In vitro model to evaluate effect of acidic pepsin on vocal fold barrier function.

The pathophysiology of laryngopharyngeal reflux (LPR) and its impact on the vocal fold is not well understood, but may involve acid damage to vocal fold barrier functions. Two different components encompass vocal fold barrier function: the mucus barrier and tight junctions. Mucus retained on epithelial microprojections protects the inside of the vocal fold by neutralizing acidic damage. Tight junctions control permeability between cells. Here we developed an in vitro experimental system to evaluate acidic injury and repair of vocal fold barrier functions. We first established an in vitro model of rat vocal fold epithelium that could survive at least one week after barrier function maturation. The model enabled repeated evaluation of the course of vocal fold repair processes. Then, an injury experiment was conducted in which vocal fold cells were exposed to a 5-min treatment with acidic pepsin that injured tight junctions and cell surface microprojections. Both of them healed within one day of injury. Comparing vocal fold cells treated with acid alone with cells treated with acidic pepsin showed that acidic pepsin had a stronger effect on intercellular permeability than acid alone, whereas pepsin had little effect on microprojections. This result suggests that the proteolytic action of pepsin has a larger effect on protein-based tight junctions than on phospholipids in microprojections. This experimental system could contribute to a better understanding of vocal fold repair processes after chemical or physical injuries, as well as voice problems due to LPR pathogenesis.

The role of Glut-1 and H+/K+-ATPase expression in hyperplasia of mice laryngeal epithelium induced by pepsin.

PURPOSE: To explore the role played by Glut-1 and H+/K+-ATPase in pepsin-induced, mouse laryngeal epithelial proliferation, growth, and development. METHODS: We established a mouse model of laryngopharyngeal reflux and measured Glut-1 and H+/K+-ATPase expression levels in mouse laryngeal epithelium treated with artificial gastric juice containing pepsin. RESULTS: Artificial pepsin-containing gastric juice induced significant hyperplastic changes in mouse laryngeal epithelium compared to control mice at 15, 30, and 45 days. Inhibition of Glut-1 expression by 2-DG significantly suppressed such hyperplasia compared to mice exposed to artificial gastric juice containing pepsin at 15, 30, and 45 days. After treatment with pepsin-containing artificial gastric juice, RT-PCR and Western blotting showed that the levels of Glut-1 and H+/K+-ATPase α, ß increased significantly. CONCLUSIONS: Pepsin-containing artificial gastric juice promoted mouse laryngeal epithelial hyperplasia associated with abnormal expression of Glut-1 and H+/K+-ATPase α, ß.

Role and mechanism of Glut-1 and H+/K+-ATPase expression in pepsin-induced development of vocal cord leukoplakia.

PURPOSE: We investigated the role of Glut-1 and H+/K+-ATPase expression in pepsin-induced development of human vocal cord leukoplakia cells (HVCLCs). Next, we analyzed the relationship between Glut-1 and H+/K+-ATPase expression with the clinicopathological features of laryngeal carcinoma. METHODS: Glut-1 and H+/K+-ATPase expression levels in HVCLCs were determined after treatment with artificial gastric juice containing pepsin and laryngeal carcinoma tissues. RESULTS: Exposure to pepsin-containing artificial gastric juice significantly enhanced the migration and proliferation of VSCLCs in a time-dependent manner. The apoptotic rate of VSCLCs decreased over time after exposure to pepsin and reached a nadir on day 7 (p < 0.01). With increasing duration of exposure to pepsin, the proportion of VSCLCs in G0/G1 phase decreased and the proportions in the S and G2/M phases significantly increased (p < 0.05). After treatment with pepsin-containing artificial gastric juice, RT-PCR and Western blotting showed that the expression of Glut-1 and H+/K+-ATPase α, ß significantly increased in HVCLCs compared to in the absence of pepsin (p < 0.05). The expression of Glut-1 and H+/K+-ATPase α, ß gradually increased from vocal cord leukoplakia (VLC) to laryngeal carcinoma (p < 0.05). Lentivirus-mediated inhibition of Glut-1 expression in VCL significantly inhibited the cells' migration and proliferation (p < 0.05) but enhanced their apoptosis (p < 0.05). Also, inhibition of Glut-1 expression resulted in an increased proportion of cells in G0/G1 phase and a significantly decreased proportion in G2/M phase (p < 0.05). CONCLUSIONS: Elevated Glut-1 expression may promote the development of VCL by upregulating laryngeal H+/K+-ATPase expression to reactivate absorbed pepsin, thus damaging the laryngeal mucosa.

Inter-Rater Variability of Reflux Finding Score Amongst Otolaryngologists.

BACKGROUND: Laryngopharyngeal reflux (LPR) is an extra-oesophageal variant of gastro-oesophgeal reflux disease. Patients often do not present with the classic reflux symptoms of heartburn or regurgitation. Accurate diagnosis of LPR can be challenging. The reflux finding score (RFS) is system based on the assessment of eight parameters seen on fibre optic laryngoscopy, used to determine the presence and the severity of laryngopharyngeal reflux (LPR). Scoring the RFS is subjective and highly dependent on the examiner's eye and experience. In this study, we investigated the inter-rater reliability between three otolaryngologists scoring a large library of video-recorded laryngoscopies for RFS. AIM: To evaluate the usefulness of RFS in daily clinical practice by assessing inter-rater reliability among otolaryngologists when interpreting a bank of identical fibre optic laryngoscopy examinations. METHOD: Three board-certified otolaryngologists with different subspecialist interests examined video-recorded fibre optic laryngoscopies of 193 patients with or without LPR symptoms and rated each video for RFS. Statistical analysis was performed. Results were compared to determine the inter-rater reliability. RESULTS: Fair to poor correlation was found between the three expert raters for total RFS score, as well as for RFS component items with nonbinary outcomes. For the dichotomous items, the inter-rater reliability was slight to moderate. Inter-rater correlation for determining whether an examination is pathological or nonpathological was fair. CONCLUSION: The RFS alone was not reliable for confirming the diagnosis of LPR, due to low inter-rater reliability and the subjective nature of the scoring system.

Dysphagia and Swallowing Disorders.

Dysphagia, defined as impairment of the swallowing process, is a common symptom and can be a significant source of morbidity and mortality in the general population. This article summarizes the causes of the condition, its prevalence, and the consequences and costs of untreated dysphagia. The aim of this article is to provide a framework for the general internist in assessing, diagnosing, and managing dysphagia in an adult patient. Basic diagnostic screening procedures and techniques for management are emphasized. A basic treatment pathway based on cause is provided for reference.

Aggravation of Reflux Finding Score (RFS) after thyroidectomy.

Laryngopharyngeal reflux (LPR) has been suggested as a possible cause of post-thyroidectomy syndrome. However, the pathophysiology and relationship between thyroidectomy and LPR have not been well investigated. We aimed to evaluate the correlation between thyroidectomy and LPR by assessing changes in LPR-related symptoms and laryngoscopic findings before and after thyroidectomy. Ninety-five patients who underwent thyroidectomy with or without central neck dissection were included. The reflux finding score (RFS) and reflux symptom index (RSI) were investigated one day before surgery and two, four, six, and twelve months after surgery. The RFS scores increased significantly after thyroidectomy and decreased to the preoperative level 12 months after surgery. The RSI scores increased after surgery and decreased gradually by 12 months postoperatively, although it was not statistically significant. The RSI and RFS scores improved with the administration of proton pump inhibitors. In conclusion, LPR-related laryngoscopic findings were exacerbated after uncomplicated thyroidectomy. Further studies using pH-monitoring and esophageal manometry are required to investigate the possible deterioration of LPR itself and the UES pressure after thyroidectomy.

Changes of Laryngeal and Extralaryngeal Symptoms and Findings in Laryngopharyngeal Reflux Patients.

OBJECTIVES/HYPOTHESIS: To assess the evolution of laryngeal and extralaryngeal symptoms and findings of laryngopharyngeal reflux (LPR) throughout a 3-month to 9-month treatment. STUDY DESIGN: Prospective Controlled Study. METHODS: One hundred twenty-seven LPR patients and 123 healthy individuals were enrolled from four European hospitals. Patients were managed with a 3-month personalized treatment considering the LPR characteristics at the impedance-pH monitoring. Regarding the clinical therapeutic response, treatment was adapted for 3 to 6 additional months. Symptoms and findings were assessed throughout the therapeutic course with the Reflux Symptom Score (RSS) and the short version of the Reflux Sign Assessment (sRSA). The relationship between patient and reflux characteristics, symptoms, and findings was assessed. RESULTS: One hundred twenty-one LPR patients completed the study. LPR patients exhibited more laryngeal and extralaryngeal symptoms and findings than healthy individuals. RSS significantly improved from baseline to 6 weeks posttreatment and continued to improve from 3 months to 6 months posttreatment. sRSA significantly improved from baseline to 3 months posttreatment. No further improvement was noted at 6 months posttreatment for pharyngeal and oral findings. Laryngeal findings continued to improve from 3 months to 6 months posttreatment. There was a significant association between patient stress level and RSS (P = .045). At 3 months posttreatment, 28.1% of patients had high or complete response, whereas 47.1% required 6 months or 9 months of treatment. Overall, 24.8% of patients had an LPR chronic course. CONCLUSIONS: Laryngeal and extralaryngeal symptoms and findings significantly improved throughout treatment in LPR patients. The improvement of laryngeal findings was slower. Regarding the low prevalence of some digestive or otolaryngological symptoms, a short version of the RSS could be developed. LEVEL OF EVIDENCE: 3 Laryngoscope, 131:1332-1342, 2021.

Presence of pepsin in laryngeal tissue and saliva in benign and malignant neoplasms.

OBJECTIVES: The current study was performed to determine the presence of pepsin in saliva and laryngeal tissue among participants with benign and malignant laryngeal neoplasms. STUDY DESIGN: Case-control study included three groups of patients with: (1) benign laryngeal neoplasms, (2) malignant laryngeal neoplasms and (3) control subjects without symptoms or signs of laryngopharyngeal reflux (LPR). METHODS: Eighty-one voluntary participants were included into study. They were recruited from a group of patients with histologically proven benign and malignant laryngeal neoplasms and in case of control subjects among patients with nasal septum deformation without symptoms of LPR. Morning saliva samples were collected preoperatively. Tumor biopsies were collected by directoscopy of larynx and the control samples from interarytenoid unit of larynx. All samples were analyzed by Enzyme-Linked Immunosorbent Assay (ELISA) and Immunohistochemistry. RESULTS: Pepsin was found in all samples of saliva and tissue biopsies in groups with malignant and benign neoplasms. The highest concentration of pepsin was found in a group of patients with malignant laryngeal neoplasms. Patients with benign laryngeal neoplasms had lower concentrations and the control subjects presented with the lowest concentration of pepsin measured from their saliva. Differences were not statistically significant. Immunohistochemical (IHC) analysis showed the largest number of high positive samples in the group of malignant lesions. CONCLUSION: These results suggest that pepsin and LPR can contribute to the development of benign and malignant laryngeal neoplasms. Further prospective studies, with far more patients, are necessary to prove the role of pepsin in multifactorial etiology of laryngeal neoplasms.

Is the pepsin immunohistochemical staining of laryngeal lesions an available way for diagnosing laryngopharyngeal reflux.

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.

Reflux finding score and reflux symptom index as potential predictors for proton pump inhibitor response in globus pharyngeus patients: A prospective study.

OBJECTIVE: To evaluate the patient-reported reflux symptom index (RSI) and the doctors-reported Reflux finding score (RFS) as potential predictors for proton pump inhibitor (PPI) response in patients with suspected lower pharyngeal reflux, presenting with globus pharyngeus as their primary complaint. METHODS: The research project was performed at the ENT department of Isala hospital Zwolle, the Netherlands. A before and after design was used for this single institution prospective exploratory study. 101 participants with globus pharyngeus symptoms as a primary complaint were included. All participants were assessed by an otorhinolaryngologist at enrollment and after eight weeks of esomeprazole use. Fiberoptic laryngoscopy was performed to document the RFS, and RSI questionnaires were self-administered by the participants. Our main outcome measurement was the patient- reported therapeutic response evaluation, that differentiated three categories: responders, partial responders and non-responders. For evaluation of the assessment tools, RFS > 7 and RSI > 13 were considered deviant. RESULTS: Among the 101 participants, 43 (42.6%) were responders, 28 (27.7%) partial responders and 30 (29.7%) non-responders. Both baseline RSI > 13 and RFS > 7 were statistically significant associated with treatment response. Also, combined into RSI/RFS baseline categories, a significant overall association between baseline scores and patient-reported treatment response was found. Patients reported success rates for deviant RSI and RFS baseline scores were 76.6% and 96%, respectively. 95.5% of patients with both deviant RSI and RFS baseline scores, reported (partial) treatment response. CONCLUSION: Both together, as well individually, pre-treatment RSI and RFS ratings can help predict treatment response of empirical PPI treatment in patients experiencing globus pharyngeus symptoms.

Optimization of Saliva Collection and Immunochromatographic Detection of Salivary Pepsin for Point-of-Care Testing of Laryngopharyngeal Reflux.

Salivary pepsin is a promising marker for the non-invasive diagnosis of laryngopharyngeal reflux (LPR). For reliable results regarding pepsin in saliva, it is critical to standardize the collection, storage, and pre-processing methods. In this study, we optimized the saliva collection protocols, including storage conditions, i.e., solution, temperature, and time, and the pre-processing filter for pepsin. Moreover, we prepared a simple immunochromatographic strip for the rapid detection of pepsin and evaluated its sensing performance. As a result, we selected a polypropylene (PP) filter as the pre-processing filter for salivary pepsin in low resource settings, such as those where point of care testing (POCT) is conducted. This filter showed a similar efficiency to the centrifuge (standard method). Finally, we detected the pepsin using gold nanoparticles conjugated with monoclonal pepsin antibody. Under optimized conditions, the lower limit of detection for pepsin test strips was determined as 0.01 µg/mL. Furthermore, we successfully detected the salivary pepsin in real saliva samples of LPR patients, which were pre-processed by the PP filter. Therefore, we expect that our saliva collection protocol and pepsin immunochromatographic strip can be utilized as useful tools for a non-invasive diagnosis/screening of LPR in POCT.

Narrow Band Imaging for lingual tonsil hypertrophy and inflammation, in laryngo-pharyngeal reflux disease.

PURPOSE: The objective of this study is to analyse the relationship between the endoscopic signs of laryngo-pharyngeal reflux, the lingual tonsil hypertrophy (LTH) and its inflammation, adopting Narrow Band Imaging (NBI) technology. METHODS: We enrolled, as cases, patients with clinical diagnosis of laryngopharyngeal reflux disease (LPRD), a Reflux Symptom Index (RSI) ≥ 13, and a Reflux Finding Score (RFS) ≥ 7. Controls were enrolled among patients who underwent the same transnasal endoscopy, equipped with NBI, with a RSI < 13 and a RFS < 7. Nasopharynx, Lingual Tonsils (LT), Hypopharynx and Larynx were evaluated by two experienced otolaryngologists, who calculated the Reflux Finding Scale (RFS) score and focused their attention on the base of the tongue, observing its surface with NBI technology. RESULTS: 82 patients with diagnosis of laryngopharyngeal reflux were enrolled as cases. Mean RFS was 11.7 (SD = 2.9). As controls, we enrolled 80 patients. Mean RFS was 2.7 (SD = 1.63). We found that RFS positivity was associated with a significant increase in LT dimension (2.5 vs 1.1, p < 0.001), with a higher LT crypt inflammation grading (1.8 vs 0.09, p < 0.001) and with a higher Roman Cobblestone pattern grading (1.48 vs 0.11, p < 0.001). A significant correlation between the crypt inflammation and the hyperemia subscore of RFS was present (r = 0.696, p < 0.0001), while it was not possible to find a correlation between crypt inflammation and either diffuse laryngeal edema (r = 0.166, p = 0.135) and posterior commissure hypertrophy (r = 0.089, p = 0.427). CONCLUSIONS: NBI allowed us to identify endoscopically the presence of enlarged lingual tonsil, crypt inflammation and superficial mucosal changes, in patients affected by LPRD. LEVEL OF EVIDENCE: 2 (prospective, case-control study).

Smoking Cessation Improves the Symptoms and the Findings of Laryngeal Irritation.

Smoking is known to increase laryngeal inflammation and laryngopharyngeal reflux (LPR), which cause laryngeal irritation. Thus, the aim of this study is to evaluate the changes in the symptoms and the findings of laryngeal irritation in smokers after a smoking cessation period. The reflux symptom index (RSI) and the reflux finding score (RFS) were used for evaluating the symptoms and findings of laryngeal irritation in smokers. Endoscopic examination of the laryngeal structures for RFS and symptom inquiry for RSI were performed at the beginning of the study and after a 2 months of cigarette cessation period. This study was carried out in 24 volunteers (14 female and 10 male), between the ages of 24 and 62 years. When we compared the results of RSI and RFS that were performed before and after the cigarette cession period, we found that there was a significant improvement both in RSI and in RFS (P < .001 and P < .001, respectively). Also, there was significant correlation between the cigarette smoking period and RFS score that was determined at the beginning of the study (P = .006, r = .54). A significant improvement was found both in RSI and in RFS after smoking cessation period, which might be the evidence of improvement in laryngeal irritation possibly caused by inflammation due to smoking and LPR. A significant positive correlation was found between smoking period and RFS, especially with vocal fold edema and posterior commissure hypertrophy.

Reflux finding score is associated with gastroesophageal flap valve status in patients with laryngopharyngeal reflux disease: a retrospective study.

Endoscopic grading of gastroesophageal flap valve (GEFV) is simple and reproducible and offers useful information for reflux activity. To investigate the potential correlation between GEFV grading and reflux finding score (RFS) in patients with laryngopharyngeal reflux disease (LPRD), 225 consecutive Patients with suspected LPRD who underwent both routine upper gastrointestinal endoscopy and laryngoscope were enrolled in our study. Patients with a RFS of more than 7 were diagnosed with LPRD. The GEFV was graded as I through IV according to Hill's classification and was classified into two groups: normal GEFV group (grades I and II) and the abnormal GEFV group (grades III and IV). The percent of GEFV grades I to IV was 39.1%, 39.1%, 12.4%, and 9.3%, respectively. Age was significantly related to an abnormal GEFV (p = 0.002). Gender, BMI, smoke and alcohol were not related to GEFV grade. Fifty-one patients (22.67%) had positive RFS. Reflux finding scores were higher in GEFV grades III and IV than I and II (p < 0.05). Endoscopic grading of GEFV is well correlated with reflux finding score in patients with LPRD. This is a simple and useful technique that provides valuable diagnostic information of LPRD.

Role of Matrix Metalloproteinases 7 in the Pathogenesis of Laryngopharyngeal Reflux: Decreased E-cadherin in Acid exposed Primary Human Pharyngeal Epithelial Cells.

Cleavage of E-cadherin and the resultant weakness in the cell-cell links in the laryngeal epithelium lining is induced by exposure to acidic contents of the refluxate. Herein, we aimed to evaluate the role of matrix metalloproteinases (MMPs) in inducing E-cadherin level changes following acid exposure to the human pharyngeal mucosal cells. E-cadherin levels were inversely correlated with the duration of acid exposure. Treatment with actinonin, a broad MMP inhibitor, inhibited this change. Immunocytochemical staining and transepithelial permeability test revealed that the cell surface staining of E-cadherin decreased and transepithelial permeability increased after acid exposure, which was significantly inhibited by the MMP inhibitor. Among the various MMPs analyzed, the mRNA for MMP-7 in the cellular component was upregulated, and the secretion and enzymatic activity of MMP-7 in the culture media increased with the acid treatment. Consequently, MMP-7 plays a significant role in the degradation of E-cadherin after exposure to a relatively weak acidic condition that would be similar to the physiologic condition that occurs in Laryngopharyngeal reflux disease patients.

Reflux and the Voice: Getting Smarter About Laryngopharyngeal Reflux.

Dysphonia is often blamed on laryngopharyngeal reflux (LPR) in the face of a normal flexible laryngoscopic examination. LPR remains primarily (and unfortunately) a diagnosis of exclusion rather than inclusion in the face of vague throat complaints and laryngeal signs attributed to reflux. LPR remains misdiagnosed and overdiagnosed as the cause of many identical, vague throat symptoms and laryngeal complaints. Despite LPR being commonly implicated as the cause of a myriad of nonspecific pharyngeal symptoms, LPR as a physiologic process is common. Whether or not it is a contributor to a patient's symptoms, especially dysphonia, is the focus of this article.

Perceptual, Aerodynamic, and Acoustic Characteristics of Voice Changes in Patients with Laryngopharyngeal Reflux Disease.

Laryngopharyngeal reflux (LPR) is an inflammatory condition suspected to be associated with dysphonia. In this study, we investigated multidimensional perceptual, aerodynamic, and acoustic voice changes in patients with clinically diagnosed LPR compared to healthy participants. We prospectively included 80 outpatients with Reflux Finding Score (RFS) >7 and Reflux Symptom Index (RSI) >13 from September 2013 to April 2016 and we compared clinical and voice quality assessments of these patients with 80 healthy participants. Statistically significant differences were found between groups with regard to Voice Handicap Index, perceptual voice quality (grades of dysphonia, roughness, strain, breathiness, asthenia, and instability), phonatory quotient, percentage jitter, percentage shimmer, peak-to-peak amplitude variation, standard deviation of fundamental frequency, and noise to harmonic ratio. Granulation score of RFS was found to affect the highest number of acoustic parameters. We did not identify significant correlation between vocal fold edema and objective voice quality measurements. This study supports that patients with LPR have significant deterioration of both subjective and objective voice quality compared to healthy participants.

Association between laryngopharyngeal reflux and benign vocal folds lesions: A systematic review.

OBJECTIVES: To investigate the role of laryngopharyngeal reflux (LPR) in the development of benign lesions of the vocal folds (BLVF). METHODS: PubMed, Cochrane Library, and Scopus were searched by three independent investigators for articles published between January 1990 and November 2018 providing substantial information about the role of LPR in the development of nodules, polyps, cysts, Reinke's edema, and sulcus vocalis. Inclusion, exclusion, diagnostic criteria and clinical outcome evaluation of included studies were analyzed using Preferred Reporting Items for Systematic Reviews and Meta-Analyses criteria. RESULTS: Of the 155 relevant publications, 42 studies were included. Thirty-five were clinical studies and seven were experimental research studying the impact of reflux on vocal fold tissue. Only seven clinical studies utilized objective LPR diagnoses (pH monitoring), suggesting an association between LPR and the development of nodules, polyps, and Reinke's edema. These studies were characterized by a substantial heterogeneity due to discrepancies in inclusion/exclusion criteria, diagnostic methods, and clinical outcome evaluation. The few basic science studies on this topic support that LPR creates an environment that may predispose to BLVF through changes in defense mechanisms of the vocal folds, cell-to-cell dehiscence, inflammatory reaction of the vocal folds, and reaction to phonotrauma. CONCLUSIONS: Caustic mucosal injury from LPR could cause increased susceptibility of the vocal fold mucosa to injury and subsequent formation of nodules, polyps, or Reinke's edema. However, the heterogeneity and the low number of high-quality studies limit the ability to draw definitive conclusions. Future clinical and experimental studies are needed to better identify the role of reflux in development of BLVF. Laryngoscope, 129:E329-E341, 2019.