Association of mRNA Expression Levels of LRP1 and Actin-Binding Proteins with the Development of Laryngeal and Laryngopharyngeal Squamous Cell Carcinoma.

We analyzed the association of the level of mRNA expression of the main endocytosis receptor LRP1 and actin-binding proteins (ezrin, profilin-1, cofilin-1, and adenylyl cyclase-associated protein 1) with the development and metastasis of laryngeal and laryngopharyngeal squamous cell carcinoma. The mRNA expression was evaluated in paired tissue samples using quantitative reverse transcription real-time PCR (RT-qPCR) and SYBR Green reagents. The study included 38 patients with stage T1-4N0-1M0 laryngeal and laryngopharyngeal squamous cell carcinoma and 10 patients with chronic hyperplastic laryngitis or grade II-III epithelial dysplasia. The expression of LRP1 in patients with laryngeal and laryngopharyngeal squamous cell carcinoma depended on the stage of the tumor process. Against the background of low expression of LRP1 mRNA, the relationship between cofilin 1 and profilin 1 expression became stronger (r=0.08; p=0.05) and a correlation between cofilin 1 and esrin expression (r=0.7; p=0.05) appeared. Studies on a larger patient cohort are required to make a definite conclusion on the role of LRP1 in the development of laryngeal and laryngopharyngeal squamous cell carcinoma.

Effects of Smoke Generated by Electrocautery on the Larynx.

OBJECTIVE: The aim of this study was to investigate effects of smoke produced by electrocautery on the laryngeal mucosa. MATERIALS AND METHODS: We used 16 healthy, adult female Wistar albino rats. We divided the rats into two groups. Eight rats were exposed to smoke for 60 min/d for 4 weeks, and eight rats were not exposed to smoke and served as controls. The experimental group was maintained in a plexiglass cabin during exposure to smoke. At the end of 4 weeks, rats were sacrificed under high-dose ketamine anesthesia. Each vocal fold was removed. An expert pathologist blinded to the experimental group evaluated the tissues for the following: epithelial distribution, inflammation, hyperplasia, and metaplasia. Mucosal cellular activities were assessed by immunohistochemical staining for Ki67. Results taken before and after effect were compared statistically. RESULTS: There was a significant difference in the extent of inflammation between the experimental group and the control group. Squamous metaplasia was detected in each group, but the difference was not significant. None of the larynges in either group developed hyperplasia. CONCLUSIONS: We showed increased tissue inflammation due to irritation by the smoke.

Laryngeal mucus hypersecretion is exacerbated after smoking cessation and ameliorated by glucocorticoid administration.

INTRODUCTION: The mechanisms underlying the effects of cigarette smoke and smoking cessation on respiratory secretion, especially in the larynx, remain unclear. OBJECTIVES: The aims of this study were to determine the effects of cigarette smoke and smoking cessation on laryngeal mucus secretion and inflammation, and to investigate the effects of glucocorticoid administration. METHODS: We administered cigarette smoke solution (CSS) to eight-week-old male Sprague Dawley rats for four weeks, then examined laryngeal mucus secretion and inflammatory cytokine expression on days 1, 28 and 90 after smoking cessation. We also investigated the effects of the glucocorticoid triamcinolone acetonide when administered on day 1 after smoking cessation. RESULTS: Exposure to CSS resulted in an increase in laryngeal mucus secretion that was further excacerbated following smoking cessation. This change coincided with an increase in the expression of mRNA for the inflammatory cytokines tumor necrosis factor and interleukin-6, as well as mRNA for MUC5AC, which is involved in mucin production. Triamcinolone suppressed CSS-induced laryngeal mucus hypersecretion and pro-inflammatory cytokine production. CONCLUSION: Cigarette smoke-associated inflammation may contribute to the exacerbated laryngeal mucus hypersecretion that occurs following smoking cessation. The inflammatory response represents a promising target for the treatment of cigarette smoke-associated mucus hypersecretion.

Expression of claudin-3 in the esophagus and larynx of rat reflux model.

OBJECTIVE: The aim of this study was to investigate the association between laryngeal expression of claudin-3 and laryngopharyngeal reflux (LPR) in a rat reflux model. METHODS: Eight Wistar rats were divided into two groups. Four rats underwent total esophageal myectomy to induce reflux, and the remainder underwent a sham operation as a control. All animals were sacrificed 12 weeks after surgery to perform tissue histology and Western blot analysis. RESULTS: Lymphocyte infiltration increased significantly in the study group in both esophageal and laryngeal samples (P=0.001, 0.002, respectively). Both esophageal and laryngeal expressions of claudin-3 were significantly lower in the study group when compared with that in the control group (P=0.045, 0.037, respectively). CONCLUSION: The results of this study suggest that a decrease in claudin-3 could be a sensitive indicator of reflux laryngitis in rats.

Vocal exercise may attenuate acute vocal fold inflammation.

OBJECTIVES/HYPOTHESES: The objective was to assess the utility of selected "resonant voice" (RV) exercises for the reduction of acute vocal fold inflammation. The hypothesis was that relatively large-amplitude, low-impact vocal fold exercises associated with RV would reduce inflammation more than spontaneous speech (SS) and possibly more than voice rest. STUDY DESIGN: The study design was prospective, randomized, and double blind. METHODS: Nine vocally healthy adults underwent a 1-hour vocal loading procedure, followed by randomization to a SS condition, vocal rest condition, or RV exercise condition. Treatments were monitored in clinic for 4 hours and continued extraclinically until the next morning. At baseline (BL), immediately after loading, after the 4-hour in-clinic treatment, and 24 hours post-BL, secretions were suctioned from the vocal folds bilaterally and submitted to enzyme-linked immunosorbent assay to estimate concentrations of key markers of tissue injury and inflammation: interleukin (IL)-1ß, IL-6, IL-8, tumor necrosis factor α, matrix metalloproteinase (MMP)-8, and IL-10. RESULTS: Complete data sets were obtained for three markers--IL-1ß, IL-6, and MMP-8--for one subject in each treatment condition. For these markers, results were poorest at 24-hour follow-up in the SS condition, sharply improved in the voice rest condition, and was the best in the RV condition. Average results for all markers and responsive subjects with normal BL mediator concentrations revealed an almost identical pattern. CONCLUSIONS: Some forms of tissue mobilization may be useful to attenuate acute vocal fold inflammation.

Can bile acids be an etiological factor for laryngeal carcinoma?

PURPOSE: The objective of this study is to show the accumulation of bile acids in laryngeal tissues of laryngeal carcinoma patients. MATERIALS AND METHODS: The present study compared the total bile acid level in the hypopharyngeal tissue, tumor tissue, and blood of 21 primary laryngeal carcinoma patients (study group) to that in the hypopharyngeal tissue and blood of 15 patients with benign laryngeal lesions (control group). RESULTS: The total bile acid level was significantly higher in the tumor and hypopharyngeal tissues of the study group than in the hypopharyngeal tissues of the control group; however, the difference in the blood total bile acid level between the 2 groups was not significant. CONCLUSION: Bile acids in reflux material accumulate in the laryngeal tissue in laryngeal carcinoma patients; therefore, bile acids should be considered a carcinogenic factor in the etiology of laryngeal carcinoma because of their mutagenicity due to DNA breaking, as they cause chronic inflammation due to intracellular accumulation.

[Optimal diagnostic criteria of ENT manifestations of GERD].

BACKGROUND: GERD has a number of extraesophageal manifestations (EEM) such as ENT, pulmonary etc. 24-hours pH monitoring in distal esophagus is widely used to confirm the diagnosis but its cut-off values for the diagnosing of extraesophageal manifestations of GERD (for example ENT) are unknown. AIM: To evaluate the optimal cut-off values for mean pH, time pH < 4 a day in the proximal esophagus and number of high gastroesophageal refluxes (HGR) in regard to presence of extraesophageal manifestations of GERD. METHODS: Ninety one GERD patients (50 men, 41 women, 42.33 +/- 16.1 y.o.) were examined using dual-probe 24-hours pH monitoring. The proximal probe was placed in the upper 1/3 part of esophagus over the upper esophageal sphincter. To confirm the presence of ENT manifestations of GERD all the patients were examined by qualified ENT-specialist; special ENT tests (laryngoscopy, pharyngoscopy with cytology and bacteriology) were performed. Toxic, allergic and infectious etiology of ENT were exclusion criteria. ROC curve analysis was used to evaluate optimal cut-off values of pH-studies. The cut-off values were chosen by the optimal diagnostic sensitivity (DSp)/specificity (DSp) ratio. RESULTS: ENT diseases were found in 59 of all the examined patients (chronic pharyngitis in 79.66% of them). HGR was found in 76.27% of patients in ENT group and in 43.75% of controls (consisted of GERD patients without signs of ENT pathology, n = 32), p = 0.0026. Mean number of HGRs was higher in ENT group compared to controls: (M +/- s) 12.51 +/- 18.56 vs 2.84 +/- 7.11 respectively, p (Mann-Whitney U-test) = 0.0003. Mean pH levels in the proximal esophagus were lower in the ENT group: (M +/- m) 6.32 +/- 0.52 vs 6.58 +/- 0.42, p = 0.011. Mean time pH <4 in the proximal esophagus differed significantly between ENT and GERD patients without ENT: 3.19 +/- 6.76 min in ENT group compared to 2.42 +/- 10.02 min in controls, p = 0.003. The calculated cut-off values for the number of high GER were 2 (DSn 71.19%, DSp 68.75%) or 3 (DSn = 61.02%, DSp = 71.88%); for mean pH in the proximal esophagus--6.3 (DSn = 75%, DSp = 51.47) or 6.4 (DSn = 68.75%, DSp = 58.82%); for time pH < 4--optimal value was 25 sec (DSn = 72.88%, DSp = 68.75%). CONCLUSIONS: Proximal pH monitoring may be useful in diagnosing extraesophageal manifestation of GERD. Optimal cut-off values of number of high GER are 2 to 3, mean pH 6.3-6.4 and time pH < 4 - 25 sec.

[Potentialities of the vegetative resonance test for diagnostics of hyperplastic processes in vocal folds].

The objective of the present study was to evaluate potentialities of the vegetative resonance test (VRT) for the elucidation of metabolic aspects of the inflammatory process in different forms of chronic vocal fold hyperplasty. The proposed diagnostic criteria characterize the inflammatory process in the larynx, specific features of metabolism in patients presenting with catarrhal and oedematopolypous laryngitis, characteristic changes in oedematofibrous and fibrous polyps. The use of VRT allowed diagnostic criteria for precarcinogenic conditions in the larynx to be developed.

Lipid laden macrophage indices and reflux finding score in canine gastroesophageal reflux model.

UNLABELLED: Laryngeal exposure to acid and aspiration of gastric contents may lead to severe respiratory disorders. This study utilizes the canine model of Gastroesophageal reflux (GER) to identify whether lower esophageal dysfunction is associated with upper and lower airway pathology. MATERIALS AND METHODS: Five mongrel dogs underwent GER-creating surgery (partial cardiomyectomy). Laryngeal reflux finding score (RFS), lipid-laden macrophage index (LLMI) and BAL fluid cell differential were obtained before and after surgery. RESULTS: Partial cardiomyectomy in dogs significantly increased the Reflux index (RI) from 0.38 +/- 0.21% to 7.56 +/- 2.89% (P = 0.048), the duration of the longest reflux episode (DLRE) from 1.22 +/- 1.19 min to 66.2 +/- 42.03 min postoperatively (P = 0.049) and the total number of episodes in 24 hr from 2.06 +/- 1.03 to a postoperative value of 19.24 +/- 4.79. There was no statistically significant change in values for RFS, LLMI, and BAL fluid cell differential after the induction of GER. CONCLUSIONS: Acid reflux to the proximal esophagus of this animal model did not cause laryngeal exposure to acid or aspiration of gastric content. The results of this study suggest that presence of GER, secondary to lower esophageal dysfunction is not necessarily associated with upper and lower airway pathology.

Estudo comparativo da concentração salivar do fator de crescimento epidérmico em indivíduos com laringite crônica por doença do refluxo gastroesofágica antes e após o tratamento: resultados preliminares / Salivary egf concentration in adults with reflux chronic laryngitis before and after treatment: preliminary results

Os mecanismos fisiopatológicos do refluxo laringofaríngeo (RLF) são pouco conhecidos. O Fator de Crescimento Epidérmico (EGF) é a proteína de produção salivar com maior ação na regeneração do epitélio da orofaringe e tubo digestivo alto, tendo sido demonstradas deficiências em sua concentração salivar em indivíduos com RLF. OBJETIVO: Comparar a concentração salivar de EGF em um mesmo indivíduo com RLF antes e após o tratamento. MATERIAL E MÉTODO: Neste estudo prospectivo doze indivíduos com DRGE e RLF de moderada intensidade tiveram sua saliva espontânea coletada antes e após o tratamento e controle da doença. A concentração salivar de EGF foi estabelecida através de exame de ELISA (Quantikine ®). RESULTADOS: Onze pacientes eram do sexo feminino e um do sexo masculino, com idade média de 49 anos. A concentração salivar de EGF pré-tratamento foi de 2.867,6pg/mL e a pós-tratamento foi 1.588,5pg/mL, sendo esta diferença estatisticamente significante (p=0,015). DISCUSSÃO E CONCLUSÕES: Apesar de a concentração salivar de EGF ser maior nos indivíduos antes do tratamento, esta não consegue alcançar àquela de uma população normal (estabelecida previamente), o que sugere uma deficiência primária deste importante fator de defesa em indivíduos com RLF.

The Laryngopharyngeal Reflux (LPR) physiopathology is still unknown. The Epidermal Growth Factor (EGF) is a biologically active salivary protein that aids in the rapid regeneration of the oropharyngeal and upper digestive tract mucosas. Salivary deficiency of this protein in patients with LPR has been demonstrated in previous studies. AIM: To compare salivary EGF concentration in patients with LPR before and after treatment. MATERIALS AND METHODS: In this prospective study twelve patients with GERD and moderate LPR were studied. Whole saliva samples were collected before and after treatment and salivary EGF concentration was determined using a commercially available ELISA kit (Quantikine ®). RESULTS: There were eleven females and one male among the patients, the mean age was 49 years. The mean pre-treatment salivary EGF concentration was 2,867.6 pg/mL and the mean post treatment EGF concentration was 1,588.5 pg/mL. This difference was statistically significant (p=0.015). DISCUSSION AND CONCLUSIONS: Although salivary EGF concentrations are higher before LPR treatment, the concentration is still much lower than the mean salivary EGF concentration in normal individuals without LPR, which suggests a primary disorder of this defense factor in individuals with LPR.

Heterotopic gastric mucosa in the cervical esophagus: could this play a role in the pathogenesis of laryngopharyngeal reflux in a subgroup of patients with posterior laryngitis?

OBJECTIVE: Acid secretion produced by a heterotopic gastric mucosal patch (HGMP) in the proximal esophagus, instead of gastric acid, may be responsible for laryngopharyngeal reflux (LPR), passing the upper esophageal sphincter. The aim of this study was to investigate the prevalence of HGMP in the proximal esophagus in patients with posterior laryngitis indicating the presence of LPR in comparison with a control group and to elucidate the possible role of this lesion in the pathogenesis of LPR. MATERIAL AND METHODS: A total of 36 consecutive patients with posterior laryngitis diagnosed on laryngoscopic examination were enrolled in the study. Esophagoscopy and ambulatory 24-h intra-esophageal dual-probe pH monitoring were performed in all patients. During endoscopy, special attention was paid to the proximal part of the esophagus, and the proximal electrode for pH monitoring was placed in this region under endoscopic view. The control group comprised 660 consecutive patients who had undergone upper gastrointestinal endoscopy for the usual indications. When HGMP was found, biopsies were taken for histological confirmation. RESULTS: HGMP was detected in 5 out of 36 patients. One out of five patients with patches was excluded from the study because the histopathology of this patient's patch revealed antral-type mucosa, which is not capable of acid secretion. Thus a total of 35 patients were included in the study, yielding a HGMP prevalence of 11.4% (4/35). Compared with the prevalence of the control group (1.6%), a significant difference was observed (p<0.005). pH monitoring showed that 45.4% of the patients had abnormal proximal acid reflux. All of four HGMP (+) patients with posterior laryngitis revealed significantly higher abnormal proximal reflux compared to the patients without patches (p<0.05). CONCLUSIONS: This first preliminary study may suggest that HGMP in the cervical esophagus could play a role in the pathogenesis of LPR, at least in a minor group of patients with posterior laryngitis, depending on its capability to produce acid in situ, although isolated proximal reflux could not be demonstrated. This finding may need to be supported by further studies with larger patient populations and using acid stimulation tests.

Salivary epidermal growth factor concentration in adults with reflux laryngitis.

OBJECTIVE: The mechanisms involved in the mucosal alterations of laryngopharyngeal reflux (LPR) have not been well established. Reports indicate a decrease in the salivary epidermal growth factor (EGF) of patients with reflux esophagitis, but there are no reports of its behavior in LPR. Our objective was to determine the salivary concentration of EGF in adults with LPR. STUDY DESIGN AND SETTING: Salivary EGF concentration of 26 patients with LPR and 20 healthy controls was determined using a commercially available ELISA kit. Patients with LPR were graded according to endoscopic and laryngoscopic criteria. RESULTS: Salivary EGF concentration was significantly lower in the LPR group when compared with controls (P = 0.002). No correlation between the severity of laryngeal findings or esophagitis and salivary EGF concentration could be determined. CONCLUSIONS: The decreased salivary concentration of EGF in adults with LPR suggests that a deficiency in this polypeptide could be associated to the disease.

Diagnosis and management of chronic laryngitis associated with reflux.

Chronic laryngitis symptoms are commonly seen in otherwise healthy people. This article reviews recent progress in our understanding and effective treatment of chronic laryngitis. Clinical experience and prospective treatment and outcome studies have demonstrated objective evidence of the efficacy of treating patients with chronic laryngitis symptoms with nocturnal antireflux precautions and acid-suppressing medications. The role of pH testing and most common errors in treatment are reviewed.

Prostaglandins in squamous cell carcinoma of the larynx: tumor and peritumor synthesis.

Prostaglandin (PG) E2, 6ketoPGF1 alpha and Thromboxane B2 (TxB2) production by the tumor, peritumor and control tissue were investigated in specimens from patients (n = 11) with squamous cell carcinoma of the larynx, in relation to the extension and infiltration of the neoplasm and to the presence of inflammation, fibrosis and necrosis. In all specimens detectable amounts of 6ketoPGF1+ and TxB2 were found, but the predominant metabolite was PGE2. No differences in the levels of TxB2 and 6ketoPGF1 alpha were observed, but the only patient with lymphnodal involvement showed the lowest levels of 6ketoPGF1 alpha both in tumor and peritumor tissue. Higher amounts (p less than 0.05) of PGE2 were synthesized by peritumor tissues in comparison to control mucosa and tumor tissue independently of the occurrence of reactive infiltration. PGs synthesis did not correlate with inflammation, fibrosis, necrosis or staging of the neoplasm. However the two cases in stage T4 showed PGE2 generation at the highest levels both in neoplastic and perineoplastic tissue. These findings indicate that in squamous cell carcinoma of the larynx an increased production of PGE2 occurs, stemming not only from inflammatory cells but at least in part from neoplastic cells. This suggests that the study of arachidonic acid metabolism may contribute to characterization of the primary cancer and lead to better understanding of the mechanisms of tumor growth and diffusion.