Role of mast cells in allergy had remained undetermined until the discovery of IgE in 1966. Then, IgE purified from many Liters of plasma, which had been donated from a patient with fatal myeloma, was distributed to researchers all over the world, and thus accelerated exploring the mechanisms involved in allergic reactions, particularly about the role of mast cells and basophils in the IgE-mediated reactions. Identification of mast cells as a progeny of a bone marrow hematopoietic stem cell in 1977 led us to successful in vitro culture of human mast cells. Along with the development of molecular biological techniques, the structure of the high affinity IgE receptor (FcεRI) was determined in 1989. These findings and subsequent investigations brought deeper understanding of IgE-mediated allergic diseases in the past half century, especially where mast cells are involved. We have now even obtained the information about whole genome expression of FcεRI-dependently activated mast cells. In sharp contrast to our comprehension of allergic diseases where IgE and mast cells are involved, the mechanisms involved in non-IgE-mediated allergic diseases or non-IgE-mediated phase of IgE-mediated diseases are almost left unsolved and are waiting for devoted investigators to reveal it.

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Interleukin-33 (IL-33), a member of the IL-1 cytokine family, is preferentially and constitutively expressed in epithelial cells, and it is especially localized in the cells' nucleus. The nuclear IL-33 is released by necrotic cells after tissue injury and/or trauma, and subsequently provokes local inflammation as an alarmin, like high-mobility group box protein-1 (HMGB-1) and IL-1α. IL-33 mainly activates Th2 cells and such innate-type immune cells as mast cells, basophils, eosinophils and natural helper cells that express IL-33R (a heterodimer of IL-1 receptor-like 1 [IL-1RL1; also called ST2, T1, Der4, fit-1] and IL-1 receptor accessory protein [IL-1RAcP]). That activation causes the cells to produce Th2 cytokines, which contribute to host defense against nematodes. On the other hand, excessive and/or inappropriate production of IL-33 is also considered to be involved in the development of such disorders as allergy. In this review, we summarize current knowledge regarding the pathogenic roles of IL-33 in the development of allergic inflammation by focusing on its effects on innate-type immune cells.

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Allergic diseases are complex diseases caused by a combination of genetic and environmental factors. To determine the genetic components of these diseases and to discover the genes and cellular pathways underlying them, a large number of genetic studies have been conducted. Progress in genetics enables us to conduct genome-wide association studies (GWASs), which is a comprehensive and unbiased approach to identify susceptibility loci for multifactorial diseases. Recent GWASs have convincingly detected a large number of loci associated with allergic diseases. Candidate genes in the susceptibility loci suggest roles for epithelial barrier functions, innate-adaptive immunity, IL-1 family signaling, regulatory T cells and the vitamin D pathway in the pathogenesis of allergic diseases. Interestingly, the IL1RL1, HLA, IL13 and C11orf30 regions are overlapping susceptibility loci among atopic dermatitis and asthma or allergic rhinitis. Although a more complete collection of associated genes and pathways is needed, biologic insights revealed by GWASs improve our understanding of the pathophysiology of human allergic diseases and contribute to the development of better treatment and preventive strategies.

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Background: Reliable symptom assessment is essential in asthma management. We developed new technology for analyzing breath sounds and assessed its clinical usefulness for monitoring asthmatic children.
Methods: Eighty asthmatic children and 59 non-asthmatic children underwent breath sound analysis in an asymptomatic state. Their asthma control was assessed by the Asthma Control Test^TM or Childhood ACT^TM scores and divided into two groups, namely, well-controlled (perfect) (n = 19) and not well-controlled (not perfect) (n = 61). Breath sounds were recorded using two sensors, located on the right anterior chest and trachea. We calculated the acoustic transfer characteristics between the two points, which indicated the relationship between frequencies and attenuation during breath sound propagation. Two indices of sound parameters, the chest wall sound index (CWI) and the tracheal sound index (TRI), were calculated from the transfer characteristics and tracheal sounds. We also developed a new parameter, the breath sound index (BSI), on a 2-dimensional diagram of CWI and TRI and tried to determine whether BSI may clarify asthma control better than CWI or TRI alone.
Results: There was a significant difference in TRI and BSI between asthmatic and non-asthmatic children (p = 0.007, p < 0.001). There was a significant difference in CWI and TRI between the well-controlled and not-well-controlled groups (p < 0.001). BSI discriminated between the two groups accurately (p < 0.001). The sensitivity and specificity of BSI for asthma control were 83.6% and 84.2%, respectively.
Conclusions: Asthma control could be evaluated using a new index calculated from breath sound analysis.

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Background: Few studies have examined the efficacy or safety of a transdermal β₂ agonist as add-on medication to long-term leukotriene receptor antagonist (LTRA) therapy in pediatric asthma patients.
Methods: In this randomized, open-label, multicenter clinical trial, children aged 4-12 years on long-term LTRA therapy were treated with tulobuterol patches (1-2mg daily) or oral sustained-release theophylline (usual dose, 4-5mg/kg daily) for 4 weeks. LTRAs were continued throughout the trial. Outcomes included volume of peak expiratory flow (% PEF), fractional exhaled nitric oxide (FeNO), clinical symptoms and adverse events.
Results: Thirty-three and 31 patients were treated with tulobuterol patches and theophylline, respectively. % PEF measured in the morning and before bedtime was significantly higher at all times in the treatment period compared with baseline in the tulobuterol patch group (p < 0.001), and was significantly higher in the tulobuterol patch group compared with the theophylline group. FeNO was similar and unchanged from baseline in both groups. There were no drug-related adverse events in either group.
Conclusions: These results suggest that short-term use of a transdermal β₂ agonist is an effective therapy for pediatric asthma without inducing airway inflammation in children on long-term LTRA therapy.

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Background: Interleukin 33 (IL-33) works as a functional mediator in allergic disease by enhancing the activity of eosinophils and inducing expression of T helper 2 (Th2)-associated cytokines. However, the role of IL-33 in pulmonary eosinophilia has not been elucidated. We investigated the levels of IL-33 in eosinophilic pneumonia (EP) together with associated cytokines, and discussed the clinical significance of IL-33 in EP.
Methods: Sera and bronchoalveolar lavage fluid (BALF) were obtained from 16 patients with EP, including acute eosinophilic pneumonia (AEP) and chronic eosinophilic pneumonia (CEP). Twelve patients with acute respiratory distress syndrome (ARDS) were also included for comparison. The concentration of IL-33 and Th2 cytokines (IL-4, IL-5, IL-13) were measured by enzyme-linked immunosorbent assay (ELISA).
Results: The concentration of serum IL-33 was significantly higher in patients with AEP than in CEP. In CEP, only patients with atopic factors showed mild increase of serum IL-33. The concentration of BALF IL-33 was also significantly elevated in AEP, however, it remained quite low in CEP. Among Th2 cytokines, IL-5 was significantly increased in both serum and BALF in AEP, and the level of IL-5 was positively correlated with that of IL-33. ARDS showed no increase of serum and BALF IL-33.
Conclusions: The remarkable increase of BALF IL-33 in AEP indicated the local production of IL-33 in lungs. IL-33 is considered to be a local key molecule for triggering pulmonary eosinophilia, together with IL-5. BALF IL-33 appears to be a useful marker for discriminating AEP from CEP and ARDS.

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Background: Dietary supplementation with unique prebiotic nondigestible carbohydrates has been shown to suppress allergy. In the present study, the prophylactic efficacy of a disaccharide β-1, 4 mannobiose (MNB) in a BALB/C mouse model of intranasally-induced pollen allergy was characterized.
Methods: Balb/c mice were pretreated with MNB orally and sensitized with pollen extract intraperitoneally and intranasally and challenged with histamine and crude pollen extract. Outcomes were measured as clinical signs, antibody isotypes, cytokine gene and protein expression patterns.
Results: The MNB-treated mice had lower sneezing frequency as compared to the positive control mice (P < 0.05). The low dose MNB-treated mice had less histamine (P < 0.05). However, the Cry j1 and Cry j 2-specific IgE, IgG, IgG1 and IgG2a antibody activity did not differ between groups (P > 0.05). The MNB-treated mice had increased IFN-γ (P < 0.05), and decreased IL-4 (P < 0.05). Mice in the high dose group had increased IL-10 (P < 0.05). However, TGF-β and IL-17 concentration did not differ between groups (P > 0.05). Both total and Cry j1 and Cry j 2-specific IgA were increased in the high dose group. Real-time RT-PCR analysis indicated that IL-4 and IL-17 mRNA expression were lower in MNB-treated mice (P < 0.05).
Conclusions: This work provides insights into using MNB as a potential prebiotic immunomodulator via decreased clinical signs, improved type1/type 2 balance, and IgA production, thus validating the potential use of MNB as a prophylactic prebiotic candidate to attenuate allergic response.

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Background: Nutritional prebiotic supplementation represents an attractive approach for interventions of allergy. In this study, the potential therapeutic effect of β-1, 4 mannobiose (MNB) in a murine model of cedar pollinosis was investigated.
Methods: Groups of Balb/c mice were intranasally sensitized to Japanese cedar pollen extract, and subsequently administered with low or high dose MNB. Both intraperitoneal and intranasal challenges were performed to monitor for clinical signs. Frequency of sneezing was recorded. Serum, spleen and Peyer's patches were collected for various biomarker analyses. Anti-allergic activity of MNB using RBL-2H3 cells was also evaluated.
Results: Significant decrease in sneezing frequency, histamine, interleukin (IL)-4 and IL-17A and increase in TGF-β and IL-10 concentration were exhibited by the MNB-treated mice. However, Cry j1 and Cry j 2-specific IgE activity remained unaltered. The high dose MNB treatment increased total IgA activity and IL-10, TGF-β and FoxP3 and decreased IL-4, IL-17A, and RORγT mRNA expression. Inhibition of activation of RBL-2H3 cells was observed via decrease in histamine, intracellular Ca²⁺ concentration, and FcεRI mRNA expression.
Conclusions: We demonstrated the immunomodulatory effects of MNB and conclude that MNB is a potential therapeutic molecular nutritional supplement candidate for treatment of pollen allergy.

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Background: Heparin is a potential anti-inflammatory drug for allergic airway inflammation. To elucidate the effects of heparin on allergic inflammation, we examined the in vivo effects of heparin on antigen-induced mucus hypersecretion and infiltration of eosinophils and neutrophils in the nasal epithelium of sensitized rats.
Methods: We induced hypertrophic and metaplastic changes of goblet cells in the nasal epithelium of ovalbumin (OVA)-sensitized rats by intranasal challenge with OVA. The effects of intranasal instillation with low molecular weight heparin (LMWH; 1-1000IU/0.1ml) on mucus production and eosinophil/neutrophil infiltration were examined.
Results: Intranasal instillation with low-dose LMWH (1-10IU/0.1ml) at 30 minutes before OVA instillation stimulated OVA-induced mucus production in the nasal epithelium of sensitized rats, whereas treatment with 100IU/0.1ml LMWH showed no effect. Intranasal instillation with high-dose LMWH (1000IU/0.1ml) significantly inhibited OVA-induced mucus production. Intranasal instillation with LMWH (1-1000IU/0.1ml) dose-dependently inhibited eosinophil and neutrophil infiltration into the rat nasal mucosa.
Conclusions: These results indicate that heparin inhibits mucus hypersecretion and infiltration of eosinophils and neutrophils in allergic inflammation, though the inhibitory effect against mucus production is obtained in high-dose heparin. Intranasal instillation with high-dose heparin may provide a new therapeutic strategy for the treatment of nasal allergic inflammation.

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Background: Cow's milk allergy (CMA) is one of the causes of gastrointestinal symptoms in neonates. A relationship between non-immunoglobulin (Ig) E mediated allergic reactions and CMA in early infancy has been proposed, but the clinical features and pathogenesis have not been established. The objective of this study is to determine the clinical characteristics of the neonates found in the earlier study to have food-related symptoms that suggested CMA.
Methods: A second questionnaire was sent to 53 NICUs, as a follow-up to the earlier study, to collect information on the background, onset age, clinical features, and results of clinical examinations.
Results: The median birth weight was 2614g and the median gestational age was 36.9 weeks. Symptoms developed within 6 days after birth in 40% of cases. Gastrointestinal symptoms were seen in 90% of cases and were mainly vomiting, bloody stool and abdominal distention. A specific IgE test, a lymphocyte stimulation test, and a fecal eosinophil test were conducted in 88%, 23% and 55% of cases, respectively, and the positive rates were 30%, 84%, and 75%, respectively. An oral food challenge (OFC) test was performed in 26% for confirmation of the diagnosis.
Conclusions: We confirmed that the clinical manifestations of food-related symptoms suggestive of CMA in neonates were distinctly different from those of common immediate type food allergy and were largely affected by underlying factors such as prematurity and gastroenterological surgery. Further OFC-based prospective accumulation of cases of CMA in neonates will be particularly important to reveal the full clinical features of this disease.

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Background: In cases using a budesonide/formoterol combination inhaler, many patients are started on fixed-dose treatment at 640/18μg (4 puffs) daily, but there are no guidelines yet regarding the step-down method when control has been maintained.
Methods: Patients with moderate asthma treated with either budesonide 400μg and salmeterol 100μg (GINA step3 group) or salmeterol/fluticasone 250 at 2 puffs (GINA step4 group) were enrolled and started on therapy of budesonide/formoterol 4 puffs. Thereafter, step-down to 2 puffs was performed if either of the following criteria was met at 8-week intervals: fractional exhaled nitric oxide (FeNO) ≤ 28 ppb plus asthma control test (ACT) score ≥ 22, or ACT score ≥ 24 at 3 consecutive visits regardless of FeNO level. Thereafter, changes in ACT score, the number of acute exacerbations and reliever use, and FeNO level were monitored through 48^th week.
Results: Fifty-one patients, 27 in step3 group and 24 in step4 group, underwent step-down. ACT scores and the number of reliever use remained stable in both groups even after step-down. In contrast, FeNO levels increased gradually in step4 group, whereas in the step3 group they increased immediately after step-down. Step-down was considered to be safely performed because the numbers of reliever use and those of moderate or more severe exacerbations during the 48-week period has not changed significantly compared to before step-down.
Conclusions: If complete control of asthma, not only of clinical symptoms but also airway inflammation, is achieved by 3-6 months of fixed-dose budesonide/formoterol 4 puffs/day, it should be possible to safely perform step-down to 2 puffs/day.

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Background: Programmed death-1 (PD-1) is a marker for human neoplastic T cells. Here, we evaluated whether or not PD-1 was also a marker for human mastocytosis, and explored the role of PD-1 in human mastocytosis cells.
Methods: Immunohistochemical analysis was used to evaluate the expression of PD-1 in clinical samples of human cutaneous mastocytosis. The expression of PD-1 in human mastocytosis cell lines was checked by RT-PCR, western blotting and flow cytometry. We stimulated human mastocytosis cell lines (LAD2 and HMC1.2) with recombinant ligand for PD-1, PD-L1 (rPD-L1), and tested the proliferative activity and the status of signal molecules by Cell Counting Kit-8 and ELISA, respectively.
Results: Ten of 30 human cutaneous mastocytosis cases (33.3%) expressed PD-1 protein. We also found that a human mastocytosis line LAD2 cells expressed PD-1 protein on their surfaces. The administration with rPD-L1 suppressed the stem cell factor-dependent growth of the LAD2 cells. And, rPD-L1 activated SHP-1 and SHP-2 simultaneously, and decreased the phosphorylation of AKT, in LAD2 cells. In contrast, we could not detect the expression of PD-1, and the significant effect of rPD-L1 on the mutated KIT-driven growth of HMC1.2 cells.
Conclusions: PD-1 could be a marker for human cutaneous mastocytosis and regulate the growth of human PD-1-positive mastocytosis cells.

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Background: Increases in allergic diseases have been reported from various epidemiological surveys. However, a few reports demonstrate the comorbidity of food allergy (FA) and allergic march. The aim of this study was to assess the prevalence and comorbidity of allergic diseases in Japanese students.
Methods: First-year students (n = 3,321; 2,209 male and 1,112 female) at Osaka University were asked about allergic diseases using postal interview sheets. Personal and family histories of doctor-diagnosed allergic diseases, clinical courses, and aggravating factors were included in the questionnaires.
Results: The lifetime prevalence of allergic rhinitis (AR), atopic dermatitis (AD), bronchial asthma (BA), and FA was 35.7%, 16.5%, 9.9%, and 7.0%, respectively. Disease-specific family histories existed for AR, AD, and BA. There was a positive correlation between the number of family histories of allergic disease and comorbidity (R = 0.370, P < 0.001). Comorbidity with AD significantly lowered the onset age of both BA (P = 0.010) and AR (P < 0.001). In addition, the onset age of AD was remarkably lowered by comorbidity with FA (P < 0.001). Comorbidity with FA was the highest risk factor for the progression of allergic march. Although most students showed improvement in AD, BA, and AR over time, the peak recurrence period was observed in adolescence.
Conclusions: These findings indicate that AD associated with FA accelerates the subsequent progression of allergic march. Early appropriate management for genetically high-risk groups is important for the prevention of allergic march.

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Background: Cluster analyses were previously performed to identify asthma phenotypes underlying asthma syndrome. Although a large number of patients with asthma develop the disease later in life, these previous cluster analyses focused mainly patients with younger-onset asthma.
Methods: Cluster analysis examined the existence of distinct phenotypes of late-onset asthma in Japanese patients with adult asthma. We then associated genotypes at the CCL5, TSLP, IL4, and ADRB2 genes with the clusters of asthma identified.
Results: Using the 8 variables of age, sex, age at onset of the disease, smoking status, total serum IgE, %FEV₁, FEV₁/FVC, and specific IgE responsiveness to common inhaled allergens, two-step cluster analysis of 880 Japanese adult asthma patients identified 6 phenotypes: cluster A (n = 155): older age at onset, no airflow obstruction; cluster B (n = 170): childhood onset, normal-to-mild airflow obstruction; cluster C (n = 119): childhood onset, the longest disease duration, and moderate-to-severe airflow obstruction; cluster D (n = 108): older age at onset, severe airflow obstruction; cluster E (n = 130): middle-age at onset, no airflow obstruction; and cluster F (n = 198): older age at onset, mild-to-moderate airflow obstruction. The CCL5-28C>G genotype was significantly associated with clusters A, B and D (OR 1.65, p = 0.0021; 1.67, 0.018; and 1.74, 0.011, respectively). The ADRB2 Arg16Gly genotype was also associated with clusters B and D (OR 0.47, p = 0.0004; and 0.63, 0.034, respectively).
Conclusions: The current cluster analysis identified meaningful adult asthma phenotypes linked to the functional CCL5 and ADRB2 genotypes. Genetic and phenotypic data have the potential to elucidate the phenotypic heterogeneity and pathophysiology of asthma.

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Background: Atopy is a phenotypically heterogeneous condition, and the extent to which atopy accounts for asthma is controversial. In this study, we aimed to identify the presence of distinct sensitization patterns to common inhaled allergens and their association with asthma, allergic rhinitis and TSLP genotypes.
Methods: We studied 1683 adults from Tsukuba, a city in central Japan and 297 adults from Kamishihoro, a cedar-free, birch-dominant town in northern Japan. Levels of total serum IgE and specific IgE antibodies towards 14 major inhaled allergens were measured. With the use of these measures, cluster analysis was applied to classify the subjects' sensitization patterns. We also examined the genetic effects of 2 TSLP functional SNPs on the development of each sensitization pattern.
Results: In the Tsukuba study, cluster analysis identified four clusters, including "Dust mite dominant", "Multiple pollen", "Cedar dominant", and "Low reactivity". In the Kamishihoro study, "Dust mite dominant", "Multiple pollen" and "Low reactivity" clusters were also identified, but a "Cedar dominant" cluster was not formed. The association with asthma was strongest for the "Dust mite dominant" cluster in both the Tsukuba and the Kamishihoro studies. In never smokers, both SNPs were associated with the "Dust mite dominant" cluster (OR > 1.2). In contrast, in current or past smokers, these alleles were inversely associated with the "Multiple pollen" cluster (OR < 0.5).
Conclusions: Cluster analysis identified the presence of distinct sensitization patterns to common inhaled allergens. TSLP may cause asthma by promoting innate allergic responses to indoor allergens and this contribution is significantly modified by smoking.

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Background: Nebulized drugs for asthma treatment are often mixed together in order to simplify inhalation regimens, although not recommended. We therefore evaluated aerosol characteristics and physicochemical stability of the admixture of an inhaled corticosteroid suspension with a beta2-agonist solution.
Methods: An 8-stage cascade impactor was used to measure the particle size distribution of admixture of Pulmicort^® Respules^® (budesonide, 0.5mg/2mL) with Meptin^® Inhalation Solution Unit (procaterol hydrochloride, 30μg/0.3mL) from a jet nebulizer, PARI LC Plus^®. Concentration of each drug was assayed with high-pressure liquid chromatography. Physicochemical compatibility was also assessed up to 24 hours after mixing.
Results: With regard to budesonide, impactor parameters such as mass median aerodynamic diameter (MMAD) and respirable mass (RM) were comparable between admixtures and single-drug preparations (2.92 ± 0.03 vs 2.99 ± 0.14μm, 146.8 ± 2.9 vs 147.6 ± 8.2μg, respectively). On the other hand, delivery rates of procaterol increased when admixed with budesonide suspension, resulting in significantly higher RM (15.1 ± 0.8 vs 10.2 ± 0.5μg, p < 0.01). Variations from initial concentration in the percentages of drug remaining at any time point were less than 10%, and there were no appreciable changes in pH of the admixtures for up to 24 hours.
Conclusions: There is a possibility that admixture might influence of aerodynamic characteristics of procaterol, but not budesonide. In vivo data will be needed for the clinical implications of our findings.

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