Journal:

Article Title: Differential regulation of metabolic, neuroendocrine, and immune function by leptin in humans

doi: 10.1073/pnas.0505429103

Figure Lengend Snippet: Leptin depletion or neutralization inhibits polyclonal T cell proliferation and mixed lymphocyte reactions (MLR), respectively. (a–c)The proliferative response of T lymphocytes to polyclonal stimuli (OKT3, PHA, and PMA/Iono) from controls is completely inhibited in medium with human serum depleted of leptin. Addition of recombinant human leptin (at 100 ng/ml final concentration) completely reverses this phenomenon. (d) Anti-leptin blocking Abs partially inhibit the antigen-specific proliferative response of T cells during MLR. HS, human serum.

Article Snippet: For leptin depletion from serum, a protein G-Sepharose affinity column (Amersham Pharmacia) was used after adhesion on G protein of a polyclonal rabbit anti-human leptin Ab (BioVendor).

Techniques: Neutralization, Recombinant, Concentration Assay, Blocking Assay

Journal: European journal of immunology

Article Title: Leptin-mediated cytokine release and migration of eosinophils: implications for immunopathophysiology of allergic inflammation.

doi: 10.1002/eji.200636866

Figure Lengend Snippet: Figure 1. Effects of leptin on viability on eosinophils. Eosinophils (5 105/well) were cultured with or without leptin (50 ng/mL) for 24 h in a 24-well plate. Apoptosis of eosinophils was assessed by the TACSTM Annexin V-FITC assay using flow cytometry. Representative dot plots of (A) medium control (CTL), and (B) leptin-treated eosinophils were shown from triplicate experiments.

Article Snippet: Anti-human leptin neutralization mAb was from R&D Systems (MN, USA) and mouse IgG1 antibody was from BD PharMingen (CA, USA).

Techniques: Cell Culture, Flow Cytometry, Control

Journal: European journal of immunology

Article Title: Leptin-mediated cytokine release and migration of eosinophils: implications for immunopathophysiology of allergic inflammation.

doi: 10.1002/eji.200636866

Figure Lengend Snippet: Figure 2. Effects of leptin on cell surface expression of ICAM-1, CD18, ICAM-3 and L-selectin on eosinophils. Eosinophils (5 105/well) were cultured with leptin (0–100 ng/mL) for 16 h in a 24-well plate. Surface expression of adhesion molecules per 10 000 viable cells was analyzed by flow cytometry as MFI; (A) representative histograms of the changes in the expression of adhesion molecules. Results of (B) ICAM-1, (C) CD18, (D) ICAM-3 and (E) L-selectin have been normalized by subtracting appropriate isotypic control and are expressed as the arithmetic mean SD of five independent experiments. * p<0.05, ** p<0.01 and *** p<0.001 when compared with medium control.

Article Snippet: Anti-human leptin neutralization mAb was from R&D Systems (MN, USA) and mouse IgG1 antibody was from BD PharMingen (CA, USA).

Techniques: Expressing, Cell Culture, Flow Cytometry, Control

Journal: European journal of immunology

Article Title: Leptin-mediated cytokine release and migration of eosinophils: implications for immunopathophysiology of allergic inflammation.

doi: 10.1002/eji.200636866

Figure Lengend Snippet: Fig. 10 shows that leptin-induced release of IL-1b and IL-6, and IL-8, GRO-a and MCP-1 was suppressed by BAY11–7082 and SB203580, and BAY11–7082, PD98059 and SB203580, respectively (all p<0.05).

Article Snippet: Anti-human leptin neutralization mAb was from R&D Systems (MN, USA) and mouse IgG1 antibody was from BD PharMingen (CA, USA).

Techniques:

Journal: European journal of immunology

Article Title: Leptin-mediated cytokine release and migration of eosinophils: implications for immunopathophysiology of allergic inflammation.

doi: 10.1002/eji.200636866

Figure Lengend Snippet: Figure 4. Effects of neutralization antibody against leptin on the biological activities of leptin on eosinophils. Leptin was incubated with or without neutralization antibody (0.1–10 lg/mL) for 15 min and then incubated with eosinophils (5 105/well) for the assay of (A) apoptosis, (B) cell surface of adhesion molecules, (C) chemokinesis and (D) induction of cytokines. Results were expressed as arithmetic mean plus SD from triplicate experiments. Mouse IgG1 was used as a nonspecific antibody for isotypic antibody control. # p<0.05; ## p<0.01 when compared with medium control (CTL); * p<0.05, ** p<0.01, *** p<0.001 when compared with leptin control (lep). Ab0.1: neutralization antibody (0.1 lg/mL); Ab1: neutralization antibody (1 lg/mL); Ab10: neutralization antibody (10 lg/mL); mIgG: mouse IgG1 (10 lg/mL).

Article Snippet: Anti-human leptin neutralization mAb was from R&D Systems (MN, USA) and mouse IgG1 antibody was from BD PharMingen (CA, USA).

Techniques: Neutralization, Incubation, Control

Journal: European journal of immunology

Article Title: Leptin-mediated cytokine release and migration of eosinophils: implications for immunopathophysiology of allergic inflammation.

doi: 10.1002/eji.200636866

Figure Lengend Snippet: Figure 5. Representative expression profiles of intracellular phosphorylation of MAPK and other serine/threonine kinases of eosinophils upon activation by leptin. Eosinophils (1 107/ well) were treated with or without leptin (50 ng/mL) for 15 min. Eosinophils were then washed and lysed. Twenty-one different intracellular phosphorylated MAPK and other serine/threonine kinases in total cellular lysate were assessed semi-quantita- tively using an antibody based Human Phospho-MAPK Array Kit. Positive and negative controls were designated at (A1, A2, A21, A22, F1, F2) and (E3–E14), respectively. Triplicate experi- ments were performed with essentially identical results. The format of the antibodies on the array are detailed in Table 2.

Article Snippet: Anti-human leptin neutralization mAb was from R&D Systems (MN, USA) and mouse IgG1 antibody was from BD PharMingen (CA, USA).

Techniques: Expressing, Phospho-proteomics, Activation Assay

Journal: European journal of immunology

Article Title: Leptin-mediated cytokine release and migration of eosinophils: implications for immunopathophysiology of allergic inflammation.

doi: 10.1002/eji.200636866

Figure Lengend Snippet: Figure 6. Activation of ERK, p38 MAPK, JAK2 and NF-jB activities in eosinophils by leptin. Eosinophils (1 107/well) were cultured with or without leptin (50 ng/mL) for different indicated incubation times. Total cellular proteins were extracted from eosinophils for the measurement of (A) phosphorylated and total p38 MAPK, (B) phosphorylated and total ERK, (C) phosphorylated and total IjBa, and (D) phosphorylated and total JAK2 by Western blot analysis. Experiments were performed in three independent replicates with essentially identical results, and representative results are shown.

Article Snippet: Anti-human leptin neutralization mAb was from R&D Systems (MN, USA) and mouse IgG1 antibody was from BD PharMingen (CA, USA).

Techniques: Activation Assay, Cell Culture, Incubation, Western Blot

Journal: European journal of immunology

Article Title: Leptin-mediated cytokine release and migration of eosinophils: implications for immunopathophysiology of allergic inflammation.

doi: 10.1002/eji.200636866

Figure Lengend Snippet: Figure 7. Effects of AG490, BAY11–7082, LY294002, PD98059, SB203580 and SP600125 on viability of leptin-treated eosino- phils. Eosinophils (5 105/well) were pretreated with AG490 (10 lM), BAY11–7082 (1 lM), PD98059 (10 lM), LY294002 (5 lM), SB203580 (7.5 lM) or SP600125 (3 lM) for 1 h followed by incubation with or without leptin (50 ng/mL) for 24 h in a 24- well plate. Apoptosis of eosinophils was assessed by the TACSTM Annexin V-FITC assay using flow cytometry. Results were expressed as arithmetic mean plus SD from triplicate experiments. ## p<0.01 when compared with medium control; * p<0.05 when compared with the leptin control. AG: AG490; BAY: BAY11–7082; PD: PD98059; LY: LY294002; SB: SB203580; SP: SP600125; DM: DMSO-

Article Snippet: Anti-human leptin neutralization mAb was from R&D Systems (MN, USA) and mouse IgG1 antibody was from BD PharMingen (CA, USA).

Techniques: Incubation, Flow Cytometry, Control

Journal: European journal of immunology

Article Title: Leptin-mediated cytokine release and migration of eosinophils: implications for immunopathophysiology of allergic inflammation.

doi: 10.1002/eji.200636866

Figure Lengend Snippet: Figure 8. Effects of AG490, BAY11–7082, LY294002, PD98059, SB203580 and SP600125 on leptin-induced cell surface expression of (A) ICAM-1, (B) ICAM-3, (C) CD18, and (D) L-selectin on eosinophils. Eosinophils (5 105/well) were pretreated with AG490 (10 lM), BAY11–7082 (1 lM), PD98059 (10 lM), LY294002 (5 lM), SB203580 (7.5 lM) or SP600125 (3 lM) for 1 h followed by incubation with or without leptin (50 ng/mL) for further 16 h. Surface expression of the adhesion molecules of 10 000 cells was assessed by flow cytometry as MFI. Results are expressed as the arithmetic mean plus SD from five independent experiments. DMSO (0.1%) was used as the DMSO control. ### p<0.001 when compared with medium control; * p<0.05, ** p<0.01 when compared with the leptin control.

Article Snippet: Anti-human leptin neutralization mAb was from R&D Systems (MN, USA) and mouse IgG1 antibody was from BD PharMingen (CA, USA).

Techniques: Expressing, Incubation, Flow Cytometry, Control

Journal: European journal of immunology

Article Title: Leptin-mediated cytokine release and migration of eosinophils: implications for immunopathophysiology of allergic inflammation.

doi: 10.1002/eji.200636866

Figure Lengend Snippet: Figure 9. Effects of AG490, BAY11–7082, LY294002, PD98059, SB203580 and SP600125 on the migration of eosinophils induced by leptin. Eosinophils (5 104 cells) were pretreated with AG490 (10 lM), BAY11–7082 (1 lM), PD98059 (10 lM), LY294002 (5 lM), SB203580 (7.5 lM) or SP600125 (3 lM) for 1 h followed by incubation with or without leptin (50 ng/mL) for further 16 h. Eosinophils were then incubated in a 48-well microchamber in the upper chambers, while the RPMI medium was placed in the lower chambers. Migration was carried out for 6 h. Results are expressed as cell number/high power field with arithmetic mean plus SD of three independent experi- ments. # p<0.05 when compared with medium control; * p<0.05 when compared with the leptin control.

Article Snippet: Anti-human leptin neutralization mAb was from R&D Systems (MN, USA) and mouse IgG1 antibody was from BD PharMingen (CA, USA).

Techniques: Migration, Incubation, Control

Journal: bioRxiv

Article Title: Obesity promotes breast epithelium DNA damage in BRCA mutation carriers

doi: 10.1101/2022.07.29.502090

Figure Lengend Snippet: ( A ) Experimental schematic showing the collection of breast adipose tissue conditioned media (CM) from lean and overweight/obese women. ( B ) MCF-10A cells were treated with CM for 24 hours. DNA damage assessed by IF (#γH2AX foci/100 cells) is shown correlated with BMI in BRCA1 +/- (n=36 CM cases) and ( C ) BRCA2 +/- (n=13 CM cases) MCF-10A cells. Spearman’s rank correlation coefficient (ρ) and associated P value are shown along with 95% confidence intervals. ( D ) DNA damage in BRCA1 +/- and BRCA2 +/- MCF-10A cells and in ( E ) primary BRCA1 +/- breast epithelial cells treated with leptin (400ng/µl) for 24 hours. ( F ) DNA damage in BRCA1 +/- MCF-10A cells after 24-hour treatment with lean CM, obese (ob) CM, or ob CM in the presence of a leptin neutralizing antibody (Lep Ab). ( G ) DNA damage in BRCA1 +/- and BRCA2 +/- MCF-10A cells and in ( H ) primary BRCA2 +/- breast epithelial cells treated with insulin (100nM) for 24 hours. ( I ) DNA damage in BRCA1 +/- MCF-10A cells after 24-hour treatment with lean CM, ob CM, or ob CM in the presence of PI3K inhibitor BKM120 (1µM). Student’s t-test was used to determine significant differences in ( D-I) . All experiments in MCF-10A cells were conducted a minimum of two times with representative results from one experiment shown. Data in primary cells were generated from cells treated in triplicate. Data is presented as mean +/- SD. * P <0.05, ** P <0.01, *** P <0.001, ns= not significant.

Article Snippet: In leptin neutralization studies, obese CM was pre-incubated with a leptin neutralizing antibody (Lep ab, 13.3μg/mL, Fisher Scientific #AF398) for 1 hour at 4°C and then cells were treated with lean or obese CM alone or obese CM + Lep ab.

Techniques: Generated