Journal: Cell stem cell

Article Title: Distinct Bone Marrow Sources of Pleiotrophin Control Hematopoietic Stem Cell Maintenance and Regeneration

doi: 10.1016/j.stem.2018.07.003

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit Anti-Leptin receptor Polyclonal Antibody, FITC , Bioss Antibodies , Cat. bs-0961R-FITC; RRID:AB_11074292.

Techniques: Staining, Recombinant, Expressing, Enzyme-linked Immunosorbent Assay, Transgenic Assay, Software, Imaging

Journal: Annals of Clinical and Translational Neurology

Article Title: Obesity and the risk of Multiple Sclerosis. The role of Leptin

doi: 10.1002/acn3.51291

Figure Lengend Snippet: (A) Body mass index (BMI) positively correlated with serum leptin levels. (B) Serum leptin levels were higher in obese patients compared to overweight and normal/underweight patients. Similarly, overweight patients showed increased serum leptin levels compared to normal/underweight patients. No statistically significant differences were found when MS and control patients were compared in each subgroup. Each circle represents values from a single individual. Data are presented as mean ± SEM. Kruskal–Wallis test of one‐way ANOVA and post hoc data analysis applying Dunn's multiple comparison test, performed to analyze differences between groups. ** P < 0.01, **** P < 0.0001.

Article Snippet: For blocking experiments human leptin receptor polyclonal antibody (MyBioSource, San Diego CA) was used at a final concentration of 20 μ g/mL; control was an irrelevant isotype‐matched antibody (R&D Systems).

Techniques: Control, Comparison

Journal: Annals of Clinical and Translational Neurology

Article Title: Obesity and the risk of Multiple Sclerosis. The role of Leptin

doi: 10.1002/acn3.51291

Figure Lengend Snippet: Lymphocyte subpopulations were isolated from fresh PBMC by magnetic separation using specific isolation kits, and leptin receptor expression was measured by RT‐PCR (A) and flow cytometry (B). Cells were cultured (5 × 10 4 cells/well) in round bottom 96‐well plates for 72 h and stimulated as follows: T cells were stimulated with soluble anti‐CD3 and soluble anti‐CD28 (both at 5 µ g/mL concentration); B cells using PMA (5 ng/mL) plus ionomycin (1 μmol/L); and monocytes were activated with 100 ng/mL of LPS. For mRNA expression, data were normalized to the amount of GAPDH, as a control housekeeping gene, using the Pfaffl method. ⁶⁵ Intra‐assay precision was determined in three repeats within one LightCycler run, and interassay variation was investigated in three different experimental runs. Variations for intertest and intratest experiments were between 5% and 7% in all cases. Flow cytometry data were acquired as described in Material and Methods. The results are expressed as Mean Fluorescence Intensity (MFI) of leptin receptor expression (Ob‐Rb; CD295). In all lymphocyte subpopulations, activation significantly increased leptin receptor expression compared to resting cells. Data represent the mean ± SEM from 25 MS patients. * P < 0.05, ** P < 0.01, **** P < 0.0001.

Article Snippet: For blocking experiments human leptin receptor polyclonal antibody (MyBioSource, San Diego CA) was used at a final concentration of 20 μ g/mL; control was an irrelevant isotype‐matched antibody (R&D Systems).

Techniques: Isolation, Expressing, Reverse Transcription Polymerase Chain Reaction, Flow Cytometry, Cell Culture, Concentration Assay, Control, Intra Assay, Fluorescence, Activation Assay

Journal: Annals of Clinical and Translational Neurology

Article Title: Obesity and the risk of Multiple Sclerosis. The role of Leptin

doi: 10.1002/acn3.51291

Figure Lengend Snippet: (A) Concentration curve of antiapoptotic effect mediated by leptin. MBP 83–102 peptide‐specific T cells were cultured in serum‐free culture medium for 24 h, in the presence and absence of different concentrations of leptin. Maximal antiapoptotic effects were seen at 250 ng/mL. Inhibition of apoptosis was leptin dependent, since leptin receptor silencing using siRNA abrogated the leptin effect. Jurkat T cells were used as positive control, with the maximum inhibition of apoptosis reached at concentrations significantly lower than those necessary to prevent apoptosis in autoreactive T cells. Data represent mean values ± SEM of triplicate cultures from five independent experiments. (B) Leptin decreases apoptosis induction in MBP 83–102 , peptide‐specific T cells from MS patients. Three days after Ag stimulation, autoreactive T cells were cultured for 24 h in serum‐free medium, with and without leptin (250 ng/mL). Both antileptin receptor and control antibodies were added at a final concentration of 20 µ g/mL, 30 min before leptin (250 ng/mL). The antiapoptotic effect of leptin was blocked by antileptin receptor mAb, but not modified by an isotype control antibody. MBP 83–102 peptide‐specific T cells in which leptin receptor was silenced using siRNA technique were included in this assay as a negative control. (C) Leptin at a concentration of 50 ng/mL decreases apoptosis induction in Jurkat T cells, cultured in conditions similar to MBP 83–102 peptide‐specific T cells. Data represent mean ± SEM from seven different experiments performed in triplicate (D) Leptin inhibited steroid‐induced apoptosis in MBP 83–102 peptide‐specific T cells. Three days after Ag stimulation, autoreactive T cells were cultured for 24 h with 10 −6 mol/L hydrocortisone in the presence and in the absence of leptin (250 ng/mL). As in the previous experiment, the antiapoptotic effect of leptin was blocked by antileptin receptor mAb, but not modified by an isotype control antibody. In panels A to D, evidence of apoptosis was evaluated by FITC‐Annexin V and PI staining and analyzed by flow cytometry. (E) Expression of the antiapoptotic molecule Bcl‐2 significantly increased in the presence of leptin. This effect was abrogated in the presence of antileptin receptor mAb, but not modified by an isotype control antibody. (F) Leptin promoted proliferation of both MBP 83–102 , and MOG 63–87 peptide‐specific T cells stimulated with increasing concentrations of the cognate antigen. Cell proliferation was assessed by measuring 3 H thymidine incorporation during the final 12 h of a 60 h culture. As in previous experiments, both antileptin receptor and control antibodies were added at a final concentration of 20 µ g/mL, 30 min before adding leptin (250 ng/mL). Data represent mean ± SEM from five different experiments. For panels B, D, and E each circle represents an individual MBP 83–102 ‐specific T‐cell line, isolated from a total of 15 MS patients. Data represent mean ± SEM. In all experiments, PBMC were isolated from patients with normal BMI, to make sure BMI did not affect cell responsiveness to leptin. Kruskal–Wallis test of one‐way ANOVA and post hoc data analysis applying Dunn's multiple comparison test were performed to analyze differences between groups. LEPR: Leptin receptor; Anti LEPR ab = antileptin receptor antibody **** P < 0.0001.

Article Snippet: For blocking experiments human leptin receptor polyclonal antibody (MyBioSource, San Diego CA) was used at a final concentration of 20 μ g/mL; control was an irrelevant isotype‐matched antibody (R&D Systems).

Techniques: Concentration Assay, Cell Culture, Inhibition, Positive Control, Control, Modification, Negative Control, Staining, Flow Cytometry, Expressing, Isolation, Comparison

Journal: Annals of Clinical and Translational Neurology

Article Title: Obesity and the risk of Multiple Sclerosis. The role of Leptin

doi: 10.1002/acn3.51291

Figure Lengend Snippet: (A) Obese MS patients showed significantly higher numbers of IL‐2, IL‐6, IL‐15, IL‐17, IFN‐γ, and TNF‐α producing cells compared to overweight and normal/underweight MS subjects. Likewise, overweight patients showed a higher number of cytokine‐producing cells compared to normal/underweight MS patients. ** P < 0.01, *** P < 0.001, **** P < 0.0001 (B) MBP 83–102 T‐cell lines isolated from normal weight MS patients, were stimulated with the cognate peptide in the presence of leptin, significantly increased the production of IL‐2, IL‐6, IL‐15, IL‐17, IFN‐γ, and TNF‐α producing cells. These effects were overcome by the addition of an antileptin receptor mAb, but not modified by an isotype control antibody. Stimulation with Ovalbumin 323–339 (20 µ g/mL), as nonrelevant peptide, showed values similar to background. In all experiments, cytokine production was assessed using ELISPOT assays. The specific number of cytokine‐producing cells was calculated by subtracting the numbers of spots obtained in 0 Ag background control cultures, from the number of spots obtained in cultures exposed to stimulating Ag. In both panels, data correspond to the number of spots per 10 5 PBMC from 30 patients, and results represent mean ± SEM. Kruskal–Wallis test of one‐way ANOVA and post hoc data analysis applying Dunn's multiple comparison test were performed to analyze differences between groups. **** P < 0.0001 Anti LEPR ab = antileptin receptor antibody.

Article Snippet: For blocking experiments human leptin receptor polyclonal antibody (MyBioSource, San Diego CA) was used at a final concentration of 20 μ g/mL; control was an irrelevant isotype‐matched antibody (R&D Systems).

Techniques: Isolation, Modification, Control, Enzyme-linked Immunospot, Comparison

Journal: Annals of Clinical and Translational Neurology

Article Title: Obesity and the risk of Multiple Sclerosis. The role of Leptin

doi: 10.1002/acn3.51291

Figure Lengend Snippet: (A) Percentage of CD4 + CD25 + FoxP3 + Treg cells from 90 treatment‐naive RRMS patients were compared to serum leptin levels. Regression analysis showed a statistically inverse correlation between serum leptin levels and the percentage of circulating Treg cells (r = −0.97, P < 0.0001). (B) Fifty thousand CD4 + CD25 + cells isolated from fresh PBMC by magnetic separation using commercially available kits (95% purity, 93% expressing FoxP3) were stimulated with soluble anti‐CD3 and anti‐CD28 (BD Bisociences), both at 5 µ g/mL concentration, in the presence and in the absence of leptin (250 ng/mL). Both antileptin receptor and control isotypes antibodies were added at a final concentration of 20 µ g/mL each, 30 min before adding leptin. Proliferation was determined on day 6 with [ 3 H]‐thymidine added during the final 18 h of culture. Proliferation of CD4 + CD25 + Foxp3 + cells was significantly inhibited after stimulation with leptin (250 ng/mL). This effect was abrogated by the addition of antileptin receptor mAb (LEPR‐ab) but not modified by the control antibody. The addition of exogenous IL‐2 (50 U/mL) reversed Treg‐cell hyporesponsiveness to anti‐CD3/anti‐CD28 stimulation in the presence of leptin. Each circle represents data from an individual patient ( n = 25). Data are presented as mean ± SEM. (C‐E) Inhibitory effects of CD4 + CD25 + FoxP3 + Treg cells were examined in 15 RRMS patients during remission on: proliferative response, and secretion of IFN‐γ, and IL‐17 by Th1 and Th17 polarized MBP 83–102 peptide‐specific T cells. For proliferation assays, CD4 + CD25 + FoxP3 + Treg cells were added together with 2 × 10 4 T‐cell‐depleted irradiated (3000 rad) accessory cells to autologous Th1 or Th17 CD4 + CD25 − MBP 83–102 ‐peptide‐specific effector cells at a ratio 1:1 (10 4 cells/well). Co‐cultures were stimulated with soluble anti‐CD3 (5 µ g/mL) together with soluble anti‐CD28 (5 µ g/mL), in the presence and in the absence of leptin, and proliferation determined in a 60‐hour assay, measuring 3 H‐thymidine incorporation. To measure IFN‐γ and IL‐17 production by CD4 + CD25 − effector cells, supernatants were removed before [ 3 H] thymidine addition, and analyzed using commercially available ELISA kits. The addition of leptin to the cultures (250 ng/mL) abrogated the inhibitory effects mediated by CD4 + CD25 + FoxP3 + Treg cells. For panels C, D, and E, each circle represents values for an individual T‐cell line (mean of triplicate cultures). Data represent mean ± SEM. Kruskal–Wallis test of one‐way ANOVA and post hoc data analysis applying Dunn's multiple comparison test were performed to analyze differences between groups. Data are presented as mean ± SEM. **** P < 0.0001.

Article Snippet: For blocking experiments human leptin receptor polyclonal antibody (MyBioSource, San Diego CA) was used at a final concentration of 20 μ g/mL; control was an irrelevant isotype‐matched antibody (R&D Systems).

Techniques: Isolation, Expressing, Concentration Assay, Control, Modification, Irradiation, Enzyme-linked Immunosorbent Assay, Comparison

Journal: Annals of Clinical and Translational Neurology

Article Title: Obesity and the risk of Multiple Sclerosis. The role of Leptin

doi: 10.1002/acn3.51291

Figure Lengend Snippet: Mean percentage inhibition of proliferative response, as well as of IFN‐γ and IL‐17 production by CD4 + CD25 + Foxp3 + Treg cells on CD4 + CD25 − MBP 83–102 ‐ and influenza hemagglutinin 307–319 ‐peptide‐specific effector T cells, derived from 15 RRMS during remission was calculated. CD4 + CD25 + FoxP3 + Treg cells were added in variable numbers together with 2 × 10 4 T‐cell‐depleted accessory cells, to a constant number of autologous Th1 or Th17 CD4 + CD25 − effector cells (10 4 cells/well) to achieve appropriate suppressor/responder ratios (1:1, 1:3, and 1:9). Co‐cultures were stimulated with soluble anti‐CD3 (5 µ g/mL) together with soluble anti‐CD28 (5 µ g/mL) in the presence or absence of recombinant human leptin (250 µ g/mL). Proliferation assays and measurement of IFN‐γ and IL‐17 production were performed as described in Figure and in Material and Methods. Proliferative response (A), IFN‐γ production (B) and IL‐17 production (C) were significantly inhibited upon the addition of CD4 + CD25 + FoxP3 + to the CD4 + CD25 − MBP 83–102 effector T cells in a 1:1ratio. Decreasing ratios of suppressor:effector cells (ratios 1:3 and 1:9) resulted in less suppression in all conditions examined. CD4 + CD25 + FoxP3 + exhibited significantly less suppressor activity on CD4 + CD25 − influenza hemagglutinin 307–319 ‐peptide‐specific effector T cells, compare with CD4 + CD25 − MBP 83–102 ‐ peptide‐specific T cells. When leptin was added to cultures, suppression mediated by Treg cells declined significantly, regardless of specific antigen. Assays were performed in triplicate, the symbols represent mean ± SEM. Percentage of CD4 + CD25 + FoxP3 + Treg‐cell inhibition in co‐cultures was defined as: [1‐(Treg:Teff values/Teff values)] × 100. The Mann–Whitney test was used to evaluate differences in CD4 + CD25 + FoxP3 + function between T‐cell lines co‐cultured at different ratios, and differences in suppressor activity exerted by Treg cells on different specific T cells. **** P < 0.0001.

Article Snippet: For blocking experiments human leptin receptor polyclonal antibody (MyBioSource, San Diego CA) was used at a final concentration of 20 μ g/mL; control was an irrelevant isotype‐matched antibody (R&D Systems).

Techniques: Inhibition, Derivative Assay, Recombinant, Activity Assay, MANN-WHITNEY, Cell Culture

Journal: Annals of Clinical and Translational Neurology

Article Title: Obesity and the risk of Multiple Sclerosis. The role of Leptin

doi: 10.1002/acn3.51291

Figure Lengend Snippet: In panels A to F cells were stimulated with soluble anti‐CD3/anti‐CD28 (5 µ g/mL each) during 6 h, in the presence and in the absence of leptin (A) Stimulation of CD4 + CD25 − MBP 83–102 effector T cells in the presence of leptin significantly increased p‐STAT3 Y705 levels. (B) In contrast, no differences were observed in CD4 + CD25 + FoxP3 + Treg cells under similar experimental conditions. (C) Stimulation of CD4 + CD25 − MBP 83–102 effector T cells in the presence of leptin, induced a significant increase in p‐ ERK1 T202/Y204 /ERK2T 185/Y187 expression. (D) Conversely, Treg cells stimulated under similar conditions showed a marked decrease in p‐ ERK1 T202/Y204 /ERK2T 185/Y187 . (E‐F) Using similar experimental conditions for both CD4 + CD25 − effector cells and for CD4 + CD25 + FoxP3 + Treg cells, a marked decrease in expression of cell cycle inhibitor p27 kip1 was observed in the former, whereas p27 kip1 was significantly increased in Treg cells. Leptin‐mediated effects were abrogated by antileptin receptor antibody, but not modified by an isotype control (20 µ g/mL). Each circle represents an individual MBP 83–102 ‐specific T‐cell line isolated from a total of 15 MS patients. Data represent mean ± SEM. Kruskal–Wallis test of one‐way ANOVA and post hoc data analysis applying Dunn's multiple comparison test were performed to analyze differences between groups. Anti LEPR ab = antileptin receptor antibody. **** P < 0.0001.

Article Snippet: For blocking experiments human leptin receptor polyclonal antibody (MyBioSource, San Diego CA) was used at a final concentration of 20 μ g/mL; control was an irrelevant isotype‐matched antibody (R&D Systems).

Techniques: Expressing, Modification, Control, Isolation, Comparison