Properties of Adenovirus Vectors with Increased Affinity to DSG2 and the Potential Benefits of Oncolytic Approaches and Gene Therapy.

Carcinomas are characterized by a widespread upregulation of intercellular junctions that create a barrier to immune response and drug therapy. Desmoglein 2 (DSG2) represents such a junction protein and serves as one adenovirus receptor. Importantly, the interaction between human adenovirus type 3 (Ad3) and DSG2 leads to the shedding of the binding domain followed by a decrease in the junction protein expression and transient tight junction opening. Junction opener 4 (JO-4), a small recombinant protein derived from the Ad3 fiber knob, was previously developed with a higher affinity to DSG2. JO-4 protein has been proven to enhance the effects of antibody therapy and chemotherapy and is now considered for clinical trials. However, the effect of the JO4 mutation in the context of a virus remains insufficiently studied. Therefore, we introduced the JO4 mutation to various adenoviral vectors to explore their infection properties. In the current experimental settings and investigated cell lines, the JO4-containing vectors showed no enhanced transduction compared with their parental vectors in DSG2-high cell lines. Moreover, in DSG2-low cell lines, the JO4 vectors presented a rather weakened effect. Interestingly, DSG2-negative cell line MIA PaCa-2 even showed resistance to JO4 vector infection, possibly due to the negative effect of JO4 mutation on the usage of another Ad3 receptor: CD46. Together, our observations suggest that the JO4 vectors may have an advantage to prevent CD46-mediated sequestration, thereby achieving DSG2-specific transduction.

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.

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.