Variability in P2X receptor composition in human taste nerves: implications for treatment of chronic cough.

Background: Antagonists to the P2X purinergic receptors on airway sensory nerves relieve refractory or unexplained chronic cough (RCC/UCC) but can evoke unwanted dysgeusias because the gustatory nerves innervating taste buds express this same family of receptors. However, the subunit composition of the P2X receptors in these systems may differ, with implications for pharmacological intervention of RCC/UCC. In most species, the extrapulmonary airway nerves involved in cough predominantly express P2X3 subunits that form homotrimeric P2X3 receptors. In contrast, most sensory nerves innervating taste buds in mice express both P2X2 and P2X3 subunits, so the majority of receptors in that system are likely P2X2/P2X3 heteromers. Methods: Since neural P2X subunit composition can differ across species, we used immunohistochemistry to test whether taste nerves in humans and rhesus macaque monkeys express both P2X2 and P2X3 as in mice. Results: In taste bud samples of fungiform papillae and larynx from humans and monkeys, all taste bud samples exhibited P2X3+ nerve fibres, but the majority lacked substantial P2X2+. Of the 35 human subjects, only four (one laryngeal and three fungiform) showed strong P2X2 immunoreactivity in taste nerves; none of the rhesus monkey samples showed immunoreactivity for P2X2. Conclusions: These findings suggest that for most humans, unlike mice, taste buds are innervated by nerve fibres predominantly expressing only P2X3 homomeric receptors and not P2X2/P2X3 heteromers. Thus, antagonists specific for P2X3 homomeric receptors might not be spared from affecting taste function in RCC/UCC patients.

Is Thyrohyoid Space Palpation a Reliable and Useful Clinical Tool Within Diagnostic Protocol for Voice Disorders?

OBJECTIVES: Palpation of the extrinsic laryngeal muscles is a common part of examination performed by otolaryngologists and speech-language pathologists on individuals presenting with voice complaints, thought to aid in diagnosis and treatment planning. While research has identified a significant relationship between thyrohyoid tension and hyperfunctional voice disorders, we are not aware of any studies exploring correlations between thyrohyoid posture during palpation and the full spectrum of voice disorders. This study aims to identify whether patterns in thyrohyoid posture at rest and during phonation can be related to stroboscopic findings and voice disorder diagnoses. METHODS: A multidisciplinary team of three laryngologists and three speech-language pathologists participated in data collection during 47 new patient visits for voice complaints. Each patient underwent neck palpation and evaluation of thyrohyoid space at rest and during phonation by two independent raters. Clinicians then used stroboscopy to rate glottal closure and supraglottic activity as part of determining primary diagnosis. RESULTS: Strong inter-rater agreement was found for ratings of thyrohyoid space posture both at rest (κ = 0.93) and during phonation (κ = 0.80). Findings revealed no significant correlations between patterns of thyrohyoid posture and laryngoscopic findings or primary diagnoses. CONCLUSIONS: Findings suggest that the presented method of laryngeal palpation is a reliable measure for assessing thyrohyoid posture at rest and during phonation. Lack of significant correlation between palpation ratings and other collected measures suggests that this method of palpation is not a useful tool for predicting laryngoscopic findings or voice diagnoses. Laryngeal palpation may still be useful in predicting extrinsic laryngeal muscle tension and guiding treatment planning; however, further research exploring the validity of laryngeal palpation as a measure of extrinsic laryngeal muscle tension is needed, as well as studies that include patient-reported measures and repeated measurements of thyrohyoid posture over time to explore whether thyrohyoid posture is impacted by other factors.

Normative Values for the Leicester Cough Questionnaire in Healthy Individuals.

INTRODUCTION: The primary self-assessment questionnaire used for patients with chronic cough is the Leicester Cough Questionnaire (LCQ). The LCQ is a validated questionnaire that ranges in total score from 3 to 21. While it is known that a higher score on the LCQ reflects a better quality of life, normative data have not been reported for this questionnaire. OBJECTIVE: The purpose of this study was to determine normative LCQ scores on a healthy population without cough. METHODS: The LCQ was distributed via electronic survey to the authors' universities, professional affiliation email lists, and personal contacts. Participants were included if they were at least 18, nonsmokers, and without abnormal cough, without pulmonary disease, and without neurological disease. Participants answered questions regarding age, gender, and race/ethnicity, and completed the 19 LCQ questions. RESULTS: One hundred forty-three (118 women) LCQ responses were analyzed. Average participant age was 47 years (SD = 13) and 133 (93%) were Caucasian. The mean LCQ Total score was 20.23 (SD = 0.85) with scores ranging from 17.05 to 21. CONCLUSIONS: This study determined the following LCQ scores should be considered normal threshold scores: Total score - 17.68, Physical domain - 5.36, Psychological domain - 5.81, and Social domain - 6.06. The findings of this study will assist clinicians in determining severity of cough impact on quality of life using the LCQ. Further research is needed to ensure more complete participant demographic representation.

Evaluation and Management Outcomes and Burdens in Patients with Refractory Chronic Cough Referred for Behavioral Cough Suppression Therapy.

PURPOSE: The purpose of this study was to investigate the typical symptoms and medical management characteristics of adult patients with refractory chronic cough (RCC) who are referred to speech-language pathology (SLP) for behavioral cough suppression therapy (BCST) in order to estimate cost-effectiveness and efficiency of current practice patterns for this population. METHODS: One hundred sixty-four (164) patients with RCC referred for BCST were surveyed. Patients completed an initial survey at BCST onset related to symptom pattern and prior treatment, including the Leicester Cough Questionnaire (LCQ). Every four to six weeks patients completed follow-up surveys to assess their response to BCST. RESULTS: Mean age was 58 years (83.5% women). The majority of patients reported their cough began two or more years prior to BCST. Approximately half (49%) reported seeing four or more physicians (including primary care physicians) and being prescribed four or more medications (57%) prior to BCST. Medications targeting post-nasal drip (72%), reflux (70%), asthma (56%), and allergies (56%) were most commonly prescribed. BCST resulted in a clinically significant improvement in 70.1% of participants. The mean change in LCQ for those who improved with BCST was 6.61. Over half (58%) reported they were quite satisfied to completely satisfied with their treatment response. The average time from enrollment to study completion was 64 days. CONCLUSION: The results of this study suggest early intervention with BCST may be a cost-effective and efficient option for patients with RCC.

Laryngeal Chemoreflex in Health and Disease: A Review.

The larynx plays a key role in airway protection via the laryngeal chemoreflex (LCR). This involuntary reflex can be evoked when hazardous substances activate mucosal receptors, which send signals to be processed within the brainstem. Although the LCR is meant to be protective, the reflex can become hyperstimulated, even to benign stimuli, which can result in pathological disorders, such as chronic cough and inducible laryngeal obstruction. In this review, we will outline the mechanism of the LCR and its associated pathological disorders.

Effect of Unilateral Cordotomy on Perception of Dysphagia.

OBJECTIVES: CO2 laser medial transverse cordotomy is a permanent static procedure performed to achieve adequate functional airway in cases of posterior glottic stenosis and bilateral vocal fold paralysis. Although it is the preferred method to manage long-term bilateral vocal fold immobility, it is widely believed that cordotomy has the potential to cause aspiration. The minimal existing data on the effect surgical enlargement of the glottic airway on swallowing function is heterogeneous. Through investigation of dysphagia after cordotomy, we hope to better understand the influence of glottic function and its role in dysphagia. The Eating Assessment Tool (EAT-10), is a validated dysphagia symptom-specific outcome measure. We hypothesized that EAT-10 scores would not change after CO2 laser cordotomy despite causing glottic insufficiency. METHODS: Retrospective review was performed on sequential patients having undergone unilateral CO2 laser cordotomy with complete pre- and postoperative EAT-10 questionnaire data available for evaluation. RESULTS: Fifteen patients were available for analysis; 10 patients underwent primary unilateral medial transverse cordotomy, 5 patients underwent revision cordotomy, and 20 unique procedures were included in the dataset. The median EAT-10 score during the visit prior to surgery was 3.5, whereas the post-surgery median score was 2. Furthermore, the median difference of 0 was statistically non-significant (P = .91). CONCLUSION: CO2 laser cordotomy does not contribute to patient-reported dysphagia despite creating glottic incompetence. This suggests vocal fold apposition may play a less significant role in normal swallowing function than widely believed.

Chemical receptors of the arytenoid: A comparison of human and mouse.

OBJECTIVES/HYPOTHESIS: The larynx is a highly responsive organ exposed to mechanical, thermal, and chemical stimuli. Chemicals elicit responses both in intraepithelial nerve fibers and in specialized chemosensory cells, including scattered solitary cells as well as taste cells organized into taste buds. Activation of both chemosensory cells and taste buds in the larynx elicit cough, swallow, or apnea with exposure to sour or bitter substances, and even by water or sweet-tasting chemicals. In an effort to begin understanding their function, we sought to compare the distribution, density, and types of chemosensory cells and chemoresponsive nerve fibers in laryngeal epithelium of humans and mice. STUDY DESIGN: Animal and human laboratory analysis. METHODS: Using immunohistochemistry, we identified taste cells and polymodal nociceptive nerve fibers in the arytenoid area of the laryngeal epithelium of the following: 1) infants undergoing supraglottoplasty for laryngomalacia, and 2) a cadaveric specimen procured from a 34-year-old donor. We then compared these findings to both preweanling and mature mouse tissue. RESULTS: Arytenoid tissue from both human and mouse contained many taste buds containing type II taste cells-bitter, sweet, or umami sensing-which were innervated by nerve fibers expressing P2X3 type adenosine triphosphate receptors. Type III cells (acid responsive) were also present, but they were fewer in human tissue than in equivalent tissue from mice. In both species, the epithelium was densely innervated by free nerve endings. CONCLUSIONS: Our findings suggest that from a standpoint of chemosensation, human and mouse larynges are biologically similar. This suggests that a murine model can be used effectively in laryngeal chemosensory research. LEVEL OF EVIDENCE: NA Laryngoscope, 130:423-430, 2020.

Voice therapy associated with a decrease in the reflux symptoms index in patients with voice complaints.

OBJECTIVES/HYPOTHESIS: Patients with muscle tension dysphonia often demonstrate an elevation in Reflux Symptom Index (RSI) and 10-item Voice Handicap Index (VHI-10) scores, and may be erroneously diagnosed with laryngopharyngeal reflux disease. In this study we assessed the effects of voice therapy on RSI and VHI-10 scores in patients with voice complaints not responsive to antireflux medications. STUDY DESIGN: Retrospective cohort study. METHODS: A study of patients was conducted at a single tertiary-care center over 1 year (January 2012-January 2013). Patients were included if they had dysphonia not responsive to proton pump inhibition, did not have neurologic or neoplastic disease, and participated in at least three voice-therapy sessions in the absence of antireflux therapy. Primary analysis assessed change in RSI scores between the initial and follow-up visits with a laryngologist. RESULTS: A total of 18 patients were included (mean age = 49.9 ± 14.5 years, 89% female, 83% with a primary complaint of dysphonia). From initial to follow-up visit, the median RSI score (18.5 [interquartile range {IQR}, 9.5-22.8] vs. 10.5 [IQR, 4.5-14]; P = .02) and median VHI-10 score (25.5 [IQR, 11.3-30.0] vs. 13.5 [IQR, 9.5-20.8]; P = .03) significantly decreased. A significant inverse correlation was found between the number of voice therapy sessions/month and change in RSI score (r = -0.4; P = .05). CONCLUSIONS: In this study of patients with muscle tension dysphonia or vocal hyperfunction not responsive to antireflux therapy, RSI and VHI-10 scores improved following voice therapy. Results suggest that self-reported symptoms typically attributed to laryngopharyngeal reflux disease may actually be secondary to inefficient voice use patterns or anxiety about dysphonia that are responsive to voice therapy. LEVEL OF EVIDENCE: 4 Laryngoscope, 129:1169-1173, 2019.

Integrating the microbiota of the respiratory tract with the unified airway model.

The unified airway model has developed from indications that the upper and lower respiratory tracts share key elements of pathogenesis. These shared traits likely extend to similar niche characteristics that support bacterial communities, and as such, we suspect that similar microbes exist on upper and lower respiratory tract epithelium. Over the past decade and a half there have been significant improvements in microbiological identification and analysis due to the development of new molecular technologies, including next-generation sequencing. In this review, we provide an overview of the modern collection and sequencing methods involved in respiratory microbiota research, and outline the specific microbial communities that have been found to be associated with the healthy and diseased human respiratory tract. Demonstration of a remarkable similarity between the upper and lower respiratory tract in terms of microbiological presence adds further corroboration to the existence of a unified airway.

Factors Associated With Infectious Laryngitis: A Retrospective Review of 15 Cases.

OBJECTIVES: To identify the culturable microbes associated with infectious laryngitis and outline effective treatment strategies. METHODS: This is a retrospective chart review of adult patients with persistent dysphonia plus evidence of laryngeal inflammation who underwent biopsy for culture at a tertiary care medical center. Demographic factors, symptoms as reported on validated patient assessment tools, past medical history, social history, culture results, and treatment duration and response were reviewed. RESULTS: Fifteen patients with infectious laryngitis were included in this study. Culture results demonstrated Methicillin-sensitive Staphylococcus aureus (MSSA), Methicillin-resistant Staphylococcus aureus (MRSA), Pseudomonas aeruginosa, Serratia marcescens, and "normal respiratory flora." In most patients, multiple courses of prolonged antibiotics were needed to treat MSSA or MRSA. Infections associated with other microbes resolved with a single course of antibiotics. CONCLUSIONS: In this population, infectious laryngitis is defined as colonization with bacteria not found in the previously characterized laryngeal microbiome of benign vocal fold lesions. In suspected cases of infectious laryngitis, culture is recommended, by biopsy if needed. For MSSA- and MRSA-associated laryngitis, an extended course of antibiotics may be necessary for symptom improvement and resolution of laryngeal inflammation. However, the optimal treatment regimen has yet to be defined and will require larger, prospective studies.

A comparison of sampling methods for examining the laryngeal microbiome.

Shifts in healthy human microbial communities have now been linked to disease in numerous body sites. Noninvasive swabbing remains the sampling technique of choice in most locations; however, it is not well known if this method samples the entire community, or only those members that are easily removed from the surface. We sought to compare the communities found via swabbing and biopsied tissue in true vocal folds, a location that is difficult to sample without causing potential damage and impairment to tissue function. A secondary aim of this study was to determine if swab sampling of the false vocal folds could be used as proxy for true vocal folds. True and false vocal fold mucosal samples (swabbed and biopsied) were collected from six pigs and used for 454 pyrosequencing of the V3-V5 region of the 16S rRNA gene. Most of the alpha and beta measures of diversity were found to be significantly similar between swabbed and biopsied tissue samples. Similarly, the communities found in true and false vocal folds did not differ considerably. These results suggest that samples taken via swabs are sufficient to assess the community, and that samples taken from the false vocal folds may be used as proxies for the true vocal folds. Assessment of these techniques opens an avenue to less traumatic means to explore the role microbes play in the development of diseases of the vocal folds, and perhaps the rest of the respiratory tract.

Laryngeal T regulatory cells in the setting of smoking and reflux.

OBJECTIVES/HYPOTHESIS: The larynx is a mucosal organ rich in lymphatic tissue that is regularly exposed to a multitude of inhaled, ingested, and refluxed microorganisms and irritants. The first line of mucosal immune defense is the barrier, including resident immune cells. T regulatory (Treg) cells are a specialized subset of CD4+ T cells that suppress or dampen immune responses to prevent damaging immunopathology. As Treg cells have been shown to preferentially accumulate at sites of infection, and Treg responses may contribute to persistence of infection by impairing antibacterial immunity, we sought to quantify these cells in laryngeal tissue exposed to smoking and reflux. STUDY DESIGN: Cross-sectional study. METHODS: Using an epigenetic assay, we quantified Treg and T cells and calculated the ratio of Treg to T cells (i.e., cellular ratio of immune tolerance [ImmunoCRIT]) in disease-free laryngeal biopsies representing four inflammatory states: 1) tobacco-exposed tissue, 2) refluxate and tobacco-exposed tissue, 3) refluxate-exposed tissue, and 4) unexposed tissue. RESULTS: There was epigenetic evidence of Treg cells in all tissues, and we found no differences in Treg cell frequency relative to smoking and reflux in laryngeal tissue collected from 42 non-treatment-seeking participants. There was a decrease in total T cell frequency and an increase in ImmunoCRIT values in smokers regardless of reflux status. CONCLUSIONS: In this study, laryngeal tissue from smokers show decreased overall T cells and increased ImmunoCRIT values. Our findings indicate that laryngeal inflammation is not directly mediated by loss of Treg cells in response to smoking and reflux in local tissue and increased ImmunoCRIT values in smokers implicate a role for this environmental exposure in modulating laryngeal immune homeostasis. More studies are indicated to explore Treg cell dysfunction in the pathophysiology of laryngeal disease. LEVEL OF EVIDENCE: NA Laryngoscope, 127:882-887, 2017.

The human laryngeal microbiome: effects of cigarette smoke and reflux.

Prolonged diffuse laryngeal inflammation from smoking and/or reflux is commonly diagnosed as chronic laryngitis and treated empirically with expensive drugs that have not proven effective. Shifts in microbiota have been associated with many inflammatory diseases, though little is known about how resident microbes may contribute to chronic laryngitis. We sought to characterize the core microbiota of disease-free human laryngeal tissue and to investigate shifts in microbial community membership associated with exposure to cigarette smoke and reflux. Using 454 pyrosequencing of the 16S rRNA gene, we compared bacterial communities of laryngeal tissue biopsies collected from 97 non-treatment-seeking volunteers based on reflux and smoking status. The core community was characterized by a highly abundant OTU within the family Comamonadaceae found in all laryngeal tissues. Smokers demonstrated less microbial diversity than nonsmokers, with differences in relative abundances of OTUs classified as Streptococcus, unclassified Comamonadaceae, Cloacibacterium, and Helicobacter. Reflux status did not affect microbial diversity nor community structure nor composition. Comparison of healthy laryngeal microbial communities to benign vocal fold disease samples revealed greater abundance of Streptococcus in benign vocal fold disease suggesting that mucosal dominance by Streptococcus may be a factor in disease etiology.

Quantification of Porcine Vocal Fold Geometry.

OBJECTIVE: The aim of this study was to quantify porcine vocal fold medial surface geometry and three-dimensional geometric distortion induced by freezing the larynx, especially in the region of the vocal folds. STUDY DESIGN: The medial surface geometries of five excised porcine larynges were quantified and reported. METHODS: Five porcine larynges were imaged in a micro-CT scanner, frozen, and rescanned. Segmentations and three-dimensional reconstructions were used to quantify and characterize geometric features. Comparisons were made with geometry data previously obtained using canine and human vocal folds as well as geometries of selected synthetic vocal fold models. RESULTS: Freezing induced an overall expansion of approximately 5% in the transverse plane and comparable levels of nonuniform distortion in sagittal and coronal planes. The medial surface of the porcine vocal folds was found to compare reasonably well with other geometries, although the compared geometries exhibited a notable discrepancy with one set of published human female vocal fold geometry. CONCLUSIONS: Porcine vocal folds are qualitatively geometrically similar to data available for canine and human vocal folds, as well as commonly used models. Freezing of tissue in the larynx causes distortion of around 5%. The data can provide direction in estimating uncertainty due to bulk distortion of tissue caused by freezing, as well as quantitative geometric data that can be directly used in developing vocal fold models.

Vocal fold myofibroblast profile of scarring.

OBJECTIVES/HYPOTHESIS: Vocal fold fibroblasts (VFF) are responsible for extracellular matrix synthesis supporting lamina propria in normal and diseased conditions. When tissue is injured, VFF become activated and differentiate into myofibroblasts to facilitate wound healing response. We investigated if vocal fold myofibroblasts can be utilized as surrogate cells for scarred VFF. STUDY DESIGN: In vitro. METHODS: Normal VFF cell lines from a 21-year-old male (N21), 59-year-old female (N59), and a scar VFF cell line from a 56-year-old female (S56) were used in this study. 10 ng/mL of transforming growth factor (TGFß1) was applied for 5 days to normal VFF. Myofibroblast differentiation was determined with immunocytochemistry and western blot, measuring alpha smooth muscle actin (α-SMA). Cell growth, proliferation, contractile properties, and gene expression profiles were evaluated. RESULTS: N21, N59, and S56 VFF presented elongated configuration. N21+ and N21- VFF demonstrated significantly greater proliferation compared to N59+, N59-, and S56 VFF at 6 days. α-SMA was expressed in all cells. Fibronectin, alpha smooth actin, connective tissue growth factor, and metallopeptidase inhibitor were the highest genes expression in VFF treated with transforming growth factor ß1 (TGFß1). At 24 hours, S56 VFF showed lower contraction compared to N21+ and N59+ VFF, but at 60 hours S56 VFF had lower collagen contraction compared to all cell groups. Highest collagen contraction matrices were measured with VFF treated with TGFß1 at 24 hours and N59- VFF at 60 hours. CONCLUSION: VFF treated with TGFß1 (myofibroblasts) appear to have similar phenotypic characteristics but different genotypic behavior compared to scar VFF. LEVEL OF EVIDENCE: N/A. Laryngoscope, 126:E110-E117, 2016.

Correlation between Reflux and multichannel intraluminal impedance pH monitoring in untreated volunteers.

OBJECTIVES/HYPOTHESIS: Although probable causative agents have been identified (e.g., refluxate components, tobacco smoke), the definitive mechanism for inflammation-related laryngeal mucosal damage remains elusive. Multichannel intraluminal impedance combined with pH monitoring (MII/pH) has emerged as a sensitive tool for diagnosis and characterization of gastroesophageal reflux disease with laryngopharyngeal manifestations. To determine the relationship between laryngeal signs and MII/pH, we examined correlations between Reflux Finding Score (RFS) ratings of videostroboscopic laryngeal examinations and findings from MII/pH. STUDY DESIGN: Correlational study. METHODS: Healthy, untreated volunteers (n = 142) underwent reflux diagnosis using data acquired from MII/pH testing. Eight trained clinicians performed RFS ratings of corresponding laryngeal examinations. Averaged RFS ratings were compared to MII/pH data using Pearson correlation coefficients. The relationship between RFS and MII/pH findings and demographic/clinical information (age, sex, smoking status, reflux) was assessed using general linear modeling. Rater reliability was evaluated. RESULTS: Posterior commissure hypertrophy was negatively correlated with minutes of nonacid refluxate (R = -0.21, P = .0115). General linear modeling revealed that 28% to 40% of the variance in ratings of ventricular obliteration, erythema/hyperemia, vocal fold edema, diffuse laryngeal edema, posterior commissure hypertrophy, and granulation/granuloma could be explained by main and interaction effects of age, sex, smoking status, and reflux. Intra- and inter-rater reliability for RFS were poor-fair. CONCLUSIONS: These results support the theory that the RFS is not specific for reflux in healthy, untreated volunteers, suggesting there may be alternate explanations for inflammatory clinical signs commonly ascribed to reflux in this population.

Characterization and comparison of bacterial communities in benign vocal fold lesions.

BACKGROUND: Benign vocal fold lesions, including cysts, nodules, polyps, and Reinke's edema, are common causes of hoarseness and subsequent voice disorders. Given the prevalence of these lesions, disease etiology and pathophysiology remain unclear and their microbiota has not been studied to date secondary to the paucity of available biopsies for investigation. We sought to characterize and compare the bacterial communities in biopsies of cysts, nodules, polyps, and Reinke's edema collected from patients in Germany and Wisconsin. These samples were then compared to the communities found in healthy saliva and throat samples from the Human Microbiome Project (HMP). RESULTS: 454 pyrosequencing of the V3-V5 regions of the 16S rRNA gene revealed five phyla that explained most of the bacterial diversity, including Firmicutes (73.8%), Proteobacteria (12.7%), Bacteroidetes (9.2%), Actinobacteria (2.1%), and Fusobacteria (1.9%). Every lesion sample, regardless of diagnosis, had operational taxonomic units (OTUs) identified as Streptococcus, with a mean abundance of 68.7%. Most of the lesions, 31 out of 44, were indistinguishable in a principal coordinates analysis (PCoA) due to dominance by OTUs phylogenetically similar to Streptococcus pseudopneumoniae. Thirteen lesions not dominated by S. pseudopneumoniae were more similar to HMP throat and saliva samples, though 12 of them contained Pseudomonas, which was not present in any of the HMP samples. Community structure and abundance could not be correlated with lesion diagnosis or any other documented patient factor, including age, sex, or country of origin. CONCLUSIONS: Dominance by S. pseudopneumoniae could be a factor in disease etiology, as could the presence of Pseudomonas in some samples. Likewise, decreased diversity, as compared to healthy saliva and throat samples, may be associated with disease, similar to disease models in other mucosal sites.

Characterization of human vocal fold fibroblasts derived from chronic scar.

OBJECTIVES/HYPOTHESIS: In vitro modeling of cell-matrix interactions that occur during human vocal fold scarring is uncommon, as primary human vocal fold scar fibroblast cell lines are difficult to acquire. The purpose of this study was to characterize morphologic features, growth kinetics, contractile properties, α-smooth muscle actin (α-SMA) protein expression and gene expression profile of human vocal fold fibroblasts derived from scar (sVFF) relative to normal vocal fold fibroblasts (nVFF). STUDY DESIGN: In vitro. METHODS: We successfully cultured human vocal fold fibroblasts from tissue explants of scarred vocal folds from a 56-year-old female and compared these to normal fibroblasts from a 59-year-old female. Growth and proliferation were assessed by daily cell counts, and morphology was compared at 60% confluence for 5 days. Gel contraction assays were evaluated after seeding cells within a collagen matrix. α-SMA was measured using western blotting and immunocytochemistry (ICC). Quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) was used to assess differential extracellular matrix gene expression between the two cell types. RESULTS: sVFF were morphologically indistinguishable from nVFF. sVFF maintained significantly lower proliferation rates relative to nVFF on days 3 to 6 (day 3: P = .0138; days 4, 5, and 6: P < .0001). There were no significant differences in contractile properties between the two cell types at any time point (0 hours: P = .70, 24 hours: P = .79, 48 hours: P = .58). ICC and western blot analyses revealed increased expression of α-SMA in sVFF as compared with nVFF at passages 4 and 5, but not at passage 6 (passage 4: P = .006, passage 5: P = .0015, passage 6: P = .8860). Analysis of 84 extracellular matrix genes using qRT-PCR revealed differential expression of 15 genes (P < .01). CONCLUSIONS: nVFF and sVFF displayed differences in proliferation rates, α-SMA expression, and gene expression, whereas no differences were observed in contractile properties or morphology. Further investigation with a larger sample size is necessary to confirm these findings.

Vocal fold fibroblasts immunoregulate activated macrophage phenotype.

Recent evidence suggests that fibroblasts play a critical role in regulating inflammation during wound healing because they express several inflammatory mediators in response to bacteria. The objective of this study was to analyze the effects of lipopolysaccharide (LPS) on the immunomodulatory properties of vocal fold fibroblasts (VFFs) derived from polyps, scar and normal tissue co-cultured with macrophages, to provide insight into their interactions during the inflammatory process. Fibroblasts were co-cultured with CD14+ monocytes and after 7 days, wells were treated with LPS for 24 and 72 h. Culture supernatants were collected and concentrations of TNF-α, IL-6, IL-8, IL-10, IL-12, IL-1ß and MCP-1 were quantified by ELISA. Normal VFF and CD14+ monocultures were used as controls. Twenty-four hours after LPS activation, macrophages co-cultured with polyp VFF had significantly increased expression of TNF-α, IL-1ß, IL-12 and IL-10 compared to controls (p<0.0001). In contrast, macrophages co-cultured with scar VFF had significantly lower expression of TNF-α, IL-1ß and IL-12 with significantly higher IL-10 compared to control (p<0.0001). After 72 h, macrophages co-cultured with polyp VFF increased expression of TNF-α, IL-1ß, IL-10, IL-6, IL-8, MCP-1 and TGF-ß (p<0.01) and macrophages co-cultured with scar VFF significantly decreased their expression of IL-1ß and IL-12 compared to control (p<0.0001). Scar VFF at both time points produced significantly lower levels of IL-8, MCP-1, IL-6 and TGF-ß compared to controls (p<0.05). Based on our findings, VFF and macrophages secrete several inflammatory mediators that modify their diverse functions. Polyp and scar VFF may play a role in regulating abnormal inflammatory responses, which could result in excessive ECM deposition that disrupts the function of the vocal folds.

Fundamental approaches in molecular biology for communication sciences and disorders.

PURPOSE: This contemporary tutorial will introduce general principles of molecular biology, common deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and protein assays and their relevance in the field of communication sciences and disorders. METHOD: Over the past 2 decades, knowledge of the molecular pathophysiology of human disease has increased at a remarkable pace. Most of this progress can be attributed to concomitant advances in basic molecular biology and, specifically, the development of an ever-expanding armamentarium of technologies for analysis of DNA, RNA, and protein structure and function. Details of these methodologies, their limitations, and examples from the communication sciences and disorders literature are presented. Results/Conclusions The use of molecular biology techniques in the fields of speech, language, and hearing sciences is increasing, facilitating the need for an understanding of molecular biology fundamentals and common experimental assays.