Comprehensive Proteomic and Metabolomic Signatures of Nontypeable Haemophilus influenzae-Induced Acute Otitis Media Reveal Bacterial Aerobic Respiration in an Immunosuppressed Environment.

A thorough understanding of the molecular details of the interactions between bacteria and host are critical to ultimately prevent disease. Recent technological advances allow simultaneous analysis of host and bacterial protein and metabolic profiles from a single small tissue sample to provide insight into pathogenesis. We used the chinchilla model of human otitis media to determine, for the first time, the most expansive delineation of global changes in protein and metabolite profiles during an experimentally induced disease. After 48 h of infection with nontypeable Haemophilus influenzae, middle ear tissue lysates were analyzed by high-resolution quantitative two-dimensional liquid chromatography-tandem mass spectrometry. Dynamic changes in 105 chinchilla proteins and 66 metabolites define the early proteomic and metabolomic signature of otitis media. Our studies indicate that establishment of disease coincides with actin morphogenesis, suppression of inflammatory mediators, and bacterial aerobic respiration. We validated the observed increase in the actin-remodeling complex, Arp2/3, and experimentally showed a role for Arp2/3 in nontypeable Haemophilus influenzae invasion. Direct inhibition of actin branch morphology altered bacterial invasion into host epithelial cells, and is supportive of our efforts to use the information gathered to modify outcomes of disease. The twenty-eight nontypeable Haemophilus influenzae proteins identified participate in carbohydrate and amino acid metabolism, redox homeostasis, and include cell wall-associated metabolic proteins. Quantitative characterization of the molecular signatures of infection will redefine our understanding of host response driven developmental changes during pathogenesis. These data represent the first comprehensive study of host protein and metabolite profiles in vivo in response to infection and show the feasibility of extensive characterization of host protein profiles during disease. Identification of novel protein targets and metabolic biomarkers will advance development of therapeutic and diagnostic options for treatment of disease.

Expanding the Health-care Pipeline through Innovation: The MCW model.

In 2016, the Association of American Medical Colleges projected a physician shortage in the United States of approximately 90,000; in the same year, the Wisconsin Hospital Association projected a shortage of 2,000 physicians in Wisconsin. The Medical College of Wisconsin has begun to address these shortages in three ways: 1) creation of immersive regional medical school campuses in Green Bay and Central Wisconsin, in partnership with rural serving health systems; 2) creation of rural-based psychiatry and family medicine residency programs in Green Bay and central Wisconsin; and 3) expansion of the scope of practice of pharmacists through creation of a new School of Pharmacy in collaboration with the Medical College of Wisconsin School of Medicine. This article will discuss those approaches, history and progress to date, principles used, and future plans to address the impending physician shortages.

Continuous monitoring of endotracheal tube position with near infrared light.

Significance: Endotracheal intubation is a common approach for airway management in critically ill patients. However, the position of the endotracheal tube (ETT) may be altered during the procedure due to head movements. Accidental displacement or dislodge of the ETT may reduce the airflow, leading to moderate to severe complications, and in some cases even fatality. Therefore, timely detection of changes in ETT position in the trachea is critical to ensure immediate and intermediate interventions to maintain the ETT in the proper position. Currently, there are no widely utilized tools for real-time monitoring of ETT positions. Aim: The goal of this study is to develop a cost-effective and easy-to-use near-infrared (NIR) device, named Opt-ETT, capable of continuously monitoring the ETT position in the trachea of a patient. Approach: A side-firing fiber is attached to the side of the ETT to illuminate the trachea tissue with NIR light, and a detector board containing five phototransistors is affixed to the chest skin to measure the intensity of diffusely transmitted light. Displacement of the ETT is estimated using second-order polynomial fitting to the ratios of the phototransistor readings. Monte Carlo simulations, ex vivo experiment on porcine tissue, and in vivo experiments using a swine model have been conducted to assess the feasibility of the device. Results: The design of the Opt-ETT device has been verified by the Monte Carlo simulations and ex vivo experiment. The estimation of displacement from in vivo experiments using the Opt-ETT exhibited a high degree of agreement with that measured by a reference sensor, with a discrepancy between -1.0 to +1.5 mm within a displacement range from -15 to +15 mm. Conclusions: The Opt-ETT device provides a potentially cost-effective solution for real-time and continuous monitoring of ETT position in patient during an intubation procedure.

17ß-estradiol Attenuates the Middle Ear Inflammatory Response to Nontypeable Haemophilus influenzae.

OBJECTIVES: 17ß-estradiol (E2) is a steroidal hormone with immunomodulatory functions that play a role in infectious and inflammatory diseases. E2 was recently identified as the leading upstream regulator of differentially expressed genes in a comparative RNA sequencing study of pediatric patients with otitis media (OM) versus OM-free counterparts and may therefore play a role in the inflammatory response to bacterial otopathogens during pediatric OM. This study examined the effect of E2 on bacterial-induced inflammatory cytokine expression in an in vitro pediatric OM model. METHODS: An immortalized middle ear (ME) epithelial cell line, ROM-SV40, was developed from a pediatric recurrent OM patient. The culture was exposed to E2 at physiological levels for 1-48 h prior to 6 h-stimulation with nontypeable Haemophilus influenzae (NTHi) whole cell lysate. TNFA, IL1B, IL6, and IL8 were assayed by qPCR and ELISA. RESULTS: E2 pretreatment (24 h) abrogated NTHi induction of IL6; a longer pretreatment (1-10 nM, 48 h) abrogated IL1B induction (p < 0.05). E2 pretreatment (5 nM, 48 h) abrogated NTHi-induced IL8 secretion (p = 0.017). CONCLUSION: E2 pretreatment partially rescued NTHi-induced cytokine production by ME epithelia. These data support a role for E2 in moderating the excessive inflammatory response to middle ear infection that contributes to OM pathophysiology. LEVELS OF EVIDENCE: NA Laryngoscope, 2024.

PANEL 3: Otitis media animal models, cell culture, tissue regeneration & pathophysiology.

OBJECTIVE: To review and summarize recently published key articles on the topics of animal models, cell culture studies, tissue biomedical engineering and regeneration, and new models in relation to otitis media (OM). DATA SOURCE: Electronic databases: PubMed, National Library of Medicine, Ovid Medline. REVIEW METHODS: Key topics were assigned to the panel participants for identification and detailed evaluation. The PubMed reviews were focused on the period from June 2019 to June 2023, in any of the objective subject(s) or keywords listed above, noting the relevant references relating to these advances with a global overview and noting areas of recommendation(s). The final manuscript was prepared with input from all panel members. CONCLUSIONS: In conclusion, ex vivo and in vivo OM research models have seen great advancements in the past 4 years. From the usage of novel genetic and molecular tools to the refinement of in vivo inducible and spontaneous mouse models, to the introduction of a wide array of reliable middle ear epithelium (MEE) cell culture systems, the next five years are likely to experience exponential growth in OM pathophysiology discoveries. Moreover, advances in these systems will predictably facilitate rapid means for novel molecular therapeutic studies.

In vivo biodistribution and ototoxicity assessment of cationic liposomal-ceftriaxone via noninvasive trans-tympanic delivery in chinchilla models: Implications for otitis media therapy.

OBJECTIVES: We report the in vivo biodistribution and ototoxicity of cationic liposomal-ceftriaxone (CFX) delivered via ear drop formulation in adult chinchilla. METHODS: CFX was encapsulated in liposomes with size of ∼100 nm and surface charge of +20 mV. 100 µl liposomes or free drug was applied twice daily in both external ear canals of adult chinchillas for either 3 or 10 days. Study groups included free ceftriaxone (CFX, Day 3: n = 4, Day 10: n = 8), liposomal ceftriaxone (CFX-Lipo, Day 3: n = 4, Day 10: n = 8), and a systemic control group (Day 3: n = 4, Day 10: n = 4). Ceftriaxone delivery to the middle ear and systemic circulation was quantified by HPLC assays. Liposome transport was visualized via confocal microscopy. Auditory brainstem response (ABR) tests and cochlear histology were used to assess ototoxicity. RESULTS: Liposomal ceftriaxone (CFX-Lipo) displayed a ∼658-fold increase in drug delivery efficiency in the middle ear relative to the free CFX (8.548 ± 0.4638% vs. 0.013 ± 0.0009%, %Injected dose, Mean ± SEM). CFX measured in blood serum (48.2 ± 7.78 ng/ml) following CFX-Lipo treatment in ear was 41-fold lower compared to systemic free-CFX treatment (1990.7 ± 617.34 ng/ml). ABR tests and histological analysis indicated no ototoxicity due to the treatment. CONCLUSION: Cationic liposomal encapsulation results in potent drug delivery across the tympanic membrane to the middle ear with minimal systemic exposure and no ototoxicity.

Chronic tympanic membrane perforation repair with a collagen-based scaffold: An in vivo model.

OBJECTIVE: The aim of this study was to assess the in vivo efficacy of a novel regenerative collagen-based scaffold developed by the Royal College of Surgeons in Ireland in a chronic tympanic membrane perforation (TMP) using a chinchilla model. METHODS: Bilateral TMPs were induced in 17 mixed gender chinchillas using tympanic membrane resection followed by a mixture of topical Mitomycin C and dexamethasone for 3 days. These were monitored with weekly otoscopy for 8 weeks. Animals were excluded if signs of infection developed in the follow up period (n = 8). At 8 weeks, intervention began and 18 TMPs were assigned to either treatment with the collagen-based scaffold (treated group) or spontaneous healing (control group). Animals were euthanized 6 weeks post-intervention. Otoscopic imaging and auditory brain response (ABR) were conducted at baseline, 8 weeks post-TMP induction and 6 weeks post-intervention. All TMPs were then evaluated at 6 weeks post-intervention and bullae underwent histologic evaluation. RESULTS: At 6 weeks post-intervention, otoscopic imaging demonstrated various degrees of healing in the treated ears. The treated group was noted to have an increased rate of healing when compared to the control group. Histologic evaluation demonstrated a variation in the degree of perforation healing within groups, with some animals in the treated group showing high levels of perforation healing. At 8 weeks after the TMP procedure, most of the animals had worsened hearing response. At 6-week post the collagen-based scaffold treatment, about 50 % (4/8) of the treated ears had improved in hearing response as compared to those of non-treated ears. CONCLUSION: Given the initial histologic evidence of partial healing in scaffold-treated ears, the post-intervention period should be extended to monitor the potential for complete healing. Given the overall positive findings related to healing with the scaffold-treated ears, this material warrants further investigation.

A novel otoferlin splice-site mutation in siblings with auditory neuropathy spectrum disorder.

We characterize a novel otoferlin mutation discovered in a sibling pair diagnosed with auditory neuropathy spectrum disorder and investigate auditory nerve function through their cochlear implants. Genetic sequencing revealed a homozygous mutation at the otoferlin splice donor site of exon 28 (IVS28 + 1G>T) in both siblings. Functional investigation showed that the intronic sequence between exons 28 and 29 was retained in the mutated minigenes that were expressed in 293T cells. Auditory nerve compound action potential recovery functions in the siblings demonstrated different rates of neural recovery, with sibling AN1 showing rapid recovery (1.14 ms) and AN2 showing average recovery (0.78 ms) compared to subjects with sensorineural hearing loss (average: adults 0.71 ms, children 0.85 ms). Differences in neural recovery were consistent with speech perception differences between the siblings. Genotype information may indicate site of lesion in hearing loss; however, additional, as yet, unknown factors may impact clinical outcomes and must be considered.

Advancing Salary Equity in Schools of Medicine in the United States.

Equity, in broad terms, is a critical issue and has been identified as an area that needs particular attention in academic medicine. Gender equity, as a subset of overall equity, has equally been shown to be lacking in academic medicine, and most medical schools and academic health systems are involved in substantive journeys to improve all dimensions of equity, diversity, and inclusion. This Invited Commentary calls for including gender-based salary equity as a foundational accomplishment for institutions seeking to achieve overall equity. In addition, the authors provide evidence and recommendations to guide institutions toward best practices in achieving salary equity. They propose 4 areas of consideration: (1) prioritization by leadership; (2) prioritization of resources to ensure success; (3) development of corrective action plans which are "automatic" and based on clear guidelines; and (4) transparency of methodology, data, and results.

Association of e-Cigarette Exposure with Pediatric Otitis Media Recurrence.

OBJECTIVE: Otitis media (OM) is a common inflammatory disease spectrum in children and a leading cause of pediatric physician visits, antibiotic prescriptions and surgery. Tobacco exposure is associated with increased risk of OM recurrence, chronicity and surgeries. Tobacco products have changed dramatically in recent years with the advent of electronic cigarettes (e-cigarettes). While users frequently perceive vape as less harmful than traditional cigarettes, burgeoning evidence supports its contribution to respiratory pathologies. The consequences of secondhand exposure, particularly among children, are understudied. The aim of this study was to examine the association of e-cigarette emissions (EE) with OM recurrence and surgeries in the US. METHODS: Questionnaire data regarding ear infections and tobacco exposure was gathered for all pediatric respondents of the National Health and Nutrition Examination Survey (NHANES) 2017 to 2018. Weighted analyzes and logistic regression models were used to assess associations. RESULTS: Data was available for 2022 participants (aged 6-17); all were included for analyzes. Tobacco exposure was observed in 42%; 9% were exposed to EE. EE contributed to risk of ≥3 ear infections (OR = 1.61, 95% CI 1.01-2.58, P = .047). After adjustment for significant covariates (race and asthma), the association fell below significance (P = .081). No other significant associations were observed between ear infections, or tympanostomy tube insertion and exposure variables (EE, gestational or other household exposure). CONCLUSIONS: Exposure to EE may confer greater risk of pediatric OM than previously identified factors such as household smoke, or gestational exposure. Further investigation of EE and its health implications in children is warranted. LEVEL OF EVIDENCE: IV.

Ensuring the future of otitis media research: Interest in and feasibility of a mentoring network program within the otitis media research community.

OBJECTIVES: The goal of this study was to assess the feasibility of the implementation of a mentoring network program based on expressed interest and the diverse composition of the otitis media research community in order to address attrition of the scientific workforce. METHODS: An online survey was sent to basic, translational, and clinical researchers with a known interest in otitis media. RESULTS: Of 509 eligible participants, 119 (23.4%) responded to the survey. Survey respondents had a diverse background by completed education, current job description, and membership in an underrepresented group in science. Most faculty respondents (76.4%) were willing to participate in the proposed program as mentors and faculty lecturers, or had early-career researchers or trainees in their research group who were willing to participate as mentees in the mentoring network. Scientific and non-scientific topics for inclusion in the training program were ranked, with immunology and inflammation, microbiology, science leadership and collaboration, mentoring, and grantsmanship as main foci of interest among respondents. CONCLUSION: Our survey results showed enthusiastic participation among responding otitis media researchers, indicating the feasibility of implementing a mentoring network program that will address workforce attrition, particularly among underrepresented groups in science.

In Vivo Optical Characterization of Middle Ear Effusions and Biofilms During Otitis Media.

Otitis media (OM), a common ear infection, is characterized by the presence of an accumulated middle ear effusion (MEE) in a normally air-filled middle ear cavity. While assessing the MEE plays a critical role in the overall management of OM, identifying and examining the MEE is challenging with the current diagnostic tools since the MEE is located behind the semi-opaque eardrum. The objective of this cross-sectional, observational study is to non-invasively visualize and characterize MEEs and bacterial biofilms in the middle ear. A portable, handheld, otoscope-integrated optical coherence tomography (OCT) system combined with novel analytical methods has been developed. In vivo middle ear OCT images were acquired from 53 pediatric subjects (average age of 3.9 years; all awake during OCT imaging) diagnosed with OM and undergoing a surgical procedure (ear tube surgery) to aspirate the MEE and aerate the middle ear. In vivo middle ear OCT acquired prior to the surgery was compared with OCT of the freshly extracted MEEs, clinical diagnosis, and post-operative evaluations. Among the subjects who were identified with the presence of MEEs, 89.6% showed the presence of the TM-adherent biofilm in in vivo OCT. This study provides an atlas of middle ear OCT images exhibiting a range of depth-resolved MEE features, which can only be visualized and assessed non-invasively through OCT. Quantitative metrics of OCT images acquired prior to the surgery were statistically correlated with surgical evaluations of MEEs. Measurements of MEE characteristics will provide new readily available information that can lead to improved diagnosis and management strategies for the highly prevalent OM in children.

Establishment of novel immortalized middle ear cell lines as models for otitis media.

Objective: Otitis media (OM) is among the most frequently diagnosed pediatric diseases in the US. Despite the significant public health burden of OM and the contribution research in culture models has made to understanding its pathobiology, a singular immortalized human middle ear epithelial (MEE) cell line exists (HMEEC-1, adult-derived). We previously developed MEE cultures from pediatric patients with non-inflamed MEE (PCI), recurrent OM (ROM), or OM with effusion (OME) and demonstrated differences in their baseline inflammatory cytokine expression and response to stimulation with an OM-relevant pathogen lysate and cytokines. Herein, we sought to immortalize these cultures and assess retention of their phenotypes. Methods: MEE cultures were immortalized via lentivirus encoding temperature-sensitive SV40 T antigen. Immortalized MEE lines and HMEEC-1 grown in monolayer were stimulated with non-typeable Haemophilus influenzae (NTHi) lysate. Gene expression (TNFA, IL1B, IL6, IL8, MUC5AC, and MUC5B) was assessed by qPCR. Results: Similar to parental cultures, baseline cytokine expressions were higher in pediatric OM lines than in HMEEC-1 and PCI, and HMEEC-1 cells were less responsive to stimulation than pediatric lines. Conclusion: Immortalized MEE lines retained the inflammatory expression and responsiveness of their tissues of origin and differences between non-OM versus OM and pediatric versus adult cultures, supporting their value as novel in vitro culture models for OM.

Association of Pepsin With Inflammatory Signaling and Effusion Viscosity in Pediatric Otitis Media.

OBJECTIVE: Otitis media (OM) is a common inflammatory disease spectrum. Cytokine signaling, neutrophil activity, and mucin hypersecretion during recurrent and chronic OM contribute to persistent, viscous middle ear (ME) effusions, hearing loss, and potential for developmental delay. Extraesophageal reflux (EER), specifically pepsin, triggers inflammatory signaling in respiratory mucosa and is associated with OM. The objective of this study was to investigate the association of pepsin with ME inflammatory signaling and the outcomes and examine causality in vitro. STUDY DESIGN: Cross-sectional study. METHODS: ME fluid (MEF) and preoperative audiometric data were collected from 30 pediatric subjects undergoing tympanostomy tube placement for recurrent OM or OM with effusion. MEF viscosity was characterized by the surgeon. Pepsin, inflammatory molecules, and mucin were assayed by enzyme-linked immunosorbent assay (ELISA). ME epithelial primary culture was exposed to 0.1 to 1 mg/ml pepsin at pH 5, 6, and 7 for 30 minutes, and cytokine expression was assayed via qPCR. RESULTS: Pepsin was observed in the MEF of 77% of patients (range 71-2,734 ng/ml). Pepsin correlated with effusion viscosity, interleukins -6 and -8, neutrophil elastase, and mucin 5B (P < .05). Pepsin-negative MEF was more frequently absent of interleukin 8 or mucin 5B (P < .05). Weak acid was generally insufficient to elicit cytokine expression in ME cells in vitro, however, pepsin induced IL6, IL8, and TNF at pH 7 (P < .05) and weak acid (pH 6) facilitated a response at lower pepsin concentration. CONCLUSIONS: Pepsin may contribute to inflammatory signaling, persistent viscous effusion, and poorer OM outcomes. LEVEL OF EVIDENCE: 4 Laryngoscope, 132:470-477, 2022.

Automated classification of otitis media with OCT: augmenting pediatric image datasets with gold-standard animal model data.

Otitis media (OM) is an extremely common disease that affects children worldwide. Optical coherence tomography (OCT) has emerged as a noninvasive diagnostic tool for OM, which can detect the presence and quantify the properties of middle ear fluid and biofilms. Here, the use of OCT data from the chinchilla, the gold-standard OM model for the human disease, is used to supplement a human image database to produce diagnostically relevant conclusions in a machine learning model. Statistical analysis shows the datatypes are compatible, with a blended-species model reaching ∼95% accuracy and F1 score, maintaining performance while additional human data is collected.