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.

Inhaled fosamprenavir for laryngopharyngeal reflux: Toxicology and fluid dynamics modeling.

Objectives: Approximately 25% of Americans suffer from laryngopharyngeal reflux (LPR), a disease for which no effective medical therapy exists. Pepsin is a predominant source of damage during LPR and a key therapeutic target. Fosamprenavir (FOS) inhibits pepsin and prevents damage in an LPR mouse model. Inhaled FOS protects at a lower dose than oral; however, the safety of inhaled FOS is unknown and there are no inhalers for laryngopharyngeal delivery. A pre-Good Lab Practice (GLP) study of inhaled FOS was performed to assess safety and computational fluid dynamics (CFD) modeling used to predict the optimal particle size for a laryngopharyngeal dry powder inhaler (DPI). Methods: Aerosolized FOS, amprenavir (APR), or air (control) were provided 5 days/week for 4 weeks (n = 6) in an LPR mouse model. Organs (nasal cavity, larynx, esophagus, trachea, lung, liver, heart, and kidney) were assessed by a pathologist and bronchoalveolar lavage cytokines and plasma cardiotoxicity markers were assessed by Luminex assay. CFD simulations were conducted in a model of a healthy 49-year-old female. Results: No significant increase was observed in histologic lesions, cytokines, or cardiotoxicity markers in FOS or APR groups relative to the control. CFD predicted that laryngopharyngeal deposition was maximized with aerodynamic diameters of 8.1-11.5 µm for inhalation rates of 30-60 L/min. Conclusions: A 4-week pre-GLP study supports the safety of inhaled FOS. A formal GLP assessment is underway to support a phase I clinical trial of an FOS DPI for LPR. Level of Evidence: NA.

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.

Amprenavir inhibits pepsin-mediated laryngeal epithelial disruption and E-cadherin cleavage in vitro.

Background: Laryngopharyngeal reflux (LPR) causes chronic cough, throat clearing, hoarseness, and dysphagia and can promote laryngeal carcinogenesis. More than 20% of the US population suffers from LPR and there is no effective medical therapy. Pepsin is a predominant source of damage during LPR which disrupts laryngeal barrier function potentially via E-cadherin cleavage proteolysis and downstream matrix metalloproteinase (MMP) dysregulation. Fosamprenavir (FDA-approved HIV therapeutic and prodrug of amprenavir) is a pepsin-inhibiting LPR therapeutic candidate shown to rescue damage in an LPR mouse model. This study aimed to examine amprenavir protection against laryngeal monolayer disruption and related E-cadherin proteolysis and MMP dysregulation in vitro. Methods: Laryngeal (TVC HPV) cells were exposed to buffered saline, pH 7.4 or pH 4 ± 1 mg/mL pepsin ± amprenavir (10-60 min). Analysis was performed by microscopy, Western blot, and real time polymerase chain reaction (qPCR). Results: Amprenavir (1 µM) rescued pepsin acid-mediated cell dissociation (p < .05). Pepsin acid caused E-cadherin cleavage indicative of regulated intramembrane proteolysis (RIP) and increased MMP-1,3,7,9,14 24-h postexposure (p < .05). Acid alone did not cause cell dissociation or E-cadherin cleavage. Amprenavir (10 µM) protected against E-cadherin cleavage and MMP-1,9,14 induction (p < .05). Conclusions: Amprenavir, at serum concentrations achievable provided the manufacturer's recommended dose of fosamprenavir for HIV, protects against pepsin-mediated cell dissociation, E-cadherin cleavage, and MMP dysregulation thought to contribute to barrier dysfunction and related symptoms during LPR. Fosamprenavir to amprenavir conversion by laryngeal epithelia, serum and saliva, and relative drug efficacies in an LPR mouse model are under investigation to inform development of inhaled formulations for LPR.

Topical Alginate Protection against Pepsin-Mediated Esophageal Damage: E-Cadherin Proteolysis and Matrix Metalloproteinase Induction.

Epithelial barrier dysfunction is a hallmark of gastroesophageal reflux disease (GERD) related to symptom origination, inflammatory remodeling and carcinogenesis. Alginate-based antireflux medications were previously shown to topically protect against peptic barrier disruption, yet the molecular mechanisms of injury and protection were unclear. Herein, Barrett's esophageal (BAR-T) cells were pretreated with buffered saline (HBSS; control), dilute alginate medications (Gaviscon Advance or Gaviscon Double Action, Reckitt Benckiser), a viscosity-matched placebo, or ADAM10 and matrix metalloproteinase (MMP) inhibitors before exposure to HBSS pH7.4 or pH4 ± 1 mg/mL pepsin for 10-60 min. Cell viability was assessed by ATP assay; mediators of epithelial integrity, E-cadherin, ADAM10, and MMPs were examined by Western blot and qPCR. Alginate rescued peptic reduction of cell viability (p < 0.0001). Pepsin-pH4 yielded E-cadherin fragments indicative of regulated intramembrane proteolysis (RIP) which was not rescued by inhibitors of known E-cadherin sheddases. Transcriptional targets of E-cadherin RIP fragments were elevated at 24 h (MMP-1,2,9,14; p < 0.01). Alginate rescued E-cadherin cleavage, ADAM10 maturation, and MMP induction (p < 0.01). Results support RIP as a novel mechanism of peptic injury during GERD. Alginate residue after wash-out to mimic physiologic esophageal clearance conferred lasting protection against pepsin-induced molecular mechanisms that may exacerbate GERD severity and promote carcinogenesis in the context of weakly acidic reflux.

The Protease Inhibitor Amprenavir Protects against Pepsin-Induced Esophageal Epithelial Barrier Disruption and Cancer-Associated Changes.

Gastroesophageal reflux disease (GERD) significantly impacts patient quality of life and is a major risk factor for the development of Barrett's esophagus (BE) and esophageal adenocarcinoma (EAC). Proton pump inhibitors (PPIs) are the standard-of-care for GERD and are among the most prescribed drugs in the world, but do not protect against nonacid components of reflux such as pepsin, or prevent reflux-associated carcinogenesis. We recently identified an HIV protease inhibitor amprenavir that inhibits pepsin and demonstrated the antireflux therapeutic potential of its prodrug fosamprenavir in a mouse model of laryngopharyngeal reflux. In this study, we assessed the capacity of amprenavir to protect against esophageal epithelial barrier disruption in vitro and related molecular events, E-cadherin cleavage, and matrix metalloproteinase induction, which are associated with GERD severity and esophageal cancer. Herein, weakly acidified pepsin (though not acid alone) caused cell dissociation accompanied by regulated intramembrane proteolysis of E-cadherin. Soluble E-cadherin responsive matrix metalloproteinases (MMPs) were transcriptionally upregulated 24 h post-treatment. Amprenavir, at serum concentrations achievable given the manufacturer-recommended dose of fosamprenavir, protected against pepsin-induced cell dissociation, E-cadherin cleavage, and MMP induction. These results support a potential therapeutic role for amprenavir in GERD recalcitrant to PPI therapy and for preventing GERD-associated neoplastic changes.

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.

Oral and Inhaled Fosamprenavir Reverses Pepsin-Induced Damage in a Laryngopharyngeal Reflux Mouse Model.

OBJECTIVE: More than 20% of the US population suffers from laryngopharyngeal reflux. Although dietary/lifestyle modifications and alginates provide benefit to some, there is no gold standard medical therapy. Increasing evidence suggests that pepsin is partly, if not wholly, responsible for damage and inflammation caused by laryngopharyngeal reflux. A treatment specifically targeting pepsin would be amenable to local, inhaled delivery, and could prove effective for endoscopic signs and symptoms associated with nonacid reflux. The aim herein was to identify small molecule inhibitors of pepsin and test their efficacy to prevent pepsin-mediated laryngeal damage in vivo. METHODS: Drug and pepsin binding and inhibition were screened by high-throughput assays and crystallography. A mouse model of laryngopharyngeal reflux (mechanical laryngeal injury once weekly for 2 weeks and pH 7 solvent/pepsin instillation 3 days/week for 4 weeks) was provided inhibitor by gavage or aerosol (fosamprenavir or darunavir; 5 days/week for 4 weeks; n = 3). Larynges were collected for histopathologic analysis. RESULTS: HIV protease inhibitors amprenavir, ritonavir, saquinavir, and darunavir bound and inhibited pepsin with IC50 in the low micromolar range. Gavage and aerosol fosamprenavir prevented pepsin-mediated laryngeal damage (i.e., reactive epithelia, increased intraepithelial inflammatory cells, and cell apoptosis). Darunavir gavage elicited mild reactivity and no discernable protection; aerosol protected against apoptosis. CONCLUSIONS: Fosamprenavir and darunavir, FDA-approved therapies for HIV/AIDS, bind and inhibit pepsin, abrogating pepsin-mediated laryngeal damage in a laryngopharyngeal reflux mouse model. These drugs target a foreign virus, making them ideal to repurpose. Reformulation for local inhaled delivery could further improve outcomes and limit side effects. LEVEL OF EVIDENCE: NA. Laryngoscope, 133:S1-S11, 2023.

Pepsinogen/Proton Pump Co-Expression in Barrett's Esophageal Cells Induces Cancer-Associated Changes.

EDUCATIONAL OBJECTIVE: At the conclusion of this presentation, participants should better understand the carcinogenic potential of pepsin and proton pump expression in Barrett's esophagus. OBJECTIVE: Barrett's esophagus (BE) is a well-known risk factor for esophageal adenocarcinoma (EAC). Gastric H+ /K+ ATPase proton pump and pepsin expression has been demonstrated in some cases of BE; however, the contribution of local pepsin and proton pump expression to carcinogenesis is unknown. In this study, RNA sequencing was used to examine global transcriptomic changes in a BE cell line ectopically expressing pepsinogen and/or gastric H+ /K+ ATPase proton pumps. STUDY DESIGN: In vitro translational. METHODS: BAR-T, a human BE cell line devoid of expression of pepsinogen or proton pumps, was transduced by lentivirus-encoding pepsinogen (PGA5) and/or gastric proton pump subunits (ATP4A, ATP4B). Changes relative to the parental line were assessed by RNA sequencing. RESULTS: Top canonical pathways associated with protein-coding genes differentially expressed in pepsinogen and/or proton pump expressing BAR-T cells included those involved in the tumor microenvironment and epithelial-mesenchymal transition. Top upstream regulators of coding transcripts included TGFB1 and ERBB2, which are associated with the pathogenesis and prognosis of BE and EAC. Top upstream regulators of noncoding transcripts included p300-CBP, I-BET-151, and CD93, which have previously described associations with EAC or carcinogenesis. The top associated disease of both coding and noncoding transcripts was cancer. CONCLUSIONS: These data support the carcinogenic potential of pepsin and proton pump expression in BE and reveal molecular pathways affected by their expression. Further study is warranted to investigate the role of these pathways in carcinogenesis associated with BE. LEVEL OF EVIDENCE: NA Laryngoscope, 133:59-69, 2023.

Alginates for Protection Against Pepsin-Acid Induced Aerodigestive Epithelial Barrier Disruption.

OBJECTIVE: Gastroesophageal reflux disease (GERD) and laryngopharyngeal reflux (LPR) are chronic conditions caused by backflow of gastric and duodenal contents into the esophagus and proximal aerodigestive tract, respectively. Mucosal barrier dysfunction resultant from the synergistic actions of chemical injury and the mucosal inflammatory response during reflux contributes to symptom perception. Alginates effectively treat symptoms of mild to moderate GERD and have recently shown benefit for LPR. In addition to forming a "raft" over gastric contents to reduce acidic reflux episodes, alginates have been found to bind the esophageal mucosa thereby preserving functional barrier integrity measured by transepithelial electrical resistance. The aim of this study was to further examine the topical protective capacity of alginate-based Gaviscon Advance (GA) and Double Action (GDA) against pepsin-acid mediated aerodigestive epithelial barrier dysfunction in vitro. STUDY DESIGN: Translational. METHODS: Immortalized human esophageal and vocal cord epithelial cells cultured in transwells were pretreated with liquid formula GA, GDA, matched viscous placebo solution, or saline (control), then treated for 1 h with saline, acid (pH 3-6) or pepsin (0.1-1 mg/ml) at pH 3-6. Endpoint measure was taken of horseradish peroxidase (HRP) allowed to diffuse across monolayers for 2 h. RESULTS: Pepsin (0.1-1 mg/ml) at pH 3-6 increased HRP flux through cultures pretreated with saline or placebo (p < 0.05); acid alone did not. GA and GDA prevented barrier dysfunction. CONCLUSIONS: GA and GDA preserved epithelial barrier function during pepsin-acid insult better than placebo suggesting that protection was due to alginate. These data support topical protection as a therapeutic approach to GERD and LPR. Laryngoscope, 132:2327-2334, 2022.

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.

RNA Sequencing and Pathways Analyses of Middle Ear Epithelia From Patients With Otitis Media.

OBJECTIVES: Otitis media (OM) is the most common pediatric diagnosis in the United States. However, our understanding of the molecular pathogenesis of OM remains relatively poor. Investigation of molecular pathways involved in OM may improve the understanding of this disease process and elucidate novel therapeutic targets. In this study, RNA sequencing (RNA-Seq) was used to discern cellular changes associated with OME compared to healthy middle ear epithelium (MEE). STUDY DESIGN: Ex vivo case-control translational. METHODS: Middle ear epithelia was collected from five pediatric patients diagnosed with OME undergoing tympanostomy tube placement and five otherwise healthy pediatric patients undergoing cochlear implantation. Specimens underwent RNA-Seq and pathways analyses. RESULTS: A total of 1,292 genes exhibited differential expression in MEE from OME patients compared to controls including genes involved in inflammation, immune response to bacterial OM pathogens, mucociliary clearance, regulation of proliferation and transformation, and auditory cell differentiation. Top networks identified in OME were organismal injury and abnormalities, cell morphology, and auditory disease. Top Ingenuity canonical pathways identified were axonal guidance signaling, which contains genes associated with auditory development and disease and nicotine degradation II and III pathways. Associated upstream regulators included ß-estradiol, dexamethasone, and G-protein-coupled estrogen receptor-1 (GPER1), which are associated with otoprotection or inflammation during insult. CONCLUSIONS: RNA-Seq demonstrates differential gene expression in MEE from patients with OME compared to healthy controls with important implications for infection susceptibility, hearing loss, and a role for tobacco exposure in the development and/or severity of OME in pediatric patients. LEVEL OF EVIDENCE: 4 Laryngoscope, 131:2590-2597, 2021.

RNA Sequencing Reveals Cancer-Associated Changes in Laryngeal Cells Exposed to Non-Acid Pepsin.

OBJECTIVE: Laryngopharyngeal reflux (LPR) is a common affliction that contributes to laryngeal inflammation, symptoms that impact quality of life, and life-threatening illnesses such as cancer. Effective treatment strategies for LPR are lacking. Pepsin is a proinflammatory and carcinogenic element of refluxate. Investigation of molecular pathways involved in pepsin-mediated damage may lead to identification of novel biomarkers and therapeutic targets for LPR. In this study, RNA sequencing was used to examine changes in human laryngeal epithelial cells following brief pepsin insult. Cells were immortalized to generate a model to aid future study of laryngeal injury and therapeutics. STUDY DESIGN: In vitro translational. METHODS: Laryngeal epithelial cells were cultured from a patient without signs or symptoms of LPR or laryngeal cancer. Cells were treated with 0.1 mg/ml pepsin for 1 hour or normal growth media (control) prior to RNA sequencing. Cells were immortalized via HPV E6/7 and characterized by microscopy, immunohistochemistry, G-banding, and soft agar assay. RESULTS: Three hundred ninety-seven genes exhibited differences in expression with pepsin treatment (P < .05). Pathway analysis revealed association with cancer and related signaling processes including dysregulation of cancer-associated molecules, Metastasis-Associated Lung Adenocarcinoma Transcript 1 and KRT82, and the long-noncoding RNA, lipoprotein receptor-related protein 1 (LRP1)-AS, which regulates the putative pepsin receptor LRP1. CONCLUSIONS: A single, brief exposure to pepsin activated cancer-associated signaling pathways in laryngeal cells in vitro, revealing novel mechanisms by which chronic reflux may contribute to carcinogenesis. The cell line developed herein represents a novel tool in which to investigate pepsin-dysregulated pathways identified by RNA sequencing and disparities of tumor proneness of laryngeal subsites. LEVEL OF EVIDENCE: N/A Laryngoscope, 131:121-129, 2021.

Analysis of Inflammatory Signaling in Human Middle Ear Cell Culture Models of Pediatric Otitis Media.

OBJECTIVES/HYPOTHESIS: Cell culture models are valuable tools for investigation of the molecular pathogenesis of diseases including otitis media (OM). Previous study indicates that age-, sex-, and race-associated differences in molecular signaling may impact disease pathophysiology. Currently, a singular immortalized middle ear epithelial (MEE) cell line exists, HMEEC-1, derived from an adult without known middle ear disease. In this study, HMEEC-1 and primary MEE cultures from pediatric patients with and without OM were stimulated with inflammatory cytokines or OM-pathogenic bacterial lysates to examine differences in the response of molecules associated with OM pathogenesis. STUDY DESIGN: Case-control series. METHODS: MEE cultures were established from patients aged <6 years: two with recurrent OM (ROM), two with OM with effusion (OME), and one patient without OM who was undergoing cochlear implant surgery control undergoing cochlear implantation (Peds CI). Primary MEE cultures and HMEEC-1 cells were stimulated with tumor necrosis factor-α, interleukin (IL)-1ß, or nontypeable Haemophilus influenzae lysate. TNFA, IL1B, IL6, IL8, IL10, and MUC5B were assayed via quantitative polymerase chain reaction. IL-8 was assayed by enzyme-linked immunosorbent assay. RESULTS: Gene/protein target expressions were frequently higher in pediatric OM lines than in HMEEC-1 and Peds CI. HMEEC-1 cells were frequently less responsive to stimuli than all pediatric lines. OME lines were often more responsive than ROM lines. CONCLUSIONS: OM may be associated with specific molecular phenotypes that are retained in primary cell culture. Adult-derived HMEEC-1 cells differ significantly in baseline expression and response of OM-associated molecules relative to pediatric MEE cells. Work is underway to immortalize pediatric OM MEE cultures as improved tools for the OM research community. LEVEL OF EVIDENCE: 4 Laryngoscope, 131:410-416, 2021.

H+/K+ATPase Expression in the Larynx of Laryngopharyngeal Reflux and Laryngeal Cancer Patients.

OBJECTIVES: The gastric H+/K+ ATPase proton pump has previously been shown to be expressed in the human larynx, however its contribution to laryngopharyngeal reflux (LPR) signs, symptoms and associated diseases such as laryngeal cancer is unknown. Proton pump expression in the larynx of patients with LPR and laryngeal cancer was investigated herein. A human hypopharyngeal cell line expressing the proton pump was generated to investigate its effects. STUDY DESIGN: In-vitro translational. METHODS: Laryngeal biopsies were obtained from three LPR and eight LSCC patients. ATP4A, ATP4B and HRPT1 were assayed via qPCR. Human hypopharyngeal FaDu cell lines stably expressing proton pump were created using lentiviral transduction and examined via transmission electron microscopy and qPCR for genes associated with inflammation or laryngeal cancer. RESULTS: Expression of ATP4A and ATP4B was detected in 3/3 LPR, 4/8 LSCC-tumor and 3/8 LSCC-adjacent specimens. Expression of ATP4A and ATP4B in FaDu elicited mitochondrial damage and expression of IL1B, PTGS2, and TNFA (P < .0001); expression of ATP4B alone did not. CONCLUSIONS: Gastric proton pump subunits are expressed in the larynx of LPR and LSCC patients. Mitochondrial damage and changes in gene expression observed in cells expressing the full proton pump, absent in those expressing a single subunit, suggest that acid secretion by functional proton pumps expressed in upper airway mucosa may elicit local cell and molecular changes associated with inflammation and cancer. LEVEL OF EVIDENCE: NA Laryngoscope, 131:130-135, 2021.

Pepsin in gastroesophageal and extraesophageal reflux: molecular pathophysiology and diagnostic utility.

PURPOSE OF REVIEW: Gastroesophageal and extraesophageal reflux are prevalent and costly diseases. Recognition of the pathogenicity of nonacid reflux has stimulated interest in alternatives to acid-targeting diagnostics and therapeutics. Pepsin is the most deleterious enzyme in refluxate, eliciting inflammatory and carcinogenic effects irrespective of acid. Its presence in all refluxate and detection in saliva have situated pepsin as the most widely researched biomarker for reflux today. This review summarizes emerging findings regarding pepsin-mediated damage during reflux and developments in pepsin-targeting diagnostics. RECENT FINDINGS: New evidence supports a role for pepsin in epithelial--mesenchymal transition, an important process in carcinogenesis and fibrosis. The first global transcriptomic analysis of pepsin-exposed laryngeal cells was described, yielding evidence of a putative airway pepsin receptor. Evaluation of pepsin diagnostics highlighted the need for rigorous validation in which pepsin concentrations are corroborated by a secondary quantitative assay, and reflux is confirmed or excluded by multichannel intraluminal impedance pH testing. Standards for sample collection and storage, and normative and pathological values are lacking. SUMMARY: Progress continues to be made in our understanding of pepsin-mediated damage with implications for novel therapeutic strategies. Salivary pepsin diagnostics continue to garner interest; however, further work appears necessary to improve their accuracy and reproducibility.

The role of pepsin in epithelia-mesenchymal transition in idiopathic subglottic stenosis.

OBJECTIVES: Idiopathic subglottic stenosis (iSGS) is commonly characterized by laryngeal fibrosis thought to arise by epithelia-mesenchymal transition (EMT) induced by chronic inflammation. Pepsin is a potent inducer of inflammation in the airways during chronic laryngopharyngeal reflux and has been observed in the subglottic mucosa of patients with iSGS, absent in normal mucosa. The aim of this study was to examine the effect of pepsin on mechanisms of EMT in laryngeal cells with implications for iSGS. STUDY DESIGN: In vitro translational research study. METHODS: Human laryngeal epithelial cell cultures were exposed to 0.1 mg/mL or 1.0 mg/mL pepsin at pH7 for 24 and 48 hours, or media pH5 ± 0.1 mg/mL pepsin for 10 minutes and harvested after 24 and 48 hours. EMT marker expression was measured by qPCR and enzyme-linked immunosorbent assays. Wound-healing scratch assay was performed on immortalized human vocal fold fibroblasts pretreated with media pH5 ± 0.1 mg/mL pepsin (10 minutes) or continuously treated with media pH7 ± 0.1 to 1 mg/mL pepsin for 24 hours. RESULTS: Pepsin yielded no effect on MMP1, MMP9, FN1, COL1A1, HAS2, or CDH1 gene expression or matrix metalloproteinase-9 or fibronectin protein expression, either alone or in the presence of weak acid. Pepsin and/or acid produced no effect on fibroblast migration. CONCLUSION: Whereas pepsin has been shown to be present in the subglottic mucosa of patients with iSGS, this in vitro acute exposure investigation does not provide evidence of a direct causal role for development of fibrosis in subglottic epithelial cell cultures. LEVELS OF EVIDENCE: NA. Laryngoscope, 130:154-158, 2020.

Correlation of salivary and nasal lavage pepsin with MII-pH testing.

OBJECTIVES: Laryngopharyngeal reflux (LPR) is a common upper airway disease. Salivary pepsin is a proposed marker for LPR; however, the optimal time for collection of specimens for pepsin detection and pepsin's presence in the oral and nasal secretions relative to concurrent multichannel intraluminal impedance-pH (MII-pH) monitoring are unknown. STUDY DESIGN: Prospective case-control study with an experimental design. METHODS: Patients undergoing MII-pH testing for evaluation of LPR and asymptomatic control subjects were selected. Nasal lavage and saliva samples were collected in the clinic prior to MII-pH probe placement. Additional saliva samples were obtained an hour after each meal and upon waking the following morning. Nasal lavage and salivary pepsin were measured by ELISA. RESULTS: Twenty-six patients undergoing MII-pH testing and 13 reflux-free control patients were enrolled. Salivary pepsin was detected in 11 of 26 patients with suspected LPR and 0 of 13 controls. Pepsin was most frequently detected in the specimen provided upon waking at an average concentration of 186.9 ng/mL. A significant correlation was observed between salivary pepsin in waking samples to MII-pH measurements, including reflux bolus duration, and proximal and distal recumbent reflux episodes (P < 0.05). A significant correlation was also observed between salivary pepsin upon waking or sinus lavage and reflux symptom index (P < 0.05). CONCLUSION: Pepsin in salivary and nasal lavage samples demonstrated an association with MII-pH-documented LPR. Pepsin detection was most frequent in morning samples, supporting use of morning salivary pepsin levels as a potential noninvasive technique for LPR diagnosis. LEVEL OF EVIDENCE: 2 Laryngoscope, 130:961-966, 2020.

Detection of Pepsin in Oral Secretions of Infants with and without Laryngomalacia.

OBJECTIVES: Laryngomalacia is a common cause of stridor in infants and is associated with laryngopharyngeal reflux (LPR). Although pepsin in operative supraglottic lavage specimens is associated with severe laryngomalacia, detection of pepsin in oral secretions has not been demonstrated in an outpatient setting. METHODS: Children <2 years old with laryngomalacia diagnosed by flexible laryngoscopy and children without stridor were selected. Oral secretion samples were obtained in clinic from all subjects. Pepsin, IL-1ß, and IL-8 enzyme-linked immunosorbent assays were performed to determine presence of LPR. RESULTS: Sixteen laryngomalacia and sixteen controls were enrolled. Pepsin was detected more frequently in oral secretions of patients with laryngomalacia (13/16) than in controls (2/16; P < .001). Four patients with laryngomalacia developed symptoms requiring supraglottoplasty. Presence and level of salivary pepsin was not significantly associated with need for surgical management, nor were the levels or presence of IL-1ß or IL-8 significantly associated with presence or level of pepsin, diagnosis of laryngomalacia, or need for operative management. CONCLUSION: Pepsin in saliva appears to be associated with laryngomalacia, suggesting a role for salivary pepsin as a noninvasive marker of LPR in patients with laryngomalacia. Future studies will determine the utility of this test in laryngomalacia.

Pepsin Triggers Neutrophil Migration Across Acid Damaged Lung Epithelium.

Pepsin represents a potential biomarker for extraesophageal reflux disease when detected in airways, however a direct role for pepsin in lung dysfunction has not been clearly established. Children experiencing gastroesophageal and extraesophageal reflux are often prescribed proton pump inhibitors (PPIs) to reduce gastric acid associated damage to esophageal and airway mucosa. The potential of pepsin and gastric fluid, from children that were either on or off PPI therapy, to cause inflammation and damage using a human in vitro co-culture model of the airway mucosa was evaluated herein. Exposure of the airway model to acidic solutions caused cellular damage and loss of viability, however, acid alone did not disrupt barrier integrity or instigate neutrophil trans-epithelial migration without pepsin. Gastric fluid from patients on PPI therapy exhibited only a slightly higher pH yet had significantly higher concentrations of pepsin and elicited more barrier disruption and neutrophil trans-epithelial migration compared to gastric fluid from patients off PPIs. Inflammatory and damaging responses observed with gastric fluid from patients on PPIs were largely driven by pepsin. These results indicate the potential for PPI usage to raise concentrations of pepsin in gastric fluid, which may enhance the pathological impact of micro-aspirations in children with extraesophageal reflux.