Novel immortalized human vocal fold epithelial cell line: In vitro tool for mucosal biology.

Study of vocal fold (VF) mucosal biology requires essential human vocal fold epithelial cell (hVFE) lines for use in appropriate model systems. We steadily transfected a retroviral construct containing human telomerase reverse transcriptase (hTERT) into primary normal hVFE to establish a continuously replicating hVFE cell line. Immortalized hVFE across passages have cobblestone morphology, express epithelial markers cytokeratin 4, 13 and 14, induced hTERT gene and protein expression, have similar RNAseq profiling, and can continuously grow for more than 8 months. DNA fingerprinting and karyotype analysis demonstrated that immortalized hVFE were consistent with the presence of a single cell line. Validation of the hVFE, in a three-dimensional in vitro VF mucosal construct revealed a multilayered epithelial structure with VF epithelial cell markers. Wound scratch assay revealed higher migration capability of the immortalized hVFE on the surface of collagen-fibronectin and collagen gel containing human vocal fold fibroblasts (hVFF). Collectively, our report demonstrates the first immortalized hVFE from true VFs providing a novel and invaluable tool for the study of epithelial cell-fibroblast interactions that dictate disease and health of this specialized tissue.

Establishment of Immortalized Laryngeal Epithelial Cells Transfected with Bmi1.

Primary laryngeal epithelial cells are essential to exploring the mechanisms of laryngeal and voice disorders; however, they are difficult to study and apply because of their limited life span. The purpose of this study was to develop a stable and reliable in vitro model for the comprehensive study of the pathogenesis of laryngeal and voice diseases. The pLVTHM-Bmi1 plasmid was constructed and used to immortalize primary laryngeal epithelial cells by lentiviral infection. The expressions of Bmi1, human telomerase reverse transcriptase (hTERT), p53, and pRB pathway proteins were detected by western blotting. Functional characteristics of the immortalized cell lines were verified by cell senescence ß-galactosidase staining, 5-ethynyl-2'-deoxyuridine cell proliferation test, and flow cytometry. We successfully introduced Bmi into human subglottic (hSG) cells and human ventricle (hV) cells. Both the human immortalized subglottic Bmi1 (hSG-Bmi1) cell line and the human immortalized ventricle Bmi1 (hV-Bmi1) cell line maintained normal epithelial morphology and divided successfully after more than 20 culture passages. As Bmi1 was overexpressed in these cells, the expression of human telomerase reverse transcriptase (hTERT) and phosphorylated Rb increased while p16 and p21 decreased. Following Bmi1-mediated immortalization, cell senescence decreased significantly, and cell proliferation was accelerated. Tumor formation was not observed for hSG, hV, or hSG-Bmi1, and hV-Bmi1 cells in nude mice. hSG-Bmi1 cells dominated by stratified squamous epithelium and hV-Bmi1 cells dominated by columnar cells were established. The new cell lines lay a foundation for the study of the pathogenic mechanisms of laryngeal and voice diseases.

Heterogeneity of Stem Cells in the Human Vocal Fold Mucosa.

1. There is growing evidence to suggest that the cells in the maculae flavae are tissue stem cells of the human vocal fold and maculae flavae are a candidate for a stem cell niche. 2. The latest research shows that the cells in the human maculae flavae are involved in the metabolism of extracellular matrices that are essential for viscoelasticity in the human vocal fold mucosa as a vibrating tissue and are considered to be important cells in the growth, development, and aging of the human vocal fold mucosa. 3. Recent evidence has indicated that the cells including vocal fold stellate cells in the maculae flavae of the human vocal fold mucosa are a functionally heterogenous population. 4. The cells in the human maculae flavae possess proteins of all three germ layers, indicating that they are undifferentiated and have the ability of multipotency. 5. The cell division in the human adult maculae flavae is reflective of asymmetric self-renewal, and cultured cells form a colony-forming unit. Therefore, the phenomenon gives rise to the strong possibility that the cells in the human maculae flavae are putative stem cells. 6. Recent research has suggested that the cells in the human maculae flavae arise from the differentiation of bone marrow cells via peripheral circulation. 7. Cultured cell populations in the human maculae flavae are roughly divided into three groups by morphological features: cobblestone-like polygonal cells, vocal fold stellate cell-like cells, and fibroblast-like spindle cells. However, at the present state of our investigation, it is difficult to clarify the stem cell system and hierarchy of stem cells in the human maculae flavae. 8. Subpopulations of cells in the maculae flavae proliferate extremely slowly and retain stem cell properties. 9. Tension caused by phonation seems to regulate the behavior and heterogeneity of the cells (mechanical regulation) in the maculae flavae of the human vocal fold. 10. The putative stem cells in the maculae flavae appear to differentiate into other kind of cells in the surrounding tissue.

[Influence of TNF-α on the immunomodulatory property of laryngeal mucosa mesenchymal stromal cells'].

Objective: To investigate the effect of tumor necrosis factor-alpha(TNF-α)on the immunoregulatory capacity of laryngeal mucosal mesenchymal stromal cells (LM-MSCs) and its potential molecular mechanism, and provide a theoretical basis for the study of chronic laryngitis. Methods: LM-MSCs were separated from epiglottal mucosa. The LM-MSCs cells were directly co-cultured with T cells in vitro to detect the immunomodulatory property of LM-MSCs. After long-term stimulation with inflammatory factors TNF-α in vitro, the differences were compared in the immunomodulatory ability of LM-MSCs between normal LM-MSCs and TNF-α stimulated LM-MSCs. The expression of general control non-repressed protein5(GCN5), FAS, FASL in normal LM-MSCs and TNF-α stimulated LM-MSCs was detected by Western blot and quantitative real-time RT-PCR(RT-qPCR). Results: After chronic stimulation of TNF-α, the RNA relative expression of GCN5 was 0.31±0.03 (3 days) and 0.53±0.06 (7 days) compared with control group, showing significant difference (F=13.45, P<0.05). The percentage of LM-MSC-induced T cell apoptosis was 6.27%±0.81% (3 days) and 4.99%±0.52% (7 days) in chronic stimulation group compared with control group 10.02%±1.02%. There is a significant difference among these groups (F=11.13, P<0.05). Moreover, the ability of LM-MSCs to induce T cell apoptosis is regulated by GCN5. Conclusion: With the chronic stimulation of TNF-α, the expression of GCN5 in LM-MSCs is decreased, thus impairing its immunoregulatory capacity.

Methodology for the establishment of primary porcine vocal fold epithelial cell cultures.

OBJECTIVE: A current lack of methods for epithelial cell culture significantly hinders our understanding of the role of the epithelial and mucus barriers in vocal fold health and disease. Our first objective was to establish reproducible techniques for the isolation and culture of primary porcine vocal fold epithelial cells. Our second objective was to evaluate the functional significance of cell cultures using an in vitro exposure to an inflammatory cytokine. METHODS: Epithelial cells were isolated from porcine vocal folds and expanded in culture. Characterization of cultures was completed by immunostaining with markers for pan-cytokeratin (epithelial cells), vimentin (stromal cells), von Willebrand factor (endothelial cell), and MUC1 and MUC4 (mucin) glycoproteins. Established epithelial cell cultures were then exposed to the inflammatory cytokine tumor necrosis factor alpha (TNF-α) for 24-hours, and transcript expression of MUC1 and MUC4 was evaluated. RESULTS: Reproducible, porcine vocal fold epithelial cell cultures, demonstrating cobblestone appearance characteristic of the typical morphology of epithelial cell cultures were created. Cells showed positive staining for pan-cytokeratin with limited expression of vimentin and von Willebrand factor. Epithelial cells also expressed MUC1 and MUC4. TNF-α significantly increased transcript expression of MUC4. CONCLUSION: Here, we present the first report of successful culture of primary porcine vocal fold epithelial cells. Cultures will provide researchers with a valuable new in vitro tool to investigate vocal fold epithelium and mucus as well as the effects of common challenges, including inflammatory cytokines, on these barriers. LEVEL OF EVIDENCE: NA Laryngoscope, 129:E355-E364, 2019.

Long-term control of laryngeal plasma cell mucositis with systemic immunosuppression.

Plasma cell mucositis (PCM) is a rare non-neoplastic plasma cell proliferative disorder of the mucous membranes, which typically presents as soft tissue lesions involving oral, upper airway or genital mucosa. Laryngeal involvement resulting in stridor has been reported in four other cases previously, with three requiring tracheostomy. We present a case of supraglottic stenosis in a 53-year-old woman presenting with dysphonia and stridor, requiring surgical resection on three occasions accompanied by tracheostomy on two occasions; biopsy was consistent with PCM. Due to relapsing disease activity, high-dose prednisolone and mycophenolate mofetil were commenced with prednisolone eventually being ceased. After 2 years of mycophenolate mofetil therapy, the patient's disease has been controlled without need for further surgical intervention. This is the first reported case of prolonged symptomatic improvement with the use of systemic immunosuppressive therapy with mycophenolate mofetil in PCM.

Activation of the Wnt/ß-Catenin Pathway by an Inflammatory Microenvironment Affects the Myogenic Differentiation Capacity of Human Laryngeal Mucosa Mesenchymal Stromal Cells.

Various microenvironments influence the multiple differentiation potential of mesenchymal stromal cells. For example, inflammatory microenvironment can suppress the myogenic differentiation capability of laryngeal mucosa mesenchymal stromal cells (LM-MSCs). The present study therefore sought to identify the underlying molecular mechanisms regulating these processes. We isolated a novel population of MSCs, LM-MSCs, from the laryngeal mucosa tissues. The cells were cultured in osteogenic, adipogenic, and myogenic differentiation media in the presence or absence of interleukin-1ß and tumor necrosis factor α (to simulate inflammatory microenvironment). The expression of active ß-catenin, p-GSK3ß, and GSK3ß were detected by western blot and real-time polymerase chain reaction. The myogenic differentiation of LM-MSCs in inflammatory microenvironment and the regulation by Dickkopf-1 (DKK1) were tested both in vivo and in vitro. Inflammatory microenvironment could suppress the osteogenesis, adipogenesis, and myogenesis of LM-MSCs. The Wnt/ß-catenin signaling pathway was activated during myogenesis in inflammatory microenvironment. The suppressed myogenic differentiation capability of LM-MSCs in inflammatory microenvironment was reversed by DKK1. By regulating the Wnt/ß-catenin signaling pathway, DKK1 can improve the myogenic differentiation of LM-MSCs in inflammatory microenvironment. Thus, the results of this study may help improve the efficacy of LM-MSCs injection therapy for vocal fold regeneration.

The Macula Flava of the Human Vocal Fold as a Stem Cell Microenvironment.

1. There is growing evidence to suggest that the cells in the maculae flavae are tissue stem cells of the human vocal fold and maculae flavae are a candidate for a stem cell niche. 2. The latest research shows that the cells in the human maculae flavae are involved in the metabolism of extracellular matrices that are essential for the viscoelasticity in the human vocal fold mucosa as a vibrating tissue, and considered to be important cells in the growth, development, and aging of the human vocal fold mucosa. 3. The cells in the human maculae flavae possess proteins of all three germ layers, indicating they are undifferentiated and have the ability of multipotency. 4. The cell division in the human adult maculae flavae is reflective of asymmetric self-renewal and cultured cells form a colony-forming unit. Therefore, the phenomenon gives rise to the strong possibility that the cells in the human maculae flavae are tissue stem cells. 5. Recent research suggests that the cells in the human maculae flavae arise from the differentiation of bone marrow cells via peripheral circulation. 6. The hyaluronan concentration in the maculae flavae is high and contains cells which possess hyaluronan receptors, indicating that the maculae flavae are hyaluronan-rich matrix, which is required for a stem cell niche. 7. A proper microenvironment in the maculae flavae of the human vocal fold mucosa is necessary to be effective as a stem cell niche maintaining the stemness of the contained tissue stem cells.

Microenvironment of macula flava in the human vocal fold as a stem cell niche.

BACKGROUND: There is growing evidence that the cells in the maculae flavae are tissue stem cells of the human vocal fold mucosa, and that the maculae flavae are a candidate for a stem cell niche. The role of microenvironment in the maculae flavae of the human vocal fold mucosa was investigated. METHOD: Anterior maculae flavae from six surgical specimens were cultured in a mesenchymal stem cell growth medium or a Dulbecco's modified Eagle's medium. RESULTS: Using mesenchymal stem cell growth medium, the subcultured cells formed a colony-forming unit, and cell division reflected asymmetric self-renewal. This indicates that these cells are mesenchymal stem cells or stromal stem cells in the bone marrow. Using Dulbecco's modified Eagle's medium, the subcultured cells showed symmetric cell division without a colony-forming unit. CONCLUSION: A proper microenvironment in the maculae flavae of the human vocal fold mucosa is necessary to be effective as a stem cell niche that maintains the stemness of the contained tissue stem cells.

Regeneration of Vocal Fold Mucosa Using Tissue-Engineered Structures with Oral Mucosal Cells.

OBJECTIVES: Scarred vocal folds result in irregular vibrations during phonation due to stiffness of the vocal fold mucosa. To date, a completely satisfactory corrective procedure has yet to be achieved. We hypothesize that a potential treatment option for this disease is to replace scarred vocal folds with organotypic mucosa. The purpose of this study is to regenerate vocal fold mucosa using a tissue-engineered structure with autologous oral mucosal cells. STUDY DESIGN: Animal experiment using eight beagles (including three controls). METHODS: A 3 mm by 3 mm specimen of canine oral mucosa was surgically excised and divided into epithelial and subepithelial tissues. Epithelial cells and fibroblasts were isolated and cultured separately. The proliferated epithelial cells were co-cultured on oriented collagen gels containing the proliferated fibroblasts for an additional two weeks. The organotypic cultured tissues were transplanted to the mucosa-deficient vocal folds. Two months after transplantation, vocal fold vibrations and morphological characteristics were observed. RESULTS: A tissue-engineered vocal fold mucosa, consisting of stratified epithelium and lamina propria, was successfully fabricated to closely resemble the normal layered vocal fold mucosa. Laryngeal stroboscopy revealed regular but slightly small mucosal waves at the transplanted site. Immunohistochemically, stratified epithelium expressed cytokeratin, and the distributed cells in the lamina propria expressed vimentin. Elastic Van Gieson staining revealed a decreased number of elastic fibers in the lamina propria of the transplanted site. CONCLUSION: The fabricated mucosa with autologous oral mucosal cells successfully restored the vocal fold mucosa. This reconstruction technique could offer substantial clinical advantages for treating intractable diseases such as scarring of the vocal folds.

Identification of distinct layers within the stratified squamous epithelium of the adult human true vocal fold.

OBJECTIVES/HYPOTHESIS: A precise molecular schema for classifying the different cell types of the normal human vocal fold epithelium is lacking. We hypothesize that the true vocal fold epithelium has a cellular architecture and organization similar to that of other stratified squamous epithelia including the skin, cornea, oral mucosa, and esophagus. In analogy to disorders of the skin and gastrointestinal tract, a molecular definition of the normal cell types within the human vocal fold epithelium and a description of their geometric relationships should serve as a foundation for characterizing cellular changes associated with metaplasia, dysplasia, and cancer. STUDY DESIGN: Qualitative study with adult human larynges. METHODS: Histologic sections of normal human laryngeal tissue were analyzed for morphology (hematoxylin and eosin) and immunohistochemical protein expression profile, including cytokeratins (CK13 and CK14), cornified envelope proteins (involucrin), basal cells (NGFR/p75), and proliferation markers (Ki67). RESULTS: We demonstrated that three distinct cell strata with unique marker profiles are present within the stratified squamous epithelium of the true vocal fold. We used these definitions to establish that cell proliferation is restricted to certain cell types and layers within the epithelium. These distinct cell types are reproducible across five normal adult larynges. CONCLUSION: We have established that three layers of cells are present within the normal adult stratified squamous epithelium of the true vocal fold. Furthermore, replicating cell populations are largely restricted to the parabasal strata within the epithelium. This delineation of distinct cell populations will facilitate future studies of vocal fold regeneration and cancer. LEVEL OF EVIDENCE: N/A.

Potential of biofluid components to modify silver nanoparticle toxicity.

Establishing realistic exposure scenarios is critical for cytotoxic investigation of silver nanoparticles (AgNP) in the gastrointestinal tract. This study investigated the potential interaction with and effect of biofluid components, namely cholic acid, deoxycholic acid and ursodeoxycholic acid, on AgNP toxicity. Two cell lines corresponding to organs related to the biofluid components were employed. These were HepG-2 a hepatocellular carcinoma derived from liver tissue and Hep2 an epithelial cell line. Physiochemical and cytotoxic screening was performed and the ability of biofluid components to modify AgNP cytotoxicity was explored. No alteration to the physiochemical characteristics of AgNP by biofluid components was demonstrated. However, biofluid component addition resulted in alteration of AgNP toxicity. Greater reactive oxygen species induction was noted in the presence of cholic acid and deoxycholic acid. Ursodeoxycholic acid demonstrated no modification of toxicity in HepG-2 cells; however, significant modification was noted in Hep2 cells. It is concluded that biofluid components can modify AgNP toxicity but this is dependent on the biofluid component itself and the location where it acts.

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.

[Myogenic differentiation of laryngeal mucosal mesenchymal stem cells].

OBJECTIVE: To investigate the myogenic differentiation of laryngeal mucosal mesenchymal stem cells (LM-MSCs) and the possibility of LM-MSCs as new alternative seed cells for laryngeal tissue engineering. METHODS: LM-MSCs were separated from normal epiglottis mucosa and the cell surface markers including CD44, CD105, CD90, CD29, CD34 and CD45 were analyzed through flow cytometry. The osteogenesis and adipogenesis differentiation of LM-MSCs were investigated by oil red staining and alizarin red S staining. Immunofluorescence staining and RT-PCR were used to detect the expressions of myogenic differentiation markers including Myod1, Myogenin and myosin heavy chain (MyHc). RESULTS: The separated LM-MSCs were in a fibrocyte-like form with long fusiform shape and grew adherent. The expression rates of cell surface markers LM-MSCs were CD44 (100.0%), CD105 (90.4%), CD90(99.9%), CD29 (93.0%), CD34 (0.4%) and CD45(1.3%) respectively. A number of beaded lipid drops and mineral deposition were observed after 14 days of adipogenesis differentiation and 21 days of osteogenesis differentiation. Myod1, Myogenin and MyHc genes appeared after 1 week and 3 weeks of myogenesis differentiation respectively. CONCLUSIONS: The LM-MSCs have the properties of mesenchymal stem cells and could be differentiated into myoblasts, providing with the possibility to repair the damaged vocal cords with LM-MSCs through tissue engineering techniques.

The use of laryngeal mucosa mesenchymal stem cells for the repair the vocal fold injury.

Stem cell transplantation is a kind of attractive and new approach that complements traditional restorative or surgical techniques for the regeneration of injured or pathologically damaged laryngeal tissues. However, the best cell delivery strategy remains to be identified. The objective of this study was to establish a new strategy to the healing of injured vocal fold, using laryngeal mucosa mesenchymal stem cells differentiating into myofibroblasts or fibroblasts and improving the reconstruction microenvironment in the vocal fold injury as a new alternative as seed cells for laryngeal tissue engineering. After isolation and expansion, cells were identified as adherent mesenchymal cells with substantial proliferation potential in vitro, and were also characterized by flow cytometry. The differentiation potential of mesenchymal cells was maintained during proliferation as confirmed by culturing for adipogenesis, osteogenesis and chondrocyte. When LM-MSC was transplanted into the injured vocal fold, it has the potent differentiated into myofibroblasts and fibroblasts, which could regulate extracellular matrix, block collagen and the fibronectin rapid increased, inhibit the rapidly decrease of elastic fiber and HA, decrease the microenvironment inflammatory reaction, and prevent the formation of vocal fold scar.

[The isolation, cultivation and identification of laryngeal mucosal mesenchymal stem cells].

OBJECTIVE: To investigate the presence of mesenchymal stem cells (MSC) in laryngeal mucosa, and to seek for the method to isolate, cultivate and identify these cells. METHODS: Normal laryngeal mucosa was obtained from patients with laryngeal carcinoma during surgery, and the generating mesenchymal cells was obtained by digestive method. The cell growth curve was evaluated by 3-(4.5-methylthiazol-2yl)-2.5-diphenyltetrazolium bromide (MTT) assay. Colony forming cell assay was used to screen different morphologic colonies and evaluate clone formation ability. Flow cytometry was performed for the expression of the cells of the 4th passage surface marker profiles. Multiple differentiation potentials were confirmed by adipogenic, osteogenic and neural lineages induction. RESULTS: MTT assay and colony forming cell assay showed that laryngeal mucosa MSC had a relatively rapid proliferation capacity and a relatively high clone formation capacity (clone formation rate 7.1%). Flow cytometry analysis revealed that the laryngeal mucosa MSC were positive for CD29 (16.9%), CD44 (97.4%), CD90 (89.5%), CD105 (85.9%), CD146 (2.5%) and stro-1 (20.4%), but negative for CD34 (1.2%) and CD45 (0.8%). Laryngeal mucosa MSC undergone adipogenic, osteogenic and neural lineages induction were positive for oil red staining, alizarin red staining and S100 staining respectively, which suggested that laryngeal mucosa MSC could differentiate into adipogenic, osteogenic and neural lineages. CONCLUSION: This study demonstrated that MSC with rapid proliferative capacity and multiple differentiation potential could be obtained from lamina propria of laryngeal mucosa.

Estudio comparativo entre las características citológicas de los epitelios nasal, faríngeo y vaginal en mujeres adultas jóvenes / Comparative study of cytological characteristics of the nasal, pharygeal and vaginal epithelium in young adult women

Determinar y comparar las características citológicas de los epitelios nasal, faríngeo y vaginal en mujeres adultas jóvenes. Estudio prospectivo y transversal de 35 mujeres no embarazadas, entre 18 y 35 años de edad, durante el período comprendido entre el 01 de enero al 01 de julio de 2004; que ingirieron o no anticonceptivos orales y a quienes se les estudiaron las citologías de los epitelios vaginal, faríngeo y nasal, según el porcentaje de índice de maduración de células exfoliadas y la cuantificación sérica de estrógeno y progesterona. En el Departamento de Obstetricia y Ginecología Hospital Chiquinquirá de Maracaibo, Estado Zulia. Se determinó que no existe diferencia significativa entre la celularidad de los epitelios estudiados. Se estableció que existe buena correlación entre las células superficiales e intermedias de los frotis de vagina, faringe y nariz durante las fases del ciclo menstrual, en todas las mujeres, aun en las que recibieron píldoras anticonceptivas. Los niveles de estrógenos y progesterona tuvieron concordancia con las fases del ciclo, con el porcentaje y tipo de células exfoliadas. Los resultados obtenidos demuestran que los epitelios nasales y faríngeos responden al influjo de hormonas ováricas similarmente como ocurre en la vagina y que estos métodos pueden ser aplicados en condiciones de difícil acceso a laboratorios hormonales o en pacientes vírgenes o con atresia/agenesia vaginal y niñas

To determine and compare the cytological characteristics of nasal, pharyngeal and vaginal epitheliums in young adult women. A prospective and transversal study of 35 non pregnant women of 18 to 35 years old during the period January 1, 2004 - November 2004; some on contraceptives pills, were studied by analysis of smear of nasal, pharyngeal and vaginal trough maturation index of exfoliated cells and serum level of estrogen and progesterone. Department of Obstetrics and Gynecology, Hospital Chiquinquira, Maracaibo, Estado Zulia. Venezuela. It was established that exists a good correlation between the superficial and intermediate cells in the different phases of the menstrual cycle and those who received oral contraceptives and the group that did not. Is established that there are correlation between the cells when compared vaginal-pharynx and vaginal-nose, in both groups. The serum levels of estrogen and progesterone were accorded to the cycle phases and with the percentage of cells in both, medicated and non medicated patients. Ours results demonstrate that the nasal and pharyngeal epitheliums respond to ovarian hormones, similar to what occurs in the vagina. It is recommended to use this method in those patients where it is need to investigate the endocrine status and where it is difficult to reach the vagina: nubile girls, vaginal agenesis, imperforate hymen; or where there is not a endocrine laboratory near

High-throughput sequencing of microRNAs in adenovirus type 3 infected human laryngeal epithelial cells.

Adenovirus infection can cause various illnesses depending on the infecting serotype, such as gastroenteritis, conjunctivitis, cystitis, and rash illness, but the infection mechanism is still unknown. MicroRNAs (miRNA) have been reported to play essential roles in cell proliferation, cell differentiation, and pathogenesis of human diseases including viral infections. We analyzed the miRNA expression profiles from adenovirus type 3 (AD3) infected Human laryngeal epithelial (Hep2) cells using a SOLiD deep sequencing. 492 precursor miRNAs were identified in the AD3 infected Hep2 cells, and 540 precursor miRNAs were identified in the control. A total of 44 miRNAs demonstrated high expression and 36 miRNAs showed lower expression in the AD3 infected cells than control. The biogenesis of miRNAs has been analyzed, and some of the SOLiD results were confirmed by Quantitative PCR analysis. The present studies may provide a useful clue for the biological function research into AD3 infection.

Physiologic cold shock increases adherence of Moraxella catarrhalis to and secretion of interleukin 8 in human upper respiratory tract epithelial cells.

Moraxella catarrhalis, a major nasopharyngeal pathogen of the human respiratory tract, is exposed to rapid and prolonged downshifts of environmental temperature when humans breathe cold air. In the present study, we show that a 26 degrees C cold shock up-regulates the expression of UspA1, a major adhesin and putative virulence factor of M. catarrhalis, by prolonging messenger RNA half-life. Cold shock promotes M. catarrhalis adherence to upper respiratory tract cells via enhanced binding to fibronectin, an extracellular matrix component that mediates bacterial attachment. Exposure of M. catarrhalis to 26 degrees C increases the outer membrane protein-mediated release of the proinflammatory cytokine interleukin 8 in pharyngeal epithelial cells. Furthermore, cold shock at 26 degrees C enhances the binding of salivary immunoglobulin A on the surface of M. catarrhalis. These data indicate that cold shock at a physiologically relevant temperature of 26 degrees C affects the nasopharyngeal host-pathogen interaction and may contribute to M. catarrhalis virulence.

[Expression pattern of MAL in normal epithelial cells, benign tumor, and squamous cell carcinoma of larynx].

OBJECTIVE: To compare the expression pattern of the MAL protein in normal and laryngeal carcinoma to derive possible implications of MAL in carcinoma development of larynx. METHOD: Use the immunohistochemical technique to analyze the distribution of MAL in normal laryngeal epithelial cells, polyp of vocal cords, laryngeal atypical hyperplasia and laryngeal squamous cell carcinoma. RESULT: MAL-like immunohistochemical reactions are strongly expressed in normal laryngeal epithelial cells and its expression is no significantly different in epithelial cells of the polyp of vocal cords. Comparatively, MAL expression is significantly down regulated in laryngeal atypical hyperplasia and laryngeal squamous cell carcinomas (P < 0.05). CONCLUSION: MAL is normally expressed in laryngeal epithelial cells and its expression changes at early stages of carcinoma development. MAL, therefore, is a potential marker for early diagnosis of laryngeal squamous cell carcinoma.