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Butyrate treatment protects OVA-challenged mice from lung inflammation. Male C57BL/6 mice were intraperitoneally (IP) injected with OVA (30 μg) + Al(OH)3 (1.6 mg) at days 0 and 7. Mice also received 250 μl of either <t>PBS</t> or butyrate (1 M) (IP) at days 0, 1, 2, 7, 8, and 9. OVA-sensitized mice were nebulized with an OVA solution (3%) for 15 min at days 14, 15, and 16. The group that received butyrate during sensitization was also treated during challenge. A control group was sensitized and challenged without OVA. Euthanasia was performed 24 h after the last challenge. Lungs were digested and cells stained with monoclonal antibodies to determine the frequency of eosinophils (CD45+CD64-Ly6G-CD11b+Siglec-F+CD11c-) by flow cytometry (A) . Bar graph shows the frequency of eosinophils in the different groups (B) . Lung tissues were also stained with hematoxylin/eosin (H E) and periodic acid–Schiff (PAS), scale bars: 100 μm. Thick and thin arrows indicate inflammatory infiltrates and mucus production, respectively (C) . Data are shown as mean ± SD. One-way ANOVA followed by Tukey's multiple comparison test were used for statistical analysis. n = 5–7 mice per group.
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1) Product Images from "Butyrate Attenuates Lung Inflammation by Negatively Modulating Th9 Cells"

Article Title: Butyrate Attenuates Lung Inflammation by Negatively Modulating Th9 Cells

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2019.00067

Butyrate treatment protects OVA-challenged mice from lung inflammation. Male C57BL/6 mice were intraperitoneally (IP) injected with OVA (30 μg) + Al(OH)3 (1.6 mg) at days 0 and 7. Mice also received 250 μl of either PBS or butyrate (1 M) (IP) at days 0, 1, 2, 7, 8, and 9. OVA-sensitized mice were nebulized with an OVA solution (3%) for 15 min at days 14, 15, and 16. The group that received butyrate during sensitization was also treated during challenge. A control group was sensitized and challenged without OVA. Euthanasia was performed 24 h after the last challenge. Lungs were digested and cells stained with monoclonal antibodies to determine the frequency of eosinophils (CD45+CD64-Ly6G-CD11b+Siglec-F+CD11c-) by flow cytometry (A) . Bar graph shows the frequency of eosinophils in the different groups (B) . Lung tissues were also stained with hematoxylin/eosin (H E) and periodic acid–Schiff (PAS), scale bars: 100 μm. Thick and thin arrows indicate inflammatory infiltrates and mucus production, respectively (C) . Data are shown as mean ± SD. One-way ANOVA followed by Tukey's multiple comparison test were used for statistical analysis. n = 5–7 mice per group.
Figure Legend Snippet: Butyrate treatment protects OVA-challenged mice from lung inflammation. Male C57BL/6 mice were intraperitoneally (IP) injected with OVA (30 μg) + Al(OH)3 (1.6 mg) at days 0 and 7. Mice also received 250 μl of either PBS or butyrate (1 M) (IP) at days 0, 1, 2, 7, 8, and 9. OVA-sensitized mice were nebulized with an OVA solution (3%) for 15 min at days 14, 15, and 16. The group that received butyrate during sensitization was also treated during challenge. A control group was sensitized and challenged without OVA. Euthanasia was performed 24 h after the last challenge. Lungs were digested and cells stained with monoclonal antibodies to determine the frequency of eosinophils (CD45+CD64-Ly6G-CD11b+Siglec-F+CD11c-) by flow cytometry (A) . Bar graph shows the frequency of eosinophils in the different groups (B) . Lung tissues were also stained with hematoxylin/eosin (H E) and periodic acid–Schiff (PAS), scale bars: 100 μm. Thick and thin arrows indicate inflammatory infiltrates and mucus production, respectively (C) . Data are shown as mean ± SD. One-way ANOVA followed by Tukey's multiple comparison test were used for statistical analysis. n = 5–7 mice per group.

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

2) Product Images from "Butyrate Attenuates Lung Inflammation by Negatively Modulating Th9 Cells"

Article Title: Butyrate Attenuates Lung Inflammation by Negatively Modulating Th9 Cells

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2019.00067

Butyrate treatment negatively impact Th9 cells in the lungs of OVA-challenged mice. Male C57BL/6 mice were intraperitoneally (IP) injected with OVA (30 μg) + Al(OH)3 (1.6 mg) at days 0 and 7. Mice also received 250 μl of either PBS or butyrate (1M) (IP) at days 0, 1, 2, 7, 8, and 9. OVA-sensitized mice were nebulized with an OVA solution (3%) for 15 min at days 14, 15, and 16. The group that received butyrate during sensitization was also treated during challenge. A control group was sensitized and challenged without OVA. Euthanasia was performed 24 h after the last challenge (A) . Lungs were digested, cells stimulated with PMA (50 ng/ml), ionomycin (500 ng/ml) and brefeldin A (5 μg/ml) for 4 h at 37°C and 5% CO 2 , and stained with monoclonal antibodies to determine the frequencies of IL-9+, IL-13+, and FOXP3+ CD4+ T cells by flow cytometry (B) . Bar graphs show the frequencies of IL-9+ (C) , IL-13+ (D) and FOXP3+ (E) CD4+ T cells in the different groups. Data are shown as mean ± SD. One-way ANOVA followed by Tukey's multiple comparison test were used for statistical analysis. n = 5–7 mice per group. NS, not significant.
Figure Legend Snippet: Butyrate treatment negatively impact Th9 cells in the lungs of OVA-challenged mice. Male C57BL/6 mice were intraperitoneally (IP) injected with OVA (30 μg) + Al(OH)3 (1.6 mg) at days 0 and 7. Mice also received 250 μl of either PBS or butyrate (1M) (IP) at days 0, 1, 2, 7, 8, and 9. OVA-sensitized mice were nebulized with an OVA solution (3%) for 15 min at days 14, 15, and 16. The group that received butyrate during sensitization was also treated during challenge. A control group was sensitized and challenged without OVA. Euthanasia was performed 24 h after the last challenge (A) . Lungs were digested, cells stimulated with PMA (50 ng/ml), ionomycin (500 ng/ml) and brefeldin A (5 μg/ml) for 4 h at 37°C and 5% CO 2 , and stained with monoclonal antibodies to determine the frequencies of IL-9+, IL-13+, and FOXP3+ CD4+ T cells by flow cytometry (B) . Bar graphs show the frequencies of IL-9+ (C) , IL-13+ (D) and FOXP3+ (E) CD4+ T cells in the different groups. Data are shown as mean ± SD. One-way ANOVA followed by Tukey's multiple comparison test were used for statistical analysis. n = 5–7 mice per group. NS, not significant.

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

Adoptive transfer of Th9 cells restores lung inflammation in butyrate-treated OVA-challenged mice. Male C57BL/6 mice were intraperitoneally (IP) injected with OVA (30 μg) + Al(OH)3 (1.6 mg) at days 0 and 7. Mice also received 250 μl of either PBS or butyrate (1 M) (IP) at days 0, 1, 2, 7, 8, and 9. OVA-sensitized mice were nebulized with an OVA solution (3%) for 15 min at days 14, 15, and 16. The groups that received butyrate during sensitization were treated during challenge. Butyrate-treated mice also received an adoptive transfer (IP) of OT-II Th0, Th2, or Th9 cells 24 h before challenge initiation. Euthanasia was performed 24 h after the last challenge (A) . Lungs were digested and cells stained with monoclonal antibodies to determine the frequency of eosinophils as shown by the bar graph (B) . Alternatively, cells were stimulated with PMA (50 ng/ml), ionomycin (500 ng/ml), and brefeldin A (5 μg/ml) for 4 h at 37°C and 5% CO 2 , and stained with monoclonal antibodies to determine the frequency of IL-9+CD4+ and IL-13+CD4+ T cells as shown by the bar graphs (C,D) , respectively. Lung tissues were also stained with hematoxylin/eosin (H E) and periodic acid–Schiff (PAS), scale bars: 100 μm. Thick and thin arrows indicate inflammatory infiltrates and mucus production, respectively (E) . Data are shown as mean ± SD. One-way ANOVA followed by Tukey's multiple comparison test were used for statistical analysis. n = 5–7 mice per group. NS, not significant.
Figure Legend Snippet: Adoptive transfer of Th9 cells restores lung inflammation in butyrate-treated OVA-challenged mice. Male C57BL/6 mice were intraperitoneally (IP) injected with OVA (30 μg) + Al(OH)3 (1.6 mg) at days 0 and 7. Mice also received 250 μl of either PBS or butyrate (1 M) (IP) at days 0, 1, 2, 7, 8, and 9. OVA-sensitized mice were nebulized with an OVA solution (3%) for 15 min at days 14, 15, and 16. The groups that received butyrate during sensitization were treated during challenge. Butyrate-treated mice also received an adoptive transfer (IP) of OT-II Th0, Th2, or Th9 cells 24 h before challenge initiation. Euthanasia was performed 24 h after the last challenge (A) . Lungs were digested and cells stained with monoclonal antibodies to determine the frequency of eosinophils as shown by the bar graph (B) . Alternatively, cells were stimulated with PMA (50 ng/ml), ionomycin (500 ng/ml), and brefeldin A (5 μg/ml) for 4 h at 37°C and 5% CO 2 , and stained with monoclonal antibodies to determine the frequency of IL-9+CD4+ and IL-13+CD4+ T cells as shown by the bar graphs (C,D) , respectively. Lung tissues were also stained with hematoxylin/eosin (H E) and periodic acid–Schiff (PAS), scale bars: 100 μm. Thick and thin arrows indicate inflammatory infiltrates and mucus production, respectively (E) . Data are shown as mean ± SD. One-way ANOVA followed by Tukey's multiple comparison test were used for statistical analysis. n = 5–7 mice per group. NS, not significant.

Techniques Used: Adoptive Transfer Assay, Mouse Assay, Injection, Staining

IL-9 treatment has no impact on IL-9+, IL-13+, and FOXP3+ CD4+ T cells in the lungs of butyrate-treated OVA-challenged mice. Male C57BL/6 mice were intraperitoneally (IP) injected with OVA (30 μg) + Al(OH)3 (1.6 mg) at days 0 and 7. Mice also received 250 μl of either PBS or butyrate (1 M) (IP) at days 0, 1, 2, 7, 8, and 9. OVA-sensitized mice were nebulized with an OVA solution (3%) for 15 min at days 14, 15, and 16. The groups that received butyrate during sensitization were also treated during challenge. Butyrate-treated mice also received either PBS or recombinant IL-9 (IP) at days 14 and 15. Euthanasia was performed 24 h after the last challenge. Lungs were digested, cells stimulated with PMA (50 ng/ml), ionomycin (500 ng/ml), and brefeldin A (5 μg/ml) for 4 h at 37°C and 5% CO 2 , and stained with monoclonal antibodies to determine the frequencies of IL-9+ (A) , IL-13+ (B) , and FOXP3+ (C) CD4+ T cells in the different groups. Data are shown as mean ± SD. One-way ANOVA followed by Tukey's multiple comparison test were used for statistical analysis. n = 5–7 mice per group. NS, not significant.
Figure Legend Snippet: IL-9 treatment has no impact on IL-9+, IL-13+, and FOXP3+ CD4+ T cells in the lungs of butyrate-treated OVA-challenged mice. Male C57BL/6 mice were intraperitoneally (IP) injected with OVA (30 μg) + Al(OH)3 (1.6 mg) at days 0 and 7. Mice also received 250 μl of either PBS or butyrate (1 M) (IP) at days 0, 1, 2, 7, 8, and 9. OVA-sensitized mice were nebulized with an OVA solution (3%) for 15 min at days 14, 15, and 16. The groups that received butyrate during sensitization were also treated during challenge. Butyrate-treated mice also received either PBS or recombinant IL-9 (IP) at days 14 and 15. Euthanasia was performed 24 h after the last challenge. Lungs were digested, cells stimulated with PMA (50 ng/ml), ionomycin (500 ng/ml), and brefeldin A (5 μg/ml) for 4 h at 37°C and 5% CO 2 , and stained with monoclonal antibodies to determine the frequencies of IL-9+ (A) , IL-13+ (B) , and FOXP3+ (C) CD4+ T cells in the different groups. Data are shown as mean ± SD. One-way ANOVA followed by Tukey's multiple comparison test were used for statistical analysis. n = 5–7 mice per group. NS, not significant.

Techniques Used: Mouse Assay, Injection, Recombinant, Staining

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Cytometry:

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    Becton Dickinson cd3 pacific blue pb
    Epratuzumab leads to decreased surface expression of the adhesion molecules CD62L and β7 integrin on CD27 negative B-cells . Comparison of the surface expression of CD62L on PBMCs from SLE patients with (grey histogram) and without (white histogram) epratuzumab incubation (a) . Monocytes (CD14 positive ) showed a moderate but non-significant reduction of CD62L, whereas this expression was not influenced on T-cells <t>(CD3</t> positive ) by epratuzumab. Notably, epratuzumab led to a significant reduction of the CD62L surface expression on B-cells ( P
    Cd3 Pacific Blue Pb, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson cd8 pb
    CD4 depletion during Mtb infection diminished the ability to <t>CD8+</t> T cells to sustain Th17-like/Th1-like and cytotoxic-like effector responses
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    Becton Dickinson 1x pbs
    Ultra-small hybrid nanoparticle stability studies. (a) Size measurements of ultra-small hybrid nanoparticles and ultra-small PLGA cores over the course of 2 weeks in both 10 mM Tris-HCl at pH 8 and <t>1X</t> PBS (n = 3; mean ± SD). (b) Absorbance at
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    Becton Dickinson anti cd4 pb
    Increased downregulation of CD127 expression and upregulation of PD-1 expression in IFN-γ-producing <t>CD4</t> + T cells. PBMCs were stimulated for 18–20 h with T . cruzi lysate or media alone. Cells were stained with FV510, CD4, CD127, CD132 and CD279 (PD-1) monoclonal antibodies followed by fixation and permeabilization for intracellular staining with anti-IFN-γ monoclonal antibody. Each symbol represents the expression of CD127 +/— CD132 + (A-C), or PD-1 (D) in IFN-γ-producing and IFN-γ nonproducing CD4 + T cells (D). Median values are indicated as horizontal lines. Comparisons between IFN-γ-producing and IFN-γ nonproducing groups were performed using paired t test. * p ≤ 0.05 and ** p ≤ 0.01 compared with IFN-γ nonproducing T cells. **** p ≤ 0.001 compared with media.
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    Epratuzumab leads to decreased surface expression of the adhesion molecules CD62L and β7 integrin on CD27 negative B-cells . Comparison of the surface expression of CD62L on PBMCs from SLE patients with (grey histogram) and without (white histogram) epratuzumab incubation (a) . Monocytes (CD14 positive ) showed a moderate but non-significant reduction of CD62L, whereas this expression was not influenced on T-cells (CD3 positive ) by epratuzumab. Notably, epratuzumab led to a significant reduction of the CD62L surface expression on B-cells ( P

    Journal: Arthritis Research & Therapy

    Article Title: Epratuzumab targeting of CD22 affects adhesion molecule expression and migration of B-cells in systemic lupus erythematosus

    doi: 10.1186/ar3179

    Figure Lengend Snippet: Epratuzumab leads to decreased surface expression of the adhesion molecules CD62L and β7 integrin on CD27 negative B-cells . Comparison of the surface expression of CD62L on PBMCs from SLE patients with (grey histogram) and without (white histogram) epratuzumab incubation (a) . Monocytes (CD14 positive ) showed a moderate but non-significant reduction of CD62L, whereas this expression was not influenced on T-cells (CD3 positive ) by epratuzumab. Notably, epratuzumab led to a significant reduction of the CD62L surface expression on B-cells ( P

    Article Snippet: The following antibodies were used: CD3-Pacific Blue (PB) or H7-allophycocyanin (APC) (BD, Clone UCHT1), CD14-PB or H7-APC (BD, Clone m5e2), CD19-phycoerythrin-cyanin 7 (PE-Cy7) (BD, Clone SJ 25C1), CD20-peridin chlorophyll protein (PerCP) (BD, Clone L27), CD62L-fluorescein isothiocyanate (FITC) (Clone 145/15, Miltenyi Biotec, Auburn, CA, USA), CD27-cyanin 5 (Cy5) (clone 2E4, kindly provided by Rene Van Lier, University of Amsterdam, The Netherlands), β7 integrin-phycoerythrin (PE) (BD, clone FIB504), β1 integrin-PE (BD, clone MAR4), CD22-PE (BD, clone S-HCL-1) and epratuzumab IgG and F(ab')2 fragment of epratuzumab (provided by UCB, Slough, UK).

    Techniques: Expressing, Incubation

    Enhanced migration of CD27 negative B-cells from SLE patients towards CXCL12 after epratuzumab incubation . To assess the migration towards CXCL12, CXCL13 and CXCR3 ligands of PBMCs from eight SLE patients, we performed transwell migration assays after epratuzumab incubation (10 μg/mL for 90 minutes). The migration of different cell types towards CXCL12 was analyzed by flow cytometry [(T-cells, CD3 positive ), (monocytes, CD14 positive ), (total B-cells CD19 positive ); CD27 negative B-cells CD27 positve B-cells] and epratuzumab incubation lead to a significantly enhanced migration of B-cells (a) (* P

    Journal: Arthritis Research & Therapy

    Article Title: Epratuzumab targeting of CD22 affects adhesion molecule expression and migration of B-cells in systemic lupus erythematosus

    doi: 10.1186/ar3179

    Figure Lengend Snippet: Enhanced migration of CD27 negative B-cells from SLE patients towards CXCL12 after epratuzumab incubation . To assess the migration towards CXCL12, CXCL13 and CXCR3 ligands of PBMCs from eight SLE patients, we performed transwell migration assays after epratuzumab incubation (10 μg/mL for 90 minutes). The migration of different cell types towards CXCL12 was analyzed by flow cytometry [(T-cells, CD3 positive ), (monocytes, CD14 positive ), (total B-cells CD19 positive ); CD27 negative B-cells CD27 positve B-cells] and epratuzumab incubation lead to a significantly enhanced migration of B-cells (a) (* P

    Article Snippet: The following antibodies were used: CD3-Pacific Blue (PB) or H7-allophycocyanin (APC) (BD, Clone UCHT1), CD14-PB or H7-APC (BD, Clone m5e2), CD19-phycoerythrin-cyanin 7 (PE-Cy7) (BD, Clone SJ 25C1), CD20-peridin chlorophyll protein (PerCP) (BD, Clone L27), CD62L-fluorescein isothiocyanate (FITC) (Clone 145/15, Miltenyi Biotec, Auburn, CA, USA), CD27-cyanin 5 (Cy5) (clone 2E4, kindly provided by Rene Van Lier, University of Amsterdam, The Netherlands), β7 integrin-phycoerythrin (PE) (BD, clone FIB504), β1 integrin-PE (BD, clone MAR4), CD22-PE (BD, clone S-HCL-1) and epratuzumab IgG and F(ab')2 fragment of epratuzumab (provided by UCB, Slough, UK).

    Techniques: Migration, Incubation, Flow Cytometry, Cytometry

    The binding capacity of epratuzumab on different PBMCs obtained from SLE patients . (a) FACS analyses were performed on PBMCs from SLE patients using PE- labeled epratuzumab. Representative histogram of the differential binding of epratuzumab on T-cells (CD3 positive , dotted line), monocytes (CD14 positive , black histogram) and B-cells (CD19 positive , black line). (b) PBMCs were incubated with (grey histogram) or without (black line) unlabelled F(ab') 2 epratuzumab fragment for 10 minutes at 4°C. PBMCs were then stained with PE labeled-epratuzumab, and epratuzumab binding analyzed on B-cells, T-cells and monocytes ( n = 3). Representative histogram of epratuzumab binding on B-cell sub-populations: CD27 negative B-cells (black line), CD27 positive B-cells (grey histogram) and T-cells (negative control, dotted line) are shown in (c) . The results of the FACS analysis (right graph), showed higher binding capacity of epratuzumab on CD27 negative B-cells compare to CD27 positive B-cells ( P = 0.0002). (d) To study the expression of CD22 on B-cells, PBMCs were stained with a mouse anti-CD22 mAb (Clone S-HCL-1), which recognizes a different epitope than epratuzumab ( n = 5) [ 16 ]. The FACS analysis demonstrated that CD22 is more highly expressed on CD27 negative B-cells compared to CD27 positive B-cells.

    Journal: Arthritis Research & Therapy

    Article Title: Epratuzumab targeting of CD22 affects adhesion molecule expression and migration of B-cells in systemic lupus erythematosus

    doi: 10.1186/ar3179

    Figure Lengend Snippet: The binding capacity of epratuzumab on different PBMCs obtained from SLE patients . (a) FACS analyses were performed on PBMCs from SLE patients using PE- labeled epratuzumab. Representative histogram of the differential binding of epratuzumab on T-cells (CD3 positive , dotted line), monocytes (CD14 positive , black histogram) and B-cells (CD19 positive , black line). (b) PBMCs were incubated with (grey histogram) or without (black line) unlabelled F(ab') 2 epratuzumab fragment for 10 minutes at 4°C. PBMCs were then stained with PE labeled-epratuzumab, and epratuzumab binding analyzed on B-cells, T-cells and monocytes ( n = 3). Representative histogram of epratuzumab binding on B-cell sub-populations: CD27 negative B-cells (black line), CD27 positive B-cells (grey histogram) and T-cells (negative control, dotted line) are shown in (c) . The results of the FACS analysis (right graph), showed higher binding capacity of epratuzumab on CD27 negative B-cells compare to CD27 positive B-cells ( P = 0.0002). (d) To study the expression of CD22 on B-cells, PBMCs were stained with a mouse anti-CD22 mAb (Clone S-HCL-1), which recognizes a different epitope than epratuzumab ( n = 5) [ 16 ]. The FACS analysis demonstrated that CD22 is more highly expressed on CD27 negative B-cells compared to CD27 positive B-cells.

    Article Snippet: The following antibodies were used: CD3-Pacific Blue (PB) or H7-allophycocyanin (APC) (BD, Clone UCHT1), CD14-PB or H7-APC (BD, Clone m5e2), CD19-phycoerythrin-cyanin 7 (PE-Cy7) (BD, Clone SJ 25C1), CD20-peridin chlorophyll protein (PerCP) (BD, Clone L27), CD62L-fluorescein isothiocyanate (FITC) (Clone 145/15, Miltenyi Biotec, Auburn, CA, USA), CD27-cyanin 5 (Cy5) (clone 2E4, kindly provided by Rene Van Lier, University of Amsterdam, The Netherlands), β7 integrin-phycoerythrin (PE) (BD, clone FIB504), β1 integrin-PE (BD, clone MAR4), CD22-PE (BD, clone S-HCL-1) and epratuzumab IgG and F(ab')2 fragment of epratuzumab (provided by UCB, Slough, UK).

    Techniques: Binding Assay, FACS, Labeling, Incubation, Staining, Negative Control, Expressing

    CD4 depletion during Mtb infection diminished the ability to CD8+ T cells to sustain Th17-like/Th1-like and cytotoxic-like effector responses

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

    Article Title: CD4+ T cells are required to contain early extrathoracic TB dissemination and sustain multi-effector functions of CD8+ T and CD3− lymphocytes

    doi: 10.4049/jimmunol.1301373

    Figure Lengend Snippet: CD4 depletion during Mtb infection diminished the ability to CD8+ T cells to sustain Th17-like/Th1-like and cytotoxic-like effector responses

    Article Snippet: The following Abs were used for culture or surface and intracellular cytokine staining for flow cytometry: CD28 (CD28.2, BD), CD49d (9F10, BD), CD3-PECY7 (SP34-2, BD), CD3-PB (SP34-2, BD), CD4-APC (L200, BD, used for measuring CD4 expression after CD4 depleting Ab treatment), CD8-PB (RPA-T8, BD), CD8-APC (RPA-T8, BD), CD8-PE (RPA-T8, BD), IFN-γ-Allophycocyanin (4S.B3, BD), IFN-γ-PE (4S.B3, BD), TNF-α-PE (MAb11, BD), TNF-α-APC (MAb11, BD), TNF-α-PB (MAb11, eBioscience), IL-17-PE (eBio64CAP17, eBioscience), IL-17-Alexa647 (eBio64CAP17, eBioscience), IL-22-biotinylated (anti-human IL-22, RD, see reference( )), Streptavidin-Pacific blue (invitrogen).

    Techniques: Infection

    Ultra-small hybrid nanoparticle stability studies. (a) Size measurements of ultra-small hybrid nanoparticles and ultra-small PLGA cores over the course of 2 weeks in both 10 mM Tris-HCl at pH 8 and 1X PBS (n = 3; mean ± SD). (b) Absorbance at

    Journal: Nanoscale

    Article Title: Ultra-Small Lipid-Polymer Hybrid Nanoparticles for Tumor-Penetrating Drug Delivery

    doi: 10.1039/c6nr04091h

    Figure Lengend Snippet: Ultra-small hybrid nanoparticle stability studies. (a) Size measurements of ultra-small hybrid nanoparticles and ultra-small PLGA cores over the course of 2 weeks in both 10 mM Tris-HCl at pH 8 and 1X PBS (n = 3; mean ± SD). (b) Absorbance at

    Article Snippet: The collected cells were washed 3 times with 1X PBS and measured on a Becton Dickinson FACSCanto II flow cytometer using the red laser and Alexa Fluor 647 filter.

    Techniques:

    Increased downregulation of CD127 expression and upregulation of PD-1 expression in IFN-γ-producing CD4 + T cells. PBMCs were stimulated for 18–20 h with T . cruzi lysate or media alone. Cells were stained with FV510, CD4, CD127, CD132 and CD279 (PD-1) monoclonal antibodies followed by fixation and permeabilization for intracellular staining with anti-IFN-γ monoclonal antibody. Each symbol represents the expression of CD127 +/— CD132 + (A-C), or PD-1 (D) in IFN-γ-producing and IFN-γ nonproducing CD4 + T cells (D). Median values are indicated as horizontal lines. Comparisons between IFN-γ-producing and IFN-γ nonproducing groups were performed using paired t test. * p ≤ 0.05 and ** p ≤ 0.01 compared with IFN-γ nonproducing T cells. **** p ≤ 0.001 compared with media.

    Journal: PLoS Neglected Tropical Diseases

    Article Title: Trypanosoma cruzi-specific IFN-γ-producing cells in chronic Chagas disease associate with a functional IL-7/IL-7R axis

    doi: 10.1371/journal.pntd.0006998

    Figure Lengend Snippet: Increased downregulation of CD127 expression and upregulation of PD-1 expression in IFN-γ-producing CD4 + T cells. PBMCs were stimulated for 18–20 h with T . cruzi lysate or media alone. Cells were stained with FV510, CD4, CD127, CD132 and CD279 (PD-1) monoclonal antibodies followed by fixation and permeabilization for intracellular staining with anti-IFN-γ monoclonal antibody. Each symbol represents the expression of CD127 +/— CD132 + (A-C), or PD-1 (D) in IFN-γ-producing and IFN-γ nonproducing CD4 + T cells (D). Median values are indicated as horizontal lines. Comparisons between IFN-γ-producing and IFN-γ nonproducing groups were performed using paired t test. * p ≤ 0.05 and ** p ≤ 0.01 compared with IFN-γ nonproducing T cells. **** p ≤ 0.001 compared with media.

    Article Snippet: Cells were stained with FV510 and anti-CD4 PB, anti-CD127 Alexa Fluor 647, anti-CD132 PE and anti-PD-1 PE-Cy7 monoclonal antibodies (BD Bioscience) for 30 min on ice followed by fixation and permeabilization for intracellular staining with anti-IFN-γ (AF488) (BD, Bioscience).

    Techniques: Expressing, Staining

    Serum cytokine levels are altered in subjects chronically infected with T . cruzi . IL-21, IL-27 and sCD127 serum levels were measured using ELISA, and IL-6 and IL-8 levels were measured using CBA. Each point represents the serum levels of IL-21 (A), IL-27 (B), IL-6 (C), and IL-8 (D). Values under the limit of detection are graphed as zero. Horizontal lines indicate median values. Black symbols indicate subjects treated with benznidazole. Based on the ELISPOT assay, responses of T . cruzi- infected subjects were used to determine IFN-γ producers (P) and IFN-γ nonproducers (NP), as described in Materials and Methods. Comparisons between P and NP for each clinical group and uninfected subjects were performed using ANOVA followed by Dunn’s multiple comparisons test. * p ≤ 0.05. (A) ### p ≤ 0.001 compared with P G0, NP G0 and P G1; (B) ## p ≤ 0.01 compared with P G0; (C) ## p ≤ 0.01 compared with P G2-G3 and NP G2-G3; (D) # p ≤ 0.05 compared with NP G0, ## p ≤ 0.01 compared with P G0. Correlation analysis between IL-6 levels and the frequency of CD127 + CD132 + memory CD4 + and CD8 + T cells (E). IL-8 levels and the frequency of CD127 — CD132 + memory CD4 + T cells (F). IL-8 levels and sCD127 (G) were assessed using Spearman correlation analysis.

    Journal: PLoS Neglected Tropical Diseases

    Article Title: Trypanosoma cruzi-specific IFN-γ-producing cells in chronic Chagas disease associate with a functional IL-7/IL-7R axis

    doi: 10.1371/journal.pntd.0006998

    Figure Lengend Snippet: Serum cytokine levels are altered in subjects chronically infected with T . cruzi . IL-21, IL-27 and sCD127 serum levels were measured using ELISA, and IL-6 and IL-8 levels were measured using CBA. Each point represents the serum levels of IL-21 (A), IL-27 (B), IL-6 (C), and IL-8 (D). Values under the limit of detection are graphed as zero. Horizontal lines indicate median values. Black symbols indicate subjects treated with benznidazole. Based on the ELISPOT assay, responses of T . cruzi- infected subjects were used to determine IFN-γ producers (P) and IFN-γ nonproducers (NP), as described in Materials and Methods. Comparisons between P and NP for each clinical group and uninfected subjects were performed using ANOVA followed by Dunn’s multiple comparisons test. * p ≤ 0.05. (A) ### p ≤ 0.001 compared with P G0, NP G0 and P G1; (B) ## p ≤ 0.01 compared with P G0; (C) ## p ≤ 0.01 compared with P G2-G3 and NP G2-G3; (D) # p ≤ 0.05 compared with NP G0, ## p ≤ 0.01 compared with P G0. Correlation analysis between IL-6 levels and the frequency of CD127 + CD132 + memory CD4 + and CD8 + T cells (E). IL-8 levels and the frequency of CD127 — CD132 + memory CD4 + T cells (F). IL-8 levels and sCD127 (G) were assessed using Spearman correlation analysis.

    Article Snippet: Cells were stained with FV510 and anti-CD4 PB, anti-CD127 Alexa Fluor 647, anti-CD132 PE and anti-PD-1 PE-Cy7 monoclonal antibodies (BD Bioscience) for 30 min on ice followed by fixation and permeabilization for intracellular staining with anti-IFN-γ (AF488) (BD, Bioscience).

    Techniques: Infection, Enzyme-linked Immunosorbent Assay, Crocin Bleaching Assay, Enzyme-linked Immunospot

    Increased frequency of TTE cells in patients with chronic Chagas disease. PBMCs were stained with FV510, CD45RA, CD8, CD4, CD127, and CD132 monoclonal antibodies and analyzed using flow cytometry. Each symbol represents the proportion of CD127 — CD132 + cells in total CD4 + CD45RA + (A) or CD8 + CD45RA + (B) T cell populations from IFN-γ producers (P) and IFN-γ nonproducers (NP), as classified according to ELISPOT responses as defined in Materials and Methods. Median values are indicated as horizontal lines. Black symbols indicate subjects treated with benznidazole. Comparisons between P and NP for each clinical group and uninfected subjects were performed using ANOVA followed by Dunn’s multiple comparisons test. (A) ## p ≤ 0.01 compared to NP G1 and NP G2-G3; ### p ≤ 0.001 compared with P G1 and NP G0; #### p ≤ 0.0001 compared with P G0 and P G2-G3; (B) ## p ≤ 0.001 compared with P G0 and NP G0.

    Journal: PLoS Neglected Tropical Diseases

    Article Title: Trypanosoma cruzi-specific IFN-γ-producing cells in chronic Chagas disease associate with a functional IL-7/IL-7R axis

    doi: 10.1371/journal.pntd.0006998

    Figure Lengend Snippet: Increased frequency of TTE cells in patients with chronic Chagas disease. PBMCs were stained with FV510, CD45RA, CD8, CD4, CD127, and CD132 monoclonal antibodies and analyzed using flow cytometry. Each symbol represents the proportion of CD127 — CD132 + cells in total CD4 + CD45RA + (A) or CD8 + CD45RA + (B) T cell populations from IFN-γ producers (P) and IFN-γ nonproducers (NP), as classified according to ELISPOT responses as defined in Materials and Methods. Median values are indicated as horizontal lines. Black symbols indicate subjects treated with benznidazole. Comparisons between P and NP for each clinical group and uninfected subjects were performed using ANOVA followed by Dunn’s multiple comparisons test. (A) ## p ≤ 0.01 compared to NP G1 and NP G2-G3; ### p ≤ 0.001 compared with P G1 and NP G0; #### p ≤ 0.0001 compared with P G0 and P G2-G3; (B) ## p ≤ 0.001 compared with P G0 and NP G0.

    Article Snippet: Cells were stained with FV510 and anti-CD4 PB, anti-CD127 Alexa Fluor 647, anti-CD132 PE and anti-PD-1 PE-Cy7 monoclonal antibodies (BD Bioscience) for 30 min on ice followed by fixation and permeabilization for intracellular staining with anti-IFN-γ (AF488) (BD, Bioscience).

    Techniques: Staining, Flow Cytometry, Cytometry, Enzyme-linked Immunospot

    Altered STAT5 phosphorylation in IFN-γ nonproducers. pSTAT5 frequency was evaluated after stimulation with rhIL-7 in total CD4 + (left) and CD8 + (right) T cell populations using flow cytometry. Each point represents the difference (Δ) in the percentage of pSTAT5 expression between rhIL-7-stimulated and unstimulated samples (A-B) or basal frequency of pSTAT5 + (C-D). Horizontal lines indicate median values. Black symbols indicate subjects treated with benznidazole. Based on the ELISPOT assay, responses of T . cruzi -infected subjects were used to determine the IFN-γ producers (P) or IFN-γ nonproducers (NP), as described in Materials and Methods. Comparisons between P and NP for each clinical group and uninfected subjects were performed using ANOVA followed by Dunn’s multiple comparisons test. * p ≤ 0.05, ** p ≤ 0.01. (A) ## p ≤ 0.01 compared with NP G1 and NP G2-G3; (B) # p ≤ 0.05 compared with NP G0, ## p ≤ 0.01 compared with NP G2-G3 and NP G1; (C) ## p ≤ 0.01 compared with NP G1 and NP G2-G3; (D) # p ≤ 0.05 compared with NP G1, ### p ≤ 0.001 compared with NP G2-G3.

    Journal: PLoS Neglected Tropical Diseases

    Article Title: Trypanosoma cruzi-specific IFN-γ-producing cells in chronic Chagas disease associate with a functional IL-7/IL-7R axis

    doi: 10.1371/journal.pntd.0006998

    Figure Lengend Snippet: Altered STAT5 phosphorylation in IFN-γ nonproducers. pSTAT5 frequency was evaluated after stimulation with rhIL-7 in total CD4 + (left) and CD8 + (right) T cell populations using flow cytometry. Each point represents the difference (Δ) in the percentage of pSTAT5 expression between rhIL-7-stimulated and unstimulated samples (A-B) or basal frequency of pSTAT5 + (C-D). Horizontal lines indicate median values. Black symbols indicate subjects treated with benznidazole. Based on the ELISPOT assay, responses of T . cruzi -infected subjects were used to determine the IFN-γ producers (P) or IFN-γ nonproducers (NP), as described in Materials and Methods. Comparisons between P and NP for each clinical group and uninfected subjects were performed using ANOVA followed by Dunn’s multiple comparisons test. * p ≤ 0.05, ** p ≤ 0.01. (A) ## p ≤ 0.01 compared with NP G1 and NP G2-G3; (B) # p ≤ 0.05 compared with NP G0, ## p ≤ 0.01 compared with NP G2-G3 and NP G1; (C) ## p ≤ 0.01 compared with NP G1 and NP G2-G3; (D) # p ≤ 0.05 compared with NP G1, ### p ≤ 0.001 compared with NP G2-G3.

    Article Snippet: Cells were stained with FV510 and anti-CD4 PB, anti-CD127 Alexa Fluor 647, anti-CD132 PE and anti-PD-1 PE-Cy7 monoclonal antibodies (BD Bioscience) for 30 min on ice followed by fixation and permeabilization for intracellular staining with anti-IFN-γ (AF488) (BD, Bioscience).

    Techniques: Flow Cytometry, Cytometry, Expressing, Enzyme-linked Immunospot, Infection

    Altered STAT5 downstream events in IFN-γ nonproducers. Bcl-2 and CD25 expression was determined using flow cytometry analysis after 48 h of in vitro stimulation with rhIL-7. Each symbol represents the IL-7-induced CD25 expression after (Δ % CD25 + ) (A-B) or the basal CD25 + expression (C-D). The difference in Bcl-2 mean fluorescence intensity (MFI) between IL-7-stimulated and unstimulated cell cultures (E-F) or the basal Bcl-2 expression (G-H) in CD4 + (left) or CD8 + (right) T cells. Horizontal lines indicate median values. Black symbols indicate subjects treated with benznidazole. Based on the ELISPOT assay, responses of T . cruzi -infected subjects were used to determine the IFN-γ producers (P) and IFN-γ nonproducers (NP), as described in Materials and Methods. Comparisons between P and NP for each clinical group and uninfected subjects were performed using ANOVA followed by Dunn’s multiple comparisons test. * p ≤ 0.05, ** p ≤ 0.01. (A) ### p ≤ 0.001 compared with NP G2-G3; (B) # p ≤ 0.05 compared with NP G2-G3; (D) # p ≤ 0.05 compared with NP G0, ## p ≤ 0.01 compared with NP G1, ### p ≤ 0.001 compared with NP G2-G3; (E, F) # p ≤ 0.05 compared with NP G0 and NP G1, ## p ≤ 0.01 compared with NP G2-G3; (G) ## p ≤ 0.01 compared with NP G0, NP G1 and NP G2-G3; (H) # p ≤ 0.05 compared with NP G0, NP G1, NP G2-G3.

    Journal: PLoS Neglected Tropical Diseases

    Article Title: Trypanosoma cruzi-specific IFN-γ-producing cells in chronic Chagas disease associate with a functional IL-7/IL-7R axis

    doi: 10.1371/journal.pntd.0006998

    Figure Lengend Snippet: Altered STAT5 downstream events in IFN-γ nonproducers. Bcl-2 and CD25 expression was determined using flow cytometry analysis after 48 h of in vitro stimulation with rhIL-7. Each symbol represents the IL-7-induced CD25 expression after (Δ % CD25 + ) (A-B) or the basal CD25 + expression (C-D). The difference in Bcl-2 mean fluorescence intensity (MFI) between IL-7-stimulated and unstimulated cell cultures (E-F) or the basal Bcl-2 expression (G-H) in CD4 + (left) or CD8 + (right) T cells. Horizontal lines indicate median values. Black symbols indicate subjects treated with benznidazole. Based on the ELISPOT assay, responses of T . cruzi -infected subjects were used to determine the IFN-γ producers (P) and IFN-γ nonproducers (NP), as described in Materials and Methods. Comparisons between P and NP for each clinical group and uninfected subjects were performed using ANOVA followed by Dunn’s multiple comparisons test. * p ≤ 0.05, ** p ≤ 0.01. (A) ### p ≤ 0.001 compared with NP G2-G3; (B) # p ≤ 0.05 compared with NP G2-G3; (D) # p ≤ 0.05 compared with NP G0, ## p ≤ 0.01 compared with NP G1, ### p ≤ 0.001 compared with NP G2-G3; (E, F) # p ≤ 0.05 compared with NP G0 and NP G1, ## p ≤ 0.01 compared with NP G2-G3; (G) ## p ≤ 0.01 compared with NP G0, NP G1 and NP G2-G3; (H) # p ≤ 0.05 compared with NP G0, NP G1, NP G2-G3.

    Article Snippet: Cells were stained with FV510 and anti-CD4 PB, anti-CD127 Alexa Fluor 647, anti-CD132 PE and anti-PD-1 PE-Cy7 monoclonal antibodies (BD Bioscience) for 30 min on ice followed by fixation and permeabilization for intracellular staining with anti-IFN-γ (AF488) (BD, Bioscience).

    Techniques: Expressing, Flow Cytometry, Cytometry, In Vitro, Fluorescence, Enzyme-linked Immunospot, Infection

    CD127 cell-surface expression is downregulated in memory CD4 + and CD8 + T cells in IFN-γ producers. PBMCs were stained with FV510, CD45RA, CD8, CD4, CD127, and CD132 monoclonal antibodies and analyzed using flow cytometry. T . cruzi- specific T-cell responses were determined using IFN-γ ELISPOT after stimulation of PBMCs with a T . cruzi lysate. Each symbol represents the number of T . cruzi -specific IFN-γ cells producing spots (CPS) by subtracting the value of wells containing media alone (A) and the proportion of CD127 +/— CD132 + cells among total CD4 + CD45RA — (B and C) or CD8 + CD45RA — (D and E) T-cell populations. Median values are indicated as horizontal lines. Black symbols indicate the subjects treated with benznidazole. The responses of T . cruzi -infected subjects were used to determine the IFN-γ producers (P) and IFN-γ nonproducers (NP) based on the ELISPOT assay, as described in Materials and Methods. Comparisons between P and NP for each clinical group and uninfected subjects were performed using ANOVA followed by Bonferroni’s multiple comparisons test. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001. ### p ≤ 0.001 compared to P G0, P G1 and P G2-G3.

    Journal: PLoS Neglected Tropical Diseases

    Article Title: Trypanosoma cruzi-specific IFN-γ-producing cells in chronic Chagas disease associate with a functional IL-7/IL-7R axis

    doi: 10.1371/journal.pntd.0006998

    Figure Lengend Snippet: CD127 cell-surface expression is downregulated in memory CD4 + and CD8 + T cells in IFN-γ producers. PBMCs were stained with FV510, CD45RA, CD8, CD4, CD127, and CD132 monoclonal antibodies and analyzed using flow cytometry. T . cruzi- specific T-cell responses were determined using IFN-γ ELISPOT after stimulation of PBMCs with a T . cruzi lysate. Each symbol represents the number of T . cruzi -specific IFN-γ cells producing spots (CPS) by subtracting the value of wells containing media alone (A) and the proportion of CD127 +/— CD132 + cells among total CD4 + CD45RA — (B and C) or CD8 + CD45RA — (D and E) T-cell populations. Median values are indicated as horizontal lines. Black symbols indicate the subjects treated with benznidazole. The responses of T . cruzi -infected subjects were used to determine the IFN-γ producers (P) and IFN-γ nonproducers (NP) based on the ELISPOT assay, as described in Materials and Methods. Comparisons between P and NP for each clinical group and uninfected subjects were performed using ANOVA followed by Bonferroni’s multiple comparisons test. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001. ### p ≤ 0.001 compared to P G0, P G1 and P G2-G3.

    Article Snippet: Cells were stained with FV510 and anti-CD4 PB, anti-CD127 Alexa Fluor 647, anti-CD132 PE and anti-PD-1 PE-Cy7 monoclonal antibodies (BD Bioscience) for 30 min on ice followed by fixation and permeabilization for intracellular staining with anti-IFN-γ (AF488) (BD, Bioscience).

    Techniques: Expressing, Staining, Flow Cytometry, Cytometry, Enzyme-linked Immunospot, Infection

    Decreased frequency of recent thymic emigrants in IFN-γ producers. PBMCs were stained with FV510, CD45RA, CD8, CD4, CD127, and CD132 monoclonal antibodies and analyzed using flow cytometry. Each symbol represents the proportion of CD4 + CD45RA + CD127 + CD132 — (A) or CD8 + CD45RA + CD127 + CD132 — (B) T-cell populations from IFN-γ producers (P) and IFN-γ nonproducers (NP), as classified according to ELISPOT responses as defined in Materials and Methods. Median values are indicated as horizontal lines. Black symbols indicate subjects treated with benznidazole. Comparison between P and NP for each clinical group and uninfected subjects were performed using ANOVA followed by Bonferroni’s multiple comparisons test. * p ≤ 0.05. (A) # p ≤ 0.05 compared with P G0, NP G0, P G1 and P G2-G3; (B) # p ≤ 0.05 compared with P G2-G3; ## p ≤ 0.01 compared with P G0; ### p ≤ 0.01 compared with P G1.

    Journal: PLoS Neglected Tropical Diseases

    Article Title: Trypanosoma cruzi-specific IFN-γ-producing cells in chronic Chagas disease associate with a functional IL-7/IL-7R axis

    doi: 10.1371/journal.pntd.0006998

    Figure Lengend Snippet: Decreased frequency of recent thymic emigrants in IFN-γ producers. PBMCs were stained with FV510, CD45RA, CD8, CD4, CD127, and CD132 monoclonal antibodies and analyzed using flow cytometry. Each symbol represents the proportion of CD4 + CD45RA + CD127 + CD132 — (A) or CD8 + CD45RA + CD127 + CD132 — (B) T-cell populations from IFN-γ producers (P) and IFN-γ nonproducers (NP), as classified according to ELISPOT responses as defined in Materials and Methods. Median values are indicated as horizontal lines. Black symbols indicate subjects treated with benznidazole. Comparison between P and NP for each clinical group and uninfected subjects were performed using ANOVA followed by Bonferroni’s multiple comparisons test. * p ≤ 0.05. (A) # p ≤ 0.05 compared with P G0, NP G0, P G1 and P G2-G3; (B) # p ≤ 0.05 compared with P G2-G3; ## p ≤ 0.01 compared with P G0; ### p ≤ 0.01 compared with P G1.

    Article Snippet: Cells were stained with FV510 and anti-CD4 PB, anti-CD127 Alexa Fluor 647, anti-CD132 PE and anti-PD-1 PE-Cy7 monoclonal antibodies (BD Bioscience) for 30 min on ice followed by fixation and permeabilization for intracellular staining with anti-IFN-γ (AF488) (BD, Bioscience).

    Techniques: Staining, Flow Cytometry, Cytometry, Enzyme-linked Immunospot