A total of 48 elderly rhinitis patients sensitized to HDMs were enrolled in 4 (Ajou, Yonsei, Korea, and Hallym University Hospitals) in South Korea, and the study flow is summarized in Fig. 1. Three patients who did not meet inclusion criteria were excluded. The patients had suffered from AR and/or bronchial asthma as defined by Allergic Rhinitis and its Impact on Asthma (ARIA) criteria and Global Initiative for Asthma (GINA), and had high serum specific IgE to Dermatophagoides pteronyssinus and/or Dermatophagoides farinae as confirmed by skin prick test and/or ImmunoCAP^® (ThermoFisher Scientific, Waltham, MA, USA). At the time of enrollment, retrospective rhinoconjunctivitis total symptom score (RTSS) related to the previous winter season was at least 8 (out of a maximum of 18). RTSS is composed of 6 rhinoconjunctivitis symptoms, sneezing, rhinorrhea, nasal pruritus, nasal congestion, ocular pruritus and watery eyes during 24 hours, each symptoms assessed by 4-point-scale (0 = no symptoms to 3 = severe).7 Patients with co-sensitization to other pollens or inhalant allergens were enrolled when: 1) typical symptoms due to co-allergens did not exist and 2) the reactions of the skin prick test for co-allergens were less than those for HDMs. We excluded the following patients who: 1) participated in other studies; 2) received immunotherapy with any allergens, SCIT with HDMs, and/or anti-IgE monoclonal antibody treatment; 3) had uncontrolled asthma, other active respiratory disease, and/or malignancy; and 4) were contraindicated to SLIT. This study was approved by the Institutional Review Board of Ajou University Hospital (Ajou IRB-MED-CT4-14-159) and each center. All the study patients gave informed consent. On the assumption that the mean change in reactivity is 0.2 in the control group and 0.3 in the optimal dose group and that the variance is 0.1 in both groups, sample size was calculated to 30 patients in the treatment group and 15 patients in the control group to satisfy α = 0.05 and a power of 90%.

The patients were randomized at the first visit (V1), and HDM-SLIT tablets (LAIS^®; Lofarma SpA, Milano, Italy) with on-demand medications were given to the treatment group, while on-demand medications alone were given to the control group. From days 1 through 4, the treatment group received 300 allergenic unit (AU) of LAIS tablet; from day 5, they took 1,000 AU of LAIS twice a week for 48 weeks. The control group received standard medications on demand according to disease control status. The standard medications were defined as unexpected use of oral antihistamine, nasal corticosteroid, eye drops and inhaled corticosteroids with long acting-β2 agonists. All patients were scheduled to visit 1 week (visit 2, V2), 3 months (84 ± 7 days, visit 3, V3), 6 months (168 ± 7 days, visit 4, V4), and 1 year (336 ± 7 days, visit 5, V5) after the randomization. All patients filled out the rhinoconjunctivitis quality of life questionnaire (RQLQ), and received rhinitis control assessment test (RCAT) and asthma control test (ACT) at V1 and V5 as previously described.789 Rhinoscopy was done by the investigators to evaluate symptoms scores. Blood samples were taken from each patient at V1 and V5 for immunological evaluation and were stored at −20°C for further immunological evaluation.

The diary was composed of questionnaire on nasal and eye symptom scores (sneezing, rhinorrhea, nasal obstruction, nasal itching sensation, tearing, eye itching and redness) ranging from 0 to 3 (0 = no symptom, 1 = mild symptom, 2 = moderate symptom and 3 = severe symptom) and medication scores (total number of antihistamine tablets, nasal sprays, and eye drops). The daily symptoms and medication scores were recorded daily on the distributed diary.

Each patient was asked to bring the diary at each visit to confirm records. The primary efficacy variable was RTSS, which is composed of 6 rhino-conjunctivitis symptoms (sneezing, rhinorrhea, nasal pruritus, nasal congestion, ocular pruritus and watery eyes). The secondary efficacy variables were medication requirements, rhinoscopy scores as evaluated by the investigators, which were composed of nasal symptoms, nasal congestion, nasal secretion and redness (ranging from 0 = none to 3 = severe), rhinoconjunctivitis quality of life by RQLQ and RCAT, and asthma control as assessed by ACT. RQLQ is composed of 7 domains (activity limitation, sleep, non-nose/eye symptoms, practical problems, nose symptoms, eye symptoms and emotional function) and 28 questions during the previous week. The RQLQ scale scores range from 1 = no impairment to 5=severe impairment. The RCAT of Korean version is composed of 6 questions regarding nasal and eye symptoms (nasal congestion, sneezing, watery eye, sleep disturbance and activity limitation; control status: 5 = never-controlled and 1 = well-controlled) during the previous week. ACT consists of 5 questions on a 5-point scale of 1 to 5 (higher scores reflecting better control state) to assess the effect of asthma on daily activities, asthma symptoms at daytime, asthma symptoms at night, use of rescue mediations and self-assessment of asthma control.

Blood samples were obtained from each patient at V1 and V5, and immunological changes were compared before and after the start of treatment using the following 2 methods: 1) changes in serum specific IgA, IgE, and IgG1/IgG4 antibodies to D. farinae as measured by enzyme-linked immunosorbent assay (ELISA); and 2) changes in basophil responses to D. farinae as evaluated by basophil activation test (BAT). The levels of serum specific IgE, IgA, and IgG1/IgG4 antibodies to D. farinae were measured by ELISA as previously described.1011

For BAT, peripheral blood samples were collected from each study patient at V1 and V5 as previously described.1213 Within 3 hours of blood sample collection in an ethylenediaminetetraacetic acid (EDTA) tube, ammonium chloride lysis buffer (0.154 M NH₄Cl, 10 mM KHCO₃, 0.1 mM EDTA, pH 7.2-7.4) was used to lyse red blood cells. Using anti-IgE antibody (1 μg/mL) and calcium inophore (1 μg/mL; Sigma-Aldrich Co., St. Louis, MO, USA), basophils were incubated as positive controls. After washing out with a phosphate-buffered saline, the resuspended cells were stained with phycoerythrin-conjugated antihuman CD203c antibody (Beckman Coulter, Marseille, France) on ice in the dark for 30 minutes. After another washing with phosphate-buffered saline, the cells were evaluated by using FACS Canto II flow cytometry (Becton Dickinson, San Jose, CA, USA) and were presented as % of cells expressing CD203c.

All statistical analyses were performed using SPSS ver. 20 (SPSS Inc., Chicago, IL, USA). The values are presented as mean ± standard deviation or median (minimal and maximal values). Student's t test was used to analyze differences between the 2 groups. Wilcoxon's signed rank test was done to compare changes in symptom/medication scores, RTSS, RQLQ, ACT, RCAT and immunologic changes between V1 and V5 in both groups. A P value of < 0.05 was considered statistically significant.

Total 45 patients were enrolled and randomized to the treatment group (n = 30), who received SLIT along with medications on demand, and the control group (n = 15) who were allowed to take medications, including antihistamines and intranasal steroids, on demand by using centralized, computer-generated randomization system. The mean age of the patients was 67.2 years (range, 60–81 years), and the male to female ratio was 22:17. Four patients in the treatment group and 2 in the control group were dropped out from the study, 1 patient was ineligible subject, 1 patient was uncooperative and 2 patients from each group withdrew informed consent. Among these 39 patients, 26 in the treatment group and 13 in the control group were analyzed. The mean age was 67.0 ± 5.8 years in the treatment group and 67.2 ± 6.5 years in the control group, with no significant difference. The baseline clinical characteristics, comorbid conditions, including asthma, skin reactivity or serum specific IgE to HDMs (as measured by ImmunoCAP), was not significantly different between the 2 groups (Table 1).

When rescue medications (including oral antihistamines, histamine eye drops, and intranasal steroids) were compared between the 2 groups, the dose of antihistamines (mg) tended to be more frequently used in the control group than in the treatment group, although there was no statistical significance (P = 0.054, Table 2).

Nasal and eye symptoms decreased significantly between V1 and V5 in the treatment group (P = 0.001 and P = 0.036, respectively), while nasal symptoms and RTSS, except for eye symptoms, decreased in the control group (P < 0.001). However, RTSS decreased significantly both in the treatment and control groups (P = 0.001 for each). The RQLQ scores tended to decrease in the treatment group without statistical significance (P < 0.05). No significant change was noted in RCAT or ACT scores between V1 and V5 in both groups (Table 2).

Rhinoscopy scores as measured by the investigators at each visit were compared between V1 and V5 in the treatment and control groups. A significant difference was noted only in changes in nasal symptom scores (P = 0.038), although nasal congestion, secretion, and redness scores were not significantly different in the 2 groups (P < 0.05, Fig. 2).

Serum specific IgE to D. farinae tended to decrease between V1 and V5 in the treatment and control groups, but no statistical significance was reached. However, the serum specific IgA levels to D. farinae decreased significantly in the treatment group (P = 0.011), while they were not in the control group. There were no significant changes in serum specific IgG1 and G4 antibodies to D. farinae between V1 and V5 in the 2 groups (Fig. 3).

In BAT, CD203 expression levels decreased significantly after incubation with 0.1 and 10 ug/mL of D. farinae at V5 compared to V1(P = 0.001 and P = 0.046, respectively, Fig. 4), while no significant changes were noted in the control group.

There was no significant difference in adverse reactions between the 2 groups. Most of the reactions were mild, recovered completely without any reported complications. Rhinitis and conjunctivitis exacerbations were reported from the treatment and control groups. The most frequent adverse reaction was upper respiratory infection in the 2 groups (10 events from 6 patients in the treatment group, 17 events from 4 patients in the control group). No systemic or severe adverse reaction was reported.