Adiponectin receptor agonist AdipoRon induces apoptotic cell death and suppresses proliferation in human ovarian cancer cells.

We tested the hypothesis that stimulation of adiponectin receptors with the synthetic agonist AdipoRon suppresses proliferation and induces apoptotic death in human high grade serous ovarian tumor cell lines and in ex vivo primary tumors, mediated by activation of 5' AMP-activated protein kinase (AMPK) and inhibition of mechanistic target of rapamycin (mTOR). We determined the effect of AdipoRon on high grade serous ovarian tumor cells lines (OVCAR3, OVCAR4, A2780) and ex vivo primary tumor tissue. Western blotting analysis was performed to examine changes in activation of AMPK and mTOR signaling and flow cytometry was utilized to examine changes in cell cycle progression. Immunofluorescence of cleaved caspase-3 positive cells and flow cytometry of annexin V positive cells were used to determine changes in apoptotic response. The CyQUANT proliferation assay was used to assess cell proliferation. AdipoRon treatment increased AMPK phosphorylation (OVCAR3 P = 0.01; A2780 P = 0.02) but did not significantly alter mTOR activity. AdipoRon induced G1 cell cycle arrest in OVCAR3 (+ 12.1%, P = 0.03) and A2780 (+ 12.0%, P = 0.002) cells. OVCAR3 and OVCAR4 cells treated with AdipoRon underwent apoptosis based on cleaved caspase-3 and annexin V staining. AdipoRon treatment resulted in a dose dependent decrease in cell number versus vehicle treatment in OVCAR3 (-61.2%, P < 0.001), OVCAR4 (-79%, P < 0.001), and A2780 (-56.9%, P < 0.001). Ex vivo culture of primary tumors treated with AdipoRon resulted in an increase in apoptosis measured with cleaved caspase-3 immunohistochemistry. AdipoRon induces activation of AMPK and exhibits an anti-tumor effect in ovarian cancer cell lines and primary tumor via a mTOR-independent pathway.

Ovarian Tumor Cell Expression of Claudin-4 Reduces Apoptotic Response to Paclitaxel.

A significant factor contributing to poor survival rates for patients with ovarian cancer is the insensitivity of tumors to standard-of-care chemotherapy. In this study, we investigated the effect of claudin-4 expression on ovarian tumor cell apoptotic response to cisplatin and paclitaxel. We manipulated claudin-4 gene expression by silencing expression [short hairpin RNA (shRNA)] in cells with endogenously expressed claudin-4 or overexpressing claudin-4 in cells that natively do not express claudin-4. In addition, we inhibited claudin-4 activity with a claudin mimic peptide (CMP). We monitored apoptotic response by caspase-3 and Annexin V binding. We examined proliferation rate by counting the cell number over time as well as measuring the number of mitotic cells. Proximity ligation assays, immunoprecipitation (IP), and immunofluorescence were performed to examine interactions of claudin-4. Western blot analysis of tubulin in cell fractions was used to determine the changes in tubulin polymerization with changes in claudin-4 expression. Results show that claudin-4 expression reduced epithelial ovarian cancer (EOC) cell apoptotic response to paclitaxel. EOCs without claudin-4 proliferated more slowly with enhanced mitotic arrest compared with the cells expressing claudin-4. Furthermore, our results indicate that claudin-4 interacts with tubulin, having a profound effect on the structure and polymerization of the microtubule network. In conclusion, we demonstrate that claudin-4 reduces the ovarian tumor cell response to microtubule-targeting paclitaxel and disrupting claudin-4 with CMP can restore apoptotic response. IMPLICATIONS: These results suggest that claudin-4 expression may provide a biomarker for paclitaxel response and can be a target for new therapeutic strategies to improve response.

A tRNA fragment, tRF5-Glu, regulates BCAR3 expression and proliferation in ovarian cancer cells.

Ovarian cancer is a complex disease marked by tumor heterogeneity, which contributes to difficulties in diagnosis and treatment. New molecular targets and better molecular profiles defining subsets of patients are needed. tRNA fragments (tRFs) offer a recently identified group of noncoding RNAs that are often as abundant as microRNAs in cancer cells. Initially their presence in deep sequencing data sets was attributed to the breakdown of mature tRNAs, however, it is now clear that they are actively generated and function in multiple regulatory events. One such tRF, a 5' fragment of tRNA-Glu-CTC (tRF5-Glu), is processed from the mature tRNA-Glu and is shown in this study to be expressed in ovarian cancer cells. We confirmed that tRF5-Glu binds directly to a site in the 3'UTR of the Breast Cancer Anti-Estrogen Resistance 3 (BCAR3) mRNA thereby down regulating its expression. BCAR3 has not previously been studied in ovarian cancer cells and our studies demonstrate that inhibiting BCAR3 expression suppresses ovarian cancer cell proliferation. Furthermore, mimics of tRF5-Glu were found to inhibit proliferation of ovarian cancer cells. In summary, BCAR3 and tRF5-Glu contribute to the complex tumor heterogeneity of ovarian cancer cells and may provide new targets for therapeutic intervention.

A method for extracting and characterizing RNA from urine: For downstream PCR and RNAseq analysis.

Readily accessible samples such as urine or blood are seemingly ideal for differentiating and stratifying patients; however, it has proven a daunting task to identify reliable biomarkers in such samples. Noncoding RNA holds great promise as a source of biomarkers distinguishing physiologic wellbeing or illness. Current methods to isolate and characterize RNA molecules in urine are limited. In this proof of concept study, we present a method to extract and identify small noncoding RNAs in urine. Initially, quantitative reverse transcription PCR was applied to confirm the presence of microRNAs in total RNA extracted from urine. Once the presence of micro RNA in urine was confirmed, we developed a method to scale up RNA extraction to provide adequate amounts of RNA for next generation sequence analysis. The method described in this study is applicable to detecting a broad range of small noncoding RNAs in urine; thus, they have wide applicability for health and disease analyses.

Claudin-4 activity in ovarian tumor cell apoptosis resistance and migration.

BACKGROUND: Claudin-4 is a transmembrane protein expressed at high levels in the majority of epithelial ovarian tumors, irrespective of subtype, and has been associated with tumor cells that are both chemoresistant and highly mobile. The objective of this study was to determine the functional role that claudin-4 plays in apoptosis resistance and migration as well as the therapeutic utility of targeting claudin-4 activity with a small mimic peptide. METHODS: We examined claudin-4 activity in human ovarian tumor cell lines (SKOV3, OVCAR3, PEO4) using in vitro caspase and scratch assays as well as an in vivo mouse model of ovarian cancer. Claudin-4 activity was disrupted by treating cells with a small peptide that mimics the DFYNP sequence in the second extracellular loop of claudin-4. Claudin-4 expression was also altered using shRNA-mediated gene silencing. RESULTS: Both the disruption of claudin-4 activity and the loss of claudin-4 expression significantly increased tumor cell caspase-3 activation (4 to 10-fold, respectively) in response to the apoptotic inducer staurosporine and reduced tumor cell migration by 50 %. The mimic peptide had no effect on cells that lacked claudin-4 expression. Female athymic nude mice bearing ZsGreen-PEO4 ovarian tumors showed a significant decrease in ovarian tumor burden, due to increased apoptosis, after treatment with intraperitoneal injections of 4 mg/kg mimic peptide every 48 h for three weeks, compared to control peptide treated mice. CONCLUSION: Claudin-4 functionally contributes to both ovarian tumor cell apoptosis resistance and migration and targeting extracellular loop interactions of claudin-4 may have therapeutic implications for reducing ovarian tumor burden.

Cricothyroid Muscle Botulinum Toxin Injection to Improve Airway for Bilateral Recurrent Laryngeal Nerve Paralysis, A Case Series.

Bilateral vocal fold paralysis most commonly results from iatrogenic trauma to the recurrent laryngeal nerve during surgical procedures in the anterior neck. Patients may require tracheostomy because of acute or gradual onset of dyspnea and airway compromise. The intralaryngeal injection of Botox has been considered as a possible therapy for these airway symptoms of bilateral vocal fold paralysis. Chronic unopposed activity of intact cricothyroid muscles could potentially result in gradual medialization of the vocal folds in patients with bilateral recurrent laryngeal nerve paralysis. This case series describes three patients who successfully underwent injections of botulinum toxin into the bilateral cricothyroid muscles to offer sustained relief of dyspnea resulting from bilateral vocal fold paralysis.

Regulation of trachebronchial tissue-specific stem cell pool size.

Tissue-specific stem cell (TSC) number is tightly regulated in normal individuals but can change following severe injury. We previously showed that tracheobronchial epithelial TSC number increased after severe naphthalene (NA) injury and then returned to normal. This study focused on the fate of the supernumerary TSC and the signals that regulate TSC pool size. We used the Keratin 5-rTA/Histone 2B:green fluorescent protein (GFP) model to purify basal cells that proliferated infrequently (GFP(bright) ) or frequently (GFP(dim) ) after NA injury. Both populations contained TSC but TSCs were 8.5-fold more abundant in the GFP(bright) population. Interestingly, both populations also contained a unipotential basal progenitor (UPB), a mitotic basal cell subtype whose daughters were terminally differentiated basal cells. The ratio of TSC to UPB was 5:1 in the GFP(bright) population and 1:5 in the GFP(dim) population. These data suggested that TSC proliferation in vivo promoted TSC-to-UPB differentiation. To evaluate this question, we cloned TSC from the GFP(bright) and GFP(dim) populations and passaged the clones seven times. We found that TSC number decreased and UPB number increased at each passage. Reciprocal changes in TSC and UPB frequency were more dramatic in the GFP(dim) lineage. Gene expression analysis showed that ß-catenin and Notch pathway genes were differentially expressed in freshly isolated TSC derived from GFP(bright) and GFP(dim) populations. We conclude that (a) TSC and UPB are members of a single lineage; (b) TSC proliferation in vivo or in vitro promotes TSC-to-UPB differentiation; and (c) an interaction between the ß-catenin and Notch pathways regulates the TSC-to-UPB differentiation process.

Roles for ß-catenin and doxycycline in the regulation of respiratory epithelial cell frequency and function.

The expression of ß-catenin-dependent genes can be increased through the Cre recombinase (Cre)-mediated elimination of the exon 3-encoded sequence. This mutant ß-catenin is termed DE3, and promotes the expression of ß-catenin-dependent genes. Our previous study used the DE3 model to demonstrate that persistent ß-catenin activity inhibited bronchiolar Clara-to-ciliated cell differentiation. The present study was designed to evaluate the roles of ß-catenin in regulating the tracheal progenitor cell hierarchy. However, initial experiments demonstrated that the tetracycline-responsive element-Cre transgene (TRE-Cre) was active in the absence of a reverse tetracycline transactivator driver or inducer, doxycycline (Dox). This spurious TRE-Cre transgene activity was not detected using the ROSA26-floxed STOP-LacZ reporter. To determine if the phenotype was a consequence of genotype or treatment with Dox, tracheal and lung specimens were evaluated using quantitative histomorphometric techniques. Analyses of uninduced mice demonstrated a significant effect of genotype on tracheal epithelial cell mass, involving basal, Clara-like cell types. The bronchial and bronchiolar Clara cell mass was also decreased. Paradoxically, an effect on ciliated cell mass was not detected. Activation of the ß-catenin reporter transgene TOPGal demonstrated that ß-catenin-dependent gene expression led to the genotype-dependent tracheal and bronchiolar phenotype. Comparative analyses of wild-type or keratin 14-rtTA(+/0)/TRE-cre(+/0)/DE3(+/+) mice receiving standard or Dox chow demonstrated an effect of treatment with Dox on basal, Clara-like, and Clara cell masses. We discuss these results in terms of cautionary notes and with regard to alterations of progenitor cell hierarchies in response to low-level injury.

ß-catenin dosage is a critical determinant of tracheal basal cell fate determination.

The purpose of this study was to determine whether ß-catenin regulates basal cell fate determination in the mouse trachea. Analysis of TOPGal transgene reporter activity and Wnt/ß-catenin pathway gene expression suggested a role for ß-catenin in basal cell proliferation and differentiation after naphthalene-mediated Clara-like and ciliated cell depletion. However, these basal cell activities occurred simultaneously, limiting precise determination of the role(s) played by ß-catenin. This issue was overcome by analysis of ß-catenin signaling in tracheal air-liquid interface cultures. The cultures could be divided into two phases: basal cell proliferation and basal cell differentiation. A role for ß-catenin in basal cell proliferation was indicated by activation of the TOPGal transgene on proliferation days 3 to 5 and by transient expression of Myc (alias c-myc). Another peak of TOPGal transgene activity was detected on differentiation days 2 to 10 and was associated with the expression of Axin 2. These results suggest a role for ß-catenin in basal to ciliated and basal to Clara-like cell differentiation. Genetic stabilization of ß-catenin in basal cells shortened the period of basal cell proliferation but had a minor effect on this process. Persistent ß-catenin signaling regulated basal cell fate by driving the generation of ciliated cells and preventing the production of Clara-like cells.

Context-dependent differentiation of multipotential keratin 14-expressing tracheal basal cells.

Multipotential (MP) differentiation is one characteristic of a tissue-specific stem cell (TSC). Lineage tracing of tracheobronchial basal cells after naphthalene (NA) injury or in the postnatal period demonstrated that basal cells were MP progenitors for Clara-like and ciliated cells. These studies, as well as reports of spatially restricted, label-retaining basal cells, and MP differentiation by human bronchial cells support the hypothesis that a TSC maintained and repaired the tracheobronchial epithelium. However, differences in basal cell phenotype (keratin [K] 5+ versus K14+), age (postnatal versus adult), health status (normal versus injured), and injury type (acid, detergent, NA) limited comparisons among studies and thus diminished the strength of the TSC argument. The finding that K14 was up-regulated after NA injury was a caveat to our previous analysis of reparative (r)K14-expressing cells (EC). Thus, the present study lineage traced steady-state (s)K14EC and evaluated differentiation potential in the normal and repairing epithelium. We showed that sK14EC were unipotential in the normal epithelium and MP after NA, sK14EC-dervied clones were not restricted to putative TSC niches, sK14EC cells were a direct progenitor for Clara-like and ciliated cells, MP-sK14EC clones accumulated over time, and sK14EC-derived Clara-like cells were progenitors for ciliated cells.

Ansa cervicalis-to-recurrent laryngeal nerve anastomosis for unilateral vocal fold paralysis: experience of a single institution.

OBJECTIVES: One treatment option for unilateral vocal fold paralysis (UVFP) is ansa cervicalis-to-recurrent laryngeal nerve (ansa-RLN) anastomosis to provide reinnervation to the affected vocal fold. The advantages of this treatment approach are that it 1) provides vocal fold tone, bulk, and tension, 2) is technically simple, and 3) does not preclude other medialization procedures. We present all patients who have undergone ansa-RLN anastomosis for UVFP at our institution. METHODS: An Institutional Review Board-approved retrospective chart review was performed to include all patients who had undergone an ansa-RLN anastomosis procedure for UVFP at our institution. Data from clinical and endoscopic laryngoscopy with stroboscopy were recorded. Statistical analysis was performed on visual and perceptual vocal data. RESULTS: A total of 46 patients were included in the study. Stroboscopic analysis and perceptual vocal evaluation was performed in a blinded fashion on the 21 patients who had preoperative and postoperative stroboscopy. Severity, roughness, breathiness, and strain all improved significantly over time. Glottic closure, vocal fold edge, and supraglottic effort all significantly improved after operation. Of the 38 patients with at least 3 months of follow-up, all except 1 demonstrated evidence of reinnervation. CONCLUSIONS: This technique for treating UVFP results in significant improvements in patients' voice and on visual examination.

Results of ansa to recurrent laryngeal nerve reinnervation.

OBJECTIVE: We sought to describe the results of ansa cervicalis to recurrent laryngeal nerve (ansa-RLN) reinnervation for unilateral vocal fold paralysis. STUDY DESIGN: A chart review was performed on patients undergoing ansa-RLN reinnervation for unilateral vocal cord paralysis at a tertiary care center. Patient perceptions of preoperative and postoperative voice quality was surveyed. Acoustic and visual parameters were assessed from videostroboscopy. RESULTS: From a total of 25 study patients, 15 patients underwent both preoperative and postoperativ video stroboscopies. In stroboscopies within 6 months, the average improvement in overall severity, roughness, and breathiness was 69, 79, and 100 percent, respectively. In stroboscopies after 6 months, the average improvement in overall severity, roughness, and breathiness was 63, 66, and 100 percent, respectively. Postoperatively, all patients had reinnervation of the vocal fold. CONCLUSIONS: Voice outcomes were improved in patients with preoperative and postoperative stroboscopies. SIGNIFICANCE: Ansa-RLN reinnervation should be considered as a treatment for unilateral vocal fold paralysis.

Vocal outcomes after laser resection of early-stage glottic cancer with adjuvant cryotherapy.

OBJECTIVE: To evaluate the vocal outcomes of patients with early-stage glottic carcinoma undergoing laser resection with adjuvant cryoablative therapy. DESIGN: Retrospective review. SETTING: Tertiary care center. Patients Twenty patients with early-stage glottic carcinoma. Intervention Treatment of early-stage glottic carcinoma with endoscopic carbon dioxide laser resection in conjunction with cryoablation. MAIN OUTCOME MEASURES: Disease-free survival and subjective and objective measures of posttreatment voice quality, based on serial videolaryngostroboscopy. RESULTS: There was 1 local treatment failure, with an overall mean disease-free follow-up of 32.6 months (range, 3-93 months). Carbon dioxide laser resection and cryoablative therapy were associated with a significant improvement in subjective voice quality (P<.001). Long-term dysphonia was uniformly improved vis-à-vis the pretreatment condition, even among patients with the most advanced disease undergoing the widest resections. Posttreatment web formation was not noted among 4 patients with anterior commissure involvement. CONCLUSIONS: Endoscopic laser laryngeal surgery performed in conjunction with cryotherapy for early-stage glottic carcinoma yielded excellent primary site control, while improving subjective and objective measures of voice quality. Combined laser surgery and cryotherapy is a possible alternative to radiotherapy for selected patients with early-stage glottic carcinoma who desire curative therapy, while optimizing vocal outcomes.

Surgical fundoplication in laryngopharyngeal reflux unresponsive to aggressive acid suppression: a controlled study.

BACKGROUND & AIMS: In patients with persistent laryngeal symptoms despite aggressive proton pump inhibitor therapy, gastroesophageal reflux disease (GERD) continues to be implicated. The role of surgical fundoplication as the definitive therapy for these patients is uncertain. METHODS: In this prospective concurrent controlled study, 72 patients with suspected GERD-related laryngeal symptoms received aggressive acid-suppressive therapy. Four-month symptomatic nonresponders (<50% improvement) with continued laryngeal inflammation and normalized esophageal acid exposure were offered laparoscopic Nissen fundoplication. The primary outcome was symptom improvement/resolution at 1 year after surgery. RESULTS: Twenty-five of 72 (35%) patients remained unresponsive after 4 months of acid-suppressive therapy. Ten patients (40%) underwent surgical fundoplication (median age, 54 y; men, 4) and 15 patients (60%) continued medical therapy (median age, 52; men, 4). The most common laryngeal symptoms were sore throat, hoarseness, and cough. pH studies at 3 and 12 months were normal in all patients after fundoplication (median % time pH < 4, .0% and .3%; respectively). One of 10 (10%) patients in the surgery group reported improvement of laryngeal symptoms at 1 year compared with 1 of 15 in the control group (6.7%) (P = 1.0). Treatment of causes other than GERD improved symptoms in an additional 2 of 10 (20%) patients in the surgical group, and 10 of 15 (66%) patients in the nonsurgical cohort. CONCLUSIONS: Surgical fundoplication does not improve laryngeal symptoms reliably in patients unresponsive to aggressive proton pump inhibitor therapy. The argument of low volume or intermittent reflux as the cause of persistent laryngeal symptoms needs to be replaced with evaluation and therapy for other potential non-GERD causes.

Chronic laryngitis associated with gastroesophageal reflux: prospective assessment of differences in practice patterns between gastroenterologists and ENT physicians.

OBJECTIVES: Ear, nose, and throat (ENT) physicians often diagnose gastroesophageal reflux disease (GERD)-related laryngitis on the basis of symptoms and laryngeal signs; and may refer patients to gastroenterologists who contend that many such patients do not have reflux. Because of this dichotomy we designed this study to assess the practice pattern differences among ENT physicians and gastroenterologists in relation to the diagnosis and treatment of patients with GERD-related laryngitis. METHODS: Separate surveys were specifically designed for ENT physicians and gastroenterologists to assess the following: the percentage of patients diagnosed with GERD-related laryngitis, dose and duration of therapy, treatment response, and other diagnostic options in nonresponders. A total of 2000 surveys were mailed randomly to members of both the American Academy of Otolaryngology Head and Neck Surgery and the American Gastroenterological Association. RESULTS: Of the total 4,000 surveys sent, 782 (39%) ENT physicians and 565 (28%) gastroenterologists responded. Most respondents (both specialties) were private practitioners (82% and 74%, respectively). From the ENT survey, the diagnosis was most commonly suspected based on the following symptoms: globus = throat clearing > cough > hoarseness. The most useful signs were laryngeal erythema and edema reported by 70% of respondents. Seventy-four percent of ENT physicians reported they made the diagnosis more on symptoms than on laryngeal signs, and initiated therapy most often with proton pump inhibitor (PPI) once daily for 2 months. Gastroenterologists were divided on pre-therapy testing, 50% reporting testing with esophagogastro-duodenoscopy followed by pH monitoring (distal > proximal) prior to therapy, while the remaining 50% reported treating empirically with PPI twice daily for 3 months. Seventy percent of gastroenterologists reported treatment response of less than 60%, while 62% of ENT physicians reported response rate of greater than 60% (p < 0.05). CONCLUSIONS: (1) Globus and throat clearing were considered the most useful symptoms in diagnosing GERD-related laryngitis, while laryngeal erythema and edema were considered the most useful signs for diagnosis and treatment of this condition by ENT physicians. However, these symptoms and signs may represent the least specific markers for reflux. (2) Many gastroenterologists perform pre-therapy testing which has low sensitivity in GERD-related laryngitis. (3) There is a dichotomy in treatment dose, duration, and perceived patient response to therapy between the two specialists. (4) Our study highlights a need for cross communication and education between these two disciplines in understanding and treating GERD-related laryngitis better.

Correlation between symptoms and laryngeal signs in laryngopharyngeal reflux.

OBJECTIVE/HYPOTHESIS: Laryngopharyngeal reflux (LPR) is diagnosed by the presence of laryngeal signs and symptoms. Some studies have noted that signs and symptoms may be nonspecific and may have poor correlation. However, many such studies were either observational or had short-term follow-up. Therefore, we conducted subgroup analysis of a prospective concurrent controlled study with a 1 year follow-up to study the correlation between signs and symptoms. STUDY DESIGN: Prospective study. METHODS: Seventy-two patients with suspected gastroesophageal reflux disease related laryngeal symptoms/signs received a 4 month trial of aggressive acid-suppressive therapy. Four month symptomatic nonresponders (<50% improvement) with continued laryngeal inflammation and normalized esophageal acid exposure were then offered laparoscopic Nissen fundoplication. The primary outcome was laryngeal symptom-sign correlation at 1 year postsurgery. RESULTS: Twenty-five of 72 (35%) patients remained unresponsive after 4 months of aggressive acid suppressive therapy. Ten (40%) patients agreed to undergo surgical fundoplication (mean age = 50, male = 4). The most common laryngeal symptoms were sore throat (40%), hoarseness (30%), and cough (20%), whereas the most common signs were medial arytenoid wall erythema/edema (60%), interarytenoid erythema (50%), and arytenoid complex erythema/edema (50%). At 1 year postfundoplication, laryngeal symptoms improved in only 1 of 10 (10%) patient, whereas signs improved in 8 of 10 (80%) patients. CONCLUSIONS: There appears to be poor correlation between signs and symptoms of LPR, particularly when monitoring therapeutic outcomes. In patients unresponsive to twice-daily proton-pump inhibitor therapy for 4 months, further aggressive therapy is unlikely to bring additional symptomatic benefit.

Prevalence of laryngeal irritation signs associated with reflux in asymptomatic volunteers: impact of endoscopic technique (rigid vs. flexible laryngoscope).

OBJECTIVES: The objectives of this study were to 1) determine the prevalence of ENT findings in the normal asymptomatic population and 2) to compare findings between flexible and rigid laryngoscopes in an attempt to increase specificity of diagnosis of reflux in endoscopic laryngeal examinations. STUDY DESIGN: Prospective study. METHODS: Fifty-two nonsmoker volunteers (24 male, 28 female), mean age of 42.7 years, with no history of ENT abnormalities or gastroesophageal reflux disease, underwent both rigid and flexible videolaryngologic examinations with a digital endoscopic unit. A group of three expert judges reviewed the oral and transnasal examinations blindly and independently for physical signs of irritation/inflammation commonly associated with reflux. RESULTS: Atleast one sign of tissue irritation was detected in 93% and 83% of the population when using a flexible and a rigid laryngoscope, respectively. Results showed a high incidence of posterior commissure bar (53.2% and 51.9%), arytenoid complex edema/erythema (76.3% and 53.2%), and pseudosulcus (37.2% and 7.7%). Most signs were more frequently detected on flexible transasal examinations than with rigid transoral examinations: posterior pharyngeal wall (<0.01), interarytenoid irritation (<0.01), arytenoids complex irritation (<0.01), ventricular obliteration (<0.01), and pseudosulcus (<0.01). CONCLUSIONS: Several signs of posterior laryngeal irritation (e.g., interarytenoid bar, erythema of the medial wall of the arytenoids), which are generally considered to be signs of laryngopharyngeal reflux, are present in a high percentage of nonsymptomatic individuals, raising question about their diagnostic specificity. In addition, these signs were more often detected with flexible than with rigid laryngoscopes, suggesting that flexible laryngoscopy is more sensitive but less specific in identifying laryngeal tissue irritation.

Laryngopharyngeal reflux: prospective cohort study evaluating optimal dose of proton-pump inhibitor therapy and pretherapy predictors of response.

PURPOSE: Laryngopharyngeal reflux (LPR) is frequently treated with empiric proton-pump inhibitors (PPI), but the optimal dosing and duration is unknown. We performed an open label prospective cohort study to evaluate whether twice-daily (BID) PPI is more effective than once-daily (QD) PPI for the treatment of LPR. METHODS: Patients diagnosed with LPR based on ear, nose, and throat (ENT) symptoms and laryngoscopy findings were enrolled. Questionnaire assessed demographics, ENT symptoms, symptom severity, and exposure to other potential laryngeal irritants. Esophageal manometry, ambulatory 24-hour pH monitoring, and upper gastrointestinal endoscopy were performed before initiation of therapy. Patients were consecutively assigned to three groups: BID PPI (lansoprazole 30 mg BID), BID PPI + H2 receptor antagonist (H2RA; omeprazole 20 mg BID + ranitidine 300 mg each night), or QD PPI (esomeprazole 40 mg QD). Greater than 50% primary symptom improvement from baseline defined symptom response. At 2 month follow-up, the same PPI dose was continued for responders, and PPIs were doubled for nonresponders for an additional 2 months. Repeat symptom assessment and laryngoscopy performed at 4 month follow-up. RESULTS: Eighty-five patients were enrolled (median age 49 years, interquartile range 44.0 - 65.0; 76% white; 34% male). Treatment groups were BID PPI for 30 patients, BID PPI + H2RA for 30 patients, and QD PPI for 25 patients. RESPONSE TO THERAPY: At 2 months, BID response occurred among 15 of 30 (50%) patients, BID + H2RA for 15 of 30 (50%), and QD for 7 of 25 (28%) (P = .03). No statistical difference found between the two BID PPI groups with and without H2RA. Among the QD group nonresponders, 7 of 13 (54%) achieved symptom response with additional 2 months of BID dosing. At 4 month follow-up, an additional 22% of responses were obtained from the two BID groups (43/60, 72%). The overall response rate for all three groups was 70% (54/77). PREDICTORS OF OUTCOME: Pretherapy interarytenoid mucosa and true vocal folds abnormalities were associated with twofold increase in symptom response (odds ratio 1.99 and 1.96, respectively, P = .017). CONCLUSION: BID PPI appears to be more effective than QD PPI in achieving clinical symptom response in suspected LPR. More response was achieved at 4 months compared with 2 months. Therefore, aggressive acid suppression with BID PPI for at least 4 months is warranted for treatment of LPR.