Journal: Nature communications

Article Title: A digital single-molecule nanopillar SERS platform for predicting and monitoring immune toxicities in immunotherapy.

doi: 10.1038/s41467-021-21431-w

Figure Lengend Snippet: Fig. 1 Digital single-molecule nanopillar surface-enhanced Raman scattering (SERS) platform for parallel counting of four types of cytokines. SEM images of a pillar array side view, b nanoboxes, and c a single nanobox on the top of a pillar; d SERS spectra of nanoboxes conjugated with 5,5-dithiobis (2-nitrobenzoic acid) (DTNB), 4-mercaptobenzoic acid (MBA), 2,3,5,6-tetrafluoro-4-mercaptobenzoic acid (TFMBA), or 2‐mercapto‐4‐methyl‐5‐ thiazoleacetic acid (MMTAA) Raman reporters; e workflow for multiplex counting of cytokines, including fibroblast growth factor 2 (FGF-2), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), and fractalkine (CX3CL1). Data from one independent experiment.

Article Snippet: FGF-2 (223-FB), G-CSF (214-CS), GM-CSF (215-GM), CX3CL1 (365-FR) cytokines; monoclonal anti-FGF-2 (MAB233), anti-G-CSF (MAB214), anti-GMCSF (MAB615), anti-CX3CL1 (MAB3652) antibodies; polyclonal anti-FGF-2 (AF233), anti-G-CSF (AF-214), anti-GM-CSF (AF-215), anti-CX3CL1 (AF-365) antibodies; and FGF-2 (DY233-05), G-CSF (DY214-05), GM-CSF (DY215-05), and CX3CL1 (DY365) ELISA kits were bought from R&D Systems.

Techniques: Multiplex Assay

Journal: Nature communications

Article Title: A digital single-molecule nanopillar SERS platform for predicting and monitoring immune toxicities in immunotherapy.

doi: 10.1038/s41467-021-21431-w

Figure Lengend Snippet: Fig. 4 Specificity of digital nanopillar SERS platform for FGF-2 cytokine detection. Representative confocal SERS images in the presence of a target FGF-2 (1031 aM), and negative controls with non-target controls b G-CSF (1031 aM), c GM-CSF (1031 aM), d CX3CL1 (1031 aM), and e PBS. The median (interquartile range) of active pillars per scanning image for FGF-2, G-CSF, GM-CSF, CX3CL1, and PBS was 72 (63.5–76.75), 1.5 (1.5–2), 2 (1–4), 0.5 (0–1.25), and 1 (1–1.75), respectively. Data from one independent experiment.

Article Snippet: FGF-2 (223-FB), G-CSF (214-CS), GM-CSF (215-GM), CX3CL1 (365-FR) cytokines; monoclonal anti-FGF-2 (MAB233), anti-G-CSF (MAB214), anti-GMCSF (MAB615), anti-CX3CL1 (MAB3652) antibodies; polyclonal anti-FGF-2 (AF233), anti-G-CSF (AF-214), anti-GM-CSF (AF-215), anti-CX3CL1 (AF-365) antibodies; and FGF-2 (DY233-05), G-CSF (DY214-05), GM-CSF (DY215-05), and CX3CL1 (DY365) ELISA kits were bought from R&D Systems.

Techniques:

Journal: Nature communications

Article Title: A digital single-molecule nanopillar SERS platform for predicting and monitoring immune toxicities in immunotherapy.

doi: 10.1038/s41467-021-21431-w

Figure Lengend Snippet: Fig. 5 Specificity of the digital nanopillar SERS platform for FGF-2 cytokine detection. Representative SEM images of pillar array incubated with FGF-2 SERS nanotags in the presence of a, b FGF-2 (1031 aM), c G-CSF (1031 aM), d GM-CSF (1031 aM), e CX3CL1 (1031 aM), and f PBS. The red circles highlight the existence of SERS nanotags. Panel b is the magnified SEM image of the red-highlighted section in a. It is noted that nanofabrication debris on the sidewall of the pillars can also be seen. Data from one independent experiment.

Article Snippet: FGF-2 (223-FB), G-CSF (214-CS), GM-CSF (215-GM), CX3CL1 (365-FR) cytokines; monoclonal anti-FGF-2 (MAB233), anti-G-CSF (MAB214), anti-GMCSF (MAB615), anti-CX3CL1 (MAB3652) antibodies; polyclonal anti-FGF-2 (AF233), anti-G-CSF (AF-214), anti-GM-CSF (AF-215), anti-CX3CL1 (AF-365) antibodies; and FGF-2 (DY233-05), G-CSF (DY214-05), GM-CSF (DY215-05), and CX3CL1 (DY365) ELISA kits were bought from R&D Systems.

Techniques: Incubation

Journal: Nature communications

Article Title: A digital single-molecule nanopillar SERS platform for predicting and monitoring immune toxicities in immunotherapy.

doi: 10.1038/s41467-021-21431-w

Figure Lengend Snippet: Fig. 6 Sensitivity for the simultaneous detection of four cytokines. Representative confocal SERS images of fibroblast growth factor 2 (FGF-2), granulocyte colony-stimulating factor (GM-CSF), granulocyte colony- stimulating factor (G-CSF), and fractalkine (CX3CL1) with the concentration of a 2.6 aM, b 26 aM, c 260 aM, d 1031 aM. Colour scale bars indicate Raman intensities from 5,5-dithiobis (2-nitrobenzoic acid) (DTNB), 4-mercaptobenzoic acid (MBA), 2,3,5,6-tetrafluoro-4-mercaptobenzoic acid (TFMBA), or 2‐mercapto‐4‐methyl‐5‐thiazoleacetic acid (MMTAA). The median (interquartile range) of active pillars per scanning image of FGF-2, G-CSF, GM-CSF, CX3CL1 for 2.6 aM: 3 (1.5–3), 1 (1–2), 2 (1–3), 2 (1–3); 26 aM: 8 (5.5–10), 10 (9–13), 7 (6–10), 8 (6–10); 260 aM: 40 (36–48), 40 (35–52), 39 (35–50), 37 (36–49); and 1031 aM: 79 (61.5–97), 78 (72–87.5), 88 (68.5–97), 79 (64–95), respectively. Data represents one experiment from three independent tests.

Article Snippet: FGF-2 (223-FB), G-CSF (214-CS), GM-CSF (215-GM), CX3CL1 (365-FR) cytokines; monoclonal anti-FGF-2 (MAB233), anti-G-CSF (MAB214), anti-GMCSF (MAB615), anti-CX3CL1 (MAB3652) antibodies; polyclonal anti-FGF-2 (AF233), anti-G-CSF (AF-214), anti-GM-CSF (AF-215), anti-CX3CL1 (AF-365) antibodies; and FGF-2 (DY233-05), G-CSF (DY214-05), GM-CSF (DY215-05), and CX3CL1 (DY365) ELISA kits were bought from R&D Systems.

Techniques: Concentration Assay

Journal: Nature communications

Article Title: A digital single-molecule nanopillar SERS platform for predicting and monitoring immune toxicities in immunotherapy.

doi: 10.1038/s41467-021-21431-w

Figure Lengend Snippet: Fig. 7 Digital nanopillar SERS assay for monitoring melanoma patients during immune checkpoint therapy. For Patient 1 who developed severe irAEs, SERS images for cytokine detection on a day 7, b day 21, c day 42, d cytokine concentration graph for fibroblast growth factor 2 (FGF-2), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), and fractalkine (CX3CL1). The two shorter horizontal lines denote the interquartile ranges (25th and 75th percentile) and the longer horizontal lines in between denote the median (50th percentile), and e LDA analysis, respectively. For Patient 6 who developed mild irAEs, SERS images for cytokine detection on f day 0, g day 21, h day 42, i four cytokine concentration graph, the two shorter horizontal lines denote the interquartile ranges (25th and 75th percentile) and the longer horizontal lines in between denote the median (50th percentile), and j LDA analysis, respectively. IPI ipilimumab, PEMBRO pembrolizumab; G3 grade 3, G2 grade 2; SD stable disease, PR partial response. For Patient 1, the median (interquartile range) of active pillars per scanning image of FGF-2, G-CSF, GM-CSF, CX3CL1 on day 7: 14 (11–22.5), 23 (21, 29), 12 (7.5–18), 17 (9–25.5); day 21: 30 (19–37.5), 33 (19–41), 26 (17.5–36.5), 29 (21–43); and day 42: 33 (16.5–58.5), 76 (64–128.5), 25 (14–39.5), 48 (26.5–73.5), respectively. For Patient 6, the median (interquartile range) of active pillars per scanning image of FGF-2, G-CSF, GM-CSF, CX3CL1 on day 0: 18 (16–23), 49 (31.5–56), 23 (17.5–28), 20 (14.5–27); day 21: 29 (24–33.5), 53 (46.5–70), 35 (25–46), 22 (19–29.5); and day 42: 13 (8–16.5), 44 (23.5–55.5), 10 (6.5–12.5), 30 (24–34.5), respectively. The data represented three technical replicates obtained from three chips. Nine images were acquired from each chip for cytokine counting. Statistical analysis was based on Kruskal–Wallis test followed by Dunn’s test to correct multiple comparisons (two-sided). Source data are provided in the Source Data file.

Article Snippet: FGF-2 (223-FB), G-CSF (214-CS), GM-CSF (215-GM), CX3CL1 (365-FR) cytokines; monoclonal anti-FGF-2 (MAB233), anti-G-CSF (MAB214), anti-GMCSF (MAB615), anti-CX3CL1 (MAB3652) antibodies; polyclonal anti-FGF-2 (AF233), anti-G-CSF (AF-214), anti-GM-CSF (AF-215), anti-CX3CL1 (AF-365) antibodies; and FGF-2 (DY233-05), G-CSF (DY214-05), GM-CSF (DY215-05), and CX3CL1 (DY365) ELISA kits were bought from R&D Systems.

Techniques: Concentration Assay

Journal: Circulation: Cardiovascular Imaging

Article Title: Molecular Imaging of VWF (von Willebrand Factor) and Platelet Adhesion in Postischemic Impaired Microvascular Reflow

doi: 10.1161/circimaging.118.007913

Figure Lengend Snippet: Figure 1. Mean (±SEM) microsphere- derived risk area from animals assigned to either (A) 30 min or (B) 45 min of coronary occlusion. C, Mean (±SEM) microvascular blood flow from myocardial contrast echocardiography (MCE) performed during coronary occlusion in the risk area and remote territories, as well as from sham-treated mice. D, Example of background-subtracted, color-coded MCE images during coronary occlusion, and time-intensity data after a destructive pulse sequence from the risk area and remote territory. For images, time after destructive pulse is at upper left and color-coded scale at bottom of the 5 s image. ADAMTS13 indicates a disintegrin and metalloprotein- ase with a thrombospondin type-1 motif member 13; LV, left ventricle; RA, risk area; and WT, wild-type. *P <0.05 vs remote territory.

Article Snippet: In Vivo Imaging Study Design Protocol 1 Closed-chest myocardial IR with 30 minutes of transient left anterior descending coronary artery (LAD) occlusion was performed in WT mice (n=40), WT mice treated with recombinant full-length human ADAMTS13, 5 μg IV (R&D Systems, Minneapolis, MN) just before IR (n=14), and ADAMTS13−/− mice (n=16).

Techniques: Derivative Assay, Sequencing

Journal: Circulation: Cardiovascular Imaging

Article Title: Molecular Imaging of VWF (von Willebrand Factor) and Platelet Adhesion in Postischemic Impaired Microvascular Reflow

doi: 10.1161/circimaging.118.007913

Figure Lengend Snippet: Figure 2. Postischemic myocardial perfusion by MCE in mice undergo- ing 30 min of ischemia and reperfusion measured in the remote region and the risk area. Data (mean±SEM) are shown for (A) myocardial microvascular blood flow (MBF), (B) microvascular blood volume (MBV), and (C) microvascular flux rate (β). ADAMTS13 indicates a disintegrin and metalloproteinase with a thrombo- spondin type-1 motif member 13. *P <0.05 vs remote territory; †P <0.05 vs WT (wild type); ‡P <0.05 only before correction for multiple comparison.

Article Snippet: In Vivo Imaging Study Design Protocol 1 Closed-chest myocardial IR with 30 minutes of transient left anterior descending coronary artery (LAD) occlusion was performed in WT mice (n=40), WT mice treated with recombinant full-length human ADAMTS13, 5 μg IV (R&D Systems, Minneapolis, MN) just before IR (n=14), and ADAMTS13−/− mice (n=16).

Techniques: Comparison

Journal: Circulation: Cardiovascular Imaging

Article Title: Molecular Imaging of VWF (von Willebrand Factor) and Platelet Adhesion in Postischemic Impaired Microvascular Reflow

doi: 10.1161/circimaging.118.007913

Figure Lengend Snippet: Figure 3. Mean (±SEM) signal enhance- ment on MCE molecular imaging after 30 min of ischemia and reperfusion for (A) VWF (von Willebrand factor) A-1 domain and (B) platelet GP (glycoprotein) Ibɑ. Data are expressed as signal difference between targeted and control microbubbles (MB). C, Examples of color-coded MCE molecular imaging in the short-axis plane for targeted and control MBs (color scale at bottom) and the microsphere-derived risk area in the same short-axis plane. *P <0.05 vs remote territory; †P <0.05 vs wild-type and ADAMTS13−/− (a disintegrin and metal- loproteinase with a thrombospondin type-1 motif member 13); ‡P <0.05 vs WT (wild type).

Article Snippet: In Vivo Imaging Study Design Protocol 1 Closed-chest myocardial IR with 30 minutes of transient left anterior descending coronary artery (LAD) occlusion was performed in WT mice (n=40), WT mice treated with recombinant full-length human ADAMTS13, 5 μg IV (R&D Systems, Minneapolis, MN) just before IR (n=14), and ADAMTS13−/− mice (n=16).

Techniques: Imaging, Control, Derivative Assay

Journal: Circulation: Cardiovascular Imaging

Article Title: Molecular Imaging of VWF (von Willebrand Factor) and Platelet Adhesion in Postischemic Impaired Microvascular Reflow

doi: 10.1161/circimaging.118.007913

Figure Lengend Snippet: Figure 5. Mean (±SEM) infarct size by triphenyltetrazolium chloride staining as a percentage of the total microsphere-de- rived risk area for mice undergoing either (A) 30 min of ischemia followed by 90 min of reperfusion or (B) 45 min of ischemia followed by 3 d of reperfusion. *P <0.05 vs WT (wild type) before Bonfer- roni correction; †P <0.05 vs WT+ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type-1 motif member 13) after Bonferroni correction.

Article Snippet: In Vivo Imaging Study Design Protocol 1 Closed-chest myocardial IR with 30 minutes of transient left anterior descending coronary artery (LAD) occlusion was performed in WT mice (n=40), WT mice treated with recombinant full-length human ADAMTS13, 5 μg IV (R&D Systems, Minneapolis, MN) just before IR (n=14), and ADAMTS13−/− mice (n=16).

Techniques: Staining

Journal: Circulation: Cardiovascular Imaging

Article Title: Molecular Imaging of VWF (von Willebrand Factor) and Platelet Adhesion in Postischemic Impaired Microvascular Reflow

doi: 10.1161/circimaging.118.007913

Figure Lengend Snippet: Figure 4. Examples of immunohistochemis- try from the sham-treated control WT (wild type) mice (anterior and posterior myocar- dial with anterior labeled as risk area) and from postischemic WT and ADAMTS13−/− (a disintegrin and metalloproteinase with a thrombospondin type-1 motif member 13) mice. The top and bottom rows show fused im- ages for platelet CD41 immunostaining (red), endothelial lectin staining (green), and nuclear counterstain (blue). The middle row shows corresponding images for only the red channel (platelet CD41) in the risk area. For the insets, arrowheads show single platelets, whereas the arrow in the ADAMTS13−/− mouse illustrates 3 platelets linearly arranged (scale bar=20 μm). MI indicates myocardial infarction.

Article Snippet: In Vivo Imaging Study Design Protocol 1 Closed-chest myocardial IR with 30 minutes of transient left anterior descending coronary artery (LAD) occlusion was performed in WT mice (n=40), WT mice treated with recombinant full-length human ADAMTS13, 5 μg IV (R&D Systems, Minneapolis, MN) just before IR (n=14), and ADAMTS13−/− mice (n=16).

Techniques: Control, Labeling, Immunostaining, Staining

Journal: Journal of cellular and molecular medicine

Article Title: The effect of ADAMTS13 on graft-versus-host disease.

doi: 10.1111/jcmm.18457

Figure Lengend Snippet: FIGURE 5 ADAMTS13 and VWF-A2 reduced donor-derived T cells in secondary lymphoid organs 24 h after bone marrow transplant. Mice were sacrificed 24 h post-transplant (n = 4). Cells were

Article Snippet: In some experiments, prior to adding Jurkat cells, histamine- stimulated HUVEC cells were treated with 1 μg/mL of recombinant human ADAMTS13 (6156- AD; R&D Systems) for 10 min at room temperature.

Techniques: Derivative Assay

Journal: Journal of cellular and molecular medicine

Article Title: The effect of ADAMTS13 on graft-versus-host disease.

doi: 10.1111/jcmm.18457

Figure Lengend Snippet: FIGURE 7 Role of VWF in the binding of T cells. (A) One hundred thousand Jurkat cells (immortalize malignant T lymphocytes) were incubated with HUVECs cells plated on 6-well plates. After 15 min incubation at 37°C, the number of adhered Jurkat cells in each well was counted using pictures taken with an inverted microscope and compared between histamine-stimulated and non-stimulated HUVECs. The effect of recombinant ADAMTS13 (1 mg/mL), recombinant VWF-A2 (1 mg/mL) and anti-αL antibodies (1:1000 dilution) on the number of adhered Jurkat cells to histamine- stimulated HUVECs were compared (n = 4, each experiment in triplicates). p-values were calculated using a one-way ANOVA test with Dunnett's multiple comparison correction compared to the number of adhered Jurkat cells to histamine-stimulated HUVECs. * p < 0.05. (B) One hundred thousand Jurkat cells were incubated in VWF-coated 96-wells plates for 15 min at 37°C. Jurkat cells, either pre-stimulated with 200 ng/mL of CCL21 or resting, were added to each well. The effect of recombinant ADAMTS13, recombinant VWF-A2 and anti-αL antibodies on the number of adhered CCL21-stimulated Jurkat cells to VWF was compared (n = 8, each experiment in triplicates). p-values were calculated using a one-way ANOVA test with Dunnett's multiple comparison correction compared to the number of adhered CCL21-stimulated Jurkat cells VWF. *p < 0.05.

Article Snippet: In some experiments, prior to adding Jurkat cells, histamine- stimulated HUVEC cells were treated with 1 μg/mL of recombinant human ADAMTS13 (6156- AD; R&D Systems) for 10 min at room temperature.

Techniques: Binding Assay, Incubation, Inverted Microscopy, Recombinant, Comparison

Journal:

Article Title: Chemically synthesized human survivin does not inhibit caspase-3

doi: 10.1110/ps.036145.108

Figure Lengend Snippet: (A) Ligation of (1–45)αCOSR and (46–142) at 1.5 h. The reaction was monitored by analytical HPLC on a Waters XBridge C18 column (4.6 × 150 mm, 3.5 μM) running a 30-min gradient of 25%–45% acetonitrile containing 0.1% TFA at a flow rate of 1 mL/min. (Insets) Mass spectra of (1–45)αCOSR and (46–142) determined by ESI-MS. (B) Ligated full-length survivin characterized by C18 RP-HPLC and ESI-MS. HPLC conditions: Waters symmetry 300 C18 column (4.6 × 150 mm, 5 μM) running a 30-min gradient of 5%–65% acetonitrile containing 0.1% TFA at a flow rate of 1 mL/min. (C) CD spectra of synthetic survivin at 5 μM in 5 mM phosphate buffer containing 0.1 mM TCEP, pH 7.5 (thin line), and at 25 μM in 5 mM phosphate buffer containing 0.1 mM TCEP and 50 μM Zn2+, pH 7.5 (thick line). (D) Representative size-exclusion chromatograms of synthetic survivin (3) and molecular mass standards (1, 2, 4, 5). Linear regression analysis of the correlation between logarithmic M r and retention time is illustrated in the inset. (E) Representative raw data from the hydrolysis of Ac-DEVD-AMC by caspase-3 in the absence and presence of different concentrations of XIAP and synthetic survivin. (F) Dose-dependent percent inhibition of caspase-3 by XIAP (filled circles), synthetic survivin without Zn2+ (empty squares), and synthetic survivin in the presence of Zn2+ (filled squares). Each curve is the mean of three independent experiments.

Article Snippet: EnzChek Caspase-3 assay kit #1 was purchased from Invitrogen; recombinant caspase-3 was obtained from Calbiochem and recombinant human XIAP, from R&D Systems.

Techniques: Ligation, Inhibition

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: The CX3C Chemokine Fractalkine Induces Vascular Dysfunction by Generation of Superoxide Anions

doi: 10.1161/01.atv.0000251535.30191.60

Figure Lengend Snippet: Figure 1. Expression of the fractalkine receptor (CX3CR1) on the surface of human umbilical vein endothelial cells (HUVECs) at baseline and after 2-hour incubation with fractalkine (FKN) assessed by flow cytometry (A, n4). Representative flow cytometry histo- grams show the right-ward shift in CX3CR1-fluorescence (fineisotype con- trol, boldanti-CX3CR1, B). After 2 hours of incubation with FKN, protein expres- sion of CX3CR1 was unchanged (A). Expression of CX3CR1 in rat aorta dem- onstrated by polymerase chain reaction (C) and Western blot (D). Stimulation with FKN (1 g/mL, 2 and 6 hours) did not induce surface expression of intercellular adhesion molecule-1, vascular cell adhe- sion molecule-1, or E-selectin on HUVECs (compared with TNF, E, n3). **P0.01 vs control.

Article Snippet: The used recombinant fractalkine was recombinant human fractalkine (365-FR, R&D Systems, Minneapolis, MN) which exerted similar ROS generation and impairment of vasorelaxation as recombinant rat fractalkine, and its activity was completely inhibited by pre-incubation of rat aortic rings with the anti-rat CX3CR1 antibody (data not shown).

Techniques: Expressing, Incubation, Cytometry, Polymerase Chain Reaction, Western Blot, Control

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: The CX3C Chemokine Fractalkine Induces Vascular Dysfunction by Generation of Superoxide Anions

doi: 10.1161/01.atv.0000251535.30191.60

Figure Lengend Snippet: Figure 2. Immunohistochemistry demon- strated endothelial and smooth muscle expression of CX3CR1 in rat aortae in the absence (A, D) or presence (B, E) of stimulation with fractalkine (FKN). Control experiments used an irrelevant IgG instead of anti-CX3CR1 (C, F), images were taken with 20-fold (A-C) and 40-fold (D-F) magnifications.

Article Snippet: The used recombinant fractalkine was recombinant human fractalkine (365-FR, R&D Systems, Minneapolis, MN) which exerted similar ROS generation and impairment of vasorelaxation as recombinant rat fractalkine, and its activity was completely inhibited by pre-incubation of rat aortic rings with the anti-rat CX3CR1 antibody (data not shown).

Techniques: Immunohistochemistry, Expressing, Control

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: The CX3C Chemokine Fractalkine Induces Vascular Dysfunction by Generation of Superoxide Anions

doi: 10.1161/01.atv.0000251535.30191.60

Figure Lengend Snippet: Figure 3. Vasoconstriction induced by incremental concentrations of phenylephrine (A) in fractalkine-pretreated (FKN, 1 g/mL, 2 hour) aortic rings. Additional increment in vascular tone by NOS inhibition (NG-nitro-L-arginine, L-NNA, 100mol/L) in slightly preconstricted aortic rings preincubated with fractalkine (FKN) for 2 hours (B). Endothelium-dependent, acetylcholine-induced vasorelaxation of aortic rings pretreated with FKN for 2 hours demonstrating ligand-dependence (anti-FKN) and receptor-dependence (anti-CX3CR1) of FKN -induced endothelial dysfunction (C). Endothelium-independent, DEA-NONOate-induced vasorelaxation (D). *P0.05, **P0.01 vs Co, n6 to 16.

Article Snippet: The used recombinant fractalkine was recombinant human fractalkine (365-FR, R&D Systems, Minneapolis, MN) which exerted similar ROS generation and impairment of vasorelaxation as recombinant rat fractalkine, and its activity was completely inhibited by pre-incubation of rat aortic rings with the anti-rat CX3CR1 antibody (data not shown).

Techniques: Inhibition

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: The CX3C Chemokine Fractalkine Induces Vascular Dysfunction by Generation of Superoxide Anions

doi: 10.1161/01.atv.0000251535.30191.60

Figure Lengend Snippet: Figure 5. Addition of the radical scaven- ger tiron reversed endothelial dysfunction induced by fractalkine (FKN) (1 g/mL, 2 hours) in isolated rat aortic rings (A, n10). Confocal microscopy of 10-m- thick aortic sections incubated with the fluorescent dye hydroxyethidium to visu- alize O2 formation throughout the vas- cular wall (B, n6). Representative con- focal images are shown (C). Aortic O2 formation detected by lucigenin- enhanced chemiluminescence was increased after 2 hours of pretreatment with FKN. Antagonism of the chemokine domain with an antagonizing antibody or inhibition of oxidases by diphenylenei- odonium chloride (DPI) significantly reduced O2 formation in aortic rings (D, Alloxanthine oxidase inhibition by allo- purionol, L-NNANOS inhibition by L-arginine analogue, E-vascular rings lacking intact endothelium following denudation, n7 to 12). O2 formation in isolated, cultured human aortic endothe- lial cells (HAEC) as well as human aortic smooth muscle cells (HASMCs) induced by either fractalkine (1 g/mL, 90 min- utes) and/or TNF (10 ng/mL, 90 min- utes) (E, n5). **P0.01 vs FKN –Ti- ron; §§P0.01 vs Co; #P0.05, ##P0.01 vs FKN.

Article Snippet: The used recombinant fractalkine was recombinant human fractalkine (365-FR, R&D Systems, Minneapolis, MN) which exerted similar ROS generation and impairment of vasorelaxation as recombinant rat fractalkine, and its activity was completely inhibited by pre-incubation of rat aortic rings with the anti-rat CX3CR1 antibody (data not shown).

Techniques: Isolation, Confocal Microscopy, Incubation, Inhibition, Cell Culture

Journal: Journal of translational medicine

Article Title: CD19/CD20 dual-targeted chimeric antigen receptor-engineered natural killer cells exhibit improved cytotoxicity against acute lymphoblastic leukemia.

doi: 10.1186/s12967-024-04990-6

Figure Lengend Snippet: Fig. 1 Preparation and mechanism of CD19/CD20 dual-targeted CAR-NK cell. ①: Ex-vivo expanded natural killer cells are derived from umbilical cord blood. ②: The mRNA encoding anti-CD19 CARs (FMC63 scFv-CD8α-4-1BB-CD3ζ) and anti-CD20 CARs (LEU16 scFv-CD8α-4-1BB-CD3ζ) were constructed by IVT. ③-④: CD19/CD20 dual-targeted CAR-NK cells were generated by simultaneous electroporation of CAR-mRNA into UCB-NK cells derived from umbilical cord blood, which specifically recognizes CD19+ and/or CD20+ ALL cells. ⑤: Once activated, CAR-NK binds to the target antigen and then lyses tumor cells by releasing perforin and IFN-γ. CAR chimeric antigen receptor, IVT in vitro transcription, UCB umbilical cord blood, NK natural killer cells

Article Snippet: To assess the cell surface expression of CAR, electroporated cells were stained with Biotinylated Human CD20 Full-Length protein (stock 50 μg/mL, 1:25 dilution, ACRO Biosystems, Beijing, CHN) for 60 min at 4 °C.

Techniques: Ex Vivo, Derivative Assay, Construct, Generated, Electroporation, In Vitro

Journal: Journal of translational medicine

Article Title: CD19/CD20 dual-targeted chimeric antigen receptor-engineered natural killer cells exhibit improved cytotoxicity against acute lymphoblastic leukemia.

doi: 10.1186/s12967-024-04990-6

Figure Lengend Snippet: Fig. 2 Ex vivo expansion and identification of UCB-NK. A Schematic diagram of ex vivo-expanded UCB-NK cells. B Morphology of NK cells at different cultured times observed via an optical microscope (Scale Bar = 200 μm). C Growth curves of UCB-NK cells from different donors. D Purity of UCB-NK cells as determined by flow cytometry. E Expression of CD19 and CD20 antigens on the surface of different acute leukemia cells. F Cytotoxic effects of UCB-NK cells on different acute lymphoblastic leukemia cells. Data are presented as mean values and S.D. of triplicate samples (*p < 0.05, **p < 0.01, ***p < 0.001, n = 3). UCB-NK, NK cells derived from umbilical cord blood

Article Snippet: To assess the cell surface expression of CAR, electroporated cells were stained with Biotinylated Human CD20 Full-Length protein (stock 50 μg/mL, 1:25 dilution, ACRO Biosystems, Beijing, CHN) for 60 min at 4 °C.

Techniques: Ex Vivo, Cell Culture, Microscopy, Flow Cytometry, Expressing, Derivative Assay

Journal: Journal of translational medicine

Article Title: CD19/CD20 dual-targeted chimeric antigen receptor-engineered natural killer cells exhibit improved cytotoxicity against acute lymphoblastic leukemia.

doi: 10.1186/s12967-024-04990-6

Figure Lengend Snippet: Fig. 3 Construction and identification of the second-generation CARs targeting CD19 and CD20 antigen. A Schematic illustration of the construction of mRNA encoding CD19 CARs and CD20 CARs utilized IVT. B Schematic diagrams of the CD19 CAR-mRNA and the CD20CAR-mRNA. FMC63(anti-CD19) scFv and LEU16 (anti-CD20) scFv were linked to the CD8α hinge and transmembrane domain, the 4-1BB (CD137) signaling domain, and the CD3 zeta signaling domain, respectively. C Denatured agarose gel electrophoresis of the CAR-mRNA. Lane (1) represents the CD19 CAR-mRNA group, lane (3) represents the CD20 CAR-mRNA group, and lane (2) represents mRNA Marker (6000nt). D Detection of CAR expression on NK cells by flow cytometry. CAR expression was detected by using one or two of four staining reagents: PE-Labeled monoclonal anti-FMC63 Antibody (column 1), Biotinylated Human CD20/MS4A1 full length protein followed by Streptavidin Protein-Alexa Fluor 647 (column 2), or FITC-Labeled Monoclonal Anti-FMC63 Antibody (column 3). The expression percentage of CAR on NK cells is noted on the right of each histogram. EGFP-transduced cells served as an additional negative control. E CD19 CARs and CD20 CARs expression kinetics on dual-target CAR-NK over 4 days. IVT in vitro transcription, scFv single-chain fragment variable regions, Cap1 Cap1 structure, UTR untranslated region

Article Snippet: To assess the cell surface expression of CAR, electroporated cells were stained with Biotinylated Human CD20 Full-Length protein (stock 50 μg/mL, 1:25 dilution, ACRO Biosystems, Beijing, CHN) for 60 min at 4 °C.

Techniques: Agarose Gel Electrophoresis, Marker, Expressing, Flow Cytometry, Staining, Labeling, Negative Control, In Vitro

Journal: Journal of translational medicine

Article Title: CD19/CD20 dual-targeted chimeric antigen receptor-engineered natural killer cells exhibit improved cytotoxicity against acute lymphoblastic leukemia.

doi: 10.1186/s12967-024-04990-6

Figure Lengend Snippet: Fig. 4 Specific cytotoxicity of CD19/CD20 dual-targeted CAR-NK cells against CD19 positive/CD20 positive acute lymphoma cells. A Detection of cytotoxicity of dual-target CAR-NK cells and single-target CAR-NK cells on acute lymphoma cells (BALL-1, REH, and Jurkat) at different E:T ratios. Data are presented as mean values and S.D. of triplicate samples (*p < 0.05, **p < 0.01, n = 3). B Death of BALL-1, REH, and Jurkat cell lines was detected by flow cytometry (CFSE/PI). Nucleated cell gates were gated according to the size and complexity of the samples (FCS-A and SSC-A, respectively). Nucleated cells were further gated in FSC-A and FSC-H to screen for single cells and exclude doublets. From the single cell gate, tumor cells were defined as CFSE+. The CFSE+PI+ population was defined as dead tumor cells from the CFSE+ gate

Article Snippet: To assess the cell surface expression of CAR, electroporated cells were stained with Biotinylated Human CD20 Full-Length protein (stock 50 μg/mL, 1:25 dilution, ACRO Biosystems, Beijing, CHN) for 60 min at 4 °C.

Techniques: Flow Cytometry

Journal: Cancers

Article Title: A Novel Allogeneic Rituximab-Conjugated Gamma Delta T Cell Therapy for the Treatment of Relapsed/Refractory B-Cell Lymphoma

doi: 10.3390/cancers15194844

Figure Lengend Snippet: Rituximab conjugation confers γδ2 T cells with CD20 binding capacity. ( a ) A representative histogram of rituximab conjugation is illustrated. DNA linker-1 and linker-2 were conjugated with γδ2 T cells and rituximab, respectively. Linker-1-conjugated γδ2 T cells and linker-2-conjugated rituximab were mixed and ACE1831, rituximab-linked γδ2 T cells, were generated through DNA hybridization. Un-conjugated γδ2 T cells and ACE1831 were stained with R-phycoerythrin-coupled anti-F(ab’)2 antibody to determine the rituximab conjugation efficiency through flow cytometry. Un-conjugated γδ2 T cells (light blue line) represent negative staining, and efficient rituximab conjugation on ACE1831 (dark blue line) is shown. Percent of Max is the highest point of each peak of the overlaid histogram derived from ACE1831 and γδ2 T cells. ( b ) CD20 binding capacity of ACE1831 and γδ2 T cells was determined through flow cytometry analysis. The cells were incubated with 0.001, 0.01, 0.1, 1, 10 and 100 μg/mL of human CD20-His recombinant protein, and the CD20-bound cell population was identified through staining with Fluorescein-coupled anti-6X His tag antibody. The study was performed in triplicate in five different experiments, and the representative results are shown. Statistical analysis was performed using the t test. **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

Article Snippet: To examine their CD20 binding capacity, the cells were incubated with 0.001, 0.01, 0.1, 1, 10 and 100 μg/mL of recombinant human CD20-His protein (ACROBiosystems, Newark, DE).

Techniques: Conjugation Assay, Binding Assay, Generated, DNA Hybridization, Staining, Flow Cytometry, Negative Staining, Derivative Assay, Incubation, Recombinant

Journal: Cancers

Article Title: A Novel Allogeneic Rituximab-Conjugated Gamma Delta T Cell Therapy for the Treatment of Relapsed/Refractory B-Cell Lymphoma

doi: 10.3390/cancers15194844

Figure Lengend Snippet: Rituximab conjugation confers γδ2 T cells with superior cytotoxicity against CD20-expressing cancer cells. ( a – c ) ACE1831 and γδ2 T cells were co-incubated with CD20-expressing cancer cells. ( a ) Daudi, ( b ) Raji and ( c ) rituximab-resistant Raji cells at effector to target (E:T) ratios of 1:1, 2:1, 5:1 and 10:1, analyzed via CellTiter-Glo ® luminescent cell viability assay after 4 h of co-incubation. ( d ) CD107a, ( e ) granzyme B and ( f ) IFNγ of ACE1831 in the absence and presence of Raji cells at an E:T ratio of 2:1 after 2 h of incubation were analyzed via flow cytometry. Each group was performed in triplicate from two donor lots, and the representative results are shown. Statistical analysis was performed using the t test. **, p < 0.01; ***, p < 0.001; ****, p < 0.0001. ( g , h ) ACE1831 and γδ2 T cells were co-incubated with ( g ) CD20-negative K562 and ( h ) donor PBMC cells at effector to target (E:T) ratios of 2:1, 5:1 and 10:1 and analyzed using a CellTiter-Glo ® luminescent cell viability assay after 4 h of co-incubation.

Article Snippet: To examine their CD20 binding capacity, the cells were incubated with 0.001, 0.01, 0.1, 1, 10 and 100 μg/mL of recombinant human CD20-His protein (ACROBiosystems, Newark, DE).

Techniques: Conjugation Assay, Expressing, Incubation, Cell Viability Assay, Flow Cytometry

Journal: Cancers

Article Title: A Novel Allogeneic Rituximab-Conjugated Gamma Delta T Cell Therapy for the Treatment of Relapsed/Refractory B-Cell Lymphoma

doi: 10.3390/cancers15194844

Figure Lengend Snippet: ACE1831 shows superior potency against CD20-expressing cancer cells in vivo. Four doses of intravenously delivered ACE1831 effectively suppressed tumor growth. Tumor-bearing SCID–Beige mice were treated with ACE1831, γδ2 T cells and a Vehicle (serum-free medium) twice per week for two weeks. ( a ) Tumor burden of mice (n = 5 per group) was determined through bioluminescence imaging. ( b ) The bioluminescence intensity of the tumor burden is presented as mean values ± SD. The difference in mean tumor burden between groups was examined using a two-way ANOVA. ***, p < 0.001. ( c ) The survival rate of mice with different treatments was analyzed using the Kaplan–Meier method. **, p < 0.01. ( d ) The body weight of each group of mice is presented as mean value ± SD.

Article Snippet: To examine their CD20 binding capacity, the cells were incubated with 0.001, 0.01, 0.1, 1, 10 and 100 μg/mL of recombinant human CD20-His protein (ACROBiosystems, Newark, DE).

Techniques: Expressing, In Vivo, Imaging

Journal: Cancers

Article Title: A Novel Allogeneic Rituximab-Conjugated Gamma Delta T Cell Therapy for the Treatment of Relapsed/Refractory B-Cell Lymphoma

doi: 10.3390/cancers15194844

Figure Lengend Snippet: T cell activation and cytotoxicity mediated by the antigen recognition of ACC-linked antibody. ( a ) Jurkat-NFAT-Luc cells were conjugated with different amounts of rituximab using ACC technology and were stained with anti-F(ab’)2 antibody to examine the levels of rituximab conjugated on Jurkat-NFAT-Luc cells. Un-conjugated Jurkat-NFAT-Luc cells (grey line) represent negative staining, and Jurkat-NFAT-Luc cells with low (light blue) and high (dark blue) rituximab conjugation are shown. Percent of Max is the highest point of each peak of the overlaid histogram. ( b ) Jurkat-NFAT-Luc cells conjugated with different amounts of rituximab were co-incubated with different Raji cell numbers (+, 5 × 10 4 ; ++, 2 × 10 5 ; +++, 5 × 10 5 ), and NFAT signaling activation was determined based on NFAT-regulated luciferase activity. Each condition was applied in triplicate in two different experiments, and the representative results are shown. Mean ± SD. Statistical analysis was performed using a t test. **, p < 0.01; ****, p < 0.0001. ( c ) The effector cells were preincubated with or without 1 μg/mL of TCRγδ blocking antibody for 1 h at 37 °C. After 4 h of co-incubation with Raji cells, cytotoxicity against Raji cells was analyzed using a CellTiter-Glo ® luminescent cell viability assay. Each condition was applied in triplicate in two different experiments, and the representative results are shown. Mean ± SD, *, p < 0.05, ***, p < 0.001. ( d ) Illustration delineating the activation of ACE1831 upon encountering CD20-expressing cancer cells.

Article Snippet: To examine their CD20 binding capacity, the cells were incubated with 0.001, 0.01, 0.1, 1, 10 and 100 μg/mL of recombinant human CD20-His protein (ACROBiosystems, Newark, DE).

Techniques: Activation Assay, Staining, Negative Staining, Conjugation Assay, Incubation, Luciferase, Activity Assay, Blocking Assay, Cell Viability Assay, Expressing