elisa kit  (Thermo Fisher)


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    Thermo Fisher elisa kit
    ALA treatment modulates cytokine secretion. Splenocytes were isolated from PBS-treated and ALA-treated NOD mice and stimulated with PMA and ionomycin. Culture medium containing secreted <t>cytokines</t> was measured by <t>ELISA.</t> The secretion of inflammatory cytokines ( A ) IFN-γ (** p = 0.0017), ( B ) TNF-α (** p = 0.0017), ( C ) IL-6 (* p = 0.0376) and ( D ) IL-17 was decreased in ALA-treated groups (** p = 0.0016). The secretion of anti-inflammatory cytokines ( E ) TGF-β (* p = 0.0313) and ( F ) IL-10 (* p = 0.0111) was increased in ALA-treated mice. Data are expressed as means ± SEM ( n = 5; * p
    Elisa Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Alpha-Lipoic Acid Inhibits Spontaneous Diabetes and Autoimmune Recurrence in Non-Obese Diabetic Mice by Enhancing Differentiation of Regulatory T Cells and Showed Potential for Use in Cell Therapies for the Treatment of Type 1 Diabetes"

    Article Title: Alpha-Lipoic Acid Inhibits Spontaneous Diabetes and Autoimmune Recurrence in Non-Obese Diabetic Mice by Enhancing Differentiation of Regulatory T Cells and Showed Potential for Use in Cell Therapies for the Treatment of Type 1 Diabetes

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms23031169

    ALA treatment modulates cytokine secretion. Splenocytes were isolated from PBS-treated and ALA-treated NOD mice and stimulated with PMA and ionomycin. Culture medium containing secreted cytokines was measured by ELISA. The secretion of inflammatory cytokines ( A ) IFN-γ (** p = 0.0017), ( B ) TNF-α (** p = 0.0017), ( C ) IL-6 (* p = 0.0376) and ( D ) IL-17 was decreased in ALA-treated groups (** p = 0.0016). The secretion of anti-inflammatory cytokines ( E ) TGF-β (* p = 0.0313) and ( F ) IL-10 (* p = 0.0111) was increased in ALA-treated mice. Data are expressed as means ± SEM ( n = 5; * p
    Figure Legend Snippet: ALA treatment modulates cytokine secretion. Splenocytes were isolated from PBS-treated and ALA-treated NOD mice and stimulated with PMA and ionomycin. Culture medium containing secreted cytokines was measured by ELISA. The secretion of inflammatory cytokines ( A ) IFN-γ (** p = 0.0017), ( B ) TNF-α (** p = 0.0017), ( C ) IL-6 (* p = 0.0376) and ( D ) IL-17 was decreased in ALA-treated groups (** p = 0.0016). The secretion of anti-inflammatory cytokines ( E ) TGF-β (* p = 0.0313) and ( F ) IL-10 (* p = 0.0111) was increased in ALA-treated mice. Data are expressed as means ± SEM ( n = 5; * p

    Techniques Used: Isolation, Mouse Assay, Enzyme-linked Immunosorbent Assay

    2) Product Images from "Heme oxygenase-1 deficiency exacerbates angiotensin II-induced aortic aneurysm in mice"

    Article Title: Heme oxygenase-1 deficiency exacerbates angiotensin II-induced aortic aneurysm in mice

    Journal: Oncotarget

    doi: 10.18632/oncotarget.11917

    Lack of HO-1 aggravates inflammatory responses in AAAs and in angiotensin II-treated macrophages A. - F. Four weeks after angiotensin II infusion, HO-1 +/+ apoE −/− and HO-1 −/− apoE −/− mouse AAA sections were immunostained with antibodies for MCP-1 A. and D. , IL-6 B. and E. , or TNF-α C. and F. Brown color indicates positive staining. G. - I. Peritoneal macrophages from HO-1 +/+ apoE −/− and HO-1 −/− apoE −/− mice (3-4 different isolates from each genotype) were stimulated with angiotensin II (10 μmol/L) and conditioned medium harvested at different time points. Concentrations of inflammatory cytokines were measured using an ELISA kit, normalized to total protein amount and expressed as pg/μg protein. G. MCP-1 ( n = 4 each genotype). H. IL-6 ( n = 3 each genotype). I. TNF-α ( n = 4 each genotype). Values are mean ± SE. * P
    Figure Legend Snippet: Lack of HO-1 aggravates inflammatory responses in AAAs and in angiotensin II-treated macrophages A. - F. Four weeks after angiotensin II infusion, HO-1 +/+ apoE −/− and HO-1 −/− apoE −/− mouse AAA sections were immunostained with antibodies for MCP-1 A. and D. , IL-6 B. and E. , or TNF-α C. and F. Brown color indicates positive staining. G. - I. Peritoneal macrophages from HO-1 +/+ apoE −/− and HO-1 −/− apoE −/− mice (3-4 different isolates from each genotype) were stimulated with angiotensin II (10 μmol/L) and conditioned medium harvested at different time points. Concentrations of inflammatory cytokines were measured using an ELISA kit, normalized to total protein amount and expressed as pg/μg protein. G. MCP-1 ( n = 4 each genotype). H. IL-6 ( n = 3 each genotype). I. TNF-α ( n = 4 each genotype). Values are mean ± SE. * P

    Techniques Used: Staining, Mouse Assay, Enzyme-linked Immunosorbent Assay

    CORM-3 reduces angiotensin II-induced inflammatory cytokine productions in HO-1 −/− apoE −/− macrophages Peritoneal macrophages from HO-1 −/− apoE −/− mice were harvested (4 different isolates) and treated with 0, 2 or 5 μmol/L CORM-3 in the presence or absence of angiotensin II (10 μmol/L). Conditioned medium were harvested 24 h later. Inflammatory cytokines were measured using ELISA kits, normalized to total protein amount and expressed as fold of control. A. MCP-1 ( n = 4). B. IL-6 ( n = 4). C. TNF-α ( n = 4). Values are mean ± SE. * P
    Figure Legend Snippet: CORM-3 reduces angiotensin II-induced inflammatory cytokine productions in HO-1 −/− apoE −/− macrophages Peritoneal macrophages from HO-1 −/− apoE −/− mice were harvested (4 different isolates) and treated with 0, 2 or 5 μmol/L CORM-3 in the presence or absence of angiotensin II (10 μmol/L). Conditioned medium were harvested 24 h later. Inflammatory cytokines were measured using ELISA kits, normalized to total protein amount and expressed as fold of control. A. MCP-1 ( n = 4). B. IL-6 ( n = 4). C. TNF-α ( n = 4). Values are mean ± SE. * P

    Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay

    3) Product Images from "A LRRK2 GTP Binding Inhibitor, 68, Reduces LPS-Induced Signaling Events and TNF-α Release in Human Lymphoblasts"

    Article Title: A LRRK2 GTP Binding Inhibitor, 68, Reduces LPS-Induced Signaling Events and TNF-α Release in Human Lymphoblasts

    Journal: Cells

    doi: 10.3390/cells10020480

    Compound 68 reduced LPS-induced TNF-α secretion. Human lymphoblasts were exposed to LPS (1 µg/mL) with or without 68 (10 µM) pretreatment for 2 h, then kept treatment for 72 h. The supernatants of culture media from each group were collected and subjected to ELISA to detect TNF-α. The TNF-α levels in each group were normalized to the total protein content from each group. * p
    Figure Legend Snippet: Compound 68 reduced LPS-induced TNF-α secretion. Human lymphoblasts were exposed to LPS (1 µg/mL) with or without 68 (10 µM) pretreatment for 2 h, then kept treatment for 72 h. The supernatants of culture media from each group were collected and subjected to ELISA to detect TNF-α. The TNF-α levels in each group were normalized to the total protein content from each group. * p

    Techniques Used: Enzyme-linked Immunosorbent Assay

    4) Product Images from "Hyperglycemia Aggravates Hepatic Ischemia and Reperfusion Injury by Inhibiting Liver-Resident Macrophage M2 Polarization via C/EBP Homologous Protein-Mediated Endoplasmic Reticulum Stress"

    Article Title: Hyperglycemia Aggravates Hepatic Ischemia and Reperfusion Injury by Inhibiting Liver-Resident Macrophage M2 Polarization via C/EBP Homologous Protein-Mediated Endoplasmic Reticulum Stress

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2017.01299

    Hyperglycemia enhances Kupffer cell (KC)-related inflammation after ischemia and reperfusion (IR). Liver partial warm IR or a sham procedure was performed in diabetic [streptozotocin (STZ)] and control (CON) mice. Six hours post-reperfusion, inflammatory gene expression in liver tissues was evaluated by quantitative RT-PCR [ (A) , n = 6/group]. Serum levels of inflammatory cytokines were measured by ELISA [ (B) , n = 6/group]. KCs from CON or STZ mice following IR or the sham procedure were cultured in vitro for 6 h. TNF-α, IL-6, and IL-10 protein levels in the culture supernatant were measured by ELISA [ (C) , n = 6/group] (* p
    Figure Legend Snippet: Hyperglycemia enhances Kupffer cell (KC)-related inflammation after ischemia and reperfusion (IR). Liver partial warm IR or a sham procedure was performed in diabetic [streptozotocin (STZ)] and control (CON) mice. Six hours post-reperfusion, inflammatory gene expression in liver tissues was evaluated by quantitative RT-PCR [ (A) , n = 6/group]. Serum levels of inflammatory cytokines were measured by ELISA [ (B) , n = 6/group]. KCs from CON or STZ mice following IR or the sham procedure were cultured in vitro for 6 h. TNF-α, IL-6, and IL-10 protein levels in the culture supernatant were measured by ELISA [ (C) , n = 6/group] (* p

    Techniques Used: Mouse Assay, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Cell Culture, In Vitro

    C/EBP homologous protein (CHOP) mediates hyperglycemic Kupffer cell (KC) pro-inflammatory activation in vitro . Diabetic [streptozotocin (STZ)] and control (CON) mice were prepared and liver partial warm ischemia and reperfusion (IR) or a sham procedure was performed. After 6 h of reperfusion, KCs were isolated and the intracellular levels of c-ATF6, ATF4, CHOP, s-XBP1, and β-actin protein were analyzed by Western blotting. Representative of three experiments (A) . Relative density ratios of target proteins in different groups to the control group (CON-Sham) were calculated [ (B) , n = 3/group]. Both CON and STZ mice were pretreated with CHOP siRNA (CHOP-siRNA) or its scramble control siRNA (SCR-siRNA) in vivo prior to IR using mannose-conjugated polymers as described in Section “ Materials and Methods .” Liver IR was performed. Six hours post-reperfusion, KCs were isolated and the intracellular levels of c-ATF6, ATF4, CHOP, s-XBP1, and β-actin protein were analyzed by Western blotting. Representative of three experiments (C) . Relative density ratios of target proteins in different groups to the control group (CON–SCR-siRNA) were calculated [ (D) , n = 3/group]. Isolated KCs from IR-stressed livers of different groups were cultured for 6 h, and TNF-α, IL-6, and IL-10 protein levels in the culture supernatant were measured by ELISA [ (E) , n = 6/group] (* p
    Figure Legend Snippet: C/EBP homologous protein (CHOP) mediates hyperglycemic Kupffer cell (KC) pro-inflammatory activation in vitro . Diabetic [streptozotocin (STZ)] and control (CON) mice were prepared and liver partial warm ischemia and reperfusion (IR) or a sham procedure was performed. After 6 h of reperfusion, KCs were isolated and the intracellular levels of c-ATF6, ATF4, CHOP, s-XBP1, and β-actin protein were analyzed by Western blotting. Representative of three experiments (A) . Relative density ratios of target proteins in different groups to the control group (CON-Sham) were calculated [ (B) , n = 3/group]. Both CON and STZ mice were pretreated with CHOP siRNA (CHOP-siRNA) or its scramble control siRNA (SCR-siRNA) in vivo prior to IR using mannose-conjugated polymers as described in Section “ Materials and Methods .” Liver IR was performed. Six hours post-reperfusion, KCs were isolated and the intracellular levels of c-ATF6, ATF4, CHOP, s-XBP1, and β-actin protein were analyzed by Western blotting. Representative of three experiments (C) . Relative density ratios of target proteins in different groups to the control group (CON–SCR-siRNA) were calculated [ (D) , n = 3/group]. Isolated KCs from IR-stressed livers of different groups were cultured for 6 h, and TNF-α, IL-6, and IL-10 protein levels in the culture supernatant were measured by ELISA [ (E) , n = 6/group] (* p

    Techniques Used: Activation Assay, In Vitro, Mouse Assay, Isolation, Western Blot, In Vivo, Cell Culture, Enzyme-linked Immunosorbent Assay

    5) Product Images from "Fermented red ginseng and ginsenoside Rd alleviate ovalbumin-induced allergic rhinitis in mice by suppressing IgE, interleukin-4, and interleukin-5 expression"

    Article Title: Fermented red ginseng and ginsenoside Rd alleviate ovalbumin-induced allergic rhinitis in mice by suppressing IgE, interleukin-4, and interleukin-5 expression

    Journal: Journal of Ginseng Research

    doi: 10.1016/j.jgr.2019.02.006

    Effect of ginsenoside Rd on the nasal allergy symptoms and allergic rhinitis (AR) biomarker expression in the nasal mucosa and blood of mice with ovalbumin (OVA)-induced AR. (A) Effect on (a) nasal allergy symptoms and (b) nasal IL-4 and (c) IL-5 levels. (B) Effect on blood (a) IL-4, (b) IL-5, and (c) IgE levels. Cytokine and IgE levels were measured using ELISA kits. The control group was treated with vehicle instead of OVA and test agents. Test agents (vehicle or ginsenoside Rd [20 mg/kg/day]) were administered orally daily for 5 days for treating mice with OVA-induced AR. Data are shown as box plots ( n = 8). Means with the same letters are not significantly different ( p
    Figure Legend Snippet: Effect of ginsenoside Rd on the nasal allergy symptoms and allergic rhinitis (AR) biomarker expression in the nasal mucosa and blood of mice with ovalbumin (OVA)-induced AR. (A) Effect on (a) nasal allergy symptoms and (b) nasal IL-4 and (c) IL-5 levels. (B) Effect on blood (a) IL-4, (b) IL-5, and (c) IgE levels. Cytokine and IgE levels were measured using ELISA kits. The control group was treated with vehicle instead of OVA and test agents. Test agents (vehicle or ginsenoside Rd [20 mg/kg/day]) were administered orally daily for 5 days for treating mice with OVA-induced AR. Data are shown as box plots ( n = 8). Means with the same letters are not significantly different ( p

    Techniques Used: Biomarker Assay, Expressing, Mouse Assay, Enzyme-linked Immunosorbent Assay

    Effects of wRG, eRG, and fRG on the nasal allergic symptoms and allergic rhinitis (AR) marker expression in the nasal mucosa and blood of mice with ovalbumin (OVA)-induced AR. (A) Effects on (a) the nasal allergy symptoms, (b) nasal IL-4 levels, and (c) IL-5 levels. (B) Effects on blood (a) IL-4, (b) IL-5, and (c) IgE levels. (C) Histological examination of nasal tissues, stained with H E. Cytokine and IgE levels were measured using ELISA kits. The control group was treated with vehicle instead of OVA and test agents. Test agents (vehicle, wRG [50 mg/kg/day], eRG [50 mg/kg/day], fRG [50 mg/kg/day], or dexamethasone [Dx, 1 mg/kg/day]) were administered orally (for wRG, eRG, and fRG) or intraperitoneally (for Dx) daily for 5 days for treating mice with OVA-induced AR. Data are shown as box plots ( n = 8). Means with the same letters are not significantly different ( p
    Figure Legend Snippet: Effects of wRG, eRG, and fRG on the nasal allergic symptoms and allergic rhinitis (AR) marker expression in the nasal mucosa and blood of mice with ovalbumin (OVA)-induced AR. (A) Effects on (a) the nasal allergy symptoms, (b) nasal IL-4 levels, and (c) IL-5 levels. (B) Effects on blood (a) IL-4, (b) IL-5, and (c) IgE levels. (C) Histological examination of nasal tissues, stained with H E. Cytokine and IgE levels were measured using ELISA kits. The control group was treated with vehicle instead of OVA and test agents. Test agents (vehicle, wRG [50 mg/kg/day], eRG [50 mg/kg/day], fRG [50 mg/kg/day], or dexamethasone [Dx, 1 mg/kg/day]) were administered orally (for wRG, eRG, and fRG) or intraperitoneally (for Dx) daily for 5 days for treating mice with OVA-induced AR. Data are shown as box plots ( n = 8). Means with the same letters are not significantly different ( p

    Techniques Used: Marker, Expressing, Mouse Assay, Staining, Enzyme-linked Immunosorbent Assay

    6) Product Images from "Ferric citrate and ferric EDTA but not ferrous sulfate drive amphiregulin-mediated activation of the MAP kinase ERK in gut epithelial cancer cells"

    Article Title: Ferric citrate and ferric EDTA but not ferrous sulfate drive amphiregulin-mediated activation of the MAP kinase ERK in gut epithelial cancer cells

    Journal: Oncotarget

    doi: 10.18632/oncotarget.24899

    Cellular amphiregulin levels in human epithelial colorectal adenocarcinoma Caco-2 cells incubated with iron compounds Quantative data measured with Thermo Scientific™ hAREG ELISA kit. Data are presented as means, n=3 ± SD. The significance of the differences is expressed as letters a-e where a: p=2,5E −7 , b: p=2,4E −5 , c: p=1,14E −10 , d: p=9.9E −7 , e: p=0.6. Differences of p
    Figure Legend Snippet: Cellular amphiregulin levels in human epithelial colorectal adenocarcinoma Caco-2 cells incubated with iron compounds Quantative data measured with Thermo Scientific™ hAREG ELISA kit. Data are presented as means, n=3 ± SD. The significance of the differences is expressed as letters a-e where a: p=2,5E −7 , b: p=2,4E −5 , c: p=1,14E −10 , d: p=9.9E −7 , e: p=0.6. Differences of p

    Techniques Used: Incubation, Enzyme-linked Immunosorbent Assay

    ADAM17 protein levels in human epithelial duodenum adenocarcinoma Caco-2 cells The cells were incubated with ferric citrate, ferric EDTA, ferrous sulfate, or control cells with no additional iron to the growth medium. ADAM17 levels were measured with the Thermo Scientific™ hTACE ELISA kit. Data are expressed as means ± SD (n=3). The significance of the differences is expressed as letters a-d where a: p=0.029, b: p=0.026, c: p=0.028, d: p=0.04, e: p=0.08. Differences of p
    Figure Legend Snippet: ADAM17 protein levels in human epithelial duodenum adenocarcinoma Caco-2 cells The cells were incubated with ferric citrate, ferric EDTA, ferrous sulfate, or control cells with no additional iron to the growth medium. ADAM17 levels were measured with the Thermo Scientific™ hTACE ELISA kit. Data are expressed as means ± SD (n=3). The significance of the differences is expressed as letters a-d where a: p=0.029, b: p=0.026, c: p=0.028, d: p=0.04, e: p=0.08. Differences of p

    Techniques Used: Incubation, Enzyme-linked Immunosorbent Assay

    7) Product Images from "Expression and role of interleukin-9 in Vogt-Koyanagi-Harada disease"

    Article Title: Expression and role of interleukin-9 in Vogt-Koyanagi-Harada disease

    Journal: Molecular Vision

    doi:

    Effect of rIL-9 on IFN-γ production. The effect of recombinant human interleukin-9 (rIL-9; 100 ng/ml) on interferon (IFN)-γ production by peripheral blood mononuclear cells (PBMCs) and CD4+ T cells from patients with active Vogt-Koyanagi-Harada (VKH) (n = 14) disease, patients with inactive VKH disease (n = 14), and normal controls (n = 13). Cells were cultured with or without rIL-9 in the presence of anti-CD3 and anti-CD28 antibodies for 3 days, and IFN-γ was measured with enzyme-linked immunosorbent assays.
    Figure Legend Snippet: Effect of rIL-9 on IFN-γ production. The effect of recombinant human interleukin-9 (rIL-9; 100 ng/ml) on interferon (IFN)-γ production by peripheral blood mononuclear cells (PBMCs) and CD4+ T cells from patients with active Vogt-Koyanagi-Harada (VKH) (n = 14) disease, patients with inactive VKH disease (n = 14), and normal controls (n = 13). Cells were cultured with or without rIL-9 in the presence of anti-CD3 and anti-CD28 antibodies for 3 days, and IFN-γ was measured with enzyme-linked immunosorbent assays.

    Techniques Used: Recombinant, Cell Culture

    8) Product Images from "Abrogation of Endogenous Glycolipid Antigen Presentation on Myelin-Laden Macrophages by D-Sphingosine Ameliorates the Pathogenesis of Experimental Autoimmune Encephalomyelitis"

    Article Title: Abrogation of Endogenous Glycolipid Antigen Presentation on Myelin-Laden Macrophages by D-Sphingosine Ameliorates the Pathogenesis of Experimental Autoimmune Encephalomyelitis

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2019.00404

    Deprivation of antigenic presentation of Mye-Galcer by D-Sphingosine. (A) Chemical sketch of D-Sphingosine. (B) Flow cytometric analysis of Mye-Galcer presentation by macrophages treated with D-Sphingosine, myelin, or the combination of D-Sphingosine and myelin. Macrophages were cultured in 10% cs-FBS media during the treatments. (C) Flow cytometric analysis of CD1d up-regulation by macrophages described in 4 (B) . (D,E) IL-17A production in myelin-laden macrophages/T cell co-culture system treated with or without D-sphingosine measured by CAB assays coupled with Flow cytometric analysis (D) and ELISA (E) . CD1d neutralizing antibody was employed as in myelin-laden macrophages/T cell co-culture system as an additional control to confirm that the increase of IL-17 is due to CD1d-mediated antigen presentation ( E right). ** p
    Figure Legend Snippet: Deprivation of antigenic presentation of Mye-Galcer by D-Sphingosine. (A) Chemical sketch of D-Sphingosine. (B) Flow cytometric analysis of Mye-Galcer presentation by macrophages treated with D-Sphingosine, myelin, or the combination of D-Sphingosine and myelin. Macrophages were cultured in 10% cs-FBS media during the treatments. (C) Flow cytometric analysis of CD1d up-regulation by macrophages described in 4 (B) . (D,E) IL-17A production in myelin-laden macrophages/T cell co-culture system treated with or without D-sphingosine measured by CAB assays coupled with Flow cytometric analysis (D) and ELISA (E) . CD1d neutralizing antibody was employed as in myelin-laden macrophages/T cell co-culture system as an additional control to confirm that the increase of IL-17 is due to CD1d-mediated antigen presentation ( E right). ** p

    Techniques Used: Flow Cytometry, Cell Culture, Co-Culture Assay, Enzyme-linked Immunosorbent Assay

    9) Product Images from "CAR-T Cells Targeting Epstein-Barr Virus gp350 Validated in a Humanized Mouse Model of EBV Infection and Lymphoproliferative Disease"

    Article Title: CAR-T Cells Targeting Epstein-Barr Virus gp350 Validated in a Humanized Mouse Model of EBV Infection and Lymphoproliferative Disease

    Journal: Molecular Therapy Oncolytics

    doi: 10.1016/j.omto.2020.08.005

    7A1-gp350CAR-T Cells Recognize and Kill gp350 + B95-8 Cells Latently Infected with EBV (A) Experimental scheme. CAR-T cells were co-cultured with B95-8 target cells at 0.1:1, 1:1, and 10:1 E:T ratios for 38–86 h. Analyses were performed to follow activation and proliferation of CAR-T cells (IFN-γ ELISA and FACS) and to assess the effects on viability of target cells (FACS). (B) IFN-ɣ detection in cell supernatants after 38 h, showing that, after co-culture, 7A1-gp350CAR-T cells produced significantly higher levels of IFN-γ than control gBCAR-T cells. ∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001 (n = 9). (C and D) Analyses of CD4 + (C) or CD8 + (D) CAR-T cells by flow cytometry to evaluate proliferation after 38 or 86 h of co-culture. The graphs show the percentages of proliferated viable lymphocytes showing loss of the CellTrace dye (n = 3). Higher 7A1-gp350CAR-T cell proliferation of was observed after 86 h of co-culture. ∗p ≤ 0.05. (E) Left: representative examples showing the remaining viable B95-8 cells (negative for the viability dye) after 38 h of co-culture with gBCAR-T (center panel) or with gp350CAR-T cells (bottom panel). B95-8 cells with no T cells are shown as a control (top panel) Right: quantified remaining viable B95-8 cells showing cytotoxic effects for co-culture at a 10:1 E:T ratio (n = 3). Cultures with no T cells were used as references. (F) Left: representative examples showing the remaining viable gp350 + B95-8 cells (negative for the viability dye) after 38 h of co-cultures with gBCAR-T (center panel) or with gp350CAR-T cells (bottom panel). B95-8 cells with no T cells are shown as a control (top panel). Right: quantified remaining viable gp350 + B95-8 cells showing cytotoxic effects for co- culture at a 10:1 E:T ratio (n = 3). ∗p ≤ 0.05 (n = 3). A summary of the descriptive statistical analyses is shown in Table S2 .
    Figure Legend Snippet: 7A1-gp350CAR-T Cells Recognize and Kill gp350 + B95-8 Cells Latently Infected with EBV (A) Experimental scheme. CAR-T cells were co-cultured with B95-8 target cells at 0.1:1, 1:1, and 10:1 E:T ratios for 38–86 h. Analyses were performed to follow activation and proliferation of CAR-T cells (IFN-γ ELISA and FACS) and to assess the effects on viability of target cells (FACS). (B) IFN-ɣ detection in cell supernatants after 38 h, showing that, after co-culture, 7A1-gp350CAR-T cells produced significantly higher levels of IFN-γ than control gBCAR-T cells. ∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001 (n = 9). (C and D) Analyses of CD4 + (C) or CD8 + (D) CAR-T cells by flow cytometry to evaluate proliferation after 38 or 86 h of co-culture. The graphs show the percentages of proliferated viable lymphocytes showing loss of the CellTrace dye (n = 3). Higher 7A1-gp350CAR-T cell proliferation of was observed after 86 h of co-culture. ∗p ≤ 0.05. (E) Left: representative examples showing the remaining viable B95-8 cells (negative for the viability dye) after 38 h of co-culture with gBCAR-T (center panel) or with gp350CAR-T cells (bottom panel). B95-8 cells with no T cells are shown as a control (top panel) Right: quantified remaining viable B95-8 cells showing cytotoxic effects for co-culture at a 10:1 E:T ratio (n = 3). Cultures with no T cells were used as references. (F) Left: representative examples showing the remaining viable gp350 + B95-8 cells (negative for the viability dye) after 38 h of co-cultures with gBCAR-T (center panel) or with gp350CAR-T cells (bottom panel). B95-8 cells with no T cells are shown as a control (top panel). Right: quantified remaining viable gp350 + B95-8 cells showing cytotoxic effects for co- culture at a 10:1 E:T ratio (n = 3). ∗p ≤ 0.05 (n = 3). A summary of the descriptive statistical analyses is shown in Table S2 .

    Techniques Used: Infection, Cell Culture, Activation Assay, Enzyme-linked Immunosorbent Assay, FACS, Co-Culture Assay, Produced, Flow Cytometry

    10) Product Images from "Benzyl Isothiocyanate Suppresses Pancreatic Tumor Angiogenesis and Invasion by Inhibiting HIF-?/VEGF/Rho-GTPases: Pivotal Role of STAT-3"

    Article Title: Benzyl Isothiocyanate Suppresses Pancreatic Tumor Angiogenesis and Invasion by Inhibiting HIF-?/VEGF/Rho-GTPases: Pivotal Role of STAT-3

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0025799

    BITC suppresses proangiogenic proteins. A . BITC down-regulates expression of HIF-α, VEGFR-2, Rho-GTPase signaling molecules. BxPC-3 or PanC-1 cells were treated with various concentrations of BITC for 24 h and for HIF-α, cells were exposed to 1% O 2 for 12 h. Whole cell lysates were prepared with urea-CHAPS buffer. Cell lysates were analyzed by SDS-PAGE followed by western blot. B . BITC down-regulates mRNA levels of VEGFR2 and MMP-2 in BxPC-3 and PanC-1 cells. Cells were treated with different concentrations of BITC and total RNA was isolated with Trizol. Total RNA was analyzed for the expression levels of VEGFR-2 and MMP-2 by RT-PCR. GAPDH was used as internal control. Quantitative analysis of mRNA expression levels were performed by Image J software and presented as bar diagram (lower panel) C . BITC inhibits HIF-1α and VEGF expression by inhibiting phosphorylation of STAT-3. 0.3×10 6 cells were plated in 6-well plate and treated with 20 ng/mL IL-6 and 10 µM BITC for 24 h. Cells were analyzed for STAT-3 (Tyr 705), STAT-3, HIF-1α, VEGF, and RhoC expression by western blot. D . STAT-3 is required for BITC mediated inhibition of HIF-1α and VEGF expression. BxPC-3 cells were transfected with 2 µg of STAT-3α over-expressing plasmid and, in another experiment, STAT-3 was either transiently silenced or permanently knocked out by shRNA. Transfected cells were treated with or without 10 µM BITC for 24 h after 48 h of transfection. Cells were lysed and analyzed by western blot. Right panel showing secreted VEGF level was evaluated in STAT-3 over-expressing or silenced BxPC-3 cells by ELISA as described above. * Values of p
    Figure Legend Snippet: BITC suppresses proangiogenic proteins. A . BITC down-regulates expression of HIF-α, VEGFR-2, Rho-GTPase signaling molecules. BxPC-3 or PanC-1 cells were treated with various concentrations of BITC for 24 h and for HIF-α, cells were exposed to 1% O 2 for 12 h. Whole cell lysates were prepared with urea-CHAPS buffer. Cell lysates were analyzed by SDS-PAGE followed by western blot. B . BITC down-regulates mRNA levels of VEGFR2 and MMP-2 in BxPC-3 and PanC-1 cells. Cells were treated with different concentrations of BITC and total RNA was isolated with Trizol. Total RNA was analyzed for the expression levels of VEGFR-2 and MMP-2 by RT-PCR. GAPDH was used as internal control. Quantitative analysis of mRNA expression levels were performed by Image J software and presented as bar diagram (lower panel) C . BITC inhibits HIF-1α and VEGF expression by inhibiting phosphorylation of STAT-3. 0.3×10 6 cells were plated in 6-well plate and treated with 20 ng/mL IL-6 and 10 µM BITC for 24 h. Cells were analyzed for STAT-3 (Tyr 705), STAT-3, HIF-1α, VEGF, and RhoC expression by western blot. D . STAT-3 is required for BITC mediated inhibition of HIF-1α and VEGF expression. BxPC-3 cells were transfected with 2 µg of STAT-3α over-expressing plasmid and, in another experiment, STAT-3 was either transiently silenced or permanently knocked out by shRNA. Transfected cells were treated with or without 10 µM BITC for 24 h after 48 h of transfection. Cells were lysed and analyzed by western blot. Right panel showing secreted VEGF level was evaluated in STAT-3 over-expressing or silenced BxPC-3 cells by ELISA as described above. * Values of p

    Techniques Used: Expressing, SDS Page, Western Blot, Isolation, Reverse Transcription Polymerase Chain Reaction, Software, Inhibition, Transfection, Plasmid Preparation, shRNA, Enzyme-linked Immunosorbent Assay

    BITC inhibits angiogenesis in HUVECs. A . BITC inhibits the secretion of proangiogenic factors from HUVECs. Cells were plated, stimulated with VEGF, and treated with BITC for 24 h. Media was collected and assayed for MMP-2 and VEGF by ELISA kit. *p
    Figure Legend Snippet: BITC inhibits angiogenesis in HUVECs. A . BITC inhibits the secretion of proangiogenic factors from HUVECs. Cells were plated, stimulated with VEGF, and treated with BITC for 24 h. Media was collected and assayed for MMP-2 and VEGF by ELISA kit. *p

    Techniques Used: Enzyme-linked Immunosorbent Assay

    BITC inhibits secretion of proangiogenic factors from BxPC-3 and PanC-1 cells under normoxic and hypoxic conditions. Serum-starved BxPC-3 or PanC-1 cells were treated with various concentrations of BITC in a 96-well plate and incubated for 24 h. For hypoxia treatment, cells were exposed to 1% pO 2 for 24 h. Culture supernatants were collected and assayed for MMP-2 or VEGF by ELISA kit -. A–B . BITC suppresses secretion of VEGF from BxPC-3 and PanC-1 cells. C–D . BITC blocks secretion of MMP-2. Values are mean ± SD of triplicates. *p
    Figure Legend Snippet: BITC inhibits secretion of proangiogenic factors from BxPC-3 and PanC-1 cells under normoxic and hypoxic conditions. Serum-starved BxPC-3 or PanC-1 cells were treated with various concentrations of BITC in a 96-well plate and incubated for 24 h. For hypoxia treatment, cells were exposed to 1% pO 2 for 24 h. Culture supernatants were collected and assayed for MMP-2 or VEGF by ELISA kit -. A–B . BITC suppresses secretion of VEGF from BxPC-3 and PanC-1 cells. C–D . BITC blocks secretion of MMP-2. Values are mean ± SD of triplicates. *p

    Techniques Used: Incubation, Enzyme-linked Immunosorbent Assay

    11) Product Images from "Labeling monocytes with gold nanoparticles to track their recruitment in atherosclerosis with computed tomography"

    Article Title: Labeling monocytes with gold nanoparticles to track their recruitment in atherosclerosis with computed tomography

    Journal: Biomaterials

    doi: 10.1016/j.biomaterials.2016.02.009

    (A) Viability of monocytes after treatment with different AuNP formulations at increasing concentrations. Viability % expressed as the ratio of living cells to total cells in the well. (B) Results from ELISA for TNF-α and IL-6 release after treatment (0.5 mg/ml) with different AuNP formulations. * P
    Figure Legend Snippet: (A) Viability of monocytes after treatment with different AuNP formulations at increasing concentrations. Viability % expressed as the ratio of living cells to total cells in the well. (B) Results from ELISA for TNF-α and IL-6 release after treatment (0.5 mg/ml) with different AuNP formulations. * P

    Techniques Used: Enzyme-linked Immunosorbent Assay

    Mouse monocytes from the spleen were cultured and evaluated in vitro for (A) viability using LIVE/DEAD assay, (B) TNF-α release using ELISA, and (C) AuNP cell uptake using ICP-OES for primary monocytes compared to the cell line.
    Figure Legend Snippet: Mouse monocytes from the spleen were cultured and evaluated in vitro for (A) viability using LIVE/DEAD assay, (B) TNF-α release using ELISA, and (C) AuNP cell uptake using ICP-OES for primary monocytes compared to the cell line.

    Techniques Used: Cell Culture, In Vitro, Live Dead Assay, Enzyme-linked Immunosorbent Assay

    12) Product Images from "Transthyretin participates in beta-amyloid transport from the brain to the liver- involvement of the low-density lipoprotein receptor-related protein 1?"

    Article Title: Transthyretin participates in beta-amyloid transport from the brain to the liver- involvement of the low-density lipoprotein receptor-related protein 1?

    Journal: Scientific Reports

    doi: 10.1038/srep20164

    Effect of TTR in Aβ peptide internalization by hepatocytes: ( A ) FAM-Aβ1-42 internalization by SAHep cells, in the absence or presence of increasing concentrations of h rTTR, as measured by flow cytometry. TTR concentrations up to 4.5–7.5 μg/mL resulted in increased Aβ internalization by cells. N = 3 for each condition. ( B ) Flow cytometry of primary cultures of hepatocytes derived from mice with different genetic TTR backgrounds; hepatocytes derived from TTR+/+ mice showed significantly more internalization of FAM-Aβ1-42 than those derived from TTR+/− and from TTR−/−. N = 11, N = 8, N = 14, N = 6 for hepatocytes derived from TTR +/+, TTR +/−, TTR −/− and h rTTR treated TTR −/− mice, respectively. ( C ) moTTR levels in supernatants of primary hepatocytes measured by ELISA confirmed the genetic reduction in TTR+/− which showed about half of the TTR in TTR+/+, while TTR−/− produced no TTR protein. N = 7 for TTR+/+ and −/− mice and N = 5 for TTR +/−.
    Figure Legend Snippet: Effect of TTR in Aβ peptide internalization by hepatocytes: ( A ) FAM-Aβ1-42 internalization by SAHep cells, in the absence or presence of increasing concentrations of h rTTR, as measured by flow cytometry. TTR concentrations up to 4.5–7.5 μg/mL resulted in increased Aβ internalization by cells. N = 3 for each condition. ( B ) Flow cytometry of primary cultures of hepatocytes derived from mice with different genetic TTR backgrounds; hepatocytes derived from TTR+/+ mice showed significantly more internalization of FAM-Aβ1-42 than those derived from TTR+/− and from TTR−/−. N = 11, N = 8, N = 14, N = 6 for hepatocytes derived from TTR +/+, TTR +/−, TTR −/− and h rTTR treated TTR −/− mice, respectively. ( C ) moTTR levels in supernatants of primary hepatocytes measured by ELISA confirmed the genetic reduction in TTR+/− which showed about half of the TTR in TTR+/+, while TTR−/− produced no TTR protein. N = 7 for TTR+/+ and −/− mice and N = 5 for TTR +/−.

    Techniques Used: Flow Cytometry, Cytometry, Derivative Assay, Mouse Assay, Enzyme-linked Immunosorbent Assay, Produced

    13) Product Images from "Protection of ripasudil, a Rho kinase inhibitor, in lipopolysaccharides-induced acute pneumonia in mice"

    Article Title: Protection of ripasudil, a Rho kinase inhibitor, in lipopolysaccharides-induced acute pneumonia in mice

    Journal: American Journal of Translational Research

    doi:

    Ripasudil prevents LPS-induced inflammatory response. A-D. The production of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6 and monocyte chemo-attractant protein-1 (MCP-1) was detected using ELISA. E-I. The production of TNF-α, IL-1β, IL-6 and MCP-1 was detected using western blot. Data were presented as mean ± SD from at least three experiments. Compared to the control group, ***P
    Figure Legend Snippet: Ripasudil prevents LPS-induced inflammatory response. A-D. The production of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6 and monocyte chemo-attractant protein-1 (MCP-1) was detected using ELISA. E-I. The production of TNF-α, IL-1β, IL-6 and MCP-1 was detected using western blot. Data were presented as mean ± SD from at least three experiments. Compared to the control group, ***P

    Techniques Used: Enzyme-linked Immunosorbent Assay, Western Blot

    14) Product Images from "β-Carotene Inhibits Activation of NF-κB, Activator Protein-1, and STAT3 and Regulates Abnormal Expression of Some Adipokines in 3T3-L1 Adipocytes"

    Article Title: β-Carotene Inhibits Activation of NF-κB, Activator Protein-1, and STAT3 and Regulates Abnormal Expression of Some Adipokines in 3T3-L1 Adipocytes

    Journal: Journal of Cancer Prevention

    doi: 10.15430/JCP.2018.23.1.37

    The effect of β-carotene on the expressions of adiponectin, monocyte chemoattractant protein-1 (MCP-1), and regulated on activation, normal T cell expressed and secreted (RANTES) in glucose/glucose oxidase (G/GO)-treated 3T3-L1 adipocytes. Cells were pretreated with β-carotene at a final concentration of 5 and 10 μM for 2 hours, followed by G/GO treatment for 24 hours (mRNA level) and 48 hours (protein level). mRNA expression of (A) adiponectin, (B) MCP-1, and (C) RANTES were determined by real-time PCR. β-Actin was used as a housekeeping gene for normalization. Protein levels in cell culture media for (D) adiponectin, (E) MCP-1, and (F) RANTES were measured using ELISA. Non, none (untreated cells); Con, control (G/GO-treated cells without β-carotene treatment). * P
    Figure Legend Snippet: The effect of β-carotene on the expressions of adiponectin, monocyte chemoattractant protein-1 (MCP-1), and regulated on activation, normal T cell expressed and secreted (RANTES) in glucose/glucose oxidase (G/GO)-treated 3T3-L1 adipocytes. Cells were pretreated with β-carotene at a final concentration of 5 and 10 μM for 2 hours, followed by G/GO treatment for 24 hours (mRNA level) and 48 hours (protein level). mRNA expression of (A) adiponectin, (B) MCP-1, and (C) RANTES were determined by real-time PCR. β-Actin was used as a housekeeping gene for normalization. Protein levels in cell culture media for (D) adiponectin, (E) MCP-1, and (F) RANTES were measured using ELISA. Non, none (untreated cells); Con, control (G/GO-treated cells without β-carotene treatment). * P

    Techniques Used: Activation Assay, Concentration Assay, Expressing, Real-time Polymerase Chain Reaction, Cell Culture, Enzyme-linked Immunosorbent Assay

    The effect of glucose/glucose oxidase (G/GO) treatment on the expressions of adiponectin, monocyte chemoattractant protein-1 (MCP-1), and regulated on activation, normal T cell expressed and secreted (RANTES) in 3T3-L1 adipocytes. Cells were treated with G/GO for the indicated time periods. mRNA expression of (A) adiponectin, (B) MCP-1, and (C) RANTES were determined by real-time PCR. β-Actin was used as a housekeeping gene for normalization. Protein levels in cell culture media for (D) adiponectin, (E) MCP-1, and (F) RANTES were measured using ELISA. * P
    Figure Legend Snippet: The effect of glucose/glucose oxidase (G/GO) treatment on the expressions of adiponectin, monocyte chemoattractant protein-1 (MCP-1), and regulated on activation, normal T cell expressed and secreted (RANTES) in 3T3-L1 adipocytes. Cells were treated with G/GO for the indicated time periods. mRNA expression of (A) adiponectin, (B) MCP-1, and (C) RANTES were determined by real-time PCR. β-Actin was used as a housekeeping gene for normalization. Protein levels in cell culture media for (D) adiponectin, (E) MCP-1, and (F) RANTES were measured using ELISA. * P

    Techniques Used: Activation Assay, Expressing, Real-time Polymerase Chain Reaction, Cell Culture, Enzyme-linked Immunosorbent Assay

    15) Product Images from "Cholinergic regulatory lymphocytes reestablish neuromodulation of innate immune responses in sepsis"

    Article Title: Cholinergic regulatory lymphocytes reestablish neuromodulation of innate immune responses in sepsis

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    doi: 10.4049/jimmunol.1100013

    Vagus nerve stimulation prevents lethal endotoxemia Mice underwent sham surgery (Control) or vagus nerve stimulation (VNS) started 20 mins before LPS (6mg/Kg, ip). (A) Serum TNF was analyzed by ELISA (n=4; One-way ANOVA with Bonferroni’s corrections). (B) Survival was analyzed for 2 weeks and no late deaths were observed (*p
    Figure Legend Snippet: Vagus nerve stimulation prevents lethal endotoxemia Mice underwent sham surgery (Control) or vagus nerve stimulation (VNS) started 20 mins before LPS (6mg/Kg, ip). (A) Serum TNF was analyzed by ELISA (n=4; One-way ANOVA with Bonferroni’s corrections). (B) Survival was analyzed for 2 weeks and no late deaths were observed (*p

    Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay

    CD3 + CD4 + CD25 − lymphocytes reestablish the anti-inflammatory potential of α7nR-agonists in splenocytes (A) Primary cultures of splenocytes from wild-type (WT) or nude mice were treated with α7nR-agonist choline (CHO) and/or LPS. (B) Splenocytes from wild-type mice were fractionated to isolate CD4 − , CD3 + CD4 + CD25 + or CD3 + CD4 + CD25 − cells, stimulated with endotoxin, and treated with choline (CHO, 20mM) or nicotine (NIC,2uM). (C) Purified CD4 − splenocytes were co-cultured with CD3 + CD4 + CD25 + or CD3 + CD4 + CD25 − splenocytes and treated with choline (CHO, 20mM) or nicotine (NIC,2uM). (D) Splenocytes from nude mice were co-cultured with CD3 + CD4 + CD25 + or CD3 + CD4 + CD25 − splenocytes from wild-type animals, stimulated with endotoxin, and treated with the indicated agonists. In all the experiments, splenocytes were pretreated with α7nR-agonists 30 mins prior LPS (100ng/mL). TNF concentrations were analyzed by ELISA in the conditioned media at 3 hours after stimulation. Graph represents the data of three different experiments in mean ± STD. * represents p
    Figure Legend Snippet: CD3 + CD4 + CD25 − lymphocytes reestablish the anti-inflammatory potential of α7nR-agonists in splenocytes (A) Primary cultures of splenocytes from wild-type (WT) or nude mice were treated with α7nR-agonist choline (CHO) and/or LPS. (B) Splenocytes from wild-type mice were fractionated to isolate CD4 − , CD3 + CD4 + CD25 + or CD3 + CD4 + CD25 − cells, stimulated with endotoxin, and treated with choline (CHO, 20mM) or nicotine (NIC,2uM). (C) Purified CD4 − splenocytes were co-cultured with CD3 + CD4 + CD25 + or CD3 + CD4 + CD25 − splenocytes and treated with choline (CHO, 20mM) or nicotine (NIC,2uM). (D) Splenocytes from nude mice were co-cultured with CD3 + CD4 + CD25 + or CD3 + CD4 + CD25 − splenocytes from wild-type animals, stimulated with endotoxin, and treated with the indicated agonists. In all the experiments, splenocytes were pretreated with α7nR-agonists 30 mins prior LPS (100ng/mL). TNF concentrations were analyzed by ELISA in the conditioned media at 3 hours after stimulation. Graph represents the data of three different experiments in mean ± STD. * represents p

    Techniques Used: Mouse Assay, Purification, Cell Culture, Enzyme-linked Immunosorbent Assay

    CD3 + CD4 + CD25 − lymphocytes reestablish vagal anti-inflammatory mechanism in Nude mice (A) Splenectomized (SPX) animals received CD3 + CD4 + CD25 − lymphocytes from wild-type (WT) or α7nR-knockout (Alpha7KO) animals. 24 hrs later, animals were treated with α7nR-agonist, choline (CHO, 400ug/kg; ip) and endotoxemia (LPS). (B) Splenectomized (SPX) animals received vehicle (control) or CD4 + CD25 − splenocytes from control animals, 24 hrs before vagus nerve stimulation (VNS) and endotoxemia (LPS). (C) Nude mice received vehicle (control), CD3 + CD4 + CD25 + or CD3 + CD4 + CD25 − lymphocytes from control wild-type animals, 24 hrs before vagus nerve stimulation (VNS) and endotoxemia (LPS). In all the experiments, serum TNF levels were analyzed by ELISA at 90 mins after the endotoxic challenge. Graphs represent the results of four different animals in mean ± STD. * represents p
    Figure Legend Snippet: CD3 + CD4 + CD25 − lymphocytes reestablish vagal anti-inflammatory mechanism in Nude mice (A) Splenectomized (SPX) animals received CD3 + CD4 + CD25 − lymphocytes from wild-type (WT) or α7nR-knockout (Alpha7KO) animals. 24 hrs later, animals were treated with α7nR-agonist, choline (CHO, 400ug/kg; ip) and endotoxemia (LPS). (B) Splenectomized (SPX) animals received vehicle (control) or CD4 + CD25 − splenocytes from control animals, 24 hrs before vagus nerve stimulation (VNS) and endotoxemia (LPS). (C) Nude mice received vehicle (control), CD3 + CD4 + CD25 + or CD3 + CD4 + CD25 − lymphocytes from control wild-type animals, 24 hrs before vagus nerve stimulation (VNS) and endotoxemia (LPS). In all the experiments, serum TNF levels were analyzed by ELISA at 90 mins after the endotoxic challenge. Graphs represent the results of four different animals in mean ± STD. * represents p

    Techniques Used: Mouse Assay, Knock-Out, Enzyme-linked Immunosorbent Assay

    16) Product Images from "Parasitic Helminth Cystatin Inhibits DSS-Induced Intestinal Inflammation Via IL-10+F4/80+ Macrophage Recruitment"

    Article Title: Parasitic Helminth Cystatin Inhibits DSS-Induced Intestinal Inflammation Via IL-10+F4/80+ Macrophage Recruitment

    Journal: The Korean Journal of Parasitology

    doi: 10.3347/kjp.2011.49.3.245

    The level of Th1-related cytokines in the spleen and MLNs. The level of cytokine production from lymphocytes in the spleen (upper) and MLNs (lower) was measured by ELISA. The lymphocyte was isolated from the spleen and MLNs, and stimulated with anti-CD3 antibody. After stimulation, the Th1 cytokine, IFN-γ, TNF-α, and IL-1β were measured. The ratio of each cytokine secretion level from non-stimulated state to stimulated state was compared with those in other groups and this ratio was used for the ANOVA ( * P
    Figure Legend Snippet: The level of Th1-related cytokines in the spleen and MLNs. The level of cytokine production from lymphocytes in the spleen (upper) and MLNs (lower) was measured by ELISA. The lymphocyte was isolated from the spleen and MLNs, and stimulated with anti-CD3 antibody. After stimulation, the Th1 cytokine, IFN-γ, TNF-α, and IL-1β were measured. The ratio of each cytokine secretion level from non-stimulated state to stimulated state was compared with those in other groups and this ratio was used for the ANOVA ( * P

    Techniques Used: Enzyme-linked Immunosorbent Assay, Isolation

    17) Product Images from "Interleukin-9 Facilitates Osteoclastogenesis in Rheumatoid Arthritis"

    Article Title: Interleukin-9 Facilitates Osteoclastogenesis in Rheumatoid Arthritis

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms221910397

    Interleukin (IL)-9 enhances TNF-α production during osteoclastogenesis in RA. ( A – F ) Cells derived from peripheral blood (PB) and synovial fluid (SF) of patients with RA were treated as indicated with macrophage colony-stimulating factor (M-CSF; 25 ng/mL), soluble receptor activator of nuclear factor κB ligand (sRANKL; 50 ng/mL), or IL-9 (100 ng/mL) for 4 days. Cell culture supernatants were analyzed for TNF-α ( A , B ), IL-1β ( C , D ), and IL-6 ( E , F ) using ELISA (mean ± SD; n = 8). Statistical analysis was performed using paired Student’s t -test comparing M-CSF/sRANKL/rIL-9-treated cells to M-CSF/sRANKL- or M-CSF-treated cells (*: p ≤ 0.05; ns–non significant).
    Figure Legend Snippet: Interleukin (IL)-9 enhances TNF-α production during osteoclastogenesis in RA. ( A – F ) Cells derived from peripheral blood (PB) and synovial fluid (SF) of patients with RA were treated as indicated with macrophage colony-stimulating factor (M-CSF; 25 ng/mL), soluble receptor activator of nuclear factor κB ligand (sRANKL; 50 ng/mL), or IL-9 (100 ng/mL) for 4 days. Cell culture supernatants were analyzed for TNF-α ( A , B ), IL-1β ( C , D ), and IL-6 ( E , F ) using ELISA (mean ± SD; n = 8). Statistical analysis was performed using paired Student’s t -test comparing M-CSF/sRANKL/rIL-9-treated cells to M-CSF/sRANKL- or M-CSF-treated cells (*: p ≤ 0.05; ns–non significant).

    Techniques Used: Derivative Assay, Cell Culture, Enzyme-linked Immunosorbent Assay

    18) Product Images from "Upregulated osteoprotegerin expression promotes lung cancer cell invasion by increasing miR-20a expression"

    Article Title: Upregulated osteoprotegerin expression promotes lung cancer cell invasion by increasing miR-20a expression

    Journal: Experimental and Therapeutic Medicine

    doi: 10.3892/etm.2021.10278

    Measurement of OPG levels in the serum of patients with NSCLC. (A) ELISA of OPG levels in the serum of 47 patients with NSCLC and 40 healthy individuals. (B) ELISA of OPG levels in the serum of 16 patients with NSCLC with distant metastasis and 31 patients with NSCLC without distant metastasis. *** P
    Figure Legend Snippet: Measurement of OPG levels in the serum of patients with NSCLC. (A) ELISA of OPG levels in the serum of 47 patients with NSCLC and 40 healthy individuals. (B) ELISA of OPG levels in the serum of 16 patients with NSCLC with distant metastasis and 31 patients with NSCLC without distant metastasis. *** P

    Techniques Used: Enzyme-linked Immunosorbent Assay

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    • human mouse tgf ãŽâ²1 elisa ready set go kit  (Thermo Fisher)
    90
    Thermo Fisher human mouse tgf ãŽâ²1 elisa ready set go kit
    Number of brain MNCs and their CNS antigen-specific autoreactive phenotype 14 d after MCAo. A , Number of MNCs infiltrating ipsilateral and contralateral hemisphere of the brain in vehicle and SNS/HPA blockade group (three individual experiments; vehicle, N = 15; SNS/HPA block, N = 20). B–D , Elispot experiments. B , Higher number of total brain MNCs responded to pMOG 35–55 stimulation with IFN-γ production (Th1 cells) per equal number of brain MNCs in SNS/HPA block group compared with the vehicle group. C , A similar effect was observed in ipsilateral hemisphere MNCs when only the SNS axis of SIDS was blocked with propranolol (two experiments; vehicle group, N = 5–8; SNS block, N = 6 or 7). D , Number of splenocytes reacting to pMOG 35–55 stimulation with IFN-γ, IL-4, or IL-17 secretion did not differ between the groups. E , F , <t>ELISA</t> experiments. <t>TGF-β1,</t> IL-10, IL-4, IL-17, and IFN-γ secretion (pg/ml) by pMOG 35–55 -stimulated brain MNCs, ipsilateral hemisphere ( E ) and by pMOG 35–55 -stimulated splenocytes ( F ).
    Human Mouse Tgf ãŽâ²1 Elisa Ready Set Go Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ifn α  (Thermo Fisher)
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    Evaluating the replication kinetics of, and level of interferon activation by, WT MHV and DUBmut in cell culture. (A) Replication kinetics of WT and DUBmut virus in DBT cells. (B) IFN-α11 mRNA levels in WT- and DUBmut-infected BMDMs were assessed at indicated time points by reverse transcription-quantitative PCR (qRT-PCR). (C) <t>IFN-α</t> protein levels in the supernatants of infected BMDMs were evaluated at the times indicated. (D) Comparison of IFN-α11 mRNA levels in B6 versus MDA5 −/− BMDMs at 12 h postinfection (h p.i.). (E) Assessment of levels of viral nucleocapsid (N) mRNA by qRT-PCR. (F) Replication kinetics of WT and DUBmut virus in BMDM cells. Data are representative of at least two independent experiments and are presented as means ± SD. Data in panels B and C were statistically analyzed using unpaired t tests. *, P
    Ifn α, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    human tim 3  (Thermo Fisher)
    93
    Thermo Fisher human tim 3
    Biochemical characterization of interactions between CEACAM1 and <t>TIM-3</t> a , hTIM-3 does not co-immunoprecipitate (co-IP) with ITGA5 despite interactions with hCEACAM1. HEK293T cells transfected with Flag–ITGA5 and HA–TIM-3 (ITGA5Tw) or Flag–CEACAM1 and HA–TIM-3 (CwTw). Immunoprecipitation with anti-HA antibody and immunoblotted (IB) with anti-Flag antibody are shown. Input represents anti-Flag immunoblot of lysates. b, Co-immunoprecipitation of human TIM-3 and CEACAM1 from activated primary human T cells after N -glycanase treatment of lystates followed by immunoprecipitation with anti-human TIM-3 antibodies (2E2, 2E12 or 3F9) or IgG as control and immunoblotted with anti-human CEACAM1 antibody (5F4). Protein lystates from HeLa-CEACAM1 transfectants treated with N -glycanase followed by immunoprecipitation with 5F4 and the immune complex used as positive control (pos). c , mTIM-3 interacts with mCEACAM1 in mouse T cells. Splenocytes from Ceacam1 4S Tg Ceacam1 −/− and Ceacam1-4L Tg Ceacam1 −/− mice cultured with anti-CD3 (1 μg ml −1 ) or anti-CD3 (1 μg ml −1 ) and anti-CD28 (1 μg ml −1 ) or medium for 96 h. Cell lysates immunoprecipitated with anti-mCEACAM1 antibody (cc1) or with mIgG and IB with 5D12 (anti-mTIM-3 antibody) are shown. Locations of mTIM-3 protein variants are indicated. CHO, carbohydrate. d , Immunoprecipitation and immunoblot as in a with tunicamycin treated, wild-type HA–hTIM-3 and Flag–hCEACAM1 co-transfected HEK293T cells. Arrowhead denotes core CEACAM1 protein. e , Potential hCEACAM1-interacting residues on hTIM-3 highlighted in blue. f , HEK293 T cells transiently co-transfected with Flag–hCEACAM1 and HA–hTIM-3 mutants. Immunoblotting of anti-HA were used to analyse hTIM-3 expression in HEK293T transfectants. Except for Pro50Ala mutation displaying enhanced overall protein expression, all other mutations in the IgV domain of hTIM-3 are equally detected by anti-HA antibody. g , Quantification of association of hTIM-3 mutants associated with wild-type hCEACAM1 shown in summing all experiments performed. Association between wild-type hCEACAM1 and hTIM-3 core protein are depicted as reference (set as 1, n = 3, mean ± s.e.m. shown, unpaired Student’s t -test). h , Immunoprecipitation with anti-Flag (hCEACAM1) and immunoblot with anti-HA (hTIM-3) or anti-Flag of wild-type hCEACAM1 and mutant hTIM-3 proteins are shown. i , Quantification of h as performed in g. j , HEK293T cells co-transfected with Flag–hCEACAM1 wild-type and HA– hTIM-3 mutants and immunoprecipitation/immunblot as in h revealing no effects of Cys52Ala or Cys63Ala mutations in hTIM-3 in affecting association with hCEACAM1 in contrast to Cys109Ala mutation of hTIM-3 that disrupts interactions with hCEACAM1. k , Potential hTIM-3-interacting-residues around the FG–CC′ cleft of hCEACAM1 highlighted in red. l , HEK293T cells transiently co-transfected with Flag–hCEACAM1 mutants and wild-type HA–hTIM-3. Immunoblot with anti-Flag antibody was used to analyse hCEACAM1 expression in HEK293T co-transfectants. All hCEACAM1 mutations in IgV domain equally detected. m . n–p , Analysis of Gly47Ala mutation of hCEACAM1 in hTIM-3 co-transfected HEK293T cells by immunoprecipitation with anti-HA (hTIM-3) and immunoblot with anti-Flag (hCEACAM1) to detect association ( n ), IB with anti-Flag to confirm similarity of hCEACAM1 transfection ( o ) and quantification of associated hCEACAM1 of n as shown in m. q–s , Analysis of hCEACAM1 mutants Asn42Ala and Arg43Ala association with hTIM-3 ( q ), similarity of transfections ( r ) and quantification of q as in n–p . Representative of four ( d, h ), three ( f, g, i, l–s ), two ( a–c ) and one ( j ) independent experiments. * P
    Human Tim 3, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    il 12p70  (Thermo Fisher)
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    Thermo Fisher il 12p70
    Rv2882c induced BMDM activation. (A) BMDMs (1 × 10 5 /well) were stimulated with 100 ng/mL LPS or 1, 5, or 10 μg/mL Rv2882c for 24 h. (A) Quantities of TNF-α, MCP-1, IL-6, IL-10, and <t>IL-12p70</t> in the culture supernatant were determined by ELISA. All data are expressed as the mean values ± SD ( n = 3). Significance levels (* p
    Il 12p70, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Number of brain MNCs and their CNS antigen-specific autoreactive phenotype 14 d after MCAo. A , Number of MNCs infiltrating ipsilateral and contralateral hemisphere of the brain in vehicle and SNS/HPA blockade group (three individual experiments; vehicle, N = 15; SNS/HPA block, N = 20). B–D , Elispot experiments. B , Higher number of total brain MNCs responded to pMOG 35–55 stimulation with IFN-γ production (Th1 cells) per equal number of brain MNCs in SNS/HPA block group compared with the vehicle group. C , A similar effect was observed in ipsilateral hemisphere MNCs when only the SNS axis of SIDS was blocked with propranolol (two experiments; vehicle group, N = 5–8; SNS block, N = 6 or 7). D , Number of splenocytes reacting to pMOG 35–55 stimulation with IFN-γ, IL-4, or IL-17 secretion did not differ between the groups. E , F , ELISA experiments. TGF-β1, IL-10, IL-4, IL-17, and IFN-γ secretion (pg/ml) by pMOG 35–55 -stimulated brain MNCs, ipsilateral hemisphere ( E ) and by pMOG 35–55 -stimulated splenocytes ( F ).

    Journal: The Journal of Neuroscience

    Article Title: Blocking Stroke-Induced Immunodeficiency Increases CNS Antigen-Specific Autoreactivity But Does Not Worsen Functional Outcome after Experimental Stroke

    doi: 10.1523/JNEUROSCI.1532-14.2015

    Figure Lengend Snippet: Number of brain MNCs and their CNS antigen-specific autoreactive phenotype 14 d after MCAo. A , Number of MNCs infiltrating ipsilateral and contralateral hemisphere of the brain in vehicle and SNS/HPA blockade group (three individual experiments; vehicle, N = 15; SNS/HPA block, N = 20). B–D , Elispot experiments. B , Higher number of total brain MNCs responded to pMOG 35–55 stimulation with IFN-γ production (Th1 cells) per equal number of brain MNCs in SNS/HPA block group compared with the vehicle group. C , A similar effect was observed in ipsilateral hemisphere MNCs when only the SNS axis of SIDS was blocked with propranolol (two experiments; vehicle group, N = 5–8; SNS block, N = 6 or 7). D , Number of splenocytes reacting to pMOG 35–55 stimulation with IFN-γ, IL-4, or IL-17 secretion did not differ between the groups. E , F , ELISA experiments. TGF-β1, IL-10, IL-4, IL-17, and IFN-γ secretion (pg/ml) by pMOG 35–55 -stimulated brain MNCs, ipsilateral hemisphere ( E ) and by pMOG 35–55 -stimulated splenocytes ( F ).

    Article Snippet: Mouse multiplex kit (Millipore) and Human/Mouse TGF-β1 ELISA Ready-SET-Go kit (Affymetrix) were used according to the manufacturer's recommendations.

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

    Evaluating the replication kinetics of, and level of interferon activation by, WT MHV and DUBmut in cell culture. (A) Replication kinetics of WT and DUBmut virus in DBT cells. (B) IFN-α11 mRNA levels in WT- and DUBmut-infected BMDMs were assessed at indicated time points by reverse transcription-quantitative PCR (qRT-PCR). (C) IFN-α protein levels in the supernatants of infected BMDMs were evaluated at the times indicated. (D) Comparison of IFN-α11 mRNA levels in B6 versus MDA5 −/− BMDMs at 12 h postinfection (h p.i.). (E) Assessment of levels of viral nucleocapsid (N) mRNA by qRT-PCR. (F) Replication kinetics of WT and DUBmut virus in BMDM cells. Data are representative of at least two independent experiments and are presented as means ± SD. Data in panels B and C were statistically analyzed using unpaired t tests. *, P

    Journal: Journal of Virology

    Article Title: Structure-Guided Mutagenesis Alters Deubiquitinating Activity and Attenuates Pathogenesis of a Murine Coronavirus

    doi: 10.1128/JVI.01734-19

    Figure Lengend Snippet: Evaluating the replication kinetics of, and level of interferon activation by, WT MHV and DUBmut in cell culture. (A) Replication kinetics of WT and DUBmut virus in DBT cells. (B) IFN-α11 mRNA levels in WT- and DUBmut-infected BMDMs were assessed at indicated time points by reverse transcription-quantitative PCR (qRT-PCR). (C) IFN-α protein levels in the supernatants of infected BMDMs were evaluated at the times indicated. (D) Comparison of IFN-α11 mRNA levels in B6 versus MDA5 −/− BMDMs at 12 h postinfection (h p.i.). (E) Assessment of levels of viral nucleocapsid (N) mRNA by qRT-PCR. (F) Replication kinetics of WT and DUBmut virus in BMDM cells. Data are representative of at least two independent experiments and are presented as means ± SD. Data in panels B and C were statistically analyzed using unpaired t tests. *, P

    Article Snippet: The secreted amount of IFN-α in culture supernatants was assayed using a mouse IFN-α ELISA kit (catalog no. BMS6027; eBioscience) according to the manufacturer’s instructions.

    Techniques: Activation Assay, Cell Culture, Infection, Real-time Polymerase Chain Reaction, Quantitative RT-PCR

    Biochemical characterization of interactions between CEACAM1 and TIM-3 a , hTIM-3 does not co-immunoprecipitate (co-IP) with ITGA5 despite interactions with hCEACAM1. HEK293T cells transfected with Flag–ITGA5 and HA–TIM-3 (ITGA5Tw) or Flag–CEACAM1 and HA–TIM-3 (CwTw). Immunoprecipitation with anti-HA antibody and immunoblotted (IB) with anti-Flag antibody are shown. Input represents anti-Flag immunoblot of lysates. b, Co-immunoprecipitation of human TIM-3 and CEACAM1 from activated primary human T cells after N -glycanase treatment of lystates followed by immunoprecipitation with anti-human TIM-3 antibodies (2E2, 2E12 or 3F9) or IgG as control and immunoblotted with anti-human CEACAM1 antibody (5F4). Protein lystates from HeLa-CEACAM1 transfectants treated with N -glycanase followed by immunoprecipitation with 5F4 and the immune complex used as positive control (pos). c , mTIM-3 interacts with mCEACAM1 in mouse T cells. Splenocytes from Ceacam1 4S Tg Ceacam1 −/− and Ceacam1-4L Tg Ceacam1 −/− mice cultured with anti-CD3 (1 μg ml −1 ) or anti-CD3 (1 μg ml −1 ) and anti-CD28 (1 μg ml −1 ) or medium for 96 h. Cell lysates immunoprecipitated with anti-mCEACAM1 antibody (cc1) or with mIgG and IB with 5D12 (anti-mTIM-3 antibody) are shown. Locations of mTIM-3 protein variants are indicated. CHO, carbohydrate. d , Immunoprecipitation and immunoblot as in a with tunicamycin treated, wild-type HA–hTIM-3 and Flag–hCEACAM1 co-transfected HEK293T cells. Arrowhead denotes core CEACAM1 protein. e , Potential hCEACAM1-interacting residues on hTIM-3 highlighted in blue. f , HEK293 T cells transiently co-transfected with Flag–hCEACAM1 and HA–hTIM-3 mutants. Immunoblotting of anti-HA were used to analyse hTIM-3 expression in HEK293T transfectants. Except for Pro50Ala mutation displaying enhanced overall protein expression, all other mutations in the IgV domain of hTIM-3 are equally detected by anti-HA antibody. g , Quantification of association of hTIM-3 mutants associated with wild-type hCEACAM1 shown in summing all experiments performed. Association between wild-type hCEACAM1 and hTIM-3 core protein are depicted as reference (set as 1, n = 3, mean ± s.e.m. shown, unpaired Student’s t -test). h , Immunoprecipitation with anti-Flag (hCEACAM1) and immunoblot with anti-HA (hTIM-3) or anti-Flag of wild-type hCEACAM1 and mutant hTIM-3 proteins are shown. i , Quantification of h as performed in g. j , HEK293T cells co-transfected with Flag–hCEACAM1 wild-type and HA– hTIM-3 mutants and immunoprecipitation/immunblot as in h revealing no effects of Cys52Ala or Cys63Ala mutations in hTIM-3 in affecting association with hCEACAM1 in contrast to Cys109Ala mutation of hTIM-3 that disrupts interactions with hCEACAM1. k , Potential hTIM-3-interacting-residues around the FG–CC′ cleft of hCEACAM1 highlighted in red. l , HEK293T cells transiently co-transfected with Flag–hCEACAM1 mutants and wild-type HA–hTIM-3. Immunoblot with anti-Flag antibody was used to analyse hCEACAM1 expression in HEK293T co-transfectants. All hCEACAM1 mutations in IgV domain equally detected. m . n–p , Analysis of Gly47Ala mutation of hCEACAM1 in hTIM-3 co-transfected HEK293T cells by immunoprecipitation with anti-HA (hTIM-3) and immunoblot with anti-Flag (hCEACAM1) to detect association ( n ), IB with anti-Flag to confirm similarity of hCEACAM1 transfection ( o ) and quantification of associated hCEACAM1 of n as shown in m. q–s , Analysis of hCEACAM1 mutants Asn42Ala and Arg43Ala association with hTIM-3 ( q ), similarity of transfections ( r ) and quantification of q as in n–p . Representative of four ( d, h ), three ( f, g, i, l–s ), two ( a–c ) and one ( j ) independent experiments. * P

    Journal: Nature

    Article Title: CEACAM1 regulates TIM–3–mediated tolerance and exhaustion

    doi: 10.1038/nature13848

    Figure Lengend Snippet: Biochemical characterization of interactions between CEACAM1 and TIM-3 a , hTIM-3 does not co-immunoprecipitate (co-IP) with ITGA5 despite interactions with hCEACAM1. HEK293T cells transfected with Flag–ITGA5 and HA–TIM-3 (ITGA5Tw) or Flag–CEACAM1 and HA–TIM-3 (CwTw). Immunoprecipitation with anti-HA antibody and immunoblotted (IB) with anti-Flag antibody are shown. Input represents anti-Flag immunoblot of lysates. b, Co-immunoprecipitation of human TIM-3 and CEACAM1 from activated primary human T cells after N -glycanase treatment of lystates followed by immunoprecipitation with anti-human TIM-3 antibodies (2E2, 2E12 or 3F9) or IgG as control and immunoblotted with anti-human CEACAM1 antibody (5F4). Protein lystates from HeLa-CEACAM1 transfectants treated with N -glycanase followed by immunoprecipitation with 5F4 and the immune complex used as positive control (pos). c , mTIM-3 interacts with mCEACAM1 in mouse T cells. Splenocytes from Ceacam1 4S Tg Ceacam1 −/− and Ceacam1-4L Tg Ceacam1 −/− mice cultured with anti-CD3 (1 μg ml −1 ) or anti-CD3 (1 μg ml −1 ) and anti-CD28 (1 μg ml −1 ) or medium for 96 h. Cell lysates immunoprecipitated with anti-mCEACAM1 antibody (cc1) or with mIgG and IB with 5D12 (anti-mTIM-3 antibody) are shown. Locations of mTIM-3 protein variants are indicated. CHO, carbohydrate. d , Immunoprecipitation and immunoblot as in a with tunicamycin treated, wild-type HA–hTIM-3 and Flag–hCEACAM1 co-transfected HEK293T cells. Arrowhead denotes core CEACAM1 protein. e , Potential hCEACAM1-interacting residues on hTIM-3 highlighted in blue. f , HEK293 T cells transiently co-transfected with Flag–hCEACAM1 and HA–hTIM-3 mutants. Immunoblotting of anti-HA were used to analyse hTIM-3 expression in HEK293T transfectants. Except for Pro50Ala mutation displaying enhanced overall protein expression, all other mutations in the IgV domain of hTIM-3 are equally detected by anti-HA antibody. g , Quantification of association of hTIM-3 mutants associated with wild-type hCEACAM1 shown in summing all experiments performed. Association between wild-type hCEACAM1 and hTIM-3 core protein are depicted as reference (set as 1, n = 3, mean ± s.e.m. shown, unpaired Student’s t -test). h , Immunoprecipitation with anti-Flag (hCEACAM1) and immunoblot with anti-HA (hTIM-3) or anti-Flag of wild-type hCEACAM1 and mutant hTIM-3 proteins are shown. i , Quantification of h as performed in g. j , HEK293T cells co-transfected with Flag–hCEACAM1 wild-type and HA– hTIM-3 mutants and immunoprecipitation/immunblot as in h revealing no effects of Cys52Ala or Cys63Ala mutations in hTIM-3 in affecting association with hCEACAM1 in contrast to Cys109Ala mutation of hTIM-3 that disrupts interactions with hCEACAM1. k , Potential hTIM-3-interacting-residues around the FG–CC′ cleft of hCEACAM1 highlighted in red. l , HEK293T cells transiently co-transfected with Flag–hCEACAM1 mutants and wild-type HA–hTIM-3. Immunoblot with anti-Flag antibody was used to analyse hCEACAM1 expression in HEK293T co-transfectants. All hCEACAM1 mutations in IgV domain equally detected. m . n–p , Analysis of Gly47Ala mutation of hCEACAM1 in hTIM-3 co-transfected HEK293T cells by immunoprecipitation with anti-HA (hTIM-3) and immunoblot with anti-Flag (hCEACAM1) to detect association ( n ), IB with anti-Flag to confirm similarity of hCEACAM1 transfection ( o ) and quantification of associated hCEACAM1 of n as shown in m. q–s , Analysis of hCEACAM1 mutants Asn42Ala and Arg43Ala association with hTIM-3 ( q ), similarity of transfections ( r ) and quantification of q as in n–p . Representative of four ( d, h ), three ( f, g, i, l–s ), two ( a–c ) and one ( j ) independent experiments. * P

    Article Snippet: Previously described vectors containing the human CEACAM1–3L variant and human TIM-3 in the pDisplay vector (Invitrogen) were used as the template for all mutations.

    Techniques: Co-Immunoprecipitation Assay, Transfection, Immunoprecipitation, Positive Control, Mouse Assay, Cell Culture, Expressing, Mutagenesis

    Structural similarities between CEACAM1 and TIM-3 IgV-like N-terminal domains and biochemical association a–c , Interaction between TIM-3–Ig fusion protein and membrane protein of 60 kDa after deglycosylation derived from surface-biotinylated TK-1 cells. TIM-3–Ig fusion proteins and human IgG-precipitated proteins were deglycosylated by PNGase F and separated by SDS–PAGE. TIM-3–Ig-binding membrane proteins detected by immunoblot. A 60-kDa membrane protein (red circles) and 32-kDa protein consistent with galectin-9 (black circles) are found specifically associated with soluble (s) TIM-3–Ig fusion protein (a, lane 5) and full-length (f) TIM-3–Ig proteins ( b , lane 5), but not with the pre-clear controls (lanes 3 and 4) or human IgG (lanes 2 and 6). c , sTIM-3–Ig and full-length (fl) TIM-3–Ig interacting proteins were de-glycosylated by PNGase F and separated by SDS–PAGE. Proteins detected by silver staining. A band of 60kDa (red circle) isspecifically associated with sTIM-3–Ig proteins (lane 2), but not with human IgG (lane 5), or TIM-1–Ig or TIM-4–Ig (lanes 3, 4, 6–10). d , Superimposition of previously described IgV-like domains of mCEACAM1 and mTIM-3 demonstrate structural similarity with a score of 2.42 by the structural alignment and root mean square deviation (r.m.s.d.) calculated by Pymol. e , Sequence alignment of the IgV-like domains of mCEACAM1 and mTIM-3 on the basis of the secondary structure alignment in d. f , Sequence alignments of IgV domain sequences of CEACAM1 and overall mTIM and hTIM family members. α helices (orange) and β strands (blue) denoted as underlined segments in hCEACAM1 and mTIM-3. β strands labelled with upper- and lower-case letters for hCEACAM1 and mTIM-3, respectively. Conserved residues are shaded red. Mutated residues are shaded violet for hCEACAM1, and green for hTIM-3. Asterisk (*) indicates positions having a single, fully conserved residue; a dagger (†) indicates conservation between groups of weakly similar residues; a double-dagger (‡) indicates conservation between groups of strongly similar residues. g , Computational modelling as defined by energy calculations (score) relative to r.m.s. values of docking models to define potential cis and trans and amino acids involved. h , CEACAM1 expression on mouse fibroblast 3T3 cells used to identify a galectin-9-independent ligand. i , CEACAM1 expression on mouse TK-1 cells as in a–c . Representative of three ( a–c, h, i ) independent experiments.

    Journal: Nature

    Article Title: CEACAM1 regulates TIM–3–mediated tolerance and exhaustion

    doi: 10.1038/nature13848

    Figure Lengend Snippet: Structural similarities between CEACAM1 and TIM-3 IgV-like N-terminal domains and biochemical association a–c , Interaction between TIM-3–Ig fusion protein and membrane protein of 60 kDa after deglycosylation derived from surface-biotinylated TK-1 cells. TIM-3–Ig fusion proteins and human IgG-precipitated proteins were deglycosylated by PNGase F and separated by SDS–PAGE. TIM-3–Ig-binding membrane proteins detected by immunoblot. A 60-kDa membrane protein (red circles) and 32-kDa protein consistent with galectin-9 (black circles) are found specifically associated with soluble (s) TIM-3–Ig fusion protein (a, lane 5) and full-length (f) TIM-3–Ig proteins ( b , lane 5), but not with the pre-clear controls (lanes 3 and 4) or human IgG (lanes 2 and 6). c , sTIM-3–Ig and full-length (fl) TIM-3–Ig interacting proteins were de-glycosylated by PNGase F and separated by SDS–PAGE. Proteins detected by silver staining. A band of 60kDa (red circle) isspecifically associated with sTIM-3–Ig proteins (lane 2), but not with human IgG (lane 5), or TIM-1–Ig or TIM-4–Ig (lanes 3, 4, 6–10). d , Superimposition of previously described IgV-like domains of mCEACAM1 and mTIM-3 demonstrate structural similarity with a score of 2.42 by the structural alignment and root mean square deviation (r.m.s.d.) calculated by Pymol. e , Sequence alignment of the IgV-like domains of mCEACAM1 and mTIM-3 on the basis of the secondary structure alignment in d. f , Sequence alignments of IgV domain sequences of CEACAM1 and overall mTIM and hTIM family members. α helices (orange) and β strands (blue) denoted as underlined segments in hCEACAM1 and mTIM-3. β strands labelled with upper- and lower-case letters for hCEACAM1 and mTIM-3, respectively. Conserved residues are shaded red. Mutated residues are shaded violet for hCEACAM1, and green for hTIM-3. Asterisk (*) indicates positions having a single, fully conserved residue; a dagger (†) indicates conservation between groups of weakly similar residues; a double-dagger (‡) indicates conservation between groups of strongly similar residues. g , Computational modelling as defined by energy calculations (score) relative to r.m.s. values of docking models to define potential cis and trans and amino acids involved. h , CEACAM1 expression on mouse fibroblast 3T3 cells used to identify a galectin-9-independent ligand. i , CEACAM1 expression on mouse TK-1 cells as in a–c . Representative of three ( a–c, h, i ) independent experiments.

    Article Snippet: Previously described vectors containing the human CEACAM1–3L variant and human TIM-3 in the pDisplay vector (Invitrogen) were used as the template for all mutations.

    Techniques: Derivative Assay, SDS Page, Binding Assay, Silver Staining, Sequencing, Expressing

    CEACAM1 is essential for TIM-3 mediated T cell tolerance a , Schematic diagram of OVA antigen-specific tolerance induction model. b , Schematic diagram of OVA immunization. c , Tracking in vivo antigen-specific T-cell responses of CFSE-labelled OT-II transgenic Rag2 −/− T cells in total lymphocyte gate of mesenteric lymph nodes, peripheral lymph node or spleen of wild-type or Ceacam1 −/− recipients after gating on CFSE-positive cells and staining for CEACAM1 in PBS and OVA 323–339 immunized mice. Hyper-responsiveness of OT-II transgenic Rag2 −/− T cells in Ceacam1 −/− mice was not due to decreased regulatory T-cell induction (data not shown) or increased initial parking on the basis of cell numbers shown. d , TIM-3 expression on CEACAM1-positive and -negative CFSE + cells as in c. e , Schematic diagram of SEB-induced T-cell tolerance model. f , mCEACAM1 and mTIM-3 expression on CD4 + Vβ8 + T cells after SEB tolerance induction. g , hCEACAM1 and hTIM-3 expression on activated primary human T cells defined by staining with indicated antibodies. h , CEACAM1 expression on TIM-3-silenced primary human T cells after re-activation by flow cytometry. Relative TIM-3, CEACAM1 or CD4 expression on T cells expressing control shRNA ( lacZ control, red) or three independent shRNAs directed at TIM3 (overlay, blue). shRNA target sequences shown. i – l , CEACAM1 and TIM-3 expression and functional consequences on T cells in HIV infection. CD4 + IFN-γ + T cells are decreased among CEACAM1 + TIM-3 + CD4 + T cells in HIV infection in response to Gag peptides ( i ). Although proportions of CEACAM1 + TIM-3 + CD8 + T cells are similar in HIV-infected and -uninfected subjects ( j ), CEACAM1 + TIM-3 + CD8 + T cells express little IFN-γ after stimulation with HIV Gag peptides or SEB relative to TIM-3 + CEACAM1 − CD8 + T cells ( k, l ). C, hCEACAM1; T, hTIM-3 ( n = 4 per group, mean ± s.e.m.). m – o , In situ proximity ligation analysis (PLA) of CEACAM1 and TIM-3. m , HEK293T cells transiently co-transfected with Flag–hCEACAM1 or HA–hTIM-3. Cells stained with DAPI (left), anti-tubulin (middle), anti-HA (rabbit) and anti-Flag (mouse) (middle right) or merged (right). Several examples of a positive PLA signal (middle right and right panels: red fluorescent dots) indicative of a maximum distance of 30–40 nm between hCEACAM1 and hTIM-3. n , Negative control, co-expression of Flag–PLK1 (protein kinase I) and HA–TIM-3 failed to generate fluorescent dots (that is, PLA negative). Cells stained with DAPI, anti-tubulin, anti-HA/anti-Flag or merged as in m. o , Negative control, co-expression of HA–ADAP (adhesion and degranulation promoting adaptor protein) failed to show a signal (that is, PLA negative) with staining as in m. p, q , CEACAM1 and TIM-3 colocalization at immunological synapse of primary human CD4 and CD8 T cells. Confocal microscopy of hTIM-3 + hCEACAM1 + primary CD4 + and CD8 + T cells forming conjugates with SEB-loaded B cells. DIC, differential interference contrast. Blue denotes B cell; red denotes CD3; purple denotes CEACAM1; green denotes TIM-3. White indicates colocalization between CEACAM1 and TIM-3 ( p ). Average Pearson correlation coefficients for CD4 + and CD8 + T cells were 0.543 and 0.566, respectively, representing strong co-localization ( q ). Data are mean ± s.e.m. and representative of five ( f, g ), four ( p, q ), three ( c, d, m–o ) and two ( h ) independent experiments. * P

    Journal: Nature

    Article Title: CEACAM1 regulates TIM–3–mediated tolerance and exhaustion

    doi: 10.1038/nature13848

    Figure Lengend Snippet: CEACAM1 is essential for TIM-3 mediated T cell tolerance a , Schematic diagram of OVA antigen-specific tolerance induction model. b , Schematic diagram of OVA immunization. c , Tracking in vivo antigen-specific T-cell responses of CFSE-labelled OT-II transgenic Rag2 −/− T cells in total lymphocyte gate of mesenteric lymph nodes, peripheral lymph node or spleen of wild-type or Ceacam1 −/− recipients after gating on CFSE-positive cells and staining for CEACAM1 in PBS and OVA 323–339 immunized mice. Hyper-responsiveness of OT-II transgenic Rag2 −/− T cells in Ceacam1 −/− mice was not due to decreased regulatory T-cell induction (data not shown) or increased initial parking on the basis of cell numbers shown. d , TIM-3 expression on CEACAM1-positive and -negative CFSE + cells as in c. e , Schematic diagram of SEB-induced T-cell tolerance model. f , mCEACAM1 and mTIM-3 expression on CD4 + Vβ8 + T cells after SEB tolerance induction. g , hCEACAM1 and hTIM-3 expression on activated primary human T cells defined by staining with indicated antibodies. h , CEACAM1 expression on TIM-3-silenced primary human T cells after re-activation by flow cytometry. Relative TIM-3, CEACAM1 or CD4 expression on T cells expressing control shRNA ( lacZ control, red) or three independent shRNAs directed at TIM3 (overlay, blue). shRNA target sequences shown. i – l , CEACAM1 and TIM-3 expression and functional consequences on T cells in HIV infection. CD4 + IFN-γ + T cells are decreased among CEACAM1 + TIM-3 + CD4 + T cells in HIV infection in response to Gag peptides ( i ). Although proportions of CEACAM1 + TIM-3 + CD8 + T cells are similar in HIV-infected and -uninfected subjects ( j ), CEACAM1 + TIM-3 + CD8 + T cells express little IFN-γ after stimulation with HIV Gag peptides or SEB relative to TIM-3 + CEACAM1 − CD8 + T cells ( k, l ). C, hCEACAM1; T, hTIM-3 ( n = 4 per group, mean ± s.e.m.). m – o , In situ proximity ligation analysis (PLA) of CEACAM1 and TIM-3. m , HEK293T cells transiently co-transfected with Flag–hCEACAM1 or HA–hTIM-3. Cells stained with DAPI (left), anti-tubulin (middle), anti-HA (rabbit) and anti-Flag (mouse) (middle right) or merged (right). Several examples of a positive PLA signal (middle right and right panels: red fluorescent dots) indicative of a maximum distance of 30–40 nm between hCEACAM1 and hTIM-3. n , Negative control, co-expression of Flag–PLK1 (protein kinase I) and HA–TIM-3 failed to generate fluorescent dots (that is, PLA negative). Cells stained with DAPI, anti-tubulin, anti-HA/anti-Flag or merged as in m. o , Negative control, co-expression of HA–ADAP (adhesion and degranulation promoting adaptor protein) failed to show a signal (that is, PLA negative) with staining as in m. p, q , CEACAM1 and TIM-3 colocalization at immunological synapse of primary human CD4 and CD8 T cells. Confocal microscopy of hTIM-3 + hCEACAM1 + primary CD4 + and CD8 + T cells forming conjugates with SEB-loaded B cells. DIC, differential interference contrast. Blue denotes B cell; red denotes CD3; purple denotes CEACAM1; green denotes TIM-3. White indicates colocalization between CEACAM1 and TIM-3 ( p ). Average Pearson correlation coefficients for CD4 + and CD8 + T cells were 0.543 and 0.566, respectively, representing strong co-localization ( q ). Data are mean ± s.e.m. and representative of five ( f, g ), four ( p, q ), three ( c, d, m–o ) and two ( h ) independent experiments. * P

    Article Snippet: Previously described vectors containing the human CEACAM1–3L variant and human TIM-3 in the pDisplay vector (Invitrogen) were used as the template for all mutations.

    Techniques: In Vivo, Transgenic Assay, Staining, Mouse Assay, Expressing, Activation Assay, Flow Cytometry, Cytometry, shRNA, Functional Assay, Infection, In Situ, Ligation, Proximity Ligation Assay, Transfection, Negative Control, Negative Staining, Confocal Microscopy

    CEACAM1 determines TIM-3 expression and function a , HEK293T cells transiently co-transfected with Flag–hCEACAM1 and wild-type or mutants of HA–hTIM-3. Flow cytometry detecting HA–hTIM-3 (detected with anti-HA) and Flag–hCEACAM1 (detected with 5F4) proteins at cell surface (top), Golgi apparatus (middle) or endoplasmic reticulum (bottom) using monensin and brefeldin A, respectively. b , Cellular distribution of wild-type or mutant hTIM-3 when co-expressed with wild-type hCEACAM1. Total counts of hTIM-3 at surface, Golgi apparatus and endoplasmic reticulum summed up to 100%. Depicted as percentage of hTIM-3. c , HEK293T cells transiently co-transfected with wild-type HA–hTIM-3 (detected with 2E2) and wild-type or mutant Flag–hCEACAM1 (detected with anti-Flag). Flow cytometry analyses as in a. d , Cellular distribution of c , as in b . Depicted as percentage of hTIM-3. e , Immunoblot for wild-type or Thr101Ile variant of hTIM-3 showing maturation status in presence of wild-type or mutated (Gln44Leu) hCEACAM1. f , Normal association of Thr101Ile variant of hTIM-3 with hCEACAM1. g , Analysis of CD4 + Vβ8 + T cells after SEB tolerance induction from experimental mice of indicated genotypes. h , Galectin-9 induction of apoptosis. Annexin V + propidium iodide staining of T H 1 cells polarized from Tim3 Tg or Tim3 Tg Ceacam1 −/− mice after treatment with galectin-9 (2 μg ml −1 ) for 8 h. Note decreased apoptosis in Tim3 Tg Ceacam1 −/− T cells. i , Schematic diagram of protocol used for protein pull-down using in-column IgV domain of GST–hTIM-3 incubated with hCEACAM1 protein derived from transfected HEK293T cells as in . j , GST or GST–hTIM-3 staining of hCEACAM1-4L–transfected Jurkat T cells. k , Wild-type CD4 + T cells stimulated with anti-CD3 and/or anti-CD28 in the presence or absence of mCEACAM1 NFc, or IgG1-Fc as control, and cells analysed for secretion of IFN-γ and IL-2. l, m , Characterization of tolerance in SEB model. Tim3 Tg (l) and Tim3 Tg Ceacam1 −/− ( m . Lymph node cells collected after SEB treatment and re-stimulated with soluble anti-CD3 at indicated doses and IL-2 measured by ELISA after 72 h. Note tolerance in Tim3 Tg but not Tim3 Tg Ceacam1 −/− mice. n = 3 per group. n , Anti-mTIM-3 blockade with 2C12 antibody of mCEACAM1 NFc or control IgG-Fc staining of CD4 + T cells from indicated genotypes expressed as levels relative to Ceacam1 −/− mice. o, p , Analysis of mTIM-3 cytoplasmic tail function in transmitting mCEACAM1-induced signals. Activated mouse CD4 + T cells from wild-type ( o ) or Ceacam1 −/− ( p ) mice were retrovirally transduced, sorted and stimulated with anti-CD3 with either human IgG-Fc (IgG, control) or mCEACAM1 N-terminal domain as NFc and TNF-α secretion assessed by ELISA after 72 h. Note ability of CEACAM1 N-terminal domain to transduce a signal associated with inhibition of TNF-α secretion in wild-type but not Ceacam1 −/− T cells. n = 3 per group. Data are mean ± s.e.m. and represent three ( f, g, k–p ) and two ( a–e, h, j ) independent experiments. * P

    Journal: Nature

    Article Title: CEACAM1 regulates TIM–3–mediated tolerance and exhaustion

    doi: 10.1038/nature13848

    Figure Lengend Snippet: CEACAM1 determines TIM-3 expression and function a , HEK293T cells transiently co-transfected with Flag–hCEACAM1 and wild-type or mutants of HA–hTIM-3. Flow cytometry detecting HA–hTIM-3 (detected with anti-HA) and Flag–hCEACAM1 (detected with 5F4) proteins at cell surface (top), Golgi apparatus (middle) or endoplasmic reticulum (bottom) using monensin and brefeldin A, respectively. b , Cellular distribution of wild-type or mutant hTIM-3 when co-expressed with wild-type hCEACAM1. Total counts of hTIM-3 at surface, Golgi apparatus and endoplasmic reticulum summed up to 100%. Depicted as percentage of hTIM-3. c , HEK293T cells transiently co-transfected with wild-type HA–hTIM-3 (detected with 2E2) and wild-type or mutant Flag–hCEACAM1 (detected with anti-Flag). Flow cytometry analyses as in a. d , Cellular distribution of c , as in b . Depicted as percentage of hTIM-3. e , Immunoblot for wild-type or Thr101Ile variant of hTIM-3 showing maturation status in presence of wild-type or mutated (Gln44Leu) hCEACAM1. f , Normal association of Thr101Ile variant of hTIM-3 with hCEACAM1. g , Analysis of CD4 + Vβ8 + T cells after SEB tolerance induction from experimental mice of indicated genotypes. h , Galectin-9 induction of apoptosis. Annexin V + propidium iodide staining of T H 1 cells polarized from Tim3 Tg or Tim3 Tg Ceacam1 −/− mice after treatment with galectin-9 (2 μg ml −1 ) for 8 h. Note decreased apoptosis in Tim3 Tg Ceacam1 −/− T cells. i , Schematic diagram of protocol used for protein pull-down using in-column IgV domain of GST–hTIM-3 incubated with hCEACAM1 protein derived from transfected HEK293T cells as in . j , GST or GST–hTIM-3 staining of hCEACAM1-4L–transfected Jurkat T cells. k , Wild-type CD4 + T cells stimulated with anti-CD3 and/or anti-CD28 in the presence or absence of mCEACAM1 NFc, or IgG1-Fc as control, and cells analysed for secretion of IFN-γ and IL-2. l, m , Characterization of tolerance in SEB model. Tim3 Tg (l) and Tim3 Tg Ceacam1 −/− ( m . Lymph node cells collected after SEB treatment and re-stimulated with soluble anti-CD3 at indicated doses and IL-2 measured by ELISA after 72 h. Note tolerance in Tim3 Tg but not Tim3 Tg Ceacam1 −/− mice. n = 3 per group. n , Anti-mTIM-3 blockade with 2C12 antibody of mCEACAM1 NFc or control IgG-Fc staining of CD4 + T cells from indicated genotypes expressed as levels relative to Ceacam1 −/− mice. o, p , Analysis of mTIM-3 cytoplasmic tail function in transmitting mCEACAM1-induced signals. Activated mouse CD4 + T cells from wild-type ( o ) or Ceacam1 −/− ( p ) mice were retrovirally transduced, sorted and stimulated with anti-CD3 with either human IgG-Fc (IgG, control) or mCEACAM1 N-terminal domain as NFc and TNF-α secretion assessed by ELISA after 72 h. Note ability of CEACAM1 N-terminal domain to transduce a signal associated with inhibition of TNF-α secretion in wild-type but not Ceacam1 −/− T cells. n = 3 per group. Data are mean ± s.e.m. and represent three ( f, g, k–p ) and two ( a–e, h, j ) independent experiments. * P

    Article Snippet: Previously described vectors containing the human CEACAM1–3L variant and human TIM-3 in the pDisplay vector (Invitrogen) were used as the template for all mutations.

    Techniques: Expressing, Transfection, Flow Cytometry, Cytometry, Mutagenesis, Variant Assay, Mouse Assay, Staining, Incubation, Derivative Assay, Enzyme-linked Immunosorbent Assay, Transduction, Inhibition

    Blockade of CEACAM1 and TIM-3 or genetic loss of CEACAM1 increases anti-tumour immunity a , Schematic presentation of antibody blockade protocol described in . b , Schematic presentation of antibody blockade protocol referred to in panel c. c , Prevention of CT26 tumour growth with indicated combinations of antibodies as in ( b ) ( n = 5 per group, post-hoc Dunnett’s correction followed by Friedman test). d , Schematic of schedule used for therapeutic antibody administration as described in e. e , Synergy of CEACAM1 and programmed death-ligand1 (PD-L1) blockade in a therapeutic protocol as described in d was performed in wild-type BALB/c mice that received a subcutaneous inoculation of CT26 tumour cells. Mean tumour size ( n = 5 per group, with linear regression analysis). Note synergistic increase in anti-tumour effect when CEACAM1 and PD-L1 co-blockade was performed. f , TILs were analysed for the relative proportion of CD4 + ( n = 4, unpaired Student’s t -test with Mann–Whitney U correction). g , Percentages of CD8 + T cells from spleen show that antibody treatments have no effects on total CD8 + T cell numbers ( n = 7/8, unpaired two-tailed t -test). h , Negative correlation of the numbers of AH1 tet + CD8 + (Pearson’s correlation coefficient, r = 0.9560, P = 0.044). i , Representative flow cytometry for tumour-specific (AH1-tetramer, tet + ) CD8 + T cells in draining lymph nodes of mice from the indicated genotypes. Data are mean ± s.e.m. and represent three ( f–i ), two ( e ) and one ( c ) independent experiments. * P

    Journal: Nature

    Article Title: CEACAM1 regulates TIM–3–mediated tolerance and exhaustion

    doi: 10.1038/nature13848

    Figure Lengend Snippet: Blockade of CEACAM1 and TIM-3 or genetic loss of CEACAM1 increases anti-tumour immunity a , Schematic presentation of antibody blockade protocol described in . b , Schematic presentation of antibody blockade protocol referred to in panel c. c , Prevention of CT26 tumour growth with indicated combinations of antibodies as in ( b ) ( n = 5 per group, post-hoc Dunnett’s correction followed by Friedman test). d , Schematic of schedule used for therapeutic antibody administration as described in e. e , Synergy of CEACAM1 and programmed death-ligand1 (PD-L1) blockade in a therapeutic protocol as described in d was performed in wild-type BALB/c mice that received a subcutaneous inoculation of CT26 tumour cells. Mean tumour size ( n = 5 per group, with linear regression analysis). Note synergistic increase in anti-tumour effect when CEACAM1 and PD-L1 co-blockade was performed. f , TILs were analysed for the relative proportion of CD4 + ( n = 4, unpaired Student’s t -test with Mann–Whitney U correction). g , Percentages of CD8 + T cells from spleen show that antibody treatments have no effects on total CD8 + T cell numbers ( n = 7/8, unpaired two-tailed t -test). h , Negative correlation of the numbers of AH1 tet + CD8 + (Pearson’s correlation coefficient, r = 0.9560, P = 0.044). i , Representative flow cytometry for tumour-specific (AH1-tetramer, tet + ) CD8 + T cells in draining lymph nodes of mice from the indicated genotypes. Data are mean ± s.e.m. and represent three ( f–i ), two ( e ) and one ( c ) independent experiments. * P

    Article Snippet: Previously described vectors containing the human CEACAM1–3L variant and human TIM-3 in the pDisplay vector (Invitrogen) were used as the template for all mutations.

    Techniques: Mouse Assay, MANN-WHITNEY, Two Tailed Test, Flow Cytometry, Cytometry

    CEACAM1 and TIM-3 heterodimerize and serve as heterophilic ligands a, b , Co-immunoprecipitation (IP) and immunoblot (IB) of wild-type hCEACAM1 and hTIM-3 in co-transfected HEK293T cells, c, d , Co-immunoprecipitation and immunoblot of wild-type hCEACAM1 and hTIM-3 mutants ( c ) or wild-type hTIM-3 and hCEACAM1 mutants ( d ) as in a and b. e , Human CEACAM1 (IgV)-TIM-3 (IgV) heterodimer structure, f, g , 2 F o — F c maps contoured at 0.9σ showing electron densities, h, i , Autoradiogram of anti-haemagglutinin (HA) (hTIM-3) immunoprecipitate from metabolic-labelled ( h ) and pulse-chase metabolic-labelled ( i ) co-transfected HEK293T cells. CHO, carbohydrate; core T, non-glycosylated hTIM-3; Cw, wild-type hCEACAM1; EndoH, endoglycosidaseH; H2-MA, HA-tagged influenza virus A M2 protein; T, hTIM-3 (Thr101Ile); Tw, wild-type hTIM-3. hTIM-3 isoforms noted. j , Quantification of densities in i ( n = 3 per group). k , Immunoblot for mTIM-3 from PBS-treated (−) or SEB-treated (+) CD4 + T cells. Labelling as in h and i. 1 , mTIM-3 expression after SEB tolerance induction, m , Column-bound glutathione S -transferase (GST)-hTIM-3 IgV-domain pull-down of hCEACAM1 detected by immunoblot. GST 2 , GST-hTIM-3 dimer. Ft, flow through, n , Suppression of mouse CD4 + T-cell proliferation by mCEACAM1 N-terminal domain-Fc fusion protein (NFc). o , Immunoprecipitation of mTIM-3 and immunoblot for BAT3 or mTIM-3 from lysates of CD4 + T cells. p, q , Proliferation of CD4 + T cells from wild-type ( p ) and CeaCAM1 −/− ( q ) mice transduced with wild-type mTIM-3 (Tw), mTIM-3 Δ252–281 (Tmut) or vector exposed to anti-CD3 and either NFc or IgG1-Fc (IgG1). Data are mean ± s.e.m. and represent five ( a, b ), four ( c, d ), three ( h-j, l, n, p, q ) and two ( k, m, o ) independent experiments. NS, not significant; * P

    Journal: Nature

    Article Title: CEACAM1 regulates TIM–3–mediated tolerance and exhaustion

    doi: 10.1038/nature13848

    Figure Lengend Snippet: CEACAM1 and TIM-3 heterodimerize and serve as heterophilic ligands a, b , Co-immunoprecipitation (IP) and immunoblot (IB) of wild-type hCEACAM1 and hTIM-3 in co-transfected HEK293T cells, c, d , Co-immunoprecipitation and immunoblot of wild-type hCEACAM1 and hTIM-3 mutants ( c ) or wild-type hTIM-3 and hCEACAM1 mutants ( d ) as in a and b. e , Human CEACAM1 (IgV)-TIM-3 (IgV) heterodimer structure, f, g , 2 F o — F c maps contoured at 0.9σ showing electron densities, h, i , Autoradiogram of anti-haemagglutinin (HA) (hTIM-3) immunoprecipitate from metabolic-labelled ( h ) and pulse-chase metabolic-labelled ( i ) co-transfected HEK293T cells. CHO, carbohydrate; core T, non-glycosylated hTIM-3; Cw, wild-type hCEACAM1; EndoH, endoglycosidaseH; H2-MA, HA-tagged influenza virus A M2 protein; T, hTIM-3 (Thr101Ile); Tw, wild-type hTIM-3. hTIM-3 isoforms noted. j , Quantification of densities in i ( n = 3 per group). k , Immunoblot for mTIM-3 from PBS-treated (−) or SEB-treated (+) CD4 + T cells. Labelling as in h and i. 1 , mTIM-3 expression after SEB tolerance induction, m , Column-bound glutathione S -transferase (GST)-hTIM-3 IgV-domain pull-down of hCEACAM1 detected by immunoblot. GST 2 , GST-hTIM-3 dimer. Ft, flow through, n , Suppression of mouse CD4 + T-cell proliferation by mCEACAM1 N-terminal domain-Fc fusion protein (NFc). o , Immunoprecipitation of mTIM-3 and immunoblot for BAT3 or mTIM-3 from lysates of CD4 + T cells. p, q , Proliferation of CD4 + T cells from wild-type ( p ) and CeaCAM1 −/− ( q ) mice transduced with wild-type mTIM-3 (Tw), mTIM-3 Δ252–281 (Tmut) or vector exposed to anti-CD3 and either NFc or IgG1-Fc (IgG1). Data are mean ± s.e.m. and represent five ( a, b ), four ( c, d ), three ( h-j, l, n, p, q ) and two ( k, m, o ) independent experiments. NS, not significant; * P

    Article Snippet: Previously described vectors containing the human CEACAM1–3L variant and human TIM-3 in the pDisplay vector (Invitrogen) were used as the template for all mutations.

    Techniques: Immunoprecipitation, Transfection, Pulse Chase, Expressing, Flow Cytometry, Mouse Assay, Transduction, Plasmid Preparation

    TIM-3 regulation of mucosa-associated inflammation requires CEACAM1 a , mCEACAM1 and mTIM-3 expression on colonic lamina propria CD4 + T cells, b , Intracellular cytokines in cells described in a. c, d , mTIM-3 ( c ) and intracellular TNF-α (d) expression in lamina propria CD4 + T cells from indicated donors, e , Body weights relative to weights on day 14 of groups in c and d . Five mice expired (†). f , Score of surviving mice of groups in e. g , Body weights of genotypes as in e. h , Score of groups described in g. i, j , Nanostring ( i ) and quantitative PCR ( j ) of lamina propria mononuclear cells. Actb , β-actin gene. All data are mean ± s.e.m. and represent six ( a ), four ( b ) and three ( c–j ) independent experiments. * P

    Journal: Nature

    Article Title: CEACAM1 regulates TIM–3–mediated tolerance and exhaustion

    doi: 10.1038/nature13848

    Figure Lengend Snippet: TIM-3 regulation of mucosa-associated inflammation requires CEACAM1 a , mCEACAM1 and mTIM-3 expression on colonic lamina propria CD4 + T cells, b , Intracellular cytokines in cells described in a. c, d , mTIM-3 ( c ) and intracellular TNF-α (d) expression in lamina propria CD4 + T cells from indicated donors, e , Body weights relative to weights on day 14 of groups in c and d . Five mice expired (†). f , Score of surviving mice of groups in e. g , Body weights of genotypes as in e. h , Score of groups described in g. i, j , Nanostring ( i ) and quantitative PCR ( j ) of lamina propria mononuclear cells. Actb , β-actin gene. All data are mean ± s.e.m. and represent six ( a ), four ( b ) and three ( c–j ) independent experiments. * P

    Article Snippet: Previously described vectors containing the human CEACAM1–3L variant and human TIM-3 in the pDisplay vector (Invitrogen) were used as the template for all mutations.

    Techniques: Expressing, Mouse Assay, Real-time Polymerase Chain Reaction

    CEACAM1 determines TIM-3 regulation of anti-tumour immune responses a , Survival curves in AOM/2.5% DSS model. b–d , Assessment of polyp numbers ( b ), polyp size ( c ) and cancer grades ( d ) in AOM/1.5% DSS model, e , Staining of CD8 + T cells associated with CT26 tumours. f , Intracellular cytokine expression in TIL subsets after anti-CD3 stimulation. g , Prevention of CT26 tumour growth in wild-type mice ( n = 5 per group). mAb, monoclonal antibody. h–k , Analysis of TILs for relative proportion of CD8 + ( h ) and CD4 + ( i ) T cells, IFN-γ + CD8 + T cells ( j ) and tumour-specific (AH1-tetramer, tet + ) CD8 + T cells in draining lymph nodes (dLN) ( k ) in groups described in g. l–n , Growth of CT26 cells (1), AH1 tet + CD8 + T cells in dLN ( m ) and TIM-3 expression on TILs ( n ) in wild-type and CeaCAM1 −/− mice. Data are mean ± s.e.m. and represent four ( e ), three ( g–k ) and two ( a–d, f, l–n ) independent experiments. * P

    Journal: Nature

    Article Title: CEACAM1 regulates TIM–3–mediated tolerance and exhaustion

    doi: 10.1038/nature13848

    Figure Lengend Snippet: CEACAM1 determines TIM-3 regulation of anti-tumour immune responses a , Survival curves in AOM/2.5% DSS model. b–d , Assessment of polyp numbers ( b ), polyp size ( c ) and cancer grades ( d ) in AOM/1.5% DSS model, e , Staining of CD8 + T cells associated with CT26 tumours. f , Intracellular cytokine expression in TIL subsets after anti-CD3 stimulation. g , Prevention of CT26 tumour growth in wild-type mice ( n = 5 per group). mAb, monoclonal antibody. h–k , Analysis of TILs for relative proportion of CD8 + ( h ) and CD4 + ( i ) T cells, IFN-γ + CD8 + T cells ( j ) and tumour-specific (AH1-tetramer, tet + ) CD8 + T cells in draining lymph nodes (dLN) ( k ) in groups described in g. l–n , Growth of CT26 cells (1), AH1 tet + CD8 + T cells in dLN ( m ) and TIM-3 expression on TILs ( n ) in wild-type and CeaCAM1 −/− mice. Data are mean ± s.e.m. and represent four ( e ), three ( g–k ) and two ( a–d, f, l–n ) independent experiments. * P

    Article Snippet: Previously described vectors containing the human CEACAM1–3L variant and human TIM-3 in the pDisplay vector (Invitrogen) were used as the template for all mutations.

    Techniques: Staining, Expressing, Mouse Assay

    CEACAM1 and TIM-3 cooperatively regulate inflammation and anti-tumour immunity a , Representative haematoxylin and eosin staining of groups described in . Scale bar, 50 μm. b , Flow cytometry for intracellular cytokine assessment of TNF-α expression from infiltrating CD4 + T cells from inflamed colonic lamina propria of Ceacam1 −/− Rag2 −/− recipients, 6weeks after transfer with naive CD4 + CD44 lo CD62L high T cells from indicated genotypes. c , Anorectal prolapse of indicated genotypes. d . Scale bar, 50 μm. e , RNA expression defined by nanostring of lamina propria mononuclear cells in indicated groups (mean of n = 3 per group). f , Schematic overview of protocol for AOM/DSS colitis-associated cancer model. g , Representative haematoxylin and eosin staining of colon from wild-type mice in AOM/1.5% DSS model. Scale bar, 50 μm; h , Representative photograph of distal colons of wild-type mice ( n = 3 per group, anorectal junction at left end) in AOM/1.5% DSS model. Vertical arrows show the sites for dissection of the polyps (black) and the vicinity of the polyps (red). i , Representative flow cytometry analyses on infiltrating lymphocytes of invading distal colonic polyps or from the vicinity of the polyps or from mesenteric lymph nodes for CD4 + and CD8 + T cells and expression of CEACAM1 and TIM-3orPD-1 and TIM-3. Note that vicinity of polyps exhibit highest numbers of T cells with an exhausted phenotype. j , Summary of flow cytometry on infiltrating lymphocytes from invading distal colonic polyps or from vicinity of polyps and from mesenteric lymph nodes for CD4 + and CD8 + T cells expressing CEACAM1 and TIM-3 or PD-1 and TIM-3 ( n = 3, median shown). k , Representative pathology in AOM/1.5% DSS model. Scale bar, 60 μm. HGD, high grade dysplasia. Representative of three independent experiments ( a–e, g–k ). Fig. 3e

    Journal: Nature

    Article Title: CEACAM1 regulates TIM–3–mediated tolerance and exhaustion

    doi: 10.1038/nature13848

    Figure Lengend Snippet: CEACAM1 and TIM-3 cooperatively regulate inflammation and anti-tumour immunity a , Representative haematoxylin and eosin staining of groups described in . Scale bar, 50 μm. b , Flow cytometry for intracellular cytokine assessment of TNF-α expression from infiltrating CD4 + T cells from inflamed colonic lamina propria of Ceacam1 −/− Rag2 −/− recipients, 6weeks after transfer with naive CD4 + CD44 lo CD62L high T cells from indicated genotypes. c , Anorectal prolapse of indicated genotypes. d . Scale bar, 50 μm. e , RNA expression defined by nanostring of lamina propria mononuclear cells in indicated groups (mean of n = 3 per group). f , Schematic overview of protocol for AOM/DSS colitis-associated cancer model. g , Representative haematoxylin and eosin staining of colon from wild-type mice in AOM/1.5% DSS model. Scale bar, 50 μm; h , Representative photograph of distal colons of wild-type mice ( n = 3 per group, anorectal junction at left end) in AOM/1.5% DSS model. Vertical arrows show the sites for dissection of the polyps (black) and the vicinity of the polyps (red). i , Representative flow cytometry analyses on infiltrating lymphocytes of invading distal colonic polyps or from the vicinity of the polyps or from mesenteric lymph nodes for CD4 + and CD8 + T cells and expression of CEACAM1 and TIM-3orPD-1 and TIM-3. Note that vicinity of polyps exhibit highest numbers of T cells with an exhausted phenotype. j , Summary of flow cytometry on infiltrating lymphocytes from invading distal colonic polyps or from vicinity of polyps and from mesenteric lymph nodes for CD4 + and CD8 + T cells expressing CEACAM1 and TIM-3 or PD-1 and TIM-3 ( n = 3, median shown). k , Representative pathology in AOM/1.5% DSS model. Scale bar, 60 μm. HGD, high grade dysplasia. Representative of three independent experiments ( a–e, g–k ). Fig. 3e

    Article Snippet: Previously described vectors containing the human CEACAM1–3L variant and human TIM-3 in the pDisplay vector (Invitrogen) were used as the template for all mutations.

    Techniques: Staining, Flow Cytometry, Cytometry, Expressing, RNA Expression, Mouse Assay, Dissection

    TIM-3 and CEACAM1 are co-expressed on T cells during induction of tolerance a, b , Tolerance induction in indicated mice. Median c.p.m., counts per minute. c, d , Responses of CFSE-labelled transgenic OT-II Rag2 −/− T cells in mesenteric lymph nodes (MLN), peripheral lymph node (LN) or spleen of wild-type (WT) or Ceacam1 −/− recipients to PBS ( n = 3 per group) or OVA ( n = 5 per group) for proliferation ( c ) and CEACAM1 or TIM-3 ( d ) expression. ND, not detectable. e , hCEACAM1 and hTIM-3 expression in co-transfected HEK293T cells. Percentage and mean fluorescence intensity (MFI) of hTIM-3 indicated. BFA, brefeldin A; ER, endoplasmic reticulum. f , hCEACAM1 and hTIM-3 expression on activated primary CD4 + human T cells. g, h , CEACAM1 + TIM-3 + CD4 + T cells ( g ) and intracellular cytokine staining for IFN-γ in CD4 + T cells after SEB stimulation ( h ) in HIV infection. C, CEACAM1; T, TIM-3 ( n = 4 per group). i , In situ proximity ligation assay of hCEACAM1 and hTIM-3 co-transfected HEK293T as in e . DAPI, 4′,6-diamidino-2-phenylindole. All data are mean ± s.e.m. and represent five ( e, f ), three ( c, d, i ) and two ( a, b ) independent experiments. * P

    Journal: Nature

    Article Title: CEACAM1 regulates TIM–3–mediated tolerance and exhaustion

    doi: 10.1038/nature13848

    Figure Lengend Snippet: TIM-3 and CEACAM1 are co-expressed on T cells during induction of tolerance a, b , Tolerance induction in indicated mice. Median c.p.m., counts per minute. c, d , Responses of CFSE-labelled transgenic OT-II Rag2 −/− T cells in mesenteric lymph nodes (MLN), peripheral lymph node (LN) or spleen of wild-type (WT) or Ceacam1 −/− recipients to PBS ( n = 3 per group) or OVA ( n = 5 per group) for proliferation ( c ) and CEACAM1 or TIM-3 ( d ) expression. ND, not detectable. e , hCEACAM1 and hTIM-3 expression in co-transfected HEK293T cells. Percentage and mean fluorescence intensity (MFI) of hTIM-3 indicated. BFA, brefeldin A; ER, endoplasmic reticulum. f , hCEACAM1 and hTIM-3 expression on activated primary CD4 + human T cells. g, h , CEACAM1 + TIM-3 + CD4 + T cells ( g ) and intracellular cytokine staining for IFN-γ in CD4 + T cells after SEB stimulation ( h ) in HIV infection. C, CEACAM1; T, TIM-3 ( n = 4 per group). i , In situ proximity ligation assay of hCEACAM1 and hTIM-3 co-transfected HEK293T as in e . DAPI, 4′,6-diamidino-2-phenylindole. All data are mean ± s.e.m. and represent five ( e, f ), three ( c, d, i ) and two ( a, b ) independent experiments. * P

    Article Snippet: Previously described vectors containing the human CEACAM1–3L variant and human TIM-3 in the pDisplay vector (Invitrogen) were used as the template for all mutations.

    Techniques: Mouse Assay, Transgenic Assay, Expressing, Transfection, Fluorescence, Staining, Infection, In Situ, Proximity Ligation Assay

    Rv2882c induced BMDM activation. (A) BMDMs (1 × 10 5 /well) were stimulated with 100 ng/mL LPS or 1, 5, or 10 μg/mL Rv2882c for 24 h. (A) Quantities of TNF-α, MCP-1, IL-6, IL-10, and IL-12p70 in the culture supernatant were determined by ELISA. All data are expressed as the mean values ± SD ( n = 3). Significance levels (* p

    Journal: PLoS ONE

    Article Title: Mycobacterium tuberculosis Rv2882c Protein Induces Activation of Macrophages through TLR4 and Exhibits Vaccine Potential

    doi: 10.1371/journal.pone.0164458

    Figure Lengend Snippet: Rv2882c induced BMDM activation. (A) BMDMs (1 × 10 5 /well) were stimulated with 100 ng/mL LPS or 1, 5, or 10 μg/mL Rv2882c for 24 h. (A) Quantities of TNF-α, MCP-1, IL-6, IL-10, and IL-12p70 in the culture supernatant were determined by ELISA. All data are expressed as the mean values ± SD ( n = 3). Significance levels (* p

    Article Snippet: Mouse TNF-α, MCP-1, IL-6, IL-10, IL-12p70, IFN-γ, and IL-2 ELISA kits were obtained from eBioscience.

    Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay