Formation of IgE-Allergen-CD23 Complex Changes in Children Treated with Subcutaneous Immunotherapy for Japanese Cedar Pollinosis.

BACKGROUND: Subcutaneous immunotherapy (SCIT) is used to treat Japanese cedar (JC) pollinosis. The formation of IgE-allergen-CD23 complex after SCIT for JC pollinosis has not yet been fully elucidated. OBJECTIVE: The objective of this study was to investigate the formation of IgE-allergen-CD23 complex after SCIT for JC pollinosis. METHODS: Eleven patients were treated with 3-year SCIT for JC pollinosis at Sa-gamihara National Hospital from 2013 to 2014. Nasal and ocular symptoms (in terms of symptom scores) during the scattering of JC pollen and immunological changes were investigated. Levels of JC pollen-specific antibodies (IgE and IgG4) were measured by ImmunoCAP assays. To detect the changes in allergen-presenting ability of B cells, the levels of IgE-allergen-CD23 complexes in serum were measured by a cell-free, enzyme-linked immunosorbent-facilitated antigen-binding assay. RESULTS: The median (interquartile range) age of the subjects was 8 (6-10) years. Three patients (27%) had comorbid atopic dermatitis, and 5 patients (45%) had comorbid bronchial asthma. Before starting SCIT, the total IgE level was 373 (75-2,870) kU/L, and the level of JC pollen-specific IgE was 77.2 (15.4-528) kUA/L. Symptom scores improved significantly from the year after treatment. JC pollen-specific IgE levels did not change after 3 years of treatment. JC pollen-specific IgG4 levels increased significantly throughout the treatment period. The levels of IgE-allergen-CD23 complexes decreased significantly after 3 years of treatment. CONCLUSION: The ability of IgE-allergen complexes to bind to CD23 decreased after SCIT, suggesting that increasing levels of IgE-blocking antibodies, including IgG4, may play an important role in the mechanism of SCIT.

Establishment of enzyme-linked immunosorbent facilitated antigen binding as a biomarker assay for Japanese cedar pollen allergy immunotherapy.

BACKGROUND: Clinical efficacy of allergen-specific Immunotherapy (AIT) towards Japanese cedar (JC) pollen allergy is firmly established but JC pollen-specific biomarker assays are lacking. Treatment-related increase of allergen-specific antibodies is a robust biomarker of successful AIT. Allergen-specific non-IgE antibodies are believed to reduce the effects of allergen exposure by competing with IgE for allergen binding, and in-vitro assays quantifying the effects of AIT-induced IgE-blocking antibodies are advantageous. A cell-free enzyme-linked immunosorbent facilitated antigen binding (ELIFAB) assay of JC pollen was established. METHODS: Serum IgE-allergen complexes were captured by immobilized recombinant CD23, and allergen-IgE-CD23 complexes were detected by a biotin-conjugated anti-human IgE antibody. Sera from JC pollen-allergic subjects without or with subcutaneous immunotherapy (SCIT) with JC pollen extract were used (n = 11/group). RESULTS: Optimal assay conditions were established at 20 µg/mL CD23 and 0.3 µg/mL JC pollen extract, and the dependency on CD23 and IgE was verified. The data show that the JC pollen ELIFAB assay is fit for purpose and demonstrates that the IgE-blocking activity is significantly increased in the JC pollen SCIT group compared with the non-treated group. CONCLUSION: The JC pollen ELIFAB assay represents a simple, cell-free biomarker assay for monitoring the development of IgE-blocking antibody activity during JC pollen AIT.

Comparison of the Allergenic Potency of House Dust Extract and House Dust Mite Allergen Extract for Subcutaneous Allergen Immunotherapy.

Subcutaneous allergen immunotherapy (SCIT) with non-standardized house dust (HD) extracts has been used in Japan since 1963 for house dust mite (HDM)-allergic patients. Since the potencies of HD extracts are unknown, the allergenic potency of HD extracts was examined by comparing with a standardized HDM allergen extracts. The major allergen content of HDM in the extracts was measured using a sandwich enzyme-linked immunosorbent assay (ELISA). The immunoglobulin E (IgE) inhibitory activities of the extracts were measured by a competitive ELISA. The extract concentrations giving 50% inhibition of IgE binding (log10 IC50) were determined from dose-response curves and defined as inhibitory activities. A linear regression line was constructed from the log10 IC50 values of the standardized HDM extract to interpolate the relative potency of the HD extract with strength of 1 : 10 w/v (HD 1 : 10). The amounts of major allergens (Der f 1, Der p 1 and Der 2) were 116.3 µg/mL in the HDM allergen extract (100000 Japanese Allergy Units [JAU]/mL) and 0.77 µg/mL in the HD 1 : 10. The inhibitory activity (log10 IC50 values) of HD 1 : 10 was 2.389 ± 0.078, indicating the allergenic potency was between 200 and 2000 JAU/mL. Based on regression analysis (R2 >0.99), the allergenic potency of HD 1 : 10 was estimated to be 842 ± 128 JAU/mL. The present study determined the major allergen content of HD extract, which contributes to its allergenic potency. The allergenic potency of HD 1 : 10 was ca. 100-fold less than that of HDM allergen extract.

An optimized protocol for the analysis of house dust mite (Dermatophagoides farinae)-induced neutrophil-dominant airway inflammation.

House dust mites (HDMs), Dermatophagoides sp., are one of the most widespread aeroallergens worldwide and cause various allergic diseases, including asthma. The pathophysiology of asthma has been intensively investigated using murine models of allergic airway inflammation induced by exposure to D. pteronyssinus. However, the pathogenic roles of D. farinae in the allergic airway inflammation remains unclear. We herein report that repetitive exposure to D. farinae resulted in neutrophil-dominant airway inflammation together with fibrotic changes and the formation of lymphoid clusters. Both type 1 and type 2 inflammatory cytokines were induced. The pathogenic changes in the airway were dependent on both the frequency and dose of D. farinae exposure. Our study provides novel procedures and insight into the pathogenesis of D. farinae-induced airway inflammation in vivo.

Effects of sublingual immunotherapy in a murine asthma model sensitized by intranasal administration of house dust mite extracts.

BACKGROUND: Sublingual immunotherapy (SLIT) has received attention as a method for allergen immunotherapy. However, the mechanism of SLIT has not yet been fully investigated. Therefore, we evaluated the effects of SLIT in a murine asthma model, sensitized by intranasal administration of house dust mite (HDM) extracts. METHODS: Female BALB/c mice were intranasally exposed to HDM for either 3 or 5 weeks (5 consecutive days per week). Mice were administered either low-dose (0.5 mg/day) or high-dose (5 mg/day) sublingual HDM extracts for 2 weeks, followed by an additional week of intranasal exposure. Airway hyperresponsiveness (AHR), bronchoalveolar lavage fluid (BALF) cell count, cytokine levels in the BALF and lymph node cell culture supernatants, and allergen-specific antibodies were measured. Lung histology was also investigated. RESULTS: In mice sensitized for 5 weeks, high-dose SLIT ameliorated AHR, airway eosinophilia and goblet cell metaplasia. In mice sensitized for 3 weeks, even low dose SLIT ameliorated AHR and airway eosinophilia. Th2 cytokine levels in culture supernatants of submandibular lymph node cells in high-dose SLIT mice decreased, whereas IL-10 levels increased. Total IgA in BALF increased in mice sensitized for 3 or 5 weeks, and high-dose SLIT also increased allergen-specific IgG2a in mice sensitized for 5 weeks. CONCLUSIONS: These data suggest that earlier induction of SLIT in HDM-sensitized mice provides superior suppression of AHR and goblet cell metaplasia. The modulation of allergen specific IgG2a and local IgA might play a role in the amelioration of AHR and airway inflammation.

The Efficacy of Sublingual Immunotherapy for Allergic Rhinitis May Vary with the Time of Day.

BACKGROUND: Sublingual immunotherapy (SLIT) is a safe and effective treatment for allergic rhinitis (AR). However, many issues regarding SLIT remain to be resolved, including the optimal timing of administration. This study investigated the effect of time of day on SLIT efficacy with the goal of optimizing the therapeutic outcome. METHODS: We performed prophylactic SLIT at different times of day (10 a.m. or 10 p.m.) in 2 mouse models of AR: an ovalbumin (OVA)-induced AR model and Cry j 1-induced AR model, and compared the effects. RESULTS: In the OVA-induced AR model, mice sublingually receiving OVA at 10 a.m. exhibited a greater decrease in total and OVA-specific IgE levels than mice treated at 10 p.m. In addition, mice treated at 10 a.m. exhibited reductions in OVA-specific IL-4, IL-10, and IL-13 production by splenocytes relative to mice treated at 10 p.m. Furthermore, we observed a more efficient capture of sublingually administered OVA in submandibular lymph nodes at 10 a.m. than at 10 p.m. in mice. Similar results were observed in the Cry j 1-induced AR model using Japanese cedar pollen extract for SLIT. CONCLUSIONS: Given the allergen-specific antibody and T cell responses, we suggest that SLIT may be more effective in the resting phase than in the active phase (note that mice are nocturnal animals). Thus, we propose that a chronotherapeutic approach should be considered for SLIT to maximize its effectiveness.

Attempt to develop taste bud models in three-dimensional culture.

Taste buds are the end organs of taste located in the gustatory papillae, which occur on the surface of the oral cavity. The goal of the present study was to establish a culture model mimicking the lingual taste bud of the mouse. To this end, three cell lines were employed: taste bud-derived cell lines (TBD cell lines), a lingual epithelial cell-derived cell line (20A cell line), and a mesenchymal cell-derived cell line (TMD cell line). TBD cells embedded in collagen gel formed three-dimensional clusters, which had an internal cavity equipped with a tight junction-like structure, a microvilluslike structure, and a laminin-positive layer surrounding the cluster. The cells with this epitheliumlike morphology expressed marker proteins of taste cells: gustducin and NCAM. TBD cells formed a monolayer on collagen gel when they were co-cultured with TMD cells. TBD, 20A, and TMD cell lines were maintained in a triple cell co-culture, in which TBD cells were pre-seeded as aggregates or in suspension on the collagen gel containing TMD cells, and 20A cells were laid over the TBD cells. TBD cells in the triple cell co-culture expressed NCAM. This result suggests that co-cultured TBD cells exhibited a characteristic of Type III taste cells. The culture model would be useful to study morphogenesis and functions of the gustatory organ.