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Nasal xenobiotic metabolizing enzymes (XMEs) are important for the sense of smell because they influence odorant availability and quality. Since the major part of the human nasal cavity is lined by a respiratory mucosa, we hypothesized that this tissue contributed to nasal odorant metabolism through XME activity. Thus, we built human respiratory tissue models and characterized the XME profiles using single-cell RNA sequencing. We focused on the XMEs dicarbonyl and l-xylulose reductase, aldehyde dehydrogenase (ALDH) 1A1, and ALDH3A1, which play a role in food odorant metabolism. We demonstrated protein abundance and localization in the tissue models and showed the metabolic activity of the corresponding enzyme families by exposing the models to the odorants 3,4-hexandione and benzaldehyde. Using gas chromatography coupled with mass spectrometry, we observed, for example, a significantly higher formation of the corresponding metabolites 4-hydroxy-3-hexanone (39.03 ± 1.5%, p= 0.0022), benzyl alcohol (10.05 ± 0.88%, p= 0.0008), and benzoic acid (8.49 ± 0.57%, p= 0.0004) in odorant-treated tissue models compared to untreated controls (0 ± 0, 0.12 ± 0.12, and 0.18 ± 0.18%, respectively). This is the first study that reveals the XME profile of tissue-engineered human respiratory mucosa models and demonstrates their suitability to study nasal odorant metabolism.

Background: In patients with iodine-negative thyroid cancer (TC), current guidelines endorse an [18F]FDG PET/CT to identify dedifferentiated sites of disease. We aimed to determine the rate of oncological management changes triggered by such a molecular imaging approach, along with the impact on outcome. Methods: 42 consecutive patients with negative findings on [131I] whole body scan were scheduled for [18F]FDG PET/CT and treatment based on PET results were initiated. To determine the impact on oncological management, we compared the therapeutic plan prior to and after molecular imaging. Based on imaging follow-up, the rate of controlled disease (CD, defined as stable disease, complete or partial response) was also recorded, thereby allowing to assess whether [18F]FDG-triggered management changes can also lead to favorable outcome. Results: We observed no alterations of the treatment plan in 9/42 (21.4%) subjects (active surveillance in 9/9 [100%]). Oncological management was changed in the remaining 33/42 (78.6%; systemic treatment in 9/33 [27.3%] and non-systemic treatment in 24/33 [72.7%]). Among patients receiving non-systemic therapy, the following changes were noted: surgery in 20/24 (83.3%) and radiation therapy in 4/24 (16.7%). In the systemic group, tyrosine kinase inhibitor (TKI) was prescribed in 8/9 (88.9%), while radioiodine therapy based on a TKI-mediated redifferentiation approach was conducted in 1/9 (11.1%). In 26 subjects with available follow-up, rate of CD was 22/26 (84.6%) and among those, 15/22 (68.1%) had experienced previous management changes based on PET/CT findings. Conclusions: In subjects with iodine-negative TC, [18F]FDG PET/CT triggered relevant management changes along with disease control in the vast majority of patients. As such, in dedifferentiated TC, [18F]FDG PET/CT may serve as a relevant management tool and therapeutic decision-aid in the clinic.

Olfaction is a multi-step process. At a peripheral level, nasal odorant metabolism contributes to olfaction via signal termination, variation, and regulation. We summarize current techniques used to investigate nasal odorant metabolism and give an outlook on future approaches, such as nasal tissue models and their potential contributions in future research directions.

The human pathogen Bordetella pertussistargets the respiratory epithelium and causes whooping cough. Its virulence factor adenylate cyclase toxin (CyaA) plays an important role in the course of infection. Previous studies on the impact of CyaA on human epithelial cells have been carried out using cell lines derived from the airways or the intestinal tract. Here, we investigated the interaction of CyaA and its enzymatically inactive but fully pore-forming toxoid CyaA-AC–with primary human airway epithelial cells (hAEC) derived from different anatomical sites (nose and tracheo-bronchial region) in two-dimensional culture conditions. To assess possible differences between the response of primary hAEC and respiratory cell lines directly, we included HBEC3-KT in our studies. In comparative analyses, we studied the impact of both the toxin and the toxoid on cell viability, intracellular cAMP concentration and IL-6 secretion. We found that the selected hAEC, which lack CD11b, were differentially susceptible to both CyaA and CyaA-AC–. HBEC3-KT appeared not to be suitable for subsequent analyses. Since the nasal epithelium first gets in contact with airborne pathogens, we further studied the effect of CyaA and its toxoid on the innate immunity of three-dimensional tissue models of the human nasal mucosa. The present study reveals first insights in toxin–cell interaction using primary hAEC.

For culture models of primary cells of the human nasal mucosa, monocultures with epithelial cells (ECs) are used as well as cocultures with ECs and fibroblasts (FBs). Well-differentiated models of the respiratory nasal epithelium can be used for ecogenotoxicological assessments, for experiments on host/pathogen interactions, or tissue engineering. However, long-term cultivation and repeated passaging may induce a loss of DNA integrity or cell functionality. The aim of this study was to evaluate these parameters in test systems created from primary nasal mucosa cells. Enzymatic and sequential cell isolation from nasal tissue was performed. EC monocultures and compartment-separated EC-FB cocultures were cultivated over three passages under air/liquid interface conditions. DNA stability and regenerative capacity at the DNA and chromosomal level as well as proliferation and cell differentiation were examined. Both methods showed equivalent levels of DNA stability and regenerative capacity over all passages. Sequential growth of the coculture provided higher cell purity, while enzymatic cell harvest was associated with FB contamination in EC culture. Mucociliary differentiation was verified with electron microscopy in both methods. Functionality measured by lipopolysaccharide stimulation of interleukins was constant over long-term cultivation. Our data confirm DNA stability in long-term cell cultivation as well as functional integrity in both culture methods. Sequential cell isolation should be favored over enzymatic isolation due to higher culture purity.Impact statementCell culture models are frequently used for ecogenotoxicological assessments, for experiments on host/pathogen interactions, or tissue engineering. However, DNA stability and functional integrity after long-term cultivation in such tissue models have not been investigated, yet. This study is the first showing systematic and evident data on DNA damage and functional aspects in primary human cell culture models of nasal epithelium.

Three-dimensional respiratory tissue models have been generated using, for example, human primary airway epithelial cells (hAEC) or respective cell lines. To investigate ciliopathies, such as primary ciliary dyskinesia, the presence of functional kinocilia in vitrois an essential prerequisite. Since access to hAEC of healthy donors is limited, we aimed to identify a respiratory epithelial cell line that is capable to display functional kinocilia on at least 60% of the apical surface. Thus, we cultured four different human respiratory cell lines with human primary airway fibroblasts under airlift conditions, characterized the morphology, and analyzed ciliary function. Only one of the tested cell lines showed beating kinocilia; however, <10% of the whole surface was covered and ciliary beating was undirected. Positive control tissue models using hAEC and fibroblasts displayed expected directed ciliary beating pattern around 11 Hz. Our data show that the available cell lines are not suitable for basic and applied research questions whenever functional kinocilia are required and that, rather, hAEC- or human induced pluripotent stem cell-derived tissue models need to be generated.Impact StatementTo study ciliopathies or Bordetella pertussisinfection in vitro, three-dimensional respiratory tissue models with functional kinocilia covering at least 60% of the model's surface are mandatory. We cultured four respiratory cell lines on a fibroblast-loaded biological scaffold and showed that none of them met this requirement. In contrast, primary airway cell-derived models sufficiently reflected the mucociliary phenotype. To further search for an alternative to primary respiratory cells, investigations on other cell lines should be conducted or even new cell lines have to be generated.

The loss of salivary gland function caused by radiation therapy of the head and neck is a serious condition and it affects a patient's quality of life. The current lack of effective therapies demands new options to be explored. This study tested whether human salivary gland epithelial cells (SGECs) could be successfully cultured on a decellularized porcine gut matrix (SIS-muc) in both mono- and coculture with microvascular endothelial cells (mvECs). By performing immunofluorescence imaging, transmission as well as scanning electron microscopy (SEM), quantitative polymerase chain reaction (qPCR), and an amylase enzyme assay, it was investigated as to what extent the three-dimensional (3D)-cultured cells could maintain their molecular differentiation and the production of working α-amylase (α-AMY) compared with two-dimensional (2D) culture. In both 3D mono- and coculture, SGECs were successfully cultured and formed acinar-like structures. Those findings were confirmed by SEM imaging. Immunofluorescence imaging revealed that 3D-cultured cells expressed α-AMY, Claudin-1 (CL-1), and water channel protein aquaporin-5 (AQP-5). Two-dimensional-cultured cells only were positive for α-AMY. Real time (RT)-qPCR analysis showed that α-AMYrelative gene expression was higher in both 3D mono- and coculture than in 2D culture. In α-AMY enzyme assay, cocultured SGECs showed about 25 times increased enzyme activity compared with 2D-cultured cells. In conclusion, the SIS-muc combined with endothelial coculture seems a suitable culture setting for the tissue engineering of functional human salivary gland tissue.

ABSTRACTZinc oxide nanoparticles (ZnO-NPs) are being used in many cosmetic products and have been shown to induce tumor-selective cell death in human head and neck squamous cell carcinoma (HNSCC) in vitro. Cetuximab is a monoclonal antibody directed against the epidermal growth factor receptor (EGFR), whose effectiveness for HNSCC, alone or in combination with cytostatic drugs, has been demonstrated intensively in the last decades. Nanoparticles are known to interact with protein structures and thus may influence their functionality. The aim of the current study was to evaluate the effect of ZnO-NPs on the antitumor properties of Cetuximab in HNSCC in vitro. Two HNSCC cell lines (FaDu and HLaC-78) were treated with 0.1, 1 or 10 μM Cetuximab as well as 0, 0.1 or 1 μg/ml ZnO-NP. Qualitative assessment of ZnO-NP was conducted via transmission electron microscopy (TEM) and immunofluorescence staining. Evaluation was done via the MTT-assay after 24, 48 and 72 hours of incubation with Cetuximab and ZnO-NPs. ZnO-NPs were shown to antagonize the anti-tumor effects of Cetuximab in a time-dependent as well as dose-dependent way. These findings suggest an inhibitory interaction of ZnO-NPs with Cetuximab, which warrants further investigation.

In vitro test systems gain increasing importance in preclinical studies to increase the predictivity and reduce animal testing. Of special interest herein are barrier tissues that guard into the human body. These barriers are formed by highly specialized tissues such as the skin, the airways, and the intestine. However, to recapitulate these tissues, researchers are currently restricted by a lack of suitable supporting scaffolds. In this study, we present biological scaffolds based on decellularized porcine gut segments that offer a natural environment for cell growth and differentiation. Employing these scaffolds, human barrier models of the skin, the airways, and the intestine that mimic the natural histological architecture of the respective tissue are generated. These models show tissue specific barrier properties, such as the stratification of the skin, the mucociliary phenotype of the airways, and polarization of the intestinal epithelium. To investigate the transport characteristics of the intestinal test system, we incubated the tissue models with fluorescein (Papp<1 × 106cm/s), propranolol (Papp>7 × 106cm/s), and rhodamin123 (ratio 2.45). The here presented biological scaffolds facilitate the in vitro generation of human barrier models that might represent useful tools for drug delivery studies.

Peripheral blood mononuclear cells (PBMCs) are the only source of human lymphoid cells routinely available for immunomonitoring of T-cell responses to microbial and tumor-associated antigens. However, previous work in mice and humans had indicated that CD4 T cells transiently lose antigen sensitivity when cellular contacts are lost (eg, by entering the circulation). Using the simple and robust protocol for resetting T cells to original reactivity (RESTORE; ie, preculturing PBMCs for 2 days at a high cell density before initiation of antigenic stimulation), we show that CD8 T-cell responses to viral and tumor-associated antigens are greatly underestimated in blood, and sometimes even remain undetected, if conventional, unprocessed PBMC cultures are used. The latter finding is particularly striking with regard to the appearance of Wilms tumor 1 protein-specific CD8 T-cell responses in leukemia patients after allogeneic bone marrow transplantation. The dramatic increase in antigen sensitivity of “restored” CD8 T cells is associated with phosphorylation of proximal T-cell receptor signaling components, and with the upregulation of genes involved in aerobic glycolysis, thereby increasing T-cell functionality. The RESTORE protocol permits a more meaningful monitoring of CD8 memory T-cell responses to viral infections and tumors and vaccination success. Furthermore, when generating T-cell lines for adoptive T-cell therapy, it avoids the loss of those clones, which strictly depend on the primed status conferred by cellular interactions in the tissue context for their initial reactivation by antigen. The data reported in this article have been deposited in the Gene Expression Omnibus database (accession number GSE63430).

Introduction Chondrogenic differentiation of adipose-derived stem cells (ASCs) has proven to be feasible. To compensate for laryngeal palsy or cartilage defects after surgery or trauma using tissue engineering, a formable and stable scaffold material is mandatory.Methods ASCs were seeded in fibrin-polyurethane scaffolds and cultured in chondrogenic differentiation medium adding the growth factors TGF-?1, TGF-?3, and BMP-2 for up to 35 days.Results Histological examination showed acid glycosaminoglycans in the extracellular matrix in all groups. Immunofluorescence presented positive staining for collagen II, aggrecan, and SOX-9 in the TGF-?1–, TGF-?3–, and BMP-2-group. With Real-time PCR analyses, chondrogenic differentiation became apparent by the expression of the specific genes COL2A1 (collagen II), AGC 1 (aggrecan), and SOX-9, whereas collagen II expression was low in all groups compared to bone marrow-derived stem cells (BMSC) due to reduced chondrogenic ability.Conclusions These findings demonstrate the general ability of ASCs to differentiate into matrix-producing chondrocytes in fibrin-polyurethane scaffolds. However, further experiments are necessary to enhance this chondrogenic potential of ASCs seeded in fibrin-polyurethane scaffolds in order to produce a suitable regeneration method for treating cartilage defects or an implantable medialization material for vocal cord palsy.

After surgery, wound healing begins with a well-orchestrated integration of several cytokines, cells, and extracellular matrix. Some studies show an involvement of stem cells in wound healing. However, little is known about the mechanism that leads to the migration of stem cells. Wound fluid (WF) with its cytokines may play an important role. We investigated in the present study the in vitroeffects of WF on adipose-derived stem cells (ADSCs). Survival, proliferation, structural integrity, changes in the multidifferentiation potential, and surface markers (cluster of differentiation [CD] 105, CD73, CD90) of ADSCs after cultivation with WF was analyzed. Further, the migration effect of WF on ADSCs was evaluated. The proliferation rate and the migration potential of ADSCs were enhanced significantly by cultivation with WF. There was also a change in the quantity of surface markers after cultivation with WF. In conclusion, in vitroexpansion of stem cells with WF proved possible. WF and its cytokines could represent one primary reason for the migration of stem cells toward the wound. Future investigation is warranted to clarify the significance of the shift in surface markers.

The 5-year survival rate of patients suffering from head and neck squamous cell carcinoma (HNSCC) is unsatisfying despite the advances in carcinoma treatment. Recent studies suggest that stem cells can be used as a gene therapy carrier for cancer treatment. Stem cells produce different cytokines such as growth factors in a paracrine manner and cancer cells may show drug resistance in the presence of such growth factors. Reports in the literature concerning treatment of cancer using bone marrow derived stem cells (BMSC) are controversial, which led us to investigate the effects of paclitaxel on human HNSCC cell lines (FaDu and HLaC 78) cultivated simultaneously with BMSC in a transwell system (co-culture). Co-culture and HNSCC cell lines were treated with 10nM of paclitaxel for 24h. Morphology, viability and apoptosis were measured by microscopy, the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, and the Annexin V-propidium iodide test. The survival of HNSCC cell lines treated with paclitaxel in co-culture increased significantly compared to control cells. Apoptosis of HNSCC cell lines in co-culture was attenuated significantly. In conclusion, BMSC increase HNSCC resistance to treatment with paclitaxel in vitro. Tumor-stroma interactions are critical components of tumor biology including tumor invasion and metastatic potential. Therefore particular attention must be paid to the complex tumor-stroma interactions to fully understand how tumor cells become chemoresistant.

Background There is an ongoing discussion concerning the potential origins of chronic rhinosinusitis with nasal polyposis (CRSwNP).Objective The aim of this study was to quantify subpopulations of T cells in peripheral blood and nasal polyps in CRSwNP to examine their influence on the etiology of this disease.Methods Tissue and blood samples were collected from 11 patients who underwent nasal sinus surgery, and these samples were analyzed by multicolor flow cytometry.Results There was a significantly lower frequency of CD4+T-helper (Th) cells and a significantly higher frequency of CD8+T cells among lymphocytes isolated from nasal polyps compared with peripheral blood mononuclear cells (PBMC). In both T-cell subpopulations, a shift mainly from naive T cells among peripheral blood lymphocytes toward an effector memory and terminally differentiated subtype predominance in nasal polyps was observed. Among CD4+T cells, the frequencies of cluster of differentiation (CD) 45RA- Forkhead-Box-Protein P3high (FoxP3high) cytotoxic T-lymphocyte-associated Protein 4high(CTLA-4high) activated regulatory T (Treg) cells, and CD45RA- Forkhead-Box-Protein P3low (FoxP3low) memory T cells were significantly increased in nasal polyps compared with PBMC.Conclusion In this study, we presented a detailed characterization of CD4+and CD8+T-cell subpopulations in patients with CRSwNP. CD8+T cells were more prominent in nasal polyps than in CD4+T cells. Both nasal CD8+T cells and CD4+T cells predominantly had an effector memory phenotype. Among CD4+T cells, activated Tregcells were increased in nasal polyps compared with PBMC. The data point toward a local regulation of T-cell composition within the microenvironment of nasal polyps, which might be further exploited in the future to develop novel immunotherapeutic strategies.

Nasal passage contributes up to 50% of total resistance in normal breathing especially in neonates who are obligatory nose breathers. Any further increase in airway resistance may lead to severe respiratory distress. Dacryocystoceles are a rare cause of nasal obstruction in neonates. We present the case of a full-term 3-day-old female infant with progressive respiratory distress due to bilateral dacryocystoceles.