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Background: Otitis media with effusion (OME) often leads to pediatric hearing loss and is influenced by innate and adaptive immune responses. Innate immunity serves as the non-specific first line of defense against OME., Methods: We induced OME in rats using ovalbumin. We administered IL-6 monoclonal antibodies intranasally to inhibit IL-6, and we injected an NF-κB inhibitor intraperitoneally to explore the role of IL-6 in innate immunity and its interaction with the NOD-like receptor signaling pathway. We analyzed RNA-sequencing data with Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathways to assess signaling pathways involved in OME. We also utilized Western blot, quantitative real-time PCR, and immunohistochemistry on middle ear samples and used microscopy to identify immune cells in ear wash fluids., Results: Our study suggests a pivotal role for IL-6 in the immune pathways of rats with OME via the regulation of CXCL1-mediated pathways. Increased levels of IL-6 and CXCL1 were observed in the middle ear tissues, and activation of the NLRP3 inflammasome in OME rats led to an immune response via NF-κB, thus promoting IL-6 and CXCL1 production, which was reduced by IL-6 antibody treatment., Conclusions: Our findings confirm that IL-6 and CXCL1 play significant roles in the innate immune response in OME in rodents, predominantly via the NOD-like receptor signaling pathway and NLRP3 inflammasome activation. This research sheds light on OME pathogenesis and its immune-related mechanisms., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

To evaluate the genetics of chronic nonsuppurative otitis media (OM). We performed a genome-wide association study of 429,599 individuals included in the FinnGen study using three different case definitions: combined chronic nonsuppurative OM (7034 cases) (included serous and mucous chronic OM), mucous chronic OM (5953 cases), and secretory chronic OM (1689 cases). Individuals without otitis media were used as controls (417,745 controls). We used immunohistochemistry (IHC) of the murine middle ear to evaluate the expression of annexin A13. Four loci were significantly associated (p < 1.7 × 10 -8 ) with nonsuppurative OM. Three out of the four association signals included missense variants in genes that may play a role in otitis media pathobiology. According to our subtype-specific analyses, one novel locus, located near ANXA13, was associated with secretory OM. Three loci (near TNFRSF13B, GAS2L2, and TBX1) were associated with mucous OM. Immunohistochemistry of murine middle ear samples revealed annexin A13 expression at the apical pole of the Eustachian tube epithelium as well as variable intensity of the secretory cells of the glandular structure in proximity to the Eustachian tube. We demonstrated that secretory and mucous OM have distinct and shared genetic associations. The association of GAS2L2 with ciliary epithelium function and the pathogenesis of dysfunctional mucosa in mucous OM is suggested. The abundant expression of annexin A13 in the Eustachian tube epithelium, along with its role in apical transport for the binding and transfer of phospholipids, indicates the role of annexin A13 and phospholipids in Eustachian tube dysfunction., (© 2024. The Author(s).)

The aim of this comprehensive review was to present the current knowledge on the role of microRNAs (miRNAs) in acute, recurrent, and chronic forms of otitis media. Special attention was focused on cholesteatoma of the middle ear. MicroRNAs modulate gene expression, which, in turn, influences the development and likelihood of the recurrence of acute and aggressive chronic middle ear inflammatory processes. Moreover, this study discusses the modulating role of a specific subgroup of noncoding RNA, circular RNA (circRNA). Recognizing the precise potential pathways and the mechanisms of their function may contribute to a better understanding of the molecular bases of middle ear diseases and identifying novel methods for treating this demanding pathology. Articles published between 2009 and 2022 were used in this analysis. In this review, we provide a complete overview of the latest progress in identifying the role and mechanisms of particular miRNAs and circRNAs in acute, recurrent and chronic forms of otitis media.

Middle ear cholesteatoma (MEC), is a destructive, and locally invasive lesion in the middle ear driven by inflammation with an annual incidence of 10 per 100,000. Surgical extraction/excision remains the only treatment strategy available and recurrence is high (up to 40%), therefore developing the first pharmaceutical treatments for MEC is desperately required. This review was targeted at connecting the dysregulated inflammatory network of MEC to pathogenesis and identification of pharmaceutical targets. We summarized the numerous basic research endeavors undertaken over the last 30+ years to identify the key targets in the dysregulated inflammatory pathways and judged the level of evidence for a given target if it was generated by in vitro, in vivo or clinical experiments. MEC pathogenesis was found to be connected to cytokines characteristic for Th1, Th17 and M1 cells. In addition, we found that the inflammation created damage associated molecular patterns (DAMPs), which further promoted inflammation. Similar positive feedback loops have already been described for other Th1/Th17 driven inflammatory diseases (arthritis, Crohn's disease or multiple sclerosis). A wide-ranging search for molecular targeted therapies (MTT) led to the discovery of over a hundred clinically approved drugs already applied in precision medicine. Based on exclusion criteria designed to enable fast translation as well as efficacy, we condensed the numerous MTTs down to 13 top drugs. The review should serve as groundwork for the primary goal, which is to provide potential pharmaceutical therapies to MEC patients for the first time in history. Video Abstract., (© 2022. The Author(s).)

This morphological and immunohistochemical study demonstrates that tumors currently known as "middle ear adenomas" are truly well-differentiated epithelial neuroendocrine tumors (NETs) composed of cells comparable to normal intestinal L cells, and therefore, these tumors resemble hindgut NETs. These tumors show consistent expression of glucagon, pancreatic polypeptide, PYY, and the transcription factor SATB2, as well as generic neuroendocrine markers and keratins. The same L cell markers are expressed by cells within the normal middle ear epithelium. These markers define a valuable immunohistochemical profile that can be used for differential diagnosis of middle ear neoplasms, particularly in distinguishing epithelial NETs from paragangliomas. The discovery of neuroendocrine cells expressing the same markers in non-neoplastic middle ear mucosa opens new areas of investigation into the physiology of the normal middle ear and the pathophysiology of middle ear disorders., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: The molecular mechanisms of acute otitis media (AOM) development, and the intercellular crosstalk within the multicellular ecosystem of AOM, are not clear., Methods: We established a model of AOM in rats (with normal rats as controls) and undertook single-cell RNA sequencing (scRNA-seq) for the middle-ear mucosa (MEM). Cell clustering and trajectory analyses were undertaken using Seurat and Monocle 2 packages in R software. Pathway analyses were done by gene set enrichment analysis (GSEA). Cell-cell interactions were inferred by CellChat. Cell scores were calculated to identify cells with dual-feature., Results: A total of 7023 cells from three samples of inflamed MEM and 5258 cells from three samples of healthy MEM underwent scRNA-seq, which identified 20 cell clusters belonging to eight major cell types. After exposure to lipopolysaccharide, the MEM underwent significant conversion of cell types characterized by rapid infiltration of macrophages and neutrophils. M2 macrophages seemed to play a key part in inflammatory intercellular crosstalk, which facilitated the maintenance and proliferation of macrophages, cell chemotaxis, and regulation of the proinflammatory activities of cytokines. Three rare cell clusters with phagocytosis-related dual-feature were also identified. They coexisted with professional phagocytes in the MEM, and displayed distinct immunoregulatory functions by maintaining a normal immune microenvironment or influencing inflammation progression., Conclusions: Macrophages might be the "master" initiators and regulators of the inflammatory response of the MEM to external stimuli. And their functions are fulfilled by a specific polarization status (M2) and sophisticated intercellular crosstalk via certain signaling pathways. Besides, the coexistence of professional phagocytes and non-professional phagocytes as well as their interplay in the MEM provides new clues for deciphering the underlying pathogenic mechanisms of AOM., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Rao, Zhong, Qiu, Cheng, Li, Zhang, Mao, Pang, Li, Song, Li, Dong, Zhang, Yu, Ren and Zhao.)

Objective: Streptococcus pneumoniae ( S.pn ) is a common respiratory pathogen and a frequent cause of acute otitis media (AOM) in children. However, little is known about the immunometabolism during AOM. This study was to assess the presence of glucose metabolic reprogramming during AOM and its underlying mechanism affecting inflammatory response and middle ear injury., Methods: The levels of glycolytic metabolism were evaluated by measuring the expression of glycolysis-related genes and the production of metabolites. HE stain, immunofluorescence, immunohistochemistry, enzyme-linked immunosorbent assay (ELISA) and Western blot were performed to measure the effect of glucose metabolic reprogramming on inflammatory response, pneumococcal clearance, hypoxia-inducible factor 1 alpha (HIF-1α) expression and cytokine secretion during AOM, respectively., Results: The analysis of microarray revealed an increase of the expression of glycolysis-related genes during S.pn -induced AOM, which was verified by real-time PCR. Increased glycolysis promoted the production of IL-1β and TNF-α and facilitated the clearance of S.pn by enhancing phagocytosis and killing capability of neutrophils, but also aggravated the middle ear injury. Furthermore, these pathogenic effects could be reversed after glycolytic inhibitor 2DG treatment. Additionally, HIF-1α was observed to involve in glycolytic metabolism during AOM., Conclusion: S.pn infection induced increased glycolysis conversion during AOM, which promoted inflammatory responses and bacterial clearance, but also aggravated tissue damage., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Fan, Ma, Ai, Ding, Li, Zhu, He, Zhang, Dong and He.)

Otitis media (OM) is the most common paediatric disease and leads to significant morbidity. Although understanding of underlying disease mechanisms is hampered by complex pathophysiology, it is clear that epithelial abnormalities underpin the disease. The mechanisms underpinning epithelial remodelling in OM remain unclear. We recently described a novel in vitro model of mouse middle ear epithelial cells (mMEECs) that undergoes mucociliary differentiation into the varied epithelial cell populations seen in the middle ear cavity. We now describe genome wide gene expression profiles of mMEECs as they undergo differentiation. We compared the gene expression profiles of original (uncultured) middle ear cells, confluent cultures of undifferentiated cells and cells that had been differentiated for 7 days at an air liquid interface (ALI). >5000 genes were differentially expressed among the three groups of cells. Approximately 4000 genes were differentially expressed between the original cells and day 0 of ALI culture. The original cell population was shown to contain a mix of cell types, including contaminating inflammatory cells that were lost on culture. Approximately 500 genes were upregulated during ALI induced differentiation. These included some secretory genes and some enzymes but most were associated with the process of ciliogenesis. The data suggest that the in vitro model of differentiated murine middle ear epithelium exhibits a transcriptional profile consistent with the mucociliary epithelium seen within the middle ear. Knowledge of the transcriptional landscape of this epithelium will provide a basis for understanding the phenotypic changes seen in murine models of OM., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2021. Published by The Company of Biologists Ltd.)

Novel environments induce a conflicting emotional approach-withdrawal state that triggers stress-related reactions. Social support through the presence of a highly familiar conspecific buffers the individual against such challenges. Although aversive events seem to be predominantly processed by the right hemisphere, this is still under debate and little is known about functional cerebral asymmetries in nonhuman primates during novelty stress, isolation and social buffering. Here we isolated adult marmoset monkeys in a new open-field arena or in their familiar home-cages to establish hemisphere activity and whether the pairmate's presence buffers the response. Monkeys socially isolated in either location had higher circulating cortisol levels than non-isolated marmosets, but different hemisphere activity patterns indicated by changes in baseline tympanic membrane temperatures (TMT). The bilateral increase in the monkeys that were isolated in the unfamiliar location may reflect an approach-withdrawal conflict. The left-sided increase in the home-cage isolation group was negatively related to cortisol release, this being potentially associated with a more proactive/approach-prone temperament. Interestingly, TMT and cortisol were unaltered when the pairmate was present. Thus, positive social interaction reduces the perceived intensity of the threat, alters hemisphere asymmetries and blocks the hormonal response to novelty stress, consistent with a buffering effect., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Otitis media (OM) is a common inflammatory disease of the middle ear cavity and mainly occurs in children. As a critical regulator of inflammation response, the nuclear factor kappa B (NF- κ B) pathway has been found to play an essential role in the pathogenesis of various human diseases. The aim of this study was to explore the potential mechanism under the inflammatory response of human middle ear epithelial cells (HMEECs). We established in vitro models of OM by treating HMEECs with lipopolysaccharide (LPS) or interleukin 17A (IL-17A). Enzyme-linked immunosorbent assay and western blot analysis were used to measure the inflammatory response of HMEECs under LPS or IL-17A stimulation. The results revealed that the concentrations of proinflammatory cytokines ( p < 0.001) and protein levels of mucin (MUC) (for MUC5AC, p = 0.002, p = 0.004; for MUC8, p = 0.004, p < 0.001) were significantly elevated by LPS or IL-17A stimulation in HMEECs. Moreover, we found that LPS or IL-17A treatment promoted the phosphorylation of I κ B α (for p-I κ B α , p = 0.018, p = 0.002; for I κ B α , p = 0.238, p = 0.057) and the translocation of p65 from cytoplasm to nucleus in HMEECs (for nucleus p65, p = 0.01; for cytoplasm p65, p < 0.001). In addition, RT-qPCR analysis revealed that long noncoding RNA (lncRNA) metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) was verified to be upregulated in LPS- or IL-17A-stimulated HMEECs ( p < 0.001). Western blot analysis and immunofluorescence staining assay revealed that that MALAT1 knockdown significantly suppressed the activation of the NF- κ B pathway by reducing phosphorylated I κ B α levels and inhibiting the nuclear translocation of p65 ( p < 0.001) in LPS- or IL-17A-stimulated HMEECs (for p-I κ B α , p < 0.001; for I κ B α , p = 0.242, p = 0.647). Silence of MALAT1 decreased the proinflammatory cytokine production and MUC protein levels ( p < 0.001). Furthermore, rescue assays revealed that the increase of proinflammatory cytokine production (for TNF- α , p = 0.002, p = 0.015; for IL-1 β , p < 0.001, p = 0.006; for IL-6, p = 0.002, p < 0.001) and MUC protein levels (for MUC5AC, p = 0.001, p < 0.001; for MUC8, p < 0.001, p = 0.001) induced by MALAT1 overexpression was neutralized by 4-N-[2-(4-phenoxyphenyl) ethyl] quinazoline-4, 6-diamine (QNZ) treatment in LPS- or IL-17A-stimulated HMEECs. In conclusion, MALAT1 promotes inflammatory response in LPS- or IL-17A- stimulated HMEECs via the NF- κ B signaling pathway, which may provide a potential novel insight for the treatment of OM., Competing Interests: The authors declare that they have no conflicts of interest., (Copyright © 2021 Xiangru Yang et al.)

Otitis media with effusion (OME) is the major cause of hearing impairment in children. miR-210 plays a critical role in inflammatory diseases, however, its role in OME is unknown. In this study, the miR-210 level in serum and middle ear effusion of is significantly down-regulated in serum, middle ear effusion from OME patients (100 cases) compared with healthy volunteers (50 cases). The expression of miR-210 is closely related to inflammatory factors and bone conduction disorder in patients with OME. In the in vitro study，the miR-210 level is significantly reduced in culture supernatant of lipopolysaccharide (LPS) treated human middle ear epithelial cells (HMEECs). miR-210 overexpression inhibited the LPS-induced in inflammatory cytokines production, cell viability reduction and cell apoptosis. Bioinformatics and dual-luciferase reporter assay showed that HIF-1a was a target gene of miR-210. The biological effects of miR-210 on cell viability, cell apoptosis and inflammation cytokines in LPS-induced HMEECs were reversed by HIF-1a overexpression. Furthermore, phosphorylation of NF-κB p65 was significantly decreased by miR-210 mediated HIF-1a in LPS-induced HMEECs. This study suggested that miR-210 may play a role in OME. Further studies are warranted to assess miR-210 as a potential target for the diagnosis and treatment of OME., Competing Interests: Declaration of competing interest The authors declare that they have no conflicts of interest to disclose., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Objective: The aim of this study was to investigate sclerostin (SOST) expression in a rat model of experimental tympanosclerosis (TS) and its possible role in the formation of TS., Materials and Methods: Thirty-four SD rats were randomly divided into 2 groups: experimental group (n = 17) and normal group (n = 17). The left tympanic cavities in the experimental group were inoculated with methicillin-resistant Staphylococcus aureus. The changes of tympanic membranes were examined and recorded under otoendoscope. Haematoxylin-eosin staining was adopted to detect the morphological changes in the tympanic membrane and middle ear mucosa. Immunohistochemistry and Western blot analysis were used to observe the expression of SOST, Wnt3a, β-catenin, and P-ERK1/2., Results: In the experimental group, sclerotic lesions were observed in 54.5% ears in the end of 6 weeks. Morphological changes such as mucosa incrassation, inflammatory cells infiltration, fibrous tissue proliferation, and interstitial tissue incrassation prominently appeared in the tympanic membrane and middle ear mucosa. SOST protein was mainly distributed in the cytoplasm of epithelial cells and gland cells, the expression of which increased significantly in the calcified experimental ears. In addition, expression levels of Wnt3a, β-catenin, and P-ERK1/2 increased significantly in the calcified group too., Conclusion: The upregulated expression level of SOST may be involved in the formation of TS, first, through the pro-phosphorylation of ERK1/2 in the inflammatory stage, and then through the enhancement of Wnt3a in the osteogenic stage., (© 2021 S. Karger AG, Basel.)

The mammalian middle ear comprises a chain of ossicles, the malleus, incus, and stapes that act as an impedance matching device during the transmission of sound from the tympanic membrane to the inner ear. These ossicles are derived from cranial neural crest cells that undergo endochondral ossification and subsequently differentiate into their final functional forms. Defects that occur during middle ear development can result in conductive hearing loss. In this review, we summarize studies describing the crucial roles played by signaling molecules such as sonic hedgehog, bone morphogenetic proteins, fibroblast growth factors, notch ligands, and chemokines during the differentiation of neural crest into the middle ear ossicles. In addition to these cell-extrinsic signals, we also discuss studies on the function of transcription factor genes such as Foxi3, Tbx1, Bapx1, Pou3f4, and Gsc in regulating the development and morphology of the middle ear ossicles., (© 2020 American Association of Anatomists.)

Electronic cigarettes (e-cigarettes) are the most widely used electronic nicotine delivery systems and are designed to imitate smoking and aid in smoking cessation. Although the number of e-cigarette users is increasing rapidly, especially among young adults and adolescents, the potential health impacts and biologic effects of e-cigarettes still need to be elucidated. Our previous study demonstrated the cytotoxic effects of electronic liquids (e-liquids) in a human middle ear epithelial cell (HMEEC-1) line, which were affected by the manufacturer and flavoring agents regardless of the presence of nicotine. In this study, we aimed to evaluate the gene expression profile and identify potential molecular modulator genes and pathways in HMEEC-1 exposed to two different e-liquids (tobacco- and menthol-flavored). HMEEC-1 was exposed to e-liquids, and RNA sequencing, functional analysis, and pathway analysis were conducted to identify the resultant transcriptomic changes. A total of 843 genes were differentially expressed following exposure to the tobacco-flavored e-liquid, among which 262 genes were upregulated and 581 were downregulated. Upon exposure to the menthol-flavored e-liquid, a total of 589 genes were differentially expressed, among which 228 genes were upregulated and 361 were downregulated. Among the signaling pathways associated with the differentially expressed genes mediated by tobacco-flavored e-liquid exposure, several key molecular genes were identified, including IL6 (interleukin 6), PTGS2 (prostaglandin-endoperoxide synthase 2), CXCL8 (C-X-C motif chemokine ligand 8), JUN (Jun proto-oncogene), FOS (Fos proto-oncogene), and TP53 (tumor protein 53). Under menthol-flavored e-liquid treatment, MMP9 (matrix metallopeptidase 9), PTGS2 (prostaglandin-endoperoxide synthase 2), MYC (MYC proto-oncogene, bHLH transcription factor), HMOX1 (heme oxygenase 1), NOS3 (nitric oxide synthase 3), and CAV1 (caveolin 1) were predicted as key genes. In addition, we identified related cellular processes, including inflammatory responses, oxidative stress and carcinogenesis, under exposure to tobacco- and menthol-flavored e-liquids. We identified differentially expressed genes and related cellular processes and gene signaling pathways after e-cigarette exposure in human middle ear cells. These findings may provide useful evidence for understanding the effect of e-cigarette exposure.

Otitis media (OM), a very common disease in young children, can result in hearing loss. In order to potentially replicate previously reported associations between OM and PLG, exome and Sanger sequencing, RNA-sequencing of saliva and middle ear samples, 16S rRNA sequencing, molecular modeling, and statistical analyses including transmission disequilibrium tests (TDT) were performed in a multi-ethnic cohort of 718 families and simplex cases with OM. We identified four rare PLG variants c.112A > G (p.Lys38Glu), c.782G > A (p.Arg261His), c.1481C > T (p.Ala494Val) and c.2045 T > A (p.Ile682Asn), and one common variant c.1414G > A (p.Asp472Asn). However TDT analyses for these PLG variants did not demonstrate association with OM in 314 families. Additionally PLG expression is very low or absent in normal or diseased middle ear in mouse and human, and salivary expression and microbial α-diversity were non-significant in c.1414G > A (p.Asp472Asn) carriers. Based on molecular modeling, the novel rare variants particularly c.782G > A (p.Arg261His) and c.2045 T > A (p.Ile682Asn) were predicted to affect protein structure. Exploration of other potential disease mechanisms will help elucidate how PLG contributes to OM susceptibility in humans. Our results underline the importance of following up findings from genome-wide association through replication studies, preferably using multi-omic datasets.

Objective: Eosinophilic otitis media (EOM) is an intractable disorder associated with bronchial asthma and chronic rhinosinusitis with nasal polyposis. Periostin is an extracellular matrix protein secreted by fibroblasts in response to interleukin (IL)-4 and/or IL-13 and is a known marker for eosinophilic disorders. We assessed serum periostin levels and expression of periostin in the middle ear mucosa according to three grade of EOM severity (grade1 to 3)., Methods: 68 patients of blood and serum samples were corrected by whom diagnose bilateral EOM in Jichi Medical University Saitama Medical Center from January 2015 to June 2017.Immunohistochemical evaluation was performed to 18 EOM middle ears mucosa samples, which cauterized in tree groups and compared to that of chronic otitis media (COM)., Results: Serum periostin levels was significantly higher in EOM patients than in COM patients (EOM, 125.0 ± 45.5 ng/mL; COM, 79.4 ± 38.3 ng/mL; P<0.0001). The expression of periostin immunopositivity in the EOM middle ear mucosa was significantly greater in severe cases (grade3 samples) than others (grade1 and grade2 samples) (P <0.001 and P = 0.011, respectively). Periostin was expressed at the lamina propria especially in severe EOM cases and the cases had little response to glucocorticoids treatment., Conclusion: This study showed that periostin in the middle ear mucosa was correlated with EOM severity, and EOM with highly expressed periostin had difficulty in glucocorticoids treatment., (Copyright © 2020. Published by Elsevier B.V.)

Objectives: To update the medical literature on recent large-scale studies employing bioinformatics data analysis tools in otitis media (OM) disease models with a principal focus on developments in the past 5 years., Data Sources: Pubmed indexed peer-reviewed articles., Review Methods: Comprehensive review of the literature using the following search terms: 'genomics, inflammasome, microRNA, proteomics, transcriptome, bioinformatics' with the term 'otitis media', and 'middle ear'. Included articles published in the English language from January 1, 2015-April 1, 2019., Implications for Practice: Large scale bioinformatics tools over the past five years lend credence to the paradigm of innate immune response playing a critical role in host defense against bacteria contributing to Otitis Media (OM) progression from acute to chronic. In total, genomic, miRNAomic, and proteomic analyses all point to the need for a tightly regulated innate immune and inflammatory response in the middle ear. Currently, there is an urgent need for developing novel therapeutic strategies to control immunopathology and tissue damage, improve hearing and enhance host defense for both acute and chronic OM based on full understanding of the basic molecular pathogenesis of OM., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Objective: The middle ear epithelium is derived from the neural crest and endoderm, which line distinct regions of the middle ear cavity. In this study, we investigated the localization of stem/progenitor cells in the middle ear mucosa of adult mice and the effects of keratinocyte growth factor (KGF) on the cell kinetics of stem/progenitor cells in vivo., Methods: In this study, after KGF-expression vector was transfected in the ear, two kinds of thymidine analogues, BrdU and EdU, were transferred at different time points. BrdU was detected by immunohistochemistry and EdU was detected by click chemistry. We also performed immunohistochemistry using anti-Keratin14 (K14) antibody (an undifferentiated epithelial cell marker), anti-p63 antibody (a stem/progenitor cell marker) and anti-acetylated α-tubulin antibody (a ciliated epithelial cell marker)., Results: A large number of EdU-positive cells were detected in the thickened mucosal epithelium of the pars flaccida and attic region at Day 1 after KGF transfection. Interestingly, in the mucosal epithelium overlying the promontory of the cochlea, many EdU-positive cells were detected. These cells were also positive for K14 and p63. The acetylated α-tubulin positive cells were reduced in the attic region at Day 1 after KGF transfection., Conclusion: These findings indicate that KGF over-expression may increase stem/progenitor cell proliferation in the mucosal epithelium not only within the attic which is typical in middle ear cholesteatoma, but also overlying the promontory of the cochlea., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Keratin (K) is the main component of the epithelial cell mesenchymal cytoskeleton, which protects the integrity of epithelial cells and maintains the function of normal epithelial cells. The expression of keratin affects epidermal proliferation and differentiation, and so as to be used as a marker for proliferation, differentiation and migration of keratinocytes. Middle ear cholesteatoma is one of the common ear diseases. In the middle ear cholesteatoma, keratinocytes over-proliferate and keratin debris accumulates. In this paper, we reviewed the recent studies on middle ear cholesteatoma and explained the possible mechanisms of keratin in the pathogenesis of middle ear cholesteatoma from the aspects of "proliferation" and " bone resorption ". At the same time, the existing problems as well as the prospect of the future research were discussed.

Bone remodeling of the auditory ossicles and the otic capsule is highly restricted and tightly controlled by the osteoprotegerin (OPG)/receptor activator of nuclear factor kappa-Β ligand (RANKL)/receptor activator of nuclear factor kappa-Β (RANK) system. In these bony structures, a pathological decrease in OPG expression stimulates osteoclast differentiation and excessive resorption followed by accrual of sclerotic bone, ultimately resulting in the development of otosclerosis, a leading cause of deafness in adults. Understanding the signaling pathways involved in maintaining OPG expression in the ear would shed light on the pathophysiology of otosclerosis and other ear bone-related diseases. We and others previously demonstrated that Ca 2+ signaling through the L-type Ca V 1.2 Ca 2+ channel positively regulates OPG expression and secretion in long bone osteoblasts and their precursor cells in vitro and in vivo. Whether Ca V 1.2 regulates OPG expression in ear bones has not been investigated. We drove expression of a gain-of-function Ca V 1.2 mutant channel (Ca V 1.2 TS ) using Col2a1-Cre, which we found to target osteochondral/osteoblast progenitors in the auditory ossicles and the otic capsule. Col2a1-Cre;Ca V 1.2 TS mice displayed osteopetrosis of these bones shown by μCT 3D reconstruction, histological analysis, and lack of bone sculpting, findings similar to phenotypes seen in mice with an osteoclast defect. Consistent with those observations, we found that Col2a1-Cre;Ca V 1.2 TS mutant mice showed reduced osteoclasts in the otic capsule, upregulated mRNA expression of Opg and Opg/Rankl ratio, and increased mRNA expression of osteoblast differentiation marker genes in the otic capsule, suggesting both an anti-catabolic and anabolic effect of Ca V 1.2 TS mutant channel contributed to the observed morphological changes of the ear bones. Further, we found that Col2a1-Cre;Ca V 1.2 TS mice experienced hearing loss and displayed defects of body balance in behavior tests, confirming that the Ca V 1.2-dependent Ca 2+ influx affects bone structure in the ear and consequent hearing and vestibular functions. Together, these data support our hypothesis that Ca 2+ influx through Ca V 1.2 TS promotes OPG expression from osteoblasts, thereby affecting bone modeling/remodeling in the auditory ossicles and the otic capsule. These data provide insight into potential pathological mechanisms underlying perturbed OPG expression and otosclerosis., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Objectives: The endoplasmic reticulum (ER) is an intracellular organelle involved in the synthesis and secretion of proteins. The ER stress response, which protects cells from cytotoxic proteins such as unfolded proteins, is related to several diseases including inflammation. In this study, we investigated the effect of ER stress on the pathophysiology of otitis media with effusion (OME)., Methods: Thirty-nine pediatric patients who were diagnosed with OME and underwent ventilation tube insertion were enrolled in this study. Exudate from the middle ear cavity was collected through ventilation insertion, and ER stress gene expression was analyzed via real-time polymerase chain reactions(PCR)., Results: There were no significant differences in ER stress-related mRNA expression between effusion culture-positive and culture-negative groups (p > 0.05). Expression of the C/EBP-homologous protein (CHOP) was higher in the otitis-prone group than in the non-otitis-prone group (p < 0.05). The most common type of fluid was mucoid, and inositol-requiring enzyme 1α expression was higher in serous fluid than in mucoid, mucopurulent, or purulent fluid (p < 0.05)., Conclusions: Endoplasmic reticulum stress-related responses are activated in pediatric OME patients, and specific ER-stress related pathways are related to both the characteristics of fluid and the frequency of OME. Thus, ER stress-related responses affect the pathophysiology of OME in pediatric OME patients., (Copyright © 2019. Published by Elsevier B.V.)

Patients with mutations in the ectodysplasin receptor signalling pathway genes - the X-linked ligand ectodysplasin-A ( EDA ), the receptor EDAR or the receptor adapter EDARADD - have hypohidrotic ectodermal dysplasia (HED). In addition to having impaired development of teeth, hair, eccrine sweat glands, and salivary and mammary glands, HED patients have ear, nose and throat disease. The mouse strains Tabby ( Eda Ta ) and downless ( Edar dl-J/dl-J ) have rhinitis and otitis media due to loss of submucosal glands in the upper airway. We report that prenatal correction of EDAR signalling in Eda Ta mice with the agonist anti-EDAR antibody rescues the auditory-tube submucosal glands and prevents otitis media, rhinitis and nasopharyngitis. The sparse- and wavy-haired ( swh ) rat strain carries a mutation in the Edaradd gene and has similar cutaneous HED phenotypes to mouse models. We report that auditory-tube submucosal glands are smaller in the homozygous mutant Edaradd swh/swh than those in unaffected heterozygous Edaradd swh/+ rats, and that this predisposes them to otitis media. Furthermore, the pathogenesis of otitis media in the rat HED model differs from that in mice, as otitis media is the primary pathology, and rhinitis is a later-onset phenotype. These findings in rodent HED models imply that hypomorphic as well as null mutations in EDAR signalling pathway genes may predispose to otitis media in humans. In addition, this work suggests that the recent successful prenatal treatment of X-linked HED (XLHED) in humans may also prevent ear, nose and throat disease, and provides diagnostic criteria that distinguish HED-associated otitis media from chronic otitis media with effusion, which is common in children., Competing Interests: Competing interestsP.S. is shareholder of Edimer Pharmaceuticals and consultant for the EspeRare Foundation. All other authors have no competing or financial interests to declare., (© 2019. Published by The Company of Biologists Ltd.)

Auditory bulla cavitation defects are a cause of otitis media, but the normal cellular pattern of bulla mesenchyme regression and its failure are not well understood. In mice, neural-crest-derived mesenchyme occupies the bulla from embryonic day 17.5 (E17.5) to postnatal day 11 (P11) and then regresses to form the adult air-filled bulla cavity. We report that bulla mesenchyme is bordered by a single layer of non-ciliated epithelium characterized by interdigitating cells with desmosome cell junctions and a basal lamina, and by Bpifa1 gene expression and laminin staining of the basal lamina. At P11-P12, the mesenchyme shrinks: mesenchyme-associated epithelium shortens, and mesenchymal cells and extracellular matrix collagen fibrils condense, culminating in the formation of cochlea promontory mucosa bordered by compact non-ciliated epithelial cells. FBXO11 is a candidate disease gene in human chronic otitis media with effusion and we report that a bulla cavitation defect initiates the pathogenesis of otitis media in the established mouse model Jeff ( Fbxo11 Jf/+ ). Persistent mesenchyme in Fbxo11 Jf/+ bullae has limited mesenchymal cell condensation, fibrosis and hyperplasia of the mesenchyme-associated epithelium. Subsequent modification forms fibrous adhesions that link the mucosa and the tympanic membrane, and this is accompanied by dystrophic mineralization and accumulation of serous effusion in the bulla cavity. Mouse models of bulla cavitation defects are important because their study in humans is limited to post-mortem samples. This work indicates new diagnostic criteria for this otitis media aetiology in humans, and the prospects of studying the molecular mechanisms of murine bulla cavitation in organ culture., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

Maternal and fetal sources of thyroid hormone are important for the development of many organ systems. Thyroid hormone deficiency causes variable intellectual disability and hearing impairment in mouse and man, but the basis for this variation is not clear. To explore this variation, we studied two thyroid hormone-deficient mouse mutants with mutations in pituitary-specific transcription factors, POU1F1 and PROP1, that render them unable to produce thyroid stimulating hormone. DW/J-Pou1f1 dw/dw mice have profound deafness and both neurosensory and conductive hearing impairment, while DF/B-Prop1 df/df mice have modest elevations in hearing thresholds consistent with developmental delay, eventually achieving normal hearing ability. The thyroid glands of Pou1f1 mutants are more severely affected than those of Prop1 df/df mice, and they produce less thyroglobulin during the neonatal period critical for establishing hearing. We previously crossed DW/J-Pou1f1 dw/+ and Cast/Ei mice and mapped a major locus on Chromosome 2 that protects against hypothyroidism-induced hearing impairment in Pou1f1 dw/dw mice: modifier of dw hearing (Mdwh). Here we refine the location of Mdwh by genotyping 196 animals with 876 informative SNPs, and we conduct novel mapping with a DW/J-Pou1f1 dw/+ and 129/P2 cross that reveals 129/P2 mice also have a protective Mdwh locus. Using DNA sequencing of DW/J and DF/B strains, we determined that the genes important for thyroid gland function within Mdwh vary in amino acid sequence between strains that are susceptible or resistant to hypothyroidism-induced hearing impairment. These results suggest that the variable effects of congenital hypothyroidism on the development of hearing ability are attributable to genetic variation in postnatal thyroid gland folliculogenesis and function.

Objectives: Unfolded proteins in the endoplasmic reticulum (ER) cause an ER stress response and can result in various pathologic conditions, including inflammation. Otitis media is the most common disease in otolaryngology and is associated with inflammation. The pathophysiology of chronic otitis media is not well understood; we therefore investigated the expression pattern of ER stress-related mRNAs in chronic otitis media., Methods: Specimens were obtained from 47 patients with chronic otitis media over a period of 2 years. Expression levels of 6 ER stress transcription factors were quantitatively assessed using real-time RT-PCR., Results: The mRNA expression of sXBP1 was significantly higher in the otorrhea present group than in the otorrhea absent group (p < 0.05). ATF6 expression was significantly higher in the ossicle destruction group than in the ossicle intact group (p < 0.05). mRNA expression of the 6 ER stress-related genes did not differ significantly between those patients with positive microbial cultures versus those with negative cultures (p > 0.05) or those with facial nerve dehiscence versus those without facial nerve dehiscence (p > 0.05)., Conclusions: sXBP1 appears to be involved in chronic otitis media-associated inflammation, including otorrhea. ATF6 is associated with the destruction of ossicles. Our results suggest that certain ER stress-related genes are expressed in chronic otitis media-associated inflammation., (© 2019 S. Karger AG, Basel.)

The skeletal structure of the mammalian middle ear, which is composed of three endochondral ossicles suspended within a membranous air-filled capsule, plays a critical role in conducting sound. Gene mutations that alter skeletal development in the middle ear result in auditory impairment. Mutations in fibroblast growth factor receptor 2 (FGFR2), an important regulator of endochondral and intramembranous bone formation, cause a spectrum of congenital skeletal disorders featuring conductive hearing loss. Although the middle ear malformations in multiple FGFR2 gain-of-function disorders are clinically characterized, those in the FGFR2 loss-of-function disorder lacrimo-auriculo-dento-digital (LADD) syndrome are relatively undescribed. To better understand conductive hearing loss in LADD, we examined the middle ear skeleton of mice with conditional loss of Fgfr2. We find that decreased auditory function in Fgfr2 mutant mice correlates with hypoplasia of the auditory bulla and ectopic bone growth at sites of tendon/ligament attachment. We show that ectopic bone associated with the intra-articular ligaments of the incudomalleal joint is derived from Scx-expressing cells and preceded by decreased expression of the joint progenitor marker Gdf5. Together, these results identify a role for Fgfr2 in development of the middle ear skeletal tissues and suggest potential causes for conductive hearing loss in LADD syndrome., (© 2018 Wiley Periodicals, Inc.)

Otits media (OM) is the most frequent indication for antimicrobial prescription to US children. Streptococcus pneumoniae (S. pneumoniae) remains one of the most common pathogens causing OM. Successful eradication of S. pneumoniae in the middle ear can be achieved by adhering to a 7-10 day regimen of oral antibiotics. However, oral drug administration is challenging for parents. Lack of adherence has been associated with treatment failure or early relapse. To overcome this challenge, we used a noninvasive formulation to achieve high transtympanic antibiotic flux and cured S. pneumoniae OM in chinchillas. The formulation consists of a thermosensitive in situ gelling hydrogel, chemical permeation enhancers, and an antibiotic. The direct transport of drugs into the middle ear produced high concentrations of ciprofloxacin (in the range of hundreds of micrograms per milliliter) within the first 24 hours of administration. Drug concentrations above the minimum inhibitory concentration (MIC) for S. pneumoniae were sustained throughout the 7-day treatment. S. pneumoniae OM in a chinchilla model was successfully eradicated, without causing tissue toxicity. Transtympanic delivery minimized systemic drug exposure, as evidenced by undetectable levels in blood, measured by high-performance liquid chromatography.

Introduction: Myringotomy for tube insertion is the most common otologic surgery. Otorrhea is a frequent complication of this procedure and, to prevent it, most surgeons strongly recommend avoiding contact with water as this is thought to adversely impact on post-operative quality of life., Objective: To understand the benefit of this recommendation., Methods: Observational study - retrospective cohort study comparing the incidence of post-operative otorrhea and its impact on patients' quality of life, in two groups of patients comprising children under 10 years of age who underwent bilateral myringotomy and tube placement for chronic otitis media with effusion between May 2011 and May 2012. One group received water protection care after surgery, the other did not. Data was collected through telephonic interview, after one year of follow up (one year after the procedure). Water exposure without protection was considered the exposure event. Incidence of otorrhea and perceived impact on quality of life were the outcome measures. Results were compared after logistic regression., Results: We included 143 children: 116 were not exposed to water without protection and 27 were exposed. In the not exposed group 36.2% had at least one episode of otorrhea, compared to 40.0% of the exposed group. Odds ratio for otorrhea on exposed was 1.21 (95% CI 0.51-2.85, p=0.6). Negative impact on quality of life was reported by parents of 48.2% on the not exposed children, compared to 40.7% on the exposed group. This difference was not significant (p=0.5)., Conclusion: We found that recommending water protection did not have beneficial effect on the incidence of otorrhea after myringotomy with tubes on chronic otitis media with effusion. However, such measures did not appear to have a negative impact on quality of life. This is a populational observational study with few cases (143 cases); these final statements would be better stated by a very large populational study with another large control group., (Copyright © 2017 Associação Brasileira de Otorrinolaringologia e Cirurgia Cérvico-Facial. Published by Elsevier Editora Ltda. All rights reserved.)

Cholesteatoma is a potentially life-threatening middle ear lesion due to the formation of an inflamed ectopic mass of keratinizing squamous epithelium. Surgical removal remains the only treatment option, emphasizing the need to gain a better understanding of this severe disease. We show for the first time that stem cells residing in cholesteatoma tissue contribute to disease progression. Cells expressing the "stemness" markers Nestin and S100B were detected in middle ear cholesteatoma and auditory canal skin. Isolated Nestin + /S100B + -cells showed the capability for self-renewal, neurosphere formation and differentiation into mesodermal and ectodermal cell types. Compared to auditory canal skin stem cells middle ear cholesteatoma-derived stem cells displayed an enhanced susceptibility to inflammatory stimuli, and this suggested a possible contribution to the inflammatory environment in cholesteatoma tissue. Cholesteatoma derived stem cells were able to differentiate into keratinocyte-like cells using factors mimicking the microenvironment of cholesteatoma. Our findings demonstrate a new perspective on the pathogenesis of cholesteatoma and may lead to new treatment strategies for this severe middle ear lesion.

Otitis media (OM) is a group of inflammatory diseases of the middle ear (ME), regardless of cause or pathological mechanism. Among the molecular biological studies assessing the pathology of OM are investigations into the expression of C-type lectin receptors (CLR) in the ME and Eustachian tube (ET). To date, nine studies have evaluated CLR expression in the ME and ET. The expression of individual CLRs in mammalian ME and ET varies by species and model of OM. Assessments have shown that the patterns of CLR expression in the ME and ET vary; that CLR expression may vary by type of OM; and that the distribution and levels of expression of CLRs may depend on the presence or absence of inflammation, with variations even within the same species and same tissue. Infection of the ME and ET with various pathogens is a common cause of all types of OM, with host responses to pathogens mediated initially by the innate immune system. CLRs are important factors in the innate immune system because they act as both adhesion molecules and as pathogen recognition receptors. The expression of CLRs in OM tissues suggests that CLRs are associated with the pathogenesis of various types of OM., Competing Interests: The authors declare no conflict of interest.

Otitis Media (OM) is characterized by epithelial abnormalities and defects in innate immunity in the middle ear (ME). Although, BPIFA1, a member of the BPI fold containing family of putative innate defence proteins is abundantly expressed by the ME epithelium and SNPs in Bpifa1 have been associated with OM susceptibility, its role in the ME is not well characterized. We investigated the role of BPIFA1 in protection of the ME and the development of OM using murine models. Loss of Bpifa1 did not lead to OM development. However, deletion of Bpifa1 in Evi1 Jbo/+ mice, a model of chronic OM, caused significant exacerbation of OM severity, thickening of the ME mucosa and increased collagen deposition, without a significant increase in pro-inflammatory gene expression. Our data suggests that BPIFA1 is involved in maintaining homeostasis within the ME under steady state conditions and its loss in the presence of inflammation, exacerbates epithelial remodelling leading to more severe OM.

The middle ear epithelium is derived from neural crest and endoderm, which line distinct regions of the middle ear cavity. Here, we investigate the distribution of putative stem cell markers in the middle ear, combined with an analysis of the location of label-retaining cells (LRCs) to create a map of the middle ear mucosa. We show that proliferating cells and LRCs were associated with specific regions of the ear epithelium, concentrated in the hypotympanum at the base of the auditory bulla and around the ear drum. Sox2 was widely expressed in the endodermally derived ciliated pseudostratified epithelium of the hypotympanum. This part of the middle ear showed high levels of Wnt activity, as indicated by the expression of Axin2, a readout of Wnt signalling. Keratin 5 showed a more restricted expression within the basal cells of this region, with very little overlap between the Sox2- and keratin 5-positive epithelium, indicating that these genes mark distinct populations. Little expression of Sox2 or keratin 5 was observed in the neural crest-derived middle ear epithelium that lined the promontory, except in cases of otitis media when this epithelium underwent hyperplasia. This study lays the foundation for furthering our understanding of homeostasis and repair in the middle ear., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

Otitis media (OM) refers to inflammatory diseases of the middle ear (ME), regardless of cause or pathological mechanism. Among the molecular biological studies assessing the pathology of OM are investigations of the expression of aquaporins (AQPs) in the ME and Eustachian tube (ET). To date, fifteen studies have evaluated AQPs expression in the ME and ET. Although the expression of individual AQPs varies by species and model, eleven types of AQP, AQP1 to AQP11, were found to be expressed in mammalian ME and ET. The review showed that: (1) various types of AQPs are expressed in the ME and ET; (2) AQP expression may vary by species; and (3) the distribution and levels of expression of AQPs may depend on the presence or absence of inflammation, with variations even in the same species and same tissue. Fluid accumulation in the ME and ET is a common pathological mechanism for all types of OM, causing edema in the tissue and inducing inflammation, thereby possibly involving various AQPs. The expression patterns of several AQPs, especially AQP1, 4 and 5, were found to be altered in response to inflammatory stimuli, including lipopolysaccharide (LPS), suggesting that AQPs may have immunological functions in OM., Competing Interests: The authors declare no conflict of interest.

Objectives: This study aimed to investigate the expression of DKK1 protein in an experimental model of tympanosclerosis and its possible role in the pathogenesis of this disorder., Methods: Forty Sprague Dawley rats were included in the study: 20 in the control group (which received no treatment) and 20 in the experimental group (which received an incision to induce tympanosclerosis). Otomicroscopy was performed to observe the development of myringosclerosis. Haematoxylin and eosin staining was performed to observe the morphological changes. Western blot analysis and immunohistochemistry were performed to assess the expression of DKK1 protein., Results: At day 15, sclerotic lesions were observed in 70 per cent of the tympanic membranes. Inflammatory infiltration and hyaline degeneration markedly appeared in the tympanic membranes and middle-ear mucosa. DKK1 protein was mainly distributed in the cytoplasm of epithelial cells, which were widely distributed in the tympanic membranes and middle-ear mucosa. The expression of DKK1 protein was significantly decreased in the calcified experimental ears., Conclusion: DKK1 protein is involved in the pathogenesis of tympanosclerosis by regulating the Wnt/β-catenin signalling pathway.

Objectives: Calgranulins (calcium-binding proteins S100A8, S100A9 and S100A12) are predominant cytoplasmic proteins of neutrophils and produced by various cells, playing multiple functions in innate immunity and the inflammatory process. Although up-regulated expression of S100A8 and S100A9 genes were observed in an animal model of otitis media (OM), their expressions have not been studied in human middle ear epithelial cells in response to the OM pathogen or in patients with recurrent or chronic OM (recurrent OM/RecOM or chronic OM with effusion/COME)., Study Design: Gene expressions were compared between Streptococcus pneumoniae (SP)-infected and non-infected human middle ear epithelial cells (HMEECs) as well as between chronic OM patients and control patients (CI)., Methods: Gene expressions were profiled by quantitative real time PCR (qPCR). S100 proteins in OM patient and CI middle ear biopsies were detected by immunostaining., Results: S100A8, S100A9 and S100A12 gene expressions were elevated in SP-infected HMEECs in time-dependent manner. S100A8 and S100A9 but not S100A12 gene expression was significantly elevated in the middle ear mucosa of OM patients. S100A8 and S100A9 protein were observed in middle ear mucosa of OM, but not CI patients. Minimal co-localization was observed between S100A8 and S100A9 with neutrophil elastase and cytokeratin in ME sections of OM patients., Conclusion: Elevated S100A8 and S100A9 gene expression in SP-infected HMEECs and in the middle ear mucosa of OM, minor co-localized with neutrophil markers suggests that middle ear epithelial cell secretion of S100A8 and S100A9 may play a role in the pathogenesis of recurrent and chronic OM., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Otitis media (OM), inflammation of the middle ear (ME), is a common cause of conductive hearing impairment. Despite the importance of the disease, the aetiology of chronic and recurrent forms of middle ear inflammatory disease remains poorly understood. Studies of the human population suggest that there is a significant genetic component predisposing to the development of chronic OM, although the underlying genes are largely unknown. Using N-ethyl-N-nitrosourea mutagenesis we identified a recessive mouse mutant, edison, that spontaneously develops a conductive hearing loss due to chronic OM. The causal mutation was identified as a missense change, L972P, in the Nischarin (NISCH) gene. edison mice develop a serous or granulocytic effusion, increasingly macrophage and neutrophil rich with age, along with a thickened, inflamed mucoperiosteum. We also identified a second hypomorphic allele, V33A, with only modest increases in auditory thresholds and reduced incidence of OM. NISCH interacts with several proteins, including ITGA5 that is thought to have a role in modulating VEGF-induced angiogenesis and vascularization. We identified a significant genetic interaction between Nisch and Itga5; mice heterozygous for Itga5-null and homozygous for edison mutations display a significantly increased penetrance and severity of chronic OM. In order to understand the pathological mechanisms underlying the OM phenotype, we studied interacting partners to NISCH along with downstream signalling molecules in the middle ear epithelia of edison mouse. Our analysis implicates PAK1 and RAC1, and downstream signalling in LIMK1 and NF-κB pathways in the development of chronic OM.

Conclusions: The heparin-binding epidermal growth factor-like growth factor (HB-EGF) plays an essential role in the development and invasiveness of cholesteatoma. This study may help to realize the molecular mechanisms underlying the pathogenesis of cholesteatoma and make HB-EGF a promising target for drug intervention of cholesteatoma., Objective: To detect HB-EGF expression in human surgical specimens of acquired middle ear cholesteatoma and analyze its functional role as a regulator of epithelial keratinocytes hyperproliferation., Methods: A total of 34 patients who underwent surgical treatment for middle ear cholesteatoma were recruited in the study. The mRNA and protein expression of HB-EGF in middle ear cholesteatoma tissues and normal postauricular skin tissues was investigated by real-time quantitative reverse-transcription-polymerase chain reaction (RT-qPCR), immunohistochemical staining, and western blot. The correlation between bone resorption degree and HB-EGF expression was also analyzed., Results: On average, compared with normal postauricular skin, expression of HB-EGF mRNA in the cholesteatoma epithelium was significantly elevated 2.41-fold by RT-qPCR, and HB-EGF protein significantly upregulated 2.32-fold by western blot. Positive HB-EGF immunostaining observed in the basal and suprabasal layers of cholesteatoma epithelium was significantly stronger than in normal postauricular skin. Meanwhile, an obviously positive correlation between HB-EGF protein expression and bone resorption degree was discovered.

High-mobility group box 1 (HMGB1) is a nuclear non-histone protein, playing a critical role as a mediator between innate and acquired immunity; when released extracellularly, it coordinates the cellular stress response (under necrosis, bacterial lipopolysaccharide stimulation) and acts as an inflammatory marker and cytokine. The aim of the study was to demonstrate whether HMGB1 is over-expressed in chronic middle-ear pathologies and whether the entity of expression and the localization are correlated with the degree of the inflammatory reaction, thus suggesting that HMGB1 may play a crucial role in chronic inflammatory disorders of the middle ear, as already demonstrated in other airway diseases. We analyzed 30 samples of middle-ear mucosa in patients affected by chronic suppurative otitis media with ear drum perforation with/without cholesteatoma and otosclerosis as control. The distribution of HMGB1 was evaluated as nuclear, cytoplasmic, and/or extracellular staining. The inflammatory cells observed in the biopsies were mostly lymphocytes and plasmacells. A statistically significant difference in inflammation score between otosclerosis and chronic otitis samples ( P < 0.01; Anova test) and between otosclerosis and cholesteatoma samples ( P < 0.05; Anova test) was observed; the HMGB1 positivity was in accordance with the density of the inflammatory infiltrate. HMGB1 is over-expressed in chronic middle-ear pathologies and may play a role in the progression of the inflammatory process from recurrent acute otitis media to chronic suppurative otitis media.

Background: The most crucial clinical problem of Eosinophilic Otitis Media (EOM) is sensorineural hearing loss. A previous report revealed that repeated antigen stimulation of middle ear causes eosinophilic inflammation not only in the middle ear but also in the inner ear., Objective: The purpose of the present study was to elucidate the mechanism of eosinophil infiltration to the inner ear in the animal model of EOM., Methods: Continuous OVA stimulation to the middle ear of guinea pigs was performed for 7 days, 14 days, and 28 days. Histological observation was made for eosinophil infiltration and morphological change of the inner ear. Immunostaining for eotaxin and RANTES was performed to study immunoreactivity of those chemokines., Results: In the 7-day stimulation side, a few eosinophils were found in the scala tympani, without obvious morphological damage of the inner ear. Moreover, immunoreactivity of both eotaxin and RANTES was significantly higher in the OVA stimulation sides than control sides. For both eotaxin and RANTES, the number of immunopositive cells was significantly increased in the 14-day stimulation side over the 7-day stimulation side., Conclusions: Eotaxin and RANTES seem to play some important roles for the eosinophil infiltration in the middle and inner ear of model animal of EOM.

Changes over time in pathogens and their antibiotic sensitivity resulting from the recent overuse and misuse of antibiotics in otitis media (OM) have complicated treatment. This study evaluated changes over 5 years in principal pathogens and their antibiotic sensitivity in patients in Korea diagnosed with acute OM (AOM) and OM with effusion (OME). The study population consisted of 683 patients who visited the outpatient department of otorhinolaryngology in 7 tertiary hospitals in Korea between January 2010 and May 2015 and were diagnosed with acute AOM or OME. Aural discharge or middle ear fluid were collected from patients in the operating room or outpatient department and subjected to tests of bacterial identification and antibiotic sensitivity. The overall bacteria detection rate of AOM was 62.3% and OME was 40.9%. The most frequently isolated Gram-positive bacterial species was coagulase negative Staphylococcus aureus (CNS) followed by methicillin-susceptible S. aureus (MSSA), methicillin-resistant S. aureus (MRSA), and Streptococcus pneumonia (SP), whereas the most frequently isolated Gram-negative bacterium was Pseudomonas aeruginosa (PA). Regardless of OM subtype, ≥ 80% of CNS and MRSA strains were resistant to penicillin (PC) and tetracycline (TC); isolated MRSA strains showed low sensitivity to other antibiotics, with 100% resistant to PC, TC, cefoxitin (CFT), and erythromycin (EM); and isolated PA showed low sensitivity to quinolone antibiotics, including ciprofloxacin (CIP) and levofloxacin (LFX), and to aminoglycosides. Bacterial species and antibiotic sensitivity did not change significantly over 5 years. The rate of detection of MRSA was higher in OME than in previous studies. As bacterial predominance and antibiotic sensitivity could change over time, continuous and periodic surveillance is necessary in guiding appropriate antibacterial therapy., Competing Interests: The authors have no potential conflicts of interest to disclose., (© 2017 The Korean Academy of Medical Sciences.)

Objective: Determine if the middle ear transmucosal nitrous oxide (N 2 O) exchange rate is affected by nasal inflammation caused by topical application of histamine., Methods: In a randomized, double-blind, crossover study, 20 adults were challenged intranasally with histamine (5 mg) and placebo on separate occasions. At each session, the subjects were fitted with a non-rebreathing mask and breathed room air for 20 minutes, 50% N 2 O:50% O 2 for 20 minutes, and 100% O 2 for 10 minutes. Throughout, heart rate, blood pressure, and blood O 2 saturation were monitored, and bilateral middle ear pressure was recorded by tympanometry every minute. The primary outcome measure was the slope of the middle ear pressure-time function for the 50% N 2 O:50% O 2 breathing period, which is a measure of the transmucosal N 2 O exchange-constant. The effects of challenge substance, session, and period on the measured vital signs and of treatment, session, ear disease history, and test ear on the pressure-time slopes were evaluated using repeated measures ANOVAs., Results: The post-challenge total symptom score and the slope of the middle ear pressure-time function were greater after histamine when compared to placebo challenge. Of the signs, only heart rate was affected, responding to challenge substance and study period., Conclusion: The transmucosal N 2 O exchange rate for the middle ear is increased during inflammation caused by nasal histamine exposure.

Acute otitis media, inflammation of the middle ear bulla, is the most common bacterial infection in children. For one of the principal otopathogens, non-typeable Haemophilus influenzae (NTHi), animal models allow us to investigate host-microbial interactions relevant to the onset and progression of infection and to study treatment of middle ear disease. We have established a robust model of NTHi middle ear infection in the Junbo mouse. Intranasal inoculation with NTHi produces high rates of bulla infection and high bacterial titers in bulla fluids; bacteria can also spread down the respiratory tract to the mouse lung. An innate immune response is detected in the bulla of Junbo mice following NTHi infection, and bacteria are maintained in some ears at least up to day 56 post-inoculation. The Junbo/NTHi infection model facilitates studies on bacterial pathogenesis and antimicrobial intervention regimens and vaccines for better treatment and prevention of NTHi middle ear infection. © 2017 by John Wiley & Sons, Inc., (Copyright © 2017 John Wiley & Sons, Inc.)

The mammalian inner ear is a complex organ responsible for balance and hearing. Sonic hedgehog (Shh), a member of the Hedgehog (Hh) family of secreted proteins, has been shown to play important roles in several aspects of inner ear development, including dorsoventral axial specification, cochlear elongation, tonotopic patterning, and hair cell differentiation. Hh proteins initiate a downstream signaling cascade by binding to the Patched 1 (Ptch1) receptor. Recent studies have revealed that other types of co-receptors can also mediate Hh signaling, including growth arrest-specific 1 (Gas1), cell-adhesion molecules-related/down-regulated by oncogenes (Cdon), and biregional Cdon binding protein (Boc). However, little is known about the role of these Hh co-receptors in inner ear development. In this study, we examined the expression patterns of Gas1, Cdon, and Boc, as well as that of Ptch1, in the developing mouse inner ear from otocyst (embryonic day (E) 9.5) until birth and in the developing middle ear at E15.5. Ptch1, a readout of Hh signaling, was expressed in a graded pattern in response to Shh signaling throughout development. Expression patterns of Gas1, Cdon, and Boc differed from that of Ptch1, and each Hh co-receptor was expressed in specific cells and domains in the developing inner and middle ear. These unique and differential expression patterns of Hh co-receptors suggest their roles in mediating various time- and space-specific functions of Shh during ear development.

Introduction: Air pollutants are associated with inflammatory diseases such as otitis media (OM). Significantly higher incidence rates of OM are reported in regions with air pollution. Diesel exhaust particles (DEPs) comprise a major class of contaminants among numerous air pollutants, and they are characterized by a carbonic mixture of polycyclic aromatic hydrocarbons (PAHs), nitro-PAHs, and small amounts of sulfate, nitrate, metals and other trace elements. DEP exposure is a risk factor for inflammatory diseases. Our previous study identified potential biomarkers using gene expression microarray and pathway analyses in an in vitro system. Although in vitro investigations have been conducted to elucidate plausible biomarkers and molecular mechanisms related to DEP exposure, in vivo studies are necessary to identify the exact biological relevance regarding the incidence of OM caused by DEP exposure. In this study, we identified potential molecular biomarkers and pathways triggered by DEP exposure in a rodent model., Methods: Transcriptomic analysis was employed to identify novel potential biomarkers in the middle ear of DEP-exposed mice., Results: A total of 697 genes were differentially expressed in the DEP-exposed mice; 424 genes were upregulated and 273 downregulated. In addition, signaling pathways among the differentially expressed genes mediated by DEP exposure were predicted. Several key molecular biomarkers were identified including cholinergic receptor muscarinic 1 (CHRM1), erythropoietin (EPO), son of sevenless homolog 1 (SOS1), estrogen receptor 1 (ESR1), cluster of differentiation 4 (CD4) and interferon alpha-1 (IFNA1)., Conclusions: Our results shed light on the related cell processes and gene signaling pathways affected by DEP exposure. The identified biomarkers might be potential candidates for determining early diagnoses and effective treatment strategies for DEP-mediated disorders., Competing Interests: The authors have declared that no competing interests exist.

Objective: Acute otitis media (AOM) causes an inflammatory response in the middle ear. We assessed differences in innate immune responses involved in bacterial defense at onset of AOM in children who were stringently defined as otitis prone (sOP) and children not otitis prone (NOP)., Study Design: Innate immune genes analysis from middle ear fluid (MEF) samples of children., Methods: Genes of toll-like receptors (TLR), nod-like and retinoic acid-inducible gene-I-like receptors, downstream effectors important for inflammation and apoptosis, including cytokines and chemokines, were studied from MEF samples by using a real-time polymerase chain reaction array. Protein levels of differentially regulated genes were measured by Luminex., Results: Gene expression in MEF among children who were sOP was significantly different in upregulation of interleukin 8, secretory leukocyte peptidase inhibitor, and chemokine (C-C motif) ligand 3, and in downregulation of interferon regulatory factor 7 and its related signaling molecules interferon alpha, Toll-like receptor adaptor molecule 2, chemokine (C-C motif) ligand 5, and mitogen-activated protein kinase 8 compared with children who were NOP. Differences in innate gene regulation were similar when AOM was caused by Streptococcus pneumoniae or nontypeable Haemophilus influenzae., Conclusion: Innate-immune response genes are differentially regulated in children who were sOP compared with children with NOP., Competing Interests: The authors have no conflicts of interest to declare pertaining to this article

The fibrocartilaginous eustachian tube is part of a system of contiguous organs including the nose, palate, rhinopharynx, and middle ear cleft. The middle ear cleft consists of the tympanic cavity, which includes the bony eustachian tube (protympanum) and the mastoid gas cells system. The tympanic cavity and mastoid gas cells are interconnected and allow gaseous exchange and pressure regulation. The fibrocartilaginous eustachian tube is a complex organ consisting of a dynamic conduit with its mucosa, cartilage, surrounding soft tissue, peritubal muscles (ie, tensor and levator veli palatine, salpingopharyngeus and tensor tympani), and superior bony support (the sphenoid sulcus)., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Conclusion: Significant expression of macrophage migration inhibitory factor and its receptor (CD74) was observed in both the middle ear and inner ear in experimental otitis media in mice. Modulation of macrophage migration inhibitory factor and its signaling pathway might be useful in the management of inner ear inflammation due to otitis media., Objectives: Inner ear dysfunction secondary to otitis media has been reported. However, the specific mechanisms involved are not clearly understood. The aim of this study is to investigate the expression of macrophage migration inhibitory factor and CD74 in the middle ear and inner ear in lipopolysaccharide-induced otitis media., Method: BALB/c mice received a transtympanic injection of either lipopolysaccharide or phosphate-buffered saline (PBS). The mice were sacrificed 24 h after injection, and temporal bones were processed for polymerase chain reaction (PCR) analysis, histologic examination, and immunohistochemistry., Results: PCR examination revealed that the lipopolysaccharide-injected mice showed a significant up-regulation of macrophage migration inhibitory factor in both the middle ear and inner ear as compared with the PBS-injected control mice. The immunohistochemical study showed positive reactions for macrophage migration inhibitory factor and CD74 in infiltrating inflammatory cells, middle ear mucosa, and inner ear in the lipopolysaccharide-injected mice.

Objective: To ascertain the expression level of discoidin domain receptors 2(DDR2) and matrix metalloproteinase-13(MMP-13) in human middle ear cholesteatoma tissues and further investigate their roles in bone destruction and correlation. Method: Using immunohistochemical S-P method to detect the expression level of DDR2 ,MMP-13 in 30 specimens with middle ear cholesteatoma epithelial tissue and 10 specimens with normal ear epithelial tissues．At the same time,the computer image analysis system was used to detect the expression of the two indexes by the quantitative analysis,analyzing their expression in middle ear cholesteatoma epithelial tissue and the correlation between the extent of bone destruction． Result: The expression of DDR2 and MMP-13 were confirmed in human middle ear cholesteatoma epithelial tissues and normal ear epithelial tissues． The mean optical density of DDR2 and MMP13 in human middle ear cholesteatoma epithelial tissues which were tested by the computer image quantitative analysis system were higher than those in normal ear epithelial tissues( P <0．05).The expression of DDR2 and MMP-13 in middle ear cholesteatoma epithelial tissues were positively correlated( r =0.738, P <0.01).In addition,the two indexes were associated and relative to the extent of bone destruction,the wider the extent of bone destruction was,the higher the expression of both indexes( P <0．05)． Conclusion: DDR2 and MMP-13 may play important roles in bone destruction of human middle ear cholesteatoma．., Competing Interests: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose., (Copyright© by the Editorial Department of Journal of Clinical Otorhinolaryngology Head and Neck Surgery.)

Objective: To detect the expression of 3-phosphoinositide-dependent kinase-1(PDK1) and nuclear factor-κB(NF-κB) in human middle ear cholesteatoma tissue and to analyze their correlation. Method: Immunohistochemical method was taken to detect the expression and location of PDK1 and NF-κB in 60 cases of cholesteatoma tissue and 30 cases of normal ear skin specimens.SPSS 19.0 software was used to analyze the data. Result: Immunohistochemistry revealed PDK1 was positive in cytoplasm and the positive expression rate in cholesteatoma was significantly higher than normal skin ( P <0.05); NF-κB expressed in the nucleus of cholesteatoma and the positive expression rate in cholesteatoma significantly higher than normal skin( P <0.05);In cholesteatoma specimens,there was a significant positive correlation between protein PDK1 and NF-κB( P <0.05). Conclusion: Abnormal expression of PDK1 and NF-κB may be related to the proliferation of cholesteatoma epithelium and they reinforce each other., Competing Interests: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose., (Copyright© by the Editorial Department of Journal of Clinical Otorhinolaryngology Head and Neck Surgery.)