Takahiro Ninomiya

Novel scoring system and algorithm for classifying chronic rhinosinusitis: the JESREC Study.

Chronic rhinosinusitis (CRS) can be classified into CRS with nasal polyps (CRSwNP) and CRS without nasal polyps (CRSsNP). CRSwNP displays more intense eosinophilic infiltration and the presence of Th2 cytokines. Mucosal eosinophilia is associated with more severe symptoms and often requires multiple surgeries because of recurrence; however, even in eosinophilic CRS (ECRS), clinical course is variable. In this study, we wanted to set objective clinical criteria for the diagnosis of refractory CRS.

Factors associated with the development and remission of allergic diseases in an epidemiological survey of high school students in Japan.

Background Allergic diseases are an important health problem for children and adults. It is important to know how allergic diseases develop and remit from infancy to adolescence. Early intervention is effective in treating allergic diseases. Objective We performed a large-scale questionnaire survey of high school students in Fukui Prefecture, Japan, and analyzed the factors associated with the development and remission of allergic diseases. Methods A total of 21,802 students participated in the epidemiologic survey, and the valid response rate was 89.3% (19,461). We applied an inverse probability weighting method with propensity scores. Results The present prevalence rate of allergic rhinitis (AR) was 19.2%. The remission rate of AR was 15.3%. Only children and firstborns had a significantly higher risk of developing symptoms of allergic diseases [only child: AR, 1.37; bronchial asthma (BA), 1.30; food allergy (FA), 1.33 and firstborn: AR, 1.38; BA, 1.10]. Constipation was an associated factor for development of atopic dermatitis (AD) (1.17) and AR (1.17), regular intake of lactic acid bacteria was not an associated factor for development of allergic diseases but was a factor for remission of AD (1.22). Hypohidrosis was an associated factor for development of AD (1.25). High academic performance was an associated factor for development of AR (1.20) but was a negative factor for development of BA (0.89). The values in parentheses are significant adjusted odds ratios. Conclusion This epidemiologic survey showed that the hygiene hypothesis and intestinal bacterial flora might influence the development of symptoms and remission of allergic diseases.

The significant expression of TRPV3 in nasal polyps of eosinophilic chronic rhinosinusitis

BACKGROUND The number of patients with eosinophilic chronic rhinosinusitis (ECRS) has been increasing in recent years in Japan. In ECRS, nasal polyps recur immediately after endoscopic sinus surgery. The molecular biological mechanism underlying the refractoriness of ECRS is unclear. METHODS Whole-transcriptome analysis with next-generation sequencing (RNA-seq) was conducted to investigate the molecular biological mechanism of ECRS. Real-time PCR, immunohistochemical staining, and immunofluorescence staining were performed to validate the results of RNA-seq. RESULTS RNA-seq analysis revealed that in the nasal polyps of ECRS, the levels of 3 transcripts were elevated significantly and those of 7 transcripts were diminished significantly. Among the genes encoding these transcripts, TRPV3 (transient receptor potential cation channel, subfamily V, member 3) was identified as the only gene that is highly expressed in ECRS nasal polyps but this genes expression was not previously detected using DNA microarray analysis in peripheral blood eosinophils. TRPV3 is newly identified here as a gene transcribed in ECRS. Our analysis also revealed that TRPV3 was highly expressed in the infiltrating eosinophils and mucosal epithelium of the nasal polyps of ECRS, and further that the more severe the refractoriness was after surgery, the higher the TRPV3 expression was in nasal polyps. CONCLUSIONS TRPV3 might play a role in the refractoriness of ECRS. Additional studies are required to evaluate the function of TRPV3 in ECRS.

Peripheral basophil reactivity, CD203c expression by Cryj1 stimulation, is useful for diagnosing seasonal allergic rhinitis by Japanese cedar pollen.

Measuring specific IgE can yield direct, accurate, and objective data. Nevertheless, clinical symptoms of allergy are often inconsistent with these data. Recently, the expression of CD203c, a surface marker of basophils, has been reported as capable of distinguishing allergic patients. This study compared specific IgE in serum and skin tests against antigen to assess CD203c as a biomarker correlated with allergic rhinitis (AR). We asked 3,453 subjects whether they experienced any AR related symptom. All subjects were assessed for six specific IgEs for common aeroallergens. Skin tests were also conducted for six aeroallergens. We observed the reactivity of peripheral basophil by measuring the levels of CD203c by Cryj1 stimulation using flow cytometry. Of the 3,453 participants, 1,987 (57.5%) possessed Japanese cedar pollen (JCP) specific IgE in their serum. Among those 1,987 JCP specific IgE positive participants, 552 (27.8%) had not experienced any allergic symptom during the JCP season. The levels of CD203c in the peripheral basophil by Cryj1 stimulation were significantly higher in SAR‐JCP subjects than in non‐SAR‐JCP subjects (Cryj1 0.5 ng/ml: 2.25 ± 0.90% vs. 60.2 ± 27.4%, p < 0.01, Cryj1 50 ng/ml: 1.89 ± 0.90% vs. 68.0 ± 21.2%, p < 0.01). Our results indicate that the levels of CD203c in peripheral basophils by Cryj1 stimulation is a more objective and reliable marker that better reflects the allergic reaction by SAR‐JCP in vivo than measuring specific IgE in serum or skin tests.

Periostin as a novel biomarker for postoperative recurrence of chronic rhinosinitis with nasal polyps

We previously reported that chronic rhinosinusitis with nasal polyps (CRSwNP) was subdivided into four chronic rhinosinusitis (CRS) subtypes using the JESREC scoring system. We sought to identify the gene expression profile and biomarkers related with CRSwNP by RNA-sequence. RNA-sequencing was performed to identify differentially expressed genes between nasal polyps (NPs) and inferior turbinate mucosa from 6 patients with CRSwNP, and subsequently, quantitative real-time PCR was performed to verify the results. ELISA was performed to identify possible biomarkers for postoperative recurrence. In the RNA-sequencing results, periostin (POSTN) expression was the highest in NP. We focused on POSTN and investigated the protein level of POSTN by immunohistochemistry and ELISA. POSTN was diffusely expressed in moderate and severe eosinophilic CRS using immunohistochemistry, and its staining pattern was associated with the severity of the phenotype of the CRSwNP (P < 0.05). There was a significant difference between the POSTN high/low groups for postoperative recurrence when the cutoff point was set at 115.5 ng/ml (P = 0.0072). Our data suggests that the protein expression level of POSTN was associated with the severity of CRSwNP, and serum POSTN can be a novel biomarker for postoperative recurrence of CRSwNP.

Influence of MILR1 promoter polymorphism on expression levels and the phenotype of atopy.

The recently identified cell surface immunoreceptor MILR1 (mast cell immunoglobulin-like receptor 1; synonyms, Allergin-1) has been shown to suppress immunoglobulin E (IgE)-mediated, mast cell-dependent responses in both mice and humans. We performed a mutation search of MILR1 together with a genetic association study to determine whether polymorphisms in MILR1 are associated with atopy in human. Mutation screening of MILR1 was performed using DNA from 146 unrelated Japanese. Genotyping of the identified polymorphisms was done with 1505 individuals from the general Japanese adult population. Atopy, as defined by positive responses for specific IgEs against at least one of the 26 common allergens, was evaluated using MAST-26. Five polymorphisms (rs6504230, c.−170_−166delAGGAA, rs8071835, rs143526766 and rs12936887) and two rare missense variants (Val273Ala and Leu311Val) were identified by mutation screening. The C allele of rs6504230 had protective effects against atopy (P=0.002). A luciferase reporter assay using the promoter region of MILR1 revealed that the C allele of rs6504230 was associated with increased expression of MILR1, which was in accordance with the results of expression quantitative trait loci analysis using human leukocytes. Our data indicates that the rs6504230 polymorphism affects MILR1 expression levels in humans, leading to a susceptibility to producing specific IgE antibodies against common allergens.

Combination therapy with montelukast and loratadine alleviates pharyngolaryngeal symptoms related to seasonal allergic rhinitis

There is growing evidence that the prevalence of allergic rhinitis is increasing, affecting 10% to 40% of the global population. Emerging evidence indicates that the clinical manifestations in united airway disease are heterogeneous, with a clinical profile often related to the individual airborne allergen. The upper and lower respiratory tracts share the same anatomical, functional, pathological, and immunological features. Some clinical features of allergic rhinitis, such as pharyngolaryngeal symptoms, also overlap with those of asthma; however, few reports have focused on the management of pharyngolaryngeal symptoms related to seasonal allergic rhinitis (SAR). Furthermore, there remains a lack of evidence showing the relationship between nasal symptoms and pharyngolaryngeal symptoms. In Japan, SAR caused by Japanese cedar (Cryptomeria japonica) pollen (SAR-JCP) is the most common allergic disease. Prophylactic treatment with antihistamine has been promoted; however, it has been suggested that leukotriene receptor antagonists might also be suitable as prophylactic agents against SAR. Our previous report showed that the combination of an antileukotriene and antihistamine was effective for treating SAR-JCP as it modified the nasal symptoms. Leukotriene receptor antagonists suppress inflammatory responses and reduce the exacerbation of asthma. We hypothesized that pharyngolaryngeal symptoms may be related to nasal symptoms. As such, we examined whether an antileukotriene and/or antihistamine could suppress pharyngolaryngeal symptoms. Patients with SAR-JCP (n 1⁄4 137) were prescribed oral montelukast (10 mg/d) once daily from the beginning of the study. They were subsequently also treated with montelukast alone if they had a visual analog scale score of less than 50 for nasal symptoms during the high pollen season. Patients with a visual analog scale score of more than 50 for all nasal symptoms during the high pollen season were subsequently enrolled in a placebo-controlled double-blind study of add-on antihistamine loratadine, as described in Figure E1 in this article’s Online Repository at www.jaci-inpractice.org. In total, 95 (69.3%) subjects were allocated to the montelukast-alone group (M group), and the remaining 42 subjects were enrolled in the placebo-controlled double-blind study, which included a montelukast þ loratadine (M þ L, n 1⁄4 21) group and a montelukast þ placebo (M þ P, n 1⁄4 21) group, as described in Table E1 in this article’s Online Repository at www.jaciinpractice.org. Nasal and pharyngolaryngeal symptoms were recorded according to the Japanese Allergic Rhinitis Standard Quality of Life Questionnaire. We summed the scores for wheezing in the throat, coughing, sputum production, hoarseness, throat itchiness, and throat soreness to obtain the total pharyngolaryngeal symptom scores (TPLSSs). In both the M þ L and M þ P groups, the TPLSSs began to increase gradually with increasing pollen dispersal during the pollen season, reaching a high level by March 9; in contrast, the TPLSSs in the M group remained low throughout the pollen season (Figure 1). The additional oral administration of loratadine or placebo was started between March 9 and 12. After 2 weeks of loratadine administration, no significant difference in TPLSSs was seen between the M and M þ L groups; however, the M þ P group still showed significantly higher TPLSSs when compared with the M group (P < .01). Although the TPLSSs in the M þ L group continued to decrease and eventually showed no significant difference with the M group, the TPLSSs of the M þ P group remained significantly higher than those of the M group until the end of the pollen season. These results indicated that loratadine may suppress the pharyngolaryngeal symptoms related to SAR-JCP. We defined the total nasal symptom scores (TNSSs) as the total scores of sneezing, rhinorrhea, nasal congestion, and nasal itchiness. The TNSSs in both the M þ P and M þ L groups peaked at around March 9 (see Figure E2 in this article’s Online Repository at www.jaci-inpractice.org). Similar to the TPLSSs, the administration of loratadine for 2 weeks decreased the TNSSs of the M þ L group; however, the TNSSs of the M þ P group were significantly higher than those of the M group (P < .001) on March 23. We analyzed the correlations between the TPLSSs and each nasal symptom score just before the start of loratadine administration. There were significant correlations between the TPLSSs and the sneezing score (R 1⁄4 0.4059; P < .0001), rhinorrhea score (R 1⁄4 0.3818; P < .0001), nasal congestion score (R1⁄4 0.5007; P < .0001), nasal itchiness score (R1⁄4 0.5663; P < .0001), and the TNSSs (R 1⁄4 0.5906; P < .0001) (Table I). These results suggested that the pharyngolaryngeal symptoms caused by SAR-JCP are closely related to nasal symptoms. Although small particles can reach the lower airway tract, where they may cause lower airway inflammation, JCP (w30 mm in diameter) becomes trapped in the larynx, where it may cause pharyngolaryngeal symptoms. Nasal congestion may enable more JCP to be trapped in the larynx. Rhinorrhea may also affect pharyngolaryngeal symptoms, because much of the fluid from the nose falls down into the larynx, which may explain how the TPLSSs and TNSSs are closely related.

DNA Methylation of Proximal PLAT Promoter in Chronic Rhinosinusitis With Nasal Polyps

Background Nasal polyps (NP) are characterized by pseudocysts derived from stromal tissue edema and cause persistent infections in patients with chronic rhinosinusitis (CRS). A low level of tissue-type plasminogen activator (gene name PLAT) is considered a cause of stromal tissue edema because of insufficient plasmin activation in NP; however, the mechanism regulating PLAT gene expression levels is still unclear. The epigenetic mechanism regulating the PLAT gene expression has been studied in other tissues. Objective We aimed to investigate the methylation levels in the proximal PLAT promoter and their effects on gene expression in NP tissue. Methods We investigated the methylation levels at 3 CpG sites in the proximal PLAT promoter regions (−618, −121, and −105 with respect to the transcription initiation site) by bisulfite pyrosequencing and their effects on the gene expression by quantitative real-time polymerase chain reaction (qPCR) in 20 paired samples of NP and inferior turbinate tissue (IT) from patients with CRS. Results The DNA methylation levels at all CpG sites were higher (P < .01), and the PLAT expression was lower (P < .001) in NP compared with IT. The methylation changes at the −618 site showed a negative correlation with the gene expression changes between NP and IT (r = −.65, P < .01). Conclusions Hypermethylation of PLAT promoter may downregulate the gene expression in NP, leading to excessive fibrin deposition by aberrant coagulation cascade. DNA methylation of proximal PLAT promoter may contribute to NP growth and have a potential as a new therapeutic target.

Expression and Functional Analysis of CST1 in Intractable Nasal Polyps

Abstract In this study, we found Cystatin SN (CST1), a type 2 cystatin subfamily member, to be highly expressed in nasal polyps from patients with intractable chronic rhinosinusitis (CRS) with nasal polyps, using a whole‐transcript analysis with next‐generation sequencing. Eosinophilic CRS (ECRS) involves nasal polyps that are refractory and recur immediately after endoscopic sinus surgery. We hypothesized that CST1 may contribute to the pathogenesis of ECRS. We examined the expression of CST1 in nasal polyps from patients with ECRS by assessing mRNA expression levels using real‐time PCR and immunohistochemistry. CST1 showed significantly greater expression in the epithelial cells of nasal polyps from patients with ECRS than in those from patients who did not have ECRS (non‐ECRS). In particular, CST1 showed very strong expression in patients with severe ECRS. The expression of CST1 may be correlated with the recurrent and refractory nature of ECRS. We examined the function of CST1 using nasal epithelial cells and nasal fibroblasts. Stimulation by a combination of IL‐4 plus double‐stranded RNA plus CST1 significantly elevated mRNA expression levels and protein levels of TSLP in nasal epithelial cells. Stimulation by TSLP or IL‐33 significantly elevated mRNA expression levels of CST1 in nasal epithelial cells. Stimulation of CST1 significantly elevated mRNA expression levels of CCL11 and POSTN in nasal fibroblasts. CST1 could amplify eosinophilic infiltration and T‐helper cell type 2 inflammation by interacting with epithelial‐derived cytokines and fibroblasts on nasal polyps. CST1 may be involved in the pathogenesis of ECRS, and may contribute to the severity and recurrence of CRS with nasal polyps after endoscopic sinus surgery.