Journal: mAbs

Article Title: Characterization of concurrent target suppression by JNJ-61178104, a bispecific antibody against human tumor necrosis factor and interleukin-17A

doi: 10.1080/19420862.2020.1770018

Figure Lengend Snippet: Summary of JNJ-8104, golimumab and CNTO 6785 in vitro target-binding kinetics and activities.

Article Snippet: The concentrations of total IL-17A in cyno and human serum were measured with similar MSD assays, except using a non-competing biotinylated mouse anti-human IL-17A antibody (Clone 4H1524.1, Rockland) for capture and a non-competing ruthenium-labeled mouse IgG1 anti-human IL-17A antibody (Clone 41802, R&D Systems) for detection.

Techniques: In Vitro

Journal: mAbs

Article Title: Characterization of concurrent target suppression by JNJ-61178104, a bispecific antibody against human tumor necrosis factor and interleukin-17A

doi: 10.1080/19420862.2020.1770018

Figure Lengend Snippet: Inhibition of in vitro inflammatory responses mediated by endogenous TNF and IL-17A. Human rheumatoid arthritis fibroblast-like synovial cells (RA-FLS) were cocultured with in vitro differentiated T h 17/T h 1 cells. Various concentrations of JNJ-8104, CNTO 9809 (parental anti-TNF), CNTO 4782 (parental anti-IL-17A) or equal-molar fixed-ratio combination of CNTO 9809 and CNTO 4782 were incubated with the coculture for 48 hours. Endogenous TNF and IL-17A-mediated production of IL-6 (a) and GROα (b) in the supernatants were measured.

Article Snippet: The concentrations of total IL-17A in cyno and human serum were measured with similar MSD assays, except using a non-competing biotinylated mouse anti-human IL-17A antibody (Clone 4H1524.1, Rockland) for capture and a non-competing ruthenium-labeled mouse IgG1 anti-human IL-17A antibody (Clone 41802, R&D Systems) for detection.

Techniques: Inhibition, In Vitro, Incubation

Journal: mAbs

Article Title: Characterization of concurrent target suppression by JNJ-61178104, a bispecific antibody against human tumor necrosis factor and interleukin-17A

doi: 10.1080/19420862.2020.1770018

Figure Lengend Snippet: Inhibition of rhTNF- and rhIL-17A-induced Cellular Influx by JNJ-8104 in the Airway Lumen of Mice. Mice were intranasally instilled with rhTNF- and rhIL-17A in combination. After 6 h their lungs were lavaged and total numbers of BAL cells (a) and neutrophils (b) were enumerated as detailed in “Materials & Methods.” Mice were injected intraperitoneally with the test mAbs or isotype control18 hours prior to cytokines challenge. Data are represented as mean ± SEM; N = 6–7 mice/group.

Article Snippet: The concentrations of total IL-17A in cyno and human serum were measured with similar MSD assays, except using a non-competing biotinylated mouse anti-human IL-17A antibody (Clone 4H1524.1, Rockland) for capture and a non-competing ruthenium-labeled mouse IgG1 anti-human IL-17A antibody (Clone 41802, R&D Systems) for detection.

Techniques: Inhibition, Injection

Journal: mAbs

Article Title: Characterization of concurrent target suppression by JNJ-61178104, a bispecific antibody against human tumor necrosis factor and interleukin-17A

doi: 10.1080/19420862.2020.1770018

Figure Lengend Snippet: Observed serum concentrations versus time profiles of Total mAb (a), total TNF (b), and Total IL-17A (c) following IV administration of golimumab (parental anti-TNF), CNTO 6785 (anti-IL-17A with identical parental Fab arms) or JNJ-8104 at the specified dose levels in cynomolgus monkeys. Data are represented as mean ± SD.

Article Snippet: The concentrations of total IL-17A in cyno and human serum were measured with similar MSD assays, except using a non-competing biotinylated mouse anti-human IL-17A antibody (Clone 4H1524.1, Rockland) for capture and a non-competing ruthenium-labeled mouse IgG1 anti-human IL-17A antibody (Clone 41802, R&D Systems) for detection.

Techniques:

Journal: mAbs

Article Title: Characterization of concurrent target suppression by JNJ-61178104, a bispecific antibody against human tumor necrosis factor and interleukin-17A

doi: 10.1080/19420862.2020.1770018

Figure Lengend Snippet: Estimated JNJ-8104, CNTO148 and CNTO 6785 cynomolgus monkey PK/TE model parameters (mean, residual standard error%) and predicted human parameters.

Article Snippet: The concentrations of total IL-17A in cyno and human serum were measured with similar MSD assays, except using a non-competing biotinylated mouse anti-human IL-17A antibody (Clone 4H1524.1, Rockland) for capture and a non-competing ruthenium-labeled mouse IgG1 anti-human IL-17A antibody (Clone 41802, R&D Systems) for detection.

Techniques:

Journal: mAbs

Article Title: Characterization of concurrent target suppression by JNJ-61178104, a bispecific antibody against human tumor necrosis factor and interleukin-17A

doi: 10.1080/19420862.2020.1770018

Figure Lengend Snippet: PK/TE model fitting of PK, total IL-17A and Free IL-17A profiles following IV administration of JNJ-8104 (a) and CNTO 6785 (b) in cynomolgus monkeys. Symbols = Observed individual data; Lines = Mean model prediction.

Article Snippet: The concentrations of total IL-17A in cyno and human serum were measured with similar MSD assays, except using a non-competing biotinylated mouse anti-human IL-17A antibody (Clone 4H1524.1, Rockland) for capture and a non-competing ruthenium-labeled mouse IgG1 anti-human IL-17A antibody (Clone 41802, R&D Systems) for detection.

Techniques:

Journal: mAbs

Article Title: Characterization of concurrent target suppression by JNJ-61178104, a bispecific antibody against human tumor necrosis factor and interleukin-17A

doi: 10.1080/19420862.2020.1770018

Figure Lengend Snippet: Comparison of observed and model-predicted Total IL-17A profiles following a single IV administration of JNJ-8104 at 0.1, 0.3, 1, 3 and 10 mg/kg (a−e) normal human subjects. Symbols = Observed individual data; Solid lines = Mean model prediction based on cyno PK/TE model parameters and allometric scaling for human PK; Dotted lines = Mean model prediction based on estimated JNJ-8104 human PK parameters from FIH data with cyno-based in vivo KD.

Article Snippet: The concentrations of total IL-17A in cyno and human serum were measured with similar MSD assays, except using a non-competing biotinylated mouse anti-human IL-17A antibody (Clone 4H1524.1, Rockland) for capture and a non-competing ruthenium-labeled mouse IgG1 anti-human IL-17A antibody (Clone 41802, R&D Systems) for detection.

Techniques: Comparison, In Vivo

Journal: Journal of Thrombosis and Haemostasis

Article Title: Development of a novel fully functional coagulation factor VIII with reduced immunogenicity utilizing an in silico prediction and deimmunization approach

doi: 10.1111/jth.15413

Figure Lengend Snippet: Schema for the incorporation of amino acid substitutions into the factor VIII (FVIII) sequence. The numbers indicate the substitutions leading to active FVIII variants in the respective rounds. The substitutions were structured in clusters. In the first round the substitutions were tested individually, whereas combinations of the most successful substitutions were tested in the second round, during design of experiment testing and in the third and final round

Article Snippet: Thrombin activation was shown via western blot with the polyclonal sheep anti‐human FVIII antibody (Cedarlane) and the donkey anti‐sheep IgG IRDye 800CW (Abm).

Techniques: Sequencing

Journal: Journal of Thrombosis and Haemostasis

Article Title: Development of a novel fully functional coagulation factor VIII with reduced immunogenicity utilizing an in silico prediction and deimmunization approach

doi: 10.1111/jth.15413

Figure Lengend Snippet: Immunogenicity scale. The scores indicate the immunogenicity of a protein calculated by the EpiMatrix system, representing >98% of the human population. The immunogenicity score for each protein is normalized to the median score that was determined for 100,000 random proteins of 1000 amino acids in length. This normalization makes it possible to compare proteins of different lengths. As shown, the score of FVIII‐19M has a lower epitope density than the median random protein score, and its score is lower than the median score for the entire human proteome

Article Snippet: Thrombin activation was shown via western blot with the polyclonal sheep anti‐human FVIII antibody (Cedarlane) and the donkey anti‐sheep IgG IRDye 800CW (Abm).

Techniques: Immunopeptidomics

Journal: Journal of Thrombosis and Haemostasis

Article Title: Development of a novel fully functional coagulation factor VIII with reduced immunogenicity utilizing an in silico prediction and deimmunization approach

doi: 10.1111/jth.15413

Figure Lengend Snippet: Specific activities of four productions of unmodified factor VIII (FVIII) and FVIII‐19M. The specific activity in % is calculated as the relation of chromogenic FVIII activity to amount of antigen. The line indicates the median of the four measurements

Article Snippet: Thrombin activation was shown via western blot with the polyclonal sheep anti‐human FVIII antibody (Cedarlane) and the donkey anti‐sheep IgG IRDye 800CW (Abm).

Techniques: Activity Assay

Journal: Journal of Thrombosis and Haemostasis

Article Title: Development of a novel fully functional coagulation factor VIII with reduced immunogenicity utilizing an in silico prediction and deimmunization approach

doi: 10.1111/jth.15413

Figure Lengend Snippet: Clotting time for FVIII‐19M, unmodified factor VIII (FVIII), moroctocog alfa, and simoctocog alfa. Different FVIII concentrations were analyzed. The measurements were performed in duplicate and the mean values are displayed. Standard deviation of each duplicate was below 6 s

Article Snippet: Thrombin activation was shown via western blot with the polyclonal sheep anti‐human FVIII antibody (Cedarlane) and the donkey anti‐sheep IgG IRDye 800CW (Abm).

Techniques: Coagulation, Standard Deviation

Journal: Journal of Thrombosis and Haemostasis

Article Title: Development of a novel fully functional coagulation factor VIII with reduced immunogenicity utilizing an in silico prediction and deimmunization approach

doi: 10.1111/jth.15413

Figure Lengend Snippet: Western blot specifically detecting the factor VIIII (FVIII) heavy and light chain. The different FVIII products were applied at a concentration of 5 U/ml. The heavy chains are indicated in red and the light chains are indicated in green. Single‐chain FVIII, consisting of heavy and light chain, is indicated in yellow, due to the overlay of green and red. MW, molecular weight

Article Snippet: Thrombin activation was shown via western blot with the polyclonal sheep anti‐human FVIII antibody (Cedarlane) and the donkey anti‐sheep IgG IRDye 800CW (Abm).

Techniques: Western Blot, Concentration Assay, Molecular Weight

Journal: Journal of Thrombosis and Haemostasis

Article Title: Development of a novel fully functional coagulation factor VIII with reduced immunogenicity utilizing an in silico prediction and deimmunization approach

doi: 10.1111/jth.15413

Figure Lengend Snippet: Factor VIII (FVIII) products activated by thrombin. Each product was applied in its non‐activated and activated form. In the non‐activated form, the typical bands for the single chain, heavy chain, and light chain were detectable. After thrombin cleavage additional bands for A1, A2, A1A2, Ba3, and A3C1C2, were detectable. MW, molecular weight

Article Snippet: Thrombin activation was shown via western blot with the polyclonal sheep anti‐human FVIII antibody (Cedarlane) and the donkey anti‐sheep IgG IRDye 800CW (Abm).

Techniques: Molecular Weight

Journal: Journal of Thrombosis and Haemostasis

Article Title: Development of a novel fully functional coagulation factor VIII with reduced immunogenicity utilizing an in silico prediction and deimmunization approach

doi: 10.1111/jth.15413

Figure Lengend Snippet: Difference between the CD4 + T cell proliferation response to dendritic cells (DCs) stimulated with IL‐Mix plus FVIII‐19M and DCs stimulated with IL‐Mix plus unmodified factor VIII (FVIII). The bars below 0 indicate a reduced CD4 + T cell response to FVIII‐19M. The lower CD4 + T cell response to FVIII‐19M compared to unmodified FVIII is significant using the Wilcoxon test ( P = .027)

Article Snippet: Thrombin activation was shown via western blot with the polyclonal sheep anti‐human FVIII antibody (Cedarlane) and the donkey anti‐sheep IgG IRDye 800CW (Abm).

Techniques:

Journal: Journal of Thrombosis and Haemostasis

Article Title: Development of a novel fully functional coagulation factor VIII with reduced immunogenicity utilizing an in silico prediction and deimmunization approach

doi: 10.1111/jth.15413

Figure Lengend Snippet: Relation between the differences in individual T cell epitope measure (iTEM) scores and proliferation. The iTEM scores for FVIII‐19M and unmodified factor VIII (FVIII) were calculated for Donor #1, #2, #3, #6, #8, and #10, based on their human leukocyte antigen (HLA)‐DR genotype. The differences of the iTEM scores were plotted against the differences in CD4 + T cell proliferation, determined in the dendritic cell (DC)‐‐T cell assay

Article Snippet: Thrombin activation was shown via western blot with the polyclonal sheep anti‐human FVIII antibody (Cedarlane) and the donkey anti‐sheep IgG IRDye 800CW (Abm).

Techniques:

Journal: PloS one

Article Title: Association of interleukin-6 signalling with the muscle stem cell response following muscle-lengthening contractions in humans.

doi: 10.1371/journal.pone.0006027

Figure Lengend Snippet: Figure 1. Circulating IL-6 and muscle IL-6 mRNA and IL-6 receptor response muscle lengthening contractions (MLC). (1a) Average serum interleukin-6 (IL-6) concentration. Time 0 hr corresponds to pre-intervention values; all other time-points correspond to post-intervention time (hr). (1b) Relative IL-6 mRNA expression, expressed as fold-change from 0 hr. (1c) Pearson correlation of serum IL-6 concentration versus muscle IL-6 mRNA (fold-change), correlation is representative of the individual data points and presented as mean values ($)6SD (error bars). (1d) Relative IL-6 receptor (IL-6Ra) mRNA expression, expressed as fold-change from 0 hr. (1e) Immunofluorescent image (406) of a muscle cross-section triple-stained for Pax7 (red), IL-6Ra (green) and nuclei (DAPI = blue); IL-6Ra staining is apparent on the sarcolemma and satellite cell membrane. White arrow denotes one of the Pax7+ nuclei which co-localized with IL-6Ra (scale bar = 100 mm). Values are reported as mean6S.E.M. Mean values represent the mean for all 8 subjects per time-point (8 samples per time-point, 40 samples total). *p,0.05 vs. 0 hr. doi:10.1371/journal.pone.0006027.g001

Article Snippet: Immunofluorescence 7 mm sections were cryosectioned and stained with antibodies against Pax7 (neat; DSHB, USA); IL-6 (500 ng/mL, MAB 2061, R&D Systems, USA); p-STAT3 (p-STAT3 Y705 1:100, Cell Signaling Technologies Inc., USA); IL-6Ra (1:50, MCA822, Serotec, UK); PCNA (1:200, ab15497, Abcam Inc., USA); and Laminin (1:1000, L8271, Sigma-Aldrich, Canada).

Techniques: Concentration Assay, Expressing, Staining, Membrane

Journal: PLoS ONE

Article Title: STAT6 knockdown using multiple siRNA sequences inhibits proliferation and induces apoptosis of human colorectal and breast cancer cell lines

doi: 10.1371/journal.pone.0207558

Figure Lengend Snippet: (A) Measure of STAT6 mRNA level in HT-29 cells 24 hours post-transfection. The graph represents the mean ± SEM of multiple independent experiments (n) obtained by real-time PCR. Results were analysed by ΔΔCt method for relative quantifications. The fold change is represented by the Y axis, and values are normalized to control cells. (B) STAT6 protein level analysis at 2, 5 and 7 days post-transfection in HT-29 cells. The graph represents the mean of the percentage of STAT6 positive cells ± SEM of multiple independent experiments (n) obtained by flow cytometry. (C) Measure of STAT6 mRNA level in ZR-75-1 cells 3 days post-transfection. The graph represents the mean ± SEM of multiple independent experiments (n) obtained by real-time PCR. Results were analysed by ΔΔCt method for relative quantifications. The fold change is represented by the Y axis, and values are normalized to control cells. (D) STAT6 protein level analysis after 4 and 7 days of transfection in ZR-75-1 cells. The graph represents the mean of the percentage of STAT6 positive cells ± SEM of multiple independent experiments (n) obtained by flow cytometry. E) Representative histograms (Control: back; NT: grey; STAT6.1: red; STAT6.4: blue) of STAT6 protein analysis at 7 days post-transfection by flow cytometry in HT-29 cells (top) and ZR-75-1 (bottom) cells. STAT6 siRNA sequences and non-targeting siRNA were used at 100 nM as the final concentration. Control cells were non-transfected cells and STAT6 siRNA sequences 1 and 4 and non-targeting siRNA are denoted as STAT6.1, STAT6.4 and NT, respectively. The number of independent experiments (n) is set out in the Fig.

Article Snippet: Cells were washed twice with PBS/0.5%BSA (Bovine Serum Albumin) and stained with anti-STAT6 APC conjugated antibody (Miltenyi Biotec, 130-104-030) (20 μl/10 6 cells) for 30 min in the dark at 4°C.

Techniques: Transfection, Real-time Polymerase Chain Reaction, Control, Flow Cytometry, Concentration Assay

Journal: PLoS ONE

Article Title: STAT6 knockdown using multiple siRNA sequences inhibits proliferation and induces apoptosis of human colorectal and breast cancer cell lines

doi: 10.1371/journal.pone.0207558

Figure Lengend Snippet: (A and B) Number of live HT-29 cells measured at 5 and 7 days post-transfection, respectively. The graphs represent the mean ± SEM of multiple independent experiments (n). (C) The graph illustrates how HT-29 cells grew over time and represents the mean ± SEM of the independent experiments (n) shown in A and B. (D and E) Number of live ZR-75-1 cells measured at 4 and 7 days post-transfection, respectively. The graphs represent the mean ± SEM of multiple independent experiments (n). (F) The graph illustrates how ZR-75-1 cells grew over time and represents the mean ± SEM of the multiple independent experiments (n) shown in D and E. The number of live cells was calculated as detailed in the material and methods using NucleoCounter NC-100. The percentage of reduction of the number of live cells was calculated by comparison between the mean of NT vs . the mean of the transfection with STAT6 siRNA sequences. STAT6 and non-targeting (NT) siRNA sequences were used at 100 nM as the final concentration. Non-transfected cells served as negative control and STAT6 siRNA sequences 1 and 4 and non-targeting siRNA are denoted as STAT6.1, STAT6.4 and NT, respectively. The number of independent experiments (n) is set out in the Fig.

Article Snippet: Cells were washed twice with PBS/0.5%BSA (Bovine Serum Albumin) and stained with anti-STAT6 APC conjugated antibody (Miltenyi Biotec, 130-104-030) (20 μl/10 6 cells) for 30 min in the dark at 4°C.

Techniques: Transfection, Comparison, Concentration Assay, Negative Control

Journal: PLoS ONE

Article Title: STAT6 knockdown using multiple siRNA sequences inhibits proliferation and induces apoptosis of human colorectal and breast cancer cell lines

doi: 10.1371/journal.pone.0207558

Figure Lengend Snippet: (A) Apoptosis analysis in HT-29 cells. (B) Apoptosis analysis in ZR-75-1 cells. In both cases, the graphs represent from left to right: early (Annexin V + /PI - ), late (Annexin V + /PI + ) and total (Annexin V + ) apoptosis. The graphs represent the mean ± SEM of multiple independent experiments (n) obtained by flow cytometry. (C) Representative flow cytometry plots in HT-29 cells. (D) Representative flow cytometry plots in ZR-75-1 cells. The X axes represent Annexin V and the Y axes represent PI fluorescence intensity. Quadrants were set according to cells independently stained with Annexin V or PI. Apoptosis was studied at 7 days post-transfection in both cell lines. Data were analysed with Flowjo Software. STAT6 siRNA sequences and a non-targeting siRNA sequence were used at 100 nM as the final concentration. Non-transfected cells served as control cells and STAT6 siRNA sequences 1 and 4 and non-targeting siRNA are denoted as STAT6.1, STAT6.4 and NT, respectively. The number of independent experiments (n) is set out in the Fig.

Article Snippet: Cells were washed twice with PBS/0.5%BSA (Bovine Serum Albumin) and stained with anti-STAT6 APC conjugated antibody (Miltenyi Biotec, 130-104-030) (20 μl/10 6 cells) for 30 min in the dark at 4°C.

Techniques: Flow Cytometry, Fluorescence, Staining, Transfection, Software, Sequencing, Concentration Assay, Control

Journal: PLoS ONE

Article Title: STAT6 knockdown using multiple siRNA sequences inhibits proliferation and induces apoptosis of human colorectal and breast cancer cell lines

doi: 10.1371/journal.pone.0207558

Figure Lengend Snippet: STAT6 siRNA transfection was carried out at day 1 of cell culture with (A) STAT6.1 and (B) STAT6.4 at 100 nM. A second transfection was carried out in both cases with STAT6.1 and STAT6.4 at the same concentration 7 days after the first transfection. The graphs represent the number of live cells over time measured at day 7 and 14 days post-first transfection counted using NucleoCounter NC-100 as detailed in the material and methods section. The mean ± SEM of 3 independent experiments is represented in each graph. Control cells were non-transfected cells and STAT6 siRNA sequences 1 and 4 and non-targeting siRNA are denoted as STAT6.1, STAT6.4 and NT, respectively. The percentage of reduction of the number of live cells was calculated by comparison between the mean of NT vs . the mean of the individual transfection with STAT6 siRNA sequences, and sequential transfection with NT (NT+NT) vs . sequential transfection with STAT6.1 and STAT6.4.

Article Snippet: Cells were washed twice with PBS/0.5%BSA (Bovine Serum Albumin) and stained with anti-STAT6 APC conjugated antibody (Miltenyi Biotec, 130-104-030) (20 μl/10 6 cells) for 30 min in the dark at 4°C.

Techniques: Transfection, Cell Culture, Concentration Assay, Control, Comparison

Journal: PLoS ONE

Article Title: STAT6 knockdown using multiple siRNA sequences inhibits proliferation and induces apoptosis of human colorectal and breast cancer cell lines

doi: 10.1371/journal.pone.0207558

Figure Lengend Snippet: (A) STAT6 expression at protein level in HT-29 cells at day 2 and 7 post-transfection measured by flow cytometry. The graph represents the mean ± SEM of multiple independent experiments (n). Data was analysed using Flowjo Software. The percentage of STAT6 positive cells is represented on the Y axis. (B) Representative histograms (Control: back; NT: grey; STAT6.1: red; STAT6.4: blue) of STAT6 protein analysis at 7 days post-transfection by flow cytometry in HT-29 cells. (C) Number of live HT-29 cells measured at day 7 post-transfection by NucleoCounter NC100. The graph represents the mean ± SEM of multiple independent experiments (n). (D) The graph illustrates how HT-29 cells grew over time and represents the mean ± SEM of the independent number of experiments shown in C. (E, F and G) The graphs show early (E) (Annexin V + /PI - ), late (F) (Annexin V + /PI + ) and total (G) (Annexin V + ) apoptosis, and represent the mean ± SEM of multiple independent experiments (n) obtained by flow cytometry. Apoptosis was studied at 7 days post-transfection. Data were analysed with Flowjo Software. STAT6 siRNA sequences and a non-targeting siRNA sequence were used at 100 nM as the final concentration. Non-transfected cells served as control cells and STAT6 siRNA sequences 1 and 4 and non-targeting siRNA are denoted as STAT6.1, STAT6.4 and NT, respectively. The number of independent experiments (n) is set out in the Fig.

Article Snippet: Cells were washed twice with PBS/0.5%BSA (Bovine Serum Albumin) and stained with anti-STAT6 APC conjugated antibody (Miltenyi Biotec, 130-104-030) (20 μl/10 6 cells) for 30 min in the dark at 4°C.

Techniques: Expressing, Transfection, Flow Cytometry, Software, Control, Sequencing, Concentration Assay

Journal: Cell reports

Article Title: High-dimensional profiling clusters asthma severity by lymphoid and non-lymphoid status

doi: 10.1016/j.celrep.2021.108974

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Anti-Human/Mouse IL-5 (TRFK5)-143Nd , Fluidigm , Cat# 3143003B.

Techniques: Purification, Control, Antibody Labeling, Flow Cytometry, Software, Staining, Blocking Assay