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Abstract

Exposure of laryngeal epithelia to pepsin during extra-esophageal reflux causes depletion of laryngeal protective proteins, carbonic anhydrase isoenzyme III (CAIII), and squamous epithelial stress protein Sep70. The first objective of this study was to determine whether pepsin has to be enzymatically active to deplete these proteins. The second objective was to investigate the effect of pH on the activity and stability of human pepsin 3b under conditions that might be found in the human esophagus and larynx. Prospective translational research study. An established porcine in vitro model was used to examine the effect of active/inactive pepsin on laryngeal CAIII and Sep70 protein levels. The activity and stability of pepsin was determined by kinetic assay, measuring the rate of hydrolysis of a synthetic pepsin-specific substrate after incubation at various pH values for increasing duration. Active pepsin is required to deplete laryngeal CAIII and Sep70. Pepsin has maximum activity at pH 2.0 and is inactive at pH 6.5 or higher. Although pepsin is inactive at pH 6.5 and above, it remains stable until pH 8.0 and can be reactivated when the pH is reduced. Pepsin is stable for at least 24 hours at pH 7.0, 37 degrees C and retains 79% +/- 11% of its original activity after re-acidification at pH 3.0. Detectable levels of pepsin remain in laryngeal epithelia after a reflux event. Pepsin bound there would be enzymatically inactive because the mean pH of the laryngopharynx is pH 6.8. Significantly, pepsin could remain in a form that would be reactivated by a subsequent decrease in pH, such as would occur during an acidic reflux event or possibly after uptake into intracellular compartments of lower pH.

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... 3 Pepsin plays a major role in the pathogenesis of LPR. Pepsin can damage the laryngeal and pharyngeal mucosa at both acidic and alkaline pH, as it shows some activity even at pH 8. 12 Johnston et al did a prospective translational study in established porcine in vitro model to examine the effect of active/inactive pepsin on laryngeal CAIII and Sep70 protein levels. They reported detectable levels of pepsin in laryngeal epithelia after a reflux event. ...
... They reported detectable levels of pepsin in laryngeal epithelia after a reflux event. 12 Normally, pepsin in this site would be enzymatically inactive, as the mean pH of the laryngopharynx is 6.8. Significantly, pepsin would be reactivated by a subsequent decrease in pH, such as would occur during an acidic reflux event or possibly after uptake into intracellular compartments of lower pH. ...
... They thus suggest that a positive pepsin test in a patient clinically suspected to have LPR can be a cost-effective, accurate, and alternative diagnostic method. 12 Another study by Alexander et al 15 suggests that salivary pepsin has a sensitivity of 78% and specificity of 53% for predicting an RFS >7. Wang et al 16 conducted a meta-analysis to assess the diagnostic value of pepsin in saliva for LPR. ...
Article
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Introduction Twenty-four-hour multichannel intraluminal impedance with double probe pH monitoring (MII-pH), though considered the most sensitive tool for the diagnosis of gastroesophageal reflux disease (GERD), is invasive, time consuming, not widely available, and unable to detect non-acid reflux. In contrast, the presence of pepsin in the saliva would act as a marker for reflux, considering that pepsin is only produced in the stomach. Objective To evaluate the predictive value of salivary pepsin in diagnosing laryngopharyngeal reflux (LPR) as suggested by the results of reflux symptom index (RSI > 13), reflux finding score (RFS > 7), and positive response to treatment with a 4-week course of proton-pump inhibitors. Methods This prospective study was done at a tertiary care hospital on 120 adult patients attending ENT OPD with clinical diagnosis of LPR. The presence of pepsin in their pharyngeal secretions and saliva using a lateral flow device, the Peptest, was compared with RSI, RFS, and with the response to medical treatment using the Chi-squared test. Results Salivary pepsin was found to be positive in 68% of the patients, with 87.5% of them showing positive response to treatment. Chi-squared analysis showed a significant association between positive salivary pepsin and RFS > 7, RSI >13, a combination of RFS > 7 and RSI > 13 as well as with response to treatment (p < 0.0001). Conclusion When considered along with the clinical indicators of RFS and RSI of more than 7 and 13, respectively, and/or with a response to treatment, a positive salivary pepsin test indicates statistically significant chance of presence of LPR.
... 35,36 It has long been understood that while the enzymatic activity of pepsin decreases with increasing pH, it remains stable at up to pH 8 and is reactivated by subsequent reduction in pH. 17,37 At the pH of the larynx and hypopharynx (pH 6.8), pepsin is endocytosed through interaction with a receptor and is retained in intracellular vesicles of pH 4-5 where it would be reactivated. 28,37 Hypopharyngeal cells exposed to pepsin for 1-24 h at neutral pH exhibit mitochondrial damage, changes in expression of genes associated with cell stress and toxicity, a proinflammatory gene expression profile similar to that observed in GERD, as well as carcinogenic changes, including dysregulation of oncogenes, cell proliferation and migration, and anchorage-independent growth. ...
... 17,37 At the pH of the larynx and hypopharynx (pH 6.8), pepsin is endocytosed through interaction with a receptor and is retained in intracellular vesicles of pH 4-5 where it would be reactivated. 28,37 Hypopharyngeal cells exposed to pepsin for 1-24 h at neutral pH exhibit mitochondrial damage, changes in expression of genes associated with cell stress and toxicity, a proinflammatory gene expression profile similar to that observed in GERD, as well as carcinogenic changes, including dysregulation of oncogenes, cell proliferation and migration, and anchorage-independent growth. 27,28,38,39 Pepsin has been implicated in epithelial-mesenchymal transition, which promotes metastasis during laryngeal cancer 12 and increases buccal pouch tumor volume after 12 weeks in vivo. ...
... 11,30,48,49,68,69 Given pepsin's role in nonacidic LPR, it has been proposed as a novel therapeutic target, especially for patients experiencing refractory symptoms on PPIs. 7,27,28,30,37 Approximately $26 billion/year is currently being spent on PPIs for the treatment of LPR, despite their poor efficacy for this patient population. 70 We and others have discussed the promise of irreversible inhibitors of peptic activity and/or receptor antagonists as potential new therapeutics for LPR. ...
Article
Airway reflux is implicated in the pathophysiology of a wide range of adult and pediatric upper and lower airway diseases. However, the diagnosis of proximal reflux–associated disease remains challenging due to evolving clinical criteria and institutional and regional variances in diagnostic practices. Evidence suggests that nonacidic contents of reflux may serve as both pathologic mediators of and biomarkers for reflux in the upper airway. Furthermore, they offer potential pharmaceutical and surgical intervention targets and are the focus of novel clinical diagnostic tools currently under investigation.
... When this is repeated with human gastric juice pepsin activity is recoverable after incubation up to pH 7.5 but completely lost at pH 8.0 (18). Studies by Johnston et al. (19) carrying out similar experiments with pepsin 3 showed no activity could be recovered after 24-hour incubation at pH 8.0. The upshot of these studies suggests that pepsin is irreversibly denatured at pH between 7.0 and 8.0. ...
... Consequently, pepsin present in a reflux episode which leaves the oesophagus can bind to the mucosa of the aerodigestive tract, remain inactive but native after neutralization by saliva and bicarbonate. The bound pepsin can be re-activated by a new reflux event if the pH is below 6.0 with resulting tissue damage (19,23). Pepsin from refluxate is recognised as a biomarker of reflux as well as in tissue damage. ...
... Pepsin from refluxate is recognised as a biomarker of reflux as well as in tissue damage. In addition to pepsin, bile acids have been strongly implicated when present in the refluxate as biomarkers of reflux and damaging agents (19,23). However, many of the papers dealing with bile acids are using assays which do not have the required sensitivity (24)(25)(26)(27)(28). ...
... Identifying dogs with naturally occurring GERD and EERD poses a diagnostic challenge as reliable, inexpensive, and minimally invasive diagnostic tests are lacking. In people, biomarkers have been effectively utilized as a part of a multimodal approach to diagnosing reflux and aspirationassociated respiratory syndromes (AARS) (8,18,20,34). Though biomarkers have been utilized effectively for a number of diseases in dogs, investigation into the biomarkers of reflux and aspiration has been rarely performed (2,5,13,22). ...
... It is possible that protein degradation, both in vivo and ex vivo, may have reduced the abundance of this protein below our limit of detection. The low abundance of pepsin A in GF and the lack of detection of pepsin A in OP swabs in vomiting/regurgitating dogs suggest this to be a less-than-ideal protein biomarker regardless of cause and represent a significant departure from the human literature (18,(34)(35)(36)(37)(38). Interestingly, pancreatic proteins were highly abundant in GF, likely suggesting that gastroduodenal reflux was a frequent occurrence in our population and supporting a biomarker study which identified bile in bronchoalveolar lavage fluid from dogs with pulmonary fibrosis (13). ...
Article
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Reflux and aspiration in people cause and exacerbate respiratory diseases in the absence of gastrointestinal signs. Protein biomarkers in humans detect extraesophageal reflux (EER) from oropharyngeal (OP) and bronchoalveloar lavage samples. Reflux likely contributes to respiratory disease in dogs. The objectives of this study were to analyze the canine gastric fluid (GF) proteome and compare this to the OP proteome in normal, vomiting/regurgitating, and coughing dogs to identify biomarkers for EER/aspiration. Twenty-three client-owned dogs were enrolled. Canine GF samples (n = 5) and OP swabs in normal (n = 6), vomiting/regurgitating (n = 7), and coughing (n = 5) dogs were within 2 weeks of sample collection. Protein digests were analyzed by liquid chromatography–mass spectrometry. Differential abundance (DA) of proteins between groups was evaluated by Fisher's exact test with p < 0.0004 significance level after correction for multiple comparisons. DA was found between all groups (p < 0.0001): GF vs. normal (n = 130 proteins), coughing vs. normal (n = 22 proteins), and vomiting/regurgitating vs. normal (n = 20 proteins). Protein abundance was highly variable between dogs. Gastrointestinal-specific proteins were found in OP swabs from vomiting/regurgitating and coughing dogs but not from healthy dogs. In conclusion, the proteomic composition of the OP varies between health and disease. The presence of gastrointestinal-specific proteins in OP of coughing dogs may suggest reflux and/or aspiration as contributing factors. The variable protein abundance warrants investigation into biomarker panels.
... Dysphonia is one of the most frequent symptoms, accounting for up to 55% of LPR patients [1,2]. Dysphonia may be attributed to pepsin-related injuries, macroscopic and microscopic histological changes on the vocal folds, which may lead to aerodynamic and acoustic measurement impairments [3][4][5]. Because evolutions of non-specific symptoms and findings are still subjective, the identification of objective indicators of the treatment effectiveness remains challenging [1]. ...
... Our data supported that acoustic measurements may be used more specifically in LPR patients with dysphonia, while they are useless in patients without self-reported dysphonia. This thought appears consistent with the basic science and clinical studies that reported macroscopic and microscopic histological changes on the vocal folds of reflux subjects that are clinically highlighted by acoustic measurements [3][4][5][6]. The physiological mechanisms involve the pepsin-related impairments of defense mechanisms of the vocal folds, including mucin production, type III anhydrase carbonic activity, growth factor secretion, which may favor the occurrence of epithelial cell dehiscence, microtraumas, inflammatory infiltrate and macroscopic lesions [3]. ...
Article
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Objectives The objective is to study the usefulness of acoustic measurements as therapeutic outcomes for patients with dysphonia related to laryngopharyngeal reflux (LPR). Methods From September 2019 to April 2021, 120 patients with LPR at the hypopharyngeal-esophageal multichannel intraluminal impedance pH-monitoring (HEMII-pH) were prospectively recruited from three University Hospitals. They were divided in two groups regarding the presence of dysphonia. The treatment consisted of a combination of diet, proton-pump inhibitors, magaldrate and alginate for 3–6 months. The following clinical and acoustic evaluations were studied regarding groups at baseline, 3- and 6-month posttreatment: reflux symptom score (RSS), reflux sign assessment (RSA), percent jitter, percent shimmer and noise-to-harmonic ratio (NHR). Results A total of 109 patients completed the evaluations, accounting for 49 dysphonic and 60 non-dysphonic individuals. HEMII-pH, gastrointestinal endoscopy, baseline clinical and acoustic features were comparable between groups. RSS and RSA significantly improved from pre- to 3-month posttreatment in both groups. Jitter, Shimmer and NHR significantly improved from pre- to 3-month posttreatment in dysphonic patients, without additional 3- to 6-month posttreatment changes. Acoustic parameters did not change throughout treatment in patients without dysphonia. Conclusion Acoustic measurements may be an interesting indicator of treatment in LPR patients who reported dysphonia. In this group of individuals, the evolution of acoustic parameters was consistent with the evolution of symptoms and findings.
... Pepsin is a digestive enzyme with maximal activity at pH 1.5-2.5; however, proteolytic activity has been demonstrated at pH levels as high at 6.5, and the enzyme remains stable up to a pH of 8 [9][10][11]. Tissue-bound pepsin within the larynx may be activated by acidic refluxate, leading to tissue damage and inflammation, while another study has demonstrated evidence of receptor-mediated endocytosis [8][9][10]. ...
... however, proteolytic activity has been demonstrated at pH levels as high at 6.5, and the enzyme remains stable up to a pH of 8 [9][10][11]. Tissue-bound pepsin within the larynx may be activated by acidic refluxate, leading to tissue damage and inflammation, while another study has demonstrated evidence of receptor-mediated endocytosis [8][9][10]. Interestingly and counterintuitively, proton pump inhibitors (PPIs) have been shown to exacerbate the pathogenic properties of pepsin, bile salts, and other gastroduodenal enzymes by creating a more alkaline milieu. ...
Article
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The purpose of this article is to review the cornerstone and most recent literature regarding laryngopharynoesophageal reflux (LPR) including epidemiological characteristics, pathophysiology, symptoms, diagnosis, and management. The role of pepsin in the pathophysiology of LPR is highlighted in addition to new diagnostic modalities and pharmacologic therapies that target pepsin.
... Pepsin is primarily active at strongly acidic pH < 4.0, and so at weakly acidic pH 5.0-6.0 remains less active. Johnston et al., have shown complete inactivation of pepsin at pH 6.5 and that pepsin at pH 7.0, although inactive, remains stable for at least 24 h at 37 °C [19]. ...
... With regard to the range of acidified pepsin selected, test solutions below pH 5.0 were not selected as previously found to induce a pronounced cell death [22,23]. Thus, we chose pH 5.0 as the most acidic in this range, pH 6.0 since, according to Johnston et al., pepsin may not be completely inactive, and pH 7.0 where pepsin is inactive but remains stable for a long period of time [19]. Similarly, test solutions above pH 7.0 were omitted because pepsin above pH 7.0 is enzymatically inactive [67]. ...
Article
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Pepsin refluxate is considered a risk factor for laryngopharyngeal carcinogenesis. Non-acidic pepsin was previously linked to an inflammatory and tumorigenic effect on laryngopharyngeal cells in vitro. Yet there is no clear evidence of the pepsin-effect on a specific oncogenic pathway and the importance of pH in this process. We hypothesized that less acidic pepsin triggers the activation of a specific oncogenic factor and related-signalling pathway. To explore the pepsin-effect in vitro, we performed intermittent exposure of 15 min, once per day, for a 5-day period, of human hypopharyngeal primary cells (HCs) to pepsin (1 mg/mL), at a weakly acidic pH of 5.0, a slightly acidic pH of 6.0, and a neutral pH of 7.0. We have documented that the extracellular environment at pH 6.0, and particularly pH 7.0, vs. pH 5.0, promotes the pepsin-effect on HCs, causing increased internalized pepsin and cell viability, a pronounced activation of EGFR accompanied by NF-κB and STAT3 activation, and a significant upregulation of EGFR, AKT1, mTOR, IL1β, TNF-α, RELA(p65), BCL-2, IL6, and STAT3. We herein provide new evidence of the pepsin-effect on oncogenic EGFR activation and its related-signaling pathway at neutral and slightly acidic pH in HCs, opening a window to further explore the prevention and therapeutic approach of laryngopharyngeal reflux disease.
... 17 Gastric juice in humans contains pepsin 7 isoforms; 1, 3a, 3b, 3c; these isoforms have different characteristics, sensitivities, and "optimal pH level" when its 9 action is at a maximum ensuring digestion occurs across a wide range of gastric pH. 18 The pepsin 3 complex is the 11 most common state and this isoform contributes to 80% of human gastric juice. 19 Gastric juice comprises water, mucus, 13 hydrochloric acid, pepsin, bile acids, and intrinsic factor. ...
Article
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Goal: The aim of this study was to investigate the pepsin values and pH results of gastric juice among the subtypes of gastroesophageal reflux disease (GERD) and functional heartburn. Background: The major destructive agents of GERD on the esophageal epithelium are gastric acid and pepsin. No precise information about pepsin concentration in gastric juice exists. Study: Ninety patients with GERD, 39 erosive reflux disease (ERD) Los Angeles (LA) grade A/B, 13 ERD LA grade C/D, 19 nonerosive reflux disease (NERD), 8 esophageal hypersensitivity, 11 functional heartburn, and 24 healthy controls were included in the study. During endoscopy gastric juices from the patients were aspirated and their pH readings immediately recorded. Gastric juice samples were analyzed using Peptest, a lateral flow device containing 2 unique human monoclonal antibodies to detect any pepsin present in the gastric juice sample. Results: The highest mean gastric pepsin concentration (0.865 mg/mL) and the lowest median gastric pH (1.4) was observed in the LA grade C/D group compared with the lowest mean gastric pepsin concentration (0.576 mg/mL) and the highest median gastric pH (2.5) seen in the NERD group. Comparing pH, the NERD patient group was significantly higher (P=0.0018 to P=0.0233) when compared with all other GERD patient groups. Conclusions: The basal gastric pepsin level in the healthy control group was comparable to literature values. There was good correlation and a significant linear relationship between the gastric pepsin level and gastric pH within the patient groups. The severity of the GERD disease is related to the lowest pH and the highest pepsin concentration in gastric juice.
... When extra-esophageal reflux reaches the laryngeal area, pepsin can be transferred into laryngeal cells via receptormediated endocytosis. [23,29] Samuels and Johnston et al found that inactive pepsin can be reactivated and damage cells in 2 ways. For example, when acidic reflux happens, the pH of the environment decreases to the appropriate range for pepsin activation. ...
Article
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The aim of this study is to explore the relationship between gastroesophageal reflux disease (GERD) and vocal fold polyps (VFPs).This is a Case-Control study and was performed with the help of The Second Affiliated Hospital of Chongqing Medical University.Twenty-seven patients with VFP and 20 controls without VFP were recruited between May and October 2018. All the subjects underwent a saliva pepsin test, completed the GerdQ questionnaire and 24-hour multichannel intraluminal impedance with pH (24-h MII-pH) monitoring. Twenty-five resected VFP specimens were examined with immunohistochemical (IHC) and double immunofluorescence (IF) staining.The incidence of GERD in the VFP group was significantly higher than that in the control group (P = .003). Patients with VFP had significantly higher GerdQ scores, pepsin concentrations, and pepsin-positive rates (P < .05). Moreover, the number of proximal and upright reflux events was significantly higher in the VFP group (P < .05). The pepsin concentration in saliva showed a significant positive correlation with the pepsin levels in tissues (r2 = 0.50, P = .011). Pepsin and TGF-β1-positive cells were colocalized with CD45RO-positive cells. IHC staining showed that the majority of VFP patients had a positive expression of pepsin (20/25, 80%) and pepsin-positive cells were found in both the squamous epithelium and mesenchymal tissues. IHC staining of TGF-β1 in VFP revealed findings similar to those of pepsin staining.GERD is an important risk factor for VFP. Pepsin may promote the aggregation of immune cells, increase the local cytokines, and promote inflammatory reaction, suggesting a potential new pathogenesis for VFP. The saliva pepsin test is a reliable method for GERD diagnosis.
... 14,21,22 From a pathophysiological standpoint, PPIs increase the pH of the gastric content and the related aerolized refluxate droplets in the upper aerodigestive tract mucosa, which may decrease the extracellular pepsin activity. 23 Because PPIs do not reduce the number and the duration of the pharyngeal reflux episodes, the gastric content (e.g., pepsin, lipase, trypsin, bile salts) may continue to refluxate through aerolized alkaline droplets in patients using only PPIs. Furthermore, it has been observed that pepsin may be internalized and reactivated into the Golgi apparatus that was associated with an intracellular toxicity on which the PPIs cannot act. ...
Article
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Objectives/Hypothesis To investigate the prevalence and features of laryngopharyngeal reflux (LPR) in patients with primary burning mouth syndrome (BMS). Study Design Prospective uncontrolled study. Methods Patients who visited our Departments of Otolaryngology-Head and Neck and Maxillofacial surgery with BMS were prospectively recruited from September 2018 to September 2020. Patients benefited from dental, maxillofacial, otolaryngological examinations, and hypopharyngeal–esophageal impedance pH-monitoring (HEMII-pH). Oral, pharyngeal, and laryngeal findings and symptoms were rated with Reflux Sign Assessment (RSA) and Reflux Symptom Score-12 (RSS-12). Patients were treated with a combination of diet, pantoprazole, and alginate. Results From the 81 included patients, 76 reported >1 pharyngeal reflux events (93.8%), accounting for 35 (46.1%), 24 (31.6%), and 17 (22.3%) acid, mixed, and nonacid LPR, respectively. Thirty-two patients had both LPR and gastroesophageal reflux disease (GERD). Thirty-eight patients benefited from pepsin saliva measurement, which was positive in 86.8% of cases. The mean scores of mouth/tongue burning, RSS-12, and RSA significantly improved from pre- to post-treatment (P < .004). At 3-month post-treatment, 62.5% of patients reported an improvement of mouth/tongue burning score. Patients with both GERD and LPR reported higher baseline RSS-12 and RSA scores. Conclusion Acid, weakly acid, and nonacid LPR may be involved in the development of BMS. The use of an appropriate treatment considering the reflux features is associated with an improvement of symptoms and findings. Level of Evidence 4 Laryngoscope, 2021
... [8] it became one of the first enzymes to be crystallized , it was using dialysis, filtration, and cooling. [9] Pepsin is most active in acidic environments between 37 °C and 42 °C. [10][11] Accordingly, its primary site of synthesis and activity is in the stomach (pH 1.5 to 2). ...
Article
Full-text available
Bio adhesive material is a protein adhesive, which has recently been highlighted as an attractive biologically based and highly functional adhesive as a new chemo enzymatic synthesis of co polypeptide. The bio adhesive polymer was prepared by modification of pepsin structure with vinyl monomer such as maleic anhydride using ceric ammonium nitrate as an initiator, then the grafted copolymer was substituted with amino drugs. This design carries controlled delivery over an extended period of time due to its digesting nature. The new drug copolymer was investigated using common spectroscopy methods, such as FTIR, ¹ H-NMR and UV Spectroscopy. The physical properties for the prepared polymers were tested. Our results show that deprotonated is required for drug copolymers when analyzed in different pH values at 37 °C in vitro study and controlled drug release was compared at zero time and after three days. The rate of hydrolysis in basic medium was higher than acidic medium. It was concluded There are several advantages of sustain release by modified drug with slow release and in vivo performance was noted to be promising using for applications of polymer used different mice and rabbits infected with bacteria and wounds.
... [8] it became one of the first enzymes to be crystallized , it was using dialysis, filtration, and cooling. [9] Pepsin is most active in acidic environments between 37 °C and 42 °C. [10][11] Accordingly, its primary site of synthesis and activity is in the stomach (pH 1.5 to 2). ...
Article
Full-text available
The International Conference of Chemistry 2020Journal of Physics: Conference Series1853 (2021) 012049IOP Publishingdoi:10.1088/1742-6596/1853/1/0120491Synthesis of peptide derivative asbioadhesive F MAAl-Salami* andRKAl-Shuwaili 11Department of Chemistry,College of Science, Mustansiriyah University, Baghdad, Iraq*Corresponding: drfiryal55@gmail.comAbstract. Bio adhesive material is a protein adhesive, which has recently been highlighted as an attractive biologically based and highly functional adhesive as a new chemo enzymaticsynthesis of co polypeptide. The bio adhesivepolymer was prepared bymodification of pepsin structure with vinyl monomer such as maleic anhydride using ceric ammonium nitrate as an initiator, then the grafted copolymer was substituted with amino drugs. This design carries controlled delivery over an extended period of timedue to its digesting nature. The new drug copolymer was investigated using common spectroscopy methods, such asFTIR,1H-NMR and UV Spectroscopy. The physical properties for the prepared polymers were tested. Our results show that deprotonated is requiredfor drug copolymers when analyzed in different pH values at 37 0C in vitrostudy and controlled drug release was compared at zero time and after three days.The rate of hydrolysis in basic mediumwashigher than acidic medium.It was concludedThere are several advantages of sustain releaseby modified drug with slow release andin vivoperformance was noted to be promisingusing forapplications of polymer used different mice and rabbits infected with bacteria and wounds.
... human diseases [1][2][3]. Recently, pepsin was considered as an enzyme suspected of being involved in gastric ulcer. According to physiologists, pepsin is one of the most important and necessary factors in the development of gastric ulcer disease in contribution with increasing gastric acidity [4]. ...
Article
Pepsin is an aspartic protease that is involved in the digestion of food in the stomach of mammals. Continuous and long-term use of therapeutic agents will cause chronic contact of the drug with pepsin, and as a result, the structure and function of enzyme may change. In this regard the interactions of isoniazid and rifampin as the first line treatments of tuberculosis with pepsin were investigated by various methods such as fluorescence spectroscopy, FTIR, molecular docking and molecular dynamics simulation. Based on the results obtained in this study, the mentioned drugs can form stable complexes with pepsin and the structure of protein changes slightly. According to the results, the major forces in the formation of the protein-drug complex are electrostatic and hydrophobic forces for isoniazid and rifampin respectively and isoniazid shows to form a stronger binding with protein. The FTIR spectrum of the protein shows that little change was occurred in the structure of pepsin in the presence of the drugs. Molecular modeling results of the binding of isoniazid and rifampin to the pepsin confirm laboratory results and show that the binding site of drugs is close to the active site of the enzyme. Also, the activity of pepsin in the presence of both drugs has significantly increased.
... 23,24 In that respect, PPIs make particularly sense in patients with acid reflux episodes in order to decrease the pepsin extracellular activity in the mucosa of the upper aerodigestive tract, the maximum pepsin activity being at pH 2.0. 25 However, recent data supported that the pepsin may be internalised and reactivated into the Golgi that is associated with an intracellular toxicity on which the PPIs cannot impact. 26 The intracellular pathway of pepsin and the potential involvement of bile salts and other gastroduodenal enzymes (active at alkaline pH) may explain the resistance to high-dose PPI therapy in some patients with acid reflux and in many patients with mixed and non-acid LPR. ...
Article
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Objective To assess the evolution of symptoms and findings of laryngopharyngeal reflux (LPR) patients according to the type of reflux (acid, nonacid, mixed and gastroesophageal (GERD)). Design Prospective uncontrolled multi‐center study. Methods One hundred and six patients with LPR have been recruited from three European Hospitals. According to the reflux characteristics at the impedance‐pH monitoring (acid, nonacid, mixed, GERD), patients received a personalized treatment based on the association of diet, pantoprazole, alginate, or magaldrate for 3 months. Reflux Symptom Score (RSS) was assessed at baseline, 6 and 12 weeks posttreatment. Reflux Sign Assessment (RSA) has been used to rate laryngeal and extra‐laryngeal findings at baseline and 12 weeks posttreatment. Overall success rate and the evolution of symptoms and findings were evaluated according to the LPR types. Results 102 LPR patients (42 acid, 33 nonacid, 27 mixed, including 49 with LPR and GERD) completed the study. RSS and RSA total scores significantly improved from baseline to posttreatment time in acid, mixed and nonacid groups. The presence of GERD in addition to LPR did not impact the clinical improvement. The 3‐month success rates of treatment ranged from 62% to 64% and there were no significant differences between groups. The success rate of patients with nonacid LPR was similar to those of patients with mixed and acid LPR. Conclusion MII‐pH is useful to specify the type of LPR and the related most adequate therapeutic regimen. Nonacid or mixed LPR similarly respond to treatment than acid LPR but require a treatment based on alginate or magaldrate covering the nonacid proximal reflux events.
... All patients underwent proton pump inhibitor treatment since the early postoperative time (rabeprazole, 20 mg once per day), as suggested in the literature. [24][25][26] All patients were trained by the speech therapist in the use and maintenance of the fistula and prosthesis. ...
Article
Objective Periprosthetic leakage represents the most demanding long-term complication in the voice prosthesis rehabilitation. The aim of this article is to discuss the various causes of periprosthetic leakage and to propose a systematic management algorithm. Study Design Retrospective cohort study. Setting Otolaryngology clinic of the University Polyclinic A. Gemelli–IRCCS Foundation. Methods The study included 115 patients with voice prosthesis who were treated from December 2014 to December 2019. All patients who experienced periprosthetic leakage were treated with the same step-by-step therapeutic approach until it was successful. Incidence, management, and success rate of every attempt are analyzed and discussed. Results Periprosthetic leakage was reported 330 times by 82 patients in 1374 clinic accesses. Radiotherapy, timing of tracheoesophageal puncture, and type of total laryngectomy (primary or salvage) did not influence the incidence of periprosthetic leakage. Salvage total laryngectomy increases the risk of more clinically relevant leakages. Conclusion By using a systematic algorithm with a step-by-step standardized approach, periprosthetic leakage management could become a less treacherous issue.
... One possible pathological mechanism by which GERD may affect AH is by the direct irritation of adenoid tissue by stomach contents, including pepsin and bile acids. Human pepsin, unlike animal pepsin, is active even at a pH greater than 4.0, does not undergo irreversible inhibition at a pH lower than 8.0 [3], and contact with it can lead to damage and remodelling of the airway mucosa [4]. However, pepsin was not detected using Western blot analysis and immunocytochemistry in adenoid tissue samples taken during adenoidectomy [5], and the possibility also remains for an impact of Helicobacter pylori transmission from the stomach to the adenoids during bouts of gastro-oesophageal reflux (GERs) [6]. ...
Objectives Recent studies have suggested that the reflux of gastric contents can cause adenoid hypertrophy (AH). The frequency of gastro-oesophageal reflux disease (GERD) in this AH population is unknown, but according to studies using pH-metry it may be as high as 65%. The aim of this study was to estimate the frequency of GERD among children with AH. Methods This was a cross-sectional, multicentre, prospective study of children with AH selected for adenoidectomy. The diagnosis of AH was made by a single laryngologist using a flexible fiberscope. All children had 24-hr multichannel intraluminal pH-impedance (MII/pH) assessment. A GERD diagnosis was made using BioVIEW software analysis after manual review by a single investigator. Results 38 consecutive patients (21 males, mean age 6.58 years) were enrolled in the study. GERD was diagnosed in 5 (13.2%) patients. A total of 1462 gastro-oesophageal reflux events (GERs) were detected by MII/pH and the majority (60.9%) were acidic. The only significant differences between the GERD-positive and GERD-negative groups were the total number of GERs, and the number of acid GERs. Conclusion It is first study using MII/pH to assess the frequency of GERD in children with AH. The data suggest that GERD in children with AH seems to be not as common as it was previously raised. Further studies are needed to confirm these results.
... The inactive pepsin at pH 6.5 can be reactivated when the pH is lowered to 2.0, retaining 70% of its original activity. Pepsin remains stable at pH 7.0 for at least 24 hours at 37 °C, retaining around 79% of its original activity after re-acidification to pH 2 (22). Pepsin as dry powder is stable for 3 years at room temperature and for extended periods of time when stored at -10 °C to -25 °C. ...
... This study explores the direct effects of different reflux substances such as pepsin, bile acid and gastric acid on the laryngeal mucosa and effectively and accurately clarifies the role of LPR to vocal cord polyp. Here, pepsin which the main invasive component of LPR [22] was detected in tissue of patients with VFPs. The positive rate of pepsin is 73.33%, confirming that laryngeal refluxes were widely present in VFPs. ...
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Purpose This study aimed to evaluate if laryngopharyngeal reflux (LPR) plays a role as a risk factor for vocal fold polyps (VFPs), and if pepsin is associated with higher oxidative DNA damage of VFPs in the presence of LPR. Methods Thirty patients with VFPs were recruited between 2017 and 2018. Prior to surgery, a laryngoscopy was performed on all subjects to evaluate VFPs. Polyp tissue and saliva samples were obtained scrupulously. Hematoxylin-eosin staining was performed for pathologic analysis. Immunohistochemistry and ELISA were used to detect pepsin in tissue and saliva of VFP patients. 8-OHdG and p-H2AX expression was detected to measure oxidative DNA damage in tissue. DNA damage was investigated in human immortalized laryngeal epithelial cells exposed to pepsin. Results The pepsin concentration in saliva was significantly higher (t = 2.38, P = .024) in the pepsin positive group. There was no significant difference in pepsin expression at different sites and pathological subtypes of VFPs. The levels of 8-OHdG and p-H2AX were significantly higher in the pepsin positive group and positively correlated with the tissue expression of pepsin. The concentration of pepsin in saliva also showed a significant correlation with 8-OHdG levels. Expression of 8-OHdG and p-H2AX, and tail moment of the comet assay were elevated in human immortalized laryngeal epithelial cells following treatment with pepsin. Conclusion Patients with VFPs have higher levels of oxidative DNA damage in the presence of pepsin reflux. Pepsin may induce DNA damage in laryngeal epithelial cells and participate in the pathogenesis of VFPs.
... This evidence suggests that the local increase of pepsin concentration could affect ocular surface Life 2020, 10, 202 6 of 8 structures and functionality though its proteolytic action and/or inflammatory stimulation. It has been clearly demonstrated that pepsin is active at an acidic pH which can be found at the ocular surface during inflammation; however, in the larynx, pepsin can also be endocytosed and activated in the lysozymes [34]. How this enzyme could damage ocular surface epithelia may only be hypothesized based on the evidence described in other mucosal lesions [35,36]. ...
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Background: patients with laryngopharyngeal reflux (LPR) showed detectable levels of tear pepsin that explain the nasolacrimal obstruction. The purpose of this study was to determine whether patients with LPR show ocular surface changes and to investigate the relationship between lacrimal pepsin concentration and ocular alterations. Methods: Fifty patients with positive endoscopic signs for LPR and an equal or higher score of 13 and 7 for Reflux Symptom Index and Reflux Finding Score were enrolled. Twenty healthy patients with no reflux disease and dry eye were included as the control group. After evaluation of ocular discomfort symptoms, the tear break-up time test, corneal staining, and tear sampling were performed. Tear pepsin levels were measured using Pep-testTM kit. Results: Patients with LPR showed ocular surface changes including epithelial damage (48%) and impairment of lacrimal function (72%). Tear pepsin levels were detectable in 32 out of 50 (64%) patients with LPR (mean ± SD: 55.4 ± 67.5 ng/mL) and in none of the control subjects. Most of the LPR patients complained of ocular discomfort symptoms, including itching (38%), redness (56%), or foreign body sensation (40%). Tear pepsin levels were significantly correlated with the severity of LPR disease and with ocular surface changes. Conclusions: A multidisciplinary approach, including ophthalmological evaluation, should be considered in order to improve the management of patients with LPR.
... 21 Moreover, it has been reported that pepsin can be endocytosed and consequently activated in the lysozymes at the larynx point. 40 The pathogenic mechanisms through which the ocular surface damage occurs could be translated by the evidence documented in other organs, including the lung and larynx. 41,42 In particular, pepsin causes a direct erosion of the mucosa that elicits hyperemia and irritative symptoms. ...
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Background: Dry eye disease (DED) is a common disorder, accounting for up to 35% of the general population. Therefore, we hypothesized that laryngopharyngeal reflux (LPR), inducing refluxate rising into airways, may involve the ocular surface and may either induce or worsen DED. Aim: To investigate the prevalence and relevance of suspected LPR in DED patients and subjects with refractive problems (RP) without DED, they were defined as non-dry eye group (NEG) in clinical practice. Methods: This retrospective study included consecutive patients evaluated because of dry eye-like symptoms at eight tertiary ophthalmological clinics. Parameters included reflux symptom index (RSI), ocular surface disease index (OSDI), symptom assessment in dry eye (SANDE) for frequency and severity, Schirmer test, tear break-up time (BUT), and Oxford grading. Results: The study included 245 subjects (72.5% females; mean age 56.3 years), 152 DED patients, and 93 sex- and age-matched NEG subjects. Pathological RSI (score>13) was detected in 80 subjects (32.6%); 68 (85%) with DED and 12 (15%) CG (OR = 8; p < .0001). In NEG, pathological RSI was associated with higher SANDE (Frequency and Severity), OSDI, and Schirmer scores (OR = 16.36; 14.51; 12.54; and 7.22, respectively. In DED patients, pathological RSI was associated with higher OSDI values (OR = 8.75). Conclusion: Patients with DED are at eight times higher risk for having pathological RSI than NEG patients. Moreover, pathological RSI was associated with more severe ocular symptoms both in DED and non-DED patients. The role of LPR in definite DED patients remains to be clarified, but this condition deserves to be investigated in managing patients with DED symptoms.
... It enters cells through endocytosis and is stored in vesicles or transported to other complex organelles (such as the Golgi apparatus), causing mitochondrial damage and promoting the expression of many tumor-related genes in a cell environment with a low pH [28]. (3) Pepsin reduces the expression of CA III and attenuates the neutralization of CA III on acid [29]. (4) It is involved in the stress response mediated by squamous epithelium stress proteins (Sep), leading to impaired laryngopharyngeal mucosal cell function [30]. ...
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Purpose Laryngopharyngeal reflux disease (LPRD) is a general term for the reflux of gastroduodenal contents into the laryngopharynx, oropharynx and even the nasopharynx, causing a series of symptoms and signs. Currently, little is known regarding the physiopathology of LPRD, and proton pump inhibitors (PPIs) are the drugs of choice for treatment. Although acid reflux plays a critical role in LPRD, PPIs fail to relieve symptoms in up to 40% of patients with LPRD. The influence of other reflux substances on LPRD, including pepsin, bile acid, and trypsin, has received increasing attention. Clarification of the substances involved in LPRD is the basis for LPRD treatment. Methods A review of the effects of acids, pepsin, bile acids, and trypsin on laryngopharyngeal reflux diseases was conducted in PubMed. Results Different reflux substances have different effects on LPRD, which will cause various symptoms, inflammatory diseases and neoplastic diseases of the laryngopharynx. For LPRD caused by different reflux substances, 24-h multichannel intraluminal impedance combined with pH-metry (MII-pH), salivary pepsin, bile acid and other tests should be established so that different drugs and treatment courses can be used to provide patients with more personalized treatment plans. Conclusion This article summarizes the research progress of different reflux substances on the pathogenesis, detection index and treatment of LPRD and lays a theoretical foundation to develop target drugs and clinical diagnosis and treatment.
... Five millilitre of bacterial culture grown at OD 600 = 1 was suspended in 0.5 ml synthetic gastric juice (150 mM of HCl, 15 mM of KCl and 0.5 mg ml À1 of pepsin) (Atkins, 1998), adjusted to pH 2.0 and mixed in a shaker under a rotation speed of 150 rpm min À1 at 37°C for 10 h. Since the enzymatic activity of pepsin is inactive at pH 6.5 or higher (Johnston, et al., 2007), the pH value was readjusted back to 7 and centrifuged to remove the precipitate. The supernatant was saved and served as the extract for testing bactericidal activity. ...
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The lactoferricin expressed in Bacillus subtilis is relatively low in yield, making it hard to apply in industrial settings. We constructed a six tandem repeat of lactoferricin cDNA driven by promoter PtrnQ. After transformation, two transformants P245 and P263 possessing a stable inheritance of plasmid and high expression of lactoferricin were selected. The bactericidal activities, 1 μl of aliquot of a total 5.5 ml of solution extracted from 5 ml of cultured P245 and P263, were equivalent to the efficacy of 238.25 and 322.7 ng of Ampicillin against Escherichia coli, respectively, and 366.4 and 452.52 ng of Ampicillin against Staphylococcus epidermidis respectively. These extracts were able to kill an Ampicillin‐resistant E. coli strain. The bactericidal activities of P245 and P263 equivalent to the efficacy of Tetracycline against Vibrio parahaemolyticus and V. alginolyticus were also determined. Moreover, the bactericidal activities of P245 and P263 were 168.04 and 249.94 ng of Ampicillin against Edwardsiella tarda, respectively, and 219.7 and 252.43 ng of Tetracycline against Streptococcus iniae respectively. Interestingly, the survival rate of E. tarda‐infected tilapia fry fed the P263 extract displayed a significantly greater than that of the fry‐fed control strain. Collectively, these B. subtilis transgenic strains are highly promising for use in animal husbandry during a disease outbreak. In this study, we generated transgenic strains of Bacillus subtilis expressing a high yield of recombinant lactoferricin, an antibacterial peptide against bacteria in a broad spectrum. Enhancement of the antibacterial capacity of these transgenic strains would reduce the cost of application and become highly promising for use in animal husbandry and fish aquaculture industry during disease outbreak.
... We collected the supernatant by 30 min centrifugation at 4000× g. After filtering the supernatant through 0.2 μm bottle filters, we lowered the pH to 2 (optimum pH for pepsin function 25 ), added pepsin to the supernatant at a final concentration of 0.01 mg/mL, and incubated it at 4°C overnight for pepsin to digest the pepsin-sensitive impurities. Next, we concentrated the voluminous crude protein solution by cross-flow filtration. ...
... It was found that the use of a head-over-heels rotation increased the bioaccessibility of sterols, compared with the commonly used orbital agitation at higher speed, due to a greater homogenization of the digestate. Furthermore, as the pepsin added in our trial was lower and its optimal activity is at pH 2 (much closer to the pH of the adult than that of the elderly), 35 at the end of digestion there will still be more protein to digest. This greater amount of protein could increase the bioaccessibility of PSs, as occurred with other lipophilic compounds. ...
Article
Elderly people suffer from a higher cardiovascular risk. Thus, the fortification of foods with plant sterols (PSs), which have a cholesterol-lowering function, could be of great interest for this target group. To date, no studies have analyzed how the gastrointestinal conditions of the elderly affect PS bioaccessibility. Therefore, this study evaluated the impact of the adaptation of the gastric phase alone and in combination with the intestinal phase on sterol bioaccessibility. For this purpose, the standardized INFOGEST 2.0 method previously adapted for sterol bioaccessibility evaluation in healthy adults was applied to PS-enriched milk-based fruit beverages, examining changes in enzyme activity, incubation time, agitation and pH, based on elderly physiology. The results suggest that the specific gastrointestinal conditions of the elderly could increase absorption of PSs, since their bioaccessibility (%) in a PS-enriched milk-based fruit beverage was significantly increased compared with that in adults (14.95 ± 0.33 vs. 7.96 ± 0.26), also indicating that these conditions increase the bioaccessibility of the beverage's own cholesterol (61.25 ± 2.91 vs. 20.86 ± 2.79). These data support the recommendation of foods of this type for the elderly who can benefit from the increase in bioaccessibility of PSs to have an improved potential cholesterol lowering effect, thus decreasing their risk of cardiovascular disease. However, the performance of subsequent in vivo tests to confirm these results is necessary.
... An antibody digestion reaction with pepsin is a time-consuming reaction and, as for other aspartic proteases, pepsin efficiency decreases at neutral pH (Johnston, Dettmar, Bishwokarma, Lively, & Koufman, 2007). Moreover, pepsin proteolytic activity varies for a series of closely related substrates of widely different susceptibility (Fruton, 2002). ...
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Antibodies are widely used in therapeutic, diagnostic, and research applications, and antibody derivatives such as F(ab′)2 fragments are used when only a particular antibody region is required. F(ab′)2 can be produced through antibody engineering, but some applications require F(ab′)2 produced from an original formulated antibody or directly from a polyclonal antibody pool. The cysteine protease immunoglobulin‐degrading enzyme (IdeS) from Streptococcus pyogenes digests immunoglobulin G (IgG) specifically and efficiently to produce F(ab′)2. Here we detail the production and purification of recombinant IdeS; its utilization to digest monoclonal or polyclonal antibodies to F(ab′)2 fragments; and F(ab′)2 purification through consecutive affinity chromatography steps. The resultant F(ab′)2 exhibit high purity, retain antigen‐binding functionality, and are readily utilizable in various downstream applications. © 2020 by John Wiley & Sons, Inc. Basic Protocol: Production and purification of F(ab′)2 fragments from monoclonal and polyclonal antibodies using IdeS Alternate Protocol: Purification of polyclonal antigen–specific F(ab′)2 fragments from human serum or secretions Support Protocol: Production and purification of IdeS
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Intraoperative aspiration is a common pulmonary complication in the surgery, anesthesia and position were main factors leading to the operative aspiration. In recent years, perioperative lung protection has attracted wide attention of thoracic surgeons and anesthetist; how to accelerate the process of postoperative rehabilitation, reduce the incidence of related complications and significantly improve the prognosis of patients, these have become a chief goal of surgical treatment. This article will center on operative aspiration and summarize it from anatomy, pathophysiology, manifestation, diagnosis, treatment and prevention.
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Backgound: Pepsin immunohistochemical (IHC) staining is a promising diagnostic approach of laryngopharyngeal reflux (LPR). The interarytenoid mucosa has been proved to be an effective biopsy area. Objectives: To investigate whether positive result of pepsin IHC staining in laryngeal lesions can predict LPR. Methods: The study included 136 patients with laryngeal cancer or vocal cord leukoplakia. 24 h multichannel intraluminal impedance-pH (MII-pH) was performed before operation, and pepsin IHC staining was performed on pathological sections after operation. The results of the two methods were compared. Results: Among the 136 patients, 101 with at least one LPR event were regarded as MII-pH positive group, and another 35 were negative. The positive rate of pepsin IHC staining was 93.1% in MII-pH positive group and 54.1% in MII-pH negative group (p < .05). If the MII-pH results were used as a reference, the sensitivity and specificity of pepsin IHC staining in the diagnosis of LPR were 93.1 and 45.7%, respectively. The consistency of pepsin IHC and MII-pH was moderate (Kappa value = 0.452). Conclusions: The sensitivity of pepsin IHC staining in laryngeal lesions for diagnosing LPR is satisfactory. The existence of false negative of MII-pH may be the main reason for the low specificity.
Article
Objective: This study explored the impact of laryngopharyngeal reflux (LPR) on quality-of-life outcomes captured by Sino-Nasal Outcome Test (SNOT-22) and Reflux Symptom Index (RSI) in patients with chronic rhinosinusitis (CRS) and patients with symptoms of LPR. Methods: In a retrospective chart review, SNOT-22 and RSI scores were analyzed in patients seen at a tertiary care center with CRS, LPR, or both CRS and LPR. SNOT-22 items were grouped into sleep, nasal, otologic, and emotional symptom subdomains. Results: A total of 138 patients (36 with CRS alone, 60 with LPR alone, and 42 with both CRS and LPR) were included. Compared to patients with CRS alone, those with CRS and LPR (CRS+LPR) had higher SNOT-22 total (50.54 ± 19.53 vs 35.31 ± 20.20, P < .001), sleep (19.61 ± 9.31 vs 14.42 ± 10.34, P < .022), nasal (17.38 ± 7.49 vs 11.11 ± 8.52, P < .001), otologic subdomains (9.17 ± 5.07 vs 5.53 ± 5.14, P < .002), and RSI (22.06 ± 9.42 vs 10.75 ± 8.43, P < .003). Patients with LPR alone had higher RSI compared to those with CRS (18.48 ± 9.77 vs 10.75 ± 8.43, P < .037). RSI and SNOT-22 scores were positively correlated irrespective of patient group (R = 0.289, P = .003). Conclusion: Compared to patients with CRS or LPR alone, those with CRS+LPR demonstrated higher RSI and total and subdomain SNOT-22 scores. Patients with LPR alone had elevated SNOT-22 despite absent endoscopic evidence of sinusitis.
Article
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, 2020
Article
Pepsin, the archetypal pepsin-like aspartic protease, is irreversibly denatured when exposed to neutral pH conditions whereas renin, a structural homologue of pepsin, is fully stable and optimally active in the same conditions despite sharing highly similar enzyme architecture. To gain insight into the structural determinants of differential aspartic protease pH stability, the present study used comparative bioinformatic and structural analyses. In pepsin, an abundance of polar and aspartic acid residues were identified, a common trait with other acid-stable enzymes. Conversely, renin was shown to have increased levels of basic amino acids. In both pepsin and renin, the solvent exposure of these charged groups was high. Having similar overall acidic residue content, the solvent-exposed basic residues may allow for extensive salt bridge formation in renin, whereas in pepsin, these residues are protonated and serve to form stabilizing hydrogen bonds at low pH. Relative differences in structure and sequence in the turn and joint regions of the β-barrel and ψ-loop in both the N- and C-terminal lobes were identified as regions of interest in defining divergent pH stability. Compared to the structural rigidity of renin, pepsin has more instability associated with the N-terminus, specifically the B/C connector. By contrast, renin exhibits greater C-terminal instability in turn and connector regions. Overall, flexibility differences in connector regions, and amino acid composition, particularly in turn and joint regions of the β-barrel and ψ-loops, likely play defining roles in determining pH stability for renin and pepsin.
Article
Objective To measure pepsin expression in patients with vocal fold leukoplakia and elucidate its clinical significance. Study Design Retrospective analysis of pathologic archive specimens. Setting Affiliated university hospital. Subjects and Methods The study included 45 patients with vocal fold leukoplakia and 19 with vocal fold polyps who underwent surgical treatment between December 2013 and July 2016. Masses were detected on both vocal cords in 5 patients with vocal fold leukoplakia and in 1 patient with vocal fold polyps. Immunohistochemistry was used to assess pepsin expression. In addition, the relationship of pepsin expression level with clinical characteristics of vocal fold leukoplakia was assessed. Results The rate of pepsin expression was high in the polyp group (75%) and the leukoplakia group (68%); however, the difference between groups was not significant ( P > .05). Pepsin expression significantly increased according to grade of dysplasia (mild, 57.1%; moderate, 88.9%; severe, 100.0%; P = .034). Similarly, the percentage of lesions that exhibited strongly positive pepsin expression increased with the grade of dysplasia (mild, 37.1%; moderate, 66.7%; severe, 100.0%; P = .005). The leukoplakia recurrence rate was higher in patients with positive pepsin expression than in patients with negative pepsin expression but without a significant difference ( P > .05). Conclusion Our study suggests that pepsin was associated with the grade of dysplasia of vocal cord leukoplakia. Further investigation with appropriate control groups and controlling for other risk factors, such as smoking or alcohol consumption, is needed.
Article
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, 2020
Article
Der gastroösophageale Reflux, auch GERD genannt (gastroesophageal reflux disease) ist mit einer Prävalenz von 20-30% der erwachsenen Bevölkerung ein weit verbreitetes Problem. Davon abzugrenzen ist der häufig in der HNO-Praxis anzutref-fende laryngopharyngeale Reflux (LPR), bei dem es sich um eine durch den Magensäurereflux bedingte Entzündung der oberen Atemwege handelt. Die Unterschiede im klinischen Bild charakterisieren die widersprüchliche Beziehung der beiden klinischen Entitäten und erschweren die Diagnosestellung. Darüber hinaus muss ein besonderes Augenmerk auf Beschwerden des Magen- und Darmtrakts gelegt und deren Behandlung nicht vernachlässigt werden. Neben diätetischen Ratschlägen zählen einige pflanzliche Arzneimittel zu den Therapieoptionen. Alginate, die ein Aufstossen verhindern, bewähren sich in der Praxis. Kombinationsarzneimittel aus der europäischen und tibetischen Tradition spielen eine wesentliche Rolle in der Behandlung, nicht nur des LPR sondern auch bei Verdauungsproblemen des oberen und unteren Verdauungstrakts, wie Reizmagen und Reizdarm. The gastroesophageal reflux, also called GERD (gastroesophageal reflux disease), is a widespread problem with 20–30% of the adult population concerned. The laryngopharyngeal reflux (LPR), which is an inflammation of the upper respiratory system due to reflux of gastric acids, is a frequently encountered problem of the gastrointestinal practitioner and should be separated from GERD. The differences in the clinical image characterize the contradictory relationship of both clinical entities and complicate the diagnosis. In addition, the focus should also be turned on eventual disorders of the gastrointestinal tract and, if necessary, they should be treated, too. Besides dietetic advice some herbal medicines belong to the therapeutic options. Alginates, which prevent reflux, have proved a great value in the practice. Multicomponent mixtures originating from the European and Tibetan herbal medicine traditions play a major role in the treatment, not only for LPR, but also for the upper and lower digestive pathologies such as functional dyspepsia and irritable colon.
Chapter
Laryngopharyngeal reflux (LPR) is associated with modifications of the macro- and microstructure of the mucosa of the human vocal folds, including epithelial cell dehiscence, microtraumas, inflammatory infiltrates, Reinke’s space dryness, mucosal drying, and epithelial thickening. These histopathological changes may lead to modification of the biomechanical properties of the vocal folds, hoarseness, and the impairment of the subjective and objective voice quality evaluations. These mucosal injuries cause increased susceptibility of the vocal fold mucosa to injury and subsequent formation of nodules, polyps, or Reinke’s edema. This chapter summarizes the current knowledge about the association between LPR and the development of benign lesions of the vocal folds.
Chapter
Gastric acid plays a very important role in the digestion of food including the denaturation of proteins in the stomach. It is released from parietal cells through the actions of the vagus nerve and hormones such as gastrin and histamine. The presence of gastric acid in the stomach lumen allows the activation of pepsinogen to pepsin, which is important for protein digestion. Gastric acid also plays a disinfectant role in the stomach; however, excess acid secretion may reflux in the esophagus and laryngopharynx and cause mucosal injury and reflux disease. Recent studies have also described a role for pepsin in reflux disease independent of gastric acid. MII-pH and salivary pepsin data appear to correlate well, suggesting pepsin in saliva may be a noninvasive diagnostic biomarker for proximal airway reflux. Currently, a myriad of pharmacological medications are used for reflux disease management; and recent studies have demonstrated symptomatic benefits with the use of Mediterranean diet. Recent studies have also explored targeting pepsin to treat reflux disease refractory to acid suppression medication. Two mechanisms by which pepsin might be targeted have been identified: irreversible inactivation and via receptor antagonism.
Chapter
This chapter reviews the research demonstrating that a mostly plant-based, Mediterranean-style diet with standard reflux precautions and alkaline water may result in improvement of reflux scores in patients with laryngopharyngeal reflux, as well as the traditional use of proton pump inhibitors and standard reflux precautions.
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.
Article
Purpose of review: This review explores the recent evidence and established scientific literature surrounding proton pump inhibitors in the context of laryngology. Recent findings: Proton pump inhibitors are often associated with gastroenterology; however, they also have a place in laryngology. Several laryngopharyngeal disorders are treated with proton pump inhibitors, though limited evidence regarding effectiveness, dosing and length of treatment exists. With the recent influx of articles reporting possible adverse effects of proton pump inhibitors, the appropriate prescribing of them has come under scrutiny. These reported risks include cancer, stroke, myocardial infarction, kidney disease and cognitive decline. It should be noted though that many of these studies by nature, are fraught with potential confounding. Regardless, clinicians ought to be aware of any risks associated with treatment regimens and prescribe the optimal dosage and duration. Summary: Proton pump inhibitor treatment should be dose-appropriate and for a limited duration. Concerning potential adverse effects, the limitations of retrospective cohort studies must be taken into consideration when reviewing the evidence.
Article
Aerodigestive disorders (AeroDs) in people encompass a wide range of clinical syndromes, reflecting the complex relationship between the respiratory and digestive tracts. In veterinary medicine, aspiration is used interchangeably with aspiration pneumonia. Although aspiration pneumonia is a common disorder in dogs, it does not reflect the breadth of AeroDs. Unfortunately, AeroDs rarely are investigated in veterinary medicine because of lack of clinical recognition, limitations in available diagnostics, and the fact that AeroDs may be caused by occult digestive disease. Recognizing patients with AerodD represents an area of significant clinical importance that may provide additional areas of clinical intervention.
Article
Aim This study explored relationships between enteral feeding and tracheal pepsin A. Background Mechanically ventilated (MV) patients receiving enteral feeding are at risk for microaspiration. Tracheal pepsin A, an enzyme specific to gastric cells, was a proxy for microaspiration of gastric secretions. Methods Secondary analysis of RCT data from critically ill, MV adults was conducted. Microaspiration prevention included elevated head of bed, endotracheal tube cuff pressure management, and regular oral care. Tracheal secretions for pepsin A were collected every 12 h. Microaspiration was defined as pepsin A ≥ 6.25 ng/mL. Positive pepsin A in >30 % of individual tracheal samples was defined as abundant microaspiration (frequent aspirator). Chi-squared, Fisher's Exact test, and generalized linear model (GLM) were used. Results Tracheal pepsin A was present in 111/283 (39 %) mechanically ventilated patients and 48 (17 %) had abundant microaspiration. Enteral feeding was associated with tracheal pepsin A, which occurred within 24 h of enteral feeding. Of the patients who aspirated, the majority received some enteral feeding 96/111 (86 %), compared to only 15/111 (14 %) who received no feeding. A greater number of positive pepsin A events occurred with post-pyloric feeding tube location (55.6 %) vs. gastric (48.6 %), although significant only at the event-level. Frequent aspirators (abundant pepsin A) had higher pepsin A levels compared to infrequent aspirators. Conclusions Our findings confirmed the stomach as the microaspiration source. Contrary to other studies, distal feeding tube location did not mitigate microaspiration. Timing for first positive pepsin A should be studied for possible association with enteral feeding intolerance.
Article
Objective The purpose of this study was to compare the diagnostic utility of pH monitoring using 24-hour esophageal pH-Impedance (HEMII-pH) testing versus pharyngeal pH (Restech) testing (Respiratory Technology Corporation, Houston, Texas) for diagnosing laryngopharyngeal reflux (LPR). Methods Retrospectively, patients were reviewed who had completed a Reflux Symptom Index (RSI) survey and stroboscopy within 60 days before or after undergoing simultaneous esophageal pH-Impedance monitoring and Restech testing. Reflux Finding Score (RFS) was determined by 4 blinded observers. 80.45% of patients were on anti-reflux medications at the time of study and had incomplete response to treatment for reflux. Improvement on reflux treatment was determined by evaluating presenting pre-pH monitoring RFS, post treatment RFS, and improvement of symptoms. Pearson correlation coefficients were calculated to assess relationships among RSI, RFS, and test results from HEMII-pH and Restech tests. Results Eighty-seven patients were included in the analysis. The inter-rater reliability of the RFS determination was 74.57%, and the intra-rater reliability was 67.00%. Subjects who had a positive RYAN Score had a significant correlation with RFS (r of 0.222 and p-value of 0.0492). There was no correlation between RFS and number or percent time of reflux events, longest event, total number of events, or percent of time at alkaline pH for either HEMII-PH or Restech test. RSI correlated better with HEMII-pH test than with Restech for percent time spent in both upright (r of 0.226 and p-value of 0.029) and supine position (r of 0.261 and a p-value of 0.032). Restech correlated better with total patient symptom Scores including cough, heartburn, burping, and throat clearing, with a r of 0.242 and a p-value of 0.048. Restech detected more percent time in reflux for total reflux, supine reflux, and upright reflux (p-value less than 0.0001). Restech also detected longer event times than Impedance (p-value of less than 0.0001). When diagnosis of LPR is based on the definition of CRC, the Sataloff Score test had 70.45% sensitivity and 80.95% specificity. The RYAN Score had a sensitivity of 72%, and a specificity of 56.45%, and the Wu Score had a sensitivity of 62.16%, and specificity of 54.05%. When the Sataloff and Wu Score were used together, the sensitivity was 71.45%, specificity 100%, positive predictive value of 100%, and a negative predictive value of 59.46%. Conclusion The amount of time of reflux events correlates with symptoms better than the number of events. The HEMII-pH test was able to detect more events of pH<4 than Restech, possibly because there might have been more acid events below than above the upper esophageal sphincter, while Restech detected more total events. Restech recorded longer event times than HEMII-pH test. Since length of time correlates with RFS (probably reflecting laryngeal inflammation), and since laryngeal clearance of acid is more similar to pharyngeal than esophageal clearance, this finding might prove valuable clinically. The Sataloff Score has a sensitivity of 70.45%, and a specificity of 80.95% and appears useful clinically to detect mild to moderate that is missed by the RYAN Score. A combination of Sataloff Score and Wu Score may be clinically valuable to identify LPR with an increased sensitivity of 71.45% and increased specificity of 100%. The Wu Score is not yet available for the general clinical use, but the Sataloff Score is.
Article
Objectives To analyze pre to posttreatment voice changes regarding the type of reflux in patients with acid, weakly acid or alkaline laryngopharyngeal reflux (LPR). Methods Patients with LPR, diagnosed using hypopharyngeal-esophageal multichannel intraluminal impedance pH-monitoring (HEMII-pH), were prospectively recruited from three University Hospitals. Patients were treated with a combination of diet, proton pump inhibitors, magaldrate and alginate for 3 months. The following clinical and voice quality outcomes were studied pre to posttreatment according to the type of reflux (acid, weakly acid, nonacid): HEMII-pH, gastrointestinal endoscopy features, reflux symptom score (RSS), reflux sign assessment (RSA), voice handicap index (VHI), perceptual voice assessment (grade of dysphonia and roughness), aerodynamic and acoustic measurements. Results From December 2018 to March 2021, 160 patients completed the evaluations, accounting for 60 acid, 52 weakly acid, and 48 alkaline cases of LPR. There were no baseline differences in clinical and voice quality outcomes between groups. RSS and RSA significantly improved from pre to posttreatment in the entire cohort and in all patient groups. VHI, dysphonia and roughness, maximum phonation time, Jitter, Shimmer and noise to harmonic ratio significantly improved from pre to posttreatment. Individuals with alkaline reflux reported better voice quality improvements as compared to acid and weakly acid reflux patients. Conclusion Patients with acid and alkaline reflux reported better posttreatment voice quality outcomes as compared to weakly acid reflux patients. Future basic science and clinical studies are needed to better understand the histological changes of the vocal folds due to reflux of varying pH types and gastroduodenal enzyme content.
Article
Lactose intolerance in infants can be overcome by adding lactase enzyme formulations to milk, hydrolysing the lactose present. Commonly, such formulations require a significant incubation period after addition to milk to allow time for hydrolysis to take place at desired temperature. Such incubation is often problematic for household conditions, and therefore, formulations with short or, ideally, no incubation period are needed. Development of such formulations requires understanding of the complex process of lactose hydrolysis within the variable temperature and pH conditions in the milk bottle and in the gastrointestinal tract. The current paper describes the application of high-resolution ultrasonic spectroscopy for detailed characterisation of the effect of pH, temperature, and proteases present in the digestive tract on hydrolysis of lactose. The results were employed in the development of a predictive model of lactose hydrolysis within conditions of the infant digestive system. The model was applied for analysis of the whole infant feeding process: from hydrolysis in the bottle, to the stomach and small intestine, and for assessment of the impact of enzyme dosage, the incubation period, and bottle temperature profile on the levels of lactose and galacto-oligosaccharides (produced during hydrolysis), released from the stomach and reaching the large intestine where symptoms of intolerance arise. The results showed that the activity of Ha-Lactase in the stomach and small intestine assists significantly in the reduction of lactose load to 10 % or less of the amount of lactose in the feed, depending on conditions.
Article
We studied rheology and nanostructures (by small angle neutron scattering) of heat-set canola seed protein gels, containing cruciferin and napin, prepared at pH 8 and pH 11. We focused on gastric and intestinal digestion of ten mm pieces, mimicking human gastro-intestinal tract. Stronger gels, prepared at pH 11 (above the IEP, isoelectric points, of both proteins), retained local folded and compact conformations, close to native. Preparation at pH 8 (below napin IEP but above cruciferin one), could destabilize conformations due to charge differences of the proteins. For preparation pH 8, proteins were almost unfolded, and the gel softer. In gastric digestion, modulus decreased for pH 8 gels, but surprisingly, increased for pH 11. We propose a competition between unfolding, increasing local interactions (hence the modulus), and enzymatic scission. Scission could be less efficient for pH 11, with less unfolded proteins and higher crosslink density, hindering enzymatic diffusion. Additional interactions could result from crossing one or two IEPs, towards gastric pH 2. For intestinal digestion, the two gels behave similar (proteins re-compaction and modulus decrease). Beyond loss of submicronic connectivity, external erosion of the gel for largest times is observed, but less on SANS, which involves the centre of the piece.
Chapter
This chapter provides a description of the gastric environment. Gastric fluids consist of numerous components either secreted by the stomach (gastric acid, enzymes, electrolytes, mucus), swallowed/ingested (saliva, food, liquids), or refluxed from the duodenum in the stomach. Gastric content volume can be described as the resultant volume in the stomach of ingested material, swallowed saliva, gastric secretions, and the emptying of gastric content in the small intestine, whereas total gastric volume also takes into account parts of the stomach void of liquid or solid material. Gastric acidity is crucial for several of the stomach's functions. In fed state, buffer capacity of gastric content is expected to initially be similar to that of the ingested meal, given the typically high meal‐to‐gastric fluid ratio. The chapter also presents the composition of the small intestinal contents under fasting and fed conditions and the luminal environment in the proximal colon.
Article
The objective of this study was to investigate the influence of particle size, texture, and gastric viscosity on food gastric disintegration and emptying rates. In vitro dynamic gastric digestion was conducted for carrots using a dynamic gastric simulation model. The changes in the texture of carrots and other foods as indicated by bulk resistance, the percent solids emptied, and the particle size distribution were used as parameters to study disintegration kinetics and emptying patterns. The influence of viscosity on food emptying was studied on both indigestible particles (amberlite beads) and digestible solids (carrots). The results indicated medium‐size carrot particles (1.40–2.00 mm) had a greater disintegration and a higher amount of emptying rate when compared to larger (2.00–3.34 mm) and smaller (1.14–1.40 mm) carrot particles. A high correlation exists between the final bulk resistance after 120 min digestion and the amount of solids emptied. Increasing viscosity up to values 8.20 Pa·s improved the particle dispersion for amberlite and carrots and increased rates of solids emptying, while further increase in viscosity hindered emptying of amberlite and carrot solids. The variable emptying rates of both indigestible and digestible solids with viscosity were described with a mathematical model based on particle‐emptying coefficient. A dynamic in vitro gastric digestion model was used to investigate how food particle size and texture, and gastric viscosity affect food disintegration and emptying. A mathematic model was proposed to predict the change in the emptying rate of solids with viscosity.
Article
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, 2022.
Article
Ethnopharmacological relevance Gastrodiae Rhizoma (GR), a well-known and commonly-used TCM (Traditional Chinese Medicine) for treating headache, dizziness, tetanus, epilepsy, and etc., has been proven to relieve chronic atrophic gastritis (CAG). Due to its complex ingredients, the active fractions responsible for the treatment of CAG remain largely unknown. Aim of the study To explore the underlying material and interpret its underlying mechanism, the therapeutic effect of extract from different polar parts of Gastrodiae Rhizoma on autoimmune CAG was studied based on the ¹H NMR metabolomics. Materials and methods The rat model of CAG was established by autoimmune method. The modeled CAG rats were then treated with 4 polar parts (T1-4 in descending polarity, corresponding to water, n-butanol, ethyl acetate and petroleum ether extracts, respectively) of Gastrodiae Rhizoma for 21 consecutive days. The stomach and serum samples were collected and then subjected to histopathology observation, biochemical measurement (MDA, SOD, GSH, NO, XOD and pepsin), ¹H NMR metabolic profiling and multivariate/univariate statistical analysis. Results The results showed that T1 had the best therapeutic effect, T2 the second, and T3 and T4 the poorest with no obvious therapeutic effect, demonstrating that the effective components of Gastrodiae Rhizoma should be compounds of high polarity. T1 achieved good therapeutic effects due to the anti-inflammatory and anti-oxidant activities, and by rectifying the disturbed energy and amino acid metabolism in CAG model. Conclusion This integrated metabolomics approach proved the validity of the therapeutic effect of extract from different polar parts of Gastrodiae Rhizoma on autoimmune CAG, providing new insights into the underlying mechanisms, and demonstrating the feasibility of metabolomics to evaluate efficacy of herbal drug, which is often difficult by traditional means.
Article
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To develop and validate an analytical procedure for the quantitation of pepsins and gastricsin in human gastric juice and to assess its potential in a controlled gastric secretory study. High performance ion-exchange chromatography was used to separate human pepsin 1, 3a, 3b, 3c and gastricsin from gastric juice. Computed chromatographic areas for each enzyme were quantified by relation to a known amount of a secondary standard porcine pepsin. The assay procedure was validated by recovery and analytical precision studies. Gastric secretions after pentagastrin and insulin stimulation from 10 patients with portal hypertension were used to assess the potential of the analytical procedure. The assay precision varied from 1.5 to 9.0% within batch and 7.5 to 18.1% between batch, with about 100% recoveries of porcine pepsin A from human gastric juice over the assay range 0.025-0.5 mg/ml. A fourfold increase in combined pepsin and gastricsin concentration was observed following pentagastrin and insulin stimulation. The mean percentage content of pepsins 3a, 3b, 3c, and 1 in non-stimulated gastric juice were 4%, 72%, 12% and 1.4%, respectively, and did not change significantly after gastric stimulation. An approximate doubling of the percentage of gastricsin (10% to 20%) relative to the pepsins was observed, however, after both insulin and pentagastrin stimulation. This procedure for quantifying individual human pepsins and gastricsin in gastric juice is simple and reliable. It may be of considerable importance in determining the mechanisms involved in the control and secretion of these digestive enzymes in man, including the effect of anti-ulcer drugs and our understanding of the pathophysiology of peptic ulcer disease.
Article
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This is the second annual report of an international collaborative research group that is examining the cellular impact of laryngopharyngeal reflux (LPR) on laryngeal epithelium. The results of clinical and experimental studies are presented. Carbonic anhydrase (CA), E-cadherin, and MUC gene expression were analyzed in patients with LPR, in controls, and in an in vitro model. In patients with LPR, we found decreased levels of CAIII in vocal fold epithelium and increased levels in posterior commissure epithelium. The experimental studies confirm that laryngeal CAIII is depleted in response to reflux. Also, cell damage does occur well above pH 4.0. In addition, E-cadherin (transmembrane cell surface molecules, which have a key function in epithelial cell adhesion) was not present in 37% of the LPR laryngeal specimens. In conclusion, the laryngeal epithelium lacks defenses comparable to those in esophageal epithelium, and these differences may contribute to the increased susceptibility of laryngeal epithelium to reflux-related injury.
Article
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The objectives of this study were to define the conditions that give rise to a stress protein response in laryngeal epithelium and to investigate whether and how stress protein dysfunction contributes to reflux-related laryngeal disease. Western analysis was used to measure stress protein (squamous epithelial proteins Sep70 and Sep53 and heat shock protein Hsp70) and pepsin levels in esophageal and laryngeal tissue specimens taken from both normal control subjects and patients with pH-documented laryngopharyngeal reflux (LPR) who had documented lesions, some of whom had laryngeal cancer. A porcine organ culture model was used to examine the effects of low pH and pepsin (0.1% porcine pepsin A) on stress protein levels. A laryngeal squamous carcinoma (FaDu) cell line was used to examine uptake of human pepsin 3b-tetramethyl-5 and -6 isothiocyanate. Sep70, Sep53, and Hsp70 were found to be expressed at high levels, and pepsin was not detected, in esophageal and laryngeal specimens taken from normal control subjects and in esophageal specimens taken from LPR patients. The patients with LPR were found to have significantly less laryngeal Sep70 (p = .027) and marginally less laryngeal Sep53 (p = .056) than the normal control subjects. Laryngeal Hsp70 was expressed at high levels in the LPR patients. The patients with laryngeal cancer had significantly lower levels of Sep70, Sep53 (p < .01), and Hsp70 (p < .05) than the normal control subjects. A significant association was found between the presence of pepsin in laryngeal epithelium from LPR patients and depletion of laryngeal Sep70 (p < .001). Using the organ culture model, we demonstrated that laryngeal Sep70 and Sep53 proteins are induced after exposure to low pH. However, in the presence of pepsin, Sep70 and Sep53 levels are depleted. Confocal microscopy analysis of cultured cells exposed to labeled pepsin revealed that uptake is by receptor-mediated endocytosis. These findings suggest that receptor-mediated uptake of pepsin by laryngeal epithelial cells, as may occur in LPR, causes a change in the normal acid-mediated stress protein response. This altered stress protein response may lead to cellular injury and thus play a role in the development of disease.
Article
The human oesophageal epithelium is subject to damage from thermal stresses and low extracellular pH that can play a role in the cancer progression sequence, thus identifying a physiological model system that can be used to determine how stress responses control carcinogenesis. The classic heat shock protein HSP70 is not induced but rather is down-regulated after thermal injury to squamous epithelium ex vivo; this prompted a longer-term study to address the nature of the heat shock response in this cell type. An ex vivo epithelial culture system was subsequently used to identify three major proteins of 78, 70, and 58 kDa, whose steady-state levels are elevated after heat shock. Two of the three heat shock proteins were identified by mass spectrometric sequencing to be the calcium-calmodulin homologue transglutaminase-3 (78 kDa) and a recently cloned oesophageal-specific gene called C1orf10, which encodes a 53-kDa putative calcium binding protein we have named squamous epithelial heat shock protein 53 (SEP53). The 70-kDa heat shock protein (we have named SEP70) was not identifiable by mass spectrometry, but it was purified and studied immunochemically to demonstrate that it is distinct from HSP70 protein. Monoclonal antibodies to SEP70 protein were developed to indicate that: (a) SEP70 is induced by exposure of cultured cells to low pH or glucose starvation, under conditions where HSP70 protein was strikingly down-regulated; and (b) SEP70 protein exhibits variable expression in preneoplastic Barrett's epithelium under conditions where HSP70 protein is not expressed. These results indicate that human oesophageal squamous epithelium exhibits an atypical heat shock protein response, presumably due to the evolutionary adaptation of cells within this organ to survive in an unusual microenvironment exposed to chemical, thermal and acid reflux stresses.
Article
Occult (silent) gastroesophageal reflux disease (GER, GERD) is believed to be an important etiologic factor in the development of many inflammatory and neoplastic disorders of the upper aerodigestive tract. In order to test this hypothesis, a human study and an animal study were performed. The human study consisted primarily of applying a new diagnostic technique (double-probe pH monitoring) to a population of otolaryngology patients with GERD to determine the incidence of overt and occult GERD. The animal study consisted of experiments to evaluate the potential damaging effects of intermittent GER on the larynx.
Article
The hydrolysis of the chromogenic peptide Pro-Thr-Glu-Phe-Phe(4-NO2)-Arg-Leu at the Phe-Phe(4-NO2) bond by nine aspartic proteinases of animal origin and seven enzymes from micro-organisms is described [Phe(4-NO2) is p-nitro-L-phenylalanine]. A further series of six peptides was synthesized in which the residue in the P3 position was systematically varied from hydrophobic to hydrophilic. The Phe-Phe(4-NO2) bond was established as the only peptide bond cleaved, and kinetic constants were obtained for the hydrolysis of these peptide substrates by a representative selection of aspartic proteinases of animal and microbial origin. The value of these water-soluble substrates for structure-function investigations is discussed.
Article
Esophageal epithelium has intrinsic antireflux defenses, including carbonic anhydrases (CAs I to IV) that appear to be protective against gastric reflux. This study aimed to investigate the expression and distribution of CA isoenzymes in laryngeal epithelium. Laryngeal biopsy specimens collected from the vocal fold and interarytenoid regions were analyzed by Western blotting and immunofluorescence. Carbonic anhydrases I and II were expressed by the majority of samples analyzed. In contrast, CA III was differentially expressed in the interarytenoid samples and was not detected in any vocal fold samples. The expression of CA III was increased in esophagitis as compared to normal esophageal tissue. Carbonic anhydrase I and III isoenzymes were distributed cytoplasmically in the basal and lower prickle cell layers. The laryngeal epithelium expresses some CA isoenzymes and has the potential to protect itself against laryngopharyngeal reflux. Laryngeal tissue may be more sensitive to injury due to reflux damage than the esophageal mucosa because of different responses of CA isoenzymes.
Article
Esomeprazole, the S isomer of omeprazole, has been shown to have higher healing rates of erosive esophagitis than omeprazole. This study compared esomeprazole with lansoprazole for the healing of erosive esophagitis and resolution of heartburn. This United States multicenter, randomized, double blind, parallel group trial was performed in 5241 adult patients (intent-to-treat population) with endoscopically documented erosive esophagitis, which was graded by severity at baseline (Los Angeles classification). Patients received 40 mg of esomeprazole (n = 2624) or 30 mg of lansoprazole (n = 2617) once daily before breakfast for up to 8 wk. The primary efficacy endpoint was healing of erosive esophagitis at week 8. Secondary assessments included proportion of patients healed at week 4, resolution of investigator-recorded heartburn, time to first and time to sustained resolution of patient diary-recorded heartburn, and proportion of heartburn-free days and nights. Esomeprazole (40 mg) demonstrated significantly higher healing rates (92.6%, 95% CI = 91.5-93.6%) than lansoprazole (30 mg) (88.8%, 95% CI = 87.5-90.0%) at week 8 (p = 0.0001, life-table estimates, intent-to-treat analysis). A significant difference in healing rates favoring esomeprazole was also observed at week 4. The difference in healing rates between esomeprazole and lansoprazole increased as baseline severity of erosive esophagitis increased. Sustained resolution of heartburn occurred faster and in more patients treated with esomeprazole. Sustained resolution of nocturnal heartburn also occurred faster with esomeprazole. Both treatments were well tolerated. Esomeprazole (40 mg) is more effective than lansoprazole (30 mg) in healing erosive esophagitis and resolving heartburn. Healing rates are consistently high with esomeprazole, irrespective of baseline disease severity.
Article
Frequency occurrence of nonacidic and nonliquid reflux events in the pharynx has not been systematically studied. The aim of the present study was to characterize the physical (liquid, gas, and mixed gas/liquid) and pH properties of the gastroesophagopharyngeal refluxate. We performed a total of 31 24-h simultaneous ambulatory pharyngoesophageal impedance and pH recordings in 11 GERD patients, 10 patients with reflux-attributed laryngitis, and 10 healthy controls. On average, the total number of reflux events (all kinds) in the pharynx was less than half of that in the proximal esophagus (18 +/- 4 vs 50 +/- 4, p < 0.01). Most of the pharyngeal reflux events were gas events and were observed in all three studied groups. Prevalence of these gas reflux events ranged between 0 and 74. The number of gas reflux events accompanied by a minor pH drop in laryngitis patients (1 (0-36)) was significantly higher than those in GERD and controls (0 (0-2) and 0 (0-1), respectively, p < 0.05). There was no significant difference in the number of nonacidic gas reflux events among the three groups (GERD: 10 (2-57), laryngitis: 11.5 (0-51), controls: 10.5 (0-27)). Impedance recording identified a total number of 566 events in the pharynx. Of these, a total of 563 events were compatible with gas reflux events, 101 events were accompanied by minor drops in intrapharyngeal pH, whereas 460 events were not accompanied by any pharyngeal pH change. Concurrent impedance and pH recordings detect significantly more events qualifying as reflux in the pharynx than pH recordings alone. A substantial majority of these events are gaseous refluxes both with and without minor pH drops. Gas reflux events with weak acidity appear to be more common among patients with reflux-attributed laryngeal lesions compared to GERD patients and controls.
Article
Proton pump inhibitors are the mainstay of medical management in gastroesophageal reflux disease. Although they provide relief from most symptoms, reflux may persist. We hypothesize that omeprazole does not reduce the total amount of gastroesophageal reflux but simply alters its pH characteristics. Six asymptomatic volunteers had combined 24-hour impedance pH monitoring before and after 7 days of omeprazole (20 mg BID). Multichannel intraluminal impedance was used to identify reflux episodes, which were classified as acid (pH < 4), weak acid (pH > 4 but decrease > 1 pH unit) and nonacid (pH > 4 and decrease < 1 pH unit) by pH measurements 5 cm above the lower esophageal sphincter (LES). A gastric pH sensor located 10 cm below the LES was used to verify the action of omeprazole. Impedance detected a total of 116 reflux episodes before and 96 episodes after omeprazole treatment. The median number of reflux episodes (18 versus 16, P = 0.4), median duration of reflux episodes (4.7 versus 3.6 minutes, P = 0.5), and total duration of reflux episodes (27.2 versus 42.4 minutes, P = 0.5) per subject were similar before and after omeprazole. Acid reflux episodes were reduced from 63% before to 2.1% after omeprazole (P < 0.0001), whereas nonacid reflux episodes increased (15% to 76%, P < 0.0001). Weak acid reflux episodes did not change (22.4% to 21.8%, P = 1.0). The proportion of reflux episodes greater than pH 4 increased from 37% to 98% (P < 0.0001). In normal subjects, omeprazole treatment does not affect the number of reflux episodes or their duration; rather it converts acid reflux to less acid reflux, thus exposing esophagus to altered gastric juice. These observations may explain the persistence of symptoms and emergence of mucosal injury white on proton pump inhibitor therapy.
Article
The objective was to investigate the potential use of pepsin and carbonic anhydrase isoenzyme III (CA-III) as diagnostic markers for laryngopharyngeal reflux disease. Prospective cell biological investigation was conducted of laryngeal biopsy specimens taken from 9 patients with laryngopharyngeal reflux disease and 12 normal control subjects using antibodies specific for human pepsin (produced in the authors' laboratory within the Department of Otolaryngology at Wake Forest University Health Sciences, Winston-Salem, NC) and CA-III. Laryngeal biopsy specimens were frozen in liquid nitrogen for Western blot analysis and fixed in formalin for pepsin immunohistochemical study. Specimens between two groups (patients with laryngopharyngeal reflux disease and control subjects) were compared for the presence of pepsin. Further analyses investigated the correlation between pepsin, CA-III depletion, and pH testing data. Analysis revealed that the level of pepsin was significantly different between the two groups (P < .001). Secondary analyses demonstrated that presence of pepsin correlated with CA-III depletion in the laryngeal vocal fold and ventricle (P < .001) and with pH testing data in individuals with laryngopharyngeal reflux disease. Pepsin was detected in 8 of 9 patients with laryngopharyngeal reflux disease, but not in normal control subjects (0 of 12). The presence of pepsin was associated with CA-III depletion in the laryngeal vocal fold and ventricle. Given the correlation between laryngopharyngeal reflux disease and CA-III depletion, it is highly plausible that CA-III depletion, as a result of pepsin exposure during laryngopharyngeal reflux, predisposes laryngeal mucosa to reflux-related inflammatory damage.
Article
The significance of gastroesophageal reflux disease (GERD) in laryngeal cancer is controversial due to disparate studies. To evaluate the overall strength of the association of GERD with laryngeal cancer, we performed meta-analysis of the original studies in literature. Meta-analysis. All studies cited on Ovid Medline (1966-June 2004), EMBASE (1980-June 2004), and Cochrane database describing GERD and laryngeal cancer were eligible for inclusion. The inclusion criteria for the study included original controlled study design and a clear documentation of the reflux prevalence in cases and controls. Statistical analysis was performed by NCSS software. Fifteen original studies were identified. Eight studies did not have control groups, two studies did not clearly document GERD prevalence in controls, and two studies were published using the same data, one of which was included in this meta-analysis. Thus, four studies qualified for inclusion for the meta-analysis. The methodologic quality in the studies was heterogeneous, not only in the evaluation of confounding risk factors such as smoking and alcohol but also in the mode of GERD diagnosis. There was also significant heterogeneity of effect of reflux among the studies (P = .001). The pooled odds ratio on the basis of fixed-effects model was 2.86 (95% CI, 2.73-2.99), and on the basis of random-effects model was 2.37 (95% CI 1.38-4.08). Our meta-analysis suggests that GERD may be a significant risk factor for laryngeal cancer. However, given the heterogeneity of the published data, future prospective controlled studies are needed.
Article
Airway symptoms are often caused by aspiration of refluxed materials into the larynx. In this study we sought to define the frequency, character, and proximal extent of refluxed contents - including nonacid reflux-in normal subjects using intraluminal impedance to improve our understanding of the relationship between reflux and aspiration. Ten subjects, who had no symptoms of gastroesophageal reflux disease or airway disease, underwent impedance/pH monitoring with a catheter that allowed simultaneous esophageal and pharyngeal monitoring. Impedance detected 496 gastroesophageal reflux episodes in the 10 subjects during 240 hours of study. The majority, 399 (81% of the total) were acid reflux episodes (pH < 4). Ninety-seven were nonacid (pH > 4). Most reflux episodes (348 of 496) reached the mid esophagus (9 cm above lower esophageal sphincter). There were 51 reflux episodes that reached the pharynx (PR). Only 13 (25%) of PR were acidic (pH < 4), while 38 were nonacid. Twenty-six PR episodes were liquid and 25 were mixed (liquid and gas). The median number of PR episodes measured with impedance was 5 (0-10). In asymptomatic subjects, most episodes of gastroesophageal reflux are acidic and reach the midesophagus. Reflux into the PR appears to be more common than previously believed, and most of these episodes are not acidic. Thus, traditional 24-hour pH monitoring may underestimate the presence of pharyngeal reflux. The combination of impedance with pH monitoring markedly enhances our ability to accurately detect potential microaspiration.
Article
Empiric proton pump inhibitor (PPI) trials have become increasingly popular leading to gastroenterologists frequently evaluating gastro-oesophageal reflux disease (GORD) patients only after they have "failed" PPI therapy. Combined multichannel intraluminal impedance and pH (MII-pH) monitoring has the ability to detect gastro-oesophageal reflux (GOR) episodes independent of their pH and evaluate the relationship between symptoms and all types of GOR. Using this technique, we aimed to characterise the frequency of acid and non-acid reflux (NAR) and their relationship to typical and atypical GOR symptoms in patients on PPI therapy. Patients with persistent GORD symptoms referred to three centres underwent 24 hour combined MII-pH monitoring while taking PPIs at least twice daily. Reflux episodes were detected by impedance channels located 3, 5, 7, 9, 15, and 17 cm above the lower oesophageal sphincter (LOS) and classified into acid or non-acid based on pH data from 5 cm above the LOS. A positive symptom index (SI) was declared if at least half of each specific symptom events were preceded by reflux episodes within five minutes. A total of 168 patients (103 (61%) females and 65 (39%) males; mean age 53 (range 18-85) years) underwent combined MII-pH monitoring while taking PPIs at least twice daily. One hundred and forty four (86%) patients recorded symptoms during the study day and 24 (15%) patients had no symptoms during testing. Sixty nine (48%) symptomatic patients had a positive SI for at least one symptom (16 (11%) with acid reflux and 53 (37%) with NAR) and 75 (52%) had a negative SI. A total of 171 (57%) typical GORD symptoms were recorded, 19 (11%) had a positive SI for acid reflux, 52 (31%) for NAR, and 100 (58%) had a negative SI. One hundred and thirty one (43%) atypical symptoms were recorded, four (3%) had a positive SI for acid reflux, 25 (19%) had a positive SI for NAR, and 102 (78%) had a negative SI. Combined MII-pH identifies the relation of reflux of all types to persistent symptoms and the importance of NAR in patients taking PPIs.