Journal: Kidney international

Article Title: Leptin induces TGF-beta synthesis through functional leptin receptor expressed by human peritoneal mesothelial cell.

doi: 10.1038/sj.ki.5000409

Figure Lengend Snippet: Figure 10 | Blocking study of TGF-b release by HPMC. Concentration of TGF-b in culture supernatant from HPMC incubated with medium containing 5.6 mM glucose, 40 mM glucose, 20 ng/ml leptin, 40 mM glucose plus 20 ng/ml leptin, or glucose-HPAC conditioned medium in the absence (white bar) presence of anti-leptin receptor antibody (crossed bar; last bar in each group; 50mg/ml), inhibitor to JAK2 (AG490; 10 mM; dotted bar; 2nd bar in each group), PKC (calphostin; 100 nM; gray bar) or both (10 mM AG490 plus 100 nM calphostin; dark bar). Data are mean7s.d. of four individual experiments.

Article Snippet: Monoclonal anti-Ob-Rb, neutralizing anti-leptin receptor antibody and leptin protein were obtained from R&D System (Minneapolis, MN, USA).

Techniques: Blocking Assay, Concentration Assay, Incubation

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: Cells

Article Title: Scrutinizing Mechanisms of the ‘Obesity Paradox in Sepsis’: Obesity Is Accompanied by Diminished Formation of Neutrophil Extracellular Traps (NETs) Due to Restricted Neutrophil–Platelet Interactions

doi: 10.3390/cells10020384

Figure Lengend Snippet: Leptin levels and impact of either exogenous leptin or endogenous leptin neutralization on neutrophil accumulation and neutrophil extracellular trap (NET) formation in liver sinusoids of lean (ND) and obese (HFD) mice. ( A ) Levels of leptin were compared between untreated (healthy) ND and HFD animals as well as endotoxemic mice, 24 h post lipopolysaccharide (LPS) injection. Some animals received a bolus of exogenous recombinant leptin (rec leptin) prior to endotoxemia induction, and subsequently neutrophil infiltration ( B ) and NET formation ( C ) in the liver vasculature were estimated. Control mice received saline. Another group of mice received mouse anti-Leptin/OB antibody to neutralize their endogenous leptin (α leptin) whereas respective controls were injected with an isotype control as detailed in . Upon induction of endotoxemia, neutrophil accumulation ( D ) and NET formation ( E ) in liver sinusoids were studied. Asterisks indicate significant differences between ND and HFD mice using unpaired two-tailed Student’s t -test (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001). Data are shown as mean ± s.d.; n ≥ 3 per group.

Article Snippet: Some groups of ND and HFD mice received i.v. leptin neutralizing antibodies (Mouse Leptin/OB Antibody, polyclonal goat IgG, R&D Systems, Minneapolis, MN, USA) via tail vein (3 μg/mouse in 0.9% saline) at 12:00 AM or 6:00 PM, and after 30 min since the induction of endotoxemia (1 mg/kg b.w.

Techniques: Neutralization, Injection, Recombinant, Control, Saline, Two Tailed Test

Journal: Immunity

Article Title: A key role of leptin in the control of regulatory T cell proliferation.

doi: 10.1016/j.immuni.2007.01.011

Figure Lengend Snippet: Figure 1. Human Treg Cells Express High ObR, and Leptin Neutralization Reverses Their Hyporesponsiveness (A) Representative flow cytometry plot of human T cells stained for CD4, CD25, and Foxp3. (B and C) Immunoblot analysis of sorted CD4+ T cells on the basis of their CD25 expression. Graphs show quantitation of Foxp3 and ObR with respect to tubulin. One representative out of five independent experiments is shown. (D) Proliferation of CD4+CD25+ Treg cells treated with recombinant human leptin (250 ng/ml) in the presence or absence of leptin mAb (10 mg/ml). The data are shown as mean ± SD (n = 5, *p < 0.0001; **p < 0.01). (E) Dose dependency of Treg cell proliferation induced by leptin mAb. Proliferation was measured after treatment with indicated doses of leptin mAb. The data are shown as mean ± SD (n = 5). (F) Proliferation of Treg cells induced by a fixed dose of leptin mAb in the presence of increasing concentration of recombinant leptin. The data are shown as mean ± SD (n = 5). (G) Proliferation of CD4+CD25 effector T cells treated with recombinant human leptin (250 ng/ml) in the presence or absence of leptin mAb (10 mg/ml). The data are shown as mean ± SD (n = 5, *p < 0.01; **p < 0.01). (H) Proliferation of CD4+CD25 effector T cells, Treg cells, and of both cell types in coculture in the presence or absence of leptin mAb (10 mg/ml). The data are shown as mean ± SD (n = 5, *p < 0.01; **p < 0.0001).

Article Snippet: Reagents, Leptin-Neutralizing Antibodies, and Leptin Measurement For in vitro blocking experiments, human leptin-neutralizing mAb (R&D Systems, Minneapolis, MN) was used at a final concentration of 0.25 to 25 mg/ml; controls were irrelevant isotype-matched antibodies (Biovendor Laboratory Medicine Inc., Brno, Czech Republic).

Techniques: Neutralization, Cytometry, Staining, Western Blot, Expressing, Quantitation Assay, Recombinant, Concentration Assay

Journal: Immunity

Article Title: A key role of leptin in the control of regulatory T cell proliferation.

doi: 10.1016/j.immuni.2007.01.011

Figure Lengend Snippet: Figure 2. Human Treg Cells Exhibit Partial Suppressive Capacity upon Leptin-mAb-Induced Proliferation (A) Proliferation of CD4+CD25 effector T cells, Treg cells, and of both cell types in coculture treated with recombinant human leptin (250 ng/ml) in the presence or absence of leptin mAb (10 mg/ml). The data are shown as mean ± SD (n = 3, *p < 0.0001; **p < 0.01). (B–I) Proliferative response (B–E) and CD25 expression analysis (F–I) of CFSE-labeled-CD4+CD25 effector T cells alone or in coculture with untreated or leptin-mAb-treated unlabeled Treg cells. The thin line represents the isotype-matched negative control and the thick line represents the CD25 stain- ing. One representative out of three independent experiments is shown (*p < 0.0001; **p < 0.01; ***p < 0.05, as compared with CD4+CD25CFSE+).

Article Snippet: Reagents, Leptin-Neutralizing Antibodies, and Leptin Measurement For in vitro blocking experiments, human leptin-neutralizing mAb (R&D Systems, Minneapolis, MN) was used at a final concentration of 0.25 to 25 mg/ml; controls were irrelevant isotype-matched antibodies (Biovendor Laboratory Medicine Inc., Brno, Czech Republic).

Techniques: Recombinant, Expressing, Labeling, Negative Control, Staining

Journal: Immunity

Article Title: A key role of leptin in the control of regulatory T cell proliferation.

doi: 10.1016/j.immuni.2007.01.011

Figure Lengend Snippet: Figure 3. Treg Cells Produce Leptin and Express High Amounts of ObR (A) Proliferation of CD4+CD25 effector T cells, Treg cells, and of both cell types in coculture in three different serum- and leptin-free media in the presence or absence of leptin mAb. The data are shown as mean ± SD (n = 6, *p < 0.01; **p < 0.001). (B) Flow cytometry plot of BrdU incorporation of CD4+CD25 effector T cells, Treg cells, and of both cell types in coculture in serum-free medium (X-VIVO), in the presence or absence of leptin mAb during anti-CD3 and anti-CD28 stimulation. One representative out of three independent exper- iments is shown (*p < 0.05; **p < 0.001). (C) Confocal microscopy of freshly isolated and 1 hr-stimulated Treg cells and CD4+CD25 effectors stained for leptin (in green) and leptin receptor (ObR) (in red). One representative out of three independent experiments is shown. (D) Immunoblot for leptin on cell lysates from Treg cells and CD4+CD25 effectors. The graph shows quantitation of leptin with respect to total ERK1/2. One representative out of three independent experiments is shown.

Article Snippet: Reagents, Leptin-Neutralizing Antibodies, and Leptin Measurement For in vitro blocking experiments, human leptin-neutralizing mAb (R&D Systems, Minneapolis, MN) was used at a final concentration of 0.25 to 25 mg/ml; controls were irrelevant isotype-matched antibodies (Biovendor Laboratory Medicine Inc., Brno, Czech Republic).

Techniques: Flow Cytometry, BrdU Incorporation Assay, Confocal Microscopy, Isolation, Staining, Western Blot, Quantitation Assay

Journal: Immunity

Article Title: A key role of leptin in the control of regulatory T cell proliferation.

doi: 10.1016/j.immuni.2007.01.011

Figure Lengend Snippet: Figure 4. Leptin-mAb-Induced Proliferation of Human Treg Cells Is IL-2 Dependent, and Foxp3 Expression Is Increased during Proliferation (A) Proliferation of CD4+CD25 effector T cells, Treg cells, and of both cell types in coculture in the presence or absence of leptin mAb and IL-2- neutralizing mAb. The data are shown as mean ± SD (n = 5, *p < 0.01, **p < 0.05, white bars; *p < 0.001, black bars; *p < 0.01, gray bars). (B) IL-2 secretion of CD4+CD25 effector T cells, Treg cells, and of both cell types in coculture in the presence or absence of leptin mAb and IL-2- neutralizing mAb. The data are shown as mean ± SD (n = 5, *p < 0.01 and **p < 0.05, white bars; *p < 0.001, black bars; *p < 0.01, gray columns). (C) Proliferation of Treg cells in the presence of either leptin mAb or recombinant IL-2 (left), stimulated or not with anti-CD3 and anti-CD28. The data are shown as mean ± SD (n = 5, *p < 0.0001; **p < 0.01; ***p < 0.05). Addition of scalar doses of recombinant leptin to proliferating Treg cells (right) stim- ulated with anti-CD3 and anti-CD28 in the presence of recombinant IL-2. The data are shown as mean ± SD (n = 5, NS, not significant). (D) Immunoblot analysis of CD4+CD25 effector T cells and Treg cells in the presence or absence of leptin mAb, at 1 hr stimulation with anti-CD3 and anti-CD28. The graph shows quantitation of Foxp3 with respect to tubulin. One representative out of five independent experiments is shown. (E) Immunoblot analysis and flow cytometry plot of CD4+CD25 effector T cells and Treg cells in the presence or absence of leptin mAb, at 12 hr stim- ulation with anti-CD3 and anti-CD28. The graph shows quantitation of Foxp3 with respect to tubulin. One representative out of five independent ex- periments is shown (*p < 0.01, as compared with anti-CD3 and anti-CD28).

Article Snippet: Reagents, Leptin-Neutralizing Antibodies, and Leptin Measurement For in vitro blocking experiments, human leptin-neutralizing mAb (R&D Systems, Minneapolis, MN) was used at a final concentration of 0.25 to 25 mg/ml; controls were irrelevant isotype-matched antibodies (Biovendor Laboratory Medicine Inc., Brno, Czech Republic).

Techniques: Expressing, Recombinant, Western Blot, Quantitation Assay, Cytometry

Journal: Immunity

Article Title: A key role of leptin in the control of regulatory T cell proliferation.

doi: 10.1016/j.immuni.2007.01.011

Figure Lengend Snippet: Figure 5. Molecular Effects of Leptin Neutralization on Human Treg Cells (A and B) Immunoblot for ObR, STAT3, and SOCS3 on CD4+CD25 T cells and Treg cells in the presence or absence of leptin mAb stimulated with anti- CD3 and anti-CD28 at 1 hr and 12 hr, respectively. Graphs show quantitation of each specific protein. One representative out of five independent experiments is shown. (C and D) Immunoblot for ERK1/2, STAT1, and p27kip1. Graphs show quantitation of each specific protein. One representative out of five independent experiments is shown.

Article Snippet: Reagents, Leptin-Neutralizing Antibodies, and Leptin Measurement For in vitro blocking experiments, human leptin-neutralizing mAb (R&D Systems, Minneapolis, MN) was used at a final concentration of 0.25 to 25 mg/ml; controls were irrelevant isotype-matched antibodies (Biovendor Laboratory Medicine Inc., Brno, Czech Republic).

Techniques: Neutralization, Western Blot, Quantitation Assay

Journal: Immunity

Article Title: A key role of leptin in the control of regulatory T cell proliferation.

doi: 10.1016/j.immuni.2007.01.011

Figure Lengend Snippet: Figure 6. In Vivo Leptin Neutralization or Congenital Leptin Deficiency Associate with Proliferation of Treg Cells (A) Proliferation measured as CFSE dilution of CFSE-labeled Treg cells obtained from WT mice and transferred into control (CTR)-Ig or mouse leptin neutralizing Ab-treated WT mice. The histogram shows the percent of CFSE+ Treg cells (gated on CD4+Foxp3+ cells) that had divided 4 and 7 days after transfer. One representative out of three independent experiments with 3 mice per group is shown (*p < 0.01; **p < 0.001). (B) CFSE dilution profile of CFSE-labeled Treg cells (gated on CD4+Foxp3+ cells) obtained from WT mice and transferred into WT or leptin-deficient ob/ ob mice, 4 and 7 days after transfer. One representative out of three independent experiments with 3 mice per group is shown (*p < 0.01; **p < 0.001). (C) CFSE dilution profile of PCC-specific CFSE-labeled AND-TCR Treg cells (gated on Va11.1+/Vb3+Foxp3+ cells) adoptively transferred into WT, ob/ob, and ob/ob treated with recombinant leptin, 4 and 7 days after transfer. One representative out of two independent experiments with 4 mice per group is shown (**p < 0.001; *p < 0.01).

Article Snippet: Reagents, Leptin-Neutralizing Antibodies, and Leptin Measurement For in vitro blocking experiments, human leptin-neutralizing mAb (R&D Systems, Minneapolis, MN) was used at a final concentration of 0.25 to 25 mg/ml; controls were irrelevant isotype-matched antibodies (Biovendor Laboratory Medicine Inc., Brno, Czech Republic).

Techniques: In Vivo, Neutralization, Labeling, Control, Recombinant

Journal: Immunity

Article Title: A key role of leptin in the control of regulatory T cell proliferation.

doi: 10.1016/j.immuni.2007.01.011

Figure Lengend Snippet: Figure 7. ObR Deficiency Increases Treg Cells Proliferative Potential, Does Not Al- ter Their Suppressive Capacity, and Im- pairs CD4+CD25 Proliferation (A) Proliferation of CD4+CD25 effector T cells from db/+ and leptin receptor-deficient db/db mice stimulated with anti-CD3 and anti- CD28. The data are shown as mean ± SD (n = 5, *p < 0.001). (B) Proliferation of Treg cells from db/+ and db/db mice stimulated with anti-CD3 and anti-CD28. The data are shown as mean ± SD (n= 5, **p < 0.01). (C) Proliferation of CD4+CD25 effector T cells from db/+ mice in the presence of increasing number of either db/+ or db/db Treg cells, stim- ulated with anti-CD3 and anti-CD28. The data are shown as mean ± SD (n = 5). (D) CFSE dilution of Treg cells (gated on CD4+Foxp3+ cells) from leptin receptor mutant NOD-Leprdb-5J/LtJ mice stimulated with mouse recombinant GAD65, after 5 days cul- ture. One representative out of five indepen- dent experiments is shown (*p > 0.01).

Article Snippet: Reagents, Leptin-Neutralizing Antibodies, and Leptin Measurement For in vitro blocking experiments, human leptin-neutralizing mAb (R&D Systems, Minneapolis, MN) was used at a final concentration of 0.25 to 25 mg/ml; controls were irrelevant isotype-matched antibodies (Biovendor Laboratory Medicine Inc., Brno, Czech Republic).

Techniques: Mutagenesis, Recombinant

Journal: Journal of neurochemistry

Article Title: Leptin induces interleukin-1beta release from rat microglial cells through a caspase 1 independent mechanism.

doi: 10.1111/j.1471-4159.2007.04559.x

Figure Lengend Snippet: Fig. 1 Expression of leptin receptors. (A) mRNA expression of the short (Ob-Ra) and long (Ob-Rb) isoforms of leptin receptors in mixed glia, microglia, neurones or astrocytes, compared with expression of b-actin mRNA. Single experiment representative of three separate experiments. (B) Ob-R immunostaining in rat microglial cultures using a specific antibody that recognizes both short- and long-isoforms of Ob-R. (a) Lectin (GSL)-IB4 staining. (b) Ob-R immunostaining. (c) Neutralization of Ob-R immunostaining in presence of 10· excess specific blocking peptide. Bar scale = 20 lm

Article Snippet: Cells were washed and Ob-R immunostaining was then carried out as previously described (Pinteaux et al. 2002), using an IgG anti-leptin receptor (Ob-R) primary antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) (1 : 500 dilution) in absence or presence of an excess (10 lg/mL) of recombinant Ob-R blocking peptide (Santa Cruz Biotechnology), and subsequent incubation with a Texas-Red-conjugated affinity-purified donkey anti-goat IgG (Chemicon, Temecula, CA, USA) (1 : 50 dilution).

Techniques: Expressing, Immunostaining, Staining, Neutralization, Blocking Assay