Cigarette Smoke Exposure to Pig Larynx in an Inhalation Chamber.

OBJECTIVES: This study investigated the effects of cigarette smoke exposure on the pig larynx using an inhalation chamber. Specifically, we compared the effects of cigarette smoke exposure from either 3 cigarettes per day (3cd) or 15 cigarettes per day (15cd) for 20 days. STUDY DESIGN: In vivo prospective design. METHODS: Female pigs were exposed via an inhalation chamber to cigarette smoke (3R4F research cigarettes) from 3cd (n = 6) or 15cd (n = 6) for 20 days. Outcomes included histopathology of vocal fold and airway tissues; gene expression of interleukins, TNF-α, and VEGF; protein levels of TNF-α and IL-6; and number of coughs recorded in the chamber. RESULTS: Pigs exposed to cigarette smoke from 15cd exhibited mild vocal fold edema as compared to the 3cd group on histopathological evaluation. There was also minimal inflammation of nasal and tracheal tissue characterized by presence of more granulocytes in the 15cd group compared to the 3cd group. Cough frequency was significantly greater for the 15cd group compared to the 3cd group. CONCLUSIONS: A custom-designed large animal inhalation chamber successfully challenged pigs repeatedly, to varying levels of cigarette smoke. Future studies will combine such low levels of smoke exposure with other common challenges such as acid reflux to understand the multifactorial causation of laryngeal pathologies.

[The expression characteristics and clinical significance of candidate molecular markers in vocal cord leukoplakia].

Objective: To investigate the expression of marker proteins in vocal cord leukoplakia, and to find markers for the early stage of diagnosis and prognosis of precancerous lesions. Methods: The study included 119 cases, 68 cases of vocal cord leukoplakia (22 cases with epithelial simple hyperplasia, 46 cases with epithelial dysplasia), and 51 cases of vocal cords benign lesions(31 cases of vocal cord polyps, 20 cases of Reinke's edema). The expression of p53, Ki-67, p21, Survivin, p16, p27, PTEN, c-Myc and vascular endothelial growth factor (VEGF) in vocal cords leukoplakia were detected, vocal cord benign lesions (vocal cord polyps and Reinke's edema) acted as controls, comparing the expression differences of different pathological tissue. Data was analyzed by SPSS 22.0 software. Results: The expression of p53, p16, Ki-67, VEGF in vocal cord benign lesions and vocal cords leukoplakia with epithelial simple hyperplasia did not show significant differences. There was a grading increase in the positive expression of p53, Ki-67 in the vocal cord leukoplakia with epithelial dysplasia contrasting to those in vocal cord benign lesions and vocal cords leukoplakia with epithelial simple hyperplasia (p53: χ(2)=13.340, P=0.002, Ki-67: χ(2)=53.386, P=0.000). The expression of p27, PTEN, c-Myc in vocal cord benign lesions and vocal cords leukoplakia with epithelial dysplasia did not show significant differences. There was a grading increase in the positive expression of p21 Survivin in vocal cords leukoplakia with epithelial dysplasia contrasting to those in vocal cord benign lesions (P<0.05). Expression of Survivin in vocal cords leukoplakia with mild-moderate epithelial dysplasia showed a significant increase than those in vocal cord benign lesions (P<0.05). The positive expression grade of p21 showed a rising trend (P=0.073) between the different grades of dysplasia. Conclusion: The positive expression grade of p53, Ki-67, p21 Survivin showed an increase in vocal cords leukoplakia with epithelial dysplasia contrasting to those in vocal cord benign lesions, which might be an implication for evaluating the diagnosis and prognosis of precancerous lesions. Expression of p21 was correlated to the degrees of dysplasia and expression of Survivin showed a significant difference in early stage of epithelial dysplasia contrasting to benign lesions.

CD34-positive fibroblasts in Reinke's edema.

OBJECTIVES/HYPOTHESIS: To elucidate whether and to what extent CD34+ fibroblasts (so-called CD34+ fibrocytes, CD34+ dendritic cells, and CD34+ stromal cells) occur in normal human vocal folds and in Reinke's edema. STUDY DESIGN: Histological study. METHODS: Conventional, immunohistochemical, and ultrastructural procedures were performed in histological blocks of 18 selected cases of Reinke's edema (with typical findings including acellular edematous spaces in the subepithelial connective tissue of vocal folds, and disarrangement of elastic, collagen, and reticular fibers). For control purposes, four normal vocal folds were analyzed. RESULTS: In normal vocal folds, most stromal cells were spindle-shaped CD34+ fibroblasts. In Reinke's edema, increased density and changes in the morphology and size of this subpopulation of fibroblasts were demonstrated in the connective tissue surrounding the edematous spaces, particularly in their borders, where together with some macrophages they formed boundaries, mimicking the walls of distended lymphatic vessels when conventional stains were used. These activated CD34+ fibroblasts acquired a dendritic morphology (with long, moniliform, often bifurcated, overlapping multipolar processes), and their cytoplasmic organelles were increased in number. In addition to CD34, they expressed vimentin, CD10 and CD99, but no α-smooth muscle actin (α-SMA), CD31, CD117, CD68, h-caldesmon, desmin, or S-100 protein. CONCLUSIONS: CD34+ fibroblasts are a major cell component in the stroma of vocal folds in Reinke's edema, and their activation, with increased density and morphologic changes around the edematous spaces, occurs without immunophenotypic transformation toward myofibroblasts (no expression of α-SMA). The mechanisms by which these cells act in Reinke's edema require further study.

Comparative study of the behavior of p53 immunoexpression in smoking associated lesions: Reinke's edema and laryngeal carcinoma.

OBJECTIVE: To assess the behavior of the immunoexpression of protein p53 in Reinke's edema and laryngeal squamous cell carcinoma. STUDY DESIGN: retrospective. METHODS: we recovered the histological paraffin blocks of patients who were subjected to Reinke's edema and laryngeal squamous cell carcinoma surgery in 2000-2011. The paraffin blocks were cut into 3-µm sections; the specimens were prepared in silanized slides (one slide for each paraffin block) and subjected to immunohistochemical reaction according to the Avidin Biotin Peroxidase method. Monoclonal primary anti-p53 antibodies were used at 1:50 dilution. Slides were examined under a light microscope at different magnitudes and results were interpreted based on the degree of brown staining in the nuclei of epithelial cells and in the extent of the fragment by using a semi-quantitative score from 0 to 3. RESULTS: 67 slides of Reinke's edema and 60 slides of laryngeal squamous cell carcinoma were included. Scores 2 and 3 for staining of the nuclei of epithelial cells were recorded for 46 slides of Reinke's edema (68.65%) and for 57 slides of laryngeal squamous cell carcinoma (95%). As to the extent of the fragment, scores 2 and 3 were recorded for 74% slides of Reinke's edema and for 95% slides of carcinomas. CONCLUSION: the positive immunoexpression for protein p53, positive in 95% carcinomas and 74% Reinke's edemas, makes us aware of the possible preneoplastic condition of the latter lesion. Further studies are needed to identify and reveal the genetic changes that lead to these results.

Reinke's edema: investigations on the role of MIB-1 and hepatocyte growth factor.

Reinke's edema is a benign disease of the human vocal fold, which mainly affects the sub-epithelial layer of the vocal fold. Microscopic observations show a strongly oedematous epithelium with loosened intercellular junctions, a disruption of the extracellular connections between mucosal epithelium and connective tissue, closely adherent to the thyroarytenoid muscle. Thickening of the basal layer of epithelium, known as Reinke's space, high deposition of fibronectin and chronic inflammatory infiltration it is also visible. We analyzed, together with the hepatocyte growth factor (HGF), the expression level of MIB-1 in samples harvested from patients affected by Reinke's edema, in order to define its biological role and consider it as a possible prognostic factor in the follow-up after surgical treatment. We observed a moderate expression of HGF in the lamina propria of the human vocal fold and in the basal membrane of the mucosal epithelium. Our finding suggests that this growth factor acts as an antifibrotic agent in Reinke's space and affects the fibronectin deposition in the lamina propria. MIB-1, on the contrary, showed a weak expression in the basement membrane of the mucosal epithelium and a total absence in the lamina propria deep layer, thus suggesting that only the superficial layer is actively involved in the reparatory process with a high regenerative capacity, together with a high deposition of fibronectin. The latter is necessary for the cellular connections reconstruction, after the inflammatory infiltration.

Edema de Reinke: estudo da imunoexpressão da fibronectina, da laminina e do colágeno IV em 60 casos por meio de técnicas imunoistoquímicas / Reinkes'edema: immunoexpression study of fibronectin, laminin and colagen IV in 60 cases by imunohistochemical techniques

Edema de Reinke é doença crônica da laringe na qual a camada superficial da lâmina própria é expandida por muco espesso conferindo-lhe aspecto gelatinoso. Relaciona-se ao tabagismo e acomete, preferencialmente mulheres, as quais apresentam a voz mais grave. Suas características histológicas nem sempre conseguem diferenciá-lo das demais lesões benignas da laringe, havendo necessidade de técnicas histológicas adicionais. Objetivos: Estudar a imunoexpressão da fibronectina, do colágeno IV e da laminina no edema de Reinke por meio de técnicas imunoistoquímicas. Estudo prospectivo. Material e métodos: Blocos histológicos de 60 casos cirúrgicos de edema de Reinke foram resgatados, submetidos a novos cortes e às reações imunoistoquímicas para fibronectina, laminina e colágeno IV pelo método da Avidina Biotina Peroxidase. Todos os pacientes eram fumantes e adultos, sendo 50 mulheres e 10 homens. Resultados: As análises da imunoexpressão da fibronectina, do colágeno IV e da laminina foram mais expressivas no endotélio dos vasos (68,33 por cento, 76,66 por cento, 73,33 por cento, respectivamente), e menos relevantes na membrana basal (25,0 por cento, 5,0 por cento e 3,3 por cento, respectivamente). Conclusões: No edema de Reinke, a imunoexpressão da fibronectina, da laminina e do colágeno IV na membrana basal não apresentam relevância, havendo predomínio desses anticorpos no endotélio do vasos.

Reinke's edema is chronic laryngeal disease in which the superficial layer of the lamina propria is expanded by thick mucus, giving it a gelatin aspect. The disease is directly related to smoking and more frequent in women, who end up having a lower tone of voice. Its histological characteristics cannot always distinguish it from other benign lesions of the larynx for which additional histological techniques are necessary. AIM: to study the immunoexpression of fibronectin, collagen IV and laminin in Reinke's edema by immunohistochemical technique. Prospective study. Materials and methods: histological blocks of 60 cases of surgical Reinke's edema were saved, submitted to new cross-sections and to immunohistochemical reactions for fibronectin, laminin and collagen IV by the Avidin-Biotin-Peroxidase method. Fragments of five normal vocal folds were used as control, removed during autopsy. All patients were chronic smokers and adults- 50 women and 10 men. Results: the immunoexpression of fibronectin, collagen IV and laminin was more important in the endothelium of blood vessels (68.33 percent, 76.66 percent, 73.33 percent, respectively) and less relevant in the basement membrane (25.0 percent, 5.0 percent and 3.3 percent, respectively). Conclusions: the immunoexpression of fibronectin, laminin and of collagen IV in the basal membrane of Reinke's edema was not relevant, with a predominance of these antibodies in the endothelium of blood vessels.

Immunohistochemical examinations of sex hormone receptors in benign vocal fold lesions.

BACKGROUND: Acquired benign vocal fold lesions are among the most common causes of voice problems. Since the local impact of estrogen and progesterone receptors in laryngeal tissue is discussed controversially, the presence of sex hormone receptors in benign vocal fold alterations needs to be clarified. GOAL OF THE STUDY: To investigate the expression of estrogen-alpha receptors (ER-alpha), estrogen-beta receptors (ER-beta), progesterone receptors (PR) and androgen receptors (AR) in acquired benign vocal fold alterations. METHODS: Laryngeal epithelial specimens of 14 patients (13 female, 1 male) taken intraoperatively were investigated using immunohistochemistry in order to objectify ER-alpha, ER-beta, PR and AR. Macroscopically and histopathologically diagnosed edemas of Reinke's space (n = 10), vocal fold polyps (n = 3) and vocal fold nodules (n = 1) were enrolled in this study. RESULTS: No specific nuclear immunohistochemical staining could be seen in the biopsies taken. Only unspecific staining patterns could be observed. CONCLUSION: Sex hormone receptors could not be detected in the specimens tested, thus, any direct influence of sex hormones on the development of benign vocal fold lesions is rather unlikely. The results of this study confirm the impact of vocal fold stress and biomechanical abnormalities on their development due to voice overstraining and abuse.

Reinkes' edema: immunoexpression study of fibronectin, laminin and collagen IV in 60 cases by immunohistochemical techniques.

UNLABELLED: Reinke's edema is chronic laryngeal disease in which the superficial layer of the lamina propria is expanded by thick mucus, giving it a gelatin aspect. The disease is directly related to smoking and more frequent in women, who end up having a lower tone of voice. Its histological characteristics cannot always distinguish it from other benign lesions of the larynx for which additional histological techniques are necessary. AIM: to study the immunoexpression of fibronectin, collagen IV and laminin in Reinke's edema by immunohistochemical technique. Prospective study. MATERIALS AND METHODS: histological blocks of 60 cases of surgical Reinke's edema were saved, submitted to new cross-sections and to immunohistochemical reactions for fibronectin, laminin and collagen IV by the Avidin-Biotin-Peroxidase method. Fragments of five normal vocal folds were used as control, removed during autopsy. All patients were chronic smokers and adults- 50 women and 10 men. RESULTS: the immunoexpression of fibronectin, collagen IV and laminin was more important in the endothelium of blood vessels (68.33%, 76.66%, 73.33%, respectively) and less relevant in the basement membrane (25.0%, 5.0% and 3.3%, respectively). CONCLUSIONS: the immunoexpression of fibronectin, laminin and of collagen IV in the basal membrane of Reinke's edema was not relevant, with a predominance of these antibodies in the endothelium of blood vessels.

Fibronectin: an interesting vocal fold protein.

A great deal of information is accruing regarding the function of the extracellular matrix. Once thought to be simply a structural entity to surround cells, it is now known to do much more. Fibronectin in particular has received specific attention. Fibronectin is a ubiquitous glycoprotein found most abundantly in the extracellular matrix of regenerating, healing, and embryonic tissue. Vast evidence supports the fact that fibronectin participates in many diverse functions throughout the body that are relevant to vocal fold biology. This article introduces the structure of fibronectin and its isoforms and provides an introduction to some of the many functions it plays. It also reviews the evidence about fibronectin's place in vocal folds and vocal fold pathology. It discusses fibronectin's presence in vocal nodules, vocal polyps, vocal scarring, and Reinke's edema, and reviews the data on its role in mucosal wave impairment. Lastly, it discusses preliminary microarray data that show gene expression for fibronectin to be upregulated in true vocal folds when compared to false vocal folds.

Electron microscopic and immunohistochemical investigation of Reinke's edema.

Examination was made of the vocal fold mucosa of Reinke's edema immunohistochemically and electron microscopically, with special attention to blood vessels in Reinke's space (RS). Ten surgical specimens of Reinke's edema were used, and the results obtained are summarized as follows. There were subepithelial vascularization and many dilated vessels in RS. The vessels had thin endothelium with many fenestrae, many vesicles, and a thickened basement membrane. Plasma exuded from the capillaries into surrounding tissue via fenestrae. These findings demonstrate increased permeability of the vessels. The vascular walls were thin, with few pericytes. The pericytes were situated away from the endothelial cells. Some endothelial cells and pericytes had degenerated, and vessel occlusion was apparent in some cases. These findings indicate the vessels had become fragile. Interstitial cells and/or inflammatory cells in RS showed stained cytoplasm with vascular endothelial growth factor. This growth factor may possibly enhance capillary permeability and form fragile vessels in RS. Fragility and alteration in permeability of the vessels are presumed to cause edema of RS, which may progress to Reinke's edema.

[Treatment of hereditary angioedema].

Hereditary angioedema is a rare familial disease caused by the defect of complement C1esterase inhibitor (C1-INH). It is characterized by recurrent acute edema of the extremities, the face, the respiratory tract and the gastrointestinal tract. Acute laryngeal edema usually produces laryngeal obstruction. Two cases have been treated since 1986, one of them had been admitted for forty-five times because of recurrent acute laryngeal edema. Investigations showed two families with a high incidence of this disease. Laboratory examination showed a remarkable decrease of C1-INH and C4. Tracheotomy is indicated in patients with laryngeal edema. Great success was achieved in two patients treated with danazol.

A histochemical study of simple laryngeal polyps.

Benign laryngeal polyps and normal larynxes have been studied by histochemical and other staining methods. These show that the polyps contain much free hyaluronic acid and 6-chondroitin sulphate. Their mode of production may be the mechanical separation of these compounds from collagen fibres by vibration. An alcian-elastic stain may be a helpful diagnostic method.