β glucan  (Millipore)


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    Name:
    Beta Glucan
    Description:
    This product is provided as delivered and specified by the issuing Pharmacopoeia All information provided in support of this product including SDS and any product information leaflets have been developed and issued under the Authority of the issuing Pharmacopoeia For further information and support please go to the website of the issuing Pharmacopoeia
    Catalog Number:
    1048288
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    Structured Review

    Millipore β glucan
    TLR2-mediated inhibition of IL-12 p75. (A) mono-DCs treated with IFN-γ or left untreated were labeled with [ 35 S]methionine and stimulated with <t>β-glucan,</t> Pam2C, R848, and their combinations. After 18 h, supernatants were immunoprecipitated with anti–IL-12 p35, anti–IL-23 p19, or anti–IL-12/23 p40 mAbs and resolved in nonreducing SDS-PAGE. (B) Cytokine production was measured by ELISA in mono-DCs 18 h after stimulation with 10 ng/ml LPS+R848, 10 μg/ml β-glucan+R848, or 10 μg/ml zymosan+R848 in the presence or absence of 50 ng/ml Pam2C (each symbol represents a single donor). (C) mono-DCs were stimulated as indicated in B in the presence or absence of 0.5, 5, and 50 ng/ml Pam2C, and IL-12 p75 was measured by ELISA (mean ± SD from triplicate cultures). (D) IL-12 p75 production was assessed by ELISA (mean ± SD from triplicate cultures) in mono-DCs stimulated with 10 μg/ml zymosan+R848 or 10 μg/ml β-glucan+R848 without or with 2, 20, and 200 ng/ml Pam3C; 1, 10, and 100 ng/ml MALP-2; or 0.5, 5, and 50 ng/ml Pam2C. (E) mono-DCs were stimulated with10 μg/ml zymosan+R848 (left) or with 0.1 μg/ml β-glucan+R848 (right) in the absence (open column) or presence (gray column) of 30 μg/ml of the neutralizing anti–IL-10 mAb 19F1, with or without 50 ng/ml Pam2C. Cytokines were measured by ELISA (mean values from duplicate cultures in a representative experiment of six performed with similar results).
    This product is provided as delivered and specified by the issuing Pharmacopoeia All information provided in support of this product including SDS and any product information leaflets have been developed and issued under the Authority of the issuing Pharmacopoeia For further information and support please go to the website of the issuing Pharmacopoeia
    https://www.bioz.com/result/β glucan/product/Millipore
    Average 97 stars, based on 19 article reviews
    Price from $9.99 to $1999.99
    β glucan - by Bioz Stars, 2020-07
    97/100 stars

    Images

    1) Product Images from "Differential regulation of interleukin 12 and interleukin 23 production in human dendritic cells"

    Article Title: Differential regulation of interleukin 12 and interleukin 23 production in human dendritic cells

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20071450

    TLR2-mediated inhibition of IL-12 p75. (A) mono-DCs treated with IFN-γ or left untreated were labeled with [ 35 S]methionine and stimulated with β-glucan, Pam2C, R848, and their combinations. After 18 h, supernatants were immunoprecipitated with anti–IL-12 p35, anti–IL-23 p19, or anti–IL-12/23 p40 mAbs and resolved in nonreducing SDS-PAGE. (B) Cytokine production was measured by ELISA in mono-DCs 18 h after stimulation with 10 ng/ml LPS+R848, 10 μg/ml β-glucan+R848, or 10 μg/ml zymosan+R848 in the presence or absence of 50 ng/ml Pam2C (each symbol represents a single donor). (C) mono-DCs were stimulated as indicated in B in the presence or absence of 0.5, 5, and 50 ng/ml Pam2C, and IL-12 p75 was measured by ELISA (mean ± SD from triplicate cultures). (D) IL-12 p75 production was assessed by ELISA (mean ± SD from triplicate cultures) in mono-DCs stimulated with 10 μg/ml zymosan+R848 or 10 μg/ml β-glucan+R848 without or with 2, 20, and 200 ng/ml Pam3C; 1, 10, and 100 ng/ml MALP-2; or 0.5, 5, and 50 ng/ml Pam2C. (E) mono-DCs were stimulated with10 μg/ml zymosan+R848 (left) or with 0.1 μg/ml β-glucan+R848 (right) in the absence (open column) or presence (gray column) of 30 μg/ml of the neutralizing anti–IL-10 mAb 19F1, with or without 50 ng/ml Pam2C. Cytokines were measured by ELISA (mean values from duplicate cultures in a representative experiment of six performed with similar results).
    Figure Legend Snippet: TLR2-mediated inhibition of IL-12 p75. (A) mono-DCs treated with IFN-γ or left untreated were labeled with [ 35 S]methionine and stimulated with β-glucan, Pam2C, R848, and their combinations. After 18 h, supernatants were immunoprecipitated with anti–IL-12 p35, anti–IL-23 p19, or anti–IL-12/23 p40 mAbs and resolved in nonreducing SDS-PAGE. (B) Cytokine production was measured by ELISA in mono-DCs 18 h after stimulation with 10 ng/ml LPS+R848, 10 μg/ml β-glucan+R848, or 10 μg/ml zymosan+R848 in the presence or absence of 50 ng/ml Pam2C (each symbol represents a single donor). (C) mono-DCs were stimulated as indicated in B in the presence or absence of 0.5, 5, and 50 ng/ml Pam2C, and IL-12 p75 was measured by ELISA (mean ± SD from triplicate cultures). (D) IL-12 p75 production was assessed by ELISA (mean ± SD from triplicate cultures) in mono-DCs stimulated with 10 μg/ml zymosan+R848 or 10 μg/ml β-glucan+R848 without or with 2, 20, and 200 ng/ml Pam3C; 1, 10, and 100 ng/ml MALP-2; or 0.5, 5, and 50 ng/ml Pam2C. (E) mono-DCs were stimulated with10 μg/ml zymosan+R848 (left) or with 0.1 μg/ml β-glucan+R848 (right) in the absence (open column) or presence (gray column) of 30 μg/ml of the neutralizing anti–IL-10 mAb 19F1, with or without 50 ng/ml Pam2C. Cytokines were measured by ELISA (mean values from duplicate cultures in a representative experiment of six performed with similar results).

    Techniques Used: Inhibition, Labeling, Immunoprecipitation, SDS Page, Enzyme-linked Immunosorbent Assay

    Role of dectin-1 and TLR2 in IL-12 and IL-23 production in mono-DCs. mono-DCs treated with IFN-γ or left untreated were labeled with [ 35 S]methionine (A) or unlabeled (B–E) and stimulated with β-glucan or Pam2C, or with a combination of β-glucan+Pam2C, with or without R848. After 18 h, supernatants were immunoprecipitated with anti–IL-12 p35, anti–IL-23 p19, or anti–IL-12/23 p40 mAbs and resolved in nonreducing SDS-PAGE (A) and tested for IL-23, IL-12 p75, and IL-10 production by ELISA (B–D) and for mRNA accumulation using the QuantiGene multiplex assay (E). Results in A are representative of four independent experiments. Results in B are mean ± SE values from mono-DCs derived from 18 donors. Results in C represent individual IL-23 levels from mono-DCs derived from 21 different donors. Results in D are mean ± SE values from five experiments. Results in E are mean ± SD ( n = 3) mRNA accumulation in cells derived from three separate donors and lysed at 3 h (right column of each triplet), 6 h (middle column of each triplet), and 12 h (right column of each triplet). Inverted triangles indicate not done.
    Figure Legend Snippet: Role of dectin-1 and TLR2 in IL-12 and IL-23 production in mono-DCs. mono-DCs treated with IFN-γ or left untreated were labeled with [ 35 S]methionine (A) or unlabeled (B–E) and stimulated with β-glucan or Pam2C, or with a combination of β-glucan+Pam2C, with or without R848. After 18 h, supernatants were immunoprecipitated with anti–IL-12 p35, anti–IL-23 p19, or anti–IL-12/23 p40 mAbs and resolved in nonreducing SDS-PAGE (A) and tested for IL-23, IL-12 p75, and IL-10 production by ELISA (B–D) and for mRNA accumulation using the QuantiGene multiplex assay (E). Results in A are representative of four independent experiments. Results in B are mean ± SE values from mono-DCs derived from 18 donors. Results in C represent individual IL-23 levels from mono-DCs derived from 21 different donors. Results in D are mean ± SE values from five experiments. Results in E are mean ± SD ( n = 3) mRNA accumulation in cells derived from three separate donors and lysed at 3 h (right column of each triplet), 6 h (middle column of each triplet), and 12 h (right column of each triplet). Inverted triangles indicate not done.

    Techniques Used: Labeling, Immunoprecipitation, SDS Page, Enzyme-linked Immunosorbent Assay, Multiplex Assay, Derivative Assay

    Induction of IL-17 and IFN-γ in human CD4 + T cells by supernatants of stimulated mono-DCs. Supernatants from differently stimulated mono-DCs were used to induce IL-17 and IFN-γ production from human CD4 + CD45RO − T lymphocytes stimulated with anti-CD3 and anti-CD28. (A) Supernatants from IFN-γ–primed mono-DCs stimulated with 200 μg/ml zymosan or 1 μg/ml LPS+R848, or from unprimed mono-DCs stimulated with 10 μg/ml β-glucan, were evaluated by ELISA for IL-12, IL23, IL-6, and IL-1β production and for the capacity to induce IL-17 and IFN-γ in naive CD4 + T cells (mean ± SE from 10 independent experiments). (B) IL-17 and IFN-γ production was determined by ELISA in naive T cell cultures in the presence of supernatants from zymosan- or β-glucan–stimulated mono-DCs, and in the presence or absence of neutralizing anti–IL-12 p75 (20C2) or anti-p40 (C8.6) mAbs (mean ± SE from seven independent experiments). (C) Effect of neutralizing anti–TGF-β or anti–IL-6, or a mixture of both mAbs on IL-17 and IFN-γ production measured by ELISA in naive T cells cultured in the presence of supernatants from zymosan-stimulated mono-DCs. (D) Effect of 10 ng/ml IL-1β on IL-17 and IFN-γ production, as determined by ELISA in naive T cells cultured in the presence of IL-23 or an IL-12–depleted supernatant from LPS+R848–stimulated mono-DCs. Supernatants were diluted to contain IL-23 at a final concentration of 15 or 1.5 ng/ml (light and dark gray, respectively). Recombinant IL-23 was used at 15 or 1.5 ng/ml (light and dark gray, respectively). C and D show the mean ± SD from triplicate cultures. Similar results were obtained in three independent experiments.
    Figure Legend Snippet: Induction of IL-17 and IFN-γ in human CD4 + T cells by supernatants of stimulated mono-DCs. Supernatants from differently stimulated mono-DCs were used to induce IL-17 and IFN-γ production from human CD4 + CD45RO − T lymphocytes stimulated with anti-CD3 and anti-CD28. (A) Supernatants from IFN-γ–primed mono-DCs stimulated with 200 μg/ml zymosan or 1 μg/ml LPS+R848, or from unprimed mono-DCs stimulated with 10 μg/ml β-glucan, were evaluated by ELISA for IL-12, IL23, IL-6, and IL-1β production and for the capacity to induce IL-17 and IFN-γ in naive CD4 + T cells (mean ± SE from 10 independent experiments). (B) IL-17 and IFN-γ production was determined by ELISA in naive T cell cultures in the presence of supernatants from zymosan- or β-glucan–stimulated mono-DCs, and in the presence or absence of neutralizing anti–IL-12 p75 (20C2) or anti-p40 (C8.6) mAbs (mean ± SE from seven independent experiments). (C) Effect of neutralizing anti–TGF-β or anti–IL-6, or a mixture of both mAbs on IL-17 and IFN-γ production measured by ELISA in naive T cells cultured in the presence of supernatants from zymosan-stimulated mono-DCs. (D) Effect of 10 ng/ml IL-1β on IL-17 and IFN-γ production, as determined by ELISA in naive T cells cultured in the presence of IL-23 or an IL-12–depleted supernatant from LPS+R848–stimulated mono-DCs. Supernatants were diluted to contain IL-23 at a final concentration of 15 or 1.5 ng/ml (light and dark gray, respectively). Recombinant IL-23 was used at 15 or 1.5 ng/ml (light and dark gray, respectively). C and D show the mean ± SD from triplicate cultures. Similar results were obtained in three independent experiments.

    Techniques Used: Enzyme-linked Immunosorbent Assay, Cell Culture, Concentration Assay, Recombinant

    2) Product Images from "CfLec-3 from scallop: an entrance to non-self recognition mechanism of invertebrate C-type lectin"

    Article Title: CfLec-3 from scallop: an entrance to non-self recognition mechanism of invertebrate C-type lectin

    Journal: Scientific Reports

    doi: 10.1038/srep10068

    a , SDS-PAGE and Western-blot analysis of rCfLec-3. Lane 1: protein molecular standard; lane 2: negative control (without induction); lane 3: induced rCfLec-3; lane 4: purified rCfLec-3; lane 5: Western-blot of rCfLec-3 with anti-CfLec-3 antibody. b , Localization of endogenous CfLec-3 in different tissues by immunohistochemistry. CfLec-3 was detected by anti-CfLec-3 antibody, and stained in red. The tissues were counterstained with haematoxylin (blue). c-f , Temporal expression of CfLec-3 mRNA relative to β-actin was analyzed by realtime PCR in scallop hemocytes after LPS ( c ), PGN ( d ), β-glucan ( e ), poly I:C ( f ) and PBS challenge ( c, d, e, f ) for 3, 6, 12, 24 and 48 h. The values are shown as mean ± SE (N = 4), student’s t -test, error-bars represent biological repeats. (*: P
    Figure Legend Snippet: a , SDS-PAGE and Western-blot analysis of rCfLec-3. Lane 1: protein molecular standard; lane 2: negative control (without induction); lane 3: induced rCfLec-3; lane 4: purified rCfLec-3; lane 5: Western-blot of rCfLec-3 with anti-CfLec-3 antibody. b , Localization of endogenous CfLec-3 in different tissues by immunohistochemistry. CfLec-3 was detected by anti-CfLec-3 antibody, and stained in red. The tissues were counterstained with haematoxylin (blue). c-f , Temporal expression of CfLec-3 mRNA relative to β-actin was analyzed by realtime PCR in scallop hemocytes after LPS ( c ), PGN ( d ), β-glucan ( e ), poly I:C ( f ) and PBS challenge ( c, d, e, f ) for 3, 6, 12, 24 and 48 h. The values are shown as mean ± SE (N = 4), student’s t -test, error-bars represent biological repeats. (*: P

    Techniques Used: SDS Page, Western Blot, Negative Control, Purification, Immunohistochemistry, Staining, Expressing, Polymerase Chain Reaction

    3) Product Images from "DNA Synthesis Is Activated in Mosquitoes and Human Monocytes During the Induction of Innate Immune Memory"

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

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.02834

    (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
    Figure Legend 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

    Techniques Used: 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
    Figure Legend 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

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

    4) Product Images from "Effects of the linker region on the structure and function of modular GH5 cellulases"

    Article Title: Effects of the linker region on the structure and function of modular GH5 cellulases

    Journal: Scientific Reports

    doi: 10.1038/srep28504

    Effect of flexibility and linker extension on the kinetic behavior of BsCel5A. The initial hydrolysis rates, V 0 , were determined in triplicate from time-courses obtained at each substrate concentration for β-glucan ( A–C ) or CMC 4M ( D–F ). For purposes of comparison, the curve corresponding to the wt enzyme was included in all graphs. The results are representative of at least five independent experiments.
    Figure Legend Snippet: Effect of flexibility and linker extension on the kinetic behavior of BsCel5A. The initial hydrolysis rates, V 0 , were determined in triplicate from time-courses obtained at each substrate concentration for β-glucan ( A–C ) or CMC 4M ( D–F ). For purposes of comparison, the curve corresponding to the wt enzyme was included in all graphs. The results are representative of at least five independent experiments.

    Techniques Used: Concentration Assay

    5) Product Images from "Evaluation of trained immunity by β-1, 3 (d)-glucan on murine monocytes in vitro and duration of response in vivo"

    Article Title: Evaluation of trained immunity by β-1, 3 (d)-glucan on murine monocytes in vitro and duration of response in vivo

    Journal: Immunology and Cell Biology

    doi: 10.1038/icb.2017.13

    Increased survival of β-glucan-trained macrophages determines increased TNFα and IL-6 levels in vitro . ( a ) Total and ( b ) live cell number normalised amount of TNFα released into cell supernatants by human macrophages after challenge with 10 ng ml −1 of LPS. ( c ) Total and ( d ) normalised murine TNFα released into supernatants. ( e ) Total and ( f ) normalised IL-6 was measured in the same supernatants. * P -values ⩽0.05.
    Figure Legend Snippet: Increased survival of β-glucan-trained macrophages determines increased TNFα and IL-6 levels in vitro . ( a ) Total and ( b ) live cell number normalised amount of TNFα released into cell supernatants by human macrophages after challenge with 10 ng ml −1 of LPS. ( c ) Total and ( d ) normalised murine TNFα released into supernatants. ( e ) Total and ( f ) normalised IL-6 was measured in the same supernatants. * P -values ⩽0.05.

    Techniques Used: In Vitro

    β-Glucan-trained macrophages express relatively low levels of phagocytic cell surface markers. Undifferentiated monocytes (Mo, day 0) or differentiated macrophages (day 7) were collected and stained with antibodies against CD11b, F4/80, MHC II and CD11c and subsequently assessed by flow cytometry. ( a ) Representative histogram plots showing the expression of the different cell surface markers in undifferentiated Mo (day 0) or day 7 differentiated macrophages initially primed with β-glucan (black), LPS (grey) or RPMI (white, control). ( b ) Percentage of positive cells for each of the four markers evaluated in monocytes and differentiated macrophages. Spleen monocytes from five mice were analysed in independent experiments for all makers. Bars and error bars represent average and s.e.m. values, respectively. * and ** P -values ⩽0.05 and 0.01, respectively.
    Figure Legend Snippet: β-Glucan-trained macrophages express relatively low levels of phagocytic cell surface markers. Undifferentiated monocytes (Mo, day 0) or differentiated macrophages (day 7) were collected and stained with antibodies against CD11b, F4/80, MHC II and CD11c and subsequently assessed by flow cytometry. ( a ) Representative histogram plots showing the expression of the different cell surface markers in undifferentiated Mo (day 0) or day 7 differentiated macrophages initially primed with β-glucan (black), LPS (grey) or RPMI (white, control). ( b ) Percentage of positive cells for each of the four markers evaluated in monocytes and differentiated macrophages. Spleen monocytes from five mice were analysed in independent experiments for all makers. Bars and error bars represent average and s.e.m. values, respectively. * and ** P -values ⩽0.05 and 0.01, respectively.

    Techniques Used: Staining, Flow Cytometry, Cytometry, Expressing, Mouse Assay

    β-Glucan facilitates the survival of differentiating monocytes in vitro . ( a ) Fold change in live cells (normalised against control cells) after priming, differentiation and LPS challenge (day 7) in the murine in vitro assays ( n =6), and ( b ) healthy volunteers ( n =8) on days 1 and 6 under the same culture conditions and treatments. ( c ) Dot plots representing the apoptotic status of murine monocytes on day 0 (untreated) and treated monocytes on day 1. Cells were stained with FITC-conjugated AnnV and PI. Late apoptotic and/or necrotic cells are gated in the top right quadrant of each of the dot plots (AnnV + PI + ). ( d ) Combined AnnV + PI + data, ( e ) AnnV − PI − data and ( f ) AnnV + PI − data obtained from three independent experiments at different time points during the differentiation assays. Bars and error bars represent averages and s.e.m., respectively. * and ** P -values ⩽0.05 and 0.01, respectively.
    Figure Legend Snippet: β-Glucan facilitates the survival of differentiating monocytes in vitro . ( a ) Fold change in live cells (normalised against control cells) after priming, differentiation and LPS challenge (day 7) in the murine in vitro assays ( n =6), and ( b ) healthy volunteers ( n =8) on days 1 and 6 under the same culture conditions and treatments. ( c ) Dot plots representing the apoptotic status of murine monocytes on day 0 (untreated) and treated monocytes on day 1. Cells were stained with FITC-conjugated AnnV and PI. Late apoptotic and/or necrotic cells are gated in the top right quadrant of each of the dot plots (AnnV + PI + ). ( d ) Combined AnnV + PI + data, ( e ) AnnV − PI − data and ( f ) AnnV + PI − data obtained from three independent experiments at different time points during the differentiation assays. Bars and error bars represent averages and s.e.m., respectively. * and ** P -values ⩽0.05 and 0.01, respectively.

    Techniques Used: In Vitro, Staining

    In vitro differentiation of murine and human monocytes after the addition of serum to cell culture media. Murine (top panels) and human monocytes (bottom panels) were isolated, treated and cultured following the schedule indicated in the timeline diagram (bottom), with images captured at day 0 (untreated) and 5 days later after priming with LPS, β-glucan or RPMI. All pictures were taken using the same magnification objective (× 40), and are representative of six others.
    Figure Legend Snippet: In vitro differentiation of murine and human monocytes after the addition of serum to cell culture media. Murine (top panels) and human monocytes (bottom panels) were isolated, treated and cultured following the schedule indicated in the timeline diagram (bottom), with images captured at day 0 (untreated) and 5 days later after priming with LPS, β-glucan or RPMI. All pictures were taken using the same magnification objective (× 40), and are representative of six others.

    Techniques Used: In Vitro, Cell Culture, Isolation

    The enhanced in vivo response in β-glucan-trained mice declines in the first 3 weeks. ( a ) Timeline of the β-glucan dosage schedule in the murine in vivo experiments. ( b ) Serum levels of TNFα, IL-6 and IL-10 in β-glucan- or PBS-treated mice in the short-term experiment and ( c ) in the long-term experiment. At least nine mice per group and two independent experiments were performed in all cases. Horizontal and error bars represent average and s.e.m. values. * and *** P -values ⩽0.05 and 0.001, respectively.
    Figure Legend Snippet: The enhanced in vivo response in β-glucan-trained mice declines in the first 3 weeks. ( a ) Timeline of the β-glucan dosage schedule in the murine in vivo experiments. ( b ) Serum levels of TNFα, IL-6 and IL-10 in β-glucan- or PBS-treated mice in the short-term experiment and ( c ) in the long-term experiment. At least nine mice per group and two independent experiments were performed in all cases. Horizontal and error bars represent average and s.e.m. values. * and *** P -values ⩽0.05 and 0.001, respectively.

    Techniques Used: In Vivo, Mouse Assay

    6) Product Images from "Impaired phagocytosis directs human monocyte activation in response to fungal derived β-glucan particles"

    Article Title: Impaired phagocytosis directs human monocyte activation in response to fungal derived β-glucan particles

    Journal: European journal of immunology

    doi: 10.1002/eji.201747224

    Effects of phagocytosis impairment on the response of human monocytes toward whole Candida albicans and the cell wall fraction composed of branched β-glucan. (A and B) Human monocytes were left untreated (ctrl) or stimulated with either heat-killed Candida albicans (HK-C.a., A) or heat-killed Saccharomyces cerevisiae (HK-S.c., B) in the presence (Cyt D) or in the absence (DMSO) of cytochalasin D. (C) Cells were stimulated with the Alkali-Soluble (AS) and Insoluble (AI) fractions, β-(1–6)-glucan and β-(1–3)/(1–6) glucan fractions from the cell wall of Saccharomyces cerevisiae after being treated with DMSO or Cyt D. (D) Data show the stimulation experiments of monocytes with two doses (1 or 10 μg/mL) of branched β-(1–3)-glucan isolated from C. albicans (β-glucan C.a.) or from S. cerevisiae (β-glucan S.c.) in the presence or in the absence of Cyt D. (E) Data show the stimulation experiments of monocytes with two doses (1 or 10 μg/mL) of insoluble linear β- (1–3)-glucan (curdlan) or of soluble branched β-(1–3)-glucan (laminarin) in the presence or in the absence of Cyt D. (F) The particulate branched β-(1–3)-glucan isolated from the cell wall of C. albicans was sonicated (Sonifier cell Disruptor B-30. Outputcontrol 4, Duty cycle 40%; 2 times 10 cycle with 5min on ice in between the two sets). Monocytes were then exposed to either sonicated or not-sonicated β–glucan in the presence or in the absence of Cyt D. (G) Cells pre-treated with DMSO or Cyt D were stimulated for 24 h with the indicated concentrations of β-glucan from C. albicans (β-glucan C.a.), HK-C.a., AS and AI fractions from S.c., or the combination of AS+AI (10+10 μg/mL). (H) Monocytes were pre-treated with either DMSO (control), or with cytochalasin D (Cyt D) or latrunculin B (Lat B) (actin polymerization inhibitors), or with jasplakinolide (Jsp, actin polymerization inducer), or with chlorpromazine (CLP, clathrin-mediated endocytic inhibitor) or the lysosomal inhibitor cloroquine (CLQ). Cells were then stimulated with either branched β-(1–3)-glucan isolated from C. albicans (β-glucan C.a.) or with heat-killed Candida albicans (HK-C.a.). For all the experiments, culture supernatants were collected after 24h and concentration of secreted TNF-α, IL-6, IL-1β and IL-10 was determined by ELISA. Data were reproducible for all the four cytokines tested even if histograms are not shown. Graphs show the mean ± SEM of at least three independent experiments. For (A, H), n = 8; for (B–G), n = 6; * p
    Figure Legend Snippet: Effects of phagocytosis impairment on the response of human monocytes toward whole Candida albicans and the cell wall fraction composed of branched β-glucan. (A and B) Human monocytes were left untreated (ctrl) or stimulated with either heat-killed Candida albicans (HK-C.a., A) or heat-killed Saccharomyces cerevisiae (HK-S.c., B) in the presence (Cyt D) or in the absence (DMSO) of cytochalasin D. (C) Cells were stimulated with the Alkali-Soluble (AS) and Insoluble (AI) fractions, β-(1–6)-glucan and β-(1–3)/(1–6) glucan fractions from the cell wall of Saccharomyces cerevisiae after being treated with DMSO or Cyt D. (D) Data show the stimulation experiments of monocytes with two doses (1 or 10 μg/mL) of branched β-(1–3)-glucan isolated from C. albicans (β-glucan C.a.) or from S. cerevisiae (β-glucan S.c.) in the presence or in the absence of Cyt D. (E) Data show the stimulation experiments of monocytes with two doses (1 or 10 μg/mL) of insoluble linear β- (1–3)-glucan (curdlan) or of soluble branched β-(1–3)-glucan (laminarin) in the presence or in the absence of Cyt D. (F) The particulate branched β-(1–3)-glucan isolated from the cell wall of C. albicans was sonicated (Sonifier cell Disruptor B-30. Outputcontrol 4, Duty cycle 40%; 2 times 10 cycle with 5min on ice in between the two sets). Monocytes were then exposed to either sonicated or not-sonicated β–glucan in the presence or in the absence of Cyt D. (G) Cells pre-treated with DMSO or Cyt D were stimulated for 24 h with the indicated concentrations of β-glucan from C. albicans (β-glucan C.a.), HK-C.a., AS and AI fractions from S.c., or the combination of AS+AI (10+10 μg/mL). (H) Monocytes were pre-treated with either DMSO (control), or with cytochalasin D (Cyt D) or latrunculin B (Lat B) (actin polymerization inhibitors), or with jasplakinolide (Jsp, actin polymerization inducer), or with chlorpromazine (CLP, clathrin-mediated endocytic inhibitor) or the lysosomal inhibitor cloroquine (CLQ). Cells were then stimulated with either branched β-(1–3)-glucan isolated from C. albicans (β-glucan C.a.) or with heat-killed Candida albicans (HK-C.a.). For all the experiments, culture supernatants were collected after 24h and concentration of secreted TNF-α, IL-6, IL-1β and IL-10 was determined by ELISA. Data were reproducible for all the four cytokines tested even if histograms are not shown. Graphs show the mean ± SEM of at least three independent experiments. For (A, H), n = 8; for (B–G), n = 6; * p

    Techniques Used: Isolation, Sonication, Concentration Assay, Enzyme-linked Immunosorbent Assay

    IκBα phosphorylation, IκBα degradation and p65 nuclear translocation in cytochalasin D/β-glucan stimulated monocytes. (A) Western blot analysis of phospho-IκBα and IκBα in cell lysates from monocytes treated for 6h with or without β–glucan C.a. in the presence or absence of Cyt D. GAPDH was used as a loading control. (B) Graphs represent the densitometric analysis of the blots. Immunoreactive bands were normalized to GAPDH. The bars represent mean values ± SEM of the analysis of 4 independent experiments. (C) Cells were treated as in (A) and nuclear translocation of p65 NF-κB was visualized by immunostaining with a specific antibody recognizing p65 and an alexa-fluor 488-conjugated secondary antibody (green). For reference the nuclei are stained Hoescht (blue). One representative image of three independent experiments is shown. (D) Quantification of nuclei with p65 translocation (3 × 200 nuclei analysed with Image J). Original magnification. 60×, scale bar: 50 μM. * p
    Figure Legend Snippet: IκBα phosphorylation, IκBα degradation and p65 nuclear translocation in cytochalasin D/β-glucan stimulated monocytes. (A) Western blot analysis of phospho-IκBα and IκBα in cell lysates from monocytes treated for 6h with or without β–glucan C.a. in the presence or absence of Cyt D. GAPDH was used as a loading control. (B) Graphs represent the densitometric analysis of the blots. Immunoreactive bands were normalized to GAPDH. The bars represent mean values ± SEM of the analysis of 4 independent experiments. (C) Cells were treated as in (A) and nuclear translocation of p65 NF-κB was visualized by immunostaining with a specific antibody recognizing p65 and an alexa-fluor 488-conjugated secondary antibody (green). For reference the nuclei are stained Hoescht (blue). One representative image of three independent experiments is shown. (D) Quantification of nuclei with p65 translocation (3 × 200 nuclei analysed with Image J). Original magnification. 60×, scale bar: 50 μM. * p

    Techniques Used: Translocation Assay, Western Blot, Immunostaining, Staining

    Analysis of ROS production in human monocytes with impaired phagocytosis mechanism upon β-glucan stimulation. Monocytes were pre-treated for 30 min with (A) the indicated concentrations of DPI (NADPH oxidase inhibitor) before being treated with either DMSO or Cyt D for 1h. Cells were then left untreated or stimulated with particulate β–glucan C.a. Luminol was added, and Reactive oxygen species (ROS) production was monitored by chemiluminescence during time (2h) and expressed as relative light units (RLU). Graphs show mean ± SEM corresponding to up to three independent experiments ( n = 3); each experimental condition was assessed in triplicate. (B) Monocytes were pre-treated with DPI, as indicated. After 30 min of incubation, cells were treated either with DMSO or Cyt D and, after an additional 1h, stimulated with β-glucan C.a. Culture supernatants were collected after 24h and secretion of TNF-α, IL-6 and IL-1β was determined by ELISA. Graphs show the mean + SEM of at least three independent experiments ( n = 8); ** p
    Figure Legend Snippet: Analysis of ROS production in human monocytes with impaired phagocytosis mechanism upon β-glucan stimulation. Monocytes were pre-treated for 30 min with (A) the indicated concentrations of DPI (NADPH oxidase inhibitor) before being treated with either DMSO or Cyt D for 1h. Cells were then left untreated or stimulated with particulate β–glucan C.a. Luminol was added, and Reactive oxygen species (ROS) production was monitored by chemiluminescence during time (2h) and expressed as relative light units (RLU). Graphs show mean ± SEM corresponding to up to three independent experiments ( n = 3); each experimental condition was assessed in triplicate. (B) Monocytes were pre-treated with DPI, as indicated. After 30 min of incubation, cells were treated either with DMSO or Cyt D and, after an additional 1h, stimulated with β-glucan C.a. Culture supernatants were collected after 24h and secretion of TNF-α, IL-6 and IL-1β was determined by ELISA. Graphs show the mean + SEM of at least three independent experiments ( n = 8); ** p

    Techniques Used: Incubation, Enzyme-linked Immunosorbent Assay

    The role of the Dectin-1/Syk/PI3K pathway in the overproduction of cytokines by monocytes impaired for phagocytosis and stimulated with β-glucan. Monocytes were pre-treated with anti-Dectin-1 Ab, anti-TLR2 Ab, anti-CR3 Ab or isotype control Abs in (A). Syk inhibitor in (B), or PI3K inhibitor (WRT; wortmannin) in (C), were used on monocytes as indicated. After the first incubation with inhibitors, cells were treated either with DMSO or Cyt D and finally stimulated with branched β-(1–3)-glucan isolated from C. albicans (β-glucan C.a.). Culture supernatants were collected after 24h and secretion of TNF-α, IL-6, and IL-1β was determined by ELISA. Graphs show the mean ± SEM of at least three independent experiments. For (A), n = 6; for (B) and (C), n = 9; * p
    Figure Legend Snippet: The role of the Dectin-1/Syk/PI3K pathway in the overproduction of cytokines by monocytes impaired for phagocytosis and stimulated with β-glucan. Monocytes were pre-treated with anti-Dectin-1 Ab, anti-TLR2 Ab, anti-CR3 Ab or isotype control Abs in (A). Syk inhibitor in (B), or PI3K inhibitor (WRT; wortmannin) in (C), were used on monocytes as indicated. After the first incubation with inhibitors, cells were treated either with DMSO or Cyt D and finally stimulated with branched β-(1–3)-glucan isolated from C. albicans (β-glucan C.a.). Culture supernatants were collected after 24h and secretion of TNF-α, IL-6, and IL-1β was determined by ELISA. Graphs show the mean ± SEM of at least three independent experiments. For (A), n = 6; for (B) and (C), n = 9; * p

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

    The role of the NLRP3-ASC-caspase-1 inflammasome in β-glucan induced IL-1β. (A and E) Monocytes were pre-treated with either z-VAD-fmk (pan-caspase inhibitor), z-YVAD-fmk (caspase-1 inhibitor), z-IETD-fmk (caspase-8 inhibitor), z-LEVD-fmk (caspase-4/5 inhibitor), MCC950 (NLRP3 inhibitor), glibenclamide (inhibitor of potassium (K+) efflux) or z-VRPR-fmk (MALT1 inhibitor) before treatment with either DMSO or Cyt D for 1 h. Cells were then stimulated with branched β-(1–3)-glucan isolated from C. albicans (β-glucan C.a.), culture supernatants were collected after 24 h and secretion of IL-1β was determined by ELISA. Graphs show the mean ± SEM of four independent experiments ( n = 8). ** p
    Figure Legend Snippet: The role of the NLRP3-ASC-caspase-1 inflammasome in β-glucan induced IL-1β. (A and E) Monocytes were pre-treated with either z-VAD-fmk (pan-caspase inhibitor), z-YVAD-fmk (caspase-1 inhibitor), z-IETD-fmk (caspase-8 inhibitor), z-LEVD-fmk (caspase-4/5 inhibitor), MCC950 (NLRP3 inhibitor), glibenclamide (inhibitor of potassium (K+) efflux) or z-VRPR-fmk (MALT1 inhibitor) before treatment with either DMSO or Cyt D for 1 h. Cells were then stimulated with branched β-(1–3)-glucan isolated from C. albicans (β-glucan C.a.), culture supernatants were collected after 24 h and secretion of IL-1β was determined by ELISA. Graphs show the mean ± SEM of four independent experiments ( n = 8). ** p

    Techniques Used: Isolation, Enzyme-linked Immunosorbent Assay

    Schematic model illustrating potential signaling pathways triggered by the binding of β–glucan to dectin-1 in human monocytes with normal (left) or impaired phagocytic (right) function. Attenuation of dectin-1 signaling is associated with the engulfment of the receptor/ligand complex. By contrast, blocking of actin-dependent phagocytosis led to increased cell activation. Black arrows depict the interactions that normally occur upon phagocytosis and β–glucan/dectin-1 interaction, whereas red arrows indicate the proposed signaling events that enhance the strength and efficacy of signal propagation.
    Figure Legend Snippet: Schematic model illustrating potential signaling pathways triggered by the binding of β–glucan to dectin-1 in human monocytes with normal (left) or impaired phagocytic (right) function. Attenuation of dectin-1 signaling is associated with the engulfment of the receptor/ligand complex. By contrast, blocking of actin-dependent phagocytosis led to increased cell activation. Black arrows depict the interactions that normally occur upon phagocytosis and β–glucan/dectin-1 interaction, whereas red arrows indicate the proposed signaling events that enhance the strength and efficacy of signal propagation.

    Techniques Used: Binding Assay, Blocking Assay, Activation Assay

    Syk activity assessement for inflammasome, and NF-κB activation in phagocytosis-impaired-monocyte stimulated with C. albicans branched β-(1–3)-glucan. (A) Monocytes treated for 6 h with β–glucan C.a. in the presence or absence of Cyt D and of the Syk inhibitor (2 μM). Nuclear translocation of p65 NF-κB was visualized by immunostaining with a specific antibody recognizing p65 and a alexa-fluor 488-conjugated secondary antibody (green). For reference the nuclei are stained Hoescht (blue). Original magnification. 60×, scalebar: 50 μMImage is representative of three independent experiments. (B) Quantification of nuclei with p65 translocation (4 × 50 nuclei analysed with Image J). (C) Cells were treated as in (A). Caspase-1 (pro-form and cleaved p20), and IL-1β (pro- and mature IL-1β) expressions were assessed by western blot. Mature IL-1β and caspase-1 were also detected in cell culture supernatants. GAPDH was used as the loading control. Blots are representative of three independent experiments.
    Figure Legend Snippet: Syk activity assessement for inflammasome, and NF-κB activation in phagocytosis-impaired-monocyte stimulated with C. albicans branched β-(1–3)-glucan. (A) Monocytes treated for 6 h with β–glucan C.a. in the presence or absence of Cyt D and of the Syk inhibitor (2 μM). Nuclear translocation of p65 NF-κB was visualized by immunostaining with a specific antibody recognizing p65 and a alexa-fluor 488-conjugated secondary antibody (green). For reference the nuclei are stained Hoescht (blue). Original magnification. 60×, scalebar: 50 μMImage is representative of three independent experiments. (B) Quantification of nuclei with p65 translocation (4 × 50 nuclei analysed with Image J). (C) Cells were treated as in (A). Caspase-1 (pro-form and cleaved p20), and IL-1β (pro- and mature IL-1β) expressions were assessed by western blot. Mature IL-1β and caspase-1 were also detected in cell culture supernatants. GAPDH was used as the loading control. Blots are representative of three independent experiments.

    Techniques Used: Activity Assay, Activation Assay, Translocation Assay, Immunostaining, Staining, Western Blot, Cell Culture

    7) Product Images from "Antibody response in silver catfish (Rhamdia quelen) immunized with a model antigen associated with different adjuvants"

    Article Title: Antibody response in silver catfish (Rhamdia quelen) immunized with a model antigen associated with different adjuvants

    Journal: Brazilian Journal of Medical and Biological Research

    doi: 10.1590/1414-431X20165281

    Serum antibody response of fish immunized with bovine serum albumin (BSA) combined with different adjuvants. A , Silver catfish were intraperitoneally injected with BSA alone (BSA+PBS, 200 µg/fish) or BSA adjuvanted with aluminum hydroxide (AlOH), Freund’s complete adjuvant (FCA), Freund’s incomplete adjuvant (FIA) and Montanide; B , BSA alone or combined with CpG 1668, CpG 2102, CpG 2133 and CpG 2143; C , BSA alone or combined with Montanide, and β-glucan (0.02; 0.06 and 0.1%). Serum samples were collected prior to (day 0) or at 14, 28, and 42 days post-inoculation to evaluate anti-BSA antibodies by ELISA. Significant differences (P
    Figure Legend Snippet: Serum antibody response of fish immunized with bovine serum albumin (BSA) combined with different adjuvants. A , Silver catfish were intraperitoneally injected with BSA alone (BSA+PBS, 200 µg/fish) or BSA adjuvanted with aluminum hydroxide (AlOH), Freund’s complete adjuvant (FCA), Freund’s incomplete adjuvant (FIA) and Montanide; B , BSA alone or combined with CpG 1668, CpG 2102, CpG 2133 and CpG 2143; C , BSA alone or combined with Montanide, and β-glucan (0.02; 0.06 and 0.1%). Serum samples were collected prior to (day 0) or at 14, 28, and 42 days post-inoculation to evaluate anti-BSA antibodies by ELISA. Significant differences (P

    Techniques Used: Fluorescence In Situ Hybridization, Injection, Enzyme-linked Immunosorbent Assay

    8) Product Images from "Direct and Intestinal Epithelial Cell-Mediated Effects of TLR8 Triggering on Human Dendritic Cells, CD14+CD16+ Monocytes and γδ T Lymphocytes"

    Article Title: Direct and Intestinal Epithelial Cell-Mediated Effects of TLR8 Triggering on Human Dendritic Cells, CD14+CD16+ Monocytes and γδ T Lymphocytes

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2017.01813

    Effects of R848-exposed intestinal epithelial cell (IEC) monolayer on dendritic cell (DC) differentiation. Peripheral blood monocytes were induced to differentiate toward DC in standard medium or in conditioned medium (CM) from Caco-2 cell-derived IEC monolayer, left untreated or stimulated with R848 (A–C) or β-glucan (A,B) . At day 5, cells were harvested and analyzed for the expression of the indicated surface markers by flow cytometry. One representative experiment out of 4 is reported in panels (A,C) . Numbers in quadrants indicate the percentages of positive cells. The percentage of CD14 + cells is reported in panel (B) , mean values ± SD from 10 independent experiments are shown. *** p
    Figure Legend Snippet: Effects of R848-exposed intestinal epithelial cell (IEC) monolayer on dendritic cell (DC) differentiation. Peripheral blood monocytes were induced to differentiate toward DC in standard medium or in conditioned medium (CM) from Caco-2 cell-derived IEC monolayer, left untreated or stimulated with R848 (A–C) or β-glucan (A,B) . At day 5, cells were harvested and analyzed for the expression of the indicated surface markers by flow cytometry. One representative experiment out of 4 is reported in panels (A,C) . Numbers in quadrants indicate the percentages of positive cells. The percentage of CD14 + cells is reported in panel (B) , mean values ± SD from 10 independent experiments are shown. *** p

    Techniques Used: Derivative Assay, Expressing, Flow Cytometry, Cytometry

    9) Product Images from "A Scallop Nitric Oxide Synthase (NOS) with Structure Similar to Neuronal NOS and Its Involvement in the Immune Defense"

    Article Title: A Scallop Nitric Oxide Synthase (NOS) with Structure Similar to Neuronal NOS and Its Involvement in the Immune Defense

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0069158

    Temporal expression of CfNOS mRNA in scallop haemocytes after LPS, PGN, β-glucan and PBS challenge. The samples were collected after the treatments for 3, 6, 12, 24 and 48 h. Data was expressed as the ratio of the CfNOS mRNA to the β-actin mRNA. The scallops injected with PBS were used as the control group. The value was shown as mean ± SD (N = 6) and bars with different letters were significantly different ( P
    Figure Legend Snippet: Temporal expression of CfNOS mRNA in scallop haemocytes after LPS, PGN, β-glucan and PBS challenge. The samples were collected after the treatments for 3, 6, 12, 24 and 48 h. Data was expressed as the ratio of the CfNOS mRNA to the β-actin mRNA. The scallops injected with PBS were used as the control group. The value was shown as mean ± SD (N = 6) and bars with different letters were significantly different ( P

    Techniques Used: Expressing, Injection

    10) Product Images from "Identifying different types of microorganisms with terahertz spectroscopy"

    Article Title: Identifying different types of microorganisms with terahertz spectroscopy

    Journal: Biomedical Optics Express

    doi: 10.1364/BOE.376584

    (a) Schematic images of molecular structures for peptidoglycan, chitin, α -glucan, and β -glucans. (b) Real (filled boxes) and imaginary (open boxes) parts of dielectric constants for peptidoglycan and polysaccharides films, measured at 1 THz.
    Figure Legend Snippet: (a) Schematic images of molecular structures for peptidoglycan, chitin, α -glucan, and β -glucans. (b) Real (filled boxes) and imaginary (open boxes) parts of dielectric constants for peptidoglycan and polysaccharides films, measured at 1 THz.

    Techniques Used:

    11) Product Images from "Identifying different types of microorganisms with terahertz spectroscopy"

    Article Title: Identifying different types of microorganisms with terahertz spectroscopy

    Journal: Biomedical Optics Express

    doi: 10.1364/BOE.376584

    (a) Schematic images of molecular structures for peptidoglycan, chitin, α -glucan, and β -glucans. (b) Real (filled boxes) and imaginary (open boxes) parts of dielectric constants for peptidoglycan and polysaccharides films, measured at 1 THz.
    Figure Legend Snippet: (a) Schematic images of molecular structures for peptidoglycan, chitin, α -glucan, and β -glucans. (b) Real (filled boxes) and imaginary (open boxes) parts of dielectric constants for peptidoglycan and polysaccharides films, measured at 1 THz.

    Techniques Used:

    12) Product Images from "Oral microbe-host interactions: influence of β-glucans on gene expression of inflammatory cytokines and metabolome profile"

    Article Title: Oral microbe-host interactions: influence of β-glucans on gene expression of inflammatory cytokines and metabolome profile

    Journal: BMC Microbiology

    doi: 10.1186/s12866-017-0946-1

    Cytotoxicity assay of cells treated with different doses of β-glucan: ( a ) OBA-9 cell (keratinocytes); ( b ) HGF-1 cell (fibroblasts). Cell viability was presented in percentage (%). n = 6
    Figure Legend Snippet: Cytotoxicity assay of cells treated with different doses of β-glucan: ( a ) OBA-9 cell (keratinocytes); ( b ) HGF-1 cell (fibroblasts). Cell viability was presented in percentage (%). n = 6

    Techniques Used: Cytotoxicity Assay

    Relative expression of genes on OBA-9 cells (keratinocytes) by quantitative PCR: ( a ) interleukin 1 alpha (IL-1-α); ( b ) interleukin 18 (IL-18); ( c ) B-cell lymphoma 2 (BCL-2); ( d ) adenovirus E1A-associated 300 kDa protein (EP 300); ( e ) prostaglandin-endoperoxide synthase 2 (PTGS-2). Dual-chamber model inoculated with A. actinomicetemcomitans and treated with different doses of β-glucan. The control group has their mean expressed equal to 1 and treated groups have their mean relative to the control group. The results were expressed by mean followed standard deviation; n = 6 and P
    Figure Legend Snippet: Relative expression of genes on OBA-9 cells (keratinocytes) by quantitative PCR: ( a ) interleukin 1 alpha (IL-1-α); ( b ) interleukin 18 (IL-18); ( c ) B-cell lymphoma 2 (BCL-2); ( d ) adenovirus E1A-associated 300 kDa protein (EP 300); ( e ) prostaglandin-endoperoxide synthase 2 (PTGS-2). Dual-chamber model inoculated with A. actinomicetemcomitans and treated with different doses of β-glucan. The control group has their mean expressed equal to 1 and treated groups have their mean relative to the control group. The results were expressed by mean followed standard deviation; n = 6 and P

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Standard Deviation

    Relative expression of genes on HGF-1 cells (fibroblasts) by quantitative PCR: ( a ) interleukin 1 alpha (IL-1-α); ( b ) interleukin 18 (IL-18); ( c ) B-cell lymphoma 2 (BCL-2); ( d ) adenovirus E1A-associated 300 kDa protein (EP 300); ( e ) prostaglandin-endoperoxide synthase 2 (PTGS-2). Dual-chamber model inoculated with A. actinomicetemcomitans and treated with different doses of β-glucan. The control group has their mean expressed equal to 1 and treated groups have their mean relative to the control group. The results were expressed by mean followed standard deviation; n = 6 and P
    Figure Legend Snippet: Relative expression of genes on HGF-1 cells (fibroblasts) by quantitative PCR: ( a ) interleukin 1 alpha (IL-1-α); ( b ) interleukin 18 (IL-18); ( c ) B-cell lymphoma 2 (BCL-2); ( d ) adenovirus E1A-associated 300 kDa protein (EP 300); ( e ) prostaglandin-endoperoxide synthase 2 (PTGS-2). Dual-chamber model inoculated with A. actinomicetemcomitans and treated with different doses of β-glucan. The control group has their mean expressed equal to 1 and treated groups have their mean relative to the control group. The results were expressed by mean followed standard deviation; n = 6 and P

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Standard Deviation

    Dual-chamber model containing OBA-9 cell (keratinocytes) in the top layer (transwell insert) and HGF-1 cell (fibroblasts) in the bottom layer inoculated with A. actinomicetemcomitans and treated with different doses of β-glucan
    Figure Legend Snippet: Dual-chamber model containing OBA-9 cell (keratinocytes) in the top layer (transwell insert) and HGF-1 cell (fibroblasts) in the bottom layer inoculated with A. actinomicetemcomitans and treated with different doses of β-glucan

    Techniques Used:

    Metabolites obtained of cell culture supernatant (HGF-1 and OBA-9 co-culture cells). ( a ) 4-aminobutyric acid; ( b ) 2-deoxytetronic acid NIST; ( c ) acetophenone NIST; ( d ) benzoic acid; ( e ) oxalic acid; ( f ) pinitol. Dual-chamber model inoculated with A. actinomicetemcomitans and treated with different doses of β-glucan. The data is expressed in relative peak heights (mAU) from HPLC-MS analysis, which are unit-less (mean followed standard deviation); n = 4 and P
    Figure Legend Snippet: Metabolites obtained of cell culture supernatant (HGF-1 and OBA-9 co-culture cells). ( a ) 4-aminobutyric acid; ( b ) 2-deoxytetronic acid NIST; ( c ) acetophenone NIST; ( d ) benzoic acid; ( e ) oxalic acid; ( f ) pinitol. Dual-chamber model inoculated with A. actinomicetemcomitans and treated with different doses of β-glucan. The data is expressed in relative peak heights (mAU) from HPLC-MS analysis, which are unit-less (mean followed standard deviation); n = 4 and P

    Techniques Used: Cell Culture, Co-Culture Assay, High Performance Liquid Chromatography, Mass Spectrometry, Standard Deviation

    13) Product Images from "Fungal β-glucan, a Dectin-1 ligand, promotes protection from Type 1 Diabetes by inducing regulatory innate immune response1"

    Article Title: Fungal β-glucan, a Dectin-1 ligand, promotes protection from Type 1 Diabetes by inducing regulatory innate immune response1

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

    doi: 10.4049/jimmunol.1400186

    Innate immune response induced using β-glucan, but not Pam3Cys or LPS, produced significant delay in hyperglycemia A) 10-week-old pre-diabetic female NOD mice were injected with β-glucan (5 μg/mouse/day), zymosan (5 μg/mouse/day), Pam3Cys (1.0 μg/mouse/day) and bacterial LPS (0.5 μg/mouse/day); for three alternate days or left untreated (control). Mice were checked every week for hyperglycemia for up to 15 weeks and mice with glucose level of 250 mg/dl for two consecutive weeks were considered diabetic. Log-rank test was performed to compare the hyperglycemia incidence in treated and control groups of mice and the statistically significant p -value is shown.
    Figure Legend Snippet: Innate immune response induced using β-glucan, but not Pam3Cys or LPS, produced significant delay in hyperglycemia A) 10-week-old pre-diabetic female NOD mice were injected with β-glucan (5 μg/mouse/day), zymosan (5 μg/mouse/day), Pam3Cys (1.0 μg/mouse/day) and bacterial LPS (0.5 μg/mouse/day); for three alternate days or left untreated (control). Mice were checked every week for hyperglycemia for up to 15 weeks and mice with glucose level of 250 mg/dl for two consecutive weeks were considered diabetic. Log-rank test was performed to compare the hyperglycemia incidence in treated and control groups of mice and the statistically significant p -value is shown.

    Techniques Used: Produced, Mouse Assay, Injection

    Fungal β-glucan induced regulatory innate immune response in DCs is Dectin-1 dependent A) DCs were generated in vitro from BM cells (BMDCs) of WT and Dectin-KO B6 mice using GM-CSF and IL-4 and left untreated or exposed to indicated agents for 48h, and cytokine levels were measured in the supernatants by ELISA. Mean±SD of values from 2 individual representative experiments carried out in triplicate are shown. Statistical significance of treated group was calculated against untreated (none) group. *, p
    Figure Legend Snippet: Fungal β-glucan induced regulatory innate immune response in DCs is Dectin-1 dependent A) DCs were generated in vitro from BM cells (BMDCs) of WT and Dectin-KO B6 mice using GM-CSF and IL-4 and left untreated or exposed to indicated agents for 48h, and cytokine levels were measured in the supernatants by ELISA. Mean±SD of values from 2 individual representative experiments carried out in triplicate are shown. Statistical significance of treated group was calculated against untreated (none) group. *, p

    Techniques Used: Generated, In Vitro, Mouse Assay, Enzyme-linked Immunosorbent Assay

    Activation of innate immune response using β-glucan prevents hyperglycemia in NOD mice A) Twelve-week old pre-diabetic female NOD mice were treated i.p. with β-glucan (5 μg/mouse/day; every other day for 30 days; total of 15 injections) or left untreated (control). Mice were checked every week for hyperglycemia and glucose level of 250 mg/dl for two consecutive weeks was considered diabetic. Log-rank test was performed to compare the hyperglycemia incidence in treated and control groups of mice and the p -value is shown. B) Euglycemic β-glucan-treated and control mice from parallel experiments were euthanized 30 days after the last injection. Pancreatic tissues were processed for H E staining to evaluate insulitis. Representative islets (left panel) and percentages of islets with different grades of insulitis plotted as bar diagram (right panel) are shown. Sections of pancreatic tissues from at least 5 mice/group were examined for insulitis and at least 180 islets/group were examined.
    Figure Legend Snippet: Activation of innate immune response using β-glucan prevents hyperglycemia in NOD mice A) Twelve-week old pre-diabetic female NOD mice were treated i.p. with β-glucan (5 μg/mouse/day; every other day for 30 days; total of 15 injections) or left untreated (control). Mice were checked every week for hyperglycemia and glucose level of 250 mg/dl for two consecutive weeks was considered diabetic. Log-rank test was performed to compare the hyperglycemia incidence in treated and control groups of mice and the p -value is shown. B) Euglycemic β-glucan-treated and control mice from parallel experiments were euthanized 30 days after the last injection. Pancreatic tissues were processed for H E staining to evaluate insulitis. Representative islets (left panel) and percentages of islets with different grades of insulitis plotted as bar diagram (right panel) are shown. Sections of pancreatic tissues from at least 5 mice/group were examined for insulitis and at least 180 islets/group were examined.

    Techniques Used: Activation Assay, Mouse Assay, Injection, Staining

    Fungal β-glucan induces regulatory innate immune response in DCs A) DCs were generated in vitro from BM cells (BMDCs) using GM-CSF and IL-4 and left untreated or exposed to indicated agents for 48h, and cytokine levels were measured in the supernatants by ELISA. Mean±SD of values from 2 individual representative experiments carried out in triplicate are shown. B) Non-activated (to detect active form) and activated (to detect total amount) culture supernatants were examined for TGF-β1 levels ELISA (upper panel) and TGF-β1 activity by bioassay using F-11 reporter cells (lower panel). TGF-β1 activity of control (none) sample was considered as 1 for calculating the fold activity. C) BMDC and splenic CD11c+ cells were exposed to indicated agents as described above, cDNA was prepared using cells collected from 18h cultures and were subjected to qualitative PCR using IDO and housekeeping gene, actin specific primer sets. Gel images from representative experiments are shown. D) cDNA prepared from splenic and BM DCs were also used in real-time qPCR for detecting IDO and actin. Expression levels were calculated relative to actin expression and the values of ligand-treated samples were compared against the value of untreated (none) sample which was considered as 1. Mean±SD values of 2 independent assays are shown. Statistical significance of treated group was calculated against untreated (none) group. *, p
    Figure Legend Snippet: Fungal β-glucan induces regulatory innate immune response in DCs A) DCs were generated in vitro from BM cells (BMDCs) using GM-CSF and IL-4 and left untreated or exposed to indicated agents for 48h, and cytokine levels were measured in the supernatants by ELISA. Mean±SD of values from 2 individual representative experiments carried out in triplicate are shown. B) Non-activated (to detect active form) and activated (to detect total amount) culture supernatants were examined for TGF-β1 levels ELISA (upper panel) and TGF-β1 activity by bioassay using F-11 reporter cells (lower panel). TGF-β1 activity of control (none) sample was considered as 1 for calculating the fold activity. C) BMDC and splenic CD11c+ cells were exposed to indicated agents as described above, cDNA was prepared using cells collected from 18h cultures and were subjected to qualitative PCR using IDO and housekeeping gene, actin specific primer sets. Gel images from representative experiments are shown. D) cDNA prepared from splenic and BM DCs were also used in real-time qPCR for detecting IDO and actin. Expression levels were calculated relative to actin expression and the values of ligand-treated samples were compared against the value of untreated (none) sample which was considered as 1. Mean±SD values of 2 independent assays are shown. Statistical significance of treated group was calculated against untreated (none) group. *, p

    Techniques Used: Generated, In Vitro, Enzyme-linked Immunosorbent Assay, Activity Assay, Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Expressing

    14) Product Images from "Impaired phagocytosis directs human monocyte activation in response to fungal derived β-glucan particles"

    Article Title: Impaired phagocytosis directs human monocyte activation in response to fungal derived β-glucan particles

    Journal: European journal of immunology

    doi: 10.1002/eji.201747224

    Effects of phagocytosis impairment on the response of human monocytes toward whole Candida albicans and the cell wall fraction composed of branched β-glucan. (A and B) Human monocytes were left untreated (ctrl) or stimulated with either heat-killed Candida albicans (HK-C.a., A) or heat-killed Saccharomyces cerevisiae (HK-S.c., B) in the presence (Cyt D) or in the absence (DMSO) of cytochalasin D. (C) Cells were stimulated with the Alkali-Soluble (AS) and Insoluble (AI) fractions, β-(1–6)-glucan and β-(1–3)/(1–6) glucan fractions from the cell wall of Saccharomyces cerevisiae after being treated with DMSO or Cyt D. (D) Data show the stimulation experiments of monocytes with two doses (1 or 10 μg/mL) of branched β-(1–3)-glucan isolated from C. albicans (β-glucan C.a.) or from S. cerevisiae (β-glucan S.c.) in the presence or in the absence of Cyt D. (E) Data show the stimulation experiments of monocytes with two doses (1 or 10 μg/mL) of insoluble linear β- (1–3)-glucan (curdlan) or of soluble branched β-(1–3)-glucan (laminarin) in the presence or in the absence of Cyt D. (F) The particulate branched β-(1–3)-glucan isolated from the cell wall of C. albicans was sonicated (Sonifier cell Disruptor B-30. Outputcontrol 4, Duty cycle 40%; 2 times 10 cycle with 5min on ice in between the two sets). Monocytes were then exposed to either sonicated or not-sonicated β–glucan in the presence or in the absence of Cyt D. (G) Cells pre-treated with DMSO or Cyt D were stimulated for 24 h with the indicated concentrations of β-glucan from C. albicans (β-glucan C.a.), HK-C.a., AS and AI fractions from S.c., or the combination of AS+AI (10+10 μg/mL). (H) Monocytes were pre-treated with either DMSO (control), or with cytochalasin D (Cyt D) or latrunculin B (Lat B) (actin polymerization inhibitors), or with jasplakinolide (Jsp, actin polymerization inducer), or with chlorpromazine (CLP, clathrin-mediated endocytic inhibitor) or the lysosomal inhibitor cloroquine (CLQ). Cells were then stimulated with either branched β-(1–3)-glucan isolated from C. albicans (β-glucan C.a.) or with heat-killed Candida albicans (HK-C.a.). For all the experiments, culture supernatants were collected after 24h and concentration of secreted TNF-α, IL-6, IL-1β and IL-10 was determined by ELISA. Data were reproducible for all the four cytokines tested even if histograms are not shown. Graphs show the mean ± SEM of at least three independent experiments. For (A, H), n = 8; for (B–G), n = 6; * p
    Figure Legend Snippet: Effects of phagocytosis impairment on the response of human monocytes toward whole Candida albicans and the cell wall fraction composed of branched β-glucan. (A and B) Human monocytes were left untreated (ctrl) or stimulated with either heat-killed Candida albicans (HK-C.a., A) or heat-killed Saccharomyces cerevisiae (HK-S.c., B) in the presence (Cyt D) or in the absence (DMSO) of cytochalasin D. (C) Cells were stimulated with the Alkali-Soluble (AS) and Insoluble (AI) fractions, β-(1–6)-glucan and β-(1–3)/(1–6) glucan fractions from the cell wall of Saccharomyces cerevisiae after being treated with DMSO or Cyt D. (D) Data show the stimulation experiments of monocytes with two doses (1 or 10 μg/mL) of branched β-(1–3)-glucan isolated from C. albicans (β-glucan C.a.) or from S. cerevisiae (β-glucan S.c.) in the presence or in the absence of Cyt D. (E) Data show the stimulation experiments of monocytes with two doses (1 or 10 μg/mL) of insoluble linear β- (1–3)-glucan (curdlan) or of soluble branched β-(1–3)-glucan (laminarin) in the presence or in the absence of Cyt D. (F) The particulate branched β-(1–3)-glucan isolated from the cell wall of C. albicans was sonicated (Sonifier cell Disruptor B-30. Outputcontrol 4, Duty cycle 40%; 2 times 10 cycle with 5min on ice in between the two sets). Monocytes were then exposed to either sonicated or not-sonicated β–glucan in the presence or in the absence of Cyt D. (G) Cells pre-treated with DMSO or Cyt D were stimulated for 24 h with the indicated concentrations of β-glucan from C. albicans (β-glucan C.a.), HK-C.a., AS and AI fractions from S.c., or the combination of AS+AI (10+10 μg/mL). (H) Monocytes were pre-treated with either DMSO (control), or with cytochalasin D (Cyt D) or latrunculin B (Lat B) (actin polymerization inhibitors), or with jasplakinolide (Jsp, actin polymerization inducer), or with chlorpromazine (CLP, clathrin-mediated endocytic inhibitor) or the lysosomal inhibitor cloroquine (CLQ). Cells were then stimulated with either branched β-(1–3)-glucan isolated from C. albicans (β-glucan C.a.) or with heat-killed Candida albicans (HK-C.a.). For all the experiments, culture supernatants were collected after 24h and concentration of secreted TNF-α, IL-6, IL-1β and IL-10 was determined by ELISA. Data were reproducible for all the four cytokines tested even if histograms are not shown. Graphs show the mean ± SEM of at least three independent experiments. For (A, H), n = 8; for (B–G), n = 6; * p

    Techniques Used: Isolation, Sonication, Concentration Assay, Enzyme-linked Immunosorbent Assay

    IκBα phosphorylation, IκBα degradation and p65 nuclear translocation in cytochalasin D/β-glucan stimulated monocytes. (A) Western blot analysis of phospho-IκBα and IκBα in cell lysates from monocytes treated for 6h with or without β–glucan C.a. in the presence or absence of Cyt D. GAPDH was used as a loading control. (B) Graphs represent the densitometric analysis of the blots. Immunoreactive bands were normalized to GAPDH. The bars represent mean values ± SEM of the analysis of 4 independent experiments. (C) Cells were treated as in (A) and nuclear translocation of p65 NF-κB was visualized by immunostaining with a specific antibody recognizing p65 and an alexa-fluor 488-conjugated secondary antibody (green). For reference the nuclei are stained Hoescht (blue). One representative image of three independent experiments is shown. (D) Quantification of nuclei with p65 translocation (3 × 200 nuclei analysed with Image J). Original magnification. 60×, scale bar: 50 μM. * p
    Figure Legend Snippet: IκBα phosphorylation, IκBα degradation and p65 nuclear translocation in cytochalasin D/β-glucan stimulated monocytes. (A) Western blot analysis of phospho-IκBα and IκBα in cell lysates from monocytes treated for 6h with or without β–glucan C.a. in the presence or absence of Cyt D. GAPDH was used as a loading control. (B) Graphs represent the densitometric analysis of the blots. Immunoreactive bands were normalized to GAPDH. The bars represent mean values ± SEM of the analysis of 4 independent experiments. (C) Cells were treated as in (A) and nuclear translocation of p65 NF-κB was visualized by immunostaining with a specific antibody recognizing p65 and an alexa-fluor 488-conjugated secondary antibody (green). For reference the nuclei are stained Hoescht (blue). One representative image of three independent experiments is shown. (D) Quantification of nuclei with p65 translocation (3 × 200 nuclei analysed with Image J). Original magnification. 60×, scale bar: 50 μM. * p

    Techniques Used: Translocation Assay, Western Blot, Immunostaining, Staining

    Analysis of ROS production in human monocytes with impaired phagocytosis mechanism upon β-glucan stimulation. Monocytes were pre-treated for 30 min with (A) the indicated concentrations of DPI (NADPH oxidase inhibitor) before being treated with either DMSO or Cyt D for 1h. Cells were then left untreated or stimulated with particulate β–glucan C.a. Luminol was added, and Reactive oxygen species (ROS) production was monitored by chemiluminescence during time (2h) and expressed as relative light units (RLU). Graphs show mean ± SEM corresponding to up to three independent experiments ( n = 3); each experimental condition was assessed in triplicate. (B) Monocytes were pre-treated with DPI, as indicated. After 30 min of incubation, cells were treated either with DMSO or Cyt D and, after an additional 1h, stimulated with β-glucan C.a. Culture supernatants were collected after 24h and secretion of TNF-α, IL-6 and IL-1β was determined by ELISA. Graphs show the mean + SEM of at least three independent experiments ( n = 8); ** p
    Figure Legend Snippet: Analysis of ROS production in human monocytes with impaired phagocytosis mechanism upon β-glucan stimulation. Monocytes were pre-treated for 30 min with (A) the indicated concentrations of DPI (NADPH oxidase inhibitor) before being treated with either DMSO or Cyt D for 1h. Cells were then left untreated or stimulated with particulate β–glucan C.a. Luminol was added, and Reactive oxygen species (ROS) production was monitored by chemiluminescence during time (2h) and expressed as relative light units (RLU). Graphs show mean ± SEM corresponding to up to three independent experiments ( n = 3); each experimental condition was assessed in triplicate. (B) Monocytes were pre-treated with DPI, as indicated. After 30 min of incubation, cells were treated either with DMSO or Cyt D and, after an additional 1h, stimulated with β-glucan C.a. Culture supernatants were collected after 24h and secretion of TNF-α, IL-6 and IL-1β was determined by ELISA. Graphs show the mean + SEM of at least three independent experiments ( n = 8); ** p

    Techniques Used: Incubation, Enzyme-linked Immunosorbent Assay

    The role of the Dectin-1/Syk/PI3K pathway in the overproduction of cytokines by monocytes impaired for phagocytosis and stimulated with β-glucan. Monocytes were pre-treated with anti-Dectin-1 Ab, anti-TLR2 Ab, anti-CR3 Ab or isotype control Abs in (A). Syk inhibitor in (B), or PI3K inhibitor (WRT; wortmannin) in (C), were used on monocytes as indicated. After the first incubation with inhibitors, cells were treated either with DMSO or Cyt D and finally stimulated with branched β-(1–3)-glucan isolated from C. albicans (β-glucan C.a.). Culture supernatants were collected after 24h and secretion of TNF-α, IL-6, and IL-1β was determined by ELISA. Graphs show the mean ± SEM of at least three independent experiments. For (A), n = 6; for (B) and (C), n = 9; * p
    Figure Legend Snippet: The role of the Dectin-1/Syk/PI3K pathway in the overproduction of cytokines by monocytes impaired for phagocytosis and stimulated with β-glucan. Monocytes were pre-treated with anti-Dectin-1 Ab, anti-TLR2 Ab, anti-CR3 Ab or isotype control Abs in (A). Syk inhibitor in (B), or PI3K inhibitor (WRT; wortmannin) in (C), were used on monocytes as indicated. After the first incubation with inhibitors, cells were treated either with DMSO or Cyt D and finally stimulated with branched β-(1–3)-glucan isolated from C. albicans (β-glucan C.a.). Culture supernatants were collected after 24h and secretion of TNF-α, IL-6, and IL-1β was determined by ELISA. Graphs show the mean ± SEM of at least three independent experiments. For (A), n = 6; for (B) and (C), n = 9; * p

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

    The role of the NLRP3-ASC-caspase-1 inflammasome in β-glucan induced IL-1β. (A and E) Monocytes were pre-treated with either z-VAD-fmk (pan-caspase inhibitor), z-YVAD-fmk (caspase-1 inhibitor), z-IETD-fmk (caspase-8 inhibitor), z-LEVD-fmk (caspase-4/5 inhibitor), MCC950 (NLRP3 inhibitor), glibenclamide (inhibitor of potassium (K+) efflux) or z-VRPR-fmk (MALT1 inhibitor) before treatment with either DMSO or Cyt D for 1 h. Cells were then stimulated with branched β-(1–3)-glucan isolated from C. albicans (β-glucan C.a.), culture supernatants were collected after 24 h and secretion of IL-1β was determined by ELISA. Graphs show the mean ± SEM of four independent experiments ( n = 8). ** p
    Figure Legend Snippet: The role of the NLRP3-ASC-caspase-1 inflammasome in β-glucan induced IL-1β. (A and E) Monocytes were pre-treated with either z-VAD-fmk (pan-caspase inhibitor), z-YVAD-fmk (caspase-1 inhibitor), z-IETD-fmk (caspase-8 inhibitor), z-LEVD-fmk (caspase-4/5 inhibitor), MCC950 (NLRP3 inhibitor), glibenclamide (inhibitor of potassium (K+) efflux) or z-VRPR-fmk (MALT1 inhibitor) before treatment with either DMSO or Cyt D for 1 h. Cells were then stimulated with branched β-(1–3)-glucan isolated from C. albicans (β-glucan C.a.), culture supernatants were collected after 24 h and secretion of IL-1β was determined by ELISA. Graphs show the mean ± SEM of four independent experiments ( n = 8). ** p

    Techniques Used: Isolation, Enzyme-linked Immunosorbent Assay

    Schematic model illustrating potential signaling pathways triggered by the binding of β–glucan to dectin-1 in human monocytes with normal (left) or impaired phagocytic (right) function. Attenuation of dectin-1 signaling is associated with the engulfment of the receptor/ligand complex. By contrast, blocking of actin-dependent phagocytosis led to increased cell activation. Black arrows depict the interactions that normally occur upon phagocytosis and β–glucan/dectin-1 interaction, whereas red arrows indicate the proposed signaling events that enhance the strength and efficacy of signal propagation.
    Figure Legend Snippet: Schematic model illustrating potential signaling pathways triggered by the binding of β–glucan to dectin-1 in human monocytes with normal (left) or impaired phagocytic (right) function. Attenuation of dectin-1 signaling is associated with the engulfment of the receptor/ligand complex. By contrast, blocking of actin-dependent phagocytosis led to increased cell activation. Black arrows depict the interactions that normally occur upon phagocytosis and β–glucan/dectin-1 interaction, whereas red arrows indicate the proposed signaling events that enhance the strength and efficacy of signal propagation.

    Techniques Used: Binding Assay, Blocking Assay, Activation Assay

    Syk activity assessement for inflammasome, and NF-κB activation in phagocytosis-impaired-monocyte stimulated with C. albicans branched β-(1–3)-glucan. (A) Monocytes treated for 6 h with β–glucan C.a. in the presence or absence of Cyt D and of the Syk inhibitor (2 μM). Nuclear translocation of p65 NF-κB was visualized by immunostaining with a specific antibody recognizing p65 and a alexa-fluor 488-conjugated secondary antibody (green). For reference the nuclei are stained Hoescht (blue). Original magnification. 60×, scalebar: 50 μMImage is representative of three independent experiments. (B) Quantification of nuclei with p65 translocation (4 × 50 nuclei analysed with Image J). (C) Cells were treated as in (A). Caspase-1 (pro-form and cleaved p20), and IL-1β (pro- and mature IL-1β) expressions were assessed by western blot. Mature IL-1β and caspase-1 were also detected in cell culture supernatants. GAPDH was used as the loading control. Blots are representative of three independent experiments.
    Figure Legend Snippet: Syk activity assessement for inflammasome, and NF-κB activation in phagocytosis-impaired-monocyte stimulated with C. albicans branched β-(1–3)-glucan. (A) Monocytes treated for 6 h with β–glucan C.a. in the presence or absence of Cyt D and of the Syk inhibitor (2 μM). Nuclear translocation of p65 NF-κB was visualized by immunostaining with a specific antibody recognizing p65 and a alexa-fluor 488-conjugated secondary antibody (green). For reference the nuclei are stained Hoescht (blue). Original magnification. 60×, scalebar: 50 μMImage is representative of three independent experiments. (B) Quantification of nuclei with p65 translocation (4 × 50 nuclei analysed with Image J). (C) Cells were treated as in (A). Caspase-1 (pro-form and cleaved p20), and IL-1β (pro- and mature IL-1β) expressions were assessed by western blot. Mature IL-1β and caspase-1 were also detected in cell culture supernatants. GAPDH was used as the loading control. Blots are representative of three independent experiments.

    Techniques Used: Activity Assay, Activation Assay, Translocation Assay, Immunostaining, Staining, Western Blot, Cell Culture

    15) Product Images from "Aureobasidium pullulans produced β-glucan is effective to enhance Kurosengoku soybean extract induced Thrombospondin-1 expression"

    Article Title: Aureobasidium pullulans produced β-glucan is effective to enhance Kurosengoku soybean extract induced Thrombospondin-1 expression

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-03053-9

    Soy isoflavones are involved in the induction of THBS1. ( A ) Mono Mac 6 cells were stimulated with a 20-fold dilution of KS-E, 20 μM Lunasin, or isoflavone mixture, saponin, and lecithin isolated from soybeans at the 1 or 10 μg/ml concentrations indicated in the Figure. ( B ) Mono Mac 6 cells were stimulated with a 20-fold dilution of KS-E, or 10 μg/ml isoflavone mixture of genistein, glycitein, and daidzein. All compounds are dissolved in 1 μl dimethyl sulfoxide (DMSO), and all cells including negative and positive (stimulation with KS-E) control cells were treated with 1 μl DMSO. ( C ) Mono Mac 6 cells were stimulated with hot water extracts prepared from whole beans as indicated in the Figure at 20-fold dilution, with or without AP-CF at the concentration of 100 μg/ml β-glucan. After 2 hours, the cells were harvested, and the THBS1 mRNA expressions were monitored using the real-time RT-PCR method. The data are represented as relative values compared with the mRNA expression in unstimulated control cells after normalization with GAPDH mRNA expression. Error bars indicate standard deviations calculated by three independent experiments.
    Figure Legend Snippet: Soy isoflavones are involved in the induction of THBS1. ( A ) Mono Mac 6 cells were stimulated with a 20-fold dilution of KS-E, 20 μM Lunasin, or isoflavone mixture, saponin, and lecithin isolated from soybeans at the 1 or 10 μg/ml concentrations indicated in the Figure. ( B ) Mono Mac 6 cells were stimulated with a 20-fold dilution of KS-E, or 10 μg/ml isoflavone mixture of genistein, glycitein, and daidzein. All compounds are dissolved in 1 μl dimethyl sulfoxide (DMSO), and all cells including negative and positive (stimulation with KS-E) control cells were treated with 1 μl DMSO. ( C ) Mono Mac 6 cells were stimulated with hot water extracts prepared from whole beans as indicated in the Figure at 20-fold dilution, with or without AP-CF at the concentration of 100 μg/ml β-glucan. After 2 hours, the cells were harvested, and the THBS1 mRNA expressions were monitored using the real-time RT-PCR method. The data are represented as relative values compared with the mRNA expression in unstimulated control cells after normalization with GAPDH mRNA expression. Error bars indicate standard deviations calculated by three independent experiments.

    Techniques Used: Isolation, Concentration Assay, Quantitative RT-PCR, Expressing

    The Syk and its downstream kinases are involved in the enhancement of the THBS1 induction after stimulation with KS-E. Mono Mac 6 cells were treated for 1 hour with piceatannol ( A ), SB203580 ( B ), or SP600125 ( C ) at the concentrations indicated in the figure. Subsequently, the cells were stimulated with a 20-fold dilution of KS-E and AP-CF containing 100 μg/ml of β-glucan. After an additional 2-hour incubation period the cells were harvested, and the expressions of THBS1 mRNA in the cells were monitored using the real time RT-PCR method. The data are represented as relative values compared with the mRNA expression in the control cells after normalization with GAPDH mRNA expression. Error bars indicate standard deviations calculated by three independent experiments.
    Figure Legend Snippet: The Syk and its downstream kinases are involved in the enhancement of the THBS1 induction after stimulation with KS-E. Mono Mac 6 cells were treated for 1 hour with piceatannol ( A ), SB203580 ( B ), or SP600125 ( C ) at the concentrations indicated in the figure. Subsequently, the cells were stimulated with a 20-fold dilution of KS-E and AP-CF containing 100 μg/ml of β-glucan. After an additional 2-hour incubation period the cells were harvested, and the expressions of THBS1 mRNA in the cells were monitored using the real time RT-PCR method. The data are represented as relative values compared with the mRNA expression in the control cells after normalization with GAPDH mRNA expression. Error bars indicate standard deviations calculated by three independent experiments.

    Techniques Used: Incubation, Quantitative RT-PCR, Expressing

    Aureobasidium pullulans -cultured fluid (AP-CF) enhances Thrombospondin-1 (THBS1) expression after stimulation with hot water extracts of Kurosengoku soybeans (KS-E). ( A ) Mono Mac 6 cells were stimulated with AP-CF or AP-CF made with ground powder of Kurosengoku soybeans (kAP-CF) as a nitrogen source, at the concentration of 100 μg/ml β-glucan, or the cells were stimulated with KS-E at 20-fold dilution. ( B , C ) Mono Mac 6 cells were treated with or without 5 mM cycloheximide (CHX) for 30 min. Subsequently, the cells were stimulated with KS-E at 20-fold dilution together with AP-CF at the concentration of 100 μg/ml β-glucan. After the incubation period indicated in the figure, the cells were harvested, and the THBS1 mRNA expressions were measured using the real-time RT-PCR method. The data are represented as relative values compared with the mRNA expression at the 0-hour time point after the normalization with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA expression. Error bars indicate standard deviations calculated by three independent experiments. ( D ) Mono Mac 6 cells were stimulated with KS-E at 20-fold dilution together with AP-CF at the concentration of 100 μg/ml β-glucan. After 24 hours, concentrations of THBS1 in the supernatant of a cultured medium of the cells were measured by ELISA. Error bars indicate standard deviations calculated by three independent experiments. The asterisk (*) and double asterisks (**) indicate p
    Figure Legend Snippet: Aureobasidium pullulans -cultured fluid (AP-CF) enhances Thrombospondin-1 (THBS1) expression after stimulation with hot water extracts of Kurosengoku soybeans (KS-E). ( A ) Mono Mac 6 cells were stimulated with AP-CF or AP-CF made with ground powder of Kurosengoku soybeans (kAP-CF) as a nitrogen source, at the concentration of 100 μg/ml β-glucan, or the cells were stimulated with KS-E at 20-fold dilution. ( B , C ) Mono Mac 6 cells were treated with or without 5 mM cycloheximide (CHX) for 30 min. Subsequently, the cells were stimulated with KS-E at 20-fold dilution together with AP-CF at the concentration of 100 μg/ml β-glucan. After the incubation period indicated in the figure, the cells were harvested, and the THBS1 mRNA expressions were measured using the real-time RT-PCR method. The data are represented as relative values compared with the mRNA expression at the 0-hour time point after the normalization with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA expression. Error bars indicate standard deviations calculated by three independent experiments. ( D ) Mono Mac 6 cells were stimulated with KS-E at 20-fold dilution together with AP-CF at the concentration of 100 μg/ml β-glucan. After 24 hours, concentrations of THBS1 in the supernatant of a cultured medium of the cells were measured by ELISA. Error bars indicate standard deviations calculated by three independent experiments. The asterisk (*) and double asterisks (**) indicate p

    Techniques Used: Cell Culture, Expressing, Concentration Assay, Incubation, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

    β-glucan is involved in the enhancement of THBS1 expression after stimulation with KS-E. ( A ) THP-1 cells were stimulated with conventional AP-CF or AP-CF made using ground powder of Kurosengoku soybeans (kAP-CF) as a nitrogen source, at the concentration of 100 μg/ml β-glucan, or the cells were stimulated with KS-E at 20-fold dilution. The cells were harvested at the time points indicated in the figure, and the THBS1 mRNA expressions in the cells were measured using the real-time RT-PCR method. ( B ) Mono Mac 6 cells were stimulated with AP-CF, AP-PG, Krestin, or laminarin at concentrations of 100 μg/ml β-glucan, with or without 20-fold dilution of KS-E. ( C ) Mono Mac cells were stimulated with a 20-fold dilution of KS-E from ground beans, whole raw beans, or whole roasted beans, with or without AP-CF at a concentration of 100 μg/ml β-glucan. After 2 hours, the cells were harvested, and then the total RNA prepared from the cells was subjected to real-time RT-PCR analysis. The data are represented as relative values compared with the mRNA expression in unstimulated control cells after normalization with the GAPDH mRNA expression. Error bars indicate standard deviations calculated by three independent experiments.
    Figure Legend Snippet: β-glucan is involved in the enhancement of THBS1 expression after stimulation with KS-E. ( A ) THP-1 cells were stimulated with conventional AP-CF or AP-CF made using ground powder of Kurosengoku soybeans (kAP-CF) as a nitrogen source, at the concentration of 100 μg/ml β-glucan, or the cells were stimulated with KS-E at 20-fold dilution. The cells were harvested at the time points indicated in the figure, and the THBS1 mRNA expressions in the cells were measured using the real-time RT-PCR method. ( B ) Mono Mac 6 cells were stimulated with AP-CF, AP-PG, Krestin, or laminarin at concentrations of 100 μg/ml β-glucan, with or without 20-fold dilution of KS-E. ( C ) Mono Mac cells were stimulated with a 20-fold dilution of KS-E from ground beans, whole raw beans, or whole roasted beans, with or without AP-CF at a concentration of 100 μg/ml β-glucan. After 2 hours, the cells were harvested, and then the total RNA prepared from the cells was subjected to real-time RT-PCR analysis. The data are represented as relative values compared with the mRNA expression in unstimulated control cells after normalization with the GAPDH mRNA expression. Error bars indicate standard deviations calculated by three independent experiments.

    Techniques Used: Expressing, Concentration Assay, Quantitative RT-PCR

    16) Product Images from "Comparative Secretome Analysis of Trichoderma reesei and Aspergillus niger during Growth on Sugarcane Biomass"

    Article Title: Comparative Secretome Analysis of Trichoderma reesei and Aspergillus niger during Growth on Sugarcane Biomass

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0129275

    Estimation of enzyme activities. Enzymatic activities (U/mL) against different substrates of A . niger and T . reesei after 24 hours (h) growth on sugarcane culm (C) and bagasse (B). a) B-glucan and xylan b) Xyloglucan, and c) CMC and Arabinan. Each bar represents the mean and the standard deviation of values from three independent experiments.
    Figure Legend Snippet: Estimation of enzyme activities. Enzymatic activities (U/mL) against different substrates of A . niger and T . reesei after 24 hours (h) growth on sugarcane culm (C) and bagasse (B). a) B-glucan and xylan b) Xyloglucan, and c) CMC and Arabinan. Each bar represents the mean and the standard deviation of values from three independent experiments.

    Techniques Used: Standard Deviation

    Schematic representation of hypothetical modes of attack of the enzyme complexes produced by A . niger (left) and T . reesei (right). One cell wall architectural unit of sugarcane (De Souza et al., 2013) is represented. Light blue: pectins; dark blue: feruloyl esters; red: hemicelluloses—beta-glucan and arabinoxylan; yellow: xyloglucan; black: cellulose microfibrils. Cell wall degradation is schematically represented in three steps for each fungi attack so as to compare the two different strategies hypothesized in this work. Whereas A . niger degrades cell wall components of different classes with approximately the same intensity, including action on feruloyl esters, pectins, hemicelluloses and cellulose, T . reesei maximizes penetration into the cell wall matrix, lacking feruloyl esterases, having limited action on pectins, but disassembling more efficiently the cellulose-xyloglucan network and attacking mainly the cellulose microfibrils.
    Figure Legend Snippet: Schematic representation of hypothetical modes of attack of the enzyme complexes produced by A . niger (left) and T . reesei (right). One cell wall architectural unit of sugarcane (De Souza et al., 2013) is represented. Light blue: pectins; dark blue: feruloyl esters; red: hemicelluloses—beta-glucan and arabinoxylan; yellow: xyloglucan; black: cellulose microfibrils. Cell wall degradation is schematically represented in three steps for each fungi attack so as to compare the two different strategies hypothesized in this work. Whereas A . niger degrades cell wall components of different classes with approximately the same intensity, including action on feruloyl esters, pectins, hemicelluloses and cellulose, T . reesei maximizes penetration into the cell wall matrix, lacking feruloyl esterases, having limited action on pectins, but disassembling more efficiently the cellulose-xyloglucan network and attacking mainly the cellulose microfibrils.

    Techniques Used: Produced

    17) Product Images from "Oral microbe-host interactions: influence of β-glucans on gene expression of inflammatory cytokines and metabolome profile"

    Article Title: Oral microbe-host interactions: influence of β-glucans on gene expression of inflammatory cytokines and metabolome profile

    Journal: BMC Microbiology

    doi: 10.1186/s12866-017-0946-1

    Cytotoxicity assay of cells treated with different doses of β-glucan: ( a ) OBA-9 cell (keratinocytes); ( b ) HGF-1 cell (fibroblasts). Cell viability was presented in percentage (%). n = 6
    Figure Legend Snippet: Cytotoxicity assay of cells treated with different doses of β-glucan: ( a ) OBA-9 cell (keratinocytes); ( b ) HGF-1 cell (fibroblasts). Cell viability was presented in percentage (%). n = 6

    Techniques Used: Cytotoxicity Assay

    Relative expression of genes on OBA-9 cells (keratinocytes) by quantitative PCR: ( a ) interleukin 1 alpha (IL-1-α); ( b ) interleukin 18 (IL-18); ( c ) B-cell lymphoma 2 (BCL-2); ( d ) adenovirus E1A-associated 300 kDa protein (EP 300); ( e ) prostaglandin-endoperoxide synthase 2 (PTGS-2). Dual-chamber model inoculated with A. actinomicetemcomitans and treated with different doses of β-glucan. The control group has their mean expressed equal to 1 and treated groups have their mean relative to the control group. The results were expressed by mean followed standard deviation; n = 6 and P
    Figure Legend Snippet: Relative expression of genes on OBA-9 cells (keratinocytes) by quantitative PCR: ( a ) interleukin 1 alpha (IL-1-α); ( b ) interleukin 18 (IL-18); ( c ) B-cell lymphoma 2 (BCL-2); ( d ) adenovirus E1A-associated 300 kDa protein (EP 300); ( e ) prostaglandin-endoperoxide synthase 2 (PTGS-2). Dual-chamber model inoculated with A. actinomicetemcomitans and treated with different doses of β-glucan. The control group has their mean expressed equal to 1 and treated groups have their mean relative to the control group. The results were expressed by mean followed standard deviation; n = 6 and P

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Standard Deviation

    Relative expression of genes on HGF-1 cells (fibroblasts) by quantitative PCR: ( a ) interleukin 1 alpha (IL-1-α); ( b ) interleukin 18 (IL-18); ( c ) B-cell lymphoma 2 (BCL-2); ( d ) adenovirus E1A-associated 300 kDa protein (EP 300); ( e ) prostaglandin-endoperoxide synthase 2 (PTGS-2). Dual-chamber model inoculated with A. actinomicetemcomitans and treated with different doses of β-glucan. The control group has their mean expressed equal to 1 and treated groups have their mean relative to the control group. The results were expressed by mean followed standard deviation; n = 6 and P
    Figure Legend Snippet: Relative expression of genes on HGF-1 cells (fibroblasts) by quantitative PCR: ( a ) interleukin 1 alpha (IL-1-α); ( b ) interleukin 18 (IL-18); ( c ) B-cell lymphoma 2 (BCL-2); ( d ) adenovirus E1A-associated 300 kDa protein (EP 300); ( e ) prostaglandin-endoperoxide synthase 2 (PTGS-2). Dual-chamber model inoculated with A. actinomicetemcomitans and treated with different doses of β-glucan. The control group has their mean expressed equal to 1 and treated groups have their mean relative to the control group. The results were expressed by mean followed standard deviation; n = 6 and P

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Standard Deviation

    Dual-chamber model containing OBA-9 cell (keratinocytes) in the top layer (transwell insert) and HGF-1 cell (fibroblasts) in the bottom layer inoculated with A. actinomicetemcomitans and treated with different doses of β-glucan
    Figure Legend Snippet: Dual-chamber model containing OBA-9 cell (keratinocytes) in the top layer (transwell insert) and HGF-1 cell (fibroblasts) in the bottom layer inoculated with A. actinomicetemcomitans and treated with different doses of β-glucan

    Techniques Used:

    Metabolites obtained of cell culture supernatant (HGF-1 and OBA-9 co-culture cells). ( a ) 4-aminobutyric acid; ( b ) 2-deoxytetronic acid NIST; ( c ) acetophenone NIST; ( d ) benzoic acid; ( e ) oxalic acid; ( f ) pinitol. Dual-chamber model inoculated with A. actinomicetemcomitans and treated with different doses of β-glucan. The data is expressed in relative peak heights (mAU) from HPLC-MS analysis, which are unit-less (mean followed standard deviation); n = 4 and P
    Figure Legend Snippet: Metabolites obtained of cell culture supernatant (HGF-1 and OBA-9 co-culture cells). ( a ) 4-aminobutyric acid; ( b ) 2-deoxytetronic acid NIST; ( c ) acetophenone NIST; ( d ) benzoic acid; ( e ) oxalic acid; ( f ) pinitol. Dual-chamber model inoculated with A. actinomicetemcomitans and treated with different doses of β-glucan. The data is expressed in relative peak heights (mAU) from HPLC-MS analysis, which are unit-less (mean followed standard deviation); n = 4 and P

    Techniques Used: Cell Culture, Co-Culture Assay, High Performance Liquid Chromatography, Mass Spectrometry, Standard Deviation

    18) Product Images from "Modulation of Myelopoiesis Progenitors Is an Integral Component of Trained Immunity"

    Article Title: Modulation of Myelopoiesis Progenitors Is an Integral Component of Trained Immunity

    Journal: Cell

    doi: 10.1016/j.cell.2017.11.034

    Training with β-Glucan Promotes a Beneficial Response to a Secondary Challenge (A) WT mice were injected with β-glucan or PBS, and after 28 days, they received a secondary challenge with LPS. (B and C) LSK, MPP, and LT-HSC cell numbers (B) and frequency of the same cells in total BM cells (C) at 24 hr after LPS injection (n = 10 mice per group). (D) Representative FACS plots and frequency of γ-H2AX-positive LT-HSCs at 24 hr after LPS injection (n = 10 mice per group, right plots and gray background). The frequency of γ-H2AX-positive LT-HSCs at 28 days after β-glucan administration in mice not injected with LPS (—) is also shown; ns = 4 and 5, left plots and white background. (E) Experimental protocol for the effect of β-glucan on the recovery of granulopoiesis after cyclophosphamide administration (4 rounds). (F and G) Total white blood cell (WBC) (F) and granulocyte (Gr1 + CD11b + ) (G) counts in the peripheral blood (n = 10 mice per group). (H) Experimental protocol for 5-FU administration. (I) Survival curves of 5-FU-treated mice treated with β-glucan or PBS control (n = 16 mice per group). Comparison of survival curves was performed by log-rank (Mantel-Cox) test, and p value is shown. (J and K) Mice were injected with β-glucan or PBS, and 7 days later, a single dose of 5-FU was administered. (J) Neutrophil numbers in peripheral blood at different time points after the administration of 5-FU (n = 5 mice per group). (K) Frequency of γ-H2AX-positive LT-HSCs 14 days after 5-FU administration (n = 10 mice per group). Data are presented as mean ± SEM. ∗ p
    Figure Legend Snippet: Training with β-Glucan Promotes a Beneficial Response to a Secondary Challenge (A) WT mice were injected with β-glucan or PBS, and after 28 days, they received a secondary challenge with LPS. (B and C) LSK, MPP, and LT-HSC cell numbers (B) and frequency of the same cells in total BM cells (C) at 24 hr after LPS injection (n = 10 mice per group). (D) Representative FACS plots and frequency of γ-H2AX-positive LT-HSCs at 24 hr after LPS injection (n = 10 mice per group, right plots and gray background). The frequency of γ-H2AX-positive LT-HSCs at 28 days after β-glucan administration in mice not injected with LPS (—) is also shown; ns = 4 and 5, left plots and white background. (E) Experimental protocol for the effect of β-glucan on the recovery of granulopoiesis after cyclophosphamide administration (4 rounds). (F and G) Total white blood cell (WBC) (F) and granulocyte (Gr1 + CD11b + ) (G) counts in the peripheral blood (n = 10 mice per group). (H) Experimental protocol for 5-FU administration. (I) Survival curves of 5-FU-treated mice treated with β-glucan or PBS control (n = 16 mice per group). Comparison of survival curves was performed by log-rank (Mantel-Cox) test, and p value is shown. (J and K) Mice were injected with β-glucan or PBS, and 7 days later, a single dose of 5-FU was administered. (J) Neutrophil numbers in peripheral blood at different time points after the administration of 5-FU (n = 5 mice per group). (K) Frequency of γ-H2AX-positive LT-HSCs 14 days after 5-FU administration (n = 10 mice per group). Data are presented as mean ± SEM. ∗ p

    Techniques Used: Mouse Assay, Injection, FACS

    IL-1β, Glycolysis, Cholesterol Metabolism, and the GM-CSF/CD131 Axis Are Involved in β-Glucan-Dependent Training in the BM (A) Cytokine and G-CSF concentrations in the BM extracellular fluid of mice at 24 hr after the administration of PBS or β-glucan (n = 10 mice per group). (B and C) Mice were injected with β-glucan in the absence (vehicle control, Ctrl) or presence of IL1RA, and BM analysis was performed 24 hr later. (B) Cell-cycle analysis in LT-HSCs. (C) The frequency of MPP subpopulations in LSK cells in the BM is indicated (n = 5 mice per group). (D) Bioenergetic extracellular flux analysis in Lin − cKit + cells sorted from mice 24 hr after β-glucan administration in the absence (vehicle control, Ctrl) or presence of IL1RA (n = 5 mice per group). Basal and maximal ECAR are indicated. (E) LSK cells were treated in vitro with IL-1β or PBS for 24 hr, and Seahorse analysis was performed (n = 5 cultures per group). Basal and maximal ECARs (left) and glycolytic reserve (right) are indicated. (F–I) Mice were injected with β-glucan on day 0. (F) Glycolysis and IL-1 were blocked by the administration of 2-DG and IL1RA, respectively, on days 0 and 1; PBS served as the vehicle control (Ctrl). (G) LSK, MPP, and LT-HSC numbers in the BM at day 7 after β-glucan administration (n = 5 mice per group). (H and I) GMP numbers in the BM (H) and frequency of GMPs within the MyP pool (I) at day 7 after β-glucan administration (n = 5 mice per group). (J and K) Mice were injected with PBS or β-glucan, and BM analysis was performed 24 hr later; representative FACS plots (J) and frequency of CD131 + LSKs, CD131 + MPPs, and CD131 + LT-HSCs (K) (n = 5 mice per group) are indicated. (L) Staining for pSTAT5 in LSK cells 24 hr after PBS or β-glucan administration. Representative FACS plots and median fluorescence intensity (MFI) are shown (n = 5 mice per group). (M–P), As indicated in (M), mice were injected with β-glucan on day 0. Cholesterol metabolism was blocked by the administration of atorvastatin on days 0 and 1; Ctrl represents the vehicle control. (N) LSK, LT-HSC, and MPP cell numbers; (O) frequency of the same cells in total BM cells; and (P) frequency of GMPs within the MyP pool at day 7 after β-glucan administration (n = 5 mice per group). (Q) Mice were injected with β-glucan on day 0. GM-CSF was blocked by specific anti-GM-CSF antibody on days 0 and 1. Immunoglobulin G (IgG) isotype served as the control. (R and S) LSK, LT-HSC, and MPP cell numbers (R) and frequency of the same cells in total BM cells (S) at day 7 after β-glucan administration are indicated (n = 5 mice per group). Data are presented as mean ± SEM. ∗ p
    Figure Legend Snippet: IL-1β, Glycolysis, Cholesterol Metabolism, and the GM-CSF/CD131 Axis Are Involved in β-Glucan-Dependent Training in the BM (A) Cytokine and G-CSF concentrations in the BM extracellular fluid of mice at 24 hr after the administration of PBS or β-glucan (n = 10 mice per group). (B and C) Mice were injected with β-glucan in the absence (vehicle control, Ctrl) or presence of IL1RA, and BM analysis was performed 24 hr later. (B) Cell-cycle analysis in LT-HSCs. (C) The frequency of MPP subpopulations in LSK cells in the BM is indicated (n = 5 mice per group). (D) Bioenergetic extracellular flux analysis in Lin − cKit + cells sorted from mice 24 hr after β-glucan administration in the absence (vehicle control, Ctrl) or presence of IL1RA (n = 5 mice per group). Basal and maximal ECAR are indicated. (E) LSK cells were treated in vitro with IL-1β or PBS for 24 hr, and Seahorse analysis was performed (n = 5 cultures per group). Basal and maximal ECARs (left) and glycolytic reserve (right) are indicated. (F–I) Mice were injected with β-glucan on day 0. (F) Glycolysis and IL-1 were blocked by the administration of 2-DG and IL1RA, respectively, on days 0 and 1; PBS served as the vehicle control (Ctrl). (G) LSK, MPP, and LT-HSC numbers in the BM at day 7 after β-glucan administration (n = 5 mice per group). (H and I) GMP numbers in the BM (H) and frequency of GMPs within the MyP pool (I) at day 7 after β-glucan administration (n = 5 mice per group). (J and K) Mice were injected with PBS or β-glucan, and BM analysis was performed 24 hr later; representative FACS plots (J) and frequency of CD131 + LSKs, CD131 + MPPs, and CD131 + LT-HSCs (K) (n = 5 mice per group) are indicated. (L) Staining for pSTAT5 in LSK cells 24 hr after PBS or β-glucan administration. Representative FACS plots and median fluorescence intensity (MFI) are shown (n = 5 mice per group). (M–P), As indicated in (M), mice were injected with β-glucan on day 0. Cholesterol metabolism was blocked by the administration of atorvastatin on days 0 and 1; Ctrl represents the vehicle control. (N) LSK, LT-HSC, and MPP cell numbers; (O) frequency of the same cells in total BM cells; and (P) frequency of GMPs within the MyP pool at day 7 after β-glucan administration (n = 5 mice per group). (Q) Mice were injected with β-glucan on day 0. GM-CSF was blocked by specific anti-GM-CSF antibody on days 0 and 1. Immunoglobulin G (IgG) isotype served as the control. (R and S) LSK, LT-HSC, and MPP cell numbers (R) and frequency of the same cells in total BM cells (S) at day 7 after β-glucan administration are indicated (n = 5 mice per group). Data are presented as mean ± SEM. ∗ p

    Techniques Used: Mouse Assay, Injection, Cell Cycle Assay, In Vitro, FACS, Staining, Fluorescence

    Single-Cell Transcriptional Analysis in LT-HSCs upon β-Glucan Administration (A–C) Single-cell qPCR in LT-HSCs isolated from mice at 24 hr after administration of PBS or β-glucan (n = 42 cells per condition). (A and B) Hierarchical clustering analysis (A) and distribution of LT-HSCs in the three identified clusters (B) at 24 hr after the administration of PBS or β-glucan. (C) Violin plots indicating genes with significantly altered expression between clusters 1 and 2. The y axis represents gene expression. The horizontal width of the plot shows the density of the data along the y axis. Color key represents the percentage of cells that express the specific gene. (D and E) Single-cell qPCR was performed in CD41 − and CD41 + LT-HSCs isolated from mice at 24 hr after the administration of PBS or β-glucan. Hierarchical clustering analysis (D) and violin plots indicating genes with significantly altered expression between CD41 + LT-HSCs from PBS and β-glucan-treated mice (E).
    Figure Legend Snippet: Single-Cell Transcriptional Analysis in LT-HSCs upon β-Glucan Administration (A–C) Single-cell qPCR in LT-HSCs isolated from mice at 24 hr after administration of PBS or β-glucan (n = 42 cells per condition). (A and B) Hierarchical clustering analysis (A) and distribution of LT-HSCs in the three identified clusters (B) at 24 hr after the administration of PBS or β-glucan. (C) Violin plots indicating genes with significantly altered expression between clusters 1 and 2. The y axis represents gene expression. The horizontal width of the plot shows the density of the data along the y axis. Color key represents the percentage of cells that express the specific gene. (D and E) Single-cell qPCR was performed in CD41 − and CD41 + LT-HSCs isolated from mice at 24 hr after the administration of PBS or β-glucan. Hierarchical clustering analysis (D) and violin plots indicating genes with significantly altered expression between CD41 + LT-HSCs from PBS and β-glucan-treated mice (E).

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

    Alterations in Lipid Metabolism in BM Progenitor Cells upon β-Glucan Administration Lin − cKit + cells were sorted from mice 24 hr after β-glucan or PBS administration, and non-targeted metabolomic and lipidomic analyses were performed (n = 4 mice per group). (A and B) Not-targeted metabolomics. (A) Volcano plots depict the comparison of metabolite abundances between cells from β-glucan- and PBS-treated mice. Altered metabolites (q value
    Figure Legend Snippet: Alterations in Lipid Metabolism in BM Progenitor Cells upon β-Glucan Administration Lin − cKit + cells were sorted from mice 24 hr after β-glucan or PBS administration, and non-targeted metabolomic and lipidomic analyses were performed (n = 4 mice per group). (A and B) Not-targeted metabolomics. (A) Volcano plots depict the comparison of metabolite abundances between cells from β-glucan- and PBS-treated mice. Altered metabolites (q value

    Techniques Used: Mouse Assay

    19) Product Images from "Corticosteroids block autophagy protein recruitment in Aspergillus fumigatus phagosomes via targeting Dectin-1/syk kinase signaling"

    Article Title: Corticosteroids block autophagy protein recruitment in Aspergillus fumigatus phagosomes via targeting Dectin-1/syk kinase signaling

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

    doi: 10.4049/jimmunol.1300132

    β-glucan surface exposure in swollen spores of A. fumigatus triggers LC3 II recruitment in fungal phagosomes A. Primary human monocytes (2 × 10 5 cells/condition) isolated from healthy individuals were infected with GFP A fumigatus swollen spores with or without laminarin (500 µg/ml) or swollen spores following overnight enzymatic digestion of β-glucan (β-glucanase) at a MOI 5: 1 for 1h. Cells were fixed, permeabilized, stained for LC3 II with the use of an Alexa 555 secondary antibody (red) and TOPRO-3 (blue, nuclear staining) and analyzed by immunofluorescence confocal microscopy. Bar, 5 µm. B The percentages of LC3 + A. fumigatus-containing phagosomes (LC3 + Aspergillus n > 150 per group) were quantified and data are presented as mean + S.E.M. of 3 independent experiments. *, P
    Figure Legend Snippet: β-glucan surface exposure in swollen spores of A. fumigatus triggers LC3 II recruitment in fungal phagosomes A. Primary human monocytes (2 × 10 5 cells/condition) isolated from healthy individuals were infected with GFP A fumigatus swollen spores with or without laminarin (500 µg/ml) or swollen spores following overnight enzymatic digestion of β-glucan (β-glucanase) at a MOI 5: 1 for 1h. Cells were fixed, permeabilized, stained for LC3 II with the use of an Alexa 555 secondary antibody (red) and TOPRO-3 (blue, nuclear staining) and analyzed by immunofluorescence confocal microscopy. Bar, 5 µm. B The percentages of LC3 + A. fumigatus-containing phagosomes (LC3 + Aspergillus n > 150 per group) were quantified and data are presented as mean + S.E.M. of 3 independent experiments. *, P

    Techniques Used: Isolation, Infection, Staining, Immunofluorescence, Confocal Microscopy

    20) Product Images from "CD14 Targets Complement Receptor 3 to Lipid Rafts during Phagocytosis of Borrelia burgdorferi"

    Article Title: CD14 Targets Complement Receptor 3 to Lipid Rafts during Phagocytosis of Borrelia burgdorferi

    Journal: International Journal of Biological Sciences

    doi: 10.7150/ijbs.7136

    Internalization of B. burgdorferi by CHO-CR3 cells is facilitated by β-glucans. (A) RAW264.7 cells were incubated with 1 mM barley β-glucan (orange histogram), Laminarin β-glucan (green histogram) or S. cerevisiae β-glucan (red histogram) and Bb914 (m.o.i. = 25) for 4 hours. Control cells (black histogram) were incubated with Bb914. (B) CHO-CR3 cells were incubated with 1 mM S. cerevisiae β-glucan and Bb914 for 4 hours. The cells were fixed, stained with phalloidin Alexa Fluor 594 and analyzed by confocal microscopy. The results presented are representative of 3 experiments preformed in triplicate.
    Figure Legend Snippet: Internalization of B. burgdorferi by CHO-CR3 cells is facilitated by β-glucans. (A) RAW264.7 cells were incubated with 1 mM barley β-glucan (orange histogram), Laminarin β-glucan (green histogram) or S. cerevisiae β-glucan (red histogram) and Bb914 (m.o.i. = 25) for 4 hours. Control cells (black histogram) were incubated with Bb914. (B) CHO-CR3 cells were incubated with 1 mM S. cerevisiae β-glucan and Bb914 for 4 hours. The cells were fixed, stained with phalloidin Alexa Fluor 594 and analyzed by confocal microscopy. The results presented are representative of 3 experiments preformed in triplicate.

    Techniques Used: Incubation, Staining, Confocal Microscopy

    21) Product Images from "Impaired phagocytosis directs human monocyte activation in response to fungal derived β-glucan particles"

    Article Title: Impaired phagocytosis directs human monocyte activation in response to fungal derived β-glucan particles

    Journal: European journal of immunology

    doi: 10.1002/eji.201747224

    Effects of phagocytosis impairment on the response of human monocytes toward whole Candida albicans and the cell wall fraction composed of branched β-glucan. (A and B) Human monocytes were left untreated (ctrl) or stimulated with either heat-killed Candida albicans (HK-C.a., A) or heat-killed Saccharomyces cerevisiae (HK-S.c., B) in the presence (Cyt D) or in the absence (DMSO) of cytochalasin D. (C) Cells were stimulated with the Alkali-Soluble (AS) and Insoluble (AI) fractions, β-(1–6)-glucan and β-(1–3)/(1–6) glucan fractions from the cell wall of Saccharomyces cerevisiae after being treated with DMSO or Cyt D. (D) Data show the stimulation experiments of monocytes with two doses (1 or 10 μg/mL) of branched β-(1–3)-glucan isolated from C. albicans (β-glucan C.a.) or from S. cerevisiae (β-glucan S.c.) in the presence or in the absence of Cyt D. (E) Data show the stimulation experiments of monocytes with two doses (1 or 10 μg/mL) of insoluble linear β- (1–3)-glucan (curdlan) or of soluble branched β-(1–3)-glucan (laminarin) in the presence or in the absence of Cyt D. (F) The particulate branched β-(1–3)-glucan isolated from the cell wall of C. albicans was sonicated (Sonifier cell Disruptor B-30. Outputcontrol 4, Duty cycle 40%; 2 times 10 cycle with 5min on ice in between the two sets). Monocytes were then exposed to either sonicated or not-sonicated β–glucan in the presence or in the absence of Cyt D. (G) Cells pre-treated with DMSO or Cyt D were stimulated for 24 h with the indicated concentrations of β-glucan from C. albicans (β-glucan C.a.), HK-C.a., AS and AI fractions from S.c., or the combination of AS+AI (10+10 μg/mL). (H) Monocytes were pre-treated with either DMSO (control), or with cytochalasin D (Cyt D) or latrunculin B (Lat B) (actin polymerization inhibitors), or with jasplakinolide (Jsp, actin polymerization inducer), or with chlorpromazine (CLP, clathrin-mediated endocytic inhibitor) or the lysosomal inhibitor cloroquine (CLQ). Cells were then stimulated with either branched β-(1–3)-glucan isolated from C. albicans (β-glucan C.a.) or with heat-killed Candida albicans (HK-C.a.). For all the experiments, culture supernatants were collected after 24h and concentration of secreted TNF-α, IL-6, IL-1β and IL-10 was determined by ELISA. Data were reproducible for all the four cytokines tested even if histograms are not shown. Graphs show the mean ± SEM of at least three independent experiments. For (A, H), n = 8; for (B–G), n = 6; * p
    Figure Legend Snippet: Effects of phagocytosis impairment on the response of human monocytes toward whole Candida albicans and the cell wall fraction composed of branched β-glucan. (A and B) Human monocytes were left untreated (ctrl) or stimulated with either heat-killed Candida albicans (HK-C.a., A) or heat-killed Saccharomyces cerevisiae (HK-S.c., B) in the presence (Cyt D) or in the absence (DMSO) of cytochalasin D. (C) Cells were stimulated with the Alkali-Soluble (AS) and Insoluble (AI) fractions, β-(1–6)-glucan and β-(1–3)/(1–6) glucan fractions from the cell wall of Saccharomyces cerevisiae after being treated with DMSO or Cyt D. (D) Data show the stimulation experiments of monocytes with two doses (1 or 10 μg/mL) of branched β-(1–3)-glucan isolated from C. albicans (β-glucan C.a.) or from S. cerevisiae (β-glucan S.c.) in the presence or in the absence of Cyt D. (E) Data show the stimulation experiments of monocytes with two doses (1 or 10 μg/mL) of insoluble linear β- (1–3)-glucan (curdlan) or of soluble branched β-(1–3)-glucan (laminarin) in the presence or in the absence of Cyt D. (F) The particulate branched β-(1–3)-glucan isolated from the cell wall of C. albicans was sonicated (Sonifier cell Disruptor B-30. Outputcontrol 4, Duty cycle 40%; 2 times 10 cycle with 5min on ice in between the two sets). Monocytes were then exposed to either sonicated or not-sonicated β–glucan in the presence or in the absence of Cyt D. (G) Cells pre-treated with DMSO or Cyt D were stimulated for 24 h with the indicated concentrations of β-glucan from C. albicans (β-glucan C.a.), HK-C.a., AS and AI fractions from S.c., or the combination of AS+AI (10+10 μg/mL). (H) Monocytes were pre-treated with either DMSO (control), or with cytochalasin D (Cyt D) or latrunculin B (Lat B) (actin polymerization inhibitors), or with jasplakinolide (Jsp, actin polymerization inducer), or with chlorpromazine (CLP, clathrin-mediated endocytic inhibitor) or the lysosomal inhibitor cloroquine (CLQ). Cells were then stimulated with either branched β-(1–3)-glucan isolated from C. albicans (β-glucan C.a.) or with heat-killed Candida albicans (HK-C.a.). For all the experiments, culture supernatants were collected after 24h and concentration of secreted TNF-α, IL-6, IL-1β and IL-10 was determined by ELISA. Data were reproducible for all the four cytokines tested even if histograms are not shown. Graphs show the mean ± SEM of at least three independent experiments. For (A, H), n = 8; for (B–G), n = 6; * p

    Techniques Used: Isolation, Sonication, Concentration Assay, Enzyme-linked Immunosorbent Assay

    IκBα phosphorylation, IκBα degradation and p65 nuclear translocation in cytochalasin D/β-glucan stimulated monocytes. (A) Western blot analysis of phospho-IκBα and IκBα in cell lysates from monocytes treated for 6h with or without β–glucan C.a. in the presence or absence of Cyt D. GAPDH was used as a loading control. (B) Graphs represent the densitometric analysis of the blots. Immunoreactive bands were normalized to GAPDH. The bars represent mean values ± SEM of the analysis of 4 independent experiments. (C) Cells were treated as in (A) and nuclear translocation of p65 NF-κB was visualized by immunostaining with a specific antibody recognizing p65 and an alexa-fluor 488-conjugated secondary antibody (green). For reference the nuclei are stained Hoescht (blue). One representative image of three independent experiments is shown. (D) Quantification of nuclei with p65 translocation (3 × 200 nuclei analysed with Image J). Original magnification. 60×, scale bar: 50 μM. * p
    Figure Legend Snippet: IκBα phosphorylation, IκBα degradation and p65 nuclear translocation in cytochalasin D/β-glucan stimulated monocytes. (A) Western blot analysis of phospho-IκBα and IκBα in cell lysates from monocytes treated for 6h with or without β–glucan C.a. in the presence or absence of Cyt D. GAPDH was used as a loading control. (B) Graphs represent the densitometric analysis of the blots. Immunoreactive bands were normalized to GAPDH. The bars represent mean values ± SEM of the analysis of 4 independent experiments. (C) Cells were treated as in (A) and nuclear translocation of p65 NF-κB was visualized by immunostaining with a specific antibody recognizing p65 and an alexa-fluor 488-conjugated secondary antibody (green). For reference the nuclei are stained Hoescht (blue). One representative image of three independent experiments is shown. (D) Quantification of nuclei with p65 translocation (3 × 200 nuclei analysed with Image J). Original magnification. 60×, scale bar: 50 μM. * p

    Techniques Used: Translocation Assay, Western Blot, Immunostaining, Staining

    Analysis of ROS production in human monocytes with impaired phagocytosis mechanism upon β-glucan stimulation. Monocytes were pre-treated for 30 min with (A) the indicated concentrations of DPI (NADPH oxidase inhibitor) before being treated with either DMSO or Cyt D for 1h. Cells were then left untreated or stimulated with particulate β–glucan C.a. Luminol was added, and Reactive oxygen species (ROS) production was monitored by chemiluminescence during time (2h) and expressed as relative light units (RLU). Graphs show mean ± SEM corresponding to up to three independent experiments ( n = 3); each experimental condition was assessed in triplicate. (B) Monocytes were pre-treated with DPI, as indicated. After 30 min of incubation, cells were treated either with DMSO or Cyt D and, after an additional 1h, stimulated with β-glucan C.a. Culture supernatants were collected after 24h and secretion of TNF-α, IL-6 and IL-1β was determined by ELISA. Graphs show the mean + SEM of at least three independent experiments ( n = 8); ** p
    Figure Legend Snippet: Analysis of ROS production in human monocytes with impaired phagocytosis mechanism upon β-glucan stimulation. Monocytes were pre-treated for 30 min with (A) the indicated concentrations of DPI (NADPH oxidase inhibitor) before being treated with either DMSO or Cyt D for 1h. Cells were then left untreated or stimulated with particulate β–glucan C.a. Luminol was added, and Reactive oxygen species (ROS) production was monitored by chemiluminescence during time (2h) and expressed as relative light units (RLU). Graphs show mean ± SEM corresponding to up to three independent experiments ( n = 3); each experimental condition was assessed in triplicate. (B) Monocytes were pre-treated with DPI, as indicated. After 30 min of incubation, cells were treated either with DMSO or Cyt D and, after an additional 1h, stimulated with β-glucan C.a. Culture supernatants were collected after 24h and secretion of TNF-α, IL-6 and IL-1β was determined by ELISA. Graphs show the mean + SEM of at least three independent experiments ( n = 8); ** p

    Techniques Used: Incubation, Enzyme-linked Immunosorbent Assay

    The role of the Dectin-1/Syk/PI3K pathway in the overproduction of cytokines by monocytes impaired for phagocytosis and stimulated with β-glucan. Monocytes were pre-treated with anti-Dectin-1 Ab, anti-TLR2 Ab, anti-CR3 Ab or isotype control Abs in (A). Syk inhibitor in (B), or PI3K inhibitor (WRT; wortmannin) in (C), were used on monocytes as indicated. After the first incubation with inhibitors, cells were treated either with DMSO or Cyt D and finally stimulated with branched β-(1–3)-glucan isolated from C. albicans (β-glucan C.a.). Culture supernatants were collected after 24h and secretion of TNF-α, IL-6, and IL-1β was determined by ELISA. Graphs show the mean ± SEM of at least three independent experiments. For (A), n = 6; for (B) and (C), n = 9; * p
    Figure Legend Snippet: The role of the Dectin-1/Syk/PI3K pathway in the overproduction of cytokines by monocytes impaired for phagocytosis and stimulated with β-glucan. Monocytes were pre-treated with anti-Dectin-1 Ab, anti-TLR2 Ab, anti-CR3 Ab or isotype control Abs in (A). Syk inhibitor in (B), or PI3K inhibitor (WRT; wortmannin) in (C), were used on monocytes as indicated. After the first incubation with inhibitors, cells were treated either with DMSO or Cyt D and finally stimulated with branched β-(1–3)-glucan isolated from C. albicans (β-glucan C.a.). Culture supernatants were collected after 24h and secretion of TNF-α, IL-6, and IL-1β was determined by ELISA. Graphs show the mean ± SEM of at least three independent experiments. For (A), n = 6; for (B) and (C), n = 9; * p

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

    The role of the NLRP3-ASC-caspase-1 inflammasome in β-glucan induced IL-1β. (A and E) Monocytes were pre-treated with either z-VAD-fmk (pan-caspase inhibitor), z-YVAD-fmk (caspase-1 inhibitor), z-IETD-fmk (caspase-8 inhibitor), z-LEVD-fmk (caspase-4/5 inhibitor), MCC950 (NLRP3 inhibitor), glibenclamide (inhibitor of potassium (K+) efflux) or z-VRPR-fmk (MALT1 inhibitor) before treatment with either DMSO or Cyt D for 1 h. Cells were then stimulated with branched β-(1–3)-glucan isolated from C. albicans (β-glucan C.a.), culture supernatants were collected after 24 h and secretion of IL-1β was determined by ELISA. Graphs show the mean ± SEM of four independent experiments ( n = 8). ** p
    Figure Legend Snippet: The role of the NLRP3-ASC-caspase-1 inflammasome in β-glucan induced IL-1β. (A and E) Monocytes were pre-treated with either z-VAD-fmk (pan-caspase inhibitor), z-YVAD-fmk (caspase-1 inhibitor), z-IETD-fmk (caspase-8 inhibitor), z-LEVD-fmk (caspase-4/5 inhibitor), MCC950 (NLRP3 inhibitor), glibenclamide (inhibitor of potassium (K+) efflux) or z-VRPR-fmk (MALT1 inhibitor) before treatment with either DMSO or Cyt D for 1 h. Cells were then stimulated with branched β-(1–3)-glucan isolated from C. albicans (β-glucan C.a.), culture supernatants were collected after 24 h and secretion of IL-1β was determined by ELISA. Graphs show the mean ± SEM of four independent experiments ( n = 8). ** p

    Techniques Used: Isolation, Enzyme-linked Immunosorbent Assay

    Schematic model illustrating potential signaling pathways triggered by the binding of β–glucan to dectin-1 in human monocytes with normal (left) or impaired phagocytic (right) function. Attenuation of dectin-1 signaling is associated with the engulfment of the receptor/ligand complex. By contrast, blocking of actin-dependent phagocytosis led to increased cell activation. Black arrows depict the interactions that normally occur upon phagocytosis and β–glucan/dectin-1 interaction, whereas red arrows indicate the proposed signaling events that enhance the strength and efficacy of signal propagation.
    Figure Legend Snippet: Schematic model illustrating potential signaling pathways triggered by the binding of β–glucan to dectin-1 in human monocytes with normal (left) or impaired phagocytic (right) function. Attenuation of dectin-1 signaling is associated with the engulfment of the receptor/ligand complex. By contrast, blocking of actin-dependent phagocytosis led to increased cell activation. Black arrows depict the interactions that normally occur upon phagocytosis and β–glucan/dectin-1 interaction, whereas red arrows indicate the proposed signaling events that enhance the strength and efficacy of signal propagation.

    Techniques Used: Binding Assay, Blocking Assay, Activation Assay

    Syk activity assessement for inflammasome, and NF-κB activation in phagocytosis-impaired-monocyte stimulated with C. albicans branched β-(1–3)-glucan. (A) Monocytes treated for 6 h with β–glucan C.a. in the presence or absence of Cyt D and of the Syk inhibitor (2 μM). Nuclear translocation of p65 NF-κB was visualized by immunostaining with a specific antibody recognizing p65 and a alexa-fluor 488-conjugated secondary antibody (green). For reference the nuclei are stained Hoescht (blue). Original magnification. 60×, scalebar: 50 μMImage is representative of three independent experiments. (B) Quantification of nuclei with p65 translocation (4 × 50 nuclei analysed with Image J). (C) Cells were treated as in (A). Caspase-1 (pro-form and cleaved p20), and IL-1β (pro- and mature IL-1β) expressions were assessed by western blot. Mature IL-1β and caspase-1 were also detected in cell culture supernatants. GAPDH was used as the loading control. Blots are representative of three independent experiments.
    Figure Legend Snippet: Syk activity assessement for inflammasome, and NF-κB activation in phagocytosis-impaired-monocyte stimulated with C. albicans branched β-(1–3)-glucan. (A) Monocytes treated for 6 h with β–glucan C.a. in the presence or absence of Cyt D and of the Syk inhibitor (2 μM). Nuclear translocation of p65 NF-κB was visualized by immunostaining with a specific antibody recognizing p65 and a alexa-fluor 488-conjugated secondary antibody (green). For reference the nuclei are stained Hoescht (blue). Original magnification. 60×, scalebar: 50 μMImage is representative of three independent experiments. (B) Quantification of nuclei with p65 translocation (4 × 50 nuclei analysed with Image J). (C) Cells were treated as in (A). Caspase-1 (pro-form and cleaved p20), and IL-1β (pro- and mature IL-1β) expressions were assessed by western blot. Mature IL-1β and caspase-1 were also detected in cell culture supernatants. GAPDH was used as the loading control. Blots are representative of three independent experiments.

    Techniques Used: Activity Assay, Activation Assay, Translocation Assay, Immunostaining, Staining, Western Blot, Cell Culture

    22) Product Images from "Varying Effects of Different ?-Glucans on the Maturation of Porcine Monocyte-Derived Dendritic Cells ▿"

    Article Title: Varying Effects of Different ?-Glucans on the Maturation of Porcine Monocyte-Derived Dendritic Cells ▿

    Journal: Clinical and Vaccine Immunology : CVI

    doi: 10.1128/CVI.00080-11

    Analysis of the phagocytic activity of β-glucan-stimulated MoDCs. Immature MoDCs were stimulated with 5 μg ( ) or 10 μg (▪) of β-glucan/ml or LPS (1 and 10 μg/ml), and the uptake of ova-dQ was assayed by flow cytometry. Mean fluorescence intensity (MFI) values were calculated by subtracting the MFI values obtained at 4°C from those obtained at 37°C. The data are shown as the means ± the SEM for four pigs. Asterisks (*) indicate a significant difference between β-glucan-stimulated MoDCs and immature MoDCs (ImDCs) (for all β-glucans P
    Figure Legend Snippet: Analysis of the phagocytic activity of β-glucan-stimulated MoDCs. Immature MoDCs were stimulated with 5 μg ( ) or 10 μg (▪) of β-glucan/ml or LPS (1 and 10 μg/ml), and the uptake of ova-dQ was assayed by flow cytometry. Mean fluorescence intensity (MFI) values were calculated by subtracting the MFI values obtained at 4°C from those obtained at 37°C. The data are shown as the means ± the SEM for four pigs. Asterisks (*) indicate a significant difference between β-glucan-stimulated MoDCs and immature MoDCs (ImDCs) (for all β-glucans P

    Techniques Used: Activity Assay, Flow Cytometry, Cytometry, Fluorescence

    Analysis of the expression of CD80/86 (top panel), CD40 (middle panel), and MHCII (bottom panel) after stimulation of immature MoDCs with 5 μg ( ) or 10 μg (▪) of β-glucan/ml or LPS (1 and 10 μg/ml). The expression of the maturation markers was assayed by flow cytometry. The data are shown as the means ± the standard errors of the mean (SEM) for four pigs. Asterisks (*) indicate a significant difference between β-glucan-stimulated MoDCs and immature MoDCs (ImDCs) ( P
    Figure Legend Snippet: Analysis of the expression of CD80/86 (top panel), CD40 (middle panel), and MHCII (bottom panel) after stimulation of immature MoDCs with 5 μg ( ) or 10 μg (▪) of β-glucan/ml or LPS (1 and 10 μg/ml). The expression of the maturation markers was assayed by flow cytometry. The data are shown as the means ± the standard errors of the mean (SEM) for four pigs. Asterisks (*) indicate a significant difference between β-glucan-stimulated MoDCs and immature MoDCs (ImDCs) ( P

    Techniques Used: Expressing, Flow Cytometry, Cytometry

    Analysis of the ability of β-glucan-stimulated MoDCs to enhance T-cell proliferation. Porcine MoDCs were left untreated or stimulated for 24 h with 5 μg ( ) or 10 μg (▪) of different β-glucans/ml or LPS (1 or 10 μg/ml) and then added to CD172a-depleted PBMC (lymphocytes). After 5 days, the cultures were pulsed with 1 μCi of [ 3 H]methyl-thymidine. T-cell proliferation was measured after an additional coculture of 18 h. The data are shown as means ± the SEM for four pigs. Asterisks (*) indicate a significant difference between β-glucan-stimulated conditions and the untreated condition (ImDCs) ( P
    Figure Legend Snippet: Analysis of the ability of β-glucan-stimulated MoDCs to enhance T-cell proliferation. Porcine MoDCs were left untreated or stimulated for 24 h with 5 μg ( ) or 10 μg (▪) of different β-glucans/ml or LPS (1 or 10 μg/ml) and then added to CD172a-depleted PBMC (lymphocytes). After 5 days, the cultures were pulsed with 1 μCi of [ 3 H]methyl-thymidine. T-cell proliferation was measured after an additional coculture of 18 h. The data are shown as means ± the SEM for four pigs. Asterisks (*) indicate a significant difference between β-glucan-stimulated conditions and the untreated condition (ImDCs) ( P

    Techniques Used:

    23) Product Images from "Autophagy Controls BCG-Induced Trained Immunity and the Response to Intravesical BCG Therapy for Bladder Cancer"

    Article Title: Autophagy Controls BCG-Induced Trained Immunity and the Response to Intravesical BCG Therapy for Bladder Cancer

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1004485

    Role of autophagy for the training of monocytes. (a) Transcriptome profiling and pathway analysis of β-glucan training of monocytes compared to LPS stimulation. Factorial design analysis was performed on genes in each K-means cluster to assess significance of response differences elicited by LPS and β-glucan (Benjamini-Hochberg (BH)-adjusted p
    Figure Legend Snippet: Role of autophagy for the training of monocytes. (a) Transcriptome profiling and pathway analysis of β-glucan training of monocytes compared to LPS stimulation. Factorial design analysis was performed on genes in each K-means cluster to assess significance of response differences elicited by LPS and β-glucan (Benjamini-Hochberg (BH)-adjusted p

    Techniques Used:

    24) Product Images from "Dendritic cell-derived VEGF-A plays a role in inflammatory angiogenesis of human secondary lymphoid organs and is driven by the coordinated activation of multiple transcription factors"

    Article Title: Dendritic cell-derived VEGF-A plays a role in inflammatory angiogenesis of human secondary lymphoid organs and is driven by the coordinated activation of multiple transcription factors

    Journal: Oncotarget

    doi: 10.18632/oncotarget.9684

    Human myeloid DCs produce VEGF-A in response to a variety of pro-inflammatory stimuli, provided PGE 2 is present in the microenvironment A. , B. DCs were stimulated for 24 hours with TLR-ligands PAM 3 CSK 4 (TLR1/2, 100 ng/ml), FSL-1 (TLR2/6, 100 ng/ml), Poly I:C (TLR3, 25 μg/ml), LPS (TLR4, 100 ng/ml) and R848 (TLR7 and TLR8, 5 μg/ml), Heat-killed S.aureus (specific for TLR2; 1:10 DC/bacteria ratio), E. coli (specific for TLR4; 1:10 DC/bacteria ratio), β-glucan (10 μg/ml), Curdlan (10 μg/ml), heat-killed C.albicans (specific for C-type lectins; 1:10 DC/fungi ratio), IL-1β (20 ng/ml), TNF-α (20 ng/ml), and necrotic cells (1:2 DC/necrotic cells ratio) in the presence or absence of PGE 2 (10 μM). VEGF-A production was evaluated in cell-free supernatants by ELISA. Data are expressed as mean + SEM ( n = 4); * p
    Figure Legend Snippet: Human myeloid DCs produce VEGF-A in response to a variety of pro-inflammatory stimuli, provided PGE 2 is present in the microenvironment A. , B. DCs were stimulated for 24 hours with TLR-ligands PAM 3 CSK 4 (TLR1/2, 100 ng/ml), FSL-1 (TLR2/6, 100 ng/ml), Poly I:C (TLR3, 25 μg/ml), LPS (TLR4, 100 ng/ml) and R848 (TLR7 and TLR8, 5 μg/ml), Heat-killed S.aureus (specific for TLR2; 1:10 DC/bacteria ratio), E. coli (specific for TLR4; 1:10 DC/bacteria ratio), β-glucan (10 μg/ml), Curdlan (10 μg/ml), heat-killed C.albicans (specific for C-type lectins; 1:10 DC/fungi ratio), IL-1β (20 ng/ml), TNF-α (20 ng/ml), and necrotic cells (1:2 DC/necrotic cells ratio) in the presence or absence of PGE 2 (10 μM). VEGF-A production was evaluated in cell-free supernatants by ELISA. Data are expressed as mean + SEM ( n = 4); * p

    Techniques Used: Enzyme-linked Immunosorbent Assay

    25) Product Images from "The fungal-specific β-glucan-binding lectin FGB1 alters cell-wall composition and suppresses glucan-triggered immunity in plants"

    Article Title: The fungal-specific β-glucan-binding lectin FGB1 alters cell-wall composition and suppresses glucan-triggered immunity in plants

    Journal: Nature Communications

    doi: 10.1038/ncomms13188

    FGB1 specifically binds to β-glucan and to the disaccharide gentiobiose. ( a ) ITC profiles showing titrations of laminarihexaose [(Glc-β-1,3-Glc) 3 ] (black line), gentiobiose (Glc-β-1,6-Glc) (blue line) and laminarin (red line) to FGB1. The laminarin titration was fitted using a model that allows binding to two equivalent and independent sites. Assuming a molecular mass of laminarin of 5 kDa the following parameters were obtained: N 1 =6.8±1.09, K 1 =2.14 × 10 6 ±1.97 × 10 7 M −1 , Δ H 1 =−508±128 cal mol −1 , Δ S 1 =27.2 cal mol −1 per degree and N 2 =1.02±0.9, K 2 =1.19 × 10 7 ±6.11 × 10 7 M −1 , Δ H 2 =−1971±251 cal mol −1 , Δ S 2 =25.7 cal mol −1 per degree. The titration of gentiobiose to FGB1 was fitted to a single-side-binding model ( N =0.854±0.243, K =8.79 × 10 4 ±6.88 × 10 5 M −1 , Δ H =−563.9±241 cal mol −1 , Δ S =19.4 cal mol −1 per degree). No binding of FGB1 to laminarihexaose was observed. Concentrations of the stock solutions are indicated. All titrations were baseline corrected and substracted with the corresponding control titration of ligand into water. Errors correspond to the s.d. of the nonlinear least-squares fit of the data points of the titration curve. ( b ) The circular dichroism-spectrum of 15 μM FGB1 (black square) shows a significant shift in the secondary structure in the presence of 1 mM gentiobiose (blue square) or 100 μM laminarin (red square). Spectra were corrected with the spectra obtained for the individual ligands (see Supplementary Fig. 4 for further details).
    Figure Legend Snippet: FGB1 specifically binds to β-glucan and to the disaccharide gentiobiose. ( a ) ITC profiles showing titrations of laminarihexaose [(Glc-β-1,3-Glc) 3 ] (black line), gentiobiose (Glc-β-1,6-Glc) (blue line) and laminarin (red line) to FGB1. The laminarin titration was fitted using a model that allows binding to two equivalent and independent sites. Assuming a molecular mass of laminarin of 5 kDa the following parameters were obtained: N 1 =6.8±1.09, K 1 =2.14 × 10 6 ±1.97 × 10 7 M −1 , Δ H 1 =−508±128 cal mol −1 , Δ S 1 =27.2 cal mol −1 per degree and N 2 =1.02±0.9, K 2 =1.19 × 10 7 ±6.11 × 10 7 M −1 , Δ H 2 =−1971±251 cal mol −1 , Δ S 2 =25.7 cal mol −1 per degree. The titration of gentiobiose to FGB1 was fitted to a single-side-binding model ( N =0.854±0.243, K =8.79 × 10 4 ±6.88 × 10 5 M −1 , Δ H =−563.9±241 cal mol −1 , Δ S =19.4 cal mol −1 per degree). No binding of FGB1 to laminarihexaose was observed. Concentrations of the stock solutions are indicated. All titrations were baseline corrected and substracted with the corresponding control titration of ligand into water. Errors correspond to the s.d. of the nonlinear least-squares fit of the data points of the titration curve. ( b ) The circular dichroism-spectrum of 15 μM FGB1 (black square) shows a significant shift in the secondary structure in the presence of 1 mM gentiobiose (blue square) or 100 μM laminarin (red square). Spectra were corrected with the spectra obtained for the individual ligands (see Supplementary Fig. 4 for further details).

    Techniques Used: Gas Chromatography, Titration, Binding Assay

    FGB1 supresses β-glucan-induced oxidative burst in barley and A. thaliana Ws-0. ( a ) Barley leaf disks react with a strong ROS burst after elicitation with 600 μM complex laminarin (red square). Incubation with 10 μM FGB1 supress ROS burst (black square). Neither 10 μM FGB1 (blue square) nor the mock water control (grey square) induce ROS production. ( b ) Leaf disks of A. thaliana ecotype Ws-0 react with ROS production after elicitation with 600 μM laminarin. Comparable to barley, Ws-0 ROS production was supressed by 10 μM FGB1. At least four independent repetitions with three different FGB1 batches were carried out and showed similar results. Error bars represent the standard error of the mean of 8–12 technical replicates (see Supplementary Fig. 10 for further details).
    Figure Legend Snippet: FGB1 supresses β-glucan-induced oxidative burst in barley and A. thaliana Ws-0. ( a ) Barley leaf disks react with a strong ROS burst after elicitation with 600 μM complex laminarin (red square). Incubation with 10 μM FGB1 supress ROS burst (black square). Neither 10 μM FGB1 (blue square) nor the mock water control (grey square) induce ROS production. ( b ) Leaf disks of A. thaliana ecotype Ws-0 react with ROS production after elicitation with 600 μM laminarin. Comparable to barley, Ws-0 ROS production was supressed by 10 μM FGB1. At least four independent repetitions with three different FGB1 batches were carried out and showed similar results. Error bars represent the standard error of the mean of 8–12 technical replicates (see Supplementary Fig. 10 for further details).

    Techniques Used: Incubation

    Model displaying the potential dual function of FGB1. Binding of FGB1 to the fungal cell wall (CW) after secretion is mediated through β-1,6-glycosidic linkages. FGB1 modulates CW polysaccharides composition. FGB1 does not protect the CW against β-1,3-endo- and β-1,3-exo-glucanase activities. Host glucanases activity release β-glucan fragments that can be sensed by a specific plant β-glucan receptor complex. Recognition leads to activation of basal defence mechanisms, such as the production of reactive oxygen species (ROS). Based on the observation that FGB1 is able to suppress β-glucan induced ROS production at substoichiometric concentrations, we hypothize that FGB1/β-glucan complexes have higher affinities to the plant β-glucan receptors than free β-glucan fragments. Binding of FGB1/β-glucan complexes to the β-glucan receptor would possibly prevent receptor/co-receptor association and defense downstream signalling.
    Figure Legend Snippet: Model displaying the potential dual function of FGB1. Binding of FGB1 to the fungal cell wall (CW) after secretion is mediated through β-1,6-glycosidic linkages. FGB1 modulates CW polysaccharides composition. FGB1 does not protect the CW against β-1,3-endo- and β-1,3-exo-glucanase activities. Host glucanases activity release β-glucan fragments that can be sensed by a specific plant β-glucan receptor complex. Recognition leads to activation of basal defence mechanisms, such as the production of reactive oxygen species (ROS). Based on the observation that FGB1 is able to suppress β-glucan induced ROS production at substoichiometric concentrations, we hypothize that FGB1/β-glucan complexes have higher affinities to the plant β-glucan receptors than free β-glucan fragments. Binding of FGB1/β-glucan complexes to the β-glucan receptor would possibly prevent receptor/co-receptor association and defense downstream signalling.

    Techniques Used: Binding Assay, Activity Assay, Activation Assay

    26) Product Images from "Dectin-1 isoforms contribute to distinct Th1/Th17 cell activation in mucosal candidiasis"

    Article Title: Dectin-1 isoforms contribute to distinct Th1/Th17 cell activation in mucosal candidiasis

    Journal: Cellular and Molecular Immunology

    doi: 10.1038/cmi.2012.1

    Dectin-1 deficiency differentially affects cytokine induction in response to β-glucan stimulation in vitro . Expression of Tnfa , Il12p35 , Il23p19 and Il10 in ex vivo Peyer's patches from naive wild-type and Dectin-1 –/– mice from ( a ) C57BL/6 or ( b ) BALB/c backgrounds, either unstimulated (−) or stimulated (+) in vitro with β-glucan for 18 h. * P ≤0.05, ** P ≤0.01 and *** P ≤0.001, unstimulated vs. stimulated Peyer's patches from either background, as indicated.
    Figure Legend Snippet: Dectin-1 deficiency differentially affects cytokine induction in response to β-glucan stimulation in vitro . Expression of Tnfa , Il12p35 , Il23p19 and Il10 in ex vivo Peyer's patches from naive wild-type and Dectin-1 –/– mice from ( a ) C57BL/6 or ( b ) BALB/c backgrounds, either unstimulated (−) or stimulated (+) in vitro with β-glucan for 18 h. * P ≤0.05, ** P ≤0.01 and *** P ≤0.001, unstimulated vs. stimulated Peyer's patches from either background, as indicated.

    Techniques Used: In Vitro, Expressing, Ex Vivo, Mouse Assay

    Dectin-1 differentially impacts the AhR/IL-22 axis in the different mouse strains. ( a , c ) Ahr expression and IL-22 production in vivo or ( b , d ) in vitro in wild-type and Dectin-1 –/– mice from the ( a , b ) C57BL/6 or ( c , d ) BALB/c mice infected i.g. with C. albicans Vir 3 at 4 dpi. Ahr and Il22 expression (real-time RT-PCR) and IL-22 production (ELISA) were performed on ex vivo Peyer's patches cells from naive (−) or infected (+) mice or in cells from naive mice stimulated in vitro with β-glucan or FICZ for 18 h. * P ≤0.05, ** P ≤0.01 and *** P ≤0.001, wild-type vs. Dectin-1 –/– mice in either background, as indicated. C. albicans ; Candida albicans ; dpi, days post-infection; FICZ, 6-formylindolo[3,2-b]carbazole; i.g., intragastrically; RT-PCR, reverse transcriptase PCR.
    Figure Legend Snippet: Dectin-1 differentially impacts the AhR/IL-22 axis in the different mouse strains. ( a , c ) Ahr expression and IL-22 production in vivo or ( b , d ) in vitro in wild-type and Dectin-1 –/– mice from the ( a , b ) C57BL/6 or ( c , d ) BALB/c mice infected i.g. with C. albicans Vir 3 at 4 dpi. Ahr and Il22 expression (real-time RT-PCR) and IL-22 production (ELISA) were performed on ex vivo Peyer's patches cells from naive (−) or infected (+) mice or in cells from naive mice stimulated in vitro with β-glucan or FICZ for 18 h. * P ≤0.05, ** P ≤0.01 and *** P ≤0.001, wild-type vs. Dectin-1 –/– mice in either background, as indicated. C. albicans ; Candida albicans ; dpi, days post-infection; FICZ, 6-formylindolo[3,2-b]carbazole; i.g., intragastrically; RT-PCR, reverse transcriptase PCR.

    Techniques Used: Expressing, In Vivo, In Vitro, Mouse Assay, Infection, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Ex Vivo, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction

    27) Product Images from "Modulation of Myelopoiesis Progenitors Is an Integral Component of Trained Immunity"

    Article Title: Modulation of Myelopoiesis Progenitors Is an Integral Component of Trained Immunity

    Journal: Cell

    doi: 10.1016/j.cell.2017.11.034

    Training with β-Glucan Promotes a Beneficial Response to a Secondary Challenge (A) WT mice were injected with β-glucan or PBS, and after 28 days, they received a secondary challenge with LPS. (B and C) LSK, MPP, and LT-HSC cell numbers (B) and frequency of the same cells in total BM cells (C) at 24 hr after LPS injection (n = 10 mice per group). (D) Representative FACS plots and frequency of γ-H2AX-positive LT-HSCs at 24 hr after LPS injection (n = 10 mice per group, right plots and gray background). The frequency of γ-H2AX-positive LT-HSCs at 28 days after β-glucan administration in mice not injected with LPS (—) is also shown; ns = 4 and 5, left plots and white background. (E) Experimental protocol for the effect of β-glucan on the recovery of granulopoiesis after cyclophosphamide administration (4 rounds). (F and G) Total white blood cell (WBC) (F) and granulocyte (Gr1 + CD11b + ) (G) counts in the peripheral blood (n = 10 mice per group). (H) Experimental protocol for 5-FU administration. (I) Survival curves of 5-FU-treated mice treated with β-glucan or PBS control (n = 16 mice per group). Comparison of survival curves was performed by log-rank (Mantel-Cox) test, and p value is shown. (J and K) Mice were injected with β-glucan or PBS, and 7 days later, a single dose of 5-FU was administered. (J) Neutrophil numbers in peripheral blood at different time points after the administration of 5-FU (n = 5 mice per group). (K) Frequency of γ-H2AX-positive LT-HSCs 14 days after 5-FU administration (n = 10 mice per group). Data are presented as mean ± SEM. ∗ p
    Figure Legend Snippet: Training with β-Glucan Promotes a Beneficial Response to a Secondary Challenge (A) WT mice were injected with β-glucan or PBS, and after 28 days, they received a secondary challenge with LPS. (B and C) LSK, MPP, and LT-HSC cell numbers (B) and frequency of the same cells in total BM cells (C) at 24 hr after LPS injection (n = 10 mice per group). (D) Representative FACS plots and frequency of γ-H2AX-positive LT-HSCs at 24 hr after LPS injection (n = 10 mice per group, right plots and gray background). The frequency of γ-H2AX-positive LT-HSCs at 28 days after β-glucan administration in mice not injected with LPS (—) is also shown; ns = 4 and 5, left plots and white background. (E) Experimental protocol for the effect of β-glucan on the recovery of granulopoiesis after cyclophosphamide administration (4 rounds). (F and G) Total white blood cell (WBC) (F) and granulocyte (Gr1 + CD11b + ) (G) counts in the peripheral blood (n = 10 mice per group). (H) Experimental protocol for 5-FU administration. (I) Survival curves of 5-FU-treated mice treated with β-glucan or PBS control (n = 16 mice per group). Comparison of survival curves was performed by log-rank (Mantel-Cox) test, and p value is shown. (J and K) Mice were injected with β-glucan or PBS, and 7 days later, a single dose of 5-FU was administered. (J) Neutrophil numbers in peripheral blood at different time points after the administration of 5-FU (n = 5 mice per group). (K) Frequency of γ-H2AX-positive LT-HSCs 14 days after 5-FU administration (n = 10 mice per group). Data are presented as mean ± SEM. ∗ p

    Techniques Used: Mouse Assay, Injection, FACS

    β-Glucan-Induced Alterations in LT-HSC Metabolic Pathways Revealed by Transcriptomic Analysis (A–F) Transcriptome analysis in LT-HSCs sorted from mice on day 7 after β-glucan or PBS administration (n = 4 mice, PBS group; n = 3 mice, β-glucan group). (A) Differential gene expression in LT-HSCs from β-glucan-treated mice as compared to PBS-treated mice. Volcano plot showing the distribution of the adjusted p values (−log(P-adj.)) and the fold changes (log 2 fold change). Significant changes are indicated in red (FDR = 0.05). (B) Top overrepresented canonical pathways showing upregulated (red) or downregulated (blue) genes in LT-HSCs from β-glucan-treated mice, as compared to PBS-treated mice. (C) Heatmap of myeloid- and lymphoid-lineage-related genes. (D) Heatmap depicting the differential gene expression of transcription regulators. Log 2 fold change in cells derived from β-glucan-treated mice, as compared to PBS-treated mice, is indicated. (E) GSEA for glycolytic genes and genes related to cholesterol homeostasis. NES, normalized enrichment score. (F) Heatmap of genes involved in glycolysis and pentose phosphate pathway (PPP) and cholesterol homeostasis in LT-HSCs from β-glucan-treated mice compared to PBS-treated mice. (G) Bioenergetic extracellular flux analysis (Seahorse) in Lin − cKit + BM progenitors sorted from mice 24 hr after β-glucan or PBS administration (n = 5 mice per group). Basal and maximal ECARs (after oligomycin) (left) and glycolytic reserve (right), calculated as the difference between basal and maximal ECARs, are indicated. (H) Glycolytic gene expression in LSKs from mice 24 hr after β-glucan or PBS administration using qPCR (ns = 4 and 5 mice). (I) Bioenergetic extracellular flux analysis of Lin − cKit + cells sorted from mice at 7 days after β-glucan administration; glycolytic reserve is shown (n = 5 mice per group). Data are presented as mean ± SEM in (G)–(I). ∗ p
    Figure Legend Snippet: β-Glucan-Induced Alterations in LT-HSC Metabolic Pathways Revealed by Transcriptomic Analysis (A–F) Transcriptome analysis in LT-HSCs sorted from mice on day 7 after β-glucan or PBS administration (n = 4 mice, PBS group; n = 3 mice, β-glucan group). (A) Differential gene expression in LT-HSCs from β-glucan-treated mice as compared to PBS-treated mice. Volcano plot showing the distribution of the adjusted p values (−log(P-adj.)) and the fold changes (log 2 fold change). Significant changes are indicated in red (FDR = 0.05). (B) Top overrepresented canonical pathways showing upregulated (red) or downregulated (blue) genes in LT-HSCs from β-glucan-treated mice, as compared to PBS-treated mice. (C) Heatmap of myeloid- and lymphoid-lineage-related genes. (D) Heatmap depicting the differential gene expression of transcription regulators. Log 2 fold change in cells derived from β-glucan-treated mice, as compared to PBS-treated mice, is indicated. (E) GSEA for glycolytic genes and genes related to cholesterol homeostasis. NES, normalized enrichment score. (F) Heatmap of genes involved in glycolysis and pentose phosphate pathway (PPP) and cholesterol homeostasis in LT-HSCs from β-glucan-treated mice compared to PBS-treated mice. (G) Bioenergetic extracellular flux analysis (Seahorse) in Lin − cKit + BM progenitors sorted from mice 24 hr after β-glucan or PBS administration (n = 5 mice per group). Basal and maximal ECARs (after oligomycin) (left) and glycolytic reserve (right), calculated as the difference between basal and maximal ECARs, are indicated. (H) Glycolytic gene expression in LSKs from mice 24 hr after β-glucan or PBS administration using qPCR (ns = 4 and 5 mice). (I) Bioenergetic extracellular flux analysis of Lin − cKit + cells sorted from mice at 7 days after β-glucan administration; glycolytic reserve is shown (n = 5 mice per group). Data are presented as mean ± SEM in (G)–(I). ∗ p

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

    Sustained Increase in Myelopoiesis upon β-Glucan Administration (A–F) WT mice were injected with β-glucan or PBS, and BM analysis was performed after 7 days. (A) LSK, MPP and LT-HSC cell numbers in the BM of mice on day 7 after administration of PBS or β-glucan (n = 6 mice per group). (B) Frequency of MPP subpopulations in the LSK cells 7 days after β-glucan or PBS administration (n = 5 mice per group). (C) Frequency of CD41 + LT-HSCs (in total LT-HSCs) on day 7 after the administration of PBS or β-glucan (n = 5 mice per group). (D) Representative FACS plots for the identification of MyP subpopulations. (E and F) GMP cell numbers (E) and frequency within the MyP pool of GMPs (Lin − c-Kit + Sca1 − CD16/32 + CD34 + ) and CMPs (Lin − c-Kit + Sca1 − CD16/32 − CD34 + ) (F) in the BM of mice on day 7 after the administration of PBS or β-glucan (n = 6 mice per group). (G–I) WT mice were injected with β-glucan or PBS, and BM analysis was performed after 28 days. (G) LSK and LT-HSC cell numbers (n = 5 mice per group). (H and I) Frequency of MPP4 cells in total BM cells (H) and GMP cell numbers in the BM (I) of mice on day 28 after the administration of PBS or β-glucan (n = 5 mice per group). (J and K) Transplantation. (J) LT-HSCs (CD45.2 + ) were sorted 28 days after β-glucan or PBS administration and transplanted to lethally irradiated SJL/BL6 (CD45.1 + ) mice. CD45.1 + BM cells were co-transplanted in order to ensure the survival of recipients. (K) Lineage output of donor LT-HSCs (CD45.2 + ) in peripheral blood of recipients at week 12 post-transplant (n = 10 recipient mice per group). Data are presented as mean ± SEM. ∗ p
    Figure Legend Snippet: Sustained Increase in Myelopoiesis upon β-Glucan Administration (A–F) WT mice were injected with β-glucan or PBS, and BM analysis was performed after 7 days. (A) LSK, MPP and LT-HSC cell numbers in the BM of mice on day 7 after administration of PBS or β-glucan (n = 6 mice per group). (B) Frequency of MPP subpopulations in the LSK cells 7 days after β-glucan or PBS administration (n = 5 mice per group). (C) Frequency of CD41 + LT-HSCs (in total LT-HSCs) on day 7 after the administration of PBS or β-glucan (n = 5 mice per group). (D) Representative FACS plots for the identification of MyP subpopulations. (E and F) GMP cell numbers (E) and frequency within the MyP pool of GMPs (Lin − c-Kit + Sca1 − CD16/32 + CD34 + ) and CMPs (Lin − c-Kit + Sca1 − CD16/32 − CD34 + ) (F) in the BM of mice on day 7 after the administration of PBS or β-glucan (n = 6 mice per group). (G–I) WT mice were injected with β-glucan or PBS, and BM analysis was performed after 28 days. (G) LSK and LT-HSC cell numbers (n = 5 mice per group). (H and I) Frequency of MPP4 cells in total BM cells (H) and GMP cell numbers in the BM (I) of mice on day 28 after the administration of PBS or β-glucan (n = 5 mice per group). (J and K) Transplantation. (J) LT-HSCs (CD45.2 + ) were sorted 28 days after β-glucan or PBS administration and transplanted to lethally irradiated SJL/BL6 (CD45.1 + ) mice. CD45.1 + BM cells were co-transplanted in order to ensure the survival of recipients. (K) Lineage output of donor LT-HSCs (CD45.2 + ) in peripheral blood of recipients at week 12 post-transplant (n = 10 recipient mice per group). Data are presented as mean ± SEM. ∗ p

    Techniques Used: Mouse Assay, Injection, FACS, Transplantation Assay, Irradiation

    Transcriptional Alterations in Hematopoietic Progenitors 28 Days after β-Glucan Administration, Related to Figure 5 LT-HSCs and MPPs were sorted from mice on day 28 after β-glucan or PBS administration and RNA sequencing was performed (n = 4 mice per group). (A and C) Differential gene expression in (A) LT-HSCs and (C) MPPs; volcano plots depicting the distribution of the adjusted p values (-log(P-adj.)) and the fold changes (log2 Fold Change). Significant changes are colored red (FDR = 0.05). (B and D) Overrepresented canonical pathways showing genes that were upregulated (red) or downregulated (blue) in (B) LT-HSCs and (D) MPPs from β-glucan–injected mice, as compared to PBS-treated mice. (E) Heatmap depicting the expression of lymphoid lineage-related genes in LT-HSCs and MPPs from β-glucan injected mice, as compared to PBS treated mice. Transcripts that are not significantly altered are shown in gray. (F) Differentially expressed genes in both LT-HSCs and MPPs. The number of upregulated (red) and downregulated (blue) genes is shown.
    Figure Legend Snippet: Transcriptional Alterations in Hematopoietic Progenitors 28 Days after β-Glucan Administration, Related to Figure 5 LT-HSCs and MPPs were sorted from mice on day 28 after β-glucan or PBS administration and RNA sequencing was performed (n = 4 mice per group). (A and C) Differential gene expression in (A) LT-HSCs and (C) MPPs; volcano plots depicting the distribution of the adjusted p values (-log(P-adj.)) and the fold changes (log2 Fold Change). Significant changes are colored red (FDR = 0.05). (B and D) Overrepresented canonical pathways showing genes that were upregulated (red) or downregulated (blue) in (B) LT-HSCs and (D) MPPs from β-glucan–injected mice, as compared to PBS-treated mice. (E) Heatmap depicting the expression of lymphoid lineage-related genes in LT-HSCs and MPPs from β-glucan injected mice, as compared to PBS treated mice. Transcripts that are not significantly altered are shown in gray. (F) Differentially expressed genes in both LT-HSCs and MPPs. The number of upregulated (red) and downregulated (blue) genes is shown.

    Techniques Used: Mouse Assay, RNA Sequencing Assay, Expressing, Injection

    GSEA in LT-HSC after 5-FU Administration, Related to Figure 5 Mice were injected with β-glucan or PBS and 7 days later a single dose of 5-FU was administered. LT-HSCs were sorted from mice on day 14 after 5-FU administration and transcriptomic analysis was performed (n = 4 mice per group). GSEA for genes related to glycolysis, OXPHOS and cholesterol biosynthesis.
    Figure Legend Snippet: GSEA in LT-HSC after 5-FU Administration, Related to Figure 5 Mice were injected with β-glucan or PBS and 7 days later a single dose of 5-FU was administered. LT-HSCs were sorted from mice on day 14 after 5-FU administration and transcriptomic analysis was performed (n = 4 mice per group). GSEA for genes related to glycolysis, OXPHOS and cholesterol biosynthesis.

    Techniques Used: Mouse Assay, Injection

    IL-1β, Glycolysis, Cholesterol Metabolism, and the GM-CSF/CD131 Axis Are Involved in β-Glucan-Dependent Training in the BM (A) Cytokine and G-CSF concentrations in the BM extracellular fluid of mice at 24 hr after the administration of PBS or β-glucan (n = 10 mice per group). (B and C) Mice were injected with β-glucan in the absence (vehicle control, Ctrl) or presence of IL1RA, and BM analysis was performed 24 hr later. (B) Cell-cycle analysis in LT-HSCs. (C) The frequency of MPP subpopulations in LSK cells in the BM is indicated (n = 5 mice per group). (D) Bioenergetic extracellular flux analysis in Lin − cKit + cells sorted from mice 24 hr after β-glucan administration in the absence (vehicle control, Ctrl) or presence of IL1RA (n = 5 mice per group). Basal and maximal ECAR are indicated. (E) LSK cells were treated in vitro with IL-1β or PBS for 24 hr, and Seahorse analysis was performed (n = 5 cultures per group). Basal and maximal ECARs (left) and glycolytic reserve (right) are indicated. (F–I) Mice were injected with β-glucan on day 0. (F) Glycolysis and IL-1 were blocked by the administration of 2-DG and IL1RA, respectively, on days 0 and 1; PBS served as the vehicle control (Ctrl). (G) LSK, MPP, and LT-HSC numbers in the BM at day 7 after β-glucan administration (n = 5 mice per group). (H and I) GMP numbers in the BM (H) and frequency of GMPs within the MyP pool (I) at day 7 after β-glucan administration (n = 5 mice per group). (J and K) Mice were injected with PBS or β-glucan, and BM analysis was performed 24 hr later; representative FACS plots (J) and frequency of CD131 + LSKs, CD131 + MPPs, and CD131 + LT-HSCs (K) (n = 5 mice per group) are indicated. (L) Staining for pSTAT5 in LSK cells 24 hr after PBS or β-glucan administration. Representative FACS plots and median fluorescence intensity (MFI) are shown (n = 5 mice per group). (M–P), As indicated in (M), mice were injected with β-glucan on day 0. Cholesterol metabolism was blocked by the administration of atorvastatin on days 0 and 1; Ctrl represents the vehicle control. (N) LSK, LT-HSC, and MPP cell numbers; (O) frequency of the same cells in total BM cells; and (P) frequency of GMPs within the MyP pool at day 7 after β-glucan administration (n = 5 mice per group). (Q) Mice were injected with β-glucan on day 0. GM-CSF was blocked by specific anti-GM-CSF antibody on days 0 and 1. Immunoglobulin G (IgG) isotype served as the control. (R and S) LSK, LT-HSC, and MPP cell numbers (R) and frequency of the same cells in total BM cells (S) at day 7 after β-glucan administration are indicated (n = 5 mice per group). Data are presented as mean ± SEM. ∗ p
    Figure Legend Snippet: IL-1β, Glycolysis, Cholesterol Metabolism, and the GM-CSF/CD131 Axis Are Involved in β-Glucan-Dependent Training in the BM (A) Cytokine and G-CSF concentrations in the BM extracellular fluid of mice at 24 hr after the administration of PBS or β-glucan (n = 10 mice per group). (B and C) Mice were injected with β-glucan in the absence (vehicle control, Ctrl) or presence of IL1RA, and BM analysis was performed 24 hr later. (B) Cell-cycle analysis in LT-HSCs. (C) The frequency of MPP subpopulations in LSK cells in the BM is indicated (n = 5 mice per group). (D) Bioenergetic extracellular flux analysis in Lin − cKit + cells sorted from mice 24 hr after β-glucan administration in the absence (vehicle control, Ctrl) or presence of IL1RA (n = 5 mice per group). Basal and maximal ECAR are indicated. (E) LSK cells were treated in vitro with IL-1β or PBS for 24 hr, and Seahorse analysis was performed (n = 5 cultures per group). Basal and maximal ECARs (left) and glycolytic reserve (right) are indicated. (F–I) Mice were injected with β-glucan on day 0. (F) Glycolysis and IL-1 were blocked by the administration of 2-DG and IL1RA, respectively, on days 0 and 1; PBS served as the vehicle control (Ctrl). (G) LSK, MPP, and LT-HSC numbers in the BM at day 7 after β-glucan administration (n = 5 mice per group). (H and I) GMP numbers in the BM (H) and frequency of GMPs within the MyP pool (I) at day 7 after β-glucan administration (n = 5 mice per group). (J and K) Mice were injected with PBS or β-glucan, and BM analysis was performed 24 hr later; representative FACS plots (J) and frequency of CD131 + LSKs, CD131 + MPPs, and CD131 + LT-HSCs (K) (n = 5 mice per group) are indicated. (L) Staining for pSTAT5 in LSK cells 24 hr after PBS or β-glucan administration. Representative FACS plots and median fluorescence intensity (MFI) are shown (n = 5 mice per group). (M–P), As indicated in (M), mice were injected with β-glucan on day 0. Cholesterol metabolism was blocked by the administration of atorvastatin on days 0 and 1; Ctrl represents the vehicle control. (N) LSK, LT-HSC, and MPP cell numbers; (O) frequency of the same cells in total BM cells; and (P) frequency of GMPs within the MyP pool at day 7 after β-glucan administration (n = 5 mice per group). (Q) Mice were injected with β-glucan on day 0. GM-CSF was blocked by specific anti-GM-CSF antibody on days 0 and 1. Immunoglobulin G (IgG) isotype served as the control. (R and S) LSK, LT-HSC, and MPP cell numbers (R) and frequency of the same cells in total BM cells (S) at day 7 after β-glucan administration are indicated (n = 5 mice per group). Data are presented as mean ± SEM. ∗ p

    Techniques Used: Mouse Assay, Injection, Cell Cycle Assay, In Vitro, FACS, Staining, Fluorescence

    Mevalonate Pathway, Related to Figure 5 Schematic depiction of the mevalonate pathway with genes significantly upregulated in LT-HSC from β-glucan–injected mice (as compared to PBS-treated mice) shown in red.
    Figure Legend Snippet: Mevalonate Pathway, Related to Figure 5 Schematic depiction of the mevalonate pathway with genes significantly upregulated in LT-HSC from β-glucan–injected mice (as compared to PBS-treated mice) shown in red.

    Techniques Used: Injection, Mouse Assay

    Single-Cell Transcriptional Analysis in LT-HSCs upon β-Glucan Administration (A–C) Single-cell qPCR in LT-HSCs isolated from mice at 24 hr after administration of PBS or β-glucan (n = 42 cells per condition). (A and B) Hierarchical clustering analysis (A) and distribution of LT-HSCs in the three identified clusters (B) at 24 hr after the administration of PBS or β-glucan. (C) Violin plots indicating genes with significantly altered expression between clusters 1 and 2. The y axis represents gene expression. The horizontal width of the plot shows the density of the data along the y axis. Color key represents the percentage of cells that express the specific gene. (D and E) Single-cell qPCR was performed in CD41 − and CD41 + LT-HSCs isolated from mice at 24 hr after the administration of PBS or β-glucan. Hierarchical clustering analysis (D) and violin plots indicating genes with significantly altered expression between CD41 + LT-HSCs from PBS and β-glucan-treated mice (E).
    Figure Legend Snippet: Single-Cell Transcriptional Analysis in LT-HSCs upon β-Glucan Administration (A–C) Single-cell qPCR in LT-HSCs isolated from mice at 24 hr after administration of PBS or β-glucan (n = 42 cells per condition). (A and B) Hierarchical clustering analysis (A) and distribution of LT-HSCs in the three identified clusters (B) at 24 hr after the administration of PBS or β-glucan. (C) Violin plots indicating genes with significantly altered expression between clusters 1 and 2. The y axis represents gene expression. The horizontal width of the plot shows the density of the data along the y axis. Color key represents the percentage of cells that express the specific gene. (D and E) Single-cell qPCR was performed in CD41 − and CD41 + LT-HSCs isolated from mice at 24 hr after the administration of PBS or β-glucan. Hierarchical clustering analysis (D) and violin plots indicating genes with significantly altered expression between CD41 + LT-HSCs from PBS and β-glucan-treated mice (E).

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

    Peripheral Blood Chimerism in Recipients of LT-HSCs Isolated from β-Glucan-Administered or PBS-Injected Mice, Related to Figure 2 Briefly, LT-HSCs (CD45.2 + ) were sorted 28 days after β-glucan or PBS administration and transplanted to lethally irradiated SJL/BL6 (CD45.1 + ) mice. The percentage of donor-derived (CD45.2 + ) cells in peripheral blood of recipient mice at 12 weeks after transplantation is shown (n = 10 recipient mice per group). Data presented as mean ± SEM.
    Figure Legend Snippet: Peripheral Blood Chimerism in Recipients of LT-HSCs Isolated from β-Glucan-Administered or PBS-Injected Mice, Related to Figure 2 Briefly, LT-HSCs (CD45.2 + ) were sorted 28 days after β-glucan or PBS administration and transplanted to lethally irradiated SJL/BL6 (CD45.1 + ) mice. The percentage of donor-derived (CD45.2 + ) cells in peripheral blood of recipient mice at 12 weeks after transplantation is shown (n = 10 recipient mice per group). Data presented as mean ± SEM.

    Techniques Used: Isolation, Injection, Mouse Assay, Irradiation, Derivative Assay, Transplantation Assay

    Alterations in Lipid Metabolism in BM Progenitor Cells upon β-Glucan Administration Lin − cKit + cells were sorted from mice 24 hr after β-glucan or PBS administration, and non-targeted metabolomic and lipidomic analyses were performed (n = 4 mice per group). (A and B) Not-targeted metabolomics. (A) Volcano plots depict the comparison of metabolite abundances between cells from β-glucan- and PBS-treated mice. Altered metabolites (q value
    Figure Legend Snippet: Alterations in Lipid Metabolism in BM Progenitor Cells upon β-Glucan Administration Lin − cKit + cells were sorted from mice 24 hr after β-glucan or PBS administration, and non-targeted metabolomic and lipidomic analyses were performed (n = 4 mice per group). (A and B) Not-targeted metabolomics. (A) Volcano plots depict the comparison of metabolite abundances between cells from β-glucan- and PBS-treated mice. Altered metabolites (q value

    Techniques Used: Mouse Assay

    Transcriptomic Analysis of LT-HSCs on Day 7 after β-Glucan Administration, Related to Figure 5 (A and B) Upstream regulator analysis in transcriptomic data using IPA. Genes regulated by the TFs (A) Cebpe and (B) Pax5. Genes with increased expression in LT-HSC from mice injected with β-glucan (as compared to cells from PBS-treated mice) are depicted in red, whereas genes with decreased expression are shown in blue.
    Figure Legend Snippet: Transcriptomic Analysis of LT-HSCs on Day 7 after β-Glucan Administration, Related to Figure 5 (A and B) Upstream regulator analysis in transcriptomic data using IPA. Genes regulated by the TFs (A) Cebpe and (B) Pax5. Genes with increased expression in LT-HSC from mice injected with β-glucan (as compared to cells from PBS-treated mice) are depicted in red, whereas genes with decreased expression are shown in blue.

    Techniques Used: Indirect Immunoperoxidase Assay, Expressing, Mouse Assay, Injection

    Administration of β-Glucan Drives Expansion of HSPC Subpopulations WT mice were injected with β-glucan or PBS, and BM analysis was performed after 24 hr. (A) Representative fluorescence-activated cell sorting (FACS) plots for the identification of hematopoietic progenitor cells. After gating for Lin − cells, LSK cells were characterized as cKit + Sca1 + cells. LSK cells subpopulations were further characterized as MPP (CD48 + CD150 − LSK), ST-HSC (CD48 − CD150 − LSK), and LT-HSC (CD48 − CD150 + LSK). (B and C) Cell numbers of LSKs, MPPs, ST-HSCs, and LT-HSCs (B) and cell percentages of the same populations in total BM cells (C) of mice at 24 hr after the administration of PBS or β-glucan (ns = 11 and 12 mice). (D) Representative FACS plots for the identification of MPP subpopulations. After gating for LSK cells, MPP4 cells are characterized as CD48 + Flt3 + CD150 − LSK, MPP3 cells are characterized as CD48 + Flt3 − CD150 − LSK, and MPP2 cells are characterized as CD48 + Flt3 − CD150 + LSK. (E) Frequency of MPP subpopulations in LSK cells in the BM of mice at 24 hr after the administration of PBS or β-glucan (n = 5 mice per group). (F and G) Representative FACS plots for the identification of CD41 + LT-HSCs (F) and frequency of CD41 + LT-HSCs (in total LT-HSCs) (G) in the BM of mice at 24 hr after the administration of PBS or β-glucan (n = 5 mice per group). Data are presented as mean ± SEM. ∗ p
    Figure Legend Snippet: Administration of β-Glucan Drives Expansion of HSPC Subpopulations WT mice were injected with β-glucan or PBS, and BM analysis was performed after 24 hr. (A) Representative fluorescence-activated cell sorting (FACS) plots for the identification of hematopoietic progenitor cells. After gating for Lin − cells, LSK cells were characterized as cKit + Sca1 + cells. LSK cells subpopulations were further characterized as MPP (CD48 + CD150 − LSK), ST-HSC (CD48 − CD150 − LSK), and LT-HSC (CD48 − CD150 + LSK). (B and C) Cell numbers of LSKs, MPPs, ST-HSCs, and LT-HSCs (B) and cell percentages of the same populations in total BM cells (C) of mice at 24 hr after the administration of PBS or β-glucan (ns = 11 and 12 mice). (D) Representative FACS plots for the identification of MPP subpopulations. After gating for LSK cells, MPP4 cells are characterized as CD48 + Flt3 + CD150 − LSK, MPP3 cells are characterized as CD48 + Flt3 − CD150 − LSK, and MPP2 cells are characterized as CD48 + Flt3 − CD150 + LSK. (E) Frequency of MPP subpopulations in LSK cells in the BM of mice at 24 hr after the administration of PBS or β-glucan (n = 5 mice per group). (F and G) Representative FACS plots for the identification of CD41 + LT-HSCs (F) and frequency of CD41 + LT-HSCs (in total LT-HSCs) (G) in the BM of mice at 24 hr after the administration of PBS or β-glucan (n = 5 mice per group). Data are presented as mean ± SEM. ∗ p

    Techniques Used: Mouse Assay, Injection, Fluorescence, FACS

    Administration of β-Glucan Promotes Cell Proliferation of LT-HSCs, Related to Figure 1 (A and B) Cell cycle analysis was performed in LT-HSC at 24h after the administration of PBS or β-glucan by staining for Ki67 and DAPI. (A) Representative flow cytometry plots and (B) frequency of LT-HSC at different phases of the cell cycle (n = 5 mice per group). Data presented as mean ± SEM. ∗ p
    Figure Legend Snippet: Administration of β-Glucan Promotes Cell Proliferation of LT-HSCs, Related to Figure 1 (A and B) Cell cycle analysis was performed in LT-HSC at 24h after the administration of PBS or β-glucan by staining for Ki67 and DAPI. (A) Representative flow cytometry plots and (B) frequency of LT-HSC at different phases of the cell cycle (n = 5 mice per group). Data presented as mean ± SEM. ∗ p

    Techniques Used: Cell Cycle Assay, Staining, Flow Cytometry, Cytometry, Mouse Assay

    28) Product Images from "Alveolar Macrophage-mediated Killing of Pneumocystis carinii f. sp. muris Involves Molecular Recognition by the Dectin-1 ?-Glucan Receptor"

    Article Title: Alveolar Macrophage-mediated Killing of Pneumocystis carinii f. sp. muris Involves Molecular Recognition by the Dectin-1 ?-Glucan Receptor

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20030932

    Effects of mannose and β-glucan receptor blockage on alveolar macrophage–mediated killing of P. carinii . Alveolar macrophages were isolated from 6–8-wk-old, male C57BL/6 mice and were pretreated with 100–600 μg/ml mannan or glucan for 30 min and thereafter added to P. carinii overnight at a final macrophage to P. carinii cyst ratio of 100:1. Controls include P. carinii cultured in the absence of macrophages but in the presence of mannan or glucan. Thereafter, RNA was isolated from the contents of each well and quantitative real time PCR for P. carinii rRNA copy number was performed. Cumulative results from four separate experiments are shown. *, significant differences between untreated and glucan-treated alveolar macrophages (P = 0.008). Data are expressed as mean percent killing ± SEM.
    Figure Legend Snippet: Effects of mannose and β-glucan receptor blockage on alveolar macrophage–mediated killing of P. carinii . Alveolar macrophages were isolated from 6–8-wk-old, male C57BL/6 mice and were pretreated with 100–600 μg/ml mannan or glucan for 30 min and thereafter added to P. carinii overnight at a final macrophage to P. carinii cyst ratio of 100:1. Controls include P. carinii cultured in the absence of macrophages but in the presence of mannan or glucan. Thereafter, RNA was isolated from the contents of each well and quantitative real time PCR for P. carinii rRNA copy number was performed. Cumulative results from four separate experiments are shown. *, significant differences between untreated and glucan-treated alveolar macrophages (P = 0.008). Data are expressed as mean percent killing ± SEM.

    Techniques Used: Isolation, Mouse Assay, Cell Culture, Real-time Polymerase Chain Reaction

    The role of the Dectin-1 β-glucan receptor in alveolar macrophage–mediated killing of P. carinii . Alveolar macrophages were isolated from 6–8-wk-old, male C57BL/6 mice and were adhered to glass coverslips. Thereafter, alveolar macrophages were stained with rat IgG (left) or the anti–Dectin-1 antibody 2A11 (right) and imaged using fluorescent deconvolution microscopy. Blue indicates DAPI-stained nucleic acid and red indicates positive Dectin-1 staining (A; ×400). In B, alveolar macrophages were pretreated with 2 μg/ml rat IgG or 2A11 for 30 min at 37°C and thereafter cocultured with P. carinii at a macrophage to P. carinii cyst ratio of 100:1. Thereafter, RNA was isolated from the contents of each well and quantitative real time PCR for P. carinii rRNA copy number was performed. *, significant differences between macrophages incubated in rat IgG or 2A11 (P
    Figure Legend Snippet: The role of the Dectin-1 β-glucan receptor in alveolar macrophage–mediated killing of P. carinii . Alveolar macrophages were isolated from 6–8-wk-old, male C57BL/6 mice and were adhered to glass coverslips. Thereafter, alveolar macrophages were stained with rat IgG (left) or the anti–Dectin-1 antibody 2A11 (right) and imaged using fluorescent deconvolution microscopy. Blue indicates DAPI-stained nucleic acid and red indicates positive Dectin-1 staining (A; ×400). In B, alveolar macrophages were pretreated with 2 μg/ml rat IgG or 2A11 for 30 min at 37°C and thereafter cocultured with P. carinii at a macrophage to P. carinii cyst ratio of 100:1. Thereafter, RNA was isolated from the contents of each well and quantitative real time PCR for P. carinii rRNA copy number was performed. *, significant differences between macrophages incubated in rat IgG or 2A11 (P

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

    29) Product Images from "DNA Synthesis Is Activated in Mosquitoes and Human Monocytes During the Induction of Innate Immune Memory"

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

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.02834

    (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
    Figure Legend 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

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

    (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
    Figure Legend 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

    Techniques Used: Incubation, Colorimetric Assay

    30) Product Images from "Characterization of a ?-glucanase produced by Rhizopus microsporus var. microsporus, and its potential for application in the brewing industry"

    Article Title: Characterization of a ?-glucanase produced by Rhizopus microsporus var. microsporus, and its potential for application in the brewing industry

    Journal: BMC Biochemistry

    doi: 10.1186/1471-2091-7-23

    Hydrolysis (μmol·min -1 ·mL -1 ) of β-glucan by the purified 1,3-1,4-β-glucanase from Rhizopus microsporus var. microsporus , in the presence of different concentrations of 1,3-1,4-β-glucan.
    Figure Legend Snippet: Hydrolysis (μmol·min -1 ·mL -1 ) of β-glucan by the purified 1,3-1,4-β-glucanase from Rhizopus microsporus var. microsporus , in the presence of different concentrations of 1,3-1,4-β-glucan.

    Techniques Used: Purification

    31) Product Images from "Activation of Vitamin D Regulates Response of Human Bronchial Epithelial Cells to Aspergillus fumigatus in an Autocrine Fashion"

    Article Title: Activation of Vitamin D Regulates Response of Human Bronchial Epithelial Cells to Aspergillus fumigatus in an Autocrine Fashion

    Journal: Mediators of Inflammation

    doi: 10.1155/2015/208491

    β -Glucan increases the expression of 1 α -hydroxylase and VDR, and vitamin D synergizes with β -glucan to induce the expression of antimicrobial peptides but attenuates β -glucan-induced expression of chemokines and cytokines in 16HBE cells. (a) β -Glucan increases the expression of 1 α -hydroxylase and VDR in 16HBE cells in a dose-dependent manner. 16HBE cells were cultured in the absence or presence of increasing doses of β -glucan as indicated for 24 h. Cells were then harvested and the protein and mRNA expression of 1 α -hydroxylase and VDR was evaluated by Western blot analysis and quantitative real-time PCR, respectively. The Western blots shown are from one representative experiment out of three, which are converted to densitometry units in graphs. Values reflect mean fold change from control and SEM of three independent experiments. Student's t -test, ∗ P
    Figure Legend Snippet: β -Glucan increases the expression of 1 α -hydroxylase and VDR, and vitamin D synergizes with β -glucan to induce the expression of antimicrobial peptides but attenuates β -glucan-induced expression of chemokines and cytokines in 16HBE cells. (a) β -Glucan increases the expression of 1 α -hydroxylase and VDR in 16HBE cells in a dose-dependent manner. 16HBE cells were cultured in the absence or presence of increasing doses of β -glucan as indicated for 24 h. Cells were then harvested and the protein and mRNA expression of 1 α -hydroxylase and VDR was evaluated by Western blot analysis and quantitative real-time PCR, respectively. The Western blots shown are from one representative experiment out of three, which are converted to densitometry units in graphs. Values reflect mean fold change from control and SEM of three independent experiments. Student's t -test, ∗ P

    Techniques Used: Expressing, Cell Culture, Western Blot, Real-time Polymerase Chain Reaction

    32) Product Images from "A mixed population of immature and mature leucocytes in umbilical cord blood results in a reduced expression and function of CR3 (CD11b/CD18)"

    Article Title: A mixed population of immature and mature leucocytes in umbilical cord blood results in a reduced expression and function of CR3 (CD11b/CD18)

    Journal: Clinical and Experimental Immunology

    doi: 10.1046/j.1365-2249.1998.00743.x

    The CR3-dependent respiratory burst is proportional to the CR3 surface density of both adult (▪) and neonatal neutrophils (□). A three-colour flow cytometry assay was used to analyse CD15 high neutrophils for the size of an intracellular respiratory burst triggered by β-glucan particles versus the surface density of CR3 expression.
    Figure Legend Snippet: The CR3-dependent respiratory burst is proportional to the CR3 surface density of both adult (▪) and neonatal neutrophils (□). A three-colour flow cytometry assay was used to analyse CD15 high neutrophils for the size of an intracellular respiratory burst triggered by β-glucan particles versus the surface density of CR3 expression.

    Techniques Used: Flow Cytometry, Cytometry, Expressing

    33) Product Images from "Nonopsonic Phagocytosis of Zymosan and Mycobacterium kansasii by CR3 (CD11b/CD18) Involves Distinct Molecular Determinants and Is or Is Not Coupled with NADPH Oxidase Activation"

    Article Title: Nonopsonic Phagocytosis of Zymosan and Mycobacterium kansasii by CR3 (CD11b/CD18) Involves Distinct Molecular Determinants and Is or Is Not Coupled with NADPH Oxidase Activation

    Journal: Infection and Immunity

    doi:

    Inhibition by polysaccharides of CR3-mediated zymosan and M. kansasii phagocytosis. CR3-transfected CHO cells were preincubated as indicated with glucose- or mannose-containing polysaccharides or β-glucan plus mannan before incubation with either zymosan (A) or M. kansasii (B). The data are expressed as the percentage of phagocytosis reported compared to control values (100%, no polysaccharide). The values are means + SEM of three to four separate experiments performed in duplicate. Statistical differences were measured for cells pretreated with sugars compared to untreated cells. ∗, P
    Figure Legend Snippet: Inhibition by polysaccharides of CR3-mediated zymosan and M. kansasii phagocytosis. CR3-transfected CHO cells were preincubated as indicated with glucose- or mannose-containing polysaccharides or β-glucan plus mannan before incubation with either zymosan (A) or M. kansasii (B). The data are expressed as the percentage of phagocytosis reported compared to control values (100%, no polysaccharide). The values are means + SEM of three to four separate experiments performed in duplicate. Statistical differences were measured for cells pretreated with sugars compared to untreated cells. ∗, P

    Techniques Used: Inhibition, Transfection, Incubation

    34) Product Images from "Crosstalk between macrophages and astrocytes affects proliferation, reactive phenotype and inflammatory response, suggesting a role during reactive gliosis following spinal cord injury"

    Article Title: Crosstalk between macrophages and astrocytes affects proliferation, reactive phenotype and inflammatory response, suggesting a role during reactive gliosis following spinal cord injury

    Journal: Journal of Neuroinflammation

    doi: 10.1186/s12974-015-0327-3

    Effects of soluble factors from previously stimulated astrocytes on primary macrophages. a Experimental set-up. Astrocytes were stimulated with conditioned medium from M1 or M2 macrophages or left unstimulated. Following a wash, conditioned medium was then collected from these and incubated with fresh primary macrophages. b , c EdU incorporation assays show medium from astrocytes previously stimulated with M2-conditioned medium has an anti-proliferative effect on both M1 and M2 macrophages following 3 days of incubation. d The anti-proliferative effect is confirmed using MTT assays. c As with M1 macrophages, the proliferation of M2 macrophages is also lower when incubated with M2-stimulated astrocyte medium. e In M1 macrophages, TNFα release following β-glucan stimulation, as measured by ELISA, is also affected, with significantly higher release following incubations with M1-stimulated astrocyte medium, and a significant decrease after incubation with M2-stimulated astrocytes medium
    Figure Legend Snippet: Effects of soluble factors from previously stimulated astrocytes on primary macrophages. a Experimental set-up. Astrocytes were stimulated with conditioned medium from M1 or M2 macrophages or left unstimulated. Following a wash, conditioned medium was then collected from these and incubated with fresh primary macrophages. b , c EdU incorporation assays show medium from astrocytes previously stimulated with M2-conditioned medium has an anti-proliferative effect on both M1 and M2 macrophages following 3 days of incubation. d The anti-proliferative effect is confirmed using MTT assays. c As with M1 macrophages, the proliferation of M2 macrophages is also lower when incubated with M2-stimulated astrocyte medium. e In M1 macrophages, TNFα release following β-glucan stimulation, as measured by ELISA, is also affected, with significantly higher release following incubations with M1-stimulated astrocyte medium, and a significant decrease after incubation with M2-stimulated astrocytes medium

    Techniques Used: Incubation, MTT Assay, Enzyme-linked Immunosorbent Assay

    35) Product Images from "C-Type Lectin in Chlamys farreri (CfLec-1) Mediating Immune Recognition and Opsonization"

    Article Title: C-Type Lectin in Chlamys farreri (CfLec-1) Mediating Immune Recognition and Opsonization

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0017089

    Temporal expression of CfLec-1 mRNA relative to β-actin was analyzed by realtime PCR in scallop hemocytes after LPS, PGN, β-glucan and PBS challenge for 3, 6, 12, 24 and 48 h. The values are shown as mean ± SE (N = 4). (*: P
    Figure Legend Snippet: Temporal expression of CfLec-1 mRNA relative to β-actin was analyzed by realtime PCR in scallop hemocytes after LPS, PGN, β-glucan and PBS challenge for 3, 6, 12, 24 and 48 h. The values are shown as mean ± SE (N = 4). (*: P

    Techniques Used: Expressing, Polymerase Chain Reaction

    36) Product Images from "Glutaminolysis and Fumarate Accumulation Integrate Immunometabolic and Epigenetic Programs in Trained Immunity"

    Article Title: Glutaminolysis and Fumarate Accumulation Integrate Immunometabolic and Epigenetic Programs in Trained Immunity

    Journal: Cell metabolism

    doi: 10.1016/j.cmet.2016.10.008

    Metabolism in Trained and Tolerant Macrophages (A) Heatmap depicting average mRNA expression of β-glucan-modulated metabolism-associated genes. The rows indicate different gene transcripts, and the columns indicate different conditions and time points. Log(e)RPKM values were Z . (B) Heatmap depicting the average metabolite intensities in the metabolomics data. The rows indicate different metabolites, and the columns indicate different conditions. The log transformed metabolite intensities were first Z . .
    Figure Legend Snippet: Metabolism in Trained and Tolerant Macrophages (A) Heatmap depicting average mRNA expression of β-glucan-modulated metabolism-associated genes. The rows indicate different gene transcripts, and the columns indicate different conditions and time points. Log(e)RPKM values were Z . (B) Heatmap depicting the average metabolite intensities in the metabolomics data. The rows indicate different metabolites, and the columns indicate different conditions. The log transformed metabolite intensities were first Z . .

    Techniques Used: Expressing, Transformation Assay

    Related Articles

    Injection:

    Article Title: CfLec-3 from scallop: an entrance to non-self recognition mechanism of invertebrate C-type lectin
    Article Snippet: .. The scallops in five groups received an intramuscular injection of 50 μL phosphate buffered saline (PBS, 0.14 M sodium chloride, 3 mM potassium chloride, 8 mM disodium hydrogenphosphate dodecahydrate, 1.5 mM potassium phosphate monobasic, pH 7.4), LPS from E. coli 0111:B4 (Sigma-Aldrich, 0.5 mg mL−1 in PBS), PGN from S. aureus (Sigma-Aldrich, 0.8 mg mL−1 in PBS), β-glucan from Saccharomyces cerevisiae (Sigma-Aldrich, 1.0 mg mL−1 in PBS), and Ploy I:C (Sigma-Aldrich, 1.0 mg mL−1 in PBS), respectively. ..

    Negative Control:

    Article Title: Variation of Neisseria gonorrhoeae Lipooligosaccharide Directs Dendritic Cell-Induced T Helper Responses
    Article Snippet: .. Poly I∶C (20 µg/ml) was used as a negative control, Staphylococcus aureus peptidoglycan (PGN, 10 µg/ml, Sigma-Aldrich) and the β-glucan curdlan (Alcaligenes faecalis , 10 µg/ml, Sigma-Aldrich) were included as positive controls for Th17 skewing. .. After 24 hours DCs were washed and incubated with allogeneic memory CD4+ T cells (ratio 1∶10).

    BrdU Incorporation Assay:

    Article Title: DNA Synthesis Is Activated in Mosquitoes and Human Monocytes During the Induction of Innate Immune Memory
    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). .. Preparations of 1 million fixed cells were processed on a Diagenode Bioruptor Pico sonicator by using five cycles of 30 s on and 30 s off.

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  • 97
    Millipore pustulan
    Hst 5 binds selectively to β-1,3-glucans in the Candida cell wall. Representative cell wall polysaccharides (1–16 mg ml −1 ) consisting of laminarin (β-1,3-glucan) (Lam, red squares), sialic acid (Sia, grey circles), mannans (Man, black diamonds), <t>pustulan</t> (β-1,6-glucan) (Pus, black squares) or untreated control (Con) were pre-incubated with BHst 5 (31 µM) then added to C. albicans cells for 1 h. Cell wall extracts were immunoblotted to detect BHst 5 (A) and quantified by dentistometry (B). Reduction in cell wall binding was accompanied by reduced candidacidal activity of Hst 5 when incubated with the same polysaccharides in a dose-dependent manner (C). The same polysaccharides were coupled to Sepharose beads and percent binding of BHst 5 was assessed by elution affinity chromatography (D). Only laminarin was found to have significant binding with Hst 5. PCW and laminarinase-treated PCW were incubated with Hst 5, and unbound Hst 5 in supernatant (S) and bound Hst 5 in pellets (P) were detected on Tricine-SDS gels (E). Laminarinase digestion of PCW prevented Hst 5 binding (E). Cell wall binding of Hst 5 was reduced in a dose-dependent manner by laminarinase digests (F), when laminarinase digests (2–8 mg ml −1 ) were pre-incubated with Hst 5, then added to C. albicans cells. Killing activities of Hst 5 were decreased against C. albicans cells grown in YPD broth with caspofungin (1 or 2 ng ml −1 ) (G).
    Pustulan, supplied by Millipore, used in various techniques. Bioz Stars score: 97/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Millipore d glucan polymers
    Identification of a novel class of <t>β-1,3-</t> d <t>-glucan</t> synthase inhibitors.
    D Glucan Polymers, supplied by Millipore, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Millipore sodium stibogluconate
    Inhibition of SHP-1 PTP activity rescues in vitro maturation of Themis −/− thymocytes Differentiation of DP thymocytes from Themis −/− or Themis +/+ to the CD4 + CD8 − stage in a two step (stimulation-rest) in vitro assay with or without the SHP-1 inhibitor Sodium <t>stibogluconate</t> (SSG). Left, two parameter plots show CD4 versus CD8 staining profiles of thymocytes at the completion of the differentiation assay. Cell recovery and % apoptotic (Annexin V + ) cells were not significantly different in similarly treated Themis −/− or Themis +/+ samples. Right, Summary of results. n=4 for each genotype (t-test 2-tailed type-2, error bars show SD). *** P
    Sodium Stibogluconate, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Hst 5 binds selectively to β-1,3-glucans in the Candida cell wall. Representative cell wall polysaccharides (1–16 mg ml −1 ) consisting of laminarin (β-1,3-glucan) (Lam, red squares), sialic acid (Sia, grey circles), mannans (Man, black diamonds), pustulan (β-1,6-glucan) (Pus, black squares) or untreated control (Con) were pre-incubated with BHst 5 (31 µM) then added to C. albicans cells for 1 h. Cell wall extracts were immunoblotted to detect BHst 5 (A) and quantified by dentistometry (B). Reduction in cell wall binding was accompanied by reduced candidacidal activity of Hst 5 when incubated with the same polysaccharides in a dose-dependent manner (C). The same polysaccharides were coupled to Sepharose beads and percent binding of BHst 5 was assessed by elution affinity chromatography (D). Only laminarin was found to have significant binding with Hst 5. PCW and laminarinase-treated PCW were incubated with Hst 5, and unbound Hst 5 in supernatant (S) and bound Hst 5 in pellets (P) were detected on Tricine-SDS gels (E). Laminarinase digestion of PCW prevented Hst 5 binding (E). Cell wall binding of Hst 5 was reduced in a dose-dependent manner by laminarinase digests (F), when laminarinase digests (2–8 mg ml −1 ) were pre-incubated with Hst 5, then added to C. albicans cells. Killing activities of Hst 5 were decreased against C. albicans cells grown in YPD broth with caspofungin (1 or 2 ng ml −1 ) (G).

    Journal: Molecular Microbiology

    Article Title: Salivary histatin 5 internalization by translocation, but not endocytosis, is required for fungicidal activity in Candida albicans

    doi: 10.1111/j.1365-2958.2010.07210.x

    Figure Lengend Snippet: Hst 5 binds selectively to β-1,3-glucans in the Candida cell wall. Representative cell wall polysaccharides (1–16 mg ml −1 ) consisting of laminarin (β-1,3-glucan) (Lam, red squares), sialic acid (Sia, grey circles), mannans (Man, black diamonds), pustulan (β-1,6-glucan) (Pus, black squares) or untreated control (Con) were pre-incubated with BHst 5 (31 µM) then added to C. albicans cells for 1 h. Cell wall extracts were immunoblotted to detect BHst 5 (A) and quantified by dentistometry (B). Reduction in cell wall binding was accompanied by reduced candidacidal activity of Hst 5 when incubated with the same polysaccharides in a dose-dependent manner (C). The same polysaccharides were coupled to Sepharose beads and percent binding of BHst 5 was assessed by elution affinity chromatography (D). Only laminarin was found to have significant binding with Hst 5. PCW and laminarinase-treated PCW were incubated with Hst 5, and unbound Hst 5 in supernatant (S) and bound Hst 5 in pellets (P) were detected on Tricine-SDS gels (E). Laminarinase digestion of PCW prevented Hst 5 binding (E). Cell wall binding of Hst 5 was reduced in a dose-dependent manner by laminarinase digests (F), when laminarinase digests (2–8 mg ml −1 ) were pre-incubated with Hst 5, then added to C. albicans cells. Killing activities of Hst 5 were decreased against C. albicans cells grown in YPD broth with caspofungin (1 or 2 ng ml −1 ) (G).

    Article Snippet: BHst 5 (20 µl) or Hst 5 (20 µl) (final concentration = 31 µM) was added to 80 µl of each polysaccharide [1.125–20 mg ml−1 in 10 mM sodium phosphate buffer, pH 7.4 (NaPB)] including laminarin (β-1,3-glucan polymer; Sigma), sialic acid (Sigma), pustulan (β-1,6-glucan polymer; Calbiochem) or mannan (mannose polymer; Sigma) and incubated with C. albicans cells for 1 h at 30°C.

    Techniques: Laser Capture Microdissection, Incubation, Binding Assay, Activity Assay, Affinity Chromatography

    Detection of the complement opsonin, iC3b on the surface of PGG β-glucan-bound cells . (A) Fluorescence intensity of BfD IV- (upper row) and anti-iC3b-stained cells (lower row) treated with PGG β-glucan (solid) in comparison to that of the vehicle-treated control cells (gray filled). Data shown here are representative of three independent experiments. (B) Confocal microscopy images of surface staining of PGG β-glucan (middle panel, in green), iC3b (third panel from left, in red), or the merged image of PGG and iC3b (far right panel) on PGG β-glucan-treated (upper row), and vehicle-treated neutrophils (lower row). Neutrophil nuclei stained with DAPI is shown in blue. Shown here are representative data from two independent experiments. (C) Direct interaction of iC3b with immobilized PGG β-glucan in serum or HI serum was evaluated by ELISA. Dextran was used as control glucan. Bars represent mean fold change values from three independent experiments. ** p ≤ 0.05 compared with PGG β-glucan-treated with serum. (D) iC3b present in the immunoprecipitated PGG β-glucan-serum complex was detected by Flow Cytometry. The histogram shows the comparison of iC3b detected on the beads pulled-down from the various glucan-serum mixtures (solid) in comparison to that from the vehicle-serum mixture (gray filled). Data shown are representative of three independent experiments.

    Journal: Frontiers in Immunology

    Article Title: Binding of Soluble Yeast ?-Glucan to Human Neutrophils and Monocytes is Complement-Dependent

    doi: 10.3389/fimmu.2013.00230

    Figure Lengend Snippet: Detection of the complement opsonin, iC3b on the surface of PGG β-glucan-bound cells . (A) Fluorescence intensity of BfD IV- (upper row) and anti-iC3b-stained cells (lower row) treated with PGG β-glucan (solid) in comparison to that of the vehicle-treated control cells (gray filled). Data shown here are representative of three independent experiments. (B) Confocal microscopy images of surface staining of PGG β-glucan (middle panel, in green), iC3b (third panel from left, in red), or the merged image of PGG and iC3b (far right panel) on PGG β-glucan-treated (upper row), and vehicle-treated neutrophils (lower row). Neutrophil nuclei stained with DAPI is shown in blue. Shown here are representative data from two independent experiments. (C) Direct interaction of iC3b with immobilized PGG β-glucan in serum or HI serum was evaluated by ELISA. Dextran was used as control glucan. Bars represent mean fold change values from three independent experiments. ** p ≤ 0.05 compared with PGG β-glucan-treated with serum. (D) iC3b present in the immunoprecipitated PGG β-glucan-serum complex was detected by Flow Cytometry. The histogram shows the comparison of iC3b detected on the beads pulled-down from the various glucan-serum mixtures (solid) in comparison to that from the vehicle-serum mixture (gray filled). Data shown are representative of three independent experiments.

    Article Snippet: For some experiments, PGG β-glucan was prepared for use by performing a buffer exchange into Dulbecco’s phosphate-buffered saline (DPBS) using 3 kDa molecular weight cut-off (MWCO) Amicon centrifugal filtration units (Millipore, Billerica, MA, USA).

    Techniques: Fluorescence, Staining, Confocal Microscopy, Enzyme-linked Immunosorbent Assay, Immunoprecipitation, Flow Cytometry, Cytometry

    Model of the binding mechanism of PGG β-glucan to complement receptors on human immune cells . PGG β-glucan has the ability to activate complement and to be opsonized by C3b, which can then be degraded to iC3b, and C3dg, which remain covalently bound. Since the different complement fragments preferentially bind to specific complement receptors, the opsonized PGG β-glucan has the potential to bind to CR1 via C3b, or CR3 via iC3b, or CR2 via C3dg. This study demonstrates that complement opsonization of soluble yeast β-glucan makes it amenable for recognition by several complement receptors on human innate and possibly, adaptive immune cells.

    Journal: Frontiers in Immunology

    Article Title: Binding of Soluble Yeast ?-Glucan to Human Neutrophils and Monocytes is Complement-Dependent

    doi: 10.3389/fimmu.2013.00230

    Figure Lengend Snippet: Model of the binding mechanism of PGG β-glucan to complement receptors on human immune cells . PGG β-glucan has the ability to activate complement and to be opsonized by C3b, which can then be degraded to iC3b, and C3dg, which remain covalently bound. Since the different complement fragments preferentially bind to specific complement receptors, the opsonized PGG β-glucan has the potential to bind to CR1 via C3b, or CR3 via iC3b, or CR2 via C3dg. This study demonstrates that complement opsonization of soluble yeast β-glucan makes it amenable for recognition by several complement receptors on human innate and possibly, adaptive immune cells.

    Article Snippet: For some experiments, PGG β-glucan was prepared for use by performing a buffer exchange into Dulbecco’s phosphate-buffered saline (DPBS) using 3 kDa molecular weight cut-off (MWCO) Amicon centrifugal filtration units (Millipore, Billerica, MA, USA).

    Techniques: Binding Assay

    Time, temperature, and serum-dependent binding of PGG β-glucan to human neutrophils and monocytes . PGG β-glucan binding to human neutrophils and monocytes was determined (A) in the presence of 2, 5, 10, 20, and 50% of serum, (B) at 10, 30, 60, and 120 min, and (C) at RT, 4 and 37°C. The MFI and percentage of BfD IV positive cells were indicated in the zebra plots, and histogram shows PGG β-glucan binding (solid) in comparison to the vehicle-control (gray filled). Data shown in each of the conditions are representative of three independent experiments performed with cells obtained from three different donors.

    Journal: Frontiers in Immunology

    Article Title: Binding of Soluble Yeast ?-Glucan to Human Neutrophils and Monocytes is Complement-Dependent

    doi: 10.3389/fimmu.2013.00230

    Figure Lengend Snippet: Time, temperature, and serum-dependent binding of PGG β-glucan to human neutrophils and monocytes . PGG β-glucan binding to human neutrophils and monocytes was determined (A) in the presence of 2, 5, 10, 20, and 50% of serum, (B) at 10, 30, 60, and 120 min, and (C) at RT, 4 and 37°C. The MFI and percentage of BfD IV positive cells were indicated in the zebra plots, and histogram shows PGG β-glucan binding (solid) in comparison to the vehicle-control (gray filled). Data shown in each of the conditions are representative of three independent experiments performed with cells obtained from three different donors.

    Article Snippet: For some experiments, PGG β-glucan was prepared for use by performing a buffer exchange into Dulbecco’s phosphate-buffered saline (DPBS) using 3 kDa molecular weight cut-off (MWCO) Amicon centrifugal filtration units (Millipore, Billerica, MA, USA).

    Techniques: Binding Assay

    Role of complement in binding of PGG β-glucan to human neutrophils and monocytes . (A) Binding of PGG β-glucan to human neutrophils and monocytes in 10% serum (upper row) was compared to binding in 10% HI serum (lower row). Histograms show PGG β-glucan binding (solid) in comparison to vehicle-control (gray filled). Bar represents mean MFI from five donors with percentage of inhibition indicated in parenthesis. ** p ≤ 0.05 compared to binding in serum. (B) Inhibitory effect of C3-inhibitor compstatin on PGG β-glucan binding in neutrophils and monocytes was measured and compared to that of control peptide-treated group. Histograms show PGG β-glucan binding in the presence of peptide control (solid) or compstatin (dotted) in comparison to vehicle-control (gray filled). Bar represents mean MFI with percentage of inhibition indicated in parenthesis. ** p ≤ 0.05 compared to binding in control peptide. Data shown are representative of at least three independent experiments performed with cells obtained from different donors.

    Journal: Frontiers in Immunology

    Article Title: Binding of Soluble Yeast ?-Glucan to Human Neutrophils and Monocytes is Complement-Dependent

    doi: 10.3389/fimmu.2013.00230

    Figure Lengend Snippet: Role of complement in binding of PGG β-glucan to human neutrophils and monocytes . (A) Binding of PGG β-glucan to human neutrophils and monocytes in 10% serum (upper row) was compared to binding in 10% HI serum (lower row). Histograms show PGG β-glucan binding (solid) in comparison to vehicle-control (gray filled). Bar represents mean MFI from five donors with percentage of inhibition indicated in parenthesis. ** p ≤ 0.05 compared to binding in serum. (B) Inhibitory effect of C3-inhibitor compstatin on PGG β-glucan binding in neutrophils and monocytes was measured and compared to that of control peptide-treated group. Histograms show PGG β-glucan binding in the presence of peptide control (solid) or compstatin (dotted) in comparison to vehicle-control (gray filled). Bar represents mean MFI with percentage of inhibition indicated in parenthesis. ** p ≤ 0.05 compared to binding in control peptide. Data shown are representative of at least three independent experiments performed with cells obtained from different donors.

    Article Snippet: For some experiments, PGG β-glucan was prepared for use by performing a buffer exchange into Dulbecco’s phosphate-buffered saline (DPBS) using 3 kDa molecular weight cut-off (MWCO) Amicon centrifugal filtration units (Millipore, Billerica, MA, USA).

    Techniques: Binding Assay, Inhibition

    Requirement of optimal percentage of serum, incubation time, and incubation temperature at the ligand level versus the cellular level . (A) PGG β-glucan-treated with PBS (PGG + PBS), PGG β-glucan and serum added separately (PGG + serum), or serum pre-opsonized PGG β-glucan (OpPGG) were added at concentration 100 μg/mL to neutrophils or to PBMC, incubated for 1 h at 37°C in 10% serum or 10% HI serum, and binding was measured by flow cytometry. (B) Binding was measured at 37°C after incubating PGG or OpPGG with the cells for 10 min or 1 h in RPMI containing 10% serum or 10% HI serum. (C) Binding was measured at 4°C after incubating PGG or OpPGG with cells as described above. Data shown in the zebra plots with MFI and percentage of BfD IV positive population are representative of three independent experiments performed with cells obtained from three different donors.

    Journal: Frontiers in Immunology

    Article Title: Binding of Soluble Yeast ?-Glucan to Human Neutrophils and Monocytes is Complement-Dependent

    doi: 10.3389/fimmu.2013.00230

    Figure Lengend Snippet: Requirement of optimal percentage of serum, incubation time, and incubation temperature at the ligand level versus the cellular level . (A) PGG β-glucan-treated with PBS (PGG + PBS), PGG β-glucan and serum added separately (PGG + serum), or serum pre-opsonized PGG β-glucan (OpPGG) were added at concentration 100 μg/mL to neutrophils or to PBMC, incubated for 1 h at 37°C in 10% serum or 10% HI serum, and binding was measured by flow cytometry. (B) Binding was measured at 37°C after incubating PGG or OpPGG with the cells for 10 min or 1 h in RPMI containing 10% serum or 10% HI serum. (C) Binding was measured at 4°C after incubating PGG or OpPGG with cells as described above. Data shown in the zebra plots with MFI and percentage of BfD IV positive population are representative of three independent experiments performed with cells obtained from three different donors.

    Article Snippet: For some experiments, PGG β-glucan was prepared for use by performing a buffer exchange into Dulbecco’s phosphate-buffered saline (DPBS) using 3 kDa molecular weight cut-off (MWCO) Amicon centrifugal filtration units (Millipore, Billerica, MA, USA).

    Techniques: Incubation, Concentration Assay, Binding Assay, Flow Cytometry, Cytometry

    Role of the alternative complement pathway in binding of PGG β-glucan to neutrophils and monocytes . (A) The effect of MgEGTA-treated serum (Mg + EGTA) on iC3b deposition on immobilized PGG β-glucan was measured by ELISA (left), and on OpPGG preparation and subsequent binding to neutrophils and monocytes (right) was measured by flow cytometry. Histograms show binding of OpPGG prepared with untreated serum (solid), OpPGG prepared with MgEGTA-treated serum (dotted) and vehicle binding (gray filled) to neutrophils and monocytes in 10% HI serum. (B) The effect of anti-Factor D Ab, 166-32 on PGG β-glucan binding to neutrophils and monocytes was measured by flow cytometry. Histograms show cell binding to PGG β-glucan in the presence of isotype control (solid) or 166-32 mAb (dotted) in comparison to vehicle-control binding (gray filled). Data shown are representative of three independent experiments with serum from three different donors. (C) Activation of classical or lectin complement pathway as measured by C4a generation upon stimulation of WB with PGG or particulate β-glucan was determined by ELISA. Bar represent mean fold change values from three independent experiments. ** p ≤ 0.05 compared to vehicle-treated WB.

    Journal: Frontiers in Immunology

    Article Title: Binding of Soluble Yeast ?-Glucan to Human Neutrophils and Monocytes is Complement-Dependent

    doi: 10.3389/fimmu.2013.00230

    Figure Lengend Snippet: Role of the alternative complement pathway in binding of PGG β-glucan to neutrophils and monocytes . (A) The effect of MgEGTA-treated serum (Mg + EGTA) on iC3b deposition on immobilized PGG β-glucan was measured by ELISA (left), and on OpPGG preparation and subsequent binding to neutrophils and monocytes (right) was measured by flow cytometry. Histograms show binding of OpPGG prepared with untreated serum (solid), OpPGG prepared with MgEGTA-treated serum (dotted) and vehicle binding (gray filled) to neutrophils and monocytes in 10% HI serum. (B) The effect of anti-Factor D Ab, 166-32 on PGG β-glucan binding to neutrophils and monocytes was measured by flow cytometry. Histograms show cell binding to PGG β-glucan in the presence of isotype control (solid) or 166-32 mAb (dotted) in comparison to vehicle-control binding (gray filled). Data shown are representative of three independent experiments with serum from three different donors. (C) Activation of classical or lectin complement pathway as measured by C4a generation upon stimulation of WB with PGG or particulate β-glucan was determined by ELISA. Bar represent mean fold change values from three independent experiments. ** p ≤ 0.05 compared to vehicle-treated WB.

    Article Snippet: For some experiments, PGG β-glucan was prepared for use by performing a buffer exchange into Dulbecco’s phosphate-buffered saline (DPBS) using 3 kDa molecular weight cut-off (MWCO) Amicon centrifugal filtration units (Millipore, Billerica, MA, USA).

    Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Cytometry, Activation Assay, Western Blot

    Role of CR3 receptor in binding of PGG β-glucan to human neutrophils and monocytes . (A) Binding of increasing concentration of PGG β-glucan (0, 10, 25, 100, 200, and 400 μg/mL) to human neutrophils (upper row) and monocytes in PBMC (lower row) was measured by flow cytometry after incubation of cells with β-glucan or vehicle at 37°C for 1 h. (B) To identify the receptor involved, binding of PGG β-glucan to human neutrophils (left) and monocytes (right) was measured in the presence of α-CD11b (upper row), α-CR3 (second row), α-CD11a (third row), or α-Dectin-1 blocking antibodies (bottom row). The MFI and percentage of β-glucan-treated, BfD IV positive cells are indicated in the zebra plots. Histograms show PGG β-glucan binding in the presence of isotype controls (solid) and blocking antibodies (dotted) compared to vehicle-control binding (gray filled). Bars represent the mean MFI of the vehicle- or PGG β-glucan-treated groups in the presence of CR3 blocking or isotype control antibodies from five donors. The percentage of inhibition (in parentheses) was indicated on the graph. ** p ≤ 0.05 compared to β-glucan-treated group in the presence of isotype controls. Data shown are representative of at least three independent experiments performed with cells from different donors.

    Journal: Frontiers in Immunology

    Article Title: Binding of Soluble Yeast ?-Glucan to Human Neutrophils and Monocytes is Complement-Dependent

    doi: 10.3389/fimmu.2013.00230

    Figure Lengend Snippet: Role of CR3 receptor in binding of PGG β-glucan to human neutrophils and monocytes . (A) Binding of increasing concentration of PGG β-glucan (0, 10, 25, 100, 200, and 400 μg/mL) to human neutrophils (upper row) and monocytes in PBMC (lower row) was measured by flow cytometry after incubation of cells with β-glucan or vehicle at 37°C for 1 h. (B) To identify the receptor involved, binding of PGG β-glucan to human neutrophils (left) and monocytes (right) was measured in the presence of α-CD11b (upper row), α-CR3 (second row), α-CD11a (third row), or α-Dectin-1 blocking antibodies (bottom row). The MFI and percentage of β-glucan-treated, BfD IV positive cells are indicated in the zebra plots. Histograms show PGG β-glucan binding in the presence of isotype controls (solid) and blocking antibodies (dotted) compared to vehicle-control binding (gray filled). Bars represent the mean MFI of the vehicle- or PGG β-glucan-treated groups in the presence of CR3 blocking or isotype control antibodies from five donors. The percentage of inhibition (in parentheses) was indicated on the graph. ** p ≤ 0.05 compared to β-glucan-treated group in the presence of isotype controls. Data shown are representative of at least three independent experiments performed with cells from different donors.

    Article Snippet: For some experiments, PGG β-glucan was prepared for use by performing a buffer exchange into Dulbecco’s phosphate-buffered saline (DPBS) using 3 kDa molecular weight cut-off (MWCO) Amicon centrifugal filtration units (Millipore, Billerica, MA, USA).

    Techniques: Binding Assay, Concentration Assay, Flow Cytometry, Cytometry, Incubation, Blocking Assay, Inhibition

    Identification of a novel class of β-1,3- d -glucan synthase inhibitors.

    Journal: Antimicrobial Agents and Chemotherapy

    Article Title: Discovery of a Novel Class of Orally Active Antifungal ?-1,3-d-Glucan Synthase Inhibitors ▿

    doi: 10.1128/AAC.00432-11

    Figure Lengend Snippet: Identification of a novel class of β-1,3- d -glucan synthase inhibitors.

    Article Snippet: Newly synthesized, radiolabeled β-1,3- d -glucan polymers were collected on the filter membrane by applying a vacuum to the plate using a MutiScreen Resist vacuum manifold (Millipore).

    Techniques:

    Inhibition of SHP-1 PTP activity rescues in vitro maturation of Themis −/− thymocytes Differentiation of DP thymocytes from Themis −/− or Themis +/+ to the CD4 + CD8 − stage in a two step (stimulation-rest) in vitro assay with or without the SHP-1 inhibitor Sodium stibogluconate (SSG). Left, two parameter plots show CD4 versus CD8 staining profiles of thymocytes at the completion of the differentiation assay. Cell recovery and % apoptotic (Annexin V + ) cells were not significantly different in similarly treated Themis −/− or Themis +/+ samples. Right, Summary of results. n=4 for each genotype (t-test 2-tailed type-2, error bars show SD). *** P

    Journal: Nature immunology

    Article Title: THEMIS enhances TCR signaling and enables positive selection by selective inhibition of SHP-1

    doi: 10.1038/ni.3692

    Figure Lengend Snippet: Inhibition of SHP-1 PTP activity rescues in vitro maturation of Themis −/− thymocytes Differentiation of DP thymocytes from Themis −/− or Themis +/+ to the CD4 + CD8 − stage in a two step (stimulation-rest) in vitro assay with or without the SHP-1 inhibitor Sodium stibogluconate (SSG). Left, two parameter plots show CD4 versus CD8 staining profiles of thymocytes at the completion of the differentiation assay. Cell recovery and % apoptotic (Annexin V + ) cells were not significantly different in similarly treated Themis −/− or Themis +/+ samples. Right, Summary of results. n=4 for each genotype (t-test 2-tailed type-2, error bars show SD). *** P

    Article Snippet: Sodium stibogluconate (CAS16037-91-5) was from EMD Millipore.

    Techniques: Inhibition, Activity Assay, In Vitro, Staining, Differentiation Assay, T-Test