The mystery behind the nostrils - technical clues for successful nasal epithelial cell cultivation.

OBJECTIVES: Research involving the nose reveals important information regarding the morphology and physiology of the epithelium and its molecular response to agents. The role of nasal epithelial cells and other cell subsets within the nasal epithelium play an interesting translational split between experimental and clinical research studying respiratory disorders or pathogen reactions. With an additional technical manuscript including a detailed description of important technical aspects, tips, tricks, and nuances for a successful culturing of primary, human nasal epithelial cells (NAEPCs), we here aim to improve the process of communication between experimentalists and physicians, supporting the purpose of a fruitful work for future translational projects. METHODS: Based on previous work on various complex culture models of subject-derived NAEPCs, this additional manuscript harmonizes previously published facts combined with own experiences for a trouble-free implementation in laboratories. RESULTS: A well-designed experimental question is essential prior to the establishment of different NAEPCs culture models. The correct method of cell extraction from the nasal cavity is essential and represent an important basis for successful culture work. Prior enzymatic processing of biopsy specimens, cell culture materials, collagenization procedure, culture conditions, and choice of culture medium are some important practical notes that increase the quality of the culture. Moreover, protocols on imaging techniques including histologic and electron microscopy must be adapted for NAEPC culture. Adapted flow cytometric protocols and transepithelial electrical resistance measurements can add valuable information. OUTLOOK: A successful culturing of NAEPCs can provide an important basis for genetic studies and the implementation of omics-science, which is increasingly receiving broad attention in the scientific community. The common aim of in vitro 'mini-noses' will be a breakthrough in laboratories aiming to perform research under in vivo conditions. Here, organoid models are interesting models presenting a basis for translational studies.

Correction: Schildgen, V., et al. Human Bocavirus Infection of Permanent Cells Differentiated to Air-Liquid Interface Cultures Activates Transcription of Pathways Involved in Tumorigenesis. Cancers 2018, 10, 410.

The authors wish to make the following correction to this paper [...].

From Submerged Cultures to 3D Cell Culture Models: Evolution of Nasal Epithelial Cells in Asthma Research and Virus Infection.

Understanding the response to viral infection in the context of respiratory diseases is of significant importance. Recently, there has been more focus on the role of the nasal epithelium in disease modeling. Here, we provide an overview of different submerged, organotypic 3D and spheroid cell culture models of nasal epithelial cells, which were used to study asthma and allergy with a special focus on virus infection. In detail, this review summarizes the importance, benefits, and disadvantages of patient-derived cell culture models of nasal- and bronchial epithelial cells, including a comparison of these cell culture models and a discussion on why investigators should consider using nasal epithelial cells in their research. Exposure experiments, simple virus transduction analyses as well as genetic studies can be performed in these models, which may provide first insights into the complexity of molecular signatures and may open new doors for drug discovery and biomarker research.

Immunohistochemical evidence of striated muscle cells within midfacial superficial musculoaponeurotic system.

INTRODUCTION: The superficial musculoaponeurotic system (SMAS) is a controversial functional fibro-adipose layer that connects the mimic muscles to the skin and is involved in a variety of facial mimic expressions. The presence of muscle fibers within SMAS fibrous septa is hypothetical. The present study analyzed SMAS fibrous septa composition for the existence of striated muscle cells. METHODS: Histological serial sections of the sample borders (n=107) of 19 in sano-resected and diagnosed cutaneous tumors of the midfacial region were investigated. Immunohistochemical (actin and myosin) and hematoxylin and eosin staining were performed to detect striated muscle cells in SMAS fibrous septa. RESULTS: A fibro-neuro-musculo-vascular functional unit within SMAS fibrous septa was demonstrated. SMAS striated muscle cells were morphologically independent from preparotideal and periorbital mimic muscles. Intraseptal blood vessels draining the superficial and deep SMAS vascular system were described. CONCLUSIONS: Striated muscle cells were demonstrated within SMAS fibrous septa. Nerve cells and vascular tissue together with the SMAS fibro-muscular meshwork demonstrated an autonomous operating functional unit that hypothetical modulated individual mimic expression contributing to the diversity of mimic expression. The SMAS develops with mimic muscle contractions as a synergetic effect during facial crease and fold formation processes.

House Dust Mite Exposure Causes Increased Susceptibility of Nasal Epithelial Cells to Adenovirus Infection.

Adenovirus (AdV) infections in the respiratory tract may cause asthma exacerbation and allergic predisposition, and the house dust mite (HDM) may aggravate virus-induced asthma exacerbations. However, the underlying mechanisms of whether and how AdV affects asthmatic patients remains unclear. To address this question, we investigated nasal epithelial cells (NAEPCs) derived from a pediatric exacerbation study cohort for experimental analyses. We analyzed twenty-one different green-fluorescent protein- and luciferase-tagged AdV types in submerged 2D and organotypic 3D cell culture models. Transduction experiments revealed robust transduction of AdV type 5 (AdV5) in NAEPCs, which was associated with an increased uptake of AdV5 in the presence of HDM. In healthy and asthmatic NAEPCs exposed to HDM before infection, we observed a time- and dose-dependent increase of AdV5 uptake associated with upregulation of entry receptors for AdV5. Furthermore, electron microscopic and histologic analyses of 3D cell cultures revealed an impairment of the respiratory cilia after HDM exposition. This ex vivo pilot study shows the impact of AdV infection and HDM exposition in a primary cell culture model for asthma.

Human Bocavirus Infection of Permanent Cells Differentiated to Air-Liquid Interface Cultures Activates Transcription of Pathways Involved in Tumorigenesis.

The parvoviral human bocavirus (HBoV) is a respiratory pathogen, able to persist in infected cells. The viral DNA has been identified in colorectal and lung tumors and thus it was postulated that the virus could be associated with tumorigenesis. This assumption was supported by the fact that in HBoV-infected patients and in an in vitro cell culture system, pro-cancerogenic and -fibrotic cytokines were expressed. In this work, it is shown by a whole transcriptome analysis that, also at the mRNA level, several pathways leading to neoplasia and tumorigenesis are significantly upregulated. In total, a set of 54 transcripts are specifically regulated by HBoV, of which the majority affects canonical pathways that may lead to tumor development if they become deregulated. Moreover, pathways leading to necrosis, apoptosis and cell death are downregulated, supporting the hypothesis that HBoV might contribute to the development of some kinds of cancer.

Pre-augmentation soft tissue expansion improves scaffold-based vertical bone regeneration - a randomized study in dogs.

OBJECTIVE: Soft tissue (ST) dehiscence with graft exposure is a frequent complication of vertical augmentation. Flap dehiscence is caused by failure to achieve tension-free primary wound closure and by the impairment of flap microcirculation due to surgical trauma. Soft tissue expansion (STE) increases ST quality and quantity prior to reconstructive surgery. We hypothesized that flap preconditioning using STE would reduce the incidence of ST complications after bone augmentation and that optimized ST healing would improve the outcome of bone regeneration. MATERIALS AND METHODS: Self-filling tissue expanders were implanted in mandibular bone defects in ten beagle dogs. After expansion, alloplastic scaffolds were placed for vertical bone augmentation in STE sites and in control sites without STE pre-treatment. ST flap microcirculation was analysed using laser Doppler flowmetry. The incidence of graft exposures was evaluated after 2 weeks. Bone formation was assessed after 2 months, using histomorphometry and immunohistochemistry. RESULTS: Test sites showed significantly less impairment of perfusion and faster recovery of microcirculation after bone augmentation. Furthermore, no flap dehiscences occurred in STE sites. Bone regeneration was found in both groups; however, significantly greater formation of new bone was detected in test sites with preceding STE. CONCLUSIONS: Preconditioning using STE improved ST healing and bone formation after vertical augmentation. The combination of STE and the subsequent placement of alloplastic scaffolds may facilitate the reconstruction of severe bone defects.

Case report of an internal granuloma investigated by light and scanning electron microscopy.

There is no doubt that the main reason for an internal grauloma is a traumatic event. The trauma may be physical or chemical as in the case of caries or coronal pulpectomy. In most of the cases it is diagnosed by hazard or, when in case of fracture or mobility, extraction is the only therapy to be performed. If diagnosed in time root canal treatment may be adequate.In the presented case no single specific event could be determined being the cause of this large internal granuloma extending from the coronal third of the root canal to the whole crown just leaving an eggshell of enamel that fractured and mimicked mobility of the whole tooth to the patient finally causing him to attend the clinic. As the patient presented severe aggressive periodontitis and mobility of all teeth it first was assumed that periodontitis was the ethiological reason in this case. Due to secondary trauma the front teeth were labially positioned thus probably being exposed to traumatic insults more frequently. Clinically the upper right medial incisor appeared discoloured darkly not showing the typical pink spot. Without any force the coronal part of the right medial incisor could be removed manually and the root was extracted using a periostal extractor. As it was not suitable to leave the patient with a missing tooth in the front the wound was sutured and as a temporary solution the tooth was reconstructed with composite intraorally and fixed to the neighbour teeth adhesively. The histopathology of the internal granuloma and the crown was investigated.

Quantification of cell fusion events human breast cancer cells and breast epithelial cells using a Cre-LoxP-based double fluorescence reporter system.

The biological phenomenon of cell fusion plays an important role in several physiological processes, like fertilization, placentation, or wound healing/tissue regeneration, as well as pathophysiological processes, such as cancer. Despite this fact, considerably less is still known about the factors and conditions that will induce the merging of two plasma membranes. Inflammation and proliferation has been suggested as a positive trigger for cell fusion, but it remains unclear, which of the factor(s) of the inflamed microenvironment are being involved. To clarify this we developed a reliable assay to quantify the in vitro fusion frequency of cells using a fluorescence double reporter vector (pFDR) containing a LoxP-flanked HcRed/DsRed expression cassette followed by an EGFP expression cassette. Because cell fusion has been implicated in cancer progression four human breast cancer cell lines were stably transfected with a pFDR vector and were co-cultured with the stably Cre-expressing human breast epithelial cell line. Cell fusion is associated with a Cre-mediated recombination resulting in induction of EGFP expression in hybrid cells, which can be quantified by flow cytometry. By testing a panel of different cytokines, chemokines, growth factors and other compounds, including exosomes, under normoxic and hypoxic conditions our data indicate that the proinflammatory cytokine TNF-α together with hypoxia is a strong inducer of cell fusion in human MDA-MB-435 and MDA-MB-231 breast cancer cells.

The ability of human periodontium-derived stem cells to regenerate periodontal tissues: a preliminary in vivo investigation.

Periodontium-derived stem cells (pdSCs) can be cultured as dentospheres and differentiated into various cells of the neuronal lineage such as glial cells, thereby demonstrating their stem cell state. This study investigated whether pdSCs could be differentiated into the osteogenic lineage and, if so, whether these cells are able to regenerate periodontal tissue in vivo in an athymic rat model. Human adult pdSCs were isolated during minimally invasive periodontal surgery and expanded in vitro. To induce osteogenic differentiation, expanded pdSCs were cultured for 3 weeks in osteogenic differentiation media. Staining for alkaline phosphatase expression was positive, suggesting osteogenic differentiation. For in vivo studies, pdSCs were delivered onto suitable collagen sponges and implanted into periodontal defects on the right buccal cortex of the mandible in 16 immunodeficient nude rats. Histologic analysis of samples from the test side revealed reformation of periodontal ligament-like tissue, collagen fibers, and elements of bone, but no functional periodontal tissue regeneration. The data show that human adult pdSCs are capable of regenerating elements of bone and collagen fibers in an in vivo animal model.

Inhibition of radiation induced migration of human head and neck squamous cell carcinoma cells by blocking of EGF receptor pathways.

BACKGROUND: Recently it has been shown that radiation induces migration of glioma cells and facilitates a further spread of tumor cells locally and systemically. The aim of this study was to evaluate whether radiotherapy induces migration in head and neck squamous cell carcinoma (HNSCC). A further aim was to investigate the effects of blocking the epidermal growth factor receptor (EGFR) and its downstream pathways (Raf/MEK/ERK, PI3K/Akt) on tumor cell migration in vitro. METHODS: Migration of tumor cells was assessed via a wound healing assay and proliferation by a MTT colorimeritric assay using 3 HNSCC cell lines (BHY, CAL-27, HN). The cells were treated with increasing doses of irradiation (2 Gy, 5 Gy, 8 Gy) in the presence or absence of EGF, EGFR-antagonist (AG1478) or inhibitors of the downstream pathways PI3K (LY294002), mTOR (rapamycin) and MEK1 (PD98059). Biochemical activation of EGFR and the downstream markers Akt and ERK were examined by Western blot analysis. RESULTS: In absence of stimulation or inhibition, increasing doses of irradiation induced a dose-dependent enhancement of migrating cells (p < 0.05 for the 3 HNSCC cell lines) and a decrease of cell proliferation (p < 0.05 for the 3 HNSCC cell lines). The inhibition of EGFR or the downstream pathways reduced cell migration significantly (almost all p < 0.05 for the 3 HNSCC cell lines). Stimulation of HNSCC cells with EGF caused a significant increase in migration (p < 0.05 for the 3 HNSCC cell lines). After irradiation alone a pronounced activation of EGFR was observed by Western blot analysis. CONCLUSION: Our results demonstrate that the EGFR is involved in radiation induced migration of HNSCC cells. Therefore EGFR or the downstream pathways might be a target for the treatment of HNSCC to improve the efficacy of radiotherapy.

Influence of a totally open oval window on bone conduction in otosclerosis.

OBJECTIVE: The aim of this study was to evaluate changes in bone conduction thresholds before, during and after total stapedectomy. STUDY DESIGN: Prospective clinical study. METHODS: In 27 ears of 26 patients undergoing stapedectomy under local anesthesia, bone conduction was measured before surgery, during surgery under open oval window conditions, and after the insertion of a steel wire connective tissue prosthesis. Statistical data analysis was performed on the audiometric results. RESULTS: Under open oval window conditions, bone conduction hearing was found to be improved between 500 and 2000 Hz, but not at 4000 Hz. After insertion of the prosthesis, an additional improvement was evident at 500 and 1000 Hz, but a loss was seen at 2000 and 4000 Hz. CONCLUSION: This is the first investigation reported in which audiometry was performed under open oval window conditions during stapes surgery. Our results demonstrate that at least part of the preoperative bone conduction hearing loss in otosclerosis must be of mechanical, but not of sensorineural origin, as already suspected by Carhart. The fixed footplate suppresses cochlear micromechanics mainly at frequencies between 500 to 2000 Hz. Furthermore, the loss in bone conduction hearing at 2000 and 4000 Hz after insertion of the prosthesis indicates that rather than the surgical procedure of total removal of the footplate, other factors such as the handling of the prosthesis or its mechanical properties after insertion cause high-frequency hearing loss after stapes surgery.

Centrosome abnormalities in head and neck squamous cell carcinoma (HNSCC).

CONCLUSIONS: Numerical and structural centrosome abnormalities play a critical role in the tumor progression of in head and neck squamous cell carcinoma (HNSCC) and may provide useful information as a prognostic factor for these patients. OBJECTIVES: Centrosome alterations are often linked with aneuploidy, cell transformation, and tumor progress. We investigated centrosome abnormalities in HNSCC and correlated these variables to clinicopathological parameters and clinical follow up data of the patients. METHODS: Retrospective analysis of numerical and structural alterations of centrosomes in tumor tissues and corresponding normal epithelium (n=50 and 31, respectively). Immunohistochemistry was performed using an anti-gamma-tubulin antibody. Image acquisition was done by an Orthoplan microscope, centrosomes were segmented interactively, and area as well as mean optical density was measured. Aneuploidy was evaluated by fluorescence in situ hybridization in a subset of cases (n=29). RESULTS: Numerical and structural centrosome abnormalities differed significantly between normal squamous epithelium and tumor cells (both P<0.0001). Especially numerical centrosome abnormalities were significantly associated with T category and tumor stage (both P<0.0001) and the occurrence of distant metastasis (P=0.002 and P=0.019, respectively). Numerical centrosome abnormalities correlated also with disease free survival of the patients (P=0.032) as well as shorter overall survival (P=0.003).

Atu027, a liposomal small interfering RNA formulation targeting protein kinase N3, inhibits cancer progression.

We have previously described a small interfering RNA (siRNA) delivery system (AtuPLEX) for RNA interference (RNAi) in the vasculature of mice. Here we report preclinical data for Atu027, a siRNA-lipoplex directed against protein kinase N3 (PKN3), currently under development for the treatment of advanced solid cancer. In vitro studies revealed that Atu027-mediated inhibition of PKN3 function in primary endothelial cells impaired tube formation on extracellular matrix and cell migration, but is not essential for proliferation. Systemic administration of Atu027 by repeated bolus injections or infusions in mice, rats, and nonhuman primates results in specific, RNAi-mediated silencing of PKN3 expression. We show the efficacy of Atu027 in orthotopic mouse models for prostate and pancreatic cancers with significant inhibition of tumor growth and lymph node metastasis formation. The tumor vasculature of Atu027-treated animals showed a specific reduction in lymph vessel density but no significant changes in microvascular density.

Intracellular localization of lipoplexed siRNA in vascular endothelial cells of different mouse tissues.

Liposomally formulated siRNA can be used for RNAi applications in vivo. Intravenous bolus administration of lipoplexed siRNA has been shown to reduce gene expression in the vascular endothelium. Here, we applied immunofluorescence staining for different endothelial markers (PECAM-1, CD34, laminin) on paraffin sections to compare the respective expression pattern with the intracellular localization of intravenously administered, fluorescently labeled siRNA (siRNA-Cy3-lipoplex). By confocal microscopy, lipoplexed siRNA-Cy3 was detected inside vascular endothelial cells in vivo, which where identified with co-staining of endothelial markers. Consequently, the finding of intracellular siRNA uptake by vascular endothelial cells correlated with RNAi based specific protein reduction in situ as revealed by PECAM-1 specific immunofluorescence staining in lung tissue sections. Therefore, by using a cell biological approach these in situ data emphasize the functional uptake of liposomal siRNA molecules in vascular endothelial cells of different mouse tissues as indicated in our previous molecular study.

Hyperbaric oxygen treatment restores sudden hearing loss in a patient with Fabry disease.

Fabry disease is an X-linked inherited disorder of glycosphingolipid metabolism due to the deficient activity of a lysosomal enzyme, alpha-galactosidase A. The resultant systemic accumulation of sphingolipids can lead to progressive and sudden hearing loss alongside renal, cardiac and cerebrovascular complications. Although replacement therapy seems to be beneficial for cochlear function, few data are available regarding treatment of sudden hearing loss. This case report describes the course of a unilateral sudden hearing loss in a young (15-year-old) male patient and its improvement following hyperbaric oxygen treatment.

The influence of measles vaccination on the incidence of otosclerosis in Germany.

The pathologic process of otosclerosis is characterized by an inflammatory lytic phase followed by an abnormal bone remodeling at very specific sites of predilection. There is a clear genetic predisposition with about half of all cases occurring in families with more than one affected member. Females are affected more frequently than males with an approximate 2:1 ratio. N, H, and F measles proteins as well as measles virus RNA have been demonstrated in osteoblasts, chondroblasts, and macrophages of the inflammatory phase of the disease. These observations merely show an association between measles viruses and otosclerosis. In the present study, we tried to prove that there is a causal relationship: voluntary measles vaccination has been available in Germany since 1974. In the absence of official data, we reconstructed the rate of vaccination coverage between 1974 and 2004 using information from the Robert Koch Institute (RKI, Berlin) and from the literature. From the German Federal Office of Statistics, we received the data of 64,112 patients who had been hospitalized between 1993 and 2004 and in whom otosclerosis (ICD-9: 387; ICD-10: H80) had been confirmed. We calculated the effect of measles vaccination on the incidence of hospital treatments for otosclerosis in the period from 1993 to 2004 in Germany. For this purpose, we divided the female and male otosclerosis patients treated as inpatients each year in the observation period into two age groups: those up to 25 years, who had in most cases been vaccinated (designated below as "vaccinated patients") and those over 25 years who mostly could not have been vaccinated (designated below as "unvaccinated patients"). We calculated the incidence of otosclerosis requiring inpatient treatment for the two age groups in each year in the period of observation. For external validation of the study results, the same analysis was carried out in all patients who received inpatient treatment for otitis media in the same period. Between 1993 and 2004 the incidence of hospital treatments for otosclerosis decreased to a significantly greater extent in the vaccinated patients than in the unvaccinated patients. The decline is much greater in men than in women. A comparable effect cannot be demonstrated in patients with otitis media. The results indicate that measles vaccination in Germany has resulted in a significant reduction in the number of hospital treatments for otosclerosis in the vaccinated age groups. We conclude that there is a causal relationship between measles viruses and the development of otosclerosis.

The influence of the footplate-perilymph interface on postoperative bone conduction.

In a prospective study, 165 total stapedectomies and 152 small fenestra stapedotomies were performed by three experienced surgeons between 2001 and 2003. In total stapedectomy, a self-made Schuknecht steel wire connective tissue prosthesis, and in stapedotomy, a 0.6-mm platinum wire Teflon piston was used. The pre- and postoperative bone conduction thresholds were compared at the frequencies 250 Hz, 500 Hz, 1 kHz, 1.5 kHz, 2 kHz, 3 kHz and 4 kHz. The postoperative bone conduction between 250 Hz and 3 kHz was significantly better in the total stapedectomy group than in the stapedotomy group. At 4 kHz, both groups showed a slight decrease in bone conduction but the difference was not statistically significant. Therefore, especially in cases with preoperative moderate sensorineural hearing loss, we recommend total stapedectomy using a Schuknecht steel wire connective tissue prosthesis, which offers a stapes-perilymph interface similar to the normal stapes.

Protecting the cochlea during stapes surgery: is there a role for corticosteroids?

The aim of the present study was to evaluate possible protective effects of corticosteroids on the inner ear after surgical trauma and to exclude any ototoxicity. A corticosteroid (triamcinolone, Volon A) was topically applied to the inner ear of guinea pigs, either by extracochlear application with permeation and diffusion through the round window membrane or by intracochlear application with direct infusion into the inner ear via a cochleostomy. Threshold and input/output functions of compound action potentials (CAPs) were determined before and after application of the corticosteroid. We found that extracochlear application of the corticosteroid induced insignificant mild shifts of mean CAP thresholds, but significantly increased mean maximal amplitudes of input/output function after the 14th day following application of the steroid. No detrimental effects on cochlear function were noted in the extracochlear group, indicating absence of ototoxicity with the concentrations used. In the intracochlear group, CAP thresholds and amplitudes of input/ output function recovered from partial hearing loss due to cochleostomy between 7 and 14 days after application of the steroid, whereas in controls without steroid application, no such recovery of hearing was detected. These results suggest that topical application of triamcinolone has no ototoxic effect and that it leads to increased recovery of cochlear functions after trauma in the guinea pig inner ear.