Journal: Frontiers in Medicine

Article Title: Glucocorticoid Effects on Tissue Residing Immune Cells in Giant Cell Arteritis: Importance of GM-CSF

doi: 10.3389/fmed.2021.709404

Figure Lengend Snippet: Reduction of CCL19 and CCL21 expression under glucocorticoid therapy. Temporal artery sections of patients were stained with (A) anti-CCL19 mAb and (B) anti-CCL21 mAB, respectively, and numbers of positive cells were determined in three sections. For each patient and control patient, positive cells were counted in three different temporal artery sections.

Article Snippet: Polyclonal Rabbit anti-cow S100 (strongly cross-reacts with human S100; DAKO Glostrup, Denmark); goat anti-mouse CCL19/MIP-3β (reacting crosswise with the human CCL19), goat anti-human CCL21/6Ckine, monoclonal mouse anti-human CD163, monoclonal anti-human GM-CSF (all from R&D Systems, Minneapolis, USA); monoclonal mouse anti-human HLA-DR (BD Biosciences, San Jose, USA); Cy2-conjugated goat anti-mouse IgG, Cy2-conjugated goat anti-rabbit IgG (both from Jackson West Grove, USA); Dako EnVision Doublestain System, AEC+ high sensitivity substrate-chromogen system, liquid DAB substrate-chromogen system (all from DAKO Carpinteria, USA); ApopTag Plus Peroxidase in situ Apoptosis Detection Kit, ApopTag Red Kit (both from Qbiogene, Illkirch, France).

Techniques: Expressing, Staining, Control

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Dendritic cells modified with 6Ckine/IFNγ fusion gene induce specific cytotoxic T lymphocytes in vitro

doi: 10.1007/s00262-007-0327-y

Figure Lengend Snippet: The expression of 6Ckine, IFNγ and 6Ckine/IFNγ fusion protein in HepG2 cells mediated by adenovirus vector (L, Ad-LacZ; C, Ad-6Ckine; I, Ad-IFNγ; F, Ad-6Ckine/IFNγ; M, marker; +, positive control; N, mock-transfection). The HepG2 cells were transfected with recombinant adenoviruses at 10 MOI or mock-transfection (N) for 48 h, and then the expression and biological activity of 6Ckine, IFNγ and 6Ckine/IFNγ were assessed. a RT-PCR analysis for 6Ckine, IFNγ and 6Ckine/IFNγ mRNA. β-actin was used as an internal control (6Ckine 405 bp, IFNγ 501 bp, 6Ckine/IFNγ 849 bp, T-bet 450 bp, and β-actin 245 bp). b Immunofluorescent staining (×200). Red fluorescence indicated positive cells. c Western blot analysis. d Interferon activity was analyzed following the protocol from NICPBP. e 6Ckine function was detected by microchemotaxis assay

Article Snippet: The RPMI 1640 containing 5% human AB serum with or without 0.1 ng/μL of recombinant human 6Ckine protein (R&D Systems, USA) was used as positive or negative controls, respectively.

Techniques: Expressing, Plasmid Preparation, Marker, Positive Control, Transfection, Recombinant, Activity Assay, Reverse Transcription Polymerase Chain Reaction, Control, Staining, Fluorescence, Western Blot

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Dendritic cells modified with 6Ckine/IFNγ fusion gene induce specific cytotoxic T lymphocytes in vitro

doi: 10.1007/s00262-007-0327-y

Figure Lengend Snippet: The influences of recombinant adenoviruses infection on the endocytosis of DCs (C, Ad-6Ckine-DCs; I, Ad-IFNγ-DCs; F, Ad-6Ckine/IFNγ-DCs; L, Ad-LacZ-DCs; N, Non-transfected DCs. The percentage of FITC-Dextran uptake by the DCs was assessed with FACS analysis after 24 h of adenoviruses infection. The data represent three separate experiments. P > 0.05 when compared among all groups

Article Snippet: The RPMI 1640 containing 5% human AB serum with or without 0.1 ng/μL of recombinant human 6Ckine protein (R&D Systems, USA) was used as positive or negative controls, respectively.

Techniques: Recombinant, Infection, Transfection

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Dendritic cells modified with 6Ckine/IFNγ fusion gene induce specific cytotoxic T lymphocytes in vitro

doi: 10.1007/s00262-007-0327-y

Figure Lengend Snippet: Chemokines secretion of DCs and the recruiting function to T cells. a and b DCs were pulsed with HepG2 cell lysates, and incubated at 37°C for 24 h. Supernatants from each DC group were harvested, and then 6Ckine (a) and RANTES (b) concentration was measured by the specific ELISA assay. The amounts of chemokines from five separate experiments were shown in nanograms per million DCs 48 h after transfection. c Microchemotaxis assay. The above-mentioned culture supernatant was collected, and the chemotaxis activity to naïve T cells was analyzed by a Transwells® apparatus. RPMI 1640 containing 5% human AB serum with or without 0.1 ng/μl of recombinant human 6Ckine protein was used as positive or negative control individually. Supernatant pre-incubated with recombinant human anti-6Ckine monoclonal antibody was used for chemotaxis blocking assay (results represent three independent experiments). *P < 0.05 when compared with other groups; **P < 0.05 compared with medium control; ***P < 0.05 compared with Ad-6Ckine/IFNγ-DCs and Ad-6Ckine-DCs, P > 0.05 compared with Ad-IFNγ-DCs Ad-LacZ-DCs and NTDCs

Article Snippet: The RPMI 1640 containing 5% human AB serum with or without 0.1 ng/μL of recombinant human 6Ckine protein (R&D Systems, USA) was used as positive or negative controls, respectively.

Techniques: Incubation, Concentration Assay, Enzyme-linked Immunosorbent Assay, Transfection, Chemotaxis Assay, Activity Assay, Recombinant, Negative Control, Blocking Assay, Control

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Dendritic cells modified with 6Ckine/IFNγ fusion gene induce specific cytotoxic T lymphocytes in vitro

doi: 10.1007/s00262-007-0327-y

Figure Lengend Snippet: Cytokine secretion of gene modified-DCs (C, Ad-6Ckine-DCs; I, Ad-IFNγ-DCs; F, Ad-6Ckine/IFNγ-DCs; L, Ad-LacZ-DCs; N, non-transfected-DCs). DCs were pulsed with 100 μg/ml of HepG2 cell lysates, and incubated for 24 h. The supernatant from each DC group was harvested, and then the level of IFNγ (a), IL-12p70 (b) and IL-10 (c) was detected by LiquidChip assay, while TNFα (d) were measured by ELISA. The values referring to the cytokine production were obtained from the average of five separate experiments. *P < 0.01 when compared with Ad-LacZ-DCs and non-transfected-DCs

Article Snippet: The RPMI 1640 containing 5% human AB serum with or without 0.1 ng/μL of recombinant human 6Ckine protein (R&D Systems, USA) was used as positive or negative controls, respectively.

Techniques: Modification, Transfection, Incubation, Enzyme-linked Immunosorbent Assay

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Dendritic cells modified with 6Ckine/IFNγ fusion gene induce specific cytotoxic T lymphocytes in vitro

doi: 10.1007/s00262-007-0327-y

Figure Lengend Snippet: Ad-6Ckine/IFNγ-DCs promote T cell activation. a Autologous T cell reaction. Following inactivation with mitomycin, antigen-loaded DCs were co-cultured with autologous T cells at a DC:T ratio of 1:20 for 96 h. 1 μCi/well of 3H-TdR was pulsed for 16 h. The values of counts per minute (cpm) were measured with a LS6500 liquid scintillation counter. Data were expressed as the stimulating indices (SIs). The results were obtained from five independent experiments. b IL-2 production of T cells. The IL-2 concentrations in the supernatants of co-cultured DCs and T cells were measured by ELISA. The results were obtained from six independent experiments. c The T-bet expression in T cells. The T-bet mRNA levels in T cells co-incubated with DCs were measured by semi-quantitative RT-PCR. The data were obtained from five separate experiments. d One of the representive experiments of RT-PCR. *P < 0.05 when compared with Ad-LacZ-DCs and NTDCs; **P < 0.05 when compared with any of other four groups

Article Snippet: The RPMI 1640 containing 5% human AB serum with or without 0.1 ng/μL of recombinant human 6Ckine protein (R&D Systems, USA) was used as positive or negative controls, respectively.

Techniques: Activation Assay, Cell Culture, Enzyme-linked Immunosorbent Assay, Expressing, Incubation, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Dendritic cells modified with 6Ckine/IFNγ fusion gene induce specific cytotoxic T lymphocytes in vitro

doi: 10.1007/s00262-007-0327-y

Figure Lengend Snippet: Cytotoxic function of T cells stimulated with Ad-6Ckine/IFNγ-DCs (C, Ad-6Ckine-DC group; I, Ad-IFNγ-DC group; F, Ad-6Ckine/IFNγ-DC group; L, Ad-LacZ-DC group; N, NTDC group). Autologous T cells were co-cultured with antigen-pulsed DCs at a DC:T ratio of 1:20 for 5 days. The cytotoxicity of activated T cells was analyzed with a CytoTox 96® Non-Radioactive Cytotoxicity Assay kit (Promega, USA), HepG2 or LoVo cells were used as the target cells (E:T = 20:1 or 50:1). The data were obtained from five separate experiments. *P < 0.05 when compared with Ad-LacZ-DCs and NTDCs groups; **P < 0.05 when compared with other four groups, respectively

Article Snippet: The RPMI 1640 containing 5% human AB serum with or without 0.1 ng/μL of recombinant human 6Ckine protein (R&D Systems, USA) was used as positive or negative controls, respectively.

Techniques: Cell Culture, Cytotoxicity Assay

Journal: Biomedical microdevices

Article Title: Confinement dependent chemotaxis in two-photon polymerized linear migration constructs with highly definable concentration gradients.

doi: 10.1007/s10544-015-9937-x

Figure Lengend Snippet: Fig. 6 Mean DC migration velocity towards the CCL21 source through 400 μm long channels of different cross-section (10×10 μm2, 15× 15 μm2, 20×20 μm2) filled with fibrillar collagen or through fibrillar collagen in the immediate vicinity of the channel structures. Constructs with 10×10 μm2 channels were tested without or with 500 nm wide slits in the channel ceilings. The asterisks show significant differences in ve- locity (p<0.01). Errors bars show the standard error of the mean (n≥5)

Article Snippet: NaHCO3 solution (7.5 %), 10x MEM solution, and collagenase (Clostridium Histolyticum, cell culture tested) were from Sigma-Aldrich, PureCol (collagen I) solution from Advanced Biomatrix (San Diego, CA), and recombinant human CCL21 from R&D Systems (Minneapolis, MN).

Techniques: Migration, Construct

Journal: Biomedical microdevices

Article Title: Confinement dependent chemotaxis in two-photon polymerized linear migration constructs with highly definable concentration gradients.

doi: 10.1007/s10544-015-9937-x

Figure Lengend Snippet: Fig. 5 Cell blockage of the microchannel results in strong accentuation of the chemokine gradient. Finite element modeling of the CCL21 chemokine concentration profile during passage of a 40 μm long cell at a speed of 6 μm/min through 10×10 μm2 channels with a length of (a+b) 400 μm or (c+d) 200 μm. The CCL21 diffusion constant is approximately 10·10-11 m2/s in aqueous media (b+d), and may be reduced to 3·10-11 m2/s through fibrillar collagen (a+c). The colored curves show the concentration profile at the indicated time points of the simulation

Article Snippet: NaHCO3 solution (7.5 %), 10x MEM solution, and collagenase (Clostridium Histolyticum, cell culture tested) were from Sigma-Aldrich, PureCol (collagen I) solution from Advanced Biomatrix (San Diego, CA), and recombinant human CCL21 from R&D Systems (Minneapolis, MN).

Techniques: Concentration Assay, Diffusion-based Assay

Journal: Biomedical microdevices

Article Title: Confinement dependent chemotaxis in two-photon polymerized linear migration constructs with highly definable concentration gradients.

doi: 10.1007/s10544-015-9937-x

Figure Lengend Snippet: Fig. 4 Mean DC migration velocities towards the CCL21 source through 10×10 μm2 channels of different lengths differ significantly (p=0.04). Error bars show the standard error of the mean (n≥4)

Article Snippet: NaHCO3 solution (7.5 %), 10x MEM solution, and collagenase (Clostridium Histolyticum, cell culture tested) were from Sigma-Aldrich, PureCol (collagen I) solution from Advanced Biomatrix (San Diego, CA), and recombinant human CCL21 from R&D Systems (Minneapolis, MN).

Techniques: Migration

Journal: Biomedical microdevices

Article Title: Confinement dependent chemotaxis in two-photon polymerized linear migration constructs with highly definable concentration gradients.

doi: 10.1007/s10544-015-9937-x

Figure Lengend Snippet: Fig. 7 Nanoslits in the channel ceiling strongly limit accentuation of the chemokine gradient in channels blocked by cells. (a+b) FEM of the CCL21 concentration profile during passage of a 20 μm long cell at a speed of 4.5 μm/min through 400 μm long channels of cross-section 20×20 μm2 using a diffusion constant of (a) 3·10-

Article Snippet: NaHCO3 solution (7.5 %), 10x MEM solution, and collagenase (Clostridium Histolyticum, cell culture tested) were from Sigma-Aldrich, PureCol (collagen I) solution from Advanced Biomatrix (San Diego, CA), and recombinant human CCL21 from R&D Systems (Minneapolis, MN).

Techniques: Concentration Assay, Diffusion-based Assay

Journal: Biomedical microdevices

Article Title: Confinement dependent chemotaxis in two-photon polymerized linear migration constructs with highly definable concentration gradients.

doi: 10.1007/s10544-015-9937-x

Figure Lengend Snippet: Fig. 8 Measured mean DC migration velocities monotonically depend on the modeled chemokine concentration across the cell ends within statistical error, independent of the degree of confinement. Vertical error bars show the standard error of the mean (n≥4; reproduced from Figs. 4 and 6). The concentration difference across a single cell, ΔCCCL21, is the mean of the values modeled using CCL21 diffusion constants of 3·10−11 m2/s and 10·10−11 m2/s (Figs. 5 and 7) with the horizontal error bars showing the span in modeled ΔCCCL21 values. The uncertainty in ΔCCCL21 for a cell in unstructured collagen is estimated to be 0.5 nM. The labels refer to the channel cross-section with all channel lengths being 400 μm except for the B10 μm / 200 μm long^ data point

Article Snippet: NaHCO3 solution (7.5 %), 10x MEM solution, and collagenase (Clostridium Histolyticum, cell culture tested) were from Sigma-Aldrich, PureCol (collagen I) solution from Advanced Biomatrix (San Diego, CA), and recombinant human CCL21 from R&D Systems (Minneapolis, MN).

Techniques: Migration, Concentration Assay, Diffusion-based Assay

Journal: Cancer Science

Article Title: CXCL12 promotes CCR7 ligand–mediated breast cancer cell invasion and migration toward lymphatic vessels

doi: 10.1111/cas.15293

Figure Lengend Snippet: The effects of CXCL12 on CCR7 ligand–dependent MDA231 cell migration. A, CXCL12‐induced cell migration assay was performed using MDA231 cells in the presence or absence of CXCL12 at indicated concentrations. The number of migrated cells to the lower wells in response to CXCL12 was analyzed. Graphs represent means ± SD of the number of cells in eight random fields per membrane in triplicate assay. * p < 0.05 by Student's t test. B, CCL21‐induced cell migration of MDA‐MB‐231. The assay was performed in the presence of CCL21 in the lower wells at indicated concentrations. One‐way ANOVA; * p < 0.05, ** p < 0.01. C, The effect of CXCL12 on MDA231 cell migration induced by a suboptimal concentration of CCL21 (50 ng/mL). The assay was performed under the indicated concentrations of CXCL12 and CCL21 added to the upper or the lower wells. One‐way ANOVA; ** p < 0.01. D, The effect of CXCL12 on CCL21‐induced cell migration in the presence of anti‐CXCR4 neutralizing antibody. CXCL12 (2.5 μg/mL) was added to the upper wells with or without anti‐CXCR4 mAb or an isotype control immunoglobulin (10 μg/mL). Mean ± SD (Student's t test; * p < 0.05, ** p < 0.01, n = 3)

Article Snippet: For detection of CCL21 and LYVE‐1 in 3D lymphatic networks, 1 μg/mL biotinylated goat anti‐human CCL21 pAb (BAF366, R&D Systems), horseradish peroxidase–conjugated streptavidin, Alexa Fluor488 Tyramide (B40953, Thermo Fisher Scientific), and 5 μg/mL eFluor660‐conjugated anti‐LYVE‐1 mAb (clone ALY7, eBioscience) were used.

Techniques: Migration, Cell Migration Assay, Membrane, Concentration Assay, Control

Journal: Cancer Science

Article Title: CXCL12 promotes CCR7 ligand–mediated breast cancer cell invasion and migration toward lymphatic vessels

doi: 10.1111/cas.15293

Figure Lengend Snippet: The effects of CXCL12 on CCR7‐mediated MDA231 cell invasion to the collagen gel. A, Parental MDA231 cells, Meta‐1, and Meta‐2 cells were pretreated with 1 μg/mL CXCL12 and subjected to a collagen gel cell invasion assay under a suboptimal concentration (10 ng/mL) of CCL21. After 15 h incubation, the number of cells on the surface of the collagen matrix and those migrated into the matrix was counted, and the percentage of cell invasion was analyzed. Data represent the mean ± SD of three independent experiments, each performed in triplicate wells (Student's t test; * p < 0.05). B, Meta‐1 cells were pretreated with CXCL12 in the presence of 10 μg/mL anti‐CCR7 mAb (left panel), anti‐CXCR4 mAb, or isotype control and were then applied to a collagen gel containing CCL21

Article Snippet: For detection of CCL21 and LYVE‐1 in 3D lymphatic networks, 1 μg/mL biotinylated goat anti‐human CCL21 pAb (BAF366, R&D Systems), horseradish peroxidase–conjugated streptavidin, Alexa Fluor488 Tyramide (B40953, Thermo Fisher Scientific), and 5 μg/mL eFluor660‐conjugated anti‐LYVE‐1 mAb (clone ALY7, eBioscience) were used.

Techniques: Invasion Assay, Concentration Assay, Incubation, Control

Journal: Cancer Science

Article Title: CXCL12 promotes CCR7 ligand–mediated breast cancer cell invasion and migration toward lymphatic vessels

doi: 10.1111/cas.15293

Figure Lengend Snippet: Expression of CXCL12 and CCL21 in MDA231‐derived tumor. A, Parental MDA231 cells (1 × 10 6 ) were injected into the thoracic mammary fat pad of female SCID mice at 9 weeks of age ( n = 3). A sham operation was performed on the contralateral side. Twelve weeks after injection, primary tumors were dissected. Serial sections of fresh‐frozen tumor specimens from tumor xenograft were subjected to immunohistochemical staining with anti‐CXCL12 (green, upper panel) and anti‐human p53 mAb (red, upper panel) or anti‐LYVE‐1 (green, lower panel) and anti‐CCL21 (red, lower panel) antibodies. Scale bar, 100 µm. B, Upper row: RT‐PCR analysis of CXCL12 and CCL21 in the parental MDA231 cell line, MDA231‐derived tumor tissue, and C57BL/6 mouse lymph node using oligonucleotide primer pairs common to mouse and human sequences. GAPDH were used as an endogenous control. Bottom: the tumor‐derived nucleotide sequence of the RT‐PCR products was determined and compared with the species‐specific nucleotide positions of the CXCL12 and CCL21 genes. C, Localization of CXCL12 expression (red) and alpha‐smooth muscle actin (α‐SMA)‐positive cancer‐associated fibroblast (green) in MDA231‐derived tumor tissue

Article Snippet: For detection of CCL21 and LYVE‐1 in 3D lymphatic networks, 1 μg/mL biotinylated goat anti‐human CCL21 pAb (BAF366, R&D Systems), horseradish peroxidase–conjugated streptavidin, Alexa Fluor488 Tyramide (B40953, Thermo Fisher Scientific), and 5 μg/mL eFluor660‐conjugated anti‐LYVE‐1 mAb (clone ALY7, eBioscience) were used.

Techniques: Expressing, Derivative Assay, Injection, Immunohistochemical staining, Staining, Reverse Transcription Polymerase Chain Reaction, Control, Sequencing

Journal: Cancer Science

Article Title: CXCL12 promotes CCR7 ligand–mediated breast cancer cell invasion and migration toward lymphatic vessels

doi: 10.1111/cas.15293

Figure Lengend Snippet: The effects of CXCL12 on CCR7 homodimerization, ligand binding, and the plasma membrane localization. A, CCR7 homodimer formation after treatment with CXCL12 in MDA231 cells. The levels of bioluminescence signals are shown for cells transfected with combinations of CCR7‐CGLuc and CCR7‐NGLuc in the presence of BSA or CXCL12. A representative experiment from at least three independent experiments is shown. Data represent mean ± SD ( n = 4). * p < 0.05 by Student's t test. B, Confocal microscopic images of MDA‐R7/X4 cells treated with or without CXCL12 for 30 min, fixed, and stained with recombinant CCL19‐Fc, biotin‐anti‐human IgG, and Alexa Fluor 647–conjugated streptavidin. The expressions of CCR7‐EGFP (green) and CCL19‐Fc (magenta) are shown. Insets show high magnification of membrane ruffles. The images were analyzed to obtain the percentage of the cells with CCL19‐Fc binding. Scale bar, 30 µm. *** p < 0.01 by Student's t test. C, Luciferase complementation assay using CCR7‐Nluc and β‐arrestin 2‐Cluc. CCL21 was added 30 min after treatment of cells with CXCL12 or a solvent, and the luminescence signal was measured at each time point indicated. Error bars indicate standard error of the mean. D, MDA‐R7/X4 cells were stimulated with or without CXCL12 for 30 min, fixed, and stained with Alexa647‐phalloidin. Fluorescence images were captured by a confocal microscopy. The expression of CCR7 (green), CXCR4 (red), and F‐actin (white) are shown. White arrowheads show CXCR4‐ and CCR7‐enriched membrane ruffles. Scale bar, 10 µm. E, The average score of CXCR4/CCR7‐enriched membrane ruffles of each cell. The cells were treated with the indicated concentrations of CXCL12 in the presence or absence of CXCR4‐specific antagonist AMD3100. The ruffling index was evaluated as 0 = no ruffles, 1 = ruffles covering less than 25% of the peripheral area, and 2 = ruffles covering more than 25% of the peripheral area. The statistical difference was determined by one‐way ANOVA and depicted with *** p < 0.01

Article Snippet: For detection of CCL21 and LYVE‐1 in 3D lymphatic networks, 1 μg/mL biotinylated goat anti‐human CCL21 pAb (BAF366, R&D Systems), horseradish peroxidase–conjugated streptavidin, Alexa Fluor488 Tyramide (B40953, Thermo Fisher Scientific), and 5 μg/mL eFluor660‐conjugated anti‐LYVE‐1 mAb (clone ALY7, eBioscience) were used.

Techniques: Ligand Binding Assay, Clinical Proteomics, Membrane, Transfection, Staining, Recombinant, Binding Assay, Luciferase, Solvent, Fluorescence, Confocal Microscopy, Expressing