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      Three-dimensional Culture of Human Airway Epithelium in Matrigel for Evaluation of Human Rhinovirus C and Bocavirus Infections

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          Abstract

          Objective

          Newly identified human rhinovirus C (HRV-C) and human bocavirus (HBoV) cannot propagate in vitro in traditional cell culture models; thus obtaining knowledge about these viruses and developing related vaccines are difficult. Therefore, it is necessary to develop a novel platform for the propagation of these types of viruses.

          Methods

          A platform for culturing human airway epithelia in a three-dimensional (3D) pattern using Matrigel as scaffold was developed. The features of 3D culture were identified by immunochemical staining and transmission electron microscopy. Nucleic acid levels of HRV-C and HBoV in 3D cells at designated time points were quantitated by real-time polymerase chain reaction (PCR). Levels of cytokines, whose secretion was induced by the viruses, were measured by ELISA.

          Results

          Properties of bronchial-like tissues, such as the expression of biomarkers CK5, ZO-1, and PCK, and the development of cilium-like protuberances indicative of the human respiration tract, were observed in 3D-cultured human airway epithelial (HAE) cultures, but not in monolayer-cultured cells. Nucleic acid levels of HRV-C and HBoV and levels of virus-induced cytokines were also measured using the 3D culture system.

          Conclusion

          Our data provide a preliminary indication that the 3D culture model of primary epithelia using a Matrigel scaffold in vitro can be used to propagate HRV-C and HBoV.

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          Most cited references41

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          Cadherin-related family member 3, a childhood asthma susceptibility gene product, mediates rhinovirus C binding and replication.

          Members of rhinovirus C (RV-C) species are more likely to cause wheezing illnesses and asthma exacerbations compared with other rhinoviruses. The cellular receptor for these viruses was heretofore unknown. We report here that expression of human cadherin-related family member 3 (CDHR3) enables the cells normally unsusceptible to RV-C infection to support both virus binding and replication. A coding single nucleotide polymorphism (rs6967330, C529Y) was previously linked to greater cell-surface expression of CDHR3 protein, and an increased risk of wheezing illnesses and hospitalizations for childhood asthma. Compared with wild-type CDHR3, cells transfected with the CDHR3-Y529 variant had about 10-fold increases in RV-C binding and progeny yields. We developed a transduced HeLa cell line (HeLa-E8) stably expressing CDHR3-Y529 that supports RV-C propagation in vitro. Modeling of CDHR3 structure identified potential binding sites that could impact the virus surface in regions that are highly conserved among all RV-C types. Our findings identify that the asthma susceptibility gene product CDHR3 mediates RV-C entry into host cells, and suggest that rs6967330 mutation could be a risk factor for RV-C wheezing illnesses.
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            Type 1 and type 2 cytokine imbalance in acute respiratory syncytial virus bronchiolitis.

            We examined the in vivo immune response of infants to natural respiratory syncytial virus (RSV) infection through analysis of cytokine levels in nasal lavage fluid and stimulated peripheral blood mononuclear cells. Eighty-eight babies with at least one parent with atopy and asthma were prospectively studied through their first winter. Twenty-eight infants had an upper respiratory tract infection where RSV was detected, of whom nine developed signs of acute bronchiolitis. Nasal lavage specimens were assayed for interferon-gamma, interleukin (IL)-4, IL-10, and IL-12 and the RSV load determined by quantitative polymerase chain reaction. Messenger RNA (mRNA) was extracted from stimulated peripheral blood mononuclear cells and interferon-gamma, IL-4, IL-12, and IL-18 mRNA levels determined by polymerase chain reaction. Cytokine profiles were analyzed in relation to clinical outcome. The IL-4/interferon-gamma ratio for infants with acute bronchiolitis was elevated in nasal lavage fluid on both Days 1-2 (p = 0.014) and Days 5-7 (p = 0.001) of the illness compared with infants with upper respiratory tract infection alone. Those with acute bronchiolitis demonstrated a higher IL-10/IL-12 ratio (p = 0.0015) on Days 1-2. IL-18 mRNA levels were reduced (p = 0.019) and the IL-4/interferon-gamma ratio elevated (p = 0.01) in stimulated peripheral blood mononuclear cells from infants with acute bronchiolitis. There was no difference in initial RSV load. These data strongly implicate excess type 2 and/or deficient type 1 immune responses in the pathogenesis of RSV bronchiolitis.
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              Lower airways inflammation during rhinovirus colds in normal and in asthmatic subjects.

              Human rhinoviruses (HRV) cause the majority of common colds and are etiologically linked with changes in lower airways physiology and asthma exacerbations. We hypothesized that changes in bronchial mucosal inflammatory cell populations may be responsible for HRV-induced changes in airway reactivity. We examined bronchial mucosal biopsies during experimental infections with HRV serotype 16 and measured changes in histamine reactivity. Seventeen adult volunteers (six atopic asthmatics) had baseline measurements of histamine reactivity and fiberoptic bronchoscopic biopsies, followed 2 wk later by viral inoculation. Further bronchial biopsies were taken on Day 4 of the infection and 6 to 10 wk later. Mast cells, eosinophils, lymphocytes, and neutrophils were quantified by immunohistochemical techniques. Infection was documented by viral culture, seroconversion, and symptoms. An increase in histamine responsiveness during the cold (p = 0.048) was accompanied by increases in submucosal lymphocytes (p = 0.050). There was a subsequent decrease in submucosal and epithelial lymphocytes in convalescence (p = 0.028; p = 0.030). There was an increase in epithelial eosinophils with the cold (p = 0.042), and in asthmatics this appeared to persist into convalescence. A peripheral blood lymphopenia correlated with increased responsiveness (r = 0.062, p = 0.014). Rhinoviral colds are associated with a bronchial mucosal lymphocytic and eosinophilic infiltrate that may be related to changes in airway responsiveness and asthma exacerbations.
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                Author and article information

                Contributors
                Journal
                Biomed Environ Sci
                Biomed. Environ. Sci
                Biomedical and Environmental Sciences
                The Editorial Board of Biomedical and Environmental Sciences. Published by Elsevier (Singapore) Pte Ltd.
                0895-3988
                2214-0190
                15 March 2018
                February 2018
                15 March 2018
                : 31
                : 2
                : 136-145
                Affiliations
                [a ]Key Laboratory of Heavy Ion Radiation Biology and Medicine of Chinese Academy of Sciences & Key Laboratory of Space Radiobiology of Gansu Province, Institute of Modern Physics, Chinese Academy of Sciences, Lanzhou 730000, Gansu, China
                [b ]Department of Pathogenic Biology, Chengde Medical University, Chengde 067000, Hebei, China
                [c ]Department for Viral Diarrhea, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China
                Author notes
                [# ]Correspondence should be addressed to HU Bu Rong hubr@ 123456impcas.ac.cn
                [^]

                These authors contributed equally to this work.

                Article
                S0895-3988(18)30031-X
                10.3967/bes2018.016
                7134816
                29606192
                dca99683-66a8-41bc-abe5-7dfa7d34548b
                © 2018 The Editorial Board of Biomedical and Environmental Sciences

                Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active.

                History
                : 17 August 2017
                : 1 December 2017
                Categories
                Article

                3d cell culture,human airway epithelium (hae),human rhinovirus c,human bocavirus,propagation

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