Journal: Journal of Exercise Rehabilitation

Article Title: Effect of combined intervention of exercise and autologous bone marrow stromal cell transplantation on neurotrophic factors and pain-related cascades over time after sciatic nerve injury

doi: 10.12965/jer.2244006.003

Figure Lengend Snippet: At early stage of axonal regeneration, combined intervention of treadmill exercise and autologous bone marrow stromal cell (BMSC) transplantation regulates induction levels of nuclear factor (NF)-κB, interleukin (IL)-6, and tumor necrosis factor (TNF)-α in the ipsilateral lumbar 4 and lumbar 5 dorsal root ganglion. (A) Western blot results on TNF-α, NF-κB, and IL-6 at 1 and 2 weeks after sciatic nerve injury. (B) Quantitative graph on TNF-α/actin ratio at week 1 and 2 later. (C) Quantitative graph on NF-κB/actin ratio at week 1 and 2 later. (D) Quantitative graph on IL-6/actin ratio at week 1 and 2 later. CONT, normal control; SS, sedentary group; SE, low-intensity treadmill exercise group; SB, BMSC transplantation group; SBE, BMSC transplantation group+treadmill exercise. * P <0.05, ** P <0.01, *** P <0.001 vs. CONT group. # P <0.05, ## P <0.01, ### P <0.001 vs. SS group. †† P <0.01 vs. SE group.

Article Snippet: Protein (20 μg) was used for Western blot analysis using anti-TrkB rabbit polyclonal antibody (1:1,000, Cell Signaling Biotechnology, Danvers, MA, USA), anti-β-actin mouse monoclonal antibody (1:1,000, Santa Cruz Biotechnology), anti-NF-κB mouse monoclonal antibody (1:1,000, Santa Cruz Biotechnology), anti-CNTF mouse monoclonal antibody (1:1,000, Cell Signaling Biotechnology), anti-NGF mouse monoclonal antibody (1:1,000, Cell Signaling Biotechnology), anti-BDNF mouse monoclonal antibodies (1:1,000, Santa Cruz Biotechnology), anti-TNF-α rabbit polyclonal antibody (1:1,000, Sino Biological, Wayne, PA, USA), anti-IL-6 rabbit polyclonal antibody (1:1,000, GeneTex Inc., Irvine, CA, USA).

Techniques: Transplantation Assay, Western Blot

Journal: Journal of Exercise Rehabilitation

Article Title: Effect of combined intervention of exercise and autologous bone marrow stromal cell transplantation on neurotrophic factors and pain-related cascades over time after sciatic nerve injury

doi: 10.12965/jer.2244006.003

Figure Lengend Snippet: At late stage of axonal regeneration, combined intervention of treadmill exercise and autologous bone marrow stromal cell (BMSC) transplantation regulates activation of proinflammatory cytokines in the ipsilateral lumbar 4 and lumbar 5 dorsal root ganglion. (A) Western blot results on tumor necrosis factor (TNF)-α, nuclear factor (NF)-κB, and interleukin (IL)-6 at 3 and 5 weeks after sciatic nerve injury. (B) Quantitative graph on TNF-α/actin ratio at week 3 and 5 later. (C) Quantitative graph on NF-κB/actin ratio at week 3 and 5 later. (D) Quantitative graph on IL-6/actin ratio at week 3 and 5 later. CONT, normal control; SS, sedentary group; SE, low-intensity treadmill exercise group; SB, BMSC transplantation group; SBE, BMSC transplantation group+treadmill exercise. * P <0.05, *** P <0.001 vs. CONT group. # P <0.05, ## P <0.01, ### P <0.001 vs. SS group.

Article Snippet: Protein (20 μg) was used for Western blot analysis using anti-TrkB rabbit polyclonal antibody (1:1,000, Cell Signaling Biotechnology, Danvers, MA, USA), anti-β-actin mouse monoclonal antibody (1:1,000, Santa Cruz Biotechnology), anti-NF-κB mouse monoclonal antibody (1:1,000, Santa Cruz Biotechnology), anti-CNTF mouse monoclonal antibody (1:1,000, Cell Signaling Biotechnology), anti-NGF mouse monoclonal antibody (1:1,000, Cell Signaling Biotechnology), anti-BDNF mouse monoclonal antibodies (1:1,000, Santa Cruz Biotechnology), anti-TNF-α rabbit polyclonal antibody (1:1,000, Sino Biological, Wayne, PA, USA), anti-IL-6 rabbit polyclonal antibody (1:1,000, GeneTex Inc., Irvine, CA, USA).

Techniques: Transplantation Assay, Activation Assay, Western Blot

Journal: Biology

Article Title: Neuroprotective, Antioxidant and Anti-Inflammatory Effect of Greek Pomegranate Seed Oil on N2a Neuroblastoma Cells and Mild Cognitive Impairment Patients

doi: 10.3390/biology14050548

Figure Lengend Snippet: The effects of PSO on LPS-induced expression of inflammatory and oxidative stress markers in N2a cells treated with PSO. ( a ) Representative Western blot images showing the expression levels of inducible nitric oxide synthase (iNOS), interleukin-1β (IL-1β), and tumor necrosis factor-alpha (TNF-α). Groups include Control, LPS (1 µg/ mL), LPS co-treatment with different concentrations of PSO (0.2, 10, and 25 µg/mL), and PSO (0.2, 10, and 25 µg/mL) (see ). ( b ) Quantification of total iNOS protein levels normalized to β-actin. ( c ) Quantification of IL-1β protein levels normalized to β-actin. ( d ) Quantification of TNF-α protein levels normalized to β-actin. Statistical significance: * p < 0.05; ** p < 0.01; **** p < 0.0001, with asterisk (*) indicating the comparison versus control group; # p < 0.05, ## p < 0.01, with (#) indicating the comparison with the LPS-treated group. Data are presented as the mean ± SD.

Article Snippet: For the detection of APP, Aβ 42 , tau, p-tau181, iNOS, IL-1β, TNF-α and SOD1, the following primary antibodies were employed: anti-APP mouse monoclonal antibody (#60342-1-Ig, Proteintech, Manchester, UK), anti-Aβ 42 rabbit monoclonal antibody (#14974, Cell Signaling Technology, Danvers, MA, USA), anti-rabbit tau monoclonal antibody (#46687, Cell Signaling Technology, Danvers, MA, USA), anti-p-tau181 rabbit monoclonal antibody (#12885, Cell Signaling Technology, Danvers, MA, USA), anti-iNOS rabbit polyclonal antibody (#18985-1-AP, Proteintech, Manchester, UK), anti-IL-1β rabbit monoclonal antibody (#L0328Y, Cusabio, Houston, TX, USA), anti-TNF-α mouse monoclonal antibody (#sc-52746, Santa Cruz Biotechnology, Heidelberg, Germany), and an anti-SOD1 rabbit polyclonal antibody (#10269-1-AP, Proteintech, Manchester, UK).

Techniques: Expressing, Western Blot, Control, Comparison

Journal: Biology

Article Title: Neuroprotective, Antioxidant and Anti-Inflammatory Effect of Greek Pomegranate Seed Oil on N2a Neuroblastoma Cells and Mild Cognitive Impairment Patients

doi: 10.3390/biology14050548

Figure Lengend Snippet: ( a ) Levels of TNF-α as measured from the implemented ELISA on patients with MCI who did not receive PSO (Controls) (n = 23) at the beginning and at the end of the trial compared to MCI patients who received no pharmacological therapy with PSO (n = 31). Results are provided with individual values scatter plots which mean values ± standard deviation (SD). All samples were analyzed at least in duplicates. Statistical analyses were performed with Graph Pad Prism 8.0 statistical software. t -test was used to examine possible differences between groups. * Differences between follow-up and baseline levels. # Differences between follow-up (12 mo.) levels of the Control group and the PSO group. *** p < 0.001, with (*) indicating differences between 12 mo. and baseline levels. ## p < 0.01, with (#) indicating differences between follow-up (12 mo.) levels of the Control and PSO groups. ( b ) Correlation analysis of the levels of p-tau181 against the levels of TNF-a in blood serum of MCI patients employed in the current study. ( c ) Correlation analysis of the ratio of p-tau181/Aβ 42 against the levels of TNF-α in blood serum of MCI patients of PSO study. The correlations were evaluated using Spearman’s rank correlation coefficients (r) and their corresponding p values. Analyses were run and graphed separately. Statistical analyses were performed with Graph Pad Prism 8.0 statistical software.

Article Snippet: For the detection of APP, Aβ 42 , tau, p-tau181, iNOS, IL-1β, TNF-α and SOD1, the following primary antibodies were employed: anti-APP mouse monoclonal antibody (#60342-1-Ig, Proteintech, Manchester, UK), anti-Aβ 42 rabbit monoclonal antibody (#14974, Cell Signaling Technology, Danvers, MA, USA), anti-rabbit tau monoclonal antibody (#46687, Cell Signaling Technology, Danvers, MA, USA), anti-p-tau181 rabbit monoclonal antibody (#12885, Cell Signaling Technology, Danvers, MA, USA), anti-iNOS rabbit polyclonal antibody (#18985-1-AP, Proteintech, Manchester, UK), anti-IL-1β rabbit monoclonal antibody (#L0328Y, Cusabio, Houston, TX, USA), anti-TNF-α mouse monoclonal antibody (#sc-52746, Santa Cruz Biotechnology, Heidelberg, Germany), and an anti-SOD1 rabbit polyclonal antibody (#10269-1-AP, Proteintech, Manchester, UK).

Techniques: Enzyme-linked Immunosorbent Assay, Standard Deviation, Software, Control

Journal: Journal of Traditional and Complementary Medicine

Article Title: Antcin-H, a natural triterpene derived from Antrodia cinnamomea , ameliorates dextran sulfate sodium-induced colitis in mice by inhibiting the NLRP3 inflammasome

doi: 10.1016/j.jtcme.2024.03.016

Figure Lengend Snippet: Antcin-H ameliorates colonic inflammation in DSS-treated mice. Protein expression levels of IL-6 (A), TNF-α (B), MCP-1 (C), CXCL1 (D) and active MPO (E) in colon were analyzed by ELISA. (F) Protein expression levels of COX-2 in colon were analyzed by Western blotting. The Western blotting images are representative of separate experiments, and the column diagram represents the fold change compared with the H 2 O + vehicle group normalized to actin analyzed by Image J. ** p < 0.01 and *** p < 0.001 compared to DSS + vehicle group.

Article Snippet: Antibodies against IL-1β and C-X-C motif chemokine ligand-1 (CXCL1) as well as ELISA kits for IL-1β, IL-6, tumor necrosis factor-α (TNF-α), monocyte chemoattractant protein-1 (MCP-1), and active myeloperoxidase (MPO) were purchased from R&D Systems (Minneapolis, MN).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: JCI insight

Article Title: Balanced engagement of activating and inhibitory receptors mitigates human NK cell exhaustion.

doi: 10.1172/jci.insight.150079

Figure Lengend Snippet: Figure 1. Prolonged stimulation through activating receptors induces NK cell exhaustion. (A) Schematic representing the in vitro model of exhaus- tion. Agonists of NKp46 (anti-NKp46) and NKG2D (MICA and MICB) were adsorbed onto tissue culture plates and used to stimulate NK cells for 7 days. Plate-bound isotype IgG served as a control. Both groups received 1 ng/mL IL-15. (B) NK cells harvested from isotype-coated and exhaustion plates (day 7) were incubated with K-562 targets for 4 hours (E/T: 2:1). Cytokine production (IFN-γ and TNF-α) and degranulation (CD107a) were measured via flow cytometry. Isotype NK cells (top row) in blue, exhausted NK cells (bottom row) in red. E/T, effector/target. (C–E) Quantification of cytokine production and degranulation as percentage of parent population (live, CD3–CD56+ cells) and mean fluorescence intensity (MFI) (n = 4). Paired t tests were used for comparisons. **P < 0.01; ***P < 0.001; ****P < 0.0001.

Article Snippet: Fluorochrome-conjugated antibodies were purchased from BioLegend (CD3 [catalog 317330], CD56 [catalog 362510], NKG2D [catalog 320806], CD96 [catalog 338418], granzyme B [catalog 515408], perforin [catalog 308112], IFN-γ [catalog 502538], TNF-α [catalog 502932], CD107a [catalog 328606], TIM-3 [catalog 345018], LAG-3 [catalog 369320], Ki67 [catalog 350515], human CD45 [catalog 304041], mouse CD45 [catalog 103149]); BD Biosciences (anti-CD3 [catalog 562406], CD16 [catalog 560717]); R&D Systems (TIGIT [catalog FAB7898A], TRAIL [catalog FAB687G]); Invitrogen (Fas-L [catalog 12-9919-42]; and Miltenyi Biotec (PD-1 [catalog 130-117-384], isotype IgG2b-PE [catalog 130-092-215], NKG2A [catalog 130-113-563]).

Techniques: In Vitro, Control, Incubation, Flow Cytometry, Fluorescence

Journal: JCI insight

Article Title: Balanced engagement of activating and inhibitory receptors mitigates human NK cell exhaustion.

doi: 10.1172/jci.insight.150079

Figure Lengend Snippet: Figure 5. Prolonged stimulation through activating receptors results in functional and proliferative defects in vivo. (A) Schematic representing exper- imental design: sublethally irradiated NSG mice were injected (i.v.) with HL-60-Luc2 cells and injected (i.v.) with NK cells 3 days later. NK cells had been incubated for 7 days on plates with either isotype IgG (control) or anti-NKp46 and MICA/B as previously described. (B) Fourteen days after NK cell injection, blood was drawn, and NK cells were counted via flow cytometry. NK cells were human CD45+CD56+CD3–. One-way ANOVA was used for comparisons (n = 8). *P ≤ 0.05. (C) HL-60 tumor burden was tracked via bioluminescent imaging (BLI), with day 21 data pictured. One-way ANOVA was used for comparisons (n = 8). ***P < 0.001. (D and E) Fourteen days after NK cell injection, NK cells were restimulated with K-562 leukemia cells for 4 hours as previously described. Ki67, IFN-γ, TNF-α, and CD107a expression was assessed via flow cytometry (D). Paired t tests were used for comparisons (n = 8). *P ≤ 0.05. Representative data depict Ki67 and IFN-γ expression following K-562 restimulation (E).

Article Snippet: Fluorochrome-conjugated antibodies were purchased from BioLegend (CD3 [catalog 317330], CD56 [catalog 362510], NKG2D [catalog 320806], CD96 [catalog 338418], granzyme B [catalog 515408], perforin [catalog 308112], IFN-γ [catalog 502538], TNF-α [catalog 502932], CD107a [catalog 328606], TIM-3 [catalog 345018], LAG-3 [catalog 369320], Ki67 [catalog 350515], human CD45 [catalog 304041], mouse CD45 [catalog 103149]); BD Biosciences (anti-CD3 [catalog 562406], CD16 [catalog 560717]); R&D Systems (TIGIT [catalog FAB7898A], TRAIL [catalog FAB687G]); Invitrogen (Fas-L [catalog 12-9919-42]; and Miltenyi Biotec (PD-1 [catalog 130-117-384], isotype IgG2b-PE [catalog 130-092-215], NKG2A [catalog 130-113-563]).

Techniques: Functional Assay, In Vivo, Irradiation, Injection, Incubation, Control, Flow Cytometry, Imaging, Expressing

Journal: JCI insight

Article Title: Balanced engagement of activating and inhibitory receptors mitigates human NK cell exhaustion.

doi: 10.1172/jci.insight.150079

Figure Lengend Snippet: Figure 6. Engagement of NKG2A during exhaustion rescues defects in cytotoxicity and cytokine production. (A) NK cells were incubated with both positive (anti-NKp46 plus MICA/B) and inhibitory (anti-NKG2A) stimuli or with positive stimuli plus isotype IgG. NK cells plated on isotype-bound cells served as a control for stimulation through the low-affinity Fc receptor CD16. NK cells were incubated with K-562 target cells (E/T 2:1), and live cell killing assays were performed using the IncuCyte platform as previously described. (B) Percentage of live targets was quantified for each group of cells at multiple time points (n = 6). One-way ANOVA with Dunnett’s multiple comparisons was used for statistical analysis (with each time point compared independently). (C and D) Expression of granzyme B (C) (n = 5) and TRAIL (D) (n = 4) was assessed via flow cytometry. One-way ANOVA with multiple comparisons was used for statistical analysis. (E) Irradiated P815 cells were coated with either anti-NKp30 and anti-NKG2A or anti-NKp30 and isotype IgG. P815 coated in isotype alone served as a negative control. NK cells were incubated with P815 target cells (E/T 2:1) for 5 days and subsequently stimulated with PMA/ionomycin for 4 hours. Following stimulation, cytokine production was assessed via flow cytometry (n = 5). One-way ANOVA with multiple comparisons was used for statistical analysis (with IFN-γ and TNF-α compared independently). *P ≤ 0.05; **P < 0.01.

Article Snippet: Fluorochrome-conjugated antibodies were purchased from BioLegend (CD3 [catalog 317330], CD56 [catalog 362510], NKG2D [catalog 320806], CD96 [catalog 338418], granzyme B [catalog 515408], perforin [catalog 308112], IFN-γ [catalog 502538], TNF-α [catalog 502932], CD107a [catalog 328606], TIM-3 [catalog 345018], LAG-3 [catalog 369320], Ki67 [catalog 350515], human CD45 [catalog 304041], mouse CD45 [catalog 103149]); BD Biosciences (anti-CD3 [catalog 562406], CD16 [catalog 560717]); R&D Systems (TIGIT [catalog FAB7898A], TRAIL [catalog FAB687G]); Invitrogen (Fas-L [catalog 12-9919-42]; and Miltenyi Biotec (PD-1 [catalog 130-117-384], isotype IgG2b-PE [catalog 130-092-215], NKG2A [catalog 130-113-563]).

Techniques: Incubation, Control, Expressing, Flow Cytometry, Irradiation, Negative Control