Journal: Journal of Natural Science, Biology, and Medicine

Article Title: Vedolizumab: A novel anti-integrin drug for treatment of inflammatory bowel disease

doi: 10.4103/0976-9668.175016

Figure Lengend Snippet: Role of integrins in pathogenesis of inflammatory bowel disease and mechanism of vedolizumab. Interaction of α4β7 and MAdCAM-1 is a crucial step activating the cascade of inflammation in inflammatory bowel disease. Vedolizumab acts by preventing this interaction impairing the inflammatory cascade

Article Snippet: Vedolizumab is a humanized version of act-1, a murine antibody originally developed in 1980s with activity against α 4 β 7 integrin heterodimer (Developed by Millennium Pharmaceuticals).

Techniques:

Journal: Nature immunology

Article Title: HIV-1 gp120 impairs B cell proliferation by inducing TGF-β1 production and FcRL4 expression

doi: 10.1038/ni.2746

Figure Lengend Snippet:

Article Snippet: The following antibodies were used for flow cytometry: anti-integrin β7 PE (clone FIB504), anti-CD10 APC (clone HI10a), anti-CD19 FITC or APC (clone HIB19), anti-CD20 APC (clone L27), anti-CD27 APC (clone M-T271), anti-CD38 APC (clone HB7), anti-CD80 PE (clone L307.4), anti-CD86 APC (clone 2331), were purchased from BD Biosciences; anti-CD23 FITC (clone M-L233) and anti-CD69 PE (clone FN50), were purchased from BD Pharmingen; anti-CD21 FITC (clone BL13) was purchased from Beckman Coulter; anti-hSiglec-2/CD22 PE (clone 219934), was purchased from R&D Systems; anti-CD307d (FcRL4) PE (clone 413D12) was purchased from Biolegend; the mAb anti-α 4 β 7 heterodimer (clone Act-1) was provided by the NIH AIDS Research & Reference Reagent Program and was PE-labeled using the LYNX Rapid Conjugation Kit (AbD Serotec).

Techniques: Chemotaxis Assay

Journal: Nature immunology

Article Title: HIV-1 gp120 impairs B cell proliferation by inducing TGF-β1 production and FcRL4 expression

doi: 10.1038/ni.2746

Figure Lengend Snippet:

Article Snippet: The following antibodies were used for flow cytometry: anti-integrin β7 PE (clone FIB504), anti-CD10 APC (clone HI10a), anti-CD19 FITC or APC (clone HIB19), anti-CD20 APC (clone L27), anti-CD27 APC (clone M-T271), anti-CD38 APC (clone HB7), anti-CD80 PE (clone L307.4), anti-CD86 APC (clone 2331), were purchased from BD Biosciences; anti-CD23 FITC (clone M-L233) and anti-CD69 PE (clone FN50), were purchased from BD Pharmingen; anti-CD21 FITC (clone BL13) was purchased from Beckman Coulter; anti-hSiglec-2/CD22 PE (clone 219934), was purchased from R&D Systems; anti-CD307d (FcRL4) PE (clone 413D12) was purchased from Biolegend; the mAb anti-α 4 β 7 heterodimer (clone Act-1) was provided by the NIH AIDS Research & Reference Reagent Program and was PE-labeled using the LYNX Rapid Conjugation Kit (AbD Serotec).

Techniques: Chemotaxis Assay

Journal: Nature immunology

Article Title: HIV-1 gp120 impairs B cell proliferation by inducing TGF-β1 production and FcRL4 expression

doi: 10.1038/ni.2746

Figure Lengend Snippet: CD80 induction by α-IgM + CpG stimulation is reduced in presence of an α 4 β 7 -reactive gp120. (a) FACS analysis of CD80 surface expression induced by α-IgM + CpG stimulation for 72h, in the presence or absence of gp120s with high (R880F) versus low (92Th14.12) affinity for α 4 β 7 . Values reported are average % reactivity normalized to CD80 expression at 72h upon B cell stimulation with α-IgM + CpG alone (100%), p <0.0001 (two way ANOVA) (s.e.m bars) (n=3). (b ) FACS analysis of CD80 surface expression over time induced by stimulation with α-IgM + CpG (24, 48, 72 h) in the presence or absence of gp120 with high affinity for α 4 β 7 (R880F) or intermediate affinity for α 4 β 7 (AN1). (c) FACS analysis of CD86 surface expression on B cells activated by α-IgM + CpG for 24, 48, 72 h, in presence of gp120s (R880F, AN1, Z185F) with different affinities for α 4 β 7 (72h). Data reported indicate MFI, and are representative of three independent experiments using different donor B cells.

Article Snippet: The following antibodies were used for flow cytometry: anti-integrin β7 PE (clone FIB504), anti-CD10 APC (clone HI10a), anti-CD19 FITC or APC (clone HIB19), anti-CD20 APC (clone L27), anti-CD27 APC (clone M-T271), anti-CD38 APC (clone HB7), anti-CD80 PE (clone L307.4), anti-CD86 APC (clone 2331), were purchased from BD Biosciences; anti-CD23 FITC (clone M-L233) and anti-CD69 PE (clone FN50), were purchased from BD Pharmingen; anti-CD21 FITC (clone BL13) was purchased from Beckman Coulter; anti-hSiglec-2/CD22 PE (clone 219934), was purchased from R&D Systems; anti-CD307d (FcRL4) PE (clone 413D12) was purchased from Biolegend; the mAb anti-α 4 β 7 heterodimer (clone Act-1) was provided by the NIH AIDS Research & Reference Reagent Program and was PE-labeled using the LYNX Rapid Conjugation Kit (AbD Serotec).

Techniques: Expressing, Cell Stimulation

Journal: Nature immunology

Article Title: HIV-1 gp120 impairs B cell proliferation by inducing TGF-β1 production and FcRL4 expression

doi: 10.1038/ni.2746

Figure Lengend Snippet: A T-dependent stimulation of B cells is suppressed by an α 4 β 7 -reactive gp120. (a) FACS analysis over time of the average % CD80 surface expression in three donors, induced by α-IgM + CpG + α-CD40 stimulation (24, 48, 72 h) in presence or absence of an α 4 β 7 -reactive gp120. Values reported are average % reactivity normalized to CD80 expression at 72h upon B cell stimulation with α-IgM + CpG + α-CD40 alone (100%), p <0.001 at 72h (two way ANOVA) (s.e.m bars) (n=4). (b) FACS analysis over time of the average % CD86 surface expression in three donors, induced by α-IgM + CpG + αCD40 stimulation (24, 48, 72 h) in presence or absence of an α 4 β 7 -reactive gp120. Values reported are average % reactivity normalized to CD86 expression at 72h upon B cell stimulation with α-IgM + CpG + α-CD40 alone (100%) (two way ANOVA) (s.e.m bars) (n=4). (c) FACS analysis for FcRL4 expression over a time course of 0–72h on freshly isolated B cells stimulated with α-IgM + CpG + α-CD40. Isotype control in shown as a grey shade. (d) FACS analysis of the average % FcRL4 and % CD80 expression induced on α-IgM + CpG + α-CD40 stimulated B cells in presence of R66M or TGF-β1. Anti-TGF-β1 was employed to block both gp120-mediated and TGF-β1-mediated effects. The average % of cells expressing FcRL4 and CD80 is reported normalized to FcRL4 and CD80 expression at 96h upon B cell stimulation with α-IgM + CpG + α-CD40 (100%), p <0.001 and p <0.0001(one way ANOVA, Bonferroni Multiple Comparison Test) (s.e.m bars) (FcRL4 n=4) (CD80 n=5). Values are normalized to % FcRL4 expression on α-IgM + CpG + α-CD40 stimulated B cells (100%). (e) CFSE assay of α-IgM + CpG + αCD40 induced B cell proliferation (1 st panel), in the presence of: an α 4 β 7 -reactive gp120 (2 nd panel), an α 4 β 7 -reactive gp120 and an anti-TGF-β1 (3 rd panel), soluble TGF-β1 (4 th panel), and soluble TGF-β1 and an anti-TGF-β1 (5 th panel) of a representative donor. Cells were cultured for 96h. (f) Division Index (FlowJo) indicating the average number of cell divisions in three independent donors, p <0.001 (two-way ANOVA) (n=3). Treatments are listed below the x-axis.

Article Snippet: The following antibodies were used for flow cytometry: anti-integrin β7 PE (clone FIB504), anti-CD10 APC (clone HI10a), anti-CD19 FITC or APC (clone HIB19), anti-CD20 APC (clone L27), anti-CD27 APC (clone M-T271), anti-CD38 APC (clone HB7), anti-CD80 PE (clone L307.4), anti-CD86 APC (clone 2331), were purchased from BD Biosciences; anti-CD23 FITC (clone M-L233) and anti-CD69 PE (clone FN50), were purchased from BD Pharmingen; anti-CD21 FITC (clone BL13) was purchased from Beckman Coulter; anti-hSiglec-2/CD22 PE (clone 219934), was purchased from R&D Systems; anti-CD307d (FcRL4) PE (clone 413D12) was purchased from Biolegend; the mAb anti-α 4 β 7 heterodimer (clone Act-1) was provided by the NIH AIDS Research & Reference Reagent Program and was PE-labeled using the LYNX Rapid Conjugation Kit (AbD Serotec).

Techniques: Expressing, Cell Stimulation, Isolation, Blocking Assay, CFSE Assay, Cell Culture

Journal: Nature immunology

Article Title: HIV-1 gp120 impairs B cell proliferation by inducing TGF-β1 production and FcRL4 expression

doi: 10.1038/ni.2746

Figure Lengend Snippet: CD80 induction by α-IgM + CpG stimulation is reduced in presence of an α 4 β 7 -reactive gp120. (a) FACS analysis of CD80 surface expression induced by α-IgM + CpG stimulation for 72h, in the presence or absence of gp120s with high (R880F) versus low (92Th14.12) affinity for α 4 β 7 . Values reported are average % reactivity normalized to CD80 expression at 72h upon B cell stimulation with α-IgM + CpG alone (100%), p <0.0001 (two way ANOVA) (s.e.m bars) (n=3). (b ) FACS analysis of CD80 surface expression over time induced by stimulation with α-IgM + CpG (24, 48, 72 h) in the presence or absence of gp120 with high affinity for α 4 β 7 (R880F) or intermediate affinity for α 4 β 7 (AN1). (c) FACS analysis of CD86 surface expression on B cells activated by α-IgM + CpG for 24, 48, 72 h, in presence of gp120s (R880F, AN1, Z185F) with different affinities for α 4 β 7 (72h). Data reported indicate MFI, and are representative of three independent experiments using different donor B cells.

Article Snippet: The following antibodies were used for flow cytometry: anti-integrin β7 PE (clone FIB504), anti-CD10 APC (clone HI10a), anti-CD19 FITC or APC (clone HIB19), anti-CD20 APC (clone L27), anti-CD27 APC (clone M-T271), anti-CD38 APC (clone HB7), anti-CD80 PE (clone L307.4), anti-CD86 APC (clone 2331), were purchased from BD Biosciences; anti-CD23 FITC (clone M-L233) and anti-CD69 PE (clone FN50), were purchased from BD Pharmingen; anti-CD21 FITC (clone BL13) was purchased from Beckman Coulter; anti-hSiglec-2/CD22 PE (clone 219934), was purchased from R&D Systems; anti-CD307d (FcRL4) PE (clone 413D12) was purchased from Biolegend; the mAb anti-α 4 β 7 heterodimer (clone Act-1) was provided by the NIH AIDS Research & Reference Reagent Program and was PE-labeled using the LYNX Rapid Conjugation Kit (AbD Serotec).

Techniques: Expressing, Cell Stimulation

Journal: Nature immunology

Article Title: HIV-1 gp120 impairs B cell proliferation by inducing TGF-β1 production and FcRL4 expression

doi: 10.1038/ni.2746

Figure Lengend Snippet: gp120 induces FcRL4 via the induction of TGF-β1. (a) Average TGF-β1 ELISA of supernatants from cultured primary B cells from three separate healthy donors stimulated with α-IgM + CpG in presence of an α 4 β 7 -reactive gp120 (R66M), p <0.0001 (unpaired t -test) (s.e.d. bars) (n=3). (b) FACS analysis of % FcRL4 and % CD80 expression on α-IgM + CpG stimulated B cells in presence of gp120 or TGF-β1. Anti-TGF-β1 was employed to block both gp120-mediated and TGF-β1-mediated effects. The average % of cells expressing FcRL4 and CD80, normalized to FcRL4 and CD80 expression at 96h upon B cell stimulation with α-IgM + CpG alone (100%), p <0.0001(one way ANOVA, Bonferroni Multiple Comparison Test) (s.e.m bars) (FcRL4 n=8) (CD80 n=5). (c) CFSE assay of α-IgM + CpG induced B cell proliferation (1 st panel), in the presence of: an α 4 β 7 -reactive gp120 (2 nd panel), an α 4 β 7 -reactive gp120 and an anti-TGF-β1 (3 rd panel), soluble TGF-β1 (4 th panel), and soluble TGF-β1 and an anti-TGF-β1 (5 th panel) of a representative donor. Cells were cultured for 96h. (d) Division Index (FlowJo) indicating the average number of cell divisions in three independent donors, p <0.001 (two-way ANOVA) (n=3). Treatments are listed below the x-axis.

Article Snippet: The following antibodies were used for flow cytometry: anti-integrin β7 PE (clone FIB504), anti-CD10 APC (clone HI10a), anti-CD19 FITC or APC (clone HIB19), anti-CD20 APC (clone L27), anti-CD27 APC (clone M-T271), anti-CD38 APC (clone HB7), anti-CD80 PE (clone L307.4), anti-CD86 APC (clone 2331), were purchased from BD Biosciences; anti-CD23 FITC (clone M-L233) and anti-CD69 PE (clone FN50), were purchased from BD Pharmingen; anti-CD21 FITC (clone BL13) was purchased from Beckman Coulter; anti-hSiglec-2/CD22 PE (clone 219934), was purchased from R&D Systems; anti-CD307d (FcRL4) PE (clone 413D12) was purchased from Biolegend; the mAb anti-α 4 β 7 heterodimer (clone Act-1) was provided by the NIH AIDS Research & Reference Reagent Program and was PE-labeled using the LYNX Rapid Conjugation Kit (AbD Serotec).

Techniques: Enzyme-linked Immunosorbent Assay, Cell Culture, Expressing, Blocking Assay, Cell Stimulation, CFSE Assay

Journal: Nature immunology

Article Title: HIV-1 gp120 impairs B cell proliferation by inducing TGF-β1 production and FcRL4 expression

doi: 10.1038/ni.2746

Figure Lengend Snippet: A T-dependent stimulation of B cells is suppressed by an α 4 β 7 -reactive gp120. (a) FACS analysis over time of the average % CD80 surface expression in three donors, induced by α-IgM + CpG + α-CD40 stimulation (24, 48, 72 h) in presence or absence of an α 4 β 7 -reactive gp120. Values reported are average % reactivity normalized to CD80 expression at 72h upon B cell stimulation with α-IgM + CpG + α-CD40 alone (100%), p <0.001 at 72h (two way ANOVA) (s.e.m bars) (n=4). (b) FACS analysis over time of the average % CD86 surface expression in three donors, induced by α-IgM + CpG + αCD40 stimulation (24, 48, 72 h) in presence or absence of an α 4 β 7 -reactive gp120. Values reported are average % reactivity normalized to CD86 expression at 72h upon B cell stimulation with α-IgM + CpG + α-CD40 alone (100%) (two way ANOVA) (s.e.m bars) (n=4). (c) FACS analysis for FcRL4 expression over a time course of 0–72h on freshly isolated B cells stimulated with α-IgM + CpG + α-CD40. Isotype control in shown as a grey shade. (d) FACS analysis of the average % FcRL4 and % CD80 expression induced on α-IgM + CpG + α-CD40 stimulated B cells in presence of R66M or TGF-β1. Anti-TGF-β1 was employed to block both gp120-mediated and TGF-β1-mediated effects. The average % of cells expressing FcRL4 and CD80 is reported normalized to FcRL4 and CD80 expression at 96h upon B cell stimulation with α-IgM + CpG + α-CD40 (100%), p <0.001 and p <0.0001(one way ANOVA, Bonferroni Multiple Comparison Test) (s.e.m bars) (FcRL4 n=4) (CD80 n=5). Values are normalized to % FcRL4 expression on α-IgM + CpG + α-CD40 stimulated B cells (100%). (e) CFSE assay of α-IgM + CpG + αCD40 induced B cell proliferation (1 st panel), in the presence of: an α 4 β 7 -reactive gp120 (2 nd panel), an α 4 β 7 -reactive gp120 and an anti-TGF-β1 (3 rd panel), soluble TGF-β1 (4 th panel), and soluble TGF-β1 and an anti-TGF-β1 (5 th panel) of a representative donor. Cells were cultured for 96h. (f) Division Index (FlowJo) indicating the average number of cell divisions in three independent donors, p <0.001 (two-way ANOVA) (n=3). Treatments are listed below the x-axis.

Article Snippet: The following antibodies were used for flow cytometry: anti-integrin β7 PE (clone FIB504), anti-CD10 APC (clone HI10a), anti-CD19 FITC or APC (clone HIB19), anti-CD20 APC (clone L27), anti-CD27 APC (clone M-T271), anti-CD38 APC (clone HB7), anti-CD80 PE (clone L307.4), anti-CD86 APC (clone 2331), were purchased from BD Biosciences; anti-CD23 FITC (clone M-L233) and anti-CD69 PE (clone FN50), were purchased from BD Pharmingen; anti-CD21 FITC (clone BL13) was purchased from Beckman Coulter; anti-hSiglec-2/CD22 PE (clone 219934), was purchased from R&D Systems; anti-CD307d (FcRL4) PE (clone 413D12) was purchased from Biolegend; the mAb anti-α 4 β 7 heterodimer (clone Act-1) was provided by the NIH AIDS Research & Reference Reagent Program and was PE-labeled using the LYNX Rapid Conjugation Kit (AbD Serotec).

Techniques: Expressing, Cell Stimulation, Isolation, Blocking Assay, CFSE Assay, Cell Culture