Journal: Journal of neuroinflammation

Article Title: Complex molecular and functional outcomes of single versus sequential cytokine stimulation of rat microglia.

doi: 10.1186/s12974-016-0531-9

Figure Lengend Snippet: Fig. 3 Expression of ROS-related molecules and contribution of NOX enzymes to myelin phagocytosis and ROS production. a Expression of ROS-related molecules. Rat microglia were stimulated with cytokines for 24 h with or without a subsequent 6-h exposure to myelin (plus or minus sign indicates presence/absence of myelin), as in Figs. 1a and 2a. b Effect of NOX inhibition on phagocytosis of myelin fragments. The activation treatments, assay, and normalization of data were the same as Fig. 2b, but now comparing the pan-NOX inhibitor, 5 μM VAS2870. c Effect of NOX inhibition on ROS levels in microglia that were treated as in Fig. 2b. As above, ROS levels were normalized to unstimulated microglia without myelin (dashed line), data are expressed as mean ± SEM (n = 6 individual cultures), and results were analyzed by two-way ANOVA with Bonferroni’s post hoc test. The comparisons are as follows: asterisk unstimulated (CTL) versus different activation states in the absence of myelin, dagger sign unstimulated (CTL) versus different activation states in the presence of myelin, number sign effects of myelin (a) or VAS2870 (b, c) within a particular activation state. One symbol p < 0.05, two symbols p < 0.01, three symbols p < 0.001

Article Snippet: To induce different activation states, microglia were exposed to recombinant rat cytokines (R&D Systems Inc., Minneapolis, MN).

Techniques: Expressing, Inhibition, Activation Assay

Journal: Journal of neuroinflammation

Article Title: Complex molecular and functional outcomes of single versus sequential cytokine stimulation of rat microglia.

doi: 10.1186/s12974-016-0531-9

Figure Lengend Snippet: Fig. 8 Transcript expression of K+ channels and SOCE-related genes is affected by the microglial activation state and myelin phagocytosis. a Single cytokines with or without myelin. Microglia were stimulated for 24 h with 20 ng/mL IFN-γ + 50 ng/mL TNF-α (I + T) or 20 ng/mL IL-4, and then treated with 25 μg/mL myelin for 6 h. b M2a→M1 paradigm. Microglia were treated with 20 ng/mL IL-4 for 2 h followed by I + T for 22 h. c M1→M2 paradigm. Cells were treated with I + T for 2 h followed by IL-4 (M1→M2a) or 20 ng/mL IL-10 (M1→M2c) for 22 h. All data are expressed as mRNA counts in 200 ng total RNA (mean ± SEM) for six individual cultures. Data were analyzed by one-way ANOVA with Tukey’s post hoc test. The dashed lines in b and c indicate expression levels in unstimulated (control) microglia. Comparisons are as follows: asterisk differences from control microglia, dagger sign CTL versus different activation states in the presence of myelin, number sign effects of myelin within a particular activation state (a) or effects of a second stimulus on the first stimulus (b and c). One symbol p < 0.05, two symbols p < 0.01, three symbols p < 0.001

Article Snippet: To induce different activation states, microglia were exposed to recombinant rat cytokines (R&D Systems Inc., Minneapolis, MN).

Techniques: Expressing, Activation Assay, Control

Journal: Pharmaceutics

Article Title: CD25-Targeted Aptamer–Drug Conjugate for the Treatment of CD25-Expressing Hematological Malignancies

doi: 10.3390/pharmaceutics18020217

Figure Lengend Snippet: The structure and characterization of CD25 aptamer. ( A ) The secondary structure of the CD25 aptamer was estimated by RNAstructure software v6.4 of Mathews Lab. The sequence of CD25 aptamer is shown with modifications indicated (Z, 5-[ N -(1-naphthylmethyl)carboxamide]-2′-deoxyuridine; N me , 2′- O -methyl nucleosides). ( B ) The binding affinity of CD25 aptamer to the CD25 recombinant protein was determined by the BLI method. Ni-NTA probes were immobilized with His-tag CD25 protein, followed by incubation with the aptamer 125 (green), 250 (yellow), or 500 nM (red). The binding signal over time is shown. Kd is expressed as mean ± SD. ( C ) The cells were stained with biotin-aptamer combined with NeutrAvidin DyLight 650 or APC-conjugated anti-CD25 monoclonal antibody (mAb), and then the specificity of the antibody and the aptamer to the cells was examined by flow cytometry (control for aptamer: DyLight 650 only; control for antibody: not stained).

Article Snippet: A 5 μg/mL recombinant human CD25 His-tagged protein solution (R&D Systems, Minneapolis, MN, USA) was immobilized onto Ni-NTA probes (Gator Bio).

Techniques: Software, Sequencing, Binding Assay, Recombinant, Incubation, Staining, Flow Cytometry, Control

Journal: Pharmaceutics

Article Title: CD25-Targeted Aptamer–Drug Conjugate for the Treatment of CD25-Expressing Hematological Malignancies

doi: 10.3390/pharmaceutics18020217

Figure Lengend Snippet: The CD25 aptamer specifically binds and internalizes into CD25-positive cells. ( A ) The cell internalization of Cy-5-labeled CD25 aptamer (red) was visualized for 0, 1, and 4 h using confocal fluorescence microscopy using CD25-positive Karpas299 and CD25-negative Daudi cell lines. The nuclei were stained with DAPI (blue). ( B ) The rate of internalized CD25 aptamer was determined using the MFI value of flow cytometry analysis at 0 to 240 min. ( C ) Cellular trafficking of the CD25 aptamer. Fluorescence microscopy visualized the lysosomal delivery of pHrodo-labeled CD25 aptamer (red) for up to 4 h.

Article Snippet: A 5 μg/mL recombinant human CD25 His-tagged protein solution (R&D Systems, Minneapolis, MN, USA) was immobilized onto Ni-NTA probes (Gator Bio).

Techniques: Labeling, Fluorescence, Microscopy, Staining, Flow Cytometry

Journal: Pharmaceutics

Article Title: CD25-Targeted Aptamer–Drug Conjugate for the Treatment of CD25-Expressing Hematological Malignancies

doi: 10.3390/pharmaceutics18020217

Figure Lengend Snippet: Effects of the CD25 aptamer on CD25/IL-2 signaling. ( A ) A competitive binding assay was performed by adding biotinylated IL-2 proteins to 96-well plates coated with CD25 proteins, in the presence or absence of the CD25 aptamer. ( B , C ) Karpas299 cells were pre-treated with the CD25 aptamer for 30 min, followed by stimulation with IL-2 for 15 min. The levels of pSTAT5 protein and TGF-β mRNA were analyzed by Western blotting and quantitative RT-PCR, respectively. ( D , E ) HuT78 cells were treated with IL-2 in the presence of either the CD25 aptamer or the anti-CD25 antibody Daclizumab. The expression of pSTAT5 was then assessed by Western blot analysis. ( F ) HuT78 cells were pre-treated with the indicated concentrations of the CD25 aptamer, stimulated with IL-2, and the secretion of IL-4 was measured as described in the Materials and Methods. Results are expressed as mean ± SD. ** p < 0.01, *** p < 0.001.

Article Snippet: A 5 μg/mL recombinant human CD25 His-tagged protein solution (R&D Systems, Minneapolis, MN, USA) was immobilized onto Ni-NTA probes (Gator Bio).

Techniques: Competitive Binding Assay, Western Blot, Quantitative RT-PCR, Expressing

Journal: Pharmaceutics

Article Title: CD25-Targeted Aptamer–Drug Conjugate for the Treatment of CD25-Expressing Hematological Malignancies

doi: 10.3390/pharmaceutics18020217

Figure Lengend Snippet: In vitro cytotoxicity of CD25 aptamer–MMAE conjugates. Karpas299 and Daudi Cells were treated with CD25-ApDC MMAE1 ( A ) or CD25-ApDC MMAE3 ( B ) for 3 days, after which cell viability was assessed, as described in the Materials and Methods. ( C ) Karpas299 and HuT78 cells were co-cultured at a 1:1 ratio for 24 h, stained with anti-CD4 and anti-CD25 antibodies, and analyzed by flow cytometry. The co-cultured cells were subsequently incubated with 45 nM CD25-ApDC MMAE3 for 24, 48, or 72 h, and analyzed again using flow cytometry. ( D ) Cells were treated with increasing concentrations of MMAE or CD25-ApDC MMAE3 for 24 h. Western blot analysis of total PRAP, cleaved PARP, total caspase-3, and cleaved caspase-3 was performed. ( E ) The cell cycle was analyzed using flow cytometry after staining with PI. Results are expressed as mean ±SD. * p < 0.05, *** p < 0.001.

Article Snippet: A 5 μg/mL recombinant human CD25 His-tagged protein solution (R&D Systems, Minneapolis, MN, USA) was immobilized onto Ni-NTA probes (Gator Bio).

Techniques: In Vitro, Cell Culture, Staining, Flow Cytometry, Incubation, Western Blot

Journal: Pharmaceutics

Article Title: CD25-Targeted Aptamer–Drug Conjugate for the Treatment of CD25-Expressing Hematological Malignancies

doi: 10.3390/pharmaceutics18020217

Figure Lengend Snippet: In vivo antitumor efficacy of CD25 aptamer–MMAE conjugates in xenograft models. Tumor growth curves were generated by measuring tumor volumes in Karpas299 tumor-bearing mice following intravenous administration of CD25 aptamer–MMAE conjugates when tumors reached an average volume of 150 mm 3 . ( A ) Red arrows indicate the time points of injection with CD25-ApDC MMAE1 at doses of 1, 2, or 4 mg/kg. ( B ) Mice were treated either four times with 4 mg/kg (red arrows) or twice with 12 mg/kg (green arrows). ( C ) Tumor-bearing mice received a single dose of 0.4, 0.8, or 1.6 mg/kg, or were administered doses three times (once per week) with 0.8 or 1.6 mg/kg CD25-ApDC MMAE3 . Data are the mean tumor volume ±SE of eight animals per group. ( D ) NOD/SCID mice were systemically inoculated with Karpas299 cells and treated intravenously with the indicated dose of CD25-ApDC MMAE1 or CD25-ApDC MMAE3 twice per week for 3 weeks. Kaplan–Meier survival curves show the percentage of survival for each group, with statistical comparison performed using log-rank tests.

Article Snippet: A 5 μg/mL recombinant human CD25 His-tagged protein solution (R&D Systems, Minneapolis, MN, USA) was immobilized onto Ni-NTA probes (Gator Bio).

Techniques: In Vivo, Generated, Injection, Comparison