunol Methods 1999;223:17-26. 18. Cistero´-Bahı´ma A, Sastre J, Enrique E, Ferna´ndez M, Alonso R, Quirce S, et al. Tolerance and effects on skin reactivity to latex of sublingual rush immunotherapy with latex extract. J Invest Allergol Clin Immunol 2004;14:17-25. 19. Bock SA, Mun˜oz-Furlong A, Sampson HA. Fatalities due to anaphylactic reactions to foods. J Allergy Clin Immunol 2001;107:191-3. 20. Bauer A, Ekanayake MS, Wigger-Alberti W, Elsner P. Oral rush desensitization to milk. Allergy 1999;54:894-5. 21. Patriarca G, Nucera E, Roncallo C, Pollastrini E, Bartolozzi F, De Pasquale T, et al. Oral desensitizing treatment in food allergy: clinical and immunological results. Aliment Pharmacol Ther 2003;17:459-65. 22. Leung DYM, Sampson HA, Yunginger JW, Burks AW, Schneider LC, Wortel CH, et al. Effect of anti-IgE therapy in patients with peanut allergy. N Engl J Med 2003;348:986-93. 23. Srivastava KD, Kattan JD, Zou ZM, Li JH, Zhang L, Wallenstein S, et al. The Chinese herbal medicine formula FAHF-2 completely blocks anaphylactic reactions in a murine model of peanut allergy. J Allergy Clin Immunol 2005;115:171-8. 24. Bucher X, Pichler WJ, Dahinden CA, Helbing A. Effect of tree pollen specific, subcutaneous immunotherapy on the oral allergy syndrome to apple and hazelnut. Allergy 2004;59:1272-6. 25. Blotter ST, Tiemessen MM, Zuidmeer L, van Leeuwen A, HoffmanSommergruber K, Bruijnzeel-Koomen CAFM, et al. Efficacy of birchpollen immunotherapy on cross-reactive food allergy confirmed by
26. 27.
28.
29.
30.
31.
skin tests and double-blind food challenges. Clin Exp Allergy 2004;34: 761-9. Eigenmann PA. Future therapeutic options in food allergy. Allergy 2003; 58:1217-23. Scurlock AM, Steele PH, Andrzejewski SS, Pons LA, Althage KA, Christie L, et al. Safety of oral peanut immunotherapy for peanut allergic patients. J Allergy Clin Immunol 2005;115:S246. Mempel M, Rakoski J, Ring J, Ollert M. Severe anaphylaxis to kiwi fruit: Immunologic changes related to successful sublingual allergen immunotherapy. J Allergy Clin Immunol 2003;111:1406-9. Skamstrup Hansen K, Vieths S, Vestergaard H, Skov PS, BindslevJensen C, Poulsen JH. Seasonal variation in food allergy to apple. J Chromatogr B Biomed Sci Appl 2001;756:19-32. La Rosa M, Ranno C, Andre´ C, Carat F, Tosca MA, Canonica GW. Double-blind, placebo-controlled evaluation of sublingual-swallow immunotherapy with standardized Parietaria judaica extract in children with allergic rhinoconjunctivitis. J Allergy Clin Immunol 1999;104: 425-32. Schocker F, Lu¨ttkopf D, Mu¨ller U, Thomas P, Vieths S, Becker W-M. IgE binding to unique hazelnut allergens: identification of non-pollenrelated and heat-stable hazelnut allergens eliciting severe allergic reactions. Eur J Nutr 2000;39:172-80.
Enrique et al 1079
32. Beyer K, Grishina G, Bardina L, Grishin A, Sampson HA. Identification of an 11 S globulin as a major hazelnut food allergen in hazelnut-induced systemic reactions. J Allergy Clin Immunol 2002;110:517-23. 33. Till SJ, Francis JN, Nouri Aria K, Durham SR. Mechanisms of immunotherapy. J Allergy Clin Immunol 2004;113:1025-34. 34. Rollinck-Werninghaus C, Kopp M, Liebke C, Lange J, Whan U, Niggemann B. Lack of detectable alterations in immune responses during sublingual immunotherapy in children with seasonal allergic rhinoconjunctivitis to grass pollen. Int Arch Allergy Immunol 2005;136:134-41. 35. Lima MT, Wilson D, Pitkin L, Roberts A, Nouri Aria K, Jacobson M, et al. Grass pollen sublingual immunotherapy for seasonal rhinoconjunctivitis: a randomized controlled trial. Clin Exp Allergy 2002;32:507-14. 36. Sicherer SH, Leung DYM. Advances in allergic skin disease, anaphylaxis, and hypersensitivity to foods, drugs, and insects. J Allergy Clin Immunol 2005;116:153-63. 37. Francis JN, Till SJ, Durham SR. Induction of IL-101CD41CD251 T cells by grass pollen immunotherapy. J Allergy Clin Immunol 2003; 111:1256-61. 38. Allam JP, Novak N, Fuchs C, Asen S, Berge´ S, Appel T, et al. Characterization of dendritic cells from human oral mucosa: a new Langerhans’ cell type with high constitutive FceRI expression. J Allergy Clin Immunol 2003;112:141-8.
Food allergy, dermatologic diseases, and anaphylaxis
J ALLERGY CLIN IMMUNOL VOLUME 116, NUMBER 5