However the mechanisms are unclear rush immunotherapy (RIT) may be effective to treat allergic diseases. until 1 yr of RIT while there were no changes in the control group. These data show that the continuous functional changes from Th2 to Th1 phenotype of the CD4(+) T cells are developed after RIT in the asthmatic children sensitized with HDM. ((or or (Table 1). At the time of the study all individuals were symptom free (for at least 4 weeks) and did not have any medication for asthma except for short acting β2-agonist for at least 4 weeks before blood sampling. The ethics committee of the Asan Medical Center Institutional Review Table approved the study and written educated consents were from the parents of all subjects. Table 1 Characteristics of individuals before rush immunotherapy Protocol of rush immunotherapy The individuals received RIT according to the protocol in our pediatric ward. The individuals were admitted for 4 days during induction period and the dosages of each injection were rapidly increased in ten-fold daily starting from 5 Therapeutic unit (TU)/mL. Then they received injections every 2 weeks till 8 weeks until maintenance was achieved (5 0 TU/mL). After then they received injections every 4 weeks. Allergen extracts were obtained from Bencard Allergie (Munchen Germany). Measurements of symptom scores and skin reactivity to and were measured. The severity of symptoms was scored on a Likert scale from 0 to 5 (0=none 1 2 3 4 and 5=very DCN severe). Asthma symptom complaints were divided into three periods: awakening (four items) daytime (six items) and nighttime (five items). The total numbers of nocturnal awakenings for asthma per week were translated into the same six-point scale as follows: no awakening=none 1 awakening=trivial 2 to 3 3 awakenings=mild 4 ABT-492 to 6 6 awakenings=moderate 7 to 10 awakenings=severe and 11 or more awakenings=very severe. Lymphocyte proliferation assay Peripheral blood mononuclear cells (PBMCs) from the patients were centrifugated by Ficoll-Hypaque gradient (Pharmacia Uppsala Sweden) and were incubated in 96 well round bottom plate at 106 cells/mL in 200 μL/well. The dosages of allergen (Allergopharma Reinbek Germany) were 1 10 30 and 50 μg/mL concentration. Proliferative responses were assayed by tritiated thymidine incorporation ABT-492 after 5 day’s culture after addition of 1 1 μCi 16 hr before cell harvesting. Flow cytometric analysis of PBMCs for intracellular cytokine production For intracellular T-cell cytokine detection we ABT-492 employed a flow cytometry which has been described elsewhere (26). PBMCs (107 cells/mL) were stimulated with phorbol myristate acetate (25 ng/mL; Wako Osaka Japan) and ionomycin (1 μg/mL; Sigma Chemical Co. St. Louis MO U.S.A.) in the presence of 2 mM/L monensin for 4 hr at 37℃ in a humidified atmosphere of 5% CO2 in air. Cells were fixed in 4% paraformaldehyde and made permeable with saponin. After blocking with 10% AB serum cells were incubated with a monoclonal mouse anti-cytokine antibody (anti-IL-5 and anti-IFN-γ: Becton-Dickinson Franklin Lakes NJ U.S.A.) at 1 mg/mL in 0.1% saponin. Twenty minutes later cells were washed twice and incubated with a fluorescein isothiocyanate- or phycoerthrin-labeled polyclonal goat anti-mouse isotype specific antibody and an anti T-cell surface marker antibody (anti-CD3 anti-CD4 and anti-CD8: Becton-Dickinson) for 30 min. Then cells were analyzed by FACSCalibur (Becton-Dickinson) and frequency of cytokine-producing cells in different subpopulation was calculated. Statistical ABT-492 analysis Wilcoxon rank sum test was applied to compare the data of two groups and Wilcoxon signed rank test was used to compare the changes of the group before and after RIT. was decreased significantly eight weeks ABT-492 after RIT and taken care of until 1 yr after RIT (Fig. 1B) however not in charge group (data not really demonstrated). Fig. 1 Respiratory sign ratings (A) and pores and skin check reactivity to (B) had been decreased considerably at eight weeks after RIT and taken care of until 1 yr after RIT in the RIT group however not in charge group. Cellular proliferation assay The proliferative response was indicated as excitement index (SI: cpm with allergen/cpm with moderate). The mobile proliferative reactions (SI) to each dosage of demonstrated dose-dependent reactions and the utmost response was within the excitement of 30 μg/mL focus. These.