Journal: Biomaterials advances

Article Title: Fabrication of heparinized small diameter TPU/PCL bi-layered artificial blood vessels and in vivo assessment in a rabbit carotid artery replacement model.

doi: 10.1016/j.msec.2021.112628

Figure Lengend Snippet: Fig. 1. Effects of AP on mitochondrial ROS production. (A) Protease (50 μg) activity assessed using a commercial protease activity kit. Trypsin (5 U) was used positive control. (B) Measurement of endotoxin level of AP and inactive AP. Escherichia coli endotoxin was used as positive control. (C) AP and inactive AP-induced mitochondrial ROS production evaluated in primary epithelial bronchial cells using confocal microscopy. (D) AP-induced mitochondrial ROS production sequentially evaluated using confocal microscopy. (E) Decrease in mitochondrial ROS production upon Mito-TEMPO treatment for 1 h evaluated in primary epithelial bronchial cells using confocal microscopy (magnification: 150 × , scale bar: 20 μm). (F) The effect of Mito-TEMPO on protease activity of AP was measured. Data from two independent experiments are shown as the mean ± standard error mean (SEM). *P < 0.05, **P < 0.01 vs. Inactive AP stimulation or endotoxin group.

Article Snippet: Human primary bronchial epithelial cells were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Activity Assay, Positive Control, Confocal Microscopy

Journal: Biomaterials advances

Article Title: Fabrication of heparinized small diameter TPU/PCL bi-layered artificial blood vessels and in vivo assessment in a rabbit carotid artery replacement model.

doi: 10.1016/j.msec.2021.112628

Figure Lengend Snippet: Fig. 2. Effects of AP on gene expression in primary bronchial epithelial cells. (A) A total of 4115 genes showing an expression change of at least 0.1 standard deviation following AP treatment were selected and clustered according to the expression level. Red and green color represent high and low expression levels, respectively, as shown in the scale bar. (B) Pathways were clustered according to the activity level calculated from the expression level of the genes included in each pathway. Red and green color represent high and low activity levels, respectively, as shown in the scale bar. The functional categories of each pathway are color- coded as shown on the side and below. In the right panel, the average activity levels of the pathways included in each functional category were compared between the two samples (1 h and 24 h after AP treatment). (C) Similarity between samples was measured according to the distribution of pathway activity. Correlation coef ficient values are displayed in color as indicated on the scale bar. (D) Distribution of the number of core differential genes isolated from each sample is shown as a Venn diagram. The ratio of common genes (dot plot) between two samples according to the number (bar plot) of core differential genes is also displayed in the lower plot. (E) GO terms enriched (FDR<0.01) in the core differential genes isolated from AP-treated samples are represented as network graph (upper panel) and tree map plot (lower panel). Each node represents a GO term, and the node size is proportional to the number of genes associated with the GO term. Closely related GO terms are indicated in the same color. Representative GO terms are indicated. (F) Relationship between AP-treated samples were measured using semantic similarity of enriched GO terms (upper panel) from the core differential genes and the distance distribution (middle panel) between core differential genes. In the upper panel, the intensity of red is proportional to the similarity between samples, as shown in the scale bar. In the middle panel, red and green color represent high and low similarity between samples in distance distribution, respectively, as shown in a time-dependent manner in the scale bar. The lower panel schematically shows the distances between samples on the network. Each sample is represented by a node. The size of the node indicates the number of core differential genes. The thicker the edge, the closer the distance between samples. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Human primary bronchial epithelial cells were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Gene Expression, Expressing, Standard Deviation, Activity Assay, Functional Assay, Isolation

Journal: Biomaterials advances

Article Title: Fabrication of heparinized small diameter TPU/PCL bi-layered artificial blood vessels and in vivo assessment in a rabbit carotid artery replacement model.

doi: 10.1016/j.msec.2021.112628

Figure Lengend Snippet: Fig. 4. Effects of AP-induced mitochondrial ROS production on epithelial cell apoptosis and epithelial permeability. (A) The change in the expression level of genes included in the apoptosis pathway according to the AP treatment is displayed as a box plot. Activity level of apoptosis pathway according to the AP treatment is also shown. (B) The distances between core differential genes induced by AP treatment and apoptosis-related genes or mitochondrial electron transport-related genes were measured in the network. (C) P53, BCL2, BAX, and active caspase-3 expression after 23 h of incubation following AP stimulation for 1 h was analyzed using western blotting (representative image). β-Actin was used as a loading control. (D) JC-1 staining for mitochondrial membrane potential (ΔΨm) on primary human bronchial epithelial cells under confocal microscopy (magnification: 60 × , scale bar: 20 μm). (E) Changes in the activity of pathways related to the cell adhesion function according to AP treatment time were measured. Data are presented as mean ± SEM. The pathways studied were the adherens junction, tight junction, gap junction, and actin cytoskeletal pathways. (F) After core differential genes in the GO terms and pathways related to the cell adhesion function were selected, the average expression level of the neighboring genes of each core differential gene was measured, and their distribution was observed as a box plot. *P < 0.05, **P < 0.01 vs. 1 h after AP stimulation group. (G) ZO-1, claudin 1, occludin, and E-cadherin expression after 23 h of incubation following AP stimulation for 1 h. (H) Monolayer permeability of primary bronchial epithelial cells is expressed as a percentage of change in FITC-dextran (Mw = 20,000 Da) fluorescence intensity following 23 h of incubation following AP stimulation for 1 h. Data from three experiments are shown as mean ± SEM. *P < 0.05, **P < 0.01 vs. control group (no AP stimulation or Mito-TEMPO pretreatment); #P < 0.05, ##P < 0.01 vs. AP stimulation group without Mito-TEMPO.

Article Snippet: Human primary bronchial epithelial cells were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Permeability, Expressing, Activity Assay, Incubation, Western Blot, Control, Staining, Membrane, Confocal Microscopy, Fluorescence

Journal: Biomaterials advances

Article Title: Fabrication of heparinized small diameter TPU/PCL bi-layered artificial blood vessels and in vivo assessment in a rabbit carotid artery replacement model.

doi: 10.1016/j.msec.2021.112628

Figure Lengend Snippet: Fig. 5. Effects of Mito-TEMPO on AP-induced changes in gene expression in primary bronchial epithelial cells. (A) In the left panel, 3742 genes showing an expression change of at least 0.1 standard deviation after AP and Mito-TEMPO treatment were selected and clustered according to the expression level. Red and green color represent high and low expression levels, respectively, as shown in the scale bar. In the right panel, the gene expression change caused by Mito-TEMPO was compared in parallel with that in the AP alone treatment group. (B) In the left panel, distribution of the number of core differential genes isolated from each sample is shown as a Venn diagram. Enriched GO terms (FDR <0.01) in core differential genes isolated from AP and Mito-TEMPO-treated samples are represented as a network graph (right panel) and dot plot (lower panel). (C) In the left panel, core differential genes selected from both AP and Mito-TEMPO treated samples were compared with those selected from AP alone-treated samples. The number of common genes between AP and Mito-TEMPO-treated samples and AP alone-treated samples is indicated in red. In the right panel, the functional association of these common genes is indicated by dots. (D) Locations of the selected core differential genes in AP alone-treated samples and in AP and Mito-TEMPO-treated samples (1 h) are displayed in the interaction network. For each sample, the size of the major node cluster composed of core differential genes and the average distance between nodes within the cluster are shown in the panel below. (E) Pathways were clustered according to the activity level calculated from the expression level of the genes included in each pathway. The functional categories of each pathway are color-coded as shown on the side and below. In the right panel, the pathway activity change caused by Mito-TEMPO was compared in parallel with that in the AP alone treatment group. The lower panel shows a correlation plot between the AP-only samples and the AP and Mito-TEMPO-treated samples. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Human primary bronchial epithelial cells were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Gene Expression, Expressing, Standard Deviation, Isolation, Functional Assay, Activity Assay

Journal: Biomaterials advances

Article Title: Fabrication of heparinized small diameter TPU/PCL bi-layered artificial blood vessels and in vivo assessment in a rabbit carotid artery replacement model.

doi: 10.1016/j.msec.2021.112628

Figure Lengend Snippet: Fig. 6. Comparative analysis with external datasets of hypoxia-treated bronchial epithelial cells. (A) Core differential genes were selected from two external datasets (GSE68378 and GSE121773) of bronchial epithelial cells exposed to hypoxia or treated with reactive oxygen species, and their distributions were plotted as Venn diagrams. (B) In the left and middle panels, enriched pathways and GO terms (FDR <0.001) associated with the core differential genes, respectively, were compared between samples. As shown in the scale bar, the significance increased as the black color increased. In the right panel, relationships between samples were measured using semantic similarity of the enriched GO terms from the core differential genes. Red color intensity is proportional to the similarity between samples, as shown in the scale bar. (C) The pattern of distance distribution in the network between core differential genes was used to measure similarity between samples. Red and green color represent high and low similarity between samples in distance distribution, respectively, as shown in the scale bar. (D) In the left panel, pathways were clustered according to the activity level calculated from the expression level of the genes included in each pathway. The functional categories of each pathway are color-coded on the side. In the middle panel, similarity between samples was measured according to the distribution of pathway activity. Correlation coefficient values are displayed in color as indicated on the scale bar. The right panel shows correlation plots between AP-treated samples (1 and 24 h) and selected external samples. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Human primary bronchial epithelial cells were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Activity Assay, Expressing, Functional Assay

Journal: Biomaterials advances

Article Title: Fabrication of heparinized small diameter TPU/PCL bi-layered artificial blood vessels and in vivo assessment in a rabbit carotid artery replacement model.

doi: 10.1016/j.msec.2021.112628

Figure Lengend Snippet: Fig. 7. Analysis of connectivity map data. (A) A total of 5170 genes showing an expression change of at least 0.1 standard deviation following treatment with five uncoupling agents (CCCP, FCCP, bongkrek acid, isorotenone, and elesclomol) were selected and clustered according to the expression level. Red and green color represent high and low expression levels, respectively, as shown in the scale bar. (B) The ratio of common core differential genes, which was adjusted for the total number of core differential genes, was compared between the connectivity map samples and the AP-treated samples. Black color intensity is proportional to the adjusted ratio of common genes between samples, as shown in the scale bar. In the right panel, the distribution of adjusted common core differential gene numbers is displayed for each individual connectivity map sample. (C) Correlation between pathway activity of individual samples in the connectivity map and that of AP- treated samples was measured. (D) Samples treated only with CCCP or FCCP were clustered according to the pathway activity level. The functional categories of each pathway are color-coded as shown on the side and below. The right panel shows a correlation plot between AP-treated samples (1 and 18 h) and CCCP- or FCCP- treated samples. (E) FCCP-induced mitochondrial ROS production was sequentially evaluated in primary epithelial bronchial cells by confocal microscopy (magnification: 150 × , scale bar: 20 μm). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Human primary bronchial epithelial cells were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Expressing, Standard Deviation, Activity Assay, Functional Assay, Confocal Microscopy

Journal: Mucosal immunology

Article Title: Neurotrophic tyrosine kinase receptor 1 is a direct transcriptional and epigenetic target of IL-13 involved in allergic inflammation

doi: 10.1038/mi.2014.109

Figure Lengend Snippet: In A–D , shown is quantitative RT-PCR analysis of NTRK1 and CCL26 transcription in TE-7 cells, EPC2 epithelial cell ALI culture, primary esophageal epithelial cells, and human bronchial epithelial cells. Cells were treated with IL-13 at 100 ng/ml for the indicated periods of time or for 6 days for EPC2 cells. The inset in C shows Western blot for NTRK1 in two independent cultures of primary esophageal epithelial cells stimulated with IL-13. In E , the effect of STAT6 gene silencing by shRNA on NTRK1 and CCL26 induction in TE-7 cells was quantified by RT-PCR. TE-7 cells were stimulated with IL-13 (1 ng/ml) for the indicated periods of time. shCtrl, control shRNA; shSTAT6, shRNA against STAT6 . Data for 3 to 4 independent experiments are presented as mean values for gene expression normalized to the level of GAPDH with standard error measurements.

Article Snippet: Normal human primary bronchial epithelial cells were purchased from Lonza (Lonza, MD, CC-2540) and cultured as previously described .

Techniques: Quantitative RT-PCR, Western Blot, shRNA, Reverse Transcription Polymerase Chain Reaction, Control, Gene Expression

Journal: Mucosal immunology

Article Title: Neurotrophic tyrosine kinase receptor 1 is a direct transcriptional and epigenetic target of IL-13 involved in allergic inflammation

doi: 10.1038/mi.2014.109

Figure Lengend Snippet: In A , shown is a Western blot analysis of primary esophageal epithelial cells pre-treated with IL-13 for 24 hr and then treated with recombinant human NGF for 0, 5, or 15 min. pNTRK1 indicates phosphorylated protein (arrow). In B , shown is Western blot analysis of TE-7 cells pre-treated with IL-13 for 24 hr and then treated with NGF for 5 min. pNTRK1 and pERK1/2 indicate phosphorylated proteins, arrow points at pNTRK1. In C , the kinetics of NTRK1 phosphorylation were assessed by Western blot. Cells were pre-treated with IL-13 for 24 hr and then treated with NGF for 5, 15 or 30 min. For A–C , phosphorylation was assessed at tyrosine residues Tyr674/675 in the catalytic domain of NTRK1. In D , kinetic analysis of EGR1 and EGR3 mRNA in TE-7 cells pre-treated with IL-13 for 24 hr followed by treatment with NGF for 1, 2, or 6 hrs was performed by RT-PCR. Fold change indicates increase over untreated (no IL-13) cells stimulated with NGF (+NGF). NGF was used at the concentration of 100 ng/ml. Data for 3 independent experiments are presented as mean value with standard error measurements; ****p < 0.0001, *p < 0.05. In E , EGR1 and EGR3 protein levels in TE-7 cells pre-treated with IL-13 for 24 hr followed by treatment with NGF were analyzed by Western blot; p38 serves as a loading control.

Article Snippet: Normal human primary bronchial epithelial cells were purchased from Lonza (Lonza, MD, CC-2540) and cultured as previously described .

Techniques: Western Blot, Recombinant, Phospho-proteomics, Reverse Transcription Polymerase Chain Reaction, Concentration Assay, Control

Journal: American Journal of Respiratory and Critical Care Medicine

Article Title: Ezrin, a Membrane Cytoskeleton Cross-Linker Protein, as a Marker of Epithelial Damage in Asthma

doi: 10.1164/rccm.201802-0373OC

Figure Lengend Snippet: Reduced serum ezrin levels of patients with asthma correlate positively with lung function and negatively with serum IL-13 and periostin levels. (A) The expression of ezrin in serum was reduced in patients with asthma (n = 59) compared with healthy control subjects (n = 18). Serum levels of ezrin were decreased according to the degree of asthma control: well-controlled group (n = 19); partly controlled group (n = 13); and uncontrolled group (n = 23). (B) The correlation between ezrin levels in exhaled breath condensate (EBC) and in serum (n = 34). (C) Ezrin mRNA expression in blood cells in severe asthma compared with healthy control subjects, by cohort (adjusted P = 0.0022). Graphs are expressed as log2 intensity robust multiarray average signals. The differences between healthy control subjects and patients with severe asthma were analyzed using the Benjamini-Hochberg method for adjusted P value/false discovery rate. (D) The correlation between EZR expression and T2 signature gene expression in IL-13–stimulated epithelial cells from patients with asthma and healthy subjects (n = 147). Ezrin gene expression is presented as log2 intensity robust multiarray average signals and the expression of the IL-13 signature genes as an enrichment score. (E) The relationship between serum ezrin and FEV1, FEV1% predicted, and FEV1/FVC (n = 44–47). (F) The concentrations of serum IgE (asthma group, n = 18; control group, n = 14), IL-13 (asthma group, n = 29; control group, n = 13), and periostin (asthma group, n = 56; control group, n = 23) were measured using ELISA (upper panels), and their relationships with ezrin were also analyzed (lower panels). Data were quantified and expressed as mean ± SD. **P < 0.01 and ***P < 0.001 compared with respective control subjects. The data were analyzed using Wilcoxon rank-sum test (A), IgE and periostin (F), Student’s t test in IL-13 (F), and Pearson’s correlation test (B and D–F). EZR = ezrin; FEV1%pred = FEV1% predicted; HC = healthy nonsmoking control subjects; MMA = mild/moderate nonsmoking asthma; ns = not significant; SAn = severe nonsmoking asthma; SAs = smokers with severe asthma.

Article Snippet: Human primary bronchial epithelial cells (PBECs; ScienCell Research Laboratories) and 16HBE cells were cultured as described previously ( 27 , 28 ).

Techniques: Expressing, Control, Gene Expression, Enzyme-linked Immunosorbent Assay

Journal: American Journal of Respiratory and Critical Care Medicine

Article Title: Ezrin, a Membrane Cytoskeleton Cross-Linker Protein, as a Marker of Epithelial Damage in Asthma

doi: 10.1164/rccm.201802-0373OC

Figure Lengend Snippet: Ezrin is expressed on exosomes secreted by bronchial epithelial cells. (A) Electron microscopic observation of whole-mounted exosomes purified from 16HBE cells. White arrows indicate exosomes. Scale bars = 1 μm (left panel) and 200 nm (right panel). (B) Intensity and size distribution of exosomes derived from 16HBE cells were measured by nanoparticle tracking analysis. Graph showing the average percentage of particles within 50- to 350-nm size in exosome. (C) Western blot analysis showed the presence of ezrin as well as exosome markers, CD9 and CD63, in exosomes isolated from 16HBE cells (Ctrl) and the IL-13 (30 ng/ml)–treated group (IL-13).

Article Snippet: Human primary bronchial epithelial cells (PBECs; ScienCell Research Laboratories) and 16HBE cells were cultured as described previously ( 27 , 28 ).

Techniques: Purification, Derivative Assay, Western Blot, Isolation

Journal: American Journal of Respiratory and Critical Care Medicine

Article Title: Ezrin, a Membrane Cytoskeleton Cross-Linker Protein, as a Marker of Epithelial Damage in Asthma

doi: 10.1164/rccm.201802-0373OC

Figure Lengend Snippet: IL-13 downregulates ezrin expression in bronchial epithelial cells via the JAK2 (Janus tyrosine kinase 2)/STAT6 (signal transducer and activator of transcription) pathway. (A) 16HBE cells were treated with IL-4 (20 ng/ml), IL-13 (30 ng/ml), and TNF-α (10 ng/ml) for 2, 6, and 12 hours, and ezrin mRNA expression was measured by quantitative RT-PCR. (B) 16HBE cells were exposed to IL-4 (20 ng/ml), IL-13 (30 ng/ml), and TNF-α (10 ng/ml) for 24, 48, and 72 hours, and ezrin protein production was determined by Western blotting. (C) Primary bronchial epithelial cells (PBECs) were stimulated with IL-13 (30 ng/ml) for 24, 48, and 72 hours. Ezrin protein was evaluated by Western blotting. PBECs were pretreated with the JAK2 inhibitor TG101348 (30 nM) for 1 hour before IL-13 (30 ng/ml) stimulation (1 h). The total phospho (p)-JAK2 protein level and p-STAT6 protein level in the nucleus were measured by Western blotting. The effect of TG101348 pretreatment on IL-13–regulated ezrin expression was evaluated by Western blotting. Data are presented as mean ± SEM of three independent experiments using one-way ANOVA followed by Student-Newman-Keuls post hoc analysis. ns = not significant. *P < 0.05, **P < 0.01 and ***P < 0.001, compared with control (Ctrl). TNF-α = tumor necrosis factor-α.

Article Snippet: Human primary bronchial epithelial cells (PBECs; ScienCell Research Laboratories) and 16HBE cells were cultured as described previously ( 27 , 28 ).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Control

Journal: American Journal of Respiratory and Critical Care Medicine

Article Title: Ezrin, a Membrane Cytoskeleton Cross-Linker Protein, as a Marker of Epithelial Damage in Asthma

doi: 10.1164/rccm.201802-0373OC

Figure Lengend Snippet: Ezrin expression and epithelial cell–cell adhesion were decreased in an ovalbumin (OVA)-treated allergic mouse model of asthma and restored by anti–IL-13 treatment. (A) Hematoxylin and eosin (H&E) staining of lung tissue in “asthma mice” (black arrows indicate bronchial epithelial cells). Representative image of E-cadherin and ZO-1 immunostaining (black arrows in the middle and bottom panels indicate their expression on the bronchial epithelial cells) was examined in saline-exposed control mice (control), OVA-treated mice (OVA), OVA + anti-IgG antibody–treated mice (anti-IgG), and OVA + anti–IL-13 antibody–treated mice (anti–IL-13), and was analyzed by Image-Pro Plus 6.0. Scale bars, 50 μm. (B) Epithelial cell–cell adherence was determined by electron microscopy (scale bars, 1 μm; white arrow). (C) Immunohistochemical analysis of ezrin expression in saline-exposed control mice (control), OVA-treated mice (OVA), OVA + anti-IgG antibody–treated mice (anti-IgG), and OVA + anti–IL-13 antibody–treated mice (anti–IL-13) (original magnification, ×400; scale bar = 100 μm; black arrow) and scored (right graph). (D) The concentrations of ezrin in BAL fluid (BALF) of OVA-treated mice (OVA), OVA + anti-IgG antibody–treated mice (anti-IgG), and OVA + anti–IL-13 antibody–treated mice (anti–IL-13), and IL-13 of asthma mice and controls were measured using an ELISA. The data are presented as mean ± SEM and were analyzed by Student’s t test (control group, n = 8–15; asthma group; n = 8–17). The correlation between ezrin and IL-13 in BALF of mice was analyzed by Pearson’s correlation test. ns = not significant. *P < 0.05, **P < 0.01, and ***P < 0.001 compared with respective controls.

Article Snippet: Human primary bronchial epithelial cells (PBECs; ScienCell Research Laboratories) and 16HBE cells were cultured as described previously ( 27 , 28 ).

Techniques: Expressing, Staining, Immunostaining, Saline, Control, Electron Microscopy, Immunohistochemical staining, Enzyme-linked Immunosorbent Assay

Journal: Clinical & Translational Immunology

Article Title: Naturally derived cytokine peptides limit virus replication and severe disease during influenza A virus infection

doi: 10.1002/cti2.1443

Figure Lengend Snippet: LAT8881 treatment of severe IAV infection reduces pulmonary immunopathology. Groups of male C57BL/6 mice received daily i.n. treatment with 20 mg kg −1 of LAT8881 or PBS alone from 1 dpi with 10 4 pfu of HKx31 IAV. BAL fluid was collected at 3 dpi. Levels of LDH (a) , ATP (b) and S100A10 (c) in BAL fluid determined by colorimetric (OD; optical density), luminescent (RLU; raw luminescence units) assays or ELISA. Data are presented as the mean with each data point representing an individual animal. n = 8 per group from one experiment. ** P < 0.005, *** P < 0.001, Student's t ‐test. Groups of male and female C57BL/6 mice received daily i.n. treatment with 20 mg kg −1 of LAT8881 or PBS alone from 1 dpi with 10 4 pfu of HKx31 IAV. Lungs underwent formalin fixation at 3 dpi and histological analysis of H&E‐stained lung tissue sections were performed. (d) Representative images at 10× magnification (scale bar = 100 μm). Lung sections were randomised and scored blind by 3 readers for (e) peribronchial inflammation (scale 0–5), (f) alveolitis (scale 0–5) and (g) epithelial damage (scale 0–4), as described in the Methods. Data are presented as the mean with each data point representing an individual animal. n = 4–8 per group from one experiment. * P < 0.05, ** P < 0.01, only PBS vs LAT8881 shown, one‐way ANOVA with Dunnett's multiple comparisons test.

Article Snippet: Human primary bronchial epithelial cells were cultured under submerged conditions on collagen‐coated flasks (Cat #A1064401; Thermo Fisher Scientific, Waltham, USA) in supplemented bronchial epithelial growth medium (BEGM; Cat #CC‐3170; Lonza, Basel, Switzerland) and were used within four passages.

Techniques: Infection, Enzyme-linked Immunosorbent Assay, Staining