scholarly journals Novel proteinase-activated receptor-2 (PAR2) antagonist C391 blocks Alternaria-induced human airway epithelial signaling and asthma indicators in murine models

2021 ◽  
Author(s):  
Candy Rivas ◽  
Michael Yee ◽  
Kenneth Addison ◽  
Marissa Lovett ◽  
Kasturi Pal ◽  
...  
Keyword(s):  
Cell Line ◽  
Mapk Signaling ◽  
Pharmacological Intervention ◽  
Airway Epithelial ◽  
Murine Models ◽  
Mucus Cell ◽  
Hek 293 ◽  
Human Airway

Background and Purpose: Despite availability of a variety of treatment options, many asthma patients have poorly controlled disease with frequent exacerbations. Proteinase-activated receptor-2 (PAR2) has been identified in pre-clinical animal models as important to asthma initiation and progression following allergen exposure. Proteinase activation of PAR2 induces intracellular Ca2+, mitogen activated protein kinase (MAPK) and -arrestin signaling the airway, leading to both inflammatory and protective effects. We have developed C391, a potent PAR2 antagonist effective in blocking peptidomimetic- and trypsin-induced PAR2 signaling in vitro as well as reducing inflammatory PAR2-associated pain in vivo. We hypothesized that PAR2 reduction with C391 would attenuate allergen-induced asthma indicators in murine models. Experimental Approach: We evaluated the ability for C391 to alter Alternaria alternata-induced PAR2 signaling pathways in vitro using a human airway epithelial cell line that naturally expresses PAR2 (16HBE14o-) and a transfected embryonic cell line (HEK 293). We next evaluated the ability for C391 to reduce A. alternata-induced asthma indicators in vivo in two murine strains. Key Results: C391 blocked A. alternata-induced, PAR2-dependent Ca2+ and MAPK signaling in 16HBE14o- cells, as well as -arrestin recruitment in HEK 293 cells. C391 effectively attenuated A. alternata-induced inflammation, mucus production, mucus cell hyperplasia and airway hyperresponsiveness in acute asthma murine models. Conclusions and Implications: To our knowledge, this is the first demonstration of pharmacological intervention of PAR2 to reduce allergen-induced asthma indicators in vivo. These data support further development of PAR2 antagonists as potential first-in-class allergic asthma drugs.

Ozone-induced release of cytokines and fibronectin by alveolar macrophages and airway epithelial cells

1994 ◽  
Vol 266 (6) ◽  
pp. L612-L619 ◽  
Author(s):  
R. B. Devlin ◽  
K. P. McKinnon ◽  
T. Noah ◽  
S. Becker ◽  
H. S. Koren
Keyword(s):  
Epithelial Cells ◽  
Cell Line ◽  
Alveolar Macrophages ◽  
Airway Epithelial Cells ◽  
Airway Hyperreactivity ◽  
Ozone Exposure ◽  
Airway Epithelial ◽  
Human Airway ◽  
Human Airway Epithelial Cells

Acute exposure of animals and humans to ozone results in decrements in lung function, development of airway hyperreactivity, inflammation, edema, damage to pulmonary cells, and production of several compounds with tissue damaging, fibrinogenic or fibrotic potential. The contribution of airway epithelial cells and alveolar macrophages to these processes is unclear. In this study we have directly exposed human alveolar macrophages and human airway epithelial cells to ozone in vitro and measured the cytotoxic effects of ozone, as well as the production of the inflammatory cytokines interleukin-6 (IL-6) and interleukin-8 (IL-8), and fibronectin, all of which are substantially elevated in the bronchoalveolar lavage fluid of humans exposed to ozone. Cells were grown on rigid, collagen-impregnated filter supports, and the interaction of cells with ozone facilitated by exposing them to the gas with medium below the support but no medium on top of the cells. The results show that, although macrophages are much more sensitive to ozone than epithelial cells, they do not produce increased amounts of IL-6, IL-8, or fibronectin following ozone exposure. In contrast, epithelial cells produce substantially more of all three proteins following ozone exposure, and both IL-6 and fibronectin are secreted vectorially. An immortalized human airway epithelial cell line (BEAS 2B) was used in these experiments since human airway epithelial cells are infrequently available for in vitro studies. Data from this study extend previous findings which suggest that the BEAS cell line is a useful model to study the interaction between airway epithelial cells and environmental toxicants.

scholarly journals Extracellular Vesicle-Mediated siRNA Delivery, Protein Delivery, and CFTR Complementation in Well-Differentiated Human Airway Epithelial Cells

Genes ◽  
2020 ◽  
Vol 11 (4) ◽  
pp. 351 ◽  
Author(s):  
Brajesh K. Singh ◽  
Ashley L. Cooney ◽  
Sateesh Krishnamurthy ◽  
Patrick L. Sinn
Keyword(s):  
Epithelial Cells ◽  
Airway Epithelial Cells ◽  
Target Cells ◽  
Airway Epithelial ◽  
Long Distance ◽  
Human Airway ◽  
Well Differentiated ◽  
Human Airway Epithelial Cells

Extracellular vesicles (EVs) are a class of naturally occurring secreted cellular bodies that are involved in long distance cell-to-cell communication. Proteins, lipids, mRNA, and miRNA can be packaged into these vesicles and released from the cell. This information is then delivered to target cells. Since EVs are naturally adapted molecular messengers, they have emerged as an innovative, inexpensive, and robust method to deliver therapeutic cargo in vitro and in vivo. Well-differentiated primary cultures of human airway epithelial cells (HAE) are refractory to standard transfection techniques. Indeed, common strategies used to overexpress or knockdown gene expression in immortalized cell lines simply have no detectable effect in HAE. Here we use EVs to efficiently deliver siRNA or protein to HAE. Furthermore, EVs can deliver CFTR protein to cystic fibrosis donor cells and functionally correct the Cl− channel defect in vitro. EV-mediated delivery of siRNA or proteins to HAE provides a powerful genetic tool in a model system that closely recapitulates the in vivo airways.

scholarly journals Forkhead Box Protein C2 (FOXC2) Promotes the Resistance of Human Ovarian Cancer Cells to Cisplatin In Vitro and In Vivo

2016 ◽  
Vol 39 (1) ◽  
pp. 242-252 ◽  
Author(s):  
Chanjuan Li ◽  
Hongjuan Ding ◽  
Jing Tian ◽  
Lili Wu ◽  
Yun Wang ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
Cancer Cells ◽  
Cell Line ◽  
Mapk Signaling ◽  
Ovarian Cancer Cells ◽  
Human Ovarian Cancer ◽  
Cancer Tissues ◽  
Mapk Signaling Pathways

Background/Aims: FOXC2 has been reported to play a role in tumor progression, but the correlations of FOXC2 with the cisplatin (CDDP) resistance of ovarian cancer cells are still unclear. The purpose of the present study is to investigate the roles of FOXC2 in the CDDP resistance of ovarian cancer cells and its possible mechanisms. Methods: Quantitative real-time PCR (qRT-PCR) was performed to detect the expression of FOXC2 mRNA in CDDP-resistant or sensitive ovarian cancer tissues and cell lines (SKOV3/CDDP and SKOV3). Gain- and loss-of-function assays were performed to analyze the effects of FOXC2 knockdown or overexpression on the in vitro and in vivo sensitivity of ovarian cancer cells to CDDP and its possible molecular mechanisms. Results: The relative expression level of FOXC2 mRNA in CDDP-resistant ovarian cancer tissues was higher than that in CDDP-sensitive tissues. Also, the expression of FOXC2 mRNA and protein in CDDP-resistant ovarian cancer cell line (SKOV3/CDDP) cell line was higher than that in its parental cell line (SOKV3). Small hairpin RNA (shRNA)-mediated FOXC2 knockdown significantly increased the in vitro and in vive sensitivity of SKOV3/CDDP cells to CDDP by enhancing apoptosis, while upregulation of FOXC2 significantly decreased the in vitro and in vivo sensitivity of SKOV3 cells to CDDP by reducing apoptosis. Furthermore, FOXC2 activates the Akt and MAPK signaling pathways, and then induced the decreased expression of Bcl-2 protein and the increased expression of Bax and cleaved caspase-3 proteins. Conclusions: FOXC2 mediates the CDDP resistance of ovarian cancer cells by activation of the Akt and MAPK signaling pathways, and may be a potential novel therapeutic target for overcoming CDDP resistance in human ovarian cancer.

scholarly journals The Luciferase-Expressing Glioma-261 Murine Models Elicit an Immune-Mediated Antitumor Response

Neurosurgery ◽  
2019 ◽  
Vol 66 (Supplement_1) ◽  
Author(s):  
Victoria E Sanchez ◽  
John Lynes ◽  
Stuart Walbridge ◽  
Xiang Wang ◽  
Nancy A Edwards ◽  
...  
Keyword(s):  
Inflammatory Response ◽  
Tumor Growth ◽  
Cell Line ◽  
Median Survival ◽  
Immune Cell ◽  
Clinical Symptoms ◽  
In Vivo Model ◽  
Murine Models

Abstract INTRODUCTION Preclinical models that accurately recapitulate the immunosuppressive properties of human gliomas are essential to assess immune therapeutics. Glioma-261 (GL261) murine glioma cells are widely used as an in Vivo model of glioma. Our group has previously shown that the red-shifted luciferase-expressing cell line, GL261 Red-FLuc, creates an inflammatory response when implanted intracranially in C57BL/6 mice. However, it remains unclear if this is particular to GL261 Red-Fluc or any GL261 cell line transfected with luciferase-expressing genes. For this reason, we have additionally explored the inflammatory response of stably transfected, monoclonal GL261-luc2 cells. METHODS To evaluate the characteristics of these various cell lines, C57BL/6 mice (n = 10 in each group) underwent stereotaxic, intracranial implantation with GL261, GL261 Red-FLuc, or GL261-Luc2 cells at doses of 5 × 104 cells/5 μL or 3 × 105 cells/5 μL. Immunohistochemistry and flow cytometry of sacrificed mouse brains assess the frequency of immune cell populations. Magnetic resonance imaging (MRI) scans were also performed to monitor relative tumor growth. Finally, in Vitro cytokine profiles were evaluated by proteome microarray. RESULTS Kaplan-Meier survival analysis demonstrated that the median survival for mice implanted with GL261 cells at 5 × 104 cells/5 μL was 21 d. However, even at a higher tumor dose (3 × 105 cells/5 μL), the GL261-Red FLuc implanted mice did not reach median survival. Mice injected with the newly transfected GL261-Luc2 cells at 3 × 105 cells/5 μL reached median survival at 23 d, but median survival was not reached for GL261-Luc2 implanted mice at 5 × 104 cells/5 μL. MRI analysis reveals clear differences in tumor growth that correspond well with the onset of clinical symptoms and median survival. In addition, proteomic analysis reveals significantly elevated inflammatory cytokines such as IFNgamma, IL-7, and TNF-alpha in the supernatant of the GL261 Red-FLuc cells and upregulated IL-1alpha in GL261-Luc2 cells. Further immune characterization is ongoing. CONCLUSION Our data suggest that both GL261 Red-FLuc and GL261-luc2 murine models create an undesirable microenvironment for tumor growth by increasing proinflammatory modulators.

scholarly journals Does the ΔF508-CFTR mutation induce a proinflammatory response in human airway epithelial cells?

2012 ◽  
Vol 303 (6) ◽  
pp. L509-L518 ◽  
Author(s):  
Thomas H. Hampton ◽  
Alicia E. Ballok ◽  
Jennifer M. Bomberger ◽  
Melanie R. Rutkowski ◽  
Roxanna Barnaby ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Inflammatory Response ◽  
Airway Epithelial Cells ◽  
Airway Epithelial ◽  
Chemokine Production ◽  
Human Airway ◽  
Δf508 Cftr ◽  
Human Airway Epithelial Cells

In the clinical setting, mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene enhance the inflammatory response in the lung to Pseudomonas aeruginosa ( P. aeruginosa ) infection. However, studies on human airway epithelial cells in vitro have produced conflicting results regarding the effect of mutations in CFTR on the inflammatory response to P. aeruginosa, and there are no comprehensive studies evaluating the effect of P. aeruginosa on the inflammatory response in airway epithelial cells with the ΔF508/ΔF508 genotype and their matched CF cell line rescued with wild-type (wt)-CFTR. CFBE41o- cells (ΔF508/ΔF508) and CFBE41o- cells complemented with wt-CFTR (CFBE-wt-CFTR) have been used extensively as an experimental model to study CF. Thus the goal of this study was to examine the effect of P. aeruginosa on gene expression and cytokine/chemokine production in this pair of cells. P. aeruginosa elicited a more robust increase in cytokine and chemokine expression (e.g., IL-8, CXCL1, CXCL2 and TNF-α) in CFBE-wt-CFTR cells compared with CFBE-ΔF508-CFTR cells. These results demonstrate that CFBE41o- cells complemented with wt-CFTR mount a more robust inflammatory response to P. aeruginosa than CFBE41o-ΔF508/ΔF508-CFTR cells. Taken together with other published studies, our data demonstrate that there is no compelling evidence to support the view that mutations in CFTR induce a hyperinflammatory response in human airway epithelial cells in vivo . Although the lungs of patients with CF have abundant levels of proinflammatory cytokines and chemokines, because the lung is populated by immune cells and epithelial cells there is no way to know, a priori, whether airway epithelial cells in the CF lung in vivo are hyperinflammatory in response to P. aeruginosa compared with non-CF lung epithelial cells. Thus studies on human airway epithelial cell lines and primary cells in vitro that propose to examine the effect of mutations in CFTR on the inflammatory response to P. aeruginosa have uncertain clinical significance with regard to CF.

In Vitro and In Vivo Inhibition of P-Glycoprotein (ABCB1) by a Novel Non-Substrate Compound: HG-829

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3986-3986
Author(s):  
Gisela Caceres ◽  
Robert W Robey ◽  
Lubomir Sokol ◽  
Kathy Rocha McGraw ◽  
William J Fulp ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Proliferation Assay ◽  
Hek 293 ◽  
P Glycoprotein ◽  
Wide Range ◽  
Xenograft Models ◽  
Substrate Inhibitor

Abstract Abstract 3986 Background: P-glycoprotein (P-gp or ABCB1) is a common cause of multidrug resistance (MDR) in cancer and leukemia that serves to extrude amphipathic drugs from the plasma membrane. P-gp is an ATP-binding cassette (ABC) transporter that exports a wide range of anti-neoplastics that are structurally or functionally unrelated. The vast majority of P-gp inhibitors tested clinically act as competitive export channel inhibitors to promote antineoplastic retention. We investigated the in vitro and in vivo effects of a novel substituted quinoline P-gp modulator, HG-829, in MDR cell lines and xenograft models. Methods: In vitro activity of HG-829 was evaluated in the K562-daunomycin-selected (K562-R) cell line and the ABCB1-transfected human embryonic kidney-293 cell line (HEK-293-B1) using a variety of P-gp substrates (daunomycin, doxorubicin, taxol, etoposide, vincristine) in a 72h MTT proliferation assay, and results compared to the effects of cyclosporine-A (CsA). Rhodamine 123 export and retention was assessed by flow cytometry. Flank injections of K562-R and parental K562-S cells in female SCID beige mice were performed for xenograft models. ANOVA and Turkey's multiple comparison tests were used to determine significant differences between groups in the proliferation assay and xenograft studies. Differences in rhodamine intracellular retention and efflux were assessed by Student's t-test. Results: Treatment with HG-829 at 0.5uM and 1uM completely reversed resistance to each of the antineoplastics studied in both the K562-R and HEK-293-B1 cell lines, but did not sensitize parental cells. HG-829 sensitized K562-R cells to daunomycin 57-fold and 97-fold at concentrations of 0.5uM and 1uM, respectively (p<0.01). Similarly, resistance to taxol (HG-829 0.5uM p<0.01 and 1uM, p<0.001), vincristine (p<0.01), and etoposide (p<0.05) were completely reversed. Comparable results were obtained with the HEK-293-B1 cell line in which HG-829 potentiated doxorubicin cytotoxicity 33- and 25-fold at concentrations 0.5uM (p<0.01) and 1uM (p<0.001), respectively. Compared to CsA, HG-829 was 3-fold more potent at equimolar concentrations. Functional studies showed that HG-829 (2.5uM) completely inhibited P-gp mediated rhodamine efflux in K562-R and the HEK transfected cells (p<0.05), but had no effect in parental cells. Pre-incubation followed by inhibitor removal showed that HG-829 inhibited rhodamine export as long as 24h (p<0.05), whereas equivalent concentration of CsA inhibited export for only 30 min. HG-829 showed no consistent effects on P-gp-ATPase activity in membrane preparations, whereas verapamil, a known P-gp substrate, displayed concentration-dependent stimulation. In mice bearing K562-R and parental xenografts, intraperitoneal administration of HG-829 significantly potentiated the antitumor activity of daunomyicin compared to vehicle without a significant increase in toxicity. Tumor volumes in HG-829 treated mice decreased to levels comparable to that of the K562-parental cohort (p<0.01 vs. vehicle). Conclusions: HG-829 is a potent non-substrate inhibitor of P-glycoprotein with a prolonged duration of action. Clinical testing of HG-829 is warranted. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Mechanisms of Zoonotic Severe Acute Respiratory Syndrome Coronavirus Host Range Expansion in Human Airway Epithelium

2007 ◽  
Vol 82 (5) ◽  
pp. 2274-2285 ◽  
Author(s):  
Timothy Sheahan ◽  
Barry Rockx ◽  
Eric Donaldson ◽  
Amy Sims ◽  
Raymond Pickles ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Infectious Clone ◽  
Human Monoclonal Antibody ◽  
Protein S ◽  
Vero Cells ◽  
Mouse Lung ◽  
Airway Epithelial ◽  
Human Airway

ABSTRACT In 2003, severe acute respiratory syndrome coronavirus (SARS-CoV) emerged and caused over 8,000 human cases of infection and more than 700 deaths worldwide. Zoonotic SARS-CoV likely evolved to infect humans by a series of transmission events between humans and animals for sale in China. Using synthetic biology, we engineered the spike protein (S) from a civet strain, SZ16, into our epidemic strain infectious clone, creating the chimeric virus icSZ16-S, which was infectious but yielded progeny viruses incapable of propagating in vitro. After introducing a K479N mutation within the S receptor binding domain (RBD) of SZ16, the recombinant virus (icSZ16-S K479N) replicated in Vero cells but was severely debilitated in growth. The in vitro evolution of icSZ16-S K479N on human airway epithelial (HAE) cells produced two viruses (icSZ16-S K479N D8 and D22) with enhanced growth on HAE cells and on delayed brain tumor cells expressing the SARS-CoV receptor, human angiotensin I converting enzyme 2 (hACE2). The icSZ16-S K479N D8 and D22 virus RBDs contained mutations in ACE2 contact residues, Y442F and L472F, that remodeled S interactions with hACE2. Further, these viruses were neutralized by a human monoclonal antibody (MAb), S230.15, but the parent icSZ16-S K479N strain was eight times more resistant than the mutants. These data suggest that the human adaptation of zoonotic SARS-CoV strains may select for some variants that are highly susceptible to select MAbs that bind to RBDs. The epidemic, icSZ16-S K479N, and icSZ16-S K479N D22 viruses replicate similarly in the BALB/c mouse lung, highlighting the potential use of these zoonotic spike SARS-CoVs to assess vaccine or serotherapy efficacy in vivo.

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