Journal: PLoS ONE

Article Title: The Binding of Monomeric C-Reactive Protein (mCRP) to Integrins αvβ3 and α4β1 Is Related to Its Pro-Inflammatory Action

doi: 10.1371/journal.pone.0093738

Figure Lengend Snippet: Wells of 96 well microtiter plate were coated with mCRP or pCRP and remaining protein-binding sites were blocked with BSA. a ) ELISA-type integrin binding assay. In a) wells were incubated with recombinant soluble αvβ3 for 2 h at 37°C in Tyrode-HEPES buffer with 1 mM MgCl 2 . b) Effect of heat treatment on mCRP binding to soluble αvβ3. We heated (90°C for 20 min) mCRP before coating wells and used for binding assays. Assays was performed as in a). c) β3- and β1-3-1-CHO cells adhere to mCRP, but β1-CHO cells did not adhere well to mCRP. mCRP was incubated with β1-CHO, β3-CHO, or β1-3-1-CHO cells for 1 h at 37°C in Tyrode-HEPES buffer with 1 mM MgCl 2 . Bound cells were quantified. d) Specificity of αvβ3 binding to mCRP. We tested if inhibitors of αvβ3 block adhesion of β3-CHO cells to mCRP. mCRP was incubated with cells for 1 h at 37°C in Tyrode-HEPES buffer with 1 mM MgCl 2 . mAb 7E3 (to human β3, 10 μg/ml) and cyclic RGDfV (specific antagonist to αvβ3, 10 μM) blocked the adhesion of β3-CHO cells to mCRP, but control purified mouse IgG (mIgG) or vehicle DMSO did not. e) Cation dependency of mCRP binding to αvβ3 . Adhesion assays were performed as described in c). mCRP was incubated with β3-CHO cells for 1 h at 37°C in Tyrode-HEPES buffer with 2 mM cations or EDTA. The coating concentration of mCRP is 50 μg/ml. The levels of adhesion in different cation conditions are statistically different. f) Alignment of β1, β3, and β1-3-1 . g) Specificity of β1-3-1 integrin binding to mCRP. We tested if anti-human β1 mAb AIIB2 blocks β1-3-1-CHO cells adhesion to mCRP. (Note: 99% of β1-3-1 is β1 and mAb AIIB2 binds to β1-3-1 and blocks its function). h) Cell adhesion to pCRP. pCRP was incubated with β1-CHO, β3-CHO, or β1-3-1-CHO cells for 1 h at 37°C in Tyrode-HEPES buffer with 1 mM MgCl 2 . Bound cells were quantified.

Article Snippet: We did not detect endotoxin in the pCRP used in this study using endotoxin detection kit (Pierce LAL Chromogenic Endotoxin Quantitation Kit, Thermo Scientific) (data not shown). mAb 7E3 (anti-human integrin β3) and mAb AIIB2 (anti-human integrin β1) hybridomas were obtained from ATCC. mAb SG73 (anti-human α4) hybridoma was a kind gift from K. Miyake (University of Tokyo).

Techniques: Protein Binding, Enzyme-linked Immunosorbent Assay, Binding Assay, Incubation, Recombinant, Blocking Assay, Purification, Concentration Assay

Journal: PLoS ONE

Article Title: The Binding of Monomeric C-Reactive Protein (mCRP) to Integrins αvβ3 and α4β1 Is Related to Its Pro-Inflammatory Action

doi: 10.1371/journal.pone.0093738

Figure Lengend Snippet: a). Adhesion of U937 cells to CRP isoforms . Wells of 96 well microtiter plate were coated with mCRP or pCRP and remaining protein-binding sites were blocked with BSA. Wells were incubated with U937 cells (10 5 cells per well) for 1 h in RPMI1640 and bound cells were quantified. b) and c). Effect of antagonists to αvβ3 and α4β1 on adhesion of U937 cells to mCRP . In b), 2.5 μM coating concentration of mCRP was used. Antibodies used were mAb 7E3 (to human β3, 25 μg/ml), mAb SG73 (to human α4, 25 μg/ml), and AIIB2 (to human β1, 25 μg/ml). “mIgG” represents purified mouse IgG used as a control. Antagonists used were cyclic RGDfV (to αvβ3, 10 μM) and BIO1211 (to α4β1, 1 μM). DMSO was used as a control. Adhesion assay was performed in RPMI. d) mCRP induces AKT activation in U937 cells, but not ERK1/2 activation. U937 cells were serum-starved and stimulated with pCRP and mCRP (100 μg/ml) and cell lysates were analyzed by western blotting. d) mCRP, and less effectively pCRP, induce chemotaxis of U937 cells in an integrin-dependent manner. Chemotaxis was measured in modified Boyden chambers (Transwells). 50 μg/ml mCRP or pCRP in 600 μl RPMI 1640 medium was placed in the lower chamber, and U937 cells (5×10 5 cells per well) were placed in the upper chamber. U937 cells were preincubated with antibodies (25 μg/ml) for 30 min at 37°C. After 4 h incubation, migrated cells were counted. e) A PI3K inhibitor, not MEK inhibitor, suppresses mCRP-induced chemotaxis of U937 cells. LY294002 (PI3K inhibitor) or PD98059 (MEK inhibitor) were added at 50 μM in the chemotaxis medium.

Article Snippet: We did not detect endotoxin in the pCRP used in this study using endotoxin detection kit (Pierce LAL Chromogenic Endotoxin Quantitation Kit, Thermo Scientific) (data not shown). mAb 7E3 (anti-human integrin β3) and mAb AIIB2 (anti-human integrin β1) hybridomas were obtained from ATCC. mAb SG73 (anti-human α4) hybridoma was a kind gift from K. Miyake (University of Tokyo).

Techniques: Protein Binding, Incubation, Concentration Assay, Purification, Cell Adhesion Assay, Activation Assay, Western Blot, Chemotaxis Assay, Modification

Journal: Nature Communications

Article Title: Rhinovirus-induced epithelial RIG-I inflammasome suppresses antiviral immunity and promotes inflammation in asthma and COVID-19

doi: 10.1038/s41467-023-37470-4

Figure Lengend Snippet: a Representative Western Blot images of secreted IL-1β (apical compartment), and pro-IL-1β, ASC, pro-caspase-1 and β-actin (cell lysates) in in vitro-cultured HBECs from control subjects ( n = 3, left panel) and patients with asthma ( n = 3, right panel). b IL-1β release to the apical compartment assessed by ELISA in in vitro-cultured HBECs from control ( n = 22, vehicle; n = 14, UV-RV-A16; n = 23, RV-A16) and asthma ( n = 17, vehicle; n = 14, UV-RV-A16; n = 18, RV-A16). c Representative confocal images of ASC speck formation in in vitro-cultured HBECs from control individuals and patients with asthma (control n = 3, asthma n = 3); scale bars: 10 μm. d Quantification of ASC specks, presented as a number of specks (mean from 5–11 equal epithelial areas from two/three technical replicates from control n = 3, asthma n = 3). e IL-1β release to the apical compartment assessed by ELISA ( n = 6, HDM; n = 7, HDM + RV-A16, HDM + RV-A16 + YVAD) in in vitro-cultured HBECs from patients with asthma in the presence or absence of caspase-1 inhibitor (YVAD). f Expression of RV-A16 positive strand (RV-A16 viral RNA) in in vitro-cultured HBECs from patients with asthma and controls ( n = 16, vehicle, ICAM-1, RV-A16, RV-A16 + ICAM-1; n = 8, UV-RV-A16) after anti-ICAM-1 treatment was assessed using RT-PCR and presented as a relative quantification (RQ = 2 -ΔΔCt ) as compared to the vehicle condition. g IL-1β release to the apical supernatants assessed by ELISA in in vitro–cultured HBECs from patients with asthma and healthy controls ( n = 10, vehicle, ICAM-1, RV-A16, RV-A16 + ICAM-1; n = 3, UV-RV-A16) in the presence or absence of anti-ICAM-1 combined with RV-A16 infection. e-g Data are presented as the percentage of the response after in vitro RV-A16 treatment. h Representative Western Blot images of RIG-I protein expression in in vitro-cultured HBECs from patients with asthma ( n = 4). i Representative confocal images of RIG-I in in vitro-cultured HBECs from patients with asthma ( n = 3); scale bars: 10 μm. j Co-immunoprecipitation (co-IP) of ASC/RIG-I complex using anti-ASC antibodies followed by RIG-I detection in the presence of HDM in in vitro-cultured HBECs from patients with asthma ( n = 4). k Co-immunoprecipitation (co-IP) of ASC/MDA5 complex using anti-ASC antibodies followed by MDA5 detection in in vitro-cultured HBECs from patients with asthma ( n = 3). l Representative Western Blot images of NLRP3 protein in in vitro-cultured HBECs from patients with asthma ( n = 4). m Representative confocal images of NLRP3 and Occludin in vitro-cultured HBECs from patients with asthma in the presence of HDM ( n = 3), scale bars: 10 μm. n IL-1β release to the apical compartment in in-vitro-cultured HBECs with/without RV-A16 and NLRP3 inflammasome inhibitor (MCC950) (control n = 3, asthma n = 3). HBECs from patients with asthma are presented in red, HBECs from control individuals are presented in blue. ( n ) indicates the number of biologically independent samples examined over at least three independent experiments. Bar graph data show mean ± SEM analyzed with one-way ANOVA (Kruskal–Wallis test), RM one-way ANOVA (Friedman test) or mixed-effects model with post-hoc analysis, as appropriate, depending on the data relation (paired or unpaired) and distribution. Source data are provided as Source Data files. anti-ICAM-1 , anti-ICAM-1 antibody; Co-IP Co-immunoprecipitation, HBECs human bronchial epithelial cells, HDM house dust mite, IC Isotype control, IP Ab antibodies used for co-precipitation, MCC950 , NLRP3 inflammasome inhibitor; RV-A16 rhinovirus A16; UV-RV-A16 UV-treated rhinovirus A16; YVAD YVAD- (caspase-1 inhibitor).

Article Snippet: Mouse IgG2a monoclonal anti-human ICAM-1 antibody (antibody R6.5) was produced from the hybridoma cells (ATCC HB-9580, mouse hybridoma).

Techniques: Western Blot, In Vitro, Cell Culture, Enzyme-linked Immunosorbent Assay, Expressing, Reverse Transcription Polymerase Chain Reaction, Infection, Immunoprecipitation, Co-Immunoprecipitation Assay

Journal: PLoS ONE

Article Title: The Binding of Monomeric C-Reactive Protein (mCRP) to Integrins αvβ3 and α4β1 Is Related to Its Pro-Inflammatory Action

doi: 10.1371/journal.pone.0093738

Figure Lengend Snippet: a). Adhesion of α4-CHO cells to CRP isoforms. Wells of 96 well microtiter plate were coated with mCRP or pCRP and remaining protein-binding sites were blocked with BSA. Wells were incubated with CHO cells that express recombinant α4β1 (α4-CHO, 10 5 cells per well) for 1 h in Tyrode-HEPES buffer with 1 mM MgCl 2 and bound cells were quantified. b). Effect of antagonists to α4β1 on adhesion of α4-CHO cells to mCRP. Experiments were performed as in a). Fifty μg/ml coating concentration of mCRP was used. Antagonists were mAb SG73 (anti-human α4, 10 μg/ml) and BIO1211 (specific antagonist to α4β1, 1 μM). “mIgG” represents purified mouse IgG used as a control.

Article Snippet: We did not detect endotoxin in the pCRP used in this study using endotoxin detection kit (Pierce LAL Chromogenic Endotoxin Quantitation Kit, Thermo Scientific) (data not shown). mAb 7E3 (anti-human integrin β3) and mAb AIIB2 (anti-human integrin β1) hybridomas were obtained from ATCC. mAb SG73 (anti-human α4) hybridoma was a kind gift from K. Miyake (University of Tokyo).

Techniques: Protein Binding, Incubation, Recombinant, Concentration Assay, Purification

Journal: PLoS ONE

Article Title: The Binding of Monomeric C-Reactive Protein (mCRP) to Integrins αvβ3 and α4β1 Is Related to Its Pro-Inflammatory Action

doi: 10.1371/journal.pone.0093738

Figure Lengend Snippet: a). Adhesion of U937 cells to CRP isoforms . Wells of 96 well microtiter plate were coated with mCRP or pCRP and remaining protein-binding sites were blocked with BSA. Wells were incubated with U937 cells (10 5 cells per well) for 1 h in RPMI1640 and bound cells were quantified. b) and c). Effect of antagonists to αvβ3 and α4β1 on adhesion of U937 cells to mCRP . In b), 2.5 μM coating concentration of mCRP was used. Antibodies used were mAb 7E3 (to human β3, 25 μg/ml), mAb SG73 (to human α4, 25 μg/ml), and AIIB2 (to human β1, 25 μg/ml). “mIgG” represents purified mouse IgG used as a control. Antagonists used were cyclic RGDfV (to αvβ3, 10 μM) and BIO1211 (to α4β1, 1 μM). DMSO was used as a control. Adhesion assay was performed in RPMI. d) mCRP induces AKT activation in U937 cells, but not ERK1/2 activation. U937 cells were serum-starved and stimulated with pCRP and mCRP (100 μg/ml) and cell lysates were analyzed by western blotting. d) mCRP, and less effectively pCRP, induce chemotaxis of U937 cells in an integrin-dependent manner. Chemotaxis was measured in modified Boyden chambers (Transwells). 50 μg/ml mCRP or pCRP in 600 μl RPMI 1640 medium was placed in the lower chamber, and U937 cells (5×10 5 cells per well) were placed in the upper chamber. U937 cells were preincubated with antibodies (25 μg/ml) for 30 min at 37°C. After 4 h incubation, migrated cells were counted. e) A PI3K inhibitor, not MEK inhibitor, suppresses mCRP-induced chemotaxis of U937 cells. LY294002 (PI3K inhibitor) or PD98059 (MEK inhibitor) were added at 50 μM in the chemotaxis medium.

Article Snippet: We did not detect endotoxin in the pCRP used in this study using endotoxin detection kit (Pierce LAL Chromogenic Endotoxin Quantitation Kit, Thermo Scientific) (data not shown). mAb 7E3 (anti-human integrin β3) and mAb AIIB2 (anti-human integrin β1) hybridomas were obtained from ATCC. mAb SG73 (anti-human α4) hybridoma was a kind gift from K. Miyake (University of Tokyo).

Techniques: Protein Binding, Incubation, Concentration Assay, Purification, Cell Adhesion Assay, Activation Assay, Western Blot, Chemotaxis Assay, Modification

Journal: Disease Markers

Article Title: Combined Analysis of Serum Alpha-Fetoprotein and MAGE-A3-Specific Cytotoxic T Lymphocytes in Peripheral Blood for Diagnosis of Hepatocellular Carcinoma

doi: 10.1155/2013/907394

Figure Lengend Snippet: Demographic features and clinicopathologic characteristics of 85 HLA-A2 positive hepatocellular cancer patients and their correlation with the frequency of MAGE-A3 + CTL.

Article Snippet: Two mL peripheral venous blood was collected from each subject and HLA phenotyping was performed using HLA-A2 fluorescein isothiocyanate (FITC) conjugated mouse anti-human monoclonal antibody (BB7.2 ATCC; Becton Dickinson, Franklin Lakes, NJ) with a FACS Calibur flow cytometer (Becton Dickinson).

Techniques: