Tina L. Samuels

Pepsin as a causal agent of inflammation during nonacidic reflux

Objective: To investigate the contribution of pepsin to inflammation attributed to nonacidic gastric reflux via analysis of inflammatory cytokine and cytokine receptor gene expression in pepsin-treated human hypopharyngeal epithelial cells in vitro. Study Design: Translational research. Setting: This study was performed in an academic research laboratory. Subjects and Methods: Human hypopharyngeal epithelial cells were incubated with or without pepsin (0.1 mg/mL) at pH 7.4, 37°C, overnight. Expression of 84 inflammatory cytokines and cytokine receptors was analyzed via RT2 qPCR array. Results: Expression of a number of inflammatory cytokines and receptors was altered in human hypopharyngeal epithelial cells following overnight treatment with pepsin at neutral pH. Greater than 1.5-fold change in gene expression was detected for CCL20, CCL26, IL8, IL1F10, IL1A, IL5, BCL6, CCR6, and CXCL14 (P < 0.05). Conclusion: Exposure of hypopharyngeal cells to pepsin in a nonacidic environment induces the expression of several pro-inflammatory cytokines and receptors, including those known to be involved in inflammation of esophageal epithelium in response to reflux and which contribute to the pathophysiology of reflux esophagitis. These data indicate that refluxed pepsin may contribute to laryngeal inflammation associated with nonacidic gastric reflux, including that experienced by patients despite maximal acid suppression therapy.

Pepsin as a marker of extraesophageal reflux.

Diagnosis of extraesophageal reflux (EER) currently relies on tools designed for diagnosis of gastroesophageal reflux. Such tools lack the sensitivity and reproducibility to detect the less frequent and mildly acidic reflux associated with upper airway disease. Pepsin has been posited to be a reliable biological marker of EER. Our aim was to present a comprehensive literature review of the use of pepsin as a diagnostic marker of EER. Two methods are typically used for detection of pepsin in the airways: enzymatic and immunologic. The limitations, advantages, and examples of use of each are discussed. Pepsin assay has been used to identify refluxate in trachea, lung, sinus, middle ear, combined sputum and saliva, and breath condensate. An immunologic pepsin assay of combined sputum and saliva was determined to be 100% sensitive and 89% specific for detection of EER (based on pH-metry), and an enzymatic test of nasal lavage fluid (100% sensitivity and 92.5% specificity) demonstrated an increased incidence of EER in patients with chronic rhinosinusitis. Pepsin assay identified tracheal pepsin to be an indicator of bronchopulmonary dysplasia and related mortality risk in ventilated preterm infants. Pepsin assay is a useful tool for correlation of reflux with airway disease and is a reliable diagnostic marker of EER.

Pepsin in nonacidic refluxate can damage hypopharyngeal epithelial cells.

Objectives: Studies using combined multichannel intraluminal impedance with pH monitoring reveal a role for nonacidic reflux in laryngopharyngeal symptoms and injury. We have discovered that pepsin is taken up by laryngeal epithelial cells by receptor-mediated endocytosis. This finding reveals a novel mechanism by which pepsin could cause cell damage, potentially even in nonacidic refluxate. The objective of this study was to determine whether pepsin, at pH 7.4 and thus in nonacidic refluxate, causes cell damage. Methods: Cultured hypopharyngeal epithelial (FaDu) cells were exposed to human pepsin (0.1 mg/mL) at pH 7.4 for either 1 hour or 12 hours at 37°C and analyzed by electron microscopy, cytotoxicity assay, and SuperArray. Results: We report mitochondrial and Golgi complex damage in cells exposed to pepsin at neutral pH, observed by electron microscopy. We also report cell toxicity of pepsin at pH 7.4, measured by a cytotoxicity assay. Furthermore, using SuperArray, we found that pepsin at pH 7.4 significantly alters the expression levels of multiple genes implicated in stress and toxicity. Conclusions: These findings are perhaps the first to explain why many patients have symptoms and injury associated with nonacidic reflux, and could have important implications for the development of new therapies for reflux, such as pepsin receptor antagonists and/or irreversible inhibitors of peptic activity.

Mucin Gene Expression in Human Laryngeal Epithelia: Effect of Laryngopharyngeal Reflux

Objectives: We sought to document the mucin gene profile in normal human laryngeal epithelium and compare it with that in patients with reflux-attributed laryngeal injury or disease. We also investigated the effect of low pH with or without pepsin on mucin messenger RNA levels in vitro. Methods: Laryngeal biopsy specimens were obtained from 3 patients with clinically diagnosed laryngopharyngeal reflux and from 2 control subjects who had no signs or symptoms of reflux. Signs and symptoms were assessed by the Reflux Finding Score and the Reflux Symptom Index, respectively. Reverse transcription–polymerase chain reaction (RT-PCR) was performed to establish the mucin gene profile. Human hypopharyngeal epithelial cells were exposed to pH 7, 5, 4, and 2 with and without pepsin (0.1 mg/mL) for 20 minutes at 37°C, and expression of selected mucins was analyzed via real-time RT-PCR. Results: Mucin 1–5, 7, 9, 13, 15, 16, and 18–20 transcripts were detected in normal laryngeal epithelium, whereas mucin 6, 8, and 17 transcripts were not. Mucins 2, 3, and 5 were expressed at reduced levels in patients with reflux-attributed laryngeal injury or disease. These mucin genes were up-regulated after exposure to low pH in vitro (p < 0.005). Pepsin inhibited this up-regulation (p < 0.001). Conclusions: Reflux laryngitis is associated with down-regulation of mucin gene expression.

Pepsin promotes proliferation of laryngeal and pharyngeal epithelial cells.

Laryngopharyngeal reflux (LPR) is thought to be a significant risk factor for laryngeal squamous cell carcinoma (SCC), but causality has never been proven. It is accepted that chronic reflux into the esophagus can induce metaplastic changes in esophageal mucosa with subsequent increased risk of esophageal adenocarcinoma, but no similar associations have been established for LPR and laryngopharyngeal SCC. The objective of this study was to test the hypothesis that reflux of pepsin into the laryngopharynx can promote carcinogenesis.

Rationale for Targeting Pepsin in the Treatment of Reflux Disease

Objectives: We undertook to 1) obtain unequivocal evidence to confirm or rebut our initial observations that pepsin is taken up by hypopharyngeal epithelial cells by receptor-mediated endocytosis, 2) investigate whether uptake of pepsin at pH 7, in nonacidic refluxate, is of pathological significance, and 3) test our hypothesis that inactive but stable pepsin (<pH 8) taken up by hypopharyngeal epithelial cells causes damage by becoming reactivated inside the cell. Methods: Human posterior cricoid biopsy specimens and cultured hypopharyngeal FaDu epithelial cells were used to perform competitive binding studies and to investigate colocalization of pepsin with clathrin, Rab-9, and TRG-46. FaDu cells were exposed to pepsin (both irreversibly and reversibly inactivated) in the presence and absence of wortmannin and dimethyl amiloride and analyzed by electron microscopy, MTT cytotoxicity assay, and Stress and Toxicity SuperArray. Results: Pepsin is unequivocally taken up by hypopharyngeal epithelial cells by receptor-mediated endocytosis. Uptake of pepsin at pH 7, in nonacidic refluxate, causes mitochondrial damage and changes the expression of several genes implicated in stress and toxicity. Irreversible, but not reversible, inhibition of peptic activity prevents these changes. Conclusions: Pepsin, at pH 7, in nonacidic refluxate, causes damage by becoming reactivated inside the cell. Irreversible inhibitors of peptic activity hold promise as a new therapy for reflux.

The role of extraesophageal reflux in medically and surgically refractory rhinosinusitis.

To clarify the relationship between chronic rhinosinusitis (CRS) and extraesophageal reflux (EER) using state‐of‐the‐art technology. We hypothesized that patients with medically and surgically refractory CRS would have a greater prevalence of EER. We also hypothesized that there would be evidence of gastric refluxate reaching the nasopharynx and paranasal sinuses.

Analysis of pepsin in tracheoesophageal puncture sites.

Objectives: Tracheoesophageal puncture (TEP) and prosthesis insertion is a well-established method of voice rehabilitation after laryngectomy. Maintenance of the prosthesis and tract can be challenging, and reflux to the TEP site has been proposed as a cause. The sites of TEP were evaluated for the presence of pepsin in tissue biopsy specimens and tract secretions to explore this association. Methods: Patients with TEP were interviewed for a history of symptoms related to reflux, medication use history, TEP voice quality, and incidence of TEP complications. Tissue biopsy specimens and tract secretions were obtained from TEP sites and analyzed for the presence of pepsin via sodium dodecyl sulfate–polyacrylamide gel electrophoresis Western blot analysis. Results: Twelve of 17 patients (47%) had some history of preoperative or postoperative symptoms of gastroesophageal reflux disease or laryngopharyngeal reflux. Pepsin was present within the TEP site in a total of 10 of 17 patients (58%; 7 of 17 tissue biopsy specimens and 6 of 7 secretion samples). There were no statistically significant associations between the presence of pepsin and sex, reflux history, use of acid suppressive medicine, or time since laryngectomy. Conclusions: Reflux with subsequent pepsin deposition into the TEP tract occurs in a majority of laryngectomy patients. Further studies on the effect of reflux on the health and function of the TEP tract are warranted.

Chronic Pepsin Exposure Promotes Anchorage-Independent Growth and Migration of a Hypopharyngeal Squamous Cell Line

Outcome Objectives (1) Investigate the role of reflux, specifically pepsin, in laryngopharyngeal carcinogenesis. (2) Evaluate effects of chronic pepsin exposure on cell migration, apoptosis, and colony-forming ability in hypopharyngeal cells. Study Design Translation research. Setting Academic research laboratory. Methods Human hypopharyngeal squamous carcinoma FaDu cells were chronically exposed to nonacidic pepsin (exposed for 24 hours, 4 times over 2 weeks at the following concentrations: 0.01 mg/mL, 0.1 mg/mL, or 1 mg/mL). Precise wounds were created in confluent cell plates, and rates of cell migration into wounds were quantified. Separately, cell viability of chronic pepsin-exposed FaDu cells acutely treated with paclitaxel was measured. Finally, a clonogenic assay was performed on these cells to measure effects of chronic pepsin exposure on colony-forming ability. Results An increased rate of relative wound density was observed in chronic pepsin-treated (0.01 mg/mL, 0.1 mg/mL) cells compared with control (P < .001), suggesting greater rates of cell migration. Pepsin-treated (0.1 mg/mL) cells demonstrated on average greater cell viability compared with control after exposure to paclitaxel, suggesting possible apoptotic resistance; however, this was not statistically significant. Chronic pepsin exposure (0.1 mg/mL, 1 mg/mL) was associated with a dose-dependent increase in colony-forming ability relative to control (P < .001). Conclusion Hypopharyngeal squamous cell line chronically exposed to pepsin demonstrated increased cell migration and colony-forming ability relative to control cells. These experiments indicate that chronic pepsin exposure acts as a promoter of tumorigenesis and metastasis of airway epithelium, suggesting a role for pepsin in laryngopharyngeal carcinogenesis attributed to gastric reflux.

The Impact of Pepsin on Human Nasal Epithelial Cells In Vitro: A Potential Mechanism for Extraesophageal Reflux Induced Chronic Rhinosinusitis.

Objectives: To describe potential mechanisms by which pepsin induces inflammation in refractive chronic rhinosinusitis (CRS). Our hypothesis was that pepsin induces mitochondrial damage and cytokine expression in human nasal epithelial cells (HNEpC) in vitro. Methods: Western blot was used to detect pepsin in sinus lavages from patients with CRS and controls. The HNEpC cells were treated with pepsin (pH 7; 0.1 mg/mL) for 1 or 16 hours and routine electron microscopy (EM) and MTT assay were performed. Cytokine ELISA was performed on media collected from HNEpC cells 16 hours following a 1-hour pepsin treatment. Results: Pepsin was detected in sinus lavages from 4 out of 6 CRS patients and 0 out of 3 controls. The EM showed mitochondrial damage in pepsin-treated HNEpC cells but not in control cells. The MTT assay demonstrated reduced mitochondrial activity in pepsin-treated HNEpC cells compared to controls (P < .001). Pepsin increased IL-1A (P = .003) and IL-6 (P = .04) expression in HNEpC cells. Conclusions: Pepsin in sinus lavages from patients with CRS is consistent with previous studies. This study reveals the damaging effect of pepsin on mitochondria in nasal epithelial cells in vitro. Cytokines previously associated with CRS were elevated following pepsin treatment of HNEpC cells in vitro. These results demonstrate mechanisms by which pepsin may potentiate CRS.