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.

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.

Gastric Acid and Pepsin Work Together in Simulated Gastric Acid Inhalation Leading to Pulmonary Fibrosis in Rats.

BACKGROUND The clinical association between gastroesophageal reflux disease (GERD) and idiopathic pulmonary fibrosis (IPF) has been known for many years, but it is still unclear. The present study investigated the association between experimentally simulated aspiration and pulmonary fibrosis. MATERIAL AND METHODS A total of 120 male Sprague-Dawley rats were randomly divided into a negative control group, a bleomycin group, and 3 simulated aspiration groups. The bleomycin group was administered a one-time intratracheal injection of bleomycin, whereas the 3 simulated aspiration groups were treated either with an intratracheal instillation of gastric fluid combined with pepsin, with pepsin alone, or with hydrochloric acid, all twice a week, and the negative control group was administered normal saline twice a week. Lung tissues were collected to evaluate pathological changes and the mRNA expression levels of connective tissue growth factor (CTGF), type I collagen, and transforming growth factor. RESULTS The results demonstrated that the degree of fibrosis in the early stage was low in each of the 3 simulated aspiration groups, but gradually increased over time. The expression levels of the downstream factor of fibrosis, CTGF, and type I collagen also reflected this trend. CONCLUSIONS The study demonstrates that aspiration of gastric contents can cause pulmonary fibrosis, and mixed aspiration of pepsin and gastric fluid can accelerate this process. This study provides strong evidence in support of a potential association between human GERD and IPF.

[Clinical significance and research progress of pepsin in laryngopharygeal reflux and laryngeal carcinoma].

Laryngeal carcinoma is a common malignancy, and the incidence of this disease is on the rise. In recent years, more and more studies of the etiology and risk factors have confirmed the correlation between laryngopharygeal reflux and the incidence of laryngeal carcinoma. Laryngopharygeal reflux is defined as reflux of the stomach contents above the upper esophageal sphincter. Stimulation and injury of acid to the esophagus and throat mucosa have now been studied more thoroughly, and pepsin plays an increasingly important role in laryngopharygeal reflux disease. The incidence of laryngopharygeal reflux in patients with laryngeal carcinoma reported in the literature was 54.0%-88.7%, mainly because of mucosal injury due to the combined effect of gastric acid and pepsin. This article reviews the significance of pepsin in laryngopharygeal reflux, its mechanism of action and related clinical detection methods.

[Extraesophageal reflux. Overview and discussion of a new method for pH monitoring]. / Oropharyngeale pH-Metrie. Überblick und Darstellung einer neuen pH-Metriemethode.

BACKGROUND: Extraesophageal reflux disease often requires diagnosis and treatment by a phoniatry or ear, nose and throat specialist. The disease needs to be differentiated from gastroesophageal reflux disease. OBJECTIVE: A new oropharyngeal pH measuring system with a single channel probe has recently been introduced. The aim of this study was to compare oropharyngeal pH-metry with the existing diagnostic methods for extraesophageal reflux disease and to present initial results in our own patients. METHODS: A literature search for oropharyngeal pH-metry was performed in the databases NHS EED, HTA, DARE, Clinical trials, Cochrane reviews and Medline/PubMed. A selective literature search was also carried out on the problem of extraesophageal reflux disease. RESULTS: Evaluation scales, trial proton pump inhibitor therapy or pH-metry, for example, can be used to diagnose extraesophageal reflux disease. pH-metry can be performed using a classical two-channel pH-metry system; a new oropharyngeal pH measuring system has recently been introduced. This new method has been evaluated in initial studies for normative data and has been compared to two-channel pH-metry. Prospective randomised studies to diagnose extraesophageal reflux disease with the new oropharyngeal pH-metry method are still lacking. DISCUSSION: Oropharyngeal pH-metry has some potential advantages compared to classical two-channel pH-metry; however, a lot of questions remain unanswered. These will be discussed and illustrated with the help of a number of own patient case reports.

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 (

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 degrees C, overnight. Expression of 84 inflammatory cytokines and cytokine receptors was analyzed via RT(2) 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.

Unexpected consequences of proton pump inhibitor use.

Proton pump inhibitors (PPIs) are among the most widely prescribed classes of medications for gastroesophageal and laryngopharyngeal reflux diseases. There is emerging evidence that the pathogenesis of disease in laryngeal mucosa is not just related to refluxed acid, but also the presence of pepsin and acidic microenvironments. The widespread use of PPIs is also calling into question potential complications of PPI use. This commentary expands upon these issues with other potential unexpected consequences, and considers the importance of determining a proper approach to patient management.

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 degrees 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 degrees 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.

A matrix effect in pectin-rich fruits hampers digestion of allergen by pepsin in vivo and in vitro.

BACKGROUND: It is a general belief that a food allergen should be stable to gastric digestion. Various acidic plant polysaccharides, including pectin, are ubiquitous in fruit matrixes and can form hydrogels under low-pH conditions. OBJECTIVE: The purpose of this study was to investigate the effect of hydrogel forming polysaccharide-rich fruit matrixes on in vivo gastric and in vitro pepsic digestion of fruit allergens. METHODS: Fruit extract proteins (kiwi, banana, apple and cherry) and a purified major kiwi allergen Act c 2 were digested with simulated gastric fluid in accordance with the US Pharmacopeia. In vivo experiments on kiwi fruit digestion were performed on four healthy non-atopic volunteers by examining the gastric content 1 h after ingestion of kiwi fruit. The Act c 2 and kiwi proteins were detected in immunoblots using monoclonal anti-Act c 2 antibodies and rabbit polyclonal antisera. RESULTS: Crude fruit extracts were resistant to digestion by pepsin when compared with commonly prepared extracts. In the gastric content of all volunteers, following kiwi fruit ingestion and immunoblotting, intact Act c 2 was detected with anti-Act c 2 monoclonal antibodies, while kiwi proteins of higher molecular weights were detected using rabbit polyclonal antisera. Addition of apple fruit pectin (1.5% and 3%) to the purified kiwi allergen was able to protect it from pepsin digestion in vitro. CONCLUSION: The matrix effect in pectin-rich fruits can influence the digestibility of food proteins and thereby the process of allergic sensitization in atopic individuals.

Essential role of pepsin in pathogenesis of acid reflux esophagitis in rats.

Pepsin, a protease activated by gastric acid, is a component of the refluxate, yet the role of pepsin in the pathogenesis of reflux esophagitis has not been well studied. In the present study, we examined the effect of pepstatin, a specific inhibitor of pepsin, on acid reflux esophagitis. Acid reflux esophagitis was induced in rats by ligating both the pylorus and the forestomach for 3 or 4 hr. Pepstatin, ecabet Na (the anti-ulcer drug), and L-glutamine were administered intragastrically after the ligation. Pepstatin or ecabet Na, given intragastrically, significantly prevented esophageal lesions, even though they did not affect basal acid secretion in pylorus-ligated rats. Pepstatin significantly inhibited pepsin activity in vivo and in vitro, while ecabet Na inhibited this activity in vitro. By contrast, L-glutamine given intragastrically aggravated the lesions in a dose-dependent manner, but even in the presence of L-glutamine the development of esophageal lesions was totally prevented by coadministration of pepstatin or ecabet Na. L-Glutamine increased the pH of gastric contents to approximately 2.0, the optimal pH for the proteolytic activity of pepsin in vitro. In addition, intragastric administration of exogenous pepsin worsened the severity of esophageal damage. These results suggest that pepstatin is highly effective against acid reflux esophagitis, without influencing acid secretion, while L-glutamine aggravated these lesions by increasing the pepsin activity by shifting the intraluminal pH to the optimal pH range for proteolytic action. It is assumed that pepsin plays a major pathogenic role in the development of acid reflux esophagitis.

Reflujo gastroesofágico y y enfermedad por reflujo gastroesofágico: límite entre la fisiología y la enfermedad / Gastroesophageal reflux and gastroesophageal reflux disease: teh limit between physiology and pathology

El reflujo gastroesofágico(RGE) es un proceso fisiológico que se manifiesta clínicamente en mayor o menor grado. La enfermedad por reflujo gastroesofágico(ERGE), entendido como el conjunto de signos y síntomas derivados del RGE, es poco frecuente. Se revisan los mecanismos fisiopatológicos implicados en estos procesos, los procedimientos diagnósticos y el tratamiento

Gastroesophageal reflux (GER)is a physiologic process that has a variable expression. We understand gastroesophageal reflux disease(GERD), as the clinical picture that appears secondary to GER. We have reviewed physiopathologic mechanisms involved, as well as the diagnosis procedures and treatment

Review article: role of pepsin and bile in gastro-oesophageal reflux disease.

Gastro-oesophageal reflux disease is defined as the presence of symptoms or lesions that can be attributed to the reflux of gastric contents into the oesophagus. Aspiration and prolonged monitoring studies in humans have shown that reflux of gastric contents is comprised of both acid and non-acid components, in healthy as well as diseased people. Methods to monitor the non-acid component of the refluxate are described in detail. Experimental models suggest that synergism between acid and pepsin and conjugated bile acids have the greatest damaging potential for oesophageal mucosa, although unconjugated bile acids may be caustic at a more neutral pH. Human studies are compatible with a synergistic action between acid and duodenogastric reflux in inducing lesions. During prolonged monitoring studies, typical gastro-oesophageal reflux disease symptoms are more related to acid reflux events than to non-acid reflux events. However, symptoms that persist during acid-suppressive therapy are often related to non-acid reflux events. The therapeutic options for the non-acid component of the refluxate, including acid suppression, prokinetics, baclofen and surgery, are discussed.

Gastric juice: a barrier against infectious diseases.

All vertebrates produce gastric acid. Its main function is inactivation of ingested microorganisms. The majority of microbiological pathogens ingested never reaches the intestine because of the gastric barrier. Although gastric hypochlorhydria is fairly common due to atrophic gastritis, gastric surgery or use of inhibitors of gastric acid secretion, the resulting susceptibility to infection has not been studied extensively. Drug-induced blockade of acid secretion leads to gastrointestinal bacterial overgrowth; the clinical significance of this is still controversial. Gastric acidity is known to protect against non-typhoid salmonellosis and cholera and it is suspected that it protects against several parasitic diseases as giardiasis and strongyloides. There is a lack of studies focusing on the impact of the gastric acidic barrier on viral infections. Concerning prion infections only a single study has been performed, demonstrating a possible role of gastric acidity in the protection against foodborne prion disease in mice. The combination of malnutrition and hypochlorhydria may contribute to the high prevalence of gastrointestinal infections in developing countries. Further studies are needed to evaluate the clinical consequences of impaired gastric acidity with respect to susceptibility to infections.

Effect of acid and pepsin on gene expression in laryngeal fibroblasts.

The aim of this study was to determine changes that momentary low pH with or without pepsin causes in gene expression in laryngeal fibroblasts. Cell cultures were established from human false vocal fold (FVF) and postcricoidal (PC) mucosae. Using a real-time polymerase chain reaction, we analyzed messenger RNA gene expression of growth factors (transforming growth factor beta1, vascular endothelial growth factor, fibroblast growth factor 2), matrix metalloproteinases (MMP-1, MMP-2), and decorin in normal media, pH 4 media, and pH 5 media with and without pepsin. The FVF fibroblast gene expression differed substantially from the PC fibroblast gene expression. No significant interaction effects for acid and pepsin were found in the FVF culture, but in PC cultures we found a significant overexpression interaction effect for vascular endothelial growth factor, fibroblast growth factor 2, MMP-1, MMP-2, and decorin. These results imply that PC tissue is more sensitive than FVF tissue to the noxious effects of gastric contents. Furthermore, there appears to be a synergistic effect for acid and pepsin exposure in the posterior larynx.

Adaptive evolution and functional divergence of pepsin gene family.

In vertebrates, a large proportion of genes is organized in gene families. Paralogous gene groups generated by gene duplication are related by homology, high degree of sequence identity and similar structural architecture of their products. Aspartic proteinases form a widely distributed protein superfamily including cathepsins, pepsins, renin and napsin. In the present study, the nucleotide sequences coding for various pepsins in 30 vertebrate species have been used to derive a gene phylogeny. Gene duplication and losses have been inferred from a reconciled tree, reconstructed by combining information from gene tree and species tree. Our findings based on the results of the relative rate ratio test and maximum likelihood analysis suggest that each round of gene duplication is characterized by adaptive evolution, although instances of evolution under positive selection have been found also long after divergence of gene families. The results of functional divergence analysis provided statistical evidence for shifted evolutionary rate after gene duplication.

Gastric luminal epidermal growth factor is affected by diet.

OBJECTIVE: Diet is an area of major interest to those investigating the causes of cancer of the oesophagus in the Transkei. This study looked at the associations between intragastric epidermal growth factor level, diet and intragastric pH. SETTING AND SUBJECTS: A dietary survey was co-ordinated with studies of gastric luminal epidermal growth factor and gastric fluid pH in 120 rural Transkeians. RESULTS: Gastric fluid epidermal growth factor was associated with low dietary intake of animal products (p = 0.002) and vegetables (p = 0.026). There was no association with pH. CONCLUSION: A dietary subgroup has been identified in the Transkei population with high levels of epidermal growth factor in the upper gastrointestinal lumen. This adds to previously demonstrated diet-related changes in the upper gastrointestinal tract in Transkei. These changes may affect the disease pattern of the population.