Middle Ear

Mice were used with Institutional Animal Care and Use Committee approvals. Muc5ac^−/− mice were generated previously^{16 (link)}. Muc5b^−/− and Muc5b^Tg mice were generated here. Muc5b protein was assessed immunohistochemically using rabbit polyclonal antisera. Ciliary beat, MCC, and transport were assessed as described previously. Lung function was measured using a head-out plethysmograph and a flexiVent (Scireq, Montreal, Quebec, Canada), and blood oxygen was assessed using a pulse oximeter. Otitis media was assessed by visual otoscopy and middle ear lavage (MEL). Pulmonary inflammation was assessed by histology and lung lavage. Lavaged leukocytes were identified by light microscopy and flow cytometry. Neutrophils, macrophages, MHC-II, and apoptotic cells, were detected using commercially available Ab’S and reagents. S. aureus was administered by 10 μl intranasal or 50 μl intratracheal inocula at 10⁷-10⁸ CFU/animal. Bacteria and bacterial DNA were isolated from MEL, lung homogenates, and lung lavage pellets. Isolated colonies were phylotyped by 16S rRNA and mecA sequencing. Kaplan-Meier (1f and 3h, l), regression (1e and 2f), one-sided t-test (1g-i, k, l; 2b-e, g; 3b, c, f, g, j, k, and 4c, d, f, g, i, j), and one-way ANOVA (3i and 4a, h, j, l) with appropriate corrections for multiple comparisons, unequal variances, and non-Gaussian distribution were carried out using GraphPad Prism v5.04 (GraphPad Software, Inc., La Jolla, CA). Full methods are found in Supplementary Information.

Patients were prospectively enrolled from the clinical practice of the senior author (v.k.a.). All subjects were outpatients who presented for otolaryngologic evaluation at a tertiary referral center between August 2010 and October 2010. All patients included in this study were at least 18 years old. Patients were diagnosed as having ETD if they had a retracted or poorly mobile tympanic membrane on pneumatic otoscopy, with a history of at least two of the following symptoms in one or both ears over the previous 1 month period: aural fullness or pressure, a sensation of clogged or muffled hearing, recurrent or persistent middle ear effusion (defined as an effusion present on examinations at least 1 month apart), or the inability to rapidly self-equilibrate middle ear pressure following changes in ambient atmospheric pressure. Abnormal impedance audiometry was used as a criterion standard to verify the diagnosis at the time of enrollment. Exclusion criteria included surgery of the head or neck within 3 months; a history of radiation therapy to the head and neck; sinonasal malignancy; evidence of acute upper respiratory infection, including sinusitis and acute otitis media; adenoid hypertrophy; nasal polyposis; cleft palate or history of cleft palate repair; craniofacial syndrome, including Down syndrome; cystic fibrosis; ciliary dysmotility syndrome; or other systemic immunodeficiency. A second group of patients who did not meet these inclusion criteria and who had presented with medical complaints not related to ETD were consecutively enrolled for use as a control group. Presenting complaints for these patients included voice disturbance, tonsil hypertrophy, and intraoral lesions. All of these patients had a normal examination of the tympanic membrane, middle ear, nasal cavity, and nasopharynx. Normal impedance audiometry was used as a criterion standard to verify the absence of ETD. Written informed consent was obtained from each subject, and approval for this study was obtained from the institutional review board of Weill Cornell Medical College.

Children in group 1, (absent/infrequent AOM group) were enrolled at 6 months of age and followed to 30 months of age; the children had no prior AOM at the time of enrollment. NP and oropharyngeal samples were obtained at 7 routine visits when the children were 6, 9, 12, 15, 18, 24, and 30 months of age. With the first and any subsequent episodes of AOM NP and oropharyngeal cultures and middle ear fluid (MEF) by tympanocentesis were obtained. A follow up visit occurred 3 weeks later after each AOM and NP and oropharyngeal samples were again collected. Two children met the definition of otitis prone (3 AOM episodes in 6 months, n=1; or 4 AOM episodes in 12 months, n=1. Data for their 3^rd / 4^th AOM episodes meeting the otitis prone definition was included in group 2 (see below).
A second cohort (Group 2) were children less than 36 months of age (otitis prone group) enrolled when they had a 3^rd AOM episode within 6 months of time or a 4th episode within 12 months of time. NP and oropharyngeal cultures and MEF by tympanocentesis were obtained at the time of AOM. NP and oropharyngeal samples were collected again at a 3-week follow up visit.
Demographic data collected included family history of AOM, daycare attendance, antibiotic exposure in the prior month, presence of upper respiratory infection, number of AOM episodes before enrollment, age of first AOM episode, and PCV-7 vaccine history. The study was approved by the University of Rochester and Rochester General Hospital IRB and written informed consent was obtained from parents before enrollment in the study.