Dilated Minute Chambers in Laryngeal Vocal Fold Polyps: Histopathological and Ultrastructural Features.

In Reinke's space of human vocal fold, type III collagen forms a three dimensional network and this contains numerous minute chambers in between these fibers. These compartments are occupied by glycosaminoglycans and glycoproteins. In laryngeal fold lesions, such as Reinke's edema and vocal fold polyps, proteoglycan (PG)/hyaluronic acid (HA) components of extracellular matrix increased. We investigated the size and quantity of the minute chambers within Reinke's space, filled with PG/HA with the aid of transmission electron microscopy. Eight vocal fold polyps and 10 mucosal biopsies (as control group) were all evaluated by light microscopy and electron microscopy. We detected that PG/HA in extracellular matrix had been increased in vocal fold lesions when compared with control group, by Alcian Blue-pH 2.5 stain. The mean volume of the chambers in Reinke's space of normal larynx was measured as 0.040233 µm2 whereas the mean volume of these chambers in vocal fold polyps was measured as 6.420221 µm2. The difference between the volumes of these chambers in vocal fold polyps and in control group was statistically significant (P = 0.001). Within these chambers PG/HA were found and PG/HA filling these chambers were increased in vocal fold polyps. We think proteoglycan and glycosaminoglycans, especially HA, play an important role in determining biochemical properties of vocal fold lesions.

Morphology of P2X3-immunoreactive nerve endings in the rat laryngeal mucosa.

The morphological characteristics of P2X3-immunoreactive nerve endings in the laryngeal mucosa were herein examined using immunohistochemistry with confocal laser microscopy. Ramified intraepithelial nerve endings immunoreactive to P2X3 were distributed in the epiglottis and arytenoid region. The axon terminals of P2X3-immunoreactive ramified endings were beaded or flat in shape. These endings were also immunoreactive to P2X2 and not identical to the nerve endings immunoreactive to Na(+)-K(+)-ATPase α3-subunit, substance P (SP), and calcitonin gene-related peptide (CGRP). P2X3-immunoreactive axon terminals were also immunoreactive to vGLUT1, vGLUT2, and vGLUT3. In addition to ramified endings, P2X3-immunoreactive nerve endings were associated with α-gustducin-immunoreactive solitary chemosensory cells and/or SNAP25-immunoreactive neuroendocrine cells. Furthermore, P2X3-immunoreactive nerve endings were also observed in the taste bud-like chemosensory cell clusters of the stratified squamous epithelium covering epiglottic and arytenoid cartilage. The P2X3-immunoreactive nerve endings that associated with sensory and/or endocrine cells and chemosensory cell clusters were also immunoreactive to P2X2, vGLUT1, vGLUT2, and vGLUT3, but not to SP or CGRP. In conclusion, P2X3-immunoreactive nerve endings may be classified into two types, i.e., intraepithelial ramified nerve endings and nerve endings associated with chemosensory cells and neuroendocrine cells.

Comparative cytotoxicity of dolomite nanoparticles in human larynx HEp2 and liver HepG2 cells.

Dolomite is a natural mineral of great industrial and commercial importance. With the advent of nanotechnology, natural minerals including dolomite in the form of nanoparticles (NPs) are being utilized in various applications to improve the quality of products. However, safety or toxicity information of dolomite NPs is largely lacking. This study evaluated the cytotoxicity of dolomite NPs in two widely used in vitro cell culture models: human airway epithelial (HEp2) and human liver (HepG2) cells. Concentration-dependent decreased cell viability and damaged cell membrane integrity revealed the cytotoxicity of dolomite NPs. We further observed that dolomite NPs induce oxidative stress in a concentration-dependent manner, as indicated by depletion of glutathione and induction of reactive oxygen species (ROS) and lipid peroxidation. Quantitative real-time PCR data demonstrated that the mRNA level of tumor suppressor gene p53 and apoptotic genes (bax, CASP3 and CASP9) were up-regulated whereas the anti-apoptotic gene bcl-2 was down-regulated in HEp2 and HepG2 cells exposed to dolomite NPs. Moreover, the activity of apoptotic enzymes (caspase-3 and caspase-9) was also higher in both kinds of cells treated with dolomite NPs. It is also worth mentioning that HEp2 cells seem to be marginally more susceptible to dolomite NPs exposure than HepG2 cells. Cytotoxicity induced by dolomite NPs was efficiently prevented by N-acetyl cysteine treatment, which suggests that oxidative stress is primarily responsible for the cytotoxicity of dolomite NPs in both HEp2 and HepG2 cells. Toxicity mechanisms of dolomite NPs warrant further investigations at the in vivo level.

Morphology and chemical characteristics of subepithelial laminar nerve endings in the rat epiglottic mucosa.

The laminar nerve endings are distributed in the laryngeal mucosa, and described as sensory receptors evoked by laryngeal pressure changes. The present study aimed to determine detailed morphological characteristics of the laryngeal laminar endings of the rat. Immunohistochemistry for Na(+)-K(+)-ATPase, α(3) subunit, showed that laminar endings were distributed in the entire laryngeal surface of the epiglottis. The parent axons of the endings were thick in diameter, and they were branched and continued to the endings. In some cases, several endings from different parent axons fused into a large complex structure of 500 µm in width. The laminar endings were also immunoreactive for vesicular glutamate transporter 1 (vGLUT1) and vGLUT2, but not for P2X(3) purinoceptor. Around the laminar endings, terminal Schwann cells with immunoreactivity for S-100 protein were closely associated with axon terminals. Use of scanning electron microscopy with alkaline maceration method showed that the terminal Schwann cells consisted of a rounded perinuclear region and lamellar cytoplasmic processes. Ultrastructurally, axon terminals with numerous mitochondria were partly covered with Schwann cell sheath, and some terminals intruded into the epithelial layer. Clear vesicles of 50 nm in diameter were also observed especially in small cytoplasmic processes of 400 nm to 1 µm in size. The results in the present study suggested that the laminar endings in epiglottic mucosa have morphological characteristics of slowly adapting mechanoreceptors and contribute to sensation of laryngeal pressure via mucosal tension.

Presence of task-1 channel in the laryngeal mucosa in the newborn lamb.

Nearly 40 potassium channels have been described in respiratory epithelial cells. Of these are found several members of the 4-transmembrane domain, 2-pore K(+) channel family (K2P family), namely Twik-1 and -2, Trek-1 and -2, Task-2, -3, and -4, Thik-1, and KCNK7. The aim of this study was to verify whether the Twik-related acid-sensitive K(+) channel, subtype 1 (Task-1) (also known as KCNK3), is present in the laryngeal mucosa in the newborn lamb. Through the use of immunohistochemistry and nested polymerase chain reaction (PCR) amplification, results indicate that Task-1 protein and mRNA are present in the laryngeal mucosa, in both the ciliated, pseudostratified columnar (respiratory) epithelium and the nonkeratinized, stratified squamous epithelium. The complete ovine Task-1 protein sequence showed high homology levels with previously reported mouse, bovine, and human Task-1 sequences. This includes a complete homology for the C-terminal amino acid sequence, which is mandatory for protein trafficking to the cell membrane. These results represent the first demonstration that Task-1, a pH-sensitive channel responsible for setting membrane potential, is present in the laryngeal mucosa of a newborn mammal.

Distribution of mucins and antimicrobial substances lysozyme and lactoferrin in the laryngeal subglottic region.

The subglottic region of the larynx is of high clinical relevance with regard to infections and malignancies. Little is known about the distribution of mucins and antimicrobial substances in this area. In this study, we have investigated the mucin distribution in the normal subglottis of the larynx. Moreover, we analysed the expression of lysozyme and lactoferrin in this area. Therefore, the subglottic region of 34 larynges was investigated immunohistochemically with different antibodies to mucins and antimicrobial substances. The epithelium reacted positive with antibodies to mucins MUC1 (34/34), 5AC (26/34), 5B (10/34), 7 (8/34), 8 (10/34) and 16 (19/34); submucosal glands were positive to mucins MUC1 (34/34), 5B (10/34), 7 (8/34), and 16 (19/34); high columnar epithelial cells and serous parts of subepithelial seromucous glands were also positive for lysozyme (34/34) and lactoferrin (34/34). The results show that human subglottic epithelium and subepithelial submucosal glands produce a broad spectrum of mucins that is almost comparable with that in other areas of the respiratory tract. We hypothesize that the mucin diversity of the subglottis has an impact on positive functional consequences during vocal production and antimicrobial defence. This antimicrobial defence is supported by synthesis and secretion of antimicrobial substances such as lysozyme and lactoferrin. Moreover, knowledge of the observed distribution pattern of mucins in the subglottis can be a useful tool for a classification of subglottic laryngeal carcinomas.

[The prevalence of E6/E7 HPV type 16 in laryngeal cancer and in normal mucosa]. / Obecnosc E6/E7 wirusa HPV16 w raku krani oraz w makroskopowo niezmienionej blonie sluzowej z marginesu operacynego.

Many authors suggest that HPV infection can play a great role in development of benign and malignant tumours of upper respiratory tract in human. The aim of this study was to determine the prevalence of E6/E7 HPV-16 in laryngeal squamous cell carcinoma, and normal laryngeal mucosa, and to analyse their correlation with sex, lymph node status, primary tumor stage, localization, and histological differentiation. HPV 16 DNA presence was analysed using PCR technique in 72 samples of laryngeal carcinoma and in samples of 50 normal mucosa. Human papillomavirus was detected in 26 (36.1%) of the 72 patients. There was no statistically significant correlation HPV positivity and clinicopathological features of the analysed group. In 5 (10%) of 50 samples of normal mucosal. HPV 16 presence in normal mucosa and in laryngeal squamous cell carcinoma was detected in 3 patients. Our observations suggest that HPV16 can play a role in pathogenesis of laryngeal cancer.

Detection of cytokeratins in normal and malignant laryngeal epithelia by means of reverse transcriptase-polymerase chain reaction.

Cytokeratins (CKs) are a subgroup of intermediate filament proteins that take part in forming the cytoskeleton. The epithelial cells in different organs express distinct CKs, and this expression may be modified during malignant transformation. Here we describe the use of reverse transcriptase-polymerase chain reaction followed by Southern blotting to determine the profile of expression of CKs in both normal and malignant laryngeal samples. Thirty-six samples were subjected to histologic examination and classified as 17 squamous cell carcinomas, 3 dysplastic lesions, and 16 normal samples. CK8 and CK19 were expressed in almost all samples, both cancerous and normal, and were therefore used to verify the integrity of RNA. Expression of CK2, CK9, and CK20 was not detected in any of the samples, normal or cancerous. CK15 and CK18 showed low sensitivity for detection of cancer (36.4% and 45.5%, respectively). CK10 showed relatively high sensitivity (91%), but only moderate specificity (69.2%). Only CK17 showed both high sensitivity and specificity (91% and 92.3%, respectively; positive predictive value, 91%). We propose that CK17 may be considered a promising candidate to use as a molecular marker for malignant transformation in laryngeal squamous cell carcinoma.

Expression of Fas and Fas ligand in esophageal tissue mucosa and carcinomas.

The Fas ligand (FasL) and its receptor Fas play a key role in the initiation of an apoptotic pathway. We describe the expression of Fas receptor and ligand pair antigens in surgical samples collected from a cohot of 89 patients compared with 89 squamous cell carcinomas (SCCs), 45 dysplasias and 42 normal mucosae of the esophagus. TUNEL method was performed in 89 SCCs. Evaluation of FasL on normal mucosae displayed a heterogeneous immunoreaction in a minority of specimens, whereas SCCs exhibited a more extended and homogeneous reactivity. Fas-positive carcinoma cells revealed frequent apoptosis. Furthermore, a significantly longer disease-free survival can be observed in patients with Fas-positive tumors than in Fas-negative carcinomas and in patients with FasL-negative tumors than in FasL-positive carcinomas. In conclusion, FasL expression may play an important role in tumor progression. On the other hand, Fas-expressing carcinoma cells were associated with frequent apoptosis. Both FasL and Fas expressions correlate with prognostic significance in esophageal SCCs.

Protective effect of 3-aminobenzamide, an inhibitor of poly (ADP-ribose) synthetase, against laryngeal injury in rats.

The effect of 3-aminobenzamide, an inhibitor of poly (ADP-ribose) synthetase activity, was evaluated in a rat model of laryngeal injury induced by endotracheal intubation for 1 h. At 1 h after extubation, the laryngeal damage was characterized by areas of mucosal necrosis, submucosal edema, swelling of subglottic glands, and submucosal infiltration of inflammatory cells. Activity of myeloperoxidase, a marker of neutrophil infiltration, was also markedly increased into the damaged tissue. Immunohistochemistry for nitrotyrosine, an index of nitrosative stress, showed an intense staining in the inflamed larynx. Treatment with 3-aminobenzamide (10 mg/kg intraperitoneally) significantly reduced the appearance of mucosal damage and was associated with a significant reduction of tissue myeloperoxidase activity and nitrotyrosine immunoreactivity in the larynx. The results of this study suggest that poly (ADP-ribose) synthetase may play a role in the inflammatory process after laryngeal intubation and extubation, and administration of 3-aminobenzamide may be a beneficial therapeutic approach.

Short- and long-term effects of irradiation on laryngeal mucosa of the rat.

Although radiotherapy is often used to treat laryngeal carcinoma, there is little information on the effects of this treatment on laryngeal structures. Rats were irradiated to the head and neck region and the larynges were studied by light- and electron-microscopy and immunohistochemistry. Ten days after irradiation, a change in the ultrastructural appearance of the granules of the subglottic glands was observed. Substance P-, bombesin- and enkephalin-like immunoreactivity was increased in local ganglionic cells and glandular nerve fibres. The mast cells were reduced in number. At examination 4 6 months after irradiation, there were no obvious differences compared with controls concerning mast-cell numbers and neuropeptide expression. The ultrastructural changes seen in the subglottic glands remained to some extent. The results show that structural changes in the subglottic glands occur concomitantly with an increased expression of certain neuropeptides in the innervation of these glands, which implies a relationship between these two parameters. The mast cells respond drastically to irradiation, but in the long run, regeneration of these cells occurs.

Laryngeal glycogenic acanthosis presenting as leukoplakia.

We report a case of an incidental finding of glycogenic acanthosis of the larynx on autopsy in a 79-year-old man who died of myocardial infarction. The lesion was grossly recognized as a white plaque (leukoplakia) on the subglottic compartment of the left side of the larynx. Histological sections revealed thickened squamous mucosa positive for abundant glycogen on periodic acid-Schiff staining. No epithelial dysplasia was noted. The patient had a history of smoking. This case represents the first report of glycogenic acanthosis involving the larynx. This benign condition should be added to the vast differential diagnosis of leukoplakia in this anatomical location.

Different glycoconjugates in the submucosal glands of the supraglottis and subglottis. Lectin histochemistry study in the hamster.

A lectin histochemistry study was performed in the supraglottic and subglottic regions of 10 hamsters. The submucosal glands were observed by light microscopy. The supraglottic submucosal glands presented numerous mucous tubules but on the other hand, the subglottic submucosal glands had serous tubules which finished at the distal portion in serous acini. The results suggest that the distribution of fucosylated-mucin and serum-type glycoproteins between the supra- and subglottic submucosal glands suggest a different viscosity and function of the mucus.

Molecular biopathology of metaplastic, dysplastic, and neoplastic laryngeal epithelium.

In a large series of more than 100 cases of laryngeal carcinomas, the presence of steroid hormone receptors was demonstrated in the cytosol and in the nuclear fraction. Their presence was confirmed by the identification of estrogen receptor isophorms and by the detection of hormone-related proteins such as ER-D5. EGFr, and cathepsin D. These molecules were also variably expressed in normal, hyperplastic, and dysplastic epithelium. These data suggest a possible role of hormone receptors during laryngeal carcinogenesis. Finally, the presence of an Angiotensin II receptor was studied in neoplastic and preneoplastic laryngeal epithelium.

Hyperplastic lesions of the larynx. Experience of the Barcelona group.

Squamous carcinoma of the larynx arises from pre-existing lesions, the so-called "preneoplastic lesions". Hyperplastic lesions represent a part of their spectrum, from both clinical and biological points of view. On morphologic grounds, the most characteristic feature with prognostic value in the evaluation of preneoplastic lesions is dysplasia. It is not only nuclear alterations that are seen in the process of malignant transformation, the cytoplasmic pattern of cytokeratins changes through neoplastic progression, with a progressive reduction of the molecular weight of the produced species. Dysplasia also associates with gross alterations of the DNA content. This is in agreement with our finding of alterations of genes participating in the control of the cell cycle, p53 and p21(WAF1/cip1). p53 overexpression is detected in non-invasive squamous lesions (even in the absence of obvious dysplasia) and p21(WAF1/cip1) shows a dramatic change in the pattern of expression in dysplastic epithelium compared with the normal. However, not all genes participating in the control of the cell cycle are altered in early lesions. Overexpression of cyclin D1, a common phenomenon in advanced carcinomas, is not likely to participate in the early phases of neoplastic development.

Laryngeal melanosis: report of four cases and literature review.

OBJECTIVE: Laryngeal melanosis is a rare condition defined by the presence of melanocytes within the laryngeal epithelial lining. Our aims were (1) to review our cases together with those in the literature, and (2) to determine whether melanocyte incidence is increased with exposure to irritant stimuli such as tobacco. METHODS: A retrospective study of all cases diagnosed with laryngeal melanosis in our hospital from January 1, 1990, to December 31, 1996, was accomplished. To determine the melanocyte incidence in the normal larynx as well as the influence of tobacco in development of laryngeal melanosis, 16 age-matched controls, 8 of whom were smokers and 8 of whom were not, were chosen, and a histochemical and immunohistochemical study was performed. The following antibodies were used: S-100 protein, CD1a, and HMB-45. A comparative study of the melanocyte incidence between patients with laryngeal melanosis and the controls was carried out. Also, a comparative study between smoking and nonsmoking patients was performed. RESULTS: Laryngeal melanosis was diagnosed in 4 patients at our hospital during this period of time. In the comparative study, the number of melanocytes in the 4 patients with laryngeal melanosis was higher than in the 8 smoking (p < 0.01, Mann-Whitney U test) and 8 nonsmoking (p < 0.01) controls, and there was a trend toward a higher number of melanocytes in the 8 smoking patients than in the 8 nonsmoking (p = 0.064) controls. CONCLUSIONS: Laryngeal melanosis was more frequent in smoking men older than 50 years. Our observations underline the association of LM with larynx carcinoma and its relation to a stimulus such as tobacco. In fact, we have found activated melanocytes in our cases of laryngeal melanosis. They were identified by immunoreactivity for HMB-45.

Cytochemical characterization of sulfo- and sialoglycoconjugates of human laryngeal glandular cells.

The composition and distribution of sulfo- and sialoglycoconjugates in human laryngeal glands have been investigated at light and electron microscopic levels by use of peroxidase-, digoxigenin-, and colloidal gold-conjugated lectins in combination with several chemical and enzymatic deglycosylation procedures. The present study reveals a variety of terminal oligosaccharide sequences in serous and mucous glands. Serous cells contained glycoconjugates with terminal Neu5Ac (alpha 2-3) Gal (beta 1-4) GlcNAc, Neu5Ac (alpha 2-6) Gal/GalNAc, Neu5Ac (alpha 2-3/6) Gal (beta 1-3 GalNAc, GlcNAc, and Gal (beta 1-4) GlcNAc sequences. Scarce SO4Gal(beta 1-3)GalNAc terminal oligosaccharide chans were detected. Serous cells show wide morphological variability of secretory granules (electron lucent, electron dense, and bizonal) with different lectin affinities. Glycoconjugates in human laryngeal mucous glands contained a variety of terminal oligosaccharide sequences including SO4Gal(beta 1-4)GlcNAc, SO4Gal(beta 1-3) GalNAc, SO4GalNAc, and Neu5Ac(alpha 2-3)GalNAc.

Thymidylate synthase activity, folates, and glutathione system in head and neck carcinoma and adjacent tissues.

BACKGROUND: Head and neck squamous cell carcinomas (HNSCC) present variable aggressiveness and chemosensitivity. Because the glutathione (GSH) system and thymidylate synthase (TS) are involved in the resistance to the main drugs used in HNSCC (cisplatin and 5-FU), we studied these systems in tumors and normal mucosae. METHODS: Tumor samples and normal adjacent mucosae were collected from 37 untreated HNSCC patients. GSH and glutathione S-transferase (GST) activity were assayed by spectrophotometry, whereas TS activity and folates were determined by radioassays. RESULTS: Mean GSH levels were higher in tumors (15.2 +/- 8.2 nmol/mg protein) than in mucosae (8.3 +/- 4.1 nmol/mg protein) (p = 0.005, paired t test). GST activity was also higher in tumors (394 +/- 194 nmol/min/mg protein) than in mucosae (261 +/- 132 nmol/min/mg protein) (p = 0.0003). TS activity was markedly higher in tumors (9.2 +/- 21.5 pmol/min/mg protein) compared to that of mucosae (0.9 +/- 1.2 pmol/min/mg protein) (p = 0.0001). Folate levels in tumors and mucosae were similar (1.2 +/- 1.1 and 0.8 +/- 0.9 pmol/mg protein, respectively; p = 0.1, NS). In relation to clinical stage and tumor size, a statistical difference was found in GSH and GST values between tumors and mucosae for stage IV and T3/T4. The increase in tumor TS compared to that of mucosae was significant for all clinical stages, tumor sizes, and nodal involvement. CONCLUSIONS: These data enhance our understanding of the enzymatic systems involved in cisplatin and 5-fluorouracil (5-FU) resistance in HNSCC and normal mucosae and may help to elucidate tumor behavior and interpatient differences in drug sensitivity.

[An observation of DNA and RNA within the cells of laryngocarcinoma by acridine orange fluorescent staining].

The shape of cells in groups of 7 individuals with normal laryngeal mucosal epithelium, 12 patients with laryngeal papilloma, 25 with atypical hyperplasia of laryngeal epithelium, 44 with laryngeal squamous cell carcinoma and DNA, RNA were observed by acridine orange fluorescent staining. The result has proved that fluorescent staining is a method of staining, which can be used to show the extent of cell differentiation. As to DNA and RNA within the cells the method has potent specificity and the shape and structure of cells and the change of the content of two kinds of nucleic acids can be demonstrated by means of the method, which is simple, easy and quick to master. The clear video can demonstrate the atypical hyperplasia of laryngeal mucosal epithelium and the changing features of DNA and RNA of laryngocarcinoma, which is of practical value of popularization in early diagnosis of laryngeal malignant tumor.