Simple, low-cost, and well-performing method, the outgrowth technique, for the isolation of cells from nasal polyps.

BACKGROUND: Epithelial cells are an important part of the pathomechanism in chronic rhinosinusitis with nasal polyps. It is therefore essential to establish a robust method for the isolation and culture of epithelial cells from nasal polyps to enable further research. In this study, the feasibility of the outgrowth technique for the isolation of the epithelial cells from the nasal polyps was evaluated. RESULTS: Using the outgrowth technique, epithelial cells could be isolated from all tissue samples. Isolated epithelial cells showed a proliferation rate of approximately 7- to 23-fold every 6 days up to the 3rd passage. Over 97% of isolated cells were shown to be cytokeratin- and p63-positive, and over 86% of them were Ki-67-positive in flow cytometry. Interleukin-33 and periostin were detectable in the supernatant. CONCLUSIONS: We introduce a simple, low-cost, and well-performing method for isolating epithelial cells from nasal polyps with the outgrowth technique.

Long-Term Cryopreservation of Nasal Polyp Tissue in a Biobank for the Isolation and Culture of Primary Epithelial Cells.

Epithelial cells may play an important role in the pathologic process of chronic rhinosinusitis with nasal polyps. Therefore, providing epithelial cells from a biobank could greatly contribute to further research. In the present work, the isolation of epithelial cells from long-term cryopreserved tissue is demonstrated. Polyp tissues were cryopreserved in a commercially available freezing medium with dimethyl sulfoxide and stored in liquid nitrogen. The outgrowth and proliferation of epithelial cells from cryopreserved tissue were evaluated and compared to that of fresh tissue. Flow cytometric analysis with anti-cytokeratin, anti-p63, and anti-Ki-67 was performed to identify epithelial cells and determine differentiation and proliferation. A functionality test was performed by determining type 2-relevant proteins, representatively thymic stromal lymphopoietin (TSLP) and periostin, using ELISA. Primary epithelial cells could be isolated from cryopreserved tissues. Cells from cryopreserved tissues showed comparable outgrowth and proliferation to that of fresh tissue. Isolated epithelial cells showed high cytokeratin, p63, and Ki-67 expression and secreted TSLP and periostin. In the present study, a method for long-term cryopreservation of polyp tissue was established, thereby enabling the isolation and cell culture of primary cell culture at a later time. Epithelial cell availability should be greatly improved by including this method in a biobank.

[Syphilis infection in the ENT area]. / Syphilisinfektion im HNO-Bereich.

The number of reported cases of syphilis has been increasing for years. The sexually transmitted disease is caused by the spirochete Treponema pallidum subspecies pallidum and progresses in different stages. Symptoms in the ENT area can occur in all stages. This means that a syphilis infection should always be considered by the ENT doctor as a differential diagnosis if the symptoms are suitable. Thus, with increasing oral sexual intercourse, the primary effect/hard chancre is more often observed in the oral cavity. In addition, symptoms can occur not only in the oral cavity, but also in the ear, nose, larynx, cervical and facial regions. The diagnosis is confirmed by direct pathogen detection or by serological detection. The spirochete cannot be cultivated. The therapeutic gold standard is the administration of benzathine penicillin G or procaine penicillin G. Doxycycline, macrolides or ceftriaxone are available as alternatives. In case of inner ear or cranial nerve involvement, the additional administration of a glucocorticoid is recommended. Before antibiotic therapy is administered, the patient must be informed about the possibility of a Jarisch-Herxheimer reaction. In cases of a syphilis infection, a collegial cooperation with the venereologists is always recommended so that an effective and comprehensive diagnosis and therapy can be carried out.

Methylation status of HPV16 E2-binding sites classifies subtypes of HPV-associated oropharyngeal cancers.

BACKGROUND: The human papillomavirus (HPV) E2 protein is a transcriptional repressor of the oncogenes E6/E7 and loss of E2 function is considered a key step in carcinogenesis. Integration of HPV into the host genome may disrupt the E2 gene. Furthermore, methylation of CpG dinucleotides in E2-binding sites (E2BSs) in the HPV upstream regulatory region may interfere with transcriptional repression of E6 and E7 by E2. The authors hypothesized that the CpG methylation status of E2BS identifies subtypes of HPV type 16 (HPV16)-associated oropharyngeal squamous cell cancers (OPSCC) in association with E2 gene integrity and viral integration. METHODS: Methylation of 10 CpG dinucleotides within the upstream regulatory region, encompassing E2BSs 1, 2, 3, and 4, was quantitatively analyzed by bisulfite pyrosequencing in 57 HPV16-associated OPSCC cases. E2 status was analyzed by gene amplification and quantitative real-time reverse transcriptase-polymerase chain reaction. Viral integration was determined by integration-specific polymerase chain reaction methods. RESULTS: Three subgroups with differential methylation at E2BS3 and E2BS 4 were identified: 1) complete methylation (>80%) associated with the presence of integrated HPV genomes with an intact E2 gene; 2) intermediate methylation levels (20%-80%) with predominantly episomal HPV genomes with intact E2; and 3) no methylation (<20%) with a disrupted E2 gene. Patients with high methylation levels tended to have a worse 5-year overall survival compared with patients with intermediate methylation (hazard ratio, 3.23; 95% confidence interval, 1.13-9.24 [P = .06]). CONCLUSIONS: Methylation of E2BS3 and E2BS4 in OPSCC is associated with E2 integrity and viral physical status. It might explain deregulated viral oncogene expression in the presence of E2. The prognostic significance of E2BS methylation for patients with HPV-associated OPSCC needs to be analyzed further.

Expression of podoplanin and prognosis in oropharyngeal cancer.

It has been shown that podoplanin expression is associated with carcinoma of the aerodigestive tract. Recent studies indicate that podoplanin may serve as a prognostic biomarker in oral carcinoma. In order to provide evidence on the role of podoplanin in oropharyngeal squamous cell carcinoma, we evaluated the prognostic impact of podoplanin in these patients. We analyzed formalin-fixed tissue samples from 107 consecutive patients with oropharyngeal squamous cell carcinoma. HPV typing and immunohistochemical staining for both p16 and podoplanin were performed. Expression of podoplanin was seen in 38.3% of all cases. We found no correlation of the podoplanin scores with either p16 expression or with HPV status. There was no significant correlation of podoplanin expression with the staging variables T, N, M, and tumor grading. Podoplanin expression did neither influence the 5-year overall survival nor the 5-year disease-free survival. Concluding, we could not find a prognostic role of podoplanin expression neither in the HPV-positive cases nor in the HPV-negative cases. It appears that podoplanin is not expressed as often in oropharyngeal cancer compared to oral cancer. We could not show any relation of lymph node metastases and podoplanin expression in this homogenous cohort of tumors.

Valosin-containing protein (VCP/p97)-expression correlates with prognosis of HPV- negative oropharyngeal squamous cell carcinoma (OSCC).

Valosin-containing protein (VCP)/p97 has been shown to be associated with antiapoptotic function via activation of the nuclear factor-[Formula: see text]B (NF[Formula: see text]B) signaling pathway and with metastasizing of tumors in several studies. VCP is located on chromosome 9p13-p12, a region often deleted in oropharyngeal squamous cell carcinoma (OSCC). The clinical significance of VCP expression in OSCC however remains unclear. In this study, expression of VCP was determined in 106 patients (77 male (71.3%) and 31 female (28.7%); age-range: 34-79 years (mean age 57 years)) by immunohistochemistry and in a subset of 15 patients by quantitative PCR. HPV-DNA was detected by polymerase chain reaction and p16INK4a immunohistochemistry. The experimental findings were correlated with clinico-pathological data and survival parameters. 47.2% of all OSCC specimens were analyzed as negative or weak staining intensity for VCP. 52.8% of all specimens showed a high staining intensity for VCP. 73.1% of all patients were tested HPV-negative, 26.9% were HPV-positive. The 5-year disease-free and overall survival probabilities of all patients were 71.2% and 55.7%, respectively. No correlation could be found between HPV-status and VCP expression. VCP overexpression in HPV-negative patients was associated with significantly better 5-year disease-free survival (86.4% vs., 45.6%, p = 0.017). The level of VCP-intensity determined by immunohistochemistry could be an additional prognostic marker in HPV-negative OSCC. VCP expression seems not to correlate with the HPV-status.