Figure 4 Anti–TGF-β and anti–PD-1 dual antibody (ab) treatment render NOD.ChgA^−/− mice susceptible to autoimmune diabetes. A: Representative FACS plots showing the percentage of indicated immune cell populations in the islets of NOD.ChgA^−/− mice either treated with four injections of anti–TGF-β ab every 3 days or left untreated (control). Infiltration of leukocytes was analyzed 15 days postinitiation of treatment. B: Quantification of the percentage of indicated cell populations among total islet cells as described in panel A. Data (mean ± SEM) are pooled from three independent experiments. Each dot represents individual mouse. C: Experimental design of the anti–TGF-β and anti–PD-1 ab treatment. D: Incidence of diabetes in 8–10 week old NOD or NOD.ChgA^−/− mice treated with anti–TGF-β either alone or in combination with anti–PD-1. Data are pooled from three independent experiments. P values were calculated by unpaired two-tailed Student t test. *P < 0.05. DC, dendritic cell; ns, not significant. More

Figure 2 The GCGR monoclonal antibody Ab-4 blocks glucagon activity at the mouse GCGR. A: Glucagon-induced cAMP accumulation in HEK293 cells expressing the mouse GGCR is inhibited by Ab-4 (K_b = 0.76 nmol/L). B: Ab-4 does not inhibit GLP-1-induced cAMP accumulation in HEK293 cells expressing the mouse GLP-1R. Figure 2. The GCGR monoclonal antibody Ab-4 blocks glucagon activity at the mouse GCGR. A: Glucagon-induced cAMP accumulation in HEK293 cells expressing the mouse GGCR is inhibited by Ab-4 (Kb = 0.76 nmol/L). B: Ab-4 does not inhibit GLP-1-induced cAMP accumulation in HEK293 cells expressing the mouse GLP-1R. More

Figure 4 BAFF-deficient (BAFF^null) and anti-BAFF antibody (Ab)–treated obese mice exhibit superior glucose metabolic control compared with WT and B^null mice. Body weights (A), GTT with AUC (B), and ITT (C) of HFD WT, BAFF^null, and B^null mice (n = 5). GTT with AUC (D), body weights (E), and fasting insulin (F) of HFD WT mice 4 weeks after they received anti-BAFF antibody or isotype control (n = 5). IgG concentration in serum (G) and VAT lysate (H) 5 weeks after anti-BAFF antibody treatment (n = 5). Cytokine concentrations in 24-h cultures of PerC (I) or spleen (J) cells stimulated with LPS from isotype control and BAFF antibody–treated mice (n = 5). K: mRNA expression in VAT from isotype control and BAFF antibody–treated mice (n = 4). Values are given as mean ± SEM. *P < 0.05; **P < 0.005; ***P < 0.0005. Figure 4. BAFF-deficient (BAFFnull) and anti-BAFF antibody (Ab)–treated obese mice exhibit superior glucose metabolic control compared with WT and Bnull mice. Body weights (A), GTT with AUC (B), and ITT (C) of HFD WT, BAFFnull, and Bnull mice (n = 5). GTT with AUC (D), body weights (E), and fasting insulin (F) of HFD WT mice 4 weeks after they received anti-BAFF antibody or isotype control (n = 5). IgG concentration in serum (G) and VAT lysate (H) 5 weeks after anti-BAFF antibody treatment (n = 5). Cytokine concentrations in 24-h cultures of PerC (I) or spleen (J) cells stimulated with LPS from isotype control and BAFF antibody–treated mice (n = 5). K: mRNA expression in VAT from isotype control and BAFF antibody–treated mice (n = 4). Values are given as mean ± SEM. *P < 0.05; **P < 0.005; ***P < 0.0005. More

FIG. 7. HMGB1 antibody (Ab) treatment regulates T-cell subpopulations. PLN and splenic T-cells originated from HMGB1 antibody–treated mice and control mice were harvested for intracellular IFN-γ staining. The percentages of CD4⁺ and CD8⁺ T-cells that are also positive for IFN-γ were determined by flow cytometric analysis. The data are presented in the table as means ± SD of three independent experiments performed. The number of CD8⁺IFN-γ⁺ T-cells (Tc1) in the PLN of antibody-treated animals was significantly higher than that of control animals (P < 0.01). A similar trend was also observed in the splenic T-cells. *P < 0.01. FIG. 7. HMGB1 antibody (Ab) treatment regulates T-cell subpopulations. PLN and splenic T-cells originated from HMGB1 antibody–treated mice and control mice were harvested for intracellular IFN-γ staining. The percentages of CD4+ and CD8+ T-cells that are also positive for IFN-γ were determined by flow cytometric analysis. The data are presented in the table as means ± SD of three independent experiments performed. The number of CD8+IFN-γ+ T-cells (Tc1) in the PLN of antibody-treated animals was significantly higher than that of control animals (P < 0.01). A similar trend was also observed in the splenic T-cells. *P < 0.01. More

FIG. 5. The effect of addition of anti-TIMP-1 antibody (Ab) (A) and anti-TIMP-2 antibody (B) on the degradation of [³⁵S]methionine radiolabeled mesangium matrix. Results are disintegrations per minute released to the culture media expressed as percentage of the total amount of count in the matrix. * P < 0.05, significantly different from corresponding samples without the addition of antibodies. FIG. 5. The effect of addition of anti-TIMP-1 antibody (Ab) (A) and anti-TIMP-2 antibody (B) on the degradation of [35S]methionine radiolabeled mesangium matrix. Results are disintegrations per minute released to the culture media expressed as percentage of the total amount of count in the matrix. * P < 0.05, significantly different from corresponding samples without the addition of antibodies. More

FIG. 5. Univariate HRs for antibody increase. All factors analyzed are depicted. ■, HR; line, 95% CI; □, P < 0.05. Ab, antibody; IE, islet equivalents; Ln, natural logarithm; pre-Tx, pretransplant. FIG. 5. Univariate HRs for antibody increase. All factors analyzed are depicted. ■, HR; line, 95% CI; □, P < 0.05. Ab, antibody; IE, islet equivalents; Ln, natural logarithm; pre-Tx, pretransplant. More

FIG. 5. Inhibitory effect of β1 integrin antibody on β-cell spreading. β-Cells in suspension were pretreated with 2 μg/ml Ha2/5 antibody (Ab), 2 μg/ml hamster IgM, or BSA (without antibody) for 1 h and then plated on poly-l-lysine (PLL)- or 804G matrix (Mx)-coated dishes in continuous presence of the antibody. Cells were fixed 5 h later. Cell area profile was measured, using ScionImage software. n = 3–4 with >100 cells analyzed per experiment. *P < 0.02 vs. poly-l-lysine control; **P < 0.04 vs. 804G matrix control. Ab, antibody; w/o, without. FIG. 5. Inhibitory effect of β1 integrin antibody on β-cell spreading. β-Cells in suspension were pretreated with 2 μg/ml Ha2/5 antibody (Ab), 2 μg/ml hamster IgM, or BSA (without antibody) for 1 h and then plated on poly-l-lysine (PLL)- or 804G matrix (Mx)-coated dishes in continuous presence of the antibody. Cells were fixed 5 h later. Cell area profile was measured, using ScionImage software. n = 3–4 with >100 cells analyzed per experiment. *P < 0.02 vs. poly-l-lysine control; **P < 0.04 vs. 804G matrix control. Ab, antibody; w/o, without. More

Figure 1 HPAP workflow. HPAP, working with nPOD, identifies organ donors of interest (recent-onset type 1 diabetes, antibody-positive donors, and control subjects). For organ donors <30 years old without diabetes, the OPO, using HPAP/nPOD protocols and reagents, screens for presence of GADAs. I... More

Figure 6 BRD2 and 4 are proximally bound to Ins1 promoter in MIN6 cells. ChIP of BRD2, 3, and 4 in MIN6 cells that were treated for 24 h with 0.1% DMSO or 1 μmol/L JQ1. The precipitated chromatin was analyzed by real-time PCR and is expressed as a percentage of the input chromatin signal. Data are mean ± SD of at least three independent experiments. *P ≤ 0.05, **P ≤ 0.01. Ab, antibody; kb, kilobase. Figure 6. BRD2 and 4 are proximally bound to Ins1 promoter in MIN6 cells. ChIP of BRD2, 3, and 4 in MIN6 cells that were treated for 24 h with 0.1% DMSO or 1 μmol/L JQ1. The precipitated chromatin was analyzed by real-time PCR and is expressed as a percentage of the input chromatin signal. Data are mean ± SD of at least three independent experiments. *P ≤ 0.05, **P ≤ 0.01. Ab, antibody; kb, kilobase. More

Figure 1 ACAM Tg mice are resistant to obesity. A: WT and ACAM Tg male C57BL/6JJcl mice were fed HFHS and standard chow (STD). B: Fat pad weight of WT and ACAM Tg mice at 30 weeks of age. C: The average size of adipocytes in epididymal adipose tissues of WT and ACAM Tg mice on HFHS diet. The size distribution of adipocytes (D), periodic acid Schiff staining (E, left panels), scanning electron micrographs (E, right panels), immunofluorescence (F, left panels), and immunoperoxidase staining (F, right panels) with ACAM Ab (antibody) and double immunofluorescence staining of F4/80 and ACAM Ab (G) in epididymal adipose tissues of WT and ACAM Tg mice on HFHS diet. All data are presented as mean ± SE. n = 8. *P < 0.05 vs. WT mice. w, weeks. Figure 1. ACAM Tg mice are resistant to obesity. A: WT and ACAM Tg male C57BL/6JJcl mice were fed HFHS and standard chow (STD). B: Fat pad weight of WT and ACAM Tg mice at 30 weeks of age. C: The average size of adipocytes in epididymal adipose tissues of WT and ACAM Tg mice on HFHS diet. The size distribution of adipocytes (D), periodic acid Schiff staining (E, left panels), scanning electron micrographs (E, right panels), immunofluorescence (F, left panels), and immunoperoxidase staining (F, right panels) with ACAM Ab (antibody) and double immunofluorescence staining of F4/80 and ACAM Ab (G) in epididymal adipose tissues of WT and ACAM Tg mice on HFHS diet. All data are presented as mean ± SE. n = 8. *P < 0.05 vs. WT mice. w, weeks. More

FIG. 4. Neutralization of MIF stimulates INS-1 cell proliferation. INS-1 cells (1 × 10⁵) were synchronized by overnight incubation in serum-free RPMI-1640 medium with 2.5 mmol/l glucose and 0.1% BSA. Glucose (11 mmol/l) was added to synchronized cells with anti-MIF monoclonal antibody or an isotype control monoclonal antibody (Con IgG). Cell proliferation rate was assessed by [³H]thymidine incorporation. Data are means ± se of triplicate assays from three separate experiments. **P < 0.001. Ab, antibody. FIG. 4. Neutralization of MIF stimulates INS-1 cell proliferation. INS-1 cells (1 × 105) were synchronized by overnight incubation in serum-free RPMI-1640 medium with 2.5 mmol/l glucose and 0.1% BSA. Glucose (11 mmol/l) was added to synchronized cells with anti-MIF monoclonal antibody or an isotype control monoclonal antibody (Con IgG). Cell proliferation rate was assessed by [3H]thymidine incorporation. Data are means ± se of triplicate assays from three separate experiments. **P < 0.001. Ab, antibody. More

FIG. 3. Inhibitory effect of anti–laminin-5 antibody on β-cell spreading and insulin secretion. Petri dishes coated with poly-l-lysine or 804G matrix (Mx) were treated with anti–laminin-5 (CM6 antibody), anti-fibronectin (fibronectin antibody), mouse IgG, or PBS-BSA (without antibody). β-Cells were incubated on these dishes for 24 h. A: Phase-contrast microscopic views. Bar = 100 μm. B: Insulin secretion in response to 2.8 and 16.7 mmol/l glucose (n = 3–5). *P < 0.05 vs. poly-l-lysine (PLL) in respective condition; **P < 0.001 vs. 804G matrix control. Ab, antibody; w/o, without. FIG. 3. Inhibitory effect of anti–laminin-5 antibody on β-cell spreading and insulin secretion. Petri dishes coated with poly-l-lysine or 804G matrix (Mx) were treated with anti–laminin-5 (CM6 antibody), anti-fibronectin (fibronectin antibody), mouse IgG, or PBS-BSA (without antibody). β-Cells were incubated on these dishes for 24 h. A: Phase-contrast microscopic views. Bar = 100 μm. B: Insulin secretion in response to 2.8 and 16.7 mmol/l glucose (n = 3–5). *P < 0.05 vs. poly-l-lysine (PLL) in respective condition; **P < 0.001 vs. 804G matrix control. Ab, antibody; w/o, without. More

FIG. 2. SK2 and -3 protein expression in mouse pancreatic islets. Shown is a Western blot of protein extracts of mouse islets with: anti-SK2 (lane 1), anti-SK2 with antigenic SK2 peptide (lane 2), anti-SK3 (lane 3), and anti-SK3 with antigenic SK3 peptide (lane 4). Note the bands corresponding to SK2 (60 kDa) and SK3 (70 kDa). Ab, antibody. FIG. 2. SK2 and -3 protein expression in mouse pancreatic islets. Shown is a Western blot of protein extracts of mouse islets with: anti-SK2 (lane 1), anti-SK2 with antigenic SK2 peptide (lane 2), anti-SK3 (lane 3), and anti-SK3 with antigenic SK3 peptide (lane 4). Note the bands corresponding to SK2 (60 kDa) and SK3 (70 kDa). Ab, antibody. More

Figure 2 UnAG and skeletal muscle inflammation. Effects of UnAG (200 μg subcutaneous injection twice per day) vs. saline (Ct) sustained 4-day treatment on the expression of IκB (A), NF-κB binding activity (B) with representative blots, and tissue expression of IL-1α (C), IL-1β (D), TNF-α (E), IL-6 (F), and IL-10 (G) measured by xMAP technology in gastrocnemius muscle. Data are mean ± SEM (n = 8–10/group). *P < 0.05 vs. Ct. Ab, antibody; a.u., arbitrary units. Figure 2. UnAG and skeletal muscle inflammation. Effects of UnAG (200 μg subcutaneous injection twice per day) vs. saline (Ct) sustained 4-day treatment on the expression of IκB (A), NF-κB binding activity (B) with representative blots, and tissue expression of IL-1α (C), IL-1β (D), TNF-α (E), IL-6 (F), and IL-10 (G) measured by xMAP technology in gastrocnemius muscle. Data are mean ± SEM (n = 8–10/group). *P < 0.05 vs. Ct. Ab, antibody; a.u., arbitrary units. More

Figure 3 VEGF neutralization in long-term high-fat–fed mice. A: Eight-week-old mice were placed on chow diet or HFD for 4 weeks and were administered a single injection of either VEGF-neutralizing or control antibody at days 0 and 3. Glucose tolerance was assessed (B) 3 days after the first single injection and again at (C) day 7, (D) day 11, (E) day 17, and (F) day 21. G: Glucose tolerance was quantified by measuring AUCs. n = 5–8 mice per group. *P < 0.05; **P < 0.01, Kruskal–Wallis test. Glucose levels are plotted as median values, and error bars are interquartile range. Ab, antibody; DXA, dual-energy X-ray absorptiometry; ITT, insulin tolerance test. Figure 3. VEGF neutralization in long-term high-fat–fed mice. A: Eight-week-old mice were placed on chow diet or HFD for 4 weeks and were administered a single injection of either VEGF-neutralizing or control antibody at days 0 and 3. Glucose tolerance was assessed (B) 3 days after the first single injection and again at (C) day 7, (D) day 11, (E) day 17, and (F) day 21. G: Glucose tolerance was quantified by measuring AUCs. n = 5–8 mice per group. *P < 0.05; **P < 0.01, Kruskal–Wallis test. Glucose levels are plotted as median values, and error bars are interquartile range. Ab, antibody; DXA, dual-energy X-ray absorptiometry; ITT, insulin tolerance test. More