Journal: Oncology letters

Article Title: Cytotoxic chemotherapy reduces T cell trafficking to the spleen by downregulating the expression of C-C motif chemokine ligand 21 and C-C motif chemokine ligand 19.

doi: 10.3892/ol.2018.9287

Figure Lengend Snippet: Figure 2. Expression of CCL21 and CCL19 in the spleen was reduced following chemotherapy. (A) Spleen cryostat sections from control mice or test mice (treated with Cis, Gem or 5‑FU) were stained for CCL21 (red) and fibroblast antibodies (ER‑TR7; green), which were imaged by confocal microscopy. Scale bar, 200 µm. Expression of (B) CCL21 and (C) CCL19 in the spleens of control and test mice in tissue homogenates was further detected by ELISA. One‑way analysis of variance, followed by Dunnett's multiple comparisons test, was used for all analyses. Error bars represent the standard error of the mean. *P<0.05, **P<0.01, ***P<0.001. n.s., not significant. Con, control; Cis, cisplatin; Gem, gemcitabine; 5‑FU, fluorouracil, CCL21, C‑C motif chemokine ligand 21; CCL19, C‑C motif chemokine ligand 19.

Article Snippet: CCL21 (catalog no. DY457; R&D Systems, Inc., Minneapolis, MN, USA) and CCL19 (cat. no. DY440; R&D Systems, Inc.) were detected using a DuoSet ELISA development kit, according to the manufacturer's protocol.

Techniques: Expressing, Control, Staining, Confocal Microscopy, Enzyme-linked Immunosorbent Assay

Journal: Oncology letters

Article Title: Cytotoxic chemotherapy reduces T cell trafficking to the spleen by downregulating the expression of C-C motif chemokine ligand 21 and C-C motif chemokine ligand 19.

doi: 10.3892/ol.2018.9287

Figure Lengend Snippet: Figure 3. Chemotherapy does not affect the homing ability of T cells to the lymph nodes or chemokine expression in the lymph nodes. (A) The localization of CFSE+ cells in the lymph nodes of control or test mice was ascertained 8 h after T cells transfer by immunofluorescence. Scale bar, 200 µm. (B) Counts of CFSE+ cells in the in the lymph nodes of control or test mice. (C) Lymph node cryostat sections from control or test mice were stained for CCL21 (red) and fibroblast antibodies (ER‑TR7; green). (D) Chemokine expression in the lymph nodes of control or test mice was quantified by reverse transcription‑quantitative polymerase chain reaction. One‑way analysis of variance, followed by Dunnett's multiple comparisons test, was used for all analyses. Error bars represent the standard error of the mean. n.s., not significant. CCL21, C‑C motif chemokine ligand 21; CCL19, C‑C motif chemokine ligand 19; CFSE, carboxyfluorescein succinimidyl ester.

Article Snippet: CCL21 (catalog no. DY457; R&D Systems, Inc., Minneapolis, MN, USA) and CCL19 (cat. no. DY440; R&D Systems, Inc.) were detected using a DuoSet ELISA development kit, according to the manufacturer's protocol.

Techniques: Expressing, Control, Immunofluorescence, Staining, Polymerase Chain Reaction

Journal: Oncology letters

Article Title: Cytotoxic chemotherapy reduces T cell trafficking to the spleen by downregulating the expression of C-C motif chemokine ligand 21 and C-C motif chemokine ligand 19.

doi: 10.3892/ol.2018.9287

Figure Lengend Snippet: Figure 4. Cytotoxic chemotherapy affects the function and survival of FRCs. (A) Spleen cryostat sections from control mice or chemotherapy‑treated mice (Cis, Gem or 5‑FU) were stained for TUNEL (red) and gp38 (green), which were imaged by confocal microscopy. Scale bar, 75 µm. (B) FRCs were co‑cultured with Cis, Gem or 5‑FU in vitro for 24 h, and then the expression in Annexin V+7‑AAD+ cells were detected via flow cytometry. (C) The percentages of Annexin V+7‑AAD+ FRCs in different groups. (D) Chemokine expression in the FRCs after 24 h of chemotherapeutic culture in vitro was quantified by reverse transcription‑quantitative polymerase chain reaction. One‑way analysis of variance, followed by Dunnett's multiple comparisons test, was used for all analyses. Error bars represent the standard error of the mean. *P<0.05; **P<0.01; ***P<0.001; n.s., not significant. FRCs, fibroblastic reticular cells; Con, control; Cis, cisplatin; Gem, gemcitabine; 5‑FU, fluorouracil CCL21, C‑C motif chemokine ligand 21; CCL19, C‑C motif chemokine ligand 19.

Article Snippet: CCL21 (catalog no. DY457; R&D Systems, Inc., Minneapolis, MN, USA) and CCL19 (cat. no. DY440; R&D Systems, Inc.) were detected using a DuoSet ELISA development kit, according to the manufacturer's protocol.

Techniques: Control, Staining, TUNEL Assay, Confocal Microscopy, In Vitro, Expressing, Flow Cytometry, Polymerase Chain Reaction

Journal: PLoS ONE

Article Title: Obesity Impairs Lymphatic Fluid Transport and Dendritic Cell Migration to Lymph Nodes

doi: 10.1371/journal.pone.0070703

Figure Lengend Snippet: A. Representative florescent micrograph (2.5× magnification) of inguinal lymph nodes harvested from control and obese mice and stained for B cells (B220; purple) and T cells (CD3; green). Note loss of follicular pattern and disorganization in obese mice. B. Representative florescent micrographs (2.5x) of inguinal lymph nodes harvested from control (left panels) and obese (right panels) wild-type mice stained for CCL21 (red) and DAPI (blue). CCL21 expression is shown on top panels with DAPI overlay on the bottom. Dotted circled represent follicular regions. Note loss of CCL21 expression gradients in obese animals. C. Representative whole mount immunofluorescent staining (Green = LYVE-1; Red = CCL21; Blue = DAPI) of ear sections from control (top) and obese (bottom). Sections are shown at 63× magnification. LYVE-1 staining is shown on the left; CCL21 is the middle panel; overlay is shown on the right panels. Dashed lines in the middle panel is the region of LYVE-1 vessel. D. Mean area of LYVE-1/CCL21 co-localization (presented as a percentage of LYVE-1 area overall) in control and obese mice. E., F. G., H. Flow cytometry analysis of T-helper cells (CD45+/CD3+/CD4+; E ), cytotoxic T cells (CD45 + /CD3 + /CD8 + ; F ); B cells (CD45 + /B220 + /CD19 + ; G ), and macrophages (CD45 +/ /B220 + /CD11b + ; H ). Mean percentage of cells from 4–5 animals per group is presented.

Article Snippet: Additionally, we analyzed the expression patterns of CCL21, a critical regulator of cell migration to the lymph node and architectural organization, , using a Rat anti-mouse mAb against CCL21 (MAB457, R&D Systems).

Techniques: Control, Staining, Expressing, Flow Cytometry

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