Expression, Distribution, and Role of C-Type Lectin Receptors in the Human and Animal Middle Ear and Eustachian Tube: A Review.

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.

The expression of thymic stromal lymphopoietin in patients and animal models with eosinophilic otitis media.

OBJECTIVE: In the present study, we aimed to clarify the expression of thymic stromal lymphopoietin (TSLP), a key trigger of Th2-type allergic disease, in the middle ear mucosa of eosinophilic otitis media (EOM). METHODS: An immunohistological study of TSLP was conducted in patients with EOM and in animal models of EOM constructed by intraperitoneal and intratympanic injection of ovalbumin for 7 and 14 days. In addition, the messenger RNA (mRNA) expression of TSLP in the middle ear mucosa of the animal models was analyzed using real-time PCR, and was compared with that of the control animals. RESULTS: Immunoreactivities for TSLP were observed in the middle ear mucosa around the tympanic ostium of the eustachian tube of patients with EOM. In the animal model, strong immunoreactivity for TSLP was also observed in the eustachian tube epithelium, and mRNA expression of TSLP in the seven-day stimulated animals was significantly higher than that in the controls. CONCLUSION: The present study indicates that the presence of epithelium-derived TSLP in the eustachian tubes plays an important role in the onset of EOM.

A Review: Expression of Aquaporins in Otitis Media.

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.

Otitis media in sperm-associated antigen 6 (Spag6)-deficient mice.

Mammalian SPAG6 protein is localized to the axoneme central apparatus, and it is required for normal flagella and cilia motility. Recent studies demonstrated that the protein also regulates ciliogenesis and cilia polarity in the epithelial cells of brain ventricles and trachea. Motile cilia are also present in the epithelial cells of the middle ear and Eustachian tubes, where the ciliary system participates in the movement of serous fluid and mucus in the middle ear. Cilia defects are associated with otitis media (OM), presumably due to an inability to efficiently transport fluid, mucus and particles including microorganisms. We investigated the potential role of SPAG6 in the middle ear and Eustachian tubes by studying mice with a targeted mutation in the Spag6 gene. SPAG6 is expressed in the ciliated cells of middle ear epithelial cells. The orientation of the ciliary basal feet was random in the middle ear epithelial cells of Spag6-deficient mice, and there was an associated disrupted localization of the planar cell polarity (PCP) protein, FZD6. These features are associated with disordered cilia orientation, confirmed by scanning electron microscopy, which leads to uncoordinated cilia beating. The Spag6 mutant mice were also prone to develop OM. However, there were no significant differences in bacterial populations, epithelial goblet cell density, mucin expression and Eustachian tube angle between the mutant and wild-type mice, suggesting that OM was due to accumulation of fluid and mucus secondary to the ciliary dysfunction. Our studies demonstrate a role for Spag6 in the pathogenesis of OM in mice, possibly through its role in the regulation of cilia/basal body polarity through the PCP-dependent mechanisms in the middle ear and Eustachian tubes.

Otitis media in a new mouse model for CHARGE syndrome with a deletion in the Chd7 gene.

Otitis media is a middle ear disease common in children under three years old. Otitis media can occur in normal individuals with no other symptoms or syndromes, but it is often seen in individuals clinically diagnosed with genetic diseases such as CHARGE syndrome, a complex genetic disease caused by mutation in the Chd7 gene and characterized by multiple birth defects. Although otitis media is common in human CHARGE syndrome patients, it has not been reported in mouse models of CHARGE syndrome. In this study, we report a mouse model with a spontaneous deletion mutation in the Chd7 gene and with chronic otitis media of early onset age accompanied by hearing loss. These mice also exhibit morphological alteration in the Eustachian tubes, dysregulation of epithelial proliferation, and decreased density of middle ear cilia. Gene expression profiling revealed up-regulation of Muc5ac, Muc5b and Tgf-ß1 transcripts, the products of which are involved in mucin production and TGF pathway regulation. This is the first mouse model of CHARGE syndrome reported to show otitis media with effusion and it will be valuable for studying the etiology of otitis media and other symptoms in CHARGE syndrome.

Failure of fluorescence to reveal middle ear penetration of quinolone drops.

OBJECTIVE: To evaluate the utility of fluorescence to assess penetration of quinolone ear drops (EDs) through tympanostomy tubes (TTs), the middle ear, eustachian tube, and into the oropharynx. DESIGN: Before-and-after trial. SETTING: Academic, tertiary care hospital. PATIENTS: Young children undergoing TT placement for otitis media and adolescents or adults undergoing repair of tympanic membrane (TM) perforations were included. INTERVENTIONS: Fluorescence of ofloxacin otic solution and serial dilutions was assessed with a Wood's lamp in vitro. Passage of ototopically administered ofloxacin into the pharynx was assessed in patients at the time of TT placement or TM repair. The oropharynx was visualized for fluorescence with a UV light for up to 2 hours after otic instillation. MAIN OUTCOME MEASURE: Oropharyngeal fluorescence. RESULTS: Ofloxacin otic fluorescence was visible at up to a 1:4 dilution. Fluorescence was confirmed in vivo by placing 1 drop of ofloxacin into the posterior pharynx and visualizing it transorally. Fluorescence was not identified in any of 20 patients after TT placement and in any of 6 patients prior to tympanoplasty. Two patients undergoing tympanoplasty reported tasting the EDs. CONCLUSION: Fluorescence is not a satisfactory method of assessing quinolone ED penetration through TTs and TM perforations, the middle ear, and into the nasopharynx.

Bacterial adherence in otitis media: determination of N-acetylgalactosamine (GalNAc) residues in the submucosal glands and surface epithelium of the normal and diseased Eustachian tube.

BACKGROUND: Acute otitis media (AOM) is the most common childhood infection caused by bacteria. The pathogenesis of AOM implicates initial adherence of a pathogen to the nasopharyngeal epithelium, which is followed by bacterial colonization of the middle ear cavity through the Eustachian tube. N-acetylgalactosamine (GalNAc) is an important constituent of mucins and GalNAc containing sugar residues seem to be essential for initial adherence of respiratory bacteria to the surface of epithelial cells. OBJECTIVE: To explore the localization of GalNAc residues, we incubated Eustachian tube sections from Streptococcus pneumoniae infected and normal control rats with seven biotinylated, GalNAc recognizing lectins: Bauhinia purpurea lectin (BPA), Psophocarpus tetragonolobus lectin (PTA), Helix aspersa lectin (HAA), Helix pomatia lectin (HPA), Phaseolus lunatus lectin (PLA), Sophora japonica lectin (SJA) and Vicia Villosa isolectin B4 (VVA-B4). RESULTS: The mucin producing epithelium and submucosal glands of the normal Eustachian tube contained GalNAc residues, as evidenced by binding of several of the lectins. Lectin binding specificity and intensity changed following acute middle ear infection. BPA was the only lectin that exclusively stained the surface epithelium and the serous acini of the submucosal glands in the infected animals, whereas no binding was detected in the normal controls. HPA, HAA, PTA and VVA-B4 binding to surface epithelial cells increased after infection, indicating an active secretion of GalNAc containing glycans. Quantitative analysis of submucosal gland staining intensity showed significantly more GalNAc residues in the normal Eustachian tube, compared to infected animals. CONCLUSION: We conclude that the mucous producing elements of the normal rat Eustachian tube contain GalNAc residues essential for respiratory pathogen adherence. In addition, the GalNAc residue specificity and reacting intensity change in relation to acute infection, which may be important in relation to subsequent development of secretory otitis media or formation of a bacterial biofilm in the middle ear. The results show that GalNAc residues increased in both the submucosal serous glands and in the surface epithelium of the Eustachian tube after middle ear infection with S. pneumoniae.

Functional study of mucus secretion of the eustachian tube in Guinea pigs.

OBJECTIVES: Our aim was to verify neural regulation of submucous gland mucus secretions in the Eustachian tubes of guinea pigs. STUDY DESIGN: Prospective animal study. METHODS: Eustachian tubes harvested from 12 guinea pigs were used for this study. For real-time resolution of pure glandular secretion, we used a modified method of single-gland optical measurement. Secretory monitoring was undertaken after each preparation with phenylephrine, isoproterenol, forskolin, and substance P. To confirm the viability of each tissue, we examined glandular secretion after treatment with carbachol. Secretory effects of each agonist were evaluated by comparing with basal secretion using a Student's t test (p < 0.01). RESULTS: The Ca-elevating agonists carbachol and substance P showed greater effects on submucous gland secretions of the Eustachian tube than the cyclic adenosine monophosphate (cAMP)-elevating agonists forskolin and isoproterenol. However, phenylephrine, although it belongs to the Ca-elevating agonist group, did not show any significant secretory effect. CONCLUSION: The optical measurement method used in this study had the merit of real-time resolution of submucous glandular secretion. Submucous glandular secretion in the Eustachian tube was regulated by both Ca- and cAMP-elevating agonists, and Ca-elevating agonists seemed to be more potent than cAMP-elevating agonists except phenylephrine. Our results suggest that not only the autonomic nerve system but also the neuropeptides such as substance P are closely related to glandular secretion in the Eustachian tube, and beta-adrenergic receptors seem to be more related to submucous glandular secretion of the Eustachian tube in guinea pig than alpha-adrenergic receptors.

Localization of aquaporins, water channel proteins, in the mouse eustachian tube.

CONCLUSION: Immunolocalization of the subtypes of water channel proteins, aquaporins (AQPs), was detected in the mouse eustachian tube (ET). AQPs are located continuously from the serous glands and the capillary vessels to the luminal side in the ET epithelium and may play an important role in the transportation of water to the surface of the ET lumen through the epithelium. OBJECTIVES: Although the water supply to the surface of the ET lumen is considered to be essential for closing of the ET, the pathway of the water in the ET is not fully understood. Since AQPs, a group of water transport proteins, have been reported to regulate water homeostasis, we examined the location of AQPs in the mouse ET. MATERIALS AND METHODS: Nine subtypes of AQPs were examined in paraffin embedded ETs of adult mice using the avidin-biotin peroxidase complex method of immunohistochemistry. RESULTS: Four subtypes of AQPs were detected in the mouse ET. AQP-1 was detected in fibroblasts, endothelial cells of capillary vessels and cartilage cells. AQP-3 was distinctly detected in the basal membrane of epithelial cells. AQP-4 was detected in the basal membrane of epithelial cells. AQP-5 was expressed in the luminal side of the ET epithelial cells and also in the apical surface of the cells of the serous glands.

Differentiation of glycans in equine guttural pouches.

The aim of this study was to investigate the carbohydrate composition of mucosubstances in the equine guttural pouches using conventional histochemical tests in conjunction with glycolytic digestion to degrade different classes of glycosoaminoglycans. In the goblet cells, the mucopolysaccharides contained chondroitin sulfate B, heparin, heparan sulfate and sialic acid residues. The acinar cells also expressed these substances (except for heparin), whereas the ductal cells produced chondroitin sulfate B and sialic acid. Neutral sugars were also found in each cell type. The diversity of the glycocomponents found in the auditory tube suggests that they may have important functional roles. Indeed, the glycosoaminoglycans provide a hydrophilic environment that prevents dehydration and desiccation of the guttural membranes during air passage. Additionally, these glycomolecules may be involved in the pathogenesis of some bacterial disease in horses, such as equine strangles.

Expression pattern of aquaporin 1 in the middle ear of the guinea pig with secretory otitis media.

OBJECTIVE: To explore the pathological change of water homeostasis in the secretory otitis media (SOM) middle ear, we observed the expression and regulation of aquaporin 1 (AQP1) in the SOM middle ear cavity. METHODS: Reverse transcriptase polymerase chain reaction (RT-PCR), Western blotting and immunohistochemical staining were used to detect AQP1 in the bullae of SOM models and normal animals. The expression patterns of AQP1 in the SOM group were compared with those in the normal animal group. RESULTS: RT-PCR and immunoblot analyses revealed that mRNAs encoding AQP1 were expressed in the middle ear membrane of the guinea pigs of both groups; AQP1 was also detected as 28-kDa proteins in both groups. Immunohistochemical analysis showed that AQP1 was localized on capillary endothelial cells and fibroblasts in the lamina propria mucosae as well as on flat and cubical epithelial cells. Quantitative analysis of RT-PCR and Western blotting revealed that AQP1 expression was higher in the SOM group than in the control group. The cellular expression of AQP1 was somewhat altered in the SOM middle ear. CONCLUSIONS: Our study suggests that AQP1 in the middle ear cavity may play a vital role in the accumulation of the effusion. It might work on the vessel-caused hydrops in the middle ear cavity.

Histologic changes in eustachian tube mucosa of rats after exposure to gastric reflux.

OBJECTIVE: Reflux is thought to be a risk factor for middle ear disease, but the mechanism underlying this relationship is unclear. In this study, we evaluated the effects of reflux on the eustachian tube (ET) mucosa. MATERIALS AND METHODS: Twenty-two healthy 150 to 220 g Wistar rats with normal middle ears were used. The animals were divided into three groups according to exposure time: 1-, 3-, and 12-week exposures. Four rats were used as a control group. An experimental model of gastroesophageal reflux was induced under general anesthesia. After exposure, the animals were sacrificed, and cross sections of the ETs were prepared. The histologic changes in the ET mucosa were observed under a light microscope. RESULTS: The density of goblet cells, numbers of lymphocytes, polymorphonuclear leukocytes, and eosinophils, subepithelial edema, subepithelial vasodilatation, subepithelial gland formation, and intraepithelial gland formation were compared among the groups. The goblet cell density and numbers of lymphocytes and polymorphonuclear leukocytes were significantly higher in the three exposure groups compared with the control group. CONCLUSION: Nasopharyngeal exposure to experimental reflux alters the ET mucosa histopathology, which suggests that gastroesophageal reflux has a role in ET dysfunction.

Sonotubometry: a useful tool to measure intra-individual changes in eustachian tube ventilatory function.

OBJECTIVE: To determine whether intra-individual changes in eustachian tube (ET) function induced by local application of a histamine phosphate solution can be detected using an improved sonotubometer. DESIGN: The function of the ET was measured with a revised sonotubometer before and after histamine was applied to the nasopharyngeal ostium of the ET. SETTING: Tertiary referral hospital. PATIENTS: Twenty-five otologically healthy adults. INTERVENTIONS: A histamine phosphate solution with a concentration of 16 mg/mL was applied to the nasopharyngeal ostium of the ET using a pressure nebulizer. MAIN OUTCOME MEASURES: The number of openings during 10 acts of swallowing. This outcome value could range from 0 to 10. The number of ET openings before and after histamine application was compared. RESULTS: The mean number of ET openings dropped dramatically: from 8.4 before application of histamine to 2.7 after application. This difference was statistically significant; there was a mean difference of 5.6 (95% confidence interval, 4.4-6.9; P < .001). CONCLUSION: Sonotubometry is capable of detecting intra-individual changes in ET function and may therefore be a very useful tool in monitoring and/or clinical research of ET dysfunction or function.

Expression of water channel proteins (aquaporins) in the rat Eustachian tube and middle ear mucosa.

CONCLUSION: Diverse expression of the different subtypes of aquaporins in different parts of the Eustachian tube and middle ear suggests region-specific functions of the aquaporins in the normal physiology of the tubotympanum and also suggests that they may play roles in the pathophysiology of otitis media. OBJECTIVES: The epithelial cells of the middle ear and Eustachian tube must maintain adequate water balance for normal function of the mucociliary system. Since aquaporins (AQPs) are known to play critical roles in water homeostasis, we investigated their expression in the tubotympanum of the rat. METHODS: The expression of AQP subtypes 1, 2, 4, 5, and 7 were examined in the rat Eustachian tube and middle ear using RT-PCR, Western blotting, and immunohistochemistry. RESULTS: Transcripts for AQP 1, 4, and 5 were detected in the Eustachian tube and middle ear. Expression of these molecules at the protein level was confirmed by Western blot analysis. Immunohistochemical analysis demonstrated that AQP 4 was localized to the basolateral membranes of ciliated epithelial cells while AQP 5 was localized to the apical surface of serous gland cells, but not goblet cells, in the rat Eustachian tube. AQP 1 was found to be expressed by the subepithelial fibroblasts.

Mechanism and rate of middle ear fluid absorption.

Several mechanisms have been suggested to explain the clearance of fluids from the middle ear. These include a pumping action through the eustachian tube, mucociliary beating through the tube, outflow of water to the blood due to osmotic gradients and an active Na(+) transport driving water absorption. In order to assess these mechanisms, the middle ear cavity of paralyzed, ventilated (eustachian tube occluded) guinea pigs was filled with fluids varying in osmotic pressure (hypotonic, isotonic, hypertonic) to which a vertical tube was attached. The change in height of fluid in the tube was taken as a measure of changes in middle ear fluid volume. A greater fluid volume reduction was seen with the hypotonic (1/5 saline) solution. A small volume increase was observed with the hypertonic solution. These results provide evidence that in these experimental conditions, water absorption due to osmotic gradients can contribute to middle ear fluid clearance.

Expression of beta-defensins in the tubotympanum of experimental otitis media.

CONCLUSION: Since the expression levels of beta-defensins 2-4 were up-regulated in experimental otitis media, they may play a protective role in the pathogenesis of otitis media. OBJECTIVES: Defensins are antimicrobial peptides that play a major role in innate immunity. The goal of this study was to identify the expression of defensins in experimental otitis media of the mouse. MATERIALS AND METHODS: The expression of three alpha-defensins (cryptdins, cryptdin-related sequences 1-C (CRS1-C), and CRS4-C) and four beta-defensins (mBD1, mBD2, mBD3, and mBD4) was investigated in the tubotympanum of experimental otitis media in mice by a semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR), and the expression levels of three beta-defensins (mBD2, mBD3, mBD4) were evaluated by Western blotting. RESULTS: The alpha-defensins were not expressed in the tubotympanum. mBD1 was expressed constitutively in normal middle ear mucosa and Eustachian tube mucosa, but up-regulated expression of mBD2, mBD3, and mBD4 was observed with RT-PCR and Western blotting in the tubotympanums in experimental otitis media, while the normal tubotympanums did not express them.

Radiopaque contrast dye in nasopharynx reaches the middle ear during swallowing and/or yawning.

CONCLUSION: Contrast dye in the nasopharynx reaches the middle ear during swallowing and yawning in normal adults. This suggests that displacement of bacteria in nasopharyngeal secretion to the middle ear may occur frequently during sleep. OBJECTIVE: The middle ear is sterile under normal conditions. The purpose of this study was to examine by means of CT whether radiopaque contrast dye in the nasopharynx would reflux into the middle ear of normal adults during swallowing and/or yawning. MATERIAL AND METHODS: Six normal adult volunteers were studied. Contrast dye was kept at the orifices of the Eustachian tube during swallowing and/or yawning by placing volunteers in either a head-down or lateral decubitus position. Reflux was determined by the presence of contrast dye in the middle ear on CT scanning of the temporal bone. RESULTS: Two of the three volunteers in each group (four out of six in total) had contrast material detected in one or both middle ear cavities.

On morphometric measurement of oxygen diffusing capacity in middle ear gas exchange.

An accurate mathematical model of transmucosal gas exchange is prerequisite to understanding middle ear (ME) physiology. Current models require experimentally measured gas species time constants for all extant conditions as input parameters. However, studies on pulmonary gas exchange have shown that a morphometric model that incorporates more fundamental physiochemical and anatomic parameters accurately simulates transport from which the species time constants can be derived for all extant conditions. Here, we implemented a variant of that model for ME gas exchange that requires the measurement of diffusional length (tau) for the ME mucosa. That measure contributes to the mucosal diffusing capacity and reflects the resistance to gas flow between air space and capillary. Two methods for measuring tau have been proposed: linear distance between the air-mucosal boundary and capillary and the harmonic mean of all contributing pathway lengths. Oxygen diffusing capacity was calculated for different ME mucosal geometries by using the two tau measures, and the results were compared with those predicted by a detailed, two-dimensional finite element analysis. Predictive accuracy was improved by incorporating the harmonic tau measure, which captures important information regarding variations in capillary shape and distribution. However, compared with the oxygen diffusing capacity derived from the finite element analysis, both measures yielded nonlinear, positively biased estimates. The morphometric techniques underestimate diffusion length by failing to account for the curvilinear gas flow pathways predicted by the finite element model.

Identification and characterization of a mucosal antimicrobial peptide expressed by the chinchilla (Chinchilla lanigera) airway.

Cationic antimicrobial peptides (APs) are produced at mucosal surfaces and play a key role as a first line of defense against infection. To understand how APs might impact disease progression in otitis media (OM), our goal was to identify and characterize APs expressed by the epithelium lining the uppermost airway of the chinchilla, the established rodent host for the study of the bacterial-viral pathogenesis in OM. Using a molecular approach, we cloned a cDNA encoding a homolog of human beta-defensin 3, designated chinchilla beta-defensin-1 (cBD-1), and found by Northern analysis expression of the corresponding mRNA in nasopharyngeal and tongue mucosae as well as skin. By reverse transcription-PCR, cBD-1 mRNA was also detected in RNA isolated from trachea, lung, and Eustachian tube tissues. The predicted mature form of cBD-1, expressed as a recombinant peptide in Escherichia coli, demonstrated bactericidal activity against the three primary opportunistic pathogens of OM as well as Candida albicans. Continued study of this and other APs will allow us to determine their role in bacterial colonization of the upper airway as well as how viruses might contribute to the pathogenesis of OM by modulating AP expression.