Journal: The Journal of Physiology

Article Title: Increased endothelial shear stress improves insulin‐stimulated vasodilatation in skeletal muscle

doi: 10.1113/JP277050

Figure Lengend Snippet: A, schematic diagram and illustration of experimental protocol 1. Human aortic endothelial cells (HAECs) were seeded and exposed to low shear stress conditions (3 dynes cm−2) for 40 h to promote cell alignment. Cells were then either exposed to increased shear stress (20 dynes cm−2) or maintained at control conditions (3 dynes cm−2) for 1 h. Following a 30 min recovery period (3 dynes cm−2) designed to mimic subsequent protocols, cells were stimulated with insulin or vehicle for 30 min and fixed in their chambers. Immunofluorescence and image analysis were performed in all conditions. The cell image displayed shows fluorescence associated with stains for phospho‐eNOS (green) and 4′,6‐diamidino‐2‐phenylindole for nuclei (blue). B, the change in the ratio of phospho‐eNOS/phospho‐MAPK upon insulin stimulation in cells previously exposed to control conditions vs. increased shear stress. The cartoon underneath further illustrates the results. IR, insulin receptor. n = 4/condition. Values are means ± SEM. * P < 0.05 vs. control condition.

Article Snippet: The cell culture schematic design image is courtesy of ibidi GmbH.

Techniques: Shear, Control, Immunofluorescence, Fluorescence

Journal: Blood Advances

Article Title: Comparison of antibody-based immunotherapeutics for malignant hematological disease in an experimental murine model ∗

doi: 10.1182/bloodadvances.2023011647

Figure Lengend Snippet: Structure and binding of Slamf7 immunotherapeutics. Shown is the molecular design of 3 different immunotherapeutic formats and binding to their specific targets. VH and VL sequences of original rat hybridoma clone 3-1-4 against Slamf7 (Biogenes) were cloned to generate the different types: (A) Schematic of chimeric Slamf7-mAb, a mAb containing mouse Fc–immunoglobulin G2a (Fc-IgG2a) and scFv of a-Slamf7. (B) Purity and integrity of antibody batches were controlled by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showing intact heavy and light chains of Slamf7-mAb corresponding to correct sizes, isotype msIgG2a served as control. (C) Antibody binding to its target Slamf7 was analyzed by flow cytometry showing fluorescence signal in MPC-11 wt (blue profile), which is decreased when incubated with MPC-11 missing Slamf7 expression (MPC-11 kd), isotype control was used as negative control (red profile). (D) Schematic bispecific molecule with scFv parts of α-Slamf7 and α-msCD3 joined via flexible glycine-serine linker (Slamf7-BiTE). (E) Purified bispecific molecules were analyzed by western blot detecting myc-tag expressed on the carboxy terminus part of Slamf7- BiTE. (F) Flow cytometry analysis showed binding to msCD3 on T cells and to Slamf7 on MPC-11 wt ([red profile] isotype control; [blue profile] Slamf7-BiTE). (G) Schematic chimeric antigen T cell receptor was cloned containing sequences of CD8a hinge and transmembrane region, CD28-, 4-1BB–, and CD3ζ-signaling domain joined by scFv of Slamf7 (Slamf7-CAR). (H) After transduction of mouse T cells, they were labeled with an antibody detecting scFv Slamf7 on cell surfaces (blue), nontransduced T cells served as controls (red). An increase in fluorescence was assumed as signals for CART constructs. Usually transduction efficiency was around 25% to 40%. (I) Legend of domains. HC, heavy chain; kDa, kilodalton; LC, light chain; Ms, mouse; VH, variable heavy chain; VL, variable light chain.

Article Snippet: VH and VL sequences of original rat hybridoma clone 3-1-4 against Slamf7 (Biogenes) were cloned to generate the different types: (A) Schematic of chimeric Slamf7-mAb, a mAb containing mouse Fc–immunoglobulin G2a (Fc-IgG2a) and scFv of a-Slamf7. (B) Purity and integrity of antibody batches were controlled by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showing intact heavy and light chains of Slamf7-mAb corresponding to correct sizes, isotype msIgG2a served as control. (C) Antibody binding to its target Slamf7 was analyzed by flow cytometry showing fluorescence signal in MPC-11 wt (blue profile), which is decreased when incubated with MPC-11 missing Slamf7 expression (MPC-11 kd), isotype control was used as negative control (red profile). (D) Schematic bispecific molecule with scFv parts of α-Slamf7 and α-msCD3 joined via flexible glycine-serine linker (Slamf7-BiTE). (E) Purified bispecific molecules were analyzed by western blot detecting myc-tag expressed on the carboxy terminus part of Slamf7- BiTE. (F) Flow cytometry analysis showed binding to msCD3 on T cells and to Slamf7 on MPC-11 wt ([red profile] isotype control; [blue profile] Slamf7-BiTE). (G) Schematic chimeric antigen T cell receptor was cloned containing sequences of CD8a hinge and transmembrane region, CD28-, 4-1BB–, and CD3ζ-signaling domain joined by scFv of Slamf7 (Slamf7-CAR). (H) After transduction of mouse T cells, they were labeled with an antibody detecting scFv Slamf7 on cell surfaces (blue), nontransduced T cells served as controls (red).

Techniques: Binding Assay, Clone Assay, Polyacrylamide Gel Electrophoresis, Flow Cytometry, Fluorescence, Incubation, Expressing, Negative Control, Purification, Western Blot, Transduction, Labeling, Construct

Journal: Blood Advances

Article Title: Comparison of antibody-based immunotherapeutics for malignant hematological disease in an experimental murine model ∗

doi: 10.1182/bloodadvances.2023011647

Figure Lengend Snippet: Mode of action of the different immunotherapeutic formats in vitro. (A) Slamf7-BiTE showed cytotoxic activity after 48 hours when incubated with unactivated mouse CD3 + T cells and MPC-11 wt at different effector-to-target ratios at a constant concentration of 10 μg/mL. Compared with Slamf7-BiTE on MPC-11 kd cytotoxic activity is reduced but increases with amount of effector T cells. (B) Increasing concentration of Slamf7-BiTE on MPC-11 wt with constant effector-to-target ratio of 10 to 1 showed an increase of cytotoxic activity but not on MPC-11 kd, same for irrBiTE, a BiTE molecule with reduced binding to MPC-11 wt or kd. (C) The role of the self-ligand Slamf7 as a negative regulator of T-cell activation was analyzed in a cytotoxic assay in which effector T cells were preincubated with Slamf7-mAb. No difference in cytotoxic activity was noticed between blocked and unblocked T cells. Using Slamf7-mAb antibody instead of Slamf7-BiTE showed no activity. (D) Further T-cell activation was followed by the T-cell activation marker CD69 in flow cytometry analysis. Activation was reduced on a low level with unblocked T cells. (E) Using a Promega Bioassay for ADCC monoclonal Slamf7-mAb lead to an increase in luminescence correlating with activation of FcγRIV receptor on effector cells in presence of target tumor cell line MPC-11 wt, which is prevented by using isotype control or MPC-11 kd. (F) Mouse CD3 + T cells transduced with CAR against Slamf7 (Salmf7-CART) showed significant cytotoxicity against MPC-11 wt after 24 hours, which is lowered when using MPC-11 kd or CD19-CART transduced cells. Data represent mean ± standard error of the mean (SEM) (n = 3). ∗ P ≤ .05; ∗∗ P < .01; ∗∗∗ P < .001. Each assay was repeated at least twice.

Article Snippet: VH and VL sequences of original rat hybridoma clone 3-1-4 against Slamf7 (Biogenes) were cloned to generate the different types: (A) Schematic of chimeric Slamf7-mAb, a mAb containing mouse Fc–immunoglobulin G2a (Fc-IgG2a) and scFv of a-Slamf7. (B) Purity and integrity of antibody batches were controlled by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showing intact heavy and light chains of Slamf7-mAb corresponding to correct sizes, isotype msIgG2a served as control. (C) Antibody binding to its target Slamf7 was analyzed by flow cytometry showing fluorescence signal in MPC-11 wt (blue profile), which is decreased when incubated with MPC-11 missing Slamf7 expression (MPC-11 kd), isotype control was used as negative control (red profile). (D) Schematic bispecific molecule with scFv parts of α-Slamf7 and α-msCD3 joined via flexible glycine-serine linker (Slamf7-BiTE). (E) Purified bispecific molecules were analyzed by western blot detecting myc-tag expressed on the carboxy terminus part of Slamf7- BiTE. (F) Flow cytometry analysis showed binding to msCD3 on T cells and to Slamf7 on MPC-11 wt ([red profile] isotype control; [blue profile] Slamf7-BiTE). (G) Schematic chimeric antigen T cell receptor was cloned containing sequences of CD8a hinge and transmembrane region, CD28-, 4-1BB–, and CD3ζ-signaling domain joined by scFv of Slamf7 (Slamf7-CAR). (H) After transduction of mouse T cells, they were labeled with an antibody detecting scFv Slamf7 on cell surfaces (blue), nontransduced T cells served as controls (red).

Techniques: In Vitro, Activity Assay, Incubation, Concentration Assay, Binding Assay, Activation Assay, Marker, Flow Cytometry, Transduction

Journal: Blood Advances

Article Title: Comparison of antibody-based immunotherapeutics for malignant hematological disease in an experimental murine model ∗

doi: 10.1182/bloodadvances.2023011647

Figure Lengend Snippet: Immuntherapeutics targeting Slamf7 lead to reduced tumor growth in a naive syngeneic mouse model. (A) Schematic presentation of a syngeneic naive mouse model using female BALB/c mice as recipients, MPC-11 wt as tumor. Tumor injection was performed subcutaneously on the right flank, followed by intravenous Slamf7-BiTE (1.25 mg/kg bw per mouse) and Slamf7-mAb injection (10 mg/kg bw per mouse), this was defined as day 0. For CART experiments, cyclophosphamide was injected intraperitoneally 1 day before IV CART infusion (1e7 transduced T cells per mouse, ∼20% transduction efficiency), followed next day by subcutaneous tumor injection. PBS injections were used as control. Tumor size and bw were measured daily. (B) Both Slamf7-mAb (violet line) and Slamf7-BiTE (red line) lead to reduced tumor growth compared to PBS group (yellow line). (C) Slamf7-BiTE (red line) but not Slamf7-mAb (violet line) treatment had influence on bw. (D) Slamf7-CART animals (green line) showed reduced tumor growth compared to animals which received nontransduced T cells (nontransduced [ntd] T cells, blue line) or PBS (yellow line). (E) Treatment of animals with CART cells had no significant influence on bw in either group. (F) At the end of experiment successful engraftment of T cell transfer controlled using blood samples of animals of ntd T cells (green), PBS (blue) and Slamf7 CART (red) for flow cytometry analysis by detecting CD3+ cells expressing scFv Slamf7 of CART receptor on cell surface. Representative histograms are shown. Results for tumor growth and bw are representative of at least 2 independent experiments with n = 5 mice per group. The data are presented as mean ± SEM, ∗ P < .05.

Article Snippet: VH and VL sequences of original rat hybridoma clone 3-1-4 against Slamf7 (Biogenes) were cloned to generate the different types: (A) Schematic of chimeric Slamf7-mAb, a mAb containing mouse Fc–immunoglobulin G2a (Fc-IgG2a) and scFv of a-Slamf7. (B) Purity and integrity of antibody batches were controlled by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showing intact heavy and light chains of Slamf7-mAb corresponding to correct sizes, isotype msIgG2a served as control. (C) Antibody binding to its target Slamf7 was analyzed by flow cytometry showing fluorescence signal in MPC-11 wt (blue profile), which is decreased when incubated with MPC-11 missing Slamf7 expression (MPC-11 kd), isotype control was used as negative control (red profile). (D) Schematic bispecific molecule with scFv parts of α-Slamf7 and α-msCD3 joined via flexible glycine-serine linker (Slamf7-BiTE). (E) Purified bispecific molecules were analyzed by western blot detecting myc-tag expressed on the carboxy terminus part of Slamf7- BiTE. (F) Flow cytometry analysis showed binding to msCD3 on T cells and to Slamf7 on MPC-11 wt ([red profile] isotype control; [blue profile] Slamf7-BiTE). (G) Schematic chimeric antigen T cell receptor was cloned containing sequences of CD8a hinge and transmembrane region, CD28-, 4-1BB–, and CD3ζ-signaling domain joined by scFv of Slamf7 (Slamf7-CAR). (H) After transduction of mouse T cells, they were labeled with an antibody detecting scFv Slamf7 on cell surfaces (blue), nontransduced T cells served as controls (red).

Techniques: Injection, Transduction, Flow Cytometry, Expressing

Journal: Blood Advances

Article Title: Comparison of antibody-based immunotherapeutics for malignant hematological disease in an experimental murine model ∗

doi: 10.1182/bloodadvances.2023011647

Figure Lengend Snippet: Immunotherapeutics targeting Slamf7 lead to reduced tumor growth in a parent-into-F1 model of stem cell transplant. (A) Schematic presentation in which female CB6F1 (H-2K bxd ) mice received total body irradiation (TBI) (9 Gy) 1 day before transplant of bone marrow of female BALB/c (H-2K d ). Three weeks after transplant, subcutaneous tumor injection of MPC-11 wt (H-2K d ) was performed on the right flank, followed by IV Slamf7-BiTE (1.25 mg/kg bw per mouse) and Slamf7-mAb injection (10 mg/kg bw per mouse) twice a week. Day of tumor injection was defined as day 0. For CART experiments, cyclophosphamide was injected intraperitoneally 1 day before IV CART infusion (1e7 transduced T cells per mouse, ∼20% transduction efficiency), followed next day by subcutaneous tumor injection. Tumor size and bw were measured daily. (B) Successful bone marrow engraftment was controlled using blood samples from animals that received transplant for flow cytometry analysis staining with H-2K b (C57BL/6) and H-2K d (BALB/c), showing just a signal for H-2K d expression in the transplanted animals (right dot plot). Animals that did not receive transplant showed expression for both major histocompatibility complex-class I markers (left dot plot). (C) At the end of experiment, engraftment of CART cells was controlled using blood samples of animals from every group. Flow cytometry analysis showed CD3 + T cells expressing scFv Slamf7-1 of CART receptor on cell surface only in Slamf7-CART group. Representative histograms from 1 animal of each group are shown. (D) Treatment of CB6F1 mice that received transplant with Slamf7-mAb (violet triangle) and Slamf7-BiTE (red rectangle) showed reduced tumor growth compared with group receiving PBS or irrBiTE as control (orange rectangle/green circle). (E) Slamf7-BiTE, Slamf7-mAb and irrBiTE treatment has an impact on bw compared with PBS group, whereas Slamf7-BiTE had the strongest. (F) CB6F1 mice treated with Slamf7-CART cells (green line) had smaller tumors as in control groups (ntd T cells, PBS). (G) bw was not influenced by treatment with Slamf7-CART cells. Representative FACS profiles are shown. Results for tumor growth and bw are representative of 5 mice per group. The data represented as mean ± SEM, P < .05.

Article Snippet: VH and VL sequences of original rat hybridoma clone 3-1-4 against Slamf7 (Biogenes) were cloned to generate the different types: (A) Schematic of chimeric Slamf7-mAb, a mAb containing mouse Fc–immunoglobulin G2a (Fc-IgG2a) and scFv of a-Slamf7. (B) Purity and integrity of antibody batches were controlled by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showing intact heavy and light chains of Slamf7-mAb corresponding to correct sizes, isotype msIgG2a served as control. (C) Antibody binding to its target Slamf7 was analyzed by flow cytometry showing fluorescence signal in MPC-11 wt (blue profile), which is decreased when incubated with MPC-11 missing Slamf7 expression (MPC-11 kd), isotype control was used as negative control (red profile). (D) Schematic bispecific molecule with scFv parts of α-Slamf7 and α-msCD3 joined via flexible glycine-serine linker (Slamf7-BiTE). (E) Purified bispecific molecules were analyzed by western blot detecting myc-tag expressed on the carboxy terminus part of Slamf7- BiTE. (F) Flow cytometry analysis showed binding to msCD3 on T cells and to Slamf7 on MPC-11 wt ([red profile] isotype control; [blue profile] Slamf7-BiTE). (G) Schematic chimeric antigen T cell receptor was cloned containing sequences of CD8a hinge and transmembrane region, CD28-, 4-1BB–, and CD3ζ-signaling domain joined by scFv of Slamf7 (Slamf7-CAR). (H) After transduction of mouse T cells, they were labeled with an antibody detecting scFv Slamf7 on cell surfaces (blue), nontransduced T cells served as controls (red).

Techniques: Irradiation, Injection, Transduction, Flow Cytometry, Staining, Expressing

Journal: International Journal of Nanomedicine

Article Title: Recent Development and Applications of Polydopamine in Tissue Repair and Regeneration Biomaterials

doi: 10.2147/IJN.S437854

Figure Lengend Snippet: Selected and Representative PDA Related Materials in Tissue Repair and Regeneration

Article Snippet: ; permission conveyed through Copyright Clearance Center, Inc. ( E ) Schematic illustration showing the PDA coating of porous microspheres and the subsequent exosome adsorption via bioinspired dopamine chemistry.

Techniques: Adhesive, Modification, Construct

Journal: International Journal of Nanomedicine

Article Title: Recent Development and Applications of Polydopamine in Tissue Repair and Regeneration Biomaterials

doi: 10.2147/IJN.S437854

Figure Lengend Snippet: ( A ) Schematic illustration of the usage and probable working principle of cross-linker adhesion system. The tissue adhesive properties of PDA NPs exhibit a correlation between higher adhesive strength and smaller particle size (* p <0.05). Reproduced with the permission from Pandey N, Soto-Garcia L, Yaman S, et al. Polydopamine nanoparticles and hyaluronic acid hydrogels for mussel-inspired tissue adhesive nanocomposites. Biomater Adv . 2022;134:112,589. Copyright © 2022, with permission from Elsevier. ( B and C ) Porous carboxymethyl chitin microspheres with PDA (CMCHm-PDA) have better hemostatic performance (clotting time and blood loss) than a wide use commercial hemostatic agents Yunnan Baiyao ® (* p <0.05). Adapted from Carbohydrate Polymers , Volume 270, Leng F, Chen F, Jiang X. Modified porous carboxymethyl chitin microspheres by an organic solvent-free process for rapid hemostasis. Pages 118348. Copyright © 2021, with permission from Elsevier.

Article Snippet: ; permission conveyed through Copyright Clearance Center, Inc. ( E ) Schematic illustration showing the PDA coating of porous microspheres and the subsequent exosome adsorption via bioinspired dopamine chemistry.

Techniques: Adhesive, Coagulation, Modification, Solvent

Journal: International Journal of Nanomedicine

Article Title: Recent Development and Applications of Polydopamine in Tissue Repair and Regeneration Biomaterials

doi: 10.2147/IJN.S437854

Figure Lengend Snippet: ( A ) PDA/heparin nanoparticles were prepared to improve the encapsulation efficiency and control BMP-2 release behavior. Reproduced from Wu Y, Li X, Sun Y, et al. Multiscale design of stiffening and ROS scavenging hydrogels for the augmentation of mandibular bone regeneration. Bioact Mater . 2023;20:111–125. Copyright © 2022 KeAi, open access. ( B ) Fabrication of bone morphogenetic protein-2 (BMP2)-functionalized 3D-printed P34HB scaffold via polydopamine surface modification. ( C ) The amount of attached BMP2 was observed to increase with the increasing initial concentration of BMP-2. ( D ) Percentage of released BMP2 from BMP2-functionalized 3D-printed P34HB scaffolds during 30 days incubation in PBS buffer. ( B – D ) Used with permission of Royal Society of Chemistry, from Zhang X, Li J, Chen J, et al. Enhanced bone regeneration via PHA scaffolds coated with polydopamine-captured BMP2. J Mater Chem B . 022;10(32):6214–6227. ; permission conveyed through Copyright Clearance Center, Inc. ( E ) Schematic illustration showing the PDA coating of porous microspheres and the subsequent exosome adsorption via bioinspired dopamine chemistry. Gao YK, Yuan ZY, Yuan XJ, et al. Bioinspired porous microspheres for sustained hypoxic exosomes release and vascularized bone regeneration. Bioact Mater . 2022;14:377–388. doi:10.1016/j.bioactmat.2022.01.041. Copyright © 2022 KeAi, open access.

Article Snippet: ; permission conveyed through Copyright Clearance Center, Inc. ( E ) Schematic illustration showing the PDA coating of porous microspheres and the subsequent exosome adsorption via bioinspired dopamine chemistry.

Techniques: Encapsulation, Control, Modification, Concentration Assay, Incubation, Adsorption

Journal: International Journal of Nanomedicine

Article Title: Recent Development and Applications of Polydopamine in Tissue Repair and Regeneration Biomaterials

doi: 10.2147/IJN.S437854

Figure Lengend Snippet: ( A ) Schematic illustration of in vivo RA therapy effect of dimethylamino group (3- dimethylamino- 1- propylamine (M) or 3, 3- iminobis (N, N- dimethylaminopropyl) ( B )) modified polydopamine (DPs). The DPs were intra articular (IA) injected into the knee joint of CIA rat and strongly bound with cfDNA to lower the expression of inflammatory factors: MMP-13, TNF-α, IL-6 and IL-1β for RA therapy. Reproduced from Chen Y, Wang Y, Jiang X, et al. Dimethylamino group modified polydopamine nanoparticles with positive charges to scavenge cell-free DNA for rheumatoid arthritis therapy. Bioact Mater . 2022;18:409–420. Copyright © 2022 KeAi, open access. ( B ) Schematic illustration of PDA@MF NPs treatment for ROS-related kidney diseases. ( C ) Bio-distribution of PDA NPs and PDA@MF NPs was examined via in vivo imaging instruments. ( D ) H&E analysis of kidneys in different groups. Adapted from Zheng B, Deng G, Zheng J, et al. Self-polymerized polydopamine-based nanoparticles for acute kidney injury treatment through inhibiting oxidative damages and inflammatory. Int J Biochem Cell Biol . 2022;143:106141. Copyright © 2022, with permission from Elsevier.

Article Snippet: ; permission conveyed through Copyright Clearance Center, Inc. ( E ) Schematic illustration showing the PDA coating of porous microspheres and the subsequent exosome adsorption via bioinspired dopamine chemistry.

Techniques: In Vivo, Modification, Injection, Expressing, In Vivo Imaging