Journal: Nature medicine

Article Title: Paradoxical effects of obesity on T cell function during tumor progression and PD-1 checkpoint blockade

doi: 10.1038/s41591-018-0221-5

Figure Lengend Snippet: a) Leptin levels in non-obese (BMI<30, n=14) and obese (BMI≥30, n=12) healthy human volunteers. b) Linear regression analysis of leptin levels and PD-1 expression on CD8+ T cells in peripheral blood of human volunteers (n=21). c) Leptin levels and d) PD-1 expression on liver CD8+ T cells in 11–12-month-old control and DIO male mice (n=4/group). e) T1-weighted MRI demonstrating subcutaneous and visceral fat (fat appears bright white, white arrows) in 19-month-old ad-libitum (AL) fed and age-matched calorie restricted (CR) male mice. f) Frequency of PD-1 expressing CD8+ T cells in the liver of 19-month-old AL and CR male mice (n=4/group). g) Frequency of PD-1+CD8+ T cells from spleens of WT and db/db (9-month-old) male mice (n=3/group). Representative flow plots and frequency of PD-1 on h) splenic (n=7 in WT group, n=4 in db/db group) and i) liver (n=3 in WT group, n=2 in db/db group) CD8+ T cells of NSG mice 13 days post-transfer. a, c-d, f-i) Data are depicted as mean ±s.e.m., with all individual points shown. One-tailed unpaired Student’s t-test p-values shown. j) Representative flow plots and frequency of PD-1 expression on splenic CD8+ T cells ex vivo stimulated with αCD3, αCD3 and recombinant mouse leptin (αCD3+rmleptin), or unstimulated for 24 hrs (n=3 technical replicates). Data are depicted as mean ±s.e.m., with all individual points shown. One-way ANOVA with Tukey post-hoc test used to compare groups. *p<0.05, **p<0.01.

Article Snippet: 2×10 5 cells were plated in each well and stimulated with 2.5 μg/mL ConA with or without the addition of 10 nM recombinant human leptin (rhleptin, Gibco LifeTechnologies) for 48 hours at 37°C in a 96-well flat bottom plate.

Techniques: Expressing, One-tailed Test, Ex Vivo, Recombinant

Journal: Arthritis Research & Therapy

Article Title: Local leptin production in osteoarthritis subchondral osteoblasts may be responsible for their abnormal phenotypic expression

doi: 10.1186/ar2925

Figure Lengend Snippet: Modulation of alkaline phosphatase, osteocalcin and collagen type 1 in OA Ob by inactivating leptin signaling . Confluent normal and OA Ob were treated for their last two days of culture with either media alone containing 0.5% FBS with or without 1,25(OH) 2 D 3 (50 nM) as per indicated for the individual markers. Cells were treated with either exogenous leptin, antibodies against leptin, tyrphostin (AG490, 100 μM) or Piceatannol (Pce, 75 μM) for 30 minutes prior to the addition of 1,25(OH) 2 D 3 except for CICP that was performed in the absence of 1,25(OH) 2 D 3 . At the end of the 48 h incubation, the supernatant was kept for osteocalcin and for collagen production, and cells were lyzed in ALPase buffer prior to measuring alkaline phosphatase activity by substrate hydrolysis. A ) Results of alkaline phosphatase activity for normal OB; B ) Results of alkaline phosphatase activity for OA OB; C ) Results of osteocalcin release by OA Ob; D ) Results of CICP production; E ) Confluent OA Ob were incubated in Ham's F12/DMEM media without serum and containing 1% ITS. Cells were treated with or without exogenous leptin, tyrphostin (AG490, 100 μM) or Piceatannol (Pce, 75 μM) for their last 48 hours of culture. Results of TGF-β1 levels in supernatants are shown. The results are the mean ± SEM of n = 4 normal and n = 9 OA Ob preparations.

Article Snippet: For the determiniation of phenotypic markers, cells were either treated with 1 μg/ml recombinant human leptin (rhleptin, Calbiochem, San Diego, California, USA), 10 μg/ml recombinant human leptin R/Fc chimera (R&D Systems, Minneapolis, MN, USA) that neutralizes the activity of rhleptin, 100 μM Tyrphostin (AG490, Sigma-Aldrich), 75 μM piceatannol (Pce, Sigma-Aldrich), or the vehicle.

Techniques: Incubation, Activity Assay

Journal: Arthritis Research & Therapy

Article Title: Local leptin production in osteoarthritis subchondral osteoblasts may be responsible for their abnormal phenotypic expression

doi: 10.1186/ar2925

Figure Lengend Snippet: Modulation of alkaline phosphatase, osteocalcin and collagen type 1 in OA Ob by inactivating leptin signaling . Confluent normal and OA Ob were treated for their last two days of culture with either media alone containing 0.5% FBS with or without 1,25(OH) 2 D 3 (50 nM) as per indicated for the individual markers. Cells were treated with either exogenous leptin, antibodies against leptin, tyrphostin (AG490, 100 μM) or Piceatannol (Pce, 75 μM) for 30 minutes prior to the addition of 1,25(OH) 2 D 3 except for CICP that was performed in the absence of 1,25(OH) 2 D 3 . At the end of the 48 h incubation, the supernatant was kept for osteocalcin and for collagen production, and cells were lyzed in ALPase buffer prior to measuring alkaline phosphatase activity by substrate hydrolysis. A ) Results of alkaline phosphatase activity for normal OB; B ) Results of alkaline phosphatase activity for OA OB; C ) Results of osteocalcin release by OA Ob; D ) Results of CICP production; E ) Confluent OA Ob were incubated in Ham's F12/DMEM media without serum and containing 1% ITS. Cells were treated with or without exogenous leptin, tyrphostin (AG490, 100 μM) or Piceatannol (Pce, 75 μM) for their last 48 hours of culture. Results of TGF-β1 levels in supernatants are shown. The results are the mean ± SEM of n = 4 normal and n = 9 OA Ob preparations.

Article Snippet: For the determiniation of phenotypic markers, cells were either treated with 1 μg/ml recombinant human leptin (rhleptin, Calbiochem, San Diego, California, USA), 10 μg/ml recombinant human leptin R/Fc chimera (R&D Systems, Minneapolis, MN, USA) that neutralizes the activity of rhleptin, 100 μM Tyrphostin (AG490, Sigma-Aldrich), 75 μM piceatannol (Pce, Sigma-Aldrich), or the vehicle.

Techniques: Incubation, Activity Assay

Journal: British Journal of Cancer

Article Title: Leptin-mediated regulation of ICAM-1 is Rho/ROCK dependent and enhances gastric cancer cell migration

doi: 10.1038/bjc.2014.70

Figure Lengend Snippet: Leptin enhanced the expression of ICAM-1 in GC cells. AGS and MKN-45 cells were treated with leptin (100 ng ml −1 ) for 24 h. The expression of ICAM-1 was measured by RT–PCR ( A ), real time RT–PCR ( B ) and WB ( C ). The cell surface expression of ICAM-1 was measured by flow cytometry ( D ), and the level of sICAM-1 in supernatant was detected by ELISA ( E ). All data shown are expressed as mean±s.d. of three independent experiments. * P <0.05, ** P <0.01.

Article Snippet: Recombinant human leptin (rhleptin), mouse anti-human ICAM-1 mAb for flow cytometry, sICAM-1 commercial ELISA (enzyme-linked immunosorbent assay) kit were obtained from R&D Systems (Minneapolis, MN, USA).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Flow Cytometry, Enzyme-linked Immunosorbent Assay

Journal: British Journal of Cancer

Article Title: Leptin-mediated regulation of ICAM-1 is Rho/ROCK dependent and enhances gastric cancer cell migration

doi: 10.1038/bjc.2014.70

Figure Lengend Snippet: Rho/ROCK pathway was involved in the leptin-induced ICAM-1 expression in GC cells. AGS and MKN-45 cells were treated with leptin (100 ng ml −1 ) for various times (0, 15, 30, and 60 min). The RhoA activity was detected by ELISA ( A ), and the phosphorylation levels of ROCK in cell lysates were determined by WB ( B ). In a separate experiment, AGS and MKN-45 cells were treated in the absence (control) or presence of leptin (100 ng ml −1 ) after 1 h pretreatment with C3 transferase (0.25 μ g ml −1 ) or Y-27632 (3.3 μ M). The inhibitors were present for the duration of leptin treatment. After 24 h, the levels of ICAM-1 ( C ), cell surface ICAM-1 ( D ), and sICAM-1 ( E ) in the presence or absence of C3 transferase or Y-27632 were determined. One representative result out of three independent experiments is shown. β -actin was used as an internal control. The results are shown as mean±s.d. * P <0.05 vs control, ** P <0.01 vs control, # P <0.05 vs control, ## P <0.01 vs the group of leptin.

Article Snippet: Recombinant human leptin (rhleptin), mouse anti-human ICAM-1 mAb for flow cytometry, sICAM-1 commercial ELISA (enzyme-linked immunosorbent assay) kit were obtained from R&D Systems (Minneapolis, MN, USA).

Techniques: Expressing, Activity Assay, Enzyme-linked Immunosorbent Assay