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Chronic rhinosinusitis with nasal polyps (CRSwNP) is characterized by type 2 inflammation with accumulation of activated group 2 innate lymphoid cells (ILC2s) and elevation of thymic stromal lymphopoietin (TSLP). A member of the TNF superfamily (TNFSF), TNFSF15, is known to induce the production of type 2 cytokines in ILC2s. Although ILC2s have been implicated in CRSwNP, the presence and role of TNFSFs in ILC2-mediated type 2 inflammation in CRSwNP has not been elucidated. Here, we investigate the involvement of TNFSFs in ILC2-mediated type 2 inflammation in CRSwNP. We found that receptor activator of NF-κB (RANK) ligand (RANK-L (TNFSF11)) was significantly elevated in nasal polyps (NPs), and that the receptor of RANK-L, RANK, was expressed on ILC2s in human peripheral blood and NPs. An agonistic antibody against RANK induced production of type 2 cytokines in human ILC2s, and TSLP significantly enhanced this reaction. The membrane-bound RANK-L was detected mainly on CD45 + immune cells, including TH2 cells in NPs. The co-culture of NP-derived ILC2s and TH2 cells significantly enhanced production of type 2 cytokines, and anti-RANK-L monoclonal antibody suppressed this enhancement. In conclusion, RANK-L, together with TSLP, may play an inductive role in the ILC2-mediated type 2 inflammation in CRSwNP.

IgG4-related disease (IgG4-RD) is a chronic fibroinflammatory disease characterized by elevation of serum IgG4 level as well as infiltration of IgG4+ plasma cells in various affected organs. The etiology of IgG4-RD is still not fully understood. Since IgG4-RD is more prevalent in the elderly, aging in itself is considered to be an important risk factor of IgG4-RD. However, the relationship between the pathogenesis of IgG4-RD and immunosenescence remains unknown. To clarify age-related features underlying IgG4-RD, we focused on T follicular regulatory (Tfr) cells, which share forkhead box P3 with regulatory T cells, since the percentage of Tfr cells is known to depend on age. Studies of blood specimens from patients with IgG4-RD and from healthy volunteers demonstrated a marked elevation of circulating Tfr (cTfr) cells in patients with IgG4-RD. Moreover, the percentage of cTfr cells was significantly correlated with various clinical parameters including the level of serum IgG4 and the number of involved organs in IgG4-RD patients. The percentages of tonsillar and blood Tfr cells were increased with aging in healthy volunteers, whereas the suppressive effect of cTfr cells on B cell function in elderly subjects was impaired in comparison with that in young subjects due to a defect in the production of a regulatory cytokine, IL-10. Given that the number of IL-10-producing cTfr cells in IgG4-RD patients was markedly increased compared with that in healthy elderly subjects, these findings suggest that an abnormal aging process of Tfr cells may be related to the pathogenesis of IgG4-RD.

Background and purpose To investigate influences of proteins involved with tumor immunity on outcomes of radiotherapy for oropharyngeal squamous cell carcinoma (OPSCC). Material and methods We performed immunohistochemical staining to examine expressions of p16 and proteins involved with tumor immunity in 92 OPSCC patients treated with radiotherapy. Results Patients with abundant infiltrating CD8-positive cells had the significantly better overall survival (OS) rate than patients with fewer CD8-positive cells (p = 0.026). Patients with higher PD-L1 expression in tumor cells (TC 1–3) had a better outcome than those with low PD-L1 expression in tumor cells (TC 0) for both OS (p = 0.019) and progression-free survival (PFS) rate (p = 0.032). Patients with high PD-L1 expression in infiltrating immune cells (IC 3) showed significantly better OS (p = 0.009) and PFS (p = 0.011) than those with low PD-L1 expression (IC 0–2). Patients with p16-negative and IC 3 showed similar OS to patients with p16-positive and IC 0–2. P16-positive tumors had a significantly higher CD8-positive cell infiltration and PD-L1 expression in tumor cells than p16-negative tumors. Conclusions In addition to tumor p16 expression, PD-L1 expression in TC and IC can be useful for predicting the response of OPSCC to radiotherapy.

Guanylate-binding protein-1 (GBP-1) is an interferon-inducible large GTPase involved in the epithelial barrier at tight junctions. To investigate the role of GBP-1 in the epithelial barrier, primary human salivary gland duct epithelial cells were treated with the the proinflammatory cytokines IFNγ, IL-1β, TNFα and the growth factor TGF-β. Treatment with IFNγ, IL-1β, or TNFα markedly enhanced GBP-1 and the epithelial barrier function, and induced not only CLDN-7 but also the tricellular tight junction molecule lipolysis-stimulated lipoprotein receptor (LSR). Knockdown of GBP-1 by its siRNA induced endocytosis of tight junction molecules, and prevented the increases of CLDN-7 and LSR with the upregulation of the epithelial barrier function induced by treatment with IFNγ or TNFα. Treatment with a PKCα inhibitor induced expression of GBP-1, CLDN-7 and LSR and enhanced the epithelial barrier function. In almost intact salivary gland ducts from patients with IgG4-related disease (IgG4-RD) indicated significant infiltration of IgG-positive plasma cells, expression of GBP-1, CLDN-7 and LSR was increased. These findings indicated that GBP-1 might play a crucial role in barrier function of normal human salivary gland duct epithelium and perform a preventive role in the duct epithelium of IgG4-RD disease.

Florid reactive follicular hyperplasia (FRFH), which is characterized by large germinal centers (GCs) within normal lymphoid follicles, is often observed in benign lesions of lymph nodes and other tissues. Because of the histologic similarity of FRFH to tumorous lesions such as follicular lymphoma, careful pathological examination is required to evaluate such lesions; however, little is known about the mechanism underlying the development of FRFH. In this study, we investigated T follicular helper (Tfh) cells in hyperplastic tonsils of patients with obstructive sleep apnea syndrome (OSA), which frequently exhibits typical FRFH. When we analyzed tonsils of OSA and recurrent tonsillitis (RT) as a control, tonsils of OSA were found to harbor Tfh cells with a nearly 3-fold higher ratio in total CD4+ T cells than that in tonsils of RT. Further analysis showed that, in comparison to Tfh cells of RT tonsils, Tfh cells of OSA tonsils were relatively tolerant to CD3-mediated activation-induced cell death (AICD) and also expressed lower levels of a Bob1 transcription coactivator and IL-4, which fosters the development of GC-B cells. Given that Bob1 controls the proliferative activity in response to CD3 stimulation and has been suggested to have a role in the production of IL-4 in Tfh cells, the unique structure of FRFH is possibly associated with the function of Bob1lo Tfh cells.

Background Atopic dermatitis (AD) is a chronic inflammatory skin disease that often cannot be completely controlled by modern medicine. Since multiple factors are intricately involved in the pathogenesis of AD, wide-ranging research is required for further advancement of AD treatment. Epidermal keratinocytes are the forefront to the external environment and play a pivotal role in the initiation of immune reaction against exogenous invasion. Objective Thymic stromal lymphopoietin (TSLP) is a keratinocyte-derived cytokine that induces differentiation and activation of type 2 helper T cells and innate lymphoid cells, cardinal effectors in pathophysiology of AD. We previously reported that ΔNp63, a p53-related molecule, regulates the expression of TSLP receptors and suggested the entity of a potential TSLP autocrine loop in the AD epidermis. In this study, we further explored the significance of p53 family transcription factors in TSLP production from human keratinocytes. Method Expression profile of p73, a p53-related molecule, was evaluated in human AD tissue by immunohistochemistry. In addition, the function of p73 in producing TSLP was investigated with in vitro cultured keratinocytes via molecular biological analysis. Results ΔNp73 was abundantly expressed in the AD epidermis and increased the release of TSLP via NF-κB activation. Furthermore, the Toll-like receptor 3 signal enhanced ΔNp73 expression and thereby induced TSLP expression. Conclusion Our results indicate that ΔNp73 is an additional participant in the mechanism of TSLP production. Amending the aberrant state of keratinocytes, represented by overexpression of ΔNp73, can be a novel therapeutic target of AD.

We present two cases of an HIV-associated parotid gland cyst. One case was a 36-year-old HIV infected woman. She was diagnosed with HIV infection and presented with slowly enlarged parotid gland cysts together with elevation of HIV viral RNA copies/mL in her serum. She was performed parotid gland biopsy under the general anesthesia. The histopathologic analysis revealed negative HIV p24-antigen in her parotid gland tissue. The other case was a 43-year-old man found his parotid gland swelling shortly after highly active antiretroviral therapy (HAART). He was diagnosed with HIV infection 2 years previously. He had started HAART several days before. He showed exceeding elevation of IgE in his serum. We treated him with medication using anti-histamic drugs for his cyst. A computed tomography scan revealed a complete response of his parotid gland cyst 4 weeks after the medication. His serum IgE level was decreased to half of the level before the medication. These findings suggested that the parotid gland swelling associated with HIV was due to various factors including immune reconstitution inflammatory syndrome (IRIS). In case such a parotid gland swelling, we could avoid invasive treatments.

Lipid mediators such as leukotrienes and lipoxines broadly regulate innate and acquired immunity, and their dysfunction causes various immune-mediated disorders. We previously reported a salient feature of arachidonate 5-lipoxyganase (Alox5), which is responsible for the production of such lipid mediators, in the regulation of high affinity antibodies in vivo. The aim of this study was to determine the functional significance of Alox5-related lipid mediators during the processes of acquired humoral responses. The results of in vitro experiments using lymphocytes in tonsils and blood specimens showed that lipoxin A4 (LXA4) and leukotriene B4 (LTB4) have the capacity to differentiate naive CD4+ T cells into T follicular helper (Tfh) cells, which activate naive B cells to form germinal centers. Such a function of LXA4 was further supported by results of in vitro studies using BML-111 and BOC-2, which are an agonist and an antagonist, respectively, of FPR2 of an LXA4-specific cell-surface receptor. The results suggest that such lipid mediators have a potential role in the development of lymphoid follicles through the regulation of Tfh cell differentiation.

An aberrant internal carotid artery (ICA) is a rare abnormality associated with life-threatening otorrhagia if inadvertently injured during middle-ear surgery including myringotomy. We present a case where a 3-year-old girl experienced massive otorrhagia following myringotomy, and computed tomographic scan showed the aberrant ICA. Bleeding was controlled by ear canal packing, but rebleeding occurred. Investigations by carotid angiography demonstrated a pseudoaneurysm of the aberrant ICA in the middle ear. We attempted surgical repair using a high-flow bypass technique; however, the bypass graft was occluded by embolic complications, and eventually, ligation of the ICA was performed, which led to the paralysis of the patient's left limbs. In this report, management of iatrogenic aberrant ICA injuries and pseudoaneurysms in the middle ear are discussed based on the case that we experienced.

Macrolide antibiotics exert immunomodulatory activity by reducing pro-inflammatory cytokine production by airway epithelial cells, fibroblasts, vascular endothelial cells, and immune cells. However, the underlying mechanism of action remains unclear. Here, we examined the effect of clarithromycin (CAM) on pro-inflammatory cytokine production, including interferons (IFNs), by primary human nasal epithelial cells and lung epithelial cell lines (A549 and BEAS-2B cells) after stimulation by Toll-like receptor (TLR) and RIG-I-like receptor (RLR) agonists and after infection by human respiratory syncytial virus (RSV). CAM treatment led to a significant reduction in poly I:C- and RSV-mediated IL-8, CCL5, IFN-β and -λ production. Furthermore, IFN-β promoter activity (activated by poly I:C and RSV infection) was significantly reduced after treatment with CAM. CAM also inhibited IRF-3 dimerization and subsequent translocation to the nucleus. We conclude that CAM acts a crucial modulator of the innate immune response, particularly IFN production, by modulating IRF-3 dimerization and subsequent translocation to the nucleus of airway epithelial cells. This newly identified immunomodulatory action of CAM will facilitate the discovery of new macrolides with an anti-inflammatory role.

Objective Adrenergic receptors (ARs) include four general types (α1, α2, β1 and β2), which are found in different target tissues. α-AR agonists are commonly used for decongestant therapy of upper airway diseases. In order to clarify the roles of AR subtypes in the upper airways, we investigated the localization of these receptors by immunohistochemistry. Methods Human turbinates were obtained after turbinectomy from 12 patients with nasal obstruction refractory to medical therapy. The specific cells expressing α- and β-AR proteins were identified by immunostaining using an anti-human AR subtype-specific antibodies (α1A-, α1D-, α2C- and β2-ARs) antibody. Results Immunohistochemical analysis revealed that immunoreactivities for α1D- and β2-ARs were densely distributed in submucosal glands. In contrast, immunoreactivities for α1A- and 2C-ARs were densely distributed in vascular smooth muscle. Conclusion Our results suggested that adrenergic receptor (AR) subtypes had different roles in upper airway diseases, such as allergic rhinitis and nonallergic rhinitis.

Allergic rhinitis (AR), the most common allergic disorder of the airway, is often accompanied by bronchial asthma. However, little is known about the mechanism by which AR advances to AR comorbid with bronchial asthma (AR+Asthma). To determine the pathophysiologic features of AR and AR+Asthma, we examined subsets of follicular helper T (Tfh) cells and regulatory B (Breg) cells in peripheral blood from AR and AR+Asthma patients. The results showed polarization of Tfh2 cells within Tfh cell subsets in both AR and AR+Asthma cases. Interestingly, the %Breg cells in total B cells were decreased in AR cases and, more extensively, in AR+Asthma cases. Moreover, we found significant correlations of fractional exhaled nitric oxide and blood eosinophil levels with the index %Tfh2 cells per %Breg cells. Our findings indicate that relative decrease in Breg cells under the condition of Tfh2 cell skewing is a putative exaggerating factor of AR to bronchial asthma.

Background Hypersecretion of mucin in the airway epithelium is an important feature of allergic airway diseases. Of the 3 cysteinyl leukotrienes (CysLTs; LTC 4 LTD 4 and LTE 4 ), only LTE 4 is sufficiently stable to be detectable in extracellular fluids. However, LTE 4 has received little attention because it binds poorly to the CysLT 1 and CysLT 2 receptors; therefore, little is known about the effects of LTE 4 on mucous secretion. Recently, studies have focused on the P2Y 12 receptor as a potential receptor for LTE 4 , because this receptor is required for LTE 4 -mediated pulmonary inflammation. In our previous study, we confirmed the expression of P2Y 12 receptor in human airway epithelial cells. To clarify the roles of LTE 4 in airway epithelial cells, we investigated mucus secretion by LTE 4 in vitro. Methods Confluent NCI-H292 cells were stimulated with LTE 4 (0.01–1 μM) for 24 h. The release and production of MUC5AC protein, a gel-forming mucin, were evaluated with an enzyme-linked immunosorbent assay. Results Western blot analysis revealed that NCI-H292 cells expressed P2Y 12 receptor protein. LTE 4 significantly induced the release of MUC5AC mucin in a dose-dependent manner. Th2 cytokines such as IL-4 (10 ng/mL) and IL-13 (10 ng/mL) accelerated the LTE 4 -induced release of MUC5AC protein. MRS2935, a P2Y 12 receptor antagonist, partially inhibited the LTE 4 -induced release of MUC5AC protein in the airway. In contrast, MK571, a CysLT 1 receptor antagonist, did not affect the release of MUC5AC protein elicited by LTE 4. Conclusions These results suggest that LTE 4 may play some important roles in allergic mucus secretion partially via activation of P2Y 12 receptor.

Objective Tracheo-innominate artery fistula (TIF) is a rare but life-threatening complication following tracheostomy or tracheoesophageal diversion (TED). Although successful surgical intervention for TIF has been reported, few studies have been performed in patients with severe motor and intellectual disability (SMID). Therefore, we aimed to analyze TIF in patients with SMID to clarify the clinical variables predicting the occurrence and adequate management for lifesaving of TIF. Methods We retrospectively reviewed the records of patients with SMID undergoing surgical tracheostomy and TED between 2006 and 2012 and identified those with TIF. When TIF occurred, we obtained the clinical status and emergency management. Results Of 70 patients who underwent tracheostomy or TED during the study period, three patients had TIFs; in one case, TIF was avoided by ligation of the innominate artery before TED. The incidence of TIF in those undergoing tracheostomy and TED was 2.3% and 7.4%, respectively. The interval between tracheostomy and TIF was 14–50 months. Conclusions Patients with SMID may have an increased risk of TIF. Prompt diagnosis and surgical intervention to control the bleeding is the only effective management at present.

Osteosarcoma is a rare but highly malignant tumor occurring most frequently in adolescents. The prognosis of non-responders to chemotherapy is still poor, and new treatment modalities are needed. To develop peptide-based immunotherapy, we previously identified autologous cytotoxic T lymphocyte-defined osteosarcoma antigen papillomavirus binding factor (PBF) in the context of HLA-B55 and the cytotoxic T lymphocyte epitope (PBF A2.2) presented by HLA-A2. PBF and HLA class I are expressed in ∼90 and 70% of various sarcomas, respectively. However, the expression status of peptide PBF A2.2 presented by HLA-A2 on osteosarcoma cells has remained unknown because it is difficult to generate a specific probe that reacts with the HLA·peptide complex. For detection and qualification of the HLA-A*02:01·PBF A2.2 peptide complex on osteosarcoma cells, we tried to isolate a single chain variable fragment (scFv) antibody directed to the HLA-*A0201·PBF A2.2 complex using a naïve scFv phage display library. As a result, scFv clone D12 with high affinity (KD = 1.53 × 10(-9) M) was isolated. D12 could react with PBF A2.2 peptide-pulsed T2 cells and HLA-A2+PBF+ osteosarcoma cell lines and simultaneously demonstrated that the HLA·peptide complex was expressed on osteosarcoma cells. In conclusion, scFv clone D12 might be useful to select candidate patients for PBF A2.2 peptide-based immunotherapy and develop antibody-based immunotherapy.

Chronic sclerosing sialadenitis, which clinically resembles a salivary gland tumor, is considered a salivary gland lesion of the IgG4-related inflammatory disease. Corticosteroids are effective for the treatment of chronic sclerosing sialadenitis, but the natural clinical course of this disease is not fully understood. We report a 67-year-old man with chronic sclerosing sialadenitis who showed lung lesions, which spontaneously regressed without steroid therapy after excision of the submandibular gland. He presented with several weeks’ history of continuous swelling in the right submandibular region. Physical examination showed a hard swelling of the right submandibular gland; the cytological finding was Papanicolaou class III. A chest X-ray demonstrated bilateral infiltration of the right middle and left lower lobes. The right submandibular gland was excised under general anesthesia for definite diagnosis, and histopathological examination revealed an IgG4-positive plasmacytic infiltration accompanied by periductal fibrosis and interlobular sclerosis, consistent with chronic sclerosing sialadenitis. Five months after operation, the lung lesions regressed spontaneously.

In this study, we report the unique role of arachidonate 5-lipoxygenase (Alox5) in the regulation of specific humoral immune responses. We previously reported an L22 monoclonal antibody with which human primary resting B cells in the mantle zones of lymphoid follicles are well-defined. Proteomics analyses enabled identification of an L22 antigen as Alox5, which was highly expressed by naive and memory B cells surrounding germinal centers. Cellular growth of mantle cell lymphoma cells also seemed to depend on Alox5. Alox5(-/-) mice exhibited weak antibody responses specific to foreign antigens at the initial and recall phases. This was probably attributable to the low number of follicular and memory B cells and the functional loss of interleukin-21-mediated responses of follicular B cells. Moreover, Alox5(-/-) mice could not fully foster the development of follicular B helper T (Tfh) cells even after immunization with foreign antigens. Further experiments indicated that Alox5 affected mortality in experimentally induced enterocolitis in germ-prone circumstances, indicating that Alox5 would endow immunologic milieu. Our results illustrate the novel role of Alox5 in adaptive humoral immunity by managing primary B cells and Tfh cells in vivo.

Objective: To determine the frequency of hearing impairment in children with congenital anomalies of the central nervous system (CNS) by using detailed audiological evaluation methods. Methods: The patients were 78 children with congenital anomalies of the CNS with a mean age of 29.5 months. They had been observed for a mean period of 38.5 months. Hearing levels were evaluated behavioral observation audiometry (BOA), visual reinforcement audiometry (VRA) and distortion product otoacoustic emissions (DPOAEs) were performed. Auditory brainstem responses (ABRs) and computed tomography (CT) scans of the temporal bone were performed in the cases in which the minimum response levels (MRLs) were above 30 dBHL. All cases were assessed in terms of developmental age. Results: A total of 14.1% (11/78) of the children with congenital anomalies of the CNS were initially diagnosed with bilateral sensorineural hearing loss (SNHL). However, the hearing levels of nine of them improved by the time of the last diagnosis. Therefore, the patients with bilateral SNHL were only 2.6% (2/ 78) of the total patients with congenital anomalies of the CNS at last diagnoses. As shown by our results, many children with bilateral SNHL at initial diagnosis showed improved ABR thresholds and behavioral hearing thresholds with age. In this series, the use of hearing aids was arranged for six patients. However, four patients stopped using hearing aids when their hearing threshold levels improved. In two cases, there were no changes in hearing levels and the children continued using hearing aids. Conclusion: Our results suggest that hearing level recovery can occur in some children with CNS anomalies. Confirmation of hearing loss in children with congenital anomalies of the CNS takes a long time. There are improvements in hearing loss during the observation period. Therefore periodic assessment of hearing is important.

Background Prostaglandin D 2 (PGD 2 ) has been thought to be a potent mediator involved in allergic rhinitis because PGD 2 has been recovered from the nasal lavage fluid of patients with allergic rhinitis after allergen provocation and because PGD 2 receptor antagonists relieved nasal allergic symptoms in an animal model of allergic rhinitis. The inflammatory effects of PGD 2 are exerted through high-affinity interactions with 2 G protein–coupled receptors: D-prostanoid receptor 1 and chemoattractant-homologous receptor expressed on T H 2 cells (CRTH2). CRTH2 may mediate the recruitment of leukocytes during a nasal allergic response. Objective To evaluate the number of CRTH2-expressing cells in allergic and nonallergic human nasal mucosa by means of immunohistochemical analysis. Methods Human turbinates were obtained after turbinectomy from 14 patients with nasal obstruction refractory to medical therapy. To identify cells expressing the CRTH2 protein, double immunostaining was performed using anti–CRTH2 antibody and monoclonal anti–leukocyte antibodies. Results The immunohistochemical study revealed that anti–CRTH2 antibody labeled eosinophils, macrophages, mast cells, T lymphocytes, epithelial cells, and submucosal glands in the nasal mucosa. CRTH2 expressions of these leukocytes in allergic nasal mucosa are significantly up-regulated compared with those in nonallergic nasal mucosa. Conclusion These results suggest that CRTH2 may play an important role in the recruitment of leukocytes into allergic nasal mucosa.

Summary Hamartoma, defined as tumour-like overgrowths of tissue, may occur in any organ in the body and is commonly seen in liver, spleen, kidney and lung, however, lingual hamartoma is a rare entity. Terminal deletion of chromosome 2q is a chromosomal abnormality causing various craniofacial malformations, and no reports can be obtained about patients who have chromosomal disorder presenting with lingual hamartoma. We describe the first case of lingual hamartoma in a patient with terminal deletion of chromosome 2, and summarize the clinical management and the histogenesis of lingual hamartoma from the literature. A 9-month-old girl who had chromosome 2q terminal deletion consulted Sapporo Medical University hospital for evaluation of upper airway obstruction. She had retractive breathing soon after birth and required treatment with continuous positive airway pressure (CPAP). Physical examination showed craniofacial abnormalities, such as round face, high palate, aural atresia, anotia and so on. Fiberoptic examination and CT scan revealed a mass on the right side of the tongue base. She underwent surgical resection and the tumour was pathologically diagnosed as hamartoma. After surgery, her respiratory condition improved to the extent where CPAP was withdrawn. There has been no tumour recurrence during the 1-year follow-up period.

Capsaicin, a type of alkaloid and the pungent component of chili peppers, is used as a therapeutic drug against allergic rhinitis and also as an index of bronchial hypersensitivity. Capsaicin receptor (TRPV1) expression has been identified in non-neuronal cells as well as neuronal cells. In our previous study, both TRPV1 protein and its gene expression on nasal epithelial cells were confirmed by immunohistochemistry and RT-PCR, respectively. In order to clarify whether or not TRPV1 acts as a functional receptor, we examined the effects of capsaicin on the production of IL-6 from primary cultured human airway epithelial cells at both protein and mRNA levels. Human nasal epithelial cells (HNECs) and normal human bronchial/tracheal epithelial cells (NHBE cells) were stimulated with increasing concentrations of capsaicin and/or pretreatment with capsazepine (TRPV1 antagonist) at 37 degrees C. The supernatant and total RNA were collected at 0, 4, 12, 24 and 48 h after treatment. IL-6 concentration and the IL-6 mRNA level were evaluated by ELISA and real-time PCR, respectively. Capsaicin (10 nM-10 muM) induced production of IL-6 from HNECs and NHBE cells and this effect was inhibited by pretreatment with capsazepine. Our findings suggest that topical application of capsaicin to the airway induces IL-6 production from respiratory epithelial cells via activation of TRPV1.

Our previous study demonstrated that the frequency of penicillin-resistant Streptococcus pneumoniae (PRSP) was lower in our district than in districts in other Japanese studies. In this study, we investigated the prevalence of erythromycin resistance. The susceptibility to erythromycin and the distribution of the macrolide-resistance genes, mefA and ermB, were examined in S. pneumoniae isolates from the upper respiratory tracts of children in four cities in the Sapporo district, Hokkaido prefecture, Japan. Of the 156 isolates, 27 (17.3%) were erythromycin-sensitive, 6 (3.9%) were erythromycin-intermediately resistant, and 123 (78.9%) were erythromycin-resistant. Fifty-nine (37.8%) had the mefA gene, 89 (57.1%) had the ermB gene, and 129 (82.7%) had the mefA and/or the ermB gene. The ermB-positive isolates tended to show high resistance to erythromycin. Erythromycin-resistant isolates and the macrolide-resistance genes were often present in infants or younger children. The frequency of erythromycin-resistant isolates in the four cities was very high, ranging from 76.3% to 83. 3%, as high as the national average. Although erythromycin-resistant isolates generally tend to show cross-resistance to penicillin, the frequency of PRSP was very low in this study, as compared with other Japanese studies. Erythromycin resistance was frequently recognized not only in PRSP but also in penicillin-sensitive S. pneumoniae (PSSP) as well. In Japan, erythromycin resistance may have already become widespread, even in local areas where penicillin resistance is not especially prevalent.

Purpose: To review 13 cases of pyriform sinus fistula (PSF) and to summarize the clinical and histologic features. Materials and methods: A retrospective review and immunohistochemical analysis of 13 cases of PSF treated in Sapporo Medical University Hospital from 1985 to 2005. This study includes 13 patients with PSF diagnosed by the existence of fistulous tract radiologically and/or pathologically. Fistulectomy was performed in all cases. Surgically obtained specimens were processed for immunohistochemistry with commercial anticalcitonin antibody. Results: The patients consisted of 4 males and 9 females, and the ages ranged from 4 to 55 years. The fistula was detected by barium study in 12 cases (92.3 %), and all cases were on the left side. Pathologic detection of the fistula was achieved in only 8 cases (69.2 %). Immunohistochemistry showed abnormal distribution of calcitonin-positive cells in 12 cases (92.3 %). No recurrence has occurred in any of the patients. Conclusions: Surgical removal of the inflammatory tissue is worthy of consideration in suspected PSF cases as in patients who exhibit similar manifestations without preoperative detection of the tract.

Gap junctions are considered to play a crucial role in differentiation of epithelial cells and to be associated with tight junction proteins. In this study, to investigate the role of gap junctions in regulation of the barrier function and fence function on the tight junctions, we introduced the Cx26 gene into human airway epithelial cell line Clau-3 and used a disruption model of tight junctions employing the Na(+)/K(+)-ATPase inhibitor ouabain. In parental Calu-3 cells, gap junction proteins Cx32 and Cx43, but not Cx26, and tight junction proteins occludin, JAM-1, ZO-1, claudin-1, -2, -3, -4, -5, -6, -7, -8, -9, and -14 were detected by RT-PCR. The barrier function and fence function of tight junctions were well maintained, whereas the GJIC was low level. Treatment with ouabain caused disruption of the barrier function and fence function of tight junctions together with down-regulation of occludin, JAM-1, claudin-2, and -4 and up-regulation of ZO-1 and claudin-14. In Cx26 transfectants, Cx26 protein was detected by Western blotting and immunocytochemistry, and many gap junction plaques were observed with well-developed tight junction strands. Expression of claudin-14 was significantly increased in Cx26 transfectants compared to parental cells, and in some cells, Cx26 was co-localized with claudin-14. Interestingly, transfection with Cx26 prevented disruption of both functions of tight junctions by treatment with ouabain without changes in the tight junction proteins. Pretreatment with the GJIC blockers 18beta-glycyrrhetinic acid and oleamide did not affect the changes induced by Cx26 transfection. These results suggest that Cx26 expression, but not the mediated intercellular communication, may regulate tight junction barrier and fence functions in human airway epithelial cell line Calu-3.

The purpose of the current study was to examine the binding of pulmonary surfactant protein A (SP-A) to TLR4 and MD-2, which are critical signaling receptors for lipopolysaccharides (LPSs). The direct binding of SP-A to the recombinant soluble form of extracellular TLR4 domain (sTLR4) and MD-2 was detected using solid-phase binding, immunoprecipitation, and BIAcore. SP-A bound to sTLR4 and MD-2 in a Ca2+-dependent manner, and an anti-SP-A monoclonal antibody whose epitope lies in the region Thr184–Gly194 blocked the SP-A binding to sTLR4 and MD-2, indicating the involvement of the carbohydrate recognition domain (CRD) in the binding. SP-A avidly bound to the deglycosylated forms of sTLR4 and MD-2, suggesting a protein/protein interaction. In addition, SP-A attenuated cell surface binding of smooth LPS and smooth LPS-induced NF-κB activation in TLR4/MD-2-expressing cells. To know the role of oligomerization in the interaction of SP-A with TLR4 and MD-2, the collagenase-resistant fragment (CRF), which consisted of CRD plus neck domain of SP-A, was isolated. CRF assembled as a trimer, whereas SP-A assembled as a higher order oligomer. Although CRD was suggested to be involved in the binding, CRF exhibited approximately 600- and 155-fold higher KD for the binding to TLR4 and MD-2, respectively, when compared with SP-A. Consistently significantly higher molar concentrations of CRF were required to inhibit smooth LPS-induced NF-κB activation and tumor necrosis factor-α secretion. These results demonstrate for the first time the direct interaction between SP-A and TLR4/MD-2 and suggest the importance of supratrimeric oligomerization in the immunomodulatory function of SP-A.

Summary Objective Colonization of middle ear pathogens is facilitated in the nasopharynx of otitis-prone children, and is associated with the development of otitis media. Recently, a new species of bacterium, Alloiococcus otitidis , is considered as one of the major middle ear pathogens. However, as far as we know, no study has been reported concerning the prevalence of A. otitidis in the nasopharynx of otitis-prone children. And, no study has been conducted on the association of A. otitidis in the nasopharynx with otitis media. Methods The frequency of A. otitidis in 83 middle ear effusions (MEE) and 56 nasopharyngeal swab (NPS) specimens from 56 children with otitis media was investigated by culture and polymerase chain reaction (PCR). Results A. otitidis was detected in 24 (28.9%) of MEE and in 6 (10.7%) of NPS specimens. When the frequency was investigated in relation to proneness to otitis media, A. otitidis was detected in 16 (64%) of 25 MEE and in 5 (29.4%) of 17 NPS specimens from otitis-prone children, whereas it was detected in 8 (13.8%) of 58 MEE and in 1 (2.6%) of 39 NPS specimens from non-otitis-prone children. The frequency of A. otitidis in both NPS and MEE specimens was significantly higher in otitis-prone than in non-otitis-prone children. Conclusions Our results suggest that colonization of A. otitidis is facilitated in the nasopharynx of otitis-prone children. And, nasopharyngeal colonization of A. otitidis may be associated with the frequency of otitis media.

Evaluation of beta-lactam susceptibility and polymerase chain reaction (PCR)-based genotyping of penicillin-binding proteins (PBP) 1A, 2B, and 2X were performed for Streptococcus pneumoniae isolates from children with otolaryngological infectious disease in the Sapporo district, Hokkaido Prefecture, Japan. Of 174 S. pneumoniae isolates, 14 (8%) were penicillin-resistant S. pneumoniae (PRSP), 87 (50%) were penicillin-intermediately-resistant, and 73 (42%) were penicillin-sensitive. Seventy-six (44%) had alterations in all of the three genes examined (pbp1a, pbp2b, and pbp2x), 81 (47%) had alterations in one or two of the genes, and 17 (10%) had no alterations. Isolates with alterations in all three genes showed low susceptibility to penicillin, while, in contrast, isolates with no alteration showed relatively high susceptibility to penicillin. Similar relationships were observed for other beta-lactams. The prevalence of PRSP in our study ranged from 5% to 12.8% (average, 8%), and there was much variation in the prevalence of PBP gene alterations among the cities. The results suggest that local differences in patterns of PBP gene alterations can be observed even at the district level. PCR-based genotyping of PBP genes is rapid, convenient, and useful to investigate genetic susceptibility to beta-lactams. Further, not only nationwide or prefectural surveys but also local surveillance at the district level is important for determining antimicrobial susceptibility status in daily practice.

Background Platelet-activating factor (PAF) has been thought to be a potent mediator of allergic rhinitis because PAF was recovered from the nasal lavage fluid of patients with allergic rhinitis after allergen provocation. Furthermore, PAF receptor antagonist attenuates the antigen-induced increase in nasal airway resistance and nasal vascular permeability in sensitized guinea pigs. Objective To clarify the expression of PAF receptor in human nasal mucosa by investigating PAF receptor messenger RNA (mRNA) expression and its protein localization using polymerase chain reaction (PCR) and immunohistochemical analyses, respectively. Methods Human turbinates were obtained after turbinectomy from 6 patients with nasal obstruction refractory to medical therapy. Total RNA was isolated from human nasal mucosa, and PAF receptor mRNA was detected in these tissues by using reverse transcriptase-PCR analysis. To identify the cells expressing PAF receptor protein, double immunostaining was performed using anti-PAF receptor antibody and monoclonal antileukocyte antibodies. Results Reverse transcriptase-PCR analysis of total nasal RNA demonstrated the expression of PAF receptor mRNA. The immunohistochemical studies revealed that anti-PAF receptor antibody-labeled eosinophils, macrophages, neutrophils, mast cells, lymphocytes, vascular endothelial cells, epithelial cells, and submucosal glands in nasal mucosa. Conclusions These results may have important clinical implications for understanding the role of PAF receptor on upper airway diseases such as allergic and nonallergic rhinitis.

This study was undertaken to investigate the expression of glucocorticoid receptor (GR), estrogen receptor (ER), progesterone receptor (PR) and androgen receptor (AR) in human palatine tonsils by RT-PCR and immunohistochemistry. Human palatine tonsils were obtained from performing tonsillectomy in three patients with recurrent tonsillitis and from one patient with tonsillar hypertrophy. By RT-PCR analysis, GR, ERα, ERβ and mRNA were detected in all four cases. However, PR and AR mRNA were found in only two cases. Antibodies to GR showed the presence of GR within all cells of tonsillar tissues with the highest quantities of GR localized in extrafollicular area. The immunohistochemical analysis of sex steroid receptor to the tonsillar tissues revealed that ERα positive cells existed in both germinal centre and extrafollicular area, and that ERβ positive cells localized in lymphoepithelial symbiosis lesion. However, neither PR nor AR positive cells were detected in any of the samples tested. These results suggest that these steroid receptors may play an important role in upper airway diseases.

BAG-1 is a multifunctional chaperone modulator may contribute to p53-mediated cell cycle arrest. We attempted to investigate whether BAG-1 expression is correlated with prognosis of laryngeal carcinoma patients after radiotherapy. Immunohistochemical analyses revealed BAG-1 expression was present in all laryngeal carcinomas examined, and its expression pattern varied, i.e. cytoplasmic, nuclear and both these staining types. Patients whose tumors predominantly express nuclear BAG-1 have a significantly poor failure-free survival rate after radiotherapy. We thus propose that nuclear BAG-1 localization is a prediction of unfavorable outcome should radiation therapy be undertaken for laryngeal carcinoma patients.

Background Although the mechanisms controlling the resolution of inflammatory processes are still not clear, it is thought that a number of inflammatory cells, including neutrophils and eosinophils, and ingestion of these cells by macrophages may be involved in the apoptotic cell process in allergic or non-allergic nasal tissues. We postulated that apoptosis of inflammatory cells may occur in vivo in nasal mucosa, thus regulating allergic and non-allergic inflammation, and to test this hypothesis we examined apoptotic cells in the nasal tissue of surgical specimens. Methods Human turbinates were obtained after conchotomy performed on patients with nasal obstruction refractory to medication. Nasal tissues were fixed in formalin and embedded in paraffin. The paraffin-embedded tissues were stained for apoptotic cells by terminal deoxynucleotidyl transferase-mediated deoxyuridine 5′-triphosphate (dUTP) nick end-labeling (TUNEL). To identify cell types undergoing apoptosis, double staining was performed by combining TUNEL and immunohistochemistry. Results The majority of TUNEL-positive cells were identified as leukocytes. Most TUNEL-positive cells found in these tissues represented granulocytes. A higher proportion of TUNEL-positive cells was found to be macrophages and most TUNEL-positive macrophages had intact nuclei and contained phagocytosed TUNEL-positive material (assumed to be apoptotic cells or bodies) in the cytoplasm. Conclusions These experiments represent the demonstration of cell type-specific apoptosis in human nasal mucosa. The results may have an important clinical implication and also promote further investigation to control the apoptosis of these cells in health and disease.

A case of a 64-year-old male with low-grade chondrosarcoma of the left posterolateral lamina of the cricoid cartilage is reported, in which a total cricoidectomy and partial resection of the left thyroid cartilage were performed. The rest of the thyroid cartilage and the arytenoid mucosa were approximated with sutures to the first tracheal ring, and a silastic T-tube was placed through the tracheostoma as a stent. The postoperative course has been successful except for the existence of a tracheal stoma and slight hoarseness. There has been no evidence of laryngeal stenosis nor recurrence at about 9 years postoperatively.

Purpose: To examine the usefulness of MR imaging for predicting local control of nasopharyngeal carcinoma (NPC) and the value of MR imaging in the newly published fifth edition of the TNM classification. Methods and Materials: We studied 29 patients with NPC with MR imaging and CT before and after treatment. Staging was done according to the fourth and newly published fifth editions of the International Union Against Cancer (UICC) staging system. The radiotherapy protocol was designed to deliver 66 to 68 Gy to the primary tumor and clinically involved nodes. Results: MR proved better than CT at identifying obliteration of the pharyngobasilar fascia, invasion of the sinus of Morgagni, through which the cartilaginous portion of the eustachian tube and the levator veli palatini muscle pass, invasion of the skull base, and metastases to lymph nodes in the carotid and retropharyngeal spaces. All seven patients without invasion of the pharyngobasilar fascia had local control. The local control rates of patients with invasion of the skull base were not good (60 to 73%). There was no apparent relationship between tumor volume determined by T1-weighted MR images and local control when the tumor volume was more than 20 cc. The newly published N staging system appears to successfully identify the high-risk group for distant metastasis as N3. In our series, four of five patients with N3 disease developed distant metastases. Conclusion: Deep infiltration of the tumor is a more important prognostic factor in NPC than tumor volume. Since the newly published T staging system requires a search for tumor invasion into soft tissue such as parapharyngeal space and bony structures, MR imaging may be indispensable for the newly published NPC staging system.

BACKGROUND Three cases with intracranial facial neurinoma underwent tumor removal and facial nerve reconstruction with or without tympanoplasty. Surgical strategy for each case was tailored to: (1) the site of main tumor mass, (2) its extension along the facial nerve, and (3) involvement of the auditory organs. METHODS Surgeries adopted in the three cases were: transpetrosal approach with intracranial-intratemporal facial nerve anastomosis, middle fossa and transmastoid approach with intratemporal facial nerve anstomosis and tympanoplasty, and middle fossa and transmastoid approach with intracranial-intratemporal facial nerve anastomosis and tympanoplasty. The greater auricular nerve was used as the nerve graft for all three cases. RESULTS In the follow-up period of 8-13 months there was no tumor recurrence; facial function was scored 20/90 in modified May's scoring system in each case, but two are still in the process of functional recovery. One of the two cases who underwent tympanoplasty showed complete recovery of hearing within 1 month, and the other showed worsened hearing, which was not serviceable at 3 months postoperatively. CONCLUSION Systematic surgical approach for tumor removal, facial nerve reconstruction, and auditory reconstruction should be considered in cases with intracranial facial neurinoma due to its varied clinical features.

A case of melanin pigmented oncocytic metaplasia is reported. A 62-year-old man presented with a history of discomfort of the ear of a 2 weeks' duration. Nasoscopic examination revealed a black nodule of about 5mm at the left Eustachian opening, and several black spots were discovered around the bilateral torus tubarius. The nodule was biopsied to determine the histology. Microscopically, there were oncocytic cells with abundant pigmented granules showing glandular pattern. Such a pigmented variant of benign oncocytic lesion is very rare.

Defensins are cysteine-rich peptides with broad-spectrum antimicrobial activity by producing channels or micropores in the membrane, and expressed in plants, insects, birds, and mammals. They play an important role in innate immune systems because of their antimicrobial activity to fungi and enveloped viruses as well as bacteria. The human palatine tonsil is thought of as an important immune barrier. Both innate and adaptive immune systems cooperate in rejection of microorganisms. We hypothesized that defensins are expressed and play some role in the tonsil. However, the expression of human defensins in tonsils has hardly been studied. In the present study, the expression profile of six human alpha defensins (hαD) and three human beta defensins (hβD) was determined by reverse transcription-polymerase chain reaction (RT-PCR) assay in 20 tonsils, 2 jejunum mucosa and 1 peripheral blood sample. The transcripts of hαD-1 through 4, hβD-1, -2, and -3 were expressed in tonsils. This result suggests that these defensins play an important role in the innate defense mechanisms in tonsils.

The three major pathogens of otitis media are Streptococcus pneumoniae, Haemophillus influenzae and Moraxella catarrhalis. Nasopharyngeal colonization of these pathogens is associated with the development of otitis media. A new species of bacterium, Alloiococcus otitidis, is detected with high frequency in the middle ear effusions (MEE) by polymerase chain reaction (PCR), however, the association of A. otitidis in the nasopharynx with the development of otitis media is still unclear. To clarify this point, we investigated the frequency of A. otitidis in MEE and nasopharyngeal swabs (NPS) of otitis-prone children, with culture and PCR. The frequency of A. otitidis in MEE was higher than that of the three major pathogens. Although our data suggest that A. otitidis colonization of the nasopharynx is associated with otitis media, the frequency of A. otitidis was higher in MEE than in NPS. This suggests that A. otitidis colonization may involve other sites as well as the nasopharynx.

To assess the dynamic changes of the autonomic neural responses evoked by vestibular-visual conflict (VVC) in the squirrel monkey, we analyzed the changes of salivary amount and sodium concentration, and the coefficient of variance (CV) of R-R intervals (RRI) along the time course of VVC exposure given in pitch plane. The sodium concentration and the amount of saliva showed clear increases at the first and second 15 min stimulation periods. The CV of RRI was found to increase over the entire periods of stimulation, and there was a significant difference between the rest value and those in stimulated periods. The correlation between the CV of RRI, and amount of salivation was highly positive. These results indicate the existence of a common pattern of autonomic neural response during VVC, and suggest usefulness of these indexes for objectively monitoring the severity of motion sickness.

FEBRUARY 2015 AB60 Abstracts S A T U R D A Y 191 Serum Zinc and Secretory IgA Levels Are Important Factors in Children with Food Allergy Yosuke Baba, MD, PhD, Reina Yokota, MD, Hiromi Yagisawa, MD, Susumu Yamazaki, MD, Asuka Ishida, MD, Eisuke Inage, MD, PhD, Mari Mori, MD, PhD, Yoshikazu Ohtsuka, MD, PhD, Toshiaki Shimizu, MD, PhD; Department of Pediatrics and Adolescent Medicine, Juntendo University Faculty of Medicine, Tokyo, Japan, Department of Pediatrics, Juntendo University Shizuoka Hospital, Shizuoka, Japan. RATIONALE: Zinc is an essential nutrient and its deficiency causes malnutrition and results in defects in innate and acquired immune responses. Also, zinc is important for highly proliferating cells, especially in the immune system and influences both innate and acquired immune functions. However, the precise roles and molecular mechanisms of zinc’s function in allergic response have not been clarified. On the other hand, the IgA antibody is massively produced in the intestinal Peyer’s patches, and the secretory IgA (sIgA) plays an important role on mucosal immune responses. It is considered that sIgA regulates the cause of allergic reactions. We studied serum zinc levels and sIgA levels in children with food allergies and studied their relationship with allergy symptoms. METHODS: It is a retrospective study using medical records of infants (from 6 months to 6 years old) who had been admitted to our hospitals. We classified the groups according to the results of physical examinations with or without allergic symptom (eczema, wheezing, food allergy). In addition, we investigated thewhite blood cell counts (eosinophils and basophils) and the serum levels of specific IgE, total IgA, sIgA, TARC (thymus and activation-regulated chemokine), and zinc. RESULTS: Children who were low levels in sIgA and serum zinc have past histories of atopic dermatitis, and their serum levels of specific IgE was significantly higher (p50.013) but their serum IgA level was significantly lower (p50.038) compared with children who does not have allergic symptoms. CONCLUSIONS: Secretory IgA levels and zinc levels are also important to the onset of allergic reactions.