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  • 97
    Millipore β glucan β glucan
    Microbicidal activity of <t>β-glucan-treated</t> neutrophils activated by different isolates of C. albicans and C. glabrata . Neutrophils (2.0×10 6 cells/ml) were previously treated or not with 3 mg/ml β-glucan and incubated with the reference strain and different isolates of (A) C. albicans and (B) C. glabrata (RVVC, VVC, and ASS; 2.0×10 7 CFU/ml) at 37°C for different times (0, 30, 60, 90, and 120 min). The quantity of viable yeast was estimated by plating the samples in Sabouraund Dextrose Agar (SDA) at 37°C for 24 h. The data are expressed as the mean ± SD of three separate experiments. * p ≤0.05, significant difference compared with the control group (yeast alone); # p ≤0.05, significant difference compared with untreated and activated neutrophils.
    β Glucan β Glucan, supplied by Millipore, used in various techniques. Bioz Stars score: 97/100, based on 48 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Thermo Fisher β glucan
    Microbicidal activity of <t>β-glucan-treated</t> neutrophils activated by different isolates of C. albicans and C. glabrata . Neutrophils (2.0×10 6 cells/ml) were previously treated or not with 3 mg/ml β-glucan and incubated with the reference strain and different isolates of (A) C. albicans and (B) C. glabrata (RVVC, VVC, and ASS; 2.0×10 7 CFU/ml) at 37°C for different times (0, 30, 60, 90, and 120 min). The quantity of viable yeast was estimated by plating the samples in Sabouraund Dextrose Agar (SDA) at 37°C for 24 h. The data are expressed as the mean ± SD of three separate experiments. * p ≤0.05, significant difference compared with the control group (yeast alone); # p ≤0.05, significant difference compared with untreated and activated neutrophils.
    β Glucan, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 91 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Megazyme β glucan
    TLC analysis of the action patterns of Cpin_6279rC on <t>β-1,2-glucan</t> and Sop n s. β-1,2-Glucan (average DP 64) ( A ), cyclic β-1,2-glucan (DP 17–24) ( B ), Sop 5 ( C ), Sop 6 ( D ), and Sop 7 ( E ) were used as substrates. Lane M contains markers (0.2% each sugar). Asterisks represent the origins of the TLC plates.
    β Glucan, supplied by Megazyme, used in various techniques. Bioz Stars score: 93/100, based on 258 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Lonza β glucan blocker
    TLC analysis of the action patterns of Cpin_6279rC on <t>β-1,2-glucan</t> and Sop n s. β-1,2-Glucan (average DP 64) ( A ), cyclic β-1,2-glucan (DP 17–24) ( B ), Sop 5 ( C ), Sop 6 ( D ), and Sop 7 ( E ) were used as substrates. Lane M contains markers (0.2% each sugar). Asterisks represent the origins of the TLC plates.
    β Glucan Blocker, supplied by Lonza, used in various techniques. Bioz Stars score: 86/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Megazyme β glucan kit
    α ( A ), β ( B ), and total ( C ) <t>glucan</t> concentrations in glucan extracts prepared from caps and stalks from Pleurotus eryngii mushrooms cultivated in various relative amounts of eucalyptus sawdust and OMSW. * p
    β Glucan Kit, supplied by Megazyme, used in various techniques. Bioz Stars score: 88/100, based on 31 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Assay.Works β glucan assay
    α ( A ), β ( B ), and total ( C ) <t>glucan</t> concentrations in glucan extracts prepared from caps and stalks from Pleurotus eryngii mushrooms cultivated in various relative amounts of eucalyptus sawdust and OMSW. * p
    β Glucan Assay, supplied by Assay.Works, used in various techniques. Bioz Stars score: 85/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Elicityl scleroglucan β glucan
    α ( A ), β ( B ), and total ( C ) <t>glucan</t> concentrations in glucan extracts prepared from caps and stalks from Pleurotus eryngii mushrooms cultivated in various relative amounts of eucalyptus sawdust and OMSW. * p
    Scleroglucan β Glucan, supplied by Elicityl, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    InvivoGen particulate β glucan
    Sts regulates ROS production downstream of fungal CLR Dectin-1. (A) Wild-type and Sts −/− BMDCs were stimulated with particulate <t>β-glucan,</t> and then levels of ROS production were assessed by luminol chemiluminescence. (B) Zymosan-induced (left) or HK (heat-killed) wild-type C. albicans -induced (right) ROS production in wild-type and Sts −/− BMDCs was assessed in the absence (−) or presence (+) of soluble β-glucan, a specific inhibitor that blocks activation of Dectin-1 signaling pathways ( 46 ). (C) ROS production in wild-type and Sts −/− BMDCs was assessed in the absence (−) or presence (+) of a blocking anti-Dectin-1 antibody. (D) Equivalent levels of surface Dectin-1 receptor expressed on wild-type BMDCs (solid red line) and Sts −/− BMDCs (dotted dark blue line), evaluated by flow cytometry with a specific anti-Dectin-1 antibody. Nonshaded lines represent a nonspecific rat IgG control. (E) Equivalent levels of zymosan stimulation-dependent downregulation of surface Dectin-1 on wild-type (closed dot) and Sts −/− (open dot) BMDCs. MFI, mean fluorescence intensity. For panels A to C, average values from at least 3 independent experiments each performed in triplicate are presented. P values were calculated by Mann-Whitney analysis.
    Particulate β Glucan, supplied by InvivoGen, used in various techniques. Bioz Stars score: 91/100, based on 24 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Novozymes β glucan enzyme
    Sts regulates ROS production downstream of fungal CLR Dectin-1. (A) Wild-type and Sts −/− BMDCs were stimulated with particulate <t>β-glucan,</t> and then levels of ROS production were assessed by luminol chemiluminescence. (B) Zymosan-induced (left) or HK (heat-killed) wild-type C. albicans -induced (right) ROS production in wild-type and Sts −/− BMDCs was assessed in the absence (−) or presence (+) of soluble β-glucan, a specific inhibitor that blocks activation of Dectin-1 signaling pathways ( 46 ). (C) ROS production in wild-type and Sts −/− BMDCs was assessed in the absence (−) or presence (+) of a blocking anti-Dectin-1 antibody. (D) Equivalent levels of surface Dectin-1 receptor expressed on wild-type BMDCs (solid red line) and Sts −/− BMDCs (dotted dark blue line), evaluated by flow cytometry with a specific anti-Dectin-1 antibody. Nonshaded lines represent a nonspecific rat IgG control. (E) Equivalent levels of zymosan stimulation-dependent downregulation of surface Dectin-1 on wild-type (closed dot) and Sts −/− (open dot) BMDCs. MFI, mean fluorescence intensity. For panels A to C, average values from at least 3 independent experiments each performed in triplicate are presented. P values were calculated by Mann-Whitney analysis.
    β Glucan Enzyme, supplied by Novozymes, used in various techniques. Bioz Stars score: 85/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Immuno Research Inc particulate β glucan
    Particulate <t>β-glucan</t> induces the differentiation of M-MDSC to antigen-presenting cells in vitro (A) Expression of CD11c, F4/80 and CD11b surface markers on M-MDSC cultured with particulate β-glucan (50 µg/ml) for 0, 5 and 7 days. Results were repeated at least three times with similar results. (B) Expression of Gr-1, CD80, CD86, MHC class II, MHC class I, and CD40 surface markers on M-MDSC cultured with particulate β-glucan (50 µg/ml) for 7 days. Histograms represent the results of three independent experiments. (C) Frequency of IFN-γ + CFSE − gated on CD4 + T cells in cultures where freshly sorted M-MDSC from the spleens of LLC-bearing mice were incubated for 5 days with CFSE-labeled CD4 + T cells in the presence of OVA (100µg/ml). Irradiated splenocytes were used as APC positive control. (D) Sorted and CFSE-labeled CD4 + OT-II T cells were co-cultured with splenic M-MDSC pre-cultured with particulate β-glucan for 7 days in the presence of OVA (50 µg/ml) for 4–5 days. The frequencies of CFSE diluted cells and IFN-γ + CD4 + T cells were shown. Irradiated splenocytes were used as APC positive control. The results are representative of at least four independent experiments. (E) Sorted and CFSE-labeled CD8 + OT-I T cells were co-cultured for 4–5 days with OVA (50 µg/ml) and splenic M-MDSC pre-cultured with particulate β-glucan for 7 days. The frequencies of CFSE diluted cells, IFN-γ + cells and Granzyme B + cells gated on CD8 + T cells are demonstrated. Results are representative of two independent experiments. *p
    Particulate β Glucan, supplied by Immuno Research Inc, 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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    90
    InvivoGen β glucan whole glucan particles
    Phagocytic ability of macrophages from individuals with different TB phenotypes. Monocyte-derived macrophages from patients at day 7 were treated with Alexa 547-beads coated with either immunoglobulin-G (IgG), trehalose 6,6'-dimycolate (TDM) or <t>β-glucan.</t> Phagocytic ability was determined by the percentage of macrophages with beads in three TB phenotypes (55 TB meningitis, 52 pulmonary TB and 56 latent TB). Bars in plots represent median values. Comparisons across three groups of TB forms or genotypes were performed by using one-way analysis of variance. On these comparisons, P -values > 0.05.
    β Glucan Whole Glucan Particles, supplied by InvivoGen, used in various techniques. Bioz Stars score: 90/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Microbicidal activity of β-glucan-treated neutrophils activated by different isolates of C. albicans and C. glabrata . Neutrophils (2.0×10 6 cells/ml) were previously treated or not with 3 mg/ml β-glucan and incubated with the reference strain and different isolates of (A) C. albicans and (B) C. glabrata (RVVC, VVC, and ASS; 2.0×10 7 CFU/ml) at 37°C for different times (0, 30, 60, 90, and 120 min). The quantity of viable yeast was estimated by plating the samples in Sabouraund Dextrose Agar (SDA) at 37°C for 24 h. The data are expressed as the mean ± SD of three separate experiments. * p ≤0.05, significant difference compared with the control group (yeast alone); # p ≤0.05, significant difference compared with untreated and activated neutrophils.

    Journal: PLoS ONE

    Article Title: β-Glucan Induces Reactive Oxygen Species Production in Human Neutrophils to Improve the Killing of Candida albicans and Candida glabrata Isolates from Vulvovaginal Candidiasis

    doi: 10.1371/journal.pone.0107805

    Figure Lengend Snippet: Microbicidal activity of β-glucan-treated neutrophils activated by different isolates of C. albicans and C. glabrata . Neutrophils (2.0×10 6 cells/ml) were previously treated or not with 3 mg/ml β-glucan and incubated with the reference strain and different isolates of (A) C. albicans and (B) C. glabrata (RVVC, VVC, and ASS; 2.0×10 7 CFU/ml) at 37°C for different times (0, 30, 60, 90, and 120 min). The quantity of viable yeast was estimated by plating the samples in Sabouraund Dextrose Agar (SDA) at 37°C for 24 h. The data are expressed as the mean ± SD of three separate experiments. * p ≤0.05, significant difference compared with the control group (yeast alone); # p ≤0.05, significant difference compared with untreated and activated neutrophils.

    Article Snippet: β-glucan β-glucan derived from Laminaria digitata was purchased from Sigma Chemical Co. (St. Louis, MO, USA; L-9634).

    Techniques: Activity Assay, Incubation

    Phagocytosis activity of β-glucan-treated neutrophils activated by different isolates of C. albicans and C. glabrata . Neutrophils (2.0×10 6 cells/ml) were previously treated or not with 3 mg/ml β-glucan and incubated for 1 h at 37°C with the reference strain and different isolates of (A) C. albicans and (B) C. glabrata (RVVC, VVC, and ASS; 2.0×10 7 CFU/ml) labeled with FITC. Phagocytosis was determined by flow cytometry, and the results are expressed as the mean fluorescence (in arbitrary units [au]) ± SD of three independent experiments. # p ≤0.05, significant difference compared with untreated and activated neutrophils.

    Journal: PLoS ONE

    Article Title: β-Glucan Induces Reactive Oxygen Species Production in Human Neutrophils to Improve the Killing of Candida albicans and Candida glabrata Isolates from Vulvovaginal Candidiasis

    doi: 10.1371/journal.pone.0107805

    Figure Lengend Snippet: Phagocytosis activity of β-glucan-treated neutrophils activated by different isolates of C. albicans and C. glabrata . Neutrophils (2.0×10 6 cells/ml) were previously treated or not with 3 mg/ml β-glucan and incubated for 1 h at 37°C with the reference strain and different isolates of (A) C. albicans and (B) C. glabrata (RVVC, VVC, and ASS; 2.0×10 7 CFU/ml) labeled with FITC. Phagocytosis was determined by flow cytometry, and the results are expressed as the mean fluorescence (in arbitrary units [au]) ± SD of three independent experiments. # p ≤0.05, significant difference compared with untreated and activated neutrophils.

    Article Snippet: β-glucan β-glucan derived from Laminaria digitata was purchased from Sigma Chemical Co. (St. Louis, MO, USA; L-9634).

    Techniques: Activity Assay, Incubation, Labeling, Flow Cytometry, Cytometry, Fluorescence

    Cytokine release by β-glucan-treated neutrophils activated by different isolates of C. albicans and C. glabrata . Neutrophils (2.0×10 6 cells/ml) were previously treated or not with 3 mg/ml β-glucan and activated or not by the reference strain and different isolates of (A) C. albicans and (B) C. glabrata (RVVC, VVC, and ASS; 2.0×10 7 CFU/ml) and 1 µg/ml LPS and cultured for 18 h. (a, a’) IL-8. (b, b’) IL-1β. (c, c’) IL-1Ra. (d, d’) TNF-α. The data are expressed as the mean ± SD of three independent experiments. * p ≤0.05, significant difference compared with the control group (neutrophils alone); # p ≤0.05, significant difference compared with untreated and activated neutrophils.

    Journal: PLoS ONE

    Article Title: β-Glucan Induces Reactive Oxygen Species Production in Human Neutrophils to Improve the Killing of Candida albicans and Candida glabrata Isolates from Vulvovaginal Candidiasis

    doi: 10.1371/journal.pone.0107805

    Figure Lengend Snippet: Cytokine release by β-glucan-treated neutrophils activated by different isolates of C. albicans and C. glabrata . Neutrophils (2.0×10 6 cells/ml) were previously treated or not with 3 mg/ml β-glucan and activated or not by the reference strain and different isolates of (A) C. albicans and (B) C. glabrata (RVVC, VVC, and ASS; 2.0×10 7 CFU/ml) and 1 µg/ml LPS and cultured for 18 h. (a, a’) IL-8. (b, b’) IL-1β. (c, c’) IL-1Ra. (d, d’) TNF-α. The data are expressed as the mean ± SD of three independent experiments. * p ≤0.05, significant difference compared with the control group (neutrophils alone); # p ≤0.05, significant difference compared with untreated and activated neutrophils.

    Article Snippet: β-glucan β-glucan derived from Laminaria digitata was purchased from Sigma Chemical Co. (St. Louis, MO, USA; L-9634).

    Techniques: Cell Culture

    Oxygen consumption by β-glucan-treated neutrophils activated by different isolates of C. albicans and C. glabrata . Neutrophils (2.0×10 6 cells/ml) were previously treated or not with 3 mg/ml β-glucan and activated or not by the reference strain and different isolates of (A) C. albicans and (B) C. glabrata (RVVC, VVC, and ASS; 2.0×10 7 CFU/ml). Oxygen consumption was monitored for 5–10 min and calculated from the polarographic recordings using an initial concentration of dissolved oxygen of 190 µM at 37°C. The data are expressed as the mean ± SD of three independent experiments. * p ≤0.05, significant difference compared with the control group (neutrophils alone); # p ≤0.05, significant difference compared with untreated and activated neutrophils.

    Journal: PLoS ONE

    Article Title: β-Glucan Induces Reactive Oxygen Species Production in Human Neutrophils to Improve the Killing of Candida albicans and Candida glabrata Isolates from Vulvovaginal Candidiasis

    doi: 10.1371/journal.pone.0107805

    Figure Lengend Snippet: Oxygen consumption by β-glucan-treated neutrophils activated by different isolates of C. albicans and C. glabrata . Neutrophils (2.0×10 6 cells/ml) were previously treated or not with 3 mg/ml β-glucan and activated or not by the reference strain and different isolates of (A) C. albicans and (B) C. glabrata (RVVC, VVC, and ASS; 2.0×10 7 CFU/ml). Oxygen consumption was monitored for 5–10 min and calculated from the polarographic recordings using an initial concentration of dissolved oxygen of 190 µM at 37°C. The data are expressed as the mean ± SD of three independent experiments. * p ≤0.05, significant difference compared with the control group (neutrophils alone); # p ≤0.05, significant difference compared with untreated and activated neutrophils.

    Article Snippet: β-glucan β-glucan derived from Laminaria digitata was purchased from Sigma Chemical Co. (St. Louis, MO, USA; L-9634).

    Techniques: Concentration Assay

    HOCl production by β-glucan-treated neutrophils activated by different isolates of C. albicans and C. glabrata determined by spectrophotometry. Neutrophils (2.0×10 6 cells/ml) were previously treated or not with 3 mg/ml β-glucan and activated or not by the reference strain and different isolates of (A) C. albicans and (B) C. glabrata (RVVC, VVC, and ASS; 2.0×10 7 CFU/ml) and read at 655 nm. The data are expressed as the mean ± SD of three independent experiments. * p ≤0.05, significant difference compared with the control group (neutrophils alone); # p ≤0.05, significant difference compared with untreated and activated neutrophils.

    Journal: PLoS ONE

    Article Title: β-Glucan Induces Reactive Oxygen Species Production in Human Neutrophils to Improve the Killing of Candida albicans and Candida glabrata Isolates from Vulvovaginal Candidiasis

    doi: 10.1371/journal.pone.0107805

    Figure Lengend Snippet: HOCl production by β-glucan-treated neutrophils activated by different isolates of C. albicans and C. glabrata determined by spectrophotometry. Neutrophils (2.0×10 6 cells/ml) were previously treated or not with 3 mg/ml β-glucan and activated or not by the reference strain and different isolates of (A) C. albicans and (B) C. glabrata (RVVC, VVC, and ASS; 2.0×10 7 CFU/ml) and read at 655 nm. The data are expressed as the mean ± SD of three independent experiments. * p ≤0.05, significant difference compared with the control group (neutrophils alone); # p ≤0.05, significant difference compared with untreated and activated neutrophils.

    Article Snippet: β-glucan β-glucan derived from Laminaria digitata was purchased from Sigma Chemical Co. (St. Louis, MO, USA; L-9634).

    Techniques: Spectrophotometry

    Myeloperoxidase activity of β-glucan-treated neutrophils activated by different isolates of C. albicans and C. glabrata (integrated light emission). The inset represents kinetic study of MPO activity of β-glucan-treated neutrophils after 20 minutes of incubation. Neutrophils (2.0×10 6 cells/ml) were previously treated or not with 3 mg/ml β-glucan and incubated with the reference strain and different isolates of (A) C. albicans and (B) C. glabrata (2×10 7 CFU/ml) for 30 min. (a,a’) ATCC. (b,b’) ASS. (c,c’) VVC. (d,d’) RVVC. After incubation, chemiluminescence was monitored for 20 min at 37°C in a microplate luminometer using luminol as a chemical light amplifier. The data are expressed as the mean ± SD of three independent experiments. * p ≤0.05, significant difference compared with the control group (neutrophils alone); # p ≤0.05, significant difference compared with untreated and activated neutrophils.

    Journal: PLoS ONE

    Article Title: β-Glucan Induces Reactive Oxygen Species Production in Human Neutrophils to Improve the Killing of Candida albicans and Candida glabrata Isolates from Vulvovaginal Candidiasis

    doi: 10.1371/journal.pone.0107805

    Figure Lengend Snippet: Myeloperoxidase activity of β-glucan-treated neutrophils activated by different isolates of C. albicans and C. glabrata (integrated light emission). The inset represents kinetic study of MPO activity of β-glucan-treated neutrophils after 20 minutes of incubation. Neutrophils (2.0×10 6 cells/ml) were previously treated or not with 3 mg/ml β-glucan and incubated with the reference strain and different isolates of (A) C. albicans and (B) C. glabrata (2×10 7 CFU/ml) for 30 min. (a,a’) ATCC. (b,b’) ASS. (c,c’) VVC. (d,d’) RVVC. After incubation, chemiluminescence was monitored for 20 min at 37°C in a microplate luminometer using luminol as a chemical light amplifier. The data are expressed as the mean ± SD of three independent experiments. * p ≤0.05, significant difference compared with the control group (neutrophils alone); # p ≤0.05, significant difference compared with untreated and activated neutrophils.

    Article Snippet: β-glucan β-glucan derived from Laminaria digitata was purchased from Sigma Chemical Co. (St. Louis, MO, USA; L-9634).

    Techniques: Activity Assay, Incubation

    Intracellular oxidant species production by β-glucan-treated neutrophils activated by different isolates of C. albicans and C. glabrata determined by flow cytometry. Neutrophils (2.0×10 6 cells/ml) were previously treated or not with 3 mg/ml β-glucan and incubated for 1 h with the reference strain and different isolates of (A) C. albicans and (B) C. glabrata (RVVC, VVC, and ASS; 2.0×10 7 CFU/ml), followed by 30 min incubation with DHR. The data are expressed as the mean ± SD of at least three independent experiments. * p ≤0.05, significant difference compared with the control group (neutrophils alone); # p ≤0.05, significant difference compared with untreated and activated neutrophils. (C and D) Representative dot plot display of FL1 (green fluorescence) vs . FL2 on a logarithmic scale. (C – (a) ATCC, (c) ASS, (e) VVC,(g) RVVC) C. albicans with untreated neutrophils. (C – (b) ATCC,(d) ASS,(f) VVC,(h) RVVC) C. albicans with neutrophils previously treated with 3 mg/ml β-glucan. (D – (a’) ATCC,(c’) ASS,(e’) VVC,(g’) RVVC) C. glabrata with untreated neutrophils. (D – (b’) ATCC,(d’) ASS,(f’) VVC,(h’) RVVC) C. glabrata with neutrophils previously treated with 3 mg/ml β-glucan.

    Journal: PLoS ONE

    Article Title: β-Glucan Induces Reactive Oxygen Species Production in Human Neutrophils to Improve the Killing of Candida albicans and Candida glabrata Isolates from Vulvovaginal Candidiasis

    doi: 10.1371/journal.pone.0107805

    Figure Lengend Snippet: Intracellular oxidant species production by β-glucan-treated neutrophils activated by different isolates of C. albicans and C. glabrata determined by flow cytometry. Neutrophils (2.0×10 6 cells/ml) were previously treated or not with 3 mg/ml β-glucan and incubated for 1 h with the reference strain and different isolates of (A) C. albicans and (B) C. glabrata (RVVC, VVC, and ASS; 2.0×10 7 CFU/ml), followed by 30 min incubation with DHR. The data are expressed as the mean ± SD of at least three independent experiments. * p ≤0.05, significant difference compared with the control group (neutrophils alone); # p ≤0.05, significant difference compared with untreated and activated neutrophils. (C and D) Representative dot plot display of FL1 (green fluorescence) vs . FL2 on a logarithmic scale. (C – (a) ATCC, (c) ASS, (e) VVC,(g) RVVC) C. albicans with untreated neutrophils. (C – (b) ATCC,(d) ASS,(f) VVC,(h) RVVC) C. albicans with neutrophils previously treated with 3 mg/ml β-glucan. (D – (a’) ATCC,(c’) ASS,(e’) VVC,(g’) RVVC) C. glabrata with untreated neutrophils. (D – (b’) ATCC,(d’) ASS,(f’) VVC,(h’) RVVC) C. glabrata with neutrophils previously treated with 3 mg/ml β-glucan.

    Article Snippet: β-glucan β-glucan derived from Laminaria digitata was purchased from Sigma Chemical Co. (St. Louis, MO, USA; L-9634).

    Techniques: Flow Cytometry, Cytometry, Incubation, Fluorescence

    (A) Human monocytes were incubated for 24 h in RPMI, β-glucan or LPS. Whereas, the former was the control, the latter two treatments represent the trained and tolerant cells, respectively. Subsequently, the cells were left for 5 days in RPMI medium with 10% human pooled serum and BrdU. Upon completion of this period, the amount of BrdU (as a parameter of endoreplication) was quantified by a colorimetric assay, and raw OD data were recorded. N = 6, Wilcoxon; * p

    Journal: Frontiers in Immunology

    Article Title: DNA Synthesis Is Activated in Mosquitoes and Human Monocytes During the Induction of Innate Immune Memory

    doi: 10.3389/fimmu.2018.02834

    Figure Lengend Snippet: (A) Human monocytes were incubated for 24 h in RPMI, β-glucan or LPS. Whereas, the former was the control, the latter two treatments represent the trained and tolerant cells, respectively. Subsequently, the cells were left for 5 days in RPMI medium with 10% human pooled serum and BrdU. Upon completion of this period, the amount of BrdU (as a parameter of endoreplication) was quantified by a colorimetric assay, and raw OD data were recorded. N = 6, Wilcoxon; * p

    Article Snippet: For experiments with BrdU incorporation, 6 million monocytes were seeded in 10-cm Petri dishes (Corning) and treated as with β-glucan, but in the presence or absence of BrdU (Sigma-Aldrich).

    Techniques: Incubation, Colorimetric Assay

    (A) The number of genomic copies of TEP1 and PPO1 following the exposure of 250,000 and 500,000 cells to Plasmodium berghei during 3 or 6 h. (B) Human monocytes were incubated for 24 h in RPMI, β-glucan or LPS (the former being the control, the latter two the trained and tolerant cells, respectively). Subsequently, the cells were left for 5 days in RPMI medium with 10% human pooled serum, and then harvested. The DNA was isolated and qPCR was run with primers for the promoter regions of TNFA, IL6, HK , and PFKP . Expression in the RPMI control group was set at 1. Relative amount of DNA of the trained (β-glucan)this and immunotolerant (LPS) groups was determined. N = 6, Wilcoxon; * p

    Journal: Frontiers in Immunology

    Article Title: DNA Synthesis Is Activated in Mosquitoes and Human Monocytes During the Induction of Innate Immune Memory

    doi: 10.3389/fimmu.2018.02834

    Figure Lengend Snippet: (A) The number of genomic copies of TEP1 and PPO1 following the exposure of 250,000 and 500,000 cells to Plasmodium berghei during 3 or 6 h. (B) Human monocytes were incubated for 24 h in RPMI, β-glucan or LPS (the former being the control, the latter two the trained and tolerant cells, respectively). Subsequently, the cells were left for 5 days in RPMI medium with 10% human pooled serum, and then harvested. The DNA was isolated and qPCR was run with primers for the promoter regions of TNFA, IL6, HK , and PFKP . Expression in the RPMI control group was set at 1. Relative amount of DNA of the trained (β-glucan)this and immunotolerant (LPS) groups was determined. N = 6, Wilcoxon; * p

    Article Snippet: For experiments with BrdU incorporation, 6 million monocytes were seeded in 10-cm Petri dishes (Corning) and treated as with β-glucan, but in the presence or absence of BrdU (Sigma-Aldrich).

    Techniques: Incubation, Isolation, Real-time Polymerase Chain Reaction, Expressing

    LAB facilitates PAMP/fungal-induced production of IL-12 and IL-23 by BMDCs. (A–C) BMDCs from WT and Lat2 −/− mice were stimulated with (A) zymosan or (B) 1×10 5 HKY, 100 ng/ml LPS, 5 µg/ml particulate β–glucan, 10 µg/ml Curdlan, 20 ng/ml Pam 3 CSK 4 or (C) 5–10 µg/ml CAWS (mannans). Cytokine levels in the supernatants were measured after 24 h incubation (B) or 48 h incubation (C). (D) BMDCs from WT and Lat2 −/− mice were stimulated for the indicated times with 1×10 5 live C. albicans yeast. RNA was isolated, cDNA was prepared and Il12b , Il12a and Il23a mRNA transcripts were detected by real-time qPCR. mRNA levels were normalized to Hprt1 . Black bars represent WT, white bars represent Lat2 −/− . (E) BMDCs from WT and Lat2 −/− mice were stimulated for the indicated times with live C. albicans yeast. Fungizone was added 2 h later and cytokine levels in the supernatants were measured after 24 h incubation. (F) BMDCs from WT and Lat2 −/− mice were stimulated with 1–1000 ng/ml LPS. Cytokine levels in the supernatants were measured after 24 h incubation. For all graphical data, results are presented as means +/− s.e.m. of three replicates and data are representative of 2–4 independent experiments. *p

    Journal: PLoS Pathogens

    Article Title: LAB/NTAL Facilitates Fungal/PAMP-induced IL-12 and IFN-? Production by Repressing ?-Catenin Activation in Dendritic Cells

    doi: 10.1371/journal.ppat.1003357

    Figure Lengend Snippet: LAB facilitates PAMP/fungal-induced production of IL-12 and IL-23 by BMDCs. (A–C) BMDCs from WT and Lat2 −/− mice were stimulated with (A) zymosan or (B) 1×10 5 HKY, 100 ng/ml LPS, 5 µg/ml particulate β–glucan, 10 µg/ml Curdlan, 20 ng/ml Pam 3 CSK 4 or (C) 5–10 µg/ml CAWS (mannans). Cytokine levels in the supernatants were measured after 24 h incubation (B) or 48 h incubation (C). (D) BMDCs from WT and Lat2 −/− mice were stimulated for the indicated times with 1×10 5 live C. albicans yeast. RNA was isolated, cDNA was prepared and Il12b , Il12a and Il23a mRNA transcripts were detected by real-time qPCR. mRNA levels were normalized to Hprt1 . Black bars represent WT, white bars represent Lat2 −/− . (E) BMDCs from WT and Lat2 −/− mice were stimulated for the indicated times with live C. albicans yeast. Fungizone was added 2 h later and cytokine levels in the supernatants were measured after 24 h incubation. (F) BMDCs from WT and Lat2 −/− mice were stimulated with 1–1000 ng/ml LPS. Cytokine levels in the supernatants were measured after 24 h incubation. For all graphical data, results are presented as means +/− s.e.m. of three replicates and data are representative of 2–4 independent experiments. *p

    Article Snippet: WT and Lat2−/− BMDCs were stimulated with heat-killed C. albicans yeast, LPS, particulate β–glucan (Sigma), curdlan, Pam3 CSK4 and CAWS (mannans).

    Techniques: Mouse Assay, Incubation, Isolation, Real-time Polymerase Chain Reaction

    Mannan-Dectin-2 signaling stimulates LAB phosphorylation. (A) BMDCs were stimulated with 1 mg/ml zymosan, zymosan extract, vehicle control, washed zymosan or Pam 3 CSK 4 for the indicated times and cells were immunoprecipitated with anti-LAB and immunoblotted with anti-phosphotyrosine. (B) BMDCs were stimulated with 1 mg/ml mannan, particulate β-glucan or Pam 3 CSK 4 for the indicated times and WCL were immunoblotted with anti-phosphotyrosine and anti-LAB. (C) BMDCs were stimulated with 10 µg/ml LPS or 1 mg/ml zymosan for the indicated times and WCL were immunoprecipitated with anti-LAB and immunoblotted with anti-phosphotyrosine and anti-LAB. (D E) BMDCs from WT, Myd88 −/− (D) and Fcer1g −/− (E) mice were stimulated for 1 min with 1 mg/ml zymosan, zymosan extract or particulate β-glucan. WCL were immunoblotted with anti-phosphotyrosine and anti-Actin or anti-LAB. (F) RAW-264 cell lines expressing Empty Vector (EV), Dectin-1 or Dectin-2 were stimulated with 1 mg/ml zymosan. Cells were immunoprecipitated with anti-LAB and immunoblotted with anti-LAB and anti-phosphotyrosine. (G–H) BMDCs were infected with scrambled shRNA control or Dectin-2 shRNA. (G) BMDCs were stained for surface expression of Dectin-2 and analyzed by flow cytometry. (H) BMDCs were stimulated with 1 mg/ml zymosan and WCL were immunoblotted with anti-phosphotyrosine and anti-LAB.

    Journal: PLoS Pathogens

    Article Title: LAB/NTAL Facilitates Fungal/PAMP-induced IL-12 and IFN-? Production by Repressing ?-Catenin Activation in Dendritic Cells

    doi: 10.1371/journal.ppat.1003357

    Figure Lengend Snippet: Mannan-Dectin-2 signaling stimulates LAB phosphorylation. (A) BMDCs were stimulated with 1 mg/ml zymosan, zymosan extract, vehicle control, washed zymosan or Pam 3 CSK 4 for the indicated times and cells were immunoprecipitated with anti-LAB and immunoblotted with anti-phosphotyrosine. (B) BMDCs were stimulated with 1 mg/ml mannan, particulate β-glucan or Pam 3 CSK 4 for the indicated times and WCL were immunoblotted with anti-phosphotyrosine and anti-LAB. (C) BMDCs were stimulated with 10 µg/ml LPS or 1 mg/ml zymosan for the indicated times and WCL were immunoprecipitated with anti-LAB and immunoblotted with anti-phosphotyrosine and anti-LAB. (D E) BMDCs from WT, Myd88 −/− (D) and Fcer1g −/− (E) mice were stimulated for 1 min with 1 mg/ml zymosan, zymosan extract or particulate β-glucan. WCL were immunoblotted with anti-phosphotyrosine and anti-Actin or anti-LAB. (F) RAW-264 cell lines expressing Empty Vector (EV), Dectin-1 or Dectin-2 were stimulated with 1 mg/ml zymosan. Cells were immunoprecipitated with anti-LAB and immunoblotted with anti-LAB and anti-phosphotyrosine. (G–H) BMDCs were infected with scrambled shRNA control or Dectin-2 shRNA. (G) BMDCs were stained for surface expression of Dectin-2 and analyzed by flow cytometry. (H) BMDCs were stimulated with 1 mg/ml zymosan and WCL were immunoblotted with anti-phosphotyrosine and anti-LAB.

    Article Snippet: WT and Lat2−/− BMDCs were stimulated with heat-killed C. albicans yeast, LPS, particulate β–glucan (Sigma), curdlan, Pam3 CSK4 and CAWS (mannans).

    Techniques: Immunoprecipitation, Mouse Assay, Expressing, Plasmid Preparation, Infection, shRNA, Staining, Flow Cytometry, Cytometry

    Viral Recovery following Alternative Treatments. Influenza virus recovery was determined by plaque assay in the different infection groups 1 week after the administration of the influenza infection. Treatment of individual groups with the β-glucan ( ), or the Pneumocystis -infected ( ), or the UV-inactivated ( ) or the uninfected (▭) lung homogenate preparations were performed 2 weeks prior to the influenza infection. The group treated with the β-glucan preparation received a second treatment 1 week prior to the influenza virus infection. This is one of three independent experiments. ***, P

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

    Article Title: Pneumocystis infection enhances antibody-mediated resistance to a subsequent influenza infection 1

    doi:

    Figure Lengend Snippet: Viral Recovery following Alternative Treatments. Influenza virus recovery was determined by plaque assay in the different infection groups 1 week after the administration of the influenza infection. Treatment of individual groups with the β-glucan ( ), or the Pneumocystis -infected ( ), or the UV-inactivated ( ) or the uninfected (▭) lung homogenate preparations were performed 2 weeks prior to the influenza infection. The group treated with the β-glucan preparation received a second treatment 1 week prior to the influenza virus infection. This is one of three independent experiments. ***, P

    Article Snippet: The β-glucan preparation was made from a β-glucan stock (Sigma Chemical Co., St. Louis MO).

    Techniques: Plaque Assay, Infection

    Reactivity of the antiβ-glucan mAbs to β-glucans of different molecular structure. Panels A: dose-effect, ELISA mAb binding curves to plastic-adsorbed laminarin ( β1,3 glucan), pustulan (linear β1,6 glucan) and C. albicans β-glucan (mixed, highly branched β1,3/β1,6-glucan). The graph illustrates the outcome in a typical experiment out of five performed with similar results. Binding is expressed as mean O.D. 405 nm readings from triplicate wells after subtraction of O.D. from the negative controls (wells reacted with no mAb or with an irrelevant mAb). SEM values were always

    Journal: PLoS ONE

    Article Title: Protection by Anti-?-Glucan Antibodies Is Associated with Restricted ?-1,3 Glucan Binding Specificity and Inhibition of Fungal Growth and Adherence

    doi: 10.1371/journal.pone.0005392

    Figure Lengend Snippet: Reactivity of the antiβ-glucan mAbs to β-glucans of different molecular structure. Panels A: dose-effect, ELISA mAb binding curves to plastic-adsorbed laminarin ( β1,3 glucan), pustulan (linear β1,6 glucan) and C. albicans β-glucan (mixed, highly branched β1,3/β1,6-glucan). The graph illustrates the outcome in a typical experiment out of five performed with similar results. Binding is expressed as mean O.D. 405 nm readings from triplicate wells after subtraction of O.D. from the negative controls (wells reacted with no mAb or with an irrelevant mAb). SEM values were always

    Article Snippet: A β-glucan preparation from Saccharomyces cerevisiae , linear β1,3,1,4 glucan from barley and dextran, an α1,6-linked polysaccharide, all from Sigma-Aldrich, were also used.

    Techniques: Enzyme-linked Immunosorbent Assay, Binding Assay

    Protection by anti-β-glucan mAbs. Panel A : Fungal burden in kidney following a systemic infection with C. albicans in anti-β-glucan mAb-treated mice. In each of these experiments, groups of three mice were passively immunized by the i.p. route with 100 µg/0.5 ml of the IgG or IgM anti-β-glucan mAb, as indicated whereas control mice (three per group) received 0.5 ml of PBS only (Experiments 1 and 2) or 100 µg/0.5 ml of an irrelevant IgG2b mAb (Experiment 3). Two hours post passive immunization, the animals were infected i.v. with C. albicans (5×10 5 , Exp 1 and 2 or 10 6 cells/mouse, Exp 3) and extent of fungal invasion was evaluated at day 2 post-challenge thruogh CFU enumeration in left kidney. The asterisks indicate a statistically significant difference (P

    Journal: PLoS ONE

    Article Title: Protection by Anti-?-Glucan Antibodies Is Associated with Restricted ?-1,3 Glucan Binding Specificity and Inhibition of Fungal Growth and Adherence

    doi: 10.1371/journal.pone.0005392

    Figure Lengend Snippet: Protection by anti-β-glucan mAbs. Panel A : Fungal burden in kidney following a systemic infection with C. albicans in anti-β-glucan mAb-treated mice. In each of these experiments, groups of three mice were passively immunized by the i.p. route with 100 µg/0.5 ml of the IgG or IgM anti-β-glucan mAb, as indicated whereas control mice (three per group) received 0.5 ml of PBS only (Experiments 1 and 2) or 100 µg/0.5 ml of an irrelevant IgG2b mAb (Experiment 3). Two hours post passive immunization, the animals were infected i.v. with C. albicans (5×10 5 , Exp 1 and 2 or 10 6 cells/mouse, Exp 3) and extent of fungal invasion was evaluated at day 2 post-challenge thruogh CFU enumeration in left kidney. The asterisks indicate a statistically significant difference (P

    Article Snippet: A β-glucan preparation from Saccharomyces cerevisiae , linear β1,3,1,4 glucan from barley and dextran, an α1,6-linked polysaccharide, all from Sigma-Aldrich, were also used.

    Techniques: Infection, Mouse Assay

    Expression of anti-β-glucan mAb epitopes in major fungal pathogens for humans. Panel A: immunofluorescence staining pattern of hyphal filaments of Aspergillus fumigatus (a, b), Cryptococcus neoformans cells (c, d) and C. albicans germ-tubes (e, f) or yeast cells (g, h) reacted with the IgG (a, c, e, g) or the IgM (b, d, f, h) anti-β-glucan mAb. Sub-panels c′ through h′ show the corresponding bright field images. Magnification: 800× times ( except A. fumigatus hyphae, magnified 400× times). Panel B: ultrathin sections from cryofixed yeast (a,b) or hyphal (c,d) cells of C. albicans after immunogold labelling with the IgG (a,c) or the IgM (b, d) mAb.

    Journal: PLoS ONE

    Article Title: Protection by Anti-?-Glucan Antibodies Is Associated with Restricted ?-1,3 Glucan Binding Specificity and Inhibition of Fungal Growth and Adherence

    doi: 10.1371/journal.pone.0005392

    Figure Lengend Snippet: Expression of anti-β-glucan mAb epitopes in major fungal pathogens for humans. Panel A: immunofluorescence staining pattern of hyphal filaments of Aspergillus fumigatus (a, b), Cryptococcus neoformans cells (c, d) and C. albicans germ-tubes (e, f) or yeast cells (g, h) reacted with the IgG (a, c, e, g) or the IgM (b, d, f, h) anti-β-glucan mAb. Sub-panels c′ through h′ show the corresponding bright field images. Magnification: 800× times ( except A. fumigatus hyphae, magnified 400× times). Panel B: ultrathin sections from cryofixed yeast (a,b) or hyphal (c,d) cells of C. albicans after immunogold labelling with the IgG (a,c) or the IgM (b, d) mAb.

    Article Snippet: A β-glucan preparation from Saccharomyces cerevisiae , linear β1,3,1,4 glucan from barley and dextran, an α1,6-linked polysaccharide, all from Sigma-Aldrich, were also used.

    Techniques: Expressing, Immunofluorescence, Staining

    Reactivity of the anti-β-glucan mAbs to the secretory material and cell wall proteins of C. albicans . Panel A: Secretion of IgG- or IgM-reactive β-glucan material during fungal growth. Serial dilutions of fungal culture supernatants obtained at different times of growth in the yeast (Y) or hyphal (H) form were adsorbed (in duplicate) onto polystirene plates and reacted with the mAbs in ELISA. The figure shows reactivity of fungal supernatants diluted 1∶100, as from one representative experiment out of two performed independently. Panel B: Western blot analysis of culture supernatants and cell wall proteins. Culture supernatants (lane 1, 50 µg and lane 2, 25 µg polysaccharide) and cell wall proteins (lane 3, SDS-extracted and lane 4 β1,3-glucanase-extracted) were reacted with the anti-β-glucan mAbs, as indicated. Calculated molecular weight for IgG-reactive bands is also indicated. MW: molecular weight markers (104.3, 97.3, 50.4, 37.2, 29.2, 20.2 kDal).

    Journal: PLoS ONE

    Article Title: Protection by Anti-?-Glucan Antibodies Is Associated with Restricted ?-1,3 Glucan Binding Specificity and Inhibition of Fungal Growth and Adherence

    doi: 10.1371/journal.pone.0005392

    Figure Lengend Snippet: Reactivity of the anti-β-glucan mAbs to the secretory material and cell wall proteins of C. albicans . Panel A: Secretion of IgG- or IgM-reactive β-glucan material during fungal growth. Serial dilutions of fungal culture supernatants obtained at different times of growth in the yeast (Y) or hyphal (H) form were adsorbed (in duplicate) onto polystirene plates and reacted with the mAbs in ELISA. The figure shows reactivity of fungal supernatants diluted 1∶100, as from one representative experiment out of two performed independently. Panel B: Western blot analysis of culture supernatants and cell wall proteins. Culture supernatants (lane 1, 50 µg and lane 2, 25 µg polysaccharide) and cell wall proteins (lane 3, SDS-extracted and lane 4 β1,3-glucanase-extracted) were reacted with the anti-β-glucan mAbs, as indicated. Calculated molecular weight for IgG-reactive bands is also indicated. MW: molecular weight markers (104.3, 97.3, 50.4, 37.2, 29.2, 20.2 kDal).

    Article Snippet: A β-glucan preparation from Saccharomyces cerevisiae , linear β1,3,1,4 glucan from barley and dextran, an α1,6-linked polysaccharide, all from Sigma-Aldrich, were also used.

    Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Molecular Weight

    Antifungal properties of the anti-β-glucan mAbs. Panel A: C. albicans growth-inhibitory activity by the mAbs. C. albicans cells were cultured at 37°C in the presence or in the absence of the indicated mAb doses and, after 4 or 18 hours of incubation, fungal growth was estimated by a standard CFU counts. Percent growth inhibition values are from three independent experiments and were calculated by comparison to fungal cultures with equal doses of heat-inactivated mAbs or with an irrelevant mAb. Statistically significant differences between growth inhibitory activities by the IgG and the IgM mAb are marked by asterisks. Panel B: ability by the mAbs to inhibit adherence of Candida to human epithelial cells. Monolayers of Hep-2 cells were put into contact with fungal cells which have been pre-treated with the IgG, the IgM or with an irrelevant mAb as indicated. After a 1 h contact at 37°C, co-cultures were gently washed to eliminate non adherent fungi. Adherent fungal cells were recovered from the wells and enumerated by a standard CFU counts. Data in the graph are from three independent experiments, each performed in triplicate.

    Journal: PLoS ONE

    Article Title: Protection by Anti-?-Glucan Antibodies Is Associated with Restricted ?-1,3 Glucan Binding Specificity and Inhibition of Fungal Growth and Adherence

    doi: 10.1371/journal.pone.0005392

    Figure Lengend Snippet: Antifungal properties of the anti-β-glucan mAbs. Panel A: C. albicans growth-inhibitory activity by the mAbs. C. albicans cells were cultured at 37°C in the presence or in the absence of the indicated mAb doses and, after 4 or 18 hours of incubation, fungal growth was estimated by a standard CFU counts. Percent growth inhibition values are from three independent experiments and were calculated by comparison to fungal cultures with equal doses of heat-inactivated mAbs or with an irrelevant mAb. Statistically significant differences between growth inhibitory activities by the IgG and the IgM mAb are marked by asterisks. Panel B: ability by the mAbs to inhibit adherence of Candida to human epithelial cells. Monolayers of Hep-2 cells were put into contact with fungal cells which have been pre-treated with the IgG, the IgM or with an irrelevant mAb as indicated. After a 1 h contact at 37°C, co-cultures were gently washed to eliminate non adherent fungi. Adherent fungal cells were recovered from the wells and enumerated by a standard CFU counts. Data in the graph are from three independent experiments, each performed in triplicate.

    Article Snippet: A β-glucan preparation from Saccharomyces cerevisiae , linear β1,3,1,4 glucan from barley and dextran, an α1,6-linked polysaccharide, all from Sigma-Aldrich, were also used.

    Techniques: Activity Assay, Cell Culture, Incubation, Inhibition

    Myeloid-Specific Deletion of SHIP-1 Improves Trained Immunity In Vivo (A) In vivo model of training by two intraperitoneal (i.p.) β-glucan injections and secondary i.p. LPS challenge for measuring serum cytokines. (B) Mice were treated according to Figure 3 A. Serum was collected after 60 min (TNFα) or 90 min (IL-1β and IL-6) of LPS challenge, and cytokines were analyzed. (C) In vivo model of training as in (A) but with secondary Candida albicans lethal infection. (D) Survival curve according to Figure 3 C. (E) In vivo model of training by a systemic infection with a low dose of C. albicans followed by a secondary lethal challenge with the same pathogen. (F) Survival curve according to Figure 3 E. (G and H) Renal cytokines on day 2 post-infection (p.i.) (G) and kidney fungal burden (H) at indicated time points p.i. were evaluated in trained mice, following model in Figure 3 E. In (B), (G), and (H), single dots correspond to individual mice. Mean ± SEM of two (B and H) or three (G) pooled experiments is shown, including at least 5 mice per condition. ∗ p

    Journal: Cell Reports

    Article Title: Targeting SHIP-1 in Myeloid Cells Enhances Trained Immunity and Boosts Response to Infection

    doi: 10.1016/j.celrep.2018.09.092

    Figure Lengend Snippet: Myeloid-Specific Deletion of SHIP-1 Improves Trained Immunity In Vivo (A) In vivo model of training by two intraperitoneal (i.p.) β-glucan injections and secondary i.p. LPS challenge for measuring serum cytokines. (B) Mice were treated according to Figure 3 A. Serum was collected after 60 min (TNFα) or 90 min (IL-1β and IL-6) of LPS challenge, and cytokines were analyzed. (C) In vivo model of training as in (A) but with secondary Candida albicans lethal infection. (D) Survival curve according to Figure 3 C. (E) In vivo model of training by a systemic infection with a low dose of C. albicans followed by a secondary lethal challenge with the same pathogen. (F) Survival curve according to Figure 3 E. (G and H) Renal cytokines on day 2 post-infection (p.i.) (G) and kidney fungal burden (H) at indicated time points p.i. were evaluated in trained mice, following model in Figure 3 E. In (B), (G), and (H), single dots correspond to individual mice. Mean ± SEM of two (B and H) or three (G) pooled experiments is shown, including at least 5 mice per condition. ∗ p

    Article Snippet: When required, BMDMs were pre-incubated for 30 min prior to β-glucan stimulation with 500 μM 5′-deoxy-5′-(methylthio)adenosine (MTA), 6 μM pargyline, 50 μM resveratrol, and 50 μM (-)-epigallocatechin-3-gallate (EGCG) (all from Sigma-Aldrich).

    Techniques: In Vivo, Mouse Assay, Infection

    Pharmacological Inhibition of SHIP-1 Enhances Trained Immunity (A) In vitro experimental model applied to mouse BMDMs, indicating when the SHIP-1 inhibitor (SHIPi) 3α-aminocholestane (3AC) was added. (B) Mouse BMDMs were incubated with the SHIPi at the indicated concentrations. TNFα production was analyzed in supernatants of β-glucan-trained cells after LPS stimulation according to Figure 4 A. Mean + SEM of four independent experiments is shown. ∗∗ p

    Journal: Cell Reports

    Article Title: Targeting SHIP-1 in Myeloid Cells Enhances Trained Immunity and Boosts Response to Infection

    doi: 10.1016/j.celrep.2018.09.092

    Figure Lengend Snippet: Pharmacological Inhibition of SHIP-1 Enhances Trained Immunity (A) In vitro experimental model applied to mouse BMDMs, indicating when the SHIP-1 inhibitor (SHIPi) 3α-aminocholestane (3AC) was added. (B) Mouse BMDMs were incubated with the SHIPi at the indicated concentrations. TNFα production was analyzed in supernatants of β-glucan-trained cells after LPS stimulation according to Figure 4 A. Mean + SEM of four independent experiments is shown. ∗∗ p

    Article Snippet: When required, BMDMs were pre-incubated for 30 min prior to β-glucan stimulation with 500 μM 5′-deoxy-5′-(methylthio)adenosine (MTA), 6 μM pargyline, 50 μM resveratrol, and 50 μM (-)-epigallocatechin-3-gallate (EGCG) (all from Sigma-Aldrich).

    Techniques: Inhibition, In Vitro, Incubation

    SHIP-1 Deletion Boosts β-Glucan-Induced Trained Immunity in Macrophages (A) SHIP-1 expression by WB, normalized to β-actin, in bone marrow-derived macrophages (BMDMs) exposed (+) or not (−) to β-glucan (whole glucan particles) for the indicated time. Representative experiment of three performed. (B) SHIP-1 protein expression in BMDMs. Representative experiment of six performed. (C) Trained immunity in vitro model in mouse BMDMs. See also Figure S1 A. (D and E) Dectin-1 expression in BMDMs before β-glucan stimulation (D) or TLR4 expression both under non-trained (left) or β-glucan-primed (right) conditions, just before LPS stimulation (E), according to Figure 1 C. FACS histograms representative of four independent experiments. See also Figures S1 B and S1C. (F) BMDMs were stimulated (+) or not (−) with β-glucan or LPS, and IL-1β (left), IL-6 (middle), and TNFα production (right) was analyzed in supernatants according to Figure 1 C. See also Figure S2 . Independent experiments (N = 4 or 5) are shown. ∗ p

    Journal: Cell Reports

    Article Title: Targeting SHIP-1 in Myeloid Cells Enhances Trained Immunity and Boosts Response to Infection

    doi: 10.1016/j.celrep.2018.09.092

    Figure Lengend Snippet: SHIP-1 Deletion Boosts β-Glucan-Induced Trained Immunity in Macrophages (A) SHIP-1 expression by WB, normalized to β-actin, in bone marrow-derived macrophages (BMDMs) exposed (+) or not (−) to β-glucan (whole glucan particles) for the indicated time. Representative experiment of three performed. (B) SHIP-1 protein expression in BMDMs. Representative experiment of six performed. (C) Trained immunity in vitro model in mouse BMDMs. See also Figure S1 A. (D and E) Dectin-1 expression in BMDMs before β-glucan stimulation (D) or TLR4 expression both under non-trained (left) or β-glucan-primed (right) conditions, just before LPS stimulation (E), according to Figure 1 C. FACS histograms representative of four independent experiments. See also Figures S1 B and S1C. (F) BMDMs were stimulated (+) or not (−) with β-glucan or LPS, and IL-1β (left), IL-6 (middle), and TNFα production (right) was analyzed in supernatants according to Figure 1 C. See also Figure S2 . Independent experiments (N = 4 or 5) are shown. ∗ p

    Article Snippet: When required, BMDMs were pre-incubated for 30 min prior to β-glucan stimulation with 500 μM 5′-deoxy-5′-(methylthio)adenosine (MTA), 6 μM pargyline, 50 μM resveratrol, and 50 μM (-)-epigallocatechin-3-gallate (EGCG) (all from Sigma-Aldrich).

    Techniques: Expressing, Western Blot, Derivative Assay, In Vitro, FACS

    SHIP-1 Regulates Molecular and Metabolic Hallmarks of Trained Immunity (A) BMDMs were exposed to β-glucan for the indicated time, and phospho-Akt, Akt, phospho-S6, phospho-4EBP1, and β-actin were analyzed by WB. Representative experiment of five performed. See also Figure S3 . (B–E) BMDMs were left untreated (dashed lines) or treated for 1 day with β-glucan (solid lines), washed, rested for 3 days, and re-plated in equal numbers for determination of extracellular acidification rate (ECAR) in a glycolysis stress test upon sequential addition of glucose, oligomycin, and 2-deoxyglucose (2DG) as indicated (B). Analysis of basal glycolysis (C), maximal glycolysis (D), and glycolytic reserve (E). See also Figure S4 . Mean ± SEM (B) or individual data (C–E) of five independent cultures are shown. (F) BMDMs were trained (+) or not (−) with β-glucan for 1 day, washed, and rested for 4 days and chromatin immunoprecipitation (ChIP) against H3K4me3 was performed in which enrichment on the TNFα promoter was analyzed using qPCR. Mean ± SEM of five independent experiments is shown. (G and H) BMDMs were incubated (+) or not (−) with the methyltransferase inhibitor 5′-deoxy-5′-(methylthio)adenosine (MTA), the histone demethylase inhibitor pargyline (G), the histone deacetylase activator resveratrol, or the histone acetyltransferase inhibitor EGCG (H) for 30 min before β-glucan training and after wash-out. TNFα production was analyzed in supernatants after LPS stimulation according to Figure 1 C. Individual data corresponding to three (G) or four (H) independent experiments are shown. In (C)–(H), ∗ p

    Journal: Cell Reports

    Article Title: Targeting SHIP-1 in Myeloid Cells Enhances Trained Immunity and Boosts Response to Infection

    doi: 10.1016/j.celrep.2018.09.092

    Figure Lengend Snippet: SHIP-1 Regulates Molecular and Metabolic Hallmarks of Trained Immunity (A) BMDMs were exposed to β-glucan for the indicated time, and phospho-Akt, Akt, phospho-S6, phospho-4EBP1, and β-actin were analyzed by WB. Representative experiment of five performed. See also Figure S3 . (B–E) BMDMs were left untreated (dashed lines) or treated for 1 day with β-glucan (solid lines), washed, rested for 3 days, and re-plated in equal numbers for determination of extracellular acidification rate (ECAR) in a glycolysis stress test upon sequential addition of glucose, oligomycin, and 2-deoxyglucose (2DG) as indicated (B). Analysis of basal glycolysis (C), maximal glycolysis (D), and glycolytic reserve (E). See also Figure S4 . Mean ± SEM (B) or individual data (C–E) of five independent cultures are shown. (F) BMDMs were trained (+) or not (−) with β-glucan for 1 day, washed, and rested for 4 days and chromatin immunoprecipitation (ChIP) against H3K4me3 was performed in which enrichment on the TNFα promoter was analyzed using qPCR. Mean ± SEM of five independent experiments is shown. (G and H) BMDMs were incubated (+) or not (−) with the methyltransferase inhibitor 5′-deoxy-5′-(methylthio)adenosine (MTA), the histone demethylase inhibitor pargyline (G), the histone deacetylase activator resveratrol, or the histone acetyltransferase inhibitor EGCG (H) for 30 min before β-glucan training and after wash-out. TNFα production was analyzed in supernatants after LPS stimulation according to Figure 1 C. Individual data corresponding to three (G) or four (H) independent experiments are shown. In (C)–(H), ∗ p

    Article Snippet: When required, BMDMs were pre-incubated for 30 min prior to β-glucan stimulation with 500 μM 5′-deoxy-5′-(methylthio)adenosine (MTA), 6 μM pargyline, 50 μM resveratrol, and 50 μM (-)-epigallocatechin-3-gallate (EGCG) (all from Sigma-Aldrich).

    Techniques: Western Blot, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Incubation, Histone Deacetylase Assay

    Adsorption of CBM30 to insoluble (I) and soluble (II) polysaccharides. In the experiment in panel I, CBM30 was incubated with insoluble polysaccharides including Avicel (A), ASC (B), BMC (C), insoluble fraction of oat spelt xylan (D), lichenan (E), agar (F), starch (G), Sephadex G-150 (H), and chitin (I). After centrifugation, proteins in the supernatant (lane 1) and the precipitate (lane 2) were analyzed by SDS-PAGE. In the experiment in panel II, affinities of CBM30-Ig (lane 1) and CBM30 (CBM30) for various soluble polysaccharides including hydroxyethylcellulose (b), methylcellulose (c), soluble fraction of oat spelt xylan (d), birchwood xylan (e), barley β-glucan (f), lichenan (g), and soluble starch (h) were analyzed by native affinity gel electrophoresis. Lane M contains bovine serum albumin as a control protein. A gel without a polysaccharide served as a reference (a).

    Journal: Journal of Bacteriology

    Article Title: Characterization of a Cellulase Containing a Family 30 Carbohydrate-Binding Module (CBM) Derived from Clostridium thermocellum CelJ: Importance of the CBM to Cellulose Hydrolysis

    doi: 10.1128/JB.185.2.504-512.2003

    Figure Lengend Snippet: Adsorption of CBM30 to insoluble (I) and soluble (II) polysaccharides. In the experiment in panel I, CBM30 was incubated with insoluble polysaccharides including Avicel (A), ASC (B), BMC (C), insoluble fraction of oat spelt xylan (D), lichenan (E), agar (F), starch (G), Sephadex G-150 (H), and chitin (I). After centrifugation, proteins in the supernatant (lane 1) and the precipitate (lane 2) were analyzed by SDS-PAGE. In the experiment in panel II, affinities of CBM30-Ig (lane 1) and CBM30 (CBM30) for various soluble polysaccharides including hydroxyethylcellulose (b), methylcellulose (c), soluble fraction of oat spelt xylan (d), birchwood xylan (e), barley β-glucan (f), lichenan (g), and soluble starch (h) were analyzed by native affinity gel electrophoresis. Lane M contains bovine serum albumin as a control protein. A gel without a polysaccharide served as a reference (a).

    Article Snippet: The affinities of these proteins for soluble polysaccharides, i.e., CMC (low viscosity [Sigma]), hydroxyethylcellulose (Fluka), methylcellulose (Nacalai Tesque), barley β-glucan (Sigma), and birchwood xylan (Sigma), were examined by native affinity gel electrophoresis as described by Meissner et al. , with some simplifications.

    Techniques: Adsorption, Incubation, Centrifugation, SDS Page, Nucleic Acid Electrophoresis

    β-Glucan treatment induces sMR production through dectin-1 engagement independently of the mouse strain and does not promote CD44 shedding. Sex- and age-matched WT and dectin-1-KO thio-Mφ were treated with β-glucan particles (50 particles/cell) in the presence and absence of 1 mg/ml glucan phosphate (β- Gluc ) or 1 mg/ml mannan ( Man ) ( A ). Sex- and age-matched WT and dectin-1-KO thio-Mφ were treated with HK C. albicans (50 particles/cell) ( B ). Analysis of cell lysates and culture supernatants by Western blotting demonstrated dectin-1-mediated enhanced sMR production in response to particulate β-glucan that could be inhibited by soluble β-glucan but not by mannan ( A ). Similarly, sMR production in response to HK C. albicans was reduced in dectin-1-deficient cells ( B ). Both C57BL/6 and BALB/c Mφ were capable of shedding MR in response to particulate β-glucan ( C ), despite the different levels of surface dectin-1 expression in each strain ( D ). CD44 could be detected in cell lysates but not in supernatants from β-glucan-treated WT Mφ, whereas cMR and sMR were detected in cell lysates and supernatants, respectively ( E ). No signal could be detected in supernatants from MR-KO cells treated with β-glucan ( F ). Data are representative of two or three independent experiments ( A–D and F ). Unt , untreated.

    Journal: The Journal of Biological Chemistry

    Article Title: Fungal Recognition Enhances Mannose Receptor Shedding through Dectin-1 Engagement *

    doi: 10.1074/jbc.M110.185025

    Figure Lengend Snippet: β-Glucan treatment induces sMR production through dectin-1 engagement independently of the mouse strain and does not promote CD44 shedding. Sex- and age-matched WT and dectin-1-KO thio-Mφ were treated with β-glucan particles (50 particles/cell) in the presence and absence of 1 mg/ml glucan phosphate (β- Gluc ) or 1 mg/ml mannan ( Man ) ( A ). Sex- and age-matched WT and dectin-1-KO thio-Mφ were treated with HK C. albicans (50 particles/cell) ( B ). Analysis of cell lysates and culture supernatants by Western blotting demonstrated dectin-1-mediated enhanced sMR production in response to particulate β-glucan that could be inhibited by soluble β-glucan but not by mannan ( A ). Similarly, sMR production in response to HK C. albicans was reduced in dectin-1-deficient cells ( B ). Both C57BL/6 and BALB/c Mφ were capable of shedding MR in response to particulate β-glucan ( C ), despite the different levels of surface dectin-1 expression in each strain ( D ). CD44 could be detected in cell lysates but not in supernatants from β-glucan-treated WT Mφ, whereas cMR and sMR were detected in cell lysates and supernatants, respectively ( E ). No signal could be detected in supernatants from MR-KO cells treated with β-glucan ( F ). Data are representative of two or three independent experiments ( A–D and F ). Unt , untreated.

    Article Snippet: For inhibition assays, thio-Mφ were preincubated with β-glucan phosphate, mannan (Sigma), Syk kinase inhibitor IV, wortmannin, Akt inhibitor VI, Raf-1 kinase inhibitor I, GM6001 (N -[(2R )-2(hydroxamidocarbonylmethyl)-4-methylpentanoyl]-l -tryptophan methylamide, a potent broad-spectrum hydroxamic acid inhibitor of matrix metalloproteases), a control for GM6001 (N-t -butoxycarbonyl-l -leucyl-l -tryptophan methylamide), cytochalasin D, latrunculin A (all from Calbiochem) or bafilomycin or chloroquine (Sigma) at the indicated concentrations for 1 h before treatment and were present during the incubation with the stimuli.

    Techniques: Western Blot, Expressing

    MR shedding is induced by zymosan, C. albicans , and A. fumigatus and requires β-glucan recognition. Thio-Mφ were incubated with zymosan (50 particles/cell; Z-50 ) for 1.5 and 3 h at 37 °C ( A ) or fixed A. fumigatus ( A. f. ) and fixed or HK C. albicans (50 particles/cell) for 3 h at 37 °C in serum-free medium ( B ). cMR and sMR levels were assessed in cell lysates and supernatants, respectively, by Western blot analysis as described under “Experimental Procedures.” All fungal particles induced MR shedding ( A and B ). MR shedding in response to HK C. albicans could be inhibited by soluble β-glucan phosphate (β- Gluc ; 1 mg/ml) but not by mannan ( Man ; 1 mg/ml) ( C ). Data are representative of three independent experiments. Unt , untreated.

    Journal: The Journal of Biological Chemistry

    Article Title: Fungal Recognition Enhances Mannose Receptor Shedding through Dectin-1 Engagement *

    doi: 10.1074/jbc.M110.185025

    Figure Lengend Snippet: MR shedding is induced by zymosan, C. albicans , and A. fumigatus and requires β-glucan recognition. Thio-Mφ were incubated with zymosan (50 particles/cell; Z-50 ) for 1.5 and 3 h at 37 °C ( A ) or fixed A. fumigatus ( A. f. ) and fixed or HK C. albicans (50 particles/cell) for 3 h at 37 °C in serum-free medium ( B ). cMR and sMR levels were assessed in cell lysates and supernatants, respectively, by Western blot analysis as described under “Experimental Procedures.” All fungal particles induced MR shedding ( A and B ). MR shedding in response to HK C. albicans could be inhibited by soluble β-glucan phosphate (β- Gluc ; 1 mg/ml) but not by mannan ( Man ; 1 mg/ml) ( C ). Data are representative of three independent experiments. Unt , untreated.

    Article Snippet: For inhibition assays, thio-Mφ were preincubated with β-glucan phosphate, mannan (Sigma), Syk kinase inhibitor IV, wortmannin, Akt inhibitor VI, Raf-1 kinase inhibitor I, GM6001 (N -[(2R )-2(hydroxamidocarbonylmethyl)-4-methylpentanoyl]-l -tryptophan methylamide, a potent broad-spectrum hydroxamic acid inhibitor of matrix metalloproteases), a control for GM6001 (N-t -butoxycarbonyl-l -leucyl-l -tryptophan methylamide), cytochalasin D, latrunculin A (all from Calbiochem) or bafilomycin or chloroquine (Sigma) at the indicated concentrations for 1 h before treatment and were present during the incubation with the stimuli.

    Techniques: Incubation, Western Blot

    Dectin-1-induced MR shedding by a non-secreted metalloprotease. GM6001 treatment inhibited MR shedding ( A and C ). Mφ were treated with the inhibitor GM6001 ( Inh ) at 1 h prior and during the 3-h treatment with HK C. albicans ( A ) or β-glucan (β- Gluc ) particles ( C ), or a GM6001 control ( Cont ) was used as a negative control. GM6001 treatment reduced TNF-α production but did not affect KC (CXCL1) production in response to HK C. albicans ( B ). MMP-8- and MMP-9-specific mRNAs in thio-Mφ treated with particulate β-glucan were quantitated ( D ). Data are representative of three independent experiments. Unt and U , untreated.

    Journal: The Journal of Biological Chemistry

    Article Title: Fungal Recognition Enhances Mannose Receptor Shedding through Dectin-1 Engagement *

    doi: 10.1074/jbc.M110.185025

    Figure Lengend Snippet: Dectin-1-induced MR shedding by a non-secreted metalloprotease. GM6001 treatment inhibited MR shedding ( A and C ). Mφ were treated with the inhibitor GM6001 ( Inh ) at 1 h prior and during the 3-h treatment with HK C. albicans ( A ) or β-glucan (β- Gluc ) particles ( C ), or a GM6001 control ( Cont ) was used as a negative control. GM6001 treatment reduced TNF-α production but did not affect KC (CXCL1) production in response to HK C. albicans ( B ). MMP-8- and MMP-9-specific mRNAs in thio-Mφ treated with particulate β-glucan were quantitated ( D ). Data are representative of three independent experiments. Unt and U , untreated.

    Article Snippet: For inhibition assays, thio-Mφ were preincubated with β-glucan phosphate, mannan (Sigma), Syk kinase inhibitor IV, wortmannin, Akt inhibitor VI, Raf-1 kinase inhibitor I, GM6001 (N -[(2R )-2(hydroxamidocarbonylmethyl)-4-methylpentanoyl]-l -tryptophan methylamide, a potent broad-spectrum hydroxamic acid inhibitor of matrix metalloproteases), a control for GM6001 (N-t -butoxycarbonyl-l -leucyl-l -tryptophan methylamide), cytochalasin D, latrunculin A (all from Calbiochem) or bafilomycin or chloroquine (Sigma) at the indicated concentrations for 1 h before treatment and were present during the incubation with the stimuli.

    Techniques: Negative Control

    Dectin-1-induced sMR production is phagocytosis-independent but requires actin polymerization. Thio-Mφ were treated with curdlan and HK C. albicans ( A ) under serum-free conditions at concentrations of four particles/cell (10 μg of curdlan would contain ∼550 particles ( 32 )) and 50 particles/cell, respectively. cMR and sMR levels were determined as described under “Experimental Procedures.” Both particles were found to enhance MR shedding ( A ). Latrunculin A inhibited the production of sMR in response to β-glucan (β- Gluc ) treatment ( B ). Higher doses of latrunculin A and cytochalasin D reduced the levels of cMR ( C ). Data are representative of three independent experiments. Unt , untreated.

    Journal: The Journal of Biological Chemistry

    Article Title: Fungal Recognition Enhances Mannose Receptor Shedding through Dectin-1 Engagement *

    doi: 10.1074/jbc.M110.185025

    Figure Lengend Snippet: Dectin-1-induced sMR production is phagocytosis-independent but requires actin polymerization. Thio-Mφ were treated with curdlan and HK C. albicans ( A ) under serum-free conditions at concentrations of four particles/cell (10 μg of curdlan would contain ∼550 particles ( 32 )) and 50 particles/cell, respectively. cMR and sMR levels were determined as described under “Experimental Procedures.” Both particles were found to enhance MR shedding ( A ). Latrunculin A inhibited the production of sMR in response to β-glucan (β- Gluc ) treatment ( B ). Higher doses of latrunculin A and cytochalasin D reduced the levels of cMR ( C ). Data are representative of three independent experiments. Unt , untreated.

    Article Snippet: For inhibition assays, thio-Mφ were preincubated with β-glucan phosphate, mannan (Sigma), Syk kinase inhibitor IV, wortmannin, Akt inhibitor VI, Raf-1 kinase inhibitor I, GM6001 (N -[(2R )-2(hydroxamidocarbonylmethyl)-4-methylpentanoyl]-l -tryptophan methylamide, a potent broad-spectrum hydroxamic acid inhibitor of matrix metalloproteases), a control for GM6001 (N-t -butoxycarbonyl-l -leucyl-l -tryptophan methylamide), cytochalasin D, latrunculin A (all from Calbiochem) or bafilomycin or chloroquine (Sigma) at the indicated concentrations for 1 h before treatment and were present during the incubation with the stimuli.

    Techniques:

    Dectin-1-mediated MR shedding is dependent on Syk and partially on Raf-1, utilizes the Akt-independent PI3K pathway, and is not affected by the presence of an agonist for TLR2. Thio-Mφ preincubated for 1 h with Syk kinase inhibitor IV ( A ), Raf-1 inhibitor I ( B ), wortmannin ( C ), or Akt inhibitor VI ( D ) were treated with particulate β-glucan (β- Gluc ; 50 particles/cell) for 3 h at 37 °C in the presence of inhibitors. cMR and sMR levels were assessed in cell lysates and supernatants, respectively, by Western blot analysis as described under “Experimental Procedures.” Thio-Mφ were treated with Pam 3 -Cys-Ser-Lys 4 (10 μg/ml) in the presence or absence of particulate β-glucan (50 particles/cell) for 3 h at 37 °C ( E ). cMR and sMR levels were assessed in cell lysates and supernatants. Data are representative of three independent experiments. Unt , untreated.

    Journal: The Journal of Biological Chemistry

    Article Title: Fungal Recognition Enhances Mannose Receptor Shedding through Dectin-1 Engagement *

    doi: 10.1074/jbc.M110.185025

    Figure Lengend Snippet: Dectin-1-mediated MR shedding is dependent on Syk and partially on Raf-1, utilizes the Akt-independent PI3K pathway, and is not affected by the presence of an agonist for TLR2. Thio-Mφ preincubated for 1 h with Syk kinase inhibitor IV ( A ), Raf-1 inhibitor I ( B ), wortmannin ( C ), or Akt inhibitor VI ( D ) were treated with particulate β-glucan (β- Gluc ; 50 particles/cell) for 3 h at 37 °C in the presence of inhibitors. cMR and sMR levels were assessed in cell lysates and supernatants, respectively, by Western blot analysis as described under “Experimental Procedures.” Thio-Mφ were treated with Pam 3 -Cys-Ser-Lys 4 (10 μg/ml) in the presence or absence of particulate β-glucan (50 particles/cell) for 3 h at 37 °C ( E ). cMR and sMR levels were assessed in cell lysates and supernatants. Data are representative of three independent experiments. Unt , untreated.

    Article Snippet: For inhibition assays, thio-Mφ were preincubated with β-glucan phosphate, mannan (Sigma), Syk kinase inhibitor IV, wortmannin, Akt inhibitor VI, Raf-1 kinase inhibitor I, GM6001 (N -[(2R )-2(hydroxamidocarbonylmethyl)-4-methylpentanoyl]-l -tryptophan methylamide, a potent broad-spectrum hydroxamic acid inhibitor of matrix metalloproteases), a control for GM6001 (N-t -butoxycarbonyl-l -leucyl-l -tryptophan methylamide), cytochalasin D, latrunculin A (all from Calbiochem) or bafilomycin or chloroquine (Sigma) at the indicated concentrations for 1 h before treatment and were present during the incubation with the stimuli.

    Techniques: Western Blot

    (A). β-1,3-glucan content was quantified using the aniline blue assay with curdlan, a β-1,3-glucan analog, as the standard. Values are expressed as relative fluorescence units per mg of mycelial tissue. The experiment was repeated three

    Journal: Molecular microbiology

    Article Title: Localization and Activity of the Calcineurin Catalytic and Regulatory Subunit Complex at the Septum is Essential for Hyphal Elongation and Proper Septation in Aspergillus fumigatus

    doi: 10.1111/j.1365-2958.2011.07886.x

    Figure Lengend Snippet: (A). β-1,3-glucan content was quantified using the aniline blue assay with curdlan, a β-1,3-glucan analog, as the standard. Values are expressed as relative fluorescence units per mg of mycelial tissue. The experiment was repeated three

    Article Snippet: To investigate if exogenous β-glucan could ameliorate the growth defect of the calcineurin mutants, β-1,3-glucan in the form of curdlan (Sigma) was supplemented at a concentration range of 0.01-0.1% in GMM medium, but we did not observe any improvement in the growth of all the mutants (data not shown).

    Techniques: Fluorescence

    dectin-1 and TLR2 participate in Mtb- induced ROS and apoptosis in PMN. A) PMN were pretreated with DHR and incubated with or without neutralizing anti-TLR2, anti-dectin-1, anti-CD11b, irrelevant IgG antibodies or the β-glucan laminarin, and then stimulated with Ra or M for 90 min at 1:2 ratio Mtb :PMN. Thereafter, the emission of oxidized DHR was evaluated by flow cytometry. Results are expressed as mean ± SE (n = 12); control (−−) vs. Ra, Ra + IgG and Ra + anti-CD11b: ***p

    Journal: BMC Infectious Diseases

    Article Title: Outbreaks of Mycobacterium tuberculosis MDR strains differentially induce neutrophil respiratory burst involving lipid rafts, p38 MAPK and Syk

    doi: 10.1186/1471-2334-14-262

    Figure Lengend Snippet: dectin-1 and TLR2 participate in Mtb- induced ROS and apoptosis in PMN. A) PMN were pretreated with DHR and incubated with or without neutralizing anti-TLR2, anti-dectin-1, anti-CD11b, irrelevant IgG antibodies or the β-glucan laminarin, and then stimulated with Ra or M for 90 min at 1:2 ratio Mtb :PMN. Thereafter, the emission of oxidized DHR was evaluated by flow cytometry. Results are expressed as mean ± SE (n = 12); control (−−) vs. Ra, Ra + IgG and Ra + anti-CD11b: ***p

    Article Snippet: The FITC-conjugated B subunit of cholerae toxin (CTB), Annexin V-FITC, the lipid raft inhibitor β-Metil-ciclodextrin (MβC), the β-glucan Laminarin, PMA, Pam3Cys, cytochalasin D, orthovanadate and amyloglucosidase (A3514, EC 3.2.1.3) were purchased from (Sigma Chemical Co., St. Louis, Mo, USA) and dihydrorhodamine 123 (DHR) was purchased from Invitrogen.

    Techniques: Incubation, Flow Cytometry, Cytometry

    Immobilised β-glucans induce Dectin-1 signalling a Soluble β-glucan binding (50 μg/ml, 10 min) to wild type and Dectin-1 -/- bone marrow-derived DC (bmDC) was assessed by flow cytometry. b, c IFN-γ-primed bmM were stimulated with soluble β-glucans (50 μg/ml) or β-glucans immobilised on either tissue culture plates ( b – plate-coated) or 0.8 μm polystyrene latex beads ( c – beads). ROS production was measured by luminol-ECL; datapoints are means of triplicate culture. d, e TNF-α production (24 h) by bmDC exposed to soluble/particulate (50 μg/ml), plate-immobilised or bead-coated soluble β-glucans was assessed by ELISA; data are expressed as means plus standard deviations of triplicate culture (*** p

    Journal: Nature

    Article Title: Activation of the innate immune receptor Dectin-1 upon formation of a "phagocytic synapse"

    doi: 10.1038/nature10071

    Figure Lengend Snippet: Immobilised β-glucans induce Dectin-1 signalling a Soluble β-glucan binding (50 μg/ml, 10 min) to wild type and Dectin-1 -/- bone marrow-derived DC (bmDC) was assessed by flow cytometry. b, c IFN-γ-primed bmM were stimulated with soluble β-glucans (50 μg/ml) or β-glucans immobilised on either tissue culture plates ( b – plate-coated) or 0.8 μm polystyrene latex beads ( c – beads). ROS production was measured by luminol-ECL; datapoints are means of triplicate culture. d, e TNF-α production (24 h) by bmDC exposed to soluble/particulate (50 μg/ml), plate-immobilised or bead-coated soluble β-glucans was assessed by ELISA; data are expressed as means plus standard deviations of triplicate culture (*** p

    Article Snippet: β-glucan immobilisation and CD45 co-immobilisation Soluble β-glucans were immobilised on tissue culture plates or large polystyrene latex beads (0.8 and 3 μm – Sigma) by incubation with PBS/EDTA containing 100 μg/ml soluble β-glucan for 1 h at 37°C.

    Techniques: Binding Assay, Derivative Assay, Flow Cytometry, Cytometry, Enzyme-linked Immunosorbent Assay

    Particulate, but not soluble, β-glucans induce Dectin-1 signalling a Size (molecular weight or diameter) of β-glucan preparations used in this study. b-e Bone marrow-derived macrophages (bmM; b-d IFN-γ-primed overnight) were stimulated with 50 μg/ml β-glucans. b TNF-α production (24 h) was assessed by ELISA; data are means plus standard deviations of triplicate culture (*** p

    Journal: Nature

    Article Title: Activation of the innate immune receptor Dectin-1 upon formation of a "phagocytic synapse"

    doi: 10.1038/nature10071

    Figure Lengend Snippet: Particulate, but not soluble, β-glucans induce Dectin-1 signalling a Size (molecular weight or diameter) of β-glucan preparations used in this study. b-e Bone marrow-derived macrophages (bmM; b-d IFN-γ-primed overnight) were stimulated with 50 μg/ml β-glucans. b TNF-α production (24 h) was assessed by ELISA; data are means plus standard deviations of triplicate culture (*** p

    Article Snippet: β-glucan immobilisation and CD45 co-immobilisation Soluble β-glucans were immobilised on tissue culture plates or large polystyrene latex beads (0.8 and 3 μm – Sigma) by incubation with PBS/EDTA containing 100 μg/ml soluble β-glucan for 1 h at 37°C.

    Techniques: Molecular Weight, Derivative Assay, Enzyme-linked Immunosorbent Assay

    CD45 and CD148 phosphatases are excluded from the β-glucan particle contact site a-c Confocal microscopy of SBPc-tagged Dectin-1-expressing RAW264.7 macrophages stimulated with zymosan (20 μg/ml, 1 min) and stained for CD45 (red) and SBPc tag (Dectin-1; green), phospho-tyrosine (pTyr; green) or active Syk (pSyk; green). z-stacks were analyzed to visualise the indicated particle contact sites (left panels) in cross-section (center panels). 3-dimensional isosurface models of the indicated contact sites were generated using ImageJ and ImageSurfer (right panels). d Resident peritoneal macrophages stimulated with WGP (20 μg/ml, 1 min) were stained (left panel) for CD45 (red) and active Syk (pSyk; green). e Dectin-1-expressing RAW264.7 macrophages stimulated with WGP (20 μg/ml, 1 min) were stained (left panel) for CD148 (red) and active Syk (pSyk; green). Isosurface models ( d, e right panels) are of the indicated particle contact sites. f bmDC were added to tissue culture plates pre-coated with HMW soluble β-glucan (cHMW) or LPS (cLPS) and/or anti-CD45 or control IgG; some bmDC were stimulated with WGP (50 μg/ml). TNF-α production (24 h) was assessed by ELISA; data are means plus standard deviations of triplicate culture (*** p

    Journal: Nature

    Article Title: Activation of the innate immune receptor Dectin-1 upon formation of a "phagocytic synapse"

    doi: 10.1038/nature10071

    Figure Lengend Snippet: CD45 and CD148 phosphatases are excluded from the β-glucan particle contact site a-c Confocal microscopy of SBPc-tagged Dectin-1-expressing RAW264.7 macrophages stimulated with zymosan (20 μg/ml, 1 min) and stained for CD45 (red) and SBPc tag (Dectin-1; green), phospho-tyrosine (pTyr; green) or active Syk (pSyk; green). z-stacks were analyzed to visualise the indicated particle contact sites (left panels) in cross-section (center panels). 3-dimensional isosurface models of the indicated contact sites were generated using ImageJ and ImageSurfer (right panels). d Resident peritoneal macrophages stimulated with WGP (20 μg/ml, 1 min) were stained (left panel) for CD45 (red) and active Syk (pSyk; green). e Dectin-1-expressing RAW264.7 macrophages stimulated with WGP (20 μg/ml, 1 min) were stained (left panel) for CD148 (red) and active Syk (pSyk; green). Isosurface models ( d, e right panels) are of the indicated particle contact sites. f bmDC were added to tissue culture plates pre-coated with HMW soluble β-glucan (cHMW) or LPS (cLPS) and/or anti-CD45 or control IgG; some bmDC were stimulated with WGP (50 μg/ml). TNF-α production (24 h) was assessed by ELISA; data are means plus standard deviations of triplicate culture (*** p

    Article Snippet: β-glucan immobilisation and CD45 co-immobilisation Soluble β-glucans were immobilised on tissue culture plates or large polystyrene latex beads (0.8 and 3 μm – Sigma) by incubation with PBS/EDTA containing 100 μg/ml soluble β-glucan for 1 h at 37°C.

    Techniques: Confocal Microscopy, Expressing, Staining, Generated, Enzyme-linked Immunosorbent Assay

    IFA staining of 1,3-β-glucan on HK B. dermatitidis . Phase-contrast pictures of HK B. dermatitidis are shown on the bottom of each pair of pictures. Corresponding IFA-stained (mouse monoclonal anti-1,3-β-glucan IgG plus goat anti-mouse IgG conjugated to Alexa Fluor 594) HK B. dermatitidis pictures are shown on the top of each pair of pictures. There was no fluorescent staining with the conjugate in the absence of the anti-glucan antibody.

    Journal: Infection and Immunity

    Article Title: Evasion of Innate Immune Responses: Evidence for Mannose Binding Lectin Inhibition of Tumor Necrosis Factor Alpha Production by Macrophages in Response to Blastomyces dermatitidis ▿

    doi: 10.1128/IAI.01185-07

    Figure Lengend Snippet: IFA staining of 1,3-β-glucan on HK B. dermatitidis . Phase-contrast pictures of HK B. dermatitidis are shown on the bottom of each pair of pictures. Corresponding IFA-stained (mouse monoclonal anti-1,3-β-glucan IgG plus goat anti-mouse IgG conjugated to Alexa Fluor 594) HK B. dermatitidis pictures are shown on the top of each pair of pictures. There was no fluorescent staining with the conjugate in the absence of the anti-glucan antibody.

    Article Snippet: 1,3-β-Glucan from baker's yeast was purchased from Sigma Chemical Co., St. Louis, MO.

    Techniques: Immunofluorescence, Staining

    (A) Effect of 1,3-β-glucan concentration on stimulation of PM for TNF-α production. The PM response to increasing concentrations of 1,3-β-glucan (μg protein/ml) is shown as increasing levels of TNF-α production. Means ± SD for triplicate determinations are shown. (B) Effect of MS on 1,3-β-glucan stimulation of PM for TNF-α production. Serum (S; 10%) inhibition of 1,3-β-glucan (62 and 125 μg/ml) stimulation of PM for TNF-α production is shown. TNF-α production by PM plus B. dermatitidis , with or without 10% MS, is also shown. TNF-α production is given as the mean ± SD for triplicate determinations.

    Journal: Infection and Immunity

    Article Title: Evasion of Innate Immune Responses: Evidence for Mannose Binding Lectin Inhibition of Tumor Necrosis Factor Alpha Production by Macrophages in Response to Blastomyces dermatitidis ▿

    doi: 10.1128/IAI.01185-07

    Figure Lengend Snippet: (A) Effect of 1,3-β-glucan concentration on stimulation of PM for TNF-α production. The PM response to increasing concentrations of 1,3-β-glucan (μg protein/ml) is shown as increasing levels of TNF-α production. Means ± SD for triplicate determinations are shown. (B) Effect of MS on 1,3-β-glucan stimulation of PM for TNF-α production. Serum (S; 10%) inhibition of 1,3-β-glucan (62 and 125 μg/ml) stimulation of PM for TNF-α production is shown. TNF-α production by PM plus B. dermatitidis , with or without 10% MS, is also shown. TNF-α production is given as the mean ± SD for triplicate determinations.

    Article Snippet: 1,3-β-Glucan from baker's yeast was purchased from Sigma Chemical Co., St. Louis, MO.

    Techniques: Concentration Assay, Mass Spectrometry, Inhibition

    Effect of coating B. dermatitidis with anti-1,3-β-glucan antibody (a-G) on TNF-α production by PM. TNF-α production by PM plus B. dermatitidis , PM plus antibody-coated B. dermatitidis , or PM plus MS (S)-coated B. dermatitidis is given as the mean ± SD for triplicate determinations.

    Journal: Infection and Immunity

    Article Title: Evasion of Innate Immune Responses: Evidence for Mannose Binding Lectin Inhibition of Tumor Necrosis Factor Alpha Production by Macrophages in Response to Blastomyces dermatitidis ▿

    doi: 10.1128/IAI.01185-07

    Figure Lengend Snippet: Effect of coating B. dermatitidis with anti-1,3-β-glucan antibody (a-G) on TNF-α production by PM. TNF-α production by PM plus B. dermatitidis , PM plus antibody-coated B. dermatitidis , or PM plus MS (S)-coated B. dermatitidis is given as the mean ± SD for triplicate determinations.

    Article Snippet: 1,3-β-Glucan from baker's yeast was purchased from Sigma Chemical Co., St. Louis, MO.

    Techniques: Mass Spectrometry

    TLC analysis of the action patterns of Cpin_6279rC on β-1,2-glucan and Sop n s. β-1,2-Glucan (average DP 64) ( A ), cyclic β-1,2-glucan (DP 17–24) ( B ), Sop 5 ( C ), Sop 6 ( D ), and Sop 7 ( E ) were used as substrates. Lane M contains markers (0.2% each sugar). Asterisks represent the origins of the TLC plates.

    Journal: The Journal of Biological Chemistry

    Article Title: Biochemical and structural analyses of a bacterial endo-β-1,2-glucanase reveal a new glycoside hydrolase family

    doi: 10.1074/jbc.M116.762724

    Figure Lengend Snippet: TLC analysis of the action patterns of Cpin_6279rC on β-1,2-glucan and Sop n s. β-1,2-Glucan (average DP 64) ( A ), cyclic β-1,2-glucan (DP 17–24) ( B ), Sop 5 ( C ), Sop 6 ( D ), and Sop 7 ( E ) were used as substrates. Lane M contains markers (0.2% each sugar). Asterisks represent the origins of the TLC plates.

    Article Snippet: CM-pachyman, CM-curdlan, lichenan, β-glucan from barley, xyloglucan (tamarind), glucomannan, arabinogalactan, arabinan, and polygalacturonate were purchased from Megazyme (Wicklow, Ireland).

    Techniques: Thin Layer Chromatography

    α ( A ), β ( B ), and total ( C ) glucan concentrations in glucan extracts prepared from caps and stalks from Pleurotus eryngii mushrooms cultivated in various relative amounts of eucalyptus sawdust and OMSW. * p

    Journal: International Journal of Molecular Sciences

    Article Title: Olive Mill Waste Enhances α-Glucan Content in the Edible Mushroom Pleurotus eryngii

    doi: 10.3390/ijms18071564

    Figure Lengend Snippet: α ( A ), β ( B ), and total ( C ) glucan concentrations in glucan extracts prepared from caps and stalks from Pleurotus eryngii mushrooms cultivated in various relative amounts of eucalyptus sawdust and OMSW. * p

    Article Snippet: Glucans Analysis The glucan content of the dried Pleurotus fruiting bodies ( ) or extracted glucans ( ) was determined using a mushroom and yeast specific β-glucan kit (Megazyme International, Wicklow, Ireland) based on a colorimetric reaction [ , ].

    Techniques:

    α ( A ), β ( B ), and total ( C ) glucan concentrations in whole mill mushrooms prepared from caps and stalks from Pleurotus eryngii mushrooms cultivated in various relative amounts of eucalyptus sawdust and OMSW. * p

    Journal: International Journal of Molecular Sciences

    Article Title: Olive Mill Waste Enhances α-Glucan Content in the Edible Mushroom Pleurotus eryngii

    doi: 10.3390/ijms18071564

    Figure Lengend Snippet: α ( A ), β ( B ), and total ( C ) glucan concentrations in whole mill mushrooms prepared from caps and stalks from Pleurotus eryngii mushrooms cultivated in various relative amounts of eucalyptus sawdust and OMSW. * p

    Article Snippet: Glucans Analysis The glucan content of the dried Pleurotus fruiting bodies ( ) or extracted glucans ( ) was determined using a mushroom and yeast specific β-glucan kit (Megazyme International, Wicklow, Ireland) based on a colorimetric reaction [ , ].

    Techniques:

    α, β, and total glucan concentrations in caps and stalks of the mushroom strain Pleurotus eryngii . * p

    Journal: International Journal of Molecular Sciences

    Article Title: Olive Mill Waste Enhances α-Glucan Content in the Edible Mushroom Pleurotus eryngii

    doi: 10.3390/ijms18071564

    Figure Lengend Snippet: α, β, and total glucan concentrations in caps and stalks of the mushroom strain Pleurotus eryngii . * p

    Article Snippet: Glucans Analysis The glucan content of the dried Pleurotus fruiting bodies ( ) or extracted glucans ( ) was determined using a mushroom and yeast specific β-glucan kit (Megazyme International, Wicklow, Ireland) based on a colorimetric reaction [ , ].

    Techniques:

    ( A ) α-glucan concentration in various Pleurotus strains; ( B ) β-glucan concentration in various Pleurotus strains and ( C ) Total glucan concentration. All concentrations are related to the percentage of dried weight. * p

    Journal: International Journal of Molecular Sciences

    Article Title: Olive Mill Waste Enhances α-Glucan Content in the Edible Mushroom Pleurotus eryngii

    doi: 10.3390/ijms18071564

    Figure Lengend Snippet: ( A ) α-glucan concentration in various Pleurotus strains; ( B ) β-glucan concentration in various Pleurotus strains and ( C ) Total glucan concentration. All concentrations are related to the percentage of dried weight. * p

    Article Snippet: Glucans Analysis The glucan content of the dried Pleurotus fruiting bodies ( ) or extracted glucans ( ) was determined using a mushroom and yeast specific β-glucan kit (Megazyme International, Wicklow, Ireland) based on a colorimetric reaction [ , ].

    Techniques: Concentration Assay

    Changes in glucan concentrations by elicitor treatments in the cultivation of Sparassis latifolia on a Pinus densiflora sawdust-based medium. G, glucuronidase; Ly, lysing enzyme; Ch, chitinase. The numbers indicate the amount of each enzyme for each bottle in mL. The values were not significantly different at the 5% level by ANOVA ( p = 0.2993 for β-glucan).

    Journal: Mycobiology

    Article Title: Enhancement of ?-Glucan Content in the Cultivation of Cauliflower Mushroom (Sparassis latifolia) by Elicitation

    doi: 10.5941/MYCO.2014.42.1.41

    Figure Lengend Snippet: Changes in glucan concentrations by elicitor treatments in the cultivation of Sparassis latifolia on a Pinus densiflora sawdust-based medium. G, glucuronidase; Ly, lysing enzyme; Ch, chitinase. The numbers indicate the amount of each enzyme for each bottle in mL. The values were not significantly different at the 5% level by ANOVA ( p = 0.2993 for β-glucan).

    Article Snippet: Assay for the glucan contents A β-glucan assay kit (Megazyme International, Wicklow, Ireland) was used to determine total, α-, and β-glucan.

    Techniques:

    Concentration of glucan in the fruit body of Sparassis latifolia cultivated on a sawdust-based medium from three kinds of conifers. The same letters on each bar indicate that the values were not significantly different by the least significant difference test at the 5% level (n = 3).

    Journal: Mycobiology

    Article Title: Enhancement of ?-Glucan Content in the Cultivation of Cauliflower Mushroom (Sparassis latifolia) by Elicitation

    doi: 10.5941/MYCO.2014.42.1.41

    Figure Lengend Snippet: Concentration of glucan in the fruit body of Sparassis latifolia cultivated on a sawdust-based medium from three kinds of conifers. The same letters on each bar indicate that the values were not significantly different by the least significant difference test at the 5% level (n = 3).

    Article Snippet: Assay for the glucan contents A β-glucan assay kit (Megazyme International, Wicklow, Ireland) was used to determine total, α-, and β-glucan.

    Techniques: Concentration Assay

    Changes in glucan concentrations by the elicitor treatments in the cultivation of Sparassis latifolia on a Larix kaempferi sawdust-based medium. G, glucuronidase; Ly, lysing enzyme; Ch, chitinase. The numbers indicate the amount of each enzyme for each bottle in mL. The same letters on each bar indicate that the values were not significantly different by the least significant difference test at the 5% level (n = 3, p = 0.0066).

    Journal: Mycobiology

    Article Title: Enhancement of ?-Glucan Content in the Cultivation of Cauliflower Mushroom (Sparassis latifolia) by Elicitation

    doi: 10.5941/MYCO.2014.42.1.41

    Figure Lengend Snippet: Changes in glucan concentrations by the elicitor treatments in the cultivation of Sparassis latifolia on a Larix kaempferi sawdust-based medium. G, glucuronidase; Ly, lysing enzyme; Ch, chitinase. The numbers indicate the amount of each enzyme for each bottle in mL. The same letters on each bar indicate that the values were not significantly different by the least significant difference test at the 5% level (n = 3, p = 0.0066).

    Article Snippet: Assay for the glucan contents A β-glucan assay kit (Megazyme International, Wicklow, Ireland) was used to determine total, α-, and β-glucan.

    Techniques:

    Changes in glucan concentrations by elicitor treatments in the cultivation of Sparassis latifolia on a Pinus koraiensis sawdust-based medium. G, glucuronidase, Ly, lysing enzyme; Ch, chitinase. The numbers indicate the amount of each enzyme for each bottle in mL. The same letters on each bar indicate that the values were not significantly different by the least significant difference test at the 5% level (n = 3, p = 0.0394).

    Journal: Mycobiology

    Article Title: Enhancement of ?-Glucan Content in the Cultivation of Cauliflower Mushroom (Sparassis latifolia) by Elicitation

    doi: 10.5941/MYCO.2014.42.1.41

    Figure Lengend Snippet: Changes in glucan concentrations by elicitor treatments in the cultivation of Sparassis latifolia on a Pinus koraiensis sawdust-based medium. G, glucuronidase, Ly, lysing enzyme; Ch, chitinase. The numbers indicate the amount of each enzyme for each bottle in mL. The same letters on each bar indicate that the values were not significantly different by the least significant difference test at the 5% level (n = 3, p = 0.0394).

    Article Snippet: Assay for the glucan contents A β-glucan assay kit (Megazyme International, Wicklow, Ireland) was used to determine total, α-, and β-glucan.

    Techniques:

    Sts regulates ROS production downstream of fungal CLR Dectin-1. (A) Wild-type and Sts −/− BMDCs were stimulated with particulate β-glucan, and then levels of ROS production were assessed by luminol chemiluminescence. (B) Zymosan-induced (left) or HK (heat-killed) wild-type C. albicans -induced (right) ROS production in wild-type and Sts −/− BMDCs was assessed in the absence (−) or presence (+) of soluble β-glucan, a specific inhibitor that blocks activation of Dectin-1 signaling pathways ( 46 ). (C) ROS production in wild-type and Sts −/− BMDCs was assessed in the absence (−) or presence (+) of a blocking anti-Dectin-1 antibody. (D) Equivalent levels of surface Dectin-1 receptor expressed on wild-type BMDCs (solid red line) and Sts −/− BMDCs (dotted dark blue line), evaluated by flow cytometry with a specific anti-Dectin-1 antibody. Nonshaded lines represent a nonspecific rat IgG control. (E) Equivalent levels of zymosan stimulation-dependent downregulation of surface Dectin-1 on wild-type (closed dot) and Sts −/− (open dot) BMDCs. MFI, mean fluorescence intensity. For panels A to C, average values from at least 3 independent experiments each performed in triplicate are presented. P values were calculated by Mann-Whitney analysis.

    Journal: mBio

    Article Title: Phagocytes from Mice Lacking the Sts Phosphatases Have an Enhanced Antifungal Response to Candida albicans

    doi: 10.1128/mBio.00782-18

    Figure Lengend Snippet: Sts regulates ROS production downstream of fungal CLR Dectin-1. (A) Wild-type and Sts −/− BMDCs were stimulated with particulate β-glucan, and then levels of ROS production were assessed by luminol chemiluminescence. (B) Zymosan-induced (left) or HK (heat-killed) wild-type C. albicans -induced (right) ROS production in wild-type and Sts −/− BMDCs was assessed in the absence (−) or presence (+) of soluble β-glucan, a specific inhibitor that blocks activation of Dectin-1 signaling pathways ( 46 ). (C) ROS production in wild-type and Sts −/− BMDCs was assessed in the absence (−) or presence (+) of a blocking anti-Dectin-1 antibody. (D) Equivalent levels of surface Dectin-1 receptor expressed on wild-type BMDCs (solid red line) and Sts −/− BMDCs (dotted dark blue line), evaluated by flow cytometry with a specific anti-Dectin-1 antibody. Nonshaded lines represent a nonspecific rat IgG control. (E) Equivalent levels of zymosan stimulation-dependent downregulation of surface Dectin-1 on wild-type (closed dot) and Sts −/− (open dot) BMDCs. MFI, mean fluorescence intensity. For panels A to C, average values from at least 3 independent experiments each performed in triplicate are presented. P values were calculated by Mann-Whitney analysis.

    Article Snippet: Particulate β-glucan, soluble β-glucan, and depleted zymosan were obtained from InvivoGen.

    Techniques: Activation Assay, Blocking Assay, Flow Cytometry, Cytometry, Fluorescence, MANN-WHITNEY

    Particulate β-glucan induces the differentiation of M-MDSC to antigen-presenting cells in vitro (A) Expression of CD11c, F4/80 and CD11b surface markers on M-MDSC cultured with particulate β-glucan (50 µg/ml) for 0, 5 and 7 days. Results were repeated at least three times with similar results. (B) Expression of Gr-1, CD80, CD86, MHC class II, MHC class I, and CD40 surface markers on M-MDSC cultured with particulate β-glucan (50 µg/ml) for 7 days. Histograms represent the results of three independent experiments. (C) Frequency of IFN-γ + CFSE − gated on CD4 + T cells in cultures where freshly sorted M-MDSC from the spleens of LLC-bearing mice were incubated for 5 days with CFSE-labeled CD4 + T cells in the presence of OVA (100µg/ml). Irradiated splenocytes were used as APC positive control. (D) Sorted and CFSE-labeled CD4 + OT-II T cells were co-cultured with splenic M-MDSC pre-cultured with particulate β-glucan for 7 days in the presence of OVA (50 µg/ml) for 4–5 days. The frequencies of CFSE diluted cells and IFN-γ + CD4 + T cells were shown. Irradiated splenocytes were used as APC positive control. The results are representative of at least four independent experiments. (E) Sorted and CFSE-labeled CD8 + OT-I T cells were co-cultured for 4–5 days with OVA (50 µg/ml) and splenic M-MDSC pre-cultured with particulate β-glucan for 7 days. The frequencies of CFSE diluted cells, IFN-γ + cells and Granzyme B + cells gated on CD8 + T cells are demonstrated. Results are representative of two independent experiments. *p

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

    Article Title: Yeast-derived Particulate β-Glucan Treatment Subverts the Suppression of Myeloid-derived Suppressor Cells by Inducing PMN-MDSC Apoptosis and M-MDSC Differentiation to APC in Cancer

    doi: 10.4049/jimmunol.1501853

    Figure Lengend Snippet: Particulate β-glucan induces the differentiation of M-MDSC to antigen-presenting cells in vitro (A) Expression of CD11c, F4/80 and CD11b surface markers on M-MDSC cultured with particulate β-glucan (50 µg/ml) for 0, 5 and 7 days. Results were repeated at least three times with similar results. (B) Expression of Gr-1, CD80, CD86, MHC class II, MHC class I, and CD40 surface markers on M-MDSC cultured with particulate β-glucan (50 µg/ml) for 7 days. Histograms represent the results of three independent experiments. (C) Frequency of IFN-γ + CFSE − gated on CD4 + T cells in cultures where freshly sorted M-MDSC from the spleens of LLC-bearing mice were incubated for 5 days with CFSE-labeled CD4 + T cells in the presence of OVA (100µg/ml). Irradiated splenocytes were used as APC positive control. (D) Sorted and CFSE-labeled CD4 + OT-II T cells were co-cultured with splenic M-MDSC pre-cultured with particulate β-glucan for 7 days in the presence of OVA (50 µg/ml) for 4–5 days. The frequencies of CFSE diluted cells and IFN-γ + CD4 + T cells were shown. Irradiated splenocytes were used as APC positive control. The results are representative of at least four independent experiments. (E) Sorted and CFSE-labeled CD8 + OT-I T cells were co-cultured for 4–5 days with OVA (50 µg/ml) and splenic M-MDSC pre-cultured with particulate β-glucan for 7 days. The frequencies of CFSE diluted cells, IFN-γ + cells and Granzyme B + cells gated on CD8 + T cells are demonstrated. Results are representative of two independent experiments. *p

    Article Snippet: On day 8 post-implantation, mice with palpable tumors were orally administered daily by gavage with 100 µl particulate β-glucan (Biothera, 800 µg/mouse in PBS suspension) or with 100 µl PBS.

    Techniques: In Vitro, Expressing, Cell Culture, Mouse Assay, Incubation, Labeling, Irradiation, Positive Control

    Particulate β-glucan treatment in vivo decreased the frequency of CD14 − HLA-DR − CD33 + CD11b + MDSC in the peripheral blood of NSCLC patients (A) Frequency of CD33 + CD11b + MDSC gated on CD14 − HLA-DR − cells in the peripheral blood of NSCLC patients (n=23) compared to age and sex matched healthy donors (n=13). (B) Frequency of CD33 + CD11b + MDSC gated on CD14 − HLA-DR − cells in the peripheral blood of NSCLC patients before and after particulate β-glucan treatment for 10–14 days (n=23). (C) IFN-γ production and proliferation (CFSE diluted cells) of allogeneic T cells (CD3 + ) cultured at 1:1 ratio with CD14 + HLA-DR + CD11b + CD33 + or CD14 − HLA-DR − CD11b + CD33 + isolated from the peripheral blood of NSCLC patients before and after particulate β-glucan treatment (n=11) (D) Relative Arginase1 mRNA expression levels in neutrophils (PMN) isolated from the peripheral blood of healthy controls, and patients with NSCLC before and after particulate β-glucan treatment. Arginase 1 relative mRNA expression significantly decreased in a cohort of 15 patients, whereas no significant change was reported in the other 20 patients (total n=35). *p

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

    Article Title: Yeast-derived Particulate β-Glucan Treatment Subverts the Suppression of Myeloid-derived Suppressor Cells by Inducing PMN-MDSC Apoptosis and M-MDSC Differentiation to APC in Cancer

    doi: 10.4049/jimmunol.1501853

    Figure Lengend Snippet: Particulate β-glucan treatment in vivo decreased the frequency of CD14 − HLA-DR − CD33 + CD11b + MDSC in the peripheral blood of NSCLC patients (A) Frequency of CD33 + CD11b + MDSC gated on CD14 − HLA-DR − cells in the peripheral blood of NSCLC patients (n=23) compared to age and sex matched healthy donors (n=13). (B) Frequency of CD33 + CD11b + MDSC gated on CD14 − HLA-DR − cells in the peripheral blood of NSCLC patients before and after particulate β-glucan treatment for 10–14 days (n=23). (C) IFN-γ production and proliferation (CFSE diluted cells) of allogeneic T cells (CD3 + ) cultured at 1:1 ratio with CD14 + HLA-DR + CD11b + CD33 + or CD14 − HLA-DR − CD11b + CD33 + isolated from the peripheral blood of NSCLC patients before and after particulate β-glucan treatment (n=11) (D) Relative Arginase1 mRNA expression levels in neutrophils (PMN) isolated from the peripheral blood of healthy controls, and patients with NSCLC before and after particulate β-glucan treatment. Arginase 1 relative mRNA expression significantly decreased in a cohort of 15 patients, whereas no significant change was reported in the other 20 patients (total n=35). *p

    Article Snippet: On day 8 post-implantation, mice with palpable tumors were orally administered daily by gavage with 100 µl particulate β-glucan (Biothera, 800 µg/mouse in PBS suspension) or with 100 µl PBS.

    Techniques: In Vivo, Cell Culture, Isolation, Expressing

    Particulate β-glucan treatment induces M-MDSC differentiation in vivo with reduced tumor growth Tumor M-MDSC sorted from C57Bl/6 LLC tumor-bearing mice (CD45.2) were treated with or without WGP for overnight and then intratumorally injected into SJL tumor-bearing mice (CD45.1). Mice were sacrificed after 7 days and single cell suspensions from tumors were stained with anti-CD45.2 or isotype control mAb (A) and CD11c and MHC class II. Cells were gated on CD45.2 + cells (B). The percentage of CD11c + MHC class II + cells was summarized. (C) M-MDSC sorted from LLC tumor-bearing mice were treated with or without WGP and then mixed with LLC cells for injection. LLC alone was used as control. Tumor growth was monitored and recorded. (D) M-MDSC sorted from LLC tumor-bearing wildtype or dectin-1 KO mice were treated with WGP and mixed with LLC cells for injection. Tumor growth was monitored. *p

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

    Article Title: Yeast-derived Particulate β-Glucan Treatment Subverts the Suppression of Myeloid-derived Suppressor Cells by Inducing PMN-MDSC Apoptosis and M-MDSC Differentiation to APC in Cancer

    doi: 10.4049/jimmunol.1501853

    Figure Lengend Snippet: Particulate β-glucan treatment induces M-MDSC differentiation in vivo with reduced tumor growth Tumor M-MDSC sorted from C57Bl/6 LLC tumor-bearing mice (CD45.2) were treated with or without WGP for overnight and then intratumorally injected into SJL tumor-bearing mice (CD45.1). Mice were sacrificed after 7 days and single cell suspensions from tumors were stained with anti-CD45.2 or isotype control mAb (A) and CD11c and MHC class II. Cells were gated on CD45.2 + cells (B). The percentage of CD11c + MHC class II + cells was summarized. (C) M-MDSC sorted from LLC tumor-bearing mice were treated with or without WGP and then mixed with LLC cells for injection. LLC alone was used as control. Tumor growth was monitored and recorded. (D) M-MDSC sorted from LLC tumor-bearing wildtype or dectin-1 KO mice were treated with WGP and mixed with LLC cells for injection. Tumor growth was monitored. *p

    Article Snippet: On day 8 post-implantation, mice with palpable tumors were orally administered daily by gavage with 100 µl particulate β-glucan (Biothera, 800 µg/mouse in PBS suspension) or with 100 µl PBS.

    Techniques: In Vivo, Mouse Assay, Injection, Staining

    Particulate β-glucan treatment in vitro subverts splenic MDSC-mediated T cell suppression (A) CFSE-labeled OT-II splenocytes co-cultured with sorted M-MDSC or PMN-MDSC from the spleens of LLC-bearing mice in the presence or absence of particulate β-glucan (100 µg/ml in the M-MDSC cultures and 50 µg/ml in the PMN-MDSC cultures) and OVA (100 µg/ml) for 3 days at 1:1 ratio. Data represent the percentage of CFSE diluted cells gated on CD4 + T cells. The experiment was repeated two times with similar results. (B) CFSE-labeled OT-I splenocytes were co-cultured with sorted M-MDSC or PMN-MDSC from the spleens of LLC-bearing mice in the presence of OVA (50 µg/ml) and particulate β-glucan (50 µg/ml) for 3 days at 1:1 ratio. Data represent the percentage of CFSE diluted cells gated on CD8 + T cells. Data is representative of three independent experiments. (C) OT-II splenocytes co-cultured with sorted M-MDSC or PMN-MDSC from the spleens of LLC-bearing mice in the presence of OVA (100 µg/ml) with or without particulate β-glucan (50 µg/ml) for 3–4 days at 1:1 ratio and stimulated with PMA/Ionomycin for intracellular IFN-γ staining. Data represent the percentage of IFN-γ + cells gated on CD4 + T cells. The experiment was repeated 3 times with similar results. (D) CFSE-labeled OT-I splenocytes co-cultured with sorted M-MDSC or PMN-MDSC from the spleens of LLC-bearing mice in the presence of OVA (50 µg/ml in M-MDSC cultures and 10 µg/ml in PMN-MDSC cultures) and particulate β-glucan (50 µg/ml) for 3 days at 1:1 ratio and then stimulated with PMA/Ionomycin for intracellular IFN-γ staining. Data represent the percentage of IFN-γ + CFSE diluted cells gated on CD8 + T cells. Results are representative of three independent experiments. (E) CFSE-labeled splenocytes from C57BL/6 mice stimulated with plate-bound anti-CD3 (5 µg/ml) and soluble anti-CD28 (2 µg/ml) (white bar) and co-cultured with sorted splenic M-MDSC or PMN-MDSC from LLC-bearing mice for 3 days with (black bar) or without particulate β-glucan (50 µg/ml) (grey bar). Data represent the frequency of CFSE diluted cells gated on CD4 + or CD8 + T cells. The experiment was repeated twice with similar results. * p

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

    Article Title: Yeast-derived Particulate β-Glucan Treatment Subverts the Suppression of Myeloid-derived Suppressor Cells by Inducing PMN-MDSC Apoptosis and M-MDSC Differentiation to APC in Cancer

    doi: 10.4049/jimmunol.1501853

    Figure Lengend Snippet: Particulate β-glucan treatment in vitro subverts splenic MDSC-mediated T cell suppression (A) CFSE-labeled OT-II splenocytes co-cultured with sorted M-MDSC or PMN-MDSC from the spleens of LLC-bearing mice in the presence or absence of particulate β-glucan (100 µg/ml in the M-MDSC cultures and 50 µg/ml in the PMN-MDSC cultures) and OVA (100 µg/ml) for 3 days at 1:1 ratio. Data represent the percentage of CFSE diluted cells gated on CD4 + T cells. The experiment was repeated two times with similar results. (B) CFSE-labeled OT-I splenocytes were co-cultured with sorted M-MDSC or PMN-MDSC from the spleens of LLC-bearing mice in the presence of OVA (50 µg/ml) and particulate β-glucan (50 µg/ml) for 3 days at 1:1 ratio. Data represent the percentage of CFSE diluted cells gated on CD8 + T cells. Data is representative of three independent experiments. (C) OT-II splenocytes co-cultured with sorted M-MDSC or PMN-MDSC from the spleens of LLC-bearing mice in the presence of OVA (100 µg/ml) with or without particulate β-glucan (50 µg/ml) for 3–4 days at 1:1 ratio and stimulated with PMA/Ionomycin for intracellular IFN-γ staining. Data represent the percentage of IFN-γ + cells gated on CD4 + T cells. The experiment was repeated 3 times with similar results. (D) CFSE-labeled OT-I splenocytes co-cultured with sorted M-MDSC or PMN-MDSC from the spleens of LLC-bearing mice in the presence of OVA (50 µg/ml in M-MDSC cultures and 10 µg/ml in PMN-MDSC cultures) and particulate β-glucan (50 µg/ml) for 3 days at 1:1 ratio and then stimulated with PMA/Ionomycin for intracellular IFN-γ staining. Data represent the percentage of IFN-γ + CFSE diluted cells gated on CD8 + T cells. Results are representative of three independent experiments. (E) CFSE-labeled splenocytes from C57BL/6 mice stimulated with plate-bound anti-CD3 (5 µg/ml) and soluble anti-CD28 (2 µg/ml) (white bar) and co-cultured with sorted splenic M-MDSC or PMN-MDSC from LLC-bearing mice for 3 days with (black bar) or without particulate β-glucan (50 µg/ml) (grey bar). Data represent the frequency of CFSE diluted cells gated on CD4 + or CD8 + T cells. The experiment was repeated twice with similar results. * p

    Article Snippet: On day 8 post-implantation, mice with palpable tumors were orally administered daily by gavage with 100 µl particulate β-glucan (Biothera, 800 µg/mouse in PBS suspension) or with 100 µl PBS.

    Techniques: In Vitro, Labeling, Cell Culture, Mouse Assay, Staining

    Particulate β-glucan reduces tumor Gr-1 + CD11b + MDSC-mediated T cell suppression (A) Tumor Gr-1 + CD11b + CD45 + MDSC sorted from LLC-bearing mice were co-cultured with CFSE-labeled OT-II splenocytes at indicated ratios, in the presence of OVA (100 µg/ml) with or without particulate β-glucan (50 µg/ml) for 3–4 days. Data represent the frequency of CFSE diluted cells gated on CD4 + T cells. The experiment was repeated twice with similar results. (B) Same cell cultures as (A) were further stimulated with PMA/Ionomycin for intracellular IFN-γ staining. The experiment was repeated twice with similar results. (C) Splenocytes of OT-I mice were co-cultured with sorted Gr-1 + CD11b + CD45 + tumor MDSC from LLC-bearing mice at indicated ratios, in the presence of OVA (50 µg/ml in M-MDSC cultures and 10 µg/ml in PMN-MDSC cultures) with or without particulate β-glucan (50 µg/ml). Data represent the percentage of IFN-γ + cells and CFSE diluted cells gated on CD8 + T cells. Results are representative of two independent experiments. *p

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

    Article Title: Yeast-derived Particulate β-Glucan Treatment Subverts the Suppression of Myeloid-derived Suppressor Cells by Inducing PMN-MDSC Apoptosis and M-MDSC Differentiation to APC in Cancer

    doi: 10.4049/jimmunol.1501853

    Figure Lengend Snippet: Particulate β-glucan reduces tumor Gr-1 + CD11b + MDSC-mediated T cell suppression (A) Tumor Gr-1 + CD11b + CD45 + MDSC sorted from LLC-bearing mice were co-cultured with CFSE-labeled OT-II splenocytes at indicated ratios, in the presence of OVA (100 µg/ml) with or without particulate β-glucan (50 µg/ml) for 3–4 days. Data represent the frequency of CFSE diluted cells gated on CD4 + T cells. The experiment was repeated twice with similar results. (B) Same cell cultures as (A) were further stimulated with PMA/Ionomycin for intracellular IFN-γ staining. The experiment was repeated twice with similar results. (C) Splenocytes of OT-I mice were co-cultured with sorted Gr-1 + CD11b + CD45 + tumor MDSC from LLC-bearing mice at indicated ratios, in the presence of OVA (50 µg/ml in M-MDSC cultures and 10 µg/ml in PMN-MDSC cultures) with or without particulate β-glucan (50 µg/ml). Data represent the percentage of IFN-γ + cells and CFSE diluted cells gated on CD8 + T cells. Results are representative of two independent experiments. *p

    Article Snippet: On day 8 post-implantation, mice with palpable tumors were orally administered daily by gavage with 100 µl particulate β-glucan (Biothera, 800 µg/mouse in PBS suspension) or with 100 µl PBS.

    Techniques: Mouse Assay, Cell Culture, Labeling, Staining

    Particulate β-glucan induces respiratory burst and apoptosis in PMN-MDSC in a dectin-1 dependent manner (A) Representative dot plots showing the frequency of annexin V + cells gated on Gr-1 high CD11b + PMN-MDSC in splenocytes of LLC-bearing mice cultured with media only or with particulate β-glucan (100 µg/ml) for indicated time. Summarized data are also shown (n=8). (B) Frequencies of annexinV + cells gated on Gr-1 high CD11b + PMN-MDSC from WT or dectin-1 KO LLC-bearing mice cultured for 18 hours with or without particulate β-glucan (100 µg/ml) (n=2). (C) Respiratory burst in PMN-MDSC sorted from spleens of LLC-bearing WT or dectin-1 KO mice stimulated with particulate β-glucan (100 µg/ml) for indicated time. Reduction of dihydrorhodamine 123 (DHR) to fluorescent rhodamine 123 (RHO) was assessed by flow cytometry. Data is representative of 6 independent experiments (WT) and two independent experiments (KO). (D) Western blot analysis of p-STAT3, p-Zap/Syk, p-Akt, p-SAPK/JNK, p-Erk1/2, p-p38, STAT3 and β-actin in PMN-MDSC sorted from the spleens of LLC-bearing mice and treated with particulate β-glucan (100 µg/ml) for 0, 15, 30 and 60 minutes. Results are representative of at least three independent experiments. *p

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

    Article Title: Yeast-derived Particulate β-Glucan Treatment Subverts the Suppression of Myeloid-derived Suppressor Cells by Inducing PMN-MDSC Apoptosis and M-MDSC Differentiation to APC in Cancer

    doi: 10.4049/jimmunol.1501853

    Figure Lengend Snippet: Particulate β-glucan induces respiratory burst and apoptosis in PMN-MDSC in a dectin-1 dependent manner (A) Representative dot plots showing the frequency of annexin V + cells gated on Gr-1 high CD11b + PMN-MDSC in splenocytes of LLC-bearing mice cultured with media only or with particulate β-glucan (100 µg/ml) for indicated time. Summarized data are also shown (n=8). (B) Frequencies of annexinV + cells gated on Gr-1 high CD11b + PMN-MDSC from WT or dectin-1 KO LLC-bearing mice cultured for 18 hours with or without particulate β-glucan (100 µg/ml) (n=2). (C) Respiratory burst in PMN-MDSC sorted from spleens of LLC-bearing WT or dectin-1 KO mice stimulated with particulate β-glucan (100 µg/ml) for indicated time. Reduction of dihydrorhodamine 123 (DHR) to fluorescent rhodamine 123 (RHO) was assessed by flow cytometry. Data is representative of 6 independent experiments (WT) and two independent experiments (KO). (D) Western blot analysis of p-STAT3, p-Zap/Syk, p-Akt, p-SAPK/JNK, p-Erk1/2, p-p38, STAT3 and β-actin in PMN-MDSC sorted from the spleens of LLC-bearing mice and treated with particulate β-glucan (100 µg/ml) for 0, 15, 30 and 60 minutes. Results are representative of at least three independent experiments. *p

    Article Snippet: On day 8 post-implantation, mice with palpable tumors were orally administered daily by gavage with 100 µl particulate β-glucan (Biothera, 800 µg/mouse in PBS suspension) or with 100 µl PBS.

    Techniques: Mouse Assay, Cell Culture, Flow Cytometry, Cytometry, Western Blot

    Particulate β-glucan-induced M-MDSC antigen-presenting function is dectin-1 and Erk1/2 dependent (A) CD4 + and CD8 + T cells were sorted from OT-II and OT-I mice, respectively, CFSE-labeled and co-cultured for 4–5 days with M-MDSC sorted from the spleens of WT or dectin-1 KO LLC-bearing mice. M-MDSC were pre-treated with particulate β-glucan for 7 days prior to co-culture with CD4 + or CD8 + T cells. The frequency of CFSE − IFN-γ + , gated on CD4 + T cells (upper panel) or CD8 + T cells (lower panel), are represented in the dot plots. Results are representative of two-independent experiments. (B) Western blot analysis of p-STAT3, p-Zap/Syk, p-Akt, p-SAPK/JNK, p-Erk1/2, p-p38, STAT3 and β-actin in M-MDSC sorted from the spleens of LLC-bearing mice and stimulated with particulate β-glucan (100 µg/ml) for 0, 15, 30 and 60 minutes. (C) CD4 + T cells sorted from OT-II mice were CFSE-labeled and co-cultured with OVA and M-MDSC pre-treated with particulate β-glucan in the presence of MEK1/2 inhibitor (PD98059) (30 ng/ml) or DMSO for 7 days. Data demonstrate the frequencies of CFSE diluted cells and IFN-γ + cells gated on CD4 + T cells. Results are representative of two independent experiments. (D) The relative mRNA expression of TNF-α, IL-12, iNOS, IL-6, and TGF-β in MDSC, sorted from the spleens of LLC-bearing mice, and incubated with particulate β-glucan for 7 days compared to its expression in freshly sorted M-MDSC. *p

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

    Article Title: Yeast-derived Particulate β-Glucan Treatment Subverts the Suppression of Myeloid-derived Suppressor Cells by Inducing PMN-MDSC Apoptosis and M-MDSC Differentiation to APC in Cancer

    doi: 10.4049/jimmunol.1501853

    Figure Lengend Snippet: Particulate β-glucan-induced M-MDSC antigen-presenting function is dectin-1 and Erk1/2 dependent (A) CD4 + and CD8 + T cells were sorted from OT-II and OT-I mice, respectively, CFSE-labeled and co-cultured for 4–5 days with M-MDSC sorted from the spleens of WT or dectin-1 KO LLC-bearing mice. M-MDSC were pre-treated with particulate β-glucan for 7 days prior to co-culture with CD4 + or CD8 + T cells. The frequency of CFSE − IFN-γ + , gated on CD4 + T cells (upper panel) or CD8 + T cells (lower panel), are represented in the dot plots. Results are representative of two-independent experiments. (B) Western blot analysis of p-STAT3, p-Zap/Syk, p-Akt, p-SAPK/JNK, p-Erk1/2, p-p38, STAT3 and β-actin in M-MDSC sorted from the spleens of LLC-bearing mice and stimulated with particulate β-glucan (100 µg/ml) for 0, 15, 30 and 60 minutes. (C) CD4 + T cells sorted from OT-II mice were CFSE-labeled and co-cultured with OVA and M-MDSC pre-treated with particulate β-glucan in the presence of MEK1/2 inhibitor (PD98059) (30 ng/ml) or DMSO for 7 days. Data demonstrate the frequencies of CFSE diluted cells and IFN-γ + cells gated on CD4 + T cells. Results are representative of two independent experiments. (D) The relative mRNA expression of TNF-α, IL-12, iNOS, IL-6, and TGF-β in MDSC, sorted from the spleens of LLC-bearing mice, and incubated with particulate β-glucan for 7 days compared to its expression in freshly sorted M-MDSC. *p

    Article Snippet: On day 8 post-implantation, mice with palpable tumors were orally administered daily by gavage with 100 µl particulate β-glucan (Biothera, 800 µg/mouse in PBS suspension) or with 100 µl PBS.

    Techniques: Mouse Assay, Labeling, Cell Culture, Co-Culture Assay, Western Blot, Expressing, Incubation

    Particulate β-glucan treatment in vivo reduces tumor burden and impacts the frequency of MDSC in spleens and tumors of LLC and E0771-bearing mice (A) C57BL/6 WT mice (n=7, 8) were injected subcutaneously (s.c) with LLC or E0771 tumor cell lines. Once palpable tumors were formed (day 8), mice were orally administered with particulate β-glucan (800 µg, daily) or PBS with a gavage needle at indicated time. Tumor diameters were measured every three days and tumor volumes were then calculated. (B) On day 32 (LLC model) or day 35 (E0771 model), mice were killed and spleens were excised and weighed. Each point in the data plot represents the spleen weight of each mouse in grams. PBS-treated group was compared to particulate β-glucan treated group (WGP) in both models (C) Tumor tissues were excised and weighted from WGP or PBS-treated mice. (D) Flow cytometry analysis of the frequencies of M-MDSC (Ly6G − Ly6C high ) and PMN-MDSC (Ly6G + Ly6C int ) in the spleens of LLC and E0771-bearing mice treated with PBS or particulate β-glucan (WGP). Cells were gated on CD11b + cells. (E) Frequencies of M-MDSC and PMN-MDSC in the tumors of LLC and E0771-bearing mice treated with PBS or particulate β-glucan (WGP). Cells were gated on CD45 + CD11b + cells. *p

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

    Article Title: Yeast-derived Particulate β-Glucan Treatment Subverts the Suppression of Myeloid-derived Suppressor Cells by Inducing PMN-MDSC Apoptosis and M-MDSC Differentiation to APC in Cancer

    doi: 10.4049/jimmunol.1501853

    Figure Lengend Snippet: Particulate β-glucan treatment in vivo reduces tumor burden and impacts the frequency of MDSC in spleens and tumors of LLC and E0771-bearing mice (A) C57BL/6 WT mice (n=7, 8) were injected subcutaneously (s.c) with LLC or E0771 tumor cell lines. Once palpable tumors were formed (day 8), mice were orally administered with particulate β-glucan (800 µg, daily) or PBS with a gavage needle at indicated time. Tumor diameters were measured every three days and tumor volumes were then calculated. (B) On day 32 (LLC model) or day 35 (E0771 model), mice were killed and spleens were excised and weighed. Each point in the data plot represents the spleen weight of each mouse in grams. PBS-treated group was compared to particulate β-glucan treated group (WGP) in both models (C) Tumor tissues were excised and weighted from WGP or PBS-treated mice. (D) Flow cytometry analysis of the frequencies of M-MDSC (Ly6G − Ly6C high ) and PMN-MDSC (Ly6G + Ly6C int ) in the spleens of LLC and E0771-bearing mice treated with PBS or particulate β-glucan (WGP). Cells were gated on CD11b + cells. (E) Frequencies of M-MDSC and PMN-MDSC in the tumors of LLC and E0771-bearing mice treated with PBS or particulate β-glucan (WGP). Cells were gated on CD45 + CD11b + cells. *p

    Article Snippet: On day 8 post-implantation, mice with palpable tumors were orally administered daily by gavage with 100 µl particulate β-glucan (Biothera, 800 µg/mouse in PBS suspension) or with 100 µl PBS.

    Techniques: In Vivo, Mouse Assay, Injection, Flow Cytometry, Cytometry

    Phagocytic ability of macrophages from individuals with different TB phenotypes. Monocyte-derived macrophages from patients at day 7 were treated with Alexa 547-beads coated with either immunoglobulin-G (IgG), trehalose 6,6'-dimycolate (TDM) or β-glucan. Phagocytic ability was determined by the percentage of macrophages with beads in three TB phenotypes (55 TB meningitis, 52 pulmonary TB and 56 latent TB). Bars in plots represent median values. Comparisons across three groups of TB forms or genotypes were performed by using one-way analysis of variance. On these comparisons, P -values > 0.05.

    Journal: Genes and Immunity

    Article Title: MARCO variants are associated with phagocytosis, pulmonary tuberculosis susceptibility and Beijing lineage

    doi: 10.1038/gene.2016.43

    Figure Lengend Snippet: Phagocytic ability of macrophages from individuals with different TB phenotypes. Monocyte-derived macrophages from patients at day 7 were treated with Alexa 547-beads coated with either immunoglobulin-G (IgG), trehalose 6,6'-dimycolate (TDM) or β-glucan. Phagocytic ability was determined by the percentage of macrophages with beads in three TB phenotypes (55 TB meningitis, 52 pulmonary TB and 56 latent TB). Bars in plots represent median values. Comparisons across three groups of TB forms or genotypes were performed by using one-way analysis of variance. On these comparisons, P -values > 0.05.

    Article Snippet: Preparation of beads for phagocytosis The procedure of coating beads was adopted from Yates et al. Carboxylate-modified silica particles (25 mg or 500 μl of 3 μm; Kisker Biotech, Steinfurt, Germany) were washed three times in 1 ml of PBS by vortexing and centrifugation at 2000 g for 1 min. Beads were incubated at room temperature in 25 mg ml−1 cyanamide (Sigma-Aldrich), which works as a cross-linker, in PBS with agitation for 15 min. Beads were washed twice in 1 ml of coupling buffer (0.1 m borate buffer, pH 8.0) and then incubated in 0.5 ml coupling buffer with 1.0 mg defatted bovine serum albumin (Sigma-Aldrich) and 0.1 mg human IgG (Molecular Probes, Eugene, OR, USA) or 0.25 mg ligands (TDM (Enzo Life Sciences) or β-glucan/whole glucan particles (Invivogen, San Diego, CA, USA)) then dispersed for 12 h with agitation.

    Techniques: Derivative Assay