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Background: Biomarkers that can predict outcome will improve the efficacy of treatment for HNSCC patients. In this regard, we retrospectively evaluated the prognostic effect of PD1, PD-L1, and CD45RO in tongue and larynx squamous cell carcinomas., Methods: FFPE tissue blocks of 63 larynx and 40 tongue squamous cell carcinoma samples were selected, cut into 3 µm sections, and immunohistochemically stained for PD1, PD-L1, and CD45RO. The slides were evaluated by an expert pathologist, and results were analysed using Chi-square, univariate, and multivariable Cox regression methods., Results: TC-PD-L1 expression (P = 0.001) and its expression intensity (P = 0.002) were significantly correlated with a higher percentage of PD-1 + tumor infiltrating lymphocytes. In univariate survival analysis, TC-PD-L1 and its expression intensity had a significant impact on both DFS (HR: 0.203; P = 0.003 and HR: 0.320; P = 0.005) and OS (HR: 0.147; P = 0.002 and HR: 0.322; P = 0.005). Based on the multivariate analysis, PD1 (DFS: HR: 3.202; P = 0.011, OS: HR: 2.671; P = 0.027) and TC-PD-L1 (DFS: HR: 0.174; P = 0.006, OS: HR: 0.189; P = 0.009) were found to be independent prognostic markers. In the second part, scoring systems were defined based on the expression status of PD1 and PD-L1. Patients with higher scores were expected to have longer DFS and OS. In multivariate analysis, the PD1/TC-PD-L1 (DFS: P = 0.001, OS: P = 0.003) scoring systems showed superior prognostic effects. Interestingly, at the highest levels of this score, none of the patients experienced recurrence or cancer-caused death., Conclusion: Collectively, this study suggests negative prognostic behaviour for TC-PD-L1 protein and introduces the PD-1/TC-PD-L1 scoring system as a strong prognostic marker in OS and DFS prediction of tongue and larynx HNSCC patients., (© 2024. The Author(s).)

Haralick texture features are used to quantify the spatial distribution of signal intensities within an image. In this study, the heterogeneity of proliferation (Ki-67 expression) and immune cells (CD45 expression) within tumors was quantified and used to classify histologic characteristics of larynx and hypopharynx carcinomas. Of 21 laryngectomy specimens, 74 whole-mount tumor slides were scored on histologic characteristics. Ki-67 and CD45 immunohistochemistry was performed, and all sections were digitized. The tumor area was annotated in QuPath. Haralick features independent of the diaminobenzidine intensity were extracted from the isolated diaminobenzidine signal to quantify intratumor heterogeneity. Haralick features from both Ki-67 and CD45 were used as input for a principal component analysis. A linear support vector machine was fitted to the first 4 principal components for classification and validated with a leave-one-patient-out cross-validation method. Significant differences in individual Haralick features were found between cohesive and noncohesive tumors for CD45 (angular second motion: P =.03, inverse difference moment: P =.009, and entropy: P =.02) and between the larynx and hypopharynx tumors for both CD45 (angular second motion: P =.03, inverse difference moment: P =.007, and entropy: P =.005) and Ki-67 (correlation: P =.003). Therefore, these features were used for classification. The linear classifier resulted in a classification accuracy of 85% for site of origin and 81% for growth pattern. A leave-one-patient-out cross-validation resulted in an error rate of 0.27 and 0.35 for both classifiers, respectively. In conclusion, we show a method to quantify intratumor heterogeneity of immunohistochemistry biomarkers using Haralick features. This study also shows the feasibility of using these features to classify tumors by histologic characteristics. The classifiers created in this study are a proof of concept because more data are needed to create robust classifiers, but the method shows potential for automated tumor classification., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

In order to study the dust exposure amount and coal dust deposition rule of coal miners under different labor intensity in coal mine environment, an airflow-particle two-phase coupling calculation model of human upper respiratory tract was established based on Euler-Lagrange framework, and the airflow field in the upper respiratory tract and the characteristics of coal dust deposition were simulated and studied. By comparing the experimental data, the relative error of simulation is in the range of 1.5 %-11.2 %. The results showed that the total deposition fraction of 1 μm dust was the smallest (0.61-1.20 %), and was relatively less affected by respiratory intensity, and the overall distribution was uniform. When the dust particle size increased to 7.07 μm, the total dust deposition fraction in the nasal cavity, pharynx and larynx was in the range of 11.10 %-20.91 %, and increased with the respiratory intensity. When the dust particle size was large, the dust particles of 20 μm and 80 μm were mostly concentrated in the front of the nasal cavity, and the deposition amount of 80 μm dust was about 99.52 %. It was found that with the increase of dust particle size or the increase of labor intensity, the possibility of dust being transported into lungs became smaller. The fitting function of 7.07 μm dust escape rate and labor intensity was obtained, for example, Y 7.07μm = 91.73-0.22n (n is labor intensity), and the escape rate of dust with 7.07 μm particle size was up to 88.90 %. Most of them escape from the upper respiratory tract and enter the lungs, which provides theoretical guidance for quantifying the accumulated dust exposure amount in the lungs and monitoring respiratory dust concentration., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Asbestos describes a group of naturally occurring fibrous silicate mineral compounds that have been associated with a number of respiratory maladies, including mesothelioma and lung cancer. In addition, based primarily on epidemiologic studies, asbestos has been implicated as a risk factor for laryngeal and pharyngeal squamous cell carcinoma (SCC). The main objective of this work was to strengthen existing evidence via empirical demonstration of persistent asbestos fibers embedded in the tissue surrounding laryngeal and pharyngeal SCC, thus providing a more definitive biological link between exposure and disease. Six human papillomavirus (HPV)-negative laryngeal (n = 4) and pharyngeal (n = 2) SCC cases with a history working in an asbestos-exposed occupation were selected from a large population-based case-control study of head and neck cancer. A laryngeal SCC case with no history of occupational asbestos exposure was included as a control. Tissue cores were obtained from adjacent nonneoplastic tissue in tumor blocks from the initial primary tumor resection, and mineral fiber analysis was performed using a scanning electron microscope equipped with an energy dispersive X-ray analyzer (EDXA). Chrysotile asbestos fiber bundles were identified in 3/6 of evaluated cases with a history of occupational asbestos exposure. All three cases had tumors originating in the larynx. In addition, a wollastonite fiber of unclear significance was identified one of the HPV-negative pharyngeal SCC cases. No mineral fibers were identified in adjacent tissue of the case without occupational exposure. The presence of asbestos fibers in the epithelial tissue surrounding laryngeal SCC in cases with a history of occupational asbestos exposure adds a key line of physical evidence implicating asbestos as an etiologic factor.

Laser induced autofluorescence (LIAF) lifetime is useful to distinguish between normal laryngeal tissues and squamous cell carcinoma (SCC) based on variations of their biochemical composition and structure alterations. LIAF was collected from samples constituted by pairs of normal and malignant tissue, which were excised from three patients. Exclusion criteria for samples harvest were: (i) macroscopic changes of normal vocal cord observed during surgery; (ii) previous surgical intervention on vocal cord, (iii) patients treated only with chemotherapy or radiotherapy for carcinoma. Inclusion conditions: men, aged 57-68, non-smokers. A pulsed laser diode excited LIAF at 375 nm and 31 MHz repetition rate; beam full-time width at half-maximum was 87 ps at an average power of 0.49 mW. Mean LIAF lifetime for normal tissues was (3.75 ± 0.49) ns and for malignant (4.37 ± 0.85) ns: it is longer in malignant than in normal tissue. Variance analysis made with Fisher's test has shown no significant difference between patients for normal tissues; the same was true for malignant. Though, when malignant tissue was compared to normal for the same patients as well as between patients, a significant difference (significance level of 5%) was evidenced. Time-resolved LIAF may allow better differentiation between normal and malignant tissues in patients diagnosed with larynx SCC., (Copyright © 2019. Published by Elsevier B.V.)

The human soft palate plays an important role in respiration, swallowing, and speech. These motor activities depend on reflexes mediated by sensory nerve endings. To date, the details of human sensory innervation to the soft palate have not been demonstrated. In this study, eight adult human whole-mount (soft palate-tongue-pharynx-larynx-upper esophagus) specimens were obtained from autopsy. Each specimen was bisected in the midline, forming two equal and symmetrical halves. Eight hemi-specimens were processed with Sihler's stain, a whole-mount nerve staining technique. The remaining eight hemi-soft palates were used for immunohistochemical study. The soft palatal mucosa was dissected from the oral and nasal sides and prepared for neurofilament staining. Our results showed that the sensory nerve fibers formed a dense nerve plexus in the lamina propria of the soft palatal mucosa. There was a significant difference in the innervation density between both sides. Specifically, the oral side had higher density of sensory nerve fibers than the nasal side of the soft palate. The mean number and percent area of the sensory nerve fibers in the mucosa of the nasal side was 78% and 72% of those in the mucosa of the oral side, respectively (P < 0.0001). The data presented here could be helpful for further investigating the morphological and quantitative alterations in the sensory nerves in certain upper airway disorders involving the soft palate such as obstructive sleep apnea (OSA) and for designing effective therapeutic strategies to treat OSA. Anat Rec, 301:1861-1870, 2018. © 2018 Wiley Periodicals, Inc., (© 2018 Wiley Periodicals, Inc.)

Background: Many of the complications of mechanical ventilation are related to inappropriate endotracheal tube (ETT) cuff pressure. The aim of the current study was to evaluate the effectiveness of automatic cuff pressure closed-loop control in patients under prolonged intubation, where presence of carbon dioxide (CO2) in the subglottic space is used as an indicator for leaks. The primary outcome of the study is leakage around the cuff quantified using the area under the curve (AUC) of CO2 leakage over time., Methods: This was a multicenter, prospective, randomized controlled, noninferiority trial including intensive care unit patients. All patients were intubated with the AnapnoGuard ETT, which has an extra lumen used to monitor CO2 levels in the subglottic space.The study group was connected to the AnapnoGuard system operating with cuff control adjusted automatically based on subglottic CO2 (automatic group). The control group was connected to the AnapnoGuard system, while cuff pressure was managed manually using a manometer 3 times/d (manual group). The system recorded around cuff CO2 leakage in both groups., Results: Seventy-two patients were recruited and 64 included in the final analysis. The mean hourly around cuff CO2 leak (mm Hg AUC/h) was 0.22 ± 0.32 in the manual group and 0.09 ± 0.04 in the automatic group (P = .01) where the lower bound of the 1-sided 95% confidence interval was 0.05, demonstrating noninferiority (>-0.033). Additionally, the 2-sided 95% confidence interval was 0.010 to 0.196, showing superiority (>0.0) as well. Significant CO2 leakage (CO2 >2 mm Hg) was 0.027 ± 0.057 (mm Hg AUC/h) in the automatic group versus 0.296 ± 0.784 (mm Hg AUC/h) in the manual group (P = .025). In addition, cuff pressures were in the predefined safety range 97.6% of the time in the automatic group compared to 48.2% in the automatic group (P < .001)., Conclusions: This study shows that the automatic cuff pressure group is not only noninferior but also superior compared to the manual cuff pressure group. Thus, the use of automatic cuff pressure control based on subglottic measurements of CO2 levels is an effective method for ETT cuff pressure optimization. The method is safe and can be easily utilized with any intubated patient.

Metalworking fluids (MWFs) are complex formulations designed for effective lubricating, cooling, and cleaning tools and parts during machining operations. Adverse health effects such as respiratory symptoms, dermatitis, and cancer have been reported in workers exposed to MWFs. Several constituents of MWFs have been implicated in toxicity and have been removed from the formulations over the years. However, animal studies with newer MWFs demonstrate that they continue to pose a health risk. This investigation examines the hypothesis that unrecognized health hazards exist in currently marketed MWF formulations that are presumed to be safe based on hazard assessments of individual ingredients. In vivo 13-week inhalation studies were designed to characterize and compare the potential toxicity of four MWFs: Trim VX, Cimstar 3800, Trim SC210, and Syntilo 1023. Male and female Wistar Han rats or Fischer 344N/Tac rats and B6C3F1/N mice were exposed to MWFs via whole-body inhalation at concentrations of 0, 25, 50, 100, 200, or 400 mg/m 3 for 13 weeks, after which, survival, body and organ weights, hematology and clinical chemistry, histopathology, and genotoxicity were assessed following exposure. Although high concentrations were used, survival was not affected and toxicity was primarily within the respiratory tract of male and female rats and mice. Minor variances in toxicity were attributed to differences among species as well as in the chemical components of each MWF. Pulmonary fibrosis was present only in rats and mice exposed to Trim VX. These data confirm that newer MWFs have the potential to cause respiratory toxicity in workers who are repeatedly exposed via inhalation.

Background: Selenium-binding protein 1 (SELENBP1) expression is reduced markedly in many types of cancers and low SELENBP1 expression levels are associated with poor patient prognosis., Methods: SELENBP1 gene expression in head and neck squamous cell carcinoma (HNSCC) was analyzed with GEO dataset and characteristics of SELENBP1 expression in paraffin embedded tissue were summarized. Expression of SELENBP1 in nasopharyngeal carcinoma (NPC), laryngeal cancer, oral cancer, tonsil cancer, hypopharyngeal cancer and normal tissues were detected using immunohistochemistry, at last, 99 NPC patients were followed up more than 5 years and were analyzed the prognostic significance of SELENBP1., Results: Analysis of GEO dataset concluded that SELENBP1 gene expression in HNSCC was lower than that in normal tissue (P < 0.01), but there was no significant difference of SELENBP1 gene expression in different T-stage and N-stage (P > 0.05). Analysis of pathological section concluded that SELENBP1 in the majority of HNSCC is low expression and in cancer nests is lower expression than surrounding normal tissue, even associated with the malignant degree of tumor. Further study indicated the low SELENBP1 expression group of patients with NPC accompanied by poor overall survival and has significantly different comparing with the high expression group., Conclusion: SELENBP1 expression was down-regulated in HNSCC, but has no associated with T-stage and N-stage of tumor. Low expression of SELENBP1 in patients with NPC has poor over survival, so SELENBP1 could be a novel biomarker for predicting prognosis., Competing Interests: The authors report no conflicts of interest.

Aim of Study: The aim was to explore the structure and functions of new target spot c-Myc target from laryngeal cancer cell. (MTLC) of c-Myc gene., Methods: This study adopted bioinformatic methods to analyze the physicochemical property, secondary structure, hydrophobic region, a transmembrane region, and prediction of functions., Results: The results showed that the whole length of the open reading frames was 708 bp, coding was 235 amino acids. This protein was a basic protein possessed two transmembrane structures and weight was about 26592.5 Da. The main elements of secondary structure were alpha-helix and random coil. MTLC was a membrane constitutive protein that possessed signal transduction and regulation may locate on karyotheca as results of subcellular localization and function prediction., Conclusion: This study has provided the theoretical basis for the further discussion of the effect and mechanism of action of MTLC in the occurrence of laryngeal cancer.

Upon viral infection, the cytoplasmic viral sensor retinoic acid-inducible gene-I (RIG-I) recognizes viral RNA to activate antiviral signaling to induce type I interferon (IFN). RIG-I-like receptors (RLRs) activate antiviral signaling in a tissue-specific manner. The molecular mechanism underlying antiviral signaling in the respiratory system remains unclear. We studied antiviral signaling in the lower respiratory tract (LRT), which is the site of many harmful viral infections. Epithelial cells of the LRT can be roughly divided into two groups: bronchial epithelial cells (BECs) and pulmonary alveolar epithelial cells (AECs). These two cell types exhibit different phenotypes; therefore, we hypothesized that these cells may play different roles in antiviral innate immunity. We found that BECs exhibited higher antiviral activity than AECs. TNF receptor-associated factor 3 (TRAF3) has been shown to be a crucial molecule in RLR signaling. The expression levels of TRAF3 and TRAF5, which have conserved domains that are nearly identical, in the LRT were examined. We found that the bronchus exhibited the highest expression levels of TRAF3 and TRAF5 in the LRT. These findings suggest the importance of the bronchus in antiviral innate immunity in the LRT and indicate that TRAF3 and TRAF5 may contribute to RLR signaling., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The major obstacle to successful chemotherapy of cancer patients is drug resistance. Previously we explored the molecular mechanisms of curcumin cross-resistance in carboplatin resistant human laryngeal carcinoma 7T cells. Following curcumin treatment we found a reduction in curcumin accumulation, and reduced induction of reactive oxygen species (ROS) and their downstream effects, compared to parental HEp-2 cells. In order to shed more light on mechanisms involved in drug resistance of 7T cells, in the present study we applied Fourier transform infrared (FTIR) spectroscopy, a technique that provides information about the nature and quantities of all molecules present in the cell. By comparing the spectra from parental HEp-2 cells and their 7T subline, we found an increase in the intensity of ester vibrational bands in 7T cells. This implied an increase in the amount of cholesteryl esters in resistant cells, which we confirmed by an enzymatic assay. Since cholesteryl esters are localized in lipid droplets, we confirmed their higher quantity and serum dependency in 7T cells compared to HEp-2 cells. Moreover, treatment with oleic acid induced more lipid droplets in 7T when compared to HEp-2 cells, as shown by flow cytometry. We can conclude that along with previously determined molecular mechanisms of curcumin resistance in 7T cells, these cells exhibit an increased content of cholesteryl esters and lipid droplets, suggesting an alteration in cellular lipid metabolism as a possible additional mechanism of drug resistance. Furthermore, our results suggest the use of FTIR spectroscopy as a promising technique in drug resistance research.

We report for the first time the implementation of transnasal, image-guided Raman endoscopy to directly assess Raman spectral properties of nasopharyngeal and laryngeal tissue in vivo during clinical endoscopic examinations. A rapid 785-nm excitation Raman endoscopy system, coupled with a miniaturized fiber-optic Raman probe, was utilized for real-time, in vivo Raman measurements of different anatomical locations in the head and neck. A total of 874 high-quality in vivo Raman spectra were successfully acquired from different anatomic locations of the nasopharynx and larynx [i.e., posterior nasopharynx (PN) (n=521), the fossa of Rosenmüller (FOR) (n=157), and true laryngeal vocal chords (LVC) (n=196)] in 23 normal subjects at transnasal endoscopy. Difference spectra and principal component analysis (PCA) were employed for tissue characterization, uncovering the tissue variability at the biomolecular level. The PCA-linear discriminant analysis (LDA) provides sensitivity of 77.0% and specificity of 89.2% for differentiation between PN and FOR, and sensitivity of 68.8% and specificity of 76.0% for distinguishing LVC and PN using the leave-one-subject-out, cross-validation method. This work demonstrates that transnasal, image-guided Raman endoscopy can be used to acquire in vivo Raman spectra from the nasopharynx and larynx in real time. Significant Raman spectral differences (p<0.05) identified as reflecting the distinct composition and morphology in the nasopharynx and larynx should be considered to be important parameters in the interpretation and rendering of diagnostic decision algorithms for in vivo tissue diagnosis and characterization in the head and neck.

Objective: The authors have previously demonstrated the H(+)/K(+)-ATPase (proton pump) in human larynx and lung glands via immunohistochemistry (IHC). The present hypothesis is that the proton pump is expressed in other seromucinous glands of the digestive tract that can be confirmed by IHC and Western blot analysis., Study Design: Prospective controlled tissue analysis study., Setting: Academic medical institution., Methods: Ten anonymous fresh-frozen donor specimens were obtained, comprising 3 submandibular glands, 4 larynges, and 3 normal stomach specimens for control. Submandibular gland sections were immunostained with 2 monoclonal antibodies selectively reactive with α or β subunits of the H(+)/K(+)-ATPase. Western blot analysis was performed on all specimens., Results: Consistent IHC staining was observed in the submandibular gland specimens for both α and β subunits. Western blot analysis revealed very strong expression for the stomach at 100 kDa, corresponding to the α protein, and weak but notable banding for all larynx and submandibular gland specimens. Similar findings were noted for the 60- to 80-kDa glycosylated β subunit protein, as well as the 52-kDa β subunit precursor for all specimens., Conclusion: The H(+)/K(+)-ATPase (proton) pump is present in the human larynx and submandibular gland although in much lower concentrations than in the stomach. Proton pump involvement in human aerodigestive seromucinous glands may have a role in protecting mucosa from acid environments (local or systemic), explain heightened laryngeal sensitivity in those patients with laryngopharyngeal reflux, and be a site of action for proton pump inhibitor pharmacotherapy.

To date, only two human laryngeal allotransplants have been reported and, although they were successful, both patients required life-long immunosuppression. A bioengineered human larynx could represent a possible alternative to allotransplantation. Human larynxes were decellularized enzymatically to obtain acellular matrices. Histological and molecular analysis demonstrated that all cellular components and nuclear material were removed. SEM showed that decellularized matrices retained the hierarchical structures of the native larynx, and mechanical tests demonstrated that the decellularization did not significantly impaired the biomechanically properties of the obtained matrices. Immunohistochemical staining found residual angiogenic factors after decellularization, and CAM analysis demonstrated that acellular laryngeal scaffolds induce a strong in vivo angiogenic response. Using a decellularization method, we are now able to obtain, in a short and clinically useful time, natural bioengineered laryngeal scaffolds which could be use for partial or total implantation in humans., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Conclusion: Transient receptor potential vanilloid (TRPV) 1, 2, 3, and 4 were expressed in the human larynx, which may act as laryngeal nociceptors perceiving luminal noxious stimuli, play an important role in thermal sensation and osmotic sensation, and are also related to some pathological conditions and prevention of aspiration., Objective: Expression of TRPV1, 2, 3, and 4 in the human larynx was analyzed., Materials and Methods: Specimens of human epiglottic epithelium obtained from six patients were used in this study. The localization of TRPV1, 2, 3, and 4 in the laryngeal epithelium was investigated by immunohistochemistry., Results: Immunohistochemical study revealed the presence of TRPV1, 2, 3, and 4 in the laryngeal epithelial cells. Chemoradiotherapy may reduce the expression of TRPV1, 2, 3, and 4, which might be a result of the mucositis and neuropathy in laryngeal epithelium.

Objective: To investigate the characteristics of freshly resected laryngeal carcinoma by Fourier transform infrared spectroscopy (FTIR)., Methods: FTIR was applied to the study of the cancerous tissues and adjacent normal tissues in 32 patients., Results: Compared with pathological diagnosis results, one benign specimen was classified as a malignant, the accuracy was 98.4%. Significant differences were seen in the FTIR spectra between the normal and malignant laryngeal tissues. The peak at 1085 cm(-1) shift to 1114 cm(-1) showed that the relative contents of DNA in laryngeal carcinoma cells was increased. The peak at 1397 cm(-1) was stronger than 1451 cm(-1) in normal tissues, while it was not obvious in cancer tissues. I(2926)/I(2870) in carcinoma cells was lower than that in normal tissues. The wave numbers of the bands of amide I and amide II, symmetric and asymmetric stretching bands of CH(3), stretching vibration bands of C-OH and NH band were shifted to higher number in cancer tissues., Conclusion: The study shows that the malignant and normal laryngeal tissues have different FTIR spectra, which are mainly due to changes in protein, nucleic acid and phospholipids. FTIR may become a new method for the diagnosis of laryngeal carcinoma in clinical practice.

Objectives: We sought to document the mucin gene profile in normal human laryngeal epithelium and compare it with that in patients with reflux-attributed laryngeal injury or disease. We also investigated the effect of low pH with or without pepsin on mucin messenger RNA levels in vitro., Methods: Laryngeal biopsy specimens were obtained from 3 patients with clinically diagnosed laryngopharyngeal reflux and from 2 control subjects who had no signs or symptoms of reflux. Signs and symptoms were assessed by the Reflux Finding Score and the Reflux Symptom Index, respectively. Reverse transcription-polymerase chain reaction (RT-PCR) was performed to establish the mucin gene profile. Human hypopharyngeal epithelial cells were exposed to pH 7, 5, 4, and 2 with and without pepsin (0.1 mg/mL) for 20 minutes at 37 degrees C, and expression of selected mucins was analyzed via real-time RT-PCR., Results: Mucin 1-5, 7, 9, 13, 15, 16, and 18-20 transcripts were detected in normal laryngeal epithelium, whereas mucin 6, 8, and 17 transcripts were not. Mucins 2, 3, and 5 were expressed at reduced levels in patients with reflux-attributed laryngeal injury or disease. These mucin genes were up-regulated after exposure to low pH in vitro (p < 0.005). Pepsin inhibited this up-regulation (p <0.001)., Conclusions: Reflux laryngitis is associated with down-regulation of mucin gene expression.

Objectives/hypothesis: The newborn is heavily dependent on voice communication and therefore has relatively higher vocal demands and expenditures than the adult, the loudness output per mass performance exceeds that of the adult, and the newborn larynx exhibits significant histological and biochemical differences. The neonatal larynx is capable of sustaining relatively greater pitch and loudness than the adult over longer periods of time. This ability may be related to a more compact arrangement of collagen within the lamina propria, less interstitial space, and a uniform distribution of hyaluronic acid (HA). As HA is the primary determinant of vocal fold viscosity and water content, the distribution of HA in the superficial portion of the neonatal vocal fold is hypothesized to be related to newborn crying endurance. Our objective was to examine the histological structure and the quantity and arrangement of HA within the lamina propria of the pediatric larynx and review the relevant physiology of hyaluronic acid and its impact on voice production., Study Design: Histological and digital subtraction analysis., Methods: Intact, neonatal larynges were sourced from fresh cadaveric specimens. Trichrome stain was used to assess the collagen content and location in the tissues. HA was stained using a colloidal iron staining technique with and without incubation with bovine testicular hyaluronidase. Average optical density was calculated in tissue before and after treatment with hyaluronidase, and the stain intensity ratio was calculated., Results: A total of 14 larynges were suitable for examination, eight males and six females. Histological examination revealed a uniform appearance of the vocal fold without evidence of a distinct vocal ligament or layered structure. Colloidal iron staining revealed an even distribution of HA throughout the vocal fold with no significant difference between quadrants. Images of the colloidal iron-stained tissue had a mean pixel intensity of 82 of 255. Slides of vocal fold tissue treated with hyaluronidase revealed a pixel intensity of 106 of 255 for a 22% mean difference in stain intensity (P < .01)., Conclusions: The identification of the layered structure of the adult lamina propria has raised a number of questions as to the development and purpose of the human larynx. Based on histological observations from the current study, possible explanations for the physiological differences include differences in the distribution and tissue concentration of HA and consequently dynamic viscosity, oncotic affinity for water, and less intercellular space in the superficial lamina propria.

We have recently demonstrated that the severity of sleep-disordered breathing in obstructive sleep apnoea hypopnoea syndrome (OSAHS) can be reduced by lowering the surface tension (gamma) of the upper airway lining liquid (UAL). Morning xerostomia (related to oral breathing during sleep) is reported by most OSAHS patients. In the present study we examine relationships between breathing route, oral mucosal 'wetness' and the gamma of UAL. We studied eight healthy subjects (age, 25 +/- 5 years [mean +/- S.D.]; body-mass index, 23 +/- 2 kg m(-2)) during a 120 min challenge of both nasal-only breathing (mouth taped) and oral-only breathing (nose clip), each on a separate day (randomized). Both oral mucosal 'wetness' (5 s contact gravimetric absorbent paper strip method) and the gamma ('pull-off' force technique) of 0.2 microl samples of UAL obtained from the posterior pharyngeal wall were measured at 15 min intervals (mouth tape removed and replaced as required). Upper airway mucosal 'wetness' increased during 120 min of nasal breathing from 4.0 +/- 0.4 (mean +/- S.E.M.) to 5.3 +/- 0.3 microl (5 s)(-1) but decreased from 4.5 +/- 0.4 to 0.1 +/- 0.2 microl (5 s)(-1) with oral breathing (both P < 0.001, repeated-measures ANOVA, Tukey's multiple comparison test, post hoc test). Concurrently, the gamma of UAL decreased from 59.3 +/- 2.2 to 51.8 +/- 0.98 mN m(-1) with nasal breathing but increased from 64.4 +/- 2.7 to 77.4 +/- 1.1 mN m(-1) with oral breathing (P < 0.001). For the group and all conditions studied, gamma of UAL values strongly correlated with upper airway mucosal 'wetness' (correlation coefficient, r2 = -0.34, P < 0.001; linear regression). We conclude that oral breathing increases and nasal breathing decreases the gamma of UAL in healthy subjects during wakefulness. We speculate that nasal breathing in OSAHS patients during sleep may promote a low gamma of UAL that may contribute to reducing the severity of sleep-disordered breathing.

The matrix metalloproteinases (MMPs) are a family of proteolytic zinc-containing enzymes, which are responsible for the breakdown of the extracellular matrix components in pathological and physiological conditions. They are involved in basement membrane disruption, stroma and blood vessel penetration, metastasis and more recently there is evidence that they participate in tumor growth and angiogenic events. Matrix metalloproteinase 2 and 9 (MMP 2 and 9) belong to the gelatinases, a subgroup of MMPs, and have the capacity to degrade the triple helix type IV collagen of basal lamina of the basement membrane. With the present study, we tried to demonstrate the expression of MMP-9 immunohistochemically, comparatively in benign, premalignant and malignant lesions of the larynx. We studied 154 laryngeal lesions including 55 squamous cell carcinomas, 8 in situ carcinomas, 54 cases of dysplasia (of low and intermediate grade), 13 papillomas and 24 cases of keratosis. Overexpression of MMP 9 was observed in 74.4% and 50% in invasive and in situ squamous cell carcinomas respectively. In dysplastic cases, in papillomas and in keratoses the percentage of overexpression was 62.9%, 61.53% and 54.16% respectively and the expression of MMP-9 was significantly higher in invasive squamous cell carcinomas compared to dysplasias (p=0.000004). Also significantly higher was the expression of MMP-9 in dysplastic cases compared to papillomas (p=0.023). The MMP-9 expression was related neither to survival nor to the other available clinicopathological parameters (tumor size, grade, clinical stage, lymph node status and patient age). In conclusion, our study indicates that the expression of MMP-9 is up-regulated in a stepwise fashion, with two main steps, the first one, when a dysplastic lesion evolves and the next one, when the dysplasia progresses to invasive carcinoma.

Objective: : To investigate the relationship between esophagoscopic findings and total acid reflux time in the upper esophagus in the patients of laryngopharyngeal reflux disease (LPRD), and to evaluate whether pH<5 holding time is appropriate for diagnosing LPRD., Method: : Esophagoscopy and 24-h pH monitoring in the upper esophagus were performed in 29 patients suspected of LPRD and 19 healthy volunteers participated as controls. The total acid reflux times below pH 4 and 5 in the upper esophagus were compared between the LPRD group and the controls. The LPRD group was divided into LPRD with reflux esophagitis (RE) and LPRD without RE, and the total acid reflux times below pH 4 and 5 of these two groups were compared., Results: : Eight of the 29 (27.6%) LPRD patients had reflux esophagitis. The LPRD group showed a significantly longer acid reflux time than the control group at pH below 5 (P=0.009) but not at pH below 4 (P=0.48). The LPRD with RE had significantly longer acid reflux times below both pH 4 and 5 in the upper esophagus compared to those of the LPRD without RE. The P values using a cut-off value of pH 5 were smaller than that of pH 4 in all statistical results., Conclusion: : The LPRD patients showed significantly longer acid reflux time in the upper esophagus than the control group. In the LPRD group, the LPRD with RE patients had more frequent acid exposure in the upper esophagus than the LPRD without RE. A pH level of 5 could be appropriate as a cutoff level for the diagnosis of LPRD in upper esophagus pH monitoring.

A neurofibroma of the larynx is a rare disease that usually involves the arytenoids and the aryepiglottic fold. Pediatric patients with a laryngeal neurofibroma often present with progressive dyspnea. A definitive diagnosis of a laryngeal neurofibroma is based on the histopathologic demonstration of the characteristic spindle cells, and a positive result in immunohistochemical staining for S-100 protein. Tumor excision of laryngeal neurofibromas by an external approach was formerly common. We present a case of a solitary laryngeal neurofibroma in a 4-year-old child that was completely excised using direct laryngoscopy and a CO2 laser. There has been no recurrence after a follow-up of 4 years. For pediatric patients with an isolated laryngeal neurofibroma, endoscopic surgery provides an alternative approach due to its safety, effectiveness, and minimal invasiveness.

The development of the glottic site, in particular of its ventral area, was studied to better understand the spreading pathways of T1, T2 cancer. Serial sections of larynges from human embryos, fetuses and adults were observed. A dorsal, a ventral and an intermediate compartment were found on the basis of their maturation schedule. A commissure muscle which develops in the anterior one third of the glottic site and wraps the connection system of vocal ligaments was recognized. The inferior paraglottic space, the compartment structures and the localization of superficial and deep blood vessels and of glands in the ventral compartment and the components of Broyles ligament were studied during ontogenesis. The compartments identified here have clinical and oncological relevance. Their detailed knowledge offers a prerequisite for planning and performing compartment conservative surgery in T1, T2 cancer, based on their spreading pathways.

Tissue microarrays are a powerful new tissue-conserving technology in the study of cancer, allowing simultaneous study of a large number of tumor specimens. We sought to ascertain the utility of tissue microarrays in head and neck cancer pathology using squamous cell carcinoma of the larynx as a model system. Whole-specimen slides from 44 different laryngeal squamous cell carcinomas were stained for p53 expression. Microarrays were then generated by taking six 0.6-mm core biopsies from each of the 44 specimens. The whole sections and the microarrays were independently scored for p53 expression. Twenty-three (53%) of the 44 tumor specimens were positive for p53. Forty-four of the 264 core biopsies (17%) were not given a score because of the lack of tumor cells. Seventy-eight percent of the individual discs on the microarray had scores in agreement with those of the whole-section slides. Among biopsy discs with tumor cells present, 94.5% were in agreement with the whole-section slide. The average probability that four randomly chosen biopsy discs, considered together, would accurately identify the presence of p53 staining in a whole section was 0.97 (95% CI.93-1.0). We conclude that tissue microarrays for squamous cell carcinomas can accurately represent immunohistochemical results of whole-slide specimens when four or more samples are used. Tissue microarrays are an important technique that may be applied to immunohistochemical studies of head and neck cancer.

Background: Laryngopharyngeal reflux disease (LPRD) can have various causes. Because LPRD differs from gastroesophageal reflux disease (GERD), the pH monitoring has to be performed directly next to the entrance of the larynx. This is now possible using a new system called pH-RESPONSE., Methods: The ambulatory pH was monitored using the double probe pH-RESPONSE in 20 patients with suspected LPRD. The number of refluxes, the number of long refluxes and the period in which the pH was below 4 were compared at the level of the larynx with the data from the esophageal electrode. The DeMeester score was also determined., Results: The system was well tolerated by 19/20 patients. In 12 patients a GERD could be proved and ten also had LPRD. Surprisingly, these patients had no typical signs such as heart burn. The number of refluxes measured in the esophagus was a third that of the larynx. The average time of pH below 4 was 30 min., Conclusion: The pH-RESPONSE easily allows ambulatory 24 -h double probe pH monitoring to diagnose LPRD. Because large differences were found between data form the esophagus from the level of the larynx, 24-h double probe pH monitoring should be a standard procedure before starting any therapy for LPRD.

Objectives/hypothesis: Head and neck cancer tumor cell invasion is responsible for both local destruction and distant metastasis. Invasion is largely mediated by matrix metalloproteases that are thought to be induced by tumor cell derived extracellular matrix metalloprotease inducer (EMMPRIN) in surrounding fibroblasts. We hypothesize that EMMPRIN is overexpressed in laryngeal cancer., Study Design: Retrospective analysis of patients with supraglottic laryngeal cancer., Methods: Total protein immunoblotting and immunohistochemical analysis of normal and malignant tissue were performed to determine EMMPRIN expression. EMMPRIN immunoreactivity in 33 patients was correlated with clinicopathological features and survival., Results: Whole-tissue lysates of tumors (n = 8) and metastatic lymph nodes (n = 2), but not normal skin (n = 8) or mucosa (n = 6), expressed significant amounts of EMMPRIN by immunoblotting. EMMPRIN membrane immunoreactivity (transmembrane EMMPRIN score) was associated with nodal positivity (P =.07), and it was a borderline significant predictor of survival (Hazards Ratio = 2.4; 95% CI, 0.88-6.55). As a categorical variable, higher transmembrane EMMPRIN score was associated with higher mortality., Conclusions: The present study helps to establish EMMPRIN as a widely expressed protein in dysplastic mucosa and supraglottic laryngeal cancer, but not in normal epithelial counterparts.

We report here the isolation and characterization of the mouse Muc5b mucin gene (mMuc5b). We determined its complete cDNA sequence, its genomic organization, and chromosomal localization. Moreover, we analyzed the expression of this gene by reverse-transcription PCR and in situ hybridization. The structure of the gene was determined from a genomic cosmid clone that encompasses the entire mMuc5b gene, including the 5'-flanking region. The mMuc5b gene spans approximately 36 kb and contains 49 exons. It is located on mouse distal chromosome 7. mMuc5b encodes at least two transcripts by alternative splicing of the second exon, the longest one being 14.9 kb in length. The deduced peptide contains 4782 amino acids. Its central region can be subdivided into 10 imperfect repeats, each composed of a cysteine-rich domain followed by a threonine, serine, and proline-rich mucin-type domain. It is flanked by cysteine-rich domains similar to cysteine-rich domains of pre-pro-von Willebrand factor. Comparison with its human homologue MUC5B revealed common features including high sequence similarities in the 5' and 3' regions, and the conservation of the genomic organization. In contrast, mMuc5b differs from its human homologue, since no highly tandemly repeated sequences could be identified within its central region. mMuc5b is expressed mainly in laryngeal mucous glands, and at a lesser extend in stomach and duodenum.

UDP-glucuronosyltransferases (UGTs) have been implicated as important detoxifying enzymes for several major tobacco carcinogens. Because the aerodigestive tract is a primary target for exposure to tobacco smoke carcinogens, the major goal of the present study was to determine whether aerodigestive tract tissues exhibit glucuronidating activity against metabolites of benzo[a]pyrene (BaP) and to explore the pattern of expression of UGT genes in a series of aerodigestive tract tissue specimens. Glucuronidation of the phenolic BaP metabolites 3-, 7-, and 9-hydroxy-BaP was observed in all upper aerodigestive tract tissue microsome specimens tested, as determined by high-pressure liquid chromatography analysis. Glucuronidating activity toward the procarcinogenic BaP metabolite trans-BaP-7,8-dihydrodiol(+/-) was also detected in aerodigestive tract tissues. By semiquantitative duplex reverse transcription-polymerase chain reaction analysis, UGT1A7 and UGT1A10 were shown to be well expressed in all aerodigestive tract tissues examined, including tongue, tonsil, floor of mouth, larynx, and esophagus. UGT1A8 and UGT1A6 were expressed primarily in larynx; no expression was observed for UGTs 1A1, 1A3, 1A4, 1A5, 1A9. Of the family 2B UGTs, only UGT2B4 and UGT2B17 exhibited significant levels of expression in aerodigestive tract tissues. Of the aerodigestive tract-expressing UGTs, only UGTs 1A7, 1A8, and 1A10 exhibited glucuronidating activity against 7-hydroxy-BaP, with UGT1A10 exhibiting the highest affinity as determined by kinetic analysis (K(m) = 49 microM). No UGT expression or glucuronidating activity was observed for any of the lung specimens analyzed in this study. These results suggest that several family 1 UGTs may potentially play an important role in BaP detoxification in the aerodigestive tract.

Background: It has been speculated that non-specific defence mechanisms of the epithelium and subepithelial seromucous glands play a role in the larynx and lungs in cases of sudden infant death., Methods: The larynx and trachea from five children who had died of sudden infant death (SID) syndrome and five control cases of comparable age were compared for the presence of lectin binding sites (12 different lectins tested)., Results: The secretory product of mucin producing cells contained carbohydrates including galactose and sialic acids. Binding sites for fucose and N-acetyl-galactosamine were only present in some of the specimens and distribution revealed no correlation between cases of SID and controls. Epithelial cells and serous cells of seromucous glands contained binding sites for sialic acid in cases of SID and controls. Moreover, binding sites for mannose were detected in these cells but were only present in SID cases. The difference between the SID and control groups as to the presence/expression of concanavalin A was highly significant., Conclusions: It is suggested that mucus hypersecretion in SID occurs in response to bacterial toxins or viral infection and is not specific. The different binding sites for mannose in cases of SID and controls could indicate differences in the production of antimicrobial peptides. A disturbed expression pattern of antimicrobial peptides in children who later succumb to SID could be responsible for an imbalance of the local microflora with a higher density of microorganisms on the mucosa. Further studies are required to elucidate the pattern of expression of antimicrobial peptides in subsequent SID victims.

Extramedullary plasmacytoma of the larynx and localized laryngeal amyloidosis are 2 uncommon disease entities that are exceedingly rare in children. We report a case of a 12-year-old girl presenting with progressive hoarseness who was subsequently found to have extramedullary plasmacytoma coexisting with localized amyloidosis involving the larynx. Results from the immunohistochemical and molecular studies showed that the tumor cells of the plasmacytoma were monoclonal (kappa-restricted), strongly supporting their neoplastic nature. The biochemical nature of the amyloid deposits was also shown to be of kappa immunoglobulin light chain, suggesting the pathogenetic relationship between the plasmacytoma and amyloid deposition in the larynx of this patient. There was no other evidence of malignancy or amyloidosis elsewhere. On a follow-up period of 4 years, this patient was well and asymptomatic. We believe that this represents the first case in the literature showing the coexistence of extramedullary plasmacytoma and localized amyloidosis of the larynx in children. This case also supports the concept that localized laryngeal amyloidosis may be a manifestation of low-grade B-cell neoplasms.

To shed light on supraesophageal complications of reflux disease, sensory innervation--particularly, distinct distribution, area, and density of sensory fibers--of the feline pharyngolaryngeal mucosa was reported. The investigations were performed by means of histochemistry (tracer techniques) and immunohistochemistry. The pharyngeal mucosa from the Eustachian cushion to the middle level of aryepiglottic fold, except the laryngeal surface of epiglottis, was supplied by the glossopharyngeal sensory fibers, whereas the laryngeal sensory fibers innervated between the apex of epiglottis and the level of the first tracheal ring in the larynx and between the middle level of aryepiglottic fold and the caudal end of piriform sinus in the pharynx. Most areas of the mucosa, except the subglottis, had unilateral innervation. The subglottis, including the caudal aspect of vocal fold and the posterior glottis, had bilateral supply with ipsilateral predominance. The density of sensory fibers in the vestibule of larynx involving the posterolateral aspect of arytenoid eminence was much heavier than the other parts. Sensory nerve fibers around the caudal pole of palatine tonsil, and in the root of tongue and the hypopharyngeal wall also were dense. Regional distribution and density of substance P and calcitonin gene-related peptide immunoreactive fibers showed almost the same pattern as did the sensory fibers.

The percentage of malignant transformation of laryngeal dysplastic lesions is difficult to estimate. There is a need for new histological markers which could enable more objective assessment of the premalignant stages of the larynx and help in estimation of the potential of future neoplastic progression. We performed a retrospective study to determine whether immunohistochemical staining for the proliferating cell nuclear antigen (PCNA), tumour suppressor gene protein p53 and antiapoptotic protein bcl-2 may be prognostic factors in laryngeal epithelial lesions. Staining was performed on 57 paraffin-embedded biopsies from patients with clinically detected precancerous stages of the larynx. Histopathologic examination revealed normal epithelium in six cases, mild dysplasia in 20 cases, moderate dysplasia in 18 cases, severe dysplasia in seven cases, CA in situ in four cases, papilloma in one case and CA invasivum in one case. The p53 count in mild and moderate dysplasia was 26.8 and 38.6%, respectively. This difference was statistically significant. There was significant correlation between PCNA and p53 scores. There was also a relationship between the scores of these markers and bcl-2 expression. In ten out of 45 cases of dysplastic lesions the invasive cancer developed in 4 years of follow-up. The correlation between PCNA score and malignant progression of the dysplastic lesions was on the statistical borderline. There was significant relationship between malignant transformation and age of the patients.

Laryngeal papillomas are benign tumors caused by human papillomaviruses types 6 and 11. This study addressed alterations in levels of signal transduction from the epidermal growth factor receptor (EGFR) in papillomas and cultured papilloma cells compared to normal tissue and cells. Mitogen-activated protein kinase (MAPK) was activated to a greater extent, phosphotyrosine was more abundant, and EGFR was overexpressed in laryngeal papillomas compared to normal laryngeal epithelium by Western blot analysis. The EGFR was 3 times more abundant in cultured papilloma cells than in normal laryngeal cells by Scatchard analysis and Western blot, without gene amplification or an increase in steady-state levels of mRNA. Following stimulation with EGF, a significant portion of the EGFR was recycled to the surface in papilloma cells, whereas in normal cells, it was not. Tyrosine kinase activity and activation of MAPK was more responsive to epidermal growth factor stimulation in papilloma cells than in uninfected primary laryngeal cells. PD153035, a specific inhibitor of the EGFR, and an EGFR-specific antibody that blocks ligand binding completely abrogated basal MAPK activation by endogenous ligands in laryngeal papilloma cells. These results demonstrated that infection of laryngeal epithelium by low-risk human papillomaviruses elevates the EGFR by posttranslational mechanisms, increasing its responsiveness to ligand-mediated activation. They also showed that MAPK activation in laryngeal papillomas depends upon ligand-mediated EGFR stimulation.

Transcription factors belonging to the family of CAAT enhancer binding proteins (C/EBPs) programme differentiation in a wide variety of cells. We asked about the expression of C/EBPs in squamous epithelium. Using immunohistochemistry, C/EBPalpha and C/EBPbeta were easily detected in tissue sections from normal larynx or laryngeal papillomas, benign tumours with a papillomavirus aetiology and characterized by abnormal differentiation. A temporal expression of these C/EBPs occured as keratinocytes differentiated. In both tissues, C/EBPbeta appeared to be exclusively nuclear. In normal tissue, the greatest amount of C/EBPbeta was present in the spinous layer, and much less occurred in the granular layer. The basal layer of the laryngeal papillomas contained the most C/EBPbeta. Less was present in the spinous layer. Little C/EBPbeta was in the granular layer. Much C/EBPalpha was in the cytoplasm in both tissues. In normal tissue, nuclear C/EBPalpha staining was virtually absent in the basal layer, and became present in the spinous layer. Nuclear C/EBPalpha was randomly distributed in all layers in papillomas. Using immunoblots and Southwestern blots, we detected abundant truncated isoforms of C/EBPalpha in the papillomas. Since differentiation of many tissues is determined by the relative amounts of different C/EBPs, our data supports a role for C/EBPs in the differentiation of squamous epithelium.

A dysmorphic infant is described who presented with laryngeal collapse leading to intubation and respiratory problems that were assigned clinically to the Sussman syndrome. The baby had repeated episodes of respiratory distress necessitating assisted ventilation. At 6 months old, uvulopharyngopalatotomy was done to enlarge the supraglottic airway without any benefit. Surgical reduction of the tongue and cricoid splitting did not ameliorate the respiratory distress; repeated extubation attempts failed with the baby developing stridor, respiratory distress, and episodes of cardiac arrest. At 10 months old he developed seizures and computed tomography showed diffuse cerebral atrophy consisted with hypoxic-ischaemic damage. He died at 17 months old. Western blots using antibodies against collagen alpha 1 (II) showed an absence of collagen type II in laryngeal tissue, which may explain the laryngeal collapse and impaired respiratory functions.

The distribution of intraepithelial nerve fibres and neuroendocrine cells within the surface and glandular epithelium of human nasal mucosa and larynx was examined using immunohistochemical techniques. Neuronal structures were immunostained for the general neuroendocrine marker protein gene-product (PGP) 9.5, and the two neuropeptides substance P (SP) and calcitonin gene-related peptide (CGRP) using immunofluorescence and streptavidin-biotin-peroxidase complex (S-ABC) methods. Intraepithelial nerve fibres with free nerve endings contained PGP 9.5 and were found within the respiratory surface epithelium of the nasal mucosa and the squamous epithelium of the larynx. A subpopulation of these nerve fibres showed positive immunoreactivties with antibodies against SP and CGRP. Nerve fibres within the ductal epithelium of subepithelial excretory ducts passing the basal membrane and reaching the luminal part were detected. These nerve fibres showed CGRP-like immunoreactivity but not for SP. A dense network of nerve fibres within the squamous surface epithelium was detected in the subglottic and epiglottic region containing CGRP and SP in a small subpopulation of nerve fibres. Single intraepithelial taste buds in the epiglottic region and neuroendocrine cells within the subglottic epithelium expressed PGP 9.5.

Background: Mucosa-associated lymphoid tissue (MALT) plays a central role in mucosal immunity. Whereas the characteristics and function of MALT in the intestine are well established, almost nothing is known about MALT in the larynx., Methods: In this study we examined the morphology and the lymphocyte subset composition of MALT in the larynges of children who had died of sudden infant death or various defined traumatic or nontraumatic causes., Results: Organized lymphoid tissue was found in the supraglottic parts of the larynx in nearly 80% of the children in both groups. This lymphoid tissue showed all morphological signs of MALT, such as typical lymphoid follicles with germinal centers, infiltration of the overlying epithelium by lymphocytes, and high endothelial venules (HEV). Thus we will use the term LALT (larynx-associated lymphoid tissue) to refer to this tissue. The lymphoid follicles of LALT contained mainly B lymphocytes with some CD4+ lymphocytes in the germinal centers. Remarkably, T lymphocytes of both subset types and B lymphocytes were observed in comparable numbers in the parafollicular area., Conclusions: We assume that LALT is a physiological structure of the larynx in young children. The morphology and the distribution of lymphocyte subsets are similar to those of MALT in the human gut. LALT may be a regular part of the mucosal immune system in young children with the role of respiratory inductive site for mucosal immunity.

The distribution and innervation of the canine laryngeal taste buds were observed using immunohistochemistry with antibodies against protein gene product 9.5 (PGP 9.5) and neurofilament protein (NFP). We also observed the immunohistochemical distribution of serotonin, tyrosine hydroxylase (TH) and various neuropeptides including calcitonin gene-related peptide (CGRP), substance P (SP), vasoactive intestinal peptide (VIP), galanin, methionine enkephalin (ENK) and neuropeptide Y (NPY). The taste buds in the canine larynx were densely distributed in the mucosa at the basal portion of the epiglottis and cuneiform process of the arytenoid cartilage. The taste cells were immunoreactive for PGP 9.5 and serotonin. The nerve fibers with immunoreactivity for PGP 9.5 in the taste buds were observed in the perigemmal region and intra- and subgemmal plexuses, and these were classified into two types based on their diameter. The thick nerve fibers corresponded to the fibers immunoreactive for NFP, while the thin nerve fibers corresponded to the fibers immunoreactive for TH and various neuropeptides. Numerous nerve fibers immunoreactive for SP and CGRP were observed in the perigemmal region, and intra- and subgemmal plexuses. A few galanin- and ENK-immunoreactive nerve fibers were also observed in the taste buds, whereas NPY-immunoreactive nerve fibers were noted beneath them. All peptide-containing fibers except for VIP-immunoreactive nerves were situated in the subgemmal regions. In conclusion, the multiple innervation to the laryngeal taste buds were documented. Thick nerve fibers are likely to be irritant receptors, while thin varicose nerve fibers seem to regulate taste buds themselves. The laryngeal taste buds may be among the important structures which are sensitive to exogeneous chemical and/or mechanical stimuli, for the protection of the airway and the regulation of the respiratory function.

The differences between the submucosal glands and globet cells of the subglottic mucosa were studied by lectin immunohistochemistry in 4 larynxes of Syrian golden hamster (Mesocricetus auratus). Submucosal glands showed intense reactivity to HPA, N-WGA and N-PNA and reactivity to PNA, UES-I, and ConA, but globet cells showed no reactivity.

Laryngeal papilloma (LP) is the most frequent benign laryngeal epithelial tumor caused by human papillomaviruses (HPV) types 6 and 11. In the present study, we were interested in whether we can find any prognostic markers which might reflect the biological behavior of the covering epithelium in LP. We focused our attention on the determination of HPV infection, the detection of p53 protein, and c-erbB-2 protein in 24 biopsy specimens of LP. We confirmed the HPV 6 and 11 etiology in 23 of 24 LP. In these lesions the overexpression of p53 protein increased with the grade of epithelial abnormalities. The distribution of positive cells changed from scattered and focal, in simple and abnormal hyperplasia, to diffuse in atypical hyperplasia. It has been shown that in the presence of HPV types 6 and 11 found in LP, p53 can still preserve its tumor suppressor activity. Infection with HPV types 6 and 11 might therefore account for the significantly lower rate of malignant transformation in LP. Two staining patterns for c-erbB-2 protein were observed in the hyperplastic epithelium covering LP: membranous and cytoplasmic. With the increasing grade of epithelial abnormalities, cytoplasmic staining became predominant, and c-erbB-2 positivity sometimes occupied the whole epithelial thickness. This may represent either an alteration in the processing stability of the c-erbB-2 mRNA, gene amplification, or even an artefact.

An immunohistochemical analysis of overexpression of epidermal growth factor receptor (EGFR), c-erbB-2, and p53 proteins was performed on 43 biopsies of laryngeal epithelial hyperplastic lesions (EHLL), classified according to the Kambic-Lenart classification, and in 11 cases of laryngeal carcinoma (SCCL). The aim of the present study was to determine whether there is a correlation between the staining patterns of these proteins and different grades of EHLL, and to reveal their possible prognostic value. We compared the staining patterns of atypical hyperplasia adjacent to cancer with the same type of lesions which have not turned malignant. p53 and EGFR overexpressions were detected in 28/54 (52%) and 33/54 cases (61%), respectively, and tend to increase with the degree of epithelial changes. The intensity of staining in various grades of EHLL adjacent to cancer was more pronounced than the same type of lesions which have not progressed to cancer. c-erbB-2 was weakly positive in the majority of cases, and changed from predominantly membranous in simple hyperplasia to cytoplasmic staining in abnormal and atypical hyperplasias. There was no significant statistic correlation between the amount of positive cells for all proteins and the grade of epithelial abnormalities. We conclude that the overexpression of each biomarker itself adds little predictive value over routine histomorphology, and cannot be regarded as a reliable prognostic factor for EHLL. However, the histologic characteristics of atypical hyperplasia together with the immunostaining patterns of EGFR and p53 up to two-thirds or more of the epithelial thickness could be considered a reliable pattern which correlates with the progression to cancer.

Areas of myxoid connective tissue were regularly found in the aryepiglottic and vestibular folds of operative resections (n = 10) or fresh postmortem specimens (n = 5) of the human larynx. Myxoid tissue was often attached to elastic cartilage (epiglottic, arytenoid, and corniculate cartilage) or spatially related to mucosal glands. This was characterized by ramified cells (myxoid cells) in an ample acidic matrix with few strands of collagen fibers. Extracellular matrix showed alcianophilia at pH 2.5 and metachromasia while tissue digestion with hyaluronidase abolished these staining reactions. Immunohistochemistry revealed reactivity of myxoid cells for S-100 alpha and S-100 beta protein, glial fibrillary acidic protein, and neuron-specific enolase. Elastic cartilage chondrocytes also stained for these markers while fibroblasts remained unstained. Reactivity for identical-neuroectodermal marker proteins of myxoid cells and chondrocytes indicated their close relationship. The presence of myxoid tissue in the larynx, as evidenced by stellate cells immunoreactive for neuroectodermal marker proteins, should be considered when using these markers in diagnostic pathology.

In our 5-year biopsy study (1991 95) 51% of keratoses and 16% of adult-onset papillomas contained certain degrees of dysplasia. Koilocytosis associated with HPV infection was prominent in papillomas and the majority of keratosis but negligible in cancer. Using mouse monoclonal antibody against human p53 we detected by immunohistochemistry mutated p53 overexpression not only in cancer but in some cases of keratoses and papillomas. CD44 is a multifunctional integral cell membrane glycoprotein and is found in many mesenchymal, epithelial and tumor cells. CD44 has a great number of splice variants, isoforms supposedly implicated in development, progression and metastasis formation of tumors. Using monoclonal antibody to CD44 our study suggests that the expression of pan CD44 at successive stages of human laryngeal carcinogenesis gives preliminary information about the selection advantage to the tumor cells.

The expression of estrogen receptors was biochemically assessed in a series of 46 cases of consecutive laryngeal carcinomas. ER-D5 and EGFr were also immunohistochemically detected in 44 and 46 cases respectively. There was no correlation between the expression of these molecules and the typical anatomo-clinical prognostic parameters of laryngeal tumors. The concomitant expression of estrogen receptors, ER-D5 and EGFr was also investigated in normal, hyperplastic and dysplastic epithelium. An increasing expression of ER-D5 and EGFr from normal to hyperplastic and dysplastic epithelium was noted. These data suggest a possible role of hormone receptors in the laryngeal carcinogenesis.

Background: The larynx is a complex organ composed of different connective tissue elements. So far, the extracellular matrix of the larynx has not been thoroughly described. Hyaluronan is a matrix polysaccharide with physicochemical effects and biological cell functions in soft connective tissues., Methods: The histochemical distribution of hyaluronan (hyaluronic acid, hyaluronate) was studied in tissue sections from various levels of the rabbit larynx by means of a hyaluronan-binding protein and avidin biotin peroxidase staining. Microwave-aided fixation was used to retain the extracellular location of hyaluronan., Results: Hyaluronan accumulated chiefly in the subepithelial lamina propria and in the connective tissue enclosing striated muscle fibres of the thyroarytenoid muscle and vocalis muscle. This localization contrasted sharply with the weak staining for hyaluronan in muscles external to the thyroid cartilage. Intensive staining for hyaluronan was found in perivascular and periglandular connective tissue, as in the vacuoles of the hyaline cartilage of the thyroid, cricoid and arytenoid cartilages, and to a lesser extent in the lacunae of the chondrocytes and in the perichondrium of the elastic cartilage of the epiglottis., Conclusions: Hyaluronan was heterogenously distributed in the rabbit larynx. It was abundant in intrinsic laryngeal muscles performing small, precise, and rapid movements and in the subepithelium at the glottic level, where it may facilitate mucosal movements. The abundant hyaluronan in the subglottic region may be involved in the control of vascular leakage and edema formation.

The present study employed immunohistochemical methods to study the cytokeratin (ck) and vimentin expression in 40 cases of laryngeal squamous cell carcinoma. Specific monoclonal isoform antibodies and mixes of antibodies vs. a specific molecule were used in order to determine what cytokeratins were present as accurately as possible. In this sampling two ck patterns were identified based on whether the ck pair 8/18 was present or not. The ck 8/18 positive cases were further broken down into three sub-groups based on the expression of one of the following: the ck 4/13 pair, ck 1/10 pair or vimentin. A statistically significant relationship was found between these sub-groups, the site at which the neoplasm arose and the tendency toward regional metastases. Moreover, it was found that the presence of ck 13 in a squamous cell carcinoma is correlated with the less aggressive forms, as indicated in the literature.

Helospectin (HS) and pituitary adenylate cyclase activating peptide (PACAP) are newly discovered peptides isolated from the salivary gland venom of the lizard Heloderma horridum and the ovine hypothalamus, respectively. They show chemical similarities to vasoactive intestinal polypeptide (VIP), appear to have similar functions and are present in gut, brain, lung, male and female genitourinary tract. In the present study, the distribution of the helospectin and PACAP-27 in the human upper respiratory system was investigated using indirect immunofluorescence and electron-microscopical ABC-pre-embedding methods. Immunohistochemistry revealed helospectin-like (HS-LI) and PACAP-like (PACAP-LI) immunoreactivity in nerve fibers in human nasal, the larynx (vocal cord, ventricular fold, epiglottis), the tongue and the soft palate mucosa. Helospectin-LI and PACAP-LI containing nerve fibers were mainly found in close association to blood vessels and glandular structures. Colocalization studies carried out by application of double immunofluorescence showed that HS and/(or) PACAP-LI coexist with VIP in apparently the same nerve fibers in the upper respiratory system, although single nerve fibers seem to exclusively express helospectin. The localization patterns of helospectin and PACAP-LI in the human upper respiratory system suggests their possible involvement in the regulation of secretory activities and local blood flow.