Receptor-mediated uptake of pepsin by laryngeal epithelial cells

OBJECTIVES: Previous data suggest a mechanistic link between exposure to pepsin and cellular changes that lead to laryngopharyngeal disorders. Initial confocal microscopy analysis of pepsin uptake by cultured hypopharyngeal epithelial cells revealed that pepsin may be taken up by a specific process. The objective of this study was to use electron microscopy to confirm the initial confocal findings and to determine whether uptake of pepsin by laryngeal epithelial cells is receptor-mediated. METHODS: Cultured human hypopharyngeal FaDu cells and human laryngeal biopsy specimens, taken from the posterior larynx of "control" patients without symptoms or findings of laryngopharyngeal reflux, were exposed to purified human pepsin 3b with or without transferrin (a marker for receptor-mediated endocytosis) in vitro. Uptake of pepsin was documented by electron microscopy. RESULTS: Pepsin co-localized with transferrin in intracellular vesicles; this finding confirms that pepsin is taken up by laryngeal epithelial cells by receptor-mediated endocytosis. CONCLUSIONS: This is a novel finding that further defines the role and mechanism of pepsin-mediated injury in laryngopharyngeal reflux. The objective of ongoing research is to identify the receptor and investigate potential antagonists as a new therapeutic option for patients with reflux-attributed disease--in particular, those patients who have persistent symptoms despite acid suppression therapy.     

Biomarkers and laryngopharyngeal reflux

Laryngopharyngeal reflux is a controversial but increasingly made diagnosis used in patients with a collection of often non-specific laryngeal symptoms. It is a clinical diagnosis, and its pathophysiology is currently poorly understood. Previous reflux research has focused on injurious agents, acid, pepsin and biomarker expression. Failure of intrinsic defences in the larynx may cause changes in laryngeal epithelia, particularly alterations in carbonic anhydrases and E-cadherin. Carbonic anhydrase III levels vary in the larynx in response to laryngopharyngeal reflux, depending on location. Expression of E-cadherin, a known tumour suppressor, is reduced in the presence of reflux. Mucin expression also varies according to the severity of reflux. Further research is required to define the clinical entity of laryngopharyngeal reflux, and to identify a definitive mechanism for mucosal injury. Understanding this mechanism should allow the development of a comprehensive model, which would enable future diagnostic and therapeutic interventions to be developed.     

Pepsin and carbonic anhydrase isoenzyme III as diagnostic markers for laryngopharyngeal reflux disease

OBJECTIVES/HYPOTHESIS: The objective was to investigate the potential use of pepsin and carbonic anhydrase isoenzyme III (CA-III) as diagnostic markers for laryngopharyngeal reflux disease. STUDY DESIGN: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.     

Functional organ preservation in laryngeal and hypopharyngeal cancer

The principles of open vs. laser microsurgical approaches for partial resections of the larynx are described, oncological as well as functional results discussed and corresponding outcomes following primary radiotherapy are opposed. Over the last decade, the endoscopic partial resection of the larynx has developed to an accepted approach in the treatment of early glottic and supraglottic carcinomas thus leading to a remarkable decline in the use of open surgery. Comparing the various surgical approaches of laryngeal partial resections, the oncological outcome of the patients, as far as survival and organ preservation are concerned, are comparable, whereas functional results of the endoscopic procedures are superior with less morbidity. The surgical procedures put together, are all superior to radiotherapy concerning organ preservation. Transoral laser microsurgery has been used successfully for vocal cord carcinomas with impaired mobility or fixation of the vocal cord, supraglottic carcinomas with infiltration of the pre- and/or paraglottic space as well as for selected hypopharyngeal carcinomas. It has been well documented that laser microsurgery achieves good oncological as well as functional results with reasonable morbidity. However, patients with those tumours have been successfully treated by open partial resections of the larynx at medical centres with appropriate expertise. The initially enthusiastic assessment of study results concerning the efficacy of various protocols of chemoradiation with the intent of organ preservation for laryngeal and hypopharyngeal carcinomas are judged more cautious, today, due to recent reports of rather high rates of late toxicity complications.     

The H+/K+-ATPase (proton) pump is expressed in human laryngeal submucosal glands

OBJECTIVES/HYPOTHESIS: Diagnosis and treatment of gastroesophageal and laryngopharyngeal reflux disease has significantly increased over recent years. The larynx is highly sensitive to the effects of LPRD and is similarly responsive to proton pump inhibitor pharmacotherapy. The hypothesis of the study was that proton pump activity exists in the human larynx and plays a functional role in normal and/or pathological laryngeal tissue. STUDY DESIGN: Pathological investigation. METHODS: Two fresh human cadaveric larynges (one male and one female larynx) were obtained as part of an exempt protocol from the Human Subjects Committee and were formalin fixed and paraffin embedded. Banked human stomach tissue was also obtained for use as comparative positive and negative control specimens. Sections were immunostained with monoclonal antibodies reactive with both alpha and beta subunits of the H+/K+-ATPase (proton) pump. Specimens were reviewed for staining pattern and intensity. RESULTS: Stomach parietal cells (known to produce gastric acid) exhibited strongly positive staining for both the alpha and beta subunits of the proton pump. There was no staining in stomach cells that were not morphologically consistent with the parietal cell. In the human larynx there were strong focal and identical staining patterns in the serous cells and ducts of the minor seromucinous glands by both alpha and beta monoclonals to the proton pump. There was variable staining in the laryngeal epithelium that was thought to be consistent with artifact staining resulting from tissue processing. CONCLUSION: The H+/K+-ATPase (proton) pump is present in serous cells and ducts of submucosal glands in the human larynx. Proton pump inhibitor pharmacotherapy may have a site of action in seromucinous glands of the human larynx, with possible relevance for patients treated for chronic laryngitis with or without laryngopharyngeal reflux disease.     

Laryngopharyngeal sensory deficits in patients with laryngopharyngeal reflux and dysphagia

There are no reliable means of quantifying the edema that results from acid exposure to the posterior larynx in patients with laryngopharyngeal reflux (LPR). However, it is possible to quantify laryngopharyngeal sensitivity in these patients by endoscopic administration of air pulses to the laryngeal mucosa in order to elicit the laryngeal adductor reflex. The purpose of this study was to determine whether patients with LPR have sensory deficits in the laryngopharynx, and whether treatment of these patients with a proton pump inhibitor (PPI) results in resolution of sensory deficits. Flexible endoscopic evaluation of swallowing with sensory testing was prospectively performed in 54 patients with dysphagia without neurologic disease and in 25 healthy controls. The laryngopharyngeal sensory level, posterior laryngeal edema, and LPR were assessed. We defined LPR as passage of food material from the esophageal inlet retrograde into the hypopharynx. Patients with LPR were placed on 3 months of omeprazole or lansoprazole and then retested. Patients without LPR were placed on H2 blockers for 3 months and then retested. In the dysphagia group, 48 of 54 patients (89%) had edema of the posterior larynx, and 42 of 54 (78%) had laryngopharyngeal sensory deficits. We noted LPR in 38 of 54 (70%). In the control group, 1 of 25 subjects (4%) had edema, sensory deficits, and LPR. The differences in incidence of edema, sensory deficits, and LPR between the dysphagia group and the control group were significant (p < .001, chi2 test). Twenty-three patients with LPR placed on a PPI returned for follow-up, with improvement in laryngeal edema in 14 of the 21 (67%) who had pretreatment edema and resolution of sensory deficits in 15 of the 19 (79%) who had pretreatment deficits. In the non-LPR, non-PPI group, 11 of 16 patients returned for follow-up, with improvement in laryngeal edema in none of the 11 and improvement in sensory deficits in 1 of the 11 (9.1%). The differences in improvement in laryngeal edema and sensory deficits between the LPR, PPI group, and the non-LPR, non-PPI group were significant (p < .01, Fisher's exact test). We conclude that patients with dysphagia and edema of the posterior larynx as a result of LPR have sensory deficits in the laryngopharynx. Treatment of these patients with a PPI appears to result in resolution of laryngopharyngeal edema and improvement of sensory deficits, both subjectively and objectively.     

Human papilloma virus and carcinoma of the laryngopharynx

The presence of human papillomavirus (HPV) genomes 6, 11, 16 and 33 in 16 patients with laryngopharyngeal carcinomas was examined by means of a polymerase chain reaction. HPV genomes were detected in 75 per cent of cases. In hypopharyngeal carcinoma, HPV 6 and 11 were present in adjacent tissue but absent from the tumour. HPV 16 and 33 were present in 40 per cent of laryngeal tumours and adjacent tissue. These results suggest that HPV 16 and 33 may be closely associated with the development of some laryngopharyngeal carcinomas.     

Expresión de E-cadherina y β-catenina en carcinomas de células escamosas de laringe e hipofaringe

Introduction and objectivesDysfunction of the E-cadherin/catenin complex is directly related to carcinogenesis and metastases development.The aim of this paper is to investigate the prognostic significance of E-cadherin and β-catenin expression in surgically treated laryngeal and hypopharyngeal squamous cell carcinomas.Material and methodsTumour tissue samples were obtained from 133 consecutive patients with squamous cell carcinomas of the head and neck: 68 of the larynx and 65 hypopharyngeal carcinomas, who underwent surgical treatment in our hospital between 2000 and 2005.E-cadherin and β-catenin expression was analysed by immunohistochemistry, quantifying the percentage of stained cells and the intensity of staining.ResultsE-cadherin and β-catenin expression was evaluable in 59 laryngeal carcinomas and in 58 cases of hypopharyngeal carcinomas.In the laryngeal tumours, a significant association was found between the low expression of membrane β-catenin with T4 tumours and tumour recurrence. In the hypopharynx there was a significant association between positive expression of nuclear β-catenin and poor histological differentiation (P = .02). In the multivariate analysis, only the presence of lymph node metastases was an independent predictive factor of decreased disease-specific survival in laryngeal squamous cell carcinomas.ConclusionsThe expression of E-cadherin and β-catenin does not show prognostic significance in laryngeal and hypopharyngeal squamous cell carcinomas over the TNM classification.     

咽喉反流与声带白斑及喉癌

咽喉反流是一组以胃食管反流为病因而产生的咽喉部病变及其相应的临床症候群.近年来,咽喉反流和喉咽部疾病之间关系日益受到耳鼻咽喉科医师的重视.本文简要综述了咽喉反流的诊断、治疗手段及咽喉反流同声带白斑、喉癌的关系,进一步对声带白斑、喉癌的病因研究.     

nm23基因产物/NDPK在喉癌、下咽癌中的表达及其临床意义

应用免疫组化LSAB方法，研究61例喉癌、下咽癌手术标本中nm23基因产物／NDPK的表达及其意义。结果显示：①NDPK／nm23和肿瘤的临床分期有良好的负对应关系，即随着临床分期的升高，NDPK／nm23表达率下降；②NDPK／nm23和肿瘤的分化程度呈正对应关系，即随着肿瘤分化程度的降低，NDPK／nm23表达下降；③NDPK／nm23的表达在淋巴结转移组明显低于无淋巴结转移组（P＜0．001）     

Activity/stability of human pepsin: implications for reflux attributed laryngeal disease

OBJECTIVES/HYPOTHESIS: 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. STUDY DESIGN: Prospective translational research study. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.     

下咽癌和喉癌的多重癌

对下咽，喉鳞癌患者的多重癌实况进行回顾性调查。方法 对１９８０年－１９９５年１６年间头颈部外科受诊者下咽癌１２５例，喉癌１８４例进行了分析，其中男２７６例，女３３例。结果３０９例患者中７９例发生多重癌。多重癌发生部位共１０１处，其中１７例三重癌，１例四重癌，的多重癌以食管癌和胃癌居多；喉癌，尤以声门型喉癌的多重癌以肺癌为主，声门上型喉癌的口咽，食管和胃多重癌的发生率较声门型喉癌为高。     

Analysis of 2,500 squamous cell carcinoma of the head and neck

The purpose of this paper is to define the prevalence of squamous cell carcinoma of the head and neck as seen at the ENT Department, Hospital de la Santa Creu i Sant Pau, Barcelona, Spain in the last years, studying 2500 cases. Relative frequencies at the various sites, age distribution, male to female ratios, TNM stage are reported. From the results we point out that laryngeal squamous cell carcinomas (1297 cases) were more than 50% of all tumors, followed by oropharygeal carcinomas, oral cavity carcinomas and hypopharyngeal carcinomas. The mean age was 61 years, and patients with nasopharyngeal carcinomas were the youngest. 92% of the patients were males, and the highest rate of males was seen in the larynx and hypopharynx (97% males). Only 9% of the patients were non-smokers and 18% were not alcohol drinkers. Hypopharyngeal squamous cell carcinomas concentrated the highest rate of patients with severe toxic consumption. A significant proportion of earlier tumors was only seen in the larynx and oral cavity (54% and 41% Stages I-II, respectively). On the other hand, hypopharyngeal carcinoma, oropharyngeal carcinoma and nasopharyngeal carcinomas were mainly diagnosed in advanced stages.     

Effect of pepsin on laryngeal stress protein (Sep70, Sep53, and Hsp70) response: role in laryngopharyngeal reflux disease

OBJECTIVES: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.     

Dx-pH监测与胃蛋白酶反流试纸条在咽喉反流诊断中的一致性分析

目的分析胃蛋白酶试纸条、Dx-pH监测及RSI（reflux symptom index）与RFS（reflux finding score）量表在咽喉反流性疾病(LPRD)中的一致性。方法回顾性分析2017年8月—2019年2月就诊于解放军总医院第六医学中心耳鼻咽喉科的110例喉科疾病住院患者的临床资料,记录患者的RSI和RSF两类量表的评分数值、Dx pH监测后的Ryan指数, 胃蛋白酶试纸盒的检测结果,用Kappa分析三者间的相关性。结果110例患者中胃蛋白酶反流检测阳性75例,阴性35例,阳性率68.18%;Dx pH检测阳性率22.73%（25/110）,两者间诊断一致率为41.82%;RSI、RFS量表诊断LPRD阳性率90%（99/110）, 与胃蛋白酶反流检测一致率74.55%;且两者间一致性较高（k=0.324）。以Dx-pH监测为参考标准,胃蛋白酶检测的敏感度为72.00% (18/25)、特异度为32.94% (28/85);以RSI、RFS量表诊断为参考标准,敏感度73.73%,特异度为81.82%, 阳性预测值为97.33%。结论RSI、RSF评分与胃蛋白酶反流试纸条结果间的一致性相较于其他检查更高,胃蛋白酶反流试纸虽然不能成为诊断咽喉反流性疾病的金标准,但是作为一项新的无创诊断技术之一, 可以联合Dx pH监测及RSI、RFS量表广泛应用于临床。     

Correlation of pH probe-measured laryngopharyngeal reflux with symptoms and signs of reflux laryngitis

OBJECTIVES/HYPOTHESIS: Laryngitis secondary to gastric acid reflux is a prevalent, yet incompletely understood, otolaryngological disorder. Further characterization of the relationship between symptoms and signs and reflux severity is needed. STUDY DESIGN: Prospective clinical trial. METHODS: Forty-two consecutive, nonsmoking patients with one or more reflux laryngitis symptoms were recruited to complete a symptom questionnaire, videostrobolaryngoscopy, and 24-hour, dual-sensor pH probe testing. Twenty-nine patients had more than four episodes of laryngopharyngeal reflux, and the remaining 13 served as control subjects. Symptom scores were produced by multiplying the severity by the frequency for the following: hoarseness, throat pain, "lump-in-throat" sensation, throat clearing, cough, excessive phlegm, dysphagia, odynophagia, and heartburn. Endoscopic laryngeal signs included erythema and edema of the vocal folds and arytenoids, and interarytenoid irregularity. RESULTS: Symptom scores varied significantly, with throat clearing being greater than the rest. None of the symptoms, except heartburn, correlated with reflux (laryngopharyngeal and esophageal) severity. Patients with worse laryngopharyngeal reflux were found to have worse esophageal reflux. Endoscopic laryngeal signs were rated as mild, on average, and did not correlate with laryngopharyngeal reflux severity. The number of laryngopharyngeal reflux episodes (per 24 h) ranged from 0 to 40 (mean number, 10.6 episodes). CONCLUSIONS: Throat clearing was the most intense symptom in the present group of patients with proven reflux laryngitis. Dual-sensor pH probe testing could not predict the severity of patient's reflux laryngitis symptoms or signs. Only the heartburn symptom correlated with laryngopharyngeal and esophageal reflux.     

原位翻转胸骨舌骨肌筋膜瓣修复喉部分切除术后缺损的疗效分析

目的探讨原位翻转胸骨舌骨肌筋膜瓣（in situ turnover sternohyoid fasciomuscular flap,ITSF）修复喉部分切除喉部缺损的可行性和效果。方法采用ITSF对因喉癌、喉咽癌实施喉部分切除术所致的喉部分缺损进行修复,观察其重建喉结构和功能的效果。结果共实施197例不同类型的喉部分切除术,同时采用ITSF一期修复喉缺损,重建喉结构,恢复喉功能。术后3例（1.5%）患者发生肌筋膜瓣坏死,其中2例坏死的ITSF自行排除,形成喉狭窄不能拔管,但保留了发音功能,另1例通过清创、抗感染和喉全切除治愈,但丧失了喉功能,其余194例患者ITSF修复手术均获得成功,喉功能得到不同程度恢复。术后初次拔除气管套管173例（89.2%）,21例（10.8%）患者初次未能拔除气管套管。初次拔管后再狭窄18例,经过支撑喉镜下激光手术治愈10例,其余8例未能拔除气管套管。在手术成功的194例患者中,总拔管率为85.1%（165/194）。结论ITSF是一种简便、易行和可靠的修复喉部分缺损的方法。     

Totale Laryngopharyngektomie und Rekonstruktion mit Jejunumsprechsiphon

Advanced laryngeal and hypopharyngeal carcinomas are associated with a poor prognosis and a pronounced loss of quality of life due to impairment of the swallowing and voice function. The fundamental therapeutic challenge is successful tumor control with concomitant rehabilitation of swallowing and voice functions. Further objectives are a low complications rate (fistula, aspiration) and prompt transfer to the adjuvant radio-oncologic therapy. With these factors in mind, the microvascular anastomosed jejunum speech siphon with a biventer rein has proven to be an effective method of reconstruction following extensive circular laryngopharyngeal resections. In this case report, a typical operative and postoperative course is presented, as are the functional results.     

The prevalence of laryngeal pseudosulcus among Japanese patients with laryngopharyngeal reflux related symptoms

Infraglottic edema extending from the anterior commissure to the posterior larynx, called the laryngeal pseudosulcus, may have some value in the diagnosing of laryngopharyngeal reflux (LPR). The purpose of this study is to evaluate the prevalence, sensitivity and specificity of this finding among Japanese patients with LPR-related symptoms. Forty-three patients diagnosed as LPR based on their symptoms and 42 control patients without LPR were enrolled. The presence of pseudosulcus was determined with transnasal fiberoptic laryngoscopy. Thirty-seven of the 43 patients with LPR and 13 of the 42 control patients had evidence of laryngeal pseudosulcus (p<0.001). The sensitivity and specificity of pseudosulcus in the symptom-based diagnosis of LPR are 86 and 69%, respectively. This study shows that laryngeal pseudosulcus is highly correlated with LPR-related symptoms. The presence of this finding is suggestive of LPR.