Regulatory T Cells in the Response to SIT in Allergic Rhinitis.

Patients The design was a one-year, randomized open-label parallel group study in which patients were randomized in a 1:1 ratio to receive SIT with Dermatophagoides pteronyssinus extract (Alutard SQ, ALK-Abello', Hørsholm, Denmark) or to observe with standard pharmacotherapy (control group).

The study participants included 50 house dust mite (HDM)-allergic children aged 9-13, who received SIT with Dermatophagoides pteronyssinus (Der p) extract for one year. These were compared with 21 HDM-allergic children aged 9-14 who served as controls. All children were of Han nationality and were recruited from the allergy clinic of Beijing TongRen Hospital. They had a history of HDM-induced moderate or severe rhino-conjunctivitis of at least three years duration. Each patient also had a positive skin prick test (SPT) result for Der p (ALK-Abello', Hørsholm, Denmark) with a wheal diameter of at least 6 mm, and were positive for specific immunoglobulin E (IgE) to Der p (Pharmacia CAP System, Pharmacia Diagnostics, Uppsala, Sweden), with a RAST value of at least 0.7 kU/L. Children with a history of asthma or atopic dermatitis were excluded. The children undergoing immunotherapy were matched to atopic donors for age, sex, SPT response to Der p, total IgE and specific IgE to Der p levels (Table 1). The study protocol was approved by our institutional review board for human studies and informed consent was obtained from all subjects.

Immunotherapy protocol Children undergoing subcutaneous immunotherapy were given Dermatophagoides pteronyssinus extract (Alutard SQ, ALK-Abello, Hørsholm, Denmark) according to a cluster protocol which we have described previously[15]. In the first six weeks, children underwent visits for up-dosing, receiving two injections one hour apart with an increasing dose every week. The highest concentration, given in the sixth week, had an allergenic activity of 100,000 SQ/ml and contained 9.8 μg/ml Der p1. After week six, the dosing interval was increased to one month and maintained until the end of the first year. For patients randomized to non-SIT group, persistent rhinitis was managed with pharmacotherapy including intranasal steroids and oral antihistamines. Intranasal steroids were kept at the same dose during the study and antihistamines were used as required. Blood samples were taken at the baseline and after one year treatment in both groups.

Clinical evaluation Symptoms of AR children were assessed by the Total 5 Symptom Score (T5SS), which includes rhinorrhea, sneezing, nasal congestion, and nasal and ocular pruritus. Each symptom was scored on a scale of 0-3 (0 = none, 3 = severe). Medication use was scored and recorded daily, as described previously [16]. Arbitrary scores were attributed to the drugs used (0.75 points for 1 puff of nasal corticosteroids and 1 points for 1 tablet of antihistamine). Patients were instructed to use local steroids only (plus antihistamines if they did not improve symptoms) and to report each administration or variation of the initial drug therapy in the diary. Patients were also instructed to stop drugs at least 7 days before blood sampling.

Flow cytometry Peripheral blood was obtained by venous puncture and collected into preservative-free heparin. Peripheral blood mononuclear cells (PBMCs) were isolated by means of Ficoll-Plaque Plus density gradient centrifugation (Amersham Biosciences, NJ, USA). For intracellular detection of cytokine, PBMCs (2 × 106 cells/mL) were stimulated with 10 μg/mL Der p1 (D. pteronyssinus major allergen 1, Indoor Biotechnologies, United Kingdom) for 24 hours. Twenty-five ng/mL phorbol 12-myristate 13-acetate (PMA), 1 μg/mL ionomycin and 1.7 μg/mL monensine were added at 37 °C for the final four hours of stimulation (all from Sigma, Missouri, USA).