scholarly journals Multiple Classes of Antiviral Agents ExhibitIn VitroActivity against Human Rhinovirus Type C

2013 ◽  
Vol 58 (3) ◽  
pp. 1546-1555 ◽  
Author(s):  
Chris Mello ◽  
Esmeralda Aguayo ◽  
Madeleine Rodriguez ◽  
Gary Lee ◽  
Robert Jordan ◽  
...  
Keyword(s):  
Kinase Inhibitor ◽  
Antiviral Agents ◽  
Airway Epithelial Cells ◽  
Human Rhinovirus ◽  
Dependent Manner ◽  
Airway Epithelial ◽  
Content Type ◽  
Type C ◽  
Rna Replicon

ABSTRACTHuman rhinovirus type C (HRV-C) is a newly discovered enterovirus species frequently associated with exacerbation of asthma and other acute respiratory conditions. Until recently, HRV-C could not be propagatedin vitro, hampering in-depth characterization of the virus replication cycle and preventing efficient testing of antiviral agents. Herein we describe several subgenomic RNA replicon systems and a cell culture infectious model for HRV-C that can be used for antiviral screening. The replicon constructs consist of genome sequences from HRVc15, HRVc11, HRVc24, and HRVc25 strains, with the P1 capsid region replaced by aRenillaluciferase coding sequence. Following transfection of the replicon RNA into HeLa cells, the constructs produced time-dependent increases in luciferase signal that can be inhibited in a dose-dependent manner by known inhibitors of HRV replication, including the 3C protease inhibitor rupintrivir, the nucleoside analog inhibitor MK-0608, and the phosphatidylinositol 4-kinase IIIβ (PI4K-IIIβ) kinase inhibitor PIK93. Furthermore, with the exception of pleconaril and pirodavir, the other tested classes of HRV inhibitors blocked the replication of full-length HRVc15 and HRVc11 in human airway epithelial cells (HAEs) that were differentiated in the air-liquid interface, exhibiting antiviral activities similar to those observed with HRV-16. In summary, this study is the first comprehensive profiling of multiple classes of antivirals against HRV-C, and the set of newly developed quantitative HRV-C antiviral assays represent indispensable tools for the identification and evaluation of novel panserotype HRV inhibitors.

scholarly journals Poly-L-Arginine Induces Apoptosis of NCI-H292 Cells via ERK1/2 Signaling Pathway

2018 ◽  
Vol 2018 ◽  
pp. 1-9 ◽  
Author(s):  
Ya-Ni Wang ◽  
Ling-Ling Zhang ◽  
Xiao-Yun Fan ◽  
Sha-Sha Wu ◽  
Sheng-Quan Zhang
Keyword(s):  
Epithelial Cells ◽  
Caspase 3 ◽  
Underlying Mechanism ◽  
Mitochondrial Pathway ◽  
Airway Epithelial Cells ◽  
Dependent Manner ◽  
Airway Epithelial ◽  
Cationic Protein ◽  
Concentration Dependent Manner

Cationic protein is a cytotoxic protein secreted by eosinophils and takes part in the damage of airway epithelium in asthma. Poly-L-arginine (PLA), a synthetic cationic protein, is widely used to mimic the biological function of the natural cationic protein in vitro. Previous studies demonstrated the damage of the airway epithelial cells by cationic protein, but the molecular mechanism is unclear. The purpose of this study aimed at exploring whether PLA could induce apoptosis of human airway epithelial cells (NCI-H292) and the underlying mechanism. Methods. The morphology of apoptotic cells was observed by transmission electron microscopy. The rate of apoptosis was analyzed by flow cytometry (FCM). The expressions of the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), Bcl-2/Bax, and cleaved caspase-3 were assessed by western blot. Results. PLA can induce apoptosis in NCI-H292 cells in a concentration-dependent manner. Moreover, the phosphorylation of the ERK1/2 and the unbalance of Bcl2/Bax, as well as the activation of caspase-3, were involved in the PLA-induced apoptosis. Conclusions. PLA can induce the apoptosis in NCI-H292 cells, and this process at least involved the ERK1/2 and mitochondrial pathway. The results could have some indications in revealing the apoptotic damage of the airway epithelial cells. Besides, inhibition of cationic protein-induced apoptotic death in airway epithelial cells could be considered as a potential target of anti-injury or antiremodeling in asthmatics.

scholarly journals MARCKS and HSP70 interactions regulate mucin secretion by human airway epithelial cells in vitro

2013 ◽  
Vol 304 (8) ◽  
pp. L511-L518 ◽  
Author(s):  
Shijing Fang ◽  
Anne L. Crews ◽  
Wei Chen ◽  
Joungjoa Park ◽  
Qi Yin ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Airway Epithelial Cells ◽  
Dependent Manner ◽  
Airway Epithelial ◽  
Mucin Secretion ◽  
Concentration Dependent Manner

Myristoylated alanine-rich C kinase substrate (MARCKS) protein has been recognized as a key regulatory molecule controlling mucin secretion by airway epithelial cells in vitro and in vivo. We recently showed that two intracellular chaperones, heat shock protein 70 (HSP70) and cysteine string protein (CSP), associate with MARCKS in the secretory mechanism. To elucidate more fully MARCKS-HSP70 interactions in this process, studies were performed in well-differentiated normal human bronchial epithelial (NHBE) cells maintained in air-liquid interface culture utilizing specific pharmacological inhibition of HSP70 with pyrimidinone MAL3-101 and siRNA approaches. The results indicate that HSP70 interaction with MARCKS is enhanced after exposure of the cells to the protein kinase C activator/mucin secretagogue, phorbol 12-myristate 13-acetate (PMA). Pretreatment of NHBEs with MAL3-101 attenuated in a concentration-dependent manner PMA-stimulated mucin secretion and interactions among HSP70, MARCKS, and CSP. In additional studies, trafficking of MARCKS in living NHBE cells was investigated after transfecting cells with fluorescently tagged DNA constructs: MARCKS-yellow fluorescent protein, and/or HSP70-cyan fluorescent protein. Cells were treated with PMA 48 h posttransfection, and trafficking of the constructs was examined by confocal microscopy. MARCKS translocated rapidly from plasma membrane to cytoplasm, whereas HSP70 was observed in the cytoplasm and appeared to associate with MARCKS after PMA exposure. Pretreatment of cells with either MAL3-101 or HSP70 siRNA inhibited translocation of MARCKS. These results provide evidence of a role for HSP70 in mediating mucin secretion via interactions with MARCKS and that these interactions are critical for the cytoplasmic translocation of MARCKS upon its phosphorylation.

scholarly journals High-throughput human primary cell-based airway model for evaluating influenza, coronavirus, or other respiratory viruses in vitro

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
A. L. Gard ◽  
R. J. Luu ◽  
C. R. Miller ◽  
R. Maloney ◽  
B. P. Cain ◽  
...  
Keyword(s):  
Influenza A ◽  
Rapid Assessment ◽  
Antiviral Agents ◽  
Antiviral Agent ◽  
Respiratory Viruses ◽  
Airway Epithelial Cells ◽  
Host Cells ◽  
Preclinical Model ◽  
Airway Epithelial

AbstractInfluenza and other respiratory viruses present a significant threat to public health, national security, and the world economy, and can lead to the emergence of global pandemics such as from COVID-19. A barrier to the development of effective therapeutics is the absence of a robust and predictive preclinical model, with most studies relying on a combination of in vitro screening with immortalized cell lines and low-throughput animal models. Here, we integrate human primary airway epithelial cells into a custom-engineered 96-device platform (PREDICT96-ALI) in which tissues are cultured in an array of microchannel-based culture chambers at an air–liquid interface, in a configuration compatible with high resolution in-situ imaging and real-time sensing. We apply this platform to influenza A virus and coronavirus infections, evaluating viral infection kinetics and antiviral agent dosing across multiple strains and donor populations of human primary cells. Human coronaviruses HCoV-NL63 and SARS-CoV-2 enter host cells via ACE2 and utilize the protease TMPRSS2 for spike protein priming, and we confirm their expression, demonstrate infection across a range of multiplicities of infection, and evaluate the efficacy of camostat mesylate, a known inhibitor of HCoV-NL63 infection. This new capability can be used to address a major gap in the rapid assessment of therapeutic efficacy of small molecules and antiviral agents against influenza and other respiratory viruses including coronaviruses.

scholarly journals Macrolides Inhibit Fusobacterium nucleatum-Induced MUC5AC Production in Human Airway Epithelial Cells

2013 ◽  
Vol 57 (4) ◽  
pp. 1844-1849 ◽  
Author(s):  
Kentaro Nagaoka ◽  
Katsunori Yanagihara ◽  
Yosuke Harada ◽  
Koichi Yamada ◽  
Yohei Migiyama ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Respiratory Tract ◽  
Fusobacterium Nucleatum ◽  
Airway Epithelial Cells ◽  
Enzyme Linked Immunosorbent Assay ◽  
Dependent Manner ◽  
Airway Epithelial ◽  
Content Type ◽  
Mucin Production ◽  
Tract Infections

ABSTRACTFusobacterium nucleatumis one of the most common anaerobic bacteria in periodontitis and is responsible for several extraoral infections, including respiratory tract diseases. In this study, we examined whetherF. nucleatuminduces mucin secretion in airway epithelial cells. We also examined the effects of macrolides onF. nucleatum-induced mucus production compared with the effects of other antibiotics that exert anti-anaerobic activities. The production of MUC5AC, the major core protein of mucin secreted from the airway surface epithelium, in bronchial epithelial cells after stimulation with culture supernatants (Sup) ofF. nucleatumwas analyzed by performing enzyme-linked immunosorbent assay and quantitative RT-PCR. The cell-signaling pathway ofF. nucleatumSup stimulation was also analyzed by Western blotting. For inhibition studies, cells were treated with azithromycin, clarithromycin, clindamycin (CLDM), and metronidazole (MTZ). TheF. nucleatumSup induced NCI-H292 cells to express MUC5AC at both the protein level and the mRNA level in both a time- and dose-dependent manner. Macrolides inhibitedF. nucleatumSup-induced MUC5AC production, while CLDM and MTZ were less effective.F. nucleatumSup induced the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), and this induction was suppressed by macrolides.F. nucleatumSup-induced MUC5AC production was blocked by the ERK pathway inhibitor U0126.F. nucleatumis likely to contribute to excessive mucin production, which suggests that periodontitis may correlate with the pathogenesis of chronic respiratory tract infection. Macrolides seem to reduce this mucin production and might represent an additional means of therapeutic intervention forF. nucleatumrespiratory tract infections other than CLDM and MTZ.

scholarly journals Differential Proinflammatory Responses to Aspergillus fumigatus by Airway Epithelial Cells In Vitro Are Protease Dependent

2021 ◽  
Vol 7 (6) ◽  
pp. 468
Author(s):  
Jessica Rowley ◽  
Sara Namvar ◽  
Sara Gago ◽  
Briony Labram ◽  
Paul Bowyer ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Aspergillus Fumigatus ◽  
Protease Inhibitors ◽  
Cell Interaction ◽  
Airway Epithelial Cells ◽  
Dependent Manner ◽  
Airway Epithelial ◽  
Proinflammatory Response ◽  
Fungal Cell

Aspergillus fumigatus is an important human respiratory mould pathogen. In addition to a barrier function, airway epithelium elicits a robust defence against inhaled A. fumigatus by initiating an immune response. The manner by which A. fumigatus initiates this response and the reasons for the immunological heterogeneity with different isolates are unclear. Both direct fungal cell wall–epithelial cell interaction and secretion of soluble proteases have been proposed as possible mechanisms. Our aim was to determine the contribution of fungal proteases to the induction of epithelial IL-6 and IL-8 in response to different A. fumigatus isolates. Airway epithelial cells were exposed to conidia from a low or high protease-producing strain of A. fumigatus, and IL-6 and IL-8 gene expression and protein production were quantified. The role of proteases in cytokine production was further determined using specific protease inhibitors. The proinflammatory cytokine response correlated with conidia germination and hyphal extension. IL-8 induction was significantly reduced in the presence of matrix metalloprotease or cysteine protease inhibitors. With a high protease-producing strain of A. fumigatus, IL-6 release was metalloprotease dependent. Dectin-1 antagonism also inhibited the production of both cytokines. In conclusion, A. fumigatus-secreted proteases mediate a proinflammatory response by airway epithelial cells in a strain-dependent manner.

scholarly journals The Nod1, Nod2, and Rip2 Axis Contributes to Host Immune Defense against Intracellular Acinetobacter baumannii Infection

2013 ◽  
Vol 82 (3) ◽  
pp. 1112-1122 ◽  
Author(s):  
Pradeep Bist ◽  
Neha Dikshit ◽  
Tse Hsien Koh ◽  
Alessandra Mortellaro ◽  
Thuan Tong Tan ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Acinetobacter Baumannii ◽  
Airway Epithelial Cells ◽  
Immune Defense ◽  
Innate Immune ◽  
Lung Epithelial Cells ◽  
Airway Epithelial ◽  
Content Type ◽  
Mitogen Activated Protein

ABSTRACTAcinetobacter baumanniiis a major extensively drug-resistant lethal human nosocomial bacterium. However, the host innate immune mechanisms controllingA. baumanniiare not well understood. Although viewed as an extracellular pathogen,A. baumanniican also invade and survive intracellularly. However, whether host innate immune pathways sensing intracellular bacteria contribute to immunity againstA. baumanniiis not known. Here, we provide evidence for the first time that intracellular antibacterial innate immune receptors Nod1 and Nod2, and their adaptor Rip2, play critical roles in the sensing and clearance ofA. baumanniiby human airway epithelial cellsin vitro.A. baumanniiinfection upregulated Rip2 expression. Silencing of Nod1, Nod2, and Rip2 expression profoundly increased intracellular invasion and prolonged the multiplication and survival ofA. baumanniiin lung epithelial cells. Notably, the Nod1/2-Rip2 axis did not contribute to the control ofA. baumanniiinfection of human macrophages, indicating that they play cell type-specific roles. The Nod1/2-Rip2 axis was needed forA. baumanniiinfection-induced activation of NF-κB but not mitogen-activated protein kinases. Moreover, the Nod1/2-Rip2 axis was critical to induce optimal cytokine and chemokine responses toA. baumanniiinfection. Mechanistic studies showed that the Nod1/2 pathway contributed to the innate control ofA. baumanniiinfection through the production of β-defensin 2 by airway epithelial cells. This study revealed new insights into the immune control ofA. baumanniiand may contribute to the development of effective immune therapeutics and vaccines againstA. baumannii.

scholarly journals In VitroAnalyses of the Effects of Heparin and Parabens on Candida albicans Biofilms and Planktonic Cells

2011 ◽  
Vol 56 (1) ◽  
pp. 148-153 ◽  
Author(s):  
Marisa H. Miceli ◽  
Stella M. Bernardo ◽  
T. S. Neil Ku ◽  
Carla Walraven ◽  
Samuel A. Lee
Keyword(s):  
Candida Albicans ◽  
Metabolic Activity ◽  
Biofilm Formation ◽  
High Dose ◽  
Dependent Manner ◽  
Planktonic Cells ◽  
Content Type ◽  
Heparin Sodium ◽  
The Individual

ABSTRACTInfections and thromboses are the most common complications associated with central venous catheters. Suggested strategies for prevention and management of these complications include the use of heparin-coated catheters, heparin locks, and antimicrobial lock therapy. However, the effects of heparin onCandida albicansbiofilms and planktonic cells have not been previously studied. Therefore, we sought to determine thein vitroeffect of a heparin sodium preparation (HP) on biofilms and planktonic cells ofC. albicans. Because HP contains two preservatives, methyl paraben (MP) and propyl paraben (PP), these compounds and heparin sodium without preservatives (Pure-H) were also tested individually. The metabolic activity of the mature biofilm after treatment was assessed using XTT [2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide] reduction and microscopy. Pure-H, MP, and PP caused up to 75, 85, and 60% reductions of metabolic activity of the mature preformedC. albicansbiofilms, respectively. Maximal efficacy against the mature biofilm was observed with HP (up to 90%) compared to the individual compounds (P< 0.0001). Pure-H, MP, and PP each inhibitedC. albicansbiofilm formation up to 90%. A complete inhibition of biofilm formation was observed with HP at 5,000 U/ml and higher. When tested against planktonic cells, each compound inhibited growth in a dose-dependent manner. These data indicated that HP, MP, PP, and Pure-H havein vitroantifungal activity againstC. albicansmature biofilms, formation of biofilms, and planktonic cells. Investigation of high-dose heparin-based strategies (e.g., heparin locks) in combination with traditional antifungal agents for the treatment and/or prevention ofC. albicansbiofilms is warranted.

scholarly journals Eosinophil Responses at the Airway Epithelial Barrier during the Early Phase of Influenza a Virus Infection in C57BL/6 Mice

Cells ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 509 ◽  
Author(s):  
Meenakshi Tiwary ◽  
Robert J. Rooney ◽  
Swantje Liedmann ◽  
Kim S. LeMessurier ◽  
Amali E. Samarasinghe
Keyword(s):  
Epithelial Cells ◽  
Influenza A Virus ◽  
Early Phase ◽  
Influenza A ◽  
Airway Epithelial Cells ◽  
Epithelial Barrier ◽  
Virus Infectivity ◽  
Airway Epithelial ◽  
In Vivo Models

Eosinophils, previously considered terminally differentiated effector cells, have multifaceted functions in tissues. We previously found that allergic mice with eosinophil-rich inflammation were protected from severe influenza and discovered specialized antiviral effector functions for eosinophils including promoting cellular immunity during influenza. In this study, we hypothesized that eosinophil responses during the early phase of influenza contribute to host protection. Using in vitro and in vivo models, we found that eosinophils were rapidly and dynamically regulated upon influenza A virus (IAV) exposure to gain migratory capabilities to traffic to lymphoid organs after pulmonary infection. Eosinophils were capable of neutralizing virus upon contact and combinations of eosinophil granule proteins reduced virus infectivity through hemagglutinin inactivation. Bi-directional crosstalk between IAV-exposed epithelial cells and eosinophils occurred after IAV infection and cross-regulation promoted barrier responses to improve antiviral defenses in airway epithelial cells. Direct interactions between eosinophils and airway epithelial cells after IAV infection prevented virus-induced cytopathology in airway epithelial cells in vitro, and eosinophil recipient IAV-infected mice also maintained normal airway epithelial cell morphology. Our data suggest that eosinophils are important in the early phase of IAV infection providing immediate protection to the epithelial barrier until adaptive immune responses are deployed during influenza.

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