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Nikolaus Poier,1,2 Johannes Hochstöger,1,2 Stephan Hackenberg,3 Agmal Scherzad,3 Maximilian Bregenzer,3 Dominik Schopper,1,2 Norbert Kleinsasser1,3 1Department of Otorhinolaryngology, Head and Neck Surgery, Kepler University Hospital, Linz 4021, Austria; 2Medical Faculty, Johannes Kepler University Linz, Linz 4040, Austria; 3Department of Otorhinolaryngology, Plastic, Aesthetic and Reconstructive Head and Neck Surgery, University of Wuerzburg, Würzburg 97080, GermanyCorrespondence: Nikolaus Poier Krankenhausstraße 9, Linz 4021, AustriaTel +43 650 2409889Email nikolaus.poier@kepleruniklinikum.atPurpose: The present study focuses on threshold levels for cytotoxicity after long-term and repetitive exposure for HUVEC as a model for the specific microvascular endothelial system. Furthermore, possible genotoxic effects and functional impairment caused by ZnO NPs in HUVEC are elucidated.Methods: Thresholds for cytotoxic effects are determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and Annexin V assay. To demonstrate DNA damage, single-cell microgel electrophoresis (comet) assay is performed after exposure to sub-cytotoxic concentrations of ZnO NPs. The proliferation assay, dot blot assay and capillary tube formation assay are also carried out to analyze functional impairment.Results: NPs showed to be spherical in shape with an average size of 45– 55 nm. Long-term exposure as well as repetitive exposure with ZnO NPs exceeding 25 μg/mL lead to decreased viability in HUVEC. In addition, DNA damage was indicated by the comet assay after long-term and repetitive exposure. Twenty-four hours after long-term exposure, the proliferation assay does not show any difference between negative control and exposed cells. Forty-eight hours after exposure, HUVEC show an inverse concentration-related ability to proliferate. The dot blot assay provides evidence that ZnO NPs lead to a decreased release of VEGF, while capillary tube formation assay shows restriction in the ability of HUVEC to build tubes and meshes as a first step in angiogenesis.Conclusion: Sub-cytotoxic concentrations of ZnO NPs lead to DNA damage and functional impairment in HUVEC. Based on these data, ZnO NPs may affect neo-angiogenesis. Further investigation based on tissue cultures is required to elucidate the impact of ZnO NPs on human cell systems.Keywords: zinc oxide, nanoparticles, cytotoxicity, genotoxicity, toxicity

Agmal Scherzad, Rudolf Hagen, Stephan Hackenberg Department of Otorhinolaryngology, Plastic, Aesthetic and Reconstructive Head and Neck Surgery, Julius Maximilian University of Wuerzburg, Würzburg 97080, GermanyCorrespondence: Agmal ScherzadDepartment of Otorhinolaryngology, Plastic, Aesthetic and Reconstructive Head and Neck Surgery, Julius Maximilian University of Wuerzburg, Würzburg 97080, GermanyEmail Scherzad_A@ukw.deAbstract: The functional role of the respiratory epithelium is to generate a physical barrier. In addition, the epithelium supports the innate and acquired immune system through various cytokines and chemokines. However, epithelial cells are also involved in the pathogenesis of various respiratory diseases, some of which are mediated by increased permeability of the mucosal membrane or disturbed mucociliary transport. In addition, it has been shown that epithelial cells are involved in the development of inflammatory respiratory diseases. The following review article focuses on the aspects of epithelial mis-differentiation, in particular with respect to nasal mucosal barrier function, epithelial immunogenicity, nasal epithelial–mesenchymal transition and nasal microbiome.Keywords: nasal mucosal barrier function, tight junction, epithelial–mesenchymal transition, microbiome

Summary: Background: SARS-CoV-2 antigen rapid detection tests (RDTs) emerged as point-of-care diagnostics alongside reverse transcription polymerase chain reaction (RT-qPCR) as reference. Methods: In a prospective performance assessment from 12 November 2020 to 30 June 2023 at a single centre tertiary care hospital, the sensitivity and specificity (primary endpoints) of RDTs from three manufacturers (NADAL®, Panbio™, MEDsan®) were compared to RT-qPCR as reference standard among patients, accompanying persons and staff aged ≥ six month in large-scale, clinical screening use. Regression models were used to assess influencing factors on RDT performance (secondary endpoints). Findings: Among 78,798 paired RDT/RT-qPCR results analysed, overall RDT sensitivity was 34.5% (695/2016; 95% CI 32.4–36.6%), specificity 99.6% (76,503/76,782; 95% CI 99.6–99.7%). Over the pandemic course, sensitivity decreased in line with a lower rate of individuals showing typical COVID-19 symptoms. The lasso regression model showed that a higher viral load and typical COVID-19 symptoms were directly significantly correlated with the likelihood of a positive RDT result in SARS-CoV-2 infection, whereas age, sex, vaccination status, and the Omicron VOC were not. Interpretation: The decline in RDT sensitivity throughout the pandemic can primarily be attributed to the reduced prevalence of symptomatic infections among vaccinated individuals and individuals infected with Omicron VOC. RDTs remain valuable for detecting SARS-CoV-2 in symptomatic individuals and offer potential for detecting other respiratory pathogens in the post-pandemic era, underscoring their importance in infection control efforts. Funding: German Federal Ministry of Education and Research (BMBF), Free State of Bavaria, Bavarian State Ministry of Health and Care.

Head and neck squamous-cell carcinoma (HNSCC) is associated with aggressive local invasiveness, being a main reason for its poor prognosis. The exact mechanisms underlying the strong invasive abilities of HNSCC remain to be elucidated. Therefore, there is a need for in vitro models to study the interplay between cancer cells and normal adjacent tissue at the invasive tumor front. To generate oral mucosa tissue models (OMM), primary keratinocytes and fibroblasts from human oral mucosa were isolated and seeded onto a biological scaffold derived from porcine small intestinal submucosa with preserved mucosa. Thereafter, we tested different methods (single tumor cells, tumor cell spots, spheroids) to integrate the human cancer cell line FaDu to generate an invasive three-dimensional model of HNSCC. All models were subjected to morphological analysis by histology and immunohistochemistry. We successfully built OMM tissue models with high in vivo–in vitro correlation. The integration of FaDu cell spots and spheroids into the OMM failed. However, with the integration of single FaDu cells into the OMM, invasive tumor cell clusters developed. Between segments of regular epithelial differentiation of the OMM, these clusters showed a basal membrane penetration and lamina propria infiltration. Primary human fibroblasts and keratinocytes seeded onto a porcine carrier structure are suitable to build an OMM. The HNSCC model with integrated FaDu cells could enable subsequent investigations into cancer cell invasiveness.

Abstract High hydrostatic pressure specifically devitalizes cells and tissues without major changes in their molecular structure. Hence, high hydrostatic pressure may enhance the development of whole-cell anti-tumor vaccines, representing tumor heterogeneity and thus (neo-) antigen diversity. Moreover, safe devitalization of tumor-infiltrated supporting tissue may facilitate reimplantation for functional reconstruction. However, precise high hydrostatic pressure thresholds for safe cancer cell killing are unknown. Here, we show that high hydrostatic pressure of at least 315 MPa is necessary to safely devitalize head and neck squamous cell cancer. A pressure of 210 MPa, which has been used frequently in cancer vaccine preparation, resulted in partial devitalization with 27% live cells in flow cytometry and 4% remaining autofluorescence in cell culture after one week. The remaining cells could form vital tumors in the chorioallantoic membrane assay. In contrast, 315 MPa killed all cells in vitro and prevented tumor outgrowth in ovo. The effectiveness of 315 MPa was attributed to the induction of DNA double-strand breaks, independent of apoptosis, autophagy, or methuosis. Furthermore, 315 MPa continued to induce immunogenic cell death. Our results demonstrate that 315 MPa of high hydrostatic pressure induces safe and sustained devitalization of head and neck cancer cells and tissues. Because of the heterogeneity in pressure resistance, we propose our approach as a starting point for determining the precise thresholds for other cancer entities. Further studies on head and neck cancer should focus on immunological co-cultures, combinations of immune checkpoint inhibition, and accurate anatomical reconstruction with pressure-treated autografts.

Abstract Seed amplification assays (SAA) are becoming commonly used in synucleinopathies to detect α-synuclein aggregates. Studies in Parkinson’s disease (PD) and isolated REM-sleep behavior disorder (iRBD) have shown a considerably lower sensitivity in the olfactory epithelium than in CSF or skin. To get an insight into α-synuclein (α-syn) distribution within the nervous system and reasons for low sensitivity, we compared SAA assessment of nasal brushings and skin biopsies in PD (n = 27) and iRBD patients (n = 18) and unaffected controls (n = 30). α-syn misfolding was overall found less commonly in the olfactory epithelium than in the skin, which could be partially explained by the nasal brushing matrix exerting an inhibitory effect on aggregation. Importantly, the α-syn distribution was not uniform: there was a higher deposition of misfolded α-syn across all sampled tissues in the iRBD cohort compared to PD (supporting the notion of RBD as a marker of a more malignant subtype of synucleinopathy) and in a subgroup of PD patients, misfolded α-syn was detectable only in the olfactory epithelium, suggestive of the recently proposed brain-first PD subtype. Assaying α-syn of diverse origins, such as olfactory (part of the central nervous system) and skin (peripheral nervous system), could increase diagnostic accuracy and allow better stratification of patients.

Immunosuppression is one key feature of mesenchymal stromal cells (MSCs) that has high expectations for therapeutic use. The influence of pro-inflammatory stimuli can modify the characteristics of MSCs and enhance immunosuppressive properties. The local postoperative environment contains cytokines, MSCs, and immune cells in high quantities, and their mutual influence is still unclear. Knowledge of in vivo processes is pivotal for potential therapeutic applications, and therefore, the aim of this study was to investigate the influence of wound fluid (WF) on the immunomodulatory potential of MSCs. CD4+ cells were co-cultured with native or WF-preconditioned MSCs for 5 days. CFSE staining revealed significant suppression of T cell proliferation after co-culture that was even more distinct in co-culture with WF-MSCs. The concentration of IDO-1, TGF-β1 and IFN-γ was higher while TNF-α was reduced in co-culture supernatants, indicating a transition to an anti-inflammatory milieu. In summary, the results provide evidence that the influence of WF alters the immunomodulatory potential of MSCs. These findings should serve as the basis for further investigations with a focus on T cell subpopulations. [ABSTRACT FROM AUTHOR]

Salivary gland tumors (SGTs) are a relevant, highly diverse subgroup of head and neck tumors whose entity determination can be difficult. Confocal Raman imaging in combination with multivariate data analysis may possibly support their correct classification. For the analysis of the translational potential of Raman imaging in SGT determination, a multi-stage evaluation process is necessary. By measuring a sample set of Warthin tumor, pleomorphic adenoma and non-tumor salivary gland tissue, Raman data were obtained and a thorough Raman band analysis was performed. This evaluation revealed highly overlapping Raman patterns with only minor spectral differences. Consequently, a principal component analysis (PCA) was calculated and further combined with a discriminant analysis (DA) to enable the best possible distinction. The PCA-DA model was characterized by accuracy, sensitivity, selectivity and precision values above 90% and validated by predicting model-unknown Raman spectra, of which 93% were classified correctly. Thus, we state our PCA-DA to be suitable for parotid tumor and non-salivary salivary gland tissue discrimination and prediction. For evaluation of the translational potential, further validation steps are necessary.

Inhalation is considered to be the most relevant source of human exposure to nanoparticles (NPs); however, only a few investigations have addressed the influence of exposing the respiratory mucosal barrier to subcytotoxic doses. In the nasal respiratory epithelium, cells of the mucosa represent one of the first contact points of the human organism with airborne NPs. Disruption of the epithelial barrier by harmful materials can lead to inflammation in addition to potential intrinsic toxicity of the particles. The aim of this study was to investigate whether subtoxic concentrations of zinc oxide (ZnO)- and silver (Ag)-NPs have an influence on upper airway barrier integrity. Nasal epithelial cells from 17 donors were cultured at the air–liquid interface and exposed to ZnO- and Ag-NPs. Barrier function, quantified by transepithelial electrical resistance (TEER), decreased after treatment with 10 µg/mL Ag-NPs, but FITC-dextran permeability remained stable and no change in mRNA levels of tight junction proteins and E-cadherin was detected by real-time quantitative PCR (RT-qPCR). The results indicate that subtoxic concentrations of Ag-NPs may already induce damage of the upper airway epithelial barrier in vitro. The lack of similar disruption by ZnO-NPs of similar size suggests a specific effect by Ag-NPs.

Background/Aim: This study aimed to assess the impact of hypoxia on epithelial-mesenchymal transition (EMT) in head and neck squamous cell carcinoma (HNSCC), focusing on the involvement of transcription factors hypoxia inducible factor 1 (HIF-1α) and Jumonji Domain-Containing Protein 1A (JMJD1A). Materials and Methods: FaDu and Cal33 cell lines were subjected to hypoxic and normoxic conditions. Cell proliferation was quantified electronically, while PCR and western blot analyses were used to measure mRNA and protein levels of HIF-1α, JMJD1A, and EMT markers. EMT was further characterized through immunofluorescence, migration, and invasion assays. Results: Hypoxic conditions significantly reduced cell proliferation after 48 hours in both cell lines. HIF1α mRNA levels increased initially during short-term hypoxia but declined thereafter, while JMJD1A mRNA levels showed a sustained increase with prolonged hypoxia. Western blot analysis revealed contrasting trends in protein levels. EMT marker expression varied markedly over time at both the mRNA and protein levels, suggesting EMT induction in hypoxia within 24 hours. Immunofluorescence, migration, and invasion assays supported these findings. Conclusion: The study provides evidence of hypoxia-induced EMT in HNSCC, although conflicting results suggest a complex interplay among molecular regulators involved in this process. [ABSTRACT FROM AUTHOR]

Objective: Confocal laser endomicroscopy (CLE) is an optical imaging technique that allows in vivo, real‐time, microscope‐like assessment of superficial lesions. Although there is substantial data on CLE use in the upper GI tract, there is limited information regarding its application in the nasal cavity and paranasal sinuses. This study aims to assess the feasibility and diagnostic metrics of CLE in the nasal cavity and paranasal sinuses regarding differentiation between healthy/benign and malignant tissue. These structures show, however, a wider variety of frequent and concomitant benign and malignant pathologies, which could pose an increased challenge for optical biopsy by CLE. Methods: We performed CLE on a case series of six patients with various findings in the nose (three chronic rhinosinusitis, adenocarcinoma, meningoenzephalozele, esthesionneuroblastoma). Forty‐two sequences (3792 images) from various structures in the nasal cavity and/or paranasal sinuses were acquired. Biopsies were taken at corresponding locations and analyzed in hematoxylin and eosin staining as a standard of reference. Three independent examiners blinded to the histopathology assessed the sequences. Results: Healthy and inflamed mucosa could be distinguished from malignant lesions with an accuracy, sensitivity, specificity, positive predictive value, and negative predictive value of 84.1%, 85.4%, 83.1%, 72.5%, and 92.1%, respectively, with a substantial agreement between raters (Fleiss κ = 0.62). Conclusion: This technique shows, despite its limitations, potential as an adjunctive imaging technique during sinus surgery; however, the creation of a scoring system based on reproducible and defined characteristics in a larger more diverse population should be the focus of further research to improve its diagnostic value and clinical utility. Level of Evidence: NA Laryngoscope, 134:4217–4224, 2024 [ABSTRACT FROM AUTHOR]