Journal: Scientific Reports

Article Title: Activation of the mTOR signaling pathway is required for asthma onset

doi: 10.1038/s41598-017-04826-y

Figure Lengend Snippet: Levels of mTOR, Th17 cells, Treg cells, and asthma-related cytokines in the serum of patients with asthma. The patients were categorized into four groups: the asthma attack group, asthma remission group, community-acquired pneumonia (pneumonia) group, and normal healthy group. The asthma group consisted of children with asthma who visited a doctor or were hospitalized from January 2014 to June 2014. The remission group comprised children who had experienced an asthma attack but were then treated with budenoside and were in asthma remission. The pneumonia group comprised children who were diagnosed with pneumonia in the same hospital in the same period. The healthy control group comprised normal healthy children who had a physical examination in the hospital in the same period. ( a ) The level of serum mTOR (pg/mL), IL-17 (pg/mL), TGF-β (pg/mL), IL-10 (pg/mL), IL-4 (pg/mL), and IFN-γ (pg/mL) detected by ELISA in the control group, the pneumonia group, the asthma attack group, and the asthma remission group. The levels of mTOR, IL-17, and IL-4 in the serum of patients in the asthma attack group were significantly higher than that in the pneumonia and control groups, whereas these elevations were not observed in patients in asthma. Similarly, the levels of TGF-β, IL-10, and IFN-γ in the serum of patients in the asthma attack group were significantly lower than that in the pneumonia and control groups, whereas reduction of these cytokines was not observed in patients in asthma. ( b ) Flow cytometric analysis of Th17 cells (CD3 + CD8 − IL-17 + ) in peripheral blood mononuclear cells (PBMC) from patients in the control group, the pneumonia group, the asthma attack, and the asthma remission group. The data was presented as the percentage of all live cells. ( c ) Flow cytometric analysis of Treg cells (CD4 + CD25 high+ CD127 low ) in PBMC from patients in the control group, the pneumonia group, the asthma attack, and the asthma remission group. ( d ) Levels of Th17 cells (CD3 + CD8 − IL-17 + ) and Treg cells (CD4 + CD25 high+ CD127 low ) detected by flow cytometry in PBMC from patients in the control group, the pneumonia group, the asthma attack group, and the asthma remission group. Th17 and Treg cell percentages in total live cells were presented. The level of Th17 was significantly higher in asthma attack patients than the rest three groups, whereas the level of Treg was significantly lower in asthma attack patients than the rest three groups. All data was presented as means ± SD.*p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: ELISA kits used were the following: Human mTOR ELISA Detection Kit, Human IL-17 ELISA Detection Kit, Human IL-10 ELISA Detection Kit, Human IL-4 ELISA Detection Kit, Human IFN-γ ELISA Detection Kit, Human TGF-β ELISA Detection Kit, Mouse mTOR ELISA Detection Kit, Mouse IL-17 ELISA Detection Kit, Mouse TGF-β ELISA Detection Kit, Mouse IL-10 ELISA Detection Kit, Mouse IL-4 ELISA Detection Kit, and Mouse IFN-γ ELISA Detection Kit, all from R&D Systems.

Techniques: Control, Enzyme-linked Immunosorbent Assay, Flow Cytometry

Journal: Scientific Reports

Article Title: Activation of the mTOR signaling pathway is required for asthma onset

doi: 10.1038/s41598-017-04826-y

Figure Lengend Snippet: Correlation of serum mTOR levels with the loss of Th17/Treg and Th1/Th2 balances in patients experiencing an asthma attack. The asthma patient group comprised children with asthma who visited a doctor or were hospitalized from January 2014 to June 2014. Serum was collected from the fasting venous blood of these patients, and mTOR as well as the cytokines were measured by ELISA. Peripheral blood mononuclear cells (PBMC) were isolated from the fasting venous blood of these patients, and Th17 and Treg were measured by flow cytometry. ( a ) Positive correlation between the amounts of serum mTOR (pg/mL) determined by ELISA and PBMC Th17 cells (%) determined by flow cytometry in patients experiencing an asthma attack. Correlation analysis performed with Pearson correlation, r = 0.960 and p < 0.001. ( b ) Negative correlation between the amounts of serum mTOR (pg/mL) determined by ELISA and PBMC Treg cells (%) determined by flow cytometry in patients experiencing an asthma attack. Correlation analysis performed with Pearson correlation, r = −0.932 and p < 0.001. ( c ) Positive correlation between the amounts of serum mTOR (pg/mL) and serum IL-17 (pg/mL) determined by ELISA in patients experiencing an asthma attack. Correlation analysis performed with Pearson correlation, r = 0.754 and p < 0.001. ( d ) Negative correlation between the amounts of serum mTOR (pg/mL) and serum TGF-β (pg/mL) determined by ELISA in patients experiencing an asthma attack, r = −0.740 and p < 0.05 by Pearson correlation analysis. ( e ) Negative correlation between the amounts of serum mTOR (pg/mL) and serum IL-10 (pg/mL), determined by ELISA in patients experiencing an asthma attack, r = −0.944 and p < 0.001 by Pearson correlation analysis. ( f ) Positive correlation between the amounts of serum mTOR (pg/mL) and serum IL-4 (pg/mL) determined by ELISA in patients experiencing an asthma attack, r = 0.855 and p < 0.001 by Pearson correlation analysis. ( g ) Negative correlation between the amounts of serum mTOR (pg/mL) and serum IFN-γ (pg/mL) determined by ELISA in patients experiencing an asthma attack, r = −0.762 and p < 0.001 by Pearson correlation analysis.

Article Snippet: ELISA kits used were the following: Human mTOR ELISA Detection Kit, Human IL-17 ELISA Detection Kit, Human IL-10 ELISA Detection Kit, Human IL-4 ELISA Detection Kit, Human IFN-γ ELISA Detection Kit, Human TGF-β ELISA Detection Kit, Mouse mTOR ELISA Detection Kit, Mouse IL-17 ELISA Detection Kit, Mouse TGF-β ELISA Detection Kit, Mouse IL-10 ELISA Detection Kit, Mouse IL-4 ELISA Detection Kit, and Mouse IFN-γ ELISA Detection Kit, all from R&D Systems.

Techniques: Enzyme-linked Immunosorbent Assay, Isolation, Flow Cytometry

Journal: Scientific Reports

Article Title: Activation of the mTOR signaling pathway is required for asthma onset

doi: 10.1038/s41598-017-04826-y

Figure Lengend Snippet: Levels of asthma-related cytokines in mouse serum and bronchoalveolar lavage fluid (BALF) determined by ELISA. ( a ) The levels of IL-17 (pg/mL) and TGF-β (pg/mL) in sera from mice in the control group and the OVA-induced asthma group, and the OVA-induced asthmatic mice treated with budenoside, rapamycin, LY294002, or triciribine. The asthmatic mice demonstrated significantly higher levels of IL-17 and lower level of TGF-β than in the control, budenoside treatment, and m-TOR pathway inhibitor treatment groups. No statistical significance was observed among those groups. ( b ) The levels of IL-17 (pg/mL), TGF-β (pg/mL), IL-10 (pg/mL), IL-4 (pg/mL), and IFN-γ (pg/mL) in BALF from mice in the control group and the OVA-induced asthma group, and the OVA-induced asthmatic mice treated with budenoside, rapamycin, LY294002, or triciribine. The asthmatic mice demonstrated significantly higher levels of IL-17 and IL-4, and lower level of TGF-β, IL-10 and IFN-γ than in the control, budenoside treatment, and m-TOR pathway inhibitor treatment groups. No statistical significance was observed among those groups. All data was presented as the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: ELISA kits used were the following: Human mTOR ELISA Detection Kit, Human IL-17 ELISA Detection Kit, Human IL-10 ELISA Detection Kit, Human IL-4 ELISA Detection Kit, Human IFN-γ ELISA Detection Kit, Human TGF-β ELISA Detection Kit, Mouse mTOR ELISA Detection Kit, Mouse IL-17 ELISA Detection Kit, Mouse TGF-β ELISA Detection Kit, Mouse IL-10 ELISA Detection Kit, Mouse IL-4 ELISA Detection Kit, and Mouse IFN-γ ELISA Detection Kit, all from R&D Systems.

Techniques: Enzyme-linked Immunosorbent Assay, Control

Journal: Oncogene

Article Title: Pancreatic β-cells activate a JunB/ATF3-dependent survival pathway during inflammation.

doi: 10.1038/onc.2011.353

Figure Lengend Snippet: Figure 1 TNF-a þ IFN-g activate JunB and c-Jun in b-cells. (a) Expression of JunB in human islets isolated from six organ donors, under control condition or following 48 h treatment with TNF-a þ IFN-g. JunB expression was determined by real time RT–PCR and is represented as fold induction compared with control (non-treated cells). ***Po0.05. (b) Time course of Jun protein expression after cytokine treatment. Cell lysates were subjected to western blotting with antibodies detecting JunB, JunD, p-c-Jun or a-tubulin as loading control. (c) INS-1E cells were infected with a recombinant adenovirus expressing either luciferase (AdLUC) or the NF-kB non- degradable inhibitor IkB(SA)2 and then exposed to TNF-a þ IFN-g. Expression of IkBa(SA)2 and JunB were determined by western blot. (d) INS-1E cells were treated with TNF-a þ IFN-g and/or the JNK inhibitor SP600125 at different time points, and p-c-Jun and a-tubulin (used as loading control) were measured by western blot. (e) Immunohistochemistry for JunB or c-Jun in pancreas section of 17-week-old NOD or BALB/c control mice. Note the increased c-Jun phosphorylation and JunB expression during insulitis in the islets of pre-diabetic NOD mice. Arrows indicate positive nuclear staining.

Article Snippet: Recombinant rat (100U/ml for INS-1E cells and 500U/ml for primary rat b-cell), mouse (500U/ml) or human (500U/ml) IFN-g (R&D Systems, Abingdon, UK), recombinant murine TNF-a (1000U/ml, Innogenetics, Ghent, Belgium), human recombinant IL-1b (50U/ml for mouse islets and 10U/ml for INS-1E cells, a kind gift from Dr CW Reinolds, National Cancer Institute, Bethesda, MD, USA), SP600125 (10 mM, Sigma, Steinheim, Germany), H89 inhibitor (15 mM, Sigma) and forskolin (20 mM, Sigma) were used.

Techniques: Expressing, Isolation, Control, Quantitative RT-PCR, Western Blot, Infection, Recombinant, Luciferase, Immunohistochemistry, Phospho-proteomics, Staining

Journal: Scientific Reports

Article Title: Engineering of anti-human interleukin-4 receptor alpha antibodies with potent antagonistic activity

doi: 10.1038/s41598-019-44253-9

Figure Lengend Snippet: Isolation and characterization of human Abs directed against IL-4Rα. ( a ) Binding activity of the isolated anti-IL-4Rα Abs to plate-coated human IL-4Rα or GST, as determined by ELISA. Data represented as mean ± SD (n = 3). ( b ) Schematic diagram of the reporter HEK-Blue TM IL-4/IL-13 cell line to monitor the biological activity of anti-IL-4Rα Abs. The details are described in the text. ( c ) IL-4Rα-blocking activity of the indicated Abs, as determined by SEAP secretion levels from HEK-Blue TM IL-4/IL-13 cells after stimulation with rhIL-4 (100 pM) in the presence of the Abs (40 and 200 nM) for 24 h. Data are presented as percentage (mean ± SD (n = 3)) in SEAP levels relative to PBS-treated samples. ( d ) Binding isotherms of the immobilized anti-IL-4Rα Abs to soluble antigen IL-4Rα, measured by bio‐layer interferometry on OctetRED96 (Fortebio). The concentrations of IL-4Rα analysed are indicated (colored).

Article Snippet: For hIL-4-mFc expression, the cDNA plasmids carrying human IL-4 (residues 1–153) genes (Sino biological Inc.,HG-11846-CM) were subcloned in frame into pcDNA3.4 vector to be expressed in the C-terminal mouse immunoglobulin Fc (hinge-CH2-CH3) fused form .

Techniques: Isolation, Binding Assay, Activity Assay, Enzyme-linked Immunosorbent Assay, Blocking Assay

Journal: Scientific Reports

Article Title: Engineering of anti-human interleukin-4 receptor alpha antibodies with potent antagonistic activity

doi: 10.1038/s41598-019-44253-9

Figure Lengend Snippet: Affinity maturation of 4 R34 and characterization of the isolated clones. ( a ) Scheme of library construction and screening of 4R34 in the format of scFab using yeast surface display technology. The indicated residues in VL-CDR3, VH-CDR2, and VH-CDR3, highlighted by “X,” were randomly mutated, while maintaining the original amino acids at each residue of 4R34 at a frequency of approximately 50%, using designed spiked oligonucleotides. Numbering is according to the Kabat definition. ( b ) Flow cytometric analysis of antigen binding and expression levels of 4R34-based scFab yeast library in each round screening by FACS. The screening conditions of antigen and sorting gate used in each round are indicated. ( c ) Comparison of association and dissociation of soluble IL-4Rα antigen at 10 nM to immobilized anti-IL-4Rα antibodies (Abs), as measured by bio‐layer interferometry. ( d ) IL-4Rα-blocking activity of the indicated Abs, as determined by SEAP secretion levels from HEK-Blue TM IL-4/IL-13 cells after stimulation with rhIL-4 (100 pM) in the presence of the Abs (20 and 100 nM) for 24 h. Data are presented as percentage (mean ± SD (n = 3)) in SEAP levels relative to PBS-treated samples.

Article Snippet: For hIL-4-mFc expression, the cDNA plasmids carrying human IL-4 (residues 1–153) genes (Sino biological Inc.,HG-11846-CM) were subcloned in frame into pcDNA3.4 vector to be expressed in the C-terminal mouse immunoglobulin Fc (hinge-CH2-CH3) fused form .

Techniques: Isolation, Clone Assay, Binding Assay, Expressing, Blocking Assay, Activity Assay

Journal: Scientific Reports

Article Title: Engineering of anti-human interleukin-4 receptor alpha antibodies with potent antagonistic activity

doi: 10.1038/s41598-019-44253-9

Figure Lengend Snippet: Engineering and characterization of 4 R34.1.19. ( a ) Library construction scheme of 4 R34.1, where the indicated residues in the VL-CDR1 and VH-CDR1 were randomized with NNK degenerate codon that encodes all 20 amino acids. ( b ) Flow cytometric analysis of antigen binding and expression levels of 4 R34.1-based scFab yeast library in each round screening by FACS. The screening conditions of antigen and sorting gate used in each round are indicated. ( c ) Binding isotherms of the immobilized anti-IL-4Rα Ab 4R34.1.19 to soluble antigen IL-4Rα, measured by bio‐layer interferometry. The concentrations of IL-4Rα analysed are indicated (colored). ( d , e ) IL-4Rα blocking activity of the indicated Abs, as determined by SEAP levels from HEK-Blue TM IL-4/IL-13 cells after stimulation with rhIL-4 (100 pM) ( d ) or rhIL-13 (1 nM) ( e ) in the presence of the Abs (20 and 100 nM) for 24 h. Data are presented as percentage (mean ± SD (n = 3)) in SEAP levels relative to phosphate buffer saline (PBS)-treated samples. Statistical analysis was performed using a two-way ANOVA followed by the Newman-Keuls post-test. * P < 0.05, ** P < 0.01, *** P < 0.001; ns, not significant versus dupilumab analogue. ( f ) Binding specificity of the indicated Abs (20 and 100 nM) for cell surface expressed IL-4Rα, as analysed in IL-4Rα-expressing THP-1 cells and IL-4Rα-deficient Molt-4 cells by flow cytometry. Representative histograms from three independent experiments are shown.

Article Snippet: For hIL-4-mFc expression, the cDNA plasmids carrying human IL-4 (residues 1–153) genes (Sino biological Inc.,HG-11846-CM) were subcloned in frame into pcDNA3.4 vector to be expressed in the C-terminal mouse immunoglobulin Fc (hinge-CH2-CH3) fused form .

Techniques: Binding Assay, Expressing, Blocking Assay, Activity Assay, Flow Cytometry

Journal: Scientific Reports

Article Title: Engineering of anti-human interleukin-4 receptor alpha antibodies with potent antagonistic activity

doi: 10.1038/s41598-019-44253-9

Figure Lengend Snippet: Inhibitory effects of anti-IL-4Rα Abs on IL-4-stimulated T cell proliferation and T H 2 differentiation. ( a ) Dose-dependent blocking effects of anti-IL-4Rα Abs on the proliferation of T cells among PHA-activated PBMCs in response to rhIL-4 (500 pM), determined by CTG assay after 72 h culture. Data are represented as mean ± SD (n = 3). ( b,c ) Inhibitory effects of anti-IL-4Rα Abs (100 nM) on the T H 2 differentiation of naïve CD4 + CD45RO − T cells from healthy donors or asthmatic patients after 7 days culture in T H 2-skewing conditions in the presence of rhIL-4 (500 pM) and anti-IL-4Rα Abs (100 nM). The number of IL-4-producing T H 2 cells were determined by ELISPOT. Quantification of spot forming T cells ( b ) and representative image both healthy donor and asthmatic patient ( c ) are shown. In ( b ), error bars, ± SD (n = 3). Statistical analyses were performed using a one-way ANOVA followed by the Newman-Keuls post-test. * P < 0.05, ** P < 0.01, *** P < 0.001; ns, not significant versus PBS-treated group.

Article Snippet: For hIL-4-mFc expression, the cDNA plasmids carrying human IL-4 (residues 1–153) genes (Sino biological Inc.,HG-11846-CM) were subcloned in frame into pcDNA3.4 vector to be expressed in the C-terminal mouse immunoglobulin Fc (hinge-CH2-CH3) fused form .

Techniques: Blocking Assay, CTG Assay, Enzyme-linked Immunospot

Journal: Scientific Reports

Article Title: Engineering of anti-human interleukin-4 receptor alpha antibodies with potent antagonistic activity

doi: 10.1038/s41598-019-44253-9

Figure Lengend Snippet: Epitope mapping of anti-IL-4Rα Abs by alanine scanning mutagenesis. ( a ) Competitive ELISA showing the percentage of bound IL-4Rα (10 and 50 nM) to plate-coated hIL-4-mFc in the presence of the indicated Abs (20, 100, and 500 nM) compared to that without the Ab competitor. Data are represented as mean ± SD (n = 3). Statistical analyses were performed using a two-way ANOVA followed by the Newman-Keuls post-test. * P < 0.05, ** P < 0.01, *** P < 0.001; ns, not significant versus dupilumab analogue. ( b ) Overall structure of the human IL-4Rα:IL-4 complex (PDB: 1IAR). Magnified section shows the residues of IL-4Rα putatively involved in IL-4 binding. ( c ) The percent relative binding of the indicated hIL-4-mFc (5 nM) and anti-IL-4Rα Abs (2.5 nM of 4 R34 and 100 pM of 4 R34.1, 4 R34.1.19 and dupilumab analogue) to IL-4Rα alanine mutants compared to that of wild-type IL-4Rα. Data are represented as mean ± SD (n = 3). Statistical analyses were performed using a one-way ANOVA followed by the Newman-Keuls post-test. * P < 0.05, ** P < 0.01, *** P < 0.001 versus binding to wild-type IL-4Rα.

Article Snippet: For hIL-4-mFc expression, the cDNA plasmids carrying human IL-4 (residues 1–153) genes (Sino biological Inc.,HG-11846-CM) were subcloned in frame into pcDNA3.4 vector to be expressed in the C-terminal mouse immunoglobulin Fc (hinge-CH2-CH3) fused form .

Techniques: Mutagenesis, Competitive ELISA, Binding Assay